Livestock Research for Rural Development 24 (4) 2012 Guide for preparation of papers LRRD Newsletter

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Effect of supplements of potassium nitrate or urea as sources of NPN on methane production in an in vitro system using molasses and Paper mulberry or Muntingia foliages as the substrate

Phonevilay Silivong, T R Preston* and Ngo Van Man**

Souphanouvong University, Lao PDR
silipvl@yahoo.co.th
* Finca Ecologica, TOSOLY, UTA (Colombia) AA#48, Socorro, Santander, Colombia
** Department of Animal Physiology and Biochemistry, 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 Paper mulberry and Muntingia foliages in an in vitro incubation system with molasses as the energy source. The incubation was for 24 h with measurements of total gas and percent methane at intervals of 6, 12, 18 and 24 hours and determination of residual unfermented substrate at the end of each interval.

Gas production, percent methane in the gas and methane produced per unit DM solubilized, at each incubation interval, were higher with urea than with potassium nitrate as NPN source. The fermentability of the substrate was higher for Paper mulberry than for Muntingia as the forage source but there were no differences between the two foliages in methane produced per unit substrate DM solubilised. 

Key words: Climate change, fermentation, greenhouse gases, incubation


Introduction

In developing improved systems for feeding live stock, account must also be taken of the impacts on the environment. It is estimated that live stock presently account for some 18% of total greenhouses gases which contribute to 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 reduces methane production in goats fed sugar cane (Nguyen Ngoc Anh et al 2010) and rice straw (Sophea and Preston 2011) as basal diets.

When urea is fed to cattle it often is recommended to dissolve the urea in molasses as one way to avoid possible toxicity problems with the urea (Preston and Leng 87, web version 2009).  The same idea should be applied 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.   

Muntingia calabura belonging to the family Elaeocarpacae grows everywhere (sandy land, humid areas, and high land area) and is well adapted to the dry season in Lao PDR. The farmers use it as shade tree around the homestead, and along the roads. It is a tall tree with a large canopy of leaves but it is not normally fed to animals (Nguyen Xuan Ba et al 2003). However,  it was shown recently to be palatable to goats supporting intakes of more than 40 g of DM/kg LW DM with a DM digestibility of 68% (Tran Trung Tuan 2008).

Paper mulberry (Broussonetia papyrifera) of the family Moraceae is a tree that is common in the northern area of  Lao growing to 15 meters tall. The leaves are variable shape (even on the same branch) (http://en.wikipedia.org/wiki/Paper_Mulberry), and contain crude protein of 20% in DM according to Inthapanya and Preston (2009). The leaves could be a potential feed resource for goats. Presently the bark of Paper mulberry is used in the handicraft industry to make paper and envelopes. The leaves would be a by-product from this process. Some farmers indicate the leaves can be used to feed to pigs. It is reported that the twigs and young leaves are consumed by deer, so it should be palatable to goats (http://en.wikipedia.org/wiki/Paper_Mulberry).

The purpose of the present study was to use a simple in vitro method to determine methane production from a substrate based on molasses as energy supplemented with protein from leaves of Paper mulberry and Muntingia and using potassium nitrate and urea as sources of non-protein nitrogen. 

Hypotheses

The hypothesis to be tested was:

         Methane production in an in vitro system will be reduced when potassium nitrate replaces urea as NPN source with substrates of foliages of Paper mulberry or Muntingia 


Materials and methods

Location and duration  
The experiment was carried out at the Animal Science laboratory of the Faculty of Agriculture and Forest Resources, Souphanouvong University, Luang Prabang province, Lao PDR. The experiment was conducted from May to June 2011. 
Treatments and experimental design 

The experimental design was a 2*2 factorial arrangement of 4 treatments with four replications of each treatment.

The factors were:

Source of NPN:
Source of foliage:

Individual treatments were:

Preparation of substrate and the in vitro system

A simple in vitro system was used (Photo 1; Inthapanya et al 2011) with recycled plastic bottles as flasks for the incubation and gas collection. The leaves from Paper mulberry and Muntingia foliages were chopped into small pieces (2-3mm) and dried in oven at 60C for 24 h 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 960 ml of buffer solution (Table 2) and 240 ml of rumen fluid from a buffalo. The rumen fluid was taken at 3.00-4.00 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 5.00 am the following  morning when the contents were filtered through  a layer of cloth before being added to the incubation bottle. 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 38C in a water bath for 24 hours. 

 Table 1. Composition of diets (%  DM basis)

 

U-MG

U-PM

KN-MG

KN-PM

Molasses

68.17

68.17

64.0

64.0

Muntingia

30.00

 

30.0

 

Paper mulberry

 

30.00

 

30.0

Urea

1.83

1.83

   

Potassium nitrate

   

6.0

6.0

Total

100

100

100

100

 

Table 2. Ingredients of the buffer solution (g/liter)

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

Source: Tilly and Terry (1963) 

 

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

 Chemical analyses 

Samples of Paper mulberry, Muntingia foliage  and residual substrate were analysed for DM, ash and N according to methods outlined in Ly and Nguyen Van Lai (1997). The residual DM in the incubation bottle was determined by filtering through cloth and drying (70C for 24 h).  Protein solubility was determined by shaking three-gramme samples with 100ml M NaCl for 3 hours, filtering through Whatman No. 4 filter paper and determining the nitrogen content of the filtrate. (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, foliage source, interaction NPN*foliages and error. 


Results and discussion

Chemical composition

The DM content of Paper mulberry was lower and ash and crude protein higher than in Muntingia (Table 3). The solubility of the protein was relatively low in the leaves of both foliages and similar to levels reported for cassava leaves (Inthapanya et al 2012).

Table 3. The chemical composition of substrate ingredients  (% in DM, except DM which is on fresh basis)

DM

N*6.25

Ash

Protein solubility, %

Molasses

80.4

5.4

10.5

 

Muntingia leaves

44.9

16.6

4.6

12.5

Muntingia stem

37.3

6.8

4.2

 

Paper mulberry  leaves

29.0

26.7

11.9

19.4

Paper mulberry stem

17.0

15.8

12.4

 

 
Gas production and concentration of methane

Gas production, percent methane in the gas and methane produced per unit substrate solubilized increased with length of incubation (Table 4; Figures 1-4). All of these criteria were lower when the NPN source was potassium nitrate rather than urea but there were no differences between the sources of the leaves (Figure 5). The percent of substrate solubilized decreased with incubation time with the rate of decrease apparently greater for nitrate than for urea (Figure 2).

 

Table 4. Mean value for gas production, percentage of methane in the gas, methane production (ml),

DM solubilized and methane production per DM solubilized according to source of  NPN and origin of the leaves

(Paper mulberry PM; Muntingia MG)

 

PM

MG

Prob.

KN

Urea

Prob.

SEM

0-6 hours

             

Gas production, ml

990

1079

0.662

812

1257

0.044

139

Methane, %

10.3

9.3

0.005

8.5

11.0

<0.001

0.2

Digested, %

73.1

68.2

0.006

72.6

68.7

0.025

1.05

Methane, ml/g DM solubilised

12.0

13.5

0.573

8.5

16.9

0.005

1.74

0-12 hours

             

Gas production, ml

1368

1054

0.166

1011

1411

0.084

150

Methane, %

11.3

10.3

0.233

9.2

12.4

<0.001

0.54

Digested, %

79.1

66.9

<0.001

74.7

71.3

0.158

1.60

Methane, ml/g DM solubilised

17.3

15.5

0.637

11.3

21.5

0.019

2.65

0-18 hours

             

Gas production, ml

1324

873

0.136

751

1446

0.030

200

Methane, %

12.0

10.8

0.436

9.3

13.5

0.020

1.13

Digested, %

74.2

58.0

<0.001

62.4

69.8

0.049

2.38

Methane, ml/g DM solubilised

19.8

15.3

0.399

10.7

24.4

0.020

3.63

0-24 hours

             

Gas production, ml

1551

1366

0.472

1159

1758

0.034

177

Methane, %

13.1

12.5

0.505

10.9

14.8

<0.001

0.64

Digested, %

68.6

57.8

0.003

60.2

66.2

0.061

2.03

Methane, ml/g DM solubilised

25.9

26.5

0.918

18.8

33.7

0.023

4.04

 

Figure 1. Effect of incubation time on gas production

 

Figure 2. Effect of incubation time on methane content in the gas

 

Figure 3. Effect of NPN source and incubation time on percent substrate solubilised

Figure 4. Effect of NPN source and incubation time on

methane production per unit substrate solubilised.


Figure 5. Effect of NPN source on methane production per unit

substrate solubilized from Paper mulberry (PM) and Muntingia (MG) leaves.

The reduction in methane production per unit substrate solubilized, with nitrate compared with urea,  is similar to the results 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. The fact that there were no differences between the leaves from Muntingia and from Paper mulberry suggests that the two sources of leaves had similar characteristics in terms of content, or absence, of anti-nutritional compounds such as cyanogenic glucosides, tannins or saponins, compounds which are known to affect methanogenesis in in vitro incubations (Cuzin and Labat 1992; Thanh et al 2011;  Inthapanya et al 2012). 

The increase in methane production with length of incubation is also in agreement with other reports from similar in vitro systems (Outhen et al 2011; Inthapanya et al 2011; Binh Phuong et al 2011 and  Thanh et al 2011). However, the slight reduction in substrate solubilized with length of incubation is in contrast with the earlier findings in the same in vitro system (Outhen et al 2011; Inthapanya et al 2011; Binh Phuong et al 2011 and  Thanh et al 2011). We have no explanation for this effect.


Conclusions


Acknowledgements

This research was submitted to Cantho University by the senior author  as part of the requirements for the MSc degree in Animal Production "Specialized in Response to Climate Change and Depletion of Non-renewable Resources". The authors acknowledge support for this research from the MEKARN project financed by Sida. Special thanks to Mr Jator and Mr Nouyang who provided valuable help in the laboratory. They also thank the staff of Department of Animal Science laboratory, Faculty of Agriculture and Forest Resource, Souphanouvong University for helping to carry out this research.


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Received 6 March 2012; Accepted 26 March 2012; Published 2 April 2012

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