Livestock Research for Rural Development 25 (1) 2013 Guide for preparation of papers LRRD Newsletter

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Methane production from Jack fruit, Muntingia, Leucaena, Gliricidia (Gliricidia sepium), Mimosa (Mimosa pigra) and Acacia auriculoformis foliages in an in vitro incubation with potassium nitrate as source of NPN

Phonevilay Silivong, Bounmar Hervaseng and T R Preston*

Souphanouvong University, Lao PDR
silipvl@yahoo.co.th
* Finca Ecologica, TOSOLY, AA#48, Socorro, Santander, Colombia

Abstract

The aim of this study was to evaluate the effect of potassium nitrate on methane production from the leaves of a range of fodder trees (Jack fruit [Artocarpus heterophyllus], Muntingia calabura, Leucaena leucocephala, Gliricidia sepium,  Mimosa pigra and Acacia auriculoformis) an in vitro incubation system. The incubation was for 24 h with measurements of total gas and per cent methane at intervals of 6, 12, 18 and 24 hours and determination of residual unfermented substrate at the end of each interval.

Gas production increased with length of incubation (Table 4) and was lower when nitrate was added to the substrate.  Percent methane in the gas and methane production increased with length of incubation and were always lower when nitrate was added to the substrate. The percentage DM solubilized and methane produced per unit DM solubilized  increased with incubation time, varied among the foliages and were lower when nitrate was present in the substrate. In the absence of nitrate, methane production from Mimosa was only some 50% of that from Gliricidia and Jackfruit.  Both percentage DM solubilized at 24h and methane production per unit DM solubilized were linearly and positively related with the solubility of the crude protein (N*6.25) in the foliages.

Keywords: climate change, digestibility, fermentation, greenhouse gases, solubility


Introduction

In developing improved systems for feeding livestock, account must therefore be taken of the impacts on the environment. It is estimated that live stock presently account for some 18% of greenhouses gases which contribute to global warming (Steinfeld et al 2006). 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 shown that nitrate reduces methane production in goats fed Muntingia and Paper mulberry (Phonevilay et al 2012); however, thee were no differences in methane production between the two foliages.

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 NO2 to NH3 thereby reducing the production of methane from CO2. Recognition that nitrate supplements in ruminant diets compete successfully for H2 and electrons (and decrease methane production) is a promising development (Leng 2008). 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).

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 (Storrs et al 2001). 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 (Nguyen Thi 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 in the mimosa leaves would confer bypass properties on the protein and that this could explain the  high nutritive value of mimosa foliage.

Gliricidia (Gliricidia sepium)  is a medium-sized tree that can grow to from 10 to 12 m high. The tree grows well in acidic soils with a pH of 4.5-6.2. The tree is found on volcanic soils in its native range in Central America and Mexico. However, it can also grow on sandy, clay and limestone soils. http://search.yahoo.com/search?p=Gliricidia&ei=UTF-8&fr=moz35  Gliricidia foliage has been used as a protein supplement for low quality forages and resulted in improved ruminant productivity (Norton 1994).

Muntingia calabura belonging to the family Elaeocarpacae grows everywhere in SE Asia (sandy land, humid areas, and high land area) and is well adapted to the dry season.  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 with a DM digestibility of 68% (Tran Trung Tuan 2008).  

The purpose of the present study was to determine the effects on methane production from the above foliages when they were incubated in an in vitro system in the presence of potassium nitrate. 


Materials and methods

Location and duration

The experiment was carried out from August to November 2012.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*6 factorial arrangement of 12 treatments with four replications of each treatment.

The factors were:

Source of NPN:

Source of foliage:

Preparation of substrate and the in vitro system

A simple in vitro system was used (Photo 1) with recycled plastic bottles as flasks for the incubation and gas collection. The leaves from the foliages were chopped into small pieces (2-3mm) and dried at 600C for 24h then ground with a coffee grinder, and mixed with the potassium nitrate (Table 1), The mixtures (12 g DM) were put in the incubator bottles  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  one 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 380C in a water bath for 24 hours. 

Table 1. Composition of substrates (% DM basis)

 

JF-KN

JF

MC-KN

MC

LL-KN

LL

GD-KN

GD

MP-KN

MP

A-KN

A

KNO3 (KN)

6

 

6

 

6

 

6

 

6

 

6

 

Jack Fruit (JF)

94

100

                   

Muntingia (MC)

   

94

100

               

Leucaena (LL)

       

94

100

           

Gliricidia (GD)

           

94

100

       

Mimosa (MP)

               

94

100

   

Acacia (A)

                   

94

100

Total

100

100

100

100

100

100

100

100

100

100

100

100



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

 

CaCl2

NaHPO4.12H2O

NaCl

KCl

MgSO4.7H2O

NaHCO3

Cysteine

 

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 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 3).

Photo 2.  Measurement of percentage of methane in the gas  

Photo 3. The substrate residue filtered though cloth

Chemical analyses

Samples of foliages were analysed for DM, ash, nitrogen solubilised 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 48h).  

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*foliage and error. 


Results and discussion

Chemical composition  

Crude protein content of the foliages was in the range of 16.4 to 25.0 with highest values for Mimosa, Gliricida and Leucana, and lower values for Muntingia, Jack fruit and Acacia (Table 3).

Table 3. the chemical composition of foliages  (% in DM, except DM which is on fresh basis)

 

DM

N*6.25

Ash

N solubility, %

Leucaena

33.3

23.8

4.81

28.1

Mimosa

37.2

25.0

6.07

12.8

Acacia

34.5

16.8

4.16

10.3

Gliricidia

24.8

23.9

5.51

21.9

Muntingia

40.8

16.3

4.55

30.2

Jack fruit

37.7

18.1

8.38

16.0

Methane production

Gas production increased with length of incubation (Table 4) and was lower when nitrate was added to the substrate.  Percent methane in the gas and methane production increased with length of incubation and were always lower when nitrate was added to the substrate (see Figures 1 and 2).  These results are similar to those reported earlier (Outhen et al (2011; Inthapanya et al 2011; Binh Phuong et al 2011; Thanh et al 2011) when nitrate was compared with urea in substrates providing some 70% fermentable carbohydrate and 30% protein-rich leaves. However, the present results appear to be the first where nitrate was supplied as an additional source of NPN in substrates composed entirely of protein-rich leaves.

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 foliages and NPN.

 

Source of leaves

 

 

K-nitrate

   

 

Acacia

Gliricidia

Jackfruit

Leucaena

Muntingia

Mimosa

Prob.

SEM

With

Without

Prob.

SEM

0-6 hours

 

 

 

 

 

 

 

 

 

 

 

 

Gas production, ml

141

346

260

298

186

165

<0.001

13.0

196

270

<0.001

7.5

Methane, %

4.4

10.4

2.5

4.5

0.0

4.1

<0.001

0.6

1.4

7.2

<0.001

0.4

Digested, %

13.7

34.5

32.8

29.0

10.0

8.8

<0.001

1.9

18.2

24.8

<0.001

1.1

Methane, ml/g DM solubilized

4.2

9.0

1.9

3.9

0.0

6.4

<0.001

0.70

1.3

7.2

<0.001

0.4

0-12 hours

 

 

 

 

 

 

 

 

 

 

 

 

Gas production, ml

190

415

378

354

224

205

<0.001

19.7

233

355

<0.001

11.3

Methane, %

5.0

14.3

9.3

5.5

3.8

4.5

<0.001

0.07

2.8

11.3

<0.001

0.4

Digested, %

15.9

36.7

35.0

31.2

12.1

11.1

<0.001

1.9

20.5

26.9

<0.001

1.1

Methane, ml/g DM solubilized

5.6

13.6

8.9

5.6

6.2

6.7

<0.001

0.90

2.5

13.0

<0.001

0.5

0-18 hours

 

 

 

 

 

 

 

 

 

 

 

 

Gas production, ml

219

458

440

389

244

236

<0.001

24.9

265.4

396.3

<0.001

14.4

Methane, %

7.8

16.6

12.4

6.6

5.1

5.5

<0.001

1.1

3.9

14.1

<0.001

0.6

Digested, %

20.1

39.7

38.3

34.6

16.3

15.2

<0.001

2.1

24.1

30.7

<0.001

1.2

Methane, ml/g DM solubilized

8.4

16.2

12.4

6.9

7.0

6.8

<0.001

1.5

3.7

15.5

<0.001

0.9

0-24 hours

 

 

 

 

 

 

 

 

 

 

 

 

Gas production, ml

260

516

534

443

269

268

<0.001

30.7

314.6

448.3

<0.001

17.7

Methane, %

13.3

21.1

16.6

13.1

6.3

8.5

<0.001

1.6

8.3

18.0

<0.001

0.9

Digested, %

20.6

41.4

40.1

35.8

16.6

15.7

<0.001

1.8

25.2

31.6

<0.001

1.1

Methane, ml/g DM solubilized

15.9

22.5

19.4

14.1

9.6

12.5

<0.001

2.7

9.3

22.0

<0.001

1.6

Total gas production, ml

810

1735

1611

1483

923

874

<0.001

82.0

1009

1469

<0.001

47.3

Total methane, ml

32.7

56.8

35.6

28.8

16.5

32.1

<0.001

4.8

15.6

52.0

<0.001

2.8

Overall methane, %

7.6

15.6

10.2

7.4

3.8

5.7

<0.001

0.8

4.1

12.7

<0.001

0.4


DM solubilized and methane produced per unit DM solubilized

The percentage DM solubilized and methane produced per unit DM solubilized  increased with incubation time, varied among the foliages and were lower when nitrate was present in the substrate (Table 4; Figure 2). In the absence of nitrate, methane production from Mimosa was only some 50% of that from Gliricidia and Jackfruit.  Both percentage DM solubilized at 24h and methane production per unit DM solubilized were linearly and possitively related with the solubility of the crude protein (N*6.25) in the foliages (Figures 3 and 4). Ho Quang Do et al (2013) reported similar relationships between CP solubility and methane production in in vitro incubations with substrates containing fish meal (low CP solubility) and peanut meal (high CP solubility) and foliages (water spinach, sweet potato vines of high CP solubility compared with cassava leaves and Sesbania sesban leaves of low solubility). The mechanisms by which the solubility of the protein might affect methane production are not understood. Ho Quang Do et al (2013) postulated that for feeds high in insoluble protein a greater proportion of the fermentable nutrients would escape from the rumen, thus resulting in decreased hydrogen production in the rumen and as a consequence a reduction in production of methane. However, research to test this hypothesis and other related mechanisms will require the use of more sophisticated analytical tools than is presently available in our laboratory. 


Figure 1. Effect of incubation time on methane production from a
range of foliages, supplemented or not with potassium nitrate
Figure 2. Effect of potassium nitrate on methane production in incubations with different
foliage leaves (in the nitrate treatment of Muntingia the level of methane was less than
5% which is the minimum that can be recorded on the Crowcom Infra-red analyser)

Figure 3. Relationship between N solubillity and methane
production per unit substrate solubilized

Figure 4. Relationship between % DM solubilized and methane
production per unit substrate solubilized


Conclusions


Acknowledgements

The authors acknowledge support for this research from the MEKARN project financed by Sida. We also thank the Faculty of Agriculture and Forest Resource, Souphanouvong University for providing the facilities to carry out this research.


References

Binh Phuong L T, Preston T R and Leng R A 2011 Mitigating methane production from ruminants; effect of supplementary sulphate and nitrate on methane production in an in vitro incubation using sugar cane stalk and cassava leaf meal as substrate. Livestock Research for Rural Development. Volume 23, Article #22. Retrieved July 15, 2011, from http://www.lrrd.org/lrrd23/2/phuo23022.ht

Ho Quang Do, Tran Duy Khoa, Trinh Phuc Hao, Preston T R and Leng R A 2013 Protein solubility of fish meal and groundnut meal and methane production in an in vitro incubation.  Livestock Research for Rural Development. Volume 25, Article #016. Retrieved , from http://www.lrrd.org/lrrd25/1/hqdo25016.htm

Inthapanya S, Preston T R and Leng R A 2011: 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. Livestock Research for Rural Development. Volume 23, Article #21. http://www.lrrd.org/lrrd23/2/sang23021.htm

Leng R A 2008 The potential of feeding nitrate to reduce enteric methane production in ruminants. A Report to the Departmernt of Climate Change Commonwealth Government of Australia. ACT Canberra Australia For paper and PPT presentation see http://www.penambulbooks.com/  

Leng R A and Preston T R 2010 Further considerations of the potential of nitrate as a high affinity electron acceptor to lower enteric methane production in ruminants. Livestock Research for Rural Development. Volume 22, Article #221. http://www.lrrd.org/lrrd22/12/leng22221.htm

Ly J and Nguyen Van Lai 1997 Laboratory manual.  http://www.mekarn.org/labman/Default.htm

Minitab 2000 Minitab Software Release 13

Norton B W 1994 Antinutritive and toxic factors in forage tree legumes. Tropical Tree Legumes in Tropical Agriculture. CAB International, pp 202-215

Nguyen Xuan Ba and Le Duc Ngoan 2003 Evaluation of some unconventional trees/plants as ruminant feeds  in Central Vietnam. In: Proceedings of Final National Seminar-Workshop on Sustainable Livestock Production on Local Feed Resources (Editors: Reg Preston and Brian Ogle). HUAF-SAREC, Hue City, 25 – 28 March, 2003. Retrieved September 28, 2003, from http://www.mekarn.org/sarec03/bahue.htm

Outhen P, Preston T R and Leng R A 2011 Effect of supplementation with urea or calcium nitrate and cassava leaf meal or fresh cassava leaf in an in vitro incubation using a basal substrate of sugar cane stalk. Livestock Research for Rural Development. Volume 23, Article #23.  http://www.lrrd.org/lrrd23/2/outh23023.htm

Silivong P, Preston T R and Man N V 2012: 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. Livestock Research for Rural Development. Volume 24, Article #69. Retrieved November 26, 2012, from http://www.lrrd.org/lrrd24/4/sili24069.htm

Steinfeld  H, Gerber P, Wassenaar T, Castel V, Rosales R, and Haan C 2006 Livestock’s long shadow. Food and Agriculture Organization of the United Nations, Rome, Italy.

Tilley J M A and Terry R  A  1963 A two stage technique for the in vitro digestion of forage crops. Journal of the British Grassland  Society 18 : 104.

Thanh V D, Preston T R and Leng R A 2011 Effect on methane production of supplementing a basal substrate of molasses and cassava leaf meal with mangosteen peel (Garcinia mangostana) and urea or nitrate in an in vitro incubation. Livestock Research for Rural Development. Volume 23, Article #98. Retrieved July 15, 2011, from http://www.lrrd.org/lrrd23/4/than23098.htm

Thu Hong N T, Quac V A, Kim Chung T T, Hiet B V, Mong N T and Huu P T 2008 Mimosa pigra for growing goats in the Mekong Delta of Vietnam. Livestock Research for Rural Development. Volume 20, Article #208. http://www.lrrd.org/lrrd20/12/hong20208.htm

Tran Triet, Le Cong Kiet, Nguyen Thi Lan Thi and Pham Quoc Dan  2007 The invasion by Mimosa pigra of wetlands of the Mekong Delta, Vietnam. [on-line]. Available from:  http://www.ento.csiro.au/weeds/pdf/mimosa_ symposium/07Trietetal.pdf

Tran Trung Tuan 2008 Effect of rumen fermentable substrate and source of bypass protein as supplements to a basal diet of Muntingia foliage on digestibility and N retention in growing goats. Miniproject, Mekarn MSc 2008-10. http://mekarn.org/msc2008-10/miniprojects/minpro/tuan.htm


Received 12 June 2012; Accepted 22 December 2012; Published 4 January 2013

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