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

Citation of this paper

Methane production in an in vitro rumen incubation is reduced when leaves from Mimosa pigra are the protein source compared with Gliricidia sepium

Phonevilay Silivong, Xaykham Onphachanh, Aloun Ounalom and T R Preston*

Souphanouvong University, Lao PDR
silipvl@yahoo.co.th
* Center for Research and Technology Transfer, 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 Mimosa and Gliricidia leaves in an in vitro incubation system with molasses as the energy source. The incubation was for 24 h with measurements of total gas and methane concentration over incubation periods (separate experiments) of 6, 12, 18 and 24 hours and determination of residual unfermented substrate at the end of each experimental period.  

Gas production, percentage methane in the gas and methane produced per unit DM solubilised for each incubation were lower with potassium nitrate  than with urea.  Methane production per unit DM solubilised was less with Mimosa than with Gliricidia in incubations over 18 and 24h, but did not differ between the two kinds of leaves  for the 6h and 12h incubations.

Keywords: climate change, fermentation, greenhouse gases, incubation


Introduction

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

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

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

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. 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 (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 would confer bypass properties on the protein in the Mimosa and that this could explain its high nutritive value.

Gliricidia (Gliricidia sepium)  is a medium-sized tree that can grow to 10 to 12 m high. The foliage has been used as a protein supplements for low quality forages and resulted in improved ruminant productivity (Norton 1994).

The purpose of the present study was to test if there were differences in methane production when leaf meals from Mimosa and Gliricidia were incubated in a rumen in vitro system using molasses as energy source and potassium nitrate or urea as sources of non-protein nitrogen.


Materials and methods

Location and duration

The experiment was carried out from November to December 2011 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*3 factorial arrangement of 6 treatments with four replications of each treatment.

The factors were:

Source of foliage:   Gliricidia (GD) or Mimosa (MP)

Source of NPN: Potassium nitrate (KN), Urea (U) or no NPN (Non).

Preparation of substrate and the in vitro system

The in vitro system (Photo 1) used recycled plastic bottles as flasks for the incubation and gas collection. The leaves from Gliricidia and Mimosa foliages were chopped into small piece (2-3mm) and dried at 60C for 24h 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 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  a layer of cloth before being added to the incubation bottles. 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 24h.

Table 1. Composition of diets (% DM basis)

 

MP-U

MP-KN

MP

GD-U

GD-KN

GD

Molasses

67.17

58.7

66

67.67

87.4

66.5

Mimosa

31

35.3

34

     

Gliricidia

     

30.5

6.6

33.5

Urea

1.83

   

1.83

   

Potassium nitrate

6

   

6

 

Total

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: Tilley 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 as separate experiments 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 2. Measurement of percentage of methane in the gas
Chemical analyses

Samples of Gliricidia and Mimosa leaves  and residual substrate were analysed for DM, ash and N according to methods outlined in Ly and  Lai (1997). The residual DM in the incubation bottle was determined by filtering through cloth and drying (70C for 24h). The solubility  of the protein in the leaves was measured by extraction in M NaCl (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, leaves, interaction NPN*leaves and error.


Results and discussion

Chemical composition

The contents of DM, ash and crude protein of Mimosa leaves were higher than  in Gliricidia (Table 3). The solubility of the protein was relatively low in the leaves of both foliages.

Table 3. The chemical composition of substrate ingredients

 

DM

N*6.25

Ash

N solubility

 

%

As % of DM

 %

Molasses

80.5

5.4

9.2

 

Mimosa leaves

45.9

25.3

4.7

16.4

Mimosa stem

47.7

15.8

3.6

 

Gliricidia  leaves

35.1

21.1

7.6

18.2

Gliricidia stem

32.6

9.2

6.1

 

Gas production and concentration of methane

Gas production, percentage methane in the gas and methane produced per unit substrate solubilised increased with incubation time and were lower when the NPN source was potassium nitrate rather than urea (Table 4: Figure 1). The increase in methane production with length of incubation is in agreement with other reports from similar in vitro systems (Outhen et al 2011, Inthapanya et al 2011, Binh Phuong et al 2011; Thanh et al 2011). 

 Methane production per unit DM solubilised was less with Mimosa than with Gliricidia for the combined incubations over 18 and 24h, but did not differ between the two kinds of leaves  for the 6 and 12h incubations (Figure 4).

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 (Gliricidia GD; Mimosa MP)

 

GD

MP

Prob.

SEM

KN

U

Non

Prob.

SEM

0-6 hours

                 

Gas production, ml

458

476

0.84

62.0

321a

526b

553b

0.088

76.0

Methane, %

7.4

10.3

0.023

0.8

5.0a

10.9b

10.6

0.001

1.0

Methane, ml/g DM solubilised

4.4

5.6

0.297

0.8

2.4

6.4

6.3

0.01

0.9

0-12 hours

                 

Gas production, ml

503

300

0.097

82.1

301

544

360

0.232

101

Methane, %

8.3

5.6

0.132

1.2

3.6a

8.8b

8.4b

0.039

1.5

Methane, ml/g DM solubilised

4.7

2.8

0.169

0.9

1.4a

5.8b

4.0b

0.042

1.2

0-18 hours

                 

Gas production, ml

813

653

0.352

117.9

718

740

741

0.991

144

Methane, %

13.8

12.7

0.322

0.8

9.9a

14.3b

15.5b

0.001

0.9

Methane, ml/g DM solubilised

12.5

8.6

0.182

2.0

8.0

11.3

12.4

0.425

2.4

0-24 hours

                 

Gas production, ml

950

767

0.345

133.6

854

826

895

0.956

164

Methane, %

13.4

12.4

0.331

0.7

9.7a

14.2b

14.9b

0.001

0.9

Methane, ml/g DM solubilised

14.1

9.8

0.183

2.2

9.2

12.3

14.3

0.411

2.7

ab Means without common superscript differ at p<9.05


Figure 1. Effect of fermentation time on gas production Figure 2. Effect of fermentation time on methane content in the gas

Figure 3. Effect of NPN source on methane production per unit substrate solubilised

 The reduction in methane production per unit substrate solubilized, with nitrate compared with urea,  is similar to that 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.  For incubations lasting 18 and 24h methane production per unit DM solubilized was lower when the substrate was Mimosa leaves compared with  Gliricidia (Figure 4).

Figure 4. Effect of source of substrate on methane production per unit substrate solubilised

There are several reports showing different rates of production of methane in in vitro rumen incubations and in in vivo studies when different sources of foliage were compared. Thus in an in vitro incubation (Silivong et al 2013), Mimosa supported the lowest rate of production of methane in a comparison with four other foliages (Artocarpus heterophyllus, Acacia auriculoformis, Leucaena leucocephala and Muntingia calabura). In a metabolism study (Kongvongxay et al 2011), partial replacement of Muntingia calabura by Mimosa led to a reduction in methane concentration in eructed gases in growing goats. A similar reduction in methane content of eructed gases in growing goats was reported by Hang et al (2012) when foliage of Melia azedarach was supplemented with Mimosa. In contrast, there is one report of  higher methane production in goats fed Mimosa as compared with Gliricidia sepium (Silivong et al 2012). Condensed tannins have been implicated as agents in forages that depress methane production (Huang et al 2011) and Thu Hong et al (2008) reported levels of condensed tannins of 4% in DM  in leaves  of Mimosa harvested after 6 weeks of re-growth. Presence of higher concentrations of cynaogenic glucosides in "bitter" cassava foliage was associated with reduced production of methane compared with sweet cassava having a lower level of these compounds (Phuong et al 2012). The solubility of the protein in Mimosa is low (Table 3; and Phonevilay et al 2013) and feeds with low protein solubility were reported to result in less methane production compared with feeds of high protein solubility (Preston et 2012; Ho Quang Do et al 2013),  perhaps suggesting that the protein in the rumen is fermented with the production of methane. Mimosa as the sole feed has supported high growth rates (of the order of 100 g/day; Thu Hong et al 2008). Protein supplements of low solubility have long been associated with improved ruminant animal productivity (Whitelaw et al 1963) and feeding cassava foliage fresh rather than sun-dried (the former has higher levels of cyanogenic glucosides; Phuong et al 2012) supported better growth in growing goats (Kounnavongsa et al 2010).

There is therefore evidence that tropical foliages differ in the extent to which they influence methane production as well as production traits in growing goats and that the two criteria (reduction in methane and increase in growth performance) appear to be positively related.


Conclusions


Acknowledgements

This research was done by the senior author as part of the requirements for the MSc degree in Animal Production submitted to Cantho University . The authors acknowledge support for this research from the MEKARN project financed by Sida. Special thanks to Mr Aloun who provided valuable help in the laboratory. I also thank the staff of Department of Animal Science laboratory, Faculty of Agriculture and Forest Resource, Souphanouvong University for providing the facilities to carry out this research.


References

Anh Nguyen Ngoc, Khuc Thi Hue, Duong Nguyen Khang and Preston T R 2010 Effect of calcium nitrate as NPN source on growth performance and methane emissions of goats fed sugar cane supplemented with cassava foliage. MEKARN Conference 2010  Live stock production, climate change and resource depletion http://www.mekarn.org/workshops/pakse/abstracts/anh_grrc.htm 

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.htm

Ffoulkes D and Preston T R 1978 Cassava or sweet potato forage as combined sources of protein and roughage in molasses based diets: effect of supplementation with soybean meal. Tropical Animal Production Volume 3,  Number 3, pp 186-192 http://www.utafoundation.org/TAP/TAP33/3_3_1.pdf

Hang B P T, Lam V and Preston T R 2012  Effect on growth of goats and enteric methane emissions of supplementing foliage of Melia azedarach with foliage of Mimosa pigra. Livestock Research for Rural Development. Volume 24, Article #227. http://www.lrrd.org/lrrd24/12/hang24227.htm
 

Ho Quang Do, Tran Duy Khoa, Trinh Phuc Hao, Preston T R and Leng R A 2013 Methane production in an in vitro rumen incubation is lower for leaves with low compared with high protein solubility.  Livestock Research for Rural Development. Volume 25, Article #134. http://www.lrrd.org/lrrd25/7/hqdo25134.htm
 

Huang X D, Liang J B, Tan H Y, Yahya R and Ho Y W 2011 Effects of Leucaena condensed tannins of differing molecular weights on in vitro CH4 production. Animal Feed Science and Technology. Volume 166, Pages 373-376

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

Kongvongxay S, Preston T R, Leng R A and Khang D N 2011 Effect of a tannin-rich foliage (Mimosa pigra) on feed intake, digestibility, N retention and methane production in goats fed a basal diet of Muntingia calabura. Livestock Research for Rural Development. Volume 23, Article #48. Retrieved June 17, 2011, from http://www.lrrd.org/lrrd23/3/sito23048.htm

Kounnavongsa B, Phengvichith V and Preston T R 2010  Effects of fresh or sun-dried cassava foliage on growth performance of goats fed basal diets of Gamba grass or sugar cane stalk. Livestock Research for Rural Development. Volume 22, Article #202. http://www.lrrd.org/lrrd22/11/boun22202.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

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

Phonevilay S, Bounmar H and Preston T R 2012: 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. Livestock Research for Rural Development. Volume 25, Article #15. http://www.lrrd.org/lrrd25/1/phon25015.htm

Phuong L T B, Preston T R and Leng R A 2012: Effect of foliage from “sweet” and “bitter” cassava varieties on methane production in in vitro incubation with molasses supplemented with potassium nitrate or urea. Livestock Research for Rural Development. Volume 24, Article #189. Retrieved June 21, 2013, from http://www.lrrd.org/lrrd24/10/phuo24189.htm

Preston T R and Leng R A 2009 Matching Ruminant Production Systems with Available Resources in the Tropics and Sub-Tropics. http://www.utafoundation.org/P&L/preston&leng.htm

Preston T R, Do H Q, Khoa T D, Hao T P 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 #16. http://www.lrrd.org/lrrd25/1/hqdo25016.htm


Silivong P, Xaykham O, Aloun O and Preston T R 2012
Effect of potassium nitrate and urea on feed intake, digestibility, N balance and methane production of goats fed a basal diet of Gliricidia (Gliricidia sepium) and Mimosa (Mimosa pigra) foliages supplemented with molasses. Livestock Research for Rural Development. Volume 24, Article #138. Retrieved June 21, 2013, from http://www.lrrd.org/lrrd24/8/phon24138.htm

Phonevilay S, Bounmar H and Preston T R 2013 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. Livestock Research for Rural Development. Volume 25, Article #15. http://www.lrrd.org/lrrd25/1/phon25015.htm

Sophea Iv and Preston T R 2011: Effect of different levels of supplementary potassium nitrate replacing urea on growth rates and methane production in goats fed rice straw, mimosa foliage and water spinach. Livestock Research for Rural Development. Volume 23, Article #71. Retrieved June 16, 2011, from http://www.lrrd.org/lrrd23/4/soph23071.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.

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

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.

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

Whitelaw F G, Preston T R and Dawson G S 1962 The nutrition of the early-weaned calf. II.  a comparison of commercial groundnut meal, heat-treated groundnut meal and fish meal as the major protein source in the diet. Animal Production Volume 4. http://www.utafoundation.org/publications/whitelaw&preston 1962.pdf

Whitelaw F G and Preston T R 1963 The nutrition of the early-weaned calf.  III. Protein solubility and amino acid composition as factors affecting protein utilisation. Animal Production. Volume 5 pp 131-145.


Received 25 May 2013; Accepted 24 June 2013; Published 1 July 2013

Go to top