Livestock Research for Rural Development 25 (1) 2013 | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
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
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.
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.
The experimental design was a 2*6 factorial arrangement of 12 treatments with four replications of each treatment.
The factors were:
Source of NPN:
KNO3 or none
Source of foliage:
Jack fruit, Muntingia, Leucaena, Gliricidia, Mimosa, Acacia
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 |
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 |
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 (70°C for 48h).
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.
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 |
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 |
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 |
Figure
4.
Relationship between % DM solubilized and methane |
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 in the foliages.
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.
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Received 12 June 2012; Accepted 22 December 2012; Published 4 January 2013