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| Photo 1. The in vitro system made from recycled "pep" water bottles |
| Livestock Research for Rural Development 27 (11) 2015 | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
The aim of this study was to evaluate the effect of an increasing level of water spinach as source of soluble protein on methane production from leaves of Bauhinia acuminata and Guazuma ulmifolia. The experimental design in an in vitro incubation was a 2*4 factorial arrangement of 8 treatments with four replications. The factors were: source of leaves (Bauhinia or Guazuma); and level of water spinach (0, 5, 15 and 25% of substrate DM). Measurements were made of total gas, methane content in the gas and DM solubilized after incubations of 6, 12, 18 and 24h.
Gas production, methane concentration in the gas and methane produced per unit substrate solubilized were increased by the length of the incubation and by increasing levels of supplementation with water spinach in the substrate on both sources of leaves. Gas production, methane concentration in the gas and methane produced per unit substrate solubilized were higher when leaf meal from Guazuma ulmifolia replaced that from Bauhinia acuminata.
Key words: gas production, greenhouse gases, protein solubility, tannins
The greenhouse gases (GHG) emissions from the agriculture sector account for about 25.5% of total global radiative forcing and over 60% of anthropogenic sources (FAO 2009). Animal husbandry accounts for 18% of GHG emissions. Emission of methane (CH4) is responsible for nearly as much radiative forcing as all other non-CO2 GHG gases combined (Beauchemin and McGinn 2005). While atmospheric concentrations of GHGs have risen by about 39% since pre-industrial era, CH4 concentration has more than doubled during this period (WHO 2009). Reducing GHG emissions from agriculture, especially from livestock, should therefore be a top priority since it could curb global warming fairly rapidly (Sejian et al 2010).
Ruminants, such as cattle, buffalo, sheep and goats, are the major contributors of total methane agricultural emissions (Leng 1993; Lassey 2007; Chhabra et al 2009). In ruminants, the H2 produced in rumen fermentation is normally removed by the reduction of CO2 to methane.
There is a need to develop feeding systems for ruminants that will result in reduced emissions of methane gas from the enteric fermentation in these animals. Previous research showed that methane production was less when fish meal rather than groundnut meal was the substrate (Preston et al 2013). The differences in methane production appeared to be related to the solubility of the protein which was 16% in fish meal compared with 70% in groundnut meal. A similar finding was reported by Silivong and Preston (2015) who showed that addition of water spinach to the substrate in an in vitro rumen fermentation increased the rate of gas production and methane content in the gas. The protein in the water spinach was highly soluble (66%).
The hypothesis that underlined the present study was that water spinach, with its high content of soluble protein, would increase the rate of fermentation and production of methane when added to forages rich in insoluble protein..
The experimental design was a 2*4 factorial arrangement of 8 treatments with four replications.
The factors were:
The in vitro system used recycled plastic bottles as flasks for the incubation and gas collection (Photo 1; Inthapanya et al 2011).
The leaves from Bauhinia and Guazuma, and leaves plus stems of water spinach, were chopped into small pieces (3-5mm) and dried at 65°C for 48h then ground with a coffee grinder, and mixed according to the proportions shown in Table 1. The mixtures (12g DM) were put in the incubation bottle with 960ml of buffer solution (Table 2) and 240ml of rumen fluid. The rumen fluid was taken at 3.00-4.00am from a buffalo immediately after the animal was killed in the LuangPrabang abattoir. A representative sample of the rumen contents (including feed residues) was put in a vacuum flask and taken to the laboratory, and stored until 5.00am, 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 bottles were incubated at 38°C in a water bath for intervals of 6, 12, 18 and 24h, with separate incubations for each interval.
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Table 1. Composition of substrates (% DM basis) |
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BA#GU |
BA#GU-WS 5 |
BA#GU-WS 15 |
BA#GU-WS 25 |
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Leaf meal# |
80 |
75 |
65 |
55 |
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Water spinach |
0 |
5 |
15 |
25 |
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Molasses |
20 |
20 |
20 |
20 |
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Total |
100 |
100 |
100 |
100 |
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# Bauhinia or Guazuma leaf meals |
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Table 2. Ingredients of the buffer solution (g/liter) |
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CaCl2 |
NaHPO4.12H2O |
NaCl |
KCl |
MgSO4.7H2O |
NaHCO3 |
Cysteine |
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0.04 |
9.30 |
0.47 |
0.57 |
0.12 |
9.80 |
0.25 |
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Source : Tilly and Terry (1963) |
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At the end of each incubation, the methane concentration in the gas was measured with a Crowcon infra-red analyser (Crowcon Instruments Ltd, UK). The residual DM in the incubation bottle was determined by filtering through cloth and drying the residue (65°C for 72 h). Solubility of the protein in the leaves was determined by shaking 3g of dry leaf meal in 100 ml of M NaCl for 3h then filtering through Whatman No.4 filter paper, and determining the N content of the filtrate (Whitelaw et al 1963).
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| Photo 1. The in vitro system made from recycled "pep" water bottles |
The samples of foliage, water spinach and residual substrate were analysed for DM, ash and N according to AOAC (1990) methods.
The data were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab Software (version16.0). Sources of variation in the model were: Foliage source, water spinach level, interaction foliage*water spinach source and error.
Percentages of crude protein, ash and protein solubility were higher in water spinach than in Bauhinia and Guazuma leave, but DM was lower. The protein content and solubility in the leaves of Guazuma were higher than in Bauhinia, but ash was lower (Table 3).
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Table 3. The chemical composition of feed (% in DM, except DM which is on fresh basis) |
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DM |
N*6.25 |
Ash |
Protein solubility, % |
Tannin, % |
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Bauhinia |
35.5 |
14.7 |
6.6 |
23.4 |
1.1 |
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Guazuma |
36.0 |
18.4 |
3.9 |
33.3 |
- |
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Water spinach |
10.6 |
18.5 |
9.7 |
66.4 |
- |
Values for the gas production, percent methane in the gas and methane produced per unit substrate solubilized increased with length of incubation time (Table 4; Figures 1-16). Gas production and percent substrate solubilized were increased by increasing the level of water spinach in the substrate, and were higher for Guazuma than Bauhinia at each incubation interval.
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Table 4. Mean values for gas production, percentage of methane in the gas, methane production (ml), DM solubilized and methane production per unit DM solubilized according to leaf source (Bauhinia and Guazuma) and level of water spinach |
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BA |
GU |
p |
WP-0 |
WP-5 |
WP-15 |
WP-25 |
p |
SEM# |
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0-6 hours |
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Gas production, ml |
363 |
483 |
<0.001 |
286d |
354c |
476b |
575a |
<0.001 |
5.1/7.2 |
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Methane, % |
8.8 |
9.9 |
<0.001 |
7.6d |
8.9c |
9.6bc |
11.4a |
<0.001 |
0.2/0.2 |
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DM solubilized, % |
53.1 |
56.3 |
<0.001 |
49.2d |
53.8c |
56.4b |
59.4a |
0.001 |
0.2/0.3 |
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Methane, ml/g DM solubilized |
5.1 |
7.2 |
<0.001 |
3.7d |
4.9c |
6.8b |
9.2a |
<0.001 |
0.2/0.2 |
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0-12 hours |
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Gas production, ml |
483 |
781 |
<0.001 |
463d |
581c |
661b |
821a |
<0.001 |
9.4/13.3 |
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Methane, % |
13.8 |
15.9 |
<0.001 |
11.1d |
13.6c |
15.1bc |
19.6a |
<0.001 |
0.3/0.4 |
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DM solubilized, % |
56.5 |
60.2 |
<0.001 |
53.2d |
57.1c |
59.9b |
63.2a |
<0.001 |
0.3/0.5 |
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Methane, ml/g DM solubilized |
10.2 |
17.5 |
<0.001 |
8.2d |
11.7c |
14.1b |
21.4a |
<0.001 |
0.4/0.6 |
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0-18 hours |
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Gas production, ml |
584 |
1023 |
<0.001 |
584d |
766c |
856b |
1009a |
<0.001 |
9.1/12.9 |
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Methane, % |
18.9 |
21.8 |
<0.001 |
16.3d |
19.5c |
21.3b |
24.3a |
<0.001 |
0.2/0.3 |
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DM solubilized, % |
59.4 |
63.5 |
<0.001 |
56.1d |
60.1c |
62.4bc |
67.0a |
<0.001 |
0.4/0.6 |
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Methane, ml/g DM solubilized |
15.8 |
29.5 |
<0.001 |
14.3d |
21.4c |
24.5b |
30.4a |
<0.001 |
0.4/0.6 |
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0-24 hour |
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Gas production, ml |
734 |
1239 |
<0.001 |
775d |
973c |
1050b |
1149a |
<0.001 |
11.3/15.9 |
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Methane, % |
24.4 |
27.9 |
<0.001 |
20.4d |
24.8c |
27.9b |
31.5a |
<0.001 |
0.2/0.3 |
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DM solubilized, % |
61.8 |
67.3 |
<0.001 |
59.0d |
64.3c |
65.2bc |
69.6a |
<0.001 |
0.4/0.6 |
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Methane, ml/g DM solubilized |
24.4 |
43.3 |
<0.001 |
22.4d |
31.6c |
37.9b |
43.6a |
<0.001 |
0.8/1.1 |
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Figure 1.
Effect of Bauhinia (BA) and Guazuma (GU) leaf meals on gas production at increasing incubation intervals |
Figure 2.
Effect of increasing level of water spinach on gas production at increasing incubation intervals |
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Figure 3.
Effect of foliages from Bauhinia (BA) and Guazuma (GU) on methane content in the gas at increasing incubation intervals |
Figure 4.
Effect of increasing level of water spinach on methane content in the gas at increasing incubation intervals |
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Figure 5.
Effect of leaf meals from Bauhinia (BA) and Guazuma (GU) on substrate DM solubilized at increasing incubation intervals |
Figure 6.
Effect of level of water spinach on substrate DM solubilized at increasing incubation intervals |
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Figure 7.
Effect of leaf meals from Bauhinia (BA) and Guazuma (GU) on methane per unit DM solubilized at different incubation intervals |
Figure 8.
Effect of increasing level of water spinach in the substrate on methane per unit DM solubilized at increasing incubation intervals |
The increases in methane concentration in the gas and per unit DM solubilized, with incubation interval, are similar to the findings reported by Outhen et al (2011), Binh Phuong et al (2011) and Silivong and
Preston (2015). This is thought to be due to methane being increasingly produced by secondary fermentation from acetate (Inthapanya et al 2011) as the incubation interval increased.
The increases in methane production with increasing proportions of water spinach in the substrate, and the higher methane production for treatments with Guazuma leaves compared with Bauhinia leaves, were closely related to the degree of solubility of the protein in these different combinations of substrate (Table 3). . A similar finding was reported by Inthapanya and Preston (2014) when cassava leaves (protein solubility 25.6%) replaced water spinach (protein solubility 66.3%) in an in vitro incubation of urea-treated rice straw.
It is not possible to differentiate between the direct effects of increased protein solubility per and the indirect effect of reducing the concentrations and the level of anti-nutritional factors (eg: condensed tannins) in the substrate (Table 3) when Bauhinia was replaced by Guazuma, and the level of water spinach was increased. The positive role of tannins in reducing the activity of methanogenic bacteria has been reported by several researchers (Goel and Makkar 2012; Soltan et al 2012).
Increasing the length of the incubation in the in vitro rumen fermentation of leaves of Bauhinia acuminata and Guazuma ulmifolia increased gas production, methane concentration in the gas and methane produced per unit substrate solubilized.
Supplementation with water spinach increased the rate of gas production, the percentage DM solubilized, and the methane concentration in the gas and methane produced per unit substrate solubilized on both sources of leaves.
Gas production, methane concentration in the gas and methane produced per unit substrate solubilized were higher when leaves of Guazuma ulmifolia replaced those from Bauhinia acuminata.
This research was done by the senior author as part of the requirements for the PhD degree in Animal Production "Improving Livelihood and Food Security of the people in Lower Mekong Basin through Climate Change Mitigation" of Nong Lam University. The authors acknowledge support for this research from the MEKARN II project financed by Sida and the help from Mr. Phonesavath, Mr. Sonephet and Mr. Khamlek in the farm and laboratory. The Faculty of Agriculture and Forest Resource, Souphanouvong University is acknowledged for providing the facilities to carry out this research.
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Received 14 September 2015; Accepted 17 October 2015; Published 1 November 2015
