AN IN VITRO INCUBATION WITH SUBSTRATES OF BAUHINIA ACUMINATA OR GUAZUMA ULMIFOLIA LEAVES AND MOLASSES
ABSTRACT
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. The result shown that 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: bauhinia acuminata, gas production, greenhouse gases, protein solubility, tannins
INTRODUCTION
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.
MATERIALS AND METHODS Location
The experiment was carried out at the Animal Science Laboratory of the Faculty of Agriculture and Forest Resources, Souphanouvong University, LuangPrabang Province, Lao PDR.
Treatments and experimental design
The experimental design was a 2×4 factorial arrangement of 8 treatments with four replications. The factors were:
Foliage source: Bauhinia acuminata and Guazuma ulmifolia (BA and GU)
Level of water spinach: 0, 5, 15 and 25% in substrate DM Preparation of substrate and the in vitro system
The in vitro system used recycled “PET” plastic bottles as flasks for the incubation and gas collection (Diagram 1).
a. Water bath
b. Fermentation bottle (1.5liters) c. Water storage reservoir (3liters) d. Gas collection bottle (1.5liters) Plastic tube (id: 4mm)
Diagram 1. A schematic view of the in vitro system to measure gas production in an in vitro incubation
A simple in vitro system was used (Diagram 1) with recycled plastic bottles as flasks for the incubation and gas collection.
- B and D bottles (1.5 liters capacity) - C bottle (3liters capacity)
The B bottle containing the substrate for fermentation was connected to the D bottle by a plastic tube (4 mm diameter). The D bottle was marked at 50ml intervals before being suspended in the C bottle containing water. Clay was used to cover the stoppers of the plastic bottle and junction of stopper and plastic tube to prevent leakage of gas. The leaves from Bauhinia and Quazuma (Photos 1 and 2), 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 960 ml of buffer solution (Table 2) and 240 ml of rumen fluid. The rumen fluid was taken at 3.00-4.00am 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 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 24 h.
Photo 1: Bauhinia acuminata Photo 2: Guazuma ulmifolia
Table 1. Composition of diets (% DM basis)
BA#GU B
A#GU-
BA#GU- BA#GU
-
W
S 5 WS 15 WS 25
Leaf meal# 80 7
5 65 55
Water spinach 0 5 15 25
Molasses 20 2
0 20 20
Total 100 100 100 100
# Bauhinia or Guazuma leaf meals
Table 2. Ingredients of the buffer solution (g/liter)
CaCl2 NaHPO4.12H2O NaC
l Cl MgSO4.7H2O NaHCO
3
Cysteine
0.04 9.30 0.47 0.57 0.12 9.80 0.25
Source: Tilly and Terry (1963)
Data collection and measurements
Gas production was measured at 4 intervals for 6, 12, 18 and 24h by water displacement (a calibrated recycled water bottle with the bottom removed) and 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 3). Residual DM in the incubation bottle was determined by filtering the incubation residues through cloth to estimate DM loss during incubation (Photo 4) and drying the residue (65°C for 72h). 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).
from recycled "pep" water bottles filtered though cloth Chemical analyses
The samples of foliage, water spinach and residual substrate were analysed for DM, ash and N according to AOAC (1990) methods. 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).
Statistical analysis
The data were analyzed by the general linear model option of the ANOVA program in the Minitab software (Minitab, 2014). In the model the sources of variation were: treatments, replicates and error.
The statistical model was:
Yijk = μ + Pi + Aj + Pi*Aj+ eijk
Where: Yijk is dependent variables μ is overall mean
Pi is the effect of foliage source
Aj is the effect of level of water spinach
(P*A) ij is the interaction between source of foliage and source of level of water spinach
eijk is random error RESULTS
Chemical composition of the feed is presented in Table 3. Percentages of crude protein, ash and protein solubility were higher in water spinach than in Bauhinia acuminata and Guazuma ulmifolia leaves, but DM was lower. The protein content and solubility in the leaves of Guazuma ulmifolia were higher than in Bauhinia acuminata, but ash was lower.
Table 3. The chemical composition of feed (% in DM, except DM which is on fresh basis)
DM N*6.25 Ash Protein solubility Tannin NDF ADF Bauhinia
leaves 40.0 15.0 21.2 23.7 1.1 43.7 32.4
Bauhinia stem 38.1 12.3 4.29 - 42.7 31.5
Guazuma
ulmifolia 36.0 18.4 3.9 33.3 -
Water spinach 10.6 18.5 9.7 66.4 42.3 33.3
Molasses 80.4 5.4 10.5
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 1-6). Gas production and percent substrate solubilized were increased by increasing the level of water spinach in the substrate, and were higher for Guazuma ulmifolia than Bauhinia acuminata at each incubation interval.
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
Foliage source
p
Level of water spinach (%)
p SEM
Interaction
BA GU WS-0 WS-5 WS-
15
WS-
25 SEM P
0-6 hr
Gas production, ml 363 483 <0.001 286d 354c 476b 575a <0.001 5.1 7.2
0.57 4 Methane, % 8.8 9.9 <0.001 7.6d 8.9c 9.6bc 11.4a <0.001 0.2 0.2
0.67 3 DM solubilized, % 53.1 56.3 <0.001 49.2d 53.8c 56.4b 59.4a 0.001 0.2 0.3
0.75 3 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 0.66 5
Gas production, ml 483 781 <0.001 463d 581c 661b 821a <0.001 9.4 13.3 0.77
2 Methane, % 13.8 15.9 <0.001 11.1d 13.6c 15.1bc 19.6a <0.001 0.3 0.4
0.56 3 DM solubilized, % 56.5 60.2 <0.001 53.2d 57.1c 59.9b 63.2a <0.001 0.3 0.5
0.75 6 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
0.67 4 0-18 hr
Gas production, ml 584 1023 <0.001 584d 766c 856b 1009a <0.001 9.1 12.9 0.68
1 Methane, % 18.9 21.8 <0.001 16.3d 19.5c 21.3b 24.3a <0.001 0.2 0.3
0.67 5 DM solubilized, % 59.4 63.5 <0.001 56.1d 60.1c 62.4bc 67.0a <0.001 0.4 0.6
0.87 2 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
0.65 3 0-24 hr
Gas production, ml 734 1239 <0.001 775d 973c 1050b 1149a <0.001 11.3 15.9 0.77
6 Methane, % 24.4 27.9 <0.001 20.4d 24.8c 27.9b 31.5a <0.001 0.2 0.3
0.83 2 DM solubilized, % 61.8 67.3 <0.001 59.0d 64.3c 65.2bc 69.6a <0.001 0.4 0.6
0.74 2 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
0.77 5
abc Mean values without common superscript differ at p<0.05, BA: Bauhinia acuminata,
GU: Guazuma ulmifolia, P: Probability value, WS: Water spinach
Object 3
Object 5
Figure 1. Effect of Bauhinia (BA) and Guazuma (GU) leaf meals on gas
Figure 2. Effect of increasing level of water spinach on gas
production at increasing incubation intervals production at increasing incubation intervals
Object 7
Object 9
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
Object 11
Object 13
Figure 5. Effect of leaf meals from Bauhinia (BA) and Guazuma (GU) on substrate DM solubilized at increasing incubation interval
Figure 6. Effect of level of water spinach on substrate DM solubilized at increasing incubation intervals
Object 15
Object 17
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
DISCUSSION
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 ulmifolia leaves compared with Bauhinia acuminata 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 acuminata was replaced by Guazuma ulmifolia, 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).
CONCLUSIONS
- 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.
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CHAPTER 3