BAUHINIA ACUMINATA AND WATER SPINACH (Ipomoea aquatica)
INTRODUCTION
Reducing GHG emissions from agriculture, especially from livestock, is a priority in order to reduce global warming (Sejian et al., 2010). Ruminants - cattle, buffalo, sheep and goats - are the major contributors of methane emissions from the agriculture sector (Lassey, 2007). There is therefore a need to develop feeding systems for ruminants that will result in reduced emissions of methane gas from the enteric fermentation in these animals. Promising ways to do this are: (i) by the feeding of biochar (Leng et al., 2012) derived from the carbonization of fibrous wastes (Orosco et al., 2018); and (ii) using as protein supplement the foliage from bitter as opposed to sweet cassava (Phuong et al., 2012). Cassava products contain cyanogenic glucosides which liberate hydrocyanic acid (HCN) when emzymically degraded. Cyanogenic glucosides exist as linamarin and lotaustralin in unbruised leaf (Makkar et al., 1995; Grainger et al., 2009). When the cellular structure is broken, the glucoside is exposed to extracellular enzymes such as linamarase which gives rise to toxic hydrocyanic acid. In studies on biodigestion of cassava residues it was shown that the HCN liberated in the digestion process was toxic to methanogenic bacteria (Smith et al., 1985;
Rojas et al., 1999). It is therefore postulated that a similar process could take place in the rumen of cattle fed cassava products, which could be an advantage as a strategy for reducing greenhouse gas emissions from ruminant animals. Cassava varieties are generally categorized into “sweet” varieties suitable for human consumption, and “bitter” varieties more appropriately used for industrial production of starch. It is understood that the ‘bitter” varieties are so-called because they have higher concentrations of cyanogenic glucosides making them potentially toxic to humans and animals. The objective of the research described in this paper was to combine both of these factors as a means to reduce methane production in goats fed foliage from the legume tree Bauhinia acuminata supplemented with foliage from water spinach.
MATERIALS AND METHODS
Treatments and experimental design
The experimental design was a 2*2 factorial arrangement of 4 treatments with four replications. The factors were: - Source of cassava leaves: Sweet (SC) or bitter (BC) variety
- Biochar: With (Bio) or without (NoBio) biochar
Table 1. Composition of substrate (% DM basis)
SC-Bio SC-NoBio BC-Bio BC-NoBio
Sweet cassava leaves 24 25
Bitter cassava leaves 24 25
Biochar 1 1
Bauhinia leaves 60 60 60 60
Water spinach leaves 10 10 10 10
Cassava root chips 5 5 5 5
Preparation of substrate and the in vitro system, data collection, measurements and chemical analyses were refered of the experiment 1 in the chapter 2.
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 cassava leaves., Aj is the effect of biochar source., (P*A)ij is the interaction between source of cassava leaves and source of biochar and eijk is random error
RESULTS
Chemical composition
Protein solubility was lowest in Bauhinia acuminata, was lower in bitter than in sweet cassava leaves and highest in water spinach leaves (Table 3).
Table 3. The chemical composition of substrate ingredients (% in DM, except DM which is on fresh basis) DM N*6.25 Ash Protein solubility Tannin NDF ADF
Bauhinia leaves 35.6 14.7 6.6 23.4 1.1 43.7 32.4
Bauhinia stem 38.1 12.3 4.29 - 42.7 31.5
Sweet cassava leaves 32.1 22.2 4.48 31.4 48.7 34.4
Sweet cassava petiole 16.8 16.7 6.39 - 48.3 38.6
Bitter cassava leaves 32.44 20.1 5.5 31.0
Cassava root chip 35.4 3.2 3.4
Water spinach 10.6 18.5 9.7 66.4
Biochar - - 38.3
The linear increase in methane concentration in the gas with duration of the incubation (Table 4) is similar to the trends observed by Outhen et al., 2011; Binh Phuong et al., 2011; Inthapanya et al., 2011 and Silivong and Preston, 2015 who used the same in vitro fermentation model. These results indicate that there is a lag time either in the growth of organisms that ferment carbohydrate to VFA and hydrogen, and/or in the growth of those that convert hydrogen to methane.
Table 4. Mean values for gas production, percentage of methane in the gas, DM digested and methane production per unit DM digested, according to source of cassava leaves (sweet SC or bitter BC) and with
(Bio) or without ( NoBio) biochar CL
p
Bio
p SEM
Interaction
SC BC Bio NoBio SEM p
0-6 h
Gas, ml 640 581 0.001 614 608 0.654 9.62 13.59 0.787
CH4, % 9.5 8.4 0.007 8.9 9.0 0.724 0.24 0.35 0.724
DM digested, % 54.9 46.4 <0.001 52 49.3 0.021 0.72 1.01 0.687
CH4, ml/g DMD 9.2 8.8 0.407 8.8 9.3 0.349 0.36 0.51 0.584
0-12 h
Gas, ml 871 815 0.001 849 838 0.382 8.76 12.40 0.921
CH4, % 17.5 16.2 <0.001 16.6 17.1 0.015 0.13 0.19 0.192 DM digested, % 61.1 50.8 <0.001 57.9 54.0 <0.001 0.46 0.65 0.061 CH4, ml/g DMD 20.9 21.7 0.156 20.4 22.2 0.006 0.39 0.55 0.265
0-18 h
Gas, ml 991 829 <0.001 945 875 0.005 14.5 20.49 0.905
CH4, % 22.9 20.7 <0.001 21.1 22.5 0.003 0.27 0.39 0.435 DM digested, % 66.2 55.0 <0.001 62.4 58.8 <0.001 0.45 0.64 0.102 CH4, ml/g DMD 28.5 26.2 <0.001 26.6 28.1 0.006 0.33 0.46 0.192
0-24 h
Gas, ml 1,438 1,314 0.002 1,410 1,341 0.056 22.9 32.44 0.792 CH4, % 28.5 25.8 <0.001 26.9 27.5 0.028 0.18 0.26 0.180 DM digested, % 72.6 59.4 <0.001 68.4 63.6 <0.001 0.53 0.75 0.499
CH4, ml/g DMD 47.1 49.3 0.048 46.7 49.7 0.012 0.70 0.99 0.920
BC: Bitter cassava leaves, Bio: Biochar, CL: Cassava leaves, P: Probability value, SEM: Standard error of the mean with dferror: 9, SC: Sweet cassava leaves.
The methane content of the gas was reduced when leaves of bitter cassava replaced leaves of sweet cassava as protein source and when 1% of biochar was added to the substrate. The magnitude of the differences was relatively small but consistent for all incubation times. The proportion of the substrate DM that was digested during the incubation was increased when biochar was included in the substrate and reduced when the protein supplement was from bitter compared with sweet cassava. Addition of biochar reduced the production of methane per unit substrate digested.
However, there was no consistent trend for effects of bitter versus sweet cassava. As was to be expected the production of methane per unit substrate digested increased linearly with duration of the incubation.
DISCUSSION
The reduction in production of methane when leaves of bitter cassava replaced those from sweet cassava has been reported in several in vitro incubations (Phuong et al., 2012; Binh et al., 2018) and appears to be a direct effect of the higher HCN levels in the bitter varieties being toxic to methanogens (Smith et al., 1985). However, the major reduction (18%) in digestibility in the 24h incubation, when leaves from bitter cassava replaced those from sweet cassava, has not previously been reported; in fact, the opposite effect was observed by Phuong et al., 2012. The logical assumption is that the reduced digestibility reflected a more general HCN toxicity on the activity of all rumen microbes. The reduction in methane production due to biochar was much less than has been observed in previous studies. A reduction of 13% was recorded by (Phuong et al., 2012; of 8%
by Vongkhamchanh et al., 2015; and 12% by Binh et al., 2018). The implication is that the sample of biochar used in this experiment may have been of inferior quality in terms of its relative surface area: weight ratio (and hence its capacity to support microbial communities in biofilms [Leng, 2017]). This stresses the need for a simpler quality test than the standard “BETS” ratio (for which the measuring equipment is not available in laboratories in Lao PDR).
CONCLUSIONS
(i) Increasing the length of the incubation from 6 to 24h in an in vitro rumen fermentation increased methane concentration in the gas and methane produced per unit substrate DM digested., (ii) The methane content of the gas was reduced when leaves of bitter cassava replaced leaves of sweet cassava as protein source and when 1% of biochar was added to the substrate. The magnitude of the reduction due to biochar was relatively small but consistent for all incubation times., (iii) The proportion of the substrate DM that was digested during the incubation was increased when biochar was included in the substrate and reduced when the protein supplement was leaf meal from a bitter compared with a sweet cassava variety.
GENERAL CONCLUSIONS
(i) It was confirmed that goats fed a tannin-rich tree foliage, such as Bauhinia acuminata, responded with improved diet digestibility, N retention and growth rate when the Bauhina acuminata was supplemented with a highly fermentable vegetable plant such as water spinach (Ipomoea aquatica)., (ii) An important negative effect was that the improvement in diet digestibility by supplementation with water spinach led to increases in methane production per unit diet DM
digested. The increase in methane production was postulated as being due to the much higher solubility of the protein in water spinach compared with the Bauhinia acuminata., (iii) The methane concentration of the gas in in vitro rumen incubations increases linearly with the length of the fermentation., (iv) Supplementing Bauhinia acuminata foliage with leaves from a bitter variety of cassava reduced the in vitro production of methane when compared with supplementation by leaves from a sweet variety of cassava., (v) It is postulated that the cyanogenic glucosides present in greater concentration in the leaves of bitter than in sweet cassava could be the reason for the reduction in methane production., (vi) Ensiled brewers’ grains fed as an additive (5% as DM) to a diet of Bauhinia acuminata improved the digestibility, N retention and growth rate of goats. The degree of improvement was greater when the Bauhinia acuminata was supplemented with cassava foliage instead of water spinach., (vii) Biochar fed at 1% of a diet of Bahinia acuminata and cassava foliage was as effective as brewers’ grains in improving the growth rate of the goats.
IMPLICATIONS
(i) Bauhinia acuminata foliage can support growth rates of >60 g/day in local goats when it is supplemented with foliage from a sweet variety of cassava foliage., (ii) Water spinach also improves growth rates of goats fed Bauhinia acuminata foliage but is not recommended as this practice will result in increased production of rumen methane., (iii) Ensiled brewers’ grains and biochar fed to goats as additives (5% for brewers’ grains; 1% for biochar) probably act as
“prebiotics” to improve growth performance and assist in detoxification in the animal of the products from enzymic breakdown of secondary plant compounds such as cyanogenic glucosides.
FURTHER RESEARCH
Cassava foliage is an important supplement to improve productivity of goats browsing on native trees and shrubs with the probability that this practice will also reduce enteric methane, an important greenhouse gas. Cassava foliage is available in large quantiles in Lao PDR when the roots are harvested for industrial starch production; It can also be produced by repeated harvest at 3-4 month intervals when the crop is grown for forage production. Varieties grown for starch production have been selected for high yield but this is also associated with higher levels of potentially toxic cyanogenic glucosides that give rise to toxic HCN when consumed by animals. Biochar is the carbon-rich byproduct of the carbonization of fibrous biomass by pyrolysis at high temperatures (700-900°C). It is an important component of strategies to reduce global warming as when applied to the soil the carbon in the biochar is not oxidized. This process is thus a natural way to sequester carbon from the atmosphere, an essential feature of activities required in order to reduce the risks of climate change. To respond to the opportunities and multiple benefits from: (i) feeding of cassava foliage as a protein supplement for goats browsing on trees and shrubs; and (ii) the associated use of biochar as a dietary additive, it is proposed that research should be prioritized to: (i) Definition of the relative roles of sweet and bitter varieties as supplements in the feeding system of goats and other ruminant animals., (ii) The production of biochar at farm level from fibrous crop byproducts (eg: cassava stems) and its role as a “prebiotic” feed additive in conserving animal health and improving productivity, especially in diets based on cassava foliage.
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