Methane production was reduced when cassava root (Manihot esculenta, Crant) was ensiled rather than dried, and when cassava leaves replaced water spinach (Ipomoea aquatic) as the protein source, in an in vitro rumen fermentation

Sangkhom Inthapanya, T R Preston¹ and Duong Nguyen Khang²

Animal Science Department, Faculty of Agriculture and Forest Resource, Souphanouvong University Lao PDR
inthapanyasangkhom@gmail.com
¹ Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV), Carrera 25 No 6-62 Cali, Colombia
² Nong Lam University, Ho Chi Minh City, Vietnam

Abstract

The purpose of the present study was to show that in an in vitro rumen fermentation methane production would be reduced when cassava leaf meal replaced water spinach as the protein source and when the carbohydrate was from ensiled rather than dried cassava root.  The treatments in a 2*3 factorial design were ensiled or dried cassava root meal with cassava leaf meal, water spinach meal or equal proportions of cassava leaf meal and water spinach meal. Urea was included in all incubations at 2% of the substrate DM. The incubation was for 24h with measurements of total gas production and methane percentage in the gas at intervals of 6, 12, 18 and 24h, with determination of residual unfermented substrate at the end. The quantity of substrate in each fermentation bottle was 12g to which were added 240 ml of rumen fluid (from a slaughtered cow) and 960 ml of buffer solution. The incubations were done in a simple in vitro system using recycled one-liter PEP water bottles with gas collection by water displacement and methane measured by an infra-red methane gas detector.

There were consistent effects at each fermentation interval for a decrease in gas production and methane content of the gas when: (i) ensiled cassava root replaced the dried root; and (ii) when cassava leaf meal replaced water spinach meal. Methane concentration in the gas increased linearly from 10-12 to 26-30% of the gas as the fermentation interval advanced from 0-6h to18-24h. Over the overall 24h fermentation the methane production per unit of substrate DM mineralized was decreased by 18% by the combination of ensiling versus drying of the cassava root and replacement of water spinach by cassava leaf meal.

Keywords: carbohydrate, drying, ensiling, HCN, methane, protein, tannin

Introduction

Greenhouse gas emissions play an important role in increasing global temperature (IPCC 2007). Agriculture produces 10-12% of total anthropogenic greenhouse gas emissions with the livestock sector contributing 44% of these emissions in the form of CH₄; the remaining sources are estimated to be 29% as N₂O and 27% as CO₂ (Gerber et al 2013). Ruminants are estimated to produce up to 95 million tonnes of methane (CH₄) annually and are implicated as a major source of greenhouse gas production (Patra 2014).

Enteric CH₄ emissions are often predicted from the chemical analysis of the diets (Hristov et al 2013; Moraes et al 2014); however, these methods do not seem sufficiently accurate and appropriate for all feeding situations.  Kebreab et al (2008) showed that CH₄ emissions inventories were more accurately estimated through diet-specific mechanistic models. Chagunda et al (2010) indicated that, in order to mitigate CH₄ emissions in a way acceptable for both the environment and animal welfare, it was important to quantify the effects of different diets on methane emissions. Other results suggest that a major difference is needed in dietary starch concentration in order to alter ruminal methanogenesis (Hassanat et al 2013).

Cassava (Manihot esculenta  Crantz) is grown in over 90 countries and is a most important food crop worldwide. It is the primary staple for more than 800 million people in the world (Lebot 2009). Of importance in a warming world is that it appears that cassava is potentially highly resilient to future climatic changes and according to Jarvis et al (2012) “could provide Africa with options for adaptation whilst other major food staples face challenges”.

Cassava foliage is considered to be a good source of bypass protein for ruminants (Ffoulkes and Preston 1978; Wanapat 2001; Keo Sath et al 2008). It has been fed as a major component of the diet for sheep (Hue et al 2008), goats (Ho Quang Do et al 2002; Dung et al 2005; Phengvichith and Ledin 2007; Seng Sokerya and Preston 2003) and cattle (Wanapat et al 2000; Thang et al 2010) in fresh, wilted or dried form.

Cassava leaves are known to contain variable levels of condensed tannins; about 3% in DM according to Netpana et al (2001) and Bui Phan Thu Hang and Ledin (2005).  Condensed tannins at moderate levels are known to have positive effects on the nutritive value of the feed by forming insoluble complexes with dietary protein, resulting in "escape" of the protein from the rumen fermentation (Barry and McNabb 1999). Numerous studies have also shown the potential of the tannin content in cassava leaves to play an anthelminthic role for the control of nematode parasites in ruminants (Seng Sokerya and Preston 2003; Seng Sokerya et al 2009; Netpana et al 2001; Khoung and Khang 2005). Condensed tannins (CT) are also reported to decrease methane production and increase the efficiency of microbial protein synthesis (Makkar et al 1995; Grainger et al 2009). Reductions of CH₄ production due to presence of  tannins of up to 13 to 16% were reported by Carulla et al (2005), Waghorn et al (2002), Grainger et al (2009) and Woodward et al (2004), apparently through a direct toxic effect on methanogens.

Water spinach (Ipomoea aquatica) plays an important role for farmers in rural areas; it is easy to cultivate and has a very high yield of biomass with a short growth period (Kean Sophea and Preston 2001). The crude protein content in the leaves and stems can be as high as 32 and 18% in dry basis (Ly Thi Luyen 2003). Water spinach is widely used for human food, but at the same time this vegetable can serve as feed for all classes of livestock.

Whitelaw and Preston (1963) fed early-weaned calves on diets in which the protein sources were of high or low solubility in M NaCl, and with well-balanced or imbalanced arrays of amino acids. They showed that a protein source with low solubility in M NaCl and a balanced array of amino acids (Peruvian fish meal) supported higher N retention  than protein sources that were highly soluble in M NaCl irrespective of their balance of amino acids (groundnut meal and enzyme-hydrolyzed fish meal). Do et al (2013) reported that methane production in an in vitro rumen fermentation was lower when the protein was of low compared with high solubility (fish meal rather than groundnut meal.
 

The protein in cassava foliage was reported to be of low solubility (31%) in contrast with that in water spinach leaves which was highly soluble (71%) (Preston et al 2013). Methane concentrations in the gas from a rumen in vitro system were higher when water spinach was the protein source compared with cassava (Silivong and Preston 2015).

The purpose of the present study was to confirm these differences in methane production from cassava and water spinach foliage when the carbohydrate source was either ensiled or dried cassava root.

Materials and methods

Location and duration

The experiment was conducted in the laboratory of Animal Science department, Faculty of Agriculture and Forest Resource, Souphanouvong University, Lao PDR, from June to July 2015.

Treatments and experimental design 

The experimental design was arranged as a 2*3 factorial in a completely randomized design with 6 treatments and 4 replications of each treatment. The factors were:

Source of carbohydrate:

• ECR: Ensiled cassava root meal
• DCR: Dried cassava root meal

Source of protein:

• CLM: Cassava leaf meal

• WSM: Water spinach meal

• CLM-WSM: Cassava leaf meal plus water spinach meal

Urea as NPN source was added to all the substrates at 2% of DM.

Experimental procedure

The in vitro system was the same as described by Sangkhom Inthapanya et al (2011).

The cassava root was chopped into small pieces around 1-2 cm of length. One half of the chopped material was ground in a liquidizer, and then stored in a plastic bag for ensiling over 7 days. The other half was dried in an oven at 80ºC for 24 hours and then ground through a 1mm sieve.   

Photo 1: Making the ensiled cassava root

Cassava leaves and water spinach foliage (leaves and petioles) were collected from farmer areas close to Souphanouvong University. They were chopped into small pieces around 1-2 cm in length, then dried in the oven at 80ºC for 24 hours before grinding.

Amounts of the substrates equivalent to 12g DM were put in the incubation bottle, followed by 0.96 liters of buffer solution (Table 2) and 240 ml of rumen fluid obtained from a cow immediately after being slaughtered. The bottles were then filled with carbon dioxide and incubated at 38 ⁰C in a water bath for 24 hours.

Data collection and measurements

During the incubation the gas volume was recorded at 6, 12, 18 and 24 hours. After each time interval, the methane concentration in the gas was measured with a Crowcon infra-red analyser (Crowcon Instruments Ltd, UK).  At the end of the incubation, the contents of the incubation bottle were filtered through cloth to determine the mineralization of the substrates.

Chemical analyses

Samples of ensiled cassava root meal, dried cassava root meal, cassava leaf meal and water spinach meal were analyzed for DM, crude protein, crude fiber and ash (AOAC 2010). The cassava root (ensiled and dried) and the cassava leaves (fresh and dried) were analyzed for hydrogen cyanide (HCN) and tannin (TNN) content according to AOAC (2010) methods.

Statistical analysis

The data were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab (2000) software (version 16.0). Sources of variation in the model were:  replicates, source of carbohydrate, source of protein, interaction between carbohydrate*protein and error.

Results and discussion

Chemical composition

The water spinach foliage had less crude fiber and more ash than the cassava leaves (Table 3).  Crude protein contents were similar for both cassava leaf and water spinach meal.

HCN and Tannin

Hydrogen-cyanide was higher in fresh than in dried cassava leaf; and in ensiled rather than dried cassava root. Condensed tannin content was lower in the cassava leaves than in the roots (Table 4).

Gas production

At all incubation times, the gas production was lower in treatments with ensiled cassava root than in those with dried root (Table 5) and was lower for treatments with cassava leaf meal alone, and for cassava leaf meal combined with water spinach meal, than for those with water spinach meal as the only protein source (Figures 1 to 4). Gas production increased from the 0-6h to the 6-12h interval and then decreased linearly to the lowest values at 18-24h.

Figure 1: Gas production (0-6h) from ensiled or dried cassava root supplemented with cassava leaf or water spinach	Figure 2: Gas production (6-12h) from ensiled or dried cassava root supplemented with cassava leaf or water spinach

Figure 3. Gas production (12-18h) from ensiled or dried cassava root supplemented with cassava leaf or water spinach	Figure 4. Gas production (18-24h) from ensiled or dried cassava root supplemented with cassava leaf or water spinach

At all incubation times, the percentage of methane in the gas was lower for cassava leaf meal or cassava leaf meal combined with water spinach meal than for water spinach meal; and for ensiled compared with dried cassava root (Figures 6-9).

Figure 5. Gas production from ensiled or dried cassava root at each fermentation interval

Methane in the gas was lower for ensiled than for dried cassava root and increased as water spinach replaced cassava leaf meal (Figures 6-9).

Figure 6: Methane in the gas (0-6h) for substrates with ensiled or dried cassava root, and those with cassava leaf or water spinach, or the combination of the two	Figure 7: Methane in the gas (6-12h) for substrates with ensiled or dried cassava root, and those with cassava leaf or water spinach, or the combination of the two

Figure 8: Methane in the gas (12-18h) for substrates with ensiled or dried cassava root, and those with cassava leaf or water spinach, or the combination of the two	Figure 9: Methane in the gas (18-24h) for substrates with ensiled or dried cassava root, and those with cassava leaf or water spinach, or the combination of the two

Figure 10: The percentage of methane in the gas at each fermentation interval for substrates with dried or ensiled cassava root

The methane concentration in the gas increased linearly with fermentation interval (Figure 10).

The proportion of the DM that was mineralized was greater with dried than with ensiled cassava root and increased as cassava leaf was replaced by waster spinach meal (Figure 11). Methane production per unit DM mineralized was greater with dried than ensiled root and increased as water spinach replaced cassava leaf meal (Figure 12).

Figure 11: DM mineralized after 24h for substrates with ensiled or dried cassava root, and those with cassava leaf or water spinach, or the combination of the two	Figure 12: Methane produced per unit DM mineralized for substrates with ensiled or dried cassava root, and those with cassava leaf or water spinach, or the combination of the two

The increase in methane production with duration of incubation, indicative of the transition to a secondary fermentation of the VFA to methane, supports the findings of Sangkhom Inthapanya et al (2011), Le Thi Binh Phuong et al (2011), Thanh et al (2011), Outhen et al (2011) and Marin et al (2014).

Replacing cassava leaf meal with water spinach meal led to linear increases in gas production of a a higher methane content. This effect is similar to that reported in in vitro incubations when water spinach replaced cassava leaf meal with rice straw-urea as the substrate (Inthapanya and Preston 2014) and when water spinach meal was added to substrates of Bauhinia and Guazima leaf meals (Silivong and Preston 2015).

The lower production of methane when the CHO substrate  was ensiled  rather than dried cassava root can be ascribed to the higher level of HCN precursors in the former, and the resultant toxic  effect of the HCN on methanogens as reported by Rojas et al (1999) and Smith et al (1985).   The increase in methane production as water spinach replaced cassava leaf meal could similarly be linked to deceasing concentration of HCN precursors and of condensed tannins in the substrates as water spinach replaced cassava leaf meal, as there are no reports of either cyanogenic glucosides or of condensed tannins in water spinach. Decreased methane production due to condensed tannins in the diet has been described by several authors (eg: Makkar et al 1995; Grainger et al 2009).

The mechanisms by which enteric methane is decreased by ensiling rather than drying of the cassava roots, and by supplementation with cassava leaves rather than water spinach, are thought to be related to the toxic effects on methanogens from cyanogenic precursors that are in greater concentration in the ensiled compared with dried cassava roots and in cassava leaves rather than water spinach. These apparent benefits in methane mitigation from low dietary concentrations of cyanogenic precursors have still to be verified in in vivo studies.

Conclusions

• There were consistent effects at each 6h fermentation interval up to 24h for a decrease in gas production and methane content of the gas when:  ensiled cassava root replaced the dried root; and when cassava leaf meal replaced water spinach meal.

  
  

• Methane concentration in the gas increased linearly from 10-12 to 26-30% of the gas as the fermentation interval advanced from 0-6h to18-24h.  Over the overall 24h fermentation the methane production per unit of substrate DM mineralized was decreased by 18% by the combination of ensiling versus drying of the cassava root and replacement of water spinach by cassava leaf meal.

Acknowledgements

This research is part of the requirement by the senior author for the degree of PhD at Nong Lam University.  The support from the MEKARN II project, financed by Sida, is gratefully acknowledged, as is the help received from the Animal Science Department, Faculty of Agriculture and Forest Resource, Souphanouvong University, Lao PDR.

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Received 6 July 2015; Accepted 14 August 2015; Published 1 September 2015

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