Citation of this paper

Methane production in rumen in vitro incubations of ensiled cassava (Manihot esculenta Cranz) root supplemented with urea and protein-rich leaves from grasses, legumes and shrubs

T R Preston¹, P Silivong and R A Leng²

Souphanouvong University, Luang Prabang, Lao PDR
¹ Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV), Carrera 25 No 6-62 Cali, Colombia
reg.preston@gmail.com
² University of New England, Armidale, NSW, Australia

Abstract

In an in vitro rumen incubation of ensiled cassava root-urea supplemented with protein and fiber from leaves of grasses, legume trees, prostrate legumes or a shrub (sweet and bitter varieties of cassava), there were close positive relationships between: (i) the percentage of methane in the gas and the rate of gas production; and ii) between methane production and the solubility of the leaf protein.

It is proposed that these relationships substantiate the hypothesis that “the presence of cyanogenic glucosides, in the cassava component of the diet, alters rumen fermentation, enhancing rumen escape of substrate (both glucogenic materials and potentially readily fermentable substrate) for digestion in the intestines followed by further fermentation in the cecum , thereby reducing the overall production of methane with concomitant improved effiency of feed utilization by the host animal. )reduces the overall rate of rumen fermentation, thus enhancing rumen escape of substrate for post-rumen digestion in the intestine and cecum, thereby reducing the overall production of methane with concomitant improved feed utilization effficiency of the host animal”.

Key words: bitter, bypass nutrients, cecum, fermentation, glucogenic, metabolism, nutrient balance, ruminants, solubility, sweet

Introduction

The hypothesis that rumen methane production is directly related to the solubility of the dietary protein was advanced by Phonethep et al (2016) to explain why supplementing foliage of Tithonia diversifolia with fresh water spinach (Ipomea aquatica) (protein solubility 67%) increased rumen methane production while the opposite effect was observed when the supplement was cassava leaves (protein solubility 21%). The proposed explanation was that for feeds in which the protein is of low solubility (ie: are rich in “bypass” or “escape” protein) the balance of feed fermented in the intestine and cecum/large intestine, relative to the rumen is increased, and that production of methane is reduced because disposal of hydrogen in fermentative degradation in the cecum-colon is dominated by acetogenesis rather than methanogenesis (Demeyer 1991; Immig 1996; Popova et al 2013; Leng 2018).

The objectives of the following experiment were to provide further evidence for the relationship between the solubility of dietary protein and methane production in ruminant feeding systems.

Materials and methods

The experiment was carried out from July to September 2018 at the Animal Science Laboratory of the Faculty of Agriculture and Forest Resources, Souphanouvong University, Luang Prabang Province, Lao PDR. Leaves from a range of grasses, prostrate and tree legumes, shrubs and a vegetable (Table 1) were compared as protein supplements in a rumen in vitro incubation of ensiled cassava root and urea simulating the feeding systems described by Phanthavong et al (2016) and Sangkhom et al (2017). Separate incubations were made for each protein source over time periods of 12, 18 and 24h, each of which was replicated three times. The in vitro system (Inthapanya et al 2011; Phanthavong et al 2018) used recycled plastic bottles as flasks for the incubation and gas collection (Photo 1). The leaves were chopped into small pieces (3-5mm) and dried at 65°C for 48h then ground with a coffee grinder prior to being mixed with the ground fresh cassava root and urea. The proportions of the leaves, of urea and of cassava root meal, in the substrates were adjusted to provide a crude protein content in the substrate DM of 14-15% (Table 1).


Photo 1. The in vitro system using recycled plastic water bottles	Diagram 1. Arrangement of the plastic bottles for the incubation and collection of the gas by water displacement

Table 1. Ingredients in the substrates, content of crude protein in the mixtures and DM mineralized after 24h
	Ingredients in the substrate, % as DM			% in substrate
Source of leaves	Leaves	Cassava root	Urea	Crude protein	DM mineralized

P. pupureum	79	20	1	14.5	59.0
P. maximum	70	28.5	1.5	14.5	55.2
B. humidicula	73	26	1	14.5	57.5
S. guiensis	64	35.5	0.5	14.6	62.7
C. ternatea	58	41.5	0.5	14.8	64.8
A. pinzoi	60	39.5	0.5	14.6	64.0
S. grandiflora	58	41.5	0.5	14.8	61.0
G. sepium	57.5	42	0.5	14.7	52.7
L. leucocephala	61.5	38	0.5	14.8	61.0
M. esculenta S	57.5	42	0.5	14.7	59.1
M. esculenta B	60	39.5	0.5	14.8	58.7
I. aquatica	67.5	32	0.5	14.9	67.5

Amounts of the mixtures (Table 1) equivalent to 12g DM were put in the incubation bottles 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 Luang Prabang 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 bottles. The remaining air in the fermentation bottle was flushed out with carbon dioxide. The bottles were incubated at 38°C in a water bath for intervals of 12, 18 and 24h, with separate incubations in triplicate for each interval.

Table 2. Ingredients of the buffer solution (g/liter)
CaCl₂	NaHPO₄.12H₂O	NaCl	Cl	MgSO₄.7H₂O	NaHCO₃	Cysteine

0.04	9.30	0.47	0.57	0.12	9.80	0.25
*Source:* Tilly and Terry (1963)

At the end of each incubation, measurements were made of total gas production (by water displacement), content of methane (by passing samples of gas through an infra-red detection meter [Crowcom Ltd., UK] and DM solubilized (by filtering the residual substrate through cloth and drying the residue residue (65°C) for 72 h. Solubility of the protein in the leaves was determined by shaking 3 g of dry leaf meal in 100 ml of M NaCl for 3h and filtering through Whatman No. 4 filter paper (Whitelaw et al 1963) prior to determining the N content of the filtrate by kjeldahl digestion (AOAC 2010).

Data were analyzed using the General Linear option of the ANOVA program in the Minitab (2014) software. Sources of variation were: source of leaves and residual error. Linear regressions were derived using the Excel software in the Microsoft Office program.

Results and discussion

The solubility of the protein in the leaves varied widely (Table 3) . The highest value was recorded for the water spinach vegetable (68.7%) followed by leaves from prostrate legumes (45-57%), then the tree legumes (35.1-46.3%), and the grasses (33.4 to 41.5%) with lowest values for the leaves of cassava (31.0 to 32.0%). The trends for gas production and the methane content in the gas were similar to those for protein solubility with the highest values for water spinach and the lowest values for the leaves of cassava.

Table 3. Mean values for production of gas, content of methane (CH₄) in the gas, and DM solubilized (sol), in rumen in vitro incubations of substrates based on ensiled cassava root-urea with protein from leaves taken from a range of grasses, prostrate and tree legumes and a shrub (cassava) (in all cases the total weight of substrate DM was 12 g; fermentation times were 12, 18 and 24h)
	Vegetable	*Grasses*			Prostrate legumes			*Tree legumes*			Shrub		p	SEM
	IA	PP	PM	BH	AP	CT	SG	*SGR*	GS	LL	ME-SW	ME-BI	p	SEM

0-12h
Gas, ml	663	553	447	593	770	817	610	510	487	343	350	313	<0.001	11.0
CH₄, %	13.0	17.3	14.0	11.7	15.7	19.7	21.0	16.0	13.7	12.7	15.3	13.7	<0.001	0.36
DM sol., %	54.5	52.4	49.3	51.9	55.7	56.7	54.3	52.9	51.3	47.1	46.2	49.1	<0.001	0.45
CH₄, ml/g DM sol.	13.2	15.3	10.6	11.1	18.1	23.6	19.7	12.9	10.8	7.7	9.7	7.3	<0.001	0.59
0-18h
Gas, ml	967	633	567	483	857	917	763	777	667	563	493	473	<0.001	23.0
CH_4,%	23.0	22.7	25.3	20.3	21.0	22.0	20.3	18.7	17.7	16.0	17.0	15.7	<0.001	0.35
DM sol., %	59.1	54.7	51.7	53.1	59.5	60.9	57.3	54.3	52.5	50.1	49.8	51.2	<0.001	0.42
CH₄,.ml/g DM sol.	31.4	21.9	23.1	15.5	25.3	27.6	22.6	22.2	18.7	15.0	14.0	12.1	<0.001	1.01
0-24h
Gas, ml	1,367	833	733	683	1,057	1,117	963	940	827	727	663	627	<0.001	27.9
CH₄, %	29.3	27.3	30.7	23.0	26.0	26.7	25.3	23.7	21.7	20.7	22.0	20.7	<0.001	0.33
DM sol., %	67.5	59.0	55.2	57.5	64.0	64.8	62.7	61.0	59.1	58.7	53.2	55.2	<0.001	0.50
CH₄,.ml/g DM sol.	49.6	32.2	34.0	22.8	35.8	38.3	32.4	30.4	25.2	21.3	22.9	19.6	<0.001	1.29
#Protein solubility, %	68.7	41.5	36.3	33.4	52.1	55.0	47.0	46.3	40.5	35.1	32.7	31.0

IA Ipomoea aquatica; PP Pennisetum purpureum; PM Panicum maximum; BH Brachiaria humidicola; AP Arachis pintoi; CT Clitoria ternatea; SG Stylosanthes guiense; SGR Sesbania grandiflora; GS Gliricidia sepium; LL Leucaena leucocephala; ME SW Manihot esculenta (Sweet var.); ME BI Manihot esculenta (Bitter var.) # Of the leaves

Figure 1. Volumes of gas produced in 24h in vitro rumen incubations of substrates based on cassava root pulp-urea with protein from leaves
from a range of grasses, prostrate and tree legumes and a shrub (sweet and bitter varieties of cassava) (see Table 3 for details)

Figure 2. Methane content of the gas produced in 24h in rumen in vitro incubations of substrates based on ensiled cassava root-urea with protein from leaves
from a vegetable, and a range of grasses, prostrate and tree legumes and a shrub (sweet and bitter varieties of cassava) (see Table 3 for details)

Figure 3. Methane produced per unit substrate DM solubilized in 24h in vitro rumen incubations of substrates based on cassava root pulp-urea with protein from leaves
from a vegetable, and a range of grasses, prostrate and tree legumes and a shrub (sweet and bitter varieties of cassava) (see Table 3 for details)

Relationships between methane content in the gas, the rate of gas production and the solubility of the leaf protein

There were positive linear relationships between: (i) the percentage of methane in the gas (and per unit substrate mineralized) and the rate of gas production (Figures 4 and 5); and (ii) between methane production and the solubility of the leaf protein (Figures 6 and 7).


Figure 4. In a 24h rumen incubation of ensiled cassava root-urea with protein from a range of leaves the methane content of the gas was positively related with the gas production	Figure 5. In a 24h rumen incubation of ensiled cassava root-urea with protein from a range of leaves the methane produced per unit DM mineralized was positively related with the gas production


Figure 6. In a 24h rumen incubation of ensiled cassava root-urea with protein from a range of leaves the methane content of the gas was positively related with the solubility of the leaf protein	Figure 7. In a 24h rumen incubation of ensiled cassava root-urea with protein from a range of leaves the methane produced per unit DM mineralized was positively related with the solubility of the leaf protein

These relationships are taken as strong supporting evidence for the hypothesis projected in this experiment, that dietary manipulations that result in reduced rates of rumen fermentation (of a diet of high fermentative potential) are associated with reduced partitioning of hydrogen to methane, and therefore an improved balance of nutrients arriving at sites of metabolism. The outcome of this dietary manipulation will then be an improvement in host animal productivity as demonstrated by Phonethep et al (2016) and Houda et al (2019). In the experiment of Phonethep et al 2016) goats fed a basal diet of foliage of Tithonian diversifolia produced less methane and retained more nitrogen (a proxy for better growth rate) when the Tithonia was supplemented with cassava foliage (protein solubility 22%) than when the supplement was water spinach (protein solubility 67%). In the experiment of Houda et al (2019), rumen fermentation was depressed by feeding an additive containing thymol (an active bactericide), gas production in vitro was depressed, and percent of methane in eructed rumen gas of dairy cows was reduced with concomitant increase in milk production.

Conclusions

In an in vitro rumen incubation of ensiled cassava root-urea supplemented with protein from leaves of grasses, legume trees, prostrate legumes or shrubs (sweet and bitter varieties of cassava), there were positive linear relationships between: (i) the percentage of methane in the gas and the rate of gas production; and (ii) between methane production and the solubility of the leaf protein.
It is proposed that these relationships substantiate the hypothesis that “the presence of cyanogenic glucosides, in the cassava component of the diet, alters rumen fermentation, enhancing rumen escape of substrate (both glucogenic materials and potentially readily fermentable substrate) for digestion in the intestines followed by further fermentation in the cecum, thereby reducing the overall production of methane with concomitant improved effiency of feed utilization by the host animal.

Acknowledgements

The authors acknowledge support for this research from the MEKARN II project financed by Sida. Special thanks to Mr Aloun who provided valuable help in the laboratory. I also thank the staff of Department of Animal Science laboratory, Faculty of Agriculture and Forest Resource, Souphanouvong University for providing the facilities to carry out this research.

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Received 1 January 2019; Accepted 25 June 2019; Published 2 July 2019

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