Citation of this paper

Effect of cassava leaf meal and coconut cake on methane production in an in vitro incubation using cassava root pulp and urea as substrate

Duong Nguyen Khang, Dang Thi Ngoc Anh and T R Preston¹

Nong Lam University, Ho Chi Minh City, Vietnam
duongnguyenkhang@gmail.com
¹ Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV), Carrera 25 No 6-62 Cali, Colombia

Abstract

The effect on methane production of supplementing cassava root pulp-urea with cassava leaf meal and coconut cake was studied in a 24h in vitro rumen incubation experiment. Measurements were made at 6h intervals of gas production, methane content of the gas. and rumen fermentation parameters (pH, ammonia, VFA and protozoal counts). Substrate DM solubilized was measured at the end of the 24h incubation. The experimental design was a 3*3 factorial with 3 repetitions.

DM solubilized was increased and gas production and the methane percentage in the gas were reduced both by cassava leaf meal and by coconut cake at each stage of the incubation. The decrease in methane percent in the gas was directly related to the proportion of dietary protein that was insoluble in M NaCl. The important feature of the in vitro incubation was the negative relationship between the percent methane in the gas and the percent DM solubilized, which was in complete contrast with the relationship with gas production which was positive. These opposing trends reflected the negative relationship between the two measures of digestion/fermentation – gas production and percent DM solubilized.

The fact that substrate digestibility (substrate solubilized) was increased by supplementation showed that supplementation with sources of bypass protein resulted in more nutrients being made available for fermentation; however, the associated decline in rate of gas production implies that not all the nutrients made available were being fermented. It is postulated that either one (or both) of the supplements contained entities that depressed slightly the fermentation (and hence the gas production). These could be the cyanogenic glucosides (precursors of HCN) in the cassava leaf meal and the lauric and myristic acids in the oil fraction of the coconut cake.

Key words: bypass nutrientes, cyanogenic glucosides, HCN, lauristic acid

Introduction

Enteric methane from ruminants is a significant greenhouse gas (Steinfeld et al 2006), estimated to represent 17 - 30% of total anthropogenic methane (Beauchemin et al 2009). It also represents a loss of dietary energy of from 2 to 12% of the gross energy intake (Johnson and Johnson 1995). These issues have led to a global search for nutritional strategies to mitigate methane emissions from ruminants.

It has been reported that cattle fed mainly on grain produce less methane than cattle grazed on pasture (Henry et al 2012). In Malawi, Maselema and Chigwa (2017) showed that in an in vitro rumen incubation, the methane produced per unit substrate DM was lower for leaves from forage trees than from grasses. Similar findings were reported by Preston et al (2019) in a rumen in vitro incubation of grasses, leaves from tree legumes and leaves from a shrub (cassava – Manihot esculenta). These authors showed that over this range of species there was a direct relationship between the per cent methane in rumen gas and the rate of gas production.

A relatively new development was the finding in the cassava starch factory in Vientiane province, Lao PDR, that the byproduct (cassava pulp) that had been “dumped” in an open pit over a 4-year period had ensiled naturally with pH lower than 3.5; and that the potential feed value was only slightly less than that of the entire cassava root (Phanthavong et al 2014). The high feeding value of the ensiled cassava pulp was confirmed in three feeding trials (Phanthavong et al 2016, 2018; Keopaseuth et al 2017) in which young cattle of the local “Yellow” breed were fed ad libitum on the ensiled pulp with added urea, supplemented with brewers’ fresh grains and rice straw. The growth rates on this feeding system were over 700g/day with DM feed conversion of less than 6:1.

In these feeding trials with cassava pulp-urea as the basal diet, the source of bypass protein was ensiled brewers’ grains. As this feed resource is not widely available the following experiment aimed to evaluate alternative sources of bypass protein from: (i) cassava foliage, in view of reports of low rumen methane production from this protein source (Phuong et al 2012; Preston et al 2019), and its successful use as a bypass protein supplement for cattle (Ffoulkes and Preston 1978, Khang and Wiktorsson 2000, Khang and Wiktorsson 2004); and (ii) coconut cake, the protein-rich byproduct from coconut oil extraction, in view of known responses of fattening cattle to byproduct meals from oil-seed extraction (Weixian et al 1994). Coconut cake has a high content of lauric and myristic acids which were recommended for methane abatement following the in vitro study of Soliva et al (2007).. Coconut cake is likely to be a good source of “bypass” protein, as the combination of the heat produced in processing, and the presence of residual oil, will tend to protect the protein against degradation in the rumen, thus conferring rumen “bypass” or “escape” properties to the protein (Preston and Leng 1987).

This study aimed to determine the effects of cassava leaf meal and coconut cake on rumen gas and methane production when used as supplements in an in vitro incubation of cassava root pulp and urea.

Materials and methods

Location

The experiment was carried out in the Research and Technology Transfer Center, Nong Lam University of Ho Chi Minh City, Vietnam from March to May 2016.

Experimental design

The in vitro rumen incubation was arranged as a 3*3 factorial in a completely randomized block design with 3 repetitions. The first factor was cassava leaf meal at 0, 5 and 10% of substrate DM; the second factor was the level of coconut cake (0, 2 and 4% of substrate DM). The basal substrate was ensiled cassava root pulp supplemented with urea (Table 1).

Table 1. Composition of the substrates (% in DM)
Cassava leaf	CL0			CL5			CL10
Coconut cake	CC0	CC2	CC4	CC0	CC2	CC4	CC0	CC2	CC4

Cassava leaf meal	0	0	0	5	5	5	10	10	10
Coconut cake	0	2	4	0	2	4	0	2	4
Urea	3.0	2.9	2.7	2.7	2.5	2.4	2.3	2.2	2.0
Cassava pulp	97.0	95.1	93.3	92.3	90.5	88.6	87.7	85.8	84.0
Crude protein, %	10.4	10.5	10.3	10.5	10.3	10.4	10.4	10.5	10.3

The in vitro procedure (Diagram 1) was that developed by Inthapanya et al (2011). In each fermentation bottle, 12 g of mixed substrate (DM basis) were placed followed by 960 ml of buffer solution (Tilly and Terry 1963) and 240 ml of rumen fluid obtained from a slaughtered cow at the local abattoir. The residual air in the fermentation bottles was flushed with carbon dioxide. The bottles were incubated at 38ºC in a water bath for 24h.

Diagram 1. A schematic view of measuring gas production in the in vitro rumen fermentation

Measurements

The gas volume was measured at 6h intervals by water displacement from the receiving bottle which was calibrated at intervals of 50ml. Methane percentage at each interval was measured by passing the gas through an infra-red meter (Crowcom Instruments Ltd, UK). At the end of each 6h period samples of substrate were withdrawn from the fermentation bottles using a syringe and needle to penetrate the wall of the bottle, subsequently closing the aperture with tape. The pH was determined immediately by pH meter; the samples were then filtered through cheese cloth and poured into centrifuge tubes containing 1 ml of 0.1N HCl for the determination of ammonia and total VFA by the standard Kjeldahl procedure. Other samples were used to determine the protozoa population by diluting 8 ml of fermented fluid with 16 ml of formol saline solution (1 part of formol 37% and 9 parts of saline 0.9% solution) and counting protozoa under light-microscopy (100x magnification) using a 0.2 mm deep Dollfus counting chamber. Four fields in the counting chamber were filled and protozoa counted, according to the method described by D’Agosto and Carneigo (1999).

DM solubilized was determined at the end of the 24h incubation period by filtering the residual substrate through 2 layers of cloth and absorbent cotton, followed by drying of the residue at 100^°C for 48h.

The DM and N in the coconut cake and cassava leaf meal were determined according to AOAC (1990). N solubility was measured by shaking a 3g sample with 100ml 1M NaCl for 3 hours, filtering through Whatman No.4 filter paper and determining nitrogen in the filtrate (Whitelaw et al 1963).

Statistical analysis

The data were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab Software (Minitab 2000). Sources of variation in the model were: levels of cassava leaf meal and coconut cake, interaction cassava leaf meal*coconut cake and error. Regression equations were calculated using the Microsoft Office Excel software.

Results

Chemical composition of substrates

The levels of protein were similar in cassava leaf meal and coconut cake, but the solubility of the protein was much lower in the latter (Table 2 ).

Table 2. Chemical composition of raw materials used in the experiment
	DM, %	% in DM				Soluble protein, % of total protein
	DM, %	CP	EE	CL	Ash	Soluble protein, % of total protein

Cassava pulp	89.8	3.32	3.28	2.42	1.62	28.7
Cassava leaf meal	91.0	21.3	3.82	6.64	6.84	32.4
Coconut cake	92.2	21.1	16.0	10.1	6.52	20.8

Effect of cassava leaf meal and coconut cake on gas production and methane content of the gas

DM solubilized was increased and gas production and the methane percentage in the gas were reduced both by cassava leaf meal and by coconut cake at each stage of the incubation (Tables 3 to 5; Figures 1-3).

Table 3. Mean values for gas volume at 6h intervals in an in vitro fermentation with (% in DM) cassava leaf meal (CL) and coconut cake (CC) as supplements to cassava pulp-urea
CL	CC			Mean#	p
CL	0	2	4	Mean#	CL	CC	*CLCC**

0-6h
0	593	590	583	589^a
5	590	573	570	578^ab
10	567	573	560	567^b
Mean#	583	578	571		0.025	0.144	0.490

6-12h
0	560	520	523	534
5	550	533	503	529
10	526	520	470	506
Mean#	546^a	524^b	499^c		0.120	0.034	0.028

12-18h
0	497	460	453	470^a
5	460	443	403	435^b
10	413	403	377	397^c
Mean#	456^a	436^b	411^c		0.002	0.012	0.122

18-24h
0	293	253	257	267
5	270	250	246	256
10	243	240	236	240
Mean#	269	248	247		0.062	0.084	0.071
#For each set of means in the same row or column, those without common superscripts differ at p<0.05

Table 4. Mean values for methane percentage in the gas in an in vitro rumen fermentation with (% in DM) cassava leaf meal (CL) and coconut cake (CC) as supplements to cassava pulp-urea
CL	CC			Mean#	p
CL	0	2	4	Mean#	CL	CC	*CLCC**

0-6h
0	16.3	16.1	15.1	15.8^a
5	16.0	15.9	14.8	15.6^a
10	15.4	14.9	14.9	15.1^b
Mean#	15.9^a	15.6^a	14.9^b		<0.001	<0.001	0.016

6-12h
0	18.7	17.1	17.4	17.7^a
5	18.4	16.4	15.4	16.7^b
10	16.8	17.1	15.1	16.3^b
Mean#	17.9^a	18.4	16.4	15.4	<0.001	<0.001	<0.001

12-18h
0	19.0	18.8	17.9	18.6^a
5	19.4	17.6	16.5	17.5^a
10	17.1	17.0	15.7	16.6^b
Mean#	18.5^a	17.8^a	16.7^b		<0.001	<0.001	0.023

18-24h
0	22.5	19.7	18.3	20.2^a
5	21.1	18.4	17.2	18.9^b
10	19.1	17.3	16.3	19.7
Mean#	20.9^a	186^b	17.3^c	18.4	<0.001	<0.001	0.315
#For each set of means in the same row or column, those with no common superscripts differ at p<0.05

Figure 1. Effect of increasing concentrations of cassava leaf meal and coconut cake on gas
production after 24h incubation of a basal substrate of cassava pulp-urea

Figure 2. Effect of increasing concentrations of cassava leaf meal and coconut cake on % DM
solubilized after 24h incubation of a basal substrate of cassava pulp-urea

Figure 3. Effect of increasing concentrations of cassava leaf meal and coconut cake on methane
content of the gas after 24h incubation of a basal substrate of cassava pulp-urea

pH, ammonia, VFA and protozoa

By the end of the 24h fermentation the pH had risen slightly and there was a slight increase due to supplementation with cassava leaf meal, but no effect of the coconut cake (Table 5). Ammonia levels in the substrate decreased over the period of incubation but there were no consistent effects of the supplements (Table 6). Concentrations of VFA decreased markedly during the incubation (Table 7) and there was a slight increase due to supplementation with coconut cake but no effect of cassava leaf meal. At the end of the first 6h fermentation the numbers of protozoa were reduced by cassava leaf meal supplementation but not by coconut cake (Table 8) and had increased by some 50% over the 24h incubation.

Table 5. Mean values for pH in the substrate after 6 and 24h incubation
CL	CC			Mean#	p
CL	0	2	4	Mean#	CL	CC	*CLCC**

6h
0	5.83	5.63	5.67	5.71
5	5.73	5.66	5.77	5.72
10	5.70	5.80	5.83	5.78
Mean#	5.76	5.7	5.76		0.070	0.110	0.002

24h
0	6.03	6.10	6.07	6.07^b
5	6.03	6.03	6.07	6.05^ab
10	6.10	6.17	6.13	6.13^a
Mean#	6.06	6.10	6.09		0.037	0.387	0.869
#For each set of means in the same row or column, those with no common superscripts differ at p<0.05

Table 6. Mean values for ammonia in the substrate in an in vitro rumen fermentation with (% in DM) cassava leaf meal (CL) and coconut cake (CC) as supplements to cassava pulp-urea
CL	CC			Means#	p
CL	0	2	4	Means#	CL	CC	*CLCC**

6h
0	55.1	59.7	52.3	33.6
5	54.1	55.1	60.7	18.7
10	54.1	56.0	56.9	24.3
Mean#	54.	56.93	56.6		0.598	0.058	0.001

24h
0	33.6	20.5	23.3	25.8^b
5	18.7	21.5	21.5	20.5^c
10	24.3	37.3	24.3	28.6^a
Mean#	25.5^ab	26.4^a	23.2^b		<0.001	0.012	<0.001
#For each set of means in the same row or column, those with no common superscript differ at p<0.05

Table 7. Mean values for VFA in the substrate in an in vitro rumen incubation with (% in DM) cassava leaf meal (CL) and coconut cake (CC) as supplements to cassava pulp-urea
CL	CC**			Mean#	p
CL	0	2	4	Mean#	CL	CC	*CLCC**

6h
0	104	105	110	105
5	104	107	110	106
10	105	105	112	107
Mean#	104^b	105^b	110^a		0.140	<0.001	0.179

24h
0	53.0	55.8	59.0	55.9
5	53.3	56.8	59.0	56.3
10	53.5	57.3	59.8	56.3
Mean#	53.3^c	56.6^b	59.3^a		0.025	<0.001	0.586
#For each set of means in the same row or column, those with no common superscripts differ at p<0.05

Table 8. Mean values for protozoa in the substrate in an in vitro rumen fermentation with (% in DM) cassava leaf meal (CL) and coconut cake (CC) as supplements to cassava pulp-urea
CL	CC			Mean#	p
CL	0	2	4	Mean#	CL	CC	*CLCC**

6h
0	1.12	1.05	1.03	1.07^a
5	0.97	0.89	0.96	0.94^b
10	0.89	0.81	0.86	0.86^c
Mean#	0.99	0.91	0.95		<0.001	0.053	0.653

24h
0	1.72	1.54	1.73	1.66
5	1.54	1.63	1.41	1.52
10	1.53	1.72	1.52	1.59
Mean#	1.59	1.63	1.55		0.161	0.522	0.092
#For each set of means in the same row or column, those with no common superscripts differ at p<0.05

Discussion

The decrease in methane percent in the gas was directly related to the proportion of dietary protein that was insoluble in M NaCl (Figure 4). A similar relationship was reported by Preston et al (2019) in a study involving a wide range of grasses, prostrate and tree legumes and shrubs (sweet and bitter varieties of cassava).

Figure 4. Relationship between insoluble protein in the
substrate and percent methane in the gas

The important feature of the in vitro incubation was the negative relationship between the percent methane in the gas and the percent DM solubilized (Figure 5), which was in complete contrast with the relationship with gas production which was positive (Figure 6).

Figure 5. Relationship between percentage methane in the gas and
the percentage substrate DM solubilized over 24h

Figure 6. Relationship between percentage methane in the
gas and the total gas production over 24h

These opposing trends result from the negative relationship between the two measures of digestion/fermentation – gas production and percent DM solubilized (Figure 7).

Figure 7. Relationship between total gas production over 24h and
the percentage of the substrate DM solubilized

The conventional approach is that gas production reflects the nutritive value of the test substance (eg: Menke et al 1979). However, our results indicate the contrary -- the greater was the gas production the greater was the percentage of substrate lost to methane with resultant decline in nutrients available for metabolism.

The fact that substrate digestibility (substrate solubilized) was increased by supplementation (Table 9) shows that supplementation with bypass protein resulted in more nutrients being made available for fermentation; however, the associated decline in rate of gas production implies that not all the nutrients made available were being fermented (hence, less gas production).

Table 9. Mean values for DM solubilized in 24h in an in vitro rumen fermentation with (% in DM) cassava leaf meal (CL) and coconut cake (CC) as supplements to cassava pulp-urea
CL	CC			Mean#	p
CL	0	2	4	Mean#	CL	CC	*CLCC**

24h
0	67.5	70.8	70.3	69.5^a
5	68.5	71.5	71.4	70.5^b
10	72.4	73.3	74.4	73.3^c
Mean#	69.5^a	71.9^b	72.0^b		<0.001	<0.001	<0.001
#For each set of means in the same row or column, those with no common superscripts differ at p<0.05

In the live ruminant some of these nutrients would escape to the lower digestive tract for more efficient enzymic digestion of protein to amino acids, while carbohydrate components could proceed to the cecum-colon for fermentation to acetic acid as postulated by Phonethep et al (2016). Thus, it would appear that one of (or both) the supplements contained entities that depressed slightly the fermentation (and hence the gas production). The cyanogenic glucosides (precursors of HCN) have been shown in many reports to depress methanogenesis (Phuong et al 2012; Phanthavong et al 2015). From the coconut cake the oil fraction could also be expected to reduce the growth of methanogens as reported by Thuy Hong et al (2018). The beneficial effects on animal production (increase in milk yield) by feeding an additive (containing the bactericide thymol) that depressed rumen gas production was recently described by Houda et al (2019) and is supportive of the above hypothesis.

Conclusions

DM solubilized was increased and gas production and the methane percentage in the gas were reduced both by cassava leaf meal and by coconut cake at each 6h interval of a 24h incubation. The decrease in methane percent in the gas was directly related to the proportion of dietary protein that was insoluble in M NaCl.
There was a negative relationship between the percent methane in the gas and the percent DM solubilized, which was in complete contrast with the relationship with gas production which was positive. These opposing trends in turn resulted from the negative relationship between the two measures of digestion/fermentation – gas production and percent DM solubilized.
Substrate digestibility (substrate solubilized) was increased by supplementation indicating that supplementation with bypass protein resulted in more nutrients being made available for fermentation; however, the associated decline in rate of gas production implies that not all the nutrients made available were being fermented.
It is postulated that either one (or both) of the supplements contained entities that depressed slightly the fermentation (and hence the gas production). These could be the cyanogenic glucosides (precursors of HCN) in the cassava leaf meal and the lauric and myristic acids in the oil fraction of the coconut cake.

Acknowledgements

The authors acknowledge the MEKARN II project financed by Sida for the support of this research. The Research and Technology Transfer Center, Nong Lam University, Vietnam provided the infrastructure and facilities.

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Received 21 June 2019; Accepted 19 July 2019; Published 1 August 2019

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