Ảnh hưởng hiệp đồng cùa lá sắn (manihot esculenta crantz), bã bia, và than sinh học (biochar) lên sự sản sinh khí methane và năng suất thú nhai lại tt tiếng anh

HUE UNIVERSITY
UNIVERSITY OF AGRICULTURE AND FORESTRY

LE THUY BINH PHUONG

SYNERGIC EFFECT OF CASSAVA (MANIHOT
ESCULENTA CRANTZ) FOLIAGE, BREWER’S GRAINS,
AND BIOCHAR ON METHANE PRODUCTION AND
PERFORMANCE OF RUMINANTS
Specialization: Animal Sciences
Code: 9620105

SUMMARY OF DISERTATION IN ANIMAL SCIENCES

HUE-2020


This dissertation is completed at: University of Agriculture and Forestry, Hue
University

Supervised by:
1. Assoc. Prof. Dr. Nguyen Huu Van
2. Dr. Dinh Van Dung

1streviewer:

…………………………………..

…………………………………………………..
2ndreviewer:

………………………………..

…………………………………………………….
3rdreviewer:

………………………………..

…………………………………………………….

The dissertation will be defended at the Council of dissertation assessment of Hue
University, 04 Le Loi Street, Hue city, at……………….…….on……/……../2020

Dissertation can be further referred at:
1. National Library
2. Center for Information and Library of Hue University of Agriculture and Forestry,
Hue University.


List of abbreviations, symbols and equivalents

ADG

Average daily gain

BG

Brewers’ grain

CP

Crude protein

CFU

Colony-forming unit

DM

Dry matter

DMI

Dry matter intake

HCN

Hydrocyanic acid

GE

Gross energy

LW

Live weight

N

Nitrogen

NDF

Neutral detergent fiber

NPN

Non-protein nitrogen

SEM
VFA

Standard error mean

Volatile fatty acid



INTRODUCTION
1. Problem statement
Cassava is perspective plant to climate change adaptation; its pests and its diseases
resistance and greater drought tolerance is a major factor in ranking cassava in the food
security of the world (Jarvis et al. 2012). In Vietnam, cassava is second crop, is grown
mainly in both at the household and small-scale processor level (Hoang Kim et al. 2000).
From the successful experiment in utilization of cassava foliage (sweet variety) as protein
source on cattle which was originally reported by Ffoulkes and Preston (1978), and then
have been successfully fed as fresh state to goat and cattle in Cambodia (See report of
Preston and Rodríguez Lylian, 2004), that make cassava foliage become important plant
protein source in ruminant diet. Nevertheless, cyanide toxin in fresh cassava foliage,
especially bitter cassava foliage, is the main obstacle for animal such as restricting the
consumption intake of ruminant or causing poisoning when they consume rapidly.

Nowadays, as the quantity of bitter cassava (high cyanide content) predominate over
sweet cassava (lower cyanide content) on the field, looking for feeding method of bitter
cassava foliage diet with minimizing negative effect of cyanide toxin will match reality
more but will a challenge.
Many studies are beginning to be interested in cyanide toxic that has certain effect on
methanogenic bacteria population by inhibited methanogenesis activity lead to diminish
methane production (Ch Olga Rojas et al. 1999; Phuong et al. 2012; Phanthavong et al.
2015). However, whether cyanide may affect overall microbial activity and impact the
rate of rumen fermentation indirectly, it is still not fully understood. Previously, the
knowledge of ruminant nutritionists focused on rumen, but the impact of cyanide on
rumen fermentation may profoundly influence lower digestive physiology of ruminant
and must be considered to fully understand when utilizing bitter cassava foliage in diet.
Even so, the challenge of bitter cassava foliage diet (high cyanide content) is a new
approach but must require the safety for animal's health. Therefore, building suitable
feeding method for fresh cassava foliage diet, particularly bitter cassava foliage, without
causing cyanide toxin is needed to utilize cassava foliage more effectively in the
ruminant feeding system.
2. The objectives
The aim of this thesis was to develop a greater understanding of both the constraints
and benefits of using cassava foliage in ruminant feeding systems. From these things can
improve the utilization of cassava foliage in ruminant feeding by enhancing its properties
as a source of bypass protein and verify the role of HCN toxin in cassava foliage on the
reduction of methane production that was built on earlier findings.
The following objectives are required to accomplish the aim of this research:
i
Determining the trend influences of HCN concentration in cassava foliage on
the characteristic of in vitro rumen fermentation such as gas and methane
production, ammonia concentration.
5

ii

iii

Building feeding method of “bitter” cassava foliage (KM 94 variety; moderate
HCN content) diet by added 4% brewers’ grain (of DM) and/or 1% biochar (as
DM), then evaluating the effects of this feeding method on growth,
digestibility/N retention, excretion of thiocyanate in urine and methane
production of cattle and goat.
Considering the benefit of brewers’ grain to “bitter” cassava foliage (KM94)
diet by examining Saccharomyces and acid lactic bacteria in fresh brewers’
grain and compare it with potential fermented cassava pulp on gas and methane
production of ruminal in vitro incubation.

3. Significance/innovation of the dissertation
This dissertation successfully demonstrated that HCN in cassava foliage is main
factor for reduction of methane production while the earlier finding could only use it as
prediction for decreased methane. Currently, the best-known cassava foliage to feed
animal is “sweet” cassava foliage with low cyanide content, my dissertation succeeds to
build feeding method for “bitter” cassava foliage diet (higher cyanide content) with
support of adding restricted brewers grain (4% of DM) and biochar (1% of DM) to feed
cattle and goats without cause HCN toxicity. Additionally, discovery of the feeding of the
bitter cassava foliage appear to modify the rumen fermentation lead to increases in
nitrogen retention associated with reduced methane production, it made a part of this
dissertation provided the implication for new approach of the proposed partial shift in
sites of digestion (from rumen to small intestine and the cecal-colon region) that
previously it is thought that only rumen fermentation have a truly symbiotic relationship
with the ruminant.
©©©©©

CHAPTER 1: LITERATURE REVIEW
The literature review consists of the key issue on:
(i) Process of rumen fermentation that related to methane production and direct its
effect on the growth of ruminant. Interpreting interaction of ruminal microorganism is
possible detoxification strategies in context of using cassava foliage that contain high
HCN content.
(ii) The utilization of some by-products in ruminant feeding such as: (i) cassava
foliage varieties as a protein source implied that HCN content as inhibition of
methanogenesis, and (ii) restricted brewer grain and biochar as additives that it is thought
cyanide detoxification in cassava foliage diet; (iii) the role of Saccharomyces in brewer’s
grain was expected as “prebiotic” to support cassava foliage diet.
All above issues were placed in integrative review via the description, interpretation
of prior research and reveal gap in literature.

6


©©©©©
CHAPTER 2. THE EFFECT OF SUPPLEMENTATION WITH “BITTER” OR
“SWEET” VARIETIES ON METHANE PRODUCTION IN AN IN VITRO
INCUBATION

Introduction
Recent industrial development of cassava root processing for extraction of starch
released source of abundant cassava pulp. Cassava pulp and other by-products of cassava
such as leaves, and stalk have potential feeding value for livestock.
Cassava by-product needs to be utilized in ruminant’s feeding system is cassava pulp,
due to its negative fermentation impact lead to polluted environment. The pulp is very
low in protein; however, the foliage is high in CP with content of more than 20% in DM
(Lukuyu et al. 2014). It was reported by Ffoulkes and Preston (1978) that fresh cassava

foliage could replace soybean meal as the only protein source in a fattening diet for cattle
based on ad libitum molasses-urea. Preston and Leng (2009) postulated that part of the
cassava leaf protein had “rumen-escape” characteristics which helped to balance the
microbial protein produced from the rumen fermentation of molasses supplemented with
urea.
Cassava products contain cyanogenic glucosides which liberate hydrocyanic acid
(HCN) when enzymatically degraded. Cyanogenic glucosides exist as linamarin and
lotaustralin in unbruised leaf (Nartey 1968). 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 bio-digestion 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. Establishing a feeding system from cassava by-product is limited the available
information on its effectiveness and the impact of different level of HCN concentration in
cassava foliage varieties on reduced methane production is not clear. Therefore, the
hypothesis of this study was to test that methane production in an in vitro rumen
fermentation would be reduced when urea-supplemented cassava root pulp was incubated
with the leaves from bitter, rather than sweet, varieties of cassava.
Materials and methods
Experimental design
The four treatments in a completely randomized design (CRD) were the leaves of a
“sweet” variety of cassava (Gon) and leaves from three bitter varieties (Japan, KM94 and
KM 140-1) with four replications. The substrates were cassava pulp and urea (Table 1).

7


The leaves were added to provide an overall level of 12.8% crude protein in substrate
DM.
Table 2.1 Composition of the substrates
Gon
Japan
DM basis, %
Cassava pulp
73.1
73.1
Cassava leaves
25
25
Urea
1.8
1.8
Fresh basis, g
Cassava pulp
10.4
10.4
Cassava leaves
12.3
9.8
Urea
0.216
0.216

KM94

KM 140-1

73.1
25
1.8

73.1
25
1.8

10.4
12.2
0.216

10.4
13.5
0.216

A simple in vitro system was used based on the procedure reported by Inthapanya et al (2011).
Material preparation
The cassava leaves (without petioles) were selected at a point approximately one
third of the height of the plant measured from the top. They were stored in plastic bags to
avoid loss of moisture. In the laboratory, the fresh leaves were chopped into small pieces
and then ground (1mm sieve). Dry cassava pulp was taken from the Wuson starch factory,
Binh Phuoc Province.
The 12 grams of substrates (Table 2.1) were mixed with 0.24 liters of rumen fluid
and followed by 0.96 liters of buffer solution (Table 2.2). This mixture was contained in
the fermentation bottle, gassed with carbon dioxide, and incubated in a water bath at 38 °C
for 24h.

Table 2.2 Ingredients in buffer solution
Ingredient
s
CaCl2
NaHPO4.12H2O
g/liter
0.04
9.3
Source: Tilley and Terry (1963)

NaCl
0.47

KCl
0.57

MgSO4.7H2O
0.12

NaHCO3
9.8

Cysteine
0.25

Measurements
The gas volume was measured by water displacement from the receiving bottle
suspended in water. The bottle was calibrated at intervals of 50ml. The methane
percentage in the gas was measured with a Crowcon meter (Crowcon Instruments Ltd,
UK). The DM and crude protein contents of the substrates were determined according to

AOAC (1990) methods. Ammonia was analysed in the filtrate after separating the solids
using a cloth filter. HCN was determined by titration with AgNO 3 after boiling the sample
in KOH to concentrate the HCN. Tannin was analyzed by the Lowenthal method
consisting of boiling the leaves in 0.1N H 2SO4, adding indigo dye and titrating with
potassium permanganate.

8


Statistical analysis
The data were analysed with the general linear model (GLM) option in the ANOVA
program of the Minitab software (Minitab 2000). Sources of variation were treatments,
and error.
Results and discussion
Chemical composition of the substrate
The cassava leaves contained a high level of crude protein (27.5-31.8% CP in DM);
the cassava pulp had less than 3% CP in DM (Table 2.3).
Table 2.3 Chemical composition of the ingredients in the substrate
Gon
Japan
KM 94 KM 140-1
Dry matter, %
24.4
30.6
24.6
22.2
Crude protein, % in DM
32.1
27.5
30

29.7
Starch, % in DM
HCN concentration, mg/kg
339
419
570
826
DM
(*) Data taken from Khempaka et al. (2009); ND= not detected

Pulp
84.4
2.5
53.5 (*)
ND

Gas production, ammonia concentration and DM mineralized
The percentage of DM mineralized (or DM solubilized) was lower in the sweet
cassava variety than in the three bitter varieties among which there were no differences.
Ammonia concentration in the fermentation medium at the end of the incubation was
higher for the sweet cassava than for the bitter varieties (Table 2.4)
Table 2.4 Mean values for gas production in 24 hours, DM mineralized and ammonia
in an in vitro rumen fermentation.
Sweet leaves
Bitter leaves
p value
Japa
KM 140- SEM
Gon
KM 94

n
1
Gas, ml/24h
425
520
515
458
39.6
0.300
DM mineralized,
33.6
26.6a
32.6 ab
38.2 b
2.5
0.044
ab
%
Ammonia mg/L
197
175
177
170
12.3
0.45
ab

Mean values in rows without common letter are different at p<0.05

Methane, HCN and condensed tannin

Methane production, expressed as percent of the gas production or per unit DM
mineralized, was lower when leaves from the bitter varieties were the protein source
compared with the sweet variety (Table 2.5). The negative relationship between HCN and
methane production indicates that it is the HCN precursors in the leaves of the bitter
varieties that are responsible for the decline in methane production (Table 2.5).

9


Table 2.5 Mean values for content of condensed tannin and HCN in the leaves of cassava
varieties, ammonia concentration and methane production per DM mineralized after 24h
incubation
Gon

Japan

KM94

a

b

b

KM 140-1
b

Methane in the gas, %
20.0
14.0

10.5
11.3
Methane
ml/g
DM
29.9a
18.3 ab 14.3 ab
11.5 b
mineralized
HCN, mg/kg DM
339 a
419 b
570 c
826 d
HCN content in treatment, mg
1.02
1.26
1.71
2.46
Condensed tannin in leaf, %
2.00
2.55
2.36
2.55
Condensed tannin in
0.06
0.08
0.07
0.08
treatment, g

abcd
Mean values in rows with common letter are different at p<0.05

SEM

p value

1.31

<0.001

3.80

0.023

31.9

0.044

0.29

0.30

Conclusions
In summary, leaves from Japan, KM94 and KM140-1 as a protein source were more
efficient in reducing methane production rather than Gon cassava leaves, in which levels
of HCN in the leaves and methane production make a negative curvilinear relationship.
Additionally, cassava leaves with higher HCN (Japan, KM140 and KM140-1) may be
suggested as potential feed for post-ruminal digestion by ammonia concentration tend to
lower in the digesta after 24h fermentation compares with Gon cassava leaf.

©©©©©
CHAPTER 3: A LOW CONCENTRATION OF BREWERS’ GRAINS IMPROVES
THE GROWTH RATE AND REDUCES THIOCYANATE EXCRETION OF
CATTLE FED CASSAVA PULP-UREA AND “BITTER” CASSAVA FOLIAGE
Introduction
Comparison of the effects of levels of HCN content in cassava leaves on in vitro
experiment (Chapter 2) only show the local effects on rumen fermentation, the need to
establish feeding trial on animal is to better understand how cassava foliage affect to
intake and growth, particularly with moderate HCN content in bitter cassava foliage diet.
However, how to manage HCN content in fresh cassava foliage diet, especially bitter
foliage variety, is still the biggest concern that it is being looked for the effective solution.
Against background of cyanide toxin in bitter leaves, we organize the experiment on
cattle with cassava pulp-urea as basal diet and compare “sweet” versus “bitter” cassava
foliage as protein source. The hypothesis is that addition of 4% of fresh brewers’ grains to
“bitter” cassava foliage may improve the growth rate of cattle and aid in the
detoxification of the HCN associated with the feeding of “bitter” cassava foliage.

10


Materials and methods
Treatments and experimental design
Twelve Laisind cattle were the experimental animals in a completely randomized
design with 3 treatments and 4 replicates. The basal diet was ensiled cassava pulp plus
0.7% urea (in DM basis of pulp) fed ad libitum together with a mineral mixture
containing 7.5% sulphur, 40% dicalcium phosphate and 52.5% sodium chloride. These
twelve cattle were spent two experimental periods as follows:
The treatments during the first 56 days (Period 1) were:
● BG-RS: Fresh brewers’ grains at 1% of live weight and rice straw 0.9% of live
weight (both on DM basis)

● CFB: Fresh foliage from a bitter variety (KM 94) of cassava at 1% of live weight
(DM basis), replacing the brewers’ grains in the BG-RS control treatment
● CFS: Fresh foliage from a sweet variety (Gon) of cassava at 1% of live weight
(DM basis).
For the 2nd period of next 56 days, the treatments BG-RS and CFS remained the same
Period 1, but treatment CFB was modified to CFB-BG treatment by adding a supplement
of brewers’ grains offered at 4% of the diet DM
Table 3.1 The chemical composition of ingredients
Cassava
pulp

Brewer’
s grain

Rice
straw

Dry matter, %
24.4
27.3
85.0
CP in DM, %
2.54
27.6
3.56
HCN, mg/kg
ND
DM
ND= not detected; CP= crude protein; DM=dry matter

Cassava foliage
Bitter (KM 94
Sweet
variety)
(Gon variety)
24.8
29.0
29.1
31.5
1331

460

Animals and housing
The 12 Laisind cattle with initial live weight from 130 to 175 kg were housed in
individual pens, with bamboo slatted floors and roof of Nipa Palm leaves. The cattle were
vaccinated against foot and mouth disease and de-wormed with Ivermectin before
starting the experiment.
Feeding and management
Fresh cassava pulp (pH 3.2) was purchased from the Wusen cassava starch factory,
Binh Phuoc province. It was stored in closed polyethylene bags prior to feeding.
Brewer’s grains were purchased from the beer factory located in Ho Chi Minh City at 03day intervals and stored in woven polypropylene bags during the 03 days. Cassava plots
of the two varieties ‘KM 94’ and ‘Gon’ were established in the University farm. Cassava
foliage took 1.5 hours to collect on field and transported it to experiment every day to
feed cattle directly. The foliage was harvested at successive periods of 4-5 months
regrowth.
Cattle were spent 1.5 months to adapt to treatments prior to starting real experiment.
For the adaptation of fresh bitter cassava foliage, sweet cassava foliage was introduced to
11

cattle firstly, then gradual replacement of sweet foliage by bitter foliage until cattle can
feed only bitter cassava foliage as a protein source. All the feeds were offered in separate
troughs as freely chosen and were feed three times a day, at 8.00 a.m., 12.00 a.m. and
5.00 p.m.
Data collection and measurements
The cattle were weighed at the beginning of the experiment and at intervals of 14
days. Feeds offered and refused were recorded daily. Samples of rumen fluid were taken
by stomach tube 03 hours after the last meal at the end of the experiment for
determination of concentrations of individual VFA (Rowe et al.1979).
On the last day of the whole experiment: (i) samples of urine were caught behind
each female cattle and avoid contaminated by feces, frozen and stored at -20°C for
thiocyanate analysis; (ii) the cattle were confined individually in a closed chamber for
successive measurements over 10 minute periods of the concentrations of methane and
carbon dioxide in mixed air and eructed gas. The procedure was that described by
Madsen et al. (2010), using an infra-red gas meter (GASMET 4030; Gasmet
Technologies Oy, Pulttitie 8A, FI-00880 Helsinki, Finland). For the cattle on treatment
CFB-BG, after first sampling of eructed gas/air, and of urine, the brewers’ grains were
removed from the diet and for 5 consecutive days they were fed only cassava pulp-urea
and bitter cassava foliage. At the end of this period the eructed gas-air was again
analyzed, and samples taken of urine for thiocyanate analysis.
Chemical analysis
Feeds offered and refused were analysed for DM and N following the procedure of
AOAC (1990). Thiocyanate in urine was measured using the procedure described in the
protocol of kit D1
Statistical analysis
The data were analysed with the general linear model (GLM) option in the ANOVA
program of the Minitab software (Minitab 2000). Sources of variation were treatments,
and error.
Results and discussion

In Period 1 the cattle fed bitter cassava foliage as sole source of bypass protein had
lower DM intake than those fed sweet cassava foliage and gained only 61g/day compared
with 383g/day for those fed sweet cassava foliage (Table 3.2a). When brewers’ grains at
4% of the diet were added to this treatment in Period 2, the DM intake increased by 47%
and the live weight gain (380g/day) did not differ from the 410g/day for cattle fed sweet
cassava foliage (Table 3.2b).

12


Table 3.2a Mean values for feed intake, change in live weight and feed conversion of Laisind
cattle in Period 1
BG-RS
CFS
CFB
SEM
p
DM intake, g/day
Cassava pulp
2401a
1834b
1891b
67.8
0.001
Urea
16.8
12.8
13.2
Brewer’s grains
690

Rice straw
1295
Cassava foliage
Sweet
1385
Bitter
587
a
b
Total DMI, g/day
4386
3219
2482c
81
0.001
a
b
DMI at % body weight
2.11
1.77
1.61b
0.06
0.001
Apparent HCN intake,
637
781
mg/day
Live weight, kg
Initial
159

146
140
11.4
0.51
Final
187
169
143
13.5
0.12
LW gain, kg/day
0.49a
0.38a
0.06b
0.05
0.001
DM conversion
9.22
8.47
abc
Mean values in rows without common letter are different at p<0.05.
LW (live weight); DMI (dry matter intake); BG-RS (brewers’ grains plus rice straw); CFS (sweet
cassava foliage); CFB (bitter cassava foliage)
Table 3.2b. Mean values for feed intake, change in live weight and feed conversion of
Laisind cattle (in Period 2)
BG-RS
CFS
CFB-BG
SEM
p

DM intake, g/d
Cassava pulp
3211a
2737b
2629b
68
0.001
Urea
22.5
19.2
18.4
Brewers grains
1541
213
Rice straw
1343
Cassava foliage
Sweet
1267
Bitter
810
a
b
Total DM, g/day
6095
4004
3652c
82
0.001
a

b
DMI at % body weight
2.93
2.17
2.34b
0.09
0.001
Apparent HCN intake,
583
1078
mg/day
Live weight, kg
Initial
187
169
143
14
0.12
Final
222
191
163
14
0.047
LW gain kg/d
0.66a
0.41ab
0.38b
0.07
0.039

DM conversion
9.75
10
10.4
1.53
0.95
ab
Mean values in rows without common letter are different at p<0.05.
LW (live weight); DMI (dry matter intake); BG-RS (brewers’ grains plus rice straw); CFS (sweet
cassava foliage); CFB-BG (bitter cassava foliage plus brewers’ grain at 4% of diet DM).

13


The excretion of thiocyanate in urine was highest in cattle fed only with bitter foliage
(CFB treatment), was reduced to half by addition of 4% brewers grains to the bitter
foliage diet (CFB-BG treatment), was present at a low level in the urine of cattle fed
sweet foliage (CFS treatment), and was not detected in the urine of cattle fed cassava
pulp-urea, and brewers’ grains at 25% of diet DM plus rice straw (Table 3.3).
Table 3.3 Mean values for thiocyanate in the urine of cattle
BG-RS
CFB
CFB-BG
CFS
SEM
p
a
b
c
90

55
12
Thiocyanate,
0
6.06
<0.001
ppm
abc
Mean without common superscript differ at p<0.05
BG-RS (brewers’ grains plus rice straw); CFB (only bitter cassava foliage as protein
source), CFB-BG (bitter cassava foliage plus 4% brewers’ grains) and CFS (only sweet
cassava foliage)

There was no effect on VFA proportions when brewers’ grains were included at 4%
of the diet on the bitter cassava foliage treatment.
Table 3.4 Mean values for VFA proportions in rumen fluid of cattle (in Period 2)
Molar %
BG-RS
CFB-BG
CFS
SEM
p
Acetic
50.4b
64.0ab
66.5a
3.19
0.024
Propionic
38.7a

24.1ab
25.5b
3.25
0.035
Butyric
10.9
11.9
7.97
1.62
0.28
Ac:Pr ratio
1.34
0.40
0.07
2.73
2.75
ab
Mean without common superscript differ at p<0.05
BG-RS (brewers’ grains plus rice straw); CFB-BG (bitter cassava foliage plus
4% brewers’ grains); CFS (sweet cassava foliage)

Levels of methane relative to carbon dioxide in mixed eructed gas-air decreased in
the order in which the cassava pulp-urea diet was supplemented with brewers’ grains,
sweet cassava and bitter cassava, respectively (Table 3.5).
Table 3.5 Mean values of methane: carbon dioxide ratios in mixed eructed gas/air of Laisind cattle
CFB-BG
BG-RS
CFS
CFB
SEM

p value
b
a
ab
b
0.0308
CH4: CO2 ratio
0.0364
0.0335
0.0310
0.00195
0.015
ab
Mean without common superscript differ at p<0.05
CFS (sweet cassava foliage); CFB (bitter cassava foliage); CFB-BG (bitter cassava foliage plus
brewers’ grains at 4% of diet)

Conclusion
Adding brewers’ grains at 4% of the diet to bitter cassava foliage, the DM intake
increased by 47% and the live weight gain (380g/day) did not differ from the 410g/day
for cattle fed only sweet cassava foliage. Besides that, adding restricted brewers’ grain
(4% of DM) in bitter foliage impact positively in reducing half urine excretion
thiocyanate compared with only bitter foliage. The ratio of methane relative to carbon
dioxide in mixed eructed gas-air decreased in order of treatments in which the cassava
14


pulp-urea diet was supplemented with brewers’ grains, sweet cassava and bitter cassava,
respectively.
©©©©©

CHAPTER 4: METHANE PRODUCTION IN AN IN VITRO RUMEN
INCUBATION OF CASSAVA PULP-UREA WITH ADDITIVES OF BREWERS’
GRAIN, RICE WINE YEAST CULTURE, YEAST-FERMENTED CASSAVA
PULP AND LEAVES OF SWEET OR BITTER CASSAVA VARIETY
Introduction
Previous studies have shown that adding small amounts (4% of diet DM) of brewers’
grain to a diet of cassava-pulp-urea-bitter cassava foliage improved DM intake and live
weight gain of cattle, and reduced excretion of urine thiocyanate (Chapter 2);
Rice wine starter culture contains yeast, to be used in alcohol fermentation of rice
wine could a hint for enhancement of nutritive value of cassava pulp. Therefore, the
comparisons fermented cassava pulp with brewer’s grain is to insight on the positive
effect of alcohol fermentation by-product on rumen fermentation, thereby looking for
potential sources of byproducts that can replace brewer's grain. Thus, the objective of the
present study was to evaluate possible alternatives to these two byproducts that could
potentially act as prebiotics in ruminant diets based on cassava root pulp and cassava
foliage of the sweet and bitter varieties.

Materials and methods
Treatments and design
Two factors were studied in an in vitro rumen incubation. The design was a 2*5
factorial with 3 replications. The factors were:
● Source of cassava foliage: sweet (Gon) or bitter (KM94) variety.
● Potential source of prebiotic (additive): No additive (CTL); Brewers’ grain (BG);
Rice wine starter culture (RWS); Cassava pulp fermented with RWS (YFCP) and
cassava pulp fermented with RWS, urea and di-ammonium phosphate (YFCP-UDAP).
The basal substrate (DM basis) was 73% ensiled cassava pulp, 2% urea and 25%
cassava leaves (sweet or bitter variety). The additives were incorporated in the substrate
at levels (DM basis) of 4%, except for the RWS which was added at 2%.
Materials
The brewer’s grains were taken directly, in the fresh state, from the brewing process

at the beer factory in Ho Chi Minh City. YFCP-U-DAP was made by fermenting fresh
cassava pulp for 7 days with 2% rice wine starter culture (RWS), 1% sodium chloride,
3% urea and 1% di-ammonium phosphate (DAP) (all on DM basis). It was stored 7 days
in closed polyethylene bags. YFCP was made by the same procedure but without urea
15


and DAP. The in vitro procedure was the same as that described by Inthapanya et al.
(2011)
Data collection
Samples of the additives were analysed prior to, and after 07 days of fermentation
for: DM and CP according to methods of AOAC (1990); pH and Brix values were made
using a digital pH meter and hand-refractometer.
For microbiological analysis: 05-gram fresh sample was diluted in Wilkin medium in
different concentrations. MRS medium was used to isolate lactic acid bacteria (LAB) at
370C. To 1-liter MRS medium (De Man, Rogosa and Sharpe agar) was added 02-gram
natamycin dissolved in 40ml sterile water to prevent the development of fungi (Dung et
al. 2007). The Sabouraud medium was used to isolate Saccharomyces at room
temperature. Colonies were counted and the results expressed as colonies per gram of
sample (CFU/g).
The volume of gas produced in the in vitro incubation was recorded by water
displacement at 7, 12 and 24 hours. The methane percentage in the gas at each of these
times was measured by infra-red meter (Crowcon Ltd, UK).
Statistical analysis
The data were analyzed with the general linear model (GLM) option in the ANOVA
program of the Minitab software (Minitab 2000). Sources of variation were source of
cassava foliage, additive, interaction forage source*additive and error.
Results and discussion
Composition of the substrates
As was expected, the supplementation of both urea and di-ammonium phosphate in

the yeast-fermented cassava pulp resulted in higher pH and crude protein than without
them (Table 4.1)
Table 4.1 Chemical composition of substrates
DM, % # Crude protein, % in DM
Fresh cassava pulp
31.2
1.89
Brewer's grain
25.8
YFCP (0 day)
29.6
2.02
YFCP (7 days)
28.5
3.01
YFCP-U-DAP (0 day)
32.2
9.34
YFCP-U-DAP
(7
29.7
10.2
days)
# Fresh basis; ## Measures % soluble sugars

Brix, % ##
2
3
6
3

pH
5
5.1
5.5
3.9
6.6

8

4.2

Gas production and methane content of the gas
Gas production was higher when the protein source was leaf from bitter as opposed
to the sweet cassava variety; and was higher when 4% brewers’ grains were added to the
substrate (Tables 4.2a, 4.2b) The exception was in the case of the rice wine starter
additive (RWS).
16


When the cassava leaf was from the sweet variety, brewers’ grains reduced the
methane in the gas, but there was no effect of the other additives. With the bitter variety
of cassava, the results were more variable.

Table 4.2a Effect of source of cassava variety on gas production and
methane percentage in the gas
Sweet
Bitter
SEM
p

0-7h
Gas, ml
660
683
16.2
0.32
Methane, %
8.53
4.73
0.26
<0.001
7-12h
Gas, ml
630
647
11.3
0.31
Methane, %
8.60
7.13
0.23
<0.001
12-24h
Gas, ml
883
790
21.6
0.006
Methane, %
8.73

10.1
0.22
<0.001
0-24h
Gas, ml
2080
2213
33.2
0.01
Methane, %
8.63
7.58
0.14
<0.001

Table 4.2b Effect of additive on gas production and methane percentage in the gas
CTL

BG

RWS

YFCP

YFCP-UDAP

0-7h
Gas, ml
600bc
725a

575c
692ab
767a
Methane, %
6.50b
5.83b
6.50b
6b
8.33a
7-12h
Gas, ml
642b
675ab
475c
650b
750a
ab
b
ab
b
Methane, %
7.67
7.17
8.17
7.33
9a
12-24h
Gas, ml
792ab
933a

708b
867a
883a
a
b
a
a
Methane, %
9.67
8
10.2
9.67
9.50a
0-24h
Gas, ml
2033b 2333a 1758c
2208ab
2400a
Methane, %
8.23ab
7.11c
8.41ab
7.86bc
8.92a
abc
Mean without common superscript differ at p<0.05
# CF*S: interaction of cassava foliage variety and supplement

SEM

p

p#

25.5
0.42

<0.001 0.024
0.003 <0.001

17.9
0.36

<0.001
0.012

0.075
0.002

34.2
0.35

0.001
0.004

<0.001
0.04

52.6
0.22

<0.001
<0.001

0.002
0.013

There were no viable Saccharomyces cells in the brewers’ grain but high populations
in the two yeast-fermented additives (Figure 4.5). There were traces of lactobacilli in the
brewers’ grains but much greater quantities in the yeast-fermented additives (Figure 4.6).
17


By contrast, in the treatments YFCP-U-DAP and RWS the fermented substrate had not
been submitted to “heat” treatment and thus the yeast in these additives was still “viable”.

Figure 4.5 Viable Saccharomyces cells in
brewers’ grain and in cassava pulp fermented
only with yeast (YFCP) or with yeast and urea
(YFCP-U-DAP) after 07 days of fermentation

Figure 4.6 Lactobacilli in brewers’ grain and in
cassava pulp fermented only with yeast (YFCP) or
with yeast and urea (YFCP-U-DAP) after 07 days of
fermentation

Conclusion
Adding 4% brewers’ grain gives the interaction with source of cassava leaves in
reducing methane when the cassava leaf source was the sweet variety but no effect when
the cassava leaf was from a bitter variety. Contrary to expectation, the other possible

alternatives (YFCP and YFCP-U-DAP and RWS) had no apparent effect on gas
production or methane content of the gas. The data on microbiology examination of
additives imply that no viable Saccharomyces and lactic acid bacteria (LAB) in brewers’
grain by distillation and acid hydrolysis is necessary for releasing β-glucan as “prebiotic”.

©©©©©
CHAPTER 5: EFFECT OF ADDITIVES (BREWER’S GRAIN AND BIOCHAR)
AND CASSAVA VARIETY (SWEET VERSUS BITTER) ON NITROGEN
RETENTION, THIOCYANATE EXCRETION AND METHANE PRODUCTION
BY BACH THAO GOATS
Introduction
In an experiment on cattle fed foliage from known “sweet” (Gon) and bitter (KM94)
varieties, as protein-rich supplements to ensiled cassava pulp-urea (Chapter 3), there were
major differences in animal response with reduced feed intake and negligible growth rate
(61 g/d) when the bitter cassava was fed compared with normal feed intake and growth
18


rate (383 g/d) when the cassava foliage was from the sweet Gon variety. The important
observation in that experiment was that the addition of a low level of brewers’ grains (4%
of diet DM) to the “bitter” cassava diet led to similar feed intakes and growth rates as for
cattle fed the sweet cassava foliage. It was hypothesized that the low-level addition of
brewers’ grains was acting as a “prebiotic” in facilitating the detoxification of the HCN
produced in the cattle fed the bitter cassava foliage. This was confirmed by analysis of
the urine taken before and after addition of brewer’s grains to the diet which showed a 50
% reduction in thiocyanate concentrations in the urine from 90 to 55 ppm.
In an initial study with a cassava-based diet fed to local Yellow cattle in Laos
(Leng et al. 2012), growth rates were increased, and the methane content of eructed gas
was reduced when 1% biochar was added to the diet. More recent studies have shown: (i)
synergistic effects from combining biochar with rice distillers’ byproduct as additives to a

cassava-based diet for fattening cattle (Sengsouly and Preston 2016); and (ii) that biochar
added to a basal diet of urea-treated cassava stems fed to goats increased daily N
retention by 46% and the biological value of the absorbed N by 12% (Thuy Hang et al.
2018). The aim of the following experiment was to test the individual and combined
effects of biochar and brewers’ grains as “prebiotics” in goats fed diets of 100% cassava
foliage. Contrasting intakes of cyanogenic glucosides were achieved by feeding: (i)
foliage of a sweet variety as the sole diet; or (ii) allowing the goats to have free access to
foliage of both sweet and bitter varieties.
Materials and methods
Experimental design
Eight male Bach Thao goats were allocated to two 4*4 Latin Square designs (Table
5.1). In the first Latin Square, the goats were fed “sweet” cassava foliage (Gon variety)
ad libitum as basal diet. In the second Latin Square, the goats had free access to foliage of
both sweet (Gon) and bitter cassava (KM94) varieties. In each square there were 4
combinations of “additives”. Each period lasted 23 days; 18 days for adaptation to the
treatments followed by collection of feces and urine over the last 5 days.
The treatments in each square were:
● CTL: No additive
● BG: Brewer’s grain (at 4% of diet DM)
● Bio: Biochar (1% of diet DM) suspended in molasses (ratio of biochar/molasses is
1:5 by weight) to prevent loss of particulate matter
● BGBio: The combination of brewers’ grains and biochar.
Table 5.1 Layout of each Latin Square
Period/go
1
2
at
1
CTL
Bio

2
BG
CTL
3
Bio
Bio
4
BGBio
BG

3

4

BGBio
Bio
BG
CTL

BG
BGBio
CTL
Bio

19


Feeds and feeding
Bitter cassava foliage was grown in a farmers’ field 30 km distant from the
University while the sweet cassava foliage was on a farm 20 km distant. They were

collected daily with total time for bitter foliage collection in the field, transportation and
feed preparation was 02 hours including: (i) spending 15-20 minutes to collect 4-6 kg
fresh foliage and removing the stems followed by: (ii) 1.5 hours for transportation by bus;
and (iii) 15 minutes for weighing the foliage according to the experimental diets. For
sweet cassava foliage, the overall time was 1.5 hours. Both cassava foliage was fed as
fresh state directly.
Brewer’s grains were purchased from the beer factory located in Ho Chi Minh City at
3-day intervals and stored in woven polypropylene bags during the 3 days. Molasses and
biochar were prepared at the beginning of the experiment and stored for use over the
whole experimental period. Biochar was made in an updraft biochar stove using rice
husks as fuel.
Animals and feeding system
The eight male Bach Thao goats had an average initial weight 16.4 ± 2.03 kg. They
were housed in raised cages with facilities for separate collection of urine and feces. Goat
was adapted to eating the cassava foliage. In both Square 1 and Square 2, sweet cassava
foliage (tender stems with petioles and leaves attached) was suspended above the feed
trough (to simulate browsing). In square 2, after 05 days with only the sweet variety, the
bitter variety was introduced and gradually increased until the goats had free access to
both the sweet and bitter varieties.
The procedure during the actual experiment was:
Day 1 to 14: The additives were offered according to the designated treatments.
Adaptation to the additives was by mixing a small amount of cassava leaves with the
additive and then removing gradually the leaves over 4-5 days until the goats were seen
to directly consume the additive (biochar was always mixed with molasses in ratio of 1:5
by weight). Brewers’ grain and biochar were placed in separate troughs so that goat can
be freely chosen according to their needs.
Day 15: Samples of rumen fluid were taken by stomach tube 2 hours after first
feeding for measurement of ammonia and molar proportions of volatile fatty acids
(VFA). The pH was measured with a digital meter and sulphuric acid added as
preservative prior to analysis of VFA.

Day 16: A sample of ample of urine was taken for determination of thiocyanate
Day 17: A closed chamber was used for methane measurement. The procedure was
that described by Madsen et al (2010), using an infra-red gas meter (GASMET 4030;
Gasmet Technologies Oy, Pulttitie 8A, FI-00880 Helsinki, Finland).
Day 18: Live weights were recorded in the morning prior to the first feeding.
Days 19-23: Sulphuric acid (10% concentration) was added daily to the urine
container (at 10% of total volume) to keep the pH under 4. The daily amounts of urine
and feces were collected and weighed, and the feces frozen at -10 0C. At the end of each
collection period: (i) the daily amounts of feces were thawed and mixed thoroughly to
20


provide a representative sample for analysis; (ii) daily collections of urine were mixed
and sub-samples taken for analysis
Chemical analyses
Samples of feed offered, feed residues and feces were determined DM and total
nitrogen content by Kjeldahl method (AOAC 1990). Condensed tannin in cassava leaves
and petioles was determined by boiling the samples in 0.1N H 2SO4, adding indigo dye
and titrating with potassium permanganate according to AOAC (2016) official method
955.35. Cyanide as equivalent hydrogen cyanic acid content (HCN) in fresh cassava
leaves and petiole was determined as per AOAC (2016), official method 915.03B.
Thiocyanate was determined following the procedure described in the protocol of kit D1
(source:
/>%20D1.pdf). Ammonia in rumen fluid was measured by a colorimetric method using UV
spectrophotometric detector at wavelength of 640 nm. Determination of molar
proportions of volatile fatty acids (VFA) was by high performance liquid chromatography
(HPLC).
Statistical analysis
The data for DM intake and N retention were analyzed using the Repeated Measures
option in the General Linear Model in the ANOVA program of the SAS Software (SAS

2010). The repeated measures were the daily DM intakes, and daily quantities of DM and
N in feces and of N in urine during days 21 to 25 of the collection period. Other data
were analyzed by the General Linear Model option in the ANOVA program of the
Minitab Software (Minitab 2016). In each model, the sources of variation were variety of
foliage, additives, interaction foliage*additives and residual error.
Results and discussion
HCN and tannin content in cassava foliage
Levels of condensed tannins were similar in leaves of sweet and bitter varieties of
cassava and were higher in leaves than in petioles (Table 5.2; Figure 5.1). Leaves of the
bitter variety had 72% more precursors of HCN than leaves of the sweet variety. HCN
precursors were higher in leaves than in petioles.
Table 5.2 Mean values for effects of cassava foliage (sweet or bitter)
on % DM, tannin and HCN equivalent in leaves and petioles
Leaf
Petiole
Swee
Bitter
t
Bitter
Sweet
p
SEM
a
a
b
b
DM, %
28.7
26.4
15.6

16.4
0.004
2.24
2.98
ab
Tannin*, %
3.18a
2.13 ab
1.9 b
0.022
0.288
HCN*, mg/kg 1282 a 745 b
296 c
415 c
<0.001
65.1
Note: % DM is on fresh basis; (*) is on DM basis

Overall intake of DM was 25% higher for the offer of mixed varieties compared with
the sweet variety alone (Table 5.4). DM intake was higher when both brewers’ grains and
biochar were added to the diet compared with the additives given separately or were not
21


given (Table 5.4). There was no interaction between source of cassava foliage and the
source of additives.

Table 5.4 Mean values (g/d) for effects of cassava foliage (bitter or sweet) and of additives (none,
biochar, brewers’ grains or both) on DM intake (DMI), apparent digestibility of DM and crude protein
(CP) and N balance

Cassava variety
Additives
BIT+SW
SW
SEM
p
Non
Bio
BG
BGBio SEM
p
e
DMI
496
408
8.35 <0.0001 449 a
436 a
441 a
482 b
8.35 0.0002
Apparent digestibility, %
DM
74.7
74.4 0.804 0.79
75.5
73.3
74.7
74.6
1.14
0.58

CP
81.9
81.2 0.563 0.43
82.8
80.3
81.7
81.4
0.796 0.19
N balance, g/d
Intake
19.1
15.3
0.39 <0.001 15.4
14.4
15.3
16
0.46 0.108
Feces
3.51
2.71
0.10 <0.001 2.91
3.11
3.13
3.29
0.137 0.28
Urine
5.85
4.57
0.27 <0.001 6.31b
4.38a

5.58b
4.56a 0.376 <0.001
a
a
a
Retention
8.68
7.14
0.45 <0.0001 7.13
7.81
7.58
9.12b 0.449 0.0032
ab
Means within main effects without common superscript differ at p=0.05
BIT+SW (mixing of bitter and sweet foliage); SW (sweet foliage alone)

N retention was increased when either brewers’ grains or biochar or both (Table 5.4)
were added to the diet. The combined effect of mixed cassava foliage with brewers’
grains and biochar represented a 58% increase in N retention compared with sweet
cassava alone and no additives.
Table 5.6 Mean values for VFA proportions (mol %), acetic: propionic ratio, rumen
ammonia, daily urine volume, daily excretion of thiocyanate (SCN) in urine and CH4:CO2
ratio in mixed eructed gas and air
Cassava foliage
Additives
SW SW-BI SEM
p
None Bio
BG BG-Bio SEM
p

Ac
81.1
77.3
3.21 0.42 76.1 75.5 83.2
82.1 4.54 0.52
Pr
9.53
11.5
1.35 0.31 10.9 11.8 9.81
9.57 1.91 0.83
Bu
9.37
11.1
2.93 0.68 13.0 12.7 7.01
8.31 4.14 0.66
Ac:Pr
9.26
9.00
1.11 0.86 9.53 7.59 9.31
10.1 1.57 0.71
NH3, mg/l
514
423
30.0 0.042 396
459
535
484
42 0.17
Urine,
ml/day

685
811
96.7 0.36
786
664
771
771
137 0.92
SCN,
mg/day
10.8
22.0
3.46 0.03 23.2
9.7
14.8
17.8 4.89 0.30
CH4:CO2
0.0422 0.0357 0.0017 0.012 0.0415 0.0355 0.0375 0.0412 0.0024 0.25
22


Thiocyanate levels in urine appeared to be reduced by the additives with the effects
being most pronounced for biochar especially in the goats fed foliage from the mixed
sweet-bitter varieties (Table 5.6). There were no indications of HCN toxicity. All the
goats were healthy
There was a negative relation between the methane: carbon dioxide ratio in mixed
eructed gas and air and N retention (Figure 5.6a). The relationship was particularly strong
when an apparent “outlier” result was removed from the data set (Figure 5.6b).
Figure 5.6a Relationship between methane: Figure 5.6b Relationship between methane:
carbon dioxide ratio in mixed eructed gas carbon dioxide ratio and nitrogen retention

and air and nitrogen retention (includes all 8 (excluding the outlier result)
goats)

Conclusion
● Rumen ammonia was lower and N retention was higher when the goats had access
to both sweet and bitter cassava foliage as opposed to the sweet variety alone.
● N retention was increased when either brewers’ grains or biochar or both were
added to the diet. The combined effect of mixed bitter and sweet cassava foliage
with brewers’ grains and biochar represented a 58% increase in N retention
compared with sweet cassava alone and no additives.
● Rumen methane production was lower when mixed bitter and sweet cassava
foliage was fed compared with sweet cassava alone.
● The reduction in rumen methane was positively correlated with N retention. It is
proposed that HCN precursors present in higher concentrations in the leaves of
bitter compared with sweet cassava leaves induced a partial shift in digestion of
nutrients from the rumen to the lower digestive tract facilitating more efficient use
of dietary protein by enzymic digestion in the small intestine and reducing the
formation of methane which is not produced when fermentation takes place in the
cecum-colon.
©©©©©
GENERAL CONCLUSIONS
In summary, the body of work presented in this dissertation has shown that
supplementing ruminant diets with cassava foliage reduces rumen methane production,
and the effect is more pronounced with varieties containing higher levels of cyanogenic
glucosides, which give rise to HCN in the rumen. However, the risk of toxicity of cyanide
in ruminants (cattle and goats) can be reduced by adding to the diet either brewer’s grains
or biochar or both as a “prebiotic” source. In the presence of these “prebiotics”, the
challenge of including foliage of bitter cassava in the diet of cattle and goat did not
negatively impact on feed intake, growth and health. In more detail, adding restricted (4%
of DM) brewers’ grain into “bitter” cassava foliage as a main protein source appeared

23


reduction of excreted thiocyanate in urine, lead to significant improvement of growth rate
of cattle compare to only “bitter” cassava foliage. Moreover, synergistic of brewers’ grain
(4% of DM) and biochar (1% of DM) as additives could show substantially same
effectiveness even when goat was fed “bitter” cassava foliage at up to 50% in the diet. On
the contrary, the feeding of the bitter cassava foliage appeared to modify the rumen
fermentation leading to an improved balance of nutrients at the whole animal level, as
manifested by the 20% increase in nitrogen retention associated with decreased production
of methane.
PUBLISHCATION LIST
Paper 1. Binh P L T, Preston T R, Van H N and Dinh V D 2018: Methane production in
an in vitro rumen incubation of cassava pulp-urea with additives of brewers’ grain, rice
wine yeast culture, yeast-fermented cassava pulp and leaves of sweet or bitter cassava
variety. Livestock Research for Rural Development. Volume 30, Article #77.
/>Paper 2. Phuong L T B, Preston T R, Van N H and Dung D V 2019: Effect of additives
(brewer’s grains and biochar) and cassava variety (sweet versus bitter) on nitrogen
retention, thiocyanate excretion and methane production by Bach Thao goats. Livestock
Research
for
Rural
Development.
Volume
31,
Article
#1.
/>
24