Nâng cao hiệu quả sử dụng bauhinia acuminate trong chăn nuôi dê ở lào (tt)

HUE UNIVERSITY
UNIVERSITY OF AGRICULTURE AND FORESTRY

PHONEVILAY SILIVONG

IMPROVED UTILISATION OF BAUHINIA ACUMINATA
FOR GOAT PRODUCTION IN LAO PDR

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

Suppervised by:
1. Associate Professor Dr. Nguyen Huu Van
2. Dr. Dương Thanh Hai
1st reviewer:……………………………………………………………………………………………………………
2nd reviewer: ……………………………………………………………………………………………………………
3rd reviewer: ……………………………………………………………………………………………………………

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

INTRODUCTION

1. PROBLEM STATEMENT
Laos is located in the central part of the Indochinese Peninsula. It is an inland state
surrounded by China, Vietnam, Cambodia, Thailand and Myanmar. Laos has a total land area of
236,800 km2. The agricultural land is limited to around 4% of total, consisting of 18
provinces/cities comprising 148 districts. Laos population has 7,028,094 people and is equivalent to
0.09% of the total world population. Laos has a distinct rainy season from May to November,
followed by a dry season from December to April. Local tradition holds that there are three seasons
(rainy, cold and hot) as the latter two months of the climatologically defined dry season are
noticeably hotter than the earlier four months. Goats are increasingly important for subsistence food
production with over 90% of the global goat population found in developing countries (Glimp,
1995; FAO, 2005; World Bank, 2013). As goats produce several livestock products with lower
inputs than cattle and buffalo, smallholder goat farmers in developing countries, particularly in Asia
and Africa, have increasingly been recruited to goat raising, with goats described as an ‘entry point’
on the ‘pathway from poverty’. Goats are considered more easily managed than cattle, especially by
resource poor farmers, including women. Goat raising offers households nutritional benefits as meat
protein for hunger alleviation, enhanced livelihoods from animal trading income, more effective
utilisation of family labour, and increased livelihood stability and resilience in rural communities
due to more self-reliance (FAO, 2005; World Bank, 2013). In Southeast Asia, goats have been of
increasing importance, particularly in countries with large Islamic populations, including Indonesia,
Malaysia, and parts of the Philippines and Thailand. However, in recent years, increasing demand
for consumption of goat meat in Vietnam and China has created opportunities for increasing
production in the Lao People’s Democratic Republic (Laos, henceforth). Currently, the government
of Laos is attempting to obtain an average meat supply for local consumption of 60kg/capita/year,
plus increased meat exports to a value of USD 50 million by 2020 (FAO, 2005). In Laos, goat
production is traditionally extensive with low inputs, and subsequently low outputs (Kounnavongsa
et al., 2010). Four major goat management systems have been described, including: free range;

semi-free range; semi-rotational grazing; and permanent grazing with or without tethering. Free
range is the most commonly observed system, although semi-free range can be found in areas where
cropping predominates (Kounnavongsa et al., 2010; Phengvichith and Preston, 2011). In most
systems, goats are herded back to the village and kept in small hutches overnight for protection,
although housing is only considered beneficial if it is kept clean (Phengsavanh, 2003). The system
used by an individual farmer will depend upon feed and labour availability plus local community
agreements, particularly related to cropping and use of common grazing areas (Kounnavongsa et al.,
2010; Phengvichith and Preston, 2011). Typically, Lao goat herds consist of 3-10 animals
(Kounnavongsa et al., 2010; Phengvichith and Preston, 2011), although there are some recent
examples of developing herds with as many as 200 animals raised on semi- and fully-commercial
farms. Approximately 551,153 goats were recorded in Laos in the 2016 agricultural census (DLF,
2016). This number is likely to be underestimated, as it is widely considered to have been
increasing rapidly due to recent expanding regional demand for goat meat, particularly from
Vietnam, with estimates that between 2,000-3,000 goats per month are being exported. Increasing
demand for consumption of goat meat in Laos and neighbouring Vietnam and China, is providing
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opportunities for smallholder farmers to increase productivity and has led to the development of
semi to full commercial production systems to capitalise on the growth in this emerging livestock
sector, particularly if biosecure transboundary trade can be enhanced (Stur et al., 2002; Windsor,
2011; Nampanya et al., 2015). However, introducing goats and expanding small goat herds where
smallholders and potential commercial operators have limited experience of small ruminants can be
exceedingly challenging. In recent years, many international development agencies have promoted
smallholder goat-raising programs with distribution of goats to untrained farmers, often
accompanied by severe mortality and morbidity problems (Windsor et al., 2017). In developing
improved systems for feeding livestock, account must also be taken of the impacts on the
environment. It is estimated that livestock presently account for some 18% of greenhouses gases
which cause global warming (Steinfeld et al., 2006). Enteric methane from fermentative rumen
digestion is the main source of these emissions. There is an urgent need to develop ways of

reducing methane emissions from ruminants in order to meet future targets for mitigating global
warming. The legume tree Bauhinia acuminata is widely distributed in many parts of Laos
specially in Luang Prabang, and it has been observed that the foliage is readily consumed by goats.
The leaves of Bauhinia acuminata have 14.5% of protein of low solubility (22%). As is the case
with foliage from most legume trees, it contains many secondary plant compounds including
tannins (Silivong and Preston, 2015). Water spinach (Ipomoea aquatica) is cultivated for human
food and also is fed to animals such as goats, pigs, ducks and rabbits. It does not appear to contain
anti-nutritional compounds and has been used successfully for goats as the only source of
supplementary protein (Phongpanith et al; 2013). It grows equally well in water or in soil. It
responds dramatically in biomass yield and protein content when fertilized. (Preston et al., 2013)
reported that the leaves contain 24% protein in dry matter (DM) and that the protein is highly
soluble (71%) and therefore easily fermentable as a source of nutrients for rumen microorganisms.
These qualities make water spinach an ideal supplement for tree foliages of low nutritive value.
Thus, (Kongmanila et al., 2007) reported that water spinach supplementation of foliages from Fig,
Jujube and Mango trees increased the DM and crude protein intake of goats, and improved the
apparent digestibility and N retention. According to Thu Hong et al., 2011, the live weight gain of
goats fed Mimosa foliage was increased 27% by supplementing with fresh water spinach. Goats fed
a sole diet of cassava foliage also responded with increased DM digestibility and N retention when
fresh water spinach was provided as a supplement (Patshoummalangsy and Preston, 2006).
Cassava (Manihot esculenta Crantz) is an annual crop grown widely in the tropical and
subtropical regions. Roots of cassava are rich in energy (75 to 85% of soluble carbohydrate) but
with minimal levels of crude protein (2 to 3% in DM). The development of the starch industry in
Lao for export to China and other neighboring countries has increased the market for cassava roots.
As a result, cassava is currently the third most important crop in Laos, after rice and maize. The
varieties used for industrial starch production are known as “bitter” varieties due to the high content
of cyanogenic glucosides that are converted into the highly toxic hydrocyanic acid when consumed
by animals and people. The cassava varieties that are planted for human consumption are known as
“sweet” varieties as they have a lower content of cyanogenic glucosides. For every tonne of roots
that are harvested there are an additional 600kg of stems and leaves. However, the farmers in the
cassava factory area have no experience in the utilization of cassava leaves as the protein

supplement to feed to animals, especially cattle. The foliage of cassava has been shown to be an
2


effective source of bypass protein for fattening steers (Ffoulkes and Preston, 1978; Keo Sath et al.,
2008; Wanapat et al., 1997). It is thus a logical forage to provide the additional protein required in
diets rich in carbohydrate but low in protein. 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 are reported to decrease rumen methane production and increase
the efficiency of microbial protein synthesis (Makkar et al., 1995; Grainger et al., 2009). Reductions
of CH4 production of 13 to 16% have been reported (Carulla et al., 2005; Waghorn et al., 2002,
Grainger et al., 2009; Woodward et al., 2004), apparently through a direct toxic effect on
methanogens. Brewers’ grains is a byproduct derived from the industrial brewing of beer. Research
with goats (Sina et al; 2017) highlighted a major interaction between the effect of the
supplementary brewers’ grains and the nature of the basal diet. The improvement in growth rate due
to addition of brewers’ grains was 130% when the basal diet was fresh cassava foliage but only
30% when the basal diet was water spinach (Sina et al., 2017). A positive approach to the problem
of how to reduce methane emissions from live stock has been to incorporate a low level (1%) of
biochar in the diet (Sangkhom et al., 2012; Leng et al., 2012a,b,c). Biochar is the product of
incomplete carbonization of fibrous biomass at high temperatures (Lehmann and Joseph, 2009). It is
a highly porous material which gives it valuable properties as a support mechanism for biofilms that
facilitate the adsorption of consortia of micro-organisms and nutrients that may prioritize
incorporation of hydrogen into volatile acids rather than methane (Leng, 2018 personal
communication).
2. THE OBJECTIVES
The study aimed at the utilization of locally available feed resources for increasing growth
performance and reducing enteric methane emissions from goats in Lao PDR. The specific
objectives were following: (i) To evaluate the of water spinach as a source of high soluble protein
and biochar on methane production in an in vitro system with substrate of Bauhinia acuminata or
Guazuma ulmifolia leaves; (ii) To evaluate the effect of water spinach as a source of high soluble

protein and biochar on feed intake, digestibility, N retention, methane emission and growth
performance of goats fed Bauhinia acuminata foliages plus molasses or dried cassava root chip as
the basal diets; (iii) To examine the effect of replacing water spinach by cassava foliage and/or
brewer’s grain on feed intake, digestibility, N retention and growth performance of goat fed
Bauhinia acuminata plus dried cassava root chip as the basal diets; (iv) To compare the sweet or
bitter of cassava leaves and biochar on gas production, methane content of the gas and methane
ml/g DM digested in an in vitro incubation.
3. THE HYPOTHESIS
(i) Water spinach, with its high content of soluble protein, would increase the rate of
fermentation and production of methane when added to forage rich in insoluble protein such as
Bauhinia acuminata or Guazuma ulmifolia leaves; (ii) The performance of growing goats fed
Bauhinia acuminata as the basal diet would be improved by supplementation with water spinach as
a rapidly fermentable protein source. Enteric methane production would be reduced by adding a low
level (1%) of biochar; (iii) Goats fed foliage of the legume tree Bauhinia acuminata would respond
positively in growth rate and feed conversion to a low-level supplement of brewers’ grains, and that
the degree of response would be greater when cassava foliage, rather than water spinach, was the
3


complementary source of protein; (iv) The methane content of the gas produced in an in vitro
fermentation would be reduced when leaves of bitter cassava replaced leaves of sweet cassava as
supplementary protein source and when 1% of biochar was added to the substrate.
4. SIGNIFICANT/INNOVATION OF THE DISSERTATION
This is the first series of study and the first sciencetific information on improving the
utilization of Bauhinia acuminata for goat production in Laos. The results presented in this
dissertation indicate that: (1) Goats fed Bauhinia acuminata responded with improved diet
digestibility, N retention and growth rate when the Bauhinia acuminata was supplemented with
water spinach; (2) But an important negative effect was that the improvement in diet digestibility by
supplementation with water spinach led to increases in methane production per unit diet DM
digested; (3) Supplementing Bauhinia acuminata foliage with leaves from a bitter variety of

cassava reduced the in vitro production of methane when compared with supplementation by leaves
from a sweet variety of cassava; (4) Ensiled brewers’ grains fed as an additive (5% as DM) to a diet
of Bauhinia acuminata improved the digestibility, N retention and growth rate of goats. The degree
of improvement was greater when the Bauhinia acuminata was supplemented with cassava foliage
instead of water spinach; (5) Biochar fed at 1% of a diet of Bauhinia acuminata and cassava foliage
was as effective as brewers’ grains in improving the growth rate of the goats.

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CHAPTER 1: LITERATURE REVIEW
There are main points following (i) Goat production in Laos; (ii) Role of goat production in
Laos; (iii) Goat nutrient and methane emission; (iv) Local available feed resources for goat in Laos.

CHAPTER 2: EFFECT OF WATER SPINACH ON METHANE PRODUCTION IN AN IN
VITRO INCUBATION WITH SUBSTRATES OF BAUHINIA ACUMINATA OR GUAZUMA
ULMIFOLIA LEAVES AND MOLASSES

INTRODUCTION
The greenhouse gases (GHG) emissions from the agriculture sector account for about 25.5%
of total global radiative forcing and over 60% of anthropogenic sources (FAO 2009). Animal
husbandry accounts for 18% of GHG emissions. Emission of methane (CH4) is responsible for
nearly as much radiative forcing as all other non-CO2 GHG gases combined (Beauchemin and
McGinn 2005). While atmospheric concentrations of GHGs have risen by about 39% since preindustrial era, CH4 concentration has more than doubled during this period (WHO, 2009). Reducing
GHG emissions from agriculture, especially from livestock, should therefore be a top priority since
it could curb global warming fairly rapidly (Sejian et al., 2010). Ruminants, such as cattle, buffalo,
sheep and goats, are the major contributors of total methane agricultural emissions (Leng, 1993;
Lassey, 2007; Chhabra et al., 2009). In ruminants, the H2 produced in rumen fermentation is
normally removed by the reduction of CO2 to methane. There is a need to develop feeding systems
for ruminants that will result in reduced emissions of methane gas from the enteric fermentation in

these animals. Previous research showed that methane production was less when fish meal rather
than groundnut meal was the substrate (Preston et al., 2013). The differences in methane production
appeared to be related to the solubility of the protein which was 16% in fish meal compared with
70% in groundnut meal. A similar finding was reported by Silivong and Preston (2015) who
showed that addition of water spinach to the substrate in an in vitro rumen fermentation increased
the rate of gas production and methane content in the gas. The protein in the water spinach was
highly soluble (66%). The hypothesis that underlined the present study was that water spinach, with
its high content of soluble protein, would increase the rate of fermentation and production of
methane when added to forages-rich in insoluble protein.
MATERIALS AND METHODS
Treatments and experimental design
The experimental design was a 2 × 4 factorial arrangement of 8 treatments with four
replications. The factors were:
- Foliage source: Bauhinia acuminata and Guazuma ulmifolia (BA and GU)
- Level of water spinach: 0, 5, 15 and 25% in substrate DM

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Preparation of substrate and the in vitro system
The in vitro system used recycled “PET” plastic bottles as flasks for the incubation and gas
collection. A simple in vitro system was used with recycled plastic bottles as flasks for the
incubation and gas collection (Diagram 1).
a.
b.
c.
d.

Water bath
Fermentation bottle (1.5liters)

Water storage reservoir (3liters)
Gas collection bottle (1.5liters)

Plastic tube (id: 4mm)

Diagram 1. A schematic view of the in vitro system to measure gas production in an in vitro incubation

- B and D bottles (1.5 liters capacity)., C bottle (3liters capacity). The B bottle containing
the substrate for fermentation was connected to the D bottle by a plastic tube (4 mm diameter). The
D bottle was marked at 50ml intervals before being suspended in the C bottle containing water.
Clay was used to cover the stoppers of the plastic bottle and junction of stopper and plastic tube to
prevent leakage of gas. The leaves from Bauhinia and Quazuma, and leaves plus stems of water
spinach, were chopped into small pieces (3-5mm) and dried at 65°C for 48h then ground with a
coffee grinder, and mixed according to the proportions shown in Table 1. The mixtures (12g DM)
were put in the incubation bottle with 960 ml of buffer solution (Table 2) and 240 ml of rumen
fluid. The rumen fluid was taken at 3.00-4.00am from the slaughter house from a buffalo
immediately after the animal was killed. 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 bottle. The
remaining air in the flask was flushed out with carbon dioxide. The bottles were incubated at 38°C
in a water bath for 24 h.
Table 1. Composition of diets (% DM basis)
BA#GU

BA#GU-WS 5

BA#GU-WS 15

BA#GU-WS 25

Leaf meal#

80

75

65

55

Water spinach

0

5

15

25

Molasses

20

20

20

20

Total

100

100

100

100

# Bauhinia or Guazuma leaf meals

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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)

Data collection and measurements
Gas production was measured at 4 intervals for 6, 12, 18 and 24h by water displacement (a
calibrated recycled water bottle with the bottom removed) and at the end of each incubation, the
methane concentration in the gas was measured with a Crowcon infra-red analyser (Crowcon
Instruments Ltd, UK). Residual DM in the incubation bottle was determined by filtering the
incubation residues through cloth to estimate DM loss during incubation and drying the residue
(65°C for 72h).
Chemical analyses
The samples of foliage, water spinach and residual substrate were analysed for DM, ash and
N according to AOAC (1990) methods. Solubility of the protein in the leaves was determined by
shaking 3g of dry leaf meal in 100 ml of M NaCl for 3h then filtering through Whatman No.4 filter
paper, and determining the N content of the filtrate (Whitelaw et al., 1963).
Statiscal analysis
The data were analyzed by the general linear model option of the ANOVA program in the

Minitab software (Minitab, 2014). In the model the sources of variation were: treatments, replicates
and error.
The statistical model was: Yijk = μ + Pi + Aj + Pi*Aj+ eijk
Where: Yijk is dependent variables., μ is overall mean., Pi is the effect of foliage source
Aj is the effect of level of water spinach., (P*A) ij is the interaction between source of foliage
and source of level of water spinach and eijk is random error
RESULTS
Chemical composition
Percentages of crude protein, ash and protein solubility were higher in water spinach than in
Bauhinia acuminata and Guazuma ulmifolia leaves, but DM was lower. The protein content and
solubility in the leaves of Guazuma ulmifolia were higher than in Bauhinia acuminata, but ash was
lower (Table 3).

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Table 3. The chemical composition of feed (% in DM, except DM which is on fresh basis)
DM

N*6.25

Ash

Protein solubility

Tannin

NDF

ADF

Bauhinia leaves

40.0

15.0

21.2

23.7

1.1

43.7

32.4

Bauhinia stem

38.1

12.3

4.29

-

42.7

31.5

Guazuma ulmifolia

36.0

18.4

3.9

33.3

Water spinach

10.6

18.5

9.7

66.4

42.3

33.3

Molasses

80.4

5.4

10.5

-

Source: Silivong et al., 2018

Values for the gas production, percent methane in the gas and methane produced per unit
substrate solubilized increased with length of incubation time (Table 4). Gas production and percent
substrate solubilized were increased by increasing the level of water spinach in the substrate, and
were higher for Guazuma ulmifolia than Bauhinia acuminata at each incubation interval.
Table 4. Mean values for gas production, percentage of methane in the gas, methane production (ml), DM solubilized and
methane production per unit DM solubilized according to leaf source (Bauhinia and Guazuma) and level of water spinach
Foliage
source
BA

Level of water spinach (%)
p

GU

Interaction
p

WS-0

WS-5

WS-15

WS-25

SEM
SEM

P

0-6 hr
Gas production, ml

363

483

<0.001

286d

354c

476b

575a

<0.001

5.1

7.2

0.574

Methane, %

8.8

9.9

<0.001

7.6d

8.9c

9.6bc

11.4a

<0.001

0.2

0.2

0.673

DM solubilized, %

53.1

56.3

<0.001

49.2d

53.8c

56.4b

59.4a

0.001

0.2

0.3

0.753

Methane, ml/g DM
solubilized

5.1

7.2

<0.001

3.7d

4.9c

6.8b

9.2a

<0.001

0.2

0.2

0.665

0-12 hr
Gas production, ml

483

781

<0.001

463d

581c

661b

821a

<0.001

9.4

13.3

0.772

Methane, %

13.8

15.9

<0.001

11.1d

13.6c

15.1bc

19.6a

<0.001

0.3

0.4

0.563

DM solubilized, %

56.5

60.2

<0.001

53.2d

57.1c

59.9b

63.2a

<0.001

0.3

0.5

0.756

Methane, ml/g DM

solubilized

10.2

17.5

<0.001

8.2d

11.7c

14.1b

21.4a

<0.001

0.4

0.6

0.674

584

1023

<0.001

584d

766c

856b

1009a

<0.001

9.1

12.9

0.681

0-18 hr
Gas production, ml

8


Methane, %

18.9

21.8

<0.001

16.3d

19.5c

21.3b

24.3a

<0.001

0.2

0.3

0.675

DM solubilized, %

59.4

63.5

<0.001

56.1d

60.1c

62.4bc

67.0a

<0.001

0.4

0.6

0.872

Methane, ml/g DM
solubilized

15.8

29.5

<0.001

14.3d

21.4c

24.5b

30.4a

<0.001

0.4

0.6

0.653

Gas production, ml

734

1239

<0.001

775d

973c

1050b

1149a

<0.001

11.3

15.9

0.776

Methane, %

24.4

27.9

<0.001

20.4d

24.8c

27.9b

31.5a

<0.001

0.2

0.3

0.832

DM solubilized, %

61.8

67.3

<0.001

59.0d

64.3c

65.2bc

69.6a

<0.001

0.4

0.6

0.742

Methane, ml/g DM
solubilized

24.4

43.3

<0.001

22.4d

31.6c

37.9b

43.6a

<0.001

0.8

1.1

0.775

0-24 hr

abc Mean values without common superscript differ at p<0.05, BA: Bauhinia acuminata, GU: Guazuma
ulmifolia, P: Probability value, WS: Water spinach

DISCUSSION
The increases in methane concentration in the gas and per unit DM solubilized, with
incubation interval, are similar to the findings reported by Outhen et al., 2011, Binh Phuong et al .,
2011) and Silivong and Preston, 2015. This is thought to be due to methane being increasingly
produced by secondary fermentation from acetate (Inthapanya et al., 2011) as the incubation
interval increased. The increases in methane production with increasing proportions of water
spinach in the substrate, and the higher methane production for treatments with Guazuma ulmifolia
leaves compared with Bauhinia acuminata leaves, were closely related to the degree of solubility of
the protein in these different combinations of substrate (Table 3). . A similar finding was reported
by Inthapanya and Preston, 2014 when cassava leaves (protein solubility 25.6%) replaced water
spinach (protein solubility 66.3%) in an in vitro incubation of urea-treated rice straw. It is not
possible to differentiate between the direct effects of increased protein solubility per and the indirect
effect of reducing the concentrations and the level of anti-nutritional factors (eg: condensed tannins)

in the substrate (Table 3) when Bauhinia acuminata was replaced by Guazuma ulmifolia, and the
level of water spinach was increased. The positive role of tannins in reducing the activity of
methanogenic bacteria has been reported by several researchers (Goel and Makkar, 2012; Soltan et
al., 2012).
CONCLUSIONS
- Increasing the length of the incubation in the in vitro rumen fermentation of leaves of
Bauhinia acuminata and Guazuma ulmifolia increased gas production, methane concentration in the
gas and methane produced per unit substrate solubilized.
- Supplementation with water spinach increased the rate of gas production, the percentage
DM solubilized, and the methane concentration in the gas and methane produced per unit substrate
solubilized on both sources of leaves.
- Gas production, methane concentration in the gas and methane produced per unit substrate
solubilized were higher when leaves of Guazuma ulmifolia replaced those from Bauhinia
acuminata.
9


CHAPTER 3:
EFFECTS OF WATER SPINACH AND BIOCHAR ON METHANE EMISSIONS AND
GROWTH PERFORMANCE OF GOAT FED BAUHINIA ACUMINATA AND MOLASSES
OR CASSAVA ROOT CHIPS AS THE BASAL DIET

INTRODUCTION
Livestock are the most important source of protein food and family cash income of farmers
in Laos, and also give manure for cropping in the rural areas. Most of the production from livestock
such as goats, cattle, pigs and poultry comes from smallholders using traditional management
systems. The main feed resources for ruminants are native grasses, legumes and tree leaves that are
available in the natural grassland and forests (Phengsavanh, 2003). The conventional feeding
system for goats in Lao PDR is based mainly on the use of natural grasses. However, in the dry
season, natural pasture decreases in nutritive value and improved grasses cannot grow. Therefore, it

is important to find an alternative feeding system because purchased supplements are too expensive
for poor farmers. On the other hand, there are many trees and shrubs available. Preston and Leng,
2009 and Leng, 1997 have emphasized that in tropical countries one of the most appropriate ways
to improve feed supplies for ruminants is through utilization of tree and shrub foliages. The
negative feature of livestock is that they contribute some 18% of the greenhouse gases that are
causing global warming (Steinfeld et al., 2006). Enteric methane from fermentative digestion is the
main source of these emissions. Thus when new or modified feeding systems are being researched
the effects of these changes on enteric methane emissions should be monitored, in view of the need
to reduce methane emissions so as to meet future targets for mitigating global warming. The legume
tree Bauhinia acuminata is widely distributed in the Luang Prabang Province and it has been
observed that the foliage is readily consumed by goats. The leaves of Bauhinia acuminata have
14.5% of protein of low solubility (22%). As is the case with most foliage from legume trees, it
contains many secondary plant compounds including tannins (Queiroz Siqueira et al., 2012). The
low solubility of the crude protein in Bauhinia acuminata is indicative of the binding action of
tannins on this nutrient source (Silivong and Preston, 2015). Daovy et al., 2007 reported that water
spinach (Ipomoea aquatica) supplementation of low quality tree foliage (from Fig, Jujube and
Mango trees) increased the DM and crude protein intake of goats, and improved the apparent
digestibility and N retention. According to Thu Hong et al., 2011, the live weight gain of goats fed
Mimosa foliage was increased by supplementing with fresh water spinach at 27% of the total DM
intake. Goats fed a sole diet of cassava foliage also responded ith increased DM digestibility and N
retention when fresh water spinach was provided as a supplement (Pathoummalangsy and Preston,
2006). Molasses is a source of highly fermentable carbohydrate (contains > 50% soluble sugars)
and is very low in crude protein “<0.5% in DM” (Ffoulkes and Preston, 1978). Cassava roots have
high levels of energy (75 to 85% of soluble carbohydrate) and minimal levels of crude protein (2 to
3% CP); they have been used as a source of readily-fermentable energy (Kang et al., 2015;
Polyorach et al., 2013; Wanapat et al., 2013a,b). A positive approach to the problem of how to
reduce methane emissions from live stock has been to incorporate a low level (1%) of biochar in the
diet (Sangkhom et al., 2012; Leng et al., 2012a,b,c). Biochar is the product of incomplete
carbonization of fibrous biomass at high temperatures (Lehmann and Joseph, 2009). It is a highly
10

porous material which gives it valuable properties as a support mechanism for biofilms that may
facilitate the adsorption of consortia of micro-organisms and nutrients (Leng, 2014). In the research
reported here, it was hypothesized that: (i) the performance of growing goats fed Bauhinia
acuminata and molasses or cassava root chip as the basal diet would be improved by
supplementation with water spinach as a rapidly fermentable protein source; and (ii) that
incorporation of a low level of biochar in the diet might reduce enteric methane emissions.
MATERIALS AND METHODS
Treatments and experimental design
The experimental plan was a 2×2 factorial arrangement in a Randomized Completely Block
Design (RCBD) with 4 treatments and there were 4 replications of the experiment 2 and 3
replications of the experiment 3.
The factors applied to a basal diet of fresh Bauhinia acuminata foliage were:
- With or without water spinach (WS and No-WS)
- With or without biochar (BC and No-BC)
Individual treatments were:
- BA = Bauhinia acuminata ad libitum
- BABC = Bauhinia acuminata ad libitum + 1% biochar on DM intake
- BAWS = 70% Bauhinia acuminata and 30% water spinach on DM basis
- BAWSBC = 70% Bauhinia acuminata and 30% water spinach + 1% biochar
Biochar was given 1% on DM intake
+ In the experiment 2: Molasses diluted with fresh water by ratio of 1:9 (1 kg of molasses
and 9 litters of fresh water) and was used as the carrier for the biochar and was given ad libitum on
all diets., In the experiment 3: Sun-dried cassava root chips were used as the carrier for the biochar.
The biochar was mixed with finely-chopped, sun-dried cassava root chips (20% biochar: 80% sundried cassava root chips) which were fed at 5% of the diet (DM basis) once daily at 7.00am.
Animals and housing
- Experiment 2: Sixteen local weaned goats with initial average body weight of
11.65±3.95kg and 5-6 months of age were used. They included 4 males (non-castrated) and 12
females. These animals were purchased from Phoukhoun District LaungPrabang Province.,

Experiment 3: Twelve weaned goats (local breed) with initial average body weight of 12.1±3.7kg
and 5-6 months of age were used. They included 8 males (non-castrated) and 4 females. These
animals were purchased from Chomphet District Laungprabang Province. In both studies, goats
were housed individual pens and made from local material such as: bamboo (dimensions of width
1 m, length 1 m and height 0.9 m) and designed to collect separately feces and urine. They were
vaccinated against Pasteurellosis, foot and mouth disease and treated with Ivermectin (1ml/20 kg
live weight) to control internal and external parasites. They were adapted to the pens and the
feeds for 10 days before starting the experiment. The experiment lasted 100 days, including the
adaptation period.
11


Feed and management
Molases were purchased from Vientiane Province, cassava root was purchased from farmers
around the Luangprabang District, chopped into small pieces and exposed to sunlight for 48 hours
to reduce the moisture to about 15%, Foliages of Bauhinia acuminata and water spinach were
collected daily from natural stands in the University campus. The biochar was produced by burning
rice husks in a top lit updraft (TLUD) gasifier stove (Olivier, 2010). It was ground to a particle size
that passes through a 1 mm sieve. The foliages were offered twice daily at 07:30 and 16:00h by
hanging in bunches above the feed trough.
Data collection and measurement
- Experiment 2: Live weight was recorded in the morning before feeding at the beginning
and at the end of the experiment and at intervals of 10 days during the experiment. Quantities of
feed offered and refused were recorded daily. Every 10 days, samples were taken for analysis of
DM and N. Samples of Bauhinia acuminata foliage offered and residues were separated into stem
and leaves (containing attached petioles). Representative samples of each component were analyzed
for DM, N and ash. Samples of rumen fluid were taken by stomach tube 2h after morning feeding
on the last day of the experiment. The pH value was measured immediately with a portable digital
pH meter. A drop of concentrated sulphuric acid was added prior to determination of ammonia by
steam distillation. Digestibility and N retention were recorded four times, over 5 days periods at 20

day intervals (after 20, 40, 60 and 80 days). In each collection period, samples of feeds offered and
refused were taken daily and bulked for the 5 days of each period. Urine was collected in buckets
containing 20ml of a solution of sulphuric acid (10% sulphuric acid concentrate + 90% distilled
water). Feces were collected daily and stored in the refrigerator at 4-8ºC and at the end of each
period, sub-samples were mixed together and ground with a coffee grinder. Samples of eructed
gases were measured on the last day of the experiment, in the morning 2h after feeding. The goats
were placed in a plastic-covered cage and after a period of 10 minutes for equilibration with the
surrounding air, the concentrations of methane and carbon dioxide were recorded over a 10 minute
period, using a GASMET 4030 meter (Gasmet Technologies Oy, Pulttitie 8A, FI-00880 Helsinki,
Finland)., Experiment 3: Live weight was recorded in the morning before feeding at the beginning
and at the end of the experiment and at intervals of 10 days during the experiment. Quantities of
feed offered and refused were recorded daily. Every 10 days, samples were taken for analysis of
DM and N. Samples of Bauhinia acuminata foliage offered and residues were separated into stem
and leaves (containing attached petioles). Representative samples of each component were analyzed
for DM, N and ash. Samples of rumen fluid were taken by stomach tube 2h after morning feeding
on the last day of the experiment. The pH value was measured immediately with a portable digital
pH meter. A drop of concentrated sulphuric acid was added prior to determination of ammonia by
steam distillation. Digestibility and N retention were recorded three times, over 5 day periods at 30
day intervals (after 30, 60 and 90 days). In each collection period, samples of feeds offered and
refused were taken daily and bulked for the 5 days of each period. Urine was collected in buckets
containing 20ml of a solution of sulphuric acid (10% sulphuric acid concentrate + 90% distilled
water). Feces were collected daily and stored in the refrigerator at 4-8ºC and at the end of each
period, sub-samples were mixed together and ground with a coffee grinder.

12


Chemical analyses
The sub-samples of feces and of feeds offered and refused were analysed for DM, N and ash
according to AOAC (1990) methods. Urine was analysed for nitrogen (AOAC 1990). Solubility of

the protein in the leaves was determined by shaking 3g of dry leaf meal in 100 ml of M NaCl for 3h
then filtering through Whatman No.4 filter paper, and determining the N content of the filtrate
(Whitelaw et al., 1963).
Statistical analysis
The data were analysed statistically as a Randomize Complete Block Design (RCBD) by
variance analysis (ANOVA) using the general linear model (GLM) procedure of Minitab software
version 16.0 (Minitab, 2014). The treatment least square means showing significant at difference at
the probability level of P<0.05 were compared Turkey’s pair wise comparison procedure.
The statistical model used in the experiments 2 and 3:
- Growth study was:
Yijk = μ + Bk+ Pi + Aj + Pi*Aj+ eijk
Where: Yijk is dependent variables., μ is overall mean., Bk is the effect of live weight., Pi is
the effect of protein source., Aj is the effect of biochar source., (P*A) ij is the interaction between
source of protein and source of biochar and eijk is random error
- Digestibility study was:
Yijk = μ + Ti + Pj +Ak + eijk
In where,

Yijk = Dependent variables., μ = Overall mean., Ti = Treatment effect (i=1-4).,
Pj = Column effect (j=1-4)., Ak = Row effect (k=1-4) and eijk = Random error

The relationship between N intake and N retention was developed by regression analyses.
The best model was selected based on adjusted R2.
The methane to carbon dioxide ratios were used to calculate the reduction of methane
production according to the formula proposed by Madsen et al., 2010:
Ratio CH4/CO2 = (a-b)/(c-d)
Where "a" is methane concentration in mixed eructed gas plus air., "c" is carbon dioxide
concentration in mixed eructed gas plus air., "b" is methane in the air in the goat shed., "d" the
carbon dioxide in goat shed air
The relationship between N intake and N retention was developed by regression analyses.

The best model was selected based on adjusted R2.
RESULTS
Chemical composition
The concentrations of crude protein and ash and the solubility of the protein were lower, and
of DM were higher, in Bauhinia acuminata than in water spinach (Table 1).

13


Table 1. Chemical composition of dietary ingredients (% in DM, except DM which is on fresh basis)
DM

N*6.25

Ash

Protein solubility

Tannin

NDF

ADF

Bauhinia leaves

40.0

15.0

21.2

23.7

1.1

43.7

32.4

Bauhinia stem

38.1

12.3

4.29

-

42.7

31.5

Water spinach

8.16

18.3

9.74

69.4

42.3

33.3

Molasses

80.4

5.4

10.5

Cassava root chips

82.4

2.81

2.23

-

-

-

-

-

38.3

-

-

-

Biochar

Source: Silivong et al., 2018

- Experiment 2:
Feed intake, growth rate and feed conversion
DM intake expressed as a percentage of live weight was not affected by supplementation
with biochar or water spinach (Table 2).
Table 2. Mean values of feed intake by goats fed Bauhinia acuminata supplemented with water spinach (WS)
or biochar (BC) or not supplemented
PS

BCS

Interaction

p
WS

No-WS

P
BC

No-BC

SEM
SEM

p

DM intake, g/d
Molasses

213

224

0.004

218

219

0.67

2.52

14.4

0.369

Bauhinia

149

229

<0.001

164

214

<0.001

3.07

35.69

0.719

Water spinach

161

0

<0.001

82.0

79.2

0.025

0.87

6.25

0.683

Biochar

2.47

2.85

<0.001

5.31

0

<0.001

0.03

0.4 1

0.371

Total

526

455

<0.001

469

512

<0.001

3.77

47.67

0.558

Per kg LW

32.9

33.1

0.21

32.8

33.2

0.016

0.13

0.6

0.923

N*6.25, % in DM

12.5

10.3

11.5

11.5

BC: Biochar, BCS: Biochar source, d: day, g gram, Kg: Kilogram, LW: live weight, P: Probability
value, PS: Protein source, SEM: Standard error of the mean with dferror: 9, WS: Water spinach.

14

Daily live weight gain and feed conversion were improved by feeding water spinach and by
supplementation with biochar (Table 3). There was a close relationship between live weight gain
and feed conversion ratio.
Table 3. Mean values for live weight, live weight change, feed DM intake and DM feed conversion for goats
fed a basal diet of Bauhinia acuminata foliage and molasses
PS

BCS

Interaction

p
WS

p

No-WS

BC

SEM

No-BC

SEM

p

Live weight, kg
In wt, kg

12.9

12.2

0.598

11.8

13.3

0.263

0.87

1.22

0.571

Fin wt, kg

18.6

15.3

0.019

16.5

17.4

0.495

0.87

1.22

0.382

LWG, g/d

51.4

28.7

<0.001

43.7

36.5

0.047

2.32

3.28

0.543

DMI, g/d

526

455

<0.001

469

512

<0.001

3.77

47.67

0.558

FCR, g/g

10.7

16.2

0.014

11.4

15.5

0.055

1.34

1.90

0.580

BC: Biochar, BCS: Biochar source, DMI: DM intake, FCR: DM feed conversion, Fin wt: Final
weight, In wt: Initial weight, Kg: Kilogram, LWG: Live weight gain, P: Probability value, PS: Protein
source, SEM: Standard error of the mean with dferror: 9, WS: Water spinach.

Apparent digestibility and N retention
Supplementing the basal diet of Bauhinia acuminata and molasses with water spinach
increased the apparent digestibility of DM, OM and crude protein, but there were no differences due
to biochar (Table 4). Daily N retention, N retention as percent of N intake and of N digested were
all improved by supplementation with water spinach. There was a tendency for biochar to improve
daily N retention (p=0.082) and this effect was significant when N retention was expressed as a
percent of N intake or of N digested.
Table 4. Mean values of apparent digestibility and N balance in goats fed Bauhinia acuminata and molasses
supplemented with water spinach (WS) and biochar (BC) or not supplemented (No-WS; No-BC)
PS

BCS

Interaction

p
WS

p

No-WS

BC

SEM

No-BC

SEM

p

Apparent digestibility, %
DM

72.0

64.5

<0.001

69.1

67.5

0.100

0.68

1.68

0843

OM

75.0

67.4

<0.001

71.5

70.9

0.579

0.74

2.10

0.591

N*6.25

69.2

43.8

<0.001

55.0

57.9

0.294

1.98

6.48

0.502

15


N balance, g/day
Intake

11.1

7.0

<0.001

8.8

9.3

0.296

0.32

1.04

0.502

Feces

3.2

2.3

<0.001

2.5

3.0

0.021

0.16

0.53

0.583

Urine

1.8

1.4

<0.001

1.4

1.8

0.007

0.09

0.29

0.371

Retention

6.1

3.4

<0.001

4.9

4.5

0.082

0.16

0.41

0.719

N retention as:
% N intake

55.0

50.0

0.008

56.2

48.8

<0.001

1.32

3.73

0.324

% N digested

76.8

72.1

0.009

77.1

71.7

0.003

1.25

3.76

0.440

BC: Biochar, BCS: Biochar source, DM: Dry matter, Kg: Kilogram, N: Nitrogen, OM: Organic
matter, P: Probability value, PS: Protein source, SEM: Standard error of the mean with df error: 9, WS: Water
spinach

Rumen ammonia, pH and methane to carbon dioxide ratio
Rumen pH did not differ among the diets (Table 5). Rumen ammonia, which was high on all
diets, was increased by supplementation with water spinach but was not affected by biochar.
Table 5. Mean values of rumen pH and ammonia, and ratio of methane to carbon dioxide in eructed breath
of goats fed Bauhinia acuminata and molasses supplemented with water spinach (WS) and biochar (BC) or

not supplemented (No-WS; No-BC)
PS

BCS

Interaction

p
WS

No-WS

Rumen pH

7.08

7.03

NH3, mg/liter

397

CH4:CO2

0.0211

p
BC

No-BC

0.465

7.04

7.06

0.756

298

<0.001

347

347

0.0292

0.043

0.0243

0.0260

SEM
SEM

p

0.05

0.063

0.727

0.999

9.70

13.71

0.935

0.641

0.003

0.0036

0.800

BC: Biochar, BCS: Biochar source, CH4: Methane, CO2: Carbon dioxide, NH3: Ammonia, pH:
Percentage of hydrogen ion, P: Probability value, PS: Protein source, SEM: Standard error of the mean
with dferror: 9, WS: Water spinach.

The ratio of methane to carbon dioxide in eructed breath of the goats was lower in the
eructed breath of the goats supplemented with water spinach and not affected by supplementation
with biochar.
- Experiment 3:

Feed intake, growth rate and feed conversion
DM intake was increased 38% by supplementation with water spinach and by 5% from
feeding of biochar (Table 6).
16


Table 6. Mean values of feed intake by goats fed Bauhinia acuminata and cassava root chips supplemented
with water spinach (WS) or biochar (BC) or not supplemented
PS

BCS

Interaction

p
WS

p

No-WS

BC

SEM

No-BC

SEM

p

DM intake, g/d
Cassava root chips

56.7

49.3

0.092

43.3

62.7

<0001

3.14

14.21

0.463

Bauhinia

350

335

0.009

361

325

<0.001

3.95

52.19

0.671

Water spinach

127

0

62.8

63.9

0.431

1.02

14.58

0.940

Biochar

2.76

3.05

5.81

0

0.38

0.478

Total

536

388

<0.001

472

451

0.009

5.81

60.01

0.572

Per kg LW

36.8

27.1

<0.001

31.8

32.2

0.244

0.26

1.79

0.609

N*6.25, % in DM

14.2

12.4

13.2

13.4

BC: Biochar, d day, BCS: Biochar source, g: gram, Kg: Kilogram, LW: Live weight, P: Probability
value, PS: Protein source, SEM: Standard error of the mean with dferror: 6, WS: Water spinach

Daily live weight gain was improved 120% by supplementation with water spinach and by
27% due to biochar (Table 7). Water spinach improved DM feed conversion by 27%. There was a
close relationship between live weight gain and feed conversion.
Table 7. Mean values for live weight, live weight change, feed DM intake and DM feed conversion for goats
fed a basal diet of Bauhinia acuminata foliage and Cassava root chips
PS

BCS

Interaction

p
WS

No-WS

p
BC

SEM

No-BC

SEM

p

Live weight, kg
In wt, kg

12.0

12.9

0.579

12.6

12.3

0.813

1.06

1.5

0.669

Fin wt, kg

17.5

15.4

0.164

17.1

15.8

0.372

0.96

1.36

0.749

LWG, g/d

60.9

27.5

<0.001

49.4

39.0

0.002

1.64

2.33

0.304

DMI, g/d

536

388

<0.001

472

451

0.009

5.81

60.01

0.572

FCR, g/g

9.0

14.8

0.041

11.0

12.8

0.481

1.68

2.37

0.852

BC: Biochar, BCS: Biochar source, DMI: DM intake, d: day, FCR: DM feed conversion, Fin wt:
Final weight, g: Gram, In wt: Initial weight, Kg: kilogram, LWG: Live weight gain, P: Probability value, PS:
Protein source, SEM: Standard error of the mean with dferror: 6, WS: Water spinach
17


Apparent digestibility and N retention
Apparent digestibility coefficients were increased by water spinach supplement for DM, OM
and crude protein and by biochar for DM and OM (Table 8). Daily N retention and N retention as
percent of N intake and N digested were all improved by supplementation with water spinach and
biochar.
Table 8. Mean values of apparent digestibility and N balance in goats fed Bauhinia acuminata and cassava
root chips supplemented with water spinach (WS) and biochar (BC)or not supplemented (No-WS; No-BC)
PS

BCS

Interaction

p
WS

p

No-WS

BC

SEM

No-BC

SEM

p

Apparent digestibility, %
DM

71.3

63.2

<0.001

68.9

65.6

0.009

0.88

2.20

0.288

OM

74.3

67.1

<0.001

71.9

69.4

0.031

0.80

2.05

0.301

N*6.25

77.0

50.7

<0.001

66.0

61.6

0.217

2.48

9.18

0.762

N balance, g/day
Intake

12.3

8.1

<0.001

10.6

9.9

0.217

0.40

1.47

0.762

Feces

3.6

2.5

<0.001

2.9

3.1

0.020

0.08

0.17

0.078

Urine

2.2

1.8

0.008

2.0

2.1

0.740

0.10

0.34

0.525

Retention

6.5

3.8

<0.001

5.7

4.7

0.023

0.30

1.11

0.614

N retention as:
% N intake

52.0

45.1

<0.001

52.3

44.8

<0.001

1.32

4.56

0.424

% N igested

73.9

66.3

<0.001

73.0

67.2

<0.001

0.74

2.21

0.515

BC: Biochar, BCS: Biochar source, DM: Dry matter, N: Nitrogen, P: Probability value,
PS: Protein source, SEM: Standard error of the mean with dferror: 6, WS: Water spinach

pH and Rumen ammonia
The rumen pH was not affected by supplements of water spinach and biochar (Table 9).
Rumen ammonia was high for all diets and was increased by supplementation with water spinach,

but was not affected by biochar. There was a close relationship between live weight gain and rumen
ammonia concentration.

18


Table 9. Mean values of rumen pH and ammonia in goats fed Bauhinia acuminata and cassava root chips
supplemented with water spinach (WS) and biochar (BC) or not supplemented (No-WS; No-BC)
PS

BCS

Interaction

p
WS

No-WS

Rumen pH

7.04

7.01

NH3, mg/liter

405

286

p
BC

No-BC

0.683

7.01

7.04

0.683

<0.001

354

337

0.422

SEM
SEM

p

0.05

0.06

0.759

14.17

24.04

0.975

BC: Biochar, BCS: Biochar source, mg: Milligram, NH3: Ammonia, P: Probability value, pH:
Percentage of hydrogen ion, PS: Protein source, SEM: Standard error of the mean with dferror: 6, WS: Water
spinach

DISCUSSION
The results of this experiment agree with the findings of: (i) Kongmanila et al., 2011 that
supplementation with water spinach increased the digestibility of Mango foliage growing goats; and
(ii) Phongpanith et al., 2013 who reported that water spinach supplementation increased the
digestibility and N retention of goats fed Muntingia foliage. Presumably the high solubility of the
protein in water spinach furnished amino acids and peptides required by micro-organisms for
efficient rumen digestion, and that these were limited in the Bauhina acuminata foliage because of
its low protein solubility. In contrast with the report by Leng et al., 2014, that biochar reduced
methane emission from cattle fed urea-treated rice straw, in the present experiment with goats fed
tree foliage there was no effect of biochar on methane emissions. However, in agreement with these
authors, the biochar had a positive effect on growth performance.
CONCLUSIONS
- Goats fed Bauhinia acuminata foliage and molasses or cassava root chip, supplemented
with water spinach, grew faster and had better DM feed conversion, and higher apparent
digestibility of DM and crude protein, and higher N retention, than goats fed only Bauhinia
acuminata foliage and molasses or cassava root chip, but were higher on daily gain and had better
DM feed conversion when goat fed Bauhinia acuminta and cassava root chips than Bauhinia

acuminata and molasses as the basal diet.
- The higher value of rumen ammonia in goats fed water spinach reflected the greater
solubility of the crude protein in the water spinach.
- Supplementing the basal diet of Bauhinia acuminata and molasses with water spinach led
to a reduction in the methane/carbon dioxide ratio in the eructed breath of the goats.

19


CHAPTER 4: EFFECT OF REPLACING WATER SPINACH (Ipomoea aquatic) BY
CASSAVA (Manihot esculenta Crantz) FOLIAGE AND/OR BREWER’S GRAINS ON FEED
INTAKE, DIGESTIBILITY, N RETENTION AND GROWTH PERFORMANCE IN GOAT
FED BAUHINIA ACUMINATA PLUS CASSAVA ROOT CHIPS AS THE BASAL DIET

INTRODUCTION
Research by Silivong and Preston (2015) showed that the growth rate of goats fed foliage of
the legume tree Bauhinia acuminata was increased by supplementation with fresh water spinach
and biochar. The protein in water spinach is very soluble (Silivong and Preston, 2015) and it is
thought that its role in improving the utilization of foliages of low digestibility, such as Bauhinia
acuminata, is because the water spinach acts as a source of readily available nitrogenous
compounds for rumen micro-organisms (Silivong and Preston 2015, 2016). The positive role of
biochar as a supplement in ruminant diets is thought to reflect another feature of ruminant nutrition,
namely as a support mechanism for biofilms that host consortia of micro-organisms facilitating the
utilization of nutrients with major benefits for the process of rumen fermentation (Leng, 2014). In
this role, it appears that biochar is acting as a “prebiotic”, by promoting synergism between
nutrients and micro-organisms in the animal’s digestive system. A similar synergism appears to be
the explanation for the beneficial effects on growth rates of cattle (Binh et al., 2017) and goats (Sina
et al., 2017) of small proportions in the diet of brewers’ grains, a byproduct derived from the
industrial brewing of beer. The research with goats (Sina et al., 2017) highlighted a major
interaction between the effect of the supplementary brewers’ grains and the nature of the basal diet.

The improvement in growth rate due to addition of brewers’ grains was 130% when the basal diet
was fresh cassava foliage but only 30% when the basal diet was water spinach (Sina et al., 2017).
The hypothesis that was tested in the present experiment was that goats fed foliage of the legume
tree Bauhinia acuminata would respond positively in growth rate and feed conversion to a
supplement of brewers’ grains, and that the degree of response would be greater when cassava
foliage, rather than water spinach, was the complementary source of protein.
MATERIALS AND METHODS
Experimental treatments and design
The basal diet was fresh foliage from the legume tree Bauhinia acuminata fed ad
libitum, supplemented with biochar (1% of diet DM) and cassava root chips (4% of diet DM).
The treatments in a 2×2 factorial arrangement were:
Source of protein-rich foliage:
- 30% of DM feed intake (Water spinach: WS)
- 30% of DM feed intake (Cassava foliage: CF)
Supplementary brewers’ grains:
- 4% of DM feed intake (Brewer’s grains: BG)
- No supplement (No-BG)
20


In the digestibility study the design was a 4×4 Latin Square with 4 male goats and 4 periods
each of 12 days: 7 days for adaptation and 5 days for collection of feed refusals, feces and urine.
The goats (local breed) weighed 15.5±0.65 kg and were 5-6 months of age. They were purchased
from farmers around Luangprabang city. They were housed individually in metabolism cages made
from bamboo (dimensions of width 0.8 m, length 0.9 m and height 1 m), designed to collect
separately feces and urine. In the growth study the design was a Randomized Completely Block
Design (RCBD) with 4 replications of the two factors in a 2×2 factorial design, with sixteen goats
(balanced males and females) with initial body weight of 14.4 ± 1.45 kg and 5-6 months of age.
They were housed in individual pens made from wood and bamboo. In both studies, the goats were
vaccinated against Pasteurellosis and Foot and Mouth disease and were de-wormed before the start

of the experiment.
Feeding and management
In both studies: Foliages of Bauhinia acuminata and water spinach (Ipomoea aquatica) were
collected daily from natural stands in and around the University campus. Cassava (Manihot
esculenta Crantz) foliage was collected daily from a demonstration plot in the Department of
Animal Science Farm. Cassava root was harvested from the demonstration plot in the department of
Animal Science Farm. It was chopped into small pieces and exposed to sunlight for 48h to reduce
the moisture to about 15%. Brewers’ grains were purchased from a brewery in Vientiane city. The
biochar was produced by burning rice husks in a top lit updraft (TLUD) gasifier stove (Olivier,
2010). It was ground to a particle size that passes through a 1 mm sieve. The biochar was mixed
with the cassava root chips and fed from a plastic bucket. Bauhinia acuminata foliage, water
spinach and cassava foliage were hung in bunches above the feed trough. Fresh feeds were offered
twice daily at 07:30 and 16:00h. Water was freely available.
Measurements
Metabolism study
Live weight was recorded in the morning before feeding at the beginning and at the end of
each period. Feeds offered, and refusals were collected daily during the 5 days of the collection
period. Urine was collected in buckets with 20 ml of a solution of sulphuric acid to ensure a pH of
less than 4 (10% sulphuric acid concentrate + 90% distilled water). Feces and urine were collected
daily and stored in the refrigerator (4-8ºC) until the end of each period, when sub-samples were
mixed together.
Growth study
Live weight was recorded in the morning before feeding at the beginning and at 10-day
intervals until the end of the 90-day experiment. Live weight gain was calculated from the linear
regression of live weight (Y) on days from the start of the experiment (X). Feed consumption was
recorded daily. Refusals were collected from individual animals every morning before offering new
feed. Samples of Bauhinia acuminata, water spinach and cassava foliage (offered and residues)
were separated into stems and leaves (containing attached petioles). Representative samples of each
component were stored at -18°C until they were analysed. Samples of rumen fluid were taken on
the last day of the experiment, using a stomach tube.

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Chemical analyses
The samples of feeds offered and refused were analysed for DM, NDF, ADF, N and ash
according to AOAC (1990) methods. The pH of rumen fluid was measured with a digital pH meter,
prior to addition of sulphuric acid for subsequent analysis of ammonia by steam distillation (AOAC,
1990) and VFA by high pressure liquid chromatography (Water model 484 UV detector; column
novapak C18; column size 3.9 mm x 300 mm; mobile phase 10 mM H2 PO4 [pH 2.5]) (Samuel et
al., 1997). Solubility of the protein in the diet components was determined by extraction with M
NaCl (Whitelaw et al., 1961).
Statistical analysis
Metabolism study
The data were analyzed by the general linear model (GLM) in the ANOVA program of the
Minitab software (Minitab, 2014).
The statistical model used in the digestibility study was:
Yijk = μ + Ti + Pj + Ak + eijk
In where, Yijk = Dependent variables., μ = Overall mean., Ti = Treatment effect (i=1-4)., Pj =
Column effect (j=1-4)., Ak = Row effect (k=1-4)., eijk = Random error
Growth study
The statistical model was:
Yijk = μ + Bk + Pi + Aj + P i*Aj+ eijk
Where: Yijk is dependent variables., μ is overall mean., Bk is the effect of live weight., Pi is
the effect of foliages source (water spinach and cassava foliage)., Aj is the effect of brewers’ grains
source., (P*A)ij is the interaction between source of foliages and source of brewers’ grains and eijk is
random error
RESULTS
Chemical composition of diet components
The low values for solubility of the protein in the leaves of Bauhinia acuminata and cassava,
and the high values for the leaves of water spinach (Table 2), are in agreement with previous

observations (Silivong and Preston, 2016) and are assumed to reflect different levels of tannin-rich
compounds in the leaves of all three species.

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Table 2. Chemical composition of dietary ingredients (% in DM, except DM which is on fresh basis)
DM

N*6.25

Ash

Protein solubility, %

NDF

ADF

Bauhinia leaves

40.0

15.0

21.2

23.7

43.7

32.4

Bauhinia stem

38.1

12.3

4.29

-

42.7

31.5

Cassava leaves

32.1

22.2

4.48

31.4

48.7

34.4

Cassava petiole

16.8

16.7

6.39

-

48.3

38.6

Cassava root chips

82.4

2.81

2.23

-

-

-

Water spinach

8.16

18.3

9.74

69.4

42.3

33.3

Brewers’ grains

28.7

27.2

38.1

-

40.7

29.5

Biochar

-

-

38.3

-

-

-

Metabolism study
The two factors had contrasting effects on digestibility of DM and on daily N retention
(Table 3). Supplementation with brewers’ grains increased the digestibility of DM but the effect
was more pronounced when cassava foliage was the source of additional protein as compared with
water spinach. Daily N retention was similar for both foliages in the absence of brewers’ grains but,
when brewers’ grains were added, N retention was greater with cassava than with water spinach.
Table 3. Mean values of apparent digestibility and N balance in goats fed Bauhinia acuminata supplemented with
water spinach or cassava foliage, with (BG) and without (No-BG) brewers’ grains
CF

WS

SEM

Items
No-BG

BG

No-BG

BG

SEM

SEM
Foliage*BG

p
P Foliage

P BG

P
Foliage*BG

0.86

0.558

<0.001

0.035

Apparent digestibility, %
DM

68.4

74.9

69.9

72.3

0.60

N balance, g/day
Intake

14.0

14.8

13.8

14.1

0.45

0.63

0.474

0.414

0.619

Feces

4.8

3.8

4.7

4.5

0.34

0.48

0.545

0.199

0.393

Urine

2.9

2.3

2.5

2.2

0.28

0.40

0.47

0.245

0.715

Retention

6.28

8.78

6.64

7.48

0.25

0.35

0.208

<0.001

0.037

N retention as:

% N intake

44.8

59.5

48.2

53.0

1.80

2.54

0.555

0.002

0.073

% N digested

68.6

79.2

73.0

78.1

2.12

2.99

0.585

0.022

0.368

BG: Brewer’s grain, CF: Cassava foliage, d: day, g: Gram, No-BG: Non-Brewer’s grain, P:
Probability value, SEM: Standard error of the mean with dferror: 0, WS: Water spinach
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