HUE UNIVERSITY
UNIVERSITY OF AGRICULTURE AND FORESTRY
NOUPHONE MANIVANH
NUTRITIVE IMPROVEMENT OF CASSAVA ROOT AND
ITS UTILISATION IN TARO FOLIAGE AND BANANA
STEMS BASAL DIETS FOR LOCAL PIG PRODUCTION
IN SMALLHOLDERS IN LAO PDR
DOCTOR OF PHILOSOPHY IN ANIMAL SCIENCES
HUE, 2019
HUE UNIVERSITY
UNIVERSITY OF AGRICULTURE AND FORESTRY
NOUPHONE MANIVANH
NUTRITIVE IMPROVEMENT OF CASSAVA ROOT AND
ITS UTILISATION IN TARO FOLIAGE AND BANANA
STEMS BASAL DIETS FOR LOCAL PIG PRODUCTION
IN SMALLHOLDERS IN LAO PDR
SPECIALIZATION: ANIMAL SCIENCES
CODE: 9620105
DOCTOR OF PHILOSOPHY IN ANIMAL SCIENCES
SUPERVISORS
1: ASSOCIATE PROFESSOR DR. LE VAN AN
2: ASSOCIATE PROFESSOR DR. TRAN THI THU HONG
HUE, 2019
GUARANTEE
I hereby guarantee that scientific work in this thesis is mine. All results
described in this thesis are righteous and objective. They have been published in
Journal of Livestock Research for Rural Development (LRRD) http://www.lrrd.org
Hue University, 2019
Nouphone MANIVANH, PhD student
i
DEDICATION
To my parents, my husband (Phoneouthai Thiphavanh), my daughter (Southida
Thiphavanh) and my son (Kanlaya Thiphavanh)
ii
ACKNOWLEDGEMENTS
The research in this PhD thesis was conducted four experiments with supported
from Mekong Basin Animal Research Network (MEKARN II) project for funding this
thesis research and the scholarship for the PhD study.
I am grateful for the support from all of those people and institutions:
I would like to express my sincere gratitude to the Mekong Basin Animal
Research Network (MEKARN II) project for funding this theses research and the
scholarship for the PhD study.
I would like to thanks the Faculty of Agriculture and Forestry, Souphanouvong
University, Luagprabang province, Laos, for allowing me study leave and helping me
to carry out the experiments.
I would like to express my cordial and faithful gratitude to my main supervisors,
Associate Professor Dr. Le Van An and co-supervisor, Associate Professor Dr. Tran
Thi Thu Hong for their support, guidance, and valuable advice for writing paper.
I would like to express deeply gratitude to Professor Dr. Thomas Reg Preston
Director of the University of Tropical Agriculture (UTA) for his good discussion,
valuable advice and useful guidance during my studies and research project.
My sincere thanks to Professor Dr. Ewadle, International Coordinator
MEKARN II project; Dr. Vanthong Phengvichith, National Agriculture and Forestry
Researcher Institute (NAFRI), Lao PDR; Dr. Kieu Borin, MEKARN II regional
coordinator for their facilitation, help and support to the whole course. Professors,
lecturers and assistant lectures in Hue University of Agriculture and Forestry and
MEKARN II program, for giving me care and useful knowledge; Dr. Vongpasith
Chanthakhoun, Dean of Faculty of Agriculture and Forestry, Souphanouvong
University for his help and encouragement.
I am also grateful to my friends on the PhD course from Cambodia, Laos and
Vietnam for their good friendship and sharing
Lastly I would like to express special thanks to my husband (Phone outhai
Tiphavanh), my parents and all my brothers and sisters for their support,
encouragements and patience.
iii
ABSTRACTS
The study was aimed at improving protein content of cassava root (Manihot
esculenta Crantz) by solid-state fermentation with yeast (Saccharomyces cerevisiae),
urea and di-ammonium phosphate (DAP) additive and its utilization as protein source
in the diets of Moo Lath Pig in Laos. Four experiments were carried out with “two in
cassava root fermentation experiments, two experiments were conducted with Moo
Lath pig using taro silage (TS) replaced by protein-enriched cassava root (PECR) as
protein sources on growing trial and digestibility. In chapter 2, experiment 1. Cassava
root was fermented with yeast, urea and DAP in a solid-state fermentation to determine
the degree of conversion of crude to true protein; and experiment 2 the limiting factor
to the synthesis of true protein from crude protein in the fermentation of cassava root
could be the decrease in pH in the fermentation substrate preventing the hydrolysis of
urea to ammonia and thus decreasing the availability of nitrogen for growth of the
yeast. The following experiment to determine the degree of conversion of crude to true
protein, pH and ammonia. In experiment 1. The experiment was arranged as a 2*3*4
factorial in a completely randomized design (CRD). The treatments were: root
processing (steamed and not steamed); DAP: 0, 1 and 2% of the substrate DM. The
fermentation was over 14 days with samples taken for determination of true and crude
protein (CP) at 0, 3, 7 and 14 days. In experiment 2. A CRD was used with 2 treatments
arranged as a 2*9 factorial. The treatments were anaerobic and aerobic fermentation.
The substrate was cassava root 93.6% + DAP 2% + urea 1.4% + yeast 3% (DM basis).
True, crude protein, ammonia and pH were measured at 0 and 3h after preparing the
substrates and every 24h until end of day 7 (0, 3h, 1, 2, 3, 4, 5, 6 and 7 day).
Experiment 1 (chapter 2). The true protein (TP) in cassava root increased with a
curvilinear trend (R2 = 0.98) from 2.30 to 6.87% in DM as the fermentation time
increased from zero to 14 days; the ratio of true to crude protein increased from 24.6 to
63.7 over the same period. Increasing the proportion of DAP from zero to 2% of the
substrate DM increased the TP from 5.6 to 7.3% in DM after 14 days of fermentation.
Steaming the cassava root prior to fermentation improved slightly (p=0.67) the
conversion of crude to TP. Experiment 2 (chapter 2). The pH decreased with
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fermentation time, according to an almost linear trend, from 5.8 immediately after
mixing the substrate, to 5.47in 3h and to 3.43 after 7 days. The level of CP after mixing
the substrate and additives was 10.35% in DM and did not change over the 7 days of
fermentation. TP in the substrate increased from 2.37 to 6.97% in DM as the
fermentation time increased from zero to 7 days. There were no differences in all these
criteria as between the aerobic and anaerobic condition, other than a tendency for the
pH to fall slightly more quickly in the first 4 days in the anaerobic condition followed
by a slower rate of fall to reach almost the same final value after 7 days, as for the
aerobic condition. Experiment 3 (chapter 3). A growth trial was conducted with 12
Moo Lath pigs with average 14.8 ±1.89 kg initial live weight in a CRD, with three
replications of four treatments. The aim of the study was to determine the effect of
replacing TS with PECR in a basal diet of ensiled banana stem (BS). There were
positive responses in dry matter (DM) intake, live weight gain, feed conversion ratio, as
the percentage of PECR in the diet was increased (zero to 15% in DM ). It was
concluded that the replacing of taro foliage silage with PECR improved the quality of
the overall diet, which resulted in higher intake, growth rate, better feed conversion
ratio and economical efficiency. Experiment 4 (chapter 4). Four castrated male Moo
Lath pig, weighing on average 15 kg were allotted at random to 4 diets within a 4*4
Latin square design, to study effects on DM intake, digestibility and N retention of
levels of protein-enriched cassava root (PECR) as 0, 25, 50 and 75% in combination
with TS as 80, 55, 30 and 5% with constant levels of ensiled banana stem 20% (all on
DM basis). PECR at 25% in a diet led to increases in feed intake, diet digestibility and
N retention in native Moo Lath pigs and PECR could be the result of its superior
biological value compared with the protein in the taro foliage. These criteria declined
linearly when the proportions of PECR were increased to 50 and 75% of the diet DM.
Key words: banana pseudo-stem, di-ammonium phosphate, probiotic, solidstate fermentation, urea, yeast, crude protein, true protein, ammonia, pH, Moo Lath pig
v
TABLE OF CONTENTS
GUARANTEE ................................................................................................................. i
DEDICATION ....................................................................................................... ii
ACKNOWLEDGEMENTS ................................................................................... iii
ABSTRACTS ....................................................................................................... iv
TABLE OF CONTENTS ....................................................................................... vi
LIST OF FIGURES ............................................................................................... xi
LIST OF PHOTO ................................................................................................ xiii
LIST OF TABLES .............................................................................................. xiii
LIST OF ABBREVIATIONS ............................................................................... xv
INTRODUCTION .................................................................................................. 1
1. PROBLEM STATEMENT ................................................................................. 1
2. OBJECTIVES ............................................................................................................. 1
3. HYPOTHESES ........................................................................................................... 3
4. SIGNIFICANCE/INNOVATION OF THE DISSERTATION.................................. 1
CHAPTER 1: LITERATURE REVIEW ........................................................................ 5
1. PIG PRODUCTION IN LAOS .................................................................................. 5
1.1. ROLE OF PIG PRODUCTION ........................................................................... 5
1.2. PIG POPULATION ............................................................................................. 6
1.3. PIG PRODUCTION SYSTEM IN LAOS ............................................................ 8
1.3.1. Commercial pig production .......................................................................... 8
1.3.2. Semi-commercial pig production ................................................................. 8
1.3.3. Smallholder pig production .......................................................................... 9
1.3.4. Main problems in smallholder pig production systems ............................ 11
1.3.5. Important points to improve smallholder pig production system ............. 11
1.4. LOCAL PIG BREEDS RAISED BY SMALLHOLDERS ................................ 12
1.4.1. Moo Lath .................................................................................................... 12
1.4.2. Moo Chid, Moo Markadon or Moo Boua................................................... 13
1.4.3. Moo Nonghad or Moo Hmong ................................................................... 14
vi
1.4.4. Moo Deng or Moo Berk ............................................................................. 14
2. REQUIREMENT OF PROTEIN AND AMINO ACID FOR GROWING PIGS .... 16
3. FEED STUFFS FOR PIG IN LAOS ........................................................................ 18
3.1. LOCAL FEED AVAILABLE FOR PIG ............................................................ 19
3.1.1. Taro (Colocasia esculenta) ......................................................................... 19
3.1.2. Cassava (Manihot esculenta Crantz) .......................................................... 22
3.1.3. Banana pseudo (Musa sapientum Linn) stems ........................................... 25
4. METHOD TO IMPROVE NUTRITIVE VALUE FOR FEED STUFF WITH LOW
PROTEIN CONTENT .................................................................................................. 26
4.1. SACCHAROMYCES CEREVISIAE (S. CEREVISIAE) .......................................... 26
4.2. SOLID-STATE FERMENTATION (SSF) ........................................................ 28
4.2.1. Application of solid-state fermentation ...................................................... 28
4.2.2. Advantage solid-state fermentation ............................................................ 28
4.2.3. Factors influencing solid-state fermentation .............................................. 29
4.2.4. Problem of solid-state fermentation............................................................ 29
4.2.5. Conditions necessary for fermentation ....................................................... 29
4.3. PROTEIN ENRICHMENT................................................................................ 30
5. UTILIZATION OF FORAGE-BASED DIET FOR PIGS ....................................... 32
5.1. EFFECT OF TARO FOLIAGE AS PROTEIN SOURCE ON FEED INTAKE
AND DIGESTIBILITY OF PIGS ............................................................................. 32
5.2. EFFECT OF TARO FOLIAGE AS PROTEIN SOURCE ON GROWING
PERFORMANCE OF PIGS ..................................................................................... 32
5.3. EFFECT OF TARO FOLIAGE REPLACING BANANA PSEUDO STEM ON
GROWTH AND DIGESTIBILITY OF PIGS .......................................................... 33
5.4. EFFECT OF ENSILED TARO FOLIAGE REPLACED BY PROTEINENRICHED CASSAVA ROOT ON GROWTH PERFORMANCE AND
DIGESTIBILITY ...................................................................................................... 34
REFERENCES ............................................................................................................. 35
CHAPTER 2 ................................................................................................................. 51
IMPROVING NUTRITIVE VALUE OF CASSAVA ROOTS ................................... 51
(Manihot esculenta Crantz) .......................................................................................... 51
vii
ABSTRACT ............................................................................................................. 51
INTRODUCTION .................................................................................................... 52
EXPERIMENT 1: ......................................................................................................... 53
MATERIALS AND METHODS .............................................................................. 53
Experimental design ............................................................................................. 53
Measurements ....................................................................................................... 55
Chemical analysis ................................................................................................. 55
Statistical analysis ................................................................................................. 55
RESULT AND DISCUSSION ................................................................................. 55
Changes in the mass of substrate during fermentation ......................................... 58
EXPERIMENT 2. ......................................................................................................... 60
MATERIALS AND METHODS .............................................................................. 60
Experimental design ............................................................................................. 60
Measurements ....................................................................................................... 61
Chemical analysis ................................................................................................. 61
Statistical analysis ................................................................................................. 61
RESULT AND DISCUSSION ................................................................................. 61
DISCUSSION ........................................................................................................... 64
CONCLUSIONS ...................................................................................................... 65
REFERENCES ......................................................................................................... 66
CHAPTER 3 ................................................................................................................. 69
REPLACING TARO (Colocasia esculenta) SILAGE BY PROTEIN-ENRICHED
CASSAVA ROOT IMPROVED THE NUTRITIVE VALUE OF A BANANA STEM
(Musa sapientum Linn) BASED DIET AND SUPPORTED BETTER GROWTH IN
MOO LATH PIG .......................................................................................................... 69
ABSTRACT ............................................................................................................. 69
INTRODUCTION .................................................................................................... 69
MATERIALS AND METHODS .............................................................................. 70
Experimental design ............................................................................................. 73
Data collection ...................................................................................................... 74
Chemical analysis ................................................................................................. 74
viii
Statistical analysis ................................................................................................. 74
RESULTS AND DISCUSSION ............................................................................... 74
DISCUSSION ........................................................................................................... 78
CONCLUSIONS ...................................................................................................... 79
REFERENCES ......................................................................................................... 79
CHAPTER 4 ................................................................................................................. 82
APPARENT DIGESTIBILITY AND N RETENTION IN GROWING MOO LATH
PIGS FED ENSILED TARO FOLIAGE (Colocasia esculenta) REPLACED BY
PROTEIN-ENRICHED CASSAVA ROOT (Manihot esculenta Crantz) ................... 82
ABSTRACT ............................................................................................................. 82
MATERIAL AND METHODS ................................................................................ 84
Experimental design ............................................................................................. 84
Measurements and data collection ........................................................................ 85
Statistical analysis ................................................................................................. 86
RESULTS AND DISCUSSION ............................................................................... 86
Chemical composition .......................................................................................... 86
Feed intake ............................................................................................................ 86
Apparent digestibility ........................................................................................... 88
Nitrogen balance ................................................................................................... 89
DISCUSSION ........................................................................................................... 90
CONCLUSIONS ...................................................................................................... 92
REFERENCES ......................................................................................................... 92
GENERAL DISCUSSION AND CONCLUSIONS .................................................... 95
1. GENERAL DISCUSSION ....................................................................................... 95
1.1. IMPROVING NUTRITIVE VALUE OF CASSAVA ROOT (MANIHOT
ESCULENTA CRANTZ) BY FERMENTATION WITH YEAST (SACCHAROMYCES
CEREVISIAE) UREA AND DI-AMMONIUM PHOSPHATE (DAP) ......................... 95
1.2. EFFECT OF THE USE OF PROTEIN ENRICHED OF CASSAVA ROOT
(MANIHOT ESCULENTA CRANTZ) ON INTAKE, DIGESTIBILITY, N BALANCE AND
GROWTH PERFORMANCE OF LOCAL PIG. ...................................................... 98
2. GENERAL CONCLUSIONS ................................................................................. 102
ix
3. IMPLICATION AND FURTHER RESEARCH.................................................... 103
3.1 IMPLICATION................................................................................................. 103
3.2. FURTHER RESEARCH.................................................................................. 104
REFERENCES ............................................................................................................. 95
PUBLISHCATION LIST ........................................................................................... 111
x
LIST OF FIGURES
CHAPTER 1 .................................................................................................................... 5
Figure 1. Number of pigs in Laos from 2010-2016 ............................................. 7
Figure 2. Characteristic of pig in northern, central and southern in 2005-2015 .. 8
CHAPTER 2 .................................................................................................................. 51
EXPERIMENT 1: .......................................................................................................... 53
Figure 1. The level of crude and true protein after fermented 14 days ............. 57
Figure 2. Curvilinear response in the true and crude protein ratio with increasing
length of fermentation........................................................................................ 57
Figure 3. Effect of level of DAP on concentration of true protein after 14 days
of fermentation................................................................................................... 58
Figure 4. Changes in the mass of substrate during the fermentation ................. 59
Figure 5. Proportion of the original substrate fermented during different stages
of the fermentation ............................................................................................. 59
EXPERIMENT 2. .......................................................................................................... 60
Figure 1. Effect of fermentation time on pH of cassava root fermented with
yeast, urea and DAP, under anaerobic and aerobic condition ........................... 63
Figure 2. Effect of fermentation on true and crude protein content of cassava
root supplemented with urea, DAP and yeast .................................................... 63
Figure 3. Distribution of the nitrogen as urea, ammonia and true protein at the
beginning and after 7 days of fermentation ....................................................... 64
CHAPTER 3 .................................................................................................................. 69
Figure 1. Effect of supplementation with PECR on DM intake of pigs by
replacing taro silage and ensiled banana stem as a basal diet........................... 75
Figure 2. Relationship between live weight gain and PECR content of the diet 76
Figure 3. Relationship between feed conversion ratio and PECR content of the
diet ..................................................................................................................... 77
xi
CHAPTER 4 .................................................................................................................. 82
Figure 1. Mean values for DM intake by pigs fed diets in which taro silage was
replaced by PECR .............................................................................................. 87
Apparent digestibility............................................................................................. 88
Figure 2. Mean values for apparent digestibility of DM and crude protein in
pigsfed diets in which taro silage was replaced by PECR ................................. 88
xii
LIST OF PHOTO
CHAPTER 1: LITERATURE REVIEW ........................................................................ 5
Photo 1. Local pigs are allowed to scavenge freely all year round .................... 9
Photo 2. Local pigs in pen ................................................................................ 10
Photo 3. Feed stuffs available in farm condition .............................................. 11
Photo 4. Moo Lath ............................................................................................ 13
Photo 5. Moo Chid, Moo Markadon or Moo Boua .......................................... 14
Photo 6. Moo Nonghad or Moo Hmong ........................................................... 14
Photo 7. Moo Deng or Moo Berk ..................................................................... 15
CHAPTER 2 ................................................................................................................. 51
EXPERIMENT 1: ......................................................................................................... 53
Photo 1. The steaming of the cassava root ....................................................... 54
Photo 2. Aerobic fermentation of the cassava root .......................................... 54
CHAPTER 3 ................................................................................................................. 69
Photo 1. Wooden boards 30cm above the base of the barrel ............................ 71
Photo 2. The bamboo strips placed above the boards ....................................... 71
Photo 3. The steaming of the ............................................................................ 71
cassava root ....................................................................................................... 71
Photo 4. Mixing cassava root with urea, di-ammonium phosphate (DAP) and
yeast .................................................................................................................. 71
Photo 5. The mixed substrate was put in bamboo baskets covered with plastic
netting ............................................................................................................... 72
Photo 6. The protein-enriched cassava root ...................................................... 72
Photo 7. Taro (Colocasia esculenta) were chopped by hand ........................... 72
Photo 8. Taro (Colocasia esculenta) were wilted for 24h to reduce the moisture
.......................................................................................................................... 72
Photo 9. Taro silage in the plastic bag .............................................................. 72
Photo 10. Ensiled taro after 14 days ................................................................. 72
Photo 11. Banana stems were chopped by hand into small pieces ................... 73
xiii
Photo 12. Ensiled banana stems in 200 liter PVC ............................................ 73
Photo 13. Housing made from local materials.................................................. 73
Photo 14. Moo Lath pig used in the experiment ............................................... 73
xiv
LIST OF TABLES
CHAPTER 1 .................................................................................................................... 5
Table 1. Number of meat consumption in 2017 of Lao PDR .............................. 6
Table 2. Statistic of livestock population in Laos (2010-2016)........................... 6
Table 3. Pig population in Laos (2005-2015) ...................................................... 7
Table 4. Classification of phenotype characteristics and reproductive
performance of native pigs produced under smallholder farm (SHPF)
conditions in Lao PDR....................................................................................... 15
Table 5. Dietary amino acid requirements of growing-finishing pigs (NRC
1998) .................................................................................................................. 17
Table 6. Chemical composition of taro (Colocasia esculenta) in DM basis ..... 20
Table 7. Planted area, yield and production of cassava root ............................. 23
Table 8. Proximate nutrient composition of Cassava root and leaves ............... 24
Table 9. Planted area, yield and production of banana ...................................... 25
CHAPTER 2 .................................................................................................................. 51
EXPERIMENT 1: .......................................................................................................... 53
Table 1. Composition of the substrates (DM basis) .......................................... 54
Table 2. Mean values for DM, OM, crude protein; true protein and ratio of
TP/CP at
different stages of the fermentations (% in DM) ............................ 56
Table 3. Effect of level of DAP on concentration of crude protein, true protein
and ratio of TP/CP
after 14 days of fermentation (% in DM) ....................... 58
Table 4. Changes in the mass of fresh (FM) and dry (DM) substrate during the
fermentation ....................................................................................................... 58
Table 5. Chemical composition (g/kg of DM)................................................... 59
EXPERIMENT 2. .......................................................................................................... 60
Table 1. Changes in pH, crude protein (CP), true protein (TP) and ammonia in
cassava root fermented with yeast, urea and DAP under aerobic or anaerobic
conditions ........................................................................................................... 62
xv
CHAPTER 3 .................................................................................................................. 69
Table 1. The chemical composition of feed ingredients (% in DM, except DM
which is on fresh basis)...................................................................................... 74
Table 2. Mean values for DM intake (g/day) by pigs fed taro silage (TS) and
ensiled banana stem (BT) supplemented with protein enriched cassava root
(PECR) ............................................................................................................... 75
Table 3. Mean values for live weight changes of growing pigs during the
experiment ......................................................................................................... 76
Table 4. Feed ingredient costs (LAK) ............................................................... 77
Table 5. Economic analysis of experimental treatments (LAK) ....................... 78
CHAPTER 4 .................................................................................................................. 82
Table 1. The chemical composition of feed ingredients (% in DM, except DM
which is on fresh basis)...................................................................................... 86
Table 2. Mean values of DM intake by pigs fed protein-enriched cassava root
(PECR) replacing taro silage with constant levels of ensiled banana stem ....... 87
Table 3. Apparent digestibility (%) of diets with PECR replacing ensiled taro
foliage with constant levels of ensiled banana stem .......................................... 88
Table 4. Mean values for N balance in pigs fed protein enriched cassava root
replacing taro silage with constant levels of ensiled banana stem..................... 89
xvi
LIST OF ABBREVIATIONS
AA
Amono acids
ADG
Average daily gain
ANOVA
Analysis of variance
ADF
Acid detergent fibre
AOAC
Association of Official Analytical Chemists
BS
Banana stem ensilage
BW
Body weight
Ca
Calcium
CF
Crude fibre
CSF
Classical swine fever
Cl
Chloride
CRD
Completely randomized design
Cm
Centimetre
CP
Crude protein
°C
Degree Celsius
DAP
Di-ammonium phosphate
DE
Digestible energy
DLF
Department of Livestock and Fisheries
DM
Dry matter
EAA
Essential amino acids
EE
Ether extract
EBS
Ensiled banana stem
FAO
Food and Agriculture Organization of the United Nation
FW
Fresh weight
FCR
Feed conversion ratio
g
Gram
GDP
Gross domestic product
h
Hour
xvii
ha
Hectare
HCN
Hydrocyanic acids
Kg
Kilogram
Lao PDR
Lao People’s democratic republic
LWG
Live weigh gain
LW
Live weigh
L
Liter
m
Meter
ME
Metabolisable energy
MAF
Ministry of Agriculture and Forestry
Mekarn
Mekong Basin Animal Research Network
N
Nitrogen
NRC
National Research Council
NAFES
National Agriculture and Forestry Extension Service
NAFRI
Institute National Agriculture and Forestry Research
NE
Net energy
NPN
None protein nitrogen
NFE
Nitrogen-free extract
F
Neutral detergent fibre
NST
Non steamed
NP
Non-protein
OM
Organic Matter
P
Phosphorus
PECR
Protein-enriched cassava root
PECP
Protein-enriched cassava pulp
pH
Power of/potential Hydrogen
Prob/p
Probability
RCBD
Randomised Complete Block Design
RDB
Rice distillers’ by-product
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