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

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) 
	 Hue University, 2019
 Nouphone MANIVANH, PhD student
DEDICATION
To my parents, my husband (Phoneouthai Thiphavanh), my daughter (Southida Thiphavanh) and my son (Kanlaya Thiphavanh)
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.
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 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, solid-state fermentation, urea, yeast, crude protein, true protein, ammonia, pH, Moo Lath pig 
TABLE OF CONTENTS
GUARANTEE	i
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
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
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
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
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)	60
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
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
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
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
Sida/SAREC Swedish international development agency-Dpartment for research Cooperation
ST
Steamed
SEM
Standard error of the mean
TS
Taro silage
T
Ton
 ... t (Saccharomyces cerevisiae), urea and di-ammonium phosphate (DAP) additive. This process of solid-state fermentation provides a means of converting cassava root into useful feed for the production of pig production. In situations where cassava root are available at low cost they can be fermented and fed safely as a protein source to pigs. However, pig performance will be improved if PECR replacing aptitude level with another protein source such as taro (Colocasia esculenta) and banana stem (Musa sapientum Linn) as energy sources. Taro (Colocasia esculenta) leaves are rich in vitamins and minerals, and are a good source of thiamin, riboflavin, iron, phosphorus, and zinc, and a very good source of vitamin B6, vitamin C, niacin, potassium, copper, and manganese. Taro is a locally available feed resource with good potential for animals, especially for pigs, because of its nutritional quality. According to Rodriguez et al., (2009), fresh leaves of Xanthosoma sagittifolium (a member of the same family as Colocacia esculenta) had a chemical composition (g/kg DM) of: crude protein, 248; crude fibre, 142; NDF, 255; ADF, 198; Ca, 17.7; P 2.0; Mg, 2.2 and K, 32.3. 
One important local feed is banana pseudo-stem (Musa sapientum Linn). Bananas are grown everywhere in Laos for human food and there is a long tradition of chopping the pseudo-stem after fruit harvest and feeding it to pigs and poultry because of pseudo-stem contain the sugar in the aqueous fraction as provided energy and another potential source of protein from green feed for livestock in Lao is taro (Colocasia esculenta). Taro leaves are rich in vitamins and minerals, and are a good source of thiamin, riboflavin, iron, phosphorus, and zinc, and a very good source of vitamin B6, vitamin C, niacin, potassium, copper, and manganese. Taro is a locally available feed resource with good potential for pigs, because of its nutritional quality. It was concluded that the Taro plant was the most promising as a source of well -balanced amino acids and digestible carbohydrate. The only negative attribute – the high level of oxalic acid has been shown to be controllable by ensiling and supplementation with a source of calcium. Therefore, feeding systems based on replacing taro (Colocasia esculenta) silage with protein-enriched cassava root (PECR) improved the nutritive value of a banana stem (Musa sapientum Linn) based diet and supported better growth in pigs. However, these local feed sould be utilize in aptitude level will be a greated potential to improve the nutritive value of local feed as diets for local pigs to improve pig production, which helps in reducing feed cost and bringing economic benefits to the farmers in rural area of Laos.
3.2. FURTHER RESEARCH 
The digestibility and N retention were improvement and growth rate from feeding the protein-enriched cassava root could be the result of its superior biological value compared with the protein in the taro foliage. The other possibility could be the increased provision of vitamins of the β-complex from the yeast in the fermented cassava root. Although there were positive responses in DM intake, live weight gain, feed conversion, apparent digestibility and N retention when protein-enriched cassava root partially replaced the other protein sources in the diets fed to pigs, the researchers were showed that the protein-enriched product could provide 25% of the dietary protein in a diets, replacing ensiled taro foliage. However, PECR should be feed appropriate level in the diet because higher proportions (more than 28%) of the protein-enriched feed in the diet led to reduced growth performance as some research reported. The finding founded feed intake appears to be a limiting factor in growing pigs. At higher rates of substitution of 50 and 75% of the taro silage by PECP, the levels of residual NPN (ammonia) and that some 30% of the original urea and DAP remains as some form of NPN possibly ranging from ammonium salts to peptides and amino acids]. May have exceeded the capacity of the gut microbes to synthesize it into amino acids with resultant toxic effects of the ammonia leading to reduced feed intake and therefore reduced growth rate. It is therefore logical to evaluate their use in systems where feed intake is normally restricted. The "problem" with pig digestive tracts is that they do not provide a sufficiently large habitat for bacteria that would have long enough to produce significant amounts of ammonia, and then convert it to amino-acids. Ammonia is smaller, more volatile and more mobile than urea. If allowed to accumulate, ammonia would raise the pH in cells to toxic levels. It can be toxic if improperly used because the idea of using urea depends on the presence of the microorganisms in the digestive system in animals., Which is working to convert urea nitrogen to protein, and animal use this protein to build their tissues. But, the digestive system of pigs where there is no microorganisms which can convert urea to protein. May this microorganisms exist in cecum but in small amount. Otherwise urea would be toxic for pigs. Pigs received 2% urea as a source of nitrogen (N) consumed less feed, gained less rapidly and required more feed per kg body weight (BW) gained as compared to those fed equivalent diet without urea. It would be of interest to evaluate other protein sources rich in this amino acid, for example the residues from fermentation and distillation of rice wine or improving the nutritive value of carbohydrate-rich (broken rice) feeds is by solid-state fermentation yeast (Saccharomyces cerevisiae, Aspergillus niger, Bacillus subtilis), urea and di-ammonium phosphate additive and evaluate them in the dite for growing pigs and pig reproduction.
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PUBLICATION LIST
I. Manivanh, N., Preston, T.R., An, L.V. and Thu Hong, T.T., 2018. Improving nutritive value of cassava root (Manihot esculenta Crantz) by fermentation with yeast (Saccharomyces cerevisiae), urea and di-ammonium phosphate. Livestock Research for Rural Development. Volume 30, Article #94. 
II. Manivanh, N., Preston, T.R., An, L.V. and Thu Hong, T.T 2018. Apparent digestibility and N retention in growing local pigs fed ensiled Taro foliage (Colocasia esculenta) replaced by protein-enriched cassava root (Manihot esculenta Crantz). Livestock Research for Rural Development. Volume 30, Article #165. Retrieved October 30, 2018, from