Làm giàu protein củ sắn bằng cách lên men với nấm men làm thức ăn cho lợn địa phương ở lào

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-ammoni

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

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

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

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.ROLEOFPIGPRODUCTION 5

1.2.PIGPOPULATION 6

1.3.PIGPRODUCTIONSYSTEMINLAOS 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.LOCALPIGBREEDSRAISEDBYSMALLHOLDERS 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

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.LOCALFEEDAVAILABLEFORPIG 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. SACCHAROMYCESCEREVISIAE (S C EREVISIAE ) 26

4.2.SOLID-STATEFERMENTATION(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.PROTEINENRICHMENT 30

5 UTILIZATION OF FORAGE-BASED DIET FOR PIGS 32

5.1.EFFECTOFTAROFOLIAGEASPROTEINSOURCEONFEEDINTAKE ANDDIGESTIBILITYOFPIGS 32

5.2.EFFECTOFTAROFOLIAGEASPROTEINSOURCEONGROWING PERFORMANCEOFPIGS 32

5.3.EFFECTOFTAROFOLIAGEREPLACINGBANANAPSEUDOSTEMON GROWTHANDDIGESTIBILITYOFPIGS 33

5.4.EFFECTOFENSILEDTAROFOLIAGEREPLACEDBY PROTEIN-ENRICHEDCASSAVAROOTONGROWTHPERFORMANCEAND DIGESTIBILITY 34

REFERENCES 35

CHAPTER 2 51

IMPROVING NUTRITIVE VALUE OF CASSAVA ROOTS 51

(Manihot esculenta Crantz) 51

ABSTRACT 51

INTRODUCTION 52

EXPERIMENT 1: 53

MATERIALSANDMETHODS 53

Experimental design 53

Measurements 55

Chemical analysis 55

Statistical analysis 55

RESULTANDDISCUSSION 55

Changes in the mass of substrate during fermentation 58

EXPERIMENT 2 60

MATERIALSANDMETHODS 60

Experimental design 60

Measurements 61

Chemical analysis 61

Statistical analysis 61

RESULTANDDISCUSSION 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

MATERIALSANDMETHODS 70

Experimental design 73

Data collection 74

Chemical analysis 74

Statistical analysis 74

RESULTSANDDISCUSSION 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

MATERIALANDMETHODS 84

Experimental design 84

Measurements and data collection 85

Statistical analysis 86

RESULTSANDDISCUSSION 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.IMPROVINGNUTRITIVEVALUEOFCASSAVAROOT(M ANIHOT ESCULENTA CRANTZ)BYFERMENTATIONWITHYEAST(S ACCHAROMYCES CEREVISIAE)UREAANDDI-AMMONIUMPHOSPHATE(DAP) 95

1.2.EFFECTOFTHEUSEOFPROTEINENRICHEDOFCASSAVAROOT (M ANIHOT ESCULENTA CRANTZ)ONINTAKE,DIGESTIBILITY,NBALANCEAND GROWTHPERFORMANCEOFLOCALPIG 98

2 GENERAL CONCLUSIONS 102

3 IMPLICATION AND FURTHER RESEARCH 103

3.1IMPLICATION 103

3.2.FURTHERRESEARCH 104

REFERENCES 95

PUBLISHCATION LIST 111

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

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) 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

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

ANOVA Analysis of variance

AOAC Association of Official Analytical Chemists

FAO Food and Agriculture Organization of the United Nation

Kg Kilogram

Lao PDR Lao People’s democratic republic

MAF Ministry of Agriculture and Forestry

Mekarn Mekong Basin Animal Research Network

NAFES National Agriculture and Forestry Extension Service NAFRI Institute National Agriculture and Forestry Research

PECR Protein-enriched cassava root

PECP Protein-enriched cassava pulp

Prob/p Probability

RDB Rice distillers’ by-product

INTRODUCTION

1 PROBLEM STATEMENT

Pig is one of the most important animals for smallholders in the uplands of Lao PDR because it can be sold when cash is needed for buying rice and other food, for paying school fees or if a household member is sick and needs medical attention and Pork used in traditional ceremonies in households Pigs can be confined in a small area, and can covert to meet a variety of crop and kitchen wastes and give a rapid return on investment (Steinfeld, 1998) About 75% of households in upland areas are raising pig in the country (FAO, 2017) Overall, native pig around 85.1% under small holder system (DLF, 2017), they are hardy and able to scavenge at least part of their feed requirements in free-range condition, Native pigs are mainly raised in extensive low-input systems that take advantage of naturally occurring feed (Kennard, 1996; FLSP, 2002) In most parts of Laos, agricultural by-products, such as rice bran, and natural grasses are the main feeds for live stock (ILRI 2002) In Lao villages, where most farmers are growing paddy rice for sale, the feed for pigs is based on rice bran, which is fed together with a small amount of green feed Thus rice bran is available in most farm households but they cannot support full performance because of their poor nutritive value (ILRI, 2002; FLSP, 2002) Since feed accounts for about 50-60% of the variable costs

of production, feed quality is crucial to the success of pig farming operations Major problems that may result from low quality feeds are poor appetite, slow growth, high feed conversion ratio, and low survival These usually develop as a result of problems on quality of raw materials, feed formulation, processing technology, storage, and feed manage The main problem is the supply of protein as soybean and fish meals are not available in rural areas and expensive (Phengsavanh and Stür., 2006)

Cassava plantation is mainly for root production The yields of root are variable depending on soil fertility, management and irrigation system Cassava root yields can be from 10 to15 tonne/ha without inputs on eroded soils (Howeler, 1991) In Laos, cassava

(Manihot esculenta Crantz) known as ‘Man Ton’, it is currently the third most important crop

in Laos, after rice and maize for smallholder farmers in remote upland areas Recently, the crop has become an important cash crop for either domestic use or for export because it can

be used for food and feed as well as for industrial processing into starch (Ministry of Agriculture and Forestry, 2013) Cassava has become a major crop in Lao PDR mainly because of the export of starch that is extracted from the cassava root There are five cassava

starch factories with a total planted area of 60,475 ha, giving an average yield of fresh roots of

27 tonnes/ha Annual production is of the order of 1.6 million tonnes (Ministry of Agriculture and Forestry, 2013) Cassava farms are needed not only for a major source of income for rural households but also for use in pig diets as energy sources because of cassava root content high levels of energy (75 to 85% of soluble carbohydrate) but low crude protein (2 to 3% CP) The root is composed of highly digestible carbohydrate in the form of starch with little fiber (Kang

et al., 2015; Polyorach et al., 2013) Solid state fermentation of the cassava root is a promising technology as this has the potential to raise the protein content to levels required to balance the carbohydrate thus presenting the opportunity to make an almost complete feed for pigs (Boonnop et al., 2009) Sengxayalth and Preston, (2017a) reported an increase in true protein from 2 to 12% in dry matter (DM) of the cassava pulp Agreement with Vanhnasin et al., (2016a) true protein increased from 2 to 7% in dry matter (DM) of the cassava root Similar findings were reported by Balagopalan et al., (1988) who developed a solid state fermentation process for the protein enrichment of cassava flour and cassava starch factory wastes using

the fungus Trichoderma pseudokonigii rifai Fermentation with yeast, bacteria has been

studied for reducing non-nutritional components, increasing the nutritive value of industrial by-products (Okpako et al 2008; Aderemi et al 2007; Tran Thi Thu Hong and Nguyen Van Ca 2013) Additional phosphate results in increased biomass growth of yeast and bacteria (Papagianni et al 1999) Huu and Khammeng, (2014) reported that when replacing maize with fermented cassava pulp containing 13% crude protein (DM basis), digestibility and N retention were similar to the control diet Protein enriched of cassava root (PECR) could provide in pig diets up to 25 to 28% of the dietary protein in a diet based on cassava pulp (or ensiled root), replacing ensiled taro foliage (Vanhnsin and Preston, 2016b) or soybean meal (Sengxayalth and Preston, 2017b) It similar to the growth response in pigs reported by Phuong et al., (2013) for cassava pulp enriched from 3 to 5.5% true protein using

agro-the fungus Aspergillus niger

The local feed used in smallholder systems for pigs include rice by-products, planted feeds and various green plant materials (ILRI 2002) However, the local feed contain low nutritive value Women typically are the key persons in this effort, and, with traditional

Center for International Agricultural Research 2010) Farmers have little knowledge on optimizing use existing feed resources, the growth rate of the pig only 100 to 120 g/day if depend on local feed staffs In commercial complete feeds, the most common protein sources

are fish meal and soybean meal These feedstuffs provide high quality protein for pigs, but they are imported and are expensive Due to their high price, such protein sources cannot be used by smallholder farmers (Phengsavanh et al., 2010) So, improving nutritive value of local feed that is abundance in their area especially the application of microorganism fermentation

it is possible to improve the nutritive value of local feed and its utilization as diets for local pigs in Laos, which helps in reducing feed cost and bringing economic benefits to the farmers

in rural area

2 OBJECTIVES

The overall aim of this thesis was to improve nutritive value of cassava roots by

fermentation yeast (Saccharomyces cerevisiae), Urea and di-ammonium phosphate additive

and its utilization as protein source in the diets of Moo Lath pigs Specific objectives were to:

 To study nutritive value of casssava root by fermentation yeast (Saccharomyces

cerevisiae), Urea and Di-ammonium phosphate additive

 To study the limiting factor to the synthesis of true protein from crude protein in the fermentation of cassava root

 To evaluate the use of protein-enriched cassava root as partial replacement of taro silage in a ensiled banana stem - based diet fed to Moo Lath pigs

• The protein-enriched cassava root as a partial replacement for taro silage in a diet will

be improve growth performance of Moo Lath pigs

4 SIGNIFICANCE/INNOVATION OF THE DISSERTATION

The innovations of the thesis study are:

This thesis is the output from four experiments; of which two experiments focus on the improving nutritive value of carbohydrate-rich (cassava root) feeds is by solid-state

fermentation Yeast (Saccharomyces cerevisiae), Urea and Di-ammonium phosphate (DAP)

additive and creating database on nutritive values, including chemical composition profile of

protein-enriched cassava root (Manihot esculenta Crantz); one on growth performance and

other one on feed intake; digestibility and N balance in local pig (Moo Lath pig) The results from experiments can help the people particularly rural farmers to understand more about the utilization of locally available feed resources for improving pig performance Despite the abundance of cassava root in Laos, the application of microorganisms, particularly yeast

(Saccharomyces cerevisiae), to enhance its nutritional and economic values to feed livestock

and, consequently, increase farming profitability, and provide jobs for the masses is yet to attract serious attention Additionally, there is very minimal biotechnological application of

yeast (Saccharomyces cerevisiae) in the production of protein-enriched agro-industrial

products in Laos This present study therefore aimed to assess the application of in yeast

(Saccharomyces cerevisiae) enriching protein content of cassava root by solid-state

fermentation This process of solid-state fermentation provides a means of converting cassava root into useful feed for the production of pig High protein forages such as taro foliage can be good sources protein in diets One important local feed is banana pseudo-stem contain the

sugar Feeding systems based on replacing taro (Colocasia esculenta) silage with proteinenriched cassava root (PECR) improved the nutritive value of a banana stem (Musa

sapientum Linn) based diet and have a great potential for increasing pig productivity The root

is composed of highly digestible carbohydrate in the form of starch with little fiber; the foliage is rich in protein and nutritive value of banana stalk could be improved by treated with indigenous microorganism And utilizing effectively those feed resources for pigs as a main protein sources (ensiled taro foliage and PECR) at small scale household in Laos Main findings were: Improving nutritive value of cassava roots by fermented with yeast, urea and DAP and PECR can be provided at highest 25% in a diet of ensiled cassava root, ensiled taro foliage and ensiled banana stem, led to increases in feed intake, diet digestibility and N retention in native Moo Lath pigs These criteria declined linearly when the proportions of PECR were 50% and 75% of the diet DM

CHAPTER 1 LITERATURE REVIEW

1 PIG PRODUCTION IN LAOS

1.1 ROLE OF PIG PRODUCTION

Pigs are widely kept throughout the country of Lao PDR, with 77 percent of all households involved in pig production (FAO, 2016) Small-holder pig farming systems play

an important role in food security and improving the livelihood of rural families They contribute a source of family income, festivals, paying a debt or as a savings bank, providing employment, buying food and medication and paying tuition fees for children) and access to markets (CelAgrid, 2006) Pigs as they adapt well to the foraging system (Phengsavanh et al., 2011) Pigs are easy to raise, easy to sell, can be confined in a small area, can covert a variety

of crop and kitchen wastes and can bring about higher incomes compared to ducks or chickens etc (Steinfeld, 1998; Ngo Thuy Bao Tran and Brian, 2005ab) Pigs manure can be used as fertilizer for cropping and animal feed which can be a source of protein for growing pigs (Lemke et al., 2002) Riethmuller et al., (2002) reported that farmers in Vietnam sell pig manure to plantations for fertilizing and improving the soil quality A survey in Laos showed that 60 pigs can produce enough manure for one hectare of fish pond in which fish stock is around 40,000 heads and can produce up to 4,000 kg of fish/year (Hoffmann, 1999) Pigs providing employment, which account for more than 50% of total family income in the remote areas of Northern Lao PDR

Devendra, (1993) added that in Southeast Asia, pig production can play three important functions, namely: (1) the diversification of resources and the reduction of socioeconomic risks, (2) the promotion of linkages between systems and resource components (land, water, crops, and animals), and (3) the generation of value added products (e.g., the recycling of fibrous crop residues to produce meat, and the use of manure) Recently, the population in Laos increase protein consumption from animal, the meat consumption is required 57 kg/capita (pork is 14.6 kg/capita) DFL, (2017) showed In 2018-2020, the needs

of the meat consumption is estimated 60-70 kg/capita, pork is needs 15.6-16.8 kg/capita, approximately of 58% of the people preferred to consume pork it high demand than other meat consumption (table1)

Table 1 Number of meat consumption in 2017 of Lao PDR

No Livestock Amount

(head)

Consumption (million kg)

Meat consumed (%)

Table 2 Statistic of livestock population in Laos (2010-2016)

No Animals Livestock population in 7 years (Thousand head)

Figure 1 Number of pigs in Laos from 2010-2016

Animal statistics from Ministry of Agriculture, Forestry and Fishery or MAF, (2015) showed that a large numbers of pigs are in the Northern region from 2005-2010 and then the population of pig was increased in the southern region between years 2011-2015 (MAF, 2017) table 3; figure 2

Table 3 Pig population in Laos (2005-2015)

Years Northern region Central region Southern region

Figure 2 Characteristic of pig in northern, central and southern in 2005-2015

1.3 PIG PRODUCTION SYSTEM IN LAOS

Pig production in Laos can be classified into three main categories:

1.3.1 Commercial pig production

Which is small compared with either smallholder or semi-commercial pig production However, during the last 10 years the number of commercial pig farms has increased 645 farms in Laos particularly near the population centres in the Mekong corridor (MAF, 2017) due to high demand for pork The pigs used are imported breeds, such as Large White, Landrace and Duroc Feeding is based on concentrate and bagged commercial feed Rice bran

is used to supplement finishing pigs Some commercial farms formulate and produce their

own feed

1.3.2 Semi-commercial pig production

Which is mostly found near population centres In this production system, breeds with imported breeds such as Landrace, Large White and Xingjin (from China) are commonly used The pigs are housed, and are fed with a mixture of locally available feedstuffs (rice bran, broken rice, cassava root and maize) and concentrate for 3-4 months to reach a marketable weight of 90-100 kg (Wilson, 2007)

cross-0 200000 400000 600000 800000 1000000 1200000 1400000 1600000

1.3.3 Smallholder pig production

Considerable numbers of pigs are raised by smallholders using three different production systems: a) free-scavenging, b) semi-scavenging and c) year-round confinement (Phengsavanh et al., 2011)

school fees Pigs can also have a social function, gift or as food during community events

Keeping scavenging pigs requires minimal inputs and un-investment, with concentrated feed

or vaccines Farmer preferred to kept native pig 1-3 head per household in the village and it

accounts for 96% of all Lao pig production Feeds are poor quality and additional feed are given occasionally, as led to growth rates tend to be slow and it may take 15 months to reach

a weight of 40-50kg (Kennard, 1996), for sows only have 1 or 2 litter per year with 6-8 piglets

per litter This system can also lead to ineffective disease control (Kagira et al., 2010), as infectious diseases of classical swine fever (CSF) (Conlan et al., 2008; Osbjer, 2006;

Phengsavanh, 2006)

Photo 1 Local pigs are allowed to scavenge freely all year round

b Semi-scavenging system:

Common in remote areas with cash crop cultivation This is commonly occurred in smallholder situation near population centers, Pigs are only allowed to scavenge freely after the main crops have been harvested During the free-scavenging period, farmers provide the pigs with a small amount of feed each day, while the main part has to be found by the pigs themselves During the crop planting season, pigs are confined either in pens or enclosures near the village or close to crop production areas (photo 2) During this period pig owner provide feed such as rice bran, maize and cassava, and green leaves which are available in the

forest Phengsavanh et al., (2011), on fallow areas, or along stream banks This system is used

for both piglet production and for fattening pigs (Phengsavanh and stur, 2006) Farmers used

native pigs combinate with improved breed’s 3-4 heads per households generally in the village with penned and are given improved feeds and accounts for 3% of all Lao pig production in this system (Vongthilath and Blacksell, 1999)

Photo 2 Local pigs in pen

c Year-round confinement system:

Common in areas close to the district centre There are two types of confinement: pens and enclosure, and pigs are commonly kept confined throughout the year The penning system is used to fatten pigs for sale, while the enclosure system is used to keep pigs away from the crops and improve village sanitation In this system, farmers use exotic and crossbred pigs, feed concentrate to both piglets and growers and provide regular vaccination

and de-worming Pigs in enclosures are fed traditional feeds such as rice bran, maze, cassava and green plant material (photo 3)

Photo 3 Feed stuffs available in farm condition

1.3.4 Main problems in smallholder pig production systems

The main problems in smallholder pig production systems in Laos are low growth rates, outbreaks of disease and high mortality of piglets These problems are common in free-scavenging systems and in semi-scavenging systems The management practices in these systems rely on traditional methods, with poor feeding and no vaccination against epidemic pig diseases The feeding system varies depending on season In the rainy season, which is the cultivation period, pigs are only fed once a day or are only fed when farmers do not work in the field In the dry season, when crop harvesting has been completed, pigs are fed twice a day and the feed mainly contains agricultural by-products Confinement in enclosures or pens allows better risk management However, a good road that improves market access may also lead to exposure to epidemics of disease due to animal movement and uncontrolled visits

from animal and meat traders (Chittavong et al., 2012; Phengsavanh et al., 2011)

1.3.5 Important points to improve smallholder pig production system

The problem of smallholder pig production as: disease epidemics, high piglet mortality, poor growth rate, and high labor demand Policies, strategies, plans, and regulations regarding pig production are developed by the Department of Livestock and Fisheries (DLF) In addition, the National Agricultural and Forestry Research Institute (NAFRI) has discovered possible ways to improve livestock production The two main

priorities for which urgent solutions are needed are nutrition and breed improvement More than 85% of small scale producers keep local breeds (DLF, 2017) which make productivities low and not attractive to farmers to invest in pig rising In terms of nutrition, the small scale producers still practice with scavenging traditional systems or semi confinement and provide animals with low nutrient diets which normally come from agriculture by-products especially the content of crude protein in the rice bran was very low indicating that the quality of the bran was low with probably a high percentage of husks from village rice mills To improve pig production performance, improving nutrition situation for pigs based on locally available

resources should be given priority High protein forages such as taro (Colocasia esculenta),

water spinach, mulberry leaves, sweet potato vine, and duckweed could be added to, or replaces other expensive sources of protein such as fish and soy bean meal Almost local feed stuffs in farm condition is energy sources such as: maize, cassava root, sweet potato, rice

bran, broken rice and banana pseudo (musa spp) stem If pigs were fed a high-quality such

that the energy supplied the authors cautioned that pigs’ intake of nenergy should be restricted

to avoid excess accumulation of body fat that will be effected to pig cost (Keonouchanh et al., 2011) One way to improve the protein content of carbohydrate-rich (cassava root) feeds is by solid-state fermentation with fungi and yeasts (Araujo et al., 2008; Hong and Ca, 2013)

1.4 LOCAL PIG BREEDS RAISED BY SMALLHOLDERS

The native breeds have been preliminarily classified into four types (Moo Lath, Moo

Chid, Moo Hmong and Moo Deng) by (Keonouchanh et al., 2011; Vongthilath and Blacksell,

1999), based on phenotypical differences recorded in a field survey focusing on general characteristics, production performance and carcass composition This classification is similar

to previously reported classes of pig breeds in Lao PDR (FAO, 2007; Wilson, 2007) The advantage of native pigs is that they are hardy, resistant to disease, achieve early sexual maturity and are adaptable to harsh rural environments with low inputs (Phengsavanh and Stur, 2006)

1.4.1 Moo Lath

The first type is mostly present in upland areas (Luang prabang, Oudomxay and Xaysomboun Province), but lso in some lowland territories (Saravane and Savannakhet Povinces) This breed is quite bigger than the first type Body length is 85-100 cm; the girth and height are 84-102 cm and 51-70 cm, respectively The ears are short and directed forward

and the face is strait Legs and the front of the face are white

The age of the first oestrus is between 189-586 days with 39 kg of body weight These varying data depend on typical extensive keeping systems, where management is nearly negligibly The weight of mature sows is about 47-61 kg, and the youngest age of first farrowing is around 360 days Depending on management systems, sows have 1.5-1.8 liters per year and 7-8 piglets per litter Normal weaning period is 60-90 days with an average of 9.5 kg of piglet's weaning weight Mature males of this type have also lower body weight (25 kg) than

females and maximum body weight of boars can reach 30-50 kg (Keonouchanh et al., 2011)

Photo 4 Moo Lath

1.4.2 Moo Chid, Moo Markadon or Moo Boua

The second type is scattered countrywide The pig is of relatively small size compared

to other types existing in the country Its body length, circumference of the girth and the height is 75-92 cm, 72 85 cm and 46-54 cm, respectively The ears are small, short and directed forward The coat colour is mainly black and legs are white Gilts show first oestrus

at the age of about 6 months (between 182-197 days) and with body weight of 21-31 kg The weight of mature sows is about 42-48 kg The age at first farrowing is approx 360 days There are 1.5 liters per year with 7-8 piglets per litter Normal weaning age is three months with an average weaning weight of the piglets of 7.8 kg Mature males have lower body weight than females, i.e the average body weight is 20.5 kg at the age of 170-200 days, and

the maximum body weight of boars is between 18-30 kg (Keonouchanh et al., 2011)

Photo 5 Moo Chid, Moo Markadon or Moo Boua

1.4.3 Moo Nonghad or Moo Hmong

This type is specifically found in the Nonghad district, Xienkhouang province It is quite big with body length of 100-105 cm, girth circumference of 115-130 cm and height of 55-76 cm The body is mostly black and in the abdominal region rose coloured It has a short and bent face, and the medium sized ears are directed forward

The age at the first oestrus is between 150-180 days with 30-40 kg of body weight The weight of mature sows is between 65 and 85 kg The age at first farrowing is 10-11 months There are 1.5-1.8 liters per year with 7-10 piglets per litter Normal weaning age of the piglets is 2-3 months and the weaning weight is 8 kg on average The maximum body

weight of boars is between 60-80 kg (Keonouchanh et al., 2011)

Photo 6 Moo Nonghad or Moo Hmong

1.4.4 Moo Deng or Moo Berk

The fourth type is e asy to identify due to its phenotype It has brown colour and is apparently larger than any other pig breed existing in Lao PDR This pig is a well-adopted and

stabilized cross-bred one (Berkshire×local pig) It is kept only in the southern part of Laos, particularly in Mounlapamok and Khong districts, Champasack Province Body length, circumference of the girth and the height are 88-120 cm, 84-116 cm and 60-70 cm, respectively It has a short and bent face, and large sized hanging ears The weight of mature sows is around 65-90 kg, and the first farrowing age is between330-360 days They have 1.5-1.8 liters per year with 7-10 piglets per litter Piglets are normally weaned at the age

of 2-3 months and a weight of 8.5 kg The maximum body weight of boars is similar to sows

(Keonouchanh et al., 2011)

Photo 7 Moo Deng or Moo Berk

Table 4 Classification of phenotype characteristics and reproductive performance of native pigs

produced under smallholder farm (SHPF) conditions in Lao PDR

Native breed

Moo chid/Maradon /Boua

Moo Lath Moo Nonghad/

Hmong

Moo Deng/Berk

Native breed

Moo chid/Maradon /Boua

Moo Lath Moo Nonghad/

Hmong

Moo Deng/Berk

Larger ears and directed forward

Large hanging ears

Body and color Black coat,

white legs

Straight face, Black coat, leg and front

of face are white

Short and bent face Mostly black coat

Brown color, bent face

Source: Keonouchanh et al., (2011)

2 REQUIREMENT OF PROTEIN AND AMINO ACID FOR GROWING PIGS

Generally, the protein supply in pig diets is formulated on the basis of crude protein (CP), which refers to the nitrogen content * 6.25 (NRC, 2012) Proteins are composed of amino acids, and a good quality protein should provide the 10 essential amino acids in proportions required for normal body functions of pigs Amino acids are critical nutrients required by all classes of pigs for maintenance, growth, gestation and lactation The quantitative requirements of amino acids in pigs varies with weight and physiological stage (table 5) In general, the first limiting amino acid for pigs is lysine In growing pigs, the lysine requirement gradually decreases with increasing age (BW) from 1.2% of DM (at <20 kg BW)

in young pigs to around of 0.7% of DM (at 100 kg BW) in older pigs (NRC, 1998)

Table 5 Dietary amino acid requirements of growing-finishing pigs (NRC 1998)

Body weight of pig (kg)

11-25 kg LW required 21% digestible protein In growing pig showed greatly ADG on the diet with 15% CP and for finisher 11% CP In the study of growing of local Mong Cai pig required a CP in diet at 13-14% in DM (Ly et al., 2003; Pham et al., 2010) Local pigs required protein lower than exotic pig, in this case, Sivilai and Preston., (2017) concluded that the growth rate and feed conversion were optimized with 12% crude protein in the diet DM, with 80% of CP derived from ensiled taro leaves and petioles Anugwa and Okwori, (2008) studies on two genetic groups of pigs {local and crossbred (local*large white)} at body weight of 7.3 and 10.2 kg, respectively and two protein levels (12 and 16% of CP) showed that local pigs performed better than crossbred on the lower CP (12%) in the diet while crossbreds performed better than local pigs on the 16% CP in diet The reported by Devendra and Clyde Parris, (1970) on the optimum protein levels for growing and finishing pigs in a tropical environment showed that there were statistically significant differences in daily live weight gain and feed conversion efficiency between pigs fed a diet containing 16% CP and those receiving 18% CP but no differences in response were found to diets containing 12 and 13% CP Feed conversion efficiency improved with increased dietary levels of CP

3 FEED STUFFS FOR PIG IN LAOS

Feedstuffs used in smallholder systems for pigs include rice by-products (e.g rice bran, broken rice and distiller’s waste), planted feeds (e.g maize and cassava), and various

green plant materials By-products and planted feeds mainly supply energy and green plant

materials (e.g sweet potato leaves, cassava leaves and taro leaves) mainly supply protein in

pig diets The composition of dietary components in selected locally available feedstuffs is presented In commercial complete feeds, the most common protein sources are fish meal and soybean meal These feedstuffs provide high quality protein for pigs, but they are imported and are expensive Due to their high price, such protein sources cannot be used by smallholder farmers Based on origin, the main feed ingredients that are used in smallholder pig

production systems in Laos can be categorised into the following groups: Rice by-products:

Rice is the main staple crop in Laos Rice by-products such as rice bran and broken rice are commonly used for feeding pigs In 2016 Laos produced 4.1 million ton of rice, which, as a by-product, provided around 4.000 ton of rice bran The use of rice bran is common in pig diets, in particular by smallholder pig producers The diet may include from 60% up to 100% rice bran (Chittavong et al., 2012; Phengsavanh et al., 2010, FAO, 2016) Broken rice has a high energy content and a low fibre content While bananas are grown for human consumption the pseudo stem is often used traditionally as part of the pig diet and is used as

an energy source in pig diets Broken rice is usually fed together with vegetables and leaves from cassava, yam, taro and sweet potato All ingredients are boiled together in water, and are mixed with kitchen waste before being fed to pigs Rice distiller’s waste is the by-product from rice wine production, and is a good energy and protein source for pigs However, this by-product is only available occasionally, and is used as a supplement for fattening pigs

3.1 LOCAL FEED AVAILABLE FOR PIG

Taro (Colocasia esculenta) and cassava root are both human consumption and animal

feeds However, there are difficulties in using as animal feed such as: boiling, fresh and ensiled Taro its content of oxalic acid cause the mouth and throat of animals consuming it to itch Oxalic acid may be present in the corm and especially in the leaves, some local farmers traditionally reduce the oxalate content by boiling While, cassava root have been used successfully for animal feed as energy sources The other plant farmers often use fresh banana stem as feed for pigs by mixing it with rice bran as energy sources for pigs in Laos

3.1.1 Taro (Colocasia esculenta)

3.1.1.1 Yield and availability

Taro (Colocasia esculenta (L.) Shott) is a member of the Araceae family (AFRIS,

2005; Lee, 1998) that originated in India and South East Asia It is presently cultivated in many tropical and subtropical countries, primarily as a food for its edible corm, and secondarily as a leaf vegetable Taro can be commonly found growing wild in the Mekong Delta, particularly on the banks of ponds and along rivers and canals Wild taro is very easy to grow, develops fastest in wet land and is highly resistant to pests and diseases Taro is a tropical food crop with high potential because of the high yield of the roots (or corms) and foliage In Laos, there are area of taro and root planted totally 189.201 ha, with yield 14.78 ton/ha (LSB, 2013) The total leaf DM biomass yield was 1.483, 1.341 and 691 kg/ha when harvested at 4, 6 and 24 weeks, providing CP 260g/kg DM, Ash 145 g/kg, 206 g/kg DM, with average tuber DM yield was 2.732 kg/ha (Kaensombath and Frankow-Lindberg, 2012) Taro foliage can be harvested at 30 days interval throughout the year with biomass yield 250 tones/ha/year of fresh matter (Ngo Huu Toan and Preston, 2007; Hang and Kien, 2012) In wet condition, taro can be harvested after 10-12 months, in dryland 12-15 months For the corm yields range from 5 to 6 t/ha, but a good crop on fertile soil is around 12 t/ha (Safo Kantaka, 2004) The average taro yield in Africa is about 5.1 t/ha as compared with 1.6 t/ha

for maize (Raemaekers, 2001)

3.1.1.2 Nutritive value

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 animals, especially for pigs, because of its nutritional quality The Taro chemical composition is variable depending on varieties, growing conditions, fertilizer application and processing Taro can be a potential protein source for animals, especially pigs due to the good nutritional quality of the leaves and foliage (DM basis): Ensiled taro foliage the crude protein (CP) content from 15% to 17%; 18.1 Ash, Dry matter (DM) 14.6 % to 26; 10.7% ether extract (EE), 39.8% nitrogen-free extract (NFE), 1.74% Ca, and 0.58% P (FAO, 1993) The chemical composition of fresh taro leaves (DM basis): 16% DM, 25% CP, 18.3% CF 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

Table 6 Chemical composition of taro (Colocasia esculenta) in DM basis

DM,% CP,% Ash,% CF,% ADF NDF pH Ca Oxalic

*Malavanh et al., (2008); ** Sivilai and Preton, (2017);*** Rodriguez et al., (2006)

3.1.1.3 Constraints of using taro (Colocasia esculenta)

Taro (Colocasia esculenta), also known as "Old Cocoyam", is a wetland crop in many

tropical and subtropical areas of the world, cultivated mainly for root (corm) production Before the corm can be eaten, the traditional practice is to cook it so as to break down the needle-like calcium oxalate crystals which are present in all parts of the plant According to