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 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 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 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, 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, and 14 days In experiment A CRD was used with 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 and 3h after preparing the substrates and every 24h until end of day (0, 3h, 1, 2, 3, 4, 5, and day) Experiment (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 (chapter 2) The pH decreased with iv 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 days The level of CP after mixing the substrate and additives was 10.35% in DM and did not change over the days of fermentation TP in the substrate increased from 2.37 to 6.97% in DM as the fermentation time increased from zero to 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 days in the anaerobic condition followed by a slower rate of fall to reach almost the same final value after days, as for the aerobic condition Experiment (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 (chapter 4) Four castrated male Moo Lath pig, weighing on average 15 kg were allotted at random to 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 PROBLEM STATEMENT OBJECTIVES HYPOTHESES SIGNIFICANCE/INNOVATION OF THE DISSERTATION CHAPTER 1: LITERATURE REVIEW PIG PRODUCTION IN LAOS 1.1 ROLE OF PIG PRODUCTION 1.2 PIG POPULATION 1.3 PIG PRODUCTION SYSTEM IN LAOS 1.3.1 Commercial pig production 1.3.2 Semi-commercial pig production 1.3.3 Smallholder pig production 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 REQUIREMENT OF PROTEIN AND AMINO ACID FOR GROWING PIGS 16 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 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 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 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 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 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 value of cassava root However, the findings reported in ecperiment (chapter 2) the data presented that ammonia levels were minimal The residual NPN it could still be in the form of urea, the hydrolysis of which by urease might be reduced due to the rapid fall in pH with the onset of fermentation However, the activity of urease is inhibited at low pH (Kay and Reid, 1934), which falls rapidly when the cassava root is fermented The pH decreased with fermentation time, it is suggested that the incomplete conversion of urea-N and ammonia-N to yeast protein was because of incomplete hydrolysis of urea to ammonia due to action of urease being inhibited by the fall in pH during the fermentation The problem confirmed, in the studies reported so far, is that not all the added nitrogenous compounds (urea and DAP) were converted to “true” protein, the levels of which never exceeded some 50 to 70% of the “crude “ protein in experiments with yeast-fermented cassava root (Vanhnasin and Preston, 2016a) and cassava root pulp (Sengxayalth et al., 2017a) It has been shown conclusively (Vanhnasin et al., 2016a; Manivanh et al., 2016; Sengxayalth and Preston, 2017a) that when cassava pulp (or cassava root) is fermented with urea, DAP and yeast, not all the NPN is converted to true protein, 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 (AA) There is evidence in humans that NPN in the form of ammonium chloride was partly converted to amino acids by the action of microbes in the small intestine (Patterson et al., 1995) and Stein et al., 1996) Colombus et al., (2014) infused urea into the cecum of pigs fed a diet deficient in the amino acid valine, and showed that it was recycled to the small intestine where it was converted by bacteria into amino acids with the result that N retention was increased These findings were corroborated by Mansilla et al., (2015) A solid-state fermentation with yeast was used to increase the protein content of cassava pulp The organism was grown in a cassava pulp medium with (% in DM) 4% urea, 1% DAP and 2% yeast, with and without steaming before fermentation The fermentation time was 0, 3, and days Fermentation increased the true protein of the cassava pulp from to 12% in DM over the day period; corresponding values for increases in crude protein were 9.5 to 18.4% It appeared that only some 60% of the NPN (urea and DAP) had been converted to yeast protein The composition of the residual NPN is not known but presumably was in the form of ammonium salts or related compounds The apparent increases in crude protein content during fermentation appeared to be due to the loss of 40% of the DM of the substrate during fermentation which resulted in “enrichment” of the crude protein fraction during the course of the fermentation There were no advantages in prior steaming of the pulp before fermentation (Sengxayalth et al., 2017a) 97 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 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) Most pigs in rural areas of Lao PDR are raised in traditional, low input, free and semi-free scavenging systems, where the pigs are allowed to scavenge freely for feed all the year round or after the main crops have been harvested (Phengsavanh et al., 2010) In Lao PDR as in most tropical countries the most widely grown crops are primarily sources of carbohydrate (eg: rice, sugar cane, cassava) Few crops are grown specifically as sources of protein As a result, protein rich feeds such as soybean meal are imported in order to produce balanced diets for livestock especially pigs and poultry An alternative approach that has been studied by several researchers is the solid state fermentation of carbohydrate-rich byproducts from these crops using combinations of fungi and yeast (Phiny et al., 2012; Phong et al., 2013; Khempaka et al., 2011; Hong and Ca, 2013) in order to enrich the content of protein In experiment (chapter 3) was evaluate the use of protein-enriched cassava root as partial replacement of Taro silage in a banana stem- based diet fed to Moo Lath pigs The result show that, there were positive linear responses in DM intake, live weight gain, feed conversion when protein-enriched cassava root partially replaced taro silage in a basal diet of ensiled banana stem fed to Moo Lath pigs The agreement with Manivanh and Preston, 2015 reported that the growth rate and DM feed conversion were increased by 46% and 5.7% by replacing Taro silage with protein-enriched cassava root The improvement in pig performance by replacing ensiled Taro foliage with protein-enriched cassava root (PECR) could be a reflection of the higher energy value in the latter (% crude fiber in DM of 3.7 in cassava root compared with 11% in Taro foliage (Hang et al., 2015) The positive effect of protein-enriched cassava root on live weight gain is similar to the growth response in pigs reported by Phuong et al., (2003) for cassava pulp enriched from to 5.5% true protein using the fungus Aspergillus niger Huu and Khammeng, (2014) 98 replaced maize with fermented cassava pulp containing 13% crude protein (DM basis) and reported similar digestibility and N retention as in the control diet Part of the improvement in 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 proteinenriched product could provide up 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) Growth rate of the pigs was increased by 16% from 150 to 175 g/day, when PECR replaced one third of the ensiled Taro foliage With complete substitution of ensiled taro foliage by PECR the growth rate decreased to 128 g/day DM feed conversion was best (3.47) with 27% of the dietary protein from PECR and poorest (4.21) when PECR was the only protein supplement (Vanhnsin and Preston, 2016b) Agreement with (Hang et al., 2018 Not yet public) using combination of two microorganisms (B.sub, A.niger) as origin yeast for fermentation cassava byproduct and replaced basal diet, the result reached higest at 25% level of N retention and Body weight and digestibility of CF reducing when increasing level of cassava byproduct from to 50% (DM) However, higher proportions of the protein-enriched feed in the diet led to reduced growth performance with almost no growth at 100% replacement of the taro/soybean protein It was reported that only 50-60% of the added non protein nitrogen (NPN) [from urea and DAP] was recovered as true protein which implied that more than one third of the original NPN remained, possibly in the form of ammonium salts or intermediary products formed in the process of yeast growth It was proposed (Sengxayalth and Preston, 2017b) The improvement in growth rate with 30% protein-enriched cassava pulp protein in the diet was the result of amino acid synthesis by bacteria in the small intestine using the residual dietary ammonia as substrate (Colombus et al., 2014) It is proposed that the improvement in growth with 30% substitution of soybean protein by PECP arose from microbial synthesis of amino acids in the intestine from dietary NPN (ammonia) present in the PECP and that the precipitous decline in feed intake and in growth rate, when the PECP exceeded 30% of the dietary protein, was the result of the presence of excess ammonia, and/or its metabolites, due to their incomplete conversion to yeast protein in the fermentation (Sengxayalth and Preston, 2017b) and that at higher levels of substitution of PECP the severe depression in feed intake and in growth was 99 due to the toxicity caused by the residual non protein nitrogen (NPN) compounds in the fermented pulp/root The same as reported of Hong et al., (2017) higher proportions of PECP in the diet (6.8 to 16.5%), there was a linear depression in live weight gain (overall reduction of 25%) It is suggested that the reduction in live weight gain with more than 4.5% PECP in the diet DM may have been caused by sub-acute toxicity caused by residual NPN compounds in the protein-enriched cassava pulp because of incomplete conversion of the NPN to yeast protein Which is supported by the studies in experiment (chapters 4) It was to be expected that the growth rate of the pigs would have declined linearly as the proportion of true protein in the diet was reduced On the contrary, the response to increasing protein-enriched cassava root level was curvilinear with feed intake and N retention increasing by 33 and 28%, respectively for the diet with 25% protein-enriched cassava root, The issues that are raised are: (i) the benefits from 25% protein-enriched cassava root (PECR) in the diet were due to the presence of live yeast (estimated to be about 1% of the DM of the PECR25 diet) acting as a probiotic and: (ii) pigs can apparently utilize small quantities of non-protein-nitrogen through absorption of the NPN from the large intestine and subsequent recycling in the blood to the small intestine where microbes use the NPN for synthesis of amino acids (Colombus et al., 2014) Amino acid synthesis from dietary NPN could be the explanation for the positive effects on growth rate at the lower level of substitution of TS protein by protein-enriched cassava pulp, since protein was set at the limiting level of 10% of diet DM At this level of dietary protein, additional amino acids resulting from microbial synthesis of amino acids from NPN could have played a critical role in increasing growth rate (Vanhnasin et al 2016b) However, at higher rates of substitution of 50 and 75% of the TS 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 has been shown conclusively (Vanhnasin et al., 2016; Manivanh et al., 2016; Sengxayalth and Preston, 2017a) that when cassava pulp (or cassava root) is fermented with urea, DAP and yeast, not all the non protein nitrogen (NPN) is converted to true protein, 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 (AA) There is evidence in humans that NPN in the form of ammonium chloride was partly converted to amino acids by the action of microbes in the small intestine (Patterson et al., 1995) and Stein et al., 1996) Colombus et al., 100 (2014) infused urea into the cecum of pigs fed a diet deficient in the amino acid valine, and showed that it was recycled to the small intestine where it was converted by bacteria into amino acids with the result that nitrogen (N) retention was increased These findings were corroborated by Mansilla et al., (2015) Amino acid synthesis from dietary NPN could be the explanation for the positive effects on growth rate at the lower level of substitution of soybean protein, since protein was set at the limiting level of 10% of diet DM At this level of dietary protein, additional amino acids resulting from microbial synthesis of amino acids from NPN could have played a critical role in increasing growth rate However, at higher rates of substitution of 60 and 90% of the soybean protein by Protein-enriched cassava pulp, the levels of residual NPN (ammonia) 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 Replacement of protein by urea in pig diets seems an unlikely posibility, although urea is essentially non-toxic to mono-gastric animals It is toxic to ruminants in excess as their digestive process readily produces ammonia from urea, and ammonia is toxic, but in small amounts is converted by gut bacteria to amino-acids The "problem" with pig digestive tracts is that they not provide a sufficiently large habitat for bacteria that would have long enough to produce significant amounts of ammonia, and then convert it to aminoacids 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 (Grimson et al., 1971) The other factors to be taken into consideration are the possible “prebiotic and probiotic” effects arising from the live yeast and lactobacilli present in the fermented cassava pulp Thiep, (2017) unpublished data reported concentrations of yeast of million CFU/g and lactobacilli 17 million CFU/g in cassava pulp fermented with yeast, urea and di-ammonium 101 phosphate (DAP) Positive effects on N retention in Moo Lath pigs were reported by Sivilai et al., (2017) when rice distillers’ by-product and brewers’ grains were fed at 4% of the diet DM Both these dietary supplements are equally rich in live yeast and lactobacilli (Thiep, 2017) unpublished data The other interesting observation from this experiment is that N retention was only marginally depressed even with as much as 75% of the diet in the form of proteinenriched cassava root, which is in marked contrast with the report of Sengxayalth and Preston, (2017b) that when 75% of the diet was in the form of Protein-enriched cassava pulp the growth rates were reduced almost to zero The difference between the two experiments was the nature of the remaining 25% of the diet DM In the research of Sengxayalth and Preston, (2017b) this was in the form of ensiled cassava root pulp whereas in the present experiment it was a combination of ensiled taro foliage (5%) and ensiled banana pseudo-stem (30%) These latter feeds would have been much richer in vitamins, minerals and trace elements than the protein-enriched cassava root which formed the balance of the diet in the experiment of Sengxayalth and Preston, (2017b) This cannot be proved in the absence of relevant biochemical analytical data in both trials, but it is an argument for the benefits of having at least a part of the dietary protein being provided by nutrient-ich foliages such as taro GENERAL CONCLUSIONS  The true protein in cassava root increased from 2.3 to 6.87% in DM as the fermentation time increased from zero to 14 days Increasing the proportion of DAP from zero to 2% of the substrate DM increased the true protein from 5.6 to 7.3% after 14 days of fermentation 30% of the original root DM was fermented in the partial conversion of root carbohydrate to true protein after 14 days of fermentation  The level of crude protein after mixing the substrate and additives was 10.35% in DM and did not change over the days of fermentation There were no differences between the aerobic and anaerobic condition, other than a tendency for the pH to fall slightly more quickly in the first days in the anaerobic condition followed by a slower rate of fall to reach almost the same final value after days, as for the aerobic condition It is suggested that the incomplete conversion of urea-N and ammonia-N to yeast protein is because of incomplete hydrolysis of urea to ammonia due to action of urease being inhibited by the fall in pH during the fermentation  There were positive linear responses in DM intake, live weight gain, feed conversion when protein-enriched cassava root partially replaced taro silage in a basal diet of ensiled banana stem fed to Moo Lath pigs Utilization of inexpensive, locally available feed resources, 102 such as taro foliage, Banana stem and cassava root, cassava root can be improving nutritive value by fermented with yeast, urea and di-ammonium phosphate (DAP), have the potential to improve the economical efficiency of smallholder pig production in Laos Enriching the protein content of cassava root (protein-enriched cassava root) by fermenting it with urea, diammonium phosphate and yeast and including the protein-enriched cassava root at 25% in a diet of ensiled cassava root, ensiled taro foliage and ensiled banana pseudostem, led to increases in feed intake, diet digestibility and N retention in native Moo Lath pigs and improvement in 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 These criteria declined linearly when the proportions of protein-enriched cassava root were increased to 50 and 75% of the diet DM It is suggested that: (i) the benefits from the 25% protein-enriched cassava root diet may have been partially a response to its content of live yeast, the other possibility could be the increased provision of vitamins of the βcomplex from the yeast in the fermented cassava root and (ii) the results support earlier research showing that pigs are able to utilize small quantities of NPN recycled to the intestine IMPLICATION AND FURTHER RESEARCH 3.1 IMPLICATION The result can be used to local feed stuffs available for pig production in Laos Cassava (Manihot esculenta Crantz) is an annual crop grown widely in the tropical and subtropical regions is one of the main food crops for smallholder farmers in remote upland areas and it is currently the third most important crop after rice The root can be used for food and feed as well as for improving animal nutrition Cassava roots have high levels of energy (75 to 85% of soluble carbohydrate) and minimal levels of crude protein From the result of this study focus on Improving the nutritive value of carbohydrate-rich (cassava root) feeds is by solid-state fermentation yeast (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 103 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 104 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 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 REFERENCES Antai, S.P and Mbongo, P.N., 1994 Utilization of cassava peels as substrate for crude protein formation Plant Foods for Human Nutrition, volume 46, pp Araujo, L.F., Silva, F.L.H., Brito, E.A., Olivera Junior, S and Santos, E.S., 2008 Enriquecimento protộico da palma forrageira com Saccharomyces cerevisiae para alimentaỗóo de ruminantes Arquivo Brasileiro de MedicinaVeterinária e Zootecnia, Volumen 60, pp 401- 407 105 Boonnop, K., Wanapat, M., Nontaso, N and Wanapat, S., 2009 Enriching nutritive value of cassava root by yeast fermentation Scientia Agricola Volume 66 no.5 Piracicaba http://dx.doi.org/10.1590/S0103-90162009000500007 Brook, E.J., W.R and Wallbridge, A., 1969 Fermentation method for protein enrichment of cassava Biotechnology and Bioengineering Volume 11 1271 Cel Agrid., 2006 Assessment of impacts of action research on pig production systems, management FAO., 2016 FAOSTAT data Food and Agriculture Organization of the United Nation http://www.fao.org/faostat FAO., 2017 FAOSTAT data Food and Agriculture Organization of the United Nation http://www.fao.org/faostat Hang, D.T, An, T.T.X., Thuong, L., Loc, N.T., Hai, V.V., Tra, T.T.T., Hai, P.V and Ngoan, L.D., 2015 Taro (Colocasia esculenta (L) Schott): biomass yield and nutritive value for pigs Livestock Research for Rural Development Volume 27, Article #109 Retrieved August 9, 2018, from http://www.lrrd.org/lrrd27/6/hang27109.html Hong, T.T.T and Ca, L.T., 2013 The protein content of cassava residue, soybean waste and rice bran is increased through fermentation with Aspergillus oryzae Livestock Research for Rural Development Volume 25, Article #132 http://www.lrrd.org/lrrd25/7/hong25132.htm Hong, T.T.T., Lien, P.T.B., Hai, D.T., Hang, P.T and Quan, N.H., 2017 Protein-enriched cassava root pulp as partial replacement for fish meal in diets for growing pigs Livestock Research for Rural Development Volume 29, Article #184 Retrieved September 11, 2018, from http://www.lrrd.org/lrrd29/9/hong29184.html Huu, H.L and Khammeng, T., 2014 Effect of yeast fermented cassava pulp (FCP) on nutrient digestibility and nitrogen balance of post-weaning pigs Livestock Research for Rural Development.Volume 26, Article #149 Retrieved February 6, 2015, from http://www.lrrd.org/lrrd26/8/huu26149.htm 106 Inthapanya, S., Preston, T.R., Phung, L.D and Ngoan, L.D., 2017 Effect of supplements of yeast (Saccharomyces cerevisiae), rice distillers’ by-product and fermented cassava root on methane production in an in vitro rumen incubation of ensiled cassava root, urea and cassava leaf meal Livestock Research for Rural Development Volume 29, Article#220 Retrieved October 28, 2018, from http://www.lrrd.org/lrrd29/12/sang29220.html Kang, S., Wanapat, M., Phesatcha, K and Norrapoke, T., 2015 Effect of protein level and urea in concentrate mixture on feed intake and rumen fermentation in swamp buffaloes fed rice straw-based diet Tropical Animal Health Production 2015; DOI: 10.1007/s11250-015-0777-8 Kay, W.W and Reid, M.A.H., 1934 The optimum buffer pH for hydrolysis of urea by urease, and the preparation of stable urease powder Biochemistry Journal 28(5): 1798 1801 William Whittle Kay and Muriel Anne Hislop Reid1 Khempaka, S., Thongkratok, R., Okrathok, S and Molee, W., 2014 An evaluation of cassava pulp feedstuff fermented with A oryzae, on growth performance, nutrient digestibility and carcass quality of broilers Journal of Poultry Science, (51), 71 - 79 Manivanh, N and Preston, T.R., 2015 Protein-enriched cassava root meal improves the growth performance of Moo Lat pigs fed ensiled taro (Colocacia esculenta) foliage and banana stem Livestock Research for Rural Development Volume 27, Article #44 http://www.lrrd.org/lrrd27/3/noup27044.html Mansilla, W.D., Columbus, D.A., Htoo, J.K and de Lang, C.F.M., 2015 Nitrogen absorbed from the large intestine increases whole-body nitrogen retention in pigs fed a diet deficient in dispensable amino acid nitrogen The Journal of nutrition, 145 (6), 1163 1169 Ministry of Agriculture and Forestry., 2013 Agriculture Statistics 2014 Vientiane: Department of Planning and Cooperation, MAF Ngo Thuy Bao Tran and Brian, O., 2005a Surveys on small-scale urban and periurban livestock production in Chau Doc Town Master thesis http://www.mekarn.org/msc200305/theses05/Trancont.htm 107 Ngo Thuy Bao Tran and Brian, O., 2005 Surveys on small-scale urban and periurban livestock production in Long Xuyen City Master thesis http://www.mekarn.org/msc2003-05/theses05/Trancont.htm Oboh, G and Akindahunsi, A.A., 2005 Nutritional and toxicological evaluation of Saccharomyces cerevisiae fermented cassava flour Journal of Food Composition and Analysis, Volume.18, pp 731 - 738 Obadina, A.O., Oyewole, O.B., Sanni, L.O and Abiola, S., 2006 Fungal enrichment of cassava peels proteins African Journal of Biotechnology Volume No 3: 302-304 Phengsavanh, P., Ogle, B., Stur, W., Frankow-Lindberg, B.E and Jan Erik Lindberg, J K., 2010 Feeding and performance of pigs in smallholder production systems in Northern Lao PDR http://lad.nafri.org.la/fulltext/2722-0.pdf Phiny, C., Preston, T.R., Borin, K and Thona, M., 2012 Effect on growth performance of crossbred pigs fed basal diet of cassava root meal and ensiled taro foliage supplemented with protein-enriched rice or fish meal Livestock Research for Rural Development Volume 24, Article #65 Retrieved August 9, 2018, from http://www.lrrd.org/lrrd24/4/phin24065.htm Phong, N.V., Ly, N.T.H, Nhac, N.V and Hang, D.T., 2013 Protein enrichment of cassava byproducts using Aspergillus niger and feeding the product to pigs Livestock Research for Rural Development Volume 25, Article #130 http://www.lrrd.org/lrrd25/7/hang25130.htm Polyorach, P., Wanapat, M and Wanapat, S., 2012 Increasing protein content of cassava (Manihot esculenta Crantz) using yeast in fermentation Khon Kaen Agriculture Journal 2012; 40 (supply 2): 178 - 182 Polyorach, S., Wanapat, M and Wanapat, S., 2013 Enrichment of protein content in cassava (Manihot esculenta Crantz) by supplementing with yeast for use as animal feed Emirates Journal Food Agriculture 2013, (25), 142 - 149 Poungchompu, O., Wanapat, M., Wachirapakorn, C., Wanapat, S and Cherdthong, A., 2009 Manipulation of ruminal fermentation and methane production by dietary saponins and tannins from mangosteen peel and soapberry fruit Animal Nutrition 2009; (63), 389 - 400 Phengsavanh, P., Ogle, B., Stur, W.E.F-L.B and Lindberg, J.E., 2011 Smallholder Pig Rearing Systems in Northern Lao PDR The Asian Australasian Journal of Animal 108 Science, 24 (Doi: 10.5713/ajas.2011.10289), 6:867-874.Technical Report No 10, August 1997, Lao Swedish Forestry Program, Louang Prabang, Lao PDR Press Washington, D.C Rodríguez, L and Preston, T.R., 2009 A note on ensiling the foliage of New Cocoyam (Xanthosoma sagittifolium) Livestock Research for Rural Development Volume 21, Article #183 http://www.lrrd.org/lrrd21/11/rodr21183.htm Sengxayalth, P and Preston, T.R., 2017a Fermentation of cassava (Manihot esculenta Crantz) pulp with yeast, urea and di-ammonium phosphate (DAP) Livestock Research for Rural Development Volume 29, Article #177 RetrievedAugust 17, 2018, from http://www.lrrd.org/lrrd29/9/pom29177.html Sengxayalth, P and Preston, T.R., 2017b Effect of protein-enriched cassava pulp on growth and feed conversion in Moo Laat pigs Livestock Research for Rural Development Volume 29, Article #178 Retrieved August 17, 2018, from http://www.lrrd.org/lrrd29/9/pom29178.html Steinfeld, H., 1998 Livestock production in the Asia and Pacific region, current status issues and trends In: hursey BS (ed), World Animal Review 90-1998/81 Animal Production and health Division, FAO (Food and Agriculture Organization of the United Nations), Rome, Italy Thongkratok, R., Khempaka, S and Molee, W., 2010 Protein enrichment of cassava pulp using micro organisms fermentation techniques for use as an alternative animal feedstuff Journal of Animal and Veterinary Advances 9(22), 2859-2862 Trang district, Takeo province Cambodia Vanhnasin, P and Preston, T.R., 2016a Protein-enriched cassava (Manihot esculenta Crantz) root as replacement for ensiled taro (Colocasia esculenta) foliage as source of protein for growing Moo Lat pigs fed ensiled cassava root as basal diet Livestock Research for Rural Development Volume 28, Article #177 Retrieved August 17, 2018, from http://www.lrrd.org/lrrd28/10/vanh28177.html Vanhnasin, P., Manivanh, N and Preston, T.R., 2016b Effect of fermentation system on protein enrichment of cassava (Manihot esculenta) root Livestock Research for Rural Development Volume 28, Article #175 Retrieved December 18, 2016, from http://www.lrrd.org/lrrd28/10/vanh28175.html 109 Wanapat, M and Kang, S., 2016 Cassava chip (Manihot esculenta Crantz) as an energy source for ruminant feeding Animal Nutrition Journal (2016), doi: 10.1016/j.aninu.2015.12.001 Wanapat, M., Pilajun, R., Polyorach, S., Cherdthong, A., Khejornsart, P and Rowlinson, P., 2013 Effect of carbohydrate source and cottonseed meal level in the concentrate on feed intake, nutrient digestibility, rumen fermentation and microbial protein synthesis in swamp buffaloes Asian-Australia 110 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 http://www.lrrd.org/lrrd30/5/noup30094.html 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 http://www.lrrd.org/lrrd30/9/noup30165.html 111 ... crude to true protein; and experiment 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... by protein- enriched cassava root (PECR) as protein sources on growing trial and digestibility In chapter 2, experiment Cassava root was fermented with yeast, urea and DAP in a solid-state fermentation... 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