HUE UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY PHONEVILAY SILIVONG IMPROVED UTILISATION OF BAUHINIA ACUMINATA FOR GOAT PRODUCTION IN LAO PDR DOCTOR OF PHILOSOPHY IN ANIMAL SCIENCES HUE, 2020 HUE UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY PHONEVILAY SILIVONG IMPROVED UTILISATION OF BAUHINIA ACUMINATA FOR GOAT PRODUCTION IN LAO PDR SPECIALIZATION: ANIMAL SCIENCES CODE: 9620105 DOCTOR OF PHILOSOPHY IN ANIMAL SCIENCES SUPERVISOR 1: ASSOC.PROF NGUYEN HUU VAN SUPERVISOR 2: DR DUONG THANH HAI HUE, 2020 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, 2020 Phonevilay Silivong, PhD student i ACKNOWLEDGEMENTS The research in this PhD thesis was conducted at the farm and laboratory of Department of Animal Science, Faculty of Agriculture and Forest Resource, Souphanouvong University, in Luangprabang Province, Lao PDR with supported from Mekong Basin Animal Research Network (MEKARN II) project for funding this theses research and the scholarship for the PhD degree I am grateful for the support from all of those people and institutions I am greatly indebted to my main supervisor, Associate Professor Nguyen Huu Van and co-supervisor, Dr Duong Thanh Hai for his mentoring and constructive advice during my studies He made me much more confident as a scientist and researcher His patience and encouragement during my illness and positive criticism made it possible to accomplish this work My special thanks to Professor Thomas Reg Preston and Professor Dr Ron Leng, my teacher and adviser, for all your valuable guidance and support during the study I would also like to extend 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 lecturers in Hue University of Agriculture and Forestry and MEKARN II program, for giving me care and useful knowledge Warm thanks are extended to my father, Mr Souk Silivong for his great help and support My mother, Ms Khampoun Silivong for her assistance and encouragement, my older brothers and sister Mrs Kesone Silivong, Mr Vone Silivong, Mr Sonexay Silivong and younger sister Mrs Bounmee Silivong for their supported and encouragement; to my wife, Mrs Souksadar Vongyalud and my son Phetsamone Silivong and my daughter Souphaphone Silivong for their love I would like to warmly thank Mr Khamlek and Mr Phonesavath, and Mr Siphone, my analysis assistants and my colleagues in the Department of Animal Science, Faculty of Agriculture and Forest Resource, Souphanouvong University All of my friends in the PhD program 2017-2020 from the three countries: Laos, Vietnam and Cambodia, for sharing the culture, friendship and creating a warm atmosphere throughout the time of the course I would like to thanks all the people who contributed to this study ii ABSTRACTS This study was aimed at the utilization of locally available feed resources for increasing growth performance and reducing enteric methane emissions from goats in Lao PDR Five experiments (two in vitro and three in vivo) were carried out and presented in four chapters of this dissertation Experiment evaluated the effect on methane production from leaves of Bauhinia acuminata and Guazuma ulmifolia of an increasing level of water spinach as source of soluble protein Experiment and were studied the effects of water spinach and biochar on enteric methane emissions and growth performance in local goats fed Bauhinia acuminata and molasses, or cassava root chip, as the basal diet Experiment aimed to determine the effect of different protein sources (cassava foliage or water spinach) with or without of brewers’ grain (5% of diet DM)) on feed intake, digestibility, N balance and growth performance of local goats Experiment was to determine the effect of biochar and leaf meal from sweet and bitter cassava leaves, on methane production in an in vitro incubation of Bauhinia acuminata and water spinach as basal substrate The main findings of the study were that: (1) Goats fed Bauhinia acuminata responded with improved diet digestibility, N retention and growth rate when the Bauhinia acuminata was supplemented with water spinach; (2) However an important negative effect was that the improvement in diet digestibility by supplementation with water spinach led to increases in methane production per unit diet DM digested; (3) Supplementing Bauhinia acuminata foliage with leaves from a bitter variety of cassava reduced the in vitro production of methane when compared with supplementation by leaves from a sweet variety of cassava; (4) Ensiled brewers’ grains fed as an additive (5% as DM) to a diet of Bauhinia acuminata improved the digestibility, N retention and growth rate of goats The degree of improvement was greater when the Bauhinia acuminata was supplemented with cassava foliage instead of water spinach; (5) Biochar fed at 1% of a diet of Bauhinia acuminata and cassava foliage was as effective as brewers’ grains in improving the growth rate of the goats The results of this study indicated that supplementation of foliage from water spinach or cassava improved growth of local goats fed Bauhinia acuminata as basal diet HCN present in the leaves of cassava could be the reason for the reduction in methane emission Ensiled brewers’ grains and biochar fed to goats as additives probably act as “prebiotics” to improve growth performance and assist in detoxification in the animal Key words: N-balance, protein solubility, bauhinia, water spinach, solubility Nbalance, rumen ammonia, biochar, prebiotics, HCN iii DEDICATION To my parents, my wife Souksadar Vongyalud, my son (Phetsamone Silivong) and daughter (Souphaphone Silivong) To: My country (Lao PDR) iv TABLE OF CONTENT GUARANTEE .I ACKNOWLEDGEMENTS II ABSTRACTS III DEDICATION IV TABLE OF CONTENT IV LIST OF FIGURES VII LIST OF TABLES .X LIST OF ABBREVIATIONS, SYMBOLS AND EQUIVALENTS XIV INTRODUCTION .I PROBLEM STATEMENT .1 THE OBJECTIVES THE HYPOTHESIS .5 SIGNIFICANT/INNOVATION OF THE DISSERTATION REFERENCES CHAPTER LITERATURE REVIEW 10 GOAT PRODUCTION IN LAO PDR 10 ROLE OF GOAT PRODUCTION IN LAOS .12 2.1 GOAT POPULATION AND DISTRIBUTION 12 2.2 GOAT PRODUCTION SYSTEMS IN SMALLHOLDER FARMS .15 2.3 GOAT BREED AND BREEDING 17 2.4 CONSTRAINTS OF GOAT PRODUCTION IN LAO 19 2.5 POTENTIAL OF GOAT PRODUCTION DEVELOPMENT IN LAO 19 GOAT NUTRITION AND METHANE EMISSION 21 3.1 NUTRIENT REQUIREMENT FOR GOAT 21 3.2 METHANE EMISSION 25 LOCAL AVAILABLE FEES RESOURCES FOR GOAT IN LAO .29 4.1 FEEDING SYSTEM .35 v REFERENCES 43 CHAPTER EFFECT OF WATER SPINACH ON METHANE PRODUCTION IN AN IN VITRO INCUBATION WITH SUBSTRATES OF BAUHINIA ACUMINATA OR GUAZUMA ULMIFOLIA LEAVES AND MOLASSES 59 ABSTRACT .59 INTRODUCTION 59 MATERIALS AND METHODS 60 RESULTS 63 DISCUSSION 67 CONCLUSIONS 68 REFERENCES 68 CHAPTER EFFECTS OF WATER SPINACH AND BIOCHAR ON METHANE EMISSIONS AND GROWTH PERFORMANCE OF GOAT FED BAUHINIA ACUMINATA AND MOLASSES OR CASSAVA ROOT CHIPS AS 71 ABSTRACT .71 INTRODUCTION 71 MATERIALS AND METHODS 73 RESULTS 78 DISCUSSION 90 CONCLUSIONS 91 REFERENCES 91 CHAPTER EFFECT OF REPLACING WATER SPINACH (IPOMOEA AQUATICA) BY CASSAVA (MANIHOT ESCULENTA CRANTZ) FOLIAGE AND/OR BREWERS’ GRAINS ON FEED INTAKE, DIGESTIBILITY, N RETENTION AND GROWTH PERFORMANCE IN GOAT FED BAUHINIA ACUMINATA PLUS CASSAVA ROOT CHIPS AS THE BASAL DIET 95 ABSTRACT .95 INTRODUCTION 95 MATERIALS AND METHODS 96 RESULTS .100 DISCUSSION 105 vi CONCLUSIONS 105 REFERENCES .105 CHARPTER EFFECT OF SWEET OR BITTER CASSAVA LEAVES AND BIOCHAR ON METHANE PRODUCTION IN AN IN VITRO INCUBATION WITH SUBSTRATES OF BAUHINIA ACUMINATA AND WATER SPINACH (IPOMOEA AQUATICA) 107 ABSTRACT 107 INTRODUCTION 107 MATERIALS AND METHODS 108 RESULTS .111 DISCUSSION 116 CONCLUSIONS 116 REFERENCES .117 GENERAL DISCUSSION AND CONCLUSIONS 120 GENERAL DISCUSSION .120 GENERAL CONCLUSIONS 123 IMPLICATIONS 123 FURTHER RESEARCH 124 REFERENCES .124 PUBLISHCATION LIST 127 vii LIST OF TABLES CHAPTER LITERATURE REVIE Table The number of goat in Lao PDR 13 Table Estimated of animals requirement (2017-2020) 20 Table Daily Nutrient Requirements for Meat Producing Goats .22 Table Nutrient Requirements of Mature Does .24 Table Important fodder tree and shrubs in the Lao PDR .30 Table Chemical composition of fodder trees and shrubs leaves 31 CHAPTER EFFECT OF WATER SPINACH ON METHANE PRODUCTION IN AN IN VITRO INCUBATION WITH SUBSTRATES OF BAUHINIA ACUMINATA OR GUAZUMA ULMIFOLIA LEAVES AND MOLASSES Table Composition of diets (% DM basis) .62 Table Ingredients of the buffer solution (g/liter) 62 Table The chemical composition of feed (% in DM, except DM which is on fresh basis) 64 Table Mean values for gas production, percentage of methane in the gas, methane production (ml), DM solubilized and methane production per unit DM solubilized according to leaf source (Bauhinia and Guazuma) and level of water spinach 65 CHAPTER EFFECTS OF WATER SPINACH AND BIOCHAR ON METHANE EMISSIONS AND GROWTH PERFORMANCE OF GOAT FED BAUHINIA ACUMINATA AND MOLASSES OR CASSAVA ROOT CHIPS AS THE BASAL DIET Table Chemical composition of dietary ingredients (% in DM, except DM which is on fresh basis) .78 Table Mean values of feed intake by goats fed Bauhinia acuminata supplemented with water spinach (WS) or biochar (BC) or not supplemented 79 Table Mean values for live weight, live weight change, feed DM intake and DM feed conversion for goats fed a basal diet of Bauhinia acuminata foliage and molasses 79 viii The methane content of the gas was reduced when leaves of bitter cassava replaced leaves of sweet cassava as protein source (Figure 1) and when 1% of biochar was added to the substrate (Figure 2) The magnitude of the differences was relatively small but consistent for all incubation times Sweet Bitter 30 Methane, % in the gas 25 20 15 10 12 18 24 18 24 Incubation, h Sweet Bitter 30 Methane, % in the gas 25 20 15 10 12 Incubation, h 114 Figure Effect of sweet or bitter cassava leaves on % methane in the gas No biochar Biochar 30 Methane, % in the gas 25 20 15 10 12 18 24 Incubation, h Figure Effect of biochar on % methane in the gas The proportion of the substrate DM that was digested during the incubation was increased when biochar was included in the substrate (Figure 3) and reduced when the protein supplement was from bitter compared with sweet cassava (Figure 4) No Biochar Biochar 80 70 DM digested, % 60 50 40 30 20 10 12 18 24 Incubation, h Figure Effect of supplementation with biochar on proportion of substrate digested 115 80 Sweet cassava Bitter cassava 70 DM digested, % 60 50 40 30 20 10 12 18 24 Incubation, h Figure Effect of bitter versus sweet cassava on proportion of substrate digested Addition of biochar reduced the production of methane per unit substrate digested (Figure 5) However, there was no consistent trend for effects of bitter versus sweet cassava (Figure 6) As was to be expected the production of methane per unit substrate digested increased linearly with duration of the incubation No Biochar Biochar Methane, ml/g DM digested 60 50 40 30 20 10 12 18 24 Incubation, h Figure Effect of supplementation with biochar on production of methane per unit substrate digested 116 Sweet cassava Bitter cassava Methane, ml/g DM digested 60 50 40 30 20 10 12 18 24 Incubation, h Figure Effect of bitter versus sweet cassava on production of methane per unit substrate digested DISCUSSION The reduction in production of methane when leaves of bitter cassava replaced those from sweet cassava has been reported in several in vitro incubations (Phuong et al., 2012; Binh et al., 2018) and appears to be a direct effect of the higher HCN levels in the bitter varieties being toxic to methanogens (Smith et al., 1985) However, the major reduction (18%) in digestibility in the 24h incubation, when leaves from bitter cassava replaced those from sweet cassava, has not previously been reported; in fact, the opposite effect was observed by Phuong et al., 2012 The logical assumption is that the reduced digestibility reflected a more general HCN toxicity on the activity of all rumen microbes The reduction in methane production due to biochar was much less than has been observed in previous studies A reduction of 13% was recorded by (Phuong et al., 2012; of 8% by Vongkhamchanh et al., 2015; and 12% by Binh et al., 2018) The implication is that the sample of biochar used in this experiment may have been of inferior quality in terms of its relative surface area: weight ratio (and hence its capacity to support microbial communities in biofilms [Leng, 2017]) This stresses the need for a simpler quality test than the standard “BETS” ratio (for which the measuring equipment is not available in laboratories in Lao PDR) CONCLUSIONS - Increasing the length of the incubation from to 24h in an in vitro rumen fermentation increased methane concentration in the gas and methane produced per unit substrate DM digested 117 - The methane content of the gas was reduced when leaves of bitter cassava replaced leaves of sweet cassava as protein source and when 1% of biochar was added to the substrate The magnitude of the reduction due to biochar was relatively small but consistent for all incubation times - The proportion of the substrate DM that was digested during the incubation was increased when biochar was included in the substrate and reduced when the protein supplement was leaf meal from a bitter compared with a sweet cassava variety REFERENCES AOAC., 1990 Official Methods of Analysis.Association of Official Analytical Chemists.15th Edition (K Helrick editor).Arlington pp 1230 Binh Phuong, L.T., Preston, T.R and Leng, R.A., 2011 Mitigating methane production from ruminants; effect of supplementary sulphate and nitrate on methane production in an in vitro incubation using sugar cane stalk and cassava leaf meal as substrate Livestock Research for Rural Development.Volume 23, Article #22.Retrieved July 15, 2011, from http://www.lrrd.org/lrrd23/2/phuo23022.htm Binh, P.L.T., Preston, T.R., Van, H.N and Dinh, V.D., 2018 Methane production in an in vitro rumen incubation of cassava pulp-urea with additives of brewers’ grain, rice wine yeast culture, yeast-fermented cassava pulp and leaves of sweet or bitter cassava variety Livestock Research for Rural Development Volume 30, Article #77 http://www.lrrd.org/lrrd30/4/binh30077.html Inthapanya, S., Preston, T.R and Leng, R.A., 2011 Mitigating methane production from ruminants; effect of calcium nitrate as modifier of the fermentation in an in vitro incubation using cassava root as the energy source and leaves of cassava or Mimosa pigra as source of protein Livestock Research for Rural Development.Volume 23, Article#21.http://www.lrrd.org/lrrd23/2/sang23021.htm 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 http://www.lrrd.org/lrrd29/12/sang29220.html Lassey, K.R., 2007 Livestock methane emission: From the individual grazing animal through national inventories to the global methane cycle Agriculture Meteorology, 142: 120-132 118 Leng, R.A., Preston, T.R and Inthapanya, S., 2012 Biochar reduces enteric methane and improves growth and feed conversion in local “Yellow” cattle fed cassava root chips and fresh cassava foliage Livestock Research for Rural Development Volume 24, Article #199 http://www.lrrd.org/lrrd24/11/leng24199.htm Leng, R.A., 2017 Biofilm compartmentalisation of the rumen microbiome: modification of fermentation and degradation of dietary toxins Animal Production Science 57(11) 2188-2203 https://doi.org/10.1071/AN17382 Minitab., 2014 Statistical Software Minitab Inc Company State College (Pennsylvania) http://www.minitab.com Olivier, P., 2010 The Small-Scale Production of Food, Fuel, Feed and Fertilizer; a Strategy for the Sustainable Management of Waste http://www.mekarn.org/workshops/pakse/html/olivier.docx Outhen, P., Preston, T.R and Leng, R.A., 2011 Effect of supplementation with urea or calcium nitrate and cassava leaf meal or fresh cassava leaf in an in vitro incubation using a basal substrate of sugar cane stalk Livestock Research for Rural Development.Volume 23, Article #23.http://www.lrrd.org/lrrd23/2/outh23023.htm Orosco, J., Patiño, F.J., Quintero, M.J and Rodríguez, L., 2018 Residual biomass gasification on a small scale and its thermal utilization for coffee drying Livestock Research for Rural Development Volume 30, Article #5 http://www.lrrd.org/lrrd30/1/jair30005.html Phuong, L.T.B., Preston, T.R and Leng, R.A., 2012 Effect of foliage from “sweet” and “bitter” cassava varieties on methane production in in vitro incubation with molasses supplemented with potassium nitrate or urea Livestock Research for Rural Development Volume 24, Article #189 Retrieved August 9, 2018, from http://www.lrrd.org/lrrd24/10/phuo24189.htm Smith, M.R., Lequerica, J.L and Hart, M.R., 1985 Inhibition of methanogenesis and carbon metabolism in Methanosarcina sp by cyanide, Journal of Bacteriology, 162, 67-71 Sejian, V., R Lal., Lakritz, J and Ezeji, T., 2010 Measurement and prediction of enteric methane emission International Journal Biometeorology DOI: 10.1007/s00484-010-0356-7 Silivong, P and Preston, T.R., 2015 Effect of water spinach and biochar on methane production in an in vitro system with substrate of Bauhinia acuminata or Bitter Neem (Azadirachtaindica) leaves Livestock Research for Rural Development.Volume 27, Article #57.Retrieved August 15, 2015, from 119 http://www.lrrd.org/lrrd27/3/sili27057.html Tilley, J.M.A and Terry, R.A., 1963 A two stage technique for the in vitro digestion of forage crops Journal of the British Grassland Society 18: 104 Vongkhamchanh, B., Inthapanya, S and Preston, T.R., 2015 Methane production in an in vitro rumen fermentation is reduced when the carbohydrate substrate is fresh rather than ensiled or dried cassava root, and when biochar is added to the substrate Livestock Research for Rural Development Volume 27, Article #208 http://www.lrrd.org/lrrd27/10/bobb27208.html Whitelaw, F.G and Preston, T.R., 1963 The nutrition of the early-weaned calf III Protein solubility and amino acid composition as factors affecting protein utilization Animal Science, Volume 5, Issue 2, pp 131-145 Published online: 01 September 2010 https://doi.org/10.1017/S0003356100021620 120 GENERAL DISCUSSION AND CONCLUSIONS GENERAL DISCUSSION Bauhinia acuminata is a species of flowering shrub native to tropical countries and is widely distributed in Luang Prabang Province It has been observed that the foliage is readily consumed by goats The leaves of Bauhinia acuminata have 14.5% of protein of low solubility (22%) As is the case with foliage from most legume trees, it contains many secondary plant compounds including tannins (Queiroz Siqueira et al., 2012; Silivong and Preston, 2015) Tannins can be advantageous through their reaction with protein to increase the degree of escape of the protein to the small intestine where it is used more efficiently (Barry, 1999) However, if present in too high a concentration they may reduce the availability of peptides and amino acids needed by the rumen biota for fermentation of the basal feed (Preston and Leng, 1987) It is presumed that the high content of soluble protein in water spinach furnished amino acids and peptides, required by micro-organisms for efficient rumen digestion, and which were in short supply in the basal diet of Mango leaves which contain protein of low solubility (Kongmanila et al., 2011) Similar benefits were reported from adding water spinach to basal diets of Muntingia calabura foliage (Phongpanith et al., 2013) and to mixtures of Tithonia diversifolia and cassava foliage (Phonthep et al., 2016) Methane emissions in eructed breath were reduced by 50% when cassava rather than Tithonia was the main foliage in the diet, and were increased when water spinach was also fed It is postulated that on such feeds the balance of feed fermented in the cecum/large intestine relative to the rumen is increased As disposal of hydrogen in fermentation degradation in the cecum-colon appears to be dominated by acetogenesis, this would account for decreased methane production on such diets (Phonethep et al., 2016), According to Porsavathdy et al., 2017, when goats were fed on a basal diet of pigeon wood foliage (Trema orientalis) and supplemented with sun-dried cassava foliage (Manihot esculenta, Crantz) or water spinach (Ipomoea aquatica), feed intake and growth rates were increased and feed conversion was better with cassava foliage The proportion of methane in eructed gas was reduced when the pigeon wood foliage was supplemented with sun-dried cassava foliage or fresh water spinach, with lower values for cassava foliage than for water spinach Feeding systems based on cassava products and by products have great potential for increasing ruminant productivity and reducing methane production The constraint to feeding cassava leaves is they can give rise to toxic hydrocyanic acid (HCN) (Van Soest, 1994) The release of free HCN is brought about by the action of 121 either the endogenous enzyme linamarase in damaged plant tissues or β glucosidases within the digestive tract of the animal The presence of cyanogenic glycosides in cassava constitutes a major limitation to the use of cassava in both human and animal food (Tewe, 1995) However, a major part of the absorbed cyanide is rapidly detoxified by conversion to thiocyanate which is excreted in the urine Owing to this rapid detoxification, it is possible for animals to ingest doses of cyanogenic glucosides only slightly less than the lethal dose over extended time periods, without harm (John et al., 1965) Factors that influence toxicity include the size and type of animal, the rate of ingestion, the type of food ingested simultaneously with the cyanogenic glycoside, presence of active degradative enzymes in the animal digestive tract and the ability to detoxify cyanide (Clarke and Clarke, 1967) Thiocyanate is a potent goitrogen and has been implicated in the aetiology of goitire in animals (Langer, 1966; Shihombing et al., 1971) An additional advantage of cassava over cereal crops such as maize is that the foliage has proved to be a valuable source of bypass protein such that the cassava plant becomes a source both of highly digestive carbohydrate (from the root) as well as protein (from the foliage) The root is composed of highly digestible carbohydrate in the form of starch with little fiber The foliage is rich in protein which, allied with low levels of tannin (Netpana et al., 2001; Bui Phan Thu Hang and Ledin, 2005), enables some of the dietary protein to escape from the rumen and, following intestinal digestion, contribute to the animal’s requirements for essential amino acids directly at the sites of metabolism (Barry, 1999) The presence of cyanogenic glucosides in cassava can be a problem but positive aspects are that they appear to be involved in a reduction in methanogenesis (Phuong et al., 2015) In recent research it has been shown that cassava foliage has been fed successfully to improve performance and reduce methane emissions in goats Sina et al., 2017 showed that when cassava foliage was supplemented with 5% brewer’s grains the growth rate of goats was increased from 70 to 160 g/day and was 50% better than with a basal diet of water spinach In this study, five experiments were carried out with the main objectives of: (i) improving the growth performance of the goat; and (ii) reducing the rumen production of methane, an important greenhouse gas contributing to global warming Two of the experiments were in vitro rumen incubations (experiments and 5); the other three experiments were combined digestibility, N-balance and growth studies In all cases foliage of Bauhinia acuminata was the basal substrate/diet In an in vitro rumen incubation with leaves of Bauhinia acuminata or Guazuma ulmifolia as the basal substrates (experiment 1), supplementation with increasing levels of water spinach increased linearly the gas production, the overall digestibility 122 of the substrate and the production of methane It was postulated that the high solubility of the water spinach protein (70%) was the reason for these effects, following the report that methane production was greater when groundnut meal (protein solubility 70%) rather than fish meal (protein solubility 16%) was the protein source in an in vitro rumen incubation (Preston et al., 2013) On all treatments, the methane concentration in the gas increased linearly as the incubation progressed up to 48h This confirms earlier findings of a linear increase in methane content of the gas as the incubation time was increased (Inthapanya et al., 2011) On the basis of this finding it is recommended that in vitro rumen incubations should not be extended beyond 24h, after which, according to Inthapanya et al., 2011, the fermentation system begins to resemble what happens in a biodigester The in vitro incubation described in the experiment provides support for the concept that non-nutritional compounds, as well as tannins, can play a role in modifying the rumen ecosystem, and especially the production of methane The leaves of “bitter” varieties of cassava are richer in cyanogenic glucosides (that in the rumen give rise to HCN) than the “sweet” varieties When leaves from these two cassava varieties were compared as sources of protein in an in vitro incubation of Bauhinia acuminata and water spinach, gas production, dry matter digested and content of methane in the gas were all decreased when the leaves were from the bitter cassava variety Two interventions - supplementation of Bauhinia acuminata with water spinach or fresh cassava foliage and manipulation of the rumen ecosystem with feed additives were studied in three feeding trials with growing goats (experiments 2, and 4) Digestibility and growth/N retention were improved in all cases by supplementation with water spinach or cassava foliage, with better results from cassava Both brewers’ grains (5% of diet DM) and biochar (1% of diet DM) had positive effects on N retention/growth rate Both effects were more pronounced when the protein supplement was cassava foliage rather than water spinach (experiment 4) It is proposed that the interaction in the degree of improved animal performance, according to whether the brewers’ grains were added to the diet with cassava foliage, compared with the diet containing water spinach, was because the brewers’ grains act as a “prebiotic” when included in diets containing potentially toxic elements such as the cyanogenic glucosides present in cassava foliage A similar explanation was proposed for the positive effects of “Kilao” (the byproduct from the fermentation/distillation process in making of “rice wine”) in increasing growth and feed conversion of cattle fed ensiled cassava root and cassava foliage (Sengsouly and Preston, 2016) Biochar was as effective as brewers’ grains in improving animal performance (experiments and 5) 123 GENERAL CONCLUSIONS - It was confirmed that goats fed a tannin-rich tree foliage, such as Bauhinia acuminata, responded with improved diet digestibility, N retention and growth rate when the Bauhina acuminata was supplemented with a highly fermentable vegetable plant such as water spinach (Ipomoea aquatica) - An important negative effect was that the improvement in diet digestibility by supplementation with water spinach led to increases in methane production per unit diet DM digested The increase in methane production was postulated as being due to the much higher solubility of the protein in water spinach compared with the Bauhinia acuminata - The methane concentration of the gas in in vitro rumen incubations increases linearly with the length of the fermentation - Supplementing Bauhinia acuminata foliage with leaves from a bitter variety of cassava reduced the in vitro production of methane when compared with supplementation by leaves from a sweet variety of cassava - It is postulated that the cyanogenic glucosides present in greater concentration in the leaves of bitter than in sweet cassava could be the reason for the reduction in methane production - Ensiled brewers’ grains fed as an additive (5% as DM) to a diet of Bauhinia acuminata improved the digestibility, N retention and growth rate of goats The degree of improvement was greater when the Bauhinia acuminata was supplemented with cassava foliage instead of water spinach - Biochar fed at 1% of a diet of Bauhinia acuminata and cassava foliage was as effective as brewers’ grains in improving the growth rate of the goats IMPLICATIONS - Bauhinia acuminata foliage can support growth rates of >60 g/day in local goats when it is supplemented with foliage from a sweet variety of cassava foliage - Water spinach also improves growth rates of goats fed Bauhinia acuminata foliage but is not recommended as this practice will result in increased production of rumen methane - Ensiled brewers’ grains and biochar fed to goats as additives (5% for brewers’ grains; 1% for biochar) probably act as “prebiotics” to improve growth performance and assist in detoxification in the animal of the products from enzymic breakdown of secondary plant compounds such as cyanogenic glucosides 124 FURTHER RESEARCH Cassava foliage is an important supplement to improve productivity of goats browsing on native trees and shrubs with the probability that this practice will also reduce enteric methane, an important greenhouse gas Cassava foliage is available in large quantiles in Lao PDR when the roots are harvested for industrial starch production; It can also be produced by repeated harvest at 3-4 months intervals when the crop is grown for forage production Varieties grown for starch production have been selected for high yield but this is also associated with higher levels of potentially toxic cyanogenic glucosides that give rise to toxic HCN when consumed by animals Biochar is the carbon-rich byproduct of the carbonization of fibrous biomass by pyrolysis at high temperatures (700-900°C) It is an important component of strategies to reduce global warming as when applied to the soil the carbon in the biochar is not oxidized This process is thus a natural way to sequester carbon from the atmosphere, an essential feature of activities required in order to reduce the risks of climate change To respond to the opportunities and multiple benefits from: (i) feeding of cassava foliage as a protein supplement for goats browsing on trees and shrubs; and (ii) the associated use of biochar as a dietary additive, it is proposed that research should be prioritized to: - Definition of the relative roles of sweet and bitter varieties as supplements in the feeding system of goats and other ruminant animals; - The production of biochar at farm level from fibrous crop byproducts (eg: cassava stems) and its role as a “prebiotic” feed additive in conserving animal health and improving productivity, especially in diets based on cassava foliage REFERENCES Barry, T.N., 1999 The implications of condensed tannins on the nutritive value of temperate forages fed to ruminants British Journal of Nutrition 81(4): 72-263 Bui Phan Thu Hang and Ledin Inger., 2005 Utilization of Melastoma (Melastoma affine, D Don) foliage as a forage for growing goats with cassava (Manihot Esculenta, Crantz) hay supplementation Proceedings International Workshop on Small Ruminant Production and Development in South East Asia (Editor: Inger Ledin), Hanoi, Vietnam, 2-4 March 2005.http://www.mekarn.org/procsr/hangctu.pdf Clacke, E.G.C and Clacke, M.L., 1967 Cyanides; Garner’s veterinary toxicology, 3rd edition, Williams and Wilkins Co; Baltimore, pp 75-80 Inthapanya, S., Preston, T.R and Leng, R.A., 2011 Mitigating methane production from ruminants; effect of calcium nitrate as modifier of the fermentation in 125 an in vitro incubation using cassava root as the energy source and leaves of cassava or Mimosa pigra as source of protein Livestock Research for Rural Development.Volume 23, Article #21 http://www.lrrd.org/lrrd23/2/sang23021.htm Johonson, R.M and Romond, W.D., 1965 The chemical composition of some tropical food plant, Manioc Tropical Science 75, 365-378 Kongmanila, D., Phommachanh, K and Preston, T.R., 2011 Effect on growth rate and digestibility in goats of supplementing a basal diet of mango foliage with fresh water spinach (Ipomoea aquatica) Livestock Research for Rural Development.Volume 23, Article #203.Retrieved December 16, 2014, from http://www.lrrd.org/lrrd23/10/daovy23203.htm Langer., 1966 Antithyroid action in rats of small dose of some naturally accurring compounds Endocrinology 79: 1117-1122 Netpana, N., Wanapat, M., Poungchompu, O and Toburan, W., 2001 Effect of condensed tannins in cassava hay on fecal parasitic egg counts in swamp buffaloes and cattle In: Proceedings International Workshop on Current Research and Development on Use of Cassava as Animal Feed T R Preston, B Ogle and M Wanapat (Ed) http://www.mekarn.org/procKK/netp.htm Preston, T.R and Leng, R.A., 1987 Matching ruminant production systems with available resources in the tropics and sub-tropics Penambul Books Armidale, Australia Web version http://www.cipav.org.co/PandL/Preston_Leng.htm Preston, T.R., Do, H.Q., Khoa, T.D., Hao, T.P and Leng, R.A., 2013 Protein solubility of fish meal and groundnut meal and methane production in an in vitro incubation Livestock Research for Rural Development.Volume 25, Article #16 http://www.lrrd.org/lrrd25/1/hqdo25016.htm Phongpanith, S., Inthapanya, S and Preston, T.R., 2013 Effect on feed intake, digestibility and N balance in goats of supplementing a basal diet of Muntingia foliage with biochar and water spinach (Ipomoea aquatica) Livestock Research for Rural Development Volume 25, Article #35 Retrieved September 1, 2018, from http://www.lrrd.org/lrrd25/2/seng25035.htm Phuong, L.T.B., Khang, D.N and Preston, T.R., 2015 Methane production in an in vitro fermentation of cassava pulp with urea was reduced by supplementation with leaves from bitter, as opposed to sweet, varieties of cassava Livestock Research for Rural Development Volume 27, Article #162 http://www.lrrd.org/lrrd27/8/phuo27162.html 126 Phonethep, P., Preston, T.R and Leng, R.A., 2016 Effect on feed intake, digestibility, N retention and methane emissions in goats of supplementing foliages of cassava (Manihot esculenta Crantz) and Tithonia diversifolia with water spinach (Ipomoea aquatica) Livestock Research for Rural Development Volume 28, Article #72 Retrieved September 1, 2018, from http://www.lrrd.org/lrrd28/5/phon28072.html Porsavathdy, P., Do, H.Q and Preston, T.R., 2017 Growth rate and feed conversion were improved, and emissions of methane reduced, when goats fed a basal diet of pigeon wood foliage (Trema orientalis) were supplemented with sun-dried cassava foliage (Manihot esculenta, Crantz) or water spinach (Ipomoea aquatica) Livestock Research for Rural Development Volume 29, Article #68 Retrieved September 7, 2018, from http://www.lrrd.org/lrrd29/4/phho29068.html Queiroz Siqueira C.F., de Vasconcelos Cabral, D.L., da Silva Peixoto Sobrinho, T.J., Cavalcanti de Amorim, E.L., de Melo, J.G., de Sousa Araújo, T.A and de Albuquerque, U.P., 2012 Levels of tannins and flavonoids in medicinal plants: evaluating bioprospecting strategies Evidence-Based Complementary and Alternative Medicine Volume 2012, Article ID 434782 pp Shihombing, D.T.H., Crownwell, G.L and Hays, V.W., 1971 Effect of added thiocyanate and iodine to corn-soybean meal diets on performance and thyroid status of pig J Anim Sci.33:1154 (Abstract) Silivong, P and Preston, T.R., 2015 Effect of water spinach on methane production in an in vitro incubation with substrates of Bauhinia (acuminata) and Guazuma ulmifolia leaves Livestock Research for Rural Development Volume 27, Article #217 Retrieved December 10, 2015, from http://www.lrrd.org/lrrd27/11/sili27217.htm Sengsouly, P and Preston, T.R., 2016 Effect of rice-wine distillers’ byproduct and biochar on growth performance and methane emissions in local “Yellow” cattle fed ensiled cassava root, urea, cassava foliage and rice straw.Livestock Research for Rural Development Volume 28, Article #178 Retrieved June 1, 2017, from http://www.lrrd.org/lrrd28/10/seng28178.html Sina, V and Preston, T.R., 2017 Effect on methane production of source of carbohydrate, and processing/variety of cassava leaf supplement, in an in vitro rumen incubation Livestock Research for Rural Development Volume 29, Article #213 Retrieved September 7, 2018, from http://www.lrrd.org/lrrd29/11/sina29213.html Tewe, O.O., 1995 Detoxification of cassava products and effects of residual toxins on consuming animals In: Root, tuber, plan tnains? and banana in animal feeding, FAO Animal Production and Health Paper, pp 81-95 Van Soest, P.J., 1994 Nutritional ecology of the ruminant nd edition Cornell University Press Ithca, USA 127 PUBLICATION LIST I Silivong, P., Preston, T.R., Van, N.H and Hai, D.T., 2018 Brewers’ grains (5% of diet DM) increases the digestibility, nitrogen retention and growth performance of goats fed a basal diet of Bauhinia accuminata and foliage from cassava (Manihot esculenta Crantz) or water spinach (Ipomoea aquatica) Livestock Research for Rural Development Volume 30, Article #55 Retrieved May 3, 2018, from http://www.lrrd.org/lrrd30/3/siliv30055.html II Silivong, P., Preston, T.R., Van, N.H and Hai, D.T., 2018 Effect of sweet or bitter cassava leaves and biochar on methane production in an in vitro incubation with substrates of Bauhinia acuminata and water spinach (Ipomoea aquatica) Livestock Research for Rural Development Volume 30, Article #163 http://www.lrrd.org/lrrd30/9/psivil30163.html 128 ... diet of foliage from Bauhinia acuminate and molasses .83 Figure Effect of water spinach on ratio of methane to carbon dioxide in eructed gas from goats fed either Bauhinia acuminata and... additive (5% as DM) to a diet of Bauhinia acuminata improved the digestibility, N retention and growth rate of goats The degree of improvement was greater when the Bauhinia acuminata was supplemented... leaf source (Bauhinia and Guazuma) and level of water spinach 65 CHAPTER EFFECTS OF WATER SPINACH AND BIOCHAR ON METHANE EMISSIONS AND GROWTH PERFORMANCE OF GOAT FED BAUHINIA ACUMINATA