Improved utilisation of bauhinia acuminata for goat production in lao pdr

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) 
Hue University, 2020
Phonevilay Silivong, PhD student
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.
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 1 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 2 and 3 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 4 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 5 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 N-balance, rumen ammonia, biochar, prebiotics, HCN
DEDICATION
To my parents, my wife Souksadar Vongyalud, my son (Phetsamone Silivong) and daughter (Souphaphone Silivong)
To:
My country (Lao PDR)
TABLE OF CONTENT
LIST OF TABLES 
CHAPTER 1. LITERATURE REVIEW
Table 1. The number of goat in Lao PDR	13
Table 2. Estimated of animals requirement (2017-2020)	20
Table 3. Daily Nutrient Requirements for Meat Producing Goats	22
Table 4. Nutrient Requirements of Mature Does	24
Table 5. Important fodder tree and shrubs in the Lao PDR	30
Table 6. Chemical composition of fodder trees and shrubs leaves	31
CHAPTER 2. EFFECT OF WATER SPINACH ON METHANE PRODUCTION IN AN IN VITRO INCUBATION WITH SUBSTRATES OF BAUHINIA ACUMINATA OR GUAZUMA ULMIFOLIA LEAVES AND MOLASSES
Table 1. Composition of diets (% DM basis)	62
Table 2. Ingredients of the buffer solution (g/liter)	62
Table 3. The chemical composition of feed (% in DM, except DM which is on fresh basis)	64
Table 4. 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 3. 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 1. Chemical composition of dietary ingredients (% in DM, except DM which is on fresh basis)	78
Table 2. Mean values of feed intake by goats fed Bauhinia acuminata supplemented with water spinach (WS) or biochar (BC) or not supplemented	79
Table 3. 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
Table 4.  Mean values of apparent digestibility and N balance in goats fed Bauhinia acuminata and molasses supplemented with water spinach (WS) and biochar (BC) or not supplemented (No-WS; No-BC)	81
Table 5.  Mean values of rumen pH and ammonia, and ratio of methane to carbon dioxide in eructed breath of goats fed Bauhinia acuminata and molasses supplemented with water spinach (WS) and biochar (BC) or not supplemented (No-WS; No-BC)	82
Table 6. Mean values of feed intake by goats fed Bauhinia acuminata and cassava root chips supplemented with water spinach (WS) or biochar (BC) or not supplemented	84
Table 7. 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 Cassava root chips	85
Table 8.  Mean values of apparent digestibility and N balance in goats fed Bauhinia acuminata and cassava root chips supplemented with water spinach (WS) and biochar (BC)or not supplemented (No-WS; No-BC)	87
Table 9.  Mean values of rumen pH and ammonia in goats fed Bauhinia acuminata and cassava root chips supplemented with water spinach (WS) and biochar (BC) or not supplemented (No-WS; No-BC)	89
CHAPTER 4. 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
Table 1. Layout of the digestibility/N retention study	97
Table 2. Chemical composition of dietary ingredients (% in DM, except DM which is on fresh basis)	100
Table 3.  Mean values of apparent digestibility and N balance in goats fed Bauhinia acuminata supplemented with water spinach or cassava foliage, with (BG) and without (No-BG) brewers’ grains	101
Table 4.  Mean values of feed intake by goats fed Bauhinia acuminata plus cassava root	102
Table 5. Mean values for live weight, live weight change, DM intake and DM feed conversion for goats fed Bauhinia acuminata supplemented with cassava or water spinach foliage, with or without brewers’ grains (interaction effects)	103
Table 6. Molar VFA proportions in rumen fluid from goats fed Bauhinia acuminata supplemented with water spinach or cassava foliage, with and without brewers’ grains	104
CHARPTER 5. 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)
Table 1. Composition of substrate (% DM basis)	110
Table 2. Ingredients of the buffer solution (g/liter)	110
LIST OF FIGURES
CHAPTER 1. LITERATURE REVIEW
Figure 1. Fermentation pathways in the rumen.	27
CHAPTER 2. EFFECT OF WATER SPINACH ON METHANE PRODUCTION IN AN IN VITRO INCUBATION WITH SUBSTRATES OF BAUHINIA ACUMINATA OR GUAZUMA ULMIFOLIA LEAVES AND MOLASSES
Diagram 1. A schematic view of the in vitro system to measure gas production in an in vitro incubation	60
Photo 1: Bauhinia acuminata	61
Photo 2: Guazuma ulmifolia	61
Photo 3. The in vitro system made from recycled "pep" water bottles	63
Photo 4. The substrate residue filtered though cloth	63
Figure 1. Effect of Bauhinia (BA) and Guazuma (GU) leaf meals on gas production at increasing incubation intervals	66
Figure 2. Effect of increasing level of water spinach on gas production at increasing incubation intervals	66
Figure 3. Effect of foliages from Bauhinia (BA) and Guazuma (GU) on methane content in the gas at increasing incubation intervals	66
Figure 4. Effect of increasing level of water spinach on methane content in the gas at increasing incubation intervals	66
Figure 5. Effect of leaf meals from Bauhinia (BA) and Guazuma (GU) on substrate DM solubilized at increasing incubation interval	67
Figure 6. Effect of level of water spinach on substrate DM solubilized at increasing incubation intervals	67
Figure 7. Effect of leaf meals from Bauhinia (BA) and Guazuma (GU) on methane per unit DM solubilized at different incubation intervals	67
Figure 8. Effect of increasing level of water spinach in the substrate on methane per unit DM solubilized at increasing incubation intervals	67
CHAPTER 3. 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
Photo 1. Goats confined in the metabolism pens	74
Photo 2. Bauhinia acuminata foliage as presented to the goats	75
Photo 3. Goats were confined in a plastic-lined cage for the measurement of the eructed gases with the Gasmet meter	76
Photo 4. Taking rumen fluid by	76
stomach tube	76
Figure 1. Effect of biochar on live weight gain of goats fed Bauhinia acuminate and molasses with and without water spinach	80
Figure 2. Effect of supplementation with water spinach and biochar, separately or together, on the growth rate of goats fed a basal diet of foliage from Bauhinia acuminata and molasses	80
Figure 3. Effect of biochar compared with water spinach on feed conversion of goats fed Bauhinia acuminata and molasses as	80
basal diet	80
Figure 4. Relationship between live weight gain and feed conversion in goats fed Bauhinia acuminata foliage and molasses supplemented or not with water spinach and biochar	80
Figure 5. Effect of biochar on N retention as % N intake of goats fed Bauhinia acuminata and molasses as basal diet with or without water spinach	82
Figure 6. Effect of biochar on N retention as % N digested of goats fed Bauhinia acuminata and molasses as basal diet with or without water spinach	82
Figure 7. Effect of supplementation with water spinach and biochar, separately or together, on rumen ammonia of goats fed a basal diet of foliage from Bauhinia acuminate and molasses	83
Figure 8. Effect of water spinach on ratio of methane to carbon dioxide in eructed gas from goats fed either Bauhinia acuminata and molasses as basal diet	83
Figure 9. Effect of biochar on ratio of methane to carbon dioxide in eructed gas from goats fed either Bauhinia acuminata and molasses as basal diet	83
Figure 10. Supplements of water spinach and biochar increased the live weight gain of goats fed Bauhinia acuminata and cassava root chips	85
Figure 11. Supplements of water spinach and biochar improved the DM feed conversion of goats fed Bauhinia acuminata and cassava root chips	86
Figure 12. Relationship between live weight gain and feed conversion in goats fed Bauhinia acuminata foliage and cassava root chips supplemented or not with water spinach and biochar	86
Figure 13. Supplements of water spinach and biochar increased the N retention by goats fed Bauhinia acuminata and cassava root chips	88
Figure 14. Supplements of water spinach and biochar improved the N retention as % of N intake by goats fed Bauhinia acuminata and cassava root chips	88
Figure 15. Supplements of water spinach and biochar improved the N retention as % of N digested by goats fed Bauhinia acuminata and cassava root chips	89
Figure 16. A supplement of water spinach increased rumen ammonia in goats fed Bauhinia acuminata and cassava root chips as basal diet	90
Figure 17. Relationship between live weight gain and rumen ammonia in goats fed Bauhinia ac ... te (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 1 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 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 5 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, 3 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 3 and 5).
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.
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.
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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
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.