Tài liệu ảnh hưởng hiệp đồng cùa lá sắn (manihot esculenta crantz), bã bia, và than sinh học (biochar) lên sự sản sinh khí methane và năng suất thú nhai lại

HUE UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY LE THUY BINH PHUONG SYNERGIC EFFECT OF CASSAVA (MANIHOT ESCULENTA CRANTZ) FOLIAGE, BREWER’S GRAINS, AND BIOCHAR ON METHANE PRODUCTION AND PERFORMANCE OF RUMINANTS DOCTOR OF PHILOSOPHY IN ANIMAL SCIENCES HUE, 2020 HUE UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY LE THUY BINH PHUONG SYNERGIC EFFECT OF CASSAVA (MANIHOT ESCULENTA CRANTZ) FOLIAGE, BREWER’S GRAINS, AND BIOCHAR ON METHANE PRODUCTION AND PERFORMANCE OF RUMINANTS SPECIALIZATION: ANIMAL SCIENCES CODE: 9620105 DOCTOR OF PHILOSOPHY IN ANIMAL SCIENCES SUPERVISOR 1: ASSOC. PROF. NGUYEN HUU VAN SUPERVISOR 2: DR. DINH VAN DUNG HUE, 2020 Declaration I declare that this dissertation is the result of my work and that it has not been presented previously as a dissertation at this university or elsewhere. To the best of my knowledge, it does not breach copyright law, and has not been taken from other sources except where such work has been cited and acknowledged within the text. All results have been published at Journal of Livestock Research for Rural Development (LRRD) http://www.lrrd.org/ Hue University, 2020 Le Thuy Binh Phuong i Dedication To my parent who spends their immense loves to me. To my husband, Than Van Dang, and my two daughters, Than Ngoc Kim Nguyen and Than Ngoc Hai An, who encouraged me to pursue my dreams. Acknowledgements ii My Ph.D. has been an amazing experience with Professor Thomas Reginal Preston. He has been teaching me how good experiment is done and how to be real researcher. I have been grown up like that, thus, I would like to thank with all my heart to his guidance. I am thankful to Professor Ron A. Leng who gave me the background knowledge in biochemistry for stimulating the ideas in research. I would like to thanks to Assoc. Prof. Nguyen Huu Van and Dr. Dinh Van Dung who gave me the most helpful advice and instructed me to complete the dissertation. I gratefully acknowledge financial support from the SIDA-financed project, MEKARN II for 3 years that made my Ph.D. work possible. My classmate in Ph.D. course, the group is source of friendship as well as good collaboration. Lastly, I would like to thank my family for all their love and encouragement. For my parents who take care of my children during course time and support me to participate in learning and research activities. Most of all for my loving, supportive, encouraging, and patient husband Dang who faithful support during the final stages of this Ph.D. is so appreciated. Thank you. iii Abstract This dissertation was aimed to develop a greater understanding of both the constraints in the presence of cyanide toxin and benefits of using cassava foliage as bypass protein in order to improve its utilization in ruminant feeding systems. The study comprised two in vitro rumen incubations, one feeding trial on cattle and a digestibility/N retention experiment on goats, in each case involving comparisons of varieties of cassava known to be rich (KM94) or poor (Gon) in cyanogenic glucosides. In the first experiment (Chapter 2), cassava foliage varieties (Japan, KM94, KM140 and Gon) with different level of cyanide concentration were considered their effect on methane production in ruminal in vitro incubation. The second experiment (Chapter 3) examined the relative responses of cattle fed cassava root pulp and urea as basal diet with foliage from “sweet” (Gon) or “bitter” (KM140) cassava foliage as protein source. The third experiment (Chapter 4) determined methane production in an in vitro rumen incubation of cassava pulp - urea with additives of brewers’ grain, rice wine yearst culture, yeast-fermented cassava pulp and leaves of sweet or bitter cassava variety. The fourth experiment (Chapter 5) measured effect of additives (brewer’s grain and biochar) on the nitrogen retention and rumen methane production when goats had access to mixed sweet and bitter varieties of cassava foliage compared with the sweet variety alone. The results of these experiments indicated that bitter cassava foliage containing high levels of cyanogenic glucosides greatly reduces methane production, compared with sweet varieties, in the rumen in vitro incubations. However, the toxicity of cyanide in vivo in ruminants (cattle and goats) can be reduced by “prebiotic” properties provided by either brewers’ grains or biochar. In the presence of these “prebiotics”, HCN-linked challenges from feeding bitter cassava leaves at up to 50% of the diet of goats did not negatively impact to feed intake, growth and animal health. On the contrary, the HCN precursors present in bitter cassava leaves may lead to a partial shift in digestion of nutrients from the rumen to the lower parts of the ruminant digestive tract leading to improvement in productivity. Key words: Prebiotic, cyanide, bitter cassava, rumen fermentation, in vitro. iv Table of Contents List of Figures..........................................................................................................ix List of Tables...........................................................................................................xi Abbreviation..........................................................................................................xiii Introduction............................................................................................................................1 1. Problem statement....................................................................................................1 2. Aim and objective of the study.................................................................................2 2.1 Aims of the study................................................................................................2 2.2 Objective of the study.........................................................................................2 3. Research hypotheses.................................................................................................2 4. Significant/Innovation of study................................................................................3 CHAPTER 1. LITERATURE REVIEW...............................................................................4 1.1 Rumen fermentation and methane production.................................................................4 1.1.1 Rumen fermentation............................................................................................4 1.1.2 Volatile fatty acid pattern....................................................................................4 1.1.3 Protein metabolism..............................................................................................8 1.1.4 Methane production.............................................................................................9 Pathway of methane production...............................................................................9 1.1.5 Effect of feeding system on rumen fermentation..............................................11 1.2 Understanding ruminal microorganism..........................................................................13 1.2.1 The self-detoxify mechanism of ruminal microbes...........................................13 1.2.2 Interaction of ruminal microorganism in biofilm formation.............................14 1.3 Using agro-industrial by-products for ruminant feeding system...................................16 1.3.1 Cassava foliage..................................................................................................17 Nutrient composition..............................................................................................19 Effect of tannin content in cassava leaves on the ruminant feeding system...........21 Cyanogenic glucosides in cassava leaf...................................................................25 New potentially application on mitigation of HCN effect.....................................29 v 1.3.2 Brewers grain.....................................................................................................29 Nutrient composition..............................................................................................30 Using brewers grain in ruminant feeding...............................................................31 1.3.3 Potential using cassava root pulp as energy source or protein enrichment source ....................................................................................................................................31 1.3.4 Biochar: application as an additive....................................................................36 1.4 Supplementary Saccharomyces cerevisiae: concept on detoxification..........................38 1.5 Conclusions....................................................................................................................40 References............................................................................................................................41 CHAPTER 2. 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.............................................................................63 Abstract................................................................................................................................63 2.1 Introduction....................................................................................................................63 2.2 Materials and methods...................................................................................................64 Location and duration.................................................................................................64 Experimental design...................................................................................................65 Material preparation...................................................................................................65 Measurements.............................................................................................................66 Statistical analysis.......................................................................................................66 2.3 Results and discussion....................................................................................................67 2.4 Conclusions and recommendation.................................................................................71 References............................................................................................................................71 CHAPTER 3. A LOW CONCENTRATION (4% IN DIET DRY MATTER) OF BREWERS’ GRAINS IMPROVES THE GROWTH RATE AND REDUCES THIOCYANATE EXCRETION OF CATTLE FED CASSAVA PULP-UREA AND “BITTER” CASSAVA FOLIAGE......................................................................................75 Abstract...............................................................................................................................75 3.1 Introduction....................................................................................................................76 vi 3.2 Materials and methods...................................................................................................78 Location and duration.................................................................................................78 Treatments and experimental design..........................................................................78 Animals and housing..................................................................................................79 Feeding and management...........................................................................................79 Data collection and measurements.............................................................................79 Chemical analysis.......................................................................................................80 Statistical analysis.......................................................................................................80 3.3 Results and discussion....................................................................................................81 3.4 Conclusions and recommendation.................................................................................90 References............................................................................................................................91 CHAPTER 4. 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....................................95 Abstract................................................................................................................................95 4.1 Introduction....................................................................................................................95 4.2 Materials and methods...................................................................................................97 Location and duration.................................................................................................97 Treatments and design................................................................................................97 Materials.....................................................................................................................97 In vitro incubation......................................................................................................98 Data collection............................................................................................................98 Statistical analysis.......................................................................................................98 4.3 Results and discussion....................................................................................................99 4.4 Conclusions and recommendation...............................................................................106 References..........................................................................................................................106 CHAPTER 5. EFFECT OF ADDITIVES (BREWER’S GRAIN AND BIOCHAR) AND CASSAVA VARIETY (SWEET VERSUS BITTER) ON NITROGEN vii RETENTION, THIOCYANATE EXCRETION AND METHANE PRODUCTION BY BACH THAO GOATS......................................................................................................109 Abstract..............................................................................................................................109 5.1 Introduction..................................................................................................................110 5.2 Materials and Methods.................................................................................................111 Location and duration...............................................................................................111 Experimental design.................................................................................................111 Feeds and feeding.....................................................................................................112 Animals and feeding system.....................................................................................113 Chemical analyses....................................................................................................114 Statistical analysis.....................................................................................................114 5.3 Results and discussion..................................................................................................115 5.4 Conclusions and recommendation...............................................................................124 References..........................................................................................................................125 CHAPTER 6. GENERAL DISCUSSION AND CONCLUSIONS...................................129 6.1 General discussion.......................................................................................................129 6.2 Conclusions..................................................................................................................134 6.3 Implication and further research..................................................................................134 References..........................................................................................................................135 PUBLICATION LIST........................................................................................................138 APPENDIX. PROTOCOL D1- DETERMINATION OF THIOCYANATE IN URINE ............................................................................................................................................139 List of Figures Figure 1.1 Pathway of VFA in metabolism...........................................................................7 viii Figure 1.2 The reaction of methane generation...................................................................10 Figure 1.3 The pathway of hexose conversation to end-products.......................................11 Figure 1.4 A sulfurtransferase reaction catalyzed by rhodanese.........................................27 Figure 1.5 The porous structure of biochar invites microbial colonization- pine saw dust-derived biochar.............................................................................................................37 Figure 2.1 Relationship between methane in the gas and HCN content in treatments.......69 Figure 2.2 Effect of HCN content in treatment on ammonia production is expressed as all data in each treatment during the in vitro fermentation..................................................69 Figure 2.3 Effect of HCN content in treatment on ammonia production is expressed as average in each treament......................................................................................................69 Figure 3.1 The negligible growth rate of Laisind cattle fed bitter cassava foliage as protein source in Period 1 was dramatically increased by adding 4% of brewers’ grains to the diet in Period 2...........................................................................................................82 Figure 3.2 Growth curves of Laisind cattle showing the change in live weight gain after introduction of 4% brewers’ grains (as % of diet DM) to those fed bitter cassava foliage...................................................................................................................................83 Figure 3.3 Mean values for VFA proportions in rumen fluid from cattle in Period 2........85 Figure 4.1 Effect of additives, and source of cassava leaf (bitter or sweet variety) on gas production after 24h fermentation...............................................................................100 Figure 4.2 Effect of stage of the fermentation on the methane content of the gas............100 Figure 4.3 Effect of additives, and source of cassava leaf on methane content of the gas after 24h fermentation........................................................................................................101 Figure 4.4 Interaction between source of cassava leaf and additive with brewers’ grains on methane content in the gas............................................................................................103 Figure 4.5 Viable Saccharomyces cells in brewers’ grain and in cassava pulp fermented only with yeast (YFCP) or with yeast and urea (YFCP-U-DAP) after 07 days of fermentation...................................................................................................................104 Figure 4.6 Lactobacilli in brewers’ grain and in cassava pulp fermented only with yeast (YFCP) or with yeast and urea (YFCP-U-DAP) after 07 days of fermentation................104 Figure 5.1 Condensed tannin in petiole and leaf from cassava foliage.............................116 ix Figure 5.2 HCN equivalent in petiole and leaf from cassava foliage...............................116 Figure 5.3 DM intake of leaf and petiole of sweet and bitter cassava varieties when the goats had free access to both (sweet+ bitter foliage) in Square 2......................................117 Figure 5.4 Individual and combined effects of additives and source of cassava foliage on N retention; (SW as Sweet; SW-BIT as Sweet and Bitter)...........................................119 Figure 5.5 Bach Thao goat from Latin Square 2...............................................................120 Figure 5.6 Methane: carbon dioxide ratios in mixed eructed gas and air in goats............120 Figure 5.7a Relationship between methane: carbon dioxide ratio in mixed eructed gas and air and nitrogen retention (includes all 8 goats)..........................................................121 Figure 5.7b Relationship between methane: carbon dioxide ratio and nitrogen retention (excluding the outlier result)..............................................................................................121 x List of Tables Table 1.1 Nutrient composition of fresh cassava leaf.........................................................20 Table 1.2 Essential amino acid profile of cassava leaf........................................................21 Table 1.3 Chemical composition of brewers’ grain............................................................31 Table 2.1 Composition of the substrates.............................................................................65 Table 2.2 Ingredients in buffer solution..............................................................................66 Table 2.3 Chemical composition of the ingredients in the substrate...................................67 Table 2.4 Mean values for gas production in 24 hours, methane in the gas and per unit DM mineralized in an in vitro rumen fermentation.............................................................68 Table 2.5 Mean values for content of condensed tannin and HCN in the leaves of sweet and bitter varieties of cassava leaves, ammonia concentration and methane production per DM mineralized after 24h incubation............................................................................68 Table 3.1 The chemical composition of ingredients...........................................................78 Table 3.2a Mean values for feed intake, change in live weight and feed conversion of Laisind cattle in Period 1.....................................................................................................81 Table 3.2b. Mean values for feed intake, change in live weight and feed conversion of Laisind cattle (in Period 2)..................................................................................................82 Table 3.3 Mean values for thiocyanate in the urine of cattle..............................................84 Table 3.4 Mean values for VFA proportions in rumen fluid of cattle (in Period 2)..........84 Table 3.5 Mean values of methane: carbon dioxide ratios in mixed eructed gas/air of cattle.....................................................................................................................................85 Table 4.1 Chemical composition of substrates....................................................................99 Table 4.2a Effect of source of cassava variety on gas production and methane percentage in the gas..........................................................................................................101 Table 4.2b Effect of additive on gas production and methane percentage in the gas.......102 Table 5.1 Layout of each Latin Square.............................................................................112 Table 5.2 Dry matter (DM) and crude protein (CP) of ingredients...................................112 xi Table 5.3 Mean values for effects of cassava foliage (sweet or bitter) on % DM, tannin and HCN equivalent in leaves and petioles........................................................................115 Table 5.4 Mean intakes of leaf and petiole for the goats in Square 2 that had free access to foliage of both sweet and bitter varieties.......................................................................116 Table 5.5 Mean values (g/d) for effects of cassava foliage (bitter or sweet) and of additives on DM intake (DMI), apparent digestibility of DM and crude protein (CP) and N balance.....................................................................................................................118 Table 5.6 Mean values for effects of additives on N retention.........................................118 Table 5.7 Mean values for VFA proportions (mol %), acetic: propionic ratio, rumen ammonia, daily urine volume, daily excretion of thiocyanate (SCN) in urine and CH4:CO2 ratio in mixed eructed gas and air.......................................................................119 xii Abbreviation ADG Average daily gain ATP Adenosine tri-phosphate ADF Acid detergent fiber BG Brewers’ grain CP Crude protein CF Crude fiber CFU Colony-forming unit DM Dry matter DMI Dry matter intake EPS Extracellular polymeric substances EE Ether extract HCN Hydrocyanic acid GE Gross energy xiii LW Live weight MOS Manna-oligosaccharide N Nitrogen NADH Nicotinamide adenine dinucleotide hydride NDF Neutral detergent fiber NPN Non-protein nitrogen RDP Rumen degradable protein Phosphoenolpyruvate PEP Standard error mean SEM UDP Un-degradable protein VFA Volatile fatty acid xiv INTRODUCTION 1. Problem statement Cassava is perspective plant to climate change adaptation; its pests and its diseases resistance and greater drought tolerance is a major factor in ranking cassava in the food security of the world (Jarvis et al. 2012). In Vietnam, cassava is second crop, is grown mainly in both at the household and small-scale processor level (Hoang Kim et al. 2000). From the successful experiment in utilization of cassava foliage (sweet variety) as protein source on cattle which was originally reported by Ffoulkes and Preston (1978), and then have been successfully fed as fresh state to goat and cattle in Cambodia (See report of Preston and Rodríguez Lylian, 2004), that make cassava foliage become important plant protein source in ruminant diet. Nevertheless, cyanide toxin in fresh cassava foliage, especially bitter cassava foliage, is the main obstacle for animal such as restricting the consumption intake of ruminant or causing poisoning when they consume rapidly. Currently, as the quantity of bitter cassava (high cyanide content) develop more predominant than sweet cassava (lower cyanide content) on the field, utilization of bitter cassava foliage in diet will match reality more; however, looking for feeding method of minimizing negative effect of cyanide toxin, from that can be utilized bitter cassava foliage in diet will more match reality but will a challenge. Many studies are beginning to be interested in cyanide toxic that has certain effect on methanogenic bacteria population by inhibited methanogenesis activity lead to diminish methane production (Ch Olga Rojas et al. 1999; Phuong et al. 2012; Phanthavong et al. 2015). However, whether cyanide may affect overall microbial activity and impact the rate of rumen fermentation indirectly, it is still not fully understood. Previously, the knowledge of ruminant nutritionists focused on rumen, but the impact of cyanide on rumen fermentation may profoundly influence lower digestive physiology of ruminant and must be considered to fully understand when utilizing bitter cassava foliage in diet. Even so, the challenge of bitter cassava foliage diet (high cyanide content) is a new approach but must require the safety for animal's health. 1 Therefore, building appropriate feeding method for fresh cassava foliage diet, particularly bitter cassava foliage, without cyanide poisoning is needed to utilize cassava foliage more effective in the ruminant feeding system. 2. Aim and objective of the study 2.1 Aims of the study The aim of this thesis was to develop a greater understanding of both the constraints and benefits of using cassava foliage in ruminant feeding systems. From these things can improve the utilization of cassava foliage in ruminant feeding by enhancing its properties as a source of bypass protein and verify the role of HCN toxin in cassava foliage on the reduction of methane production that was built on earlier findings. 2.2 Objective of the study The following objectives are required to accomplish the aim of this research: (i) Determining the trend influences of HCN concentration in cassava foliage on the characteristic of in vitro rumen fermentation such as gas and methane production, ammonia concentration. (ii) Considering the benefit of brewers’ grain to “bitter” cassava foliage (KM94) diet by examining Saccharomyces and acid lactic bacteria in fresh brewers’ grain and compare it with potential fermented cassava pulp on gas and methane production of ruminal in vitro incubation. (iii) Building feeding method of “bitter” cassava foliage (KM 94 variety; moderate HCN content) diet by added 4% brewers’ grain (of DM) and/or 1% biochar (as DM), then evaluating the effects of this feeding method on growth, digestibility/N retention, excretion of thiocyanate in urine and methane production of cattle and goat. 3. Research hypotheses The research hypothesizes following: (i) Higher HCN content in bitter cassava foliage would be more effective in reducing methane production in both rumen in vitro incubations and in vivo experiment rather than foliage from a sweet variety (low HCN content). 2 (ii) By added 4% brewers’ grain and/or 1% biochar in bitter cassava foliage (KM94 variety) diet would lead to: (1) improving the growth rate of cattle fed a basal diet of cassava pulp-urea; and (2) increasing N-retention, reducing thiocyanate in urine of goats having free access to both bitter and sweet cassava foliage. 4. Significant/Innovation of study This dissertation successfully demonstrated that HCN in cassava foliage is main factor for reduction of methane production while the earlier finding could only predict the role of HCN for decreased methane. Currently, the best-known cassava foliage to feed animal is “sweet” cassava foliage with low cyanide content, my dissertation succeeds to build feeding method for “bitter” cassava foliage diet (higher cyanide content) with support of adding restricted brewers grain (4% of DM) and biochar (1% of DM) to feed cattle and goats without cause HCN toxicity. Additionally, discovery of the feeding of the bitter cassava foliage appear to modify the rumen fermentation lead to increases in nitrogen retention associated with reduced methane production, it made a part of this dissertation provided the implication for new approach of the proposed partial shift in sites of digestion (from rumen to small intestine and the cecal-colon region) that previously it is thought that only rumen fermentation has a truly symbiotic relationship with the ruminant. CHAPTER 1. LITERATURE REVIEW 3 1.1 Rumen fermentation and methane production 1.1.1 Rumen fermentation Understanding rumen fermentation is an important step in applying the basis knowledge to improve rumen function and utilizing feed efficiency. Rumen microbial fermentation is crucial for growth and production of ruminants. Although there are many kinds of microbes have been found in the digestion of ruminant, yet it is thought that only ruminal microbes have a truly symbiotic relationship with the host until now. Individual rumen microbial species have developed in a complex process of evolution extending over a long period and provide nature's best example of microbial symbioses. Rumen ecosystem is a self-contained ecosystem, the feed is fermented by ruminal microbes to end products along with microbial biomass, is the source of energy and protein to respond to essential nutritional needs for ruminant. With diverse anaerobic microbes in the rumen, ruminant is able to degrade the complex fiber source to provide nutrient essential is readily digested by the host while this is completely restricted on non-ruminant (Owens and Basalan 2016). The rumen contains a variety of microorganism including main groups such as bacteria, fungi, and protozoa. In which bacteria is considered majority microbes with a diversity of bacteria genera that its classification base on the preference for certain substrates. There are three forms for the distribution of bacteria in the rumen, free-floating bacteria constitute a minor component (~30 %), and bacteria adhere on feed particle account for the largest population with 70%. Both bacteria groups mainly participate in the digestion of feed. The last distribution form is bacteria that attach to the ruminal epithelial cells. They do not make any significant contribution on feed digestion, but it is assumed that they may scavenge oxygen to maintain an anaerobic medium. Otherwise, epimural bacteria produce urease enzyme to hydrolyze urea (Nagaraja et al. 2016), this allows ruminant can use urea more efficient than non-ruminant. The cellulose-degrading bacteria are able to degrade cellulose, one of the components in the cell wall of plant. They include Ruminococcus albus, Fibrobacter succinogenes and Ruminococcus flavefaciens (Valente et al. 2016). Cellulolytic bacteria prefer neutral pH between 6 and 9 is for best maintenance and growth, with pH 4