Luận án tiến sĩ: Tác dụng synergic của sắn, bã bia, biochar lên sản xuất methane và hiệu suất vật nuôi nhai lại - Đại học Nông Lâm Huế 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 DOCTOR OF PHILOSOPHY IN ANIMAL SCIENCES HUE, 2020 luan an 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. NGUYEN HUU VAN SUPERVISOR 2: DR. DINH VAN DUNG HUE, 2020 luan an 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.org/ Hue University, 2020 Le Thuy Binh Phuong i luan an 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 luan an My Ph. 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. 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.

My classmate in Ph. 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. is so appreciated. iii luan an 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 luan an Table of Contents List of Figures.ix List of Tables. Aim and objective of the study.1 Aims of the study.2 Objective of the study. Significant/Innovation of study.1 Rumen fermentation and methane production.2 Volatile fatty acid pattern.9 Pathway of methane production.5 Effect of feeding system on rumen fermentation.2 Understanding ruminal microorganism.1 The self-detoxify mechanism of ruminal microbes.2 Interaction of ruminal microorganism in biofilm formation.3 Using agro-industrial by-products for ruminant feeding system.19 Effect of tannin content in cassava leaves on the ruminant feeding system.21 Cyanogenic glucosides in cassava leaf.25 v luan an New potentially application on mitigation of HCN effect.30 Using brewers grain in ruminant feeding.3 Potential using cassava root pulp as energy source or protein enrichment source .4 Biochar: application as an additive.4 Supplementary Saccharomyces cerevisiae: concept on detoxification.

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.2 Materials and methods.64 Location and duration.3 Results and discussion.4 Conclusions and recommendation. 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 vi luan an 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.3 Results and discussion.4 Conclusions and recommendation. 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.2 Materials and methods.97 Location and duration.97 Treatments and design.97 In vitro incubation.3 Results and discussion.4 Conclusions and recommendation. EFFECT OF ADDITIVES (BREWER’S GRAIN AND BIOCHAR) AND CASSAVA VARIETY (SWEET VERSUS BITTER) ON NITROGEN vii luan an RETENTION, THIOCYANATE EXCRETION AND METHANE PRODUCTION BY BACH THAO GOATS.2 Materials and Methods.111 Location and duration.111 Feeds and feeding.112 Animals and feeding system.3 Results and discussion.4 Conclusions and recommendation.

GENERAL DISCUSSION AND CONCLUSIONS.3 Implication and further research. PROTOCOL D1- DETERMINATION OF THIOCYANATE IN URINE. 139 List of Figures Figure 1.1 Pathway of VFA in metabolism.2 The reaction of methane generation.10 viii luan an Figure 1.3 The pathway of hexose conversation to end-products.4 A sulfurtransferase reaction catalyzed by rhodanese.5 The porous structure of biochar invites microbial colonization- pine saw dust-derived biochar.1 Relationship between methane in the gas and HCN content in treatments.2 Effect of HCN content in treatment on ammonia production is expressed as all data in each treatment during the in vitro fermentation.3 Effect of HCN content in treatment on ammonia production is expressed as average in each treament.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 2.2 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.3 Mean values for VFA proportions in rumen fluid from cattle in Period 2.1 Effect of additives, and source of cassava leaf (bitter or sweet variety) on gas production after 24h fermentation.2 Effect of stage of the fermentation on the methane content of the gas.3 Effect of additives, and source of cassava leaf on methane content of the gas after 24h fermentation.4 Interaction between source of cassava leaf and additive with brewers’ grains on methane content in the gas.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.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.1 Condensed tannin in petiole and leaf from cassava foliage.2 HCN equivalent in petiole and leaf from cassava foliage.116 ix luan an 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 2.4 Individual and combined effects of additives and source of cassava foliage on N retention; (SW as Sweet; SW-BIT as Sweet and Bitter).5 Bach Thao goat from Latin Square 2.6 Methane: carbon dioxide ratios in mixed eructed gas and air in goats.7a Relationship between methane: carbon dioxide ratio in mixed eructed gas and air and nitrogen retention (includes all 8 goats).7b Relationship between methane: carbon dioxide ratio and nitrogen retention (excluding the outlier result).121 x luan an List of Tables Table 1.1 Nutrient composition of fresh cassava leaf.2 Essential amino acid profile of cassava leaf.3 Chemical composition of brewers’ grain.1 Composition of the substrates.2 Ingredients in buffer solution.3 Chemical composition of the ingredients in the substrate.4 Mean values for gas production in 24 hours, methane in the gas and per unit DM mineralized in an in vitro rumen fermentation.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.1 The chemical composition of ingredients.2a Mean values for feed intake, change in live weight and feed conversion of Laisind cattle in Period 1. Mean values for feed intake, change in live weight and feed conversion of Laisind cattle (in Period 2).3 Mean values for thiocyanate in the urine of cattle.4 Mean values for VFA proportions in rumen fluid of cattle (in Period 2).5 Mean values of methane: carbon dioxide ratios in mixed eructed gas/air of cattle.1 Chemical composition of substrates.2a Effect of source of cassava variety on gas production and methane percentage in the gas.2b Effect of additive on gas production and methane percentage in the gas.1 Layout of each Latin Square.2 Dry matter (DM) and crude protein (CP) of ingredients.112 xi luan an Table 5.3 Mean values for effects of cassava foliage (sweet or bitter) on % DM, tannin and HCN equivalent in leaves and petioles.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.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.6 Mean values for effects of additives on N retention.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 luan an 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 luan an 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 luan an 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.