SYNERGIC EFFECT OF CASSAVA (MANIHOT ESCULENTA CRANTZ) FOLIAGE, BREWER’S GRAINS, AND BIOCHAR ON METHANE PRODUCTION AND PERFORMANCE OF RUMINANTS

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 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 Problem statement Aim and objective of the study .2 2.1 Aims of the study 2.2 Objective of the study .2 Research hypotheses .2 Significant/Innovation of study CHAPTER LITERATURE REVIEW .4 1.1 Rumen fermentation and methane production .4 1.1.1 Rumen fermentation 1.1.2 Volatile fatty acid pattern 1.1.3 Protein metabolism 1.1.4 Methane production 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 v New potentially application on mitigation of HCN effect 29 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 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 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 vi 3.1 Introduction 76 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 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 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 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 Figure 1.2 The reaction of methane generation 10 viii Madsen, J., Bjerg, B S., Hvelplund, T M., Weisbjerg, R and Lund, P., 2010 Methane and carbon dioxide ratio in excreted air for quantification of the methane production from ruminants Livestock Science 129, 223–227 Minitab 2016 Minitab user's guide Data analysis and quality tools Release 13.1 for windows Minitab Inc., Pennsylvania, USA Nguyen Thi Thu Hong, Nguyen Thi Ngoc Trang, Duong Nguyen Khang and Preston, T R., 2018 Supplementing water spinach (Ipomoea aquatica) with foliage of Mimosa pigra or Leucaena leucocephala and coconut oil; effects on N retention and enteric methane emissions in growing goats; Livestock Research for Rural Development Volume 30, Article #207 http://www.lrrd.org/lrrd30/12/hong30207.html Onwuka, C F I., Akinsoyinu, A O and Tewe, O O., 1992 Role of sulphur in cyanide detoxification in ruminants Small Ruminant Research 8(4): 277-284 Phanthavong, V., Sangkhom, I., Preston, T R., Dung, D V and Ba, N X., 2018 Effect of leaves from sweet or bitter cassava and brewers’ grains on methane production in an in vitro rumen incubation of cassava root pulp-urea Livestock Research for Rural Development Volume 30, Article #167 http://www.lrrd.org/lrrd30/9/phant30167.html 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 http:// www.lrrd.org/lrrd28/5/phon28072.html 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 Preston, T R and Leng, R A., 1987 Matching Livestock Systems to Available Resources in the Tropics and Subtropics Penambul Books, Australia Web version http://www.cipav.org/P&L/preston&leng.htm Preston, T R., 2015 The role of biochar in farming systems producing food and energy from biomass In: Geotherapy: Innovative Methods of Soil Fertility Restoration, Carbon Sequestration and Reversing CO2 Increase (Editor: Thomas J Goreau) CRC Press, Tayler and Francis Group, Boca Raton, Florida USA SAS 2010 Statistical Analysis Software, SAS/STAT | SAS https://www.sas.com/en_us/ software/stat.html 124 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 http://www.lrrd.org/lrrd28/10/seng28178.html Silivong, P and Preston, T R., 2016 Supplements of water spinach (Ipomoea aquatica) and biochar improved feed intake, digestibility, N retention and growth performance of goats fed foliage of Bauhinia acuminata as the basal diet Livestock Research for Rural Development Volume 28, Article #98 http://www.lrrd.org/lrrd28/5/sili28098.html 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 http://www.lrrd.org/lrrd30/3/siliv30055.html Sina, V., Preston, T R and Tham, T H., 2017 Brewers’ grains have a synergistic effect on growth rate of goats fed fresh cassava foliage (Manihot esculenta Crantz) as basal diet Livestock Research for Rural Development Volume 29, Article #137 http:// www.lrrd.org/lrrd29/7/sina29137.html 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, page 67-71 Thuy Hang, L T., Preston, T R., Leng, R A and Ba, N X., 2018 Effect of biochar and water spinach on feed intake, digestibility and N-retention in goats fed urea-treated cassava stems Livestock Research for Rural Development Volume 30, Article #93 http://www.lrrd.org/lrrd30/5/thuyh30093.html Vongkhamchanh, B., Preston, T R., Leng, R A., An, L V and Hai, D T., 2018 Effect of biochar on growth performance of local “Yellow” cattle fed ensiled cassava roots, fresh brewers’ grains and rice straw Livestock Research for Rural Development Volume 30, Article #168 http://www.lrrd.org/lrrd30/9/bobby30168.html 125 CHAPTER GENERAL DISCUSSION AND CONCLUSIONS 6.1 General discussion The important of cassava in developing countries is the reflection of its high adaptation to climate change (Jarvis et al 2012), high disease resistance and being the source of an important export commodity (starch) as well as contributing to the livelihood of poor people Therefore, in promoting an increasing role for cassava production in tropical countries, attention should be paid on several main issues: (i) increasing the utilization of the byproducts from cassava starch factories; (ii) utilizing the residual foliage and stems of cassava available at the time of root harvest; (iii) managing the cyanogenic glucosides present in highest concentration in the leaves; and (iv) optimizing the use of cassava foliage as a source of bypass protein in ruminant feeding systems by cultivating and using it as a specialist forage crop The foliage from “sweet” varieties of cassava, which contain low levels of cyanogenic glucosides (the precursors of HCN) has been shown to be an effective source of bypass protein in ruminant feeding systems (Ffoulkes and Preston 1978; Wanapat et al 1997; Promkot and Wanapat 2003; Sath et al 2008) However, the “sweet” varieties are less productive in leaves and roots compared with those that contain higher levels of HCN-precursors Therefore, there is a major incentive to develop ways in which the potential toxicity of the higher-yielding cultivars can be managed so that their yield advantages can be accommodated in livestock feeding systems It is often recommended that post-harvest bitter cassava foliage can be utilized after ensiling or drying However, the disadvantage is that under these conditions it is easy to generate mycotoxins during storage Phanthavong et al (2018) reported the presence of mycotoxin contaminants in mixed feeds containing 30% cassava foliage ensiled for 03 weeks Such feeds, rich in protein and moisture, provide habitat of rich nutrient content for growth of fungi that give rise to mycotoxins However, the feeding of fresh cassava foliage to animals, particularly of the bitter variety, can lead to poisoning because of the presence of cyanogenic glycosides that give rise to HCN 126 when digested in the animal This is the biggest obstacle to the widespread use of cassava leaves as feed Therefore, greater insight on the impact of bitter cassava leaves on rumen fermentation, from the point of view of minimizing HCN toxicity, will be a useful contribution that will help to orientate future research activities This thesis aims to increase our knowledge on the effect of cassava foliage on rumen methane production and on growth of ruminant animals Each experiment follows a logical sequence in which account was also taken of the implications from related pioneering experiments being done by colleagues The first rumen in vitro experiment (Chapter 2) demonstrated that the higher levels of HCN precursors in Japan, KM94 and KM140-1 varieties were associated with reduced production of methane when compared with leaves from a sweet variety (Gon) This result can be evaluated in the context of previous findings (Phuong et al 2012; Outhen et al 2011) that bitter cassava leaves in the fresh state reduced methane production compared with dried cassava leaves; and that drying the leaves reduced the level of HCN precursors The effect of the HCN precursors in reducing rumen methane is consistent with results of the in vitro experiment (Chapter and 4) and the in vivo trials (Chapter and 5) The role of tannins to form tannin-protein complexes and/or inactivate proteolytic enzymes has been demonstrated by many research (Kuma and Singh 1984; Wanapat 1995; Gerlach et al 2018; Ravindran 1993), thus the tannin content of plants is seen as an element that can facilitate the bypass (or escape) of protein from the rumen to be better utilized in the lower gut However, our analyses (Chapter 2) indicated tannin content was similar in leaves from different cassava varieties, and that ammonia concentration in rumen fluid was more closely related (negatively) with levels of HCN precursors than with tannins This finding is supported by earlier results from Majak (1991) that the HCN released in rumen fluid negatively affected microbial activity resulting in decreased gas production We therefore hypothesize that higher levels of HCN precursors in cassava leaves inhibits not only methanogenesis but has an adverse effect on overall microbial activity leading to increased escape of nutrients for digestion/fermentation in the lower regions of the ruminant digestive tract This concept is in line with the “new paradigm” proposed by Phonethep et al (2017) that diets rich 127 in “escape” nutrients would also result in reduced overall production of methane in view of the finding that fermentation in the cecum-colon was dominated by acetogenesis and not methanogenesis (Leng 2018) On the basis of these results, and the inferences from them, the experiment described in Chapter was designed to compare the effect of sweet and bitter cassava foliage as a combined source of bypass protein and fiber on the growth rate of cattle given an intensive fattening diet based in cassava root pulp and urea The cattle were challenged with three sources of protein: (i) brewers’ grain and rice straw as a positive control treatment; (ii) foliage of a cassava variety (KM94) with medium level of HCN precursors; and (iii) foliage of a sweet cassava variety (Gon) with low level of HCN precursors The results were dramatic: cattle fed the “bitter” cassava foliage did not grow whereas growth was normal when the cassava foliage was of the sweet variety Experiences related to us by colleagues (Phanthavong et al 2016) indicated that derived cattle fed bitter cassava foliage exhibited a “craving” to eat brewers’ grains Bearing in mind that brewers’ grains had been identified as a potential source of βglucan, an established “prebiotic” derived from barley cell wall (Delaney et al 2003) and yeast cell wall (Hojjatollah Shokri et al 2009), we hypothesized that the experience quoted by Phanthavong et al (2016) was an example of “self-medication” such that the cattle had become aware (from animals in the adjacent pens) that brewer’s grains were an antidote to the “HCN” toxicity caused by the cyanogenic glucosides in the bitter cassava foliage We therefore tested the effect of adding 5% brewers’ grains to the diet containing foliage of bitter cassava There was an immediate response in increased intake of the leaves and in the growth rate of the cattle The urine excretion of thiocyanate (the degradation product of detoxification of HCN) was reduced by half The knowledge that restricted amounts of brewer’s grain could serve as an antidote to the HCN precursors in leaves of bitter cassava leave suggested a positive line of research to follow; namely, where to find (or how to produce) products derived from grain/yeast cell wall fermentation According to these ideas, experiment (Chapter 4) was planned to compare other cell wall fermentation products as alternative sources of additives that would simulate 128 the effect of brewers’ grains in rumen in vitro incubations The additives tested in a comparative study with brewers’ grains as control treatment were: rice wine starter culture (RWS), yeast-fermented cassava pulp (YFCP) and cassava pulp fermented with yeast, urea and di-ammonium phosphate (YFCP-U-DAP) The basal substrates in the in vitro incubations were: cassava pulp, urea, and leaves of either sweet or bitter cassava varieties There was an interaction in treatment effects as none of the supplements affected rumen methane production when bitter cassava leaves were the source of protein In contrast, brewers’ grains reduced methane production only when leaves of sweet cassava were the protein source The other additives had no effect on rumen methane irrespective of the source of cassava leaves On the one hand, these results confirmed the findings reported in Chapter that HCN precursors reduce rumen methane production On the other hand, the lack of effect of the various fermentation products confirmed that the “prebiotic” effect from plant cell walls (i.e.: the presence of β-glucan and/or related compounds) required acid-hydrolysis of the cell walls as would normally be achieved when the fermentation products in a beer distillery are distilled to separate the alcohol We hypothesize that brewers’ grains, and similar fermentation/distillation by products, eg: “Hem” the byproduct from production of rice wine are effective in reducing methane production (Sengsouly and Preston 2016; Inthapanya et al 2016) and in detoxifying HCN (Chapter 3), because of the “prebiotic” effect of the β-glucan resulting from the fermentation/distillation of the cereal and/or yeast cell walls that occurs in ethanol production from barley and or rice The knowledge that the yeast in brewers’ grains is “deactivated” as is the yeast in “Hem” (presumably due to the elevated temperature involved in the distillation process), implies that the beneficial effects of these two byproducts in the rumen and in the animal are the results of improved habitat supporting diverse microbial communities in biofilms (Leng 2017) Increasing the understanding of the role of microbial communities in biofilms in the rumen and in the animal is important in order to develop improved nutritional strategies for the use of foliage from “bitter” varieties of cassava Thus the fourth experiment (Chapter 5) aimed to measure the response of goats to contrasting levels of cyanogenic 129 glucosides by offering: (i) foliage of a sweet variety as the sole diet; or (ii) allowing the goats to have free access to the foliage of both sweet and bitter varieties Brewers’ grains and biochar in research of Thuy Hang et al (2018) were offered as contrasting sources of prebiotics in both “cassava systems” The experimental design (a separate Latin square for each cassava system; with the same four prebiotic treatments in each) was a compromise between the need to economize on resources but guaranteeing adequate statistical control Such a changeover design can be criticized when the treatments involved may well have carry-over effects when changing treatments within the same animals On the other hand, the design permitted contrasting intakes of cyanogenic glucosides but with the goats in “control” of what they found convenient to consume In fact, the balance between sweet and bitter cassava foliage, freely chosen by the goats, was close to 50:50 It is concluded that this selection balance was a further example of “self-medication” or “self-satisfaction” on the part of the goats as demonstrated by the 25% increase in DM intake and the 22% increase in N retention recorded for the freely-accessed combination of high and low HCN-potential diets It is hypothesized that the nutritional advantage to the goats of consuming 50% of their diet as bitter cassava was manifested through a partial shift of digestion from the rumen to the lower digestive tract (small intestines and cecum-colon) thus improving the balance of nutrients available to the animal by reducing losses from methane production in the rumen As expected, the response to the prebiotic additives was more variable than in previous experiments; however, the results indicate overall beneficial effects from the additives in increasing N retention, reducing methane production and excretion of thiocyanate The effect of adding both brewers’ grains and biochar in combination with mixed sweet and bitter cassava foliage was a 58% higher N retention than on the control treatment of only sweet cassava and no additives An important finding was the negative relationship between methane production and N retention, confirming the original comments of Johnson et al (1993) (cited by Johnson and Johnson 1995) regarding the potential gain in dietary net energy by reducing enteric methane production 6.2 Conclusions 130 In summary, the body of work presented in this dissertation has shown that supplementing ruminant diets with cassava foliage reduces rumen methane production, and the effect is more pronounced with varieties containing higher levels of cyanogenic glucosides, which give rise to HCN in the rumen However, the risk of toxicity of cyanide in ruminants (cattle and goats) can be reduced by adding to the diet either brewer’s grains or biochar or both as a “prebiotic” source In the presence of “prebiotic”, challenges of bitter cassava leave in the feeding system of goat and cattle did not negatively impact to feed intake and animal’s health In more detail, adding restricted (4% of DM) brewers’ grain into “bitter” cassava foliage as a main protein source appeared reduction of excreted thiocyanate in urine, lead to significant improvement of growth rate of cattle compare to only “bitter” cassava foliage Moreover, synergistic of brewers’ grain (4% of DM) and biochar (1% of DM) as additives could show substantially same effectiveness even when goat was fed “bitter” cassava foliage at up to 50% in the diet On the contrary, the feeding of the bitter cassava foliage appeared to modify the rumen fermentation leading to an improved balance of nutrients at the whole animal level, as manifested by the 20% increase in nitrogen retention associated with decreased production of methane 6.3 Implication and further research The dissertation is the process with step-by-step experiments has provided evidence for the benefits of restricted amounts (4 %) of brewers’ grains and/or biochar (1%) as a means of avoiding risks of HCN toxicity in ruminant feeding systems using bitter cassava foliage as the source of bypass protein However, the implications of the proposed partial shift in sites of digestion (from rumen to small intestine and the cecalcolon region) must be substantiated in long-term feeding trials Research is also needed to monitor how modifications in the rumen fermentation (eg: increased escape of digestible organic matter) affect fermentation in the cecum References Delaney, B., Nicolosi, R J., Wilson, T A., Carlson, T., Frazer, S., Zheng, G H., Hess, R., Ostergren, K., Haworth, J., Knutson, N., 2003 Beta-glucan fractions from barley and oats are similarly antiatherogenic in hypercholesterolemic Syrian golden hamsters Journal of Nutrition 133(2): 468-475 131 Ffoulkes, D and Preston, T R., 1978. Cassava or sweet potato forage as combined sources of protein and roughage in molasses based diets: effect of supplementation with soybean meal http://www.cipav.org.co/TAP/TAP/TAP33/3_3_1.pdf Gerlach, K., Pries, M., Südekum, K-H., 2018 Effect of condensed tannin supplementation on in vivo nutrient digestibilities and energy values of concentrates in sheep Small Ruminant Research 161: 57-62 Hojjatollah Shokri, Farzad Asadi and Ali Reza Khosravi, 2008 Isolation of β -glucan from the cell wall of Saccharomyces cerevisiae Natural Product Research: Formerly Natural Product Letters, 22:5, 414-421 Inthapanya, S., Preston, T R and Leng, R A., 2016. Ensiled brewers’ grains increased feed intake, digestibility and N retention in cattle fed ensiled cassava root, urea and rice straw with fresh cassava foliage or water spinach as main source of protein Livestock Research for Rural Development Volume 28, Article #20. http://www.lrrd.org/lrrd28/2/sang28020.htm Jarvis, A., Ramirez-Villegas, J., Herrera Campo, B.V., Navarro-Racines, C., 2012 Tropical Plant Biology Volume 5, Issue 1, Page 9-29 https://doi.org/10.1007/s12042012-9096-7 Johnson, K A and Johnson, D E., 1995 Methane emissions from cattle. Journal of Animal Science 73:2483-2492 Kuma, R., Singh, M., 1984 Tannins: their adverse role in ruminant nutrition Journal of Agriculture and Food Chemistry 32, pages 447-458 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 Leng, R A., 2018 Unravelling methanogenesis in ruminants, horses and kangaroos: the links between gut anatomy, microbial biofilms and host immunity Animal Production Science, 58, 1175–1191 Perspectives on Animal Biosciences https://doi.org/10.1071/AN15710 Majak, W., 1991 Metabolism and absorption of toxic glycosides by ruminants Journal of Range Management, 45(1):67-71 Phanthavong, V., Preston, T R., Vorlaphim, T., Dung, D V and Ba, N X., 2018 Fattening “Yellow” cattle on cassava root pulp, urea and rice straw: completely mixed ration system with cassava foliage as protein supplement compared with feeds not mixed and brewers’ grains as protein source. Livestock Research for Rural Development Volume 30, Article #169. Retrieved January 4, 2019, from http://www.lrrd.org/lrrd30/10/phan30169.html 132 Phanthavong, V., Preston, T R., Viengsakoun, N and Pattaya, N., 2016 Brewers' grain and cassava foliage (Manihot esculenta Cranz) as protein sources for local “Yellow” cattle fed cassava pulp-urea as basal diet Livestock Research for Rural Development Volume 28, Article #196 http://www.lrrd.org/lrrd28/11/phan28196.html 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 http:// www.lrrd.org/lrrd28/5/phon28072.html 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.  Retrieved July 25, 2019, from http://www.lrrd.org/lrrd23/2/outh23023.htm 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 19, 2018, from http://www.lrrd.org/lrrd24/10/phuo24189.htm Promkot, C and Wanapat, M., 2003 Ruminal degradation and intestinal digestion of crude protein of tropical protein resources using nylon bag technique and three-step in vitro procedure in dairy cattle Livestock Research for Rural Development Volume 15, Article #81. http://www.lrrd.org/lrrd15/11/prom1511.htm Ravindran, V., 1993 Cassava leaves as animal feed: Potential and limitations Journal of the Science of Food and Agriculture 61(2): 141-150 Sath, K., Borin, K and Preston, T R., 2008 Effect of levels of sun-dried cassava foliage on growth performance of cattle fed rice straw Livestock Research for Rural Development Volume 20, from http://www.lrrd.org/lrrd20/supplement/sath2.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 January 2, 2019, from http://www.lrrd.org/lrrd28/10/seng28178.html Sivilai, B., Preston, T R., Leng, R A., Hang, D T., and Linh, N Q., 2018 Rice distillers’ byproduct and biochar as additives to a forage-based diet for growing Moo Lath pigs; effects on growth and feed conversion Livestock Research for Rural Development Volume 30, Article #111 http://www.lrrd.org/lrrd30/6/lert30111.html 133 Sina, V., Preston, T R and Tham, T H., 2017 Brewers’ grains have a synergistic effect on growth rate of goats fed fresh cassava foliage (Manihot esculenta Crantz) as basal diet Livestock Research for Rural Development Volume 29, Article #137 Retrieved August 16, 2018, from http://www.lrrd.org/lrrd29/7/sina29137.html Thuy Hang, L T., Preston T R., Ba, N X., and Dung, D V., 2018 Digestibility, nitrogen balance and methane emissions in goats fed cassava foliage and restricted levels of brewers’ grains Livestock Research for Rural Development Volume 30, Article #68. Retrieved January 3, 2019, from http://www.lrrd.org/lrrd30/4/thuy30068.html Wanapat, M., Pimpa, O., Petlum, A and Boontao U., 1997 Cassava hay: A new strategic feed for ruminants during the dry season Livestock Research for Rural Development Volume 9, Article #18.http://www.lrrd.org/lrrd9/2/metha92.htm Wanapat, M., 1995 The use of local feed resources for livestock production in Thailand Proceedings of the International Conference on Increasing Animal Production with Local Resources (Editor: Guo Tingshuang) China Forestry Publishing House, Ministry of Agriculture, China 134 PUBLICATION LIST Paper 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 Paper Binh P L T, Preston T R, Duong K N and Leng R A 2017: 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 Livestock Research for Rural Development Volume 29, Article #104 http://www.lrrd.org/lrrd29/5/phuo29104.html Paper 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 Paper Phuong L T B, Preston T R, Van N H and Dung D V 2019: Effect of additives (brewer’s grains and biochar) and cassava variety (sweet versus bitter) on nitrogen retention, thiocyanate excretion and methane production by Bach Thao goats Livestock Research for Rural Development Volume 31, Article #1 http://www.lrrd.org/lrrd31/1/ phuong31001.html 135 APPENDIX PROTOCOL D1- DETERMINATION OF THIOCYANATE IN URINE (Source: http://biology-ssets.anu.edu.au/hosted_sites/CCDN/protocols/Protocol %20D1.pdf) Components A Protocol D1 instructions for thiocyanate analysis of urine B Thirty (30) flat-bottomed plastic bottles with screw capped lids C Three (3) graduated ml plastic pipettes D Ten (10) white standard papers with thiocyanate equal to 10 ppm E One hundred (100) yellow indicator papers glued to strips of clear plastic STORE IN FREEZER Stable for one month only at room temperature F Colour chart with ten (10) shades of colour which correspond to 0-100 ppm thiocyanate G Bottle containing 0.5 g potassium permanganate Method (Complete steps to quickly as the enzyme acts rapidly to release HCN) Urine samples should either be fresh or stored in the freezer for up to months Prepare a sulphuric acid solution, add 5.5 ml of concentrated (96%) sulphuric acid to 100 ml water in a beaker slowly with stirring TAKE CARE! heat is produced not add water to acid! Prepare a permanganate solution by dissolving 100 mg potassium permanganate in ml water This is stable for months if stored at room temperature and away from direct sunlight Follow sketch Use plastic pipette to add 1.0 ml urine to a flat-bottomed plastic bottle followed by three (3) drops of sulphuric acid solution from a second plastic pipette and mix Add three (3) drops of permanganate solution from a third plastic pipette and mix gently IMMEDIATELY add a yellow indicator paper attached to a plastic strip so that the paper does not touch the liquid in the bottle When not in use STORE INDICATOR PAPERS IN FREEZER IMMEDIATELY close the bottle with a screw capped lid The magenta colour should disappear A positive and negative control should be run for each set of experiments a For a negative control, prepare another sample as shown in sketch but with 1.0 ml water instead of urine b For a positive control follow sketch Place a white standard paper disc in the bottle Add 1.0 ml water, three drops of sulphuric acid solution, mix and add three drops of permanganate solution IMMEDIATELY add the yellow indicator paper and IMMEDIATELY close the bottle with a screw capped lid and carefully mix to avoid wetting the paper The magenta colour may remain for some time (wash the pipettes thoroughly to remove urine, sulphuric acid and permanganate) Allow the bottles to stand for 16-24 hr at room temperature 10 Open the bottles and match the colour of the indicator papers against the shades of colour on the colour chart supplied 11 Read from the colour chart the thiocyanate content in ppm in the urine sample (see 16b) Check that the negative control is zero and that the positive control gives a colour of about 10 ppm THIS SECTION TO BE FOLLOWED IF YOU HAVE A SPECTROPHOTOMETER 12 For each sample, carefully remove the plastic backing sheet from the indicator paper 13 Place the paper in a test tube and add 5.0 ml of water measured accurately 136 14 Leave the test tube at room temperature for about 30 with occasional gentle stirring 15 Measure the absorbance at 510 nm of the solution, subtract the value of the negative control 16 The thiocyanate content in ppm is calculated by the equation1 a thiocyanate content (ppm) = 78 x absorbance b thiocyanate content in µmol/Lt = thiocyanate content (ppm) x 17.2 17 The thiocyanate content obtained for the same sample of urine, from both measurements 11 and 16 should be about the same Also check the standard value agrees using both methods Troubleshooting The thiocyanate content of the white standard paper should be about 10 ppm Possible problems could be: • If the indicator paper is left at room temperature it gradually becomes darker and after one month its colour will be around 1-2 ppm on the colour chart • If the indicator paper has been left in bright sunlight it becomes bleached on one side and is no good • Permanganate solution has decomposed which would give a low result Make up a new solution • Sulphuric acid solution was not made up properly or may be too dilute Make up a new solution • If you use a bottle which is not gas tight (e.g the screw cap is cracked) then gas could escape and this would give a low result • As stated in steps & 7, it is important to add the indicator paper immediately after the permanganate and then immediately close the lid otherwise there will be a loss of gas which would give a low result Reference Haque, R and Bradbury, J H., 1999 Simple kit method for determination of thiocyanate in urine Clinical Chemistry, 45, 1459-1464 Correspondence Konzo Prevention Group, Research School of Biology, Australian National University, 46 Sullivans Creek Rd, Acton, ACT, 2601, Australia Email: konzo@anu.edu.au Web: http://biology.anu.edu.au/hosted_sites/CCDN 137 138 ... 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. .. 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. .. 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