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

This dissertation was aimed to develop a greater understanding of both theconstraints in the presence of cyanide toxin and benefits of using cassava foliage asbypass protein in order to

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

I declare that this dissertation is the result of my work and that it has not been presentedpreviously as a dissertation at this university or elsewhere To the best of myknowledge, it does not breach copyright law, and has not been taken from other sourcesexcept where such work has been cited and acknowledged within the text All resultshave been published at Journal of Livestock Research for Rural Development (LRRD)http://www.lrrd.org/

Hue University, 2020

Le Thuy Binh Phuong

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

My Ph.D has been an amazing experience with Professor Thomas Reginal Preston Hehas been teaching me how good experiment is done and how to be real researcher I havebeen 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 inbiochemistry for stimulating the ideas in research

I would like to thanks to Assoc Prof Nguyen Huu Van and Dr Dinh Van Dung whogave 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 goodcollaboration

Lastly, I would like to thank my family for all their love and encouragement For myparents 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

This dissertation was aimed to develop a greater understanding of both theconstraints in the presence of cyanide toxin and benefits of using cassava foliage asbypass 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 ofvarieties of cassava known to be rich (KM94) or poor (Gon) in cyanogenic glucosides

In the first experiment (Chapter 2), cassava foliage varieties (Japan, KM94, KM140and 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 dietwith 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 yearstculture, yeast-fermented cassava pulp and leaves of sweet or bitter cassava variety Thefourth experiment (Chapter 5) measured effect of additives (brewer’s grain and biochar)

on the nitrogen retention and rumen methane production when goats had access tomixed sweet and bitter varieties of cassava foliage compared with the sweet varietyalone

The results of these experiments indicated that bitter cassava foliage containing highlevels 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-linkedchallenges from feeding bitter cassava leaves at up to 50% of the diet of goats did notnegatively impact to feed intake, growth and animal health On the contrary, the HCNprecursors present in bitter cassava leaves may lead to a partial shift in digestion ofnutrients from the rumen to the lower parts of the ruminant digestive tract leading toimprovement in productivity

Key words: Prebiotic, cyanide, bitter cassava, rumen fermentation, in vitro

Table of Contents

List of Figures

List of Tables

EPS Extracellular polymeric substances

LW Live weight

NADH Nicotinamide adenine dinucleotide hydride

NDF Neutral detergent fiber

Standard error mean

1 Problem statement

Cassava is perspective plant to climate change adaptation; its pests and its diseasesresistance and greater drought tolerance is a major factor in ranking cassava in the foodsecurity of the world (Jarvis et al 2012) In Vietnam, cassava is second crop, is grownmainly 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 inCambodia (See report of Preston and Rodríguez Lylian, 2004), that make cassavafoliage become important plant protein source in ruminant diet Nevertheless, cyanidetoxin in fresh cassava foliage, especially bitter cassava foliage, is the main obstacle foranimal such as restricting the consumption intake of ruminant or causing poisoningwhen they consume rapidly Currently, as the quantity of bitter cassava (high cyanidecontent) develop more predominant than sweet cassava (lower cyanide content) on thefield, utilization of bitter cassava foliage in diet will match reality more; however,looking for feeding method of minimizing negative effect of cyanide toxin, from thatcan be utilized bitter cassava foliage in diet will more match reality but will achallenge

Many studies are beginning to be interested in cyanide toxic that has certain effect

on methanogenic bacteria population by inhibited methanogenesis activity lead todiminish methane production (Ch Olga Rojas et al 1999; Phuong et al 2012;Phanthavong et al 2015) However, whether cyanide may affect overall microbialactivity and impact the rate of rumen fermentation indirectly, it is still not fullyunderstood Previously, the knowledge of ruminant nutritionists focused on rumen, butthe impact of cyanide on rumen fermentation may profoundly influence lower digestivephysiology of ruminant and must be considered to fully understand when utilizing bittercassava foliage in diet Even so, the challenge of bitter cassava foliage diet (highcyanide content) is a new approach but must require the safety for animal's health

Therefore, building appropriate feeding method for fresh cassava foliage diet,particularly bitter cassava foliage, without cyanide poisoning is needed to utilizecassava 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 constraintsand benefits of using cassava foliage in ruminant feeding systems From these thingscan improve the utilization of cassava foliage in ruminant feeding by enhancing itsproperties as a source of bypass protein and verify the role of HCN toxin in cassavafoliage 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/or1% biochar (as DM), then evaluating the effects of this feeding method ongrowth, digestibility/N retention, excretion of thiocyanate in urine andmethane 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)

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 cattlefed a basal diet of cassava pulp-urea; and (2) increasing N-retention,reducing thiocyanate in urine of goats having free access to both bitter andsweet cassava foliage

4 Significant/Innovation of study

This dissertation successfully demonstrated that HCN in cassava foliage is mainfactor for reduction of methane production while the earlier finding could only predictthe role of HCN for decreased methane Currently, the best-known cassava foliage tofeed animal is “sweet” cassava foliage with low cyanide content, my dissertationsucceeds to build feeding method for “bitter” cassava foliage diet (higher cyanidecontent) 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 ofthe 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 proposedpartial shift in sites of digestion (from rumen to small intestine and the cecal-colonregion) that previously it is thought that only rumen fermentation has a truly symbioticrelationship with the ruminant

CHAPTER 1 LITERATURE REVIEW

1.1 Rumen fermentation and methane production

1.1.1 Rumen fermentation

Understanding rumen fermentation is an important step in applying the basisknowledge to improve rumen function and utilizing feed efficiency Rumen microbialfermentation is crucial for growth and production of ruminants Although there aremany kinds of microbes have been found in the digestion of ruminant, yet it is thoughtthat only ruminal microbes have a truly symbiotic relationship with the host until now.Individual rumen microbial species have developed in a complex process of evolutionextending 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 ruminalmicrobes to end products along with microbial biomass, is the source of energy andprotein to respond to essential nutritional needs for ruminant

With diverse anaerobic microbes in the rumen, ruminant is able to degrade thecomplex fiber source to provide nutrient essential is readily digested by the host whilethis is completely restricted on non-ruminant (Owens and Basalan 2016) The rumencontains a variety of microorganism including main groups such as bacteria, fungi, andprotozoa In which bacteria is considered majority microbes with a diversity of bacteriagenera that its classification base on the preference for certain substrates There arethree 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 largestpopulation 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 donot make any significant contribution on feed digestion, but it is assumed that they mayscavenge oxygen to maintain an anaerobic medium Otherwise, epimural bacteriaproduce urease enzyme to hydrolyze urea (Nagaraja et al 2016), this allows ruminantcan 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 of less

than 5.5, it would affect fiber digestibility Cellulolytic bacteria release cellulaseenzyme that can catalyze the β-1, 4-glycosidic bonds of cellulose to provide glucose for

its growth Amylolytic bacteria mainly include Streptococci bovis, Bacteriodes ruminicola, Ruminobacter amylophilus, Selenomonas ruminantium, Succinomonas amylolítica These bacteria can debranch starch to produce monosaccharide and ferment them to end products such as VFA, formate, and acid lactic Streptococci bovis

can change its metabolite to produce acid lactic as a final product when there is muchhighly fermentable ingredient such as grain or concentrate in diet, is the cause ofacidosis in the rumen (Castillo-Gonzáleza et al 2014) The pH value, in this case, candrop to lower 5.5, causes extreme inhibition of cellulolytic bacteria To avoid this case,the requirement of gradually introducing fermentable carbohydrate to animal and thebalance ratio of starch and cellulose in feeding system is necessary For proteolyticactivity, there are many strain and species of rumen contain a different type ofproteolytic enzyme They include ciliate protozoa, bacteria, and anaerobic fungi thathave been found to be proteolytic (Wallace 1996) In which, the main proteolytic

bacteria have been reported to include Bacteroides amylophilus, Bacteroides rutminicola, and Butyrivibrio fibrisolvens The proteolytic activity has also been reported in Streptococcus bovis and Prevotella albensis (reviewed by Castillo-

Gonzáleza et al 2014) This bacteria group is major responsible for dietary proteinbreakdown while protozoa proteolysis both particulate feed protein of appropriate sizeand also bacteria protein (Wallace 1996) Some report showed that proteolytic bacteriawere affected in population decline when the presence of condensed tannin in the diet(McSweeney 1999; Min et al 2003) However, Min et al (2003) also reported thatmicrobial protein outflow to the abomasum was unchanged in this case This meansthat although tannin binding protein to reduce the activity of microbial enzymes, and toreduce the growth rate of proteolytic bacteria but increasing by-pass protein to respond

to demand for animal protein

Fungi represent a small proportion, approximately 8% of the biomass in the ruminalecosystem (Jenkins et al 2008) The rhizoidal development of fungi cell allows them topenetrate plant tissue better than bacteria and protozoa, which would weaken the

structure of plant and greater in degradation of forage Ruminal fungi produce phenolicesterases (p-coumaroyl and feruloyl) that can break cross-linkages betweenhemicelluloses and lignin, which allow the fungus would have increased access tohemicelluloses (Nagaraja 2016) Thus, fungi degrade cellulose more efficient than themain species of ruminal cellulolytic bacteria Most of the ruminal fungi can use di ormonosaccharide as substrate effectively

Protozoa can contribute up to 50% of biomass in the rumen, it can be eliminatedfrom ruminal environment but does not affect much ruminal fermentation Newbold et

al (2015) demonstrated that elimination of ciliate protozoa increases microbial proteinsupply by up to 30% and reduces methane production by up to 11% Protozoa act asmicro-ruminants continually engulfing and digesting both small feed particles andbacteria (Owens et al 2016), by this way, defaunation is applied to increase the cause

of increasing bacteria density Ruminal protozoa harbor methanogen on the outsidesurface and inside the cell (Nagaraja 2016), metabolism of protozoa also producesmethanogen’s substrate such as H2 for reducing CO2 to form methane Thereby, it isconsidered that the removal of protozoa decreases methanogenesis due to reduction ofavailable H+ for methanogens (Mosoni et al 2011)

In term of overall biochemical metabolism, ruminal microbes secrete enzyme thathydrolysis all macromolecule such as polysaccharide, protein, lipid and othercompounds to monomer that after then fermented to the intermediate substrate (VFA,ammonia, ATP ) The main purpose of rumen fermentation is to generate energy formaintenance and synthesis processes of microbial polymers which leads to thesynthesis of more microbial cells which in turn increases available protein to the animal(Phuong 2012)

1.1.2 Volatile fatty acid pattern

When ruminal microbes fermented soluble sugar, they produce VFAs and ATP that isconsidered energy source and is re-utilized for maintenance and growth of microbes.Acetic acid, butyric acid, and propionic acid are the majority component of VFAs andlargely part was absorbed via rumen wall as free form After passing into hepatic portalblood, they circulate as anion having a net negative electrical charge (acetate,

propionate and butyrate) at blood pH (Voan Soest 1982) Acetate may enter mainlyfatty synthesis via actyl-CoA intermediate than is ketone body due to it must not passthrough this stage metabolism, while partly butyrate is interconverted to ketone bodies(acetoacetate, β-hydroxybutyrate) in the liver, the excessive accumulation of ketonebodies is result in ketosis as a pathological condition of the ruminant Propionic acid isconcerned as precursor of glucose synthesis with 80% propionate into blood transferred

to hepatic for gluconeogenesis (Van Soest 1982) Preston and Leng (1987) cited thatpropionate may contribute 80-90% of the glucose synthesized in sheep on roughagediets (Cridland 1984) With by-product diet or dry pasture, poorly absorbed glucosethus gluconeogenesis plays the major role to provide glucose needed for ruminant,while some starch escape fermentation in grain-based diets can be digested in the smallintestine

Figure 1.1 Pathway of VFA in metabolism

PEP=phosphoenolpyruvate Source: Voan Soest (1982)Therefore, the ratio of acetic and propionic (Ac/Pro) is calculated as indicating aparameter for animal production The higher acetate concentration may require fillingthe gap by propionate and high Ac/Pro may be an indication of the insufficient forgluconeogenesis

1.1.3 Protein metabolism

The ruminal microbes are likely to utilize non-protein nitrogen source (NPN) such

as urea to contribute ammonia pool in the rumen Level of ammonium in rumen regard

to microbial output due to microbes is likely to convert ammonium to protein forsynthesis microbial polymer The low ammonium indicated nitrogen shortage tomicrobes lead to low fermentation rate, in contrast, excessive ammonium is result inammonium toxicity for the animal Nevertheless, in order to utilize NPN effectively, theconversation of ammonium to microbial protein requires the availability of ATP energygenerated by fermentation of carbohydrates In other words, it requires the balancebetween carbohydrate and NPN in diet In term of N digestion from non-NH3N flowinto small intestine, mainly in duodenum, account for 65% of nitrogen from animal’sfeeding (MacRae and Ulyatt 1974) due to the extent of conversation of protein to NH3-

N in rumen is faster than using NH3-N of ruminal microbes into pathway of microbialprotein synthesis (Ulyatt et al 1975) Approximately 60% of amino acids absorbedthrough the small intestine is from a bacterial protein, and the remaining 40% is fromruminal un-degraded dietary protein (Wattiaux 1991) In addition, the ruminant can useeffective sources of bypass protein from by-product source

The term of bypass protein in rumen fermentation is defined protein escape thedegradation of ruminal microbes Two important factors influencing amount of proteinbypassing degradation in the rumen are the length of time spent in the rumen andfermentability of protein (Miller 2012) Leng et al (1981) indicated that bypass protein

in ruminant diet was postulated on stimulating feed intake, influencing the efficiency ofmicrobial cell yield and digestion in small intestine, providing amino acids post ruminaldigestion which are used efficiently, and in addition to increasing the total energyintake With too soluble protein as sole diet in rumen, dietary protein can be lost due tolargely part of essential amino acid is fermented by microbes, and microbial proteinwould escape rumen to lower digestion to compensate protein needed of the animal,meanwhile, the by-pass protein can provide essential amino acids that are synthesizedinto animal tissues, via absorption from digested feed

Non-degradable and degradable protein play an important role in rumen functionand animal efficiency Although it has not been well defined the desirable proportion ofnon-degradable and degradable protein in ruminant feeding, but there was quite evident

to believe that diet must contain sufficient both proteins to rise efficient productivity

(Miller 2012) There are many studies to be interested in searching the most effectiveratio of rumen degradable protein and un-degradable protein (RDP: RUP) Wang et al.(2008) and Tacoma et al (2017) did not found the significant difference among ratios

of RDP: RUP on milk yield, milk composition, and dry matter intake, but reducing theratio of RDP: RUP reduced N excretion in urine and faeces lead to enhance theefficiency of N utilization Savari et al (2018) suggested that an RDP: RUP ratio of65:35 could be adequate for cows in early lactation with an average milk production of

44 kg and a DMI of 25kg

1.1.4 Methane production

Methane gas is produced from rumen fermentation by ruminal microbes.Domesticated ruminant represents a loss of 2–15% of the gross energy (GE) intake bymethane production (Holter and Young 1992), therefore being one of most importantfactors involve to inefficiencies in ruminant production systems (Moss et al 2000)

In the rumen, methanogens are a large and diverse group of Archaea By isolationmethod, it is classificated such as Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrecibacter millerae, Methanobrevibacter olleyae, Methanobacterium formicicum, Methanobacterium bryantii, Methanosarcina barkeri, Methanosarcina mazai and Methanomicrobium mobile (Qiao et al 2014).

Overall, the methanogen can be divided into two groups: H2/CO2 and consumers with different level of energy yielding (-130.7 kJ/mol substrate and -32.3kJ/mol substrate respectively) The distribution of methanogen is diverse, it is assumedthat they are free-swimming in fluid or attach to digested solid or attach to protozoa(Morgavi et al 2010)

acetate-Pathway of methane production

The pathway of methanogenesis has yet to be fully defined due to the diversemicrobes in rumen create overall synergistic and antagonistic interactions However, ithad been known that formate, carbon dioxide, methanol, and acetate derived fromcarbohydrate fermentation to be concerned as terminal electron receptor for hydrogen

to form methane (Figure 1.2) Based on biochemistry pathway, hexose metabolism viathe Emden-Meyerhof-Parnas pathway (EMP) which produces pyruvate as an

intermediate associated with cofactor NADH generation (Leng 2011) In methaneproduction, CO2 substrate is concerned to as an electron acceptor to form methane, thispathway predominates in metabolism of hydrogenotrophic methanogen This bacteriumgroup also use formate as an important electron donor and estimated up to 18% of themethane produced in the rumen Many of involved syntrophs are able to produce bothH2 and formate, and most of methanogenic partners are able to oxidise both substrates

to methane (Leng 2014) Acetate as substrate produce methane through the acetoclastic

pathway by Methanosarcina group but in terms of energy order, the energy level of methane production from acetate is very low, lead to Methanosarcina population is limited in rumen (Morgavi et al 2010) Furthermore, largely acetate is absorbed into

bloodstream of animal, thus, the hydrogen would be contributed mainly by CO2 tomethanogenesis (Galand et al 2014)

Figure 1.2 The reaction of methane generation

The process of methane production requires in low hydrogen partial pressure,which is necessary for continual fermentation in the rumen (Figure 1.3) However, theinhibition of methanogenesis would redirect available hydrogen into alternative energy-yielding metabolic pathways which are expected to improve the productivity ofruminant but do not adversely affect ruminal metabolism Martinez-Fernandez et al.(2016) had a comprehensive assessment on methanogenesis inhibition by addingdifferent levels of chloroform on steer fed roughage hay versus hay: concentrate, theresult showed that increasing chloroform level would enhance the expel of hydrogenbut there was no effect both on dry matter intake and on fiber degradation The criticalissue was found in this study is that expelled H2 per mole of decreased methane waslower on steer fed roughage hay alone in diet compared to hay: concentrate Theevaluation of rumen microbial response in this study showed that decreasing of inArchaea and in Synergistetes for both diets accompanied with increasing in

Bacteroidetes (the bacteria involve to propionate production), but did not changefibrolytic bacteria, fungi, and protozoa These results can conclude that hydrogen wasredirected into other products than into CH4 and H2, probably in microbial protein, it can

be expected to improve performance of animal Furthermore, using roughage hay indiet is the suggestion in slow fermentation that creates a condition for microbesutilizing H2 more effectively as a reduction of methane production, meanwhile, highlyfermented concentrates in diet would high partial pressure of H2

Figure 1.3 The pathway of hexose conversation to end-products

(Source: Bernalier et al 1999)

1.1.5 Effect of feeding system on rumen fermentation

Feeding system has generally the direct effects on the rumen fermentation patternbecause of the various chemical compositions among the ingredients and then hasimpact on ruminant performance Nolan and Dobos (2005) identified that the feature offresh forage diet is highly soluble protein and carbohydrate that it would increase thecrude protein degradation, meanwhile, crude protein in dried-forage and cereal grain isgenerally less degradable protein and is considered as bypass protein (or un-degradableprotein) Beside the balance of energy and protein in rumen fermentation, along withsoluble characteristic of feed, perspective of feed utilization is also depended on the

activity of microbe’s consortia (communication of bacteria groups and fungi) when it wasapplied to issues related to increasing fiber digestibility (Firkins 2010)

Asizua et al (2018) compared three feeding systems that has using agro-industrialbyproduct in steers, particularly those that are cereal based: (1) natural sole grazing(control), (2) control plus concentrate supplement (composition g/kg DM: 375 maizebran, 559 brewer's spent grain, 62.5 molasses and 3.75 NaCl), and (3) feedlot systemswhere steers were fed total mixed ration (TMR) comprising g/kg DM: 200 maize stove,

300 maize bran, 447 brewers’ spent grain, 50 molasses and 03 NaCl The higherammonia-nitrogen (NH3-N) concentration in sole grazing steers compared with grazingplus concentrate supplement and feedlot system reflected the deviation betweendigestible energy and nitrogen metabolism in the rumen, or the imbalance ratio of C:N.Highly inclusion of maize bran and molasses as fermentable carbohydrate in feedlotsystem showed cause of higher VFA and lower pH within the range for sub-acuteruminal acidosis (SARA) of 5.0–5.6 compared with sole grazing This experiment alsoshowed that the lower degradation of DM, CP and NDF in feedlot system and grazingplus concentrate supplement compare to sole grazing is as a result of highly solublecarbohydrate intake It is well known that declined cellulolytic bacteria activity caused

by pH value in rumen drop to 6.5 on high-grain diets (Krause et al 2003) Theseshowed that critical factor affected on rumen fermentation is mainly the type of feedrather than feeding system

Fibrous carbohydrate (green grass, rice straw…) is one of critical requirement ofruminal microbes, forage is incorporated with concentrate tend to increase protein,energy, mineral and vitamin Nevertheless, the feeding of high concentrate in diet inprolongation is cause of VFA accumulation and lower pH value Therefore,determination of appropriate ratio of forage and concentrate (F: C) is being looked for.Most studied showed that increasing ratio of F: C in diet corresponded to highermethane production (Benchaar et al 2001, Aguerre et al 2011, Seon-Ho et al 2018).The diets that being rich in fiber content have tend to increase acetate productionbecause of abundant acetate producing bacteria (Marques et al 2017) In contrast, non-

fiber carbohydrate in diet oriented mainly propionate production and resulting in lower

of methane emission (Valadares et al 1999)

1.2 Understanding ruminal microorganism

1.2.1 The self-detoxify mechanism of ruminal microbes

Grazing forage-based ruminant is likely to get plant toxins such as tannins,alkaloids, goitrogens, gossypol, saponins, glucosinolates, mimosine, fluoroacetate,cyanogens, and mycotoxins However, the ruminant is less susceptible to toxins thanmono-gastric due to fermentation in the rumen can reduce toxicity by microbialmetabolism in the foregut (Leng 2017) With good fiber digestion, ruminal microbesare likely to detoxify many substances prevalent in certain plants and herbs that canprove toxic for non-ruminants (Owens and Basalan 2016) These authors are thoughtthat the suggested detoxification by ruminal microbial requires ingestion of low dosetoxin and gradual increasing its concentration, it facilitates microbes can adapt andincrease biochemistry of metabolism for detoxification or be increasing the sorption oftoxins to prevent animals from choosing poison feed This was demonstrated in series

of experiments using nitrate as fermentative N replace to urea in cattle (Sangkhom et al.2012; Sophal et al 2013) and goat (Trinh Phuc Hao et al 2009; Silivong et al 2011;Sophea et al 2011) without manifestation of poisoning during the experiment Gradualadaptation of nitrate in diet allows ruminal microbes to convert nitrate to ammoniumdirectly Smith (1992) reviewed that ruminant can increase tolerance of some planttoxic when they have gradually adapted to the toxin such as nitrate, nitrite,nitropropanoic acid, oxalate, prussic acid (cyanogenic glycosides), sulfate and sulfide,some alkaloids (e.g., mimosine) and, perhaps, even some mycotoxins This author alsomentioned that activated charcoal has been effectively used to adsorb some kinds ofpoisons and prevent their absorption by animal In summary, rumen microorganisms arelikely to establish a detoxification mechanism; however, to be achieved high efficiencyrequires the coordination of microorganisms to act in concert

1.2.2 Interaction of ruminal microorganism in biofilm formation

The requirement of microbes, in general, to attach to solid materials, become sessileand form biofilms was recognized in the 1970s, largely through the laboratories ofProfessors Costerton (Costerton 2007) The natural microorganism has two types ofgrowth: (1) a unicellular life phase, the cells are free-swimming (planktonic), (2) amulticellular life phase in organized consortia within biofilms encased in self-producedextracellular polymers (EPS) which constituent of complex mixture ofexopolysaccharides, nucleic acids, proteins, and other compounds (Leng 2017) Theinduced signals are emitted by an individual cell of microorganism being a form of cell-to-cell communication for formation and growth of biofilm (Berlanca and Guerrero2016) The activity of microorganisms in biofilm is an integration of metabolicprocesses, generate intermediate such as H2, H2S, NH3, several organic compounds,electron acceptors (O2, SO42-, CO2, etc.), waste products, and other substances thatestablishes the driving forces that lead to the formation of the chemical gradients andallowing molecular diffusion (Stewart et al 2008) According to Leng (2011), thegrowth of biofilm includes the following phases:

1 Initial (reversible) attachment of cells to the surface by adhesions, receptors and specific mechanisms that rely on physical-chemical forces such as van-der Waals forces

non-2 Irreversible attachment by production of EPS resulting in more firmly adhered

3 Maturation I Early development of biofilm architecture

4 Maturation II Maturation of biofilm architecture, attachment of other organisms,competition, organization to create pores, channels

5 Dispersion of single cells from the biofilm

Biofilm does not exist forever, at the end of the decline phase, the biofilm breaks downand releases single cells The consecutive releasing of the previous biofilms facilitatesthe formation of subsequent surface links and formed new biofilm (Berlanca andGuerrero 2016)

The biofilm formation in the rumen is similar to the above description; biofilm mode

of living enhances the rate of reaction related to fermentation through the self-organised

and closely associated assembly of sessile bacteria/Archaea attached to the matrix(Leng 2017) It is inevitable for the growth of ruminal microbes and ruminant’s health.Leng (2014) stated that highly solubilization of feed organic matter requires theattachment of many different ruminal microorganisms on the surface of feed particle toaccess the nutrient, particularly adhere to the damaged edge by chewing of ruminant;consequently, EPS membrane is formed In this population, fungi grow deeper into theplant structure, especially also at previously damaged sites of feed particle, but closelyassociated with EPS of fermentative biofilm Fungi weaken plant structure byhydrolytic enzymes such as cellulases, hemicellulases, proteases, amylases, feruloyland p-coumaryl esterases, various disaccharidases, pectinases, and exonucleases.Consequently, reducing the size of the feed particle and creating the opportunity forother microorganisms to access substrate fermentation In biochemistry activity ofruminal biofilm, the sugar or starch is glycolyzed to form volatile fatty acids (VFAs), Ncompound convert to ammonium which is used to synthesize bacteria cell The pressure

of H2, is mostly produced in VFAs formation, play the key role in methane production.The high pressure of H2 allows diffusing the outer layer of the biofilm, makingmethanogenesis prefer to embed on this layer to take their H2 substrate However, notonly methanogenesis is concentrated in the outer layer of biofilm, but alsomethanotrophic group used methane as substrate for methane oxidation To beeffective in capturing methane substrate, methanotrophic would be distributed close tomethanogenesis that may be located in the outer layer biofilm where having the highestmethane pressure (Leng et al 2011) Although diffusion of intermediate compoundssuch as VFAs, amino acids and ammonia (NH3), and gases such as CH4, H2 and carbondioxide (CO2) could get into or out of biofilm but the concentration of this material can

be expected more higher in a matrix of biofilm than in external rumen fluid (Leng2014) Therefore, both the methanogenesis and methanotrophic tend to attach in biofilmthan free-swimming (planktonic) in rumen fluid The absence of protozoa did notaffect fermentation activity, as well as the ruminant's health, was found via de-faunaprotozoa process by oil (Beauchemin and McGinn 2006) The single protozoa groupwas reviewed the most in many studies on biofilm protozoan They included naked

amoebae, heterotrophic flagellates, testate amoebae, foraminiferans (in marinehabitats), heliozoans and ciliates (Arndt et al 2003) There are arguments on the impact

of protozoa on biofilm formation Many studies reported that protozoa reduced biofilmdevelopment due to protozoa reduced the thickness of mature multispecies biofilms atsteady-state (Huws et al 2005) In addition, protozoa have known as predation ofbacteria in the rumen and Huws et al (2005) also discovered that the rates of ingestion

by A castellanii were estimated 90 bacterial cells amoeba-1 h-1 Nevertheless, Arndt et

al (2003) reviewed that protozoa presence very abundance on biofilm of the widerange of substrate such as stone, macrophage, animal, water Rychert and Neu (2010)demonstrated that protozoa from healthy activated sludge initially disturbed the biofilmdevelopment but later they could stimulate its growth Leng (2017) reviewed that theprotozoa spend part of their time as free-swimming organisms after the host animal hasingested a meal, and the remaining time associated with digesta particles or attached tothe rumen epithelium It is thought that protozoa may attach to the plant material on thesurface of the biofilm and they can use soluble sugars from rumen fluid directly asenergy metabolism after they are released from biofilm and particles

1.3 Using agro-industrial by-products for ruminant feeding system

Utilizing rational agricultural by-products in animal husbandry is key to achieveeconomically livestock production The logical coordination of by-products in thefeeding system in the best possible way would be overcoming nutritional deficienciesand/or anti-nutrition factor in each of feeds, thereby be improved ruminantperformance In this system of feeding, the ruminants can continuous free choice ofavailable feed for the demand of productivity or animal’s self-medical

A large amount of agro-industrial by-products have exposed in Vietnam annuallysuch as cassava leaf, cassava pulp, brewer’s grain They have seriously pollutedenvironment by pulp fermentation and burning foliage In fact, these availableingredients can be formulated to ration of ruminant and these are generally cheaperthan a commercial feed Additionally, cassava by-product source is accessed easily by afarmer after harvesting cassava root Thereby, the establishment of feeding system from

agro- industrial by-products are rather advantageous for small scale household, theimportant is that how to design the feeding system from by-products to matchnutritional requirement and the response of animal on the intake.

1.3.1 Cassava foliage

Cassava foliage is recognised as a locally available resource for animal feeding with

a high edible biomass yield (Khang et al 2005), a valuable source of protein, varyingfrom 2.24 to 2.84 tones CP/ha (Dung et al 2005; Khang et al 2005) and a highconcentration of minerals and vitamins (Chadha 1961)

The actual yield of cassava leaves depends on the way that these leaves areharvested Maximum stem growth is at sixth month after planting, after which DMaccumulation is redirected from growing stems and leaves to that of roots (Wargiono1982) The potential yield of cassava leaves varies considerably, depending on cultivarage, the age of the plant, plant density, soil fertility, harvesting frequency and climate(Ravindran 1993) It is recommended that the harvesting leaves can be started when theplant is 04 months old without any effect on average total biomass and storage rootyields, the harvesting of leaves also did not result in significant effects on both heightand stem diameter compared with the un-harvested plant (Munyahali et al 2017).Actually, the farmer usually trimmed leaves on stems to 40 cm prior to tuber harvestand after then leaves were chopped by hand or by a stationary forage chopper forfurther processing of feed

To be classified as bitter (high cyanide content) or sweet (low cyanide content) cassavaare generally depended on the two cynogenic glucosides, including linamarin (accountfor 95%) and lotaustralin (account for 5%) that present in the parts of cassava(Siritunga and Sayre 2003) The broken cell wall of plant would liberty linamarin tocontact endogenous linamarase and then would release free-hydrocyanic acid (HCN)(Maherawati et al 2017) Thus, so-called “sweet” variety is considered safe for humanconsumption purpose by only basis treating (e.g peeling and cooking), while "bitter"variety must be treated by eliminating the cyanogen or least reduce them tophysiologically tolerable levels (Nicolau 2016), must not exceed 10 mg HCNequivalent/kg dry weight as recommended by FAO/WHO (CX/ CF 13/7/10, 2013,

reported by Åkesson 2013) Åkesson (2013) also reported that the classification ofcassava leaf was based on the HCN content with lower 50 mg/kg fresh matter is forsweet leaf and higher is for bitter leaf However, the HCN content depends upon soil,fertilizer and climate, therefore, the HCN content for sweet leaf may greater comparewith above data On cassava field, the farmer classifies cassava foliage based onbitterness in plants: “sweet” cassava leaf with lower HCN precursor content wasplanted mainly for human consumption and “bitter” cassava leaf with higher HCNprecursor content is for industrial starch processing.

In Vietnam, the newest updated data in 2017 by FAOSTAT reported that the yield ofcassava was 19,28 ton/ha This yield accompanied with potential to use cassava leaf asprotein source for ruminants However, the research of cassava foliage in ruminantfeeding system in Vietnam is less widely published and mainstream A comprehensivestudy on using cassava leaves as protein source in ruminant feeding system in Vietnamwas published by Thang (2010) the author evaluated using protein source from cassavafoliage in case to be associated with another protein source (legume foliage orstylosanthes) would improve live weight gain of cattle when compared with control orwith fed cassava alone Or useful combination in a mixture of cassava foliage as proteinsource and cassava meal as energy to demonstrate the relationship of balance protein-energy in the diet of cattle, the highest digestibility, and average daily gain was found inboth high crude protein and metabolizable energy diet Additionally, this author alsorevealed that supply extra energy could overcome the negative effect of cyanide toxin

in cassava foliage Another point of interest from rumen fermentation is that the effect

of cyanide toxin in cassava leaf on reducing methane production, as mentioned in the

overall research of Phuong (2012a on cattle and 2012b on in vitro rumen fermentation).

The result showed that fresh cassava foliage could diminish methane compared withcassava leaf meal (to be dried), “bitter” leaf reduced methane production compares with

“sweet” one Hieu et al (2014) conducted the comparison among no cassava forage(control), supplement 20% dried cassava foliage, 20% ensiled cassava forage and 20%fresh cassava forage in elephant grass as basal diet of Laisind (Sindhi-Yellow) femalecattle The results showed that DMI and the digestibility of DM, OM, CP, NDF

improved when supplementing cassava forage compared with control Supplementingcassava forage also resulted in increasing ammonia nitrogen concentration in rumenafter 03 hours of feeding Supplementation of cassava forage (dried, ensiled or freshstatus) mitigated rumen methane production (L/kg DMI) compare with control Eventhough cassava foliage is used in ruminant feeding system but its effect on rumenmetabolism and growth of animal still has many questions of interest.

Nutrient composition

Nutritional value of cassava leaves could difference among leaves varieties,harvesting interval, harvesting time, soil and fertilizer (Table 1.1) Dry matter (DM),neutral detergent fiber (NDF), acid detergent fiber (ADF) and total tannin content incassava foliage is higher with longer the cutting interval, in contrast, lower crudeprotein (CP) and HCN content was found at later harvesting time, at 09 and 06 monthsafter planting compared with 03 months (Hue 2012) A similar finding ofPhengvilaysouk and Wanapat (2008), the fiber content, NDF and ADF and ADL ofharvested leaves at 04 months old was higher but lower in crude protein (CP) than that

of harvested leaves at 02 months old

There are many varieties of cassava grown with different nutrient composition.Different nutrient compositions can also be seen on the same leaf variety amongexperiments, probably due to soil, fertilizer, climate or stage of maturity (Ravindran 1993)

Table 1.1 Nutrient composition of fresh cassava leaf

Cassava

leaves

variety

Cassava leaf in Vietnam

KM94 18.6-20.7 36.4-39.4 20.7-28.5 2.86-4.36 489-730 (a) Hue (2012) KM98-7 18.6-20.5 36.5-39.4 26.8-28.3 2.76-4.20 359-607 (a)

Local 18.5-20.8 38.1-43.2 28.2-31.6 2.74-4.13 307-552 (a)

Cassava leaf in Cambodia

Unknown

Chhay Ty (2003)

Note: (a) mg/kg as fresh basis; (b) mg/kg DM

Cassava leaves are rich in protein with an average of around 30%, but widevariability range from 14.7% to 40% of DM have been reported by Lancaster andBrooks (1983) Eggum (1970) has studied 60 cassava leaves varieties and has reportedthat 85% of CP fraction is true protein The amino acid profile showed that cassava leafhas high in valine and leucine content, but sulfur-containing amino acid is mostrestricted in cassava leaf

Table 1.2 Essential amino acid profile of cassava leaf (g/kg total

The data were taken from Diasolua Ngudi et al (2003)

Effect of tannin content in cassava leaves on the ruminant feeding system

Tannins can be defined as any phenolic compound of moderately high molecularweight containing sufficient phenolic hydroxyls and other suitable groups to formstrong complexes effectively with protein and other macromolecules (Van Soest et al.1987) Because of the great structural diversity, therefore, the classification of tannin isbased on its chemical properties Hydrolysable tannin (HTs) is recognized by fractionalhydrolysis into smaller structure in treatment with hot water or with tannases(Khanbabaee and van Ree 2002) Structure of hydrolysable tannin constituent of acarbohydrate core with phenolic carboxylic acids bound by ester linkages whistle non-hydrolysable tannin (condensed tannins- CT) is oligomeric and polymericproanthocyanidins, which can be only produced anthocyanidins on acid degradation(Hervás et al 2000) Tannin can combine with dietary protein to form tannin-proteincomplexes or inactivation of proteolytic enzymes (Kumar and Singh 1984; Gerlach et

al 2018) lead to the protein can apparently bypass the ruminal fermentation and makebetter utilization for lower gut (Ravindran 1993; Wanapat 1995) Theodoridou et al.(2010) reported that Sainfoin condensed-tannin diminished NH3-N concentration inrumen fluid due to reducing N solubility, consequently, increasing the non-NH3 flowmigrate to duodenum (Barry and McNabb 1999, Orlandi et al 2015) However, usingtannin in feeding system can have a beneficial or detrimental effect, this was notedmainly on the amount ingested The interaction of tannin to reduce protein degradation

in rumen was identified even at low dose (e.g Hervas et al 2000; 1g/100g of tannicacid treated-soybean meal or 17.3g/kg DM of condensed-tannin in sheep’s feedingobserved in the study of Gerlach et al 2018) The neutral ruminal environment(pH=6.8) is an advantage to form the complexes of tannin-protein but to be weak and to

be hydrolyzed in abomasum with acidic environment (pH = 2- 3) (Vissers et al 2017).The dietary of total condensed tannin at the medium concentration (3-4% of DM basis)have known beneficial effect on the absorption of essential amino acid in duodenum,increased sheep’s performance but no effect on feed intake (Barry and McNabb 1999).However, depressing of feed intake and nutritional digestion was found whenconcentration of dietary condensed tannin was exceeding 5% of dry matter (Naumann

et al 2017) Hervás et al (2000) found that no effect on extent of dry matterdegradation in rumen at low dose of tannic acid and only to be reduced when reaching10% of commercial tannic acid was used to treat soybean meal, moreover, the negativeeffect of intestinal digestion of the non-degraded protein was noted at 20% and 25% ofcommercial tannic acid Besides that, Gerlach et al (2018) found that organic matter

digestibility diminished drastically with condensed tannin at 21% and 28% of DM in in vivo experiment The depressed absorption in duodenum at high tannin concentration

was assumed by the inhibition of tannin on the capability of endogenous enzymes tosplit proteins into peptides and amino acids and impede their absorption (Frutos et al.2004; Waghorn 2008) Increased secretion of digestive mucus may lead to endogenousprotein loss (Orlandi et al 2015) Recently, some report showed that presence of tannin

in feeding system may affect the N retention, particularly on urinary excretion Powell

et al (2009) tested the effect of silage from alfalfa (ALF) with silage from low-tanninbirdsfoot trefoil (LTBT), high-tannin birdsfoot trefoil (HTBT), or o-quinone containingred clover (RCL) on lactating Holstein dairy cows, there was no difference of fecal Namong diets but HTBT and ALF got the result in higher urinary N compare with LTBTand RCL However, N retention was not observed in this experiment Ebert et al.(2017) compared the levels of condensed-tannin extract (0, 0.5 and 1% of DM)supplemented in beef steer, the result showed that urinary N as a proportion of total Nexcretion linearly decreased when adding condensed-tannin but no difference in

retained nitrogen Whilst Orlandi et al (2015) showed that the increasing of Acaciamearnsii tannin extract (i.e 9, 18 or 27 g/kg of total dietary DM) supplemented inrestricted feeding of steer gave increasing fecal N and reducing urinary N excretion.This result reflected consistently in shifting partly of urinary N into fecal N as anecessity on N balance of body that found in report of Theodoridou et al (2010) ButAcacia mearnsii tannin extract increased linearly retained N as improving the efficiency

of N utilization by steers Similarly, Pathak et al (2017) also found a linear increasing

of the percentage of N retention in lamb supplemented condensed tannin at differentconcentration (at 1, 1.5 and 2% of DM)

The content of tannin in cassava leaves was reported at medium concentration, rangefrom 2 to 4% of DM basis (Elly Roza et al 2013; Hue 2012) and has also reported toincrease with maturity (Ravindran and Ravindran 1988) and vary among varieties(Padmaja 1989) With this feature, cassava leaves were used in ruminant feedingsystem as bypass protein to improve the animal’s performance Elly Roza et al (2013)showed that the increasing of cassava leaves flour level increase NH3 production, drymatter digestibility and organic matter digestibility of rumen liquid On productivityaspect, Wanapat et al (2003) reported that condensed tannin in cassava hay could formtannin-protein complexes to provide bypass protein source to digestive small intestine,lead to positive effect such as reducing concentrate requirements, and increase milkyield and composition Besides that, some studies have reported the tannin in cassavaleaf could control the parasite with the result of Seng Sokerya (2009) was found thatfresh cassava leaf feeding reduced worm fecundity, while feeding ensiled cassava alsoreduced the worm burdens, but only of haemonchus contortus

Tannin is also interested in reduction of methane production when was supplied inruminant’s diet, thus, tannin might be potentially useful as rumen manipulating agentsHowever, its impact on methane production is highly variable due to there are manyfactors to contribute such as origin, concentration, molecular structure, and dosage oftannin (Aboagye et al 2018) The presence of higher condensed tannin (CT) in sericealespedeza (17.7% of DM) in Angora goat’s diet was result in lower methane emissionexpressed as both quantity per day or relative DMI compare with 5.5 % CT in

crabgrass/tall fescue, the result on growth of animal is also positively with dry matterintake (1.11 vs 0.67 kg/d) and digestible DMI (0.71 vs 0.51 kg/d) were greater forsericea lespedeza than for crabgrass/tall fescue (Puchala et al 2005) Similarly, highlevel of tannin in diet at 163 g/day CT or 326 g/day CT initially, reduced to 244g/day CT by day 17 also give reduced methane 14 and 29%, respectively, however,there was negative effect on DMI and milk production, especially at high dose of CT(Grainger et al 2009) There is an inconsistency in the effects of tannin on reduction ofmethane, perhaps low levels of tannin in the diet, its effectiveness would be unclear.Tavendale et al (2005) explored that CT at 1% of DM in forage legumes Lotus pedunculatus gave lower methane production compared with Medicago sativa (lucerne;

CT < 0.1 % of DM), but after addition of polyethylene glycol (PEG) as a tannin

binding reagent methane production was increased for L pedunculatus (17%, p < 0.001) but not for M sativa It can be seen that diminished

methane by feeding CT-binding salivary protein can reduce ruminant’s intake(Naumann et al 2017), alternatively, CT may also be associated with lesions of the gutmucosa lead to decrease the absorption of the other nutrients (Reed 1995) andendogenous protein loss (Orlandi et al 2015) However, compared with hydrolysable

tannin (HT), the stronger affinity for proteins of CT can restrict its absorption in thedigestive tract and can reduce potential toxicity to the animal For this reason, Aboagye

et al (2018) examined the long-term effects of feeding HT with or without CT on

showed that tannin type and dose did not affect to daily methane but tended to decreasemethane production compared with control HT alone or in combination with CT can

be added at 1.5% DM do not also give negatively affecting performance The result

plants that contain both CT and HT to enhance the reduction of methane production in

in vitro incubation, the result also revealed that methane production was reduced with

increment CT and HT content in plant while depressing this effect by absence of tannin.The inhibition of tannin on methane production was demonstrated by Aboagye et al.(2018), oligomeric CT fractions 1–7mDP was added to pure culture of

Methanobrevibacter ruminantium strains, as the common methanogens found in

grazing animals, as determined by culture (Jarvis et al 2000) and molecular methods(Whitford et al 2001) The result showed that polymeric CT fraction have ability to be

inactive against both the Mbb ruminantium strains This author also concluded that the

activity of polymeric CT fraction in pure methanogen culture may be quite differencewith impact of CT in plant in those obtained in mixed ruminal culture, wheremetabolism of methanogen is influenced by action of other microorganism such ascellulolytic bacteria Therefore, action of CT on methanogens is attributed to indirecteffects by reduction of hydrogen concentration, which is considered as their substrate,this related to a depression of fibre digestion in the rumen by presence of tannin (Patraand Saxena 2011)

Cyanogenic glucosides in cassava leaf

A concern of using cassava leaves in animal feeding system is the presence of nutrition that limits the usability of leaves, especially cyanide toxin The presence ofcyanogenic glucosides, linamarin, and lotaustralin, the bonds of which are hydrolysedwhen tissues plant are damaged or disrupted, linamarin is converted to linamarasewhich catalyze cyanogenic glucosides to free hydrogen cyanide (HCN) (Onwuka et al.1992) In animal and human, the cyanide inhibits oxygen transportation by linkinghaemoglobin to form cyanohemoglobin If exposure of prolonged cyanide will lead toclinical sign such as labored breathing, restlessness, tremors, groaning, terminal colonicconvulsions and opisthotonus in affected animals (Kutay et al 2017) The neutral pH inrumen and ruminal microorganism secretes enzymes that accelerate the hydrolyzing ofcyanogenic-glycosides and release HCN, thereby, the ruminant is more susceptible topoisoning than non-ruminant (Ludgate and Scholz 1992) The lower ruminal pH incattle fed high grain diet was found that reducing the action of microbial enzymaticactivity and releasing of HCN (Patel et al 2014) Some recent literature, however,assume that the activity of ruminal microorganism is likely to participate todetoxification of some toxins if getting the advantageous factors (will be mentionedlater)

anti-There are many reports showing the tolerance of ruminant on HCN content inforage The lethal dose of cyanide for cattle and sheep was reported about 02 mg/kgbody weight (Gurnsey et al 1977; Clarke et al 1981), while Shirley and Cunha (2012)also reported that ruminant may tolerate considerable amount of HCN if grazed foragedoes not exceed 15-50mg HCN/kg of body weight per day Wargiono and Sudaryanto(2002) manifested that small ruminant fed cassava leaves where the HCN intake was 15mg/kg body weight, had no significant effect on dry matter intake and average dailyweight gain These reports showed that there is a wide range in HCN tolerance ofruminant and it depends on the feeding system When feed choice is abundant lead tofeeding rate slowly and reduces the absorption of HCN toxin In view of the writer'spoint, the level of HCN tolerance on ruminant requires increasing gradually levels inadaptation period as the critical key associated with facilitating the availability of freechoice in feeding systems to avoid fast ingestion of animal.

Detoxification of HCN toxin:

The mechanisms of physiological self-medical involved in detoxification ofcyanogenic compound could be via many pathways, yet primarily by reaction withthiosulfate to form thiocyanate Sulfurtransferases enzyme, to be called rhodaneses,was found in both plant and mammals This enzyme is presented in many mammalianorgans with varying concentrations and to be richest in liver and kidney (Hol et al.1983), particularly bovine liver (Russell et al 1978) This enzyme is thought to belikely paving for the transfer of a sulfur atom from appropriate sulfur donors to

nucleophilic sulfur acceptors, at least in vitro (Most and Papenbrock 2015; Knowles

1988) Described by Gliubich et al (1996), the forming Rhod-S is played as anintermediary for the formation of sulfite and thiocyanate, which is established in Figure1.4

Figure 1.4 A sulfurtransferase reaction catalyzed by

rhodanese (adapted from Most and Papenbrock 2015)Mechanism of thiocyanate formation in liver is the interpretation for detoxification

of cyanide compound that ended by excreting thiocyanate in urine This process also

has been proved on the metabolism of cyanide poisoning resistance by Pseudomonas aeruginosa when they protected aerobic respiration (Cipollone et al 2008) This

conversion represents a 200-fold reduction in cyanide compound toxicity (Ravindran1993) Smith (1988) is referred to Blackley and Coop (1949) for requirement ofapproximately 1.2 gram of sulfur to detoxify 1.0 gram of HCN Thus, the demand forthe sulfur source is necessary for improving the utilization efficiency of cassava leaves

in diet Onwuka et al (1992) have demonstrated that dietary cyanide intake of 246-248mg/kg diet was detoxified and metabolized into 2.80-4.01 mg thiocyanate/100 mlruminant fluid Similarly, Silva et al (2017) were successfully in detoxification ofcyanide toxic in sheep by treated with sodium thiosulfate when sheep ingested cassavawastewater This author also observed that without treated sodium thiosulfate, the sheepwho received dose 0.46 and 0.34 mg HCN/kg body weight excreted strongly positivethiocyanate at 5 minutes after production, moderately positive after 24 hours andnegative at 48h This means that the sheep is ably recovered spontaneously bythiocyanate forming in liver occurred rapidly after 24-48 hours ingestion

The detoxification of cyanide toxin in rumen may be expected by some bacteria viatheir β-cyanoalanine formation (Knowles and Bunch 1986; McSweeney et al 2002).Although this hypothesis has not been demonstrated, yet the reaction of cyanide withcysteine to form β-cyanoalanine by catalysis of β-cyanoalanine synthase that wasshown in the study of Yi et al (2012)

Reduction of methane production: There is limited information available on the

impact of cyanide on reduction methane production in rumen fermentation Moststudies of cyanide inhibited methane production were recognized on anaerobic process

of bio-digester, sludge or wastewater Cyanide was the cause of inhibition ofmethanogenic activity by 50% during the exposure stage in the anaerobic process instudy of Ch Olga Rojas et al (1999) Gijen et al (2000) suggested to consider the cause

of inhibited methanogens by cyanide, sludge activity tests showed that whenhydrogenotrophic methanogens that were incubated to acid formic as the substrate wasless sensitive to cyanide compared to acetoclastic methanogens Similarly, Gupta et al.(2016) reported that the result of microbial community analysis using 16srRNA gene

PCR-DGGE showed bacterial profile having a maximum match with C diolis that have

been known as highest hydrogen producer, in wastewater reactor added cyanide Itleads to confirm that hydrogen utilizing methanogens were more cyanide tolerant thanacetate utilizing methanogens These results are an affirmation for previousexplanations is that hydrogen cyanide is a potent inhibitor of acetoclastic methanogenswhich are slow-growing and mainly present in such ecosystems as bio-digester, but italso inhibits to hydrogenotrophic Archae (Cuzin and Labal 1992; Annachhatra andAmonkuew 2001) which are the main producers of methane in the rumen, although thetoxicity to this group of methanogens is low (Smith et al 1985) The higher CN-inhibition in the case of acetate metabolism may be explained by the sensitivity ofcarbon monoxide dehydrogenase enzyme (Eikmanns et al 1985) which involved inhydrogenotrophic methane production is less sensitive to cyanide

More recently, there are many results showed that methane production was reduced

in response to the “bitter” cassava leaf treatment compared with “sweet” cassava leafvariety (Phuong et al 2012; Phanthavong et al 2015) Effect of methane reduction alsopronounced by fresh leaf state rather than drying leaf (Phommasack Outhen et al 2011;Phuong et al 2012), which is known to have reduced HCN content by sun-dried orensiling (Phuc et al 1995) HCN toxin in cassava foliage was thought the main factor

to decrease ruminal methane production, but there is not yet evidence to demonstratethe relationship between HCN concentration and reduction of methane in rumenfermentation

New potentially application on mitigation of HCN effect

Sornyotha et al (2010) were found the combination of xylanase from Bacillus firmus K-1 and xylanase and cellulase from Paenibacillus curdlanolyticus B-6 at the

ratio of 1:9 increased the hydrolyzed cassava cortex-cell wall, result in releasinglinamarase enzyme and low cyanide content in starch production

Recently, some studies have indicated that the small amount of brewer’s grain added incassava foliage feeding system of ruminant could confer a major benefit on growthperformance and health The reason for benefit of brewer’s grain on cassava foliagefeeding system has unknown yet, but this is promising to realize the possible effect ofprobiotic or prebiotic presence in brewer’s grain to cassava leaf, particularly “bitter” leaf

1.3.2 Brewers grain

Brewers grain (BG) production depends on the demand of beer consumption Theprocess of beer manufacturing produce brewer’s grain residue is rich in protein andfiber, approximately 20 and 70% of dry matter, respectively (Cooray et al 2017) Thisprocess includes: (1) the digestion of starch by the enzyme in the malting step forsteeping, germination and kilning; (2) mashing malt to make fermentable sugar bytreating hot water at 70-740C; (3) the conversion of sugar to alcohol by yeast duringfermentation of wort cooling (Ferreiraa et al 2010) After collecting the beer, the yeastdeveloping in the fermentation process is removed and dried and can be sold asbrewer’s yeast (Westendorf and Wohlt 2002) Yeast produces not only ethanol andcarbon dioxide but also other compounds (higher alcohols, organic acids, esters,aldehydes, ketones, sulfur compounds) which play a key role on the sensorial profile ofbeer (Pinho et al 2006) Beer processing is similar to make rice wine, the highlypopular traditional beverage in Vietnam, which is involved in use of starter culture.Yeast, mould and bacteria in starter could convert starch to fermentable sugar andsubsequently is alcohol and organic acid (Dung 2004)

Brewing yeast has many important properties such as fast growth, good ability toproduce ethanol and a tolerance for several medium stress, such as high ethanol

concentration and low oxygen levels (Piskur and Langkjaer 2004) Saccharomyces was

identified as the major factor in starter culture brewing, it is classified as

Saccharomyces sensu stricto includes Saccharomyces bayanus, Saccharomyces cariocanus, Saccharomyces cerevisiae, Saccharomyces kudriavzevii, Saccharomyces mikatae and Saccharomyces paradoxus (Kurtzman and Robnett, 2003).

on dairy cattle is 0.2% of diet dry matter, while BG can provide around 0.38% sulfur(of DM) for ruminant (NRC 2001) In addition, sulfur source can be supplied based onsulfur-amino acid in common feed BG contain both essential (including lysine,histidine, methionine, phenylalanine, and tryptophan) and non-essential (includingalanine, serine, glycine, and proline) amino acid (Cooray et al 2017) Compared withsoybean meal, BG has a more favorably balanced amino acid profile in the ruminal un-degraded protein (Cozzi and Polan 1994) Thus, BG is commonly used as a by-passprotein in ruminant feeding system

Table 1.3 Chemical composition of brewers’ grain

Parameter Content (% of DM) Publication