Aim and objective of the study
Aims of the study
The aim of this thesis was to develop a greater understanding of both the constraints and benefits of using cassava foliage in ruminant feeding systems From these things can improve the utilization of cassava foliage in ruminant feeding by enhancing its properties as a source of bypass protein and verify the role of HCN toxin in cassava foliage on the reduction of methane production that was built on earlier findings.
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 on growth, digestibility/N retention, excretion of thiocyanate in urine and methane production of cattle and goat.
Research hypotheses
(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 cattle fed a basal diet of cassava pulp-urea; and (2) increasing N-retention,reducing thiocyanate in urine of goats having free access to both bitter and sweet cassava foliage.
Significant/Innovation of study
This dissertation successfully demonstrated that HCN in cassava foliage is main factor for reduction of methane production while the earlier finding could only predict the role of HCN for decreased methane Currently, the best-known cassava foliage to feed animal is “sweet” cassava foliage with low cyanide content, my dissertation succeeds to build feeding method for “bitter” cassava foliage diet (higher cyanide content) with support of adding restricted brewers grain (4% of DM) and biochar (1% of DM) to feed cattle and goats without cause HCN toxicity Additionally, discovery of the feeding of the bitter cassava foliage appear to modify the rumen fermentation lead to increases in nitrogen retention associated with reduced methane production, it made a part of this dissertation provided the implication for new approach of the proposed partial shift in sites of digestion (from rumen to small intestine and the cecal-colon region) that previously it is thought that only rumen fermentation has a truly symbiotic relationship with the ruminant.
LITERATURE REVIEW
Rumen fermentation and methane production
Understanding rumen fermentation is an important step in applying the basis knowledge to improve rumen function and utilizing feed efficiency Rumen microbial fermentation is crucial for growth and production of ruminants Although there are many kinds of microbes have been found in the digestion of ruminant, yet it is thought that only ruminal microbes have a truly symbiotic relationship with the host until now. Individual rumen microbial species have developed in a complex process of evolution extending over a long period and provide nature's best example of microbial symbioses. Rumen ecosystem is a self-contained ecosystem, the feed is fermented by ruminal microbes to end products along with microbial biomass, is the source of energy and protein to respond to essential nutritional needs for ruminant
With diverse anaerobic microbes in the rumen, ruminant is able to degrade the complex fiber source to provide nutrient essential is readily digested by the host while this is completely restricted on non-ruminant (Owens and Basalan 2016) The rumen contains a variety of microorganism including main groups such as bacteria, fungi, and protozoa In which bacteria is considered majority microbes with a diversity of bacteria genera that its classification base on the preference for certain substrates There are three forms for the distribution of bacteria in the rumen, free-floating bacteria constitute a minor component (~30 %), and bacteria adhere on feed particle account for the largest population with 70% Both bacteria groups mainly participate in the digestion of feed. The last distribution form is bacteria that attach to the ruminal epithelial cells They do not make any significant contribution on feed digestion, but it is assumed that they may scavenge oxygen to maintain an anaerobic medium Otherwise, epimural bacteria produce urease enzyme to hydrolyze urea (Nagaraja et al 2016), this allows ruminant can use urea more efficient than non-ruminant.
The cellulose-degrading bacteria are able to degrade cellulose, one of the components in the cell wall of plant They include Ruminococcus albus, Fibrobacter succinogenes and Ruminococcus flavefaciens (Valente et al 2016) Cellulolytic bacteria prefer neutral pH between 6 and 9 is for best maintenance and growth, with pH of less than 5.5, it would affect fiber digestibility Cellulolytic bacteria release cellulase enzyme 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 much highly fermentable ingredient such as grain or concentrate in diet, is the cause of acidosis in the rumen (Castillo-Gonzáleza et al 2014) The pH value, in this case, can drop to lower 5.5, causes extreme inhibition of cellulolytic bacteria To avoid this case, the requirement of gradually introducing fermentable carbohydrate to animal and the balance ratio of starch and cellulose in feeding system is necessary For proteolytic activity, there are many strain and species of rumen contain a different type of proteolytic enzyme They include ciliate protozoa, bacteria, and anaerobic fungi that have 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 protein breakdown while protozoa proteolysis both particulate feed protein of appropriate size and also bacteria protein (Wallace 1996) Some report showed that proteolytic bacteria were 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 that microbial protein outflow to the abomasum was unchanged in this case This means that although tannin binding protein to reduce the activity of microbial enzymes, and to reduce 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 ruminal ecosystem (Jenkins et al 2008) The rhizoidal development of fungi cell allows them to penetrate plant tissue better than bacteria and protozoa, which would weaken the structure of plant and greater in degradation of forage Ruminal fungi produce phenolic esterases (p-coumaroyl and feruloyl) that can break cross-linkages between hemicelluloses and lignin, which allow the fungus would have increased access to hemicelluloses (Nagaraja 2016) Thus, fungi degrade cellulose more efficient than the main species of ruminal cellulolytic bacteria Most of the ruminal fungi can use di or monosaccharide as substrate effectively
Protozoa can contribute up to 50% of biomass in the rumen, it can be eliminated from ruminal environment but does not affect much ruminal fermentation Newbold et al (2015) demonstrated that elimination of ciliate protozoa increases microbial protein supply by up to 30% and reduces methane production by up to 11% Protozoa act as micro-ruminants continually engulfing and digesting both small feed particles and bacteria (Owens et al 2016), by this way, defaunation is applied to increase the cause of increasing bacteria density Ruminal protozoa harbor methanogen on the outside surface and inside the cell (Nagaraja 2016), metabolism of protozoa also produces methanogen’s substrate such as H2 for reducing CO2 to form methane Thereby, it is considered that the removal of protozoa decreases methanogenesis due to reduction of available H + for methanogens (Mosoni et al 2011)
In term of overall biochemical metabolism, ruminal microbes secrete enzyme that hydrolysis all macromolecule such as polysaccharide, protein, lipid and other compounds to monomer that after then fermented to the intermediate substrate (VFA,ammonia, ATP ) The main purpose of rumen fermentation is to generate energy for maintenance and synthesis processes of microbial polymers which leads to the synthesis of more microbial cells which in turn increases available protein to the animal(Phuong 2012)
When ruminal microbes fermented soluble sugar, they produce VFAs and ATP that is considered 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 and largely part was absorbed via rumen wall as free form After passing into hepatic portal blood, they circulate as anion having a net negative electrical charge (acetate, propionate and butyrate) at blood pH (Voan Soest 1982) Acetate may enter mainly fatty synthesis via actyl-CoA intermediate than is ketone body due to it must not pass through this stage metabolism, while partly butyrate is interconverted to ketone bodies (acetoacetate, β-hydroxybutyrate) in the liver, the excessive accumulation of ketone bodies is result in ketosis as a pathological condition of the ruminant Propionic acid is concerned as precursor of glucose synthesis with 80% propionate into blood transferred to hepatic for gluconeogenesis (Van Soest 1982) Preston and Leng (1987) cited that propionate may contribute 80-90% of the glucose synthesized in sheep on roughage diets (Cridland 1984) With by-product diet or dry pasture, poorly absorbed glucose thus 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 small intestine
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 a parameter for animal production The higher acetate concentration may require filling the gap by propionate and high Ac/Pro may be an indication of the insufficient for gluconeogenesis
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 for synthesis microbial polymer The low ammonium indicated nitrogen shortage to microbes lead to low fermentation rate, in contrast, excessive ammonium is result in ammonium toxicity for the animal Nevertheless, in order to utilize NPN effectively, the conversation of ammonium to microbial protein requires the availability of ATP energy generated by fermentation of carbohydrates In other words, it requires the balance between carbohydrate and NPN in diet In term of N digestion from non-NH3N flow into small intestine, mainly in duodenum, account for 65% of nitrogen from animal’s feeding (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 microbial protein synthesis (Ulyatt et al 1975) Approximately 60% of amino acids absorbed through the small intestine is from a bacterial protein, and the remaining 40% is from ruminal un-degraded dietary protein (Wattiaux 1991) In addition, the ruminant can use effective sources of bypass protein from by-product source.
The term of bypass protein in rumen fermentation is defined protein escape the degradation of ruminal microbes Two important factors influencing amount of protein bypassing degradation in the rumen are the length of time spent in the rumen and fermentability of protein (Miller 2012) Leng et al (1981) indicated that bypass protein in ruminant diet was postulated on stimulating feed intake, influencing the efficiency of microbial cell yield and digestion in small intestine, providing amino acids post ruminal digestion which are used efficiently, and in addition to increasing the total energy intake With too soluble protein as sole diet in rumen, dietary protein can be lost due to largely part of essential amino acid is fermented by microbes, and microbial protein would escape rumen to lower digestion to compensate protein needed of the animal, meanwhile, the by-pass protein can provide essential amino acids that are synthesized into animal tissues, via absorption from digested feed
Non-degradable and degradable protein play an important role in rumen function and animal efficiency Although it has not been well defined the desirable proportion of non-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 effective ratio 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 the ratio of RDP: RUP reduced N excretion in urine and faeces lead to enhance the efficiency of N utilization Savari et al (2018) suggested that an RDP: RUP ratio of 65:35 could be adequate for cows in early lactation with an average milk production of
44 kg and a DMI of 25kg.
Methane gas is produced from rumen fermentation by ruminal microbes. Domesticated ruminant represents a loss of 2–15% of the gross energy (GE) intake by methane production (Holter and Young 1992), therefore being one of most important factors involve to inefficiencies in ruminant production systems (Moss et al 2000).
In the rumen, methanogens are a large and diverse group of Archaea By isolation method, 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 acetate- consumers with different level of energy yielding (-130.7 kJ/mol substrate and -32.3 kJ/mol substrate respectively) The distribution of methanogen is diverse, it is assumed that they are free-swimming in fluid or attach to digested solid or attach to protozoa (Morgavi et al 2010)
The pathway of methanogenesis has yet to be fully defined due to the diverse microbes in rumen create overall synergistic and antagonistic interactions However, it had been known that formate, carbon dioxide, methanol, and acetate derived from carbohydrate fermentation to be concerned as terminal electron receptor for hydrogen to form methane (Figure 1.2) Based on biochemistry pathway, hexose metabolism via the Emden-Meyerhof-Parnas pathway (EMP) which produces pyruvate as an intermediate associated with cofactor NADH generation (Leng 2011) In methane production, CO2 substrate is concerned to as an electron acceptor to form methane, this pathway predominates in metabolism of hydrogenotrophic methanogen This bacterium group also use formate as an important electron donor and estimated up to 18% of the methane produced in the rumen Many of involved syntrophs are able to produce both
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 than mono-gastric due to fermentation in the rumen can reduce toxicity by microbial metabolism in the foregut (Leng 2017) With good fiber digestion, ruminal microbes are likely to detoxify many substances prevalent in certain plants and herbs that can prove toxic for non-ruminants (Owens and Basalan 2016) These authors are thought that the suggested detoxification by ruminal microbial requires ingestion of low dose toxin and gradual increasing its concentration, it facilitates microbes can adapt and increase biochemistry of metabolism for detoxification or be increasing the sorption of toxins 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 Gradual adaptation of nitrate in diet allows ruminal microbes to convert nitrate to ammonium directly Smith (1992) reviewed that ruminant can increase tolerance of some plant toxic 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 also mentioned that activated charcoal has been effectively used to adsorb some kinds of poisons and prevent their absorption by animal In summary, rumen microorganisms are likely to establish a detoxification mechanism; however, to be achieved high efficiency requires 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 sessile and form biofilms was recognized in the 1970s, largely through the laboratories ofProfessors Costerton (Costerton 2007) The natural microorganism has two types of growth: (1) a unicellular life phase, the cells are free-swimming (planktonic), (2) a multicellular life phase in organized consortia within biofilms encased in self-produced extracellular polymers (EPS) which constituent of complex mixture of exopolysaccharides, nucleic acids, proteins, and other compounds (Leng 2017) The induced 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 Guerrero
2016) The activity of microorganisms in biofilm is an integration of metabolic processes, generate intermediate such as H2, H2S, NH3, several organic compounds, electron acceptors (O2, SO4 2-, CO2, etc.), waste products, and other substances that establishes the driving forces that lead to the formation of the chemical gradients and allowing molecular diffusion (Stewart et al 2008) According to Leng (2011), the growth of biofilm includes the following phases:
1 Initial (reversible) attachment of cells to the surface by adhesions, receptors and non- specific mechanisms that rely on physical-chemical forces such as van-der Waals forces.
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 down and releases single cells The consecutive releasing of the previous biofilms facilitates the formation of subsequent surface links and formed new biofilm (Berlanca and Guerrero 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 the attachment of many different ruminal microorganisms on the surface of feed particle to access the nutrient, particularly adhere to the damaged edge by chewing of ruminant;consequently, EPS membrane is formed In this population, fungi grow deeper into the plant structure, especially also at previously damaged sites of feed particle, but closely associated with EPS of fermentative biofilm Fungi weaken plant structure by hydrolytic enzymes such as cellulases, hemicellulases, proteases, amylases, feruloyl and p-coumaryl esterases, various disaccharidases, pectinases, and exonucleases. Consequently, reducing the size of the feed particle and creating the opportunity for other microorganisms to access substrate fermentation In biochemistry activity of ruminal biofilm, the sugar or starch is glycolyzed to form volatile fatty acids (VFAs), N compound 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, making methanogenesis prefer to embed on this layer to take their H2 substrate However, not only methanogenesis is concentrated in the outer layer of biofilm, but also methanotrophic group used methane as substrate for methane oxidation To be effective in capturing methane substrate, methanotrophic would be distributed close to methanogenesis that may be located in the outer layer biofilm where having the highest methane pressure (Leng et al 2011) Although diffusion of intermediate compounds such as VFAs, amino acids and ammonia (NH3), and gases such as CH4, H2 and carbon dioxide (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 (Leng
2014) Therefore, both the methanogenesis and methanotrophic tend to attach in biofilm than free-swimming (planktonic) in rumen fluid The absence of protozoa did not affect fermentation activity, as well as the ruminant's health, was found via de- fauna protozoa process by oil (Beauchemin and McGinn 2006) The single protozoa group was reviewed the most in many studies on biofilm protozoan They included naked amoebae, heterotrophic flagellates, testate amoebae, foraminiferans (in marine habitats), heliozoans and ciliates (Arndt et al 2003) There are arguments on the impact of protozoa on biofilm formation Many studies reported that protozoa reduced biofilm development due to protozoa reduced the thickness of mature multispecies biofilms at steady-state (Huws et al 2005) In addition, protozoa have known as predation of bacteria 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 wide range of substrate such as stone, macrophage, animal, water Rychert and Neu (2010) demonstrated that protozoa from healthy activated sludge initially disturbed the biofilm development but later they could stimulate its growth Leng (2017) reviewed that the protozoa spend part of their time as free-swimming organisms after the host animal has ingested a meal, and the remaining time associated with digesta particles or attached to the rumen epithelium It is thought that protozoa may attach to the plant material on the surface of the biofilm and they can use soluble sugars from rumen fluid directly as energy metabolism after they are released from biofilm and particles.
Using agro-industrial by-products for ruminant feeding system
Utilizing rational agricultural by-products in animal husbandry is key to achieve economically livestock production The logical coordination of by-products in the feeding system in the best possible way would be overcoming nutritional deficiencies and/or anti-nutrition factor in each of feeds, thereby be improved ruminant performance In this system of feeding, the ruminants can continuous free choice of available feed for the demand of productivity or animal’s self-medical
A large amount of agro-industrial by-products have exposed in Vietnam annually such as cassava leaf, cassava pulp, brewer’s grain They have seriously polluted environment by pulp fermentation and burning foliage In fact, these available ingredients can be formulated to ration of ruminant and these are generally cheaper than a commercial feed Additionally, cassava by-product source is accessed easily by a farmer after harvesting cassava root Thereby, the establishment of feeding system from agro- industrial by-products are rather advantageous for small scale household, the important is that how to design the feeding system from by-products to match nutritional requirement and the response of animal on the intake.
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, varying from 2.24 to 2.84 tones CP/ha (Dung et al 2005; Khang et al 2005) and a high concentration of minerals and vitamins (Chadha 1961).
The actual yield of cassava leaves depends on the way that these leaves are harvested Maximum stem growth is at sixth month after planting, after which DM accumulation is redirected from growing stems and leaves to that of roots (Wargiono
1982) The potential yield of cassava leaves varies considerably, depending on cultivar age, 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 the plant is 04 months old without any effect on average total biomass and storage root yields, the harvesting of leaves also did not result in significant effects on both height and 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 harvest and after then leaves were chopped by hand or by a stationary forage chopper for further processing of feed.
To be classified as bitter (high cyanide content) or sweet (low cyanide content) cassava are generally depended on the two cynogenic glucosides, including linamarin (account for 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 to contact endogenous linamarase and then would release free-hydrocyanic acid (HCN)(Maherawati et al 2017) Thus, so-called “sweet” variety is considered safe for human consumption purpose by only basis treating (e.g peeling and cooking), while "bitter" variety must be treated by eliminating the cyanogen or least reduce them to physiologically tolerable levels (Nicolau 2016) , must not exceed 10 mg HCN equivalent/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 of cassava leaf was based on the HCN content with lower 50 mg/kg fresh matter is for sweet 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 compare with above data On cassava field, the farmer classifies cassava foliage based on bitterness in plants: “sweet” cassava leaf with lower HCN precursor content was planted mainly for human consumption and “bitter” cassava leaf with higher HCN precursor content is for industrial starch processing.
In Vietnam, the newest updated data in 2017 by FAOSTAT reported that the yield of cassava was 19,28 ton/ha This yield accompanied with potential to use cassava leaf as protein source for ruminants However, the research of cassava foliage in ruminant feeding system in Vietnam is less widely published and mainstream A comprehensive study on using cassava leaves as protein source in ruminant feeding system in Vietnam was published by Thang (2010) the author evaluated using protein source from cassava foliage in case to be associated with another protein source (legume foliage or stylosanthes) would improve live weight gain of cattle when compared with control or with fed cassava alone Or useful combination in a mixture of cassava foliage as protein source 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 in both high crude protein and metabolizable energy diet Additionally, this author also revealed 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 with cassava 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) female cattle The results showed that DMI and the digestibility of DM, OM, CP, NDF improved when supplementing cassava forage compared with control Supplementing cassava forage also resulted in increasing ammonia nitrogen concentration in rumen after 03 hours of feeding Supplementation of cassava forage (dried, ensiled or fresh status) mitigated rumen methane production (L/kg DMI) compare with control Even though cassava foliage is used in ruminant feeding system but its effect on rumen metabolism and growth of animal still has many questions of interest.
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 in cassava foliage is higher with longer the cutting interval, in contrast, lower crude protein (CP) and HCN content was found at later harvesting time, at 09 and 06 months after planting compared with 03 months (Hue 2012) A similar finding of Phengvilaysouk and Wanapat (2008), the fiber content, NDF and ADF and ADL of harvested 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 among experiments, probably due to soil, fertilizer, climate or stage of maturity (Ravindran 1993)
Table 1.1 Nutrient composition of fresh cassava leaf
CP, % ADF, % NDF, % Tanin, % HCN Publication
Canh Nong 25,28 717,59 (b) Loc and An
Cassava leaf in Southern-Western, Nigeria
Note: (a) mg/kg as fresh basis; (b) mg/kg DM
Cassava leaves are rich in protein with an average of around 30%, but wide variability 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 reported that 85% of CP fraction is true protein The amino acid profile showed that cassava leaf has high in valine and leucine content, but sulfur-containing amino acid is most restricted in cassava leaf.
Table 1.2 Essential amino acid profile of cassava leaf (g/kg total protein)
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 molecular weight containing sufficient phenolic hydroxyls and other suitable groups to form strong complexes effectively with protein and other macromolecules (Van Soest et al.
1987) Because of the great structural diversity, therefore, the classification of tannin is based on its chemical properties Hydrolysable tannin (HTs) is recognized by fractional hydrolysis into smaller structure in treatment with hot water or with tannases (Khanbabaee and van Ree 2002) Structure of hydrolysable tannin constituent of a carbohydrate core with phenolic carboxylic acids bound by ester linkages whistle non- hydrolysable tannin (condensed tannins- CT) is oligomeric and polymeric proanthocyanidins, which can be only produced anthocyanidins on acid degradation (Hervás et al 2000) Tannin can combine with dietary protein to form tannin-protein complexes or inactivation of proteolytic enzymes (Kumar and Singh 1984; Gerlach et al 2018) lead to the protein can apparently bypass the ruminal fermentation and make better utilization for lower gut (Ravindran 1993; Wanapat 1995) Theodoridou et al.
(2010) reported that Sainfoin condensed-tannin diminished NH3-N concentration in rumen fluid due to reducing N solubility, consequently, increasing the non-NH3 flow migrate to duodenum (Barry and McNabb 1999, Orlandi et al 2015) However, using tannin in feeding system can have a beneficial or detrimental effect, this was noted mainly 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 tannic acid treated-soybean meal or 17.3g/kg DM of condensed-tannin in sheep’s feeding observed 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 when concentration 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 matter degradation in rumen at low dose of tannic acid and only to be reduced when reaching 10% of commercial tannic acid was used to treat soybean meal, moreover, the negative effect of intestinal digestion of the non-degraded protein was noted at 20% and 25% of commercial 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 to split proteins into peptides and amino acids and impede their absorption (Frutos et al. 2004; Waghorn 2008) Increased secretion of digestive mucus may lead to endogenous protein 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-tannin birdsfoot trefoil (LTBT), high-tannin birdsfoot trefoil (HTBT), or o-quinone containing red clover (RCL) on lactating Holstein dairy cows, there was no difference of fecal N among diets but HTBT and ALF got the result in higher urinary N compare with LTBT and 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 N excretion linearly decreased when adding condensed-tannin but no difference in retained nitrogen Whilst Orlandi et al (2015) showed that the increasing of Acacia mearnsii tannin extract (i.e 9, 18 or 27 g/kg of total dietary DM) supplemented in restricted 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 a necessity on N balance of body that found in report of Theodoridou et al (2010) But Acacia 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 different concentration (at 1, 1.5 and 2% of DM)
Supplementary Saccharomyces cerevisiae: concept on detoxification
In the relationship of microbial rumen on the process of digestibility feed, probiotic has been studied in recent years with the objective to manipulate the microbial ecosystem to enhance rumen fermentation characteristic Fuller (1989) mentioned that probiotic mostly consist of Lactobacillus species, Bifidobacterium species,
Streptococcus species, yeasts, and molds was used as probiotic, in which yeast as Saccharomyces, mold (mainly as Aspergillus oryzae) and Lactic Acid Bacteria group
(LAB) was interested in many studied about their mode action on rumen fermentation. Most of these probiotics are microorganisms traditionally grown in fermented foods (Araújo et al 2012) Recently, Saccharomyces cerevisiae is particularly attentive on mechanism of detoxification that it applies not only on animals but also on humans There have been many studies growing interested in the influence of inactive yeast on rumen fermentation One of the major components of yeast and yeast cell wall products are polysaccharides such as α-D-glucan and β-D-glucan, constituting up to90% of the cell wall dry weight The inner layer of yeast cell walls consists of insoluble β-glucan (30–35%), the middle layer is soluble β-glucan (20–22%), and the external layer consists of glycoprotein 30% (Tokunaka et al 2002) The structure of glucans consists of β-1, 3 glucans with β-1, 6 branch linkages, and both endo and exo β-1, 3 glucanases are produced during yeast autolysis (Halasz and Lasztity 1991) The hydrolyzing process of yeast includes: (i) yeast was inactivated by heating in a water bath for 1.5 h at 80°C followed by freeze-drying (Jiang et al 2017) and (ii) then processing with acid-alkaline treatment (Oeztuerk et al 2016) β –glucan present not only in cell walls of yeast, fungi, and some bacteria but also in endosperm cell walls of cereal grain such as oats and barley (Volman et al 2008) The β-glucans are becoming very popular in the animal feed industry because of their potential beneficial effects on animal health and growth performance They have been shown to adsorb or bind toxins, viruses, and pathogenic bacteria (Vetvicka et al 2014) Suzuki et al (1990) suggested that feeding β-1,3-glucans improves immune response through receptor-mediated interactions with Mcells, which are specialized epithelial cells used for transporting macromolecules in the Peyers patches, resulting in increased cytokine production and resistance to infection The second most important component of yeast cell walls are a concern to be Mannans, it is linked by α-1,6 bonds in a branch structure, with side chains consisting of mannose units linked to the backbone with α-1,2 bonds (Halasz and Lasztity 1991) Phosphorus is connected to mannans with amount varies from 0.04 to 4.4% Mannanoligosaccharides (MOS) serve as prebiotics, or sources of nutrients for select microbes in the gastrointestinal tract, that can lead to providing a probiotic effect (Spring et al 2015) Garcia Diaz et al (2018) reported that diets containing MOS and live yeast+ MOS enhanced the health of the ruminal epithelium of sheep by reducing the thickness of the stratum corneum, and diets containing live yeast and live yeast+MOS decreased the incidence and severity of hepatic abscesses.
Recently, the aspect of the positive mechanism of S cerevisiae on binding mycotoxin has been reviewed in the literature The cell wall of S cerevisiae consists of the network of β-1, 3 glucan backbone with β-1, 6 glucan side chains, which is in turn attached to highly glycosylated mannoproteins which make the external layer (Kollar et al 1997) The proteins and glucans provide numerous easily accessible binding sites with different binding mechanisms such as hydrogen bonding, ionic or hydrophobic interactions (Huwig et al 2001) Binding of different mycotoxins such as aflatoxin,ochratoxin and zearalenone and minimize their adverse effects on animal health and performance have been reported earlier and the binding has been attributed to cell wall glucans in case of ochratoxin and zearalenone (Bejaoui et al 2004; Yiannikouris et al.2004; Shetty and Jespersen 2006) Strains of S cerevisiae have also been shown to bind ochratoxin A (Bejaoui et al 2004) and zearalenone (Yiannikouris et al 2004) and the binding has been attributed to the glucan components in both cases Binding seems to be a physical phenomenon with cells were treated at 52, 55 and 60 °C for 5 to 10 minute or 120 °C for 20 minutes, it makes binding significantly higher quantities of aflatoxin B1 than their viable counterpart (Shetty et al 2007) Likewise, when the cells were treated with 2 M HCl for 01 hours, up to 2-fold increase in binding was observed. The results obtained show that some strains of S cerevisiae, viable or non-viable, are effective aflatoxin binders and these properties should be considered in the selection of starter cultures for relevant indigenous fermented foods where high aflatoxin level is a potential health risk It opens up the potential for mycotoxin management and further on another toxin.
Conclusions References
Methane production plays the role important for rumen fermentation and affects directly to the growth of ruminant The reduction of methane has significant in utilizing the energy of feed intake more effectively In the methods of diminishing methane production, “bitter” cassava foliage is evaluated more effective than “sweet” cassava foliage, yet the concentration of cyanide in cassava leaves is rarely measured so that it is still poorly understood the relationship between cyanide and decreased ruminal methane However, methanogen bacteria not only act as an individual process but also relate to overall microbial activity as integration of metabolic process Consequently, a more direct evaluation of “bitter” cassava foliage in ruminant diet and its effect on ruminal parameter is required to truly understand the impact of cyanide in cassava foliage to ruminant’s feeding system.
Although “sweet” cassava foliage was demonstrated its effectiveness on health safety and productivity of ruminant, nevertheless, feeding “bitter” cassava foliage will a challenge with building feeding method and looking for the support from additives that it is thought cyanide detoxification As mentioned, adding brewer grain and/or biochar as the detoxifying support of cyanide toxin into “bitter” cassava foliage has not been explored in depth, yet the insight of this support will be more significantly on promoting the utilization of cassava by-product Furthermore, this information will help toxicologist to develop detoxification method of cyanide on animal and human more effectively.
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METHANE PRODUCTION IN AN IN VITRO FERMENTATION
Introduction
Recent industrial development of cassava root processing for extraction of starch released source of abundant cassava pulp Cassava pulp and other by-products of cassava such as leaves, and stalk have potential feeding value for livestock Cassava by-product needs to utilize in ruminant’s feeding system is cassava pulp, due to its negative fermentation impact lead to polluted environment Cassa pulp represents approximately 10 to 15% of the original root weight (Khempaka et al 2007) On a dry matter (DM) basis, cassava pulp contained 70% starch, 1.70% ash, 1.55% crude protein(CP), 27.8% crude fiber (CF) and 0.12% ether extract (EE) (Sriroth et al 2000) The pulp is very low in protein; however, the foliage is high in CP with content of more than 20% in DM (Lukuyu et al 2014) It was reported by Ffoulkes and Preston (1978) that fresh cassava foliage could replace soybean meal as the only protein source in a fattening diet for cattle based on ad libitum molasses-urea Preston and Leng (2009) postulated that part of the cassava leaf protein had “rumen-escape” characteristics which helped to balance the microbial protein produced from the rumen fermentation of molasses supplemented with urea.
Cassava products contain cyanogenic glucosides which liberate hydrocyanic acid (HCN) when enzymatically degraded Cyanogenic glucosides exist as linamarin and lotaustralin in unbruised leaf (Nartey 1968) When the cellular structure is broken, the glucoside is exposed to extracellular enzymes such as linamarase which gives rise to toxic hydrocyanic acid In studies on bio-digestion of cassava residues it was shown that the HCN liberated in the digestion process was toxic to methanogenic bacteria (Smith et al 1985; Rojas et al 1999) It is therefore postulated that a similar process could take place in the rumen of cattle fed cassava products, which could be an advantage as a strategy for reducing greenhouse gas emissions from ruminant animals. Sweet cassava variety are generally categorized into “sweet” varieties suitable for human consumption The “bitter” varieties more appropriately used for industrial production of starch It is understood that the ‘bitter” varieties are so-called because they have higher concentrations of cyanogenic glucosides making them potentially toxic to humans and animals Establishing a feeding system from cassava by-product is limited the available information on its effectiveness and the impact of different level of HCN concentration in cassava foliage varieties on reduced methane production is not clear Therefore, the hypothesis of this study was to test that methane production in an in vitro rumen fermentation would be reduced when urea-supplemented cassava root pulp was incubated with the leaves from bitter, rather than sweet, varieties of cassava.
Materials and methods
The in vitro experiments were conducted in the laboratory of Nong Lam University,
Ho Chi Minh city, Viet Nam, in December 2014.
The four treatments in a completely randomized design (CRD) were the leaves of four cassava varieties (Gon, Japan, KM94 and KM 140-1) with four replications The substrates were cassava pulp and urea The leaves were added to provide an overall level of 12.8% crude protein in substrate DM.
Table 2.1 Composition of the substrates
A simple in vitro system was used based on the procedure reported by Inthapanya et al (2011).
The cassava leaves were from plants five months old growing in different locations in Cam My, Dong Nai province Leaves (without petioles) were selected at a point approximately one third of the height of the plant measured from the top They were stored in plastic bags to avoid loss of moisture In the laboratory, the fresh leaves were chopped into small pieces and then ground (1mm sieve) Dry cassava pulp was taken from the Wuson starch factory, Binh Phuoc Province.
Rumen fluid was taken from a Holstein male animal immediately after it was slaughtered at the local abattoir Rumen fluid was filtered directly through 2 layers of cloth to contain in thermal flask to keep warm, then moving quickly to laboratory for mixing The 12 grams of substrates (Table 2.1) were mixed with 0.24 liters of rumen fluid and followed by 0.96 liters of buffer solution (Table 2.2) This mixture was contained in the fermentable bottle, gassed with carbon dioxide, and incubated in a water bath at 38 ° C for 24h.
Table 2.2 Ingredients in buffer solution
Ingredient s CaCl 2 NaHPO 4 12H 2 O NaCl KCl MgSO 4 7H 2 O NaHCO 3 Cysteine g/liter 0.04 9.3 0.47 0.57 0.12 9.8 0.25
The gas volume was measured by water displacement from the receiving bottle suspended in water The bottle was calibrated at intervals of 50ml The methane
UK) The DM and crude protein contents of the substrates were determined according to AOAC (1990) methods Ammonia was analysed in the filtrate after separating the solids using a cloth filter HCN was determined by titration with AgNO3 after boiling the sample in KOH to concentrate the HCN Tannin was analyzed by the Lowenthal method consisting of boiling the leaves in 0.1N H2SO4, adding indigo dye and titrating with potassium permanganate.
The data were analysed with the general linear model (GLM) option in the ANOVA program of the Minitab software (Minitab 2000) Sources of variation were treatments, and error.
Y is the observation random variable representing the response for cassava varieties. à is the overall mean.
Ti term the treatment effect (i=1-4). eij is random error.
Chemical composition of the substrate
The cassava leaves contained a high level of crude protein (27.5-31.8% CP in DM);the cassava pulp had less than 3% CP in DM (Table 2.3) The starch residue in cassava pulp is abundant, therefore, it was used as an energy source effectively in ruminant feeding system by adding non-protein source such as urea (Phanthavong et al 2016) The concentration of HCN in Gon variety is lowest among varieties, therefore, it does not taste bitter like the rest of three cassava varieties (Japan, KM94 and KM140-
1) In cassava field, Gon variety is called “sweet” cassava and is grown for human consumption Japan, KM94 and KM140-1 varieties was called “bitter” varieties and appropriately used for industrial production of starch.
Table 2.3 Chemical composition of the ingredients in the substrate
Gon Japan KM 94 KM 140-1 Pulp
HCN concentration, mg/kg DM 339 419 570 826