2000.From the successful experiment in utilization of cassava foliage sweet variety as proteinsource on cattle which was originally reported by Ffoulkes and Preston 1978, and thenhave be
Trang 1HUE 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
Trang 2This dissertation is completed at: University of Agriculture and Forestry, Hue University
Supervised by:
1 Assoc Prof Dr Nguyen Huu Van
2 Dr Dinh Van Dung
1streviewer: ………
………
2ndreviewer: ………
………
3rdreviewer: ………
………
The dissertation will be defended at the Council of dissertation assessment of Hue University, 04 Le Loi Street, Hue city, at……….…….on……/…… /2020
Dissertation can be further referred at:
1 National Library
2 Center for Information and Library of Hue University of Agriculture and Forestry, Hue University
Trang 3List of abbreviations, symbols and equivalents
Trang 51 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 proteinsource on cattle which was originally reported by Ffoulkes and Preston (1978), and thenhave been successfully fed as fresh state to goat and cattle in Cambodia (See report ofPreston and Rodríguez Lylian, 2004), that make cassava foliage become important plantprotein source in ruminant diet Nevertheless, cyanide toxin in fresh cassava foliage,especially bitter cassava foliage, is the main obstacle for animal such as restricting theconsumption intake of ruminant or causing poisoning when they consume rapidly.Nowadays, as the quantity of bitter cassava (high cyanide content) predominate oversweet cassava (lower cyanide content) on the field, looking for feeding method of bittercassava foliage diet with minimizing negative effect of cyanide toxin will match realitymore but will a challenge
Many studies are beginning to be interested in cyanide toxic that has certain effect onmethanogenic bacteria population by inhibited methanogenesis activity lead to diminishmethane production (Ch Olga Rojas et al 1999; Phuong et al 2012; Phanthavong et al.2015) However, whether cyanide may affect overall microbial activity and impact therate of rumen fermentation indirectly, it is still not fully understood Previously, theknowledge of ruminant nutritionists focused on rumen, but the impact of cyanide onrumen fermentation may profoundly influence lower digestive physiology of ruminantand must be considered to fully understand when utilizing bitter cassava foliage in diet.Even so, the challenge of bitter cassava foliage diet (high cyanide content) is a newapproach but must require the safety for animal's health Therefore, building suitablefeeding method for fresh cassava foliage diet, particularly bitter cassava foliage, withoutcausing cyanide toxin is needed to utilize cassava foliage more effectively in theruminant feeding system
2 The objectives
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 things canimprove 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 thereduction of methane production that was built on earlier findings
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
Trang 6ii Building feeding method of “bitter” cassava foliage (KM 94 variety; moderate
HCN content) diet by added 4% brewers’ grain (of DM) and/or 1% biochar (asDM), then evaluating the effects of this feeding method on growth,digestibility/N retention, excretion of thiocyanate in urine and methaneproduction of cattle and goat
iii 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.
3 Significance/innovation of the dissertation
This dissertation successfully demonstrated that HCN in cassava foliage is mainfactor for reduction of methane production while the earlier finding could only use it asprediction for decreased methane Currently, the best-known cassava foliage to feedanimal is “sweet” cassava foliage with low cyanide content, my dissertation succeeds tobuild feeding method for “bitter” cassava foliage diet (higher cyanide content) withsupport of adding restricted brewers grain (4% of DM) and biochar (1% of DM) to feedcattle and goats without cause HCN toxicity Additionally, discovery of the feeding of thebitter cassava foliage appear to modify the rumen fermentation lead to increases innitrogen retention associated with reduced methane production, it made a part of thisdissertation provided the implication for new approach of the proposed partial shift insites of digestion (from rumen to small intestine and the cecal-colon region) thatpreviously it is thought that only rumen fermentation have a truly symbiotic relationshipwith the ruminant
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CHAPTER 1: LITERATURE REVIEW
The literature review consists of the key issue on:
(i) Process of rumen fermentation that related to methane production and direct itseffect on the growth of ruminant Interpreting interaction of ruminal microorganism ispossible detoxification strategies in context of using cassava foliage that contain highHCN content
(ii) The utilization of some by-products in ruminant feeding such as: (i) cassavafoliage varieties as a protein source implied that HCN content as inhibition ofmethanogenesis, and (ii) restricted brewer grain and biochar as additives that it is thought
cyanide detoxification in cassava foliage diet; (iii) the role of Saccharomyces in brewer’s
grain was expected as “prebiotic” to support cassava foliage diet
All above issues were placed in integrative review via the description, interpretation
of prior research and reveal gap in literature
Trang 7CHAPTER 2 THE EFFECT OF SUPPLEMENTATION WITH “BITTER” OR
“SWEET” VARIETIES ON METHANE PRODUCTION IN AN IN VITRO
INCUBATION
Introduction
Recent industrial development of cassava root processing for extraction of starchreleased source of abundant cassava pulp Cassava pulp and other by-products of cassavasuch as leaves, and stalk have potential feeding value for livestock
Cassava by-product needs to be utilized in ruminant’s feeding system is cassava pulp,due to its negative fermentation impact lead to polluted environment The pulp is verylow 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 cassavafoliage could replace soybean meal as the only protein source in a fattening diet for cattlebased on ad libitum molasses-urea Preston and Leng (2009) postulated that part of thecassava leaf protein had “rumen-escape” characteristics which helped to balance themicrobial protein produced from the rumen fermentation of molasses supplemented withurea
Cassava products contain cyanogenic glucosides which liberate hydrocyanic acid(HCN) when enzymatically degraded Cyanogenic glucosides exist as linamarin andlotaustralin in unbruised leaf (Nartey 1968) When the cellular structure is broken, theglucoside is exposed to extracellular enzymes such as linamarase which gives rise totoxic hydrocyanic acid In studies on bio-digestion of cassava residues it was shown thatthe 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 takeplace in the rumen of cattle fed cassava products, which could be an advantage as astrategy for reducing greenhouse gas emissions from ruminant animals
Cassava varieties are generally categorized into “sweet” varieties suitable for humanconsumption, and “bitter” varieties more appropriately used for industrial production ofstarch It is understood that the ‘bitter” varieties are so-called because they have higherconcentrations of cyanogenic glucosides making them potentially toxic to humans andanimals Establishing a feeding system from cassava by-product is limited the availableinformation on its effectiveness and the impact of different level of HCN concentration incassava 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 incubatedwith the leaves from bitter, rather than sweet, varieties of cassava
Materials and methods
Experimental design
The four treatments in a completely randomized design (CRD) were the leaves of a
“sweet” variety of cassava (Gon) and leaves from three bitter varieties (Japan, KM94 and
KM 140-1) with four replications The substrates were cassava pulp and urea (Table 1)
Trang 8The leaves were added to provide an overall level of 12.8% crude protein in substrateDM.
Table 2.1 Composition of the substrates
The 12 grams of substrates (Table 2.1) were mixed with 0.24 liters of rumen fluidand followed by 0.96 liters of buffer solution (Table 2.2) This mixture was contained inthe fermentation bottle, gassed with carbon dioxide, and incubated in a water bath at 38°Cfor 24h
Table 2.2 Ingredients in buffer solution
in KOH to concentrate the HCN Tannin was analyzed by the Lowenthal methodconsisting of boiling the leaves in 0.1N H2SO4, adding indigo dye and titrating withpotassium permanganate
Trang 9Statistical analysis
The data were analysed with the general linear model (GLM) option in the ANOVAprogram of the Minitab software (Minitab 2000) Sources of variation were treatments,and error
Results and discussion
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)
Table 2.3 Chemical composition of the ingredients in the substrate
(*) Data taken from Khempaka et al (2009); ND= not detected
Gas production, ammonia concentration and DM mineralized
The percentage of DM mineralized (or DM solubilized) was lower in the sweetcassava variety than in the three bitter varieties among which there were no differences.Ammonia concentration in the fermentation medium at the end of the incubation washigher for the sweet cassava than for the bitter varieties (Table 2.4)
Table 2.4 Mean values for gas production in 24 hours, DM mineralized and ammonia
in an in vitro rumen fermentation.
Sweet leaves Bitter leaves
ab Mean values in rows without common letter are different at p<0.05
Methane, HCN and condensed tannin
Methane production, expressed as percent of the gas production or per unit DMmineralized, was lower when leaves from the bitter varieties were the protein sourcecompared with the sweet variety (Table 2.5) The negative relationship between HCN andmethane production indicates that it is the HCN precursors in the leaves of the bittervarieties that are responsible for the decline in methane production (Table 2.5)
Trang 10Table 2.5 Mean values for content of condensed tannin and HCN in the leaves of cassava
varieties, ammonia concentration and methane production per DM mineralized after 24h
incubation
Gon Japan KM94 KM 140-1 SEM p valueMethane in the gas, % 20.0a 14.0 b 10.5 b 11.3 b 1.31 <0.001Methane ml/g DM
mineralized 29.9a 18.3 ab 14.3 ab 11.5 b 3.80 0.023HCN, mg/kg DM 339 a 419 b 570 c 826 d 31.9 0.044HCN content in treatment, mg 1.02 1.26 1.71 2.46
Condensed tannin in leaf, % 2.00 2.55 2.36 2.55 0.29 0.30Condensed tannin in
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CHAPTER 3: A LOW CONCENTRATION OF BREWERS’ GRAINS IMPROVES THE GROWTH RATE AND REDUCES THIOCYANATE EXCRETION OF CATTLE FED CASSAVA PULP-UREA AND “BITTER” CASSAVA FOLIAGE
Introduction
Comparison of the effects of levels of HCN content in cassava leaves on in vitroexperiment (Chapter 2) only show the local effects on rumen fermentation, the need toestablish feeding trial on animal is to better understand how cassava foliage affect tointake and growth, particularly with moderate HCN content in bitter cassava foliage diet.However, how to manage HCN content in fresh cassava foliage diet, especially bitterfoliage variety, is still the biggest concern that it is being looked for the effective solution.Against background of cyanide toxin in bitter leaves, we organize the experiment oncattle with cassava pulp-urea as basal diet and compare “sweet” versus “bitter” cassavafoliage as protein source The hypothesis is that addition of 4% of fresh brewers’ grains to
“bitter” cassava foliage may improve the growth rate of cattle and aid in thedetoxification of the HCN associated with the feeding of “bitter” cassava foliage
Trang 11Materials and methods
Treatments and experimental design
Twelve Laisind cattle were the experimental animals in a completely randomizeddesign with 3 treatments and 4 replicates The basal diet was ensiled cassava pulp plus
0.7% urea (in DM basis of pulp) fed ad libitum together with a mineral mixture
containing 7.5% sulphur, 40% dicalcium phosphate and 52.5% sodium chloride Thesetwelve cattle were spent two experimental periods as follows:
The treatments during the first 56 days (Period 1) were:
● BG-RS: Fresh brewers’ grains at 1% of live weight and rice straw 0.9% of live
weight (both on DM basis)
● CFB: Fresh foliage from a bitter variety (KM 94) of cassava at 1% of live weight
(DM basis), replacing the brewers’ grains in the BG-RS control treatment
● CFS: Fresh foliage from a sweet variety (Gon) of cassava at 1% of live weight
(DM basis)
For the 2 nd period of next 56 days, the treatments BG-RS and CFS remained the same
Period 1, but treatment CFB was modified to CFB-BG treatment by adding a supplement
of brewers’ grains offered at 4% of the diet DM
Table 3.1 The chemical composition of ingredients
Cassavapulp
Brewer’
s grain
Ricestraw
Cassava foliageBitter (KM 94
variety)
Sweet (Gon variety)
HCN, mg/kg
ND= not detected; CP= crude protein; DM=dry matter
Animals and housing
The 12 Laisind cattle with initial live weight from 130 to 175 kg were housed inindividual pens, with bamboo slatted floors and roof of Nipa Palm leaves The cattle werevaccinated against foot and mouth disease and de-wormed with Ivermectin beforestarting the experiment
Feeding and management
Fresh cassava pulp (pH 3.2) was purchased from the Wusen cassava starch factory,Binh Phuoc province It was stored in closed polyethylene bags prior to feeding.Brewer’s grains were purchased from the beer factory located in Ho Chi Minh City at 03-day intervals and stored in woven polypropylene bags during the 03 days Cassava plots
of the two varieties ‘KM 94’ and ‘Gon’ were established in the University farm Cassavafoliage took 1.5 hours to collect on field and transported it to experiment every day tofeed cattle directly The foliage was harvested at successive periods of 4-5 monthsregrowth
Cattle were spent 1.5 months to adapt to treatments prior to starting real experiment.For the adaptation of fresh bitter cassava foliage, sweet cassava foliage was introduced to
Trang 12cattle firstly, then gradual replacement of sweet foliage by bitter foliage until cattle canfeed only bitter cassava foliage as a protein source All the feeds were offered in separatetroughs as freely chosen and were feed three times a day, at 8.00 a.m., 12.00 a.m and5.00 p.m.
Data collection and measurements
The cattle were weighed at the beginning of the experiment and at intervals of 14days Feeds offered and refused were recorded daily Samples of rumen fluid were taken
by stomach tube 03 hours after the last meal at the end of the experiment fordetermination of concentrations of individual VFA (Rowe et al.1979)
On the last day of the whole experiment: (i) samples of urine were caught behindeach female cattle and avoid contaminated by feces, frozen and stored at -20°C forthiocyanate analysis; (ii) the cattle were confined individually in a closed chamber forsuccessive measurements over 10 minute periods of the concentrations of methane andcarbon dioxide in mixed air and eructed gas The procedure was that described byMadsen et al (2010), using an infra-red gas meter (GASMET 4030; GasmetTechnologies Oy, Pulttitie 8A, FI-00880 Helsinki, Finland) For the cattle on treatmentCFB-BG, after first sampling of eructed gas/air, and of urine, the brewers’ grains wereremoved from the diet and for 5 consecutive days they were fed only cassava pulp-ureaand bitter cassava foliage At the end of this period the eructed gas-air was againanalyzed, and samples taken of urine for thiocyanate analysis
Chemical analysis
Feeds offered and refused were analysed for DM and N following the procedure ofAOAC (1990) Thiocyanate in urine was measured using the procedure described in theprotocol of kit D1
Statistical analysis
The data were analysed with the general linear model (GLM) option in the ANOVAprogram of the Minitab software (Minitab 2000) Sources of variation were treatments,and error
Results and discussion
In Period 1 the cattle fed bitter cassava foliage as sole source of bypass protein hadlower DM intake than those fed sweet cassava foliage and gained only 61g/day comparedwith 383g/day for those fed sweet cassava foliage (Table 3.2a) When brewers’ grains at4% of the diet were added to this treatment in Period 2, the DM intake increased by 47%and the live weight gain (380g/day) did not differ from the 410g/day for cattle fed sweetcassava foliage (Table 3.2b)