Comparison of a two-stage technique using rumen inocula of sheep and an enzymatic method for predicting digestibility of food resources in Sika deer, Cervus nippon, habitats

NII-Electronic Library Service

BiosphereConservatjon9₍₂₎:29-37,2009

Comparison

of a

Two-stage

rllechnique

Using

Rumen

Inocula

of

Sheep

and an

Enzymatic

Method

for

Predicting

Digestibility

of

Food

Resources

in

Sika

Deeg

Ciervus

nilp!pon,

Habitats

Norio

lbkitai,

Emi

Sakatai

Akiko

Tlakli2,

Masaki

Nedui,

Tlsutomu

Konnoi,

and

Teruaki

Ibkita3

iDepartment

qfAnimal 5lrienee,IVipponfeterinaty andLgi2i Sbience(j7tiversiijl

Adiisaghino-shi,lblyol80-8602,Japan

2Fkecudy

qfAgriculture,7bkyoUhiversity

ofAgriculture

and fechnolognFuchu-shi,7bkyol83-8509,.Japan

3Department

qf'AnimalSciences,feikyoUhivensityqf'Science and fechnology

Uenohara-shi,lhmanashi409-O193,Jopan

Abstract Digestibilityoffbod resources inSikaDeerhabitat were mcasured using in vifrz] rnethods using rumen inocutaof deerand sheep oracellulelitic enzyme. Experimeritalsamples were collectedbothinwarm

ternperate

(1

50-900m above sea level)and coel temperate

(900-1500m

above sealevel>zones inthe

mountain-ous of Tanzawa,A totaLof 150test_plantsconsisted of 39sampLes of38 species insummer and 111samples of

65species inwinter, Thetest_plantsconsjstedof leavesand stems fromforbsand grasses,leaves,deadleaves,

twigs,and barkfromevergreen or deciduousbroadleavedshrubs and trees.Drymatter digestibility

(DMD)

measured bythetwo-stepmethed

(a

seriesofjncubations with mmen inoculaand acidic _pepsinso]ution) using rumen inoculaof SikaDeerranged from23.5Y6forlcafinPolygonumfilijZ)rmeto83.8%ofleafinlsolp,rum stoionij2iruminsummer and fromO.3% ofdeadleafinRrgesscrenata to76,6%ofleaves in Ctiidomine,17exuo-sa inwinter. Comparedwith theseresults,DMD measured bythemethod usingsheep rumen inoculawas fitted

for_predictinginvitro DMD used deerrumen fluidand highlycorrelatcd with each _pa{tof thevalues. DMD as-sayed bytheenzymatic method, combining aseriesofincubationsusingthe acid-pepsin treatmentand cel-]uloliticdigestion.was correlated with thesamples collected inwinter and inthecoo]temperateareain

sum-mer, butdidnot fitwith thesamples fromthewarm temperatearea insummer. Ourexperimental results showed thatin vimo methods using sheep rumen inoculaareuscfu1inpredictinginvitroDMD ofdeer, and the

enzymatic method isalso able tomeasure DMD of foodreseurces inwinter deerhabitat,Theresultsare also available toevaluatethenutritive_qualityofplant resources eaten byfree-rangingdeer.

Keywords: digestibility,pepsin-cellulaseassay, rumen jnoculum,sheep, sika deer,

INTRODUCTION

Free-rangingdeeracquire all oftheirnutntional neeessities fromthefoodresources intheirhabitats

throughoutthe_year

(Vttn

SoestI982).The_qualityof

feedisbasical]yexpressed as nutritive value and

measured chemica[ composition, energy content and utilization fbradvantages of hostanimal. Food

di-gestibilityisan important_guidelinefor

understand-ingthenutritive value of fbod becausethevalue

re-flectsa combjnation of fbodcharacteristics and animal _perfbtmance

(Ulyatt

1973),Itwill provideus

to

be

very usefu1

in

investigating

both

habitat

man-agement and

deer

nutrition.

The digestibilityof _plantseaten bySikaDeer,

Cemsttsning7on, _providesimportantnutritional

infoT-mation thatrelates toanimal _growth,maintenance and reproduction

(Vati

Soestl982).Althoughseveral

methods of measuring invivo

digestibility

of

feed

havebeen developedand designedfordomestic

BiosphereCenservation9(2),2009

minants such ascattleand sheep

(Church

1984),itis

diMcult

toadapt thesemethods forwild deerinthe

field

_CRobbins

1993)becauseitisnot_possibleeither

tomeasure foodintake_{quantitatively}or tocollectali

feces.Forcaptive deer,researchers and managers must feedacertainnumber ofdeer ina_proper facil-ityand must _preparcand control largeamounts of foodmaterials forfeedingexperiments. The

conven-tionalmethod of

determining

digestibility

bytotal fecaleollection method

(Minson

19g5)isthemost preciseand reliable method forruminants,

but

it

re-quiresconsiderable, time,laborand money. Thereis

a _greatneed fora simple and accurate method for evaluating _thedigestibilityof_plantresources eaten

bydeer.

Tilleyand Terry

(1963)

introduceda two-stage

technique,which substitutes theinvitro digestionof foragecrops forthedigestionintherumen and the

hind

digestive

tractsof rurninants. Minson and

McLeod

₍₁₉₇₂₎

developedthistechniquefor

esti-mating the

digestibility

ofa

large

numbers of

forage

samples, Although Tilleyand Tlerry

(1963),

and Min-son and McLeod

(1972)

used rumen inoculaintheir methods, Gotoand Minson

(1977)

verified the

pre-dictionof digestibilityof_grassesusing a

pepsin-cel-lulaseassay, which allowed them toestimate the di-gestibilityvalue without utilizing any rumen

inoculum,

Eachof thesethreeinvitro methods is

commonly used toestimate the_drymatter digestibil-ityof foragesamples of domesticruminants. These

invitro methods havetheadvantage that:thesample amounts required are very small

(0,5g

each), the

measurement isinexpensive,and the_predicted val-ues relatetothe

digestibility

invivo

in

each run.

InSikaDeer habitats,

typically

inwarm and cool

temperateareas on mountain sides,natural food

re-sources mainly consist of forestunderstory

vegeta-tion,treeleaves,twigsand bark

(Furubayashi

and

Maruyama 1977;Furubayashi1995).

Seasonal

dif ferencesin_plantgrowthand chemical composition may afftctdigestibilityvalues

(Grime

l979;Wailmo

l977).Thus the digestibilityof food resources in deerhabitatmay vary considerably compared with

food

provided

for

domestic

ruminants. Therefbre,it isnecessary toverify theconformity ofthe

digest-ibilityvalues obtained

by

each method

in

order to

determinedifferencesin_plantresources from

differ-entsarnpling sitesand seasons.

The purposeof thisstudy was toevaluate thcin

vitro drymatter

digestibility

ofplant sarnples

com-prisingthenatural fbodresources of deerinwarrn

and cool temperate areas throughout_theyear,

by

means of invitromethods using deerand sheep

ru-men

inocula.

We also attempted to_predjctthe di-gestibilityof foodsamples using theartificial

enzy-rriatic

(Onozuka-cellulase)

assay, and we compared

thetwo methods.

MATERIALS AND METHODS

Ptantsample coiiection and chemicat analysis Allplantsamples used inthis experiment were collected fromSikaDeer habitatsof theTanzawa mountains insummer

(July

]999)and inwinter

(De-cember 1998-March1999).Sampleswerc divided intowarin

(ca

200-900m)and cool temperate

(900-1500m)areas

based

on theelevation ofthe

collec-tionsites, Sarnpleswere also _groupedaccording to

theirmajor species and morphological

characteris-ties.Eachsample was

dried

inaforced-draughtoven at 60℃ toconstant weight and then_groundthrough

a 1mm screen inaWileymillbefbreanalysis,Crude

protein

(CP)

was deterrninedaccording tothe

meth-od reported bytheAssociationof OrncialAnalytical Chemists

_(1984),

and aciddetergentfiberand lignin

bythemethod ofGoering and VanSoest

(1970),

Lfeasurements

ofdy

ntatter digestibility

I}ivitrodrymatter digestibilityofall samples used

inthisexperiment was measured using thetwo-stage

techniqueintroducedbyTilleyand Terry

(1963),

Therumen fiuiduscd inthefirstincubationwas

ob-tainedfromtherumens of two or threeadult Sika

Deertakcn

during

legaL

hunting

during

the sarne

pe-riods of _plantsample collection. Rumen inocula were also collected fromthreedomesticSuffolk

NII-Electronic Library Service

BiosphereConservation9_(2),2oo9

sheep fittedwith rurnen cannula. Thesesheep were

keptinindividualcages and fed_goodqualityalfalfa

hayfreely.In

both

of theabove invitromethods,

O.5gef sample was _placedinaNarugentube

(Naru-genCo,Ltd.)towhich 50rnlof buffersolution and

rumen fluid

(4:1,

vfv) containing O.5mlofruniinant artificial saliva was added. Then,each tube was capped with arubber _plugwith an exhaust valve for

gas release, and incubated

in

a water

bath

at

39

℃

fbr48h.Thefirstincubationtubewas centrifuged at 3,OOOrpmfbr15minand theupper layerdiscarded.

Then,50mlof O.INhydrochloricacid containing

O.2%_pepsinselution was added totheresidue ofthe firstincubationinthetubeand incubatedinthesame manner as thefirstincubation,Afterthe second

in-cubation, indigestible

_parts

were collected by

cen-trifugationand driedat60℃ over night,Digestibitity was calculated asthedifferencebetweentheoriginal sample wcight and

losses

fromthesecond

incuba-tion.

Thepepsin-cellulaseassay _procedurefbllowedthe

method introducedbyGotoand Minson

(1977).

A O,5gsample was used inthesame manner as men-tionedabove and 50ml of O,1Nhydrochloricacid

containing O,2% _pepsinsolution was added; thefirst incubationwas at

39

℃

for

48h.

After

the

first

incu-bation,residues were collected bycentrifugation and 50mlof acetic buffersolution containing 2.5%

cel-lulase

_(Onozuka

cellulase,P-1500,YakultCo.Ltd,) were added and incubatedunder thesame conditions

as the firstincubation.Indigestible_partswere

col-lectedand counted forthecalculation of

DMD,

Each proeessof incubationand assay of each

sample was conducted intriplicate.

StatistiealanaC}{sis

Analysisof statistical differencesand linear

re-gressionsbetweenmeasurements were rnade using

multiple testsaccording to theanalysis of variance

and correlation functionas describedbyYoshida

(1980).

RESUUI'SANDDISCUSSION

Crude

protein,acid

detergent

fiber

and

lignin

con-tentof thediffbrent_groupsof _pLantsamples used in thisexperiment were all measured

(see

Table1),The

crude _proteincontent of 39 samples insummer ranged t'rorn12.9%to16.7%and thatof 111

sam-plesinwinter ranged from7,O% to16,8%.The

crude _pToteincontent ofwinter samples were mainly

basedon thediffbrenceofplant _partsand were high-er values in]eavesthanthoseintwigs,deadleaves,

andbarks,

Mean values of invitrodrymatter digestibility

(IVDMD)

of the_plantgroupsinsumrner and winter

were measured inthreedifferentways

(see

Tables2

and

3).

Themean IVDMD yatuesofsummer

sam-ples

ranged from49.0%to71.6%with deerrumen

inocula,from50.4%to77.8%with sheep rumen in-oeula, and from47,1%to66,8%with

pepsin-cellu-lase,Themean IVDMD values of winter samples

ranged from22.7% to _69.19,6with deerrumen inoc-ula, from34.6%to83.3%with sheep rumen inocula,

and from33,O% to80,7% with _{pepsin-cellulase,} When themean IVDMD values of allsamples mea-sured using sheep rumen inoculaand

pepsin-cellu-lase

assay were cornpared with IVDMD values from

the method using deerrumen inocula,itwas ob-served thatthemethod using sheep rurnen

inocula

overestimated thevalues with deerones except

for

theleavesofevergreen monocot and twigs

ofdccid-uous broadleavedtreesinsummer. Similarresults were also

found

theIVDMD values bythe

pepsin-celluage assay except fbrthe_plantsamples from

barkand twigsof

deciduous

broadleaved

trees

ob-tainedfromwarm temperateareas insummer and

cooltemperateareas inwinter.

RelationshipsbetweenIVDMD measured

by

deer

mmen inoculaand DMD measured bysheep rumen

inoculaor enzyme cellulase were examined

(see

Figures lto4).Theslopcs of rcgression equations

were similar for_plotsinwarm and cool temperate

areas insummer, and thecorre]ation coeMcients ef linearregressions between

digestibility

BiosphcrcConservation9_(2),2009

Table1.Mean val ues')ofcrude _protein,aciddctcrgentfiber,and ligriincontentofthe_plantsamples.

Season/arealplantgroup Part Cp2}ADF])ADL4)No. offamilyNo,ofspeclcsNo,ofsamplcs

Summer

Warm temperatearea

(200-900m

above sea level)

Deciduousbroadleavedherb5} Leaf

Deciduousbreadleavedtree Leaf

Cooltemperatearea

(900-1500m

above sea level)

Deciduousbroadleavedherb Leaf

Evergreenmonocotolydon6) Leaf

Deciduous broadleavedtree Twiglleaf

16,215.5 16.312.913.4 15,611.6 8.629,813.8 5.76.1 4,84.49.5 75 1034 108

1235

u8

123s

Winter

Warm temperatearea

(150-900m

above sea level)

Evergreenmonocotolydon

Monocotolydon

Deciduous

broadleaved

herb

Evergreenbroad]eaved tree Deciduousbroadleavedtree Deciduous

broadleaved

tree

LeafLeafLeailstem

LeafTWigBark

Cooltempcratearea

(900-1500m

above sea

level)

Evergr¢enrnonocotolydon Leaf

Deciduous

broadleavedtree Dead

leaf

Decidueus broadleavedtree [[kNig

Deciduousbroadleavedtree Bark

16.415.516,8 9.9 8.6 8.3 13.5 7.0 7.7 8.4 40.030.916,134.841,438.1 38.938.641.745.4 4.8 6.5 4.718,3IS,7122 6.918.517,121.8 136563 27IS10 156663 29216 310131274 5152913 1)%onadrymatterbasis 2)Crude_protein 3)Aciddetergentfiber 4)AclddetcrgentligrTin

5)lnclidingKudzu_(Puerariathunbergiana)_(Onesample)

6)IncludingTwagarami(SchizqphaTagmah),drangeoides)(Onesample)

ments made byIVDMD using deerrumen fluidand

by

thatusing sheep rumen

fluid

were O.9464

(p<O,05)

and O.95I6

(P<O.05),

respectively

(Fig.

1).

Theslope of theregression linewas steeper forthe

cool temperate area than tbrthewarm temperate

area, Therefore,theIVDMD measured bydeeT

ru-men

inocula

and theDMD measured bythe

pepsin-cellulase assay were correlated forthe_plantsofthe cool temperatearea

(r=O.7455,

P<O.05),

but

there

was no significantcorrelation forleavesinthewarm

temperatearea

(Fig.

2).IVDMD measured bythe

deerrumen inoculaforwinter

food

resources, both inwarm and cool temperateareas, with thevalues fromthesheep rumen _procedureand theenzyme as-say mcthod

(Figs.

3and 4).

In vitro method using rumen inoculais

theoreti-cally dernonstratedinvivo digestionof digestive

tractsinruminants and theextent ofdigestiblerates

relatively depends on theactivity ofmicroorganisms inthe rumen fiuid

(Tilley

and Terry1963).Inthe

pepsin-cellulaseassay, an enzyme

ofmicro-NII-Electronic Library Service

BiosphcreConservation9_(2),2009

Table2,Dry]nattcrdigestibilityi)ofsurnmcT _plantsmeasured bythreedifllerentmethods.

Plant_group Part[[Xvo-stage

meth-od using deer rumen

inocula

Two-stage meth-odusingshccp rumen inocula Pepsin-cellulase assay No.ofsamples

Wtirmtemperatearea

(200-900m

above sea level)

Dcciduousbroadleavedherb2)Leaf 50,9a3)tlO.3

Deciduousbroadteavedtrec Leaf 62.3a±13.8

Cooltemperatearea

(900-1500m

above sea level)

Deciduousbroadleavedherb Leaf 7e.7a± 8.1

Evergreenmonocotolydon2) Leaf 54,5U± 7,7

Deciduousbroadleavedtree TX-･ig 48.9a± 8.0

s8.4b ± 102 692h ±16.8 76.9b ± 10,5 57,9M±13.3 50.4"± 8.0 60,2b±11,2 66.6'in ± 13.5

66,8"

± 12,6 47.lh± 7.4 56.5a± 4.2 118 1235 1)`vaonadrymatterbasis 2)SeefootnotcillTable1.

3)Meanlstandarddeviation,MeansinarowwithdifferentsupeTscriptsdiffbr{P<O.05},

Table3.Drymatterdigcstibjlityi)ofwinter_plantsmeasurcd bythreediffercntmethods.

Plant_group PartTwo-stagcmeth-od usjng deer

rumen

inocula

TWo-stage meth-od using sheep rumen

inocula

Pepsin-cellulase assay No,ofsamples

SVarmtemperatearea

(150-900m

abovc sca level)

Evergreenmonocotolydon Leaf Monocoto]ydon Leaf DeciduousbroadleavedherbLeaD'stem Evcrgreenbroadleavedtree Leaf DeciduousbroadleavedtreeTWig DcciduousbroadleavedtreeBark 27,7a2)± 3.0 68.7a± 5.0 69.1"± 6.5 41.4"± 13,3 36.4"± 16.8 495"± 9,8

Cooltemperatearea

(900-1

500m abovc sea Ievel)

Evergreenmonocotolydon2) Dcciduousbroadleavcdtrec Deciduousbroadleavedtree Deciduousbroadleavedtree

LeafDcad leaf TWigBark 22.7"± 4,5 34,9a± 17.3 31.2a± g.8

32.0"

± rl.9 39.7b±

3,9

81.4b± 6.9 83.3b± 6.4 so,gh ± 13.0 4s.4b ± 16.0 59.lb± ll.6

34,6b

± 2.8 46,8b±16.2 41.6b± 7.9 4o.9b ± 12,8

37.2b

± 1,9 75.0C± 9.7 8o.7bt

8.1

sO.2b±10.5 44.lb±162 s6.6ab ± 15.7 33,ob± 3,3 44.4b±19.7 40.8bth7.9 43.2b±11,6 310131274 51529l3 l)"Monadryrnatterbasis 2)Meantstmldarddeviatioll,Meansinarowwithdifflrrentsuperscriptsdiffbr(?<e.OS).

organisms intherumen

(Goto

and Minson1977).In

our results, digestibility,as measured byusing sheep rumen fluid,was higher

(except

fortheleaves

ofev-ergreen monocotyledons and thetwigsof deciduous

trees)

in

winter thanthatfbundbyusing deerrumen

inocula.Thisresult might beleadthatsheep rumen

fiuidcontains 15_generaand 34species of

microor-ganisms

when sheep are_givenregular feeds.Inwild

SikaDeer only one _genusand 7species of

microor-ganismswere foundintherumen fiuid

(Imai

and

Katsune 1977).Plantmaterials used inthis experi-ment would havemuch more _potentialdigestibility

fbrruminants, butSikaDeer could not utilize bythe

limitationrclatingtospecies and activities

ofmicro-organisms

(Church

1984;Minson 1990).Although theactivityof rumen bacteriarnay serve to

Biesphere　Consen・ation 　9_（2），2009 コ … 芸 ⊆   ∈ 量 」 g 葛 二 雪 （ 巴 避 ＝ 舅 芻 h 。 皿 翌 2 毎 ∈ ヒ 竃 わ 魯 go 8e　「 　　　1 　　 　1 　 　 　170 fie 5e 40 ：e 20 d9 o ー ト ー IIII 「 1 」 ー ． ． 鹵 」 ー 」 ． ．_． ー ト ー ー −₁

ー

「−_． ＝ 11 」 汚 闢 5 o10 尸．．tr　− ＿＿＿．，」r．，　＿一 ’．rttrrr，」冨凵」 20　　　 　3D　 　　　40　　　 　sa　　 　　60　 　　　70　　　 　SD

加vifredry　matter 　eligegt｝bili之y（覧）usingsheoplumen 刊uidgo100

Fig　L　Relationship　ofin 　vitro　dly　matter 　digestibili重_y　as　fbund　using 　deer　and 　sheep 　ru一 men　fluids　for　suminer　tbod　resources，

羣 モ_」 霎 ⊃ 」 ＝ 舘 Ψ 響 … 籌 ｛ 醤 》 嗣 窪 暑  畠 喝_」 婁 。」 ∈_〉 七 ミ 湿 ミ 「 ． ． ． − − 「 ． ． ． 」 ． ． −．_−． − − ． − ． 「 ． −．₁

−

I ー ト ー ー ． − 」 ． ． ． 1 ． ． − 0 0 0 0 G O O g S 7 6 5 4 3 20 10 o1 〆 ▲ 日 ’ ’ 贋 　　　　’ 　 　 　 　 ’ 　 　 　 ’ 　 　 ’ 　’ 　’ ’ 　　　　　　タ 　　　　　　ヲ”_贋 　　　　　　　▲ ●　　，， y＝O．737x＋17．呂 　 　　　 　　　 　　　 　　　 　　　　 　　　 　　　 　　　 ■　　’ 　 　 　 ’ ▲ 42．l ＞O．05

L

− −L −一一L − 一一一．一一．一．一＿．一一＿．L−．＿＿，⊥．一＿一．＿L −一一一 一 o10 　　　　　20　　　　　30　　　　　40 　　　　　50 　　　　　60 　　　　　70　　　　　BO

　　　 Dry　matterdigestlbllihy （覧）measetedby 　peps｝n−ce：Luls3eassaygo100

Fig　2　Realtjonship　betweett　dly　mattcr　digosdbility_（％）as　found　using 　dcer　rumen 　fiu−

NII-Electronic Library Service BiosphereConservation9_(2),2009 pu.= ¢ [eElle-sue'gc..h.e'g?-geseljs

::I

7e1 ' t60 t l

lse

l'

4o

L

t30 V [ 20 to o r -- th.-1.. Fig] o102e 3o 4o so 6o 7o so ihntrodryntatterdlgest[biilty(li)u$Ingsheeprvmenfiuld

.Relationshipbetweenim,itrodrymatter digestibil sheep rLtn]enfluidsforwinterfoodresources.

ulLmrrrugo loe

ity_(%) as found using deerand

tsEg 7oi:.. 6e.:.RsL.' 50sseen 40ts-isgE 3esE go

Fse

Lt

i 2o

r4I

ae

L

oole 2o 3o 4o se 6o 7o so ge loe Orvmatterdigestib"itv(-)moeserecibypepsin-cellulaseassffy

Fig4.Reiatiomshipbetweeninvitro drymatter digestibil{tyC%)asfo

men fiuidsand enzyme-cellulase forwinter foodresources.und

using deer

ru-35

BiosphereConservation9(2),2009

dowll

and digest_piantmaterials

(Tchimura

et al,

2004;Orskov1982),thiswas notclearinour experi-ment. Inthe_{pepsin-cellulase}assay,digestibility

val-ues were not correlated

for

_plantsamples

(especially

theleavesof deciduousbroadleavedherbsand de-ciduous broadleavedtrces)collected insummer.

Al-though thcsematerials were characterized as being highin_proteinand lowinfiber,thecorrelation coee ficientsbetwccnmeasurernents of above samples

were lowintheexperiment. The_{pepsin-cellulase} method was lessaccurate in

_predict{ng

IVDMD of

legume

herbage

_(Terry

et al. 1978)beeauschigh proteincontent depressesceilulase activity to de-gradefibercomponents. TheCellulase-Onozuka

en-zyme, which was used fbrthe _{pcpsin-cellulase}assay

inthisexperiment, exhibits low_proteaseactivityand

doesnot havcenough functiontobreakdown

di-etary components highin_protein.Inmateria]s low in_proteinand h{gh infibersuch as deadleaves,

twigsand

bark

of

deciduous

broadleavedtrees

col-lectedinwinter, theIVDMD values obtained bythe pepsin-cellulaseassay methods were correlated to

thealternativcmethod.

Inthepresentstudy,wc showed thatthetwo-step

method, using sheep rumen inoculaand acidic

pep-sin solution, could estimate theIVDMD of Sika

Deerwith sjgnificantcorrelation forvarious fbod

re-sources indeerhabitats.The _{pepsin-cellulase}assay

was also a reliablc method fbrevaluating theDMD

ofplant materials eaten bySikaDeerinwinter.

ACKNOWLEDGEMENTS

We gratefu11yacknowledge the assistance of the

rncmbcrs of thcAtsugibranch,Kanagawa Hunting Association,inthecapture of Sikadeer.We also

thankMr. M. Nakamura, _presidentof theTanzawa

NatureConservationAssociation,for

his

financial

support.

R.EFERENCES

Asseciationof OthcialAnalytiealChemists

(1984)

OMcial Methods ofAnalysis, Association

cial AnalyticalChcmist.Arlington,Virginia. Church,D.C.

_(]984)

Livestockfeedsand feeding.O and BBooks,Oregon.

Furubayashi,K.

₍₁₉₉₅₎

Foodavailability of sika

deer

(Cem?tts

nippon)

during

the_growingseason

inFudakakc, Tanzawa mountains. Wijdlife

servation Japan.1:

97-106,

(In

Japanesewith an

Englishabstract)

Furubayashi,K.and N.Maruyama.

(1977)

Food

habitsof sika deerinFudakake,Tanzawa

tains,Japan,JoumalofMammalogy. 7:55-63.

(In

]apanesewith an Englishabstract)

Goering,H.K.and P.J.VtinSoest

(1970)

Forage

beranalyscs. AgricultureHandbook No,379,

USDA,

Goto, I.and D.J.Minson

(1977)

Predictionof the drymatter digestibilityof tropical_grassesusing a

pepsin-ccllulaseassay,

Animal

FeedScienceand

Technology,2:247-253.

Grime,J.P,

₍₁₉₇₉₎

Plantstrategies and vegetation processes.JohnWileyand Sons,New Ybrk.

Ichimura,Yl,H.Yano,T,Takano,S.Koike,YL bayashi,K.Tanaka,N. Ozaki,M, Suzuki,H. daand M. YAmanaka.

(2004)

Rumen microbes

and fermentationofwild sika deeron the tokopeninsulaof Holdcaidolsland,Japan.

]ogicalRcscarch,19:389-395.

Imai,S.and M,Katsuno

(1977)

lntroductionson thc identificationmethods of mimen microorganisms.

JournaEof VeterinariansAssociationof Miyagi

Prefecture.30:3-24.

(ln

Japanese)

Minson,D.J.

₍₁₉₉₀₎

ForageinRuminantNutrition. Academic Press,London.

Minson, D.J.

(1985)

The measurement

tyand voluntary intakeof forageswith confined

animals. In:Wheeler,J.L.and R.D.Mochric

(cd),

Forageevaluation: conccpts and tcchniques,

monwcalth Scientificand IndustrialResearch

ganization,Arrnida]c.p.I59-176.

Minson, D.J.and McLeod, M.N.

(1972)

The invitro

technique:itsmodification fbTestimating

sam-NII-Electronic Library Service

BiospheTeConservation9_(2),20e9

ples.Divisionof tropical_pasturestechnical_papeT No, 8.Commonwealth Scientillcand Industrial

ResearchOrganization.p.3-15.

Orskov,E.R,

₍₁₉₈₂₎

Rumen microorganisms and

theirnutrition,In:Orskov,E,R.

(ed),

Protein

tritioninruminants. Academic Press,London,

pp.19-40.

Robbins,C,T.

₍₁₉₉₃₎

Wildlifefeedingand nutrition.

AcademicPress,SanDiego.

Terry,R.A.,D,C.Mundelland D.F.Osbourn

(1978)

Comparisonof two invitro _proceduresusing men liquor-pepsinor _{pepsin-cellulasc}

for

tionof fbragedigestibility.Journalof British

GrasslandSociety.33:13-18.

Til]ey,J.M.A.and R.A.Terry

(1963)

A two stage

technique

for

the invitro

digcstion

of

forage

crops, JournalofBritish Grass]andSociety.18:

I04-111,

Ulyatt,M.J.

₍₁₉₇₃₎

The feedingvalue of herbage.

In:Butler,G.W.and Bailey,R.W.

(ed),

Chemistry

and biochemistryof herbage.Academic Press,

London.pp,131-179,

VanSoest,P.J,

₍₁₉₈₂₎

Thefree-ranginganimal. In:

VanSoest,P.J.

(ed),

Nutritionalecology ofthe

minant: ruminant metabolism, nunitional

gies,thecellulolyticfemnentationand the

tryof

forages

and _plant

fibers.

Comell

University

Press,Ithaca.pp.309-324.

Wallmo,

O,C.,L.H, Carpenler,W,L,Re.aelin,R.B, Gilland D.L.Baker

(1977)

Evaluationof deer

habitaton a nutritional basis.Journalof Range

Management. 30:122-127.

YOshida,M,

(1980)

Design ofexperiments for

mul husbandry.Youkendo,[Ibkyu,

(ln

Japanese)