Citation
The Effect of trace minerals on cellulose digestion as studied in the artificial rumen

Material Information

Title:
The Effect of trace minerals on cellulose digestion as studied in the artificial rumen
Creator:
Sala, Juan Carlos, 1930- ( Dissertant )
Davis, George K. ( Thesis advisor )
Arrington, Lewis Roberts ( Reviewer )
Becker, R. B. ( Reviewer )
Shirley, Ray L. ( Reviewer )
Stearns, T. W. ( Reviewer )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
1957
Language:
English
Physical Description:
viii, 72 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Animal science ( jstor )
Cattle ( jstor )
Cellulose digestion ( jstor )
Cobalt ( jstor )
Digestion ( jstor )
Food rationing ( jstor )
In vitro fertilization ( jstor )
Inoculum ( jstor )
Rumen ( jstor )
Rumen microorganisms ( jstor )
Animal Science thesis Ph. D
Cellulose ( lcsh )
Dissertations, Academic -- Animal Science -- UF
Rumination ( lcsh )
Trace elements ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Abstract:
The feeds normally consumed by cattle, sheep and other ruminants contain relatively large amounts of celluloses and hemi-celluloses. Utilization of feeds of this nature by ruminants is possible because of the activity of numerous microorganisms living in the rumen of the host animal. The animal is dependent upon the microorganisms for the breakdown of complex compounds into simple substances which can be utilized by the animal. Therefore, nutrition of cattle and sheep involves not only feeding of the animal but also the providing of conditions for optimal activity of the desirable types of microorganisms. Not only are rumen microorganisms beneficial to the animal in breakdown of complex polysaccharides, but they are also responsible for the synthesis of proteins and amino acids, vitamins, especially those of the B complex group and in the formation of volatile fatty acids. The recognition of the significant role played by microorganisms in the nutrition of ruminant animals has created the need for more Critical study of rumen activity and of factors which affect this activity. Several in vitro techniques have been developed and are useful in such studies. These methods have made possible the study of cellulose digestion and other studies of microbial activity under controlled conditions. Among the dietary factors which are known to affect the activity of rumen microorganisms, particularly cellulose digestion, are the type and amount of carbohydrate, fat, nitrogenous compounds, the amount of vitamins, minerals, hormones, antibiotics, and certain unidentified factors. Data on specific effects of different levels of trace minerals on cellulose breakdown and on the possibility of using the artificial rumen to improve rations high in fiber or cellulose for feeding monogastric animals are scant in the literature existing today. This dissertation reports the results of investigations designed to determine: (1) the effect of different levels of copper, cobalt, iron, manganese, molybdenum and sine on cellulose digestion, using an artificial rumen, (2) the effect of different types of diets, fed to cattle, upon the digestion of two purified celluloses in an artificial rumen, (3) the biological feeding value for rats and chicks of rations high in a purified cellulose after being fermented in an artificial rumen.
Thesis:
Thesis (Ph. D.)--University of Florida, 1957.
Bibliography:
Includes bibliographical references (leaves 61-71).
General Note:
Typescript.
Statement of Responsibility:
by Juan Carlos Sala.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
029813387 ( AlephBibNum )
36467266 ( OCLC )
ACE8240 ( NOTIS )

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THE, EFFECT OF TRACE MINERALS
ON CELLULOSE DIGESTION AS. STUDIED.
IN THE ARTIFICIAL RUMEN







JUAN: CARLOS SALA






A OfSSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
INPAJRTI FULFILLMENT OF THE REQUIREMiENTS FOR THE
Dl5dREE OF DOdCTOR OF VMLOSOMHY






1g11V A 9,7
iiiii


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Me wrl r vishds to *wes his stRaer appreietatiL
ts 4 thX M w havm 1e atiibat to the prletai and
sadtMU af the retMek udt to the prqpratton of this
dtertattes. B ag en *4iAly grate to Dr. george K.
BuAhO. Idwo, as blut~lr of the ntrvtisy samitta, has
pOdu ths enetmi nd wrmided the amesury faelliti for
its.n..t
I, t hi. bie prvtileTl to have had Desters L. RI
Artl.tea, 3* B i e, 9. 3. Mal, 2 1 L. lt iriw and T. V.
M. it as amiws hi.s p wwrvty emmtte., ema Dr.
ri 3. S -tM~ alS e ameeMr *t the wmittee.
lEa.. wLtSar As .-ntetS to P ete@ a. F. AMiwi ad
,3 Ii eit the utel Y mrtwrr 3>ise Jepertant for
inMaw the .pmattas Ia pranerettam tf the rm. I iAstula.



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BaXt. t Ihr helpftil simsgnthte ia proern lag ut typlag

tk. inuxMrpt.


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fleaeer, Itn m eanssregeIst sad eaitsakes tkveeghnt his

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tsialqas L .uta. eaf Kimeo o IIra
ft4 Used AI StudterR *n i s f lera
SISSA*YLhedtyps-8esp** ep'u see

a ta t n p Artifi eta n tn .
S Sat .t Yt.n Dste Faststrai a om lZ.

0et rts aos, ws @s ama
.je...i et em te a o .r

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A : ....ettE: t ~ a.. . . .

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ltteNt of raee Min rate a fts Ctlut DilP ti
vaB t"i Artieial Beas . . .. .
OI alt ....... ..... 6 S ,
Hilr a * e e * e e, I
Ipper t . .. . * .



teat af a t olf Xse sew1e'te "r 'f
BMS t S S S I S S C : :

wa .41.. direetta . .
Fdif T ulsa I tai.$e ea Oata" a*t1 he w .
NllAtug Vnta th A40e44 Rm

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at


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L, e Jt iet Oaltat tw Ued in the artificial
Wfme. .. .. . ...... 2
S mwsrtratniUam t eal atA lJ the ram
fltrate at ster* fed praettel dieta . .
S. fi alX aauiya t of ]iflled eslltelea
(Lphm 1a4d the esidA after fIrIate-
tM. ath. artWiitsi raea .... . 2
4* emdUlm deatf daets f toU rt .. . 2
5: .S :ah.lsua 4tids eo diet f"I to rate 2,
S*. ditt.oe. di.t fISd to Ll* . . . 31
f, e 4l amtraly.t df Uta fed to 6 hs . 32
S .* BStat t etlt uI a diesteioa e tie types
of pw m l t1 amie is th USrtifiitUl
,.,. .. .' .o
= =t me"se 35es atd
~rl...~e.. . um N II

ii .i :i. *:.... .. ..
fiS tt)tamits StaeS| itta dihuemat esen et
tidal the ans stapis saws
i'. :: : ". ..* .......... *
"',. : '" . '.' .a .

.i';, .' . .. s
Rimrr strate o*f eartease *f data to "*
i k ir.r-. ... .....t ,.. as6



vt

I; .:,., ,,,,. : .
H~wZ ". .': % :i!':.,:: ";i "". ..






viA


TMW.E 9Pge
13. metlt obtoAmd with dirnt 1al of
r un the Imn j Ciyarlt range
t ., .. *... *.*.*. . S
14. Effeaoto fra Ien dlgetAla ot tw type
pariie as the pr arlf
nums and tamels .ft u ate a .t
form"e.. . . . . 41
15. bmary r af amltrL er wurleams f data is.
tt. 14... . . * ... 4
18. neselt. obtalaS vitk difleat leo et.
ira uuOlt thM Bnsa' ltipe xagq test a 4S
1t. .tfe t Minm drestn .tto .tws .
*t perlftd t, +- rthe artif
M] m ea. fed
diffUh t arew. . . . .. 44
16. bimufy .f ealyu ef vr.eaet data a

19S. 5relt ebtajald with difrhweat leveLs .


20. Mnt etf MuldaOnj 4 f ist et t S
t lid. fet atrqimm vi aIeft. tiu i u o 0
W.es of AS 0the


21. -of, an Lys" 4i- *f IVAW 40 4ia i .*:




.......
as. meet., ir$tw& <'lf W. M i f 8 :..-...*'

** * * *4. .t SOW antekia e fl t.

Ad d""

.. . : . ..;: .'" .. .i i
i : : ]i :!:i
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Viii



-8aw


l-mi udem te Dae au tAUPi raw*


8SeO Wet eof seIX.i tIwrmsttmd Ia the
eftfetatX wems spa the growth rate .9
Mril t ats p p, S S . S. S p U S P U *


N t. it-, t f ellttiesm teetd Li th
Wstifet vnas %poa tkhe grith rate of
B eatis'i ~.pa ti*. *, t* **** *o*


"" a. the gaoth Irarte of
b4 ,..:: B* * *

St asetrta .9 min. fl.te ruthe, e t

WS as a o tf a frme *ft tUe vrath


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fo teed.s &am yr .m t hy eattue, amp atd
*ethr rdJukts* emafta selativty 1ave o emat f
ell4Urt s had Mi-nlalea. tItsstha of ts* e t.
atre byr nmrtmis t poMtWs booms atf ti sttvtt& r of
imMarus staigaaista liviap sA the raXss .1 tb. hamr
aunt.an o a sit si duipsmet yen tohe s n tr
the brb1u at 1 Lawt .ped tati .at. M* *s
whiitah be attttie by the antmi* Sloadorn, miitfle
Of eat tle IS sheep Lmei* Mt ety heimg ef the aSdta0
het AtIM the prvliia *f wauitlat fme pka uasi AmttEtWilty


Iet alp m remi am a uLatSiunB Wmit Iait ae AA s

-.. ueq.. .. : uhI. e l at.e e1 ptataM. 'uf '.'.
Ar, vitaita, eqaeialy these of te B :ma e"
..a t tb&a mlw. t.im I stat LAe atty aid

m .m. .Ad h eI t" a:. .. O:

.^^d ^kjh~ffc^^A J lkLk ^.i^L~b. Jk. . . . .b *** : .. : :.. .: ".
ItM tte A JM.. k thisd atib l
Vp t . I ...I. .

. ..i" .. ".. *..: ".:r *" *:* A *'.''










atudiuL, hea thd. hav pea aSrtsblo the lstAr of
etUlnl dtrt .a al ther stiue of lsterbial activity
eMair Elatrll emditian.
assel tith tsers whAI are knw M to affect
lbt awitfv MMs A aimsWauLMm potAtitealy Gellutl
:gtm e the type l mat lf ea r drate, fat
a ttgsmsns onesnmrb, the nwt of vitma., a*imrals,








f..s ai .tJose hIN!e Di t t
.,.6 t... Uttf s: fttigr b t. A t t y


t.hAU digutM Itt dtemiOaMs (Ii lb efftt at dte iRnt
* ... *':' Ikr i *gta t.Jta f wng i) iA we r nt





Me em e iamii Ogesa.es uuiM am artit"at aes,


O; tAs tA4. d aSIS at lg rthjed lhl *utem j a ax rpt
.,.n (.... a s, it, thi "a wos e ratw and



r ...
SSvII'.m ae" pvi fud aitnal after r bt as

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...;. .. . .
M. [':." .


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1ESSrAV E AtR-X


S.a4rpwn ard Ia tftty akeaNlyXi
and Rm A ittivtty

hPr the studyr *t amR a.rn--ahaswr, arew
altivdity amd the maW~ f rs iAVE a ie the aUsTiity
the air eMara, a musr Ea d t*mnt rmtPodp have bem
M.aud Ment af the n_ tahauu h *asbd tho ga t
irhi odf statutaeiamo alNLfa t ad am sttAlassM eat
stAinIneamse miadsita. I rameNt yeas -" Aaa fl
A.lA k a dn* tqS b 6vlpd.
Swuh&A mr-atrtewaotu atsm -t.



tJpst ef Imale Afl the ms, mthe Mo &e Amd the
at,:dm M b. m''M mm6 t tye a.
&the as pasIrslataIj, "dastius a s1en Maw-watns m
Ir trhe lrdaelt eat aSd UqlAs, ciudt aiw l&Sa Xat b
aI3 a afttefl tOt es6 m t1" flte *pit
ntw te" of tkhe ma. stat.. La late t AtM was
petm td oat that If At hIs I-I the aik mattmr .d


:a : -.a:.... w* a n.. .i a::



l *. " '. .. . .-..... .
|t*
^^^^ *^m^Wr^ ^Wy fK^^BBt ^^^^^'l^^y ^WWJWR' ^f~^^pBWH(p BMH~P' ''I''" "









emiselO, alt gitt Ui ~a ldie_ at other eXtagated fore
(4). th sairta the Lffeet of diet am the rmaM miroefler
ntte a~ (U, 19) aai ma t of itSedphiUel4 buterla
aas felitiint Swainytil bf.or *
04~S se ta the aM f e esagtairL per wit et



l. OwtUit b*nf kpra lr frohl a n ebat at.r oft
: Ithe remnewa. wrenwt La the rstima was drlbpa sa t.
kedlye dta n a prettnLW haM MtUs has bens shem to
eathe hbtehast s to hilla (448, ). In
; '"iS. "tAl. () rprd that ae esntat
a m a3 4 dfslflt La ehbat etatd 30.) blioa

A : .a S . lLt 4 bltMs x tO *ra u be baa nwsa

aMS asn .ai0ti1 d pttat type* et iats fed to


^* St U0 methods jao e bet we by mssal wkerk to
4.ria r S lfle A. es.I as ,U, bhdst4 ita.
boa .0 s ae





.n.ea ..a .. ..e mat w..


Wl 0 "a ha a a o&e t os i

~id *1 3 temple st ainar
"m m m NN'N' m mm m m mmm mm:" -f )"l """R L JX flH:r ": L :'X .m . ..
o. ....... i ...r "^W 1 w ^ -. .. _. il it -----












.........
W.^ i:.:" "*." "" "l " ) h "
..:: .. . ".:iif a j *" ? . . ..


;;a jJ:l.:m m: ms i m m mm mJm:'mm ktm mmm m mmm mmm m' mmfm IR
^Ss1^1^^^-! u ^^^ ^^^^^^ Xs~rr JB'- ---Kf ---X ^^mK XSHHr *












Jjg L !!:!:^ ^ ;*s^^iP Ip 'F"^ T ^ w ^ ^WI^^frP ^ J >^ ^^ ^^i^ ?^








thais rrmia 4allu eaIred Uad bU at bere eallfated ti
krdm r-f SewUntso.
Mwu pwe utarS et oMtO. r e ia eums hae be"n
IXlated Ma the onm a the Np.rt hr 8~ .tr*4 at .L
(5) jet ot the m i mbew i eUUN atl.s be!'te.wt.
tillha tX eIr a dit lm f t kI. .. .,artsta fa e.t



mltwmre ft rmnUd atesenaai. o1 e4atn wnt aImsa" ;,
pRhanas soaft pdemsem sns ftr the. Urn flas sa dlk:
alm bhe reported (5)). A demRpbths o#f 9 tyipS *f
tie. IzBH s- t shoe with iI a Upeut to Ahle t im.en. .i
Iras rt IAdin statn m alh bee m ie hI W ft Mt 5.a.n



ana t I.mei t.i 4.lMeSt a m .iiri a s. beei .f ... .

oat ap . .I .I flv1us..* it a m o sANim
wseam mn.rsh .m. n t s iMML' O'l us aAAi'
W A r.. .4.


o toily)*



41- 04. a lle page
. ...a.t.j aS *fl i l ..... a e... ... .e. S''
fgs . :a.w ;u;l";. t:





: E''a... i2
..H . . . ..... : : .










tyips 9N amU, tb*a, awIr artle d~ea, al of whah vre
atel to diegttiam ft ftbr, has be reported by Haktanaen
aM ktX (AS). aPw LeaswI Awrt, straight reds eaurriim
Ia l*alal dit sarS to be tirtaat aand ~ s~a-
miua, ateat ama s prm sag betyrie, msetio
"S last4 aside was lsi teaLate
Atbqbg thea OAthet have pnveotd venable








";": SIM b. S l~ i to a em h tri the
.*.,. Shl 4 _atept ...t ldwttlaud **S*4WSM tue ad nWdt
': the ll t the utia ttt lmes, ad tia uset O
f,:',,, ",





L the 2rn u Se a ptiai esatLttios as estabisaed

a.as *.sk weOW at tesp W tut So to
: .: .S. .. fikl .. I te :a '" t"e e"


.i".. .r1t. SiO.@ be mttlre, ir of m nafa








fai a gala sad toatively eofu an.a o
. .:s:i; ... ,a . t. .. . .. ., "*... .
o r.. .: a*: .. p .i la to '.







43 . a .
i a p ^ -',;. ,: .:..':. .kj.... ikk... : .. .
''':'; B :: ": : ""








As rnly as 1b84 Ta ppetc r (eited by BUrrnghk an
5i11. 2i) iLas ted xmn ms tets fts ea ttU* iL eoleed jars
at roee tmiperstwxe a served that raMe fiMt deireased
after ftem days. In later werl he te batd rams etntats
Ja tka at 537 C. sad eobmrved after 14 dayr tahbatiot
that U8 per uat af the *NNW flber 4ilspp mnxd. IWhi
haXreotea was a46t befhe lambatitn, .Wlttal our
poeitasa d14d t take pluae.
V' ger t aL. (185) addd Ursa Ut nmsa sOtate aSt
after LatMatit at S70 C. dEtLras id tanzgsE* attges:
mlasriagr iS this vw the eav4erstnI to MteO d s a Ak.
w arkerw (91, $S) resulted as tetratext it thest
malia ad IabatS the .tUthrStt t iS* Ce La a vwat Iath
AF' 2 to 4 Moun sar S en atqbNt a0.l10 Sbt pe a"
was eMd t at%* th e*ffet sf p, ty4p -e---& atb
pIarsrt, test s gussoa eviswrimatd aS is.E
someatratits a the alstf ity nf wwiMaia. "a 5: ':.
thesta -tmeth we IrttiSLmti d Ji the l* at s lweat ~eF
duftaeMt of the a. pBdsenta as a the itw amOtt .'
:





spoters atiatR sa wbtOtAe witSt at at.
I t of L ( UI u.f:lt 1. ~s a: ::':
,ia t nt.. i "I"- r M. ".: "i a "M ".:""il ""-'*f^

e to ." .*A. ": '... 1': 104i
.*5 " : ad :lW.r!:: ':ltt Y: O"i: ::':d.,



!u ..:d i "i :
... .. .. .... ... :, Ii









0e1ttui v the strtaiA ramn a~pLes at ,Q000 r.pan.
fr fivo nLmates Oae per seat maltoe ead 0.05 per eat
iea weo added. ad the .mn3-otein aitregeu deters.nat.
SI snwlp' was M fad r tear hot ur at W C. and the
tnaptetin atroge det taltlioas were ado again.
A rather ews teated tehaiiqe has b-a neggsted by
SBW (ft) wit& sasilted of a glass jar supmaded in a
Mater bfth at a "enatolSed *twratune. A growth smdiun wa
added to the jar aM" upetd Iis the growth adinL was a
flat., awb thi.g whit the dftlerat bayedata of
emateflths e*WA4 be dilyMed. the glass was prwovded with
a 4 14M at ltuts fotr the ofiaglr, tliAtg ad
flr diitties * hofet. bfe atertul wvas kept Ia


weta A::Natue attm lal m ta a
....:..': ... .jh h M blh r ^r imuuft h t r! a B ta i


~i Srtdae fbs digeseths by swam khla. etso t*shiqa
|w,:I tio- the ll tal r"w diseMt bhr s I(4
"l iS '" ''^: " '"" .;: "

S I' 'd it t 'mAeS' Sa the faqust astrtilisatla
low i As atttil4 tono bms as the pevmubte
b t t hee. aeinad (S). It assist. a a
i;.ix ...ii.f T j.u.. ^lkhJauto -* ka, l k l UC

is fth IsU ae sMtOMn takes Akes within
.0 Sm e-1 then pp Ws wnv 40these.
.:4: fl ta:;m:s J! tuit, *.ee. 0) ba .im m iaitesstte eaitles

."'SWM4 *SS. aabMated 406w

::.... .. x. : = . ....... .. .t ......t ....J
,...,U '. .. "::,. ;' .,, B 'W


''"". 1 :. :. : ;'j .:E: ." .* .. ....


* 1j ,ti i ,,l, , ,",, ,,l , ,,, . . .. ,
b :!!it":::i:.;,.: i . = i r








ibid. The rate of di iategratlen is usd as a measun of
e Ulle i yti activity
Burrmai~ t aJ (S, s, a S, s) develop
as artiflelal ma which oadiated of 5MQ at o.y 1,000 a
sis fermutatime flasks i aeaated ts a wat bath at 4 ,.
h fluk 'vwa fitted with a thr bolea. n+r sterpp Wr f
the ganmsn with waaboa dioxide and for the reagtatisa of
he pig of @the eoatts, Fmen tatiats m e s rderd at
oat4imoass for 6 -hm pwriod. e ite ntm fo tlh flrst
feumitatioa m ue d dimt tly tr the eam but fr tih
amuses~ w pereds the itmamlsm onauated of Uhaf t1 the
1retda ot the prMesdi frntwmai.ktat cO~itta- dI1ostiai
was detaestd lmiatlUry ad e stains ae md a t t h
Ibgasluas sad ad ef a**h4 emIafttlSa pYisL ,
eatr a setbad to iwn tMhe istatft .. .
dwvalpmd hr weken y s i. (ia 14K, 1$, ) .ita iA .
rsum Lqd was sfatvtifged at Mot weeds d te t.-
saiisinm the udsam..w.tm v.. aw q*imd m a m h UP:O
uhswhate hefUae Sluttan seetatabg *seS 3S5 0*t ase



w. e & .a..dp.i.. wAS C f l ~~ii: l..m.::;:.. 1



.mo P.0 d anness* am SA 1t
91 : t 4 ." .: ". ." :i ..
.. .. : .s. .. ..il; .:.: . :. ::. ..
... .. ....... ... : ,::. :. .. .:i ii. iS.. '... : i, ::::
+ ^ *^ :*-* **' ". *> '**" '* * "* .. :i;.^a! : < ': iiJl






10


At tpeiMty-lty tle sam tiu, Cheag .t al. (34)
dml01eped a teedmiLq based on the uo of a walked
*rs ja of rmma atirnesyaataM for the study of
dtXll Ns.l getitea J K. The r trained rena sonteats
Wer.e Mtrtaifed at hih speeds sad the sedimnt was washed
seWra tIe with distilled water er a phaphate buater.
ste C al mdstoit wie sipstndd in a osqpes ux trient
fatten aud the p adjusted to 7. his wa followed by
tamltatila ter 14 lurea at ir C. and wter anaaeebie
eOtndilttSa previd lb erasbo dioxide. The se of wuahe
.. m sauieaaw k mm l w i t possible to We an aiealna whieh
s , e suly free of atiri elts.
Sbt .Mlt te iqte han boeea riteised beraes.
S !::t LA the iatl aro lm m ar mad the Ianlow of saliv
A": aS m Wleni ot invests td ed*pe.st. of fersetation
AbaSS anp sot be dApLIeMttt* Although them itr tslamis
.he i, thUe as of the avtUifita t L ar=
~ t-''t e:e prigMtd aro usefl. Liatentin.L
t'. e .hiety eand a, f



alth asot he iartsMi teeha StUdio lte hatve
:* hS p0911tih iy eaeam. beai e ee of the TWAU
:?::';":: : ;ils.:i .:i : i .i i*4 iI t, Cieft^ l*l IiftlllJte
.*"" .* W)c :.e 4b a.fl pes.. a. basir
aaemu..s.. SE SPUR eatUS am samade ea a rm

H~iii' ." i .t l:i :.i" ". ."t ."
1.,* :, ; ,i,," ,, ,,
t" .',".. :..4,g .









dtdtry rentals. f mi of fiattated f atIals ffters a
muE o twiyta t*he tatd, p Ity dl aMd tareb al
mwm 'that sIe v witMS the S at W s a imR ete a imea


"ul t ate tO ON *

bvwt ef%riw DU wU ,




e muw aw t we a mmat tt tey. at a .i a ta


an boa #hiMS moo dtlar Iutps I Sntik
.cb Mdrata, tat peats srpnapeteul Mttaeps, asti
ttaka Mii am A oa aMte maaS f ats .


- of. -' .e pes t de enS ws;...


u s am ea ne a wFNe.S
tnta e r 0t .afss

S. a .i



a t . . . A .: ...",,i
.. .... . .... .4, . .. .i
S.. m a ,. ... ..."... ,.. :***










.Aly y a a ft eaf M eat msea a disease, but al o a
tlAght e:mar e .wertaia stas t the diet of r muu t
MW ld- to amo9alliUtlee Prhap some of the beut-kawm
.mLM| ai deas mss hr messes of mlybdamu and

Saaidm. am ewae, aKeeta that are sentiLal may
h te: ts prels Ie* t in *easal ; pmtls are appr and


O. *1I the staMue of the t eft o.t sainsal. in rmsn
n ttie s ly a few ha b bfT n M d r.laUtt to the eOffet
t ,.s mnt sa ts meMn, uAd rma bastetra.
:t uha e h*am that -aUsla ask, watr extracts
*;*f XN rat, trtUd tlttrf. slua bIs, "aw al
"msta"sa a. atntaee wt- le Ottra st -f
'0% aaM :;a"".M"A ae ,: tan "eas d the dsPAUti




ue-r Dee 9@-i, Sa"meoas"'ae. tha.ta ti
f... .' a ".. :i':i.n, ,, n ).





mao (3m) m at ma:.y

i,,",ta .. ..... 'i fci. fc t^. i .i..* .. .. t et w a n t n ( ).
..,MWWM uuet' :mus a ir t.






It:
S" i....... u. .*. . ..
it .~... .m. 40 lo:: .. w 4ated ina a tSe

.. . .. .. i.. .
iM:." 0 ...: "



a .~ 4... . .... .: .
TI.7
:ii'p : '". : ". .:f .H; ,, :^ ^ ^i, ,,


'i:
i~~~ai I~r~~3rf~~r iliI~a








tmromm in livweeiht 4ins o*f eattIU. Th tmas maineal
nmixtw uaed s the astuadin esoa ted of eMlt, .ur,
AssOssa, irA, lodiAe, and saltr. I father wk, (IL),
thLore mIati tih dbene tmlts Uar t ead that ilaXes was
of o plqppewmt beanit uta &A5A to a ratiMU. *1i4 Stuedd
trnes miAra or ro4 qu4 Lt i Ad har.
Othar w ofthr ave reported usfaveanh results fIt
feeding tree a ritm S nws t tal. (9n) Dead ttat *ta
bet sealv retinix a srtirf lagXey semwerd 4-na Gee
atfe Ja a.d saw at a ~elm rat whet trPa s aestl
addM. l Umes$ aI. (ll) sad abitt au Ri ms (I)
repeated that swithew 4alta ash Ma, a 11.te Ma alsoel t
state LApeved tM dAlL."Mabit *t lew quaiUty peaktrie



:.t.a. e .a.a. .. am a.u, .n .
nt. tet.* e s "s tat me ".st :l be"sfwe Nm

9-ttet *mqbu, at .t s.M
alats, Imemn e At rnst Wn tenting amPt4
a t ... .. *,, i,
Ba 4 t Uy wibnt 4Waea


S .. . ... .t

..i.t. A am:. n I& e1111aI I
t e s ::s ,:,m;; ,u:





*. ". .. "" .. :.....":: : : .. .sij :" -
.. ..... ....
Joe.....0 iii s!ow-
', IM .... ... : ...' : ',
. &A lp: =
r "ur~ lE ."i
; Ci IEIFampp::lWr
J P:::XE T";-17









tam 0L pP.ea ehalt atd between 100 am 1,000 p.pW.
.Mtbdemaus *owwth et the bastfi was dtflAitely iahibited
hr 4000 pppa. Mi 1 SS popn sopper, 1,000 p.pa. eoalt
2j" 30AS lPM alpbdea6tsl. alabar~ t Atl (IS) xrported
-t ultu- diS*ties by xamp Voraanraniu ib lit
VW SuAtly dLp MlM p.*opa, of added halt aad
s.saly daprasd hr 100 p.p.Ia
,:,a ss. untts M at l ithium have Mt bewa foud to
e eeet SA tO sfULaMtaw wrester aeflaSe nigCstles vhea
Viog petauiar satd sdla were present La the Ldim (S.)


S.rwaN that the Dtm t ef ebrtehyrate ad fat upen
. o r U rlatd to the other atriats
; i uty mt muMt oft sf ad fiber digestetd i the ren has
::.:: .. .. ", .. :
.g 11 01 ..h' to. Sla betolma S tO o5 per e ot (53 4).e
S4a pU ntOge 1 s attested by .A3 other laridiata is the
S..:s. ant ts aaah e.eydrtes have. .
E s b ai'....: a . "si s a

*'M'i "*taee A ^th bit m t et ec digated
4 S. Steb3MM tlt (CA) bt thtu the naelatratieo

,,r es p.;..ua. .... n...... .s. s3A Att wts
:i fad lin.~ ...4pm eat. f t r: (4* Loe that
'.:i"sp. S S'S "a t ,.. lea to a st. a,;n. "K a
I;to 4 iinWiida q utt;ly l;ame Ut I .
ease VIS-a. a me r
aA:'i::i ':.: ::" i:e f I.':u'.". t': ".. ." -- - l w- X
',.. ii j, : :::.. .;,, ...:i '...: .;. S ...
S : .'55 :a l:''" 2;. ::::::':: t... :








tWhen


digestioa i


marge muts of
iA the ratts, a
ebtaind (1, 12,


readily available udah drat


s0, 44, S, 6. 8, ti, 6e).


Ski is du tohe wt pgreme tf the atnmeepaats. As the
rumm ftr soll ealrbhytrate (S), 0t. wex (44)
swwid that sIitius of 4 pweads of lasm to a titlmtr
leapadu heer alstwa ter deir ews apparently daeree
tie u tee*tA*tlAty *et rne pertel, ltera msd erM a tfow.
Vrk silvtas the bastrial peps~ a ti afttlestN.
by ecrbhydrate has beesn yprt*r b itr'r t..y- (IW1 A
Sher obasrrv that the eiUttle Of poWds of Suwr to a
he rMtUa ratedd AhA a 10M per a0Ot tarme r ia the bar
teeX pdtta eer that f her M' u. a0 starMt w .
emd at the asm sat*, eyr a mikt iaurmasm As bal teria


ttu w has bw am *a t e eO ffeto* te oa
a.Ss esgesisma tlut f.j.. (18.) ISt za's..t
.*ws. m Naw las4 wvflas *-Sm S 3 fl.;f:l Ft|
A t.st se. a finre, b ate wa ... .....t
peat m .k aaear ss .u t3t. qwa 2a6

as. .eS the .... ...t ..M. ..".S b
0.. w .. ".. :.. .
,' U s,.
eo flbum meetus sue emStS as1 &. s
al:^'-^

S... .r. . .i.

I .;. : : .. .










fSt attlisatt n* adieatioma that valerit and uproie asid
*at te -a l4ser tant lte-btyrLa and ie-valelie aside
tlereased the rate* aof eel.Uo digentona ad amania
wULt atc~U jA~.ie ha b.ee shun by Brn tley et al. (14,




..at a ubas rf eyae s the lttUinatelt of aitroge as
e, emMu ry the. mtaelts hUs bee Oi interest to the
1i6n8sM wakel aot easy frna the poiat of viw of protein
Sithe 4 itat Eot their eff et o other t tivitie perfoed
t.. W :iAtAl t LA the wmeu partielarly eelluloe


.. O* tie e6 ayr goorte af the ee oft an-proteia
::::li::::a fl0 r rmlent was tlat pUbtshlid b grt t *t l. (58)
L, S .I.. "l. t m. d ja n as mema eafnate iA a rataio for
Ma"y ka.-. i obtainS nstt. wa. i terrted
W Os-at saiteew Ame thn* asarm S eAs saenveted
mtiamwa ataW isat MiMaIl prOtta. Seixe the
WthE s m et maw oth. eqiutmuats have eatlAmed
S:. e; s I, n Ira r E.


S::: : .J s .. 0a0a"m mitsimus wtay



s:i. .t .:. 's at ..l d
i:'ti. O : ....



.lH -* '..* .... .. ,
' "
"
xy !, i+:" '
i: ':" ;: ":i:+i "ii i: . "








ftdbatuff. (*8, 48, 73, 74, 74,, l7, 101). Littnga oft
-anestas .gaMie at mt a g ni. malm Oalts ha. &lo best
* howa, by ia .itn wrbe, to sn as Oanesw of t itrne
for the ruse bastwia (10)
GO* of tOAE ewtitLion neenu-my forf Lotint stULA*
attes of riponteia attloiess .o. by .t .n..l Ast
to th usemt a bn pw itets atloas, It laxg ammat fet
tw tira pietaet are prnmmnt i the rMtia, little emaMNa
fet mwpmatetia aiea lte pReti t will take p a. (1Wi1a
iawral rp rts have aov that hatraens rmmb
attht th0 rat. *e 4at aiteo 0, of 7, *a, a, 0)
t i tiSea of *2 rs to a <4et atatMa 8,1M Ir PeM iat .i
artta taseisraet ta eslHt*a geCetSlr ta 1a1w U..-
sat to U.7? pm oat (87). Bmseh a"d e at a (a
AtMUd t" Wt :t ".. *.t9Mat a tIen k 4 *
tautly rj dtlta it ,ntas te an ,tts lit':
aq EIn a s e es tems abeut UM ,pr et ; M ..
matte 4A ..tlhle thk ha. Xt as ..lli a tu;e

ats e to tmI o....... ... Sa, n. : cu
an 4 p-tde otitf Lathef d ti se pen .. :
. . . . a o :: .. : a e,


ts w... *." -to.-

No .: a" a" 9-flu1t w a tI I
4 ,I F . . a i. . ... .
.* . *. . . ":. .

... . ;..... .. . . .......
'. .i :. ..l






.1


etIUttai Al-4it digtiea has been S anm hby the Aa
ate t*alqf useed by S laeat (11}).


na ratr.-nT wide a. a*ataetl. ltl inmom liv ateek
eatiuas ba prmnated latmtst la studies" of the offset of
attletAt p wa sMmUM funttiha nbe usbstaane Ln Sertain
*eAeS AMndr dAtevainld eOaditla have ixoeamd the
S amiat of ,lie dgewteS, It Us ir. a der eas has
xumwrft Vanirgesk at als (104) yOrtAed that p4BaolhA,
tbwee BLm aMd nanala ufinirtaadt the 4eleeUlLyttl
a-titr ed sea e te atn.i ebte&l oeat advrse.ly
tS the atAtty*, CaioxtraoysiIa (Arewnoa) has
t an .toi 4dt4wi the aetiTity of r sn eu oaitm
1.:' S:t t.1. ".ti.a "( )* rtt (16) h AnNm


1 Jr:

..' Ss6 it (Si ) ebse)ed that lt aMitL e of o1
.. e e dietptUhtt to a S a* artitfital
.... M: i:;;.. > : :.. ;, >;** .
m mt Mas the diAtSa etlulten a average of,


:~b'.S" A, itet l34 bieti, MM,, ratitaMe,
'A.;b S. a sMA: Sture 1 Lt itO t m be reported to
:. ..ji as e aaaamst .A e (il. w u.


J. -"..a: i .p :.:/ ..* .*
AA-
-.cu. I.".... ,.. .
P A. ,







19



he literatIe rXUttiat to ot he apeatt of

n ttast matritin has ben gsellantl eiwkwei by others,

Th readr is iefnred to p tr pIltminA by DAatil *a

ebtma (), Mard (4 1), fll. at Phitlkpma (4),

Ges (1)., Nastim ), (5),), W. Karate (U1) at

omn (e9).































.4
i9

... .. ,... i






A.1
S .... ... ." .:.




iSi
... : ";"*:"" ," ."": "/':' .



": .. .:. .* *: 'L : *' ::f :':'I"f "iil

U : :: ". r .. m :@ : :::: [:
,A" ":"" . :,;, .+ : i .,' ~
+, i . . :. ". ; ,::+i',.ii ; +.:t! ::.,a











MPIMTAL PROCEDURE


tW type of puriied selulee werev fsrated i an
satei taL em wad eoatroled eeaditions to detamin
the H tes oef tlve~ry eaaatratlta of certain minerals
pVn th eIxtat of -aUtlea di4esttioa samples of rnea
eamints Vwee Obtaised hu a ftAstated steer and frea
asiIt..s" tau1gkttd at a Leal p king heas. Ro enstent
we. filtend to rvm veliAd material, ad the filtrate
ASAt wth esi t n ad a a aitrient fmlutle for ermatatieo
is attiiftata ~umas Th ateat t etf efluleo dicautit
: s 'te..a::: m4 by aLmeit analysis fer eellmlese befor and
.r. t Sa"nt.. a by fetein trials with rats and kiekst.




S1; eat*^ r sm Vta this ,stow as a*ffeWt ef timeth
%-i





.. ;.. h:.met~a a @Atted ls Ierl umr
!i^:i-AIn..s:-. .t;*^..:^.fc eistatei n obtainS Iite aM stulated0


R.h a uawu. anstu*"".* e
.t.. aa teMsat SN L. (awfle eat fnt a*d
a d.ta.... :) i: :........ua :t ae *e x .w e ..te



::t 5" e a" a a:" ""a ..'n s.. e"
i~~l^^^^S^^Bll i~tBBB^^~ ii Yt* IS' i irwi bo at t" wS




44, md.;. : ,r".
tQ.;"1 i: : .









ste lb. frimpSd ets Lbs ieed ltrn pulp, 400 lb,.
gread anra etr, Lb, Sit. tt eia labi. 4 dM turtaoat
ree phakatr rl h water ad additia e de teirst
14ik phwhate tMd s"lt on avaabl *o the asatI.L A
Puet"M puig 4.rnfbd by steask4t stit. () wus ant t
tht wIa ifSura. Vhl sMe rM te te in1 a on 1 1 .,

eIntat waft witke an Iarly atteanet, %WaRpr hSelm*



$A bud t.e sta tahtea at a 2a at
s watan te* vn** ussvk tth a 1 ,:... a ss. *.


a. a.. ..s...A a ..'a..'....."..:.' at A f a a at s A

thee. ath e. estae.4.ta s :'uM '"0 "i" "e" s" "e"
.es--- u an a e -s-- se ,i eWms..M .... *





sur. ci .hsqgubt emnn ts iset u s .: .
lSo hw d. ,a eam m t att P" *

S.rtti Was UWe "misse .w. b *ii': "

:. muim h Whi.i.. I i
flapsIe ftI I ae. A 1* U. s.:: ; m ... F NU&

a S 1. ;"00 .b UN f A. a ..! a.
..t..pt ..a w&. JdiA^^ktt,,^1,





....
.... ...... ... '. '
... .B .. ... ...n.a"m IAIid 1n"_ .:;i.....




.. . . .. ... .


l.. . . . .. . .. . .. . .. . C. g 4':.. .:: h. .. ..h .. .. .. . ...* .. ^.. : S j .. ::.
" ..r a :.[.. ....., : '.q .. .,, l" ..' .'1 ", ,:* : :" ,,T' ..:% ":!".: ;"" ; ." : ;R !7 TI', ,r ,:~ji. .; i:ii i .;;;~ ::' 'iir =:
~ ~ ~ i ::.: :::I.:.r:. ....... : ,:;.:[ .:.i:






22






NUtSIN mzarzamS DUD 3V SE ARTIFICIAL RUIN


Watitatt aJltiatAn A alustia Bb
-aft* lke.L 4* /L

ta .eS i.e


E. I .4L.t .l
",'. : *". '. a^ |

M% gi 0s 04.


. ... --. ;.. 4 6 M
Ge as Give





lirl. I tll



F.r. taA I&- *t A*
." *.* .: : ". ..:" "






Ellll-^ ,: l: .
IS^&'-I^ ^tiw~ M~lir t di*rtioa of








AIpklhast2 wfs ns d ad resd la the" t Lodi*. Before
the rmea fIltrat w a"dg the t#a* ia othCl sea nts
ha ben warad ln a mastant tweraturs water bath at
400 C. for 1s Jwtsnt
Zrdat.nLy after the hAtrnate was Ua, mmestd
mxidt- Va uIdB tat, the sattKc fe S sixatte. CtLlta
w tabma ad pR d teaSd lh p8 o.t the sttw ow
adj~uwstt o 7.1 Lth 41 Nra wbn f2 Wnmers e ni
Atmt was hea teS toI study the taflhM ii eo at vrhmiti
sltarats oa the dt o6taXt e, S l e.


Ia TaI I ae AM stint aIsItes ntat
ueni and vatrss ats. M A a w N A the sta"d
*I the OfttSt *t sabt ers. :, .r:. : "molt..e


n.tsmOh It awfst -Ato udal the ei thelr t mq. the .att
- dWmtt.s SMOIte. lllllrA s u hr u re li I :




l~ t 1 atmts A btba the t.: r:.
test Se iatmmft. its itlmat 'a 4s.t ta 4 te flfl


- MOSS M dawns S'." o S SS*i n* as *t IM
'a. wSS La asli~sat msm g tha
git to 84 L*t a-- tjnin1u flrtMId a* Siw


S i : :
te' ri a
^^. .^ ^^'^ ^^^^^^t^ ^^^P^^^^^^^^^^ ^*^j^^l^'l^^^y l^^^^.1^^jj~,* *_ *":j'J:g::









aesatrattsae of each elementa
Ob4t (0b94*.?1 0) 0, 1, i, 10, 25, 50, 100 and 250
popalks
Copper (CW 4.SgO)u 0, 1 1, 0, 2 0, 40, 0o, 8 and
100 p.p.a.
IMrA (Pe047lp,0iD) 0, 6, 10, 80, 100, 250 and 500


; O0) O 0, 1, 8, 10, 50, 100 and 250 p.p.m.

iV(1b4,2.Sa), 0, 1, $, 10, 25, 50, 100 and 250
pep.p.
Sit S (saW),~TKO) 0, 1, S, 10, 2S, 50, 100 and 250
p.pa.
All the soItis nd t we reweAt grue.
Aa aditlsal wrrramet was run using wedia mlphate

I.. (eS ). at a auage tratten giving 500 p.pe.a of mudiu and
,45" pi.peOs. net fate in erder to test the fftei t of the
aIIeS.llj Smf ii hIose
i ... l be X, data In given fOr tMhe seasatMatite of
t :!e '; e: a teaLUts that Vwe f9ma the ser filtrate of
sem. sthe n4ed in these studies. All thi maples wnre
ain.-,,ii. mi,, u peam trie anlil aittr aid it the r ir"
i:. 3..i. amnefls. Oepr tand *iae wier determined acting

Same .(W *dalt wsin aittsoeet alt (96), rea I sing
4.1 t:.mathl:.in. (:.), fmeia-as u0ing periodate (g.)

... th.atr e tamna altride (MS).
j..!lp ? i.: .. .11~llf HIW^ W .. ^ ~ ~ ^^ ^^ ^- ^ r^^^^^* ^i ^^^^^^^ ^ -










-ATL OF IrUa h IN U! EWIS FIWIA O
aOGBXII P afiX 21 tEL




as ,..at ,, a*ft o.Sd
Co~lt . .. . . *. 0. t* O.eMt

biOer . . . . .. *. I 0.3 O.1Ln
MfM-# * ** * to" 0*41

%, a a .. a. ... sf a.ta .A






ktarrustba.I aent ai rlu a llolhliet
mib V at. t. .. .e a. t ..o









w a







j, :
. . . . . . 0.

B ill .b. Ea.. sA"* ... . k I r 1. 1...o. '.. ".

p"id.''"a S ""ta.t" ad.f
isii Mmd sMwM mXit S mit u ssl .flk


an ..a ..t en ..

vh 11ets0Neari We -,-V







14.;';. .. N r 4N
: .H ,"''ir i :::: .'ll I i,. ":: .:A





.. ..
.. ..^^ -^ ^"- i l b : ... . ...: ..:...,:, : :::::.. :. '" . 46 l":.: :::m{ :







.- . . . . .... .... ... .
i^ ;i:::^ .... ** i, __: *: < :** ::-.;:^ r ": ...:" ;.:. .:.i'* : **'..: :. : "..:: l.:?3:










lXteat 4detmiwAtia, STi remata; I tabes wre laabated
fa a wtr bfth at 40 Co. ter 34 heWs, afttr wt tim the
K m df mm *lu d ea 1 4* eL 1 N H 4 added to step

0tmua1i tiat ..a d5 ett amli orM kept 01ld 'La a
: m at a t tU 9Xai is w"a dete imiode, astiL
the SiLrSL*X1tr.i aeid proinduxw it dxrpqtoa ad ysrnard

(U)h S7e p i wdund xa3 ut *,th SifRl ea applied as u
felsesw the ei n tel aftwr tdiesiste, wer treastfrred to
:i: :' ,.

:ss*W .:.W 4 5 S . . ravity.

'; ~:,:s ; mIaata a mw 4deelSted &ad 1 of glaSial

Ss 1.5 at n trat altrie. s were

H.S t" ss. .d.. e t%*es ws thus heated ia a
hAu watO bafta ftr n 8 aimntes am th sentents filtred
*m..:: KxS" h"#. haAb e so :l mid was" transtan d

S4 at ., els ,A a d tate ad w ighd,
i it W k i r ,

~..a; ait.l. tu.. at r .*., ...tl and .t ed
i K ." .: ,.;" !... Eli-. ". .... '. .. ,"
.=L. .... I. At h.. t

I.; ;:: ": ;::i:: ..'s a. s. ta. as th I t of a.l.u.a.
f: "oti .; :.:j ..l &.:,, ... .... : .

..:= .: ..





..........




4K K :;: KJ: ,,, ;:t' : :
Erm:i!E lli :. "" u.






27

Vei af at. and Chkiek to bWtemsni the Extent of
CalUlere P telHteM in the Artifileal Rem


In this stadl, narm katnts wer Obtain"d eaty tfro
the St tlhted ster. sin b exe prdestere of prepreaV the
runa tmpAt iua we fluewd ua aWbv esMa that awrg
qsatitle w're u9 d aMd antitlat sl tiena f tOfll 1 aI
us. .
fo a 6-itew Flereaeo fteak -& tmdd d 2W0 giam *.
Alphaeil, 1 Uter of strieust -amlities (Tom*X X4, 5 utfe
ot dtIatllId water and the misture earned tr 1U aint. Is
a water bat at 4a0 C., %hsm I lItU f nrom fMtUtt wu.-s
addd, man& d41l4e behl.d thre9,04 A atdjuted to ...



ireseated ntaswl wm ma lnt ta &w Sti paeuaMLa pS ::
iated at ao r A hanr *at w. SA thmA a Ver ......
:" : %1. .; .. "..' "
a11t. .tis iatI was at a t tbiipiMk sitl tl
mevalae ..t oathe s ta S "a .gest di
aMine, as. .as. ne.m a.sa, a a :.


as.. fat ...a. ":m5ts h' em :: S
ia "le fl ,th. aI WtOi the aal i e 0 :
................... ..'.. .. ..






.. .. .* 4,










TABLE 3
OagQ.A AtALTS OF PRIEDl CELLULOSE (ALPACEL) AND TH
S13BSM W FwUrNTAIONm IN TE ARTIFICIAL RUMEN

ahraeatod Permenated
($2 ($5)
maa 7.S1 0,7 11.63 0o.8
AM 0.10 0.0 8.42 0.65
ebd ?er 87.1 2 1.92 43.6 0.86
ai ll ,I. 4eft OwIO
t Petait 0.0 0.00 4.o8 t 0.90
tbkr xtrt 0.1 1 O..Q 0.8 0.23
hitxeges ire. xthtet 84.80 t 2.5 25.06 1.76
^ *; --' -- ^ -. - .. .- . .
:'" nmes ea: stadtad dertattla.

tat:fla& tth:t -O.n*ae hatbred and twenty
fmian ti at thle L mEarTnl trait, fre the Nltritien
Zibta wy flnh6k eiea r were tad in thee experiaats. They
w;*' b ested i I is eops of 3 er 4 Is galvanised metal wire e*as
.... 3t tb t t tral, 60 irats, reakMge in e afroa

. . ...
to. i8 8 wa r n sitsS** ,e-e*dix. to weiskit aad sex
*i:-" "4. '!;" ..' I .. *
Ait* a *miSmS* t 0 rtte baok (1s Sa"ts ad 10 faleo).
age hi the S diet. of takel 4. ft dietary groups
4.;ga p feMr Mt AAt ('it.), )i per seat AliPaeIL
,N.<.-. :owkt AU6NM. exNTtNd 1 the mm mmrtimfifial





;fu . . ....). M y p..I !w.
INtAWW cas9 iStrWiat bottle. Oewth was
m" mmf mb mk L f M
m ft" n#A*





29


TAIBL 4
CMw PmON OF DIETS Fb TO RATS


agredliatt Coetrel Diet 2 Diet 1
(1) (1) (1)
raMod fellow sera Mo 00 0400 4,00
Dry vhole aslk 35,06 i5.0 5.400
saybean oei aral 13.00 U1200 U.N0
eroN 16.00 $.00 I800
cas ta 6.70 4,70 eye
Altalta Ist sral 1.5so 1 .0O
Iatt 0.40 0.40 6.0
Cm om 0.40 04U 0.40
Alphieel 4.00 16.00 -
Feremmtd AlpbmslX --*- -0-** LA*










aincan AizrcX o1 BurS D mm w2 muA


otxrel


AM a


3M'S'


S.. .. .. .: . ...



...
to's



": ti: i H:; 'I::."It'::li Hij

S. . ... .. ,

,, ., : ".".:.i; ":" ,'i"i.,,A B









eamiflnatl per"ed.
Ie aprmIakt was repeated at a later Utie usa the
SaM SlWer ef raft and dietary tfkatnats.
SM rests oft ebmieal analfyis (86) of the 8 diets
ar Mdutnre, adI eirede ftOer erad pwoteta, ether eutrmet,
a itmeen w entrent an re rdd in table .
t.inAlMtrii.iMlAabAsa.-io9n handred and twenty
wtrned chhAs (OuraiS a flit. myl.ath BeIk seruabred) ore
dewr 44, were ob XlaS Inst a 1oel tmee n tntl hatteuy at





.- .l ratld wba a rd M he1a b se lk 4 tar
S*:*". :: ',a WWW $ t periods so seampeitim
A& d A l abL Th . :try SO .p
.. ..t ",r t am eurat: (Ms* 1" ", U pper cent Alph'ap
Ibt {d H par -elt A1h04t fa lited IA thO Artif1e9al
.' ;,. :: S S m..5:. a1' t .. " y. ".w... ". W"7 0?



kM* w b -* tb. water ui aA at al a ine a@ a
t::nP &vaw m nd hirt wwkl eight** -
,adan~db~ate ab.H.Hh...JSrsA w. sJ a
;:;, a t s a .... ::.. .. .. .
Ha1t- fl.1 a .ult .S e,.h..e..


.ii .. ... .....

i i:,ii,:i,::::: Id a .' li R t m
H HEE .l:"a :a a eed.a"lt


N H]FK
!..H ,,::,










TABLE

O7 BiZW ME


wt *fI3m


Ingrdi4ato Coantsol bDit Diet 3
-`------- -~--------------------------------PQ-------------yj

11um Wlf.w ern ar .00 1.e0 Ues.o

yb~ea ai t s 39.00 .89.0 U.0

AMlatfa Sat mCl .00 3.00 ts6t

la m a 0 8.00 aB.LU.

Dried whey 00 3.00 SW00

bam mo MI 15.80 na l0




AUtpha*el wl l 1,80 .

slnt alO ,h0 a. O-n ta




Dietary 8a 1 ULOs Rpua *d ASt .D


"' ta I 4 'l a,


a-


kj
.. ... ..... .. ..
..F U'
": ..~~~ ~.. ',;:::!;I :i


: ::, ii":: ;; i,
: .. i;;.i ,i: :.;i .i ii
... :..;"i.': ,i


m*:


...
jrt
'".
;;'19 ;::;i
' ';'










TA2.I 7
OerLm a AIALTa f OF BIE PFD TO CHICE


Oestril Diet I Diet 3

(1) (1) (1)
t ts*Ltm. 5gl0 1,9U 1,S60

i:. -.5- 12.47 8.48
in otlA. 2.X1 311.77 33.82
,....tra t .' .. 1 6
"n"s: W"-"40t "S 4."" 4312
J A : .A 0- . N


tfatistiL.4 AmlTsiE
hstatLat tneatmt of data foUwed the method


4 W. t....t f:r a..OORafn. r syiis f ivaam

l tint) fas &r SwamBs LttpI. rawa. test (48). LU

samb tet Us duess te Mtefiem wahik values obtaiand
it b. hw tWaitl sbts -f s0 ittleaatly 4ffftmet ha
a th" ..

* '.."" ; . .. .. "
j:. *:: . .,.. * . . ,















.. .'.. .. .." .:.
. ... ..
....3~~C :: liibii ..ii:. :lr~ ltXJ ii~ ~) Fi









Iar m s Ill IEW


Mlfntt Ot ase ~owr e ea l11. $*gMa*.a
Webt the Artifitau hams

Ia. fteS tash Is tIh 4data sad statiatlAl
elyais am m fi are ntbitet tUhat wn obtaa ma t the
stay of the 4ma11me et Ver Isles elOt .6 f..,
int s a .te, ltl.hmm, M at sta em the igetlea 40!..
seflit er t a Ifalmijstmi La the wrtlfsteal VWs. flo "
futOs i wrt9Mit ssflehs, W-lhra fte.at. Mallei, wei
sdl to 0w"S atimel .tr. mam fatrte tres aitalts
nsm tiathy her, pua -ra he ad pwoem "w
ourl ts tke iM .ta tv. R*If4taUwit :tan uerna .- *

us As *he stea et eia in. ** -irik to fte she at he
t.. typse S. ae,.l.n. .. .. tiheleted en the m.. '

an by aIaw*s sartlf w- test* I. w Mne: W fU ,
the n Wt f,'fl m*e, n ar e m a


*8a3 s gUss.ee i 1s rili tt as e ::. p'
S. ; .. : -.. : .p

S. .. . "2:i::'i:.+ .*i+il
Stamt s atS Sf t Iftas a I4H A l s.


a*







34


alSt .. la, tMe Vass ia2U eataaS mall Smats
09 tasm uasw. eaArbttlod-d b the rma filtrate plu
Mittal kamn amrats of the salemt beim stt led.

Akl441a bule or, 9, and 10 aeo pre atl the
4tB e-.atIk A" th str Of e *hlt. It.H avera pratape
Et.. S s.. ."aS d i. 1, 10, 5, 0, 5, 100, nd 250
p" I pa.t *f Oatt wo sed, were gn esaoaty t S79 73,
l 9, 8, I*, U St a Ie uirttlye. .e 4tia 1
IM:b lf9 sehaln hts k isntla.e hut as iaurnia levels
s damn we. aMsto, aab easities rmsattd ia a

aUiMtt la (P te- them 0.01), IA the Imrt of
S:.:'eS"t s.. 4 ,


: Er. '.l. () who u :d .with lrna-Fl as
..! -... -- -.
s000e A est. M a M d"1at1ag u typo




ttIu. .d nes nutl iny hA .the a4t0Jinta

Silmt ttf ulg Sflaqa e*Sthait 3etre t at




4>W V 0* PbS' .0 X. lb) flt vhat daily. 1A00
&A4 JA i e aieea t, toad in a saiat
I ..ii.' ...' n p a ....t as an
.d,:, .W" ..to 3... ....0 ,
. .,b.. .... .... p M Ot1 ft10







--



a a
A' i.,: .i. : ..... " '


. ..... a . .




4V..
.,.......... ".... r ).it Li rrpL lrt M lft a.t



...... !*":.'i ::"!.:;:."' i. :.li .. lf8 lS i;^. *" I" :





^^^^^^-'^*Hlllj-- *'**ifBI~kf --&jlxrX- KK




















OLWEiLoa uVnr WH ARncu mA a AK 38008TU



Plruenage at Gall llIx B&
.. ... .. ... ......... OF C "u m |hii VM nmnmnni M 1'


I ,. = i....n.... a C.lt,1s D c in
MW -l.M M A. FO aa gA. A.l
. ... ..... add $ --_ ....--"--~r
"Mfta p~~4 ~iZY~~~ Bi)III~
QaJ1; rl~m1 .00


7741





n,i

4A.3
3.4




'As


Itx







te




nu.
4,.


75., Nbt




sad ae:.
1,1410 5a *4

S.i OSA


N
. . . .. . : "* : "
.., .* ," :. .- *

": :- *:~ ~ ~: .;* : .:E*.x. :
: . *:. .,;. :
.~ 'w.' i

. E::
:::



'. "*'vi
.. ., :, i .
:. : A ....
". .,. ?. ". .. i:,l ..li ,,E


i::;;:i'";.. :: ";*i

I ,' ,, li!ii ," !

: : I : :. :EE:+EE::"" i


r-----


*


abutlrnr. #1


.nnnt.lU


~uaylpea


an...-

Al-a .


0

10






as
SO


14



1.1


.as.









14.0

1.


. *


MUMMM--------- --~--~~- -------


. .. m- . .. . . --- ... .. .... .. ....-------- - -- -- ---------










TAKLE
9a1rY OP AKALYhIr OF VAwAI 0 DMAA IN TAB a

SsWtee YaruatAaia S___

7.47
M Mi t" 1 11.22

NwSI a) 2 80.45
1W4N 7 oU.,s

Uwur (b) 21 3.14

.t*la y a garflat, P blu tha 0 .01.




STALs as





''. alo0 100 $ 1t0 5 1 0
*----
SM M Mat As





.vt .: ... .
:1n.7ii::^: ::^': "









of 20 gaflou. Conall wekters (7, 6), n a series a st uditA
avestigatiMpa bined halu .g in sheep, featr tiht as uM
au 0.1 e t of eobt per 100 bs. of body wevogt daily,
eamud senly d-epressd appetite and lwas. a bhey ,ritt.
fro the r*eats of this ianetIatUea, the
probab~ity f depenra tg snonl beteral amti.a ti ni
rnms by r thepe trat t with Oit lUee 4sn ef
emalt sms very "rote.
an .--he data Obtatad ia the tkdr .f t his
eleit are presetA a tf bless U1, 1g, ead .L Sihe isa
pareetagnu e*f -liX-t d-a-td hOm 0O, i, A, S, 1rS 40,
6,, S0, Mad 100 p.ps, *9t empper uMle dded, War" a.ga.t *1!
Sta 7.e T, n, f 17, t, Y, s, amft iaptt, .... ...
."* *- . . ." .. ...: ...' ".'
additier f 1 *p p.a. f elpper hld M Asfim n a, t. w :.'i
.- .. ... :.i :.' :.: .[.
Aeeuad evels eage the Yet teha 4gggaggg iuman
Inti at fl papea. I eMp Sect $ per 5 ia art naIbn Mu
wh o to .... ..... -.1 0 :;: A,,i:


: i ..: :::
,"ab to. 9.f l .: "' ...0.4 '' ,k K


No ao AaS wi n r am& e



. he* a.t a .. .. a aM .u : ...
as 1 as a.aim mu s.s (Setas a .
': .. :. . .. ... .
WB" ""- i l" i .( t.i ;..


1 . ': .'.. ; | i:x iiiE:'..
"^: ": "- n. ".. ^ ". " : : ..::*. 'Ii .. :.
! .. .... ." *. !* y i i' i" "r" "": '.":l i i y: ^! ^ ^^ ^ ^
.. ..- .. ..- .:..."" j ,i ..'. ".. ii .. ^ -i :
fl H X..... : E M ".. ..- '..Rlfe' ;.. .. .N I .,,. i( B ".~K ......... ** ..:. :: ::. "':!! iill" ii:,. ': i 'll:.. ': i
















Mu W cm r I BI DRMI2 OF n0 TYPES 07 PURIn
K SLMA I 951X SoIueIL n6NBI2 AND IOCULUM


IN A .-000 _uI
:; FMr. AWAI.S PS BIFrSMW FOaf
".._. ta" o af. l e Digested

PPa n pea' Pasture

t b b W
.,. ,: ..... .' J A. a.r. As 8.P. A.

.' 8.3 66. 80.7 80S. 75.3 75.4


5 *4 72.6 755 l0.0 U.1 65.4

.It 54. 54.8 64.1 8.6 56.1 56.5
*::;: :- "
s 3o IS.8 2..O 1I.8 14.6 25.8 15.4
$A 4e L S.7 8.A 17.3 9.1

T ;::." .. 4.* 100, 7.7
To s et.9 41 so$ t..2 8.6

A0 t 3*4 S$ I U. 6.* 8514
:i ',**.: .... '.1 l"* :ii. i. i.. i?.i 1---
.s:! ; p ::r at... .a l .
":M *t Mau ek *h aei
... .. .. . ..:


i;, .. a:, :':, : .l ,, Y . ep .
.. .. .. .. . .




jri :; :; ii.. .... .
i.A .. .. .. ..S..... ,. .
2 ...i,,:











TABLE U
MruaT AMA LTIIa Or TAIAmI Or MDIM Zl 2a U

se8 oS YeMtfartt D 3a
TtaA4l saDF

fliuitnr 1 2 89,
laek a ea*"

I wiit .. *,1i
ammw x Lftml 8 U,3
Irxer (b) 24 s.,I

ag.. y .tgatIfla t,. P I,@" the c. I






-ssanM Sn uanVrm smW. saxag 1 -L.
-j LI1Ul2Zr


-LedS
a'ir
P~liWS* 1


ao " 40


o88 s


131 t"w ..U.. .: .......



M.I
S . .. . :. :.


"" : .:' H ..I



"r ." "," : ..!' i ",


I









stAWtr iL dry lat a qrtnleat musat of ashydroam. eppr
m te. Gea O at d Wafter U1 dars with darateristio symptoms
ifla toe t hese bseeo n by reider the other steer failed
to d.vaM p aS text *mpat ms. A maxixml eonaoatration of
qpsl atei4Sy P4 p.p,,. of pepper enwIrd iln the rMn
alats. otf then ste-er, smmdie that thek had a rxwen
sAlie of io g oles.
e aXah i& sta tdi*s reported this investigation
MWpt Set pepper is tesi la t that it decrease selwl e
s t ln bam it Slpp P re* t in t the rom at
RuAnt eable to these ued i& the artifitial rmna,
bl fI diont. ether the aa oaffset upa ellslone digest on


',!hr,=:== .... :^. .^i:. i* Ifa m- . *- f.. J,
.ig dMat. .Matb4 ia the stdy Of the e ffst
:.:.-tte. idigaL lsa stA la n pinted la Tables 14,
'- U, iS. l1tm data iiItate that Ara has vey little
0101t 0 a o1lb 4tgsatbAI hosewr, the statistic al
E l H1f "I S that the mrvMtd Sfe .s eOpprifmately 5
t., he en.t X.... t 0 p.p.s 0... .us, o.
1t es0 an h .0U) M* lan t.m (s) aar t*9so



!iii:i li At, A =t t
H fl bit c 1.0W0 p.a. of in. t the .rtifioeel ram
jtlfh SIr ' .. y l h asq. ins sat to battle ao reports of
H ;,ii; ; ===!= ==.;,?.. ..b s ti..tm=



EEiH E ..:1 1 "" ." Ex'. i i,,;.:" :; ..iiiii = ..= = .























a t.a .1 .

Imun mm.' WJ

s~ V= .*m AAMfp pgg(fgg


. .. . . .... i-.. ; .^ ... ... ., .....^P... ... ^:ir'. .r ( . ... .. T..I. .
74 l4,om o. lf A '1440a1 l




-A -- itit 9priu^7 LATS IS?



.. .. n* 19n4. 1, .01 6 ,



s 7t4 7"*4 7S 7fi 0Y a.t



Ieso 41 7,4 A 1 4A n'S O .




11 "04 it.s 7.., ; Pi 78I *b.
T ..i.m i :.r. n-inr __f- -- - l_-- _- --. ~ '--' : *I -:1 -""- Y_:'^**- l ^' ;-


tsterw .1 btJ1 rl


dCy


a-u


"*


ta


,* .. -** ;- .. .x
*, : ." .. :: ." i, .

.. .. .

.'." .. :." :... ". i;
'*16.

::. : ..
li 4 l ... ::: ..-.::. .. ;..
" ": ... ,** ":: ::.. ::: '*
: :1.




JI.;
".~ i .': .':.
i







;. ;iik
S t .: :: .... ii.:i
" .i; .. .

;. !: i] !-, ,: 7,

i~iil:, .i
i :: .. ..i I" :':.. ,

" :" ";::i;
: :F. .. .: i

: :;: :
;; E


-:

::

;:
:'

1



.~





i
i i
..~
I' i '

I':
:ii

':
.1.
l.i


i








42






Mau~ i~s


ast OW AMALTRS Or RIAM L OF D= IN TABLE 14


ats.1 of variatia D Ms
----ii -N >*i- !-. ..-. ii>> i i 111 i m i i> n. ... i. i iri i 11 a ,


iCtt
4^jHE
* .. . ^^ f'vf :^B




'." .. .

,. :


:: ". :. *

'::' i;,* ; '' -
"-.: "'.







." *
..:. :











.i5
! .."' L" ""*
:.* ..... Xff

!... i ;.: . ". .' S .






::!!::i?:::- : : ...I.
*'. : ,." : .. :....^ ^ ^
" ": : "- ..:i .. ; . '



'.. .... ... ^
.:: :: r :.''* ^'" r BW ^^:"











,:: : ;i ,':
I.;;;:" . :, : *a



































:y -.:.. . "* ... .,,








,,,. ::: : :.'E./ ,' .
...: .:/ . .. .. '
;,:., ,, .." .. .... ,,, ...











































: : ; . - ..i .: ... .. ** *
^ ; ... ... .


I


a loml


t


X4.46


41.47

Ob.s


0.49


"WtOLy a~gtilem, P L U thaa 0.01






sEo" 16


C-- --


Sg. 'a uO xI




n.4 MaiL.M.L Mtit


5 0 10


fl~~;~t~YE Asa;, fl.m f Af~as.


thAl l ai IiS -l

! Py~S~rnt


C'--'--- --~- Mlk








*-I Tabm 17, 16, od t 19th data o"
steard*t fora row et ofe a*t"*y vith imeass1sse.11 1, "0"
mrbw1 Althalo f tet* Obo tht Ipps* oftmgns nnme etl
digheth Mr by pw aoat ad 100 P*~peas deeaet9t1ra
40ato Tbef "Wows mee *ea eleatue Matfute la

IN 1 -M "09tl mat"Ot ett t Skiatk tiwe oftrt re9 lH

offeevt of slowgms e% 0*e die of 441 U*1-eM by the.

eatle edir hs ap~eJAs the litoato**w



WIN"& 0" of lla omgest ftn Obth 20 ., wd 1,80 8
of, IW n 48ps 0.0t1 addedm sipeaa wsit Is ppe#a00e

?I* 'rs' Y5 4* V61,10# f& a 8 eutblakyv

...... ..e .bee .t .h Oswto pmpe*, 4w srawte mowe Ws'
now s"'Osa ab10 st0stal agtteaSS Jjjawea~jjjjorW H
effutat meaht ahetratest t abtt erat
ala**w mowkpets ~e P.A 10 8 awtme
As~~~j 1" vt~t~laa dmsH k~tsa







44















ItC ern mxs unczriae D anxoau-w8.





PfM'lS#1tL.)partr D tS
Pi, a A t. r i, Y BFP.tM F AGS,
01, L 8.. 1.


V 7.7 fl.1 7.s 7.4 771.6 78.9

I fl.0 7,S 76.0 77.4 78.6 86.7

I 7t1, W.S 74.8 15.6 71.0 71.5



to 7. 7t.? 7"8. 74.6 718. 75.7

S 74.?7 7P 798, 71.5 7I.l 75.1

S 14.0 77. 7lJ 18.1 7647 75.2
tsr. n4r -.,s s .,o '. 7


.. .-----r ...... .-- ,"-- ; ,- ............ ..-r*- . .. ... .- -~-- ~ ~ ----


, ..." "
- ..: .


. . ..


Sp-n4t


.a*1 fle


b~ilkrF~e.


* 31 iper .et Alpblat.


.. .. . .... .
..

EX
A Es..i.' X
*::''?:""






-. ..... .. .... ..
. .1, .. : ..
. ...







M. .. : :"
;' 'l; i l: '








.:..:: ^ ::*: ..
:. ::i ., J .. i .











TABLEU 1
MMIAT OF ALULTI OF VRl OF DMA IX TARJM 17







sTfat


rrerx (ab) 21 0.4
6b til0t.x LaI l



ushly .Aiwaititust P less tam 5*#.


'gOlSaCsrla y5


9KM~


_ 77 7 7 - - - 7- 7a


Ie in


a mf


U S I


... .. : . :. "



S. i ;
S" ..." .. i.
.. . i ii
: i .: .: .
.
:"~~~~~ :... ..:. ..


t~PB~a:


a MHIM~
Sfrt f W


i
P


~gtt~~
]P~s~EQ~w


















TARMA 20

arn o imawmBU woN D=Iezm OF wo TYP E OF PURIFIED
IZJOnS Mlg A"TI ICIAL UMeM AIiD DI3OM
FS AEMISLE IE DMUUEt FOlRMES


UP....e stage of tlul Dirgted

*atht Pbaela Paare
a441 6,b. A. 8l.b A e b AsS


St'o 71.5 Ti.t 77.0 I81.I 8m.

t 66 4 70.8 71.6 76.8 60.7 71.5

10 67.5 70.1 791. 75.7 81.2 79.4

SU.64 67.7 7T.O IS.l 7n.5 78.0V

4 a'J 5.60 J S .8 47 78. 75.1

W1 S N0e t8. 7S.4 u.S, 75.1 72.7

t1 5.1 0.4 78.S 71.6 71.4 716,

00 AS7 .S 74.4 8*.1 70.1 S6.1


'':! '
II.::
r;ir:
;;l;ii...
w:.;
tg .;';
Yli:::::I ;r ;
,

:


*WNM * MIXIM6-


il~ise. **


.:: : ..:' :::." .




; .: ." :





,.. .... .
.i" ^ ." ..
:i : .:

" : ;ii;,.ii .:;'l ,.











MAAr OF A Mru 8 OF TARIAME OF DA I TAlE 30

eaWt of Yarlatten IF Ms

Tatal 47


Pagmola atd Ftature 1 X40l
"'rx4a).
buwrn x lo w4 .

gaitetrtt P leb tha .

r ,lfimkt. P 1 an 0.1


-Ungi


~iss^


wasawr'.ss ,M


Ip


zin a@r


-S. ;
* 0


fl-p, .

-Madianno~rr


& "-', I..'i
m ...... . ..... . ..


a
:::!%,A. .L'


7-...~-`~--3--1.~~-.~`~..I---~.~I


.










.4w*( IuP ..laflirMO at an ILa.

Se :emalts paenatmed omr mb mess that

asL
e bItn .hrt ,r diaSm soeS b* r a thlyhdfau tAiumty is
S:sm by a I Lkatmrer'enm with tle activity t the tea
SM. ..... as.. sn .a at. ,l ir ps.stsr, L
m--*:16 :t t"he auliduiama eetet *. the terae ts 1U0 p.p.,. er
ea i t.he.ry matterO .ie w..d. he ntertn to irte a
ed.: f .ttn.;e.... f Af th.n Uo p.p.sf. a the ,i is o estate.

S."'.. :::t. : Wais 12.. d4. U:. SO.N . .. ri lt.
.. lll;,:/... ...,;,..:,. .... .. . e'. 1S81 tla T La a A X P! r) lt>
flU neste iei. wt*v of Lia the stflyr f

a a i* Iwo" ." : ap man ea ntaa

4 -H a,,,, is, as, so,, u as an p pen. wee ap ni-*
". .if l":. .", ., U." I l 4W n*al t nsuus.".y.
^.*:..,;. '... *
:*"'x x '' ." .i.A : :::. .....ig^ Biilu^ "" """ .. .. .. ., "? "',F 'R. t .h


S ':n' a:.' : :an. ... .. A. Ii





...'. '" d:o.ut Asopo
"ti M.tWO:: .7. :. ".I:t "o .t a

f. afr..ft 1h a& m L"s tat "

"t' '.i j g I..r .H i :.. "



..j.i.... a.. ... .




ON' ""I" bided
jj"iN" ;pb? e^s*&dfl a .efaJ.




i id
*S^i-jl^i-'"!: **"."'in* *w^'^j:^ _&^LjlEjJ~^^~flwm*^&Hj 'H^^K.J~tnhsnL jlw in^^u^j^bt4hjkn'^f
"12"~i-'jiii i~lIIBBP P '^W IIwlllBB.^ 'iS^^ ^ wf 9v-^ ^p w;^ ^ ^






48








TABLE sa

WW 09 S3S Wof a W WN orPTBo TSf w i
assumes value u aZzrzoze. am l a/e nannu
PM AN FED SIWU M MOMAS

... .. .n.. . .- 0. ... . S


a.?? A. ad. A. ad." A.
8 9848 at iPat at 80 st ft
N 9I8 A* a*,* t,*4 96,4 t,*

8 t.i* *.st 0TS.l 04 78.8 6At





as m.a ese 77sa aba usm seta
n 7 46fi fit 7MA5 1#4 :.


6a 448 44*8 8I i.At J, ilI 88 8

AtW bV 7TI PS 8 4 LI LA

a" *Am. 44 4r *a XS
--m M0MON--111011--M NO cii N


*wa orw et


a n.... r.
as-i.


I


I


It.

.ii. .


1 T.
RJ.PI
K;
* ..u
ijt i
t..;"
udhY: ': '
:~.~. '*: ji:~?
" S











&SBUE A4
SST .r AIALSz5 YTAlIES Or Ma IN TAB LE 28


n- at.i


--IS3l breD
f .. :. . .
:-*:*:M* f t h)- b i


DI1 NB


14.U
$U.|2


!.1
so


4.45


"W tr S*R-L a"to. P l.s than 0.01.r


TABLE IS
aveM RMM vmK .. m.LS or Me
-' * h ^ '- . '6 a.


~~~WrIY S-~-~iIZE t. 77<.Ya


ne 1ee 1 o s


a' I 1 0


-i MAMA


tbe ams tite are


a,.









.emtqatlwo. lS daily AntOs of t1he m.A-tratt Ime

lfta aMi f~im al te 4 Lbs. dIs the euaev.tatl ArldL
Pa tMyr, the onee hi the bmme of my AL i eOm t, va
tht ti* ~wa hlad nut yet dwlapt at the himA~s of
the PfM psaRrM lA thi e hft rl h bave bemsl l uitMlM
to the aM b th tie tthe :m a lrpet NewII, It .
Umvol tf stma fe dah ba, gIe a eui M Ita M t1 ,at bt.

La Su mill of 1.00 p0Aup, *Ai iAs vry iA.

S.MS -sr aflins the e m e~lt se. *1
.Was .maydni a '1" ASm ,r: ts L he am ,,,. ,
.tina MOM n. aita LA th lavA elift M4 s e0 0

as=as stst, At Iw sea. u. a I. t.!m.es usnt
"d fsAhe Ies #a- Otto effeets, this, test
9eas no: s I&t v 800& S" 0*0 t of. 494.3 &A popoo
a uSt. .... .t. .at.


.e.. .. a.i.e.u ..
*-* .t .-4 0flagi ai W, .

S. :i :
.aW '..'. ...'b.a .na..i.I.

S. .i.. .. .....

S* *. " .*' "I."*:.; 7.. *:.: ,
a, .. . i..... .K i,,;i 'ra4 :ii
: l .....' !.K H : ...; .... :* ..:.. .:::%
.. ^^ ^ M ^ Wi -- ;l ....: .. .. .; .. ::. .. : ...:,,,.. :, :i :.! Sif, ::..'': ^, : f .i
... "^ .' .. "l 'i : '. ^i; iii'? i ";;;;'i.[ i,;,

, i:,i :,,,.^ !^W i :. ".;. .... ' : ,, ": : ",i
J~~h^^J^^^ ^^^^^^^ft^& ^i~~f ^^^^^^L^^u ^^^^&^jJC^^.^a^l^^^b mL^j^^ I~~i^H~i '. '^"'i'"ii'^5:*'.'"':
w^ ^vy; I^^y ^ ^^ ...>^ ^f ;^^ i^^ M^ti ^ jM^^i^^ '^qii^ . ^T^' **j^^|^:' *
*teE^^^^^II^ ^kft ^^L~JI^^^^ ^^^Jk j^^^ -j~b ^k.^ J~~k^ '^^^^^' dute' ^udim^^^Il :Jbh A^K^^ ^^~l~jiiijaaJ' .i ::: {!: :::..::y
^^ b .A^ ,^t^^ ^jj^ Jbl.'f^J^c^ku |^ .^^|^^_^ Jfjj& JifijiMi^L ^J ^^je^^ ^il ;:* 'xy.^:^!!::!1::.:
BE'jB:^'idll'r*^*^H^EHIKte ^^B~~^^^^^ B^^ ^^^^^^-^^ '^^^HHH^k *. i""ii*^"x^~^itf"^' *










thewi mts.u ies r rad filtrate. ot filtrate.

fla the I d4ithr at see of efrage mre nt eseatally
dAffuiwat Ih their effet pma eatUl-e divetise. In me

"SNo "bhl so, fitrate ftw the priod OR timthy har
mmeas raised t i AeetRa of Lesl eUll ul thu the
Mr IrmU ef aInm filtraUte the oesA mr tf h doeormad

S M .lea M. la thuk trEl i aa uwamm
At *h edt fete di4oartia prLed the pi vales were
nwdsrunS toi fall I thel rage tf 6M8 to t65.
v toa tiw ae a l withi the mal rap mS*Iting ai toh




A' .0 14 Artifl ai al amm.
;et . .ta d d to st the




Stat .t the mtisse *le tfaesmtod eaflu oes
::" U .v t w et a ian" 4 ai fbei
. : ', i :.:.. : .:.
iiH..".::::.:. ::::: li:H >)! Itii r -ftf fm n L to -






-a 4LS is the gaerith dati za.teeS

SH a te s:te of pevat *ea, rat.
"':l.: '0iU h q wt. 2: "u" .Ga


.. I H.. .... ...... i
i.:..?: M T" ..





.i. '::... i :. :.. t L t ...
.:.. ....i.. .i:": .. E H --B I KB I r







. .. ... ....:: .






89






TABLE I2


uum mu mu n, ora, m S
SFB7 OF Q Cml PERE1 1..B XI IN M AMIF...



Veeks a& MtD
t -- ------------


haittra

I


S


tOfa Wala, 6


(1o rats)
463
18.1
0t.

188.6
War~
8884


c .,


184.8


t0 sills)
#.4
M MkLs




N *99


247.l


. M.. 2S.a
IUT



af t l I t r a . S. 1 ...


qU swp


aCasken~1 trr Mit


1 s- nas*.A


: ... ...


. .: : :
S. .
S S .. : .. .. : .: : :
: ., ,,,, : .: :..I m:" ' *... . .. : ....
ii "'.
;" ,.in ii "'ii'.I


III
All










'pA



Ma..

tT'



*I S.
7 .. ......I




V ... ;hi
i,
ii
;*il;
,:~ VS
V"


"


V..


:: ::


*
I;
i..
I






4






TAWLE 2

WI!m O I'Cmajm P IW Is 88 E ARTIFICIAL
-1 WOW GO MA Olt"F Ft A MS



vWeSgts I arums
Voo ots sAt Pit ab

U( st1 ) (s10 ats) (10 rats)


il .S t 5.2

I. I1s.a 14.3

t IIr -I m-

S;:o .c 4 *W 1.t yr Tbig !
: I .a 11


i C




;m I I m
wet


dl',
74')
IF,
.2
.tI
.1


1W.4


.M
Iii

tti~:T
]l a ,


U*l.*


14.7

May
a.

'itt


ulls.


C 5*fl hstS Aiiait.






.m .. ... ..
.. : :i ., : l t ::.." :: ".. w ... m m im i m mHm m




AI'I m7 m' m mI
m :]E ] m E i 1N mH:;m:i ::E m m: m


.;I~ r ; )H m m lmmmm
:m :)) ~ ~ ~ ~ ~ : mmm mm m mmm
m(((mmm :E m :EE )mmm mm m E mm m m m Emm:H m


:* "'* .




S" 'i : : l i
3:r9 : iii i i
m' m
A. .
,:vm
K,


' -' .-~-- "-. .._ ;t ,__ ,,,. p .... . .. .... ..... .... . .... ... W


Y..YY.~--~ I~-iili-lil- .i ~L;_-lliI*I liY I_~i-l-~L~ ..~YII1~ ~.~.-


















PIIQI ~ adp~a ~IN 3 x an l



aVJAd I:




wateek *a IL d

MAl". (2 UtW 3410




aa

a rIA
-~~~~~ ~ b--- mm ~ -' "


I


a .


-il:




...,


kmb 1t 4en




i' ee


.. ... .... ....

.01A. lit Ao

:.I :''Ij. .. .. ... ..II


g A,,


C :: -:

~' ' . ..... A H
Ar

..... .......
,;.. ........ ......~


S... .. . : ...
. *'*"i :, : i ,, :.. ,i' : . ,, .. *,: .. W -: "


5 L'.;;!Il;


H..
















"t X9,Batt a


1 0
jIi i.gl lo .


;i1 80,1 .21
*'Ra: i m&U fe e
markit 8lU Yff.R












txS 0W IN I W ,O2
.". t .... i' . ..7 *
... .. :.. .. : . .



... .... ". ... ..







HE" ... i ll X6
sttt eIs at
SO, Sii: W i : .... .. ..
Edi~i,,"'~i ":'' "" ...:: ': ""emI]e l


wpg - "; ;; :
f;--:uit ~iiY.,i. CY- ..I.~.LV. ~..L.-L-IY ..~
'l'fin`~ c~""r.""l-~-- ~--~--;~- -
"H P t2


-I
u,


P -lIs tIm e6l.'


16tit
I UO I








Matter vain as tadn ..m.d IUd tU r. better thei
the rat. he rowth f th kt rats aMnd OhAsh uue a te
mataLn dits we gaiflst ure ter (P leX tlhn .01).
thoa growth f rats and a saed.m emamse. the p .tl
d4tI i etMd S s wth ot I eak a"d fat. eedim
the entn4 diet "Webiry mlaed t the sweeter





earnedd A the vats rais the 8 aixthee pwimtX
pwtrsl tn nai *8 Ao. iadAted that hAks 4d.d
d3art the trets. a 1. ue th dUed w"thlA the. DIt
* drs f 9e*e am tt omfe#sri tMat Mth W a ft e4 to
-m ass tuen tuhe di"t.


flte*t itmmanwa La a astlSat ea Mat
Oit p,Le l Aatt- ndameS asta, a *A W



sems as a. a a -i assihe Mto a t. ^
IlftaNi t tgg * a i *M t a g iagg try rU tB;
ear fsne t ... .

m e a *e n .-: ." . . . .
imstblwhg Aet Ar X i;uukb"t U A U cN F
MO nsnw e .,,ua,,, d s.; .s.S.
S, ...... .. .: ..II-



II
S, ,IM~I .
.. 1
,=, H ,,wi y ,=,, ,,,,| P,,B ,H,
.... .. .. : i
j^^^^^^^^R ^^^^^^^^^B^^^^^^B<^B^R^H i~ikK fl^^--- ---- T *U1^J~IH~lJIIIIIIIIIII~bJilH^ ----|^ x. lij '''i~i" *j'""*lik-l1 '**,,,1
~^^^ W^^^ ^^s ^^yv ^^H ^W~pjip w W~ y- m ^^M^ IMR E'' P~l '^I RIWPWWIVIM p'- |KW91.... 'jls:,,.,,.,," 'h i +, :::::iii~i~ 'l








U




lroat .i nwm ftatbSt, b ttv eatrl *f the pi, et

mn: other f rlan.


IM p! Vlun n fMdod af rs the 24,lur 24tat a"

SSon is ths no" et of I MSAd s.a, prd pmabrt




4u otitf at thu w hANtete.
..- ... .





:.. .....

AlH.' .i
. i".. . .. ..
': "




. *" .." .












i.. : ;, ,. *, , .















.. .i. :' ": ".


S -. .. .:t .-.
.d ':r E :i : :... :
-!;' . .... ".,.

























,.. i': ...: :"" .. .. " ,'. .
,. ,,.,... ......,..: .,. .',, ... .. ..
~ ~ ~ ~~~~~~~ "".Fi,!.,:":. .:., .













Aa artifittt1a uMa was Ud t* taWy the eff1et *f
embmt, empprf Amr, anmgaee, trbdm an sM aL *a toh
dtrstita f te tmks oef priced et Uto La tohe pwe e
ef rc t n msae trea stais *eAw d4ftemest
reshape. mher wmtet ef lltieel dlreti t wa dtamtwAr
haMuS ly ad the t aemn ine St t* NuatenS l ..... o
diestLaBd hA feitlia twait with rats iand *sl*. O.mtSs 'm
wht11kh a iad. the snlets a*: l::I i:. the :


t.. a. SdAltia e. 1 p9p.a.Y e e abt had .



"nadi 'a .ut.awt p ne;. bi pea MAil.',
agrese eat US p.p a. atasavir dereen d tan
wQMK^H ,MPW.

bb 9 e".ep p'v *"th:e sev : m:S
neasktas addS La the git~tsut -ae h a Wn a' "


...Bi., W ".:*A.
: . .., w e. ...... '" :. "Jl

4 .. p Ia .
. . ... '.i; 'iii iIiii:I;X ii. : -ii .:ii.'i*'^ i1^:











I. i a41ittuft e 1 Md S p.p.* *ef Sire LM

itaILmM ap t4he ma0t et o llfleer diMgeted, but the so

spMu AlAM dAsseiYs the. diqestis to ahbot 41 per mat

St tt btaeSul la 1as ws addtd. At 100 pp.u. saty

t s 1 te asrlt et t mhe mue tatema teak ,pla.

4, aNtly 2P.p.a d ineatwr a atatta eOt

ugl i a9Ht wtBUetlys t taew ast ivmiss dtwatt. i Lavels

s 4-4 Ap pOp.a4 1 mapma haI It*s.

.* aw adtUM t iora 9 to see p.p.a. svt enga

I N: ha 65 aS .M&af aeFa Ar lAiM r Sadalr u buts J aib t
4', nnawpc --AA H -- ainwh uu. stt agy, a t

fl MW- depe tts ths ta smeed by above lmantsw

a .. .A ittle *w a I a MI Sod aWate Ls. hls d a.
.i ..... ".. : :"
:: s.O te at *p fas. used.



.4j
w1 p 4UMta:,:.u PM|rem imtl AM set, ttR et tIhe

S Sa I f S4xSS I :
. i.. i..: .



E :". .'" . .u..e S a w w s i




'4 I M:': 'U, '7
4 1 ..... = ..' .:.: : .. ... .... '.



S.. ... .... .. ....





*, 1" .. ..
aJ-l' ..+!.ajic",: '. :.":"':'.' +..::'BIf^" .. ) 1 '" ^ m H^ B ^ ^ ^ i;^ ^ ^^ ^ V ^ W :"^^ :^ ^^ "^^ jB W^ ^ ~ ^ ^ ^ ^ ^ ^ w *~~












LI-A*I b BS onSB


1. Artaf C., V. ahnmks, P. artdlpk ,ad I I. We btthlae
S.wnw oil diftitat fatstS I Wd S marieIV"


s1o tser P. UX. ht alterat Nist me ls n aOtN

4 eattie. lutei 16SF o.

So. BWr r. 194 tweto aweterospteat
of the Man .Asnt
Apist Bler Ja, U0fl". "
4. Bbant P. aSd 8. I. Naria. 1941. Sh .4ef the
r the atiatawy traft .tf jiMta
1ago IN to -.utaN"*. .
gI&a the em No|6fl adlPtl No ti-


S. Pms il. & Il. & Nainmfie L R. Atfim .sE. 5.


v. b n LI. m&r .i .. *
B. N7Lib ii i a i3



.J n.. . ...:a
.: : : . .:... ..' .E ,



:. :. : 'I .. .*.. . : ..; .: .
.. .... ..-.:.:.. .:' .

5:". ..... ...
69':m
Si *
% * .; .* *;' :::.:i::













i 1*. Iko 194. weMr at md to
AfliI M or' tel l sea.
AS.


a. sa. X.


wamp s rn
Sonnset .2


nof m sad r.inrt

I At*txJnne.ii1


.. Steas i .,l Uth, n. U mf T AsILn td t. .u

a *.S.t. 3.. VaImb1t. ea, et I.
S i* 4. asi, d, d by nMa

Ree m-Aer nw


1~


IL.. i


:..






"'' *;
': '
, . : .: :..
t".,: :,,,"" ".












. . :


31..11d w .


;'f: r
;t;i;
;;

:t '. 1.'
i;li
r....i
~.;...
;i.
i;liiiiii"r:;;;; ~~ii;~
.r...





S8


0a Breoks, C. C., s. B. Garar, rL. lk hrl n V.L L
WC1J.t e-.asrn r.n ratk
airmnaian Joarnal at Sait batiw ia ew.
11. 2rr9tO Mo P. O11. Ae 6D d1retertist"e ft the
dlffrent bttberia preset Lm the ri"n of tt*
*o a seUtt ratnta. J-aral an aML al.. ,

S,. Bryat, n. P. d LX As. Bhker. 1U3. Ctua tta stied
a d mem a r .t ti ea st t~ r Itie
Jeerwal fS55y f Jml, mmm 17m



t S ttle 3I @U L 5, .. '. *





mm m' m m m
"2 Snmjim s V. 4 O, ta. 6i Mogld if N dda O.



a. n h 'itl* * Iu.I PI,6I: IWIIl S'i




s9 b w s." *V:. .' U.''..U"".,,,6". S,
1. SO lSam aS m islm s I s .o
A- -


*&? 't'ta lift




4fi t ^i~ii Jr
iy11i~~ul^ X* :&HIU ? ININK^ N f* f Mllte*:461k'"


bun-


Vt,)'


ad


'III











64






WA. VewN P. Glra* "sd 3l. Ld Betk. 1948.

X sof WaLfa ash a a watwr entrt of
tlfalla 9lqtn LA etfl.. Jlanal
.Ai X fOdIn 1a 2 L.


SfntV, V.i. MB. N. Mgeagtm amd R. X.
1 X. a ?T ialm *t Gera stra k pn

rh re 4iOtiof &a at tl. lJernl 1 Ani-ulm





uglaesh. 98. Cll~Et dissttan tapfeod and
04r .iPtyW s lmht: a art if itaul ri .











..* m f' iNMMr iCma.6 x.
asHitL^f- e W eat ie dl ese tj by sF tv B


w Aav JT Pa 6eAm*ma It a. Me11. a


= mn t l t4 ea pdbte4a ric

1iembaoy s eftwa lsta as ao rtgiict0

dhmft Seni0 et A n a"- Sio 19 5As-S30.




j-^Mifri iHM~iitH~ikiikiiui~kd JRJ jiHM~i~ mfjD~tttdERII &^^l~A JB~tt


:





,; i






.r: r








r .

-" : "' :i
r;

c
::;

:'?`:
r.r;.: :;.


:i. -





:;:,. :- .^ ,
. :: .
':'" '" ':








E:':S... '. t. .





i :' -:: i ,,:i .. ... . .
: .




.'.
(I, i,: '" ,:,
,4 t*:. :': ".















qIt. #nl.i:'!: :


trg li a.M, ~~. amta l

a.. Wl eyat h.Ia. ., Wee ?fk,






SI


S, avia, r I*, re.ll Jg. Lw ee aA.a1 S. Is
ite wna mnosnVUM r*aO IamWBEMfad e. v rimfI
gayms. 251. -RSM. ae
&"ose ad$7aMtte. JtwnSit "try
satene $, F y77.


to. o b. b... Bblii& 1R*. a.
.t et tt. halift H-I' A mi'sy


3N".Wo-


~.a e tfaarti a u h~ Sl f


41. abtaN, D CG
meas


Si
11


A- "A.lAA U.iw "pt s* '-Mi^
42. jlats I.t, I.adL<>1 A.!. N faBsafcj
?6ineal Into #


U4. I stu.w.y.. a4*4
44.Nmms.
"i erl A6Te0


lMnjtlte


45 N..Jv..L. Ltlwb ie... 4.
X i t i I fi M l kf iI lJI ft A *i m III I I ^ U I
1..L11ansr Z'fl.A
a--A

fXL-4J =tlFtj^m

-.i.
^E'^^ P^^^^^ff'^.^r'^ff .'T^^^^^^W_~y .'^^f^B^^lf^BP^^^ 'Ir^^ll-t a2^P'Tf

^w*-^pRui u pw.^^P^;- Imjlwifr' s^^i <.sY


** ..'.
S.::.


:. F : :. ..
/ .: . N '" :":. :
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JiXMS I 1 164.
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An l Stenes 18, 417-424.
103. TamuR, J.. Rat L. IL. itakell. 1948. CIaentrtiten of
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PAGE 1

THE EFFECT OF TRACE MINERALS ON CELLULOSE DIGESTION AS STUDIED IN THE ARTIFICIAL RUMEN JUAN CARLOS SALA A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA August, 1957

PAGE 2

ACKNOWLEDGMENTS The writer wishes to express his sincere appreciation to all those who have contributed to the planning and conduction of the research and to the preparation of this dissertation. He is especially grateful to Dr. George K. Davis, who, as chairman of the supervisory committee, has guided this research and provided the necessary facilities for its completion. It has been a privilege to have had Doctors L. R. Arrington, R. B. Becker, L. E. Mull, R. L. Shirley and T. W. Stearns as members of his supervisory committee, and Dr. T. J. Cunha as ex officio member of the committee. The writer is indebted to Doctors C. F. Simpson and W. R. Dennis of the Veterinary Science Department for performing the operation in preparation of the rumen fistula. The staff and graduate students of the Animal Nutrition Laboratory offered personal assistance and cooperation. Mr. J. T. McCall gave technical advice in laboratory analyses and Mr. J. C. Dollahon offered help and counsel in statistical analyses. Acknowledgment is made of the financial assistance and encouragement from Nicolini Hnos., S.A., Lima, Peru. 3tf??Z ii

PAGE 3

iii Sincere appreciation is also expressed to Mrs. Frank Beazlie for her helpful suggestions in preparing and typing the manuscript. The writer is especially indebted to his wife, Eleanor, for her encouragement and assistance throughout his graduate study.

PAGE 4

TABLE OF CONTENTS Page ACKNOWLEDGMENTS ii LIST OF TABLES vi INTRODUCTION 1 LITERATURE REVIEW 3 Techniques Used in Studying Rumen Microflora and Rumen Activity ...... 3 Bacteriological techniques . 3 In vitro techniques: Artificial rumen .... 6 In vivo techniques 10 Effect of Various Dietary Factors Upon Cellulose Digestion 11 Effect of minerals on rumen activity ..... 11 Effect of carbohydrates and fats on rumen activity 14 Effect of nitrogenous compounds on rumen activity 16 Effect of antibiotics, hormones and vitamins on rumen activity . 18 EXPERIMENTAL PROCEDURE 20 Preparation of Artificial Rumen Inoculum .... 20 Preparation of Nutrient Solutions ........ 23 Determination of Effect of Minerals on Cellulose Digestion by Rumen Inoculum 25 Use of Rats and Chicks to Determine the Extent of Cellulose Fermented in the Artificial Rumen. . 27 Feeding trial with rats 28 Feeding trial with chicks 30 Statistical Analysis 32 iv

PAGE 5

Page RESULTS AND DISCUSSION 33 Effect of Trace Minerals on Cellulose Digestion Using the Artificial Rumen 33 Cobalt 34 Copper 37 Iron . 40 Manganese 43 Molybdenum 43 Zinc 48 Sodium sulfate • • 51 Effect of type of cellulose and source of rumen inoculum upon cellulose digestion .... 51 Feeding Value for Rats and Chicks of Cellulose Following Fermentation in the Artificial Rumen . . 52 SUMMARY 59 LITERATURE CITED 61

PAGE 6

LIST OF TABLES TABLE Page 1. Nutrient solutions used in the artificial rumen ............... 22 2. Concentration of elements in the rumen filtrate of steers fed practical diets ... 25 3. Chemical analysis of purified cellulose (Alphacel) and the residue after fermentation in the artificial rumen 28 4. Composition of diets fed to rats ..... . . 29 5. Chemical analysis of diets fed to rats .... 29 6. Composition of diets fed to chicks . ..... 31 7. Chemical analysis of diets fed to chicks ... 32 8. Effect of cobalt upon digestion of two types of purified cellulose using the artificial rumen and inoculum from animals fed different forages ...... . 35 9. Summary of analysis of variance of data in Table 8 36 10. Results obtained with different levels of cobalt using the Duncan's multiple range test 36 11. Effect of copper upon digestion of two types of purified cellulose using the artificial rumen and inoculum from animals fed different forages 38 12. Summary of analysis of variance of data in Table 11 39 vi

PAGE 7

vii TABLE Page 13. Results obtained with different levels of copper using the Duncan's multiple range test 39 14. Effect of iron upon digestion of two types of purified cellulose using the artificial rumen and inoculum from animals fed different forages 41 15. Summary of analysis of variance of data in Table 14 42 16. Results obtained with different levels of iron using the Duncan's multiple range test . 42 17. Effect of manganese upon digestion of two types of purified cellulose using the artificial rumen and inoculum from animals fed different forages .............. 44 18. Summary of analysis of variance of data in Table 17 ........ 45 19. Results obtained with different levels of manganese using the Duncan's multiple range test 45 20. Effect of molybdenum upon digestion of two types of purified cellulose using the artificial rumen and inoculum from animals fed different forages . ...... 46 21. Summary of analysis of variance of data in Table 20 47 22. Results obtained with different levels of molybdenum using the Duncan's multiple range test 47 23. Effect of zinc upon cellulose digestion of two types of purified cellulose using the artificial rumen and inoculum from animals fed different forages 49 24. Summary of analysis of variance of data in Table 23 . 50

PAGE 8

viii TABLE Page 25. Results obtained with different levels of zinc using the Duncan's multiple range test 50 26. Effect of cellulose fermented in the artificial rumen upon the growth rate of male ratd 53 27. Effect of cellulose fermented in the artificial rumen upon the growth rate of female rats ........ c ....... . 54 28. Effect of cellulose fermented in the artificial rumen upon the growth rate of chicks ........* ..... 55 29. Summary of analysis of variance of the growth data of rats 56 30. Summary of analysis of variance of the growth data of chicks 56

PAGE 9

INTRODUCTION The feeds normally consumed by cattle, sheep and other ruminants contain relatively large amounts of celluloses and hemicelluloses. Utilization of feeds of this nature by ruminants is possible because of the activity of numerous microorganisms living in the rumen of the host animal. The animal is dependent upon the microorganisms for the breakdown of complex compounds into simple substances which can be utilized by the animal. Therefore, nutrition of cattle and sheep involves not only feeding of the animal but also the providing of conditions for optimal activity of the desirable types of microorganisms. Not only are rumen microorganisms beneficial to the animal in breakdown of complex polysaccharides, but they are also responsible for the synthesis of proteins and amino acids, vitamins, especially those of the B complex group and in the formation of volatile fatty acids. The recognition of the significant role played by microorganisms in the nutrition of ruminant animals has created the need for more Critical study of rumen activity and of factors which affect this activity. Several in vitro techniques have been developed and are useful in such

PAGE 10

studies. These methods have made possible the study of cellulose digestion and other studies of microbial activity under controlled conditions. Among the dietary factors which are known to affect the activity of rumen microorganisms, particularly cellulose digestion, are the type and amount of carbohydrate, fat, nitrogenous compounds, the amount of vitamins, minerals, hormones, antibiotics, and certain unidentified factors. Data on specific effects of different levels of trace minerals on cellulose breakdown and on the possibility of using the artificial rumen to improve rations high in fiber or cellulose for feeding monogastric animals are scant in the literature existing today. This dissertation reports the results of investigations designed to determine: (1) the effect of different levels of copper, cobalt, iron, manganese, molybdenum and sine on cellulose digestion, using an artificial rumen, (2) the effect of different types of diets, fed to cattle, upon the digestion of two purified celluloses in an artificial rumen, (3) the biological feeding value for rats and chicks of rations high in a purified cellulose after being fermented in an artificial rumen.

PAGE 11

LITERATURE REVIEW Techniques Used in Studying Rumen Microflora and Rumen Activity For the study of rumen microorganisms, rumen activity and the many factors which affect the activity of the microflora, a number of different methods have been employed* Most of these techniques have included the usual methods of staining, isolation and cultivation, and microscopic examination. In recent years new in vit;ro and in vi,vo techniques have been developed. Bacteriological techniques . — Differential counts of rumen microorganisms were used in early studies. Baker (2, 3) using the iodine staining technique, reported two types of organisms in the rumen, the iodophilic and the aniodophilic. He considered two types of iodophilic forms; the free population, consisting of microorganisms suspended in the ruminal and caecal liquids, and a fixed population which was attached to and acting upon the structural materials of the rumen contents. In later work it was pointed out that fixed forms were the most active and included the following morphological forms: curved rods and vibrios, very small coccoids, larger coccoids, giant

PAGE 12

coccoids, and giant ellipsoidal and other elongated forms (4). In studying the effect of diet on the rumen microflora, Bortree et al . (18, 19) also made use of iodophilic bacteria as indicator organisms. The use of the number of microorganisms per unit of rumen content became popular, when a technique for counting the microorganisms present in the rumen was developed. The number of bacteria per gram of fresh rumen contents of healthy animals on practical farm rations has been shown to be in the neighborhood of 50 to 100 billion (48, 49). In later work, Gall et al . (50) reported that rumen contents from an animal deficient in cobalt contained 80.2 billion microorganisms per gram, while the contents from a normal animal contained 54.6 billion. This technique has been used extensively when comparing different types of diets fed to cattle or sheep. Culture methods have been used by several workers to isolate rumen bacteria. As early as 1928, Bechdel et al . (5) isolated a microorganism, identified as Flavobacterium iitaxjifflen, from a Hoi stein cow receiving a diet highly deficient in the vitamin B complex. This bacterium was grown on plain nutrient agar, free of vitamin B complex and when fed to rats as dried bacterial cells, it was found to be highly potent in the vitamin B complex. When the Holstein cow was changed to a diet containing B complex vitamins,

PAGE 13

this organism disappeared and has not been isolated since from rumen contents. Many pure cultures of other species have been isolated from the rumen since the report by Bechdel et al . (5). Most of these have been cellulose decomposing bacteria. Twenty type cultures of rumen microorganisms have been isolated and described by Gall and Huhtanen (47). Bryant (21) has isolated 51 different kinds of bacteria from 102 cultures of rumen microorganisms. Isolation of an anaerobic, propionic acid producing coccus from the rumen of sheep has also been reported (69). A description of 33 types of rumen microorganisms of sheep with respect to shape, siae, and gram and iodine staining, has been made by Moir and Masson (88). In addition, some observations on motility, formation of spores and other relevant properties were recorded. The anaerobic forms which digest cellulose have been classified into five wide categories: actinomycetes, thermophilic sporeformers, non-sporeforming rods, cocci and mesophilic sporeformers (67). Recently, Bryant and Burkey (22) made a detailed study of cultural methods for rumen bacteria, and isolated 896 strains, all of which were strict anaerobes except the facultative anaerobes of the genus Streptococcus. The morphological types cultured included cocci, rodshaped organisms, spirilla and spirochetes. Isolation of five

PAGE 14

typos of small, thin, curved, motile rods, all of which were active in digestion of fiber, has been reported by Huhtanen and Gall (65). Three larger, short, straight rods occurring in chains, which appeared to be important and were nonsporeforming obligate anaerobes producing butyric, acetic and lactic acids were also isolated* Although these methods have provided valuable information on certain aspects of the rumen microflora and rumen activity, a complete study of the rumen population can not be made by standard bacteriological procedures. Some organisms require special techniques, and the many morphological types of organisms in the rumen make such studies muoh more complex. This has led to a search for the subsequent development of additional techniques, two of which are the use of the artificial rumen, and the use of fistulated animals. In vitro techniques: artificial rumen. — The artificial rumen as an in vitro technique is a method of providing, as nearly as possible, conditions similar to those existing in the rumen. The optimal conditions as established and recognized by most workers are: temperature of 39 to 40° C, pH of 6.0 to 7.2 and anaerobic conditions using carbon dioxide or nitrogen gas. This method of studying rumen activity offers a quick and relatively inexpensive means of obtaining information.

PAGE 15

As early as 1884 Tappeiner (cited by Burroughs and Hall, 26) incubated rumen contents from cattle in closed jars at room temperature, and observed that crude fiber decreased after a few days. In later work he incubated rumen contents in vitro at 37° C. and observed after 14 days of incubation that 23 per cent of the crude fiber disappeared. When chloroform was added before incubation, cellulose decomposition did not take place. Wegner et al . (105) added urea to rumen contents and after incubation at 37° C. determined inorganic nitrogen measuring in this way the conversion to protein. English workers (91, 92) collected rumen ingesta, strained it through muslin and incubated the filtrate at 39° C. in a water bath for 2 to 4 hours under one atmosphere of air. This procedure was used to study the effects of pH, type of carbohydrate present, temperature, gaseous environment, and urea concentration on the activity of rumen organisms. Both of these methods were criticised for the lack of movement or diffusion of the end products as in the live animal. In 1948, Marston (82) reported the use of a technique which consisted of a closed jar containing the rumen contents. The jar was equipped with a mechanical stirrer for movement of the ingesta and a gas inlet and outlet system to maintain an anaerobic condition. McNaught (84) and McNaught et al . (85) modified this technique by

PAGE 16

centrifuging the strained rumen samples at 2,000 r.p.m. for five minutes. One per cent maltose and 0.05 per cent urea were added and the no nprotein nitrogen determined. The sample was incubated for four hours at 39° C. and the non-protein nitrogen determinations were made again. A rather complicated technique has been suggested by Louw (78) which consisted of a glass jar suspended in a water bath at a controlled temperature. A growth medium was added to the jar and suspended in the growth medium was a Visking casing through which the different by-products of fermentation could be dialyzed. The glass was provided with several inlets and outlets for the gassing, sampling and for the addition of buffers. The material was kept in constant motion by use of a stirrer. Huhtanen et al . (66) described a miniature artificial rumen technique for studying fiber digestion by rumen bacteria. The technique is similar to the artificial rumen devised by Louw (78) except that it is smaller, and the frequent neutralization is eliminated. An artificial rumen known as the permeable type, has also been described (46). It consists of a cellophane sack containing the inoculum and substrate immersed in the medium. The reaction takes place within the sack through which fermentation products may diffuse. South African workers (62) have determined cellulose digestion by suspending cotton threads in incubated rumen

PAGE 17

fluid. The rate of disintegration is used as a measure of cellulolytic activity. Burroughs et al . (23, 24, 25, 31, 32, 33) developed an artificial rumen which consisted of 500 ml. or 1,000 ml. size fermentation flasks incubated in a water bath at 40° C. Each flask was fitted with a three hole rubber stopper for the gassing with carbon dioxide and for the regulation of the pH of the contents. Fermentations were carried out continuously for 36"hour periods. The inoculum for the first fermentation was used directly from the rumen, but for the successive periods the inoculum consisted of half of the residue of the preceding fermentation. Cellulose digestion was determined chemically and gram stains were made at the beginning and end of each fermentation period. Recently a method to improve the inoculation was developed by workers in Ohio (13, 14, 16, 17). Strained rumen liquid was centrifuged at high speeds and the sediment containing the microorganisms was suspended in an isotonic phosphate buffer solution containing 0.05 per cent cysteine. The culture medium was composed of cellulose, urea, minerals and glucose. The fermentations were carried out in liter low actinic Erlenmeyer flasks which were arranged in a water bath with the temperature controlled at 38 to 39° C. Flasks were gassed individually with carbon dioxide. The length of fermentation periods ranged from 24 to 48 hours.

PAGE 18

10 At approximately the same time, Cheng et al . (34) developed a technique based on the use of a washed suspension of rumen microorganisms for the study of dellulose digestion in vitro . The strained rumen contents were centrifuged at high speeds and the sediment was washed several times with distilled water or a phosphate buffer. The final sediment was suspended in a complex nutrient solution and the pH adjusted to 7. This was followed by fermentation for 24 hours at 39° C. and under anaerobic conditions provided by carbon dioxide. The use of washed cell suspensions makes it possible to use an inoculum which is more nearly free of nutrients. The in vitro techniques have been criticised because a shift in the microflora may occur and the inflow of saliva and the movement of ingesta and end-products of fermentation in . vivo may not be duplicated. Although these criticisms may be valid, the use of the artificial rumen in many experiments has provided much useful information. In , vivo techniques . — The wide variety and number of in vivo studies related to rumen digestion, which include feeding and digestion trials and experiments with fistulated animals, will not be reviewed here. Studies which have been particularly valuable have made use of the rumen fistula (40, 99). This device makes possible an easier collection of rumen contents from animals on a known

PAGE 19

11 dietary regime. The use of fistulated animals offers a means of studying the chemical, physical and microbial changes that occur within the rumen and also offers a means of forced feeding materials which otherwise are difficult to get into the rumen. Effect of Various Dietary Factors Upon Cellulose Digestion Results of the numerous experiments concerning rumen activity have demonstrated that many factors are capable of effecting a change in rumen activity. Among those which have been studied are dietary intakes of minerals, carbohydrates, fats, protein, non-protein nitrogen, antibiotics, hormones and certain unidentified factors. The determination of these factors and their control which may be used to provide optimum conditions for proper rumen function have been the goals of research workers. Many of these factors which are known to alter microbial digestion in the rumen do not operate independently. As a rule all of these factors or conditions should be expected to have an interaction or interrelationships. Effect of minerals on rumen activity . — At the present time the importance of the minerals in the nutrition of the ruminant is well known. Diseases have been reported from many areas as a result of deficiencies in minerals. Not

PAGE 20

12 only may a lack of an element cause a disease, but also a slight excess of certain metals in the diet of ruminants may lead to abnormalities. Perhaps some of the best-known examples are diseases caused by excesses of molybdenum and selenium. In some cases, elements that are essential may be toxic when present in excess; examples are copper and cobalt. Of all the studies of the effect of minerals in rumen nutrition, only a few have been made relative to the effect of these elements on the rumen, and rumen bacteria. It has been shown that alfalfa ash, water extracts of alfalfa, yeast, dried distiller's solubles, mineral mixtures, molasses ash, autoclaved water extract of cow manure, autoclaved rumen liquid all increased the digestion of cellulose (15, 16, 29, 32, 25, 72). To sic and Mitchell (103) considered that cobalt may be more necessary for the rumen microorganisms than for the ruminant. In cobalt deficient sheep the efficiency of digestion of nitrogen free extract and ether extract was reduced but digestion of fiber was not reduced (6). The number of bacteria present in rumen contents from normal sheep is almost double that of cobalt deficient sheep (50). Klosterman et al . (72) found that an addition of a trace mineral mixture to a ration of soybean oil meal, corn and cob meal, and poor quality hay resulted in a marked

PAGE 21

13 increase in liveweight gains of cattle. The trace mineral mixture used in these studies consisted of cobalt, copper, manganese, iron, iodine, and sulfur. In further work (71), they confirmed the above results and found that molasses was of no apparent benefit when added to a ration which included trace minerals or good quality mixed hay. Other workers have reported unfavorable results from feeding trace minerals. Plumlee et al . (93) found that twin beef calves receiving a ration largely composed of corn cobs ate less and grew at a slower rate when trace minerals were added. Tillman et al . (102) and Gossett and Riggs (52) reported that neither alfalfa ash nor a complete mineral mixture improved the digestibility of low quality prairie hay. By using in vitro techniques, it has been found that additions of molasses ash, phosphorus and iron stimulated cellulose digestion. The statement has also been made that factors other than iron, phosphorus, sodium, potassium, calcium, magnesium, chlorine and sulfur are involved in rumen physiology (32). McNaught et al . (85), using the diminution of non protein nitrogen in rumen liquid when incubated in vitro as an index of bacterial growth, without actually counting the bacteria, found that the microorganisms were able to tolerate 100 p. p.m. iron, 10 p. p.m. copper, somewhat less

PAGE 22

14 than 10 p. p.m. cobalt and between 100 and 1,000 p. p.m. molybdenum. Growth of the bacteria was definitely inhibited by 1,000 p. p.m. iron, 25 p. p.m. copper, 1,000 p. p.m. cobalt and 2,000 p. p.m. molybdenum. Salsbury et al . (95) reported that cellulose digestion by rumen microorganisms in vitro was slightly depressed by 12 p. p.m. of added cobalt and strongly depressed by 100 p. p.m. Rubidium, cesium and lithium have not been found to be effective in stimulating greater cellulose digestion when ample potassium and sodium were present in the medium (63) . Effect of carbohydrates and fats on rumen activity.— It appears that the effect of carbohydrate and fat upon cellulose digestion is closely related to the other nutrients present. The amount of crude fiber digested in the rumen has been reported to range between 70 to 85 per cent (53, 54). This percentage is affected by all other ingredients in the ration (80) . Small amounts of readily available carbohydrates have been shown to increase the amount of cellulose digested (1, 83, 35). Hoflund et al . (62) found that the concentration of glucose for optimum cellulose digestion in vitro varied between 0.1 and 0.2 per cent. In vivo work (44) showed that the addition of 2 pounds of molasses to a grain and alfalfa hay ration fed to dairy cows, apparently increased the digestibility of crude protein, cellulose and crude fiber.

PAGE 23

15 When large amounts of readily available carbohydrate are included in the ration, a decrease in crude fiber digestion is obtained (1, 12, 30, 44, 56, 62, 68, 77, 80). This is due to the preference of the microorganisms in the rumen for soluble carbohydrate (56). In vivo work (44) showed that additions of 4 pounds of molasses to a timothylespedeza hay mixture for dairy cows apparently decreased the digestibility of crude protein, cellulose and crude fiber, Work involving the bacterial population as affected by carbohydrate has been reported by Bortree et al . (18). They observed that the addition of 3 pounds of sugar to a hay ration resulted in a 100 per cent increase in the bacterial population over that of hay alone. When starch was used at the same rate, only a slight increase in bacterial population was obtained. Little work has been done on the effect of fats upon rumen microorganisms. Swift et al . (100) fed rations containing fat levels varying from 3 to 10 per cent. Corn oil replaced cerelose on a caloric basis. Utilization of protein and energy was similar in all trials reported. The addition of 10 mg. corn oil to a 25 ml. artificial rumen increased the breakdown of cellulose, while levels of 20 to 80 mg. resulted in a decrease (20). Willev et al . (108) reported that adding 5 per cent cottonseed oil to a practical fattening ration for steers resulted in a more efficient

PAGE 24

16 feed utilization. Indications that valeric and caproic acids and to a lesser extent iso-butyric and iso-valeric acids increased the rate of cellulose digestion and ammonia utilization in vitro has been shovm by Bent ley et al . (14, 16). Effect of nitrogenous compounds on rumen activity. — For a number of years the utilization of nitrogenous compounds by the ruminants has been of interest to the research worker not only from the point of view of protein synthesis but for their effect on other activities performed by the microflora in the rumen, particularly cellulose digestion. One of the early reports of the use of non-protein nitrogen for ruminants was that published by Hart et al. (58) in 1939. Using urea and ammonium carbonate in a ration for dairy heifers, they obtained results which were interpreted to mean that nitrogen from these compounds was converted by the microorganisms into microbial protein. Since the time of this report, many other experiments have confirmed this conclusion (24, 105, 106, 107). Several reports have appeared in the literature showing that cattle can use no nprotein nitrogenous compounds among which are: ammoniated beet pulp, ammoniated cane molasses, ammoniated condensed distillers molasses solubles, urea, ammoniated industrial by-products and other ammoniated

PAGE 25

17 feedstuffs (38, 45, 73, 74, 79, 87, 101). Nitrogen of numerous organic and inorganic ammonium salts has also been shown, by , J,n v^ro work, to serve as sources of nitrogen for the rumen bacteria (10) , One of the conditions necessary for efficient utilization of non-protein nitrogenous compounds by the ruminant is the use of a low protein ration. If large amounts of natural protein are present in the ration, little conversion of non-protein nitrogen into protein will take place (106). Several reports have shown that nitrogenous compounds affect the rate of cellulose digestion (11, 27, 28, 30, 57) . The addition of urea to a diet containing 0.136 per cent of nitrogen, increased the cellulose digestion from 17.8 per cent to 38.7 per cent (57). Burroughs and Gerlaugh (28) studied the effect of soybean oil meal upon corn cob and timothy hay digestion in a fattening type ration for steers. They found an average increase of about 15 per cent in dry matter digestion when soybean oil meal was added. In later work (30) the addition of skimmilk to a diet containing corn cobs fed to steers increased roughage digestion, but only when 4 pounds of starch in the diet were present. Similar results were obtained when casein was added to a diet low in protein (27) . Indications that urea was more effective than soybean, linseed, cottonseed and corn gluten meal in

PAGE 26

18 stimulating cellulose digestion has been shown by the in vitro technique used by Belasco (11). Effect of antibiotics, hormones, and v itamins on rumen activity . — The wide use of antibiotics in some livestock rations has promoted interest in studies of the effect of antibiotics upon rumen function. These substances in certain cases and under determined conditions have increased the amount of cellulose digested, but as a rule a decrease has occurred. Y/asserman et al . (104) reported that penicillin, streptomycin, and neomycin stimulated the cellulolytic activity of rumen microorganisms; Chloromycetin adversely affected the activity. Chlortetracycline (Aureomycin) has been shown to depress the activity of rumen microorganisms and cellulose digestion (75, 76). Prescott (94^ has shown that terramycin, chlotetraoycline, bacitracin and penicillin reduced the utilization of non-protein nitrogen by the rumen bacteria. Brooks et al . (20) observed that the addition of 10 to 20 meg. of diethyl stilbestrol to a 25 ml. artificial rumen increased the digestion of cellulose an average of 9 per cent. Riboflavin, vitamin B 12 # biotin, PABA, pyridoxine, folic acid and a mixture of B vitamins have been reported to increase cellulose digestion in vitro (13, 55).

PAGE 27

19 The literature relating to other aspects of ruminant nutrition has been excellently reviewed by others. The reader is referred to papers published by Doetsch and Robinson (39), Edwards (41), Elsden and Phillipson (42), Goss (51), Hastings (59), Huffman (64), Marston (81) and Owen (90) .

PAGE 28

EXPERIMENTAL PROCEDURE Two types of purified cellulose were fermented in an artificial rumen under controlled conditions to determine the effect of varying concentrations of certain minerals upon the extent of cellulose digestion. Samples of rumen contents were obtained from a fistulated steer and from animals slaughtered at a local packing house. Rumen contents were filtered to remove solid materials, and the filtrate mixed with cellulose and a nutrient solution for fermentation in the artificial rumen. The extent of cellulose digestion was determined by chemical analysis for cellulose before and after fermentation and by feeding trials with rats and chicks. Preparation of Artificial Rumen Inoculum Rumen contents were obtained from a fistulated, 2-year-old Jersey steer. With this steer the effect of timothy and pangolagrass hay on cellulose digestion was studied. The hay was fed once a day about 8:00 A.M. (sufficient for adlibitum feeding). An interval of 10 days occurred between the change from the timothy to the pangolagrass hay before rumen contents were obtained again. In addition to the hay, the steer was fed daily 5 lbs. of a concentrate mixture composed of: 200 lbs. of cottonseed meal (41 per cent protein), 20

PAGE 29

21 200 lbs. crimped oats, 200 lbs. dried citrus pulp, 400 lbs. ground snapped corn, 10 lbs. salt, and 5 lbs. defluorinated rock phosphate. Fresh water and additional defluorinated rock phosphate and salt were available to the animal. A plastic plug described by Hentsahel _e_t_kl. (61) was used in the rumen fistula. When securing the rumen samples the contents were withdrawn in early afternoon, usually before the concentrate was fed. Samples of rumen contents were also obtained from 24 mature beef type steers, slaughtered at a local abattoir. The rumen contents were removed within 5 to 10 minutes after the animals were slaughtered. These steers had been raised on pasture and received a variety of diets. The effect of these rations on cellulose digestion was compared to the studies with the fistulated steer. The ingesta from all steers wa3 strained through 4 layers of cheesecloth to remove the undigested solid materials, Three hundred milliliters of the filtrate were added to a 1 liter Erlenmeyer flask that contained 6 gm. of a purified cellulose, 100 ml. of distilled water and 200 ml. of a nutrient media recorded in Table 1. The nutrient media contained urea and many inorganic salts. As an element was investigated, it was deleted from this media. Two different sources of purified cellulose, known as Solka-Floc^ and ^•Solka-Floc BW-40, Brown Co., Berlin, N. H.

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22 TABLE 1 NUTRIENT SOLUTIONS USED IN THE ARTIFICIAL RUMEN Constituent

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23 9 Alphacel, were used and compared in these studies. Before the rumen filtrate was added, the flask and other contents had been warmed in a constant temperature water bath at 40° C. for 15 minutes. Immediately after the filtrate was added, carbon dioxide was bubbled into the mixture for 5 minutes. Aliquots were taken and pH determined. The pH of the mixture was adjusted to 7.2 with 4N Na 2 C<^ when necessary. The rumen inoculum was then ready to study the influence of various minerals on the digestion of cellulose. Preparation of Nutrient Solutions In Table 1 are shown 2 nutrient solutions containing urea and various salts. Solution A was used in the studies of the effect of cobalt, copper, iron, manganese, molybdenum and ainc upon artificial rumen digestion of cellulose. Solution B was used to study the efficiency of the artificial rumen in digesting cellulose for use by monogastric animals. In the use of nutrient solution A for the study of the particular element of interest, the element was deleted from the nutrient media, but added in various concentrations to the artificial rumen inoculum as described below. The elements studied were prepared in sufficient concentrations so that 1 ml. added to 24 ml. of rumen inoculum contributed the following oo ^vj 2Nutriti °nal Biochemical s Corporation, Cleveland *8, Ohio.

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24 concentrations of each element: Cobalt (Co30 4 .7H 2 0): 0, 1, 5, 10, 25, 50, 100 and 250 p. p.m. Copper (Cu30 4 .5H 2 0): 0, 1, 5, 10, 20, 40, 60, 80 and 100 p. p.m. Iron (Fe30 4 .7H 2 0): 0, 5, 10, 50, 100, 250 and 500 p. p.m. Manganese (Mn30 4 .H 2 0): 0, 1, 5, 10, 50, 100 and 250 p. p.m. Molybdenum (Na 2 Mo0 4 .2H 2 0): 0, 1, 5, 10, 25, 50, 100 and 250 p. p.m. Zinc (ZnS0 4 .7H 2 0): 0, 1, 5, 10, 25, 50, 100 and 250 p. p.m. All the salts used were reagent grade. An additional experiment was run using sodium sulphate (Na 2 S0 4 ), at a concentration giving 500 p. p.m. of sodium and 1045 p. p.m. of sulfate in order to test the effect of the sulfate and sodium ions. In Table 2, data are given for the concentration of the above elements that were found in the rumen filtrate of some steers used in these studies. All the samples were digested using perchloric and nitric acid in the micro Kjeldahl apparatus. Copper and zinc were determined using zincon (83); cobalt using nitroso-R salt (96), iron using Ortho-phenanthroline (96), manganese using periodate (96) and molybdenum using thiocyanatestannous chloride (96).

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25 TABLE 2 CONCENTRATION OF ELEMENTS IN THE RUMEN FILTRATE OF STEERS FED PRACTICAL DIETS BjIKfc P.P.M. Cobalt 0.06 * 0.02 a Copper 0.36+0.13 Iron 2.55 ± 0.51 Manganese 1.90+0.41 Molybdenum 0.076 + 0.02 Zinc 1.17 + 0.39 a Mean values and standard deviation. Determination of Effect of Minerals on Cellulose Digestion by Rumen Inoculum To a 75 ml. Pyrex test tube was added 1 ml. of a solution that contained the particular concentration of the element being studied, and 24 ml. of the rumen inoculum containing the substrate, nutrient solution and rumen filtrate. These tubes were fitted with two-hole rubber stoppers and glass tubing used as an inlet and outlet for carbon dioxide. Carbon dioxide was continuously bubbled through the mixture to maintain anaerobic conditions and to provide agitation. Eighteen to 24 fermentation tubes were connected in series by the use of short pieces of rubber tubing. In all tests, two samples were retained for initial

PAGE 34

26 cellulose determination. The remaining tubes were incubated in a water bath at 40° C. for 24 hours, after which time the pH was determined and 1 ml. of IN ^££4 added to stop fermentation. The digested samples were kept cold in a refrigerator at 38° F. until cellulose was determined, using the acetic-nitric acid procedure of Crampton and Maynard (37). This procedure was slightly modified and applied as follows: the samples, after digestion, were transferred to 50 ml. centrifuge tubes, and centrifuged for 1 minute at approximately 2,000 r.p.m. or 1,100 times gravity. The supernatant solution was decanted and 12 ml. of glacial acetic acid and 1.5 ml. of concentrated nitric acid were added to each residue. The tubes were then heated in a boiling water bath for 20 minutes and the contents filtered while hot through Gooch crucibles. The residue was transferred from the tubes and washed with 95 per cent ethyl alcohol, dried in an oven at 80° C, cooled in a dessicator and weighed, ashed in a muffle furnace at 675° C, cooled and weighed. The differences in weight between the acid-alcohol extracted dry residue and the ash were taken as the weight of cellulose present in the sample.

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27 Use of Rats and Chicks to Determine the Extent of Cellulose Fermented in the Artificial Rumen In this study, rumen contents were obtained only from the fistulated steer. The same procedure of preparing the rumen inoculum was followed as above except that larger quantities were used and nutrient solution B of Table 1 was used. To a 6-liter Florence flask were added 200 grams of Alphacel, 1 liter of nutrient solution (Table 1), 3 liters of distilled water and the mixture warmed for 15 minutes in a water bath at 40° C. Then 1 liter of rumen filtrate was added, carbon dioxide bulfoled through, pH adjusted to 7.2 with 4 N Na 2 C0 3 if needed, and fermented for 24 hours. A mechanical stirrer was used throughout this period. The fermented material was placed in a shallow porcelain pan, dried at 80° C. for 36 hours and ground through a Wiley mill. This material was used in rat and chick diets to evaluate the extent of the artificial rumen digestion. Aliquot samples of the material were also analyzed for moisture, ash, crude fiber, crude protein, ether extract and nitrogen free extract. Results of the analyses are recorded in Table 3. The methods used in the analyses were those recommended by the Association of Official Agricultural Chemists (86) .

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28 TABLE 3 CHEMICAL ANALYSIS OF PURIFIED CELLULOSE (ALPHACEL) AND THE RESIDUE AFTER FERMENTATION IN THE ARTIFICIAL RUMEN Unfermented Fermented (%) (%) Moisture 7.81 ± 0.73 a 11.63 + 0.82 a Ash 0.10 * 0.02 8.42 ± 0.85 Crude Fiber 57.72 + 1.92 49.96 ± 0.82 Crude Protein 0.00 + 0.00 4.38 + 0.90 Ether Extract 0.17 + 0.09 0.55 + 0.23 Nitrogen Free Extract 34.20+2.52 25.06+1.76 ^Iean values and standard deviation. Feeding trials vith rats.— One hundred and twenty weanling rats of the LongEvans strain, from the Nutrition Laboratory stock colony were used in these experiments. They were housed in groups of 3 or 4 in galvanized metal wire cages, For the first trial, 60 rats, ranging in age from 23 to 25 days were randomized according to weight and sex into 3 treatments of 20 rats each (10 males and 10 females) . They were fed the 3 diets of Table 4. The dietary groups received 4 per cent Alphacel (control), 15 per cent Alphacel (diet 2) and 15 per cent Alphacel fermented in the artificial rumen (diet 3) . The rats of diets 2 and 3 were fed the same weight of feed as eaten ad libitum by the control group. Fresh water was provided from glass watering bottles. Growth was determined from weekly weights made throughout the

PAGE 37

TABLE 4 COMPOSITION OF DIETS FED TO RATS Ingredient

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30 experimental period. The experiment was repeated at a later time using the same number of rats and dietary treatments. The results of chemical analysis (86) of the 3 diets for moisture, ash, crude fiber, crude protein, ether extract, and nitrogen free extract are recorded in Table 5. feeding trial with clicks . — One hundred and twenty unsexed chicks (Cornish x White Plymouth Rock corssbreed) one day old, were obtained from a local commercial hatchery at two different periods. Initially, 60 chicks were randomized according to weight into 3 treatments of 20 chicks each. A commercial temperature-controlled battery was used to house each dietary group during the 4-week feeding period. The composition of the diets is given in Table 6. The 3 dietary groups received 15 per cent sucrose (control), 15 per cent Alphacel (diet 2) and 15 per cent Alphacel fermented in the artificial rumen (diet 3) . The diets were fed ad libitum and weekly records of feed consumption were taken. Fresh water was provided at all times. Growth was determined by weekly weights. The experiment was repeated at a later time using the same number of chicks and dietary treatments. The results of chemical analysis of the 3 diets for moisture, ash, crude fiber, crude protein, ether extract and nitrogen free extract are recorded in Table 7.

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31 TABLE 6 COMPOSITION OF DIETS FED TO CHICKS Ingredients

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32 TABLE 7 CHEMICAL ANALYSIS OF DIETS FED TO CHICKS Control Diet 2 Diet 3 (%) (%) (%) Moisture

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RESULTS AND DISCUSSION Effect of Trace Minerals on Cellulose Digestion Using the Artificial Rumen In Tables 8 through 25 the data and statistical analysis summaries are presented that were obtained in the study of the influence of various levels of cobalt, copper, iron, manganese, molybdenum, and zinc on the digestion of cellulose by microorganisms in the artificial rumen. Two forms of purified cellulose, SolkaFloe and Alphacel, were used in each mineral study. Rumen filtrate from animals consuming timothy hay, pangolagrass hay and pasture was used in the study of each element and in the study of the two types of cellulose. The values tabulated are the means of four to six observations in each case. The data were evaluated for significance by analysis of variance (F test) and by Duncan's multiple range test. When comparing some of the results obtained with in vivo work, the assumption was made that an animal weighing 1,000 lbs. has a rumen capacity of 20 gallons. The results recorded in these tables represent changes caused by the addition of certain trace minerals. The trace elements, present in the rumen inoculum (Table 2), were transferred with the filtrate to the fermentation 33

PAGE 42

34 mixture. Thus, the rumen inoculum contained small amounts of trace minerals contributed by the rumen filtrate plus additional known amounts of the element being studied. Cobalt .— In Tables 8, 9, and 10 are presented the data obtained in the study of cobalt. The average percentages of cellulose digested when 0, 1, 5, 10, 25, 50, 100, and 250 p. p.m. of cobalt were added, were approximately 79, 79, 73, 68, 46, 28, 16, and 6 respectively. The addition of 1 p. p.m. of cobalt had no influence; but as increasing levels of the element were added, each addition resulted in a significant depression (P less than 0.01), in the amount of the cellulose digested. Salsbury et al . (95) who worked with Solka-Floc as the source of cellulose and a dialyzing membrane* type artificial rumen found that 12 p. p.m. of cobalt depressed, and 100 p. p.m. of cobalt significantly decreased cellulose digestion. McNaught et al. (85) reported that 10 and 100 p. p.m. of cobalt in an artificial rumen resulted in a slight and tremendous decrease respectively in the utilization of non-protein nitrogen. In feeding experiments Becker et al (9), observed no toxic effects when cattle received 0.5 grams of cobalt per 1,000 lbs. of body weight daily. This amount of cobalt, if given at one time, would give a maximum concentration of approximately 6 p. p.m. in the rumen, calculated for a 1,000-lb. animal having a rumen capacity

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35 TABLE 8 EFFECT OF COBALT UPON DIGESTION OF TV/O TYPES OF PURIFIED CELLULOSE USING THE ARTIFICIAL RUMEN AND INOCULUM FROM ANIMALS FED DIFFERENT FORAGES Cobalt added, p. p.m.

PAGE 44

36 TABLE 9 SUMMARY OF ANALYSIS OF VARIANCE OF DATA IN TABLE 8 Source of Variation DF MS Total 47 Substrates 1 11.22 Inoculum 2 7.20 Error (a) 2 80.45 Levels 7 5,258.36** Source x Levels 14 0.98 Error (b) 21 2.14 Highly significant, P less than 0.01. TABLE 10 RESULTS OBTAINED WITH DIFFERENT LEVELS OF COBALT USING THE DUNCAN'S MULTIPLE RANGE TEST 3 Levels, added p. p.m. 250 100 50 25 10 5 1 Means, per cent Cellulose digested 5.96 16.21 27.70 46.36 68.46 73.41 78.71 78.85 a Any two means not underscored by the same line are significantly different (P less than 0.01). Any two means underscored by the same line are not significantly different,

PAGE 45

37 of 20 gallons. Cornell workers (7, 8), in a series of studies investigating blood changes in sheep, found that as much as 0.16 gm. of cobalt per 100 lbs. of body weight daily, caused severely depressed appetite and losses in body weight. From the results of this investigation, the probability of depressing normal bacterial action in the rumen by therapeutic treatment with quite large doses of cobalt seems very remote. Copper . --The data obtained in the study of this element are presented in Tables 11, 12, and 13. The average percentages of cellulose digested when 0, 1, 5, 10, 20, 40, 60, 80, and 100 p. p.m. of copper were added, were approximately 78, 78, 71, 58, 17, 9, 7, 6, and 5 respectively. The addition of 1 p. p.m. of copper had no influence, but with increasing levels of copper the cellulose digested decreased until at 20 p. p.m., only about 22 per cent as much cellulose was digested as at or 1 p. p.m. of added copper. These data are similar to those obtained by McNaught et al . (85), who reported that 10 and 25 p. p.m. of copper in an artificial rumen resulted in a slight and tremendous decrease respectively in the utilization on non-protein nitrogen. Copper has been demonstrated to be toxic to cattle. Kidder (70) fed 1 gm. of anhydrous copper sulfate per 100 lbs. of body weight to a steer pasturing on Saint Augustine grass and it died after 122 days. Henson (60) fed two

PAGE 46

38 TABLE 11 EFFECT OF COPPER UPON DIGESTION OF TrfO TYPES OF PURIFIED CELLULOSE USING THE ARTIFICIAL RUMEN AND INOCULUM FROM ANIMALS FED DIFFERENT FORAGES Copper

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39 TABLE 12 SUMMARY OF ANALYSIS OF VARIANCE OF DATA IN TABLE 11 Source of Variation

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40 steers in dry lot an equivalent amount of anhydrous copper sulfate. One died after 91 days with characteristic symptoms similar to those observed by Kidder; the other steer failed to develop any toxic symptoms. A maximum concentration of approximately 45 p. p.m. of copper occurred in the rumen contents of these steers, assuming that they had a rumen volume of 20 gallons. The in vitro studies reported in this investigation suggest that copper is toxic in that it decreases cellulose digestion. However, if copper was present in the rumen at levels comparable to those used in the artificial rumen, harmful effects other than an effect upon cellulose digestion may be expected. Iron . --The data obtained in the study of the effect of iron on cellulose digestion are presented in Tables 14, 15, and 16. These data indicate that iron has very little effect on cellulose digestion; however, the statistical analysis shows that the observed decrease of approximately 5 per cent, at the levels of ICC to 500 p. p.m. of iron, was significant (P less than C.01). McNaught et al . (85) reported that a level of 1,000 p.p.m. of iron in the artificial rumen significantly decreased nonprotein nitrogen utilization. In practical feeding of iron salts to cattle no reports of toxicity symptoms have appeared in the literature.

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41 TABLE 14 EFFECT OF IRON UPON DIGESTION OF WO TYPES OF PURIFIED CELLULOSE USING THE ARTIFICIAL RUMEN AND INOCULUM FROM ANIMALS FED DIFFERENT FORAGES Iron

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42 TABLE 15 SUMMARY OF ANALYSI3 OF VARIANCE OF DATA IN TABLE 14 Source of Variation DF MS Total 41 • Substrate 1 19.34 Inoculum 2 34.48 Error (a) 2 41.47 Levels 6 41.20** Source x Level 12 0.99 Error (b) 18 0.49 **Highly significant, P less than 0.01. TABLE 16 RESULTS OBTAINED WITH DIFFERENT LEVELS OF IRON USING THE DUNCAN'S MULTIPLE RANGE TEST* Levels, added p. p.m. 500 ' 250 100 50 5 10 Means, percent Cellulose digested 72.41 72.48 73.75 77.60 77.70 77.71 77.85 a Any two means not underscored by the same line are significantly different (P less than 0.01). Any two means underscored by the same line are not significantly different

PAGE 51

43 Manganese .-In Tables 17, 18, and 19 the data are recorded for results of the study with manganese. These observations show that 1 p. p.m. of manganese Increased cellulose digestion by 2 per cent and 100 p. p.m. decreased it by 2 per cent. These changes were statistically significant. In comparing the effect of manganese with the effect of similar concentrations of cobalt, copper and zinc, manganese has very little practical effect. This is the first report of the effect of manganese on the digestion of cellulose by the artificial rumen. No report of toxicity of manganese in cattle feeding has appeared in the literature. Mol vbdenum . The data obtained in the study of the effect of molybdenum on cellulose digestion by the artificial rumen are presented in Tables 20, 21, and 22. The average percentages of cellulose digestion with 0, 5, 10, 20, 40, 60, 80, and 100 p. p.m. of added molybdenum were approximately 77, 76, 75, 74, 71, 70, 68, and 66 respectively. The decreases observed at the 20 p. p.m. and greater concentrations of molybdenum were statistically significant. However, the effect of comparable concentrations of cobalt, copper and isinc were much greater. McNaught et al . (85) reported that, in the artificial rumen using the utilization of non-protein nitrogen as an index, rumen bacteria would tolerate from 100 to 1,000 p. p.m. of molybdenum, but the utilization of

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44 TABLE 17 EFFECT OF MANGANESE UPON DIGESTION OF TWO TYPES OF PURIFIED CELLULOSE USING THE ARTIFICIAL RUMEN AND INOCULUM FROM ANIMALS FED DIFFERENT FORAGES Manganese

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45 TABLE 18 SUMMARY OF ANALYSIS OF VARIANCE OF DATA IN TABLE 17 Source of Variation DF MS Total 41 Substrate 1 29.17 Inoculum 2 49.32 Error (a) 2 S.66 Levels 6 5.70** Source x Level 12 0.44 Error (b) 18 0.41 Highly significant, P less than 0.01. TABLE 19 RESULTS OBTAINED WITH DIFFERENT LEVELS OF MANGANESE USING THE DUNCAN'S MULTIPLE RANGE TEST a Levels, added p.p.m. 100 250 50 10 5 1 Means, per cent Cellulose digested 75.36 75.88 76.05 76.50 76.53 77.25 78.31 a Any two means not underscored by the same line are significantly different (P less than 0.01). Any two means underscored by the same line are not significantly different.

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46 TABLE 20 EFFECT OF MOLYBDENUM UPON DIGESTION OF TWO TYPES OF PURIFIED CELLULOSE USING THE ARTIFICIAL RUMEN AND INOCULUM FROM ANIMALS FED DIFFERENT FORAGES Molybdenum added p.p. in.

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47 TABLE 21 SUMMARY OF ANALYSIS OF VARIANCE OF DATA IN TABLE 20 Source of Variation DF MS Total 47 Substrate 1 5.81 Inoculum 2 672.67* Pangola vs. Pasture 1 4.28 2 (Timothy) vs. Pangola and Pasture 1 1340.26* Error (a) 2 34.69 Levels 7 98.09** Source x Level 14 8.18** Error (b) 21 0.75 Significant, P less than 0.05. **Highly significant, P less than 0.01. TABLE 22 RESULTS OBTAINED WITH DIFFERENT LEVELS OF MOLYBDENUM USING THE DUNCAN'S MULTIPLE RANGE TEST* Levels, added p. p.m. loo 80 60 40 20 10 5 Means, per cent Cellulose digested 65.65 68.01 69.60 71.41 73.58 75.51 75.90 76.70 a Any two means not underscored by the same line are significantly different (P less than 0.01). Any two means underscored by the same line are not significantly different,

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48 non-protein nitrogen was significantly decreased at a level of 2,000 p. p.m. of molybdenum. The results presented for molybdenum suggest that the "teartness" disease caused by a molybdenum toxicity is not caused by an interference with the activity of the rumen bacteria. The disease occurs in cattle grazing pastures, in which the molybdenum content of the forage is 100 p. p.m. or less in the dry matter. This would be expected to give a concentration of less than 10 p. p.m. in the rumen contents. Zinc . — Data in Tables 23, 24, and 25 represent results of trials using various levels of sine in the study of cellulose digestion by the artificial rumen. The average percentages of cellulose digested when the zinc concentrations were 0, 1, 5, 10, 25, 50, 100, and 250 p. p.m. were approximately 79, 79, 79, 75, 60, 32, 7, and 4 per cent respectively. The addition of 1 and 5 p. p.m. of zinc had no influence, but the 50 p. p.m. level decreased the digestion to approximately 41 per cent of that obtained when no zinc was added. At 100 p. p.m. only about 10 per cent of the normal fermentation took place. This is the first report of the effect of zinc on the digestion of cellulose by the artificial rumen. Skipper (97) fed a concentrate containing 2,000 p. p.m. of zinc to young dairy calves from one through 27 weeks of age and observed no influence on growth or in several blood

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49 TABLE 23 EFFECT OF ZINC UPON DIGESTION OF WO TYPES OF PURIFIED CELLULOSE USING THE ARTIFICIAL RUMEN AND INOCULUM FROM ANIMALS FED DIFFERENT FORAGES Z inc added

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50 TABLE 24 SUMMARY OF ANALYSIS OF VARIANCE OF DATA IN TABLE 23 Source of Variation DF MS Total 47 Substrate 1 14.08 Inoculum 2 62.82 -Error (a) 2 6.69 Levels 7 1,479.5 * Source x Level 14 6.12 Error (b) 21 4.35 Highly significant, P less than 0.01. TABLE 25 RESULTS OBTAINED WITH DIFFERENT LEVELS OF ZINC USING THE DUNCAN'S MULTIPLE RANGE TEST 3 Levels, p. p.m. 250 100 50 25 10 5 1 Means, per cent Cellulose digested 4.33 7.10 32.5 60.0 75.5 78.8 79.2 79.5 a Any two means not underscored by the same line are significantly different (P less than 0.01). Any two means underscored by the same line are not significantly different,

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51 constituents. The daily intake of the concentrate was increased from 1 to 4 lbs. during the experimental period. Possibly, the reason for the absence of any ill effects was that the rumen had not yet developed at the beginning of the feeding program and the bacteria may have become adjusted to the zinc by the time the rumen developed. However, the level of zinc fed should have given a concentration of zinc in the range of 100 p. p.m., which is very high. Sodium sulfate . — Since the cobalt, copper, iron, manganese, molydenum and zinc ions were added in the above artificial rumen studies in the form of the sulfate or sodium salts, it was considered necessary to evaluate sodium and sulfate ions for possible effects. To do this, tests were made in which 500 p. p.m. of sodium and 1,045 p. p.m. sulfate ions as I^SO^ were added to each articial rumen digestion tube. Eight determinations were made on both the purified Solka-Floc and Alphacel cellulose, using the timothy, pangola and pasture rumen inoculum. These high levels of sodium and sulfate ions had no effect on the extent of cellulose digestion. Therefore, the effect of cobalt, copper, iron, manganese, molybdenum and zinc, were due to the elements themselves. Effect of tvoe of cellulose and source of rumen jnoculum upon cellulose digestion . The Solka-Floc and Alphacel were digested to the same extent in the presence of

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52 the various minerals and rumen filtrates. The filtrates from the 3 different sources of forage were not essentially different in their effect upon cellulose digestion. In one case, Table 20, filtrate from the period of timothy hay consumption caused the digestion of less cellulose than the other sources of rumen filtrate. The reason for the decreased digestion in this trial is unknown. At the end of each digestion period the pH values were recorded and observed to fall in the range of 6.0 to 6.5. These figures are all within the normal range existing in the rumen, pH 6.0 to 7.2. Feeding Value for Rats and Chicks of Cellulose Following Fermentation in the Artificial Rumen Results of the feeding trials designed to study the feeding value for rats and chicks of cellulose following fermentation are recorded in Tables 26 through 30. A test of the feeding value of fermented cellulose appeared desirable because of the potential application of this procedure in improving rations high in crude fiber for monogastric animals. A statistical analysis of the growth data indicated that fermentation of cellulose in the artificial rumen had no beneficial influence on the rate of growth of the rats and chicks. However, in all cases with male and female rats, as well as the chicks, the fermented cellulose produced

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53 TABLE 26 EFFECT OF CELLULOSE FERMENTED IN THE ARTIFICIAL RUMEN UPON THE GROWTH RATE OF MALE RATS TriaTT Weeks on Diet Weights in GramF Control

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54 TABLE 27 EFFECT OF CELLULOSE FERMENTED IN THE ARTIFICIAL RUMEN UPON THE GROWTH RATE OF FEMALE RATS

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55 TABLE 28 EFFECT OF CELLULOSE FERMENTED IN THE ARTIFICIAL RUMEN UPON THE GROWTH RATE OF CHICKS

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56 TABLE 29 SUMMARY OF ANALYSIS OF VARIANCE OF THE GROWTH DATA OF RATS Source of Variation

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57 better gains. The chicks responded slightly better than the rats. The growth of the rats and chicks consuming the control diets was significantly greater (P less than 0.01) than growth of rats and chicks consuming the experimental diets. The increased growth of chicks and rats consuming the control diet probably resulted from the greater proportion of available energy in the control diets. The appetites and health of both the chicks and rats appeared normal throughout the feeding periods. No mortality occurred in the rats during the 2 six-week experimental periods. In Table 28 it is indicated that 7 chicks died during the tests. In all cases they died within the first 3 days of feeding and it appeared that death was due to causes other than the diets. Muhrer et al. (89) fed a ruminant type ration, following fermentation in an artificial rumen, to a limited number of pigs. The results indicated that such a ration could be improved by fermenting some ingredients of the ration. Comparing these results with the data above, it is indicated that the artificial rumen could be used to improve diets high in cellulose for monogastric animals, but modification of the technique is necessary to make possible more satisfactory results. Such modification could involve a longer fermentation period, increase or decrease the

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58 amount of rumen filtrate, better control of the pH, and possibly other factors. The pH values recorded after the 24-hour fermentation periods were in the range of 5.3 and 5.7, and probably within this range the low pH was harmful to the cellulolytic activity of the rumen bacteria.

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SUMMARY An artificial rumen was used to study the effect of cobalt, copper, iron, manganese, molybdenum and zinc on the digestion of two kinds of purified cellulose in the presence of rumen inoculum from animals consuming different roughages. The amount of cellulose digested was determined chemically and the feeding value of fermented cellulose was determined by feeding trials with rats and chicks. Conclusions which may be made from the experiments are summarized in the following statements. 1. The addition of 1 p. p.m. of cobalt had no influence upon the amount of cellulose digested, but as increasing levels of the element were added, each addition resulted in a significant depression. Ten p. p.m. slightly depressed and 100 p. p.m. strongly depressed cellulose digestion. 2. One p. p.m. of copper added to the substrate and reaction medium in the artificial rumen had no influence in the amount of cellulose digested, but with increasing levels the cellulose digested decreased greatly, until at 20 p. p.m., only 22 per cent as much cellulose was digested as at or 1 p. p.m. of added copper. 59

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60 3. The addition of 1 and 5 p. p.m. of zinc had no influence upon the amount of cellulose digested, but the 50 p. p.m. level decreased the digestion to about 41 per cent of that obtained when no zinc was added. At 100 p. p.m. only about 10 per cent of the normal fermentation took place. 4. Twenty p. p.m. and greater concentrations of molybdenum significantly decreased cellulose digestion. Levels of 5 and 10 p. p.m. of molybdenum had no influence. 5. The addition of iron up to 500 p. p.m. and manganese up to 250 p. p.m. slightly decreased cellulose digestion, but the decrease was much less than that caused by above elements. 6. The addition of sodium and sulfate ions had no influence upon the amount of cellulose digested. 7. There was no difference in the amount of cellulose digested when Solka-Floc or Alphacel were used as substrates. Three different sources of rumen inoculum did not affect the rate of cellulose digestion. 8. No significant beneficial effects were obtained as measured by growth when rats and chicks were fed a purified source of cellulose fermented in an artificial rumen for 24 hours.

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LITERATURE CITED 1. Arias, C, V7. Burroughs, P. Gerlaugh and R. M. Bethke. 1951. The influence of different amounts and sources of energy upon in vitro urea utilization by rumen microorganisms. Journal of Animal Science 1 0. 683-692. 2. Baker, F. 1942. Normal rumen microflora and microfauna of cattle. Nature H9, 220. 3. Baker, F. 1943. Direct microscopical observations upon the rumen population of the ox. I. Qualitative characteristics of the rumen population. Annals of Applied Biology 10, 230-239. 4. Baker, F. and S. T. Harris. 1947. The role of the microflora of the alimentary tract of hervibora with special reference to ruminants. 2. Microbial digestion in the rumen (and caecum) with special reference to the decomposition of structural cellulose. Nutrition Abstracts and Reviews 12, 3-12. 5. Bechdel, S. I., PI. E. Honeywell, R. A. Ditcher and LI. H. Knutsen. 1928. Synthesis of vitamin B in the rumen of the cow. Journal of Biological Chemistry 8,0 . 231-238. 6. Becker, D. E. and S. E. Smith. 1949. The metabolism of cobalt in lambs. Journal of Animal Science 8, 615. 7. Becker, D. E. and S. E. Smith. 1951. The level of cobalt tolerance in yearling sheep. Journal of Animal Science IP., 266-271. 8. Becker, D. E. and S. E. Smith. 1951. The metabolism of cobalt in lambs. Journal of Nutrition 13, 87-100. 9. Becker, R. B., •. M. Neal, P. T. D. Arnold and L. L. Rusoff . 1938. Annual Report Florida Agricultural Experiment Station, pp. 62-63. 61

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62 10. Belasco, I. J. 1954. New nitrogen feed compounds for ruminants. A laboratory evaluation. Journal of Animal Science 13, 601-610. 11. Belasco, I. J. 1954. Comparison of urea and protein meals as nitrogen sources for rumen microorganisms: urea utilization and cellulose digestion. Journal of Animal Science 13., 739-747. 12. Belasco, I. J. 1956. The role of carbohydrates in urea utilization, cellulose digestion and fatty acid formation, Journal of Animal Science IS, 496-508. 13. Bentley, 0. G., R. R. Johnson, S. Vanecko and C. H. Hunt. 1954. Studies on factors needed by rumen microorganisms for cellulose digestion in vitro . Journal of Animal Science l£, 581-593. 14. Bentley, 0. G., R. R. Johnson, T. V. Hershberger, J. H. Cline and A. L. Moxon. 1955. Cellulolytic factor activity of certain short chain fatty acids for rumen microorganisms in vitro . Journal of Nutrition 52, 389-400. 15. Bentley, 0. G., A. Latona, P. DePaul and C. H. Hunt. 1951. Factors affecting the digestibility of cellulose of poor quality hay. Journal of Animal Science 10, 1038. 16. Bentley, 0. G., A. Lehmkuhl, R. R. Johnson and A. L. Moxon. 1954. The stimulatory effect of certain fatty acids on i,n vitro cellulose digestion by rumen microorganisms. Journal of Animal Science 13 . 1015. 17. Bentley, 0. G., S. Vanecko, C. H. Hunt and A. L. Moxon. 1953. Nutritional requirements of rumen microorganisms for cellulose digestion in vitro . Journal of Animal Science 12, 908. 18. Bortree, A. L., K. M. Dunn, R. E. Ely and C. F. Huffman. 1946. A preliminary report on the study of factors influencing rumen microflora. Journal of Dairy Science 2£, 542-543. 19. Bortree, A. L., C. K. Smith, B. C. Sarkar and C. F. Huffman. 1948. Types and numbers of microorganisms in the rumen contents of cattle being fed natural and semisynthetic rations. Journal of Animal Science 1, 520-521.

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63 20. Brooks, C. C, G. B. Garner, M. E. Muhrer and W. H. Pfander. 1953. The, effect of fat and diethyl stilbestrol on cellulose digestion by sheep rumen microorganisms. Journal of Animal Science 12, 909. 21. Bryant, M. P. 1951. Some characteristics of the different bacteria present in the rumen of cattle on a constant ration. Journal of Animal Science 10 . 1042. 22. Bryant, M. P. and L. A. Burkey. 1953. Cultural methods and some characteristics of some of the more numerous groups of bacteria in the bovine rumen. Journal of Dairy Science J6, 205-217. 23. Burroughs, •., C. Arias, P. Gerlaugh and R. M. Bethke. 1950. The use of an artificial rumen in studying urea utilization by microorganisms taken from the paunch of cattle. Journal of Animal Science J[, 650-651. 24. Burroughs, W., C. Arias, P. DePaul, P. Gerlaugh and R. M. Bethke. 1951. In yitro observations upon the nature of protein influences upon urea utilization by rumen microorganisms. Journal of Animal Science ifi, 672-682. 25. Burroughs, W., N. A. Frank, P. Gerlaugh, and R. M. Bethke. 1950. Preliminary observations upon factors influencing cellulose digestion by rumen microorganisms. Journal of Nutrition JO, 9-24. 26. Burroughs, W. and G. Hall. 1954. Principles and theory of rumen function and rumen nutrition. Proceedings SemiAnnual Meeting Nutrition Council. Published by American Feed Manufacturers Association, Chicago, Illinois. 27. Burroughs, W., L. S. Gall, P. Gerlaugh and R. M. Bethke. 1950. The influence of casein upon roughage digestion in cattle with rumen bacteriological studies. Journal of Animal Science 9, 214-220. 28. Burroughs, •., and P. Gerlaugh. 1949. The influence of soybean oil meal upon roughage digestion in cattle. Journal of Animal Science 8, 3-8.

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64 29. Burroughs, W., P. Gerlaugh and R. M. Bethke. 1948. Influence of alfalfa ash and water extract of alfalfa upon roughage digestion in cattle. Journal of Animal Science J7, 522. 30. Burroughs, •., P. Gerlaugh, B. H. Edgington and R. M. Bethke. 1949. The influence of corn starch upon roughage digestion in cattle. Journal of Animal Science 8, 271-278. 31. Burroughs, W., H. G. Headley, R. M. Bethke and P. Gerlaugh. 1950. Cellulose digestion in good and poor quality roughages using an artificial rumen. Journal of Animal Science 9, 513-522. 32. Burroughs, W., A. Latona, P. DePaul, P. Gerlaugh and R. M. Bethke. 1951. Mineral influences upon urea utilization and cellulose digestion by rumen microorganisms using the artificial rumen technique. Journal of Animal Science 10, 693-705. 33. Burroughs, W., J. Long, P. Gerlaugh and R. M. Bethke. 1950. Cellulose digestion by rumen microorganisms as influenced by cereal grains and protein rich feeds commonly fed to cattle using an artificial rumen. Journal of Animal Science 9, 523-530. 34. Cheng, E. •., G. Hall and •. Burroughs. 1955. A method for the study of cellulose digestion by washed suspensions of rumen microorganisms. Journal of Dairy Science 18, 1225-1230. 35. Clark, R. and J. I. Quinn. 1951. Studies on the alimentary tract of the Merino sheep in South Africa. The effect of supplementing poor quality grass hay with molasses and nitrogenous salts. Onderstepoort Journal of Veterinary Research J25, 93-103. 36. Cochran, W. G. and G. M. Cox. 1950. Experimental Designs. John Wiley and Sons, Inc., New York, New York. 37. Crampton, E. W. and L. A. Maynard. 1938. The relation of cellulose and lignin content to the nutritive value of animal feeds. Journal of Nutrition 15 . 383-395.

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65 38. Davis, R. F., R. H. ?7asserman, J. K. Loosli and C. H. Crippin. 1955. Studies on the availability of nitrogen from various ammoniated products for rumen bacteria and dairy cattle. Journal of Dairy Science J38, 677-687. 39. Doetsch, R. N. and R. Q. Robinson. 1953. The bacteriology of the bovine rumen: A review. Journal of Dairy Science 16_, 115-142. 40. Dougherty, R. W. 1955. Permanent stomach and intestinal fistulas in ruminants, some modifications and specifications. Cornell Veterinarian _4j>, 331-357. 41. Edwards, D. C. 1955. Review of the progress of dairy science. Physiology of dairy cattle. The biochemistry and microbiology of the rumen. Journal of Dairy Research J22, 232-247. 42. Elsden, S. R. and A. T. Phillipson. 1948. Ruminant digestion. Annual Review of Biochemistry 17 . 705-726. 43. Federer, \I. T. 1955. Experimental Designs. (Theory and Application). The Macliillan Co. New York, New York. 44. Foreman, C. F. and H. A. Herman. 1953. Effects of carbohydrate feeding levels on roughage digestion in dairy cattle. Missouri Agricultural Experiment Station, Research Bulletin, 535. 45. Frye, Jr., J. B., B. T. Parham, B. L. Kilpatrick and L. L. Rusoff. 1954. A comparison of cottonseed meal, urea, and ammoniated molasses (327<>) in the ration of dairy heifers. Journal of Dairy Science £Z, 657. 46. Gall, L. S. and W. L. Claws. 1951. The bacteriology of the artificial rumen. Bacteriological Proceedings 1951 . 20-21. 47. Gall, L. S. and C. N. Huhtanen. 1951. Criteria for judging a true rumen organisms and a description of five rumen bacteria. Journal of Dairy Science 34, 353-362.

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66 48. Gall, L. S., C. N. Stark and J. K. Loosli. 1947. The isolation and preliminary study of some physiological characteristics of the predominating flora from the rumen of cattle and sheep. Journal of Dairy Science 30, 891-899. 49. Gall, L. S., •. Burroughs, P. Gerlaugh and B. H. Edgington. 1949. Rumen bacteria in cattle and sheep on practical farm rations. Journal of Animal Science 8, 441-449. 50. Gall, L. S., S. E. Smith, C. N. Stark, D. E. Becker and J. K. Loosli. 1949. Rumen bacteria in cobalt deficient sheep. Science 109 . 468-469. 51. Goss, H. 1943. Some peculiarities of ruminant nutrition. Nutrition Abstracts and Reviews 12, 531-538. 52. Gossett, J. W. and J. K. Riggs. 1956. The effect of feeding dehydrated alfalfa leaf meal and trace minerals to growing beef calves fed poor quality prairie hay. Journal of Animal Science .15, 840845. 53. Gray, F. V. 1947. The digestion of cellulose by sheep. The extent of cellulose digestion at successive levels of the alimentary tract. Journal of Experimental Biology .24., 15-19. 54. Hale, E. B., C. W. Duncan and C. F. Huffman. 1940. Rumen digestion in the bovine with some observations on the digestibility of alfalfa hay. Journal of Dairy Science .23, 953-967. 55. Hall, G., E. T ,7. Cheng and W. Burroughs. 1955. B vitamins stimulatory to cellulose digestion by washed suspensions of rumen microorganisms. Proceedings of the Iowa Academy of Science .62, 273-278. 56. Hamilton, T. S. 1942. The effect of added glucose upon the digestibility of protein and fiber in rations for sheep. Journal of Nutrition .23, 101-110. 57. Harris, L. E. and H. H. Mitchell. 1941. The value of urea in the synthesis of protein in the paunch of the ruminant. Maintenance. Journal of Nutrition 22, 167-182.

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67 58. Hart, E. B., G. Bohstedt, H. J. Deobald and M. I. Wegner. 1939. The utilization of simple nitrogenous compounds such as urea and ammonium carbonate bygrowing calves. Journal of Dairy Science 22, 785-798. 59. Hastings, E. G. 1944. The significance of the bacteria and the protozoa of the rumen of the bovine. Bacteriological Reviews 8, 235-254. 60. Henson, Jr., J. N. 1949. Interelationship of copper and molybdenum upon phosphorus metabolism and alkaline blood phosphatase levels in cattle. Master of Science thesis, University of Florida. 61. Hentschel, A. F., R. N. Berry and C. F. Huffman. 1954. A plastia plug for use in the bovine rumen fistula. Michigan State College Veterinarian li, 77-81. 62. Hoflund, S., J. F. Quinn and R. Clark. 1948. Studies on the alimentary tract of Merino sheep in South Africa. The influence of different factors on the rate of cellulose digestion (a) in the rumen and (b) in the ruminal ingesta studied in vitro . Orders tepoort Journal of Veterinary Science and Animal Industry 23., 395-409. 63. Hubbert, Jr., F., E. W. Cheng and W. Burroughs. 1956. Effect of cesium, lithium and rubidium on cellulose digestion by rumen microorganisms. Journal of Animal Science 15, 1246. 64. Huffman, C. F. 1953. Ruminant Nutrition. Annual Review of Biochemistry 22, 3S9-422. 65. Huhtanen, C. N., and L. S. Gall. 1953. Rumen organisms. I. Curved rods and a related rod type. Journal of Bacteriology j35, 548-553. 66. Huhtanen, C. N., R. K. Saunders and L. S. Gall. 1954. Fiber digestion using the miniature artificial rumen. Journal of Dairy Science .37, 328-335. 67. Hungate, R. E. 1950. The anaerobic mesophilic cellulolytic bacteria. Bacteriological Reviews 14, 1-49. 68. Hunt, C. H., 0. G. Bentley, T. V. Hershberg and J. H. Cline. 1954. The effect of carbohydrates and sulfur on B vitamins synthesis, cellulose digestion, and urea utilization by rumen microorganisms in vitro . Journal of Animal Science 13, 570-580.

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68 69. Johns, A. T. 1951. Isolation of a bacterium, producing propionic acid from the rumen of sheep. Journal of General Microbiology j>, 317-325. 70. Kidder, R. W. 1949. Symptons of induced copper toxicity in a steer. Journal of Animal Science &, 623-624. 71. Klosterman, E. W., 0. G. Bentley, A. L. Moxon and L. E. Kunkle. 1956. Relationships between level of protein, molasses, trace minerals and quality of hay in ration for fattening cattle. Journal of Animal Science 15, 456-463. 72. Klosterman, E. W., L. E. Kunkle, 0. G. Bentley, and W. Burroughs. 1953. Supplements to poor quality hay for fattening cattle. Ohio Agricultural Experiment Station, Research Bulletin 732. 73. Knodt, C. B., J. B. Williams and J. Brumbaugh. 1950. Ammoniated cane molasses in the rations of dairy calves. Journal of Animal Science 9, 661-662. 74. Knodt, C. B., J. B. Williams and J. Brumbaugh. 1951. Ammoniated cane molasses and similar products in the rations of dairy calves. Journal of Dairy Science 34, 1042-1046. 75. Lambert, M. R. and I* L. Jacobson. 1956. The effect of chlortetracycline feeding on in vitro cellulose digestion by rumen microorganisms. Journal of Animal Science 15., 509-514. 76. Lodge, J. R., J. T. Miles, N. L. Jacobson and L. Y. Quinn. 1956. Influence of aureomycin on in vitro cellulose digestion by bovine rumen microorganisms. Journal of Dairy Science 19, 303-311. 77. Louw, J. G., S. I. Boderstein and J. L. Quinn. 1948. The digestibility, for sheep, of the cellulose in a poor hay, as affected by supplements of a mixture of concentrates and green feed. Onderstepoort Journal of Veterinary Science and Animal Industry .23, 239-259. 78. Louw, J. G., H. W. Williams and L. A. Maynard. 1949. A new method for the study in vitro of rumen digestion. Science 110, 478-480. 79. Magruder, N. D., C. D. Knodt and P. S. Williams. 1953. Ammoniated industrial by-products in dairy heifer rations. Journal of Agricultural and Food Chemistry 1, 944-946.

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69 80. Mangold, E. 1934. The digestion and utilization of crude fiber. Nutrition Abstract and Reviews 3, 647-656. 81. Marston, H. R. 1939. Ruminant nutrition. Annual Review of Biochemistry 8, 557-578. 82. Marston, H. R. 1948. The fermentation of cellulose in vitro by organisms from the rumen of sheep. Biochemical Journal 42., 564-574. 83. McCall, J. T. 1956. A method for the determination of copper and zinc in animal tissues. Master of Science thesis, University of Florida. 84. McNaught, M. L. 1951. The utilization of non-protein nitrogen in the bovine rumen. 7. A qualitative and quantitative study of the breakdown of carbohydrate which accompanies protein formation in bovine rumen contents during in vitro incubation. Biochemical Journal 19, 325-332. 85. McNaught, M. L., E. C. Owen and J. A. B. Smith. 1950. The utilization of nonprotein nitrogen in the bovine rumen. 6. The effect of metals on the activity of the rumen bacteria. Biochemical Journal 46, 36-48. 86. Methods of Analysis of the Association of Official Agricultural Chemists. 1950. 7th. ed. Published by the Association of Official Agricultural Chemists. Washington, D. C. 87. Millar, H. C. 1944. Ammoniated sugar-beet pulp as a new nitrogenous feed for ruminants. Journal of Dairy Science 27, 225-241. 88. Moir, R. J. and M. J. Masson. 1952. An illustrated scheme for the microscopic identification of the rumen microorganisms of sheep. Journal of Pathology and Bacteriology 64, 343-850. 89. Muhrer, M. E., G. B. Garner and W. H. Pfander. 1953. Improving a swine ration containing roughage and urea by fermenting with rumen microorganisms. Journal of Animal Science 12, 926. 90. Owen, E. C. 1954. Physiology and biochemistry of rumination. Journal of Dairy Research J21, 408-453.

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70 91. Pearson, R. M. and J. A. B. Smith. 1943. The utilization of urea in the bovine. 2. Conversion of urea for ammonia. Biochemical Journal j37, 148-152. 92. Pearson, R. M. and J. A. B. Smith. 1943. The utilization of urea in the bovine. 3. The synthesis and breakdown of protein in rumen ingesta. Biochemical Journal IZr 153-164. 93. Plumlee, M. P., R. Totusek and W. M. Beeson. 1953. The effect of adding trace minerals to rations of identical twin beef cattle. Journal of Animal Science 12, 928. 94. Prescott, J. M. 1953. Effects of diet and antibiotics on utilization of non-protein nitrogen. Rumen microorganisms. Journal of Agricultural and Food Chemistry 1, 894-896. 95. Salsbury, R. L., C. K. Smith and C. F. Huffman. 1956. The effect of high levels of cobalt on the in vitro digestion of cellulose by rumen microorganisms. Journal of Animal Science 15., 863-868. 96. Sandell, E. B. 1950. Colorimetric determination of trace metals. III. Inter science Publishers, Inc., New York, New York. 97. Skipper, F. H. 1951. The effect of varying levels of copper, molybdenum, and zinc in the diet upon the growth and certain blood constituents of young dairy calves. Master of Science thesis, University of Florida. 98. Snedecor, G. W. 1946. Statistical methods. 4th. ed. The Iowa State College Press. Ames, Iowa. 99. Stoddard, G. E., N. N. Allen, •. H. Hale, A. L. Pope, D. K. Sorensen and W. R. Winchester. 1951. A permanent rumen fistula cannula for cows and sheep. Journal of Animal Science JJ3, 417-423. 100. Swift, R. W., J. W. Bratzler, W. H. James, A. D. Tillman and D. C. Meek. 1948. The effect of dietary fat on utilization of the energy and protein of rations by sheep. Journal of Animal Science 7, 475-485. 101. Tillman, A. D. and R. W. Swift. 1953. The utilization of ammoniated industrial by-products and urea by sheep. Journal of Animal Science 12, 201-212.

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71 102. Tillman, A. D. # C. F. Chapel, R. J. Sirny and R. MacVicar. 1954. The effect of alfalfa ash upon the digestibility of prairie hay by sheep. Journal of Animal Science IS, 417-424. 103. To sic, J. and R. L. Mitchell. 1948. Concentration of cobalt by microorganisms and its relation to cobalt deficiency in sheep. Nature 162 . 502-504. 104. Wasserman, R. H. # C. •. Duncan, E. S. Churchill and C. F. Huffman. 1952. The effect of antibiotics on in vitro cellulose digestion by rumen microorganisms. Journal of Dairy Science .35, 571-580. 105. Yfegnar, M. I., A. N. Booth, G. Bohstedt and E. B. Hart. 1940. The in vitro conversion of inorganic nitrogen to protein by microorganisms from cows rumen. Journal of Dairy Science 13., 1123-1129. 106. Wegner, M. I., A. N. Booth, G. Bohstedt and E. B. Hart. 1941. Preliminary observations on chemical changes of rumen ingesta with and without urea. Journal of Dairy Science 24, 51-56. 107. Wegner, M. I., A. N. Booth, G. Bohstedt and E. B. Hart. 1941. The utilization of urea by ruminants as influenced by the level of protein in the ration. Journal of Dairy Science 24., 835-844. 108. V/illey, N. B., J. Z. Riggs, R. If. Colby, 0. D. Butler and R. Reiser. 1952. The influence of fat and energy in the ration upon feedlot performance and carcass composition of fattening steers. Journal of Animal Science JL1, 705-711.

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BIOGRAPHICAL SKETCH Juan Carlos Sala was born March 27, 1930, at Lima, Peru. He was graduated from La Salle School of Lima, Peru, in December, 1946. He entered the Escuela Nacional de Agricultura of Lima in April, 1948. In November, 1950, he received a scholarship from the University of Florida, and transferred to this school in February 1951, obtaining the degree of Bachelor of Science in Agriculture with high honors in February, 1952. He received the Master of Science degree in Agriculture in August, 1953, from the University of Florida. Following his graduation in 1953, Mr. Sala returned to Lima, Peru and worked for two years with Nicolini Hnos. S.A. as a Nutrition Adviser. He was granted a leave of absence in September, 1955, and re-entered the University of Florida for predoctoral studies. During his graduate studies he held a Research Assistantship in the Department of Animal Husbandry and Nutrition. He is a candidate for the degree of Doctor of Philosophy in August, 1957. He is a member of Gamma Sigma Delta, Sigma Xi, Phi Kappa Phi, the American Society of Animal Production and the American Dairy Science Association. 72

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This dissertation was prepared under the direction of the chairman of the candidate's supervisory committee and has been approved by all members of that committee. It was submitted to the Dean of the College of Agriculture and to the Graduate Council, and was approved as partial fulfillment of the requirements for the degree of Doctor of Philosophy. August 10, 1957 Dean, College of Agriculture Dean, Graduate School SUPERVISORY COMMITTEE: Chairman A> X< bliA^<$b~ ^, )£vu«J)(L

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