EFFECT OF ROUGHAGE LEVEL IN THE RATION ON PRODUCTION
OF SHORT CHAIN ACIDS AND SULFUR AMINO ACIDS
IN RUMEN FERMENTATION

Uy
Roy S. Emery

A THESIS
Subm itted to th e School of G raduate S tu d ie s of
M ichigan S ta te U n iv e rsity o f A g ric u ltu re and Applied S cience
in p a r t i a l f u lf illm e n t o f th e req u irem en ts
f o r th e degree of
DOCTOR OF PHILOSOPHY

Department of D airy
1955

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ACKNOWLEDGEMENTS

The au th o r e x p re sse s h is s in c e re a p p re c ia tio n to D r. C.
P. Huffman, D airy D epartm ent, and D r. C. K. Sm ith, Department
o f M icrobiology and P u b lic H ealth , f o r t h e i r guidance and
a s s is ta n c e throughout t h i s work and f o r t h e i r c r i t i c a l read in g
of t h i s m an u scrip t.
G ra titu d e i s ex p ressed to D r. L. F. W o lterin k , Department
o f Physiology and Pharmacology, whose s tim u la tin g le c t u r e s and
su g g e stio n s g r e a tly c o n trib u te d to th e i n i t i a t i o n and p ro g re ss
o f t h i s in v e s tig a tio n .
Thanks are extended to Dr. R. L. S alsb u ry and D r. G. M.
Ward f o r t h e i r h e lp fu l c r itic is m s and aid in v a rio u s phases
o f th e in v e s tig a tio n .

The w r ite r i s in d eb ted to Mr. C. W.

F i f i e l d and Miss E sth er M. Smith f o r a id w ith th e m icro ra d io ­
au to g rap h s.
The w r ite r w ishes e s p e c ia lly to acknowledge h is g r a titu d e
and in d eb ted n ess to h is w ife , R uth, whose s a c r i f i c e s and en­
couragement made t h i s study p o s s ib le .

ii

EFFECT OF ROUGHAGE LEVEL IN THE RATION ON PRODUCTION
OF SHORT CHAIN ACIDS AND SULFUR AMINO ACIDS
IN RUMEN FERMENTATION

by
Roy S. Emery

AN ABSTRACT
Subm itted to th e School of Graduate S tudied of1
M ichigan S ta te U n iv e rsity o f A g ric u ltu re and Applied S cience
in p a r t i a l f u lf illm e n t of th e req u irem en ts
fo r th e degree of

DOCTOR OF PHILOSOPHY

Department of D airy
Year

Approved

C.^h.

1955

f-3 «
Roy S. Emery

Rumen l iq u id from 5 bovines (re c e iv in g hay alone or 75%
of th e t o t a l d ig e s tib le n u tr ie n ts as c o n c e n tra te ) was in cu b ated
w ith s u b s tr a te and s3 5 -la b e le d in o rg a n ic s u l f a t e .

Two t r i a l s

used th e r a t i o n of th e donor as s u b s tr a te and two used a sub­
s t r a t e of mixed a l f a l f a meal and c o n c e n tra te f o r b o th hay
in o c u la and g ra in in o c u la .

In each t r i a l , a s e p a ra te ferm en­

t a t i o n f la s k was in cu b ated f o r each time o f 0 . 5, 1 , 1 . 5, 2 ,
2 .5 , and 3 h o u rs.

Each ferm ent was f r a c tio n a te d in to p ro te in

s u l f u r , f r e e o rg an ic s u lf u r and in o rg a n ic s u l f a t e .

The p ro ­

t e i n and f r e e o rg an ic s u lf u r f r a c tio n s were f u r th e r se p a ra te d
by chrom atography.
to ta l a c tiv ity .

G lu tath io n e accounted fo r 2 to k% of th e

The mean p a r t i t i o n of in c o rp o ra te d a c t i v i t y

in a t o t a l o f bk f la s k s f o r a l l tim es on b o th tre a tm e n ts was
62 % e y s t( e ) in e and 385? m ethionine p lu s an u n id e n tif ie d f r a c ­

tio n .

E x p o n en tial e q u a tio n s f i t t e d to th e t o t a l in c o rp o ra tio n s

f o r each com bination of s u b s tra te and inoculum are p re se n te d .
The c o n c e n tra tio n o f a c e tic , p ro p io n ic , and b u ty ric a c id s
in 3 bovine rumens was determ ined a t v a rio u s tim es a f t e r fe e d ­
ing e i t h e r an a l l hay r a t i o n or one c o n s is tin g la r g e ly o f con­
c e n tr a t e s .

Two t r i a l s were conducted on each r a t i o n .

The

c o n c e n tra tio n o f each a c id f i r s t in c re a se d and th e n d ecreased
w ith tim e a f t e r fe e d in g .
tu rn o v e r c u rv e s.

The v alu es o b tain ed were f i t t e d to

A cetic a c id disappeared from th e rumen a t

th e r a t e of 3 . 3^ /h o u r and b u ty ric ac id a t th e r a t e of 2.7%/
hour.

The sta n d a rd e r r o r was 0 .3 $ .

The d isap p earan ce r a t e

o f p ro p io n ic a c id on th e high c o n c e n tra te r a ti o n appeared to
iv

Roy S. Emery

be ^ .6 # /h o u r v e rsu s 3 .2 # /h o u r on th e hay r a ti o n .

The r a t e s

f o r co n v ersio n o f fe e d to sh o rt ch ain a c id s ranged from 35
to 8H $/hour.
P ooling d a ta from th e h t r i a l s , th e amounts o f a c id s per
100 gm. o f rumen liq u id which were produced from one pound of
t o t a l d ig e s tib le n u tr i e n t s w ere, 0 .2 to 0 .67 a c e tic , 0 .02 to
0 .2 6 p ro p io n ic , and O.OU to 0.21 b u ty r ic .
co n fid en ce le v e l ra n g e s.

These a re th e 95#

The average rumen in th e s e t r i a l s

was e stim a te d to c o n ta in 70 Kilograms o f li q u id .

Using th e se

v a lu e s , i t was computed t h a t th e cow o b ta in s 3 to 13# of i t s
energy from s h o rt ch ain a c id s .

v

TABLE OF CONTENTS

Page
INTRODUCTION

1

REVIEW OF LITERATURE . ................. . ..............................................

2

In flu e n c e o f C o n cen trate on Rumen D ig e stio n and
Passage .........................................................................................
2
In flu e n c e o f C o n cen trate on th e Rumen M icrobiota . . .
6
Rumen P ro d u ctio n o f S hort Chain Acids . . . . . . . .
9
A bsorption and U t i l iz a t i o n o f S hort Chain Acids
. . . 19
Rumen S y n th e sis o f C arbohydrate ........................................... 27
Rumen S y n th e sis of P r o t e i n ...................................................... 30
PART I . INCORPORATION OF INORGANIC SULFATE BY RUMEN
MICROBIOTA IN V IT R O ..........................................

38

P re lim in a ry Experim ent
........................................................ 38
F erm en tatio n Technique ............................................................ 38
Hydrogen S u lfid e P ro duction ............................................... Hi
G lu tath io n e P r o d u c tio n ................................................................ k2
M icroradioautographs .......................... . . . . . . . . .
W3
P r in c ip a l E x p e rim e n t.........................................................................W5
P r o c e d u r e ..........................................................................................*+5
R e s u l t s .............................
50
PART I I . PRODUCTION AND ABSORPTION OF ACETIC, PROPIONIC,
AND BUTYRIC ACIDS IN THE BOVINE RUMEN................................... 56
P r o c e d u r e .......................................................................
56
R e s u lts
...............................................................................................59
DISCUSSION . . .

..................................................................................6»+

SUMMARY.......................................................................................................70
LITERATURE CITED...................................................................................... 73
APPENDIX....................................................................................................... 89

vi

INTRODUCTION
Ruminants have long been known f o r t h e i r a b i l i t y to
u t i l i z e roughage.

With th e advent of in te n s iv e anim al p ro ­

d u c tio n and in c re a se d c o n c e n tra te fe e d in g , anom alies have
been noted in rumen fe rm e n ta tio n .

These in c lu d e changes in

th e p ro d u ctio n of sh o rt chain a c id s which are a major source
of energy f o r th e cow a s w ell as an end product of th e f e r ­
m en tatio n .

Thus, in fo rm atio n concerning amounts and ty p es

of th e se a c id s i s im p erativ e to a b e t t e r u n d erstan d in g of
feed u t i l i z a t i o n .
Large amounts of p ro te in a re sy n th esized in th e rumen.
S ince m ethionine appears to be a lim itin g f a c to r in t h i s
s y n th e s is , a study of th e form ation of s u lf u r amino a c id s
might w e ll lead to in c re a se d p ro d u c tio n by ru m in an ts.
The purpose of t h i s study i s to determ ine th e q u a n tity
of s h o rt ch ain a c id s and s u lfu r amino a c id s made a v a ila b le to
th e rum inant as a f f e c te d by th e r a t i o of hay to c o n c e n tra te
in th e r a t i o n .

1

REVIEW OF LITERATURE

The re c e n t review s on rum inant n u t r i t i o n by Huffman
(1953) and Owen (195*0 e s ta b lis h th e im portance of m ic ro b ia l
d ig e s tio n f o r econom ical roughage u t i l i z a t i o n and f o r anim al
p ro d u c tio n .

D oetsch and Robinson (1953) review ed th e b a c te ­

rio lo g y o f th e rumen w h ile J a r r ig e (1953) gave p a r t i c u l a r
a t t e n t i o n to th e d e g ra d a tio n of carb o h y d rates and th e r e s u ltin g
p ro d u c ts.

The l i t e r a t u r e d iscu ssed here p e r ta in s to th e dynam­

i c s of rumen fe rm e n ta tio n as in flu e n c e d by th e r a ti o n and
p o rtra y e d by th e p ro d u c tio n of s h o rt ch ain ac id s and th e syn­
th e s i s o f p r o te in .
In flu e n c e o f C o n cen trate on Rumen D ig e stio n and Passage
The fundam ental problem of rum inant n u t r i t i o n i s to
enhance th e n a tiv e a b i l i t y of c a t t l e to u t i l i z e co arse feed
(Huffman, 1953)*

According to P h illip s o n (1953)» carb o h y d rates

can e i t h e r enhance o r d ep ress roughage u t i l i z a t i o n depending
on t h e i r q u a n tity and q u a lity .

J a r r ig e (1953) l i s t s re s e a rc h

over f i f t y y e a rs d e a lin g w ith th e in flu e n c e of so lu b le carb o ­
h y d ra te on roughage u t i l i z a t i o n .
S w ift e t a l . (19*+7) re p o rte d a 5 to 10$ d e p re ssio n of
crude f i b e r d i g e s t i b i l i t y when corn s ta rc h or sugar formed
some 8 to 15$ of a r a t i o n based on equal p a r ts of mixed hay
and co rn .

This could not be confirm ed by Dowe e t a l . (1955)

3
who fe d over 80$ corn in th e r a ti o n but no pure s ta rc h .
However, Burroughs ejt a l . (19*+9) o b tain ed a 35$ d e p re ssio n
of o rg an ic m atter d i g e s t i b i l i t y w ith a r a tio n of corncobs,
skim m ilk and 30$ s ta rc h but only a
s ta r c h le v e l was reduced to 18$.

d e p re ssio n when th e

S im ila r d ata were o b tain ed

w ith a r a ti o n o f s ta r c h , a l f a l f a hay, and corncobs.

According

to Louw and Van d er Wath (19*+3), supplem entation of a hay
r a ti o n w ith corn a t th e 8 to 33$ le v e l depressed c e llu lo s e
d ig e s tib ility .
A wood m eal, stu d ie d by K o istin en (19^8), which was 50$
d i g e s t i b le when fe d w ith hay or lim ite d c o n c e n tra te , became
v i r t u a l l y in d ig e s tib le when fed w ith s ila g e .

B alch and John­

son ( 1950) found th a t c o tto n th re a d suspended in th e rumen
was a tta c k e d s e v e ra l tim es f a s t e r on an a l l hay r a ti o n th an
on one c o n tain in g over 25$ c o n c e n tra te .
Foreman and Herman (1953) in c re a se d c e llu lo s e d ig e s tio n
somewhat by adding 1 to 2 pounds of m olasses to a b a s a l r a ti o n
of about *+ p a r ts hay to 1 c o n c e n tra te but t h i s d i g e s t i b i l i t y
was u s u a lly d ep ressed when th e m olasses le v e l was such th a t
th e r a t i o n co n tain ed 30 to *+0$ t o t a l c o n c e n tra te .
v a rie d w ith th e type o f hay fe d .

The r e s u l t s

I t should be noted th a t th e

w ater so lu b le carb o h y d rate of roughages can range from *+ to
30$ of th e d ry m atter depending on s p e c ie s , method o f c u rin g ,
s ta g e of growth and o th e r f a c to r s as re p o rte d by W aite and
Boyd (1953).

k
Hale et. a l . (19*+0, 19*+7)* u t i l i z i n g th e li g n in r a t i o
tech n iq u e to compare rumen and f e c a l d ig e s tio n , showed th a t
th e rumen accounts fo r th e g re a te r p a r t of d ig e s tio n in bovines
fe d a l f a l f a hay.

This has been confirm ed and extended to o th e r

fe e d s as review ed by J a r r ig e (1953)*

The abomasal f i s t u l a

tech n iq u e f o r m easuring forestom ach d ig e s tio n was used by
Heald (1953)? who found th a t about kO% of th e in g e ste d xylan
was d ig e s te d in th e forestom ach, and by W eller and Gray (195*0*
who found t h a t n e a rly a l l of th e s ta rc h in chopped p o ta to e s
was d ig e s te d in th e forestom ach.
B aleh et. ^ 1 . (1951) s t a t e th a t th e fe rm e n ta tio n and
d ig e s tio n of fe ed in rum inants i s a fu n c tio n of i t s r a t e of
passage through th e re tic u lo -ru m e n , i t s r a t e of d is s im ila tio n
by th e m icro b io ta and f i n a l l y , th e r a t e o f a b so rp tio n or p as­
sage of th e fe rm e n ta tio n p ro d u cts.
Some 2 to 5$ of th e s ta in e d hay p a r t i c l e s p re se n t in th e
rumen a t a given tim e d isap p eared in th e next hour according
to th e d a ta of Balch (1950).

F in er p a r t i c l e s , such as c o tto n ­

seed h u lls o r ground hay passed somewhat f a s t e r (5 to 10$ /
h o u r), but only i f long hay or o th e r roughage was a ls o p re s e n t.
M oreover, by re c la im in g th e sta in e d p a r t i c l e s in th e fe c e s and
ex p ressin g reco v ery as p ercentage of th e t o t a l , he found th a t
about 20 hours were re q u ire d f o r passage from th e rumen to th e
fe c e s.

S tain ed p a r t i c l e recovery appeared to be e x p o n e n tia l

approaching an asym ptote; th e r a t e of th e e x p o n e n tia l was
ag ain some 2 to 5$/h o u r.

E a r li e r , Moore and W inter (193*0

5
found t h a t f e r r i c oxide passed from rumen to fe c e s in 9 to 13
hours and sm all ru b b er rin g s in 10 to 20 h o u rs.

A fte r th e

i n i t i a l app earan ce, reco v ery was ag ain roughly e x p o n e n tia l
approaching an asym ptote a t 2 to 5$/hour.

The work o f B alch

§ t a l . ( 1950, 1952b , 1953) showed th a t such f a c to r s as th y ro x ­
in e a d m in is tr a tio n , r a t i o n s , r e s t r i c t e d w ater in ta k e and o th e rs
had l i t t l e e f f e c t on t h i s r a t e .

The r a tio n s stu d ie d in clu d ed

a l l hay, eq u al p a r ts o f hay and c o n c e n tra te , hay p lu s mangolds,
and f i n e l y ground hay.
The d a ta of Agrawala et, a l . (1953) and of Chance et, a l .
( 1953a9 b) f o r d ry m atter passage from th e rumen measured
between 6 and 2*+ hours a ls o in d ic a te s a r a t e of 2 to 5$ /h o u r.
The d isap p earan ce was much f a s t e r between 0 and 6 hours a f t e r
, fe ed in g due to d ig e s tio n as ex p lain ed by Hale e t a l . (19*+7),
who showed th a t rumen d ig e s tio n i s v i r t u a l l y completed in 12
hours a f t e r feed in g and over h a lf com pleted in 6 h o u rs.

Balch

and Johnson (1950) found c o tto n th re a d d is s ip a tin g in th e
v e n tr a l p o rtio n of th e rumen a t as much as 50$ /h o u r a f t e r a
20 hour la g p hase.

According to B alch et, a l . (1951), passage

through th e re tic u lo -o m a s a l o r i f i c e i s la rg e ly r e s t r i c t e d to
f in e p a r t i c l e s in a 90 to 95$ aqueous suspension.
The duodenal or abomasal f i s t u l a p re s e n ts an o th er way of
m easuring passage from th e forestom achs.

In t h i s way, P h i l l i p -

son ( 19*+8 , 1952a) measured a passage of k67 to 186U m l./h o u r
in sheep consuming 900 to 1700 gm. of dry m a tte r/d a y ; th e
la r g e r volume was a s s o c ia te d w ith h e a v ie r fe e d in g .

C onsidering

6
th e t o t a l washed fe ed re sid u e in 2k hour fe c e s c o lle c tio n s
and in duodenal c o n te n ts from a sheep m aintained on hay, i t
was c a lc u la te d t h a t 10.7 l i t e r s of m a te ria l passed through
th e duodenum d a ily .

Masson and P h illip s o n (1952) c a lc u la te d

t h a t tw o -th ird s o f t h i s m a te ria l was g a s t r ic ju ic e and onet h i r d omasal c o n te n ts .

T heir c a lc u la tio n s were based on th e

c h lo rid e c o n ten t of th e g a s tr i c ju ic e , omasal c o n te n ts , and
abomasal c o n te n ts .

Passage of omasal c o n te n ts was stim u la te d

somewhat by c o n c e n tra te but was la r g e ly Independent of fe e d in g .
This su g g ests t h a t passage o f omasal c o n te n ts corresponds to
about 2 to 5# /h o u r of th e mass of th e t o t a l rumen in g e s ta which
i s in agreement w ith th e passage o f m arkers from th e rumen.
The in c re a se d c o n c e n tra tio n of in o rg an ic io n s in omasal
liq u id over rumen l iq u id found by G arton (1951) in d ic a te s
a b so rp tio n of w ater from th e omasum.

Gray e t a l . (195^)

confirm ed t h i s and c a lc u la te d from th e lig n in r a t i o s in rumen
and omasal c o n te n ts th a t 33 to 6k% of th e w ater and ko to 69$
of th e v o l a t i l e f a t t y ac id i n th e rumen d ig e s ta e n te rin g th e
omasum i s absorbed th e r e .

Using th e same te c h n iq u e , th ey were

ab le to show th a t l i t t l e or no rumen d ig e s ta passed d i r e c t l y
to th e abomasum v ia th e omasal o r i f i c e and t h a t w ater was not
p r e f e r e n t i a l l y passed from th e omasum to th e abomasum.
In flu e n c e of C o n centrate on th e Rumen M icrobiota
S ince forestom ach d ig e s tio n seems to occur la r g e ly through
a c tio n of th e rumen m ic ro b io ta , as p o in ted out by H astin g s

7
( l ^ V ) , we might expect a q u a lita tiv e or q u a n tita tiv e change
i n t h i s m icro h io ta w ith changes in d ig e s tio n .

H astin g s (19*+^)

n o te s changes in th e q u a n tity of v a rio u s ty p es of p rotozoa
w ith marked changes in fe e d , but c o n sid e rs th e popular id ea
th a t b io lo g y of th e d ig e s tiv e t r a c t v a r ie s w idely w ith th e
normal changes in r a t i o n as unfounded.

Baker (19^3) concluded

from h is e x te n siv e m icroscopic o b se rv a tio n s th a t th e rumen
m icro b io ta i s p e r s i s t a n t and s ta b le w ith broad q u a li ta t iv e
f e a tu r e s which a re independent of th e n a tu re of th e d i e t .
However, Louw and Van der Wath (19^3) in c re a se d th e rumen
b a c t e r i a l count by p ro v iding 8 to 22% corn in a r a ti o n of low
q u a lity hay and meat m eal.

C e llu lo se d ig e s tio n was not aug­

mented by th e in c re a se d p o p u la tio n and g re a te r c o n c e n tra tio n s
of corn d ep ressed b a c t e r i a l numbers.

B o rtre e e t a l . (19^6)

o b tain ed a 100$ in c re a s e in th e io d o p h ilic m ic ro b ia l p o p u latio n
of th e bovine rumen when 3 pounds of glucose were ad m in istered
in a d d itio n to th e u s u a l feed in g of hay.

S ta rc h ad m in istered

under s im ila r circu m stan ces produced a n e g lig ib le e f f e c t ;
q u a lity of roughage or p a stu re in flu e n c e d counts somewhat.
W alter (1952) found no a p p re c ia b le change in d i r e c t m icro­
sco p ic counts on rumen in g e s ta when b a rle y supplem ented th e
b a s a l of a l f a l f a hay f o r sheep.
Some q u a l i t a t i v e d iffe re n c e s among c e r ta in in d ic a to r
organism s were observed by Pounden and Hibbs (19^8) when th ey
examined th e rumen m icro b io ta of c a lv e s fe d v a rio u s p ro p o rtio n s
o f hay and g ra in .

The broad appearance of th e rumen m icro b io ta

8
and th e im portance of th e in d ic a to r organism s i n rumen f e r ­
m en tatio n were n ot re p o rte d .

U tiliz in g both c u l t u r a l and

m orphological te c h n iq u e s , G all (19*+9) found g re a te r numbers
of f a s t growing organism s w ith in c re a sin g amounts of g ra in
in th e r a t i o n b ut few q u a lita tiv e d iffe re n c e s were n o ted .
Foreman and Herman (1953) conducted d ir e c t m icroscopic
counts on th e rumen in g e s ta of 12 cows in 3 groups fe d d i f f e r ­
en t roughages.

Four le v e ls of cane m olasses were fe d w ith in

each group on a r o t a t i o n b a s is .

The b a s a l r a ti o n co ntained

about k p a r ts of hay and 1 p a rt c o n c e n tra te .

The t o t a l co u n ts,

which were grouped in to 10 m orphological c la s s e s , showed a
p e r s i s t a n t in c re a s e from about 67 x 10^ to 106 x 10^ organism s
per gm. o f m oist in g e s ta as m olasses supplem entation in c re a se d
from 0 to

pounds per day.

The numbers of p rotozoa were con­

s i s t e n t l y 200 to 300 thousand per ml. of rumen f l u i d .

The

s in g le co cci c o n s titu te d about 70$ of th e p o p u la tio n .

The

sh o rt ro d s which c o n s titu te d about 20 to 25$ of th e p o p u la tio n
tended to in c re a s e s l i g h t l y w ith 1 or 2 pounds of m olasses,
but were d ep ressed by b pounds.
d i s t i n c t l y dep ressed by m olasses.

Long rods and d ip lo c o c c i were
However, th e q u a l i ta t iv e

and q u a n tita tiv e changes in th e p o p u latio n were as g re a t w ith
changes in roughage as w ith th e m olasses supplem entation.
W illiam s e t a l . (1953) found th a t 1 p a r t of s ta r c h added
to 1 or 2 p a r ts of o a te n c h a ff c o n tain in g 5$ crude p r o te in
d ecreased th e numbers of f r e e rumen b a c te r ia by 50$ or more,
b u t i f th e p r o te in source was corn g lu te n m eal, t h i s d e p re ssio n

9
la r g e ly d isap p ea red .

The high s ta r c h , low p ro te in r a t i o n

produced a pleom orphic ro d in long ch a in s c lu s te re d on s ta rc h
g ra n u le s .

This ro d la r g e ly d isap p eared as p r o te in in c re a se d

and was ab sen t on low s ta rc h r a ti o n s .

An in c re a s e in th e

p ro p o rtio n of g ra m -p o sitiv e organism s was a lso noted on th e
h ig h s ta r c h , low p ro te in r a tio n .
The rumen m ic ro flo ra from cows fe d a l f a l f a hay an d /o r
c o n c e n tra te m ixture o r wheat straw was stu d ie d by Bryant and
Burkey (1953a, 1953b) u sin g both m orphological and c u lt u r a l
methods.

Q u a lita tiv e ly , th e m ic ro flo ra were la r g e ly th e same

w ith numbers in c re a s in g on th e c o n c e n tra te r a ti o n ; however,
th e y estim ated t h a t only 10# of th e t o t a l p o p u la tio n a re
c u ltu re d by th e p re se n t in v i t r o tech n iq u es and c a l l a tte n tio n
to th e pleom orphic n a tu re of many of th e rum inal m icroorgan­
ism s.

The f l o r a was more complex on th e a l f a l f a hay co n tain in g

r a ti o n s th an on th e r a tio n s of c o n c e n tra te or wheat straw .
C e llo b io se ferm enting organism s were depressed by th e concen­
t r a t e r a t i o n and s ta rc h ferm en ters were augmented; hydrogen
s u lf id e producing organism s were augmented somewhat by th e
straw r a t i o n .

V a r i a b i li t y was a lso noted in th e f l o r a of

d i f f e r e n t anim als on th e same r a ti o n .
Rumen P ro d u ctio n of S hort Chain Acids
The e a rly l i t e r a t u r e on t h i s s u b je c t, review ed by P h i ll i p son (1 9 ^7 ), d a te s back to lSQW when Tappeiner found la rg e quan­
t i t i e s of v o l a t i l e a c id formed in th e bovine rumen.

However,

10
th e com position of t h e i r m ixture was not known u n t i l Elsden
e t a l . ( I 9H6) developed a method f o r t h e i r s e p a ra tio n on
s i l i c a g e l.

These w orkers found an average of 329 g®. of

v o l a t i l e ac id i n th e re tic u lo -ru m e n of 2 oxen and an average
of

gm. in t h a t of H sheep.

These a c id s c o n siste d of about

67$ a c e t i c , 19$ p ro p io n ic and 1*+$ b u ty r ic .

This i s a t v a ria n c e

w ith th e d a ta of M arston (19^8) who found about equal p o rtio n s
o f a c e tic and p ro p io n ic a c id s produced from fe rm e n ta tio n of
c e llu lo s e by rumen m icro b io ta i n v i t r o .
Rumen in g e s ta from s e v e ra l cows examined by Stone (19*+9)
th ro u g h a 9 hour p e rio d follow ing e a tin g , co n tain ed 133 to 162
m illig ram e q u iv a le n ts f o r se v e ra l hours a f t e r e a tin g .

The

r e s id u a l feed in 100 ml. of in g e s ta removed 1 hour a f t e r feeding
hay and g ra in and in cu b ated a t 39°C. was converted to v o l a t i l e
ac id a t th e r a t e of about 60$/h o u r w ith a t o t a l p ro d u ctio n of
20 to 25 m illig ram e q u iv a le n ts of v o l a t i l e a c id .

This amounts

to some 12 to 15 g m ./ li t e r rumen in g e s ta .
C a rr o ll and Hungate (195*+) p o in ted out th a t p ro d u c tio n of
v o l a t i l e a c id can n ot be determ ined d i r e c tl y in the rumen
because a b so rp tio n d e c re a se s th e a c id c o n c e n tra tio n sim u ltan e­
o u sly w ith i t s in c re a s e through fe rm e n ta tio n .

Moreover, rumen

m icro b io ta d is s o c ia te d from th e a b so rp tiv e and s e c re to ry
a c t i v i t i e s o f th e h o st may soon develop abnormal c o n d itio n s .
These w orkers a ls o noted a r i s e in th e v o l a t i l e a c id ity of
rumen in g e s ta du rin g th e f i r s t s ix hours a f t e r fe e d in g .

The

average amount of a c id produced in 1 hour of in v i t r o in c u b a tio n

11
w ith rumen c o n te n ts from hay fed s te e r s was 1.37 m illig ram
e q u iv a le n ts per 100 gm.

The corresponding amounts w ith a

g ra in d i e t and a p a s tu re d ie t were 2.01 and 0.91 r e s p e c tiv e ly .
The average p ro p o rtio n s of a c e tic , p ro p io n ic and b u ty ric a c id s
i n th e m ixture formed during 6 t r i a l s w ith hay fe d anim als
were 61$, 20$, and 19$ r e s p e c tiv e ly .

I t was estim ated th a t

66$ o f th e t o t a l d ig e s tib le energy in th e r a ti o n i s p resen ted
to th e rum inant as v o l a t i l e a c id s .

In hay fe d s t e e r s , a c e tic

a c id c o n trib u te d U2$ o f t h i s energy; b u ty ric a c id , 32$ and
p ro p io n ic a c id , 26$ .
These e stim a te s o f C a rr o ll and Hungate (195*+) were based
on th e ex p e rim e n ta lly determ ined r a t e s of a c id p ro d u c tio n and
e s tim a te s o f t o t a l rumen mass.

The fe ed was converted to

v o l a t i l e a c id s a t th e r a t e of about *+0$/hour.

They considered

t h i s r a t e as a minimum and po in ted out th a t th e b e s t way of
a s c e rta in in g r a t e of p ro d u c tio n of a product in th e rumen
would be to c o n s tru c t tim e -c o n c e n tra tio n curves and c a lc u la te
th e i n i t i a l r a t e .

They p o in ted out t h a t a very la rg e volume

of d a ta would be re q u ire d .
Gray e t a l . (1951b) incubated wheaten and a l f a l f a hay
w ith l i q u i d rumen c o n te n ts in v i t r o .

The y ie ld of v o l a t i l e

a c id s was about o n e -fo u rth of th e s u b s tr a te or 50$ of th e
d i g e s t i b le m a tte r.
^6$ a c e t ic ,

The a c id s produced from wheaten hay were

p ro p io n ic and 13$ b u ty ric w hile th o se from

a l f a l f a were 59$, 27$, and 1 *4$ r e s p e c tiv e ly .

12
D ealing w ith sheep fe d wheaten hay, Gray and P ilg rim
(1951) found v o l a t i l e a c id c o n c e n tra tio n s in th e rumen liq u id
of 8 .7 m illig ram e q u iv a le n ts/1 0 0 ml. a t th e time of fe ed in g
which in c re a s e d to 20.6 s ix hours a f t e r feeding and re v e rtin g
to th e i n i t i a l c o n c e n tra tio n in about 2k h o u rs.

In sheep fed

a l f a l f a hay, th e corresponding i n i t i a l v alu e was 9*3 in c re a s in g
to 2 5 .5 fo u r hours a f t e r fe e d in g .

Moreover, th e p ro p io n ic a c id

c o n c e n tra tio n in c re a se d f a s t e r th an th e a c e tic a c id c o n c e n tra ­
t io n and th u s , i t was concluded th a t p ro p io n ic a c id was p ro ­
duced i n g r e a te r q u a n titie s th an a c e tic a c id and was a ls o
absorbed f a s t e r sin c e th e c o n c e n tra tio n re v e rte d to th e p re ­
feed in g l e v e l .

This h y p o th esis was te s te d by Gray and P ilg rim

(1952a) by in v i t r o in c u b a tio n of rumen m icrobiota w ith wheaten
hay c h a ff .

V o la tile a c id p ro d u ctio n was la r g e ly completed

w ith in 7 i hours and alth ough th e p ro d u ctio n of p ro p io n ic acid
exceeded t h a t of a c e tic a c id a t f i r s t , th e p ro p o rtio n of th e
a c id s in th e f i n a l m ixture was about 50# a c e tic , k0% p ro p io n ic
and 10# b u ty r ic .
The v o l a t i l e a c id s of rumen f l u i d from hay fed sheep were
more com pletely analyzed by Gray e t a l . (1951a) who found 2 to
3# v a le r ic a c id , 0 .3 to 0.6# is o b u ty ric a c id , and 0 .5 to 1.0#
ca p ro ic a c id in a d d itio n to th e u su a l a c e tic , p ro p io n ic and
b u ty ric a c id s .

Using C ^ - la b e le d a c e tic and p ro p io n ic a c id s ,

th ey found th a t th e carboxyl carbon of a c e tic a c id occurred
to an a p p re c ia b le e x te n t in b u ty ric and v a le r ic a c id s and
a ls o to some e x te n t i n p ro p io n ic a c id .

The carboxyl carbon

13
o f p ro p io n a te appeared in v a le r ic but not in b u ty ric a c id .
The a b i l i t y of C lo strid iu m to r e v e r s ib ly in te re o n v e ft a c e tic ,
p ro p io n ic and b u ty r ic a c id s was more f u l l y ex p lain ed by Wood
e t a l . (19*+5) and Stadtman and B arker (19**9).
E l-S h azly (1952) sim ultaneously o b tain ed s im ila r r e s u l t s
by u sin g a new and v ery s e n s itiv e gas p a r t i t i o n method of
chrom atography.

He found somewhat h ig h er p ro p o rtio n s of

is o b u ty r ic a c id (1 .5 to 2 .8 molar$ of th e v o l a t i l e acid
m ixture) and showed th a t most of th e v a l e r ic a c id i s branched
ch a in .

He a ls o showed t h a t th e se branched chain a c id s are

formed la r g e ly by m ic ro b ia l decom position of p r o te in .

This

work was continued by Annison (195*0 vho i d e n ti f ie d 2-m ethylb u ty ric a c id among th e branched chain ac id s in th e rumen.
Coon et, a l . (1952a, 1952b) showed th a t t h i s a c id could a r is e
from is o le u c in e in th e r a t li v e r and could y ie ld a 2-carbon
k eto g en ic compound and a 3-carbon glucogenic compound w ith
p ro p io n ic a c id as an in te rm e d ia te .
According to Annison (195*0* th e t o t a l v o l a t i l e a c id
c o n c e n tra tio n in th e rumen d ec lin ed e x p o n e n tia lly a t th e r a t e
of 3^/hour in f a s te d sheep, but th e branched ch ain a c id s
rem ained alm ost c o n sta n t a t 1 .5 to 3 .5 m illigram e q u iv a le n ts /
100 ml. rumen in g e s ta .

The branched chain a c id s in c re a se d as

th e n itro g e n c o n ten t of th e r a tio n in c re a s e d .

A r a ti o n of 2 .5

p a r ts ground corn and 1 p a rt hay was converted to v o l a t i l e
a c id a t a r a t e between 30 and 80^ /h o u r.

Ik
P h illip s o n (1952b) stu d ie d p ro d u ctio n of v o l a t i l e a c id s
on a s im ila r r a t i o n v i t h s im ila r r e s u l t s .

The molar composi­

t i o n of th e v o l a t i l e a c id s was about 60# a c e t ic , 30# p ro p io n ic ,
7# b u ty ric and k% h ig h er a c id s 8 hours a f t e r fe e d in g .

The

r e l a t i v e amount o f p ro p io n ic a c id in c re a se d a f t e r feed in g and
hence, was e i t h e r produced f a s t e r or absorbed slow er th an
a c e tic a c id .

L a c tic a c id c o n c e n tra tio n s as high as 630 m i l l i ­

grams/100 ml. rumen l i q u id were found in lambs fe d a corn
ra tio n .

Waldo and S c h u ltz (1955) o b tain ed v a lu e s of 0 .3 to J+l

m illigram # l a c t i c a c id in rumen liq u id from hay fe d s t e e r s .
These v a lu e s in c re a se d to as much a s 58 m illigram # when 7-5
pounds o f g ra in was in clu d ed in th e r a t i o n .

Maximum c o n c e n tra ­

t io n in th e rumen occu rred 1 hour a f t e r feeding hay or oneh a lf hour a f t e r feed in g g ra in .

D isappearance was e q u a lly

ra p id d e c lin in g to o n e -h a lf of peak c o n c e n tra tio n in le s s
th an 2 h o u rs.

However, when P h illip s o n (1952b) drenched 2

lambs w ith l a c t a t e , d isap p earan ce of l a c t a t e from th e rumen
of one lamb was much slow er th a n in th e o th e r.

Waldo and

S c h u ltz (1955) found t h a t 1 pound of glucose in c re a se d th e
l a c t i c a c id c o n c e n tra tio n i n th e rumen much more th a n 1 pound
of s ta r c h , c e llu lo s e , o r pyruvic a c id .

Both groups o f in v e s ­

t i g a t o r s propose t h a t l a c t i c a c id i s r a p id ly converted prim a­
r i l y to p ro p io n ic a c id in th e rumen.

This would e x p la in th e

v ery hig h ferm en tab le carbohydrate c o n c e n tra tio n which i s
a p p a re n tly re q u ire d in th e rumen to produce a d e te c ta b le con­
c e n tr a tio n of l a c t i c a c id .

Johns (1952) and G u tie rre z (1953)

15
i s o l a t e d b a c te r ia from th e rumen v h ic h d is s im ila te l a c t a t e
w ith p ro d u c tio n of p ro p io n ic and a c e tic a c id s .
Washed su sp en sio n s o f rumen b a c te r ia p repared by Robinson
et. a l . (1955) produced l a c t i c a c id from m altose and c e llo b io s e
and to a sm aller e x te n t from g lu co se, p y ru v a te , or m alate, but
n o t from x y lo se o r a ra b in o se .

When th e sm aller organism s were

e lim in a te d by u sin g lower c e n tr if u g a l f o r c e , l a c t i c a c id was
produced a t pH 5 .0 or 8 .0 but not a t pH 6 .9 .
The molar com position of sh o rt ch ain a c id s was about 6%
a c e t i c , 20% p ro p io n ic , and

b u ty ric in a number of cows fed

a c o n tro l r a t i o n of hay, s ila g e , and c o n c e n tra te (Tyznik, 1951).
The molar r a t i o s of a c e ta te and p ro p io n ate were u s u a lly re v e rse d
2 to 3 weeks a f t e r r e s t r i c t i n g roughage and l i b e r a l l y feed in g
c o n c e n tra te or f in e ly ground hay.

I t was hypothesized t h a t th e

p ro d u c tio n of a c e ta te was suppressed in fav o r of p ro p io n ate
p ro d u c tio n .
Card and S c h u ltz (1953) stu d ie d p ro d u c tio n of v o l a t i l e
f a t t y a c id s in 28 cows on w idely d if f e r e n t r a tio n s and found
a s ig n i f i c a n t d iffe re n c e in th e molar p ercen tag e of each ac id
due to r a t i o n .

The molar % com position of th e t o t a l v o l a t i l e

f a t t y a c id s f o r a c e t i c , p ro p io n ic , and b u ty ric a c id s re s p e c ­
t i v e l y were as fo llo w s :

mixed hay - 60 . 0 , 2 1 .0 , 1 9 .0 ; hay

p lu s g ra in - 5 7 .7 , 1 8 .8 , 2 3 .5 ; p a stu re - 5 5 .6 , 1 7 .9 , 2 6 .5 ;
p a s tu re p lu s g ra in - 5 3 .0 , 2 0 .3 , 26.7 and; g ra in alone - M-7.3,
2 3 .2 , 2 9 .5 .

Roughage from a l a t e r d a te of c u ttin g tended to

in c re a s e th e p ro p o rtio n of a c e tic and d ecrease th e p ro p o rtio n

16
of b u ty r ic a c id which could be ex p lain ed by decreased p ro te in
(E l-S h a z ly , 1952).

The t o t a l v o l a t i l e ac id c o n c e n tra tio n was

h ig h e s t on th e a l l c o n c e n tra te r a tio n and low est on th e pas­
tu re .

S c h u ltz (1951*) found th e t o t a l v o l a t i l e a c id c o n c e n tra ­

t i o n in 8 bovine rumens was depressed about 25# by a 16 hour
f a s t and th e r e l a t i v e p ro p o rtio n of p ro p io n ic a c id was a lso
d e p re sse d .

This i s in agreement w ith th e d a ta of R obertson

and Thin (1953).
Johns (1953) found t h a t glucose in th e rumen of sheep
q u a n tita tiv e ly y ie ld e d a m ixture of a c e t ic , p ro p io n ic and
b u ty ric a c id s w ith in an hour.

Both jln v i t r o and %n v iv o ,

a c e ta te p ro d u c tio n was much g re a te r th an p ro p io n ate o r b u ty ra te p ro d u c tio n .

Under th e same c o n d itio n s , g ly c e ro l was

a tta c k e d more slow ly y ie ld in g alm ost e n t i r e l y p ro p io n ic a c id .
According to S i j p e s t e i j n and E lsden (1952), su c c in a te a lso
q u a n tita tiv e ly y ie ld s p ro p io n ate in th e rumen.
The p ro d u cts of i n v i t r o f erm en tatio n re p o rte d by Me
Naught (1951) were d i f f e r e n t f o r rumen m icro b io ta from cows
on p a s tu re th an f o r m icro b io ta from hay and g ra in fe d cows.
The p ro d u c ts a ls o v a rie d w ith s u b s tr a te .

P a stu re m icro b io ta

a c tin g on m altose or arab in o se produced a c id s w ith a molar
com position pf about *+6# a c e ta te , H6# p ro p io n a te , and 8#
b u ty ra te w hile th e h a y -c o n c e n tra te m icro b io ta produced a
corresp o n d in g com position of 53# > 32#, and 15#.

Xylose

y ie ld e d more t o t a l a c id th a n m alto se, but th e com position
was th e same.

A carbon balance study showed th a t 2U to 25#

17
of th e m alto se, a ra b in o s e , or xy lo se carbon was converted to
v o l a t i l e a c id .

L a c tic a c id accounted f o r 6 to 10$ of th e

m altose carbon w h ile only a tr a c e or no l a c t i c a c id was p ro ­
duced from a rab in o se or x y lo se .

This was confirm ed by Heald

( 1952) , except f o r la r g e r p ro p o rtio n s of a c e ta te from x y lo se ,
and extended to g lu cu ro n o lacto n e w ith s im ila r r e s u l t s .

Heald

( 1953) e stim a te d t h a t pentosans form 20 to 25$ of th e dry
m a tte r i n common rum inant r a ti o n s .
S uspensions o f rumen b a c te r ia prep ared by D oetsch e t a l .
(1953) ferm ented c e llo b io s e or pyruvate y ie ld in g a m ixture of
a c id s w ith a molar com position of about 6k% a c e tic , 21$ p ro p i­
o nic and 15$ h ig h er a c id s ; glucose and m altose y ie ld in g 36$
a c e t ic , *+0$ p ro p io n ic and 2*+$ higher a c id s ; xylose y ie ld e d
ko% a c e t i c ,

30$ p ro p io n ic and 30$ h ig h er a c id s and su c c in a te

y ie ld e d only p ro p io n ic a c id .

L a c ta te and o x a la c e ta te were

a tta c k e d slow ly w ith only a tr a c e of a c id p ro d u c tio n .

P ro p i­

o nate y ie ld e d a tr a c e of a c e ta te ; a c e ta te was not a tta c k e d ;
b u ty ra te was converted q u a n tita tiv e ly to a c e ta te .

B eta-hydro-

x y b u ty ra te y ie ld e d some a c e ta te w hile m alate was converted to
a c e ta te and p ro p io n a te .
by S iro tn a k

Manyof th e se r e s u l t s were confirm ed

et. a l . (195^) and were extended, using th e same

te c h n iq u e , to a la n in e which was not a tta c k e d , fum arate which
was d is s im ila te d w ith o u t a c id p ro d u c tio n , and to L -a s p a rta te
which appeared to be deam inized to su c c in a te and th en d e c a rb o x y lated to p ro p io n a te .

16
Gray and P ilg rim (1952b) examined th e f a t t y a c id s p ro ­
duced i n v i t r o from th e c e llu lo s e and h e m i-c e llu lo se of
w heaten hay and found approxim ately equal p a r ts of a c e tic and
p ro p io n ic a c id s and a sm all amount of b u ty ric a c id produced
from e i t h e r o r b o th su b stan ce s.

B u ty ric a c id seemed to come

only from p r o te in so u rces which a ls o y ie ld e d r a th e r la rg e
amounts of a c e tic a c id .

This p a tte r n i s found ag ain in th e

work o f B elasco (195*0 where only a c e ta te and p ro p io n ate w ith
a tr a c e of b u ty ra te were formed by th e a c tio n of rumen m icro­
b io ta in v i t r o on c e llu lo s e and s ta r c h .

P ro te in n itro g e n was

re p la c e d by n o n -p ro te in n itro g e n in t h i s work and as th e con­
c e n tr a tio n of th e l a t t e r in c re a s e d , more v a le r ic and b u ty ric
a c id s were formed.
Tyznik e t a l . (195*+) te s te d th e e f f e c t of v a rio u s sub­
sta n c e s on sh o rt ch ain a c id p ro d u c tio n when re p la c in g c e ll u ­
lo s e on an eq u al carbon b a s is .

M altose or s ta rc h in c re a se d

t o t a l v o l a t i l e a c id p ro d u c tio n w ith a 100$ in c re a s e in th e
p ro p o rtio n of b u ty ric a c id .

Glucose alone or w ith c e llu lo s e

gave th e same p ro p o rtio n s of v o l a t i l e a c id s as c e llu lo s e alone
b u t s i g n i f i c a n t l y in c re a se d th e t o t a l a c id p ro d u c tio n .

L a c tic

a c id in c re a se d th e p ro p o rtio n of p ro p io n ic a c id and in c re a se d
t o t a l a c id p ro d u c tio n but depressed c e llu lo s e fe rm e n ta tio n
above th e 25$ carbon replacem ent l e v e l .

R eplacing 25$ of th e

carbon w ith s ta r c h , l a c t i c a c id , m alto se, or sodium pyruvate
y ie ld e d v ary in g amounts of v a le r ic a c id amounting to from 1
to 7.5$ of th e t o t a l v o l a t i l e a c id .

A ddition of a c e t ic ,

19
p ro p io n ic and b u ty ric a c id s to re p la c e 25$ of th e carbon had
no e f f e c t on t o t a l v o l a t i l e a c id p ro d u c tio n .
A bsorption and U t i l iz a t i o n of S hort Chain Acids
The l i t e r a t u r e d e a lin g w ith a b so rp tio n of v o l a t i l e a c id s
was review ed by P h illip s o n (191+7) who, c itin g e a r l i e r work
done by h is group, concludes t h a t more th an 5 gm. of v o l a t i l e
a c id p er hour a re absorbed from th e bovine rumen.

Pfander

and P h illip s o n (1953) p o in t out t h a t , although numerous ex­
p erim en ts have been conducted on a b so rp tio n r a te s of sh o rt
c h a in a c id s , none of them s u f f i c ie n tl y sim u late normal con­
d i t io n s to allow d ed u ctio n s concerning q u a n titie s of th e se
a c id s norm ally absorbed.
McClymont (1951) re p o rte d a maximum a r t e r i a l v o l a t i l e
a c id l e v e l of 1 .2 to 2 .3 m illigram e q u i v a l e n t s / l i t e r 2 to 5
hours a f t e r fe e d in g .

This d e c lin e d to 0 .3 to 0 .9 m illig ram

e q u iv a le n ts / l i t e r 16 hours a f t e r feeding w ith a molar com­
p o s itio n of over 90$ a c e tic a c id .

S im ila r v a lu e s were found

by Reid (1950) in sheep.
The p e r ip h e r a l blood and venous blood d ra in in g th e rumen
in lb sheep on p a s tu re was examined by K iddle ejt a l . (1951).
P e rip h e r a l blood co n tain ed O.H to 1 .7 m illig ram e q u iv a le n ts
v o l a t i l e a c id / l i t e r c o n s is tin g la r g e ly of a c e tic w hile venous
blood d ra in in g th e rumen co n tain ed 1 .7 to 8 .6 m illig ram eq u iv ­
a le n ts v o l a t i l e a c i d / l i t e r w ith a molar com position of 57 to
95$ a c e t i c , 10 to 33$ p ro p io n ic and 3 to 16$ b u ty ric a c id .

20
C raine and Hansen (1952) o b tain ed s im ila r v a lu e s in venous
blood d ra in in g th e rumens of a d u lt g o a ts.

Even a f t e r c o rre c ­

t i o n f o r th e a c e tic a c id in p e rip h e ra l blood, th e p ro p o rtio n
of a c e tic a c id in blood d ra in in g th e rumen was always h igher
th an i n rumen in g e s ta .

Lymph c o lle c te d from th e th o ra c ic

d u ct i n H anim als co n tain ed the same c o n c e n tra tio n of v o l a t i l e
a c id s as a r t e r i a l blood.

However, when Masson and P h illip s o n

(1951) co n tin u ed t h i s work by measuring th e d isappearance of
in d iv id u a l a c id s from em ptied, washed rumens lig a te d a t th e
oesophagus and om asal-abom asal o r i f i c e , they found t h a t a l l
a c id s d isap p eared a t about th e same r a t e .

The rumen tended

to b u ffe r a t pH 7 .5 to 7 .8 by in flu x of c h lo rid e s and CO2
from b lo o d .

A d m in istra tio n of b u ty ra te and p ro p io n ate to

normal anim als had l i t t l e e f f e c t on th e a r t e r i a l v o l a t i l e
a c id c o n c e n tra tio n w h ile a c e ta te caused a th r e e - f o ld in c re a s e .
These anomalous r e s u l t s were p a r t i a l l y c l a r i f i e d when
P ennington (1952, 195*+) found rumen e p ith e liu m m etabolizing
b u ty ra te much more r e a d ily th an p ro p io n ate or a c e ta te .

Ketones

were produced from b u ty ra te and a c e ta te , but not p ro p io n a te .
P ro p io n ate m etabolism was in c re a se d by in c re a sin g C02-te n s io n
up to 20$.

M alonate in h ib ite d u t i l i z a t i o n of each a c id and

caused p ro d u c tio n of su c c in a te from p ro p io n a te .

The ammonium

io n d ep ressed m etabolism of p ro p io n a te , but not b u ty r a te ,
p o s s ib ly by b lo ck in g th e tr ic a r b o x y lic a c id c y c le . ' This
d e p re s s io n occu rred a t ammonium le v e ls which o fte n occur in
th e rumen a f t e r fe e d in g .

R obertson and Thin (1953) confirm ed

th e e x tra -ru m in a l n a tu re of ketone body p ro d u c tio n .

21
Scharabye (1951) conducted s im ila r s tu d ie s using r a tio n s
o f chopped or unchopped hay, crushed o a ts , and lin s e e d meal.
The p re feed in g v o l a t i l e acid c o n c e n tra tio n in p o r ta l blood
was double t h a t of a r t e r i a l blood.

A fte r fe e d in g , th e p o r ta l

le v e l ro s e about 50# p a r a lle lin g th e in c re a se d c o n c e n tra tio n
o f v o l a t i l e a c id s in th e rumen w hile th e a r t e r i a l le v e l ro se
only s l i g h t l y .

I n je c tio n of a c e tic a c id in to th e rumen in ­

creased th e c o n c e n tra tio n in p o r ta l blood but not in venous
blood d ra in in g th e l i v e r .

Thus, th e v o l a t i l e a c id s were

la r g e ly removed from p o r ta l blood by th e l i v e r .

The p o r ta l

glycem ia showed l i t t l e change a f te r feed in g and th e p o r ta l a r t e r i a l d iffe re n c e s were n e a rly z e ro .

O ral a d m in is tra tio n

of 1 to 2 gm. of g lu co se/k ilo g ram body w eight r e s u lte d in a
sm all and t r a n s i t o r y hyperglycem ia.

The very la rg e molar

r a t i o of absorbed v o l a t i l e ac id to absorbed glucose le a v e s
l i t t l e doubt t h a t v o l a t i l e ac id s c o n s titu te th e g re a te r source
of c a lo r ie s p assin g from th e d ig e s tiv e t r a c t to th e l i v e r .
Moreover, glucose i s absorbed more ra p id ly from th e abomasum
of sheep th an from th e abomasum of non-rum inants (R eid; 1950,
1952).

W eller and Gray (195*0 observed only a 1 0 -fo ld in c re a s e

in s ta r c h passage through th e abomasum when s ta rc h in g e s tio n
was in c re a s e d 1 0 0 -fo ld .

The s ta r c h was not a tta c k e d in th e

abomasum.
Johnson (1951) in fu se d n e u tra liz e d v o l a t i l e ac id m ixtures
in to th e rumens o f f a s te d g oats to dem onstrate th e r a t e s of
a b s o rp tio n w ith o u t exceeding p h y s io lo g ic a l c o n c e n tra tio n s .

22
He concluded t h a t when rumen v o l a t i l e a c id a n a ly s is shows 65
m olar # a c e tic a c id , 20# p ro p io n ic a c id and 15# b u ty ric a c id ,
th e amounts a c tu a lly absorbed a re probably about 60# , 20# and
20# r e s p e c tiv e ly .

This was confirm ed and extended by Pfander

and P h illip s o n (1953) showing th a t a c e tic a c id d isa p p e a rs from
th e rumen i n th e g r e a te s t q u a n tity due to i t s high co n c en tra­
t i o n , b u t p ro p io n ic and p a r ti c u l a r ly b u ty ric a c id s d isap p ea r
a t f a s t e r r a t e s and c o n trib u te equal or g re a te r amounts of
energy to th e rum inant.

This ag rees w ith th e co n clu sio n s of

C a r r o ll and Hungate (195*0 , Gray e t a l . (1951b), Gray and
P ilg rim (1951, 1952a, 1952b), and McNaught (1951).
A rumen liq u id t o n i c i t y of 0.165 molar was re p o rte d by
P a rth a s a ra th y and P h illip s o n (1953).

A bsorption of c h lo rid e

and a c e ta te b ut n o t sodium and potassium was in h ib ite d by
0.002 m olar m ercuric c h lo rid e .

A cetate was absorbed f a s t e r

a t a low er pH (confirm ing r e s u l t s of D a n ie lli et, a l . , 19*+5)
b u t could n ot be absorbed a g a in s t a c o n c e n tra tio n g ra d ie n t.
An a rte rio -v e n o u s d iffe re n c e in sheep head tis s u e of 2
to 3 m illig ram % f o r a c e ta te compared to le s s th a n 2 m illig ram
% f o r g lucose was re p o rte d by Reid (1950) who concluded th a t
a c e ta te might be u t i l i z e d in p re fe re n c e to glucose by rum inant
tis s u e .

J a r r e t t and P o tte r (1950) showed th a t 27 to b0% of

th e a c e ta te in je c te d in tra v e n o u sly in to p h lo riz in iz e d sheep
could g iv e r i s e to su g ar.

This sugar was e x c re te d in th e

u rin e sin c e p h lo r iz in blocked r e s o r p tio n .
was a ls o e x te n siv e under th e se c o n d itio n s .

Form ation of ketones
J a rre tt et a l.

23
(1952), fo llo w in g th e tim e -c o n c e n tra tio n curves f o r blood
v o l a t i l e a c id s in je c te d in tra v e n o u s ly , found p ro p io n ate d i s ­
ap p earing more r a p id ly th an a c e ta te or b u ty ra te .

A cetate

caused no s ig n if ic a n t glycem ia w hile p ro p io n ate was markedly
g lu co g en ic.

A cetate and b u ty ra te alone were markedly k e to -

g e n ic , b u t sim ultaneous in je c tio n of p ro p io n ate prevented
t h e i r k e to n ic e f f e c t .

>r

Pennington (1952, 195^) found th a t rum inant l i v e r and
kidney in v i t r o could u t i l i z e a c e ta te and b u ty ra te and to a
l e s s e r e x te n t, p ro p io n a te .

Kidney was p a r ti c u l a r l y a c tiv e

w h ile h e a rt muscle weakly u t i l i z e d a c e ta te and b u ty r a te , but
not p ro p io n a te .
Less th a n % of th e in je c te d a c e ta te (500 m illig ra m s/
kilogram body w eight) appeared in th e u rin e of 16 normal ewes
s tu d ie d by Pugh and S c a ris b ric k (1952).

The h a lf-tim e fo r

d isap p ea ran ce of blood a c e ta te in normal sheep was 25 m inutes
v e rsu s 60 m inutes in k e to n ic sheep.

A llen et, a l . (1955) p o in t

out t h a t both h e p a tic and e x tra -h e p a tic c e l l s , in c lu d in g even
b r a in , can o x id iz e both sugars and f a t t y a c id s .

They demon­

s tr a te d a b a s a l f a t t y a c id catab o lism o ccu rrin g g e n e ra lly in
t i s s u e s which was not suppressed even by abundant p resen ce of
c a rb o h y d ra te .

B en tley e t a l . (195*+a) found 1 pound of l a c t i c ,

a c e t i c , o r p ro p io n ic a c id e q u iv a le n t e n e r g e tic a lly to 2 .7 5 ,
1 .6 , or 1.*+ pounds o f corn when fed to sheep.
I n d ir e c t evidence f o r th e e s s e n t i a l i t y of a c e ta te in
fo rm a tio n of m ilk f a t s t a r te d accum ulating when Powell (19^1)

2k

found th a t a r a t i o n of se v e re ly r e s t r i c t e d or v ery f i n e l y
ground roughage d ep ressed th e f a t co n ten t of m ilk.

F u rth e r

stu d y of t h i s phenomenon by A llen and co-w orkers was review ed
by Tyznik (1951).

The d epressed f a t co n ten t caused by r e ­

s t r i c t e d roughage-high c o n c e n tra te r a ti o n s or by f i n e l y ground
roughage could be re v e rse d by o r a l a d m in is tra tio n of sodium
a c e ta te .

Although Tyznik (1951) found a lowered rumen concen­

t r a t i o n of a c e tic a c id on th e se r a t i o n s , both th e rumen f e r ­
m en tatio n and th e b u t t e r f a t t e s t of most cows spontaneously
r e v e r te d to normal w ith in a few weeks.
B alch e t a l . (1952a, 195^a, 195^b) stu d ie d th e d e p re ssio n
o f th e f a t c o n ten t of m ilk a s s o c ia te d w ith changing cows from
dry feed to p a s tu re .

They co n sid ered t h i s an e f f e c t of th e lack

of co a rse m a te ria l in young p a s tu re .

According to th e s e work­

e r s , th e s o lid s not f a t co n ten t i s not a ffe c te d by a r e s t r i c t e d
roughage r a t i o n .

High c o n c e n tra te r a tio n s depressed th e f a t

c o n te n t re g a rd le s s of t h e i r p ro te in c o n te n t; however, th e
e f f e c t became l e s s sev ere l a t e r in la c t a t i o n .

Hay consumption

had to be l e s s th a n 12 pounds per day per cow to produce th e
e f f e c t , but d e p re ssio n was not fu rth e re d by d ec reasin g hay
in g e s tio n below 8 pounds.
M ilk f a t p ro d u c tio n was stim u la te d 3.2$ in cows fe d 350
gm. of a c e ta te d a ily by Z e lte r (1952).

P yruvate fe ed in g a lso

caused some s tim u la tio n over th e c o n tro ls w hile no s ig n if ic a n t
in c re a s e in p ro d u ctio n could be a tt r i b u t e d to b u ty ra te .

Van

S o est e t a l . (195*+) confirm ed th e se r e s u l t s using cows whose

25
m ilk f a t p ro d u c tio n had been depressed by a r e s t r i c t e d rough­
ag e-h ig h c o n c e n tra te r a ti o n and found th a t th e in c re a se d p ro ­
d u c tio n accounted fo r a l l o f th e carbon ad m in istered as a c e ta te .
However, M ille r and A llen (1955) could not fin d s ig n if ic a n t
d if f e r e n c e s i n e i t h e r th e f a t t e s t or th e amount o f m ilk
s e c re te d between c o n tro l cows fe d hay p lu s corn s ila g e and
16 pounds o f c o n c e n tra te d a ily and experim ental cows fed th e
same r a t i o n p lu s one pound of sodium a c e ta te .

The experim ent

could d e te c t d iffe re n c e s of 0.1# in f a t t e s t or 0 .8 pounds of
m ilk /d ay .
This i n d i r e c t evidence f o r a c e ta te u t i l i z a t i o n in m ilk
f a t s y n th e s is i s augmented by mammary gland s tu d ie s review ed
by Popjak (1952).

F o lle y and French (19^8) re p o rte d th a t

rum inant l i v e r s l i c e s do not u t i l i z e a c e ta te b e tt e r th an r a t
l i v e r s l i c e s , but rum inant kidney and mammary tis s u e s do have
a g r e a te r a c e ta te u t i l i z i n g a b i l i t y th an e q u iv a le n t non-rum in an t t i s s u e s and even show a g re a te r u t i l i z a t i o n of a c e ta te
th a n g lu c o se .

Ruminant mammary tis s u e u t i l i z e s a c e ta te but

n o t glucose w ith an R.Q. g re a te r th an 1.

Non-ruminant mammary

ti s s u e u se s only g lucose w ith an R.Q. g re a te r th an 1.

When

Popjak et, a l . (1951a, 1951b) in je c te d la c ta t in g g o ats w ith
c a rb o x y -G ^ -a c e ta te , th e maximum s p e c if ic a c t i v i t y of th e
plasma f a t a c id o ccurred in 2*+ hours whereas s p e c if ic a c t i v i t y
of m ilk f a t was maximum in 3 to k hours and was s e v e ra l tim es
g r e a te r th an th e plasma f a t maximum.

During th e f i r s t 12 hours

a f t e r i n j e c t i o n , a c t i v i t y of th e 8 to 12 carbon a c id s in m ilk

26

exceeded t h a t f o r th e 1*+ to 16 carbon a c id s .

H alf-tim e f o r

th e d isap p earan ce of blood a c e ta te was 50 m inutes v e rsu s H
hours f o r th e v o l a t i l e a c id s of m ilk.

Only th e odd-numbered

carbons of a c e ta te , b u ty r a te , and ca p ro ate were a c tiv e and
th e carb o x y l-carb o n of ca p ro ate was more a c tiv e than carbons
3 or 5.

About bo# o f th e m ilk b u ty ra te came from a c e ta te v ia

b e ta co n d en satio n and 60 # from some n o n -is o to p ic ^-carb o n
compound.

Higher a c id s were formed e x c lu siv e ly by a stepw ise

co n d en satio n of a c e ta te w ith c a p ro a te .

F if ty p ercen t of th e

n o n -o x id iz e d , la b e le d a c e ta te was used f o r milk f a t s y n th e s is .
Cowie et, a l . (1951b) c ir c u la te d a c e ta te through an i s o ­
la te d bovine udder and recovered bo% of i t in th e m ilk f a t
along w ith tr a c e s in m ilk c h o le s te r o l and la c to s e .

S ince

b u ty ric a c id s p e c if ic a c t i v i t y corresponded w ith th a t of th e
o th e r a c id s , th e U-carbon p re c u rso r of m ilk b u ty ra te noted
above came from blood.

Carbon dioxide in c o rp o ra tio n in to m ilk

f a t and la c to s e was n e g lig ib le .

Shaw and P e te rse n (19*+3)

showed t h a t th e bovine mammary gland r e a d ily u t i l i z e s b e ta hy d ro x y b u ty ric a c id and Kumar and Lakshmanan (195*0 showed
t h a t t h i s a c id i s probably th e H-carbon p re c u rso r of m ilk
b u ty ra te h y p o th esized by Popjak (1952).
Glucose was more e f f e c tiv e than a c e ta te as a p re c u rso r
f o r m ilk f a t in th e r a b b it mammary te s te d by Popjak e t a l .
(1953) b u t th e s y n th e s is was s t i l l v ia pyruvate and some 2carbon u n i t .

G ly cero l was a lso formed much more slow ly from

a c e ta te th an g lu co se.

27
McClymont (1951) found *+0 to 80$ of th e a c e ta te removed
from blood c ir c u la tin g through th e i n t a c t , l a c t a ti n g bovine
mammary.

A r t e r i a l a c e ta te le v e ls were not a ffe c te d by th e

high c o n c e n tr a te - r e s tr ic te d roughage r a ti o n although th e milk
f a t t e s t was d ep ressed .

H yperinsulinism or delayed m ilking

a ls o f a i l e d to a f f e c t a c e ta te u t i l i z a t i o n .

A high c o r r e la tio n

was found between CC>2 p ro d u ctio n and a c e ta te u t i l i z a t i o n by
th e mammary t i s s u e even when excess a c e ta te was ad m in iste red .
I t was concluded t h a t th e v o l a t i l e a c id o f m ilk i s independent
o f blood v o l a t i l e f a t t y a c id and th a t a c e ta te i s used in th e
mammary gland f o r e ith e r s y n th e s is or u s e fu l o x id a tiv e meta­
b olism .
B ryant and D oetsch (195l+> 1955) and B en tley e t a l . (195*+b)
have found t h a t a m ixture of th e branched-chain v o l a t i l e ac id s
and s tr a ig h t- c h a in 5 to 8 carbon a c id s norm ally p re se n t in th e
rumen i s re q u ire d fo r growth of some rumen m icroorganism s and
f o r optimum c e llu lo s e d ig e s tio n and n itro g e n u t i l i z a t i o n .
Rumen S y n th e sis o f C arbohydrate
S ev eral rumen m icroorganism s, examined by Baker (19*+2),
s y n th e siz e d io d in e s ta in in g p o ly sa c c h a rid e .

These organism s

were not s u b je c t to p e p tic or t r y p t i c d ig e s tio n , but were
d ig e s te d in la rg e q u a n titie s by th e c i l i a t e p ro to zo a .

Baker

and H a rris s (19*47) review ed th e su b je c t of s y n th e s is by rumen
m ic ro b io ta .

A rumen p rotozoan, c u ltu re d by Hungate (19*+3),

produced a re s e rv e io d in e s ta in in g p o ly sacch arid e 2 to 7 hours

28
a f t e r exp osu re to c e l l u l o s e .

The p o ly sa cc h a r id e d isap peared

w ith in 12 hours a f t e r removing th e c e l l u l o s e .

Van der Wath

(19*+8) confirm ed th e a b i l i t y o f rumen m icro b io ta to s y n th e s iz e
io d o p h ilic p o ly sa c c h a r id e from s ta r c h .
Heald (1951) prepared r e l a t i v e l y pure b a tch es o f rumen
m icroorganism s from hay fe d sheep and found th a t th e y co n ta in ed
*+ to % g lu c o s e -y ie ld in g -c a r b o h y d r a te on th e dry b a s is .

A

maximum o f 5 gm. o f g lu c o se per day p assed from th e abomasum
o f hay fe d sheep assuming th a t a l l abomasal p assage was rumen
Masson and P h illip s o n (1952) showed th a t tw o -th ir d s

in g e s t a .

o f t h i s m a te r ia l was g a s t r ic j u ic e w hich would r e v is e th e
e s tim a te t o about 2 gm.

T his amounts to a n e g lig i b l e p o r tio n

o f th e t o t a l fe e d en erg y .

W eller and Gray (195*0 used th e lig n in r a t i o technique
w ith rumen and abomasal f i s t u l a e to show th a t s ta rc h was
la r g e ly d estro y ed in th e omasum along w ith th e p ro to zo a.
S ta rc h p assin g through th e abomasum in c re a se d from 1 gm. per
day when 3 gm. were fe d to only 8 gm. when 1^8 gm. were fe d .
D e s tru c tio n o f s ta rc h in th e rumen and omasum was alm ost com­
p le te .

Heald (1951) found th e ferm en tab le, g lu c o se -c o n ta in in g

carb o h y d rate c o n ten t of th e rumen m icro b io ta in c re a se d from a
p re feed in g le v e l of 2 to 7% to 7 to 21$ of th e dry m atter 2
hours a f t e r fe e d in g .

This re tu rn e d to th e p re feed in g le v e l

w ith in 6 to 8 h o u rs.

C e n trifu g a tio n showed t h a t t h i s s y n th e s is

was la r g e ly a fu n c tio n o f th e in te rm e d ia te and la rg e sized
organism s.

29
H o lo tr ic h c i l i a t e s from th e rumen o f hay fe d sheep d e p o s it
a p o ly sa c c h a r id e w hich Oxford (1951) and Masson and Oxford

(1951) found to be s ta r c h .

T his s t a r c h , which cou ld be p re­

pared w ith a 1$ y ie ld from the dry m atter o f s tr a in e d rumen
l iq u id from sheep grazed on s t a r c h - f r e e sp rin g g r a s s , was
s y n th e s iz e d in v i t r o from g lu c o s e , f r u c t o s e , s u c r o s e , in u lin
and b a c t e r i a l le v a n w ith 15 to 21$ y i e l d s and from c e llo b io s e
w ith a 6% y i e l d , but n ot from m a lto se .
McNaught (1951) reco v ered 15 t o 39$ o f th e carbon o f
m a lto s e , a r a b in o se , or x y lo s e as b a c t e r ia l p o ly sa cc h a r id e
when th e y were ferm ented in v i t r o by rumen m icr o b io ta .

Me

Naught e t a l . (195M found p o ly sa cc h a r id e accou n tin g fo r 32$
o f th e rumen b a c t e r i a l dry m atter and 62$ o f th e p r o to z o a l
dry m a tter .

Sugden (1953) found th a t c e r t a in o l i g o t r i c h pro­

to z o a co u ld not form p o ly sa c c h a r id e s from sim ple su gars or
c e l l u l o s e when t h e ir sy m b io tic b a c t e r ia were e lim in a te d by
stre p to m y c in .
R obinson e t a l .

(1955) concluded from t h e ir work w ith

washed su sp e n sio n s o f rumen m icro b io ta th a t p o ly sa c c h a r id e
s y n t h e s is i s probably a mechanism w hich e n a b le s th e m icro b io ta
to s t o r e a r e s e r v e en ergy su p p ly .

They f a i l e d t o o b ta in t h i s

s y n t h e s is w ith m a lto se s u b s tr a te w hich con firm s th e work o f
Masson and Oxford (1951).

30
Rumen S y n th esis of p ro te in
The l i t e r a t u r e concerning u re a as a p ro te in replacem ent
f o r ru m inants has been review ed by Reid (1953)•

According to

t h i s rev iew , Zuntz recognized th e v alue of u re a as a p a r t i a l
p r o te in replacem ent f o r rum inants in 1891.

H art e t a l . (1939)

confirm ed th e v alu e o f sim ple n itro g en o u s compounds as p ro te in
s u b s t i tu t e s fo r rum inants and considered th e rumen m icrobiota
re s p o n s ib le f o r t h e i r u t i l i z a t i o n .

The l i t e r a t u r e reviewed

h ere p e r ta in s to th e r o le of rumen m icrobiota in t h i s p ro te in
s y n th e s is .
Pearson and Smith (19*+3b) concluded th a t p ro te in sy n th e sis
d u rin g rumen fe rm e n ta tio n , in vivo or in v i t r o , i s accompanied
by p r o te in catab o lism and e ith e r process may predom inate. . The
amount of s y n th e s is during th e f i r s t two hours of in cu b atio n
o f c e n trifu g e d rumen liq u id w ith 0.3$ s ta rc h and u re a added
was e q u iv a le n t to 8 m illigram s N/100 gm. rumen liq u id which
would amount to some ^50 gm. of p ro te in in the in t a c t bovine
rumen.

S y n th e sis was stopped by l e s s than 0.1$ sodium f lu o r id e

in d ic a tin g i t s m ic ro b ia l n a tu re .

S tarch had a r e l a t i v e l y high

s tim u la to ry e f f e c t on s y n th e s is compared to o th e r compounds
s tu d ie d w h ile 1$ g e la tin markedly in h ib ite d s y n th e s is .
a c id s had r e l a t i v e l y l i t t l e e f f e c t .

Amino

Burroughs e t a l . (1951)

examined th e e f f e c t of v a rio u s p ro te in s on u re a u t i l i z a t i o n
d u rin g rumen fe rm e n ta tio n in v i t r o and concluded th a t the
n itro g e n req u irem en ts of rumen m icro b io ta are r e s t r i c t e d to
ammonia.

31
About 5-0$ of th e t o t a l rumen n itro g e n i s m icro b ial accord­
ing to Gray et, a l . (1953).

Moreover, th e r a t i o of t o t a l n i t r o ­

gen to l i g n i n i s n e a rly th e same in th e rumen as in th e fodder
a t a l l tim es a f t e r feed in g so n e t lo s s of n itro g e n from th e
rumen occurs only w ith lig n in passage to th e lower alim en tary
tra c t.

McDonald (195*0 pointed out th a t th e in s p lu b le p ro te in ,

z e in , does not a l t e r th e ammonia c o n c e n tra tio n in th e rumen
w h ile th e so lu b le p r o te in s , c a se in and g e la tin , cause marked
in c r e a s e s .

When sheep were fed a p a r t i a l l y p u rifie d r a tio n

to which z e in c o n trib u te d 9*+$ of the t o t a l n itro g e n , approxi­
m ately **0$ o f th e z e in was converted to m icro b ial p ro te in .
Using s te e r s fe d n a tu r a l and p u rifie d r a ti o n s , Agrawala
e t a l . (1953) and Duncan et. a i . (1953) stu d ie d p ro te in synthe­
s i s by th e rumen passage technique of Hale et. a l . (19*+0, 19*+7).
Minimum v a lu e s f o r p ro te in sy n th e sis v a rie d from 33 to 109 gm.
w h ile c o n sid e ra b le amino a c id s y n th e sis was dem onstrated on
th e r a ti o n co n ta in in g only n o n -p ro te in n itro g e n .

Smith (1953)

confirm ed th e s y n th e s is of ly s in e u t i l i z i n g th e same technique
w ith n a tu r a l r a ti o n s , b ut could not dem onstrate an accum ulation
of m ethionine in th e rumen.
McNaught (1951) found th a t cooked s ta rc h provided a b e tte r
source of energy f o r p r o te in sy n th e sis than raw s ta rc h ; a r a b in o se, x y lo s e , f r u c to s e , and c e llo b io s e were p a r tic u la r ly good
energy so u rces w ith m altose and glucose ranking as in te rm e d ia te ;
g lu c u ro n ic , a c e t ic , p ro p io n ic , b u ty r ic , c i t r i c , m alic, t a r t a r i c ,
fu m aric , s u c c in ic , l a c t i c , and beta-h y d ro x y b u ty ric a c id s and

32
mannose or sorbose were e ith e r poor o r in e f f e c tiv e so u rces.
B a c te r ia l p ro te in accounted fo r 7 to 18# of th e carbon of
m alto se, a ra b in o se , or x ylose ferm ented in v i t r o by rumen
m ic ro b io ta .

A rias et, aji. (1951) noted th a t sm all amounts of

so lu b le carb o h y d rates (0 .1 to 0 .2 gm./lOO ml. volume in th e
fe rm e n ta tio n f la s k ) aided c e llu lo s e d ig e s tio n which in tu rn
y ie ld e d energy f o r p ro te in s y n th e s is .

S alsbury (1955) con­

firm ed t h i s s tim u la to ry e f f e c t of 0 .1 to 1% so lu b le carbohy­
d r a te s .
A low r a t e o f sugar and c e llu lo s e fe rm en tatio n by rum inal
c o n te n ts from sheep f a s te d ^8 hours was re p o rte d by Quin e t a l .
(1951)*

The fe rm e n ta tio n a b i l i t y re v e rte d to normal f a s t e r

w ith a low p r o te in r a ti o n than w ith a high p ro te in r a tio n .
B e ll jgt, a l . (1953) s tu d ie d r e te n tio n of u rea n itro g e n by
s t e e r s fed d if f e r e n t carbohydrates concluding th a t u re a n i t r o ­
gen was u t i l i z e d w ith eq u al e f f ic ie n c y in r a tio n s of d if f e r e n t
c e r e a l g ra in s b ut w ith l e s s e f fic ie n c y in m olasses co n tain in g
r a ti o n s .
W alter (1952) s tu d ie d th e e f f e c t of a number of carbohy­
d r a te s on th e magnitude of rumen fe rm en tatio n in v i t r o and
found t h a t a c t i v i t y w ith sucrose s u b s tra te exceeded s l i g h t l y
t h a t w ith glucose which was s e v e ra l tim es g re a te r th an a c ti v i t y
w ith x y lo s e , h e m ic e llu lo se , and s ta rc h s u b s tr a te s .

When b a rle y

supplem ented th e a l f a l f a r a tio n of the ovine source of rumen
m ic ro b io ta , fe rm e n ta tio n on a l l s u b s tra te s was in t e n s i f ie d .
According to Reid (1953), e f f i c i e n t p ro te in sy n th e sis from

33
n o n -p ro te in n itro g e n depends upbn an optim al balance between
ammonia l i b e r a t i o n and carbohydrate fe rm e n ta tio n .
Reed e t a l . (19*+9) found th a t th e d i g e s t i b i l i t y and b io ­
lo g ic a l v alu e o f rumen b a c te r i a l p ro te in fo r r a t s a re 62 to
65$ and 79 to 80$ compared to 101$ and 80$ fo r c a s e in .

Crude

p r o te in c o n s titu te d ^8 to 51$ of th e dry m atter of th e b a c te ­
r i a l p re p a ra tio n .

McNaught (195*0 confirmed th ese v alu es and

found a d i g e s t i b i l i t y of 91$ fo r th e p ro te in of rumen protozoa.
The amino ac id com position of rumen b a c te r i a l p ro te in ,
stu d ie d by Holmes e t a l . (1953), v a r ie s l i t t l e i f any w ith the
change from dry feed to p a s tu re .

The amino acid com position

o f rumen b a c t e r i a l p r o te in i s s im ila r to recorded v alu es f o r
a number of m icroorganism s excepting f o r a somewhat higher
c o n te n t o f a rg in in e , h i s t i d i n e , tryptophane and glutam ic acid
and lower co n ten t of is o le u c in e .

The content of le u c in e ,

th re o n in e , p h en y lalan in e and p a r tic u l a r ly m ethionine and
is o le u c in e was su b -o p tim al f o r maximum n u t r i t io n a l v alue as
a ls o noted by Reed et. a l . (19*+9).

Duncan e t a l . (1953) con­

firm ed th e low m ethionine content of rumen b a c te r ia l p ro te in
but found somewhat h ig h er p ro p o rtio n s of is o le u c in e , le u c in e ,
p h en y lalan in e and th re o n in e w ith lower p ro p o rtio n s of a rg in in e ,
h i s t i d i n e and try p to p h an e.

The m ethionine co n ten t of rumen

dry m atter was very low as re p o rte d by Chance et. a l . (1953b)
and Smith (1953).
I n v e s tig a tio n s of th e s u lfu r amino acid requirem ents of
ru m in an ts began when Huffman and Duncan (19*+0) f a ile d to

s tim u la te m ilk p ro d u ctio n w ith c y stin e supplem entation to a
legume hay r a t i o n .

However, Smuts e t a l . (19*+1) found th a t

sup p lem en tatio n of a l f a l f a w ith c y stin e in creased r e te n tio n
of th e a l f a l f a n itro g e n in lambs.

A lfa lf a c o n ta in s 0.3$

s u lf u r according to D avis g t a^. (195*+) and Lewis (195*0
c i t e s v a lu e s fo r s u l f a t e - s u lf u r in fo ra g es ranging from 0 .3
to 1.0$ of th e dry m a tte r.
pared to o th e r fe e d s .

A ll of th e se are high v alu es com­

L o o sli and H a rris (19*+5), Jones and

Haag (19*+6), S ta rk s e t a l . (195*0 and Noble e t a l . (1955)
have dem onstrated in c re a se d n itro g e n r e te n tio n and/or growth
or p ro d u c tio n when s u lf u r supplemented b a sa l r a tio n s c o n ta in ­
ing e i t h e r l e s s th an 0.1$ or u n sp e c ifie d amounts of t o t a l
s u lf u r .

M ethionine s u lf u r i s s l i g h tl y more v alu ab le than

in o rg a n ic s u lfu r according to L o o sli and H a rris (19*+5).

U t il ­

i z a t i o n of f i e l d pea o r a l f a l f a p ro te in by sheep was stim u la te d
by m ethionine supplem entation as re p o rte d by Klosterm an e t a l .
(195*0 who noted th a t th e p ro te in s of th e se fe ed s a re d e f ic ie n t
in m ethionine.

Hunt e t a l . (195*0 re p o rte d a g re a te r in v i t r o

a c t i v i t y of rumen m icro b io ta when m ethionine or sodium s u lf a te
su p p lied t h e i r s u lf u r th an when c y s tin e or elem ental s u lfu r
served t h i s fu n c tio n .
Block and S tek o l (1950), Block e t a l . (1951), and Block
( 1953) dem onstrated conversion of in g ested ra d io a c tiv e , in o r­
ganic s u l f a t e to c y s tin e and m ethionine in th e rumen.

The

m ilk p r o te in , which a ls o contained ra d io a c tiv e c y stin e and
m eth io n in e, a tta in e d a maximum a c t i v i t y in 2 to 3 days.

The

35
r a t i o s o f c y s tin e n itro g en -m eth io n in e n itro g e n in rumen m icro­
b i o t a , serum album in, and m ilk p ro te in were about l s l , 6:1 and
1 :5 r e s p e c tiv e ly and, th e r a t i o s o f c y s tin e a c tiv ity :m e th io n in e
a c t i v i t y follow ed th e same p a tte r n .

M ethionine and c y stin e

v e re a p p a re n tly formed in equal amounts in th e rumen.

Since

Lewis (195*0 found t h a t s u lf a te d isa p p e a rs from th e rumen w ith
a h a lf-tim e o f l e s s th an 2 hours accompanied by a sim ultaneous
in c re a s e i n rumen s u l f i d e , s u lf a te u t i l i z a t i o n must proceed
la r g e ly v ia s u l f i d e .

Rumen raicro b io ta degrades c y ste in e to

ammonia, s u l f i d e , C02 , and a c e tic acid according to S iro tn ak
e t a l . (1953) and Lewis (195*0.
Rumen s u l f a t e and s u lfid e c o n c e n tra tio n s are norm ally
about 0 .1 5 and 0 .0 8 m illig ram eq u iv alen ts/1 0 0 ml. as re p o rte d
by P a te l (1952) and Lewis (195*+).

Weir and Rendig (195*+)

re p o r t serum in o rg a n ic s u lfu r v alu es o f 0.02 m illig ram $ in ­
c re a s in g to k m illig ram % as s u lf u r in g e s tio n was in c re a se d .
About 70$ o f th e in o rg a n ic , ra d io a c tiv e s u lf a te fed w ith
s ila g e to cows by Keener e t a l . (1953) was recovered in th e
u rin e along w ith 1 to 2$ in th e milk and th e rem ainder in th e
fe c e s .

Kulwich e t a l . (195*0 o b tain ed a s im ila r s u lf a te p a r­

t i t i o n i n sheep fed soybean m eal, but when u re a provided the
d ie ta r y n itro g e n , only h a lf of th e a c t i v i t y appeared in u rin e
in d ic a tin g in c re a se d in c o rp o ra tio n by rumen m icro b io ta.
The review s by B e rsin (1950) and Fromageot (1953) re v e a l
th e c e n tr a l r o le of s u lf u r and s u lfu r amino ac id s in m etabolism .
This i s p a r t i c u l a r l y tru e fo r c y s te in s u lf in ic ac id which appears

36

to be a key in te rm e d ia te among s e v e ra l m etabolic a c id s and
amino a c id s .

These amino a c id s in clu d e hypotaurine and ta u rin e

which a re u rin a ry e x c re tio n products and are a lso sy n th esized
by th e ch ick embryo (Chatagner e t a l . , 1955; Lowe and R o b erts,
1955; M acklin e t a l . , 1955).

Their c h a r a c t e r is t ic s a re sim ila r

to th e u n id e n tif ie d compounds sy n th esized by b a c te r ia noted by
Cowie e t a l . (1950, 1951a).

Block e t a l . (1952) d isc u ss the

r o l e of g lu ta th io n e in tr a n s f e r of amino ac id s to p ro te in .
Horowitz (19^7) is o la te d c y s ta th io n in e and showed th a t
N eurospora s y n th e s iz e s m ethionine from in o rg an ic s u lf u r v ia
c y ste in e , c y s ta th io n in e , and homocysteine c o n se c u tiv e ly .

Cowie

e t a l . (1950, 1951a, 1952), B olton e t a l . (1952), W ijesundra
and Woods (1953)? and th e work of o th e rs reviewed by them
showed th a t th e same g e n e ra l mechanism a p p lie s in o th er m icro­
organism s.

Hess (1952), A rn stein (1953) and t h e i r l i t e r a t u r e

review s show e s s e n t i a l l y th e same mechanism in r a t tis s u e only
re v e rs e d ; th a t i s , m ethionine goes to homocysteine to c y s ta ­
th io n in e .
To w hatever e x te n t E sc h e ric h ia c o ll metabolism p a r a l le ls
th a t o f rumen m ic ro b io ta , th e follow ing f a c ts found by Cowie
e t a l . (1950, 1951a, 1952, 19 5*0, and B olton e t a l . (1952)
apply to t h i s problem :

In o rg an ic s u lf a te of media and c e l l s

i s in in sta n ta n e o u s e q u ilib riu m w hile the o rg a n ic , cell-b o u n d
s u lf u r amounts to 3 .5 m illigram s S/ml. c e n trifu g e d c e l l s and
i s s ta b le ; in o rg a n ic s u lf a te i s not bound by non-growing c e ll s
and s u lf u r in c o rp o ra tio n by growing c e ll s i s d i r e c tl y c o r re la te d

37
w ith p r o te in s y n th e s is provided adequate s u lfu r i s p re se n t;
g lu ta th io n e accounts f o r 25 to 2,0% of th e o rg a n ic , cell-hound
s u lf u r and a c ts as re s e rv e s u lfu r fo r c e l l growth in th e
absence o f an e x te r n a l supply; c y ste in e and s u l f i t e s u lfu r
markedly su ppress w hile m ethionine and homocysteine s u lfu r
only s l i g h t l y suppress in c o rp o ra tio n of s u lf a te s u lfu r in to
c e l l s ; i t i s concluded th a t s u lf a te s u lfu r i s in co rp o rated
as c y s te in e v ia s u l f i t e w hile m ethionine i s formed more slowly
v ia c y s te in e .

i

PART I

INCORPORATION OF INORGANIC SULFATE BY
RUMEN MICROBIOTA IN VITRO
P relim in a ry Experim ents
F erm en tation T echnique.

This experim ent was i n i t i a t e d

in 1953 w ith th e o b je c t o f measuring c e l l growth and p r o l i f ­
e r a t io n during in v i t r o ferm en ta tio n s by rumen m icro b io ta .

This i n i t i a l study u t i l i z e d th e fe rm en tatio n method d esc rib ed
by S alsb u ry (1955* pp. 73-76) w ith th e m ineral complex m odified
by s u b s titu tin g c h lo rid e s a l t s fo r the e q u iv a le n t s u lf a te s .
About 0 .8 gm. of a i r dry a l f a l f a l e a f meal was placed in s id e
th e d ia ly z in g sacs w ith 25 ml. of c h e e se -c lo th s tra in e d rumen
liq u id from f i s t u l a s te e r C7*+l re c e iv in g an a l f a l f a hay r a tio n .
C aution was used in t h i s and th e follow ing experim ents to avoid
d e la y , tem p eratu re change, and a i r exposure in tr a n s f e r r in g
in o c u la from th e rumen to th e fe rm en tatio n v e s s e l.

These in -

o cu la were always c o lle c te d about 2 hours a f te r fe ed in g .
Follow ing in o c u la tio n , th e d ia ly z in g sac was suspended in
175 ml. o f m ineral complex co n tain in g about 2 .5 m icro cu ries
of s 3 5 -ia b e le d in o rg a n ic s u lfa te /m l.

The c a r r ie r s u lf a te

c o n c e n tra tio n was l e s s th an U.5 x 10 9 m oles/m l.

One complete

assembly was incu b ated a t *+0°C. fo r each time of 1 , 2 , *+, and
6 hours and two com plete assem blies were r e f r ig e r a te d as
c o n tr o ls .

38

39
A fte r in c u b a tio n , th e r e s id u a l in o rg an ic s u lf a te a c ti v i t y
was exchanged out by d i a l y s i s a g a in st d i l u te ammonium s u lf a te
a t 6°C.

Thick smears o f th e d ialy zed fe rm en tatio n re sid u e

were s h ie ld e d so a c o n sta n t area was exposed and counted under
an end-window, G-M d e te c to r .

These counts are p resen ted in

Table 1, page *t0.
The c o n tro l f l a s k s , d esig n ated as 0 and O ', were exposed
to th e r a d io a c tiv e m ineral mix fo r 1 and 3 hours re s p e c tiv e ly
b efo re exchange a g a in s t ammonium s u lf a t e .

The a c ti v i t y in th e

c o n tro ls was a p p a re n tly in o rg an ic s u lf a te as dem onstrated by
th e chrom atographic r e s u l t s .

This dem onstrated incom plete

exchange w ith n o n -lab e le d s u lf a te in s p ite of th e f a c t th e
ammonium s u lf a te s o lu tio n s were changed re p e a te d ly u n t i l
p r a c t i c a l l y no exchange a c ti v i t y could be d e te c te d .

For t h i s

and o th e r te c h n ic a l re a so n s, th e d ia ly z in g sac was not used
a fte r th is t r i a l .

A supplem ental experim ent dem onstrated th a t

in o rg a n ic s u lf a te e q u ilib r a te s acro ss th ese v isk in g tubing
d ia ly z in g sacs a t a r a t e of 2\% per m inute.

T herefore, the

ap parent r a t e o f s u l f a t e in c o rp o ra tio n i s a fu n c tio n of both
th e r a t e o f bin d in g by the c e l l s and th e r a t e of s u lf a te d i f ­
fu s io n in to th e fe rm e n ta tio n sac.
The rem aining fe rm e n ta tio n re sid u e s were hydrolyzed w ith
6m HC1, th e excess ac id removed by ev ap o ratio n , and th e amino
a c id s s e p a ra te d ch ro m atographically on f i l t e r paper as d e s­
c rib e d by P a tto n (1950) w ith th e ex cep tio n th a t la r g e r paper
and c y lin d e rs were used .

The developed chromatograms were cut

TABLE 1

SULFUR ACTIVITY IN DIALYZED FERMENTATION RESIDUES

In c u b a tio n
Time
(Hours)

R a d io a c tiv ity
in th e C e lls
^et

2R e la tiv e

^ R elativ e A c tiv itie s
C vstine
--------

Methionine
--------

0

11.13

--------

0*

23.03

--------

0

0

1

1U.88

^1 .9

M-.7

0

2

23.66

66.7

8.2

5.8

U

3H.00

95.8

H8.7

9.0

6

35.^9

100.0

26.5

11.9

1. Counts per second c o rre c te d f o r n a tu r a l background a c t i v i t y .
2. Each count as i t s p ercen tag e of the s ix hour count.
3. Area under curves o b tain ed by graphing a c ti v i t y v ersu s Rf
v a lu e .

kl
in to s t r i p s and the s t r i p s counted as d escrib ed by W interin gf
ham e t a l . (1952). M ethionine y ie ld e d an Rf of 0.85 under
th e se c o n d itio n s .

While a l l of the s t r i p s from incubated

f la s k s co n tain ed a p p reciab le a c t i v i t y near Rf 0 .8 and Rf 0 .2 ,
over 95# o f th e a c t i v i t y in the c o n tro ls was below Rf 0 .2 .
Although t h i s c o n c lu siv e ly dem onstrated m ethionine s y n th e s is ,
c y s tin e was o b lite r a te d by in o rg an ic s u lf a te .

The chromato­

graphing^ p ro cess was re p e a te d using a so lv en t m ixture of 80#
m ethanol, 18# w a te r, and 2# g la c ia l a c e tic acid which gave an
Rf o f 0 .7 f o r m ethionine and an Rf t a i l i n g from 0 .5 f o r c y s tin e .
In o rg a n ic s u lf a te d id not move above Rf 0 .2 .

The r e l a t i v e

a c t i v i t i e s corresponding to m ethionine and c y stin e are p re ­
sen ted in Table 1, page *+0.
Hvdroeen S u lfid e P ro d u ctio n .

This experim ent was con­

ducted to measure any lo s s of ra d io a c tiv e s u lfu r as fre e
hydrogen s u lf id e .

A 25 ml. p o rtio n of c h e e se -c lo th s tra in e d

rumen l i q u id from hay fed rumen f i s t u l a cow A55 was added to
each of two Uo ml. c e n trifu g e tubes co n tain in g 1 gm. o f a l f a l f a
l e a f meal and 750 m icro cu rie s of
o rg an ic s u l f a t e .

as 1 x 1 0 moles of i n ­

The tu b es were closed w ith sto p p e rs c o n ta in ­

ing c a p il l a r y tu b es jo in ed to a m anifold.

A ir was drawn

through th e m anifold a t a s l i g h tl y reduced p re ssu re and washed
through c u p ric a c e ta te .

A fte r 3 hours in cu b atio n a t *+0°C.,

th e fe rm e n ta tio n was stopped by cooling and the s u lfid e o x i­
d ized to s u lf a te w ith hydrogen peroxide.

The cu p ric oxide-

c u p ric s u l f a t e re sid u e was th en d isso lv e d in h y d ro ch lo ric

h2
a c id .

T h is s o lu tio n contained le s s than O.W m icrocuries of

s35.
G lu tath io n e P ro d u c tio n .

This experim ent was concerned

w ith th e form i n which th e ra d io a c tiv e c y s tin e and c y ste in e
o ccu rred in th e c e l l s .

Tw enty-five ml. p o rtio n s of cheese­

c lo th s tr a in e d rumen liq u id from rumen f i s t u l a s te e r C707
(re c e iv in g a r e s t r i c t e d roughage-high co n c en trate r a tio n )
were added to 50 ml. Erlenmeyer f la s k s co n tain in g 0.73 gm.
of c o n c e n tra te m ix tu re.

S u f f ic ie n t s3 5 -ia b e le d s u lf a te was

added to g iv e an a c t i v i t y o f 7.** m icro cu ries/m l.

One f la s k

was th en incu b ated

a t Uo°G. fo r each of th e follow ing tim es;

0 9 0 .5 , 1, 1 .5 , 2 ,

3, k and 6 hours.

The fe rm e n ta tio n
s u f f i c i e n t e th a n o l
e th a n o l.

was stopped by pouring the ferm ent in to

to y ie ld a f i n a l c o n c e n tra tio n of 80$

A fter a d d itio n o f 0.002 moles of s u lf u r ic ac id and

k to 6 m illig ram s of c y s te in e hy d ro ch lo rid e as c a r r i e r , th e
p ro te in was removed by f i l t r a t i o n .

S u lfh y d ra l compounds were

p r e c ip ita te d from th e f i l t r a t e as t h e i r cadmium s a l t s (Waelch
and R itte n b e rg , 19*+1) and the s u lfh y d ra l compounds re d iss o lv e d
by p r e c ip ita tin g cadmium as i t s s u lf id e .
tio n was assayed fo r r a d io a c t iv it y .

The r e s u ltin g so lu ­

The p ro te in was hydrolyzed

and assayed fo r r a d io a c tiv ity along w ith the combined f i l t r a t e s .
The n o n -p ro te in s u lfh y d ra l compounds, recovered in t h i s manner,
accounted fo r 2 .6 to 3 .7% of th e t o t a l a c t i v i t y re g a rd le s s of
in c u b a tio n tim e.

The a c t i v i t y of th e p ro te in h y d ro ly sa te s

a f t e r rem oval o f in o rg a n ic s u lf a te ranged from 2,b% of the

t o t a l a c t i v i t y a f t e r 0 .5 hour in c u b a tio n to 1 9 . a f t e r **
hours in c u b a tio n .
The s u lfh y d ra l compounds were sep arated chromat©graph­
i c a l l y on paper by th e method of M ille r and Rockland (1952).
S trong n in h y d rin re a c tio n s were o b tain ed a t th e p o s itio n s
occupied by c y s tin e and g lu ta th io n e w ith a f a in t e r re a c tio n
a t th e c y s te in e lo c a tio n .

S ^ ^ - a c tiv ity was la r g e ly concen­

t r a t e d a t th e g lu ta th io n e lo c a tio n , but a c ti v i t y as in o rg an ic
s u l f a t e co in c id in g w ith th e c y ste in e lo c a tio n made a q u a n tita ­
t i v e i n t e r p r e t a t i o n im p o ssib le.
M icro rad io au to g rap h s.

This experim ent was undertaken to

v is u a lly dem onstrate in c o rp o ra tio n o f s u lf a te in to th e c e l l s
o f rumen m icro b io ta and to d is tin g u is h th e m orphological types
re s p o n s ib le fo r t h i s in c o rp o ra tio n .
A 25 ml. p o rtio n o f c h e e se -c lo th s tra in e d rumen liq u id
from rumen f i s t u l a s te e r C707 (re c e iv in g a r e s t r i c t e d rough­
ag e-h ig h c o n c e n tra te r a tio n ) was added to a Uo ml. c e n trifu g e
tube c o n ta in in g 0.73 gm*

th e c o n c e n tra te mix and 2 x 10"?

moles of s 3 5 -la b e le d in o rg a n ic s u lf a te co n tain in g 0 .6 m i ll i c u r ie s of r a d io a c t iv i t y .

This tube was then incubated fo r

3 .5 hours a t *+0°C. and th e fe rm e n ta tio n stopped by adding 10
m l. o f WlO (NHif) 2S0if in 0.5M aqueous NaF.

The tube was cooled

in ic e w a te r, c e n trifu g e d f o r 30 m inutes a t 1,2^0 x G .; the
re s id u e was resuspended in 30 ml. of th e s u lf a te - f lu o r id e
s o lu tio n , and c e n trifu g e d fo r 30 m inutes a t l,2U o x G.

A

sm all amount of th e w h ite , c e ll-c o n ta in in g la y e r on to p of

M+

th e re s id u e was removed w ith a tr a n s f e r loop and spread on a
m icroscope s l i d e .

A c o n tro l tube co n tain in g s35 was processed

in e x a c tly th e same way except th a t th e f lu o r id e - s u lf a te so lu ­
t i o n was added im m ediately a f te r in o c u la tio n and th en t h i s
tube was r e f r i g e r a te d w hile th e experim ental tube was in cu b ated .
The smears o f in cu b ated and c o n tro l c e ll s were flame fix e d
and th e n th e s lid e s were suspended in M/100 I^SO]^ in 80J& e th ­
anol f o r 15 m inutes follow ed by r in s in g w ith d i s t i l l e d w ater.
B efore th e washing pro cedure, both smears y ie ld e d over 10,000
counts per minute w ith a d efin ed counting procedure, w hile
a f t e r w ashing no a c t i v i t y could be d e te c te d in th e c o n tro l
smear and th e smear o f incubated c e ll s s t i l l y ie ld e d over
10,000 co u n ts per m inute.
These smears were th en s ta in e d by the method of Gram and
c ro ss-h a tc h e d by c u ttin g s t r i p s about 1 m illim e te r wide out
of th e smear a t 2 m illim e te r in t e r v a l s .

This l e f t a s e r ie s

o f sm all r e c ta n g le s w ith *+ to 20 o i l immersion f i e l d s per
re c ta n g le which made i t p o s sib le to a c c u ra te ly lo c a te a given
fie ld .

These smears were th en clip p ed in a p p o sitio n to n u clear

tra c k p la te s (NTB)*, exposed fo r 1 week and th e NTB-plates
developed as d escrib ed by F itz g e ra ld e t a l . (1953).

He s ta te s

th a t t h i s f in e g rain ed photographic em ulsion produces a la t e n t
image p o rtra y in g th e r a d ia tio n source on the smear w ith re a so n ­
ab le f i d e l i t y on th e c y to lo g ic a l le v e l.

However, in t h i s work

*NTB-5 m icrons; Emulsion No. *+97, 9*+3“ 52; Eastman Kodak
Company; R o ch ester, New York.

*♦5
th e edges of th e image are b lu rre d sin ce a 5 micron th ic k n e ss
cannot be sh arp ly focused under th e o i l immersion o b je c tiv e .
F ig u re 1, page U6, p o rtra y s a f i e l d from th e m icro b ial
sm ear, th e image t h i s f i e l d produced on an NTB-plate and a
r e p r e s e n ta tiv e f i e l d from th e NTB-plate which was exposed to
th e c o n tro l smear.

I t can be concluded th a t many o f th e rumen

m icroorganism s in c o rp o ra te d ra d io a c tiv e s35 as a r e s u l t of th e
in v i t r o fe rm e n ta tio n and th a t th e la r g e r b a c te r ia and protozoa
e i t h e r d id not in c o rp o ra te th e s35 or th e in c o rp o ra tio n was
lo c a liz e d y ie ld in g an image appearing much lik e sm all b a c te r ia .
R e so lu tio n was not adequate to d is tin g u is h b a c te r ia l morphology
and many of th e sm aller rumen m icroorganism s do not s ta in or
photograph w e ll.
On th e b a s is of th e se p re lim in a ry fin d in g s , an in v e s tig a ­
t i o n o f th e e f f e c t of roughage le v e l in th e r a ti o n on incorpo­
r a ti o n o f in o rg an ic s u lf a te by rumen m icrobiota was undertaken.
P r in c ip a l Experiment

P ro ced u re.

In o c u la fo r use in th ese s tu d ie s were obtained

from 3 m ature s te e r s (C707, C7*+l, and CS126) and 2 mature tw in
cows (T1 and T2) which were f i t t e d w ith th e p l a s t i c f i s t u l a
plug d e sc rib e d by H entschl e t a l . (195^)•

C707> CS126, and T1

re c e iv e d a r e s t r i c t e d roughage-high c o n c en trate r a t i o n w herein
3 to 6 pounds of a l f a l f a hay re p re se n te d th e roughage and the
c o n c e n tra te was re p re s e n te d by 10 to 22 pounds of a m ixture
c o n ta in in g 77$ ground co rn , 20$ soybean o i l meal, 1$ Ca2HP0i+,

a

b

c
F igure 1.

M icroradioautographs

a. A f i e l d from the smear of
incubated m icroorganism s,
Gram s ta i n . (x*+70)
b. L atent image produced by
th e f i e l d in a on an NTBp la te . (x*+70)
c . L aten t image produced by
a r e p re s e n ta tiv e f i e l d
on th e smear of c o n tro l
m icroorganism s. (x**70)

*+7
J.% NaCl, 1% CaCO^ p lu s v ita m in

A and ir r a d ia te d y e a s t .

C7^1

and T2 r e c e iv e d an a l l hay r a tio n c o n s is tin g o f 30 to 3U
pounds o f a l f a l f a .

In o cu la were o b tain ed by s tr a in in g co n ten ts

from th e v e n t r a l p o r tio n o f th e rumen through c h e e s e - c lo th in to
f l a s k s p r e v io u s ly flu s h e d w ith C02 , warmed to *+0 to U3°C,
w it h in an in s u la te d ca rry in g c a s e .

In the f i r s t 2 t r i a l s , 25 ml. p o rtio n s of ino cula from
the a l l hay or high co n c en trate fed donors were t r a n s f e r r e d
w ith a graduated c y lin d e r to a s e r i e s of 50 ml. Erlenmeyer
f l a s k s c o n tain in g 1 gm. of a l f a l f a l e a f meal as s u b s tr a te
f o r th e a l l hay in o c u la , or 0.73 gnu of the co n cen trate mixture
fo r th e high c o n c e n tra te in o c u la .

These ferm entation f la s k s

had p re v io u s ly been flu sh e d w ith CO^, warmed to ho°C. and
about 0 .1 ml. of S-^^-labeled inorgan ic s u lf a te in about 0.05M
HC1 added, giving an a c t i v i t y of about 10 m icro curies/m l.

The

c a r r i e r s u l f a t e amounted to l e s s than I x 10“ ^ moles per f l a s k .
Following in o c u la tio n , the f l a s k s were stoppered, ro ta te d
to mix, and placed i n a ho°C. w ater b ath .

At 15 minute i n t e r ­

v a l s , th e c o n te n ts of the f l a s k s were r o t a t e d to mix and
p re ssu re r e li e v e d by b r i e f l y loosening the sto p p e rs.

Approx­

im ately 3 minutes were re q u ire d to in o c u la te the e n t i r e s e r i e s
of f l a s k s .

The order of in o c u la tio n and the a p p ro p ria te zero

time fo r in c u b a tio n was noted.

One f l a s k f o r the hay s e r ie s

and one f o r th e high c o n c en trate s e r i e s was removed a t each
of th e follow ing tim e s; 1, 1.5? 2, 2 .5 , and 3 hours.

A fter

in c u b a tio n , the c o n te n ts from th ese f l a s k s were immediately

H8
poured in to 80 ml. of alco h o l and th en processed as d escrib ed
in Table 2, page *+9.
In th e second 2 t r i a l s , 25 ml. p o rtio n s of in o cu la from
th e a l l hay (T l) , or high c o n c en trate (T2) fed donors were
handled in th e same manner except th a t a l l fla s k s contained
0 .8 7 gm. o f a m ixture of the co n c en trate and a l f a l f a le a f
meal i n p ro p o rtio n s o f 0 .7 3 /1 .

A lso, 0 .% of tympanol* was

added to th e in o c u la to f a c i l i t a t e han d lin g .

The r a d io a c tiv ity

as s 3 5 -la b e le d in o rg a n ic s u lf a te in th e se t r i a l s was about 27
to Ho m icro cu rie s p er ml.

An 0.5 hour in cu b atio n time was

a ls o in clu d ed in th e second 2 t r i a l s .

Assays f o r th e in o rg a n ic ,

w a te r-s o lu b le s u lf a te co n ten t of the in o cu la and s u b s tra te s by
th e method o f Lewis (195H) showed th a t the rumen liq u id from
Tl co n tain ed 5*H x 10"S m o les/m l., th a t from T2 contained 11.0
x 10”® m o les/m l., th e a l f a l f a le a f meal contained 2 .9 x 10“ 5
m oles/gm ., and th e c o n c e n tra te m ixture contained 7.9 x 10“?
moles/gm.
The chem ical procedures are d escrib ed in Table 2, page H9.
S e le c tio n o f 80% e th a n o l as th e p ro te in p r e c ip ita n t was based
on th e fin d in g s o f Pearson and Smith (19H3a).
Assays fo r r a d io a c t iv it y were conducted by p lacin g 3 ml.
of th e s o lu tio n of known volume in a 5 ml. beaker under a G-M
d e te c to r w ith a window th ic k n e ss of le s s than 2 m illigram s per
square c e n tim e te r.

The same geometry and tube was used

♦ S ilic o n e a n ti-fo a m p rep a ra tio n ; J en -S a l L a b o ra to ries;
Kansas C it y , M isso u ri.

TABLE 2
FLOW DIAGRAM FOR PARTITION OF s35-ACTIVITY

25 ml. o f Incubated Ferment
1. Pour Lnto oO ml. of 9& to 99% eth an o l
c o n ta in in g 0.05M H2S 0 i ./li t e r .
2 . R inse fe rm e n ta tio n f la s k s w ith 25 ml.
o f 80# e th a n o l and add to 1.
R e frig e ra te f o r 1 to *+ hours a t 5°C.
F i l t e r in to 250 ml. graduated f la s k and
wash w ith 80 to 100 ml. o f 80# e th a n o l.

5.
6.

_________ L
r
F i l t r a t e and

7.
8.

£.
Make to v o lu m e w itn M/1 HC1.
Remove 3 ml. f o r assay of
r a d io a c t iv i t y (n o n -p ro te in
s u lfu r).
Add 5 ml. o f 20# BaCl2 and
allow to s e t t l e .
F i l t e r through d ry paper

i---- '—

1

R esidual
F iltra te
In o rg an ic
Remove
3
ml.
f
o
r
9.
S u lfa te
assay o f ra d io ­
a c t i v i t y (o rg an ic
s u lfu r).
10 . Add 20# HpSOj* to
p r e c i p it a t e excess
BaClp.
11. F i l t e r and chromatograph
filtra te .

12.

13.
lb.
15.
16 .

1
P ro te in Residue
T ransfer w ith paper to
a 250 ml. Erlenm eyer.
Add *+0 ml. of 6M HC1
and cover w ith beaker.
Hydrolyze b hours a t
120°C. in au to clav e .
C oncentrate to about
10 ml. in 2 to 3 hours
on hot p la te .
F i l t e r in to a 100 ml.
v o lu m etric f la s k and
wash to volume.

i----------------------

Humln

P r o te in H vd rolvsate

17. Remove 3 ml. f o r assay of r a d io a c tiv ity
and fo r chrom atographic assay (amino acid
and in o rg a n ic s u l f u r ) .
18. Add 2 ml. o f 20# BaCl2 .
19. F i l t e r through dry paper.
1

S u lfa te -F re e P ro te in
H ydrolysate
20. Remove 3 ml. fo r
assay of r a d io ­
a c tiv ity .

----------- 1
R esidual Ino rg an ic
S u lfa te ______

50
throughout each t r i a l .

C o rre ctio n s fo r background a c ti v i t y

were determ ined a t 1 to 2 hour in te r v a ls and su b tra c te d from
th e raw counts to o b ta in th e n et co unts.

The n et counts per

second (C .P .S .) o b tain ed in t h i s manner were p ro p o rtio n a l to
th e c o n c e n tra tio n of s35 in m icrocurie s/m l.

No d iffe re n c e s

in s e lf - a b s o r p tio n could be d e te c te d between th e s o lu tio n s
used in th e s e ex p erim en ts.

Net C .P.S. tim es the ml. of so lu ­

t i o n in a given f r a c tio n was th en recorded as the r e la ti v e
a c t i v i t y in t h a t f r a c tio n a f te r a p p lic a tio n of ap p ro p iate
d i l u ti o n f a c t o r s .

The combined a c t i v i t y from ste p s 6 and 17

of Table 2 , page *+9, c o n s titu te s th e t o t a l recovered a c tiv i ty
of which th e o th er f r a c tio n s were expressed as a p ercen tag e.
Chrom atographic s e p a ra tio n of th e amino ac id s in ste p s
11 and 17 of Table 2 was c a rrie d out on f i l t e r paper by the
method of P a tto n (1950).

The a c t i v i t y on th e chromatograms

was assayed as d esc rib ed by W interingham (1952) and the a c tiv ­
i t y of th e liq u id f r a c tio n s p a r titio n e d according to th e d i s ­
t r i b u t i o n of a c t i v i t y on the chromatograms.
R e s u lt s .

The t o t a l in c o r p o r a tio n o f la b e le d - s u lf a t e in t o

th e o rg a n ic s u lfu r f r a c t io n s i s p resen ted in Table 3, page 51.
The e x p o n e n tia l eq u a tio n s f i t t e d to th e se v a lu e s g r a p h ic a lly
and th e s t a t i s t i c s a p p lie d t o th e s e eq u a tio n s as d esc rib ed by
S te v e n s (1951) are p resen ted in Table *+, page 52.
page 53, v i s u a l l y p r e s e n ts the same e q u a tio n s.

F igu re 2,

Table 5, page

5^+, shows th e p a r t it i o n o f in co rp o ra ted a c t i v i t y o f th e chroma­
to g r a p h ic f r a c t i o n s .

These f r a c t io n s were not ch rom atograp h ically

51
TABLE 3

TOTAL INCORPORATION OF LABELED-INORGANIC SULFATE

In c u b a tio n
Time
( t h o u rs)

^•Organic A c tiv ity
T o tal Recovered A c tiv ity
Hay inoculum

(100) s At
G rain inoculum

Means o f f i r s t 2 t r i a l s w ith in d iv id u a l s u b s tr a te s :
1*0

2.6

1 .5

3 .0

*+.3
12.6

2 .0

8.1

12.2

2 .5

7 .8

11.V

3 .0

6.2

15 .H

Means of second 2 t r i a l s w ith mixed s u b s tr a te s :

1.

0 .5

6.39

3.78

1 .0

1 6 . Ik

5.80

1 .5

13.29

5.18

2 .0

18.68

7.35

2 .5

2*+. 79

7.63

3 .0

27.03

9.88

The combined a c t i v i t i e s from ste p s 9 and 20 o f Table 2.

TABLE b

ASYMPTOTIC REGRESSION EQUATIONS FOR INCORPORATION OF SULFATE

E q u ation Form:

At = A.^ - Aje"p t

Aj. = % o f t o t a l a c t i v i t y which i s in co rp o rated a t tim e t
A^ = % o f th e t o t a l a c ti v i t y which would be in co rp o rated
in an i n f i n i t e tim e (th e asym ptote)
e

z base of th e n a tu r a l system of logarithm s (2.71828)

r

9 th e r a t e o f in c o rp o ra tio n o f in o rg an ic s u lf a te in to

t

s hours o f in c u b a tio n

(1 0 0 )r

o rg an ic s u lfu r

9 # /h o u r co n v ersion of in o rg an ic s u lf a te rem aining to

be converted to organic s u lfu r a t th a t tim e.

R atio n o f
Inoculum
Donor

S u b s tra te

A ll Hay

A lfa lf a
Meal

A ll Hay

Standard E rro r
of
r
E stim ate
Ai

r

D.F.

10.5

O.b

3

1.79

l.b

0 .7

A lf a lf a
37.5
Meal and
C o n cen trate

Q.b

b

2.88

2 .0

1.1

0 .6

3

2.7*+

2.2

1.1

A lf a lf a
11.6
High
C o n cen trate Meal and
C o n cen trate

0.52

b

1.09

0.8

O.b

Mean o f A ll Treatm ents

0M

lb

Concen­
High
C o n cen trate t r a t e

16.5

0.b5

53
Figure 2.

Asymptotic Curves For Incorporation of S u lfate

Individual Substrates
Grain Inoculum
A^=l6 .5-16 .'5e“®*^
P

o p©•H
>
CJ cfl P
O O
cfl 03 <*
P IH
C0
o ©-P
p«
f-ciH O
O 0) H
o ,o
G «0«H
Mi-3 O

Inoculum

/
a
(t ) Hours Incubation

Mixed Substrates
3r
<M >» 2c
o p
•H /5
cs C
D
o P •H
■H(# -P
P Cp O
«J i~)
/«•H
O 03
P« I C f l 9
f-i 'CJ P
O <DO
6
O
H
fl © 5
<P
Mcia
cfl O
4
-p
-*5
a

Hay Inoculum
.
At =37.5-37. J e'0 -1^

^ Grain Inoculum
At = 11.6-11.6e-0-52t

( t ) Hours Incubation

TABLE 5
ACTIVITY RECOVERED IN ORGANIC FRACTIONS AS PERCENT OF THE
TOTAL RECOVERED ACTIVITY
In c u b atio n Time
(Hours)

C ysteine
/C y s tin e

M ethionine

^U n id en tified

0 .V8

0 .U5
0.78
O.83
1.00
1 .1 6
2 .3 0

G rain Inoculum-Mixed S u b s tra te :
0 .5

2.85

1 .5

3.0V
4.W8
3 .9 9
4 .0 6

1.0

2.0

2 .5
3.0

3.91

1.13
1.31

1.88
2 .V8

3.5 1*

Hay Inoculum-Mixed S u b s tra te :
0 .5
V.87
1 .0

1 .5
2 .0

2 .5

0.76

1 2 .3 2

2.13

1 .6 8

8.89

2.71
V.37
5.63
7.89
3V.3

1.69
2.56
3.1V
5.39
21.8

23.5$

lV.9$

1 1 .ib
1 6 . 0V

13.77
T o ta l— —™ ------90.0
P ercen t
D i s tr ib u tio n — 6 1 . 6$
3 .0

In c u b a tio n Time
(Hours)

C ysteine
/C y stin e

G rain In o cu lu m -In d iv id u al S u b s tra te :
1.0

1 .5

2.0

2.6
8.1

7.2

8.8

2 .5
9.0
3 .0
Hay In o cu lu m -In d iv id u al S u b s tra te :
1.0
2.0

1 .5

2.4
5.3

2 .5
3 .0

2'1
*+.3

T o ta l---- -------- -—---- —-----P ercen t D i s t r i b u t io n - — -. 6 2 . 2$
Mean o f A ll T r i a l s —- - - - - - • 61.9$
1.

M ethionine/
lU n id e n tifie d
-

1.8

4 .5
5.1
2.6

6 .5
1.2
0.6
2.8

4 .7

2.0

31.8
37 M
3 8 . 15?

R* i s g re a te r th a n 0 .9 on paper chromatography w ith 80$
P henol.

55
pure and co n tain ed sm aller amounts of a c t i v i t y as u n id e n tifie d
compounds.

PART II

PRODUCTION AND ABSORPTION OF ACETIC, PROPIONIC,
AND BUTYRIC ACIDS IN THE BOVINE RUMEN
Procedure

Twin H o ls te in cows, Tl and T2, weighing 1050 and 1110
pounds and a Guernsey s t e e r , C707, were used in t h i s experim ent.
The s te e r weighed 1^20 pounds when used fo r th e r e s t r i c t e d
roughage t r i a l and 1500 pounds fo r th e a l l hay t r i a l .

A ll

th re e anim als were f i t t e d w ith screw cap, p la s ti c f i s t u l a
plugs d e sc rib e d by H entschl et. a l . (195*0 which f a c i l i t a t e d
sampling o f th e rumen c o n te n ts .

Tl was in th e second month

of l a c t a t i o n and T2 was d ry .
The ex p erim en ta l r a t io n s were fed tw ice d a ily at 8 A.M.
and 3 P.M. fo r at l e a s t th r e e weeks b efo re each t r i a l .

A lf a lf a

hay was used f o r th e roughage and th e co n cen tra te m ixture was
th e same as d e sc r ib e d in th e procedures fo r Part I .

For the

r e s t r i c t e d roughage t r i a l s , Tl r e c e iv e d a r a tio n o f 28 pounds
o f c o n c e n tr a te p lu s k pounds o f hay and C707 r e c e iv e d 10 pounds
o f c o n c e n tr a te p lu s U pounds o f hay.

For the a l l hay t r i a l s ,

T2 re c e iv e d a r a tio n of 30 pounds of hay and C707 re ceiv ed 2U
pounds of hay.
On th e days o f th e exp erim en t, fe e d in g was r e s t r ic t e d to
2 hours fo r th e a l l hay t r i a l s and 3/*+ hour fo r th e h igh con­
c e n t r a t e - r e s t r ic t e d roughage t r i a l s .

56

Water was p erm itted at

57
w i l l d u rin g th e next 2*+ hours.

During the feeding p erio d ,

Tl a te 11 pounds of c o n c e n tra te and 2 pounds of hay, T2 a te
8 pounds of hay, C707 a te 5 pounds of c o n c e n tra te and 2 pounds
of hay f o r th e r e s t r i c t e d roughage t r i a l and 6 pounds of hay
fo r th e a l l hay t r i a l .
The tim e halfw ay between i n i t i a t i o n and te rm in a tio n of
feed in g was tak en as 0 hour.

The sample recorded as 0 hour

was tak en j u s t p r io r to fe e d in g .

Samples of th e liq u id in

th e m iddle of th e v e n tr a l sac of th e rumen were taken a t
d e f in i t e i n t e r v a l s a f t e r 0 hour and s tra in e d through cheese­
c lo th in to a v o lu m etric f la s k co n tain in g 50$ s u lf u r ic acid
eq u al to 3$ o f i t s volume.

This volume of s u lf u r ic acid

lowered th e pH to 1 .5 to 2 .0 and y ie ld e d a f i n a l sample con­
ta in in g about 91. 3$ rumen liq u id on a dry m atter fre e b a s is .
Most o f th e suspended s o lid s in th e i n i t i a l sample were p re ­
c i p i t a t e d by th e s u lf u r ic a c id .

These samples were sto red at

6°C. u n t i l analyzed which was always le s s th an a month.
A c e tic , p ro p io n ic , and b u ty ric a c id s were determ ined by
d i r e c t chromatography of rumen liq u id on s i l i c a g el as described
by Harper (1953) w ith m o d ific a tio n s.

T h irteen gm. of dry s i l i c ­

ic a c id (M allin k ro d t s p e c ia l chrom atographic No. 28^7, fu rth e r
graded by suspending 2 tim es in an equal volume of w ate r,
allow ing to s e t t l e 30 m inutes and d isc a rd in g th e su p ern atan t)
and 7 .5 ml. of 1.6m potassium phosphate pH 6.3 b u ffe r were
th o ro u g h ly mixed in a m ortar and th en about 50 ml. of ch lo ro ­
form (U .S.P. washed 3 tim es w ith an equal volume of w ater and

58
f i l t e r e d to remove suspended w ater) were used to form a s lu rry .
This s l u r r y was poured in to a 38 m illim e te r diam eter column
w ith a f r i t t e d g la s s f i l t e r o v e rla id w ith f i l t e r paper to form
th e bottom .

The column was tapped to remove a i r bubbles and

th en k ep t covered w ith chloroform added slowly from a sep ara­
to ry fu n n e l.

A f i l t e r paper d isc 38 m illim e te rs in diam eter

was p laced on th e b u ffe r s e c tio n .

The sample se c tio n was not

added u n t i l th e chloroform le v e l was about 1 m illim eter above
t h i s p ap e r.
The s u lf u r ic acid s e c tio n of Harper (1953) was om itted
sin ce i t had no e f f e c t on th e r e s u l t s in t h i s experim ent.

The

sample s e c tio n was p rep ared by weighing 6 to 7 gm. of rumen
l iq u id d i r e c t l y on to 10 gm. o f dry s i l i c i c acid on a watch
g la s s .

The s i l i c i c ac id and sample were then q u a n tita tiv e ly

tr a n s f e r r e d to a m ortar and thoroughly mixed.

F if ty ml. of

chloroform were used to form a s lu r r y and used as a v e h ic le
to t r a n s f e r t h i s s e c tio n to the column.
E lu a te f r a c tio n s o f ap p ro p iate siz e as determ ined by
p re lim in a ry t r i a l s were c o lle c te d s ta r tin g im m ediately a f te r
adding th e sample s e c tio n .
hour.

The flow r a te was about 150 m l./

Four columns were run sim ultaneously.

F u rth er a d d itio n s

of s o lv e n t were made u sing a se p a ra to ry funnel a d ju ste d to keep
5 to 10 ml. o f so lv e n t covering the column.

A 50 ml. a d d itio n

of pure chloroform was follow ed by 50 ml. p o rtio n s of 1$, 2.5$>
5$, 10% and 20$ n -b u ta n o l in chloroform s o lu tio n s .

A d d itio n al

20$ n -b u ta n o l in chloroform was used to give a t o t a l e lu a te

59
volume o f 500 ml.

Blank d e te rm in a tio n s were made w ith each

b atch o f re a g e n ts and ap p ro p iate c o rre c tio n s made.
R e su lts
The chrom atographic procedure was te s te d w ith pure acids
a lo n e , and added to rumen liq u id .

Recovery in *+ t r i a l s w ith

0 .5 to 3 .8 m illim o les of added a c e tic acid was 93$ w ith a
stan d ard d e v ia tio n o f 8$.

Recovery of 0 .2 to 0 .5 m illim oles

of b u ty ric ac id was s im ila r ly 96+ 6$ .

Threshold volumes fo r

b u ty r ic , p ro p io n ic , and a c e tic ac id s were about 70, 200, and
280 m l., b ut v a rie d somewhat w ith changes in re a g e n ts and the
amount of sh o rt ch ain acid ap p lied to th e column.

The v alues

re p o rte d as b u ty ric ac id could include tra c e s of higher a c id s.
F ig u re 3> page 60, p o rtra y s th e re g re s s io n of rumen con­
c e n tr a tio n s on tim e a f t e r feeding fo r a c e tic , propionic,, and
b u ty ric a c id s .

Table 6, page 61, l i s t s the estim ated amounts

per 100 ml. of rumen liq u id of a c e tic , p ro p io n ic, and b u ty ric
a c id s produced from each pound of t o t a l d ig e s tib le n u tr ie n ts
in g e ste d as th e a l l hay r a ti o n or the high co n cen trate r a tio n
on th e day of th e experim ent.

I t was assumed th a t th e hay

co n tain ed 50$ t o t a l d ig e s tib le n u tr ie n ts and the c o n c en trate
75$.

Table 7, page 61, g iv es th e r a t e s a t which th e acid s

d isap p ea red from th e rumen.

The re g re s s io n eq uations are

summarized in Table 8 (Appendix) and Table 9 (Appendix) gives
th e c o n c e n tra tio n s o f a c e tic , p ro p io n ic , and b u ty ric acid s
fo r each cow, r a t i o n , and sample tim e.

60

F ig u re 3.

C o n cen tra tio n o f Short Chain Acids in th e Rumen
A fte r F eeding.

Millimoles

of Acid/100

grams of Rumen L iquid

A cetic A cid-A ll Hay R atio n ; At s9 * 5 9 e"°* ^ ^ ^ -1 .9 0 e *^*^2^

-A cetic Acid-High C oncentrate R ation;
At=7 . l6 e - ° • °3 1 1 t- 2 . 1 6 e '° • 838*
£.5-

-B utyric Acid-High C oncentrate R ation;
At = 2 .3 5 e-°-028^ -0 .7 3 e -° * 7 3 3 t

-^ B u ty ric A cid-A ll Hay; A^=1.63e“^«®2l+8t.Q#22e“^ * 3 ^ 't

C

^P ro p io n ic A cid-A ll Hay R ation;
_____________
At :2 .2 8 e -0 -0322t -0 .3 6 e -0 -lt50t

"Propionic A cidHigh C o n cen trate R atio n ;

A t:2 .3 6 e-0 -^ ?t.0.itle -0.276t
*
Hours A fte r Feeding

61

TABLE 6

THE AMOUNT OF ACETIC, PROPIONIC, AND BUTYRIC ACIDS PRODUCED
FROM ONE POUND OF TOTAL DIGESTIBLE NUTRIENTS
Acid

A ll Hay R ation

^mM/100g.

%

R e s tric te d Roughage Pooled
-High C oncentrate
Data
R ation
From Both
R ations
1mM/100g.
2mM/100g.
%

A cetic

0.52

68.4

0.30

65.2

.2 4 -.6 7

P ro p io n ic

0.15

19.7

0.06

13.0

.0 2 -.2 6

B u ty ric

0.09

11.8

0.10

21.7

.0 4 -.2 1

T o tal

0.76

--------

0.46

--------

1.
2.

_— »

M illim o les of acid /1 00 grams of rumen liq u id .
Range a t 95$ confidence le v e l.

TABLE 7
THE DISAPPEARANCE RATES FOR ACETIC, PROPIONIC, AND BUTYRIC
ACIDS FROM THE RUMEN
A ll Hay R ation

Acid

R e s tr ic te d Roughage
R ation

$ /H r .

1S.E .

%/ET,

l-S.E.

A c e tic

3 .5

0 .4

3.1

HA
.
' o

P ro p io n ic

3.2

0 .5

4 .6

B u ty r ic

2 .5

0 .5

2 .8

1.

S tan d ard E rro r

Pooled
Data
From Both
R ation s

%/Hr. 1S.E.
3 .3

0 .3

0 .9

------

------

0.5

2.7

0 .3

62
The type e q u a tio n used to d e sc rib e the d ata is s
At =
w here,

- V -k2*

A. = th e c o n c e n tra tio n o f th e acid in m illim oles per
100 gm. o f rumen liq u id ;
A0 = th e d isap p earance re g re s s io n e x tra p o la te d to 0
tim e; t h i s could re p re s e n t th e amount of acid
which would he p re se n t i f th e fe rm en tatio n were
completed w ithout any disappearance of the ac id ;
Ap = (A0-A+.) when t i s 0; t h i s should re p re se n t the
amount of acid produced from the c u rre n t feeding
in m illim o les/1 0 0 gm. of rumen liq u id ;
e = base of th e n a tu ra l system of logarithm s (2.71828);
= r a te o f disappearance of the acid from the rumen;
lOOki = $ /h o u r disappearance of the acid p resen t
a t any given tim e;
k2 = r a te of acid production approaching AD which
would re p re s e n t the r a te of conversion of f e r ­
m entable feed in to i t s acid product;
t

z tim e a f t e r feeding in hours.

A g rap h ic p o rtra y a l of t h i s equ atio n i s given in Figure
*+, page 63.
The n a tu r a l lo g arith m s of th e a c e tic acid c o n c e n tra tio n s
from 3 hours a f t e r fe e d in g , and of th e propionic and b u ty ric
acid c o n c e n tra tio n s from 3.5 hours a f te r fe ed in g , through the
f a s tin g p erio d were f i t t e d to a re g re s s io n on time by the
method of l e a s t squares as d escrib ed by Snedecor ( l ^ h ) .

The

0 hour e x tra p o la tio n o f t h i s re g re s s io n i s th e n a tu ra l logarithm
of A0 and th e slope i s k1 .

The c o n c e n tra tio n s of a c e tic , pro ­

p io n ic , and b u ty ric a c id s determ ined between 0 and 3 or 3*5
hours a f t e r feed in g were su b tra c te d from the AQ value ad ju sted
f o r tim e a f t e r feed in g according to th e disappearance re g re s s io n .

F ig u re H.

A G raphic P o rtra y a l of th e Type Equations
At « A0e*kl t -Ape -k2t

C o n cen tra tio n

-kit

0 .5 t

Time

6W
The n a tu r a l lo g arith m s of th e se d iffe re n c e s were f i t t e d to a
re g re s s io n a g a in s t tim e as b e fo re .

The slope of t h i s second

r e g re s s io n e q u a tio n i s k2 and th e 0 hour e x tra p o la te i s the
n a tu r a l lo g arith m o f Ap .

The t e s t s f o r s ig n ific a n c e were

c a r r ie d out on th e lo g a rith m s.
There were no s ig n if ic a n t d iffe re n c e s a t th e 5% le v e l
between any o f th e d isappearance r a te s although the pooled
r a t e f o r a c e tic acid approached s ig n ific a n c e over th a t fo r
b u ty r a te .

The disap p earance r a t e fo r propionic acid w ith

th e r e s t r i c t e d roughage r a ti o n approached s ig n ific a n c e over
th a t w ith th e a l l hay r a ti o n .

The r a te s of production of the

a c id s were not s ig n i f i c a n t ly d if f e r e n t in any case and ranged
from 27 to 83^ /h o u r.

The amounts of each acid produced from

a pound of T.D.N. were not s ig n if ic a n t between th e r a tio n s .
However, a c e tic acid p roduction from hay approached s i g n i f i ­
cance over th a t from th e high c o n c en trate r a tio n .

DISCUSSION

The energy su p p lied to the cow by th e sh o rt chain acid s
can be computed from th e d a ta in Table 6, page 6 l.

The aver­

age anim al i n t h i s experim ent weighed 1270 pounds.

C a rro ll

and Hungate (195*0 concluded th a t rumen co n ten ts c o n s titu te
1*$ of th e bovine body w eight.

The average weight of th e

rumen c o n te n ts in t h i s experim ent would then be 178 pounds
which would c o n ta in about 70 kilogram s of rumen liq u id (dry
m atter f r e e ) .

Computing from th e 95$ confidence le v e l ranges

given in Table 6, a pound of t o t a l d ig e s tib le n u tr ie n ts y ie ld s
168 to U69 m illim o les of a c e tic a c id , l*f to 182 m illim oles
p ro p io n ic a c id , and 28 to l*+7 m illim o les b u ty ric a c id .

Con­

v e r tin g th e v a lu e s to kilogram c a lo r ie s , a t o t a l energy of
55.0 to 2 3 2 .3 K c a l./lb . of t o t a l d ig e s tib le n u tr ie n ts i s
o b ta in e d , 35*2 to 98.3 are re p re se n te d by a c e tic a c id , 5.1 to
66.9 by p ro p io n ic a c id , and 1^.7 to 77.1 by b u ty ric a c id .

If

one pound of t o t a l d ig e s tib le n u tr ie n t i s e q u iv a le n t to 181*+
c a lo r ie s (Brody, 19*+5) 9 th e cow o b ta in s 3 to 13$ of i t s energy
from s h o rt ch ain a c id s .
A c o n sta n t rumen volume was assumed in computing th e se
r e g r e s s io n e q u a tio n s .

Ir r e g u la r f lu c t u a t io n s in volume would

in c r e a s e th e standard erro r and h en ce, be co r re cted fo r by the
range in 3 to 13$ e s tim a t e .

However, i f rumen volume in c r e a s e s

a p p r e c ia b ly and r e g u la r ly a f te r e a tin g due to s a liv a t io n ,

65

66

d rin k in g , o r in f lu x of w ater from th e t i s s u e s , th ese estim ates
would be to o low.

S im ila rly , i f rumen volume decreases appre­

c ia b ly w ith f a s t i n g , as suggested by th e d a ta of W eller and
Gray (195*+), b o th th e d isappearance r a te s and the energy
e s tim a te s are b ia se d downwards.
The carbon balan ce s tu d ie s of McNaught (1951) and of
Heald (1952) in d ic a te a 25 to 50$ conversion of feed energy
to sh o rt ch ain a c id energy.

C a rro ll and Hungate (195*+) e s t i ­

mate t h a t sh o rt ch ain a c id s account fo r 66$ of the a v a ila b le
feed energy w hile pfander and P h illip s o n (1953) imply a
s im ila r v a lu e .

These e stim a te s were based on d a ta obtained

under extrem ely abnormal c o n d itio n s.

The 3 to 13$ estim ate

in t h i s experim ent i s co n sid erab ley lower than the v alu es
c ite d above and i s probably b iased downwards.

However, t h i s

method u t i l i z e d normal f i s t u l a t e d c a t t l e under p h y sio lo g ic a l
c o n d itio n s .
I f a method fo r d eterm in atio n o f rumen volume a t freq u en t
i n t e r v a l s were a v a ila b le , th e accuracy of th ese estim a te s
would be g r e a tly enhanced.

Perhaps t h i s could be accomplished

w ith dye d i l u t i o n tech n iq u es s im ila r to those used fo r blood
volume d e te rm in a tio n s .

Accuracy would be fu rth e r enhanced by

in c re a s in g feed in g e s tio n during the lim ite d feeding period
and low ering th e 0 hour c o n c e n tra tio n of rumen a c id s .

Both

o f th e se o b je c tiv e s could be accomplished by once d a ily feed in g .
E rro r o c c a s io n a lly e n te re d th e chrom atographic d eterm in atio n s
th ro u g h p ip e ttin g s o lid s from the bottom of th e f la s k along

67
w ith rumen l i q u i d .

This could have been avoided by a p relim ­

in a ry s e p a ra tio n .
A cetate p ro d u c tio n from hay approached a s ig n if ic a n tly
g r e a te r v alu e th an th a t from g ra in .

The disappearance of

a c e ta te approached a s ig n if ic a n tly f a s t e r r a te than th a t fo r
b u ty ra te which i s in agreement w ith Pfander and P h illip s o n
(1953)-

Although r a t i o n had no s ig n if ic a n t in flu en ce on the

r a t e s , p ro p io n ic acid disappearance was f a s te r w ith th e high
c o n c e n tra te th an w ith the hay r a ti o n .

The molar r a t i o of

a c e tic to p ro p io n ic ac id was reduced by the high co n cen trate
r a t i o n b u t, th e re d u c tio n was not as marked as th a t re p o rte d
by Tyznik (1951).
M icroscopic exam ination of rumen co n ten ts from anim als
on th e a l l hay and th e r e s t r i c t e d roughage-high co n cen trate
r a tio n s f a i l e d to re v e a l any ap p reciab le or c o n s is te n t d i f f e r ­
en ces.

This does not p reclude th e p o s s i b i li t y of le s s obvious

m orphological d if f e r e n c e s .

Smears of th e in o cu la and ferm ents

from th e i n v i t r o experim ents showed no r e a d ily d e te c ta b le or
c o n s is te n t d iffe re n c e s w ith in cu b atio n tim es under 3 hours.
Longer in c u b a tio n tim es o fte n re s u lte d in a d is s o lu tio n of
p ro to zo a and an in c re a s e in gram -p o sitiv e ro d s.

C a rro ll and

Hungate (1951*), using a s im ila r ferm en tatio n tech n iq u e, found
th a t c e r t a i n p h y s io lo g ic a l c h a r a c te r is tic s which changed a f te r
2 to b hours in c u b a tio n were q u ite co n stan t before t h i s tim e.
They a ls o found b u ffe r s o lu tio n s were unnecessary fo r sh o rt
tim e in v i t r o fe rm e n ta tio n s.

The v alu es obtained between 2

68

and 3 hours i n Experiment I . were more e r r a t i c than values
w ith s h o rte r in c u b a tio n tim es.

More accu rate estim a te s could

probably have been o b tain ed by using s h o rte r in cu b atio n p erio d s.
Robinson et. a l . (1955) found th a t 1,952 x G. f o r 10 min­
u te s sedim ented most rumen m icrobiota although more were s e d imented by 5,782 x G.

The l,2*+0 x G. used in th e p re p a ra tio n

o f m icro rad io au to g rap h s f o r Experiment I was probably somewhat
low but th e 30 minute c e n tr if u g a tio n time used should p a r t i a l l y
o f f s e t th e lower fo rc e .
The d eco m p o sitio n o f c y s t ( e ) in e and m ethionine noted by
O lc o tt and F raenk el-C onrat (19**7) and Van H alteren (1951)
co m p lica ted th e amino a c id p a r t it io n d a ta .

C y s t(e )in e and

i t s d eco m p o sitio n p rod ucts moved as much as h a lf-w a y to the
s o lv e n t fr o n t w ith th r e e areas o f peak c o n c e n tr a tio n .

Meth­

io n in e formed 2 or more peaks nearer th e so lv e n t fr o n t than
c y s t(e )in e .

Another d i s t i n c t r a d io a c tiv e area appeared very

near th e s o lv e n t fr o n t and was e ith e r in to o low a con cen tra­
t io n to v i s i b l y r e a c t w ith ninh yd rin or was not an am ino-su lfur
compound.

The a c t i v i t y cou ld be d iv id ed in to a c y s t ( e ) in e

f r a c t io n c o n ta in in g la r g e ly c y s t ( e ) in e a c t i v i t y and a sim ila r
m ethion in e f r a c t io n .

Both f r a c t io n s were probably contam inated

w ith sm a lle r amounts o f u n id e n t ifie d compounds.

More e x te n s iv e

se p a r a tio n was n e ith e r w arranted nor p r a c t ic a l fo r t h i s stu d y.

Lewis (195*0 found th a t rumen microorganisms ra p id ly
reduce s u lf a te to s u l f i d e .

Only a tr a c e of hydrogen s u lfid e

was found in t h i s experim ent b u t sm all amounts of d isso lv ed

69
s u lf id e of h ig h s p e c if ic a c t i v i t y might have contam inated the
c y s t( e ) in e an d /o r m ethionine f r a c tio n s .

The p o s s i b i lit y of

s u lf a te in c o rp o ra tio n v ia s u lfid e needs fu rth e r in v e s tig a tio n .
The s y n th e s is of m ethionine and c y s t(e )in e found in th i s
experim ent ag rees w ith th e r e s u l t s of Block and S tekol (1950),
Block e t a l . (1951, 1953), and Duncan e t &1. (1953).

Cys-

t ( e ) i n e appeared to be formed a t a f a s te r r a te th an m ethionine,
b u t th e s e d a ta were e n t ir e l y in c o n c lu siv e .

Of th e la b e le d -s u l-

f a t e in c o rp o ra te d during the f i r s t 3 hours of in cu b atio n ,
about tw o -th ird s was found in th e c y s t(e )in e and o n e -th ird in
th e m ethionine f r a c t i o n .
Reed e t a l . (19^9) and Block et. a l . (1951) found equal
amounts o f c y s t( e ) in e and m ethionine s u lfu r in the rumen
m ic ro b io ta .

The d a ta in t h i s experim ent do not c o n f lic t

w ith th e e a r l i e r work sin ce m ethionine could w ell be forming
a t a slow er r a t e th an th e c y s t(e )in e and s t i l l e v e n tu a lly
a t t a i n an eq u al s ta t u s provided c y s t(e )in e form ation ceases
b efo re t h a t of m ethionine.

A lso, as much as 30$ of m icrobial

s u lf u r may occur as g lu ta th io n e (Cowie et, a l . , 195*+) which
may have been l o s t in th e f r a c tio n a tio n procedures of Reed
e t a l . (19^9) and Block e t a l . (1951).

The d a ta presen ted

here su pport th e h y p o th esis th a t in o rg an ic s u lfa te i s f i r s t
in c o rp o ra te d in to c y s t( e ) in e which i s then converted to
m ethionine (Cowie ejt a l . , 195^; Block, 1953).
The o v e r - a l l r a t e o f s u lf a te in c o rp o ra tio n was about *+8$
/h o u r.

The r a t e fo r th e c o n c e n tra te m icrobiota was s l ig h tly

70

but n o t s i g n i f i c a n t l y f a s t e r th an th a t fo r the hay m icrobiota.
The c o n c e n tra te m icro b io ta a c tin g on a co n c en trate s u b s tra te
in c o rp o ra te d more a c t i v i t y than the hay m icrobiota actin g on
a hay s u b s tr a te .

This can be a t l e a s t p a r t i a l l y explained by

a g r e a te r d i l u t i o n o f th e la b e le d - s u lf a te by the s u lf a te
a lre a d y p re se n t in th e hay s u b s tr a te .

When the s u b s tra te s

wer e a m ixture o f hay p lu s c o n c e n tra te , the hay m icrobiota
in c o rp o ra te d more a c t i v i t y th an th e c o n c en trate m icrobiota.
Since Cowie et, a l . (195*0 found th a t s u lf a te in c o rp o ra tio n by
E s c h e ric h ia c o li i s d i r e c t l y p ro p o rtio n a l to c e l l p r o li f e r a ­
tio n , and sin ce th e s u lf a te content of the mixed s u b s tra te s
was e q u a l, th e h ig h er in c o rp o ra tio n of a c ti v it y by in o cu la
from a hay fed cow would seem to re p re s e n t g re a te r c e l l pro­
life ra tio n .

However, t h i s could re p re s e n t e ith e r a g re a te r

number of c e l l s used as in o cu la or a s h o rte r g en e ratio n tim e.
Where s u lf a te c o n c e n tra tio n of s u b s tra te s i s not eq u al,
in c o rp o ra tio n o f a c t i v i t y i s a fu n c tio n of both c e ll p r o l i f ­
e r a tio n and th e s p e c if ic a c t i v i t i e s of th e in o rg an ic s u lf a te
in th e fe rm e n ts.

C o n tro l o f in o cu la siz e by use of c e n trifu g e d

c e l l su sp en sio n s and c o n tro l of s p e c ific a c tiv i ty by use of
s u lf a te f r e e s u b s tr a te s would provide more accu rate d a ta .
A lthough, rumen m icro b io ta from cows fed a l l hay may d i f f e r
somewhat from th e rumen m icro b io ta of high co n c en trate fed
cows in t h e i r a b i l i t y to u t i l i z e s u l f a te , the r a t e and the
p a tte r n o f s u lf a te u t i l i z a t i o n i s predom inatly s im ila r in
b o th c a s e s .

SUMMARY

Rumen liq u id from 5 bovine s (re c e iv in g e ith e r hay alone
or 1% of th e t o t a l d ig e s tib le n u tr ie n ts as co n cen trate) vas
in cu b ated w ith s u b s tr a te and s 3 5 - iab eled in o rg an ic s u lf a te .
Two t r i a l s used th e r a t i o n of the donor as s u b s tra te and two
used a s u b s tr a te of mixed a l f a l f a meal and co n cen trate fo r
b o th hay in o c u la and g ra in in o c u la .

In each t r i a l , a sep arate

fe rm e n ta tio n f la s k was incubated fo r each time of 0 . 5 , 1 , 1. 5,
2, 2 .5 , and 3 h o u rs.

Each ferm ent was fra c tio n a te d in to pro­

t e i n s u l f u r , f r e e o rg an ic s u lfu r and in o rg an ic s u lf a te .

The

p ro te in and f r e e o rg an ic s u lfu r f r a c tio n s were fu rth e r sepa­
ra te d by chrom atography.
of th e t o t a l a c t i v i t y .

G lutathione accounted fo r 2 to
The mean p a r ti t i o n of in co rp o rated

a c t i v i t y i n a t o t a l of U1* f la s k s fo r a l l tim es on both t r e a t ­
ments was 62% c y s t( e ) in e and 38$ m ethionine plus an u n id e n ti­
fie d f r a c t i o n .

E x p o n en tial eq u atio n s f i t t e d to th e t o t a l

in c o rp o ra tio n s fo r each com bination of s u b s tra te and inoculum
are p re se n te d .
The c o n c e n tra tio n of a c e tic , p ro p io n ic , and b u ty ric acid s
i n 3 bovine rumens.was determ ined a t v a rio u s tim es a f te r feed ­
ing e i t h e r an a l l hay r a ti o n or one c o n s istin g la r g e ly of
c o n c e n tra te s .

Two t r i a l s were conducted on each r a tio n .

The

c o n c e n tra tio n o f each acid f i r s t in creased and then decreased
w ith tim e a f te r fe e d in g .

The v alu es obtained were f i t t e d to

72

tu rn o v er c u rv e s.

A cetic acid disappeared from the rumen a t

the r a t e of 3 . 3$ /h o u r and b u ty ric acid a t the r a te of 2.7%/
hour.

The stan d ard e r r o r was 0.3%.

The disappearance r a te

of p ro p io n ic a c id on th e high co n c en trate r a tio n appeared to
be *f.6$ /h o u r v e rsu s 3 .2 $/hour on the hay r a tio n .

The r a te s

fo r co n v ersio n of feed to sh o rt chain acid s ranged from 35
to 8H $/hour.
P o o lin g d a ta from th e U t r i a l s , the amounts of ac id s per
100 gm. of rumen l iq u id which were produced from one pound of
t o t a l d i g e s t i b le n u tr ie n ts w ere, 0 . 2*+ to 0.67 a c e tic , 0.02 to
0 .2 6 p ro p io n ic , and 0.0*+ to 0.21 b u ty ric .
confid en ce le v e l ra n g e s.

These are the 95%

The average rumen in th ese t r i a l s

was e stim a te d to c o n ta in 70 kilogram s of liq u id .

Using these

v a lu e s , i t was computed th a t th e cow o b ta in s 3 to 13$ of i t s
energy from sh o rt ch ain a c id s .

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H eald, p. J . The assessm ent of g lu co se-co n tain in g substances
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195^

The Metabolism of sh o rt-c h a in f a t t y acids
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P fan d er, W. H. and A. T. P h illip s o n . The r a te s of abso rp tio n
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1953
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P h i llip s o n , A. T.

19**7

The r o le of the m icro flo ra of th e alim en­
ta r y t r a c t of h erb iv o ra w ith sp e c ia l re fe re n c e to
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N u tr. Abs. and R ev s., 12: 12-18,

P h illip s o n , A. T. A method of measuring th e flow of d ig e s ta
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19V8
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P h illip s o n , A. T.

1952a

The passage of d ig e s ta from th e abomasum
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P h illip s o n , A. T.

1952b

The f a t t y a c id s p re se n t in the rumen of
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P h illip s o n , A. T. D ig e stio n of c e llu lo s e by the rum inant.
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1953
P opjak, G. The m etabolism of f a t in th e mammary gland and
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1952
a p p lic a tio n of is o to p ic t r a c e r s . N utr. Abs. and
R ev., 21: 535*553.

85
P opjak, G. , T. H. French and S. J . F o lle y . U tiliz a tio n of
1951a
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Popjak, G. , T. H. F rench, G. D. Hunter and A. J . D. M artin.
1951b
Mode o f fo rm ation o f milk f a t t y acid s from a c e ta te
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Popjak, G. , G. D. Hunter and T. H. French. B io sy n th esis of
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m ilk -fa t in th e r a b b it from a c e ta te and glucose.
The mode o f conversion of carbohydrate to f a t .
Biochem. J . , 2+s 238-21+7.
Pounden, W. D. and J . W. Hibbs. The in flu en ce of th e r a t i o
19*+8
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19*+1
Pugh, P. S . and R. S c a ris b ric k . A cetate metabolism in ovine
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1952
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1951
X V III. The e f f e c t of fa s tin g on the a c tiv i t y of
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19“t9
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1953
R eid, R. t , S tu d ie s on th e carbohydrate metabolism of sheep.
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1950
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1952
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181-195.
1953

86
Robinson, R . Q., R. N. D oetsch, F. M. S iro tn ak and J . C.
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1953
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19^1
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2*+7-

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19^9
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1951
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1955
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1952
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195*+
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Ruminal f l o r a s tu d ie s in sheep. IV. The in flu en ce
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1952

P a r tic ip a tio n o f a c e tic and b u ty ric acid in the
lip o g e n e s is o f th e m ilk cow. Comp, r e n d ., 23i+s
567-569.

APPENDIX

89

90

TABLE 8

SUMMARY OF REGRESSIONS OF ACETIC, PROPIONIC, AND BUTYRIC ACID
CONCENTRATIONS IN THE RUMEN ON TIME AFTER FEEDING

A ll Hay R atio n
A cetic Acid

P ro p io n ic Acid

B u ty ric Acid

L°ge AQ

2.2602

0.8256

0.^915

L°ge Ap

0 . 6¥+l

-1.0103

-1 .1 5 1+5

kl
k2

0.0353

0.0322

0 . 021+8

0.5226

O.M+99

0.3511

S tandard E rro r of E stim ate
R eg ressio n of
Log^e Xo on t

0.1230

0 . 13^2

O.1308

kl

0.00^1

0 . 00^8

0 . 00^6

Lo&e Ao
D egrees of
Freedom

0.0*+97

0.0607

0.0592

16

Ik

lU

R eg ressio n of
Log^e Ap on t

0.9739

0.5712

0 A 966

k2

0.293S

0.2372

O.IU98

L°ge Ap

0.3872

O.kOOk

0.3355

Degrees of
Freedom

6

3

6

91
TABLE 8

(Continued)

SUMMARY OF REGRESSIONS OF ACETIC, PROPIONIC, AND BUTYRIC ACID
CONCENTRATIONS IN THE RUMEN ON TIME AFTER FEEDING

R e s tr ic te d Roughage-High C oncentrate R ation
A cetic Acid

P ropionic Acid

B u ty ric Acid

1.9683

0.8593

0.8562

0.7710

-0.8915

- 0.3201

0.0311

0 . 0*t65

0 . 028 k

0.8379

0.2763

0.7335

S tandard E rro r of E stim ate
0 . 13^8

0.2197

0.1279

0.0050

0.0089

0.0052

0 .0 6 1 3

0 . 1161+

0 .0 6 8 0

12

10

10

0.6509

1.U099

0.9568

0 . 266*+

0.6753

0.3520

0 .2 5 5 1

0 .9 3 3 8

0 .6 3 9 3

6

5

8

92
TABLE 9

THE CONCENTRATION OF ACETIC, PROPIONIC, AND BUTYRIC ACIDS
IN RUMEN LIQUID
(M illim o les Acid /1 0 0 gm. Rumen Liquid-*-)
Hours
A fter
Feeding

A ll Hay R atio n
A cetic Acid

P ro n io n ic Acid

B u ty ric Acid

C707

T2

C707

T2

0707

T2

0 .0

5.87

8.32

1.66

2.07

l.Cfc

1 .k7

2 .0

8.76

7.69

2.0U

1.90

1.32

l.*tl.

2 .5

8.50

7.35

2.22

1.73

1.W7

1 .38

2.22

-------------

1M

-------------

7.78

-------------

1 .7 7

3 .0
3.25

1A 0

3 .5

8.71

8.35

2.36

1.76

i .5 o

1.6 6

*♦.0

8.19

7.97

2.13

1.79

1.38

1M

5.0

8.06

8.33

2.03

2.01

1.7k

l.k l

6 .0

8.08

8.27

1 .80

1 .95

1.52

l.kQ

6.57

-------------

1 .59

--------

1.2k

1.71

--------

1.15

10.25
10.5

6.90

12.5

-------------

6.18

-------------

I .k 2

-------------

i*+.5

5.*+l

-------------

1.37

-------------

1 .00

18.0

-------------

6.38

-------------

1.56

19.5

k.2 1

-------------

1.09

23.5

--------

5.36

— ——-

2H.0

3.07

1.

0.82

1.2k
■

— ———

1.03
0.96

1.37

--------

1.19
0 .68

Sample weighed on to column co n tain ed 91.3# rumen l i q u i d
on a dry m a tte r f r e e b a s is ; th e v a lu e s re p o rte d h ere a re
c o rre c te d to 100# rumen l i q u i d .

93
TABLE 9

(Continued)

THE CONCENTRATION OF ACETIC, PROPIONIC, AND BUTYRIC ACIDS
IN RUMEN LIQUID
(M illim o les Acid /ICO gm. Rumen Liquid^-)

Hours
A fte r
Feeding

R e s tr ic te d Roughage-High C oncentrate R ation
A cetic Acid
C707

P ronionic Acid

T1

C707

T1
2.25

0 .0

H.9

5.*+o

1.31

1 .0

6 .2

------------

l.S*

6 .H

5.^2

I.W3

2 .0

6.**7

5.62

2 .5

6.75

3 .0

B utyric Acid
C707

T1

1 .8

2.15

1.73

------------

2.15

1.80

1.69

1.^9

2.07

1.77

2.17

6.20

1.71

2 . 3U

1.87

2.12

6.77

6.26

1.58

2.25

1.89

2.3^

3.75

------------

5.93

------------

2.18

------------

2.18

U.O

6 .5

5-69

1.63

2.13

1.70

2.15

5.0

------------

7.17

2.29

------------

2.52

6 .0

6.57

--------

1.58

--------

2.13

-----------

8 .0

5.97

------------

1.5^

1.72

------------

10.75

------------

it. 88

-----------

1.65

------------

1.83

12.0

5.6*+

------------

1.21

------------

i.7 1

---« = ■

13.0

------------

3.36

18.25

------------

3.96

——- —

1.23

--------

1 J +5

2*t.O

3.38

3.81

0.53

1.09

1.07

1.36

1

1.38

1.05

Samnle "Welshed on to column co n tain ed 91*3$ rumen liq u id
on a d ry m atter fre e b a s is ; th e v alu es re p o rte d here are
c o rre c te d to 100$ rumen liq u id .