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A
STU D Y
OF
OF
WI TH
THE
SHEEP FED
PART I C ULAR
A
AMINO
ih e s i s
ACI D
STA T U S
S I L AG E ,
REFERE�JC E
TO
f'1ETHION INE
s u b mi t t ed for th e
d egre e o f D oc t or of P h il o s oph y
a t Ma s s e y U niv er sity ,
N e w Z ealand
by
E . Mar g ar e t Gil l
June,
1976
(o �b. )0�5
c;., L
MASSEY UNtVO:Slll
LIB !tAU
11·
11,703
i
A B S TRACT
The a m i n o a c i d s ta tus o f s h e e p f e d s il a ge appears t o b e a n
imp o r t ant f a c t o r i n c o n t r ol l ing v o l unt a r y i nt ake .
Th e bas i c a i m o f t h e
e xperiment s desc r i b e d i n t h i s t h e s i s , was t o s t u d y th e d i g e s t i o n a n d
util i s at i o n o f s il a g e , i n r e s p o n s e t o i n c r eased av a i l ab i l i t y o f
meth i o n i n e and p r o t e in .
T h e t r e a tm e nts c h o s e n to inc r e a s e p r o t e i n
avai l abil i t y , w e r e f ormal in-tr e a t m e n t o f s i lage
( Expt .
1
)
and s up p l e-
ments of f o r mal d eh y d e-t r e a t e d c a s e i n and a readily av a i l a b l e e n e r g y
s o ur c e
(Expt. 2) .
inc r e as e d by
I/P
Meth i o n i n e a v a i l a b i l i t y o n untr e a t e d s i lage was
inf u s i o n in Ex p t . 1 , and m e t h ioni ne avail a b il i t y o n
all t h r e e t r e a t ments was inc r e ased by d u o d e nal infusion i n E xp t .
2.
T h e s e tre a t m e n t s p r o v i d e d f r o m 0 . 8 t o 6 . D g/day o f meth i onine av a i l able
for a b s o r p t i on , and f r om
t h e d uo d e n u m .
33
to 1 6 7 g/d a y of total amino ac ids sn t e r i n g
T h e r e s p o n s e to i nc r eas e d m e t h i onine av a i l abilit y wa s
meas u r ed i n t e r ms o f
N
b a lanc e , pl asm a amino ac id c onc e nt r a tion and t h e
pe r c e n tag e o f L -me th ioni n e-C 1 4 ( U) oxidis e d to c a r b o n d i oxide .
In t a k e was r e s t r ic t e d t o n e a r m a i ntenanc e , but th e amo unt o f f e r e d
was s e ldom c om p l e t e l y e a t e n .
Th e r e w e r e no tre3tment d i f f e renc e s i n
i nt a k e i n E xp t . 1 , b u t i n take o f th e b as al sil age d i e t dec r e ased i n
E xp t . 2 .
Th is d ec r e a s e was p a r t l y p r e v e nted b y th e t w o s upple m e n t s ,
and intake was i nc r e a s e d b y d uo d enal i n f u s i o n of L -methion ine o n a l l
t r e a t ment s .
T h e d i f f e r e nc es i n amino ac id i n take account e d for 67 % o f
t h e d if f e r e nc e i n flow o f amino ac i ds i n t o t h e d u o d enum i n t h e d a t a f r o m
b o th exp e r imen t s .
;;::i�
I n a d d i t i o n , th e
suppl ement i ncre a s e d t h e
a
f l ow o f amino a c i d s into t h e d u o d e n J� � b � i n c r e as i n g t h e c o nv e r s i o n o f
no n- p r ot e i n-ni t r o ge n t o p r o t ei n -n i t r o g e n i n the r umen .
A n adequ a t e
s up p l y o f amino a c i d s t o th e t i s s u e s is r e q u i r e d f o r e f f i c ient u t i l i sa t i o n of a f e e d .
T h u s , h i gh a m i n o aGid and water s o l u b l e sug a r
iL
c o n t e n ts w i l l i mp r o v e th e n u t r i t i v e v al u e o f sil a g e , by i n c r e asi n g t h e
f low o f a m i no a c i d s i n t o t h e d u o d e n um .
N b a l a nc e i n c r e ased, and to tal p l asma amino acid c o nc e n t r a t i o n
d ec r�ased � i n r esp ons� t o me th i o n i n e i n f u si o n on a l l t h ree t r e a tm e n t s
i n E xp t .
2.
Th e se r e sul t s su g ge st th a t m e t h i o n i n e w a s l i m i t i n g t i ssue
p r o t e i n sy n th e sis .
In t a ke , was a l so d e c r ease d , b u t to a g r e a t e r e xt e n t
a t l o w e r l ev e l s o f d u o d e n al amino ac i d f l ow i n E xp t . 2.
H o w ev e r, t h e r e
was n o d e p r essio n o f i n t a k e i n E xp t . 1 , d esp i te l o w f l ow r a t e s o f amin o
acids i n t o the d u o d e n u m .
Th i s a p p e a r e d t o b e due t o a l ow e r r equi remen t
f o r m e t h i o n i n e i n t h e sh e e p i n E xp t . 1 , c o mpared t o E xp t .
2.
i ii
ACKNOWL ED G Ef'1f:NTS
T h is T h e s is w a s m a d e p o s s i b l e t h r o u g h r ec e i pt o f a C omm o n w e alth
a n d by p r o v i s i o n o f f ac i l it i e s,
Sc h o l a r s h i p,
f r om A p p l i e d B i oc h em i str y D iv i s i o n,
and Or R .W .
B ai l e y ) .
O .S . I . R .
and f i n a nc i a l a s s i st a nc e
( D i r ect o r s :
Or G .W .
B utl e r
T h i s h e l p i s g r at e f u l l y ac k n o wl e d g e d .
C o m p l et i o n o f th e T h esis w o u l d n ot h av e b e e n ac h i e v e d w it h o ut the
s u p p o rt of m a n y p e o p l e , t o o n um e r o u s to m e nt i o n i n d i v i d u a l l y .
H o w e v er,
t h is s u p p o rt w as g r eat ly apprec i at e d .
P a rt ic u l a r t h anks a r e due t o :
M y s u p e rvi s o r s :
I .M.
B r o o k e s,
Or M . J .
Ulyatt,
O.S. I .R. ,
a n d D r s G . A . Wick h am a n d
M as s e y U n i ver sit y .
O r T . N.
B a r r y, I n v e rm ay Res e arch C e nt r e,
A .R . C . ,
H ami lt o n an d Or A . W.F.
D a ve y,
Mo s gi el ,
Or J . E .
Ma s s e y U ni v e r s it y ,
Wo l f f,
Ru a kur a
f o r supp l ies
o f s ilage .
T h e " v o l u n t e e rs" w h o h el p e d pac k, t r an sp o rt an d st o r e the si l a;;e .
O r J . E . W ol f f a n d Mr
P. R .
Jenk ins f o r e n a b l in g th e a n al y s i s o f
m ethi o ni n e s p ec i f ic activ it i e s t o b e c o n duct ed at R u aku r a .
T h e st aff o f t h e R um in a nt Physio l o gy a n d N ut rit io n g r oups,
D .S . I . R .,
f o r h e l pf u l discu s si on,
a dv ic e a n d pr o vi s i o n of f ac i l it i e s,
T h e sta f f o f th e A n alytic a l a n d I n st r um e nt Wo r k sho p G r ou p s ,
D .S . I . R . ,
A . B .D . ,
A.B.D. ,
f o r m a i nt a ining m y s a n it y d u r i n g th e t ri a l s a n d t r i b u l at i o ns
o f r u nn i n g an am i n o ac id an a l y s e r,
T h e st af f of
A p p l i e d M athematics D i v i s i o n, D . S . I . R .
f o r stat ist ic a l
help .
T h e L i b r a r y s t a f f at O.S . I . R .
st a f f at O . S . I . R .
f o r p at i e nt h e l p,
a n d oth e r memb e r s
f o r h e l pfu l d isc us s i o n a n d i nt e r e st ,
of
iv
My T y p is t , Lo r r a in e , for h e r s p e e d and e f f ic ienc y , and m y f l a t m a t e s ,
pas t and p r esent , a n d frie nd s , f o r t h e i r c o nviction t h a t th is
c o nc l usio n would be r e a c h ed .
Tab l e o f Cont e n t s
i
Ab s t r ac t
Ac knowl ed gemen t s
iii
List o f A b b r evi a t i o ns
vii
1
PR EFACE
CH A PT E R
1
- Li t er a t ure R ev i ew :
1 .1
N met abolism in the r umen
3
1 .2
N ent ering the d uoden um
7
1 .3
Amino acid meta b o l i sm i n t h e
a n im al
9
Metab o l ism of S - c o ntai n i n g
amino acid s
14
A � i n o acid req u i r ements o f
r um i n ants
19
1 .6
Fo r mal d eh y d e-trea tme nt o f
pro t e i ns
29
1 .7
T h e f ermen tation of silage
a n d i t s effec t o n i n t ake
33
Co n c l usion
40
1 .4
1 . 5-
1 .8
CH A PT E R
2
-
T ec h n i q ues f o r S t ud y i n g A m i n o Acid Metab o l ism in
R um i n a n ts:
2.1
I n t r o d uc t i o n
42
2.2
M easu remen t of d u od e n al f l ow
43
2.3
A m i n o acid anal y sis a n d
measur e�en t of spec i f ic
ac t i v i ties
48
M easu r emen t of m ic r o b i al
p r o tein syn thesis
55
2.4
CH A PT E R
3
-
T h e E f f ec t o f Fo rmal i n -t r eatment and Supp l emen tati o n
o f S i l a ge D i et s o n t h e Di g est i o n of S i l ag e b y Sh eep :
3.1
I n tr od uc t i o n
60
3.2
M etho d s
61
CH A P T E R 4
-
3.3
R e sults
69
3 .4
Discussion
75
T h e E f f ec t of Fo rma l i n-t r e a t me n t and Supp l e me n t a t i on
o f S il a g e Di e t s on t h e U t i l i sation o f N b y S h e e p :
4.1
I n t r o duc t i o n
92
4.2
M e t h ods
93
4.3
R esu l ts
98
4.4
Discussi on
1 02
G E N E R A L DI S CU S S I O N
117
R E F E R E NCE S
1 20
A P P E NDICE S
1 32
vii
A B B R E VIA T I O NS
AA
a m i n o ac i d
C
c ar b on
OAP
2 : 6 diami no p imel ic ac i d
OM
d r y mat t e r
DNA
d e o x y r i b o n u c l e i c ac i d
I /P
i n trape r i t o n e al
ME
m e t a b o l i s ab l e e n er g y
MHA
m e t h i o n i n e - h yd r o x y - an al ogue
N
nitrogen
NA
n u c l eic ac i d
N A AN
n o n-am i n o ac i d-N
NPN
n o n-p r o t e in-N
OM
o r g anic mat t e r
RNA
r i b o nuc l e ic ac i d
S
s u l ph u r
S A.
s p e c if ic ac t i v i t y
WSC
wat e r s o lu b l e c ar b o hyd r at e s
w/v
w e i g h t p e r v ol u m e
w/w
w e i g h t p e r w e i gh t
1
T h e use o f d omes t i c an imals t o p r o v ide reso u r c es f o r th e h um a n
p o pu l a t i o n h as b e e n a p ar� of agric u l t u r e for s o m e 1 0 , 00 0 y e ar s .
H owev e r , t h e i n c r e ase i n t h e w o r l d 's p o p u l a t ion a n d h e n c e th e r e l at iv e
d e c r e ase i n l and and f o o d reso u r c es emph asise th e n e e d f o r i nc r e asi n g
t h e e f f ic i e n c y o f f o o d p r o d uc t i o n .
R um i n ants ma ke an i m p o r t a n t c o n t r i b u t i o n to th e e f f ic i e n t
u t i l isa t i o n o f reso urc e s , b y th e d i gest i o n of p l ant c e l l w a l l c ar b o­
h y drates a n d b y th e c o n v e rsi on o f non-p r o t e in-n i t r o g e n ( NPN ) c o m p o u nds
to meat , w o o l and m i l k .
Th e i n i tial c o n v e rsion of NPN to p r o t e i n is
ac h i e v e d b y m i c r o b e s in th e rum e n .
H ow ev e r , the e f f ic i e n c y of m i c r o b i a l
p r o t e i n sy n t h esis i n sa t isf y i n g th e a m i n o a c i d r e q ui re m e n ts o f
r umi nan t t i ssues h as o n l y rec e n t l y b e c o m e t h e sub j e c t o f d e t a i l e d stud y.
T h e main reaso n f o r th e d e l a y i n stud y i n g r uminan t ami n o a c i d requ i re­
m e n ts was t h is abi l i t y o f the mic r o b es to modify d i e t ary pro t e i n .
H e nc e , ami n o ac id r e q u i r eme nts m ust b e spec i f i e d at a d u od enal r a t h e r
t h a n a diet a r y , lev e l f o r ruminan ts.
Th is h as o n l y r e c e n tly b ecome
p ossi b l e , t h r o ugh the use o f sh e e� p r e p a r e d with re-e n t r a n t c an n u l ae
i n t h e pro ximal d u o d e n um ( A sh , 1 9 6 2 ).
Numer ous workers h av e suggested th at m e th ioni n e is t h e f i rs t
limi ting ami no ac id f o r l i v e-we i g h t g a i n a n d wool g r o w th o n a v arie t y
o f ruminant d i e ts ( N im r ic k e t al . , 1 9 7 0;
A rmst r o n g a n d A nnison , 1 9 7 3;
H u tton and A n n iso n , 1 9 7 2;
H ar r ison et a l . , 1 9 73 ).
I n ad d i ti o n ,
B ar r y e t al . ( 1 9 7 3 ) r e p o r t e d th a t the i n t a k e of some sil a g e s w as
i n c r e ased i n r esp o nse t o i n t r ap e r i to n e al i n j ec t ions o f m e t h i o n i n e .
H e nc e , th e a v a i la b i l ity of m e th i o n i n e o n suc h f eeds a p p e ars t o l i m i t t h e
e f f ic i enc y o f t h e i r u t i l isat ion .
Th is th esis w i l l e x a m ine t h e u t i l isat i o n o f n i t r o g e n and a m i n o
a c i d s , wi th p a r tic u l ar emphasis o n m e th i o n ine , b y sh e e p f e d sil a g e ,
2
al o n e , and w i t h s u ppl e me nt s t o i n c r ea s e t h e av ail ab i l i t y o f m e t hi o n in e
and t o t al ami n o ac i d s t o t he t i s s u e s .
C H A PTER
L I T ERATURE
1
REV I EW
fig.
-
1.1
N
recycling in ruminants
SALIVARY
NITROGEN
RUMEN
LIVER
(-.__
T
J
UREA
AMI"lONIA
N
T
E
s
�
· r---.:...�
...
N
<E:
H
E
s
EN D O G E N O U S P R OTEI N
N
S E C R ETI O N
E
s
AMIN
ACID
NH3
AC OS
T
T
I
Af'"IINO
)AmNO ACID
AND UREA
SYNTHESIS
INTESTINAL WALL
OR LIVER
I
s
3
1
rH trooen
.1
"
m et abol i s m
in
the r umen
Th e m e tab o l i sm o f n itro g e n ( N ) i n r u m inants h as r ec e i v e d
c o n s i d erab l e a tten t io n f r o m b i och emist s , m ic rob i o l o g i s t s a n d n u t r i t ion­
i s t s , and n u m e r ous r e v i e ws o n v ar io us a s p e c ts of t h e s u bjec t h av e b ee n
wri t t e n ( e . g . Waldo , 1 9 6 8 ;
B ar t l e y , 1 97 1 ) .
S m i th , 1 9 69 ;
H e lmer and
I n a d d i t i o n , q u a n t i t a t i v e models of N m e t ab o l i s m in
sheep hav e bae n �r o p o s e d ( N o l an , 1 9 75 ;
a n d Nol an , 1 9 7 6 ) .
A ll i s on , 1 9 7 0 ;
N o l a n et a�. , 1 9 76 ;
Mazanov
T h us , o n l y a b r i ef o u tline of t h e a s p ec t s of N
·me tabolism which a r e p e r t i ne n t to the p r e s e n t exp e r im e n t s w i l l b e giv e n
h e re .
A simplified diag r a m of the main N t r a n s a c t i o n s i n she e p i s g i v e n
i n F ig. 1 . 1 .
T h e m a i n r e ac t ions o c c u r r i ng i n the rumen c an b e
s ummarised a s f o l l o ws .
A.
�-?ntering the rume n
N
may e n t e r the r u me n i n th e di e t , i n s aliva or b y t r a n s f e r ac r o s s
t h e rumen wall .
The
majo r c om p o n e n t s o f t h e N f r ac ti o ns o f h e r b age a r e
ami�o ac ids , p resent e i ther as fre e ami n o acids o r as par t o f pro t e i n
m o l e cules .
Howe v e r , s i l age d i f f e r s f r o m f resh f e e ds i n h a v i n g
r e l a t i v e l y h i gh ammoni a , ami n e a n d am i d e conte n t s ( H ugh es , 19 70 ) .
Th e
N e n t e ring the r umen in s al i v a i s als o m a i n l y i n t h e f o rm o f NPN , wit h
ur e a acc o unt i n g f o r 60-70 % o f the t o t a l N s ec re t e d ( S ome rs , 1 961 a ) .
H oweve r , t h e p r o p o r t i o n o f u r e a i n t o t a l s al iva -N
·i ntake ( S o me rs, 1 9 6 1 c ) .
is a f f e c t e d b y N
S o m e r s ( 1 9 61 b ) h ad e arlie r s h o wn t h a t wh i le
d ai l y sec r e tio n o f ure a-N was l i n e arly r e l a t e d to N i n t ake , d aily
s ec re tion of t o t al s al iv a -N
did n o t f o l l o w a simple l inear rel a t i o ns h i p .
S al i v a ure a-N c onc e n t r a t ion appeared t o f o l low b l o o d ur ea-N c o n c en t r a ti o n ,
b u t n o s i mpl e r e l a t i on s h i p b e t w e e n t h e s e two fac tors w as e v i d e n t
4
( S o mers , 1 9 61b i .
N o l an e t al .
r el a t i ve i m p o r t a nc e o f s al i v a -N
t h e r umen wa l l .
(1973 ) and Th o r n t o n ( 1 9 7 0 ) s t res s e d t h e
c om p a r e d t o trans f e r o f u r e a ac r o s s
Ho u p t a n d H o upt ( 1 9 68 ) f o u n d a p o s i t i v e l i near
r e l a t i o ns h i p b e t we e n ne t t r a n s f e r o f u r e a across the r umen wall and t h e
conc e ntr ation g r ad i e n t b e twe e n plasma a n d r um en , b u t o th er work e r s h a v e
r e p o r t e d l imi t s t o t h i s tran s f er ( Wes t o n a n d H o g a n , 1 9 6 7;
V e rc o e ,
1969 ) .
B.
Protein and am i n o acid d egr a d a t i o n
Th e d ie t a r y p r o t e i n may e i t h e r b e d e g r a d e d t o ami n o aci d s , f at t y
aci d s and ammo n i a , o r i t may l e a v e th e r um e n u n d e g r ad e d .
Di f f e r e nc e s
in t h e rate o f r um i n al f e rm e n ta t i o n of p r o t e ins h ave b e e n r e c o r d e d b y
McO o n ald ( 1 952 ) and H e nderic k x and M�r ti n ( 1 9 63 ) , who s h o w e d th a t h i gh
s o l ub i l i t y was c o r r e l ated w i th a rapid rate of f e r men t a t io n .
Th e
s o l ub i l i t y o f d ie t a r y p r o te i n , and h e nce th e r a t e o f r um i n a l d e g r ad a t i o n
h as b e e n d e c r e a s e d b y h e at tre atme n t o f th e p r o t e in ( T a gari e t a l . , 1 9 62 )
and b y t r e a tm e n t w i t h t a nn i n s o r f o rm aldeh y d e ( Tagari e t al . , 1 9 6 5;
F e r g uson e t al . , 1 9 6 7;
O f f er et al . , 1 9 7 1 ) .
De g r ad a t i o n o f d i e t a r y
p r o t e i n r e l e a s e s am i n o a c i d s , b u t o n l y v e r y l o w l e v e l s o f f r e e a m i n o
a c i d s h ave b e e n f o u n d i n the r u m e n ( L e w i s , 1 9 55;
1 9 67 ) .
Wr i g h t a n d H u n g a t e ,
A mi n � acid s ma y b e d e aminated w i th r e l e a s e of ammo n i a ( Lew i s
a n d E8ery , 1 9 62 ) or as s i m il a t e d d irec t l y i n t o b ac t e r i a l p r o t e i n ( N o l an
e t al . , 1 9 7 6 ) .
Some ami n o acid s are m o r e r a p i d l y d e a m i n at e d th a n o t h e r s
( Lew i s and Eme r y , 1 9 62; Ch al u p a , 1 9 74 ) and d e ami n a t i o n r a t e s al s o d i f f e r
w ith t h e s tr a i n o f b ac t e r ia i n v o l v e d ( S c h e i f i n ge r e t a l . , 1 9 7 5 ) .
Sch e i f in g e r e t al .
( 1 9 75 ) a l s o r e p o r t e d th a t wh i l e i n d iv i d ual s t r a i n s
o f b acte r i a d o n o t d e am i n ate a l l th e amino a c i d s , me th i o n i n e w a s u n i q u e
i n b e i n g metab o l i s e d b y a l l s t rains .
I t was d e g r a d e d b y 25% of t h e
s t r a i n s t e s te d , a n d s yn t h e s i s e d b y th e o t h e r 75% .
I n a d d i t i o n , S a l i s b ur y
5
e t al .
( 1 970 ) sugge s t e d t h a t me t h i o nine c o u l d b e s t o r e d b y b ac t e r ia
t h r o u gh c o nv e r s ion to t h e s u l p hoxide when m e th i onine c o nc e n tr a t i o n s we r e
r e l a t i v e l y h i g h , with s ub s e q u e n t r e c o n v e r s i o n to m e t h i o nine when t h e
m e th i o n ine c o n cent r a t i o n dec r e as e d .
C.
M i c rob ial pr o t e in syn t h e s i s
Mic r o b i al p r o t e i n may b e s y n th e sised d i rec t l y f r om am i n o ac ids o r
f rom ammon i a .
There i s s t i l l c o n s ide r ab l e d is p u t e o v e r t h e r e l a t iv e
imp o r tance of these precurs o r s , w i th s ome w o rkers f av o u r ing s y n t h e s is
f r om ammonia ( B r yan t and R o b i n s o n , 1 9 63;
1 96 6 ;
P o r t ugal a n d S u t h e rlan d ,
Wal k e r end N ader , 1 9 6 8 ) wh i l e o th e rs s uppo r t th e im p o r tance o f
ass i m i lat i o n o f amino acids ( P r es c o tt , 1 9 61 ;
e t al . , 1 9 7 6 ) .
Landis , 1 9 63;
N o l an
Various meth o d s f o r m e as u r i n g the r a t e o f p r o t e i n
s y nthesis h av e b een p r o p o s e d , and t h e s e w i l l b e d i s c us s e d i n C h a p t e r 2 .
A n adequate s u p p l y o f e n e r g y i s also r e quired f o r e f f i ci e n t p r o t e i n
s y n t h esis , a n d t o p re v e n t br e akdo w n o f pro t e in wh ich h as a l ready b e e n
s y n th es is e d.
H e nde r i c k x ( 1 9 6 1 ) s u gg e s t e d t h a t b r e a k d o w n of mic r o b i al
pr o t e i n w i t h i n the r umen migh t p r o v ide ener g y for p r o te i n s y n th e s is
d u r i n � p e r iods of l o w e n e r g y av ai l a b i l ity .
The i n e f f ic ienc y of th is
p r oc e s s of rec y cling of N w i th i n t h e r umen was p o i n t e d out b y N o l an and
Leng ( 1 972 ) and Nolan et al .
( 1 976 ) .
Th e e f fic i enc y of an e n e r g y s o urc e in inc reasi n g m ic ro b ial pr o t e in
s yn th e sis w i l l depend o n i t s rate o f u t i lis ation b y t h e microbes ,
-r e l a t i v e t o t h e rate of ammo n i a r e l e ase f r o m the N s o u r c e ( H e lme r and
Bar tl e y , 1 97 1 ) .
Thus , s ta r c h h as b ee n found to be m o r e e f f ec t i v e than
x y l a n , p ec tin or the simple s u g ar s , wh ich i n turn a r e m o r e e f fec t i v e
than c e l l u l o s e o r h emic el l u l o s e i n p r omoting u r e a u t i l isat i o n in v i t r o
( B e l a sc o , 1 9 5 6 ;
Hende r i c kx a n d M a r t i n , 1 9 6 3 ) .
Howev e r , d i f f e r e n c e s
6
e x i s t b e t w e e n s t arc h from d i f f e r e n t s o u r c e s .
B l o o mf i e l d e t al .
( 1 958 )
repo r t ed a 98 % i nc r e as e i n u r e a u t i l i s at i o n with t h e a d d i t i o n o f c o rn
s tarc h , wh ile w he a t s tarch o n l y p r o d u c e d an 8 1 % i ncre as e , and s o lu b l e
s tarch a 5 5 % i nc re as e .
A r i as e t al .
( 1 9 5 1 ) sugg e s t e d th a t th e r um en
mic r o b ial popul a t i o n u t i l is e s urea mor e e f f i c ie n t l y i f sm al l amo u n t s o f
rea d i l y av a i l a b l e e n e r gy s uc h as s uc r os e o r s tarch a r e p r o v i d e d , w h e n
c e l l u l os e i s t h e m a i n e ne r gy s o u rc e .
L e w i s a n d M c O o n a l d ( 1 9 58 ) f o und
s t arch o r g ras s l e v a n t o provide f o r e f f i c i e n t u t i l isation of ammo n i a
r e le a s ed f r om c as e i n .
Th e y s t res s ed the i m p o rtance o f a s t e ad y r e l e a s e
o f e n e r gy a t a r a t e s im i l ar t o t h e r a t e o f ammonia p r o d uc t i o n .
e t al .
Oj o r d e v ic
( 1 974 ) c om m e n t e d o n the hi gh d e g r e e of plant pro te i n d e g r a d a t i o n
��
a t a l ow l ev e l o f distsP� e n e r g y ( 2 3 0 0 koal / k g die t ) .
a
�9��6k
Th e y f o und maximum
p ro tei n s y n thes i s a t Pff e n e r g y l e v e l o f 2 9 0 0 kcal/ kg d i e t .
A t h i gh e r
e n e r g y l e v e l s prote i n s y n thes is d e c re as e d again , b u t n o adequate
e xplanat i o n for this was g iv e n .
D.
N l e a v i n g t he r umen
N
may l e ave th e ruman i n the form o f m i c robial p r o t ei n , d i e t a r y
p ro t e i n , a m i n o a c i d s and o t h e r N P N c o mp o u n d s .
The mo d i f ic a t i on o f
d i e t a r y p r o t e i n b y the m ic robes i n th e rumen dec r e a s e s the i mp o r t anc e
o f d ietar y ami n o ac i d c omp o s i ti o n i n the n u t r i t i o n of t he r u minan t , and
p l ac e s more emph as i s o n t h e c ompo s i t i o n o f t h e amino ac i d s l e av i n g th e
rume n .
A f t er l e av i n g t h e rume n , n u t r i e n t s f l ow i n to t h e o m a s um a n d
abo mas um p r io r t o e n te r i n g t h e d u o d e n um .
Wh ile s ome c h an g e s in t he
n i t r o g e n o u s c o mp o n e n t s o f th e d i g e s t a will t ake p l ac e i n t h e abom as u m ,
d u e t o lys i s o f p r o t o z o a ( Be e v er e t a l . , 1 9 7 3 ) and t h e add i t ion o f
e n d o g e no us s e c r e t i o n s ( H a r r o p , 1 9 7 4 ) , mos t o f th e w o r k on i n tes t i nal
d ige s t io n of a m i n o a c i d s h as s t u d i e d the f l o w of am i n o ac i d s i n t o the
7
duodenum.
This
is
due to the introduction of re-entrant cannula e which
ca n be ins erted in the proxima l duodenum
1 .2
N enterinq
( A s h, 1 9 62 ) .
the duodenum
Since the introduction of duodena l re-entrant cannula e,
a ttention
has been given to the proportion of dieta ry-N in the total-N entering
the duodenum on na tura l feeds s uch as
pas ture and ha y
1 97 1 ;
Sutton et a l.,
1 973 ;
Coelh o da Silva et a l.,
et a l.,
1 975 ) .
Ha rris on et a l.
( 1 9 73 )
( 1 975 )
Wa lker et a l.
( as
non-a mmonia-N
19 75 ;
reported a ra nge of
37 - 8 5%
of
dried a nd frozen
4 2 - 74 %
microbia l
a percenta ge of tota l N entering the duodenum )
( 1 973 )
compa ris on of s even pas ture and ha y diets .
Proud
a mino a cid-N entering the duodenum on gra s s
fed fres h,
in a
compa red total
as
s ilage or
He recorded 1B.Bg a mino a cid-N/ da y entering the s ma ll
a fter drying.
intes tine
et a l.,
Walker
reported a range of
ba cterial-N in the total-N a t the duodenum on fres h,
diets .
( Hutton
of s heep fed dried gra s s ,
wilted s ila ge.
A rms trong
( 1 9 73 )
a nd only 12.3
g/ day for s heep fed
s ugges ted that the low value for s ilage
was .due to inefficient us e of s ila ge-N within the rumen.
Differences in the N a nd energy compos ition of herbage,
va riety a nd s ta ge of
ma turity ha ve been found to ha ve
on the diges tion of N by ruminants
1 974 ;
MacRae
a nd Ulyatt,
dieta ry-N content,
1 9 74 ;
( Egan, 1 9 74 ;
Hume a nd Purs er,
a
1 9 74 ) .
NPN,
Increa s ed
a nd a high concentra tion of water s oluble ca rbohydra tes
diges ted in the s toma chs .
duodenum is
marked effect
Ulyatt a nd MacRa e,
genera lly increas ed the yield of microbia l protein/ 100g
protein s ynthes is
due to
is
However,
OM a pparently
an increas ed rate of microbia l
only beneficia l if the tota l protein entering the
increas ed.
Thus ,
on diets with a high ratio of protein
increa s ed diges tion of dieta ry protein in the intestines
ma y be
:
8
more beneficia l
than increas ed rumina l diges tion.
Cha lmers a nd Synge
( 1 954 )
s tudied the effect of duodena l
supplementa tion of protein on the efficiency of
N
utilis a tion.
found differences between different protein s ources ,
They
s ugges ting tha t
the quality of protein entering the duodenum ha d a n importa nt effect
on
N
meta bolis m in the a nima l.
Thus ,
cha nges
in the balance of dieta ry
and microbia l protein entering the duodenum might be expected to a lter
the qua lity of protein a va ila ble to the a nima l.
amino a cid compos itions
and t hus
the es s entia l
of herba ge a nd microbial protein a re s imilar,
the amino a cid compos ition of duodena l diges ta from s heep fed
different herbages
Wes ton,
1 9 70;
et al.,
1 975 ) .
is
a ls o s imila r
.
Ma cRa e et a l.,
( Clarke
1972 ;
amino a cids
et al.,
1 9 66 ;
Ha rris on et a l.,
Microbial protein does
methionine a nd lys ine th a n herbage,
Hoga n a nd
1 973 ;
Ulyatt
ha ve a higher content of
a nd th e concentra tions
of thes e
have been s hown to increa s e on diets s upporting a high rate
of microbia l protein s ynthes is
et a l.,
However,
1 97 3 ;
and Sch elling
Ulyatt et a l.,
( 1 9 68 )
( Coelho
1 975 ) .
da Silva et a l.,
However,
did report differences
compos ition where the feed protein was
1 972 ;
Ha rris on
Poley a nd Trenkle
( 1 963 )
in duodenal a mino a cid
very different in compos ition to
microbia l protein.
The qua lity of protein a t the duodenum will depend on its
to fulfill the metabolic requirements
individua l a mino a cid requirements
determined,
acids
for rumina nts
ha v e s o fa r been
However,
( USD A , 1 9 68 )
pork a nd mutton amino a cid compos itions
which s ugges ts that the proportions
required for tis s ue protein s ynthes is by pigs
s imilar.
but s ince few
a ny definition of a high qua lity protein ca n only rela te to
monoga s tric a n ima ls .
are s imila r
of the a nima l,
capa city
Hence,
a protein which is
of a mino
a nd s heep a re a ls o
of a high qua lity for pigs ,
would
9
p res umably a ls o be of a high
synthes is ,
1.3
A.
qua lity for rumin a n t tis s ue protein
when s upplied at a duoden a l level.
A min o a cid meta bolis m with in
the a n ima l
I n troduction
The meta bolis m of in dividua l a min o a cids
I
cons idera ble deta il s in ce th e work of Ros e
s upply of leucin e,
tryptopha n ,
growth,
is oleucin e,
threon in e,
( 1938)
lys in e,
N
ba la n ce in
referred to a s
been s tudied in
s h owed tha t a dieta ry
methion in e,
his tidin e a n d a rgin in e was
or the ma in tena n ce of
h a ve genera lly been
valin e,
has
ra ts .
required for optima l
Thes e a min o a cids
the "es s en tial a min o a cids ",
t heir requiremen t for protein s yn thes is
This
adoption of
the term "non-es s en tial" to refer to a min o
Chris tens en
cla s s ifica tion of ess en tia l amin o a cids
( 1 964 )
pla y more domin a n t roles
in meta bolis m.
s ugges ted that "dis pen s a ble" a min o a cids
than
a cids
poin ted out tha t "es s ential" amin o a cids
"n on -es s entia l",
a n d s ome of
was
a n d he referred to the
tis s ue
led to the
rate s ufficien t to meet the requiremen t.
of prime importa n ce when in s hort s upply,
amin o a cids
s in ce
ca n n ot be met by mammalian
synthes is .
be s yn thes is e d a t a
phenyla la n in e,
which coul d
However,
were on ly
the "n on-es s en tial"
Harper
( 1974)
a more a ppropria te term
N
of th es e amin o a cids
as
"n on -s pecific"-N.
It has
been kn own for more tha n
twen ty yea rs
th a t rumin a l micro­
orga n is ms could s yn thes is e the ten es s en tia l amin o a cids
( Loos li
et a l.,
1949).
a s s umed
to be
Tis s ue a min o a cid metabolis m in
s imila r to that of monogas trics ,
o f the ten
a min o acids
by rumin a n t tis s ues .
Since then ,
the rumin a n t was
un til Oown es
( 1961)
verified tha t n in e
es s en tial for rat growth could n ot be s yn thes is ed
However,s ome s yn thes is
of a rgin in e did take pla ce.
pos trumin a l a dmin is tration of amin o a cids
has
con firmed
10
s ome o f th e assume d s im i l a r i t i e s b e tw e e n ruminants and m o n o g as t r ic s .
H o w e v e r , s p e c i e s d i f f e r e nc e s do e x is t .
R e s ults f rom m o n o g a s t r i c
s pe c ies o t h e r t h a n t h e r a t , i n d i c a t e t h a t d i e tary g l y c i n e m ay b e
r e q u i r e d b y t h e c h i c k ( McOo n a l d e t al . , 1969 ) , wh i l e d i e t a r y a r g i n i n e
m a y n o t b e nec e s s a r y f o r h umans ( I rw i n a n d H e g s t e d , 1971 ) .
T h u s i t i s app are n t t h a t f al s e a s s ump t ions m a y b e m ade b y e x t r a­
· p o l a t i n g r e s u l t s f r om o n e s p ec i e s t o a n o th e r .
H o w ev e r , o w i n g t o t h e
l ac k o f d a t a o n r u m i n a n t amino ac id m e t ab o l is m , t h e f o l l ow i n g d i s c u s s i o n
w i l l inc l u d e r e f e r e nc e s t o mono g a s t r i c met a b o l is m .
B.
F ac t o r s a f f e c t i ng c o nc e n t r a t ions o f amino ac ids in p l a s m a
Th e c o nc e n t r a t i o n s o f i n d i v i d u al a m i n o ac ids in pl asma a r e t h e n e t
r e s u l t o f i npu t s s uc h as i n t es tinal a b s o r p t io n and tis s ue p r o t e i n b re ak ­
d own,and u t il i s a t i o n f o r t i s s u e p r o t e i n s y n th e sis and m2tab o l ic
r e ac t ions .
Th e r e l a tive rates o f t h ese reac tions are aff e c t e d b y many
f ac t ors , which c an be b r o ad l y c l as s i f i e d i n t o :
( 1 ) ph y s i o l o g i c al ,
(2) n u t r i t i o n al .
(1) Phys iol o gic al f ac tors :
M e t ab o l is m of amino ac i d s by th e l iv e r and
u p t ake o f amino ac i ds by t h e i n d iv i d u a l t i s s ues fo r p r otei n s y n th es is ,
r e s u l ts i n d i f f e r e n t co nc e n t r a t ions o f f r e e amino a c i d s in b l o o d s am p l e s
t a k e n f r o m v ar i o u s p a r t s o f th e b o d y ( Re i l l y a n d Fo r d , 1971;
e t al . , 1973 ) .
Wo l f f
T h u s c o n c e n t r a t i o n s i n p o r t al plasma a r e h igh , wh i l e
th o s e o f j ugu l a r o r c a r o t i d p l asma a r e l o we r .
Dif f e r e n c es w i t h i n b l oo d ,
b e twe e n p l as ma-f r e e and c e l l amino ac id c o n c e n t r a t i o n s h av e a l s o b ee n
f o un d ( B u r ac zews k a e t a l . , 1972 ) i n d i c a t i n g e i th e r d i f f e re n t r a t e s of
m e t a b o l i s m b e tw e e n the two c om partmen ts , o r s elec t i v e u p t a k e by t h e
c ells .
A
d i s t in c t i o n s h o u l d a l s o b e m a d e b e tween "f r e e " and p r o t e in­
b ound ami n o acids . i n the p l asma .
Th e fo l low ing d i s c u s s i on s w i l l r e f e r
11
t o f r e e pl asma amin o ac i d s unl e s s oth e rw i s e s pec i f i e d .
T h e u tilis ation of ami n o ac i d s b y t h e t is s u e s i s m ar k e d l y a f f e c t e d
b y t h e ac t ion o f h or mon e s ( Har pe r , 1969;
M u nro a n d Portu g a l , 1972)
s inc e c e r t ain h ormon es c an i nf l u e nc e :
( a ) the r a t e of prot ein s y n t h e s i s and m e t abol ic i n t e r ac tion s w i t h i n t h e
t is s u e ,
( b ) the r a t e of upt a ke of am ino a c i ds by th e t i s s u e s ,
( c ) t he r a t e of prot ein d e g r ad a t i on ,
( d ) the r a t e of s y n t h es i s of non- e s s e n t ial amino a c i d s within th e t i s s u e
( Munro and Por t u gal , 1972).
Mos t a t t e n t ion has be e n g i v e n to the
e f f e c t s of g row th hormone ,
•
th y r ox i ne and i n s u l i n ( M u n r o , 1964;
1972).
H a r p e r , 1969;
C ahil l e t al . ,
G r owth hormone a n d i ns u l in inc r e as e d prot e i n s ynth e s is , thus
i nc r e as i ng the u t i l i s a t i on o f amino ac ids f rom the pl as ma , and h e nce
dec r e as ing the i r con c e n t r at i on .
Thy rox i ne c au s e d lo ss of body prot e i n ,
w i th r e l e a s e of ami n o ac i d s i n t o t h e plas ma , inc r e a s i n g pl asma
c onc entr at ions .
D i f f erenc e s i n the r a t e of uptake of i n d i v i d u a l amino ac ids by the
t is s ues as a .r e s ul t of a g e of an a nimal m igh t a l s o be e x pe c t e d t o a f f ec t
plas m a ami no ac i d c onc e n t r a t ions .
Ol t j en e t al .
(1969) f ou n d m a r k e d
s ex a n d a g e d i f f er e n c e s i n t h e plasma amino ac i d conc e n t r a t ions of
calv e s , wh i l e Oav e y et a l .
(1973) foun d s i g n i f ic a n t age e ff e c ts onl y
· i n l e uc i n e and l ys i n e conc e n t rat ions .
H owev e r , di f f e r i ng r a t e s of
grow th be t we e n animals of s im ilar age hav e be en s hown to a f f ec t p l asma
amino a ci d conc e n t r a t ions ( Wind e l s et al . , 1971;
B a l i n g et al . , 1972).
Tot a l pl asma amino a c i d s w e r e n e g a t iv e l y cor r e l a t e d w i th l iv e -w e i gh t
gain .
12
(2)
Nutritiona l factors:
The a bsorption of a mino a cids into the blood-
strea m would be expected to a ffect plasma con centra tions,
cha nges occur due to utilisa tion
( Hume
et a l . ,
1972;
but some
of a min o a cids by the gut tissue
Wolff et a l. ,
1973).
A da ptation
of individual a mino a cids ha s a lso been reported,
to high in ta kes
sin ce initial
respon ses d isappea red with continuous infusion of the a mino a cid.
However,
repea ted a dministration of methionin e led to persistent cha n ges
in the pla sma a minogra m
in total protein
I
,
I
( Synderman
et a l.,
1968).
The effect of cha nges
intake would a lso be expected to a lter plasma a min o
a cid concen tra tions,
a nd n umerous workers ha ve reported increased plasma
a min o a cid concen tration s in response to increa sed in take in mon oga strics
..
( Win dels
et al.,
1971;
I n creased plasma
Oavey et al. ,
1973;
Itoh et a l.,
a min o acid concentrations in
a bsorption of prot8in in
1974).
response to increased
ruminants ha s a lso been recorded,
with the
change in essen tial a mino a cid concentrations being grea ter tha n that of
dispensable amino acids
( A mos
et a l.,
1972;
Potter a nd Bergen,
1974).
Evidence for protein synthetic mecha nisms ada pting to the supply
of a mino a cid substra tes has been reported for mon ogastric a nima ls
( Mun ro, 1969;
Clemens,
be mediated by a
1972;
Wa n n emacher,
hormonal response to diet.
1972)
but this response ma y
A decrea se in cata bolism of
liver proteins a ppea rs to occur in con dition s of protein deficien cy
( Stephen
a nd Waterlow,
1966),
a nd Wa terlow
( 1969)
proposed tha t protein
homeostasis is the result of the ba la n ce between ra tes of cata bolism
a n d syn thesis of body protein.
The ra te of protein syn thesis in monogastrics is stimulated by a n
increa se i n ca rbohydra te inta ke,
with a con sequen t decrea se i n plasma
essentia l a min o a cid concen tra tion s
et a l.,
1967).
A
( Mun ro
a n d Thomson ,
1953;
Swen dseid
.
simila r respon se to intra ven ous a n d intra-arterial
13
energy in fus ion
ha s
1 9 74 )
Es kela n d et al.,
s ources .
a ls o been
reported for sheep
with differing res pon s es
( Potter.
1 968 ;
et a l.,
to different energy
Glucos e res ulted in th e greates t decreas e in plas ma es s entia l
a mino a cid con cen tra tion,
res pectively.
followed by propion a te,
Es kela n d et a l.
( 19 74 )
a cetate a n d
butyra te
reported a clos e correla tion
between decreas ed es s en tia l a mino a cid con cen tration a n d increa s ed N
bala n ce va lues ,
s ugges tin g tha t the decreas e in plas ma a min o a cids
res ulted from in creas ed protein s yn thes is .
Th ey s ugges ted that,
s in ce
different tis s ues
meta bolis e each energy s ource at a differen t r a te
( Mayfield
1968 ) ,
et a l.,
glucos e a n d the volatile
s timula te protein syn thes is
fa tty a cids
to a differen t degree.
The effect of energy in s timula ting protein s yn thes is
th rough a
reduced requiremen t for glucon eogenes is ,
of a min o a cids
is a bs orbed
production
for protein s ynth es is .
from
Since very little glucos e,
th e rumin a nt diges tive tract
( 1 970 )
g lucos e production was
( 1 970 )
Bergman et a l.
ha ve s hown tha t only
20
( 1 967 )
p er
s e,
( Lin ds a y, 1 9 70 ) ,
to
4 0%
is
es s en tial.
of the daily
obtained from a bs orbed propion a te,
and Len g et a l.
may occur .
an d hence th e releas e
of glucos e from propion a te a nd a mino acids
Bergma n et a l.
would
although both
s howed tha t
50%
of
g lucose ca rbon
could come from propion a te durin g periods o f pea k rumen
fermentation .
Wolff a n d Bergma n
( 1 9 73 )
reported between
g lucos e bein g derived from pla s ma a mino a cids
Ford
( 1 971 )
from a min o a cids .
production
rate,
res pon s e to an
orga n ic a cids
and
30 %
of
Reilly a nd
of glucos e derived
They a ls o found a n increas e in total glucos e
a n d in glucos e production rate from a mino a cids ,
in crea s ed rate of a mino
� cid
a bs orption.
Thus ,
in
a mino or
could be expected to make a n importan t con tribution to
g lucon eogenes is ,
intakes .
in fed s heep.
28%
reported a n a pproximate va lue of
11
particularly during fas tin g,
or on
n ea r-mainten a n ce
Fig.
(a)
1.2
structurG
C H 3
-
S
-
of
C H
2
sulphur amino acids and
-
C H
2
-
(b)
H S - C H
I
2
I
f�ETHIONINE
2
- c - c
I
H
0 0 H
C YSTEINE
N
(c)
s
- c
H
c
H
- c H -
I
2
2
- c
I
N H
(d)
(e)
H
0 0 C
C H
I
N H
2
2
-
- C
C
I
H
H
I
2
- c
H
2
H - C 0 0 H
c 0 0 H
CYSTATHIONINE
2
N H
I
H
2
- C
H
2
- S - S
-
C H
2
N H
2
I
- C - C 0 0 H
I
H
2
compounds
N H
2
I
C - C 0 0 H
H
N H
related
-
S
0
3
CYSTINE
H
TAURINE
Fig.
1 .3
convorsion of methionine to cysteine
�
'
� f 1E TH IO N I NE
Protein
�
ATP
�
s-adenosyl
methionine
--------�CH3
Transmethylation
\/'
rea ctions
s-adenosyl
homocysteine
L-HDr·10CYSTEINE �
'*"*
I
k---
�
Serine
CYSTATHIONINE
*-)(··X·
CYSTEINE
1
+
V
cl.-I<ETOBUTYRIC
ACID
+
TAUR WE
*
enzymes
are N5-methyltetrahydrofolate
methyltransferase
**
and
homocysteine
betaine-homocysteine
enzyme
is
cystathionine
enzyme
is
cystathionine
synthase
'd -lyase
methyltransferase
14
1 .4
Meta b o lism o f S -c o ntain i ng ami n o ac i d s
Meth io n i n e and cys t ( e ) i n e are th e �ain S-amino ac ids w h ich oc c u r
n atu r ally i n p r o te i n s .
�rm�
C ysti n e is inssr�sratod in� p r ote i n b y
o xidation o f th e th i o l g r o up o f c ys te i n e .
Th i s le a d s t o th e f o r matio n
o f d i-sulp h i d e b r i d g e s b e twee n p r o te i n c h ains ( Mah ler and C o r d e s , 1 9 7 1 ) .
M eth i o n ine h as a fiv e c ar b o n ( C ) s t r u c tu r e ( Fig . 1 . 2a ) w h i c h c an n o t b e
s y nth e sis e d b y m ammalian tis s u e .
C ys tei n e h a s only th r e e C ato m s
( Fig . 1 . 2 b ) a n d can b e s y n th e s is e d f r om meth i o nine .
Th e path w a y f o r
cyste i n e s y nth es i s f r om meth io n i n e i n v olves th e f o llo w i n g s te p s
( Fig. 1 . 3 ) :
( 1 ) a n i n i ti al tr an s meth ylati o n r e action, with ac tiv ati o n o f m e t h i o n i n e
t o S-ad e n o s y l m e th i o n ine ( catalys e d b y m e th i o n ine a d e n o s yltr ans f e r a s e ) ,
followed b y th e f ormation of L -h omoc y s te i n e ,
( 2 ) h o moc y s te i n e the n u n d e r g o e s an i r r ev e rs ible r e action with s er i n e ,
t o f orm c y s tath io n i n e , wh ich contains two ami n o g r o ups ( F i g . 1 . 2 c )
( catalys e d b y c y s tath i o n i n e s y nth as e ) ,
( 3 ) cystath i o n i n e is th e n c o nv e r te d to c yste i n e , w i th loss o f o n e
molec ule o f ammo n i a , and f orm atio n o f �-ketobutyric ac i d ( catal y s e d b y
cystath i o n i n e - �- l y as e ) ,
( 4 ) o x idation o f cys te i n e to cystine ( Fi g . 1 . 2 d ) m a y ta k e p la c e ,
(5 ) c o nv e r s i o n o f c y steine to tau r i n e ( Fi g . 1 . 2 e ) i s an alte rnative
r eaction f o r c ys tein e .
While d etai le d s tud i e s o n th e e n z ym e s r e g ulating th e s e p ath w ays r e late
mainly to r at liv e r p r e p arations , th e s e e n z ym e s h ave als o b e e n f o und i n
t h e t i s s u e s o f r um i n a nts ( M udd et al. , �965;
Radcl iffe and E g a n , 1 9 7 4 ) .
15
A.
Metabolis m bf methion ine
Methion ine is
required for three main functions :
(1)
protein s yn thes is ,
(2)
con vers ion t o S-aden os yl methion ine,
which is
the domina nt methyl
group donor in biologica l s ys tems ,
(3)
con vers ion to cys tein e via cys ta thionin e.
The dis tribution
on the
of methion ine between thes e functions
a va ila bility of
i ncrea s es ,
( Gra u
the requirement
However,
as
of methionine
a nd Ka mei,
1 950 ) .
This
decreas e is
pres umably due to
for protein s ynthes is in crea s ing a t a
( i . e.
2
a nd 3
inta ke,
a bove ) .
greater rate in
tha n the extra -protein function s
The level of cys tein e in the diet
als o been s hown to a ffect methionine requiremen t.
d emons trated by Woma ck a n d Ros e
effect,
( 1 941 )
wh ereby cys teine could pa rtially repla ce the dieta ry methion ine
Womack et a l.
.
s imila r "methion ine s pa ring" effect of cy s tein e when
m en ts
This was firs t
who s howed a "methion ine s pa ring"
in maintain in g the growth ra te of rats .
in terms of
N
ba la nce,
but no effect on
a n y other dis pen s a ble a min o acid was
B.
protein intake
the percenta ge requiremen t for methionin e
res pons e to increas ed protein
has
Thus ,
the a bs olute requirement for methion ine for protein s yn thes is
a ls o in creas es .
d ecreas es
other a mino a cids .
will depen d
( 1 95 3 )
foun d a
mea s urin g require­
methion in e requiremen t by
a pparen t.
Reg ula tion of methion in e meta bolis m
The con vers ion of methion in e to cys tein e in volves
h omocys tein e,
prior to its con vers ion to cys ta thion in e
Supplementa tion of low methion in e diets
( Fig. 1 . 3 ) .
with cys tein e has
d ecreas e the concentration of cys ta thionine s yn tha s e in
the con centra tion of
the forma tion of
been s hown
the liver,
beta in e-homocys teine methyltran s fera s e was
to
while
16
u n af f e c t e d ( F i � k e l s t e i n a nd l'lud d , 1 9 67;
Shannon e t a l. , 19 7 2 ) .
b e t a i n e-h omoc y s t e i ne m e th yJ. t r ans f e r as e is o n e of t w o e n z ym es w h i c h
c at a l y s e r e -m e th y l a t i o n o f m e t h io n i ne f r o m h omoc y s t e i n e ( F i g . 1 . 3 ) and
th e a b o v e r e s u l t would s ug g e s t an i n c r e a s e d recyc l i n g cf h �m o c y s t e i n e
t o m e t h io n ine ( 1 b , F i g . 1 . 3 ) .
Th e r e l a t i v e dec r e as e i n c ys t a th i o n i n e
s yn t h a s e w o u l d dec r e as e th e p r o p o r t i o n o f m e t h i o n i n e c o n v e r t e d t o
c ys t e i n e , a nd thus e x p l ai n t h e "me th i o n i n e s p aring" e f f e c t o f c ys t e i n e .
F i n k e l s t e i n and Mudd ( 1 9 67 ) h a d s u g g e s t e d t h a t me t h y l a t i o n o f
5
h omoc y s t e in e b y N -me th y l te t r a h y d r o f o l a t e ( N-mTHF ) w as n o t v e r y
i m p o r t an t , a n d th e r e f o r e d i d n o t a s s a y this e n z yme .
H ow e v er , i n a l a t e r
p a p e r ( F i n k e l s te i n e t al . , 1 9 7 1 ) t h e y c o nc l uded t h a t N-mT H F activ it y was
s ig n i f i c a n t l y affec t e d b y d i e t a r y m e t ab o l i tes and c o u l d c o n t r i b u t e to
r e g u l a t i o n o f me th i o n i n e m e t a b o l is m .
F i n k e l s tein et al .
( 1 971 ) found
t h a t h i gh p r o t e i n d ie t s c a us e d d ec r e a s e d s ynth e s i s o f t h i s e n z ym e , and
C e l l o a nd F i n k e l s t e i n ( 1 9 75 ) r e p o r ted an inc r e a s e d s y n th e s is o f N-mTHF
e n z yme in r e s p o n s e to l o w p r o t e i n d i e t s .
Th is s u gg e s ts an e f f ec t of
p r o t e i n i n dec r easing m e th i o n i n e r e ge n e r a t i o n d u r i ng h i gh av ailab i l i t y
o f amino a cid s .
Th e r e s p o n s e o f l i v e r e n z ymes to amino ac id s up p l y h as m a i n l y b e e n
s t u d i e d u s i n g in v i t r o p re p ar a t i o ns , h owev e r , is o t o p es h av e b e e n u s e d
f o r i n v i v o s t udies o n t h e r e g ul a t i o n of m e t h i o n i n e m e t a b o l i s m .
e t al .
( 1 9 63 ) f o u n d t h a t c o ns id e r ab l E r e c yc l ing o f m e t h i o n i ne c an o c c u r
i n t h e l i v e r , s ince v e r y l i t t l e l a b el f r o m methio n i n e-C H 3
·
Edwa rds
i nc o r p o r a t e d i n t o t i s s u e p r o t e in .
14
c was
Th e y s u g g e s t e d t h a t appr o x imat e l y
h al f o f t h e m e t h i o n i n e e n t e r i n g t i s s u e p r o t e i n f i r s t u n d e rw e n t t ransmeth y l a t i o n r e ac t i o n s , r e g a i n i n g a me thyl group from e n d o g e no u s
m e th i o n in e .
A h i gh p e r c e n t a g e of t h e meth yl l a b e l w as r e c o v e r e d i n t h e
l iv e r , b e c aus e s o m e o f t h e m e th y l g r o ups d o n a t e d b y m e th i o n i n e a r e
l i k e l y t o b e u s e d f o r th e s y n th es i s o f c h o l i n e and c r e a t i n i n e ( S t e k o l
17
e t al . , 1 9 58 ) .
F in kels t ein and M u d d ( 1 9 67 ) al s o f o u n d a d e c reas e in t h e
c bnc e ntrat io n o f me t hio n ine ac tiv atin g e n z ymes in the l iv e r of r ats f e d
d ie t s l o w in me th io n ine .
Th ese e n z ymes ar e invo l v e d i n th e initial
format io n of S -ad e n o syl meth ionin e , w h i c h ac ts as a m e t h y l d o n o r �
F in k e ls tein and Mud d ( 1 9 67 ) s u gg e s te d t h at t h e s e c h an g e s in e n zyme
c o nc e ntratio n s r e g u l at e d the dis t r ib u t io n o f methionine b e t w een p r o t e in
s yn th esis and t rans m e th y l ation.
C.
Meth io n in e as a d o n o r of meth yl gr o ups and s u lph u r
Th e r e l ativ e imp o r t ance o f t h e e x t r a-p r otein f u nc tio n s of
m eth io nine , c om p ar e d to thos e of o th e r amin o ac id s , was show n by A g uil ar
e t al .
( 1 9 72 ) wh o s tu d ie d t h e e f f ic ie n c y o f ut ilis atio n o f th e e s s e n t ial
amin o 2c id s in r ats .
T h e y f o u nd t h at l ab e ll ed m e th io n ine gave t h e
l o wes t val u es f o r inc o r p o rat ion in to tis s ue p r o t e in , and t h e h ig h e s t
p erc e ntage o x id atio n , wh e n f e d at a g iv e n p r o p o r t i o n o f its r e qu ir eme n t .
A c omparis o n o f th e meth y l -an d c ar b o x y l-lab e l led is o t o p es o f m e t h io nin e
s h ow e d a h igh e r p e r c e nt a ge o x idat io n of t h e c arbo x y l - t h an the m e th y l-C .
I n additio n , h e av y l ab e ll ing of s e rine f r om meth y l-lab e ll e d meth io n in e
in dic ated t h � imp o r tanc e o f meth io n in e as a m e t h y l g r o u p d ono r ( A gu ilar
e t al . , 1 9 7 4 ) .
Me th io nin e al s o ac t s as a s o u r c e o f S f o r th e s y n th e s is of o th e r S­
c o ntaining o r g anic c ompounds ( Fig . 1 . 3 ) .
R e is e t al .
( 1 973) reported
inc r e as e d p l as m a c o nc e n t r atio ns o f me th io n in e , c y s tin e , tau r ine and
.c ys tathio n ine in r e s p o n s e to ab o m as al inf us io n of meth ionine in s h e e p .
Howe v e r , T ao e t al .
( 1974 ) d id n o t o b s e r v e an y s ignif ic an t inc r e as e in
p l as m a c ys t ine af t e r meth io n ine inf us io n in t o the jug u l ar v e in o f s h e e p .
S h e e p plasm a-f r ee c ys tine c o nc e n t r atio ns h av e g e n e r al l y b e e n f o u n d t o b e
l ow :
0 . 1 4 - 0 . 4 8 m g/ 1 0 0ml p l asma ( Wil liams e t al . , 1 9 7 2 ;
T ao e t al . ,
1 9 74 ) , and w e r e n o t inc r eas e d by inf u s io n s o f lar g e amo un t s o f c ys t in e
( Reis e t al . , 1 9 73 ) .
T h is s ugges t s t h at t h e s h e e p is ab l e t o r e m o v e
18
l arge amo u nts o f c yst ine from the plasma qui t e rap i d l y.
Th is may be
partl y d u e to rap i d bin d i ng of c ystine t o p l asma pro t ei ns wh ich can
later b e rel eased as requ ire d ( Oownes,1961 ) .
Part o f the increased
cystine l o ad app ears to be c o nverted to taurine, sinc e taurine
conc e ntrat ion increase d mark e dly in resp o nse to c yst i n e inf usio n .
H owever, t h e increase i n taurine in resp o nse to m eth i o nine i n f u sion was
l ess mark e d, indic ating that the sh eep was u nabl e to rapid l y c o n v ert
e xc ess me t h ionine to cystine ( Re is et al., 1973 ) .
Reis et al .
( 1973 )
suggested that the c o nv e rsion of meth i o n i n e t o c yst e i n e was limited b y
decrease d availabil ity o f serine ( reac tion 2, Fig. 1. 3 ) si n c e p l asma
conc e ntration of serine decrease d with m e t h i onine infusio n.
,
While the c o n v ersion of meth i o n{n e to c ysteine is imp ortan t f o r
t h e synth e sis o f wo ol protein, o n l y o n e sp ec ific p h ysiol ogic al functio n
o f taurine has been rec ord ed.
acids, wh ich are important in
( Jac obso n and Smith , 1968 ) .
Taurine is k nown to c o njugate with b il e
the
intestinal absorpt i o n of fat
Taurine was shown to be p rese n t in m ost
o rgans of the b ody ( Sturman, 1973 ) , but sup p l emental taurine giv e n to
humans, was largel y exc reted in the urin e ( sturmar1 et al., 1975 ) .
Wh i t tle and Smith ( 1974 ) sh owed that taurine synth esis was increased b y
S
restric t ion i n rats, b u t the add i tional taurine was also large l y
e xcreted i n th e uri ne.
D.
Influence of me thionine on the plasma c once ntrations o f oth e r amino
ac ids
The effec t of p lasma infusio ns of m e t h ionine on t h e c o nc e n t rat i o ns
of o th er amino ac i d s in the plasma has b e e n st udied b y a n umber o f
workers.
While th e respo nse measured,large l y depend s o n t h e quantity
and qual i t y o f die t ary p rotein av ailab l e to the an imal, some sp e c i f ic
19
ac t i o n s o f metn ion in e are appare n t .
T h us , an inc reas e d c o nc e n t rati o n
o f � -ami n obu tyric ac id, i n r e s p o nse t o m e th ionine i n f u s ion, is
t h o u g h t to indic ate s y nth e s is o f this am ino ac id f r om m e th io n i ne, and
s ome e v id e nc e o f th e rev e r s e r eac t ion occ u r r ing in rats has als o b ee n
pres e n ted ( Syn d erman et al . , 1968;
E d war ds e t al. , 1 972 ) .
S ynde rman
( 1968) als o r e po r t e d dec r eas e d plas ma c o n c e n t rat i ons o f the
e t al .
b ranc h ed c h ain am ino ac i d s ( valine, is oleuc ine and leuc i n e ) , t y r o s ine
and p h e nylalan i n e in r e s p o n s e to meth ionine in rat s .
H owever, Canolty
and Nas s e t ( 1975 ) sh owed i n c reas e d plas ma leuc ine and i s oleuc ine and
d ec reas ed h istidine and th r e o n i n e .
r·
Wo rk with s h e e p and lambs has s h ow n
d e c r e ases i n plasma c o nc e n trat ions of th r e o n in e , gly c i n e, lys ine, s e r i n e ,
glu tamic ac id, v alin e , le u c i n e and is ole u c i n e ( Sch elling, 1968;
a n d T u nks, 1971;
Re is e t al . , 1973;
Tao e t al . , 1974 ) .
Re is
H ow e v e r, t h e
quan t i t y and quali ty o f p r o t e i n available t o the an imal will als o a f f e c t
plas ma am i n o ac i d c o n c e n t rat ions, and exp r e s s i n g i n d i v i dual am ino ac i d s
a s ratios t o , o r p e r c e n tages o f other am ino ac ids may b e m o r e meaning­
f ul.
Thus, Papas et a l .
( 1973) r e p o r t ed t h e e f f e c t o f o ral and
ab omas al s u ppleme nts of m e th io n in e in te rms of plasma me t h i o n i n e : valin e
rat i o s .
T he se were unaf f ec t e d b y oral me th ionine, o r MH A , wh i le
ab omas al infus i o n s ign i f i c antly inc reas e d t h e m e t h i o n i n e : valine rat i o .
1 . 5
A mino ac id requ i reme nts of r uminants
Knowledge of th e n u t r i e n t requ i r ements of an an imal e nables t h e
pr ov i s ion o f n u t r i e n t s t o b e o p timised, b e tw een maximum p r o d u c t i o n and
economic i n p ut �
Quali tat iv e as s essm ent o f the amino ac i d ( s ) mos t
l ike l y to l imit produc t i o n may lead to u n c o n t rolle d s up plem e n tat i o n o f
a d i e t and th is may lead t o p r o blems of toxic i t y .
Th us, quan t i tat i v e
as s e s s ment of r e quiremen ts is neces sary f o r e f f ic i e n t animal pr oduc t i o n .
Wh ile the amino ac id requ i rements o f n on-ruminants hav e r e c e i v e d
20
c o nsiderab l e attention ( e. g.
A . R. C . ,
1967 ;
I rwin and H e gste d , 1971 )
t h e capac i t y o f the rum ina+ mic ro-organisms to utilise
NPN
in the
syn t h e sis o f amin o ac ids ( L o osl i e t al. , 1949) negate s th e re l e v anc e o f
d i e tary am ino ac id requirements f o r rumi nants.
Henc e , some. knowledge
o f t h e ami n o ac ids availab l e f or absorp t ion , is a pre-re quisi te f or t h e
assessm e n t of re qu irements.
Th is has o n l y been ac h ie v e d rec e n tl y ,
through th e use o f sh e e p p r e pare d with re-entran t c annulae i n t h e
proximal duodenum and t erminal i l e um ( B rown et al. , 1968 ).
I nt e rest i n
th e asse ssm ent o f amino ac id re quireme � t s f or ruminan ts h as also b e e n
stimulated b y rec ent work on t h e manipu l ation of rumen f erme n t a t i o n .
T h r e e main meth o ds hav e been use d f or c on tro l l ing the q u a n t i t y an d
quality
of
p r o tein entering the d u o d e n u m :
( 1 ) nutri e n t b y-pass o f the rume n , using the o eso phageal-gro o v e re f l e x
( % rskov and B e n z i e , 1969) ,
( 2 ) pro tec t i o n o f pro teins from micro b ial attac k , e. g. b y reac tion with
f o rmal d eh y d e o r tannins ( Tagari et al . , 1965 ;
Ferguson e t al . , 1967 ;
O f f e r et al . , 1971 ) ,
( 3 ) promo t i n � natural fac tors wh i c h decrease microb ial attac k , e. g.
increased f l ow o u t of the rumen.
T h us , a quan ti tativ e assessm e n t of the ami no ac ids requ ired f o r
me tab ol ism b y ruminant tissues , is esse n t i al to maxim ise p o st-ruminal
p ro t ein u t i l isat ion.
D e f i n i t ion and classi ficat i o n of t h e amino ac ids i n t o e sse n t ial
· and d isp e nsable, h as b e e n d isc ussed ab o v e, and b e tween spec i es d i f f ere nces
i n amino ac i d me tab o l ism were also c o nsi d ered .
Wh ile th e re qu irements
f o r e sse n tial amino ac ids m ust b e i n d i v i d ual l y spec i f i e d , t h e non­
spec i f ic i t y of d isp ensab l e amino ac ids al l ows f o r their re qu irem ent t o
b e state d so l e l y in terms o f
N
and C .
H e nc e only th e re qu iremen ts f o r
e sse n tial am ino ac ids w i l l b e d i sc u ssed b e l ow.
21
A.
R espons e to am i n o ac id imb a l anc ed d i e t s
Harp e r ( 1964) d ef ine d amino ac id i m b alanc e as "a c h ange in t h e
prop ortions o f amino ac ids in a d i e t, wh i c h res ults i n a depress ion in
f o o d intake or growth rat e, that can b e c ompletely prev ented b y a
s upplement of th e ind i s p ensab le amino aci d pres ent in leas t amo u n t in
th e diet in re lat i o n to the amount re qu ire d for o p t imum growth ".
This
d e f ini t ion ignore s any c hange i n th e pro portional requirements f or amino
ac ids, in res p ons e t o d i f f ering levels o f p rotein intake.
The
m e tabolism o f methionine h a s b een sh own t o change with increas ing
av ailab i l i t y ( F ink e l s t e in e t al . , 1971;
Aguilar e t al . , 1974 ) .
In
add i t ion, Peng e t al. ( 1975 ) re p orted that intake depre s s i on in rats f e d
amino ac i d imbalanc e d d i e ts, increas ed with increas ed ov eral l pro t e in
s ynth e s is.
Henc e , d isc ussion o f amino aci d imbalanc e in the pres ent
work, will as s ume th e d e f ini tion of Harp er (1964 ) , with an ad d i t i o nal
Qrov is o that amino acid imbalanc e may aris e in res p ons e to a change in
th e rate of t i s s u e pro t e in s ynth esis and / or the l e v e l of prp t e in
avai lab i l i ty.
Harpe r e t al .
(1970 ) s h owed th a t d e c reased v o l untary intake and
growth rate in rats res ul t e d from the ad d i t i on of some amino ac ids to a
l ow pro t e in diet .
The add i t ion o f th e s e amino ac ids decreas e d t h e
perc entage o f th e f irs t limiting amino ac i d in t h e d i e t .
A d d i t i o n of
th is amino ac id res t ore d the balanc e, and t h us inc reas e d intake.
Wh il e
vari ous t h e ories on th e mech ani s m of t h e i n take ·re s p ons e to am ino ac id
imbalanc e d diets hav e b een proposed , no one theory has y e t b een
acc ep ted.
S imson and B o o th (1974) sugge s t e d that the d e l e t e ri o u s e f f e c ts
o f a d i e t l ow in h is tidine , c o ul d ini tial l y be buf f ered b y th e
u t i l isat i on o f h is t i dine from endo geno us p rotein, b u t this wo uld l ater
ac c entuat e the d e f ic ienc y respons e to a h i s tidine-fre e d ie t .
S ims on
and B ooth ( 1974) al s o s ugges ted that b i o c h e mical c h ange s in re s pons e t o
22
an imb alanc ed die t occ u r wit h in 2h of _ its ingesti o n , and th ese c h anges
w e r e involved in l a t e r c o n d itio ned rejec tio n o f the diet.
P e ng and
Har p e r ( 1969 ) sh owed that ch anges in liv er c o ncent rations o f amino acids
w e re not respo nsib l e for d e c r e ase d in take of imbalanc ed die t s , sinc e ,
wh i l e liv er t r y p t o p h an c o nc e n t r ation d e c r e ased on a t r y p t o ph an­
d e f ic ien t diet , l i v e r th r eonine c o nc e n tration inc r eased on a th r e onine­
d e f icient die t.
I n take d e c r e ased in b o th c ases.
N o d a ( 1975) p r o p ose d that b lood ammonia l e v e ls regu l a t e d th e
i n t a k e o f amin o acid imbal anc e d die ts , b u t f ur t h e r p r o o f is stil l
r e quir e d.
Noda ( 1975 ) h ad suggested that d e t o x ific atio n o f ammo nia
p r io r to its e n t r y in to t h e u r e a c y c l e c o u l d be a rate-limiting ste p ,
a nd thus inc rease th e c o nc e n t ration o f ammonia in th e b l o o d.
e t al.
Prior
( 1975) did find a th ree to f o u r f o l d inc r e ase in b l o o d u r e a
d u ring t h e feeding of an amino acid imbal anc e d diet.
R o ge rs and L e u ng ( 1973) imp l icat e d the p r e p yrif orm c o r t e x and
m e dial amygdal a of the b rain in monito ring the fac tors resp o nsib l e f o r
t h e d e p ression o f f o o d intake i n resp o nse t o diets d e f ic i e n t i n a n am i n o
a c id , b u t not to diets with exc ess amino ac ids.
While most o f this w o r k
o n amino ac id imb al anc ed diets has b e e n rec o r ded using non-ruminant
animals , R ogers and Egan ( 1975 ) sh owed that p re-r uminant lambs r esp o nd e d
in a similar manner to amino ac id imb al anc ed diet s .
H o w ev e r , with
r uminan ts the imb al anc e must occur at a d u o d e n al, rath e r than a d i e t a r y
l e v e l.
B a r r y et al.
( 1973 ) r e p o r ted th a t l ow intake of silage by sh e e p
c o ul d b e inc re ased b y intraperitoneal supplemen tation with m e thionine.
This suggests that f e r m e n tation o f sil age in th e rumen p r o d u c e d an amino
ac id imbalanc e in the sh e e p , b u t that b al anc e c ould b e r esto r e d b y
P l a s ma meth i on ine
c o nce t rat i o n
'--
a..
Meth i o n i n e avai lab le f or
a b s o rpt i o n
F iq.
1 .4
P las ma r e spo nse c ur v e
23
addition o f meth i o n ine .
Thus , th e quan t i t y o f methio n i n e e n t erin g t h e
duode num a p p e ared t o b e l imiting t h e util isation o f o th er ami n o ac ids
w i th th ese sh e e p .
O t h er workers h av e also reported m e t h i o n i n e a s
l imit ing � r o t e i n synth esis i n sh e e p and l ambs ( Reis, 1967;
Grac e v a ,
1969;
Th ese
Wakeling and L e wis, 1 970;
D ove and Rob ards, 1974) .
results are further sup p orted b y c ompariso ns of tissue pro t e i n anal ysis
w i th duod en a l ami n o ac i d f l o w , which sh ow ed that the duodenal f low o f
S-amino ac ids was m ost l i kel y to l imi t tissue pro t e i n sy n t h e sis ( H o gan ,
1970;
B.
Hut t o n and A nniso n , 1972;
Armstro n g and Annison , 1973 ) .
Methods f or quant i tativ e assessme n t o f amino ac id requir e m e n t s
Th e A RC ( 1965 ) manual o n nutri e n t re quirements f or ruminants does
n o t giv e in d iv idua l amino ac id requiremen t s , sinc e these cannot b e
e xpressed i n d ietary terms.
H o w e v er, t h e ARC ( 1967) manual o n p ig
re quirements d o es giv e amino ac id requiremen ts, ex pressi ng i n d iv i d ual
amino ac id c o n c e n trat i o ns as a perc e n t age of the OM in th e d i e t .
Th ese
v alues are d ef ined as giv ing maximum re sp o nse , b ut t h e wri ters
c o nc luded t h e sec t i o n b y su�gest ing t h e use of respo nse curv es to d e f ine
amino ac id requiremen ts over a range o f resp o nses .
The resp o nse c urv es
suggested , are p l o ts o f the · resp onse , e xpressed in t erms o f one c h osen
cri t eri o n , v ersus incre asing absorb ab l e l ev e l s of the am ino ac i d i n
q uestion ( F ig . 1. 4) �
W h i l e the l e v e l o f absorbable amino ac i d i s
usual l y t a k e n to m e a n d ietary in take i n monogastrics; it rel ates to amino
acids e n t ering the d uo d enum in ruminants.
I n most i nstanc es, such c urv es
are c ompose d of two lines, h aving d i f f eren t sl o pes, and the " bre ak­
poi n t " wh ere these two l ines intersec t , i s d ef i ned as the requirement.
The criteri o n use d to me asure the resp o nse will d e p e n d o n th e
se nsi t i v i t y required , and th e degree o f c om p l exity o f t h e experi m e n t
t o b e und ertaken .
24
T h e c r i teria usually c o n sidere d are as f o ll ows :
( 1 ) p rod uc tion resp o nses suc h as wo ol growt h , l iv e-we i gh t gain and
m ilk y ield,
( 2 ) f ac torial assessm ent by carcass analys is,
( 3 ) N balance,
(4) p l asma amino ac i d c on c e n trat ion,
(5 ) t h e use of isotopically l ab e l l e d amino ac ids.
( 1 ) Produc t ion responses :
W h i l e so me at t e n t i o n has b e e n giv e n t o
assessing indiv i d ual ami n o ac i d requirements b y measurement o f
p ro d u c tion resp o nses ( C h ung e t al . , 1973 ) th ese c ri t e ria are n o t v e ry
se nsi t ive, and a cons i d e rab l e t ime d e lay is i n v ol v ed .
Th ese . m e t h o d s
are t h eref ore o f more use for th e d e tec t i o n o f limiting arnino ac ids,
and f o r assessi ng t h e o v e ral l qual i t y of a p ro t ei n ( Jac quot and P ere t,
1972 ).
(2) F ac torial assessment :
A more rap id m e t h o d for assessing i n d i v i d ual
amino ac id re q u i r em e n ts for pro d uc ti o n, is t h e calc ulat ion of t h e
amo u n t of each amino ac i d d e p osi ted in t h e m eat, wo ol or milk p ro te in
synt h e sised .
Williams e t al .
( 1954) c ompare d estimates of ami n o ac i d
requ i remen ts o f monogastric s, calculated e i th e r b y anal ysis o f t h e wh o l e
c arc ass, or b y n u tri t io n experiments .
Th e y f o u nd go o d agre ement
b etwe e n th e two sets o f val u e s f or rats, but f ewer data were availab l e
for p igs.
Th e am ino ac ids wh ich sh ouJed c l o sest agre e m e n t were l e u c i n e ,
i so l e uc ine, thre onine and valine, wh ich hav e n o f unc t i o n other than
synth e sis of t i ssue p ro tein, wh ile resul ts f o r c yst e i n e and meth io n ine
were c onsist entl y l ower wh e n estimat e d . from c arcass analysis.
This
migh t b e expec t e d b e c ause o f th e h igh requ irements f o r meth io n i n e f o r
e xtra-prot e i n f unctions e . g. m e th y lat ion reac t ions.
H u tton and A n n ison
(1972) calc ulat e d fac t o rs f or the utilisat i o n o f amin o ac ids b y p igs ,
25
f r o m t h e c a rc as s c o n c e n t r a t i o n e x p r e s s e d a s a p e rc e n t a g e o f f o o d
r e q u i r e m e n t f o r in d i v i d u a l a m i n o a c i d s .
T h e y th e n u s e d t h e s e f a c t o r s
t o c a l c u l a t e d u o d e n a l am i n o a c i d r e q u i r e m e n ts f o r t h e y o u n g s t e e r ,
t h e c o n c e n t r a t i o n s o f am i n o a c i d s
c o n v e r s i o n f ac t o r s f r o m p i gs ,
i n t h e c a r c as s .
H ow e v e r ,
from
the use of
i g n o r e s a n y d i f f e r e nc e s i n th e
m e t a b o l is m o f a m i n o a c i d s b y r u m i n a n t and n o n - r um i n a n t t i s s u e s .
A r ms t r o ng a n d A n n i s o n
( 1 973 )
a l s o u s e d a f ac t o r i a l a p p r o ac h
r e quir ements f o r meth i o n ine ,
t ak i n g m a i n t e n a n c e ,
accou n t .
c y s t e i n e and t h r e o n i n e f o r 4 5 k g s h e e p ,
wool growth a n d tissue protein d e p os i t i o n i n t o
T h e i r a p p r o ac h
us e d
th e a p p a r e n t d i g e s t i o n o f e n e r g y t o
. e s t i m a t e t i s s u e p r o t e i n s y n th e s i s ,
r e gar d i ng
to c alculate
a n d r e q u i r e d n um e r o u s a s s ump t i o n s
t h e u t i l i s a t i o n o f e �e r g y .
I n addi tion t h e r e
is p r o b ab l y n o t
s u f f i c i e n t e v i d e nc e o n th e m e t a b o l i s m o f t h e s e am i n o a c i d s i n r u m i n a n t s ,
t o a l l o w e s t i m a t i o n o f th e i r r e q u i r e m e n t f o r mai n t e n a n c e b y t h i s m e th o d .
(3) N
b a l a nc e :
A d e f i c i e nc y o f a n a m i no a c i d w h i c h
l im i ts t i s s u e
p r o t e i n s y n th e s i s w i l l l e ad t o a n e xc e s s o f t h e r e m a i n i n g e s s e n t i al a m i n o
a c id s ,
wh i c h w i l l b e c o n v e r t e d t o u r e a i n t h e l i v e r ,
e x c r e t e d i n th e u r i n e
u r e a c o nc e n t r a t i o n ,
( Prior
1 9 75 ) .
( 1 975)
t h e e f f ic i e n c y of
used
N
Th us ,
N
or the daily amount of
g i v e s o me i n d i c a t i o n of
W a l k e r and K i r k
et al . ,
meas urement o f plasma
retained by t h e anima l ,
c an
u t i l is a t i o n o f t h e am i n o ac i d s .
b a l a n c e meas u r em e n t s
r e q u i r em e n t s o f l am b s f e d c o w ' s m i l k ,
and subsequently
t o assess meth ionine
a n d f o u nd t h a t m e t h i o n i n e s u p p l e­
m e n t a t i o n w as n e c e s s a r y t o o b t a i n m a x imum
N
b a l an c e o n l o w o r m e d i um
p r o t e i n i n t ak e s .
(4)
P l as m a a m i n o ac i d c o n c e n t r a t i o n :
$ c h e l l i ng ( 1 9 6 8 )
proposed a
w o r k i n g m o d e l f o r p l as m a e s s e n t i a l a m i n o a c i d c o n c e n t r a t i o n s i n c o r p o r a t­
i n g t h r e e i nc r e as i n g l e v e l s o f r e s p o n s e :
26
{a)
a b as al ,
o r m i n im a l ,
c oncentration
( wh ich
is different for each
a m i n o a c i d ) i n d ic a t i n g a d � f i c i e n c y o f th a t a m i n o ac i d ,
w h i c h d e c r e as e s
th e e f f i c i e n c y o f p r o t e i n s y n t h e s i s ,
(b)
t h e op timal c onc entration ,
wh i c h s u p p o r ts t h e mos t e f f ic i e n t
utilisation ,
(c )
excess c o n c e n t r a t io n ,
w h i c h a g a i n d e c r e as e s e f f ic i e n c y o f p r o t e i n
synth esis .
F i t t i n g t h e s e c o n c e n tr a t i o n l e v e l s
(Fig. 1 .4 ) ,
th e i n t r o d uc t i o n t o th is s e c t i o n
i.e.
the f i r s t
at the p o i n t o f inter­
meeting the requirement and
(c )
o n t h e s ec o n d i n t e r s ec t i n g
abov e t h e r equirement .
acids
for predicting requ irement ,
i n c r e as i n g l e v e l s o f :
p o s t ruminal amino
1 9 74 ) .
on
(b)
line ,
( Ch alupa
(a)
p l ac e s
b e l o w th e r e q u i r em e n t ,
i n t e r s e c t ing l ine ,
s ec t i o n ,
t o a r e s pons e curve as d e s c r i b e d in
acids
a n d C h and l e r ,
H owev e r ,
S e v e r a l w o r k e r s h a v e u s e d p l as m a a m i n o
b y m e a s u r i n g t h e r e s p o n s e t o g r ad u a l l y
intake for r ats
for c al v es
1 9 72 ;
( W i l l i am s
( 1 970)
1 965 ) ,
and Scott ,
and S m i th ,
A rms t r o n g a n d A nnis o n ,
Wakeling and Lewis
s e n s i t i v i t y o f t h i s m e th o d ;
( Z immerman
1 9 74 )
1 973 ;
and
and sheep
Tao e t al . ,
c r i t i c i s e d t h e l ac k o f
s i nc e n u m e r o u s o th e r f ac t o r s w i l l a l s o
a f f e c t p l as m a a m i n e ac i d c o n c e n t r a t i o n s .
(5)
T h e u s e o f i s o t opi c a l l y l a b e l l e d a m i n o a c i d s :
a c i d s h av e b e e n u s e d t o m e a s u r e
t o c arbon d i o x id e ,
p l as m a ,
( 1 97 2 )
s i nc e e x c e s s c o n c e n t r a t i o n s of a n a m i n o a c i d i n th e
w i l l r e s u l t in
an i n c r e a s e i n o x i d a t i o n .
s tated t h e advanta�es of t h i s
N
i t t a k e s l e s s t i me t h a n t h e m e a s u r e­
t r a n s i e n t c h a n g e s i n p l a s m a am i n o a c i d s
a c i d c o nc e n t r a t i o n s .
Chalupa and Ch andler
t e c h n i q u e o v e r o th e r m e t h o d s o f
b a l a n c e or p r o d uc t i o n r e s p o n s e s ,
p l a s m a amino
C-la b e l l ed amino
t h e d e g r e e o f o x i da t i o n o f a n a m i n o a c i d
a s s e s s i n g amino acid requirements :
ment of
14
and i t is l e s s
a f f ec t e d b y
t h a n d i r ec t m e as u r em e n t o f
27
( 1 9 73 )
B r o o k es e t al .
es timated t h e l ys in e r e q u i rement o f s h e e p
b y p 1 a s m a a m l· n o ac l· d c o n c e n t r a t l· o n s ,
s im i l a r v a l u e s
for
e ac h m e t h o d .
injec tion of l ysinehours .
1 973 )
used
·
( unpubli s h ed
T h e y f o und
•
( 1 973 )
used a s ingle
o b s e r v a t i o n s c i t e d A r ms t r o n g a n d
t h e s am e p r i n c i p l e t o m e a s u r e me t h i o n i n e r e q u i r e­
14
c -l a b e l l e d
a n d p l o t t e d s pe c i f i c r ad i o a c t i v i t y o f
f i n d t h e a s y m p t o t ic
·
B ro o k e s e t a l .
b u t u s e d a c o n t i n u o us i n f u s i o n of
5-6 h o u r s ,
c oxldat l o n
c a n d m e as u r e d o x i d a t i o n o v e r t h e f o l l o w i n g s ix
M i tc h e l l e t a l .
Annis o n ,
men t ,
14
14
a nd
value
( A n nis o n
14
co
aminG
2
t h e s e v a l u e s a g a i n s t a b s o r b a b l e m e th i o n i n e ,
measu reme n ts
( A r ms t r o n g
f u r t h e r r e f i n e d th e
leuc i n e a n d lys ine
and A n n i s o n ,
use of
14
to
1 967 ) and t h e p e r c e n ta g e o f
et a l . ,
good agreement with
for
a g a i n s t t i me ,
i n f us ed r a d i o a c t i v i t y e x p i r e d d u r i n g t h e i n f u s i o n p e r i o d .
m e n t wh i c h w e r e in
acids
Plotting
gave e s t imates of r e quire-
I
t h o s e f r om p l a s m a amino a c i d
1 9 73 ) .
N e al e a n d W a t e r l o w
( 1 9 74 )
c -l a b e l l e d a m i n o a c i d s f o r e s t i m a t i n g
requirements i n rats ,
l ab e l f r o m t h e b o d y , u n d e r c o n d i t i o n s
b y determi n i n g total l o s s o f
a p p r o ac h i n g ma i n t e n a n c e .
H owev er ,
t h e i r e s t im a t e s w e r e h i g h e r t h a n t h o s e o b t a i n e d b y g r o w th o r N b al a n c e
m e th o d s .
acids ,
Annison
poi nted out
( 1 975 ) ,
that
wh i l e d is c u s s i n g t h e u s e o f
the s ensi tivit y and accuracy
w o u l d d e p e n d o n th e p a t h w a y s
d i f f e renc e s in
h av e b e e n s h o w n
35
14
c - l a b e l l e d am i n o
of this
o f o x i d a t i o n o f e a c h am i n o ac i d ,
technique
and large
t h e r a t e s of o x i d a t i o n o f i n d i v i d u a l a m i n o a c i d s �y �h�Q�
( B l ac k
e t al . ,
1 968 ) .
s-l a b e l l e d m e t h i o n i n e h as a l s o b e e n u s e d t o a s s e s s m e th i o n i n e
r e q u i r em e n t , s i n c e s u r p l us m e th i o n i n e m a y b e c o n v e r t e d t o t a u r i n e a n d
exc r e t e d i n t h e u r i n e .
Merc e r a n d M i l l e r
e s t i m a t e m e th i o n i n e r e q u i r em e n t o f
( 1 9 73 )
g r o w i n g l am b s ,
u s e d t h i s m e th o d t o
a n d f o u n d s im i l ar
r e s u l t s t o t h o s e d e t e r m i n e d b y p l a s m a u r e a m e a s u r em e n t s .
Tab l e 1.1
Es timates o f meth ion ine requireme nts by sheep
M ethod o f estimation
P l asma amino ac i d
c on c e ntra t i o n
Oxidation to
14
c o2
3 5s excre t i o n
Requireme n t
(mg/kg• 75/d ay)
Re f ere nce
(
(
(
105-125
1
207
Wake l i ng et al. (1970 )
(
(
(
120-130
Arms tro ng
130-160
A n n i s o n (1975)
170
1 re f ers to i n f u s e d me thionine o n l y .
Re is e t al. (1973 )
&
A n nison (1973 )
Merc e r and Miller (1973 )
28
C.
E s t i m a t e s o f m e t h i o n i n e r eq u i r e m e n t s o f s h e ep
V a r i o u s e s t i ma t e s o f th e d u o d e n a l m e t h i o n i ne r e q u i r e m e n t s o f s h e e p
h av e b e e n m a d e ,
1.1.
and th e s e a r e c o m p a r e d i n T a b l e
W h i l e th e e f f e c t
o f m e t ab o l ic b o d y w e i gh t i s t a k e n i n t o c o n s i d e r at i o n i n s t a t i n g · th e s e
r e q u i r e me n t s ,
c onsiderab l e v ar i a b i l i t y migh t b e e x p e c t ed to a r i s e f r o m
d i f f e r e n t e n v i r o nm e n t a l c o n d i t i o n s a n d r a t e o f wo o l g r o w th b e tw e e n t h e
e xperimen t s .
n ote that
W i th r e g a r d t o t h i s l a t t e r a s p e c t ,
th e A us t r a l i a n w o r k e r s
( Reis
e t al . ,
r e q u i r e me n t t h a n th e B r i t i s h w o r k e r s .
I
I.
f
I
I
s h e e p for
1 9 73 )
r e c o r d e d a h i gh e r
T h e i m p o r t a n c e o f wo o l t o th e
A us t r al i a n s h e e p f a r m e r h as p r o b ab l y l e d
·
it is interesting to
to s e l e c t i o n o f A us t r a l i a n
a h i g h r a t e o f w o o l p r o d uc t i o n .
M e th i o n i n e c an b e c o n v e r t e d t o c y s t ( e ) i n e
( F i g . 1 .3 )
a n d t h u s th e
r e q u i r e m e n t f o r m e t h i o n i n e w i l l al s o d e p e n d o n th e a v a i l a b i l i t y o f
c y s t ( e ) i n e r e l a t i v e t o i ts r e q u i r e m e n t .
T h u s th e r e q u i r e me n t s f o r
m e th i o n i n e a n d c y s t ( e ) i n e a r e f r e q u e n t l y e x p r e s s e d a s t o t a l S - a m i n o
a c id
requirements .
A r ms t r o n g a n d A n n i s o n
( 1 973 )
u s e d th e k n o wn v a l u e s
f o r a p p a r e n tl y d i g e s t e d e n e r g y t o c al c ul a t e t i s s u e p r o t e i n d e p o s i t i o n
a n d h e nc e
a f ac t o r i al e s t i m a t e o f S - a m i n o a c i d r e q u i r e m e n t s ,
v ar i e t y o f d i e ts .
m gI k g
.
75;
s ug g e s t e d
day ,
a n d f r om th e s e v a l u e s ,
gave v alues o f
82
-
A rm s t r o n g a n d A n n i s o n
1 27
( 1 9 73 )
t h a t wh i l e S-ami n o a c i d s s h o u l d n o t l i m i t w o o l g r o w th
f or a g e d i e ts ,
t h is
T h e s e c a l c ul a t i o ns
on a
t h is may n o t b e
t r ue f o r s i l a g e diets .
t ec h n iq u e h as a l r e a d y b e e n c r i t i c i s e d ,
on
The accuracy of
b u t th e r e l a t i v e
d i f f e r e n c e s b e t w e e n d i e ts d o s u p p o r t o t h e r s t u d i e s i n s u g � e s t i n g th a t
s i l a g e m a y b e a f e e d i n wh i c h
l im i t i n g .
the S-amino acids are particul arly
29
1.6
A.
Fo rmaldeh y d e - treatmen t of pr o t e i ns
The t h eo ry f o r the r eac t i on of p r o t eins with formaldehyd e
of
Treatmen t
p r ot eins w i th al d eh y d es i s a c ommon prac t ic e i n the
Leath er I ndust r y , wh ere the r eac t i o n of a l d eh ydes w ith th e su rfac e of
t h e f ib r o u s p r o t e ins results in a t o u g h er mater ial , w i th d ec reased water
sensi t i v i t y an d inc r eased resistan c e t o the ac tion of enzymes and
c h em ical r eagents ( Wal k er , 1 9 64 ) .
F r enc h and Edsal l ( 1 94 5 ) str essed
the v ariet y an d complex i t y o f possi b l e reac tions between f o rmal d eh yd e
and p rotei ns , w i th t h e optimum c on d i t i o ns f o r fo rmat i o n and b r eakd own
of t h e b onds, b ei n g d ependen t o n p H an d tem p erat u r e .
They quo t ed
evi d e nc e f or th r e e types of b on d i n g b etween f o rmald eh yde and p r o tein :
( 1 ) a l o ose r ev ersi b l e c omb i nat io n , wh ere f o rmald eh yde can b e r emov ed
b y w a sh in g ,
(2 ) b onds wh ic h can be b roken by c o n t i n uous wash i n g o v er a p er i o d of
days,
(3)
a
stab l e c h em i c al c omplex which d o es not r ead i l y d issoc iate .
F renc h and Edsall ( 1 94 5 ) c o u l d n o t sp ec ify t h e var i o u s c h emical g r o u p s
invo l v ed i n eac h t y p e o f b o n d ing , b ut t h ey d id revi ew ev id ence f o r t h e
existence o f meth y l ene b r id g es b etween reac tive g r o u p s suc h as t h e free
ami n o g r o u p of lysine, and sulfh yd r yl g r o u p s .
e. g .
�
R
�
NH 2
R
+
R
<
HCHD
SH
--
-)
N
I
CH 2
R
�
I
s
Wal k er ( 1 964 ) also sug g ested t h e format ion o f meth y l en e b rid g es ,
and h e p o inted o u t that suc h f o rmal deh yde-p r o t ei n c o m p l exes w ould b e
l ess stab l e u n d er ac id than al kal i n e c o n d i t i o ns.
Th u s , l i b erat i o n o f
formal deh yde b y dist i llat i o n with d i l u t e ac id is o f ten used t o d et ermi n e
t h e d egree o f c omb i nat i o n wi th p r o tei n .
H owev er , acid distillation i s
30
I.
I
n o t a l ways
reliable ,
B owes e t al .
s i nc e s o m e i r r e v e r s i b l e b i n d i n g m a y t a k e p l ac e .
( 1965 )
e s t i m a t e d b o u n d f o r m a l d e h y d e b y i s o t o p ic m e t h o d s ,
a n d r e p o r t e d th a t a s m a l l p r o p o r t i o n o f t h e f o r m a l d e h y d e b ec am e m o r e
f i rml y b o u n d d u r i n g a c i d t r e a tm e n t .
appeared t o b e bound
T h i s f r ac t i o n o f t h e f o r m a l d e h y d e
to o nl y one g r ou p ,
was markedly de c reas e d .
s i nc e t h e d e g r e e o f c r o s s l i n k i n g
( 1 965)
B o w es e t a l .
found
that ,
i n c o n t r as t
t o e a ri i e r r e p o r ts ,
r e v e r s al o f t h e b o n d s w i t h t h e a m i n o g r o u p s d i d
occur ,
( 1 9 68 )
and Neissner
b inding,
r e p o r t e d t h a t a f t e r r e v e r s a l o f f o r m a l d eh y d e -
by boiling in water ,
c as e i n mo l e c u l e .
there appeared
T h e i s a n d L am s
( 1 943 )
to b e n o ch ange i n th e
showed a pH
r e v e r s i b i l i t y w i th
.4 5%
r e l e as e o f b i n d i n g wh e n th e p H w a s d e c r e as e d f r o m e l e v e n t o e i g h t
and
E 6%
( 1 958 )
wh e n t h e p H w a s d e c r e as e d f r o m e l e v e n to t w o .
r e p o r t e d that b inding of amino groups
tc
t ak e p l a c e a t a n ac i d p H u n d e r s o m e c o n d i t i o n s .
t h a t b o th t h e r e ac t i v i t y o f
the protein ,
r e ac t i o n w i th f o r ma l d e h y d s, c a n a f f e c t
the
and
H ow e v e r , M e l l o n
f o rm a l d e h y d e c o u l d a l s o
It would thus app e a r ,
th e c o n d i t i o n s f o r i t s
th e t y p e o f b o n d f o r m e d
a n d t h us
e a s e o f r e v e r s i b i l i ty .
T h e r e v e r s a l o f f o r m a l d e h y d e- p r o t e i n b o n d s w i t h d e c r e as i n g p H w a s
t h e b as i s f or f o rma l d e h y d e - t r e a t m e n t o f p r o t e i ns f o r f e e d i n g t o
r um i n a nts .
F o r m a l d e h y d e- t r e a tm e n t w o u l d d e c r e a s e t h e s o l u b i l i t y o f t h e
p r o t e i n i n t h e r um e n ,
b u t the b o n d s would b e reverse d i n the low pH o f
t h e a b omas u m a n d t h u s
p e r m i t e n z y m ic d i g e s t i o n in th e i n t e s t i n e s
( Ferguson
c on d i t ions
et al . ,
1 9 67 ) .
Howeve r ,
th e e f f e c t o f d i f f e r i n g r e a c t i o n
on t h e t y p e of b o n d f ormed ,
h as l e d t o d e t a i l e d s t u d i e s o n
t h e r e v e r s i b i l i t y o f f o r ma l d eh y d e- p r o t e i n b o n d s .
H e ms l e y e t a l .
s t u d i e d th e e f f ec ts o f v a r y i n g t h e r e ac t i o n c o n d i t i o n s ,
t h e o p t i m u m d e g r e e o f b i n d i n g w a s w i th
l evel ,
1%
f o rmaldeh y d e .
and f o u n d t h a t
B e l o w th is
th e r e w as i n c r e as i n g m i c r o b i a l a t t ac k i n th e r u m e n ,
h i gh e r d e g r e e o f b i n d i n g l e d t o d e c r e as e d
N
( 1 973 )
while a
d i g e s t i b i l i t y i n th e
31
i n t e s tine s .
The r a t e and e x tent of the protec t i o n r e ac ti o n were f o u n d
t o i n c r e a s e w i t h c o nc e n t r at i o n o f f ormal deh yde s o l u t i o n , w i th d ec r e a s i n g
v olume o f formal deh y d e u s e d , a n d with i n c r e as i n g reac tion time .
Hems l e y e t al .
(1973) a l s o n o ted u n p u b l is h e d o b s e r v a t ions b y
D . M . Wal k e r where f ormaldeh y d e-tr e ated c a s e i n was f ed t o r a t s .
No
d i f f e rence i n g r ow t h r a t e w a s rec orded , c ompared t o untreated c asein ,
wh e n th e l evel o f b o und f o rm aldeh y d e was l e s s t h a n 1% .
a r e i n c o n tras t w i t h th o s e o f Wac h ira e t al .
T h e s e r e s ul t s
( 1 974) wh o f ed c as ein
whic h h ad b een tre a t ed with D . B g f ormal d e h y d e/ 1 0 0 g crude p r otei n , t o
rats , a n d f ound a d e c r e a s e i n growth r a t e w i t h t h e treated v e r s u s
u n t r e a ted c as e i n .
H owe v e r , t h e s e w o r k e r s app l i e d t h e formal deh y d e i n
v er y l o w v olume s , a n d d i d n o t m e as ure t h e amount o f b o und f o rmaldeh y d e .
H o v e and L oh r e y ( 1 9 7 6 ) a l s o r e p o r t e d dec reased d i g e s t i b i l i t y o f
f ormaldeh y de-t r e a t e d c a s e i n in t h e i r r a t gr owth trial s .
Th e s e w o r k e r s
s u g g e sted that m o r e stab l e s ec on d a r y c om p l e x e s h ad b e e n f o rmed d ur i n g
t h e d r y i n g proc ess i n c l u d e d i n th e f o r m al d e h y d e-tr eatment o f th e
protein .
Howe v e r , M ac R a e e t al .
( 1972) f e d this same f o rmaldeh yde­
t r e a t ed p r otein to s h e e p , and o b s e rved a s ignif i c ant inc r e as e i n
a p p a r e n t absorp tion o f amino ac i d s f r o m t h e s m a l l i n t e s t i n e with
f ormaldeh y d e�t r e atme n t , r e s u l ting i n a h ig h e r o v e r al l r e t e n t i o n o f
N.
With r eference t o t h e e a r l i e r evidence o f F r e nc h a n d E d s al l ( 1 94 5 ) ,
wh o s ug g e s ted t h r e e grad ati o ns i n t h e s t ability o f b o nd i n g b e t w e e n
f ormaldeh y d e and p r o t e i n , t h is s e c o nd a r y c ompl e x i n g may b e r e v e r s i b l e
i n t h e s h e ep , b u t n o t i n t h e rat , due t o p h ys i o l o g ic al d i f f erenc es
b e t w e e n th e two d i g e s t i v e s y s tems .
Thus , wh i l e t h e c h emis t r y o f t h e r e ac t ions i n v o l v e d i n f o r m al­
deh y d e-t r e atme n t of p r o t e i n s is n o t y e t c l ear , t h e f o l l o w i n g s ummar y
c a n b e made .
32
( 1 ) T h e r e appears t o b e a n o p t i m um l e v e l o f f ormal d e h y d e - t r e a t m e n t t o
e ns u r e a de q u a t e p r o t ec t i o n f r om micro b i a l attac k i n t h e r um e n , w i th o u t
af f e c t i n g t h e d i ges t ib i l i ty i n t h e i n t e s t ines .
H e m s l e y e t al .
( 1 9 73 )
p ro p o s e d a n o pt imum l ev e l of 1 % b o und f o rmaldeh yd e .
( 2 ) T h e c o nce n t r a t i o n of f orma l d e h y d e s o l u t io n , v o l um e o f a p p l ic a t i o n
a n d d e g r e e of d r ying h av e a l s o b e e n s h own to affec t b i n d i n g and r e v e rsi b i l i ty ( H e ms l e y El t al . , 1 9 7 3 ;
H o v e a n d L oh r e y , 1 9 76 ) .
( 3 ) T h e r e v er s a l of f ormal d e h y d e - b ind ing w i l l b e a f f ec t e d b y the d e g r ee
o f b i nd i n g , and is p r o b ab l y a l s o d e pe n d e n t on pH and t i me s pe n t in t h e
dige s t ive t r ac t ( Fr e nch a n d E d s a l l , 1 94 5 ) .
B.
A n i�al pr o d uc t ion respo n s e s t o fo r m aldehyd e-tr ea t e d pr o t e i ns
T h e w o rk b y F e r g u s o n e t a l .
( 1 9 67 ) wh ich res t im u l a t e d i n t e res t in
t h e us e o f t re a t e d p r o t e i n s , r e p o r t ed a r e s p o n s e i n w o o l growth t o
f o rmaldeh y d e-tre atme n t o f c a s e i n .
S i nc e t h e n , var i o us w o r k e rs h av e
r ec o r d e d s imilar inc r e as e s in w o o l gr owth ( H u ghes and W i l l iams , 1 9 7 0 ;
L an g l a nds , 1 9 7 1 ;
B a rr y , 1 9 7 2 ) .
B ar r y et al .
( 1 9 7 3 ) r e p o r t e d i nc r e as e d
• 1
wo o l g ro wt h w i th formald e h y d e- t r e a tm e n t o f S l..c
a ge .
H o w e v e r , r e p o r t s of
no r e s pons e to f orma l d eh y d e- t r e a t m e n t als o a p p e ar i n the l i t erature
( e . g . Clark e t a l . , 1 9 7 1 ;
Sc h mi d t e t al . , 1 9 7 2 ;
Wac h i r a e t al . , 1 9 7 4 ) .
P o s i t i v e r e s p o n s e s appear t o b e l e ss p r e d i c tab l e a t h i g h e r l ev e l s o f
e n e r g y i n t a ke , and wh e n t h e u n t r e a t e d d i e t has a h i gh c r u d e p r o t e i n
c o n t e n t ( S h arma e t al . , 1 97 4 ;
Wac h i r a e t al . , 1 9 74 ) .
I n a d d it i o n ,
P i s u l e w s k i and R ys ( 1 9 7 5 ) p o i n t e d o u t t h e imp o r t an c e o f c o ns i d e r ing t h e
q u al i t y o f a p r o t �in i n r e l a t i o n t o t h e a nimal ' s amino a c id requirements .
Th e s e w o r k e r s f o u n d n o b e n e f ic ial effect on N r e t e n t i o n i n
s h e e p , f r o m f o r m ald e h y d e-t r e a tme n t o f H o r s e B e ans .
Mac R a e e t a l .
( 1 9 7 2 ) f o u n d t h a t t h e dail y f l o w o f amin o a c i d s i n t o
t h e d u o d e n u m w a s inc r e a s e d w i t h f o rmaldeh yd e-tr e a t e d o v e r u n t r e a t e d
33
c as e i n .
A p pa re n t amino a cid abs o r p t i o n f r om the i n tes t ines was a l s o
i nc r e as e d .
Sh a rma e t al .
( 1 9 74 ) f o un d s im i l ar r e s u l t s w i t h f o rmal d e h y d e­
tr e at e d c as ein , b u t v e r y l i t t l e e f f e c t on f l ow from f o r ma l d e h y d e-t r e at e d
r ap e s e e d m e a l .
Th e y s ugges t e d t h a t t h is c o uld be d ue t o t h e h i gh f l o w
r a t e o f a m i n o a c ids i n t o t h e d u od e n um on u n t reated r a p e s e e d meal .
A
d e pre s s io n i n o v e r a l l di ge s t i o n i n th e r u m e n when f e e d i n g f o rm al d eh y d e­
t re a t e d s u p p l e m e n t s h as b e e n r e p o r t e d ( F a i c h n e y and Wes t o n , 1 9 71 ;
H ems l e y
�. ,
1 9 70 ) .
F a i c h n e y ( 1 9 7 5 ) f o und a d e c rease i n t h e m e an
r e te n t i on t ime i n t h e rume n , with f o rmald eh yde -t r e a tme n t o f a c o nc e n t r a t e
d i e t , b u t b o th the s olid a n d p a r t ic ul ate p h ases we r e r e t a i n e d l o n g e r in
the ab omas u m a n d small i n t e s t i n e s .
T h e i m p o rtanc e o f abom a s al p H w a s
s u gg e s ted b y F a ic h n e y ( 1 9 74 ) , wh o f o u n d a h i gher p e r c e n tage o f ami n o and p e p t i d e-N i n t h e abomasal-N o f a s h e e p w i t h a l ower abom asal p H ,
wher e a s a h igh e r d e g r e e o f f o rmal d e h y de-p r o te i n c o m p l e x i n g w as f o u n d in
a sh e e p w i th a h i gh e r ab omas al pH .
This d at a mig h t s ug g e s t t h a t pH is
a ma j o r f ac t or in th e rev e r s a l o f f o r ma l d e h y de-bi n d i n g , b u t the d a t a
r ec o r d e d w e r e o n l y o b tained f r om t w o s h ee p .
T h e o v e r a l l e f f e c t o f f o rmald e h y d e- t r e a tment ap p e a r s t o b e a
dec r e as e d s ol u b i l i t y i n th e rum e n , r e s u l t i n g in inc reas ed f l ow o f
p r o t e i n i n t o t h e d u o d e n um .
obviously d epend on :
th e av a i l ab i l i t y o f the ami n o ac i d s f o r a b s o r p t io n ,
i . e . r ev e r s al o f b i nd ing ;
d ie t ;
T h e adv an tages o f this i nc r e as e w i l l
t h e f l o w o f amino ac ids o n t h e u n treated
and th e p r op o r ti o ns o f i n d i v i d ual am ino ac i d s p r e s e n ted f o r
abso r p tio n , i n r e l a t i o n t o t h e p r o t e i n r e q u i r em ents of t h e a nimal .
1 .7
T he f e rm e n ta t i o n of s il age a n d i t s e f f e c t on i n t a k e
The e f f i c i e n t u s e o f h e rb a g e f o r r u m i n a n t p r o d u c t i o n r e q u i r e s i t s
c o ns e rv a t i o n d u r i ng p e r i o d s o f rap id growth , t o e n able s u p p l em en tar y
f e e d i n g d ur i n g p e r i o ds o f r e s t r i c t e d · g r o w th .
The main aims are to s ta r t
34
w i th a c r o p of h i gh n u t r i t i v e v a l u e , a n d t o mai ntain t h i s q u a l i t y
d u r i n g th e c o n s e r v a t i o n p r o c ess .
Th e two m ain c ons e r v a t i o n proc e s s e s
i n c ur r e n t p r ac t ice are :
( 1 ) f i eld d r y i n g of a c r o p f o r s t o r age as h a y ,
(2) a n a e r o b i c f e rme n tati o n t o p r o duce s i l a g e .
S inc e one o f t h e k e y f ac t o rs i n t h e e f f ic i e n t c o n s e r v a t i o n o f g r a s s i s
t h e w eath e r , s i l age has t h e advan tage o v e r h ay , in t h a t the g r a s s c an
b e c u t and r emov ed f rom t h e f i e l d i n o n e o p e r ation .
H ow e v e r , a h i gh
q ua l i t y f e e d is of l it t l e v a l u e unless it is ac c e p t ab l e to the a n im al ,
and t h e v o l u n t a r y i n t a ke o f s i l a g e b y r u m i n a n t s h as f r e q u e n t l y b e e n
. f o und t o b e l o w e r t h an t h a t o f h ay m ade f ro m th e s ame c r op ( e . g .
Mur doc h , 1 9 64 ;
1 9 70 ;
Thom s o n , 1 9 66 ; · We l lm an , 1 9 6 6 ;
O e m a r q u i l l y and J a r r i g e ,
1 9 70 ) .
B i s h o p a 1 1d K e n t i s h ,
W h i l e c o ns i d e r able res e arch o n
t h e e n s i l a g e p r o c e s s h as l e d t o a n a p p r e c i a ti o n o f t h e o p t im um
c on d i t i o ns f o r f e rme n t a t i o n ,- th e f ac t o rs i n v o l v ed i n l i m i t i n g i n t a k e
h ave n o t b e en c l e ar l y e s t abl i s h e d .
A.
T h e f e r m e n t a t i o n of s ilage
B arn e t t ( 1 9 54 ) o u t l i ned f o u r main p h a s es i n e n s il i n g :
( 1 ) t h e r e s p i r a t i o n o f p l a n t c e l l s , l e a d i n g t o th e f o rm ation o f c ar b o n
d ioxi d e a n d wat e r f r om w a t e r s o l u b l e c ar b o h y d r ates ,
( 2 ) t h e p r o d u c t i o n of ac e t ic acid b y b ac t e rial ac t i o n ,
( 3 ) t h e p r o d uc t i o n o f l a c t ic ac id b y b ac t e ri a util i s i n g s o l u b l e
c ar b o h y d r at es ,
( 4 ) a q ui e s c e n t p h as e .
T h e s e f o ur may b e f o l l ow e d b y a f i f th ph a s e o f c l o s t ri d ial f e rm e n t a t i o n
if t h e c r o p i s p o o r l y f e r m e n t e d .
C l o s t r i d i um spp. c on v e r t am ino ac i d s
t o ammonia a n d ami n e s , a n d l ac tic ac id t o b u t yric a c id , with a
resul t an t i nc r e as e i n pH ( Wh i t t e n b u r � e t al . , 1 9 6 7 ) .
T h us , a h i gh
35
q u a l i t y s ilage h as b e e n c l a s s i f i e d as h a v i n g :
(1) a l ow b ut y r ic a c i d c o n c e n tr a t ion ( l e s s t h a n 0 . 3% ) ,
( 2 ) a l o w ammon i a c onc e n t r a t i o n ( l e s s than B% o f t o tal N ) ,
( 3 ) a p H o f approx imat e l y 4. 2 o r l ess ( M c D o nald and Wh i tt e nb ur y , 1 9 7 3 ) .
Wh i l e a l o w ammo n i a c o nc e n t r a t i o n r e p r e s e n ts l i t t l e b r e akdown o f
ami n o ac i d s , 40% o f th e o r i g i n a l p r o t e i n m ay b e b r o k e n d o w n , e v e n i n a
w e l l p r e s e rved s i l age ( M c D o n a l d e t al . , 1 9 69 ) .
Th e i n i t ial p r o t e o l y s is
a p p e ars t o be d u e t o a u t o l ys i s b y p l a n t e n zymes ( K e mb l e , 1 9 5 6 ) and t h is
c an b e l im i t e d by rapid e�c l u s i o n of o x y g e n from the s i l o .
An
a n a e r o b i c e n v i r o n m e n t also f a v o u r s a l ac tic ac i d f e rm e n t a t io n , and t h i s
w i l l r a p i d l y d e c r e a s e p H , th e r e b y p r e v e n t i n g c l o s t r i d i a l g r o wth
( M ac ph e r s o n and V i o l an t e , 1 9 6 6 ;
R ux t o n e t al . , 1 9 7 5 ) .
D e g r ad a t i o n o f
th e f r e e amino ac i d s i s b r o u g h t a b o u t mai n l y b y c l oit r i d i a with L-s a r i n e
a n d L-a r g inine b e i n g t h e onl y amino ac ids to b e s i g n i f i c a n t l y d e g r a d e d
b y l ac t i c ac id b ac t e r i a ( M c D o n a l d a n d Wh i t t e n b u r y , 1 9 73 ) .
Decarb o x y­
l a t i o n o f ami n o ac ids b y c l o s t ri d i a l e ads t o th e f o rmat i o n . o f ami n e s ,
a n d H u gh e s ( 1 9 7 0 ) f o u n d t h e am inas , h i s tamine , t r y p t am i n e , t y r am i n e ,
e t h an o l amin e , o r n i th in e , p u t r e s c i n e and c ad a v e r i n e p r e s e n t in s i l a g e .
T h e e f f ec t i v e n e s s o f a d ec r ease in pH i n i n h i b i t i n g c l o s t r i d i a
w i l l b e i n c r e a s e d b y a l o w m o is t u r e c o n t e n t o f th e h e rb age ( Wats o n a n d
N ash , 1 9 60 ) and a �igh c o nc e n t r a t i o n o f w a t e r s o l u b l e c ar b o h y d r a t e
( Jones , 1 970) .
T h e r a t i o o f w a t e r s o l u b l e c arboh y d r at e ( WSC ) : p r o t e i n
i n t h e h e r b a g e is a l s o i m p o r t an t , s in c e a l o w WS C : p ro t e i n r a t i o w i l l
p r o v i d e s ub s t r a t e s w h i c h fav o u r th e growth o f c l o s t r i d i a r a t h e r th a n
l ac t o b ac il l i .
T h is w o u l d l e ad t o t h e u n d e s i r a b l e p r o d u c t i o n of amm o n i a
a nd b u t y r ic a c id .
Kemb l e ( 1 9 56 ) succ e s s f u l l y e ns i l e d g r a s s o f a h i gh
p r o t e i n c o n t e n t b y add i t i o n o f g l u c o s e i n c o m b i n a t i o n w i th a n i n n o c u l u m
o f l a c t o b ac i l l i .
36
T h e main c on s i d e r a t ions f o r s ucc e s s ful ensiling o f h e r b age are
t h us t o e n s ur e an a n ae r o b ic e n v i r o nme n t and to prevent c l os t r i d i al
p r ol i f e r a t i o n .
Th e l a t t e r c an b e s t b e ach i e v ed b y e n s i l i n g h e r b age o f
a h i gh OM p e r c e nt a ge , a n d a h i gh WSC : p r o t e i n rat io .
H owev e r , n u m e r o u s
c h emi c al a d d i t i v es h av e b e e n s u g g e s t e d a s a means to f ac i l i tate
s uc c e s s f u l f e rme n t a t i o n .
B.
C h e mic al a i ds t o e n s i l i ng
Mc O o n ald and Wh i t t e n b u r y ( 1 9 73 ) d iv i d e d silage a d d i t iv es i n t o two
main g r o u ps :
( 1 ) s t im u l ants ,
( 2} inhib i t o r s .
( 1) S t imul ants :
S t imulants a r e c ompounds added prio r t o e n s i l i n g w i th
t h e aim o f p r o m o t i n g a l ac t i c ac i d t y p e of f ermentat i o n .
Examp l e s o f
t h es e a r e c ompo unds h i gh i n c ar b o h yd r a t e ( e . g . m o l as s e s ) , o r i n n oc u l a
o f l ac tob a c i l l i .
Th e advan t a g e s o f t h e s e additives h av e al r e a d y b e e n
d i sc u s s ed i n r e l a t i o n t o th e i r e f f e c t o n th e WSC : p r o t e i n r a t i o o f t h e
herbage .
( 2) I nh ib i t o r s :
I n h i b i tors o n th e o th e r h and , may e i t h e r c ompl e t e l y
i nh i b i t b a c t e r i al ac tiv i t y , o r s el ec t i v e l y p r event d e l e t e r i o us f e rmen­
t at i o n .
The main i n h i b i tors i n c ommo n u s e are sodium m e tab i s u l ph i t e ,
f o rmic ac i d a n d t h e m o r e rec e n t l y i n t r o d u c e d formal d e h y d e .
( a ) S o l i u m m e t c b is u lph i t e .
Th is i s us e d a t c o nc e n t r a t i ons wh ich d o n o t
· c omp l e t e l y inh i b i t f e rmentati o n .
Z e l t e r ( 1 9 60 ) f o u n d t h a t wh i l e
metab i s u l p h i t e c on s i d e r ab l y r e d uc ed b u t yric ac id p r o d uc t i o n , it d i d n o t
marke d l y p r e v e n t p r o t e o l ys is .
M u r d oc h e t al .
( 1 9 5 6 ) r e p o r ted t h a t
metab i s u l p h i t e d ec r e as e d t he f o r m a t i o n of v o latile ac i d s and b a s e s , b u t
h ad l i t t l e e f f e c t o n th e p r o d uc t i o n o f l ac tic ac id .
( b ) F o rmic a c i d .
Th e u s e o f m in e r al ac ids t o p r o d u c e a r a p i d i n i t i a l
37
d r o p i n p H , an a th u s i n h ib i t clos t r i d i a l f e rmentatio n , h as now l a r g e l y
I n the U K ,
b e e n r e p l aced b y a p p lic a t ion of f o rmic a c i d to the h e r b age .
8 5% f o rmic ac i d is n o r m a l l y u s e d , at 0 . 2 7% o f th e h e r b age O M .
d ec r e as e s pH t o 4 . 6 - 4 . 8 ( M c D o n a l d a n d Wh i tt e n b u r y , 1 9 73 ) .
This
H e n d e rs o n
and M c D o n ald ( 1 9 7 1 ) co mp ared t h r e e l e v e l s o f f o rmic acid a d d i t i o n t o
g r as s :
0 . 2 2% , 0 . 34 % and 0 . 5 1 % .
A t h i g h er l e vels o f applic a t i o n , th e
f ormic ac i d a p p e a r e d t o affe c t the s t r uc tu r e of the g r ass .
No intake
res p o n s e t o f o rmic acid-tre a tm e n t w a s o b s e r v e d , b u t t h e c o n t r o l s i l a g e s
.
il¥4
were t h ems e l v e s r e a d i l y c o ns umed . (W al d o e t al . , 1 9 7 0 ; A C as t l e and
(
(
)
Wats o n , 1 9 7 3 ) f o und th e f ol l o w i n g adv a n t ag e s of formic ac i d add e d p r i o r
t o e n s i l in g :
d e c re as e d pH , i n c r e a s e d d i ges t i b il i t y , d e c r e a s e d l e v e l s o f
b ut y r ic ac id , ac e t i c ac id a nd ammo n i a , � Hld i ncreased v o l u n t a r y i n t a k e .
.
H owev e r. , f o rmic ac i d is expe n s i v e , and und e r s ome c o n d i tions , l o s s es o f
up
t o 50% o f th e ac i d h av e b e e n r e p o r t e d ( H e nders o n a n d Mc D o nald , 1 9 7 1 ) .
( c ) F o r � a l d e hyd e .
F o rmaldeh y d e is a s ub s t a n c e in c o mmon us e in M e d ic i n e
and i n t h e L e a t h er I ndus try , b ec e u s e o f i t s b ac t e r i o s t atic p r o p e r ties ,
and i t s a b i l i t y to r e ac t with p r o t e i n s and t h us p r o t e c t th e m f r om
mic r o b ial f e rm e n ta t i o n .
F o r m aldeh y d e h as b e e n u s e d t o pro t e c t d ie t a r y
p r o t e i n s f r om d e gr a d a t i o n in th e r ume n , s i n c e t h e b o nds a r e s ub s e q ue n t l y
r e v e r s e d a t t h e l o w pH i n t h e abomas um .
Th us , t h e p r o tein is e v a i l a b l e
f or d i ges t i o n a n d a b s o r p t io n in th e i n t es t ines ( F e rgus on e t al . , 1 9 67 ) .
H e n c e f o rmaldeh y de h as b e en a p p l i e d t o h e r b age in a n e f f o r t t o p r ev e n t
p ro t e i n de gr ad a t io n d u r i n g e ns i l i n g .
H o we v e r , r e s p o n s e to f o rmal deh yde
. t r e a t m e n t o f s i l age h as been v ar i e d .
I nc r e as e d i n take in r e s po n s e t o
f ormal d eh y d e- t r e at m e n t o f s i l age was repo r t e d by B a r r y e t a l .
( 1 9 73 )
and V a l e n t ine a n d R ad c l i f f e ( 1 9 75 ) , wh i l e B r own and V al e n t i n e ( 1 9 72 )
r epo r t e d d e c r e a s e d i nt a k e a t h igh l ev e l s o f applic ati o n .
B r own and
V al e n t ine ( 1 9 72 ) s u gges t e d that th e l a c k o f res p o n s e i n th e i r e x p e r i m e n t
c oul d h av e b e e n d u e t o f r e e f o rmal d e h y d e p r e s ent i n t h e s i l a g e , s i n c e
38
N e um a r k e t al .
( 1 9 64 ) n o t e d t h a t f r e e f o r m a l d e h y d e p r e s e n t i n o r a d d e d
t o s il a g e dec r e as e d i n take�
Som e free f o r m al d eh y d e m i g h t be expec t e d
t o o c c ur i n a l l f o r m a l d e h y d e - t r e a t e d s i l a g e s , s i nce Th e is and L ams
( 1 94 3 ) r e p o r t e d th a t r e v e r s a l o f f o rmaldeh y d e - b in d i n g w i th · p r o t e i n
o c c urred w i th a grad u a l d ec r e as e i n p H .
H e n c e t h e d e c r ease i n p H i n
t h e s ilage s t ac k m i gh t b e e x p e c t e d t o r e l e a s e f r e e f o rma l d e h y d e a n d
protein .
B arry and F e n n e s s y ( 1 9 7 3 ) r e p o r t e d t h a t s e c o n d a r y f e rm en t a t i o n o n
o p e n ing o f t h e s tac k was � r e a t e r i n form a l d e h yd e-t r e a t e d t h a n u n t r e a t e d
s il ages .
C o m b i n a t i o ns o f a d d i t i v e s h a v e b e e n s u gges t e d t o o v e rc ome
t h is problem o f s e c o n d a r y f e rme n t at i o n , a n d B arry ( 1 9 7 5 ) c om p a r e d t h e
e f f ec ts of f o r ma l d e h y d e , a n d formald eh y d e / ac id ( f o rmic o r s u l ph u r i c )
m i x t u re s .
I n t ake w a s h i gh e s t on f o rmal d e h y d e /f o rmi c ac i d-t r e a t e d
s il a ges , and wh i l e i n t a ke o f u n t r e a t e d a n d f o rm al d eh yd e-t re at e d s il a ges
d e c r e as e d with t i m e after o p e n i n g the s t ac k , addition o f f ormic ac i d
p re v e nt e d th i s d e c r e ase .
e v id e nc e .
H o wev e r , s e c o n d a r y f e rme n t a t i o n was s t i l l i n
V al e n t i n e and B r own ( 1 9 73 ) al s o f o und
a
s i g n i f ic an t i nc r e ase
i n i ntake o f f o rmal d eh y d e / f o rmic ac i d s i l ag e o v er u n t r e a ted s i l a g e ,
w h i l e n e i th e r f o rmaldeh y d e n o r f o rmic ac i d a l o n e s i g n i f i c an t l y i n c r e as ed
i n take o v e r u n t r e a t e d s il a g e .
C.
F �c t o r s a f f ec t i ng i n t a k e of s i l age
( 1 ) G e ne ra l :
Much o f t h e w o r k o n s il a g e q u a l i t y h as b e e n c o n f i n e d t o
b ioch emic al a n d mic r o b i o l o g i c al s t udies o f f e rme n t a t i o n w i t h i n t h e
s il age s t ac k , and h as n o t c o ns i d e r e d v ol u n t ary i n t a k e b y th e a n i m al .
T h us , wh i l e a h i gh qua l i t y s il a g e h as b e e n d e f in e d as h av in g a p H o f
4 . 2 o r l e s s ( McDo n a ld a n d Wh i t t e n b u r y , 1 9 7 3 ) various w o r kers h av e
r e p o r t e d t h a t s i l ag e s w i t h a l o w p H ad v e r s e l y af f e c t i n take ( H u t t o n e t
al . , 1 970 ;
Mc L e o.d e t al . , 1 9 7 0 ;
W i l k ins e t a l . , 1 9 7 1 ) .
H u tch i n s o n and Wil k i n s , 1 9 7 1 ;
T h i s a p p ears t o b e partly d ue t o a d ir ec t e f f e c t
39
o f h i gh a c i d c � nc e n t ra t i o n ( � c l e o d e t al . , 1 9 70 ) , b u t a l o w pH a l s o
i n d i c a t e s c o n s i d e r a b l e f e r m e n t a t i o n o f w a t e r s o l u b l e c ar b o h y d r a t e s
( B r o w n a n d R a d c l if f e , 1 9 72 ) , a n d t h i s m ay adv e r s e l y a f f e c t th e b al a nc e
o f N and e n e r g y m e t a b o l i s m i n th e rumen .
( 2 ) T h e e f f ec t of f o rm and c o r c e n t r a t i o n o f N and e n e rgy o n s i l age
intake :
T h e c omp o s i t i o n o f t h e N frac t i o n of s i l ag e d i f f e r s f r om t h a t
o f p a s t u r e a n d h a y , i n h av i n g r e l a t i v e l y h i gh ammo n i a , amine and amide
c o n t e n t s ( H u gh es , 1 9 7 0 ) .
H i gh amm o n i a c c n c e n t r a t i o n s i n s i l age h a v e
b e e n c o r r e l at e d w i t h l o w i n t akes ( H a r r i s a n d Raymo n d , 1 9 63 ;
e t al . , 1 9 71 ) .
Wil kins
C as tl e and W a ts o n ( 1 9 69 ) f o und t h a t t o tal i n t ak e o f a
m i x e d r a t i on was a l s o aff e c t e d b y s il age-N c on t e n t .
Th e y c o mp a r e d
v ol u n ta r y i n take o f s il a g e s o f l o w ( 8 % ) a n d h i gh ( 1 6 %) d i g e s t i b l e c r u d e
p r o t e i n c o n t e n t , and f o und t h a t t o tal OM i n take w a s ·l o w e r o n t h e s i l ag e
w i t h th e h i gh e r N c on t e n t .
T h e e f f e c t o f t h e N c o n t e n t o f t h e c o nc e n­
t r a t e p a r t o f mixed r a t i o n s was s t ud i e d by Murdoch ( 1 9 64 ) .
He f o und
that i n t a k e of s il a g e d e c r e as e d t o a l e s s e r e x t e n t w i t h a h i gh p r o t e i n
than a low protein c oncentrate .
S i nc e th e N c o n t e n t o f s i l a g e i s
u s u a l l y o f a low t r u e p r o t e i n p e rc ent ag e , t h e s e r e s ul ts s u g g e s t t h a t t h e
f orm , r a t h e r t h a n the quanti t y o f N intake , appeers to exert a m a j o r
e f f e c t o n s il age i n take .
E f f ic i e n t u t i l is a t i o n o f N P N in t h e r um en is d e p e n d e n t o n t h e
a v a i l ab i l i t y of e n e r g y ( H e n d e r i c kx a n d M a r t i n , 1 9 63 ) .
Since the
c o n c e n t r a t i o n o f w a t e r s o l u b l e c arboh y d r a t es in s i l age i s g e ne ra l l y l o w
( M c D o n a l d e t al . , 1 9 69 ) , i n c r e as ed u t i l iiat i o n o f N m a y b e � c h i e v ed b y
i n c r e as e d e n e r g y a v a i l ab i l i t y .
Th us , i n c r e as e d N r e t e n t i o n in r e s p o n s e
t o s up p l em e n t a t i o n of s il a g e w i t h e n e r g y h as b e e n r e c o r d e d ( O urand
e t al . , 1 9 68 ;
Th o ms o n , 1 9 6 8 ;
G r i f f i t h s e t a l . , 1 9 73 ) .
Th e e f f ec t o f
s il a g e -N and e n e r g y c o n t e n ts o n s il a g e i n t ake , t h u s a p p e a rs t o b e d u e
40
t o t h e p r om o t i o n o f m i c r o b i a l p r o t e in s y n th esis and h e n c e th e t o t a l
amo u n t o f p r o t e i n e n t e r i n g the d uo d e n u m .
( 3 ) Th e e f f ec t of d uodenal pr o t e in av a i l a b i lity on s il age i n t ak e :
E gan
( 1 9 6 5 ) s u g g e s t e d that v o l u n t a r y i n t ake of l ow p r o t e i n r o u gh a g e s may
p r im ar i l y be l i m i t e d by a d e f i c i e n c y of p r o t e i n ab s o r b ed by the anima l .
I nc r e a s e d growth r ates in r e s p o n s e to p r o t e i n s u p p l e m e n t a t i o n o f
an imals f e d s il a g e h av e b e e n r e p o r ted ( F orbes and I r w i n , 1 9 70 ;
1 9 73 ) .
O r e nnan ,
H o w ev e r , H u tch i ns o n e t al . ( 1 9 7 1 ) f o un d no i n t a k e r e s p o n s e to
d u o d e nal l y i n f u s e d c a s e i n o n a 1 . B 6%N s i l age , altho ugh N r e t e n t i on was
inc r e as e d .
I nc r e as e d intake i n r es p o ns e to f o r malin-treatme n t o f h e r b age p r i o r .
t o e n s i l i n g h a s b ee n r e c o r d e d ( B a r r y e t a l . , 1 9 73 ) .
S i n c e t h e main
e f f ec t o f f o r ma l i n-treatment i s t o p r e v e n t protein d e g r ad a t i o n d u r i n g
e ns i l i n g , th i s r e s u l t s u g g e s ts a p o s i t i v e intake r e s p o n s e t o i n c r e a s e d
p r o t e i n a v a i l ab i l i t y .
I nc r e a s e d in t a k e s o f s ilage in r e s p o n s e t o
i n t r a p e r i t o n e a l methionine h a v e also b e e n r e p o r t e d ( B er r y e t a l . , 1 9 73 ;
K e l l y and Thomas , 1 9 7 5 ;
B a r r y , 1 9 76 ) .
Th is r e s u l t s u g g e s ts th at
f e e d i n g t h e untreated silages t o s h e e p induced a s ta t e o f meth i o n i n e
imbal anc e .
M e t h i o n i n e h as f r e q ue n t l y b e e n s u ggested a s t h e f i r s t
l im i t i n g ami n o a c i d f o r ruminants ( N i m r i c k e t al . , 1 9 7 0 ;
A nn i s o n , 1 9 7 2 ;
A rm s t r o n g and A n n i so n , 1 9 73 ;
H u t t o n and
Harrison et al . , 1 973 ) .
H e nc e th e av a i l a b i l i t y of amino acids , and partic u l a r l y m e t h i o n i n e ,
a p p e ars t o l i m i t i n take in s ome s i l a g e s .
1 .8
C o nc l u s i o n
Meth ionine appears to b e t h e f i r s t l imitin g ami n o ac id f o r l i v e­
w e i g h t gain and wo o l growth i n r uminants o n a v a r i e t y o f f e e d s .
In
p ar t ic ul a r , s ome s i lages p r o d uc e a v e r y l o w level o f S-amino acids
e n t e r i n g th e d u o de num .
S i nc e l o w i n t a k e of silage h as i n s ome c a s e s
41
b een i n c r e a s ed . b y s up p l emental m e t h i o n i n e , i t is s u g g e s ted t h a t f e e d i n g
s ilage may p r o d u c e a m e th i o n i n e imbalanc e .
An imb al anc e s ta t e
indicates t h a t t h e c omp lement o f am ino ac ids absorbed f r o m t h e d u o d e n u m
d o es not c o r r e s p o nd t o th e c omplement r e q uired b y t h e t i s s u e s .
Th u s �
s ilage appears t o h a v e a m a r k e d e f f e c t o n e i th e r t h e c ompos i t i o n o f
duode nal amino ac ids , o r the r e q u i r eme n t f o r meth i o n i n e r e l a t i v e t o
t btal ami n � acid requ i r ements .
Th � a m i n o ac i d c ompos i t i o n o f d u oden�l
d i g e s t a r e mains r e lativ e l y c o n s t a n t i n s h e e p f e d natural d i e ts , s inc e
mic robial p r o t e i n is o f relativ e l y c o n s t a n t am ino acid c omp o s i t i on and
is s im i l a r to t h a t of h e rbage p r o t e i n .
F e e d i n g s i l a g e may c h a n g e t h e
c ompos i t i o n o f d u o d e n a l am ino ac ids , b u t a more l i k e l y e x p l a n a t i o n of
th e imb al a nc e s t a t e , i s a change i n th e r e l ativ e requi rements f o r
meth i o n i n e and o t h e r amino ac ids , d u e t o a d ec r eas ed t o tal a v a i l ab i l i t y
in t h e s m a l l i n t e s t ine .
T h e p e rc e n ta g e o f t h e av a i l a b l e me t h i o n ine
wh ich is u s e d for p r o t ein s y n th e s i s , c om p ared to th e p e r c e n t a g e us e d
f o r trans m e th y l a t i o n r e ac tions a n d c o n v e rs io n t o c ys te i n e , c h an ges with
t h e avail a b i l i t y o f methionine and other amino ac i ds .
H e nc e , a d e c r e a s e
in th e avail a b i l i t y o f t o t a l ami no ac i d s m a y increas e t h e p e r c e n tage o f
meth i o n i n e r e q u i red .
Thus , th e e x p e r ime nts t o b e r e p o r t e d w e re undertaken t o s t u d y t h e
e f f ec ts o f i n c r e a s i n g the availab i l i t y o f to tal p r o tein , and m e th i o n i n e
alone , t o s h e e p f e d s i l ag e .
utilis a t i o n o f
N
T h e e f f e c t s were s tu d ied i n t e r ms o f t h e
a n d amino ac ids b y t h e s h e e p .
. total p r o t e in was i n c r eased in t h r e e w a y s.:
the s il a g e ;
T h e avai l a b i l i t y o f
b y f o r ma l i n-tre a tm e n t o f
s up p l em e n tation with f o r m a l deh yd8-treated c as e i n ;
s u p p l e m e n t a t i o n with
a
and
read i l y avai l a b l e energy s o urc e , t o s t imulate
mic r o b i a l protein s y n t h es is and thus increase th e t o t al f l o w o f amino
ac ids into th e d u o d e n u m .
M e t h i o n i n e av a i l a b ility was i n c r e a s e d b y
i n f u s i o n i n t o th e . p e r i toneal c av i t y o r d u o d enum .
CHAPTER
TECH N I Q U E S
FOR
S T UD Y I N G
A M I NO
2
AC I D
M E T A B OL I SM
IN
R UM I N A N T S
42
2.1
I n tr o d uc t i o n
T h e p r ec� ing
c h a p t e r h as s h own that a study o f a m i n o ac id
metab o l ism in rumina n ts r e q u i re s meas urement of t h e daily f l ow o f
amin o ac i d s into th e d uo d e nu m .
T h e ac c u r a c y o f m e a s u r i n g th e f l ow
o f d i g esta into t h e d u o d e n um h as b e e n greatly f ac i l i tated b y th e u s e
o f s h e e p p r epared w i t h r e-e n t ran t c annulae i n the p r oximal duod e n um
( Ash , 1 9 62 ) .
These c an n u l ae e n a b l e total c o l l e c t i o n of d i g e s t a and
thus a r e p resenta tiv e s am p le c a n be o b tained .
C a l c u l a t i o n o f f l ow
r ate also requires r e f e r e n c e to an indige s t i b l e marker s in c e
c o l l e c ti o n proc e d u r e s f r e q u e n t l y d e p r e s s f l o w .
T h e reasons f o r th e
c h o i c e o f s pe c i f ic s amp l i n g s y s t em used in the e x p e riments t o b e
r e p o r ted w i l l b e d i s c us s e d b e l o w .
A n a l y s is o f t h e amin o a c i d c ompos i t i o n of th e s am p l e s o b t a i n e d ,
w as c ar r i e d out o n a s t a n d a r d amino ac id anal yser e m p l o y i n g t h e ion­
e xc h ange p r inc iple o f am ino ac i d analysis , f i r s t i n t r o d uc ed by M o o r e
a n d S te i n ( 1 9 5 1 ) .
S inc e th e f i r s t c h r omatog raph i c c o n d i t i o n s w e r e
r e p o r t e d , nume r o us m o d i f i c a t i o n s h a v e b e e n made , and s p e c i f i c
p r oc e d u r e s for t h e m e as u r e m e n t o f g r o u p s o f amino acids h av e b e e n
p ro p o s e d ( e . g . J e p p s s o n a n d Karlss o n , 1 9 7 2 ) .
I n t h e e x p e r im e n t s t o b e
d es c r i b e d , meas urem e n t o f m e th i o n ine s p e c i f i c rad i o ac t i v i t y was
r eq u i r e d .
Th e s e an alys e s w e r e als o c o nduc ted o n a n am ino ac i d anal y s e r
and t h e r e asons f o r th i s c h o ic e w i l l b e d i s c u s s e d b e l ow .
T h e amino acids e n t e r in g th e duod enum w i l l b e s u p p l i e d e i t h e r b y
d i e t a r y p r o te i n wh i c h h as e s c a p e d rumen f e rmentat i o n , o r b y m i c r o b i al
p r o t e i n s y nth e s ised i n t h e r um e n .
Th e e f f e c t o f e n er g y and N
m e t ab o l i s m in t h e r um e n o n mic r ob ial p r o t e i n s y n t h e s is h as a l ready b e e n
d i s c u s s ed, and thus t h e a d v an t a g e s o f b e i n g able t o measure i t s r at e o f
s y n th esis are a p p ar e n t .
N um e r o us meth ods f o r q ua n t if y i n g m i c r o b ial
43
p r o t e in s y n t h e s i s h av e b e e n pro p o s e d , b u t there are s t i l l c on s i d e r a b l e
d r a w b a c k s t o t h e i r rout ine· u s e , and t h e s e will also b e d i s c u s s e d .
2.2
Measurement of d uod enal f l o w
Th e meas urement o f f l ow o f n u t r i e n t s into t h e d u o d e n u m h a s two
main r e q u i r e m e nt s .
The first is o b taining an accurate m e a s u r e m e n t of
d i ge s ta f l ow , and the s e c o n d is t h e c o l l e c tion of a r e p r e s e n ta t i v e
s amp l e o f d i g e s t a .
S e v e r a l rev i ew s of t h e techniques i n v o l v e d i n
m e as u ring f l ow i n t o th e duodenum h a v e b e e n publish e d rec e n t l y ( K o tb ,
1 9 72 ;
E n g e l h ard t , 1 974 ;
Mac R a e , 1 9 74 ;
MacRae , 1 9 75 ;
F a ic h ne y , 1 9 75 ) .
Thus , o n l y t h e main c o n s id e r a t i o n s af f e c t i ng th e c h o i c e o f m e th o d f o r
t h e e x p e r i m e n t s t o be report ed , w i l l b e d i s c uss ed .
A.
G e n e r a l c o n s i d e rations
T h e c o l l e c t i o n of s amp l e s f o r measuring th e c om p o s i t i o n o f d i g e s t a
e n t e r ing t h e duode num m u s t b e m a d e p r i o r t o a n y abs o r p t i o n or s e c r e t i o n
w i t h i n th e s m a l l in tes t ine .
H o w e v e r , s i nce the abomas u m i s a s a c u l a r
organ , with n u m e r o u s f o lds d i v i d in g t h e c o ntents , c o l l ec t i o n o f a
r e p r e s e n t a t i v e s ample o f abomasal d i g e s ta is diffi cu l t .
Th e r e f o r e , t h e
mos t s u i t ab l e s ampling s i t e ap p e a r s t o b e imme d iate l y a f t e r th e e n t r y
o f d i ge s t a i n t o the duode num , p r i o r to entry o f s e c r e t i o n s o f b il e a n d
panc reat i c j u ic e .
T h e e a s e of c ol l ec t i o n o f d igesta a n d m e a s u r e m e n t o f
f l o w h a s b e e n g r e a t l y i n c r e a s e d b y th e p r e paration o f s h e e p w i t h
re-e n trant c annulae in th e p r o x im a l d u o d e num ( Ash , 1 9 62 ) .
T h e s e c annulae
e n a b l e th e c o l l ec t ion o f the t o t a l d i g e s t a entering t h e d uo d e num , t h e
r e m o v a l o f a s u b s ample a n d t h e r e t u rn o f digesta v i a t h e d i s t a l c a n n u l a .
F a i l u r e t o r e t urn digesta to t h e animal has been s h o w n t o increase f l ow
( H o gan and Ph i l l ipson , 1 96 2 ) .
H ar r is and P h i l l i p s o n ( 1 9 6 2 ) th e r e f o r e
s u g g e s te d t h a t an indi g e s t i b l e m a r k e r b e used , t o c o r r e c t t o t a l f l o w t o
44
1 00% rec o v e r y � f t h e marke r .
T h e y a l s o s ug ges ted th at the v o l um e o f
subsamp l e t a k e n f o r anal ysis b e r e p l a c e d w i th d o n o r d i g e s t a p r i o r t o
return o f t h e c o l l e c t e d digesta to t h e a n imal .
H owev e r , a numb e r o f
workers h av e r e p o r t e d t h at this m e t h o d o f t o tal c o l l e c t i o n · t e n d s to
depress d i g e s t a f l ow ( T opps e t al . , 1 9 6 8 ;
M ac R ae and A r ms t r o n g , 1 9 69 ;
Mac Rae e t a l . , 1 9 72 ) .
N ic h o l s o n and S u t to n , 1 9 69 ;
K l o o s t e r e t al .
( 1 9 7 2 ) f o und t h a t t h i s depre s s i o n · i n f l o w was onl y temporar y , and
c ontinu a t i o n o f t h e c o l le c t i o n f o r 7 2 h s h o w e d a r e c o v e r y i n flow r ate .
S ince th e c ol l ec t io n and return of d i g e s ta is extrem e l y t e d i o u s , s e v e r a l
attem p ts t o c o l l e c t and samp l e d i g e s t a a u tomatical l y h av e b e e n made .
Very l i t t l e d a t a from the u s e o f automa t ic s amplers h as as y e t b e e n
p u b l i s h e d ( Tas e t al . , 1 9 74 ;
Mac R a e , 1 9 7 5 ) and t h e s e s amp l ers are n o t
generally av ailab l e .
An a l ternat iv e pr ocedure is t o take s p o t s amp l e s .
Th e s e h a v e t h e
advantage o v e r c o n t in u o us sampling t e c h n iques i n that t h e y a r e l e s s
traumatic to t h e animal and less l ab o r i o us f o r th e e x p e rime n t e r .
Mac R ae
and U l y a t t ( 1 9 7 2 ) c omp ared t h e use o f s p o t and c o nt i n uo us s am p l i n g
tech n i q u e s a n d f o u n d c l ose a g reeme nt b e tween the two m e t h o d s, a l t h o u gh
more v ar i a b i l i t y was ap pare n t wh e n es t imating flows from s p o t s amp l in g .
I n g e n e r al , s p o t s ampl ing t e c h n i q u e s a r e u s e d for s h e e p p r e p a r e d with
s im p l e T-s h a p e d c a n n u l ae .
These c an n u l ae are eas ie r t o imp l an t and
main tain th an re-e n t rant c ann u l ae .
H o we v e r , sheep w i th re-e n t ra n t
c annulae were u s e d f o r th e p r e s e n t e x p e rime n ts ( C h ap t ers 3 a n d 4 ) , a n d
a v a r i a t i o n o f the s p o t sam p l i n g t e c h n i que w a s emp l o y e d ( Wa l k e r e t a l . ,
1 975 ) .
T w e l v e s p o t s amples were c o l l e c ted o v er a p e r i o d o f t h r e e days .
There w e r e f o u r sampling times p e r d a y , with a 6h interval b e tween e ac h
samp l i n g t ime .
i n t e rv al .
D a i l y groups o f s am p l i n g times were s e p a r a t e d b y a n B h
H e nc e , i f samples o n t h e f i r s t d a y were c o l l e c t e d a t 1 0 am ,
Fig. 2 . 1
proximal end
of c annula
Duod enal cannula
b etween s a mpl i ng
c an n ul a
of
------�
D i r e c t i o n of flow
D ir e c t i o n of
flow
Fig. 2 . 2 Co llec tion of
d i ge s t a s ampl e
(f
(i)
45
4 pm , 1 0 p m and 4 a m , the n e x t s am p l e w o u l d b e c o l l ec te d a t 1 2 n o o n .
The u s e o f re-e n t r a n t c an n u l ae f o r s p o t s amp l i n g e n a b l e s t h e
c o l l e c t i o n of the total d i g e sta f l owing d u r i n g t h e s amp l i n g p e r i o d .
A s t h e d u o de n um is a t u b u l a r o r g a n , it was c o n s i d e r e d t h a t th e r e w o u l d
n o t b e a m a r k e d d i f f e r e n c e i n f l o w r a t e b e tween t h e s o l i d and l i quid
p h a s e s o f diges t a .
Hence it was c o nsidered that o n l y o n e m a r k e r t h at
as s o c iated w i th e i t h e r th e l i q u i d o r s o l i d phas e , w as r e q ui r e d t o
d e te rmine b o th
OM
and t o t a l f l o w rates .
S ince some d i f f i c u l t y h ad
e a r l i e r b e e n e x p e r i e n c e d i n f i n d in g s u i t able s o l i d phase markers ,
( Mac Rae and U l y at t , 1 9 7 2 ) , a l i q ui d p h as e marker ( C r EDTA ) was c h os e n .
( We l l e r and P i l g r i m , 1 9 74 ;
Wa l k e r et al . , 1 9 7 5 ) .
C r was anal y s e d b y
atomid a b s o rption s p ec t r o p h o t6m e t r y .
B.
A c c u r acy of method and m o d i f i c a t i ons int roduc e d
I n E x p t . 1 , s p o t s amp l e s o f 4 0 g were taken a t e ac h s amp l i n g time
and th e n b u l k e d .
This t ec h n iq u e was modified p r i o r to E x p t . 2 , t o
o b ta i n a more r e p r e s e n t a t i v e s am p l e .
This mod i f ic at i o n i n v o l v e d
r e p l ac e m e n t of the tub i n g c o n n e c t i n g the t w o e n d s o f t h e r e-entrant
c a nn u l a , with a Perspex c o n n e c t i n g p i e c e .
Th is h ad two s ide-arms , one
for c o l l e c ti o n and one for r e t u r n of digesta ( F i g . 2 . 1 ) .
Samp l e s c o u l d
t h u s b e c o l l e c ted with o u t d i s c o n n e c t i o n o f the c a n n u l a at each s amp l i n g
t im e .
T o e n s u r e c o l l e c t i o n o f t o tal c o n tents , a b al l o o n ( F i g . 2 . 2
was i n s e r t e d t h r o u gh ( a ) a n d i n f l ated with air f r om a s y r i n g e ( d ) .
c
)
This
s y r i n g e was attac h e d t o a s h o r t l e n gth o f t u b i n g w h i c h w a s e n c l o s e d
within the balloon .
I nf l a t i ng t h e b a l l o o n b lo c k e d o f f t h e d is t al e nd
o f th e c a n n u l a f o rc ing d i g e s t a to f low th r o u gh ( b ) .
A l e n g th o f L a t e x
c o l o s t o m y t u b i n g ( e ) was a t t ac h e d to ( b ) and c a r r ied t h e d u o d enal d i g e s t a
i n t o a p l as t i c c ol l e c t i o n b o t t l e ( f ) .
T h e t u b i n g c o l l a p s e d w h e n emp t y ,
.
I
I
F ig .
2.3
Return of c o nten t s a f t er
s u b s ampl i ng
46
.
and thus s imul a t e d t h e c o n d i tions o f f l ow t h r o u gh th e d u o d e n um .
A f ter
250ml o f d ig e s t a h a d c o l l e� t e d in ( f ) , t h e b alloon was remo v e d and th e
r u b b e r p l u gs r e p l a c e d i n ( a ) and ( b ) .
s ub s ample ta k e n .
The digesta was mix e d a n d a 2 5m l
T h e c o llec ti o n b o t t l e w a s th e n s t o p p e r e d · a n d p l ac e d
°
in a b as i n o f w arm wat e r , to r e h e a t t h e d i g e s t a t o 3 9 C , p r i o r t o r e t u r n
to t h e animal .
The c o l l e c t i o n b o t t l e ( f ) w as th en attac h e d t o a
.
f i e x i b l e p l a s t i c t u b e ( F i g . 2 . 3 ) wh i c h in turn was c o nnec t e d t o th e
re-e n t r y arm o f th e c o nnec t i n g p i e c e ( a ) b y a rub b e r s l e e v e .
R e turn o f
t h e d i g e s t s was t h u s ac h i e v e d b y inv e r t i n g the plas tic b o t t l e a n d
remo v i n g t h e small r u b b e r s t opper ( i ) .
was c o n t r o l l e d b y a c l amp ( j ) .
Th e r a t e o f r e t u r n o f d i g e s t a
Th i s t e c h n ique enab l e d the c o l l e c t io n
o f a l a r g e r v o l um e o f samp l e , wh ich c o ul d t h e n b e s u b s am p l e d t o r e d u c e
the e r r o r a s s o c i a t e d with e ach samp l e .
B y t h is p r o c e d u r e o n l y 2 5 g
d i g e s t a were r e mo v e d a t e a c h s amp l i ng t ime , and in to tal t h i s c ompri s e d
less th an 1 . 5% of t h e daily f l o w .
I t was c o nside r e d that re p l a c e me n t
o f t h e v o l ume s am p l e � with d o n o r d i ge s ta was not n e c e s s a r y , s i nc e 6h
si.(aess t >�e
,,
elap s e d b e tw e e n &aefi s am p l i n g t im�
I n a d d i t i o n , th e r e t u r n o f d o n o r
d i g e s t a w h i c h h as b e e n fro z e n a n d t h a w e d , t h u s denaturing t h e p r o t e i n ,
migh t h av e a m ark e d e f f e c t o n ami n o ac i d d i g e s t i o n and ab s o r p t i o n .
T h e s am p l e s c o ll e c ted in Ex p t . 1 , we r e b ulked o v e r a l l f o r e ac h
s h e e p o n each tre atmen t , giv ing o n l y t h r e e estimates o f f l o w p e r
treatm e n t .
C o n s i d e rable v ar i a t i o n b e t w e e n replicates w a s o b s e r v e d i n
Exp t . 1 , a n d s am p l e s in Ex p t . 2 w e r e b ul k e d o n _ a dail y b as is .
This
s h o u l d h av e e n ab l e d e s timation o f t h e r e p e atabi l i t y o f m e as u r em e n ts .
Howev e r , d a i l y intakes d u r i n g f l ow measuremen ts v aried c o ns i de r a b l y a n d
this p r o d u c e d large d i f f e r e n c es i n t h e f l o w estimates rec o r d e d .
O n th e
c o n t r o l d i e t , i n t a k e v aried b y as muc h as 200g b e tween c o n s e c u t i v e d a y s .
47
Th us , estimation o f t h e accuracy o f f l ow m e as urement was o n l y
c al c ul ate d f o r s h e e p 1 1 5 7 o n r e p l icates 1 and 2 , and f o r s h e e p 1 3 0 3 o n
r e p l ic ates 3 and 4 , w h e n t h e i n t a k e of t h e s e sheep remained
a p p r oxima t e l y c o ns t a n t .
S i n c e these two s h e e p were b o t h b e i n g
s up p l emented w i th f o r m al d e h y d e-treated c as e i n d u r i n g t h e s p e c i f i e d
p e riods , a c o m b i n e d s t andard e r r o r ( S E ) w a s c al c ul a t e d .
The S E o b t a i n e d
w i th a m e a n o f 4 2 9 gD M/ day w a s + 9 . i g/ day w h i � h ind i c a t e s a h i gh d e g� e e
o f r ep e at a b i l i t y .
C.
A n alys i s o f C r EDTA
P r i o r t o the s ta r t o f t h e f ir s t e x p e r i m ent , two methods of
p r e p aring d i g e s ta s am p l e s f o r at omic ab s o r p ti o n anal y s i s of Cr w e r e
tested.
I n t h e f i r s t m e t h o d , t h e samp l e s w e r e ash ed i n a f u r n ac e , p r i o r
t o o xidation w i th p o tass ium b r omate , a n d t h e y were s u b s e q u e n t l y
d i s s o l v e d i n p h o s p h o ric acid-man ganese s u l p h ate s o l u t i o n ( W i l l iams
e t al . , 1 9 62 ) .
In the s ec o n d m e th o d , the s upernatant was p r e p a r e d b y
c e n t r i f u g a t i o n o f t o t al d i ge s t a a t 3 B , DD D g , followed b y f i l t r a t i o n
thiough Wh atman N o . 1 f i l te r p aper .
a s h i n g m e t h o d of W i l l iams e t al .
P r e p ar ation o f t h e samp l e b y t h e
( 1 9 62 ) o n l y gav e a 7 1 . 3% (± 4 . 0 )
r ec o v e r y o f added C r . A n a l y s is o f Cr in t h e s upernatan t , p r e p a r e d b y
c e ntrif u g a t i o n gav e a r e c o v e r y o f 96 . 6% ( ± 4 . 1 ) , and a f t e r was h i n g t h e
p e l l e t t h r e e t i m e s i n w a te r , and ash i n g , no Cr w a s d e t e c t e d .
Hence
f ur t h e r t e s ts were c o n d u c t e d o n s upernatan t mate r i al .
T h e r o u tine s t andards u s e d a potas s i u m dic h r omate s o l u t i o n , w h e r e
C r i s p r e s e n t i n a d i f f e r e n t oxidation s ta t e ( c i 6+ ) f r om C r c om p l e x e d
3
t o EDTA ( C r + ) .
T o t e s t t h e v al i dity o f t h e dic h r omate s tandar d s ,
alterna tiv e s tandards were made up from c h r omium p o tass ium s u l p h a t e
( c r 3+ ) b u t n o d i f f e r e n c e w a s d e t e c ted .
M e as urement of C r w i th a n d
48
w i t h o u t E D TA a l s o h ad n o e f f e c t .
Wil l i ams e t al .
( 1 962 ) suggested the
add i t i o n o f 1 0 00ppm c a l c ium t o s t andards a n d s amp l e s , t o o v e r r i d e a n y
i n t e r f e r e n c e from c al c i um p r e s e n t i n t h e diges t a .
The effect of
c a l c ium d uring t h e s e t e s ts was f ou nd t o b e v ariabl e , b u t t�e p r e s e n c e
o f c al c i u m a p p e a r e d to m a i n t a i n a c o ns tant recov e r y .
Th e f ol l owing g u i d e l i n e s f o r the meas urement o f C r b y atomic
a b s o r p t i o n s p e c t r o p h o t o m e t r y were adop ted :
( 1 ) p r e p a r ation o f s am p l e s b y c e n t rifugation ( 3B , O O O g ) f o l l o w e d b y
filtratio n ,
( 2 ) d i l u t i o n o f s am p l e s and s t and ards with c alc ium , t o a f i n a l s o l u t i o n
o f 1 00 0 p p m c al c i u m ,
( 3 ) m e as u r ement o f th e c o r r e s p o n d i ng infus ates wi th each b atch o f
s amp l es .
2.3
A.
Amino acid analys is and measurement o f spe c i f i c ac t i v i t i e s
G e n e r a l amino a c i d an alys i s
R o u t ine measurement o f amino ac i d c om p o s i t i o n i s n o w m a i n l y
c o n d uc t e d on automated ami no ac i d an a � ys e rs .
A wide range o f
i n s truments are av ailat l e , w i t h adap t a tions f o r. s p e c i a l anal y t i c a l
r e q u i rements ( P o r t e r e t al . , 1 9 68 ) .
T h e b a s i c p r inc i p l e of th e s e
anal y s e r s i s t h e d i f f e r e n t i a l s e p aration o f individual a m i n o ac i ds o n a
c o l um n o f ion-exchange r e s i n , b y e l u t i o n w i th b uf f e rs o f d i f f e ri n g p H
and i o n i c c o n c e n t r a t i o n .
T h e c h romatograp h ic c o n d i t i o n s a r e c h anged t o
ach ie v e t h e partic u l ar anal y s i s require d · ( e . g . p r o t e i n h yd r o l ys ate o r
p h y s i o l o g ic al f l ui d analysis ) .
Howev e r , analys is o f s pe c i f i c amino
acids by s uc h automated p r oc e d ures re quire s c o n s i d e r a b l e m a n i p u l a t i o n
o f t h e c h romatographic c o nd i t ions to ac h ie v e s e p a r a t i o n i n a mi nimum
t ime .
I n add i t i o n the v ol um e o f sam p l e wh ich c an b e an al y s e d, and t h e
49
.
r a n g e of c onc e ntration wh ich c a n b e a c c urat e l y d e te c t e d, a r e l i m i t e d b y
th e s p e c i f ic a t i o ns o f t h e i n s trum e n t �
Thus , f o r anal y s is o f m e t h i o n i n e
s p e c ific a c t i v i t ies in the e x p e r iments to b e describ e d , c o ns i d e r a t i o n
was f i rs t giv e n to o th e r me t h o ds o f me as uring m e th i o n i n e c o n c e n t r at i o n .
B.
M e a s u r eme n t of meth ionine spe c i f i c ac t i v i ty
Th e c o nc e n t ration o f m e t h i o n ine i n s h e e p p l asma i s v e r y l o w , and
s in c e the isotope used i n th e p r e sent e x p e r iments ( L-m e t h i o n i n e -C 1 4 ( U ) )
was v e r y e xpe n s iv e , the s pe c i f ic ac tiv i t y ( S A ) o f me t h i o n i n e i n t h e
p l a s m a s ampl e s was also very low .
Th u s a t e c h n ique c ap a b l e of
analysing a r e l a tiv e l y l arge v o l ume of samp l e wes r e q u i re d .
S �e c i f ic methods f o r th e d e t e rmi n aI tion of S- ami n o ac ids h av e b e e n
p r o p o s e d ( L aR u e , 1 9 6 5 ;
K y r i ac ou , 1 9 6 6 ;
R u b lev , 1 9 7 3 ) .
H owev e r , s in c e
t h e r e are nume ro us transformations b e tween individual amino ac ids , t h e
meas u reme n t o f met� i o n i n e S A r e q u i r e s p rior s eparation f r o m th e o t h e r
amin o ac i d s .
Thus , the c h o i c e o f me t h o d f o r de te rmi n i n g S A is l imi t e d
to t h e v ar i o u s c h r omat o g raph ic m e th o d s .
W i t h paper a n d t h i n l a y e r
c � romatograph y and e l e c troph o re s is , amino acid c o nc e n t r a t i o n i s m e as u r e d
b y a c o l o u r r e ac t i o n o n th e s ta t i o n a r y phas e .
reag e n t f o r c o lour d e v e lopme n t i s n i n h yd rin .
r e l e as es
c o2 ,
meth i o n i n e .
The mo s t c om mo n l y us e d
H owev e r , th i s r e ac t i o n
t h e l o s s o f w� ich w o u l d aff e c t t h e S A o f 1 4 c l ab e l le d
T h us , ide n tif i c a t i o n o f t h e l ab e l l e d am i n o ac i d e i t h e r
inv o l v e s t h e r unning o f two ide n t ic a l c h romatograms , o f wh i c h o n e i s
dev e l o p e d a n d s o e n ab l e s l o c ati o n o f t h e amino acid p o s i t i o n o n the
s ec o n d , which can then b e u s e d for s c i n t i l l ation c o u n t ing , or
alte rnativ e l y , aut o r ad i o gr ap h y c an b e used to d e t e c t r ad i o ac t i v i t y .
A f t e r d e t e c t i o n of the p os i t io n r e qui red , t h at amino a c i d is e l u t e d
f r o m th e s up p o r t m e d ium a n d th e ac ti v i ty m e asured i n a s c i n t i l l a t i o n
c o u n t e r ( Me i s t er , 1 9 65 ) .
Th e p r o b l e m of r unning t w o c h r o m a t og r ams c a n b e
50
avoided b y th in l a y e r c h roma t o g raph y o f th e dansyl d e r iv a t i v e s o f amino
acids ( A ir h a r t e t a l . , 1 9 73 � s ince their c o nc e ntrat i o n c an b e m e as u r e d
b y f l u o r e s c e n c e und e r UV l i gh t ( We b e r , 1 9 5 2 ) .
Howe v e r , o n l y s m a l l
v o lumes o f s amp l e c a n b e app l i e d t o p a p e r o r th in l a y e r p l at e s and th us
t h e s e m e t h ods are not s u itable f o r anal ysis o f meth i o n i n e SA i n the
present s amples .
Th e u s e o f i o n-e xc h ange or gas c h romatogr aph y to me as ur e S A s h as
th e adv a n t age that t h e amino acids are e l u t e d from the s ta t i o n a r y p h a s e
p r i o r t o c o l o u r dev e l o pmen t .
T h e e l u e n t c a n th ere f o re b e s p l i t i n t o
t w o s t r e ams , o n e f o r c o l o ur d e v e l op m e nt , and one f o r radio ac t i v e
counting .
Thus , s i n c e s tandard ami n o a c i d analysers w e r e a v a i l ab l e , i t
w a s dec id e d t o meas ure meth i o n i n e S A using t � e s p ec i f ic S-�m i n o ac i d
proc e d u r e of J e p p s s o n a n d Karlsson ( 1 9 7 2 ) .
T h e Bec kman anal y s e r at
A p p l i ed B i och e m i s t r y Divisi o n , D . S . I . R . , wh ich was u s e d for r o ut i n e
amino ac i d anal ysis c annot b e read i l y mod i f i e d to c o l l e c t p a r t o f t h e
c ol u mn e f f l u e n t f o r c o unting o f rad i oac tiv i t y .
Thus , a J e o l ( J L C 6 A H )
amin o ac i d anal y s e r at Ruakura A n imal R e s e arch Centre , H am i l t o n was
used f o r the m e as u r e m e n t of methionine S A s .
C.
D i s c r epancy b e tween ami no acid anal y s e r s
Th e c h romato g r a p h i c procedure o f J e p p s s o n and K ar l s s o n ( 1 9 7 2 ) ,
.
wh i c h was u s e d f o r m e as uring m e th i o n i n e SA o n the J e o l , o n l y s e p ar a t e d
t h e f o l l owing amin o acids :
v al ine , c y s te i n e , meth i o n i n e , i s o l e u c i n e ,
leuc i ne , t y r o s ine and ph e n y l a l an i n e .
Th e r e f ore a c ompl e t e
p h y s i o l o g i c al f l u i d analysis f o r d up l i c a t e p l asma s am p l e s f o r each
sheep f r om each r e p l ic ate was als o run .
g iv e n
in
T h e details o f E xp t . 1 are
Chapter 3 ( E x p t . 1 ) , b u t t h e basic design inv o l v e d t h r e e
treatments ( u n t r e a t e d s ilage , formaldeh yde-treated s il a g e , a n d u n t r e a t e d
T ab l e
A c ompa r i s o n o f th e o b s e r v e d pl asma meth i o n in e
2.1
c o n c e n tr a t i o n s as m e a s u r e d o n a J e o l o r a B e c kman
( i n par e n th e s e s ) a m i n o a c i d analys e r .
R ep.
Untreated
1
s il a g e
Treated
s il a g e
Untreated
R ep.
1
R ep. ;3
2
2
9 . 6±1 . 7 ( 7 . 5 )
6 . 7±0 . 7 ( 6 . 0 )
1 1 .. 2;t1 . 6 ( 9 . 6 )
9 . 6±1 . 9 ( 7 . 2 )
1 2 . 4±2 . 0 ( 1 0 . 3 )
1 3. 7;t2 . S ( 9 . 4 )
1 1 . 8±1 . 2 ( 1 9 . 0 ) 1 1 . 7±2 . 1 ( 1 6 . 4 )
s il a g e
(i-Jm o l e/1 )
20. 0;t1 .. 2 ( 25. 5 )
+ met
1
2
f o r e x p e r i m e n t a l de s i gn ,
+
see Ch apter 3 ,
Expt .
1.
t h e S E o f the mean .
Table
2.2
A
c ompa r is o n o f th e c o r r e c t e d pl asma meth i o n i n e
c o n c e n t r a t i o n f o r t h e J e o l ami n o a c i d a n a lys e r , w i t h th e
o b s e r v e d v a l u e f r o m t h e B e c kman a m i n o ac i d an alys e r (i n
pa r e n t h e s e s ) .
R ep.
Untreated
9.2
T re a ted
9.2
s il a g e
s il a g e
Untreated
1
12.1
s ilage
1
R ep.
(7.5)
5 . 5 ( 6. 0)
1 1 . 3 ( 9. 6 )
(7.2)
1 2. 8 ( 1 0. 3 )
14.5 (9.4)
( 1 9.0)
1 1 . 9 ( 1 6.4 )
22 . 6 ( 25 . 5 )
2
Rep.
3
+ met
1
v al u e s f r o m Jeol a m i n o ac i d anal y s e r , c o r r e c t e d w i th
equation :
Y = 1 . 281
X -
3 . 062
wh e r e
Y
th e r e g r es s i o n
i s t h e c on c e n t ration m e a s u r e d o n
th e B e c km a n
A m i n o ac i d c o n c e n t r ati o n (Hm o l e/L )
200
o n B e c kman
1 60
1 20
+
+
+
Y
80
+
+
+
=
1 . 2 8 1 X - 3 . 062
r = 0 . 99
40
0
40
80
1 20
1 60
Amino ac i d c o nc e n t r a t i o n (�m o l e/L )
on Jeol
F ig. 2 . 4
R egress i o n r e l a t i o n s h ip b e t w e e n plasma amino ac id
c on c e n t r a t i o n s m e as u r e d on a J e o l or B e c kman amino
ac i d analys e r .
200
51
s il age
+
i n t r ap eritoneal ( I / P ) m e th io n i ne i n f us i o n ) , w i th t h ree s h e e p
p e r t r e a tment i n a L a t i n Squ a re d e s i g n, giv i n g n i ne s e p ar a t e sampl es .
Th e p h y s i o l o g i c al f l uid anal y s i s was c ar r i e d o u t on a B ec kman 1 2 0C
ana l ys e r u s i n g a two c o l umn p r o c edure .
Howe ver , th e c o nc e n t ra t i o ns of
t h e s ix amino acids ( c y s te i n e c o n c e n t r atio ns we r e t o o l o w t o m e as u r e )
meas u red o n b o th anal y s e rs d i f f e r e d marked l y with th e i ns t r um e n t us e d .
T h e v al u e s o b t ained on the B ec kman we r e h i gh e r t h an t h o s e o b ta i n e d o n
th e J e o l , f o r a l l amino ac i d s exc ept methio n i ne in th e p l a s m a o f s h e e p
on t u o o f the tr e at ments .
A r e g r e s s i o n r e l ations h i p w a s f it t e d b e tw e e n
the B e c kman v a l ues ( Y) a n d t h e J e o l v al ues ( X ) , us i n g the c o nce n tr a t i o n s
o f each o f th e six ami n o ac i ds , from e ach o f th e n i n e samp l e s a n al ys e d .
This r e g r e s s i o n r e l a t i o n s h i p is g iv e n in
Fig. 2.4.
I
T h e correlation
c o e f f i c i e n t o f 0 . 99 ind icat e s a c o ns t an t r e l ationsh i p b e t w e e n t h e two
analys ers , independent o f th e amino acid c o mp ar e d .
H o wev e r , th e
c o nc e n t r a t ion o f me thio nine i n th e pl asma o f s h e ep r e c e iv i n g I / P
me th i o n i n e inf usion did n o t f ol l o w th e gene r al t r e nd ( Tab l e 2 . 1 ) .
This
is n o t ap p a r e n t f r o m F i g . 2 . 4 , owing t o th e low c onc e n trat i o n o f
m e th i o n i n e in s h e e p plas m a .
C o r r e c t i o n of plasma me t h ion i n e
c o nc e n t r a t ions us i n g t h e r e g r e s s i o n r e l at i o nship ( Y
=
1 . 281 X - 3 . 062 )
( Ta b l e 2 . 2 ) p r oduc e d l i ttle c h an g e in th e ac tual v al u e .
Th u s , t h e
meth i o n i n e c o n c e n t r a t i o n in t h e p l asma of s h eep fed t reate d and
u n t r e ated s il age r e mai n e d h i g h e r from th e Jeol than f rom t h e B e c kman ,
this d i f f e re n c e b e i ng o n l y s l i gh t l y g reate r than th e S E .
B y c o n t r as t ,
the p l as m a me t h i o n i ne c o nce n t ration i n s h e e p rec e i v i n g I / P inf u s io n o f
met h i o n in e , r e mai n e d h ig h e r o n th e B e c kman t h a n t h e J e ol .
Thus , t h e r e
appe a r t o be t w o s e parate a s p e c t s of t h e d i f f e r e n c e s be twe e n
anal y s e r s .
Th e f i r s t i s a d ifference i n a l l ami n o a c ids w h ich c an b e
d es c r ib e d b y t h e r e gr e s s i o n r e l a tionship i n F i g . 2 . 4 .
The s e c o n d i s a
Tab l e 2 . 3
T h e e f f ec t o f f r e e z e-dryi ng on plasma am i n o ac i d
c o nc e n t r a t i o n (I�mo l e/1 )
1
Untreated s i l age
( Rep. 2 )
Untreated sila e
+ m e t ( R ep. 1
B e f o re
After
Before
After
V al in e
85.4
84 . 0
111.6
1 04 . 4
M e t h i o n i ne
18.3
1 8.4
4.9
5. 6
I soleucine
. 40 . 8
45. 7
42.4
42 . 8
L e uc i n e
69 . 0
75.2
65. 1
60 . 0
T yr o s i n e
27.4
30 . 6
30.4
32.4
P h e n y l al an i n e
34 . 9
41 . 0
21 . 9
24 . 3
T ab l e 2 . 4
C o mpa r i s o n o f c o l o ur f ac t o rs, denoting th e e f f ic i e ncy
of n i nhyd r i n reac t i o n f r o m J e o l a n d B ec kman amino ac i d
a nalysers
B ec kman
J e o l 1 9 74
J e o l 1 9 76
V al i ne
1 . 05
+
. 028
0 . 92
+
. 048
1 . 44 +
Methionine
1 . 02
+
. 03 9
0 . 87
+
. 046
0 . 84
+
I s o l eu c i n e
0 . 84
+
. 1 OB
0 . 82
+
. 0 57
1.16
+
. 1 07
L e uc in e
1 . 01
+
. 04 6
0 . 7 5 + . 056
1 .02
+
. 069
Tyrosine
1 .01
+
. 04 0
0 . 79 + . 0 50
1 . 01 + . 0 8 9
P h e n y l a l anine
1 . Q1
+
. 05 1
0 . 82
1 . 01
+
. 03 1
+
•
•
118
071
. 076
52
tre atment e f f e c t o n t h e d if f e r e n c e in me t h i o n ine c on c e n t r a t i o n b e tw e e n
analysers .
To t r y and f i n d t h e reasons f o r th e s e d i fferenc es , t h e f o l l o w i n g
p oi n ts of p r o c e d u r e w i l l b e d is c ussed r e l a t i v e t o e ac h amino ac i d
anal ys e r :
( 1 .) method
of
s amp l e pre p aration ,
( 2 ) s amp l e s t orage t ime ,
( 3 ) meth o d o f c a l c u l at i n g results ,
( 4 ) d i f f e r e n t c h r o m a t o g raphic c o nd it ions .
( 1 ) Meth o d o f s ampl e pr eparati on :
Th e v o l u m e o f s am p l e a p p l i e d t o t h e
r es i n on t h e J e o l a m i n o ac id a n a l y s e r is a c o ns tant D . Bml .
T h e s am p l e s
f o r anal y s i s o f m e t h i o n i n e SAs were the r e f o re firs t f r e e z e-d r i e d , and
then re-c o ns t i t u t e d in 1 ml of D . D 1 N H C l t o enable the a n a l y s i s of a
l arger v o l ume o f p l asma .
were not f r e e z e-d ri e d .
The s amples a p p l ied t o t h e B e c kman c o l umns
Howev e r , anal y s i s of duplicate s am p l es b e f o r e
a n d a f t e r f re e z e-d r y i n g indicated no mar k e d e f f e c t o f t h i s t r e a t m e n t
( Table 2 . 3 ) .
( 2 ) Time o f s t orage :
.
A l l p l asma samp l e s were s t o r e d a t -20 0 C f o r at
l e as t s i x m o n th s p r i or t o anal y s is �
Th e c omplete p h y s i ol o g i c a l f l uid
anal ys is ( B e c kman ) was c o nduc t e d s ome months after t h e m e as ureme n t o f
methionine SAs ( Jeol ) .
H ow e v e r , the c o n c e ntrations me as ured o n t h e
J e o l were l ow e r t h a n t h ose meas ured o n th e Bec kman , a n d t h u s l o s s o f
amino acids d u r i n g s t o r age c an n o � expl a i Q th e ov e r� l l c o n c e n t r a t i o n
d i f f e r e n c e b e t w e e n ana l ys ers .
S torage l o s s c o uld e x p l a i n t h e s l i gh t
d i f f e re nc e b e t w e e n analysers i n the meth i o nine c o nc e n t rations i n th e
p l asma o f s h e e p f e d t r e ated and untre a t e d s i lages , s i n c e meth i o n i n e h as
b e e n s h own t o b e p articular l y s u s c e p t i bl e to degrad a t i o n o n s to r a g e .
53
H o w e v e r , the m e th i o n ine c o n c e n tr ation in the pl asma of s h e e p
rec e iv in g I/P meth i o ni n e w a s h i g h e r o n th e Bec kman t h a n t h e J e o l , and
c a n n o t th e re f o r e be at t r i b u t e d to loss with s t o r ag e .
( 3 ) � alc u l at i o n of r e s u l ts :
Reac tion o f amino acids with n i nh yd r in is
r e c o rded as a s e r i e s o f p e a k s , the areas o f which r e p r e s e n t the
c o n c e ntrat ion o f the c o r r e s p o n d i n g am i n o ac i ds .
C al c u l at i o n o f t h e
are as und e r t h e p e a ks was c a r r i e d out b y a comp u t e r f o r th e me t h i o n i n e
SA
analys i s ( J e o l ) and b y h an d f o r th e ph y s i o l o g ic al f l u i d anal y s is
( B e c kma n ) .
R e c al c u l at i o n b y h a n d of r an d o m l y c h os e n c om p u t e d anal yse s
f ro m the J e o l , i n d i c at e d n o c o ns i s t e n t d i f f e r e n c es b e tween c o m p u t e r and
h a n d c al c ulatio n .
Calc u l a t i o n o f the amino ac i d c o n c e n t rat i o ns f rom th e
p e a k are a , inv o l v e d the u s e o f a c onv e rs i o n f ac t o r , r e l ate d to t h e a r e a
o f a n i n t e rnal stan dard ( �l e u c i n e ) .
Norl euc i n e ( N L ) ( 0 . 1 00 jJ m o l e ) w a s
app l i ed t o t h e column w i th e ac h s ampl e , a n d also w i th a s t andard m i x of
am i n o a c i ds .
r e q u i red .
This s t andard mix c o ntained 0 . 1 00 � mo l e of e a c h amino ac i d
T h e rel a t i o n s h i p b e tw e e n t h e peak ar e a f o r a s p e c i f i c ami n o
ac i d in th e s t anda r d a n d t h e a r e a und e r th e p e a k f o r N L , g a v e t h e
e f f i c i e nc y of ninh y d r i n reac t ion f o r e ach amino ac i d , r e l a t i v e t o NL .
Th e r a tio of amino acid area t o N L are a i n the s am p l e c o u l d th e n be
d i v ided b y the rat i o i n the s t andard , and thus c o r r e c t e d for the
e f f ic i e n c y of ninh y d rin reac t i o n f o r e ac h amino ac id .
The r a t i o s
o b t a i n e d f rom c h romatog r a ph i n g a s t and ar d mix o f the s ix a m i n o ac ids
r e l e v ant t o th i s d is c us s i o n , s h o u l d b e c l o s e to 1 . 00 .
o b t a in e d f o r e ach anal y s e r a r e p r es e n t ed i n Tab l e 2 . 4 .
The a c t u a l v a l u e s
Th e v al u es
ob t a i n e d a t th e time o f anal y s is o n the J e o l ( 1 9 74 ) w e r e g e n e r a l l y l o w e r
th a n thos e o b t ai n e d on t h e B e c kman , a n d l a t e r v al ues o b tained o n the
Je o l .
Th e d i f f e r e n c e i n c o l o u r f ac t o r s f o r the Jeol as measured i n 1 9 74
54
and 1 9 7 6 , toge ther with the l o w e r conc e n trat ions d e t e r m i n e d on t h e J e o l
t h a n t h e B e c k m a n s u g g e s t s th at t h e c o l o u r f ac tors u s e d i n 1 9 74 m a y n o t
h a v e been v al i d .
The d i f f e r e nc e s b e t w e e n the t w o J e o l s t andards a r e
n o t c o ns i s t e n t for all the amino acids , a n d t h u s can n o t e x pl a i n th e
r e g r e ss i o n r e l a t i o n s h i p f o u n d f o r all amino ac ids b e t w e e n t h e two
an alys ers .
Howev e r , th e c o l o u r f ac to r for me th ionine was not
s ig n i f i c a n t l y d if f e rent as m e a s u r e d i n 1 9 74 o r 1 9 76 , and t h us the
m e th i o n i n e SA meas u rements a p p e a r to be v a l i d , at l e as t f o r th e s h e e p
o n t r e ate d a n d unt r eated s i l a g e .
( 4 ) C h ro m a togr aph ic pr oc edur e :
T h e J e o l analys e s us e d two b uf f e r s o f
p H 2 . 82 a n d 3 . 6 6 , w i th n o t e m p e r a ture c h ange .
T h e B e c kman anal y s e s f o r
,
the ic idic and neut ral amino ac i d s u � e d b u f f ers of p H 3 . 2 5 and 4 . 3 0 ,
°
with a tenperat ure change f r o m 3 0 - 6 2 . 5 C .
Th us th e e l u t i o n t imes f o r
t h e i nd i v i d ual amin o ac i ds d i f f e r e d b e tween t h e two i n s t r um e n ts .
Henc e ,
i f u n i d e n t i f i e d ninh ydrin-po s i t i v e c o m p o unds were al s o p r e s e n t i n t h e
p l a s c. a s amples , th e y migh t h av e b ee n e l u t ed f ro m th e t w o i n s truments in
d i f f e re n t r e g i o ns of th e c h r o m a t o g r ap h .
Th e elution of s uc h a c ompound
with meth i o n in e , i n plasma f r om s h e e p r ec e i v ing I / P m e th i o n i ne , o n the
B ec kman , m igh t exp l a i n t h e h i g h e r c o nc entration meas u r e d f o r s h e e p
r e c e i v i n g th i s treatment , o n t h e B e c kman c ompared t o t h e J e o l .
O x i d at i o n
o f t h e S-amino ac ids p r i o r t o analys is d i d n o t r e v e a l a n a d d i t io nal
peak i n p l ac e of me thionine .
I n add i t i o n , attempts t o s e p a r a t e and
r e-c h r omatograp h th e me t h i o n i n e p e a k f rom the B ec kman ana l y s is w e r e
uns uc c ess f ul , part l y as a r e s u l t o f t h e v e r y l o w c o n c e n t r a t i o n o f
meth i o n i n e i n t h e p l asm a .
T h us , t h e p resenc e o f a n i m p ur i t y
c o n t r i b u t i n g t o th e meth i o n i n e p e a k i n th e p h ys i o l o g i c al f l u i d a n a l y s i s
f ro m s h e e p r e c e iving I / P m e t h i o n i n e i n f u s io n , remains a p o s s i b l e
e x p l a na t i o n .
55
No d e f i n i�e c o nclus ions on t h e d i s c re p ancy in a m i n o a c id
conc e n trations as d e te rmined on t h e B e c kman and Je ol amino ac i d
anal y s ers can b e g i v en .
H o w e v e r , the most l i k e l y ex p l a na t i o n f o r t h e
dif f e re nc e in m e t h io n i n e c o n c e n t r a t i o n s ap p e ars t o b e part l y d u e to a
stor age e f f ec t , and p a r t l y t o an u nid e n t i f i e d c omp o u n d c o n t r i b u t i ng t o
th e �eth i o nine c on c entr ation i n s h eep r e c e i v ing I /P i n f us i o n as
dete rmine d on the B ec kma n .
H e nc e th e c o nc e ntrations d e termihed f o r
meth i o n i n e SA a p p e a r t o b e v alid .
Th e c o nc en trations of t h e o t h e r
amin o ac i d s w e r e d e termined t o s t u d y t h e e f f ec t of th e v a r i o us
treatme n t s , a n d th u s c o mpar i s ons b e t w e e n s amples d e t e rmin e d o n t h e s ame
anal yser are also v alid , with th e q u a l i fic ation that th e m e th i o nine
peak may not be pu r e .
2.4
Meas u reme n t o f mic r o b i a l pro t e i n synth esis
Seve ral m e th o d s for measuring the p r o p o rtion of mic r o b i al -N in th e
total pro tein a v a i l ab l e to the animal at t h e duo denum h a v e b e e n
pro p o s e d .
D i r e c t e s t imation may b e ac h i ev e d b y meas u r ing t h e d e gr e e o f
inc o r p oration o f a s pecific c omp o n e n t i n t o mic robial p r o t e i n ,
T h is
estimation r e q u ires ac c urate meas u r e m e n t o f the c o nc e ntr a t i o n o f t h e
comp o n e n t c h o s e n, i n a pure s ampl e o f r umen mic ro-o r g a n isms .
Th is
samp l e must n o t b e c o n t aminated b y p l an t m a te r ial and mus t b e
r e p r e s e n t a t i v e o f t h e micro-organisms p r e s e n t in t h e rumen .
Separation
i s a c h i e v e d b y dif f eren tial c entr i f ug a tion o f a l a r g e v o l u m e o f d i ge s t a .
R e s u s pens ion and r e c e n t r i f u g ation o f t h e b ac terial l a y e r i s n e c e s s a r y
to e n s u r e pu r i t y , and examination w i t h a m i c ros c o p e t o c h e c k f o r
c o n t aminatio n with plant material s h o u l d a l s o b e mad e .
mus t be d is c a r ded ,
I m p ur e s amples
Th e s eparation o f a pure samp l e i s a d i f f i c u l t , b u t
es s e n tial par t o f a n y m e th o d f o r m e as u r i n g microb ial p r o t e i n s y nt h e s is .
56
T h e micro b es are u s u a l l y s e p arated from r u m e n d i gesta , s i n c e i t i s m o r e
qiff icult to o b tain an acc e p tab l e prepar at i o n o f d u o d e nal d i ges t a, d u e t o
t h e s mall s i z e of p l ant particles ( Ul ya t t e t al . , 1 9 7 5 ) and to l y s i s o f
micr o-or g a nis m s i n t h e acid c o ndi tions o f t h e ab omas um ( B e ev e r e t a l . ,
1 974 ) .
T h e s ample s h o u l d also b e prepared f rom fresh dige s t a r a t h e r
t h a n dige s t a w h ich h as b e en f r o z e n , w i th c o nsequent l ys i s o f m i c r o b i a l
c el l s .
Th e c once ntration o f th e marker i n t o t al d i g e s t a is a l s o m e as u re d ,
and t h us t h e p r o p o r t ion of microb ial-N in t o tal diges t a-N i s g i v e n b y : .
conc e n tration o f marker /
g
diges ta-N
c o n c e ntra t ion o f m arker /
g
mic r o b i al-N
Th e main mar k e r s w h ich have b een used are :
( 1 ) 2 : 6 d i am i n opime l ic acid ( OA P ) ,
( 2 ) nucl e ic ac ids ( N A ) ,
35
( 3 ) l abe l l e d S g r o u p s ( s ) .
( 1 ) O i ami n opim e l ic acid :
Th e pres ence o f t h e amino a c i d O A P i n
b a c t e r ial c e l l wal l s was f i r s t r e c o r d e d b y Work ( 1 9 5 1 ) .
S ynge ( 1 9 5 3 )
then reported that OAP was n o t p r e s e n t i n p l ant mate r i al a n d th i s l e d t o
the use o f OAP as a mar k e r f o r r umen m i c r o b ial p r o te i n sy n t h e s i s .
How e v e r , O A P i s n o t p r e sent in p r o t o z o a ( R o s e , 1 9 6 8 ) and t h us o n l y
b ac t erial p r o t e i n s ynth e s is i s measured .
I n additio n , th e r e i s
c o n s i derab l e v ariat ion i n t h e c o nc e n t r a t i o n o f O A P b e tween b ac te r i al
s p e c ies ( We l l e r e t a l . , 1 9 58 ) .
Furth e G O A P is ab s e n t from s ome b a c t e r i a
( R os e , 1 9 6 8 ) , thus , a h i gh p r o l i f e r at i o n o f th e s e bac teria c o u l d l e a d
to a n und e res t imat i on o f s y n th e s i s .
( 2 ) N uc l e ic ac ids :
Sm i t h and Mc A l lan ( 1 9 7 0 ) s h owed t h a t t h e amo u n ts o f
NAs e n t e r i n g t h e s mall intes tine o f r um i na n ts b o r e l i t tle r e l a t i o n t o
dietary i n takes o f NAs .
Th e y o b s e r v e d a l m o s t c o m pl e t e b r e a k d o w n o f
57
d i e t a r y NAs i n the r umen, and c o n c l uded that duode nal NAs w e re almost
ent i r e l y m ic r o b ial i n o r i g i n .
S m i th ( 1 9 75 ) pointed o u t t h a t th e
v ar i a tion i n R N A c o nc e n t r a t i o n o f b ac t e r ia was much l o we r t h an t h e
varia tion i n O A P c o n c e n t r ati o n .
T h e c o nc e n t r a t i o n o f R N A also appeared
t o be l e s s va r i ab l e th an the c o nc e n tration o f D N A ( Mc A l l an and S mith ,
1 9 7 2 ) and thus the use o f R N A as a mar k e r was p r e f e r r e d to DNA o r t o t a l
NAs .
Hriwe ver ; t h i s meth o d ass umes c o m p l e t e d e g r a d a t i o n o f d i e t a r y N A
i n t h e r ume n .
This h y p o t h es i s was t e s t e d b y a d d i t i o n o f p u r e N A s t o
t h e r umen ( Sm i t h and Mc A l l an , 1 9 7 0 ) b u t t h e f a t e o f d i e t a r y NAs i n
i ns o l ub l e plant mate rial h as not b e en c l ear l y e s t a b l i s h e d .
( 3 ) L ab e l l e d S :
Nader and Walker ( 1 9 7 0 ) s howed that b ac te r ial
synthesis o f S- ami n o
acids was m o re - important t h an d i re c t i nc o r p o r a t i o n
o f th e p r e formed S-amino
ac ids i n t o b ac ter ial p r o t e i n .
�
was t h e b a s is o f meth ods u s i n g inf u s i o n of N a 5 s o
4
mic r o b i al pr o t e i n s y nth e s is ( B e e v e r et al . , 1 9 74 ;
1 97 5 ) .
Th is f i nd i n g
to meas u r e rumen
Wal ker and Nader ,
H o wev e r , other w o r k e r s h a v e s u g g e s t e d th a t ami n o a c i ds per s e ,
including
th e S- amino
p r o t e i n ( L and i s , 1 9 63 ;
acids , are an important p r e c u r s o r o f mic r o b i a l
N o l a n e t a l . , 1 9 76 ) .
Thus , f u r t h e r w o r k
ap p e ars to be n e c e s s ar y b e f o r e a n evaluation of t h e v a l i d i t y of
.
35
t e c h ni q u e s us i n g
S l a b e l c a n b e made .
B e e v e r e t al .
( 1 9 74 ) u s e d a c on tinuous inf u s i o n o f N a
label the meth i onine s y n thes i s e d i n microbial p r o t e i n .
� 5 so4
to
Th e y sampled
duode nal dige s t a and m e as ure d SA o f m e th io n i n e i n t o t a l di g e s t a and i n a
sample o f mic r o bes , prep ared f r om d uo denal d iges t a .
Knowledge o f th e
p e r c entage of m e t h i o n i n e in t h e m i c r o b e s , and in total d u o d e nal dige s t a
allowed c alcul ation o f t h e p r o p o r t ion o f mic r o b i a l p r o tein a s d e sc r i b e d
ab ov e .
H owev e r , s e para t ion o f a pure and r e p re s e n ta t i v e s am p l e of
m ic r o bes from d uode nal d i ges t a is a v e r y d i f f ic u l t p r o c e d u r e , as
Table 2 . 5
T r e atment
C a l c u l a t i o n of m i c ro b i a l pro t e i n synthesis in s h e ep f e d
35
s i l age , u s i ng
S m ar k e r (f o r E xpt . 1 ) .
3 5s S A i n
D ige s t a
{dQmL9
wet wt . }
3 5 s S A i n R umen
Mic r o b i a l V o l u rr e
s {dpmL�9
ill
21
T o t al
F l ow o f
Amino
Ac ids
{gZd ay}
T urnover
C o ns t ant
{ d- 1 }
Microbial
Protein
Flow
(gLday}
1 . 95
29 . 2
47.8
U n t re a t e d S i l age
Rep.
1
382
11.2
Rep.
2
378
4. 5
4.7
1 . 94
62. 2
46. 2
Rep.
3
926
8. 1
1 .6
2.18
32. 4
32. 8
T r e ated S i l age
Rep.
1
777
12.3
4.2
0 . 58
1 2.5
57. 6
Rep .
2
399
5.7
4.0
2 . 42
55. 1
67. 8
Rep.
3
565
4.6
5.6
1 . 93
1 07 . 8
72. 0
U n t r e at e d S i l age
+
Meth ionine
Rep.
1
562
7. 3
1 . 92
39.6
65 . 2
Rep.
2
743
6.4
1 .35
3 0. 6
52 . 9
Rep.
3
428
3.2
2.18
64 . 0
33. 0
2.7
1 For experimental design , see Chapter 3 , Expt . 1
58
disc us s e d e ar l i e r , a n d t h e determi n a t i o n o f meth ionine S A s i s a c om p l e x
a n d tedious p r o c e s s .
Wal k e r and Nader ( 1 9 7 5 ) al s o m e as u r e d microbial p r o t e in s y n th e s is
�
b y c o n t in uous i n f u s i o n of N a 5 so •
4
Th e y m e as ured
c o n tents and in a p u r e mic rob ial p r e p ar a t i o n .
35
s S A i n t o t a l r umen
The t u r n o v e r c o ns ta n t
.
t a was e a 1 c u 1 a t e d f r a m th e r a t e o f d e c r e as e l. n S A
f o r 3 5 S l. n rumen d lges
a f t e r termination o f the l a b e l l e d i n f u s i o n .
Rumen v o l ume was a l s o
d e t e r mine d a n d h e n c e to tal mic r o b i al-S f l o w/ day was c a l c ul a t e d as :
35
s S A / g wet w t . rumen c o n t e n t s x r um e n v ol ume ( 1 ) x t u r n ov e r c o ns tant
35
s S A / � g m i c r o b ial-S
Wal k e r and N a d e r ( 1 9 7 5 ) then as s u� e d a c o ns tant N : S r a t i o i n m i c r o b ial
p r o t e i n of 1 3 : 1 , and thus c al c ul a t e d m i c r o b ial protein f l ow p e r day .
Howev e r , t h e r e is dispute o v e r th e c o n s t a n c y of th is r a t io , and v al ue s
r a n g i n g f r om 1 0 . 7 : 1 t o 2 1 . 1 : 1 h av e b e e n r e p o r ted ( H e n d e r ic k x e t al . ,
1 9 72 ;
B i r d , 1 9 73 ) .
A n o t h e r s o u r c e o f e r r o r in th is m e t h o d i s t h e
c o n t r i b ut io n o f r e c yc l ing of l a b e l l e d S t o and w i th i n � th e r u m e n d u r ing
th e d e c ay per i o d .
This would give a low t u r nov e r c o n s t a n t and h en c e
u n d e r e s t i mate p r o t e in s yn th e s i s .
O r D . J . Wal k e r had been wo r k i n g at A p pl ied B i o c h e m i s t r y D iv i s i o n ,
o . s . I . R . j u s t p r i o r to th e s tart o f t h e p res ent w o r k , and h e n c e th e
m e th od o f Wal ker and Nader ( 1 9 7 5 ) was u s e d t o e s t imate m i c r o b i al p r o t e i n
f l o w d u r i n g E x p t . 1 ( C h ap t e r 3 ) .
The r e s u l t s obtai ned a r e r e p o r te d i n
T ab l e 2 . 5 to h igh l i gh t s ome o f th e p o s s i b l e s ources o f e r r o r i n t h i s
meth o d .
F o u r separ a te f i gures a r e r e q u i r e d for c al c u l a t i o n o f m i c r o b i a l
p r o t e in f l o w b y this meth o d .
w i l l affec t t h e f i nal resul t .
A n e r r o r i n any o n e o f t h e s e m e as u r em e n t s
T h e r e s u l ts s h owed c o n s i d e r a b l e v ar i a t i o n
w i th s ome v a l ues b e ing h i gh e r t h a n th e t o tal f l o w of p r o te in p e r d a y ,
-
59
and h ence b e i n g imp o s s i b l e .
Ove res timation o f m ic r o b i al p r o t e i n
s y n t h es is m a y be d u e to imp ure m i c r o b i a l p r e para tio n s whic h w o u l d g i v e
a l o w v a l u e f o r th e S A o f t h e m i c r o b e s a n d h e n c e a h i gh v a l u e f o r t o tal
r ume n- S, a nd t h us m i c r o b ial pro t e i n s yn th e s i s .
The v al u e for m i c r o b ial
s y n t h es is on t r e a t e d s il age R e p . 1 is c o n s i d e r a b l y l o wer than any of
the o th e r s , r e f l ec t i ng a l o w turnover c o ns t a n t f o r t h is a n imal .
This
m a y i nd ic ate a h i g h d e g r e e o f S r e c yc l ing i n th is a n imal .
Vario us c omparisons of mic r o b ial p ro t e i n s y nth e s is as e s t i m a t e d b y
s ome o f t h e s e meth o d s w i t h i n the s ame expe r i ments h av e b e e n made ( H o g an
and Weston , 1 9 72 ;
H arr ison e t a l . , 1 9 73 ;
a n d S m ith , 1 9 7 4 ;
W a l k e r and Nad e r , 1 9 7 5 ) .
c on s ider a b l e v aria tion .
B e ev e r e t a l . ,
1 9 74 ;
Mc A l l a n
T h e res u l ts s h owed
I
H e n c e , s ince t h e s e meth ods a l s o i n v o l v e c o mpl e x
and t im e-c onsumin g p roc e d u r e s, and use a s s u m p tions wh i c h m a y not b e v al i d ,
no att emp t t o e s t im a te m ic ro b ial p r o t e i n s y n the s is w a s made in E x p t . 2 .
C H A P TER
THE
EFFECT
OF
D I E TS
F O RMAL I N-TR E A TME N T
ON
TH E
D I GEST I ON
3
AND
OF
S U P P L EM E NTAT I O N
SIL AGE
BY
S� E E P
OF
SILAGE
60
3.1
I n t r od uc t1on
N um e r o u s workers h av e r e p o r t e d low intakes o f s i l a g e c ompared t o
h ay made f r o m the s ame pas t u r e ( Murdoc h , 1 9 64 ;
T h oms o n , 1 9 6 6 ;
Wellman , 1 9 6 6 ;
C amp l i n g , 1 9 66 ;
B is h o p and Kentish , 1 9 7 0 ) . · H o w e v e r ,
w h i l e ac e tate , b u t y r at e , ammo n i a and qmi n e s h a v e a l l b ee n s u g g e s t e d as
p o s s i b l e i n t a k e depressants ( H a r r is and R aymond , 1 9 63 ;
1 9 64 ;
Mc l e o d e t al . , 1 9 70 ;
Neumark et al . ,
W i l kins e t al . , � 9 7 1 ) , n o s i n g l e c om p o n e n t
o f s il a g e h as been f o und to b e a c ommon f ac t or i n l i m i t i n g i n t a k e .
B ar r y e t a l .
( 1 9 7 3 ) s t u d i e d th e intake o f pas t u r e s ilage b y s h e ep ,
a n d r e p o r t e d an inc r e ased i n t a k e i n r e s p o n s e to b o th i n t r a p e r i t o n e a l
( I / P ) meth i o n ine, and f o rmal in-tr eatment of s ilage .
F o rmalin
( f ormil d e h y d e + fo rmic ac id )- t r e a tme nt o f s il ag e was i n t r o d u c e d as a
means of p r o tec ting th e h e r b age p r o t e i n f r o m degrad a t i o n d u r i n g e n s i l i n g ,
and thus o f increasing th e p r o t e i n intake o f th e a n i m a l .
S ince
f o rmal d e h y d e h as a l s o b e e n a p p l i ed t o p r o t e ins to p r o t e c t t h e m f r o m
d e gr ad a t i o n in the r umen ( F e r gu s o n e t al . , 1 9 67 ) , f o rmal in�treatment of
s il age m i gh t also b e expec t e d t o i n c r ease th e f low o f p r o t e i n into th e
d u o d e n um .
H e nc e t h e f l ow o f m e th io n i n e into th e d u o d e n u m w o u l d a l s o b e
i n c r e a s e d b y f o rmal i n-treatm e n t of s i l age .
Th e r e s u l t s o f B a r r y e t a l .
( 1 9 73 ) s u g g e s t th e r e f o r e , t h a t f e e d i n g u n t r e ated s il a g e to s h e e p may
adve r s e l y a f f e c t th e relat i o n s h ip b e tween th e p e rc e n t a g e o f meth i o n i n e
i n t o t a l amino ac i d s e n t e r i n g t h e d uod e n um , relat i v e t o th e p e r c entage
of m e th i o n i n e requi r e d b y th e t i s s u e s .
T h e f i r s t exper iment t o b e reported was th ere f o r e u n d e r t a k e n t o
s tu d y t h e e f f ec ts o f f o rmal i n-treatment a n d I / P m e t h i on i n e i n f u s i o n , o n
N d i g e s t i o n i n s h e e p f e d a b as a l s i lage d i e t .
Th e m a i n e f f e c t o f
f o rmal i n-treatment was t o i n c r e as e th e f l o w of ami n o a c i d s i n t o t h e
duodenum .
I n Exp t . 2 , oth e r m e th o d s of i n c r eas i n g t h e f l o w o f amino
ac i d s into t h e duode num w e r e s tu d i e d in s h ee p f e d s i l a g e .
Th e two
61
t r e a t me n t s c h os e n were :
( a ) s u p p l e m e n t a t i o n with f o r m a l d e h y d e- t r e a t e d
c as e i n and ( b ) i n f us ion i n t o th e r um e n , o f a readily av a i l a b l e e n e r g y
sourc e .
The f o rmal deh yde-t r e a t e d c as e i n w a s c h o s e n t o e n s u r e t h a t a
h i gh l e v e l o f am ino ac ids e n t e r e d th e d u o d e num .
The e n e r g y s o u r c e was
c h o s e n as a p o s s i b l e means o f i n c r e as in g microbial p r o t e i n s y nthes i s ,
s inc e t h e water s o l ub l e c a r b o h y d r a t e f r ac tion of s i l a g e i s l ow , c om p a r e d
t b t h a t o f h e rbage ( Mc Oonald e t a i . , 1 9 6 9 ) .
( s i l age a l o n e , s i l age + p r o t e i n and s il a g e
The t h r e e t r e atments
+
e n e r g y ) were aimed at
p r o d uc i n g th r e e d i f ferent f l o w rates of amino ac i d s i n t o t h e d u o d e n u m .
Th is w o u l d enab l e N diges t i o n and u t i l i s a t ion to b e c om p a r e d a t
d i f f e r e n t l e v e l s o f duodenal f l o w r at e , and thus give s om e i n d i c a t i o n
of o p t imum l e v e l s of s upp l emen t a t i o n .
Th e e f f e c t o f th e s e t h r e e t r e atments ( E xp t . 2 ) in inc r e as i n g t h e
f l o w o f m e th i o n i n e into the d u o d e n um was a l s o s t udied ( s e e f o l l o w i n g
C h a p t e r ) , a n d t h u s each o f t h e t r e atments w a s also g i v e n i n c o m b i n a ti o n
w i t h d uo d e n al i n f u s ion o f L-meth i o n i n e .
3.2
Meth ods
S h e ep :
M a t u r e New Zealand R omney w e t h ers were u s e d in b o th ex p e r imen t s .
T h e w e i g h t range was 28-44k g in E x p t . 1 a n d 3 1 -41 k g i n E xp t . 2 .
The
s h e e p w e r e ke p t i n metab o l i s m c r a t e s unde r c on t inuous a r t i f i c ial l i gh t ,
.
and at a cons tant temperature o f 1 8
±
0
2 C , with f r e e ac c es s t o wate r .
Each s h e e p was p r epared w i th a rumen c a n n u l a and a re-e n t r a n t c annula
in t h e p r oximal d uo d enum ( B r own e t al . , 1 9 6 8 ) .
F eed :
The two s i l ages f o r E x p t . 1 , u n t r e a t e d and f o rmal in-t r e ated ,
were p r e pared at I nv e rmay R es e arch C e n tr e , Mos giel .
Th e f o r m a l i n-
t r e a t e d s il ag e was p repared b y a p p l ic a t i o n of 40% ( w/ v ) f o rmaldeh y d e
( at a r a t e o f 4 . 7 6 t/ ton of f resh g r a s s ) a n d 8 5% ( w/w ) f o rmic ac i d ( at
a rate o f 1 . 0 0 1 / t o n o f f re s h grass ) , t o g r as s during h ar v es t i n g .
The
"
O e s ign o f Expt . 1 .
Tab l e 3 . 1
Sheep
,..."'
... """' b�rs .
R ep. 1
S ilage
R ep.
2
R ep. 3
U n t r e ated
786
647
1 1 57
Treated
731
786
441
647
731
786
Unt r e ated
Fig .
3.1
+
m e t h ionine
S egu e nc e o f s ampl i ng, E xp t . 1 .
P r e l im i n a r y
1 4 days
period for
ad j u s tm e n t
to feed .
7 days
B al an c e
c o l l ec t i o n .
4 days
S am p l i n g o f p l asma
and e x p i r e d a i r .
9 days
I nf u s i o n o f
C r E O T A and
c o l l e c t i o n of
d u o d e n a l d igesta
s amp l e s .
62
I
I
�
I
s il a g e used i n this experime nt was s e n t t o Palmers ton N o r th in b u l k ,
and w e i gh e d i n t o daily rations o f 3 . 2 k g o n arriv al . T h e s e r a t i o ns w e r e
°
s to r e d at -20 C un t i l o n e day p r i or t o u s e . R e f us a l s w e r e w e i g h e d
°
d ai l y a n d s to r e d at �20 C p r i o r t o d e t e rmination o f OM .
Th e s h e S p h ad
f re e acc e s s to salt licks during t h e e x p e r iment .
The s il a g e us e d in Ex p t . 2 was c u t and e n s il e d at M as s e y N o . 3
Dair y Un i t .
Th e silage to b e u s e d was c u t from t h e s t ac k , p ac k e d i n t o
°
d ai l y r a t i o n s o f 3 . 2 k g , a n d s t o r e d at -20 C a l l i n t h e s am e day .
Th e
d ai l y rat i o n s were remov e d f rom t h e f r e e z e r one day p r i o r t o f e e di n g .
20g o f a m i n e r a l m i x ( S ummit P r o d u c ts ) was applied t o t h e f e ed e ach
day .
°
Dai l y r e f u s a l s were dried at 1 1 0 C for 48h prior t o w e igh i n g .
Th es e wei gh t s were c o rrec t e d f o r t h e l o s s o f 1 B g v o l a t i l e acids per
1 00 g oven-DM ( v ol a t i l e acids m e as u r e d b y t o l uene d i s ti l l a t i o n ) , to
c alc ulate true OM i n take .
The d a i l y ration o f s i l age f o r each e x periment was s p r e ad o n a
c o ntinuous b e l t f e e d e r b e twe e n 8 a n d 9 a . m . each d a y .
This delivered
a p p r o xima t e l y e q ual amo unts o f f e e d e ac h h o u r .
D e s ign :
I n t h e f i r s t e x perime n t , a 3X3 L a t i n S q u a r e was ch o s e n , w i t h
t h r e e treatments ( u ntreated s i l a g e , f o rmalin-treated s il a g e and
u n t r e ated s i l a g e plus I / P m e th i o ni n e ) , and three s h e e p p e r t r e atmen t .
H o we v e r , a f t e r t h e s econd r e p l i c a t e h ad b e e n compl e t ed , s h e e p 64 7 a n d
7 3 1 were l os i n g c onsiderable q u an t i ti e s o f duodenal f l u i d f rom a r o u n d
t h e i r re-e ntrant cannulae .
attempt t o r ec t i f y th is .
S h e e p 647 d i e d u n d e r anae s t h e s i a d u ring an
Sheep 731 was also replaced for t h e t h ird
r e p l i c ate , s inc e th e loss o f f l ui d would h av e introduc e d error i n f l o w
meas u r eme n t .
Th e r e f ore a b a l anc e d L a t i n S q uare was n o t p o s s i b l e .
T ab l e 3 . 1 gives t h e layout o f s h e e p numb e r s in eac h r e p l i c a t e .
Tab l e 3 . 2
D e s ign o f Exp t . 2 .
T h e t r e a t m e n t s were :
Control ( c )
C o n t r o l + f o r mal deh yde-treated c as e i n ( C + P )
C o n t r o l + s t arch /s uc r o s e i n f u s i o n ( C + E )
M e th i o nine i n f u s i o n ( + M )
S h e ep N o s .
821
1 1 57
1 3 03
c + E
c + E + M
Rep.
Rep.
1
c
2
c + M
c + p
c + p + M
Rep.
3
c + E
c
c + p
Rep.
4
c + E + M
c + M
c + p + M
Rep.
5
6
c + p
c + p + M
c + E
c
Rep.
c + E +
f\1
c + M
F ig. 3 . 2
S egu e n c e o f s ampl i ng, E xpt . 2 .
N
Preliminary
0
period for
M
13 days
E
adjustment
T
H
to feed.
I
0
7 days
B a l an c e ,
c o l l e c ti o n ,
'
�+"
N
I
-
0
� CFEDT-A .
3 days
6 days
Collection of
d u o d e n al sampl es .
>
Lr E.OTA
,flr'"'"',!);O(")
N
E
...
P r e l im i n a r y
period for
adjus tment
t o me t h i o n ine .
+
M
E
T
7 days
H
I
B al anc e ,
c o l l e c t io n
�
i�f:IISj_.QJl
Gf: ErED=tA .
3 days
Collection of
duodenal
diqe s t a s am pl e s .
0
,..
�
Cr- E D TA
'"�� 10() '
N
I
N
E
63
E ac h replicate las t e d f o r 34 days , with a 14 day " r ec o v e r y "
wAM ea.d.. $ Allep w�ts
&_,/ ufl.u. .fr-e.-<;1.
p e r i o d o f ad l i b . f e e di n g b e t w e e n each r e p l ic a t �
{Xls��e tJ r J�:Jrtl SS
T h e s e q uenc e o f
s amp l i n g w i t h i n e a c h r e p l ic a t e i s g i v e n i n F i g . 3 . 1 .
The s ec o n d experiment c o n s i s t e d o f two s up e r impos e d 3 X 3 L a t i n Sq uar e s .
T h e f i rs t L atin Square i nv o l v e d f e e d i n g th e c o n t r o l s i l age plus
s u p p l ements .
Th e s e cond Latin S q uare was
a
r e p e at of t h e s ame t reat-
ments � b u t with an addi t i o n a l d uo d e n al s u p p lement o f L -meth i o n i n e t o
e ach s h e e p .
Th e experi m e n t a l d e s ign i s g i v e n i n T a b l e 3 . 2 .
T h e f i rs t rep l i c at e o f o n e L at i n S q uare was immed iate l y f o l l o w e d
b y t h e f i r s t r e p l ic a te o f th e s e c o nd Latin S quare .
Th i s r e p l i c a te w a s
{!tAt.U·nfl ci..a /1 )
f o l l o wed b y a " rec o v ery " p e r i o d o f ad l i b , f e e d in g f o r 1 4 d ay J(, and
the s eq u e n c e th e n r e p e a t e d f o r t h e next two repl ic ates .
Th e f i r s t
L at i n S q u a r e ( n o me thio n i n e ) c o n s i s ted o f r e p l ic a t e s 1 , 3 a n d 5 , each
o f wh ich l asted 23 days .
Th e s ec ond L at i n Sq uare ( + m e th io nine )
c on s i s ted o f rep lic a tes 2 , 4 and 6 , each o f wh ich l as t e d 1 6 days .
Th e
s e q u e n c e o f sam p l i n g w i t h in r e p l ic a tes is g i v e n in F" i g . 3 . 2 .
I n E x p t . 1 , th e b al anc e c o l l ec tions were made a f t e r an adap tation
p er i o d of f o urt een d ays .
A p r el iminary p e r i o d of t h i s l e n g th was n o t
p o s s i b l e i n . Exp t . 2 , owing t o th e s h o r t a g e of f e e d .
H e nce , t h e adap-
t ati o n p e r i o d to f e e d i n rep l ic a t e s 1 , 3 and 5 was l i m i t e d to t h i r t e e n
days , a n d t h e adap t ation p e r i o d t o meth i o nine · in r e p l i c ates 2 , 4 a n d 6
was l imi t e d to s i x d ays .
Th e v al i d i t y o f this ad a p tat i o n p e ri o d was
as s e s s ed b y monitoring t h e d a i l y c h anges i n p l asma amino ac i d c o nc e nt r a t i ons and th ese results a re p r e s e n t e d and disc u s s e d in t h e n e xt
c h ap t e r .
S uppl ements :
Th e two s up p l e m e n t s given in Exp t . 2 w e r e f o rmaldeh yde-
t r e a t ed c a s e in and an i n f us i on o f read i l y availab l e ener g y .
Th e f o rm-
aldeh y d e- t r eated c a s ein was f r o m th e · same b a tch as th a t u s e d b y Mac R a e
et al. ( 1 9 72 ) .
T h e da il y r a ti o n of f o rmal d e h y d e - t r e a t e d c a sein ( 4 5 gDM )
64
was s p r e a d e v e nly over the s il a g e on t h e b el t f e e d e r .
H o w e v e r , an
es t i m a t e d 5g/ d a y w e re not e a ten by th e s h e e p , a n d th i s amo un t i n c r e as e d
w i t h inc r e as i n g s i l age ref u s als .
1 : 1 mix o f s t a rc h and s uc ro s e .
T h e d a i l y s u p p l em e n t o f e n e r g y was a
S t arc h h as b e e n s h ow n to b e m o r e
e f f e c t i v e t h a n s imple s ugars , x y l a n o r pec t i n i n prom o t i n g u r e a
u t il i s a t i o n i n t h e r u m e n ( H e n tclr i c kx a n d M a r t i n , 1 9 6 3 ) .
T h e s up p leme n t
w as i n f us e d c o n t i n u o us l y i n t o th e r um e n , b o th t o ens u r e · an acc u rate
s u p p l y o f e n e r g y and t o p r e v e n t p o s s i b l e m i c r o b ial d e g r a d a t i o n o n
c o n t ac t w i th t h e s i l age .
Th e s o l u b le s t arch us e d ( T B 8 70 , N Z S t arch
C o . ) f o r me d a ge l a t a c onc e n t r a t i o n greater th an 5% ( w /v w a t e r ) .
Thus,
t o a v o id i n f u s ing l arge volumes o f wa t e r , s u c r o s e ( B D H , L a b . G r ad e ) was
s ub s t i t u t e d f o r S O % of t h e t o tal e n�r g y i n f u s e d per d a y .
The i n f us i o n
was p r e p a r e d e v e r y second d a y , b y add i n g 1 00g s t arch a n d 1 00 g s u c r o s e
t o 2 1 o f b o il i n g wat er .
Th i s s o l u t i o n was i n f u s e d i n t o t h e r u m e n a t
a p p r o x i ma t e l y 7 5 0 m l /day w i t h a p e r i s t a l tic pump .
Th e s h e e p o n t h e
o th e r t w o tre a tments rec eiv e d a s i mil a r v o l ume o f w a t e r p e r d a y , t o
e q u a l i s e a n y e f fec ts o f t h i s v o l u m e o n th e anim al .
Th e t wo
�r-MS
J.e'o'eh
of s up p l e me n t giv e n , w e r e c h o sen t o p r o v i d e two
d i f f e r e n t flow rates o f am i n o ac i d s i n t o t h e d u o d e n u m , r e l a t i v e to th e
c o n t ro l .
Mac R a e e t al . ( 1 9 7 2 ) r e p o r t e d th a t th e c o m p l e t e 6 0 g o f
f o rm al d e h yde- t r e a t e d c as e in s u p p l emen t r e a c h e d t h e d u o d e n u m o n a d r i e d
grass die t .
T h e 4 5 g g i v e n i n t h i s e x p e r iment , w a s a p p r o x i m a t e l y e q u a l
t o t h e b a s al f l ow of amino a c i d s i n t o t h e d u o d e num o n u n t r e a t e d s i l a g e
in Expt . 1 .
The�e i� little
M e t h i o n i n e i n f us i o n :
e r i t o ne al l y .
��entiteti�s infsP�otisn
on the
utilisation
T h e L- m eth i o n i n e in E x p t . 1 was i n f u s e d i n trap-
A n I / P c a th e t e r ( 1 . 5 mm i n t e rnal d i ame t e r , s i l as t ic ) was
65
i ns e r ted as f o l l ows .
Th e sh e ep was a n ae s th e ti s ed , a n d a small i nc i s i on
made thro u gh the s k i n , in th e r e g i o n o f the lower ab dome n .
One end o f
the c ath e t er w a s introduced i n t o t h e p e r i t o neal c a v i t y t h r o u gh a 1 2
gauge nee d l e and t h e free end was t h e n d r awn s u bc u taneousl y , b y· means
o f a 2 50 c m long s tainless s t e el n e e d l e , to emerge through the s k in
l e v e l with th e b ac k b one .
Th e inc i s i o n was then s u t u r e d .
A continuous
infus i o n w a s maintained with a p e r is t a l t ic p ump , u s i n g t u b i n g
c ali b r at e d t o d e l i v e r 1 50ml / day .
Th e i n f u s ate was made u p i n 4 1
b atc h e s , e ach s t erilised b y m i l l i p o r e f i l t e r in g .
T h e L-me t h i o n i n e was
diss o l v ed i n 0 . 7 4% s a l in e , t o g i v e a s o l u t i o n isotonic w i th b l o o d .
The
. c o n c e n t r a t i o n o f methi onine ( 6 . 7g / l ) was c alc ulated to p r ov i d e 1 g
L -me t h i o n i n e / d a y .
I n p r ac tic e � th e f o l l o w i n g amo u n t s were i n f us ed :
S h e e p 647 i n replica te 1 rec e i v e d D . 9 7 g /d a y
"
731
"
"
2
"
D . 94 g/d ay
"
786 "
"
3
"
1 . 0 7 g/day
Th e L -meth i o nine in E x p t . 2 was infused into t h e d i s t a l end of the
duode nal r e-ent r ant c annulae .
Th e m e t h i o n i n e was infused a s a 2%
s ol u t ion w h ich was made up i n 61 b a t c h e s .
E ac h b a t c h was s t e r i l is e d
b y m i l l i p o r e f i l teri ng , and div id e d i n t o s e par ate f l as k s f o r each s h e e p .
°
Thes e w e r e k e p t in a r e f r i g e r a t o r a t -4 C d uring i n f us i o n .
Th e
i nfus ion w as ma intained w i t h a peris tal tic p ump and t u b i n g c al i b rated
t o d e l i v e r 8 5ml / day ( i . e . 1 . 7 g meth i o n i n e / d ay ) .
H o wev e r , t h e p ump
did n o t m aintain a c o ns tant rate in r e p l ic ates 2 and 4 and t h e r e w e r e
f l uc t ua t i o ns i n t h e d a i l y meth ionine i n f u s i o n .
T h e average v a l u es f o r
each s h e e p d u r i n g th e c o l l ec t i o n o f s ampl e s were :
66
1 . 96 g/da ; for s h e e p 8 2 1 )
1 . 77 g/day for sheep 1 1 5 7 ) R e p . 1
1 . 9 7 g/day for s h e e p 1 3 0 3 )
1 . 64 g/day for sheep 8 2 1 )
1 . 4 7 g/day for s h e e p 1 1 5 7 ) R e p . 2
1 . 64 g/day for s h e e p 1 3 0 3 )
1 . 8 8 g/day for sh e e p 8 2 1
1 . 85 g/day for s h e e p 1 1 5 7
1 . 84g/day for s h e e p 1 3 0 3
B al a nce c o l l e c tions :
s e v e n days .
Rep . 3
F aeces and urine w e r e c o l l ec te d f o r a p e r i od o f
F aeces were c o l l ec t e d u s i n g a h a rness a n d c o l l e c t i o n b a g ,
wh il e urine w a s allowed t o r un f r e e l y i n t o a c o l l e c t i o n v e ss e l
c o n t aini n g 25ml o f c o nc entra ted h y dro c h l o r ic acid , t o p r e v e n t l o s s o f
ammo n ia .
anal ys is .
A 1 0% s u b sam p l e o f faec es was t a k e n d a i l y and b ul k ed f o r
Th e remaind e r was dried in
an
°
oven at 1 1 0 C to determine
Dail y s ub s am p l e s ( 1 %) o f urine w e r e a l s o t a k e n .
OM .
Th e s e s u b s amp l e s w e r e
°
b u l k e d ov e r t h e s e v en d a y p e r i o d , and s t o r e d at -20 C p r i o r t o anal ysis .
Expi red a i r was c o l l ec t ed i n E x p t . 1 u s ing the r e s p i r a t i o n h o o d
o f U l y att ( u n p ublished ) and meth ane d e termi ned o n a n a l i q u o t u s i n g a n
Aero graph 660 gas c h roma to g r aph .
me tabol i s a b l e e n e r g y ( M E ) i n t ake .
This al l o wed c a lc u l a t i o n o f
M e t h an e p rod uc t i o n i n E x p t . 2 was
c a l c ul a t e d from t h e regres s i on e q u a t i o n o f B l axter and C l a p p e r t o n
( 1 9 6 5 ) i . e . M e th an e pro duc t i o n ( k c a l/1 00 kc a l intake )
=
3 . 6 7 + 0 . 0 6 20
w h e r e 0 i s th e app arent d i g e s t i b i l i t y c o e f f icient f o r ene r g y .
Mark e r a n d d igesta s ampl e c o llec tion :
T h e tec h n i q u e s u s e d are g i v e n in
d e t a i l in C h a p ter 2 , but a b r ief s ummary w i l l be giv e n h e r e .
C h romium
e th y l ene diamine t e tra a c e t i c ac i d ( C r EDTA ) ( B in n e r t s e t a l . , 1 9 6 8 )
was cont i nu o u s l y i n f us e d into t h e rumen o v e r a p e r i o d of s ix d a y s .
Twe l v e s t eady s t ate duode nal s am p l e s w e r e c ol l ec ted as d e s c r i b e d i n
C h a� ter 2 .
A l l t w e l v e s am p l e s were b ul k e d in E x p t . 1 , b u t th e s a m p l e s
i n E xp t . 2 w e r e b u l ked o n a dai l y b a s i s .
OM p e rc e n t a g e w a s d e t e rm i n e d
67
°
on 1 0 g a l i q uo t s by d r y i n g f o r 2 4 h a t 1 1 0 C .
F u rth e r a l i q u o ts w e r e
c en t r i f u g e d twice a t 3 8 , 0 0 0 g f o r 1 5 min and th e s u p e r n a ta n t s to re d f o r
c h r o m ium analys is .
at - 2 0
0
c.
The r e m a i n i n g sam p l e � a s f r e e z e-dried and s t o r e d
Rumen s am p l e s w e r e c o l l e c t e d o n th e las t d a y of each
repl icate in E x p t . 1 , and o n t h e l as t day o f r e p l ic ates 2 , 4 and 6 in
E xp t . 2.
The s ampl e s w e r e s t rained th r o u g h ch e e s e-cl o th , and p re s e r v e d
b y a d d i ti o n of 3ml 1 0 N H s o t o 1 5ml r umen l i q u or, p r i o r to s to r ag e a t
2 4
°
-20 C .
C h e m i c al a n alys i s :
An a l y s i s o f
OM ,
N , e n e r g y , l ac tic and a c e t i c ac id
c o n t e n ts for E x p t . 1 were c o n d uc ted at I nv e rmay ( s e e B a rry , 1 9 7 5 ) .
T h e r ema i n der o f th e an a l y s e s f o r E xp t . 1 s il a g es , and the an a l y s i s o f
t h e c ontr o l s i l age f o r E x p t . 2 , w e r e c ar r i e d o u t a t Palme r s t o n N o r t h .
OM p e r c e n t ages of th e s i l a ges were m e a s u r e d b y dis t i l l a t i o n � i t h
t o l u e n e ( B arry , 1 9 74 ) .
A sam p l e o f f r esh s i l a g e w a s s q u e e z e d t h r o u gh
c h e e s ec l o th and th e j uic e used f o r th e d e t e rm i nat i o n o f pH .
S u b s am p l e s of t h e f e eds w e r e f r e e z e-d ried and g r o u n d , p � i o r to f u r t h e r
c h e m i c al anal y s i s .
Wat e r s o l u b l e s u gars w e r e anal y s e d b y t h e a n th r o n e
me th o d of Bail e y ( 1 9 64 ) .
Anal y s i s f o r
OM ,
N and e n e r g y was c o n d u c t e d
i n a s im i l ar manner for t h e f e e d s , d u o d e nal c o n t e nts a n d f aeces .
an a l y s es were c o nduc ted o n g r o und , f r e e z e- d r i e d ma te r i a l .
The
All
OM
p e rc e ntag e s o f this mat e r ial were d e t ermined by d r y ing i n a n o v e n a t
0
1 1 0 C for 24h . OM was d e termined b y as h in g f o r 5h in a f u r nace at
°
550 C . T o tal N was d e t e r mi n e d o n d r i ed s am p l e s ( 0 . 1 4 g ) and on 0 . 5ml
u r i n e sam p l es with a T e c h nic o n autoanalys e r , a f t e r p r i o r d i ges t io n b y
the K j e l d a h l m e t hod .
G r o s s e n e r g y was det ermi ned o n a n ad iabatic b omb
c al o r ime t e r ( G a l l e n k amp ) .
t o d e terminat i o n of e n e r g y .
U r i n e s amples ( 1 0m l ) were f r ee z e-d r i ed p r i o r
Th e am ino ac id c o n t e n t of f e e d and
d u o d e nal d i g e s t a was me a s u r e d o n h y d r o l ys a t e s o f the f r e e z e-d r i e d
68
m a t e r i a l , with a B e c kman 1 20 C amino a c i d anal y s e r .
H yd ro l ys is was
°
c arried out w i th excess 6N HCl at 1 1 0 C f o r 22h , in s e a l e d e v ac uated
tubes , a n d after rotary e v a p o r a t i o n , t h e h ydrol ysates were t a k e n up in
5ml p H 2 . 2 b u f f e r .
Ch romium . d e t e rminations were made on th e s up er-
n atants of d u od e nal d i g es t a , w i th an atomic abs o r p t i o n s p ec t r o p h o t o m e t e r
( Techtron ) .
Th e samples were d i l uted 1 i n 3 w i th 1 50 0 p p m c al c i u m .
R u m e n amm o hia was meas ured b y t h e ·mic r o d i f f u s i o n me t h o d o f C o n w a y
( 1 9 5 7 ) o n s trained samples o f r u m e n c o n t e nts , pres e r v e d b y t h e add i t i o n
o f 1 0 N H s o saturated w i th Mg S 0 • T o tal v o l a t i l e f at t y ac id c o nc e n­
4
2 4
t r a t i o n was d e t e rmined b y t i t t r a t i o n with N /2 5 NaOH a f t e r d i s t i l l a t i o n
i n a Mar k h am still .
The m o l a r p r o p o r t i ons o f v o l at i l e f at t y ac ids
w e r e d e t e rmined on an Aerograph 6 60 gas c h romatograph .
C a l c u l a t i o n o f duodenal f l o w :
Th e d a i l y f l o w o f OM i n t o th e duodenum
was c a l c u l a ted from th e known rate o f i n f u s i o n of c h r o m i um ( X p p m/d a y )
a n d t h e m e asured c o nc e ntra t i o n o f c h r om i um a t the d u o d e num ( Yppm /m l
s u pernatan t ) .
i.e.
OM f l o w ( g /day )
S t at i s t i c al analys i s :
=
X
y(
x
OM%
1 0 0-Dr�% )
Th e r e p l ac ement o f two o f t h e s h e ep i n t h e th ird
r e p l i c at e of Expt . 1 gave a t o tal o f f i v e s h e e p used i n this e x p e r imen t .
T h us , f o u r d e grees of f r e e d o m m u s t b e a l l o c a t e d t o t h e s ums o f s q u a r e s
f o r a n i m a l v ariatio n .
The t o t a l d e g r e e s o f freedom i n a 3 X 3 L a t i n
S q uare a r e e i gh t , and w i t h t w o d e gr e e s o f f r eedom f o r t h e e f f ec t o f
r e p l i c at e s , a n d two d e grees o f f r eedom f Q r tr eatment e f f ec ts , n ? d e gr e e s
o f f r e e d o m are l e f t f o r t h e e r r o r t e rm .
WU't
confour-deJ
bcj
H e n c e comp a r i s o ns b e tw e e n
treatmentsA��ld ��uiPe th e u s e o f animal e f f e c t s as t h e e r r o r t e rm .
T h i s ass umes that th e e f f e c t o f animal v ariation is n o t s i g n i f i c an t .
Th is assumption is inval i d a t e d b y t h e r e s u l t s of E x p t . 2 , w h e r e
s i g n i f ic ant d i f f�renc es b e t w e en an imals were recorde d .
Th us , n o v a l i d
Table
3. 3
An alys is o f v ar i a n c e for E xpt .
Degr e e s
E f f ec t
f r e e d om
4
Replic ates
T re a tm e n ts
M e th i o n i n e
I n t e r ac t i o n
I
I
I
I --
1I
I
2.
(T)
(M)
( T XM )
2
2
1
An i m a l s
2
E rror
6
T OTAL
17
of
Tab l e 3 .4
Th e c o mpo s i t i o n of t h e s il age s f e d in E x p t s 1 and 2 .
Exp t . 1
Exp t . 2
U n t r e ated
Treated
Control
S i l age
S i l age
Silage
T o l u e ne OM %
20 . 0
20.3
21 . 9
O rg a n ic ma t ter
( % OM )
87 . 9
86.9
87.7
Nitr o gen
( % OM )
A mi n o ac id N
( % t o tal N )
2 . 42
50 . 9
2 . 24
90. 7
2 . 51
74 . 6
E ne r g y
( kca l/g )
4 . 48
4 . 34
4 . 27
W a t e r sol u b le
s u g a r ( % OM )
1 . 33
1 . 94
2 . 68
A c e t i c ac id
( % OM )
3.6
2.4
NO
L a c t i c ac i d
( % OM )
3.1
2.5
NO
pH
4.6
4.5
4.1
Table 3. 5
T h e amino a c i d c o mpos i t i o n of th e silage s f e d i n
E xpts 1 and 2 , c ompar e d w i t h th e amino ac i d c o mpos i ti o n
o f the f o rm n l d ehyd e- t r e at e d c a s e i n and an a v e r age h e rbage
( e xpr e s s e d as g i n d i v i d u al amino acids/1 0 0g t o t a l amino
ac i d s ) .
E xp t . 1
Exp t . 2
Untreated
Treated
C ontrol
Treated
S i l age
S il age
S il age
Casein
arg
3.4
5.6
2.8
3.5
4.8
h is
4.2
2.0
3.2
2.6
2.1
ile
6. 9
5.5
5.4
4.9
5.6
leu
11.7
9.9
9.1
8.9
8.8
lys
3.5
3.6
4.6
6.6
7.2
met
1.7
1 .8
1 .8
2.6
2.4
phe
7. 0
6.4
7.2
5.0
5.7
thr
3.6
6.3
5.8
4.0
5o9
v al
8.8
6.5
7.1
5.7
6.4
ala
1 1 .7
8.0
10.8
2.7
7. 1
asp
10.2
11.3
1 1 .4
7.1
11.1
glu
9.7
11.2
1 0. 1
23 . 2
12.8
gly
7.2
6.9
7.4
1 .8
6.1
pro
5.7
6.7
5.6
10.0
ser
4.6
5.4
5.0
5.9
3.1
2.8
tyr
1
2
TR .
5.4
H er b age
L! 0
2
1
2
5.5
4.2
D a t a f r om M a c R a e a nd U l y a t t ( 1 9 74 ) f o r a v e r a g e of t h r e e h e r b ag e s .
V al u e f o r t y r o s i n e t a k e n f o r u ntr e a t e d c as e i n , s i n c e th e p r e s e nc e o f
f o r m a l d eh y d e d e c r e as e s th e r e c o v e r y o f t y r o s i ne af t e r h y d ro l y s is .
{}1/}.L� eA al.) 1�'72.) '
69
s tatis tic a l anal y s i s c o u l d b e carried o u t for Expt . 1 .
Expt . 2 c o nsis t e d o f two , 3 X 3 L at i n S q uares , one w i t h m e th i o nine
s up p l emen tation and th e o t h e r w i th no m e th i o nine s uppl ement at i o n .
S i g n i f i c a n t d i f f e r e n c e s were detected b y A n a l ys is o f V a r i anc e a s
d et a i l e d i n Tab l e 3 . 3 .
I nd i v idual d i f f e r e nc es b e tween p r o t e i n and e n e r g y s u p p l em e n t s and
the c o nt r o l , were a s s e s s e d by comparing t h e " l e as t s i g ni f ic ant
d i f f e re n c e " ( L S D ) w i th the t r e atment means .
j2
c al c ulated as :
where
SE
r
x
t
0
�
The L SD ( a t P
<.
0 . 0 5 ) was
S E is t h e s t andard e r r o r o f t h e tr eatme n t m e ans ,
r is th e numb e r o f means u s e d t o c omp u t e t h e S E ,
t
3.3
0
?�
i s t h e " t " v a l u e at t h e 5% level of s i gnif ic anc e .
Res u l t s
The c h emic al c o mp o s i t ions o f t h e s il a g e s fed in E x p t s . 1 a n d 2 a r e
g i v e n i n Table
3.4.
T h e w a t e r s o l u b l e s u g a r c o nt e n t o f the h e r b a g e
e n s i l e d i n Expt . 1 w as 1 6 . 2% ( B arr y , 1 9 7 5 ) .
A compar ison o f th i s
c o nc entr a t i o n c ompar ed t o th o s e o f th e t r e a te d and u n t r e a ted s i l ag e s
( Tab l e 3 . 4 ) 1 indica t es that c o n s id e r a b l e f e rmenta t i o n o f th e c a r b o­
h ydrate f r ac t i o n t o o k p l ac e in b o th s il a g e st acks .
in
Th e ami n o ac i d - N
th e t o t al N f r ac t i o n was marke d l y h i gh e r o n th e t r e a t ed than
u n t r e ated s il ag e .
T h e amino ac i d-N c o nc e n t r a t ion o f th e c on trol s i l a ge
in E x p t . 2 was b e tw ee n those of th e t r e a t e d and u n t r e a t e d s i l a g e s
( Expt . 1 ) .
The amino acid c o m p o s i t i o n s o f t h e s i l ages f r om b o th e x p e r i m e n t s
a n d f o r t h e formal d e h y d e-treated c as e in s up p l ement ( E x p t . 2 ) a r e g i v e n
in Table 3 . 5.
S ince t h e amino a c i d c om p o s i t i o ns o f th e o r i g inal
h e rbages w e re n o t a v a i l a b l e , t h e s i l ag e c o mpositions a r e compared w i th
t h e a v e r a g e amino ac i d c ompo s i t io n of th r e e h e rbages , taken f r o m
The OM, energy, ni trog e n ,
Table 3 . 6
non a�ino aoid N
i n t a k e s o f s h e ep f e d s i l age (g/d ay) .
E xpt . T r e a tm e n t
!:!.£·
Meth i o n i n e
S i l age OM
i n take
Mean
T o tal OM
i ntake
,
and amino a c i d
N i t r ogen
intake
Amino
acid
intake
Mean
Mean
599
13.9 )
1 5 . 3 ) 1 4 .·5
14.4 )
45 )
4 9 ) 47
46 )
61 )
72 ) 72
83 )
Mean
Untreated
1
Rep .
Rep .
Rep .
1
2
3
574 )
63 1 ) 5 9 9
593 )
1
2
3
480 )
5 6 1 ) 564
650 )
564
1 o.8 )
1 2 . 6 ) 1 2.7
14.6 )
640 )
6 33 ) 6 2 0
587 )
620
15.5 )
1 5.3 ) · 1 5.0
14.2 )
49 )
4 9 ) 48
45 )
41 3
41 3
1 0.4
48
Treated
Rep.
Rep .
Rep.
Untreated
Rep.
Rep .
Rep .
1
2
3
C o n tr o l
2
+
protein
651
688
22 . 2
1 14
+
energy
576
669
14.5
65
+
587
587
14.7
68
118
C ontrol
<
* p
**
+
p
+
protein
+
672
71 0
23 . 0
+
energy
+
64 7
734
16.2
0 . 71
.
74
'2 . 5
SE
1 7.4
T r e atme n t s i gn i f i c a nc e
**
**
**
Meth i o n i n e s i gn i f ic an c e
�-
**
**
A n imal s i gn i f i c ance
**
*
**
0 . 05
<. o .
01
V o l L n tary
Expt .
OM
i n tak�
( g/d ay )
2
•
•
0
500
•
•
•
•
••
•
0
••
-0
••
•
•
3001
700
500
300
700
0
0
0
500
0
D ooo
0
0
oD
DD 0
OD 0
0 o ••
•
0
300
=
10
F ig.
3.3
C h a nge
in
• • 11 •
20 a
Time ( d ay s )
v o l untar�
OM
•
••
•
•
•
•
0
0
•
•
0
�
c ontrol
•
c o ntrol + m e t
1:::..
+
A.
+ protein
0
+ energy
p rotein
+
met
•
+ energy + met
0 40
30
i n t a k e with t ime - E xp t . 2
70
Mac R a e a n d U l y a t t ( 1 9 74 ) .
T h e c ompos i t io n o f t h e t r e ated s il a g e
{ E x p t . 1 ) was c l os e r to th a t of h e r b a g e th a n t o t h e untr e a t e d s i l a ge .
C o n s i d e r a b le d if f e � enc e s b e t ween t h e c o mpo s i tion of t h e c o n tr o l
s i l a g e ( E x p t . 2 ) a n d f o rmal d ehyde-tre a t e d c asein , w e r e rec o rd e d f o r
alan i ne , aspar t a t e , gl u tama t e , g l y c i n e and proline .
The
OM ,
N and amin o ac i d ( A A ) i n t a k e s f o r b o th e x p e r i m e n t s are
giv e n in Tab l e 3 . 6 .
The main d i f f ere n c e in Exp t . 1 was a n i nc r e ased A A
intake o n tre a t e d s ilage , d u e to t h e h i gh e r c o n c e ntr ation o f amino
ac i d s in the s i lag e .
Th e intakes
in
Tab l e 3 . 6 r e p r es e n t th e mean d a i l y i n t a k es
c al c u l a t e d f r o m th e total i n t ake o v e r t h e l as t three days o f e a c h
repl i c ate .
Th is c o�parison w a s c h os e n , b e c ause the i n t a k e s i n E x p t . 2
c h a n g e d w i th t ime a f t e r the s ta r t o f e ach r e plic a t e .
for t h e m e an d a ily
OM
Separate plots
intake on e a c h t r e a tm e nt , a r e g i v e n i n F i g . 3 . 3 .
The i n takes ( T ab le 3 . 6 ) for repl i c a t e s 1 , 3 and 5 w e r e c al c u l a t e d o v e r
time ( a ) and f o r r e plic ates 2, 4 a n d 6 o v e r time ( b ) .
I t c an b e s e e n
from F i g . 3 . 3 th at there was a g r a d u a l dec rease i n i n t ake o f th e c o n t r o l
s i l a g e af ter t h e f i r s t t e n d a ys o f f e e d i n g .
Th is d e c l i n e c o n t i n ued
until the s tar t of the L -me th io n i n e i nf us i o n , wh e n in take b e gan to
inc r e ase .
Howev e r , during the t i m e of i n f u s ion measured , i n take did
not r e tu r n to th e l ev e l at the s t a r t o f the exp e r ime n t .
S up pl e m e n t at i o n
with p r o t e in a n d e n e r gy mai n tained in t a k e a t a h i gh e r o v e r a l l l ev e l ,
but s o�e d e c r e as e i n in take s t i l l o c c u r r e d prior to m e th io n i n e i n f u s i o n .
OM i ntakes in Tab l e 3 . 6 s h o w e d a s i g n i f ic an t increas e i n r e s po n s e t o
s u p p l eme n t a t i o n ( P ' 0 . 0 1 ) a n d t o me t h io n i n e ( P
<
0 . 01 ) .
T h ere was
also a s i gnif icant e f f e c t of animal v a r i a t i o n on OM i n take ( P
The i ndiv idual data for each s h e e p a r e g i v e n in A p p e n d ix 1 .
<
0 . 01 ) .
Th e d a y
to d a y varia � i o n in intake r es u l t e d i n d i f f e renc e s i n i n t a k e b e t w e e n
peri o d s o f b a l a n c e c o l l e c t i o n and d u o d e n al f low meas u r em e n t ( s e e
Table
The metabo l is�ble eneroy i n take
3,?
of
sheep fed fornalin-treated s i l ag�untreated s i l age,
and
untrea ted silage diets suppl e�ented w i th f o rmaldehyde-treated casei�energy and pos trum i nal
(I/P and duodenal) methionine compared to the estimated maintenance
tA!l.i·
Ii£·
T r ea tme n t
Methionine
� excretion 1
ME intake
(kca l/day)
(kcalL1 00kcal
GE intakel
. 2
�
c
excret�on
(kcalL1 00kcal
GE i n takel
�
�
�
re u
q irem e n t .
!:!LQ.
�
'
�
Maintenance
r
kcalME
Mean
Me a n
2.6
1 350
5,7
2.8
1 320
·
5, 7
2,5
1 350
Untreated
1
e
e ,
R p,
R p
Rep ,
-�
Rep.
Rep ,
Rap .
2
3
1 644 )
1 6 1 4 ) 1 586
1 501 )
7,9
5,3
9.2 ) 6,0
3,5
1
2
3
1 627 )
1435 ) 1 577
1 670 )
7.8
8.1
6.2
2.8
l
1 562 )
1 573 ) 1 567
1 565 )
8,0
7,9
5. 4
3_, 7
)
1 235
1 629
8,2
6,4
NO
3,0
1 295
ND
2.8
1 320
1 669
6,4
ND
3,3
1 295
U ntrea ted
Rep,
2
3
Rap ,
R ep ,
2
+
1
-
Control
+ protein
+ energy
control
+
1 458
8,3
ND
2,6
1 350
+ p r o tein
+
2000
NO
3,0
1 295
+ energy
+
1 983
8,5
8,4
ND
3,1
1 295
Calculated f rom the regression equation of 8laxtar and Clapparton
CH
2
3
4
e x c r e t i o n ( kcal /1 00kcal
where
0
is
the
G E intake )
( 1 965)
calculated
from ARC
values
the feeds.
NO · no t determined
3 , 67
+
0, 0620
apparent diges t i b i l i t y coefficient
measured in respiration apparatus
for
=
for
Expt.
( 1965 ) 1
for e n e r g y .
1
Nutrient requirements
for
ruminants using the M/0
t
•
Table
3 ,8
The e f f e c t o f formalin-treatment o f s i l age and s
uppl
s m l (I/P
ementation
formaldehyde-treated casein, e n e rgy, and po t ru
rumen ammonia c o n c e n t r a t i o n and v o l 3 t i l e f a t ty a c i d
with
E xpt .
lli:.·
Treatment
Methionine
Total
of
untreated s i l age diete
or
duodenal ) methionine on
concentration
l'lolar
vrA
concn,
ina
and mo l a r pr opo r t i o ns .
proportio ns
(%)
Bu y a e
�
-N
NH3--"
(�2
-
�
Propionate.
P'lean
Mean
79, 2 )
7 4 , 3 ) 77 , 8
80,0 )
11.1 )
15.2 ) 13,3
12.5 )
9,5 ) 8,9
7,5
9,0 )
74 , 2 ) 58 , 5
62 , 3 )
83,5 )
86.2 ) 8 5 , 6
87, 1 )
11,0 )
7,5 ) 8,8
7,8 )
5.5
6 ,4 ) 5 , 7
5,2
NO
72, 6 )
82,9 ) 80,7
86,7 )
86,9 )
76, 6 ) 8 1 , 4
80,7 )
6,7 )
15,1 ) 10,8
10,7 )
6.4
8,7 ) 7,9
8,7
NO
69 . 0
77 , 2
85,5
66, 6
60,8
61 , 8
21,5
22,0
22,0
9,3
12.8
1 1 ,9
(meguiv/1)
Mean
t r t
a c
1 00nl.)
Untreated
Rep,
Re p ,
Re p ,
59,5 )
75.8 ) 66.0
52,6 )
1
2
3
�
Rep,
Rep,
Rep,
-
1
2
3
U t rea te
Re , 1
n
d
2
2
3
Control
+
+ p l' o t e i n
+
+ energy
+
SE
1,1
T r e a t m e n t s ignificance
Animal
s i gnificance
NO
not determined
NS
n o t s i g n i f icant
1,8
NO
+
p
Rep.
Rep,
5
..
..
1 ,9
1.1
0,8
NS
NS
NS
NS
NS
NS
.
I
14,7
1 ,6
1 3 ,4
4
0 , 87
NS
..
The effec t o f formalin-treatment o f s i l age, and s uppl ementation o f untreated s i l age diets
Table 3 , 9
with
the
�
!!.E....
formaldehyde-treated casein, energy and pos truminal (1/P
per C en t age of OM, N and energy intakes appa r e n t ly diges ted
Treatment
Methi onine
Ornanic matter
�
N i tr oge n
�
and
in
d uodenal)
methionine on
t he stomach
of
sheep.
�
�
Untreated
Rep.
Rep,
Rep.
�
Rep.
Rep.
Rep.
1
52 . 7
2 .
59, 2
) 60,9
70,7 )
3
15,8
)
51 , 7
28. 1
) 29,9
55.9
)
)
45,8
l
6 5 . 6 ).
30,2
57.7
1
35,9
)
-37 , 0
)
2
45,7
) 44 , 7
}- 2 2 . 7
39,7
3
52.4
)
-2 1 . 4
- 9.6
)
49.8
3,9
)
45,4
) 21.8
56 , 5 · ) 56 , 9
U n�
Rep. 1
Rep, 2
Rep. 3
2
)
t ro l
protein
e n e rg y
) 39.9
+
46.5 )
59 . 5
)
67.6 )
57,9
17.0
44 , 4
l
68 , 8
)
)
C on
...
50 , 3
-1 0 . 3
+
-
54 . 7
15.0
51,4
-
57, 7
- e. ?
53,1
+
44,7
Control
+
54 , 7
4,8
49,9
+
p ro t e i n
+
50 , 7
3,3
43,8
+
energy
+
48,7
-2 2 . 4
40,9
·
3 .4
1,7
SE
Tre a m n t
t e
sig n if ica nc e
Meth ionine signific ance
NS
NS
*
NS
2,8
NS
NS
71
·
A p p e ndix 1 ) . H e n c e d i r e c t c ompar is o n s b e t w e e n b al a n c e a n d f l o w d a t a
canr o t b e made .
The adequacy o f t h e v a r i o us di e t s i n m e e t i n g t h e e n e r gy r e q u ire­
men t s o f t h e s h ee p for ma i n t enance are g i v e n in Tab l e 3 . 7 .
Th e m ain­
te nanc e r eq u i r eme n ts f o r m e t a b o l i s ab l e e n e r g y ( ME ) w e re c alc u l a t e d f r o m
tab l e s i n th e A R C ( 1 9 6 5 ) manual o n n u t r i e n t r e q u i r e m e n t s o f r u m i n a n t s .
Th e s e de p e nd o n t h e M/D v a l u e ( Mc alME / kg OM ) of th e f e ed .
I t w o uld
appe ar t h a t t h e o nl y t r eatm e n t wh ich d id no t p r ov i d e t h e s h e e p w ith a
gre a t e r t h an m a i n t e nanc e in t ake o f e n e r g y , was the c o n t r o l s il a g e i n
E x p t . 2 a nd t h is w a s v e ry c l os e t o ma in t e n a nce .
ME i n takes i n Exp t . 2 w e re c al c u l a t e d u s i n g a v a lue f o r m e th a n e
exc r e t i o n cal c u l a t e d f r om th e r e gr e s s i o n e q ua t i o n o f B l a x t e r a n d
C l a p p e r t o n ( 1 9 65 ) .
Th i s i s c ompared w i t h t h e meas ur e d v a l u e f o r m e t h a n e
exc r e t i o n in E x p t . 1 .
T h e d i f f e r e n c e r e c o r d ed c o n t r i b u t e s o n l y a 3 %
err o r to t h e ME i n t ake e s t im ated .
Tab l e 3 . 8 p r e s e n t s t h e e f f e c t o f f o rmalin-treatment o f s i l age an d
s u p p l e me n t a t i o n w i t h f o rmal d e h y d e-t r e a te d c as e i n , e n e rgy a n d p o s t r u m i n a l
meth i o n i n e i n f us i o n o n r u m e n amm o n i a c o n c e n trations and v o l a t i l e f a t t y
ac i d ( V F A ) c o n c e n t r a t i o ns a n d m o l a r p r o p o r t i o n s . T o t a l V F A c o nc e n t r a t i o n
was s i gn i f i c a n t l y i nc r e as e d by s up p l e m e n t a t ion ( P ( 0 . 01 ) i n E x p t . 2 , a n d
the e f f e 6 t of a n i m a l v ar i a t i o n was a l s o s i g n i f i c a n t ( P ( 0 . 0 5 ) . T� e r e w a s a
mar k e d d i f f e r e nc e i n t h e molar p r o p o r t i o n s of V F A s b e twee n th e two
expe rime n t s .
The p r o p o r t i o n of ac e t a t e was h i g h e r a n d th e p r o p o r t i on
of p r o p i o nate l o w e r in Exp t . 1 t h a n E x p t . 2 .
Th e r e w e re n o s i g n i f i c a n t
diff e r enc e s i n r um e n ammonia c o n c e n t r a t io n in Exp t . 2 .
Th e e f f e c t of t h e t r e a t m e n t s o n t h e p e rcentage o f n u t r i e n t s
dige s te d i n th e s t o mach r e g i o n i s g i v e n in Table 3 . 9 .
Th e m ai n
dif f e re nc e in b o th exp e rime n ts was in t h e d i g e s t i o n o f N i n t h e s t omach .
Table 3 . 1 0
T h e e f f e c t o f f o rm a l i n - t r e a t m e n t of
s i l age a n d t h e s uppl e­
m e n t a t i o n o f u n t r e a t e d s i l age d i e t s w i t h f o rm a l d e h yd e ­
t r e a t e d c a s e i n , e n e r qy a n d p o s t r u m i n a l
( I/P
and duodenal)
m e t h i o n i n e o n t h e i n t a k e a n d d u o d e n a l f l o w o f n o n- a m i n o
-N (�J A A N) .
acid
E xp t .
Treatment
Me t h i o n i n e
NAAN
intake
No .
1
Duodenal
N A I\ N
D u ode nal N A A N :
N A A N i n take
Mean
Mean
U n tr e a ted
1
2
3
6.7 )
7.5 ) 7. 1
7. 0 )
4.1 )
3.6 ) 3.4
2.6 )
0 . 61 )
0 . 48 ) 0.49
0 . 37 )
1
2
3
1.0 )
1 1 ) 1 2
1.5 )
5.6 )
4.1 ) 4.9
3.0 )
5.6
4. 1
3.0
8.1 )
8. 1 ) 7.9
7.5 )
4.5 )
4.2 ) 3 . 8
2.6 )
0 . 56 )
0 . 52 ) 0 . 4 8
0 . 35 )
C o nt r o l
2.7
3.1
1.16
+
protein
3.6
1 3
0 . 35
+
energy
3.7
3.9
1 . 01
Rep .
Rep .
Rep .
Treated
Rep .
Rep .
Rep .
U n t r e a te d
Rep .
Rep .
Rep .
2
•
•
)
) 4.2
)
+
1
2
3
•
Control
+
3.8
4.4
1.14
+
protein
+
3.7
2. 1
0 . 56
+
energy
+
4.1
5.1
1 . 23
SE
0.15
0 . 35
0.11
T reatment s i gn i f i c a n c e
*
**
**
M e th i o n i n e s i g n i f i c a n c e
**
-l<"*
NS
I nteraction significance
NS
NS
NS
A n i rn a l s i g n i f i c an c e
**
NS
NS
Table
3.11
The effect of form"l in-treatment of silage and the suppleme n tation of untreated silage diets with
formaldehyde-treated c asein, eneroy and postruninal ( I/P and duodenal) methionine on the flow of N and
amino acids (AA) into.
�·
Treatment
Me th ionine
No.
the duodenul"l.
�
�
�
Duode nal
�
Duodenal
AH:
AA ir.t a!.<e
�
Mean
Duodena l
AA :
�
Mean
Ouadeilal N
�
Duodenal AA
ao % total r�
Mean
Untreated
Rep.
Rep .
Rep.
�
Rep.
Rep.
Rop.
1
2
3
49 ) 4 7
46 )
45 )
47.0 )
46.2 ) 42.2
32.8 )
1 . 06 )
0 . 94 ) 0 . 90
a. 11 )
3,4 )
3,0 ) 2.8
2.1 )
11.7 )
1 1 .0 ) 10.2
7.8 )
65.4
67. 2 ) 66 . 6 .
67. 3
1
2
3
61 )
72 ) 72
82 )
57.6 )
6 7 . 8 ) 65 , 8
72.0 )
0 . 94 )
0 . 94 ) 0 . 92
o . 88 )
5.3 )
5.4 ) 5.2
4.9 )
14.8 )
1 5 . 3 ) 1 5.4
16.0 )
62 . 3
70 . 9 ) 6 8 . 4
72.0
46 )
45 ) 4 2
42 )
65.2 )
52 . 9 ) 5 0 . 4
33.0 )
1 .42 )
1 .18 ) 1 . 13
0 . 79 )
4.2 )
3.5 ) 3.3
2.3 )
14,9 )
12.7 ) 1 1 . 8
7.9 )
70 . 0
66 . 6 ) 6 7 . 8
66 . 8
72 . 1
Untreated
Rep.
Rep.
Rep.
2
+
1
2
3
Control
-
48
49.6
1 . 07
4.9
11.0
protein
+ energy
Control
-
114
110.5
0 . 97
5.1
18.8
94 . 0
-
67
76.6
1 .14
5.4
15.1
81 . 2
+
65
59. 5
0 . 08
4.1
14.0
68 . 9
+
+
118
125.7
1 . 07
5.6
21.8
92 . 3
+
74
98 . 6
1 , 36
6.3
21.0
75. 1
6.0
o . os
0.33
NS
NS
NS
+
+
protein
energy
SE
Treatment s ignificance
Methionine significance
I n teraction signif icance
Animal significance
2.5
**
**
**
**
NS
NS
NS
*
NS
**
NS
**
*
0 . 92
1.3
**
**
**
*
NS
NS
NS
**
.
A STUDY OF THE AMINO ACID STATUS OF SHEEP FED
SILAGE , WITH PARTICULAR REFERENCE TO METHI ONINE
E.
MARGARET GILL
The uni t s omi t t ed trom Tab l e s 3 ,
g/d s.y
1 0 and 3 . 1 1
should b e
72
H ow e v e r ,
1
t h e d i ges t i o n o f O M a n d e n e r g y i n t h e s t o m a c h s i n E x p t .
w e r e a l s o d e c r e as e d w i t h t r e a t e d c o m p a r e d t o u n t r e a t e d s i l a g e .
There
w a s a n e t a d d i t i o n o f N t o t h e r um e n o n t r e a t e d s i l a g e a n d a n e t l o s s
o n u n treated s ilage in Exp t .
1.
I n Exp t .
2
there was a s i g n if i c a n t
d i f f e r e n c e b e t w e e n t h e e f f ec t s o f p r o t e i n a n d ene r g y s u p p l e m e n t a t i o n
o n N d i g e s tion in t h e r u m e n
( P <: 0 . 0 5 ) .
Energy supplementation
r e s u l t e d i n n e t ad d i t i o n o f N t o t h e r u me n ,
wh i l e t h e
p r o t e i n s u p p l e-
ment r e s u l ted i n a n e t l o s s .
C om p a r is o ns o f t h e i n t a k e s o f a m i n o a c i d s and n o n- a m i n o a c i d - N
( NAAN ) ,
and
w i th t h e i r f l o w i n t o t h e d u o d e n um a r e g i v e n i n T a b l e s
3.11 .
N A AN
Th e main t r e a t m e n t d i f f e r e n c e i n E x p t .
was the decrease in
b e t w e e n t h e m o uth a n d d u o d e n u m o n u n t r e a t ed s i l a g e ,
t h e i n c r e a s e i n NAAN
Thus ,
1
3.10
c ompared t o
b e t w e e n m o u th a n d d u o d e n u m o n t r e a t e d s i l a g e .
t h e r a t i o o f N A AN
a t t h e d u o d e n um t o th e N A A N
c o ns i d e r a b l y h i gh e r o n t r e a t e d t h an u n t r e a t e d s il a g e .
intake was
F o r m a l i n-
t r e a t m e n t o f s i l a ge a l s o i n c r e a s e d t h e t o t a l f l ow o f a m i n o a c i d s
t h e d u o d e n um o v e r u n t r e a t e d s i l a g e ,
on treated silage.
Howe v e r ,
d es p i t e lower i n t a k e s o f t o t a l N
th e r e w a s n o t r e at m e n t e f f e c t o n th e p e r-
c e n t a g e o f a m i n o ac i d s i n th e t o t a l N e n t e r i n g th e d u o d e n � m .
o f d u o d e n a l a m i n o ac i d s
into
Th e r a t i o
to amino a c i d i n t a k e a l s o s h o w e d no d i f f e r e n c e
b e t w e e n t r e a tm e n t s .
T h e r e g r e s s i o n r e l a t i o n s h i p b e tw e e n a m i n o ac i d i n t a k e a n d t h e f l o w
o f a m i n o a c i d s i n t o th e d u o d e n um w a s p l o t t e d f o r t h e d a t a f r o m b o t h
e x p e r im e n t s
(Fig. 3 . 4 ) .
i ndic a t i n g that
2
T h e c or re l a t io n c o efficient
67 % ( r )
( r ) was 0 . 8 2
o f th e v a r i a b � l i t y i n d u o d e n a l a m i n o ac i d
f l o w w as d u e t o v ar i a t i o n i n a m i n o a c i d i n t a k e .
In Expt.
2,
t h e f o r m a l d e h y d e-t r e a t e d c as e i n s i g n i f i c a n t l y d e c r e a s e d
the r atio of t h e NAA N
e n t e r i n g t h e d u o d e n um t o N A A N
w h i l e e n e r g y s up p l e m e n t a t i o n h ad n o e f f e c t
intake
( Table 3 . 1 0 ) .
( P <: 0 . 0 1 )
Mst�i�nin�
F l m�
of
acids
a r.+ i n o
i n t o d u o d e n u m (g/d ay)
1 60
y
7 . 0 7 + D . 9 5 6X
=
r
=
0.82
1 20
lil
0 11
!J.
4
BD
e
0
0
+
•
$
40
0
0
� +
0
1::,
A
0
�
0
Ami no
F ig.
3o4
Th e
and d uo d e nal
r e l a t i o n s h ip b e t w e e n
a�i no
ac
id
E xpts e
f low for
)
c o n t .v o l + m e t
)
+
+
+
p r o tein
protei n
energy
1
and
2
+
me t
+ e n e r g y + me t
1 60
amino ac i d in take
all
)
control
i n t a k e ( o/d ay )
ecid
)
u n t r e a t e d + me t
1 20
80
40
�
untr e ated
tre a t e d
t r e a tments
in
)
)
)
)
Expt.
1
Exp t .
2
Table 3 , 1 2
The amino a c i d c ompo s i t i o n o f m i c r o b i a l pr o t e i n, and th e ef fec t
o f f o rmal in-treatment of s i l age, and s uppl e m e n t a t i o n o f u n t r e a t e d s i l age
d i e t s w i th f o rmaldehyde-treated c a s e in, energy and po s truminal ( I/P a n d duodena l) m e th i o n i n e o n duodenal amino acid compo s i t i o n ( I n d i v i d ual
amino a c i d s as a % of
the t o t a l amino acids),
--
E x pt . 2
Expt, 1
U n t r e a ted T re a ted U n t r e a ted
C o n t rol
+ P r o te i n
+ E n e rg�
+ met
.
Control + Protein
�
�
+ E n e rg�
�
.§!.
T r e a tm e n t
Meth i o n i n e
S i�n i f ic ance
Sign i f icance
8acte r i a
1
Protozoa
1
erg
4.3
4,9
4,5
4,7
4,4
4, 6
4,8
4,3
4,8
0 , 06
**
NS
5 , 0- 5 , 3
3 , 7- 4 , 8
his
1,9
1 ,8
2.0
2.2
2.4
2.9
2.3
2.4
2.6
0,14
NS
NS
1 . 8- 2 , 0
1 , 6- 2 . 2
ile
5,9
5,8
5,7
5,7
5.2
5,4
5.2
5,1
5,2
0 , 09
NS
NS
5 , 2- 6 , 3
5 . o- 6 . 3
lou
0,9
9,2
9,9
0,5
0,7
0,1
0,0
0,4
0,2
0, 21
N!;
N!j
7 . 7 - 11 , 7
7 . '• - 11 . 2
l ya
6,8
5.9
6,9
6,5
6,7
6,7
.6 , 3
6,6
6,6
0 , 09
NS
NS
7 , 6- 9 , 0
9 , 4 -1 3 , 0
met
2.1
2.2
2.1
2.3
2.4
2.4
2,4
2.5
2.4
0 , 04
NS
N5
2 , 0- 2 . 2
1 . 3- 1 . 9
N5
4 , 4- 5 , 1
5 , o- 6 , 0
.
ph e
6.1
6, 2
6,0
6,2
5,6
5.8
6.2
5,4
5,8
0 , 09
**
thr
5.6
5.6
5,8
6,5
5.4
6,3
6,2
5,2
6,0
0,16
**
NS
6 . 0- 7 , 8
4 , 8- 6 , 5
val
6,5
6,5
6,4
6,2
6,4
6.2
6,3
6,3
6,3
0 , 09
N5
N5
4 , 7- 5 . 2
4 . 0- 4 , 2
al a
7,3
7.1
7,1
6,2
5.3
7,3
6,7
4,9
7.2
0,13
**
NS
7 . 2- 7 , 6
4 , 4- 6 . 1
asp
11.2
11.2
11.2
11.7
9,7
11.8
11,3
9.5
11.7
0 , 21
**
NS
1 1 . 1 -1 2 , 1
1 1 . 9-1 3 . 5
g lu
1 2. 5
12.6
12.3
12.8
17,8
13.1
12.5
18.0
13,0
0 , 20
**
NS
1 3 . 5-1 4 , 4
1 5 . 0-1 6 . 3
g ly
6,3
6.3
6.1
6.6
4,7
6.8
6. 5
4.6
6,6
0,18
**
NS
6 , 0- 7 , 6
4 , 1- 5,5
pro
5.4
5.6
5,5
. 4. 7
6.8
4.1
5.2
7.4
4,5
0.17
**
*
2 , 4- 2 . 9
2 . 9- 6 . 0
sar
5,3
5.4
5.7
4,8
5,0
4.8
4,4
4,7
4,8
0 , 04
**
**
4 . 2- 5 , 1
4 , 4- 5 , 1
tyr
3,6
3.6
4,0
5,3
4.6
4,7
4,6
4,6
4,9
0,15
NS
N5
4 . 1- 4,5
4 , 2- 5 , 2
1
.
Taken from Bergen
�
( 1 968 )
I
St'jllrfltc a ,r.Jiy
of du�dt.Mo / ,ff} Jv ItA 1nf.t,.keinfld&i>lR e:i�nifisa.ntly
tho Fetio of duodonel
S4f';J/t-wreY�f�{,qn (P<:. . 01)
ho
�t.
N)j,t)
Wit S
()
dooPoa.ood
0
73
decNPSnl hj
AA to AA intBIW .
t� ( o.os)A and t h e r e was fa s i g n i f ic an t t r e atme n t/m e t h i o n i n e i n t e rac t i o n ( P < 0 . 0 1 ) , i . e . wh i l e d uo d e nal A A to
AA
i n t a k e d ec r e a s e d i n
r e s p o ns e t o me t h ionine o n th e c o n t r o l s il a g e alone , i t i n c r e a s e d i n
r es p o ns e t o me t h i o n i n e w h e n t h e s i l age w a s s up p lemen t e d w i t h e i t h e r
e n e r g y or p r o t e i n .
T h e p e r c e n t a g e of amino a c i ds in t h e t o t al N
e n t e r i n g t h e d u o d e num was s i g n i f i c a n t l y inc reas ed b y p r o t e i n and e n e r g y
s u p p l eme n t a t i o n ( P < 0 . 0 1 ) and s i g n i f i c a n t l y d ec r e a s e d b y me th i o n i n e
infus ion (P
<
0 . 05) .
The amino ac id c ompo s i t i o n o f d u o d e n a l d i g e s t a in E x p t s . 1 and 2
· i s c ompar e d w i t h t h e amino ac i d c om p o s i t i o n o f mic ro b i a l p r o t e i n
( B e r g e n e t al . , 1 9 68 ) i n Tab l e 3 . 1 2 .
Th e r e were no m ar k e d t r e a t m e n t
d if f e renc e s i n d u o d e nal a m i n o a c i d c o mp o s i t i o n in Ex p t . 1 .
In Expt. 2 ,
p r o t e i n s u p p l e m e n t a t i o n o f t h e c o n t r o l s i l a ge r e s u l t e d i n s i gni f ic a n t
d ec r e as e s i n t h e p e r c e n t a g e s o f a r g i n ine , p h e n y l al a n i n e , t h r e o n i n e ,
alan i n e , a s p a r t ate and g l y c i n e ( P < 0 . 0 1 ) and s i g n i f i c a n t i n c r e a s e s i n
g l u t a mate , p r o l i ne and s e r i n e ( P
<.
0 . 01 ) .
Energy supplementation
r es u l t ed i n a s i gni f ic a n t d e c r e a s e i n t h e p e r c e n tage o f p h e n y l al a n i n e
( P < 0 . 0 5 ) a n d a s i gni f ic an t i nc r e a s e in t h e p e r c e n t a g e s o f alan i n e a n d
s er i n e ( P < 0 . 0 1 ) .
Meth i o n i n e i n f us i o n gav e a s i g n i f i c a n t i n c r e a s e i n
t h e p e rc e n tage o f p ro l i n e ( P
<
0 . 0 5 ) a n d a s ig n i f i c a n t d e c r e as e i n th e
perc e nt a ge of s er i n e ( P < 0 . 0 1 ) .
T h e re was a l s o a s i gn i f ic a n t i n t e r-
ac t i o n b e t w e e n m e th i o ni ne and t h e s u p plem ents i n th e p e r c e n tage o f
serine ( P < 0 . 05) .
H owev e r , a b s o l u t e c h anges were s m a l l a n d w e r e
p r o b a b l y o f l i t t le b io l o g ic al s i g n i f icanc e .
Th e compo s i t i o n o f t h e
mic r o b ial pro t e i n was inc l u d e d t o g i v e a n i n d i c a t i o n o f t h e r e l a t i v e
c o n t r i b u t i o n o f mic r o b ial and f e e d p r o t e i n a t t h e d u o d e n um .
Table 3 . 1 3
The e f f e c t o f formalin-treatment o f s i l age and supplementation
of
untreated s i l age d i e t s with
f o rmal dehyde-treated c a s e i n, energy and po s t ruminal ( I/P and duode n al) methionine o n the pe rcentage
o f 0�1, O M , N a n d eneroy, and
E xpt .
�·
Treatment
Methionine
the pe rcentooe o f duodonal
OM
OM
-
�
z mfa k e
N, dige s t e d in the intes tir1e s .
�
Mean
�
�
Mean
�
duodenal
Mean
/�
Mea11
Untreated
Rep.
1
28.6
)
18.7
)
Rep.
2
20 . 0
) 20.4
14.1
)
Hep.
�
1 2. 5
)
0
)
Rep.
1
50.8
)
37.6
)
Rep.
2
38.7
) 41.8
20.3
)
R ep .
3
35. 8 )
19.1
)
35.8
)
27.6
R ep .
1
2
25.3
) 26 . 9
13.8
Rep.
3
19.6
)
�
l1nlrc.:ttod
R ep .
2
10.9
67.1
17.1
)
14.2
) 10.7
52 . 3
)
69 . 8
) 47.5
)
63 . 0
4 1 . !J
)
73.0
) 81 .4
59 . 5
o.8
)
2� . �
39.4
)
1 00 . 0
22.6
) 27.6
72.2
20 . 7
)
71 . 9
)
6Li , 4
)
25.6
)
51.8 )
) 1 5. 8
1 1 .2
) 13.1
42.5 )
)
19.4
25.7
)
58 . 2
)
65 . 6
)
52.2
+
6.0
2.4
)
61.5
37.9
)
59 . 1
35.9
Control
-
40.3
28.4
27.9
76 . 3
+ protein
-
29 . 1
22.8
23.7
62.2
73 . 1
+ energy
-
29 . 3
25.7
22.6
77.5
70 . 0
Control
+
32 · ''
22.1
25.8
GS.O
G8 . 1
+ p r o tein
+
36.5
28.9
31.1
?�.s
73 . Sf
+ energy
+
42.9
32.3
28.0
90 . 4
80 . 6
5[
Treatment s i g n i ficance
:5 . 1
NS
1 .0
�.2
NS
NS
., . r..,
IJS
68 . 6
L.�
115
Methionine s i gnificance
NS
NS
NS
NS
IJS
Ani�1�l s i g n i f ic�nce
*
**
NS
rJc
''
I '"
Ts�le
3.14
The ef fec t of formalin-tre atment of s i l age and suppl ementation o f un treated s i l age d i e t s
l. r 1 : : . t. 1 : r l ' · ; · · · ' · i n
,
nnr�r 'rt ;,r.d pn · : t. 1 1 1m i n: • l
0�, energy and N, and an N balance.
E>ot.
�
Treatment
Me thionine
Q!l%
(1/r
,, nd rlund • ! ll,, l ) mnt.h i n n i r 1 1 •
Q!l%
�
Mean
nn
l l11'
�
:lpp,, r r n t
!:!%
Mean
w i th
farral dehyje-
rl i nr:: t i l, i l i h
nf
N b a l a �o c e
Mean
l.>JL!cl
h e an
Untre ;::. ted
Rep.
Re� .
r� c. ; J •
71 . 3 )
63.0 )
73 . 3 ) 7 1 . 2
70 . 1 )
69.1 )
66.4 )
6 3 . (, ) 6 7 . 4
6� . 1 )
73 . 7 )
66 . 2 ) 70 . 5
71 . 5 )
69 . 6
)
62.3
)
67.J
)
70 . 9 )
74 . 0 )
71 . 0
)
71 . 1
)
1
68.8 )
2
3
70 . 6 ) 68 . 5
66 . 0 )
1
70 . 6 )
07. � )
68 . 4
64.3 )
66.4
63. 0
70 . 8
:; . 1 )
) 67 . 6
-1 . 4 ) 1 . 1
1 .6 )
Treated
Rop .
R r: p .
ll t..: f.J ·
2
3
)
51.0 )
6U.4
58 . 1
U n t r f"! ;} tf"!d
Rep.
Rep.
r-u�p .
1
70 . 5 ) 70 . 5
3
70. 0 )
Control
+
protein
+ e:ner g y
Control
+
protein
+ ener gy
+
2
-
-
-
73 . 4 ) 7 3 . 7
73 . 6 )
67 . 8 ) 7 0 . 0
70.9 )
6 9 . 6 ) 69 . 3
67.5 )
3.3 )
1 . 6 ) 2.5
2.6 )
3.2 )
1 . 9 ) 2.8
3.2 )
67 . 8
75 . 5
72 . 6
66 . 0
71 . 7
79 . 3
76.3
74 . 8
3.5
72 . 9
79 . 6 1
75.7
66 .
4
0.1
-0 . 6
+
70.8
76 . 7
75.1
69 . 7
+
72 . 3
79.8
77 . 1
75.8
5.3
+
73 . 7
80 . 1
76 . 9
67.5.
4.0
SE
0 . 97
o . 58
0.81
Treatment significance
NS
**
NS
Methionine significance
NS
NS
NS
1 . 25
2,3
0 . 67
**
*
NS
-
f1f'l ,
74
Tab l e 3 . 1 3 giv e s t h e e f f e c t o f f o rmal i n-treatme n t o f s i l ag e a n d
s up p l em e n t a t i o n of u n t r e a t e d s i l a g e w i th p r o t e i n , e n e r g y a n d meth i o n i n e
o n t h e p e r c e n t age o f n u t r i e n t i n t a k e and d uo d e n al
in t h e intestines.
Th e p e r c e n t ag e o f
N
N
wh ich w a s d i g e s t e d
entering the duodenum , which
w a s d i ges t e d i n t h e int e s t i n e s in E xp t . 1 , w a s sim i l ar o n all t r e a t m e n t s
i n r e p l i c ates 1 and 2 , b u t t h e r e w a s a m a r k e d d ec r e a s e on u n t r e a t e d
s i l a g e al o ne and � i t h m e t h i o n i n e i n f u s io n i n r e p l ic a t e 3 .
Th i s
d if f e r e nc e in r e p l i c at e 3 was al s o a p p a r e n t in th e p e r c e n tage o f i n t a k e
w h i c h was d i g e s t ed i n th e i n t e s t i n e s , f o r a l l n u t r i e n ts .
Th e v a l u e s
f o r d i ges t ion o f O M a n d e n e r g y w e r e p ar t i c u l a r l y l o w a n d s u g g e s t th a t
meas u r eme n t o f d uo d e nal f l ow i n t h e s e two s h e e p was i n acc u r a t e .
H owe v e r , f o rmalin-t r e atme n t o f s il a g e s t i l l appear e d t o i n c r ease t h e
perc e n tage of OM , O M , e n e r g y and
N
i n take wh ich was d ig e s t e d i n th e
i nt e s t i ne s .
I n E x p t . 2 th e r e was no e f f ec t o f s up p lemen t a t i o n o r meth i o n i n e
i n f u s i o n o n t h e perc e n t a g e o f i n t a k e dig e s t e d in t h e i n t e s t ine s .
Ho we v e r , t h e re was a s i g n i f ic a n t d i f f e renc e b e t we e n anim als i n t h e
perc e n tage o f O M ( P < 0 . 0 5 ) a n d O M in take ( P < 0 . 0 1 ) d i g e s t e d i n t h e
intes tines
•
.
The appar e n t d i ges t i b i l i t i e s and
s up p l e me n t s are giv e n i n T ab l e 3 . 1 4 .
N
b al ance o f t h e s ilage d i e t s and
I n E xp t .
1,
N
d i g e s t i b i l i t y was
The a v e r age N b a l a n c e
l owe r on treated s i l a g e t h a n un t r e at e d s i l age .
app e a r ed t o i n c r e a s e i n r e s p o ns e t o b o th t r e atme n t o f s i l a g e a n d
me th i o ni n e , b u t t h e r e was c o ns i d erable v a r i a t ion w i t h i n t r e atme n t s .
In E x p t . 2 , th e r e w a s n o s i g n i f i�ant e f f e c t o f m e t h i o n i n e i n f us io n
on
N
dig e s t i b il i t y .
E n e r g y and p r o t e i n s up pleme n t s b o th inc reas e d O M
d i g e s t i b i l i t y ( P < 0 . 0 1 ) a n d p r o t e i n inc r e as e d
N
d i g e s t i b i l it y .
N
b a l a n c e was s i g n i f ic a n t l y i n c r e a s e d b y s up p lemen t s ( P < 0 . 0 5 ) a n d b y
75
meth i o n i n e ( P < 0 . 0 1 ) .
3.4
Di s c u s s i o n
A . I n tro d uc t i o n
T h e r e p o r t b y B ar r y e t al . ( 1 9 73 ) th a t b o t h f o rm a l i n- t r e a t m e n t o f
s ilage a n d I / P m e th i o n i n e s u p p l e m e n t a t i o n o f u n t r e a t e d s i l age i n c r e a s e d
i n take i n s h e e p , s u g g e s ts t h a t t h � am i n o ac i d s t a t u s o f s h e e p f e d
s il ag e m a y b e a n i m p o r t ant f ac t o r i n c o n t r o l l i n g i n t a k e .
T h u s , th e
main aim o f E x p t . 1 was to c om p a r e th e e f f ec ts of f o rm a l i n-t r e a tm e n t o f
s il a g e a n d I / P m e t h i o n i n e s u p p l e m e n t a t i o n o n th e d i g e s ti o n a n d u t il is­
a t i o n o f N by s h e e p .
Th e f l ow r a t e s o f amino ac i d s i n t o th e d u o d e n um
i n E xp t . 1 o n l y r a n g e d from 3 3- 7 2 g/ d a y and th us two o t h e r m e th o d s o f
f u r t h e r i n c r e a s i ng t o t al ami n o ac i d a v a i l a b i l i t y w e r e s t u d i e d i n E x p t .
2.
T h e s e w er e :
s u p p l ements of f o rmal d eh yd e- treated c as e i n , t o e n s u r e a
h i gh f l o w o f amino ac i d s i n t o th e d u o d e n um, and rum inal inf u s i o n o f
r e a d i l y a v a i l a b l e e ne r g � t o s ti m u l a t e m i c r o b ial p r o t e i n s y n th e s i s .
An
i n c r e a s e d f l o w o f t o t a l ami n o ac i d s i n t o th e d uo d e n u m is us u al l y
a s s o c i a t e d w i th a n inc r eased f l o w o f m e t h i o nine .
Th us , t h e e f f ec t o f
i nc r e as i n g m e t h i o n i n e t h r o u g h i nc r eas i n g th e to tal f lo w o f a m i n o ac i d s ,
was a l s o c om p a r e d w i t h d i r e c t i n f u s i o n o f m e th i o n i n e i n t o th e p e r i t o n e a l
c av i t y ( E x p t . 1) o r d u o d e nu m ( E x p t . 2 ) .
B . F eed c ompo s it io n
F o rmalin- t r e a t m e n t of s il a g e i n E x p t . 1 did n o t a p p e a r to d e c r e a s e
th e f e rme n t a t i o n o f s o l u b l e c a r b oh y d r a t es , s inc e th e w a t e r s ol u b l e s u g ar
c on t e n t o f b o th s i l a g e s was low c om p a r e d t o th at o f h e r b a g e ( T ab l e 3 . 4 ) .
H owe v er , th e h i gh e r amino ac id c o n t e n t of t r e a t e d s il age i n d ic a t e d
i nh i b i t i o n of p r o t e i n and/ o r amino ac i d d e grad a t i o n d u r i n g e n s i l i n g .
Th e ami n o a c i d c om p o s i t io n o f t h e o r i g i nal h e r b ag e s e ns il e d was n o t
availab l e , b u t s in c e th e am i n o ac id c ompos i t io n o f h e r b a g e s i s f a i r l y
c on s t a n t ( Mac R a e a n d U l yat t , 1 9 74 ), t h e c om p o s i t i o n o f th e t r e a t e d a n d
76
u n t r e ated s il�ges w a s c ompar e d w i th t h e c o mp o s i ti o n o f p r o t e i n f r om
s im i l ar h er b a g e ( T a b l e 3 . 5 , U l y a t t e t al . , 1 9 7 5 ) .
Th i s c o m p ar i s o n
i nd i c ated t h a t t h e a m i n o a c i d c om p l em e n t o f t r eated s i l age w a s p r o b a b l y
c l o s e r t o t h a t o f h e r b a g e th a n t o u n t r ea t e d s ilage .
H e n c e f o rma l in­
t r e a t m e n t did a pp e a r t o b e e f f ec t i v e i n p ro t e c t i n g h e r b a g e p r o t e i n f r om
d e g r ada t i o n du r i n g e ns i l i n g .
Th e ami n o ac i d c ompos i t i o n o f th e c o n t r o l
s i l a g e ( E x p t . 2 ) w a s c l o s e r t o t h a t o f u n t r e a t e d s il a g e ( E x p t . 1 ) t h a n
t o h e rbage ( T a b l e 3 . 5 ) .
The ma i n d i f f e r e n c e b e tw e e n t h e c o n t r o l s ilage ( E xp t . 2 ) a n d t h e
u n t r e ated and t r e a t e d s i l a g e s ( E x p t . 1 ) , was i n th e c o nc e n t r a t i o n o f
amin o ac i d s ( Ta b l e 3 . 4 ) .
Th u s , w h i l e o n l y 50 . 9% o f th e to tal
N
in
u n t r e ated s il a g e was i n th e f o r m o f amino a c i d s , 74 . 6% o f th e
N
was in
t h e f o rm o f ami n o ac i d s i n t h e c o n tr o l s il a g e ( Exp t . 2 ) .
Th e m a i n
e f f e c t o f f o rma l i n- t re atm e n t o n s i l a g e c om pos i t i o n w a s t o inc r e a s e t h e
ami n o ac i d c o n t e n t .
S uc h d i f f e r e nc e s in ami n o acid c o n t e n t b e t w e e n t h e
t w o d i f f e r e n t u n t r e a t e d s i l a g e s , m a y p a r t i a l l y explain some of t h e
r e p o r ted v a riab i l i t y i n r e s p o n s e s to fo rmal i n- t r e a t m e n t o f s i l a g e
( V a l e n ti n e and B ro w n , 1 9 7 3 ;
B a r r y a n d F e n n e s s y , 1 9 73 ) .
H o w ev e r , t h e
s il a g e s u s ed i n E x p t s . 1 and 2 w e r e m ade f r o m d i f f e r e n t h e r b a g e s, a n d
t h e amino ac i d c o n t e n t o f th e o r i ginal h e r b a ges w a s u n k n o w n .
·
The r e l a t i v e p r o p o r t i o n s o f p r o t e i n a n d f r e e am ino a c i d s in th e
s il a g e ar e n o t re p o r t e d , s i n c e c o n tradic t o r y r e s ul t s w e r e o b t a i n e d .
E x t r ac t io n of t h e s il age " p r o t e i n " w i th s o d i um d o d e c y l b e n z e n e s u l p h o n a t e
( SOS ) was a t t e mpt e d , b u t n o p r o t e i n w a s d e t ec te d .
e x t r actio n o f t h e
"r�PN"
O n th e o th e r h a n d ,
in alc o h o l i n d i c a t e d th at t h e t o tal amino a c i d
c o n t e n t w a s d e r i v e d f ro m p r o t e i n .
C . I n take
I n b o th e x p e r im e n t s , i n t ak e s were r e s t r ic te d , s o that c omp a r i s o ns
b e twe e n t r e a t me nt s c o u l d b e m ade a t th e s am e l e v e l o f i n t a k e .
The
77
l imit o n s i l age i n t a k e was c h o s e n t o p r o v i d e n u t r i e n ts a t a l e v el
s ligh t l y a b o v e mai n t e n a nc � .
H ow ev e r , th e d a i l y r a t i o n was s el d om
c ompl e t e ly e at e n , r e f u s al s b e i n g p a r t ic u l arly h i gh o n th e c on t r o l
s i l a g e i n Exp t . 2 .
M e t ab o l i s ab l e e n e r g y ( M E ) i n t a k e s w e r e . c o m p a r e d
w i th mai n t e nanc e r eq u i reme n t s f o r M E , a s p r o p o s e d b y th e A RC ( 1 9 65 )
manual o n th e n u t r i e n t r e q u i r e m e n t s f o r ruminants ( T a b l e 3 . 7 ) .
Th is
i n d i c a t e d th a t i n t a k e s i n E x p t . 1 · we r e s ti l l s l i gh t l y a b o v e m a i n t e n a nc e ,
wh i l e th e i n t ake o f th e c o n t r o l s il a g e i n Ex p t . 2 w as v e r y n e a r to
m a i n t e n an c e .
The s u p p l eme n t s i n Exp t . 2 inc reas e d i n take t o g r e a t e r
t h a n m ai n t e nanc e .
T h e d ai l y o u t p u t o f m e t h ane was m e a s u r e d i n E x p t . 1 b u t n o t E xp t .
2.
M e t h a n e o u t p u t was c al c u l a t e d f o r b o th experime n t s and c om p a r e d
w i th t h e o b s e rv e d v a l u e s f r o m E x p t . 1 .
Wh ile th e r e was s o m e d i f f e r e n c e
b e tw e e n t h e c al c u l a t e d a n d o b s e r v e d v a l u e s , t h e e r r o r wh ich th is w o u l d
i n t r o d uc e int o th e c al c u l a t i o n o f M E i n t a k e , would b e sm all ( app rox . 3 % )
d u e t o t h e l o w l e v e l o f me t h a n e o u t p u t c ompared t o t o t a l M E i n t a k e .
R e f u s als in E x p t . 1 w e r e l o w and no t r eatme n t d i f f e r e nc e s i n OM
i n t ake we r e appar e n t ( Tab l e 3 . 6 ) .
How ev e r , B ar r y ( 1 9 7 5 , 1 9 7 6 ) did
r e p o r t i n c r e ased i n t ake o f t h e s e s ame s i l ages in r e s p o n s e t o f o rm alin­
t r e atme n t and I/P m e t h i o n i n e i n j ec t i o n .
Th e h yp o t h e s is t h a t th e amino
ac id s t a t us of i n d iv i d u a l s h e e p w i l l h av e a mar k e d e f f e c t on t h e i n t ak e
o f s il a g e , is dis cus s e d i n C h ap t e r 4.
Th e increas e d c o nc e n t r a t i o n o f
a m i n o ac i ds in t r e a t e d s i l a g e d i d res u l t in a h i gh e r i n t a k e o f amino
a c i d s r e l a t i v e to u n t r e a t e d s i l a g e .
In E x p t . 2 , t h e r e was a s i g ni f ic an t e f f ec t o f b o th s up p l e m e n t a t i o n
and m e t h i o nine i n f u s i o n o n s il a g e OM i n t a k e ( P < 0 . 0 1 ) .
T h e i nt ake o n
th e c o n t r o l s il a g e was mai n t ai n e d n e a r t o t h e maximum f o r t h e f i r s t t e n
d a y s ( F i g . 3 . 3 ) a n d t h e n s h o w e d a m a r k e d d e c l i n e u n t i l t h e s ta r t o f
m e th i o n i n e inf us i o n .
D u r i n g i n f us ion , i n take inc r e a s e d r a p id l y , b u t
78
u n d e r th e l im i t s i mp o s e d b y
th e e x p e r i m e n t ,
intake did not r e tu r n to
t h e s am � l e v e l as a t th e s t a r t o f s i l a g e f e e d i n g .
i n t a k e w a s s i g n i f ic a n t l y r e d uc e d ,
p r o t e i n s u p p l em e n t a t i o n
Th e d e c l i n e i n
b u t n o t e l im i n a t e d b y e n e r g y a n d
(Fig. 3 . �
a n d i n c r e a s e d i n t a ke
r e s p ons e to me th i o nine was s ti l l p r e s e n t .
in
A similar d e c l i ne in i n t a k e
w i th t i m e o n u n t r e a t e d s i l a g e d i e t s w a s r e c o r d e d b y B a r r y e t a l .
a� d B a r r y
I /P
( 1 976 ) .
( 1 97 3 )
Th is d e c l i n e w a s a t l e a s t p a r t i al l y p r e v e n t e d b y
m e t h i o n i n e a n d f o rm a l i n- t r e a t m e n t o f s i l ag e .
Th e s e r e s u l t s s u g g e s t
t h a t th e p e r c e n t a g e o f m e t h i o n i n e i n t h e t o t a l a m i n o a c i d s a b s o r b e d b y
the animal o n th e c ontrol s i l a g e ,
d i d n o t c o rres pond t o th e p erc e n t a g e
o f m e t h i o n i n e i n t h e t o t al a m i n o a c i d s r e q u i r e d b y th e a n i m a l .
an a m i n o a c i d i m b a l a nc e w a s p r o d uc e d .
Thus ,
B al an c e a p p e a r e d t o b e p a r t i a l l y
r e s t o r e d a t h i g h e r l e v e l s o f a m i n o ac i d a v a i l a b i l i t y ,
i.e.
w i th p r o t e i n
and ene r g y s up p lementation .
Th e r e w as a s i g n i f i c a n t d i f f e r e n c e b e t w e e n s h e e p i n v o l u n t a r y O M
intake i n Exp t .
2 (P
<
0 .01 ,
1).
A p p endix
Th is d i f f e r e n c e m a y b e d u e
t o d i f f e r e nc�s i n a m i n o ac i d s t a t u s b e t w e e n s h e e p a n d t h i s
d i s c u s s e d i n t h e f o l l o w i n g c h ap t e r ,
i n r e l a t i o n t o th e
treatment effect on intake in Expt .
1,
on intake in Exp t .
point is
absence of a
c o m p a r e d to t h e t r e a t m e n t e f f e c t
2.
Th e i n t a k e d i f f e r e n c e s i n Exp t .
2
m i g h t b e e x p e c t e d t o h av e a n
i mp o r t a n t e f f e c t o n o th e r p a r am e t e r s m e as u r e d ,
t h r o u gh
a direct
i n f l u e n c e o n t h e r a t e o f f l o w o f n u t r i e n t s t h r o u g h t h e d i g e s t i v e t r ac t
( G r o v um
and H e c ke r ,
1 9 73 ;
U l y a t t a n d M ac R a e ,
1 9 74 ) .
H e nc e ,
t h e e f f ec t
o f t h e t h r e e s u p p l e m e n t s i n i n c r e a s i n g i n t a k e s h o u l d b e r em e m b e r e d
d u r i n g t h e f o l l o w i n g d i s c us s i o n o n th e d i g e s t i o n o f s i l a g e .
79
D.
D i ge s t i o n o f s i l age
(1 )
R um e n v o l a t i l e f a t ty ac i d a n d a mm o n i a c o nc e n t r a t i o ns
T h e r e � e r e no m a r k e d t r e at m e n t d i f f e r e nc e s in r um e n v o l a t i l e f at t y
(VFA )
acid
VFA
1 ( T abl e 3 . 8 ) .
c o n c e n t r ation i n Exp t .
2,
I n Exp t .
t o tal
c o nc e n t r a t i o n i n t h e r u rn e n w a s s i g ni f i c a n t l y i n c r e as e d b y b o th
( P < 0 . 01 ) .
e n e r g y a n d p r o t e i n s u p p l em e n t � t i o n
The h i gh e r i n t a k e s b n
t h e s e s u p p l em e n ts w o u l d h av e c o n t r i b u t e d t o t h i s i n c r e as e i n
co n c e n t r a t i o n and
i n additi o n ,
rapidly d e g rad e d t o
VFAs .
total
B ath
VFA
t h e e n e r g y s up p l e m e n t w o u l d h a v e b e e n
T h e r e w a s n o s i g n i f i c a n t . t r e a tm e n t e f f e c t o n
rume n amm o n i a c o n c e n t r a t i o n ,
b e tw e e n a n i m a l s i n b o t h
VFA
b u t th e r e w a s a s i g n i f i c an t d i f f e r e n c e
amm o n i a a n d
VFA
(P < 0.01 ) .
c oncentrat i o n
Th e
,
and a m m o n i a c o nc e n t r a t i o n s w e r e l o w e r t h a n th o s e r e c o r d e d b y
and R o o k
may b e due
( 1 965 )
to t h e
and Chalmers
( 1 9 63 )
r e l a t iv e l y l o w i n t a k e s ,
also f o r s il age d i e t s .
Th is
and t o t h e c o n t i n u o u s f e a d i n g
s y s t e m u s e d i n th i s e x p e r i m e n t .
T h e h i gh
diets
(e . g .
r um i n al p r o p o r t i o n o f a c e t a t e o n s i l a g e c o m p a r e d t o o t h e r
ac e t a t e
60 %
o n p as t u r e ,
U l y a t t and H e n d e r s o n ,
affec t th e amino a c id n utrit ion o f ruminants ,
p r o t e i n s y nth e s i s h as b e e n f o u n d
e ne r g y s o u rc e s
( Po tter
et al . ,
s ince t h e r a t e o f
to v a r y i n r e s p o n s e
1 9 68 ;
1 968 )
may
t is s ue
to d i f f e r e n t
E s k e l and e t a l . ,
1 9 74 ) .
I n t r a-
a r t e r i al i n f u s i o n o f p r o p i o n a t e l e d t o a g r e a t e r d e c r e as e i n p l as m a
essential
am i n o a c i d c o nc e n t r a t i o n th a n a s im i l a r i n f us i o n o f
o r b u ty r a t e .
H ow e v e r ,
acetate
a m i n o ac i d s r a t h e r t h an e n e r g y ap p e ar t o l i m i t
t h e r a te o f l i v e -w e i g h t g a i n of a n i m a l s f e d s i l ag e
H e nc e th e h i g h p r o p o r t i o n o f ac e ta t e m a y o n l y b e
wh i c h p r o m o t e a h i gh f l o w o f
( M c C a r r ic k , 1 9 66 ) .
1-blevarrf.
iHlpef'te�Flt,\ o n
am i n o ac i d s i n t o t h e d u o d e n u m .
s il a g es
80
T h e r e w e r e m a r k e d d i f f e r e nc e s b e tw e e n t h e m o l ar p r o p o r t i o n s o f
1
a c e t a t e a n d p r o p i o n a t e b e tw e e n E x p t s .
and propionate l o w e r in E xp t .
1
and
2
w i th ac e t a t e b e i n g h i g h e r
c ompared t o Exp t .
2.
d i f f e r e n c e s i n t h e V F A c omp o s i t i o n o f th e t w o s i l a g e s .
These may reflect
However ,
c om p l e t e a n a l y s i s o f t h e v o l a t i l e ac i d s w a s n o t c o n d uc t e d o n t h e s e c o n d
s ilage ,
s i n c e t h e m e t h o d w a s n o t a v a i l a b l e a t P a l me r s t o n N o r th .
c h a n g e in t h e p r o p o r t i o n of
propionate in th e t otal V F A s migh t also
h av e h ad a n e f f e c t o n t h e a m i n o a c i d n u t r i t i o n o f th e a n im a l s ,
g l uc o n e o g e n e s i s p l a y s
B e r gm a n e t al .
( 1 9 70 )
Th e
since
a v i t a l r o l e i n e n e r g y m e t ab o l i s m i n r um i n an t s .
20 - 4 0 %
h av e s h o w n t h a t
of d a i l y g l uc o s e
p ro d u c t i o n w a s o b t a i n e d f r o m a b s o r b e d p r o p i o n at e ,
and a decrease in
p r o p i o n a t e av a i l ab i l i t y m i g h t b e e x p e c t e d t o i n c r e a s e th e c o n t r i b u t i o n
o f a m i n o ac i d s t o g l uc o s e p r o d uc t i o n .
acids h as been r e p o r t e d
to
1 973 ;
1 97 1 ) .
Reilly and F o rd ,
r um i n a n ts f e d s i l a g e i s n o t
( 2)
G l u c o s e produc t i o n f r o m amin o
1 1 - 3 0% ( W o l f f
range from
However ,
a n d B e r g ma n ,
d a t a o n g l uc o n e o g e n e s i s i n
yet availabl e .
Appa r e n t d i g e s t i o n o f n u t r i e n t s i n th e s t o m ac h s
T h e p e rc e n t a g e o f N i n t a k e w h i c h w as d i g e s t e d i n t h e s t o m a c h
r e g i o n in Exp t .
1,
appeared to differ between the treated and untreated
s i l a g e s ( T ab l e 3 . 9 ) .
N t o t h e rume n ,
O n th e t r e a t e d s i l a g e th e r e w a s
wh i l e o n the untreated s il age ,
a net addition of
th e r e w as a n e t l o s s .
A
s im i l a r e f f ec t o f f o r m a l i n- t r e a t m e n t o f s i l a g e was r e p o r t e d b y T h o m s o n
et al.
( 1 9 73 ) .
D e s p i t e t h e d i f f e r e n c e s i n t h e · ami n o a c i d - N p r o p o r t i o n
i n t o t al N i n t h e t w o s i l ag e s ,
N
t h e p e r c e n t a g e s o f am i n o ac i d- N i n t o t a l
e n t e r i n g th e d u o d e n u m w e r e n o t a p p r ec i a b l y d i f f e r e n t b e t w e e n t r e a tm e n t s .
A c o m p a r i s o n o f t h e am i no a c i d - N
c o n s um e d ,
( AAN )
a n d e n t e r i n g t h e d u o d e n um ,
a n d n o n-am i n o a c i d- N
( NAAN )
s h o w e d th a t th e m a i n e f f e c t o f
f o rm a l in- t r e a t m e n t o n N d i g e s t i o n i n t h e s t o m ac h s ,
occ urred in the NAAN
81
f rac ti o n .
Th e ratio o f N A A N a t the d u o d e n u m to N A A N i n t h e f ee d , was
h ig h e r on the treated s il a g e th a n u n t r e a t e d sil age .
T h i s m ay b e d u e t o
t h e l o w N A A N i n t ake o n th e t r e a t e d s il age ( 1 . 2g /day , T a b l e 3 . 1 0 )
c om p a r e d t o t h e u n t r e a t e d s i l a g e ( av . 7 . 2 g / d ay ) .
A p o s s i b l e e x p l an­
a t i o n m i g h t be that amm o n i a p r o d uc t i o n in the r um en f r o m t r e a t e d s il a g e
w a s low , a nd m i g h t t h e r e f o r e f av o u r th e t r a n s f e r of u r e a a c r o s s th e
r �m e n wall f r o m th e p l as ma t o t h e rum en ( H o u p t and H o u p t � 1 9 6 8 ) .
The
h i gh e r i n t a k e o f N A A N o n t h e u n t r e a t e d s i l a g e , o n t h e o th e r h a n d , w o ul d
l e ad t o a h igh e r ammo n i a c o n c e n t r a t i o n w i th in the rum e n , and this m i gh t
r e s u l t i n a b s o r p t i o n o f ammo n i a f r o m t h e r u men .
Th e a p p a r e n t d i g e s ti o n o f O M and e n e r g y in th e s t o mach r e gi o n ,
a l s o a p p e a r e d to b e l o w e r on f o rmalin-tr e a t e d than o n u n t r e a t e d s i l ag e .
Th i s m i g h t s u gge s t t h a t s o m e f r e e f o rmaldeh y d e was p r e s e n t i n t h e
s il a g e , t h u s d e p r e s s i n g mic r o b i al ac t i v i t y with in th e r ume n ,
or ,
a l t e r n a t i v e l y , t h e r a t e o f p as s age t h r o u gh the rumen m i g h t h av e b e e n
i n c r e as e d .
H owev e r , th e r e was c o ns i d e ra b l e variab i l i t y w i th i n t r e a t­
men t s .
I n E x p t . 2 , th e r e was a s i gn i f i c ant e f f e c t o f s u p p l e m e n t s o n t h e
p e r c e n t age o f N i nt a ke d i g e s t e d in t h e s t omachs ( P � 0 . 0 5 , T a b l e 3 . 9 ) .
T h is was d u e to a s i g n i f i c a n t d if f e r e n c e b e tween th e n e t add i t i o n o f N
t o t h e r um e n with t h e e n e r g y s uppleme n t , and a n e t l o s s af N f r o m t h e
r um e n w i t h th e p r o t e i n s u p p l e m e n t .
N e i t h e r s uppleme n t was s i gn i f ic an t l y
d i f f e r e n t f r o m th e c on t ro l s il a g e alo n e .
C om �aring t h e r a t i o s o f A A N
a n d N A A N i n f e e d and a t th e d uo d e n um ( Tab l e s 3 . 1 0 and 3 . 1 1 ) s h ow e d t h a t
t h e m a i n e f f e c t o f p r o t e i n s up p l em e n t a t i o n was on d i g e s t i o n o f N A A N .
T h e f o r m a l d eh y d e c a s e i n s u p p l e m e n t s i g n i f i c antly d e c r e as e d t h e f l o w o f
N A A N r e l a t i v e to c o n t r o l s i l a g e a l o n e ( P ( 0 . 0 1 T a b l e 3 . 1 0 ) .
The flow
o f N A A N i n t o t h e d u o d e n um o n t h e c o n t rol p l us pro t e i n d i e t w a s als o l e s s
82
t h a n t h e c o rr e s p o n d i n g N A A N i n t a k e , s u gges t i n g th a t f o rm al d e h yd e-c a s e i n
migh t h a v e i n c r e a s e d t h e ab s o r p t i o n o f ammo nia f r o m th e r um e n .
no e v i d e n c e f o r t h i s is availab l e .
H o w ev e r ,
Th e i n c reased n e t l o s s o f N A A N i n
t h e s t omach r e g i o n o n c a s e i n s up p l eme n t ed s ilage , c o n t r i b u t e d t o t h e
s i g n i f i c a� t i n c r e a s e i n p e r c e n t a g e o f ami n o ac ids i n t h e t o t a l N
e n t e r i n g th e d uo d e num ( P < 0 . 0 1 ) r e l a t i v e t o b o th t h e c o n t r o l a n d
e n e r g y s u p p l e m e n t e d animal s .
Th e p e r c e n t a g e o b tained ( m e an 9 3 % ) was
c ons i d e r a b l y h i gh e r th an th e 79 . 5 % o b t a i n e d by MacRae et al . ( 1 9 72 ) wh o
s up p l e m e n t e d a d r i e d grass d i e t w i t h 6 0 g/d a y o f th e s am e f o r m a l d eh y d e­
t r e a t e d c as e i n .
E n e r g y s up p l em e n t a t i o n i n E x p t . 2 appeared to inc r e a s e t h e r a t i o
o f d u o d e n al A A t o A A i n t ake , b u t th e d if f e r e n c e was n o t s i g n i f ic an t .
Th e r e w a s a s ig n i f i c an t d i f f e r e n c e b e t we e n animals ( P < 0 . 0 1 , Tab l e
3.11 ).
Th is m i gh t s u g g e s t th a t t h e e n e r g y s upplement w a s s t im u l a t i n g
mic r o b i a l p r o t e i n s y n th es i s a n d e v i d e n c e f o r t h is c o m e s f r o m t h e
inc r e as e d p r o p o r t i o n o f ami no ac i d s i n t h e to tal N e n t e r i n g t h e d uo d e num
( Table 3 . 1 1 ) .
Th is r a t i o was s i g n i f i c a n t l y i n c r e a s e d b y e n e r g y s up p l e­
m e n t a t i o n r e l a t iv e t o t h e c o n t r o l , i n d ic at i n g that th e s ta r c h / s u c r o s e
i n f u s i o n w a s e f f e c t i v e in i n c r e a s i n g t h e m i c r o b ial u t i l i s a t i o n o f N A A N
within the rumen .
T h e r e was also a s i g n i f ic a n t i n t e r ac t i o n b e t w e e n th e e f f e c t o f
s u p p l e m e n t at i o n wi t h e n e r g y and p r o t e i n , and t h e e f f e c t o f m e t h i o n i n e
inf u s i o n o n t h e r a t i o o f A A e n t e r i n g t h e d u od e num to A A i n t a k e
(P
<
0 . 01 ) .
This r a t i o d e c r eas e d i n r e s p o n s e to m e th io n i n e i n f u s i o n
i n s h e e p f e d c o n t r o l s ila ge a l o n e , a n d inc r e as e d in r e s p o n s e to
m e th i o n i n e i n f us i o n i n s h e ep f e d s up p l em e n t e d s il age d i e t s .
This
r e s u l t s u g g e s t s d i f f e r e nt e f f e c ts o f m e th i o n i n e o n N r e c y c l i n g to t h e
r ume n, o n s u p p l em e n t e d and u n s u p p l e m e n t e d d i e ts .
T h i s i n t u r n m i gh t
83
r ef l e c t c h anges i n th e d i s t r ib u t i o n o f m e t h i o nine b e tw een i t s r e q u i r e­
m e n t for p r o t e i n s y nth e s i s , and f o r e x t r a- p r o t e i n f u n c t i o n s ( A g u il a r
e t a l . , 1 9 72 ) , d e p e n d e n t on the l e v e l o f am ino ac ids a v a i l a b le t o t h e
t i s s ues .
How e v e r , no d e f i n i t e e x pl an a t io n c an b e giv e n .
( 3 ) F l ow o f n i t roge n o u s c ompo und s i n t o th e d u o d e num
F o rma l i n- t r e a t m e n t of s il age ( Ex p t . 1 ) incr eas ed t h e f l o w o f t o t al
N a n d am i n o ac i d s i n t o th e d uo d e n um c o mpar e d to u n t r e a t e d s i lag e .
The
o n l y appr e c ia b l e d i f f e r e n c e i n i n t ake b e tw e e n t h e t r e a t e d a n d u n t r e a t e d
s il a g e s w a s i n ami n o a c i d i n take , w i t h th e e n ergy i n t a k e o f t r e a t e d
s i l a g e b e i ng s l i gh t l y l o w e r ( Tab l e 3 . 6 ) .
Th us , t h e i n c r e a s e d f l ow o f
ami no ac i d s i n t o t h e d uo d e n u m on t r e a t e d s il age app e a r s t o r es u l t f rom
b o th the inc r e as e d ami n o ac i d i n t a k e , due to protec t i o n of the h e r b a g e
p ro t e in a n d/o r ami n o a c i d s , f rom d e g r a d a t i o n during e n s il i n g , a n d t o
t h e d ec re a s e d p e rc e n t a g e o f N i n t ake appar e n tly d iges t ed i n th e s t omach
region.
T h e r es ul t s f r o m E x p t . 1 d o n o t p r o v i d e any evid e n c e a s t o wh e t h e r
t h e f o rmalin-t r e a t m e n t c o n t i n ued t o p r o t e c t the h e r b a g e p r o t ein f r o m
m i c r o b ial d e g r adat i o n i n th e rum e n .
P r o t ec t ion of p r o t e i n b y f o rm a l­
d eh y d e is ge n e r al l y t a k e n t o me an rev e rs i b l e b i nding o f t h e f o r mal­
d eh y d e to re·a c t i v e g r o u p s i n th e p ro t e i n .
T h r e e t y p e s of b o nd a r e
f o r m e d b etwe e n f o r m al d e h y d e and p r o te i n , a n d a t l e as t o n e o f t h e s e c an
b e r e vers e d b y a d e c r e a s e i n pH ( F r e n c h a n d Edsal l , 1 94 5 ) .
Wh i l e p r o­
t e c t ion of d i e t a r y p r o t e ins by r e ac ti o n w i t h formal d e h y d e h as b e e n
f o u n d t o p ro t e c t t h e p r o te i n f r o m rum e n m i c r o b ial d e gr ad a ti o n ( F e r g us dn
e t al . , 1 9 67 ;
Mac R a e e t al . , 1 9 7 2 ) , t h e d e c reas e in p H d u r ing e n s i l i n g
m i gh t b e e x p e c t e d t o r e v e r s e s o m e o f t h e f o rmal d eh y d e b o nd s .
H o w ev e r ,
a c i d t r e a t m e n t o f f o rmaldeh y d e-b o und p r o t e i ns h as al s o b e e n f o u nd t o b i n d
p a r t of t h e p r o te i n mo r e fi rmly ( B ow es et a l . , 1 9 65 ) .
Th e eff e c t o f th e
d ec r ease i n p H d u r i n g e ns i l i n g , o n f o r mal i n-treatment o f s i lage m a y t h u s
84
r e l e ase s ome f ree f o rmaldeh y d e and p r o t e i n , wh i l e a n o th e r part of t h e
prot ein
may
become
i r r e v e rs i b l y
bound .
H e nc e , Th o m s o n e t al .
( 1 9 7 3 ) f o und t h a t f o r m a l d e h yde- t r e a t m e n t o f s i l a g e d i d inc r e a s e th e
d ie t a r y N reac h i n g th e i n t e s t i ne s , b u t at t h e e xpens e of d e c r e a s e d
mic r o b i a l pro t e i n s ynth e s i s and d e c r e ased avai l ab i l i t y of t h i s N f o r
d i g e s t i o n i n t h e i n te s t i n e s .
A t tempts to meas u r e p r o te i n s y n t h e s i s i n
Ex p t . 1 w e re r e p o r t ed i n C h a p t e r 2 , b u t th e res u l t s w e r e i nc o n c l u s iv e .
H ow e v e r , dec r e as e d d i g e s t i b i l i t y i n t h e r umen was r e c o rd e d ( Ta b l e 3 . 9 ) .
Th e d i ges t i b i l i t y o f N i n t h e i n te s t i n e s ( Table 3 . 1 3 ) w i l l b e d is c us s e d
i n d e t a i l b e l o w b u t n o d el e t e r i o us e f f ec t o f f o rma l i n-tr e a tm e n t was
apparent .
Th e l e v el of f o rmali n-t r e a t m e n t used by T h o ms o n et al .
( 1 97 3 ) was not reported .
H owev e r , B r o w n and V a l e n t i n e ( 1 9 7 2 ) r e p o r t ed
t h a t f o rma l i n a ppl i c a t i o n r a t es o f 3 . 2 % o f the
OM
d e c reas e d
ad
lib .
i n t a ke r el a t i v e t o u n t r e a t e d s i l age, and th e s e w o r ke r s s u g g e s t e d t h a t
f r e e f o rmaldeh yde pres e n t i n th e s i l a g e c o uld h av e h ad an a dv e r s e e f f e c t
o n t h e r umen m i c r o b ial p o p u l at i o n .
R e v e r s al of b in d i n g i n th e s i l a g e
s t a c k w o u l d also h av e i n c r e a s e d t h e c o n t e n t o f f r e e f o rmal d e h y d e i n th e
s i l ag e a n d c o u l d h av e a d v e r s ely affec t e d d i ges t i b i l i t y i n th e i n tes­
t i n e s as d isc u s s e d ear l ie r .
T h e f a t e o f f r ee f o rmal d eh y d e w i t h i n t h e
d i g e s t i v e trac t d o e s n o t a p p e ar t o h a v e b e e n s t ud i e d .
L o w e r l e v e ls o f
f o rmal i n appl i c a t i o n t o s i l age h av e b e e n r e p o r t e d t o i nc r e as e l ive­
w e i gh t gain and w o o l g r o w t h ( B a r r y et al . , 1 973 ;
B a r r y , 1 9 75 ) .
Th us
d e l e t e r i o us e f f ec t s of the r e v e r sal of f o rm ald e h y d e-b i n d i n g i n t h e
s i l ag e s t ac k m a y o nl y b e a p p are n t a t h i gh r a t es o f a p pl ic a t io n .
Th e
l e v e l of appl i c a t i o n u s e d i n Ex p t . 1 ( 40% a t 4 . 7 6 1/ t o n f r e sh g r a s s ) d id
i n c r e as e the f l ow o f a m i n o a c i d s i n t o th e d uode n um ( T a b l e s 3 . 1 1 ) and
d i d not affect th e d i g e s t i o n of N i n t h e i n t es t ines ( Tab l e 3 . 1 3 ) .
This
s u g g e s ts th a t t h e main e f f e c t o f t h e f o rmal i n-t r e at m e n t o f s i l a g e f o r
t h i s exp e r im e n t was i n i n c r e as i n g ami n o ac i d i n t ake th r o u gh p r o t ec t i o n
85
o f t h e h e r b a g e p r o tein from d e g r a d a t i o n during ens i l i n g .
I n E x p t . 2 , th e f l ow . o f t o t a l N i n t o t h e duode n um was s i gnific antl y
i n c r e a s e d b y b o th supplements a n d meth ionine infus io n ( P
3.11).
<
0 . 0 1 , Tab l e
T h e f l ow o f amino acids i n t o t h e duoden um was a l s o s i gnific-
antly i n c r e a s e d b y b o t h s u p p l e m e n t s r e lative t o the c on t r o l , and b y th e
p r o t e in s u pplement r e l ativ e t o th e e n e r g y s u pplement
( P < 0 .01 ) .
M e th io n i n e i n f us i o n h a d no s i g n i f ic a n t effect on the f l o w o f amino
acids i n t o the d uode num .
H e n c e the proportion of amino ac ids i n t o tal
N at t h e d u o d e n um d ec r eased i n r e s p o n s e to meth ionine i n f us i o n
(P
<
0 . 05 ) .
Th e reas o ns f o r th i s d e c r e a s e a r e unkno w n .
I t migh t b e
s ug g e s t e d th a t increas e d i n t a k e c o u l d h av e influenced t h is ratio b y
a f f e c t i n g r e t e n t i o n time i n th e r umen ( Gr o v um and H e c k e r , 1 9 7 3 ) , b u t
th e w i th i n t r eatment d i f f e r e nces i n t h e ratio were l o w , d e s p i t e a h i gh
v ar i a t i o n in intake ( A p p endix 1 ) .
The e f f e c t o f intake o n t h e flow o f amino ac i d s i n t o t h e duode n um
w as also s t u d i e d through t h e r a t i o s o f duod enal A A t o A A intake , and
d uode nal AA t o N intake ( Ta b l e 3 . 1 1 ) .
Th e l atter r a t i o sh owed more
v ariability than the f o rmer, s u g g e s t i n g that the intake o f amino ac i d s
h ad a g r e a t e r e f f ec t on th e flow o f a m i n o ac ids i n t o t h e d uo d e num , than
did t h e t o t a l N intak e .
Th e l o w range of t h e ratio d uo d e n al AA t o A A
i n t a k e o v er t h e t w o exp e rime nts , l e d t o th e plottin g o f th e r e g r e s s i o n
b et w e e n amino a c i d int ake and duodenal ami n o a c i d f l o w ( F i g . 3 . 4 ) .
Th e
p o s i t i v e r e l ationship indic a ted that increas ed- ami n o a c id i n take
i n c r e a s e d the flow of amin o ac i d s into th e duodenum .
Howe v e r , t h i s
r el a t i o n s h i p applied o n l y t o b e t w e e n t r eatment d i f f e r e nc e s , and n o t t o
w ith i n t re atment d if f e r e nc e s .
t-d"l.on Sh tp
(/al't;;. .f, u- n
O . B,� , i n d i c a t e d th a.t diffeiellces 1 h n a m i n o a c i d i n t a k e
t/55()Ctaled writ.
VaNa ftin
FS§PSooion ,
was
T h e c o rrelat ion c oe f f i c i e n t f o r th e
aGG9unts9
r
..
=
fgrA6 7� of th e
diffoponsssA i n
amino acid f l ow i n t o th e
Table
3.15
The c o n c e n t r a t i o n of
a m i n o ac i d 5
in
fpods,
a nd t h e OM i n t a t<. e requ i r e d t o pr oduce
S i l ages
(ExBt s . 1
Untreated
Treated
and
1 DOg
the
r�tio
of
amino a c i d s
duodennl
am i n o ac i d s :
e n t e r i ng the
d u o d enL..n
�mina o�id
intake,
oer day.
2)
�I
�
!:! 1
!::!21.
�
�
I
6.0
15.0
1 3.0
I
2.7
I
667
� 1
D r i ed
2
Dr ied
Cr <J::; ::;
3
f"'lanawa
nyc:qr .:..�.::;
4
!!!b.lli 1
Red
�
22.1
14.4
Amino .J. C i d
c o nc e n t ra t i o n
i n feed (%
•
Rat1o
oF
of
7.8
OM )
AA!
AAD
0 . 70
OH i n t a k e required
ta � r oUuctl 1 0 0 y
a ;""l ino a c i d ::;; at
Data
3
4
"
"
from H a r r i s o n �·
"
"
MacRae
0 . 92
( 1 973 )
�· ( 1 97 2 )
Coelho d a
Silva
et a l .
l'lacRae and U l y a t t
1 . 03
830
856
1425
duodenum ( gDM/d )
1 1 .7
1 2. 7
( 1 974 )
( 1 97t• )
o.88
758
0.93
827
18.7
1 .02
525
14.5
0.95
742
1
C l ov l :r
0.63
11e
1 . 05
661
86
duodenum.
Th � r e g r e s s i o n w as c al c ul a t e d f o r a l l treatments i n E x p t s . 1
and 2 , s ince no t r e atment . e f f e c ts were s ignificantly d i f f e r e n t from t h e
o v e r a l l r e gression .
Howe v e r , e n e r g y s up p l ementation i n Ex p t . 2 did
a p p e ar t o increas e th e c o n v e rs ion of N A A N t o micro b i al p r o t e i n , as
d i s c u s s e d earl i e r .
Thus , v a riation i n e n e r g y i n t ake/ would also h a v e
c o n t r i b u t e d to t h e d i f f e r e nc e s in f l o w o f amino ac ids i n t o t h e duod e n u m .
Th e ratios of d uo d e nal amino ac ids t o amino ac id i n t a k e o n f r esh a n d
d r i e d pas ture/ a n d h a y d i e ts ( MacRae e t al . , 1 9 72 ;
1 9 73 ;
Mac Rae and U l y a t t , 1 9 74 ;
H a r r i s o n e t al . ,
C o elh o d a S i l v a e t al . , 1 9 73 , are
c ompared with th e data f r o m E x p ts . 1 and 2 , in Table 3 . 1 5 .
Th e ratio
v ar i e d from 0 . 6 o n a diet o f wh ite c l o v e r , to 2 . 7 on a diet o f l ow N
h a y s up p l emented with u r e a .
The v al u e s ob taine d f o r s i l a g e in t h e s e
e x p e r iments were s i milar t o t h o s e ob tained o n Manawa r y e grass ( MacRae
and U l y at t , 1 9 7 4 ) ;
d r i e d grass ( MacRae e t al . , 1 9 72
C o e l h o da S i l v a
e t al . , 1 9 73 ) and o n f r e s h r e d c l over ( H ar r i s o n e t al . , 1 9 7 3 ) .
C al c u l a t i o n of th e OM intake required to p r o d uc e 1 0 0 g amino acids
e n tering the d u o d e num was made using these ratios and th e c o n c e n t r a t i o n
o f a m i n o ac ids in t h e individ ual feeds ( Ta b l e 3 . 1 5 ) .
Th is c al c u l a t i o n
was l i m i t e d to t h e feeds h av in g s imilar rat ios to s i l a g e , and t o th e
two f e e d s with e x t r eme r a t i o s o f duod enal amino acids t o amino acid
i n ta k e .
The c a l c u l a t i o n s h o w e d that th e OM intake r e q u i r e d t o p r o d u c e
1 0 0 g amino acids e n t e r i n g th e duod enum p e r day , w as cons i d e r a b l y l e s s
o n t h e f r esh f ee d s a n d h a y than on s i l a g e , e s p e c i a l l y u n t r e a t e d s i l a g e .
H e nc e , t h e total intake o f OM r e quired to p r o v i d e th e animals w i th t h e
s ame amount of p r o t e i n , w o u l d h av e to b e h i gh er w i th s i l a g e , than t h e
o th e r feeds c ompare d .
S i l a g e s with a h i g h amino ac id c o n t e n t , o r a
r a t i o of duod enal amino acids to amino acid intak e , wh ich is g r e a t e r
t h an o n e ( e . g . d u e t o a h i gh W S C c o n t e n t ) , would a p p e a r t o maintain t h e
amin o ac id s t atus o f th e animal at a b alanc ed l e v el .
However , silages
87
o f a low ami n o ac i d c o n t ent , or
a
l ow ratio o f duod e n al A A t o A A i n t a k e ,
woul d l im i t t h e availab i l i t y o f amino acid s t o th e a n i ma l .
I f th e s e
s il a ges a l s o c ontain c ompone nts which a r e l i kel y t o d e p r e s s i n take ,
e . g . h i gh ac e tate , b u t y rate o r ammo n i a l e v e l s ( McOo n a l d and Wh i t t e n b u r y ,
1 9 73 ) , th e am i n o acid s tatus o f t h e a n imals would b e f u r t h e r d e c r e as e d ,
and an a m i no acid imbal ance migh t occ u r ( s ee Ch apter 4 ) .
( 4 ) C ompo s i t i o n o f d u o d e n al am i n o acids
Th e c o m p o s i t i o n o f duod enal ami n o acids was not m ar ke d l y a f fec t e d
b y f o rmal in-t r e atme nt o f s i l a g e i n E x p t . 1 ( Table 3 . 1 2 ) .
The p r o p or-
t i o n s of ami n o ac i d s in d uo d enal dige s t a f r om Exp t . 1 , were s imilar t o
thos e o f m ic r o b ial pro t ein and/o r th e feed amino ac i d c o n t e n t .
Th e
p r o p o r t i o n o f meth i o n i n e in th e t o tal d u o d enal ami no acids of s h eep f e d
u n tr e ated s i l a ge ( 2 . 1 % ) was with i n t h e ran g e , of 1 . 1 - 2 . 9 % , r e p o r t e d
b y H arris o n e t al . ( 1 9 7 3 ) f o r f o ur f r e s h a n d dried f o r ag e d i e t s a n d h a y .
This sugg e s ts that the produc ti o n of a meth ionine imb alanc e i n s h e ep f e d
s i l a g e , i s d u e t o a n i nc r eas e i n t h e p ropo r t ion of m e th i o n ine i n th e
t o t a l ami no ac ids required b y th e animal , rather than t o a d e c r e a s e i n
I
I.
I
th e pro po r t i o n o f m e th i o n i n e available t o t h e animal .
A me thod f o r e s timating th e relative c o n trib u t i o n s o f m i c r o b i a l
a n d d i e t a r y p r o t ein to the amino ac ids e n t e r ing the d uo d e n um , b as e d o n
th e known ami n o ac i d p r ofil e s o f f e e d , mic r o bi al a n d e n d o g en o u s p r o t e i n
w a s rec e n tl y p r opo s ed b y E v ans e t al .
( 1 975) .
Th e r e l e v a n t amino acid
p r o f i l e s were fed into a c o m p ut e r , and used to find the r e l at i v e p r o p o r t i o n s of d i e t a r y and microb i a l p r o t e i n wh i c h wou l d g e n e r a t e th e known
ami n o ac i d compos i t i o n o f d u o d e n al d i g e s ta .
The pos s ib il i t y o f i n d iv -
idual d i e tary amino ac ids r e ac h i n g th e duod e n um would i n t r o d u c e e r r o r
i n t o this computat i o n .
Th i s p r o gramme was not av ail a b l e .
H o we v er , a
comp a r i s o n o f duodenal diges ta amino acid c ompo s i t i o n w i th th e
88
c ompos i t i o n o f m ic r o b i al ( 8 e r g e n e t al . , 1 9 68 ) a n d f ee d p r o t e i n ( Ta b l e
3 . 5 ) c an g i v e s ome q u a l i t a t i v e i n d ic a t i o n o f the b a l anc e b e t w e e n mic ro­
b ial and d i e tary p r o t e i n at th e d u o d e n u m .
A comp a r i s o n of t h e d uo d e n a l
a m i n o ac i d c ompos i t i o n on th e c o n trol s i l a ge ( E x p t . 2 ) w i t h t h a t of th e
u n t r e a t e d s i l a g e ( E x p t . 1 , T a b l e 3 . 1 2 ) , sh owed th e main d i f f e r e nces t o
b e i n c r e as es i n th e c o n c e n t r a t i o n o f th r e o n i ne a n d t y r o s i ne , and
d e c r e a s e s i n th e c o n c e n tra t i o n o f · proline and alani n e .
A l a n i ne and
p r o l i n e c o n c e n t r a t i o n s were h i gh e r in th e f e eds than i n mic r o b i al
prot e i n , wh i l e t h r e o n i n e and t y r o sine c o n c e n t r ations w e r e l ow e r in
u n t r e a t e d s i l a g e th an i n mic r o b i a l p r o t e i n o r the c o ntrol s i l a g e .
Th is
migh t s u g g e s t a h i g h e r perc e n ta g e c o n t r i b u t i o n of mic r o b ial p r o t ein to
the d u o de nal ami n o acid c o n t e n t o f th e c o n t r ol s i la g e .
H ow ev e r , th e
rate o f m i c r o b i a l p r o t e in s y n th e s i s was n o t meas u r e d i n E x p t . 2 , and the
d ata f o r microb ial protein s y nth esis i n E x p t . 1 w e r e inconcl u s i v e ( s e e
Chapter 2 ) .
P r o tein s u p p l emen tation in E x p t . 2 h ad a s i g n i f ic a n t e f f e c t o n t h e
c o nc e n t r a t i o n s o f a r g i n ine , ph e n y l al a n i n e , th reon i n e , alanine ,
aspart a t e , g l y c i n e , g l u tam a t e , p r o l i n e and s e r i n e i n d u o d e n a l d ig e s ta
( P ( 0 . 0 1 , Table 3 . 1 2 ) .
M o s t o f t h e s e ch anges c an b e traced t o th e
c omp o s i t i o n of the c as e in , c o mpared t o th e amino acid c ompos i t i o n o f th e
s i l a g e ( T able 3 . 5 ) .
Th e ch anges in a r g i n i n e and s e r ine c o n c e n t r a t i o n
c o r r e s p o nd to d if f e r ences b e tw e e n c as e in and mic r o b ial p r o t e i n c ompos­
ition ( B e r g e n e t a l . , 1 9 68 ) .
Thus , t h e r e i s f ur th e r e v i d e n c e to
indicate that a h i gh p r o p o r t i o n o f th e f o rmaldeh y d e-treated c as e in was
r e ac h i n g th e d u o d e n u m .
The magn i t ud e o f t h e s e d i f f erences was g r e a t e r
than t h o s e rec o rd e d b y Mac R a e e t al . ( 1 9 7 2 ) w h o u s e d the s a m e
f o r maldeh yde-treated c as e i n to s up p l e m e n t a dried grass d i e t .
Th e s e d i f f e r e n c e s i n amino acid c om p o s i tion d u e t o th e f o rmal d e h y d e­
treated c as e i n migh t s u g g e s t th a t s u p p l e m e n tation o f s i l a g e d i e ts w i th
89
f o rmald eh yde-tr eated p r o t e i n s o f c o m p o s i t i o n c h o s en t o c omplement
m ic r o b i al protein in m e e ti n g th e am ino ac i d req uireme n t s o f th e animal ,
wo u l d i n c r eas e th e e f f i c ien c y o f u t i l i s a t i o n o f th e amino acids
abs orbed .
( 5 ) I n t e s t i n a l d ige s t i o n
I n Ex p t . 1 , t h e p e rc e n t a ge o f n u t r i e n t in take d i g e s t e d i n th e
i n t e s t i nes was i n c r eas e d b y f or mal in-treatment of s il a g e ( Ta b l e 3 . 1 3 ) .
Th i s would b e e x p e c t e d f r om t h e d ec re a s e d d i g e s tion w i th i n th e s t o m a c h s
( Table 3 . 9 ) .
T h e apparent d i g e s t i b i l i ty o f th e N e n t e r i n g th e d u o d e n um
w as h i gh, and n o t advers ely a f f e c t e d b y f o rmal i n-treatment o f t h e s i l a g e ,
s u g g es t ing th at i r r e v e r s i b l e b i n ding o f f o rmal d eh yd e t o th e h e r b age
p r o t e i n was n o t an imp o rtant factor .
I n E x p t . 2 , th ere were no s i g n i f i c an t e f f e c ts o f e i t h e r s u p p l e m e n­
tation or m e th i o n i n e o n th e p e r c e n t a g e o f OM , O M , e n e r g y or N i n t a k e
d i g e s t e d in t h e i n t e s t ines .
H o we v e r , th e r e w a s a s i g ni f i c a n t e f f ec t o f
animal variation on th e perc entage of OM ( P < 0 . 0 5 ) and OM (P
<
0 . 01 )
intake d i g e s t e d i n th e intes tines .
T h e appar e n t d i g e s t i b i l i t y o f th e N e n tering th e d u o d e n u m was n o t
s i gn i f i c a n t l y d i f f e r e n t b e tw e e n treatments ( Table 3 . 1 3 ) .
Wh i l e th i s
m i gh t s u g g e s t that the formaldeh y d e-b i n d i n g t o cas e i n was c o mp l e t e l y
r e v e rs i ble , t h e l a r g e d i f f e r e n c e i n t h e p e r c e n t age o f amino ac ids i n t h e
total N e n t e r i n g th e d uodenum , b e tw e e n th e c on trol a n d p r o t e i n s up p l e­
m e n t e d diets , migh t h a v e m a s k e d any d i f f e re n c e b e tw e e n tre atme nts , s in c e
amino acids a r e m o r e d i ge s t i b l e th an n o n-ami no ac id-N i n t h e i n t e s t i n e s
( Mac R ae e t al . , 1 9 72 ) .
90
( 6 ) Appa r e n t d iges tib il ity i n t h e wh o l e d iqestive t r a c t
I n Expt . 1 , there was
a
dec rease in ap pare nt d i g e s t i b il i t y o f N i n
r e s p o ns e t o f o rmal i n-t r eatm e n t of s i l a g e ( T able 3 . 1 4 ) .
dec r e as e w as r e p o r t e d by Barry and F e nnes s y ( 1 9 73 ) .
A s imilar
Th is d e c r e a s e in
apparent dige s tib i l i ty o f N , appeared to be due to a dec r e ase in the N
d i g e s te d i n t h e s t om2c h s .
T h e r e w as a net addition o f N t o th e r umen
on t r ea t e d s i l age c ompared t o untreated s i l age ( Tab l e 3 . 9 ) .
D i ges t i o n
of N i n t h e i n tes t i nes was n o t adv e r s e l y affected b y formal in-t r e a t m e n t
( T ab l e 3 . 1 3 ) .
Th u s , since f o rmal in-t r eatme nt inc r e a s e d t h e flow o f
ami n o acids i n to t h e d u o d e n u m ( Ta b l e 3 . 1 1 ) , tre atme n t of s il age would
ap p e ar to inc r eas e the ab s o r p tion of amino acids , r e l ativ e to u n t r e a t e d
sil a g e .
Amino acid-N w i l l b e util is e d , mo r e effic i e n t l y t h an N P N
ab s o r b e d f r om t h e r ume n, and h e nce tre atment o f s i l a g e migh t b e e x p e c t e d
to i nc r e a s e N b al anc e .
very s l i gh t .
H o w e v e r , t h e d i f f e r enc e b e t w e e n t r eatme n ts was
This paramete r w i l l b e d i s c u s s e d in m o r e d e t a il i n th e
foll owing ch a p ter .
No treatment e f f e c t on the appar e n t d i g est i b il i t y
o f OM , O M o r e nergy was rec o rd e d i n E x p t . 1 .
I n E x p t . 2 , th e o v e rall d i g e s t i b i l i ty of OM was s i g n i fican t l y
(P
<
0 . 0 1 ) inc reas e d b y b o t h e n e r g y a n d p r o tein s u p p l ementation ( Ta b l e
3.14).
T h is i ncrease i s p r o b ab l y d u e t o t h e dige s t i b l e n a ture o f t h e
s u p p l emen ts .
Howev e r , n o s i g n i f i c an t dif f e r enc e in e n e r g y dig e s t i b i l i t y
w a s rec o r d e d i n r e s p o n s e to eith e r e n e r g y o r protein s up p l ementat i o n .
Th i s may b e d u e t o t h e h igh e r SE f o r e nerg y than O M d i ges t i b il i t y .
P r otein s uppl emen t a t i o n significantly increas e d N d i g e s t i b i l i t y
(P
<
0 . 01 ) .
S inc e int e s tinal d i g e s t i b il i t y o f N s h o w ed n o s ig n i f i c a n t
t r e a tment dif f erenc e , i nc r e a s e d N d i g e s t i b i l i t y is p r es umab l y d u e t o
t h e inc r e ased l o s s of n o n-amino acid- N from the r u m e n wh e n s i l a g e w a s
s u p p l emen ted w i th f o rmaldeh y d e-t r e a t e d cas ein ( Ta b l e 3 . 1 0 ) .
N b alanc e
91
was a l s o s i gnific antly inc r e as e d b y p r o t e i n s up plementation ( P < 0 . 0 5 )
b u t N i n t ak e w as c o ns iderabl y g r e a t e r w i th th e p r o t e i n s u p p l emented
d ie t .
Thus , the inc reased N intake c o n tributed b y th e f o rmal deh y d e ­
t r e a t e d c as e i n w a s n o t u t i l i s e d as e f f ic i e ntly a s the b a s a l s il age-N .
This p o i n t and th e effect o f me t h i o n i ne i n f usion i n inc r e a s i n g N
b al a n c e w i l l b e d i s c ussed i n d e t ai l i n th e next chapte r .
C H A PT E R
THE
EFFECT
OF
D I ET S
F O RMA L I N-TR E A TM E N T
ON
THE
A ND
UT I L I S AT I O N
4
S UPPLEMENTA T I O N
OF
N
BY
SHEEP .
OF
SILAGE
92
4.1
I n troduc ti o n
Th e h y p o t h e s i s th a t me t h i o n in e is t h e f i r s t limiting amino ac i d o n
a v a r i e t y o f ruminant d i e ts , h as b e e n p r o p o s e d b y a n u m b e r o f w o r k e rs
( e . g . N imric k e t al . , 1 9 7 0 ;
A nn is o n , 1 9 73 ;
H u tton a n d A n n i s o n , 1 9 72 ;
H arris o n et al . , 1 9 73 ) .
A r�s t r o n g and
Ev idenc e f o r inc r e a s e s i n l i v e­
w e i gh t gain and w o o l gro wth i n r es p o n s e to supp l emental me t h i o n i n e
administered p o s t r uminal l h h as b e en r e p o r t e d ( R e is , 1 9 6 7 ;
1 971 ;
Dove and R o b ards , 1 9 74 ) .
Wrigh t ,
P r o d uc t i o n res ponses s uc h as t h e s e ,
i n d i c a t e th a t meth i o n i n e m a y l i m i t effic i e n t u t il i s a t i o n o f a m i n o ac i d s .
I n a d d i t ion , B a r r y e t al . ( 1 9 7 3 ) h av e r e p o rted inc reas ed i n t a k e s o f
s il a g e i n r e s p o n s e t o i n trap eritone al i n j ec t i o n o f meth i o n i n e .
Such a
r e s po n s e suggests that f ee d i n g s il age to s h e e p may place th e animal i n
a s t ate of amino acid im bal a nce .
S i n c e microb ial p r o t e i n is o f
r e l a t i v e l y c o nstant c o m p o s i t i o n and is s imilar t o h e rb age p r o t e i n , t h e
ami n o a c i d c o mp o s i tion o f d u o d e n al d i g e s t a remains relati v e l y c o n s t a n t
i n s h eep fed d i f f e r e n t n atu r al d i e t s ( C la r k e e t al . , 1 9 66 ;
W e s t o n , 1 9 70 ;
H arrison et a l . , 1 9 73 ;
U l y a t t e t al . , 1 9 7 5 ) .
H o gan a n d
H e nc e ,
t h e amino ac id s t a t us o f r umin ants appears t o d e p end m o r e o n th e
quanti t y , ' th an th e qual i ty o f amino acids available t o th e animal .
The
f l o w o f amino acids i n t o t h e duode num o f s h e e p fed s i l a g e , i s g e n e r al l y
l o w e r th an p a s t u re-f ed animals ( Arms t r o n g , 1 9 73 ;
Th oms o n e t al . , 1 9 7 3 ) .
S i l a g e , theref o r e , appe ars t o b e a s u i t a b l e b as al d i e t f o r a s t u d y o f
th e metabolism o f meth i on i n e, relative t o t h e availab i l i t y o f o th e r
amino acids i n s h eep .
Thus , the e x p e r i m e n t s t o b e r e p o r t e d c o m p a r e d
t h e e f f e c t o f c h anging the a b s o l u t e and p e r c e n tage ( o f t o ta l amino
ac i d s ) availab i l i t y o f meth i onine t o s h e e p .
T h e utilisation of
me t h i onine was measured b y v ar i o us p h y s i o l o g i c al parame t e r s wh ich h av e
b e en used t o s tu d y amino ac i d m e t a b o l i s m i n n o n-ruminant s .
93
I n Expt .
1,
th e m e t h i o n i n e available t o t h e s h e e p was incre as e d
e i th er b y I/P infus ion o f � e th i onine, or b y f eeding f o rmalin- t r e a t e d
silage .
F o rmal in-t reated s i l a g e h as b e e n s h own to i n c r e a s e i n t a k e and
N r e t e n t i o n o v e r untreated s i l age ( B arry e t al . , 1 9 7 3 ) s u g g e s t i n g t h a t
it i n c r e a s e d th e flow o f amino ac i d s i n t o t h e d u o d e n u m ( s e e C h a p t e r 3 ) .
Th e r e s p o n s e to th e s e t r e a t m e n t s was meas u r e d in terms o f N b alanc e ,
f r e e p l as m a amino ac id c onc e n t rations and t h e oxid a t i o n o f m e th i o n i n e
t o c a r b o n dioxide .
I n a d d i t io n , L-me thionine-C 1 4 U , w a s u s e d t o o b tain
i n f o rm a ti o n o n meth i o n i n e m e t a b o l is m in ruminants .
Th e s ec o nd
e x p e r im e n t was und e r t a k e n t o p r o v i d e add i t io nal data o v e r a w i d e r range
o f d u o d e nal amino acid flow r a tes .
The t h r e e treatments c h o s e n t o
p r o v id � t h e v arying l e v e l s o f me t h i onine w e r e :
a b a s al untreat e d s i l ag e
d ie t , a n d s u p p l ementation o f t h i s d i e t w i th e i t h e r f o rmaldeh yde-t r e a t e d
c as ei n o r a r e adily av ailab l e e n e r g y sourc e .
I n ad d i t io n , e a c h o f
t h e s e t r e a tm e n ts w a s s up p l e m e n t e d with a d u o d enal i n f u s i o n o f
L-m e th i o n in e .
Only N b al anc e and f r e e plasma amino acid c o nc e n t r a t i o ns
w e r e us e d t o s t udy res p o n s e t o t h e s e treatments , d u e t o the c o s t o f
L-me t h i o n i n e-C 1 4 ( u ) .
4.2
Methods
D e t a il s of th e s h e ep , t r e atments , c o ll ec tion a n d anal y s is o f
d i g e s t a s amples h av e b e e n g i v en in C h ap t e r 4 .
Th e o n l y additio n al
m e t h o d s u s e d were r e l a t e d t o th e measurement of meth i o n i n e metab o l is m .
C ol l e c t i o n o f pl asma and expi red air sampl e s : · c o l l ec t i o n o f p l as m a and
e x p i r e d air samples ( Ex p t . 1 ) f o l l owed immediately after t h e p e r iod of
c ol l e c t i o n o f faec es and u r ine s amples for b a l anc e m e as u remen t .
L-m e th i o n ine-C 1 4 ( U ) ( N ew E n gl and N uc l e a r ) was inf u s e d c o n tinuous l y i n t o
t h e r igh t j ugular v e i n .
j ugular v e i n .
A s ampling c ath e t e r was i n s e r t e d i n t h e l e f t
Th e c ath e ters w e r e 4 5c m l o n g ( 1 mm i n t ernal diam e t e r ,
P o r t e x ) and 1 Dc m were i n s e r t e d i n t o th e v ei n on t h e d a y p r ior t o
94
i n f us i o n .
T h e c a t h e t e r s w e r e f i l l e d . with h e paran i s e d s a l i n e ( 2 5 0 I U/ml )
to p r e v e n t th e f o rmation b f b l o o d c l o ts w i t h i n th e c ath e t e r .
Th e f r e e
e n d o f t h e c a t h e t e r was attac h e d t o w oo l o n th e b ac k o f th e s h e ep .
S amples c o uld th u s b e taken with o u t d is t u r b i n g th e animal .
infus i o n s o l u tions c o n t ained approximately 8 0
�C .
l
The
L-m e th i o n i n e-C 1 4 ( U )
i n 200ml s t e r i l e s aline and were i n f u s e d o v e r 1 2h ( s e e s ec t i o n o n
me th i o n i n e me t ab o l is m ) .
A l l t u b i n g used i n t h e inf us i o n w a s f l u s h e d
with alc o h o l , f o l l owed by s t e r i l e s a l i n e p r io r t o us e .
P e r i s ta l t ic
p umps w i th t u b i n g c alibrated to d el iv er 1 50ml/day , w e r e u s e d t o
a dmi n i s t e r th e i n f u s io n .
Th e i n f u s i o n s o l ution w a s p l ac ed o n a b alanc e
w:#.,;-.
t o r e c o rd the inf usion rate J;ifilh'efil>l A e ach s ampling p e r io d .
D u r i n g an
infus i o n , the s h e ep was p l ac e d i n a m e t a b o l i s m c r ate with i t s h e ad
e nc l o s e d i n a Pers pex r e s p i r a t i o n h o o d , s ealed by a r u b b e r c o l l a r
( Ul y a t t , u np ub li sh e d ) .
Th e s h e e p h ad f r e e ac cess to water , a n d w a s able
t o s t and o r s i t .
Hourly rations were g i v e n to th e an imal t h r o u gh the
top o f t h e h o od .
T h e total f l o w rate o f e x p ired air was m e as u r e d with
a gas f l owm e t er ( P ar kinson C owan ) .
Temp e r a ture , vapour p r e s s ur e and
atmo s p h e r ic p r e s s u r e were me as u r e d c o n t i n u o usly and u s e d t o c o r r e c t gas
v o l um e s t o s t andard tem p e r a t u r e and p r e s s u r e .
C ar b o n d i o x i d e c o n t e n t
w a s d e t e rm i n e d o n an aliquo t , with a n I n f r ared g a s an a l y s e r ( G r u b b
Pars o ns ) .
A s e c o n d a l i q u o t w a s b u b b l ed t h r o ugh 1 N NaOH i n a O r e s c h e l
w ash b o t t l e , t o a b s o r b c a r b o n d i o x i d e f o r the meas u r e m e n t o f s p ec if ic
ac t i v i t y ( S A ) .
T h e wash b o t t l e s w e r e c h anged at time o f w i t h d rawal o f
b l o o d s am p l e s , i . e . , a t 5 , 1 5 , 3 0 , 6 0 , 9 0 , 1 20 , 1 50 , 1 80 , 2 1 0 , 2 4 0 min
after th e s ta r t o f the infus i on , and t h e r eaf ter at h o u r l y i n t e r v als f o r
a f u r t h e r 8h .
B l ood samp l e s ( 1 0ml ) w e r e w i thd rawn i n t o h e p a r i n i s e d
t u b e s and c e n t rifuged a t
6 , 000 g f o r 20 min t o s e parate p l asma . Th e
°
p l asma s am p l e s f ro m E x p t . 1 w e r e s to re d at -20 C p r i o r t o p r o t e i n
precipitation .
H owev e r , i n th e f i r s t r e p l i c ate o f E x p t . 2 , th i s m e th od
Table
4.1
T h e e f fec t
of b l o o d s ampli ng o n t h e pac k e d c e l l
volumes of
th e sh eep
Rep.
S h e ep N o .
I
I
I
I
64 7 ( 44 1 ,
73 1 { 1 1 57 ,
786
Rep.
R ep .
3)
3)
1
( E xpt .
1)
Rep .
2
Rep .
3
29. 5
28. 5
17.5
23 . 0
24 . 0
21 . 5
29.5
29 . 5
32. 5
32 . 0
28 . 0
26 . 0
25.0
28. 5
25.0
25 . 0
25.0
22 . 5
95
o f s t o r a g e was f o u n d t o r e s u l t i n the d isappear anc e o f f r e e c ys teine
f rom t h e p l asma .
H e nc e , lat e r s amples w e r e s ep ar a t e d into p r o tein-f r e e
p l asma , a n d protein p r e c ip i t a t e p r i o r t o s torag e .
I n all c as e s , p r o t e in
w as p r e c i p i t ated b y addition o f an equal v o l ume o f 1 0% s ul p h o s a l i c y l i c
ac i d t o th e plasma .
f u g a t i o n at
Th e p r o t e i n p r e c i p i t a t e w a s t h e n r e m o v e d b y c en t r i-
6 , 000 g f o r 1 5 mi� and th e s u p e rnatant s to r e d at -20
0
c.
Th e t o t al v o l ume o f b l o od s ampled from each s h eep d ur i n g a
r e p l i c a t e in E x p t . 1 was a p p r o x imately 200ml .
I n o r d e r t o c h e c k wh e th e r
r em o v al o f th is v o l ume af f e c ted t h e animal , p ac ke d c e l l v o l um e s w e r e
d e t e rmined at 0 and 1 2h af t e r t h e s ta r t o f infus i o n f o r e ach s h ee p .
Th e s e a r e given in T ab l e 4 . 1 .
S ampling h ad l i t tl e e f f e c t o n packed c e l l
v o l ume.
In E x p t . 2 , c a th e t e rs were i n s e r t e d into a j u gular vein o f each
s h e e p , i n a simi l a r manner t o th o s e in E xp t . 1 , t h r e e d ays p r i o r t o t h e
s t a r t o f methionine infus io n .
S amples ( 1 0ml ) w e r e t a k e n o n e a c h o f t h e
f o l l ow i n g two days , and d a i l y a f t e r th e s t art o f meth ionine i n f u s i o n .
Plasma amino ac id c o nc e n tr a t i o n :
Th e total f r e e p l asma amino ac ids i n
s am p l e s f � om Exp t . 1 were d e t e rmined ci n a B ec kman 1 2 0C amino · acid
anal y s e r using a two c o lumn proc e d ur e .
Prior t o E x p t . 2 , this
ins t r u m e n t was modified by the addition o f a L o c a r te aut omatic l o ad e r .
Th e l o ad e r nec es s itated th e us e o f one c o l umn p r o c e dures , and wh i l e
this m o d i f ication h ad b ee n c o m p l e t e d f o r p r o t e i n h yd r o l y s at e analysis ,
th e r e s in for p h y s i o l o gical f l u i d ana lysis was n o t avail ab l e at th e
s ta r t o f E x p t . 2 .
Th us , p l asma amino acid anal y s e s in E x p t . 2 w e r e
c ar r i e d o u t on th e p h y s i o l ogic al ac idic a n d n e u t r a l r e s i n f ro m th e
o r i g i n a l B ec kman amino ac i d analys e r .
S eparate d e te rmination o f th e
bas ic amino ac ids was n o t p o s s ib l e , d u e t o incomp a t abil i t y b e tween th e
L o c a r t e s ys tem and th e B ec kman b as ic amino acid c o l umn .
SA
of pl asma m e t h i o n i n e
( d pm/iJmole )
0
0
1 2 , 0 00
0
0
0
0
s , oo o
4 , 000
I n f usion r ate was :
7 , 3 54 , 750 d pm/h
0
2
4
6
8
10
Time a f t e r s tart of i n f u s i o n ( h )
F iqo
4. 1
Th e change in S A o f pl asma m e th i o n i n e with time after s tart of
i n f u s ion of L-meth i o n i ne-C 1 4 (U )
12
96
The m e th i o ni n e S As i n E x p t . 1 w e r e measured o n a Jeol-JLC 6 A H amino
acid an2l y s e r , u s i n g the s p e c i f ic 5-amino ac id pr oc e d u r e o f J e p p s o n a n d
Karl s s o n ( 1 9 72 ) .
T h is anal y s i s w a s d i s c u s s e d i n C h apter 2 .
P lasma u r e a c o r c e n t r it i o n :
P l asma u r e a c o n c entration was m e a s u � e d b y
m
reac t i o n w ith d iac e t yl monoxi�e o n a T e c h n i c o n autoanalys e r .
S c i n t i l l a t i o n c o un t i ng:
c o 2 trapped i n 1 N NaOH f r o m t h e gas wash
b o t t l e s was p r e c ip i t at ed as B a c o 3 , b y add i t ion of exc ess B aC 1 2 •
p rec i p i t a t e was c o l le c t e d o n Wh atman N o . 5 4 1 paper .
Th e
A f t e r w as h i n g w i th
hot w a t e r and d r ying with ac e t o n e , a w eigh ed s ampl e o f t h e B ac o 3
( ap p r oximately 1 5D m g ) was s us pe n d e d i n the gel sc i n t i l l a t i o n mix of
· C l u l e y ( 1 9 6 2 ) ( 0 . 3% ( w/v ) d i p h e n y l o x a z o l e ( P P D ) i n t o l u en e , +0 . 4%
4
A eros i l ) , and 1 c ac tivity c o u n t e d in a Pac kard l i q u i d s c i n t i l l a t i o n
c o un t e r .
The e f fic i e n c y o f c o u n t i n g f o r t h e set w e i g h t o f B ac o
3
was
c al c u l a ted as d e tailed by C l u l e y ( 1 9 6 2 ) .
T h e radi o a c t i v i ty i n p l as m a m e th i o n i n e was de te rmined o n f r ac t io n s
o f t h e c o l umn e f f l u e n t f r om t h e J e o l ami n o a c i d anal y ser .
The
f r ac t i o ns were add e d t o o n e o f t h e f o l lowing s c i n t i l l a t i o n w i x e s , and
counted o n a B ec kman or Packard l i q u i d s c i ntillation c o u n t e r .
S c i n t i l l a t i o n mix 1 :
0 . 5% P P O + 0 . 02% POPOP i n Tri ton/T o l u e ne ( 1 : 2 )
S c i n t il l a t i o n mix 2 :
0 . 4 % Omnifluor i n Tri t o n/T o l u e ne ( 1 : 2 )
A n e x t e rnal s t a ndard of H e xadec ane-C 1 4 was used t o p l o t t h e d e g r e e o f
quenc h in g f o r each c o unt e r , a n d t h u s th e e f f ic ienc ies o f c o un t i n g w e r e
c alc u l ated .
C alc�lations
M e th i o ni n e me t a b o l i s m :
t ime f o r e ach s h eep .
SAs o f plasma m e th ionine w e r e p l o t t e d agains t
Th e p l o t f o r s h eep 6 4 7 , r e p l ic a t e 1 i s giv e n i n
F i g . 4 . 1 ( th e c ompl e t e data f o r all � h e e p i s giv e n i n A p p e n d ix 3 ) .
DE2rnLm9
c
0
200
0
0 0
1 50
0
1 00
0
0
0
0
0
0
0
0
0 0
0
0
0
0
0
0
0
50
0
0
I nfus i o n r a t e was :
0
6 , 2 06 , 1 1 0 d p m/h
0
0
0
5
15
10
20
Ti m e (h )
Figo 4 . 2
Th e c h a nge i n S A o f C 0
with t i�e a f t e r t h e s t a r t of
2
infusion of L-meth i o n in e-C 1 4 ( U )
25
97
Wh i l e it c an �e s e e n t h a t s o m e of t h e data in the e a r l y p a r t of this
partic ular c u r v e are mis s i n g ( d us t o l o s s of samp l e s on th e amino acid
analys e r ) , it would appear that S A increas ed expo n e n t i a l l y w i th time ,
t o a p l a t e a u at appro ximatel y 3h .
Th e small i nc r e a s e a b o v e th is .
p l at e a u v a l u e c an b e a t t r i b u t e d t o t h e r e c y c l i n g o f l a b el l e d m e t h i o n i n e
w ith i n the anim al .
Th e s h a p e of this c u r v e can th e n b e d e f i n ed b y t w o
parame ters w h ich d e s c r i b e meth i o n i n e metab o l i s m �
The f i r s t i s t h e
pla t e a u S A ( S A00 , d p m/�mo l e ) , and f r o m t h i s t h e m e th io n ine t ur no v er r a t e
c an b e calc ulated .
i .e.
Turnover rate = I n f us i o n r a t e o f L-meth ionine- C 1 4
S A ( d p m �mole )
T h e s econd is t h e r a t e c o ns tant ( k ) .
o f t h e c u r v e r e p res e n ted b y i n ( S A 00 1 9 63 ) .
Where SA
t
is th e S A a t time t .
This was calc ul a t e d a s th e s l o p e
S A ) ver sus time ( t ) ( R i ggs ,
t
T h e s i z e o f t h e methionine p o o l
w as a l s o c al c u l ated from the r e l a t i onsh i p :
O x id a t ion o f m e t h i o n ine :
Pool s i z e = T u r n o v e r r a t e / k .
Th e f i r s t s h e e p was inf u s e d c o ntinuo u s l y f o r
2 4 h t o try a n d d e t e r mi n e th e time taken t o reach a p l a t e au v al u e o f
14
c o 2 S A ( F ig . 4 . 2 ) . A p l at e au w a s n o t r each e d w i t h i n 2 4 h i n d ic atin g
14
r ec y c l in g o f
c within t h e animal . How ever , the rate of i n c r e a s e
a p p e a r e d t o ch ange a t 1 1 - 1 2h .
Th us , t o ob tain a s t andard compar is o n ,
s ub s e q u e n t inf usions w e r e limited t o 1 2h d uration , and S A 00 calc u l a t e d
as t h e av e r age of th e v a l ues for t h e l a s t th r e e h o urs o f i n f us i o n .
S A 00 o f c o
2 a n d the S A oe o f meth i o n i n e w e r e t h en u s e d t o c al c u l a t e t h e
p e rc e n tage of expired
i.e.
The
% of co
c o2
wh ich w as d e r i v e d from m e th i o nin e .
deri v e d f r om meth i o nine = S A � of e xpi r e d c o (dpm/mgC )
2
S A 00 of p l asma m e t ( d p m/mgC )
14
c expi re d/min
and % meth ionine o x i d i s e d t o C 0 2 = dpm
X 1 00
14
dpm
c infused /min
2
Table 4 . 2
amino
s i l age , and
u n t r e a t ed s i l a o e d i e t s s upplemented with
and po s t ru�inal
E xpt .
�
QCids i n t o t h e duodenum i n she8p f e d f o r m a l i n - t r e a t e d
T h e f l o w of
Treatment
(I/P
and duodenal
Methionine
)
into
UutHJl?num
th e
�
Mothionino u v u i l o b l e
f o r absorotion
�
�
Untreated
Rep.
1
47.6
Rep.
2
46.2
3
32.8
Rep.
Treated
1
R p.
2
67 . 8
3
72.0
e
Ra�.
Untreated
Rep .
Rep.
Re p .
2
57 . 6
Re p .
+
1
65.2
2
52.9
3
33.0
NS
P "-
)
)
)
65.8
)
)
)
50.4
meth i o n i n e
�
Mean
1.2
o.a
0.8
1.4
1 .5
1.7
2.5
1 .9
1 .9
)
)
)
o.9
)
)
)
1 .5
)
)
)
2.1
2.4
1. 7
2.4
2.4
2.2
2.4
3.8
3.6
5.8
)
2. 1
)
2.3
)
4.4
-
49.6
1.1
2.2
-
110.5
2.7
2.4
+ energy
-
74 . 1
1.7
;{ . J
Control
+
60.3
3.2
5.3
+ p r o t ein
+
125.7
s.o
4.0
+ energy
+
98.6
4.2
4.3
0.18
0 . 06
**
rJS
**
Methionine s i g n i f icance
P "-
42.2
:g
total a m i n o acids
Control
Tre�tmcnt e iQ n i f i c n n c e
**
)
)
)
in
+ protein
SE
*
eneroy
methionine .
F l o w of .:.mina
acids
s i l age, u n t r e a t e d
formald ehyde- t r e a ted c a s e i n ,
0 . 05
0 . 01
not s i g n i f i c a n t
6.0
**
NS
**
98
Avai l abil i ty
ci f
me t h i o n i n e f o r a b s o rpt i o n :
The meth i o n i ne a v ai l a b l e
f o r a b s o r p t i o n f r om th e intestines was c al c ulated b y a d d i n g th e d ai l y
amo u n t o f meth i o n i n e infused , t o th e daily flow of m e th io n i n e i n t o th e
duod enum .
The I/P meth ionine was assumed t o b e ab s o r b e d t o th e s ame
e x t e n t as the meth i o ni n e en t e r i n g the d u o d e num , sinc e n o d a t a on t h i s
were a v a i l ab l e .
Statistic a l analys i s :
Th is was i d e n t ic a l t o the a n a l y s i s d e s c r i b e d i n
Chapter 3 .
4 .3
Results
T h e m a in a i m o f E x p t . 1 was to c ompar e th e e f f e c ts o f f o r m a l in­
t rea tment o f s i l ag e , and intraper itoneal i n f us ion o f L-me th i o n i n e o n
t h e u t ili s ation o f N b y s h ee p .
In Expt.
2
the effec ts o f s u p p l e m e n t i n g
an u n t r e a t e d s il a g e d i e t w i t h f o rmal d eh yd e-treated c a s e i n and e n e r g y
were comp a r ed w i th t h e e f f ec t o f d u o d e n a l infusion o f L-meth i o n i n e o n
t h e u t ilis a ti o n o f N .
The e f fec t of the t r eatme n ts o n th e f l ow of ami n o acids i n t o t h e
d uode num , and o n th e total meth i o n i n e a v a i l a b l e f o r a b s o r p t io n , a r e
g iv e n in T able 4 . 2 .
I n Expt . 1 , th e f low of t o t a l amino �cids i n to th e
d uo d e num was h i gh e r o n f o rmalin-treated th an untr e a t ed s i l ag e , w h i l e
m eth i o n i n e infus i o n appeared t o h av e no e f f ec t .
Th e p e r c e n t a g e o f
meth i o n i n e i n t h e t o tal ab s o r b a b l e amino acids remained th e s am e o n
t r e a t ed a n d u n t r e a t e d s il a ges , b u t w a s i n c reas ed b y I/P i n f u s i o n .
Exp t .
2,
In
t h e f l ow o f to tal ami n o acids i n t o th e d u o d en u m w a s s i g n i f ic­
antl y inc r e as e d by b o th p r o t e i n and e n e r g y s u pplemen ta t i o n r e l a t i v e to
uns u p p lem e n t ed s i l a g e ( P Z 0 . 0 1 ) , wh i l� p r otein a l s o s i gnific a n t l y
i nc r e ased t h e f l o w o f amino a c i d s i n t o th e d u od e n um r e l a t i v e t o e n e r g y
s uppleme n t at i o n ( P � 0 . 0 1 ) .
M e t h i o n i n e i n(usion d i r e c t l y i n c r e a s e d
The e f f e c t of
Table 4 .3
f o r ma l i n -t r e a tment of
s i laqe, and s uppl eme n ta t i o n o f un t r e a ted s i l age d i e t s
formaldehyde-treated casein, e n e rgy and po s t ruminal
with
(I/P
and duo denal) methionine o n
t h e concen t r a t i o n o f m e t h i o n i n e and total amino acids i n sheep plasma
U!lli
�
Me t h i o n i n e ·
Treatment
PlaGm<J
llill thionine
concentration
i0ililil
1
T o t a l ol�sma amino
acid concentration
(pmo l e/1 )
ea n
%
met
amino a c i d s
�
Moan
M
in
t o t a l pl asma
U n t r eated
Rep.
1
7.5
)
Re p .
2
6.7
9.6
)
)
3626
)
) 9.0
2761
)
)
3066
)
Rep
.
3
7.9
)
3084
)
2240
2728
) 2684
0.3
)
0.4
0.2
) 0.3
Treated
Rep .
1
7.2
Rep .
2
10.3
Rep.
3
9.4
Untreated
Rep.
Rep.
Rep.
2
0.4
) 0.3
0.4
+
1
19.0 )
1 8 68 )
2
16.4 ) 20.3
2075
3
25.5 )
2056 )
-
Control
-
+ protein
+ m
c er uy
)
1.1
2000
o.8
)
1 .0
1.3
1
14.2
2036
16.9
1 987
0.9
14.�
2046
0.7
0.7
Control
+
31 . 4
1 673
2.0
+ protein
+
33.4
1 3 66
2.5
+ energy
+
35.7
1617
1.8
66
SE
NS
Treatment s i gn i f icance
-
Me t h i o n i ne s i y n i f icance
Replicate
1 11
0.2
3 1 51
*
sign i f icance
n
r o t a l11 p l asma am i o ac i d s in ( x p t .
2 only
rt!pr�st!ntb
2.3
0.13
NS
liS
**
**
NS
*
I' rea acidic a1 n .J n � u l r a l amino a c i d s .
Table
4.4
T h e e f f e c t o f formalin-treatment o f s i l a9e and
the
s ueelementat i o n o f u n t r e a t e d s i l a o e d i e t s w i t h
formuldeh:tde-t r e a t e d c o 5 e i n1 ener9� ond Eos l ruminal
c o nc o n t r n t i o n s of
i r l d l v i d u G l umino ac i d s i n the
! ILP
Ex�t.
Untreated
and duodenal
t o t a l El asma
au1ino
)
me th ionine an th e �e r c e n t a r e
t
IJ C i d s .
1
Untreated + met
Treated
�
�
�
8.5
6.8
4.3
4.8
6.3
1 1 .7
6.9
7.6
5.0
6.5
8.2
3.3
4.4
2.4
2.6
3.7
5.0
4.5
6.3
13.1
6.1
7.1
9.7
G.O
9.9
0.2
0.2
0.1
3.0
2. 4
2.1
�
�
ornithine
8.2
4.6
�
5.0
�
9.9
lysine ·
6.7
5.8
7.6
h i stidine
2.7
5.5
arginine
5.B
6.1
taurine
0.3
0.7
o.8
�
.B.!!h1.
3.8
2.5
2.8
2.2
4.7
4.1
2.2
1 .4
1.9
10.1
4.9
5.1
4.6
5.8
6.8
3.5
4.3
4.0
p r o l ine
2.1
3.1
5.1
2.8
4.0
4.1
6.0
2.6
4.2
g l u tamic acid
9.6
1 6. 0
11.7
7.0
6.9
7.9
16.1
9.6
13.1
c i trulline
5.3
4.8
3.9
3.6
5.9
4.2
6.5
5.9
6.0
glycine
23 . 6
28.6
28 . 7
24 . 8
28 . 2
27 . 2
20 . 5
32.7
1 6. 8
alanine
7.6
threonine
serine
r
7.4
7.5
6.1
6.3
7.6
s.o
6.5
8.7
6.5
4.8
7.4
6.5
s.o
4.9
5.7
Tr.
Tr.
Tr .
Tr.
Tr.
Tr .
v a l ine
6.5
5.0
cysteine
Tr.
Tr.
Tr .
cystath ionine
Tr .
Tr.
Tr.
Tr.
Tr.
Tr.
0.4
0.2
0.6
methionine
0.2
0.3
0.4
0.2
0.4
0.4
1.1
0.8
1.3
�l e u c i n e
3.1
2.1
3.0
2.0
3.0
2.6
2.4
2.3
3.5
leucine
3.9
3.0
4.4
3.4
4.3
4.6
3.9
2.7
3.6
tyrosine
1.6
1.5
1 .6
1.2
1.8
2.2
1 .6
1 .4
2.0
ph e n y l a l anine
1 .0
1.2
1 .4
0.9
1.3
1.6
2.1
1 .8
2.0
To�l� 4 . 4
c o n t inued
Expt. 2
C on tr o l
+ p r o te i n
+ energy
Significance
- I!' S t
�
�
�
- met
+ met
g_
taurine
0,6
s.o
0,6
3.7
0,6
4,9
0,49
threonine
3.0
1 .9
5,6
3.1
4,3
2.2
0,21
Treatment
(T)
Methionine
(M)
Reelicc:te
I n terac tion (T��)
NS
**
N5
**
**
N5
:.s
-
*
1:s
serine
8,1
5.0
6,4
5,7
7,1
4.2
0 , 35
N5
**
p r o l ine
4,8
3.7
7.9
8,7
4.1
4,2
0 , 35
**
N5
N5
r;5
4,5
8.3
4.3
5,2
4,8
6.1
1 , 26
N5
NS
N5
::5
5,0
7,8
5.6
7,4
5.1
7,0
0 , 39
NS
**
N5
1.5
**
**
N5
1:5
*
1:s
gl utanic
acid
c i trulline
£: l y c i ne
45,8
37 . 9
35.8
25,6
41.8
36.4
1 . 51
alanine
11.0
1 1 .3
8,8
12,1
1 1 ,4
13,9
0 . 51
NS
*
**
val ir.e
6.2
5.5
10.9
9,6
8,7
6,5
0 , 29
**
N5
1:s
C } s t e i ne
1 .3
1,6
1.2
2.0
1.0
1,8
0,10
N5
**
**
*
c y s t a t h ionine
Tr.
o.8
Tr,
0.6
Tr,
0.6
0 , 04
NS
iHC·
*
�;s
*
r:s
;r e t h i o n i n e
0,7
2,0
0,9
2,9
0.7
2.3
0.13
N5
**
�leucine
2.3
2,6
3,1
3.0
3,0
3,0
0 , 06
**
NS
**
N5
l e uc i ne
3,9
2,9
5.2
4,6
4,0
3.1
0 , 08
**
**
**
1.5
0 . 22
**
**
NS
;�s
NS
**
NS
1·1 5
tyro s i ne
phenylalanine
2.2
1.8
3.3
2.6
3.6
2,1
4.9
3,4
2.8
1,7
3,6
2.6
0.17
99
avail able me th ioni n e , b u t did n o t s i g n i f ic a n tl y i nc r e a s e t h e t o t a l flow
of amino acids .
Th e perc e n t age of me t h i o n i n e in t o t al ab s o r b a b l e amino
acids was t h e r e f o r e i nc r e as e d b y m e th i o n i n e infus io n , but i t was
unaf f ec t e d b y s uppl emen tation with p r o t e i n or ene r g y .
Th e d a t a o n f r e e p l asma meth ionine a n d to tal amino acid c o n c e nP l a s m a meth i o n i n e c o nc e n t r a t i o n
trati ons are p r e s e n t e d i n Table 4 . 3 .
w as incre ased b y I /P inf u s i o n , b u t u n af f e c t e d by f o rmali n-t r e a tm e n t o f
s il a g e i n Ex p t . 1 .
T h e c o nc entration o f t o t al ami n o a c i d s in t h e
p l asma w a s l ow e r i n res p o n s e t o meth i o n i n e i nf us i o n c ompared t o t h e
c on c e n tration i n sh e e p f e d treated s i l ag e , b ut th e c on c e nt r a t i o n o n
· untre ated s il a g e was b e tween t h e v a l u e s f o r t h e o th e r two treatme n t s .
Th e free p l asma ami n o acid c o nc e n t r a t i o ns from E x p t . 2 , i n T a b le
4 . 3 , are th e av erage of s amples taken tw o d a ys prior t o , and t h e 1 5 th
a nd 1 6 th d ays a f t e r , the s tart o f m e t h i o n i n e infus io n .
P l asma
m e th i o nine c o n c e n t r a t i o n was s i gnific a n t l y i nc r eas e d by d u o d e nal
m e th i o nine infusion ( P
<
0 . 0 1 ) , but unaf f ec t ed by p r o t e i n or e n e r g y
s up p l e men tatio n . ''T o tal!' p l asma amino acid c o nc e ntratio n in E xp t . 2
r e f e r s o n l y to acidic and n e u tral amino ac i d s .
d e c r e ased b y m e t h i o n ine i n f u s i o n ( P
o f t h e supple me n ts .
<
Th is was s i g nific a n t l y
0 . 0 1 ) , and unaf f e c ted b y e i t h e r
Th e percentage o f m e t h i onine i n th e " t otal" p l asma
amino ac i d s was s i g n i f i c a n t l y increas e d by d uodenal m e t h i o n in e i n f us i o n
(P
<
0 . 01 ) .
Tab l e 4 . 4 gives th e e f f e c t o f f o rmal i n-treatment and s u p p l e m e n tation
of s i l ag e o n th e p e r c e n t a g e c o n t r i b u t i o n s o f ind i v i d u al amino ac i d s to
the t o tal amino ac i d s i n t h e plasma .
T h e o n l y marked c o n s i s t e n t
d i f f e r ences in r e s p o n s e t o intrape r i t o n eal m e t h i o n i n e i n E x p t . 1 w e r e
i nc r e ases i n th e p e r c en tages of me t h i o n in e , tauri n e , c y s tathioni n e ,
c it r u l lin e , and ph e n ylalanine and a d e c r e a s e i n t h e p e r c e n t ag e o f
UNtVER.SJJl
LIB RAn
MASSEY
Table 4 . 5
The e f f e c t o f d u r a t i o n o f meth ionine infusion on th e
/
perc en tage o f i n d i v i d ual amino ac i d s i n the t o tal pl asma
amino ac ids - E xpt . 2 ( R ep. 1 ) .
Day
0
1
2
3
4
5
6
met
0.8
2.2
2. 5
3.3
3.2
4.6
4.8
ser
7.4
6.1
5.8
5.4
4o0
3.7
5.5
7.0
5.8
5.5
7.6
7.7
7.4
6.4
7. 6
5.5
5.2
6.3
7. 1
7. 1
6.8
4.0
3.3
4.2
5.8
5. 6
6.9
5.1
C o n t r o l + pr o tein
met
0. 8
2.3
2.8
3.0
3.4
s er
6.4
1 .9
4.9
5.8
5.8
5.3
5.4
3.3
5. 1
9.0
8.4
9.4
1 0. 8
9.6
10.0
10.2
1 1 .6
10.6
1 0.4
10.6
11.6
10.5
11.3
6.0
6.9
7.2
8.2
8.8
8.9
9.2
met
0.8
2.1
2.9
3.7
3.5
NO
5.3
s er
7. 6
6.4
5.7
5. 1
5.0
NO
5. 6
7.0
5.1
3.6
5.0
5.1
NO
6. 1
9.2
6.1
6.3
5.9
8.5
NO
7. 1
4.4
4.5
3.6
4.8
5.9
NO
7. 6
Control
ile
+
leu
v al
t yr
ile
+
+
ph e
leu
v al
t yr
+
phe
C o n trol
ile
+
+
leu
v al
tyr
+
phe
energy
Table 4 . 6
/
The e f f e c t of formalin-treatment of silage and I P methioni�e i n f u s i o n t o sheep fed untreated sil aoe.
methionine
(E xpt . 1).
Expt.
I
1
---
T r e a t me n t
� Methionine
Meth�Qnine
Turnovl!r
R .::� t e
( mmole7h )
�
U n t r e ated
1\l'p ,
1
0.43
1 \�!).
2
0 . �4
Rep.
3
0 . 55
�2 �
Oxidised to
)
)
)
)
o . �1
£9.z
1G.D
14.7
15.3
Methionine
Pool
From
Methionine
�
Meaf'1
)
o . u7o
)
)
1 5. 3
)
)
)
15.6
)
0. 050
0 . 078
)
)
)
0. 066
P l asma
f'le: t r ionine
(flnole )
( flmo l e )
646
Calculated
Tot-:!1
5ize
477
on metabolie� gf
f'l e a n
Mean
T o t a : :-o· .� s c l e
F.e:. t� i o :--d 1•::
( ·· .. a 1
.)
r•.ean
)
5 62
I
19.9
576
I
l
605
I
18.7
558
1 02 6
I
23.5
599
---
�
t �cp.
1
0 . 53
Rep.
2
0.67
Rep.
3
D. 7;2
)
)
)
13.3
0 . 64
)
17.5
16.0
0 . 073
l
0 . 1 08
0. 1 1 9
)
)
)
531
0 . 1 00
)
245
939
Ll n t re a t ed + me t
Rep.
1
0.68
Rep.
2
0 . 86
3
0 . 79
R ep .
)
)
)
)
18.3
0 . 78
18.8
19.9
)
)
)
)
0 . 1 01
19.0
0 . 1 32
0 . 1 56
)
)
l
0 . 1 30
1 089
953
)
1 00
s erine .
I n E�pt .
2
,
1 5 d ays o f duode nal methionine i n f us i o n s i g n if­
ic an t l y inc reased t h e percentages o f t a u r i n e , citrul l i n e , c y s t at h i o n ine ,
c y s t e i n e , meth i o n i n e , tyrosine and p h e n y l a l anine ( P ( 0 . 0 1 ) and
s i gnif i c a n t l y decreased th e p e r c e n t a g e s of t h r e o n in e , s e r i n e , g l y c i n e ,
v al i ne and l e u c i n e ( P < 0 . 0 1 ) .
icantly i n c r eased ( P
<
0 . 05 ) .
T h e p e rc e n t age of alanine was s i gn i f­
T h e r e w as a significant e f f e c t o f
r e pl i c a t e s o n th e perc e ntages o f seri n e , c ys t eine , l e uc ine and i s o­
l e u c i n e ( P < 0 . 0 1 ) and alanine , and m e th i o n i n e ( P < o . o s ) .
Th e r e was
also a s i gnific a n t inter action b e tw e e n the effects o f s u p p l e me n t a t i o n
a n d me t h i o nine o n th e perc entage o f s e r i n e a n d c y s te i n e ( P < o . o s ) .
T h e c o nc e n t r a t i o n of individual amino ac ids in t h e p l as m a a f t e r
o n e day o f me t h i o n i n e infusion w a s al s o m e as ured , a n d is g i v e n i n
Appendix 4 .
One i n t e r e s t i n g f e a t u r e o f t h e s e c o n c e n t r a t i o n s i s th e
d is app earance o f t a u r i n e from th e plasma after 2 4 h o f me th i o n i n e
i nfusi o n .
The e f f e c t s o f duration o f inf us i o n o f meth i o n i n e o n t h e p e r c e n t­
ages o f i n dividual amino acids i n th e p l asma was measured d u r i n g t h e
f irst r e p l icate o f E xp t . 2 ( T a b l e 4 . s ) t o as s e ss t h e v a l i d i t y o f o n l y
six days p r e l im i n a r y p e riod f o r ad j us tm e n t to meth i o n i n e i n f u s i o n .
O n l y t h e perc e n t a g e s o f meth i o n i n e , s e r i ne and th e b r an c h e d c h a i n and
aromatic amino ac ids a r e giv e n , as th e y were c o n s i d e r e d to be r e pr e s e n t­
a tive o f all the amino acids .
A n i n f u s i o n o f L-me th ionine-C 1 4 ( U ) was used to e s timate p arameters
o f m e th i o n ine metabolism in E x p t . 1 .
4.6.
C 02 ,
The results are given i n Table
T h e r at e o f t urnover o f m e th i o n i n e , th e percentage o x i d i s e d t o
th e perc e n tage o f
c o2
derived f r om m e t h ionine and t h e me t h i o nine
pool size were all increased b y I/P i n f us i o n of meth i o n i n e r e l a t i v e to
Table.
4,
7
"
The e f f e c t of f o r m a l i� t r eatment of s i l age ,
and supplementation
of
treated cas ei n1 ene r�� and eo s t ruminal (l(P and ducdenzl} meth ion i ne
u n t r e a t e d s i l age d i e t s with f o r m a l d ehydeon ur i n a r� N1 d a il� N r e t e n t io n and
el o�m<:J Ur UU C 0 1 1C W 1 l r '-l l 1 o n .
E xpt,
!!.e.&.
T r eatment
Meth i o n i ne
N intake (nt::da�)
U r i na r� N
(ot::da�l
�
�
N retention
IilZii:l1
Plasma urea c o nc n .
lm�Nz1 OOml)
�
�
Untreated
Rep.
Rep.
Rep,
�
Rep,
Rep.
Rep.
13,9 )
15,3 ) 14,5
1� .4 )
1
2
3
10.8 )
12,6 )
14,6 l
1
2
3
Rep.
nup.
2
8,4 \
3.1 )
-1 .4 )
1.6 )
1.1
5,9 )
5,7 ) 5,9
)
1.6 ) 2,5
2.6 )
7,3 )
8,4 ) 6.8
4,8 \
3.2 )
1 . 9 ) 2. 7
-0 . 6
6.1 l
3.3
15
12 )
14
12
12
14
14
)
) 13
)"
+
Untreated
Rep.
12,7
7.2 )
1 2 . 3 ) 9,3
14,8 )
14,8 ) 14.0
1
2
3
13,2 )
- ---
Control
-
+ protein
3.2 l
9
1 2 l 10
10
10.4
8,7
21,8
12.3
14.1
10.2
1.5
12
2.3
14
3,5
13
16
+ energy
-
Control
+
14.7
7, 5
+ prot ei n
+
22.6
1 1 ,8
5,3
17
+ energy
+
15.9
7.1
4,0
13
0.36
0 , 67
0 . 64
SE
Treatment o i g n i f icanca
**
**
f"'qt.l d u t d ! la
N�,
..
n
l rJI I l f l u aiiLQ
. . *
..
0 , 94
NS
N!.i
1 01
u n t r e a t e d s i l a ge alone .
Th e v a l u e s ob t a i n e d o n formalin- t r e a t e d s i l a g e
w e r e intermedia te b e twe e n · u n t r e a t e d s i l a g e al o n e , and i n t r ap e r i t oneal
m e t h i o n i n e i n f u s io n .
I ndividual sheep S A data and th e v al u es for th e
t u r n o v e r c o n s t an ts are giv e n i n A p p e ndix 3 .
C alcul a t i o n o f t h e s i z e o f
t h e m e th i o n i n e p o o l f r om th e s e d at a , c o u l d only b e made f o r
s e v e n o f th e s h e e p . Th is w as d u e t o l o s s o f some plas m a s amples
c o l l e c t e d d u r i n g the early p a r t o f th e i n f u s i o n .
Th e pool s i z e s
o b t ai n e d , w e r e c ompared with t h e c alc u l a t e d val ues f o r t o tal p l asma and
muscle m e t h ionine .
T h e s e were c al c ul a t e d by assuming b l o o d v o l ume to
b e 5% o f b o dy we igh t and total m u s c l e weight to be 4 5% o f e m p t y b o d y
w e i g h t ( H arper , 1 9 69 ) .
Plasma m e t h io n i n e c o nc e ntrations w e r e me as u r e d ,
and t h e m e th i o ni n e c o n t e n t o f mus c l e was t a k e n as 0 . 58% ( U SDA , 1 9 68 ) .
T o tal plasma meth ionine was c o ns i d er a b l y lower than t h e v al u e s o b tained
f r o m t h e d ata , while t o t al mus c l e me t h i o n i n e was o n e th o us and times
h i gh e r .
Th e approximate pool sizes f or plasma 2nd mus c l e m e t h i o n i n e
w e r e c al c u l at e d as s uming b l o o d v o l ume to b e 5% o f b o d y weigh t , a n d t o t a l
m u s c l e w e i gh t to be 4 5% of e m p t y b o d y weigh t ( H ar p e r , 1 9 69 ) .
Pl���a
��thignin�
�gn�gntratione . �oro �aaourad, and tho �othionino oontont of
"'uoolo \:t!OO
tolwn . oo
0a 5@%
(USD/\, 196@) .
U r i n ar y-N , daily N r e t e n t i o n and p l asma urea c o n c e n t r a t i o n o n
f o rmalin-treated silage and u n t r e a ted s ilage suppleme n t e d with p r o t e i n ,
e n e r g y and methionine are g i v e n i n T ab l e 4 . 7
•
.
U r i nar y-N appeared t o b e
d e c r e as e d b y f ormal i n-t reatment o f s i l age i n Exp t . 1 , b u t N r e t e n t i o n
a n d p l as m a urea d i d n o t show a n y c o ns i s t e n t dif f e r e n c e b e tw e e n t r e a t­
ments .
I n E x p t . 2 , s upplementation o f u n t r e ated s i l a g e with p r o t e i n
s i gnific an t l y increased both u r i nary-N ( P
Z
0 . 01 ) a n d N r e t e n t i o n
( P Z 0 . 0 5 ) b u t h a d n o s i g n i f ic an t e f f e c t o n plasma u r e a c o n c e n t r a t io n .
N b a lance
4
( g/Da y )
*
+
3
2
+
1
0
1 00
-s o
1 50
D u od enal m e t h i o n i n e
75
( mg/k g • )
-1
+
+
-2
F ig.
4.3
N
b a l a nce
flow
r e s p o n se
i n t o th e
to
d u o d e num
�
untreated
*
t r e ated
@
untreated + met
i n c r eas ing
( Expt. 1 )
me t h i o n i n e
N b al an c e
( g/d a y )
8
A
6
•
6.
4
•
6.
• •
2
0
•
6.
A
•
0
•
0
0
1 00
200
0
400
300
D u o d e n a l meth i o n ine
75
( mg/kg • )
-2
0
-4
0
0
•
6.
A
0
•
F igo
4.4
�
c on t r o l
c on t r o l
+
+ p r o t e in
+ protein
+
+
energy
energy
N b al a n c e r e spo n s e t o i n c r e as ing m e th io n ine
f l o w i n t o th e d u o d e n u m
( Expto 2 )
met
+
+
met
met
Plasrn a m e th i o n i n e
c onc n . ( � mole/ 1 )
30
20
+
50
0
�
lit
•
+
10
*
+
1 DO
1 50
+
unt r eated
•
treated
$
untreated + m e t
200
Duod e n a l m e t h i o n i n e
( m g / k g . 75 )
F ig.
4.5
P l asma m e th i o n i n e c o n c e n t r a t i o n response to
i n c r e a s i n o d uo d e nal meth i o n in e ( E x p t o 1 )
Plasma methionine
c o nc n . ( � m o l e/1 )
50
•
40
•
...
•
•
Duod e nal m e th i o n i n e
75
( mg/kg " )
F ig.
4.6
P l as ma m e t h i o n i n e c o n c e n t r at i o n r e spo n s e
t o i n c r eas i ng d u o d e n a l m eth i o n ine ( E x p t . 2 )
1 --
% me th i o ni n e
o x id s e d t o c o
2
20
18
+
16
+
+
14
+
�
u n t r e at e d
*
treated
u n t r e a t e d + me t
12
0
50
1 DO
1 50
200
D u o d enal meth i o n i n e
75
( mg/kg Q )
F igo
4o7
P erc e n tage of meth i o ni n e o x i d i s ed to C 0 2 in
r e spo n s e t o i n c r e a s i ng m e th i o n ine f l ow i n t o
t h e d u o d e n um ( E xpt . 1 )
1 --
1 02
E n e r g y s u p p leme n t a t i o n h ad no s i gnific ant e f f ec t o n u r i n ar y- N , N
r e t e n t io n or p l asma u r e a c o nc e n tr a t i o n .
D u o d e n al i n f u s i o n o f m e t h i o n i n e
s ig n i f ic a n tly d ec r e a s e d u r i n a r y N ( P ( 0 . 0 1 ) a n d s i g n i f i c a n t l y
i nc r e a s e d N r e t e n t i o n ( P < 0 . 0 1 ) , b ut h ad no s i gn i f ic ant e f f ec t _ o r
plasma urea concentration .
F i gs . 4 . 3 - 4 . 7 g i v e t h e r e s p o n s e in N b a l anc e , p l a s ma me th i o n i n e
a nd p e r c e n t age o f m e t h i o n i n e o x i d is e d to C O , t o i n c r eas i n g l e v e l s o f
2
75
m e t h i o ni n e/kg "
e n t er i n g t h e d u o d enum . A l l th e p arame t e r s m eas u r e d
i nc r e as e d in r e s p o ns e t o i n c r e a s i n g m e t h i o n i n e av a i l ab i l i ty .
H owev e r ,
m at h e matic al anal y s is o f t h e c ur v e s d i d n o t d e t ec t any p o i n t s o f
i n f l ec t i o n .
N b al a n c e ( F i g s . 4 . 3 a n d 4 . 4 ) inc r e a s e d f r o m n e g a t i v e t o
p os i t i v e v al u e s in r e s p o n s e t o i nc r e a s i n g d uo d e nal m e th i o n i n e i n b o th
e x p e r i m en t s .
Th e d a t a w e r e c o n s i d e r e d t o o v a r i ab l e f o r acc u r at e
p r e d i c t i o n of t h e m e t h i o n i n e r e q ui r e d t o p r o d uc e z e r o N b alanc e , b u t
t h i s a p p e a r e d t o f al l w i th i n a range o f 50 - 1 0 0mg d u o d e nal me t h i o n i n e/
k g " 7 5 , i n Ex p t .
2.
1,
a n d 1 00 - 2 0 0mg d u o d enal m e t h i o ni ne/k g "
75
in Exp t .
Th e d ec l i n i n g i n ta k e s d u r i n g R e ps . 1 and 3 i n E x p t . 2 r e s u l t e d i n
m ar k e d l y d if f e r e nt i n t a k e s d u r i n g p e r i o d s o f b al a n c e and f l o w m eas u r ements .
Thus , N b a l a n c e v a l ues f o r F i g . 4 . 4 w e r e r ec a l c u l a t e d o n t h e
b a s i s o f a c tual N i n ta k e d ur i n g f l o w meas u r e me n ts .
4.4
A.
Discus sion
I n t r o d uc t i o n
E x p t . 1 wa s u n d e r t ak e n to c ompare th e e f f e c ts o f f o rmal in-tr e a t m e n t
o f s i l age , and I/P s up p l e m e n t a t i o n of u n t r e a t e d s i l age , o n t h e d i g e s t i o n
a n d u t il i s ation of N i n s h e e p .
T h e s e t r e atments h ad b e e n sh o w n t o
i n c r e a s e t h e v o l u n t a r y i n t a k e o f a b a s al s i l ag e d i e t , a n d th e m a i n aim
o f th e e x p e rime n t w a s t o i n v e s t i g ate p o s s i b l e f ac t o r s c o n t r o l l i n g t h i s
i nc r e as e .
Sinc e th e main e f f e c ts o f f o r m a l i n-t r e a tm e n t and m e th i o n i n e
1 03
i n f u s i o n w e r e · t o inc r e as e t h e m e t h i o n i ne and total am i n o ac ids
a v a i l a b l e to th e s h e e p , al t e r n a t i v e m e th o d s o f f u r th e r i nc r ea s i n g amin o
aci d avai l ab i l i ty w e r e s tu d i e d i n E x p t . 2.
Th u s , f o r m a l d e h y de-t r e a t e d
c as e i n and a n i ntra-r uminal i n f u s i o n o f r e ad i l y av a i l a b l e e n e r g y{ w e r e
u s ed t o s u pplement a b as al s il a g e d i e t .
The effec t s o f th e v a r i o us
t r e a t m e n t s on t h e d i g e s t i o n o f s il a g e h a v e al ready b e e n d i s c u s s e d
.( C h a p t e r 3 )j a n d th u s th e f o l l o w i n g d is c u s s i o n wi l l c e n tr e on t h e
u ti l i s a t i o n of meth i o n i n e , r e l a t i v e t o th e p r e s enc e o f o th e r amino
acid s .
B.
M e th i o n ine and to tal am i n o a c i d s b e f o r e and a f t e r a b s o rpt i o n
I n E x p t . 1 , th e to tal f l ow of am i n o a c i d s in t o th e d u o d e num w a s
i nc r e a s e d b y f o rmal i n-t r e atme n t of s i l ag e a n d th e a b s o r p t i o n o f
meth i o n i n e alo n e , w a s i n c r e a s ed b y I/P i n f u s i o n o f m e t h i o n i n e t o sh e e p
f ed u n t r e at e d s ilage ( Ta b l e 4 . 2) .
H o w e v e r , t h e t o t a l f l ow o f ami n o
a c i d s o n l y ran g e d f r o m 3 2 . 8 - 7 2 . 0 g/day , and t h e m e th i o n i n e av a i l a b le
f or a b s o r p t i on r a ng e d f r o m D . B - 2 . 5 g/day .
I n Expt. 2 , the daily f low
o f a m i no ac ids i n t o t h e d uo d e n u m o n u n tr e a t e d s i l a g e w a s i n c r e a s e d b y
b o th f o rm aldeh y de-t r e a t e d c a s e i n a n d i n t r a ruminal i n f u s i o n o f e n e r g y
( Tab l e 4 . 2 ) .
E ac h t r e a t m e nt w a s a l s o s up p l em e nt e d w i t h a d uo d e n al
i n f u s i o n o f meth ionine .
Th e r a n g e of f l o w o f total am ino a c i d s and o f
t h e m e th i o n ine avail a b l e f o r a b s o r p t i o n w e r e b o th g r e a t e r t h a n f o r
E xp t . 1 .
i . e . 33 . 1 - 1 67 . D g am i n o acids/day , and D . B - 6 . D g
meth i o n i n e/day .
Th e q u a n t i t y o f ami n o a c i d s a b s o r b ed f r o m th e i n t e s t i n e s was n o t
meas u r ed , b u t i t was ass umed th at t h e . am o u n t abs o r b e d w o u l d i n c r e a s e
w i th i nc r e as i n g f l o w r a t e s o f a m i n o a c i d s i n t o th e d u o d e n u m .
A r ms t r o n g
( 1 97 3 ) d i d r e p o r t a s i g n i f ic an t dec r e a s e i n t h e a p p ar e n t d i g es t i b i l i t y
o f m e th i o n ine i n th e small i n te s ti n e · w h e n i n t ake w a s i nc r e as e d f r o m
I.
I
1 04
900 - 1 4 0 0 g/d a y .
H ow e v e r , th e d e c r e a s e i n d i ges t i b il i t y o n g r o und an d
pell e ted g r a s s was o n l y 7 . 5% , and t h u s th e ab s o l u t e amo u n t a b s o r b e d
would s t i l l h a v e inc r e as ed .
and c as e i n ( Ex p t .
2�
F o rmald e h y d e-b ind ing t o s il a g e ( E x p t . 1 )
appear e d to b e l a r g e l y r e v e r s e d p r i o r t o d i g e s t i on
i n t h e i n t e s t i n e s , s i nce t o t al i n t es t i nal N d i g es t i o n was n o t a d v e r s e l y
aff e c ted b y th e f o rm a l d e h y d e -t r e atme n t ( C h ap t e r 3 , T a b l e 3 . 1 3 ) .
D u r i ng a b s o r p t i o n o f amino a c i d s f r o m th e d uo d e n u m , s o me c h a n g e s in
conc e n t r a t i o n o c c u r , d u e to t h e u t i l i s a t i o n of ami n o a c i d s b y the gut
t is s u e ( H ume e t al . ,
1972;
W o l f f e t al . , 1 9 73 ) .
I n a d d i t i o n , m e ta-
b o l i s m of amin o ac i d s by the l iv e r , and u p t a k e of amino ac i d s b y th e
indi v id u a l tis s u e s f o r p r o t e i n s yn t h e s i s , r e s u l t i n d i f f e r e n t amino
acid c o nc e n tr a t i o n s i n p l as m a taken f r o m p o r tal , j u g ul a r o r c ar o t id
b lo o d v e s s els ( R e i l l y a nd F o rd , 1 9 7 1 ;
Wol f f e t a l . , 1 9 7 3 ) .
J u g ul a r
b l o o d s a m p l e s w e r e t a k e n i n t h e p r e s e n t w o r k, and s o metab o l i s m o f amino
ac i d s in the t i s s u e s prior t o meas u r e me n t i n j u g u l a r p l as m a will af f e c t
t h e c o nc e n tr a t i o n s r e l a t i v e to t h e am o u n t a b s o r b e d .
Uti l i s a t i o n o f ami n o ac i d s b y th e t i s s u e s c a n b e b r o a d l y d i v i d e d
i n t o t w o c at e g o r i e s .
( 1 ) T h e f i r s t i s p r o t e i n s y n t h e s is , w h i c h r e q u i r e s a p a r t ic u l a r c om p l ement o f am i n o a c i d s , c o r r e s p o n d i n g to t h e c o mpos i t i o n of t h e p r o t e i n
f o rm e d .
Th e r a t e o f p r o te i n s yn th e s i s w i l l b e l i m i t e d b y th e av ail-
abil i t y of the amino acid wh ich is p r es e n t in the l o w e s t c o n c en t r a t i o n
r el a t i v e t o th e c o n c e n t r a ti o n r e q u i r e d .
Th us , an i n a d e q u a t e s u p p l y o f
o n e a m i n o a c i d w i l l l i m i t th e u t i l i s a t i o n o f o t h e r s .
Rate o� tissue
prot e in s y nth e s i s h a s a l s o b e e n f o u n d t o v ary in r e s p o n s e t o t o tal
ami n o ac i d av a i l a b i l i t y ( M un r o , 1 9 69 ;
C l emens , 1 9 7 2 ) , and t h e ac t i o n s
o f g r owth h o rmo n e , i ns u l i n a n d t h y r o x i n e ( Mun r o , 1 9 64 ;
1 964 ) .
Cahill et al. ,
1 05
( 2 ) T h e s ec o nd c a t e g o r y c ov e r s t h e r e q u i r e m e n t s f o r i n d i v i d u a l a m i n o
a c i d s f o r s p e c i f i c f unc t ions .
Meth i o n i n e i s r e q u i r e d as a d o n o r o f
m e t h y l g r o ups and a l s o f o r c o n v e r s i o n to c ys t e i n e ( F in k e l s t e i n a n d
Mu d d , 1 9 6 7 ) .
Th e f ac t o rs c o n t r o l l in g t h e d i s trib u t i o n of s p ec i f i c amino ac i d s
b e t w e e n th ese f unc t i o ns h a v e n o t y e t b e e n c l ea r l y d e f i n e d .
However ,
s ome 6f th e d i e t ary fac t o rs i n v o l v e d in th e r e gul a t i o n o f m e th i o ni n e
m e t a b o l i s m h av e b e e n s t u d ie d .
I nc r e a s e d av a i l a b i l i t y o f c y s t ( e ) in e h a s
b e e n f o u n d to d ecr e a s e c o nv e rs i on o f meth i o n i n e t o c y s te i n e ( S h a n n o n
e t al . , 1 9 7 2 ) ;
l o w ami n o a c i d i n t ake i n c r e a s e d r e g e n e ra t i o n o f
meth i o ni ne f r o m h om o c y s t e i n e ( F i n k e l s te i n and Mud d , 1 9 67 ) ;
and low
m e t h i o n i n e i n t a ke d ec r e as e d th e u s e of m e t h i o n i ne as a meth y l g r o u p
d o n o r ( F i n ke l s t e in a nd M u d d , 1 9 67 ) .
Th es e s t u d i e s w e r e c o n d uc t e d o n
i n v i t r o l iv e r pr e pa r at i o ns f r om m o n o g as t r i c animal s .
The impo r tanc e
o f c y s t ( e ) i ne i n w o o l g r ow th ( R o s s , 1 9 6 1 ) w o ul d b e e x p e c t e d to p r o d u c e
mar k e d d i f f e r e nc es i n S-am i n o a c i d m e t ab o l i s m in s h e e p, c om pared t o
mo n o g as tr ic s .
Howev e r , t h e r e s u l ts q u o t e d d o emph a s i s e t h a t a n umb e r
o f f a c t o r s i n f l uenc e m e t h io n i n e m e t ab ol i s m, and th at o u r und e r s t a n d i n g
o f t h e s e f ac to rs i s s t i l l i n c o mp l e t e .
I n E x pt . 1 , p l a s m a meth i o n i n e c o n c e n t r a t i o n a p p e a r e d t o b e
inc r e as e d b y I /P me t h i o n ine i n f us i o n , b u t n o t b y f o rm alin- t r e a t m e n t o f
silage ( Table 4 . 3 ) .
To tal p l asma ami no ac i d s w e r e l ower o n u n t r e a t e d
s i l a g e p l u s meth ion i n e , t h a n t r e a t e d s i l a g e , w h i l e th e r e w a s c o ns i d e r­
a b l e vari a b il i t y i n th e v a l u e s f o r u n t r e a t e d s i l a g e a l o n e .
Th e s e
r e s u l ts s u g g e s t that t h e q u a n t i t y o f me th i o ni n e a v a i l a b l e t o th e t i s s ue s
on u n tr e a t e d s ilage w a s l i m i t i n g t i s s u e p r o t e i n s yn t h e s i s .
Meth i o n i n e
i n f u s i o n p o s s i b l y s t im u l at e d p r o t e i n s yn th e s is b y r e m o v i n g amino ac i d s
f r o m th e p l asm a .
1 06
I n E x p t . ·2 , n e i t h e r th e c o nc e n tr at i o n of meth io ni n e , n o r " to tal "
amino ac i d s i n the p l asma w as a f f e c t e d b y s uppleme n t a t i o n w i th e n e r g y
o r p r o te i n .
H oweve r , plasma m e th io n i n e was increas e d ( P .( 0 . 0 1 ) a n d
" to t a l " p l asma ami n o a c i d c o n c e n t r a t i o n w a s d e c r e a s e d ( P <. 0 . 0 1 ) b y
d uo d e nal m e t h i o nine i n f u s io n , o n a l l treatments .
Th e d ec r e as e i n
'' t o t a l " p l asma ami n o ac i d c o n c e n t r a tions o n all t r e a t m e n t s s ug g e s ts
that ma x i mum t i s su e p r o t e i n s y n t h e s i s was s till l i m i t e d b y m e th i o n i n e
avai l a b i l i t y a t 2 . 7 g meth i o n e/day ( + pro t e in , Tab l e 4 . 2 ) .
Th e c o nc e n­
t r at i o n o f " t o t al" amino ac i d s in t h e plasma was n o t s i g n i f ic a n t l y
affec t e d b y tr eatme n t d e s p i t e a two-fold i n c r ease i n t h e f l o w o f amino
a c i d s into the d u o d e n um .
Th i s s u g g e s ts th a t th e a d d i t i o nal amino
acids wer e e i t h e r b ei n g e F f i c i e n t l y util i s ed o r r a p i d l y d e amin ate d .
The a b o v e res u l t s s u g g e s t t h a t the av ailabil i t y o f meth i o n i n e wa s
l ow e r th a n th at r e q u ir e d f o r maximum tis s u e protein s y n th e s i s , even a t
t h e h i gh e r f l o w r a t e s o f ami n o ac i d s i n t o th e duo d e n u m .
B a r r y e t al .
( 1 973 ) f o u n d no b e ne f ic i al e f f ec t o f I /P m e th i o n i n e s up p l e m e n t a t i o n o f
f ormalin- t r eated s i l age o n i n t a k e o r l i v e-weight g a i n , b ut w o o l g r ow th
was i n c r e as ed .
Th e h i gh me th i o n i n e r eq u i rement f o r w o o l g r owth b y
she e p , m a y inc r ease t h e i r s u s c e p t i b i l i ty t o meth i o n i n e imb al anc e , at
low l e v e l s of amino ac i d av a i l ab il i t y .
I/P i n j ec t i o n o f m e th i o n i n e t o
s h e e p f e d u n t r e a t e d s i l a g e p r od uc e d a g r e a t e r inc r e a s e i n w o o l g r owth
than f o r m a l in-treatment o f s i l a g e , b u t f o rmalin- t r e a t m e n t p r o d uc e d a
g r e a t e r i n c re a s e i n i n t a k e th an m e t h i o n i n e infus i o n ( B ar r y e t al . ,
1 9 73 ) .
Th i s s u gges t s th a t f e e d i n g u n t r e a t e d s i l a g e t o s h e e p m a y
p r o d u c e a n imb alanc e s i t u at i o n wh ich c a n b e par t i a l l y al l e v i a t e d b y
meth i o n i n e i n f u s i o n .
A m i n o a c i d b al a nc e c an als o b e imp r o v e d b y
i nc r e as e d av ailab i l i ty o f t o t al a m i n o ac id s .
Th u s , an imb alanc e
s it u a t i o n i s m o r e l i ke l y to o c c u r at low f l o w r a t e s o f t o t a l amino ac i d s
i n t o th e d u o d e num .
Th i s m i g h t i n d ic a t e t h a t th e e x t r a-p r o t e in r eq u i r e-
1 07
m e n t f o r m e th i o n in e is p r o p o r t i o n al l y h i gh er, than at h i gh e r f l o w r a t e s
o f t o tal a � i n o ac i d s i nt o th e d u o d e n um ( Grau a n d Ka�e i , 1 9 5 0 ) , o r
al t e r n at i v e l y t h a t h i g h e r a v a i l a b i l i t y o f total amino ac i d s r e d u c e s
the e f fec t o f a l o w meth i o n i n e p r o p o r t io n .
H owev e r , i n E xp t . Z , .i n tak e
s ti l l d e c r eas e d s l i gh t l y , at h i gh e r f l o w rates of �uodenal amino ac i d s .
This may s ug g e s t t h a t th e e x t r a-p r o t e i n r e q u i r eme n t s f o r m e t h i o n i n e
w e r e h i gh e r f o r th e s h e e p i n Ex p t . 2 th an i n E xp t . 1 , wh e r e no i n t a k e
r e s p o n s e was r e c o r d e d .
Th is w i l l b e d i s c u s s e d l a t e r , i n r e l a t i o n t o N
b al a nc e .
The p l as m a m e t h i o n i n e c o nc e n t r a t i o n s o n the b as al s il a ge d i e t w e r e
h i gh e r i n E x p t . 2 t h an E x p t . 1 .
S i n c e th e d e c r ea s ed i n t a k e in E x p t . 2
( C h a p t e r 3 ) s ug ges t s th a t m e th i o n i n e was m o r e lim i t i n g in E x p t . 2 th a n
E xp t . 1 , t h is d i f f e r ence may r e f l e c t e i t h e r a g r e a t e r a d a p t a t i o n to l ow
meth i o n i n e i n E x p t . 1 , o r a d ec r e as e in m e t h i o n i n e l o s s w i t h s t o r a g e o f
pl as m a s amp l e s .
Th i s l a t t e r p o s s i b i l i t y a r i s e s f r om th e t ime d el a y
b e t w e e n c o l l e c t i on and anal y s i s o f s am p l e s in Exp t . 1 ( 1 y e ar ) c om p a r e d
t o E x p t . 2 ( 1 m o nth ) .
Th e " to t a l " a m i n o a c i d c o n c e n t r a t i o n in p l a s m a
a l s o d i f f e r e d b e tw e e n th e tw o e x p e r i m e n t s , b u t th i s was d u e t o t h e
omis s ion o f th e b a s i c amino ac i d s i n E x p t . 2 .
C.
T h e e f f e c t o f t r eatm e n t s o n i n d i v i d u al amino acid p r opo r tions i n
t h e pl asma
The e f f ec t of t re a tm en t s on th e c o n c e n tr a tio n of i n d i v i d u a l ami n o
ac i d s i n t h e pl a s ma was a l s o m e as u r e d ( Tab l e 4 . 4 ) .
S i n c e p l a s m a ami n o
a c i d c onc e n tr a t i o n s a r e r ead i l y af f ec t e d b y n umer o u s n u t r i t i o n a l a n d
phy s i ol o g i c al f ac t o r s ( Swen d s e i d e t al . , 1 9 67 ;
O l t j e n e t al . , 1 9 69 ;
S yn d e r man e t al . , 1 9 6 8 ;
Amos e t al . , 1 9 72 ) , few co n c lus i on s c an b e d r aw n
f ro m c h a n g e s i n i n d i v i d u a l p l a s m a a m i n o acid c o nc e n tr a t i o n s .
H owev e r ,
1 08
o b s e rv at i o n s on th e e f f ec t o f i n c r e a s e d m e th ionine av a i l a b i l i t y o n th e
c o n c e n t r a t i o n o f o th e r p l as m a amino a c i d s m ay giv e s o m e i n d i c a t i o n o f
s pec if ic ac t i o ns o f m e t h io n i n e .
Th e d i f f e r e nc e s i n p e rc e n t a g e c o nc e n t r a t i o n s of s e r ine , alan i n e ,
c y s tath i o n i n e , m e t h i on ine , l euc i n e and i s o l e uc i n e b e tw e e n r e p l ic at e s in
Ex p t . 2 w e r e s i g n i f i c an t .
Taur i n e c o n c e n t r ation was t o o l o w t o be
m e as ured i n th e p l a s ma samp l e s f rom r e p l i c a te 1 , E x p t . 2 , but i t s c o n­
c en t r a t i o n c o ul d b e meas u r e d af t e r f o u r t o s e v e n
i n f u s io n .
d a y s o f m e th i o n i n e
T a u r i n e w as p r e s e n t i n t h e p l a s m a o f th e s h e e p p ri o r t o
m e t h i o n i n e i n f u s i o n i n r e p l ic a t e s 2 and 4 .
Th e s e r e s u l t s s u g g e s t t h a t
th e r e - was a c ar r y o v e r e f f e c t f r o m th e p r e c e d ing m e th i o n i n e i n f u s i o n
p eriods .
Th i s e f f ec t a p p e ar e d t o b e s im i l a r on a l l t r e atm ents , a n d
t h us it w o u l d h a v e h ad no e f f ec t o n th e t r e atment c o m p ar i s o n a s a n a l y s e d
b y t h e A n a l y s i s o f V arianc e .
Th e p e r c e n t a g e s o f c y s t ei n e , c y s t a th i o nine and t a u r i n e w e r e s i gnif­
i c an tly i n c r e a s e d b y m e th i o n ine i n f u s i o n ( P < 0 . 0 1 ) .
Th is s u g g e s t s
t h a t c o n s i d e r a b l e c o nv e r s i o n of m e th i o n i n e to cys t e i n e d i d take p l ac e .
C y s tath i o n i n e i s an i n t e rm e d i ar y i n th e c o n v e r s i o n o f m e th i o n i n e t o
c ys t eine , and t a u r i n e i s a p r o d u c t o f th e m e tabolism o f c y s t e i n e
( Ch ap t e r 1 , F i g . 1 . 2 ) .
Th e p l a s ma amino ac id c o n c e n t r at i o n s af t e r o n e
d a y of m e th i o n i n e i n f u s i o n w e r e a l s o m eas u r ed in Ex p t . 2 ( A p p e n d i x 5 ) .
Th e s e w e r e i n t e r e s t i n g in r e s p e c t o f th e c h ange i n t a u r i n e c o n c e n t r a t i o n .
I n r e p l i c a t e s 2 a n d 4 , t a u r i n e w a s p r e s e n t p r i o r to m e t h i o n in e i n f u s i o n ,
b ut h ad d i s a p p e ared f r o m th e s am p l e t a k e n 24h a f t e r t h e s ta r t o f
i n f us i o n .
I t r e ap p e ar e d a f t e r f ou r d a y s o f infu s i o n .
Th e r e was al s o a s i g n i f ic a n t i n t e r ac t i on b e tw e e n s up p l em e n t s and
me t hi o ni ne i n f usion o n th e p er c e ntage o f c y s t e i n e and s er i n e i n th e plas m a
1 09
(P
<
0 . 0 5 ) . Th e d e c r ea s e i n s e r i n e p e r c e n t a g e in r e s p o n s e t o m e t h i o n i n e
i nf u s i o n , w as l es s o n the p r o t e i n s up p le m e n t e d di e t , th a n o n t h e oth e r
two t r e atme nts . S i n c e s e r i ne i s r e qu i r ed f o r the c o n v e r s ion o f m e th i o ni n e
t o c y s te i n e , t h i s m i g h t s u g g e s t a d ec re a s e d p e rc e n ta g e c o n v e r s i o n o f
m e t h i o nine t o c ys t e in e a t high e r f l ow s o f d u od e nal amin o ac i d s .
H o we v e r ,
t h e p lasma c y s t eine p e r c e n ta g e gave a g r e a t e r r e s p o n s e t o m e th i o n i n e
i n f u s i on o n th e p r o tein s u p p l em e n t ed t h a n the c on tr o l d i e t . R e i s et a l .
( 1 9 7 3 ) als o r e p o r t e d d e c re a s e d p l asma s e r i n e c o nc e n t r at i o ns , and
i n c r e a s e d taur i n e a n d c ys t ath i o n i n e c o nc e n t r ation s , in r e s p o n s e to
p o s t r u mi n a l me th i o n i n e i n f us i o n . S imilar ch anges to th o s e r ec o r d e d f o r
t h e o th e r amin o a c i d s h av e a l s o b e e n r e p o r t e d ( S ynd e rman et al . , 1 9 68 ;
·
T a o e t al . , 1 9 74 ) .
S u p p l ement ati o n with p r o t e i n and e n e r g y ( Exp t . 2 ) h ad a s i g n i f i c a n t
e f f e c t o n th e p e r c e n t a g e o f th r e o n ine , g l y c i n e , v al i n e , is£l e uc i n e ,
l e u c i n e a nd t y r o s i n e ( P ( 0 . 0 1 ) .
Th e s e c h a n ge s g e n e ral l y r e f l e c t th e
d i f f e r e n c e s in d uo d e n al amino a c i d p r o p o r ti o n s b e tw e e n t r e a tm e n t s .
H owev e r , the inc r e a s e s i n valin e , i s o l e u c i n e and l e u c i n e a r e n o t r e l a t e d
t o d u o d e n a l amino ac id f l o w .
S i nc e th e m ain r e q u i r e m e n t f o r th e s e amino
ac ids is for th e s y n t h e s i s o f tissue p r o t e i n , th e s e inc r e as e s m a y
i n d i c a t e a c h a n g e i n th e r ate o f ti s s u e p r o t e i n s y n t h e s i s r e l a t i v e t o
amino ac i d availab i l i t y , a s d u o d e nal amino a c i d f l ow r a t es inc r e ase .
T h e c h an g e s in th e p r o p o r tions o f p l asma amino ac i d s w i th t ime
a f te r the s t ar t of m e th i o n i n e i n f u s i o n w e r e also meas u r e d ( T a b l e 4 . 5 )
in Expt. 2 , Rep . 1 .
Th e g r e a t e s t c h a n g e s a p p e ar e d to o c c ur d u r i n g th e
f irs t two days of i n f us io n , a n d the p e r c e n tages w e r e r e l a t i v e l y c o n s t a n t
after four days of infusion.
T h i s s u g� e s ts t h e r e f o r e , that th e p e r i o d
o f s i x d a y s ad j u s tm e n t t o meth i o n i n e infus i o n w a s o f s uf f ic i ent l e n g th .
110
D.
M e tab o l ism of m e th i o n i ne
Meth i on i n e meta b o l i s m was s t u d i e d th r o ugh the u s e of l ab el l ed
meth i o ni n e .
C o n tinuous i n f u s i o n o f L-me t h i o n i ne-C 1 4 ( U ) o v e r a p e r i o d
o f 1 2h, e n a b l e d es timat i o n of meth i o n i n e t u r n o v e r r a t e , t h e perc e nt a g e
o f m e t h i o n i n e o x i d i s ed t o c o , th e p e r c e n tage o f c o d e r i v e d f r o m
2
2
meth i o nin e a n d t h e s i z e o f th e p o o l w i th wh i ch i n f u s e d meth i o n i n e w a s
in e q u i l i b r i um ( T�b l e 4 . 6 ) .
The I/P meth i o nine i n f u s i o n s u p pl i e d an
a v e r a g e of 1 . 2 g meth i o n i n e/d ay ( i . e . 0 . 3 3 mmo l e/h ) m o r e than the
u n t r e ated s i l a g e al o n e .
T h i s i n f u s i o n i n c r e as e d the m e t h i o n i n e turn­
o v e r rate b y a n av e rage o f 0 . 2 7 mmo l e/h .
Th e treated s i l a g e p r o v id e d
a n a d d i t i o nal 0 . 6g/d a y ( i . e . 0 . 1 7 mmol e/h ) w h i c h inc r e as e d m e th i o nine
tur no ver b y an average o f 0 . 1 3 mmo l e/h .
T h e s e f ig u r e s s u ppo r t th e
h yp o t h e s i s th a t meth i o n i ne a b s o rp t i o n i n c r e a s e d w i th b o th i n c r e a s e d
f low o f amino acids i n t o th e d u o d e n um , a n d i n f u s i o n o f meth i o n i n e i n t o
t h e p e r i t o neal c av i ty .
E s t i m a t i o n of the s i z e o f th e me th i o n i n e pool , f r o m pl asma
m e th i o n i n e SAs , could only be mad e for
s e v en · of
the
sheep .
However ,
the s i z e o f p o o l appeared to i n c r e a s e f r om 5 62 - 1 026�mo l e , in r e s p o n s e
t o I /P me t h i o n i n e i n f us i o n .
I n o r der t o g a i n some u n d e r s t a n d i n g o f t h e
s o ur c e o f meth i on i n e c o n t r i b u t i n g t o t h i s p o o l , es tima t e s o f th e t o t a l
p l a s m a a n d mus c l e m e t h i o n i n e w e r e mad e , u s i n g data o n b o dy w ei gh t a n d
p l �s m a me t h i o n ine c o nc e n t r at i o n .
m e th i o n i n e was 2 1 �moles .
T h e a v e r a g e val u e f o r t o t a l p l asma
Th i s s u gges ts that the t u r no v e r rates meas u r e d
( Tab l e 4 . 6 ) di d n o t r e f e r s o l e l y t o t u r n o v e r of meth i o nine in th e
p l as m a , b u t th a t an a d d i t i o n a l p o o l o f meth i onine was a l s o i n v o l v e d .
The m e th i o nine pool s i z e o b t a i n e d f r o m the d a ta was o n l y 0 . 1 % o f t o t a l
m usc l e m e t h i o n i ne ( av . 5 7 8mm o l e ) .
H o w e v e r , m u s c l e a p p e a r s to c o ns i s t
o f b o th m e tab o l ic al l y a c t i v e and inac t i v e amino a c i d p o ols ( Munr o ,
111
1 9 64 ) .
H e nc e � the meth i o n i n e p o o l t o wh ich the t u r n o v e r r a t e a p pl i e s ,
may b e c ompos e d of pl as ma m e th i o ni n e , l i v e r meth io ni n e a n d/or f r ee
mus c l e me t h i o n i ne .
I
I.
I
The p e r c e n tage of meth i o n i n e w h i c h was oxid i s e d to c o
meas u r e d , a n d was i n c r e as e d b y I/P i n f us i o n of m e th i o n i n e .
2
was a l s o
Th is m ay
ind ic a te e i th e r th a t meth i o n i n e was b e i n g s up p l i e d i n exces s o f r e q u i r emen t , o r t h a t u til i s a t i o n o f m e th i o n i n e f o r n o n- p r o t e i n func t i o n s w a s
inc r e as ed uJith i nc r e as e d me t h i o n i n e av ail ab i l i t y .
D u r i n g t h e c o nv e r s i o n
o f m e t h i o nine t o c y s te i n e , t h e c ar b o x y l m o i e t y is c o n v e r t e d i n t o
�
- k e t o b u t y r i c ac i d , which m a y th e n b e o x i d i s e d a n d
14
co
2
expired .
A g u i l a r e t al . ( 1 9 74 ) r e p o r t ed ch a n g e s in t h e me t a b o l i s m o f m eth i o n i n e
b y r at s w i th i n c r e as i n g perc e n t a g e o f meth i o nine i n th e d i e t .
They
obs e r v ed t h a t i n c o r p o r at i o n o f t h e m e t h y l g r o up o f m e t h i o n i n e i n t o
pro t e i n s i nc r e as ed as m e t h i o nine i n c r e as ed , w h i l e i n c o r p o r a t i o n of th e
c ar b o x yl g ro up rem a i n e d unch ang e d .
T h u s , o x i d ati o n o f the c ar b o xyl
gro u p i nc r e as e d mo r e r ap i d l y t h a n o x i d ati o n o f the m e th yl g r o up wh i c h
was i n c o r p o r a t e d i n t o o t h e r a m i n o ac i d s a n d h e nc e i n t o p r o t e ins .
Th e p e r c e n tage of c o
I /P i n f u s i o n .
d e r i v e d f r om m e t h i o n i n e a l s o i nc r e as e d w i th
T h e a v e r a g e f o r a l l tr e atm ents w a s 0 . 1 % at a t u r nov e r
rate of D . 0 1 g/h .
1 9 63 )
2
C o rr e s p o n d i n g v a l u e s f o r g l uc o s e ( B e r gman e t al . ,
w e r e a t u r n o v e r r a t e o f 4 g/h g i v i n g a p e r c e n t a g e of c o
f r o m gluc o s e o f 1 0% .
2
derived
Thus , o x i d a t i o n o f m e t h i o n i n e makes a c om p ar a t i v e l y
min o r c o n t r ib u t io n t o t o t a l o x i d a t i o n i n t h e s h e ep .
E.
O v e r a l l N me ta b o l i sm
Th e c h a n g e s i n t h e rate of t i s s u e p r o t e i n s y n th es i s w h ich h av e
alr e a d y b e e n d isc u s s e d, w i l l a l s o a f f ec t d a i l y N r e te n t i o n b y th e s h e e p .
I n E x p t . 1 , f o r mal i n-t r e a t m e n t o f s i l ag e r e s ul t e d i n n e t a d d i t i o n o f N
112
t o t h e rumen , . c o m p a r e d t o n e t ab s o r p t i o n o n u n t reated s il a g e ( C h ap t e r 3 ,
Tab l e 3 . 9 ) .
Thus , the b al a n c e of ami n o a c i d s abs o rb e d , to amm o n i a
ab s o r b ed woul d h av e b e e n h i gh e r o n th e t r e a t e d than u n t r e a t e d s i l ag e .
H e nc e , u r i nary-N a p p e a r e d to d ec r e a s e i n s h e ep fed u n t r e a t e d c o m p a r e d
to t r ea t e d s i l a g e .
H o w ev e r , N r e t e n t i o n a n d p l asma u r e a c o n c e n t r at i o n
d i d n o t s h ow a n y c o n s i s te n t t r e a t m e n t d i f f e r en c e ( Ta b l e 4 . 7 ) .
I n Exp t . 2 , u r i n a r y-N was s i g n i f i c a n t l y i nc r e a s e d b y s u p p l e m e n t a t i o n
w i th f ormal d e h y d e- t r e a t e d c a s e i n o v e r u n t r e a t e d s i l a g e a l o n e .
Th i s w a s
p o s s i b l y due t o inc r e a s ed d i g e s t i o n o f N i n th e s tomach r e g i o n i n
r e s p o n s e to th e p r o t e i n s u p p l ement ( C h ap t e r 3 , Tab l e 3 . 9 ) .
I n a d d i t ion ,
th e u t i l i s a t i o n of abs o r b e d amino a c i d-N i s l imi ted b y i ts b a l a n c e o f
ami n o ac ids , r el a t i v e t o t h e pro p o r t i o n s r e q u i red f o r t i s s ue p r o t e i n
s y n t h es i s .
Th e e f f e c t of m e t h i o n i n e i nf u s i on i n i n c r eas i n g t is s u e
p r o t e i n s y nth e s i s h as a l r ead y b e e n d i s c u s s e d .
Th us t h e d e c r e a s e i n
u r i n a r y-N in r e s p o n s e to m e t h i o n i n e i n f u s i o n, c an b e a t t r i b u t e d t o
i nc r eased e f f i c i e n c y o f u t i l i s a t i o n o f a m i n o ac i d s f o r t is s ue p r o t e i n
s yn t h e s i s .
U r i n ar y-N o n t h e p r o t e i n s u p p l em e nted d i e t w a s o n l y s l i gh t l y
d e c r eased b y d uo d e n al m e t h i o n i n e i n f u s i o n , b u t N i n t a k e h ad inc r e a s e d
o n a l l . tr eatme nt s .
N b al an ce was s i g n i f i c an tly inc r e a s e d b y b o th
p r o t ei n s up p l e me n t ati o n ( P � 0 . 0 5 ) and b y m e t h i o n i n e i n f u s i o n ( P � 0 . 0 1 ) .
T h i s r e s u l t g iv es f u r t h e r ev idenc e that t i s s u e p r o t e i n s y n t h e s is w a s
i n c r e as e d b y b o th inc r e a s e d total amino ac i d , a n d m e th i o n i n e , a v a i l ­
ability .
E n e r g y s u p p l e m e n t a t i o n h ad n o s i g n i f i c a n t e f f e c t o n N b al anc e o r
u r i n ar y-N , d e s p i t e a n i n c r e a s e d f l o w o f ami n o ac ids i n t o th e d u o d e n um .
Th i s may r e f l e c t d ec r e as e d a b s o r p t ion o f ammonia f r o m th e r ume n , d u e to
m o r e e f f i c i e n t u t i l i s ati o n of N P N in th e r um e n ( C h a p t e r 3 ) .
113
T h e r e we r e no s i g n i f i c a n t d i f f er enc e s in plas m a u r e a c onc e n tr a t i on .
This res u l t s u g g e s t s th a t p l asma u r e a c o n c entrati o n i s n o t a s u f f ic ­
ien t l y s e n s i t i v e p ar ame t e r f o r a s s e s s i n g t h e N s t a t u s o f r um i n a n ts .
Thi s was r e inf o r c e d b y d a i l y meas u r em e n t o f p l asma u r e a c o n c e n tr a t i o n
a f t e r th e s tart o f m e th i o n i n e i n f us io n .
The effect of duration of
meth i o n i n e inf u s i o n o n th e p e r c e n t a g e c o n c entrati o n o f ami n o ac i d s i n
t h e p l as m a has a lr e a d y b e e n d is c u s s e d ( Tab l e 4 . 5 ) a n d thes e s h o w e d a
g rad u al c h ange with t ime a f t e r t h e s ta r t o f inf us i o n .
Howe v e r , p l a s m a
urea c o n c e ntrations d i d n o t s h ow any c o n s is t e n t t r e n d s .
F.
R equ i r ements for meth i o n i n e
O n e o f th e aims o f t h e s e e x p e r i m e n t s was to e s t imate m e th io ni n e
r e q u i reme n t f o r th e s h e e p .
T h e p ar a m e t e r s t o b e u s e d f o r t h is e s tim�
a ti o n wer e :
( a ) N bal a nc e ,
( b ) p l as m a meth i o ni n e ,
( c ) oxidation of methionine t o
co2 •
Thes e p a r a m e t e r s h av e b e e n u s e d to es timate amino a c i d r eq u i r eme n ts i n
n o n- r umi n ants ( Z immerma� a n d Sco t t , 1 9 65 ;
N e ale a n d Wate r l o w , 1 9 74 )
b y p l o t t i n g - inc reas i n g a m i n o ac i d i n take v e r s us r e s p o ns e .
Thes e
" res p o ns e c ur v e s " g e n e r al l y t ake th e f o r m o f two i n t e r s e c t i n g l i n e s ,
with t h e p o int o f i n t er s e c t i o n b e i n g d e s i g n a t e d as th e o p t imum r eq u i r e­
ment .
A b o v e t h i s p o i nt , an i n c r eas e d r e s p o n s e in t e rms of N b a l anc e
r e q u i r es a greater i nc r em e n t in meth i o n i n e i n take, th an the s ame s i z e o f
i nc r e ase b elow t h e p o i n t o f i n t e r s e c t i o n .
Howeve r , th e r e s u l t s r e p o r t e d
h er e f o r r umina nts, e m p h as i s e t h e l im i t s o f s uch m e t h o d s f o r es t i m a t i n g
meth i on i n e r e q u i r e m e n t b y h i g h l i gh t ing t w o f ac to r s i n p ar t i c ular ,
which s h o u l d b e d e f i n e d f o r a n y e s t im atio n o f r e q u i r em e n t .
114
( 1 ) P hys i o l ogic al s t ate :
A c om p a r i s o n o f the dail y N r e t e n t i o n b e t w e e n
E x p t . 1 a n d 2 , s h o w e d a m a r k e d d i f f e r e nc e i n t h e q uant i ty o f N r e t a i n e d
a t l o w l e v els o f meth ionine availab i l i t y .
4 . 3 and 4 . 4 .
Th is may b e s e e n from F i g s .
I n Ex p t . 1 , d u o d e n a l f l ow r a tes of 4 7 and 6 0 m g
75
m e th i onine /k g " / d a y , g a v e N b a l anc es o f -1 . 2 a n d +1 . 6 g/ d a y r e � p � c tivel y .
I n Ex p t . 2 , the d e c l i n i n g i n take o f t h e c o n t r o l s i l ag e i n r ep s .
1 and 3 , r e s u l t e d i n mar k e d l y d i f f e r e n t i n takes d u r i n g c o l l ec t i o n o f
f ae c e s an d u r i n e s amples f o r b a l anc e meas u rements , c o m p a r e d to i n t a k e s
dur i n g c o l lec tion o f duod enal d i ge s t a samples f o r flow me as urements .
H ow e v e r , i n takes i n � e p . 5 w e r e s im i l ar d uring b o t h p e r i od s , a n d a f l ow
75
r ate o f 9 7mg / k g • / d ay gave a value o f -1 . 2 g N re tained / d a y . S uc h
d i f f e renc e s b e tween experiments may indic ate t h a t th e s h e e p in E xp t . 2
required m o r e m e th i o n ine to r e ac h a p o s i t i v e N b al anc e , t h a n th e s h e e p
in Expt. 1 .
Th i s p o s s i b i l i t y c o u l d explain the o b s e r v e d i n take
resp o ns e in Ex p t . 2 , wh i l e n o s uc h r e s p o n s e was apparent i n Ex p t . 1 .
A h i g h e r r equirement for meth i o n i n e would h av e b e e n mo r e l i ke l y to
res u l t i n an imbalance s i tuati o n .
N o d e f i n i t e evidenc e f o r a h i g h e r m e t h i o n ine r e q u i r e m e n t i n E x p t .
2 is avai l a ble , b u t two f ac t ors wh i c h c o u l d h ave c on t r i b u t e d , w i l l b e
c o ns i d ere d .
T h e f i r s t is th e s e as o n o f t h e y e ar d ur i n g wh i c h t h e
expe r ime n t s w e r e c o nduc ted , i n r e l a t i o n t o wool g r owth .
Expt. 1 was
hw
c on d u c ted from M a y t o S e p temb e r , a p e r i o d of relativel y � w o o l
grow t h , a n d E x p t . 2 from S e p tember t o January , a p e r i o d o f r e l a t iv e l y
A.A
-� w o o l growth ( R y der and S t e p h e ns o n , 1 9 6 8 ) . S i n c e w o o l g rowth h as a
h igh r e q u i rement f o r c ys t ( e ) i n e ( Ro s s , 1 9 6 1 ) , th e requi re�e n t f o r
meth i o n i n e f o r c o nv e r s i o n t o c ys t e i n e ( at a c o nst ant c y s t e i n e a v aila b i l i t y ) would b e e x p ected t o inc r e as e .
A s e c ond f ac t o r which c o u l d h a v e i n f l uence d r e q u i r e me n t b e tw e e n
expe r imen t s, was a d i fference i n th e prev i o us nutr i ti o n al h is to r y o f t h e
115
animals .
The " s h e e p in E x p t . 2 w e r e s ub j e c t e d to c o n s i d e r a b l e n u t r i­
tional d e p r i v a t i o n s i x w e e ks p r i o r to th e s tart o f th e e x p e r i me n t .
This was c aus e d b y t h e s h e e p r e f us i n g t o e a t a n a l t e r n ativ e b at c h o f
sil a g e .
S ix s h e e p w e r e o f f e r e d th i s p a r t ic ular s i l a g e , and all i n t a k e s
d ec r eased r ap i d l y f o r t wo w e e k s .
H o w e v e r , th is s ame s il a g e was a g a i n
o f f e r e d t o th r e e o f th e s e s h e e p a f t e r s i x m o n ths o f g o o d n u t r i t i o n .
Two o f t h e s h e e p m a i n t a i n e d an i n t ake o f g r eater t h a n 6 0 0 g DM/d a y o v e r
a p e r i o d o f s e v e r a l we e ks .
Th e i n take o f t h e third sh e e p , a n d o f a n
a d d i t i o n a l s h e e p d e c r � as e d , b u t in th e f i r s t c as e was r es t o r e d b y
d u o d e n al m e t h i o n i n e inf us io n , and i n th e s e c o nd b y d a i l y s u p p l e m e n t a t i o n
w i t h f o r m a l d eh y d e-t r ea t e d c a s e i n , g i v e n i n t o t h e r u m e n .
Th es e r es ul t s ,
t o g e t h e r w i th t h e s i gn i f i c an t d i f f e r � n c es b e tween animals in v o l u n t a r y
i n take i n E x p t . 2 , s u g g e s t t h a t t h e p r e v i o u s nutr i t i o nal h is t o r y o f
i n d i v i dual animals , wi t h r e s pec t to p r o t e i n s t atus , w i l l h av e a m a r k e d
e f f e c t o n the r e s p o n s e o f th e s h eep t o l o w meth io n i n e av ai l ab i l i t y .
( 2 ) A v ail a b i l i ty o f oth e r amino acids :
D u r i n g th e d i s c us s i o n o f
r e s u l t s i n th i s c h a p te r , r e f e r e n c e h as f r e q ue n t l y b e e n made to t h e e f F ec t
o f t h e av ailab i l i t y of o t h e r amino ac i d s o n meth i o n i n e r e q u i r e m e n t .
T h e b alan c e . i n the u t i l i s a t i o n o f m e t h i o n i n e for t i s s u e p r o t ein
s yn t h esis , o r e x tra-pr o t e i n f u n c t i o n s h as b e en shown t o c h a n g e w i th
i nc r e as i n g i nt a ke o f amino acids and me t h i o nine f o r m o n o g as t r ic s ( G r a u
and K ame i , 1 9 5 ;
F i nke l s t e i n and Mud d , 1 9 67 ;
A gu i l a r e t al . , 1 9 7 4 ) .
S h a n n o n e t al . , 1 9 7 2 ;
W h i l e f e w d a t a o n meth i o n i n e me t a b o l is m i n
r um i n ants are a v ai l ab l e , a s im i l ar s i t u a t i o n appears t o e x i s t .
H ence
a p o i n t o f in t e r s e c t i o n i n a r e s p o n s e c u r v e , may s i m p l y i n d i c a t e a
c h a n g e i n the f u n c t i o n f o r w h i c h meth i o n i n e is b e i n g util i s e d .
In this
r es p e c t , d if f e r e nt r e q u i r ements h a v e b e e n p re d i c t e d f r o m d i f f e r e n t
parameters i n th e s ame e x p e riment ( Ta o e t al . , 1 9 74 ) .
116
T he ''response c ur v e s " ( F i g s . 4 . 3 - 4 . 7 ) , f o r
N
b alanc e , p l asma
me t h i o n i n e and perc e n t a g e o x i d a t i o n of meth i o n i n e t o c o , w i th
2
inc r e as i n g levels of d u o d e nal me th i o n i n e p e r u n i t of m e t ab o l ic b o d y
w e i gh t , s h o w t h a t t h e s e p aram e t e rs d i d r e s p ond to i n c r e a s e d m e th i o n i n e
availability.
d e t e c t ed .
H ow e v e r , n o c l e a r c u t p o in t s o f i n t e r s e c t i o n w e r e
I n p ar t i c ul a r , r e s p o n s e in the p e r c e n t a g e o f meth i o n i n e
oxidised t o co
2
ap p e a r e d t o b e l in e ar .
r e p o r ted b y A g u i l a r e t a l . ( 1 9 72 ) .
A s imilar r e s u l t f o r r a t s w a s
A g u i l a r e t al . ( 1 9 74 ) s u g g e s t e d
t h a t s ome feature o f th e m e t a b o l i s m of m e t h i o n i n e a f f e c t e d o x i d a t i o n ,
and t h us p r ev e n t ed the p r e s e n c e o f a c le a r l y d e f i n e d p o i n t o f i n t e rs ec ti o n .
T h e l evel and t y p e o f p r o d u c t i o n r e s p o n s e sh o ul d a l s o b e c o n s i d e r e d
a s a c r i t e r io n w i t h wh i c h t o d e f i n e re q u i r eme n t .
Th e s e p o i n t s a r e
emp h a s is e d b y t h e w i d e r a n g e o f meth i o n i n e r e q u i r ements r e p o r t e d i n th e
l i t e r a t u r e , f r o m 1 0 5 - 1 2 5 mg m e t h i o n i n e e n t e r i n g the � u o d e n u m p e r u n i t
o f m e t ab o l ic l i ve-w e igh t p e r d ay ( W a k e l i n g ��� . , 1 9 70 ) , t o 2 0 7 m g
s up p l eme n t al m e th i o n i ne p e r u n i t of metab o l ic l i v e-w e i gh t p e r day ( R e i s
e t al . , 1 9 73 ) .
Th u s , t h e me t h i o n i n e r e q u i r e m e n t o f s h e e p a p p e ar s t o b e
a f f e c t ed b y a v ar i e t y o f f ac t o r s which s h o u l d b e d e f i n e d f o r a n y
es t i m a te o f r e q ui r e m e n t .
GENERAL
D I SC U S S I O N
117
The amino acid s tatus o f s h e e p f e d s il a ge ap p e crs t o b e a n
impor tant factor in r e g u l a t i n g v o l u n tary intake .
T h u s , E x p t . 1 was
u nd e r t a k e n to c o mpare the e f f ec ts o f f o r malin-treatment o f s i l ag e , and
I /P m e t h i o nine i n f us i on , on th e d i g e s t i o n and uti l i s a ti o n o f s ilage by
shee p .
In E x p t . 2, two a l t e r n at i v e m e th o d s of inc r e as i n g d uo d e nal amin o
acid f l ow were s t u d i e d .
I n a d d i t io n , each of th e t r eatments w a s s u p p l e­
m e n t e d w i t h d u o d enal methionine , to c omp a r e the u t il i s a t i o n o f
meth i o ni n e a t v a r y i n g l evels o f d u o d e n al amino ac i d f l o w .
T h e main d if f e r e nc es in c h emic al c om p o s ition b e tween s i l a g e and
h erbage , a r e th e low water s o l u b l e c a r b o h y drate c o n t en t , and h ig h
· p ro p o r t i o n of N P N i n th e t o t a l
N
f r a c t io n .
Th ese d i f f e r e n c e s w i l l
c omb i n e t o limi t th e f l o w of amino a c i d s into the d u o d e n um , s i nc e
read i l y av ailab l e e n e r g y is r e q u i r e d f o r e f f ic i ent u t i l i s a t i o n o f N P N
b y r umen mic ro-organisms ( B e l as c o , 1 9 5 6 ;
H e n d e r i c k x and Mar t i n , 1 9 63 ) .
B l o o mfi e l d �. , 1 9 58 ;
Th e t h r ee tr eatments ;
f o rmal i n- t r eatm e n t
o f s i l age ( E x p t . 1 ) , f o rmald e h y d e- t r e a t e d c a s e i n and e n e r g y s u p p l ements
( Exp t . 2 ) , all i n c r e ased the f l o w o f ami n o acids into the d u o d e n u m , t o
a max imum o f 1 6 7 g /d a y .
A c o mparis o n o f t h e d a t a f r o m b o th e x p e r iments
i ndic ated that amino acid i n t ake acc o u n t e d for 67% o f th e v a r i a t i o n in
d uode nal amino ac id f l o w .
H e nc e , th e degree o f amino acid d e g r a d a t i o n
d urin g ens i J in g , ap p e ars to b e a k e y f ac tor in d e t e r m i n i n g i t s
effic ienc y of u t i l i s a t i o n .
F o rmal i n- t r e atment of s i l age p r e v e n t e d th e
d egrad ati o n of p r o t e i n and amino ac i d s i n the s i l a g e s tack ( B a r r y and
F enne s s y , 1 9 72 ) .
A c o m p a r i s o n o f th e OM i n take r e q � i r e d to s up p l y 1 0 0 g am i n o a c i d s /
day , e n t e r i n g th e d u o d e num , o n s i l a g e , c o mpared t o o th e r d i e ts ,
indic ated th e n e e d f o r a rel ativ e l y h i gh i n take o f s il ag e t o m a i n t a i n
this f l ow .
I n E x p t . 2 , intake o f th e c o n tr o l s i l a g e decr eas e d w i th
118
time .
Th i s d e c r e as e was p r e v e n t e d b y b o th t h e forma l d e h yd e-tr e a t e d
c a s e i n a n d ene r g y s u p p l ements .
T h e f o r m al d e h y d e-tr e a t e d c as e i n was
l ar g e l y p r o tec t e d f r om d e gr ad a t i o n in th e rume � , b ut appeared t o b e
d i g e s ted i n th e int e s tines .
S u p p l ementation with e n e r g y a l s o i n c r e a s e d
t h e f l ow o f amino ac ids i n t o t h e duoden um , b y inc r e a s i n g t h e c on v e r s i o n
o f N P N t o p r o t e in w i th i n t h e r umen .
H e nc e , t h e amino ac id s t atus o f
t h e s h eep was i n c r e a s e d b y t h e s e t w o tre atme n t s , and i n take was main­
tai n e d .
T h us , the u t i l i s at i o n o f s ilages w i t h a l ow amino a c i d c o n t e n t
may b e im p r o v e d b y s up p l ementation w i th '' p r o t ected-p r o t ein " or a s o u r c e
o f r e adily av a i l a b l e ene r g y .
The d e c r e a s e i n i n t a k e i n E xp t . 2 was r ev ers e d b y d u o d e n a l s u p p l e­
me n t a t ion �ith meth i o n i n e s s u gg e s t i n g th a t s il age-f ed s h e e p may b e i n
an a mino a c i d imbal anc e w i th r e s p e c t to meth i oni n e .
N bal anc e w a s
s i g n i f ican tly i n c r e a s e d b y d u o d e nal meth i o n i n e a n d t o ta l p l asma a m i n o
acid c onc e n tra t i on w a s d e c r e ase d .
Th us , the availab i l i t y o f meth i o n ine
appe a r ed to l i m i t t is s u e protein s y n th e s i s .
Howe v e r , N bal ance at t h e
s ame l ev e l of meth i o n ine availab i l i t y was h i gher i n E x p t . 1 , c om p a r e d
t o Expt. 2 .
T h is s ug g e s t s that a h i gher pro p orti o n o f th e av a i l a b l e
meth i onine w a s us ed f o r t i ssue p r o t e i n s y n th e s is i n Ex p t . 1 .
S inc e
inta k e was mai n tained on all treatme n ts i n E x p t . 1 , b u t d e c r e as e d o n
t h e c o nt r o l s i l age i n Ex p t . 2 , th e s usc e p tib i lity o f s h e e p t o m e t h i on i n e
i mb a l ance a p p e ars t o d e p e nd o n t h e r a t e o f t i ssue p r o t e in s y nth e s i s i n
th e a nimal .
I ntake w as a l s o part ial l y maint ained b y t h e two s u p p l e­
ments in E x pt . 2 , a n d h e n c e meth i o n i n e i mb a l a nc e appears t o b e m o r e
s e v e r e a t l ow e r l e v e l s o f duodenal amino ac i d s .
Henc e , t h e p a r t ic u l a r p r o b l em o f s il ag e , i n h a v i n g l o w ami n o a c id ,
and r e ad i l y av ailab l e e n e rgy c o n t e n ts , appears to l i m i t th e f l o w o f
ami n o ac i d s . i n t o th e du o d enum .
Low av a i l a b i l ity o f amino a c ids t o t h e
119
tiss ues , may then r e s u l t in a s t a t e
� urn m a y l im i t intake .
of
m e th ionine imb alanc e , w h ich i n
The s usc e p tib i l i t y o f s h e e p t o m e t h i o n i ne
imb alanc e , d e p e n d s o n t h e p r ote i n s ta t us o f ind i v i d u al animal s .
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W I L K I N S , R . J . , HUTC H I NS O N , K . J . , W I L S O N , R . F . and H A R R I S , C . E . ( 1 9 7 1 )
J , a g r i c , Sc i . ( Camb , ) J..2 5 3 1 - 538 ,
W I L L I AMS , H . H . , C U R T I N , L . V . , A B R A H AM , J . , L O O S L I , J . K . and M A Y N A R D ,
L . A . ( 1 9 54 ) J , b i o l . Ch e m , 2 0 8 277-2 8 6 .
W I L L I AMS , C . M . , DAV I D , D . J . a n d I I SMA A , D . ( 1 9 62 ) J . a g ric . Sc i . ( C amb . )
5 9 3 8 1 -3 8 5 .
W I L L I AMS , A . J . , L E N G , R . A . a n d S T E P H E NS ON , S . K . ( 1 9 7 2 ) A u s t . J . b i o l ,
S c i . 2 5 1 2 59W IL L I A MS , A . P . and S M I TH , R . H . ( 1 9 74 ) 8 r , J , N ut r , 2£ 4 2 1 -4 3 3 ,
W I ND E L S , H . F . , M E A D E , R . J . , N ORDSTR OM , J . W . and S T O C K L AND , W . L . ( 1 9 71 )
J . A n im , S c i . ]2 2 68 -2 7 6 ,
WOL F F , J . E . and B E R G f'lA N ,
E.N.
( 1 9 7 2 ) A m . J , P h ysi o l . 2 2 3 4 5 5-4 6 0 ,
WOL F F , J . E . , B E R GMA N , E . N . and W I L L I A MS , H . H . ( 1 9 7 2 ) A m , J . P h y s i o l .
2 2 3 4 3 8-44 6 .
WOMA C K , M . W . and RO S E , W . C . ( 1 94 1 ) J . b i o l . C h e m . 1 4 1 3 7 5-3 7 9 .
WOMA C K , M . W . , MAR T I N , H . A . a n d L I N , P . M . ( 1 9 5 3 ) J . N ut r . 4 9 5 1 3- 5 2 6 ,
WORK ,
E.
( 1 9 51 ) B i o c h em , J , � 1 7- 2 3 .
WR I G H T , O , E , and H L N G A T E , R . E . ( 1 9 6 7 ) A p p l , M i c ro b ial , 1 5 1 4 8-1 5 1 .
Z EL T E R ,
s.z.
( 1 9 60 ) P r oc . V I I I th I n ternat l , G r ass l d , C o n� r . pp . 505-509 .
Z IMME RMAN , R . A . and S C O T T , H . M . ( 1 9 65 ) J . Nut r . 8 7 1 3- 1 8 .
APPENDI C E S
1 32
APPEND I X
Silage OM in ta kes
A.
-
1
E xpt . 2 .
Mean d a ily i n take d u r i ng s e v e n day b a l a n c e pe riod (gDM/day) .
S h e e p numbers
No me thionine
T re atme n t
+
m e th io n i n e
821
1 1 57
1 3 03
821
1 1 57
1 303
C ontrol
+ p r o tein
443
3 66
637
409
597
675
648
608
696
688
663
701
energy
61 1
629
652
594
667
681
+
B . M e an d aily in ta ke d u r ing three day c o l l ec t ion
of
d u o d enal d igests
s ampl es (qDM/d ay) .
S h e e p n umb ers
T r eatment
+ m e th i o n i n e
No methionine
821
1 1 57
1 303
§11
1 1 51.
1 3 03
C o n t rol
+ p r o tein
335
2 93
61 0
51 5
559
688
61 0
641
701
660
654
701
+ energy
461
587
679
572
678
691
1 33
APPENDIX
2
A n a lys es of V a rianc e - E xp t . 2
S o u rc e :
Tr eatment
x
Rep .
Trtmt .
Me t .
T x M
Ans .
Err.
R e p l ic ates
Treatment
M e t h i o nine
Met I n t e r action
,C, n ima l s
Error
Mean S q u a r e
MS
S i g n i f ic ance
Sig.
p <. 0 . 0 1
P < 0 . 05
N o t s i gn i f i c a n t
**
*
NS
�
S i l age
0 �1
MS
Si g .
Trtm t .
Me t .
4 5 4 53 . 50
3 58 2 3 . 2 2
T
X �1
A ns .
Err.
I n take
7 0 1 6 . 99
NS
**
*
91 88 . 47
2662 . 8 1
NS
N I n take
MS
Sig.
3 . 28
1 5 5 . 74
NS
**
85. 2
561 2 . 4
22. 22
*
2 . 84
NS
22. 55
*
3 . 03
fl. A I n t a k e
MS
Sig.
NS
**
555 . 6
*"*
1 02 . 7
NS
4 53 . 7
**
38 . 0
Total rumen V F A c o nc e n t r a t i o n
MS
Sig.
93 . 4 6
**
205 . 02
**
23 8 . 64
3 . 37
1 1 . 42
1 8 . 46
**
Molar pr opo r tion o f acetate
MS
Sig.
2 . 55
29 . 30
NS
NS
NS
Molar pr opo r tion o f propi o n a t e
MS
Sig .
5 . 35
NS
0 . 22
NS
7 . 66
7 . 66
NS
Molar pr opo r t ion o f b u t:t:r ate
MS
Sig.
0 . 66
NS
9 . 91
NS
1 . 05
NS
4 . 20
NH -N c o nc e ntration in r um e n
-3
MS
S ig .
7.8 . 69
**
1 . 93
NS
8 2 . 34
**
1 . 69
1 34
Trtmt .
R ep.
Met.
T
x
M
A
ns .
Err .
� OM d ige s t e d i n s t omach
MS
Sig.
3 2 . 06
NS
3 7 . 56
NS
68 . 0 6
NS
142.72
NS
54 . 3 9
0 . 87
NS
1 20 . 1 2
NS
1 1 8. 40
NS
261 . 3 1
NS
45.49
9 1 5 . 54
54 . 08
NS
387 . 92
NS
1 4 94 . 2 9
1 75 . 60
0 . 72
NS
� en e rgJ:: d ige s t ed i n st omach
MS
Sig.
1 9 . 90
NS
d
� N
i o e s t e d i n s t omach
MS
Sig.
309 . 93
NS
*
*
Non-arnino a c i d-N e n t e ri ng th e d u o d e n u m
MS
Sig .
9.1
0.3
NS
9.3
1 .8
NS
3.6
NS
0 . 72
1 . 30
0 . 37
NS
**
1 . 23
0.13
0 . 23
NS
0.25
NS
0 . 23
NS
0 . 07
1 2 65 . 0 5
NS
73 . 8 3
NS
570 . 3 7
NS
21 2 . 78
**
**
Non-c:rn i n o a c i d-N in take
MS
Sig.
0 . 23
NS
0 . 70
**
*
R a t i o o f d uo d e n al N A A-N t o N A A-N i n t a k e
MS
Sig .
Amino
MS
Sig .
0 . 02
NS
ac i d s
enterino
53 1 . 8 5
NS
0 . 82
**
th e duo d e n um
59 7 8 . 7 7
**
R a t i o o f d uo d e n a l A A to A A i n t a k e
MS
S ig .
o . o8
0 . 23
0.013
5. 1 8
0 . 67
3 . 97
NS
4.31
NS
5 . 07
4 . 00
NS
1 6 1 . 34
1 0 . 55
0 . 00 2
NS
*
**
3 . 38
NS
o . oo
NS
0 . 88
NS
*
97. 1 5
70 . 4 1
0 . 20
0 . 52
NS
**
R a t i o o f d u o d e n a l A A to N i n t a k e
MS
Sig .
0 . 62
NS
N e n t er i ng t h e d u o d e n um
MS
Sig.
AA
1 1 . 29
NS
a s � t o tal
MS
S ig .
N
**
**
e n t e r ing the d u o d e n um
6 . 67
NS
664 . 73
**
9 1 . 58
*
**
1 35
Reo.
Trtmt.
Me t .
D u o d enal an i n o ac i d s ( e xpr e s s ed as a
T
£if
X
�1
u.
062
NS
0 . 318
**
Err .
o f t h e t o t al )
3.£9.
MS
Sig.
Ans .
0 . 01 4
0 . 04 0
NS
NS
0 . DOt�
0 . 024
0 . 02 7
0.1 12
NS
H is
MS
Sig .
0 . 1 74
0 . 051
0 . 1 61
NS
0 . 09 1
NS
NS
NS
NS
0 . 346
0 . 1 51
0 . 24 5
NS
NS
0 . 04 5
0 . 01 6
0 . 361
o . ooo
Ile
MS
S ig .
*
NS
NS
0.117
0 . 073
0 . 04 3
Leu
MS
Sig.
0 . 1 09
NS
NS
NS
NS
NS
0 . 267
�
MS
Sig.
0 . 1 96
NS
0.111
0 . 0 94
NS
NS
0 . 027
0 . 014
0 . 01 1
NS
0 . 3 89
0 . 04 4
0 . 004
0.01 1
*
Met
MS
Sig .
0 . 082
*
NS
NS
0 . 00 1
NS
NS
Phe
MS
Sig.
0 . 1 42
NS
0 . 64 1
**
0 . 014
0 . 027
NS
NS
0 . 34 7
0 . 00 7
0 . 394
0. 053
0 . 1 44
0 . 1 48
0 . 01 2
0 . 09 1
NS
0 . 04 4
NS
0 . 00 5
0 . 24 5
NS
NS
0 . 51 7
0 . 09 6
0 . 1 80
0 . 02 7
NS
0 . 336
NS
0. 261
0 . 027
0 . 136
NS
NS
*
Th r
MS
Sig.
0 . 2 02
NS
1 . 684
**
NS
NS
NS
V al
MS
Sig.
0 . 81 3
**
0 . 01 7
NS
0 . 005
NS
Ala
MS
Sig.
o. 651
*
7 . 1 04
**
*
�
MS
Sig.
0 . 351
NS
8 . 376
**
NS
Glu
MS
Sig.
0.317
NS
50 . 6 1 6
**
0 . 51 7
NS
0. 228
1 36
lli
MS
S ig .
x
M
Ans .
Err .
0 . 094
NS
0 . 01 1
NS
1 . 732
0 . 1 99
1 2 . 494
1 . 1 25
0 . 01 2
NS
1 . 94 1
0 . 1 69
0 . 202
0 . 096
0 . 1 42
0 . 069
0 . 03 7
NS
0 . 008
0 . 602
NS
0 . 204
NS
0 . 1 09
NS
0 . 261
NS
0 . 1 21
NS
0 . 1 37
�
T r tmt .
Met .
0 . 046
NS
7 .; 8 3 5
**
0 . 598
NS
T
*
Pro
MS
Sig.
**
*
**
Ser
MS
Sig .
**
**
"**
*
TFo s i ne
�lS
Sig .
�
Dr'l
MS
Sig .
� m�
MS
Sig .
i n t a k e d i9es t e d i n i n t e s t i n e s
45 . 59
NS
8 5 . 83
NS
23 . 53
NS
2 1 6 . 25
NS
4 64 . 8
56. 8
79 . 75
NS
7 83 . 65
1 9 . 85
7 3 . 62
NS
269 . 39
NS
60 . 6 1
1 63 . 3 1
NS
544 . 2 2
NS
340 . 70
54 . 69
NS
89 . 45
NS
28 . 77
*
i n t c; k e d ige s t e d i n i n t e s t i n e s
36 . 7 5
NS
64 . 1 5
NS
8 3 . 20
NS
"**
� e n e rg:t i n t a k e d i o e s t e d i n i n t e s t i n e s
MS
Si g .
�
284 . 7 8
NS
55 . 8 9
NS
N i n t a k e d i o es t e d in i n t e s tine s
MS
Sig.
�
22 . 9 3
�J S
1 1 7.42
NS
4 63 . 75
NS
70 . 47
NS
d u o d e n a l N d ige s t ed i n i n t e s t i n e s
MS
Sig .
1 5 . 40
NS
5 8 . 64
NS
78 . 5 0
NS
Aeea re nt d ige s t i b i l i t:t o f O M
24 . 7 1 2
0 . 620
MS
NS
Sig .
NS
Aeea r e n t d ige s ti b i l i t:t o f
MS
Sig.
3 . 4 07
NS
9 . 680
NS
2 . 565
NS
2 . 007
NS
5. 362
3. 309
NS
0.21 1
NS
0 . 09 1
NS
2 . 037
2 . 44
NS
o . 77
NS
3 . 92
OM
23 . 9 24
**
Aeea re nt d ige s t i b i l i t:t o f e n ergt
�1 5
Sig .
2 . 08
NS
1 4 . 39
NS
1 0 . 89
NS
1 37
�
Met .
T r tmt .
T
x
M
Ans .
Err .
A eea r e n t d i9e s t i b i l i t� o f N
1"15
0 . 367
N
1 2 6 . 34
1 6 . 63
**
NS
Sig .
NS
3 . 57
1 . 24
NS
NS
0.46
0 . 68
NS
NS
0 . 06
0 . 40
NS
NS
1 . 55
0.14
**
NS
9 . 36
b a l anc e
MS
S ig .
7.46
1 8 . 93
2 5 . 92
*
NS
**
2. 67
M e t ava i l a b l e f o r a b s o ret i on
MS
Sig .
�
4 . 01
0 . 53
M e t i n t o t a l du o d enal
MS
Si g .
0 . 599
NS
2 2 . 98
**
NS
**
0. 1 9
AA
0 . 64 3
2 1 . 56
**
NS
0.14
Plas ma m e t c o nc e nt r at i o n
MS
Sig .
Total
1 47 . 83
*
�
NS
1 5 2 1 . 68
**
91 . 93
1 0. 38
NS
20 . 4 0
NS
amino ac i d c o nc e n t r a t i o n
olasma
MS
Sig.
1 0 . 45
52751 . 53
NS
391 0 3 . 69
1 1 44 94 7 . 3 6
1 59 96 . 77
8 9 4 78 . 8 0
**
NS
0 . 1 24
9 . 976
0 . 04 1
0 . 64 4
NS
**
NS
*
NS
2 5 9 68 . 5 1
NS
me t i n t ot a l el a s m a amino ac i d s
MS
Sig .
�
0 . 8 62
*
0 . 1 09
i n d i v i d ual a11ino acids in t o t a l el a s m a am i n o a c i d s
T a u r i ne
MS
Sig .
3 . 68
NS
3 . 00
NS
2 0 . 89
**
5 . 62
3 . 29
NS
NS
0 . 76
0. 1 7
NS
NS
1 . 45
Th r
MS
S ig .
0 . 73
NS
5 . 48
**
1 6 . 44
**
0 . 25
Ser
MS
Sig.
4 . 75
**
1 .18
0 . 66
3 2 . 49
NS
2 . 77
2 . 89
*
*
0.16
1 . 64
0.37
NS
NS
NS
2 1 . 78
**
0 . 38
Pro
�1 5
Sig .
NS
**
0 . 75
Glu
MS
Sig.
7 . 72
NS
4 . 04
NS
1 7 . 80
NS
3 . 47
NS
1 1 . 49
NS
9 . 49
1 38
R ep .
Trtm t .
---
Met .
T
x
M
Ans .
Err.
C i trulline
MS
S ig.
1 . 34
0 . 37
NS
NS
2 1 . 34
**
0 . 44
6 . 25
*
0 . 92
NS
9 . 00
49 . 35
1 3 . 60
Glv
MS
S ig .
25. 20
NS
201 . 7 1
**
273 . 00
**
NS
NS
3 . 47
5 . 47
NS
NS
0 . 85
2 . 29
NS
NS
0 . 64
0.12
Ala
MS
Si g .
13.17
*
7 . 22
NS
1 8 . 61
*
1 . 56
V al
MS
Sig.
0.48
29 . 27
NS
**
1 . 37
0 . 06
8 . 82
**
0 . 50
�
MS
Sig.
**
NS
0 . 84
**
*
0 . 06
NS
C;ts tath i o n i n e
MS
S ig .
0 . 09
0 . 02
NS
NS
0 . 50
0 . 63
0 . 97
**
o . 01
0 . 04
NS
NS
0 . 04
0 . 30
0 . 03
o . os
1
01
**
0 . 04
NS
0.12
0 . 76
0 . 29
NS
NS
0.10
0 . 07
NS
NS
2 . 79
3 . 47
NS
NS
0 . 28
7 . 00
NS
NS
0 . 01
Ile
MS
Sig.
**
**
0 . 03
NS
**
NS
Leu
MS
Sig.
0 . 40
**
3 . 91
**
3 . 29
**
•
l.:iE.
MS
S ig .
0 . 50
3 . 62
NS
**
0.13
0 . 66
NS
NS
5 . 23
**
Phe
MS
S ig .
4 . 81
**
0.17
Uri nar;t-N
MS
S ig .
1 . 74
NS
2 8 . 36
**
1 1 . 68
**
0 . 78
P l asma urea c o nc en t rat ion
MS
S i g.
4 . 84
NS
23 . 22
NS
2 . 83
NS
5 . 29
1 39
APPEND I X
pl as m a
SA
of
A.
U n t r e a ted
me t h i o n i n e - Expt . 1
R ep.
Time
�h ) :
3
R ep.
1
2
Rep .
0
0
0
o.os
4 p 093
1 , Cl 1 7
44 1
0.25
9 , 799
4 , 1 68
3p087
7 , 373
5, 451
0
0, 50
1
1 5 , 668
1.5
1 4 , 455
6,080
2
1 4 , 2 30
8 , 90 3
2.5
1 7 ' 433
3
1 6 , 79 2
9 , 877
3. 5
14,013
7 , 852
6, 227
4
1 4 , 6 51
1 1 , 466
6, 598
1 0 , 077
6, 752
7 , 61 3
9, 1 97
8
9 , 967
8, 755
9
1 1 , 1 32
9, 373
I
9 , 51 6
5
6
7 ' 543
7
Pl3teau
1 1 ' 938
11
1 6, 3 1 0
8,718
12
1 6 t 545
7,281
v al u e
����
Inf
J�;p=���
1 2 , 335
10
( d p m/ 1-1 mo l e )
( d p m /h )
-1
c o n s t an t ( h )
r �ta
1 5 p 9 57
1 1 , 1 64
8 , 66 5
6 , 8 9 9 , 040
6 , 0 68 , 04 0
1 .14
4 , 755, 240
Th e t ur n o v er c o n s t a n t f o r R e p . 1 was n o t c al c u l ated ,
owin g t o the l ow c or r e l a t i o n c o e f f i c i e n t ( 0 . 54 ) .
0. 85
3
1 40
B.
Treate d
R ee . 1
0
Ree .
2
0
o . oa
848
·j 6 •j .
0 . 25
4 , 204
1 ,219
0.5
6 , 1 47
5 , 890
2,112
1
7 , 348
6 , 61 7
3 , 7 59
1.5
9 , 3 30
7 , 665
2
1 3 , 4 43
9 , 286
2.5
1 0 , 255
1 1 , 0 71
3
1 6 , 4 79
6 , 08 1
4
1 6, 520
5
1 8 , 0 08
7 , 928
6 , 676
7 , 921
6
1 1 , 61 3
7
7 , 679
8
9
1 0 , 2 83
10
1 1 , 7 64
9 , 223
6 , 459
6, 987
6, 591
7 , 789
11
12
14, 303
14
1 0 , 884
18
1 1 , 9 58
19
1 8 , 9 00
20
1 2 , 7 56
22
1 4 , 20 6
24
1 9 , 3 27
Plat e a u v a l ue ( d p m/ !Jmole )
I nf u s ion r a te ( d pm/h )
c o nstant (h
6 , 7 97
4 , 240
3.5
�QUQo;:
3
0
T ime {h ) � 0
ko.fe
f.t:to;:
R ee .
-1 )
8 , 562
1 1 , 7 69
7 , 845
7 , 09 2
6 , 206, 1 1 0
5 , 230 , 500
5 , 1 27 , 3 9 0
0 . 84
2 . 74
0 . 77
1 41
C.: o
Untr e ated + met
Ree . 1
Time { h ) : 0
n
0
n n
u . uo
0 . 25
4 , 540
R ee .
2
0
0
332
777
9 , 1 59
1 , 537
4 , 523
1.5
5 , 1 87
3 , 886
2
5 , 260
4 , 594
6 , 653
4 ' 1 40
2.5
1 1 , 1 05
3
1 0 , 31 7
3 , 1 42
3.5
1 0 , 28 0
4 ,422
4
1 1 ' 72 5
5
1 0 ,451
1 0 , 2 94
6
1 1 '471
6 , 664
7
1 1 , 800
5 , 784'
8
1 1 , 999
7 , �82
6 , 80 1
9
9 , 51 4
6 , 058
9 , 1 88
6 , 1 25
10
5 , 378
4 , 996
5 , 521
9 , 1 79
11
6, 021
3 , 79 7
1 0 , 79 3
6 , 527
5 , 43 6
7 , 354 , 7 5 0
5 , 6 1 4 , 620
4 , 3 0 9 , 44 0
0. 79
0 .82
12
Plateau value ( d p m/1-Jmol e )
I n f u s i o n rate ( d p m/h )
RG;:te c ons tant ( h -1 )
��PASY9�
3
4 , 1 41
0.5
1
R ee .
The turnover cons tant f o r Rep. 1 w as not calcul ated ,
owing to the limited numb er of d ata availab l e .
1 42
A P P E ND I X
4
P l asma am i n o acid c o n c e n t r a t i o n s ( as a % of total plasma amino acids )
a f t e r 1 d a y o f meth ionine infus i o n - E x p t . 2
C o ntro l
+ Protein
+
E n e rqy
2.8
Tr .
4.4
Tr .
2.5
Ser
6. 3
6.4
6. 1
Pro
3.3
5o4
Glu
4.9
5.4
3.4
4.4
Citrulline
5.3
5.6
5.3
Gly
49. 4
36. 7
51 . 7
Ala
10.1
9.6
9.6
V al
5.3
8.9
5.5
C ys t a th i o nine
0.5
Tr �
Tr .
fl1 e t
1.8
2.1
1.7
Ile
2.4
3.4
2. 6
L eu
3.2
5.0
2.7
Tyr
2.8
4.5
2. 7
Phe
2.0
2.6
1.7
Taur i n e
Tr.
Thr
Total
( fJmole/1 )
1 449
1 4 24
1 957