Effect of certain amino acid, aspirin and zinc supplements on the zinc-deficiency syndrome of chicks
given four protein sources
by Susan Lynne Smith Davis
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE in Home Economics
Montana State University
© Copyright by Susan Lynne Smith Davis (1970)
Abstract:
Four basal protein rations, either low in available zinc (sesame meal) or low in zinc casein-gelatin, egg
albumen and soybean meal) were fed to day-old chicks for three weeks. Supplements of zinc, histidine,
glutamic acid or aspirin, or a combination of zinc and histidine or aspirin were fed. Chick weights, leg
deformities (indicated by leg score and length to width ratio), and tibia zinc and nitrogen content were
determined. Tibia of chicks given sesame, 0 and 60 mg/kg zinc and histidine, or histidine, 30 mg/kg
zinc; casein-gelatin, 0 and 10 mg/kg zinc and histidine, or histidine, 5 mg/kg zinc rations were
separated into four fractions on the basis of probable time of deposition of collagen. The nitrogen of
each fraction was measured. The hydroxyproline content of the tibia of chicks given sesame rations
with and without histidine was determined as a measure of collagen.
Growth of chicks fed the basal plant protein rations was better than chicks fed basal animal protein
rations. Additions of 60 mg/kg zinc to sesame, 10 mg/kg zinc to casein-gelatin and 10 mg/kg zinc to
egg albumen rations was sufficient to prevent zinc deficiency symptoms.
Addition of 1 percent histidine to the basal sesame rations decreased leg deformities but had little effect
on growth. Addition of 1 percent glutamic acid did not prevent zinc deficiency symptoms.
One percent histidine added to casein-gelatin rations had little effect on either criteria.
Short tibia deformity of chicks fed sesame rations was alleviated with 1 percent aspirin, but growth of
chicks was depressed.
Addition of 1 percent histidine to sesame rations resulted in a significant increase in total tibia nitrogen
content, concomitant with decreases in leg deformities, but growth was only moderate. Zinc or zinc and
histidine additions significantly improved growth and leg deformities but not tibia nitrogen content.
Tibia nitrogen of casein-gelatin fractions were increased only with. 10 mg/kg zinc.
The addition of histidine to the basal sesame rations also resulted in increases in hydroxyproline.
However, both the increases in tibia nitrogen of fractions and hydroxyproline disappeared when values
were calculated per gram of bone or nitrogen. STATEMENT OF PERMISSION TO COPY
In p r e s e n tin g t h is th e s i s in p a r t i a l f u lf il lm e n t of th e re q u ir e ­
m e n ts f o r a n a d v a n c e d d e g r e e a t M ontana S t a t e U n i v e r s i t y , I a g r e e t h a t
t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e , f o r i n s p e c t i o n .
I fu rth e r
a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y in g o f t h i s t h e s i s f o r s c h o l a r l y
p u r p o s e s may b e g r a n t e d b y my m a j o r p r o f e s s o r ,
th e D ire c to r of L ib r a r ie s .
or,
i n h i s a b s e n c e , by
I t i s u n d e r s t o o d t h a t any c o p y in g o r p u b l i ­
c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a in s h a l l n o t be a llo w e d w ith o u t
my w r i t t e n p e r m i s s i o n .
S ig n a tu re
D a te
EFFECT OF CERTAIN AMINO ACID, ASPIRIN AND ZINC SUPPLEMENTS ON THE
ZINC-^DEFICIENCY SYNDROME OF CHICKS GIVEN FOUR PROTEIN SOURCES
by
SUSAN LYNNE SMITH DAVIS .
A t h e s i s s u b m itte d to th e G ra d u a te F a c u lty i n p a r t i a l
f u l f i l l m e n t of th e req u irem e n ts f o r th e degree
- of
MASTER OF SCIENCE
f„
Home E conom ics
A pproved:
Head
I a j o r D e p a r tm e n t
C h a irm a n , E x a m in in g C om m ittee
G r a d u a t e Dean
MONTANA STATE UNIVERSITY
Bozeman, M ontana
A u g u s t , 1970
ill
ACKNOWLEDGMENTS
I w o u ld l i k e t o e x t e n d my s i n c e r e t h a n k s t o :
D r. J a n e L e a s e f o r h e r p a t i e n c e an d g u i d a n c e e x t e n d e d t o me as
a g r a d u a t e s t u d e n t and f o r h e r a s s i s t a n c e w i t h my t h e s i s ;
D r. M a r j o r i e R e i s e r f o r m aking my g r a d u a t e w o rk p o s s i b l e ;
D r. Sam uel R o g e rs f o r h i s a s s i s t a n c e i n c h e m i s t r y ;
Mr, J o h n N a e g e l e f o r h i s a s s i s t a n c e w i t h t h e t e c h n i c a l e x p e r i m e n t s ;
an d t o my f a m i l y f o r t h e i r u n d e r s t a n d i n g an d e n c o u r a g e m e n t .
iv
TABLE OF CONTENTS
Page
I .
III.
IV .
V.
I
I
2
REVIEW OF LITERATURE . . . . . .
........................................... . . . .
C o m p o s it io n o f Bones ................................................................................. .
C a u s e s o f Z i n c D e f i c i e n c i e s R e s u l t i n g i n Bone D e f o r m i t i e s .
Compounds w h ic h A l l e v i a t e Z i n c D e f i c i e n c y Symptoms . . . .
ro co in co
II.
INTRODUCTION. ....................................................................................... ....
I m p o r t a n c e o f t h e S t u d y . .............................................................................
P u r p o s e o f t h e S tu d y . ............................. . . . . . . . . . . .
METHODOLOGY
S ampI e * . . . . * * . . # . . . . . . .
. . . .
. . .
. .
Chxck C a r e . . . . . . . . . . . a . . . . . . . . . . . .
C r i t e r i a f o r M e asu re m e n t . . . . . . . . . . . . . . . . .
11
11
II
11
RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . .
R e su lts. . . . . . . . . . . . . .
........................................ . . .
D isc u ssio n . . . . . . . . . . .
........................ . ........................
16
16
28
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS................................................ 37
Su
m
m
a
r
y
37
Cone I u s x ons . . . - . a . . . . . . . . .
. . . . . . . . . .
38
R e c o m m e n d a tio n s .
. . . .
39
APPENDIX:
METHODS OF ANALYSES.
LIST OF REFERENCES
. . . . . . . . . . . . . . . . .
40
53
V
LIST OF TABLES
T able
Page
■I -
C o m p o s it io n o f B a s a l D i e t s ................................................
II.
A d d itio n s to th e B asal D ie ts .
III.
IV .
V.
V I.
V II.
V III.
„
. . . . . . . . . . . . . .
12
14
M o r t a l i t y o f 20 C h ic k s G iv e n S e v e r a l P r o t e i n S o u r c e s
f o r T h r e e Weeks . ....................................................................
C r i t e r i a Mean t o J u d g e t h e E x t e n t o f Z in c D e f i c i e n c i e s
a t T h r e e Weeks i n C h ic k s Fed S e v e r a l P r o t e i n S o u r c e s .
18
.
19
N i t r o g e n C o n t e n t Mean o f F r a c t i o n s o f C h ic k T i b i a Fed
S e v e r a l P r o t e i n S o u r c e s S u p p le m e n te d w i t h Z i n c a n d / o r
..................................................... .
H istid in e . . . . . . . . . .
25
N i t r o g e n C o n t e n t Mean i n F r a c t i o n s o f C h ic k T i b i a Caps
Fed S e v e r a l P r o t e i n S o u r c e s S u p p le m e n te d w i t h Z i n c
a n d / o r H i s t i d i n e . . . ..................................................................................
27
N i t r o g e n C o n t e n t M eal p e r Gram o f T i b i a and Caps o f
C h ic k s Fed S e v e r a l P r o t e i n S o u r c e s S u p p le m e n te d w i t h
Z in c a n d /o r H i s t i d i n e . . . .
................... . . . . . . . .
29
H y d r o x y p r o l i n e Mean i n F r a c t i o n s o f T i b i a o r Caps T aken
fro m C h ic k s Fed Sesame R a t i o n s . . ........................ . . . . .
30
vi
LIST OF FIGURES
F ig u re
1.
2,
Page
' E f f e c t o f T e x a s 61 Sesame M eal Fed t o C h i c k s
........................
17
E f f e c t o f F e e d i n g Egg Albumen R a .tio n s t o C h ic k s ■ . . . .
21
v ii
ABSTRACT
F o u r b a s a l p r o t e i n r a t i o n s , e i t h e r low i n a v a i l a b l e z i n c ( se s a m e
m ea l) o r low i n z i n c . ( c a s e i n - g e l a t i n , eg g a lbum en and s o y b e a n m eal)
w ere fe d to d a y -o ld c h ic k s f o r t h r e e w eeks.
S u p p le m e n ts o f z i n c ,
h i s t i d i n e , g l u t a m i c a c i d o r a s p i r i n , o r a c o m b i n a t i o n o f z i n c and
h i s t i d i n e o r a s p i r i n w ere f e d .
C h ic k w e i g h t s , l e g d e f o r m i t i e s
( i n d i c a t e d by l e g s c o r e an d l e n g t h t o w i d t h r a t i o ) , and t i b i a z i n c
and n i tr o g e n c o n te n t w ere d e te rm in e d .
T i b i a o f c h i c k s g i v e n s e sa m e ,
0 and 60 m g/kg z i n c and h i s t i d i n e , o r h i s t i d i n e , 30 m g/kg z i n c ; c a s e i n g e l a t i n , 0 a n d 10 m g/kg z i n c and h i s t i d i n e , o r h i s t i d i n e , 5 mg/kg z i n c
r a t i o n s w e r e s e p a r a t e d i n t o f o u r f r a c t i o n s on t h e b a s i s o f p r o b a b l e
t im e o f d e p o s i t i o n o f c o l l a g e n .
The n i t r o g e n o f e a c h f r a c t i o n was
m easured.
The h y d r o x y p r o l i n e c o n t e n t o f t h e t i b i a o f c h i c k s g i v e n ,
s e sa m e r a t i o n s w i t h and w i t h o u t h i s t i d i n e was d e t e r m i n e d a s a m e a s u re
of c o lla g en .
G rowth o f c h i c k s f e d t h e b a s a l p l a n t p r o t e i n r a t i o n s was b e t t e r
th a n c h ic k s fe d b a s a l a n im a l p r o t e i n r a t i o n s .
A d d i t i o n s o f 60 mg/kg
z i n c t o s e s a m e , 10 m g/kg z i n c t o c a s e i n - g e l a t i n and 10 m g/kg z i n c t o egg
a lb u m e n r a t i o n s was s u f f i c i e n t t o p r e v e n t z i n c d e f i c i e n c y symptom s.
A d d itio n o f I p e r c e n t h i s t i d i n e to t h e b a s a l sesam e r a t i o n s
d e c r e a s e d l e g d e f o r m i t i e s b u t h a d l i t t l e e f f e c t on g r o w t h . A d d i t i o n
o f I p e r c e n t g l u t a m i c a c i d d i d n o t p r e v e n t z i n c d e f i c i e n c y symptoms.
One p e r c e n t h i s t i d i n e a d d e d t o c a s e i n - g e l a t i n r a t i o n s h a d l i t t l e e f f e c t
on e i t h e r c r i t e r i a .
S h o r t t i b i a d e f o r m i t y o f c h i c k s f e d s e sa m e r a t i o n s was a l l e v i a t e d
w i t h I p e r c e n t a s p i r i n , b u t g r o w th o f c h i c k s was d e p r e s s e d .
A d d i t i o n o f I p e r c e n t h i s t i d i n e t o s e sa m e r a t i o n s r e s u l t e d i n a
s i g n i f i c a n t i n c r e a s e in t o t a l t i b i a n i t r o g e n c o n te n t , c o n c o m ita n t
w i t h d e c r e a s e s i n l e g d e f o r m i t i e s , b u t g r o w th was o n l y m o d e r a t e . Z i n c
o r z i n c and h i s t i d i n e a d d i t i o n s s i g n i f i c a n t l y im p ro v e d g r o w th and l e g
d e fo rm itie s b u t not t i b i a n itro g e n c o n te n t.
T ib ia n itr o g e n of c a se in g e l a t i n f r a c t i o n s w e r e i n c r e a s e d o n l y w i t h . 10 mg/kg z i n c .
The a d d i t i o n o f h i s t i d i n e t o t h e b a s a l s e sa m e r a t i o n s a l s o r e s u l t e d
i n i n c r e a s e s i n h y d r o x y p r o l i n e . H ow ever, b o t h t h e i n c r e a s e s i n t i b i a
n i t r o g e n o f f r a c t i o n s an d h y d r o x y p r o l i n e d i s a p p e a r e d when v a l u e s w e r e
c a l c u l a t e d p e r gram o f b o n e o r n i t r o g e n .
I.
INTRODUCTION
" A r t h r i t i s " p r o p e r l y d e f i n e d , means a n i n f l a m m a t i o n o f a j o i n t and
c a u s e s t h e l o s s o f many man h o u r s i n i n d u s t r y a l o n e .
a rth ritis,
R h e u m a to id
t h e m o st d a m aging o f a l l fo rm s o f a r t h r i t i s h a s an e s t i m a t e d
3 ,0 0 0 ,0 0 0 to 5 ,0 0 0 ,0 0 0 v ic tim s .
The c a u s e and c u r e r e m a i n unknown; t h e
symptoms i n c l u d e a c u t e an d p a i n f u l i n f l a m m a t i o n o f s e v e r a l o r a l l m a j o r
jo in ts
(1 ,2 ).
S p o n t a n e o u s r e m i s s i o n o f a r t h r i t i c .symptoms o f t e n o c c u r ;
t h i s makes i t d i f f i c u l t t o a s s a y t r e a t m e n t .
E x p e r i m e n t a l a n i m a l s c o u l d p r o v e u s e f u l i n d e t e r m i n i n g w h ic h d r u g s
a c tu a lly a l l e v i a t e th e a r t h r i t i s
sym ptom s.
To d a t e , a r t h r i t i s symptoms
h a v e n o t b e e n p r o d u c e d i n e x p e r i m e n t a l a n i m a l s by d i e t a r y m eans.
R e c e n t l y , h o w e v e r , i t h a s b e e n shown t h a t c h i c k s f e d c e r t a i n l o w - z i n c
d i e t s h a v e l e g d e f o r m i t i e s w h ic h r e s e m b l e form s o f r h e u m a t o i d a r t h r i t i s .
I n c l u s i o n o f h i g h d i e t a r y l e v e l s o f t h e amino a c i d h i s t i d i n e ,
d ecreased
t h e o c c u r r e n c e and s e v e r i t y o f l e g d e f o r m i t i e s i n c h i c k s g i v e n l o w - z i n c
s e sa m e an d i s o l a t e d p r o t e i n d i e t s b u t g r o w th o f t h e z i n c c o n t e n t o f t h e
t i b i a was n o t a f f e c t e d
(3 ,4 ,5 ).
P re lim in a ry exp erim en ts su g g e ste d t h a t in c lu s io n o f h i s t i d i n e a ls o
le d to an in c re a s e d n itr o g e n c o n te n t of th e t i b i a
(6 ).
C ollag en i s
m ain n i t r o g e n - c o n t a i n i n g c o n s t i t u e n t o f b o n e and c a r t i l a g e .
th e
V a ria tio n s
i n c o l l a g e n c o n t e n t o r c o m p o s i t i o n h a v e b e e n fo u n d when s e v e r e c r i p p l i n g
d e f o r m i t i e s o f b o n e s an d j o i n t s o c c u r r e d i n humans ( 7 ) .
The p u r p o s e o f t h i s s t u d y i s :
I.
To d e t e r m i n e t h e e f f e c t o f f o u r p r o t e i n s o u r c e s , low i n a v a i l ­
a b l e z i n c , on c h i c k body w e i g h t ,
l e g d e f o r m i t i e s an d t i b i a z i n c c o n t e n t .
2
2.
To d e t e r m i n e t h e e f f e c t on body w e i g h t , l e g d e f o r m i t i e s and
t i b i a z i n c c o n t e n t w hen v a r i o u s am ounts o f z i n c o r h i s t i d i n e o r a
c o m b i n a t i o n o f z i n c and h i s t i d i n e a r e a d d e d .
3.
To d e t e r m i n e t h e e f f e c t on body w e i g h t , l e g d e f o r m i t i e s and
t i b i a z i n c c o n t e n t when a n a n t ! a r t h r i t i c a g e n t a l o n e a n d w i t h added
z in c i s added to sesam e r a t i o n s .
4.
D e p e n d in g u p o n t h e r e s u l t s o f o b j e c t i v e s on e and tw o, t o d e t e r
m ine t h e e f f e c t o f v a r i o u s z i n c - a m i n o a c i d t r e a t m e n t s on t h e t o t a l
n i t r o g e n c o n t e n t o f c h i c k t i b i a e an d f o u r c h i c k t i b i a f r a c t i o n s .
5.
To a n a l y z e f r a c t i o n a t e d c h i c k t i b i a f o r h y d r o x y p r o l i n e as a
m easure of c o ll a g e n i f a d i f f e r e n c e in p e r c e n t n i t r o g e n i s found.
II.
REVIEW OF LITERATURE
Z i n c d e f i c i e n c y symptoms c a n b e p r o d u c e d i n r a t s , p o u l t r y , p i g s
and h u m a n s .
bone.
Of t h e s e ,
o n l y young f o w l d e v e l o p d e f o r m i t i e s o f t h e l e g
J a p a n e s e q u a i l e x h i b i t e d p o o r g r o w t h , a b n o rm a l t i b i a e and p o o r
fea th erin g
(8 ).
P o u l t s , w h ic h w e r e f e d a r a t i o n d e f i c i e n t i n z i n c ,
d ev e lo p e d an e n la rg e d hock c o n d it io n .
D e fo rm itie s in c h ic k s a re
c h a r a c t e r i z e d by s h o r t e n e d , t h i c k e n e d l e g b o n e s w i t h e n l a r g e d h o c k s and
r e s e m b l e t h e in fla m m e d j o i n t s o f human a r t h r i t i s
(9 ).
C o m p o s itio n o f Bones
Bone i s composed o f t h r e e m a j o r c o m p o n e n ts :
two o r g a n i c and one
in o rg a n ic „
O r g a n ic
M u c o p o l y s a c c h a r i d e s . — M u c o p o l y s a c c h a r i d e s , a n o r g a n i c bone
c o m p o n e n t, com poses t h e g r o u n d s u b s t a n c e o f b o n e c a l l e d m ucoid ( 1 1 ) .
I t i s a n i t r o g e n o u s s u b s t a n c e w h ic h i s a c i d i n n a t u r e .
C o l l a g e n . — C o l l a g e n , a l s o a n i t r o g e n o u s b o n e c o m p o n e n t, i s t h e
c h i e f o r g a n i c p o r t i o n o f b o n e and c a r t i l a g e ( 1 2 ) .
I t i s p a r t of th e
b o n e m a t r i x w i t h i n w h ic h g r o w th i n l e n g t h o c c u r s .
C a l c i f i c a t i o n of
b o n e may de p e n d upon a n i n t e r a c t i o n b e tw e e n c o l l a g e n f i b r i l s
m u co p o ly sa cc h a rid e s (1 2 ).
show
and
Any c h a n g e i n e a r l y c o l l a g e n f o r m a t i o n c o u ld
up i n m ore m a t u r e b o n e s i n c e c a l c i f i c a t i o n i s c o m p l e t e o n ly i n
m ature bone.
4
The s t r u c t u r a l p r o p e r t i e s o f t h e b o n e d e p e n d on t h e way i n w h ic h
th e b a s i c e le m en ts ( c a l c i f i e d c o lla g e n f i b r i l s )
tissu e
a re d i s t r i b u t e d in th e
(1 4 ).
A r e c e n t s tu d y s u g g e s te d t h a t one of t h e f u n c tio n s o f th e z in c c o n t a i n i n g enzym e, a l k a l i n e p h o s p h a t a s e ,
sy n th e sis
(13).
i s im p o rta n t f o r c o lla g e n
The enzyme f u n c t i o n s i n t h e m a t u r a t i o n o f t h e c h i c k
e p i p h y s e a l p l a t e c a r t i l a g e and i s m a r k e d ly d e c r e a s e d i n a z i n c
d e fic ie n c y
(13).
■
The m e a s u re m e n t o f c o l l a g e n i s a t w o - s t e p p r o c e s s :
(I)
e x tra c tio n ;
and (2) t h e d e t e r m i n a t i o n o f h y d r o x y p r o l i n e .
C o llag en v a r ie s in e x t r a c t a b i l i t y w ith age.
C o llag en e x tr a c te d
w i t h NaC l w i l l c o n t a i n t h e m o st r e c e n t l y s y n t h e s i z e d m o l e c u l e s and t h e
n a rro w e st age ran g e (1 5 ).
in a u to c la v in g ,
When h e a t e d a b o v e a c e r t a i n
te m p e ra tu re , as
t h r e e s tr a n d e d c o ll a g e n m o le c u le s can be red u ced to a
m i x t u r e o f random c o i l c o m p o n e n ts c a l l e d g e l a t i n
w i l l e x tr a c t th e o ld e r f i b r i l s
(16).
A c id s o l u t i o n s
i n a d d i t i o n t o t h e ne w e r on e s ( 1 7 ) .
. D e te rm in a tio n o f h y d r o x y p ro lin e as a m easure o f c o ll a g e n c o n te n t
i n c o m p a r is o n t o t h e t o t a l n i t r o g e n o f t h e - f r a c t i o n , i s b a s e d on
r e s e a r c h u s i n g b o t h c o l o r i m e t r i c m eth o d s ( 1 8 ,3 7 )
and an am ino a c i d
a n a ly z e r.
■ L a th r y ism , a bone d i s o r d e r i n c h i c k s , i s
c a u s e d by i n g e s t i o n o f
t h e p e a , L a t h y r u s O d o r a t u s ; i t i s t h o u g h t t o b e due t o f a i l u r e t o fo rm
norm al i n s o l u b l e c o lla g e n (1 9 ).
C o llag e n fo rm a tio n i s n o t i n t e r f e r e d
5
w ith in la th y ris m , b u t th e c o lla g e n , a lre a d y p r e s e n t,
e x t r a c t a b l e (20) .
i s more
The f a i l u r e c o u l d b e due t o a l o s s o f i n t r a m o l e c u l a r
c r o s s l i n k i n g , p r e v i o u s l y form ed i n t e r m o l e c u l a r b o n d s b e i n g d i s r u p t e d ,
or p o s s ib le a l t e r a t i o n in c o lla g e n .
C a lc iu m , c o p p e r an d z i n c i o n s p a r t i a l l y r e l i e v e l a t h y r i s m symptoms
(21).
C o l l a g e n fro m c o p p e r - d e f i c i e n t c h i c k s was more s o l u b l e t h a n
c o l l a g e n fro m c o n t r o l c h i c k s , r e s u l t i n g i n b o n e a b n o r m a l i t i e s
(2 2 ).
A p p r o x i m a t e l y 65 p e r c e n t o f t h e b o n e i s i n o r g a n i c and i s te rm e d
th e bone s a l t p o r ti o n .
The b o n e s a l t s
and p h o s p h a t e a s h y d r o x y a p a t i t e .
c o n s i s t p r i m a r i l y of ca lc iu m
M agnesium , s o d iu m , c i t r a t e and
c a r b o n a t e i o n s a r e p r e s e n t i n m in o r am o u n ts ( 1 2 ) .
The i n o r g a n i c
m a t e r i a l s a r e d e p o s i t e d i n t h e c o l l a g e n m a t r i x a s g r o w th p r o g r e s s e s .
The z i n c - c o n t a i n i n g enzym e, a l k a l i n e p h o s p h a t a s e , i s
t h o u g h t t o be
a c t i v e i n p ro m o tin g d e p o s i t i o n o f th e i n o r g a n ic c o n s t i t u e n t s
(12).
A lthough a l k a l i n e p h o s p h a ta s e l e v e l s a r e red u ced i n z i n c - d e f i c i e n t
ra ts
(23),
th e l e v e l of d i e t a r y z in c does n o t a f f e c t th e p e rc e n ta g e of
bo n e a s h i n t h e c h i c k .
C a u se s o f Z in c D e f i c i e n c i e s R e s u l t i n g i n Bone D e f o r m i t i e s
Z in c R eq u irem en t .
The z i n c r e q u i r e m e n t o f t h e n o r m a l c h i c k i s g i v e n a s a p p r o x i m a t e l y
35 m g/kg o f fo o d ( 2 4 ) .
B e c a u s e o f v a r i o u s f a c t o r s , a s d i s c u s s e d b e lo w ,
t h i s r e q u ir e m e n t can v a ry g r e a t l y .
6
E f f e c t o f P h y t i c A cid
P h y t i c a c i d i s a p h o s p h a t e e s t e r o f i n o s i t o l f o u n d i n p l a n t s and
o c c u r s a s a c a l c i u m , m agnesium c o m p le x .
By b i n d i n g a v a i l a b l e z i n c i n
p l a n t p r o t e i n s , c h i c k s f e d t h e s e p r o t e i n s may become z i n c d e f i c i e n t (25)
P l a n t p r o t e i n r a t i o n s . — Some s e s a m e m e a ls h a v e z i n c p r e s e n t a s a
com ponent o f o r a s s o c i a t e d w i t h a n i n s o l u b l e c a l c i u m ° m agnesium •
p h y t a t e co m p le x ( 2 6 ) .
of z in c ,
is used a s a so u rce of p r o te in ,
a b n o rm a litie s,
c h ic k s
When a s e sa m e m e a l , T exas 61 c o n t a i n i n g 150 ppm
(39),.
leg d e fo rm itie s , fe a th e r
low t i b i a z i n c c o n t e n t , and p o o r g r o w th o c c u r r e d i n
I t r e q u i r e d 60 mg/kg o f a d d e d z i n c t o p r e v e n t t h e d e f i ­
c i e n c y sym ptom s.
T h i s m e a l , t h e r e f o r e , n o t o n ly makes z i n c u n a v a i l a b l e
(b ec a u se o f th e p h y t ic a c id c o n te n t) b u t a ls o has a h ig h c a p a c ity to
b in d added z in c .
A lth o u g h a u to c l a v i n g th e m eal does n o t s i g n i f i c a n t l y
r e d u c e t h e p h y t i n c o n t e n t , c h i c k s f e d t h i s m e a l showed l i t t l e s i g n s o f
a z in c d e f ic ie n c y .
The z i n c was t h o u g h t t o b e r e l e a s e d t h r o u g h o t h e r
means t h a n t h e d e c r e a s e o f p h y t i c . a c i d
(27).
.S o y b e a n m e a l i s a n o t h e r p r o t e i n . i n g r e d i e n t o f c h i c k r a t i o n s and
c o n t a i n s a b o u t 40 p e r c e n t a s much z i n c o r p h y t a t e p h o s p h o r u s a s t h e
sesam e m eal ( 2 8 ),
S o y b e a n p r o t e i n s make z i n c a v a i l a b l e by means o t h e r
t h a n t h r o u g h r e l e a s i n g i t fro m t h e p h y t a t e com plex ( 2 6 ) .
When f e d t o
c h i c k s , no a b n o r m a l i t i e s and s l i g h t l y p o o r g ro w th r e s u l t e d .
I s o l a t e d s o y p r o t e i n , h o w e v e r, h a s a h i g h a f f i n i t y
f o r z i n c and
i n c r e a s e s t h e amount o f z i n c n e c e s s a r y t o p r e v e n t d e f i c i e n c y symptoms
(2 9 ,3 0 ).
I n t h e p r o d u c t i o n o f t h e I s o l a t e d soy p r o t e i n , t h e com ponents
7
o f t h e s o y b e a n m e a ls t h a t make z i n c a v a i l a b l e a r e t h o u g h t t o b e l o s t ( 2 6 ) .
From 1 5 -4 0 m g/kg o f ad d e d z i n c a r e n e c e s s a r y f o r n o r m a l g r o w th o f c h i c k s
f e d i s o l a t e d so y p r o t e i n .
A nim al p r o t e i n r a t i o n s . — The p r e s e n c e o f p h y t i c a c i d i s n o t
n e c e s s a ry f o r p ro d u c tio n of a zin c d e fic ie n c y .
A nim al p r o t e i n d i e t s ,
w h ic h a r e low i n z i n c p r o d u c e s e v e r e z i n c d e f i c i e n c y sym ptom s.
C h ic k s g i v e n eg g w h i t e r a t i o n s grew p o o r l y , d e v e l o p e d open s o r e s
an d b l e e d i n g , b u t d i d n o t h a v e l e g d e f o r m i t i e s ( 3 1 ) .
C a s e i n - g e l a t i n r a t i o n s , w h ic h h a v e a low z i n c c o n t e n t , do n o t b i n d
z in c in th e r a t i o n .
Leg d e f o r m i t i e s , p o o r w e i g h t g a i n , an d p o o r
f e a t h e r i n g h a v e b e e n fo u n d when c h i c k s a r e g i v e n a c a s e i n - g e l a t i n d i e t ,•
Some r e s e a r c h e r s h a v e f o u n d t h a t 0 . 4% -2.0% p h y t i c a c i d , when a d d e d t o
c a s e in d i e t s reduced th e z in c a v a i l a b l e to th e c h ic k ( 3 0 ,3 2 ,3 3 ) .
O th ers
fo u n d t h a t t h e a d d i t i o n o f 0.5% p h y t i c a c i d t o a c a s e i n h y d r o l y s a t e
d i e t d id n o t red u ce a v a il a b l e z in c (3 1 ).
C a s e in h y d r o l y s a t e d i e t s
c a u s e p o o r g r o w th and a b n o rm a l f e a t h e r i n g i n c h i c k s b u t n o t l e g
d e fo rm itie s
(23).
From 9 t o 20 m g/kg o f a d d e d z i n c w e r e fo u n d t o be
s u f f i c i e n t f o r n o r m a l g r o w th ( 3 0 , 3 1 ) .
E f f e c t o f M in e ra ls
C a l c iu m . — H ig h d i e t a r y c a l c i u m d e c r e a s e s t h e a v a i l a b i l i t y o f z i n c
t o t h e c h i c k when a p h y t a t e - c o n t a i n i n g r a t i o n i s f e d . • I n v i t r o s t u d i e s
showed t h a t z i n c • c a l c i u m • p h y t a t e c o m p le x es a r e m ore i n s o l u b l e t h a n
z i n c p h y t a t e c o m p le x es a t t h e i n t e s t i n a l pH o f t h e c h i c k ( 2 8 ) .
8
M agnesium . — S i n c e t h e c h e m i c a l b e h a v i o r o f m agnesium i s s i m i l a r t o
t h a t o f c a l c i u m , t h e e f f e c t o f ad d e d d i e t a r y m agnesium was a l s o s t u d i e d
(31).
I n c r e a s e d m agnesium i n some s e sa m e d i e t s d e c r e a s e d t h e z i n c
a v a i l a b l e t o t h e c h i c k , b u t a v a i l a b l e z i n c was n o t d e c r e a s e d on a
s o y b e a n m e a l r a t i o n u n t i l 60 mg/kg o f z i n c w e re a d d e d w i t h 3 ,0 0 0 m g/kg
o f m agnesium .
Up t o 3 ,0 0 0 mg/kg o f a d d e d m agnesium d i d n o t d e c r e a s e
th e z in c a v a i l a b l e to c h ic k s fe d c a s e in (n o n -p h y t a t e c o n ta in in g )
C o p p e r, M anganese and V i t a m in
. — T h i s can p a r t i a l l y b e e x p l a i n e d
by t h e f a c t t h a t c o p p e r and m an g a n e se d e f i c i e n c i e s ,
bone d e fo r m itie s
(2 2 ,3 1 ).
am o u n ts o f V i t a m in
ra tio n s
i n t h e m s e lv e s c a u s e
S u p p le m e n ts o f c o p p e r , m a n g a n e se o r v a r y i n g
d id n o t p re v e n t th e leg d e fo rm itie s of th e z in c -
d e f i c i e n t c h ic k .
Compounds Which A l l e v i a t e Z in c D e f i c i e n c y Symptoms
Eth y le n e d ia m in e te tr a a c e tic
E t h y l e n e d i a m i n e t e t r a a c e t i c (EDTA) , a c h e l a t i n g a g e n t , when added
t o e i t h e r s e s a m e o r i s o l a t e d so y p r o t e i n d i e t s , i n c r e a s e d g ro w th and
reduced le g d e fo r m itie s .
S i n c e EDTA, i t s e l f ,
d o es n o t s t i m u l a t e g r o w t h ,
i t was t h e o r i z e d t h a t . EDTA makes p h y t a t e o r p r o t e i n - b o u n d z i n c more
a v a ila b le
(3 8 ,3 9 ).
A n t i - A r t h r i t i c A g e n ts
S e v e ra l a n t i - a r t h r i t i c a g e n ts
( i . e . , c o r t i s o n 'e , a s p i r i h , in d o m e th -
a c i n , p h e n y lb u ta zo n e ) hav e been added to i s o l a t e d soy p r o t e i n d i e t s
(34)
9
A l t h o u g h t h e y p r e v e n t e d some l e g d e f o r m i t i e s , t h e y a l s o h a d h a r m f u l
s i d e e f f e c t s su ch as h i g h m o r t a l i t y r a t e s o r p e r f o r a t e d c h ic k g i z z a r d s .
H istid in e
I t i s p o s s i b l e t h a t h i s t i d i n e p la y s a r o l e in c o r r e c t i n g z in c
d e f i c i e n c y symptoms t h r o u g h t h e v a r i a t i o n i n t h e amount o f s o l u b l e
c o lla g e n ,
s in c e c o lla g e n c o n te n t i s
d e fo rm itie s
th o u g h t to be r e l a t e d to c r i p p l i n g
(10).
One p e r c e n t h i s t i d i n e ,
a d d e d t o i s o l a t e d so y p r o t e i n d i e t s ,
d e c r e a s e d t h e s e v e r i t y o f b o n e d i s o r d e r s i n c h i c k s g i v e n an i s o l a t e d
so y p r o t e i n d i e t .
I t d i d n o t c o u n t e r a c t . t h e low w e i g h t g a i n o r low
t i b i a - z i n c c o n te n t (3 1 ).
When a d d e d t o a se sa m e m e a l d i e t ,
f ic a n t l y decreased leg d e fo r m itie s .
how ever, w ere n o t a f f e c t e d
i t sig n i­
W e ig h t g a i n o r t i b i a z i n c c o n t e n t ,
(35).
Z i n c d e f i c i e n c y symptoms c a u s e d by c a s e i n - g e l a t i n d i e t s w e re n o t
c o u n t e r a c t e d by h i s t i d i n e
(29).
A r e c e n t s t u d y s u g g e s t e d t h a t t h e t i b i a e o f c h i c k s g i v e n a s e sa m e
m e a l d i e t low i n a v a i l a b l e z i n c , s u p p l e m e n t e d w i t h h i s t i d i n e ha d a
h i g h e r n i t r o g e n c o n te n t th a n t i b i a o f c h ic k s n o t g iv e n su p p le m e n ta ry
h istid in e
(2 9 )•
S i n c e a d i f f e r e n c e i n g ro w th due t o h i s t i d i n e
su p p le m e n ta tio n d id n o t o ccu r,
th e in c r e a s e in n itr o g e n c o n te n t of
t h e t i b i a e may b e due s p e c i f i c a l l y t o c h a n g e s i n b o n e s t r u c t u r e r a t h e r
t h a n a g e n e r a l i n c r e a s e i n body n i t r o g e n .
10
I n an e f f o r t t o d e t e r m i n e how h i s t i d i n e c o u n t e r a c t e d l e g d e fo rm ­
i t i e s , h i s t i d i n e was a d d e d t o a Ca • Mg • Zn p h y t a t e c o m p le x , i s o l a t e d
f ro m s e sa m e m e a l an d l a b e l e d w i t h z i n c ^
(3) .
The low a b s o r p t i o n o f
Z n ^ f ro m t h e p h y t a t e c o m plex by t h e c h i c k was n o t i n c r e a s e d by
in c lu sio n of h is tid in e .
I t was h y p o t h e s i z e d t h a t h i s t i d i n e p r e v e n t s
l e g d e f o r m i t i e s by o t h e r means t h a n by r e l e a s i n g z i n c fro m a p h y t a t e
com plex ( 2 9 ) .
R e c e n t w o rk showed t h a t 2 p e r c e n t o f d i e t a r y a r g i n i n e added t o eg g
a lbum en r a t i o n s h a s b e e n fo u n d t o h a v e a s y n e r g i s t i c e f f e c t on t h e z i n c
d e f i c i e n c y symptoms ( 3 6 ) .
Low z i n c , 2 p e r c e n t ( a d d e d ) a r g i n i n e d i e t s ,
p r o d u c e d n o t o n l y m ore s e v e r e l e g d e f o r m i t i e s , b u t a l s o p o o r e r g ro w th
and l o w e r t i b i a - z i n c c o n t e n t ( 3 6 ) .
of a rg in in e is
1 .2 p e r c e n t.
The c h i c k ' s d i e t a r y r e q u i r e m e n t
T h e s e d e f o r m i t i e s w e re c o r r e c t e d by t h e
a d d itio n of I p e rc e n t h i s t i d in e .
I ll.
METHODOLOGY
S amp I e
Two f a c t o r i a l e x p e r i m e n t s w e r e c o n d u c t e d u s i n g day o l d W h ite Rock
( f ) X C o r n i s h (M) c h i c k s
I
w ith o u t d i f f e r e n t i a t i n g a c c o rd in g to sex .
The c h i c k s w e r e r a n d o m ly d i s t r i b u t e d i n t o d u p l i c a t e g r o u p s o f 10 f o r
t h e f i r s t e x p e r i m e n t a n d 10 o r 13 f o r t h e s e c o n d .
C h ic k C a re
T he c h i c k s w e r e h o u s e d i n a s t a i n l e s s s t e e l b a t t e r y
c o n t a m i n a t i o n f ro m e n v i r o n m e n t a l z i n c .
g i v e n ad l i b i t u m .
2
t o m in im iz e
Feed and d e i o n i z e d w a t e r w e r e
E ach c a g e o f c h i c k s was w e ig h e d a s a g ro u p f o r t h e
f i r s t two w eeks an d i n d i v i d u a l l y t h e t h i r d w eek.
A, p . 4 2 ) w e r e d e t e r m i n e d a t t h e same t i m e .
Leg s c o r e s (A p p e n d ix
C h ic k s w e r e s a c r i f i e d a t
3 w eeks o f a g e .
R a tio n s w ere com prised o f f o u r b a s a l p r o t e i n s o u r c e s , sesam e,
c a s e i n - g e l a t i n , eg g album en and s o y b e a n , p l u s t h e n e c e s s a r y v i t a m i n s
and m i n e r a l s ( T a b l e I ) .
A d d itio n s, h i s t i d i n e ,
z i n c , a s p i r i n and
g l u t a m i c a c i d , a r e shown i n T a b l e I I .
C r i t e r i a f o r M e asu re m e n t
The c r i t e r i a u s e d t o d e t e r m i n e t h e e f f e c t s o f f e e d i n g t h e v a r i o u s
r a t i o n s f o r t h r e e w eeks w e r e : .
^ O b t a i n e d fro m Q u a l i t y H a t c h e r y , B i l l i n g s , M o n ta n a .
2
P e t e r s i m e I n c u b a t o r Company, G e t t y s b u r g , O hio.
12
TABLE I .
C o m p o s it io n o f B a s a l D i e t s
T exas 61
. Sesame
g
T e x a s 61 s e s a m e m ealg
C a se in , v ita m in f r e e
G e la tin ^
Egg w h i t e
5
Soybean m eal
V i t a m in m ix^ _
MHA (Ca s a l t ) '
C h o l i n e c h l o r i d e (70%)
V i t a m in D&
S a n to q u in ^
Corn o i l l O
S a ltsll
MgCO3
B io tin
L y sin el^
Sucrose
300
——
—
—
—
5 .0
——
3 .0
1 .5
2 .5
9 4 .0
6 0 .1
—
9 .0
5 2 4 .9
1 0 0 0 .0
C a se in G e la tin
g
—
230
. 100
—
—
5 .0
3 .3
3 .0
1 .5
2 .5
. 9 6 .0
6 0 .1
1 .5
—
—
4 9 7 .1
1 0 0 0 .0
S o y bean
M eal
g
—
—
—
—
400
5 .0
3 .3
3 .0
1 .5
2 .5
9 6 .0
6 0 .1
—
—
—
4 2 8 .6
1 0 0 0 .0
Egg
W h ite
g
—
t—
— —
—
200
—
5 .0
—
3 .0
1 .5
2 .5
9 6 .0
6 0 .1
1 .5
1 0 .0
—
6 2 0 .4
1 0 0 0 .0
T e x a s 61 s e s a m e m e a l e x t r a c t e d w i t h p e t r o l e u m e t h e r , t w i c e .
N u t r i t i o n a l B i o c h e m i c a l s C o r p o r a t i o n , C l e v e l a n d , O h io .
3
P h a r m a c e u t i c a l g r a d e g e l a t i n , P . L e i n e r & S o n s , A m e ric a , I n c . , S t .
C l a i r , M ich ig an .
^Egg w h i t e s o l i d s
( a l b u m e n ) , A rm our, C h i c a g o , I l l i n o i s .
^50 p e r c e n t p r o t e i n ,
Bozeman, M o n ta n a .
c o m m e rc ia l s o y b e a n m e a l , F a r m e r 's E l e v a t o r ,
The v i t a m i n m ix c o n t a i n e d :
( i n gm.) v i t a m i n B ^
( a s 0.1% m i x ) , 4;
m e n a d io n e b i s u l f i t e s o d iu m 0 , 9 ; b i o t i n , 0 . 0 4 ; p y r i d o x i n e * HC1, 1 . 0 ;
f o l i c a c i d , 1 . 0 ; m l a c l n , 1 0 . 0 ; r i b o f l a v i n , 2 . 0 ; D-Ca p a n t o t h e n a t e , 6 , 0 ;
t h i a m i n m o n o n i t r a t e , 2 . 0 ; v i t a m i n A ( 2 5 0 ,0 0 0 lU/gm) 8; v i t a m i n E (250
I U / g m ) , 40.
The b a s i c m ix was made t o 1000 gm w i t h s u c r o s e .
^ C a lc iu m s a l t o f m e t h i o n i n e h y d r o x y a n a l o g u e .
8V i t a m in D , 1000 I U / m l .
9
'
S a n t o q u i n , M o n san to Company, S t , L o u i s , i n c o r n o i l , 0 . 0 5 g /m l.
10M
.
M azda.
(co n tin u ed )
13
F o o tn o te s T able I
11
(co n tin u ed )
The s a l t mix c o n t a i n e d :
( i n gm) CaHPO^ • ZH20 , 5 4 4 0 ; CaCO^, 2984
K2HPO^, 2222; NaCL, 1200; MgCO^, 2 5 ; Fe c i t r a t e , 6 6 . 6 ; MnSO^ ' H 2O,
6 6 . 6 ; K I , 0 . 5 2 ; CuSO • 5H_0, 6 . 6 8 .
12
^
Z
B i o t e i n , 40 mg p e r 100 gm s u c r o s e .
13
L - I y s i n e • HC1, N u t r i t i o n a l B i o c h e m i c a l s , C l e v e l a n d , O hio.
14 '
TABLE I I .
A d d itio n s to th e B a s a l D ie ts ( a t th e expense o f su c ro se ) .
P r o t e i n Source
H istid in e ^
1%
2
Z inc
mg/kg
A sp irin ^
1%
G l u t a m ic A c id ^
1%
60
SESAMEEXPERIMENT I
+
0
+
30
0
+
30
+
•0
4*
EXPERIMENT 2
CASEIN-GELATIN
EXPERIMENT I
EXPERIMENT 2
0
10
+
0
+
'5
+
0
10
20
EGG ALBUMEN
SOYBEAN MEAL
■
+
0
+
10
+
20
+
0 .
^ H i s t i d i n e HC1, m o n o h y d r a te , N u t r i t i o n a l B i o c h e m i c a l s , C l e v e l a n d , O h io .
2
3
4
ZnCpg m ix , ZnCO^ i n s u c r o s e ; I gm = 10 mg/kg o f Zn f o r 10 k g r a t i o n .
A sp irin , S t. Josephs.
L - g l u t a m i c a c i d , N u t r i t i o n a l B i o c h e m i c a l s , C l e v e l a n d , O h io .
15
1)
Body w e i g h t ;
2)
Leg s c o r e s a s i n d i c a t i v e o f d e f o r m i t y (A p p e n d ix A, p . 4 2 ) ;
3)
R a tio of le n g th to w id th o f th e t i b i a
4)
A n a l y s i s o f t i b i a f o r z i n c (A p p e n d ix A, p . '4 5 ) ;
5)
A n a l y s i s f o r t o t a l n i t r o g e n (A p p e n d ix A, p . 46)5 and
6)
A n a ly sis of fo u r f r a c ti o n s of each s e t of t i b i a e f o r n itr o g e n
( A p p e n d ix A, p .
4 4 );
' ( A p p e n d ix A, p . 4 7 ) .
The s i g n i f i c a n c e o f t h e s e c r i t e r i a w e r e d e t e r m i n e d s t a t i s t i c a l l y w i t h
D u n c a n 's m u l t i p l e c h o i c e t e s t . ^
3
S t e e l , R o b e r t G .D ., James H. T o r r i e , 1960. P r i n c i p l e s and
■P r o c e d u r e s o f S t a t i s t i c s , (New Y o rk : M c G ra w -H ill Book C o , ) , pp 1 0 7 -1 0 9 .
4
The a u t h o r w o u ld l i k e t o a c k n o w le d g e D r. E r v i n P . S m ith f o r h i s
h e lp w ith th e s t a t i s t i c a l t e s t in g .
IV .
RESULTS AND DISCUSSION
R e su lts
The p u r p o s e o f t h i s s t u d y was t o d e t e r m i n e t h e r e s u l t s o f f e e d i n g
s e s a m e , c a s e i n - g e l a t i n , eg g album en an d s o y b e a n r a t i o n s , w i t h z i n c o r
am ino a c i d
a n a ly se s,
su p p le m e n ts,
t o c h i c k s , and t o d e t e r m i n e , by v a r i o u s
th e deg ree of d e fo rm ity , i f any, th a t r e s u l te d .
The f o l l o w i n g r e s u l t s a r e b a s e d upon t h e num ber o f c h i c k s w h ic h
w e r e a l i v e a t t h r e e w eeks ( T a b l e I I I ) .
The o n ly m o r t a l i t y o f c h i c k s
o c c u r r i n g on b a s a l s e s a m e r a t i o n s was w i t h s e sa m e and a s p i r i n .
The
m o r t a l i t y o f c h i c k s on egg album en and c a s e i n - g e l a t i n w i t h o u t z i n c was
h ig h .
The a d d i t i o n o f z i n c t e n d e d t o c o r r e c t t h i s .
P r o t e i n Sources
S e s a m e . — C h ic k s g i v e n t h e b a s a l d i e t ( e x p e r i m e n t s I and 2)
e x h i b i t e d low body w e i g h t , and s e v e r e l e g d e f o r m i t i e s
F ig u re I ) .
ra tio s.
(T a b le I I I ,
Leg d e f o r m i t i e s w e r e a l s o e v i d e n t by low l e n g t h t o w i d t h
T ib ia z in c c o n te n t, a lth o u g h h ig h e r th an t h a t o f th e d a y -o ld
c h i c k was s t i l l low .
The a d d i t i o n o f 60 m g/kg o f z i n c s i g n i f i c a n t l y i n c r e a s e d t h e w e i g h t
o f t h e c h i c k s and d e c r e a s e d t h e l e g s c o r e s
(ex p e rim e n t I ) .
S u p p l e m e n ta l
z i n c was d e p o s i t e d i n t h e t i b i a e a s e v i d e n c e d by t h e r e l a t i v e l y h i g h
t i b i a z in c c o n te n t.
L e n g th t o w i d t h r a t i o was s i g n i f i c a n t l y g r e a t e r .
C a s e i n - G e l a t i o n . — C h ic k s f e d t h e b a s a l r a t i o n s g rew p o o r l y , ha d
s e v e r e l e g d e f o r m i t i e s an d v e r y low t i b i a z i n c c o n t e n t ( e x p e r i m e n t I ) .
The l e n g t h t o w i d t h r a t i o was h i g h ( T a b l e I V ) .
17
F ig u re I
E f f e c t o f T e x a s 61 Sesame M eal Fed t o C h i c k s .
D e f o r m i ty a t Arrow.
Severe
18
TABLE I I I .
M o r t a l i t y o f 20 C h ic k s G iven. S e v e r a l P r o t e i n S o u r c e s f o r
T h r e e Weeks,
P r o te i n Source
Added Z i n c
E x p e r im e n t I
E x p e r im e n t 2
. mg/kg
. %
%
0
0
0
SESAME
60
0
Sesame +
0
0
0
1% H i s t i d i n e
30
0
S e s + G l u t A c id
0
0
Sesam e +
0
5
1% A s p i r i n
30
20
CASEIN0
45
3 I/
GELATIN
10
0
C a se in -G e l +
0
35
11 I /
1% H i s t i d i n e
5
20
0
43 I /
EGG ALBUMEN
10
. 5
20
0
Egg Albumen
0
29 I /
+
10
0
1% H i s t i d i n e
20
I
0
SOYBEAN
0
)
■
5
0
Soy + H i s t i d i n e ________ 0________ I_____________________
I / P e r c e n t a g e s fro m t h e s e r a t i o n s w e r e b a s e d on 2 6 , 2 6 , 23 and 24
c h ic k s r e s p e c t i v e l y .
19
TABLE IV .
C r i t e r i a Mean t o J u d g e t h e E x t e n t o f Z i n c D e f i c i e n c i e s a t
T h r e e Weeks i n C h ic k s Fed S e v e r a l P r o t e i n S o u r c e s .
P ro te in
S ource
EXPERIMENT I
Added
Z inc
W eight
mg/kg
8
I/
2/
Z in c
T o tal
N itro g en
% I/
19Se 2 /
291b
23 7 c
306a
3 .6
1 .0
1 .2
1 .5
9 .1 2 a
1 1 .8 8 c
1 1 .8 9 c
1 1 .7 3 c
103
3 7 5 .5
1 3 9 .0
2 9 5 .0
5 .4 lb c
5 . 38bc
5 . 69a
5 .2 6 cd
0
0
30
0
10
0
5
209d
10 7g
115f
85h
287b
106 g
194e
3 .1
1 .4
1 .0
2 .7
1 .1
3 .0
1 .6
9 .2 6 a
1 4 . 55g
1 3 . 63e
1 4 .37f
1 0 .4 9 b
1 2 .9 4 d
1 2 . 81d
7 0 .0
1 0 2 .5
7 3 .5
3 8 .0
3 0 7 .5
5 3 .0
1 1 7 .5
4 . 94f
4 . 39g
5 , 09ef
5 . 1 9 de
5 . 31bcd
5 . 33bcd
5 .4 6 b
EXPERIMENT 2
0
173e
3 .2
1 1 .0 5 a
6 4 .0
Sesame +
1% H i s t i d i n e
Sesame + 1%
G l u t . A c id
Sesame +
1% A s p i r i n
CASEINGELATIN
G as-G el +
.1% H i s t i d i n e
Egg Albumen
+
1% H i s t i d i n e
SOYBEAN
S o y bean +
1% H i s t i d i n e
1/w. R a t i o
0
60
0
30
SESAME
SESAME
Sesame + 1%
H istid in e
CAS-GEL
C a s - G e l + 1%
H istid in e
EGG
ALBUMEN
T ib ia
Leg
Score
-
5 . 60a
0
0
190b
197a
2 .4
2 .0
1 2 . 49b
1 2 . 24b
8 0 .5
2 4 2 .5
5 .56ab
5 . 18d
0
0
10
20
0
10
20
0
16 I d
58g
116f
122f
6 Sg
121f
13 I e
177c
1 .8 5
1 .4
.3 3
.30
-.9 0
.60
.2 6
.7 .
1 2 .8 9 c
1-5.8 4 fg
1 5 . 50de
1 5 . 73ef
1 6 .1 2 gh
1 6 . 34h
1 5 . 36.d
1 3 .7 7 c
2 1 3 .0
35
1 1 0 .0
2 3 0 .0
4 7 .0
1 2 5 .0
2 3 1 .5
8 9 .0
5 .2 0 d
4 .5 7 s
4 . 70 fg
4 . 70fg
4 . 7 4 e fg
4 . 85ef
4 . 90e
5 . 14bc
0
184b
• 16
1 3 .3 5 c
. 8 6 .0
5 . 28cd
P e r c e n t a g e was b a s e d on d r i e d , f a t - f r e e b o n e s .
P ( < 0 . 5 ) w i t h D u n c a n 's M u l t i p l e Range T e s t . V a lu e s f o l l o w e d by t h e
same l e t t e r s w i t h i n t h e same colum n a r e n o t s i g n i f i c a n t l y d i f f e r e n t .
20
C h ic k body w e i g h t , l e g s c o r e and t i b i a l e n g t h t o w i d t h r a t i o w e r e
s i g n i f i c a n t l y im p ro v e d w i t h t h e a d d i t i o n o f 10 mg/kg o f z i n c ,
m ent I ) .
(ex p e ri­
S u p p l e m e n t a l z i n c was d e p o s i t e d i n t h e t i b i a a s shown by t h e
s i g n i f i c a n t l y h i g h e r t i b i a z in c c o n te n t (T a b le I V ) .
I n e x p e r i m e n t 2 , a d i f f e r e n t l o t o f c a s e i n was u s e d ,
sequent a n a ly sis,
t h e c a s e i n h a d a l a r g e r amount o f z i n c .
On s u b ­
As a r e s u l t ,
c h ic k s g iv en th e b a s a l r a t i o n d id n o t e x h ib i t d e fo r m itie s or poor
g ro w th .
T h e re fo re , a d is c u s s io n of th e s e r e s u l t s i s n o t in clu d e d in
th e fo llo w in g s e c tio n s .
Egg A lbum en. — V ery p o o r g r o w th and o n l y s l i g h t l e g d e f o r m i t i e s
w ere o b ta in e d w ith th e b a s a l r a t i o n .
Leg d e f o r m i t i e s may n o t h a v e
d e v e l o p e d b e c a u s e o f t h e p o o r g r o w th ;
th e h ig h le n g th to w id th r a t i o
m ig h t a l s o b e a t t r i b u t e d t o p o o r g r o w t h .
T i b i a z i n c c o n t e n t was v e r y
lo w .
A n o th er c o n d it io n , b a l d n e s s , o c c u rre d (F ig u re 2 ) ;
a l l t h e f e a t h e r s on t o p o f t h e i r h e a d .
d if f e r e n c e in th e deg ree of b a ld n e s s ;
th e c h ic k s l o s t
The a d d i t i o n o f z i n c made no
t h e c o n d i t i o n may h a v e b e e n
r e la te d to o th e r r a tio n in g re d ie n ts .
W ith t h e a d d i t i o n s o f 10 m g/kg o f z i n c , c h i c k body w e i g h t was
s i g n i f i c a n t l y i n c r e a s e d (T a b le I V ) .
The i n t a k e o f a d d e d z i n c was
e v i d e n t by i n c r e a s e d t i b i a z i n c c o n t e n t .
The a d d i t i o n o f 20 mg/kg o f
z i n c d i d n o t im p ro v e g r o w th o v e r t h a t w i t h 10 mg/kg z i n c n o r d i d i t
in c r e a s e th e t i b i a c o n te n t.
.
Even w i t h a d d e d z i n c , c h i c k s d i d n o t grow
w e l l and le n g th to w id th r a t i o s w ere h ig h .
21
F ig u re 2.
E f f e c t o f F e e d in g Egg Albumen R a t i o n s t o C h i c k s .
22
S o y b e a n . — C h ic k s f e d b a s a l r a t i o n s h a d l i t t l e
s i g n s o f l e g d e fo rm ­
i t i e s b u t d i d n o t d e p o s i t i n t h e t i b i a much z i n c o v e r t h e amount
(28 mg/kg)
fo u n d i n d a y - o l d c h i c k s .
H i s t i d i n e A d d itio n to B a s a l R a tio n s
S e s a m e . — The a d d i t i o n o f I p e r c e n t h i s t i d i n e i n e x p e r i m e n t I s i g n i ­
f i c a n t l y im p ro v e d body w e i g h t , l e g s c o r e s and l e n g t h t o w i d t h r a t i o s
(T ab le I V ) .
The t i b i a z i n c c o n t e n t was low .
m ent 2 ( T a b l e IV) showed s l i g h t l y
and l i t t l e
The r e s u l t s fro m e x p e r i ­
im p ro v e d g ro w th and l e g d e f o r m i t i e s
d if f e r e n c e in th e t i b i a - z i n c c o n te n t.
C a se in -G e latin
Growth o f c h i c k s was s i g n i f i c a n t l y i n c r e a s e d ( T a b l e I V ) .
z i n c c o n t e n t was l i t t l e
T ib ia
a f f e c t e d and l e g d e f o r m i t i e s o r l e n g t h t o w i d t h
r a t i o s w ere n o t changed.
Egg Albumen
C h ic k w e i g h t , l e g s c o r e s ,
le n g th to w id th r a t i o s or t i b i a z in c
c o n te n t w ere l i t t l e changed.
Soybean
C h ic k g r o w th o r l a c k o f l e g d e f o r m i t i e s o r t i b i a z i n c c o n t e n t was
little
a lte re d .
23
G lu t a m ic A c id
Sesam e. — L i t t l e d i f f e r e n c e in w e i g h t, le g s c o r e s ,
ra tio s
le n g th to w id th
o r t i b i a - z i n c c o n t e n t was f o u n d .
Z i n c i n C o m b in a tio n and Amino A c id s
Z in c and H i s t i d i n e . — The c o m b i n a t i o n o f I p e r c e n t h i s t i d i n e and
30 m g/kg o f z i n c r e s u l t e d i n s i g n i f i c a n t i n c r e a s e s i n c h i c k w e i g h t , and
in le n g th to w id th r a t i o s
d ecreased ;
(T a b le IV ).
Leg d e f o r m i t i e s w e r e s i g n i f i c a n t l y
t h e s e im p ro v e m e n ts w e r e c o m p a r a b le t o t h o s e r e s u l t i n g fro m
t h e a d d i t i o n o f 60 m g/kg o f z i n c .
A l t h o u g h an i n c r e a s e i n t i b i a z i n c
c o n t e n t was f o u n d , i t was n o t s o g r e a t a s t h a t o f t h o s e c h i c k s g i v e n
60 m g/kg o f z i n c .
ratio s.
L e g s , h o w e v e r , w e r e c o m p a r a b le i n l e n g t h t o w i d t h
.
The a d d i t i o n o f I p e r c e n t h i s t i d i n e p l u s 5 mg/kg o f z i n c s i g n i ­
f i c a n t l y im p ro v e d w e i g h t and l e g s c o r e s .
H ow ever, g r o w th o r t i b i a z i n c
c o n t e n t was n o t s o g r e a t a s t h a t o f c h i c k s g i v e n 10 m g/kg o f z i n c .
The a d d i t i o n o f I p e r c e n t h i s t i d i n e and 10 m g/kg o r 20 mg/kg o f
z i n c d i d n o t s i g n i f i c a n t l y im p ro v e t h e v a r i o u s c r i t e r i a o v e r t h a t w h e re
o n l y z i n c was s u p p l e m e n t e d .
P e r c e n t N i t r o g e n p e r Gram o f Bone ( T a b l e IV)
S e s a m e . — The a d d i t i o n o f I p e r c e n t h i s t i d i n e t o t h e b a s a l d i e t
s i g n i f i c a n t l y im p ro v e d t h e n i t r o g e n c o n t e n t o f c h i c k t i b i a e . ( e x p e r i m e n t
I).
The a d d i t i o n o f z i n c o r z i n c and h i s t i d i n e h a d l i t t l e
e ffe c t.
24
The a d d i t i o n o f g l u t a m i c a c i d o r a s p i r i n t o t h e b a s a l d i e t lo w e r e d
th e n itr o g e n c o n te n t.
The g r e a t e s t i n c r e a s e i n p e r c e n t a g e o f n i t r o g e n
came fro m t h e a d d i t i o n o f 30 mg/kg o f z i n c t o t h e b a s a l d i e t p l u s
a sp irin .
C a s e i n - G e l a t i n . — The a d d i t i o n o f 5 m g/kg z i n c p l u s I p e r c e n t
h i s t i d i n e s i g n i f i c a n t l y in c re a s e d th e n itr o g e n c o n te n t of th e t i b i a e
over th e b a s a l r a t i o n .
Egg A lbum en. — The p e r c e n t n i t r o g e n o f t h e b o n e was l o w e r t h a n t h a t
of th e c h ic k s g iv en o th e r p r o te i n b a s a l r a t i o n s .
The a d d i t i o n s o f
h i s t i d i n e w i t h 10 o r 20 m g/kg o f z i n c w e r e t h e o n ly s u p p l e m e n t s t h a t
made a s i g n i f i c a n t i n c r e a s e i n n i t r o g e n .
S o y b e a n . — The a d d i t i o n o f h i s t i d i n e t o t h e b a s a l d i e t h a d l i t t l e
e f f e c t upon th e n i t r o g e n c o n te n t of th e t i b i a e .
N i t r o g e n C o n t e n t o f C h ic k T i b i a e F r a c t i o n s
( T a b le V)
S a l t S o l u b l e . — The a d d i t i o n o f h i s t i d i n e p l u s 30 m g/kg o f z i n c made
a la rg e s ig n i f ic a n t in c re a s e in th e n itro g e n c o n te n t.
The a d d i t i o n
o f h i s t i d i n e o r 60 m g/kg o f z i n c made s m a l l e r , n o n - s i g n i f i c a n t i n c r e a s e s .
A u t o c l a v a b I e . — No s i g n i f i c a n t c h a n g e s i n m i l l i g r a m s o f n i t r o g e n
w e re f o u n d ; a s i g n i f i c a n t d e c r e a s e i n p e r c e n t a g e o f t o t a l n i t r o g e n
o c c u r r e d w i t h 60 m g/kg o f z i n c o r h i s t i d i n e p l u s 30 m g/kg o f z i n c ,
NaQH. — S i g n i f i c a n t i n c r e a s e s w e r e n o t e d w i t h ' t h e a d d i t i o n o f 60
mg/kg o f z i n c o r h i s t i d i n e p l u s 30 m g/kg o f z i n c .
TABLE V.
N i t r o g e n C o n t e n t Mean o f F r a c t i o n s o f C h ic k T i b i a e Fed S e v e r a l P r o t e i n S o u r c e s S u p p l e ­
m e n te d w i t h Z i n c a n d / o r H i s t i d i n e .
P ro te in
S o u rc e
Added
Z in c
mg/kg
S a l t S o lu b le '
mg
% I/
9 .9 0b C d -j^
o
1
4 . 8 0 ah ■
60
o
■
Sesam e+1%
1 2 .95abc
H i s t i d i n e . J Q •■■ 1 8 . 7 0 a
SESAME.
CASEIN-...
-Q .
GELATIN
c a s - g e l ■ . 10 .0
.
Cas-Gel+I Z H i s t ; ‘ ' '5 '
I/
2/
3/
1 4 .2 5 a
1 4 ; 90a
1 5 .4 0 a
1 7 .2 0 a
• 2 ,4 5 d 4 ■
1 3 . IO ahc ■1 0 .8 5 a
' 3 . 4 0 d ' ’ ‘ ' 1 3 ; 50a
6 .55cd'
1 4 .0 5 a
A u to c la v a b le
mg
% I/
3 2 . 50ab
3 0 . 75ab
3 3 . 20ab '
3 6 .3 0 a b ;
NaOH S o l u b l e
mg
% I/
4 6 . 7 5 a 9 .9 5 b c
3 1 .9 5 b 2 6 . 00a
'4 0 .8 0 * 1 3 .20bc
' 3 2 ; 6 5 b ' 1 8 .9 5 a b
1 4 .3 0 b c
2 6 .4 5 a
1 5 . 45bc
1 7 .1 0 b '
E la stin
% I/
mg
1 7 .2 5 cd e'
2 6 .50bc '
2 3 . 6 5 cd
3 7 .0 5 a b ' '
2 8 . 60b c - 1^
1 2 . 4 5 b c^-/
1 1 .9 0 d e -/
4 6 .9 0 a
3 8 .55ab 1 7 . 90ab ' 1 4 . 75bc 4 2 . 7 5 a '
1 2 .4 5 c ■ ' 4 9 ; 7 5 a ' 3 . 7 0 c ' ' 1 4 . 5 0 b c 6 . IOe
21.7Q bc ' 5 0 ; 7 5 a ' 4 . 5 5 c ' ' ' 1 0 . 4 0 c ' 1 1 . 60de
Sum
mg
2 5 .1 4 a 6 9 . 5 0 b cde
2 6 .7 5 a 9 7 . 75abc
2 8 .4 0 a 8 3 . OOabcd
3 3 . 3 5 a 1 1 1 . OOab
5 5 . 4 0 cde
3 4 . 8 5 a 1 2 0 .6 5 a
2 2 .5 0 a 2 5 . 65e
2 4 .7 5 a 4 4 . 40de
P e r c e n t o f t h e sum o f m i l l i g r a m s o f n i t r o g e n .
P ( < 0 .0 5 ) w i t h D u n c a n 's M u l t i p l e Range T e s t . V a l u e s f o l l o w e d by t h e same l e t t e r s w i t h i n t h e
same column a r e n o t s i g n i f i c a n t l y d i f f e r e n t .
O nly on e a n a l y s i s .
26
E l a s t i n . — S i g n i f i c a n t d i f f e r e n c e s w e re n o t fo u n d i n e i t h e r th e
t o t a l n itr o g e n o f th e f r a c ti o n s o r th e p e rc e n ta g e o f n itr o g e n .
H ow ever,
30 m g/kg o f z i n c and h i s t i d i n e te n d e d t o b e t h e h i g h e s t .
Sum. — W h ile t h e c h a n g e s w e re n o t s i g n i f i c a n t , i n c r e a s e s i n
n i t r o g e n w e re n o t e d when 60 m g/kg o f z i n c , h i s t i d i n e ,
o r h i s t i d i n e p lu s
30 m g/kg w e re a d d e d .
C a s e in -G e la tin . — S ig n if ic a n t in c r e a s e s in n itr o g e n o c c u rre d in
e v e r y f r a c t i o n and th e sum w i t h t h e a d d i t i o n o f 10 m g/kg o f z i n c .
H ow ever, t h e p e r c e n t a g e o f n i t r o g e n w as n o t s i g n i f i c a n t l y d i f f e r e n t
among t h e f r a c t i o n s
N i t r o g e n C o n te n t o f C h ic k Cap F r a c t i o n s
( T a b le VI)
Sesam e
S a l t S o l u b l e . — N e a r ly s i g n i f i c a n t i n c r e a s e s w e re n o t e d when 60
m g/kg o f z i n c o r h i s t i d i n e and 30 m g/kg o f z in c w e re a d d e d t o t h e b a s a l
ra tio n s .
A u t o c l a v a b I e . — No s i g n i f i c a n t c h a n g e s i n n i t r o g e n o c c u r r e d w i t h
th e v a r io u s d i e t tr e a tm e n ts .
NaOH. — The a d d i t i o n o f h i s t i d i n e and h i s t i d i n e p l u s 30 m g/kg o f
z in c to th e b a s a l r a t i o n cau se d s i g n i f i c a n t in c r e a s e s in n itr o g e n .
E l a s t i n . — T h e re was n o t enough r e s i d u e r e m a in in g a f t e r t h e f r a c ­
t i o n a t i o n p r o c e d u r e t o b e a n a ly z e d a s e l a s t i n .
TABLE V I,
N i t r o g e n C o n te n t Mean i n F r a c t i o n s o f C h ic k T i b i a e Caps F ed S e v e r a l P r o t e i n S o u rc e s
S u p p le m e n te d w i t h Z in c a n d / o r H i s t i d i n e ,
P r o t e i n S o u rc e
Added Z in c
m g/kg
SESAME
S a l t S o l u b le
mg
Z I/
o ............ 1 0 .6 0 b c
■
6 0 .............. 1 5 . 70ab
......... o .............. '1 1 . 9 0 b c
Sesam e + IZ
H is tid in e
■ 30 ............ 1 5 .6 5 a b
CASEIN' ‘4 .8 4 d
0
GELATIN ............
io
■■ ■ 1 9 .5 0 a
■■■ o .............. ■ 5 .5 0 d
C a s -G e l + 1% '
5
H is tid in e
7 . 65cd
..
I/
2]
.
.
. . .
^ '
----'
'
■' •
;
A u to c la v a b le
mg
Z I/
2 0 .8 8 a
3 3 :9 5 a
1 8 :3 6 a
5 2 .1 5 a ■
1 5 .6 5 a ' 4 5 .9 0 a "
2 0 .9 5 a ■ 4 7 .7 5 a
1 6 .5 8 a ' 1 9 .5 5 a
1 7 . 6 4 a ' 6 8 .0 5 a
1 5 .7 5 a
5 5 :9 3 a ■
1 7 .9 6 a
2 7 .8 5 a
NaOH S o l u b le
mg
Z I/
6 0 .8 6 a
9 . 30bc
6 1 .9 6 a ■'1 6 . 5 0 a b '
5 9 .6 6 a
1 9 .0 0 a '
5 6 .3 3 a
1 8 .3 5 a
6 6 . 48a
5 .2 5 c ;
6 1 .5 8 a
2 3 .0 0 a
6 7 .6 4 a
5 .5 0 c
6 4 .8 2 a ■ 7 . 30bc
1 7 .4 4 a
1 9 .6 7 a
2 4 .6 9 a
2 2 . 84a
1 6 .9 4 a
2 3 .7 9 a
1 6 .6 3 a
1 7 .2 2 a
P e r c e n t o f t h e sum o f m il li g r a m s o f n i t r o g e n .
P (-?0.05) w i t h D u n c a n 's M u l t i p l e R ange T e s t . V a lu e s f o llo w e d by t h e same l e t t e r s w i t h i n
t h e sam e colum n a r e n o t s i g n i f i c a n t l y d i f f e r e n t .
28
C a s e in -G e la tin
The a d d i t i o n o f 10 m g/kg o f z i n c t o t h e b a s a l r a t i o n i n c r e a s e d t h e
n i t r o g e n c o n t e n t o f c h ic k t i b i a .
little
changes.
The o t h e r d i e t t r e a t m e n t s c a u se d
S i g n i f i c a n t i n c r e a s e s d i d n o t a p p e a r i n p e r c e n ta g e
o f n i t r o g e n o f c h ic k t i b i a e cap f r a c t i o n s .
N i t r o g e n F r a c t i o n s p e r Gram o f Bone and Cap
The n i t r o g e n f o r e a c h f r a c t i o n was c a l c u l a t e d p e r gram o f b o n e ,
n o s i g n i f i c a n t d i f f e r e n c e s w e re fo u n d ( T a b le V I I ) .
No s i g n i f i c a n t d i f f e r e n c e s w e re fo u n d when n i t r o g e n p e r gram o f
cap w e re c a l c u l a t e d
( T a b le V I I ) .
H y d r o x y p r o lin e
Sesam e
T h e re was a te n d e n c y f o r h y d r o x y p r o l in e t o i n c r e a s e i n c e r t a i n
t i b i a an d cap f r a c t i o n s when h i s t i d i n e w as added t o t h e b a s a l sesam e
ra tio n .
H o w ev er, when t h e c a l c u l a t i o n s w e re b a s e d on t h e n i t r o g e n
c o n t e n t p e r w e ig h t o f b o n e o r c a p , t h e d i f f e r e n c e te n d e d t o d i s a p p e a r
( T a b le V I I I ) .
D is c u ss io n
The r e a c t i o n o f c h ic k s t o d i e t s low i n a v a i l a b l e z i n c h a s b e e n
shown t o v a r y .
C h ic k s f e d v e g e t a b l e p r o t e i n
(se sa m e m e a l and i s o l a t e d
29
TABLE V I I .
P ro te in
S o u rc e
SESAME
Sesam e + 1%
H is tid in e
CASEINGELATIN
C a s -G e l +
1% H i s t i d i n e
SESAME
Sesam e + 1%
H is tid in e
CASEINGELATIN
C a s -G e l +
1% H i s t i d i n e
I/
N i t r o g e n C o n te n t Mean p e r Gram o f T i b i a e and Caps o f C h ic k s
Fed S e v e r a l P r o t e i n S o u rc e s S u p p le m e n te d w i t h Z in c a n d /o r
H is tid in e .
B ones
A u t o c la v a b le
S o l u b le
mg
Added
Z in c
m g/kg
S a lt
S o l u b le
mg
0
60
0
30
0
10
0
5
2 .8 6 a I /
2 . 66a
2 .6 8 a
3 .0 0 a
1 .9 5 a
1 .9 0 a
' 1 .7 5 a
2 .0 5 a
9 . 70a
5 .7 0 a
7 .1 0 a
5 . 80a
1 0 .9 0 a
6 . 80a
6 .3 5 a
7»2 5 a
. 42a
.5 3 a
. 44a
. 44a
.3 9 a
»48a
.3 7 a
. 50a
Caps
1 .0 4 a
1 .8 0 a
1 .6 9 a
1 .3 0 a
1 .5 6 a
1 .6 6 a
1 .6 0 a
1 .8 8 a
0
60
0
30
0
10
0
5
NaOH
S o l u b le
mg
E la s tin
mg
2 .7 0 a
4 .7 0 a
2 .6 5 a
2 .1 5 a
1 0 . 60a
2 .6 0 a
1 .8 5 a
1 .5 0 a
. 465a
. 48a
. 495a
. 600a
.7 1 0 a
. 620a
. 300a
. 365a
T o ta l
mg
1 4 .5 5 a
1 3 .3 5 a
1 3 .0 0 a
1 2 .5 9 a
3 3 .3 4 a
1 1 .9 5 a
1 0 .2 5 a
1 1 .2 0 a
-
,3 7 a
.5 8 a
.7 0 a
. 51a
. 40 a
. 56a
. 40a
. 48a
P ( < 0 ,0 5 ) .
V a lu e s f o llo w e d by t h e sam e l e t t e r s w i t h i n t h e same
colum n a r e n o t s i g n i f i c a n t l y d i f f e r e n t .
TABLE V I I I .
H y d r o x y p r o lin e Mean i n F r a c t i o n s o f T i b i a e o r Caps T aken fro m C h ic k s Fed
Sesam e R a t i o n s .
. S a l t S o l u b le
P i o t e i n S o u rc e
Added Z i n c ' '
m g/kg
.
. 'T i b i a '
mg
T o ta l'
mg
6 .1 2 0 .6 1 8 ' 0 .0 0 1 6 '. '9 5 5 .0
0 .6 8 7 0 .0 0 1 8
9 4 2 .5
Ol
CO
SESAME■‘
' .' o ..............
S e sa m e '+ '1% ' H i s t '
' 0 ..............
T o ta l ' N '
mg- .
mg
A u to c la v a b le
_
. .C ap.
■ SESAME
'■
Sesam e +'1% H i s t '
O
1 7 .3 1 .6 3
O.............. '1 9 . 9 ' 1 .6 7
' 0 : 0 0 7 0 - 1415.0' 0 :0 0 7 3 1 7 8 7 .5
NaOH
' 'N ' ' ' T i b i a ' ' T o t a l
mg
mg'
- mg
2 9 : 3 8 ' 0 :2 5 6 ' ' 6 4 :1 2
2 8 .3 8 0 .1 9 8 1 1 4 .9
Cap
4 1 .6 7 ' 0 :3 7 9
'4 5 .0 8
3 8 .9 3 ' '0 .3 7 5 ' 1 0 8 .0
' ' N
mg
T ib ia
mg
6". 44 0 .0 1 7 2
8 .7 0 0 .0 2 4 1
Cap
4 .8 3 0 .0 1 2
5 .6 8 0 .0 2 2 .
31
s o y p r o t e i n ) , e x h i b i t e d p o o r g ro w th , low t i b i a z in c a n d l e g d e f o r m i t i e s
( 9 ,3 9 ) .
C h ic k s f e d s o y b e a n m e a l h a d o n ly a p o o r w e i g h t g a i n .
C h ic k s f e d a n im a l p r o t e i n
itie s
(c a s e in -g e la tin )
and p o o r w e ig h t g a in ( 2 9 ) ,
e x h i b i t e d l e g d e fo rm ­
F e e d in g egg album en r a t i o n s r e s u l t e d
i n p o o r g r o w th , open s o r e s , b l e e d i n g a n d a h i g h m o r t a l i t y r a t e
(3 4 ).
P a r t I - W e ig h t, Leg D e f o r m i ti e s and T i b i a Z in c C o n te n t
B a s a l r a t i o n s . — I n t h i s s t u d y , g ro w th o f c h ic k s on p l a n t p r o t e i n s
low i n a v a i l a b l e z i n c (se sa m e ) o r low i n z in c (so y b e a n ) was p o o r , b u t
n o t as
p o o r a s on r a t i o n s low i n z in c c o n t a i n i n g a n im a l p r o t e i n
g e l a t i n and e g g a lb u m e n ) .
p l a n t an d a n im a l p r o t e i n s
(c a s e in -
Leg d e f o r m i t i e s , h o w e v e r, o c c u r r e d w i t h b o t h
( c a s e i n - g e l a t i n and sesam e) w h i l e c h ic k s f e d
egg album en and s o y b e a n d e v e lo p e d few l e g d e f o r m i t i e s .
C h ic k s fe d
egg album en r a t i o n s d i d n o t d e v e lo p open s o r e s b u t d i d h a v e a h ig h
m o rta lity r a te .
A d d i t io n o f h i s t i d i n e . — Leg d e f o r m i t i e s a r e ju d g e d b o t h on th e
s h o r t n e s s o f t h e . t i b i a a n d . t h e d e g r e e o f d e f o r m ity o f t h e j o i n t s .
H e a v ie r c h ic k s h a v e m ore p r e s s u r e on t h e i r t i b i a .
I f a l e g d e f o r m ity
w e re t o o c c u r , i t m ig h t b e m ore s e v e r e b e c a u s e o f th e g r e a t e r ad d ed
w e ig h t.
C h ic k s g iv e n se sa m e r a t i o n s show ed b o th ty p e s o f d e f o r m i t i e s ,
a s shown by t h e d e c r e a s e d l e n g t h and w i d t h r a t i o s and l e g s c o r e s .
F e e d in g c a s e i n - g e l a t i n r e s u l t e d i n o n ly s e v e r e j o i n t d e f o r m i t i e s s i n c e
l e n g t h t o w id th r a t i o was n o t d e c r e a s e d fro m when z i n c w as added
( T a b le I V ) .
When I p e r c e n t h i s t i d i n e was a d d e d t o t h e b a s a l sesam e
32
r a t i o n , le g d e fo rm itie s
im p ro v e d .
( b o th t y p e s ) w e re p r e v e n t e d a n d . g ro w th som ew hat
The a d d i t i o n o f I p e r c e n t h i s t i d i n e t o c a s e i n - g e l a t i n d id
n o t p r e v e n t d e f o r m i t i e s o r g r e a t l y i n c r e a s e g ro w th .
p ro m o te g ro w th i n l e g s ,
H i s t i d i n e c o u ld
le n g th w is e , and d e c re a s e j o i n t d e f o r m itie s .
The r e a s o n why h i s t i d i n e d id n o t im p ro v e l e g d e f o r m i t i e s r e s u l t i n g fro m
f e e d i n g lo w - z i n c c a s e i n - g e l a t i n r a t i o n s i s unknown.
had l i t t l e
H is tid in e a d d itio n s
e f f e c t on g ro w th o f c h ic k s f e d egg album en and so y b e a n
ra tio n s ,
A d d itio n o f g lu ta m ic a c i d . — G lu ta m ic a c i d , a m e t a b o l i t e o f
h i s t i d i n e , when ad d ed t o b a s a l se sa m e r a t i o n s , d id n o t a c t a s h i s t i d i n e .
Leg d e f o r m i t i e s w e re s t i l l s e v e r e b u t w e ig h t and t i b i a z i n c c o n te n t
was n o t i n c r e a s e d .
A p p a r e n tly h i s t i d i n e e x e r t e d i t s
e f f e c t b e fo re i t
was c a t a b o l i z e d t o g lu ta m ic a c i d .
A n o th e r m e t a b o l i c p ath w ay f o r h i s t i d i n e i s by d e c a r b o x y l a t i o n t o
h ista m in e .
H is ta m in e was s u c c e s s f u l a t a much lo w e r l e v e l (0,1% ) th a n
h i s t i d i n e when u s e d a s a s u p p le m e n t f o r t h e p r e v e n t i o n o f l e g d e fo rm ­
itie s
( 4 ) ; i t c o u ld b e t h a t t h e e f f e c t o f h i s t i d i n e i s o c c u r r i n g
th r o u g h m e t a b o l i c c o n v e r s io n t o h i s t a m i n e .
A d d itio n o f z i n c . — I t was e x p e c te d t h a t a d d i t i o n o f z in c t o th e
b a s a l r a t i o n s w o u ld i n c r e a s e g ro w th an d t i b i a z in c c o n t e n t ; h o w e v e r,
t h i s was n o t u n if o r m ly t h e c a s e .
I n c r e a s e d g ro w th and t i b i a z in c d id
o c c u r f o r c h ic k s f e d b a s a l se sa m e o r c a s e i n - g e l a t i n d i e t s when t h e
f u l l r e q u i r e m e n t o f z i n c , 60 m g/kg o r 10 m g /k g , r e s p e c t i v e l y , was
33
added.
O n e - h a lf t h e z i n c ' r e q u ir e m e n t was ad d ed w i t h h i s t i d i n e t o
d e te r m in e i t s p o s s i b l e s y n e r g i s t i c e f f e c t .
The r e s u l t s
fro m f e e d i n g
h i s t i d i n e and 30 m g/kg z i n c t o sesam e w e re a s g r e a t a s f e e d i n g 60 m g/kg
z in c o r h i s t i d i n e a lo n e ,
No s y n e r g i s t i c e f f e c t was n o t e d w i t h c a s e i n -
g e l a t i n s i n c e 5 m g/kg z in c and h i s t i d i n e d i d n o t e q u a l t h e r e s u l t s fro m
f e e d i n g 10 m g/kg z i n c .
The a d d i t i o n o f z i n c t o egg album en r a t i o n s a l s o r e s u l t e d i n
i n c r e a s e d body w e ig h t a n d t i b i a z in c c o n t e n t .
A p p a r e n tl y , t h e a d d i t i o n
o f 10 m g/kg was s u f f i c i e n t t o m eet t h e z i n c r e q u i r e m e n t.
When 20 m g/kg
o f z in c was a d d e d , no f u r t h e r i n c r e a s e i n g ro w th r e s u l t e d b u t z in c was
a b s o rb e d a s shown by i n c r e a s e d t i b i a z i n c c o n t e n t .
No s y n e r g i s t i c -
e f f e c t s o f a d d e d h i s t i d i n e w e re n o t e d i n t h e w e ig h t g a in o f c h ic k s f e d
egg a lb u m e n .
A d d i t io n o f a s p i r i n . — A s p i r i n i s w id e ly u s e d a s a p a i n a l l e v i a t o r
i n human a r t h r i t i s .
A s p i r i n was u s e d ,
th e r e f o r e , b e c a u se th e le g
d e f o r m i t i e s fo u n d w i t h se sa m e r a t i o n s w e re s i m i l a r i n p a r t t o th o s e
o f human a r t h r i t i s .
ra tio n s .
A s p i r i n d e p r e s s e d g ro w th o f c h ic k s , f e d sesam e
I n c o n t r a s t t o t h e o t h e r r a t i o n s s u p p le m e n te d w i t h z i n c , 30
m g/kg z i n c d id n o t r e l i e v e t h i s d e p r e s s i o n .
The s h o r t t i b i a d e f o r m ity
o f c h ic k s d id n o t a p p e a r , b u t i t i s q u e s t i o n a b l e w h e th e r t h e j o i n t
d e f o r m it y was r e a l l y p r e v e n t e d s i n c e t h e c h ic k s d id n o t grow .
O th e r w o r k e rs (3 4 ) fo u n d few l e g d e f o r m i t i e s i n c h ic k s g iv e n a
b a s a l i s o l a t e d so y p r o t e i n d i e t p l u s a s p i r i n .
p e r f o r a t e d g i z z a r d s w e re f o u n d .
G row th was p o o r and
W h ile t h e c h ic k s g iv e n s e sa m e , p l u s
34
a s p i r i n o r a s p i r i n p l u s 30 m g/kg z i n c d i d n o t h a v e p e r f o r a t e d g i z z a r d s ,
th e y a l s o d i d n o t grow .
The t i b i a z i n c c o n t e n t was n o t i n c r e a s e d e v e n
w i t h t h e a d d i t i o n o f 30 m g /k g .
A p p a r e n tl y , sesam e an d a s p i r i n i n ■com bin
a t i o n k e p t ad d ed z i n c fro m b e in g d e p o s i t e d i n t h e t i b i a .
L ack o f
g ro w th w as n o t t h e c o n t r o l l i n g f a c t o r , h e r e , b e c a u s e c h ic k s g iv e n egg
alb u m e n r a t i o n s d i d n o t grow b u t t h e s u p p le m e n ta l z in c was r e f l e c t e d
i n i n c r e a s e d t i b i a z in c c o n t e n t .
P a r t I I - T o t a l N i t r o g e n and N i t r o g e n o f T i b i a F r a c t i o n s
T o t a l n i t r o g e n .'— W h ile a s p i r i n may h a v e b e e n i n h i b i t i n g z in c u p ­
ta k e ,
t h e n i t r o g e n c o n t e n t o f t h e t i b i a was n o t c o m p a ra b ly a f f e c t e d .
The t i b i a n i t r o g e n c o n t e n t o f c h ic k s f e d t h e b a s a l se sa m e p l u s a s p i r i n
r a t i o n h a d a lo w e r t i b i a n i t r o g e n th a n c h ic k s g iv e n c a s e i n - g e l a t i n
who grew p o o r l y .
When 30 m g/kg z in c w as a d d e d , t h e t i b i a n i t r o g e n
i n c r e a s e d s i g n i f i c a n t l y , a lm o s t t o t h a t o f th e o t h e r se sa m e d i e t s .
T h is i n c r e a s e was n o t d u e t o an i n c r e a s e i n l e g s i z e s i n c e t h e l e g
s i z e s o f c h ic k s f e d n o a d d ed z in c and a s p i r i n w e re c o m p a ra b le t o th o s e
f e d 30 m g/kg ad d ed z i n c ( 1 .0 v s . 0 .9 gm) .
S in c e a d i f f e r e n c e i n
g ro w th d u e t o z i n c s u p p le m e n ta ti o n d i d n o t o c c u r , t h e i n c r e a s e i n
n i t r o g e n c o n t e n t may b e due s p e c i f i c a l l y
to changes i n bone s t r u c t u r e
r a t h e r th a n a g e n e r a l i n c r e a s e i n body n i t r o g e n .
I n c o n s t r a s t , added, z in c in ' eg g album en r a t i o n s was r e f l e c t e d
i n i n c r e a s e d t i b i a z i n c c o n t e n t , b u t t i b i a n i t r o g e n re m a in e d low .
It
35 .
a p p e a rs t h a t t i b i a z in c u p ta k e h as l i t t l e
e f f e c t on t o t a l n i t r o g e n
o r v ic e v e rs a ,
■
The a d d i t i o n o f h i s t i d i n e d id n o t a lw a y s r e s u l t i n an i n c r e a s e i n
t o t a l n itro g e n .
O nly i n c h ic k s f e d se sa m e p l u s h i s t i d i n e d id t h e t i b i a
n itro g e n in c r e a s e .
When h i s t i d i n e was a d d e d t o c a s e i n - g e l a t i n , egg
album en o r s o y b e a n r a t i o n s , n o s i g n i f i c a n t c h a n g e s i n t i b i a n i t r o g e n
re s u lte d .
G lu ta m ic a c i d i n s e sa m e , a l s o , d i d n o t c a u s e an i n c r e a s e . i n t i b i a
n itr o g e n c o n te n t.
T i b i a n i t r o g e n w a s , i n f a c t , much l o w e r , a lth o u g h
t h e l e g w e ig h ts w e re c o m p a ra b le t o t h e b a s a l r a t i o n
( 2 ,2 v s , 2 .3 gm) .
N i t r o g e n o f f r a c t i o n s . — When c h ic k t i b i a w e re f r a c t i o n a t e d , any
i n c r e a s e s i n n i t r o g e n som ew hat r e f l e c t e d d e c r e a s e s i n o c c u r r e n c e o f
le g d e fo rm itie s .
The a d d i t i o n o f 60 m g/kg o r h i s t i d i n e p l u s 30 m g/kg
z in c t o se sa m e s i g n i f i c a n t l y d e c r e a s e d 'l e g d e f o r m i t i e s .
S lig h t
i n c r e a s e s i n th e n i t r o g e n o f t h e t h r e e f r a c t i o n s o c c u r r e d w ith th e
a d d itio n o f z in c o r h i s t i d i n e .
L a r g e r i n c r e a s e s o c c u r r e d w ith h i s t i d i n e
p lu s z in c , r e f l e c t i n g th e d e c re a s e in le g d e f o r m itie s .
changes in a u to c la v a b le f r a c tio n s
No s i g n i f i c a n t
(se sa m e ) w e re fo u n d , b u t t h i s may
b e b e c a u s e e a c h f r a c t i o n e x t r a c t e d c o n ta i n e d d i f f e r e n t am ounts o f
s o lu b le p r o te in .
S a l t s o l u b l e f r a c t i o n s c o n ta i n t h e m o st r e c e n t l y
fo rm e d c o l l a g e n .
A u to c la v in g i n v o lv e d m e l ti n g t h e ,s e c o n d a r y s t r u c ­
t u r e o f c o l l a g e n r e s u l t i n g i n g e l a t i n , and NaOH e x t r a c t s t h e o l d e r
c o lla g e n f i b r i l s .
36
Leg d e f o r m i t i e s d id n o t 'a p p e a r u n t i l t h e s e c o n d w eek o f c h ic k
g r o w th .
C h ic k s w e re t a k e n a t t h r e e w e e k s .
B e c a u se o f t h i s , i t was
assum ed t h a t th e n e u t r a l s a l t s o l u b l e f r a c t i o n o r a u t o c l a v a b l e w o u ld
show d i f f e r e n c e s i n am ount o f c o l l a g e n i f l e g d e f o r m i t i e s made a .
d iffe re n c e .
T h i s , h o w e v e r, d id n o t h a p p e n ,
■ S lig h t i n c r e a s e s i n t i b i a n i t r o g e n o f t h e s a l t s o l u b l e , NaOH
s o l u b l e and e l a s t i n
f r a c tio n s o c c u rre d w ith th e a d d itio n s of h i s t i d i n e
t o se sa m e r a t i o n s .
D i f f e r e n c e s i n h y d r o x y p r o l in e a l s o o c c u r r e d w ith
s a l t . s o l u b l e an d NaOH s o l u b l e f r a c t i o n s .
H ow ever, when m i l li g r a m s o f
n i t r o g e n o r m ic ro g ra m s o f h y d r o x y p r o l in e w e re c a l c u l a t e d p e r gram o f
b o n e, th e s e d if f e r e n c e s d is a p p e a re d .
I t c o u ld b e t h a t t h e l o s s o f 25
p e r c e n t n i t r o g e n d u r in g f r a c t i o n a t i o n was enough t o d e s t r o y d i f f e r e n c e s
o r t h e d i f f e r e n c e s w e re s p r e a d among t h e f r a c t i o n s a n d d id n o t a p p e a r .
The a d d i t i o n o f h i s t i d i n e o r h i s t i d i n e a n d z in c on t h e o t h e r hand t o
c a s e i n - g e l a t i n r a t i o n s d id n o t i n c r e a s e n i t r o g e n o f b o n e o r f r a c t i o n s .
The r e s u l t s fro m t h e se sa m e i n d i c a t e t h a t s h a r p e r d i f f e r e n c e s
m ig h t b e o b t a i n e d w i t h m ore e x p e r i e n c e i n f r a c t i o n a t i o n m ethods and
m ore s o p h i s t i c a t e d e q u ip m e n t.
V.
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
Summary
F o u r b a s a l p r o t e i n r a t i o n s , e i t h e r low i n a v a i l a b l e z in c (se sa m e
m ea l) o r low i n z in c ( c a s e i n - g e l a t i n , e g g album en a n d s o y b e a n m e a l) ,
w e re f e d t o d a y - o l d c h ic k s f o r t h r e e w e e k s .
S u p p le m e n ts o f z i n c ,
h i s t i d i n e , g l u ta m i c a c i d o r a s p i r i n , o r a c o m b in a tio n o f z in c and
h is tid in e ,
o r . a s p i r i n w e re f e d .
C h ic k w e i g h t s , l e g d e f o r m i t i e s
( i n d i c a t e d by l e g s c o r e a n d l e n g t h t o w id th r a t i o ) , and t i b i a z in c
and n i t r o g e n c o n t e n t w e re d e te r m in e d .
T i b i a cof c h ic k s g iv e n s e sa m e ,
0 and 60 m g/kg z i n c an d h i s t i d i n e , o r h i s t i d i n e , 30 m g/kg z i n c ;
c a s e i n - g e l a t i n , 0 and 10 m g/kg z i n c and h i s t i d i n e o r h i s t i d i n e , 5 m g/kg
z i n c r a t i o n s w e re s e p a r a t e d i n t o f o u r f r a c t i o n s on t h e b a s i s o f
p r o b a b l e tim e o f d e p o s i t i o n o f c o l l a g e n .
was m e a s u re d .
The n i t r o g e n o f e a c h f r a c t i o n
The h y d r o x y p r o l in e c o n t e n t o f th e t i b i a o f c h ic k s g iv e n
sesam e r a t i o n s w i t h and w i t h o u t h i s t i d i n e was d e te r m in e d a s a m e a su re
o f c o lla g e n .
G row th of. c h ic k s f e d t h e b a s a l p l a n t p r o t e i n r a t i o n s was b e t t e r
th a n c h ic k s f e d b a s a l a n im a l p r o t e i n r a t i o n s .
A d d i t io n s o f 60 m g/kg
z i n c t o s e s a m e , 10 m g/kg z i n c t o c a s e i n - g e l a t i n and 10 m g/kg z in c t o
egg alb um en r a t i o n s w as s u f f i c i e n t t o p r e v e n t z in c d e f i c i e n c y sym ptom s.
A d d itio n , o f I p e r c e n t h i s t i d i n e t o t h e b a s a l se sa m e r a t i o n
d e c re a s e d le g d e f o r m itie s b u t had l i t t l e
e f f e c t . o n g r o w th ,
A d d itio n
o f I p e r c e n t g l u ta m i c a c i d d i d n o t p r e v e n t z in c d e f i c i e n c y sym ptom s.
38
One p e r c e n t h i s t i d i n e a d d e d t o c a s e i n - g e l a t i n r a t i o n s h a d l i t t l e
e ffe c t
on e i t h e r c r i t e r i a .
S h o r t t i b i a d e f o r m it y o f c h ic k s f e d ‘se sa m e r a t i o n s was a l l e v i a t e d
w i t h I p e r c e n t a s p i r i n , b u t g ro w th o f c h ic k s .w a s d e p r e s s e d .
A d d i t io n o f I p e r c e n t h i s t i d i n e t o sesam e r a t i o n s r e s u l t e d i n
a l a r g e s i g n i f i c a n t i n c r e a s e i n t o t a l t i b i a n i t r o g e n c o n t e n t , concom­
i t a n t w i t h d e c r e a s e s i n l e g d e f o r m i t i e s , b u t g ro w th was o n ly m o d e r a te .
Z in c o r z i n c and h i s t i d i n e a d d i t i o n s s i g n i f i c a n t l y im p ro v e d g ro w th and
le g d e f o r m itie s b u t n o t t i b i a n itr o g e n c o n te n t.
T ib ia n itro g e n of
c a s e i n - g e l a t i n f r a c t i o n s w e re i n c r e a s e d o n ly w i t h 10 m g/kg z in c .
The a d d i t i o n o f h i s t i d i n e t o t h e b a s a l sesam e r a t i o n s a l s o r e s u l t e d
in in c r e a s e s in h y d ro x y p ro lin e .
H o w ev er, b o t h t h e i n c r e a s e s i n t i b i a
n i t r o g e n o f f r a c t i o n s an d h y d r o x y p r o l in e d i s a p p e a r e d when v a lu e s w e re
c a l c u l a t e d p e r gram o f b o n e o r n i t r o g e n .
C o n c lu s io n s
Sesam e a n d c a s e i n - g e l a t i n r a t i o n s f e d t o c h ic k s r e s u l t e d i n p o o r
g r o w th , l e g d e f o r m i t i e s an d low t i b i a z i n c c o n t e n t .
W ith so y b e a n and
egg album en r a t i o n s , p o o r g ro w th and low t i b i a z in c c o n t e n t , b u t few.
l e g d e f o r m i t i e s w e re o b t a i n e d .
A d d i t io n o f 60 m g/kg z i n c t o sesam e
r a t i o n s p r e v e n t e d l e g d e f o r m i t i e s an d g r e a t l y i n c r e a s e d gro w th , and
t i b i a z in c c o n te n t.
The r e s u l t s fro m f e e d i n g h i s t i d i n e and 30 m g/kg z i n c
t o se sa m e r a t i o n s w e re a s g r e a t a s f e e d i n g 60 m g/kg z i n c .
A lth o u g h
10 m g/kg z i n c a d d ed t o c a s e i n - g e l a t i n an d egg album en r a t i o n s
39
p r e v e n t e d z i n c d e f i c i e n c y sym ptom s, no. s y n e r g i s t i c e f f e c t w i t h h i s t i d i n e
and o n e - h a l f t h e z i n c r e q u ir e m e n t o c c u r r e d .
h is tid in e ,
little
A d d i t io n o f I p e r c e n t
a l o n e , t o sesam e r a t i o n s d e c r e a s e d l e g d e f o r m i t i e s , b u t h a d
e f f e c t on c h ic k s f e d c a s e i n - g e l a t i n , egg album en and so y b e a n
ra tio n s .
G lu ta m ic a c i d , a m e t a b o l i t e o f h i s t i d i n e , d i d n o t p r e v e n t
z i n c d e f i c i e n c y sym ptom s.
One p e r c e n t a s p i r i n , ad d ed t o sesam e r a t i o n s
d e p r e s s e d g ro w th b u t t h e s h o r t t i b i a d e f o r m it y d id n o t a p p e a r ; th e
a d d i t i o n o f 30 m g/kg z i n c d i d n o t h e l p .
T o t a l n i t r o g e n was o n ly s i g n i f i c a n t l y i n c r e a s e d w i t h t h e a d d i t i o n
o f I p e r c e n t h i s t i d i n e t o sesam e r a t i o n s .
N i t r o g e n o f f r a c t i o n s was
i n c r e a s e d w i t h h i s t i d i n e , z i n c o r z i n c and h i s t i d i n e a d d i t i o n s t o se sa m e
and 10 m g/kg z i n c ad d e d t o c a s e i n - g e l a t i n r a t i o n s .
One p e r c e n t h i s t i d i n e
a d d e d t o se sa m e r a t i o n s i n c r e a s e d h y d r o x y p r o l in e c o n t e n t o f c h ic k t i b i a
fra c tio n s .
R ecom m endations f o r F u r t h e r R e s e a rc h
F u r t h e r r e s e a r c h i s recom m ended t o a n a ly z e t h e s a m p le s ta k e n
fro m c h ic k s f e d se sa m e a s p i r i n r a t i o n s s i n c e a l a r g e i n c r e a s e i n t o t a l
n i t r o g e n r e s u l t e d w i t h t h e a d d i t i o n o f 30 m g/kg z i n c t o t h e b a s a l
se sa m e a s p i r i n r a t i o n s and i t i s unknown w h a t c a u s e d t h e i n c r e a s e .
More r e s e a r c h on h y d r o x y p r o l in e c o n t e n t o f c o ll a g e n f r a c t i o n s i s
recom m ended t o d e te r m in e i f t h e i n c r e a s e s i n n i t r o g e n a r e c a u s e d by
changes in c o lla g e n .
40
. APPENDIX A .
METHODS OF ANALYSES
41
FLOW SHEET FOR PREPARATION OF TIBIAE FOR ANALYSES
LEG SCORES
PREPARATION OF TIBIAE
LENGTH - WIDTH RATIO
ZINC - T h re e r i g h t t i b i a e
ASH
T h re e l e f t t i b i a e - NITROGEN
KJELDAHL
T h re e o t h e r l e f t t i b i a e - COLLAGEN ANALYSIS
I
FRACTIONATION
HYDROXYPROLINE ON A, B, C
42
.
LEG SCORES
C h ic k t i b i a e w e re s c o r e d w i t h t h e f o ll o w i n g d e s i g n a t i o n s :
0 - n o rm a l l e g d e v e lo p m e n t;
1 - s l i g h t d e f o r m it y i n one l e g ;
2 -
s l i g h t d e f o r m ity i n b o th l e g s ;
3 - s e v e r e d e f o r m ity i n one l e g ; and
4 - s e v e r e d e f o r m ity i n b o t h l e g s .
The d e g r e e o f l e g d e f o r m ity i s d e te r m in e d by t h e s h o r t n e s s o f t h e
t i b i a a n d t h e k n o b s on t h e t i b i a j o i n t ,
s c o r e s was c o n s id e r e d s i g n i f i c a n t .
A d i f f e r e n c e o f one i n l e g
43
PREPARATION OF TIBIAE
1.
T h re e c h ic k s w e re ran d o m ly s e l e c t e d fro m e a c h c a g e .
The l e f t
t i b i a w e re u s e d f o r n i t r o g e n a n a l y s i s , t h e r i g h t f o r z i n c .
Z in c and N i t r o g e n
2.
The e x tr a n e o u s f l e s h was rem oved a n d a f t e r d i p p in g i n b o i l i n g
d e i o n i z e d w a t e r f o r I m in u te , a l l e x c e s s t i s s u e , i n c l u d i n g th e
e p ip h y s e a l p l a t e c a r t i l a g e
3.
( c a p s ) rem oved.
The t h r e e b o n e s fro m e a c h c a g e w e re w ra p p e d t o g e t h e r i n p a p e r
t o w e li n g , c r u s h e d and s e c u r e l y t i e d ; e x t r a c t i o n i n a s o x y l e t
e x t r a c t o r f o r 20 h o u r s w i t h 95% a l c o h o l , th e n f o r 20 h o u r s w i t h
e t h y l e t h e r f o llo w e d .
4.
The e x t r a c t e d b o n e s w e re d r i e d a t a c o n s t a n t t e m p e r a t u r e o f 5 0 ° ,
c o o le d i n d e s i c c a t o r s and w e ig h e d .
44
LENGTH AND' WIDTH RATIO
1,
A l l t h e t i b i a e , im m e d ia te ly a f t e r c l e a n i n g ( s t e p .2, p g . 42) w e re
i n d i v i d u a l l y m e a s u re d w i t h c a l i p e r s f o r l e n g t h an d w i d t h ,
2.
The r a t i o o f l e n g t h t o w id th f o r t h e b o n e was d e te r m in e d by
d i v i d i n g t h e l e n g t h by t h e w id th .
45
ANALYSIS FOR BONE ZINC
I-
S in c e th e b o n e i s
t h e m ain s t o r a g e o rg a n f o r z i n c , t h e a b i l i t y o f
c h ic k s t o t a k e up z i n c on v a r i o u s r a t i o n s i s r e f l e c t e d i n t i b i a e
z in c c o n t e n t .
2.
The e x t r a c t e d b o n e s
( r i g h t t i b i a , s t e p 4 , p g . 43) w e re a s h e d a t
5 5 0 ° C f o r 24 h o u r s , c o o le d i n d e s i c c a t o r s and w e ig h e d ,
3.
The a s h , d i s s o l v e d i n 0 .1 N HCl was a n a ly z e d f o r z in c by an
a to m ic a b s o r p t i o n m e th o d .
A P e r k in - E lm e r 29O-B S p e c tr o p h o to m e te r
was u s e d ; t h e w ave l e n g t h was 2138 A; a s f u e l , t h e o x i d a n t was
a ir .,
M e t a l l i c z i n c was d i s s o l v e d i n 0 .1 HCl f o r t h e s t a n d a r d .
The c o n c e n t r a t i o n was v a r i e d fro m 0 t o 5 ppm o v e r 0 t o 100%
tra n s m is s io n .
46
NITROGEN ANALYSIS
B o n es— M ic ro K j e l d a h l M ethod
D ig e s tio n :
Io
The f a t e x t r a c t e d l e f t t i b i a e
( s t e p 4 , p . 43) w e re g ro u n d i n a
M ic ro W ile y M i l l t o a f i n e p o w d e r.
2.
An a l i q u o t o f p o w d ered b o n e ( 0 .1 0 - 0 .3 5 gm) was d i g e s t e d w i t h 6 ml
o f c o n c e n t r a t e d E^SO^ and 3 -4 H e n g a r c r y s t a l s u n t i l t h e s o l u t i o n
was c l e a r ,
3.
A f t e r b o i l i n g 1 5 -2 0 m in u te s l o n g e r , t h e s o l u t i o n w as c o o le d and
2 -3 ml o f d e io n i z e d w a t e r was a d d e d , m ixed th o r o u g h ly and a llo w e d
to c o o l.
D is tilla tio n :
4.
I n t r o d u c e c o o le d s a m p le th ro u g h f u n n e l o f K i r k - t y p e M ic ro K j e l d a h l ■
d is tillin g
5.
a p p a ra tu s .
S lo w ly add 10 m l o f 30% NaOH,
A pply c u r r e n t s lo w ly and b r i n g w a t e r t o b o i l .
M a in t a in h e a t s o
w a te r b o i ls s t e a d i l y w ith o u t tu rb u le n c e .
6.
15 m l o f d i s t i l l a g e i s c o l l e c t e d i n 20 ml o f s a t u r a t e b o r i c a c i d .
7.
Add 2 -3 i drops,, m ixed i n d i c a t o r I / t o t h e s o l u t i o n w h ic h tu r n s
v i o l e t on c o n t a c t w i t h NH, OH.
4
8.
T i t r a t e sa m p le w i t h 0.2N HCl t o g r a y e n d p o in t o r u n t i l I m ore d ro p
tu rn s
I/
s o lu tio n a f a i n t v io le t.
The i n d i c a t o r was 0 .2 m e th y l r e d (2 m l)
: 0 ,2 m e th y le n e b l u e ( I m l) .
47
COLLAGEN FRACTIONATION'
The g e n e r a l m eth o d f o r f r a c t i o n a t i o n was t h a t o f O 'D e l l ,: ejt„ a l . ,
f o r d e te r m i n in g c o l l a g e n c o n t e n t o f a o r t i c t i s s u e fro m c o p p e r d e f i c i e n t
c h ic k s .
The l e f t o f t h r e e o t h e r c h ic k s w e re ta k e n f o r c o l l a g e n a n a l y s i s .
The t i b i a e w e re s e p a r a t e d fro m t h e e p i p h y s e a l p l a t e c a r t i l a g e
w ith a k n i f e .
(cap s)
The e x c e s s t i s s u e was rem oved fro m b o t h c a p s and b o n e s .
A.
S a l t S o l u b le
I.
A f t e r c r u s h i n g t h e t h r e e t i b i a e o r t h e s^.x c a p s , th e y w e re homog­
e n iz e d i n 15 ml o f I M NaCI , u s i n g a V ir tis h o m o g e n iz e r , f o r
a p p r o x im a te ly 5 m in u te s o r u n t i l a u n ifo r m s u s p e n s i o n was o b t a i n e d .
2;
The h o m o g en a te was r i n s e d i n t o a 250 m l b e a k e r w i t h 10 ml o f I M
N a C l.
3.
T he com bined h o m o g en a te a n d r i s e w e re s t i r r e d w i t h m ix e r s i n th e
b e a k e r s i n an i c e b a t h f o r 8 h o u r s .
4.
The r e s u l t i n g s u s p e n s i o n was c e n t r i f u g e d a t 5 ,0 0 0 X g f o r I h o u r ,
a f t e r w h ic h t h e s u p e r n a t a n t w as d e c a n te d i n t o an e v a p o r a t i n g d i s h .
5.
The r e s i d u e w as w ash ed tw ic e w i t h 5 m l o f w a t e r , c e n t r i f u g e d and
t h e w a s h in g co m b in ed w i t h t h e s u p e r n a t a n t from s t e p 4 .
6.
The s u p e r n a t a n t and w a s h in g s w e re d r i e d by e v a p o r a t i o n a t room
t e m p e r a t u r e f o r 3 6 -4 8 h o u r s .
7.
The d r i e d s u p e r n a t a n t was r e h y d r a t e d t o a volum e o f 10 m l and
d e s ig n a te d th e s a l t s o lu b le f r a c t i o n .
48
B.
8.
C o lla g e n S o l u b le a f t e r A u to c la v in g
The c e n t r i f u g e d r e s i d u e , s t e p 5 , was e x t r a c t e d t w i c e by s h a k in g
w i t h 5 m l o f 2 : 1 c h lo r o f o r m m e th a n o l, c e n t r i f u g e d , an d t h e s u p e r ­
n a t a n t s w e re d e c a n te d and d i s c a r d e d .
9.
The r e s i d u e was r e h y d r a t e d w i t h 5 m l o f w a t e r , a u t o c l a v e d f o r 3
h o u r s a t 1 2 0 ° C, c e n t r i f u g e d a t 5000 X g f o r 10 m in u te s and th e
s u p e r n a t a n t d e c a n te d .
10.
S te p 9 was r e p r e a t e d w i t h 4 m l o f w a t e r , and t h e s u p e r n a t a n t s
co m b in ed .
11.
The r e s i d u e w as w ash ed w i t h I m l o f w a t e r ; t h e w a s h in g s com bined
w i t h t h e s u p e r n a t a n t s and made t o a volum e o f 10 m l w i t h w a t e r .
C.
12.
Sodium H y d ro x id e S o l u b le C o lla g e n
.
The r e s i d u e , s t e p 1 1 , was h e a t e d i n a IOO0C w a t e r b a t h f o r I h o u r
w i t h 5 m l o f 0 .1 NaOH.
13.
A f t e r c e n t r i f u g i n g an d d e c a n t i n g , t h e p r o c e d u r e was r e p e a t e d f o r
15 m i n u t e s .
14.
The r e s i d u e was w ash ed th o r o u g h ly w i t h 5 ml o f w a t e r t o rem ove
so d iu m h y d r o x id e .
15.
The e x t r a c t s and w a s h in g s w e re com bined and made t o a volum e o f
15 m l.
D . . E la s tin
16.
The r e s i d u e , c o n s id e r e d t o b e e l a s t i n , was a i r - d r i e d and g ro u n d
by m o r t a r and p e s t l e t o a f i n e p o w d e r.
49
NITROGEN ANALYSES ON COLLAGEN FRACTIONS
A l i q u o ts o f e a c h f r a c t i o n a s f o l l o w s , w e re u s e d f o r n i t r o g e n
d e te rm in a tio n .
D ig e s tio n :
Io
4 ml s a l t s o lu b le ■
or
2 ml a u to c la v a b le
or
3 m l NaOH s o l u b l e
or
, 1 0 0 0 - o3000 gm e l a s t i n
2.
Add t h e sam e am ount o f c o n c e n t r a t e d H^SO^ a s t h e m l o f sa m p le
(6 m l f o r e l a s t i n )
and 3 -4 H e n g a r c r y s t a l s .
3.
Same a s s t e p 3 u n d e r b o n e n i t r o g e n , p . 46.
4.
C ool t o room te m p e r a t u r e and add same am ount o f d e i o n i z e d w a t e r a s
th e re i s a c id .
C o o l.
D is tilla tio n :
5.
I n th e d i s t i l l a t i o n p r o c e d u r e ( s t e p 5 - 9 , p . 4 6 ) , 0 .0 2 N HCl was u s e d
<
50
HYDROXYPROLINE
H y d ro ly sis
Io
O n e - h a lf ml o f e a c h f r a c t i o n fro m p a g e 48 e x c e p t e l a s t i n , w ere
com bined w i t h 0 ,5 m l c o n c e n t r a t e d HCl i n C a r iu s t u b e s ,
2,
A vacuum was c r e a t e d i n t h e tu b e s u s i n g a vacuum pum p,
W h ile
h o l d i n g t h e vacuum on t h e t u b e s , t h e tu b e s w e re s e a l e d o f f w ith
a to rc h .
3.
The tu b e s w e re p l a c e d i n an oven f o r 24 h o u r s a t 100°C ,
C o lo rim e tric T e st
1,
The h y d r o l y s i s tu b e s w e re o p ened and t h e h y d r o l y s a t e d e c a n te d
i n to a t e s t tu b e .
2,
The t u b e s w e re r i n s e d s e v e r a l tim e s w i t h d e io n i z e d w a t e r ; t h e
w a s h in g s w e re com bined w i t h t h e h y d r o l y s a t e and b r o u g h t t o a
volum e o f 5 m l.
3,
S e v e r a l d ro p s o f 0.02% m e th y l r e d i n d i c a t o r w e re a d d e d and th e
pH a d j u s t e d t o 6 -7 w i t h 2 .5 N NaOH.
S ta n d a r d s
1,
0 .0 1 3 1 gm o f h y d r o x y p r o l in e (3 7 ) w as d i s s o l v e d i n t o 100 ml o f
d e io n iz e d w a te r.
2.
From t h i s s t o c k , s e v e r a l d i l u t i o n s w e re made and t h e f i n a l
c o n c e n t r a t i o n s a r e a s f o ll o w s :
51
Sta n d a rd
A
B
C
D
E
C o n c e n t r a ti o n g /m l
h y d r o x y p r o l in e
1 0 .4 8
3 .6 3
2 .6 2
1 .9 6 5
1 .3 1
Sam ple S iz e
1.
S a l t S o l u b le F r a c t i o n — 2 ml o f h y d r o l y s a t e o r I m l + I m l w a t e r w e re
a n a ly z e d .
2.
A u t o c la v a b le F r a c t i o n — A f t e r a 100 f o l d d i l u t i o n o f t h e h y d r o l y s a t e ,
2 m l w e re ta k e n
3.
fo r a n a ly s is .
NaOH F r a c t i o n — 0 .5 m l, I ml o f 2 m l o f h y d r o l y s a t e w e re made up
to 2 ml f o r a n a ly s is .
C o lo rim e tric T e st
P ro c ed u re :
1.
The h y d r o x y p r o l in e was o x i d iz e d w i t h c h lo r a m in e T.
2.
One m l o f a s o l u t i o n , 1 .4 4 gm c h lo r a m in e T d i s s o l v e d i n 2 ml m e th y l
c e l l o s o l v e and made f r e s h d a i l y , was ad d ed t o e a c h s a m p le .
3.
The c o n t e n t s w e re m ixed by s h a k in g v i g o r o u s l y and a llo w e d t o s t a n d
20 m in u te s a t room t e m p e r a t u r e w i t h p e r i o d i c s h a k in g .
4»
One m l o f a 70% s o l u t i o n o f p e r c h l o r i c a c id was a d d e d ; t h e tu b e s
w e re a g a in s h a k e n v i g o r o u s l y and a llo w e d t o s t a n d f o r 5 m in u te s .
5.
One m l o f p - d im e th y la m in o b e n z a ld e h y d e , 20% i n m e th y l c e l l o s o l v e ,
was ad d ed and the. m ix t u r e was s h a k e n u n t i l c l e a r .
6.
The tu b e s w e re p l a c e d i n a 60° w a t e r b a t h f o r 20 m in u te s th e n
c o o le d i n ta p w a t e r f o r 5 m in u t e s .
52
7.
The p e r c e n t t r a n s m i t t a n c e , c h a n g ed t o 0 ,D , and com pared t o a
s t a n d a r d c u rv e a s e s t a b l i s h e d a b o v e , was r e a d i n t h e S p e c t r o n i c
20 a t 557 mu.
8.
The g o f h y d r o x y p r o l in e w e re fo u n d by p l o t t i n g
on a s t a n d a r d c u r v e .
t h e O.D. v a lu e s
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