Agronomic potential of sainfoin (Onobrychis viciaefolia) for Montana
by John L Holden
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE in Agronomy
Montana State University
© Copyright by John L Holden (1963)
Abstract:
A study was initiated to evaluate the potential of sainfoin for production in Montana.
A search of the literature revealed sainfoin to be a deep-rooted, long-lived, tetrapoid, perennial legume
particularly adapted to dry, well-drained calcareous soils. It is reported not to cause bloat and
outyield-ed all other legumes at the Montana Experiment Station when harvested for only one cutting
of hay.
Research was undertaken to evaluate the seedling emergence as affected by seeding depth, the protein
quality of the seed, and the comparative seasonal chemical composition of sainfoin to other legumes
including estrogenic activity.
The experiment on seeding depth indicated that sainfoin will allow a higher percentage of seedling
emergence than alfalfa from a depth of three inches or less.
The protein quality of sainfoin seed is similar to that of soybean oil meal and warrants consideration as
a source of supplemental protein for livestock feeding.
The seasonal protein content of sainfoin was found to decrease with advancing maturity and dry matter
percentage increases. A high negative correlation (-.89) was calculated for protein as related to dry
matter percentage in legumes which could lead to a quick, comparative method of calculating crude
protein content of legume forages. Sainfoin matures at about the same rate as alfalfa. It has a higher
leaf to stem ratio than alfalfa and is lower in crude fiber and protein. Sainfoin has a high percentage of
nitrogen free extract in comparison to other legumes which, coupled to its lower protein content, makes
it worthy of consideration as a silage crop.
All three stages of red clover and the regrowth stage of alfalfa produced a significantly higher
estrogenic response than any of the other legumes. Sainfoin did not exhibit a measurable amount of
estrogenic activity at any stage of growth.
Sainfoin possesses potential for both forage and send production in Montana, especially in dryland
areas where one cutting of hay is harvested, or on irrigated land where a high-yielding silage crop is
desired. AGRONOMIC POTENTIAL OF SAINFOIN
f ONOBRYCHI S VICiAEFOL-IA) FOR MONTANA
by
JOHN Lo HOLDEN
A t h e s i s s u b m i t t e d t o t h e G r ad u at e F a c u l t y i n p a r t i a l
f u l f i l l m e n t of the requirem ents fo r the degree
of
MASTER OF SCIENCE
in
Agronomy
Approved:
/V"1
Heads Major Department
Chairman, Examining C o m m it t e e .
•!
Dean, G ra d u a te D i v i s i o n
MONTANA STATE COLLEGE
Bozeman, Montana
May, 1963
Jl
-IiiACKNOWLEDGMENT
The a u t h o r w is h es t o acknowledge t h e c o u n s e l and a d v i ce o f P r o f e s s o r
Ro be rt F. Eslick-, un d er whose g u id a n c e t h i s s t u d y was c o n d u c t e d .
Appre­
c i a t i o n i s e x p r e s s e d t o A s s i s t a n t P r o f e s s o r A r th u r S. H ov er sla n d of th e
Animal S c ie n c e Department f o r h i s h e l p and encouragement d u r i n g t h e b i o ­
a s s a y s t u d y and t o Dr. Oscar 0. Thomas, Dr. E r h a r d t R. Hehn, and Dr. Lark
P. C a r t e r f o r s e r v i n g as members o f t h e g r a d u a t e comm it tee .
S p e c i a l acknowledgment i s due Dr. R ic h a r d R. Roehm and Mrs. Helen L.
M ay fie ld o f Home Economics R e s e a r c h f o r t h e I r g e n e r o u s c o o p e r a t i o n and
use o f t h e i r sm al l animal l a b o r a t o r y f a c i l i t i e s .
The a u t h o r a l s o w is h e s t o th a n k P r o f e s s o r Fred S. W i l l s o n , head o f
t h e Animal S c ie n c e D ep ar tm en t, f o r use o f t h e f a c i l i t i e s i n t h e Animal
N u t r i t i o n L ab or at or y.-
- iv TABLE OF CONTENTS
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ACKNOWLEDGMENT
LIST OF TABLES
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LIST OF FIGURES .
ABSTRACT
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INTRODUCTION
LITERATURE REVIEW .
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SEEDING DEPTH . .
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R e s u l t s and D i s c u s s i o n » . .
12
M a t e r i a l s and Methods
15
PROTEIN QUALITY OF SAINFOIN SEED
. . =
15
R e s u l t s and D i s c u s s i o n . . «
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SEASONAL CHEMICAL COMPOSITION . ,
19
M a t e r i a l s and Methods
M a t e r i a l s and Methods
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R e s u l t s and D i s c u s s i o n . . .
ESTROGENIC ACTIVITY ........................
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R e s u l t s and D i s c u s s i o n „ .. ,
34
M a t e r i a l s and Methods
SUMMARY o o o o o o e e o o e o
47
LITERATURE CITED
49
-V -
LIST OF TABLES
Page
Ta bl e
P e r c e n t a g e o f v i a b l e s a i n f o i n and a l f a l f a s ee d s
which g e r m i n a t e d and emerged from v a r i o u s s e e d ­
ing d e p t h s o o o o o o o o o o o o o o o o o o o o o o o o e o t O O O O O O
T ab l e I I .
13
Comp os it ion o f r a t i o n s used t o e v a l u a t e p r o t e i n
o o o o
q u a l i t y o f s a i n f o i n seed meal
. 0 0 0 0 0 0 0 0 *
0 0 '
o o o o
Average r a t w e i g h t g a i n s and f eed e f f i c i e n c y
d u r i n g a fee d t r i a l con d u ct ed u s i n g pigweed
seed m e a l s s a i n f o i n seed meal and soybean o i l
meal as p r o t e i n s o u r c e s .
T ab le I I I .
0 0 0 0 0 0 * 0 0
Growth s t a g e s o f 8 legumes and o r c h a f d g r a s s on
o o o
v a r i o u s h a r v e s t d a t e s a t Bozeman i n 1962=„
Tabl e
o o o o o
Tabl e Va
H ei g h t i n i n c h e s o f 8 legumes and o f c h a r d g r a s s
on v a r i o u s h a r v e s t date's a t Bozeman in 1962=«„ < O O O O O
Ta b le
Dry m a t t e r c o n t e n t o f 8 legumes and o r c h a r d g r a s s on v a r i o u s h a r v e s t d a t e s a t Bozeman in
1962======#==o======*========================="
T ab le VII =
Crude p r o t e i n c o n t e n t o f 8 legumes and o r c h a r d g r a s s on v a r i o u s h a r v e s t d a t e s a t Bozeman i n
0 0 0
1962 ======== 0 0 0 0 0 0
0 0 0 0 0
24
0 0 " O O O O O O O O
Ta b le VIII= L e a f - s t e m com par iso n o f 7 legumes and o r c h a r d g r a s s a t hay s t a g e a t Bozeman i n 1962===============
Ta b le IX=
Ta bl e X=
Ta bl e XI=
26
N u t r i e n t c o m p o s i t i o n o f l e a f and stem s e p a r a ­
t i o n s of a l f a l f a and s a i n f o i n hays grown a t
Bozeman i n 1962oooo =o =o =o = =========== ==== ====== =====
27
E strogenic a c t i v i t y of v a rio u s le v e ls of d i ­
e t h y l s t i l b e s t r o l added t o a c o n t r o l r a t i o n = = == ==== ==
37
E strogenic a c t i v i t y of th r e e stag es of a l f a l f a
w i t h d i f f e r e n t , s t o r a g e p r o c e d u r e s and s a i n f o i n
s e e d , when fed w i t h o u t e x t r a c t i o n as p a r t o f th e
t mouse r a t i o n = o = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =
= = = =
38
-v iPage
E strogenic a c t i v i t y of in c re a s in g le v e ls of
e x t r a c t from v e g e t a t i v e s t a g e s o f a l f a l f a and
. r e d c l o v e r u s i n g B i c k o f f F r e s h Forage E x t r a c t i o n
Method
. . . o o . . . . . . . . . . . . . . . . . . . . . . . . . i ) . .
40
Table X III. E strogenic a c t i v i t y , of th r e e " s t a g e s of a l f a l f a
d u r i n g two t r i a l s u s i n g t h e Canadi an E x t r a c t i o n
Method. . . . . . . . . o . . . . . . . . . . . . . . . . . . s ' . . . . . . . . . 6 . . . ^ . . .
42
E s t r o g e n i c a c t i v i t y o f t h r e e s t a g e s o f legumes
and s a i n f o i n seed u s i n g t h e Canadian E x tr ac ti o n "
Method . . . . . . o . . o o . . . . . . . . . . . . . ^ . . . . . . . . . . . . . . . . . . . . .
43
E s t r o g e n i c a c t i v i t y comp ariso n o f B i c k o f f F r e s h
Forage E x t r a c t i o n Method w i t h Canadian Dry Fo rage
E x t r a c t i o n Method on hay s t a g e f o r a g e o f v a r i o u s
legumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
44
Estimated d i e t h y l s t i l b e s t r o l equivalency of red
c l o v e r and a l f a l f a i n micrograms p e r pound o f
dry m a tte ro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
46
Ta b l e X I I .
Ta b l e XIV.
Ta b l e XV.
Ta b l e XVI.
-viiLIST OF FIGURES
Page
Figure I .
D i e t h y i s t i l b e s t r o l dose r e s p o n s e cur ve and r e g r e s s i o n
o f U t e r i n e w e i g h t as p e r c e n t a g e o f body w e i g h t on
micrograms o f d i e t h y l s t i l b e s t r o I i n f eed a t l e v e l s
o f O t o .06 micrograms p e r gram o f f e e d . . . . . . o.„-. . . . .
36
-v iiiABSTRACT
A s t u d y was i n i t i a t e d t o e v a l u a t e t h e p o t e n t i a l o f s a i n f o i n f o r p r o ­
d u c t i o n . i n Montana.
A s e a r c h o f t h e l i t e r a t u r e r e v e a l e d s a i n f o i n t o be a d e e p - r o o t e d ,
l o n g - l i v e d , t e t r a p o i d j p e r e n n i a l legume p a r t i c u l a r l y ad ap te d t o d r y , w e l l drained calcareous s o i l s .
I t i s r e p o r t e d n o t t o cause b l o a t and o u t y i e l d ed a l l o t h e r legumes a t t h e Montana Experiment S t a t i o n when h a r v e s t e d f o r
o n l y one c u t t i n g o f hay.
R e s ea r c h was u n d e r t a k e n t o e v a l u a t e t h e s e e d l i n g emergence as a f f e c t ­
ed by s e e d i n g d e p t h , t h e p r o t e i n q u a l i t y o f t h e s e e d , and t h e co m p ar at iv e
s e a s o n a l ch em ic a l c o m p o s i t i o n of s a i n f o i n t o o t h e r legumes i n c l u d i n g e s t r o
genic a c tiv ity ^
The e x p e r i m e n t on s e e d i n g d e p t h i n d i c a t e d t h a t s a i n f o i n w i l l all ow
a h i g h e r p e r c e n t a g e o f s e e d l i n g emergence t h a n a l f a l f a from a d e p t h of
th r e e inches or l e s s .
The p r o t e i n q u a l i t y o f s a i n f o i n seed i s s i m i l a r t o t h a t o f soybean
o i l meal and w a r r a n t s c o n s i d e r a t i o n as a s o u r ce o f s u p p le m e n ta l p r o t e i n
f o r l i v e s t o c k f e e d i rig. '
The s e a s o n a l p r o t e i n c o n t e n t o f s a i n f o i n was found t o d e c r e a s e w i t h
adva nc ing m a t u r i t y and d r y m a t t e r p e r c e n t a g e i n c r e a s e s .
A h ig h n e g a t i v e
c o r r e l a t i o n ( - . 8 9 ) was c a l c u l a t e d f o r p r o t e i n as r e l a t e d t o d r y m a t t e r
p e r c e n t a g e i n legumes which coul d l e a d t o a q u i c k , c o m p a r a ti v e method o f ' ,
c a l c u l a t i n g cru de p r o t e i n c o n t e n t o f legume f o r a g e s . S a i n f o i n m a tu r es a t
abo ut t h e same r a t e as a l f a l f a .
I t has a h i g h e r l e a f t o stem r a t i o thana l f a l f a and i s lower i n cru de f i b e r and p r o te i n - . S a i n f o i n has a h ig h p e r ­
c e n t a g e o f n i t r o g e n f r e e e x t r a c t i n comparison t o o t h e r legumes which,
cou pl ed t o i t s lower p r o t e i n c o n t e n t , makes i t wor thy o f c o n s i d e r a t i o n as •
a sila g e crop.
All t h r e e s t a g e s o f r e d c l o v e r and t h e r e g r o w th s t a g e o f a l f a l f a pro*duced a s i g n i f i c a n t l y h i g h e r e s t r o g e n i c r e s p o n s e t h a n any o f t h e o t h e r
legumes. S a i n f o i n d i d n o t e x h i b i t a m e a s u r a b l e amo.unt o f e s t r o g e n i c a c t i v
i t y a t any s t a g e of growth.
S a i n f o i n p o s s e s s e s p o t e n t i a l f o r b o t h f o r a g e and send p r o d u c t i o n in
Montana, e s p e c i a l l y i n d r y l a n d a r e a s where one c u t t i n g o f hay i s h a r v e s t e d
or on i r r i g a t e d la nd where a h i g hW y ie ld i n g s i l a g e cr o p i s d e s i r e d .
INTRODUCTION
Dr= R= G= S t a p l e d o n s famous B r i t i s h g r a s s s p e c i a l i s t s has s a i d ,
"No
g r a s s l a n d i s w o r th y o f t h e name and h a r d l y w o r th b o t h e r i n g w i t h u n l e s s a
legume i s a t work=
Find o r b r e e d t h e r i g h t legume f o r e v e r y c o r n e r o f the'
wo rld and you have d ev el o p ed good g r a s s l a n d i n e v e r y c o r n e r o f t h e world="
The " r i g h t " legume f o r Montana would combine t h e c h a r a c t e r i s t i c s of
high n u t r i e n t p ro d u ctio n , n o n -b lo a tin g , p a l a t a b i l i t y , com petitive a b i l i t y ,
s e e d l i n g v i g o r , ample seed p r o d u c t i o n , lo n g l i f e , d i s e a s e and d r o u g h t r e ­
s i s t a n c e , w i n t e r h a r d i n e s s , arid a d a p t a b i l i t y t o our s o i l s =
Our p r e s e n t '
legumes have some o f t h e s e a t t r i b u t e s b u t n o t a l l = B i r d s f o o t t r e f o i l i s
l o n g - l i v e d , s e m i - w i n t e r h a r d y , sometimes h i g h y i e l d i n g , somewhat d r o u g h t
resistant,
a l k a l i t o l e r a n t and has been r e p o r t e d n o t t o cau se b lo a t=
How­
e v e r , i t i s v e r y slow t o e s t a b l i s h , may be somewhat l a c k i n g i n p a l a t a b i l ­
i t y , and w i l l n o t w i t h s t a n d g r a z i n g un d er d r o u g h t c o n d i t i o n s =
arid a l s i k e c l o v e r ar e s h o r t - l i v e d and n o t d r o u g h t r e s i s t a n t =
Red c l o v e r
White c l o v e r
i s lew y i e l d i n g and i t s c l o s e r e l a t i v e , L a d i n o , l a c k s w i n t e r h a r d i n e s s , and
ha s h i g h m o i s t u r e and f e r t i l i t y r e q u i r e m e n t s =
The s w e e t c l o v e r s are b i e n ­
n i a l , s u s c e p t i b l e t o t h e s w e e t c l o v e r w e e v i l and c o n t a i n t h e u n d e s i r a b l e
compound coumarin=
A l f a l f a i s good from al m o s t a l l s t a n d p o i n t s e x c e p t
t h a t i t may r e p r e s e n t a s e r i o u s b l o a t h a z a r d i n p a s t u r e ' s =
S a i n f o i n (On ob rvchis v i c i a e f o l i a ) i s a d e e p - r o o t e d , l o n g - l i v e d , t e t r a
p o i d , p e r e n n i a l legume i n t r o d u c e d from Europe which comes c l o s q t o p o s s e s s
in g a l l t h e d e s i r e d c h a r a c t e r i s t i c s o f a legume f o r Montana=
t i c u l a r l y ad ap te d t o d r y c a l c a r e o u s s o i l s =
I t -is p a r ­
P i p e r and M a n s f i e l d s t a t e ,
-2 •!'■that s a i n f o i n has n e v e r been known-to cau se b l o a t ° "
I t s common name
s a i n f o i n , Fr ench f o r ! ' h e a l t h y hay"', and t h e L a t i n s p e c i e s hah$ On.bbjrychis,
meaning " t h a t f o r which a s s e s b r a y " , may a p t l y d e s c r i b e i t s f o r a g e q u a l ­
ities.
These f a c t o r s were t h o u g h t t o make s a i n f o i n wo rth y o f more d e t a i l e d
e v a l u a t i o n as a p o s s i b l e legume f o r p r o d u c t i o n i n Montana.
LITERATURE REVIEW
The s a i n f o i n p l a n t c o n s i s t s o f 3 t o 32' s t o u t , e r e c t stems which
a r i s e from a b r an che d crown (40)„
15 l e a f l e t s o
''The l e a v e s ar e o d d - p i n n a t e w i t h 13 t o
The r o s e c o l o r e d ( r a r e l y w h i t e ) f l o w e r s a r e i n an e r e c t ,
c l o s e raceme 2 t o 5 i n c h e s I o n g 0
The r o o t s ar e Repor te d t o r e a c h a d i ­
ameter o f 2 i n c h e s and e x t e n d t o a d e p t h o f 20 f e e t o r m o r e «
o f s a i n f o i n p r o b a b l y d a t e s back about 400 y e a r s .
The c u l t u r e
I t was f i r s t c u l t i v a t e d
i n F r a n c e ; t h e f i r s t d e f i n i t e r e c o r d a c c o r d i n g t o Vianne was i n 1 5 8 2 . " ( 3 4 )
At p r e s e n t s a i n f o i n i s commonly grown i n p a r t s o f England and F r a n c e .
S a i n f o i n ( On obr vch is a l t i s s i m a ) comes from t h e Near E a s t e r n C e n t e r o f O r i ­
gin.
S e v e r a l s p e c i e s o c c u r w i l d i n t h a t a r e a and i n t h e s o u t h e r n h a l f o f
Europe and e x t e n d e a s t w a r d toward R u s s i a where e x t e n s i v e r e s e a r c h i s b e ­
i n g done on s a i n f o i n f o r f o r a g e ( 2 5 ) ,
(34).
In E h g l a n d , i n f o r m a t i o n o b t a i n e d from p r o d u c t i o n o f s a i n f o i n hay,,
which i s h i g h l y p r i z e d by r a c i n g s t a b l e s f o r f e e d i n g brood mares and c o l t s ,
has le d t o i t s use on much d r y c a l c a r e o u s l a n d , which p r e v i o u s l y had been
n e a rly v a l u e le s s (34).
Numerous p o p u l a r a r t i c l e s have been p u b l i s h e d i n t h e United S t a t e s on
s a i n f o i n ' s v a l u e as a bee f o r a g e ( l ) ,
(ll),
(24), (25),
t i a l f o r l i v e s t o c k u t i l i z a t i o n (3) b u t v e r y l i t t l e
(31) arid p o t e n ­
s a i n f o i n i s being
grown a t p r e s e n t .
The two v a r i e t i e s o f s a i n f o i n grown i n England, common arid g i a n t ,
may have p o s s i b i l i t i e s f o r p r o d u c t i o n i n Montana.
They a r e p a r t i c u l a r l y
w e l l a da pt ed t o d r y , w e l l - d r a i n e d c a l c a r e o u s s o i l s w i t h a low w a t e r t a b l e
i'
”4 (26)o
P e l l e t 9 ( 3 1 ) , r e p o r t e d them e x t r e m e l y d r o u g h t and c o l d r e s i s t a n t .
Common d i f f e r s from g i a n t s a i n f o i n i n h a v i n g f i n e r s t e m s , a more p r o s ­
t r a t e growth h a b i t , g r e a t e r p e r s i s t e n c y o f s t a n d , and i n a b i l i t y to f lo w er
twice a season (18).
The f i r s t c u t t i n g o f g i a n t i s u s u a l l y t a k e n f o r hay
and t h e second l e f t f o r seed p r o d u c t i o n ( 3 ) .
G ia n t i s u s u a l l y seeded as
a s h o r t r o t a t i o n c r o p , 2 t o 3 y ea r s ', and common as a lo n g r o t a t i o n c r o p ,
4 to 7 years.
A f t e r 7 o r 8 y e a r s o f s a i n f o i n p r o d u c t i o n , good s o i l s b e ­
come s i c k i n a manner ann al o go us t o -"clover . s i c k " s o i l s anti must be r e ­
seeded t o a n o t h e r c r o p ; h ow eve r, t h i s d o e s n ’t o cc u r on t h e d r y e r , l e s s
f e r t i l e s o ils (34).
E s l i c k (15) r e p o r t e d a s t a n d a t H a l l , Montana, which
has been i n e x i s t e n c e s i n ’c e 1898=
Both g i a n t and common s a i n f o i n are r e ­
p o r t e d t o be s i m i l a r t o a l f a l f a i n t h e i r a b i l i t y t o w i t h s t a n d f r e q u e n t
close g razin g or c lip p in g (16).
S a i n f o i n i s a d a p t a b l e t o good i r r i g a t e d s o i l s ( 3 4 ) .
Re s ea r c h by
Cooper (10) a t t h e Montana A g r i c u l t u r a l Experiment S t a t i o n showed t h a t
s a i n f o i n o u t y i e l d e d a l l o t h e r legume s p e c i e s when compared w i t h f i r s t c u t ­
t i n g hay y i e l d s .
The n u t r i t i v e c o n t e n t o f a p l a n t , w i t h i n l i m i t s , i s t h e f i n a l d e t e r ­
min ing f a c t o r i n i t s v a l u e as a f o r a g e c r o p .
Norton (29) s t a t e s ,
"There
ar e two f a c t o r s which g r e a t l y i n f l u e n c e t h e f e e d i n g v a l u e o f a l f a l f a hay;
namely, t h e p r o p o r t i o n o f l e a v e s t o stems and t h e crude p r o t e i n c o n t e n t . "
M o r r i s o n ' s Feeds and F ee d i n g (27) d o e s n ' t l i s t t h e ch em ic a l c o m p os it io n
of sa in fo in .
Baker ( 4 ) , i n Eng la nd , found t h a t l i k e most f o r a g e s ,
-5 s a i n f o i ' n ha s 3 lower cru de p r o t e i n c o n t e n t , and a h i g h e r cru de f i b e r con­
t e n t as t h e p l a n t advances toward m a t u r i t y .
t o be somewhat v a r i a b l e ,
The'.mineral c o n t e n t was found
The r a t i o o f l e a f d r y m a t t e r t o t o t a l d r y ma t­
t e r decre ase s with m a tu r ity of the p la n t (4 ),
The l e a f c o m p o s i ti o n i s
f a i r l y c o n s t a n t i r r e s p e c t i v e o f growth s t a g e , number o f c u t t i n g s and s e a ­
son; w i t h l e a f f i b e r c o n t e n t b e i n g r e m a r k a b l y c o n s t a n t i n c o n t r a s t to
t h a t o f stem, where i t i n c r e a s e s w i t h m a t u r i t y o f th e p l a n t .
The l e a f
c o n t a i n s more cru de p r o t e i n , e t h e r e x t r a c t , and m i n e r a l m a t t e r , p a r t i c u ­
l a r l y c a l c i u m , t h a n t h e stem, (4)
Changes which occ ur in' t h e c o m p os it io n
o f s a i n f o i n ar e p o s t u l a t e d t o be due t o v a r i a t i o n i n stem c o m p o s i ti o n and
leaf-stem r a t i o .
S a i n f o i n i s n o t c o n s i d e r e d t o be a p a r t i c u l a r l y r i c h
s o u r c e of b e t a - c a r o t e n e ( 4 ) ,
Baker (4 ) l i s t s t h e f o l l o w i n g as th e a v e r ­
age c o m p o s i t i o n o f 35 samples o f s a i n f o i n hay grown Tn En gl and ,
For com­
p a r i s o n , d a t a on a l f a l f a from M o rr iso n (27) i s g i v e n .
Co mposit ion o f Dry M a t t e r
C onstituent
Sainfoin
%
M o is tu r e
Crude P r o t e i n
Ether Extract
Crude F i b e r
N, F r e e E x t r a c t
T o t a l Ash
S ilica
Calcium
Phosphorous
P o ta s s iu m
Chlorides'
0.0
15 .0
1,8
3 0 .8
45.5
6.9
0.8
1 .8 4
0.63
1 .5 2
0.53
A lfalfa
%
9.5
15 .3
1 .9
2 8.6
36.7
8.0
— =, = = =
1.47
0 .2 4
1.97
= = = = =
Nor ton (29) r e p o r t s 4 0 , 4 as t h e av er a ge l e a f p e r c e n t a g e o f f i r s t
“ 6 “
c u t t i n g a l f a l f a hay i n comp ariso n t o 3 2 . 3 as t h e avera ge l e a f p e r c e n t a g e
o f s a i n f o i n hay g i v e n by Baker ( 4 ) .
In r e c e n t y e a r s t h e e s t r o g e n i c a c t i v i t y o f f o r a g e s h as r e c e i v e d
co n sid e ra tio n in ev a lu a tin g forages.
The t e r m " e s t r o g e n " can be d e f i n e d as any compound o r s u b s t a n c e
which i s c a p a b l e o f c a u s i n g s e x u a l deve lo pm ent i n t h e female? i n c l u d i n g
changes i n the' v a g i n a l e p i t h e l i u m , h y p e r t r o p h y o f t h e u t e r u s and mammary
g l a n d s , and t h e develo pme nt o f female s e c o n d a r y sex c h a r a c t e r i s t i c s ( 4 4 ) ;
E s t r o g e n i c s u b s t a n c e s were f i r s t d i s c o v e r e d i n p l a n t s i n 1926 by Loewe
(38).
S in c e t h a t time a p p r o x i m a t e l y 50 s p e c i e s of p l a n t s have been shown
to possess varying degrees of e s tro g e n ic a c t i v i t y (32);
I n t e r e s t in f o r ­
age e s t r o g e n s was renewed i n 1941 when w id e s p r e a d b r e e d i n g d i s o r d e r s of
sheep i n Western A u s t r a l i a were found t o be caused by t h e h i g h e s t r o g e n i c
c o n t e n t o f s u b t e r r a n e a n c l o v e r (T r i f o l i u m s u b t e r r a n e a n ) . 1 S in ce t h a t t i m e ,
o t h e r f o r a g e p l a n t s which have been found t o p o s s e s s e s t r o g e n i c compounds
o f v a r y i n g a c t i v i t y ar e r e d c l o v e r fT r i f o l i u m p r e t e n s e ) , a l f a l f a (Medic ago s a t i v a ) , whitfe c l o v e r (T r l f o l i u m r e p e n s ) $ s t r a w b e r r y c l o v e r (Trl fo. Iium f r a g e f e r u m ) , Ladino c l o v e r (T r i f o l i u m r e p e n s , l a t u m ) , and b i r d s f o o t
trefoil
( Lot us c o r n r o u l a t u S ' ) 1 3 8 ) .
c o n t a i n e s t r o g e n i c ^qmppunds ( 3 8 ) .
Soybean o i l meal i s a l s o r e p o r t e d t o
R e d - c l o v e r i s most c o n s i s t e n t l y r e p o r t ­
ed as h a v i n g e s t r o g e n i c a c t i v i t y a t a l l s t a g e s o f gr owth;
b r e e d i n g d i s o r d e r s i n sheep i n Ohio and Oregon ( 9 ) .
I t has caused
A l f a l f a v a r i e t i e s ar e
r e p o r t e d t o have w i d e . d i f f e r e n c e s i n e s t r o g e n c o n t e n t ( 3 2 ) .
Youngman (44)
r e p o r t s Ladak a l f a l f a i n Washington t o have i n c r e a s i n g e s t r o g e n i c a c t i v i t y
Jl
(I
- 7 -
with m a tu rity o
Hay samples o f a l f a l f a ,
a l s i k e c l o v e r ? w h i t e c l o v e r and
r e d c l o v e r have been r e p o r t e d t o p o s s e s s e s t r o g e n i c a c t i v i t y (38)°
A lfalfa
s i l a g e has been found t o p o s s e s s s i g n i f i c a n t l y g r e a t e r e s t r o g e n i c a c t i v i t y
t h a n f r e s h a l f a l f a o r a l f a l f a hay (32)-,- ( 4 4 ) /
White c l o v e r , b i r d s f o o t
t r e f o i l s arid a l f a l f a have been r e p o r t e d t o be h i g h e s t i n e s t r o g e n c o n t e n t
d u r i n g e a r l y s p r i n g growth ( 3 8 ) .
The e s t r o g e n i c a c t i v i t y o f f o r a g e s seems
t o v a r y w i t h v a r i e t y , s t a g e o f g r o w th , number o f c u t t i n g s and e n v i r o n ­
ment ( 3 2 ) ,
(44).
V ar io us c o n f l i c t i n g t h e o r i e s e x i s t as t o t h e s i t e o f p r o d u c t i o n o f
estrogens in p la n ts .
E s t r o g e n s o cc u r e a r l i e s t i n t h e c h l o r o p l a s t f r a c t i o n ' ,
so t h i s would seem t h e most l i k e l y s i t e o f p r o d u c t i o n ( 2 3 ) .
The e s t r o g e n i c compounds which have been i s o l a t e d and i d e n t i f i e d are
the f l a v i n o i d s , g e n i s t e i n , i t s glycoside g e n is te n , diadzein,- i t s glycoside
d i a d z i n , f o r m o n o n e t i n , b i o c h a n i n A and a coumafi n - l i k e compound, c o u m e s t r o l .
These a l l have s i m i l a r che m ica l s t r u c t u r e s which d i f f e r m a i n l y i n number
and l o c a t i o n o f h y d r o x y l and methoxyl grou ps ( 3 8 ) .
C o u m e st r o l, which has
been i s o l a t e d i n a l l legumes t e s t e d , p o s s e s s e s a h i g h e r e s t r o g e n i c a c t i ­
v i t y t h a n any o f t h e o t h e r compounds (9),.
Q u a n t i t a t i v e and q u a l i t a t i v e d e t e r m i n a t i o n o f e s t r o g e n s i n p l a n t and
animal m a t e r i a l can be acco mplish ed by che m ic a l methods and b i o - a s s a y
procedures.
Chemical p r o c e d u r e s ar e ti m e consuming, e s p e c i a l l y w it h p l a n t
m a t e r i a l s , s i n c e ch r o m a tr o g r a p h y and a c c u r a t e a n a l y t i c a l methods are n e c e s ­
sary'.-
When t h e che m ic a l and p h y s i c a l p r o p e r t i e s o f e s t r o g e n s ar e no t
- 8 -
d e s i r e d , a g r o s s q u a n t i t a t i v e e s t i m a t e o f t h e e s t r o g e n i c a c t i v i t y can be
o b t a i n e d by use o f b i o - a s s a y t e c h n i q u e s ( 3 8 ) .
With f o r a g e s , t h i s i n v o l v e s
e x t r a c t i o n o f t h e e s t r o g e n w i t h a s o l v e n t and t h e n addi ng t h e e x t r a c t t o
a c o n t r o l r a t i o n which i s fed t o immature female mice o r r a t s .
There i s
no d i f f e r e n c e i n r e l i a b i l i t y o f r e s u l t s , b u t mice are l e s s e x p e n s i v e .
an im al s a r e fed from t h r e e t o t e n days and t h e n s a c r i f i c e d .
The
The r e s u l t a n t
u t e r i n e w e i g h t i n c r e a s e i s used as a m e a s u r e . o f e s t r o g e n i c a c t i v i t y .
The
r e s u l t s ar e u s u a l l y s t a t e d as e q u i v a l e n t t o t h e amount o f d i e t h y l s t i I b e s t r o l which would cause t h e same u t e r i n e r e s p o n s e ( 3 8 ) .
B i c k o f f ' e t al>' (8)
found t h a t acetone-, a l c o h o l , o r a 2 :1 b e n z e n e - a l c o h o l m i x t u r e are e q u a l l y
e f f e c t i v e i n e x t r a c t i n g e s t r o g e n i c compounds from f r e s h o r d r i e d Ladino
clover.
Drying a f o r a g e can ca us e up t o a 75% l o s s i n e s t r o g e n i c a c t i v i t y .
E s t r o g e n i c compounds a r e f a i r l y s t a b l e a f t e r e x t r a c t i o n ( 9 ) .
In a d d i t i o n t o a f f e c t i n g r e p r o d u c t i o n , p l a n t e s t r o g e n s have been ob­
s e r v e d t o a f f e c t t h e c o m p o s i t i o n and p r o d u c t i o n o f m i l k and t o s t i m u l a t e
growth i n c a s t r a t e d male an im al s (9 )°
B i c k o f f (9) s t a t e s ,
t h a t we may need two s e p a r a t e t y p e s o f f o r a g e s .
" I t now a p p e a r s
One o f t h e s e would be a
l o w - e s t r o g e n f o r a g e f o r b r e e d i n g s t o c k , b e ca u se t h e n a t u r a l l y - o c c u r r i n g
e s t r o g e n s ar e n o t d e s i r a b l e h e r e .
The second ty p e would be a h i g h - e s t r o g e n
f o r a g e f o r f a t t e n i n g s t e e r s , w e t h e r s and p o u l t r y . "
Data has n o t been pub­
l i s h e d c o n c e r n i n g t h e e s t r o g e n i c a c t i v i t y o f s a i n f o i n o r s a i n f o i n se e d .
E f f i c i e n t p r o d u c t i o n o f legume f o r a g e s i s d ep en d e n t upon s u c c e s s f u l
stand e s ta b lis h m e n t.
Among f a c t o r s i n f l u e n c i n g e s t a b l i s h m e n t are seed
s i z e , seed v i a b i l i t y , r a t e o f s e e d i n g , d e p t h of s e e d i n g and v a r i e t y .
-9S a i n f o i n seed i s prod uced by t h e p l a n t i n o n e - s e e d e d pods which ar e
d e s c r i b e d by P i p e r (34) as b e i n g "brown, i n d e h i s c e n t , l e n t i c u l a r , arid r e ­
t i c u l a t e d on t h e s u r f a c e . "
a l l y compressedo
A c t u a l l y , t h e pod i s b ea n - s h ap ed and b i l a t e r ­
I t h as a rough n e t v e i n e d a p p e a r a n c e , and i s sometimes
r e f e r r e d t o as a " c o c k s h e a d "• when i t has s p i n e s p r o t r u d i n g from t h e v e n t r a l
edge o r k e e l .
Evidence i n d i c a t e s t h a t t h e s p in e d c h a r a c t e r i s t i c i s con­
t r o l l e d by orie p a i r o f genes w i t h s p i n y do minant t o s p i n e l e s s ( 4 0 ) ,
S a i n f o i n grown f o r seed p r o d u c t i o n may prod uce 25 t o 30 b u s h e l s o f
seed i n t h e pod p e r a c r e ( 3 ) ,
Like a l f a l f a , i t i s en to m op h al ou s , r e q u i r ­
in g i n s e c t c r o s s - p o l l i n a t i o n f o r maximum seed s e t ( 1 5 ) ,
The podded seed
weighs abo ut 30 pounds p e r bushel.,' o f which o n e - t h i r d , by w e i g h t , i s pod
arid s h r i v e l e d s e e d s ( 4 0 ) .
S a i n f o i n seed rem a in s v i a b l e un d er h ig h d r y ­
in g t e m p e r a t u r e s a f t e r h a r v e s t .
I t i s s a f e t o s t o r e a t 12% m o i s t u r e and
i s r e p o r t e d i n England t o l o s e i t s v i a b i l i t y a f t e r t h r e e y e a r s s t o r a g e ( 3 0 ) .
S a i n f o i n seed i s s o l d c o m m e r c i a l l y i n two f o rm s, m i l l e d and u n m i l l e d .
M i l l e d seed has t h e pod removed and i s k i d n e y - s h a p e d , v a r y i n g from y e l l o w ­
i s h g r e e n t o d a r k brown i n c o l o r .
S a i n f o i n seed i n t h e pod weighs 23
grams p e r 1000 and m i l l e d seed weighs 15 grams p e r 1000 ( 3 9 ) .
i s o n a l f a l f a seed weighs 2 grams p e r 1000 ( 3 7 ) .
In compar­
G e r m in a ti o n i s more u n i ­
form from m i l l e d seed due t o r e d u c t i o n o f h ar d se eds d u r i n g m i l l i n g and
removal o f t h e seed pod which has been pr oved t o p r o v i d e m e c h a n ic a l r e s i s ­
t a n c e t o r a d i c l e emergence ( 4 0 ) .
a longer p eriod (39).
U n b i l l e d seed r e t a i n s i t s v i a b i l i t y f o r
” 10“
The recommended s e e d i n g r a t e f o r a l f a l f a and s a i n f o i n i n England i s
22 and 40 pounds o f m i l l e d seed p e r a c r e , r e s p e c t i v e l y (30)„
In Montana
t h e recommended s e e d i n g r a t e f o r a l f a l f a i s 10 t o 12 pounds p e r a c r e .
P i p e r (34) s t a t e s t h a t s a i n f o i n s h o ul d be seeded a t a d e p t h of oneh a l f in c h o r m o r e .
The most s a t i s f a c t o r y s e e d i n g d e p t h f o r s ma ll seeded
legumes and g r a s s e s i n t h i s c o u n t r y i s 0 . 5 in c h (5 ).
Two i n c h e s i s th e
i
recommended s e e d i n g d e p t h f o r soybeans(28)>6 E r i c k s o n (13) found t h a t as
d e p t h o f s e e d i n g i n c r e a s e d , t h e adv an ta ge o f l a r g e seed o v er smal l seed
i n s t a n d e s t a b l i s h m e n t o f a l f a l f a became i n c r e a s i n g l y a p p a r e n t .
!
Thi s
would' cau se one t o wonder j u s t what t h e optimum s e e d i n g d e p t h f o r s a i n ­
f o i n mi ght b e .
S a i n f o i n seed r e q u i r e s i n b c u l a t i o n f o r p r o p e r n i t r o g e n
f i x a t i o n and growth on s o i l s - which had n o t p r e v i o u s l y grown s a i n f o i n ( I ) .
P i p e r (34) recommends s p r i n g seeding,- s i n c e f a l l s e e d i n g s ar e more ap t t o
1
w interkill.
Woodman (42) g i v e s t h e ch em ic a l c o m p o s i t i o n o f s a i n f o i n seed as
:!
I
follows 5
Moisture
Crude P r o t e i n
Ether E xtract
N= F r e e E x t r a c t
Crude F i b e r
Ash
Calcium
Phosphorous
Chlorides
Unmilied seed
M i l l e d seed
%
%
12.01
2 6.38
5 =96
33.74
17.86
4.05
1 .0 2
.91
.11
8 .9 8
36 =63
7 .2 8
34.06
9 .4 6
3.58
.2 4
1.15
.11
For a long time p r o t e i n has been t h e most e x p e n s iv e i n g r e d i e n t and a
11lim itin g fa c to r of liv e sto c k r a ti o n s .
Soybean' o i l meal i s commonly used
as a p r o t e i n supp lem ent i n l i v e s t o c k r a t i o n s ,
i n c l u d i n g dog and c a t f o o d s ,
b e ca us e o f i t s h i g h p r o t e i n c o n t e n t and e s s e n t i a l amino a c i d b a l a n c e i n
r e l a t i o n to c e re a l g ra in s .
Recent r e s e a r c h i n d i c a t e s t h a t t h e l i m i t i n g
amino a c i d f o r soybean o i l meal i s m e t h i o n i n e , b u t even w i t h o u t th e ad­
d i t i o n o f m e t h i o n i n e , i t i s one o f t h e b e s t q u a l i t y p l a n t p r o t e i n s a v a i l ­
able fo r l i v e s t o c k feeding (2 ).
(27) as b e i n g a bo u t 44
%
Soybean o i l meal i s l i s t e d by Morrison
cr ude p r o t e i n .
M i l l e d s a i n f o i n seed compares
f a v o r a b l y t o soybean o i l meal i n p r o t e i n p e r c e n t a g e , and m i g h t be con­
s i d e r e d f o r commercial p r o d u c t i o n i f i t was e q u a l t o soybean o i l meal i n
amino a c i d b a l a n c e .
Soybean o i l meal s e l l s c o m m e r c i a ll y a t t h e p r e s e n t
time f o r $83 a t o n .
Woodman (42) i n d i c a t e d t h a t lambs d i d v e r y w e l l on
u n m i l l e d s a i n f o i n seed as a p r o t e i n supp lem ent and would have eq u al ed
g a i n s o f lambs on l u c e r n e and c l o v e r seed i f t h e pod had bee n removedi
The s e a r c h o f l i t e r a t u r e on s a i n f o i n i n d i c a t e d t h a t i n f o r m a t i o n was
l a c k i n g i n t h e a r e a s o f recommended s e e d i n g depth,- p r o t e i n q u a l i t y o f
t h e s e e d , arid c o m p a r a ti v e s e a s o n a l ch em ic a l c o m p o s i ti o n i n c l u d i n g e s t r o ­
genic a c t i v i t y .
sainfoin.
R e s ea r c h was u n d e r t a k e n t o e v a l u a t e t h e s e q u a l i t i e s of
SEEDING DEPTH
M a t e r i a l s and Methods
Because o f t h e e x c e p t i o n a l l y l a r g e s i z e o f s a i n f o i n seed and l a c k o f
i n f o r m a t i o n r e g a r d i n g p r o p e r s e e d i n g d e p t h , an e x p e r i m e n t was d e s i g n e d t o
e v a l u a t e t h e e f f e c t o f s e e d i n g d e p t h on t h e r e s u l t i n g s e e d l i n g emergence
of sainfoin.
One hundred a l f a l f a s e e d s and an e q u a l number o f s a i n f o i n s eed s were'
p l a n t e d a t e i g h t d i f f e r e n t d e p t h s , s t a r t i n g a t o n e - h a l f in c h and i n c r e a s ­
in g i n o n e - h a l f in c h i n c r e m e n t s t o f o u r inches., i n s o i l , on a bench i n th e
greenhouse.
A s p l i t - p l o t e x p e r i m e n t a l d e s i g n was used w i t h s e e d i n g d e p t h s
a s s i g n e d a t random t o t h e main p l o t s and s p e c i e a t o su;hpl ot sv
Three r e p ­
l i c a t i o n s were p l a n t e d . ' G e r m in a ti o n p e r c e n t a g e . o f t h e seed was o b t a i n e d
before p la n tin g .
R e l a t i v e ' r a t e o f emergence, was n o te d and r e s u l t s were
compiled as p e r c e n t a g e o f v i a b l e seed em er g in g. .
A l f a l f a , was in c l u d e d i n
t h e e x p e r i m e n t as a chec k.
R e s u l t s and D i s c u s s i o n
The g e r m i n a t i o n p e r c e n t a g e o f s a i n f o i n seed and a l f a l f a seed was 78
and 95 r e s p e c t i v e l y .
The s a i n f o i n seed had been s t o r e d f o r t h r e e y e a r s
which r e f u t e s d a t a by Owen (30) t h a t i n d i c a t e d s a i n f o i n seed l o s e s i t s
v i a b i l i t y during t h a t perio d of time.
The a l f a l f a s e e d l i n g s emerged a day e a r l i e r t h a n t h e s a i n f o i n s e e d ­
l i n g s a t d e p t h s o f s e e d i n g t o two i n c h e s .
t h e f a s t e r em ergence.
S a i n f o i n a t g r e a t e r d e p t h s had
The s a i n f o i n s e e d l i n g s emerged w i t h t h e h y p o c o t y l
p u s h in g t h e c o t y l e d o n s s t r a i g h t up t h r o u g h t h e s o i l as d i d a l f a l f a .
This
i
-13Tabl e 1»
r -
■■ ■_
P e r c e n t a g e o f v i a b l e s a i n f o i n and a l f a l f a s ee d s which g er m i n a te d
and emerged from v a r i o u s s e e d i n g d e p t h s , l /
r—
-
■ n,.-,
Seedi ng
depth in
inches
—
P e r c e n t age o f
s a i n f o i n s ee d s
emerging
P e r c e n t age o f
a l f a l f a seeds
emerging
8 8 .5 a b
60.2 a
1.0
97.0 a
57.9 a ■
1. 5
7 6 .9 a b
56.3 a
2.0
88;. 9 a b
37.2 a b .
2.5
6 3 .7 b c '■
22.8 b c
3.0
38.9 c
17 .6 b c
3.5
9.4 d
1. 1 C
4.0
1.-3 d
1.4 c
.5
l/
L
Values u n d e r l i n e d w i t h i n a s e e d i n g d e p t h are s i g n i f i c a n t l y d i f f e r e n t '
a t P — o05 o.
Values w i t h i n t h e legume f o ll o w e d by t h e same l e t t e r 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 a t P = .05 ( 1 2 ) .
Il >1
-14i s d i f f e r e n t from some le g u m e s , such as b e a n s , i n .which t h e h y p o c o t y l i s
s h a r p l y b e n t arid p u l l s t h e c o t y l e d o n s t o t h e s o i l s u r f a c e .
The r e s u l t s o f t h e e x p e r i m e n t a r e shown i n Tabl e I as p e r c e n t a g e o f
v i a b l e s e e d s which emerged from t h e s o i l as s e e d l i n g s .
A l f a l f a had an emergence o f 6 0 . 2
%
which is. comparable t o f i e l d emergence.
from t h e O'.5 in'ch s e e d i n g d e p t h
S a i n f o i r i e x h i b i t e d good emergence
from t h e f i r s t f o u r s e e d i n g depths-, f a i r emergence i n t h e 2 . 5 and 3 in ch
seeding, d e p t h s and p oor i n t h e 3 . 5 and 4 in c h d e p t h s .
.In co m p ar is on , a l ­
f a l f a had good emergence i n t h e f i r s t t h r e e ^seeding d e p t h s y f a i r emergence
i n t h e 2> 2^5 and 3 in c h s e e d i n g d e p t h s and poor i n t h e 3 . 5 and 4 in ch
depths.
The e x p e r i m e n t was d e s i g n e d t o p l a c e maximum p r e t i s i o n i n s t a t i s t i c a l
a n a l y s i s on the. s e n d in g d e p t h by legume i n t e r a c t i o n .
f e r e n c e g r e a t e r t h a n P = .05 e x i s t e d between legumes.
A significant d if ­
S ee di n g d e p t h d i f ­
f e r e n c e s were s i g n i f i c a n t a t P = .01 w i t h t h e i n t e r a c t i o n h a vi n g s i g n i f i ­
cance g r e a t e r t h a n P = . 0 5 .
'
Thi s e x p e r i m e n t i n d i c a t e s t h a t s a i n f o i n i s s u p e r i o r t o a l f a l f a in
s e e d l i n g emergence and w i l l prod uce a g r e a t e r p e r c e n t a g e o f s e e d l i n g s t h a n
a l f a l f a from a s e e d i n g d e p t h o f 3 i n c h e s o r l e s s .
I
PROTEIN QUALITY OF SAINFOIN SEED
M a t e r i a l s and Methods
The e x p e r i m e n t h e r e i n d e s c r i b e d was d e s i g n e d t o compare t h e p r o t e i n
q u a l i t y ( e s s e n t i a l amino a c i d b a l a n c e ) o f s a i n f o i n seed (On1ObrvCh i s
v i o i a e f o l i a ) and pigweed seed ( Amaranthus r e t r o f l e x u s ) w i t h t h a t of s o y ­
bean o i l meal (G ly ci n e max) and t o measure i t s e f f e c t on r a t growth and
fe e d c o n s u m p t i o n o
F i f t e e n t h r e e - w e e k - o l d female w h i t e r a t s were d i v i d e d i n t o t h r e e
e q u a l w e ig h t groups o f f i v e r a t s each=
R a t i o n s were f o r m u l a t e d t o c o n t a i n 10% cru de p r o t e i n , on t h e b a s i s
o f work by Rama Rao et_ al_» (35) which i n d i c a t e d t h a t t h e minimum r a t r e ­
q u i r e m e n t o f p r o t e i n f o r maximum n i t r o g e n r e t e n t i o n i s 10% and t h e . m in iv
mum r e q u i r e m e n t f o r maximum growth i s 8=8%= Crude p r o t e i n c o n t e n t s of
u n h u l l e d s a i n f o i n s e e d , soybean o i l meal and pigweed seed u se d i n th e
r a t i o n s were 24=6%, 42.0% and 15=0% r e s p e c t i v e l y =
Table I I shows t h e r a t i o n s used f o r t h e e x p e r i m e n t .
Ta bl e II=
Comp os it ion o f r a t i o n s used t o e v a l u a t e p r o t e i n q u a l i t y of
s a i n f o i n seed me al.
Pigweed seed
meal r a t i o n
S a i n f o i n seed
meal r a t i o n
%
Soybean o i l
meal r a t i o n
■ ■ ’ %
1.0
O
Ingredients
1 =0
U=S=P= 14 s a l t mix
4 =0
4 =0
4 .0
Alpha c e l l u l o s e
5.0
5 =0
5 .0
Corn o i l
1 0 .0
10 =0
10 .0
Corn S t a r c h
13.3
39.3
5 6.2
P r o t e i n s o u r ce
67=7
4 0 ,7
23.8.
Vitamin premix
'•
I
Jl
"16“
Each gr ou p o f r a t s was fed ad l i b i t u m f o r 28 d a y s .
Weights were
t a k e n a t abo ut 10 d ay i n t e r v a l s d u r i n g t h e f e e d i n g t r i a l and fee d con­
sumption was n o t e d .
R e s u l t s and D i s c u s s i o n
The soybean o i l meal r a t i o n and s a i n f o i n seed meal r a t i o n produced
s i g n i f i c a n t l y (P = . 0 5 ) g r e a t e r r a t g a i n s t h a n t h e pigweed seed meal r a ­
t i o n d u r i n g t h e l a s t I 7 days o f t h e f e e d i n g t r i a l
(Table I I I ) .
There was
no s i g n i f i c a n t d i f f e r e n c e between w e ig h t g a i n s o f r a t s fed th b s a i n f o i n
and pigweed r a t i o n s d u r i n g t h e f i r s t I l days o f t h e e x p e r i m e n t ? nor b e ­
tween w e i g h t g a i n s from t h e s a i n f o i n and soybean r a t i o n s t h r o u g h o u t th e
trial.
Ta b le I I I .
Average r a t w e i g h t g a i n s and fee d e f f i c i e n c y d u r i n g a feed
t r i a l cond uct ed u s i n g pigweed seed m e a l, s a i n f o i n seed meal
and soybean o i l meal, as p r o t e i n s o u r c e s . l /
T r ea tm e n t
■ •
In t.
w t.
11 day
gain
Weights
11-21
r day
gain
t a k e n in- grams
21-28 F inal
Total
( day ■ wt.
gain
gain
Avg.
daily
■
G rs.
fe e d
per
100
grs-.
gain
Soybean o i l
meal r a t i o n
5 6 .2
38.6a
28.1a
14.2a
136,7
80'. 5 a
2.88a
39.6
S a i n f o i n seed
meal r a t i o n
55.9
3 1 .5ab
301.4 a
10.5a
128.3
72.4a
2.59a
460
Pigweed seed
meal r a t i o n
5 7 .1
20'. 3b
11.0b
3.3b
91 .7
34 .-6b
1.24b
643
I/
D i f f e r e n c e s among r a t i o n s ar e s i g n i f i c a n t a t P = .05 i f n o t . f o l l o w e d
by t h e same l e t t e r ( 1 2 ) .
Kumpta and H arp er (22) s t a t e d t h a t ,
"A f a l l i n food i n t a k e can u s u a l ­
l y be d e t e c t e d w i t h i n a s h o r t time a f t e r an animal has been fed ad l i b i t u m
s
-17a d i e t i n which an amino a c i d imb ala nc e h as b e e n . c r e a t e d ° ”
w i t h t h e r a t s on t h e pigweed seed r a t i o n .
Thi s o c c u r r e d
They a t e a t o t a l o f 1112 grams
o f f e e d compared t o 1595 grams o f f e e d f o r t h e r a t s on soybean and 1664
grams o f f e e d f o r t h e r a t s on s a i n f o i n .
They were a l s o more nervous arid
i r r i t a b l e t h a n t h e r a t ' s i n t h e o t h e r two g r o u p s .
Thi s a g r e e s w i t h p r e v i o u s
r e s e a r c h by Young and Dunn ( 4 3 ) .
The h i g h e r f e e d consumption and lower f e e d e f f i c i e n c y o f t h e r a t s on
s a i n f o i n as compared t o t h o s e on soybean may have been due t o th e pods on
t h e s a i n f o i n seed which were ground w i t h t h e seed and mixed as p a r t of
the r a t i o n .
These pods were u n p a l a t a b l e and t h e r a t s w as te d p a r t of them.
The pods a r e h ig h i n f i b e r (42) and s ho u ld c o n t a i n l e s s e n e r g y thari an
e q u a l amount i n w e ig h t o f co r n s t a r c h which was t h e i r c o u n t e r p a r t i n t h e
soybean o i l meal r a t i o n .
The a u t h o r s u r m i s e s , as d i d Woodman arid Evans ( 4 2 ) , t h a t m i l l e d s a i n ­
f o i n seed w i t h t h e pods removed would compare much more f a v o r a b l y t o s o y ­
bean o i l meal w i t h r e s p e c t t o p r o t e i n q u a l i t y .
In Eng la nd, s a i n f o i n i s r e p o r t e d t o produce about 600 pounds o f m i l l e d
seed p e r a c r e ( 3 ) .
Compared t o t h e p r e s e n t p r i c e o f soybean o i l me al, t h i s
would be w o r t h abodt $24 p e r a c r e .
year.
S a i n f o i n seed coul d be h a r v e s t e d e v e r y
The v a l u e o f two y e a r s ' p r o d u c t i o n o f s a i n f o i n seed would be e q u a l
t o a 24 b u s h e l wheat cr op i n an a l t e r n a t e y e a r - summer ^al-Iow c ro pp in g
syst em.
The v a l u e o f t h e seed as a p r o t e i n su pplement f o r t h e l i v e s t o c k
i n d u s t r y p l u s t h e f o r etge p r o d u c t i o n o f t h e p l a n t makes s a i n f o i n wor thy
o f c o n s i d e r a t i o h f o r p r o d u c t i o n i n Montana.
SEASONAL CHEMICAL COMPOSITION
M a t e r i a l s and Methods
In o r d e r t o o b t a i n a com parison o f s a i n f o i n w i t h o t h e r f o r a g e l e ­
gumes, weekly h a r v e s t s were made from e i g h t legumes and one g r a s s grown
i n p l o t s l o c a t e d a p p r o x i m a t e l y f i v e m i l e s w e s t of Bozeman oh t h e Montana
A g r i c u l t u r a l Experi ment S t a t i o n , Crops and S o i l s F i e l d R e s ea r c h L a b o r a t o r y .
The f o r a g e s compared were s a i n f o i n fO n o b r v c h i s -v i c i a e f o l i a ) . Ladak a l f a l f a
(Medicago s a t i v a ) , t e t r a p o i d al si k 'e c l o v e r (T r i f o l i u m h v b r i d i u m ) , Kenland
r e d c l o v e r (T r i f o l i u m p r a t e n s e L . ) . Ladino c l o v e r (T r i f o l i u m r e p e n s )■,' Tana ,
b i r d s f o o t t r e f o i l (Lotus, c o r n i c u l a t u s ) , c i c e r mil'kvetch ( A s t r a q u l u s d l c & f ).
s i c k l e m i l k v e t c h ( A s t r a q u l u s f a l c a t u s ) and o f c h a r d g r a s s (D a c t v l i s glomera-ta) =
The h a r v e s t s were made on F r i d a y a f t e r n o o n each week.
c u t t i n g was 2 i n c h e s .
The h e i g h t of
He ight^and s t a g e o f m a t u r i t y were n o te d a t time of
harvest.-' ' The samples were s e l e c t e d a t random frotn t h e p l o t s , p l a c e d in
p a p e r bags and oven d r i e d i n t h e f o r a g e d r y e r a t abo ut 68° C f o r t h r e e
days.
Leaf and stem s e p a r a t i o n s were made a t t h e bay s t a g e .
Ladino c l o ­
v e r was n o t i n c l u d e d i n t h e comp ariso n u n t i l August 3, when samples were'
. i t a k e n on r e g r o w t h from t h e hay c u t t i n g . Dry m a t t e r p e r c e n t a g e was d e t e r ­
mined and samples ground t h r o u g h a 1 /3 2 in c h s c r e e n i n a Wiley m i l l f o r
cru de p r o t e i n d e t e r m i n a t i o n by t h e K j e l d a h l method.
Samples o f l e a f and
stem s e p a r a t i o n from a l f a l f a and s a i n f o i n were an al y ze d by t h e Chem istry
Re s ea rc h L a b o r a t o r y f o r cr ud e f i b e r , e t h e r e x t r a c t , and t o t a l as h .
gen f r e e e x t r a c t was c a l c u l a t e d u s i n g t h e o t h e r v a l u e s .
N itro­
"
20
-
R e s u l t s and D i s c u s s i o n
The r e l a t i v e r a t e o f m a t u r i t y o f s a i n f o i n i n r e l a t i o n t o o t h e r l e ­
gumes and o r c h a r d g r a s s i s shown i n Ta bl e IV=
s t a g e one day e a r l i e r t h a n a l f a l f a .
S a i n f o i n r e a c h e d t h e hay
I t d i d n o t e x h i b i t l e a f l o s s i n com­
p a r i s o n t o a l f a l f a whi ch, a t Bozeman, n o r m a l l y l o s e s some o f i t s lower
l e a v e s b e f o r e r e a c h i n g t h e h ay s t a g e .
S a i n f o i n and t h e two m i l k v e t c h e s
were l e s s a f f e c t e d by f r o s t i n t h e f a i l t h a n t h e o t h e r legumes.
Sainfoin
was t h e t a l l e s t o f a l l legumes d u r i n g t h e development o f t h e f i r s t c u t ­
t i n g , T ab l e V.
Comparative s e a s o n a l v a r i a t i o n i n d r y m a t t e r i s shown i n Ta bl e VI=
V a r i a t i o n s from normal f o r some d a t e s may be a t t r i b u t e d t o r a i n and m o i s - •
t u r e on the f o r a g e when t h e samples were h a r v e s t e d .
S a in fo in did not ex­
h i b i t as h i g h a d r y m a t t e r p e r c e n t a g e as m ig ht be e x p e c t e d c o n s i d e r i n g i t s
h e i g h t and l a r g e , r a n k growing s te m s.
The p r o t e i n c o n t e n t o f s i c k l e m i l k v e t c h was c o n s i d e r a b l y h i g h e r t h a n
t h e o t h e r legumes t h r o u g h o u t t h e e x p e r i m e n t , Ta bl e V II .
S a i n f o i n and
a l f a l f a had a lower p r o t e i n c o n t e n t t h a n p r e v i o u s l y r e p o r t e d ( 4 ) , ( 2 7 ) .
T h i s could have been due t o e n v i r o n m e n t a l , v a r i e t y , o r i n o c u l a t i o n e f f e c t s .
The t r e f o i l p l a n t s were y e l l o w and app ea re d t o be s u f f e r i n g from improper
in o c u la tio n or a lack of n itr o g e n .
T h i s co u ld ac co un t f o r i t s r a p i d d e c l i n e
-in p r o t e i n c o n t e n t .
The p r o t e i n and d r y m a t t e r comp ari so ns were n o t s t a t i s t i c a l l y an al y ze d
b eca use j u s t one sample was t a k e n i n t h e f i e l d t o o b t a i n t h e d a t a of
Table' IV.
Date of
Harvest---.
5-18
5-25
6-1
6-8
6-15
6-20
6-22
O Ag
6-27
6-29
7- 2
7-5
7-6
7-14
7- 20
7-27
Growth s t a g e s o f 8 legumes and o r c h a r d g r a s s on v a r i o u s h a r v e s t d a t e s a t Bozeman
in 1 9 6 2 .1 /
.
-.rc, ,c „ ^
Cicer
Red
Sickle
Ladino
Clover M ilkM ilk S a i n f o i n Clover
vetch '
vetch
F i r s t Cro D Growth
DDG
ve g .
veg.
veg.
veg.
veg.
v eg. *
v eg.
veg.
veg.
veg.
veg.
veg.
veg.
veg.
-- —
veg.
veg.
veg.
veg.
veg.
1 s t buds
veg .•
™““
veg.
1 s t bloom v eg.
1 s t buds
veg.
veg.
veg.
1 s t bloom adv. bud 1 s t buds
1 s t l e a f l o s s 1 s t bud 30% blooir veg.
=C=C=
<===
= ==
===.
75% bloom ===
1 s t buds
1 s t bloom Fullbloom 1 s t bud 20% bloom pre-bloom 1 s t bloom
A lfalfa
5% bloom
35% bloom
===
==—
65% bloom
5% pods
■ 25% pods
50% pods
Alsike
C lov er
B ir d s f o o f
Trefoil
/- . ■
O rch ard grass
veg.
veg.
boot
head in g
hea di n g
p r e -bloom
blooming
a sea
«=C=€=
= ==.,
CO«==C=C=CC
cods form
blooming
75% bloom 75% pods adv.bud fu ll blo om 50% pods 75% bloom ™=blooming
<===
===G™
™=fullbloom
fullbloom ' ™=™*
=C=C=
—
===
===
===
50% bloom C==C=
100%
podE
40%
d
.
f
.
15% d . f . * 35% pods 50% bloom
75% d . f .
s.f.*
50% d . f . 100% pods 15% pods 80% d . f .
s.f.
90% pods ==•=
s.f.
s.f.
s.f.
75% d . f .
75% pods 90% d . f . s . f .
s.f.
s
.
f
.
s
.
f
.
s
.
f
.
s.f.
100% pods
95% d . f .
S .f O
Req rowth Aft e r C u t t i r
veg.
25% bloom v eg.
5% bloom veg.
veg.. 10% bloom veg.
veg."
8 -3 ' .
8-10
15% bloom veg.
veg. 20% bloom veg.
veg.'
5% bloom 40% pods veg.
50% bloom ' veg.
10%. bloom 90% pods v eg.
10% bloom 35% bloom veg. .
8-17 ; i5% bloom
40% bloom
70% bloom ! ve g .
;20% bloom 50% bloom veg.
20% bloom 100% pods veg.
8-24
s.f.
ve g .
30% d . f .
veg.
50% bloom
35% bloom 100%bloom veg.
15% d . f .
8-31
60% d i f . 45% d . f .
9-7 " . 5.6% d , f .
s.f.
veg.
60% d . f .
veg.
75% d . f .
veg.
s.f.
ve g .
85% d . f . I veg.
90% d . f . 90% d . f .
9-14 . 65% d . f .
- 95% d . f .
veg.
.s.f... ■ v e q ■ 95% d .f . .
frosted
. s.f.
D . f . * - 90% d . f... - :vea-o 9-21
T7 Underscored s t a g e s i n d i c a t e d a t e s and s t a g e s c o n s i d e r e d t o be c o r r e c t f o r ha yi ng ;
samples f o r l e a f - s t e m ■s e p a r a t i o n s were t a k e n on t h e s e d a t e s .
*
v eg. = v e g e t a t i v e s t a g e . d . f = = d r i e d f l o w e r s a f t e r - b l o o m i n g , s . f . -■= seeds forming,
p . f . = pods fo r m i n g .
T ab le V°
■...
H e ig h t i n in c h e s o f 8 legumes and o r c h a r d g r a s s on v a r i o u s h a r v e s t d a t e s a t
Bozeman i n 1 9 6 2 o l / - , ~
.■ '
■ ,
,
,
Date o f
H a r v e s t ’ - AlfaTfa-.
in.
12
16
19
22
25
29
“
32
32
=
=
34
36
36
36
5-8
5-25
6=1
6-8
6=15
6-20
6-22
6-26
6-27
6-29
7-2
7-5
746
7- 1 4
7-20
7-27
8-3
8-10
8-17
8-24 .
8-31
9=7
9-14
9-21
Alsike B ird sfo o t
Cicer
Red
Cl o v e r T r e f o i l ' M i l k v e t c h -,Clover
. ' in.
4
9
12
14
20
24
24
24
=
24
24
24
24
14
16
21
22
21 .
18
18
16
/.
l/
7
9
11
10
10 •
10
9
9
iV •'
in.
5
9
12
14
17
20
21
£22
24
24
.24
24
13
13
13
14
14
13
13
13
Sickle
Ladino
M il k v e tc h S a i n f o i n Glover
F i r s t Crop Growth
in.
in.
6
8
10
9
15
15
20
19
21
22
21
27
24
29
30
22
£4
=
24
25
33
29
26
33
29
26
33
29
26
33
29
in.
5
7
11
15
16
Regrowth i a f t e r c u t t i n g
2
16
5
20.
7
4
24
.6
9
10
8
24
10
6
24
10.
8
24
8
23
9
11
23
9
‘ '• 'i'.
Values u n d e r l i n e d a r e h e i g h t s a t hay s t a g e .
in.
8
12
16
20
23
33
34
- ~
36
=
=
38
38
38
38
in.
='
=
9
12
17
18
22
20
20
19,
7
10
10
12
11
10
10
9
Orchard=
"g ra s s
-■
_
=
14
-
.
in.
12
14
19
24
30
44
47
48
'48
48
48
48
7
12
13
14
13
13
13
12
&
I
legumes and o r c h a r d g r a s s on v a r i o u s h a r v e s t d a t e s
T a b l e .VI
•
%
5 “ 18
5=25 ■
6 “1
6-"8
6-15
6-20
6-22
6-26
6-27
6-29
7;-2
7-5
7-6
7- 14
7- 20
7-27
__
23;9
%
%
15.5
14.3
14.4
16.1
16 .3
18.5
19.1
--- — =
-
i—l
= = = =
— —
CM
18 .2
21.9
—
CO
28.4
3 1 .2
21 .1
2 4 .4
24.8
29,6
2 9 .3
3 5 .5
' 38.0
4 5 .0
-
= •’
27.2
=
= —
33 .2
2 3 ,0
—™—™
22.0
2 2 .5
24.5
2 4 ,3
2 3 .3
2 3 .2
23 .6
2 5 .4
20 .9
™—- 23.. 8
2 4 .9
27;3
2 7 .4
2 6 .3
16.0 .
17.-7
18.4
2 2 .7
2 1 .9
27,5
26.7
32 .5
24 .6
25.0 '
26.3
2 9 .0
2 3 .8
28U3
2 8 .5
2 9 ,3
Regrowth a f t e r c u t t i n g
22.8
27..0
19,3
19.7
17.-2
23..-9'
21.3
2 3 ,0
14.9
28.0
2 2 ;0
25.5
2 7 .0
25.0
18 .3
2 6 .3
2 7 .3
2 7 .9
24.0
28.-4
27 .5
2 6 .6
35.6
3 1 .2
^r==-
= = ™ «
33.2
31 .5
31;9
30.3
———
.
r '
-
2 8 ,1
2 7 .3
30.5
30 .5
26;6
30 .0
3 1 .3
42.3.
•
Values u n d e r l i n e d a t e d r y m a t t e r p e r c e n t a g e a t hay s t a g e ,
/
■
16.0.
14.3
1475
15.3
16.4
--= C = C =
%
21.6
14.7
19.2
19.5
24. 3
_ _ _ _
20.9
27.3'
29,5
■.r'-V_
39.1
36. 0
— —— —
29-. 0
----- =
34 .4
37.-5
33.3
34. 7
23.-6
— = =
.
- ■ i”
■%
%
%
F i r s t cr o p growth
10.1
1 8 .0
23 .7
14.4
1 5 .8
17.8
13.7
13.-7
15.9
14 .2
14 .4 .
17.6
13 .7
1 8 .2
1815
-™L
16 .2
2 2 .9
2317
—
•' ■
l/
17.1
1 5 .0
12.9
. 13.3
11.9
■=*■™=
15.5
16.8
14 oS
15.9
17.. 0
18.9
8—3
1S -IQ$-17
'8-24
8“ 3l
9479- 1 4 .
9^21
.
%
%
20.9
20.0
20,9
2612
24; 0
2 6 ,5
29.4
3 1. 2
—
—
17.-4..
2 0. 6
16.6
1915
17.5
24.6
2 5, 9
2 4. 7
4 8, 2
38.9
33.7
49.1
27 .4
2 4, 8
28,9
33.0
29 .6
30.9
35.1
3 2. 2
A
GO
I
T able VII-,
Date o f
Harvest-
Crude’ p r o t e i n c o n t e n t o f 8 legumes and o r c h a r d g r a s s on v a r i o u s h a r v e s t
d a t e s at. Bozem an.in 1962°l /
A lfalfa
'%
Alsike B ird sfo o t
C lo v e r T r e f o i l
%
5- 1 8
5-25
6-1
6-8
6-15
6-20
6-22
6-26
6-27
6-29
7- 2
7-5
7-6
7-14
7-20
7-27
26.3
23.5
2 0 .2
2 2 .2
18.6
2 2 .8
22.6
22.8
21.5
19.5
14.3
1 6 .7
8- 3
8-10
8-17
'8-24
8-31
9-7
9-14
9-21
\J
21 .9
18.0
16.9
1 3 .4
1 2 .3
11.5
8.3
= —™ —
— c= — ™
14b5
8 .8
%
Cicer
Red
Sickle
Ladino
M i lk v e tc h Clove r M il k v e tc h S a i n f o i n Clover
%
%
%
%
%
F i r s t crop growth
31.8
2 1 .3
23. 5
33 .3
22.0
30 .7
2 9 .6
21. 9
————
2 0 .0
22.5
18.9
2 7.9
2 3 .7
2 5 .0
2 0.6
15. 4
20.2
17.6
19.1
14.9
-------— —
“ ———
= =■——
14 .7
1 3 .4
15 .8
13.4
™—™—
— C=
=■———
12.3
■
-- . -----19.2
14.0
T l.4
20.2
9 .2
14.9
14.6
7.9
——— —
™—
™ *=™ —
9 .9
12.9
11.0
1 1 .4
15.4
14.5
12.9
13.6
7.0
6 .8
6 .1
6 .1
1 3 .3
10.8
1 0 .3
11 .0
11.2
22.4
20.2
18.9
19.7
17.1
17.1
16.5
12.5
23.9
22,6
22.6
20.6
2 0 .2
18 .0
1 8 .0
15.8
9 .9
7.5
6 .8
8. 1
8.6
6 .8
6.6
6.4
Regrowth a f t e r
27.4
19.5
27 .6
14.5
25.0
17 .3
22.2
15.8
2 2 .2
16.5
19.5
16.5
17.6
1 5 .8
17 .3
14 .3
= - = -
™“
%
19.7
14.9
12.3
9.7
8.8
7.7
——
—
6 .9
8. 1
~ a ——
--- -----------
“
O r ch ar d qrass
= e = ,= ™
12.5
10.5
12a
12.5
17.6
16.2
16.7
16.7
cutting
31 .8
2 8 .5
29:6
2 7 .4
2 4.6
25 .5
2 5.5
2 3 .5
Values u n d e r l i n e d ar e p r o t e i n p e r c e n t a g e s a t hay s t a g e .
9 .4
7.7
9 .2
6.6
21 .9
18.2
15. 8
15. 8
15.6
13.6
12.1
11. 0
™—— —
6 .8
7 .0
9.2
6.6
21.3
20. 4
21.5
19.5
18.2
19.5
17.6
16.9
16.2
14.7
14. 0
12.7
14. 0
12.5
11.6
9 .9
i
ro
-25T a b l e s VI and V I I .
A c o r r e l a t i o n c o e f f i c i e n t o f - . 8 9 was c a l c u l a t e d f o r
the r e l a t i o n of p ro te in content to dry m atter percentage.
All p r o t e i n ’
v a l u e s ha v i n g a c o r r e s p o n d i n g d r y m a t t e r p e r c e n t a g e o f 20 o r more were
used i n t h i s a n a l y s i s .
T h i s a g r e e s w i t h p r e v i o u s r e s e a r c h which has
shown t h a t p r o t e i n c o n t e n t d e c r e a s e s and d r y m a t t e r p e r c e n t a g e i n c r e a s e s
with p la n t m a tu rity (4),
(19),
(33),
(36).
The h ig h c o r r e l a t i o n of p r o ­
t e i n content to dry m a tte r percentage in d i c a te s t h a t the p r o te i n content
o f legume f o r a g e s mi gh t be a c c u r a t e l y p r e d i c t e d from d r y m a t t e r p e r c e n t a g e
when t h e d r y m a t t e r p e r c e n t a g e exc e ed s 20'.
Leaf and stem c o m p o s i t i o n s o f f o r a g e s i n t h e hay s t a g e ar e shown in
Tabl e V I I I .
Ladirio c l o v e r was n o t i n c l u d e d bec a u se i t i s alm os t a l l l e a v e s
i f t h e p e t i o l e i s c o n s i d e r e d p a r t o f t h e l e a f f r a c t i o n as i t was w i t h t h e
other forages.
S i c k l e m iIkV etch had a h i g h e r p e r c e n t a g e o f l e a v e s th a n
any o f t h e o t h e r f o r a g e s .
M il k v e tc h l e a v e s a l s o c o n t a i n a h i g h e r p e r c e n t ­
age o f t o t a l p l a n t p r o t e i n t h a n any o f t h e o t h e r f o r a g e l e a v e s .
The l e a f
percentage of s a in f o in i s higher than in d ic a te d in previous re s e a rc h , 43.8
as compared t o 3 2 . 3 as r e p o r t e d by Baker ( 4 ) .
Th is m i g h t be ac co un ted f o r
by h i s i n c l u s i o n o f t h e i n f l o r e s c e n c e i n t h e stem p o r t i o n , whereas i t was
included in the l e a f p o r ti o n in t h i s experiment.
The a l f a l f a l e a f p e r ­
c e n t a g e was n e a r l y t h e same as shown i n p r e v i o u s r e s e a r c h , 39.1 as compared
t o 4 0 . 4 r e p o r t e d by Norton ( 2 9 ) .
The n u t r i e n t c o n t e n t o f s a i n f o i n hay and i t s l e a f and stem s e p a r a ­
t i o n s , as compared t o c o r r e s p o n d i n g a l f a l f a v a l u e s , i s r e p o r t e d i p Tabl e IX.
Table V I I I .
Lea f- Ste m comparison o f 7 legumes and o r c h a r d g r a s s a t hay s t a t e a t BOzeman
. i n 1962.
Ledume
B ird sfo o t Cicer
Red
Sickle
S a in ­ O rc ha rd T r e f o i l M ilk ve tch Clover M ilk ve tc h f o i n
grass
A lfalfa
Alsike
Cl o v er
6-27
5%
Bloom
7-2
Full
Bloom
6- 2 0
75%
Bloom
7-5
50%
Bloom
6=29
Full
Bloom
32
24
20
24
24
, 23.7
8. 6
14.5
2 1 .3
7 .0
14.6
1 8.2
6.6
11 .5
18.6
6. 6
13.3
Leaf d r y m a t t e r %
Stem d r y m a t t e r %
Total dry m atter %
2 7 .2
29.1
2 8 .3
2 5 .3
18.1
21 .9
18.4
18.6
1 8.5
l e a v e s by w ei g h t
39.1
5 3 .2
6 3 .9
77 .6
V
Date o f h a r v e s t
Sta ge o f growth
H ei g h t
Leaf p r o t e i n %
Stem p r o t e i n %
Total p ro te in %
%
to ta l protein
in leaves
6-27
Pods
Forming
6-26
50%
Bloom
6-27
Bloom
Stage
29
34
48
18 .0
6.4
11 .4
26.1
9 .4
19.2
2 0 .2
6 .1
12. 2
9.4
4. 1
6.9
2 3 .8
23 .7
23 .7
28.1
26 ;4
27.2
35.9
2 9 .4
3 3.2
28 .3
2 7 .3
2 7 .3
41. 5
36. 4
39.1
4 2 .5
56.1
4 6 ,2
58. 6
43.8
53.6
6 7 .2
78 .5
72 ,9
79.7
72.5
73.0
%
-2 7 Tabl e IXo
N u t r i e n t c o m p o s i t i o n o f I q a f and sje'ni s e p a r a t i o n s o f a l f q l f a
and s a i h f e i n bays grown sit Bozeman 'in 1^62.
Lqaves
Sainfoin
• A lfalfa 1
' " % : % ■
Crude P r o t e i n
Ether Extract
Crude F i b e r
No' F r e e E x t r a c t
T o t a l Ash
2 2 .9
2 .9
16 .2
41.6
1 0 .4
1,9^0
.2.3
15.0
50.4
6 .9
Stems
Sainfoin
. %
A lfalfa
%
Crude P r o t e i n
Ether E x tract
Crude F i b e r
No F r e e E x t r a c t
T o t a l Ash
8.6
1.1
43.5
■ 35.1
6.0
•
5 .7
1.0
3 4 .3
4 7 .9
5.7
Total
A lfalfa
%
Crude P r o t e i n
Ether E xtract
Crude F i b e r
N; F r e e E x t r a c t
T o t a l Ash
1 4 .2
1.8
32 o8
37.6
7.7
Sainfoin
%
11 .5
1. 6
2 5 .8
4 9 .0
6.2
■
'_, ■
-2 8 S a i n f o i n had a lower p r o t e i n and cru de f i b e r c o n t e n t t h a n a l f a l f a which
r e s u lt e d in a higher n itro g en fre e e x t r a c t value for s a in fo in .
These
r e s u l t s a g r ee w i t h t h o s e r e p o r t e d by Baker (4) and M o r r is o n ( 2 7 ) ; and
i n d i c a t e t h a t s a i n f o i n mi gh t have p o s s i b i l i t i e s as a s i l a g e legume, due
t o i t s w id e r c a r b o h y d r a t e - p r o t e i n r a t i o .
This also p la c e s a higher value
on s a i n f o i n hay which e v i d e n t l y c o n t a i n s more n u t r i e n t s t h a n i t s l a r g e ,
r o u g h , stemy ap p e a r a n c e i n d i c a t e s .
The d a t a o b t a i n e d comparing t h e s e a s o n a l che m ica l c o m p o s i t i o n of
s a i n f o i n t o o t h e r legumes grown i n Montana i n d i c a t e s t h a t s a i n f o i n may be
of sim ilar n u tr ie n t v alu b .
I
ESTROGENIC ACTIVITY.
M a t e r i a l s and Methods
The f o r a g e s p e c i e s used f o r t h i s e x p e r i m e n t were t h e same as t h o s e '
used f o r s e a s o n a l ch em ic a l composition=
Three h a r v e s t s were made from t h e same p l o t s on t h d e x p e r i m e n t a l
f arm :
Io
V egetative -
h a r v e s t e d a t a c o n s t a n t d a t e -= June 20'=
2=
Hay stag e. -
h a r v e s t e d as s p e c i e ma tured t o p r o p e r s t a g e
f o r hay=
3=
Regrowth s t a g e = h a r v e s t e d a t a c o n s t a n t d a t e
August 15=
The samples were c o l l e c t e d i n p l a s t i c bags and f r e s h f r o z e n as soon
as p o s s i b l e t o p r e v e n t a l t e r a t i o n o f e s t r o g e n i c compounds w i t h i n t h e
plants.
They were s t o r e d i n a f r o z e n s t a t e a t a p p r o x i m a t e l y - 2 0 b C. u n t i l
used f o r b i o - a s s a y i n J a n u a r y ,
Female m i c e , 19 t o 21 d ays old were o b t a i n e d from t h e V e t e r i n a r y Re=
s e a r c h L a b o r a t o r y and used as t h e b i o - a s s a y animal f o r d e t e r m i n i n g t h e .
;
e stro g en ic a c t i v i t y of the fo rag es.
The b a s i c mouse r a t i o n c o n s i s t e d o f :
1=5% v i t a m i n premix
■
I
4.0% U=S=P= 14 s a l t mix
5.0% a l p h a c e l l u l o s e
10=0% c o r n o i l
'I
j
20.0% m i l k c a s e i n
;
59.5% co r n s t a r c h
i
None o f t h e s e i n g r e d i e n t s were r e p o r t e d t o p o s s e s s e s t r o g e n i c
I
I
j
I
- 30 activity=
A p r e t r i a l using varying l e v e l s of d i e t h y l s t i l b e s t r o l in d ic a te d
t h a t t h e maximum v a r i a t i o n i n i n i t i a l w e i g h t s o f mice which w i l l produce
a c c u r a t e d a t a i s 3 grams.
A 7 day f e e d i n g p e r i o d was found t o be n e c e s ­
s a r y t o o b t a i n a m e a s u r a b le u t e r i n e growth from an e s t r o g e n i c f e e d .
Thi s
i s i n c o n f l i c t w i t h d a t a by S w i e r s t r a ( 3 8 ) , which i n d i c a t e d t h a t maximum
u t e r i n e growth c o u l d be o b t a i n e d in. a 3 day p e r i o d .
S t r a i n s of mice may
d i f f e r i n t h e i r r e s p o n s e t o e s t r o g e n i c s t i m u l a t i o n and t h e r a t e o f r e ­
sponse must be d e t e r m i n e d b e f o r e r e l i a b l e r e s u l t s can be o b t a i n e d .
This
s t r a i n o f mice r e q u i r e d 60 grams o f mouse r a t i o n t o fee d f i v e mice 7 days
i n comparison t o 50 grams o f c o n t r o l r a t i o n used by B i c k o f f (6) f o r t h e
same p e r i o d .
In t h e f o l l o w i n g e x p e r i m e n t s , t h e mice were weighed t o t h e n e a r e s t
o n e - h a l f gram and s e p a r a t e d i n t o grou ps o f e q u a l body w e i g h t .
v a r i a t i o n i n i n i t i a l w e ig h t was all ow ed i n t r e a t m e n t g r o u p s .
ment was a l l o t t e d f i v e m i c e .
A two-gram
Each t r e a t ­
The mice were s e l e c t e d a t random from t h e
d e s i r e d w e i g h t g r o u p s and d i s t r i b u t e d among..treatment grou ps so t h a t t h e
i n i t i a l w e ig h t o f each t r e a t m e n t gr ou p was as n e a r l y e q u a l as p o s s i b l e .
Each group o f f i v e mice was housed t o g e t h e r i n w ir e l a b o r a t o r y cages f o r
t h e d u r a t i o n of t h e s i x t o seven day f e e d i n g p e r i o d .
The mice were fed
t h e t e s t d i e t i n e q u a l i n c r e m e n t s ea ch d ay u n t i l 60 grams o f th e t e s t
d i e t were consumed by t h e f i v e mice=
Ch loroform was used t o s a c r i f i c e
t h e mice and t h e f r e s h l y . e x c i s e d u t e r i were trimmed, b l o t t e d on t i s s u e
p a p e r and weighed on a R o l l e r - S m i t h b a l a n c e .
.I
=■31 The f i r s t group o f fe e d t r e a t m e n t s was p r e p a r e d w i t h i n c r e a s i n g
l e v e l s o f a d i e ' t h y l s t i l b e s t r o l ( DoE=S. ) t o e s t a b l i s h a u t e r i n e dose r e ­
sponse cu r v e f o r t h e s t r a i n o f mice u s e d .
A stock s o lu ti o n of d i e t h y l -
S t i l b ^ s t r o l was made by mix ing 5 mg. o f t h e d ru g w it h 500 ml. o f
ethanol.
95%
One ml. o f t h i s s o l u t i o n when added t o 100 grams o f mouse.' r a t i o n -
g i v e s a Ool u g / g r . c o n c e n t r a t i o n o f d i e ' t h y l s t i l b e s t r o l =
The s t i l b e s t r o l r a t i o n s were p r e p a r e d by p i p e t t i n g t h e d e s i r e d amount
o f d i e t h y l s t i l b e s t r o l s t o c k s o l u t i o n i n t o 20 ml. o f 95% e t h a n o l and mix­
in g t h i s w i t h 60 grams o f mouse r a t i o n .
The r a t i o n s were d r i e d i n open
a i r a t room t e m p e r a t u r e f o r 48 h o u r s and t h e n fed t o t h e m i c e .
The mouse
r a t i o n s fed i n t h i s t r i a l c o n t a i n e d t h e f o l l o w i n g micrograms o f d i e t h y l =
s t i l b e s t r o l p e r gram o f r a t i o n :
.02,
.04,
. 0 6 , =08, and 0 . 1 and t h e con­
t r o l r a t i o n was t r e a t e d w i t h p l a i n e t h a n o l .
An e x p e r i m e n t was d e s i g n e d t o d e t e r m i n e i f i n c l u s i o n o f a c t u a l f o r a g e
i n t h e r a t i o n would pr od uc e a u t e r i n e r e s p o n s e i n d i c a t i n g e s t r o g e n i c a c t i v ­
ity .
The r a t i o n c o n s i s t e d o f 20 grams o f d r i e d ground f o r a g e mixed w i t h
40 grams o f mouse r a t i o n .
Three s t a g e s ( v e g e t a t i v e , hay and r e g r o w th ) o f
a l f a l f a were fed w i t h two methods o f s t o r a g e .
d r i e d when c o l l e c t e d arid t h e n s t o r e d .
ary a f t e r storage in a frozen s t a t e .
One gfoup o f s t a g e s was
The o t h e r g r oup .w as d r i e d i n F e b r u ­
S a i n f o i n seed was i n c l u d e d i n t h i s
e x p e r i m e n t t o se e i f i t would e x h i b i t e s t r o g e n i c a c t i v i t y i n t h e n a t u r a l
form.
T h i s e x p e r i m e n t produ ced n o n - s i g n i f i c a n t r e s u l t s and d e m o n s t r a te d
t h e need f o r use of s o l v e n t e x t r a c t i o n q e t ^ o d s .
I
-3 2 The f i r s t e x t r a c t i o n p r o c e d u r e used was a method d e s c r i b e d by B ic k off e t ' al.
(6) f o r e x t r a c t i n g e s t r o g e n i c compounds from f r e s h f o r a g e =
F r e s h f r o z e n f o r a g e was ground t h r o u g h t h e s au s ag e g r i n d e r i n th e
Meats L a b o r a t o r y .
A 400-gram p o r t i o n was removed f o r m o i s t u r e d e t e r m i n a ­
t i o n and 100 grams were e x t r a c t e d .
The e s t r o g e n i c e x t r a c t was p r e p a r e d
by m a c e r a t i n g t h e 100 grams o f ground p l a n t t i s s u e w i t h 350 ml. o f a c e to n e
i n a Waring commercial b l e n d e r f o r two m i n u t e s .
The e n t i r e c o n t e n t s o f
t h e b l e n d e r bowl were p l a c e d i n a Buchner f u n n e l and s u c t i o n f i l t e r e d
t h r o u g h Whatman No. I f i l t e r p a p e r .
The f i l t e r cake was washed w i t h ah
a d d i t i o n a l 100 ml. o f a c e to n e and t h e combined f i l t r a t e was c o n c e n t r a t e d
o v e r low h e a t t o a volume o f about 30 ml.
Th is aqueous c o n c e n t r a t e was
t r a n s f e r r e d t o a s e p a r a t o r y f u n n e l and e x t r a c t e d t w ic e w i t h 50 ml. of
ethyl ether.
The e t h e r e x t r a c t s were p u t i n 120 ml. b o t t l e s and allowed
t o e v a p o r a t e under a hood w i t h a s u c t i o n f a n .
The b o t t l e s were corked and
p u t i n a f r e e z e r u n t i l a d d i t i o n t o t h e mouse b i o - a s s a y r a t i o n .
The r e s i d u e
i n s i d e t h e b o t t l e was d i s s o l v e d i n 100 ml. o f 95% e t h a n o l and added t o t h e
mouse r a t i o n a t t h e d e s i r e d l e v e l .
An e x p e r i m e n t was d e s i g n e d t o e s t a b l i s h a dose r e s p o n s e curve w i t h
v a r y i n g l e v e l s o f e x t r a c t from r e d c l o v e r and a l f a l f a i n t h e v e g e t a t i v e
stage.
The r a t i o n s were p r e p a r e d by adding t h e d e s i r e d amount o f e x t r a c t
t o 20 ml. o f 95% e t h a n o l and mixing t h o r o u g h l y w it h 60 grams o f r a t i o n .
The m i x t u r e was al low ed t o d r y f o r 48 h o u r s i n open a i r and fed t o th e
mice.
B i c k o f f et. a l .
(.6) recommended a l e v e l o f 8 t o 10 grams o f f o r a g e
-3 3 e x t r a c t p e r 100 grams o f mouse r a t i o n ' .
L e v e ls o f e x t r a c t from 5 , 10, 15,
20, 40 and 80 grams o f f o r a g e p e r 100 grams o f mouse r a t i o n were f e d .
N o n - s i g n i f i c a n t r e s u l t s were o b t a i n e d from t h i s e x p e r i m e n t ; so a m o d i f i ­
c a t i o n o f t h e e x t r a c t i o n p r o c e d u r e used by S w i e r s t r a (38) was used in
s u b s e q u e n t t r i a l s , h e r e a f t e r r e f e r r e d t o as t h e Canadiah Method -and i s
d e s c r i b e d below.
Samples o f f r o z e n f o r a g e were d r i e d i n t h e f o r a g e d r y e r a t 68° '6.
fo r th ree days.
M o is tu r e p e r c e n t a g e s were c a l c u l a t e d arid t h e d r i e d Varh-
p l e s ground t h r o u g h t h e I / 3 2 in c h s c r e e n i n t h e Wiley m i l l .
S i x t y gfams o f ground f o r a g e were mixed w i t h 200 ml. o f ac e to n e and
150 ml. o f 95% e t h a n o l .
T h i s was r e f l u x e d o v er low h e a t i n a iBOO ml-,
b e a k e r f o r one hour and s u c t i o n f i l t e r e d t h r o u g h Whatman No. I p a p e r i n
- a Buchner f u n n e l .
The f i l t e r cake r e s i d u e was mixed w i t h 150 ml. of
a c e to n e and 150 ml. o f 95% e t h a n o l and r e f l u x e d f o r one more h o u r .
was s u c t i o n f i l t e r e d by t h e same p r o c e d u r e as above .
Th is
The f i l t r a t e s were
combined and e v a p o r a t e d o ve r low h e a t t o a p p r o x i m a t e l y 30 ml.
Th is e x t r a c t
was mixed w i t h 60 grams o f mouse r a t i o n and d r i e d f o r 48 h o u r s b e f o r e b e i n g
fed t o t h e m i c e .
T h i s method produced m e a s u r a b le r e s u l t s when used i n an
experiment with th r e e stag es of a l f a l f a .
The hay s t a g e o f a l f a l f a which
had been s t o r e d i n a d r y form from h a r v e s t u n t i l e x t r a c t i o n was in c l u d e d
i n t h e above e x p e r i m e n t t o check t h e e f f e c t of s t o r a g e on e s t r o g e n i c a c t i ­
vity.
E x t r a c t s o f a l l legume f o r a g e s t a g e s p l u s s a i n f o i n seed were mixed
i n t o mouse r a t i o n s .
O r c h a r d g r a s s e x t r a c t was n o t i n c l u d e d bec au se of a
-3 4 shortage of mice.
The r a t i o n s were d i v i d e d a c c o r d i n g t o f o r a g e growth
s t a g e s and f e d t o mice w i t h i n a tw o-gram"weight gr o up .
A diethylstilbes-
t r o l c o n t r o l r a t i o n o f .0067 u g / g r . o f mouse r a t i o n was fed. d u r i n g t h i s
e x p e r i m e n t as a check a g a i n s t t h e p r e v i o u s l y e s t a b l i s h e d d i e t h y l s t i I b e s t r o l r e s p o n s e cur ve =
The hay s t a g e s of a l l n in e foracjes were a l s o t e s t e d u s i n g t h e Bic ko f f e t aT. e x t r a c t i o n method ( 6 ) . . The r a t i o n s were prepa red , havi ng a
l e v e l o f 27 . 7 5 grams o f e x t r a c t p e r 100 grams o f r a t i o n .
T h i s gave a
comparison o f t h e B i c k o f f and Canadian p r o c e d u r e s f o r o b t a i n i n g e s t r o g e n i c ■
e x t r a c t s from f o r a g e s .
The r e s u l t s o f t h e s e e x p e r i m e n t s where a p p l i c a b l e ^ were s t a t i s t i c a l l y
a n a l y z e d u s i n g Duncan’s m u l t i p l e r a n g e t e s t ( 1 2 ) .
C o r r e l a t i o n s were d e ­
t e r m i n e d on t h e r e l a t i o n o f mouse body w e ig h t t o u t e r i n e w e i g h t i n e s t r o g e n i c a l l y s t i m u l a t e d and u n s t i m u l a t e d mi ce.
E s t r o g e n i c a c t i v i t y o f f o r a g e s where a p p l i c a b l e was c a l c u l a t e d by
r e g r e s s i o n as e q u i v a l e n t t o t h e micrograms o f d i e t h y l s t i l b e s t r o l p e r pound
o f d r y m a t t e r r e q u i r e d t o prod uce an e q u a l r e s p o n s e i n t h e b i o - a s s a y
anim als.
R e s u l t s arid D i s c u s s i o n
A ta b le of r e s u l t s i s not p resen ted fo r the p r e t r i a l .
However-, v a l ­
u a b l e e x p e r i e n c e and i n f o r m a t i o n f o r s u c c e e d i n g t r i a l s was o b t a i n e d from
it.
data.
The r a n g e i n i n i t i a l w e i g h t o f t h e mice was t o o wide t o g ive r e l i a b l e
E rror in form ulating l e v e ls of d i e t h y l s t i l b e s t r o l fo r the r a t i o n
“ 35“
a l s o d e c r e a s e d t h e a c c u r a c y o f t h e experiment=
The d a t a d i d i n d i c a t e t h a t
t h r e e grams was t h e maximum v a r i a t i o n i n i n i t i a l w e i g h t s o f mice w i t h i n
an e x p e r i m e n t which would g iv e r e l i a b l e r e s u l t s =
f o r a m e a s u r a b le amount o f u t e r i n e growth=
Seven days yrere r e q u i r e d
The mice r e q u i r e d 12 grams o f
f eed each f o r a s e v e n - d a y f e e d i n g p e r i o d = T h i s knowledge was e s s e n t i a l
to proper conduction of following t r i a l s =
A dose r e s p o n s e cu r v e was e s t a b l i s h e d u s i n g i n c r e a s i n g l e v e l s of
d i e t h y l s t i l b e s t r o I ( F i g u r e I=)=
The u t e r i n e w e i g h t s i n c r e a s e d up t o th e
=06 ug/gr= o f c o n t r o l r a t i o n l e v e l and s t a r t e d t o d e c l i n e i n t h e two h i g h ­
e r l e v e l s f e d (Tab le X) =
A c o r r e l a t i o n was c a l c u l a t e d on t h e r e l a t i o n o f
i n i t i a l w e i g h t o f mice t o t h e r e s u l t a n t u t e r i n e w e i g h t a f t e r t r e a tm e n t=
There was no s i g n i f i c a n t r e l a t i o n s h i p o f body w ei g h t t o u t e r i n e w ei g h t
o f mice on c o n t r o l r a t i o n s c o n t a i n i n g no e s t r o g e n i c s t i m u l a t i o n =
A sig­
n i f i c a n t p o s i t i v e c o r r e l a t i o n c o e f f i c i e n t o f =44 ( s i g n i f i c a n t a t P = =05)
was c a l c u l a t e d f o r r e l a t i o n o f body w e i g h t t o u t e r i n e w e i g h t o f mice on
ra tio n s'c o n ta in in g d ie th y ls t i l b e s t r o l =
shown i n F i g u r e 1°
The r e g r e s s i o n l i n e f o r t h i s i s
Al l f o l l o w i n g d a t a was s t a t i s t i c a l l y a n a l y z e d . u s i n g
u t e r i n e w e i g h t as p e r c e n t a g e o f body w e i g h t which was c o n s i d e r e d th e
most a c c u r a t e i n d i c a t i o n o f t h e e s t r o g e n i c a c t i v i t y o f a t r e a tm e n t=
The e x p e r i m e n t h a v i n g a c t u a l ground f o r a g e as p a r t o f t h e mouse r a ­
t i o n d i d n o t p r o du ce any s i g n i f i c a n t e s t r o g e n i c s t i m u l a t i o n (Table Xl)=
There co ul d have been an a l t e r a t i o n o f e s t r o g e n i c a c t i v i t y due t o t h e
d i f f e r e n c e i n methods o f d r y i n g whicft had been used on t h e a l f a l f a b u t
none occurre d=
S a i n f o i n seed was 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 i n e s t r o g e n i c
-3 6 -
dose
response
curve
U t e r i n e w e i g h t as
percentage of
body w ei g h t
regression
Micrograms o f DcE=S= p e r gram o f c o n t r o l r a t i o n
F i g u r e 1=
D i e t h y l s t i l b e s t r o l dose r e s p o n s e cu rv e and r e g r e s s i o n of
u t e r i n e w e ig h t as p e r c e n t a g e o f body w e ig h t on micrograms
o f d i e t h y l s t i l b e s t r o l i n fe e d a t l e v e l s o f O t o =06 m i c r o grams p e r gram o f feed=
-3 7 Tabl e Xo
Estrogenic a c t i v i t y of v ario u s l e v e ls of D i e t h y l s t i l b e s t r o l
added t o a c o n t r o l r a t i o n =
Mic r o grams
Do-Eo-So p e r
gram o f r a t i o n
A c tu a l
No. o f mice
Average
Average u t e r i n e
Amount o f
per
u t e r i n e w t. w e i g h t as p e r DoE=So i n f eed T r e a tm e n t i n m i l l i g r a m s c e n t a g e o f body
-we I q h t
5
11.7
.09740
.02
o12
5
48.42
.34000
.04
.24
5
53.34
.37857
06
81.40
.57115
69.08
.54719
5 4 .0
.40005
5
08
00
.0
<o
CO
Control
5
01
.60
5
“3 8 Table' XIo
E strogenic a c t i v i t y of th r e e stag es of a l f a l f a with d i f f e r e n t •
s t o r a g e p r o c e d u r e s and s a i n f o i n seed when fed w i t h o u t e x t r a c ­
t i o n as p a r t o f t h e mouse r a t i o n =
Trea tme nt
A c tu a l grams o f
Dry M a t t e r i n
Fe-ed
Number o f
Mice p e r
T r ea tm e n t
Average
Average
U t e r i n e Weight U t e r i n e Weight
i n M i l l i g r a m s as P e r c e n t a g e
■
' of Body Wt=
A l f a l f a f r o z e n , s t o r e d and t h e n d r i e d
Veg= s t a g e
20
5
10 =32
=07381
Hay s t a g e
20
5
9 .9 2
=06815
Regrowth s t a g e
20
5
11 =24
=07497
A l f a l f a d r i e d im m e d i a te l y a f t e r h a r v e s t
and s t o r e d
Veg= s t a g e
20
5
10 =54
.06522
Hay s t a g e
20
5
11 =74
.07535
Regrowth s t a g e
20
5
10 =56
=07273
20
5
11 =76
=08885
0
5
11 =48
=08234
S a i n f o i n seed
Control
l/
D i f f e r e n c e s between t r e a t m e n t s were n o t s i g n i f i c a n t a t P = .0 5 .
“3 9 a c t i v i t y from any o f . t h e a l f a l f a t r e a t m e n t s =
The mice wa sted a l a r g e
amount o f t h e f i b r o u s m a t e r i a l i n t h e r a t i o n s which would te n d t o r e d u c e
t h e a c c u r a c y of t h e d e t e r m i n a t i o n even i f e s t r o g e n i c a c t i v i t y had been
observed.
No s i g n i f i c a n t d i f f e r e n c e s i n u t e r i n e r e s p o n s e between legumes or
t r e a t m e n t l e v e l s o c c u r r e d when s i x d i f f e r e n t l e v e l s o f v e g e t a t i v e s t a g e s
o f a l f a l f a and r e d c l o v e r were t e s t e d u s i n g t h e B i c k o f f e x t r a c t i o n p r o ­
ce dur e (6) as p a r t o f t h e mouse r a t i o n , Table X I I .
B i c k o f f ejt aT. (6)
recommends t h a t t h e e x t r a c t from 8 t o 10 grams o f f o r a g e -be added t o each
100 grams o f r a t i o n .
L e v e ls o f e x t r a c t ra n g ed from 5 grams p e r 100 grams
o f r a t i o n t o 80 grams p e r 100 grams o f r a t i o n w i t h o u t p r o d u c i n g an i n d i c a ­
t i o n of e s tr o g e n ic a c t i v i t y .
Red c l o v e r i n t h e v e g e t a t i v e s t a g e was l a t e r
shown t o have t h e h i g h e s t e s t r o g e n i c a c t i v i t y o f a l l t h e s t a g e s of f o r a g e s
(Table XIV).
The f a i l u r e o f t h e B i c k o f f e x t r a c t i o n p r o c e d u r e (6) could
have been due t o many f a c t o r s .
E s t r o g e n i c compounds o c c u r as min ute quan­
t i t i e s i n f o r a g e even though t h e i r e f f e c t on anim als i s sometimes q u i t e
im portant.
E s t r o g e n s ar e e a s i l y d e s t r o y e d by h e a t and t h i s could have
occurred during e x t r a c ti o n .
The h e a t was -kept as low as p o s s i b l e t o p r o ­
duce e v a p o r a t i o n o f t h e a c e to n e and a l c o h o l , b u t may have been t o o high
at tim es.
E x t r a c t s from t h r e e s e p a r a t e e x t r a c t i o n s o f r e d c l o v e r were used
i n t r y i n g t o e s t a b l i s h a dose l e v e l f o r t h e B i c k o f f e x t r a c t i o n p r o ce d u r e
(6) and none o f them p ro du ced a r e s p o n s e i n d i c a t i n g t h e p r e s e n c e o f e s t r o ­
genic a c t i v i t y .
At l e a s t one o f t h e s e , e x t r a c t s s hou ld have had a smal l
amount o f e s t r o g e n i c a c t i v i t y when compared t o t h e r e s u l t s o b t a i n e d from
-
/•
-
-4 0 Tabl e XIIo
E s t r o g e n i c a c t i v i t y o f i n c r e a s i n g l e v e l s o f e x t r a c t from
v e g e t a t i o n s t a g e s o f a l f a l f a and r e d c l o v e r u s i n g B ic k o f f
F r e s h Fo rage E x t r a c t i o n M et ho d .l /
Level o f e x t r a c t A c tu a l grams
Number o f Average
Average u t e r fed p e r 100 g r s .
of dry m atter
p i c e p e r u t e r i n e wt.
i n e wt.. as
o f c o n t r o l r a t i o n e x t r a c t ',in fe e d t r e a t m e n t i n m i l l i g r a m s p e r c e n t a g e o f
bodv weig ht
A lfalfa■
5 grams
00 .8 8
5
10.48
o07527
10 grams
I o75
5
11.12
.08184
15 grams
2 .6 3 ,
5
14.0 4
.10373
20 grams
3 .5 0
5
11.52
.08372
40 grams
7.00
5
11.22
.07985
80 grams
14.0 0
4
11.70
.08526
5 grams
00.75
5
16.30
.10249
10 grams
1 .5 0
5
12.38
.09206
15 grams
2 .2 5
5
10.84
.08534
20 grams
3.00'
5
11.4 2
.07885
40 grams
6 .0 0
5
12.80
.09306
80 grams
12.0 0
5
12.04
.08605
5
11.70
.09740
Red Clove r
Control Ration
l/
0
D i f f e r e n c e s between c r o p s , c r o ps and c o n t r o l , o r t r e a t m e n t l e v e l s
were n o t s i g n i f i c a n t a t P = . 0 5 .
-.41t h e Canadian e x t r a c t i o n method (38) which i s a much l e s s r e f i n e d t e c h - ' •
nique.
These r e s u l t s w i l l be d i s c u s s e d l a t e r .
Two s e p a r a t e t r i a l s were r u n w i t h t h r e e s t a g e s o f a l f a l f a u s i n g t h e
Canadian e x t r a c t i o n p r o c e d u r e ( 3 8 ) .
S ig n if ic a n t d iffe re n c e s in estro g en ic
a c t i v i t y were produced b o th t i m e s showing t h e r e g r o w th sta ge, o f a l f a l f a t o
be h ig h i n e s t r o g e n i c a c t i v i t y (Tab le X I 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
o c c u r r e d between t h e two methods o f s t o r a g e used on t h e hay s t a g e .
The r e p e a t a b i l i t y of" a p r o p e r l y co n du ct ed b i o - a s s a y p r o c e d u r e i s
shown i n Ta b le X I I I .
The n e x t e x p e r i m e n t c o n t a i n i n g e i g h t legutne f o r a g e s a t t h r e e s t a g e s
o f growth ( v e g e t a t i v e , hay and r e g r o w t h ) and s a i n f o i n seed e x t r a c t e d by
t h e Canadian method (38) a l s o p r od uce d p o s i t i v e r e s u l t s (Ta b le XIV).
The
t h r e e growth s t a g e s o f r e d c l o v e r ( v e g e t a t i v e , hay and r e g r o w t h ) and a l ­
f a l f a i n t h e r e g r o w t h s t a g e produ ced s i g n i f i c a n t d i f f e r e n c e s i n e s t r o g e n i c
activity.
Red c l o v e r i n t h e v e g e t a t i v e s t a g e was s i g n i f i c a n t l y more e s t r o ­
g e n i c t h a n t h e o t h e r two s t a g e s o f r e d C lov er o r t h e a l f a l f a r e g r o w th s t a g e .
S a i n f o i n f o r a g e e x t r a c t i n any o f t h e t h r e e s t a g e s and s a i n f o i n seed d i d
n o t show a s i g n i f i c a n t r e s p o n s e .
Al l t h e mice on t h e v e g e t a t i v e and r e -
growth s t a g e s o f s i c k l e m i l k v e t c h d i e d w h i l e on e x p e r i m e n t .
T h i s was due
t o an un d e t e r m in e d s u b s t a n c e i n t h e s i c k l e m i l k v e t c h e x t r a c t .
S i g n i f i c a n t d i f f e r e n c e s r e s u l t e d from t h e e x p e r i m e n t c o n t a i n i n g t h e
n i n e f o r a g e s i n t h e hay s t a g e u s i n g t h e B i c k o f f e x t r a c t i o n method (6)
(Tab le XV) .
However, t h e s e were n o t r e l i a b l e when. compared t o t h e same
f o r a g e u s i n g t h e Canadian e x t r a c t i o n p r o c e d u r e ( 3 8 ) .
Red c l o v e r shows
-4 2 Tabl e XIII=
T r ea tm e h t
E s t r o g e n i c a c t i v i t y o f t h r e e s t a g e s o f a l f a l f a d u r i n g tiW
t r i a l s u s i n g . t h e Canadian E x t r a c t i o n Metho d= !/
A c tu a l grams
of dry m atter
e x t r a c t 'In) fee d
Number o f
mice p e r
treatm ent
I— 1|
I
Trial
Average
u t e r i n e wt=
in m illigrams ,
Average u t e r ­
in e wt . as
percentage of
. body weight
.
A lfalfa
*
Veg= s t a g e
60
5
8 .7 4
=06455 b
Hay s t a g e
60
5
10=14
.07969 b
Regrowth s t a g e
60
5
19 =46
=13977 a
0
5
11 =48
=08234 b
60
5
9 =84
=07590 b
Control
Hay s t a g e
(summer d r i e d )
■Trial No=- 2
A lfalfa
Veg., s t a g e
60
5
11 =48
=08834 b
■Hay s t a g e
60
5
10 =94
.07643 b
''R'egm&wth s t a g e
60
5
15 =54
=11341 a
l/
Values f o l l o w e d by same T e t f e r ar e n o t s i g n i f i c a n t a t P = =05="
Tabl e XIV°
E s t r o g e n i c a c t i v i t y o f - th re e s t a g e s o f legumes and s a i n f g i n seed u s i n g t h e
Canadian E x t r a c t i o n Method.%/
. . . . . . .
T r ea tm e n t
Date o f Stage o f
H a r v e s t Growth
Red c l o v e r
A lfalfa
C i c e r mv.
Ladino c l o v e r
Alsike clover
Sainfoin
B. t r e f o i l
S i c k l e mv.
6-11-62
6-11-62
6= h -62
6=11-62
6=11-62
6=11-62
6-11-62
6-11-62
Red c l o v e r
S a i n f o i n seed
A lfalfa
C i c e r my.
Ladino c l o v e r
B. t r e f o i l
S i c k l e mv.
Alsike clover
Sainfoin
.0067 D .E.S.
6=29-62 f u l l bloom
—C= ==———™——
6=27-62 5% bloom
7-5-62 50% bloom
7-3=62 f u l l bloom
6-20-62 75% bloom
6-27-62 pod forming
7-2=62 f u l l bloom
6-26=62 50% bloom
control ra tio n
A lfalfa
Red c l o v e r
Sainfoin
B. t r e f o i l
Alsike clover
C i c e r mv.
Ladino c l o v e r
S i c k l e mv.
8 - 21 - 62
8 - 21 - 62
8-21-62
8-21-62
8-21-62
8 - 21 - 62
8-21-62
8-21-62
. 7
17
Dry M a t t e r
Percentage
veg.
veg.
veg.
veg.
v eg.
veg.
veg.
veg.
15% bloom
50% bloom
10% bloom
90% pods
10% bloom
veg.
35% bloom
veg.
13.8
17.8
1 5 .5
11 .5
12.0
17.1
14.1
20.3
30.8
— ——
33:3
24.4
2 3 .6
18.6
27.2
22 .1
28.8
2 7 .2
2 2 .3
22 .1
2 5 .3
2 0 .7
1 9 .2
17 .8
.25.0
Number o f Mice1 Average U t e f P e r T r ea tm e n t
i n e Wt. i n
Milligrams
.
5
22 .3 4
5
11.48
5
7 .9 8
5
7.36
5
7 .7 4
5
7.64
6 .2 0
5
Mice a l l d ie d —- ===
Average U t e r i n e Wt= as P e r c e n t a g e o f Body
Weight
.20845 a
.08834 b
.06594 b
.06179 b
.06057 b
.05682 b
.05000 b
5
5
5
5
5
4
5
5
5
5
18.20
12.6 8
10.94
11.46
10.86
9 .4 8
7. 56
9 .5 8
9 .8 4
17.50
.12257
.08176
.07643
.07253
.07032
.06975
.06697
.06657
.06650
.15735
a
b
b
b
b
b
b
b
b
5
5
5
5
5
5
5
Mice a l l d i e d
15.54
13.84
10.54
9 .1 8
9 .2 2
9 .1 0
8.00
====
.11341
.11273
.07972
.06883
.06875
.06533
>05965
a
a
b
b
b
b
b
Values w i t h i n a growth s t a g e f o l l o w e d by t h e same l e t t e r ar e n o t s i g n i f i c a n t a t P = .05 .
Values u n d e r l i n e d ar e s i g n i f i c a n t t o P = .05 w i t h i n t h e i r p a r t i c u l a r l e g i m e .
1
6
1.
“44=
Tabl e XV,
E s t r o g e n i c a c t i v i t y com par iso n o f B i c k o f f F r e s h Forage
E x t r a c t i o n Method w i t h Cana di an Dry Forage E x t r a c t i o n
Method on hay f o r a g e s t a g e o f v a r i o u s l e g u m e s . l /
Tr ea tm e n t
A c tu a l grams
Number o f Average.
' Average u t e r of dry matter
mice p e r u t e r i n e w t.
in e wt . as
e x t r a c t i o n f eed t r e a t m e n t i n m i l l i g r a m s p e r c e n t a g e o f
body weight.. .
BiCkoff E x t r a c t i o n Method
27 .7 5 grams o f f o r a g e p e r
100 grams o f c o n t r o l r a t i o n
•
A lfalfa
5.25
5
10.76
.08772 a
Control ■
O
5
11.4 8
.08234 a b
Sainfoin
4.63
5
7.88
.07099 a b c
Ladino c l o v e r
4.10
5
9.06
.06908 a b c
B, t r e f o i l
2.87
4
.9 .4 3
=06714 b c
S i c k l e mv.
4.42
5
9.18
.06487 b c
Orchardgrass
6 .4 3
5
8.4 8
i 06445 b c
C i c e r my.
3-. 88
5
7.98
.06273 c
Red c l o v e r
4.79
5
8.4 8
.06264 c
Alsike clover
3.54
5
7 .5 0
.05670 c
Canadian Drv E x t r a c t i o n Method
Red c l o v e r
60
5
18.20
.12257 a
A lfalfa
60
5
10 ,9 4
.07643 b
C i c e r mv.
60
5
11.46
.07253 b
Ladino c l o v e r ■
60
5
10.86
.07032 b
B. t r e f o i l
60
5
9.48
.06975 b
S i c k l e mv.
60
5
7.5 6
.06697 b
Alsike clo v er
60
5
9 .5 8
.06657 b
Sainfoin
60
5
9 .8 4
.06650 b
I/
Values fo ll o w e d by same l e t t e r . a r e n o t s i g n i f i c a n t a t P = .05 T
-4 5 no e s t r o g e n i c a c t i v i t y i n t h e f i r s t p r o c e d u r e and e x h i b i t e d s i g n i f i c a n t
a c t i v i t y i n t h e second (Can adi an) pro ced ur e=
The a u t h o r would recommend
use o f t h e Canadian method i f f u r t h e r r e s e a r c h i s con d uc te d w i t h f o r a g e
e s t r o g e n s as i t r e q u i r e s l e s s p r e c i s e che m ic a l methods.
The e s t i m a t e d d i e t h y l s t i l b e s t r o l e q u i v a l e n t p o t e n c y o f t h e e s t r o g e n i c
p r o d u c i n g f o r a g e s was c a l c u l a t e d by r e g r e s s i o n ( F ig u r e I ) ,
The r e g r e s s i o n
l i n e was c a l c u l a t e d u s i n g t h e r e s u l t s o f t h e f i r s t t h r e e l e v e l s of d i e t h y l s t i l b e s t r o l on t h e dose r e s p o n s e cu r ve and t h e c o n t r o l r a t i o n .
The d i ­
e t h y l s t i l b e s t r o I check used i n th e e x p e r i m e n t w i t h a l l s t a g e s o f f o r a g e s
was compared t o t h i s l i n e and t h e n was used as p a r t o f t h e r e g r e s s i o n
f o r m u l a t o c a l c u l a t e t h e e s t i m a t e d micrograms o f d i e t h y l s t i l b e s t r o I p e r
pound o f fe e d needed t o prod uce t h e same r e s p o n s e as p r odu ced by th e f o r ­
age (Table XVl)=
Red c l o v e r i n t h e v e g e t a t i v e s t a g e e x p r e s s e d a s i g n i f i ­
c a n t l y h i g h e r e q u i v a l e n t i n micrograms o f d i e t h y l s t i l b e s t r o I p e r pound of
d r y m a t t e r t h a n any o f t h e o t h e r f o r a g e s t a g e s which p o s s e s s e d e s t r o g e n i c
activity.
These v a l u e s . a r e s i m i l a r t o t h o s e o b t a i n e d i n o t h e r r e s e a r c h
(2 1 ), (3 2 ), (3 8 ).
S a i n f o i n seed and f o r a g e i n t h r e e gro wt h s t a g e s d i d n o t show any
s i g n i f i c a n t e s t r o g e n i c a c t i v i t y when compared w i t h t h e o t h e r legumes.
-4 6 “
Tabl e XVI=
Estimated d i e t h y l s t l l b e s t r o I equivalency of red clover
a n d , . a l f a l f a . i n micrograms p e r . pound o f d r y m a t t e r . l /
S ta g e o f growth
■and legume
Red c l o v e r
veg =
A l f a l f a regrowth
, Red c l o v e r
Red c l o v e r
l/
E s t i m a t e d p o t e n c y i n micrograms
per. pound o f d r y m a t t e r
6 =78 a
2=12
hay
1=87 b
regrowth
1=31 b
b■
Values f o l l o w e d by same l e t t e r a r e n o t s i g n i f i c a n t a t P - =05.
SUMMARY
S a i n f o i n i s a d e e p - r o o t e d , l o n g - l i v e d , t e t r a p o i n d p e r e n n i a l legume
p a r t i c u l a r l y ad a p t e d t o d r y , w e l l - d r a i n e d c a l c a r e o u s s o i l s .
I t is reported
n o t t o ca us e b l o a t and o u t y i e l d e d a l l o t h e r legumes a t t h e Montana A g r i c u l ­
t u r a l Expe rim ent S t a t i o n when h a r v e s t e d f o r o n l y one c u t t i n g o f hay.
An e x p e r i m e n t was cond uct ed comparing s a i n f o i n and a l f a l f a s e e d l i n g
emergence from e i g h t d i f f e r e n t s e e d i n g d e p t h s .
S a i n f o i n was found t o p r o ­
duce a s i g n i f i c a n t l y h i g h e r p e r c e n t a g e o f s e e d l i n g s t h a n a l f a l f a from a
s e e d i n g d e p t h o f t h r e e i n c h e s or l e s s .
S e e d l i n g emergence o f b o th s p e c i e s
s i g n i f i c a n t l y d e c r e a s e d from a s e e d i n g d e p t h o f two in c h e s o r more.
The
v a r i e t y by s e e d i n g d e p t h i n t e r a c t i o n excee ded P - . 0 5 .
T h r e e - w e e k - o l d female w h i t e r a t s were used i n a f e e d i n g t r i a l t o e v a l ­
uate the p r o t e i n q u a l i t y of s a in f o i n seed.
S a i n f o i n seed meal was found
t o be comparable i n p r o t e i n q u a l i t y t o soybean o i l meal and t h u s w a r r a n t s
c o n s i d e r a t i o n as a s o u r c e o f s u p p l e m e n t a l p r o t e i n f o r l i v e s t o c k f e e d i n g .
An e x p e r i m e n t was c a r r i e d o u t t o compare t h e s e a s o n a l chem ica l compo­
s i t i o n o f s a i n f o i n w i t h Ladak a l f a l f a , t e t r a p o i d a l s i k e c l o v e r , Tana b i r d s =
foot t r e f o i l ,
c i c e r m i l k v e t c h , Ladino c l o v e r , r e d c l o v e r , s i c k l e m i l k v e t c h
and o r c h a r d g r a s s .
The d a t a was c o l l e c t e d from weekly samples o f f o r a g e
t a k e n from p l o t s a t t h e Montana A g r i c u l t u r a l Experiment S t a t i o n , Crops and
S o i l F i e l d R e s ea r c h L a b o r a t o r y .
The s e a s o n a l p r o t e i n c o n t e n t o f s a i n f o i n
was found t o d e c r e a s e w i t h adva nc ing m a t u r i t y a t about t h e same !,ate as
the o ther cro p s.
decreased.
Dry m a t t e r p e r c e n t a g e i n c r e a s e d as p r o t e i n p e r c e n t a g e
A h i g h n e g a t i v e c o r r e l a t i o n ( - . 8 9 ) was c a l c u l a t e d f o r th e
\
-48r e l a t i o n of p ro te in to dry m a tter percentage.
T h i s coul d l e a d t o a q u i c k ,
c o m p a r a ti v e method o f e s t i m a t i n g cr u de p r o t e i n c o n t e n t o f legume f o r a g e s .
S a i n f o i n m a t u r e s a t abo ut t h e same r a t e as a l f a l f a .
I t has a h i g h e r l e a f
t o stem r a t i o t h a n a l f a l f a and i s lower i n cru de f i b e r and p r o t e i n .
Sain­
f o i n has a h i g h p e r c e n t a g e o f n i t r o g e n f r e e e x t r a c t i n comparison t o a l - '
f a l f a whic h, cou pl ed w i t h i t s lower p r o t e i n c o n t e n t and h i g h y i e l d , makes
i t w or th y o f c o n s i d e r a t i o n as a s i l a g e c r o p .
A b i o - a s s a y f o r e s t r o g e n i c a c t i v i t y u s i n g immature female w h ite mice
was cond uct ed w i t h t h r e e growth s t a g e s ( v e g e t a t i v e , h a y , and r e g r o w t h ) of
t h e same legumes used f o r co m par iso ns o f s e a s o n a l ch em ic a l c o m p o s i t i o n .
The f o r a g e samples were c o l l e c t e d and s t o r e d i n a f r o z e n s t a t e u n t i l ex'- •
traction.
Two methods o f e x t r a c t i o n were u s e d .
The e x t r a c t s were added
t o a c o n t r o l r a t i o n and fed t o t h e mice f o r seven d a y s .
"
The Canadian
method of E x t r a c t i o n pr oved t o be more r e l i a b l e t h a n t h e B i c k o f f method
for e x tra c tin g fresh forage.
All t h r e e s t a g e s o f r e d c l o v e r and t h e r e ­
growth s t a g e o f a l f a l f a prod uced a s i g n i f i c a n t l y h i g h e r e s t r o g e n i c r e s p o n s e
t h a n any o f t h e o t h e r le g u m e s .
S a i n f o i n d i d n o t e x h i b i t a m e a s u r a b le a -
mount o f e s t r o g e n i c a c t i v i t y i n any s t a g e o f growth.
S a i n f o i n p o s s e s s e s p o t e n t i a l f o r b o th f o r a g e and seed p r o d u c t i o n i n
Montana, e s p e c i a l l y i n d r y l a n d a r e a s where one c u t t i n g o f hay i s h a r v e s t e d
o r on i r r i g a t e d la nd where a h i g h - y i e l d i n g s i l a g e cr o p i s d e s i r e d .
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