Bovine plasma analysis for progesterone: a quantitativemethod employing gas liquid chromatography

Item Type text; Thesis-Reproduction (electronic)

Authors Glenn, Lloyd Wayne, 1943-

Publisher The University of Arizona.

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BOVINE PL A SM A ANALYSIS F O R P R O G E S T E R O N E .

A Q U A N T IT A T IV E M ETH O D E M PL O Y IN G GAS

LIQUID CH RO M A TO G RAPH Y

by

L lo y d W ayne G lenn

A T h e s i s S u b m it te d to th e F a c u l ty of the

D E P A R T M E N T OF DAIRY SC IE N C E

In P a r t i a l F u l f i l lm e n t of the R e q u i r e m e n t s F o r th e D e g re e of

M A STE R OF S C IE N C E

In the G ra d u a te C o llege

T H E UNIVERSITY O F ARIZO N A

1 9 6 7 •

STATEM ENT BY AUTHOR

T h is th e s i s h a s b e e n s u b m i t te d in p a r t i a l f u l f i l lm e n t of r e q u i r e m e n ts fo r an a d v a n c e d d e g r e e a t The U n iv e r s i ty of A r iz o n a and is d e p o s i te d in the U n iv e r s i ty L i b r a r y to be m a d e a v a i la b le to b o r r o w e r s u n d e r r u le s of the L i b r a r y .

B r i e f q u o ta t io n s f r o m th is t h e s i s a r e a l lo w ab le w ithou t s p e c ia l p e r m i s s i o n , p ro v id e d th a t a c c u r a t e a c k n o w le d g m e n t of s o u r c e is m a d e . R e q u e s ts fo r p e r m i s s i o n fo r e x ten d e d q u o ta t io n f r o m o r r e p r o d u c t io n of th is m a n u s c r i p t in w hole o r in p a r t m a y b e g ra n te d by the h e ad of the m a jo r d e p a r tm e n t o r the D ean of the G ra d u a te C o lleg e w hen in h is ju d g m e n t the p ro p o s e d u se of the m a t e r i a l is in the i n t e r e s t s of s c h o la r s h ip . In a l l o th e r i n s t a n c e s , h o w e v e r , p e r m i s s i o n m u s t be o b ta in e d f r o m the a u th o r .

SIGNED:

A P P R O V A L BY THESIS D IR E C T O R

T h is th e s i s h a s b e e n a p p ro v e d on the d a te shown below :

u . M. 5 1 V i i D a i ry S c ie n c e D e p a r tm e n t H ead

„ - l i A •S ' S * - 6 7D ate

ACKNOWLEDGMENTS

The a u th o r w is h e s to th a n k D r . G. H. S to t t f o r h is a id in d e s ig n

ing th e p r o j e c t and c o n t in u e d s u p p o r t th a t m a d e th is r e s e a r c h p o s s ib le .

S p e c ia l th a n k s a r e e x te n d e d to M r . F r a n k W hiting and M r . E . H,

P a s q u a l w ith o u t w h o se t e c h n ic a l a id th is p r o j e c t w ou ld h av e b e e n m u c h

m o r e d if f ic u l t and t im e c o n su m in g .

S e v e r a l p e r s o n s lo a n e d e q u ip m e n t f o r th is r e s e a r c h and the

a u th o r w is h e s to th a n k D r . W. H. B ro w n and D r . J . W. S tu l l f o r the

u se of t h e i r gas c h r o m a t o g r a m and o th e r e q u ip m e n t . A ls o , a p p r e c i a

t io n to M r . L e o n a r d S tr a n g of the D a i r y R e s e a r c h C e n te r f o r a id in

h e a t d e te c t io n and c a r e of th e co w s.

My d e e p e s t a p p r e c ia t io n and g r a t i tu d e to m y w ife , L i s a , fo r

h e r u n d e r s ta n d in g p a t ie n c e and e n c o u r a g e m e n t th a t m a d e i t p o s s ib le

to f in i s h th is r e s e a r c h .

TABLE OF CONTENTS

P a g e

T iT 1 I ‘ 1 j F TJ" I R o e o o o o o e o o o o o e e o o o o o o o o o e e e o o e o e o o o e e e o o e

i C * j a e o e o o e e e . e o e o o o o o e o o o o e d a e o o e e e e o e o e o e o o e e e e o o o

T~^J * I ̂R 1 XT T C ' I * T C*)lSJ o o o o o e e e e o o o o e o o e o o o o t t o o o o e e e o e o e e e o o o e e o o i

REV IEW OF L IT E R A T U R E o o t i o o e o o o e o e o o o o o o o o o o o o o o o o e o o

S e a s o n a l V a r ia t io n in B r e e d in g E f f ic ie n c y « o , .............. 3A s s a y M eth o d s fo r P r o g e s t e r o n e . „ „ , * , , „ . . . . . . . . . „ . . . . 4

B io a s S y o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o c o . o « o o o o « o o 3C h e m ic a l ^4eth.OdS o o o e o o e o e o o o o 6 o o o o o o © o e » o o o o o e o » 5

Q u a n t i ta t io n of 3?r o g e s t e r o n e o o o o o o o p o o o © © © © © © © © © © © © © © © 7C^as C h ro m a to g ra p h y 8 o o o o » o © © © * » o * o © o « © © o o o o o o o o © o a p © 3

E X P E R IM E N T A L P R O C E D U R E . . . . . . . . . . . . . . o . _____. . . . . . . . 10

G e n e r a l d. © . . . . . . . . . . . . . . . . . . . © . . . . . . . . . . . © . . . . . © . . . 10P r o g e s t e r o n e D e te r m in a t io n . . 11T h in L a y e r C h ro m a to g ra p h y (T L C ) ©. ©. . ©. © © . © © © © . 12Cjas C h ro m a to g ra p h y Ĉ3X-/(v) . © . . . . © e . o . © . © . . . . © © © . . . © . . 13P e r C en t R .ecovery a p o . . © © . © © . © © © © © © . . © . © © . . . . . . © © . © . 14

R E S U L T S AND DISCUSSION o o . © . . © © © © . © © . . . © © . © © © . © © © © © . . © . 16

E v a lu a t io n of 3S4 e tliods . © o © © © © © © © © © © © © © © . © © © © © © . © © © . © o © 16P e r i p h e r a l P r o g e s t e r o n e in E x p e r im e n ta l Cows » . . « . ©©. © 19

O v a r e a to m iz e d Cows - P r e - h e a t s t r e s s , P r e -o v 3* r e c t o m y © . © © © . © . . © © © . . . © . © o © © © © © © © . © © . © © 2 0

O v a r e c to m iz e d Cows - P o s t - h e a t s t r e s s # P o s t -o v a r e c to m y . © © © © © © © . © © . © © © © o o © © © © . © © . © © . © © © 20

C o n tro l Cows - P r e - h e a t s t r e s s ........... . . ©©! , * © © © ........... 21C o n tro l Cows - P o s t - h e a t s t r e s s . © ©.»©.© © © . © © © © © © © . © 21

V

T A B L E OF C O N T E N T S- - C on tinued

P a g e

S jR, "Y" o o e e o e o e o o o o o o o o o o o o o a o o e o e o e e o o e e o o e o o o e o o e o o 2 4

J&. JP E N e e o o o o o o o o o o o o o o o e o o o e o o o o o o o e o o o o e o o o o o o o o o o 23

JB X!B 3LT O Cf JR. j A. H "Y" o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o 32

LIST OF FIG U R E S

F ig u r e P a g e

lo S ta n d a rd C u rv e fo r D e te r m in a t io n of P r o g e s t e r o n e . „ „ . . , . 26

2 o varec toxn ized . ow S o o o o o o o o o o o o o o ^ o a o ^ o o o o o o o o o 0 0 0 * 0 9 27

3 o Ov a r e C t O im Z e d (Z) O W S o o e e e o e o e o o o o o e e o o e o o e o o o o o o o o o o o 2 S

o CZ# Ont r 01 CZ# O W S o o e o o o e o o o ' o o o o o o o o e o o o e o o o o o e o o o o o o o o o o o o 2 ̂

5 o Ẑ< Ont r OI CZ# O S o o o o o o o o o o o o o e o o o o o o o o o o e o o o o o o o o o o o o o o o 30

6o A v e r a g e s of C o n tro l Cows and O v a r e a to m iz e d Cows 0000*0 31

v i

A B S T R A C T

A te c h n iq u e w as d e v e lo p e d th a t co u ld q u a n t i ta te s u b - m i c r o g r a m

a m o u n ts of p r o g e s t e r o n e f r o m bo v in e b lo o d p l a s m a . U sin g e th e r e x t r a c

t io n , t h i n - l a y e r c h ro m a to g r a p h y and g as c h r o m a to g r a p h y , n a n o g r a m

a m o u n ts of p r o g e s t e r o n e w e r e m e a s u r e d w ith e x c e l le n t p r e c i s i o n and

r e p r o d u c a b i l i ty .

Six cow s w e r e a s s ig n e d to one of tw o g r o u p s . One g ro u p of t h r e e

cow s h a d t h e i r o v a r i e s r e m o v e d to e l im in a te a p o s s ib le s o u r c e of p r o g e s

t e r o n e . The o th e r t h r e e w e r e le f t in t a c t an d u s e d as c o n t r o l s . A ll s ix

cow s w e r e b le d f r o m th e ju g u la r v e in on days 1, 5 , 10, 15, 20 (day 1 is

th e day of e s t r u s ) p r i o r to b e in g h e a t s t r e s s e d o r o v a r e c to m iz e d . In

e f fe c t , e a c h cow a c te d a s h e r own c o n t r o l . A llow ing a 30 day p e r io d fo r

the o v a r e c to m i z e d cows to h e a l , th e y w e r e th en p la c e d in a c o n t r o l le d

e n v i ro n m e n t c h a m b e r f o r a 24 h o u r p e r io d . T he cow s w e r e ag a in b le d

a t 5 day i n t e r v a l s f o r 30 d a y s , s t a r t i n g th e day th e y c a m e out of th e h e a t

b a r n . T he c o n t r o l cows w e r e a ls o b le d a t f ive day in t e r v a l s f o r 30 day s

a f t e r h e a t s t r e s s i n g . T h is w as done to d e te r m in e i f t h e r e w a s an i n c r e a s e

in p r o g e s t e r o n e due to the s t r e s s e f fe c t and not d i r e c t ly c o n t r ib u ta b le to

the o v a r i e s . A ll c o w s , b o th c o n t r o l an d o v a r e c to m i z e d , sh o w ed the

e f fe c t of h e a t s t r e s s . S om e cow s h a d a d e la y e d r e s p o n s e to the e l e v a te d

t e m p e r a t u r e .v ii

E v id e n c e s u g g e s t s th a t h e a t s t r e s s does i n c r e a s e th e p ro d u c t io n

of a d r e n a l p r o g e s te r o n e , and th e p o s s ib i l i ty of an a d r e n a l r e l a t io n s h ip

to lo w e r e d b r e e d in g e f f ic ie n c y m a y e x i s t in a r e a s w h e re h igh a m b ie n t

t e m p e r a t u r e s a r e c o m m o n .

INTRODUCTION

C o n s id e r a b le r e s e a r c h h a s sh o w n .th a t c a t t le a r e in f lu e n c e d in

t h e i r r e p r o d u c t iv e b e h a v io r by s e a s o n a l and t e m p e r a t u r e c h a n g e s . A

w ide ra n g e of in f o r m a t io n is a v a i l a b le on d i f f e r e n t a s p e c t s of m a le

b o v in e f e r t i l i t y r e l a t e d to a m b ie n t t e m p e r a t u r e . R e c e n t in v e s t ig a t io n s ,

h o w e v e r , in d ic a te th a t th e cow , r a t h e r th a n the b u l l , is th e m a jo r c a u s e

of lo w e r e d s e a s o n a l f e r t i l i t y in A r iz o n a .

B a s ic kn o w led g e on th e e x a c t p h y s io lo g ic a l c a u s e of th is lo w e re d

f e r t i l i t y i s l im i t e d . Such in f o r m a t io n is n e c e s s a r y f o r an u n d e r s ta n d in g

of th e p r o b le m , and a s a m e a n s of d e te r m in in g how to im p r o v e r e p r o d u c

t iv e e f f ic ie n c y in a r e a s of th e w o r ld w h e r e h igh t e m p e r a t u r e s a r e

c o m m o n .

A r e c e n t in v e s t ig a t io n h a s show n a s ig n i f ic a n t i n c r e a s e in

p r o g e s t in c o n te n t of the a d r e n a l g lands of bo v in e u n d e r co n d i t io n s of

t h e r m a l s t r e s s . It s e e m s e v id en t th a t if th e h ig h e r l e v e l s of a d r e n a l

p r o g e s t e r o n e a r e b e in g s e c r e t e d in to th e b lo o d , th is cou ld a l t e r the

n o r m a l r e p r o d u c t iv e a c t iv i ty and b e r e s p o n s ib l e f o r low b re e d in g

e f f ic ie n c y of c a t t l e d u r in g h o t w e a th e r . H o w e v e r , i t i s e s s e n t i a l to

d e m o n s t r a t e th is r e l a t i o n s h ip of h e a t s t r e s s and h igh b lo o d le v e l s of

p r o g e s t e r o n e to r e p r o d u c t iv e f a i l u r e , if th is i s to be u s e d a s a lo g ic a l

e x p la n a t io n .

S tu d ie s r e l a t e d to th e s e c r e t i o n and m e ta b o l i s m of p r o g e s t e r o n e

in th e p e r i p h e r a l b lo o d h a v e b e e n h a m p e r e d b y th e la c k of c h e m ic a l

m e th o d s s u f f ic ie n t ly s e n s i t iv e and s p e c i f ic to q u a n t i ta te th e h o rm o n e

w ith r e l a t i v e e a s e and r e p r o d u c a b i l i ty .

W ith th is in m in d , the p r e s e n t s tu d y w as d e s ig n e d to :

(1) D evelop a t e c h n iq u e , u s in g g as c h r o m a to g r a p h y , to m e a s u r e

q u a n t i ta t iv e ly p e r i p h e r a l b lo o d le v e l s of p r o g e s t e r o n e in b o v in e .

(2) D e te r m in e i f in d u c e d t h e r m a l s t r e s s in b o v in e w il l e f fe c t

p e r i p h e r a l b lo o d p r o g e s t e r o n e .

(3) D e te r m in e th e e n d o c r in e s o u r c e of p e r i p h e r a l b lo o d

p r o g e s t e r o n e u n d e r s t r e s s c o n d i t io n s .

REV IEW O F L IT E R A T U R E

S e a s o n a l V a r ia t io n in B r e e d in g E f f ic ie n c y

C o n s id e ra b le r e s e a r c h h a s show n th a t c a t t le a r e in f lu e n c e d in

t h e i r r e p r o d u c t iv e b e h a v io r by s e a s o n a l and t e m p e r a t u r e c h an g e s (3 S 4 ,

10, 12, 23, 25, 26, 28, 30, 34, 39, 4 0 , 47 , 48 , 50 , 53, 54, 57, 61, 62 ,

63, 64, 65 , 66, 67, 70). A w ide r a n g e of in f o r m a t io n i s a v a i la b le on

d i f f e r e n t a s p e c t s of m a le bo v in e f e r t i l i t y r e l a t e d to a m b ie n t t e m p e r a t u r e

(12, 39 , 67). I t w ould a p p e a r f r o m r e c e n t in v e s t ig a t io n s by S to tt and

c o - w o r k e r s (61 , 62, 63, 64, 70) th a t the cow r a t h e r th a n th e b u l l is the

m a j o r c o n t r ib u to r to low s e a s o n a l f e r t i l i t y . K now ledge of the b a s i c

p h y s io lo g ic a l c a u s e s of th is lo w e re d f e r t i l i t y of the f e m a le i s l im i t e d (17 ,

18, 22, 61, 62).

S om e w o r k e r s h av e show n th a t d u r in g c r i t i c a l p e r io d s of e a r ly

p r e g n a n c y , e x c e s s iv e p r o g e s t e r o n e in p r o p o r t io n to e s t r o g e n can c a u s e

a d i s r u p t io n of c le v a g e (14), too r a p id p a s s a g e of th e f e r t i l i z e d ov u m

th ro u g h th e o v id u c t (2 , 17), la c k of im p la n ta t io n (63), an d f in a l ly ,

a b n o r m a l p la c e n ta t io n (56).

H o w a r th e t a l . (38) f e l t th a t th e s p e r m w e r e a f fe c te d by th e h ig h e r

body t e m p e r a t u r e of th e f e m a le c a u s in g a s m a l l e r n u m b e r of s p e r m to be

t r a n s f e r r e d up the o v id u c t and fe w e r s p e r m in the zo n a p e l lu c id a of th e

r e c o v e r e d e g g s .

M o s t r e s e a r c h w o r k e r s a g r e e th a t th e m a jo r s o u r c e of p r o g e s t e r o n e

in th e e a r l y s ta g e s of p re g n a n c y (up to p la c e n ta t io n ) is th e c o rp u s

l e u te u m (24, 32, 33, 51 , 59)- G o m es (31) r e p o r t e d the m a j o r s o u r c e

of p r o g e s t e r o n e th ro u g h o u t p r e g n a n c y in the cow is the c o rp u s lu te u m ,

b u t th a t o v a r i e s and a d r e n a l s m ig h t c o n t r ib u te a s ig n i f ic a n t a m o u n t ,a n d

b ody f a t m a y s t o r e the h o rm o n e to be u s e d as n e e d e d . H o w e v e r , i t h a s

b e e n e s t a b l i s h e d th a t th e a d r e n a l g lan d s s e c r e t e s ig n i f ic a n t a m o u n ts of

p r o g e s t e r o n e (5 , 6, 7, 13, 15, 33, 51, 59). M oody (50) found th a t u n d e r

p e r io d s of h e a t s t r e s s th e a d r e n a l g lan d s c o n ta in e d up to tw ic e t h e i r

n o r m a l l e v e l w ith no i n c r e a s e in p r o g e s t e r o n e f r o m th e c o rp u s le te u m .

It m ig h t b e e x p e c te d th a t th e a d r e n a l g lan d s co n ta in m o r e p r o g e s t e r o n e ,

s in c e p r o g e s t e r o n e is an i n t e r m e d ia t e of c o r t i s o n e p ro d u c t io n . C o r t i s o n e

is p ro d u c e d in the c o r t e x of th e a d r e n a l u n d e r s t r e s s c o n d i t io n s . I t i s ,

t h e r e f o r e , n e c e s s a r y to d e m o n s t r a t e th a t th is ad d ed p r o g e s t e r o n e f inds

i t s w ay in to th e p e r i p h e r a l b lo o d and i s r e s p o n s ib le f o r th e r e p r o d u c t iv e

f a i l u r e s found in A r iz o n a d u r in g th e s u m m e r m o n th s .

A s s a y M e th o d s fo r P r o g e s t e r o n e

S tu d ie s r e l a t e d to the s e c r e t i o n and m e ta b o l i s m of p r o g e s t e r o n e in

the p e r i p h e r a l b lo o d h a v e b e e n h a m p e r e d by th e la c k of c h e m ic a l m e th o d s

s u f f ic ie n t ly s e n s i t iv e an d s p e c i f ic to q u a n t i ta te the h o rm o n e w ith r e l a t i v e

e a s e and r e p r o d u c a b i l i ty . P r e s e n t m e th o d s r e q u i r e th e u se of l a r g e

q u a n t i t ie s of b lo o d p l a s m a (58).

B i o a s s y : Som e b io lo g ic a l a s s a y s a r e v e r y s e n s i t i v e , b u t la c k

s p e c i f i c i ty , q u a n i ta t io n , and h av e u n d e f in ed l im i t s of e r r o r (21, 37). T he

a s s a y s a r e b a s e d on a s u b je c t iv e end p o in t su c h as c o m p le te p r o g e s t a t i o n a l

p r o l i f e r a t i o n , u te r in e g r a n u l o s a / m u c o s a a r e a , c a r b o n ic a n h y d ra s e

a c t iv i ty , p r e g n a n c y m a in ta in a n c e , o r p a r t u r i t i o n d e lay . T h e s e m e th o d s

h av e in d ic a te d p r o g e s t e r o n e l e v e l s in b io lo g ic a l f lu id th a t c an n o t be

v e r i f i e d b y c h e m i c a l a s s a y (20, 21, 58).

C h e m ic a l M e th o d s : In r e c e n t y e a r s c o n s id e r a b le p r o g r e s s h a s b e e n

m a d e in th e d e v e lo p m e n t of c h e m ic a l m e th o d s to d e te r m in e p r o g e s t e r o n e

(19, 20, 44 , 58).

E x t r a c t io n of p r o g e s t e r o n e f r o m p l a s m a h a s g e n e r a l l y b e e n

a c c o m p l i s h e d by th e u se of o rg a n ic so lv e n ts su c h a s e t h e r , m e th y le n e

c h lo r id e , a c e to n e , o r e t h e r - c h l o r o f o r m (29 , 46 , 52, 68 , 69 , 73). Z a n d e r

(74) h a s show n th a t p r e - t r e a t m e n t of p l a s m a s a m p le s w i th s o d iu m

h y d ro x id e as p r o p o s e d b y S h o r t (58) im p r o v e s th e p r e c i s i o n of the f in a l

p r o g e s t e r o n e q u a n t i ta t io n . In a d d i t io n , so d iu m h y d ro x id e d e c r e a s e s the

f o r m a t io n of s o l v e n t - p l a s m a e m u ls io n d u r in g e th e r e x t r a c t i o n .

One of the d i f f ic u l t ie s in v o lv e d in so lv e n t e x t r a c t i o n is th e g r e a t

q u a n t i ty of l ip id e x t r a c t e d . To o b ta in a m o r e n e a r ly p u r e e x t r a c t f r o m

p l a s m a s e v e r a l te c h n iq u e s h a v e b e e n u s e d . A. O. L u r ie e t a l . (46)

p u r i f i e d the e x t r a c t f r o m p l a s m a by c h ro m a to g r a p h y th ro u g h m in ia tu r e

C e l i te c o lu m n s u s in g t r i m e t h y l p e n ta n e as th e m o b i le p h a s e and 90%

m e th a n o l as the s t a t i o n a r y p h a s e . T h is s te p r e m o v e d a l a r g e q u a n t i ty

of p ig m e n te d m a t e r i a l and c h o le s t e r o l , N ie l l j a t a l . (52) f u r t h e r

c le a n e d th e e th e r e x t r a c t w ith w a sh in g s of 1 /10 v o lu m e of w a te r b e f o r e

e v a p o r a t in g to d r y n e s s . A n o th e r c l e a n - u p p r o c e d u r e p r o p o s e d by

W il l ia m s (69) w a s to ta k e th e d r i e d , c ru d e l ip id s a m p le in to s o lu t io n in

b e n z e n e and c h r o m a to g r a p h on s i l i c a c id c o lu m n s .

S e p a r a t io n and a d d i t io n a l p u r i f i c a t io n of th e s t e r o i d s by c o u n te r -

c u r r e n t d i s t r ib u t io n h a s b e e n u t i l i z e d in on ly one ro u t in e m e th o d (45),

A p p l ic a t io n of th is m e th o d a p p e a r s to b e l im i t e d by th e r e l a t i v e ly long

t im e r e q u i r e d to s e p a r a t e and p u r i f y a s in g le s a m p le .

U se of p a p e r c h r o m a to g r a p h ic te c h n iq u e s in th e s e p a r a t io n and

id e n t i f ic a t io n of a d r e n a l s t e r o id s (8, 9) h a s e v id en t a d v a n ta g e s , b u t

a g a in is l im i t e d by the t im e in v o lv e d .

T h i n - l a y e r c h r o m a to g r a p h y h a s r e c e n t ly co m e on th e s c e n e as a

te c h n iq u e th a t w i l l s u p p le m e n t o r ev en r e p l a c e p a p e r c h r o m a to g r a p h y .

The s p e e d (2 0 -40 m i n , ) c o m p a r e d to th a t of p a p e r c h r o m a to g r a p h y (4-

24 h r . ) , th e w ide r a n g e of d i s t in c t r e s o lu t io n s (5 -5 0 0 ug. )„ and the

n o t ic e a b ly s h a r p s e p a r a t i o n of c o m p o u n d s a r e d i s t in c t a d v a n ta g e s of

t h i n - l a y e r c h r o m a to g r a p h y (36). T he m e th o d h a s b e e n t e s t e d f o r a g r e a t

n u m b e r of s t e r o id s in v a r io u s s o lv e n t s y s t e m s and c o m p le te d a ta a r e

a v a i l a b le f o r th e p r e g n a n e s e r i e s (41, 42 , 43).

W a r n e r - C h i l c o t t (68) d i s s o lv e the e th e r e x t r a c t i o n r e s id u e in

c h lo r o f o r m and app ly i t to a t h i n - l a y e r p la te c o a te d w ith S i l ic a G el G

c o n ta in in g f l u o r e s c e in . The p la te is d e v e lo p e d u s in g B e n z e n e - e th y l

a c e ta te (32). W il l ia m s (69) u s e s th e s a m e type of p l a t e s , b u t h a s found

a s a t u r a t e d c h a m b e r of c h lo r o f o r m : a b so lu te e th a n o l ( 9 : l » v /v ) u s e f u l

f o r s t e r o i d s e p a r a t i o n . A w ide r a n g e of s o lv e n t s y s t e m s a r e a v a i la b le

( fo r s p e c i f ic so lv e n ts s e e r e f e r e n c e s 9» 27, 29, 36, 41 , 42 , 43 , 45 , 46 ,

52, 68, 69, 73).

Q u a n t i ta t io n of P r o g e s t e r o n e

T he a 4 - e n e - 3 - o n e g ro u p in g of the s t e r o i d n u c le u s m a y be

q u a n t i t a te d by i t s s t r o n g a b s o r b a n c e of UV l ig h t a t 2 4 0 .m u in a lc o h o l ic

s o lu t io n s (10, 11, 16).

O th e r s p e c t r o p h o to m e t r i c a s s a y s h a v e b e e n r e v ie w e d by Z a n d e r (74)

and a r e b a s e d on a b s o r b a n c e of UV l ig h t a t w ave le n g th s o th e r th an 240 m u

by p r o g e s t e r o n e d e r i v i t i e s . T h e s e in c lu d e the p r o g e s t e r o n e

b i s - t h i o s e m i c a r b a z o n e a t 300 m u , the p r o g e s t e r o n e b i s - d in i t r o p h e n y l -

h y d ra z o n e a t 380 m u , and th e s u l f u r i c a c id - e th a n o l c h r o m a g e n a t 290 m u .

A s Z a n d e r (74) p o in te d o u t , th e s e m e th o d s h a v e the a d v a n ta g e of s h if t in g

the Q u a n t i ta t io n w ave le n g th aw ay f r o m th a t of i n t e r f e r i n g m a t e r i a l s ,

b u t l a c k s p e c i f i c i ty and m a k e i t d if f ic u l t to id e n t i fy th e i s o l a t e d m a t e r i a l

a s p r o g e s t e r o n e . The l i m i t of s e n s i t i v i t y of the s p e c t r o p h o to m e t r i c

m e th o d s r a n g e f r o m 0 .5 to 1 .0 ug if m i c r o - c e l l s a r e u s e d . The le v e l s

of p r o g e s t e r o n e in p e r i p h e r a l b lo o d a r e e x p e c te d to be in th e 0. 1 ug

r a n g e , m u c h be low th e r a n g e of the s p e c t r o p h o to m e te r .

The double i s o to p e d e r iv a t iv e (DID) p r in c ip le g e n e r a l l y in v o lv e s14

th e a d d i t io n of an i s o to p ic a l ly la b le d s t e r o i d (C ) to the unknown

s a m p le . The unknow n s t e r o i d i s th e n e x t r a c t e d and p u r i f i e d a long w ith

th e la b le d s t e r o i d an d a r e b o th c o n v e r te d to a d e r iv a t iv e u s in g a r e a g e n t

3 3la b le d w ith a s e c o n d i s o to p e , T r i t i u m (H ). The a m o u n t of H in the

14f in a l d e r iv a t iv e q u a n t i ta te s the a m o u n t of d e r iv a t iv e f o r m e d and th e C

m e a s u r e s p r o c e d u r a l l o s s e s .

R io n d e l l (55) f i r s t a p p l ie d the DID p r o c e d u r e to p r o g e s t e r o n e

3 35d e te r m in a t io n u s in g p r o g e s t e r o n e - H w ith t h io s e m i c a r b a z i d e - S as the

d e r iv a t iv e f o r m in g ag en t . W o o le v e r an d G old fe in (72) c o n v e r te d

4p r o g e s t e r o n e to A p r e g n e n e - 2 0 D - o l - 3 - o n e (20B -o l) u s in g t r i t i a t e d

14s o d iu m b o r o h y d r id e and C - p r o g e s t e r o n e as the iso to p e s o u r c e . T h is

m e th o d is r e p o r t e d s e n s i t iv e to 0 .0 1 ug of p r o g e s t e r o n e , bu t i t w as

found th a t the t im e (8-19 days) and p e r c e n t r e c o v e r y w e r e the b ig d r a w

b a c k s w ith th is m e th o d (71).

G as C h ro m a to g ra p h y

As r e c e n t ly as 1962 F u t t e r w e i t , e t aJL (29) r e p o r t e d th e f i r s t u s e

of g as c h ro m a to g r a p h y fo r th e q u a n t i ta t iv e a s s a y of p r o g e s t e r o n e . H ow

e v e r , i t w a s a d i f f ic u l t , t im e c o n su m in g m e th o d w ith l e s s s e n s i t iv i ty th a n

a v a i la b le m e th o d s . In 1964, Y annone, M . E . e t aL (73) r e p o r t e d the

f i r s t h ig h ly s e n s i t iv e gas c h r o m a to g r a p h y a s s a y fo r p r o g e s t e r o n e .

T h is m e th o d is r e p o r t e d s e n s i t iv e to l e s s th a n 0. 02 ug of p r o g e s t e r o n e .

S ince th a t t im e m a n y w o r k e r s h av e r e p o r t e d e x c e l le n t u se of gas

c h ro m a to g r a p h y fo r th e q u a n t i ta t io n of p r o g e s t e r o n e (29, 46 , 52, 60,

68, 69). G as c h ro m a to g r a p h y s e e m s to o f fe r s p e c i f i c i ty th a t c an n o t

b e show n by any of th e b e f o r e m e n t io n e d te c h n iq u e s .

A w ide r a n g e of c o lu m n p a ck in g s and t e m p e r a t u r e s h ave b e e n

u s e d f o r the q u a n t i ta t io n of p r o g e s t e r o n e . E s t e r g r e e n (27) r e p o r t e d

r e s o lu t io n s of p r o g e s t e r o n e a t 0. 05 ug w ith th e u s e of 6 - f o o t g la s s

c o lu m n s p a c k e d w ith A n a k r o m ABS 80 -1 0 0 m e s h fo r the s u p p o r t and 1%

S E -3 0 l iq u id p h a s e , and a c o lu m n t e m p e r a t u r e of 210° C. L u r ie (46)

r e p o r t e d d e te c t io n of 0. 02 to 0. 22 ug ad d ed to m a le p o o le d p l a s m a w ith

c o lu m n s p a c k e d w ith 3% X E -6 0 on G as C h ro m P of 8 0 -1 0 0 m e s h

( P e r k i n - E l m e r ) , b u t ad d ed th a t th e m e th o d te n d s to o v e r - e s t i m a t e th e

am o u n t of p r o g e s t e r o n e in the 2 0 -80 n a n o g r a m r a n g e . N e i l l (52) r e p o r t e d

the s m a l l e s t a m o u n t of p r o g e s t i n th a t c o u ld be d e te c te d w ith gas c h r o m a

to g ra p h y w a s n e a r 0 .0 1 ug on 1% S E -3 0 on 6 0 -8 0 m e s h C h r o m p o r t 3X

(M ic ro T ek I n s t r u m e n t I n c . ) . C o iled p y r e x g la s s c o lu m n s 6 f e e t x .125"

O. D. w e r e u s e d w ith a co lu m n t e m p e r a t u r e of 250° C. W a r n e r - C h i l e o t t

(68) u s e d c o lu m n s 33" x .1 2 5 " O. D. p a c k e d w ith 1% X E -6 0 on C h r o m o s o r b

G (1 0 0 -1 2 0 m e sh ) DMCS t r e a t e d b u t d id n ' t g ive th e lo w e r ra n g e of

d e te c t io n .

E X P E R IM E N T A L P R O C E D U R E

G e n e r a l

One of the m a in p u r p o s e s of th is s tu d y w as to d e t e r m in e the e n d o

c r in e s o u r c e of p r o g e s t e r o n e u n d e r c o n d it io n s of s t r e s s . S ix c o w s , 4

H o ls te in and 2 G u r n s e y ’s , w e r e a s s ig n e d to one of two g r o u p s . One

g ro u p of 3 cow s h a d t h e i r o v a r i e s r e m o v e d to e l im in a te th is as a p o s s ib le

s o u r c e of p r o g e s t e r o n e . T h is u n i l a t e r a l o v a r e c to m y w as a c c o m p l i s h e d

by a s im p le l a p a r o to m y , c la m p in g off th e o v a r i e s f o r a p p r o x im a te ly 30

m in u te s , an d th e n te a s in g aw ay w ith the f i n g e r s . T he o th e r t h r e e , a l l

of th e H o ls te in b r e e d , w e r e le f t in ta c t and u s e d as c o n t r o l s . The t h r e e

o v a r e c to m i z e d cow s (G -2 5 4 , G -2 6 6 , H -710) w e r e b le d f r o m the ju g u la r

v e in on days 1, 5, 10, 15, 20 (day 1 is the day of e s t r u s ) p r i o r to h a v in g

t h e i r o v a r i e s r e m o v e d . A llow ing a 30 day p e r io d f o r h e a l in g , the cow s

w e r e th e n p la c e d in a c o n t r o l l e d e n v i ro n m e n t c h a m b e r ( t e m p e r a t u r e x

101° C h u m id i ty x 44%) f o r a 24 h o u r p e r io d . T hey w e r e b le d a t 5 day

in t e r v a l s f o r 30 d a y s , s t a r t i n g the day th e y c a m e ou t of the h e a t c h a m b e r ,

to d e t e r m in e i f t h e r e w as an i n c r e a s e in p r o g e s t e r o n e due to the s t r e s s

e f fe c t and n o t d i r e c t l y c o n t r ib u ta b le to the o v a r i e s .

The c o n t r o l cow s (H -5 6 4 , H -7 0 6 , H -708) w e r e a ls o b le d f r o m th e

ju g u la r v e in on days 1, 5 , 10, 15, 20 (day 1 is th e day of e s t r u s ) p r i o r

10

11

to h e a t s t r e s s . On th e day of n e x t e s t r u s th e y w e r e p la c e d in the

c o n t r o l le d e n v i r o n m e n t c h a m b e r ( t e m p e r a t u r e x 103° C h u m id i ty x 44%)

fo r a 24 h o u r p e r io d to h e a t s t r e s s .

P e r i p h e r a l b lo o d w as c o l le c te d f r o m th e ju g u la r in c le a n g la s s

b le e d in g b o t t l e s . T he b o t t l e s c o n ta in e d S o d iu m H e p a r in (N B C ), 1 u n it

p e r 1 m l of b lo o d , d ry e d down to r e d u c e h e m o ly s i s . A p p ro x im a te ly

20 m l of b lo o d w as c o l le c te d a t e a c h b le e d in g , spun down w ith in one h o u r ,

and th e p l a s m a d ra w n off. No a t t e m p ts w e r e m a d e to co o l the b lood

w h ile b e in g t r a n s p o r t e d f r o m th e f a r m to th e l a b o r a to r y . F r o m the 200

m l of b lo o d , a p p r o x im a te ly 8 0 -9 0 m l of p l a s m a w as o b ta in e d , p la c e d in

p la s t i c v i a l s , an d f r o z e n u n t i l n e e d e d f o r a n a ly s i s .

P r o g e s t e r o n e D e te r m in a t io n

It w a s e s s e n t i a l to d ev e lo p a te c h n iq u e f o r m e a s u r i n g s u b m i c r o g r a m

am o u n ts of p r o g e s t e r o n e , b e f o r e the r e m a in in g p o r t io n of the e x p e r im e n t

c o u ld be c a r r i e d ou t. T he g e n e r a l p r o c e d u r e , u s e d f o r d e te r m in a t io n of

p r o g e s t e r o n e w as a c o m p o s i te of s e v e r a l p u b l i s h e d te c h n iq u e s (58, 68,

69, 72, 74).

The f r o z e n p l a s m a s a m p le s w e re th a w ed a t r o o m t e m p e r a t u r e .

T w en ty m l of th e p l a s m a w a s p la c e d in s to p p e r e d c e n t r i fu g e tu b e s , and

500 la m b d a of p r o g e s t e r o n e 4 - C ^ (0. 1 m i c r o c u r i e s ) w as ad d ed to the

s a m p le . A ls o , 1 m l of 5N aq u eo u s S o d iu m H y d ro x id e , and 1 m l of to lu e n e

12

w e r e ad d ed and m ix e d th o ro u g h ly . The p l a s m a w as th e n w a sh e d 8 t im e s

w ith an e q u a l v o lu m e of e t h e r . T he e th e r e x t r a c t i o n w as p o u r e d in to a

ro u n d b o t to m f l a s k , p la c e d in a 70° C w a te r b a th and d r i e d u n d e r a

v a c u u m . T he s id e s of th e ro u n d b o t to m f l a s k w e r e th e n w a sh e d down

w ith e th e r w h ich w as e v a p o r a te d . T he p r o c e s s w as r e p e a t e d tw ic e in

o r d e r to c o n c e n t r a t e the e x t r a c t a t th e b o t to m of th e f l a s k . T he r e s id u e

w as th e n pu t in to s o lu t io n w ith , 5 m l of m e th a n o l : c h lo r o f o r m 1:1 (v:v)

and s p o t te d on t h i n - l a y e r p l a t e s . A f te r th e o r ig i n a l . 5 m l h a d b e e n

sp o t te d , a n a d d i t io n a l .5 m l of m e th a n o l : c h lo r o f o r m w as ad d ed to the

ro u n d b o t to m f la s k an d s p o t te d .

B e c a u s e of the s e n s i t iv i ty of p r o g e s t e r o n e d e t e r m in a t io n s to a lm o s t

any k in d of c o n ta m in a n t , a l l so lv e n ts w e r e r e d i s t i l l e d b e f o r e u s e .

T h in L a y e r C h r o m a to g r a p h y (T LC )

T he t h i n - l a y e r p la te s w e r e p r e - c o a t e d w ith S i l i c a G el F 2 5 4 (E .

M e r c k AG ° D a r m s ta d t G e rm a n y and d i s t r i b u t e d by B r in k m a n I n s t r u

m e n t Co. ). The p la te s w e r e d iv id ed in to five e q u a l l a n e s an d s c o r e d to

p r e v e n t o v e r - l a p of th e s a m p l e s . T he p l a s m a e x t r a c t w a s s p o t te d on

3 la n e s (the two o u te r and th e m id d le ) , an d a p r o g e s t e r o n e s t a n d a r d

s p o t te d on the r e m a in in g tw o.

The c h r o m a t o g r a m w a s th e n d e v e lo p e d in an e q u i l ib r a te d ta n k c o n

ta in in g B e n z e n e :E th y l a c e ta te 4:1 (v :v). The c h r o m a to g r a p h w as c o m p le te d

in a b o u t 45 m in u te s . T he p la te w as th e n ta k e n out of the ta n k , a l lo w ed to

13

d r y , and th e n r e p l a c e d in th e ta n k a n o th e r 30 m in u te s to f u r t h e r m o v e

th e p ig m en tso T he p la te w a s th e n r e m o v e d , a l lo w ed to d ry , an d the

p o s i t io n of the p r o g e s t e r o n e s t a n d a r d s w e r e d e t e r m in e d u n d e r UV l ig h t

( M in e r a - l i g h t , U l t r a - V io l e t P r o d u c t s , Inc . , San G a b r ie l , C a l i fo rn ia ) .

The c o r r e s p o n d in g a r e a s in th e l a n e s co n ta in in g the s a m p le e x t r a c t w e r e

m a r k e d , s c r a p e d in to a h e a p , and p la c e d in a 15 m l c e n t r i fu g e tu b e .

A f te r the S i l ic a G el w as t r a n s f e r r e d to th e tu b e , 1 m l of e th a n o l w as

ad d ed , s h a k e n th o ro u g h ly f o r one m i n u t e , and th e n c e n t r i f u g e d f o r 15

m in u te s a t 2 5 ,0 0 0 r p m . T he e lu a te w as c o l le c te d in a 2 .5 m l s to p p e r e d ,

t a p e r e d c e n t r i fu g e tube and d r i e d u n d e r a s t r e a m of n i t r o g e n . The p r o c e s s

w a s th e n r e p e a t e d one m o r e t im e to be s u r e th a t a l l th e f r e e p r o g e s t e r o n e

h a d b e e n r e c o v e r e d f r o m the S i l ic a G el. T he s id e s of th e s m a l l tube

w e r e th e n w a s h e d 2 t i m e s w ith e th a n o l and th e s o lv e n t w as e v a p o r a te d

e a c h t im e to c o n c e n t r a t e th e r e s id u e a t th e b o t to m of the tu b e . T he p l a s m a

e x t r a c t w a s th e n r e a d y f o r gas c h r o m a to g r a p h y .

Gas C h ro m a to g ra p h y (GLC)

A f te r e x t r a c t i o n and p u r i f i c a t io n , p r o g e s t e r o n e w a s id e n t i f ie d and

q u a n t i t a te d , u s in g a M i c r o - T e k m o d e l DSS 170 Gas C h r o m a to g r a p y w ith

a dua l h y d ro g e n f la m e d e t e c to r . T h is m o d e l w as f u r t h e r m o d if ie d b y th e

in s t a l l a t i o n of a g la s s in je c t io n s y s t e m . T he c o lu m n s w e r e p a c k e d w ith

3% S. E . 30 on A e r o p a c k 30 of 8 0 /1 0 0 m e s h p r e p a r e d in b u lk by W ilk en s

14

I n s t r u m e n t and R e s e a r c h Inc . , W alnu t C r e e k , C a l i fo rn ia . F o r the

p u r p o s e of o b ta in in g the d a ta f o r th is s tu d y , a 3 ’ p y r e x g la s s c o lu m n

w ith a n OD of 1 /8 " w e r e p a c k e d w ith 3% SE 30 on A e r o p a c k 30 8 0 /1 0 0

m e s h and c o n d it io n e d o v e rn ig h t a t 260° C and a n i t r o g e n c a r r i e r p r e s s u r e

of 30 p s i . W hen ru n n in g s a m p l e s , th e oven w as k e p t a t 25 5 ° C, the

o °d e t e c to r a t 255 C, an d th e v a p o r iz a t io n c h a m b e r a t 315 C. N i t ro g e n

flow w as 20 p s i . H y d ro g e n 25 p s i , an d O xygen . 2C F H .

S a m p le s f r o m T L C s e p a r a t i o n w e r e ta k e n in to s o lu t io n in 20 u l of

c h lo r o f o r m and 1-5 u l in j e c te d onto th e c o lu m n . T he s a m p le s w e r e

in t r o d u c e d th ro u g h a r u b b e r d i a p h r a m w ith a H a m il to n M i c r o - s y r i n g e

onto th e c o lu m n . The p r o g e s t e r o n e p e a k w as r e c o r d e d a t th e o p t im a l

a t te n u a t io n to p ro d u c e an e a s i l y m e a s u r e d p e a k h e ig h t . B e f o r e an d a f t e r

e a c h g ro u p of s a m p l e s h a d b e e n a n a ly z e d , know n q u a n t i t ie s of

p r o g e s t e r o n e w e r e c h r o m a to g r a p h e d an d th e s t a n d a r d c u r v e d e r iv e d w as

u s e d to q u a n t i ta te th e p e a k h e ig h ts r e c o r d e d in th e p l a s m a e x t r a c t

( F ig u r e 1).

P e r C en t R e c o v e ry

B e f o re q u a n t i ta t in g th e p l a s m a e x t r a c t by G L C , 100 la m b d a of e a c h

s a m p le w a s p la c e d in co u n tin g v ia ls to d e te r m in e p e r c e n t r e c o v e r y of

p r o g e s t e r o n e . The s a m p l e s in th e co u n tin g v ia l s w e r e d r i e d , and 15 m l

of P P O - P O P O P w as ad d ed b e f o r e p la c in g in the cou n tin g c h a m b e r . The

s c i n t i l l a t o r f lu id w a s p r e p a r e d by add ing 5 g r a m s of P P O (2, 5 -

D ip h en y lo x az o le ) an d 0. 3 g r a m s of D im e th y l P O P O P (1» 4 - 5 i s - 2 - ( 4 -

m e t h y l - 5 - P h e n y lo x a z o ly e ) - B en z e n e ) to one l i t e r of to lu e n e . L iq u id

s c in t i l l a t io n coun ting w as done in a P a c k a r d T r i - C a r b L iq u id S c in t i l l a

t io n S p e c t r o m e t e r ( P a c k a r d I n s t r u m e n t C o . ) f o r 10 m in u te s . T he C P M

of th e s a m p le w a s d iv id ed by th e C P M of th e S ta n d a rd to d e t e r m in e

p r o c e d u r a l l o s s .

R E S U L T S AND DISCUSSION

T he la c k of an a c c e p ta b le m e th o d to d e t e r m in e b o v in e s y s te m ic

b lo o d p r o g e s t e r o n e f o r c o m p a r a t iv e p u r p o s e s n e c e s s i t a t e d f i r s t the

d e v e lo p m e n t of a d e p en d a b le p r o c e d u r e th a t c o u ld b e u s e d ro u t in e ly f o r

a n a ly s i s .

E v a lu a t io n of M ethods

F o llo w in g th e p r o c e d u r e of W o o le v e r and Goldfe-in (71), 0, 1

m i c r o c u r i e s of p r o g e s t e r o n e - 4 - C ^ w a s ad d ed to th e p l a s m a in o r d e r

to m e a s u r e r e c o v e r y . T he ad d i t io n of 1 m l 5N aq u eo u s S o d iu m H y d ro x id e

to p l a s m a g r e a t l y im p r o v e d th e r e c o v e r y of l a b le d p r o g e s t e r o n e . The

ad d i t io n of 1 m l of to lu e n e to p l a s m a co n ta in in g S o d ium H y d ro x id e u s u a l ly

r e s u l t e d in a g u m m y m a s s th a t w as e a s i e r to e x t r a c t w ith e t h e r . E x t r a c

t io n w ith e t h e r d id no t l e a d to f o r m a t io n of e m u ls io n s an d th is gave th e

14b e s t p e r c e n t r e c o v e r y of p r o g e s t e r o n e - 4 - C a d d e d to th e s a m p l e s .

S e v e r a l m e th o d s w e r e t r i e d , b u t th e s e a m o u n ts w h ich gave th e g r e a t e s t

r e c o v e r y of s t e r o i d w e r e a d o p te d f o r ro u t in e u s e .

A n a d d i t io n a l p u r i f i c a t io n s te p w as found n e c e s s a r y s in c e p ig m e n ts

w e r e v i s ib le and gas c h r o m a to g r a p h y of th e r e s id u e r e v e a l e d v e r y l a r g e

p e a k s and a c o n s id e r a b le am o u n t of n o n - s p e c i f i c b a c k g r o u n d m a t e r i a l

16

17

w h ich r e q u i r e d a p ro lo n g e d p e r io d to e lu te f r o m the g as c h r o m a to g r a p h y

co lu m n . T h i n - l a y e r c h ro m a to g r a p h y w as d e c id e d on fo r th is a d d i t io n a l

c l e a n - u p b e c a u s e of th e s p e e d and s h a r p s e p a r a t i o n of co m p o u n d s .

The s m a l l r e s id u e o b ta in e d a f t e r e th e r e x t r a c t io n w a s e f f ic ie n t ly

h a n d le d by th e S i l ic a G el F 2 5 4 w ith l i t t l e e v id e n c e of o v e r lo a d in g . In

th e few c a s e s th a t show ed e v id e n c e of o v e r lo a d in g , th e s a m p le s w e re

r e - c h r o m a t o g r a p h e d . T he s y s t e m of B e n z e n e :E th y l a c e ta te 4:1 (v:v) n o t

only s e p a r a t e d p r o g e s t e r o n e f r o m c h o l e s t e r o l , b u t a l s o p ro v e d h ig h ly

s u c c e s s f u l in r e m o v in g th e n o n - s p e c i f i c b a c k g ro u n d m a t e r i a l .

S e v e r a l m e th o d s f o r r e c o v e r i n g th e s t e r o i d f r o m t h i n - l a y e r s i l i c a

ge l w e r e t r i e d . L u r ie £ t a l . (46) p r o p o s e d s c r a p p in g th e s i l i c a ge l in to a

h e a p , ta k in g i t up by v a c u u m in to a t a p e r e d g la s s tu b e p lu g g ed w ith g la s s

w oo l. A f te r th e s i l i c a g e l w as t r a n s f e r r e d to th e g la s s tu b e , 5 m l of.

c h l o r o f o r m / e t h e r 1:1 w as e lu te d th ro u g h i t to r e c o v e r th e p r o g e s t e r o n e .

The m e th o d d e c id e d on, th a t of s c r a p p in g the s i l i c a g e l off in to a

c e n t r i fu g e tube an d e lu t in g th e s t e r o i d w ith e th a n o l , c o m p a r e d f a v o ra b ly

w ith the m e th o d of ta k in g the s i l i c a g e l up b y v a c u u m and p r o v e d to b e l e s s

t im e c o n s u m in g .

I t w as o p t im is t i c a l ly h o p e d by so m e w o r k e r s in th e p a s t th a t s o m e

s im p le and r a p id e x t r a c t i o n m e th o d of s t e r o id s f r o m b io lo g ic a l f lu id s

w o u ld su ff ic e to p r e p a r e th e s a m p le s fo r a p p l ic a t io n to c h r o m a to g r a p h ic

c o lu m n s . T h i s , h o w e v e r , h a s no t p r o v e d to be th e c a s e , and i t is now

18

e v id e n t th a t the s m a l l q u a n t i t ie s of s t e r o i d p r e s e n t in th e s e f lu id s a r e

c o m p le te ly o b s c u r e d by the a c c o m p a n y in g m a t e r i a l .

G as c h ro m a to g r a p h y w as d e c id e d on as a m e th o d of q u a n t i ta t io n

s in c e i t gave e x c e l l e n t s p e c i f ic i ty and r e p r o d u c ib i l i t y u s in g a p r o g e s t e r o n e

s t a n d a r d . H o w e v e r , th e G. L. C. h a d to be f u r t h e r m o d if ie d w ith a g la s s

in je c t io n s y s t e m to m e a s u r e s u b - m i c r o g r a m a m o u n ts of p r o g e s t e r o n e .

The s m a l l e s t a m o u n t of p r o g e s t e r o n e s t a n d a r d th a t cou ld be s a t i s f a c t o r i l y

m e a s u r e d w ith a f l a m e io n iz a t io n d e t e c to r w as 10 n a n o g r a m s . T he

m e a s u r e m e n t of s m a l l e r a m o u n ts w e r e n o t r e p r o d u c ib le . E s t i m a t e s of

p r o g e s t e r o n e in th e s a m p l e s c a n be o b ta in e d by u s in g p e a k h e ig h t a lone

and w as found to be a s a c c u r a t e a s p e a k h e ig h t x p e a k w id th a t h a l f

h e ig h t ( F ig u r e 1). T he r e t e n t io n t im e of p r o g e s t e r o n e on s u c c e s s iv e

in je c t io n s w a s h ig h ly r e p r o d u c ib le an d th is ad d ed co n f id e n ce to the

id e n t i f i c a t io n of e a c h p eak . S u c c e s s f u l q u a n t i ta t io n of s u b - m i c r o g r a m

a m o u n ts of p r o g e s t e r o n e on th e g as c h r o m a to g r a p h r e q u i r e s th e u se of a

s h o r t co lu m n to o b ta in s h a r p m e a s u r a b l e p e a k s . S in ce th e e f f ic ie n c y of

a s h o r t (3 ‘) c o lu m n is l im i t e d , th e h o r m o n e to be a s s a y e d h a d to be

i s o l a t e d w ith a m in im u m a m o u n t of i n t e r f e r r i n g s u b s ta n c e .

T h r e e p e r c e n t SE 30 w a s found to b e th e m o s t s a t i s f a c t o r y

s t a t i o n a r y p h a s e f o r th e c h ro m a to g r a p h y of p l a s m a e x t r a c t s s in c e m o s t

of th e n o n - s p e c i f i c b a c k g r o u n d m a t e r i a l w a s e lu te d a f t e r the p fe rg e s te ro n e

p e a k . The h e ig h t of th e p e a k cou ld be m e a s u r e d w ith co n f id e n ce w hen

s u b - m i c r o g r a m a m o u n ts w e r e p r e s e n t .

19

The s e v e r a l s te p s of th is m e th o d h a v e b e e n e x a m in e d and i ts o v e r

a l l r e s u l t s in d ic a te th a t i t i s s u i ta b le fo r m e a s u r e m e n t of p r o g e s t e r o n e

in th e p l a s m a . The m e th o d i s s im p le and r a p id enough to q u a n t i ta te

m a n y s a m p le s in one day . The g r e a t ly i n c r e a s e d s e n s i t iv i ty in the

m e a s u r e m e n t of p r o g e s t e r o n e by the u se of gas c h ro m a to g r a p h y h a s b e e n

a n o ta b le a d v an ta g e of th is m e th o d . T he u s e of gas c h r o m a to g r a p h y fo r

th e m e a s u r e m e n t of p r o g e s t e r o n e in p l a s m a r e q u i r e d c a r e f u l e x t r a c t i o n

and p u r i f i c a t io n of the s t e r o i d .

P e r i p h e r a l P r o g e s t e r o n e in E x p e r im e n ta l Cows

A f te r a m e th o d f o r m e a s u r i n g n ano g r a m a m o u n ts of p r o g e s t e r o n e

h a d b e e n d e v e lo p e d , i t w as th e n n e c e s s a r y to d e t e r m in e if in d u c ed

t h e r m a l s t r e s s in b o v in e w ould e f fe c t p e r i p h e r a l b lo o d p r o g e s t e r o n e . I t

w as a l s o e s s e n t i a l to d e m o n s t r a t e th e e n d r o c r in e s o u r c e of p e r i p h e r a l

b lo o d p r o g e s t e r o n e u n d e r s t r e s s c o n d i t io n s .

To do t h i s , e x p e r im e n t a l cow s w e r e e x p o se d to h ig h t e m p e r a t u r e

and r e l a t i v e h u m id i ty in the c o n t r o l l e d e n v i r o n m e n t c h a m b e r . R e s p o n s e

w as n o te d in th e w ay of e le v a te d body t e m p e r a t u r e , a p p a r e n t d i s c o m f o r t

and i n c r e a s e d r e s p i r a t i o n r a t e s . Cows p la c e d in th e h e a t c h a m b e r by

th e m s e lv e s w en t off fe e d f o r the 24 h o u r p e r io d , w h ile th o s e cows p la c e d

in th e c h a m b e r w ith a n o th e r cow s e e m e d to c o n su m e th e r e g u l a r a m o u n t

of f e e d . T h is m ig h t p o s s ib ly be a s t r e s s e f fe c t not a n t ic ip a te d in the

e x p e r i m e n t a l d e s ig n .

20

I t w as found th a t t h e r e w as l e s s h e m o ly s i s of th e b lo o d p l a s m a if

i t w a s c o l le c te d in g la s s b o t t l e s co n ta in in g d r i e d s o d iu m h e p a r in , 1 u n it

p e r 1 m l of b lo o d . T he l e s s th e b lood w as sh a k e n , and the s o o n e r i t w a s

c e n t r i fu g e d a f t e r c o l le c t io n , th e m o r e i t a id e d in r e d u c in g h e m o l y s i s .

O v a r e c to m iz e d Cows - P r e - h e a t s t r e s s , P r e - o v a r e c t o m y ( F ig u r e s

2 and 3): I t h a s b e e n show n in the p a s t th a t m o s t s p e c i e s of a n im a ls

r e a c h a p e a k of p r o g e s t e r o n e abou t day 15, w h en th e c o rp u s lu te u m i s

fu l ly d e v e lo p e d .

Cow 254 s e e m e d to show a n e a r l y a b n o r m a l ly h ig h le v e l of p r o g e s t e r

one e a r l y in h e r c y c le . Cow 266 show s a p e a k of p r o g e s t e r o n e on day 20

to w a rd s the end of h e r c y c le . T h e s e r e s u l t s a r e c o n t r a r y to w ha t h a s

b e e n a s s u m e d in the p a s t , th a t day 15 i s w hen the p r o g e s t e r o n e p e a k is

r e a c h e d . T h e s e two cow s s e e m to show t h e i r p e ak of p r o g e s t e r o n e p r o d u c

t io n a ro u n d day 1 o r e s t r u s . T h is c o u ld be n o r m a l s in c e o u r know ledge

of th e c o w 's p r o g e s t e r o n e s e c r e t i o n d u r in g a c y c le is l im i t e d . Cow 710

show s a p r o g e s t e r o n e p ro d u c t io n c l o s e r to th a t s e e n in o th e r s p e c i e s .

O v a r e c to m iz e d Cows - P o s t - h e a t s t r e s s , P o s t - o v a r e c t o m y ( F ig u r e s

2 and 3): Cows in the o v a r e c to m i z e d a n d h e a t s t r e s s e d g ro u p a l l sh o w ed a

r e s p o n s e to the h e a t s t r e s s . Cow 254 an d cow 266 sh o w ed an obvious

im m e d ia t e r e s p o n s e and a g r a d u a l t a p p e r in g to 20 d a y s . Cow 710 d id n ' t

show a n im m e d ia te i n c r e a s e in p r o g e s t e r o n e u n t i l day 15. It is i n t e r e s t

ing to no te th a t on day 30 a l l cow s s e e m to be m u c h h ig h e r , in d ic a t in g a

r e s i d u a l o r d e la y e d e f fe c t of th e h e a t s t r e s s .

C o n tro l Cows - P r e - h e a t s t r e s s ( F ig u r e s 4 and 5): The c o n t ro l

c o w s » p r e - h e a t s t r e s s , on th e a v e r a g e sh o w ed th e c y c le ( le v e ls of

p r o g e s t e r o n e ) th a t h a s b e e n s e e n in o th e r s p e c ie s of f a r m a n i m a l s „

A g a in , t h e r e a r e d e v ia t io n s f r o m th e e x p e c te d 15 day h ig h le v e l of

p r o g e s t e r o n e an d is m o s t p ro b a b ly due to s a m p l in g e r r o r . S ig n if ic an t

is the fa c t th a t a g a in two cow s (564 and 708) sh o w ed h ig h le v e l s of

p e r i p h e r a l p l a s m a p r o g e s t e r o n e e a r l y in th e c y c le . A ls o , the c o n t ro ls

and o v a r e c to m i z e d cows p r e - h e a t s t r e s s , show a h ig h e r l e v e l of

p r o g e s t e r o n e on day 5, th a n th e y do on day 1 o r 10.

C o n tro l Cows - P o s t - h e a t s t r e s s : Cows 564 and 706 ag a in show an

im m e d ia te e f fe c t of h e a t s t r e s s w ith cow 708 show ing a d e la y e d e f fe c t .

A g a in n o te th a t on day 30 a l l cow s s e e m to h a v e a h ig h e r p r o g e s t e r o n e

le v e l th a n n o r m a l .

A ll c o w s , b o th c o n t r o l an d o v a r e c to m i z e d , sh o w ed th e e f fe c t of

h e a t s t r e s s . S om e cow s h a d a d e la y e d r e s p o n s e . A p o s s ib le e x p la n a t io n

of th e s e v a r i e d r e s u l t s i s th a t s in c e the b lo o d s a m p le s w e r e ta k e n e v e r y

five d a y s , the p e a k of p r o g e s t e r o n e p ro d u c t io n w as r e a c h e d s o m e w h e re

b e tw e e n one of th e s e f ive day i n t e r v a l s . E v e r y c o w 's c y c le is of a

d i f f e r e n t le n g th , and 17-25 days is c o n s id e r e d n o r m a l . So i t is p o s s ib le

th a t e v e r y c o w 's c o rp u s lu te u m m a t u r e s e a r l i e r o r l a t e r th a n day 15.

Cows c u l le d f r o m the h e r d w e r e u s e d in th e o v a r e c to m i z e d g ro u p . T h ey

22

w e r e h a r d b r e e d e r s o r r e p e a t b r e e d e r s » w h ich m a y a c c o u n t f o r th is

w ide v a r i a n c e in r e s u l t s .

The a v e r a g e s f o r th e s ix cow s p r e - h e a t s t r e s s e d an d p r e -

o v a r e c to m i z e d is a s fo l lo w s :

T he l im i t e d d a ta in th is s tu d y s u g g e s t s th a t p r o g e s t e r o n e c o n c e n t r a

t io n is i n c r e a s e d in th e p e r i p h e r a l b lo o d by t h e r m a l s t r e s s . S ince th e

o v a r i e s w e r e r e m o v e d in h a lf of the e x p e r im e n t a l c o w s , i t s e e m s

e v id e n t th a t th is i n c r e a s e h a d to c o m e f r o m th e a d r e n a l g la n d s . T h is i s

i n a g r e e m e n t w ith M oody (50) who found th a t h e a t t r e a t e d a n im a ls h a d ,

on the a v e ra g e , abou t tw ice th e p r o g e s t e r o n e c o n c e n t r a t io n in the a d r e n a l s

a s th e c o r r e s p o n d in g c o n t r o l g ro u p .

S e v e r a l c o n c lu s io n s can lo g ic a l ly be b u i l t f r o m th e s e o b s e r v a t io n s

th a t w ou ld p o s s ib ly e x p la in r e d u c e d b r e e d in g e f f ic ie n c y d u r in g ho t

w e a th e r .

One th e o r y is th a t h ig h le v e l s of p r o g e s t e r o n e a t th e t im e of e s t r u s

s e e m s to s p e e d up th e t im e of o v u la t io n and s h o r te n s th e d u ra t io n of

e s t r u s (H a n s e l e t a h , 3.5). T h is g iv es tw o p o s s ib le r e a s o n s fo r lo w e r e d

f e r t i l i t y d u r in g t im e s of h ig h a m b ie n t t e m p e r a t u r e : (a) s h o r t e n e d

d u r a t io n of e s t r u s r e s u l t s in f a i l u r e of o b s e r v a t io n of e s t r u s , (b) th e t im e

Day No X n g / m l

15

101520

113138118144117

of o v u la t io n i s h a s te n e d , r e s u l t in g in i m m a t u r e f e r t i l i z e d ov u m r e a c h in g

th e u t e r u s too e a r l y , r e s u l t i n g in e m b ry o n ic d e a th .

A l l i s to n and U lb e rg (2) b y th e u se of o v a - t r a n s f e r te ch n iq u e hav e

i l l u s t r a t e d th e in a b i l i ty of n o r m a l f e r t i l i z e d ova f r o m t e m p e r a t u r e -

s t r e s s e d ew es to d ev e lo p in th e u t e r u s of a n o n - s t r e s s e d ew e . T hey

s u g g e s t th a t the s p e e d the e m b ry o t r a v e l s th ro u g h the o v id u c t m a y be

i n c r e a s e d in th e c a s e of the s t r e s s e d ew e and the e m b r y o n ic m o r t a l i t y

m a y be due to " i m m a t u r e " e m b r y o s r e a c h in g th e u t e r u s . T h is is in

a g r e e m e n t w ith Dutt (17) th a t the c r i t i c a l p e r io d is the t im e w hen the

f e r t i l i z e d ova i s in th e o v id u c t .

A s tu d y b y W il l ia m s (70) of o v u la t io n t im e w ith cows in c e n t r a l

A r iz o n a d u r in g th e f i r s t tw o w e ek s in S e p te m b e r found no e v id e n c e of

a b n o r m a l ly e a r l y o v u la t io n . H o w e v e r , he d id o b s e r v e th a t th e cow s w e r e

v e r y in a c t iv e d u r in g the h e a t of th e day . If we a s s u m e th a t the i n c r e a s e d

le v e l s of p r o g e s t e r o n e o b s e r v e d in th e b lo o d p l a s m a in th is s tudy c a m e

f r o m th e a d r e n a l g lan d s d u r in g p e r io d s of t h e r m a l s t r e s s , we cou ld

c o n je c tu r e a s h o r t e n e d d u ra t io n of e s t r u s as o b s e r v e d by H a n s e l j e t a l . (3 5 ) .

C o r r e l a t i n g the in a c t iv i ty of cow s d u r in g th e h e a t of th e day w ith the

p o s s ib i l i ty of a s h o r t e n e d d u ra t io n of e s t r u s , th e f a i l u r e of d e te c t io n

of e s t r u s s e e m s p la u s ib le .

SUMMARY

An e s s e n t i a l p a r t of th is r e s e a r c h w as to d ev e lo p a te c h n iq u e f o r

m e a s u r i n g q u a n t i ta t iv e ly p e r i p h e r a l b lo o d p r o g e s t e r o n e . The s e v e r a l

s te p s of th is m e th o d h a v e b e e n e x a m in e d and i t s o v e r - a l l r e s u l t s

in d ic a te th a t i t i s s u i ta b le f o r m e a s u r e m e n t of p r o g e s t e r o n e in the

p l a s m a . The m e th o d is r e l a t i v e ly s im p le and r a p id enough to

q u a n t i ta te m a n y s a m p le s in one day . T he g r e a t l y i n c r e a s e d s e n s i t iv i ty

in th e m e a s u r e m e n t of p r o g e s t e r o n e by th e u se of g as c h r o m a to g r a p h y

h a s b e e n a n o ta b le a d v an ta g e of th is m e th o d .

The p r e s e n t s tudy w as d e s ig n e d to in v e s t ig a te the p o s s ib i l i ty of

a h o r m o n a l im b a la n c e due to e le v a te d body t e m p e r a t u r e s d u r in g the

t im e of e s t r u s and s h o r t ly t h e r e a f t e r . P e r i p h e r a l p l a s m a p r o g e s t e r o n e

in n o n - h e a t s t r e s s e d a n im a ls w as c o m p a r e d to th a t of h e a t s t r e s s e d

a n im a l s . One g ro u p of cow s w as o v a r e c to m i z e d to e l im in a te the o v a r i e s

a s a p o s s ib le s o u r c e of p r o g e s t e r o n e . B o th g ro u p s of cow s w e r e h e a t

s t r e s s e d and p l a s m a p r o g e s t e r o n e c o m p a r e d .

P r o g e s t i o n c o n c e n t r a t io n in the p l a s m a w e re on the a v e r a g e h ig h e r

f o r th e two g ro u p s of h e a t s t r e s s e d a n im a l s . The m e a n r e c o v e r y of a l l

s a m p le s w as 4 1 .9 % a w ith a r a n g e of 1 6 .8 -8 8 .0 % .

E v id e n c e s u g g e s t s an i n c r e a s e d p r o g e s t e r o n e p ro d u c t io n by the

a d r e n a l s u n d e r s t r e s s c o n d i t io n s .

24

A P P E N D IX

1H9I3H

>1

d

300

2 5 0

200

1 5 0

100

50

5 0 100 150 200

N A N O G R A M S

Figure I. Standard Curve for Determination of

P ro gestero n e .

Day Cow N o .254 Cow No. 266 Cow No. 710No. % R e co v ery 3- n g /m l^ % R ecovery^ n g / rnl 3̂ % R eco v ery 3- n g /m l^ X n g /m l

P r e - O v a r e c t o m i z e d and P r e - H e a t S t r e s s e d

1 3 3 .2 142 67. 3 26 3 7 .4 55 745 23. 7 181 6 1 .8 34 44. 7 58 91

10 77. 5 21 78. 0 28 2 5 .7 134 6115 4 5 .9 35 7 5 .9 31 - - 3320 6 6 . 5 32 88. 0 98 5 9 .0 102 77

O v a re c to m iz e d and H ea t S t r e s sed1 24. 2 289 26. 5 85 4 3 .2 40 1385 20. 3 161 • 2 6 .4 210 5 2 .3 41 137

10 16 .8 192 36. 9 391 5 5 .3 30 20415 5 7 .2 184 34. 9 219 55. 7 116 17320 26. 5 108 2 1 .9 228 3 7 .9 165 16730 2 2 .9 218 6 9 .4 292 33. 2 151 220

a P e r c en t r e c o v e r y of p r o g e s te r o n e 4 - C ^ f r o m p la s m a

^ N a n o g ra m s (10~^) of p r o g e s t e r o n e / m l of p la s m a

F ig u r e 2. O v a re c to m iz e d cow s.N

NA

NO

GR

AM

S28

400

3 0 0

100

5 0

5 10 15 20 I 5 10 15 20 30

DAYS OF CYCLE

Figure 3 . Ovarectomized Cows.

Day Cow No. 564 Cow No. 706 Cow No. 708No. % R ecovery^ n g / m l ^ % R ecovery^ n g /n il^ % R ecovery^ n g / m l^ X n g /m l

P r e - H e a t S t r e s s e d1 3 3 .8 220 50. 2 95 4 9 .8 142 1525 6 2 .8 73 20 . 6 307 43. 3 177 185

10 5 9 .5 66 28. 0 300 42. 2 158 17515 76. 5 130 23. 7 504 4 8 .6 135 25620 2 5 .5 139 29 . 2 207 4 3 .6 129 158

H eat S t r e s s e d1 23. 2 122 25. 0 321 3 3 .2 142 1945 33. 8 197 30. 5 206 - - 202

10 2 3 .4 213 26. 1 253 3 5 .7 171 21215 2 6 .4 114 7 3 .6 449 5 7 .0 137 23320 3 3 .3 210 28. 1 229 42. 1 158 19930 3 4 .5 268 2 1 .6 154 50. 1 141 188

a P e r c en t r e c o v e r y of p r o g e s t e r o n e 4 - C ^ f r o m p la s m a

^ N a n o g ra m s ( 10~^) of p r o g e s t e r o n e / m l of p la s m a

F ig u r e 4. C o n tro l cow s. NN O

NA

NO

GR

AM

S30

600

5 6 4 7 0 6 7 0 8

5 0 0

4 0 0

3 0 0

200

100

0 1

10 15 20 Heats t r es s

DAYS OF CYCLE

10 15 2 0 30

Figure 5. C o n tro l C o w s .

NA

NO

GR

AM

S31

O v a r e c t o m i z e d

Control

2 5 0

# *

150

5 10 15 2 0 I 5 10 15 2 0 30s t r e s s

DAYS OF CYCLE Figure 6. Averages of Control Cows and Ovarec

tomized Cows.

B IB L IO G R A PH Y

1. A ld redp J„ , S tob , M» , and A n d r e w s » F , N. E f fe c t of H igh T e m p e r a t u r e , C h lo r p r o m a z in e and P r o g e s t e r o n e upon E m b r y o n ic S u r v iv a l in the M o u se . J . A n im a l S c i. » 20 :964 . 1961. (A b s t ra c t )

2. A l l i s to n , C. W. , and U lb e rg , L . C. E a r l y P r e g n a n c y L o s s e s inS heep a t A m b ie n t T e m p e r a t u r e s of 70 F an d 90 F as D e te r m in e d by E m b r y o T r a n s f e r . J . A n im a l S c i. » 20:608. 1961.

3. A n d e r s o n , J . S e a s o n a l V a r ia t io n in th e R e p ro d u c t iv e C ap a c i ty ofth e B u ll . J . A g r . S c i. 35 :184 . 1945,

4. A s d e l l , S. A. C a t t le F e r t i l i t y and S te r i l i ty . L i t t l e , B ro w n and Co. ,B o s to n 6, M a s s . 1955.

5. B a l f o u r , W. E . , C o m lin e , R . S. , and S h o r t , R . V. S e c r e t io n ofP r o g e s t e r o n e by th e A d r e n a l G land . N a tu r e , 18Q: 1480. 1957.

6 . B a l fo u r , W. E . , C o m lin e , R. S. , an d S h o r t , R . V. C hanges in theS e c r e t io n s of 2 0 aH y d ro x y P r e g n - 4 - e n - 3 - o n e by the A d r e n a l G landof Young C a lv e s . N a tu r e , 183:467. 1959.

7. B e a l l , D. The I s o la t io n of P r o g e s t e r o n e and 3 :2 0 -A l lo p re g n a n o le n ef r o m Ox A d r e n a l s . B io c h e m . J , , 32 :1957 . 1938.

8 . B ra n to n , C. , G r i f f i th , W. S. , N o r to n , H. W. , an d H a l l , J . G. T heIn f lu en ce of H e r e d i ty and E n v i r o n m e n t on the F e r t i l i t y of D a iry C a t t le . J . D a i ry S c i. , 39 :933 . 1956. (A b s t r a c t )

9. B u r to n , R. B . , Z a f f a ro n i , A. , and K e u tm a n n , E . H. P a p e r c h r o m ofS te r o id s . II. C o r t i c o s t e r o id s a n d R e la te d C o m p o u n d s . J . B io l . C h em . , 188:763.

10. B u sh , I. E . M eth o d s of P a p e r C h ro m a to g ra p h y of S te ro id s A p p l ic a b leto the Study of S te r o id s in M a m m a l ia n B lood an d T i s s u e s . B io c h e m . J . , 50 :370 . 1952.

11. B u s h , I. E . C h ro m a to g ra p h y of S te r o id s . P e r g a m e n P r e s s , NewY o rk . 1961.

32

33

12. Casady-g R . B . , M y e r s , R„ M. and L e g a te s , J . E„ The E f fe c t ofE x p o s u r e to H igh A m b ie n t T e m p e r a t u r e s on S p e r m a to g e n e s i s inD a i ry B u ll . J . D a i ry Sci. , 36 :14 . 1953.

13. C u p p s , P . T. and B r i g g s , J. R. M e ta b o l i s m of P r o g e s t e r o n e byA d r e n a l G land H o m o g e n a te s f r o m B u l l s . J . D a i ry S c i. , 47 :1964 .

14. D a n ie l , J . C. , J r . and L e v y , J . D. A c tio n of P r o g e s t e r o n e as aC lev ag e In h ib i to r of R a b b i t O va in V i t ro . J . R e p ro d . F e r t i l .7 :323. 1964.

15. D uncan , G. W . , B o w e r m a n , A . N. , A n d e r s o n , L . L . , H e a r n , W. R.and M e la m p y , R. M. F a c t o r s In f lu en c in g In V i t ro S y n th e s is of P r o g e s t e r o n e . ' J . E n d o c r . 1961.

16. D is z a , J . P . , H e l l e r , M. and B e r n s t e i n , S. U l t r a V io le t A b s o r p t io n , In L . L . E n g e l , E d . P h y s i c a l P r o p e r t i e s of th e S te ro id H o r m o n e s . M a c M il l ia n Co. , New Y o rk .

17. D u tt , R . H. C r i t i c a l P e r i o d f o r E a r l y E m b ry o M o r ta l i ty in E w esE x p o s e d to H igh A m b ie n t T e m p e r a t u r e s . J . A n im a l S c i. , 22;713„1963.

18. D u tt , R . H. D e t r im e n ta l E f fe c ts of H igh A m b ie n t T e m p e r a t u r e son F e r t i l i t y and E a r l y E m b ry o S u r v iv a l in S heep . In t. J .B io m e te o r . , 8 :47 , No. 1. 1964.

19- E d g a r , D. G. The P r o g e s t e r o n e C o n ten t of B ody F lu id s and T i s s u e s . J . E n d o c r . , 10:54. 1953.

20. E d g a r , D. G. The C h e m ic a l A s s a y of P r o g e s t e r o n e . B io c h e m . J . ,54 :50 . 1953.

21. E d g a r , D. G. , F lu x , D. S. and R o n a ld so n , J . W. C o m p a r i s o n of -C h e m ic a l an d B io lo g ic a l E s t im a t i o n of P l a s m a P r o g e s t i n . J . E n d o c r . , 19:44. 1959-

22. E l - S a y e d , F . I, A Study of Low B r e e d in g E f f ic ie n c y in D a i ry CowsE x p o s e d to .H ig h A m b ie n t T e m p e r a t u r e s . T h e s i s , U n iv e r s i ty of A r iz o n a , T u c s o n , A r iz o n a . 1963.

23. E r b , R. E . , A n d r e w s , F . N. and H il to n , J . W. S e a s o n a l V a r ia t io nin S e m a n Q u a li ty of D a i ry B u l ls . J . D a i ry S c i. , 25 :815 . 1942.

34

24. E r b 9 R„ E . an d S to r m s h a n k , F„ P r o g e s t i n s in C o r p o r a L u te a .O v a r ie s and A d r e n a l s a f t e r E s t r u s and B r e e d in g of N o r m a l and A b n o r m a l C ow s. J . D a iry S c i. , 44 :888 . 1961.

25. E r b , R . E . an d W ald o , D. R . S e a s o n a l C h an g es in F e r t i l i t y ofD a i ry B u l ls in N o r t h - W e s t e r n W ash in g to n . J . D a i ry S c i. , 35 :245 . 1952.

26. E r b , R. E . , W ilb u r , J . W. , and H il to n , J . H. Som e F a c t o r sA ffec tin g B r e e d in g E f f ic ie n c y in D a iry C a t t le . J . D a i ry S c i. , 23 :549. 1940. (A b s t ra c t )

27. E s t e r g r e e n , V. L. P e r s o n a l C o m m u n ic a t io n s . D ep t, of A n im a lS c i. , W ash in g to n S ta te U n iv e r s i ty , P u l l m a n , W ash in g to n , 1966.

28. F r y e r , H. C. , M arion, G. B . and F a r m e r , E . L= N o n r e tu r n R a teof A r t i f i c a l ly I n s e m in a t e d D a i ry Cows a s A ffe c te d by A ge of S e m e n , B r e e d of B u ll an d S e a so n . J . D a iry S c i . , 41 :987 . 1958.

29. F u t t e r w e i t , W. , M cN iv en , N . , an d D o r fm a n , R . G as C h r o m a to g ra p h ic Id e n t i f i c a t io n of P r o g e s t e r o n e in H u m an P r e g n a n c y P l a s m a . B io c h e m . B io p h y s , A c ta . , 71 :474. 1963.

30. G a n g w a r , P . C. , B r a n to n , C e c i l and E v a n s , D. L. R e p ro d u c t iv eand P h y s io lo g ic a l R e s p o n s e s of H o ls te in H e i f e r s to C o n t ro l le d and N a tu r a l C l im a t ic C o n d it io n s . J . D a i ry S c i. , 48 :222 . 1965.

31. G o m e z , W. R , an d E r b , R. E . P r o g e s t e r o n e in B ov ine R e p r o d u c tion : A R ev iew . J . D a i ry S c i . , 4 8 :314 . 1965.

32. G o m e z , W. R. , E s t e r g r e e n , V. L . , F r o s t , O. L . , and E r b , R . E .P r o g e s t i n L e v e ls in J u g u la r an d O v a r ia n V enous B lo o d , C o r p o r a L u te a and O v a r ie s of the N o n p re g n a n t B o v in e . J . D a iry Sci. , 4 6 :553 . 1963.

33. G o r s k i , J . , E r b , R . E . , D ic k so n , W. M . and B u t l e r , H, C.S o u r c e s of P r o g e s t i n s in th e P r e g n a n t Cow. J . D a i ry S c i. , 41 :1380 . 1958.

34. H a fe z , E . S. E . . R e p ro d u c t io n in F a r m A n im a ls . L e a and F e b i g e r ,P h i l a d e lp h ia . 1962.

35. H a n s e l , W. and T r i m b e r g e r , G. W. The E f fe c t of P r o g e s t e r o n e onO v u la tion T im e in D a i ry H e i f e r s . J . D a iry S c i. , 35 :65 . 1952.

35

36. H a r a , S. , T a n a k a , H. , and T a k e u c h i , M . S y s te m a t ic A n a ly s isof T h in L a y e r C h ro m a to g ra p h y . C h em . P h a r m . B u ll . , 12:626. 1964.

37. H o o k e r , C. W. and F o r b e s , T. R. A B i o - a s s a y f o r M in u te A m o u n tsof P r o g e s t e r o n e . E n d o c r in o lo g y 41 :158 . 1948.

38. H o w a r th , B. , A l l i s to n , C. W. and U lb e rg , L. C. I m p o r ta n c e ofU te r in e E n v i r o n m e n t of R ab b i t S p e r m P r i o r to F e r t i l i z a t i o n . J . A n im a l S c i. , 24 :1027. 1965.

39. J o h n s to n , J . E . an d B r a n to n , C. E f fe c ts of S e a s o n a l C l im a t icC hanges on C e r ta in P h y s io lo g ic a l R e a c t io n s , S e m e n P r o d u c t io n and F e r t i l i t y of D a i ry B u l l s . J . D a i ry S c i. , 36 :934 . 1953.

40. J o h n s to n , J . E . , M cD o w e ll , R. E . , S h ro d e , R . R. an d L e g a t e s ,J . E . S u m m e r C l im a te and I ts E f fe c ts on D a i ry C a t t le in the S o u th e rn R eg io n . S o u th e rn C o o p e r a t iv e S e r i e s B u ll . No. 63.Ju n e 1959o

41. L i s b o a , B . P . S e p a r a t io n and C h a r a c t e r i z a t i o n of 4 - 3 - K e to' S te ro id s of th e P r e g n a n e S e r i e s by M e a n s of T h in L a y e r C h r o m a

to g ra p h y . A c ta E n d c o r . , 43 :47 . 1963.

42. L i s b o a , B . P . T he A p p l ic a t io n of T h in L a y e r C h ro m a to g ra p h y toS te ro id s of th e A n d ro s ta n e S e r i e s . J . C h ro m a to g . , 13:391. 1964.

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