Chromosome studies of domestic animals

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This thesis, having been approved by the special Faculty Committee, is accepted by the Graduate School of the University of Wyoming , in partial fulfillm ent of the requirements fo r the degree of

M a ste r o f Sc ie n ce___ __________ Dean of the Graduate School.

T in t 0 .

Aug u s t 21, 1950

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

C H R O M O S O M E D O M E S T IC

S TU D IE S

OF

A N IM A L S

by H a rry

P.

Savery

A T h e s is S u b m itte d to th e D e p a r t m e n t o f A n i m a l P r o d u c tio n a n d th e G r a d u a te S c h o o l o f th e U n i v e r s i t y o f W y o m in g in P a r t i a l F u l f i l l m e n t o f R e q u ir e m e n ts f o r th e D e g r e e o f M a s t e r o f S c ie n c e

O BW ttTY □ F THE U N IV E R S IT Y OF W YO M ING LARAM IE

U n i v e r s i t y o f W y o m in g L a r a m i e , W y o m in g A u g u s t,. 1950

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

UMI Number: EP20620

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i 1EBARD ? w U i

ACKNOW LEDGEM ENTS

T h e a u th o r w is h e s to a c k n o w le d g e th e c o u r te s ie s a n d fin e c o o p e ra tio n he r e c e iv e d f r o m M r . a t th e U n i v e r s i t y F a r m , D r .

J im D a v id s o n ,

sheep h e rd s m a n

C . L . D a v is , h e a d o f th e B . A . I .

P a t h o lo g ic a l L a b o r a t o r y in D e n v e r , C o lo r a d o , in c o lle c tin g th e m a t e r i a l u s e d in th is s tu d y ; an d K a y S tu d io in p r in t in g th e p ic tu r e s u s e d as r e f e r e n c e s .

S in c e r e g r a titu d e is a ls o e x p r e s s e d to D r .

C . B . R o u b ic e k , o f th e d e p a r t m e n t , f o r h is c o n s u lta tio n a n d a d v ic e .

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T A B L E OF C O NTENTS

Page I N T R O D U C T I O N .........................................................................................................

1

R E V I E W O F L I T E R A T U R E ...............................................................................

2

S h e e p (O v is a r i e s ) a n d G o a t ( C a p r a h i r c u s ) ...................................

2

S w in e (S us s c r o f a ) ...........................................................................................

4

C a t t le (B o s t a u r u s ) ...........................................................................................

5

O th e r S p e c ie s a n d T e c h n i q u e s ..................................................................

6

M A T E R I A L S A N D T E C H N I Q U E S ..................................................................

11

M a t e r i a l s .................................................................................................................

11

F i x a t i v e s .................................................................................................................

12

P r o c e d u r e s .............................................................................................................

13

S t a i n s ..........................................................................................................................

14

P h o t o m i c r o g r a p h y ...........................................................................................

15

O B S E R V A T I O N S ........................................................................................................

18

D I S C U S S I O N .................................................................................................................

25

S p e r m a t o g e n e s i s ................................................................................................

25

S U M M A R Y ......................................................................................................................

35

S E L E C T E D R E F E R E N C E S ...............................................................................

37

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LIST OF FIG URES

P la te I.

II.

III.

Page F ig u r e a .

T y p i c a l tu b u le o f a 9 7 - d a y - o l d C o lu m b ia r a m

.

20

F i g u r e b.

T y p i c a l tu b u le o f 1 0 4 - d a y - o ld C o lu m b ia r a m

.

20

F ig u r e a.

T y p i c a l tu b u le o f 1 1 8 - d a y - o ld C o lu m b ia r a m

.

21

.

21

F ig u r e

b.

T y p ic a l tu b u le o f 1 5 0 - d a y - o l d C o lu m b ia r a m

F ig u r e

a.

T y p i c a l v ie w o f tu b u le s o f th e

9 7 -d a y -o ld

C o lu m b ia r a m ............................................................................................ F i g u r e b.

T y p i c a l v ie w o f tu b u le s o f th e 1 0 4 - d a y - o ld

C o lu m b ia r a m ............................................................................................ IV .

F ig u r e

a.

T y p i c a l v ie w o f tu b u le s o f th e

b.

T y p ic a l v ie w o f tu b u le s o f th e

F ig u r e

a.

24

S e c tio n o f a tu b u le s h o w in g s p e r m a to g o n ia ,

p r i m a r y s p e r m a to c y te s , a n d S e r t o li c e lls . . . . . . . . F i g u r e b.

24

1 5 0 -d a y -o ld

C o lu m b ia r a m ............................................................................................ V.

23

1 1 8 - d a y - o ld

C o lu m b ia r a m ............................................................................................ F ig u r e

23

26

S e c tio n o f a tu b u le s h o w in g d iv id in g p r i m a r y

s p e r m a to c y te s , s e c o n d a r y s p e r m a to c y te s , an d s p e r m a t i d s ....................................................................................................

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26

V

P la te V I.

V II.

Page F i g u r e a . E a r l y p ro p h a s e s t a g e ...........................................................

28

F i g u r e b. M id - p r o p h a s e s t a g e ...........................................................

28

F i g u r e a . P r i m a r y s p e r m a to c y te in e a r l y m e ta p h a s e d i v i s i o n ............................................................................................................. F ig u r e s b - f .

P o l a r v ie w s o f p r i m a r y s p e r m a to c y te s in

th e m e ta p h a s e d i v i s i o n .......................................................................... V III.

IX .

F ig u r e s a - d.

29

P o l a r v ie w s o f p r i m a r y s p e r m a to c y te s

in th e m e ta p h a s e s t a g e ..........................................................................

31

F i g u r e e . L a t e d ia k in e s is s h o w in g t e t r a d s ................................

31

F i g u r e a . P r i m a r y s p e r m a to c y te in e a r l y a n a p h a s e s ta g e

32

F ig u r e s b - c.

P r i m a r y s p e r m a to c y te s in la t e a n a p h a s e

s t a g e ................................................................................................................. F i g u r e d. X.

29

C h r o m a t id b r i d g e ...................................................................

32 32

F i g u r e a . L a t e a n a p h a s e s ta g e w it h th ic k e n in g of a c h r o m a t ic f i g u r e ................................................................................... F ig u r e b.

34

P r i m a r y s p e r m a to c y te s in v a r io u s a n a p h a s e

s t a g e s ..................................................... . , ........................................................

34

F i g u r e c.

P r i m a r y s p e r m a to c y te in te lo p h a s e s ta g e . . .

34

F i g u r e d.

N e w ly f o r m e d s e c o n d a r y s p e r m a to c y te

34

. . . .

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IN T R O D U C T I O N

C y to lo g ic a l in v e s tig a tio n s b e c o m e m o r e im p o r t a n t as a n im a l b r e e d in g p la n s f o r o u r d o m e s tic a n im a ls , n a m e ly , b e e f, s h e e p , a n d s w in e , b e c o m e m o r e c o m p le x .

A th o ro u g h u n d e rs ta n d in g o f th e

c h r o m o s o m e c o m p le x h e lp s us to d e te r m in e w h e n a n im a ls r e a c h s e x u a l m a t u r i t y , w h a t th e e ffe c ts o f m a n a g e m e n t a n d n u t r it i o n on s e x u a l m a t u r i t y a r e , w h a t th e b r e e d in g a b i li t i e s o f a n im a ls a r e , w h a t m a tin g s a r e f e a s ib le , an d w h e th e r th e o ff s p r in g o f s u c h m a tin g s w i l l b e c a p a b le to r e p r o d u c e t h e m s e lv e s .

A c r i t i c a l e x a m in a t io n o f th e

c h r o m o s o m e c o m p le x d u r in g th e d iv is io n c y c le s s h o u ld a ls o y ie ld s ig n if ic a n t in f o r m a t io n c o n c e r n in g th e p e c u l i a r it ie s o f h e r e d i t a r y t r a n s m is s io n . T h e p u rp o s e o f th is s tu d y w a s t w o - f o ld :

(1 ) to s tu d y th e c h r o m o ­

s o m e c o m p le x o f d o m e s tic a n im a ls , an d (2 ) to s e t up th e n e w c y to ­ lo g ic a l la b o r a t o r y e q u ip m e n t o n ly r e c e n t l y a c q u ir e d b y th e A n im a l P r o d u c tio n D e p a r t m e n t .

T h e f i r s t p u rp o s e is s e lf - e x p la n a t o r y ; th e

s e c o n d p u rp o s e e n t a ile d s e ttin g up th e n e w B a u s c h a n d L o m b c o m p o u n d r e s e a r c h m ic r o s c o p e a n d c a m e r a , d e t e r m in in g w h ic h te c h n iq u e s w e r e b e s t s u ite d to th is ty p e o f s tu d y , a n d r e v ie w in g c o m p r e h e n s iv e ly l i t e r a t u r e p e r t a in in g to th is f i e l d .

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2

REVIEW OF L IT E R A T U R E

D i f f i c u l t y in c o u n tin g th e h ig h c h r o m o s o m e n u m b e r in th e c h r o m o ­ s o m e c o m p le x , p lu s i m p r o p e r te c h n iq u e s , p r o b a b ly a c c o u n ts f o r th e v a r ia t io n s in c h ro m o s o m e co u n ts r e p o r t e d b y d if f e r e n t in v e s t ig a t o r s .

S h ee p (O v is a r i e s ) a n d G o a t ( C a p r a h ir c u s ) A h m e d ( 1 ) , w o r k in g w it h t e s t i c u l a r m a t e r i a l f r o m L e i c e s t e r r a m s , r e p o r t e d a d ip lo id c h ro m o s o m e n u m b e r of 5 4 .

H e n o te d th a t

th e c h ro m o s o m e s v a r i e d in s iz e a n d s h a p e , a n d th a t th e s e x c h r o m o ­ s o m e s c o n s is te d o f a l a r g e X w it h a m e d ia n c e n t r o m e r e , a n d a s m a l l e r Y w ith a s u b t e r m in a l c e n t r o m e r e .

U s in g v a r io u s f ix a t iv e s ,

A h m e d o b ta in e d h is b e s t r e s u lt s w ith th e fo llo w in g : M in o u c h i A -

1 p a r t 2% O s m ic a c id 2 p a r ts 1% C h r o m ic a c id 2 p a r t s 3% P o ta s s iu m b ic h r o m a te T h is w a s d ilu te d w it h d i s t i l l e d w a t e r in a 1:1 r a t i o .

M in o u c h i B -

1 p a r t 2% O s m ic a c id 4 p a r ts 1% C h r o m ic a c id T h is w a s d ilu te d w ith d i s t i l l e d w a t e r in a 4 :1 r a t i o .

T h e p e r io d o f f ix a t io n w a s b e tw e e n 15 a n d 18 h o u r s , a n d th e s e c ­ tio n s , w h ic h w e r e c u t 16 m ic r o n s th ic k , w e r e s ta in e d w it h g e n tia n v io le t . B u t a r i n ( 7 ) , w o r k in g w it h A r k a r a n d f a t - r u m p e d s h e e p , r e p o r t e d a c h ro m o s o m e c o u n t o f f r o m 54 to 56 f o r th e A r k a r , a n d f r o m 52 to 54

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3

f o r th e f a t - r u m p e d .

I n b o th c h ro m o s o m e c o m p le x e s , he o b s e r v e d 3

p a ir s o f l a r g e c h r o m o s o m e s ,

12 p a ir s o f i n t e r m e d ia t e c h r o m o s o m e s ,

a n d 12 p a ir s o f s m a l l c h ro m o s o m e s .

T h e m a j o r i t y o f th e c h r o m o ­

s o m e s w e r e r o d -s h a p e d . G a te s ( 1 0 ) , in r e p o r t in g on c h ro m o s o m e n u m b e r s in m a m m a ls a n d m a n , g a v e th e s h e e p an d g o a t a d ip lo id c o u n t o f 6 0 .

H e o b s e rv e d

th a t th e c h ro m o s o m e n u m b e r s in p la c e n ta l m a m m a ls a r e g e n e r a lly m o r e th a n d o u b le th o s e fo u n d in m a r s u p i a l s , a n d th a t th e u n g u la te s h a v e a h ig h e r n u m b e r th a n th e p r im a t e s . W o r k in g w ith t e s t i c u l a r m a t e r i a l f r o m th e S a a m e n g o a t, a n d th e M e r i n o , K a r a k u l a n d C o r r i e d a l e s h e e p , M a k in o (1 4 ) r e p o r t e d a d ip lo id n u m b e r o f 6 0 a n d a h a p lo id n u m b e r o f 30 f o r th e g o a t.

F o r th e

s h e e p he r e p o r t e d a d ip lo id n u m b e r o f 5 4 a n d a h a p lo id n u m b e r o f 2 7 . M a k in o o b s e r v e d th a t t h e r e w e r e 2 9 h o m o lo g o u s p a i r s o f a u to s o m e s in th e g o a t, a n d a n e q u a l p a i r o f s e x c h r o m o s o m e s .

The X -c h ro m o ­

s o m e w a s th e l a r g e s t , w h ile th e Y - s h a p e d e le m e n t r e p r e s e n t e d th e s m a lle s t in th e g ro u p .

I n th e c h ro m o s o m e c o m p le x o f th e s h e e p , he

o b s e r v e d 3 h o m o lo g o u s p a ir s o f s t e lo m it ic V - s h a p e d c h r o m o s o m e s , an d 23 h o m o lo g o u s p a ir s o f t e l o m i t i c r o d -s h a p e d c h r o m o s o m e s .

M a k in o

a ls o r e p o r t e d th a t t h e r e w a s a n u n e q u a l p a i r o f th e X - a n d Y - c h r o m o som es.

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N o v ik o v ( 1 7 ) , r e p o r t in g on th e s p e r m a to g e n e s is o f th e h y b r id s o f th e E u r o p e a n M o u flo n a n d th e M e r i n o s h e e p , o b s e r v e d a d ip lo id c h ro m o s o m e n u m b e r of 60. P c h a k a d z e ( 2 1 ) , w o r k in g w ith f a t - t a i l e d s h e e p f r o m G e o r g ia , r e p o r t e d a h a p lo id n u m b e r o f 3 0 .

H e a ls o o b s e r v e d th a t th e c h r o m o ­

s o m e s w e r e l a r g e l y s p h e r ic a l w ith 2 l a r g e V - s h a p e d o n e s .

S w in e (Sus s c r o f a ) In r e p o r t in g on th e c h ro m o s o m e c o m p le x o f th e p ig , C r e w a n d K o l l e r (8 ) g a ve a d ip lo id c o u n t o f 3 8 .

T h e y o b s e r v e d th a t th e lo n g e s t

c h r o m o s o m e w a s 7. 5 m ic r o n s lo n g , w h ile th e s m a lle s t w a s 1. 5 to 2 m ic r o n s lo n g .

T h e y o b ta in e d t e s t i c u l a r m a t e r i a l f r o m 8 la r g e W h ite

b o a r s th a t r a n g e d f r o m 8 m o n th s to 2 y e a r s in a g e .

T h e m a t e r ia l w as

f ix e d in M in o u c h i's a n d C h a m p y 's , w ith o r w ith o u t a c e t ic a c id . s e c tio n s w e r e c u t f r o m

The

16 t o 18 m ic r o n s t h ic k , a n d w e r e s ta in e d w it h

g e n tia n v io le t . G a te s (1 0 ) r e p o r t s a d ip lo id co u n t o f 38 f o r th e d o m e s tic p ig . H i l l e s b r a n d ( 1 1 ) , w o r k in g w ith G e r m a n E d e ls c h w e in , im p r o v e d L a n d s c h w e in , a n d B e r k s h i r e b r e e d s , r e p o r t e d th e d ip lo id c h r o m o ­ s o m e n u m b e r in a l l w a s 3 8 , a n d th e h a p lo id n u m b e r w a s 19. B r y d e n ( 6 ) , w o r k in g w it h l a r g e w h ite Y o r k s h i r e b o a r s r a n g in g in age f r o m 8 w e e k s to 2 y e a r s , fo u n d a c h r o m o s o m e n u m b e r o f 38 f o r th e p ig .

T h e t e s t is w a s s e c tio n e d a n d f ix e d in 2 B D , 2 B E ( L a C o u r

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5

1 9 3 1 ), 2 B D a n d C a r n o y , m e d iu m F l e m m i n g 's , a n d s tr o n g F l e m m i n g 's . W a x w a s u s e d f o r e m b e d d in g .

T h e s e c tio n s w e r e c u t f r o m

m ic r o n s t h ic k ; th e n , s ta in e d w it h g e n tia n v io le t .

10 to 16

B ry d e n re p o rte d

th a t he o b ta in e d h is b e s t r e s u lt s f r o m s e c tio n s fix e d in 2 B D an d C a r n o y , o r f r o m e it h e r o f th e F l e m m in g 's f i x a t iv e s . W o r k in g w it h t e s t i c u l a r m a t e r i a l f r o m a 1 0 -m o n th o ld P o la n d C h in a b o a r , W o d s e d a le k (2 5 ) r e p o r t e d a d ip lo id n u m b e r o f 20 c h r o m o ­ som es.

T h e t e s t is w a s r e m o v e d f r o m th e l i v e a n im a l a n d s m a l l

p ie c e s w e r e p la c e d in B o u in 's , c o r r o s iv e -a c e tic .

Z e n k e r 's , T e l ly e s n i c k y 's , a n d

S e c tio n s w e r e c u t f r o m 4 to 12 m ic r o n s t h ic k .

W o d s e d a le k o b ta in e d h is b e s t r e s u lt s f r o m m a t e r i a l f ix e d in B o u in 's f lu id , an d s ta in e d w ith H e id e n h a in 's i r o n - h e m a t o x y l i n , w it h a c id fu c h s in as a c o u n te r s ta in .

H e a ls o r e p o r t e d fa v o r a b le r e s u lt s f r o m

D e l a f ie l d 's h e m a t o x y lin , w ith e o s in as a c o u n te r s ta in .

C a ttle (B o s t a u r u s ) G a te s (1 0 ) r e p o r t s a d ip lo id c h r o m o s o m e c o u n t o f 6 0 f o r c a ttle and y ak . W o r k in g w ith d o m e s tic c a t t le , M a k in o (1 5 ) fo u n d 60 d ip lo id c h r o m o s o m e s f o r c a t t le . r o d -s h a p e d .

H e o b s e r v e d th a t a l l c h r o m o s o m e s w e r e

I n th is s a m e r e p o r t , M a k in o o b s e r v e d 4 8 d ip lo id c h r o m o ­

s o m e s f o r th e z e b u , 40 r o d -s h a p e d , a n d 8 V - s h a p e d o n e s .

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P k h a k a d z e (2 2 ) , a d ip lo id c o u n t o f 5 8 .

r e p o r t in g on th e c h ro m o s o m e s o f b u ffa lo , g a ve D i s s i m i l a r i t y in b o th n u m b e r a n d m o r p h o lo g y ,

P k h a k a d z e b e lie v e s , is th e r e a s o n f o r th e f a i l u r e o f c a t t le - b u f f a lo c ro s s e s . W o r k in g w it h y a k , Z u i t i n (2 6 ) r e p o r t e d th a t th e d ip lo id n u m b e r o f c h ro m o s o m e s w a s 6 4 .

H e n o te d c o n s id e r a b le v a r i a t i o n in th e s iz e

o f th e c h r o m o s o m e s a n d r e p o r t e d th e s m a l l e r c h ro m o s o m e s la y in th e c e n te r.

H e a ls o n o te d th a t th e c h r o m o s o m e s w e r e u s u a lly r o d -s h a p e d ,

b e a n -s h a p e d , o r V -s h a p e d .

L a t e r Z u i t i n (2 7 ) g a ve a d ip lo id n u m b e r

o f 60 f o r th e y a k , a n d n o te d th a t th e X - c h r o m o s o m e o f th e s e x c o m p le x w a s c u r v e d a n d e lo n g a te d .

O th e r S p e c ie s a n d T e c h n iq u e s R e p o r t in g on th e c h r o m o s o m e s o f m a m m a ls a n d m a n , G a te s (1 0 ) g a v e th e fo llo w in g d ip lo id c o u n ts : M a r s u p ia ls A r m a d i ll o s H o rs e s P e c c a r ie s Foxes Dogs M ic e R a ts S q u ir r e ls

2 2 (s o m e 12 o r 14)

60 60 30 34 78 40 42 2 8 t o 62

W o r k in g w it h th e c o lla r e d p e c c a r y , K r a l l i n g e r (1 2 ) fo u n d a h a p ­ lo id c h ro m o s o m e c o u n t o f 15 a n d a d ip lo id c h ro m o s o m e c o u n t o f 3 0.

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7

M a k in o (1 3 ) g a ve th e fo llo w in g d a ta on d ip lo id c h r o m o s o m e n u m b e rs : S i lv e r fo x J a p a n e s e m in k G u in e a p ig N u tr ia

38 38 64 42

(3 3 Y - s h a p e d , 5 r o d -s h a p e d ) (1 6 V -s h a p e d , 22 r o d -s h a p e d ) (2 V - s h a p e d , 6 2 r o d -s h a p e d ) (3 9 V -s h a p e d , 3 r o d -s h a p e d )

M a k in o 's b e s t p r e p a r a t io n s w e r e o b ta in e d b y u s in g as f ix a t iv e s , F l e m m i n g 's s tro n g s o lu tio n c o n ta in in g n o a c e t ic a c id , a n d C h a m p y 's m ix tu r e .

F o r s ta in in g , M a k in o r e c o m m e n d s H e id e n h a in 's i r o n -

h e m a t o x y lin m e th o d , w it h lig h t g r e e n as a c o u n te r s ta in . M e la d z e ( 1 6 ) , r e p o r t in g on th e c h ro m o s o m e n u m b e r o f th e d o m e s tic a s s , g a v e a d ip lo id c h r o m o s o m e c o u n t o f 6 0 .

U s in g t e s t i c ­

u l a r m a t e r i a l , he o b ta in e d b e s t r e s u lt s f r o m A l le n 's f i x a t iv e , a n d h e m a t o x y lin a s a s ta in . B a c h h u b e r ( 2 ) , r e p o r t in g on v a r io u s te c h n iq u e s w h ile w o r k in g w ith r a b b it t e s t e s , fo u n d th e fo llo w in g te c h n iq u e s b e s t f o r c h r o m o ­ s o m e s tu d ie s .

F o r s tu d y in g a c c e s s o r y c h r o m o s o m e s a n d th e c h r o m a -

t o id b o d y , he u s e d B o u i n ' s - - 100 c c .

of B o u in 's m a d e u p o f 75 p a r t s

o f f o r m a l i n a n d 10 p a r ts o f g l a c i a l a c e t ic a c id . g r a m s o f c h r o m ic a c id a n d 3 g r a m s o f u r e a . tio n to 37 d e g r e e s , he a d d e d th e t is s u e .

T o th is he a d d e d 1. 5

A f t e r h e a tin g th e s o lu ­

B a c h h u b e r r e p o r t e d t h a t th is

f ix a t iv e m a d e th e c h ro m o s o m e s s ta n d ou t b e t t e r a n d c l e a r e r .

For

s ta in in g , he r e c o m m e n d e d H e id e n h a in 's i r o n - h e m a t o x y l i n a n d a c id fu c h s in o r o ra n g e G .

In m a k in g s m e a r p r e p a r a t io n s , B a c h h u b e r o b ta in e d

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8

h is b e s t r e s u lt s f r o m fix in g tis s u e in s tro n g F l e m m i n g 's o r B o u in 's , a n d s ta in in g w ith i r o n - h e m a t o x y lin , a n d c o u n te r s ta in in g w ith lic h t g r u n . T h e te c h n iq u e u s e d b y P a i n t e r (1 9 ) in h is s tu d y on f ix in g t e s t i c u l a r m a t e r i a l o f m e n , m o n k e y s , b a ts , d o g s , a r m a d i l l o s , h o r s e s , a n d o p o s s u m s h a s p r o v e d m o s t s u c c e s s fu l.

T h is te c h n iq u e fo llo w s

d ir e c tly : T h e fix in g f lu id , A l l e n 's m o d if ic a t io n o f B o u in 's

f lu id ,

c o n s is ts

of*. S o lu tio n A , S a tu r a te d aq u eo u s s o lu tio n o f p i c r i c a c id , C . P .................................................. 75 c c. F o r m a l i n ( f u l l s tr e n g th p u r e ) . 25 cc. G l a c i a l a c e tic a c i d .........................10 c c. S o lu tio n B , C h r o m ic a c id c r y s t a ls C . P . . 1 . 5 g r a m s U r e a c r y s t a ls C . P ..........................2 . 0 g r a m s J u s t b e fo r e u s in g , s o lu tio n A is w a r m e d to 35 d e g r e e s in a w a t e r b a th ; th e n w h ile th e s o lu tio n is b e in g s t i r r e d , f i r s t th e c h r o m ic a c id , an d th e n u r e a a r e a d d e d .

A t th e t i m e o f f ix a t io n , th e t e m p e r a t u r e o f

th e f l u i d is th a t o f b lo o d h e a t, r o u g h ly 38 d e g r e e s C e n t ig r a d e , o r ju s t b e lo w .

T h e t e m p e r a t u r e is n e v e r a llo w e d to go h ig h e r th a n 38 d e g r e e s . T h e s te p s in f ix a t io n a r e : 1.

C a s t r a t e th e a n im a l w ith o u t th e use o f a n a e s th e tic s ,

a n d i m m e d i a t e l y p la c e th e s m a ll p ie c e s in th e f ix in g f lu id . 2.

T e a s e o r f r a y o u t th e tu b u le s in th e f ix in g f lu id .

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

3.

F i x th e tis s u e f o r tw o o r t h r e e h o u rs , no lo n g e r .

4.

R in s e in 5% a lc o h o l (2 0 0 c c . ) tw o o r t h r e e t im e s

w it h in a 1 2 - h o u r p e r io d . 5.

P la c e th e tis s u e in a 25 c c .

c a p a c ity v e s s e l, a n d b y a

d r o p m e th o d , a d d 10% a lc o h o l w h ile g e n tly a g ita t in g th e f lu id w ith a i r o r a p lu n g e r . 6. w a s h e d o u t.

K e e p th e tis s u e in 10% a lc o h o l u n t il th e p i c r i c a c id is

L i t h i u m c a r b o n a te , in a r a t i o o f 1 c c . s a t u r a t e d a q u eo u s

s o lu tio n to 50 c c . a lc o h o l, a d d e d to th e a lc o h o l w i l l f a c i l i t a t e in th e r e m o v a l o f th e p i c r i c a c id . 7.

G r a d u a lly d r o p in to th is s o lu tio n 5 0 % a lc o h o l u n t il a

c o n c e n tr a tio n o f 4 0 % is r e a c h e d . 8.

E i t h e r p r o c e d u r e A o r B m a y be u s e d n e x t: A.

L e t th e tis s u e h a r d e n a w e e k o r te n d a y s w h ile

in c r e a s in g th e c o n c e n tr a tio n o f th e a lc o h o l to 7 5 % o r 8 0 % . H e r e , to o , th e d r o p m e th o d is r e c o m m e n d e d . B.

A f t e r p la c in g th e tis s u e in a 10 c c . c a p a c ity

v e s s e l, d r o p i n a m i x t u r e c o n s is tin g of e q u a l p a r t s o f 50% a lc o h o l a n d s t r a w - c o l o r e d a n ilin e o il.

D a m p w e a th e r m a y

c lo u d th e s o lu tio n , b u t a fe w d ro p s o f 9 5 % a lc o h o l w i l l c le a r th e s o lu tio n . 9.

A d d a m ix t u r e o f e q u a l p a r t s o f 70% a lc o h o l a n d a n ilin e

o i l u n t il i t r e p la c e s th e p r e v io u s m i x t u r e .

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

10

10.

B y th e d ro p m e th o d , a d d p u r e a n ilin e o i l u n t il th e

f o r m e r m ix t u r e is r e p la c e d .

L e a v e th e tis s u e in th is u n t il i t is w e l l

c le a r e d . 11.

R e p la c e th e p u r e a n ilin e o il w ith o il o f w in t e r g r e e n .

12.

T o c a r r y th e tis s u e th r o u g h th e v a r io u s s te p s to p u re

p a r a f f i n , s t a r t w it h 1 p a r t p a r a f f i n a n d 8 to 10 p a r ts o i l o f w i n t e r g re e n .

U s in g 8 to 10 b o tt le s , in c r e a s e th e p e r c e n ta g e o f p a r a f f i n

u n t il th e tis s u e h as p a s s e d th r o u g h s e v e r a l c h an g es o f p u r e p a r a f f in . T h e tis s u e s h o u ld be in e a c h b o ttle te n m in u te s . 13.

W h e n s e c tio n in g th e t is s u e , 4 to 8 m ic r o n s t h i c k is

s u f f ic ie n t f o r m a n , d o g s , h o r s e s , a n d m o n k e y s ; a n d 7 to 8 m ic r o n s th ic k fo r op o ssu m s. 14.

S ta in w it h m o d if ic a t io n o f i r o n - h e m a t o x y lin . M o r d a n t:

2% aq u eo u s s o lu tio n o f f e r r i c - a l u m . (U s e u n o x id iz e d c r y s t a ls . )

S ta in :

0 . 5% aq u eo u s s o lu tio n o f h e m a t o x y lin ( H a m e r 's ) .

15.

M o r d a n t th e s e c tio n f r o m 4 5 to 90 m in u te s .

16.

W a s h th e s lid e in d i s t i ll e d w a t e r , u s in g 2 o r 3

17.

S ta in th e tis s u e f r o m 30 m in u te s to 2 h o u r s .

18.

D i f f e r e n t i a t e , u n d e r th e m ic r o s c o p e , w ith 0 . 1 to 0 . 2%

chan g e s .

s o lu tio n o f f e r r i c a lu m .

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11

P a i n t e r o b s e rv e d th a t th e p r e s e r v a t io n w i l l be s p o tte d , b u t s e a r c h in g w i l l y ie l d w e l l - p r e s e r v e d c e lls f o r a l l p h a s e s o f s p e r m a t o ­ g e n e s is .

M A T E R I A L S A N D T E C H N IQ U E S

T h e m a t e r i a l u s e d in t h is s tu d y c o n s is te d o f t e s t i c u l a r m a t e r i a l f r o m f o u r yo u n g C o lu m b ia r a m s , f r o m w e l l - m a t u r e d H a m p s h ir e a n d R a m b o u ille t r a m s , f r o m S h o r th o r n , H e r e f o r d , a n d A b e r d e e n - A n g u s b u lls , a n d f r o m a n u n id e n tifie d s w in e b r e e d .

M a te r ia ls T h e f o u r C o lu m b ia r a m s , o b ta in e d f r o m th e f l o c k m a in t a in e d a t th e U n i v e r s i t y f a r m , w e r e p h y s ic a lly n o r m a l in e v e r y r e s p e c t , e x c e p t f o r s u ch u n d e s ir a b le c h a r a c t e r is t i c s a s p o o r h e a d s , T h e y r a n g e d in ag e f r o m 9 7 d a y s to 150 d a y s . f r o m th e l iv e a n im a l b y th e h e r d s m a n , M r .

T h e t e s t is w a s r e m o v e d

D a v id s o n , a n d s m a ll

p ie c e s w e r e im m e d i a t e l y p la c e d in a f i x a t iv e . u s e d w e r e B o u in 's ,

c r o o k e d le g s , e tc .

T h e v a r io u s f i x a t iv e s

Z e n k e r 's , a 10% s o lu tio n of F o r m a l i n , a n d a 1%

aq u eo u s s o lu tio n o f o s m ic a c id .

L e s s th a n f iv e m in u te s la p s e d f r o m

th e t i m e th e t e s t e s , w h ic h a l l a p p e a r e d g r o s s ly n o r m a l, w e r e r e m o v e d u n t il th e s m a l l p ie c e s w e r e in th e f ix a t iv e s ; t h e r e f o r e , t h e r e w a s h a r d l y t i m e f o r a n y h a r m f u l p a th o lo g ic a l ch an g es to h a v e ta k e n p la c e .

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

12

A l l t e s t i c u l a r m a t e r i a l s o th e r th a n th e C o lu m b ia r a m s w e r e c o n tr ib u te d b y D r .

C . L . D a v is , o f th e B . A . I . P a t h o lo g ic a l

L a b o r a t o r y in D e n v e r , C o lo r a d o , w ith th e c o o p e ra tio n o f th e S w ift a n d C o m p a n y P a c k in g H o u s e a b a t t o ir .

T h e tis s u e w a s s h ip p e d i n 70%

a lc o h o l a f t e r b e in g f ix e d in B o u in ’ s.

F ix a tiv e s 1.

B o u in 's P i e r o - F o r m a l • P i c r i c a c id , s a t u r a t e d aq u eo u s s o lu tio n . . . . 75 p a rts F o r m a l i n .......................................................................... 2 5 p a r ts A c e t ic a c id ( g l a c i a l ) ............................................................... 5 p a rts *

2.

Z e n k e r 's B ic h r o m a t e o f p o t a s s i u m ................................ 2. 5 g ra m s B ic h lo r id e o f m e r c u r y ( C o r r o s i v e s u b lim a te ) . 5. 0 g ra m s S o d iu m s u l f a t e ........................................................... 1 .0 g r a m W a te r 10 v o lu m e s G l a c i a l a c e t ic a c i d ...................................................... 100 c c.

3.

4.

F o r m a l i n , 10% F o r m a lin ,c o m m e r c ia l W a te r

......................................

10 v o lu m e s 90 v o lu m e s ^

O s m ic a c id , 1% a q u eo u s s o lu tio n O s m ic a c i d ................................................................... 1 g ra m W a t e r ............................................................................................... 100 c c . 4

1. M . F . G u y e r , A n i m a l M i c r o l o g y ( U n iv e r s i t y o f C h ic a g o P r e s s , C h ic a g o , Illin o is ^ 1 9 3 9 ), p . 9 2.

Ib id . , p. 8.

3.

L o c c it .

4.

O p.

c it . , pp. 2 4 0 - 4 1 .

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

13

P ro c e d u re s 1.

T is s u e f ix e d in B o u in 's ( s lo w an d f a s t m e th o d ): F ix . . . . 12 to 2 4 h o u r s , a c c o r d in g to s iz e o f tis s u e W ash . . . 7 0 % a lc o h o l, s e v e r a l ch an g es D e h y d r a te 8 5 % a lc o h o l (2 to 6 h o u r s ) (1 to 2 h o u r s ) 9 5 % a lc o h o l (1 2 to 2 4 h o u rs ) (1 h o u r) 100% a lc o h o l (1 2 t o 2 4 h o u r s , 2 c h a n g e s ) (1 to 2 h o u r s ) In filtr a te 1 p a r t t o lu o l, 2 p a r t s 100 % a lc o h o l (h h o u r s ) E q u a l p a r t s to lu o l a n d 100 % a lc o h o l (6 to 12 h o u r s ) 3 p a r ts t o lu o l a n d 1 p a r t 100% a lc o h o l (6 to 12 h o u rs ) P u r e t o lu o l, 2 c h an g es (8 to 12 h o u rs e a c h ) M i x t u r e o f 50 p a r t s s o ft p a r a f f i n a n d 50 p a r ts Em bed t o lu o l (1 h o u r o r m o r e ) S o ft p a r a f f i n (1 h o u r) H a r d p a r a f f i n (2 h o u rs o r m o r e ) E m b e d fr o m h a rd p a r a ffin

2.

T is s u e f ix e d in Z e n k e r 's : F i x .................. 12 to 24 h o u r s , a c c o r d in g to s iz e o f tis s u e W ash . . . . R u n n in g w a t e r (1 2 t o 2 4 h o u r s ) D e h y d r a t e . 3 5 % a lc o h o l (2 to 3 h o u r s ) 5 0 % a lc o h o l (2 to 6 h o u r s ) 7 0 % a lc o h o l p lu s io d in e , u n t il no m o r e io d in e is ta k e n up 8 5 % a lc o h o l (2 to 6 h o u r s ) 9 5 % a lc o h o l (2 t o 6 h o u r s ) 100% a lc o h o l (6 to 8 h o u r s ) In filtr a te S a m e p r o c e d u r e as th a t u s e d f o r B o u in 's and em b ed

3.

T is s u e f ix e d in 10% F o r m a l i n ; F i x ..... 2 4 h o u rs D io x a n e 1 . 2 h o u rs D io x a n e 11 . 4 h o u rs o r o v e r n ig h t P a r a f f i n 1an d 11 . . 3 to 5 h o u rs

4.

T is s u e f ix e d in 1% O s m ic a c id : F i x .................. 2 4 h o u rs W ash . . . . R u n n in g w a t e r (2 4 h o u r s ) D e h y d ra te , in filtr a te , and e m b e d . S a m e p r o c e d u r e as t h a t u s e d w ith B o u in 's

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14

S ta in s S ta in s u s e d on a l l m a t e r i a l s in th is s tu d y w e r e g e n tia n v io le t , a c e t o - c a r m i n e , a n d H e id e n h a in 's i r o n - h e m a t o x y lin . u s e d w e r e e o s in , o ra n g e G , an d c o n g o -r e d .

C o u n te r s ta in s

D i f f e r e n t i a t i n g a g e n ts

u s e d w e r e i r o n - a l u m , a n d a s a t u r a t e d s o lu tio n o f p i c r i c a c id . P r o c e d u r e f o r S ta in in g w ith H e id e n h a in 's I r o n - H e m a t o x y l i n 1.

R e m o v e p a r a f f i n in x y l o l (1 0 to 15 m in u te s ) .

2.

A b s o lu te a lc o h o l (1 m in u te ).

3.

9 5 % a lc o h o l (1 m in u t e ) , th e n d i s t i l l e d w a t e r (2 m in u te s ) .

4.

T r a n s f e r f r o m w a t e r to i r o n - a l u m (3 0 m in u te s to 12 h o u r s ).

5.

W a s h th o r o u g h ly in w a t e r u n t il a l l a lu m is w a s h e d out

(1 0 m in u te s to 1 h o u r ). 6.

S ta in in 0 . 5% h e m a t o x y lin (1 h o u r ).

7.

R in s e in ta p w a t e r (5 m in u te s ) .

8.

E i t h e r p r o c e d u r e A o r B m a y be u s e d : A.

P la c e s e c tio n s in a s e c o n d s o lu tio n o f i r o n - a l u m .

d e s ta in s , a n d th e a m o u n t o f t im e r e q u i r e d v a r i e s . to 30 m in u te s m a y be r e q u ir e d .

A m in u te o r tw o

A good p r o c e d u r e to f o llo w is th a t of

removing the slid e from the iron-alum occasion ally, to rin se it in

w a t e r , a n d to e x a m in e th e s lid e u n d e r th e m ic r o s c o p e f o r p r o p e r d i f ­ f e r e n t ia t io n .

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

T h is

15

B.

P la c e s e c tio n s in a s a t u r a t e d s o lu tio n o f p i c r i c a c id .

T h e t i m e o f d if f e r e n t ia t io n v a r i e s . q u ir e d in th is s tu d y .

F ro m

1 h o u r to 3 h o u rs w a s r e ­

A good p r o c e d u r e is to g e n tly a g ita t e th e s lid e s

a b o u t e v e r y 30 m in u te s u n t il, a f t e r r in s in g th e m in w a t e r , th e y h a v e a m e d iu m - g r a y c o lo r . 9.

W a s h in ru n n in g w a t e r (1 h o u r o r u n t il a l l th e d if f e r e n t ia t in g

s o lu tio n is r e m o v e d ). 10.

W ip e o ff e x c e s s w a t e r ; t r a n s f e r s lid e to 9 5 % a lc o h o l, th e n

t r a n s f e r to a b s o lu te a lc o h o l, a n d f i n a l l y to x y l o l (1 m in u te e a c h ). 11.

M o u n t f r o m x y lo l.

P h o to m ic r o g r a p h y T h e p h o to m ic r o g r a p h ic e q u ip m e n t u s e d c o n s is te d o f th e B a u s c h a n d L o m b L c a m e r a , w it h c o m p o u n d m ic r o s c o p e a n d i l l u m i n a t o r .

The

c a m e r a , m ic r o s c o p e , a n d i l lu m i n a t o r a r e s e t up to fu n c tio n as one u n it.

C o r r e c t a d ju s tm e n t a n d a lig n m e n t a r e m o s t n e c e s s a r y f o r s u c ­

c e s s fu l p h o to m ic r o g r a p h y . A lig n in g th e C a m e r a a n d M ic r o s c o p e .

L o o s e n th e s c r e w s s e c u r ­

in g th e L - s h a p e d lo c a tin g s t r i p , a n d th e n lo o s e n th e k n u r le d h e a d s of th e c la m p s th a t s e c u r e th e m ic r o s c o p e t o th e c a m e r a s lid e b a s e , an d t u r n th e c la m p s o u tw a r d .

N e x t , s e t th e m ic r o s c o p e on th e c a m e r a

s lid e b a s e b e tw e e n th e c la m p s , w ith th e to e s o f th e m ic r o s c o p e b a s e to u c h in g th e L - s h a p e d lo c a tin g s t r i p .

W it h th e c a m e r a h ig h on th e

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16

c a m e r a b a r , m o v e th e s lid e b a s e a g a in s t it s s to p a n d t ig h te n th e c la m p s c r e w s .

S e t up th e i l l u m i n a t o r a n d a d ju s t i t so th a t th e lig h t

w i l l f a l l on th e m ic r o s c o p e m i r r o r .

N e x t, p la c e a s p e c im e n s lid e

on th e m ic r o s c o p e a n d b r in g i t in to fo c u s w it h a lo w p o w e r o b je c tiv e (1 0 X ). A f t e r th e s p e c im e n s lid e h as b e e n b ro u g h t in to s h a r p fo c u s , r e ­ m o v e th e e y e p ie c e , a n d in it s p la c e , i n s e r t th e l i g h t - t i g h t c o n n e c to r. L o o s e n th e c la m p s c r e w th a t s e c u r e s th e s lid e b a s e , a n d , b y m o v in g th e s lid e b a s e , lo c a te th e m ic r o s c o p e d i r e c t l y b e lo w th e c a m e r a f r o n t so th a t th e l i g h t - t i g h t c o n n e c to rs on th e c a m e r a f r o n t a n d th e m i c r o ­ sco p e w i l l e n t e r one a n o th e r .

W h e n th e tw o l i g h t - t ig h t c o n n e c to rs

a p p e a r to be in l i n e , s lo w ly lo w e r th e c a m e r a f r o n t u n t i l th e l i g h t - t i g h t c o n n e c to r on th e m ic r o s c o p e e n te r s th e l i g h t - t i g h t c o n n e c to r on th e c a m e ra fro n t.

A f t e r th e s e s te p s h a v e b e e n ta k e n , s e c u r e th e lo o s e n e d

s c r e w s , m a k in g c e r t a in th a t th e to e s o f th e m ic r o s c o p e b a s e to u c h th e L - s h a p e d lo c a tin g s t r i p . B y s e ttin g th e s h u tte r on " t i m e , " a n d o p e n in g i t , th e f i e l d o f v ie w , in fo c u s on th e m ic r o s c o p e ,

c a n be s e e n on th e g ro u n d g la s s

v ie w in g s c r e e n o f th e camera. T h e im a g e on th e v ie w in g s c r e e n p r o b a b ly w i l l n o t b e v e r y s h a r p u n le s s one m a k e s fin e a d ju s tm e n ts on th e m ic r o s c o p e .

C o r r e c t a li g n ­

m e n t o f th e c a m e r a a n d m ic r o s c o p e c a n be a s c e r t a in e d b y m o v in g th e

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17

c a m e r a up a n d d o w n th e c a m e r a b a r , w h ile n o tin g w h e th e r th e ends of th e g ro u n d g la s s s c r e e n a r e e q u id is ta n t to th e c i r c u l a r f i e l d o f v ie w . A lig n in g th e I l l u m i n a t o r a n d M ic r o s c o p e .

S in c e th is a lig n m e n t

is done b y v is u a l m e th o d s , i t is n e c e s s a r y to s e p a r a te th e m ic r o s c o p e f r o m th e c a m e r a .

T h is is done b y r a is in g th e c a m e r a f r o n t , lo o s e n ­

in g th e s lid e b a s e c la m p , a n d p u llin g th e s lid e b a s e o u tw a r d so th a t th e m ic r o s c o p e is out f r o m u n d e r th e c a m e r a .

N o w r e m o v e th e l i g h t ­

tig h t c o n n e c to r f r o m th e m ic r o s c o p e , a n d i n s e r t in it s p la c e th e e y e ­ p ie c e .

N e x t c la m p th e i l lu m i n a t o r to th e s lid e b a s e a t s u c h p o s itio n

a n d d is ta n c e f r o m th e m ic r o s c o p e th a t th e im a g e o f th e lig h t s o u r c e , w h e n fo c u s e d on th e m i r r o r ,

w i l l be la r g e e n o u g h to f i l l th e o p e n in g

o f th e s u b s ta g e c o n d e n s e r. A t t h is p o in t i t is n e c e s s a r y to i n s e r t a d e n se f i l t e r (n o t g ro u n d g la s s ) in th e p a th o f lig h t f r o m th e i l l u m i n a t o r .

I t is a ls o n e c e s s a r y

to h a v e a s m a l l p ie c e o f fin e w i r e to u s e as a p o in t e r .

T h is p o in te r

is p la c e d a t th e c e n te r o f th e le n s of th e i l lu m i n a t o r c o n d e n s e r.

The

s u b s ta g e c o n d e n s e r , w ith th e i r i s d ia p h r a g m o p e n e d w id e , is fo c u s e d to im a g e th e p o in te r in th e f i e l d o f v ie w .

W h ile fo c u s in g th e s u b s ta g e

c o n d e n s e r, one s h o u ld tilt th e m icroscope m irror to bring the pointer in th e c e n t e r o f th e f i e l d .

A f t e r t h is h a s b e e n d o n e , th e o c c u la r e y e ­

p ie c e is r e m o v e d a n d a p in h o le c a p , m a d e b y fo r m in g a s m a l l p ie c e s o f t i n f o i l , 3 in c h e s s q u a r e , dow n o v e r th e o p e n in g o f th e tu b e .

By

p ie r c in g a s m a l l h o le in th e c e n t e r w it h a n e e d le p o in t th e lig h t s o u rc e

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

18

is s e e n in th e le n s .

W h e n th e im a g e o f th e lig h t s o u rc e is lo c a te d ,

th e i l l u m i n a t o r is r a is e d o r lo w e r e d an d t i l t e d u n t il th e p o in te r is fo u n d in th e c e n t e r o f th e f i e l d , a n d th e im a g e of th e l ig h t s o u r c e f i l l s th e a p e r t u r e .

O n c o m p le tio n o f th e s e s te p s , th e i l lu m i n a t o r an d th e

m ic r o s c o p e a r e a lig n e d . T h e fo llo w in g ta b u la tio n g iv e s th e o u ts ta n d in g s ta in s u s e d a n d s u g g e s te d f i l t e r s :

S ta in s

F ilte r s

A c id F u c h s in e A zu re 1 B a s ic F u c h s in e C a r m in e C r y s t a l V i o le t E o s in e Y H e m a t o x y lin ( H e id e n h a in 's ) M e th y l G re e n M e th y l V io le t M e th y le n e B lu e O ra n g e I I S a fr a n in e 0 S u d an I I I S udan I V

B B B B B G B F B D C G G G

& & & & & & &

G E G G G H E

& E & G & H & H & H

O B S E R V A T IO N S

W o r k in g w it h t e s t i c u l a r m a t e r i a l f r o m fo u r yo u n g C o lu m b ia r a m s , r a n g in g in age f r o m 9 7 to 150 d a y s , i t a p p e a r s th a t th e m in im u m age f o r s tu d y in g c y t o lo g ic a lly m e io t ic c e l l d iv is io n in s h e e p is 150 d a y s . A t th is ag e t h e r e a r e a c t iv e l y d iv id in g p r i m a r y and s e c o n d a r y

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19

s p e r m a to c y te s , an d s p e r m a tid s in v a r io u s t r a n s f o r m a t i o n s ta g e s . In th e y o u n g e r a n im a ls , 9 7 ,

1 0 4 , a n d 118 d a y s o ld , th e p r e d o m in a t in g

c e lls a r e p r i m a r y s p e r m a to c y te s in th e g r o w th s ta g e , o r in o c c a s io n a l m e io t ic d iv is io n s ( P la t e s I a n d I I ,

F ig u r e s a a n d b ).

A h m e d (1 ) r e p o r t e d th a t 6 - m o n t h - o ld L e i c e s t e r r a m s s h o w e d n o s p e r m a to g e n e s is a t a l l ,

o r o n ly v e r y fe w p r i m a r y s p e r m a to c y te s .

H o w e v e r , B u t a r i n (7 ) d e s c r ib e d th is s p e r m a to g e n e s is in 6 - m o n t h - o ld fa t-ru m p e d ra m s .

In th is p a r t i c u l a r s tu d y , p r e p a r a t io n s f r o m th e

y o u n g e r a n im a ls w e r e fo u n d t o be th e m o s t s a t is f a c t o r y ,

s in c e th e

c h r o m o s o m e s te n d e d to be s p r e a d o u t m o r e a n d th e c e lls w e r e l a r g e r . T h e m a t e r i a l o b ta in e d f r o m D r . D a v is w as e x a m in e d , b u t co u n ts a n d m o r p h o lo g ic a l s tu d ie s c o u ld n o t be a s c e r t a in e d w it h a c c u r a c y due to th e c lu m p in g o f th e c h r o m o s o m e s .

I t is th o u g h t th a t th is c lu m p in g

is a r e s u l t o f th e e x c ita b le s ta te th e a n im a ls w e r e in p r i o r to k il l i n g . A n d p e r h a p s t h e r e w a s to o g r e a t a la p s e of t i m e f r o m th e k i l l i n g u n t il th e r e m o v a l o f th e t e s t i s , a n d it s b e in g p la c e d in a f i x a t iv e .

P a in te r

(1 9 ) s tr e s s e s th a t th e a n im a l s h o u ld be in a s ta te o f r e s t s e v e r a l d a y s p r i o r to o b ta in in g th e m a t e r i a l ,

an d th a t e v e r y e f f o r t s h o u ld be m a d e

to p reserv e tbe tissu e as soon as p o s s ib le a f t e r it s b lo o d s u p p ly is c u t o ff. T h e tis s u e s b e s t p r e p a r e d in t h is s tu d y w e r e th o s e f ix e d in B o u in 's a n d s ta in e d w it h H e id e n h a in 's ir o n - h e m a t o x y l i n w ith no c o u n te r ­ s ta in .

T h e tis s u e w a s s e c tio n e d f r o m 6 to 8 m ic r o n s t h ic k .

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

For

D e s c r ip tio n of P la te I

F ig u r e a.

T y p ic a l tu b u le o f a 9 7 - d a y - o ld C o lu m b ia r a m , X 1 2 1 2 . 5.

F i g u r e b.

T y p ic a l tu b u le of 1 0 4 - d a y - o ld C o lu m b ia r a m , X 1 2 1 2 . 5.

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PLATE

FI GURE

I

b

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D e s c r ip tio n of P la te II

F ig u r e a.

T y p i c a l tu b u le of 1 1 8 - d a y - o ld C o lu m b ia r a m X 1 2 1 2 . 5.

F i g u r e b.

T y p i c a l tu b u le o f 1 5 0 - d a y - o ld C o lu m b ia r a m X 1 2 1 2 . 5.

N o te s p e r m .

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PLATE

II

FIGURE

FIGURE

______

a

b

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22

d if f e r e n t i a t i o n , i t w a s fo u n d th a t a f r e s h s a t u r a t e d s o lu tio n o f p i c r i c a c id l e f t th e c y to p la s m c l e a r , an d as a r e s u l t , th e c h r o m o s o m e s w e r e s h a r p a n d c le a r .

T h e ir o n -a lu m

p la s m w ith a f a i n t s ta in .

d e s ta in te n d e d to le a v e th e c y to ­

In u s in g p i c r i c a c id as a d if f e r e n t ia t in g

a g e n t, i t is im p o r t a n t th a t th e s o lu tio n be f r e s h ; th a t i s , n o t u s e d c o n ­ tin u o u s ly o v e r a lo n g p e r io d .

I t is im p o r t a n t , to o , th a t th e s lid e s be

w a s h e d in ru n n in g w a t e r u n t i l th e p i c r i c a c id is w a s h e d f r o m th e s e c tio n s ; an h o u r o r m o r e is r e c o m m e n d e d . T h e m o s t s a t is f a c t o r y p h o to m ic r o g r a p h ic p la te s w e r e o b ta in e d b y u s in g a n o i l im m e r s i o n o b je c tiv e ( 9 7 X ) w it h a lo w p o w e r a m p lip la n le n s in th e l i g h t - t i g h t c o n n e c to r.

B & E W r a t t e n !tM " F i l t e r s w e r e

u s e d an d th e b e llo w s d r a w , th e d is ta n c e f r o m the e y e p ie c e to th e p la n e o f th e g ro u n d g la s s s c r e e n , w a s m a in t a in e d a t 20 in c h e s , e x c e p t in P la t e s I I I a n d I V , F ig u r e s a a n d b , w h e re th e b e llo w s d r a w w a s 10 in c h e s . C a u tio n s h o u ld be o b s e r v e d to p r e v e n t o v e r - m a g n if ic a t io n .

I t is

r e c o m m e n d e d th a t a f t e r th e f i e l d o f v ie w h as b e e n lo c a te d on th e v ie w ­ in g s c r e e n o f th e c a m e r a , th a t th e c a m e r a b a c k be m o v e d up a n d dow n th e c a m e r a b a r , a n d s h a r p fo c u s in g done w it h th e fin e a d ju s t m e n t u n t il th e im a g e a p p e a r s s h a r p a n d c le a r .

A t th is p o in t, o r b e llo w s d r a w ,

th e n e g a tiv e s h o u ld be e x p o s e d .

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D e s c r ip tio n of P la te III

F ig u r e a.

T y p ic a l v ie w o f tu b u le s o f th e 9 7 - d a y -o ld C o lu m b ia r a m , X 2 5 0 .

F ig u r e b.

T y p ic a l v ie w o f tu b u le s o f th e 1 0 4 -d a y o ld C o lu m b ia r a m , X 2 5 0 .

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

PLATE III

FIGURE

a

FIGURE

b

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

D e s c r ip tio n of P la te IV

F ig u r e a.

T y p ic a l v ie w o f tu b u le s o f th e 1 1 8 -d a y o ld C o lu m b ia r a m , X 2 5 0 .

F i g u r e b.

T y p ic a l v ie w o f tu b u le s of th e 1 5 0 -d a y o ld C o lu m b ia r a m , X 2 5 0 .

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

PLATE

IV

FIGURE

a

FIGURE

b

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25

D IS C U S S IO N

Spe r m a to g e n e s is T h e g la n d u la r e le m e n t, s e m in ife r o u s tu b u le s , a r e c o n v o lu te d tu b u le s fo u n d th ro u g h o u t th e t e s t is ( P la t e s I I I and I V , F ig u r e s a a n d b ). T h e s e tu b u le s a r e m a d e up o f s e v e r a l l a y e r s o f e p i t h e li a l c e l l s , w h ic h , th r o u g h m u l t i p l ic a t i o n a n d m a t u r a t io n , p ro d u c e s p e r m a to z o a . yo u n g c e l l s ,

The

o r s p e r m a to g o n ia , a r e fo u n d ly in g n e x t to th e b a s e m e n t

m e m b r a n e w h ic h s u rro u n d s th e tu b u le s .

I n t e r m in g le d w it h th e s p e r m a ­

to z o a a r e th e n u t r ie n t a n d s u p p o rtin g S e r t o li c e lls ( P la t e V , F i g u r e a ). W h e n th e s p e r m a to g o n ia e n la r g e s o m e w h a t, th e y a r e t e r m e d p r i m a r y s p e r m a to c y te s .

T h e s e p r i m a r y s p e r m a to c y te s , th r o u g h

g r o w th a n d m e io t ic n u c le a r d iv is io n , p u s h in w a r d to w a r d th e lu m e n . I t is d u r in g th is m e io t ic d iv is io n th a t th e d ip lo id c h ro m o s o m e n u m b e r o f th e s p e r m a to g o n ia is re d u c e d to th e h a p lo id n u m b e r fo u n d in th e s e c o n d a r y s p e r m a to c y te s , a n d f i n a l l y th e s p e r m a t id s .

A f t e r one c o m ­

p le te m e io t ic d iv is io n , th e tw o n e w c e lls a r e k n o w n as s e c o n d a r y s p e r m a to c y te s .

T h e s e s e c o n d a ry s p e r m a to c y te s i m m e d i a t e l y d iv id e ,

b y m i t o s i s , in to f o u r s p e r m a t id s , w h ic h th r o u g h t r a n s f o r m a t i o n f o r m s p e r m ia ( P la t e I I ,

F i g u r e b ).

C h r o m o s o m e B e h a v io r d u r in g M e i o s i s .

T h e n u c le u s o f th e you ng

s p e r m a to g o n ia a p p e a r s a s a h o m o g e n e o u s m a s s ; in d iv id u a l c h r o m o ­ s o m e s a r e in d is t in g u is h a b le .

G r a d u a lly , th is m a s s b e g in s to b r e a k up

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D e s c r ip tio n of P la te V

F ig u r e a.

S e c tio n o f a tu b u le s h o w in g s p e r m a to g o n ia ( S p g .), p r i m a r y s p e r m a to c y te s (S p c .

1), a n d S e r t o li

c e lls ( S e r . ) , X 2 4 2 5 .

F ig u r e b.

S e c tio n o f a tu b u le s h o w in g d iv id in g p r i m a r y s p e r m a to c y te s (S p c .

1), s e c o n d a r y s p e r m a to c y te s

(S p c . 2 ), a n d s p e r m a tid s ( S p t . ) , X 2 3 7 5 .

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

P L ATE V

SPC-

FIGURE

a

' ^

\5

r-

1

SPC

FIGURE

b

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

a n d th e c h r o m o n e m a ta b e c o m e m o r e d is t in c t and a p p e a r as lo n g th r e a d s ( P la t e V I , F i g u r e a ). g u is h a b le on th e th r e a d s .

Th e c h ro m o m e re s a r e e a s ily d is tin ­

T h e c h r o m o n e m a ta th e n p a s s th ro u g h

s y n a p s is b y a s s o c ia tin g l a t e r a l l y w ith t h e i r h o m o lo g u e s ( P l a t e V I , F i g u r e b ).

W h ile th is p a ir in g is in p r o g r e s s , th e c h r o m o n e m a ta b e ­

g in to s h o r te n a n d th ic k e n .

A t th is s ta g e th e t h ic k th r e a d s a r e p r e s e n t

in m o n o p lo id n u m b e r , a n d e a c h is b iv a le n t , h o m o lo g o u s c h r o m o s o m e s (S h a r p ( 2 3 ) ) .

s in c e i t c o n s is ts o f tw o

S h o r tly , e a c h m e m b e r o f th e

s y n a p s e d p a ir b e c o m e s d o u b le , r e s u lt in g in b iv a le n ts in th e f o r m o f t e t r a d s o f c h r o m a t id s .

O ne p a i r o f th e c h r o m a t id s n o w b e g in s to

s e p a r a te f r o m th e o th e r p a i r , b u t c o m p le te s e p a r a tio n is p r e v e n te d b y c h ia s m a t a , th a t i s , b y th e c r o s s in g o v e r a r e a s .

T h e t e t r a d s c o n tin u e

to s h o r te n a n d th ic k e n a n d a d en se m a t r i x c o lle c ts a b o u t th e m . I t w a s o b s e r v e d th a t d u r in g th e e a r l y m e ta p h a s e 1 s ta g e , th e s p in d le o r a c h r o m a t ic f i g u r e , a p p e a r s a n d th e t e t r a d s b e c o m e lo c a te d in a p la n e a t th e e q u a to r w it h t h e i r k in e to c h o r e s fa c in g th e p o le s ( P la t e V I I , F i g u r e a ).

D u e to th e dense m a t r i x , th e d o u b le n e s s o f e a c h

c h r o m a t id is n o t e v id e n t.

T h e a p p e a r a n c e o f th e t e t r a d s a t th is s ta g e

d ep en d s on th e p o s itio n o f th e k in e to c h o r e s .

I n th is s tu d y i t w a s n o te d

th a t th e p o s itio n o f th e k in e to c h o r e s w a s t e r m i n a l , s u b t e r m in a l, and m e d ia n , r e s u lt in g in r o d , J , a n d V - s h a p e d c h ro m o s o m e s ( P l a t e V I I , F ig u r e s b a n d c ).

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D e s c r ip tio n of P la te VI

F ig u r e a.

E a r l y p ro p h a s e s ta g e .

N o te th e lo n g

s le n d e r th r e a d s , X 2 4 2 5 .

F i g u r e b.

M id - p r o p h a s e s ta g e .

N o te th e p a r t i a l l y

synapsed th re a d s , X 2 4 2 5 .

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PLATE

VI

FIGURE

FIGURE

a

b

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D e s c r ip tio n of P la te VII

F ig u r e a.

P r i m a r y s p e r m a to c y te in e a r l y m e ta p h a s e d iv is io n .

F ig u r e s b - d.

N o te th e a c h r o m a t ic f i g u r e , X 2 4 2 5 .

P o l a r v ie w s o f p r i m a r y s p e r m a to c y te s

in th e m e ta p h a s e d iv is io n .

N o te th e sh ap e and

lo c a tio n o f th e c h r o m o s o m e s , X 2 4 2 5 .

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29

PLATE

FIGURE

FIGURE

VII

a

c

FIGURE

b

FIGURE

d

1 FIGURE

e

FIGURE

f

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30

S in c e th e p o la r v ie w o f the p r i m a r y s p e r m a to c y te in a m e ta p h a s e s ta g e p r e s e n te d th e c h r o m o s o m e c o m p le x c l e a r e r , i t w a s u s e d f o r c o u n tin g .

N u m e r o u s c e lls w e r e s tu d ie d u n d e r o il im m e r s i o n , an d

c o u n ts ta k e n r a n g e d f r o m 25 to 3 2 , w ith th e g r e a t e s t n u m b e r o f c e lls s h o w in g c o u n ts o f 26 a n d 2 7 .

B y a v e r a g in g th e co u n ts m a d e , a h a p lo id

n u m b e r o f 2 7 w a s a tta in e d ; th u s , a d ip lo id c o u n t o f 5 4 . a g r e e s w ith B e r r y (3 ) a n d M a k in o ( 1 4 ) .

T h is c o u n t

I t w a s a ls o n o te d t h a t th e

l a r g e r c h r o m o s o m e s o c c u p ie d th e p e r ip h e r y o f th e c o m p le x , w h ile th e s m a l l e r c h ro m o s o m e s o c c u p ie d th e c e n t e r ( P la t e V I I , F ig u r e s b - f) ( P l a t e V I I I , F i g u r e s a - d ). D u r in g th e e a r l y a n a p h a s e s ta g e , th e c e l l e lo n g a te s , th e t e t r a d s b e g in to s e p a r a t e , a n d th e d yad s m o v e to th e o p p o s ite p o le s ( P l a t e I X , F ig u r e s a - d ).

W h a t a p p e a r s to be a c h r o m a t id b r id g e in th e f i r s t

a n a p h a s e s ta g e w a s fo u n d in th e 9 7 - d a y o ld a n im a l s tu d ie d ( P l a t e I X , F ig u r e d ).

A lth o u g h s u ch b r id g e s a r e k n o w n to be c o m m o n in p la n ts ,

th e y a r e o n ly o c c a s io n a lly r e p o r t e d in a n im a ls . r e p o r t e d s u c h b r id g e s in s w in e .

C r e w a n d R o l l e r (8 )

T h e y r e p o r t e d th a t s u c h b r id g e s a n d

a c c e n t r ic f r a g m e n ts r e s u l t f r o m th e f o r m a t io n o f d ic e n t r ic c h r o m a t id s th r o u g h c r o s s in g - o v e r o f a r e l a t i v e l y in v e r t e d s e g m e n t; h e n c e , i t c an be a s s u m e d th a t th e s w in e s tu d ie d w a s h e te ro z y g o u s f o r a n in v e r s io n in a t le a s t one o f its c h r o m o s o m e s . T h e p e d ig r e e o f th e 9 7 - d a y o ld a n im a l u s e d in th is r e p o r t w a s s tu d ie d a n d i t w a s fo u n d th a t h is d a m w a s o u t o f c r o s s - b r e d p a r e n ts

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D e s c r ip tio n of P la te VIII

F ig u r e s a - d.

P o l a r v ie w s o f p r i m a r y s p e r m a to c y te s

in th e m e ta p h a s e s ta g e , X 2 4 2 5 .

F i g u r e e.

L a t e d ia k in e s is s h o w in g t e t r a d s .

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31

PLATE

VIII

FIGURE

a

FIGURE

b

FIGURE

c

FIGURE

d

A

V

FIGURE

_________

e

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

D e s c r ip tio n of P la te IX

F ig u r e a.

P r i m a r y s p e r m a to c y te in e a r l y a n a p h a s e s ta g e , X2425.

F ig u r e s b - c.

P r i m a r y s p e r m a to c y te s in la t e a n a p h a s e

s ta g e , X 2 4 2 5 .

F i g u r e d.

C h r o m a t id b r id g e , X 2 4 2 5 .

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PL ATE

IX

FI GURE

FIGURE

c

FIGURE

b

d

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33

a n d h is s i r e w a s ou t o f a c r o s s - b r e d d a m . r i s e to a v e r y h e te ro z y g o u s in d iv id u a l.

S u ch b r e e d in g w o u ld g iv e

C h r o m o s o m e a b e r r a t io n s su ch

a s th e s e g e n e r a l l y g iv e r i s e to u n a c c o u n ta b le c h a r a c t e r is t i c s in p ro g e n y . A f t e r th e c h ro m o s o m e s a r r a n g e th e m s e lv e s a t th e p o le s , th e s p in d le f i b e r s g ro u p to g e th e r a t th e e q u i t o r i a l p la n e a n d b e g in to th ic k e n ( P la t e X , F i g u r e a ).

D u r in g t h is la t e a n a p h a s e s ta g e , th e

c e l l b e g in s to d iv id e b y f u r r o w in g , a n d th e c e l l m e m b r a n e is f o r m e d ( P l a t e X , F i g u r e s b a n d c ). W h e n th e c e l l m e m b r a n e is f o r m e d , th e tw o n e w c e lls f o r m e d a r e k n o w n as s e c o n d a r y s p e r m a to c y te s ( P la t e X , F ig u r e d ).

These

p a s s th r o u g h a lo n g g r o w th p e r io d , w h ic h is e v id e n t b y th e la r g e n u m b e r s p r e s e n t in th e tu b u le s .

D u r in g th is g r o w in g s ta g e , th e

s e c o n d a r y s p e r m a to c y te has a g r e a t e r v o lu m e of n u c le a r m a t e r i a l th a n a t a n y p r e v io u s t i m e ; th u s r e v e r s in g th e u s u a l C / N r a t i o . C h r o m o s o m e B e h a v io r d u r in g M i t o s i s .

T h e d y a d s n e x t lo c a te

th e m s e lv e s w ith t h e i r k in e to c h o r e s a t th e e q u a to r s o f th e n e w ly f o r m e d a c h r o m a t ic f ig u r e s . II,

Im m e d i a t e l y p a s s in g th r o u g h a m i t o t i c m e ta p h a s e

a n d an a n a p h a s e I I , th e y th e n p a s s in to th e te lo p h a s e I I s ta g e .

Upon

c o m p le tio n o f th e n e w c e ll m e m b r a n e s , th e f o u r n e w c e lls a r e k n o w n as s p e r m a t id s .

B e c a u s e o f th e s c a r c it y o f c e lls in th e m e ta p h a s e I I

a n d a n a p h a s e I I s ta g e s , i t is a s s u m e d th a t th e t i m e f o r th is d iv is io n is v e r y s h o r t.

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D e s c r ip tio n of P la te X

F ig u r e a.

L a t e a n a p h a s e s ta g e w ith th ic k e n in g o f a c h r o m a t ic fig u r e , X 2 4 2 5 .

F i g u r e b.

P r i m a r y s p e r m a to c y te s in v a r io u s a n a p h a s e s ta g e s .

F i g u r e c.

N o te th e f u r r o w in g , X 2 4 2 5 .

P r i m a r y s p e r m a to c y te in te lo p h a s e s ta g e .

N o te

f o r m a t io n of c e l l m e m b r a n e , X 2 4 2 5 .

F i g u r e d.

N e w ly f o r m e d s e c o n d a r y s p e r m a t o c y t e , X 2 4 2 5 .

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34

PLATE

FIGURE

FI GURE

X

a

FIGURE

b

c

F I G UR E

d

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35

T h e n e w ly f o r m e d s p e r m a tid s a r e n o w lo c a te d n e a r th e lu m e n o f th e tu b u le .

T h e in d iv id u a l c h ro m o s o m e s a r e lo s t in a d e n se

c h r o m a t in n e tw o r k .

T h e c h r o m a t in n e t w o r k , o r n u c le u s , lo c a te s

i t s e l f a t one e n d o f th e c y to p la s m . m a s s a t it s p o s t e r io r .

T h e c y to p la s m f o r m s a n e lo n g a te d

A f t e r th e d e v e lo p m e n t of a m o t ile a x i l , a n d

th e s lu ffin g o ff o f th e e lo n g a te d m a s s o f c y to p la s m , th e s p e r m a t id is k n o w n a s a s p e r m a to z o o n ( P la t e I I ,

F i g u r e b ).

A f t e r th e t r a n s f o r m a ­

tio n s d e s c r ib e d h e r e in , th e y o u n g s p e r m a to g o n ia f o r m m a t u r e s p e r m s w ith th e h a p lo id c h r o m o s o m e c o u n t.

SUMMARY

1.

T e s t i c u l a r m a t e r i a l f r o m fo u r yo u n g C o lu m b ia r a m s r a n g in g

in ag e f r o m 9 7 to 150 d a y s o ld w a s u s e d in t h is s tu d y . 2.

T h e t e s t i c u l a r m a t e r i a l w a s im m e d i a t e l y p la c e d in one o f th e

v a r io u s f ix a t iv e s u s e d . 3.

B o u in ’ s f i x a t i v e , w ith H e id e n h a in 's ir o n - h e m a t o x y li n f o r

s ta in in g , a n d p i c r i c a c id f o r d if f e r e n t ia t io n g a v e e x c e lle n t p r e p a r a t io n s f o r c h r o m o s o m e s tu d ie s . 4.

A c c o r d in g to th is s tu d y , th e m in im u m age f o r s tu d y in g

s p e r m a to g e n e s is in s h e e p w a s e s t a b lis h e d a t fiv e m o n th s . 5.

C o r r e c t a lig n m e n t b e tw e e n c a m e r a , m ic r o s c o p e , a n d i l l u ­

m in a t o r w e r e e le m e n t a r y f o r s u c c e s s fu l p h o to m ic r o g r a p h y .

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36

6.

S h a r p , c le a r fo c u s in g p r e v e n te d o v e r - m a g n if ic a t io n .

7.

T e r m in a l,

s u b t e r m in a l, a n d m e d ia n k in e to c h o r e s w e r e

o b s e r v e d in th e c h r o m o s o m e c o m p le x . 8.

I t w a s n o te d th a t th e l a r g e r c h r o m o s o m e s o c c u p ie d th e

p e r ip h e r y o f th e c o m p le x , w h ile th e s m a l l e r c h ro m o s o m e s o c c u p ie d th e c e n t e r , d u r in g th e m e ta p h a s e s ta g e . 9.

I n one in d iv id u a l, t h e r e a p p e a r e d to be a c h r o m o s o m e b r id g e ,

th u s i t m a y be a s s u m e d th a t a c h r o m o s o m e p a i r w a s h e te r o z y g o u s f o r a n in v e r s io n . 10.

C r i t i c a l e x a m in a t io n r e v e a le d th e h a p lo id c h ro m o s o m e

n u m b e r to be 2 7 .

T h e c h ro m o s o m e s d i f f e r e d in s iz e a n d s h a p e .

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37

SELECTED REFERENCES

1-

A h m e d , I . A . , 1 T h e S tr u c tu r e an d B e h a v io r o f th e C h r o m o s o m e s o f S h e e p d u rin g M it o s is and M e i o s i s , " P r o c e e d in g s o f th e R o y a l S o c ie ty o f E d in b u r g , 6 0 : 2 6 0 - 6 9 . ( 1 9 3 9 - 4 0 S e s s io n )

2.

B a c h h u b e r , L . J . , " T h e B e h a v io r o f th e A c c e s s o r y C h r o m o s o m e s , a n d o f th e C h r o m a to id B o d y in th e S p e r m a to g e n e s is o f th e R a b b it, " B io lo g ic a l B u lle t in , 3 0 : 2 9 4 - 3 0 5 , 1 91 6 .

3.

B e r r y , R . O . , " S tu d ie s on th e C h ro m o s o m e N u m b e r s in S h e e p an d G o a t, a n d S h e e p -G o a t H y b r i d s , " J o u r n a l of H e r e d it y , 2 9 : 3 4 3 - 5 0 , 1938.

4.

B e r r y , R . O , , " T h e C h r o m o s o m e C o m p le x o f D o m e s t ic S h e e p , " J o u r n a l o f H e r e d it y , 3 2 : 2 6 1 - 6 7 , 1 94 1 .

5.

B r e m e r , J . L . , a n d W e a t h e r f o r d , H . L . , H is to lo g y , P h ila d e lp h ia : T h e B la k is t o n C o m p a n y , S ix th E d it io n , pp. 4 7 9 - 5 0 0 , 1 9 4 4 .

6.

B r y d e n , W m . , " T h e C h r o m o s o m e s o f th e P ig , " C y to lo g ia , 5 : 1 4 9 - 5 3 , 1 93 3 .

7.

B u t a r i n , N . S. , " T h e C h r o m o s o m e C o m p le x of th e A r k a r a n d th e F a t - r u m p e d S h e e p ," T r u d y , K i r g i z , k o m p l. E k s p . , 4 : ( 3 ) 7 7 - 8 0 , 1 9 3 3 -3 4 . C it e d f r o m A n im a l B r e e d in g A b s t r a c t s , 4 : ( 1 ) 4 5 , 1 9 3 6 .

8.

C r e w , F . A . E . , and K o l l e r , P . C . , " C y t o g e n e t ic a l A n a ly s is o f th e C h r o m o s o m e s in th e P i g , " P r o c e e d in g s o f th e R o y a l S o c ie ty o f E d in b u r g , 5 9 : 1 6 3 - 7 5 . (1 9 3 8 - 3 9 S e s s io n )

9.

D u k e s , H . H . , T h e P h y s io lo g y of D o m e s t ic A n i m a ls , S ix th E d it io n , It h a c a , N e w Y o r k : C o m s to c k P u b lis h in g C o m p a n y , In c . , pp. 7 7 6 -7 8 1 , 1947.

10.

G a t e s , R . R . , " C h r o m o s o m e N u m b e r s in M a m m a ls a n d M a n , " S c ie n c e , 9 6 : 3 3 6 - 3 7 ,

11.

1942.

H il l e s b r a n d , P . , " S tu d ie s in th e C h r o m o s o m e s o f T h r e e D i f f e r e n t B r e e d s o f D o m e s tic P i g s , " D i s s e r t a t i o n , U n i v e r s i t y o f B r e s l a u , p p . 2 1 - 2 2 , 1 9 3 6 . C it e d f r o m A n i m a l B r e e d in g A b s t r a c t s , 5 : 1 6 2 - 6 3 , 1 93 7 .

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

39

24.

S h a r p , L . W . , F u n d a m e n ta ls o f C y to lo g y , N e w Y o r k : M c G r a w H i l l B o o k C o m p a n y , F i r s t E d it io n , pp. 1 0 2 -1 1 7 , 1 9 4 3 .

25.

W h it e , M . J . D . , A n im a l C y to lo g y a n d E v o lu t io n , C a m b r id g e : U n i v e r s i t y P r e s s , F i r s t E d it io n , pp. 7 0 - 9 8 , 1 9 4 8 .

26.

W o d s e d a le k , J . E . , " S p e r m a to g e n e s is o f th e P ig w ith S p e c ia l R e fe r e n c e to th e A c c e s s o r y C h r o m o s o m e s , " B io lo g ic a l B u l l e t i n , 2 5 :8 2 5 , 1 9 1 2 -1 3 .

27.

Z u i t i n , A . I . , " O n th e C h r o m o s o m e s o f th e Y a k , " C . R . (D o k l) A c a d e m y o f S c ie n c e , U R S S , N . S. , 4 : 8 1 - 8 3 , 1 9 3 5 . C it e d f r o m A n i m a l B r e e d in g A b s t r a c t s , 4 ;( 1) 3 3 , 1 93 6 .

28.

Z u i t i n , A . I . , " N e w D a ta on th e C h r o m o s o m e N u m b e r in Y a k , " C . R . (D o k l) A c a d e m y o f S c ie n c e , U S S R , N . S . , 1 9 : 2 0 1 - 0 2 , 1 9 3 8 . C ite d f r o m A n i m a l B r e e d in g A b s t r a c t s , 7 : 1 1 8 - 1 9 , 1939.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.