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Some aspects of reproduction in female Microtus townsendii MacFarlane, Joan Damon 1977

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6 * V SOME ASPECTS OF REPRODUCTION I N FEMALE MICROTUS TOWNSENDII JOAN DAMON MACFARLANE B . S . , Simmons C o l l e g e , 1958 THESIS SUBMITTED I N P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE THE FACULTY OF GRADUATE STUDIES ( D e p a r t m e n t o f Z o o l o g y ) We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA A p r i l , 1977 f*^)joan Damon MacFarlane, 1977 by i n In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced d e g r e e at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Depar tment o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depar tment o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date ABSTRACT I n 1966 and 1 9 6 7 , d a t a o b t a i n e d f r o m a b r e e d i n g c o l o n y o f M i c r o t i a s  t o w n s e n d i i t o w n s e n d i i , s u p p l e m e n t e d w i t h f i e l d d a t a , have b e e n u t i l i z e d t o examine t h e f o l l o w i n g a s p e c t s o f r e p r o d u c t i o n : o n s e t o f p u b e s c e n c e i n f e m a l e ; n a t u r e o f t h e e s t r o u s c y c l e ; o v u l a t o r y p a t t e r n ; p r e g n a n c y and e v e n t s f o l l o w i n g p r e g n a n c y . O n s e t o f p u b e s c e n c e : V a g i n a l p e r f o r a t i o n o c c u r r e d w i t h t h e g r e a t e s t f r e q u e n c y be tween days 30 and 45 o f age (Range 19-82) among i s o l a t e d l i t t e r f e m a l e s . T h e r e was no p r e d i c t a b l e r e l a t i o n s h i p be tween t h e i n i t i a l o c c u r -r e n c e o f p e r f o r a t i o n and f i r s t e s t r u s i n a n i m a l s s t u d i e d (Range 1-36 d a y s ) . F i e l d f e m a l e s became p e r f o r a t e w i t h t h e g r e a t e s t f r e q u e n c y be tween 15 and 20 da ys ( u t i l i z i n g l a b o r a t o r y g r o w t h c h a r t ) . S i x o f s even f e m a l e s i n t h i s r a n g e d e m o n s t r a t e d b o t h v a g i n a l p e r f o r a t i o n and e s t r u s by smear . Inasmuch a s f i e l d s t a t i s t i c s o f age o f s e x u a l m a t u r i t y v a r y g r e a t l y i n M i c r o t u s on s e v e r a l e c o l o g i c a l b a s e s ( K r e b s and M y e r s , 1 9 7 4 ) , t h e s e d a t a a r e o f m i n i m a l s i g n i f i c a n c e i n t h e f i e l d . N a t u r e o f t h e e s t r o u s c y c l e : On t h e b a s i s o f v a g i n a l smear s t u d i e s and c o r r e l a t e d h i s t o l o g i c a l e x a m i n a t i o n o f o v a r i e s and r e p r o d u c t i v e t r a c t s , M . t o w n s e n d i i a p p e a r s t o be an i n d u c e d o v u l a t o r . Of 466 smear d a y s o f i s o l a t e d f e m a l e s : 57 p e r c e n t o f t h e smears were e s t r u s ; 35 p e r c e n t d i e s -t r u s ; 8 p e r c e n t p r o e s t r u s and m e t e s t r u s . C y c l i c p e r i o d i c i t y o f e s t r u s was f r o m 2 - 7 . 5 d a y s . No o v u l a t i o n , c o r p o r a l u t e a o r c o r p o r a a l b i c a n t i a were o b s e r v e d i n any unmated f e m a l e . A n a l y s i s o f v a r i a n c e d e m o n s t r a t e d s i g n i f -i c a n t d i f f e r e n c e be tween e s t r u s and t h e o t h e r two s t a g e s , b u t no s i g n i f i -c a n t d i f f e r e n c e be tween d i e s t r u s and a n e s t r u s e x c e p t f o r t h e l a r g e r i i d i e s t r u s mean maximum o v a r i a n d i a m e t e r . I n t e r s t i t i a l t i s s u e a p p e a r a n c e and number o f medium s i z e d f o l l i c l e s a r e p o s e d as e x p l a n a t i o n f o r t h i s d i f f e r e n c e . Two m e t e s t r u s f e m a l e s a p p e a r e d mos t s i m i l a r t o o b s e r v a t i o n s o f e s t r u s . The i n f e r e n c e i s made t h a t s t a g e s r e f l e c t l o w t o h i g h e s t r o g e n l e v e l s i n t h e o r d e r : a n e s t r u s , d i e s t r u s , m e t e s t r u s , e s t r u s . A n e s t r o u s f e m a l e s g i v e n P . M . S . became p e r f o r a t e and 7 o f 9 were i n e s t r u s 51 -53 h o u r s l a t e r . T h i s s u p p o r t s t h e e x i s t e n c e o f a p r i o r e x t r i n s i c a l l y b a s e d F . S . H . b l o c k . C o i t u s - i n d u c e d o v u l a t i o n : No unmated f e m a l e s e x a m i n e d h a d o v u l a t e d ; t h r e e f e m a l e s o v u l a t e d a f t e r sperm i n t h e v a g i n a l smear were o b s e r v e d . E v i d e n c e , t h o u g h n o t c o n c l u s i v e , i s s t r o n g l y p r e s u m p t i v e i n s u p p o r t o f c o i t u s - i n d u c e d o v u l a t i o n . P r e g n a n c y a n d e v e n t s f o l l o w i n g p r e g n a n c y : T h r e e d a t e d p r e g n a n c i e s were o b t a i n e d w i t h g e s t a t i o n p e r i o d s o f 2 1 , 2 3 , and 24 d a y s . L i t t e r s i z e ^ w a s 8 . 4 8 o f l a b o r a t o r y l i t t e r s (n = 1 3 ) ; 6 o f p r e g n a n t f i e l d f e m a l e embryo c o u n t s (n = 2 0 ) ; 5 . 4 1 o f f i e l d f e m a l e s w h i c h had l i t t e r s i n t h e l a b o r a -t o r y (n = 1 2 ) . A l l p r e g n a n t f e m a l e s d e m o n s t r a t e d d i e s t r u s v a g i n a l smea r s ; two i n s t a n c e s o f p o s t p a r t u m e s t r u s were documen ted . TABLE OF CONTENTS Page L I S T OF TABLES v i i i L I S T OF GRAPHS x L I S T OF PLATES x ACKNOWLEDGMENTS x l DEDICATION x i l C h a p t e r 1. INTRODUCTION 1 GENERAL INFORMATION 1 PURPOSE OF THIS STUDY 3 LITERATURE REVIEW OF REPRODUCTION OF MICROTUS TOWNSENDII TOWNSENDII (BACHMAN). . . 3 LITERATURE REVIEW OF REPRODUCTION I N THE GENUS MICROTUS 5 Age o f S e x u a l M a t u r i t y 5 N a t u r e o f t h e E s t r o u s C y c l e . 6 P r e g n a n c y and E v e n t s F o l l o w i n g P r e g n a n c y . . . . . . . . 10 2. MATERIALS AND METHODS 13 TRAPPING 13 L i v e T r a p p i n g 14 M a n u a l C a p t u r e 14 HOUSING AND MAINTENANCE OF ANIMALS . . . 14 P h o t o p e r i o d 15 T e m p e r a t u r e 15 D i e t 15 i i i i v Chapter Page Cages 15 I s o l a t e d a n i m a l s 15 P a i r e d a n i m a l s and unweaned l i t t e r s . . . 16 A n i m a l H e a l t h ' 16 W e i g h t s . . . 17 TECHNICAL PROCEDURES AND EQUIPMENT USED I N THE STUDY . . 17 V a g i n a l Smear T e c h n i q u e . . . . . . . 17 V a g i n a l Smear E x a m i n a t i o n . . . 18 A n i m a l S a c r i f i c e 18 P r e p a r a t i o n o f T i s s u e s f o r H i s t o l o g i c a l E x a m i n a t i o n 18 F i x a t i o n 18 Embedding , 18 T i s s u e s e c t i o n i n g and s t a i n i n g 19 H i s t o l o g i c a l e x a m i n a t i o n . 19 P h o t o m i c r o g r a p h y 19 S p e c i f i c H i s t o l o g i c a l P r e p a r a t i o n o f R e p r o d u c t i v e Organs E x a m i n e d 19 O v a r i e s , u t e r u s , f a l l o p i a n t u b e s 19 V a g i n a 20 HISTOLOGICAL MEASUREMENTS AND OBSERVATIONS 20 O v a r i e s 20 Maximum o v a r i a n d i a m e t e r 20 F o l l i c l e s 20 C o r p o r a l u t e a 22 I n t e r s t i t i a l t i s s u e 22 U t e r u s 22 V C h a p t e r Page F a l l o p i a n Tube 23 V a g i n a 23 MALES U T I L I Z E D 23 S P E C I F I C STUDY METHODS 23 O n s e t o f P u b e s c e n c e 23 N a t u r e o f t h e E s t r o u s C y c l e 25 A d m i n i s t r a t i o n o f P . M . S . t o A n e s t r o u s F e m a l e s 25 P r e g n a n c y and E v e n t s F o l l o w i n g P r e g n a n c y . . . 25 3 . RESULTS 27 OBSERVATIONS OF THE ONSET OF PUBESCENCE I N FEMALE MICROTUS TOWNSENDII TOWNSENDII . ' . 27 L i t t e r F e m a l e s 27 F i e l d F e m a l e s 30 NATURE OF THE ESTROUS CYCLE I N MICROTUS TOWNSENDII. TOWNSENDII 34 E s t r o u s C y c l e as D e t e r m i n e d by V a g i n a l Smears 34 H i s t o l o g i c a l E x a m i n a t i o n o f O v a r i e s and R e p r o d u c t i v e T r a c t s i n E a c h o f t h e Commonly O b s e r v e d V a g i n a l Smear S t a g e s : 39 O v a r i e s 43 Mean number o f d e v e l o p i n g f o l l i c l e s l a r g e r t h a n 250 m i c r a 47 Mean number o f a n t r a l f o l l i c l e s l a r g e r t h a n 350 m i c r a 47 Mean number o f a t r e t i c f o l l i c l e s g r e a t e r t h a n 250 m i c r a 47 Mean maximum o v a r i a n d i a m e t e r 47 Mean maximum o v a r i a n f o l l i c l e d i a m e t e r 47 Summary o f a n a l y s i s o f v a r i a n c e 49 A n e s t r o u s o v a r i e s 49 v i C h a p t e r Page D i e s t r o u s o v a r i e s 50 E s t r o u s a n i m a l s 50 M e t e s t r o u s o v a r i e s 50 F a l l o p i a n t u b e s 55 U t e r i n e h o r n 56 V a g i n a 57 O v a r i a n and R e p r o d u c t i v e T r a c t Response o f N i n e A n e s t r o u s F e m a l e s t o P . M . S . . . . . . 58 O b s e r v a t i o n s o f B e h a v i o r a l E s t r u s i n M i c r o t u s t o w n s e n d i i 60 M a t i n g b e h a v i o r 61 V a g i n a l smear s t a g e and b e h a v i o r a l e s t r u s 61 R e l a t i o n s h i p be tween b e h a v i o r a l e s t r u s and o v u l a t i o n 53 OBSERVATIONS OF PREGNANCY AND EVENTS FOLLOWING PREGNANCY I N MICROTUS TOWNSENDII 63 B r e e d i n g S e a s o n 63 G e s t a t i o n P e r i o d . . , 64 B i r t h and S u r v i v a l o f L i t t e r s 64 B e h a v i o r D u r i n g and F o l l o w i n g P r e g n a n c y . . . 67 The E s t r o u s C y c l e D u r i n g P r e g n a n c y and t h e P o s t p a r t u m P e r i o d . 68 R e m a t i n g A f t e r P r e g n a n c y . . . 68 4. DISCUSSION 69 ONSET OF PUBESCENCE I N MICROTUS TOWNSENDII TOWNSENDII . . . 69 NATURE OF THE ESTROUS CYCLE I N MICROTUS TOWNSENDII TOWNSENDII 74 E s t r o u s C y c l e as D e t e r m i n e d b y V a g i n a l Smears 74 C o r r e l a t i o n o f E s t r o u s C y c l e Smear S t a g e w i t h R e p r o d u c t i v e T r a c t H i s t o l o g i c a l E x a m i n a t i o n 76 v i i Page COITUS-INDUCED OVULATION 84 PREGNANCY AND EVENTS FOLLOWING PREGNANCY . . . . 86 SUMMARY 88 BIBLIOGRAPHY 93 L I S T OF TABLES T a b l e Page . I . Sperm M a t u r a t i o n S t a t u s B a s e d on C a u d a l E p i d i d y m i s Smears o f 26 F i e l d M a l e s i n J u l y 1966 24 I I . V a g i n a l P e r f o r a t i o n Age and W e i g h t and Age o f F i r s t E s t r u s i n L a b o r a t o r y B o r n F e m a l e s , 31 I I I . O c c u r r e n c e o f V a g i n a l P e r f o r a t i o n i n 53 C a p t u r e d F i e l d F e m a l e s D i s t r i b u t e d i n t o 10-Gram W e i g h t I n c r e m e n t s f r o m 5 grams t o 4 4 . 9 grams 32 I V . O v a r i a n H i s t o l o g y o f S e v e n P e r f o r a t e F i e l d F e m a l e s o f 16 t o 2 4 . 9 grams i n W e i g h t D e m o n s t r a t i n g E s t r o u s C y c l e Smear S t a g e o f E s t r u s o r P r o e s t r u s . . . . 33 V . E s t r o u s C y c l e S t a g e s as D e t e r m i n e d by D a i l y V a g i n a l Smears o f S e x u a l l y M a t u r e I s o l a t e d L a b o r a t o r y Housed M i c r o t u s t o w n s e n d i i 36 V I . P e r C e n t o f E s t r o u s C y c l e S t a g e s R e p r e s e n t e d i n 466 Days o f C o n s e c u t i v e V a g i n a l Smears o n T w e l v e L a b o r a t o r y F e m a l e s 37 V I I . Smear S t a g e s O b s e r v e d on Days P r e c e d i n g and F o l l o w i n g E s t r u s i n T w e l v e L a b o r a t o r y F e m a l e s 40 V I I I . F r e q u e n c y and P e r C e n t o f O c c u r r e n c e o f E s t r o u s C y c l e D u r a t i o n i n 40 C y c l e s O b s e r v e d i n T w e l v e L a b o r a t o r y F e m a l e s 42 I X . T a b u l a t i o n o f O v a r i a n H i s t o l o g i c a l Measuremen t s o f O v a r i e s o f F e m a l e s S a c r i f i c e d i n E s t r o u s C y c l e S t a g e s o f A n e s t r u s , D i e s t r u s , E s t r u s and M e t e s t r u s 44 X . A n a l y s i s o f V a r i a n c e o f S e l e c t e d Means o f T a b l e 8 46 X I . A n a l y s i s o f V a r i a n c e o f Means o f U t e r i n e H o r n E p i t h e l i a l C e l l H e i g h t and F a l l o p i a n Tube E p i t h e l i a l C e l l H e i g h t i n A n e s t r o u s , D i e s t r o u s and E s t r o u s F e m a l e s 48 X I I . O v a r i a n and R e p r o d u c t i v e T r a c t Response o f N i n e A d u l t I m p e r f o r a t e F e m a l e s t o P r e g n a n t M a r e ' s Serum 59 X I I I . M a t i n g D a t a o f M i c r o t u s t o w n s e n d i i F e m a l e s . . . . 62 v i i i i x T a b l e Page X I V . P r e g n a n c y D a t a and Average . L i t t e r S i z e o f L a b o r a t o r y C o l o n y F e m a l e s 65 X V . P r e g n a n c y W e i g h t and Embryo C o u n t s o f 20 P r e g n a n t F i e l d F e m a l e s 66 L I S T OF GRAPHS G r a p h Page 1. G r o w t h C u r v e o f 20 F e m a l e M i c r o t u s t o w n s e n d i i f rom Wean ing t o Day 65 o f Age . . . 28 2 . F r e q u e n c y o f O c c u r r e n c e o f V a g i n a l P e r f o r a t i o n R e l a t e d t o A n i m a l Age 29 3 . F r e q u e n c y o f O c c u r r e n c e o f V a g i n a l P e r f o r a t i o n R e l a t e d t o A n i m a l W e i g h t 29 4 ; E s t r o u s C y c l e D u r a t i o n i n Days o f 40 C y c l e s O b s e r v e d i n 12 L a b o r a t o r y F e m a l e s 41 L I S T OF PLATES P l a t e Page 1. V a g i n a l Smear S t a g e o f E s t r u s (x400) 38 2 . " H e a v y " V a g i n a l Smear S t a g e o f D i e s t r u s (x400) 38 3 . " L i g h t " V a g i n a l Smear S t a g e o f D i e s t r u s (x400) 38 4 . O v a r y o f A n e s t r o u s Female 230 (x40) 51 5 . V a g i n a l C r o s s S e c t i o n o f A n e s t r o u s Fema le 78 (x400) 51 6 . O v a r y o f F e m a l e 266 i n D i e s t r u s (x40) 52 7 . V a g i n a l C r o s s S e c t i o n o f Fema le 266 i n D i e s t r u s ( x 4 0 0 ) . . . . 52 8. O v a r y o f Fema le 411 i n E s t r u s (x40) 53 9 . V a g i n a l C r o s s S e c t i o n o f Fema le 411 i n E s t r u s ( x 4 0 0 ) . . . . . 53 1 0 . O v a r y o f Fema le 234 i n M e t e s t r u s (x40) . . . 54 1 1 . V a g i n a l C r o s s S e c t i o n o f F e m a l e 234 i n M e t e s t r u s (x400) . . . 54 x ACKNOWLEDGMENTS I w o u l d l i k e t o e x t e n d my g r a t i t u d e t o a l l t h e p e o p l e who a s s i s t e d me w i t h t h i s p r o j e c t . P a r t i c u l a r l y I t h a n k D r . M a r y T a y l o r f o r h e r p a t i e n c e and s u p p o r t t h r o u g h o u t t h e e x t e n d e d d u r a t i o n o f t h i s p r o j e c t . D r . Dean F i s h e r and D r . H a r o l d N o r d a n c o n t r i b u t e d m o r a l s u p p o r t t h r o u g h t h e i r many ' d r o p - i n ' v i s i t s t o t h e V i v a r i u m l a b o r a t o r y a s w e l l a s t e c h n i c a l s u p p o r t a s Commi t t ee members. D r s . H e l e n and D e n n i s C h i t t y a s s i s t e d by s h a r i n g M i c r o t u s f i e l d and l a b o r a t o r y c a r e i n f o r m a t i o n w i t h me. I r e c e i v e d f e l l o w s h i p s u p p o r t f r o m a t e a c h i n g a s s i s t a n t s h i p and summer r e s e a r c h g r a n t s f r o m t h e C a n a d i a n g o v e r n m e n t . x i DEDICATION TO A MOUSE On T u r n i n g Up i n H e r N e s t W i t h t h e P l o u g h , November 1785 Wee, s l e e k i t , c o w r i n , t i m ' r o u s b e a s t i e , O, wha t a p a n i c ' s i n t h y b r e a s t i e ! Thou need n a s t a r t awa sae h a s t y W i ' b i c k e r i n g b r a t t l e ! I was be l a i t h t o r i n a n ' cha se t h e e , W i ' m u r d e r i n g p r a t t l e ! I ' m t r u l y s o r r y m a n ' s d o m i n i o n Has b r o k e n N a t u r e ' s s o c i a l u n i o n . A n ' j u s t i f i e s t h a t i l l o p i n i o n W h i c h makes t h e e s t a r t l e A t me, t h y p o o r e a r t h - b o r n compan ion And f e l l o w m o r t a l ! . . . B u t M o u s i e , t h o u a r t no t h y l a n e , I m p r o v i n g f o r e s i g h t may be v a i n : The b e s t - l a i d schemes o ' m i c e a n ' men Gang a f t a g l e y , A n ' l a e ' s us n o u g h t b u t g r i e f a n ' p a i n , F o r p r o m i s e d j o y ! - R o b e r t B u r n s And my c h i e f a s s i s t a n t s : M a u r e e n , G a r t h , B r u c e and K e i t h C u t h b e r t . x i i Chapter 1 INTRODUCTION GENERAL INFORMATION In 1966 and 1967, when I c o l l e c t e d data f o r t h i s p r o j e c t , repro-duction with the genus Microtus was of growing concern to e c o l o g i s t s , a g r i c u l t u r a l i s t s and laboratory animal breeders. The c y c l i c population f l u c t u a t i o n s exhibited by these animals have r e s u l t e d i n f i e l d i n v e s t i -gations of various aspects of vole reproductive biology which might pro-vide an explanation f o r the c o n t r o l mechanism or mechanisms responsible for t h i s e c o l o g i c a l phenomenon (Clarke, 1955; Hoffman, 1958; Houlihan, 1963; C h i t t y , 1960; C h r i s t i a n and Davis, 1966; Krebs at a l . , 1973; Anderson, 1976). To date, the r o l e played by reproductive processes i s s t i l l of major concern to population e c o l o g i s t s . Data are a v a i l a b l e at present which asc e r t a i n that two reproductive v a r i a b l e s are major d r i v i n g forces behind Microtus population c y c l e s . These are the length of breeding season and the age of sexual maturity (Krebs and Meyers, 1974). However, the factor or factors which bring about v a r i a t i o n s i n these parameters are unknown. Since the natural habitat of most members of t h i s genus are f i e l d s and meadows, and t h e i r preferred d i e t i s the vegetation therein, voles pose a threat to a g r i c u l t u r a l crops, e s p e c i a l l y i n times of high popula-t i o n density (Cowan and Guignet, 1965:213). Much ea r l y research on reproduction was dire c t e d toward providing a g r i c u l t u r a l i s t s with i n f o r -mation on vole breeding habits (Hamilton, 1941; Bodenheimer, 1949). 1 2 The use o f t h e v o l e as a l a b o r a t o r y a n i m a l f o r e x p e r i m e n t a l s t u d i e s n o t n e c e s s a r i l y r e l a t e d t o r e p r o d u c t i o n has a l s o p r o d u c e d b a s i c r e p r o d u c -t i v e s t u d i e s r e s u l t i n g f r o m t h e e s t a b l i s h m e n t and m a i n t e n a n c e o f b r e e d i n g c o l o n i e s o f t h e s e s m a l l a n i m a l s ( S e l l e , 1928; R a n s o n , 1934 ; P o i l e y , 1949 ; C h i t t y , D . , 1 9 5 7 ) . I n g e n e r a l , l i t e r a t u r e r e p o r t s c o n c e r n e d w i t h t h e r e p r o d u c t i v e b i o l o g y o f v a r i o u s s p e c i e s o f M i c r o t u s , b a s e d on d a t a o b t a i n e d f r o m o b s e r -v a t i o n s o f a n i m a l s i n l a b o r a t o r y c o l o n i e s , p r o v i d e more c o n c l u s i v e e v i d e n c e o f r e p r o d u c t i v e e v e n t s t h a n t h o s e b a s e d on l i v e t r a p p e d o r k i l l e d f i e l d a n i m a l d a t a . F o r e x a m p l e , age o f s e x u a l m a t u r i t y / e s t r o u s c y c l e componen t s , d u r a t i o n o f g e s t a t i o n , o c c u r r e n c e o f p o s t p a r t u m e s t r u s o r l a c t a t i o n e s t r u s c a n o n l y be a c c u r a t e l y s t u d i e d i n t h e l a b o r a t o r y s i n c e d a t a c a n be p r e -c i s e l y d e t e r m i n e d o n l y b y c o n t i n u o u s m o n i t o r i n g o f a n i m a l s o f known age and s e x u a l h i s t o r y , an a p p r o a c h w h i c h c a n n o t be a c c o m p l i s h e d i n t h e f i e l d . T h r o u g h o u t t h e p a s t t e n y e a r s , many l a b o r a t o r y - b a s e d s t u d i e s have been p u b l i s h e d i n t h e l i t e r a t u r e w h i c h have f o c u s e d upon b a s i c f e m a l e r e p r o d u c t i v e p h y s i o l o g y i n t h e genus M i c r o t u s as an end i n i t s e l f . T h i s has r e s u l t e d i n many c o m p r e h e n s i v e c o n t r o l l e d s t u d i e s o f an e x p e r i m e n t a l n a t u r e as o p p o s e d t o t h e p r i m a r i l y d e s c r i p t i v e r e p r o d u c t i v e s t u d i e s a v a i l -a b l e p r i o r t o t h i s t i m e ( B r e e d , 1 9 6 9 ; . R ichmond and Conaway, 1969 ; C l u l o w and M a l l o r y , 1970 ; K i r k p a t r i c k and V a l e n t i n e , 1 9 7 0 ; C r o s s , 1972 ; C l u l o w and M a l l o r y , 1974 ; G r a y e t a l . , 1 9 7 4 a ) . O t h e r l a b o r a t o r y s t u d i e s have c o n s i d e r e d t h e e f f e c t o f v a r i o u s e x t r i n s i c f a c t o r s on f e m a l e r e p r o d u c t i v e p h y s i o l o g y . Some o f t h e s e a r e : t h e p r e s e n c e o f f r e s h g r e e n v e g e t a t i o n ( P i n t e r and N e g u s , 1 9 6 5 ; B e r g e r and N e g u s , 1974 ; B e r g e r e t a l . , 1 9 7 7 ) ; ma l e pheromone e f f e c t s ( H a s l e r and N a l b a n d o v , 1974 ; C l u l o w and C l a r k e , 1968 ; S t e h n and R i c h m o n d , 1 9 7 5 ; M i l l i g a n , 1 9 7 6 a , b ) ; p h o t o p e r i o d ( B r e e d 3 and Clarke, 1970); v a r i a t i o n i n quantity of mating stimulation (Gray et a l . , 1974b; Davis et a l . , 1974). L i t e r a t u r e sources c i t e d are not meant to be exhaustive, but rather, to provide the reader with an o r i e n t a t i o n to the types of approaches i n studies of the reproductive biology of members of t h i s genus. PURPOSE OF THIS STUDY The purpose of t h i s study was to decipher and describe c e r t a i n events of the reproductive cycle of females of the species Microtus town- sen d i i townsendii. This was approached by e s t a b l i s h i n g a breeding colony and u t i l i z i n g f i e l d data for supplementation and comparison. Aspects investigated i n t h i s study were: the onset of pubescence; the nature of the estrous cycle; pregnancy and events following pregnancy. The major area of concentration i s concerned with the nature of the estrous cycle i n Microtus townsendii. LITERATURE REVIEW OF REPRODUCTION OF MICROTUS  TOWNSENDII TOWNSENDII (BACHMAN) At the time of t h i s study's inception, information on the reproduc-t i v e h i s t o r y of t h i s -species was l i m i t e d to the following f i e l d data: Young number one to nine, average four; the breeding season extends from March to September (Cowan and Guignet, .1956). Within the past ten years, several Microtus townsendii studies have been done at the University of B r i t i s h Columbia through the I n s t i -tute of Animal Resource Ecology. While several of these have studied various reproductive parameters, t h e i r basic focus has been an attempt to c l a r i f y population dynamics of Microtus and more p a r t i c u l a r l y , the r o l e of genotypic v a r i a t i o n as a possible population c o n t r o l mechanism 4 (LeDuc and Krebs, 1975; Krebs et a l . , 1976; Anderson, 1976). Probably the greatest importance of specific reproductive data resulting from their research i s to demonstrate that there are no consistent reproductive f i e l d data as such for Microtus townsendii for the following parameters: juvenile growth rate; age at puberty; pregnancy rate (determined by per'cent of lactating females); length of breeding season. A l l may vary both from pop-ulation to population and within populations based on: (a) population cycle phase of given population studied; (b) common environmental effects within a given population; (c) season of the year observed. Their data prevent generalization inasmuch as they cannot be abstracted from the season, year, population, and cycle phase from which they were obtained. Parameters pertinent to this study are included as evidence of observed variation range: Age of sexual maturity as measured by mean weight: 31 grams in Spring, 1972 to 71 grams in Winter, 1973 (LeDuc and Krebs, 1975); Breeding season: 12 months in 1971-1972 to 8 months in 1972-1973 (LeDuc and Krebs, 1975); Pregnancy rate as determined by per cent of lactating females: 22 per cent in Summer, 1971 on Study grid H to 45 per cent on study grid G in Summer, 1971 (LeDuc and Krebs, 1975). Litter size i s a parameter which does not appear to be linked to population dynamics in Microtus (Krebs and Myers, 1974). The l i t t e r size determined on the basis of autopsy mean by. Anderson (1976) was 4.88. Litter size mean based on observed births was 4.03. Both of these figures are consistent with the mean observed l i t t e r size of 4 (Cowan and Guignet, 1956). 5 LITERATURE REVIEW OF REPRODUCTION I N THE GENUS MICROTUS A s p e c t s o f r e p r o d u c t i o n o f c o n c e r n t o t h i s s t u d y a r e : age o f s e x u a l m a t u r i t y ; n a t u r e o f t h e e s t r o u s c y c l e i n M i c r o t u s ; p r e g n a n c y and e v e n t s f o l l o w i n g p r e g n a n c y . The f o l l o w i n g i s a summary o f p r e s e n t i n f o r m a t i o n a v a i l a b l e i n t h e l i t e r a t u r e r e l a t i v e t o e a c h o f t h e s e a r e a s . Age o f S e x u a l M a t u r i t y The age o f s e x u a l m a t u r i t y o f an a n i m a l h a s been d e f i n e d by L o n g and Evans (1922) as t h e e a r l i e s t s t a g e a t w h i c h an a n i m a l i s c a p a b l e o f p r o d u c i n g y o u n g . These a u t h o r s , i n t h e i r i n v e s t i g a t i o n o f r e p r o d u c t i o n o f f e m a l e R a t t u s n o r v e g i c u s , o b s e r v e d t h a t o f 193 r a t s , 46% o v u l a t e d a t t h e same t i m e v a g i n a l p e r f o r a t i o n o c c u r r e d ; 22% w i t h i n t h e n e x t f o u r d a y s ; t h e r e m a i n d e r be tween d a y s s i x t o f i f t e e n . I n M i c r o t u s a g r e s t i s B r a m b e l l and H a l l (1939) o b s e r v e d v a g i n a l p e r f o r a t i o n t o o c c u r b y day 20 i n mos t a n i m a l s , y e t no v i a b l e p r e g n a n c i e s r e s u l t e d i n a n i m a l s w h i c h c o u l d have c o n c e i v e d be tween days 20 and 30—an i n d i c a t i o n o f i n a b i l i t y o f a n i m a l s t o r e p r o d u c e d u r i n g t h i s p e r i o d . G r e e n w a l d (1957) s t a t e s t h a t young M i c r o t u s c a l i f o r - n i c u s o c c u r i n f i e l d p o p u l a t i o n s w i t h c o r p o r a a l b i c a n t i a y e t w i t h o u t u t e r i n e s c a r s o r mammary g l a n d d e v e l o p m e n t . He s u g g e s t s t h i s i s i n d i c a t i v e o f s t e r i l e c y c l e s w h i c h r e f l e c t a " t u n i n g - u p " s t a g e b e f o r e t h e p i t u i t a r y -g o n a d a l r e l a t i o n s h i p i s w e l l e s t a b l i s h e d . Cowan and A r s e n a u l t (1954) d e s c r i b e t h e o c c u r r e n c e o f b e h a v i o r a l e s t r u s as e v i d e n c e d by o b s e r v a t i o n o f c o i t u s be tween age d a y s 22 and 24 and y e t no c o n c e p t i o n s o c c u r r e d u n t i l age d a y s 35 and 36 i n M i c r o t u s o r e g o n i . They s u g g e s t t h e e x i s t e n c e o f a s t e r i l e p e r i o d o f f r o m f i v e t o f o u r t e e n d a y s f o l l o w i n g p u b e r t y i n t h i s a n i m a l . F rom t h e above i n f o r m a t i o n , i t a p p e a r s t h a t i n M i c r o t u s , v a g i n a l 6 p e r f o r a t i o n , b e h a v i o r a l e s t r u s and o v u l a t i o n a r e n o t n e c e s s a r i l y i n d i c a t i v e o f t h e age t r u e s e x u a l m a t u r i t y o c c u r s a s d e f i n e d b y L o n g and E v a n s . These s i g n s a r e r e f l e c t i v e o f i n i t i a l g o n a d a l h o r m o n a l changes i n d i c a t i v e o f p u b e r t y . N a t u r e o f t h e E s t r o u s C y c l e To s t u d y t h e e s t r o u s c y c l e o f an a n i m a l , f i r s t t h e c o n s t i t u e n t components o f t h e c y c l e must be d e t e r m i n e d . L o n g and E v a n s (1922) i n t h e i r s t u d y o f t h e n o r m a l e s t r o u s c y c l e o f R a t t u s n o r v e g i c u s s t a t e t h a t v a g i n a l smear s t a g e s c o n s t i t u t e t h e o n l y r e l i a b l e method o f r e c o g n i z i n g v a r i o u s s u b d i v i s i o n s o f t h e c y c l e i n t h e l i v i n g a n i m a l . The v a g i n a l smear method was f i r s t d e m o n s t r a t e d b y S t o c k h a r d and P a p i n i c o l a o u (1917) i n t h e g u i n e a p i g . They d e t e r m i n e d t h a t v a g i n a l s e c r e t i o n s r e f l e c t e d c h a n g e s i n t h e v a g i n a l e p i t h e l i u m c o r r e l a t e d w i t h v a r i o u s components o f t h e e s t r o u s c y c l e . I n summary, t h e s e components have b e e n i d e n t i f i e d s e q u e n t i a l l y a s : p r o e s t r u s , t h e s t a g e o f r a p i d G r a a f i a n f o l l i c l e d e v e l o p m e n t , i d e n t i f i e d i n v a g i n a l smears p r i m a r i l y by t h e a p p e a r a n c e o f many n u c l e a t e d e p i t h e l i a l c e l l s , few c o r n i f i e d c e l l s o r l e u k o c y t e s , a s m a l l amount o f o r no mucus ; e s t r u s , t h e s t a g e i m m e d i a t e l y p r e c e d i n g o v u l a t i o n and p o s s i b l y i n c l u s i v e o f o v u l a -t i o n a t t h i s t e r m i n a t i o n ; i d e n t i f i e d i n v a g i n a l smears c h a r a c t e r i s t i c a l l y by t h e a l m o s t e x c l u s i v e a p p e a r a n c e o f l a r g e , o f t e n t a t t e r e d c o r n i f i e d c e l l s , u n n u c l e a t e d o r w i t h p y k n o t i c n u c l e i ; m e t e s t r u s , p e r i o d i m m e d i a t e l y f o l l o w i n g o v u l a t i o n d u r i n g w h i c h c o r p o r a l u t e a o r g a n i z a t i o n o c c u r s , and c h a r a c t e r i z e d b y a v a g i n a l smear c o n t a i n i n g many l e u k o c y t e s and c o r n i f i e d c e l l s ; d i e s t r u s , t h e s t a g e w h i c h r e f l e c t s t h e l u t e a l s t a g e o f t h e c y c l e a t w h i c h t i m e c o r p o r a l u t e a a r e a c t i v e , and r e f l e c t e d by t h e p r e s e n c e o f mucus i n v a g i n a l s m e a r s , s c a t t e r e d l e u k o c y t e s and c e l l d e t r i t u s a n d o f t e n c o r n i f i e d c e l l s i n e a r l y d i e s t r u s and n u c l e a t e d e p i t h e l i a l c e l l s i n l a t e 7 d i e s t r u s . D i e s t r u s i s t e r m i n a t e d w i t h a t r e s i a o f c o r p o r a l u t e a i n t h e n o n -p r e g n a n t a n i m a l o r a t p o s t p a r t u m i n t h e p r e g n a n t a n i m a l . A t t h i s t i m e t h e c y c l e r e t u r n s t o p r o e s t r u s and i s r e p e a t e d u n l e s s t h e a n i m a l goes o u t o f b r e e d i n g c o n d i t i o n and i n t o a n e s t r u s , a c o n d i t i o n i n r o d e n t s g e n e r a l l y c h a r a c t e r i z e d by v a g i n a l i m p e r f o r a t i o n i n w h i c h no v a g i n a l smear e x a m i n a -t i o n i s p o s s i b l e . A l l e n (1922) a l s o d e m o n s t r a t e d t h i s a p p r o a c h t o be a r e l i a b l e method f o r d e t e r m i n a t i o n o f e s t r o u s c y c l e s t a g e i n Mus m u s c u l u s . S i m i l a r smear s t a g e s have been d e s c r i b e d f o r many mammals w i t h m i n o r v a r i -a t i o n s . E a r l y p a p e r s w h i c h d e s c r i b e d e s t r o u s c y c l e s t a g e s d i d so i n s p o n -t a n e o u s o v u l a t o r s s i m i l a r t o t h o s e m e n t i o n e d . C o n f u s i o n has d e v e l o p e d due t o t h e a p p l i c a t i o n o f t h e same e s t r o u s c y c l e t e r m i n o l o g y t o t h e v a g i n a l smear a p p e a r a n c e o f n o n - s p o n t a n e o u s o r i n d u c e d o v u l a t o r s . The i n d u c e d o v u l a t o r w i l l o n l y o v u l a t e i f c o i t u s o r s i m i l a r v a g i n a l c e r v i c a l s t i m u l a -t i o n o c c u r s . T h e r e f o r e , i n t h e i s o l a t e d i n d u c e d o v u l a t o r t h e v a g i n a l smear s t a g e s o f d i e s t r u s and m e t e s t r u s w o u l d n o t be e x p e c t e d t o o c c u r ; o r , s h o u l d t h e y o c c u r , one w o u l d n o t e x p e c t t o o b s e r v e , on h i s t o l o g i c a l e x a m i n a t i o n o f t h e o v a r i e s , o v u l a t i o n o r e v e n t s s u b s e q u e n t t o o v u l a t i o n . A l l s t a g e s d e s c r i b e d b y A l l e n (1922) i n Mus m u s c u l u s have been d e s c r i b e d i n t h e v a g i n a l smears o f unmated known m i c r o t i n e i n d u c e d o v u l a -t o r s . I n a c o l o n y o f M i c r o t u s a g r e s t i s h o u s e d a t O x f o r d , C h i t t y and A u s t i n (1957) o b s e r v e d c l e a r e v i d e n c e o f r e g u l a r v a g i n a l c y c l e s a l t h o u g h n o t a s c l e a r l y d e f i n e d a s i n t h e common l a b o r a t o r y r o d e n t s . On t h e b a s i s o f r e c e p t i v i t y t h e y d e t e r m i n e d a c y c l e w h i c h a p p e a r e d t o be r e p e a t e d e v e r y t h i r d o r f o u r t h d a y . I n a s e c o n d c o l o n y o f M i c r o t u s a g r e s t i s h o u s e d i n L o n d o n , t h e same p a p e r d e s c r i b e s a v a g i n a l smear p a t t e r n o f p r o l o n g e d 8 p e r i o d s o f c o r n i f i c a t i o n i n t e r r u p t e d o c c a s i o n a l l y by a d i e s t r o u s i n t e r v a l and o v a r i a n h i s t o l o g i c a l e x a m i n a t i o n o f t h e s e a n i m a l s w h i c h d e m o n s t r a t e d no c o r p o r a l u t e a o r t u b a l o v a . B r e e d (L967) u t i l i z e d t h e c o l o n y o f M i c r o - t u s a g r e s t i s a t O x f o r d and d e m o n s t r a t e d by c o n t r o l l e d m a t i n g e x p e r i m e n t s and s u b s e q u e n t o v a r i a n h i s t o l o g i c a l e x a m i n a t i o n t h a t t h e s e a n i m a l s were i n d u c e d o v u l a t o r s . I n a c o l o n y o f M i c r o t u s o c h r o g a s t e r , R ichmond and Gonaway (1969) o b s e r v e d g r e a t v a r i a t i o n i n t h e n a t u r e o f t h e v a g i n a l smear c o r r e l a t e d w i t h h o u s i n g v a r i a t i o n s f o r a d u l t f e m a l e s . T h i s v a r i a t i o n r a n g e d f r o m i m p e r f o r a t i o n t o p r o e s t r u s , e s t r u s and d i e s t r u s s m e a r s . The e s t r u s s t a g e was common i n mos t f e m a l e s . V i a c o n t r o l l e d m a t i n g e x p e r i m e n t s , t h e y d e m o n s t r a t e d M . o c h r o g a s t e r t o be an i n d u c e d o v u l a t o r . I n o n l y two f e m a l e s d i d t h e y o b s e r v e c y c l i c phenomena and t h e y s t a t e t h a t t h e excep- . ' t i o n s a r e p r o b a b l y r e s p o n s i b l e f o r many l i t e r a t u r e r e p o r t s t h a t m i c r o t i n e s have v a g i n a l c y c l e s s i m i l a r t o t h o s e o f t h e l a b o r a t o r y mouse . Zondek and Sulman (1940) o b s e r v e d t h a t i s o l a t e d M i c r o t u s g u e n t h e r i f e m a l e s demon-s t r a t e d c o n t i n u o u s l y t h e c h a r a c t e r i s t i c v a g i n a l smear s t a g e o f d i e s t r u s . H o w e v e r , when M i c r o t u s g u e n t h e r i f e m a l e s were ma ted and s a c r i f i c e d f o r o v a r i a n h i s t o l o g i c a l e x a m i n a t i o n , B o d e n h e i m e r and Su lman (1946) o b s e r v e d t h e v a g i n a l smear s t a g e o f e s t r u s . a n d t h e p r e s e n c e o f c o r p o r a l u t e a on h i s t o l o g i c a l e x a m i n a t i o n . I s o l a t e d f e m a l e s d e m o n s t r a t e d n e i t h e r e s t r u s n o r c o r p o r a l u t e a . The a u t h o r s c o n c l u d e t h a t i n d u c e d o v u l a t i o n o c c u r s i n t h i s s p e c i e s . K i r k p a t r i c k and V a l e n t i n e (1970) s t a t e t h a t t h e y o b s e r v e d i n M i c r o t u s p i n e t o r u m a l l smear s t a g e s r e p o r t e d t o e x i s t i n t h e house mouse as d e s c r i b e d by A l l e n ( 1 9 2 2 ) , a l t h o u g h t h e y do n o t d e s c r i b e c y c l i c phenomena. V i a c o n t r o l l e d m a t i n g e x p e r i m e n t s t h e y a l s o d e t e r m i n e d M i c r o -t u s p i n e t o r u m t o be an i n d u c e d o v u l a t o r . I n summary t h e n , a l l smear s t a g e s d e s c r i b e d f o r s p o n t a n e o u s 9 o v u l a t o r s o c c u r i n M i c r o t u s , b u t n o t n e c e s s a r i l y i n t h e same s e q u e n t i a l p a t t e r n n o r w i t h c y c l i c p e r i o d i c i t y . M e t e s t r u s and p r o e s t r u s o c c u r r a r e l y b u t d i e s t r u s and e s t r u s smears a r e p r e s e n t t h e m a j o r i t y o f t h e t i m e . The p a r t i c u l a r smear e x h i b i t e d a p p e a r s t o be more dependen t upon e x t r i n s i c e n v i r o n m e n t a l f a c t o r s t h a n upon i n t r i n s i c r e p r o d u c t i v e r h y t h m . A t t h e i n i t i a t i o n o f t h i s p r o j e c t i n 1 9 6 6 , my a s s u m p t i o n was t h a t t h e M i c r o t u s  t o w n s e n d i was a s p o n t a n e o u s o v u l a t o r . A t t h a t t i m e two s p e c i e s were s t a t e d t o be i n d u c e d o v u l a t o r s : M i c r o t u s c a l i f o r n i c u s ( G r e e n w a l d , 1 9 5 6 ) , and M i c r o t u s g u e n t h e r i (Bodenhe imer and S u l m a n , 1 9 4 6 ) . Two o t h e r s p e c i e s were s t a t e d t o be s p o n t a n e o u s o v u l a t o r s , M i c r o t u s oeconomus ( H o y t e , 1955) and M i c r o t u s a r v a l i s ( D e l o s t , 1 9 5 5 ) . B o t h t y p e s o f o v u l a t i o n were s t a t e d t o o c c u r i n M i c r o t u s a g r e s t i s ( C h i t t y and A u s t i n , 1 9 5 7 ) . I n o r d e r t o o b t a i n c o n c l u s i v e e v i d e n c e o f i n d u c e d o v u l a t i o n , t h e c r i t e r i a s u g g e s t e d b y G r e e n w a l d (1956 :214) s t a t e : "The o n l y p o s i t i v e p r o o f o f i n d u c e d o v u l a t i o n i s t h r o u g h c o p u l a t i o n , o r c e r v i c a l s t i m u l a t i o n , com-b i n e d w i t h d a t a f r o m u n s t i m u l a t e d c o n t r o l s . O t h e r l i n e s o f e v i d e n c e , t h o u g h s u g g e s t i v e a r e n o t c o n c l u s i v e . " B r e e d (1967) s t a t e s t h a t i n t h e genus M i c r o t u s , e v i d e n c e f o r s p o n t a n e o u s o v u l a t i o n i s a t p r e s e n t o n l y c i r c u m s t a n t i a l . O b s e r v a t i o n s o n b e h a v i o r and v a g i n a l smear changes a r e n o t s u f f i c i e n t p r o o f f o r i t s o c c u r r e n c e . R ichmond and Conaway (1969) a g r e e w i t h B r e e d and a l s o q u e s t i o n t h e n a t u r e o f t h e e v i d e n c e upon w h i c h i n v e s t i g a t o r s have s u g g e s t e d t h e o c c u r r e n c e o f s p o n t a n e o u s o v u l a t i o n i n M i c r o t u s . To d a t e , t h o s e v o l e s f o r w h i c h c o n c l u s i v e e v i d e n c e o f i n d u c e d o v u l a t i o n has b e e n p u b l i s h e d , u s i n g t h e c r i t e r i a o f G r e e n w a l d (1956) a r e : M i c r o t u s g u e n t h e r i (Bodenhe imer and S u l m a n , 1 9 4 6 ) ; M i c r o t u s c a l i f o r n i c u s ( G r e e n w a l d , 1 9 5 6 ) ; M i c r o t u s a g r e s t i s ( B r e e d , 1 9 6 7 ) ; M i c r o t u s o c h r o g a s t e r 10 (Richmond and Conaway, 1 9 6 9 ) ; M i c r o t u s p e n n s y l v a n i c u s ( C l u l o w and M a l l o r y , 1 9 7 0 ) ; M i c r o t u s p i n e t o r u m ( K i r k p a t r i c k and V a l e n t i n e , 1 9 7 0 ) ; M i c r o t u s  montanus (Gray e t a l . , 1 9 7 4 a ) . No s t u d i e s d i s a g r e e w i t h t h e i m p l i e d s u g -g e s t i o n o f B r e e d (1967) t h a t i n d u c e d o v u l a t i o n may be t h e p r i m a r y r e p r o -d u c t i v e mechan i sm o f t h e w h o l e g e n u s . P r e g n a n c y and E v e n t s F o l l o w i n g P r e g n a n c y A s p e c t s o f p r e g n a n c y and e v e n t s f o l l o w i n g p r e g n a n c y r e s e a r c h e d i n t h e l i t e r a t u r e f o r t h i s s t u d y i n c l u d e l e n g t h o f g e s t a t i o n and l i t t e r s i z e , o c c u r r e n c e o f p o s t p a r t u m e s t r u s , and o c c u r r e n c e o f l a c t a t i o n a n e s t r u s . The g e s t a t i o n p e r i o d and a v e r a g e l i t t e r s i z e i s known f o r s e v e r a l s p e c i e s o f M i c r o t u s . I n summary, t h e g e s t a t i o n p e r i o d i s o f a p p r o x i m a t e l y t h r e e w e e k s ' d u r a t i o n : M i c r o t u s c a l i f o r n i c u s 21 d a y s ( S e l l e , 1 9 2 8 ) ; M i c r o - t u s a g r e s t i s 21 days (Ranson , 1 9 3 4 ) ; M i c r o t u s p e n n s y l v a n i c u s 21 da ys ( P o i l e y , 1949) ; M i c r o t u s o r e g o n i 23 J 5-24 3 s da ys (Cowan and A r s e n a u l t , 1954) ; M i c r o t u s o r c a d e n s i s 2 0 - 2 1 days ( C h i t t y , - 1 9 5 7 ) ; M i c r o t u s o c h r o g a s t e r 2 1 . 5 days (Richmond and Conaway, 1 9 6 9 ) . The a v e r a g e l i t t e r s i z e o f t h e s p e c i e s l i s t e d r a n g e s f r o m 2 . 9 5 t o 4 . 0 . . The o c c u r r e n c e o f a p o s t p a r t u m e s t r u s i n t h e i m m e d i a t e p e r i o d f o l l o w i n g p a r t u r i t i o n h a s been r e p o r t e d f o r s e v e r a l s p e c i e s o f v o l e s : M i c r o t u s a g r e s t i s (Ranson , 1934); M i c r o t u s o r e g o n i (Cowan and A r s e n a u l t , 1954); M i c r o t u s c a l i f o r n i c u s ( G r e e n w a l d , 1957); M i c r o t u s o c h r o g a s t e r (Richmond and Conaway, 1 9 6 9 ) ; and M i c r o t u s p i n e t o r u m ( K i r k p a t r i c k and V a l e n t i n e , 1 9 7 0 ) . Among t h o s e s p e c i e s o f M i c r o t u s t h a t have b e e n s t u d i e d , t o t h i s a u t h o r ' s k n o w l e d g e , a l l e x p e r i e n c e a p o s t p a r t u m e s t r u s . T h e r e i s s t i l l q u e s t i o n as t o w h e t h e r o r n o t l a c t a t i o n a n e s t r u s o c c u r s i n f e m a l e s w h i c h f a i l t o mate a t p o s t p a r t u m e s t r u s . A l s o , t h e r e i s a q u e s t i o n as t o wha t mechanism may be r e s p o n s i b l e f o r t h i s phenomenon. 11 B r a m b e l l and H a l l (1939) s u g g e s t e d t h a t t h e c o r p o r a l u t e a w h i c h f o r m e d a t p o s t p a r t u m i n M i c r o t u s a g r e s t i s w o u l d become t h e c o r p o r a l u t e a o f l a c -t a t i o n and t h e a n i m a l s w o u l d n o t r e t u r n t o e s t r o u s c o n d i t i o n u n t i l l a c t a -t i o n c e a s e d . B r e e d (1969) d e m o n s t r a t e d t h a t no c o r p o r a l u t e a f o r m a t p o s t -p a r t u m i n unmated M i c r o t u s a g r e s t i s . H e l e n C h i t t y (1957) s t u d i e d f r e q u e n c y i n t e r v a l s o f l i t t e r s o f t h e same s p e c i e s and c o n c l u d e d t h e r e was no e v i d e n c e o f l a c t a t i o n a l a n e s t r u s . H o w e v e r , she s u g g e s t e d t h a t t h e p o s s i b i l i t y o f i m p l a n t a t i o n d e l a y , i n a s m u c h as h e r d a t a s u g g e s t e d l i t t e r s were b o r n o f p o s t p a r t u r i e n t a n i m a l s a t p e r i o d s l o n g e r t h a n t h e e x p e c t e d 2 1 - d a y g e s t a -t i o n p e r i o d . I n t h e Montane v o l e , M . m o n t a n u s , G r a y e t a l . (1974a) o b s e r v e d d a t a a l s o b a s e d on s t u d y o f l i t t e r i n t e r v a l s w h i c h s u g g e s t t h a t e s t r u s s u p p r e s s i o n o c c u r s d u r i n g l a c t a t i o n f o l l o w i n g p o s t p a r t u m e s t r u s . R ichmond and Conaway (1969) a l s o s t u d i e d l i t t e r i n t e r v a l s o f M i c r o t u s  o c h r o g a s t e r . They o b s e r v e d t h a t 61.5% o f t h e a n i m a l s d i d n o t show a l a c -t a t i o n a l p r o l o n g a t i o n o f g e s t a t i o n and t h a t t h e r e m a i n d e r o f t h e a n i m a l s may r e f l e c t t h a t f e r t i l e m a t i n g s d i d n o t o c c u r a t p a r t u r i t i o n . H o w e v e r , t h e y do s u g g e s t t h a t a d e g r e e o f e s t r u s s u p p r e s s i o n may o c c u r a b o u t m i d -l a c t a t i o n on t h e b a s i s o f l i t t e r i n t e r v a l s . E x p l a n a t i o n f o r t h i s t h e y s u g g e s t may be due t o s u b l i m i n a l s t i m u l a t i o n by t h e o r i g i n a l mate r a t h e r t h a n due t o l a c t a t i o n a l b l o c k . C r e d e n c e i s g i v e n t o t h i s h y p o t h e s i s b y t h e w o r k s o f M i l l i g a n and M a c K i n n o n ( 1 9 7 6 ) . T h e i r work p r o v i d e s e v i d e n c e w h i c h d e m o n s t r a t e s p r e g -n a n c y f a i l u r e i n M . a g r e s t i s c a n be due t o i n e f f e c t i v e m a t i n g s t i m u l u s w h i c h f a i l s t o e v o k e s u f f i c i e n t l e v e l s o f p r o l a c t i n and l u t e i n i z i n g hormone t o s u s t a i n c o r p o r a l u t e a f u n c t i o n . The work o f G r a y e t a l . (1974b) d e m o n s t r a t e s t h a t s u b l i m i n a l m a t i n g s t i m u l a t i o n may a l s o b l o c k o v u l a t i o n . They s t a t e t h a t i t i s p r o b a b l e t h a t e a c h s p e c i e s has a p a r t i c u l a r m a t i n g 12 p a t t e r n r e l a t i v e t o e i t h e r q u a l i t y o r q u a n t i t y o f c o i t u s , o r b o t h , w h i c h must o c c u r i n o r d e r f o r t h e f e m a l e t o e v e n o v u l a t e . A n o t h e r p o s s i b l e e x p l a n a t i o n f o r o b s e r v e d phenomena i s t h e o c c u r -r e n c e o f p r e g n a n c y b l o c k on a p h e r o m o n a l b a s i s . T h r e e s p e c i e s o f M i c r o t u s have d e m o n s t r a t e d p r e g n a n c y f a i l u r e when f e m a l e s were e x p o s e d t o s t r a n g e m a l e s w i t h i n 48 t o 72 h o u r s f o l l o w i n g an e f f e c t i v e . m a t i n g : M . a g r e s t i s ( C l u l o w and L a n g f o r d , 1 9 6 8 ) ; M . p e n n s y l v a n i c u s ( C l u l o w and L a n g f o r d , 1 9 7 1 ) ; M . o c h r o g a s t e r ( S t e h n and R i c h m o n d , 1 9 7 5 ) . H o w e v e r , i t i s u n l i k e l y t h a t t h i s w o u l d a c c o u n t f o r a p p a r e n t l a c t a t i o n a l b l o c k o r e s t r u s s u p p r e s s i o n u n d e r t h e u s u a l l a b o r a t o r y s i n g l e s t u d m a t i n g r e g i m e . I n summary, t h e r e i s e v i d e n c e t o s u g g e s t t h a t i t may be i m p o s s i b l e f o r f e r t i l e m a t i n g s t o o c c u r d u r i n g a t l e a s t some p o r t i o n o f l a c t a t i o n , w h e t h e r t h e c a u s e o f t h i s phenomenon i s i m p l a n t a t i o n d e l a y , s u b l i m i n a l m a t i n g s t i m u l a t i o n o r h o r m o n a l l a c t a t i o n a l b l o c k due t o i n t r i n s i c r e p r o -d u c t i v e mechanisms o r e x t r i n s i c e n v i r o n m e n t a l f a c t o r s , p o s s i b l y o f p h e r o m o n a l n a t u r e , i s a t p r e s e n t unknown. C h a p t e r 2 MATERIALS AND METHODS T h i s s t u d y i s b a s e d p r i m a r i l y o n o b s e r v a t i o n s o f two g r o u p s o f f e m a l e a n i m a l s c o n f i n e d i n t h e l a b o r a t o r y : l a b o r a t o r y b o r n l i t t e r s and a d u l t f i e l d a n i m a l s . F i e l d d a t a o b t a i n e d d u r i n g t h e s t u d y a r e i n c l u d e d when p e r t i n e n t t o l a b o r a t o r y r e s u l t s . TRAPPING A l l a n i m a l s were c a p t u r e d i n L a d n e r , B . C . on a d e l t a a r e a a t t h e mouth o f t h e F r a s e r . R i v e r . A n i m a l s f o r t h i s s t u d y were c a p t u r e d w h i l e i n b r e e d i n g c o n d i t i o n . B r e e d i n g s e a s o n f o r M i c r o t u s t o w n s e n d i i (Bachman) e x t e n d s f rom M a r c h t o Sep tember (Cowan and G u i g n e t , 1 9 6 5 ) . I n w i n t e r 1971-1972 t h e r e i s e v i d e n c e t h a t some M i c r o t u s t o w n s e n d i i r e m a i n e d i n b r e e d i n g c o n d i t i o n t h r o u g h o u t t h e w i n t e r , w h i l e i n 1 9 7 2 - 1 9 7 3 , t h e same s t u d y p o p u l a t i o n c e a s e d b r e e d i n g f o r a f o u r month p e r i o d (Leduc and K r e b s , 1 9 7 5 ) . I n 1967 i n t h i s s t u d y a r e a , a n i m a l s c a p t u r e d p r i o r t o F e b r u a r y were o b s e r v e d t o be i n a n e s t r u s . D u r i n g two s p e c i f i c p e r i o d s , 169 a n i m a l s were c a p t u r e d : May 1966 t o Sep tember 1966; l a t e F e b r u a r y 1967 t o J u n e 1 9 6 7 . Of t h e s e a n i m a l s , 52 were k i l l e d i m m e d i a t e l y and t h e i r r e p r o d u c t i v e t r a c t s e x a m i n e d ; 117 a n i m a l s were k e p t i n t h e l a b o r a t o r y f o r s t u d y and b r e e d i n g p u r p o s e s . T r a p p i n g methods u t i l i z e d were l i v e t r a p p i n g and manua l c a p t u r e o f a n i m a l s . 13 14 L i v e T r a p p i n g A n i m a l s were t r a p p e d i n b o t h L o n g w o r t h and Sherman l i v e t r a p s . A t r a p l i n e o f 30 t r a p s was c h e c k e d d a i l y . A l l t r a p p i n g was done i n c u l -t i v a t e d f i e l d s o f h a y and c l o v e r c r o p s . T r a p s u c c e s s was p o o r a l t h o u g h t r a p s were p l a c e d n e a r t u n n e l o p e n i n g s i n runways and c o v e r e d w i t h h a y . T h i s me thod n e t t e d a p p r o x i m a t e l y one a n i m a l p e r 30 t r a p n i g h t s . B a i t d i d n o t i m p r o v e t r a p s u c c e s s . B a i t s t r i e d i n c l u d e d r o l l e d o a t s and p e a n u t b u t t e r . A p i e c e o f c a r r o t o r t u r n i p was added t o e a c h t r a p t o p r e v e n t d e h y d r a t i o n o f c a p t u r e d a n i m a l s . T r a p s u c c e s s i m p r o v e d when 1 0 - f o o t - l o n g t a r p a p e r s t r i p s were p l a c e d on t o p o f s e v e r a l t r a p s s e t i n r u n w a y s . A n i -m a l s m i g r a t e d t o t h e t a r p a p e r a r e a s as e v i d e n c e d by new t u n n e l d i g g i n g , new l a v a t o r y a r e a s , and f r e s h l y c l i p p e d g r a s s i n t h e c o v e r e d r u n w a y s . One t i m e a week t a r p a p e r s were moved a p p r o x i m a t e l y 20 f e e t f r o m p r e v i o u s l o c a -t i o n s and t r a p s : r e s e t . D u r i n g t h e month o f M a r c h 1 9 6 7 , 44 f e m a l e s were o b t a i n e d f r o m one f i e l d u s i n g t h i s m e t h o d . M a n u a l C a p t u r e F l e e i n g a n i m a l s were e a s i l y c a p t u r e d by hand w h i l e r o u t i n e a g r i c u l -t u r a l f i e l d o p e r a t i o n s were i n p r o g r e s s ( e . g . , d i s k i n g , p l o w i n g , hay b a i l i n g ) . Recommended f i e l d e q u i p m e n t f o r t h e method i n c l u d e s l e a t h e r g l o v e s . T h i s method o f a n i m a l c a p t u r e p r o v i d e d g r e a t e r numbers o f a n i m a l s t h a n l i v e t r a p p i n g . On one o c c a s i o n , i n a w e l l t u n n e l e d f i e l d , 70 a n i m a l s were c o l -l e c t e d by t h i s me thod i n t h r e e h o u r s . HOUSING AND MAINTENANCE OF ANIMALS A n i m a l s were h o u s e d i n a c o n t r o l l e d e n v i r o n m e n t room i n t h e Z o o l o g y V i v a r i u m a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a . 15 P h o t o p e r i o d A r t i f i c i a l d a y l i g h t was p r o v i d e d w i t h a 200-watt b u l b . An a u t o m a t i c I n t e r m a t i c t i m e s w i t c h was s e t so a n i m a l s r e c e i v e d 16 h o u r s o f l i g h t a n d 8 h o u r s o f d a r k n e s s p e r 24 h o u r s . B a k e r and Ranson (1932) f o u n d 15 h o u r s o f l i g h t p e r 24 h o u r s s u f f i c i e n t t o m a i n t a i n M i c r o t u s a g r e s t i s i n b r e e d i n g c o n d i t i o n t h r o u g h o u t t h e y e a r . T e m p e r a t u r e Range o f t e m p e r a t u r e i n t h e c o n t r o l l e d e n v i r o n m e n t room was f r o m 24.4 t o 25.6 d e g r e e s C e n t i g r a d e . T h i s i s w i t h i n t h e r a n g e C h i t t y (1957) c o n s i d e r s a d e q u a t e f o r l a b o r a t o r y m a i n t e n a n c e o f v o l e b r e e d i n g c o n d i t i o n . D i e t S t a n d a r d B u c k e r f i e l d ' s Mouse P e l l e t s - U . B . C . F o r m u l a were p r o v i d e d a d l i b i t u m i n a d d i t i o n t o t h e f o l l o w i n g : a l f a l f a h a y , a p p r o x i m a t e l y 20 grams p e r week; w a t e r , a d l i b i t u m w i t h s t a n d a r d l a b o r a t o r y d r i p b o t t l e s ; l o c a l c l o v e r s , g r a s s e s and b u t t e r c u p s , p e r i o d i c a l l y when i n s e a s o n ; g e r -m i n a t e d wheat,"'" p e r i o d i c a l l y when l o c a l p l a n t s n o t a v a i l a b l e . Cages I s o l a t e d a n i m a l s . A n i m a l s were c o n t a i n e d i n d i v i d u a l l y i n m e t a l c i r u l a r b a s i n s 12 i n c h e s i n d i a m e t e r and 6 i n c h e s d e e p . These b a s i n s were p l a c e d i n t i e r e d D e x i o n r a c k s o f h e a v y w i r e mesh . A p p r o x i m a t e l y lh i n c h e s o f s awdus t was u s e d as an a b s o r b e n t i n t h e b o t t o m o f c a g e s . Once e a c h e a c h week a n i m a l s were p l a c e d i n c l e a n s t e r i l i z e d b a s i n s c o n t a i n i n g f r e s h s a w d u s t . P i n t e r and Negus (1965) p r e s e n t e d e v i d e n c e t h a t s u p p l e m e n t a l f e e d i n g o f g e r m i n a t e d whea t i n c r e a s e d number o f l i t t e r s and o f f s p r i n g o f M i c r o t u s m o n t a n u s . 16 Paired animals and unweaned l i t t e r s . For breeding purposes and rearing of l i t t e r s , eight large cages were constructed containing glass sides measuring 27 inches by 13h inches and varying from 16 to 24 inches in height. These large containers had 'a-inch wire screen tops and alu-minum tray bottoms. Galvanized sheet metal nest boxes were added to the cages. Also added were a small amount of cotton batting to nest boxes, a l f a l f a hay, and standard drip water bottles. Approximately lh inches of sawdust was used as absorbent in the bottom of these cages. Once a week a l l components of the cages were removed. The cages were washed at this time and fresh sawdust and a l f a l f a hay added. Nest boxes were replaced undisturbed. Animal Health Any obviously i l l animals were removed from the colony immediately and destroyed. The majority of the animal deaths which occurred demon-strated no obvious pathology on autopsy; therefore, definite cause of death cannot be stated. The most common infectious condition observed was one resulting in an eye exudate followed by loss of eye and generally death. Approximately 12 animals with these symptoms were destroyed or died during the study. The exudate was cultured in the Bacteriology Department of the University of British Columbia and found to contain a sulfonamide-sensitive strain of Escherichia c o l i . As no major outbreak occurred, treatment was not attempted. Overgrown incisors posed a f a i r l y common problem. These were observed most commonly in animals recently captured, i.e., during the f i r s t few weeks of/.laboratory confinement. This problem was solved by tooth-clipping to above the pulp line with toe-nail clippers (suggested 17 by l o c a l v e t e r i n a r i a n ) . Weights A l l animals were weighed weekly on day of cage cleaning. An Ohaus Triple-Beam balance scale was used. TECHNICAL PROCEDURES AND EQUIPMENT USED IN THE STUDY Vaginal Smear Technique The back of the vole i n the region of the scapulae was grasped with the l e f t hand. The thumb and f o r e f i n g e r held both sides of the cra-nium, thus immobilizing the head and neck. The r i g h t hand was used to aspirate f l u i d from the vagina. Approximately one drop of water was placed i n a f i n e l y drawn glass pipette with an aspir a t o r top. The pipett e was placed at the vaginal o r i f i c e . Water was expressed r a p i d l y into the vagina and aspirated. An attempt was made not to i n s e r t the pi p e t t e i n t o the vagina i n order to avoid trauma of the vaginal wall and the p o s s i b i l i t y of inducing ovulation. Vaginal f l u i d obtained was dropped on a clean glass s l i d e . Smears were a i r d r i e d and then f i x e d with absolute methyl alcohol f o r f i v e minutes. Sl i d e s were then placed i n Giemsa stock s o l u t i o n d i l u t e d with 75 volumes of d i s t i l l e d water. S l i d e s remained i n t h i s s o l u t i o n f o r 15 minutes f. then were rins e d i n tap water and a i r d r i e d . Routinely, on the suggestion of Dr. W. K. Whitten (pers. comm.), ten days of smears were taken on one s l i d e and stained and read every ten days. In order to obtain ten smears per s l i d e , s l i d e s were ru l e d o f f i n t o ten sections l i g h t l y with .a diamond marking p e n c i l . 18 V a g i n a l Smear E x a m i n a t i o n V a g i n a l smears were e x a m i n e d w i t h an A m e r i c a n O p t i c a l Company s t a n d a r d m o n o c u l a r compound l i g h t m i c r o s c o p e a t 40x and lOOx m a g n i f i c a t i o n . The G e i m s a s t a i n a l l o w s a f a i r l y r a p i d d i s c r i m i n a t i o n as t o t h e age and s t a g e o f d e v e l o p m e n t o f e p i t h e l i a l c e l l s i n t h e smea r . The n u c l e i o f s m a l l r o u n d n u c l e a t e d e p i t h e l i a l c e l l s s t a i n a deep p i n k - p u r p l e d y e ; t h e c y t o p l a s m s t a i n s b l u e . I n l a r g e r and more i r r e g u l a r l y shaped e p i t h e l i a l c e l l s , b o t h t h e c y t o p l a s m and n u c l e i a r e more l i g h t l y c o l o r e d t h a n i n t h e y o u n g e r n u c l e a t e d c e l l s . The l a r g e i r r e g u l a r l y s h a p e d c o r n i f i e d c e l l s g e n e r a l l y c o n t a i n p y k n o t i c n u c l e i and r a r e l y t a k e any s t a i n ' , a l t h o u g h o c c a s i o n a l l y t h e c y t o p l a s m s t a i n s a p a l e b l u e . Mucus s t a i n s a p a l e p i n k c o l o r and l e u k o c y t e n u c l e i a deep b l u e t o b l a c k . A n i m a l S a c r i f i c e The v o l e t o be s a c r i f i c e d was p l a c e d o n i t s abdomen o n a f l a t s u r -f a c e . A b l u n t o b j e c t ( e . g . , s c a l p e l h a n d l e ) was h e l d a c r o s s t h e u p p e r c e r v i c a l v e r t e b r a e . W i t h r a p i d and f i r m p u l l o f t h e t a i l and s i m u l t a n e o u s downward and f o r w a r d movement o f t h e b l u n t o b j e c t , c e r v i c a l d i s l o c a t i o n was e f f e c t e d . P r e p a r a t i o n o f T i s s u e s f o r H i s t o l o g i c a l  E x a m i n a t i o n F i x a t i o n . A f t e r r e m o v a l f r o m a n i m a l , t i s s u e s were i m m e d i a t e l y p l a c e d i n B o u i n ' s s o l u t i o n and l e f t i n t h i s s o l u t i 6 n f o r 24 t o 72 h o u r s . They were t h e n p l a c e d i n 70% e t h y l a l c o h o l u n t i l embedded i n p a r a f f i n . E m b e d d i n g . T i s s u e s were p a s s e d t h r o u g h s o l u t i o n s as f o l l o w s : 95% e t h y l a l c o h o l ; 100% e t h y l a l c o h o l ; o n e - h a l f 100% e t h y l a l c o h o l and 19 o n e - h a l f X y l e n e m o r d a n t s o l u t i o n ; 100% X y l e n e ; P a r a p l a s t p a r a f f i n embedding medium (two c h a n g e s ) . P a r a f f i n embedded t i s s u e s were t h e n p l a c e d i n p l a s t i c d i s p o s a b l e m o l d s and c o o l e d f o r s e c t i o n i n g . E q u i p m e n t w h i c h f a c i l i t a t e d t h e embedding p r o c e s s w a s : an a u t o m a t i c E l l i o t T i s s u e P r o c e s s o r ; a vacuum embedder ; T i s s u e - t e k T h e r m o e l e c t r i c C e n t e r . T i s s u e s e c t i o n i n g and s t a i n i n g . A l l embedded t i s s u e s were s e c t i o n e d w i t h a S p e n c e r r o t a r y m i c r o t o m e a t 7 m i c r a o f t h i c k n e s s . T i s s u e r i b b o n s were p l a c e d on g l a s s s l i d e s and s t a i n e d w i t h a r o u t i n e h e m a t o x y l i n ( D e l a f i e l d ' s ) and e o s i n s t a i n . H i s t o l o g i c a l e x a m i n a t i o n . A W i l d b i n o c u l a r l a b o r a t o r y m o d e l com-p o u n d m i c r o s c o p e w i t h a b u i l t - i n l i g h t s o u r c e was u t i l i z e d f o r e x a m i n a t i o n o f t i s s u e s ( l O x e y e p i e c e o b j e c t i v e and 4 x / 1 0 x / 4 0 x and lOOx n o s e p i e c e o b j e c -t i v e s ) . Measu remen t s were made w i t h a 10mm o c u l a r m i c r o m e t e r . P h o t o m i c r o g r a p h y . A Z e i s s a u t o m a t i c " U l t r a p h o t 1 1 " m i c r o s c o p e w i t h camera a t t a c h m e n t was u t i l i z e d . B l a c k and w h i t e / p h o t o g r a p h s were t a k e n on 4 - i n c h b y 4 - i n c h p h o t o g r a p h i c p l a t e s t h r o u g h a g r e e n i n t e r f e r e n c e f i l t e r . S p e c i f i c H i s t o l o g i c a l P r e p a r a t i o n o f  R e p r o d u c t i v e Organs E x a m i n e d O v a r i e s , u t e r u s , f a l l o p i a n t u b e s . O v a r i e s were removed t o g e t h e r w i t h - t h e f a l l o p i a n t u b e and u p p e r p o r t i o n o f e a c h u t e r i n e h o r n . The c o n -n e c t i v e t i s s u e w h i c h h e l d t h e s t r u c t u r e s i n a n a t o m i c a l p o s i t i o n was n o t d i s t u r b e d . These t i s s u e s were o r i e n t e d when embedded so t h a t t h e o v a r i e s were f o r e m o s t when s e c t i o n e d . C o m p l e t e o v a r i a n s e r i a l s e c t i o n s , i n as much as t e c h n i c a l l y p o s s i b l e , were o b t a i n e d . S e c t i o n s were c o n t i n u e d i n 20 o r d e r t o i n c l u d e r e p r e s e n t a t i v e s e c t i o n s o f b o t h f a l l o p i a n t u b e and u t e r i n e h o r n . T h u s , i n e x a m i n a t i o n , a known p o r t i o n o f f a l l o p i a n t u b e and u t e r i n e h o r n c o u l d be compared i n e a c h c a s e . V a g i n a . The e n t i r e v a g i n a was removed and o r i e n t e d when embedded so t h a t r e p r e s e n t a t i v e s e c t i o n s c o u l d be c u t i n c r o s s s e c t i o n . HISTOLOGICAL MEASUREMENTS AND OBSERVATIONS The f o l l o w i n g c r i t e r i a were u t i l i z e d f o r o v a r i a n and r e p r o d u c t i v e t r a c t measurements and o b s e r v a t i o n s . O v a r i e s Maximum o v a r i a n d i a m e t e r . The l a r g e s t o v a r i a n s e c t i o n o n s l i d e s o f s e r i a l o v a r i a n s e c t i o n s was m e a s u r e d a t 40x m a g n i f i c a t i o n f o r e a c h o v a r y . D i a m e t e r s p e r p e n d i c u l a r t o e a c h o t h e r were m e a s u r e d and a v e r a g e d . The a v e r a g e d i a m e t e r f o r e a c h o v a r y was t h e n a v e r a g e d t o g i v e one v a l u e t o e a c h a n i m a l . F o l l i c l e s . F o l l i c l e s were c l a s s i f i e d a s d e v e l o p i n g , a n t r a l , o r a t r e t i c on t h e f o l l o w i n g b a s e s : D e v e l o p i n g E a r l y immature S i z e r a n g e f r o m 75-125 m i c r a i n d i a m e t e r , c o n s i s t s . . 6 f ;oocy te s u r r o u n d e d b y a w e l L -d e v e l o p e d s i n g l e l a y e r o f c u b o i d a l e p i t h e l i u m . L a t e immature S i z e r a n g e f r o m 1 2 5 - 2 5 0 m i c r a ; p r o g r e s s i v e l a y e r s o f c e l l s f o r m i n g g r a n u l o s a p r e s e n t ; s m a l l a r e a s o f p i n k s t a i n i n g l i q u o r f o l l i c u l i o c c u r i n s m a l l p o c k e t s on o p p o s i t e s i d e s o f 21 ovum at upper size limit Mature Size range 250 micra and up; several layers of granulosa cells present; increased amount of liquor f o l l i c u l i present; ovum not com-pletely surrounded by liquor f o l l i c u l i Mitotic figures present in granulosa of developing f o l l i c l e s in most cases. Antral Antral f o l l i c l e s were considered to be those which con-tained an ovum surrounded by a corona radiata. The ovum was completely surrounded by liquor f o l l i c u l i except at the area of the discus proligerus. The presence of a stalk was not considered a necessary criterion to place the f o l l i c l e in the category of antral f o l l i c l e s . Atretic Decreased amount of cytoplasm in granulosa cells resulting in apparent disorganization of granulosa c e l l nuclei and basal layer of granulosa appears mainly as deeply staining nuclei; granulosa basement c e l l membrane not clearly delineated and in some cases ' apparent ingrowth of theca interna i s evident; liquor f o l l i c u l i , i f present, appears flocculent rather than fine and granular as i s evident in healthy f o l l i c l e s . F o l l i c l e diameters were measured from granulosa basement membrane to granulosa basement membrane on the opposite side of the f o l l i c l e . The average measurement of two perpendicular diameters was calculated (the theca externa was not used as a basis of measurement due to the fact i t 22 o f t e n a p p e a r e d t o merge w i t h t h e s t r o m a l i n t e r s t i t i a l t i s s u e , m a k i n g measurement o f i t s b o r d e r d i f f i c u l t ) . A l l f o l l i c l e s were measu red a t lOOx m a g n i f i c a t i o n . The l a r g e s t f o l l i c l e d i a m e t e r measu red i n e i t h e r o v a r y o f e a c h a n i -m a l , r e g a r d l e s s o f t h e c a t e g o r y o f f o l l i c l e , was r e c o r d e d ; a l l d e v e l o p i n g f o l l i c l e s 200 m i c r a o r l a r g e r were m e a s u r e d ; a l l a n t r a l f o l l i c l e s were m e a s u r e d ; a l l a t r e t i c f o l l i c l e s w i t h a d i a m e t e r 250 m i c r a o r l a r g e r were m e a s u r e d . C o r p o r a l u t e a . P e r p e n d i c u l a r d i a m e t e r s o f c o r p o r a l u t e a were measured f r o m o u t e r a s p e c t o f t h e c a e x t e r n a l a y e r . These were a v e r a g e d t o g i v e one v a l u e p e r c o r p u s l u t e m . The t o t a l number o f c o r p o r a l u t e a p r e s e n t i n b o t h o v a r i e s o f a g i v e n a n i m a l was r e c o r d e d . I n t e r s t i t i a l t i s s u e . The c e l l u l a r m a t e r i a l f o r m i n g t h e s t r o m a o f t h e o v a r y was n o t m e a s u r e d . H o w e v e r , d e s c r i p t i v e c o m p a r i s o n o f r e l a t i v e amounts o f s t r o m a l c e l l s i n a n i m a l s s t u d i e d was r e c o r d e d . U t e r u s Two p e r p e n d i c u l a r measurements o f t h e d i a m e t e r o f t h e u t e r i n e h o r n f r o m s e r o s a t o s e r o s a were a v e r a g e d whenever p o s s i b l e . M a g n i f i c a -t i o n was 4 0 x . T h i s measurement was i m p o s s i b l e i n t h e c a s e s o f s e v e r a l a n i m a l s due t o t h e f a c t t h a t t h e h o r n had been s e c t i o n e d o b l i q u e l y r a t h e r t h a n c r o s s - s e c t i o n a l l y . Measu remen t s were made o f t h e l i n i n g e p i t h e l i a l c e l l s a t a m a g n i -f i c a t i o n o f lOOOx. D e s c r i p t i o n o f t h e a p p e a r a n c e o f t h e u t e r i n e h o r n was made f r o m m a g n i f i c a t i o n s o f 40x and lOOOx. 23 F a l l o p i a n Tube Measuremen t o f t h e h e i g h t o f t h e l i n i n g e p i t h e l i a l c e l l s o f t h e f a l l o p i a n t u b e were made f r o m t h e same a r e a o f e a c h t u b e , a s j u d g e d b y a p p e a r a n c e . M a g n i f i c a t i o n o f lOOOx was u s e d . E s t i m a t e s o f t h e amount o f d i l a t a t i o n o f t h e t u b e i n v a r i o u s a n i m a l s r e p o r t e d were made o n t h e b a s i s o f a p p e a r a n c e . V a g i n a E p i t h e l i a l c e l l l a y e r s were c o u n t e d . H o w e v e r , due t o t h e v a r i a -t i o n o f o r i e n t a t i o n d u r i n g embedding and t h e r e f o r e s e c t i o n i n g , t h i s s t a t i s -t i c was n o t c o n s i d e r e d r e l i a b l e and t h e r e f o r e n o t u t i l i z e d . MALES U T I L I Z E D A p p r o x i m a t e l y 15 s e x u a l l y m a t u r e a d u l t m a l e s were h o u s e d i n t h e l a b o r a t o r y t h r o u g h o u t t h i s s t u d y . The m a j o r i t y o f t h e s e a n i m a l s were f i e l d a n i m a l s . New f i e l d m a l e s were p e r i o d i c a l l y added as r e p l a c e m e n t s when d e a t h s o c c u r r e d o r l a c k o f r e p r o d u c t i v e p e r f o r m a n c e was o b s e r v e d . F o l l o w i n g i s a d e s c r i p t i o n o f t h e c r i t e r i a u t i l i z e d t o d e t e r m i n e a d u l t m a l e s e x u a l m a t u r i t y . I n J u l y 1 9 6 6 , 25 f i e l d m a l e s w i t h d e s c e n d e d t e s t e s were s a c r i f i c e d . Smears f r o m t h e c a u d a l e p i d i d y m i s were t a k e n , a n d , o n t h e b a s i s o f t h e p r e s e n c e o r a b s e n c e o f m a t u r e s p e r m a t o z o a , s e x u a l m a t u r i t y was d e t e r m i n e d . Smear t e c h n i q u e u t i l i z e d was a h e m o t o x y l i n and e o s i n s t a i n as s u g g e s t e d by C h r i s t i a n (.1950) . The r e s u l t s o b t a i n e d a r e shown i n T a b l e 1. S P E C I F I C STUDY METHODS O n s e t o f P u b e s c e n c e The r e l a t i o n s h i p o f v a g i n a l p e r f o r a t i o n t o a n i m a l a g e , w e i g h t and TABLE 1 SPERM MATURATION STATUS BASED ON CAUDAL EPIDIDYMIS SMEARS OF 26 FIELD MALES IN JULY 1966 Number of Animals Animal Weight Number of in Which Mature in grams Animals Sperm Observed 0-29.9 4 None 30-39.9 7 5 a 40-49.9 7 6 b 50-59.9 4 4 60-69.9 4 4 Large, apparently immature sperm observed in smears of 2 animals. One no sperm. 25 f i r s t estrus were studied i n the following manner. Twenty laboratory born animals were weaned and i s o l a t e d i n separate cages. Average weaning weight f o r the 20 females was 15.1 grams; range: 11.4 to 20.0 grams. Animals were checked d a i l y f o r the presence of a perforate vagina. Females were weighed on the day vaginal p e r f o r a t i o n was observed. A vaginal smear was performed on a l l animals at t h i s time to ascertain estrous cycle stage. Oh 12 females, v a g i n a l smears were performed d a i l y from the day of vaginal p e r f o r a t i o n u n t i l an estrous smear was observed. Nature of the Estrous Cycle Vaginal smears were performed d a i l y on 12 i s o l a t e d adult l i t t e r females. T h i r t y i s o l a t e d females were k i l l e d i n various smear stages, and t h e i r ovaries and reproductive t r a c t s h i s t o l o g i c a l l y examined. Analysis of variance was performed on h i s t o l o g i c a l measurements of anestrus (n=10), estrus (n=9), and diestrus (n=9) females. Administration of P.M.S. to Anestrous  Females Pregnant Mare's Serum contains a gonadotropin s i m i l a r to FSH which w i l l produce ovarian f o l l i c u l a r maturation i n the immature r a t within 48 to 72 hours (Rowlands, 1944). Pregnant Mare's Serum does not provoke ovulation i n the majority of instances (Rowlands, 1944; Cole, 1930). The response of adult anestrous females to t h i s drug was tested by intramuscular i n j e c t i o n followed by s a c r i f i c e at 51 to 53 hours post administration. Nine imperforate anestrous adult females were given an i n j e c t i o n of eith e r 25 I.U. or 50 I.U. of "Wellcome" brand Pregnant Mare's Serum Gonadotrophin. A l l animals u t i l i z e d were anestrous animals which had been perforate a t some time during c a p t i v i t y and imperforate f o r at le a s t three weeks preceding P.M.S. administration. 26 O v a r i e s a n d r e p r o d u c t i v e t r a c t s were s e r i a l l y s e c t i o n e d f o l l o w i n g s a c r i f i c e and o b s e r v a t i o n s and measurements e f f e c t e d s i m i l a r t o t h o s e d e s c r i b e d i n t h e p r e v i o u s s u b s e c t i o n e n t i t l e d " H i s t o l o g i c a l Measu remen t s and O b s e r v a t i o n s . " P r e g n a n c y and E v e n t s F o l l o w i n g  P r e g n a n c y T h r e e d a t e d p r e g n a n c i e s w e r e o b t a i n e d b y t h e f o l l o w i n g m e t h o d : f e m a l e s were p a i r e d u n t i l sperm were o b s e r v e d i n a v a g i n a l smear ; ma l e r e m o v a l was e f f e c t e d u n t i l a l i t t e r was o b s e r v e d . L i t t e r d a t a a r e c o m p i l e d f r o m : 13 l a b o r a t o r y b o r n l i t t e r s o f l i t t e r f e m a l e s ; 20 f i e l d f e m a l e embryo c o u n t s ; 12 f i e l d f e m a l e w h i c h had l i t t e r s i n t h e l a b o r a t o r y . Chapter 3 RESULTS OBSERVATIONS OF THE ONSET OF PUBESCENCE IN FEMALE MICROTUS TOWNSENDII TOWNSENDII L i t t e r Females L i t t e r s were weaned at 15 t o 17 days of age. Although suckled to the time they were weaned, l i t t e r animals were f r e q u e n t l y observed t o n i b -b l e a t food p e l l e t s and take water from d r i p b o t t l e s s e v e r a l days p r i o r to t h i s . Females of t h i s species are e a s i l y sexed a t weaning. The membrane which covers the v a g i n a l o r i f i c e appears as a p a l e blue crease p o s t e r i o r t o the u r i n a r y p a p i l l a . Average weaning weight f o r 20 l a b o r a t o r y females was 1 5 . 1 grams; range: 1 1 . 4 to 2 0 . 0 grams. Laboratory growth curve f o r these animals may be observed i n Graph 1. The age range of 20 l a b o r a t o r y females on the day of v a g i n a l per-f o r a t i o n was from 19 days to 82 days; the highest frequency of p e r f o r a t i o n occurred between days 30 and 45 (Graph.2 ) . The weights of these females at t h i s time ranged from 1 9 . 0 grams t o 5 3 . 8 grams; the g r e a t e s t number of animals became p e r f o r a t e between 30 and 3 9 . 9 grams of weight (Graph 3 ) . V a g i n a l smears, taken on the day of v a g i n a l p e r f o r a t i o n f o r each of the 20 l a b o r a t o r y females, demonstrated the smear stage of d i e s t r u s . Three of these animals became imperforate a f t e r i n i t i a l p e r f o r a t i o n f o r periods of time l i s t e d : animal 294 was p e r f o r a t e f o r 2 days f o l l o w e d by 17 days of i m p e r f o r a t i o n ; animal 295 was p e r f o r a t e f o r 5 days f o l l o w e d by 5 days of i m p e r f o r a t i o n ; animal 244 was p e r f o r a t e f o r 2 days f o l l o w e d by 14 days of i m p e r f o r a t i o n . A l l animals remained p e r f o r a t e a f t e r 27 28 55-t-50J-4 5 1 15 25 35 45 55 Days of Age Graph 1 Growth Curve of 20 Female Microtus townsendii from Weaning to Day 55 of Age Note. At least 10 animals weighed for each plotted point on graph, a Pooled data of 20 animals. 29 4 Age of Animals (days) Graph 2 Frequency of Occurrence of Vaginal Perforation Related to Animal Age 12 + Weight of Animal (grams) Graph 3 Frequency of Occurrence of Vaginal Perforation Related to Animal Weight 30 " r e - p e r f o r a t i o n . " The age o f 11 f e m a l e s a t t h e t i m e o f f i r s t v a g i n a l e s t r u s and t h e t i m e i n t e r v a l be tween i n i t i a l v a g i n a l p e r f o r a t i o n and f i r s t e s t r u s i s p r e -s e n t e d i n T a b l e 2 . A s may be o b s e r v e d , a l l a n i m a l s d e m o n s t r a t e d f i r s t e s t r u s w i t h i n t h e f i r s t 11 weeks o f a g e . W i t h i n t h i s g r o u p o f a n i m a l s , t h e o c c u r r e n c e o f f i r s t e s t r u s i s d i s t r i b u t e d t h r o u g h o u t t h e p e r i o d f r o m 35 days t o 80 d a y s o f a g e . D a t a o f t h e t i m e i n t e r v a l be tween v a g i n a l p e r -f o r a t i o n and f i r s t e s t r u s d e m o n s t r a t e t h e s e two e v e n t s a r e n o t s i m u l t a n e o u s i n any i n s t a n c e ; t h e r a n g e was f r o m 1 day t o 36 d a y s w i t h o n l y 2 o f t h e a n i m a l s , o r 18%, d e m o n s t r a t i n g l e s s t h a n a 6 -day i n t e r v a l . The w i d e v a r i -a t i o n o f b o t h age o f f i r s t e s t r u s and i n t e r v a l o f days be tween v a g i n a l p e r -f o r a t i o n and f i r s t e s t r u s d e m o n s t r a t e s t h e r e i s no p r e d i c t a b l e r e l a t i o n s h i p i n M i c r o t u s t o w n s e n d i i w i t h r e f e r e n c e t o t h e s e two v a r i a b l e s i n t h e a n i m a l s s t u d i e d . Of t h e 30 l a b o r a t o r y f e m a l e s d i s c u s s e d , o n l y one o f 19 w h i c h r e m a i n e d i n t h e c o l o n y (#445 was d e s t r o y e d a t t h e t i m e o f v a g i n a l p e r f o r a -t i o n ) f a i l e d t o d e m o n s t r a t e a t l e a s t one e s t r u s smear a t some t i m e a f t e r v a g i n a l p e r f o r a t i o n . T h i s a n i m a l , #260, d i d n o t d e m o n s t r a t e an e s t r u s smear i n 74 c o n s e c u t i v e d a y s a f t e r p e r f o r a t i o n o r o n smears t a k e n s p o r a d i -c a l l y a f t e r t h i s t i m e . H o w e v e r , t h e a n i m a l d i d r e m a i n p e r f o r a t e and smear s t a g e was a l w a y s c l a s s i f i e d a s d i e s t r u s . F i e l d F e m a l e s F i f t y - t h r e e f i e l d f e m a l e s were e x a m i n e d a t c a p t u r e f o r t h e p r e s e n c e o f v a g i n a l p e r f o r a t i o n . The d a t a p r e s e n t e d i n T a b l e 3 d e p i c t t h e r e s u l t s o b s e r v e d . F rom t h e d a t a i t i s a p p a r e n t t h e r a n g e i n w h i c h t h e g r e a t e s t d i f f e r e n c e o c c u r r e d be tween p e r f o r a t i o n and i m p e r f o r a t i o n was i n t h e 31 TABLE 2 VAGINAL PERFORATION AGE AND WEIGHT AND AGE OF FIRST ESTRUS IN LABORATORY BORN FEMALES Age Day Weight Day Animal Perforation Perforation Number Observed Observed Age Day F i r s t Estrus Observed Time Interval i n Days between Vaginal Perforation and F i r s t Estrus 209 210 240 242 244 259 268 295 425 456 457 260 261 269 294 296 426 453 454 455 43 38 47 38 18 59 43 20 34 36 48 40 82 54 19 70 66 32 45 25 45.0 gm 42.1 gm 39.5 gm 35.7 gm 19.0 gm 39.2 gm 39.0 gm 21.0•gm 36.2 gm 32.5 gm 37.7 gm 37.0 gm 53.8 gm 31.5 gm 20.0 gm 39.0 gm 41.6 gm 31.5 gm 40.0 gm 22.5 gm 49 61 80 72 45 76 79 49 35 38 65 Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown 6 23 33 34 27 17 36 29 1 2 17 Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown 32 r a n g e o f 15?.to 2 4 . 9 g r a m s . Of t h e 14 a n i m a l s e x a m i n e d , 64% were p e r f o r a t e and 36% i m p e r f o r a t e . The g r o w t h c u r v e o f l a b o r a t o r y f e m a l e s (Graph 1) i n d i c a t e s a c o m p a r a b l e age r a n g e o f f r o m 15 t o 20 d ay s o f age f o r l a b o r a -t o r y f e m a l e s o f t h i s w e i g h t . I n t h i s r a n g e one w o u l d e x p e c t i n i t i a l v a g i n a l p e r f o r a t i o n t o o c c u r w i t h t h e g r e a t e s t f r e q u e n c y . N i n e t y - f i v e p e r c e n t o f t h e 17 f e m a l e s i n t h e w e i g h t r a n g e o f 25 t o 3 4 . 9 grams were p e r f o r a t e a s were e i g h t y p e r c e n t o f t h e 20 a n i m a l s i n t h e w e i g h t r a n g e o f 34 t o 4 4 . 9 g r a m s . T a b l e 3 O c c u r r e n c e o f V a g i n a l P e r f o r a t i o n i n 53 C a p t u r e d F i e l d F e m a l e s D i s t r i b u t e d i n t o 10-Gram W e i g h t I n c r e m e n t s f rom 5 grams t o 4 4 . 9 grams Weight Range Total Number of Animals Number Perforate Number Imperforate 5 - 1 4 . 9 grams 2 0 2 1 5 - 2 4 . 9 grams 14 9 5 2 5 - 3 4 . 9 grams 17 16 1 3 5 - 4 4 . 9 grams 20_ 16 4 Totals 53 41 12 V a g i n a l smears were p e r f o r m e d on 7 f i e l d f e m a l e s i n t h e w e i g h t r a n g e o f 15 t o 2 4 . 9 g r a m s . These a n i m a l s were t h e n s a c r i f i c e d and t h e i r o v a r i e s and r e p r o d u c t i v e t r a c t s h i s t o l o g i c a l l y e x a m i n e d i n o r d e r t o d e t e r m i n e w h e t h e r o v u l a t i o n , f e r t i l i z a t i o n o r i m p l a n t a t i o n h a d o c c u r r e d i n any o f t h e s e a n i m a l s . T a b l e 4 p r e s e n t s t h e r e s u l t s o f t h i s e n d e a v o r . I t i s e v i -d e n t t h a t 6 o u t o f 7 o f t h e s e a n i m a l s e x h i b i t e d a n e s t r o u s v a g i n a l smear a t a w e i g h t w h i c h s h o u l d r e f l e c t c l o s e p r o x i m i t y t o t h e e x p e c t e d t i m e o f TABLE 4 OVARIAN HISTOLOGY OF SEVEN PERFORATE FIELD FEMALES OF 16 TO 24.9 GRAMS IN WEIGHT DEMONSTRATING ESTROUS CYCLE SMEAR STAGE OF ESTRUS OR PROESTRUS Animal Number Capture Weight i n gm Vaginal Smear Stage Maximum Ovarian Diameter i n mm Largest Ovarian F o l l i c l e i n micra Number of Ovarian F o l l i c l e s Number of Corpora Lutea a Dev. Antral A t r e t i c 611 16.0 Proest. 0.63 142 0 0 0 0 610 18.5 Estrus 1.15 338 3 9 4 0 609 19.0 Estrus 1.09 360 10 4 8 0 614~ 20.0 Estrus 1.28 360 19 4 12 0 616 b 20.0 Estrus 0.97 315 0 0 1 0 617 b 20.0 Estrus 1.07 330 2 4 3 0 615 21.0 Estrus (SPERM) 1.81 675 26 1 2 5 Developing. One ovary destroyed a c c i d e n t a l l y while processing t i s s u e s . LO 34 i n i t i a l v a g i n a l p e r f o r a t i o n . O v u l a t i o n and f e r t i l i z a t i o n were o b s e r v e d t o have o c c u r r e d i n o n l y one a n i m a l : #615. T h i s a n i m a l was n o t a v i r g i n a s sperm were o b s e r v e d i n t h e i n i t i a l e s t r o u s v a g i n a l smear . H i s t o l o g i -c a l e x a m i n a t i o n o f t h e f a l l o p i a n t u b e s d e m o n s t r a t e d t h e p r e s e n c e o f f i v e t w o - c e l l e d t u b a l o v a . F i v e c o r p o r a l u t e a w h i c h were c e n t r a l l y h o l l o w and h e m o r r h a g i c were o b s e r v e d i n t h e o v a r i e s . A l s o o b s e r v e d were an i n c r e a s e d number o f d e v e l o p i n g f o l l i c l e s , l a r g e r maximum f o l l i c l e s i z e , and l a r g e r maximum o v a r i a n d i a m e t e r t h a n o b s e r v e d i n any o f t h e o t h e r a n i m a l s . A l t h o u g h o n l y one o v a r y was e x a m i n e d f o r a n i m a l s 617 and 6 1 6 , t h i s i s c o n -s i d e r e d a d e q u a t e e v i d e n c e o f no o v u l a t i o n , a s t h e o v a r i e s o f a l l p r e g n a n t a n i m a l s e x a m i n e d d e m o n s t r a t e d a d i s t r i b u t i o n o f c o r p o r a l u t e a be tween t h e two o v a r i e s . C o r p o r a a l b i c a n t i a were n o t o b s e r v e d i n any o f t h e a n i m a l s . NATURE OF THE ESTROUS CYCLE I N MICROTUS TOWNSENDII TOWNSENDII E s t r o u s C y c l e as D e t e r m i n e d b y  V a g i n a l Smears D a i l y v a g i n a l smears were p e r f o r m e d o n 12 l a b o r a t o r y b o r n , i s o l a t e d , v i r g i n f e m a l e s f o r c o n s e c u t i v e d a y s a f t e r an e s t r o u s smear was d e m o n s t r a t e d . D a t a o b t a i n e d f r o m 10 o f t h e a n i m a l s were r e c o r d e d f r o m t h e day o f i n i t i a l e s t r o u s smear ; d a t a f r o m two o f t h e a n i m a l s were r e c o r d e d f r o m unknown t i m e w i t h r e f e r e n c e t o t h e f i r s t e s t r o u s smear ( a n i m a l s #200 and #454) . D a t a o b t a i n e d may be f o u n d i n T a b l e 5 . Smear s t a g e s s i m i l a r t o t h o s e r e p o r t e d by A l l e n (1922) i n Mus  m u s c u l u s were o b s e r v e d i n M i c r o t u s t o w n s e n d i i . S t a g e s o t h e r t h a n t h e s t a g e o f e s t r u s were n o t a s c l e a r l y d e f i n e d as i n M u s . The m a i n c e l l t y p e o b s e r v e d i n e s t r u s was t h e c o r n i f i e d e p i t h e l i a l c e l l . O t h e r v a g i n a l smear components were g e n e r a l l y n o n - e x i s t e n t o r m i n i m a l . To d e t e r m i n e w h e t h e r 35 o r n o t c y c l i c phenomena were o c c u r r i n g , I d e f i n e d " a c y c l e " as t h e p e r i o d f r o m t h e f i r s t d a y o f a n e s t r o u s smear t o t h e f i r s t d a y o f a s u b s e q u e n t e s t r o u s smear ( T a b l e 5 ) . E s t r u s was t h e mos t common s t a g e o b s e r v e d i n l a b o r a t o r y f e m a l e s . E s t r o u s p e r i o d s measured r a n g e d f r o m one day i n d u r a -t i o n t o 31 d a y s d u r a t i o n . T a b l e 6 i l l u s t r a t e s p o o l e d d a t a o f o b s e r v a t i o n s o f 466 d a y s o f c o n s e c u t i v e v a g i n a l smears o f 12 l a b o r a t o r y f e m a l e s . O f t h e s e s m e a r s , 56.65% d e m o n s t r a t e d t h e smear s t a g e o f e s t r u s . The n e x t mos t f r e q u e n t smear s t a g e o b s e r v e d , a s i l l u s t r a t e d by T a b l e 6 , was d i e s -t r u s w h i c h o c c u r r e d i n 34.98% o f t h e 466 smears e x a m i n e d . Two t y p e o f d i e s t r o u s smears were commonly o b s e r v e d . The " l i g h t " d i e s t r o u s smear was p r i m a r i l y a c e l l u l a r w i t h few l e u k o c y t e s , few e p i t h e l i a l components and o n l y t r a c e s o f mucus and c e l l d e b r i s p r e s e n t . The more u s u a l " h e a v y " d i e s t r o u s smear had as i t s m a i n component mucus w h i c h g e n e r a l l y c o n t a i n e d l e u k o c y t e s t r a p p e d w i t h i n i t g i v i n g t h e a p p e a r a n c e o f s w i r l s t o t h e smea r . E p i t h e l i a l componen t s , when p r e s e n t , were p r i m a r i l y n u c l e a t e d e p i t h e l i a l c e l l s . O b s e r v a t i o n o f t h e s t a g e s o f p r o e s t r u s and m e t e s t r u s was i n f r e -q u e n t . As may be o b s e r v e d i n T a b l e 6 , o f 466 smear o b s e r v a t i o n s , p r o e s t r u s was o b s e r v e d on 3.64% o f t h e smears and m e t e s t r u s o b s e r v e d o n 4.73% o f t h e smears e x a m i n e d . A n y , o r a l l o f t h e p o s s i b l e v a g i n a l smear components were g e n e r a l l y p r e s e n t i n p r o e s t r o u s and m e t e s t r o u s s m e a r s . The d i s t i n -g u i s h i n g c h a r a c t e r i s t i c w h i c h p r o m p t e d a d i a g n o s i s o f p r o e s t r u s was l a r g e number s , o c c a s i o n a l l y s h e e t s , o f s m a l l n u c l e a t e d e p i t h e l i a l c e l l s . The d i s t i n g u i s h i n g c h a r a c t e r i s t i c o f m e t e s t r u s was m a s s i v e l e u k o c y t o s i s i n a smear w h i c h c o n t a i n e d l a r g e numbers o f c o r n i f i e d e p i t h e l i a l c e l l s . Any q u e s t i o n a b l e smear was c o n s i d e r e d t o be d i e s t r u s . A p p e a r a n c e o f t h e two smear s t a g e s d e m o n s t r a t e d t h e m a j o r i t y o f t h e t i m e s s t u d i e d , e s t r u s and d i e s t r u s , may be o b s e r v e d i n P l a t e s 1, 2 , and 3 . TABLE 5 ESTROUS CYCLE STAGES AS DETERMINED BY DAILY VAGINAL SMEARS OF SEXUALLY MATURE ISOLATED LABORATORY' HOUSED MICROTUS TOWNSENDII Smear Days Animal' Number 12345678910 20 123456789 12345678930 40 123456789 12345678950 12345678960 . 123456789?0 80 123456789 210 DPEEEEDEEE EDDDDEEEEE EEEEEEE 209 DPEEEEDDDD DDDDDPEEEE EEEDDDDPEE EEEEEEEE 242 DPEEEEEEEE EEEEEEEEEM MDDDDDPDDD EEEDEEEEEE EEEEEEEEEE EEDPE 244 DMEDDDEEEE MDDDDDDEDD EEEMEMMEMM MEEEEEEEEE EEEEEEEEEE MEEEEEEEEE EEEDEEEEEE EEEEEEEEE 259 DEDEDDPDDE EMPDDDDDDD EEDEDDDDDD EEDEDDD 268 DEDDDDDDDD DDDDDDDDDD EEEEEEEEEE EEEEE 295 DEDDDDEEEM EEEEEEEEEE EEEEEEEEED DEEEEEEEEE EEEEE 425 DEEEDEDEDD EDEDEEDDED DDDDEEMDDD DDDDDDDDDD 456 DEEEEEEEED DDDDDDDD -Not Done- ; :EE EEEEEE —40 days l a t e r — EEEEEEEE EEEEEEEEEE EEEEEEE 457 MPEEEEEEEE EEEEMDEDDD EEED 200 MPEEEMDDPE EEEDDDPEMD DDDDDDPEED DDEEEEEEED DD 454 DDDDDEEEEM EEEPDDMEEE EEDDDPPM Note. E = Estrus; D = Diestrus; P = Proestrus; M = Metestrus, cn TABLE 6 PER CENT OF ESTROUS CYCLE STAGES REPRESENTED IN 466 DAYS OF CONSECUTIVE VAGINAL SMEARS ON TWELVE LABORATORY FEMALES Total Days of Consecutive Smears Per Cent of T o t a l Days Smear Stage of: Animal Number Estrus Diestrus Metestrus Proestrus 210 27 74 22 — 4 209 38 55 37 — 8 242 55 71 20 4 5 244 79 71 16 13 — 259 37 27 65 3 5 268 35 46 54 — — 295 45 82 16 2 425 40 30 68 2 — 456 18 44 56 — — 457 24 67 21 8 4 200 41 41 44 5 10 454 28 43 35 11 11 Averages 466 56.65 34.98 3.64 4.73 38 • » P l a t e 1 V a g i n a l Smear S t a g e o f E s t r u s (x400) 2 f t - ~ J -' •! " I — * U ! .* -« i» if VT . ' <• - . .A P l a t e 2 P l a t e 3 "Heavy" V a g i n a l Smear S t a g e o f D i e s t r u s (x400) " L i g h t " V a g i n a l Smear S t a g e o f D i e s t r u s (x400) 39 S e q u e n t i a l l y , t h e s t a g e o f p r o e s t r u s w o u l d be e x p e c t e d t o o c c u r p r i o r t o e s t r u s . E s t r u s w o u l d be f o l l o w e d by m e t e s t r u s . I n o r d e r t o s t u d y smear s e q u e n c e , T a b l e 7 was c o n s t r u c t e d f r o m T a b l e 5 . E x a m i n a t i o n o f t h i s t a b l e i l l u s t r a t e s t h a t t h e smear s t a g e o b s e r v e d p r e c e d i n g and f o l l o w i n g e s t r u s w a s , i n o v e r 60% o f t h e c a s e s , r e c o r d e d as d i e s t r u s ; 64% on t h e day p r e c e d i n g and 68% o n t h e day f o l l o w i n g e s t r u s . A p r o e s t r o u s smear was o b s e r v e d on t h e day p r e c e d i n g e s t r u s i n o n l y 21% o f t h e c y c l e s , and m e t e s t r u s o b s e r v e d i n 30% o f t h e c y c l e s f o l l o w i n g e s t r u s . The a p p e a r a n c e o f a m e t e s t r u s smear p r i o r t o e s t r u s on e i g h t o c c a s i o n s i s w o r t h y o f comment; a g a i n e m p h a s i z i n g t h e n o n s e q u e n t i a l n a t u r e o f t h e s e s t a g e s i n M . t o w n s e n d i i . F rom t h e m a t e r i a l t h u s f a r , i t i s e v i d e n t t h a t t h e e s t r o u s c y c l e o f M i c r o t u s t o w n s e n d i i i s n o t t y p i c a l o f t h a t o f M u s . I n e x t a s k e d t h e q u e s t i o n o f w h e t h e r o r n o t t h e r e w a s , i n f a c t , a c y c l e . T h e r e f o r e G r a p h 4 was c o n s t r u c t e d f r o m T a b l e 8. G r a p h 4 i l l u s t r a t e s t h a t t h e m a j o r i t y o f a n i m a l s d i d i n f a c t d e m o n s t r a t e c y c l i c phenomena r e p e a t e d a p p r o x i m a t e l y e v e r y 2 t o 7 . 5 d a y s . The c r i t e r i o n f o r " a c y c l e " was o b s e r v a t i o n o f f i r s t day o f an e s t r o u s smear t o f i r s t day o f a s u b s e q u e n t e s t r o u s smear r e g a r d l e s s o f t h e smear s t a g e s r e c o r d e d i n be tween t h e s e two o b s e r v a t i o n s . H i s t o l o g i c a l E x a m i n a t i o n of . O v a r i e s and  R e p r o d u c t i v e T r a c t s i n E a c h o f t h e  Commonly O b s e r v e d V a g i n a l Smear  S t a g e s T h i r t y a n i m a l s were i s o l a t e d i n s e p a r a t e cages and o b s e r v e d f o r a t l e a s t t h r e e weeks p r i o r t o s a c r i f i c e . B o t h l a b o r a t o r y b o r n and f i e l d c a p t u r e d f e m a l e s were u t i l i z e d , i n t h i s p o r t i o n o f t h e s t u d y . A l l a n i m a l s were o b s e r v e d t o be p e r f o r a t e a t some t i m e p r i o r t o s a c r i f i c e . A n i m a l s were s a c r i f i c e d i m m e d i a t e l y a f t e r d e s i r e d v a g i n a l s t a g e was i d e n t i f i e d . 40 TABLE 7 SMEAR STAGES OBSERVED ON DAYS PRECEDING AND FOLLOWING ESTRUS IN TWELVE LABORATORY FEMALES Day Preceding Estrus Day Following Estrus Number Proestrus Metestrus Diestrus Proestrus Metestrus Diestrus 210 1 0 2 0 0 2 209 3 0 0 0 0 2 242 2 0 2 0 1 2 244 0 5 4 0 6 3 259 0 0 7 0 1 6 268 0 0 2 0 0 1 295 0 1 3 0 1 2 425 0 0 8 0 1 7 456 0 0 1 0 0 1 457 1 0 2 0 1 2 200 4 0 1 0 2 3 454 _ 0 2 _JL 1 1 _1 Subtotals 11 8 33 1 14 32 Totals 52 477 Per Cent of Total 21 15 64 2 30 68 C y c l e L e n g t h (days) G r a p h 4 E s t r o u s C y c l e D u r a t i o n i n Days o f 40 C y c l e s O b s e r v e d i n 12 L a b o r a t o r y F e m a l e s 42 TABLE 8 FREQUENCY AND PER CENT OF OCCURRENCE OF ESTROUS CYCLE DURATION IN 40 CYCLES OBSERVED IN TWELVE LABORATORY FEMALES Length of Cycle i n Days Number of Per Cycle Occurrences Totals Number of Occurrences as a Per Cent . of Total Number of Cycles Observed 2 4 10 3 5 12.5 4 8 20.0 5 4 21 10.0 6 2 5 7 3 7.5 8 2 5 9 0 0 10 1 8 2.5 11 2 5.0 12 1 2.5 13 1 ,2.5 14 2 5.0 15 0 6 0 16 0 0 17 0 0 18 0 0 19 2 5 20 1 3 2.5 21 1 2.5 22 0 0 23 0 0 24 0 0 25 0 • 1 0 26 0 0 27 0 0 28 1 1 2.5 43 A s p r o e s t r u s and m e t e s t r u s were r a r e l y o b s e r v e d , t h e y were n o t c o n s i d e r e d as t y p i c a l s t a g e s . Two m e t e s t r o u s a n i m a l s were i n c l u d e d i n h i s t o l o g i c a l e x a m i n a t i o n s i n o r d e r t o o b s e r v e w h e t h e r o r n o t t h e a p p e a r -ance o f a m e t e s t r o u s smear on t h e day f o l l o w i n g an e s t r u s smear d o e s , a s i n M u s , i n d i c a t e t h e o c c u r r e n c e o f o v u l a t i o n . S t a g e s R e p r e s e n t e d Number o f A n i m a l s A n e s t r u s 10 D i e s t r u s 9 E s t r u s 9 M e t e s t r u s 2 P r e s e n t a t i o n o f h i s t o l o g i c a l r e s u l t s o b t a i n e d f rom o b s e r v a t i o n s o f t h e o v a r i e s and r e p r o d u c t i v e t r a c t s o f t h e s e a n i m a l s w i l l be made o n a c o m p a r a t i v e b a s i s by o r g a n s . O v a r i e s . The o v a r i e s were o f p r i m a r y s i g n i f i c a n c e t o t h i s s t u d y and t h u s w i l l be d i s c u s s e d f i r s t . A t a b u l a t i o n o f h i s t o l o g i c a l m e a s u r e -ments o f o v a r i e s o f j f e m a l e s s a c r i f i c e d i n e s t r o u s c y c l e s t a g e s i s p r e s e n t e d i n T a b l e 9 . A l t h o u g h s i g n i f i c a n t f o l l i c l e s i z e i n d i c a t i v e o f o v a r i a n a c t i v i t y was c o n s i d e r e d t o be l a r g e r t h a n 250 m i c r a , i t may be o b s e r v e d f r o m t h e c o l u m n o f l a r g e s t o v a r i a n f o l l i c l e s t h a t 350 m i c r a and l a r g e r r e p r e s e n t s t h e minimum a p p a r e n t p r e o v u l a t o r y f o l l i c l e s i z e ( e x c e p t f o r a n i m a l 89 i n w h i c h t h e l a r g e s t f o l l i c l e s i z e was 285 m i c r a ) . An a n a l y s i s o f v a r i a n c e was p e r f o r m e d on s e l e c t e d means p r e s e n t e d i n T a b l e 10 i n o r d e r t o d e t e r m i n e t h e p r e s e n c e o f r e a l d i f f e r e n c e among t h e means o f t h e t h r e e g r o u p s s t u d i e d w i t h r e f e r e n c e ' t o t h e v a r i a b l e s p r e s e n t e d . M e t e s t r o u s a n i m a l s were n o t i n c l u d e d i n t h i s a n a l y s i s as t h e i r number was l i m i t e d t o two a n i m a l s . The s t a t i s t i c a l summary o f t h e TABLE 9. TABULATION OF OVARIAN HISTOLOGICAL' MEASUREMENTS OF OVARIES OF FEMALES SACRIFICED IN ESTROUS CYCLE STAGES OF ANESTRUS, DIESTRUS, ESTRUS, AND METESTRUS Estrous Cycle Stage Animal Number Number of Ovarian F o l l i c l e s Larger Than .250 micra:. . Number of Ovarian F o l l i c l e s Larger Than 350 micra Maximum Ovarian Diameter i n mm Largest Ovarian F o l l i c l e i n micra Dev. Ant. Atr. Total Dev. Ant. Atr. Total Anestrus 2 7 5 2 14 0 4 0 4 1.37 412 5 3 5 5 13 0 4 0 4 1.37 390 71 9 7 4 20 0 5 1 6 1.24 412 73 6 4 7 17 2 4 2 8 1.28 420 78 9 4 4 17 1 2 1 4 1.26 390 81 4 7 4 15 0 4 1 5 1.26 398 89 5 0 0 5 0 0 0 0 0.96 285 92 2 7 5 14 0 3 0 3 1.07 405 118 9 5 8 22 3 5 2 10 1.24 435 230 9 _1 _0 10 _2 _1 JO 3 1.13 398 Totals 63 45 39 147 8 32 7 47 Averages 6.3 4.5 3.9 14.7 0.8 3.2 0.7 4.7 1.22 394.5 Diestrus 419 4 6 0 10 ' 1 3 0 4 1.35 398 231 7 4 2 13 2 2 0 4 1.35 375 252 14 5 2 21 1 2 0 3 1.44 368 260 7 3 2 12 2 3 1 6 1.15 473 296 11 5 1 17 2 5 1 8 1.55 443 244 7 1 1 9 2 1 1 4 1.18 405 255 5 10 3 18 1 7 1 9 1.20 4.3 258 5 5 5 15 0 1 2 3 1.35 398 266 12 4 _6 22 _0 _4 _0 _4 1.44 360 Totals 72 43 22 137 11 28 6 45 Averages 8.0 4.7 2.4 15.2 1.2 3.1 0.7 5.0 1.33 404 ' lb TABLE 9—Continued Estrous Cycle Stage Animal Number Number of Ovarian F o l l i c l e s Larger Than 250 micra Number of Ovarian F o l l i c l e s Larger Than 350 micra Maximum Ovarian Diameter in mm Largest Ovarian F o l l i c l e in micra Dev. Ant. Atr. Total Dev. Ant. Atr. Total Estrus 209 8 4 2 14 2 4 1 7 1.46 443 210 14 6 3 23 0 6 1 7 1.44 480 211 8 5 5 18 0 5 1 6 1.45 398 253 12 6 9 27 1 6 2 9 1.63 548 269 5 8 1 14 1 7 0 8 1.54 413 294 10 6 5 21 5 6 2 13", 1.54 435 411 10 6 7 23 3 6 7 16 1.64 585 416 6 8 5 19 1 8 4 13 1.79 443 283 7 _5 2 12 _0 _5 1 6 1.48 563 Totals 80 54 39. 171 13 53 19 85 Averages 8.9 6.0 4.3 19.0 1.4 5.9 2.1 9.4 1.55 479 Metestrus 114 7 7 4 18 0 6 1 7 1.57 450 234 _6 _4 _7 17 _0 _4 _4 _8 1.68 510 Totals 13 11 11 35 0 10 5 15' Averages 6.5 5.5 5.5 17.5 0 5.0 2.5 7.5 1.63 480 Ul TABLE 10 ANALYSIS OF VARIANCE OF SELECTED MEANS OF TABLE 8 Means T a b l e S o u r c e s o f V a r i a t i o n V a r i a b l e A n . D i . E s t . S S . D F . MS. F . Maximum o v a r i a n d i a m e t e r G r o u p s E x p e r i m e n t a l e r r o r : T o t a l s Maximum o v a r i a n f o l l i c l e d i a m e t e r Groups E x p e r i m e n t a l e r r o r T o t a l s Number o f d e v e l o p i n g f o l l i c l e s l a r g e r t h a n 250 m i c r a G r o u p s E x p e r i m e n t a l e r r o r T o t a l s Number o f a n t r a l f o l l i c l e s l a r g e r t h a n 350 m i c r a G r o u p s E x p e r i m e n t a l e r r o r T o t a l s Number o f a t r e t i c f o l l i c l e s l a r g e r t h a n 250 m i c r a Groups E x p e r i m e n t a l e r r o r T o t a l s 1.22 1.34 1 .55 3 9 4 . 5 0 4 0 3 . 6 7 4 7 8 . 6 7 6 . 3 0 6 . 6 0 3 . 2 0 3 . 1 1 3 . 9 0 2 . 4 4 8 . 8 9 5 . 8 9 4 . 3 3 .5372 2 .4113 25 . 26859 1 6 . 3 2 5 0 8 .01645 .9485 27 3 9 3 1 5 . 5 0 0 0 2 1 9 6 5 6 . 2 5 0 0 0 7 .66930 6 4 0 7 4 . 5 0 0 0 25 2 5 6 2 . 9 8 0 0 0 1 0 3 3 8 7 . 0 0 0 0 27 3 6 . 9 8 8 3 2 2 2 0 . 9 0 8 9 25 2 5 7 . 8 9 7 1 27 4 5 . 5 8 6 5 2 6 7 . 3 7 7 8 25 1 1 2 . 9 6 4 3 27 1 7 . 7 3 4 9 2 1 4 7 . 1 2 2 2 25 1 8 . 4 9 4 1 3 2 .09296 2 2 . 7 9 3 2 5 8 .45726 2 . 6 9 5 1 1 8 .86746 1 .50682 5 .88489 1 6 4 . 8 5 7 1 27 47 a n a l y s i s o f v a r i a n c e i s p r e s e n t e d i n T a b l e s 10 and 1 1 . I n t h e t e s t , u t i l i z e d , an F v a l u e o f g r e a t e r t h a n 3 . 4 0 r e p r e s e n t s a s i g n i f i c a n t d i f -f e r e n c e be tween t h e means e x a m i n e d . S t a t i s t i c a l c o m p u t e r a n a l y s i s was p e r f o r m e d by M r . C l i f f o r d Weedman o f Comps tad I n c . , L a J o l l a , C a . A com-p a r a t i v e d i s c u s s i o n o f r e s u l t s o b t a i n e d f o l l o w s . Mean number o f d e v e l o p i n g f o l l i c l e s l a r g e r t h a n 250 m i c r a . A n a l y -s i s o f v a r i a n c e i n d i c a t e s t h e r e i s no s i g n i f i c a n t d i f f e r e n c e among t h e means o f t h e t h r e e g r o u p s w i t h r e f e r e n c e t o t h i s v a r i a b l e (F = 2 . 0 9 ) . Mean number o f a n t r a l f o l l i c l e s l a r g e r t h a n 350 m i c r a . A n a l y s i s o f v a r i a n c e d e m o n s t r a t e s t h e r e i s no s i g n i f i c a n t d i f f e r e n c e be tween t h e a n e s t r o u s and d i e s t r o u s a n i m a l s ; t h e r e i s a s i g n i f i c a n t d i f f e r e n c e be tween t h e numbers o f t h e s e f o l l i c l e s o b s e r v e d i n t h e e s t r o u s a n i m a l and e i t h e r o f t h e o t h e r g r o u p s (F = 8 . 4 6 ) . Mean number o f a t r e t i c f o l l i c l e s g r e a t e r t h a n 250 m i c r a . S t a t i s t i -c a l a n a l y s i s d e m o n s t r a t e s t h e r e i s no s i g n i f i c a n t d i f f e r e n c e among any o f t h e t h r e e g r o u p s ; a p p a r e n t l y random f l u c t u a t i o n s o f numbers o f a t r e t i c f o l l i c l e s o c c u r w i t h i n and be tween t h e s e g r o u p s (F = 1 . 5 1 ) . Mean maximum o v a r i a n d i a m e t e r . R e s u l t s d e m o n s t r a t e t h e s e o v a r i e s come f rom d i f f e r e n t p o p u l a t i o n s o f a n i m a l s as t h e r e i s a s i g n i f i c a n t d i f -f e r e n c e be tween any p a i r o f means (F = 1 6 . 3 2 ) . Mean maximum o v a r i a n f o l l i c l e d i a m e t e r . R e s u l t s d e m o n s t r a t e t h e r e i s a s i g n i f i c a n t d i f f e r e n c e be tween t h e mean maximum f o l l i c l e d i a m e t e r o f t h e e s t r o u s o v a r i e s and t h e mean maximum f o l l i c l e d i a m e t e r s o f e i t h e r o f t h e o t h e r two means . Howeve r , t h e r e i s no s i g n i f i c a n t d i f f e r e n c e be tween t h e a n e s t r o u s and d i e s t r o u s mean maximum o v a r i a n f o l l i c l e d i a m e t e r s 48 TABLE 11 ANALYSIS OF VARIANCE OF MEANS OF UTERINE HORN EPITHELIAL CELL HEIGHT AND FALLOPIAN TUBE EPITHELIAL CELL HEIGHT IN ANESTROUS, DIESTROUS, AND ESTROUS FEMALES Variable Means Table An. Di. Est. Sources of V a r i a t i o n SS. DF. MS. F. Uterine horn e p i t h e l i a l c e l l height i n micra Groups Experimental error T o t a l 8.32 10.84 15.73 266.2761 2 133.13803 33.63491 98.9582 25 3.95833 365.2343 27 Fa l l o p i a n tube e p i t h e l i a l c e l l height i n micra Groups Experimental error 10.88 10.93 14.13 63.6526 2 31.82629 13.75237 57.8560 25 2.,31424 To t a l 121.5086 27 49 (F = 7 .67 ) . Summary o f a n a l y s i s o f v a r i a n c e . A random d i s t r i b u t i o n o f d e v e l o p i n g f o l l i c l e s o f a s i z e g r e a t e r t h a n 250 m i c r a i s e v i d e n t be tween t h e t h r e e g r o u p s o f a n i m a l s s t u d i e d . A l s o , t h e d i s t r i b u t i o n o f a t r e t i c f o l l i c l e s o f a s i z e g r e a t e r t h a n 250 m i c r a a p p e a r s t o be r andom. L a r g e a n t r a l f o l l i c l e s o f a s i z e g r e a t e r t h a n 350 m i c r a a r e f o u n d t o be p r e s e n t i n a s i g n i f i c a n t l y g r e a t e r number i n t h e e s t r o u s a n i m a l s t h a n i n e i t h e r t h e a n e s t r o u s o r d i e s t r o u s a n i m a l s . Howeve r , t h e r e a p p e a r s t o be no s i g n i f i c a n t l y d i f f e r -e n t number o f t h e s e f o l l i c l e s be tween t h e l a t t e r two g r o u p s . The maximum f o l l i c l e d i a m e t e r i s a l s o s i g n i f i c a n t l y d i f f e r e n t f o r t h e e s t r o u s a n i m a l s , b u t n o t s i g n i f i c a n t l y d i f f e r e n t f o r t h e a n e s t r o u s and d i e s t r o u s a n i m a l s . The o n l y a r e a a n a l y z e d w h i c h d e m o n s t r a t e s a s i g n i f i c a n t d i f f e r e n c e among a l l t h e s e means i s t h a t o f t h e maximum o v a r i a n d i a m e t e r . D e s c r i p t i v e s i m i l a r i t i e s among t h e t h r e e g r o u p s i n c l u d e : no a n i m a l d e m o n s t r a t e d r e c e n t o v u l a t i o n , f o r m i n g c o r p o r a l u t e a o r a p p a r e n t s e c r e t o r y c o r p o r a l u t e a ; t h e o v a r i e s o f a l l a n i m a l s c o n t a i n e d d e v e l o p i n g , a n t r a l and a t r e t i c f o l l i c l e s o f v a r i o u s s i z e s l i s t e d i n T a b l e 4 , e x c e p t a n i m a l 89 w h i c h was m i s s i n g any a n t r a l o r a t r e t i c f o l l i c l e s o f a s i z e g r e a t e r t h a n 250 m i c r a and a n i m a l 230 w h i c h was m i s s i n g any a t r e t i c f o l l i c l e s o f a s i z e g r e a t e r t h a n 250 m i c r a ; c o r p o r a a l b i c a n t i a were o b s e r v e d i n no a n i m a l . D e s c r i p t i v e d i f f e r e n c e s o b s e r v e d i n t h e t h r e e g r o u p s a r e i n c l u d e d i n t h e d i s c u s s i o n w h i c h f o l l o w s . A n e s t r o u s o v a r i e s (see P l a t e 4 ) . A c o n s p i c u o u s l a c k o f e a r l y d e v e l o p i n g f o l l i c l e s o f l e s s t h a n 250 m i c r a i n d i a m e t e r was o b s e r v e d i n t h e s e o v a r i e s . Many p r i m a r y o o c y t e s were o b s e r v e d i n t h e o v a r i a n c o r t e x b u t v e r y few o f t h e s e were s u r r o u n d e d by more t h a n one o f two l a y e r s o f 50 granulosa cel l s . Many small atretic f o l l i c l e s were observed which appeared to be f i l l e d in with connective tissue: degenerate ova were contained within these f o l l i c l e s . I n t e r s t i t i a l cells were packed closely together between f o l l i c l e s . Examination of this tissue under o i l immersion demon-strated many closely packed cells containing l i t t l e apparent cytoplasm. Few blood vessels or lymphatic spaces were observed in the stroma. Diestrous ovaries (see Plate 6). More intermediate sized f o l l i c l e s in the range of 75 to 250 micra were observed in the ovaries of diestrous animals than in those of the anestrous animals. Two animals (#231 and #245) demonstrated i n t e r s t i t i a l tissue similar to that of the anestrous animals when ovaries were examined under o i l immersion. The i n t e r s t i t i a l tissue of the other animals examined were more typical of those of the estrous animals. That i s , the tissue was more diffuse, and more cellular cytoplasm was visible. Estrous animals (see Plate 8). Large lymph spaces were seen throughout the stroma and also many apparently dilated blood vessels. A large amount of i n t e r s t i t i a l tissue was present in the stroma which, under o i l immersion, appeared to be organized into cords and contained cells exhibiting obvious cytoplasm. More frequent mitoses were observed in the granulosa cells of developing f o l l i c l e s than were observed in either of the other two groups. Metestrous ovaries (see Plate 10). The two animals studied demon-strated ovaries containing large lymph sinuses and blood vessels in addi-tion to stromal i n t e r s t i t i a l tissue similar to that described for estrous animals. Neither of these animals had ovulated. Neither f o l l i c l e size P l a t e 4 P l a t e 5 V a g i n a l C r o s s S e c t i o n o f A n e s t r o u s Female 78 (x400) 52 P l a t e 6 Ova ry o f Female 266 i n D i e s t r u s (x40) P l a t e 7 V a g i n a l C r o s s S e c t i o n o f Female 266 i n D i e s t r u s (x400) V a g i n a l C r o s s S e c t i o n o f Female 411 i n E s t r u s (x400) P l a t e 10 Ova ry o f Female 234 i n M e t e s t r u s (x40) P l a t e 11 V a g i n a l C r o s s S e c t i o n o f Female 234 i n M e t e s t r u s (x400) 55 n o r number d e m o n s t r a t e d s i g n i f i c a n t d i f f e r e n c e f r o m t h a t o f t h e e s t r o u s o v a r i e s . F a l l o p i a n t u b e s . Two d i s t i n c t d i v i s i o n s o f t h e f a l l o p i a n t u b e were o b s e r v e d i n M i c r o t u s t o w n s e n d i i . The u p p e r p o r t i o n c o n s i s t e d o f a t u b e l i n e d w i t h a s i n g l e l a y e r o f c i l i a t e d c u b o i d a l e p i t h e l i a l c e l l s . The d i s t a l p o r t i o n o f t h e t u b e a p p e a r e d t o c o n t a i n many s u b m u c o s a l p r o -j e c t i o n s i n t o t h e t u b e l u m e n . T h i s p o r t i o n o f t h e t u b e was l i n e d w i t h c l o s e l y a p p r o x i m a t e d , c i l i a t e d , c o l u m n a r e p i t h e l i a l c e l l s . Measuremen t s o f t h e f a l l o p i a n t u b e were made o f t h e c i l i a t e d c u b o i d a l c e l l s o f t h e p r o x i m a l end o f t h e t u b e . The a p p e a r a n c e o f t h e f a l l o p i a n t u b e was s i m i l a r f o r b o t h t h e a n e s t r o u s and d i e s t r o u s g r o u p s o f a n i m a l s . The a v e r a g e measurement o f t h e h e i g h t o f t h e t u b e f o r b o t h o f t h e s e g r o u p s was 1 0 . 9 m i c r a . A n a l y s i s o f v a r i a n c e p e r f o r m e d on t h e means o f t h e t h r e e g r o u p s o f a n i m a l s ( T a b l e 11) d e m o n s t r a t e s a s i g n i f i c a n t d i f f e r e n c e i n t h e a v e r a g e e p i t h e l i a l c e l l h e i g h t be tween t h e e s t r o u s a n i m a l s and e i t h e r t h e g r o u p o f a n i m a l s i n a n e s t r u s o r d i e s t r u s ; h o w e v e r , t h e r e i s no s i g n i f i c a n t d i f f e r e n c e be tween t h e e p i t h e l i a l c e l l h e i g h t o f t h e l a t t e r two g r o u p s (F = 1 3 . 7 5 ) . One m a j o r d e s c r i p t i v e d i f f e r e n c e o b s e r v e d be tween e s t r o u s a n i m a l s and e i t h e r o f t h e o t h e r g r o u p s o f a n i m a l s was i n t h e amount o f c y t o p l a s m p r e s e n t i n t h e l i n i n g c e l l s . I n t h e e s t r o u s a n i m a l s more c u l l u l a r c y t o -p l a s m was e v i d e n t . The o t h e r o b s e r v a b l e d i f f e r e n c e was i n t h e s i z e o f t h e f a l l o p i a n t u b e l u m e n . T h e r e was a c o n s p i c u o u s d i l a t i o n o f t h e u p p e r p o r t i o n o f t h e t u b e and l e s s s u b m u c o s a l p r o j e c t i o n s p r e s e n t i n t h e l o w e r p o r t i o n o f t h e t u b e i n t h e e s t r o u s a n i m a l s . I n one a n i m a l , #411, a v e r y g r e a t l y d i s t e n d e d u p p e r t u b a l p o r t i o n was o b s e r v e d . T h i s was a l s o o b s e r v e d i n a n i m a l #234, one o f t h e m e t e s t r o u s a n i m a l s s t u d i e d . 56 The m e t e s t r o u s a n i m a l s had f a l l o p i a n t u b e s mos t c l o s e l y c o r r e s -p o n d i n g t o t h o s e d e s c r i b e d f o r t h e e s t r o u s a n i m a l s . U t e r i n e h o r n . O b s e r v a t i o n s made i n c l u d e t h e h e i g h t and n a t u r e o f t h e u t e r i n e e p i t h e l i u m ; t h e n a t u r e o f t h e o r g a n i z a t i o n o f t h e submucosa and i t s g l a n d s ; and t h e o r g a n i z a t i o n o f t h e m u s c l e l a y e r s o f t h e u t e r u s . The mean c r o s s s e c t i o n a l d i a m e t e r o f t h e a n e s t r o u s and d i e s t r o u s u t e r i n e h o r n was s m a l l compared t o means f o r t h e e s t r o u s a n i m a l s ( 0 . 5 8 mm f o r a n e s t r o u s a n i m a l s ; 0 . 7 6 mm f o r d i e s t r o u s a n i m a l s ; 1.2 mm f o r e s t r o u s a n i m a l s ) . The e p i t h e l i a l l i n i n g c e l l s o f t h e u t e r u s were l o w c u b o i d a l i n t h e a n e s t r o u s a n i m a l s . C e l l s c o n t a i n e d s p h e r i c a l n u c l e i and a t h i n a p i c a l l y d i r e c t e d b o r d e r o f c y t o p l a s m . F i v e o f t h e n i n e d i e s t r o u s a n i m a l s e x a m i n e d d e m o n s t r a t e d a l i n i n g e p i t h e l i u m t y p i c a l o f t h a t d e s c r i b e d f o r t h e a n e s t r o u s f e m a l e s . F o u r o f t h e d i e s t r o u s a n i m a l s d e m o n s t r a t e d an e n d o m e t r i a l l i n i n g composed o f t a l l c o l u m n a r c e l l s w i t h o b l o n g n u c l e i c o n t a i n i n g an o b v i o u s a p i c a l b o r d e r o f c y t o p l a s m . E s t r o u s f e m a l e s a l s o d e m o n s t r a t e d t h e a p p e a r a n c e d e s c r i b e d f o r t h e l a t t e r d i e s t r o u s a n i m a l s . A n a n a l y s i s o f v a r i a n c e was p e r f o r m e d o n mean e p i t h e l i a l c e l l h e i g h t o b t a i n e d f o r e a c h g r o u p o f a n i m a l s ( T a b l e 1 1 ) . A s i g n i f i c a n t d i f f e r e n c e was o b s e r v e d among t h e t h r e e g r o u p s (F = 3 3 . 6 ) . The v a l u e o f t h e s e r e s u l t s a p p l i e d t o t h e d i e s t r o u s f e m a l e s i s q u e s t i o n a b l e . A s s t a t e d , t h i s g r o u p o f a n i m a l s d e m o n s t r a t e d e n d o m e t r i a l l i n i n g a p p e a r a n c e v a r i a t i o n s o f e s t r u s o r a n e s t r u s . D i e s t r u s a p p e a r s t o have no d i s t i n c t i v e i d e n t i f y i n g c h a r a c t e r i s t i c a s r e f l e c t e d v i a t h e u t e r i n e h o r n . The submucosa o f t h e a n e s t r o u s a n i m a l c o n t a i n s many c o n n e c t i v e t i s s u e n u c l e i and w h a t . a p p e a r t o be c o l l a g e n o u s f i b r i l s a r r a n g e d i n a d i s o r g a n i z e d f a s h i o n . G l a n d c e l l s a p p e a r t o be a l m o s t t o t a l l y w i t h o u t c y t o p l a s m and a p p e a r as s c a t t e r e d c i r c l e s o f n u c l e i i n t h e submucosa . 57 U t e r i n e m u s c l e l a y e r s i n t h e a n e s t r o u s f e m a l e s c o n t a i n e d numerous n u c l e i and l i t t l e c y t o p l a s m . The f i v e d i e s t r o u s f e m a l e s w i t h l o w c u b o i d a l e p i t h e -l i a l l i n i n g d e m o n s t r a t e d a submucosa s i m i l a r t o t h a t d e s c r i b e d f o r t h e a n e s t r o u s f e m a l e s . The o t h e r f o u r d i e s t r o u s f e m a l e s d e m o n s t r a t e d t h e p r e s e n c e o f s m a l l s u b m u c o s a l g l a n d s ; t h e c e l l s o f w h i c h a p p e a r e d t o c o n t a i n c y t o p l a s m . The g e n e r a l s t a t e m e n t c o u l d be made t h a t t h e e s t r o u s a n i m a l s d e m o n s t r a t e an i n c r e a s e i n c e l l u l a r c y t o p l a s m i n a l l l a y e r s o f t h e u t e r i n e w a l l . The submucosa i s e n l a r g e d i n t h e s e a n i m a l s ; c o n n e c t i v e t i s s u e c e l l s a p p e a r t o c o n t a i n more c y t o p l a s m and i n c o n t r a s t t o the , :sparse. , . . . small^. j.and i n a c t i v e a p p e a r i n g g l a n d s p r e s e n t i n t h e a n e s t r o u s a n i m a l , t h e g l a n d s i n t h e submucosa o f t h e e s t r o u s a n i m a l a r e l a r g e and numerous . C u b o i d a l c e l l s w i t h o b v i o u s c y t o p l a s m a r e o b s e r v e d ; p i n k s t a i n i n g s e c r e t i o n s were f r e q u e n t l y o b s e r v e d i n t h e g l a n d u l a r l u m e n . M u s c l e l a y e r s a r e w e l l o r g a -n i z e d and a r e t h i c k e r t h a n t h o s e o b s e r v e d i n t h e a n e s t r o u s a n i m a l . V a g i n a . V a g i n a l c e l l w a l l l a y e r s were c o u n t e d and means r e c o r d e d f o r e a c h o f t h e t h r e e g r o u p s . No s t a t i s t i a l a n a l y s i s was p e r f o r m e d o n t h e s e d a t a a s t h e number o f l a y e r s a p p e a r e d t o v a r y d e p e n d i n g on t h e o r i e n t a t i o n o f t h e s e c t i o n o f v a g i n a l w a l l m e a s u r e d . The d e s c r i p t i v e d a t a p r e s e n t e d d e m o n s t r a t e s t h e r e i s a v i s i b l e d i f f e r e n c e be tween t h e v a g i n a l l i n i n g o f a n i m a l s w i t h i n t h e t h r e e g r o u p s . A m a j o r o b s e r v a t i o n a l d i f f e r e n c e be tween t h e v a g i n a e o f t h e a n e s -t r o u s a n i m a l s and t h o s e o f e i t h e r o f t h e o t h e r g r o u p s was t h a t i n t h e f o r m e r g r o u p t h e r e were l e s s c o n v o l u t i o n s i n t h e w a l l ; a t h i n submucosa ; t h e l i n i n g r e s t r i c t e d t o two o r t h r e e l a y e r s o f r e l a t i v e l y a c y t o p l a s m i c c e l l s . D i e s t r o u s a n i m a l s d e m o n s t r a t e d an e n l a r g e d submucosa ; i n f o l d e d w a l l s ; s e v e r a l b a s a l l a y e r s o f l i n i n g t o p p e d by l a y e r s i n w h i c h l e u k o c y t i c i n f i l t r a t i o n was o b v i o u s . I n a l l c a s e s t h e p r e s e n c e o f m a s s i v e numbers 58 o f l e u k o c y t e s was o b s e r v e d i n t h e l u m e n . The o v e r a l l v a g i n a l h i s t o l o g y o f t h e s e a n i m a l s more c l o s e l y r e s e m b l e d t h a t o f t h e e s t r o u s a n i m a l s t h a n o f t h e a n e s t r o u s a n i m a l s . The u p p e r l a y e r s o f t h e v a g i n a l l i n i n g e p i t h e -l i u m c o n t a i n e d c o r n i f i e d c e l l s i n a l l e s t r o u s a n i m a l s e x a m i n e d . The v a r i -a t i o n be tween e s t r o u s a n i m a l s a p p e a r e d t o be due t o t h e t h i c k n e s s o f t h i s c o r n i f i e d l a y e r . One o f t h e a n i m a l s , #296, has o n l y a t h i n l a y e r o f s u r -f a c e c o r n i f i e d c e l l s w h i c h was h e a v i l y i n f i l t r a t e d w i t h l e u k o c y t e s . O t h e r w i s e , l e u k o c y t e s were n o t o b s e r v e d i n t h e c o r n i f i e d l a y e r s . Howeve r , i n s e v e r a l f e m a l e s l e u k o c y t e s were p r e s e n t i n a l a y e r i m m e d i a t e l y b e l o w t h e c o r n i f i e d l a y e r s . I n b o t h o f t h e m e t e s t r o u s a n i m a l s h e a v y l e u k o c y t i c i n f i l t r a t i o n o f t h e l i n i n g e p i t h e l i u m o c c u r r e d . A p p e a r a n c e o f s e v e r a l v a g i n a l c r o s s s e c t i o n s and r e l a t e d l a r g e s t o v a r y o f f e m a l e s s a c r i f i c e d i n smear s t a g e s o f a n e s t r u s , d i e s t r u s , e s t r u s and m e t e s t r u s may be o b s e r v e d i n t h e l i s t e d p l a t e s : a n e s t r u s , P l a t e s 4 and 5; d i e s t r u s , P l a t e s 6 and 7; e s t r u s , P l a t e s 8 and 9; and m e t e s t r u s , P l a t e s 10 and 1 1 . O v a r i a n and R e p r o d u c t i v e T r a c t Response  o f N i n e A n e s t r o u s F e m a l e s t o P . M . S . E i g h t o f t h e n i n e f e m a l e s were p e r f o r a t e w i t h i n 53 h o u r s and a l l d e m o n s t r a t e d t h e smear s t a g e o f e s t r u s a t t h i s t i m e e x c e p t #291 w h i c h d e m o n s t r a t e d a p r o e s t r o u s smear . O n l y one a n i m a l r e m a i n e d i m p e r f o r a t e a f t e r a d m i n i s t r a t i o n o f t h e d r u g . T h i s a n i m a l , #286, h a d o v a r i e s w h i c h a p p e a r e d a b n o r m a l w i t h s m a l l a t r e t i c f o l l i c l e s p r i m a r i l y i n e v i d e n c e f o f t e n p a r t i a l l y f i l l e d w i t h c o n n e c t i v e t i s s u e , and had many a b n o r m a l a n t r a l f o l l i c l e s w h i c h c o n t a i n e d g r a n u l o s a c e l l s i n w e b - l i k e f a s h i o n t h r o u g h o u t t h e l i q u o r f o l l i c u l i . R e s u l t s o b t a i n e d may be o b s e r v e d i n T a b l e 1 2 . Maximum f o l l i c l e s i z e s i n e a c h o f t h e o v a r i e s was n o t i c e a b l y l a r g e r t h a n t h a t o b s e r v e d TABLE 12 OVARIAN AND REPRODUCTIVE TRACT RESPONSE OF NINE ADULT IMPERFORATE FEMALES TO PREGNANT MARE'S SERUM Observed Vaginal Number of Ovarian Smear Response F o l l i c l e s Larger E p i t h e l i a l Height to P.M. S. Than 350 micra Average Maximum i n micra Capture uvarian r u i i i c i e Animal Weight P. M.S. Death i n Diameter Size Uterine F a l l o p i a n Number i n gms Dose 0 hr 24 hr 51-53 hr Dev . Ant Atr. Total i n mm i n micra Horn Tube' . 213 37.1 25 I.U. I a D b E C 0 24 6 30 1.96 638 14.4 13.6 274 30.0 25 I.U. I I E 0 15 2 17 1.85 720 18.4 20.0 279 58.5 25 I.U. I • P d E 0 10 1 11 1.55 585 16.0 13.6 290 33.5 25 I.U. I I E 0 5 1 6 1.54 600 13.6 16.0 291 26.5 25 I.U. I I P 0 17 0 17 1.55 20". 0 16.0 16.0 250 44.5 25 I.U. I I E 0 19 0 19 1.72 623 20.0 16.0 286 34.0 25 I.U. I I I H i s t o l o g i c a l examination demonstrated abnormal f o l l i c l e s . 276 35.8 50 I.U. I I E 2 13 1 16 1.70 638 17.6 12.8 254 58.0 50 I.U. I I E 0 26 3 29 2.16 765 15.2 13.6 Averages 0. 25 16 .1 1.7 18.1 1.75 639 16.9 15.2 I = imperforate. D = diestrous. E = estrous. P = proestrous. U3 60 i n t h e n o n - t r e a t e d e s t r o u s f e m a l e . The number o f f o l l i c l e s l a r g e r t h a n 350 m i c r a i n d i a m e t e r i s a l s o much g r e a t e r t h a n was o b s e r v e d i n o v a r i e s o f f e m a l e s s a c r i f i c e d w h i l e i n t h e v a g i n a l smear s t a g e o f e s t r u s . O v u l a t i o n o c c u r r e d i n none o f t h e a n i m a l s . One u n u s u a l o b s e r v a -t i o n was t h e e x i s t e n c e o f e x t r a v a s a t i o n o f b l o o d i n s e v e r a l f o l l i c l e s o f f o u r o f t h e n i n e a n i m a l s . I n a l l c a s e s , t h e s e f o l l i c l e s a p p e a r e d t o be a n t r a l f o l l i c l e s i n e a r l y a t r e t i c s t a g e s and c o n t a i n i n g d e g e n e r a t i n g o v a . H i l l and W h i t e (1933) o b s e r v e d s i m i l a r a p p e a r i n g f o l l i c l e s i n r a b b i t s and s t a t e t h a t u n o v u l a t e d h e m o r r h a g i c f o l l i c l e s a r e an a b n o r m a l f o r m o f f o l l i -c l e r e g r e s s i o n . L u t e i n i z a t i o n , c h a r a c t e r i z e d b y t h e p r e s e n c e o f e n l a r g e d c e l l s s i m i l a r t o t h o s e o b s e r v e d i n c o r p o r a l u t e a o f p r e g n a n t f e m a l e s was o b s e r v e d i n t h e i n t e r s t i t i a l t i s s u e s u r r o u n d i n g f o l l i c l e s and o c c a s i o n a l l y i n a t r e t i c f o l l i c l e s . The o c c u r r e n c e o f l u t e i n i z a t i o n f o l l o w i n g P . M . S . a d m i n -i s t r a t i o n was d e s c r i b e d by C o l e and H a r t ( 1 9 3 0 ) . L a r g e numbers o f a n t r a l o v a r i a n f o l l i c l e s were o b s e r v e d w i t h v e r y few o f e i t h e r d e v e l o p i n g o r a t r e t i c ones p r e s e n t . The a v e r a g e o v a r i a n d i a m e t e r s i z e i s e n l a r g e d o v e r t h a t o f t h e u n t r e a t e d e s t r u s a n i m a l s ( 1 . 7 5 as compared t o 1.4 mm). The maximum f o l l i c l e s i z e i s g r e a t l y i n c r e a s e d when compared w i t h t h a t o f t h e u n t r e a t e d e s t r o u s a n i m a l (639 as compared t o 479 m i c r a ) . O b s e r v a t i o n s o f B e h a v i o r a l E s t r u s i n M i c r o t u s t o w n s e n d i i No i m m e d i a t e c o p u l a t i o n was o b s e r v e d upon p a i r i n g an a d u l t ma l e and f e m a l e , e v e n i n t h o s e i n s t a n c e s i n w h i c h t h e p a i r h a d p r e v i o u s l y s u c c e s s f u l l y c o p u l a t e d . C h a s i n g o f t h e f e m a l e b y t h e m a l e was f r e q u e n t l y o b s e r v e d . T a b l e 12 p r e s e n t s a l l v e r i f i e d m a t i n g d a t a o b t a i n e d i n t h i s 61 s t u d y . I n f o u r o f t h e s e v e n r e c o r d e d raatings, c o p u l a t i o n o c c u r r e d w i t h i n t h e f i r s t two d a ys a f t e r i n t r o d u c t i o n . M a t i n g b e h a v i o r . . The t y p e o f b e h a v i o r o b s e r v e d upon i n t r o d u c t i o n o f a m a l e t o a f e m a l e cage was t h e same i n a l l c a s e s w i t h m i n o r v a r i a t i o n s . The f e m a l e , when a p p r o a c h e d by t h e m a l e , w o u l d e x h i b i t d i s t u r b a n c e by m a k i n g h i g h p i t c h e d n o i s e s and c h a t t e r i n g s o u n d s . A t t h e c o n t i n u e d a p p r o a c h o f t h e m a l e , t h e r e was t e e t h b a r i n g and l u n g e s a t a p e r s i s t e n t m a l e by t h e f e m a l e . No a c t u a l i n j u r i e s were s e e n as a r e s u l t o f t h e s e a p p a r e n t l y sham a t t a c k s . G e n e r a l l y c h a s i n g w o u l d o c c u r f o r a s h o r t t i m e p r i o r t o d e m o n s t r a t i o n o f a g g r e s s i v e b e h a v i o r by t h e f e m a l e . I n e v e r y i n s t a n c e o b s e r v e d , r e g a r d l e s s o f t h e o f t e n much s m a l l e r s i z e o f t h e f e m a l e , t h e ma le w o u l d r e t r e a t . The m o r n i n g f o l l o w i n g t h e f i r s t day o f i n t r o d u c t i o n t h e two a n i m a l s were o f t e n o b s e r v e d h u d d l e d t o g e t h e r i n some a r e a o f t h e cage o r s h a r i n g t h e n e s t box w i t h o u t a p p a r e n t a g g r e s s i o n . H o w e v e r , i n no i n s t a n c e d i d v a g i n a l smears d e m o n s t r a t e t h e p r e s e n c e o f s p e r m a t o z o a a t t h i s t i m e . V a g i n a l smear s t a g e and b e h a v i o r a l e s t r u s . T a b l e 13 i n d i c a t e s t h a t f e m a l e s have been o b s e r v e d t o mate b o t h when an e s t r o u s v a g i n a l smear (6 i n s t a n c e s ) and when a d i e s t r o u s v a g i n a l smear (1 i n s t a n c e ) was o b s e r v e d t h e p r e c e d i n g d a y . Two r e c o r d e d i n s t a n c e s i n a n i m a l s ma ted f o r 11 and 13 days (#456 and #242, r e s p e c t i v e l y ) o f c o n s t a n t e s t r o u s v a g i n a l smears f a i l e d t o d e m o n s t r a t e any o c c u r r e n c e o f m a t i n g . F rom t h e s e d a t a i t i s a p p a r e n t an e s t r o u s smear does n o t n e c e s s a r i l y i n d i c a t e t h e p r e s e n c e o f b e h a v i o r a l e s t r u s ; t h a t i s , w i l l i n g n e s s o f t h e f e m a l e t o c o p u l a t e , a t l e a s t i m m e d i a t e l y . A l s o , b e h a v i o r a l e s t r u s may be p r e s e n t when t h e v a g i n a l smear s t a g e a p p e a r s t o be more t y p i c a l o f d i e s t r u s t h a n o f e s t r u s . 62 TABLE 13 MATING DATA OF MICROTUS TOWNSENDII FEMALES Date of Animal Vaginal Subsequent Smear Number • Smear Pattern Time Between Introduction of Male and Mating Animal Disposition 60 4-•16-67 E 4-17 E 4-18 E S 4-19 D 2 days Male removed when sperm observed. Litter born 5-8-67. 60 5-•23-67 E 5-24 E 5-25 E S 5-26 D 2 days Male removed when sperm observed. Litter born 6-10-67. 64 5-•14-67 D 5-15 D 5-16 DS 2 days Dead in cage 5-29; uterus non-pregnant at autopsy. 295 10--6-67 10-7 10-8 10-9 10-10 2 days Killed 10-12; centrally E E' E" D D hollow developing cor-' pora lutea observed in ovaries. -259 10--6-67 10-7 10-8 10-9 10-10 3 days Killed 10-10; ovaries D D D D & D demonstrate no ovula-tion has occurred. 457 9--14-67 9-15 9-16 9-17 9-18 9-19 Some time On 9-27 smear demon-D E D D D E less than strated presence of many 9-20 9-21 9-22 9-23 9-24 13 days red blood c e l l s and E E D D D headless sperm—indica-9-25 9-26 9-27 9-28 9-29 tive of prior missed D D D S D D mating. Ki l l e d 10-6; 9-30 10-1 10-2 10-3 10-4 ovaries demonstrated no E ' E E E D recent ovulation had 10-5 10-6 occurred; 7.regressing E E S corpora lutea observed. 10 9--19-67 9-15 9-16 9-17 9-18 9-19 11 days Kil l e d 9-26; tissues E E D D D D accidentally destroyed 9-20 9-21 9-22 9-23 9-24 in processing. D D E D D 9-25 9-26 E S D Note. E = estrus; D = diestrus; = presence of sperm. Date male introduced also. 63 R e l a t i o n s h i p be tween b e h a v i o r a l e s t r u s and o v u l a t i o n . V a g i n a l smear d o c u m e n t a t i o n o f m a t i n g was a s s o c i a t e d w i t h o v u l a t i o n i n o n l y t h r e e documented i n s t a n c e s , o b s e r v e d i n T a b l e 1 3 . A n i m a l #60, i n b o t h i n s t a n c e s r e c o r d e d , had a l i t t e r f o l l o w i n g t h e r e m o v a l o f t h e ma le o n t h e day s p e r m a -t o z o a were o b s e r v e d i n t h e v a g i n a l smear . A n i m a l #295 e x h i b i t e d t h e p r e -s ence o f f o r m i n g c o r p o r a l u t e a on h i s t o l o g i c a l e x a m i n a t i o n . A n i m a l #457 had o v u l a t e d a t some t i m e p r i o r t o s a c r i f i c e . Howeve r , t h e h i s t o r y o f t h e d a t a o f m a t i n g f o r t h i s a n i m a l i s n o t c o m p l e t e , t h e r e f o r e t h e mean ing o f t h e r e g r e s s i n g c o r p o r a l u t e a o b s e r v e d a t h i s t o l o g i c a l e x a m i n a t i o n i s n o t c l e a r . The o v a r i a n f o l l i c l e s a t t h e t i m e o f d e a t h a p p e a r t o be s i m i l a r t o t h o s e o f t h e e s t r o u s a n i m a l s and no p r e o v u l a t o r y i n c r e a s e i n s i z e i s e v i d e n t . The p o s s i b i l i t y e x i s t s t h a t b o t h t h i s a n i m a l and a l s o a n i m a l #259 were s a c r i f i c e d t o o soon a f t e r m a t i n g f o r p r e o v u l a t o r y changes a n d / o r o v u l a t i o n t o have o c c u r r e d . The o v a r i a n f o l l i c l e s o f a n i m a l #295 a l s o a p p e a r e d t o be more t y p i c a l by v i r t u e o f s i z e and number t o t h o s e o f e s t r o u s a n i m a l s and no p r e o v u l a t o r y g r o w t h s p u r t o f f o l l i c l e s was o b s e r v e d . OBSERVATIONS OF PREGNANCY AND EVENTS FOLLOWING PREGNANCY I N MICROTUS TOWNSENDII I n t h i s s t u d y , t h r e e g r o u p s o f p r e g n a n t a n i m a l s were o b s e r v e d : a n i m a l s b r e d i n t h e l a b o r a t o r y ; p r e g n a n t f i e l d a n i m a l s b r o u g h t i n t o t h e l a b o r a t o r y and m a i n t a i n e d t h e r e ; and p r e g n a n t a n i m a l s c a p t u r e d i n t h e f i e l d and s a c r i f i c e d i m m e d i a t e l y f o r u t e r i n e embryo c o u n t . B r e e d i n g S e a s o n Cowan and G u i g n e t (1965) s t a t e t h a t t h e b r e e d i n g s e a s o n o f M i c r o - t u s t o w n s e n d i i e x t e n d s f r o m March , t o September i n B r i t i s h C o l u m b i a . F i e l d d a t a o b t a i n e d i n my own s t u d y i n d i c a t e t h a t i n 1967 b r e e d i n g began i n 64 e a r l y F e b r u a r y i n t h e V a n c o u v e r a r e a . A f e m a l e , c a p t u r e d on M a r c h 2 , 1 9 6 7 , was i n l a t e p r e g n a n c y , a judgment b a s e d o n a u t e r i n e w e i g h t o f 7 grams and t h e p r e s e n c e o f l a r g e e m b r y o s . Two a d u l t f e m a l e s c a p t u r e d i n m i d -F e b r u a r y had p e r f o r a t e v a g i n a e . A j u v e n i l e w e i g h i n g 19 grams was c a u g h t o n M a r c h 1 1 , 1967 , and a n o t h e r w e i g h i n g 18 grams was c a u g h t s e v e n d ay s l a t e r . The l a b o r a t o r y g r o w t h (Graph 1) i n d i c a t e s t h a t t h e s e a n i m a l s were b o r n a p p r o x i m a t e l y F e b r u a r y 20 and M a r c h 1, r e s p e c t i v e l y . A g e s t a t i o n p e r i o d o f 21 t o 24 da ys (see s u b s e c t i o n f o l l o w i n g e n t i t l e d " G e s t a t i o n P e r i o d " ) p l a c e s t h e c o n c e p t i o n d a t e s o f t h e s e v o l e s i n l a t e J a n u a r y o r ; e a r l y F e b r u a r y . A n i m a l s m a i n t a i n e d i n t h e a r t i f i c i a l l y c o n t r o l l e d l a b o r a t o r y e n v i -ronment b r e d t h r o u g h o u t t h e y e a r (see p a r t u r i t i o n d a t e s i n T a b l e 1 4 ) . G e s t a t i o n P e r i o d O n l y t h r e e d a t e d p r e g n a n c i e s were o b s e r v e d i n t h i s s t u d y . A n i m a l #60 y i e l d e d two d a t e d p r e g n a n c i e s o f 21 d ay s and 23 d a y s f o r two l i t t e r s ( T a b l e 1 4 ) . The ma le was removed f r o m a n i m a l #10 on day o f l i t t e r o b s e r -v a t i o n . P o s t p a r t u m e s t r u s m a t i n g r e s u l t e d i n a s u b s e q u e n t l i t t e r 24 d ay s l a t e r . B i r t h and S u r v i v a l o f L i t t e r s Embryo c o u n t s o f 20 p r e g n a n t f i e l d a n i m a l s a v e r a g e d s i x p e r p r e g n a n c y , r a n g i n g be tween f i v e and s e v e n ( T a b l e 1 5 ) . The a v e r a g e s i z e o f 13 l a b o r a t o r y b r e d l i t t e r s was 4 . 8 4 a n i m a l s p e r l i t t e r w i t h a r a n g e o f two t o s i x a n i m a l s ( T a b l e 1 4 ) . A l t h o u g h f i e l d a n i m a l s were c o l l e c t e d f o r t h e l a b o r a t o r y d u r i n g t h e summer b r e e d i n g s e a s o n , o n l y 12 l i t t e r s were s u b s e q u e n t l y b o r n o f f i e l d b r e d f e m a l e s . The t w e l v e l i t t e r s c o n -s i s t e d o f 65 o f f s p r i n g , o r an a v e r a g e l i t t e r s i z e o f 5 . 4 1 . ' a n i m a l s , r a n g i n g 65 TABLE 14 PREGNANCY DATA AND AVERAGE L I T T E R S I Z E OF LABORATORY COLONY FEMALES A n i m a l Number ' D a t e o f P a r t u r i t i o n Number o f L i t t e r A n i m a l s Number o f L i t t e r A n i m a l s Weaned M F Comments 10 1 0 - 1 7 - 6 6 5 2 3 M a l e removed day l i t t e r b o r n . M a t i n g o c c u r r e d a t p o s t - p a r t u m e s t r u s . L i t t e r N o . 2 b o r n 24 d ay s l a t e r . 1 1 - 1 0 - 6 6 5 N S a NS M a l e added day l i t t e r o b s e r v e d ; l i t t e r d e s t r o y e d . 1 2 - 2 8 - 6 6 5 1 0 M a l e added p r i o r t o w e a n i n g o f l i t t e r . 1 -23-67 5 1 3 M a l e added a f t e r w e a n i n g . 3 - 4 -67 4 1 3 M a l e added a f t e r w e a n i n g . 4 - 1 4 - 6 7 6 3 3 M a l e added a f t e r w e a n i n g . 5 - 2 3 - 6 7 b 1 C 0 0 60 2 - 2 2 - 6 7 6 3 3 3 - 2 8 - 6 7 5 2 3 5 - 8-67 6 2 4 Sperm o b s e r v e d i n v a g i n a l smear 4 - 1 8 - 6 7 ; ma l e r e m o v e d ; i n d i c a t e s 2 1 - d a y g e s t a t i o n p e r i o d . 6 - 1 7 - 6 7 2 2 Sperm o b s e r v e d i n v a g i n a l smear on 5 - 2 5 - 6 7 ; ma l e r emoved ; i n d i c a t e s 2 3 - d a y g e s t a t i o n p e r i o d . 64 1 0 - 1 3 - 6 6 5 2 2 3 - 7-67 5 3 2 295 9 - 4 -67 _4 . NS NS T o t a l s 63 20 27 T o t a l number o f a n i m a l s w h i c h s u r v i v e d w e a n i n g e q u a l s 4 7 . N o t e . P e r c e n t o f l i t t e r a n i m a l s 74 .6%. A v e r a g e l i t t e r s i z e = 6 3 / 1 3 , w h i c h s u r v i v e d t o w e a n i n g = 4 7 / 6 3 o r o r 4 . 8 4 a n i m a l s p e r l i t t e r . a .None s u r v i v e d . b N o t i n c l u d e d i n s t a t i s t i c s o f a v e r a g e l i t t e r s i z e . c N o t i n c l u d e d i n s t a t i s t i c s a s p r o b a b l y f e m a l e d e s t r u c t i o n o c c u r r e d p r i o r t o o b s e r v a t i o n . 66 TABLE 15 PREGNANCY WEIGHT AND EMBRYO COUNTS OF 20 PREGNANT F I E L D FEMALES A n i m a l Number Fema le W e i g h t U t e r i n e a Wexght C o r r e c t e d A n i m a l W e i g h t M i n u s U t e r i n e W e i g h t Embryo C o u n t 100A 3 0 . 5 0 . 1 9 3 3 0 . 2 6 88 3 0 . 5 0 . 1 7 0 3 0 . 3 6 92A 3 1 . 9 0 . 7 4 6 3 1 . 2 6 608 3 3 . 0 N D b ND 6 109 3 6 . 5 1 .920 3 4 . 6 6 500 4 1 . 0 ND ND 7 79 4 4 . 2 2 . 0 3 5 4 2 . 2 7 606 4 4 . 6 2 . 8 0 0 4 0 . 8 6 1 0 2 ° 4 6 . 4 1 .459 4 4 . 9 5 111A 4 7 . 5 0 . 2 0 5 4 7 . 3 6 120 4 9 . 1 4 . 2 1 2 4 4 . 9 6 127 5 1 . 2 0 .292 5 0 . 9 6 305 5 2 . 0 ND ND 5 304 5 3 . 0 6 . 8 9 0 4 6 . 1 5 502 5 3 . 3 ND ND 7 605 5 4 . 0 ND ND 6 600 5 7 . 7 ND ND 5 9 8 A C 6 3 . 7 1 .000 6 2 . 7 7 508 6 3 . 8 ND ND 6 501 6 6 . 0 ND ND 6 T o t a l 120 N o t e . A v e r a g e Embryo Coun t = 1 2 0 / 2 0 , o r 6 / p r e g n a n c y . I n g r ams . ND = No t d o n e . O b s e r v e d t o be l a c t a t i n g a t t i m e o f c a p t u r e . 67 f rom two t o s e v e n p e r l i t t e r . A l l o f t h e f i e l d b r e d l i t t e r s were b o r n b e t w e e n one and f i v e da ys a f t e r c a p t u r e . S i n c e none was b o r n be tween f i v e and 25 days a f t e r c a p t u r e , a b o r t i o n o r r e s o r p t i o n p r o b a b l y o c c u r r e d i n t h e m a j o r i t y o f p r e g n a n t f i e l d f e m a l e s i n t r o d u c e d t o t h e l a b o r a t o r y . V a g i n a l b l e e d i n g o c c u r r e d i n n i n e a d u l t f e m a l e s d u r i n g t h e f i r s t t h r e e weeks i n c a p t i v i t y . S i n c e new f i e l d f e m a l e s were h a n d l e d o n l y once a week, a t t h e t i m e c a g e s were c h a n g e d , v a g i n a l b l e e d i n g may have o c c u r r e d more e x t e n s i v e l y t h a n my r e c o r d s i n d i c a t e . A dead embryo was o b s e r v e d i n one cage c o n c u r r e n t w i t h t h e v a g i n a l b l e e d i n g . H o w e v e r , s e v e r a l a n i -m a l s e x h i b i t e d a w e i g h t l o s s t h a t r e m a i n e d s t a b l e f o r s e v e r a l weeks s u b -s e q u e n t t o b l e e d i n g . None o f t h e a n i m a l s was k i l l e d t o examine f o r t h e p r e s e n c e o f r e s o r b i n g e m b r y o s . S e v e n t y - f i v e p e r c e n t o f t h e l a b o r a t o r y b r e d a n i m a l s s u r v i v e d u n t i l w e a n i n g ( T a b l e 1 4 ) , y e t o n l y 52.5% o f t h e f i e l d b r e d , l a b o r a t o r y b o r n l i t t e r a n i m a l s s u r v i v e d u n t i l w e a n i n g (34 o f t h e 65 l i t t e r a n i m a l s ) . B o t h l a b o r a t o r y b r e d and f i e l d b r e d f a t a l i t i e s among l i t t e r s were e i t h e r e a t e n b y t h e f e m a l e , o r were f o u n d dead e i t h e r w i t h o r w i t h o u t i n j u r y m a r k s . B e h a v i o r D u r i n g and F o l l o w i n g P r e g n a n c y F e m a l e s were more h o s t i l e when p r e g n a n t o r l a c t a t i n g t h a n t h e y were when n o t r e p r o d u c i n g . P r e g n a n t f e m a l e s o r f e m a l e s w i t h young d i d n o t h e s i t a t e t o a t t e m p t t o b i t e when h a n d l e d o r t o make l o u d c h i r p i n g n o i s e s when d i s t u r b e d . Two l i t t e r s o f a n i m a l #10 were damaged: one t o t a l l y and one w i t h o n l y one l i t t e r a n i m a l r e m a i n i n g . . I n b o t h i n s t a n c e s m a l e s were i n t h e cage w i t h t h e l i t t e r . S u b s e q u e n t l y , m a l e s were added o n l y a f t e r a l i t t e r was r e m o v e d . F i e l d b r e d f e m a l e s g e n e r a l l y d e s t r o y e d t h e i r l i t t e r s i f t h e i r c a g e s were d i s t u r b e d by my a t t e m p t s t o t a k e c o u n t s 68 of l i t t e r s at postpartum. The Estrous Cycle During Pregnancy  and the Postpartum Period Vaginal smears of a l l pregnant animals examined demonstrated a heavy diestrous smear stage. A l l postpartum animals examined at the time of l i t t e r weaning ( f i e l d females not examined) demonstrated the stage of dies t r u s , except animals #60 and #295 which demonstrated estrous smears. Vaginal smears were taken f o r f i v e days a f t e r and i n c l u s i v e of the day of p a r t u r i t i o n on animal #295. On the day of p a r t u r i t i o n , only one v i a b l e o f f s p r i n g was observed. Smears on each of the f i v e days demonstrated the stage of estrus. Throughout t h i s period, the one l i t t e r animal remained a l i v e ; l a c t a t i o n obviously occurred throughout the period, and l a c t a t i o n anestrus d i d not occur i n t h i s animal during the period observed. Remating A f t e r Pregnancy A l l animals that reproduced i n the laboratory remated at l e a s t once during the study. Animal #295 remated on October 8, 1967, and ovarian and f a l l o p i a n tube h i s t o l o g i c a l examination showed that ovulation and f e r t i l i z a t i o n had occurred (Table 13). Seven of the twelve f i e l d bred females which had l i t t e r s i n c a p t i v i t y were mated at, or subsequent to, the time of l i t t e r weaning. No sperm were ever observed i n p e r i o d i c v a g i -na l smears of these animals, nor were any l i t t e r s born to these females subsequently. Chapter 4 DISCUSSION ONSET OF PUBESCENCE IN MICROTUS  TOWNSENDII TOWNSENDII The age range w i t h i n which v a g i n a l p e r f o r a t i o n occurred i n most of the l a b o r a t o r y females was from 30 t o 45 days of age w i t h age v a r i a t i o n of from 18 t o 66 days. There was an observed discrepancy between female weights a t the time of v a g i n a l p e r f o r a t i o n of f i e l d captured animals when compared t o l a b o r a t o r y females. Females i n the f i e l d became p e r f o r a t e a t an e a r l i e r age than those born and housed i n the l a b o r a t o r y . Nine of f o u r -teen observed f i e l d animals, or 64% of the f i e l d animals observed, were p e r f o r a t e w i t h i n the weight range of 15 to 2 4 . 9 grams. However, i n the l a b o r a t o r y only four o f twenty, or 20%, of the females were p e r f o r a t e i n t h i s weight range, although i n d i v i d u a l s i n both groups demonstrated a wide range of weight at p e r f o r a t i o n . F i e l d animals a l s o demonstrated f i r s t e s t r u s , based on v a g i n a l smear, c l o s e r t o the time of v a g i n a l p e r f o r a t i o n than l a b o r a t o r y animals. S i x of the seven f i e l d females i n the .15 to 2 4 . 9 gram weight range were both p e r f o r a t e and had estrous v a g i n a l smears. The remaining female had a v a g i n a l smear t y p i c a l of p r o e s t r u s . In 11 l a b o r a t o r y females on which d a i l y v a g i n a l smears were obtained subsequent t o p e r f o r a t i o n , none demon-s t r a t e d an estrous smear w i t h i n t h i s weight range. There was a wide v a r i a t i o n of occurrence day of e s t r u s w i t h i n the sample of animals s t u d i e d . The minimum age v a g i n a l p e r f o r a t i o n was observed i n t h i s species i s c o n s i s t e n t w i t h data recorded f o r s e v e r a l other m i c r o t i n e s . 69 70 P e r f o r a t i o n has been o b s e r v e d i n M i c r o t u s a g r e s t i s f e m a l e s i n t h e l a b o r a -t o r y as e a r l y as 19 da ys o f age (Ranson , 1 9 3 4 ) . One M i c r o t u s c a l i f o r n i c u s f e m a l e was o b s e r v e d i n t h e f i e l d t o be p e r f o r a t e , have d e v e l o p i n g c o r p o r a l u t e a , sperm i n t h e u t e r u s and t w o - c e l l e d t u b a l o v a l i n t h e f a l l o p i a n t u b e s a t an age e q u i v a l e n t o f 15 d a y s ( G r e e n w a l d , 1 9 5 7 ) . G r e e n w a l d s t a t e s t h a t t h e d i s p l a y o f s e x u a l p r e c o c i t y e x h i b i t e d by M i c r o t u s i s u n e q u a l e d among mammals. N e i t h e r v a g i n a l p e r f o r a t i o n n o r f i r s t e s t r u s v i a v a g i n a l smear p r o v i d e s i n f o r m a t i o n r e l a t i v e t o t h e more i m p o r t a n t r e p r o d u c t i v e phenomenon s i g n i f i e d b y t h e end o f p u b e s c e n c e ; t h a t i s , t h e a b i l i t y t o m a i n t a i n a v i a b l e p r e g n a n c y . One f i e l d f e m a l e w i t h a w e i g h t e q u i v a l e n t t o a 20 day o l d l a b o r a t o r y f e m a l e was o b s e r v e d t o have o v u l a t e d , ma ted and had f i v e d e v e l o p i n g c o r p o r a l u t e a and t w o - c e l l e d t u b a l o v a . H o w e v e r , t h e s e d a t a do n o t n e c e s s a r i l y mean t h a t t h e a n i m a l w o u l d be c a p a b l e o f m a i n t e n a n c e o f a v i a b l e p r e g n a n c y , and i n d e e d , a s G r e e n w a l d (1957) o b s e r v e d i n young M i c r o t u s c a l i f o r n i c u s , t h e p r e g n a n c y may t e r m i n a t e due t o f a i l u r e o f i m p l a n t a t i o n and a s t e r i l e b r e e d i n g c y c l e o c c u r . Inasmuch as no c o r p o r a a l b i c a n t i a were o b s e r v e d i n young f i e l d f e m a l e s d e t e r m i n e d by w e i g h t t o be a t o r b e l o w t h e age o f p u b e s c e n c e , t h e r e i s no d i r e c t e v i d e n c e t o s u p -p o r t t h e o c c u r r e n c e o f i m p l a n t a t i o n f a i l u r e o r s t e r i l e b r e e d i n g c y c l e s i n M i c r o t u s t o w n s e n d i i . The y o u n g e s t p r e g n a n t f i e l d f e m a l e h a d a w e i g h t o f 3 0 . 2 grams o r l a b o r a t o r y age e q u i v a l e n t o f 25 d a y s . T h e r e a p p e a r s t o be a w e i g h t gap be tween t h e t i m e mos t f i e l d a n i m a l s became p e r f o r a t e and d e m o n s t r a t e d e s t r o u s smears and t h e t i m e v i s i b l e embryo c o u n t s were p o s s i -b l e . T h i s t i m e p e r i o d i s i n t h e o r d e r o f f r o m 5 t o 15 d a y s . C o n s i d e r a -t i o n o f f e r t i l i z a t i o n - i m p l a n t a t i o n t i m e and t i m e n e c e s s a r y f o r embryo d e v e l o p m e n t t o a t . l e a s t m i n i m a l v i s i b l e s t a g e d i m i n i s h e s t h e s i g n i f i c a n c e 71 o f t h i s t i m e i n t e r v a l . On t h i s b a s i s , t h e e x i s t e n c e o f s t e r i l e b r e e d i n g c y c l e s i s n o t i m p l i e d i n M i c r o t u s t o w n s e n d i i o n t h e b a s i s o f t h i s s t u d y . O b s e r v e d d i f f e r e n c e s be tween t i m e o f s e x u a l m a t u r i t y o f l a b o r a t o r y v e r s u s f i e l d f e m a l e s a r e p r o b a b l y r e l a t e d t o t h e d r a s t i c a l l y d i f f e r e n t e n v i r o n m e n t a l e x p o s u r e s o f t h e two p o p u l a t i o n s . G r e e n w a l d (1957) r e p o r t s t h a t even w i t h i n t h e same f i e l d p o p u l a t i o n o f M i c r o t u s c a l i f o r n i c u s i n two s u c c e s s i v e y e a r s , t h e age a t w h i c h mos t a n i m a l s r e a c h e d s e x u a l m a t u r i t y v a r i e d f rom an a v e r a g e e q u i v a l e n t o f 41 d a y s i n 1952 t o 29 days i n 1953 a n i m a l s c a p t u r e d i n t h e same s t u d y a r e a and t h e same s e a s o n . LeDuc and K r e b s (1975) r e p o r t a mean w e i g h t v a r i a t i o n a t p u b e r t y , i n t h e same p o p u l a -t i o n o f M . t o w n s e n d i i , o f 35 grams ( S p r i n g 1972) t o 71 grams ( W i n t e r 1 9 7 2 - 1 9 7 3 ) . W i t h i n t h e l a b o r a t o r y , a n i m a l h o u s i n g r e m a i n e d c o n s t a n t w i t h m i n i -m a l v a r i a t i o n s . F e m a l e s r e m a i n e d i s o l a t e d b o t h f r o m m a l e s and f r o m e a c h o t h e r , a l t h o u g h o n t h e s i d e o f t h e room w i t h o t h e r f e m a l e s . I f t h e l a b o -r a t o r y e n v i r o n m e n t had been d e s i g n e d t o enhance r e p r o d u c t i v e a c t i v i t y , i t i s p r o b a b l e t h a t t h e o c c u r r e n c e o f v a g i n a l p e r f o r a t i o n and f i r s t e s t r u s w o u l d have been b o t h e a r l i e r and more n e a r l y s i m u l t a n e o u s . The f o l l o w i n g f a c t o r s a r e known t o enhance r e p r o d u c t i v e a c t i v i t y : s e n s o r y s t i m u l i m o d i f i c a t i o n (Richmond and Conaway, 1 9 6 9 ) ; p r e s e n c e o f m a l e s , e s p e c i a l l y s t r a n g e m a l e s (Richmond and Conaway, 1969 ; H a s l e r and N a l b a n d o v , 1 9 7 4 ) ; h o u s i n g i n m i x e d m a l e , f e m a l e g r o u p s ( P a s l e y and M c K i n n e y , 1 9 7 3 ) . A b r i e f r e v i e w o f s e v e r a l l a b o r a t o r y s t u d i e s c o n c e r n e d w i t h . t h e e f f e c t o f t h e s e v a r i a b l e s f o l l o w s . R ichmond and Conaway (1969) d e m o n s t r a t e d t h a t f r e q u e n t m o d i f i c a -t i o n s o f h o u s i n g o f M i c r o t u s o c h r o g a s t e r l a b o r a t o r y f e m a l e s w o u l d s t i m u ^ l a t e some d e g r e e o f i n c r e a s e d r e p r o d u c t i v e a c t i v i t y r a n g i n g f r o m p e r f o r a t i o n 72 t o e s t r o u s v a g i n a l smear and i n c r e a s e d r e c e p t i v i t y t o m a l e s . These h o u s i n g m o d i f i c a t i o n s were a r r a n g e d so as t o p r o v i d e f o r e i t h e r d i r e c t o r i n d i r e c t s e n s o r y s t i m u l i be tween f e m a l e s and o t h e r f e m a l e s o r m a l e s . The mos t p r o f o u n d s t i m u l u s t o i n c r e a s e d r e p r o d u c t i v e a c t i v i t y was t h e p r e s e n c e o f m a l e s . They a l s o o b s e r v e d f e m a l e s became a n e s t r o u s and i m p e r f o r a t e when h o u s e d i n i s o l a t i o n w i t h o u t t h e s e n s o r y s t i m u l i o f o t h e r a n i m a l s o r w i t h o u t m o d i f i c a t i o n o f e n v i r o n m e n t a l s t i m u l i . W h i l e t h e p r e s e n c e o f m a l e s h a s an e n h a n c i n g e f f e c t upon t h e ; a t t a i n m e n t o f s e x u a l m a t u r i t y , a l l m a l e s do n o t have e q u a l e f f e c t . H a s l e r and N a l b a n d o v (1974) o b s e r v e d a s i g n i f i c a n t s t a t i s t i c a l d i f f e r e n c e i n t h e age o f v a g i n a l p e r f o r a t i o n i n M . o c h r o g a s t e r among t h r e e g r o u p s o f f e m a l e w e a n l i n g s : e a r l i e s t when p a i r e d w i t h a d u l t m a l e s ; i n t e r m e d i a t e when p a i r e d w i t h n o n - l i t t e r w e a n l i n g m a l e s ; l a t e s t when p a i r e d w i t h l i t t e r -mate m a l e s . I n a d d i t i o n , t h e d e l a y be tween f i r s t p e r f o r a t i o n and f i r s t l i t t e r was s i g n i f i c a n t l y l o n g e r f o r t h e g r o u p p a i r e d w i t h l i t t e r m a t e m a l e s t h a n f o r t h e o t h e r two g r o u p s . P a s l e y and M c K i n n e y (1973) have d e m o n s t r a t e d t h a t s t i m u l i a s s o c i -a t e d w i t h a l l f e m a l e g r o u p s f r o m w e a n l i n g t o s e x u a l m a t u r i t y a p p e a r s t o r e t a r d o v u l a t i o n i n d u c t i o n and t h e d e v e l o p m e n t o f r e p r o d u c t i v e o r g a n s i n M i c r o t u s p e n n s y l v a n i c u s u n d e r l a b o r a t o r y c o n d i t i o n s ; n a m e l y , e a r l i e r age a t v a g i n a l p e r f o r a t i o n and a p p a r e n t c l o s e p r o x i m i t y o f f i r s t e s t r u s t o t h e t i m e o f v a g i n a l p e r f o r a t i o n . I t i s e v i d e n t t h a t e n v i r o n m e n t h a s a s i g n i f i c a n t e f f e c t upon t h e s t a t u s o f r e p r o d u c t i v e m a t u r i t y o f M i c r o t u s . These l a b o r a t o r y s t u d i e s p r o v i d e i n f o r m a t i o n w h i c h may e x p l a i n my o b s e r v e d d i f f e r e n c e s be tween young f i e l d f e m a l e s c a p t u r e d w i t h i n t h e w e i g h t r a n g e where s e x u a l m a t u r i t y w o u l d be f i r s t e v i d e n c e d and t h e g r o u p o f i s o l a t e d l a b o r a t o r y f e m a l e s . 73 T h i s s t a t e m e n t i s b a s e d o n t h e a s s u m p t i o n t h a t w e a n l i n g s i n n a t u r e a r e e x p o s e d t o s t r a n g e m a l e s , n o t c o n f i n e d t o a l l f e m a l e g r o u p s , and s u b j e c t t o d a i l y v a r i a t i o n i n e n v i r o n m e n t a l s t i m u l i . H o w e v e r , i n a s m u c h as e c o l o g i -c a l s t u d i e s have d e m o n s t r a t e d t h a t t h e age o f s e x u a l m a t u r i t y i n w i l d M i c r o t u s i s v a r i a b l e and d e p e n d e n t on s e v e r a l e n v i r o n m e n t a l b a s e s ( K r e b s and M y e r s , 1 9 7 4 ) , i s o l a t e d f i e l d s t a t i s t i c s have l i t t l e s i g n i f i c a n c e . K e l l e r and K r e b s (1970) o b s e r v e d t h a t t h e m e d i a n w e i g h t a t s e x u a l m a t u r i t y o f M . p e n n s y l v a n i c u s and M . o c h r o g a s t e r was h i g h e r a t p o p u l a t i o n p e a k t h a n i n t h e o t h e r p h a s e s , b u t e q u a l i n t h e i n c r e a s e and d e c l i n i n g p h a s e s . T h i s i n d i c a t e s an i n c r e a s e d age a t s e x u a l m a t u r i t y i n a s s o c i a t i o n w i t h i n c r e a s e d p o p u l a t i o n d e n s i t y . However , b a s e d on l a b o r a t o r y e x p e r i -ments o f f a c t o r s a f f e c t i n g s e x u a l m a t u r i t y , i t w o u l d a p p e a r t h a t i n c r e a s e d c o n t a c t w i t h m a l e s and g r e a t e r e n v i r o n m e n t a l s t i m u l a t i o n w o u l d o c c u r w i t h i n c r e a s e d d e n s i t y w h i c h w o u l d r e s u l t i n an e a r l i e r age a t s e x u a l m a t u r i t y . P o s s i b l y , i n h i g h p o p u l a t i o n d e n s i t y , s t r e s s r e l a t e d mechanisms s u p e r c e d e r e p r o d u c t i v e m e c h a n i s m s . Howeve r , two s e a s o n a l l y d e p e n d e n t e x t r i n s i c f a c t o r s s t u d i e d i n t h e l a b o r a t o r y do c o r r e l a t e w e l l w i t h t h e o b s e r v a t i o n s o f LeDuc and K r e b s ( 1 9 7 5 ) . On t h e i r c o n t r o l g r i d t h e y o b s e r v e d a two y e a r s e a s o n a l p a t t e r n o f w e i g h t a t t h e age o f s e x u a l m a t u r i t y i n M . t o w n s e n d i i o f l o w e s t S p r i n g w e i g h t s , i n t e r m e d i a t e Summer w e i g h t s and h i g h e s t W i n t e r w e i g h t s . P h o t o -p e r i o d has been d e m o n s t r a t e d b o t h i n t h e l a b o r a t o r y and i n a w i l d p o p u l a -t i o n t o a f f e c t t h e r e p r o d u c t i v e s t a t u s o f M i c r o t u s . The c r i t i c a l p o s i -t i v e e f f e c t a p p e a r s t o o c c u r a t 14 h o u r s o f l i g h t ; w i t h l e s s l i g h t t h e r e i s a s i g n i f i c a n t d e c r e a s e i n r e p r o d u c t i v e t r a c t a c t i v i t y ( B r e e d and C l a r k e , 1970a; B r e e d , 1 9 7 2 ) . The o t h e r s e a s o n a l l y dependen t v a r i a b l e w h i c h has been d e m o n s t r a t e d t o enhance r e p r o d u c t i v e t r a c t a c t i v i t y i s 74 c o n s u m p t i o n o f f r e s h g r e e n f o l i a g e , p a r t i c u l a r l y y o u n g , b y f e m a l e s ( P i n t e r and N e g u s , 1965 ; B e r g e r and N e g u s , 1974; B e r g e r e t a l . , 1 9 7 7 ) . Inasmuch as t h i s p a r a m e t e r i s o f g r e a t i n t e r e s t t o b o t h e c o l o g i s t s and r e p r o d u c t i v e p h y s i o l o g i s t s , and i s c o n s i d e r e d t o be a ma j o r d r i v i n g f o r c e b e h i n d p o p u l a t i o n c y c l e s ( K r e b s and M y e r s , 1 9 7 4 ) , f u r t h e r r e s e a r c h i s needed t o c l a r i f y f a c t o r s a f f e c t i n g t h e age o f s e x u a l m a t u r i t y . E s p e -c i a l l y i n d i c a t e d a r e s t u d i e s w h i c h w o u l d t e s t t h e a p p l i c a b i l i t y o f l a b o r a -t o r y s t u d y r e s u l t s i n w i l d p o p u l a t i o n s . NATURE OF THE ESTROUS CYCLE I N MICROTUS TOWNSENDII TOWNSENDII E s t r o u s C y c l e as D e t e r m i n e d by  V a g i n a l Smears E a r l y on i n t h i s s t u d y , v a g i n a l smears d e m o n s t r a t e d a p a t t e r n n o t t y p i c a l o f w e l l s t u d i e d s p o n t a n e o u s o v u l a t o r s s u c h as R a t t u s n o r v e g i c u s (Long and E v a n s , 1922) and Mus m u s c u l u s ( A l l e n , 1 9 2 2 ) . E s t r o u s s t a g e s d i d n o t f o l l o w t h e r e p e t i t i v e s e q u e n t i a l p a t t e r n o f p r o e s t r u s , e s t r u s , m e t e s -t r u s and d i e s t r u s e x h i b i t e d by R a t t u s and M u s , a l t h o u g h e a c h o f t h e s e s t a g e s was o b s e r v e d i n some f e m a l e s . Of t h e 466 s t u d y smear d a y s , a n i m a l s r e m a i n e d i n t h e s t a g e o f e s t r u s 57% o f t h e t i m e . The r e m a i n d e r o f t h e t i m e p r i m a r i l y d i e s t r o u s smears were o b s e r v e d . Smears c l a s s i f i e d as d i e s t r u s were o b s e r v e d 35% o f t h e d a y s s t u d i e d and t h o s e o f p r o e s t r u s o r m e t e s t r u s o n l y 8% o f t h e d a y s . C y c l i c phenomena were o b s e r v e d t o o c c u r i n M i c r o t u s t o w n s e n d i i i f a " c y c l e " i s d e f i n e d as day one o f c o r n i f i e d e s t r o u s v a g i n a l smear t o t h e s u b s e q u e n t day one o f a c o r n i f i e d smear , as f i r s t s u g g e s t e d by B r e e d ( 1 9 6 7 ) . M i c r o t u s t o w n s e n d i i d e m o n s t r a t e d a 2 - 7 . 5 day c y c l i c p e r i o d i c i t y . Howeve r , w i d e i n d i v i d u a l v a r i a t i o n was e v i d e n t among t h e 12 a n i m a l s o b s e r v e d . S t i l l , t h e s e d a t a do s u g g e s t a 75 c y c l i c n a t u r e t o t h e o c c u r r e n c e o f e s t r u s i n t h i s s p e c i e s . The o b s e r v e d e s t r o u s c y c l e p a t t e r n , o r l a c k o f p a t t e r n , i s t y p i c a l o f t h a t d e s c r i b e d f o r s e v e r a l known M i c r o t u s i n d u c e d o v u l a t o r s . E v i d e n c e o f i r r e g u l a r c y c l i c v a g i n a l smear changes and p r i m a r y p r e s e n c e o f smear s t a g e s o f e s t r u s and d i e s t r u s i n unmated f e m a l e s h a s b e e n o b s e r v e d i n M . a g r e s t i s ( B r e e d , 1 9 6 7 ) , M . o r c h r o g a s t e r (Richmond and Conaway, 1 9 6 9 ) , M . p i n e t o r u m ( K i r k p a t r i c k and V a l e n t i n e , 1 9 7 0 ) , M . p e n n s y l v a n i c u s ( C l u l o w and M a l l o r y , 1 9 7 0 ) , M . montanus (Gray e t a l . , 1 9 7 4 a ) . W i t h i n e a c h l a b o r a -t o r y c o l o n y t h e r e a p p e a r s -to be w i d e i n d i v i d u a l v a r i a t i o n w i t h some a n i -m a l s d e m o n s t r a t i n g l o n g p e r i o d s o f p r i m a r i l y e s t r o u s s m e a r s , o t h e r s p r i m a r i l y d i e s t r o u s smears and o t h e r s i r r e g u l a r i n t e r v a l s o f t h e s e two s m e a r s . I n t h i s s t u d y , t h e same t y p e s o f i n d i v i d u a l and g r o u p v a r i a t i o n among f e m a l e s was o b s e r v e d , w h i c h s u g g e s t s t h e p o s s i b i l i t y t h a t M . t o w n -s e n d i i i s a l s o an i n d u c e d o v u l a t o r on t h e b a s i s o f c o m p a r a t i v e v a g i n a l smear s t u d y . S e q u e n t i a l c y c l i c v a g i n a l s m e a r s , s u c h a s have been r e p o r t e d f o r Mus and R a t t u s , have been r e p o r t e d f o r M i c r o t u s by C h i t t y and A u s t i n (1957) and R i c h m o n d and Conaway ( 1 9 6 9 ) . The l a t t e r a u t h o r s o b s e r v e d c y c l i c p a t t e r n s i n o n l y two o f t h e f e m a l e s s t u d i e d . B r e e d (1967) r e p e a t e d smear s t u d i e s on t h e c o l o n y o f M i c r o t u s a g r e s t i s u t i l i z e d by C h i t t y and A u s t i n and o b s e r v e d no r e g u l a r v a g i n a l c y c l e s . B r e e d (1967) r e c o r d e d a 2 -5 day p e r i o d i c i t y f r o m f i r s t d a y o f e s t r u s t o s u b s e q u e n t n e x t f i r s t day o f e s t r u s . G r a y e t a l . (1974a) a l s o r e c o r d e d modes o f e s t r u s p e r i o d i c i t y w h i c h v a r i e d b a s e d on t h e e x p e r i m e n t a l c o n d i t i o n i n w h i c h M . montanus was p l a c e d . T h e i r i s o l a t e d v i r g i n f e m a l e s , t h e c o n d i t i o n o f f e m a l e s i n t h i s s t u d y , d e m o n s t r a t e d a m o d a l e s t r u s p e r i o d i c i t y o f 5 d a y s . T h e r e a r e , t h e n , p u b l i s h e d d a t a w h i c h s u p p o r t my o b s e r v a t i o n o f t h e c y c l i c n a t u r e o f 76 v a g i n a l smear changes i n M i c r o t u s . The e v i d e n c e d " c y c l e s " d i f f e r f r o m t h o s e o f known s p o n t a n e o u s o v u l a t o r s i n t h a t component measurements a r e o n l y c o n c e r n e d w i t h t h e o c c u r r e n c e o f e s t r u s p e r i o d i c i t y . The phenomenon o f e s t r u s p e r i o d i c i t y has b e e n o b s e r v e d t o o c c u r i n t h e r a b b i t , a known i n d u c e d o v u l a t o r ( H a m i l t o n , 1951) . H a m i l t o n o b s e r v e d a c y c l i c e s t r u s p e r i o d i c i t y o f 2 t o 4 d a y s i n t h e s e a n i m a l s . She o b s e r v e d v a g i n a l smear v a r i a t i o n r e l a t i v e t o t h e p e r c e n t a g e o f n u c l e -a t e d e p i t h e l i a l c e l l s compared t o t h e numbers o f c o r n i f i e d c e l l s p r e s e n t and o b s e r v e d , v i a n o n q u a n t i t a t i v e b i o a s s a y o f e s t r o g e n t i t e r , t h a t t h e r e was a h i g h e r b l o o d e s t r o g e n l e v e l a t t h e t i m e t h e v a g i n a l smear c o n t a i n e d t h e g r e a t e s t number o f c o r n i f i e d c e l l s . I t i s p r o b a b l e t h a t t h e smear s t a g e o f e s t r u s i n M i c r o t u s i s a l s o r e f l e c t i v e o f e l e v a t e d e s t r o g e n l e v e l . Howeve r , e s t r o g e n l e v e l i n s u f f i c i e n t t o evoke v a g i n a l c o r n i f i c a t i o n , y e t a d e q u a t e l y h i g h t o m a i n t a i n v a g i n a l p e r f o r a t i o n , i s r e f l e c t e d i n M i c r o t u s n o t b y n u c l e a t e d smear , b u t r a t h e r m a i n l y b y t h e m u c i f i c a t i o n and l e u k o -c y t o s i s t y p i c a l o f t h e d i e s t r o u s smear . M e y e r and A l l e n (1933) d e m o n s t r a t e d t h a t changes i n d u c e d b y v a g i n a l m u c i f i c a t i o n v e r s u s c o r n i f i -c a t i o n and r e l a t e d t o t h e e s t r o g e n l e v e l o f o v a r i e c t o m i z e d m i c e and g u i n e a p i g s g i v e n exogenous e s t r o g e n ; l o w d o s e s a r e r e f l e c t e d by m u c i f i c a t i o n and h i g h e r d o s e s r e f l e c t e d by c o r n i f i c a t i o n . I t i s p r o b a b l e t h a t endogenous e s t r o g e n l e v e l s a r e a l s o r e f l e c t e d p r i m a r i l y b y v a g i n a l m u c i f i e d d i e s t r o u s smears i n M i c r o t u s . P r o e s t r o u s and m e t e s t r o u s smears p r o b a b l y r e f l e c t e s t r o g e n l e v e l s h i g h e r t h a n t h o s e r e f l e c t e d by t h e d i e s t r o u s smear s t a g e b u t a r e s u b c o r n i f y i n g i n e f f e c t . C o r r e l a t i o n o f E s t r o u s C y c l e Smear  S t a g e w i t h R e p r o d u c t i v e T r a c t  H i s t o l o g i c a l E x a m i n a t i o n H i s t o l o g i c a l s t u d y o f t h e r e p r o d u c t i v e t r a c t o f f e m a l e s k i l l e d i n 77 e a c h o f t h e s t a g e s o f t h e e s t r o u s c y c l e a s d e t e r m i n e d by v a g i n a l smear o n l y y i e l d e d i n f o r m a t i o n r e l a t i v e t o t h e f o l l i c u l a r p h a s e o f t h e c y c l e . No o v u l a t i o n , c o r p o r a l u t e a o r c o r p o r a a l b i c a n t i a were o b s e r v e d i n any o f t h e a n i m a l s s t u d i e d . These d a t a c o r r e l a t e w i t h v a g i n a l smear o b s e r v a t i o n s t o p r o v i d e s u g g e s t i v e e v i d e n c e t h a t M . t o w n s e n d i i does n o t o v u l a t e s p o n t a -n e o u s l y , a t l e a s t u n d e r t h e e x p e r i m e n t a l c o n d i t i o n s o f t h i s s t u d y . I n o r d e r t o a s c e r t a i n t h e r e l a t i o n s h i p be tween v a g i n a l changes and o v a r i a n a c t i v i t y and t o compare v a g i n a l changes w i t h changes e v i d e n t i n t h e f a l l o p i a n t u b e s and u t e r i , I p r e p a r e d b o t h d e s c r i p t i v e and s t a t i s t i -c a l a n a l y s i s o f h i s t o l o g i c a l d a t a . The r a t i o n a l e f o r t h i s a s p e c t o f t h e s t u d y was b a s e d o n t h e a s s u m p t i o n t h a t i f t h i s a n i m a l i s an i n d u c e d o v u l a t o r , h i s t o l o g i c a l c o m p a r i s o n w o u l d d e m o n s t r a t e : o v a r i a n a c t i v i t y t y p i c a l o f t h a t o f o t h e r i n d u c e d o v u l a t o r s , • . d e s c r i p t i v e and s t a t i s t i c a l i n f o r m a t i o n w h i c h w o u l d d e m o n s t r a t e d i f f e r e n c e s among t h e a n e s t r o u s , d i e s -t r o u s , m e t e s t r o u s and e s t r o u s o v a r i e s and r e p r o d u c t i v e t r a c t s w h i c h w o u l d r e f l e c t p r o g r e s s i v e l y i n c r e a s i n g e s t r o g e n p r o d u c t i o n and e f f e c t i n t h e o r d e r l i s t e d ( l o w e s t i n a n e s t r u s t o h i g h e s t i n e s t r u s ) . To my k n o w l e d g e , t h i s i s t h e f i r s t h i s t o l o g i c a l s t u d y o f t h i s n a t u r e t o be r e p o r t e d f o r M i c r o t u s . S t a t i s t i c a l v a r i a t i o n o f o v a r i a n d a t a among t h e t h r e e g r o u p s f o r w h i c h s u f f i c i e n t a n i m a l numbers were a v a i l a b l e , t h a t i s , a n e s t r o u s , d i e s -t r o u s and e s t r o u s o v a r i e s , d e m o n s t r a t e d t h a t a l l a n i m a l s a p p e a r e d t o have t h e same b a s e l i n e o f r e p r o d u c t i v e a c t i v i t y . No d i f f e r e n c e was o b s e r v e d among t h e t h r e e g r o u p s o f f e m a l e s w i t h r e f e r e n c e t o t h e mean numbers o f d e v e l o p i n g a n t r a l f o l l i c l e s l a r g e r t h a n 250 m i c r a o r t h e mean number o f a t r e t i c f o l l i c l e s l a r g e r t h a n 250 m i c r a . A p p a r e n t l y , n e i t h e r d i f f e r i n g numbers o f d e v e l o p i n g f o l l i c l e s n o r a t r e t i c f o l l i c l e s was r e s p o n s i b l e f o r 78 o b s e r v e d v a g i n a l d i f f e r e n c e s among t h e t h r e e g r o u p s . Numbers o f a t r e t i c f o l l i c l e s and l a r g e G r a a f i a n f o l l i c l e s have been r e p o r t e d t o o c c u r i n t h e unmated e s t r o u s r a b b i t ( H a m i l t o n , 1 9 5 1 ) , t h e e s t r o u s v o l e , M . a g r e s t i s ( B r e e d , 1969) , and i n t h e e s t r o u s 1 3-rlined g r o u n d s q u i r r e l ( C i t e l l u s  T r i d e c e m l i n e a t u s ) ( F o s t e r , 1934) , a l l i n d u c e d o v u l a t o r s . F o s t e r a l s o o b s e r v e d i n t e r m e d i a t e d e v e l o p i n g f o l l i c l e s a s w e l l a s a t r e t i c f o l l i c l e s i n a n e s t r o u s g r o u n d s q u i r r e l s t a k e n f r o m t h e f i e l d i n a n e s t r o u s c o n d i t i o n . However , c o m p a r a b l e w i t h my o b s e r v a t i o n s i n M . t o w n s e n d i i , he o b s e r v e d e s t r o u s f e m a l e s t o have t h e l a r g e s t p r e o v u l a t o r y f o l l i c l e s . I n t h i s s t u d y , e s t r o u s f e m a l e s d e m o n s t r a t e d a s i g n i f i c a n t l y h i g h e r mean number o f f o l l i c l e s g r e a t e r t h a n 350 m i c r a , and a s i g n i f i c a n t l y l a r g e r mean maximum o v a r i a n f o l l i c l e d i a m e t e r and mean o v a r i a n d i a m e t e r t h a n a n i -m a l s i n t h e o t h e r two g r o u p s . My o b s e r v a t i o n o f l a r g e s t p r e o v u l a t o r y o v a r i a n d i a m e t e r i n e s t r o u s f e m a l e s has a l s o been d i r e c t l y o b s e r v e d i n R a t t u s (Long and E v a n s , 1922) and Mus ( A l l e n , 1922) and i n d i r e c t l y i n t h e 13 l i n e d g r o u n d s q u i r r e l by a. s t a t e d t r i p l e i n c r e a s e o f e s t r o u s o v a r i a n w e i g h t compared w i t h t h e a n e s t r o u s f e m a l e ( F o s t e r , 1 9 3 4 ) . F a l l o p i a n t u b e e p i t h e l i a l h e i g h t was a l s o s i g n i f i c a n t l y h i g h e r f o r e s t r o u s f e m a l e s t h a n f o r t h e o t h e r two g r o u p s . D e s c r i p t i v e h i s t o l o g i c a l e x a m i n a t i o n i n d i c a t e d i n c r e a s e d a c t i v i t y o f t h e u t e r i n e h o r n and v a g i n a s i m i l a r t o t h a t d e s c r i b e d f o r known e s t r o u s s p o n t a n e o u s o v u l a t o r s . They were s i m i l a r t o t h e changes i n t h e r e p r o d u c t i v e t r a c t o f a n e s t r o u s 1 3 - l i n e d g r o u n d s q u i r r e l s w h i c h o c c u r r e d upon a d m i n i s t r a t i o n o f exogenous e s t r o g e n b y F o s t e r ( 1 9 3 4 ) . B r e e d (1972) o b s e r v e d s t a t i s t i c a l l y d i f f e r e n t o v a r i a n and r e p r o -d u c t i v e t r a c t r e s p o n s e i n w i l d f e m a l e M . a g r e s t i s e x p o s e d t o F a l l p h o t o -p e r i o d compared t o t h o s e e x p o s e d t o Summer p h o t o p e r i o d . The F a l l g r o u p d e m o n s t r a t e d l i g h t e r o v a r i a n and u t e r i n e w e i g h t s , a s m a l l e r mean number 79 o f G r a a f i a n f o l l i c l e s and a s m a l l e r mean maximum f o l l i c l e s i z e . S e v e r a l members o f t h i s g r o u p had c l o s e d v i g i n a e f and he s t a t e s g r o u p members a p p e a r e d t o be g o i n g i n t o a n e s t r u s . The o t h e r g r o u p d e m o n s t r a t e d s i g n i f i -c a n t l y l a r g e r v a l u e s f o r a l l measurements d e s c r i b e d , had p e r f o r a t e v a g i n a e w i t h c o r n i f i e d w a l l s , t y p i c a l o f e s t r u s . B r e e d ' s work p r o v i d e s r e s u l t s s i m i l a r t o mine o f o b s e r v e d d i f f e r e n c e s be tween e s t r o u s and o t h e r g r o u p e d f e m a l e s . The s t a g e o f e s t r u s i n M . t o w n s e n d i i a p p e a r s t o r e f l e c t maximum p r e o v u l a t o r y o v a r i a n a c t i v i t y . T h i s i s c o n s i s t e n t w i t h t h e d a t a o f e s t r u s phenomena p r e s e n t e d i n a l l mammalian e s t r o u s c y c l e s r e s e a r c h e d i n t h e l i t e r a t u r e . I n t h e l i t e r a t u r e , no d e s c r i p t i v e h i s t o l o g i c a l d a t a were l o c a t e d w h i c h d e s c r i b e t h e s t a g e o f d i e s t r u s i n M i c r o t u s . Inasmuch as t h i s s t a g e does n o t r e p r e s e n t t h e o v a r i a n l u t e a l s t a g e o f d i e s t r u s i n a s p o n t a n e o u s o v u l a t o r , h i s t o l o g i c a l d a t a o n d i e s t r u s i n t h e s e mammals c a n n o t be u t i l i z e d . B r e e d (1967) s t a t e s l a r g e G r a a f i a n and a t r e t i c f o l l i c l e s were common i n unmated r e p r o d u c t i v e l y a c t i v e M . a g r e s t i s e x h i b i t i n g v a r i e d smear t y p e s . I n M . t o w n s e n d i i , t h e s t a g e o f d i e s t r u s was s i g n i f i c a n t l y d i f f e r e n t f rom t h e s t a g e o f e s t r u s o n a l l measu red o v a r i a n p a r a m e t e r s e x c e p t mean numbers o f d e v e l o p i n g and a t r e t i c f o l l i c l e s l a r g e r t h a n 250 m i c r a . However , t h i s g roup d i f f e r e d s i g n i f i c a n t l y f r o m a n e s t r o u s f e m a l e s on o n l y one measured p a r a m e t e r , t h a t o f mean maximum o v a r i a n d i a m e t e r . Inasmuch a s t h e s e two g r o u p s d e m o n s t r a t e p r i m a r i l y s i m i l a r o v a r i a n h i s t o l o g y , t h e q u e s t i o n i s p o s e d as t o what m i g h t a c c o u n t f o r t h e d i f f e r e n c e i n mean maximum o v a r i a n d i a m e t e r be tween t h e two g r o u p s . On t h e b a s i s o f d e s c r i p t i v e h i s t o l o g i c a l d a t a , t h e r e a p p e a r t o be two p o s s i b l e e x p l a n a t i o n s f o r t h e d i f f e r e n c e . F i r s t , s e v e n o f t h e n i n e 80 d i e s t r o u s females studied and a l l o f the estrous females demonstrate an a c t i v e i n t e r s t i t i a l stroma which i s w e l l organized i n t o cords, w e l l vascu-l a r i z e d and apparently secretory on the b a s i s of the presence of w e l l developed c e l l s containing much cytoplasm. The anestrous o v a r i e s , and two of the nine d i e s t r o u s ovaries had i n t e r s t i t i a l stroma which d i d not appear to be secretory, had poor v a s c u l a r i z a t i o n and contained t i g h t l y packed acytoplasmic c e l l s . Inasmuch as t h i s t i s s u e , i n r a b b i t s , has been demon-s t r a t e d to be s t e r o l s y n t h e s i z i n g under the i n f l u e n c e of gonadotrophic hormones (Oxender, M i l l e r , and Hafs, 1971) and may produce estrogen but c o n c l u s i v e l y produces progesterone and androgens (Guraya, 1973), i t i s probable that hormone production by t h i s t i s s u e i s at l e a s t p a r t i a l l y responsible f o r p r o l i f e r a t i o n of the v a g i n a l mucosa i n the d i e s t r o u s females. Observation of evident i n t e r f o l l i c u l a r stroma i n M. c a l i f o r n i c u s has been reported by Greenwald (1956). However, he s t a t e s that t h i s stroma i s most obvious i n the anestrous and proestrous ovary and t h a t i t s c e l l s are never secretory i n appearance. Hammond and Marshall (1930) s t a t e t h a t i t i s s i g n i f i c a n t t h a t i n the r a b b i t , the f e r r e t and the c a t , a l l induced ovulators, there i s a large amount of i n t e r s t i t i a l t i s s u e . To date, the r e l a t i o n s h i p o f t h i s t i s s u e t o the reproductive mechanism of induced o v u l a t i o n has not been i n v e s t i g a t e d . Secondly, the number of small intermediate s i z e d f o l l i c l e s (75-250 micra) appeared to be more numerous i n the d i e s t r o u s o v a r i e s than i n the anestrous o v a r i e s , although these f o l l i c l e s were not counted. These may account f o r ovarian and v a g i -n a l d i f f e r e n c e s observed i n t h i s group when compared with the anestrous group. Examination of the reproductive t r a c t s of d i e s t r o u s females revealed no s i g n i f i c a n t d i f f e r e n c e between d i e s t r o u s and anestrous females 81 o n t h e b a s i s o f m e a s u r e d f a l l o p i a n t u b e e p i t h e l i a l h e i g h t . D e s c r i p t i v e l y , h o w e v e r , f o u r o f t h e n i n e f e m a l e s d e m o n s t r a t e d t h e h i g h c y t o p l a s m i c c o l u m -n a r e p i t h e l i u m , w e l l d e v e l o p e d m u s c l e l a y e r s , w e l l d e v e l o p e d g l a n d s t y p i c a l o f t h e e s t r o u s u t e r u s and f i v e o f t h e n i n e d e m o n s t r a t e d t h e l o w e n d o m e t r i u m , t h i n m u s c l e c o a t , l a c k o f p r e s e n c e o f m u c o s a l g l a n d s d e s c r i b e d f o r t h e u t e r i n e h o r n o f a n e s t r o u s f e m a l e s . V a g i n a l d e s c r i p t i v e h i s t o l o g y p l a c e d t h e g r o u p o f d i e s t r o u s f e m a l e s more c l o s e l y w i t h t h e g r o u p o f e s t r o u s f e m a l e s t h a n w i t h t h e a n e s t r o u s f e m a l e s i n t h a t t h e y d e m o n s t r a t e d t h e e n l a r g e d submucosa , s e v e r a l b a s a l c e l l l i n i n g l a y e r s , and i n most i n s t a n c e s heavy c o r n i f i e d t o p l a y e r s , a l t h o u g h t h e y d i f f e r e d f r o m t h e e s t r o u s f e m a l e s i n a s m u c h as t h e c o r n i f i e d t o p l a y e r s c o n t a i n e d h e a v y l e u k o c y t i c i n v a s i o n . T h a t o b s e r v e d d i f f e r e n c e s i n t h e r e p r o d u c t i v e t r a c t s o f f e m a l e s i n e s t r u s and a n e s t r u s c o u l d be due t o v a r y i n g e s t r o g e n l e v e l s was demon-s t r a t e d b y F o s t e r (1934) i n t h e 1 3 - l i n e d g r o u n d s q u i r r e l . He o b s e r v e d t h a t f a l l o p i a n t u b e h e i g h t was l o w d u r i n g a n e s t r u s and t a l l and c i l i a t e d d u r i n g e s t r u s . When he a d m i n i s t e r e d exogenous e s t r o g e n t o a n e s t r o u s f e m a l e s , he o b s e r v e d t h e same e f f e c t on t h e u t e r i n e h o r n , t h a t i s , t r a n s -f o r m a t i o n f r o m a n e s t r o u s a p p e a r a n c e t o e s t r o u s a p p e a r a n c e . U n f o r t u n a t e l y , t h e g r o u n d s q u i r r e l does n o t d e m o n s t r a t e a d i e s t r o u s v a g i n a l smear . S e v e r a l s t a t e m e n t s a b o u t d i e s t r o u s f e m a l e s on t h e b a s i s o f F o s t e r ' s work and my d a t a c a n be s u g g e s t e d : t h e d i e s t r o u s s t a g e r e p r e s e n t s a r a n g e o f b l o o d l e v e l e s t r o g e n t i t e r w h i c h , o n i t s u p p e r end i s r e f l e c t e d b y e s t r o u s t y p e u t e r i n e changes and o n i t s l o w e r end b y a n e s t r o u s changes i n t h e u t e r u s . The two t y p e s o f d i e s t r o u s smears " l i g h t " and " h e a v y " d e s c r i b e d i n C h a p t e r 3 may a l s o r e f l e c t l o w e r and h i g h e r e s t r o g e n e f f e c t r e s p e c -t i v e l y . E x a m i n a t i o n o f l a r g e s t o v a r y and c o m p a r a b l e v a g i n a l mucosa o f a d i e s t r o u s a n i m a l (No. 2 6 6 , P l a t e s 6 and 7) d e m o n s t r a t e s b o t h t h e o v a r y 82 and the vaginal mucosa of t h i s animal appear more s i m i l a r to that of the anestrous female (Plates 4 and 5) than the estrous female (Plates 8 and 9). U t i l i z i n g the diestrous stage as demonstrative of the greatest v a r i a t i o n i n estrogen t i t e r , i t appears that vagina and uterus are more s e n s i t i v e r e f l e c t o r s of ovarian estrogen e f f e c t than the f a l l o p i a n tube i n M. town- s e n d i i . The ovaries of the two animals s a c r i f i c e d i n the smear stage of metestrus resembled those of the estrous group of females as d i d the appearance and l i n i n g e p i t h e l i a l c e l l height of the f a l l o p i a n tube. On f i v e observed instances a metestrous smear was observed p r i o r to a day of estrus and on ten occasions on the day following estrus. Only on one occasion was a metestrous smear observed within a diestrous i n t e r v a l . Since neither of the other two groups, that i s diestrous or anestrous females, demonstrated an estrous f a l l o p i a n tube estrogenic e f f e c t , i t i s presumed that t h i s group of females r e f l e c t s ovarian estrogen production most near to that of estrous females. The active appearance of a cross section of the la r g e s t ovary of a female i n metestrus and r e l a t e d vaginal cross section (Plates 10 and 11) would Indicate;, that a'-higher estrogen l e v e l i s i n e f f e c t than that observed i n the diestrous female (Plates 6 and 7). The l a t t e r female more c l o s e l y resembles the ovarian and vagi-na l mucosa of the anestrous female (Plates 4 and 5). Anestrous females, as a group, represent s i m i l a r descriptions of the ovaries and reproductive t r a c t s described i n the l i t e r a t u r e f o r both spontaneous and induced anestrous ovulators. That i s , ovaries and repro-ductive t r a c t s represent minimal a c t i v i t y , both m i t o t i c and secretory. At the i n i t i a t i o n of t h i s p r o j e c t , most f i e l d females were observed to become anestrous sh o r t l y a f t e r entering the laboratory. For the most 83 p a r t t h e y r e m a i n e d i n a n e s t r o u s c o n d i t i o n w h i l e i n t h e l a b o r a t o r y . A t t h i s t i m e t h e work o f R ichmond and Conaway (1969) was u n p u b l i s h e d . These a u t h o r s d e m o n s t r a t e d t h e i m p o r t a n t e f f e c t on r e p r o d u c t i v e a c t i v i t y o f e n v i -r o n m e n t a l v a r i a t i o n and p a r t i c u l a r l y , o f e x p o s u r e t o m a l e s . They s t a t e . t h a t t h e i r f e m a l e M . o c h r o g a s t e r i n i s o l a t i o n r a r e l y were p e r f o r a t e . G i v e n t h i s i n f o r m a t i o n , and t h e i n f o r m a t i o n t h a t a l l f e m a l e s on w h i c h t h i s p a r t o f t h e s t u d y was b a s e d were i s o l a t e d , i t i s s u r p r i s i n g t h a t a s u f f i -c i e n t number o f f e m a l e s were p e r f o r a t e and d e m o n s t r a t e d r e p r o d u c t i v e a c t i v -i t y t o p e r f o r m t h i s s t u d y . R ichmond and Conaway (1969) a l s o d e m o n s t r a t e d t h a t cage c l e a n i n g was s u f f i c i e n t , i n some o f t h e i r a n i m a l s , t o evoke e s t r u s . W e e k l y cage c l e a n i n g and d a i l y v a g i n a l smear e x a m i n a t i o n may have p r o v i d e d enough e n v i r o n m e n t a l s t i m u l u s t o p r e v e n t my t o t a l c o l o n y o f M . t o w n s e n d i i f r o m e n t e r i n g t h e s t a t e o f a n e s t r u s . B r u c e (1966) s t a t e s t h a t t h e e f f e c t o f t h e ma le on t h e o c c u r r e n c e o f e s t r u s i s due t o t h e s t i m u l a t i o n o f FSH s e c r e t i o n . R ichmond and Conaway (1969) a g r e e b u t e n l a r g e t h i s h y p o t h e s i s f r o m male e f f e c t o n l y t o i n c l u d e any m i l d l y s t r e s s f u l e n v i r o n m e n t a l c o n d i t i o n w h i c h may a l s o be a c a u s a t i v e f a c t o r f o r h y p o t h a l a m i c s t i m u l a t i o n and s u b s e q u e n t FSH s e c r e t i o n and r e s u l -t a n t e s t r u s . I n t h i s s t u d y , f i e l d f e m a l e s i n a n e s t r o u s p e r i o d s f o r l o n g e r t h a n t h r e e weeks were g i v e n i n j e c t i o n s o f P r e g n a n t M a r e ' s Se rum. T h i s d r u g i s a known a n a l o g o f FSH and e v o k e s a s i m i l a r r e s p o n s e ( C o l e and H a r t , 1 9 3 0 ) . E i g h t o f t h e n i n e a n e s t r o u s f e m a l e s became p e r f o r a t e and s e v e n o f t h e s e d e m o n s t r a t e d t h e v a g i n a l smear o f e s t r u s , one o f p r o e s t r u s w i t h i n 53 h o u r s . O v a r i e s o f t h e s e f e m a l e s , e x c e p t t h e n o n - r e s p o n s i v e f e m a l e , d e m o n s t r a t e d m u l t i p l e l a r g e f o l l i c l e s , l a r g e r t h a n t h e maximum d i a m e t e r s r e c o r d e d f o r t h e a n e s t r o u s f e m a l e s ( T a b l e 1 2 ) . T h i s l e n d s s u p -p o r t t o t h e i d e a t h a t t h e a n e s t r o u s c o n d i t i o n i n t h e s e x u a l l y m a t u r e , 84 unmated i s o l a t e d , M i c r o t u s f e m a l e r e p r e s e n t s f a i l u r e o f adequa t e l e v e l s o f FSH t o " p r i m e " t h e a n i m a l s f o r o v u l a t i o n . F u r t h e r r e s e a r c h i s n e c e s -s a r y u t i l i z i n g b i o a s s a y t e c h n i q u e t o measure w h i c h e n v i r o n m e n t a l s t i m u l i a r e mos t e f f e c t i v e i n e l i c i t i n g maximum FSH r e l e a s e i n M i c r o t u s f e m a l e s . COITUS-INDUCED OVULATION D i s c u s s i o n o f t h e n a t u r e o f t h e e s t r o u s c y c l e i n M . t o w n s e n d i i has t h u s f a r p r e s e n t e d p r e s u m p t i v e e v i d e n c e t h a t t h i s a n i m a l i s an i n d u c e d o v u l a t o r o n t h e b a s i s o f v a g i n a l smear, p a t t e r n and h i s t o l o g i c a l e x a m i n a -t i o n o f t h e o v a r i e s a n d r e p r o d u c t i v e t r a c t s o f unmated f e m a l e s . P r o v i s i o n o f c o n c l u s i v e e v i d e n c e o f i n d u c e d o v u l a t i o n i n M . t o w n s e n d i i w o u l d n e c e s -s i t a t e random d i v i s i o n o f a l a r g e sample o f e s t r o u s f e m a l e s i n t o a ma ted and unmated g r o u p . S i m u l t a n e o u s l y , a f t e r a g i v e n p e r i o d o f t i m e , a l l f e m a l e s w o u l d be s a c r i f i c e d and t h e i r o v a r i e s e x a m i n e d f o r t h e p r e s e n c e o f c o r p o r a l u t e a . A l l unmated f e m a l e s w o u l d be e x p e c t e d t o l a c k e v i d e n c e o f o v u l a t i o n a t t h i s t i m e and a l l , o r m o s t , o f t h e ma ted f e m a l e s w o u l d have o v u l a t e d , have fo rmed c o r p o r a l u t e a , and have f e r t i l i z e d o v a p r e s e n t i n t h e f a l l o p i a n t u b e s . T h i s a p p r o a c h was n o t p o s s i b l e i n t h i s s t u d y due t o t h e s m a l l s i z e o f t h e b r e e d i n g c o l o n y . H o w e v e r , m a t i n g d a t a a v a i l a b l e i n t h i s s t u d y , a l t h o u g h l i m i t e d , s u p p o r t t h i s c o n c l u s i o n . No c o r p o r a l u t e a were o b s e r v e d i n unmated f e m a l e s , b u t were o b s e r v e d i n some mated f e m a l e s , d i r e c t l y o r i n d i r e c t l y . One f e m a l e k i l l e d two d a y s a f t e r sperm were i d e n t i f i e d i n t h e v a g i n a l smear had o v u l a t e d and fo rmed c o r p o r a l u t e a . On t h e b a s i s o f o b s e r v a t i o n o f s u b s e q u e n t l i t t e r s , two o t h e r f e m a l e s , s u b s e q u e n t t o sperm i n v a g i n a l smear and m a l e r e m o v a l , must have o v u l a t e d and f o r m e d c o r p o r a l u t e a . F i e l d d a t a s u p p o r t t h e s e l i m i t e d l a b o r a t o r y d a t a i n t h a t o f t h e 85 s e v e n f e m a l e s c a p t u r e d w h i c h d e m o n s t r a t e d e s t r o u s s m e a r s , t h e o n l y one w i t h c o r p o r a l u t e a was one w i t h sperm i n t h e smear and f i v e t w o - c e l l e d t u b a l o v a p r e s e n t i n t h e f a l l o p i a n t u b e s . The r e l a t i o n s h i p o f v a g i n a l smear t y p e t o t h e p a t t e r n o f o v u l a t i o n was f i r s t i m p l i e d t o be r e l a t e d t o e n v i r o n m e n t a l c o n d i t i o n s by C h i t t y and A u s t i n ( 1 9 5 7 ) . R ichmond and Conaway (1969) o b s e r v e d t h a t by v a r y i n g h o u s i n g c o n d i t i o n s , and p a r t i c u l a r l y i n t h e p r e s e n c e v e r s u s a b s e n c e o f m a l e s , t h e y were a b l e t o w i t n e s s g r e a t v a r i a t i o n i n v a g i n a l smears f rom a n e s t r o u s t h r o u g h e s t r o u s s m e a r s . They d i d n o t o b s e r v e t h e o c c u r r e n c e o f s p o n t a n e o u s o v u l a t i o n s . K i r k p a t r i c k and V a l e n t i n e ( 1 9 7 0 ) , G r a y e t a l . ( 1 9 7 4 a ) , and M i l l i g a n ( 1 9 7 4 ) , h o w e v e r , have o b s e r v e d o c c u r r e n c e s o f s p o n -t a n e o u s o v u l a t i o n i n M . p i n e t o r u m , M . mon tanus , and M . a g r e s t i s , r e s p e c -t i v e l y . The ma jo r p a t t e r n o f o v u l a t i o n was i n d u c e d i n a l l o f t h e s e s p e c i e s . H o u s i n g , i n t h e s e r e p o r t s , i n v o l v e d unmated f e m a l e s h o u s e d i n i m m e d i a t e p r o x i m i t y t o s e n s o r y s t i m u l i o f m a l e s b u t s e p a r a t e d p h y s i c a l l y f r o m m a l e s . I n o t h e r w o r d s , b o t h s p o n t a n e o u s and i n d u c e d o v u l a t i o n have been demon-s t r a t e d i n t h e genus M i c r o t u s u n d e r c o n t r o l l e d l a b o r a t o r y c o n d i t i o n s . The p r e v a l e n t mechan i sm i n t h o s e s p e c i e s t h u s f a r s t u d i e d i s c o i t u s - i n d u c e d o v u l a t i o n . The v a l u e o f i n d u c e d o v u l a t i o n a p p e a r s p r i m a r i l y t o be t h a t o f e f f i c i e n c y i n a s m u c h a s t h i s mechan i sm s y n c h r o n i z e s semen d i s p o s i t i o n , t r a n s -p o r t , and c a p a c i t a t i o n , as w e l l a s o f o v u l a t i o n , p r o d u c t i o n and r e l e a s e o f hormones r e s p o n s i b l e f o r c r e a t i n g o p t i m a l c o n d i t i o n s f o r o v i d u c t a l m o t i l i t y and s e c r e t i o n , and e v e n t u a l l y f o r f e r t i l i z a t i o n ( J o c h e l , 1 9 7 4 : 5 2 4 ) . O t h e r e f f i c i e n c y v a l u e s w h i c h he d e s c r i b e s a r e t h a t t h e mechan i sm o f c o i t u s - i n d u c e d o v u l a t i o n , by p r o v i d i n g pe rmanen t s t i m u l a t i o n f o r f o l l i c l e g r o w t h , has a s s u r e d t h e s p e c i e s o f enough e s t r o g e n p r o d u c t i o n and r e l e a s e 86 t o b o t h a t t r a c t m a l e s and t o i n s u r e t h e i r r e c e p t i o n . J o c h l e (1973) l i s t s t h r e e c a t e g o r i e s o f c o i t u s - i n d u c e d o v u l a t o r s : c o m p l e t e r e f l e x o v u l a t o r s ; f a c u l t a t i v e r e f l e x o v u l a t o r s ; s p o n t a n e o u s o v u l a t o r s i n w h i c h c o i t u s h a s -t e n s o v u l a t i o n . A p p a r e n t l y M i c r o t u s c o m p r i s e s a new c a t e g o r y ; t h a t o f f a c u l t a t i v e s p o n t a n e o u s o v u l a t o r . P a r t i c u l a r a d a p t i v e s i g n i f i c a n c e o f i n d u c e d o v u l a t i o n i n M i c r o t u s s u g g e s t e d by G r e e n w a l d (1956) i s r e l a t e d t o t h e c y c l i c p o p u l a t i o n f l u c t u a t i o n s o f members o f t h i s g e n u s . I n t h e l o w p o p u l a t i o n d e n s i t y y e a r s , i n d u c e d o v u l a t i o n w o u l d mos t e f f i c i e n t l y a s s u r e t h a t a p r o d u c t i v e m a t i n g w o u l d o c c u r , w h i c h w o u l d i n s u r e s u r v i v a l o f t h e s p e c i e s . PREGNANCY AND EVENTS FOLLOWING PREGNANCY P r e g n a n c y d a t a a r e l i m i t e d due t o t h e s m a l l b r e e d i n g c o l o n y and t h e i n c r e a s e d i n c i d e n c e o f l i t t e r d e s t r u c t i o n a s s o c i a t e d w i t h f e m a l e d i s -t u r b a n c e o r m a l e i n f l u e n c e on a new l i t t e r . O n l y t h r e e p r e g n a n c i e s , d a t e d on t h e b a s i s o f sperm i n t h e v a g i n a l smear , were o b t a i n e d , w i t h g e s t a t i o n p e r i o d s o f 21, 23, and 24 d a y s . W h i l e t h i s sample s i z e o f t h r e e i s t o o l i m i t e d t o c a l c u l a t e an a v e r a g e g e s t a t i o n p e r i o d i n t h i s s p e c i e s , t h e r a n g e i s a p p r o x i m a t e d a t 21 t o 24 d a y s . T h i s i s w i t h i n t h e r a n g e o f g e s -t a t i o n p e r i o d c i t e d i n t h e l i t e r a t u r e f o r a l l s t u d i e d s p e c i e s o f M i c r o t u s (see C h a p t e r 1). L i t t e r s i z e d a t a a r e a v a i l a b l e f r o m t h r e e s o u r c e s i n t h i s s t u d y : l a b o r a t o r y c o l o n y a n i m a l s , f i e l d a n i m a l s p r o d u c i n g l i t t e r s i n t h e l a b o r a -t o r y , and f i e l d a n i m a l embryo c o u n t s . The a v e r a g e o f c o u n t s f r o m t h e s e t h r e e s o u r c e s y i e l d s an a v e r a g e l i t t e r s i z e o f 5.40 w i t h a r a n g e o f f r o m 1 t o 7. T h i s i s a l a r g e r l i t t e r s i z e t h a n t h e a v e r a g e o f 4 r e c o r d e d by Cowan and Leduc and K r e b s (1975) f o r M . t o w n s e n d i i and a l s o l a r g e r t h a n 87 t h e a v e r a g e l i t t e r s i z e o f s i x o t h e r s t u d i e d s p e c i e s o f 3 . 4 2 (see C h a p t e r 1 ) . A l l o b s e r v e d f e m a l e s d u r i n g p r e g n a n c y d e m o n s t r a t e d t h e smear o f d i e s t r u s , v e r y s i m i l a r t o t h a t d e s c r i b e d f o r unmated a n o v u l a t o r y i s o l a t e d f e m a l e s ( see C h a p t e r 3 ) . O n l y one f e m a l e was smear t e s t e d a t p a r t u r i t i o n and f o r f o u r s u b s e q u e n t d a y s . T h i s f e m a l e d e m o n s t r a t e d e s t r o u s smears t h r o u g h o u t t h i s p e r i o d . P o s t p a r t u m e s t r u s b y smear d i d o c c u r i n t h i s f e m a l e and i n one o t h e r f e m a l e . The l a t t e r f e m a l e ' s mate was removed o n t h e day o f p a r t u r i t i o n , and a s e c o n d l i t t e r was b o r n 24 d a y s l a t e r . The o c c u r r e n c e o f p o s t p a r t u m e s t r u s i n t h e genus p r o v i d e s t h e p o s -s i b i l i t y f o r a l l s e x u a l l y m a t u r e f e m a l e s t o be c o n s t a n t l y p r e g n a n t t h r o u g h -o u t t h e b r e e d i n g s e a s o n , t h e r e b y p r o d u c i n g t h e maximum number o f o f f s p r i n g p o s s i b l e , a p o p u l a t i o n e c o l o g i s t ' s n i g h t m a r e . The maximum number o f a n i -m a l s w h i c h c o u l d r e s u l t f r o m t h i s c o n t i n u o u s p o s t p a r t u m e s t r o u s m a t i n g o f one f e m a l e and h e r . m a t u r e p r o g e n y d u r i n g one b r e e d i n g s e a s o n w o u l d e q u a l a p p r o x i m a t e l y 1 ,033 a n i m a l s . A p p a r e n t l y M . t o w n s e n d i i f e m a l e s i n n a t u r e a r e u n a b l e t o r e a l i z e t h i s r e p r o d u c t i v e p o t e n t i a l , a t l e a s t a t t h e t i m e o f t h i s s t u d y . Of 36 f i e l d f e m a l e s s a c r i f i c e d d u r i n g b r e e d i n g s e a s o n , 64% o f t h o s e o f 30 gram w e i g h t and above and 16% o f t h o s e o f 40 gram w e i g h t and above d i d n o t d e m o n s t r a t e v i s i b l e u t e r i n e e m b r y o s . P o s s i b l e e x p l a n a t i o n s o f t h i s o b s e r -v a t i o n i n c l u d e : some o f t h e f e m a l e s were i n e a r l y p r e g n a n c y m a k i n g v i s i b l e embryo c o u n t i m p o s s i b l e ; p r e g n a n c y b l o c k o c c u r r e d s i m i l a r t o t h a t demon-s t r a t e d by C l u l o w . a n d C l a r k e ( 1 9 6 8 ) , C l u l o w and L a n g f o r d ( 1 9 7 1 ) , and S t e h n and Richmond (1975) i n M . a g r e s t i s , M . p e n n s y l v a n i c u s , M . o r c h r o g a s t e r , F i g u r e b a s e d on t h e f o l l o w i n g a s s u m p t i o n s : 1 8 9 - d a y b r e e d i n g s e a s o n , 2 1 - d a y g e s t a t i o n p e r i o d , 2 1 - d a y m a t u r a t i o n p e r i o d , 2 m a l e s and 2 f e m a l e s p e r l i t t e r . 88 r e s p e c t i v e l y ; l a c t a t i o n a l a n e s t r o u s o r e s t r o u s s u p p r e s s i o n may have been i n e f f e c t i n some f e m a l e s . P r e g n a n c y f a i l u r e has b e e n i m p l i c a t e d as a p o s s i b l e s i g n i f i c a n t c o n t r i b u t o r t o o b s e r v e d p o p u l a t i o n c y c l e s i n M i c r o t u s ( M a l l o r y , 1 9 7 2 ) . P r e g n a n c y f a i l u r e due t o e n v i r o n m e n t a l d i s r u p t i o n was o b s e r v e d i n t h i s s t u d y . M o s t p r e g n a n t f i e l d f e m a l e s unde rwen t embryo r e s o r p t i o n when b r o u g h t i n t o t h e l a b o r a t o r y . A v e r y e f f e c t i v e c a u s e o f p r e g n a n c y b l o c k , d e m o n s t r a t e d i n s e v e r a l l a b o r a t o r y e x p e r i m e n t s , i s t h e p h e r o m o n a l l y i n d u c e d B r u c e e f f e c t . A l t h o u g h no e v i d e n c e o f t h i s e f f e c t was o b t a i n e d i n t h i s s t u d y , and t h i s phenomenon has n o t b e e n d e m o n s t r a t e d t o be o p e r a t i v e i n w i l d p o p u l a t i o n s , i t s e x i s t e n c e p o i n t s t o t h e n e e d f o r a s t a b l e s o c i a l e n v i r o n m e n t i n o r d e r f o r e f f e c t i v e l i t t e r p r o d u c t i o n t o o c c u r i n M i c r o t u s . I f , as K r e b s e t a l . (1973) p r o p o s e , d i s p e r s a l i s c r i t i c a l t o p o p -u l a t i o n r e g u l a t i o n i n M i c r o t u s , t h e n s o c i a l e n v i r o n m e n t a l d i s r u p t i o n b y a d u l t s l e a v i n g and e n t e r i n g p o p u l a t i o n s must a l s o o c c u r . T h i s d i s r u p t i o n w o u l d be e x p e c t e d t o f a v o r p r e g n a n c y f a i l u r e w h i c h w o u l d i n t u r n f a v o r r e t u r n t o e s t r u s and r e m a t i n g . Inasmuch as p r e g n a n c y f a i l u r e w o u l d have a s e l e c t i o n v a l u e f o r t h e new m a t e , i t i s n o t t o o f a r a f i e l d t o s p e c u l a t e t h a t t h i s p r o c e s s may i n d e e d p l a y a s i g n i f i c a n t r o l e i n p o p u l a t i o n d y n a -m i c s , p a r t i c u l a r l y w i t h r e f e r e n c e t o f a c t o r s w h i c h w o u l d f a v o r g e n o t y p i c and p h e n o t y p i c s h i f t s . SUMMARY I . O n s e t o f p u b e s c e n c e i n M . t o w n s e n d i i : A . Twenty l a b o r a t o r y l i t t e r f e m a l e s became p e r f o r a t e be tween 19 and 82 days o f age ; t h e g r e a t e s t number became p e r f o r a t e be tween 30-45 o d a y s . E l e v e n o f t h e s e f e m a l e s d e m o n s t r a t e d f i r s t e s t r u s by v a g i n a l 89 smear 1-36 days a f t e r i n i t i a l p e r f o r a t i o n ; a l l w i t h i n t h e f i r s t 11 weeks o f a g e . T h e r e was no p r e d i c t a b l e r e l a t i o n s h i p be tween t h e s e v a r i a b l e s i n t h e a n i m a l s s t u d i e d . B . P u b e r t y i n f i e l d f e m a l e s , a s measu red b y v a g i n a l p e r f o r a t i o n , o c c u r r e d w i t h g r e a t e s t f r e q u e n c y be tween 1 5 - 2 1 . 9 grams w h i c h i s b a s e d on e x a m i n a t i o n o f 53 f i e l d f e m a l e s . S i x f i e l d f e m a l e s i n t h e r a n g e o f 1 5 - 2 4 . 9 grams w h i c h i s 1 5 - 2 0 d a y s o f age b a s e d on l a b o r a t o r y g r o w t h c h a r t d e m o n s t r a t e d e s t r u s v a g i n a l s m e a r s . C . D i s c u s s i o n o f o b s e r v a t i o n s : 1. L a b o r a t o r y f e m a l e s were n o t i n an e n v i r o n m e n t w h i c h w o u l d enhance r e p r o d u c t i v e a c t i v i t y ; t h e y were i s o l a t e d , i n c l o s e p r o x i m i t y t o o t h e r f e m a l e s , n o t i n c o n t a c t w i t h m a l e s , and h o u s i n g m o d i f i c a t i o n s were f e w . A l l o f t h e s e f a c t o r s have b e e n d e m o n s t r a t e d i n l a b o r a t o r y s t u d i e s t o e f f e c t r e p r o d u c t i v e a c t i v i t y n e g a t i v e l y . 2 . F i e l d f e m a l e s t a t i s t i c s o f age o f s e x u a l m a t u r i t y have l i t t l e s i g n i f i c a n c e a b s t r a c t e d f r o m p o p u l a t i o n c y c l e p h a s e , s e a s o n o f t h e y e a r and t h e p a r t i c u l a r u n i q u e p o p u l a t i o n f r o m w h i c h t h e y were d e r i v e d . N a t u r e o f t h e e s t r o u s c y c l e : A . E s t r o u s c y c l e d e t e r m i n e d b y v a g i n a l smear : 1. On t h e b a s i s o f v a g i n a l smear s t u d i e s and c o m p a r i s o n w i t h s i m i l a r s t u d i e s o f s e v e r a l known s p o n t a n e o u s and i n d u c e d o v u l a t o r s , M . t o w n s e n d i i a p p e a r s t o be an i n d u c e d o v u l a t o r . 2 . Of t h e 466 s t u d y smear d a y s , a n i m a l s r e m a i n e d i n t h e smear s t a g e o f e s t r u s 57 p e r c e n t o f t h e t i m e ; i n d i e s t r u s 35 p e r c e n t o f t h e t i m e ; i n p r o e s t r u s and m e t e s t r u s combined 8 p e r c e n t 90 o f t h e t i m e . T h e r e was some e v i d e n c e o f c y c l i c p e r i o d i c i t y when a c y c l e i s d e f i n e d as f i r s t d a y o f an e s t r o u s smear t o t h e s u b s e q u e n t n e x t f i r s t day o f an e s t r o u s smear . T h i s p e r i o d i c i t y was 2 - 7 . 5 days i n d u r a t i o n . B . C o r r e l a t i o n o f v a g i n a l smear s t a g e w i t h r e p r o d u c t i v e t r a c t h i s t o l o g y : 1. H i s t o l o g i c a l s t u d y o f r e p r o d u c t i v e t r a c t s o f f e m a l e s k i l l e d i n t h e v a g i n a l smear s t a g e s o f e s t r u s , d i e s t r u s , a n e s t r u s and m e t e s t r u s y i e l d e d i n f o r m a t i o n o n l y a b o u t t h e f o l l i c u l a r s t a g e o f t h e c y c l e . No o v u l a t i o n , c o r p o r a l u t e a o r c o r p o r a a l b i c a n t i a were o b s e r v e d i n any a n i m a l s s t u d i e d ; on t h e b a s i s o f p u b l i s h e d h i s t o l o g i c a l s t u d i e s of ' i n d u c e d and s p o n t a n e o u s o v u l a t o r s and o n c o m p a r a t i v e h i s t o l o g i c a l e x a m i n a t i o n o f o v a r i e s and r e p r o d u c t i v e t r a c t s o f unmated f e m a l e s , M . t o w n - s e n d i i a p p e a r s t o be an i n d u c e d o v u l a t o r . 2 . A n a l y s i s o f v a r i a n c e f o r t h e a n e s t r u s (n = 1 0 ) , e s t r u s (n = 9) and d i e s t r u s (n = 9) g r o u p s d e m o n s t r a t e d no s i g n i f i c a n t d i f -f e r e n c e among t h e g r o u p s w i t h r e f e r e n c e t o t h e mean number o f d e v e l o p i n g f o l l i c l e s l a r g e r t h a n 250 m i c r a n o r t h e mean number o f a t r e t i c f o l l i c l e s l a r g e r t h a n 250 m i c r a . The e s t r u s g r o u p d e m o n s t r a t e d s i g n i f i c a n t l y g r e a t e r mean number o f f o l l i -c l e s l a r g e r t h a n 350 m i c r a , l a r g e r mean maximum o v a r i a n f o l l i c l e d i a m e t e r , m e a n - o v a r i a n d i a m e t e r , and f a l l o p i a n t u b e e p i t h e l i a l h e i g h t t h a n t h e o t h e r two g r o u p s . Howeve r , t h e d i e s t r u s and a n e s t r u s f e m a l e s d i f f e r e d s i g n i f i c a n t l y on o n l y one o f t h e s e m e a s u r e m e n t s , t h a t o f mean o v a r i a n d i a m e t e r . Two p o s s i b i l i t i e s f o r t h e l a t t e r d i f f e r e n c e a r e p r o p o s e d : 91 o n e , t h e d i e s t r u s f e m a l e p o s s e s s e d a w e l l d e v e l o p e d a p p a r -e n t l y s e c r e t o r y i n t e r s t i t i a l t i s s u e whe reas t h e a n e s t r u s f e m a l e s i n t e r s t i t i a l t i s s u e was a v a s c u l a r and c o n t a i n e d t i g h t l y p a c k e d a c y t o p l a s m i c c e l l s ; t h e d i e s t r u s f e m a l e s a p p e a r e d t o c o m p a r a t i v e l y have a l a r g e r number o f i n t e r m e d i a t e f o l l i c l e s t h a n t h e a n e s t r u s f e m a l e s (75-125 m i c r a ) . O b s e r v a t i o n s o f r e p r o d u c t i v e t r a c t : The u t e r u s and v a g i n a were t h e mos t s u b t l e i n d i c a t o r s o f v a r i a t i o n i n e s t r o g e n ] l e v e l . D e s c r i p t i v e l y , a l l e s t r u s f e m a l e s d e m o n s t r a t e d s i m i -l a r h i s t o l o g i c a l o b s e r v a t i o n s t y p i c a l o f m i t o t i c and s e c r e -t o r y a c t i v i t y . A l l a n e s t r u s f e m a l e s d e m o n s t r a t e d a m i t o t i c , a s e c r e t o r y and a t r o p h i c a p p e a r i n g v a g i n a e and u t e r i . F o u r d i e s t r u s f e m a l e h a d u t e r i and v a g i n a e s i m i l a r t o e s t r u s f e m a l e s ; , f i v e r e s e m b l e d a n e s t r u s f e m a l e s . A l l d i e s t r u s f e m a l e s had f a l l o p i a n t u b e e p i t h e l i u m t y p i c a l o f a n e s t r u s f e m a l e s . The r e p r o d u c t i v e t r a c t s o f two m e t e s t r u s f e m a l e s r e s e m b l e d e s t r u s f e m a l e s i n a p p e a r a n c e , i n c l u d i n g f a l l o p i a n t u b e e s t r o g e n e i c e f f e c t . The i n f e r e n c e i s made t h a t t h e s e f o u r s t a g e s r e p r e s e n t v a r i o u s e s t r o g e n l e v e l s f r o m l o w e s t t o h i g h e s t : n a m e l y , a n e s t r u s ; d i e s t r u s ; m e t e s t r u s ; e s t r u s . Response o f a n e s t r u s f e m a l e s t o . P . M . S : M o s t f i e l d f e m a l e s became a n e s t r u s when b r o u g h t i n t o t h e l a b o r a t o r y . N i n e f i e l d f e m a l e s , i m p e r f o r a t e f o r a t l e a s t 3 weeks p r i o r t o P . M . S . a d m i n i s t r a t i o n , were g i v e n an i n j e c t i o n o f t h e F . S . H . a n a l o g . E i g h t o f t h e a n i m a l s were p e r f o r a t e w i t h i n 53 h o u r s a f t e r a d m i n i s t r a t i o n ; 7 d e m o n s t r a t e d t h e smear s t a g e o f e s t r u s ; 1 o f p r o e s t r u s . T h e i r a n e s t r u s c o n d i t i o n was n o t r e l a t e d t o 92 an i n t r i n s i c reproductive defect, and therefore must be based on e x t r i n s i c environmental f a c t o r or factors which r e s u l t " i n an F.S.H. block. I I I . Coitus-induced ovulation: One laboratory female k i l l e d two days a f t e r sperm were observed i n the vaginal smear had ovulated and formed corpora lu t e a . No unmated females examined had ovulated or demonstrated corpora l u t e a . Of 7 f i e l d females with estrus smears at the time of capture, the only one with corpora lutea and tubal ova, was one with sperm i n the i n i -t i a l smear. Although t h i s study does not present conclusive evidence of induced ovulation, on the basis of vaginal smear patterns, ovarian h i s t o l o g i c a l examinations, and l i m i t e d mating data, the evidence i s strongly presumptive.. IV. Pregnancy and events following pregnancy: A. Three dated pregnancies were obtained with gestation periods of 21, 23, and 24 days. B. Mean l i t t e r s i z e was 4.84 of laboratory born l i t t e r s (n = 13); 6 of pregnant f i e l d female embryo counts (n =20); 5.41 of f i e l d females which had l i t t e r s i n the laboratory (n = 12). L i t t e r s i z e i s larger than that recorded for other studied Microtus and larger than that recorded for M. townsendii by Cowan and Guignet (1956) and Anderson (1976). C. A l l pregnant females demonstrated the smear stage of d i e s t r u s . D. Post partum estrus was evidenced by two females; one by vaginal smear and one by producing a l i t t e r subsequent to male removal on the day of p a r t u r i t i o n . E. No evidence r e l a t i v e to the occurrence of l a c t a t i o n anestrus was obtained i n t h i s study. BIBLIOGRAPHY A l l e n , E . 1 9 2 2 . The o e s t r u s c y c l e i n t h e mouse . Am. J . A n a t . 3 0 : 2 9 7 - 3 7 2 . A n d e r s o n , J . L . 1976 . P h e n o t y p i c c o r r e l a t i o n s among r e l a t i v e s and v a r i -a b i l i t y i n r e p r o d u c t i v e p e r f o r m a n c e i n p o p u l a t i o n s o f t h e v o l e , M i c r o -t u s t o w n s e n d i i . P h . D . T h e s i s . U n i v e r s i t y o f B r i t i s h C o l u m b i a . A r o n , C . , J . A j c h , J . R o o s . 1 9 6 6 . T r i g g e r i n g o f o v u l a t i o n by c o i t u s i n t h e r a t . I n t . R e v . C y t o l . 2 0 : 1 3 9 - 1 4 3 . B a k e r , W. D . , and R . M . R a n s o n . 1 9 3 2 . F a c t o r s a f f e c t i n g t h e b r e e d i n g o f t h e f i e l d mouse ( M i c r o t u s a g r e s t i s ) . P r o c . R . S o c . 1 1 0 : 3 1 3 - 3 2 2 ; 1 1 2 : 3 9 - 4 6 . B e r g e r , P . J . , and N . C . N e g u s . 1974 . I n f l u e n c e o f d i e t a r y s u p p l e m e n t s o f f r e s h l e t t u c e o n o v a r i e c t o m i z e d M i c r o t u s mon tanus . J . Mamm. 5 5 : 7 4 7 - 7 5 0 . B e r g e r , P . J . , E . H . S a n d e r s , P . D . G a r d n e r , N . C . N e g u s . 1 9 7 7 . P h e n o l i c p l a n t compounds f u n c t i o n i n g as r e p r o d u c t i v e i n h i b i t o r s i n M i c r o t u s  mon tanus . S c i e n c e 1 9 5 : 5 7 5 - 5 7 7 . B o d e n h e i m e r , F . S . 1 9 4 9 . P r o b l e m s o f v o l e p o p u l a t i o n s i n t h e M i d d l e E a s t . R e s e a r c h C o u n s e l o f I s r a e l , A z r i e l P r i n t i n g W o r k s , J e r u s a l e m . B o d e n h e i m e r , F . S . , and F . S u l m a n . 1 9 4 6 . 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