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Ovary, reproduction, and productivity of female columbian black-tailed deer Thomas, Donald Charles 1970

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THE OVARY, REPRODUCTION, AND PRODUCTIVITY OF FEMALE COLUMBIAN BLACK-TAILED DEER  by  DONALD CHARLES THOMAS B.A., U n i v e r s i t y o f Saskatchewan, 19': 1 Honours, 1962.  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n the Department of Zoology  We a c c e p t t h i s t h e s i s as conforming r e q u i r e d standard  THE  to the  ••JNIVER5ITY OF BRITISH CQLUMBL "FEBRUARY, 1970  In p r e s e n t i n g an the  this thesis  in p a r t i a l  advanced degree at the U n i v e r s i t y Library  shall  f u l f i l m e n t o f the requirements f o r of B r i t i s h  make i t f r e e l y a v a i l a b l e  I f u r t h e r agree tha  permission  Columbia,  f o r reference  I agree  that  and study.  f o r e x t e n s i v e copying o f t h i s  thesis  f o r s c h o l a r l y purposes may be granted by the Head o f my Department o r by  h i s representatives.  of  t h i s t h e s i s f o r f i n a n c i a l gain  written  permission.  Department o f  ZOOLOGY  The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  Date  I t i s understood  FEBRUARY, 1970  Columbia  shall  that  copying o r p u b l i c a t i o n  not be allowed without my  ii  ABSTRACT Ovarian changes, patterns of reproduction, and a g e - s p e c i f i c product i v i t y of female Columbian b l a c k - t a i l e d deer (Odocoileus hemionus columbianus) on Vancouver Island were elucidated l a r g e l y by examination of s e r i a l , s t a i n e d sections of ovaries from UUU females. Well-defined, ing  8 or 9-day, f o l l i c u l a r  season i n November and e a r l y December.  cycles occur during the breed-  Many f o l l i c l e s r u p t u r i n g at f i r s t  o v u l a t i o n are asynchronous and of extreme s i z e s .  R e l a t i v e l y small (4-5 mm  ),  s h o r t - l i v e d (8-day) corpora l u t e a develop a f t e r f i r s t o v u l a t i o n ; these r a p i d l y regress a f t e r second o v u l a t i o n , while the corpora l u t e a of pregnancy grow 3  to 100 mm  , w i t h i n 5-8 days. The changes i n ovarian structures during the breeding season were  ascertained i n 12 females, whose f o l l i c l e s the cleavage stage of f e r t i l i z e d  ova.  and corpora l u t e a were dated by  These provided  criteria  for  estimating  o v u l a t i o n dates i n other females. I n each c y c l e , about 50% of the adult females ovulated w i t h i n an day period.  The mean date of f i r s t o v u l a t i o n i n each of the 5 years  8-  was  approximately November 16; the second o v u l a t i o n followed a f t e r a remarkably short period of 8-9 days.  About 96% of the females conceived at second  o v u l a t i o n and U% on subsequent ovulations.  A high proportion of ' s i l e n t heats'  accompany f i r s t o v u l a t i o n , as i n d i c a t e d by l a c k of sperm on four of s i x ova. Growth curves, based on fetuses conceived at second o v u l a t i o n , provided  the conception dates of two l a t e c o n c e i v e r s — t h o s e  than one generation may  containing more  of regressed corpora l u t e a of non-pregnancy.  Females  cycle a t l e a s t f i v e times i f pregnancy does not occur. Accessory corpora l u t e a develop i n large and small unruptured f o l -  l i c l e s , i n small ruptured  follicles,  and i n r e g r e s s i n g corpora l u t e a .  They  iii  o c c u r r e d i n 4-7% of f e m a l e s between f i r s t and second o v u l a t i o n ^ and i n 3 6 % of pregnant females. Minimum l o s s o f ova was 8 . 3 % i n a l l f e m a l e s t h a t produced c o r p o r a l u t e a and A-3%  i n f e m a l e s t h a t became p r e g n a n t , of w h i c h 3.1,% was  moribund  fetuses. C o r p o r a l u t e a o f pregnancy, c o r r e s p o n d i n g t o the number o f f e t u s e s , d e v e l o p i n t o d i s t i n c t i v e s c a r s , w h i c h p e r s i s t f o r the l i f e of the doe. Pregnancy r a t e s of the p r e v i o u s season, as w e l l as t h e l o n g term p r o d u c t i v i t y of  i n d i v i d u a l s and the p o p u l a t i o n , were e s t i m a t e d from t h e s e . The average number of v i a b l e f e t u s e s per doe i n c r e a s e d p r o g r e s -  s i v e l y f r o m 0.91  i n y e a r l i n g s t o 1.81  t h e r e a f t e r decreased.  i n the 5-5 t o 6.5 a g e - c l a s s e s , and  These changes i n f e r t i l i t y w i t h age were m i r r o r e d  changes i n w e i g h t and g i r t h .  by  The r e l a t i v e c o n t r i b u t i o n o f fawns by each  a g e - c l a s s i n the p o p u l a t i o n p r o g r e s s i v e l y d e c r e a s e d w i t h age. f e m a l e s of r e p r o d u c t i v e age produced about 137  fawns.  One  hundred  iv  TABLE OF CONTENTS Page 1.  INTRODUCTION  2.  THE STUDY AREA  A  3.  THE BLACK-TAILED DEER OF NORTHWEST BAY. .  5  4..  THE REPRODUCTIVE CYCLE OF DEER  6  5.  METHODS  7  5. 5. 5. 5. 5. 5. 6.  >  1 C o l l e c t i o n Of The M a t e r i a l 2 Weights And Measurements 3 Age Determination A H i s t o l o g i c a l Technique 5 Recovery Of Ova 6 Microscopy  -  7 8 9 10 13 1-4  RESULTS  SECTION 1.  15 STRUCTURE OF THE FETAL OVARY  15  1. 1 Cortico-Medullary R e l a t i o n s h i p s 1.2 The Surface E p i t h e l i u m 1.3 The Germ C e l l 1 . 4 F o l l i c u l a r Development SECTION 2.  1  '  17 18 18 20  STRUCTURE AND FUNCTION OF THE OVARY IN FAWNS, YEARLINGS, AND ADULTS  .  22  CHAPTER 1.  GENERAL MORPHOLOGY OF THE DEER OVARY  22  CHAPTER 2.  THE SURFACE EPITHELIUM  23  CHAPTER 3.  THE OVARIAN FOLLICLE  .  27  3. 1 H i s t o l o g i c a l Components Of The F o l l i c l e . . . . . . . . . 3.2 The Preovulatory F o l l i c l e .3.3 Normal A t r e s i a Of F o l l i c l e s . . 3. A A t y p i c a l A t r e s i a Of F o l l i c l e s 3. 5 Polyovular F o l l i c l e s 3. 6 F o l l i c u l a r Cysts 3. 7 P r i m o r d i a l F o l l i c l e s . 3. 8 T e r t i a r y F o l l i c l e s .• • • CHAPTER A- THE FORMATION AND DEGENERATION OF CORPORA LUTEA. .  27 30 36 4-6 4-7 4-8 4-9 51 73  4...1 "A- 2 4-. 3  The Problem Of Mechanically-Ruptured F o l l i c l e s The Growth Rate Of Corpora Lutea Temporal Changes I n The R a t i o Between The Volumes Of Corpora Lutea Of The F i r s t And Second Generation. . . .  73 75 77  V  Page 4. 4 4. 5 4- 6 4- 7 4. 8 4. 9 4-10 4-. 11  E s t i m a t i n g The Age Of Corpora Lutea H i s t o l o g y Of Young Corpora Lutea The Early-Degeneration Of Corpora. Lutea From the F i r s t Ovulatory Cycle The Fate Of F i r s t - C y c l e Corpora Lutea Corpora Lutea In The Pregnant Doe Accessory Corpora Lutea Scars Derived From Corpora Lutea Corpora Lutea And Scars In Macroscopic Sections  SECTION 3.  77 81 91 94 96 101 104112  THE BREEDING SEASON AND THE LENGTH OF THE ESTROUS CYCLE .  3. 1 3. 3. 3. 3.  The Frequency Of Ruptured F o l l i c l e s And Corpora Lutea In P e r i o d i c Samples 2 Dates Of Ovulation Estimated From Ovarian Structures . . . 3 The Estimated Dates Of Conception Determined From F e t a l Growth Curves 4- The Occurrence Of Corpora Lutea Of Non-Pregnancy In Late Breeders , 5 Dates Of P a r t u r i t i o n  SECTION 4-. 4.. 4. 44.  1 2 3 4  SECTION 57.  7. 4 7. 5 7. 6 7. 7 7. 8 7. 9 7.10 7.11 7.12 8.  130 132 134  .  LITERATURE CITED  134 134 138 14.0 144  .  148  General Comments Development Of The F e t a l Ovary E s t i m a t i n g The Age Of Ovarian Structures By Use Of Segmentation Stages Of Ova And F e t a l Growth Curves. . . . The Weight Of Ovaries And Ovarian Function The Structure And Function Of F o l l i c l e s The Corpus Luteum Ovarian Scars The Breeding Season The Length Of The Estrous Cycle In Deer F e r t i l i t y And P r o d u c t i v i t y Of Northwest Bay Deer R e l a t i v e To Other Populations The E f f e c t Of Age On F e r t i l i t y The E f f e c t Of Size On F e r t i l i t y  SUMMARY  APPENDIX  127  THE EFFECT OF SIZE ON FERTILITY  DISCUSSION AND CONCLUSIONS 7. T 7. 2 7. 3  117 124-  THE EFFECT OF AGE ON FERTILITY AND PRODUCTIVITY Numbers Of Embryos And Fetuses Numbers Of Corpora Lutea Numbers Of Scars Derived From Corpora Lutea Of Pregnancy The P r o d u c t i v i t y Of The Population  116  148 152  ;  . . .  153 155 156 166 172 175 178 182 186 187 .  •  190 194 204  vi  LIST OF TABLES Table 1. 2.  Page The number, age,and c o l l e c t i o n period of deer whose ovaries were studied  8  Measurements of deer fetuses and c h a r a c t e r i s t i c s of t h e i r ovaries a t selected periods during g e s t a t i o n  15  Per cent frequency of one Class I V , two Class I V , and one Class IV plus one Class I I I f o l l i c l e s i n preovulatory yearl i n g s Nov. 6 - Dec. 6, 1963-1966  56  4-. The per cent frequency of adult does,in s i x age classes,cont a i n i n g only one or two Class I V f o l l i c l e s , p r i o r t o f i r s t ovulation  56  3.  5. 6. 7.  The number and c o n d i t i o n of 25 ovulated ova recovered from the oviducts and u t e r i of 19 does, 1964.-67  66  Stages i n the advanced degeneration of f i r s t - c y c l e l u t e a (CL 1)  95  corpora  C h a r a c t e r i s t i c s of s t r u c t u r e s i n gross sections of preserved ovaries  114  8.  The i n t e r v a l i n days between f i r s t and second ovulations. . .  9.  The a g e - s p e c i f i c pregnancy r a t e and fetus production r a t e of females i n the 1963-64 season The a g e - s p e c i f i c o v u l a t i o n r a t e , based on corpora l u t e a , of a l l females c o l l e c t e d from 1963 to 1967  10. 11.  The o v u l a t i o n r a t e of age-classes samples from 1963 t o 1967.  123 135 137  of females i n combined 137  «  12.  13.  14-. 15.  The a g e - s p e c i f i c average number of scars of 5 months derived from corpora l u t e a of pregnancy i n the reproductive seasons from 1962 to 1967  139  The estimated mean number of l i v e fawns born to each female, i n s p e c i f i c age-classes,based on adjusted counts of f e t u s e s , corpora l u t e a , and scars..  14-3  The t o t a l and r e l a t i v e p r o d u c t i v i t y of each age-class a t Northwest Bay  14-3  A comparison between the o v u l a t i o n r a t e i n females from above and below 2000 f e e t a t Northwest Bay from 1963 to 1966 .  150  vii  LIST OF FIGURES Figure 1.  Page The age-specific weight of paired ovaries from deer c o l l e c t e d i n November and early-December from 1963 to 1966  24  Changes i n the weight of ovaries from four age-classes of deer c o l l e c t e d between October and June, 1963-64 . . . .  25  The a g e - s p e c i f i c number of p r i m o r d i a l f o l l i c l e s , p e r m i l l i m e t e r of outer c o r t e x , i n 10 p. sections from the ovaries of deer c o l l e c t e d i n November and early-December, 1963-67  50  The a g e - s p e c i f i c number of Class 1 (1-2 mm diam) f o l l i c l e s i n both ovaries of females c o l l e c t e d i n November and e a r l y December, 1963-67  53  The frequency of the l a r g e s t f o l l i c l e , and i t s state of maturity, i n fawns, y e a r l i n g s , and adults p r i o r to f i r s t ovulation of the current breeding season. . . . .  55  The r e l a t i o n s h i p between the l a r g e s t a c t i v e f o l l i c l e and the l a r g e s t a t r e t i c f o l l i c l e i n p a i r s of ovaries from preovulatory females  59  The r e l a t i o n s h i p between the l a r g e s t a c t i v e , second-cycle f o l l i c l e and the l a r g e s t a t r e t i c f o l l i c l e of previous c y c l e s .  60  The r e l a t i o n s h i p between the volume of the l a r g e s t a c t i v e second-cycle f o l l i c l e and the volume of the l a r g e s t f i r s t cycle corpus luteum (CL 1) i n y e a r l i n g and adult females . .  63  The regressions of the volumes of the l a r g e s t t h i r d - c y c l e f o l l i c l e on the l a r g e s t second-cycle corpus luteum (CL 2) i n y e a r l i n g and adult females .  65  The growth r a t e of the l a r g e s t t h i r d - c y c l e f o l l i c l e ( i n each doe) i n which the i n t e r v a l since second ovulation was estimated from the cleavage stage of ova  71  Seasonal changes i n the volume of the l a r g e s t f o l l i c l e i n pregnant females  74  12. - The growth r a t e of second-generation corpora l u t e a (CL 2) whose ages were estimated from the cleavage stage of ova . .  76  2. 3.  4.  5.  6.  7. 8.  9.  10.  11.  13.  Temporal changes i n the r a t i o between the volume of the l a r g e s t second-generation corpus luteum (CL 2) and the volume of the l a r g e s t s h r i n k i n g , f i r s t - g e n e r a t i o n corpus luteum (CL 1)  78  vlii  Figure 1-4.  15-  16.  17.  18.  19-  20.  21 .  22.  23. 24-. 25.  Page The volume of d e g e n e r a t i n g f i r s t - c y c l e c o r p o r a l u t e a (CL 1) accompanying s e c o n d - c y c l e c o r p o r a l u t e a (CL 2) whose ages were e s t i m a t e d by 1) the c l e a v a g e s t a g e of ova and 2) s e v e r a l c r i t e r i a .  93  The volumes of c o r p o r a l u t e a of pregnancy (CLP) a t f i v e s t a g e s of g e s t a t i o n  97  The a g e - s p e c i f i c volumes of s c a r s 5 and 17 months o l d d e r i v e d f r o m c o r p o r a l u t e a of pregnancy  108  R e g r e s s i o n of the average number of c o r p o r a l u t e a of pregnancy s c a r s on the age of f e m a l e s a t the p r e v i o u s b r e e d i n g season  113  The o v u l a t i o n i n c i d e n c e on weekends i n November and December, 1963  early119  The o v u l a t i o n i n c i d e n c e based on d a i l y samples o b t a i n e d d u r i n g November and early-December f r o m 1963 t o 1967. . . .  121  F r e q u e n c y d i s t r i b u t i o n s and the d e r i v e d s t a t i s t i c s of e s t i m a t e d d a t e s of f i r s t and second o v u l a t i o n s i n combined samples f r o m the b r e e d i n g seasons of 1963 t o 1966  126  The r e l a t i o n s h i p between h i n d - f o o t l e n g t h and 1) the d a t e of c o l l e c t i o n i n 1963-64-, and 2) the g e s t a t i o n age o f fetus  1  28  The r e g r e s s i o n o f f e t a l forehead-rump l e n g t h on 1) the d a t e of c o l l e c t i o n and 2) the g e s t a t i o n age of f e t u s  131  The f r e q u e n c y d i s t r i b u t i o n ( a n d d e r i v e d s t a t i s t i c s ) o f d a t e s on w h i c h fawns were tagged f r o m 1960 t o 196-4  133  The age s t r u c t u r e o f f e m a l e s (fawns e x c l u d e d ) t a k e n f r o m the Northwest Bay p o p u l a t i o n between 1960 and 1966  14-1  S t a t i s t i c s f o r hind-foot l e n g t h , chest girth,and dressed w e i g h t of e i g h t a g e - c l a s s e s o f f e m a l e s c o l l e c t e d d u r i n g November and early-December, 1963-66  14-5  26.  Schematic r e p r e s e n t a t i o n o f f o l l i c l e  . .  163  27.  Schematic r e p r e s e n t a t i o n o f r e p r o d u c t i v e c y c l e s i n d e e r . . .  181  growth and a t r e s i a .  ix  LIST OF PLATES Plate  Page  1.  Development of the F e t a l Ovary  16 •  2.  H i s t o l o g y of the F e t a l Ovary  19  3.  The Components of F o l l i c l e s  4- .  The Pre-and Post-ovulatory Oocyte  34-  5-  Preovulatory and Degenerate Oocytes  38  6.  M e i o t i c D i v i s i o n i n the Oocyte.  39  7.  The F o l l i c u l a r Wall  43  8.  Hyperplasia and Metaplasia of Tissue i n A t r e t i c F o l l i c l e s . .  45  9.  A t y p i c a l A t r e s i a of F o l l i c l e s  68  •  29  10.  The Ovulated Ovum  11.  Development of Corpora Lutea From the F i r s t Ovulatory Cycle .  84-  12.  Degeneration of the Corpus Luteum From the F i r s t Ovulatory Cycle.  85  13.  •  E a r l y Development of Corpora Lutea From the Second Ovulatory Cycle  14-. .1516.  69  86  Changes i n Corpora Lutea During Pregnancy  99  Scars Derived From Corpora Lutea  110  Color Photomicrographs  116  of Ovarian Structures  X  L I S T OF APPENDICES  Appendix 1.  2.  3.  4.  5.  6. 7.  8.  Page The e a r l y c l e a v a g e of f e r t i l i z e d mammalian s p e c i e s  ova i n s e l e c t e d 204  The volume of g r o w i n g c o r p o r a l u t e a of the second o v u l a t o r y c y c l e ; the volume of d e g e n e r a t i n g c o r p o r a l u t e a of the f i r s t o v u l a t o r y c y c l e (CL 1 ) ; and the r a t i o of the l a r g e s t CL 1 ( i n the same p a i r of o v a r i e s ) ; a t i n t e r v a l s f o l l o w i n g second o v u l a t i o n  205  The t i m i n g and v a r i a b i l i t y of the b r e e d i n g seasons f r o m 1963 t o 1967,based on s t a t i s t i c s d e r i v e d f r o m f r e q u e n c y d i s t r i b u t i o n s of d a t e s of o v u l a t i o n  206  A g e - s p e c i f i c pregnancy r a t e and f e t u s - p r o d u c t ! o n N o r t h w e s t Bay f e m a l e s , 1963 - 66  207  rate  of  The r e l a t i o n s h i p between the number of c o r p o r a l u t e a (CL) of t h r e e s i z e s and the number of f e t u s e s i n 55 p r e g n a n t does  208  The a g e - s p e c i f i c o v u l a t i o n r a t e of f e m a l e s t h a t o v u l a t e d i n November and early-December  209  had  Temperature and p r e c i p i t a t i o n d e v i a t i o n s f r o m the normal f o r each month f r o m 1963 t o 1967 a t the Nanaimo a i r p o r t , B r i t i s h Columbia  210  The l o s s of ova a t t h r e e s t a g e s of the r e p r o d u c t i v e c y c l e , as r e v e a l e d by the r a t i o between the number of f e t u s e s and the number of c o r p o r a l u t e a  211  xi  ACKNOWLEDGEMENTS  I am i n d e b t e d t o my r e s e a r c h s u p e r v i s o r , D r . I a n McTaggart-Cowan, Dean o f Graduate S t u d i e s , who i n i t i a t e d s t i m u l a t i o n , and r e s e a r c h f u n d s . of my. r e s e a r c h committee: of the Department  t h i s study, p r o v i d e d  S p e c i a l thanks go t o the other members  D r s . D. C h i t t y , V/. S. Hoar, and Ii. D. F i s h e r , a l l  o f Zoology; Dr. W. D. K i t t s ,  Department  o f Animal  and Dr. P. J . Bandy, Animal Resource E c o l o g y and the B r i t i s h F i s h and W i l d l i f e  Branch.  phase o f t h i s study. agreed  guidance,  Science;  Columbia  Dr. Bandy was e s p e c i a l l y h e l p f u l i n t h e w r i t i n g  Dr. J . F. B e n d a l l , Department  o f Zoology, k i n d l y  t o s e r v e as p r o tern S u p e r v i s o r i n t h e absence o f Dr. Cowan and added  h e l p f u l suggestions. The B r i t i s h Columbia F i s h and W i l d l i f e  Branch p e r m i t t e d me t o  c o l l e c t female deer and, thanks t o Dr. Bandy, t o use v a l u a b l e equipment i n their research  laboratory.  I am g r a t e f u l f o r funds r e c e i v e d from Canadian I n d u s t r i e s L t d . , the Canadian W i l d l i f e  S e r v i c e , and the U n i v e r s i t y o f B r i t i s h  Columbia.  Many people a s s i s t e d i n t h e c o l l e c t i o n o f specimens i n c l u d i n g Mr. W. Tremblay, Mr. L. Berget, Mr. W. A. Low, Mr. Don Blood, Mr. G. Smith, and my f a t h e r , Mr. P. Thomas.  The f i r s t mentioned  was e s p e c i a l l y h e l p f u l .  I  thank Dr. Bobbi Low f o r the l i n e drawings h e r e i n and Mr. W. A. Low f o r h e l p f u l suggestions.. I a p p r e c i a t e most o f a l l t h e h e l p c o n t r i b u t e d  by my w i f e A l i c e ,  who typed t h i s t h e s i s and p r o v i d e d a p r o p i t i o u s environment f o r t h e c o n c e p t i o n and l o n g g e s t a t i o n o f t h i s study.  1  1.  Introduction. Population s i z e i s mediated or regulated through the r e l a t i v e  a c t i o n of two a n t i t h e t i c a l processes, reproduction and m o r t a l i t y .  The part  that each of these i n e x t r i c a b l y - r e l a t e d processes plays i n determining animal numbers i s i m p e r f e c t l y known f o r a l l species, but most population r e g u l a t i o n t h e o r i s t s (Lack, 195-4; P i t e l k a et a l . , 1955; Wynne-Edwards, 1962; G h i t t y , 196-4; C h r i s t i a n and Davis, 1964) recognize that changes i n n a t a l i t y are important, although l e s s important than changes i n m o r t a l i t y .  The  accu-  mulated information gained from studies of reproductive f u n c t i o n w i l l event u a l l y e l u c i d a t e the r o l e of reproduction i n the r e g u l a t i o n of animal numbers. This i s a study of reproduction i n Columbian b l a c k - t a i l e d deer, a species whose numbers f l u c t u a t e remarkably i n response to h a b i t a t changes (Cowan, 1945; Dasmarm and Hines, 1959; Gates, 1968; Smith, 1968). R e l a t i v e l y l i t t l e i s known about the reproductive physiology of w i l d ungulates.  Knowledge of reproductive f u n c t i o n s has come l a r g e l y from  studies of common l a b o r a t o r y and domestic species, yet a r t i f i c i a l s e l e c t i o n and unnatural h a b i t a t have undoubtedly modified or dampened the n a t u r a l r e productive rhythms of these species.  I n s p i t e of adaptive s p e c i a l i z a t i o n s  i n the reproductive processes of many mammalian species, the basic neuroendocrine system i s thought to be s i m i l a r i n a l l .  I n s i g h t s i n t o the mecha-  nism of reproductive f u n c t i o n i s obtained through comparative  studies of  many species. " I t i s evident that the e l u c i d a t i o n of the d i v e r s e p a t t e r n of mammalian reproduction i s dependent upon research from a comparative viewpoint" (Anderson and Melampy, 1966).  In. t h i s study, the p a t t e r n of reproduction i n female deer w i l l be examined l a r g e l y through h i s t o l o g i c a l a n a l y s i s of t h e i r o v a r i e s , and the r e s u l t s w i l l be compared with those f o r other species.  2  N a t a l i t y rates i n n a t u r a l populations are d i f f i c u l t to determine, although good estimates can be obtained by k i l l i n g large numbers of pregnant females, a method c o s t l y to both the population and to the i n v e s t i g a t o r . Since the i n i t i a l discovery by Cheatum (194-9) that estimates of f u t u r e and past reproductive performance could be obtained by examining the ovaries of deer, the method has been applied to a v a r i e t y of species with varying success.  Notable among these are studies on w h i t e - t a i l e d deer (Cheatum and  Severinghaus, 1950; Gibson, 1957; Ransom, 1967), b l a c k - t a i l e d deer  (Taber,  1953; Golley, 1954), mule deer (Robinette et a l . , 1955), e l k (Halazon  and  Buechner, 1956; Morrison, 1960), moose ( P i m l o t t , 1959; Simkin, 1967), and caribou (McEwan, 1962).  I n most of the above studies the ovaries  ex-  \7ere  amined macroscopically and i n s u f f i c i e n t information was obtained on the o r i g i n s and v a r i a t i o n s of the various ovarian s t r u c t u r e s .  I t i s imperative  to f i n d q u a n t i t a t i v e r e l a t i o n s h i p s between corpora l u t e a and f e t u s e s , and between corpora l u t e a and corpora l u t e a scars.  In t h i s study, the r e l a t i o n -  ship between morphology of the ovary and reproductive f u n c t i o n i s ascertained. A g e - s p e c i f i c reproductive f u n c t i o n i s l i t t l e known i n n a t u r a l populations because of the d i f f i c u l t y of a c c u r a t e l y determining age.  Pre-  vious studies of deer reproduction have r e l a t e d f u n c t i o n to age estimated the r e l a t i v e l y inaccurate tooth wear and eruption method.  by  I n t h i s study,  many aspects of reproductive f u n c t i o n are r e l a t e d to age determined accura t e l y from sections of teeth (Low and Cowan, 1963; Ransom, 1966; 1966; E r i c k s o n and S e l i g e r , 1969).  Gilbert,  I n a d d i t i o n , t h i s study i s f a c i l i t a t e d  by  two recent research p r o j e c t s on the r e l a t i o n s h i p s between deer and the e n v i ronment of the Northwest Bay study area.  One researcher examined the vege-  t a t i o n of a s e r i e s of age-classes f o l l o w i n g logging and estimated the use deer of each s e r a i stage (Gates, 1968).  by  Another i n v e s t i g a t o r examined the  e f f e c t of hunting and changes i n vegetation on the q u a n t i t y and q u a l i t y of the  3  population (Smith, 1968).  Their data enabled me to examine the reproductive  performance of i n d i v i d u a l s and groups of i n d i v i d u a l s i n terms of known changes i n the population and i n terms of c e r t a i n aspects of the environment. The s p e c i f i c o b j e c t i v e s of t h i s study are: 1.  To determine, at various stages of the annual reproductive c y c l e , the morphological and h i s t o l o g i c a l features of the deer ovary, with emphasis on the growth and a t r e s i a of f o l l i c l e s and corpora l u t e a .  2.  To determine the length of the breeding season, incidence of r e p e t i t i v e breeding, length of the estrous c y c l e , m o r t a l i t y r a t e of ova, and f e t a l growth patterns.  3.  To determine a g e - s p e c i f i c reproductive r a t e s of female Columbian b l a c k - t a i l e d deer i n the study region and  there-  by the age of i n i t i a l breeding, age of maximum f e r t i l i t y , and the e f f e c t of age on i n t r a - u t e r i n e 4-.  mortality.  To evaluate the methods of gross and microscopic  ovarian  a n a l y s i s as a means of determining past, present  and  f u t u r e n a t a l i t y of a population of deer. 5.  To evaluate some of the f a c t o r s that a f f e c t the f e r t i l i t y of the population..  4  2.  The Study A r e a . R e p r o d u c t i o n i s i n f l u e n c e d by t h e h a b i t a t , w h i c h  i n turn i s  i n f l u e n c e d by g e o l o g i c a l , t o p o g r a p h i c a l , and c l i m a t i c f a c t o r s . d e s c r i p t i o n of the study r e g i o n f o l l o w s .  More d e t a i l e d i n f o r m a t i o n , espec-  i a l l y on t h e v e g e t a t i o n , may be found i n Gates (1968) and Smith N o r t h w e s t Bay i s an u n i n h a b i t e d  (1968).  a r e a o f about 135 square m i l e s i n  the E n g l i s h m a n R i v e r watershed on t h e e a s t e r n B. C.  A brief  s l o p e o f Vancouver I s l a n d ,  E l e v a t i o n s range from 4-00 f e e t i n t h e e a s t , t o n e a r l y 6000 f e e t  (Mt. Arrowsmith) i n t h e w e s t , a l t h o u g h most o f t h e a r e a l i e s between 1000 and 3000 f e e t .  P h y s i o g r a p h i c a l f e a t u r e s above 2000 f e e t a r e i n c l u d e d i n t h e  Vancouver I s l a n d Land f o r m , w h i l e those below 2000 f e e t , f o r m i n g p a r t o f t h e C o a s t a l Trough, a r e c l a s s i f i e d as Nanaimo Lowlands ( H o l l a n d , 1964). e n t i r e r e g i o n d i p s toward t h e n o r t h e a s t , b u t  The  t h i s f e a t u r e i s somewhat obscured  by r i d g e s and r i v e r v a l l e y s t h a t d i s s e c t t h e r e g i o n .  There i s a r e m a r k a b l e  p r e c i p i t a t i o n g r a d i e n t f r o m t h e h i g h e l e v a t i o n o f t h e w e s t e r n border t o t h e low e l e v a t i o n o f t h e e a s t e r n s i d e .  The w e s t e r n p o r t i o n ( i n t h e C o a s t C l i m a t i c  R e g i o n ) r e c e i v e s more t h a n 100 i n c h e s y e a r l y , whereas t h e e a s t e r n edge ( i n t h e Southwest C o a s t C l i m a t i c R e g i o n ) r e c e i v e s o n l y 30 t o 4-0 i n c h e s . r e g i o n , however, r e c e i v e s f r o m 4-0 t o 60 i n c h e s a n n u a l l y . higher  e l e v a t i o n s are blanketed  Most o f t h e  I n w i n t e r , the  i n deep snow,but a t l o w e l e v a t i o n s snow  cover i s s p o r a d i c and u n p r e d i c t a b l e . K r a j i n a ' s (1964) b i o g e o c l i m a t i c c l a s s i f i c a t i o n o f t h e g e n e r a l s t u d y a r e a i n c l u d e s two r e g i o n s s u b d i v i d e d  i n t o t h r e e zones:  a Coastal  Douglas F i r Zone a t e l e v a t i o n s up t o 1500 f e e t ; a C o a s t a l Western Hemlock Zone a t e l e v a t i o n s between 1500 and 3500 f e e t ; and a M o u n t a i n Hemlock Zone a t e l e v a t i o n s between 3700 and 6000 f e e t . a t i o n s have been d e s c r i b e d  Subzones and t h e i r p l a n t a s s o c i -  by K r a j i n a (1964).  Douglas F i r (Pseudotsuga  m e n z i e s i i v a r . m e n z i e s i i ) i s t h e most common c l i m a x s p e c i e s f o l l o w e d by  5  (Tsuga h e t e r o p h y l l a ) , western red cedar (Thuja p l i c a t a ) , balsam f i r grandis) and lodgepole pine (Pinus contorta) (Gates, 1968).  (Abies  Clear-cut  patch-logging of Northwest Bay commenced i n 1939 and has continued to the present, thereby c r e a t i n g a great number of s e r a i stages. Logging started e a r l i e r , and has been more extensive, at lower elevations.  For t h i s reason, and because of geoclimatic f a c t o r s , the age  and composition of the vegetation v a r i e s considerably w i t h i n the study area. Smith (1968) d i v i d e d the area i n four zones and c a l c u l a t e d the percentage of various s e r a i stages i n each zone.  C h a r a c t e r i s t i c s of c e r t a i n s e r a i  stages and t h e i r r e l a t i v e use by deer are presented by Gates (1968).  He  was  able to c a l c u l a t e the s e r a i stage composition that was optimal f o r deer. Reproductive performance of deer from d i f f e r e n t portions of the study area w i l l be evaluated i n terms of the above r e s u l t s . The b l a c k - t a i l e d deer i s the only wild ungulate present.  Mountain  l i o n s are t h e i r c h i e f n a t u r a l predator, with black bears and eagles c o n s t i t u t i n g a minor t h r e a t to fawns.  Each autumn a more important predator,  takes s e v e r a l hundred deer from the population.  man,  Hunting i s allowed w i t h i n  the logging c l a i m only on weekends and h o l i d a y s because loggers are present during the week.  The hunting season begins i n e a r l y September and runs to  the end of November.  A n t l e r l e s s deer may be taken only during the l a s t  three to f i v e weekends of t h i s period.  3.  The Black-Tailed Deer Of Northwest Bay. Measurements of reproductive output are most meaningful when  r e l a t e d to the stage of population growth or d e c l i n e .  During t h i s study the  Northwest Bay deer population was d e c l i n i n g p r i m a r i l y because of a d e t e r i o r a t i o n i n h a b i t a t and, secondarily,because of hunting (Smith, 1 9 6 8 ) .  The  number of deer removed s t e a d i l y increased from a low of 120 i n 1 9 5 6 , t o  6  approximately  700 p e r year i n 1962 and 1963. I n c l u d e d i n t h e t o t a l f o r t h e  l a t t e r y e a r were 78 f e m a l e s k i l l e d excluded,  t h e peak k i l l  e x p r e s s l y f o r t h i s study.  I f these a r e  by h u n t e r s was 1962. I n 1961-62, Gates  (1968),  on t h e b a s i s o f p e l l e t - g r o u p c o u n t s , e s t i m a t e d w i n t e r and summer d e n s i t i e s o f 50 and 67 deer p e r square m i l e w i t h i n a 10 square m i l e p o r t i o n o f Northwest Bay.  S i n c e 1962-63, t h e number o f deer k i l l e d has s t e a d i l y d e c l i n e d t o a  low o f 142 i n 1968.  Although  some o f these changes r e f l e c t changes i n  h u n t i n g p r e s s u r e , t h e y a l s o i n d i c a t e changes i n p o p u l a t i o n s i z e . The p o p u l a t i o n has been e x t e n s i v e l y e x p l o i t e d f o r many y e a r s . (1968) e s t i m a t e d  t h a t , i n each year from i960 t o 1962,  Smith  hunting m o r t a l i t y  accounted f o r 2 0 % o f t h e p o p u l a t i o n and n a t u r a l m o r t a l i t y removed an a d d i t i o n a l 10%. was  He concluded  t h a t p r i o r t o and d u r i n g t h e p e r i o d o f t h i s s t u d y  there  no o v e r a l l s i g n i f i c a n t change i n t h e age s t r u c t u r e o f t h e p o p u l a t i o n .  There was, however, a g r a d u a l d e c l i n e i n average w e i g h t o f deer w i t h i n s e x and  age c l a s s e s .  The above f i n d i n g s and o t h e r s a l l i n d i c a t e t h a t i t was a  d e c l i n i n g p o p u l a t i o n , b o t h w i t h r e s p e c t t o numbers and p h y s i c a l c o n d i t i o n , f r o m w h i c h t h e samples f o r t h i s s t u d y were drawn. The d e c l i n e i n t h e p o p u l a t i o n may have been a b e t t e d by two w i n t e r s (1964.-65 and 1965-66) o f extreme s n o w f a l l and a summer (1967) t h a t was warmer and d r i e r than u s u a l .  4..  The R e p r o d u c t i v e  C y c l e Of Deer.  Some f e a t u r e s o f t h e r e p r o d u c t i v e c y c l e i n t h e Columbian b l a c k t a i l e d deer have been summarized by Cowan (1956), Brown (1961), Ommundsen (1967), and West (1968).  Deer a r e p o l y t o c o u s  (may r e l e a s e more than one  ovum i n an e s t r o u s p e r i o d ) and m u l t i o v u l a r (may pass through c y c l e s i f c o n c e p t i o n does n o t o c c u r ) .  several estrous  E s t r u s l a s t s 24- t o 36 h o u r s and t h e  7  l e n g t h o f t h e e s t r o u s c y c l e i n c a p t i v e b l a c k - t a i l e d d e e r i s 22 t o 28 days. B r e e d i n g u s u a l l y o c c u r s i n November and December b u t may extend i n t o  March.  Fawns o f 0. hemfonus columbianus r a r e l y c o n c e i v e e x c e p t i n c a p t i v i t y .  The  m a j o r i t y o f y e a r l i n g s produce o n l y one faun,whereas two, or o c c a s i o n a l l y j t h r e e fawns.  o l d e r does produce one,  I m p l a n t a t i o n o f embryos o c c u r s 27 t o 30  days a f t e r c o n c e p t i o n (Cheatum and Morton, 194-2; Hudson and Browman, 1959)G e s t a t i o n a v e r a g e s about 203 days i n c o n f i n e d b l a c k - t a i l e d d o e s , b u t between 183 and 212 days in  (Cowan, 1956; Brown, 1 9 6 l ) .  varies  Most fawns a r e born  June.  5. 5. 1  Methods. C o l l e c t i o n Of T i s s u e s . Most o f t h e t i s s u e s used i n t h i s s t u d y were o b t a i n e d f r o m female  d e e r t a k e n by h u n t e r s d u r i n g t h e r e g u l a r f a l l h u n t i n g seasons f r o m 1963 t o 1967.  R e p r o d u c t i v e t r a c t s , w e i g h t s , body measurements, and t h e l o w e r jaw  were o b t a i n e d f r o m each deer a t a h u n t e r - c h e c k s t a t i o n o p e r a t e d by t h e B r i t i s h Columbia F i s h and W i l d l i f e Branch.  Because  t h e s t a t i o n was l o c a t e d  on t h e o n l y open a c c e s s r o a d i n t o t h e s t u d y a r e a , a l l h u n t e r s were stopped and p r o v i d e d w i t h d i a g r a m s , and i n 1 9 6 3 , w i t h p h o t o g r a p h s , so t h e y c o u l d  identify  t h e female r e p r o d u c t i v e t r a c t and l e a v e i t i n t a c t i n t h e deer f o r l a t e r r e moval a t t h e s t a t i o n . When a r e p r o d u c t i v e organ was removed, t h e appearance mammary g l a n d , u t e r u s , and o v a r i e s was n o t e d .  of the  I f surface ovulation  sites  were p r e s e n t , t h e i r l o c a t i o n and c o l o r a t i o n were noted on a s m a l l d r a w i n g of the ovary.  The f r e s h w e i g h t o f each u t e r u s , severed a t m i d - c e r v i x , was  also obtained. From October 1963 t o June 1964, an a d d i t i o n a l 77 f e m a l e deer were c o l l e c t e d p e r i o d i c a l l y throughout a r e p r o d u c t i v e c y c l e (from p r e - o v u l a t i o n t o  8  post-partum).  These c o l l e c t i o n s were supplemented by 10 animals taken  during the winter and spring of 1965 and 1966.  In a d d i t i o n , f i v e animals  r a i s e d at the U n i v e r s i t y of B r i t i s h Columbia, whose reproductive h i s t o r i e s were known, provided comparative information.  Two were adult females t r e a t -  ed w i t h e s t r a d i o l i n an experiment i n v o l v i n g the h i s t o l o g y of the p i t u i t a r y gland, two were used to obtain known-age fetuses f o r a study by Ommundsen (1966), and one was an i n j u r e d animal of 9 months.  A t o t a l of 4-61 females  were a v a i l a b l e f o r study.(Table l ) . I n a d d i t i o n , ovaries from 15 fetuses were sectioned. Table 1.  The number, age,and c o l l e c t i o n period of deer whose ovaries were studied. Year 1963 1963-64 19641965 1965 1965 1966 1967  Fawns  Yrlg.  Adults  Total  Nov-Dec Dec-June Nov-Dec Jan Nov Apr-May Nov Nov-Dec Other s  249 9 0 15 0 0 1 1  26 16 28 0 21 .3 8 12 2  73 62 4-0 2 51 5 26 20 7  123 77 87 2 87 8 3433 10  Subtotals  59 .  116  286  4-61  Period  Fawns: 0 to 1 years; Yearling ;s: 1 to 2 years; Adults : 2+ ;  5. 2  Weights And Measurements. The t o t a l weight of deer was measured to the nearest pound on a  beam balance or a platform scale and no adjustment was made f o r l o s s of blood.  Weight of the animal,with a l l v i s c e r a removed (dressed w e i g h t ^ was  also measured t o the nearest pound on a plajtform or beam scale.  I f the  l i v e r remained i n the c a r c a s s , 2 pounds were subtracted, and i f the heart remained, the measured weight was reduced by 1 pound.  The f r e s h weight  9  of t h e r e p r o d u c t i v e t r a c t , u t e r u s , f e t u s , mammary g l a n d , l i v e r ,  heart,  k i d n e y s , and s p l e e n were measured t o t h e n e a r e s t gram on a t r i p l e beam balance.  P o s t - f i x a t i o n weights of the u t e r u s , f e t u s , o v a r i e s , p i t u i t a r y  g l a n d , a d r e n a l g l a n d s , and t h y r o i d glands were measured on e l e c t r i c  balances  t o a d e g r e e o f p r e c i s i o n a p p r o p r i a t e t o t h e w e i g h t o f t h e o b j e c t (See " H i s t o l o g i c a l Technique" f o r f i x a t i v e s  used).  H i n d - f o o t l e n g t h I s t h e d i s t a n c e between the t i p o f t h e h o o f and the t i p o f t h e c a l c a n e o u s measured i n m i l i m e t e r s , • w i t h a s t e e l r u l e . G i r t h i s the circumference  of t h e a n i m a l  j u s t p o s t e r i o r to the  f o r e l e g s , measured t o t h e n e a r e s t m i l i m e t e r w i t h a s t e e l r u l e a t a t e n s i o n of 5 pounds (measured on a s p r i n g s c a l e , o f 10 pounds c a p a c i t y ,  attached  to the r u l e ) . Crown-rump and forehead-rump l e n g t h s a r e the d i s t a n c e i n a s t r a i g h t l i n e f r o m t h e crown (embryo) or f o r e h e a d  ( f e t u s ) t o t h e rump,  measured t o t h e n e a r e s t m i l i m e t e r w i t h a f l e x i b l e s t e e l r u l e .  5. 3  Age  Determination. The ages o f deer were e s t i m a t e d  when t h e lower  by the wear and e r u p t i o n method  jaw was removed f r o m t h e d e e r a t t h e c h e c k - s t a t i o n .  l a t e r date, the assigned  At a  ages were r e c h e c k e d by t h e same method, e x c e p t  t h a t known-age jaws were used c o m p a r a t i v e l y  t o i d e n t i f y age c l a s s e s .  Be^  cause t h e above method was n o t a c c u r a t e f o r deer o l d e r t h a n two y e a r s , t h e t o o t h - s e c t i o n method (Low and Cowan, 1963)  was used t o d e t e r m i n e the age o f .  a l l a d u l t s , many y e a r l i n g s , and some fawns used i n t h i s s t u d y . c u r a c y o f the t o o t h - s e c t i o n method was confirmed  The a c -  through examination  of  10  teeth removed from known-age deer from Northwest Bay.  5. 4- H i s t o l o g i c a l Technique. 5. L\. 1  Ovaries.  F i x a t i o n was based on the intended  treatment.  Bouin's f l u i d was used from 1963 to 1966,when the ovaries were t h i n sectioned, and e t h y l a l c o h o l - f o r m a l i n - a c e t i c acid (AFA) was used to harden the t i s s u e s f o r macroscopic a n a l y s i s i n 1967.  The l a t t e r , a c o l o r l e s s  f i x a t i v e , d i d not mask any color inherent i n the t i s s u e .  In c o n t r a s t ,  the p i c r i c acid i n Bouin's f l u i d colored the t i s s u e a b r i g h t yellow,which masked yellow and orange pigments important i n the macroscopic i d e n t i f i c a t i o n of ovarian s t r u c t u r e s .  A f t e r a few days i n e i t h e r f i x a t i v e , t h e  ovaries were t r a n s f e r r e d to 70% ethanol f o r storage. Gross sections of ovaries were obtained i n a manner described by Cheatum (194-9). c i s i o n s about  A sharp s c a l p e l was used to make a s e r i e s of p a r a l l e l i n 1 mm apart throughout the ovary.  These i n c i s i o n s d i d not  sever the h i l a r portion,so that a book of sections 1 mm t h i c k was produced. A l l s t r u c t u r e s were measured and described as the ovary was s l i c e d .  Some  ovaries, cut t r a n s v e r s e l y i n the above manner, were subsequently dehydrated, cleared, and embedded i n paraplast f o r the preparation of t h i n microscopic s e c t i o n s .  Because the above technique was time-consuming, most of  the comparisons between gross sections of ovaries and microscopic sections were made on small pieces of t i s s u e removed from the t h i c k s e c t i o n s .  Small  blocks of ovarian t i s s u e , c o n t a i n i n g p e r t i n e n t or perplexing s t r u c t u r e s , were e a s i l y processed using r o u t i n e h i s t o l o g i c a l methods. s t r u c t u r e s i n a p a i r of ovaries were processed one or two s l i d e s .  together,and  Several such a l l placed on  Blocks of t i s s u e were cut i n t o various s i z e s and  shapes to i d e n t i f y each s t r u c t u r e . P r i o r to 1967, whole ovaries were sectioned s e r i a l l y i n t h e i r  11  e n t i r e t y , s o t h a t i n t e r n a l morphology was n o t d i s r u p t e d . i n d i c a t e d t h a t s u i t a b l e s e c t i o n s c o u l d n o t be o b t a i n e d h i s t o l o g i c a l procedures.  T r i a l runs using  routine  I t was n e c e s s a r y t o f o l l o w d e h y d r a t i o n  w i t h two  immersions i n m e t h y l berizoate f o r a t o t a l o f 4-0 h o u r s , b e f o r e f i n a l c l e a r i n g i n benzene.  T h i s t e c h n i q u e i s recommended  fibrous tissue.  The embedding medium was  Embedded r o t a r y microtome.  o v a r i e s were s e c t i o n e d Initially,  cause t h e d i a m e t e r o f t h e embedded e v e r y o o c y t e was sampled.  paraplast. a t 8 or 10 u on a  standard  o v a r i e s were c u t a t 10 u, and i n c o n s e c u t i v e  o r d e r , e v e r y t e n t h s e c t i o n was p l a c e d  doubled  by Humason (196.2) f o r tough,  on l a b e l l e d a l b u m i n i z e d  slides.  o o c y t e was s l i g h t l y g r e a t e r  Be-  t h a n 100 u,  S u b s e q u e n t l y , t h e i n t e r v a l between s e c t i o n s was  ( e v e r y 2 0 t h s e c t i o n was t a k e n ) t o r e d u c e t h e number o f s e c t i o n s .  S e r i a l s e c t i o n s o f o o c y t e s f r o m medium and l a r g e f o l l i c l e s were  obtained  because t h e appearance o f t h e o o c y t e i s o f d i a g n o s t i c v a l u e i n i n t e r p r e t i n g the s t a g e o f f o l l i c l e m a t u r a t i o n and t h e h e a l t h o f t h e o o c y t e . A few e x t r a s e r i a l s e c t i o n s o f each o v a r y were t a k e n f o r s p e c i a l s t a i n i n g methods.  C o r p o r a l u t e a f r o m a l l c o l l e c t i o n s were s t a i n e d i n the  same s o l u t i o n s and p r o c e s s e d i n a few h o u r s , so t h a t t i n c t o r i a l between c o r p o r a  l u t e a could  be a t t r i b u t e d t o d i f f e r e n c e s i n t h e t i s s u e and  not t o d i f f e r e n t batches o f s t a i n . trichrome G.  S l i d e s were s t a i n e d w i t h Masson's  (Humason, 1962) or a m o d i f i e d  Massons t r i c h r o m e  Regaud s or W e i g e r t ' s h e m a t o x y l i n was used. 1  Selected  s t a i n e d w i t h p e r i o d i c a c i d - S c h i f f (PAS), PAS and Masson's c a r b o l f u c h s i n , methylene b l u e , t o l u i d i n e blue,and 5. 4-. 2  Uteri.  differences  c o n t a i n i n g orange s l i d e s were . trichrome,  aldehydethionin.  U t e r i were f i x e d i n B a k e r ' s f o r m a l i n m i x t u r e  (Humason, 1962), t h e n t r a n s f e r r e d t o AFA.  Small rectangular  pieces of the  v e n t r o - l a t e r a l w a l l between the f i r s t and second c a r u n c l e s were removed f r o m each u t e r u s  and p l a c e d  i n 70% e t h a n o l .  Duplicate h i s t o l o g i c a l  sections,  12  8 u i n t h i c k n e s s , were prepared and s t a i n e d , using a combination of PAS and Masson's trichrome s t a i n , or Masson's trichrome alone.  5. 4-- 3  Oviducts.  Oviducts from deer that were near o v u l a t i o n , or had  r e c e n t l y ovulated, were sectioned and checked f o r the presence of sperm and ova.  Oviducts were dehydrated  and embedded i n a f l a t , c o i l e d  state so  that a minimum number of sections were required to permit a search f o r the ovum.  Sections were cut a t 10 u, and every f i f t h one was placed on  a s l i d e and stained with Masson's trichrome or PAS combined with Masson's trichrome.  The l a t t e r mixture was preferred because the zona p e l l u c i d a of  the ovum stained r e d , making the search f o r ova r e l a t i v e l y easy.  When  an ovum was found, a d d i t i o n a l sections of i t were obtained from stored oviduct s e c t i o n s . Few ova were found i n the 196-4 c o l l e c t i o n , probably because they were a c c i d e n t a l l y washed out when the reproductive t r a c t s were immersed i n the f i x a t i v e .  I n 1965 and 1966,the oviduct t e r m i n i were t i e d with f i n e  s t r i n g before f i x a t i o n , r e s u l t i n g i n a higher recovery r a t e of ova.  5. -4. 4- Vaginal smears.  Vaginal smears were obtained to r e l a t e  changes i n the surface of the vagina t o ovarian f u n c t i o n and to determine, from the presence of sperm, i f the doe had r e c e n t l y copulated.  Smears  were obtained by passing a s l i d e over the surface of the v a g i n a l e p i t h e l i u m 2 cm from the c e r v i x .  E a r l y i n t h i s study,smears were a i r - d r i e d or f i x e d  for a few minutes i n absolute ethanol and l a t e r stained w i t h Weigert's hematoxylin and ponceau 2R - acid f u c h s i n , but they were not s a t i s f a c t o r y because of c e l l shrinkage and inadequate d i f f e r e n t i a t i o n of c e l l u l a r components.  I n 1967,smears were f i x e d i n equal parts of ether and 95%  ethanol and kept i n t h i s f i x a t i v e u n t i l they were stained w i t h H a r r i s '  13  h e m a t o x y l i n and  5. 4-. 5  the Schoor I I I . s t a i n ( d e N e f f ,  Teeth.  1965).  R o o t s of t e e t h were d e c a l c i f i e d f o r 2 t o 4- days i n  a m i x t u r e o f 25% f o r m i c a c i d and  5% f o r m a l i n .  After neutralization i n  aqueous l i t h i u m c a r b o n a t e the r o o t s were d e h y d r a t e d , c l e a r e d , imbedded i n p a r a p l a s t , and on s l i d e s and  sectioned  a t 10 or 12 u.  E v e r y 2 5 t h s e c t i o n was  placed  l a t e r s t a i n e d f o r 30 t o 4-0 m i n u t e s i n H a r r i s ' h a e m a t o x y l i n .  Some s l i d e s were s t a i n e d i n t h i o n i n , methylene b l u e , t o l u o d i n e c r y s t a l v i o l e t , w i t h r e s u l t s comparable or s u p e r i o r t o those  blue,  and  obtained  with hematoxylin s t a i n i n g .  5.  5.  R e c o v e r y Of Ova. The  transported  reproductive  t r a c t s of recent  t o the l a b o r a t o r y , and  f r o m the u t e r u s . needle, attached The  .  The  o s t i a l end  o v u l a t o r s were k e p t c o o l ,  f l u s h e d a f t e r r e s e c t i o n of the  of the tube was  tubes  secured around a number  t o a 10 ml s y r i n g e c o n t a i n i n g a 0.9%  22  NaCl s o l u t i o n .  c o n t e n t s of the t u b e , f l u s h e d w i t h 2 ml of f l u i d , were c o l l e c t e d i n a  watch g l a s s and  m i c r o s c o p i c a l l y examined w i t h t r a n s m i t t e d  the f i r s t f l u s h i n g was flushing  unsuccessful,  l i g h t a t 25X.  s e v e r a l more were t r i e d w i t h  increased  pressure. The u t e r i n e h o r n s were f l u s h e d w i t h a g r e a t e r volume of the  tion.  The  If  connective  t i s s u e s e p t a between h o r n s were c u t so t h a t the h o r n  c o u l d be l i g a t u r e d near the u t e r i n e body. s e v e r e d , and  solu-  the s y r i n g e n e e d l e was  The  t i p of the u t e r i n e h o r n  was  i n s e r t e d t h r o u g h the w a l l of the h o r n  a t the l i g a t u r e . Segmenting ova sank t o the bottom of the g l a s s , whereas most o f the c e l l u l a r d e b r i s i n o v i d u c t s and u t e r i n e h o r n s f l o a t e d h i g h e r f l u s h i n g medium.  I f excessive  c e l l u l a r m a t e r i a l was  i n the  e n c o u n t e r e d , some of i t  was c a r e f u l l y aspirated to f a c i l i t a t e the search f o r ova. When f i r s t sighted, ova were described  i n regard  to the stage of segmentation, the  presence of sperm, and general c y t o l o g i c a l f e a t u r e s , a f t e r u h i c h they were t r a n s f e r r e d i n s a l i n e I n a p i p e t t e to a w e l l s l i d e .  Then the ova were  viewed under higher m a g n i f i c a t i o n , stained with d i l u t e s o l u t i o n s of t o l u i d i n e blue, ponceau 2R - acid f u c h s i n , a l c i a n blue and hematoxylin, and then temporarily mounted i n p h y s i o l o g i c a l s a l i n e on a glass s l i d e .  5. 6  Microscopy. A m a g n i f i c a t i o n of 25 was s a t i s f a c t o r y f o r most i d e n t i f i c a t i o n  procedures,including  the l o c a t i o n of oocytes and ova. Linear and area  measurements were made a t a l l magnifications with a 20 x 20 u n i t ocular grid.  The volumes of n e a r l y - s p h e r i c a l objects were c a l c u l a t e d using the  formula V = 4/3 If (r^ x dimension r a d i i .  X r^) where r j _ , T^, and r ^ represent the three-  The volumes of non-spheroid objects were obtained  by the  grid method, where V = A x d, where A (area) was determined a t i n t e r v a l s throughout the structure by counting g r i d i n t e r s e c t i o n s , and d (depth) was the i n t e r v a l between sections i n which A was determined.  Usually,every  s e c t i o n on the s l i d e was used i n determining volumes,but f o r large s t r u c t u r e s , A was determined from every 2nd, 4-th, or 8th s e c t i o n on the s l i d e .  Suf-  f i c i e n t p r e c i s i o n i n the measurement of volume was achieved i f A was determined i n a t l e a s t 10 sections,and  a t l e a s t 40 to 50 grids covered  the s t r u c t u r e i n the s e c t i o n i n which i t s area was greatest.  In practice,a  m a g n i f i c a t i o n and s e c t i o n i n t e r v a l was chosen t o meet the above r e q u i r e ments, grids were counted, t o t a l l e d , then m u l t i p l i e d by a f a c t o r dependent on the magnification and the s e c t i o n i n t e r v a l . Compound binocular microscopes equipped with ocular scales were used f o r d e t a i l e d h i s t o l o g i c a l evaluation and measurement of s t r u c t u r e s .  15 6.  RESULTS.  SECTION 1.  STRUCTURE OF THE  FETAL OVARY.•  Development of the f e t a l o v a r y and p l e of 14- f e t u s e s and and  one  p o s t n a t a l fawn.  o o g e n e s i s were s t u d i e d i n a sam-  Measurements of the  fetuses,  some c h a r a c t e r i s t i c s of t h e i r o v a r i e s , a r e r e c o r d e d i n Table 2.  l e a s t v a r i a b l e of the t h r e e f e t a l measurements i n the t a b l e , l e n g t h , was obtained  used t o e s t i m a t e  f r o m a doe  f e t a l age.  hind-foot  known-age f e t u s , U34,  One  The  r a i s e d i n c o n f i n e m e n t a t the U n i v e r s i t y of  was  British  0 Columbia.  A l t h o u g h the f e t u s of 102 days was  of s i m i l a r age  f r o m a w i l d doe,  o v a r i e s were e q u i v a l e n t deer (Table Table 2.  s l i g h t l y l a r g e r than a f e t u s  the w e i g h t and d e g r e e of development of i t s  t o t h a t of f e t u s e s 122  t o 14-4- days o l d f r o m w i l d  2). Measurements of deer f e t u s e s and c h a r a c t e r i s t i c s of ovaries at selected periods during gestation.  O v a r i e s (both)  Fetus Specimen No. T32 T27 T36 • T37 • H U34T43 T52 T84  T65 T66  T76 T77 T70  BG  Age (Days) 6468 73 79 98 102 122 123 14-4170 177 194 202 2041-2PP  FR (mm) 79 89 112  126  166 187 225 222 301 350 366 4-03 4-60 445  FR - Forehead-rump len£ HF P  - H i n d - f o o t l e n gth - Primordial  their  HF (mm) 20 24 31 37 60 71 92 93 127 170 182 211 225 227 239  Wt (g) 20  26 53 63 151 257 417 442 858 1395 1674 2103 2852 2670 2200  Wt (mg) 4.8 8.0 16.5 12.1 15.4 29.2 18.0 25-0 29.5 29.2 42.4 31.0 33.9 42.7 28.0 Py S T PP  Diam (u) Largest Sex C e l l 14 12 11 12 18 29 25 20 28 35 30 65 33 72 60  -  Primary Secondary Tertiary P o s t - p a r turn  Diam L a r g e s t F o l l i c l e (u) and type  32P 31P 28P 35Py 60S 43 S 125T 104T 220 T 150T  F a c i n g page 1 6  Plate 1 Development of the F e t a l Ovary ( A l l c r o s s s e c t i o n s of o v a r i e s a t  10X)  1. Two a b n o r m a l l y - f u s e d o v a r i e s i n a f e t u s about 98 days a f t e r i t s conception. The c e n t r a l w h i t e r e g i o n i s the mesonephric r u d i m e n t t h a t g i v e s r i s e t o the r e t e a p p a r a t u s . Note the l a r g e blood v e s s e l s i n the c o r t e x . 2. An o v a r y i n a f e t u s 102 days o l d f r o m a doe r a i s e d i n c a p t i v i t y . r e t e o v a r i ( c e n t r a l e l o n g a t e s t r u c t u r e ) has condensed and a s m a l l medulla i s evident (white r e g i o n ) .  The  3. An o v a r y i n a f e t u s about 122 days o l d . The c o r t e x i s l a r g e r and c o n t a i n s l a r g e v e s s e l s . . Growth of the c o r t e x produces a mushrooml i k e form. U. An o v a r y i n a f e t u s about \L\L, days o l d . The w i d t h of the c o r t e x , r e l a t i v e t o the m e d u l l a , i s l e s s t h a n i n younger o v a r i e s . 5. An o v a r y i n a f e t u s about 170 days o l d . The m e d u l l a , c o n t a i n i n g a round r e t e o v a r i , i s l a r g e and f u r t h e r d i f f e r e n t i a t e d ( w h i t e r e g i o n s ) . F o l l i c l e s , t o the s e c o n d a r y s t a g e , are abundant i n the c o r t e x . 6.  An o v a r y of a p o s t - p a r t u m fawn. The c o r t e x i s h y a l i n i z e d and appears d a r k i n the photograph. The c o r t i c o - m e d u l l a r y b o r d e r i s marked by small t e r t i a r y f o l l i c l e s .  17  I n g e n e r a l t h e r e was  a progressive increase,with age,in  weight, of o v a r i e s , s i z e of the l a r g e s t sex c e l l , follicle.  The  two  l . l ) , b u t t h e y may  and  s i z e of the l a r g e s t  o v a r i e s of the 98-day f e t u s were p a r t i a l l y f u s e d have s e p a r a t e d  l a t e r i f d e a t h had  s e r i e s , t o the term f e t u s , i s p r e s e n t e d  the  below.  Cortico-Medullary Relationships. I n the e a r l i e s t stage a v a i l a b l e , a f e t a l age  c o r t e x was  dominant and  connective  t i s s u e f i b e r s supported  i n c l u d i n g sex c e l l s . age  (Plate  not i n t e r v e n e d . The i n -  f e r r e d sequence o f o v a r i a n development, f r o m the e a r l i e s t stage of  1. 1  the  s t r u c t u r a l l y uniform  o f 98 d a y s , when i t was  ( P l a t e 1. 1 ) .  The  throughout.  clumps and  The m e d u l l a was  of 6 4 d a y s , the A l o o s e network o f  w h o r l s of e p i t h e l i a l  not c l e a r l y d i s c e r n i b l e u n t i l a f e t a l  composed l a r g e l y o f a d i f f u s e r e t e o v a r i i  growth of the m e d u l l a was  p r o g r e s s i v e up t o a f e t a l  o f 177 d a y s , when i t reached i t s maximum s i z e r e l a t i v e t o the ( P l a t e 1.1-5).  C o i n c i d e n t w i t h growth of the m e d u l l a ,  i a l c e l l s f r o m the zone and connective  tissue.  cells,  was  age  cortex  a l o s s of e p i t h e l -  an i n c r e a s e i n the amount and d i f f e r e n t i a t i o n o f  There was  a marked i n c r e a s e i n the number and  hyalini-  z a t i o n o f f i b e r s d u r i n g the l a s t month of g e s t a t i o n . D u r i n g e a r l y growth, the o v a r i a n m e d u l l a merged i n t o the c o r t e x , b u t a t 170 d a y s , when the c o r t e x was m a r c a t i o n was  sharp ( P l a t e 1. 5).  outer  r e l a t i v e l y t h i n n e s t , the  de-  From 73 t o I 4 4 days o f a g e , t h e r e was  marked l o b u l a t i o n o f the l o o s e l y k n i t o u t e r c o r t e x ( P l a t e 2. 2, 7, and I n o l d e r f e t u s e s (170 days t o term), the c o r t e x became f i b r o u s and  a  8).  a layer  o f f i b e r s , the t u n i c a a l b u g i n e a , formed under the s u r f a c e e p i t h e l i u m .  A  s m a l l i n c r e a s e i n the c o r t i c a l t h i c k n e s s , d u r i n g the f i n a l p r e n a t a l month, produced an o v a r y w i t h c o r t e x and m e d u l l a p r o p o r t i o n a t e t o t h a t o f a fawn o f 5 months.  18  1. 2  The  Surface The  Epithelium.  s u r f a c e e p i t h e l i u m c o n t r i b u t e d c e l l s t o the i n t e r i o r f o r the  e n t i r e p e r i o d of development,but p r o l i f e r a t i o n was 14-4- days a f t e r c o n c e p t i o n , a n d had 6).  g r e a t e s t f r o m 98  to  v i r t u a l l y ceased a t term ( P l a t e 2.2  and  I n the f e t u s of 177 d a y s , i n v a g i n a t i o n s of e p i t h e l i u m were r e s t r i c t e d  t o l o c a l i z e d a r e a s of the o v a r i a n s u r f a c e .  Some i n v a g i n a t i o n s a t the peak  of p r o l i f e r a t i o n i n v o l v e d dozens of c e l l s , but more commonly o n l y a s m a l l number entered  the c o r t e x a t one  location.  U s u a l l y , t h e e p i t h e l i u m was  s i n g l e l a y e r of c e l l s , but i n some a r e a s l a y e r i n g o c c u r r e d  w i t h subsequent  wedging of c e l l s i n t o the stroma.  A f t e r the t u n i c a a l b u g i n e a was  established  s u r f a c e e p i t h e l i a l c e l l s commonly  entered  (170 and  177 d a y s ) , t h e  the u n d e r l y i n g t i s s u e a t a t a n g e n t .  when p r o l i f e r a t i o n of the e p i t h e l i u m was entered  the e p i t h e l i u m became s t r e t c h e d and  1. 3  In mid-gestation  The  well  ovaries,  greatest, c e l l s occasionally  m e i o t i c prophase i n the s u r f a c e l a y e r .  f e t a l development,the e p i t h e l i u m was  a  From 14-4- t o 177 days of  a smooth l a y e r of c u b o i d a l c e l l s , b u t f l a t t e n e d i n o v a r i e s of term f e t u s e s .  Germ C e l l . The  i n Table 2.  growth of the germ c e l l i n a s e r i e s of f e t u s e s i s summarized O n l y a r e l a t i v e l y s m a l l number of oogonia were c l e a r l y d i s -  t i n g u i s h a b l e f r o m o t h e r e p i t h e l i a l c e l l s i n f e t u s e s 64- t o 79 days o l d . Most of t h e s e a c i d o p h i l i c c e l l s measured 5 t o 8 u i n d i a m e t e r , b u t some a t t a i n e d d i a m e t e r s o f 14- P-.  I n the 98-day f e t u s , o o g e n i c c e l l s were more  numerous i n the o u t e r c o r t e x , w h e r e most ranged between 7 and  15  u.  By t h i s s t a g e , some germ c e l l s had reached the r e s t i n g phase of m e i o s i s  and  were e n c i r c l e d by a few e p i t h e l i a l c e l l s ( P l a t e 2. l ) . There were many m u l t i n u c l e a t e oogonia and  o o c y t e s , b u t i t was  not a s c e r t a i n e d i f these  o r i g i n a t e d f r o m m i t o t i c d i v i s i o n of o n l y the n u c l e u s , o r  from coalescence  of  F a c i n g page 19  Plate 2 H i s t o l o g y o f t h e F e t a l Ovary. 1. The c o r t e x o f t h e o v a r y i n a f e t u s about 98 days o l d . S i n g l e and m u l t i n u c l e a t e oogonia ( l a r g e d a r k a r e a s ) occur i n l o o s e l y o r g a n i z e d c o n n e c t i v e t i s s u e . Lower r i g h t i s a l a r g e blood v e s s e l (X4OO). 2. The o u t e r c o r t e x o f an o v a r y i n a f e t u s 102 days o l d t a k e n f r o m a c a p t i v e doe. Two i n v a g i n a t i o n s o f t h e g e r m i n a l e p i t h e l i u m a r e e v i d e n t . Note t h e c o r d s o f c e l l s (X4OO). 3. The same o v a r y as 2.2 t o show n u c l e i o f oogonia m e i o s i s (X800).  i n prophase o f  4. Same as 2. 3. Note t h e t e l e p h a s e ( l e f t c e n t e r ) o f m i t o s i s and o t h e r n u c l e i i n prophase o f m e i o s i s (X4OO). 5. The deep c o r t e x o f t h e same o v a r y t o i n d i c a t e t h e i n c r e a s e d amounts of c o n n e c t i v e t i s s u e and t o i l l u s t r a t e t h a t many oogonia n u c l e i have reached t h e d i c t y a t e stage o f m e i o s i s (X400). 6. The s u r f a c e e p i t h e l i u m and s u b j a c e n t c o r t e x o f an o v a r y i n a f e t u s about 122 days o l d . The w h e e l - l i k e cord c o n t a i n s many oogonia i n prophase o f m e i o s i s . T h i c k e n i n g o f t h e g e r m i n a l e p i t h e l i u m ( t o p l e f t ) i s t h e f i r s t stage i n t h e i n v a g i n a t i o n o f c e l l s i n t o t h e c o r t e x (X600). 7. The o u t e r c o r t e x , i n c l u d i n g t h e g e r m i n a l e p i t h e l i u m , o f a f e t a l o v a r y about 122 days o l d . Clumps ( o r c o r d s ) o f c e l l s , i n c l u d i n g m u l t i n u c l e a t e oogonia w i t h condensed n u c l e i , a r e numerous. The p o o r l y d e f i n e d d a r k g r e y a r e a (lower c e n t e r ) i s a blood v e s s e l (X4OO). 8. The same s e c t i o n as 2. 7 b u t deep i n t h e c o r t e x where oogonia and p r i m a r y o o c y t e s a r e l o o s e l y e n c i r c l e d by e l o n g a t e p r e - g r a n u l o s a c e l l s (X800). 9. The c o r t e x o f an o v a r y about 144 days o l d . There i s a marked i n c r e a s e i n t h e amount o f c o n n e c t i v e t i s s u e between t h e s e x c e l l s t h a t range i n development f r o m oogonia t o p r i m a r y o o c y t e s (X400). 10. An o v a r y f r o m a p o s t n a t a l fawn t o i l l u s t r a t e a t e r t i a r y f o l l i c l e , two a t r e t i c f o l l i c l e s ( b l a c k s t r u c t u r e s ) , and i n c r e a s e d amount o f c o n n e c t i v e t i s s u e (X400).  20  c y t o p l a s m ( P l a t e 2. 1, 7 and 9 ) . i n older f e t a l  M u l t i n u c l e a t e s t r u c t u r e s were l e s s common  ovaries.  I n f e t a l o v a r i e s 102, 122,and 123 days o l d , t h e r e were a l l s t a g e s of s e x - c e l l development, f r o m oogonia t o t h e p r i m a r y oocyte stage 2.2-8).  (Plate  The germ c e l l s o c c u r r e d i n clumps or c o r d s i m m e d i a t e l y below the  e p i t h e l i u m but t h e y were more s c a t t e r e d deep i n t h e c o r t e x .  A great  number o f sex c e l l s i n the o u t e r c o r t e x were i n the m e i o t i c prophase maturation d i v i s i o n ,  whereas most of those o c c u r r i n g deep i n the c o r t e x  were i n t h e p r i m a r y oocyte s t a g e and were e n c l o s e d i n p r i m o r d i a l Many oogonia and p r i m a r y o o c y t e s were a t r e t i c .  follicles.  I n the 1 4 4 - d a y f e t a l  o v a r i e s t h e r e were s t i l l many n u c l e i i n m e i o t i c d i v i s i o n , around  of  especially  t h e p e r i p h e r y , but a g r e a t e r p r o p o r t i o n o f t h e germ c e l l s had  en-  l a r g e d t o the p r i m a r y oocyte s t a g e and \^ere e n c i r c l e d by f o l l i c u l a r c e l l s . V i r t u a l l y a l l o f the sex c e l l s i n o v a r i e s 170 and 177 days o l d were i n the d i c t y a t e phase of the f i r s t m e i o t i c d i v i s i o n . ensheathed  i n epithelial  follicles.  c e l l s and  Most p r i m a r y o o c y t e s were  some were c o n t a i n e d i n secondary  I n p e r i n a t a l o v a r i e s t h e r e was  c o n s i d e r a b l e growth o f the  mary o o c y t e and i n c r e a s e d numbers o f a t r e t i c  oocytes.  pri-  The m a j o r i t y o f  o o c y t e s , l o c a t e d i n p r i m o r d i a l or p r i m a r y f o l l i c l e s , r a n g e d between 15 and 20 u i n diameter.  L a r g e o o c y t e s i n secondary and  t e r t i a r y f o l l i c l e s were en-  v e l o p e d i n a zona p e l l u c i d a , w h i c h formed around  o o c y t e s as s m a l l as 22 u , i f  t h e o o c y t e was r i n g e d w i t h f o l l i c u l a r c e l l s .  1. A  Follicular  Development.  The s i z e and type o f t h e l a r g e s t f o l l i c l e i n each f e t u s i s summ a r i z e d i n Table 2.  The e a r l i e s t  f o l l i c l e , termed p r i m o r d i a l , c o n s i s t e d o f  one c o n t i n u o u s l a y e r o f f l a t t e n e d e p i t h e l i a l Epithelial  c e l l s around  the sex  cell.  c e l l s formed an i n c o m p l e t e r i n g around p r i m a r y o o c y t e s as  early  as 102 days i n a f e t u s from a doe r e a r e d i n c a p t i v i t y (U34).  There were  many p r i m o r d i a l f o l l i c l e s i n f e t u s e s 122 and 123 days o l d from w i l d d e e r . At t h i s stage  t h e e p i t h e l i a l or g r a n u l o s a c e l l s were t h i n l y d i s t r i b u t e d  over t h e 20 t o 25 u o o c y t e , w h i c h had reached t h e d i c t y a t e or r e s t i n g phas of m e i o s i s .  A t 144- d a y s , many f o l l i c l e s were surrounded by c u b o i d a l  g r a n u l o s a c e l l s and t h e r e b y had reached t h e p r i m a r y - f o l l i c l e s t a g e . Secondary f o l l i c l e s , those w i t h more than a s i n g l e l a y e r o f granulosa c e l l s , to 177 d a y s .  b u t no lumen, o c c u r r e d i n f e t a l o v a r i e s o l d e r t h a n 170  These f o l l i c l e s a t t a i n e d d i a m e t e r s o f 43 t o 60 u, c o n t a i n e d  o o c y t e s o f 30 t o 35 P-, and were s t i l l a t t h e p r i m o r d i a l or p r i m a r y s t a g e of development.  A s i m i l a r p i c t u r e o f f o l l i c l e development p r e v a i l e d  i n p e r i n a t a l o v a r i e s , e x c e p t t h a t some f o l l i c l e s , d e e p i n t h e c o r t e x , had reached  t h e v e s i c u l a t e or t e r t i a r y f o l l i c l e s t a g e .  These a t t a i n e d  d i a m e t e r s o f 220 u , c o n t a i n e d o o c y t e s up t o 72 u , and were enveloped i n t h i c k l a y e r s o f g r a n u l o s a and t h e c a l c e l l s  ( P l a t e 1. 6).  Many o f  the l a r g e f o l l i c l e s were d e g e n e r a t e and some were r e p l a c e d w i t h h y a l i n c o n n e c t i v e t i s s u e ( P l a t e 2. 1 0 ) .  22  SECTION 2.  STRUCTURE AND FUNCTION OF THE OVARY IN FAWNS, YEARLINGS, AND ADULTS.  Chapter 1.  General Morphology Of The Deer Ovary. Deer ovaries l i e 5 to 10 cm l a t e r a l to the t i p s of the u t e r i n e  horns.  Except f o r the h i l a r connection, the surface of the ovary i s exposed  to the f l u i d s of the body c a v i t y because there i s no p e r i t o n e a l capsule or ovarian bursa.  Ovaries of prepubertal deer are f l a t t e n e d ovoid s t r u c t u r e s  with a smooth surface.  At puberty, ovaries become much enlarged  and  f l a t t e n e d i f many small f o l l i c l e s but no l a r g e f o l l i c l e s have developed. At the other extreme, the development of one l a r g e f o l l i c l e i n a small ovary r e s u l t s i n a s p h e r i c a l form. the normal o u t l i n e -of the ovary.  Large f o l l i c l e s protrude s l i g h t l y from E x t r u s i v e or herniated f o l l i c l e s , and  the  corpora l u t e a and corpora l u t e a scars .that develop from them, create protuberances of various shapes, i n c l u d i n g s p h e r i c a l , b u l b u l a r , and mushroom. In a d d i t i o n to e x t r u s i v e f o l l i c l e s and l a r g e p r o t r u s i v e corpora l u t e a , there are smaller surface features that vary considerably i n form, size,and c o l o r .  Often, they form small protuberances l e s s than 2 mm i n  diameter, which l i e i n the center of small depressed regions on the ovarian surface.  They are cream, yellow, orange, red, or r a r e l y , black.  represent newly-ruptured  Black ones  f o l l i c l e s or the surface o v u l a t i o n scars of former  corpora l u t e a of pregnancy.  A l l of the s t r u c t u r e s i n d i c a t e the point of  an o v u l a t i o n , recent or o l d , and t h e i r c o l o r i s i n d i c a t i v e of t h e i r Young a c t i v e corpora l u t e a are represented  age.  by red protuberances and older  corpora l u t e a by cream, yellow, or orange bulbs of t i s s u e on the ovarian surface.  Pale orange or yellow protuberances represent degenerate corpora  l u t e a or t h e i r scars.  O c c a s i o n a l l y , p i t s are found on the surface  of the ovary that i n d i c a t e former' ruptures of small, p a r t l y - e x t r u s i v e  23  follicles. The w e i g h t of deer o v a r i e s was the doe.  Within age-classes,  r e l a t e d t o the season and  the w e i g h t s of o v a r i e s d u r i n g the  season changed i n s i g n i f i c a n t l y f r o m 1963  t o 1967.  age  breeding  Consequently, ovarian  w e i g h t s f r o m a l l y e a r s were combined t o show more a c c u r a t e l y the s h i p between age and There was b i n e d 7.5  t o 9-5  relation-  weight.  a g e n e r a l i n c r e a s e i n s i z e , w i t h age, up t o the com-  a g e - c l a s s , a l t h o u g h a s i g n i f i c a n t d i f f e r e n c e between  a d j a c e n t means o n l y o c c u r r e d  between ages 1.5  and  2.5  (Fig- 1).  There  v i r t u a l l y no d i f f e r e n c e i n the w e i g h t of l e f t and r i g h t o v a r i e s f r o m age-class.  of  was  any  I n b r e e d i n g - s e a s o n c o l l e c t i o n s , mean w e i g h t s f o r 278 p a i r s of  o v a r i e s f r o m deer o f a l l ages were 62.3  g ( i t ) and  62.9  g (rt).  F i g . 2 shows s e a s o n a l changes i n the w e i g h t of o v a r i e s f r o m f o u r age-classes  of d e e r .  c o l l e c t i n g period.  P l o t t e d v a l u e s a r e averaged f o r each a g e - c l a s s  The d a t a were n o t t r e a t e d s t a t i s t i c a l l y because of s m a l l  sample s i z e s but c e r t a i n t r e n d s were a p p a r e n t .  I n fawns t h e r e was  decrease i n o v a r i a n weight from f a l l to mid-winter. i n c r e a s e d i n w e i g h t f r o m October t o m i d - w i n t e r and c o n s i s t e n t f e a t u r e o f a d u l t o v a r i e s was tween e a r l y May  and mid-June.  volume o f c o r p o r a l u t e a and 2) June t h e r e was two  a  of y e a r l i n g s  then s t a b i l i z e d .  A  the marked i n c r e a s e i n w e i g h t  be-  an i n c r e a s e i n the  an i n c r e a s e i n f o l l i c u l a r a c t i v i t y .  In  no i n c r e a s e i n o v a r i a n w e i g h t o f f e m a l e s 2 y e a r s o l d , because g i v e n b i r t h and  contained  shrunken  lutea.  Chapter 2. The source  Ovaries  T h i s i s a t t r i b u t e d t o 1)  o f the t h r e e i n the sample had  corpora  per  The  Surface  Epithelium.  s u r f a c e e p i t h e l i u m was  examined because i t i s a p o t e n t i a l  o f sex c e l l s i n p o s t n a t a l o v a r i e s , and i t s c o n t r i b u t i o n o f germ c e l l s  The a g e - s p e c i f i c weight of paired ovaries from deer c o l l e c t e d i n November and e a r l y December from 1963 to 1966. (Mean, S^-t^s* s, range, and sample s i z e ) .  F i g . 1.  36  * 95% confidence limit  28 26  24 22 20 18 16 14 12 10  8  22  6  1  4 2  I  16  |41 0.5  12 15  28 53  77 1  1.5  '2.5  3.5  4-5  5-5 '. AGE (YEARS)  6.5  7.5-9.5  10.5S+  — j  1  0  N  1  .  D  1  J  : J'  MONTH  1  1 M  ; A  i  ~ M  i  7 J  ( i f any) may be r e l a t e d to the estrous c y c l e (Brambell, 1956; Harrison, 1962). In ovaries of fawns from 4 to 6 months of age, the surface e p i t h e l i a l c e l l s were u s u a l l y f l a t t e n e d and contained elongate, tened n u c l e i .  flat-  I n some areas, the epithelium was stretched u n t i l i t  c o n s t i t u t e d l i t t l e more than a membrane with sparse, t h i n n u c l e i . O c c a s i o n a l l y , no epithelium was found.  At dips i n the surface, a t  i n v a g i n a t i o n s , and between lobes, the c e l l s were cuboidal or even columnar.  Obviously, t h e i r shape i s dependent upon surface tensions.  Because breeding-season ovaries enlarged as a r e s u l t of f o l l i c l e growth, the surface epithelium was g e n e r a l l y i n a stretched s t a t e .  Compared  to other e p i t h e l i a l c e l l s , the surface c e l l s were hyperchromatic, e s p e c i a l l y when i n a stretched c o n d i t i o n . There were few i n v a g i n a t i o n s of surface epithelium i n t o the cortex of fawn ovaries.  Invaginations were more numerous i n ovaries  with l a r g e numbers of p r i m o r d i a l f o l l i c l e s i n the outer cortex.  Commonly,  the epithelium was s t r a t i f i e d a t p i t s i n the surface and a t i n v a g i n a t i o n s . There appeared to be c e l l and nuclear enlargement as the e p i t h e l i a l c e l l s became incorporated i n t o the stroma below the t u n i c a albuginea. Some of the.invaginated c e l l s seemed to develop i n t o l a r g e c e l l s resembling oogonia, but i f oogenesis occurred a f t e r b i r t h , i n s i g n i f i c a n t numbers of sex c e l l s were i n v o l v e d . Invagination of the epithelium took several forms.' The simplest was a budding inward of one or several c e l l s , and these wedged between the f i b e r s of the t u n i c a albuginea.  More complex  i n v a g i n a t i o n s r e s u l t e d i n a s e r i e s o f t u b u l e s , i n or below t h e t u n i c a , termed s u b - s u r f a c e fawn o v a r i e s c o n t a i n e d  c r y p t s by H a r r i s o n and Mathews (1951).  Some  l o n g t u b u l e s below t h e t u n i c a , s i m i l a r t o those  noted i n the badger ( N e a l and H a r r i s o n , 1958). The appearance o f t h e s u r f a c e e p i t h e l i u m o f y e a r l i n g o v a r i e s was s i m i l a r t o t h a t o f fawn o v a r i e s , e x c e p t t h a t i t was more c o n s i s t e n t l y a s i n g l e l a y e r o f f l a t t e n e d c e l l s w i t h few i n v a g i n a t i o n s i n t o t h e c o r t e x . ing  the breeding  season,the s u r f a c e e p i t h e l i u m o f y e a r l i n g s and a d u l t s was  t h i n or h y a l i n i z e d .  On p o r t i o n s o f t h e s u r f a c e o f many o v a r i e s / t h e r e was a  t h i c k zone o f h y a l i n i z e d f i b e r s .  E p i t h e l i a l i n v a g i n a t i o n s were r a r e i n  a d u l t d o e s , a l t h o u g h s e v e r a l s m a l l i n v a g i n a t i o n s were found i n one f e m a l e 5-5 y e a r s o l d .  Chapter 3. 3. 1  The O v a r i a n  Follicle.  H i s t o l o g i c a l Components Of The F o l l i c l e . 3. 1. 1  The Antrum.  I n t h e antrum of t h e deer f o l l i c l e , t h e r e  i s a f i n e i n t e r c o n n e c t i n g network o f membranes or f i b e r s s p h e r i c a l a c i d o p h i l i c b o d i e s up t o 3 u i n d i a m e t e r .  containing  These membranes  or f i b e r s a r e c o n t i n u o u s w i t h , and may o r i g i n a t e f r o m , t h e o u t e r granulosa  c e l l membranes.  Because t h e a c i d o p h i l i c s p h e r u l e s  o r i g i n a t e at the antro-granulosa products of granulosa  cells.  boundary, t h e y may be  its fluid  t h e antrum.  seem t o  degradation  I n s m a l l f o l l i c l e s t h e network f i l l s  the antrum b u t i n l a r g e f o l l i c l e s i t g e n e r a l l y o c c u p i e s one-half  l e s s than  As a h e a l t h y f o l l i c l e i n c r e a s e s i n s i z e ,  becomes p r o g r e s s i v e l y more b l u e when s e c t i o n s a r e s t a i n e d  i n a n i l i n e blue.  Dur-  This o b s e r v a t i o n , i n a d d i t i o n to p o s i t i v e p e r i o d i c  a c i d - S c h i f f (PAS) and "metachromatic r e a c t i o n s , i n d i c a t e t h a t  28  mucopolysaccharides  3. 1. 2  or g l y c o g e n a r e p r e s e n t i n the f l u i d .  The Oocyte.  I n fawns, a l l or a l m o s t a l l sex c e l l s have  reached t h e p r i m a r y oocyte s t a g e and a r e i n p r i m o r d i a l f o l l i c l e s .  As the  g r a n u l o s a c e l l s i n c r e a s e i n number, t h e o o c y t e i n c r e a s e s i n s i z e from 30 u to about 100 u,and t h e r e a f t e r growth v i r t u a l l y c e a s e s . The zona p e l l u c i d a , 4 t o 7 u t h i c k , i s t r a v e r s e d a t r i g h t a n g l e s by p r o c e s s e s t h a t presumably p r o v i d e communication  between t h e g r a n u l o s a  c e l l s and t h e o o c y t e ( P l a t e 3 : 4 and 8 ) . E x c e p t f o r t h e s e a l m o s t subm i c r o s c o p i c p r o c e s s e s , t h e zona p e l l u c i d a i s a homogenous a n i l i n e b l u e and P A S - p o s i t i v e l a y e r i n some p r e p a r a t i o n s . I n o t h e r m a t e r i a l the membrane appears t o be l a m i n a t e d ( P l a t e 3 : 4 and 8 ) . There i s an a c i d o p h i l i c  zone  n e x t t o t h e n u c l e a r membrane o f t h e oocyte and an o u t e r a n i l i n e - p o s i t i v e layer.  3. 1 . 3  The Membrana G r a n u l o s a .  Supposedly,the  granulosa c e l l s i n  mammalian f o l l i c l e s o r i g i n a t e f r o m t h e g e r m i n a l e p i t h e l i u m ( B r a m b e l l , 1956), but i n deer t h e y o f t e n appear Whatever t h e i r o r i g i n ,  t o d e v e l o p from s u r r o u n d i n g s t r o m a l c e l l s .  t h e f l a t t e n e d c e l l s d i v i d e , become c u b o i d a l or even  columnar, and e v e n t u a l l y s t r a t i f y i n t o s e v e r a l l a y e r s as t h e c o n t a i n e d o o c y t e enlarges.  Follicular  f l u i d f i r s t appears i n f o l l i c l e s w i t h a d i a m e t e r o f  about 200 u and forms p o o l s i n 300 u f o l l i c l e s .  A t one p o i n t on t h e  membrana g r a n u l o s a o f v e s i c u l a t e f o l l i c l e s , a mound o f c e l l s , t h e cumulus oophorus, p r o j e c t s i n t o t h e antrum. outermost s u r f a c e o f t h e f o l l i c l e ,  Although u s u a l l y l o c a t e d opposite the t h e cumulus oophorus i s o c c a s i o n a l l y  s i t u a t e d on t h e s i d e o f t h e f o l l i c l e or even a d j a c e n t t o t h e f o l l i c u l a r and ovarian surface. The v e s i c u l a t e n u c l e i o f g r a n u l o s a c e l l s a r e g e n e r a l l y 5 t o 7 u  F a c i n g page 29  P l a t e 3. The Components o f F o l l i c l e s . 1. P r i m o r d i a l ( l o v e r l e f t ) , p r i m a r y and s e c o n d a r y f o l l i c l e s . The n u c l e o l u s i n t h e n u c l e u s o f the oocyte has a w h e e l - l i k e s t r u c t u r e (X4-00) . 2. An o o c y t e c o n t a i n i n g s e v e r a l b a s o p h i l i c i n c l u s i o n s . The i n c l u s i o n s o c c u r r e d i n most f o l l i c l e s between t h e p r i m a r y and s m a l l t e r t i a r y s t a g e s (X200). 3. The o o c y t e w i t h i n t h i s p r i m a r y f o l l i c l e b a s o p h i l i c i n c l u s i o n (X800).  c o n t a i n s one s p h e r i c a l  U- A l a y e r e d zona p e l l u c i d a c o n t a i n i n g g r a n u l o s a c e l l p r o c e s s e s and a d i s c o n t i n u o u s c h r o m o p h i l i c i n n e r l a y e r . A p e r i v i t i l l i n e space o c c u r s between t h e zona p e l l u c i d a and the v i t i l l i n e membrane e n c l o s i n g t h e ooplasm (X2000). 5. Two o o c y t e s i n a t e r t i a r y f o l l i c l e . i n many deer (X80).  Such p o l y o v u l a r f o l l i c l e s  occurred  6. A C a l l - E x n e r body on t h e i n n e r s u r f a c e o f t h e membrana g r a n u l o s a . These s t r u c t u r e s , o f unknown s i g n i f i c a n c e , were numerous i n some f e m a l e s  (X400).  3 7. The cumulus oophorus and oocyte i n a 67 mm f o l l i c l e i n a doe on t h e verge o f f i r s t o v u l a t i o n . The oocyte and s u r r o u n d i n g r i n g o f c e l l s i s v i r t u a l l y f r e e f r o m t h e cumulus oophorus (X200). 8. A p o r t i o n o f an oocyte t o i n d i c a t e l a y e r i n g o f t h e zona p e l l u c i d a and the appearance o f t h e ooplasm. The outer, ooplasm was f i n e l y g r a n u l a r i n some o o c y t e s o f f o l l i c l e s near r u p t u r e whereas t h e c e n t r a l ooplasm ( l o w e r l e f t ) was c o a r s e l y g r a n u l a r (X2000).  30  i n diameter, average j u s t under 6u, and large n u c l e i adjacent to the lumen may excede 8 u.  There i s a gradient of i n c r e a s i n g nuclear s i z e s from the  basement membrane to the lumen, w i t h those i n the cumulus oophorus being the l a r g e s t .  I n mature f o l l i c l e s some of the c e l l s near the lumen have  b a s o p h i l i c cytoplasm, whereas most others have a c i d o p h i l i c cytoplasm. T i n c t o r i a l d i f f e r e n c e s between c e l l s were apparent when the granulosa was stained i n f a s t green, or methyl blue and orange G. C i r c u l a r groupings of granulosa c e l l s around a small c e n t r a l r e g i o n occurred i n many secondary and t e r t i a r y f o l l i c l e s (Plate 3:6). These Call-Exner bodies (Harrison, 1962) were numerous i n a l l the f o l l i c l e s i n some ovaries,but t h e i r s i g n i f i c a n c e could not be ascertained. Granulosa c e l l s were d i v i d i n g i n a l l a c t i v e f o l l i c l e s , except those near o v u l a t i o n .  The thickness of the membrana granulosa was  maximal (50 t o 60 n) i n small t e r t i a r y f o l l i c l e s but decreased to as l i t t l e as one c e l l i n thickness (5 u) i n c y s t i c f o l l i c l e s and i n very large f o l l i c l e s on the verge of rupture.  Although the thickness of the granulosa  was u s u a l l y quite uniform around the f o l l i c l e , i t was t h i c k e r i n a r e g i o n where the f o l l i c l e was unable t o s t r e t c h , such as adjacent to another follicle.  3.  2  The Preovulatory F o l l i c l e . To f u l l y i n t e r p r e t c y c l i c f o l l i c u l a r development, i t was necessary  to d i f f e r e n t i a t e f o l l i c l e s that were on the verge of p h y s i o l o g i c a l rupture from those undergoing e a r l y degeneration.  By p h y s i o l o g i c a l rupture, I  mean normal o v u l a t i o n , presumably mediated by the sequential s e c r e t i o n of hormones from the p i t u i t a r y gland, as opposed to the' "mechanical" or n e c r o t i c rupture of a f o l l i c l e .  Since there were progressive h i s t o l o g i c a l  changes i n large f o l l i c l e s , culminating i n o v u l a t i o n , i t was p o s s i b l e to  31  d i s t i n g u i s h those near o v u l a t i o n , although the exact i n t e r v a l to rupture could not be estimated.  Because ovaries of 367 females were c o l l e c t e d  throughout the breeding season, November 6 to December 6, i t followed t h a t , at the time of death,several females were w i t h i n one day of o v u l a t i o n . doe was undoubtedly  One  on the verge of o v u l a t i o n , as i n d i c a t e d by abundant  sperm i n i t s reproductive t r a c t .  F o l l i c l e s judged to be near rupture were  studied and provide the f o l l o w i n g d e t a i l s .  3. 2. 1  The Antrum.  Antra of most f o l l i c l e s near o v u l a t i o n contained  a f l u i d t h a t was p o s i t i v e f o r a n i l i n e blue,but i n some f o l l i c l e s the f l u i d had a cloudy-grey appearance caused by d e t r i t u s from degenerate granulosa cells.  I n some preovulatory f o l l i c l e s , an a c i d o p h i l i c and PAS-positive  f l u i d , which had the t i n c t o r i a l p r o p e r t i e s of vascular f l u i d , was produced around the periphery of the antrum.  Large f o l l i c l e s near o v u l a t i o n con=-  tained an accumulation of a c i d o p h i l i c d r o p l e t s along the surface of the stratum granulosum.  The o r i g i n and chemistry of these d r o p l e t s i s not  known but s i m i l a r spherules are commonly associated with t h e c a l blood vessels that loop i n t o the granulosa below the cumulus oophorus.  3. 2. 2  The Oocyte.  I n c l u d i n g the zona p e l l u c i d a , the oblate oocyte  of preovulatory f o l l i c l e s measured about 125 x 105 x 105 i n the three planes, approximately the same s i z e as oocytes i n small t e r t i a r y f o l l i c l e s .  Con-  sequently, s i z e was of no d i a g n o s t i c value i n determining the degree, of f o l l i c u l a r maturation.  The zona p e l l u c i d a was 4- to 5 }X t h i c k i n most  h e a l t h y f o l l i c l e s , b u t was t h i c k e r around a t r e t i c oocytes.  In sections  stained w i t h Masson's trichrome c o n t a i n i n g ponceau 2R, an outer l a y e r of the zona was p o s i t i v e ' f o r a n i l i n e blue,but the inner l a y e r was Often the inner l a y e r was discontinuous  acidophilic.  and varied i n thickness (Plate 3.4.).  32  The l i g h t l y - a c i d o p h i l i c cytoplasm of oocytes u s u a l l y appeared uniform and f i n e l y granular.  Occasionally,there were splotchy regions  i n the cytoplasm and the c e n t r a l p o r t i o n was more c o a r s e l y granular (Plate 3. 8 ) .  S e r i a l sections revealed that, p r i o r to o v u l a t i o n , most n u c l e i  were l a r g e , v e s i c u l a t e , and i n the r e s t i n g phase of meiosis.  This was  the state of the n u c l e i i n oocytes of l a r g e f o l l i c l e s i n a doe already inseminated (Plate 4. 4) • N u c l e i reached a maximum s i z e of 45 x 38 x 38 p., but averaged 38 x 32 x 32u. The nucleolus and clumps of chromatin were present on the inner surface of the membrane but otherwise the nucleus was clear.  virtually  P r i o r to the alignment of chromosomes at metaphase, the chromatin  became condensed i n t o one or two l a r g e r clumps that were often associated w i t h i n f o l d i n g of the nuclear membrane next to the zona p e l l u c i d a .  3. 2. 3  The Granulosa.  The cumulus oophorus of f o l l i c l e s near ovula-  t i o n were e i t h e r f r e e from the mural granulosa or were held i n tenuous bond (Plate 3. 7 ) .  Loosening of the cumulus was accomplished through a u t o l y s i s  of granulosa c e l l s at i t s base.  C e l l a u t o l y s i s u s u a l l y created s e v e r a l  elongate spaces p a r a l l e l to the granulosa l a y e r .  Fusion of these r e s u l t e d  i n a f r e e - f l o a t i n g outer p o r t i o n which contained the oocyte, or the degene r a t i n g cumulus oophorus was held i n place by an outer f i b r i l l a r network. Further degeneration of the cumular granulosa proceeded from the periphery towards the oocyte.  A d e f i n i t e r i n g of a t r e t i c c e l l s , and u l t i m a t e l y a  space, was commonly found a few c e l l s d i s t a n t from the oocyte.  Cells re- '  maining around the oocyte contained l a r g e r n u c l e i (to 8 u) and greater amounts of f i n e l y - g r a n u l a r , a c i d o p h i l i c cytoplasm.  I n some f o l l i c l e s j u s t  p r i o r to o v u l a t i o n , the c e l l s remaining around the oocyte r a d i a t e d out on tapered s t a l k s of cytoplasm (corona r a d i a t a formation).  Oocytes trapped i n  33  ruptured f o l l i c l e s were s t i l l e n c i r c l e d by attached granulosa c e l l s (Plate 4. 7-9),and f r e e granulosa c e l l s were found i n one oviduct. C e l l d i v i s i o n occurred i n the cumulus oophorus a f t e r i t had v i r t u a l l y ceased i n the remainder of the granulosa.  Cessation of m i t o s i s  then proceeded from the periphery of the cumulus towards the oocyte, where i t occurred u n t i l , and during, corona r a d i a t a formation.  The l a t t e r  process  precedes o v u l a t i o n by only a few hours i n other species (Brambell, 1956). In the membrana granulosa, c e l l d i v i s i o n s r a r e l y occurred i n the l a s t day or two p r i o r to the o v u l a t i o n of f i r s t - c y c l e f o l l i c l e s , but they were more common i n second-cycle f o l l i c l e s near rupture. In f o l l i c l e s near rupture, the membrana granulosa ranged i n t h i c k ness from s e v e r a l ranks of c e l l s , to a s i n g l e l a y e r of c e l l s ( i n very l a r g e follicles).  Generally, the granulosa layer was t h i c k e r and more a c t i v e i n  preovulatory, second-cycle f o l l i c l e s than i n f i r s t - c y c l e f o l l i c l e s .  Prior  to ovulation,the granulosa often became a p s e u d o s t r a t i f i e d l a y e r of columnar cells.  The outer c e l l s , r e l a t i v e to the membrane, were attached to i t by  long s t a l k s of cytoplasm (Plate 7. 5). N u c l e i i n these columnar c e l l s were opposed to the membrana propria,thereby c r e a t i n g a nucleus-free zone adjacent to the membrane.  The surface c e l l s , not attached to the membrane,  were more rounded and some contained b a s o p h i l i c cytoplasm. In preovulatory f o l l i c l e s , numerous a c i d o p h i l i c granules or d r o p l e t s were produced a t the surface of the granulosa.  These d r o p l e t s or granules  were secreted by the granulosa c e l l s ; r e s u l t e d from granulosa c e l l a u t o l y s i s ; or were vascular i n o r i g i n .  At the border between the f o l l i c u l a r f l u i d and  the membrana granulosa, c l e a r spaces the s i z e of granulosa c e l l s u s u a l l y occurred.  These spaces i n d i c a t e d the a u t o l y s i s of surface c e l l s .  I n ad-  d i t i o n to normal l i q u i f a c t i o n of surface c e l l s /there were u s u a l l y some i n d i c a t i o n s of c e l l death, t y p i c a l of e a r l y f o l l i c u l a r a t r e s i a .  These  Facing page 34 Plate 4 The Pi-e-and Post- Ovulatory Oocyte. ( a l l photomicrographs of sections at 400X) 1.  An oocyte i n a f o l l i c l e on the verge of rupture i n a doe approaching the f i r s t o v u l a t i o n of the season. Note the r i n g of c e l l s around the oocyte and the v e s i c u l a t e nucleus w i t h i n i t .  2. An oocyte i n a large growing f o l l i c l e . The d i v i d i n g c e l l s around the oocyte are forming i n t o a corona r a d i a t a . 3. An oocyte i n a growing f o u r t h - c y c l e f o l l i c l e an estimated 12 days a f t e r second o v u l a t i o n . 4. An oocyte i n a f o l l i c l e near rupture. The female was w i t h i n hours of f i r s t o v u l a t i o n of the season. Note the l a m e l l a r nature of the zona p e l l u c i d a . 5. An oocyte i n a f o l l i c l e w i t h i n hours of rupture. The female, approaching second o v u l a t i o n , contained sperm i n i t s oviducts. cumulus oophorus was detached from the mural granulosa.  The  6. The nucleolus and chromatin i n the nucleus of an oocyte i n a r a p i d l y growing 24 mm f o l l i c l e of the second estrous c y c l e . 7. A degenerate oocyte trapped i n a small newly ruptured f o l l i c l e . Two of the three f o l l i c l e s ruptured at f i r s t ovulation i n t h i s female aged 2.5 years,were small, out of phase, and probably were not v i a b l e . The zona p e l l u c i d a i s t h i c k ; the ooplasm i r r e g u l a r i n o u t l i n e and hyperchromatic. 8. An oocyte trapped i n a large newly-ruptured f o l l i c l e of the f i r s t ovulatory c y c l e . The nucleus of the oocyte i s large and v e s i c u l a t e . There i s no i n d i c a t i o n of nuclear maturation. 9 . A s e c t i o n through the nucleus of an oocyte trapped i n a newly-ruptured f o l l i c l e of the f i r s t ovulatory c y c l e . The nuclear membrane i s i n d i s t i n c t but the chromatin has not clumped i n t o one mass. The n u c l e i of the granulosa c e l l s surrounding the oocyte are v e s i c u l a t e but those of the mural granulosa are condensed.  35  included cytoplasmic v a c u o l a t i o n and a u t o l y s i s of c e l l s below the surface, formation of pyknotic n u c l e i i n c e l l s on the surface, abnormal enlargement and clumping of c e l l s , and the appearance of a c e l l that produced abundant fine fibers.  The l a t t e r c e l l s produced a f i b r o u s sheath around the de-  generating cumulus oophorus i n some f o l l i c l e s . Hyperchromasia of granulosa c e l l s occurred i n some preovulatory follicles  (Plate 7.-4).  More intense s t a i n i n g was associated with cyto-  plasmic shrinkage, increased g r a n u l a t i o n , and nuclear condensation.  Some  n u c l e i changed from the round, v e s i c u l a r type of growing f o l l i c l e s to oblate, a l m o s t - s o l i d s t r u c t u r e s . In newly-ruptured f o l l i c l e s , c e l l and nuclear condensation ( r e s u l t i n g i n hyperchromasia) commonly occurred during the metaplasia of granulosa c e l l s to l u t e a l  3.  2. 4  The Theca.  cells.  In a l l l a r g e f o l l i c l e s the theca i n t e r n a thinned  to about 20 p., but i t became as t h i n as one or two c e l l s (5 to 6 u) i n some very l a r g e f o l l i c l e s of the f i r s t estrous c y c l e . i n the area below the cumulus oophorus.  I t was often t h i c k e s t  Most of the oblate or f u s i f o r m  c e l l s contained i r r e g u l a r l y - s h a p e d , oblate n u c l e i .  The vacuolated cyto-  plasm of t h e c a l c e l l s i n growing f o l l i c l e s became more dense and acidop h i l i c i n some f o l l i c l e s j u s t before o v u l a t i o n . C e l l d i v i s i o n s were r a r e i n the t h e c a l c e l l s of preovulatory f o l l i c l e s . . As o v u l a t i o n neared, the vessels i n the theca i n t e r n a became l a r g e and distended with blood, causing them to push i n t o the granulosa l a y e r (Plate 7. 5 ) .  P r o l i f e r a t i o n of vessels i n t o the granulosa  was most extensive below the present or former s i t e of the cumulus oophorus (Plate 7. 3 ) .  The granulosa c e l l s around these evaginating vessels were  columnar and scattered between them were h i g h l y a c i d o p h i l i c spherules ranging from 1 to 8 u i n diameter.  These spherules were a l s o located w i t h i n the  36  v e s s e l s , where they presumably o r i g i n a t e d .  i  Their s i g n i f i c a n c e i s not  known, but they were common i n degenerate f o l l i c l e s . The only s i g n i f i c a n t change noted i n the theca externa of preovulatory f o l l i c l e s , w a s marked enlargement of the blood v e s s e l s that served the f o l l i c l e .  3. 3  Normal A t r e s i a Of  Follicles.  F o l l i c l e a t r e s i a or death, as revealed by c y t o l o g i c a l characteri s t i c s , occurred at a l l stages of f o l l i c l e development.  Only a small  f r a c t i o n of f o l l i c l e s developed past the p r i m o r d i a l stage of development. A t r e s i a of p r i m o r d i a l f o l l i c l e s was i n d i c a t e d by cytoplasmic changes i n the oocytes, i n c l u d i n g :  hypochromasia of the ooplasm, formation  of l a r g e vacuoles, enlargement or shrinkage, and i n c l u s i o n s .  Concomitant  changes i n the nucleus included enlargement, clumped chromatin (pyknosis), d i s i n t e g r a t i o n of chromatin, and eventual breakdown of the nuclear membrane.  C y t o l y s i s of the f o l l i c u l a r c e l l s around the oocyte occurred at  an e a r l y stage of degeneration.  In some instances the f o l l i c u l a r c e l l s were  resorbed l e a v i n g the oocyte surrounded zona p e l l u c i d a u s u a l l y developed i n a zona p e l l u c i d a degenerated  only by stromal c e l l s .  around the oocyte. slowly.  A thick  Oocytes incorporated  I f a t r e s i a occurred before zona  p e l l u c i d a formation, the oocyte autolysed, l e a v i n g a space surrounded  by a  few f l a t t e n e d f o l l i c u l a r c e l l s . ; The space was f i l l e d with an a n i l i n e p o s i t i v e f l u i d coagulum, and e v e n t u a l l y by stromal connective t i s s u e . E a r l y i n d i c a t i o n s of a t r e s i a i n primary and secondary  follicles  included, the formation of s p h e r i c a l a c i d o p h i l i c i n c l u s i o n s i n the ooplasm (Plate 3. 2 and 3 ) , v a c u o l a t i o n of the ooplasm, and the absence of m i t o s i s i n the granulosa.  As a t r e s i a progressed, the oocyte was replaced by h y a l i n  m a t e r i a l and u l t i m a t e l y by stromal connective t i s s u e .  37  The s e q u e n t i a l a t r e s i a of l a r g e t e r t i a r y f o l l i c l e s was  arbi-  t r a r i l y d i v i d e d i n t o e i g h t s t a g e s so t h a t r e f e r e n c e c o u l d be made t o a p a r t i c u l a r s t a g e i n subsequent  d i s c u s s i o n s , and p r o g r e s s i v e changes i n the  components o f the d e g e n e r a t i n g f o l l i c l e c o u l d be i n t e r r e l a t e d . f o l l i c l e s q u i c k l y passed  t h r o u g h the f i r s t few stages of t h i s  but l a t e r s t a g e s o c c u p i e d a c o n s i d e r a b l e t i m e .  Apparently, classification,  The sequence of a t r e s i a  i n f o l l i c u l a r components of medium and l a r g e f o l l i c l e s a r e d e s c r i b e d as follows: 3. 3. 1  The Antrum.  A c t i v e t e r t i a r y f o l l i c l e s were  spheroid,whereas  a t r e t i c f o l l i c l e s , because of reduced f l u i d p r e s s u r e , were misshapen by o t h e r a c t i v e f o l l i c l e s or c o r p o r a l u t e a . I n c o n t r a s t t o the c l e a r , homogenous, a n i l i n e - p o s i t i v e f l u i d h e a l t h y f o l l i c l e s , the f l u i d i n a t r e t i c f o l l i c l e s appeared ( f r o m c e l l d e t r i t u s ) , c o a g u l a t e d , and a c h r o m a t i c . more d e g e n e r a t e , t h e  granular, " d i r t y "  As t h e f o l l i c l e became  i n t r a f o l l i c u l a r r e t i c u l a r network became t h i c k e r ,  d i s c o n t i n u o u s , and i n t e n s e l y a c i d o p h i l i c .  Loss of the f l u i d s  p r o p e r t i e s , the most u s e f u l d i a g n o s t i c c h a r a c t e r i s t i c , was as the t r a n s i t i o n a l s t a g e . e s s e n t i a l l y achromatic. as s t a g e one of a t r e s i a . the shrunken lumen.  of  tinctorial  e v i d e n t as  By t h e t h i r d s t a g e o f a t r e s i a , t h e f l u i d  C o a g u l a t i o n of t h e f l u i d  early  was  c r e a t e d f i s s u r e s as e a r l y  By s t a g e 5,there were w h o r l s o f g r e y d e t r i t u s i n  E v e n t u a l l y , t h e lumen was f i l l e d w i t h f i b r o b l a s t s  and  t h e i r products.  3.  3.  2  The Oocyte.  The d e g e n e r a t e  oocytes of l a r g e f o l l i c l e s  i n t e n s e l y and t h e endoplasmic r e t i c u l u m i n the ooplasm became more The appearance o f v a c u o l e s i n the ooplasm i n d i c a t e d e a r l y a t r e s i a .  stained  prominent. As  a t r e s i a p r o g r e s s e d , t h e s e e n l a r g e d t o g i v e the c y t o p l a s m a c o a r s e l y v a c u o l a t e d appearance ( P l a t e s 5 and 6 ) .  Huge v a c u o l e s or spaces developed  in a  F a c i n g page 38 Plate 5 P r e o v u l a t o r y and Degenerate Oocytes. 1. The cumulus oophorus and c o n t a i n e d oocyte i n a 59 mm- f o l l i c l e on the verge of r u p t u r e . The f o l l i c l e was i n a y e a r l i n g a p p r o a c h i n g f i r s t 5  ovulation  (X200).  2. Higher m a g n i f i c a t i o n of the above. The s t a l k e d c e l l s o f the c o r o n a r a d i a t a c o n t a i n p o l a r n u c l e i opposed t o the o o c y t e . The c e l l s of the c o r o n a r a d i a t a were s t i l l d i v i d i n g as i n d i c a t e d by the m i t o t i c f i g u r e .  (X400). 3 3. A d e g e n e r a t e oocyte i n an a t r e t i c 41 mm follicle. The f o l l i c l e , h a v i n g f a i l e d t o r u p t u r e a t f i r s t o v u l a t i o n 1 t o 2 days p r e v i o u s l y , was d e s t i n e d t o l u t e i n i z e and become an a c c e s s o r y corpus luteum. 3 4. A p e c u l i a r c o n f i g u r a t i o n of c h r o m a t i n i n the oocyte of a 43 mm active follicle. The o v a r i e s of t h i s doe c o n t a i n e d a n e w l y - r u p t u r e d f o l l i c l e t h a t was a t r e t i c p r i o r t o r u p t u r e (X800). 5. A b a s o p h i l i c zona, o f unknown s i g n i f i c a n c e , a d j a c e n t t o the n u c l e u s of an o o c y t e . The 33 mm^ f o l l i c l e c o n t a i n i n g the o o c y t e was i n the o v a r i e s of a y e a r l i n g a p p r o a c h i n g i t s f i r s t o v u l a t i o n (X800). 6. A d e g e n e r a t e o o c y t e , c o n t a i n i n g two p o l a r b o d i e s , i n an f o l l i c l e ( s t a g e 3 t o 4 of a t r e s i a ) ( X 2 0 0 ) .  atretic  7. N u c l e a r f r a g m e n t a t i o n i n a oocyte l o c a t e d i n a 1 1 9 mm^ l u t e i n i z e d follicle. F i r s t o v u l a t i o n o c c u r r e d about 6 days p r e v i o u s l y ( X 4 0 0 ) . 3 8. L a r g e v a c u o l e s and the d e g e n e r a t e n u c l e u s i n an oocyte of a 33 mm growing f o l l i c l e i n a doe 2.5 y e a r s o l d p r i o r t o f i r s t o v u l a t i o n of the  season  (X4OO).  9. An e n l a r g e d , fragmented, and v a c u o l a t e d oocyte i n a s m a l l ( 1 . 5 mm diam) follicle. C e l l d i v i s i o n s s t i l l o c c u r r e d i n the s u r r o u n d i n g g r a n u l o s a (X400).  10.  T w o - c e l l e d o o c y t e i n a s m a l l ( 1 . 5 mm diam) a t r e t i c f o l l i c l e ( s t a g e 5 or 6 o f a t r e s i a ) i n a doe a p p r o a c h i n g f i r s t o v u l a t i o n o f the season ( X 4 0 0 ) .  11.  I n v a s i o n o f the oocyte by p h a g o c y t i c c e l l s i n an a t r e t i c f o l l i c l e  (X400).  F a c i n g page 39 Plate 6 M e i o t i c D i v i s i o n I n t h e Oocyte. 1.  N u c l e a r c o n f i g u r a t i o n i n t h e oocyte o f a f o l l i c l e w i t h i n a fe\^ hours o f o v u l a t i o n ( X 4 O O ) .  2.  Metaphase o f m e i o s i s I i n a 6 7 mm^ f o l l i c l e  e s t i m a t e d t o be  on t h e verge o f r u p t u r e  (X400).  3. E a r l y t e l o p h a s e o f m e i o s i s I i n a f o l l i c l e i n the T r a n s i t i o n a l of m a t u r a t i o n (X800). 4. L a t e t e l o p h a s e of m e i o s i s I i n a. 51 mnP a t r e t i c f o l l i c l e of a t r e s i a (X400).  Stage  (Stage 2  5. Higher m a g n i f i c a t i o n o f 6 . 4 (X2000). 6. The f i r s t p o l a r body and c h r o m a t i n o f t h e secondary 2 atretic follicle (X2000). 7.  oocyte i n a S t a g e -  E a r l y metaphase o f m e i o s i s I I i n a Stage 3 a t r e t i c f o l l i c l e . p o r t i o n o f t h e f i r s t p o l a r body i s e v i d e n t (X2000).  8. L a t e metaphase o f m e i o s i s I I i n a s m a l l a t r e t i c f o l l i c l e The f i r s t p o l a r body i s d e g e n e r a t e (X400).  A  (Stage 5)•  9- Higher m a g n i f i c a t i o n o f . 6 . 8. I n t h e o r i g i n a l photomicrograph t h e metaphase s p i n d l e was c l e a r l y e v i d e n t as were c e l l p r o c e s s e s t h r o u g h the zona p e l l u c i d a (X2000).  3  10.  L a t e anaphase or e a r l y t e l o p h a s e o f m e i o s i s I I i n a 30 mm a t Stage 4 o f a t r e s i a ( X 4 0 0 ) .  11.  A t w o - c e l l e d oocyte i n a s m a l l f o l l i c l e a t Stage  6  follicle  of a t r e s i a  (X400).  few oocytes. Nuclear changes occurred i n the degenerate oocyte i n c l u d i n g : abnormal enlargement of the nucleus, abnormal clumping of chromatin, breakdown of the nuclear membrane, and l o s s of chromatin (Plates 5 and 6). During the breeding season, many oocytes i n a t r e t i c f o l l i c l e s passed through meiosis and a few d i v i d e d . ( P l a t e 6).  At stage one of a t r e s i a , the  nuclear membrane started to r e t r a c t towards the zona p e l l u c i d a .  By the  second stage of a t r e s i a , the oocyte was i n , or had completed, the . f i r s t maturation d i v i s i o n .  The f i r s t polar body, enclosed i n a membrane,  was expelled from the ooplasm and l a y j u s t i n s i d e the zona p e l l u c i d a .  In  some oocytes the remaining chromosomes i n the ooplasm divided again by the 4-th or 5th stage of a t r e s i a . pseudocleavage  A few divided i n t o two c e l l s , a process termed  rather than parthenogenesis (Ingram,  1962).  I n a t r e t i c oocytes, the borders of the zona p e l l u c i d a often became i n d i s t i n c t and p i t t e d with f i s s u r e s .  The membrane, 3 to 5 p. t h i c k  around a c t i v e oocytes, often thickened to 5 to 8 p. around a t r e t i c oocytes. Eventually,  f i s s u r e s allowed phagocytic c e l l s to enter the ooplasm and  destroy i t (Plate 5. 11). Other i n d i c a t i o n s of f o l l i c u l a r a t r e s i a i n the oocytes, included thickening and r e t r a c t i o n of the v i t e l l i n e membrane and severance of g r a n u l o s a - c e l l processes from the zona p e l l u c i d a .  3.3.  3  The Granulosa.  The p h y s i o l o g i c a l state of large t e r t i a r y  (Graafian) f o l l i c l e s was best evaluated by exajnination of the membrana granulosa.  I n the 1 to 8 scale of a t r e s i a , the e a r l y stages were defined  l a r g e l y by the appearance of the granulosa.  Three c h a r a c t e r i s t i c s of the  membrana granulosa s i g n i f i e d e a r l y a t r e s i a .  These were:  l ) Absence  of m i t o s i s i n a l l granulosa c e l l s , i n c l u d i n g those of the cumulus oophorus  41  (and the corona r a d i a t a ) .  2) Shrinkage of the cytoplasm and n u c l e i v/ith  concomittant hyperchromasia.  3)  The appearance of new c e l l types on the  surface of the granulosa l a y e r . The l a s t d i v i s i o n s occurred i n c e l l s near the oocyte.  In f o l l i c l e s  i n the t r a n s i t i o n a l stage of development, c e l l d i v i s i o n s were numerous i n the terminal p o r t i o n of the cumulus oophorus.  I n f o l l i c l e s i n the f i r s t  stage of a t r e s i a , m i t o s i s were found only i n c e l l s of the corona r a d i a t a . Thereafter c e l l d i v i s i o n s  ceased.  C e l l s i n the cumulus oophorus dissapeared r a p i d l y d u r i n g the e a r l y stages of a t r e s i a .  Large lacunae occurred a t the base of the cumulus  oophorus i n f o l l i c l e s i n the t r a n s i t i o n a l phase.  A r i n g of degenerate c e l l s  a l s o occurred a few c e l l s d i s t a n t from the oocyte.  By the f i r s t stage of  a t r e s i a , the cumulus- oophorus was detached from the mural granulosa, or was n e a r l y detached, and many c e l l s were undergoing a u t o l y s i s .  By the  second stage of a t r e s i a , 20 t o 50% of the c e l l s were undergoing and many c e l l s contained pyknotic n u c l e i .  autolysis  I n some f o l l i c l e s , a c i r c u l a r  a c e l l u l a r zone ringed the band of a c t i v e c e l l s around the oocyte. 10 t o 4-0% of the c e l l s remained by the t h i r d stage of a t r e s i a . i n g c e l l s , except those of the corona r a d i a t a  ( i f present),  and enclosed i n a p e r i p h e r a l network of f i n e f i b e r s .  Only  The remain-  were.degenerate  By the f o u r t h  stage  of a t r e s i a only a few clumps of degenerate c e l l s remained i n a coagulum of f i b e r s and c e l l d e t r i t u s . arrangement  A t h i n l a y e r of c e l l s , o c c a s i o n a l l y  (Plate 5. 1 - 3 ) , s t i l l persisted  around the oocyte.  i n radial Only  scattered,small clumps of degenerate c e l l s remained a t the f i f t h stage of . a t r e s i a , and only one l a y e r of c e l l s e n c i r c l e d  the oocyte.  A small per-  centage of f o l l i c l e s s t i l l contained t i n y c l u s t e r s of c e l l s i n f o l l i c l e s i n the s i x t h stage of a t r e s i a . Changes i n the mural granulosa a t the various stages of a t r e s i a  U2  a r e i l l u s t r a t e d i n P l a t e 7.  S h r i n k a g e and h y p e r c h r o m a s i a  o f c e l l s and  n u c l e i were f i r s t e v i d e n t i n f o l l i c l e s i n t h e t r a n s i t i o n a l s t a g e . o f c e l l s became d i s o r g a n i z e d as c e l l s l o s t t h e i r attachments membrane and t o one a n o t h e r .  The l a y e r  t o t h e basement  C e l l d e a t h proceeded f r o m t h e antrum towards  the basement membrane and t h e f i r s t new c e l l t y p e s , i n d i c a t i v e o f a t r e s i a , o c c u r r e d a l o n g t h e l u m i n a l border o f t h e g r a n u l o s a .  These c e l l  types  included a fiber-producing c e l l , c e l l s with pyknotic n u c l e i , c e l l s  exibiting  k i o l y c y t o t i c a t y p i a , m u l t i n u c l e a t e c e l l s , and o t h e r c e l l s w i t h f e a t u r e s w h i c h u s u a l l y s i g n i f y c e l l death  ( N i e b u r g s , 1967).  A s m a l l number o f these  abnormal c e l l t y p e s , and i n c r e a s e d  i n d i c a t i o n s o f c e l l p a t h o l o g y i n t h e r e m a i n d e r o f t h e membrana g r a n u l o s a , were t y p i c a l o f t h e s t a g e o f a t r e s i a d e s i g n a t e d " A t r e s i a 1".  Fiber  p r o d u c t i o n c o n t i n u e d u n t i l a c o n t i n u o u s , t h i n , r e t i c u l a r network r e s t e d on the s u r f a c e of the degenerating granulosa l a y e r ( s t a g e " A t r e s i a 2").  At  t h i s s t a g e , t h e i r r e g u l a r l y - s h a p e d n u c l e i were c o n c e n t r a t e d c l o s e t o t h e basement membrane.  I n l a r g e f o l l i c l e s , a u t o l y s i s of the outer  cells  c o n t i n u e d u n t i l o n l y one l a y e r o f c e l l s remained c l o s e l y a t t a c h e d t o t h e t h i c k e n e d basement membrane.  At t h i s t h i r d stage o f a t r e s i a , t h e f i b r o u s  network was t h i c k , c o n t a i n e d dead c e l l s  ( o f t e n clumped), and c e l l remnants.  In medium-sized f o l l i c l e s , t h e s e v e r a l - r a n k e d d e g e n e r a t e c e l l s appeared t o develop i n t o f i b r o b l a s t - l i k e c e l l s aligned p a r a l l e l to the f o l l i c u l a r  wall.  In l a r g e r f o l l i c l e s , f i b r o b l a s t i c c e l l s developed  f r o m , or r e p l a c e d , t h e  g r a n u l o s a c e l l s a t t h e f o u r t h stage o f a t r e s i a .  The t h i c k n e s s o f these  c e l l s v a r i e d f r o m one t o s e v e r a l .  They produced a l a y e r o f amorphous h y a l i n  t i s s u e , t h e t h i c k n e s s o f w h i c h was p r o p o r t i o n a l t o t h e t h i c k n e s s o f t h e layer of f i b r o b l a s t i c c e l l s . h y a l i n i z a t i o n o f t h e former  These d e p o s i t i o n s , a l o n g w i t h f u r t h e r t h e c a i n t e r n a and s h r i n k a g e o f t h e f o l l i c l e ,  caused t h e w a l l o f t h e s t r u c t u r e t o become p r o g r e s s i v e l y t h i c k e r  (stages  F a c i n g page 43 P l a t e 7. 1.  The F o l l i c u l a r  Wall.  The membrana g r a n u l o s a , t h e c a i n t e r n a , and a p o r t i o n o f t h e t h e c a e x t e r n a o f a growing f o l l i c l e . The c e l l d i v i s i o n s i n t h e g r a n u l o s a (two i n t h e photograph) and t h e t h i n l a y e r o f p o o r l y d e f i n e d t h e c a i n t e r n a c e l l s a r e c h a r a c t e r i s t i c o f growing f o l l i c l e s ( X 4 O O ) . .  3 2. The w a l l o f a 59 mm f o l l i c l e i n the t r a n s i t i o n a l stage o f development. The e n l a r g e d blood v e s s e l s i n t h e t h e c a i n t e r n a , a l a y e r o f u n a t t a c h e d g r a n u l o s a , and t h e columnar and p o l a r c e l l s i n t h e m u r a l g r a n u l o s a are t y p i c a l o f " t r a n s i t i o n a l " f o l l i c l e s (X800). 3. Another v i e w of t h e above f o l l i c l e . The b l o o d v e s s e l s o f the t h e c a i n t e r n a have i n v a g i n a t e d i n t o t h e b a s a l r e g i o n o f the d e g e n e r a t e cumulus oophorus. P o l a r , columnar c e l l s r a d i a t e o u t f r o m t h e vessel loops ( X 2 0 0 ) . 4. The w a l l of a 50 rnrrP f o l l i c l e between t h e t r a n s i t i o n a l stage and s t a g e one o f a t r e s i a . Many n u c l e i o f b o t h t h e membrana g r a n u l o s a and t h e t h e c a i n t e r n a a r e condensed i n t h i s f o l l i c l e w h i c h was e s t i m a t e d t o be on t h e verge o f r u p t u r e ( X 4 O O ) . 5.  The w a l l of a f o l l i c l e i n t h e t r a n s i t i o n a l phase (between growth and a t r e s i a ) o f development. Note t h e " s t a l k e d " columnar g r a n u l o s a c e l l s c o n t a i n i n g p o l a r n u c l e i and t h e l a r g e b l o o d ' v e s s e l i n t h e t h e c a i n t e r n a (X800).  6. The w a l l o f a 62 mnr f o l l i c l e i n t h e f i r s t stage o f a t r e s i a . Format i o n o f t h e r e t i c u l a r network on t h e s u r f a c e o f the membrana g r a n u l o s a t y p l i f i e s t h i s s t a g e . Such f o l l i c l e s may r u p t u r e a t f i r s t e s t r u s or l u t e i n i z e , and become an a c c e s s o r y c o r p u s luteum.  3 7. The w a l l o f a 4 4 mm f o l l i c l e i n t h e second s t a g e o f a t r e s i a . There i s a c o n t i n u o u s zone o f f i b e r s on t h e d e g e n e r a t e membrana g r a n u l o s a ( X 4 0 0 ) .  3  8. The w a l l o f a 22 mm . f o l l i c l e i n t h e t h i r d s t a g e of a t r e s i a . Death o f g r a n u l o s a c e l l s has r e s u l t e d i n a wide zone o f f i b e r s and d e g e n e r a t e cells (X400).  3  9. The w a l l o f a 7 mm f o l l i c l e i n t h e f o u r t h stage o f a t r e s i a . Autol y s i s o f the g r a n u l o s a c e l l s i s n e a r l y complete. The b a s a l l a y e r o f c e l l s i s s t i l l i n t a c t (X800). 10.  The w a l l o f a f o l l i c l e i n t h e f o u r t h t o f i f t h stage o f a t r e s i a . the m i t o s i s ( r i g h t c e n t e r ) i n t h e c e l l s a l i g n e d p a r a l l e l t o t h e t h i c k e n e d and h y a l i n i z e d f o l l i c u l a r w a l l (X800).  Note  11.  The w a l l o f a 1.8 mnr f o l l i c l e i n t h e f i f t h s t a g e o f a t r e s i a . A c l u s t e r of d e g e n e r a t e g r a n u l o s a c e l l s p e r s i s t i n t h e lumen. The former t h e c a l l a y e r s ( l e f t t h i r d o f t h e photograph) i s h y a l i n i z e d . The l i g h t e r zone ( c e n t r a l o n e - q u a r t e r ) i s composed o f f i b r o b l a s t s and c o l l a g e n i c i n t e r c e l l u l a r m a t e r i a l s e c r e t e d by t h e f i b r o b l a s t s (X800).  12.  The w a l l o f a f o l l i c l e i n t h e s i x t h s t a g e o f a t r e s i a . A t h i c k l a y e r of c o n n e c t i v e t i s s u e ( l e f t t w o - t h i r d s o f photograph) s u r r o u n d s a s m a l l lumen. F i b r o b l a s t s a r e s t i l l s e c r e t i n g amorphous t i s s u e (X400).  3  s i x and  seven of a t r e s i a ) .  The  i n t o active,spindle-shaped retarded  transformation  of d e g e n e r a t e g r a n u l o s a c e l l s  c e l l s r e s e m b l i n g f i b r o b l a s t s ( A t r e s i a 4)  i n l a r g e f o l l i c l e s i n which a v e r y t h i n (one  g r a n u l o s a l a y e r was  present.  c a t i v e of abnormal e n d o c r i n e f u n c t i o n  Such f o l l i c l e s , u s u a l l y  indi-  1956).  f i b r o b l a s t or f i b r o b l a s t - l i k e c e l l s l i n i n g the lumen a t  f o u r t h s t a g e of a t r e s i a , o c c a s i o n a l l y d i v i d e d  and  s e m b l i n g young l u t e a l c e l l s  P l a t e 8. 1 ) .  and  of  ( M o u l t o n , 1961), a r e termed " c y s t i c "  because of t h e i r slow d e g e n e r a t i o n ( B r a m b e l l , The  to three c e l l s )  F o l l i c l e s with only a single layer  granulosa c e l l s persisted f o r long periods.  was  hyperplasia  ( P l a t e 7. 10 and  enlarged i n t o c e l l s r e This hypertropy  of t i s s u e u s u a l l y o c c u r r e d i n o n l y a p o r t i o n of  degenerate f o l l i c l e .  the  Y e l l o w pigment, a p p a r e n t l y a p r o d u c t of  d e g e n e r a t i o n , formed on d e g e n e r a t i o n of the n e o p l a s t i c  the  lipoid  tissue.  About the f i f t h s t a g e of a t r e s i a , a p e c u l i a r l a r g e r i n g , or semispherical, follicles  s t r u c t u r e o f a c t i v e and  ( P l a t e 8. 3-6).  A p p a r e n t l y i t o r i g i n a t e d f r o m a r e t r a c t i o n of  most of the former g r a n u l o s a to one Spindle-shaped,fibroblastic to a l e s s e r extent,  dead c e l l s d e v e l o p e d i n some  l o c a t i o n on the w a l l of the  c e l l s p r o l i f e r a t e d around the p e r i p h e r y  the i n n e r  surface  of the s t r u c t u r e .  m a i n i n g i n t e r n a l c e l l s were d e g e n e r a t e . and  amorphous m a t e r i a l was  3. 3. 4  The  Theca.  and,  Most of the  re-  Peculiar basophilic, fibrous,  produced i n the m i d d l e of the t i s s u e l a y e r .  C e l l s o f the t h e c a i n t e r n a began d e g e n e r a t i n g  l a t e r than t h o s e o f the g r a n u l o s a . included  follicle.  I n d i c a t i o n s o f a t r e s i a i n the  l ) a v a s c u l a r i t y , 2) appearance of l e u c o c y t e s ,  theca  3) l o o s e n i n g  of  f i b e r s because of reduced f l u i d p r e s s u r e , 4) t h i c k e n i n g and h y a l i n i z a t i o n of f i b e r s , a n d  5) i n c r e a s e d  amounts of amorphous h y a l i n t i s s u e .  I n s m a l l f o l l i c l e s , a band of h y a l i n t i s s u e , termed a  "glassy"  F a c i n g page 45 Plate 8 Hyperplasia  and M e t a p l a s i a  of T i s s u e  i n Atretic Follicles.  1. E a r l y p r o l i f e r a t i o n o f f i b r o b l a s t i c - l i k e c e l l s i n an a t r e t i c  follicle  (X4OO).  2. The n e c r o t i c t i s s u e r e s u l t i n g f r o m t h e d e g e n e r a t i o n of a s m a l l neoplasm such as p i c t u r e d i n 8. 1. Some y e l l o w pigment i s s e c r e t e d around t h e d e g e n e r a t e , v a c u o l a t e d c e l l s (X800). 3. A c i r c u l a r s t r u c t u r e i n a f o l l i c l e i n t h e f i f t h  stage of a t r e s i a  (X4O).  4. A p o r t i o n o f t h e s t r u c t u r e i n 8.3 a t h i g h e r m a g n i f i c a t i o n . The o u t e r c e l l s a r e f i b r o b l a s t i c - l i k e ( l e f t s i d e o f photograph) and the i n n e r c e l l s a r e degenerate, g r a n u l o s a - l i k e c e l l s . Between the' a f o r e m e n t i o n e d layers i s peculiar, structureless, black-stained material (X4OO). 5. A s i m i l a r b u t l a r g e r and more d e g e n e r a t e s t r u c t u r e than t h e one shown i n 8.3 above ( X 4 O ) . r  6. A p o r t i o n o f the above s t r u c t u r e a t h i g h e r m a g n i f i c a t i o n  (X4OO).  3 7. E a r l y l u t e i n ! z a t i o n o f a 16 mm f o l l i c l e about one d a y a f t e r f i r s t ovulation. A c t i v e c e l l s occur i n b o t h t h e membrana g r a n u l o s a ( t o r i g h t o f dashes) and the t h e c a i n t e r n a . A c e l l i n telophase of m i t o s i s i s e v i d e n t i n t h e t h e c a i n t e r n a (X800).  3  8. E a r l y l u t e i n i z a t i o n o f a 12 mm f o l l i c l e 1 t o 2 days f o l l o w i n g f i r s t ovulation. A metaphase o f m i t o s i s i s p r e s e n t i n t h e t h e c a i n t e r n a (to l e f t o f dashes) (X800).  3  9. L u t e i n i z e d w a l l o f a 41 mm a t r e t i c f o l l i c l e about 2 days a f t e r f i r s t ovulation. The l o c a t i o n o f t h e former membrane p r o p r i a i s i n d i c a t e d by two dashes ( r i g h t c e n t e r ) . I t i s evident t h a t both the membrana g r a n u l o s a and t h e t h e c a i n t e r n a undergo l u t e i n i z a t i o n (X800). 10. The same l u t e i n i z e d f o l l i c l e as i n 8. 9. Most o f t h e l u t e a l t i s s u e o r i g i n a t e d f r o m g r a n u l o s a c e l l s (X400).  46  membrane, formed around t h e lumen. g l a s s y membrane has become f o l d e d  Small a t r e t i c f o l l i c l e s i n which the i n t o v a r i o u s shapes a r e termed  "ribbon  atretic" I n l a r g e r f o l l i c l e s , h y a l i n i z a t i o n of the theca progressed ( s t a g e s f i v e t o . s e v e n ) u n t i l a s o l i d , t h i c k , amorphous band o f a n i l i n e p o s i t i v e t i s s u e surrounded t h e s h r i n k i n g lumen. the h y a l i n t i s s u e g r a d u a l l y  3. 4  Complete r e s o r p t i o n o f  ensued, l e a v i n g no t r a c e o f t h e former  follicle.  A t y p i c a l A t r e s i a Of F o l l i c l e s . F o l l i c l e s i n U>5% o f 312 f e m a l e deer underwent an a t y p i c a l f o r m o f  atresia.  I t o c c u r r e d i n f e m a l e s o f a l l ages and a t any s t a g e o f t h e b r e e d -  i n g season,but was most e x t e n s i v e i n a fawn o f 5 months. o n l y two or t h r e e f o l l i c l e s i n a p a i r o f o v a r i e s  I n some f e m a l e s ,  were i n v o l v e d  but i n  o t h e r s many f o l l i c l e s underwent a t y p i c a l changes. F o l l i c l e s i n e a r l y stages of a t y p i c a l a t r e s i a contained a c e n t r a l j s t r u c t u r e l e s s mass o f d e g e n e r a t e t i s s u e .  Occasionally, t h i s  c e n t r a l a r e a , c o r r e s p o n d i n g t o t h e f o r m e r antrum, was f i b r i n o u s , w h i c h s u g g e s t s t h a t i t was d e r i v e d 4-).  The p e r i p h e r a l  a t l e a s t i n p a r t from the blood  p o r t i o n of these e a r l y - a t r e t i c f o l l i c l e s  many l e u c o c y t e s arranged i n c i r c u l a r p a t t e r n t h e c a l l a y e r s , f i b r o b l a s t s , and v a s c u l a r l i f e r a t i n g and, i n p l a c e s , necrotic region  contained  by c o l l a g e n i c f i b e r s o f t h e  elements.  extended a s h o r t d i s t a n c e  ( P l a t e 9. l ) . S c a t t e r e d  ( P l a t e 9. 3 and  The l a t t e r were p r o i n t o the c e n t r a l  around and ahead o f t h e s e  invading  elements,were m o b i l e p h a g o c y t i c c e l l s t h a t i n v a d e d t h e c e n t r a l t i s s u e mass ( P l a t e 9. 4- and 5). Phagocytosis of material  i n t h e c e n t r a l zone c o n t i n u e d u n t i l  the phagocytes remained and t h e n t h e r e g i o n was chromophobic,except f o r the e v e n l y d i s t r i b u t e d b a s o p h i l i c n u c l e i .  The l a s t s u r v i v i n g c e l l s had  only  c h a r a c t e r i s t i c s of n e u t r o p h i l s , each with two or three s p h e r i c a l chromatin masses (Plate 9 ) . Concurrently, the inflammation spread outward to i n v o l v e a l l of the former theca and p o r t i o n s of the surrounding stroma.  Portions  of the t h e c a l connective t i s s u e were replaced by h y p e r p l a s t i c c e l l s and multinucleate giant c e l l s ; then by achromatic t i s s u e w i t h numerous, vacuol a t e d and polymorphonuclear tissue.  c e l l s ; and f i n a l l y by f i b r o u s connective  I n some f o l l i c l e s , most of the p e r i p h e r a l p o r t i o n was l a r g e l y  acromatic and contained b i z a r r e vacuolated c e l l s amongst the f i b e r s , d e t r i t u s , and a c t i v e connective t i s s u e c e l l s (Plate 9- 7 ) . As the whole s t r u c t u r e shrank to a small s i z e , i t became a patch of white or cream c e l l s or lacunae surrounded  and interspersed by h i g h l y  v a r i a b l e amounts of h y a l i n i z e d or collagenated connective t i s s u e f i b e r s . In h y a l i n i z e d s t r u c t u r e s , the c e l l spaces were smaller and contained yellow or orange-brown c e l l remnants.  C e l l s i n loose non-encapsulated  were l a r g e ; the contents achromatic and degenerate..  structures  Later,the connective  t i s s u e capsules were resorbed and e v e n t u a l l y the lacunae was replaced by connective t i s s u e stroma.  Some of the above-mentioned features are i n d i -  c a t i v e of a chronic inflammatory c o n d i t i o n (Koss., 1968).  3. 5  Polyovular F o l l i c l e s . A small proportion of f o l l i c l e s contained more than one oocyte.  Most of them were i n the e a r l y p r i m o r d i a l stage, and probably d i d not proceed past that stage.  Nevertheless, s e v e r a l f o l l i c l e s c o n t a i n i n g two or  r a r e l y three or f o u r , oocytes had reached'the (Plate 3.5).  small t e r t i a r y f o l l i c l e stage  The l a r g e s t polyovular f o l l i c l e , c o n t a i n i n g four oocytes, was  1.3 mm i n diameter. The greatest number of primary or l a r g e polyovular f o l l i c l e s , 18, occurred i n the ovaries of a doe 2.5 years o l d .  This doe had the l a r g e s t  AS  ovaries and greatest number of 1 to 2 mm (diameter) f o l l i c l e s of a l l f e males c o l l e c t e d .  I t s ovaries contained innumerable polynuclear  and polyovular p r i m o r d i a l f o l l i c l e s . oocytes were numerous.  oocytes  Tubules containing as many as 10  The second l a r g e s t number of polyovular f o l l i c l e s ,  eight, occurred i n the ovaries of a y e a r l i n g .  Only a f r a c t i o n of the  polyovular f o l l i c l e s i n each ovary can be found by r o u t i n e examination of every tenth s e c t i o n , because the p r o b a b i l i t y of s e c t i o n i n g two or more oocytes with one s l i c e decreases as the s i z e of the f o l l i c l e increases. Consequently, determination of the occurrence of polyovular f o l l i c l e s i n each ovary e n t a i l s examination  of every tenth s e c t i o n through each of  hundreds of f o l l i c l e s . At l e a s t one oocyte i n each a c t i v e polyovular f o l l i c l e was normal i n appearance ,but most supernumary oocytes were small and retarded, i n d e v e l opment.  They were often located i n the mural granulosa or i n a poorly de-  veloped cumulus oophorus. ing,  I n many instances,the f o l l i c l e s were s t i l l grow-  as i n d i c a t e d by m i t o s i s . Polyovular f o l l i c l e s occurred i n female deer of a l l ages but were  most frequent i n young animals and i n ovaries with a great number of small follicles.  When sex c e l l s are concentrated, there i s a greater chance that  f o l l i c u l a r c e l l s w i l l form a r i n g around, two or more oocytes.  3. 6  F o l l i c u l a r Cysts. Two types of f o l l i c u l a r c y s t s were found.  The f i r s t type, derived  from normal f o l l i c l e s i n the ovarian cortex and l i n e d by a s i n g l e l a y e r of granulosa c e l l s , was rare i n deer of Northwest Bay.  One occurred i n an a d u l t  doe c o l l e c t e d on October 30, p r i o r to the breeding season.  Another occurred  i n a doe on the verge of i t s f i r s t o v u l a t i o n of the current season. lumena of both f o l l i c l e s were c l e a r and achromatic.  The  The  second type o f c y s t was  columnar e p i t h e l i u m , c o n t a i n e d the mesovarium.  f l u i d , and  i n form, l i n e d w i t h  o c c u r r e d i n the h i l a r  ( S t e r n b e r g , 1963).  metaplasia  great-  epithelium  The neoplasms, w h i c h d i d not appear t o i n t e r f e r e w i t h  r e p r o d u c t i o n , occurred  i n 1.8%  about 200 ml of f l u i d ,  occurred  Primordial  or n e o p l a s i a of c o e l o m i c  ciliated  r e g i o n or i n  These s t r u c t u r e s , termed s e r o u s cystadenomas, v a r i e d  l y i n s i z e and r e p r e s e n t e d  3. 7  follicular  (7/398) of a l l does. i n a p r e g n a n t doe  The  largest, containing  c o l l e c t e d on May  7.  Follicles  P r i m o r d i a l f o l l i c l e s , which occurred  i n a narrow zone  immediately  below the t u n i c a a l b u g i n e a , were counted i n one 10 p. ( t h i c k ) , m i d - h o r i z o n t a l s e c t i o n o f each ovary. dial  f o l l i c l e s was  The  circumference  o f the zone c o n t a i n i n g the  primor-  c a l c u l a t e d f r o m the d i a m e t e r o f the s e c t i o n ( i f s p h e r o i d )  or measured d i r e c t l y w i t h a c a l i b r a t e d o c u l a r s c a l e , i f the s e c t i o n had r e c t a n g u l a r or i r r e g u l a r shape. the p r i m o r d i a l f o l l i c l e s was index of f o l l i c u l a r  per l i n e a r mm  c l a s s e s , and were n o t  o f the f o l l i c u l a r  Values  a p r o g r e s s i v e d e c l i n e i n the number o f  primor-  ( F i g . 3).  There was  a .significant  i n the number o f f o l l i c l e s between the 1.5  between the l a t t e r and  the 3.5  age  class.  and  de-  the 2.5  age  Other d i f f e r e n c e s  significant. The i n d e x o f p r i m o r d i a l - f o l l i c l e numbers was  e s p e c i a l l y i n young a n i m a l s . f r o m 2.8  count t o o b t a i n an zone.  d i a l f o l l i c l e s w i t h i n c r e a s i n g age (P < 0.05)  containing  o v a r i e s were averaged.  G e n e r a l l y t h e r e was  crease  l e n g t h o f the zone ( i n mm)  d i v i d e d i n t o the f o l l i c u l a r  occurrence  'obtained f o r the two  The  a  t o 27.4- i n y e a r l i n g s .  The  i n d e x v a r i e d f r o m 1.2  I t i s d i f f i c u l t to  two-dimension i n d i c e s of f o l l i c u l a r s i n c e the t h i r d d i m e n s i o n  highly variable, t o 17.5  i n fawns and  convert,accurately,these  numbers t o a b s o l u t e numbers f o r each  must be c o n s i d e r e d .  C a l c u l a t i o n s i n d i c a t e an  ovary  Fig.. 3.  30  11 10  The age-specific.number of p r i m o r d i a l f o l l i c l e s per mm of o u t e r c o r t e x i n 10 u s e c t i o n s from the o v a r i e s of deer c o l l e c t e d i n November and early-December, 1963-67. (Mean, 95% c o n f i d e n c e l i m i t s , standard d e v i a t i o n and sample s i z e ) .  32  9 28 PH co &q  o.  1—t  o  24 !—I  n  20  17  1  §  17  I  O  c  T  2 1 •  "oTF  —1—  2.5  4.5  4 4  6.5  8.5 • AGE (YEARS)  10.5  L2.5  14. 5  16.  18.5 o  average number of about 50,000,and a range of from 8,000 t o 200,000 p r i m o r d i a l f o l l i c l e i n each o v a r y of fawns 5 months o l d . A l l p r i m o r d i a l f o l l i c l e s were counted i n e v e r y t e n t h s e c t i o n of one  o v a r y f r o m the o l d e s t d e e r , aged 19.5  years.  W i t h due  the s i z e of p r i m o r d i a l f o l l i c l e s , s e c t i o n t h i c k n e s s c a l c u l a t e d t h a t the o v a r y c o n t a i n e d secondary f o l l i c l e s . the doe  contained  j u s t over 1000  considerably  15  I t was  estimated  earlier  ovary,  that  a s t o r e of from 10,000 t o 200,000 o o c y t e s , w i t h a  Based on the above s t a t i s t i c s the average fawn had  t i m e s more o o c y t e s t h a n the o l d e s t The  535 p r i m o r d i a l f o l l i c l e s and  oocytes.  to  i n t e r v a l , i t was  Assuming e q u a l numbers of f o l l i c l e s i n the o t h e r  each fawn o v a r y c o n t a i n e d mean of 50,000.  and  consideration  average s i z e and  100  doe.  appearance of p r i m o r d i a l f o l l i c l e s  varied  between i n d i v i d u a l s of a l l a g e - c l a s s e s , b u t d i f f e r e n c e s were  not a p p a r e n t l y  l i n k e d t o s t a g e s of the e s t r o u s  cycle.  However, as  the  b r e e d i n g season p r o g r e s s e d t h e r e appeared to be a g r e a t e r number o f l a r g e sex c e l l s i n p r i m o r d i a l f o l l i c l e s .  Many of the l a r g e o o c y t e s were  i n a t h i c k zona p e l l u c i d a .  3. 8  enclosed  ••  Tertiary Follicles. To f a c i l i t a t e the a n a l y s i s and d i s c u s s i o n o f " t e r t i a r y  follicles,  t h e y were a r b i t r a r i l y d i v i d e d i n t o the f o l l o w i n g s i z e c l a s s e s : Class I Class II Class I I I Class IV 3.  8.  1  Class I T e r t i a r y F o l l i c l e s .  0.5- 4.1 4-2-20 21 -30 > 30  mm mrn^ mmo mm 3  C l a s s I f o l l i c l e s were numerous  i n b r e e d i n g - s e a s o n o v a r i e s , but t h e i r i m p o r t a n c e t o the r e p r o d u c t i v e i s n o t known. l i c l e s was  U n l i k e s m a l l e r f o l l i c l e s , growth and  cyclic.  process  a t r e s i a of Class I f o l -  52  The r e l a t i o n s h i p between t h e numbers o f C l a s s I f o l l i c l e s and t h e age o f t h e f e m a l e was i n v e s t i g a t e d i n 101 p a i r s o f o v a r i e s f r o m t h e b r e e d i n g season o f 1963. Both a c t i v e and e a r l y - a t r e t i c f o l l i c l e s were counted.  The  r e s u l t s ( F i g . A) i n d i c a t e t h a t C l a s s I f o l l i c l e s were most numerous i n deer 2.5  y e a r s o l d , and l e a s t numerous i n does o l d e r t h a n 9 y e a r s .  The number o f  C l a s s I f o l l i c l e s i n y e a r l i n g s was s i g n i f i c a n t l y l o w e r t h a n i n any a g e - c l a s s e x c e p t . t h e o l d e s t group (P < 0.05). the  No C l a s s I f o l l i c l e s were p r e s e n t i n  o l d e s t doe (19-5 y e a r s ) , and another o l d doe c o n t a i n e d o n l y two such  follicles. A g e n e r a l d e c r e a s e i n t h e number o f C l a s s I f o l l i c l e s o c c u r r e d a f t e r t h e b r e e d i n g season.  I n fawns, t h e number o f C l a s s I f o l l i c l e s d e -  c r e a s e d s i g n i f i c a n t l y f r o m a mean o f 50 i n November (N = 23), 16, i n e i g h t fawns c o l l e c t e d f r o m December t o June.  t o a mean o f  I n y e a r l i n g s , t h e average  number d e c r e a s e d f r o m 38 i n November (N = 2 5 ) , t o 23 (N = 1 3 ) , i n p o o l e d samples f r o m December t o June.  C l a s s I f o l l i c l e s were n o t counted i n  o v a r i e s o f a d u l t s c o l l e c t e d f r o m December t o May, but a p r o g r e s s i v e d e c r e a s e i n f o l l i c u l a r numbers was a p p a r e n t . A f t e r n o t i n g t h e r e l a t i o n s h i p between s i z e o f a few o v a r i e s and t h e i r f o l l i c u l a r development,  I h y p o t h e s i z e d t h a t t h e s i z e o f t h e o v a r y was  l a r g e l y d i c t a t e d by t h e number o f C l a s s I f o l l i c l e s and n o t by t h e number or size of larger f o l l i c l e s .  T h i s h y p o t h e s i s was t e s t e d i n a sample o f 24-  p a i r s o f o v a r i e s f r o m fawns.  There was a s i g n i f i c a n t c o r r e l a t i o n ( r = 0.89)  between t h e t o t a l number o f s m a l l f o l l i c l e s i n a p a i r o f o v a r i e s and t h e w e i g h t o f t h e two o v a r i e s .  Thus, o v a r y s i z e d i f f e r e n c e s between fawns, as'  measured by w e i g h t , was l a r g e l y due t o d i f f e r e n c e s i n t h e number o f Class I f o l l i c l e s .  This r e l a t i o n s h i p a l s o applied t o the ovaries of  older deer, but t o a l e s s e r extent.  F i g . -4.  The age-specific number of Class  1 (1-2 mm diam) f o l l i c l e s i n both ovaries of females c o l l e c t e d i n November and e a r l y December, 1963-67.  .24  100 • 90 23 80 CO I—I  10  70  w O  13  60  25  -I  1 95%  confidence.limit (Sx-t.os)  50 CO  co 40  <  1-1 o  o  Sample s i z e Standard d e v i a t i o n  • Mean  6  -I  30  o s  20 10 -  0.5  1.5  2.5  AGE (YEARS)  -4-5  5-5-8.5  9-5S+  54  3. 8. 2  F o l l i c l e s P r i o r To F i r s t and Second Ovulation.  The frequency-  d i s t r i b u t i o n s of the l a r g e s t f o l l i c l e i n p a i r s of ovaries c o l l e c t e d during the breeding season,revealed d i f f e r e n c e s i n f o l l i c u l a r development between fawns, y e a r l i n g s , and adults ( F i g . 5).  The histograms f o r fawns and y e a r l i n g s  include- animals c o l l e c t e d from November 6 to December 5.  The histogram f o r  adults i n c l u d e s animals c o l l e c t e d between November 6 and November 23,when most were approaching t h e i r f i r s t o v u l a t i o n of the season. was 11-20 mm  3  i n fawns, 31-4-0 mm  3  i n y e a r l i n g s , and 4-1-50 mm  The mode c l a s s 3  i n adults.  3 None of the f o l l i c l e s i n fawns exceeded 50 mm , whereas f o l l i c l e s i n y e a r l i n g s 3 and adults attained volumes up to 100 mm  .  The l a r g e s t f o l l i c l e i n some year-  l i n g s d i d not exceed 20 mm^, but a l l adults developed at l e a s t one f o l l i c l e . l a r g e r than 20 mm  i n each c y c l e .  Many of the large f o l l i c l e s i n the ovaries had stopped growing, as i n d i c a t e d by c e s s a t i o n of c e l l d i v i s i o n i n the granulosa and other c y t o l o g i c a l c h a r a c t e r i s t i c s ( " e a r l y - a t r e t i c " and " t r a n s i t i o n " f o l l i c l e s i n F i g . 5). fawns w i t h l a r g e f o l l i c l e s e x i b i t e d unusual ovarian a c t i v i t y .  Two  The ovaries  of one animal contained a 50 mm  e a r l y - a t r e t i c f o l l i c l e ( A t r e s i a l ) , and a 3 small, newly-ruptured f o l l i c l e that s t i l l contained the oocyte. A 4-4 mm 3  t r a n s i t i o n a l f o l l i c l e and eight 4 to 25 mm  f o l l i c l e s , i n abnormal states of  atresia,occurred i n another fawn. Although more than one Class IV f o l l i c l e ( > 30 mnP) never developed i n fawns, two Class IV f o l l i c l e s were found i n 13% of y e a r l i n g s i n ''. 1963, and i n 11% of a l l y e a r l i n g s c o l l e c t e d from 1963 to 1966 (Table 3 ) . From 1963 to 1964, the frequency of y e a r l i n g s w i t h two Class IV f o l l i c l e s or one Class IV plus one Class I I I f o l l i c l e decreased. P r i o r to f i r s t o v u l a t i o n , two Class IV f o l l i c l e s occurred f r e quently i n females 2.5 years old and older.  In adult females, the f r e -  quency of two Class IV f o l l i c l e s increased w i t h age up to 4«5 years,and then  55  Fig.  The f r e q u e n c y o f t h e l a r g e s t f o l l i c l e , and i t s s t a t e o f m a t u r i t y , i n f a u n s , y e a r l i n g s , and a d u l t s p r i o r t o f i r s t • o v u l a t i o n o f t h e c u r r e n t b r e e d i n g season.  FOLLICULAR STATE [  40 Fawns n = 45  35 30  | Early-Atretic Transitional Active  25 20 15 10 5  35  -I  30  Adults n = 95  25 20 15 10  5 010  1120 VOLUME  21-  30  J l-  40  4'i-  50  60  b l -  70  HL 7180  81-  90  91100  (MM ) OF THE LARGEST FOLLICLE I N EACH FEMALE 3  56  Table 3.  Pe r c e n t f r e q u e n c y o f one C l a s s I V , two C l a s s I V , and one C l a s s I V p l u s one C l a s s I I I f o l l i c l e s i n p r e o v u l a t o r y y e a r l i n g s , Nov. 6-Dec. 6, 1963-1966.  Per c e n t Frequency  Year  One C l a s s I V follicle ( >30.mm )  Two C l a s s I V follicles  14 2  69 73 71 100  13 07 07  47  72  11  Sample S i z e 1 5  1963-66  3  3  16  1963 1964 1965 1966  13 00 00 00  50  4  (Table 4 ) .  d e c r e a s e d t o a lower l e v e l i n o l d e r a n i m a l s Table 4 .  One C l a s s I V & One Class I I I f o l l i c l e ( > 30mm +20-30mm3)  The p e r c e n t f r e q u e n c y o f a d u l t d o e s , i n s i x age c l a s s e s , c o n t a i n i n g o n l y one or two C l a s s I V f o l l i c l e s , p r i o r t o f i r s t o v u l a t i o n .  Per c e n t Age-class ( y r ) One C l a s s I V F o l l i c l e * Two C l a s s I V F o l l i c l e s Sample S i z e  f r e q u e n c y i n age-- c l a s s  2.5  3.5  4.5  5.5  52  42 58 19  ' 15 85 13  33 . 67 12  48  29  6.5-8.5  9.5&^  33 67 15  25 75 4  > 30mm  3  I n many i n s t a n c e s , e s p e c i a l l y i n young and v e r y o l d p r e o v u l a t o r y ; females,  t h e two C l a s s I V f o l l i c l e s  y e a r l i n g s were b o t h f o l l i c l e s  were o u t o f phase.  r a t e d i n t h e same stage o f development.  three of the others, the l a r g e r f o l l i c l e was d e f i n i t e l y a t r e t i c  O n l y i n one o f f i v e  o f t h e two was more advanced,- and one  (Stage 2 o f a t r e s i a ) .  C l a s s I V f o l l i c l e and one C l a s s I I I f o l l i c l e . were o n l y s l i g h t l y o u t o f phase.  In  Two y e a r l i n g s each had one I n both i n s t a n c e s the f o l l i c l e s  About o n e - h a l f  ( 2 1 / 4 O )  o f t h e two C l a s s I V  57  follicles  i n a d u l t s were a t t h e same stage o f m a t u r a t i o n b u t o t h e r s were  out o f phase by as much as f o u r s t a g e s i n t h e a c t i v e , t r a n s i t i o n a l , 1, a t r e s i a 2, etc. c l a s s i f i c a t i o n . licles  U s u a l l y , however, t h e o u t - o f - p h a s e f o l -  were i n t h e t r a n s i t i o n a l or e a r l y - a t r e t i c s t a g e i . e . one or two  s t a g e s o u t o f phase.  C l a s s I I I or I V , e a r l y - a t r e t i c f o l l i c l e s  3 9 % o f a d u l t o v a r i e s (37 o f 9 5 ) . the  atresia  l a r g e s t one i n t h e o v a r i e s .  occurred i n  U s u a l l y t h e e a r l y - a t r e t i c f o l l i c l e was When f o l l i c l e s  were o u t o f phase,  often  o n l y one r u p t u r e d t o form a c o r p u s l u t e u m and t h e o t h e r developed i n t o an a c c e s s o r y c o r p u s luteum.  T h i s phenomenon was more common i n young f e m a l e s  (1.5 t o 3.5 y e a r s ) t h a n i n o l d e r does. ruptured a t the f i r s t  Occasionally, a t r e t i c  follicles  ovulation.  There were n o t a b l e d i f f e r e n c e s between t h e f i r s t and second f o l l i c u l a r c y c l e s i n t h e volume o f t h e l a r g e s t f o l l i c l e i n each doe just prior to ovulation.  F o l l i c l e s i n f e m a l e s on t h e verge o f second  o v u l a t i o n a t t a i n e d s m a l l e r b u t l e s s v a r i a b l e volumes approaching f i r s t ovulation.  than f o l l i c l e s  i n does  F o l l i c l e s near f i r s t r u p t u r e ranged i n volume  3 f r o m about 20 t o 100 mm , whereas f o l l i c l e s of  i n seven f e m a l e s on t h e v e r g e  3 3 second o v u l a t i o n ranged between 35 and 51 mm , and averaged 45 mm.. -a  The s m a l l e s t p r e - o v u l a t o r y f o l l i c l e i n t h e l a t t e r group, 35 mm , was i n t h e o v a r i e s o f an i n s e m i n a t e d y e a r l i n g i n e s t r u s . Females a p p r o a c h i n g second o v u l a t i o n , l i k e t h o s e a p p r o a c h i n g f i r s t o v u l a t i o n , contained various combinations of Class I I , I I I ,  and I V f o l l i c l e s .  The most f r e q u e n t c o m b i n a t i o n i n a d u l t s was two C l a s s I V f o l l i c l e s .  Also  of f r e q u e n t o c c u r r e n c e was a s i n g l e C l a s s I V f o l l i c l e , and one C l a s s I V f o l l i c l e p l u s one C l a s s I I o r I I I f o l l i c l e .  58  3. 8. 3 Preovulatory  The R e l a t i o n s h i p Between Active and A t r e t i c F o l l i c l e s i n Female Deer.  development was  The occurrence and nature of c y c l i c f o l l i c u l a r  i n v e s t i g a t e d by comparing the volume of the l a r g e s t a c t i v e  f o l l i c l e i n each female to the volume of the l a r g e s t a t r e t i c f o l l i c l e (presumably from the previous c y c l e ) .  C y c l i c development would be i n d i c a t e d  by a r e c i p r o c a l r e l a t i o n s h i p between the l a r g e s t a c t i v e and l a r g e s t a t r e t i c f o l l i c l e s i n a p a i r of ovaries.  In a d d i t i o n , the a t r e t i c f o l l i c l e s  become p r o g r e s s i v e l y more a t r e t i c as they decreased i n s i z e , w h i l e successive,active  f o l l i c l e became l a r g e r .  should the  Both of the above r e l a t i o n s h i p s  were found i n adults and y e a r l i n g s c o l l e c t e d during October, and i n a l l females c o l l e c t e d during the breeding season ( F i g . 6 ) . The r e c i p r o c a l r e l a t i o n s h i p between the l a r g e s t a c t i v e and  the  l a r g e s t a t r e t i c f o l l i c l e was most evident i n a d u l t s , i n c l u d i n g those c o l l e c t e d i n October, and l e a s t evident i n fawns and progressive adults.  a t r e s i a of the s h r i n k i n g f o l l i c l e was  some y e a r l i n g s .  The  also most evident i n  Some fawns and a few y e a r l i n g s did not produce f o l l i c l e s  larger  3 than 10 to 20 mm , which r e s u l t e d i n a group of i n t e r c e p t s near the o r i g i n i n F i g . 6. 3. 8. 4  The A t r e s i a of F o l l i c l e s A f t e r F i r s t and Second  Ovulation.  The volume of the l a r g e s t a t r e t i c f o l l i c l e p l o t t e d against the volume of the l a r g e s t second-cycle (growing) f o l l i c l e i n the same p a i r of ovaries i s presented i n F i g . 7. of time because one,  The volume of the l a r g e s t a c t i v e f o l l i c l e i s a measure two, or three f o l l i c l e s begin growth soon a f t e r ovu-  l a t i o n and continue to grow f o r several days u n t i l second o v u l a t i o n .  It is  evident i n F i g . 7 that a l l f i r s t - c y c l e f o l l i c l e s l a r g e r than about 20  mm ,  which f a i l e d to ovulate at f i r s t estrus, became p a r t l y l u t e i n i z e d by the  3 time the l a r g e s t second-cycle f o l l i c l e had reached about 10 mm  (about 2  3  59  Fig.  6.  The r e l a t i o n s h i p between the l a r g e s t a c t i v e f o l l i c l e and the l a r g e s t a t r e t i c f o l l i c l e i n p a i r s of ovaries, from preovulatory females. The degree of a t r e s i a from 1 (early a t r e s i a ) to 8 ( l a t e a t r e s i a ) , and a t r a n s i t i o n a l stage ( t ) i s i n d i c a t e d (L = l u t e i n i z e d f o l l i c l e ) .  Adults Nov. 6-23  50 H • .  o  3  .t  •H rH rH O  AO  1 1 - 2  50 -  Fawns Nov. 6-Dec. 5  4.0  2  PH  30 -|  •t  o  1-2  <  20  30  2-3  -H •P CD •  3  -p M (D  3  20 5-6  4  2 t  -1 .3  t  U0  10  10 •  5-6  '•. .2-3 2  '5  — i —  20  10  50  40  30  10  60  20  — i  r—  50  40  30  90  75  <  604  Yearlings Nov. 6-Dec. 5  50-  Adults and Yearlings ( a Oct. 1963  1-2  50  1-2  4.0-  AO  1-2  2-3 • 1  30-  20-  t  20  •» «  *  t  *  . 4 4.  1  •  .4-5  .  2-5  •  10  \ •  6.  3-4  " 5-6 .  .5  6 .5  ' *  "T"  -7  7  I  30 40 50 LARGEST ACTIVE FOLLICLE (MM3<) 10  2-3  30  20  J  I  1  60  "lO  20  ~~30  4~0  50"  )  Fig.  7.  The r e l a t i o n s h i p between t h e l a r g e s t a c t i v e , secondc y c l e f o l l i c l e and the l a r g e s t a t r e t i c f o l l i c l e o f p r e v i o u s c y c l e s (L = l u t e i n i z e d f o l l i c l e ) .  50  D  L  60  °  1  4.0 o  O  L  30  L  0  20 10  ®  3  O  L  oo o —  I  10  —  Yearlings  L  0  o —  1  I  20  o 1  30  (185 mm) 3  50  B  1 —  60  70  1  —  L  (120 mm3)  80 70  1  -40  J  ^ 6 0  Adults  o M  5  0  o ^ AO o  43  3  L  H  E-i  § 30 EH  20  L  |L L  O  10  0°oa 10  L  o 20  oo  L  30  4.0  50  60  70  LARGEST ACTIVE SECOND-CYCLE FOLLICLE  (MM ) 3  Luteinized Non-luteinized  61  days a f t e r o v u l a t i o n ) .  Two l a r g e f o l l i c l e s i n d i c a t e d i n F i g . 7 u n d o u b t e d l y  would have l u t e i n i z e d i n a n o t h e r d a y or two. The i n c i d e n c e o f C l a s s I I I and C l a s s I V a t r e t i c f o l l i c l e s i n y e a r l i n g s w i t h young f i r s t - c y c l e c o r p o r a l u t e a was 31% ( 5 / l 6 ) . I n a l l ovaries i n which l u t e i n i z e d f o l l i c l e s occurred, a l u t e i n z e d f o l l i c l e was t h e l a r g e s t a t r e t i c f o l l i c l e .  I n a d u l t s , t h e r e was an i n d i -  c a t i o n t h a t some l u t e i n i z e d f o l l i c l e s i n c r e a s e d i n volume a f t e r began,because two a t t a i n e d volumes o f 185 and 120 mm  luteinization  ( F i g . 7 ) . They were  l a r g e r t h a n any p r e o v u l a t o r y f o l l i c l e s . L u t e i n i z e d f o l l i c l e s o c c u r r e d i n 4-2% (5/12) o f y e a r l i n g s w i t h f i r s t - g e n e r a t i o n corpora l u t e a estimated  t o be o l d e r t h a n 2 days.  Volumes  3 o f t h e l u t e i n i z e d f o l l i c l e s were 58, 3 1 , 29, 9 and 6 mm .  A second  l u t e i n i z e d f o l l i c l e o c c u r r e d i n o n l y one o f t h e y e a r l i n g s and i t was a small f o l l i c l e . The i n c i d e n c e o f l u t e i n i z e d f o l l i c l e s  (accessory corpora  f o l l o w i n g f i r s t o v u l a t i o n i n a d u l t does was 48.5% (16/33).  lutea)  Be cause  l u t e i n i z a t i o n was n o t e v i d e n t f o r a d a y or two f o l l o w i n g o v u l a t i o n , o n l y  3 those w i t h c o r p o r a l u t e a l a r g e r t h a n 10 mm , and s e c o n d - c y c l e l a r g e r t h a n 5 mm , were i n c l u d e d i n t h e sample. 62.5%  follicles  The i n c i d e n c e was h i g h e s t  (10/16) i n 1963 when r e p r o d u c t i v e r a t e s were h i g h e s t . A f t e r f i r s t o v u l a t i o n , m u l t i p l e l u t e i n i z e d f o l l i c l e s occurred i n  6 o f 17 a d u l t f e m a l e s w i t h l u t e i n i z e d f o l l i c l e s . contained  F i v e o f the females  two t o f o u r l u t e i n i z e d f o l l i c l e s and t h e f i f t h f e m a l e had I 4  accessory corpora l u t e a i n i t s ovaries.  T h i s e x c e p t i o n a l female was t h e  3  o n l y one w i t h two C l a s s I V ( >30 mm ) l u t e i n i z e d  follicles.  A s l i g h t l y higher i n c i d e n c e of l u t e i n i z e d f o l l i c l e s occurred i n a d u l t f e m a l e s o v u l a t i n g o n l y one f o l l i c l e a t f i r s t e s t r u s t h a n i n those o v u l a t i n g two or t h r e e f o l l i c l e s .  The r e s p e c t i v e f r e q u e n c i e s were 5U%  62  (N=13) and  45%  (N=20).  After the second ovulation i n y e a r l i n g s and a d u l t s , a wave of a t r e s i a swept the remaining second-cycle f o l l i c l e s .  These were r a r e l y  3  l a r g e r than 10 mm  and soon shrank t o an i n s i g n i f i c a n t s i z e .  Often,the  h i g h l y a t r e t i c f o l l i c l e s of previous cycles were l a r g e r than those o r i g i n a t i n g from second-cycle f o l l i c u l a r  development.  In a d u l t s , only one Class IV f o l l i c l e (75 mm ) f a i l e d to rupture 3  and i t developed i n t o an accessory corpus luteum (the doe ruptured only one f o l l i c l e a t second o v u l a t i o n ) . luteinize. only  11%  One other large f o l l i c l e f a i l e d to  A f t e r the second o v u l a t i o n , l u t e i n i z e d f o l l i c l e s occurred i n  (5/44-)  of the a d u l t s .  Most of these were small (Class I ) f o l l i c l e s .  A f t e r second o v u l a t i o n , no a t r e t i c f o l l i c l e s l a r g e r than 11 mrrr 3 occurred i n 15 yearlings,and the mean s i z e was l e s s than 5 mm . 3.8. 5  The Growth of Second-Cycle F o l l i c l e s R e l a t i v e to the Growth  of F i r s t - C y c l e Corpora Lutea. After the f i r s t o v u l a t i o n , growth of corpora l u t e a was contemporaneous  with growth of the f o l l i c l e s that were destined to  ovulate a t the next estrus.  The r e l a t i o n s h i p between the volumes of the  l a r g e s t f i r s t - c y c l e corpus luteum and the l a r g e s t second-cycle f o l l i c l e was s i m i l a r i n y e a r l i n g s and i n adults,but v a r i a b i l i t y was greater i n y e a r l i n g s and the two curves d i f f e r e d s l i g h t l y (Fig.. 8 ) . r e l a t i o n s h i p , a c t i v e corpora l u t e a were d i s t i n g u i s h e d  In this  from early-degenerate  ones,for the l a t t e r were vacuolate and s h r i n k i n g i n volume p r i o r to second estrus. The curves i n F i g . 8 represent the i n t e r r e l a t i o n s h i p between two growth curves; one f o r f o l l i c l e s and one f o r corpora l u t e a .  Early  growth of the corpus luteum was f a s t e r than that of the f o l l i c l e , b u t a f t e r 3  reaching a volume of about 7+0 to 50 mm ^the corpus luteum stopped growing  8.  The r e l a t i o n s h i p between t h e volume o f t h e l a r g e s t a c t i v e s e c o n d - c y c l e f o l l i c l e and t h e volume o f t h e l a r g e s t f i r s t c y c l e corpus l u t e u m (CL 1) i n y e a r l i n g and a d u l t f e m a l e s . . (Curves f i t by e y e ) .  ®  Active  CL 1  64  whereas t h e f o l l i c l e c o n t i n u e d t o grow. were near second o v u l a t i o n l u t e a ( F i g . 8).  Most f o l l i c l e s l a r g e r t h a n 30 mm  and u s u a l l y t h e y accompanied  degenerate corpora  I n t h e l a s t 1 t o 3 days p r i o r t o second o v u l a t i o n , many 3  of t h e s e c o r p o r a l u t e a shrank i n volume from 4-0-50 mm t h i s reason, the curve i s f i t t e d  3  t o 25-4-0 mm . F o r  o n l y t o c o o r d i n a t e s r e p r e s e n t e d by a c t i v e  corpora l u t e a . In y e a r l i n g females, the rate of f o l l i c u l a r the c o r p u s l u t e u m a t an e a r l i e r s t a g e than i n a d u l t s . difference  growth exceded t h a t o f I attribute this  t o l e s s e r c o r p u s l u t e u m development i n y e a r l i n g s  than i n a d u l t s .  The d i f f e r e n c e , however, may n o t be r e a l because o f t h e s m a l l for  sample  size  yearlings. 3. 8. 6  The Growth o f T h i r d - C y c l e F o l l i c l e s R e l a t i v e t o t h e Growth  of Second-Cycle C o r p o r a L u t e a .  The r e l a t i o n s h i p between t h e volumes o f  c o r p o r a l u t e a and f o l l i c l e s , a f t e r second o v u l a t i o n contrasts  of the current  r e m a r k a b l e w i t h t h e r e l a t i o n s h i p between t h e s t r u c t u r e s  f i r s t ovulation.  season, after  The l a r g e s t s e c o n d - c y c l e c o r p u s l u t e u m grew a t about t w i c e  the r a t e o f t h e l a r g e s t t h i r d - c y c l e f o l l i c l e  ( F i g . 9 ) . Moreover, t h e 3  c o r p o r a l u t e a c o n t i n u e d t o grow u n t i l many a t t a i n e d  volumes o f 100 t o l60mm .  By t h i s s t a g e , some o f t h e f o l l i c l e s were i n e a r l y s t a g e s o f a t r e s i a and were succeeded by a f o u r t h g e n e r a t i o n o f f o l l i c l e s . .  The growth o f t h e l a r g e s t  f o l l i c l e , r e l a t i v e t o t h e growth o f t h e l a r g e s t c o r p u s l u t e u m , was greater i n y e a r l i n g s females than i n a d u l t s 3. 8. 7  ( F i g . 9).  D e t e r m i n i n g t h e Growth Rate o f F o l l i c l e s and C o r p o r a L u t e a .  I n p a s t s e c t i o n s ,the growth o f f o l l i c l e s r e l a t i v e t o 'the growth o f t h e c o r p o r a l u t e a was p r e s e n t e d b u t no d a t a were o b t a i n e d on t h e growth r a t e o f f o l l i c l e s and c o r p o r a l u t e a .  This.information  was p r o c u r e d by an  65 The r e g r e s s i o n s o f the volumes of the l a r g e s t t h i r d c y c l e f o l l i c l e on the l a r g e s t s e c o n d - c y c l e c o r p u s l u t e u m .(.CL 2). i n y e a r l i n g and a d u l t f e m a l e s . ( R e g r e s s i o n s f i t "by l e a s t - s q u a r e s method t o a c t i v e f o l l i c l e s only).E  Active  ©  Atretic Follicle  1 The F i r s t Stage Of Follicular Atresia 2 A L a t e r Stage Of Follicular Atresia  Yearlings n = 15  0.35x - 0.15 0.98  "I  1  1  1  1  1  1  I  1  ' 32  1  1  64  I  I  1  1  1  128  96  1+ ©  y  =  O.4I6X  +  0.91  r = 0.91  I  Follicle  1 1 1 1 1—1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 32 64 96 128 160 LARGEST  CL  2  (MM )' 3  r-  66  i n d i r e c t method o f e s t i m a t i n g t h e age o f ova r e c o v e r e d f r o m t h e o v i d u c t s and uteri. The o v u l a t o r y c y c l e from w h i c h t h e ova were d e r i v e d , t h e number of ova found i n each  r e p r o d u c t i v e t r a c t , t h e c e l l s t a g e , t h e presence or  absence iof sperm on t h e ovum, t h e l o c a t i o n o f t h e ovum, and the method o f r e c o v e r y are p r e s e n t e d i n Table 5.  T a b l e 5.  The number and c o n d i t i o n of 25 o v u l a t e d ova r e c o v e r e d from t h e o v i d u c t s and u t e r i o f 19 does, 1964-67.  Specimen No.  Ovulatory Cycle  No. Ova Recovered  Location  X62  First  1  Oviduct  K9 Zll  iiII  Z24  Second  Z26  II II  V21 V19  II  Z28 X61 Z23 K73 Z15 Z22 V20 Vll V30 V28 V24 V27  First Second II  it  First  n  Second II  II  it ti n OS Fl *  1 1 1 2 2 2* 1 1 2 1 1 1 2 2 1 1 1 1  Cell Stage 1 1 1 1 1 . 1 1-2 2  II II  n  1! 1 1 II II II II  4 4  II II II II  4-8  5  6 8 8-16 16 32-64  II  11  Uterus  iiII  64-128  Biastula  Sperm  Recovery Method  Nil  OS  1 1 n  11 11 11 FL  Few Many  11  ti  OS  Few Nil Few Many  11 11 11 11  •  Many  ti 11 11 11  Oviduct Sections F l u s h i n g of Oviduct or Uterus One Fragmented  P e r t i n e n t o b s e r v a t i o n s r e l a t i n g t o t h e above ova a r e as f o l l o w s : l)  I n a l l s i x i n s t a n c e s i n which two ova were f o u n d , both were a t t h e same s t a g e o f development.  11 11 11 n 11 n Fl  11 n 11 11 11  67  2.  Ova from four of s i x deer, which ovulated a t f i r s t estrus, had no sperm attached to the zona p e l l u c i d a (Plate 10. 2-4). Nevertheless, oviducts of one of the four contained a twoc e l l e d ovum with fragmented n u c l e i ' ( P l a t e 10. 4 ) . Manysperm were attached to the zona p e l l u c i d a of the other two ova from the f i r s t ovulatory cycle and they had divided i n t o f i v e and s i x c e l l s .  In contrast, abundant sperm was  attached to the zona p e l l u c i d a of a l l ova from the second estrus. 3.  Ova containing as many as 16 c e l l s were found i n the oviduct, whereas the three ova i n more advanced stages of segmentation were located i n the uterus.  4.  Although more ova were obtained by s e c t i o n i n g oviducts than by f l u s h i n g them, the l a t t e r method i s more e f f i c i e n t and the only p r a c t i c a l means of obtaining ova from the uterus.  The ages of segmenting ova were estimated from r a t e s of cleavage i n the ova of other mammals because the r a t e has not been determined i n deer.  Ova of a d i v e r s i t y of mammalian species segment a t about the same  r a t e (See Appendix 1 f o r cleavage r a t e s , and Hamilton and Laing, 1946, f o r a review).  The average of the r a t e s of cleavage i n the goat, sheep, and  cow probably provide the best estimate f o r deer because these three domestic species most c l o s e l y resemble deer i n the length of the estrous c y c l e , time of i m p l a n t a t i o n , and g e s t a t i o n period. i n these species i s as f o l l o w s :  The approximate average r a t e  F a c i n g page 68  Plate- 9 Atypical  3  Atresia  of F o l l i c l e s  1.  A t y p i c a l a t r e s i a of a 1 2 mm follicle. A zone of e l o n g a t e f i b r o b l a s t - l i k e c e l l s occur between the c e n t r a l degenerate a r e a ( b l a c k m a t e r i a l on r i g h t s i d e of photograph) and the t h e c a e x t e r n a (X800).  2.  The w a l l of a f o l l i c l e u n d e r g o i n g a t y p i c a l a t r e s i a . The c e n t r a l r e g i o n of the f o l l i c l e ( r i g h t ) c o n t a i n s l e u c o c y t e s . The o u t e r zone ( f a r l e f t ) i s the t h e c a e x t e r n a . The r e g i o n c o r r e s p o n d i n g I n l o c a t i o n t o the former s t r a t u m granulosum and t h e c a i n t e r n a c o n t a i n s f i b r o b l a s t i c c e l l s , blood v e s s e l s and l e u c o c y t e s ( X 2 0 0 ) .  3. F i b r o b l a s t i c c e l l s (one i n metaphase), degenerate c e l l s , c e l l d e b r i s and l e u c o c y t e s occur i n the r e g i o n of the former w a l l of a f o l l i c l e undergoing a t y p i c a l a t r e s i a ( X 4 O C ) . 1  R  4-. L e u c o c y t e s and amorphous m a t e r i a l i n the c e n t r a l p o r t i o n o f a a t an e a r l y stage of a t y p i c a l a t r e s i a (X800). 5.  follicle  P h a g o c y t i c c e l l s are d e s t r o y i n g an o o c y t e . A p o r t i o n o f the ooplasm i s v i s i b l e (lower r i g h t ) and a p o r t i o n o f the t h i n zona p e l l u c i d a i s e v i d e n t (X800).  6. L e u c o c y t e s occur among s i n g l e and l a r g e m u l t i n u c l e a t e c e l l s a t a l a t e r s t a g e of a t y p i c a l a t r e s i a (X800). 7. An advanced s t a g e s of d e g e n e r a t i o n . C o n n e c t i v e t i s s u e c e l l s occur between the d e g e n e r a t e c e l l s which c o n t a i n p a l e cream or y e l l o w pigment ( X 4 O O ) . 8.  A s c a r r e s u l t i n g from a t y p i c a l f o l l i c u l a r a t r e s i a . White or cream spaces a r e l o c a t e d i n a network o f c o n n e c t i v e t i s s u e c e l l s and f i b e r s (X4OO).  9. A s m a l l f o l l i c l e i n advanced s t a g e s of abnormal a t r e s i a . N e c r o t i c t i s s u e ( b l a c k ) s t i l l o c c u r s i n the c e n t e r of the f o l l i c l e . Scar t i s s u e c o n t a i n i n g w h i t e c e l l spaces has formed around the p e r i p h e r y of the f o l l i c l e (X40). 10.  S e v e r a l f o l l i c l e s (appear as w h i t e a r e a s i n the photograph) have undergone a t y p i c a l a t r e s i a i n t h i s o v a r y (X5).  F a c i n g page 69  P l a t e 10 The O v u l a t e d Ovum. 1.  A o n e - c e l l e d ovum f l u s h e d f r o m t h e o v i d u c t , f i x e d i n 5% f o r m a l i n , and s t a i n e d w i t h a d i l u t e s o l u t i o n o f t o l u i d i n e b l u e ( X 4 O O ) .  2. S e c t i o n o f a o n e - c e l l e d ovum d e r i v e d f r o m t h e f i r s t o v u l a t o r y c y c l e . Note t h e l a c k o f sperm and t h e d i s c o n t i n u o u s c h r o m o p h i l i c band i n the zona p e l l u c i d a (X800). 3. A d e g e n e r a t e o n e - c e l l e d ovum f r o m t h e f i r s t o v u l a t o r y c y c l e . was found on t h e zona p e l l u c i d a ( X 4 O O ) . 4.  No sperm  A t w o - c e l l e d ovum f r o m t h e f i r s t o v u l a t o r y c y c l e . No sperm was found on t h e zona p e l l u c i d a and development had p r o b a b l y c e a s e d . Two n u c l e i a r e p r e s e n t i n each c e l l ( X 4 O O ) .  5. A s i x - c e l l e d ovum f r o m t h e f i r s t o v u l a t o r y c y c l e . Note t h e chromop h i l i c l a y e r i n the zona p e l l u c i d a and t h e numerous sperm on t h e s u r f a c e o f t h e zona p e l l u c i d a (X800). 6. A o n e - c e l l e d ovum f r o m t h e second o v u l a t o r y c y c l e . The b l a c k o b j e c t i n t h e ooplasm i s t h e sperm head. Only a few sperm were a t t a c h e d t o the zona p e l l u c i d a ( X 4 O O ) . 7. A f o u r - c e l l e d ovum f r o m t h e second o v u l a t o r y c y c l e . abundant on t h e zona p e l l u c i d a ( X 4 O O ) .  Sperm a r e  8. A f i v e - c e l l e d ovum f r o m t h e second o v u l a t o r y c y c l e . Note t h e abundant sperm and t h e columnar e p t h e l i u m i n t h e o v i d u c t ( X 4 O O ) .  70  No. of c e l l s 1 2 4-8 9- 16 17- 64 65-128  Time s i n c e o v u l a t i o n (days) 0-1 1.5 2 3 4 5  The above r a t e s were a p p l i e d o n l y t o f e r t i l i z e d ova shed a t the second e s t r u s of the season.  3. 8. 8  The Rate o f F o l l i c u l a r  Growth.  The growth r a t e o f the  l a r g e s t , t h i r d - c y c l e f o l l i c l e , from 1 t o 5 days a f t e r s e c o n d - c y c l e o v u l a t i o n , was determined i n 12 does ( F i g . 1 0 ) .  I n t h e s e , the e l a p s e d time s i n c e  o v u l a t i o n was e s t i m a t e d from the c l e a v a g e s t a g e of r e c o v e r e d ova.  Growth  was a p p a r e n t l y r a p i d and l i n e a r from 1 t o 5 days p o s t - o v u l a t i o n . as 4 t o 5 d a y s , f o l l i c l e s grew t o a volume of 30 mm  As e a r l y  (near o v u l a t o r y s i z e  i n some f e m a l e s ) . The growth r a t e of s e c o n d - c y c l e f o l l i c l e s was n o t d e t e r m i n e d because o n l y two f e r t i l i z e d ova were o b t a i n e d f r o m f i r s t - c y c l e and t h e s e ova were thought t o be r e t a r d e d i n development. s i z e d , t h a t the r a t e o f f o l l i c u l a r growth was  ovulations,  I t i s hypothe-  similar i n d i f f e r e n t estrous  c y c l e s , and t h e r e f o r e the r a t e o b t a i n e d f o r t h i r d - c y c l e f o l l i c l e s a p p l y t o the f i r s t and second Follicular  would  cycles.  growth d u r i n g t h e b r e e d i n g season seemed t o be  i n d e p e n d e n t o f c o r p o r a l u t e a development.  T h i r d - c y c l e f o l l i c l e s developed  c o i n c i d e n t a l l y w i t h s e c o n d - g e n e r a t i o n c o r p o r a l u t e a d e r i v e d f r o m secondcycle f o l l i c l e s .  A l t h o u g h most o f the f e m a l e s c o n c e i v e d a t second  f o l l i c u l a r growth and development became a t r e t i c , a new  c o n t i n u e d . . As the t h i r d - c y c l e  ( f o u r t h ) c y c l e of f o l l i c l e s grew d e s p i t e  and l a r g e a c t i v e c o r p o r a l u t e a .  estrus,  follicles  pregnancy  71  F i g . 10. The growth r a t e o f the l a r g e s t t h i r d - c y c l e f o l l i c l e ( i n each doe) i n w h i c h the i n t e r v a l s i n c e second o v u l a t i o n was e s t i m a t e d f r o m the c l e a v a g e s t a g e o f ova ( r e g r e s s i o n f i t t e d by l e a s t s q u a r e s ) .  O Yearling  l  1 .  1  2  1  3  1  A  DAYS SINCE SECOND OVULATION  r  5  72  3. 8. 9 the  S e a s o n a l Changes i n F o l l i c l e s •  volume of the l a r g e s t f o l l i c l e i n the o v a r i e s o f fawns.  sample  occurred i n  In a large  (N = 4-6) of o v a r i e s c o l l e c t e d i n November, t h e mean volume of the  l a r g e s t f o l l i c l e was 18 mm^.  By December, the mean volume was  and by J a n u a r y t o March, i t was 1 mm  3  old,  S e a s o n a l changes  (N - A) •  c o l l e c t e d i n June, c o n t a i n e d a 30 mm  3  (N =  and a t h i c k - w a l l e d  a t r e t i c f o l l i c l e , i n d i c a t i v e o f renewed p r o d u c t i o n of l a r g e f o l l i c l e s e a r l y as May.  C y c l i c development  of one l a r g e f o l l i c l e  o c c u r r e d i n y e a r l i n g and a d u l t s i n October.  3),  However, a f e m a l e 1 y e a r  active f o l l i c l e  3  5 mm  as  ( C l a s s I I I or IV)  Thick-walled a t r e t i c  follicles  i n d i c a t i v e of f o l l i c u l a r development i n September and even August, were a l s o present.  A C l a s s I I I or I V r u p t u r e d f o l l i c l e , w h i c h s t i l l  c o n t a i n e d the  a t t a c h e d o o c y t e , was p r e s e n t i n a doe c o l l e c t e d October Aearly-atretic f o l l i c l e  A Class IV,  was near r u p t u r e i n a n o t h e r doe c o l l e c t e d i n October.  F o l l i c u l a r development d u r i n g t h e f i r s t t h r e e c y c l e s of t h e b r e e d i n g season was d e s c r i b e d p r e v i o u s l y . at  Because most f e m a l e s c o n c e i v e d  second e s t r u s and few deer were o b t a i n e d a f t e r December 6, l i t t l e  m a t i o n was p r o c u r e d on the n a t u r e o f c y c l e s a f t e r the t h i r d .  infor-  Only one  doe  ( c o l l e c t e d November 28, 1965) c o n t a i n e d two g e n e r a t i o n s o f c o r p o r a l u t e a and a n e w l y - r u p t u r e d f o l l i c l e .  I n t h i s doe, the second g e n e r a t i o n corpus  l u t e u m was more d e g e n e r a t e (by 1 t o 2 days) t h a n were f i r s t - c y c l e c o r p o r a l u t e a a t second o v u l a t i o n .  Another l a r g e , t h i r d - c y c l e f o l l i c l e had r u p t u r e d  i n a doe c o l l e c t e d on November 27, 1965.  Perhaps t h i s was a m e c h a n i c a l •  r u p t u r e because the s e c o n d - g e n e r a t i o n c o r p o r a l u t e a were s i m i l a r i n appearance t o normal c o r p o r a l u t e a of the same age i n p r e g n a n t f e m a l e s . The u t e r i n e h i s t o l o g y of t h e doe was c h a r a c t e r i s t i c o f a p r e g n a n t doe , r a t h e r t h a n one which had j u s t o v u l a t e d . Reduced f o l l i c u l a r development d u r i n g mid-pregnancy  i s also  e v i d e n t i n the graph d e p i c t i n g the volume o f the l a r g e s t f o l l i c l e i n a p a i r  73  of ovaries a t f i v e periods d u r i n g g e s t a t i o n ( F i g . 1 1 ) .  There were s i g n i f i -  cant d i f f e r e n c e s i n the s i z e s of the l a r g e s t f o l l i c l e s between December and February, and between March and May (P<0.05).  The volume of the.  3 l a r g e s t f o l l i c l e i n females i n March was 37 mm , whereas many f o l l i c l e s  3  exceeded 50 mm  i n May and June.  In pregnant females c o l l e c t e d between mid-December and mid-June, there was u s u a l l y a r e c i p r o c a l r e l a t i o n s h i p between the volumes of the l a r g e s t a c t i v e and the l a r g e s t a t r e t i c f o l l i c l e i n a p a i r of ovaries.  This  form of c y c l i c a l development was l e s s evident from January to March than i n December, May, and June.  Many of the l a r g e f o l l i c l e s i n females c a r r y -  i n g fetuses were i n the t r a n s i t i o n a l or e a r l y - a t r e t i c stage of development. Apparently, mature f o l l i c l e s p e r s i s t f o r longer periods i n pregnant females during the winter months than during the breeding  season.  Four females c o l l e c t e d during the winter and s p r i n g of 1963-64 were not v i s i b l y pregnant.  The ovaries of only one of these, a doe aged  7 y e a r s , c o l l e c t e d on May 14, contained no corpora l u t e a and e x i b i t e d negligible follicular activity.  The other three females,collected i n  February and March,were not v i s i b l y pregnant but they were e i t h e r i n e a r l y stages of pregnancy or were s t i l l c y c l i n g .  A l l three contained l a r g e  f o l l i c l e s a t various stages of development and degeneration. Chapter 44..1  The Formation And Degeneration Of Corpora Lutea.  The Problem Of Mechanically-Ruptured  Follicles.  In deer ovaries i t was often d i f f i c u l t to d i s t i n g u i s h f o l l i c l e s ovulated at estrus from f o l l i c l e s ruptured a t other times.  F o l l i c l e s may  rupture a t any time of the year and a t any stage of the f o l l i c u l a r  cycle.  These "mechanical" ruptures were e s p e c i a l l y common between ovulations and were a p o t e n t i a l source of e r r o r i n the i n t e r p r e t a t i o n of ovarian f u n c t i o n s .  F i g . 11. Seasonal changes i n the volume of the l a r g e s t f o l l i c l e i n pregnant females.  8  80  14  70' A  S 6o1  Sample s i z e Range • Standard d e v i a t i o n 95% confidence l i m i t  E-i  I  50'  Mean  40 o  30  20J  T  1-3  V  g 10' Dec.  20  13  — I  Fe D. 2 70  1  1  119  May 10 168  Mar. 22  June 12 200  DATE OF COLLECTION A V G . GESTATION AGE  75  Mechanically-ruptured f o l l i c l e s were examined i n an attempt to i d e n t i f y d i s t i n c t i v e features. 1.  P e r t i n e n t observations were as f o l l o w s :  Commonly, the granulosa was a t r e t i c i n mechanicallyruptured f o l l i c l e s but i t was a l s o a t r e t i c i n some f o l l i c l e s ruptured during p h y s i o l o g i c a l o v u l a t i o n , e s p e c i a l l y those of the f i r s t c y c l e .  2.  C e l l d i v i s i o n i n the theca, and to a l e s s e r extent i n the granulosa ( i f a c t i v e ) , was c h a r a c t e r i s t i c of a l l ruptured f o l l i c l e s , even those o c c u r r i n g i n pregnant does.  3.  During the breeding season the s i z e of ruptured f o l l i c l e s was one of the best c r i t e r i a f o r separating ruptures a t estrus from ruptures at other times.  From the s i z e of the  collapsed s t r u c t u r e , and the degree of f o l d i n g , i t was p o s s i b l e to estimate whether the pre-ruptured f o l l i c l e was r e l a t i v e l y small or l a r g e .  During the breeding  season,  most f o l l i c l e s ruptured at estrus were l a r g e . 4.  Non-extrusion of the oocyte was c h a r a c t e r i s t i c of many .mechanically-ruptured f o l l i c l e s but i t a l s o occurred I n f o l l i c l e s ruptured a t o v u l a t i o n .  In summary, f o l l i c l e s ruptured outside of estrus could o f t e n , but not always, be d i s t i n g u i s h e d from those ovulated a t e s t r u s .  4. 2  The Growth Rate Of Corpora Lutea. The growth r a t e of second-generation corpora l u t e a was determined  by the cleavage stage of the ova i n 12 females (data i n Appendix 2 ) . E a r l y growth of corpora l u t e a was r a p i d and the maximum growth r a t e was attained i n about 2 . 5 to 3 days ( F i g . 1 2 ) .  The volumes of corpora l u t e a  estimated to be 3 days old were omitted from the curve f i t t i n g because  12.  The growth r a t e o f s e c o n d - g e n e r a t i o n c o r p o r a lutea "" (GL 2),whose ages were e s t i m a t e d f r o m the c l e a v a g e s t a g e o f ova. -  The l a r g e s t CL 2 i n a p a i r of ovaries. Considered a t y p i c a l and omitted from the curve f i t t i n g .  77  t h e y were a t y p i c a l l y l a r g e c o r p o r a l u t e a . A growth c u r v e f o r f i r s t - g e n e r a t i o n c o r p o r a l u t e a was n o t o b t a i n e d but t h e i r g r o w t h , d u r i n g the f i r s t 2 t o 3 days, was of the n e x t g e n e r a t i o n .  expected  A f t e r 2 t o 3 d a y s , the growth of  to f o l l o w  first-generation  c o r p o r a l u t e a ceased and a f t e r a n o t h e r 2 t o 3 days t h e y s t a r t e d  4. 3  that  to  degenerate.  Temporal Changes I n The R a t i o Between The Volumes Of S u c c e s s i v e Corpora  Lutea.  Because s e c o n d - g e n e r a t i o n  c o r p o r a l u t e a a r e growing r a p i d l y w h i l e  f i r s t - g e n e r a t i o n c o r p o r a l u t e a a r e s h r i n k i n g r a p i d l y i n s i z e , the r a t i o between the two i s a s e n s i t i v e i n d i c a t o r o f the e a r l y age of corpora l u t e a .  The r a t i o between the volumes o f t h e l a r g e s t corpus l u t e u m  each g e n e r a t i o n was  established  ova were r e c o v e r e d ovulation  was  second-generation  f o r each o f 12 f e m a l e s f r o m w h i c h  ( F i g . 13 and Appendix 2 f o r d a t a ) .  e s t i m a t e d f r o m the d e v e l o p m e n t a l  segmenting  The time e l a p s e d  changes r a p i d l y i n t h e f i r s t 3 days a f t e r o v u l a t i o n . a f t e r 3 days,but  since  stage o f the ova.  The r a t i o i n the volumes o f the two g e n e r a t i o n s of c o r p o r a  begins to d e c e l l e r a t e  of  lutea  The r a t e of change  o b v i o u s l y the r a t i o c o n t i n u e s t o  i n c r e a s e p a s t 5 days (the r a t i o o f t e n exceeded 50 a t an e s t i m a t e d 10 days after ovulation).  The r a t i o method i s u n d o u b t e d l y  o f e l a p s e d time s i n c e l u t e a and f o l l i c l e s ,  second  ovulation  because t h e r e i s  w i t h i n f e m a l e s i n the s i z e of f o l l i c l e s  4. U  E s t i m a t i n g The Age Of Corpora Five  criteria  a more a c c u r a t e measure  t h a n a r e the growth c u r v e s f o r c o r p o r a l e s s v a r i a t i o n between f e m a l e s and c o r p o r a l u t e a .  Lutea.  were used t o e s t i m a t e the age o f c o r p o r a l u t e a  than  78  F i g . 13.  Temporal changes i n t h e ' r a t i o between the volume of the l a r g e s t s e c o n d - g e n e r a t i o n corpus l u t e u m (GL 2) and the volume o f the l a r g e s t s h r i n k i n g , f i r s t - g e n e r a t i o n corpus l u t e u m (CL 1) (time estimated from ova-segmentation s t a g e s ) .  T I M E  SINCE SECOND OVULATION (DAYS)  79  derived from the second ovulatory c y c l e . 1.  The cleavage r a t e of ova.  2.  The volume of the corpus  3.  The volume and degree of maturation of f o l l i c l e s of succeeding  luteum.  cycles. 4.  The volume and h i s t o l o g i c a l appearance of the degenerating corpus luteum of the preceeding c y c l e .  5.  The r a t i o betv/een the volumes of successive corpora l u t e a .  Of the f i v e c r i t e r i a , the cleavage r a t e of ova vas the standard and provided a base f o r the q u a n t i t a t i v e components of the other methods.  Corpora l u t e a ,  whose ages were estimated by the cleavage r a t e of o v a , w i l l henceforth be termed "known-age" corpora l u t e a . The ages of second-cycle corpora l u t e a , t o 5 days of age,were e s t i mated p a r t l y by o b j e c t i v e methods (the four curves f o r growth and degeneration of corpora l u t e a and f o l l i c l e s ) and p a r t l y by s u b j e c t i v e methods (the h i s t o l o g i c a l comparison  of corpora of unknown age to corpora l u t e a of known age).  The ages of corpora l u t e a older than 5 days were estimated s u b j e c t i v e l y by the h i s t o l o g i c a l appearance of f o l l i c l e s and corpora l u t e a .  The usefulness of f o l -  l i c l e s i n determining the stage of the estrous c y c l e a f t e r 4 to 5 days was pendent on f i n d i n g the l e n g t h of the estrous c y c l e .  de-  Data presented i n the next  s e c t i o n i n d i c a t e that the average i n t e r v a l between f i r s t and second o v u l a t i o n was 8 days. I assumed that.successive f o l l i c u l a r c y c l e s were the same l e n g t h and t h a t f o l l i c u l a r growth and maturation r a t e s were equivalent i n the consecutive cycles.  I f t h i s assumption i s v a l i d , i t f o l l o w s that the average i n t e r v a l be-  tween f i r s t and second o v u l a t i o n , 8 days, was a l s o the l e n g t h of the f o l l i c u l a r c y c l e s preceeding f i r s t o v u l a t i o n and succeeding second o v u l a t i o n . Thus, the stage of a f o l l i c u l a r c y c l e , and thereby the age of corpora l u t e a , was estimated from the s i z e of f o l l i c l e s and t h e i r degree of maturation i n r e l a t i o n to an  80  8-day c y c l e . During the f i r s t 3 days,the age of corpora l u t e a derived from the f i r s t o v u l a t i o n was  estimated by: l )  comparing the volumes of the  corpora l u t e a to the growth curve f o r second-cycle corpora l u t e a , 2)  com-  paring the volumes of second-cycle f o l l i c l e s to the growth curve f o r t h i r d c y c l e f o l l i c l e s , and 3)  h i s t o l o g i c a l comparison of the corpora l u t e a to  known-age corpora l u t e a of the second c y c l e .  The ages of older corpora  l u t e a from the f i r s t o v u l a t i o n were estimated by comparing the volumes of second-cycle f o l l i c l e s to the growth curve of t h i r d - c y c l e f o l l i c l e s , the degree of second-cycle f o l l i c u l a r maturation,and  the h i s t o l o g i c a l  appearance of the corpora l u t e a . B l a s t o c y s t s recovered from f i r s t - c y c l e ovulations were not v a l i d i n d i c a t o r s of the age of corpora l u t e a . i n d i c a t e d by the ova.  Corpora l u t e a were often older .than  Because the three one-celled ova apparently were  not f e r t i l i z e d , development may have ceased.  A comparison of these three  corpora l u t e a with those derived from the second c y c l e , whose ages were estimated by b l a s t o c y s t development, revealed that one was about one day old and the other two were about 1.5 days o l d .  S i m i l a r l y , the two-celled  ovum (apparently not f e r t i l i z e d ) was retarded i n development.  Although  two-celled ova occur i n most mammals about 1.5 days a f t e r o v u l a t i o n , the corpus luteum associated with t h i s two-celled ovum was comparable i n development to an older corpus luteum. the f i r s t c y c l e was a l s o retarded.  Development of f e r t i l i z e d ova from  On the b a s i s of h i s t o l o g i c a l character-  i s t i c s ,the corpora l u t e a corresponding to the ova c o n t a i n i n g 5 and 6 c e l l s were estimated to be 6 and 3 to A days old, r e s p e c t i v e l y , r a t h e r than 2 days old as i n d i c a t e d by the ova.  81  4- 5 H i s t o l o g y Of Young C o r p o r a 4. 5. 1  Lutea.  The R e c e n t l y - R u p t u r e d  Follicle.  Three m o r p h o l o g i c a l  c h a r a c t e r i s t i c s were e v i d e n t i n s e c t i o n s o f r e c e n t l y - r u p t u r e d f o l l i c l e s viewed a t l o w m a g n i f i c a t i o n ; a r u p t u r e d o v a r i a n s u r f a c e n o t y e t b r i d g e d by new c o n n e c t i v e t i s s u e , a f o l d e d f o l l i c u l a r w a l l , and an o b l i t e r a t e d or much-reduced antrum. I n f o l l i c l e s w h i c h had j u s t r u p t u r e d , the w a l l was  moderately  f o l d e d around a s m a l l c e n t r a l lumen c o n t a i n i n g a few blood c e l l s .  The  membrana g r a n u l o s a , f i r m l y a t t a c h e d t o t h e membrana p r o p r i a , resembled preovulatory granulosa.  B u t i n most r u p t u r e d f o l l i c l e s , t h e a t t a c h e d  sa c e l l s c o n t a i n e d e l o n g a t e condensed n u c l e i and h y p e r c h r o m a t i c ( P l a t e 13. 2 ) . O f t e n , t h e  g r a n u l o s a c e l l s around f r e e o o c y t e s  granulo-  cytoplasm trapped  i n r u p t u r e d f o l l i c l e s were l e s s c h r o m a t i c , l e s s g r a n u l a r , and t h e n u c l e i l e s s condensed, than t h e c e l l s i n t h e m u r a l g r a n u l o s a ( P l a t e 4. 9 ) .  Cell  d i v i s i o n s o c c u r r e d i n f r e q u e n t l y i n t h e membrana g r a n u l o s a and t h e t h e c a i n t e r n a u n t i l s e v e r a l hours a f t e r o v u l a t i o n .  About t h i s t i m e , t h e membrana  g r a n u l o s a , c o n t a i n i n g b o t h v e s i c u l a t e and condensed n u c l e i , u s u a l l y became d i s s o c i a t e d f r o m t h e membrana p r o p r i a .  At t h i s stage, c e l l  division  o c c u r r e d more f r e q u e n t l y i n t h e c e l l s , w h i c h ranged between 7 and 10 u i n diameter  ( o n l y s l i g h t l y l a r g e r than p r e o v u l a t o r y g r a n u l o s a  cells).  I n most r u p t u r e d f o l l i c l e s , t h e appearance o f t h e t h e c a l c e l l s was unchanged,but i n a few  t h e c y t o p l a s m became g r a n u l a r and  hyperchromatic,  some n u c l e i changed f r o m o b l a t e v e s i c u l a t e t o t h e s p h e r i c a l condensed  type,  and m i t o s i s o c c u r r e d .  ,  I n most r u p t u r e d f o l l i c l e s , engorged blood v e s s e l s o c c u r r e d a l l a l o n g t h e i n n e r border o f t h e t h e c a i n t e r n a d a r y between i t and t h e membrana g r a n u l o s a  t o f o r m a d i s t i n c t i v e boun( P l a t e 13. 2 ) . The l a r g e s t  v e s s e l s , l o c a t e d i n t h e f o l d s o f t h e f o l l i c u l a r w a l l , o c c a s i o n a l l y bulged  82  i n t o the granulosa or i n t o the c e n t r a l c a v i t y i f the granulosa l a y e r was detached from the membrana p r o p r i a .  Apparently, many hours elapsed before some  of the turgid blood vessels ruptured and the collapsed f o l l i c l e developed i n t o a corpus hemorrhagicum.  Free blood c e l l s and vascular f l u i d formed  a c e n t r a l c l o t or coagulum i n these f o l l i c l e s . The above d e s c r i p t i o n of newly-ruptured  f o l l i c l e s applied to  second-cycle ruptures and some f i r s t - c y c l e ruptures.  But other f o l l i c l e s  that ruptured a t f i r s t estrus were a t r e t i c and therefore e x i b i t e d d i f f e r ent c h a r a c t e r i s t i c s .  I n females o v u l a t i n g two f o l l i c l e s a t f i r s t e s t r u s ,  often one was a c t i v e and the other was i n e a r l y stages of a t r e s i a . major d i f f e r e n c e s encountered  The  i n ruptured a t r e t i c f o l l i c l e s of the  f i r s t estrous are l i s t e d below:  1.  The w a l l of a t r e t i c f o l l i c l e s d i d not always f o l d but remained q u i t e r i g i d , a l t h o u g h the volume of the f o l l i c l e decreased considerably.  2.  The amount of membrana granulosa remaining i n the f o l l i c l e a f t e r rupture was h i g h l y v a r i a b l e .  I n some f o l l i c l e s  almost a l l of i t was extruded from the f o l l i c l e a t o v u l a t i o n . Presumably,the granulosa l a y e r was a t r e t i c and f r e e from the membrana p r o p r i a a t the time of rupture.' 3.  Many of the granulosa and t h e c a l c e l l s were dying.  4-.  Some f o l l i c l e s were r e l a t i v e l y avascular and i n many, a corpus hemorrhagicum apparently d i d not form f o l l o w i n g rupture.  4-. 5. 2  One Day After Ovulation.  day old s t i l l had l ) an i r r e g u l a r shape, blood and f l u i d coagulum,  Corpora l u t e a estimated to be one 2) a small lumen c o n t a i n i n g a  3) prominent f o l d i n g of the former membrana  83  g r a n u l o s a , and 4) a l o o s e l y - k n i t appearance.-  F i v e known-age c o r p o r a  f r o m t h e second o v u l a t o r y c y c l e averaged 6 mm  3  s t r u c t u r e was  pear or f l a s k - s h a p e d .  f r o m an i n c r e a s e i n the number and accumulation  Commonly, the  The i n c r e a s e i n s i z e r e s u l t e d  partly  s i z e of c e l l s , and p a r t l y , f r o m an  of f l u i d , i n c l u d i n g b l o o d .  s u r f a c e r u p t u r e was  i n volume.  lutea  The l a t t e r o c c u r r e d because the  b r i d g e d by a t h i n l a y e r of c o n n e c t i v e  tissue.  E x t e n s i v e p r o l i f e r a t i o n of t h i n - w a l l e d blood v e s s e l s was t i v e c h a r a c t e r i s t i c s o f these c o r p o r a l u t e a .  Large  a distinc-  s i n u s o i d s , l o c a t e d i n the  a p i c e s o f the t h e c a l i n v a g i n a t i o n s , extended n e a r l y t o t h e c e n t e r of the collapsed structure.  R u p t u r e of one or more of these v e s s e l s r e s u l t e d  i n m a s s i v e blood p e r f u s i o n throughout corpus luteum.  t h e c e n t r a l r e g i o n of the young  The r e s u l t a n t c l o t c o n t a i n e d l a r g e numbers o f l e u c o c y t e s  ( P l a t e 13. 3 ) . Pronounced f o l d i n g o f the membrana g r a n u l o s a o c c u r r e d 1 day ovulation.  The  granulosa c e l l s a t t a i n e d diameters  of 13-14  s t i l l d i s t i n g u i s h a b l e from t h e c a l l u t e i n c e l l s ( P l a t e The number of l u t e a l c e l l s per f i e l d 50, and  t h e r e was  about  one  dividing  u and  were  11.2).  o f v i e w a t 1250X was  c e l l i n each f i e l d  about  o f v i e w a t 500X.  N u c l e i o f t h e g r a n u l o s a c e l l s were s m a l l and most were v e s i c u l a t e . n u c l e i were u s u a l l y c o n f i n e d t o the more c e n t r a l r e g i o n s o f - t h e l u t e u m , where e l o n g a t e and h y p e r c h r o m a t i c  after  Condensed  corpus  cells-occurred.  The g e n e r a l boundary between the g r a n u l o s a and  the t h e c a was  still  e v i d e n t ( P l a t e 11. 2) but, e s p e c i a l l y a t i n v a g i n a t i o n s , t h e c a l c e l l s were mingled  among g r a n u l o s a c e l l s t o c r e a t e a d i f f u s e boundary.  c e l l s c o n t a i n e d l a r g e , i r r e g u l a r - s h a p e d n u c l e i and  Many t h e c a l  sparse cytoplasm.  Cell-  d i v i s i o n was more f r e q u e n t i n the t h e c a than i n the membrana g r a n u l o s a . Many o f - t h e d i v i d i n g c e l l s , e i t h e r young l u t e a l c e l l s or f i b r o b l a s t s , were e l o n g a t e and h y p e r c h r o m a t i c  (Plate l l . - ' l ) .  I n two c o r p o r a l u t e a , abundant  F a c i n g page 84  P l a t e 11  Development o f Corpora L u t e a From t h e F i r s t O v u l a t o r y C y c l e . ( A l l photomicrographs  X800).  1. A c o r p u s l u t e u m one d a y a f t e r o v u l a t i o n . A p o r t i o n of a t h e c a l i n v a g i n a t i o n (running d i a g o n a l l y through the photograph), c o n t a i n s e l o n g a t e , d i v i d i n g c e l l s , and c e l l s w i t h i r r e g u l a r l y - s h a p e d n u c l e i . Other c e l l s a r e d e r i v e d from t h e g r a n u l o s a . 2. The edge o f a corpus l u t e u m about one d a y o l d . The c e l l s of b o t h the g r a n u l o s a ( t o r i g h t o f dashes) and t h e t h e c a a r e u n d e r g o i n g lutein!zation. C e l l d i v i s i o n s a r e b e g i n n i n g i n b o t h zones (note anaphase i n t h e c a ) . 3. C e n t r a l r e g i o n of a corpus l u t e u m about one d a y o l d . F o l d s o f g r a n u l o s a c e l l s a r e s e p a r a t e d by c o n n e c t i v e t i s s u e membranes ( v i s i b l e i n c e n t e r of p h o t o g r a p h ) . The c y t o p l a s m o f t h e c e l l s i s sparse. 4. A corpus l u t e u m ( f i r s t c y c l e ) e s t i m a t e d t o be 1.5 days o l d . There i s e x t e n s i v e p r o l i f e r a t i o n o f blood v e s s e l s ( l a r g e one a l o n g l e f t b o r d e r o f p h o t o g r a p h ) . The c e l l i n prophase i s p r o b a b l y an endothelial c e l l . Note t h e l a r g e r and more g r a n u l a r l u t e a l c e l l s . 5. A corpus l u t e u m about 2 days a f t e r o v u l a t i o n . The l u t e a l c e l l s a r e l a r g e r and t h e i r b o r d e r s a r e more d i s t i n c t . Connective t i s s u e c e l l s are f o r m i n g between t h e l u t e a l c e l l s . 6. A c o r p u s l u t e u m 2.5 t o 3 days a f t e r o v u l a t i o n . Connective t i s s u e , i n c l u d i n g blood v e s s e l s ( l o w e r l e f t ) and c a p i l l a r i e s , has i n s i n u a t e d between t h e l u t e a l c e l l s . 7. A c o r p o r a l u t e u m 2.5 t o 3 days a f t e r o v u l a t i o n . u p p e r ' c e n t e r ) has v i r t u a l l y ceased. 8.  Mitosis (telophase  A c o r p u s l u t e u m e s t i m a t e d t o be 3 days o l d . Many l u t e a l c e l l s have v a c u o l a t e d c y t o p l a s m , and a few a r e d e g e n e r a t e . H i g h l y c h r o m o p h i l i c c e l l s w i t h condensed n u c l e i o c c u r ( l o w e r l e f t . ) .  9. A corpus l u t e u m 4 t o 5 days o l d . and s h r i n k a g e o f c e l l s .  There i s e x t e n s i v e v a c u o l a t i o n  10. A corpus l u t e u m about 6 t o 7 days o l d . Note t h e e x t e n s i v e v a c u o l a t i o n and s h r i n k a g e o f c e l l s . C e l l s and n u c l e i a r e p o o r l y d e f i n e d . 11. A corpus l u t e u m i n an i n s e m i n a t e d y e a r l i n g doe on t h e verge o f second o v u l a t i o n o f t h e season. A l t h o u g h some c e l l s a r e v a c u o l a t e d and shrunken and l e u c o c y t e s occur ( l o w e r l e f t ) , many c e l l s appear a c t i v e and c e l l d i v i s i o n s t i l l o c c u r s ( t e l o p h a s e upper c e n t e r ) . 12. A f i r s t - c y c l e corpus l u t e u m i n a y e a r l i n g f e m a l e h a v i n g j u s t o v u l a t e d a t second e s t r u s . Many c e l l s and n u c l e i a r e p o o r l y d e f i n e d because they are i n e a r l y stages of a u t o l y s i s .  Plate  12  F a c i n g page 85  D e g e n e r a t i o n of the Corpus Luteum From the F i r s t O v u l a t o r y C y c l e .  • 3 1. J u s t a f t e r the second o v u l a t i o n , a 58 mm degenerate corpus luteum from the f i r s t c y c l e s t i l l c o n t a i n s most of i t s l u t e a l c e l l s (X800).  3  2. A p o r t i o n of a 7.2 mm d e g e n e r a t e c o r p u s l u t e u m one day second o v u l a t i o n . Few c e l l s r e m a i n (X800).  after  3  3. A p o r t i o n of a 5-5 mm d e g e n e r a t e c o r p u s l u t e u m two days a f t e r second o v u l a t i o n . H y a l i n c o n n e c t i v e t i s s u e i s f o r m i n g between c e l l s u n d e r g o i n g a u t o l y s i s (X800). 4.  A p o r t i o n o f a 4 . 7 mm d e g e n e r a t e c o r p u s l u t e u m 3 days a f t e r s e c o n t o v u l a t i o n . A c t i v e l u t e a l c e l l p e r s i s t s i n the c e n t e r of the s t r u c t u r e ( r i g h t ) . The normal s t a t e of d e g e n e r a t i o n i s p i c t u r e d t o the l e f t (X80). 3  3  5. A p o r t i o n of a 2.8 mm d e g e n e r a t e c o r p u s l u t e u m 4 days a f t e r second ovulation. H y a l i n c o n n e c t i v e t i s s u e forms wide bands between shrunken c e l l spaces c o n t a i n i n g n u c l e i (X800). -  3  6.  A p o r t i o n of a 0.8 mm d e g e n e r a t e c o r p u s l u t e u m about 10 days a f t e r second o v u l a t i o n . L a r g e , s p h e r i c a l v a c u o l e s have formed i n the p e r s i s t i n g c e l l s (X800).  3  7. A p o r t i o n of a 2.0 mm d e g e n e r a t e c o r p u s l u t e u m about 10 days second o v u l a t i o n . S m a l l , a n g u l a r c e l l s and l e u c o c y t e s occur i n the v a c u o l a t e d , h y a l i n i z e d s t r u c t u r e (X800).  after  3 8. A s e c t i o n o f a 0.3 mm s c a r of a c o r p u s l u t e u m o f non-pregnancy i n a non-pregnant doe c o l l e c t e d May I 4 . T h i s female c o n t a i n e d n i n e similar structures i n i t s ovaries. The p i c t u r e d s c a r was e s t i m a t e d t o be from the s e c o n d - l a s t o v u l a t o r y c y c l e of the p a s t b r e e d i n g season (X800).  3  .9-  A p o r t i o n of the 0.5 ronr s c a r o r i g i n a t i n g from the youngest c o r p u s l u t e u m o f non-pregnancy i n the doe d e s c r i b e d i n 12.8. Y e l l o w pigment o f t e n forms around t h e s p e r i c a l v a c u o l e s e v i d e n t i n the s c a r (X800).  3  10. A 0.07 mm s c a r i n a doe k i l l e d May 13The s c a r was d e r i v e d from a c o r p u s l u t e u m of non-pregnancy of the p r e v i o u s November and was t h e r e f o r e about 5-5 months o l d . Note the l a r g e v a c u o l e s (X800).  3  11. A 0.05 mm corpus l u t e u m o f non-pregnancy s c a r about 11 months o l d . A few s m a l l , t h i c k - w a l l e d b l o o d v e s s e l s o f t e n occur around the p e r i p h e r y of t h e s e s c a r s . The c e l l spaces a r e bordered by b r i l l i a n t y e l l o w pigment (X200).  3  12. A O . O 4 mm corpus l u t e u m o f non-pregnancy s c a r about 12 months o l d . T h i s one has a h y a l i n c a p s u l e ( r i g h t and l o w e r r i g h t ) around a p o r t i o n o f the c e n t r a l r e g i o n (X200).  Facing page 86 P l a t e 13 ' E a r l y Development of Corpora Lutea From the Second Ovulatory Cycle. ( A l l photographs X800). 1. The c e n t r a l r e g i o n of a corpus luteum about one day o l d . Nuclei of the granulosa c e l l s are becoming v e s i c u l a t e . Many leucocytes are scattered amongst the l u t e a l c e l l s . 2. The w a l l of a newly-ruptured f o l l i c l e . The granulosa c e l l s (to l e f t of dashes) contain mostly condensed n u c l e i . A large blood v e s s e l and a m i t o t i c f i g u r e occur i n the theca i n t e r n a (to r i g h t of dashes). 3. The c e n t r a l region of a corpus luteum one day a f t e r ovulation. n u c l e i of l u t e a l c e l l s are now v e s i c u l a t e . Large numbers of leucocytes ( r i g h t side of photograph) are present.  Most  -4. The p e r i p h e r a l r e g i o n of a corpus luteum about 2 days old to show the p r o l i f e r a t i o n of connective t i s s u e c e l l s (elongate and angular c e l l s between enlarged l u t e a l c e l l s ) . C e l l s are d i v i d i n g r a p i d l y at t h i s stage (prophase i n upper l e f t corner). 5. The c e n t r a l region of a corpus luteum about 2 days o l d . Connective t i s s u e , associated with the e n d o t h e l i a l system, i s p r o l i f e r a t i n g between groups of l u t e a l c e l l s (Prophase at top of photograph). 6. A corpus luteum about 4- days a f t e r ovulation. c e l l s have peripherally-vacuolated cytoplasm.  Many of the l u t e a l  7. A corpus luteum 5 to 6 days a f t e r ovulation. The cytoplasm of the l u t e a l c e l l s i s e x t e n s i v e l y vacuolated. A c e n t r a l zone of f i n e granular cytoplasm contains a g o l g i zone and an e c c e n t r i c a l l y positioned nucleus. 8. A corpus luteum estimated to be 7 days o l d . Large vacuolated c e l l s occur and smaller c e l l s described i n 13.7 are present. 9. A corpus luteum estimated to be 8 to 10 days o l d . Angular hyperchromatic c e l l s with condensed n u c l e i occur at t h i s stage. 10. A corpus luteum estimated to be 11 to 12 days o l d . Portions of two hyperchromatic c e l l s containing condensed n u c l e i are present along the r i g h t edge of the photograph.  87  l e u c o c y t e s o c c u r r e d i n the t h e c a l l a y e r s . C o l l a g e n i c f i b e r s f r o m the t h e c a e x t e r n a formed a t r i a n g u l a r wedge of t i s s u e a t the base of each t h e c a l f o l d . each f o l d developed  The c e n t r a l p o r t i o n of  i n t o f i b r o u s tongues of t i s s u e termed t r a b e c u l a e .  These t r a b e c u l a , b e a r i n g l a r g e blood v e s s e l s , extended t o the c e n t e r of the corpus l u t e u m and  became.the source of the c o n n e c t i v e t i s s u e framework of  the g l a n d .  4.  5. 3  One  And  One-Half Days A f t e r O v u l a t i o n .  days o l d a t t a i n e d volumes of 12 mm c e n t r a l amorphous coagulum. 4-0 per f i e l d field  t h e widened t r a b e c u l a e .  F i b r o b l a s t - l i k e c e l l s extended i n t o t h e The rounder  about two  Days A f t e r O v u l a t i o n .  granulosa  There was  extensive  4).  Corpora l u t e a e s t i m a t e d t o be  days o l d were compact s t r u c t u r e s w i t h u n i f o r m round or o b l a t e shapes. c o n n e c t i v e t i s s u e b r i d g e over the s u r f a c e r u p t u r e a r e a was  thicker.  3  known-age c o r p o r a l u t e a averaged 23 mm , and ranged f r o m 8 t o 38 mm t i s s u e p r o t r u d e d from t h e o v a r i a n s u r f a c e of some o v a r i e s . t i s s u e s was  per  granulosa c e l l s , c o n t a i n i n g  p r o l i f e r a t i o n o f r e l a t i v e l y l a r g e blood v e s s e l s ( P l a t e 11.  Two  approximately  the f r e q u e n c y o f - ' d i v i s i o n s was  v e s i c u l a t e n u c l e i , a t t a i n e d d i a m e t e r s up t o 15 p..  5. 4  a  Many c e l l d i v i s i o n s o c c u r r e d a l o n g t h e edges o f  c e l l zone f r o m the t r a b e c u l a e .  4.  1.5  , were more rounded, and c o n t a i n e d  The d e n s i t y of l u t e a l c e l l s was  o f v i e w a t 1250X, and  o f view a t 500X.  Corpora l u t e a  fluid.  • The l u t e a l c e l l s a t t a i n e d d i a m e t e r s  average number o f c e l l s per f i e l d  The Eight  .  Luteal  The f o l d i n g o f  l e s s o b v i o u s on the second day than on the f i r s t .  c e n t r a l lumena were bounded by young c o n n e c t i v e t i s s u e c e l l s  3  2  The s m a l l , and  contained  ' o f 20 p.,  w h i c h reduced  o f view ( a t 1250X) t o about 20.  the  88  P r o l i f e r a t i o n o f l u t e a l c e l l s reached approaching  a maximum i n c o r p o r a  2 days ( f o u r - c e l l e d ovum).  f i g u r e s per f i e l d  lutea  I n t h e s e , an average o f s i x m i t o t i c  o f v i e w was common,but by about 2.5 days ( 8 - c e l l e d  the number o f c e l l d i v i s i o n s d e c r e a s e d  t o about two.  The l a r g e s t  ovum),  luteal  c e l l s , o c c u r r i n g around the p e r i p h e r y o f the s t r u c t u r e , were v a c u o l a t e d . Small c e l l s w i t h granular cytoplasm occurred i n the c e n t r a l r e g i o n s . Elements o f t h e t h e c a , i n c l u d i n g f i b r o b l a s t s , were found out the corpus luteum. hyper chroma t i c  through-  Many o f t h e t h e c a l c e l l s were s m a l l , e l o n g a t e , and  ( P l a t e 13. 4-) •  Some of them were p r o b a b l y a s s o c i a t e d w i t h  t h e r e t i c u l o e n d o t h e l i a l system. The l a r g e blood s i n u s e s were fewer i n number b u t t h e y were a s s o c i a t e d w i t h blood pigment.  still  Medium-sized v e s s e l s e n t e r e d t h e gland v i a  the t r a b e c u l a e and d i v i d e d i n t o s m a l l e r v e s s e l s t h a t i n s i n u a t e d between groups o f l u t e a l  4-. 5- 5  cells.  Three Days A f t e r O v u l a t i o n .  Three known-age c o r p o r a l u t e a o f  3 the second c y c l e averaged 70 mm , but u n d o u b t e d l y  t h e y were e x c e p t i o n a l l y  3 l a r g e and t h e t r u e mean volume was p r o b a b l y between 4-0 and 50 mm l u t e a l c e l l s o c c u r r e d throughout form  appearance.  in a field field  Enlarged  the oblate s t r u c t u r e , g i v i n g i t a s o l i d , u n i -  The d e n s i t y o f l u t e a l c e l l s was reduced  t o about 14- c e l l s  o f v i e w a t 1250X, and an average o f two d i v i s i o n s occured i n a  o f view a t 500X.  diameters  .  o f 24-  A l a r g e p r o p o r t i o n of the c e l l s , which a t t a i n e d  contained small vacuoles.  The c e n t r a l lumen, w h i c h c o n t a i n e d f i b r o b l a s t s , was s m o o t h l y bounded by young c o n n e c t i v e t i s s u e .  The corpus luteum was u n i f o r m l y smooth  around t h e p e r i p h e r y , t h e t r a b e c u l a e h a v i n g t h i n n e d c o n s i d e r a b l y . T h i n - w a l l e d blood v e s s e l s o f medium s i z e o c c u r r e d around t h e periphery.  F i b r o b l a s t s and amorphous c o n n e c t i v e t i s s u e r e p l a c e d many o f t h e  89  l a r g e s i n u s o i d s found i n younger c o r p o r a l u t e a . c o n n e c t i v e t i s s u e developed  Other zones of l o o s e  a t the expense of l u t e a l c e l l s .  Increased  numbers of c o n n e c t i v e t i s s u e c e l l s c r e a t e d a p r i m i t i v e network amongst the luteal cells  ( P l a t e 11.  6).  The c h a r a c t e r i s t i c s of c o r p o r a l u t e a o f the same age f r o m the f i r s t o v u l a t o r y c y c l e were comparable t o the above, e x c e p t t h a t a l m o s t a l l c e l l d i v i s i o n had  ceased  vacuolated cytoplasm  4.  5. 6  and a n g u l a r or s t e l l a t e c e l l s w i t h c h r o m o p h i l i c ,  o c c u r r e d f o r the f i r s t time  Four Days A f t e r O v u l a t i o n .  ( P l a t e 11. 8 ) .  Corpora l u t e a o f the f i r s t  second c y c l e s d i f f e r e d i n s i z e and h i s t o l o g i c a l appearance f r o m t h i s  and stage  onward. Corpora l u t e a f r o m the second o v u l a t o r y c y c l e averaged between 60 and 70 mm  .  Two  known-age c o r p o r a l u t e a , s u p p o r t i n g a m u l t i - c e l l e d  (16 t o 64 c e l l s ) b l a s t o c y s t l o c a t e d i n the u t e r u s , a t t a i n e d volumes of  74  '3 and 78 mm  .  A small f l u i d - f i l l e d  lumen o c c u r r e d i n one of these.'  The  i n c r e a s e d average c e l l s i z e was r e f l e c t e d by a l u t e a l c e l l d e n s i t y (per field  of v i e w a t 1250X) of 10.  B u t , t h e 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 s were  the v i r t u a l l a c k of c e l l d i v i s i o n s and and  the h i g h degree of c e l l v a c u o l a t i o n  even c e l l d e a t h , r e s u l t i n g I n i n t e r c e l l u l a r spaces ( P l a t e 13. 6).  few of the c e l l s c o n t a i n e d s o l i d , i r r e g u l a r - s h a p e d n u c l e i and c h r o m a t i c but v a c u o l a t e d c y t o p l a s m .  A new  A  deeply  c e l l type w i t h a c e n t r a l , a c i d o -  p h i l i c , g r a n u l a r zone and a v a c u o l a t e d o u t e r zone, made i t s f i r s t appearance.  T h i s c e l l type may  i n d i c a t e t h a t the b l a s t o c y s t has reached  the  u t e r u s and h e r e a f t e r I w i l l term them ' c o n c e p t i o n c e l l s ' . " The.bands o f c o n n e c t i v e t i s s u e were w i d e r , more f i b r o u s and collagenic. diameter.  The major blood v e s s e l s were t h i c k e r - w a l l e d but reduced S m a l l v e s s e l s and c a p i l l a r i e s o c c u r r e d t h r o u g h o u t  the  in  structure.  90  Most c o r p o r a l u t e a d e r i v e d 30 and 50 mm , 3  and c o n t a i n e d wide bands o f c o n n e c t i v e t i s s u e between t h e  vacuolated c e l l s i n the r e g i o n  k. 5- 8 containing  f r o m t h e f i r s t c y c l e ranged between  ( P l a t e 11. 9 ) -  Considerable a u t o l y s i s of l u t e a l  o f t h e c o n n e c t i v e t i s s u e bands, r e s u l t e d  S i x t o 8 Days A f t e r O v u l a t i o n .  follicles  cells  i n cellular  debris.  Corpora l u t e a i n ovaries  o f the s u c c e e d i n g c y c l e i n t h e t r a n s i t i o n a l  of development, were e s t i m a t e d t o be 6 t o 8 days o l d .  stage  A l t h o u g h lumena i n  3 t h e s e were g e n e r a l l y cavity.  s m a l l , one c o n t a i n e d a 39 mm , f l u i d - f i l l e d  central  The c y t o l o g y o f most s e c o n d - g e n e r a t i o n c o r p o r a l u t e a o f t h i s age  was s i m i l a r t o c o r p o r a l u t e a 5 days o l d .  Some l a r g e , v a c u o l a t e d , l u t e a l  cells  were much l a r g e r , a t t a i n i n g d i a m e t e r s o f 30 ; i ( P l a t e 13. 8 ) . About o n e - h a l f of t h e c e l l s were o f t h e c o n c e p t i o n t y p e . From 6 t o 8 days a f t e r f i r s t o v u l a t i o n , progressively  more d e g e n e r a t e .  Sections of corpora l u t e a a t low  appeared h y p o c h r o m a t i c because o f c e l l v a c u o l a t i o n , ( P l a t e 11. 1 0 ) . B a s o p h i l i c ,  t h e c o r p o r a l u t e a became magnification  s h r i n k a g e , and a u t o l y s i s  s t e l l a t e c e l l s w i t h condensed n u c l e i and  leucocytes occurred. N i n e c o r p o r a l u t e a i n d e e r on t h e v e r g e o f second  ovulation  3 averaged 32.6 mm .  O b v i o u s l y some r e d u c t i o n  i n volume o c c u r r e d  maximum development a t about U t o 5 days a f t e r  after  ovulation.  Hemorrhagic c o r p o r a l u t e a o c c u r r e d i n two d o e s , i n c l u d i n g one i n estrus. had  The o v a r i e s  o f t h e l a t t e r were hyperemic and edematous and b l o o d  r e c e n t l y escaped i n t o t h e c e n t r a l lumen o f t h e c o r p u s luteum.  a c t i v e , and d i v i d i n g c e l l s o c c u r r e d t h r o u g h o u t t h e s t r u c t u r e even though o v u l a t i o n was i n c i p i e n t , a s i n d i c a t e d  Young,  ( P l a t e 11. 1 1 ) ,  by the t h i n - w a l l e d  w i t h f r e e o o c y t e s and by t h e p r e s e n c e o f sperm i n t h e o v i d u c t s .  follicles  91  4. 5. 9 second  Nine t o 15 Days A f t e r O v u l a t i o n .  Corpora l u t e a o f t h e  ovulatory c y c l e , c o n t a i n i n g l a r g e e a r l y - a t r e t i c f o l l i c l e s of the  t h i r d c y c l e and young growing f o u r t h - c y c l e f o l l i c l e s , were e s t i m a t e d t o be 9 t o 12 days o l d . 1.  C h a r a c t e r i s t i c s o f t h e s e c o r p o r a l u t e a were as f o l l o w s :  The l u t e a l c e l l s were s l i g h t l y l a r g e r ; many a t t a i n e d d i a m e t e r s o f 30 _u and a few reached  2.  35 P-.  About o n e - h a l f o f t h e l u t e a l c e l l s were o f t h e c o n c e p t i o n type and most o t h e r s were l a r g e and v a c u o l a t e d .  The c h r o m o p h i l i c ,  a n g u l a r c e l l s , c o n t a i n i n g condensed n u c l e i , were more common ( P l a t e 13. 9 ) . Honeycomb-like v a c u o l a t i o n o c c u r r e d i n many of the l u t e a l 3.  cells.  The t h i n n e r and m o r e - f i b r o u s t r a b e c u l a e c o n t a i n e d  elongate  spaces. 4.  A t h i c k l a y e r o f c o l l a g e n i c f i b e r s bordered ( i f present).  F i b r o b l a s t s and amorphous c o n n e c t i v e t i s s u e  partially filled 5.  t h e c e n t r a l lumen  t h e c e n t r a l lumen.  The i n t e r c e l l u l a r network o f c o n n e c t i v e t i s s u e ,  including  c a p i l l a r i e s , more d e f i n i t e l y s e p a r a t e d n e a r l y e v e r y l u t e a l cell."  4". 6.  The E a r l y D e g e n e r a t i o n Cycle.  Of Corpora L u t e a From The F i r s t  Ovulatory  C o r p o r a l u t e a d e r i v e d from t h e f i r s t o v u l a t o r y c y c l e degenerated rapidly after  the rupture  w i t h newly-ruptured was  of second-cycle f o l l i c l e s .  In five  females  f o l l i c l e s , t h e average volume o f e i g h t c o r p o r a l u t e a  3 3 28.7 mm , a s compared t o 32.6 mm i n females j u s t p r i o r t o second  ovulation. averaged  But,one d a y a f t e r second  o n l y about 15 mm  3  ovulation, degenerating corpora l u t e a  3 and were as s m a l l as 5-4- mm .  Two days a f t e r  92  ovulation the corpora l u t e a were shrunken to an average volume of about 5 mm  but thereafter shrinkage was slower ( F i g . 14- and Appendix 2 ) . Two curves d e p i c t i n g the volume of degenerate corpora l u t e a are  presented i n F i g . 14.  One i s based on known-age corpora l u t e a and the other  i s based on corpora l u t e a whose ages were estimated by the p r e v i o u s l y outlined i n 4. 4. comparison  criteria  Included i n one of the above methods was a  of degenerate corpora l u t e a of unknown age to degenerate  l u t e a dated by the cleavage stage of the ova.  corpora  Both curves i n F i g . 14  i n d i c a t e an extremely rapid shrinkage d u r i n g the f i r s t 2 to 3 days a f t e r second ovulation.  Reasons f o r the f a s t i n i t i a l shrinkage and slower  shrink-  age a f t e r 3 days was evident i n h i s t o l o g i c a l sections. Immediately a f t e r the rupture of the second-cycle f o l l i c l e , most c e l l s of the corpus luteum were s t i l l i n t a c t , although they were shrunken, vacuolated, and i n e a r l y stages of a u t o l y s i s .  One day a f t e r second  ovulation,  c y t o l y s i s of c e l l s was extensive and only a l i m i t e d number of r e l a t i v e l y i n t a c t c e l l s remained. l e a s t affected.  C e l l s i n the c e n t r a l p o r t i o n of the structure were  The connective t i s s u e of the trabeculae formed wide bands  of t i s s u e between groups of c e l l s .  Two days a f t e r second o v u l a t i o n , the  connective t i s s u e formed large a c e l l u l a r zones i n the structure and a network of h y a l i n i z e d t i s s u e started to develop.  Remains of degenerate c e l l s were  present i n most corpora l u t e a , but i n some more-degenerate s t r u c t u r e s , only c e l l spaces containing c e l l u l a r d e b r i s remained. the center of a few scars that were 2 days o l d .  Active c e l l s p e r s i s t e d i n Three days a f t e r second  o v u l a t i o n , the bands of h y a l i n t i s s u e between the c e l l remnants were wider and by the f o u r t h to f i f t h day only a reduced number of shrunken lacunae p e r s i s t e d .  cell  Five days a f t e r the i n i t i a l rapid degeneration, almost  the e n t i r e s t r u c t u r e was h y a l i n i z e d . throughout these and older scars.  Less degenerate c e l l s were scattered  C e l l s bordering the c e n t r a l lumen were  93  .Fig.  601  14.  The volume o f d e g e n e r a t i n g f i r s t - c y c l e c o r p o r a l u t e a (CL 1) accompanying s e c o n d - c y c l e c o r p o r a l u t e a (CL 2) whose ages were e s t i m a t e d by 1) t h e c l e a v a g e stage of ova,and 2) s e v e r a l c r i t e r i a (see t e x t ) .  94  s t i l l a c t i v e i n a few corpora l u t e a of non-pregnancy.  The above changes  are i l l u s t r a t e d i n P l a t e 12.  4.. 7  The Fate Of F i r s t - C y c l e Corpora Lutea. Stages i n the advanced degeneration of corpora l u t e a derived from  the f i r s t ovulatory c y c l e are recorded i n Table 6.  The average age of the  scars i s the i n t e r v a l between the mean date of the c o l l e c t i o n period and November 25, the mean date of second-cycle ovulation.  The i n i t i a l degenera-  t i o n of the f i r s t - c y c l e corpora l u t e a begins at second  ovulation.  Shrinkage of the scars was gradual but continuous from November to March, a f t e r which the average volumes of those measured was about constant 3 at 0.04  to 0.05 mm  .  Apparently,the scars o c c a s i o n a l l y persisted f o r about  2 years,but some were barely d i s c e r n a b l e a f t e r 12 months.  Exluded  from  the above samples,were degenerate f i r s t - c y c l e corpora l u t e a that underwent secondary l u t e i n i z a t i o n .  These,of course,were much l a r g e r .  General changes i n the h i s t o l o g y of the degenerating corpus luteum i n c l u d e d : hyalin tissue,  2)  1) a r e d u c t i o n i n the amount and s t a i n a h i l i t y of the a r e d u c t i o n i n the number of degenerate c e l l s and  lacunae, 3) an increase i n the amount of yellow pigment associated with the degenerate c e l l s and lacunae, and to the scar.  4) a r e d u c t i o n i n the stromal response  Some scars older than 120 days contained vacuoles and  spaces bordered by b r i l l i a n t yellow pigment.  cell  Others, with l e s s pigment,  appeared as white spaces i n a.pale h y a l i n s t r u c t u r e .  Almost a l l the scars  were located near the ovarian surface and could be traced to a surface scar of h y a l i n t i s s u e .  Although most surface scars were small plugs of h y a l i n  t i s s u e , some were l a r g e r than the i n t e r n a l scar.  Some of the above changes  i n degenerating f i r s t - c y c l e corpora l u t e a are i l l u s t r a t e d i n P l a t e 12. Degenerate corpora l u t e a that developed i n t o accessory corpora  Table 6.  Mean Coll. Date 30 Nov. 5 Dec. 12 Dec. 2  Feb. 21 Mar, 10  May 13 June 20 Nov.  Stages i n the advanced d e g e n e r a t i o n o f f i r s t - c y c l e c o r p o r a l u t e a (CL l ) and t h e i r characteristics.  Avg CL 1 ' Age Scar of CL 1 (mm^) scars (x and (days) range) 3.38 (1.25 4.0) 1.95 10 (1.13.5) . 1.02 10 (0.142.3) 0.07 •(.0369 0.09) 0.04 (0.010.09) 0.04 167 • .. • (0.010.08) 0.05 201 (0.030.07) 0.05 360 (0.020.10) . 118  Sample Size 14 12  10  18  17  37 8  10  histological  H i s t o l o g i c a l C h a r a c t e r i s t i c s H y a l i n i z e d network o f f i b e r s around c e l l l a c u n a e c o n t a i n i n g n a t u r a l y e l l o w orange pigment and c e l l remnants. A few v a c u o l a t e d c e l l s r e m a i n t r a p p e d i n amorphous a n i l i n e - p o s i t i v e c o n n e c t i v e t i s s u e . C e l l s and l a c u n a e fewer. C e l l a u t o l y s i s n e a r l y complete. Amorphus bands o f c o n n e c t i v e t i s s u e more e x t e n s i v e b u t t h e r e a r e p a t c h e s o f n o n - h y a l i n i z e d c o n n e c t i v e t i s s u e c e l l s and f i b e r s . C e l l l a c u n a e s c a t t e r e d throughout a n i l i n e - p o s i t i v e , h y a l i n e c o n n e c t i v e t i s s u e . C e l l remnants and w a l l s c o n t a i n y e l l o w pigment. Outer c a p s u l e o f f i b e r s reduced. Two have a c t i v e l u t e a l c e l l s i n c e n t e r . Less a n i l i n e - p o s i t i v e c e n t r a l core o f amorphous c o n n e c t i v e t i s s u e w h i c h cont a i n s l a c u n a e and some y e l l o w m a t e r i a l . I n some t h e r e i s a p a l e , more h y a l i m band around the c e n t r a l c o r e o f l a c u n a . Scar i s surrounded by compressed a c i d o p h i l i c stromal c e l l s . As above, but whole s t r u c t u r e l e s s c h r o m a t i c , e x c e p t f o r l a c u n a e , x^hich may be b r i l l i a n t y e l l o w . Less s t r o m a l r e s p o n s e . Scar i s l o c a t e d near s u r f a c e o f ovary and may be t r a c e d t o t h e s u r f a c e where a r u p t u r e s c a r i s u s u a l l y presen' H y a l i n e core o f s c a r w a s t i n g or b e i n g r e s o r b e d by s t r o m a l c e l l s . A group of y e l l o w pigmented c e l l s or l a c u n a e o c c u r s i n , or a d j a c e n t t o , degenerate hyaline tissue. As i n p r e c e e d i n g s t a g e . S t r u c t u r e c a n u s u a l l y be t r a c e d t o t h e s u r f a c e o f t h e o v a r y where a h y a l i n e s c a r may be p r e s e n t . Some have a c e n t r a l c o r e o f h y a l i n e t i s s u e , w i t h y e l l o w c e l l s i n a r i n g around i t . I n o t h e r s , a l m o s t a l l amorphous c o n n e c t i v e • t i s s u e has been r e s o r b e d and a group o f y e l l o w c e l l s l i e s i n t h e stroma near t h e s u r f a c e of the ovary.  96  l u t e a (Type 2) did not f o l l o w the above pattern of r e s o r p t i o n .  Upon  degeneration,they developed i n t o miniature corpora l u t e a of pregnancy scars (Chapter 4-. 9 ) .  4. 8  Corpora Lutea In The Pregnant Doe. Corpora l u t e a at f i v e stages of pregnancy were obtained from  p e r i o d i c sampling of the population i n 1963-64.  Only one doe i n the  December sample contained v i s i b l e embryos but other a d u l t females i n the sample were assumed to be pregnant because t h e i r u t e r i were enlarged, convoluted, and distended with f l u i d .  4. 8. 1  Size.  Corpora l u t e a increased i n s i z e d u r i n g g e s t a t i o n ,  except f o r a period near mid-gestation when the mean volume was l e s s than at any other period ( F i g . 1 5 ) .  The apparent r e d u c t i o n i n s i z e could be  a t t r i b u t e d to chance because the mean volume was not s t a t i s t i c a l l y d i f f e r e n t from the means f o r the December and January-February  collections.  In May  and June, however, there was a s i g n i f i c a n t increase (P < 0.05) i n the s i z e of corpora l u t e a , i n comparison to the three e a r l i e r sampling periods. Three does c o l l e c t e d i n February,which were not v i s i b l y pregnant, contained smaller than average corpora l u t e a . separate designations i n F i g . 15.  These corpora l u t e a received  In a d d i t i o n , the r a p i d l y - r e g r e s s i n g  corpora l u t e a i n two p o s t - p a r t u r i e n t does were not included i n the s t a t i s t i c s f o r the June c o l l e c t i o n period. Primary corpora l u t e a i n pregnant females ranged i n volume from 3 13 to 156 mm  .  The smallest one was undoubtedly  second smallest was 46  mm^.  exceptional because the  Fig.  150 -j UO  15.' The volumes o f c o r p o r a l u t e a o f pregnancy (CLP) a t f i v e s t a g e s o f g e s t a t i o n .  •  No v i s i b l e  conceptual  O  Post-Parturant Range  130 120 110  A  100  1  Standard d e v i a t i o n 35% c o n f i d e n c e limit  90  Mean  PH  O  o  10  80 i  S 70. 1-1 o >  ©  60  50  "i5  Sample s i z e  26  AO  Xi  o  p  •rH  P P,  30 •  CD O  C  20 •  O  O  20  20  30  NOVEMBER  "75 20 I T DECEMBER  20 10 < ;u ^ JANUARY  10  20 29 FEBRUARY  10  20 MARCH  o 31  10 20 • 30 • APRIL  10  20 MAY  10 20 JUNE  98  4. 8. 2 and  Histology.  I n the f o l l o w i n g account of the h i s t o l o g i c a l  c y t o l o g i c a l changes i n corpora l u t e a during gestation, the stage of  gestation i s the i n t e r v a l between the mean c o l l e c t i o n - p e r i o d date and November 25, the mean date of conceptions.  Corpora l u t e a i n two does with  r e l a t i v e l y small fetuses were excluded from the f o l l o w i n g d e s c r i p t i o n s . Reference should be made t o Plate 14 before reading the d e s c r i p t i o n s of corpora l u t e a . Corpora l u t e a from the December 12 c o l l e c t i o n period were, on the average, about 17 days o l d .  The younger ones i n the group contained  poorly  defined, l i g h t l y a c i d o p h i l i c , vacuolated c e l l s with v e s i c u l a t e n u c l e i . A f a i r number of conception c e l l s , w i t h c e n t r a l a c i d o p h i l i c zones containing an e c c e n t r i c a l l y located n u c l e i and g o l g i apparatus,occurred throughout the gland.  Also present,were small numbers of angular, deeply  chromatic c e l l s with condensed n u c l e i (Plate I4. l ) . Older corpora l u t e a i n the group contained v a r i a b l y s i z e d , l i g h t l y a c i d o p h i l i c c e l l s i n c l u d i n g l a r g e r (to 35 p),  more-vacuolated c e l l s .  amounts of yellow l i p o i d pigment.  Some of these contained  small  Up t o 40% of the l u t e a l c e l l s i n these  older corpora l u t e a were v a r i a b l y s i z e d , angular or s t e l l a t e , hyperchromatic c e l l s with p e r i p h e r a l vacuoles.  About 9 or 10 l u t e a l c e l l s , i n c l u d i n g n u c l e i ,  occurred i n a f i e l d of view a t 1250X.  P r a c t i c a l l y every c e l l was bordered  by connective t i s s u e c e l l s , i n c l u d i n g those of the vascular  system.  Corpora l u t e a i n deer from the February 2 c o l l e c t i o n period were about 69 days o l d .  The average l u t e a l c e l l was much l a r g e r than those of the  previous c o l l e c t i o n period.  Only four to s i x l u t e a l c e l l s , i n c l u d i n g n u c l e i ,  occurred i n a f i e l d of view a t 1250X. only s l i g h t l y l a r g e r (39 p).  However, the l a r g e s t c e l l s were  Most c e l l s were l a r g e , spheroid or oblate,  l i g h t l y stained and contained small p e r i p h e r a l vacuoles and nuclei.  Only a small proportion  semi-vesiculate  of the spheroid c e l l s contained deeply  F a c i n g page 99 P l a t e 14 Changes i n Corpora L u t e a D u r i n g Pregnancy ( a l l p h o t o m i c r o g r a p h s X800). 1. A s e c t i o n o f a c o r p u s l u t e u m about 25 days a f t e r c o n c e p t i o n . Most l u t e a l c e l l s c o n t a i n s m a l l v a c u o l e s t h r o u g h o u t . A n g u l a r chromop h i l i c c e l l s ( l o w e r l e f t ) a r e common. The l a r g e c e l l ( l o w e r c e n t e r ) c o n t a i n s some y e l l o w pigment. 2. A s e c t i o n o f a c o r p u s l u t e u m 62 days a f t e r c o n c e p t i o n . Note t h e p e r i p h e r a l v a c u o l a t i o n of the l a r g e c e l l s . Some n u c l e i a r e condensed. 3. A s e c t i o n o f a c o r p u s l u t e u m about 72 days a f t e r c o n c e p t i o n . the a n g u l a r c e l l s c o n t a i n i n g condensed n u c l e i .  Note  4. A s e c t i o n o f a c o r p u s l u t e u m about 119 days a f t e r c o n c e p t i o n . There i s a r i n g o f e x t e n s i v e v a c u o l a t i o n i n most c e l l s . Condensed n u c l e i occur i n some s p h e r o i d c e l l s (upper r i g h t ) . Some c e l l s have died. 5. A s e c t i o n o f a c o r p u s l u t e u m about 159 days a f t e r c o n c e p t i o n . e n l a r g e d c e l l s c o n t a i n g r a n u l e s around t h e n u c l e i .  Some  6. A s e c t i o n o f a c o r p u s l u t e u m about 171 days a f t e r c o n c e p t i o n . few c e l l s a r e b a s o p h i l i c and c o n t a i n condensed n u c l e i (upper right).  A  7. A p o r t i o n o f a c o r p u s l u t e u m about 200 days a f t e r c o n c e p t i o n (near t e r m ) . I n a d d i t i o n t o t h e l a r g e a n g u l a r c e l l s t h e r e a r e s m a l l c e l l s (one a t t h e r i g h t b o r d e r and two a t t h e t o p , j u s t l e f t of c e n t e r ) . 8. A p o r t i o n o f a c o r p u s l u t e u m i n a doe near p a r t u r i t i o n . are i n v a r i o u s s t a g e s o f d e g e n e r a t i o n .  A l l cells  3 9. A s e c t i o n o f a degenerate,22 mm c o r p u s l u t e u m i n a doe t h a t had g i v e n b i r t h a few days p r e v i o u s t o c o l l e c t i o n . The c e l l s a r e comp l e t e l y v a c u o l a t e d . A p o r t i o n o f a t h i c k - w a l l e d blood v e s s e l i s evident. '  3  10. A p o r t i o n o f a d e g e n e r a t e 12 mm c o r p u s l u t e u m i n a second doe w h i c h had g i v e n b i r t h a few days p r e v i o u s t o b e i n g k i l l e d . This s t r u c t u r e i s more d e g e n e r a t e than t h e one above. O n l y a few degenerate c e l l s s u r v i v e (lower c e n t e r ) . A t h i c k - w a l l e d blood v e s s e l was c u t i n c r o s s s e c t i o n ( r i g h t ) .  100  a c i d o p h i l i c cytoplasm.'  A n g u l a r , c h r o m o p h i l i c c e l l s w i t h condensed  were not numerous e x c e p t i n zones u n d e r g o i n g t i s s u e network was  more e x t e n s i v e .  t i s s u e c e l l s separated f i b e r s surrounded Corpora  cell autolysis.  cells.  A t h i c k l a y e r of c o l l a g e n i c  t h e l a r g e blood v e s s e l s . l u t e a f r o m the c o l l e c t i o n p e r i o d c e n t e r e d on March 21 As i n the p r e v i o u s group,most o f the c e l l s were  l a r g e , c o n t a i n e d v e s i c u l a t e n u c l e i and numerous v a c u o l e s . contained a d i s t i n c t  one-tenth  p e r i p h e r a l r i n g of v a c u o l e s and  others contained  t o o n e - t h i r d o f the c e l l s c o n t a i n e d d e e p l y c h r o m o p h i l i c  s m a l l , a n g u l a r , and Corpora  cytoplasm,  A s m a l l p r o p o r t i o n o f c e l l s were  l u t e a i n does c o l l e c t e d around May widespread  10 were about 167  days  i n t h e s e , e s p e c i a l l y i n the c e n t e r o f  The r e l a t i v e p r o p o r t i o n s o f the c e l l t y p e s v a r i e d . Deeply a c i d o -  p h i l i c c e l l s , w i t h condensed, c r i n k l y n u c l e i and v a c u o l a t e d comprised  From  chromophilic.  A u t o l y s i s o f c e l l s was  the g l a n d .  Many c e l l s  l a r g e v a c u o l e s , y e l l o w pigment, and a c i d o p h i l i c g r a n u l e s .  condensed n u c l e i , and p e r i p h e r a l v a c u o l e s .  old.  connective  I n p l a c e s , two or t h r e e c o n n e c t i v e  adjacent l u t e a l  were about 117 days o l d .  relatively  The  nuclei  20 t o 80%  o f the l u t e a l  c e l l population.  be most numerous i n r e g i o n s u n d e r g o i n g  cytoplasm,  These c e l l s appeared t o  c e l l autolysis.  The l i g h t l y a c i d o -  p h i l i c c e l l s w i t h v e s i c u l a t e - n u c l e i were e x t e n s i v e l y v a c u o l a t e d . amongst t h e l a r g e c e l l s were s m a l l c e l l s w i t h l a r g e v a c u o l e s . o f c e l l a u t o l y s i s , s p h e r u l e s o f c y t o p l a s m were common.  Scattered  In regions  The w a l l s o f the  major blood v e s s e l s were t h i c k . Corpora l u t e a f r o m the c o l l e c t i o n p e r i o d c e n t e r e d on June 13 were, about 200 days o l d .  Two  does had  w i t h i n a few days o f p a r t u r i t i o n . t u r i t i o n c o n t a i n e d c e l l s up t o 4-7  g i v e n b i r t h and most o f the o t h e r does were Corpora  l u t e a i n does a p p r o a c h i n g  i n diameter,whereas c e l l s i n  c o l l e c t i o n s d i d n o t exceed about 39 _u.  par-  earlier  The m a j o r i t y o f c e l l s were l a r g e ,  101  f a i n t l y a c i d o p h i l i c , vacuolated  ( e s p e c i a l l y around the p e r i p h e r y ) , and  c o n t a i n e d s e m i - v e s i c u l a t e or c r i n k l y , condensed n u c l e i . the p e r i p h e r y o f t h e gland c o n t a i n e d y e l l o w pigment.  A few c e l l s around  Up t o o n e - t e n t h  of the  c e l l p o p u l a t i o n were l a r g e , d e e p l y - a c i d o p h i l i c c e l l s w i t h condensed n u c l e i . Up t o 30% of the l u t e a l c e l l p o p u l a t i o n were s m a l l , c h r o m o p h i l i c c e l l s i n w h i c h l a r g e v a c u o l e s a p p a r e n t l y developed  as the c e l l aged.  The e x c e e d i n g l y d e g e n e r a t e c o r p o r a l u t e a i n the two p o s t - p a r t u r i e n t females  c o n t a i n e d o n l y a few shrunken,  vacuolated c e l l s .  Consequently,the  3 c o r p o r a l u t e a were shrunken t o volumes of 22 mm pigment was  p r e s e n t i n the d e g e n e r a t e ,  3 and 12 mm  Fine, yellow,  vacuolated c e l l s that p e r s i s t e d ,  e s p e c i a l l y t h o s e trapped i n h y a l i n c o n n e c t i v e t i s s u e . d e g e n e r a t e corpus luteum,  .  I n the s m a l l e r more  c o n s i d e r a b l e g r a n u l a r pigment was  the i n s i d e of a t h i c k , f i b r o u s , o u t e r c a p s u l e .  present  along  A l s o p r e s e n t I n the  c o l l a g e n i c c a p s u l e , were t h i c k - w a l l e d blood v e s s e l s w h i c h f o r m e r l y served t h e corpus luteum.  S m a l l e r , t h i c k - w a l l e d v e s s e l s occurred i n the  former  c e l l u l a r p o r t i o n o f the s h r i n k i n g s t r u c t u r e s . 4. 9  Accessory Corpora  Lutea.  L u t e a l t i s s u e s o c c u r r i n g i n o v a r i a n s t r u c t u r e s t h a t d i d not t r i b u t e v i a b l e ova a r e termed a c c e s s o r y or secondary  corpora l u t e a .  occur o n l y i n o v a r i e s c o n t a i n i n g p r i m a r y c o r p o r a l u t e a . l u t e a are d e r i v e d from f o l l i c l e s ova).  conThey  (Primary corpora  c o n t r i b u t i n g v i a b l e , or p o t e n t i a l l y v i a b l e ,  I c l a s s i f i e d a c c e s s o r y c o r p o r a l u t e a i n deer i n t o t h r e e t y p e s , based  on t h e i r  source. Type 1 a c c e s s o r y c o r p o r a l u t e a o r i g i n a t e d i n u n r u p t u r e d  especially i n large f o l l i c l e s z a t i o n o f the f o l l i c u l a r f o l l i c l e was  follicles,  that f a i l e d to rupture at o v u l a t i o n .  w a l l began 1 t o 2 days a f t e r o v u l a t i o n .  s m a l l , o n l y the w a l l or a p o r t i o n o f the w a l l was  Luteini-  Unless  the  composed o f  102  luteal tissue.  These s t r u c t u r e s  were p r e v i o u s l y d e s c r i b e d i n 3. 8. L\.  Type 2 a c c e s s o r y c o r p o r a l u t e a d e v e l o p e d i n d e g e n e r a t e ,  first-  c y c l e c o r p o r a l u t e a f r o m c e l l s t h a t f a i l e d t o d e g e n e r a t e , or f r o m c e l l s t h a t became h y p e r p l a s t i c lutea.  d u r i n g t h e e a r l y growth o f s e c o n d - c y c l e c o r p o r a  A few a c t i v e c e l l s p e r s i s t e d  i n g corpora l u t e a .  i n t h e c e n t r a l r e g i o n s o f some d e g e n e r a t -  There was no sharp d e m a r c a t i o n between a c t i v e c e l l s and  a t r e t i c c e l l s i n some e a r l y - d e g e n e r a t e c o r p o r a l u t e a , b u t a f t e r 8 t o 12 d a y s , the a c t i v e t i s s u e formed a d e f i n i t e b a l l w i t h i n t h e s c a r .  The s m a l l ,  s p h e r i c a l s t r u c t u r e s were surrounded by s c a r t i s s u e t h a t could the  s u r f a c e o f t h e ovary.  compact,  be t r a c e d t o  The amount o f l u t e a l t i s s u e i n t h e 29 s c a r s  f r o m j u s t a few c e l l s , t o a volume o f 0.87  mm . 3  Type 3 a c c e s s o r y c o r p o r a l u t e a o r i g i n a t e d  i n small f o l l i c l e s  r u p t u r e d about t h e same time as t h e p r i m a r y f o l l i c l e s . l u t e i n i z e d but r a r e l y contributed  varied  v i a b l e ova.  These s m a l l  that  follicles  Type 3 a c c e s s o r y c o r p o r a  3 l u t e a , w i t h volumes o f l u t e a l t i s s u e t o 16 mm , were t h e l a r g e s t o f t h e t h r e e types.  Because t h e y were i n d i s t i n g u i s h a b l e f r o m , and r a r e l y o v e r l a p p e d i n  size with  s m a l l , p r i m a r y c o r p o r a l u t e a , an a r b i t r a r y d e c i s i o n on t h e  d e m a r c a t i o n s i z e was made.  I f a corpus l u t e u m w i t h an o v u l a t i o n  s c a r was  l e s s t h a n o n e - q u a r t e r t h e volume o f t h e l a r g e s t c o r p u s l u t e u m i n t h e same p a i r o f o v a r i e s , i t was c o n s i d e r e d a c c e s s o r y . smallest  I n pregnant animals,the  p r i m a r y c o r p u s l u t e u m was about o n e - q u a r t e r o f t h e s i z e o f t h e  l a r g e r c o r p u s l u t e u m i n t h e same f e m a l e . The  i n c i d e n c e o f t h e t h r e e t y p e s o f a c c e s s o r y c o r p o r a l u t e a was  r e l a t e d t o t h e age o f t h e doe and t o t h e c h r o n o l o g y o f t h e e s t r o u s Following  f i r s t - c y c l e ovulation,  lutea i n yearlings  cycles.  t h e i n c i d e n c e o f Type 1 a c c e s s o r y c o r p o r a  and a d u l t s was UU% and 4-8%, r e s p e c t i v e l y .  l u t e a l t i s s u e d e v e l o p e d , i t was always i n t h e l a r g e s t a t r e t i c  I f accessory follicle,  103  a l t h o u g h s m a l l e r f o l l i c l e s i n t h e same p a i r o f o v a r i e s l u t e i n i z e d occasionally.  A f t e r f i r s t - c y c l e o v u l a t i o n t h e r e was no Type 3 a c c e s s o r y  c o r p o r a l u t e a i n 50 f e m a l e s , a l t h o u g h some c o r p o r a l u t e a were between one- . q u a r t e r and o n e - h a l f t h e s i z e o f t h e l a r g e r c o r p o r a l u t e a i n t h e same p a i r of  ovaries. After  second o v u l a t i o n ,  TyP  1 a c c e s s o r y c o r p o r a l u t e a were  e  n o t n e a r l y as common as f o l l o w i n g f i r s t o v u l a t i o n , and t h e y o c c u r r e d i n small f o l l i c l e s ovulation,  (see 3. 8. 4 ) .  mostly  I n the f i r s t 2 weeks f o l l o w i n g second  Type 2 secondary c o r p o r a l u t e a o c c u r r e d i n a bout 41% (7/17) o f  the does w i t h degenerate  f i r s t - c y c l e corpora l u t e a .  l u t e a l t i s s u e , i n o t h e r w i s e degenerate  The i n c i d e n c e of a c t i v e  c o r p o r a l u t e a , w a s 3 1 % (9/29)-  Usually,  o n l y one o f two c o r p o r a l u t e a o f non-pregnancy i n a p a i r o f o v a r i e s c o n t a i n e d active luteal tissue.  Type 3 a c c e s s o r y c o r p o r a l u t e a o c c u r r e d i n o n l y  3.2% (2/62) o f t h e f e m a l e s w i t h s e c o n d - c y c l e c o r p o r a l u t e a e s t i m a t e d t o be between 2 and 14 days o l d . Both a c c e s s o r y c o r p o r a l u t e a were i n t h e o v a r i e s of females  2.5 y e a r s o l d . Four o t h e r deer c o n t a i n e d c o r p o r a l u t e a between  one-quarter  and o n e - h a l f t h e volume o f t h e l a r g e r c o r p o r a l u t e a i n t h e same  pair of ovaries. The i n c i d e n c e o f a l l t y p e s o f a c c e s s o r y c o r p o r a l u t e a i n pregnant does was 3 5 . 9 % (19/53).  They o c c u r r e d i n f e m a l e s o f a l l ages, b u t t h e  l a r g e r Type 3 s t r u c t u r e s were more p r e v a l e n t i n young and o l d does.  Of  3  the f i v e l a r g e s t a c c e s s o r y c o r p o r a l u t e a , one was 16 mm  i n volume and t h e  3  o t h e r f o u r measured between 2.4 and 8.4 mm . 3 w e r e . l e s s than  1 mm  i n volume.  Most a c c e s s o r y c o r p o r a l u t e a  The l a r g e s t one o c c u r r e d i n a non-  pregnant female c o l l e c t e d on March 24. Apparently, a l l 3 types o f accessory corpora l u t e a survived t o the t e r m i n a t i o n o f pregnancy.  As pregnancy proceeded,  t h e source (and t h e r e f o r e  t y p e ) o f t h e a c c e s s o r y c o r p o r a l u t e a became i n c r e a s i n g l y more d i f f i c u l t t o  104  determine. form.  Ovulation stigmas healed and the corpora l u t e a became modified i n  For example, a few months a f t e r i n i t i a l development of Type 2  accessory corpora l u t e a , hypertropy of the l u t e a l c e l l s and atrophy of the surrounding scar t i s s u e made diagnosis d i f f i c u l t .  Only a b a l l of l u t e a l  t i s s u e remained i n the stroma, not u n l i k e Type 1 accessory corpora l u t e a a f t e r the f o l l i c l e was resorbed. was d i f f i c u l t corpora l u t e a .  I f a surface rupture scar was present, i t  to d i s t i n g u i s h some Type 2 s t r u c t u r e s from Type 3 accessory I n a l l three types,the l u t e a l c e l l s resembled those of the  primary corpora l u t e a .  4. 10 Scars Derived From Corpora Lutea. The term corpus a l b i c a n s was o r i g i n a l l y used t o describe scars r e s u l t i n g from a degenerative corpus luteum of the human menstrual cycle (Harrison, 1962).  The term was also applied to the scar derived from a  human corpus luteum of pregnancy.  The s t r u c t u r e i s h y a l i n i z e d and appears  c o l o r l e s s i n f r e s h , f i x e d , and r o u t i n e l y stained (hematoxylin and eosin) sections.  I n many species, the term corpus a l b i c a n s has been applied to a l l  types of scars derived from corpora l u t e a and a l s o to scars r e s u l t i n g from the  h y a l i n i z a t i o n of f o l l i c l e s  i n advanced stages of a t r e s i a .  I n the cow,  the  scar, developing from a corpus luteum of pregnancy, i s termed a corpus  rubrum because of i t s high content of reddish pigment (Hammond, 1927). •In deer, the d i f f e r e n t types of scars o r i g i n a t i n g from corpora l u t e a may be c l a s s i f i e d as f o l l o w s : 1.  Scars derived from corpora l u t e a of non-pregnancy. 1. 1  Scars derived from degenerate, f i r s t c y c l e corpora l u t e a .  1. 2  Scars derived from degenerate corpora l u t e a of the second, t h i r d , e t c . c y c l e i f pregnancy doesn't occur.  1. 3  Scars derived from accessory corpora l u t e a of the f i r s t ovulatory c y c l e (and subsequent cycles i f pregnancy  105  does not occur). 2.  Scars derived from corpora l u t e a of pregnancy. 2. 1  Scars derived from normal corpora l u t e a of pregnancy.  2. 2  Scars derived from aberrant corpora l u t e a (often associated with the i n t r a - u t e r i n e m o r t a l i t y of zygotes).  2. 3  Scars derived from accessory corpora l u t e a .  The c h a r a c t e r i s t i c s of each type are presented i n the f o l l o w i n g pages.  4. 10. 1  Scars not Derived From L u t e a l S t r u c t u r e s . The most numerous  ovarian scars r e s u l t from the normal a t r e s i a of f o l l i c l e s .  They are charac-  t e r i z e d by l a r g e amounts of amorphous, h y a l i n connective t i s s u e , which gives them a white appearance i n f r e s h and f i x e d m a t e r i a l . However, they are a n i l i n e p o s i t i v e when stained w i t h trichrome methods.  I n microscopic s e c t i o n s ,  or upon hasty examination of stained s e c t i o n s , these scars can be confused with scars derived from corpora l u t e a of non-pregnancy.  However, the l a t t e r  always contain degenerate l u t e a l c e l l s , or evidence of these c e l l s , such as c e l l lacunae.  O c c a s i o n a l l y , a few of the major blood v e s s e l s , which f o r m e r l y  served l a r g e f o l l i c l e s , p e r s i s t f o r long periods. Abnormal or n e c r o t i c a t r e s i a of f o l l i c l e s r e s u l t e d i n scars cont a i n i n g v a r i a b l e amounts of connective t i s s u e associated w i t h degenerate c e l l s and t h e i r products.  As the n e c r o t i c s t r u c t u r e regressed, p e c u l i a r  creamy-white m a t e r i a l was produced i n spaces between the c o l l a g e n i c f i b e r s and f i b r o b l a s t s (Plate 9. 9-10).  4. 10. 2  Scars Derived From Corpora Lutea of Non-Pregnancy.  The scars  r e s u l t i n g from the degeneration of f i r s t - c y c l e corpora l u t e a , were described p r e v i o u s l y i n . 4 . 6 and 4- 7.  They may be confused w i t h s i m i l a r h y a l i n s t r u c -  tures that form i n very a t r e t i c f o l l i c l e s , but the scars derived from  106  f i r s t - c y c l e corpora l u t e a always contained remnants of l u t e a l c e l l s and the s t r u c t u r e often extended to the surface of the ovary, where some scar t i s s u e was evident.  I n a d d i t i o n , corpora l u t e a scars were u s u a l l y s o l i d  s t r u c t u r e s , whereas scars r e p l a c i n g f o l l i c l e s contained a c e n t r a l lumen , which g r a d u a l l y decreased i n s i z e .  Scars r e s u l t i n g from the second and  subsequent o v u l a t i o n s , which d i d not r e s u l t i n conception, were not common i n the ovaries because most does conceived a t second estrus.  The scars were  s i m i l a r i n appearance to scars r e s u l t i n g from f i r s t - c y c l e corpora l u t e a , except they were l a r g e r a t equivalent stages of r e g r e s s i o n .  The ovaries of  one non-pregnant doe, c o l l e c t e d May 14, contained t e n 0.1 to 0.8 mm  3  scars  from the previous breeding season. Scars r e s u l t i n g from accessory corpora l u t e a of the f i r s t cycle were small and soon disappeared.  They were u s u a l l y crescent shaped and had no  connection to the surface of the ovary.  Apparently,the s i z e of the scar was  p r o p o r t i o n a l t o the s i z e of the accessory corpus luteum from which i t was derived.  The l a r g e s t a c t i v e accessory corpus luteum (Type l ) contained  3  11.3 mm  of l u t e a l t i s s u e .  L\. 10. 3  Corpora Lutea of Pregnancy Scars.  Corpora l u t e a that  supported a pregnancy f o r an extended period of time, degenerated i n t o scars w i t h d i s t i n c t i v e c h a r a c t e r i s t i c s .  Following the b i r t h of the fawn  i n June, the corpus luteum r a p i d l y regressed,and w i t h i n a few days i t was o n e - f i f t h t o one-tenth of i t s p r e - p a r t u r i t i o n volume. partum, the structure had a volume of about 3.4- mm former s i z e ) , and a l l l u t e a l c e l l s had been resorbed.  At 3.5 months post(one t h i r t i e t h of i t s • The structure of the  scars 5 t o 6 months a f t e r i n i t i a l degeneration w i l l be described i n d e t a i l , because most of the samples came from deer k i l l e d i n November and e a r l y December.  107  Because c o r p o r a l u t e a never or r a r e l y developed i n w i l d fawn d e e r of t h e s t u d y a r e a , l a r g e s c a r s found i n f e m a l e s between 2.0 and 2.4 y e a r s of age were d e r i v e d from c o r p o r a l u t e a o f pregnancy o f t h e p r e v i o u s November t o June r e p r o d u c t i v e season.  The f o l l o w i n g d e s c r i p t i o n o f s c a r s  was f r o m f e m a l e s 2.4 t o 2^5 y e a r s o l d a t the time o f c o l l e c t i o n i n November or e a r l y December. The mean volume o f 49 s c a r s was 2.24 +.0.10 mm  (Fig. 16).  3 Excluded  f r o m t h e above sample were a l l s c a r s l e s s t h a n 1.0 mm , a l l o f  w h i c h a r e b e l i e v e d t o be s c a r s d e r i v e d from a c c e s s o r y c o r p o r a l u t e a .  3 I n c l u d e d i n t h e above sample were s i x l a r g e s c a r s o f 3.5 t o 4>8mm , w h i c h may have a r i s e n from l a r g e c o r p o r a l u t e a or f r o m a l a t e  parturition.  B o t h a r e p o s s i b i l i t i e s , f o r some y e a r l i n g s developed l a r g e c o r p o r a l u t e a o f pregnancy and some were l a t e o v u l a t o r s .  E x c l u d i n g these e x c e p t i o n a l l y l a r g e  s c a r s lowered t h e mean s c a r volume t o 2.08 + 0.06 mm  (N = 4 3 ) .  There was a g r a d u a l i n c r e a s e i n t h e s i z e o f s c a r s e s t i m a t e d 5 t o 6 months o l d , a s t h e age o f t h e f e m a l e s i n c r e a s e d n i f i c a n t d i f f e r e n c e occurred  ( F i g . 16).  t o be  A sig-  between t h e volume o f s c a r s i n young does  (2.5 and 3.5 y e a r s o l d ) and o l d does ( o l d e r t h a n 7 y e a r s ) ( A n a l y s i s o f v a r i a n c e F = 7.35, P < 0.01 w i t h 6 and 213 d f ) . A macroscopically v i s i b l e surface protrusion, u s u a l l y located i n the c e n t e r o f a s m a l l depressed a r e a , marked t h e l o c a t i o n o f most s c a r s o f 5 months.  The p r o t r u s i o n , composed o f a s o l i d h y a l i n mass o f c o n n e c t i v e  t i s s u e , was c o n n e c t i v e stroma.  t o t h e main body o f t h e s c a r l o c a t e d deeper i n t h e  The m a j o r i t y o f these s c a r s , l o c a t e d i n t h e o u t e r c o r t e x , were  o b l a t e s p h e r o i d i n f o r m ; o t h e r s were d i t o r t e d by f o l l i c l e s and c o r p o r a lutea. *  Unless  otherwise  the s t a n d a r d  s t a t e d t h e p l u s and minus f i g u r e a f t e r a mean r e p r e s e n t s  error.  F i g . 16.  The a g e - s p e c i f i c volumes o f s c a r s 5 and 17 months o l d d e r i v e d f r o m c o r p o r a l u t e a o f pregnancy. S c a r s 5 months o l d a r e i n t h e upper row. (Mean, 95% c o n f i d e n c e l i m i t s , s t a n d a r d d e v i a t i o n , r a n g e , and sample s i z e ) .  24  49 ^  4'  37 5 months  B  £  3'  o 00  1y months  1  V 3  24  2  2.5  "3TT  4.5  5.5 AGE OF DOE (YEARS)  6.5  7.5-9.5  10.5&+-  o ax  109  The  t y p i c a l s c a r of 5 months was  outer connective  ensheathed i n an  t i s s u e capsule of c o l l a g e n i c f i b e r s .  t h i c k around' some s c a r s but was  The  aniline-positive, capsule  was  b a r e l y d i s c e r n i b l e around o t h e r s .  Located  i n the i n n e r p o r t i o n of t h i s zone were l a r g e , c o i l e d , t h i c k - w a l l e d blood v e s s e l s w h i c h passed t h r o u g h the c a p s u l e a l o n g one  s i d e o f the s c a r  extended a s h o r t d i s t a n c e towards the h i l a r p o r t i o n of the ovary.  and These  were f o r m e r l y the l a r g e p e r i p h e r a l blood v e s s e l s o f the c o r p u s luteum. The  c e n t r a l p o r t i o n of the s c a r was  composed of h y a l i n c o n n e c t i v e t i s s u e  i n t e r s p e r s e d w i t h s m a l l , t i g h t l y - c o i l e d blood v e s s e l s and The  l i p o g e n i c pigment.  l a r g e r , o u t e r blood v e s s e l s were u s u a l l y a c i d o p h i l i c ,  o c c a s i o n a l l y , p o r t i o n s of i n d i v i d u a l v e s s e l s f a i l e d t o s t a i n . l a r g e v e s s e l s s t i l l contained  the  the  In sections stained  the c e n t r a l blood v e s s e l s , h y a l i n m a t e r i a l , and  stained red.  Most o f  blood c e l l s but u s u a l l y the lumena o f  s m a l l e r more c e n t r a l a c i d o p h i l i c v e s s e l s were o c c l u d e d . w i t h PAS,  but  pigment, a l l  I n s e c t i o n s s t a i n e d i n combined PAS and Massons's  the r e d s c a r s c o n t r a s t e d m a r k e d l y a g a i n s t the b l u e stroma  trichrome,  ( P l a t e 16).  A l i p o g e n i c pigment, o v a r i a n f u s c i n , i d e n t i f i e d by the c a r b o l f u s c h s i n technique  (Thompson, 1966), was  observed i n s c a r s o f 5 months.  The pigment i s P A S - p o s i t i v e , but the r e a c t i o n i s n o t s p e c i f i c t o the pigment.  I n s e c t i o n s s t a i n e d w i t h Masson's t r i c h r o m e , the pigment r e t a i n e d  i t s i n t r i n s i c c o l o r , orange brown ( P l a t e 1 6 ) . • The  o v a r i e s of does 3.5  above d e s c r i p t i o n and  years old contained  These r e p r e s e n t c o r p o r a l u t e a o f pregnancy I n  the does when t h e y were between 1.5  and 2.0  y e a r s o l d , the s i z e o f 21 s c a r s e s t i m a t e d 3  the  o t h e r s i m i l a r but more d e g e n e r a t e s c a r s t h a t were  o b v i o u s l y 17 months o l d .  mm .  scars f i t t i n g  years o l d .  I n females  t o be 17 months o l d was  3.5 1.17+0.07  T h i s i s s i g n i f i c a n t l y s m a l l e r (P < 0.001) t h a n the s c a r s o f 5 months  i n f e m a l e s aged 2.5  years, although  the r a n g e s o v e r l a p .  The  s i z e of scars  F a c i n g page  Plate Scars Derived  15  From Corpora L u t e a  1.  A scax d e r i v e d f r o m a corpus l u t e u m of pregnancy. About 5 months have e l a p s e d s i n c e the c o r p u s l u t e u m began t o d e g e n e r a t e ( X 2 0 ) .  2.  P o r t i o n s of a d j a c e n t s c a r s d e r i v e d f r o m c o r p o r a l u t e a of pregnancy. Note the chromophobic h y a l i n i z e d r e g i o n s around the p e r i p h e r a l blood v e s s e l s of the s c a r 17 months o l d (on the l e f t ) and the s m a l l amount of amorphous c o n n e c t i v e t i s s u e w i t h i n the s c a r . The t i s s u e between the s c a r s i s p a r t of a band of c o l l a g e n i c f i b e r s around the s c a r of 5 months (on the r i g h t ) . The l a t t e r s c a r c o n t a i n s amorphous c o n n e c t i v e t i s s u e between the blood v e s s e l s ( X 4 I ) .  3.  A s c a r aged 5 months d e r i v e d f r o m a p a r t l y - h e r n i a t e d c o r p u s luteum. A t h i c k band of h y a l i n i z e d c o n n e c t i v e t i s s u e marks the former r u p t u r e s i t e on the s u r f a c e of the o v a r y ( X 4 I ) .  4.  A s c a r of 5 months d e r i v e d f r o m a c o m p l e t e l y - h e r n i a t e d of pregnancy ( X 4 I ) .  5.  Four o l d s c a r s d e r i v e d f r o m c o r p o r a l u t e a of pregnancy. The t h r e e scars,composed a l m o s t e n t i r e l y of a c h r o m a t i c h y a l i n t i s s u e ^ a r e p r o b a b l y between 1 0 and 17 y e a r s o l d ( X 2 0 ) .  6.  The c e n t r a l r e g i o n of a s c a r of 5 months t o demonstrate the t h i c k w a l l e d v e s s e l s (two i n the r i g h t h a l f of the photograph) and the amorphous c o n n e c t i v e t i s s u e c o n t a i n i n g n u c l e i , pigment and v a c u l e s (X800).  corpus luteum  7. A s e c t i o n of a d e g e n e r a t e c o r p u s l u t e u m i n the o v a r i e s of a doe k i l l e d November 9The l a r g e i r r e g u l a r - s h a p e d c e l l s are l a d e n with granules. The slow and abnormal f o r m of d e g e n e r a t i o n i s a t t r i b u t e d t o f e t a l m u m m i f i c a t i o n (X800). 8. A 0.9 mm , h y a l i n - t y p e scar b e l i e v e d to represent a corpus luteum t h a t f a i l e d because of embryonic or f e t a l m o r t a l i t y . A few t h i c k w a l l e d blood v e s s e l s occur around the p e r i p h e r y o f a c e n t r a l mass of amorphous h y a l i n t i s s u e (X80). 3  110  111  estimated  t o be 17 months o l d , i n does o l d e r than 3.5  i n F i g . 16. the age  There was  o f does.  years, i s  presented  a g r a d u a l i n c r e a s e i n the s i z e of these  I n s p e c t i o n of F i g . 16 r e v e a l s  scars with  t h a t , f o r each age-group  t h e r e was  a s i g n i f i c a n t d i f f e r e n c e between s c a r s of the two ages.  t h e r e was  c o n s i d e r a b l e o v e r l a p i n s i z e s between s c a r s w i t h i n an  and  However,  age-class,  t h e r e f o r e n o t a l l s c a r s 5 months o l d can be d i s t i n g u i s h e d , o n the  of s i z e a l o n e , f r o m Surface  older scars. s c a r s m a r k i n g the r u p t u r e s i t e of s t r u c t u r e s 17 months o l d ,  were s i m i l a r - t o those less stainable.  5 months of age,  but t h e y v/ere g e n e r a l l y s m a l l e r  because of t h e i r g r e a t e r  or  follicular  Compared t o the younger s c a r s , the d e m a r c a t i o n of the 17-month  s c a r f r o m the s u r r o u n d i n g  stroma was  o f an o u t e r c o l l a g e n i c c a p s u l e .  The  more d e f i n i t e .  There was  no  indication  o u t e r blood v e s s e l s were s m a l l e r , more  occluded,  and  old.  l a r g e , p e r i p h e r a l blood v e s s e l s , f o r m e r l y c o n t i n u o u s  The  and  Most s c a r s aged 17 months were d i s t o r t e d by f o l l i c l e s  c o r p o r a l u t e a , e s p e c i a l l y i n young a n i m a l s activity.  basis  l e s s s t a i n a b l e than corresponding  v e s s e l s i n . s c a r s 5 months with  the  major o v a r i a n v e s s e l s i n the h i l u m , were more c o i l e d and r e t r a c t e d . h y a l i n c e n t r a l p o r t i o n o f the s c a r was commonly c o n t a i n e d  fissures.  much r e d u c e d , l e s s c h r o m a t i c ,  This r e d u c t i o n of t i s s u e placed  thick-walled, central vessels i n close proximity. more d e e p l y s t a i n e d pigment was  The and  .  the s m a l l ,  O n l y a s m a l l amount o f  p r e s e n t i n the s c a r .  The pigment was  of  d i v e r s e shapes, whereas pigment i n s c a r s aged 5 months s c a r s were o f t e n sheroid. I n f e m a l e s aged 4-5  y e a r s , which c o n t a i n e d  f i v e or s i x s c a r s i n  t h e i r o v a r i e s , the s c a r t h a t had degenerated most and  appeared o l d e r t h a n  t y p i c a l s c a r s o f 17 months,was assumed t o be 29 months o l d . t h e s e s c a r s were s m a l l e r , c o n t a i n e d pigment, were more v a c u o l a t e d  and  little  Generally  or no h y a l i n c o n n e c t i v e  l e s s chromatic  t i s s u e or  t h a n younger s c a r s , and  112  the p e r i p h e r a l vessels were u s u a l l y achromatic.  I t was apparent that  scars 27 months old could not alx-zays be d i s t i n g u i s h e d from those of 17 months. In older does,there were scars 4 I , 53, 65, e t c . months o l d . 3 most of these scars exceeded 0.5  mm  Since  i n volume,, i t was apparent that degenera-  t i o n and absorption of the scar proceeded at a slow r a t e a f t e r the second year.  From the progression of changes i n the scars, i t appeared that a l l scars  derived from corpora l u t e a of pregnancy p e r s i s t e d i n the ovaries f o r the l i f e of the doe.  I f t h i s was true, and the pregnancy r a t e of does did not  change appreciably with age, then a r e g r e s s i o n of the average number of scars per age-class  on age should r e v e a l a strong c o r r e l a t i o n .  Such was  the case (r = 0.998)'('Fig. 17). 4. 11 Corpora Lutea And Scars In Macroscopic Sections. At c e r t a i n periods of the reproductive c y c l e , each major type of ovarian s t r u c t u r e (corpora l u t e a , degenerate corpus luteum of non-pregnancy, accessory corpus luteum, scar of a corpus luteum of pregnancy, a t r e t i c f o l l i c l e ) was e a s i l y i d e n t i f i e d i n gross sections by c o l o r and c h a r a c t e r i s t i c s (Table 7).  size  At other times during the breeding season,  mistakes i n i d e n t i t y were made,as' revealed by examination structures i n thin,stained sections.  of the same  The ages of s t r u c t u r e s i n Table 7  were estimated from t h e i r volumes and t h e i r h i s t o l o g i c a l appearance i n thin sections. A c t i v e corpora l u t e a were d i s t i n c t i v e s t r u c t u r e s but at l e a s t one small newly-ruptured  f o l l i c l e was missed i n the macroscopic s e c t i o n s .  First-  c y c l e corpora l u t e a i n ' e a r l y stages of degeneration were d i s t i n c t , but those older than 4 or 5 days were s i m i l a r i n c o l o r (orange) and s i z e to pregnancy scars of 5 months.  As a r e s u l t , three d e g e n e r a t e , f i r s t - c y c l e corpora l u t e a  F i g . 17  R e g r e s s i o n o f .the-, average number o f c o r p o r a l u t e a o f p r e g n a n c y s c a r s on the age o f females a t the p r e v i o u s b r e e d i n g season. (Sample s i z e by each p o i n t ) .  28 1  © l ©  26 • I  ©  24' 22 1  I  I  2.5  1  I  4.5  1  1  6.5  1  1  8.5  I  I  10.5  «  1  1  12.5  AGE OF DOE AT THE PREVIOUS BREEDING SEASON  1  14.5  I  I  16.5  1  !—  18.5  Table 7. C h a r a c t e r i s t i c s o f s t r u c t u r e s i n g r o s s s e c t i o n s o f p r e s e r v e d Structurei  Color  0-1 B zones & j streaks. ¥ periph. t i s s .  W  5x4  Bulb-shaped or f l a t t e n e d . Folded w a l l . Cent, lumen  Color  See • j Corresp. CL 1  abund ant B streaks  few B streaks  4-7x 2-5  5-8x 3-7  See Corresp. CL 1 Form  R=red,  |  0=orange,  C PaY  6-7x  j  3-4x2-3  j  1  Second-Cycle 1-2  S i ze(two ] 3 - 4 X diam mm) j 1-3  j  4-6  0-1  wc  7-8.5x 5.5-7  2.5-3x 1.5-2  5-8 CO, PaO  9+ PaO, PaOY YO  1.5-2x 1-2  2x1-2  9+ w  c  5-6 •months DeO 0 PaO  7-10x 1.5-2.5 7-8 • x l - 2  Oblate, Same Reduced S o l i d , spher., or lumen. oblate as f l a s k - s h a p - Compact s p h e r o i d prev. ed . Lumen stage u s u a l l y sm. 3 occas. l a r g e Y=yellow, C= cream, W^white, B=Black,  Scar Of Pregnancy 17-18 months 0 PaO  l-2x 0.5-1  A c c e s s o r y Corpus Luteum Various Same as c o r r e s p . C L l o r CL2.  Various  Oblate Oblate Same as Same as spheroid s p h e r o i d p r e v i o u s •previous or or stage stage spherical spherical  Corpus Luteum 5-8 3-4 W C  3-4 DeC, CY, C. Y, PaO  C  Oblate spher. o r S o l i d j oblate flask-shaped. Folding indisSpher i t i n c t . Sm. lumen  W  1-2  6-8 .j  2-3  S i z e ( t w o ] 3-5x1-3 diam mm) j  Structure j Age(days)j  ]De;g e n e r a t i n g F i r s t - C y c l e Corpus Luteum  F i r s t - C y c l e Corpus Luteum  Age(days)j  ovaries.  Type 1 h o l l o w . Spher. t o c r e s c . Shaped. Type 2 & 3 o b l a t e or spher.  JAtretic F o l l i c l e 29-30 ! Various months \ | P e r i p h e r y W. j Center n o r m a l l y PaO ! clear. Center ] o f some PaY or Y PaO | Avg 1 i  Various  j  Spher., o b l a t e , U s u a l l y Flattened flattened flask-shaped. and i R a d i a l patoften i t e r n of blood curved 1 ! vessels & ! pigment. De^deep, Pa=pal e. Spher. = s p h e r o i d ,  Large lumen surrounded by a white capsule t o a s o l i d mass of w h i t e t i s s u e . sm. = s m a l l .  115  were i n i t i a l l y mistaken f o r pregnancy scars.  Four other s t r u c t u r e s i n  gross sections were i n d i s t i n c t but were estimated t o be corpora l u t e a of non-pregnancy.  This i d e n t i f i c a t i o n was confirmed i n t h i n sections.  In  a d d i t i o n , two u n i d e n t i f i e d , c o l o r e d s t r u c t u r e s noted i n t h i c k sections were i d e n t i f i e d i n t h i n sections as degenerate corpora l u t e a of non-pregnancy. At l e a s t one small,degenerate corpus luteum of non-pregnancy,found sections,was missed i n macroscopic sections.  i n thin  Many pregnancy scars were  not v i s i b l e i n gross sections. Accessory corpora l u t e a e x i b i t e d the same texture and color as primary corpora l u t e a i n the same p a i r of ovaries.  Because of l i m i t e d  development of l u t e a l t i s s u e , they were not confused w i t h primary corpora l u t e a , nevertheless, some small ones would c e r t a i n l y be missed I n macroscopic s e c t i o n s . I t was not always possible to estimate the age of pigmented corpora l u t e a of pregnancy scars i n macroscopic sections.  The ovaries of  does older than 4- years u s u a l l y contained three to s i x pigmented scars.  The  l a r g e r , more rounded and deeply pigmented scars were assumed to be 5 months o l d , but they c l e a r l y graded i n t o older scars.  One scar of 5 months was  b a r e l y v i s i b l e i n the ovaries of a female aged 2.5 years.  P r i o r to  macroscopic s e c t i o n i n g , one surface corpus luteum of pregnancy scar was i n c o r r e c t l y i d e n t i f i e d as a p a r t l y herniated corpus luteum.  Only  pigmented pregnancy scars were v i s i b l e i n gross sections (Plate 16). Several black, s p h e r i c a l , objects were c o r r e c t l y i d e n t i f i e d as hemorrhagic f o l l i c l e s .  F o l l i c l e s undergoing normal a t r e s i a had an outer  white capsule of v a r i a b l e thickness and an empty or p a r t l y - f i l l e d lumen. A few a t r e t i c f o l l i c l e s contained a pale, yellow-orange  central region  ( i d e n t i f i e d i n t h i n sections as n e c r o t i c tissue),.which tended to form a hollow sphere i n large f o l l i c l e s (See F o l l i c l e A t r e s i a ) .  F a c i n g Page  Plate  16  C o l o r Photomicrographs Of O v a r i a n 1.  2.  116  Structures  Two g e n e r a t i o n s of c o r p o r a l u t e a . The most r e c e n t (upper l e f t ) i s about 2 days o l d . The d e g e n e r a t i n g . , f i r s t - g e n e r a t i o n c o r p u s l u t e u m c y s t i c and f i l l e d w i t h b l o o d . (Masson's t r i c h r o m e , X16).  was  A c o r p u s l u t e u m of pregnancy about 7 days a f t e r o v u l a t i o n and a shrunken c o r p u s l u t e u m f r o m the f i r s t o v u l a t i o n . ( M a s s o n s t r i c h r o m e , X16). 1  3.  A t i n y s c a r f r o m the c o r p u s l u t e u m of non-pregnancy about 12 months a f t e r i t s i n i t i a l r e g r e s s i o n . Note the y e l l o w pigment i n the c e l l remnants. Such s c a r s u s u a l l y c o n t a i n more h y a l i n c o n n e c t i v e t i s s u e t h a n i s p r e s e n t i n t h i s s c a r . ( M a s s o n ' s t r i c h r o m e , X160).  4.  C o r p o r a l u t e a of pregnancy s c a r s aged 5 months (On the l e f t ) and 17 months (on the r i g h t ) . Note the c a p s u l e of c o l l a g e n i c f i b e r s around t h e younger s c a r . A p o r t i o n of a l a r g e f o l l i c l e i s a l s o p r e s e n t . (Masson's t r i c h r o m e , X16).  5-  A c o r p u s l u t e u m of pregnancy s c a r 5 months o l d . The c e n t r a l p o r t i o n of the s c a r i s magenta when s t a i n e d i n a c o m b i n a t i o n of p e r i o d i c a c i d S c h i f f and Masson's t r i c h r o m e . (X16).  6.  Pregnancy s c a r s of 5 months (bottom) and 17 months ( t o p ) . The c o i l e d , t h i c k - w a l l e d blood v e s s e l s are c l e a r l y e v i d e n t . The younger s c a r c o n t a i n s g r e a t e r amounts of h y a l i n c o n n e c t i v e tissue.(Masson's t r i c h r o m e , X32).  7.  O v a r i a n f u s c i n pigment i n a pregnancy s c a r aged 5 months. The s e c t i o n was s t a i n e d i n c a r b o l f u s c h i n w h i c h i s s p e c i f i c f o r l i p o g e n i c pigment (XI600).  8.  Pigment i n a pregnancy s c a r of 5 months s t a i n e d i n p e r i o d i c a c i d . Schiff. The pigment i s m o s t l y i n the f o r m of s p h e r u l e s . Connective t i s s u e f i b e r s are s t a i n e d l i g h t l y . (X1600).  9.  This h y a l i n - t y p e scar i s b e l i e v e d to represent a corpus luteum t h a t f a i l e d because of embryonic or f e t a l m o r t a l i t y . The few, p e r i p h e r a l blood v e s s e l s are a c h r o m a t i c .  10.  A t h i c k s e c t i o n t h r o u g h an o v a r y f i x e d i n A.F.A. The pigmented s t r u c t u r e - below the l a r g e f o l l i c l e i s a pregnancy s c a r of 5 months. Blood v e s s e l s and r e g r e s s i n g f o l l i c l e s are a l s o e v i d e n t i n t h i s s e c t i o n . (X8).  117  SECTION 3.  THE BREEDING SEASON AND THE LENGTH OF THE ESTROUS CYCLE. I t was imperative t o define,accurately,the breeding season f o r two  main reasons: 1.  To determine the i n t e r v a l between f i r s t and second o v u l a t i o n .  2.  To determine the mean date of conceptions so that the age of embryos, corpora l u t e a , and other structures could be estimated.  In t u r n , i t was necessary to establish,accurately,the i n t e r v a l between o v u l a t i o n s , so that successive corpora l u t e a and f o l l i c l e s could be f i x e d i n a framework of time. Information on the dates of ovulations and the i n t e r v a l between successive ovulations was obtained from f i v e sources : 1)  The f r equency of  females having ovulated f o r the f i r s t and second time i n samples obtained i n November and e a r l y December.  2)  The dates of f i r s t and second o v u l a t i o n  estimated from c h a r a c t e r i s t i c s of corpora l u t e a and f o l l i c l e s . dates of conception estimated from f e t a l growth curves. of degenerate corpora l u t e a i n l a t e breeders.  5)  A)  3)  The  The occurrence  The dates of p a r t u r i t i o n  estimated from records of tagged fawns. I t was p o s s i b l e to estimate the l e n g t h of the i n t e r v a l between the f i r s t and second ovulations by the f i r s t two methods because,apparently,the f i r s t o v u l a t i o n was never s u c c e s s f u l .  Because large numbers of deer were  obtained throughout the breeding season, ovulation-frequency curves were constructed f o r f i r s t and second ovulations and they were compared statistically.  F o r t u n a t e l y , most of the females ovulated f o r the f i r s t time  w i t h i n a span of about 2 weeks and f o r the second time w i t h i n a shorter span. Because the breeding season changed i n s i g n i f i c a n t l y from year to year (except f o r one year),the samples from 5 years were combined to increase the  118  sample s i z e .  3. 1  The Frequency Of Newly-Ruptured Periodic  F o l l i c l e s And C o r p o r a L u t e a I n  Samples.  Most o f the o v a r i e s came f r o m deer k i l l e d by h u n t e r s on weekends and h o l i d a y s i n November and e a r l y December.  Because the d a i l y sample s i z e  i n a g i v e n y e a r was  s m a l l , the average o v u l a t i o n i n c i d e n c e f o r t h e two week-  end days was used.  Even t h e n t h e sample s i z e was i n a d e q u a t e f o r some week-  ends of some y e a r s .  O v u l a t i o n i n c i d e n c e c u r v e s f o r f i r s t and second  ovula-  t i o n s were e s t a b l i s h e d f o r a l l does ( y e a r l i n g s and o l d e r ) and f o r a d u l t does (over 2 y e a r s ) .  The sample s i z e of the former group was l a r g e r but i n c l u d e d  a s m a l l and v a r i a b l e p e r c e n t a g e of l a t e o v u l a t o r s and n o n - b r e e d e r s . t i o n , the l e n g t h o f the e s t r o u s c y c l e may i n older females.  In addi-  be more v a r i a b l e i n y e a r l i n g s t h a n  S e p a r a t e graphs were c o n s t r u c t e d f o r does w i t h d e f i n i t e  c o r p o r a l u t e a and f o r does w i t h e i t h e r c o r p o r a l u t e a or n e w l y - r u p t u r e d f o l l i c l e s , b e c a u s e some of the l a t t e r c o u l d be m e c h a n i c a l r u p t u r e s . Fig.  18 shows the p e r c e n t a g e of does h a v i n g o v u l a t e d f o r t h e f i r s t  and second time a t f o u r p e r i o d s  (weekends) d u r i n g November o f 1963.  The  c u r v e was smoothed t h r o u g h the p o i n t s because o f the s m a l l sample s i z e f o r the c o l l e c t i o n p e r i o d s November 16-17  and 23-24-  The p e r i o d o f f i r s t o v u l a t i o n extended f r o m t h e f i r s t few days of N o v e m b e r , u n t i l about November 29, when a l l does i n a sample o f 21 had By November 9-10,  ovulated.  o n l y about 12% had o v u l a t e d but the i n c i d e n c e i n c r e a s e d t o  about 60% by t h e f o l l o w i n g weekend.  I t i s deduced f r o m t h e . g r a p h t h a t 50% o f  a l l does o v u l a t e d f o r the f i r s t time between November 9 and November 23. About 50%. o f t h e a d u l t does o v u l a t e d f o r the f i r s t time between November 10 and November 18. The second o v u l a t i o n s o f t h e season began about November 15  and,  F i g . 18  The o v u l a t i o n i n c i d e n c e on weekends i n November and e a r l y December, 1963. (Sample s i z e i n d i c a t e d ) .  NOVEMBER  120  presumably,extended  i n t o t h e f i r s t week o f December.  I t i s deduced from  the graph ( F i g . 18) t h a t about 50% o f a l l does o v u l a t e d f o r t h e second between November 17 and November 27.  time  Second o v u l a t i o n s began i n a d u l t f e m a l e s  about November 17, and reached t h e 50% i n c i d e n c e l e v e l by November 25.  About  50% o f t h e a d u l t does o v u l a t e d w i t h i n a span o f 9 d a y s . In  t h e y e a r s 1964- t o 1967, t h e d a t e s of f i r s t and second  were s i m i l a r t o t h o s e o f 1963.  Curves f i t t e d  ovulations  t o t h e a v a i l a b l e samples  i n d i c a t e . t h a t t h e d a t e on w h i c h t h e o v u l a t i o n i n c i d e n c e reached 50% v a r i e d w i t h i n 2 days over t h e 4- year p e r i o d f r o m 1963 t o 1966. Because t h e b r e e d i n g season v a r i e d o n l y s l i g h t l y f r o m year t o y e a r , the r e s u l t s f o r a l l 5 y e a r s were combined t o i n c r e a s e t h e sample s i z e . 1963  t o 1967 samples were a v a i l a b l e f o r each d a y ( e x c e p t December 4-) from  November 6 t o December 6. based  From  The o v u l a t i o n i n c i d e n c e c u r v e s f o r a l l  does,  on a sample o f 298 f e m a l e s , were s i m i l a r t o t h o s e f o r t h e 1963  sample.  The c u r v e s i n d i c a t e t h a t f i r s t o v u l a t i o n s began i n e a r l y November, reached t h e 50% i n c i d e n c e l e v e l by November 16, and t e r m i n a t e d about November 28 a t t h e 96 t o 9 7 % i n c i d e n c e l e v e l ( F i g . 1 9 ) .  The r e m a i n i n g 3 t o  4-% r e p r e s e n t s l a t e o v u l a t o r s and n o n - o v u l a t o r s ( m o s t l y y e a r l i n g s ) .  The  d a t a i n d i c a t e d an e a r l y peak i n o v u l a t o r y a c t i v i t y between November 5 and November 8, f o l l o w e d by a c e s s a t i o n o f a c t i v i t y u n t i l November 12.  Because  t h e r e was l i t t l e i n d i c a t i o n o f a s i m i l a r b i m o d a l d i s t r i b u t i o n o f o v u l a t i o n d a t e s i n t h e c u r v e f o r s e c o n d - c y c l e o v u l a t i o n s , t h e i r r e g u l a r i t y was a t t r i b u t e d l a r g e l y to sampling e r r o r .  For t h i s reason,the curve f o r a d u l t s  i s smoothed t h r o u g h t h e p o i n t s . About 50% o f a l l does o v u l a t e d f o r t h e f i r s t time between November 13 and November 21,and 7 5 % o v u l a t e d between November 12 and November 27. A d u l t does-completed began  f i r s t o v u l a t i o n * b y November 26.  Second o v u l a t i o n s  about November 17 and t e r m i n a t e d e a r l y i n December.  About 50% o f a l l  F i g . 19-  —i—  A  —r-  6  The ovulation incidence based on d a i l y samples obtained during November and e a r l y December from 1963 to 1967.  10  12 NOVEMBER  U  16  20  22  24  26  30  2 4 DECEMBER  122  does o v u l a t e d 75% o v u l a t e d  f o r t h e second t i m e between November 18 and November 26,and between November 18 and December 1.  A d u l t does completed  second o v u l a t i o n around December 2. Because t h e sample s i z e f o r many i n d i v i d u a l days were s m a l l , mean o v u l a t i o n i n c i d e n c e v a l u e s f o r p e r i o d s  o f 2, 3, and 4 days were c a l c u l a t e d ,  and  t h e mean d a t e s f o r each p e r i o d were weighted a c c o r d i n g  for  each d a y o f t h e p e r i o d .  The e f f e c t  of these running  r e d u c t i o n i n v a r i a t i o n about t h e c u r v e f i t t e d was  placed  on o b t a i n i n g an a c c u r a t e  estimate  t o t h e sample s i z e averages was a  t h r o u g h t h e p o i n t s . Emphasis o f t h e mean d a t e s o f f i r s t and  second o v u l a t i o n because these d a t e s were n e c e s s a r y t o d a t e o v a r i a n and  structures  fetuses. Curves f o r t h e f r e q u e n c y o f d e f i n i t e c o r p o r a  l u t e a per u n i t of  t i m e g e n e r a l l y p a r a l l e l e d , b u t on t h e average l a g g e d about one d a y b e h i n d , the o v u l a t i o n i n c i d e n c e c u r v e s f o r f e m a l e s w i t h c o r p o r a ruptured was  follicles.  greatest  The gap between t h e c u r v e s ,  l u t e a and newly-  which a r e n o t i l l u s t r a t e d ,  (up t o 2 d a y s ) i n t h e mid p o r t i o n o f t h e c u r v e s —  a t the  peak o f f i r s t and second o v u l a t i o n . The  length of the estrous  c y c l e , t h e average i n t e r v a l between  f i r s t and second o v u l a t i o n s , was deduced, f r o m t h e o v u l a t i o n i n c i d e n c e based on combined samples f r o m 1963 t o 1 9 6 7 . I n t e r v a l s o f does w i t h c o r p o r a  l u t e a and r u p t u r e d  follicles  based on t h e i n c i d e n c e  and does w i t h  corpora  l u t e a o n l y , a r e t a b u l a t e d f o r a l l deer,and a d u l t d o e s , a t o v u l a t i o n l e v e l s o f 25, 50, and 75%, u s i n g r u n n i n g 1, 2, 3,  and U days (Table 8 ) .  curves  incidence  averages f o r time groupings o f  I t i s deduced f r o m t h e c u r v e s t h a t t h e  i n t e r v a l between the f i r s t and second o v u l a t i o n s  i s 8 o r 9 days . The  r e a s o n s f o r a c c u r a t e l y * e s t i m a t i n g t h i s i n t e r v a l appear i n t h e D i s c u s s i o n .  Table 8.  The i n t e r v a l i n days between f i r s t and second ovulations.  Ovulation Incidence Level (%)* A l l does (1.5 years old and old er)  25 50 75 Means  J ] ! j  I n t e r v a l Between Ovulations Based on CL & NRF** Time grouping (days)-"-I--X1 2 3 4  1 10.0  j j !  8.0 8.0 8.7  10.0 9.0 9.0 • 9.3  10.0 9-0 7.0 8.7  9.0 9.0 10.0 9.3  Ovulation Incidence L e v e l (%) 25 50 75 Means  | | | !  I n t e r v a l Between Ovulations Basec on CL only. Time groupin g (days) 1 2 3 4  | 7.0 j 8.0  9.0 9.0 8.0 8.7  9-5 8.5 6.5 8.2  8.5 9.0 8.5 8.7  ] 7.0 i 7.0 ] 6.5  8.5 8.0 8.0  8.0 7.5 7.0  7.5 7.5 7.5  !  8.2  7.5  7.5  i j 7.5 7  5  1  Adult does (2.5 years old and old er)  25 50 75 Means  | j  7.0 7.0  10.0 9.0 9-0  i  7.5  9-3  !  8  -  5  9-5 8.5 8.5  9.0 8.0 8.5  25 50 75  8.8  8.5  Means  6.8  The i n t e r v a l between the curves was determined a t these three a r b i t r a r i l y - c h o s e n l e v e l s . Ovulation incidence curves were established f o r corpora l u t e a plus newly-ruptured f o l l i c l e s (CL & NRF) and f o r corpora l u t e a (CL) alone. The curves were f i t t e d to weighted running averages of o v u l a t i o n incidences f o r periods of 1, 2, 3 and 4 days.  ro  124  3. 2  Dates Of O v u l a t i o n E s t i m a t e d From O v a r i a n S t r u c t u r e s . C e r t a i n a s p e c t s o f t h e b r e e d i n g season were more p r e c i s e l y de-  f i n e d from o v u l a t i o n d a t e s e s t i m a t e d from c h a r a c t e r i s t i c s of c o r p o r a l u t e a and f o l l i c l e s , as d e s c r i b e d p r e v i o u s l y i n Chapter 4.  Estimated dates of  f i r s t and second o v u l a t i o n f o r each o f the b r e e d i n g seasons f r o m 1963 t o 1967 were p l o t t e d i n h i s t o g r a m form. the  The f r e q u e n c y d i s t r i b u t i o n s r e v e a l e d  following points: 1.  F i r s t and second o v u l a t i o n s u s u a l l y began about November 5 to  2.  6 and November I 4 t o 16, r e s p e c t i v e l y .  The d a t e s of f i r s t o v u l a t i o n s spanned a g r e a t e r time p e r i o d t h a n t h e d a t e s o f second  3.  ovulations.  On t h e a v e r a g e , y e a r l i n g s o v u l a t e d l a t e r i n t h e season t h a n a d u l t s and t h e i r d a t e s o f o v u l a t i o n were more v a r i a b l e .  4.  The b r e e d i n g season o c c u r r e d a t about t h e same time i n each o f t h e f i v e y e a r s e x c e p t i n 1967, when i t was l a t e r .  5.  There was a t e n d e n c y f o r o v u l a t i o n s t o d e c r e a s e i n number d u r i n g , and f o r a d a y or two a f t e r , t h e weekend h u n t i n g p e r i o d s .  Means, s t a n d a r d d e v i a t i o n s , a n d s t a n d a r d e r r o r s were c a l c u l a t e d the  f r e q u e n c y d i s t r i b u t i o n s f o r second o v u l a t i o n (Appendix 3 ) .  mean d a t e o f f i r s t o v u l a t i o n was November 1 7 + 1 November 16 + 1 f o r 24 a d u l t does. November 16 +  1 f o r 24 a d u l t does.  from  I n 1963 t h e  f o r a l l 32 does and  The mean d a t e o f second o v u l a t i o n was The mean d a t e o f second o v u l a t i o n , was  November 22 + 1 f o r a l l . 2 0 does and November 23 + 1 f o r 17 a d u l t does.  Be-  cause o f t h e n a t u r e o f the s a m p l i n g d u r i n g November, t h e above mean d a t e s of  f i r s t and second o v u l a t i o n s a r e c o n s i d e r e d t o be, r e s p e c t i v e l y , one and  two days e a r l y . Frequency d i s t r i b u t i o n s were a n a l y s e d s t a t i s t i c a l l y t o d e t e r m i n e , if,  i n some y e a r s , t h e b r e e d i n g season was e a r l i e r or l a t e r t h a n i n o t h e r  125 years.  Tests between the means derived from the d i s t r i b u t i o n s f o r f i r s t  o v u l a t i o n were not made because of the small sample s i z e f o r most years, the o b v i o u s l y high v a r i a b i l i t y , and the bias caused by the method of sampling. The mean dates of second o v u l a t i o n were compared, but the comparisons were not v a l i d i n a l l cases because a l l the does ( e s p e c i a l l y y e a r l i n g s ) had not completed second o v u l a t i o n by the end of the sampling period. For example, when a l l does were considered there was a s i g n i f i c a n t d i f f e r e n c e between the mean o v u l a t i o n dates of 1963 and 1964- (P < 0.05), but the d i f f e r e n c e between the means f o r adults was not s i g n i f i c a n t .  Because  a few adults had not completed second o v u l a t i o n by the end of the sampling periods i n 1963, I concluded that no s i g n i f i c a n t d i f f e r e n c e existed between o v u l a t i o n dates i n 1963 and 1964. Further comparisons between years were made a f t e r the d i s t r i b u t i o n of dates of f i r s t o v u l a t i o n was s h i f t e d 8 days forward and added to the d i s t r i b u t i o n of dates f o r second o v u l a t i o n i n the same year.  This technique,  based on an 8-day i n t e r v a l between ovulations, increased the sample s i z e f o r each year and p a r t i a l l y compensated f o r l a t e ovulators.  Tests between  means revealed that the combined d i s t r i b u t i o n s of 1967 d i f f e r e d from the combined d i s t r i b u t i o n s of a l l other years except 1963.  I n the samples  obtained, the 1965 d i s t r i b u t i o n was almost s i g n i f i c a n t l y d i f f e r e n t from d i s t r i b u t i o n s of 1963, 1964,and 1966,and i t was s i g n i f i c a n t l y d i f f e r e n t from that of 1967.  •  •  '  *  The frequency d i s t r i b u t i o n s f o r f i r s t o v u l a t i o n from 1963 to 1966 were combined because there was no s t a t i s t i c a l d i f f e r e n c e between them. S i m i l a r l y , d i s t r i b u t i o n s f o r second o v u l a t i o n were combined ( F i g . 20). The s t a t i s t i c s derived from these combined d i s t r i b u t i o n s i n d i c a t e that, over the four year period, the mean dates of f i r s t and second o v u l a t i o n s were November 14 to 15 and November 23 r e s p e c t i v e l y .  The apparent i n t e r v a l  126 Fig.' 20.  Frequency d i s t r i b u t i o n s and the derived s t a t i s t i c s of estimated dates of f i r s t and second ovulations i n combined samples from . the breeding seasons of 1963 to 1966.  127  between ovulations was 8 days. adults.  Yearlings ovulated s l i g h t l y l a t e r than  The standard d e v i a t i o n of the dates of f i r s t o v u l a t i o n was n e a r l y  twice that of the dates of second-ovulation.  The mean date f o r the second  o v u l a t i o n was s l i g h t l y e a r l y because a few does had not completed second o v u l a t i o n at the termination of the sampling period. the  For t h i s reason,  a c t u a l d i s t r i b u t i o n of second ovulations i s more c l o s e l y approximated  by s h i f t i n g the frequency d i s t r i b u t i o n of f i r s t o v u l a t i o n forward (  l a t e r i n the season ) 8 days and adding i t to the d i s t r i b u t i o n f o r  second o v u l a t i o n .  The r e s u l t a n t frequency d i s t r i b u t i o n i s s l i g h t l y over-  represented i n l a t e dates. The d i f f e r e n c e between the 1967 mean date of second ovulations f o r a l l does, and the corresponding mean f o r combined samples from the other four years, was h i g h l y s i g n i f i c a n t (P < 0.001). S i m i l a r l y , t h e mean f o r adults from 1967 was very s i g n i f i c a n t l y d i f f e r e n t from, the mean f o r adults from the  combined samples from the other four years ( P < 0.01).  3. 3  The Estimated Dates Of Conception Determined From F e t a l Growth Curves. Some information on the breeding season and length of the estrous  c y c l e was obtained from f e t a l growth curves,used i n conjunction with ovarian analysis.  Ommundsen (1966), who studied the r e l a t i v e growth of fetuses  c o l l e c t e d f o r t h i s study, found that h i n d - f c o t length c o r r e l a t e d best with mean developmental ranks based on numerous morphological c r i t e r i a .  The  c u r v i l i n e a r r e l a t i o n s h i p between hind-foot length ( p o s t - f i x a t i o n ) and the date of f e t a l c o l l e c t i o n s i n 1963-64 i s presented i n F i g .  21.  Because the  mean date of second o v u l a t i o n i n 1963 was November 25, the growth of the hind f o o t was a l s o expressed i n terms of the mean gestation age.  I n addi-  t i o n to fetuses from the 1963-64 season, there were a few from the 1964-65 and 1965-66 reproductive seasons, and known-age fetuses from two does r a i s e d i n  F i g . 2 1 . The r e l a t i o n s h i p between hind-foot length-and 1) the date of c o l l e c t i o n i n 1963- 6-4, and 2) the' gestation age of the f e t u s . The l a t t e r was c a l c u l a t e d from November 25, the mean date of conceptions i n 1963. Also included are two known-age fetuses and samples from 1965 and 1 9 6 6 . ( E y e - f i t curve).  230 210  n Known-age Fetus O Fetus from a y e a r l i n g A Conceived on a cycle subsequent to Cycle 2. 54 Specimen no.  190170  w 150 EH  O  S3  S 130i rH  g  110  a  90-  I  a  70  3  50-  o  -p  u ra  30-  •H  MEAN GESTATION AGE  10  50  60  70 80 90 100 110 120 .130 1-40 150 160 170 M _> , i_ 10 20 30 10 20 29 iO 20 30 10 20 30 10 20 JANUARY FEBRUARY MARCH APRIL MAY DATE  190  200 10  210  . 20 JUNE  220  30  ro oa  129  captivity.  Only fetuses conceived  at the second o v u l a t i o n i n  1963,  as determined by ovarian a n a l y s i s , were included i n the regression. two does (T54 and T56),that conceived  ThuSj  i n a cycle subsequent to the second,  were omitted. Measurements of known-age fetuses from captive females were i n close agreement with the growth curve f o r fetuses from w i l d deer.  The  hind-foot lengths of the fetuses from two does c o l l e c t e d i n 1965 were a l s o i n close agreement with the extrapolated growth curve f o r the 1963-64 samples. This was expected- because the•breeding about the same time each November.  seasons of 1963  and 1964  occurred  at  That the m a j o r i t y of points f o r  fetuses from the 1965-66 reproductive season f e l l above the curve, was i n accordance with  a. s l i g h t l y e a r l i e r breeding season i n 1965.  A growth curve f o r forehead-rump length was established to determine i f the r e l a t i o n s h i p between the points f o r the various samples and curve was  s i m i l a r to that f o r hind-foot length.  a l l respects ( F i g . 22).  The pattern was  the  similar i n  No complex curve f i t t i n g was necessary because the  l i n e through the points was a simple l i n e a r r e g r e s s i o n .  The l i n e i s  described by the equation y = 2.57x - 83.6, where y i s the forehead-rump length and x i s the mean g e s t a t i o n age.  Forehead-rump length was more  v a r i a b l e than hind-foot length i n s p i t e of the measurements f o r twins averaged and represented  by a s i n g l e point.  Based on the growth curve f o r the hind f o o t , a y e a r l i n g doe conceived  being  (T54)  January 10, 46 days a f t e r the average date of conceptions i n 1963.  Because i t s ovaries contained  one corpus luteum and three corpora l u t e a  of non-pregnancy, the doe probably conceived  on i t s f o u r t h c y c l e .  I f the  doe ovulated f o r the second time on November 25, then i t underwent two more c y c l e s i n a period of 46 days.  I t i s p o s s i b l e that t h i s doe started to  c y c l e l a t e i n the breeding season as do many y e a r l i n g s which are a t t a i n i n g  130  puberty.  Thus,the i n t e r v a l between ovulations was,at most,23 days. An a d u l t doe (T56) conceived about January 1, 36 days l a t e r than  the average doe.  Because i t s ovaries contained two corpora l u t e a and seven  corpora l u t e a of non-pregnancy, i t i s l i k e l y that t h i s doe conceived on e i t h e r the f o u r t h or f i f t h c y c l e , with the l a t t e r being more probable.  I f the  doe conceived f o r the second time on November 25, then i t underwent two or probably three c y c l e s i n a period of 36 days. Both females (T54 and T56) are important f o r they provided an i n d i c a t i o n of the l e n g t h of estrous c y c l e s subsequent to the f i r s t .  3.4  The Occurrence  Of Corpora Lutea Of Non-Pregnancy In Late Conceivers Or  Non-Conceivers. Some data on the timing of breeding and the length of the estrous c y c l e were obtained from four does that were not pregnant or contained no 3  v i s i b l e conceptus.  An a t r e t i c , 81 mm  corpus luteum and three very small  corpora l u t e a of non-pregnancy were located i n the ovaries of a doe 2 . 5 years of age c o l l e c t e d on February 3 .  Because the corpora l u t e a of non-pregnancy  were very small (0.08, 0.06 and 0.02 mm ), 3  e i t h e r the embryo had been  resorbed or the female had stopped c y c l i n g s e v e r a l weeks p r e v i o u s l y , and the corpus luteum was p e r s i s t e n t .  The doe passed through three or probably  four c y c l e s before becoming t e m p o r a r i l y pregnant or ceasing to c y c l e . The ovaries of a y e a r l i n g taken on February 4 contained two 3 r e l a t i v e l y small corpora l u t e a (69 and 32 mm ), a very small corpus luteum 3 of non-pregnancy, and a l a r g e (5-5 mm ), h y a l i n i z e d corpus luteum of non- • pregnancy. I t was deduced that t h i s animal became pregnant on c y c l e two, but there was zygote m o r t a l i t y , corpus luteum degeneration, and subsequent development of the two e x i s t e n t corpora l u t e a . The ovaries contained a 30 3 mm , Stage 1, a t r e t i c f o l l i c l e , which (having f a i l e d to rupture) i n d i c a t e d  131  F i g . 22. The r e g r e s s i o n of f e t a l forehead-rump length on 1) date of c o l l e c t i o n and 2) the gestation age of f e t u s . (See F i g . 21) .  420'  -jO  4.00380-  Known-age fetus Fetus from a y e a r l i n g Conceived on a cycle subsequent to cycle 2  5 4 specimen no.  360'  340 320' 300'  280 260' EH O  240 220'  Pi I  200' 1 so-  i l 6o'140  :120H  :  100  80'  :-6o  4°"  50 60  70  0 20 30 JANUARY  80 10  MEAN GESTATION AGE (DAYS)  "90 20  100 T"  28  FEBRUARY  110  10  120  20  MARCH DATE  30  130 140  10  150  160  20 30 APRIL  170  10  180 190 200  20  . MAY  30  10  JUNE  132  t h a t o v u l a t i o n s had ceased or d i d n o t occur w i t h each f o l l i c u l a r A y e a r l i n g , c o l l e c t e d on March 24, c o n t a i n e d a 114 mm  3  cycle.  corpus  3 l u t e u m , an a c c e s s o r y corpus l u t e u m o f 16 mm , and f o u r c o r p o r a l u t e a o f non-pregnancy.  The doe had p r o b a b l y c y c l e d f i v e t i m e s .  Follicular 3  development  i n c l u d e d a waning Stage 3 f o l l i c l e o f 19 mm , and a growing  3 f o l l i c l e o f 19 mm . A non-pregnant d o e , a p p r o a c h i n g 7 y e a r s o f age,taken on May 14, c o n t a i n e d no c o r p o r a l u t e a and t e n c o r p o r a l u t e a o f non-  3 p r e g n a n c y , r a n g i n g i n volume f r o m 0.1 t o 0.8 mm .  A t an average o f two  c o r p o r a l u t e a per c y c l e , t h i s doe had passed t h r o u g h f i v e c y c l e s b e f o r e o v u l a t i o n s ceased many weeks p r i o r t o c o l l e c t i o n . 3. 5  Dates Of P a r t u r i t i o n . Each June, from 1959 t o 1964, crews o f t h e B r i t i s h Columbia  Fish  and W i l d l i f e Branch tagged newly-born fawns a t Northwest Bay. The f r e q u e n c y d i s t r i b u t i o n and d e r i v e d s t a t i s t i c s o f t h e t a g g i n g d a t e s f o r a l l seasons b u t t h e f i r s t , when t h e s e a r c h f o r fawn began l a t e , a r e shown i n F i g . 23.  D u r i n g t h i s p e r i o d , i n s i g n i f i c a n t d i f f e r e n c e s o c c u r r e d between  the d i s t r i b u t i o n s  f o r each y e a r .  The mean d a t e o f t h e combined  distribution,  June 1 4 , i s 203 days l a t e r t h a n t h e mean d a t e o f s e c o n d - c y c l e o v u l a t i o n s f o r the p e r i o d 1963-66.  I f t h e average fawn was tagged w i t h i n t h r e e days o f  b i r t h , t h e average g e s t a t i o n p e r i o d under n a t u r a l c o n d i t i o n s i s 200 t o 203 days,or equal t o that f o r c a p t i v e deer.  The s t a n d a r d d e v i a t i o n s o f  f a w n - t a g g i n g d a t e s (5 days) was s i m i l a r t o t h e s t a n d a r d d e v i a t i o n o f 6 days f o r t h e d a t e s o f second o v u l a t i o n s from 1963 t o 1966.  133  F i g . 23.  The frequency d i s t r i b u t i o n ( a n d derived s t a t i s t i c s ) o f dates on w h i c h fawns were tagged f r o m 1960 t o 1964-.  4  6  8  10  12  14  16  JUNE  x = 14 s = 5 n = 207  ±0.37 (SE)  18  20  22  24  26  28  30  134  SECTION 4.  THE EFFECT OF AGE ON FERTILITY AND  PRODUCTIVITY.  I n f o r m a t i o n on the a g e - s p e c i f i c f e r t i l i t y of the p o p u l a t i o n came f r o m c o u n t s of f e t u s e s , c o r p o r a l u t e a , and s c a r s d e r i v e d f r o m c o r p o r a l u t e a o f pregnancy.  C o r p o r a l u t e a r a r e l y d e v e l o p i n w i l d fawns, a l t h o u g h l a r g e  and r u p t u r e d f o l l i c l e s a r e common d u r i n g the b r e e d i n g season.  Only one  s m a l l s c a r i n d i c a t i v e of a former c o r p u s l u t e u m of non-pregnancy, 70 p a i r s o f o v a r i e s from y e a r l i n g s .  was noted i n  None o f the u t e r i f r o m fawns or s h o r t  y e a r l i n g s e x i b i t e d any i n d i c a t i o n o f a p r e v i o u s pregnancy, nor were a c t i v e c o r p o r a l u t e a ever e n c o u n t e r e d .  Thus, fawns a r e e x c l u d e d f r o m the f o l l o w i n g  results.  4. 1  Numbers Of Embryos And F e t u s e s . F e t u s e s were o b t a i n e d f r o m 61 f e m a l e s c o l l e c t e d a t p e r i o d i c  v a l s d u r i n g the g e s t a t i o n a l p e r i o d .  inter-  The m a j o r i t y of samples came f r o m  the 1963-64 r e p r o d u c t i v e season, but a few were t a k e n i n t h e f o l l o w i n g seasons.  two  C o l l e c t i o n s f o r each p e r i o d were made w i t h i n a p e r i o d o f 10 days.  R e s u l t s o f t h e s e c o u n t s i n d i c a t e a h i g h pregnancy r a t e i n a l l does, e x c l u d i n g fawns, and maximum f e t u s p r o d u c t i o n i n f e m a l e s 5.5  t o 8.5  years o l d . A l -  though sample s i z e s were s m a l l f o r each a g e - c l a s s , t h e r e was a g e n e r a l i n c r e a s e i n the pregnancy r a t e w i t h age, up t o 3.5  y e a r s , and an i n c r e a s e  i n the f e t u s p r o d u c t i o n r a t e w i t h age, up t o 5.5 y e a r s (Appendix 4.).  I n samples f r o m a l l y e a r s , t h e pregnancy r a t e was 84.6% 91.7% i n f e m a l e s 2.5  y e a r s o l d , and 97.2%  i n yearlings,  i n females older than 3 y e a r s .  I n samples f r o m 1963-64, the pregnancy r a t e s were s i m i l a r t o the above. O n l y one f e m a l e , o l d e r , t h a n 3 y e a r s , was n o t p r e g n a n t i n a sample o f 33 does (Table 9).  135  Table 9-  The a g e - s p e c i f i c p r e g n a n c y r a t e and f e t a l r a t e o f f e m a l e s i n 1963-64Conception Age ( y r ) 0.5 1.5 2.5 3.5 4.5 5.5-6.5 8.5 & +  Sample Size 8 10 8 6 8 11 8  Per Cent Pregnant 0.0 80.0 87.5 100.0 100.0 90.9 100.0  Fetuses Per Doe 0.00 0.90 1.38 1.67 1.60 1.91 1.75  I n samples f r o m a l l y e a r s , o n l y the f e t a l r a t e i n y e a r l i n g s (0.92  + 0.I4) 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 t h e r a t e s i n t h e o t h e r a g e - c l a s s e s .  However, t h e r e was a s i g n i f i c a n t d i f f e r e n c e (P < 0.05) between t h e mean f e t a l r a t e f o r does 2.5 t o 4.5 y e a r s o f age (I.46 + 0.12, N = 24) and t h e r a t e f o r does 5.5 t o 8.5 y e a r s o l d (1.93 + 0.16, N = 14). I n samples f r o m 1963-64, t h e r e was a g r a d u a l i n c r e a s e i n t h e f e t u s r a t e , w i t h age (Table 12). age-group.  The maximum r a t e was a t t a i n e d i n t h e 5.5 t o 6.5  The o n l y s e t o f t r i p l e t f e t u s e s o c c u r r e d i n a female  6.7  years  old. O n l y t h r e e o f 92 f e t u s e s were o b v i o u s l y d e f e c t i v e .  A mummified  f e t u s , a n d a second l a r g e f e t u s a t t a c h e d t o t h e p l a c e n t a by a t h i c k g e l a t i n o u s u m b i l l i c a l c o r d , o c c u r r e d i n t h e u t e r u s o f a doe 3.6 y e a r s o l d . I t i s d o u b t f u l i f t h e l a t t e r f e t u s would have been born i n a v i a b l e dition.  con-  One member o f a t w i n p a i r , i n a doe o f 9.7 y e a r s , was i n an  advanced s t a g e o f r e s o r p t i o n .  4. 2  Numbers Of Corpora  Lutea.  I n a sample o f 56 f e m a l e s , t h e  number o f c o r p o r a l u t e a l a r g e r  3 t h a n 30 mm  corresponded  e x a c t l y t o t h e number o f f e t u s e s .  One doe o f 3.5  136  y e a r s c o n t a i n e d two l a r g e c o r p o r a l u t e a i n i t s o v a r i e s and o n l y f e t u s i n i t s u t e r u s , but another doe  one  (2.5 y e a r s o l d ) c o n t a i n e d a second 3  f e t u s t h a t was r e p r e s e n t e d by an e x c e p t i o n a l l y s m a l l (12.8 luteum.  mm  ) corpus  The n e x t s m a l l e s t corpus l u t e u m r e p r e s e n t e d by a f e t u s was 49  3 mm  i n volume ( t h e s m a l l e r of two), and the s m a l l e s t s i n g l e corpus luteum was 3 mm  .  73  I n o t h e r does, s m a l l c o r p o r a l u t e a w i t h volumes of 16, 13, 8, 6,and  3 4 mm  were n o t r e p r e s e n t e d by f e t u s e s .  These, and many o t h e r s m a l l e r l u t e a l  b o d i e s , were a c c e s s o r y c o r p o r a l u t e a and were excluded from comparisons between the number of c o r p o r a l u t e a and  the number of f e t u s e s .  Because  two  of the 84 f e t u s e s i n the above sample were d e f i n i t e l y moribund and a t h i r d 3 p r o b a b l y would have d i e d , each corpus luteum l a r g e r than 30 mm r e s e n t e d by O.96  healthy fetuses.  was  rep-  T h i s f i g u r e i s c o n s e r v a t i v e because  many o f the c o l l e c t i o n s were t a k e n e a r l y i n the p e r i o d o f g e s t a t i o n . P r i o r t o the stage when embryos became m a c r o s c o p i c a l l y v i s i b l e (about 25 d a y s ) , I e s t i m a t e d the o v u l a t i o n r a t e from counts of c o r p o r a lutea.  A c c e s s o r y c o r p o r a l u t e a , those l e s s than o n e - q u a r t e r  of the  volume of the l a r g e s t corpus luteum i n the same p a i r of o v a r i e s , were excluded.  Because most f e m a l e s o f Northwest Bay c o n c e i v e d a t second e s t r u s ,  i t was i m p o r t a n t t o determine  i f f i r s t - c y c l e c o r p o r a l u t e a c o u l d be used t o  e s t i m a t e the o v u l a t i o n r a t e a t second e s t r u s .  I n 64 females c o n t a i n i n g  c o r p o r a l u t e a from b o t h c y c l e s , t h e r e were 93 f i r s t - c y c l e c o r p o r a l u t e a and 97 s e c o n d - c y c l e , c o r p o r a l u t e a , an i n s i g n i f i c a n t d i f f e r e n c e (P = X  0.8,  =0.08 w i t h one d f ) . Unequal numbers o f c o r p o r a l u t e a from the  two c y c l e s were most common i n young and o l d does. Thus, the number o f c o r p o r a l u t e a i n females h a v i n g o n l y o v u l a t e d once,was added t o those h a v i n g o v u l a t e d t w i c e , t o i n c r e a s e the sample s i z e and p l a c e more c o n f i d e n c e i n the mean o v u l a t i o n r a t e s f o r each age (Table  10).  class  137  Table 10.  The a g e - s p e c i f i c o v u l a t i o n r a t e , based on c o r p o r a l u t e a , o f a l l f e m a l e s c o l l e c t e d f r o m 1963 t o 1967.  Age (years) 1.5 2.5 3.5 -4.5 5-5 6.5 7.5-10.5 11.5 & +  Sample Size 41 35 13 12 11 11 13 10  Corpora Lutea per doe 1.12  SE 0.05 0.10 0.15 0.15 0.16 0.12 0.10 0.18  1.51 1.54. 1.50 1.91 1.82 1.85  1.90  I n a l l t h e samples f r o m 1963 t o 1967, t h e o v u l a t i o n r a t e was  1.12 per y e a r -  l i n g doe, about 1.52 per doe 2.5 t o 4-.5 y e a r s o l d and 1.82 t o 1.91 per doe older than 5 years. The o v u l a t i o n r a t e d i d n o t d e c l i n e i n f e m a l e s over 11.5 y e a r s o f age.  Because o f t h e s m a l l sample s i z e s o n l y t h e mean r a t e f o r y e a r l i n g s  differed  s i g n i f i c a n t l y from t h e means f o r o t h e r f e m a l e s .  The means f o r  samples f r o m each o f t h e b r e e d i n g seasons i s i n Appendix 6. sample s i z e s were i n c r e a s e d  by g r o u p i n g a g e - c l a s s e s ,  When t h e  t h e e f f e c t o f age  on t h e o v u l a t i o n r a t e was more c l e a r l y d e m o n s t r a t e d ( T a b l e 1 1 ) .  T a b l e 1 1 . The o v u l a t i o n r a t e o f a g e - c l a s s e s o f f e m a l e s i n combined samples f r o m 1963 t p 1967.  N X  s SX S55«t.05 "  1.5 41 1.12 0.33 0.05 0.10  2.5-4.5 60 1.52 0.57 0.07 0.14  Age o f doe ( y r ) 5.5-6.5 22 1.86 0.47 0.10 0.21  7.5-10.5 13 1.85 0.38 0.10 0.22  * 95% c o n f i d e n c e l i m i t s o f t h e means  11.5&+ 10 1.90 0.57 0.18 0.41  5.5&t45 1.87 0.46 0.07 0.14  138  I t i s o b v i o u s i n the above Table t h a t the o v u l a t i o n r a t e of yearling"females differed group 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 o l d e r does; t h a t the 2.5  s i g n i f i c a n t l y f r o m o t h e r groups;  and  to  4.5  t h a t groups o f does  o l d e r t h a n 5 y e a r s d i f f e r e d i n s i g n i f i c a n t l y f r o m one a n o t h e r .  The  above  c o u n t s r e p r e s e n t the o v u l a t i o n r a t e of those h a v i n g o v u l a t e d and does not n e c e s s a r i l y r e p r e s e n t the o v u l a t i o n r a t e o f a l l females i n each age For example, about 20%  4. 3  class.  of the y e a r l i n g s d i d n o t o v u l a t e .  Number Of S c a r s D e r i v e d From Corpora L u t e a Of Pregnancy. A measure of p a s t r e p r o d u c t i v e performance was  o b t a i n e d by  count-  i n g s c a r s 5 months o l d t h a t r e s u l t e d f r o m c o r p o r a l u t e a of pregnancy. U n t i l t h e y were about 8 months o l d , s c a r s f r o m the p r e c e e d i n g r e p r o d u c t i v e season were r e a d i l y d i s t i n g u i s h a b l e f r o m o l d e r s c a r s but t h e r e a f t e r , s e p a r a t i o n was  more d i f f i c u l t .  Because the m a j o r i t y of females  obtained f o r  t h i s s t u d y were c o l l e c t e d 5 t o 6 months a f t e r p a r t u r i t i o n , the c o u n t s  of s c a r s  a r e b e l i e v e d t o be a c c u r a t e . S c a r s r e s u l t i n g f r o m c o r p o r a l u t e a o f non-pregnancy, a c c e s s o r y c o r p o r a l u t e a , and a b e r r a n t c o r p o r a l u t e a were e x l u d e d . atypical scars, containing l i t t l e  The  last-mentioned  or no pigment and few blood v e s s e l s , were  b e l i e v e d t o be a s s o c i a t e d w i t h f e t a l m o r t a l i t y .  They were q u i t e r a r e .  A l l r e c e n t s c a r s l e s s than o n e - h a l f t h e s i z e o f the l a r g e s t s c a r , i n the same p a i r o f o v a r i e s , were e x c l u d e d  because p r o b a b l y t h e y developed  from  3 accessory corpora l u t e a .  A l l t h o s e e x c l u d e d were l e s s t h a n 1.0  I n combined samples f r o m t h e b r e e d i n g seasons o f 1963 average number o f s c a r s 5 months o l d I n c r e a s e d years 2.00  (Table 12).  i n volume.  t o 1966,  s t e a d i l y w i t h age up t o  (age a t t h e p r e v i o u s b r e e d i n g s e a s o n ) , and s c a r s per female  mm  the 4-5  t h e r e a f t e r remained near  I n combined samples f r o m a l l y e a r s , t h e r e  were s i g n i f i c a n t d i f f e r e n c e s (P < 0.05)  between the means of a g e - c l a s s e s  up  Table 12.  The a g e - s p e c i f i c average number o f s c a r s o f 5 months d e r i v e d f r o m c o r p o r a l u t e a o f pregnancy • i n t h e r e p r o d u c t i v e seasons f r o m 1962 t o 1966.(Sample s i z e s a r e i n p a r e n t h e s e s and s t a n d a r d e r r o r s a r e p r e s e n t e d where a p p l i c a b l e ) . The Average Number o f S c a r s p e r Female i n t h e Season I n d i c a t e d Age* 1.5 2.5 3  -5  4.5 5.5 6.5 7.5-10.5 11.5-18.5  *  1962-63 1.09+0.10 (33) 1.46+0.14 (13) 1.86+0.14 (7) . 1.94+0.06 (16) 2.00+0.00 (10) 2.00 (2) 2.00 (6) 1.60 (5)  1963-64 '  •  0.94 (17) 1.43 (7) 1.73 (11) 2.00 (1) 1.40 (5) 2.00 (2) 1.75 (4)  1964-65 0.74 (17) 0.86 (7) 1.40 (5) 2.00 (2) 1.50 (2) 2.00 (2) 1.60 (5) 1.50 (2)  :  1965-66 0.33 (3) 1.20 (5) 1.50 (2) 1.50 (2) 2.00 (5) 2.00 (3) 2.00 (1)  The age o f t h e females a t t h e time o f t h e c o n c e p t i o n s scars.  1962  t o 1966  1  0.97+0.06 (70) 1.28+0.09 (32) 1.68+0.10 (25) 1.90+0.07 (21) 1.82+0.08 (22) 2.00+0.00 (6) ' 1.89+0.08 (18) 1.63+0.18 (8) that eventually r e s u l t e d i n the  UJ  UO  to L\,5  years. Over the 4-year period there was an o v e r a l l general d e c l i n e i n  the average number of scars per doe i n each age c l a s s up to 4.5 years. d e c l i n e i n scar production was most evident i n y e a r l i n g s . the means between years w i t h i n an age-class were not  The  Differences i n  statistically  s i g n i f i c a n t , except between 1962 and 1964 i n the 1.5 age-group.  However,  the sample s i z e s f o r i n d i v i d u a l age-classes and i n d i v i d u a l years were generally  inadequate. The long-term reproductive performance of the population was  revealed by the r e l a t i o n s h i p between the age of the doe and the average number of corpora l u t e a of pregnancy scars per doe, because the scars p e r s i s t e d f o r the l i f e of the doe.  This r e l a t i o n s h i p i s represented  by  the equation y = 1.885x - 2.326 where y i s the mean number of scars per doe and x i s the age of the female  4. 4  at the previous breeding season.  The P r o d u c t i v i t y Of The Population. The p r o d u c t i v i t y or b i r t h r a t e of the population was estimated  by  r e l a t i n g a g e - s p e c i f i c r a t e s of reproduction to the age s t r u c t u r e of the population.  In t h i s paper, p r o d u c t i v i t y i s defined as the average number  of young (at b i r t h ) produced by each female of reproductive age.  As defined  above, p r o d u c t i v i t y i s equivalent to the "General F e r t i l i t y Rate" used by demographers to measure human reproductive r a t e s , except that the base i s 100 females instead of 1000. The age s t r u c t u r e of the population was estimated from the age s t r u c t u r e of deer k i l l e d by hunters  (Fig. 24).  determined by the tooth s e c t i o n method.  The ages of these deer were  I t was necessary to pool samples  from s e v e r a l years to obtain an adequate sample.  Young females,  especially  F i g . 24. The age s t r u c t u r e of females (fawns excluded) taken from the Northwest Bay population between 1960 and 1966.  40  1. 1963-66 Hunter n = 336  30  EH  Sample  20  o  o  10  cy  n n  n  EH  20  2. 1963-65 Personal 78 n  Sample  10  1.5  2.5 3.5  4.5  5.56.5  n  7.5 8.5 9.510.5  AGE-CLASS (YEARS)  _a  12.5  C L  U.5  16.5 19.5  U2  y e a r l i n g s , are probably over-represented t h e y are l e s s wary than o l d e r does.  i n the h u n t e r  T h i s c o n t e n t i o n i s supported  by the age d i s t r i b u t i o n of f e m a l e s k i l l e d 24)•  sample, because  s p e c i f i c a l l y f o r t h i s study ( F i g .  Most o f t h e s e were t a k e n a t n i g h t and  o u t s i d e o f the h u n t i n g  when f e m a l e s were l e s s wary. N e v e r t h e l e s s , t h i s d i s t r i b u t i o n may towards o l d e r , more p r o d u c t i v e , f e m a l e s was  i d e n t i f i e d by the presence  season,  be b i a s e d  because i n many i n s t a n c e s a  doe  of a fawn.  An e s t i m a t e of the mean number of v i a b l e fawns produced by each a g e - c l a s s was  o b t a i n e d by computing the weighted  average r a t e s of f e t u s  p r o d u c t i o n , ovulation,and scar p r o d u c t i o n (Table 13). amalgamation, each of the r a t e s was reduced implantation  fetal mortality.  The  observed  P r i o r to t h e i r  by 5% t o account f o r p o s t f e t a l m o r t a l i t y of 3.3%  was  i n c r e a s e d t o 5% because some f e t u s e s were c o l l e c t e d e a r l y i n g e s t a t i o n . The r e l a t i v e p r o d u c t i v i t y of each a g e - c l a s s i s the p r o d u c t o f t h e per c e n t f r e q u e n c y o f the a g e - c l a s s i n t h e p o p u l a t i o n and  the mean number  o f l i v e fawns born t o each female i n the a g e - c l a s s (Table 14-) .  The sums  of the r e l a t i v e p r o d u c t i v i t i e s , 136.7, i s the number o f v i a b l e f e t u s e s produced by 100 f e m a l e s  of b r e e d i n g age.  The r e l a t i v e c o n t r i b u t i o n o f each  a g e - c l a s s t o the t o t a l a n n u a l p r o d u c t i o n i s more r e a d i l y comprehended i f the c o n t r i b u t i o n of each a g e - c l a s s i s expressed p r o d u c t i o n ( T a b l e 14-).  I t i s apparent  young t o the p o p u l a t i o n and decreases 4-.5  and  of t o t a l  t h a t y e a r l i n g s c o n t r i b u t e the most  t h a t the c o n t r i b u t i o n o f i n d i v i d u a l  w i t h age t h e r e a f t e r .  5.5  as a p e r c e n t a g e  age-classes  A l t h o u g h i t i s n o t e v i d e n t i n the T a b l e ,  a g e - c l a s s e s c o n t r i b u t e d about e q u a l numbers o f fawns.  the  143  Table 13 .  The e s t i m a t e d mean number o f l i v e fawns born t o each female i n s p e c i f i c a g e - c l a s s e s based on a d j u s t e d counts o f f e t u s e s , c o r p o r a l u t e a , and s c a r s , ( S a m p l e size i nparentheses).  Age Class (yr)  Adjusted Fetal Rate  1.5  0.87 (13)  2.5 3.5 4.5 5.5-6.5  1.59 ( 6) 1.59 ( 9) 1.82 (12)  1  1.26 (12)  AdjustedOvulation Rate 2  0.90 (41) 1.31 (35) 1.42 (13) 1.39 (12) 1.86 (22)  Adjusted Scar Rate  Weighted Average Rate 4  0.92 1.22 1.60 1.80 1.77  0.91 1.27 1.55 1.64 1.81  3  (70) (32) (25) (21) (28)  (124) ( 79) ( 44) ( 42) ( 42)  1.  The t o t a l number o f f e t u s e s ( b o t h v i a b l e and moribund) d i v i d e d by t h e t o t a l number o f f e m a l e s , and t h e r e s u l t reduced 5% t o account f o r f e t a l m o r t a l i t y .  2.  C a l c u l a t e d from t h e o v u l a t i o n r a t e ( o f those o v u l a t i n g i n t h e f a l l ) t i m e s t h e p e r c e n t pregnant (84.6% o f y e a r l i n g s , 91.7% o f 2.5 y e a r s , and 9 7 . 2 % o f 3.5 y e a r s and o l d e r f e m a l e s , (App e n d i x 4) t i m e s a 5% r e d u c t i o n t o a c c o u n t f o r f e t a l m o r t a l i t y .  3.  The p r e g n a n c y - s c a r r a t e ( s c a r s 5 months o l d ) r e d u c e d by 5% t o account f o r f e t a l m o r t a l i t y .  .4.  Weighted a c c o r d i n g t o t h e sample s i z e .  Table 1 4 .  Age Class (yr)  1.5 2.5 3.5 4.5 5.5-6.5 7.5 & + Totals  The t o t a l and r e l a t i v e p r o d u c t i v i t y o f each a g e - c l a s s a t Northwest Bay.  Per c e n t F r e q o f t h e Age-Class i n the P o p u l a t i o n  30 21  15 9 14 11  100  . L i v e Fawns Born p e r Doe  0.91  1.27 1.55 1.64 1.81 1.76  "Productivity per Age-Class p e r 100 Does  27.3 26.7 23.2 14.8 25.3 19.4 136.7  Relative Productivity p e r A g e - C l a s s (%)  20.0 19.5 17.0 10.8 18.5 14.2 100.0  :  144-  SECTION 5-  THE EFFECT OF SIZE ON FERTILITY. I hypothesized that w i t h i n an age-class, l a r g e r does would have  a higher reproductive r a t e than smaller ones.  The s i z e of females was  evaluated from measurements of the hind f o o t , c h e s t . g i r t h , and weight.  dressed  In samples from the breeding seasons of a l l years, 1963 to  1967,  the hind-foot length increased with age up to 2.5 years, and t h e r e a f t e r r e mained almost constant ( F i g . 25).  Both chest g i r t h and dressed weight i n -  creased with age to 5-5 years, although s i g n i f i c a n t d i f f e r e n c e s occurred only to age 2.5  years.  The r e l a t i o n s h i p between s i z e of doe and o v u l a t i o n r a t e , i n 1963, was explored.  In these t e s t s , dressed weight, g i r t h , and length of hind f o o t ,  were used to separate each age-class into' two groups of equal s i z e .  I f , for  any doe, at l e a s t two of the three measurements were greater than the medians for  the a t t r i b u t e s , then i t was placed i n the " l a r g e " category.  ovulated twice and the number of f i r s t - c y c l e corpora  I f a doe  had  l u t e a d i f f e r e d from  the number of second-cycle corpora l u t e a ( t h i s r a r e l y occurred), then the average of the two was used. A l l eight y e a r l i n g s that ovulated contained j u s t one current corpus luteum, however, s i x of the eight does were l a r g e r than the average of a l l y e a r l i n g s c o l l e c t e d during the 1963 breeding season.  A greater percent-  age of the smaller y e a r l i n g s had not ovulated by the termination of the hunting season on December 1..  The f e r t i l i t y of y e a r l i n g s weighing l e s s than  55 pounds ( f i e l d - d r e s s e d weight) i n November was low; those weighing l e s s than 50 pounds seldom produced corpora l u t e a .  In c o n t r a s t , some y e a r l i n g s with  dressed weights over 70 pounds produced two corpora l u t e a . corpus luteum developed,  I f only one  there was u s u a l l y a second l a r g e f o l l i c l e  present,  i n d i c a t i n g that the y e a r l i n g was n e a r l y capable of o v u l a t i n g two ova.  Of  U5  5.  S t a t i s t i c s f o r h i n d - f o o t l e n g t h , c h e s t g i r t h and d r e s s e d w e i g h t o f e i g h t a g e - c l a s s e s o f females • c o l l e c t e d d u r i n g November and early-December, 1963-66. (Mean, 95% c o n f i d e n c e l i m i t s , s t a n d a r d d e v i a t i o n , r a n g e , and sample s i z e ) .  AO  CK5  1.5  2*. 5  3T5 AGE  Z?5  5?5  6.5-7.5  8.5&f  U6  the two y e a r l i n g s weighing i n excess of 80 pounds ( f i e l d dressed), one had ovulated two ova and the other one contained two large f o l l i c l e s near rupture. Thus, the growth r a t e of y e a r l i n g s had an important bearing on t h e i r f e r t i l i ty. Twelve females, 2.5 years old, were divided i n t o equal-sized large and small groups. •The mean o v u l a t i o n r a t e s f o r the r e s p e c t i v e large and small groups were 2.08 + 0.20 and 1.58 + 0.33 (NS). An i n v e s t i g a t i o n of the r e l a t i o n s h i p between the s i z e of females 2.5 years old and the number of scars derived from corpora l u t e a of pregnancy of the previous reproductive season, revealed that large does had an i n s i g n i f i c a n t l y greater mean number of scars than small does.  Within samples from  each year, does were divided i n t o two almost numerically-equal groups on the basis of the three s i z e a t t r i b u t e s .  This procedure removed the p o s s i b i l i t y  of bias created by changes i n weight and f e r t i l i t y between years.  I n the  aggregated r e s u l t s f o r a l l years,the mean f o r 25 l a r g e females was 1.00 + 0.08. scars as compared to 0.85 + 0.10 scars f o r 27 small does (NS). In older does, d i f f e r e n c e s between l a r g e and small females were g e n e r a l l y not s t a t i s t i c a l l y s i g n i f i c a n t because of small sample s i z e s , nevertheless, some trends were apparent.  For example, i n 1963, s i x l a r g e  does 3.5 years old averaged 1.67 scars from the previous reproductive season, whereas seven small does averaged 1.29 scars.  In the above sample, the four  l a r g e s t averaged 2.00 scars per doe, whereas the four smaller ones averaged 1.25 scars.  S i m l l a r l l y , i n 1964,five l a r g e females of 4.5 years averaged  2.00 scars from the previous reproductive season, whereas four small does of the same age averaged 1.25 scars. Over a period of years, changes i n weight may r e f l e c t changes i n fertility.  From 1964 to 1966 there was a s i g n i f i c a n t o v e r a l l decrease i n  the weight of young (to 3.5 years), male deer at Northwest Bay (Smith, 1968).  147  He found a s i m i l a r , b u t i n s i g n i f i c a n t , d e c l i n e i n t h e - w e i g h t o f female fawns and y e a r l i n g s .  D u r i n g t h e p e r i o d 1962 t o 1966, t h e r e was a d e c r e a s e i n  f e r t i l i t y , w h i c h was most e v i d e n t i n young f e m a l e s .  For i n s t a n c e , from  1963-64 t o 1966-67 t h e mean number o f pregnancy s c a r s , i n f e m a l e s 2.5 y e a r s old,  d e c r e a s e d as f o l l o w s :  1.09, 0.94, 0.74 and 0.33.  These s c a r s r e p r e -  sented c o r p o r a l u t e a o f pregnancy i n y e a r l i n g s i n t h e p r e v i o u s r e p r o d u c t i v e season.  S i m i l a r , b u t l e s s d r a m a t i c r e d u c t i o n s a l s o o c c u r r e d i n t h e n e x t two  age-classes.  I n f e m a l e s 4-5 y e a r s o f age and o l d e r , t h e mean number o f s c a r s  changed as f o l l o w s :  1.92, 1.83, 1.69, and 1.91.  The above r e s u l t s  indicate  t h a t , w i t h i n young a g e - c l a s s e s o f f e m a l e d e e r , f e r t i l i t y i s d i r e c t l y r e l a t e d to  s i z e o r growth r a t e .  148  7.  DISCUSSION AND CONCLUSIONS.  7. 1  General Comments. I n t h i s study, the d e s c r i p t i v e phase was an e s s e n t i a l prelude to  l a t e r sections i n which the p a t t e r n and l e v e l of reproduction were examined. I t was necessary to disentangle the complex events occurring at d i f f e r e n t phases of the sexual c y c l e before the ovaries could y i e l d r e l i a b l e information. Many of the previous studies of ovarian f u n c t i o n have been based on inadequate knowledge about the basic microanatomy of the dynamic ovary. Previous h i s t o l o g i c a l studies of the deer ovary (Cheatum, 194-9; Golley, 1954; Gibson, 1957;  Trauger and Haugen, 1965)  a l s o s u f f e r because of inadequate  sample s i z e s , undated s t r u c t u r e s , and samples from deer of unknown age.  I  circumvented these problems, to some extent, by obtaining s e r i a l sections from a large, number of ovaries; by obtaining ova from the reproductive t r a c t ; by d e f i n i n g , w i t h i n narrow l i m i t s , the period of ovulations and and by determining  conceptions;  the age of the females from tooth sections.  In previous i n v e s t i g a t i o n s of the deer ovary, only a few dozen p a i r s of ovaries were sectioned i n t h e i r e n t i r e t y .  The r e s u l t s of t h i s study are  based on s e r i a l , h i s t o l o g i c a l sections of 4-44 p a i r s of ovaries, and both gross and t h i n  sections of 33 p a i r s . Descriptions of s t r u c t u r e s are v i r t u a l l y useless unless they can be  r e l a t e d temporally to v i t a l reproductive events, such as o v u l a t i o n or parturition.  The chronology of events i n the ovaries, i n c l u d i n g the quanti-  t a t i v e changes i n the s i z e of s t r u c t u r e s , was ascertained from cleavage stages of ova obtained from the reproductive t r a c t . —  The f i r s t attempt to obtain ova  by f l u s h i n g them out of the oviduct a t the f i e l d s t a t i o n —  of inexperience and poor working c o n d i t i o n s .  f a i l e d because  Next, the oviducts were sectioned  (a l a b o r i o u s technique), but ova were found i n only a small proportion of them.  In the f i n a l year, I discovered that ova could be flushed from cooled  149  oviducts and u t e r i i n the l a b o r a t o r y , up to 2 days a f t e r they were c o l l e c t e d at the f i e l d s t a t i o n . The ova provided the best information a v a i l a b l e on the stage of the cycle i n the n a t u r a l population, although there are undefined inherent i n the method (discussed l a t e r ) .  sources of error  These ova a l s o provided valuable  information on the frequency of f e r t i l i z a t i o n a t the two o v u l a t i o n s , and concrete evidence f o r ' s i l e n t heats' i n deer. Emphasis was placed on determining the mean dates of f i r s t and second o v u l a t i o n , because they defined the length of the f i r s t estrous c y c l e and provided a basis f o r f e t a l growth curves.  I was able to a s c e r t a i n , w i t h i n  narrow l i m i t s , the period of f i r s t and second o v u l a t i o n s , p r i m a r i l y because l a r g e numbers of ovaries were obtained from throughout the breeding season, and because I was able to estimate dates of o v u l a t i o n u s i n g c r i t e r i a e s t a b l i s h ed from s t r u c t u r e s dated by means of ova.  The timing of conceptions c o r r e s -  ponded c l o s e l y to that of second o v u l a t i o n s , because over 95%> of the females conceived a t second o v u l a t i o n . D e f i n i t i o n of the period of conceptions permitted a s t a r t i n g point f o r curves d e p i c t i n g the growth of fetuses. exclude fetuses conceived  Fortunately, I was able to  on cycles subsequent t o the-second.  The ovaries  of females h o s t i n g these fetuses contained more than one generation of scars from corpora l u t e a of non-pregnancy.  The f e t a l growth curves, the f i r s t f o r  the species and f o r a n a t u r a l population of deer, were used t o estimate the conception dates of two l a t e breeders — before becoming pregnant.  females that had cycled s e v e r a l times  The curves, i n conjunction with counts of scars from  corpora l u t e a of non-pregnancy, provided a means of estimating the length of estrous cycles subsequent t o the second. P e r i o d i c sampling of the population during the g e s t a t i o n period provided i n f o r m a t i o n on the r e l a t i o n s h i p between corpora l u t e a and f e t u s e s , and  150  on the amount of p r e n a t a l m o r t a l i t y .  Estimates of both are required before  corpora l u t e a and t h e i r scars can be used to estimate f e t u s and fawn production. Fortunately, accessory corpora l u t e a can be excluded from primary ones by s i z e 3 alone.  Remarkably, i n pregnant females, the number of corpora l u t e a over 30  mm  equalled the number of f e t u s e s . Included i n the sample,was a female with two fetuses and only one 3 corpus luteum l a r g e r than 30 mm. The second corpus luteum occupied only 3 16 mm , nevertheless, i t was c l a s s i f i e d as a primary corpus luteum because i t s volume was j u s t over 25% of the volume of the l a r g e r one. corpus luteum was represented by 0.99  Thus, each primary  fetuses (85:84-).  Ovarian a n a l y s i s , i n conjunction w i t h counts of ova, embryos, and f e t u s e s , i s the only means of determining the r a t e and timing.of p r e n a t a l mortality.  The observed percentage of moribund f e t u s e s , 3.4%,  was  arbitrarily  increased to 5%,because c o l l e c t i o n s were made throughout g e s t a t i o n . The ovary may be l i k e n e d to a kymograph, which records past reproductive events.  Thus, the number of corpora l u t e a of non-pregnancy and preg-  nancy are f a i t h f u l l y recorded, the l a t t e r f o r the l i f e of the female.  The  number of c y c l e s experienced by a doe p r i o r to conception or reproductive f a i l ure was estimated from the number of scars derived from corpora l u t e a of nonpregnancy.  U s u a l l y , only scars from the previous reproductive season are  present, because of t h e i r r a p i d degeneration.  In c o n t r a s t , scars derived from  corpora l u t e a of pregnancy record the past reproductive h i s t o r y of the female. However, only scars from the immediate-past breeding season can be d i s t i n guished with c e r t a i n t y from older scars. I t soon became evident that accurate measures of f u t u r e , present,and past p r o d u c t i v i t y could be obtained by microscopic examination of o v a r i e s . Along with means of determining age of the females, the method i s a u s e f u l t o o l i n s t u d i e s of comparative p r o d u c t i v i t y .  One advantage i s that  151  reproductive performance can be examined i n r e t r o s p e c t — a f t e r some change i n the population or the environment has occurred.  In a d d i t i o n , the long term  reproductive output of i n d i v i d u a l s and the population can be compared quant i t a t i v e l y to a t h e o r e t i c a l or e c o l o g i c a l standard.  Perhaps, information ob-  tained from ovarian a n a l y s i s i s one of the best means of evaluating the s u i t a b i l i t y of a p a r t i c u l a r environment f o r a population. Gross a n a l y s i s of o v a r i e s , although much quicker than microscopic a n a l y s i s , provides considerably l e s s information. year may  P r o d u c t i v i t y of the current  be obtained from counts of corpora l u t e a but only a crude estimate of  p r o d u c t i v i t y i n the immediate past season i s obtained from counts of pigmented scars.  The l a t t e r u s u a l l y exceed counts of corpora l u t e a of pregnancy by 10  to 18% (Golley, 1957;  Brown, 1961; Teer et a l . , 1965).  In some ovaries, scars  from more than one season are present; i n others the most recent scars are indistinct.  In a d d i t i o n , the r e g r e s s i n g corpora l u t e a of non-pregnancy are  s i m i l a r , f o r a b r i e f period, to scars of pregnancy.  Both s t r u c t u r e s produce  pigment and some i n d i c a t i o n of the age of the scars can be obtained by noting their color. L i t t l e information on the extent of repeat breeding can be obtained by gross a n a l y s i s .  A f t e r reviewing the l i t e r a t u r e , Teer et a l . (1965) con-  cluded that corpora l u t e a of o v u l a t i o n , accessory corpora l u t e a , and l u t e i n i z e d s t r u c t u r e s were r e l a t i v e l y uncommon i n most c e r v i d s . i n v e s t i g a t i o n s may  other  More d e t a i l e d  show, as I have, that these s t r u c t u r e s are common.  In the f o l l o w i n g pages, c e r t a i n aspects of t h i s study are discussed more f u l l y and reproductive functions i n deer are compared with those of other species.  152  7. 2  Development Of The F e t a l Ovary. To my  The  knowledge t h i s i s the f i r s t s t u d y of f e t a l deer o v a r i e s .  sequence of development c o r r e s p o n d s c l o s e l y t o t h a t of man,  (Macaca m u l a t t a ) (van Wagenen and (van Tienhoven, 1968).  Simpson, 1965), and  the d o m e s t i c  cow  I n d e e r , f o l l i c u l a r development b e g i n s e a r l i e r  proceeds more s l o w l y t o the t e r t i a r y f o l l i c l e primates.  the monkey  but  s t a g e t h a n i t does i n the  T e r t i a r y f o l l i c l e s do not exceed 0.25  mm  deer o v a r i e s but t h e y a t t a i n d i a m e t e r s of 1 t o 4 mm  i n diameter in' p e r i n a t a l i n man.  I n d e e r , as i n  o t h e r s p e c i e s , most t e r t i a r y f o l l i c l e s i n p e r i n a t a l o v a r i e s are  atretic.  Development of the c o r t e x and m e d u l l a i n deer o v a r i e s f o l l o w s the p a t t e r n s d e s c r i b e d  f o r man  and  i n v e s t m e n t appears e a r l i e r and  i s more e x t e n s i v e i n d e e r .  a r e p a r t i t i o n e d by a c o n n e c t i v e albuginea  the monkey,except t h a t c o n n e c t i v e  F e t a l ovaries  t i s s u e network a t 64 days and  I s w e l l d e v e l o p e d by 170 days.  tissue  the t u n i c a  In contrast, connective  tissue  p r o l i f e r a t i o n i n the human o v a r y i s n o t r a p i d u n t i l a f t e r 180 days,and t u n i c a albuginea i s not w e l l developed u n t i l b i r t h 1965).  (van Wagenen and  I n f e t a l deer o v a r i e s , development of the f i b r o u s t u n i c a  the  Simpson,  albuginea  v i r t u a l l y suspends i n v a g i n a t i o n s of the s u r f a c e e p i t h e l i u m one month p r i o r to b i r t h .  Oogenesis e s s e n t i a l l y ceases a t about the same t i m e .  p a t t e r n i s s i m i l a r t o t h a t of man, continues  This  but i n the macaque monkey,ovogenesis  i n t o the f i r s t few p o s t - p a r t u m months.  I n d e e r , as i n the monkey, t h e r e are numerous m u l t i n u c l e a t e and m u l t i o v u l a r sex c e l l s . 123 days a f t e r  They are most.abundant i n d e e r o v a r i e s 98  to  conception.  I t i s c o n c l u d e d t h a t the g e r m i n a l t h a t d e v e l o p i n t o sex c e l l s .  However, one  epithelium contributes  cannot make f i n a l  cells  conclusions  .153  about d e t a i l e d embryological processes from h i s t o l o g i c a l study alone, as warned by Franchi et a l .  (1962).  The f i n d i n g that the ovaries of a f e t u s from a captive doe were l a r g e r , f u r t h e r developed, and contained greater numbers of sex c e l l s than f e t a l ovaries of equal age i n w i l d does, i s worth f u r t h e r i n v e s t i g a t i o n . There i s l i t t l e doubt that the penned doe was better nourished than i t s w i l d counterparts.  The f e t a l ovary, which undergoes progressive developmental  changes, may be a more s e n s i t i v e i n d i c a t o r of the maternal environment, and u l t i m a t e l y the p h y s i c a l environment of the female host, than the fetus as a whole.  Presumably, the ovary i s a low p r i o r i t y organ i n the d i s t r i -  bution of. energy f o r development. Study of the f e t a l ovary not only f a c i l i t a t e s comprehension of the h i s t o l o g y and f u n c t i o n of the p o s t - n a t a l ovary, but i n some instances, provides information concerning endocrine changes i n the female host. the  For example,  presence of an important F.. S . H . - l i k e compound i n the blood of the preg-  nant mare was deduced from examination of f e t a l ovaries.  No unusual endo-  c r i n e a c t i v i t y was indicated by the f e t a l deer ovary.  7. 3  Estimating The Age Of Ovarian Structures From Ova And F e t a l Growth Curves. D e s c r i p t i o n s of s t r u c t u r e s are of l i t t l e value unless they can be  r e l a t e d to a reference p o i n t , such as o v u l a t i o n or p a r t u r i t i o n .  For t h i s  reason, considerable e f f o r t was d i r e c t e d i n t o means of estimating the ages of corpora l u t e a and f o l l i c l e s .  The ages of ovarian s t r u c t u r e s were  estimated from standard growth curves based on the volumes and h i s t o l o g i c a l appearance of corpora l u t e a and f o l l i c l e s i n females from which ova were recovered.  I assumed that the r a t e of cleavage of deer ova i s equivalent  to about the average of the r a t e s i n the cow, sheep, and goat (Appendix 1 ) .  154 Comparable stages of cleavage i n r e l a t i o n to time and Enders,  occur i n the r a t (Schlafke  (1967).  L i t t l e i s known about the v a r i a b i l i t y i n cleavage r a t e s because i t i s d i f f i c u l t to determine the moment of o v u l a t i o n .  Austin (1957) observed  considerable v a r i a t i o n a f t e r the 1 - c e l l stage i n the f i e l d vole (Mlcrotus agrestis).  However, more v a r i a t i o n i s expected i n a l i t t e r - b e a r i n g species  because of maturation d i f f e r e n c e s among ova and asynchronous rupture of follicles.  The v a r i a b i l i t y between the species l i s t e d i n Appendix 1 i s not  l a r g e considering the source of v a r i a b i l i t y between o v u l a t i o n and the point i n overt estrus to which the stages were timed. Ova containing as many as 16 c e l l s occurred i n the oviducts of deer.  I n most domestic species of ungulates,the  ova enter the uterus  3 to 4 days a f t e r o v u l a t i o n a t about the 8-celled stage (Hartmann, 1962).. More recent studies i n d i c a t e that some ova containing only 3 or 4 c e l l s enter the uterus as e a r l y as 2 days a f t e r o v u l a t i o n (Austin, 1957; and Day,  1966).  Oxenreider  Three ova c o n t a i n i n g more than 32 c e l l s were recovered  from  the uterus, but e a r l i e r stages may have been present because few u t e r i were flushed f o r ova. The ages of ovarian s t r u c t u r e s , a f t e r about the f i r s t month of pregnancy, can be estimated from f e t a l growth curves.  Previous to t h i s  study there were no f e t a l growth curves f o r b l a c k - t a i l e d deer.  Most i n -  v e s t i g a t o r s r e l i e d on the f e t a l growth curve f o r w h i t e - t a i l e d deer, developed by Cheatum and Morton (1946), to estimate the ages of fetuses.  I was  able  to construct f e t a l curves with the aid of information obtained from the ovaries.  I t was simply necessary to define the period and mean date of  second ovulations and then use only fetuses derived from the second o v u l a t i o n i n formulation of the curves,i.e. females with more than one generation of corpora l u t e a of non-pregnancy were excluded.  Sample s i z e s were l a r g e ,  155 consequently,the mean date of second ovulations and the mean s i z e s of fetuses from second o v u l a t i o n are r e l i a b l e s t a t i s t i c s .  The accuracy of  the curves i s confirmed somewhat, by the close f i t of the two sets of known-age f e t u s e s . The curve based on hind-foot length i s preferred to that f o r g i r t h or weight because the hind f o o t i s the l e a s t v a r i a b l e of the three measurements (weight i s the most v a r i a b l e ) . F e t a l growth curves are not r e l i a b l e i n d i c a t o r s of f e t a l age i n the l a t t e r stages of g e s t a t i o n because of d i f f e r e n c e s i n growth r a t e , d i f f e r e n c e s between singletons and twins, and d i f f e r e n c e s between sexes (Ommundsen,  1967).  For example, there was a s i g n i f i c a n t d i f f e r e n c e i n  weight between male (heavier) and female fetuses i n the sample taken j u s t p r i o r to p a r t u r i t i o n i n June.  7. 4  The Weight Of Ovaries And Ovarian Function. The weight of deer ovaries varied with age of female and with  the season.  In samples from the breeding season, there was a general  increase i n ovarian weight, w i t h age, up to 7.5 ovaries of old females were s l i g h t l y l i g h t e r .  to 9-5  years.  The  Excluding fawns, the  curve d e p i c t i n g a g e - s p e c i f i c changes i n ovarian weight c l o s e l y p a r a l l e l s the corresponding curve f o r the b i r t h r a t e .  Perhaps, a good estimate of  the r e l a t i v e f e r t i l i t y of age classes of deer can be obtained from ovarian weight alone. The sharp increase i n ovarian weight i n pregnant females i n May and June i s caused l a r g e l y by Increased f o l l i c u l a r a c t i v i t y , and to a l e s s e r extent by enlargement of corpora l u t e a .  A decrease i n ovarian  weight during the winter i s caused by reduced f o l l i c u l a r a c t i v i t y . There was an i n s i g n i f i c a n t d i f f e r e n c e between the weights of  156  the l e f t and r i g h t o v a r i e s , w h i c h i s i n k e e p i n g w i t h the t h a t the two  o v a r i e s f u n c t i o n randomly.  I n some s p e c i e s , one  c o n t r i b u t e s s i g n i f i c a n t l y more ova t h a n the other  7. 5  The  S t r u c t u r e And  Function  Of  observation ovary  ( A s d e l l , 1964).  Follicles.  P r i o r t o t h i s s t u d y , v i r t u a l l y n o t h i n g was f o l l i c u l a r c y c l e i n any w i l d u n g u l a t e .  known about the  In deer, a well-defined  folli-  c u l a r c y c l e o c c u r s i n w h i c h f o l l i c l e s d e v e l o p f r o m an i n s i g n i f i c a n t s i z e t o m a t u r i t y I n about 8 d a y s .  T h i s r a p i d growth of f o l l i c l e s , s t a r t i n g  i m m e d i a t e l y a f t e r o v u l a t i o n , has (1921) and  been r e p o r t e d  species.  Corner  Hammond (1927) b e l i e v e d t h a t f o l l i c l e s i n the p i g and  grew t o o v u l a t o r y s i z e over a p e r i o d (about 6 weeks).  of two  More r e c e n t l y , i t was  c y c l e s occur w i t h i n each 21-day e s t r o u s Rajakoski,  i n few  1961).  a f t e r e s t r u s and  The  or t h r e e e s t r o u s  discovered  t h a t two  cow  cycles follicular  c y c l e i n c a t t l e (Bane  and  f i r s t growth s t a r t s on the t h i r d or f o u r t h  day  the second s t a r t s about 12 t o 14 days a f t e r e s t r u s .  A  wave of a t r e s i a sweeps a l l but one f o l l i c l e about 5 t o 7 days a f t e r ovulation.  Therefore,  the b a s i c l e n g t h of the f o l l i c u l a r c y c l e i n c a t t l e  i s about 9 d a y s , because o v u l a t i o n o c c u r s on day 2 of t h e e s t r o u s  cycle.  Loeb (1911) observed two waves of f o l l i c u l a r growth w i t h i n the 18-day c y c l e i n the g u i n e a p i g .  Estrous  c y c l e s as s h o r t as 10 days have been  r e c o r d e d f o r the h o r s e , a l t h o u g h the average l e n g t h of t h e c y c l e i s 22 days ( B e r l i n e r , 1959).  A l l of t h e s e o b s e r v a t i o n s  d e f i n e d f o l l i c u l a r c y c l e s may species.  The  s u g g e s t t h a t two w e l l -  occur w i t h i n the e s t r o u s  c y c l e of some  s h o r t c y c l e i n deer i s c h a r a c t e r i z e d by a s m a l l ,  l i v e d corpus l u t e u m and growth b e g i n n i n g  an 8-day f o l l i c u l a r c y c l e , w i t h  immediately a f t e r ovulation.  short-  follicular  157  Several weeks p r i o r to the breeding season, s i n g l e l a r g e f o l l i c l e s form and degenerate i n a c y c l i c manner, but during the breedi n g season two large f o l l i c l e s u s u a l l y develop.  Thus, as the breeding  season approaches, the f o l l i c u l a r change i n adults i s not so much i n the s i z e of large f o l l i c l e s produced, but i n the number.  Some of the large  f o l l i c l e s rupture p r i o r to the breeding season but no corpora l u t e a develop. C y c l i c f o l l i c u l a r development continues throughout g e s t a t i o n , but i t i s dampened during the mid-portion of pregnancy. the f o l l i c u l a r c y c l e may be extended during t h i s period.  The length of Invariably,  only one l a r g e a c t i v e f o l l i c l e i s present a t a time i n females containi n g v i s i b l e embryos. There i s l i t t l e information on f o l l i c u l a r patterns i n pregnant ungulates.  C y c l i c production of f o l l i c l e s , a t i n t e r v a l s corresponding  to the length of the estrous c y c l e s , occur i n pregnant r a t s and guinea pigs (Perry and Rowlands, 1962).  .'Precocious' r i p e n i n g of f o l l i c l e s  occurs during the f i r s t 35 days of pregnancy i n the goat (Harrison, 1948b).  I n the mare, the c y c l i c production of f o l l i c l e s and corpora  l u t e a i s most prevalent i n the second and t h i r d month of pregnancy (Amoroso e t a l . , 194-8).  Post-conception o v u l a t i o n s , which r e s u l t i n  corpora l u t e a , occur i n the A f r i c a n elephant (Perry, 1953), e l k (Halazon and Buechner, 1956; Morrison, 1960), and the mare ( B e r l i n e r , 1959).  Some of the l a r g e f o l l i c l e s rupture i n pregnant deer but corpora  l u t e a do not develop. F o l l i c l e s i n deer ovaries grow to ovulatory s i z e during the l a s t 6 weeks of pregnancy, and the presence of l a r g e r e g r e s s i n g f o l l i c l e s i n d i c a t e s that r e l a t i v e l y - s h o r t f o l l i c u l a r c y c l e s occur.  This  resurgence of f o l l i c u l a r a c t i v i t y during the l a t t e r stages of g e s t a t i o n  158  has not been reported i n a r t i o d a c t y l s , although i t occurs i n the porcupine  (Mossman and Judas, 194-9) • I n e l k , f o l l i c l e s decrease i n  average s i z e during g e s t a t i o n (Morrison, 1960).  Pre-partum enlargement  of f o l l i c l e s does not occur i n the caribou (McEwan, 1962), and there i s l i t t l e f o l l i c u l a r development i n the l a t t e r two-thirds of pregnancy i n the cow (Hammond, 1927). The h i s t o l o g y and cytology of growing f o l l i c l e s i n deer are quite s i m i l a r to the d e s c r i p t i o n s f o r other l a r g e mammals (Brambell, 1956;  Harrison, 1962).  The changes i n the f o l l i c l e i n the l a s t day or  two p r i o r to o v u l a t i o n were examined c l o s e l y .  The preovulatory f o l l i c l e  i n deer ovaries has many of the c h a r a c t e r i s t i c s of f o l l i c l e s i n the ovaries of other species known to be i n estrus. n i f y i n g f o l l i c u l a r maturation were: oophorus,  Characteristics sig-  1) loosening of the cumulus  2) increased v a s c u l a r i z a t i o n ,  3)  c e s s a t i o n of m i t o s i s  i n granulosa c e l l s , except those immediately around the oocyte, and 4-) t h i n n i n g of the ovarian and f o l l i c u l a r w a l l .  Probably the most u n i v e r s a l  c h a r a c t e r i s t i c i s c e l l a u t o l y s i s i n the cumulus oophorus. ing  This loosen-  of the oocyte occurs j u s t p r i o r to o v u l a t i o n i n guinea pigs (Myers  et a l . , 1936; Hartman, 1962), r a t s (Odor, 1955; Hartman, 1962), swine (Spalding e t a l . , 1955), c a t t l e (Corner, 1920), r a b b i t s , and man (Brambell^, 1956).  A u t o l y s i s of the c e l l s of the cumulus oophorus a l s o  occurs i n a t r e t i c f o l l i c l e s of deer, i n c l u d i n g those that l u t e i n i z e . Blandau (1966a) reported that d i s p e r s i o n of c e l l s d i d not occur i n l a r g e , c y s t i c f o l l i c l e s which did not ovulate i n the r a t .  Associated-  w i t h cumulus oophorus d i s p e r s i o n i n some preovulatory f o l l i c l e s of deer, i s formation of the corona r a d i a t a .  Apparently, t h i s s t r i k i n g r a d i a l  159 arrangement o f g r a n u l o s a c e l l s around t h e oocyte o c c u r s i n most mammals ( B r a m b e l l , 1956).  The e n t i r e p r o c e s s o f cumulus oophorus a u t o l y s i s and  corona r a d i a t a f o r m a t i o n may be i n d u c e d a n t e r i o r p i t u i t a r y (Blandau,  by l u t e i n i z i n g hormone f r o m t h e  1965b).  P r o l i f e r a t i o n o f v a s c u l a r l o o p s i n t o t h e membrana g r a n u l o s a below the cumulus oophorus o c c u r s i n most l a r g e f o l l i c l e s o f d e e r , b u t e n l a r g e as o v u l a t i o n n e a r s .  the loops  T a l l , columnar c e l l s w i t h l e s s dense  r a d i a t e o u t f r o m these l o o p s , as i n t h e cow ( C o r n e r , 1920).  cytoplasm  A marked i n c r e a s e  i n the v a s c u l a r i t y of the f o l l i c l e , r e s u l t i n g i n v a s c u l a r p r o j e c t i o n s i n t o the m u r a l g r a n u l o s a , s i g n i f i e s i n s i p i e n t o v u l a t i o n .  Hartman (1962) b e l i e v e s  t h a t i n c r e a s e d v a s c u l a r i t y o f t h e f o l l i c l e i s one o f t h e b e s t i n d i c a t o r s o f i m p e n d i n g o v u l a t i o n i n man.  A c c o r d i n g t o B u r r and D a v i s  (1951), t h e r e i s  increased p e r m e a b i l i t y of c a p i l l a r i e s p r i o r to o v u l a t i o n i n the r a b b i t . The  s i z e o f f o l l i c l e s and t h e t h i n n e s s o f f o l l i c u l a r and o v a r i a n  w a l l s are of d i a g n o s t i c value i n c o n j u n c t i o n w i t h other f e a t u r e s .  Follicles  t h a t have extended through t h e - f i b e r s o f t h e t u n i c a a l b u g i n e a a r e d e s t i n e d t o r u p t u r e e i t h e r p h y s i o l o g i c a l l y or m e c h a n i c a l l y .  Enlargement or l u t e i n i -  z a t i o n o f t h e c a c e l l s does n o t occur i n deer p r i o r t o o v u l a t i o n , a l t h o u g h i t does occur i n some o t h e r s p e c i e s ( H a r r i s o n , 1962). S i g n i f i c a n t l y , i f two l a r g e f o l l i c l e s a r e p r e s e n t d u r i n g t h e b r e e d i n g season, one o f them i s o f t e n o u t o f phase w i t h t h e o t h e r . b o t h do n o t always r u p t u r e a t t h e same t i m e .  Evidently,  A l t h o u g h t h e r e may have been  an i n t e r v a l o f a d a y o r two between some o f t h e r u p t u r e s , n e v e r t h e l e s s , b o t h f o l l i c l e s develop i n t o corpora l u t e a .  Gibson  (1957) r e l a t e s one i n s t a n c e o f  a 1 t o 2 d a y i n t e r v a l between r u p t u r e s i n w h i t e - t a i l e d d e e r .  McEwan (1962)  b e l i e v e s t h a t t h e a c c e s s o r y c o r p o r a l u t e a i n c a r i b o u .are produced f r o m asynchronous r u p t u r e o f f o l l i c l e s .  However, t h e r e a r e many i n s t a n c e s i n w h i c h  16o  one of the large f o l l i c l e s does not rupture at f i r s t ovulation. f o l l i c l e s i n v a r i a b l y develop i n t o accessory corpora l u t e a .  Such  Thus, a l l large  f o l l i c l e s , a n d many smaller ones which develop during the breeding season, are capable of producing l u t e a l t i s s u e . The f o l l i c l e s which rupture a t second o v u l a t i o n are mature and a t the same stage of development presumably because the s t a r t of t h e i r growth i s synchronized by the f i r s t o v u l a t i o n .  Thus, most of the ova shed are v i a b l e ,  whereas many of the ova shed a t f i r s t o v u l a t i o n are probably incapable of producing embryos,even i f provided w i t h a s u i t a b l e environment.  The  s e l e c t i v e advantage of the second o v u l a t i o n i s a t once apparent. In deer,the a t r e s i a of t e r t i a r y f o l l i c l e s i s a normal, c y c l i c , seasonal process. As i n most mammals (Brambell, 1956), the form of f o l l i c u l a r a t r e s i a depends p r i m a r i l y on the stage of development.  Detection of  e a r l y a t r e s i a was stressed because i t was soon apparent that some of the f i r s t - c y c l e f o l l i c l e s were obviously a t r e t i c p r i o r to rupture.  The e a r l i e s t  stages of a t r e s i a are i d e n t i f i e d by m u l t i p l e c h a r a c t e r i s t i c s , i n v o l v i n g a l l of the f o l l i c u l a r components.  However, the most d i a g n o s t i c t i s s u e s are the  membrana granulosa, the vascular system i n the theca i n t e r n a , and the zona p e l l u c i d a and i t s attachments t o the surrounding granulosa c e l l s .  There i s  some v a r i a b i l i t y between f o l l i c l e s i n the component that f i r s t s i g n i f i e s commencement of a t r e s i a .  This v a r i a b i l i t y probably accounts f o r the disagree-  ment among h i s t o l o g i s t s regarding the component which f i r s t e x i b i t s characteri s t i c s of a t r e s i a (Ingram, 1962).  I n deer f o l l i c l e s , t h e granulosa u s u a l l y  signified atresia f i r s t . Oocytes i n l a r g e a t r e t i c f o l l i c l e s commonly underwent meiotic d i v i s i o n and even divided i n t o two c e l l s .  Hafez (1961) has seen as many as  5 c e l l s i n these "fragmented" u n f e r t i l i z e d ova,but he d i s a s s o c i a t e s them from parthenogenic development.  Mann (1924-) noted that u n f e r t i l i z e d ,  161  segmented ova c l o s e l y resembled normal f e r t i l i z e d one or more signs of  ova, although most showed  degeneration.  Hypertropy and hyperplasia of c e l l s occur  i n s i d e the glassy  membrane of a small proportion of f o l l i c l e s i n advanced stages of a t r e s i a . These c e l l s are s i m i l a r i n appearance to l u t e a l c e l l s but they are smaller, hence  I term them " s e m i - l u t e i n i z e d " c e l l s .  Upon degeneration,  the c e l l s  produce some yellow pigment which i s probably a product of l i p i d degeneration.  The p e c u l i a r c i r c u l a r grouping of c e l l s i n some a t r e t i c f o l l i c l e s  occur i n other mammals besides deer.  may  Mossman and Judas (194-9) described  concentric l a y e r s of young i n t e r s t i t i a l t i s s u e containing p a r t l y - l u t e i n i z e d c e l l s i n f o l l i c l e s i n the porcupine.  They described these c e l l s as being  c y t o l o g i c a l l y t r a n s i t i o n a l between the granulosa of e a r l y - a t r e t i c f o l l i c l e s and young l u t e i n c e l l s .  They c l a s s i f i e d the s t r u c t u r e s as one type of  accessory corpora l u t e a .  Such s t r u c t u r e s have been termed"corpora l u t e a  atretica"(Brambell,1956), a term a l s o applied to f u l l y l u t e i n i z e d f o l l i c l e s . I t h i n k the term i s misleading and should be abandoned.  I consider  s t r u c t u r e s containing t y p i c a l l u t e a l c e l l s , a s corpora l u t e a or corpora l u t e a .  only  accessory  The m a t e r i a l i n the center of the w a l l s of these c i r c u l a r  s t r u c t u r e s has not been described previously,and remains u n i d e n t i f i e d . I t i s n a t u r a l l y black or i s stained black by Masson's trichrome. I t was shown t h a t a t r e s i a of f o l l i c l e s took two forms i n deer. a t y p i c a l form of degeneration i n the l i t e r a t u r e ,  The  i n v o l v i n g leucocytes, i s u n l i k e any described  unless the fatty-degeneration forms of a t r e s i a described  by h i s t o l o g i s t s around 1900  (reviewed  by Brambell, 1956)  are equivalent  processes. There i s l i t t l e information i n the l i t e r a t u r e on the r a t e at which f o l l i c l e s degenerate because of the obvious d i f f i c u l t y of d a t i n g events.  I  was able to obtain information on t h i s t o p i c by e s t a b l i s h i n g the r e l a t i o n s h i p  162 betv/een the d e g e n e r a t i o n of l a r g e f o l l i c l e s of f o l l i c l e s .  Because the r a t e of f o l l i c u l a r  s e g m e n t a t i o n s t a g e s of o v a , i t was atresia.  The  and  the growth of the n e x t c y c l e  growth was  estimated from  p o s s i b l e t o e s t i m a t e the r a t e o f  g e n e r a l r e l a t i o n s h i p , s i m p l i f i e d and  follicular  diagrammed i n F i g . 26,  is  e x i b i t e d by does o f a l l ages f r o m 6 weeks p r i o r t o the b r e e d i n g season t o end  of g e s t a t i o n , a l t h o u g h dampened f o l l i c u l a r  m i d d l e h a l f of g e s t a t i o n . follicles  For  t h a t the f o l l i c l e p a s s e s t h r o u g h the f i r s t t h r e e  a t r e s i a i n one  s t a g e s (seven and e a r l i e r stages,  season.  s t a g e s of  A l t h o u g h i t i s n o t e v i d e n t i n the f i g u r e , the l a s t  e i g h t ) occupy s e v e r a l weeks,and t h e i r d u r a t i o n ,  two  unlike  i s l a r g e l y dependent on the s i z e of the d e g e n e r a t i n g f o l l i c l e .  S t u r g i s (194-9) has  conducted one  atresia.  The  of the few  general  s a t i s f a c t o r y s t u d i e s on the  rate  t e m p o r a l changes i n the r e g r e s s i n g f o l l i c l e of  d e e r are s i m i l a r t o those he d e s c r i b e s The  that  or two d a y s , but t h e r e a f t e r the r a t e of a t r e s i a becomes  p r o g r e s s i v e l y slower.  of f o l l i c u l a r  the  the purposes of the f i g u r e , i t i s assumed  grow a t the same r a t e t h r o u g h o u t d i f f e r e n t c y c l e s of the  I t i s evident  follicles  a c t i v i t y occurs during  the  progressive  the  i n the monkey.  d e c l i n e i n the numbers o f  primordial  w i t h age ( i n deer) i s comparable t o the s i t u a t i o n i n o t h e r  species.  The d e c l i n e i n numbers i s n o t as r a p i d i n deer as i n s m a l l mammals,but t h e l a t t e r have s h o r t c o n t i n u o u s c y c l e s , w h e r e a s the d e e r i s a s e a s o n a l b r e e d e r . There i s a c o n t i n u o u s d e c l i n e i n the number of o o c y t e s , w i t h t i m e , i n  man  ( B l o c k , 1952), the r a t • (Mandl and  and  K r o h n , 1961). follicles  I n the l a t t e r two  i s exponential.  Shelton, species,the  1959), and  the mouse (Jones  d e c l i n e i n numbers of  I n d e e r , as i n o t h e r s p e c i e s , t h e r e i s  i n d i v i d u a l v a r i a t i o n i n the number of o o c y t e s .  For example, i t was  t h a t fawns have f r o m 8,000 t o 200,000 p r i m o r d i a l f o l l i c l e s  primordial great calculated  i n each o v a r y .  Hafez (1961) e s t i m a t e d t h a t h e i f e r s of 3 months c o n t a i n 100,000 t o 300,000 oocytes i n t h e i r o v a r i e s .  N e o f o r m a t i o n of o o c y t e s a f t e r b i r t h i s i n s i g n i f i c a n t ,  163  F i g . 26.  Schematic r e p r e s e n t a t i o n o f f o l l i c l e growth and a t r e s i a . Degeneration curves f o r f i v e s i z e s of f o l l i c l e s are presented w i t h time r e l a t i o n s g i v e n f o r one. 'The o t h e r c u r v e s r e p r e s e n t s t a g e s o f a t r e s i a and a t r a n s i t i o n a l (T) s t a g e .  7-  10  1  20  :  »  30  VOLUME LARGEST ACTIVE FOLLICLE  1  40  1  50  1—  60  164 i f i t occurs. exhausted  There i s no i n d i c a t i o n t h a t t h e s t o c k o f o o c y t e s i s ever  i n deer as i t i s i n man ( B l o c k , 1952). The o l d e s t doe, 19.5 y e a r s ,  s t i l l h a r b o r e d about 2,000 o o c y t e s . The number o f s m a l l f o l l i c l e s  g r e a t l y i n f l u e n c e s the weight of  o v a r i e s , e s p e c i a l l y , i n fawns b u t t h e y have no b e a r i n g on t h e f e r t i l i t y o f t h e doe.  Fawns and f e m a l e s aged 2.5 y e a r s c o n t a i n e d t h e g r e a t e s t numbers o f  small f o l l i c l e s i n o l d does.  and t h e r e was a d e f i n i t e r e d u c t i o n i n s m a l l f o l l i c l e numbers  Perhaps,the number o f s m a l l f o l l i c l e s may i n d i c a t e t h e type or  amount o f g o n a d o t r o p i c hormone s t i m u l a t i o n .  The f a c t t h a t fawns and f e m a l e s  aged 2.5 y e a r s c o n t a i n g r e a t numbers o f s m a l l f o l l i c l e s , b u t fewer  large  f o l l i c l e s , c o u l d be a t t r i b u t e d t o an i m b a l a n c e o f f o l l i c l e - s t i m u l a t i n g hormone (FSH) and l u t e i n i z i n g hormone ( L H ) . I t was noted c o n t a i n e d a l a r g e number o f s m a l l f o l l i c l e s , r e l a t i v e l y small. small f o l l i c l e s  t h a t i f an i n d i v i d u a l  t h e l a r g e s t f o l l i c l e s were  I n c o n t r a s t , some y e a r l i n g s and o l d does w i t h v e r y few  c o n t a i n e d one or two r e l a t i v e l y l a r g e  follicles.  Seemingly p e c u l i a r t o d e e r , i s t h e f o r m a t i o n o f abundant c h r o m o p h i l i c bodies i n oocytes l o c a t e d i n s m a l l f o l l i c l e s . to  These i n c l u s i o n s , w h i c h seem  o r i g i n a t e i n the n u c l e u s and spread outward, a r e u n l i k e s t r u c t u r e s i n  o o c y t e s o f t h e mouse ( G r e s s o n , 1933), oppossum ( M a r t i n e z - E s t e v e , 1 9 4 2 ) , and monkey ( P o l l a k , 1926).  Because t h e I n c l u s i o n s occur i n f o l l i c l e s  with  r a p i d l y - p r o l i f e r a t i n g g r a n u l o s a , t h e y may n o t s i g n a l t h e o n s e t o f a t r e s i a as suggested  by Ingram (1962).  The i n c l u s i o n s a r e s i m l l i a r t o t h e t i n y  n u c l e o l a r e x t r u s i o n s i n t h e r a t ( G r e s s o n , 1933),but i n deer t h e y a r e many times l a r g e r .  The i n c l u s i o n s may be a s s o c i a t e d w i t h t h e s h i f t i n g o f t h e  nucleus from the center to the p e r i p h e r y of the oocyte.  I n some f o l l i c l e s ,  about 1.0 mm i n d i a m e t e r , t h e n u c l e u s seems t o d i s a p p e a r t e m p o r a r i l y . I n r a t s , the. p r e o v u l a t o r y f o l l i c l e i s about 0.9 mm i n d i a m e t e r when t h e s h i f t o f t h e nucleus occurs.  B r a m b e l l (1956) d e s c r i b e d f a t t y y o l k spheres i n t h e ooplasm  165 that 'pushes' the nucleus to the periphery. Call-Exner bodies, which occurred i n the granulosa of many deer f o l l i c l e s , were extremely numerous i n the f o l l i c l e s of a few females.  The  s i g n i f i c a n c e of these s t r u c t u r e s , which occur i n the r a b b i t , p r a i r i e dog, c a t , whales, and man, i s not known (Brambell,  1956;  Harrison,  1962).  They were most  numerous i n young deer whose ovaries contained a large number of small follicles.  P o s s i b l y , they s i g n i f y a r e l a t i v e l y high s e c r e t i o n of f o l l i c l e -  s t i m u l a t i n g hormone  (FSH)..  Polynuclear oocytes and polyovular f o l l i c l e s are found i n the ovaries of most mammalian species (Brambell,  1956).  Gibson  (1957) concludes  that polyovular f o l l i c l e s occur r a r e l y i n deer, however, they were common i n the b l a c k - t a i l e d deer of Northwest Bay. encountered was 1.2  The l a r g e s t polyovular f o l l i c l e  mm i n diameter, f a r below o v u l a t o r y s i z e .  Hundreds of  l a r g e f o l l i c l e s were examined f o r m u l t i p l e oocytes and none was found.  Even  i f polyovular f o l l i c l e s reached o v u l a t o r y s i z e , i t i s u n l i k e l y that the second or t h i r d oocyte would be v i a b l e , because, i n most small f o l l i c l e s , only one oocyte i s of normal s i z e .  Nevertheless, embryos i n excess of corpora  l u t e a have been a t t r i b u t e d to polyovular f o l l i c l e s i n the w i l d Norway r a t (Davis and H a l l ,  1950), and  r a b b i t s ( A l l e n , Brambell and M i l l s ,  1947).  Three  instances of p o l y o v u l a t i o n have been reported i n w h i t e - t a i l e d deer by Hesselton  (1967).  Other examples of s i n g l e corpora l u t e a and twin deer  fetuses are known (Robinette et a l . , 1955;  Brown, 196l),but l i k e l y the twins  were homozygous or two corpora l u t e a had fused. F o l l i c u l a r cysts are r a r e i n the ovaries of Northwest Bay deer. noted by Ingram  (1962),  As  the cysts are l i n e d w i t h a s i n g l e l a y e r of epithelium  and t h e i r c a v i t y contains achromatic f l u i d .  He states that they often  develop when the ovary i s stimulated by a high t i t r e of gonadotrophic hormone.  Such cysts are e a s i l y confused'with some large f o l l i c l e s of the f i r s t  166  f o l l i c u l a r c y c l e i n which the membrana granulosa t h i n s t o one l a y e r of c e l l s . I t i s thought, however, that a l l of the l a t t e r f o l l i c l e s e i t h e r ovulate or become accessory corpora l u t e a . epithelium) i s more common. a pregnant doe.  The second type of cyst (derived from coelomic  One e x c e p t i o n a l l y large cyst was recovered from  There i s no mention of f o l l i c u l a r cysts i n the l i t e r a t u r e on  the deer ovary. The 'cysts' found by Gibson (1957) i n the ovaries of whitet a i l e d deer were undoubtedly  scar t i s s u e t h a t formed a f t e r the r e g r e s s i o n of  p a r t l y - e x t r u s i v e corpora l u t e a . I concur with the opinion of Morrison (i960), who examined e l k o v a r i e s , that information on the reproductive status of herds can be obtained from a n a l y s i s of f o l l i c u l a r development p r i o r to the breeding season.  The  3 number, size, and appearance of f o l l i c l e s over about 10 mm estimate of the number that w i l l rupture.  provides a crude  Animals e x i b i t i n g very l i t t l e  f o l l i c u l a r a c t i v i t y ovulate l a t e i n the season or not a t a l l . 7. 6  The Corpus Luteum. These r e s u l t s are the f i r s t proof that corpora l u t e a of short dura-  t i o n occur i n ungulates.  Buechner et a l .  (1966) suggested  that corpora  l u t e a of short d u r a t i o n might occur i n the Uganda kob,and Perry (1953) bel i e v e d that " r e l a t i v e l y s h o r t - l i v e d " corpora l u t e a occurred i n the A f r i c a n "elephant (not an ungulate).  I n t e r e s t i n g l y , Short and Buss (1965) were unable  to detect progesterone i n corpora l u t e a of the elephant.  According to Perry  and Rowlands (1962), i t i s w e l l established that corpora l u t e a of non-pregnancy are r e l a t i v e l y i n a c t i v e i n the r a t , mouse, and p o s s i b l y i n many other small rodents.  The s h o r t - l i v e d corpora l u t e a derived from the f i r s t o v u l a t i o n 3  grow to a volume of about 50 mm  i n a span of 2 or 3 days and they  begin to shrink a f t e r another 2 or 3 days.  The f u n c t i o n a l l i f e of the f i r s t  corpus luteum i n deer i s probably 3 to 6 days.  Growth ceases between the  second and t h i r d day and the s t r u c t u r e shows h i s t o l o g i c a l i n d i c a t i o n s of  167 d e g e n e r a t i o n 1 t o 2 days b e f o r e second  ovulation.  U s u a l l y , sheep do  o v u l a t e f o r 2 t o 7 days a f t e r exogenous p r o g e s t e r o n e i s w i t h d r a w n  not  (Robinson,  1959), and the l e v e l of p r o g e s t e r o n e i n the b l o o d f a l l s r a p i d l y 2 days b e f o r e e s t r u s i n c y c l i n g sheep ( R o b i n s o n , 1966).  S i m i l a r l y , e n u c l e a t i o n o f the  b o v i n e corpus luteum r e s u l t s i n e s t r u s 2 t o 5 days l a t e r Hansel,  (Hammond, 1927;  1966). The e x t r e m e l y r a p i d d e g e n e r a t i o n o f the f i r s t - c y c l e corpus  a f t e r second  luteum  o v u l a t i o n i s an o u t s t a n d i n g f e a t u r e of the r e p r o d u c t i v e c y c l e .  I t i s more r a p i d t h a n i n the cow  (McNutt, 1924.) or p i g ( C o r n e r ,  1919).  B o l i n g (1942) noted a r a p i d d e c r e a s e i n the volume of the p r e v i o u s  corpus  l u t e u m i n the f i r s t 12 hours a f t e r the onset of e s t r u s i n the r a t .  Greep  (1938) showed t h a t i n j e c t i o n s of l u t e i n i z i n g hormone caused  rapid  r e g e n e r a t i o n of p e r s i s t e n t c o r p o r a l u t e a i n hypophysectorn!zed  rats.  Simi-  l a r l y , the r e g r e s s i o n of c o r p o r a l u t e a of pseudopregnancy i n the r a b b i t facilitated  was  by v e r y s m a l l doses o f exogenous l u t e i n i z i n g hormone (Stormshak  and C a s i d a , 1964).  The r a p i d a u t o l y s i s of l u t e a l c e l l s  accounts  t h e sudden d e c r e a s e i n the volume of c o r p o r a l u t e a i n d e e r . days a l l but a few c e l l s d e g e n e r a t e  for  Within three  completely.  The c u r v e s e s t a b l i s h e d f o r growing and d e g e n e r a t i n g f o l l i c l e s  and  c o r p o r a l u t e a p r o v i d e a q u a n t i t a t i v e method f o r e s t i m a t i n g the age o f s t r u c t u r e s and t h e s t a g e o f t h e e s t r o u s c y c l e .  The c u r v e based on the r a t i o o f the  volumes o f the a c t i v e and r e g r e s s i n g c o r p o r a l u t e a u n q u e s t i o n a b l y p r o v i d e s t h e b e s t e s t i m a t e o f the e a r l y age o f s e c o n d - c y c l e c o r p o r a l u t e a . curves developed,  t h i s one  i n the s i z e of f o l l i c l e s  is  Of the f o u r  l e a s t s u b j e c t t o v a r i a t i o n s between a n i m a l s  and c o r p o r a l u t e a .  There i s l e s s v a r i a t i o n i n the  s i z e o f s t r u c t u r e s w i t h i n f e m a l e s t h a n between them. r e l a t i o n s h i p s between the f i r s t and  second  In establishing  temporal  c y c l e s , the growth r a t e s o f f o l -  l i c l e s and c o r p o r a l u t e a were assumed t o be e q u i v a l e n t .  There i s g e n e r a l  agreement t h a t the e a r l y growth o f c o r p o r a l u t e a a s s o c i a t e d w i t h pregnancy,  168  pseudopregnancy,  and non-pregnancy  i s t h e same w i t h i n s p e c i e s t h a t range i n  s i z e f r o m t h e mouse t o t h e e l e p h a n t ( P e r r y and Rowlands, G e n e r a l l y , t h e e a r l y development  1962).  o f t h e corpus l u t e u m o f pregnancy  i n deer i s s i m i l a r , i n b o t h r a t e o f growth and t i s s u e changes, t o t h e c y c l i c c o r p o r a l u t e a o f t h e p i g ( C o r n e r , 1921), goat ( H a r r i s o n , 194.8b), sheep ( R o b i n s o n , 1959), and t h e cow (Hammond, 1927).  I n d e e r , t h e corpus l u t e u m o f  pregnancy grows r a p i d l y i n t h e f i r s t 4- o r 5 days and i t i s n e a r l y f u l l y grown i n 8 days.  Maximum development i s reached i n 10 t o 20 d a y s .  The above  p a t t e r n o f growth i s t y p i c a l o f t h e c o r p o r a l u t e a of. pregnancy and nonpregnancy i n many s p e c i e s . A t y p i c a l , i s the decrease i n the s i z e of corpora l u t e a a t midpregnancy, and e s p e c i a l l y t h e sudden i n c r e a s e i n s i z e i n t h e l a t e p r e n a t a l months.  The c o r p o r a l u t e a o f most a r t i o d a c t y l s p e c i e s a t t a i n maximum s i z e  w i t h i n t h e f i r s t month and r e m a i n a t t h a t s i z e u n t i l f i n a l r e g r e s s i o n o c c u r s ( B r a m b e l l , 1956; H a r r i s o n , 1962).  However, c e l l s h r i n k a g e (and perhaps  cor-  pus l u t e u m s h r i n k a g e ) has been r e p o r t e d by the 24-th day i n t h e monkey ( C o r n e r , 194-5), and between t h e 3 5 t h and t h e 4-5th d a y i n t h e goat 194-8a).  (Harrison,  Corpora l u t e a r e m a i n a t about t h e same s i z e t h r o u g h o u t g e s t a t i o n  i n t h e c a r i b o u (McEwan, 1962), cow ( C o r n e r , 1921; Hammond, 1927), and t h e A f r i c a n e l e p h a n t (Buss and S m i t h , 1966).  Trauger and Haugen (1965)  stated  t h a t "the average s i z e o f t h e c o r p o r a l u t e a a p p a r e n t l y i n c r e a s e d d u r i n g pregnancy" i n t h e w h i t e - t a i l e d d e e r .  I n the s p e c i e s , the l u t e a l c e l l s en-  l a r g e d d u r i n g g e s t a t i o n and a t t a i n e d t h e i r maximum s i z e j u s t p r i o r t o p a r t u r i t i o n ( G i b s o n , 1957).  I observed s i m i l a r changes I n b l a c k - t a i l e d  deer.  - T i s s u e changes d u r i n g e a r l y development  o f t h e c o r p u s l u t e u m were  r e m a r k a b l y s i m i l a r t o d e s c r i p t i o n f o r t h e p i g ( C o r n e r , 1919), monkey ( C o r n e r ,  169  194-5), horse  (Harrison, 194-6), man (Corner, 1955) and, e s p e c i a l l y , the  goat (Harrison,  194-8).  My observation that hemorrhage of large amounts  of blood occurs several hours a f t e r o v u l a t i o n coincides with Corner's (194-6) i n t e r p r e t a t i o n . However, i n deer ovaries a large proportion of the f o l l i c l e s which rupture a t f i r s t o v u l a t i o n do not develop i n t o corpora hemorrhagica.  I n t h i s respect they are s i m i l a r to most  ruptured f o l l i c l e s i n man (White e t a l . ,  1951).  The amount of blood  associated with the young corpus luteum i s h i g h l y v a r i a b l y among mammals (Harrison,  194-8a,  b).  The r e l a t i v e c o n t r i b u t i o n s of the t h e c a l and granulosal l a y e r s to the l u t e a l t i s s u e , i s a l s o v a r i a b l e among mammals.  I n deer, both l a y e r s  contribute c e l l s but the granulosa layer appears to contribute the bulk of the l u t e a l t i s s u e , as i t does i n most a r t i o d a c t y l s (Harrison, Nevertheless,  194-8a).  i n deer, the t h e c a l layer apparently contributes a l l of the  l u t e a l t i s s u e i n f i r s t c y c l e f o l l i c l e s i n which the granulosa i s degenerate and i s extruded a t rupture.  Also, considerably m i t o t i c a c t i v i t y  was noted i n the c e l l s of the theca i n t e r n a located along the edges of the invaginated f o l d s of t h e c a l t i s s u e .  In a d d i t i o n , c e l l s located i n  the theca externa were converted i n t o l u t e a l c e l l s as was noted i n the porcupine by Mossman and Judas  (194-9).  The f a t e of the c e l l s from the  r e s p e c t i v e l a y e r s could not be followed with c e r t a i n t y past the second or t h i r d day.  In contrast to most d e s c r i p t i o n s , m i t o t i c d i v i s i o n s occurred f r e q u e n t l y i n developing corpora l u t e a .  They occurred from o v u l a t i o n  u n t i l about the f o u r t h day, although o c c a s i o n a l l y they were observed a t l a t e r stages.  Changes i n the number of d i v i s i o n s and the s i z e of the l u t e a l  170  c e l l s provide  a q u a n t i t a t i v e means of e v a l u a t i n g the age of corpora l u t e a .  The number of c e l l s i n a c e r t a i n f i e l d of view provides a b e t t e r i n d i c a t i o n of c e l l s i z e than do  measurements of c e l l diameters.  Bassett (194-9) found  that the m i t o t i c . a c t i v i t y of the c e l l s i n r a t corpora l u t e a p r o g r e s s i v e l y decreased with time,whereas i n deer i t increased to a peak at about 1.5 days a f t e r o v u l a t i o n and decreased sharply t h e r e a f t e r . The corpora l u t e a of deer are u s u a l l y of the compact type, as i n the sheep, pig,and goat (Harrison, 194-Sa). However, some corpora l u t e a with l a r g e c e n t r a l lumena, s i m i l a r to those of the cow (Hammond, 1927), monkey (Corner,  194-5) and man (Corner, 1956), develop a f t e r the f i r s t ovulation.  These  ' c y s t i c ' corpora l u t e a form i n f o l l i c l e s which were abnormally l a r g e at rupture.  I n these f o l l i c l e s , the t h i n granulosa and theca i n t e r n a l a y e r s  produce only a band of l u t e a l t i s s u e around a l a r g e c a v i t y before development ceases.  Many of the very l a r g e f o l l i c l e s that produce these corpora l u t e a  are a t r e t i c p r i o r to rupture. The cytology of the corpus luteum of pregnancy g r a d u a l l y changed during gestation.  Of s i g n i f i c a n c e , are the "conception c e l l s " which seem to  s i g n i f y presence of the b l a s t o c y s t i n the uterus.  These c e l l s , which are  most prominent 5 to 10 days a f t e r conception, have been noted i n young corpora l u t e a i n the pregnant cat (Dawson, 194-6), goat (Harrison, 1948b) and the p i g (Corner, 1919).  As noted by Gibson (1957) i n the w h i t e - t a i l e d  deer, there was a gradual increase i n maximum c e l l s i z e d u r i n g gestation. She also noted the l a r g e vacuoles i n the c e l l s , l a t e I n gestation. i s c e l l u l a r enlargement j u s t p r i o r to b i r t h , i s not known.  Why  there  The s i g n i f i c a n c e  of the d i f f e r e n t c e l l types was not apparent, although small c e l l s with chromophilic cytoplasm and condensed n u c l e i may be t h e c a l c e l l s .  Hyper-  chromasia i n l a r g e c e l l s seemed to be associated w i t h c e l l death. Accessory corpora l u t e a occur i n most species of mammals (Brambell,  171  1956; H a r r i s o n , 1962) and i n deer, they develop from three d i s t i n c t sources. The f i r s t type develops i n small f o l l i c l e s which rupture about the time of normal o v u l a t i o n .  Because they r a r e l y overlap i n s i z e w i t h primary corpora  l u t e a , they are a p o t e n t i a l source of error i n the determination of o v u l a t i o n r a t e from counts of primary corpora l u t e a .  I n deer, few e r r o r s w i l l be  made i f corpora l u t e a smaller than one-quarter the s i z e of the l a r g e s t corpus luteum i n the same p a i r of ovaries are c l a s s i f i e d as accessory.  Accessory  corpora l u t e a i n many species are e a s i l y confused w i t h primary ones.  In elk,  they o r i g i n a t e i n f o l l i c l e s which rupture i n the pregnant female (Halazon and Buechner, 1957; Morrison, i960).  P i m l o t t (1959) a r b i t r a r i l y defined  accessory.corpora l u t e a , i n moose,as those l e s s than h a l f the s i z e of the l a r g e r corpus luteum i n the same p a i r of ovaries. s i z e was diameter, whereas I used volume.  However, h i s c r i t e r i o n of  The accessory corpora l u t e a which  occur i n red deer (Cervus elaphus) are also much smaller than the primary ones (Douglas, 1966). McEwan (1962) suggested t h a t , i n caribou, accessory corpora l u t e a developed i n f o l l i c l e s which ruptured out of phase.  His d e s c r i p t i o n  bf o v u l a t i o n s i t e s suggests that some accessory corpora l u t e a may develop i n the r e g r e s s i n g corpus luteum of the f i r s t ovulatory c y c l e .  I have termed  these Type 3 accessory corpora l u t e a .  Gibson (1957) termed them "corpora  l u t e a aberrans" i n w h i t e - t a i l e d deer.  This term was used o r i g i n a l l y by  Corner (1945) t o describe a p e r s i s t e n t corpus luteum which formed from a corpus luteum of estrus I n the monkey. Although represented by a rupture s i t e on the surface of the ovary, Type 3 accessory corpora l u t e a are small and cannot be confused w i t h primary corpora l u t e a i n b l a c k - t a i l e d deer. The second type of accessory corpus luteum, which develops i n unruptured f o l l i c l e s , i s the most common i n deer and i n most other mammals. I t i s more common a f t e r the f i r s t o v u l a t i o n than a f t e r the second, and forms i n f o l l i c l e s that are out of phase or are too small t o rupture.  172  When formed i n l a r g e f o l l i c l e s these accessory corpora l u t e a may  represent  intermediate stages between the production of one, two, or three corpora lutea.  7. 7  Ovarian Scars. S i g n i f i c a n t l y , scars produced by corpora l u t e a of pregnancy can be  used to estimate, accurately,the b i r t h r a t e of the previous reproductive season.  In t h i n , stained sections the most recent scars are r e a d i l y d i s -  tinguished from older ones and from scars from other sources.  The r a t e s  obtained from counts of scars derived from corpora l u t e a of pregnancy must be reduced by the amount of i n t r a u t e r i n e m o r t a l i t y .  An estimate of the  l a t t e r i s obtained from c o l l e c t i o n s taken during the g e s t a t i o n a l period.  This  technique i s valuable because the hunting season of many deer herds occurs p r i o r to or during the breeding season, and i t i s during the hunting season that l a r g e samples of ovaries may be e a s i l y obtained.  Further, the technique  permits one to compare the reproductive output of i n d i v i d u a l animals f o r at l e a s t two successive years.  That i s , the number of a c t i v e corpora l u t e a i s  compared to the number of scars from the previous breeding  season.  Pregnancy scars p e r s i s t i n the ovaries f o r the l i f e of the doe.  It  i s therefore p o s s i b l e to estimate the t o t a l past fawn production of any f e male.  An estimate of v i a b l e fawn production i s obtained by a d j u s t i n g the  r a t e by the amount of f e t a l m o r t a l i t y .  In a d d i t i o n , the v a r i a b i l i t y i n  f e r t i l i t y between i n d i v i d u a l s can be r e a d i l y ascertained. Furthermore,  a  r e g r e s s i o n of scar numbers with age produces a curve that describes the long term f e t u s production of the population.  Scar production by deer of Northwest  Bay i s described by the equation y = 1.885x - 2.326 where y i s the number of scars and x i s the age of the doe a t the previous conception. 1.885, i s the r a t e a t which scars are produced.  The slope,  173  Counts of pregnancy s c a r s i n c o n j u n c t i o n w i t h age i s a means of. q u a n t i t a t i v e l y comparing the l o n g - t e r m ,  e c o l o g i c a l p r o d u c t i v i t y o f a popu-  l a t i o n t o a t h e o r e t i c a l c u r v e d e p i c t i n g the p h y s i o l o g i c a l r e p r o d u c t i v e  po-  t e n t i a l of the s p e c i e s .  long  I t a l s o has a p p l i c a t i o n i n comparisons of the  term p r o d u c t i v i t y between two p o p u l a t i o n s .  I t i s the o n l y t e c h n i q u e a p p l i c a -  b l e t o deer by w h i c h p a s t p r o d u c t i v i t y of a p o p u l a t i o n can be P r i o r t o t h i s s t u d y , i t was  deterimed.  n o t known t h a t s c a r s d e r i v e d f r o m  c o r p o r a l u t e a o f pregnancy p e r s i s t e d i n the o v a r i e s f o r the l i f e of f e m a l e deer.  Although  Gibson counted as many as 18 pigmented s c a r s i n the  ovaries  of w h i t e - t a i l e d d e e r , some o l d f e m a l e s c o n t a i n e d  few s c a r s , s u g g e s t i n g  pregnancy s c a r s d i d not p e r s i s t i n t h e s e .  (1965) a l s o found a l i n e a r  r e l a t i o n s h i p between age and o f moose o v a r i e s . (Mackintosh,  Simkin  the number of pigmented s c a r s i n s t a i n e d s e c t i o n s  Pregnancy s c a r s p e r s i s t f o r many y e a r s i n the  194-6) and  that  the cow  (McNutt, 1924).  S c a r s may  cetacea  p e r s i s t f o r many  y e a r s i n numerous o t h e r s p e c i e s but few p e o p l e have i n v e s t i g a t e d them w i t h s u f f i c i e n t thoroughness. I agree w i t h G i b s o n (1957) t h a t , i n d e e r , o n l y the pregnancy s c a r s o f the p r e v i o u s b r e e d i n g  season a r e d i s t i n c t f r o m o l d e r s c a r s .  I n some  f e m a l e s t h e r e i s a d e f i n i t e sequence o f s c a r s of d i f f e r e n t ages,but i n o t h e r s the p e n u l t i m a t e age  there i s a progressive decrease i n :  connective  tissue fibers,  hyalin material, 5)  s e t o f s c a r s cannot be d i s t i n g u i s h e d f r o m o l d e r s c a r s .  4-)  2)  1)  the e x t e r n a l c a p s u l e  the amount o f pigment,  3)  of  the amount o f  the number o f s m a l l , c e n t r a l l y - l o c a t e d blood vessels,-  the s t a i n a b i l i t y o f the blood v e s s e l s w h i c h p e r s i s t .  The  t h a t r e m a i n i n o l d s c a r s a r e the t h i n - w a l l e d blood v e s s e l s .  only tissues I t i s probably  e l a s t i n i n the w a l l s o f these v e s s e l s w h i c h makes them so p e r s i s t a n t . i s probably  With  the most r e s i s t a n t o f a l l the body p r o t e i n s t o c h e m i c a l  Elastin  change  174  (Ham  and  L e e s o n , 1961). There appear  deer and  t o be some b a s i c d i f f e r e n c e s between w h i t e - t a i l e d  b l a c k - t a i l e d deer i n the r a t e of c o r p u s l u t e u m d e g e n e r a t i o n .  (1957) noted t h a t p i g m e n t - b e a r i n g l u t e i n c e l l s r e t a i n e d t h e i r c e l l and  n u c l e i f o r a t l e a s t one  t a i l e d deer.  and  l u t e a 2 weeks a f t e r p a r t u r i t i o n , and  deer.  Few  c e l l s were p r e s e n t in. the  l u t e a l c e l l s had  C e l l a u t o l y s i s i s much more r a p i d i n b l a c k - t a i l e d  G o l l e y (1954-) r e p o r t e d  none a r e p r e s e n t 5  t h a t i n b l a c k - t a i l e d deer a l l  d i s a p p e a r e d f r o m the pregnancy s c a r s w i t h i n 2 months.  t h i s s t u d y the c o r p o r a l u t e a of o n l y one doe The  corpora  s c a r s aged 6 months were composed l a r g e l y  l u t e a l c e l l s r e m a i n a few days a f t e r b i r t h and  months p o s t - p a r t u m .  outlines  one-half years a f t e r p a r t u r i t i o n i n white-  In a d d i t i o n , l a r g e vacuolated  of pigmented l u t e a l c e l l s .  Gibson  cause o f t h i s r e t a r d e d  r e g r e s s i o n was  perhaps remnants of a r e s o r t i n g f e t u s .  contained  In  l a r g e , granulated  cells.  p r o b a b l y some m a t e r i a l i n the  uterus,  C o r p o r a l u t e a p e r s i s t i n mule deer  w h i c h have r e s o r b e d embryos or have a b o r t e d ( R o b i n e t t e  e t a l . , 1955).  There  are a l s o d i f f e r e n c e s between the two  s p e c i e s i n the r a t e of r e g r e s s i o n  c o r p o r a l u t e a of non-pregnancy.  year a f t e r t h e i r i n i t i a l r e g r e s s i o n  One  of in  b l a c k - t a i l e d d e e r , the s t r u c t u r e s are composed o f a s m a l l amount of h y a l i n t i s s u e , a few  c e l l spaces surrounded by f i l m s of y e l l o w pigment, and  s m a l l , t h i c k - w a l l e d blood v e s s e l s .  a  few  I n w h i t e - t a i l e d d e e r , s t r u c t u r e s of  the  3 same age  a r e 10 t i m e s l a r g e r (O.4O  scattered  mm  ) and  c o n t a i n pigmented l u t e a l  cells  throughout h y a l i n connective t i s s u e . I cannot agree w i t h B r a m b e l l (1956) t h a t the r e t r o g r e s s i v e p r o c e s s e s  a r e e s s e n t i a l l y s i m i l a r whether the c o r p o r a l u t e a are o f o v u l a t i o n pregnancy.  He n o t e d , however, t h a t d e t a i l e d s y s t e m a t i c  l u t e u m r e g r e s s i o n a r e n o t numerous. non-pregnancy r e g r e s s  The  or  a c c o u n t s of c o r p u s  c o r p o r a l u t e a of o v u l a t i o n  or  r a p i d l y i n deer because no o u t e r c o n n e c t i v e t i s s u e  c a p s u l e i s produced and  the b l o o d v e s s e l s a r e few  and  thin-walled.  The  175  dominant h y a l i n c o n n e c t i v e t i s s u e i s r e s o r b e d q u i t e r a p i d l y . pigment i s produced  around t h e p e r i p h e r y o f d e g e n e r a t i n g c e l l s b u t i t i s n o t  i n t h e f o r m o f g r a n u l e s as i s t h e pigment produced l u t e a o f pregnancy.  Some y e l l o w  i n degenerating  corpora  C l e a r l y , t h e t y p e o f s c a r w h i c h forms i s dependent on  the s i z e and f u n c t i o n a l l i f e span o f t h e corpus luteum.  The l a r g e r t h e  s t r u c t u r e , t h e g r e a t e r t h e number, s i z e , and t h i c k n e s s o f t h e s u p p o r t i n g b l o o d v e s s e l s , and t h e g r e a t e r t h e d e p o s i t i o n o f pigment upon r e g r e s s i o n .  Thus,  s c a r s b e l i e v e d t o be a s s o c i a t e d w i t h embryonic m o r t a l i t y a r e i n t e r m e d i a t e i n c h a r a c t e r i s t i c s between s c a r s d e r i v e d f r o m c o r p o r a l u t e a o f non-pregnancy and those o f pregnancy. S c a r s d e r i v e d from a c c e s s o r y c o r p o r a l u t e a have a l l t h e c h a r a c t e r i s t i c s o f t h e c o r r e s p o n d i n g s c a r s f r o m t h e p r i m a r y c o r p o r a l u t e a w i t h which t h e y were a s s o c i a t e d .  Thus, t h e i r age i s e s t i m a t e d s o l e l y on t h e b a s i s o f  h i s t o l o g i c a l c h a r a c t e r i s t i c s s i n c e t h e y v a r y markedly  i n size.  Most o f them  a r e o f an i n s i g n i f i c a n t s i z e and c o u l d never be confused w i t h s c a r s d e r i v e d from primary corpora l u t e a .  However, s c a r s a r e p r o b a b l y r o u g h l y p r o p o r -  t i o n a t e i n s i z e t o t h e l u t e a l mass f r o m which t h e y a r e d e r i v e d and i t was noted e a r l i e r t h a t Type 3 a c c e s s o r y c o r p o r a l u t e a o c c a s i o n a l l y i n s i z e with primary corpora l u t e a .  overlapped  Any s c a r l e s s t h a n o n e - t h i r d t h e s i z e  o f t h e l a r g e r s c a r o f t h e same age i n t h e same p a i r o f o v a r i e s was c o n s i d e r e d t o be d e r i v e d f r o m a n a c c e s s o r y corpus  7. 8  luteum.  The B r e e d i n g Season. The  t i m i n g and v a r i a b i l i t y o f c o n c e p t i o n s and b i r t h s i s i m p o r t a n t ,  f o r i t r e l a t e s t o n a t u r a l s e l e c t i o n and t o f a c t o r s t h a t l i m i t p o p u l a t i o n s i z e . Presumably, s e a s o n a l b r e e d e r s produce t h e i r young a t a time o f y e a r d u r i n g w h i c h e n v i r o n m e n t a l f a c t o r s a r e o p t i m a l f o r t h e s u r v i v a l o f t h e young and, to a l e s s e r extent, the a d u l t s .  As p o i n t e d o u t by S a d l e i r  (1969), t h e r e i s  176  l i t t l e i n f o r m a t i o n on the r e l a t i v e e f f e c t s of environmental stresses on surv i v a l at various stages of the reproductive c y c l e . On the average, the breeding season of w h i t e - t a i l e d and mule deer i s e a r l i e r and more condensed at higher l a t i t u d e s than at lower l a t i t u d e s (Einarson, 1956;  Severinghaus and Cheaturn, 1956).  v a l i d f o r the Columbian b l a c k - t a i l e d deer.  These observations are  Most conceptions occur i n the  l a s t 2 weeks of November at Northwest Bay (49° 10' N l a t i t u d e ) and between 4-6° and 47° N l a t i t u d e i n Washington State (Brown, 1961).  Between 39°  and  42° N l a t i t u d e i n C a l i f o r n i a , the mean dates of conceptions f o r 5 populations of the subspecies varied from November 15 to December 25 ( B i s c h o f f , 1957). Ultimately,  the timing of the breeding season i s l i n k e d unquestionably to  b i o c l i m a t i c factors,and l a t i t u d e i s only a crude measure of r e g i o n a l differences.  The l a t e r and more v a r i a b l e breeding season i n southern deer,  u s u a l l y can be explained by these b i o c l i m a t i c f a c t o r s . are produced  climatic  Usually, the fawns  s h o r t l y a f t e r the s t a r t of the season of accelerated p l a n t growth,  which occurs as e a r l y a s . A p r i l i n some l o c a t i o n s and as l a t e as J u l y i n others. In northern l a t i t u d e s , there i s , or has been, considerable s e l e c t i o n pressure to produce and maintain a r e l a t i v e l y short breeding season i n deer.  I n the present study only two of 49 does conceived l a t e ( e a r l y i n  January), although three others were s t i l l c y c l i n g i n February and March. i s u n l i k e l y that they conceived.  It  A l l other females conceived at second ovu-  l a t i o n between about November 15 and December 5.  About 75% of the pregnant  does of 0. h. column!anus i n Washington State conceived w i t h i n a period of 15 days (Brown, 1961).  Breeding seasons of s i m i l a r l e n g t h occur i n mule deer  ( C h a t t i n , 1948; Robinette and Gashwiler, 1950; Lassen et a l . , 1952) w h i t e - t a i l e d deer (Cheatum and Morton, 1946; Ransom, 1966b).  Shaw and McLaughlin,  and 1951;  The fawns of l a t e breeders probably do not survive except  i n years when environmental c o n d i t i o n s are n e a r l y optimal.  A fawn born i n  177  mid-July  i n t h e Cedar Creek e n c l o s u r e  S i m i l a r l y , a fawn e s t i m a t e d  i n Oregon soon d i e d  (Hines,  1968).  t o have been b o r n i n August a t N o r t h w e s t Bay  was abandoned and near d e a t h when found i n November. The  s m a l l amount o f v a r i a b i l i t y i n t h e t i m i n g o f f i v e  successive  breeding  season (from 1963 t o 1967) a t N o r t h w e s t Bay i n d i c a t e s t h a t t h e  proximal  cause o f t h e b r e e d i n g  seasonally constant  season i s some e n v i r o n m e n t a l f a c t o r t h a t i s  (such as p h o t o p e r i o d ) ,  or t h e season i s c o n t r o l l e d by  endogenous rhythms w i t h w h i c h e n v i r o n m e n t a l f a c t o r s may i n t e r a c t . s y n c h r o n i z a t i o n o f most r e p r o d u c t i v e  c y c l e s c a n be e x p l a i n e d  The  by e x t e r n a l  factors. Although the t i m i n g of the breeding l a r g e l y by p h o t o p e r i o d , I n 1967 t h e b r e e d i n g  season may be c o n t r o l l e d  b i o c l i m a t i c f a c t o r s may m o d i f y t h e s y n c h r o n i z a t i o n .  season was l a t e .  The e a r l y p o r t i o n o f t h e s p r i n g  (March and A p r i l ) o f 1967 was c o l d e r and w e t t e r  t h a n n o r m a l , whereas t h e  summer (June t o September) was a b n o r m a l l y warm and d r y .  The c l i m a t e may  have a f f e c t e d t h e a n i m a l s d i r e c t l y , or i n d i r e c t l y t h r o u g h changes i n t h e vegetation.  C a p t i v e , f e m a l e w h i t e - t a i l e d deer on a low l e v e l o f n u t r i t i o n c o n -  c e i v e d about 10 days l a t e r t h a n w e l l - n o u r i s h e d  f e m a l e s (Verme, 1965)-  Some y e a r l i n g s a r e l a t e b r e e d e r s because t h e y a r e j u s t . a t t a i n i n g p u b e r t y and presumably have m a r g i n a l  endocrine stimulus.  a l l degrees of s e x u a l i t y , from a nonovulatory that of adults. f a i l to conceive,  s t a t e , t o f e r t i l i t y equal t o  Generally, small y e a r l i n g s are anovular, even a l t h o u g h  they ovulate.  undergo s e v e r a l o v u l a t o r y c y c l e s w i t h o u t l i n g s i n an e n c l o s e d  Yearlings express  l a t e b r e e d e r s , or  P o s s i b l y , some y e a r l i n g s  experiencing a true estrus.  Year-  herd o f b l a c k - t a i l e d d e e r i n Oregon were t h e l a s t t o  bear fawns each y e a r ( F i n e s , 1968).  178  7. 9  The Length Of The Estrous Cycle In Deer. In captive deer, the i n t e r v a l between copulations v a r i e s from 24-  to 29 days (Cheatum and Morton, 194-2; Cowan, 1956; Haugen, 1959).  I n two  0. h. columbianus females, estrus was apparently detected by behavioral changes every 22 to 25 days (West, 1968).  Previous to t h i s study, no i n f o r -  mation was obtained on the i n t e r v a l between successive ovulations i n f r e e ranging deer.  Therefore, i t was s t a r t l i n g to f i n d that the i n t e r v a l between  f i r s t and second o v u l a t i o n was only 8 or 9 days.  I have a t e n t a t i v e explana-  t i o n f o r the discrepancy between t h i s new f i n d i n g , and the r e s u l t s of previous workers. . Female deer i n the study area apparently never conceive on t h e i r f i r s t ovulatory c y c l e but most conceive at second ovulation.  P r i o r to f i r s t  o v u l a t i o n , sperm could not be detected i n the v a g i n a l smears of females whose f o l l i c l e s e x i b i t e d the t y p i c a l c h a r a c t e r i s t i c s of an animal i n estrus.  On  t h i s observation, and because ova of only two of s i x females were inseminated at f i r s t o v u l a t i o n , i t i s p o s s i b l e that a large percentage of females may not come i n t o f u l l estrus a t t h i s time.  This phenomenon, known as ' s i l e n t  heat' or 'quiscent estrus' i s r e l a t i v e l y common i n domestic sheep (Robinson, 1959), and i t i s known to occur i n domestic c a t t l e (Hammond, 1927), pigs (Pomery, i 9 6 0 ) , goats (Fraser, 1968), and horses ( B e r l i n e r , 1959).  There i s  a l s o some evidence that s i l e n t heats occur i n free-ranging Uganda kob (Buechner e t a l . , 1966), e l k (Morrison, i 9 6 0 ) , moose (Simkin, 1965), A f r i c a n elephants (Perry, 1953), hedgehogs,and bankvoles (Perry and Rowlands, 1962). In sheep, s i l e n t heats are common a t the beginning and a t the end of the breeding season ( A s d e l l , 1964).  In c a t t l e , s i l e n t heats are common i n the  e a r l y post-partum phase, p a r t i c u l a r i l y i n older cows (Fraser, 1968).  Hansel  et a l . (1961) determined that cows i n subestrus were f e r t i l e , i f inseminated. However, f e r t i l i z e d ova of the f i r s t c y c l e i n deer do not develop i n t o v i a b l e  179  embryos. I n animals w i t h a short breeding may  be l i k e n e d t o the r e a t t a i n m e n t  season, such as d e e r , each season  of p u b e r t y ,  comes i n t o f u l l r e p r o d u c t i v e performance. the t o t a l g o n a d o t r o p i c  content  i n t h a t each f e m a l e g r a d u a l l y  S t u d i e s on the s e a s o n a l  of p i t u i t a r y glands f r o m mule deer i n d i c a t e  t h e r e i s a g r a d u a l i n c r e a s e i n hormone l e v e l s i n the gland and f a l l  ( G r e i s e r and  Browman, 1956).  In animals  a p p a r e n t l y there i s s u f f i c i e n t gonadotropic and  d u r i n g the summer  experiencing s i l e n t  heat,  s t i m u l a t i o n t o evoke o v u l a t i o n  growth o f the c o r p u s l u t e u m but i n s u f f i c i e n t s t i m u l a t i o n t o e l i c i t  behavior. and  changes i n  However, some of the w i l d deer e x p e r i e n c e  t h e i r ova a r e f e r t i l i z e d .  Nevertheless,  these  estrus at f i r s t  soon f a i l and  estrous  ovulation  the f e m a l e  undergoes a second o v u l a t i o n , w h i c h u s u a l l y r e s u l t s i n pregnancy. E v i d e n t l y , deer undergo o v u l a t o r y or e s t r o u s c y c l e s o f two lengths.  The  s h o r t 8 t o 9 day f i r s t  c y c l e s o f 24 t o 28 days.  Short  ( o v u l a t o r y ) c y c l e may  (8 t o 9-day) f o l l i c u l a r  or more  be f o l l o w e d  by  c y c l e s f o l l o w second  o v u l a t i o n , so t h a t , p o t e n t i a l l y , a s e r i e s of o v u l a t o r y c y c l e s o f 8 t o 9 days i s possible. may  I f c o n c e p t i o n does not occur a t second o v u l a t i o n , the female  c o n t i n u e i n 8 t o 9-day f o l l i c u l a r  second, t h i r d , or f o u r t h f o l l i c u l a r w i t h each o f these f o l l i c u l a r and  24 t o 28 days.  One  cycles with estrus occurring  cycle.  cycles.  O v u l a t i o n may,  This hypothesis  not,  I n the Uganda kob,  t h a t the l e n g t h o f the e s t r o u s c y c l e was  e x p l a i n s c y c l e s of  s h o r t c y c l e s and  Buechner e t a l . (1966) 20 t o 26 days.  cycles  However, t h e i r every  12 t o 13 d a y s , and  s h o r t e r c y c l e s o f 5 t o 7 d a y s , i n w h i c h some e s t r o u s  t i v i t y was  ( v i s i t s t o the t e r r i t o r i a l b r e e d i n g  s h o r t e r , sub-estrous  8,  concluded  d a t a c l e a r l y I n d i c a t e some e s t r o u s c y c l e s ( r e s u l t i n g i n c o p u l a t i o n )  present  occur  would a l s o e x p e c t c y c l e s of 16 days.  Some o t h e r s p e c i e s o f mammals e x p e r i e n c e of d i f f e r e n t lengths.  or may  every  grounds).  c y c l e s were c o n f i n e d t o f e m a l e s a t t a i n i n g  ac-  Most o f puberty.  these  180  Remarkably, a few short c y c l e s were observed i n gravid females.  Estrous  cycles of three lengths occur i n confined populations of the tree shrew (Conaway and Sorenson, 1966). Obviously,  the length of an estrous cycle i s l a r g e l y dependent on  the f u n c t i o n a l l i f e of the corpus luteum.  I t i s w e l l documented that pro-  gesterone prevents o v u l a t i o n (Rothchild, 1 9 6 6 ) .  Known cycles i n deer may  be  explained on the basis of a "basic f o l l i c u l a r c y c l e " of 8 days, which i s modif i e d by the f u n c t i o n a l l i f e of the corpus luteum ( F i g . 27).  Short c y c l e s  occur i f the f u n c t i o n a l l i f e - s p a n of the corpus luteum i s l e s s than that of the follicle.  Long c y c l e s apparently occur i f the corpus luteum i s a c t i v e  for a period s u f f i c i e n t to block the o v u l a t i o n of two cycles of f o l l i c l e s . Presumably, o v u l a t i o n and estrous occur at the end of the t h i r d  follicular  c y c l e i n captive females because the corpus luteum i s no longer f u n c t i o n a l . T h e o r e t i c a l l y , intermediate-length  cycles should occur i f the corpus luteum  i s f u n c t i o n a l f o r more than 8 days but l e s s than perhaps 13 to 15 days. c e i v a b l y , a deer could experience cycles of a l l three lengths.  Con-  I t has been  shown t h a t , i n seasonal breeders, the i n t e n s i t y and duration of estrus i n creases with each c y c l e as the animal comes i n t o f u l l reproductive t i o n (West, 1968;  condi-  Fraser, 1 9 6 9 ) -  The i n t e r v a l between the f i r s t and second ovulatory c y c l e i n w i l d deer i s short but the length of subsequent cycles i s not known.  Only f i v e  females f a i l e d to conceive at second o v u l a t i o n and these animals, e s p e c i a l l y the three y e a r l i n g s , may have started to cycle l a t e i n the season. l e s s , one a d u l t apparently underwent two or probably three (subsequent to the second) i n 36 days.  Neverthe-  cycles  A y e a r l i n g doe probably cycled  twice (3rd and 4-th cycle) i n a minimum period of 4-6 days.  These scanty  observations i n d i c a t e that the length of the t h i r d and f o u r t h c y c l e s i n deer probably l i e s somewhere between 12 and 23 days.  F i g . 27.  1.  Schematic r e p r e s e n t a t i o n of reproductive c y c l e s i n deer. The bars represent the f u n c t i o n a l l i f e span of corpora l u t e a (CL) and f o l l i c l e s ( F o i l ) .  Short Cycle ( f i r s t c y c l e i n w i l d ; subsequent cycle i n w i l d ? ; f i r s t c y c l e i n some captive deer?)  j 5-6 days |  CL Foil  8 days  0  0  E or SE NP  E P  Long Cycle (captive deer; second and subsequent c y c l e i n w i l d deer?) U  - 1i  CL Foil 0  0  E P  E P  Intermediate-Length Cycle ( t h e o r e t i c a l ; second and subsequent cycles i n wild?)  8  CL Foil  0  8  0 E P  NP  "0 = Ovulation E = Estrus P = Pregnancy may  occur  SE - S i l e n t estrus NP = Pregnancy does not occur  182 Captive deer may experience a short cycle at the s t a r t of the breeding season, but i t would probably go undetected.  West (1968) noted a  s l i g h t peak of a c t i v i t y 10 days p r i o r to the f i r s t d e f i n i t e estrus i n a captive y e a r l i n g doe.  This a c t i v i t y may have i n d i c a t e d a s i l e n t heat.  However, there i s some evidence that s i l e n t heats, accompanied by corpus luteum formation, do not always occur i n captive females.  No corpora  l u t e a of non-pregnancy were present i n the ovaries of two captive animals that became pregnant. The l e v e l of n u t r i t i o n may be an important f a c t o r .  Conceptions  i n penned w h i t e - t a i l e d deer on a high l e v e l of n u t r i t i o n were d i s t r i b u t e d bimodally w i t h an i n t e r v a l of about 30 days between the modes.  Most con-  ceptions i n females on l o w - l e v e l n u t r i t i o n occurred between the mode dates of the high-plane females (Verme, 1965).  P o s s i b l y the low plane females  experienced a s i l e n t heat whereas the high plane animals did not. The long cycles i n captive animals may be likened to the pseudopregnant cycles of small mammals.  In confined deer, some stimulus may  production of l a r g e , l o n g - l i v e d corpora l u t e a .  cause  I t i s w e l l known that female  mice housed i n groups can experience long pseudopregnant  c y c l e s , even i n the  absence of mating (the LeeBoot e f f e c t , Parkes, 1962).  7. 10  F e r t i l i t y And P r o d u c t i v i t y Of Northwest Bay Deer R e l a t i v e To Other Populations. The Northwest Bay population has the highest f e r t i l i t y of a l l  i n v e s t i g a t e d populations of 0. h. columbianus.  The pregnancy r a t e was 85% •  i n y e a r l i n g s , 92% i n females 2.5 to 3.0 years o l d , and 97% i n females older than 3 years.  The above r a t e i n adults was duplicated by herds i n C a l i f o r n i a  ( B i s c h o f f , 1958) but lower r a t e s were recorded i n Washington State (Brown, 1961).  Fetus production by females of Northwest Bay was higher than i n the  183  other populations. per doe. O.45  The  For example, i n 1963-64, y e a r l i n g s averaged 0.90  c o r r e s p o n d i n g r a t e s i n C a l i f o r n i a and Washington were 0.36  r e s p e c t i v e l y (Bischoff,.1958;  of N o r t h w e s t Bay,  1.68  Brown, 1961).  per f e m a l e , was  C a l i f o r n i a h e r d s ( B i s c h o f f , 1958) population  fetuses  and  The  a l s o higher the 1.32  and  f e t a l rate i n a l l adults  than the 1.62  recorded i n  l i s t e d f o r the Washington  (Brown, 1961). Comparisons between the o v u l a t i o n r a t e s of v a r i o u s h e r d s i n d i c a t e  d i f f e r e n c e s i n the p o t e n t i a l p r o d u c t i o n environmental conditions.  A d u l t Columbian b l a c k - t a i l e d deer i n the  r e g i o n , i n Washington S t a t e , and  (1.67  The  study  i n C a l i f o r n i a have s i m i l a r o v u l a t i o n r a t e s .  I n the above o r d e r , the r a t e s were 1.67, 1958).  of f e t u s e s under the e x i s t i n g  1.57  (Brown, 1961)  and  1.70  (Bischoff,  o v u l a t i o n r a t e i n 45 a d u l t f e m a l e s t a k e n t h r o u g h o u t g e s t a t i o n  corpora  l u t e a per f e m a l e ) was  e q u a l t o the r a t e i n 105  a d u l t females  c o l l e c t e d d u r i n g the b r e e d i n g season. Pregnancy, f e t a l , and  o v u l a t i o n r a t e s of some h e r d s of mule  w h i t e - t a i l e d deer are s i m i l a r t o the r a t e s i n the s t u d y h e r d , but i n the r a t e s a r e much h i g h e r  (Robinette  B a n a s i a k , 1964;  e t a l . , 1965;  Hesselton  e t a l . , 1955;  Julander  Ransom, 1967b).  p a r t i c u l a r i l y i n w h i t e - t a i l e d d e e r , p u b e r t y and  and others  e t a l . , 1961 ;  I n mule d e e r ,  f u l l reproductive  and  output  i s a t t a i n e d much e a r l i e r t h a n i n Columbian b l a c k - t a i l e d d e e r . The  e f f i c i e n c y of the r e p r o d u c t i v e  t i l i t y of a p o p u l a t i o n .  p r o c e s s i s one measure o f the  I n d e e r , the e f f i c i e n c y of the  post-implantation  g e s t a t i o n a l phase i s r e f l e c t e d i n the amount of i n t r a u t e r i n e m o r t a l i t y . 3.3%  fer-  Only  o f the f e t u s e s examined i n t h i s s t u d y were moribund, w h i c h i s s i m i l a r t o  the 2.6%  r e c o r d e d by Brown (1961).  These e s t i m a t e s  are c o n s e r v a t i v e  the f e t u s e s were c o l l e c t e d a t i n t e r v a l s t h r o u g h o u t g e s t a t i o n .  because  However,  ob-  s e r v a t i o n s on the e x t e n t of i n t r a u t e r i n e m o r t a l i t y i n d e e r have shown t h a t  184 o n l y a s m a l l p r o p o r t i o n of i t o c c u r s i n the l a s t t h r e e - q u a r t e r s of the gest a t i o n a l p e r i o d (Taber, 1953;  R o b i n e t t e e t a l . , 1955;  e t a l . , 1965;  None of the 243  (1953) and 1000  Ransom, 1967).  Brown, 1961;  f e t u s e s examined by Taber  B i s c h o f f (1958) i n C a l i f o r n i a were d e f e c t i v e .  About 2% of over  mule deer f e t u s e s were moribund when c o l l e c t e d ( B i s c h o f f ,  Robinette  et a l . , 1955;  populations  N e l l i s , 1968).  Plesselton  1958;  D e f e c t i v e f e t u s e s are a l s o r a r e i n  of w h i t e - t a i l e d deer (Teer e t a l . , 1965;  Ransom, 1967).  I n the above d i s c u s s i o n , o n l y o b v i o u s l y p a t h o l o g i c a l embryos and f e t u s e s were c o n s i d e r e d .  I n most s p e c i e s , t h e g r e a t e s t l o s s of ova  p r i o r t o the s t a g e a t w h i c h embryos a r e m a c r o s c o p i c a l l y v i s i b l e . t h i s l o s s may  be e s t i m a t e d  corpora l u t e a .  occurs In deer,  by comparing the number of f e t u s e s t o the number o f  I t i s assumed t h a t p o l y o v u l a r f o l l i c l e s a r e r a r e , monozygotic  t w i n n i n g i s r a r e , and  t h a t a l l p r i m a r y c o r p o r a l u t e a p r e s e n t i n the  ovaries  a r e produced by f o l l i c l e s t h a t c o n t r i b u t e d ova a t the o v u l a t i o n i n w h i c h conception  occurred. From my  o b s e r v a t i o n s , and  deer r e p r o d u c t i o n , i t i s concluded  the o b s e r v a t i o n s  of o t h e r i n v e s t i g a t o r s of  t h a t the above assumptions a r e v a l i d .  l a r g e p o l y o v u l a r f o l l i c l e s o c c u r r e d i n more than 4OO I examined. 0 954)  N e i t h e r was  p a i r s of o v a r i e s t h a t  t h e r e any e v i d e n c e o f monozygotic t w i n n i n g .  r e p o r t e d one i n s t a n c e i n w h i c h one  corpus l u t e u m was  two abnormal f e t u s e s i n a Columbian b l a c k - t a i l e d doe.  has r e c o r d e d  concluded  associated with  suspected  774 i n four  In w h i t e - t a i l e d deer, Hesselton  t h r e e cases i n w h i c h the number o f c o r p o r a l u t e a was  t h a n the number o f f e t u s e s .  Golley  I n a sample o f  v i a b l e f e t u s e s i n Utah mule d e e r , monozygotic t w i n n i n g was i n s t a n c e s ( R o b i n e t t e e t a l . , 1955).  No  one  (1967) less  Because the f e t u s e s were of d i f f e r e n t sex,  he  t h a t the supernumary f e t u s e s a r o s e f r o m p o l y o v u l a r f o l l i c l e s .  Teer e t a l . , (1965) observed fewer c o r p o r a l u t e a t h a n embryos i n 2.5% pregnant females.  Nevertheless,  both events are u n q u e s t i o n a b l y  of  525  r a r e and  may  be i g n o r e d i n comparisons between the numbers o f c o r p o r a l u t e a and  fetuses.  185  The t h i r d assumption i s v a l i d i f accessory corpora l u t e a are excluded from the counts of corpora l u t e a .  In deer, t h i s separation can be made on the  basis of s i z e . Remarkably, i n t h i s study, the number of corpora l u t e a l a r g e r than 30 mm  3  (92), i n females pregnant when c o l l e c t e d , equalled the number of  fetuses.  Because three of the fetuses were moribund, each corpus luteum  was represented on the average by 0.97 v i a b l e fetuses.  I n a d d i t i o n , one of  the non-pregnant females (a y e a r l i n g ) had been temporarily pregnant e a r l i e r i n the season, as revealed by a l a r g e f i b r o u s scar i n i t s ovaries.  Therefore,  the minimum l o s s of embryos, a f t e r approximately the implantation stage,was 4.3% (4-/93). In l a t e November or December c o l l e c t i o n s , the three females that d i d n ' t become pregnant would have contained four corpora l u t e a .  Thus,  on the average, each corpus luteum i n f a l l c o l l e c t i o n s was subsequently represented by no more than 0.92 t o t a l l o s s of ova ivras 8.3%.  v i a b l e fetuses.  Therefore, the minimum  The above c a l c u l a t i o n s , and others s p e c i f i c  to age-classes, are summarized i n Appendix 8. The o v e r a l l l o s s of ova i n females of Northwest Bay i s lower than the l o s s i n other populations of deer.  In pregnant females,, the number of  corpora l u t e a u s u a l l y exceeds the t o t a l number of fetuses by 3% to 12% (Taber, 1953; Robinette et a l . , 1955; B i s c h o f f , 1958; Taber and Dasmann, 1958; Brown, 1961; Teer et a l . , 1965; N e l l i s , 1968).  The l o s s of embryos a f t e r the  implantation stage was 4-3% i n t h i s study, 6.9% i n the same subspecies i n Washington State (Brown, I961),and 8.8% i n mule deer i n Utah (Robinette et a l . , 1955).  The c a l c u l a t e d d i f f e r e n c e between the ovulation r a t e i n samples  from the breeding season and the maximum production of v i a b l e fetuses i n these females was 8.3% i n the study herd, 14-5% i n Washington deer (calculated from the data of Brown, 1961), and 16.5% i n mule deer i n Utah (calculated from the data of Robinette et a l . , 1955).  Loss of ova i n the f i r s t month,is determined  186  from the r a t i o of the t o t a l number of embryos (which reached the implantat i o n stage) to the number of corpora l u t e a . study was about 4-%. tilized.  The pre-implantation l o s s i n t h i s  Some of i t represents l o s s of ova that were never f e r -  The minimum pre-implantation l o s s i n deer of Washington State v/as  5.1% (Brown, 1961) and i n Utah deer i t was 7.5% (Robinette et a l . ,  7. 11  1955).  The E f f e c t Of Age On F e r t i l i t y . The f e r t i l i t y  of female deer a t Northwest Bay g r a d u a l l y increase  with age from 1.5 years to 5-5-6.5- years and t h e r e a f t e r decreased s l i g h t l y . A s i m i l a r trend was noted i n w h i t e - t a i l e d deer by Severinghaus and Cheatum (1956).  Nevertheless, females apparently reproduce to f u l l c a p a c i t y almost  u n t i l they become d e c r e p i t from old age.  Semi-tame deer aged 17 to 21 years  have been known to produce a t or near c a p a c i t y v i r t u a l l y u n t i l t h e i r death (Cowan, 1956; Severinghaus and Cheatum, 1956).  Reduced pregnancy r a t e s ,  fetus production, and o v u l a t i o n r a t e s i n old females have been noted i n Columbian b l a c k - t a i l e d deer (Taber and Dasmann, 1958; Brown, 1961) and mule deer (Robinette et a l . , 1955).  The l a t t e r found that the l o s s of ova  was greater i n old does than i n 'prime' females.  Cheatum and Morton (194-6)  observed that old does were productive on good range but r e l a t i v e l y unproductive on poor range. The age composition of the female component of the population has an important bearing on p r o d u c t i v i t y .  The r a t e of increase of a population  (excluding m o r t a l i t y and d i s p e r s i o n ) i s profoundly influenced by the time at which puberty and f u l l reproductive c a p a c i t y i s a t t a i n e d .  In t h i s r e s -  pect, the e c o l o g i c a l or r e a l i z e d n a t a l i t y of the Northwest Bay population i s considerably l e s s than the p a r t i a l reproductive p o t e n t i a l of the species, as e x i b i t e d by females kept i n c a p t i v i t y under somewhat l e s s than optimum condtions.  For example, there i s record of a semi-tame doe producing 18 fawns  187  i n her f i r s t 10.5 years of l i f e (Cowan, 1956).  Captive female Columbian  b l a c k - t a i l e d deer are capable of producing one fawn a t about one year of age (Rampont, 1926; Shantz, 194-3, quoted i n Brown, 1961; Cowan and Wood, 1955) and t h e r e a f t e r twins are u s u a l l y produced.  Excluding fawns, the above  r a t e of fetus production would y i e l d a p r o d u c t i v i t y of 200 fa\ms per 100 f e males of reproductive age.  In the study region, the corresponding produc-  t i v i t y was estimated to be 137 fawns per 100 females. F u l l p r o d u c t i v i t y was attained even l e s s slowly i n populations of the  same subspecies i n Washington (Brown, 1961) and C a l i f o r n i a (Taber, 1953;  B i s c h o f f , 1958).  Puberty, and f u l l reproductive c a p a c i t y ( f o r a p a r t i c u l a r  environment) i s reached more q u i c k l y i n some populations of mule deer (Robinette et a l . , 1955; Julander et a l . , 1961), and much e a r l i e r i n some populations of w h i t e - t a i l e d deer (Morton and Cheatum, 194-6, 194-6, G i l l , 1956; Robb, 1959; Haugen and Trauger, 1962; Banasiak, 1964; Hesselton e t a l . , 1965; Nixon, 1965; Ransom, 1967).  Studies of both n a t u r a l and confined  populations of deer have shown that n u t r i t i o n i s a key f a c t o r i n the r a t e at which females a t t a i n puberty and f u l l reproductive c a p a c i t y (Morton and Cheatum, 1946; Cheatum and Severinghaus, 1950; Taber , 1953; Robinette et a l . , 1955, Cowan, 1956; G i l l , 1956; Taber and Dasmann, 1958; Brown, 1961; Julander et a l . , 1961; Severinghaus and Tanck, 1964; Teer et a l . , 1965; Verme, 1965; Murphy and Coates, 1966).  In deer, the age a t which puberty and f u l l  reproductive output i s attained i s undoubtedly dependent on the r a t e of growth, the subject of the next t o p i c .  7. 12  The E f f e c t Of Size On F e r t i l i t y . There i s l i t t l e i n f o r m a t i o n i n the l i t e r a t u r e on the e f f e c t s of  s i z e , w i t h i n age c l a s s e s , on f e r t i l i t y .  The r e s u l t s of t h i s study c l e a r l y  i n d i c a t e that w i t h i n an age c l a s s at Northwest Bay, l a r g e females are more  188  f e r t i l e than small females.  Differences i n f e r t i l i t y were most evident i n  females up to 4 years o l d , e s p e c i a l l y i n y e a r l i n g s . 2/3  Those l e s s than about  the average maximum weight of adults are u n l i k e l y to reproduce; those  equal i n weight to adults reproduce at the r a t e of a d u l t s . Several i n v e s t i g a t o r s have shown that the f e r t i l i t y of deer i n d i f f e r e n c e herds, or w i t h i n one herd over a period of time, c o r r e l a t e s w e l l with d i f f e r e n c e s i n p h y s i c a l a t t r i b u t e s . Robinette et a l . (1955) reported a s i g n i f i c a n t l y lower pregnancy and f e t a l r a t e i n y e a r l i n g mule deer dying from m a l n u t r i t i o n than i n those dying v i o l e n t l y .  G i l l (1956) found a good cor-  r e l a t i o n between s i z e and p r o d u c t i v i t y of w h i t e - t a i l e d deer i n several r e gions of West V i r g i n i a . of mule deer was  The per cent d i f f e r e n c e i n weight between two herds  exceeded by the per cent d i f f e r e n c e i n the o v u l a t i o n r a t e  (Julander et a l . , 1961).  Bansiak (1964) ranked w h i t e - t a i l e d deer from f i v e  regions i n Maine on the basis of three s i z e measurements and found that the o v u l a t i o n r a t e c o r r e l a t e d quite w e l l with the ranks.  As the s i z e of deer  i n the Seneca Army Depot increased, the p r o d u c t i v i t y of the herd also i n creased (Hesselton et a l . , 1965).  Nixon (1956) a l s o found a good c o r r e l a t i o n  between p r o d u c t i v i t y and the s i z e of deer i n various herds i n Ohio.  Similar  r e s u l t s were reported by Teer et a l . (1965) a f t e r a study of the small w h i t e - t a i l e d deer of the Central Mineral Basin i n Texas.  The p r o d u c t i v i t y  of subspecies of deer that are not g e n e t i c a l l y i s o l a t e d apparently w e l l with m u l t i p l e p h y s i c a l v a r i e n t s over extensive geographical  correlates regions.  In a l l of the above studies on n a t u r a l populations, d i f f e r e n c e s i n s i z e were a t t r i b u t e d to n u t r i t i o n a l d i f f e r e n c e s . Growth r a t e d i f f e r e n c e s between and w i t h i n species, or even w i t h i n a population, have an important bearing on p r o d u c t i v i t y . showed t h a t , w i t h i n a subspecies, weight) was retarded  Bandy (1965)  the r a t e of growth (as measured by t o t a l  on a low plane of n u t r i t i o n , thereby i n c r e a s i n g the  age  189  at which the u l t i m a t e maximum s i z e was a t t a i n e d .  Murphy and Coates (1966)  noted that growth of fawns was retarded when they were fed low p r o t e i n d i e t s . When fawns of a r e l a t i v e l y small and unproductive w h i t e - t a i l e d deer herd ( i n the Central Adirondacks, New York) were transferred to c o r r a l s i n a l e s s harsh environment, they grew l a r g e r and became much more productive than t h e i r counterparts i n the o r i g i n a l environment (Severinghaus and Tanck, 1964). The p a r a l l e l decrease i n both the s i z e and p r o d u c t i v i t y of youngdeer d u r i n g the course of t h i s study i s i n accord w i t h the above r e s u l t s . I t i s hypothesized that the p r o d u c t i v i t y of a deer population could be e s t i mated quite a c c u r a t e l y from s i z e measurements taken i n November - i f the exact age of each female was determined.  I t would be necessary to e s t a b l i s h  the r e l a t i o n s h i p between the s i z e v a r i a b l e and the reproductive r a t e , w i t h i n each age-class (older age-classes could be grouped).  Large samples would  be required to e s t a b l i s h accurate r e l a t i o n s h i p s , however, a s t a r t could be made from i n f o r m a t i o n obtained i n t h i s study.  190  8.  SUMMARY O v a r i a n changes, p a t t e r n s o f r e p r o d u c t i o n , and a g e - s p e c i f i c p r o -  d u c t i v i t y o f f e m a l e Columbian b l a c k - t a i l e d deer ( O d o c o i l e u s hemionus c o l u m b i a n u s ) on Vancouver examination of o v a r i e s .  I s l a n d were e l u c i d a t e d l a r g e l y by h i s t o l o g i c a l The o v a r i e s o f 444 f e m a l e s , r a n g i n g i n age f r o m  64  days p o s t - c o n c e p t i o n t o 19-5 y e a r s , were s e c t i o n e d a t i n t e r v a l s o f 90 p. or 190 p; a n o t h e r 33 p a i r s were examined  i n t h i c k , u n s t a i n e d s e c t i o n s and  sequently i n h i s t o l o g i c a l sections.  sub-  R e p r o d u c t i v e f u n c t i o n was r e l a t e d t o  s p e c i f i c ages as d e t e r m i n e d f r o m s t a i n e d s e c t i o n s o f t e e t h . The f o l l o w i n g i s a l i s t of the most i m p o r t a n t f i n d i n g s . 1.  Oogenesis v i r t u a l l y ceases i n t h e f e t a l o v a r y 30 days b e f o r e b i r t h . O v a r i e s i n a f e t u s f r o m a c a p t i v e doe were l a r g e r and f u r t h e r d e v e l o p e d t h a n t h o s e i n f e t u s e s o f the same age f r o m w i l d f e m a l e s .  2.  W e l l - d e f i n e d f o l l i c u l a r c y c l e s o c c u r t h r o u g h o u t most of t h e y e a r , i n c l u d ing  the p e r i o d of g e s t a t i o n .  They occur a t 8-day i n t e r v a l s d u r i n g t h e  b r e e d i n g season and t h e i r growth seems t o be i n d e p e n d e n t o f l u t e a l f u n c tion.  The  average s i z e o f the l a r g e s t f o l l i c l e i n p r e g n a n t f e m a l e s i n -  c r e a s e d s i g n i f i c a n t l y f r o m 21 mm  3  May and 3.  i n F e b r u a r y and March t o 45 mm  3  in  June.  F o l l i c l e s a t f i r s t o v u l a t i o n v a r i e d c o n s i d e r a b l y i n s i z e and i n s t a g e o f m a t u r a t i o n ; many were a t r e t i c a t r u p t u r e .  Large f o l l i c l e s that f a i l e d to  r u p t u r e became p a r t l y l u t e i n i z e d ; t h e s e and s m a l l e r ones o c c u r r e d i n 47% of 4.  the f e m a l e s a f t e r f i r s t  ovulation.  A p p a r e n t l y , pregnancy never ensues i n November.  Even f e r t i l i z e d  f r o m t h e f i r s t o v u l a t i o n o f t h e season  ova soon cease development.  The. p r e s e n c e o f  sperm on o n l y two o f s i x ova s u g g e s t s . t h a t ' s i l e n t h e a t s ' o c c u r f r e q u e n t l y at f i r s t  ovulation.  191  5.  R e l a t i v e l y s m a l l c o r p o r a l u t e a (45 mm  ) d e v e l o p a f t e r the f i r s t  but t h e y cease growth a f t e r 2-3 d a y s , b e g i n t o d e g e n e r a t e a f t e r 5 or 6 d a y s , and r a p i d l y r e g r e s s a f t e r second 6.  ovulation, about  ovulation.  C o r p o r a l u t e a of the second o v u l a t i o n , which succeeded the f i r s t by  8-9  3 d a y s , r a p i d l y grow t o 100 mm c e i v e d a t second 7.  .  N i n e t y - s i x per c e n t o f the f e m a l e s con-  ovulation.  The b r e e d i n g season o c c u r r e d a t about the same t i m e i n each of t h e 5 y e a r s , e x c e p t a f t e r the h o t , d r y summer of 1967, when i t was 3-4- days  8.  late.  F i r s t and second o v u l a t i o n s u s u a l l y began about November 5 and November 14, r e s p e c t i v e l y .  About 50%, o f the a d u l t does o v u l a t e d f o r the f i r s t  t i m e between November 10 and November 18, and f o r the second time between November 17 and November 25.  On the a v e r a g e , y e a r l i n g s o v u l a t e d  l a t e r t h a n a d u l t s and t h e i r d a t e s of o v u l a t i o n were more v a r i a b l e . 9.  S c a r s o f c o r p o r a l u t e a o f non-pregnancy  i n l a t e and n o n - c o n e e i v e r s ,  i n d i c a t e t h a t f e m a l e s c y c l e a t l e a s t f i v e t i m e s i f pregnancy does n o t occur. 10. ' There were 93 f i r s t - c y c l e c o r p o r a l u t e a and 97 c o r p o r a l u t e a f r o m t h e second c y c l e i n f e m a l e s t h a t had o v u l a t e d t w i c e a t the time o f c o l l e c t i o n . Thus, f u t u r e p r o d u c t i o n o f f e t u s e s can be e s t i m a t e d i n f e m a l e s h a v i n g o v u l a t e d o n l y once a t t h e t i m e of c o l l e c t i o n . 11.  The q u a n t i t a t i v e and q u a l i t a t i v e changes i n o v a r i a n s t r u c t u r e s d u r i n g the  b r e e d i n g season were a s c e r t a i n e d i n 12 f e m a l e s whose f o l l i c l e s and  c o r p o r a l u t e a were d a t e d by t h e c l e a v a g e s t a g e o f f e r t i l i z e d second o v u l a t i o n ) .  ova (shed a t  I t was assumed t h a t deer ova segment a t about the  same r a t e as the ova o f d o m e s t i c u n g u l a t e s .  Dates o f o v u l a t i o n f o r a l l  f e m a l e s c o l l e c t e d I n November and e a r l y December were e s t i m a t e d f r o m c r i t e r i a f o r m u l a t e d f r o m t h e 12 f e m a l e s .  192  12.  Corpora l u t e a o f pregnancy c o n t a i n e d d i s t i n c t i v e c e l l s s h o r t l y a f t e r  con-  c e p t i o n ; p e r s i s t e d t o p a r t u r i t i o n ; i n c r e a s e d i n s i z e d u r i n g t h e l a s t few  3 weeks o f t h e 200-day g e s t a t i o n p e r i o d ; and those l a r g e r t h a n 30 mm corresponded i n number t o t h e f e t u s e s i n pregnant f e m a l e s . 13.  Only t h r e e o f 92 f e t u s e s (3.4%) were d e f e c t i v e , b u t t h i s e s t i m a t e i s c o n s e r v a t i v e because samples were o b t a i n e d t h r o u g h o u t g e s t a t i o n .  The  minimum l o s s o f ova i n f e m a l e s t h a t became pregnant was 4.3%; i t was 8.3% i n a l l f e m a l e s t h a t produced 14.  c o r p o r a l u t e a d u r i n g t h e b r e e d i n g season.  Three t y p e s o f a c c e s s o r y c o r p o r a l u t e a occur i n d e e r : unruptured f o l l i c l e s , pregnancy,  Type 1 d e v e l o p s i n  Type 2 forms i n r e g r e s s i n g c o r p o r a l u t e a o f non-  and Type 3 d e v e l o p s i n s m a l l f o l l i c l e s  about t h e time o f o v u l a t i o n .  t h a t r u p t u r e a t or  Any c o r p o r a l u t e a l e s s t h a n o n e - q u a r t e r the  volume o f t h e l a r g e s t corpus l u t e u m i n a p a i r o f o v a r i e s , were d e f i n e d as a c c e s s o r y c o r p o r a l u t e a . 15.  They o c c u r r e d i n 3 6 % i n p r e g n a n t f e m a l e s .  F e t a l growth c u r v e s f o r h i n d - f o o t l e n g t h and g i r t h were e s t a b l i s h e d , u s i n g o n l y f e t u s e s c o n c e i v e d a t second o v u l a t i o n s , t h e mean d a t e o f w h i c h was known. of  Then t h e c u r v e s were used .to d e t e r m i n e t h e c o n c e p t i o n d a t e s  two f e m a l e s t h a t c o n c e i v e d on l a t e r c y c l e s .  The l i m i t e d i n f o r m a t i o n  obtained from these suggest t h a t the l e n g t h of e s t r o u s c y c l e s to 16.  subsequent  t h e second, was between 12 and 23 days..  Corpora l u t e a o f pregnancy r e g r e s s r a p i d l y a f t e r p a r t u r i t i o n , but p e r s i s t as d i s t i n c t s c a r s f o r t h e l i f e o f t h e f e m a l e . r e p r o d u c t i v e performance  Thus, t h e l o n g - t e r m  o f t h e p o p u l a t i o n was r e v e a l e d by t h e l i n e a r  r e l a t i o n s h i p between t h e age o f t h e doe and t h e average number o f s c a r s per doe.  However, o n l y s c a r s up t o about 8 months o f age a r e r e a d i l y  d i s t i n g u i s h e d from older s c a r s .  The r a t e o f s c a r r e g r e s s i o n i s i n v e r s e l y  p r o p o r t i o n a l t o t h e age o f t h e doe.  193  17.  Some o f the o v a r i a n s t r u c t u r e s , such as r e g r e s s i n g c o r p o r a l u t e a on pregnancy, a c c e s s o r y c o r p o r a l u t e a , and  non-  s c a r s of c o r p o r a l u t e a of  pregnancy, c o u l d n o t be i d e n t i f i e d w i t h c e r t a i n t y i n t h i c k ,  unstained  sections. 18.  Reproductive and  r a t e s were o b t a i n e d f r o m counts of f e t u s e s , c o r p o r a  corpora l u t e a scars.  d i s t r i b u t i o n , to estimate 19.  These were u s e d , i n c o n j u n c t i o n w i t h the  and  f r o m 0.91  i n y e a r l i n g s t o 1.81  t h e r e a f t e r i t decreased.  increased progressively,  i n the 5-5  and  6.5  age-classes,  However, the r e l a t i v e c o n t r i b u t i o n of  f e t u s e s f r o m each a g e - c l a s s d e c r e a s e d w i t h age. r e p r o d u c t i v e age produce about 137 20.  age  the p r o d u c t i v i t y o f the p o p u l a t i o n .  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Reproductive physiology of vertebrates.  W. B.  Van Wagenen, G. and M. E. Simpson. 1965. Embryology o f t h e Ovary and t e s t i s o f Homo.sapiens and Macaca m u l a t t a . Y a l e U n i v . P r e s s , New Haven. Verme, W. J . 1965. R e p r o d u c t i o n s t u d i e s on penned w h i t e - t a i l e d d e e r . J . W i l d l . Mgmt. 29:74-79West, N. 0. 1968. The l e n g t h o f t h e e s t r o u s c y c l e i n t h e Columbian b l a c k - t a i l e d deer or Coast deer ( O d o c o i l e u s hemionus c o l u m b i a n u s ) . B.Sc. T h e s i s , U n i v . o f B r i t i s h Columbia, Vancouver. W i n t e r s , L.. M., W. W. Green and R. E. Comstock. 1942. P r e n a t a l development of t h e b o v i n e . M i n n e s o t a Agr. Exp. S t a . Tech. B u l l . No. 151. W h i t e , R, F., A. T. H e r t i g , J . Rock and E. Adams. 1951. H i s t o l o g i c a l and h i s t o c h e m i c a l o b s e r v a t i o n s on t h e corpus l u t e u m o f human pregnancy w i t h s p e c i a l r e f e r e n c e t o c o r p o r a l u t e a a s s o c i a t e d w i t h e a r l y normal and abnormal ova. C o n t r i b . Embryol.34:55-74W h i t e , M. 1966. P o p u l a t i o n e c o l o g y o f some w h i t e - t a i l e d d e e r i n S o u t h T e x a s . Ph.D. T h e s i s , Purdue U n i v . Wynne-Edwards, V. C. 1962. A n i m a l d i s p e r s i o n i n r e l a t i o n t o s o c i a l beh a v i o u r . Hafner Pub. Co.  Appendix 1.  The e a r l y c l e a v a g e o f f e r t i l i z e d ova i n s e l e c t e d mammalian s p e c i e s .  Species and Ref  Age Ref Point C EE C C  Cattle* Cattle^ Goat SheepT: Swine? Swine 3  SE^  Elapsed 1-cell 33 23-52 30 0-38 0-51 36-54  time ( h r ) f r o m r e f e r e n c e p o i n t t o r e c o v e r y a t v a r i o u s c e l l 2-cell 50-62 40-56 30-48 38-39 51-66 60-69  3-to 4 - c e l l 44-66  60 42 66-72 60-90  5-to 8 - c e l l 62-64 46-96 85 44 90-110 75-96  9-to 1 6 - c e l l  stages-.-* Morula  110 71-141 98 .65-77 96+  134 ' 144 120-140 96 110-114  O v u l a t i o n o c c u r s about 24-30 h r a f t e r the s t a r t o f e s t r u s and about 10-15 h r p o s t - e s t r u s i n t h e cow (Ref 2 ) ; about 30-36 h r a f t e r t h e s t a r t o f e s t r u s i n t h e goat ( S a l i s b u r y , 1961); about 24 h r s a f t e r the s t a r t o f esfcrus i n t h e sheep ( A s d e l l , 1964); and about 30 h r a f t e r t h e s t a r t o f e s t r u s i n t h e p i g (Ref 6 ) . Reference. 1. 2. 3. 4. 5. 6.  W i n t e r s e t a l . , 1942. H a m i l t o n and L a i n g , 1964. Amoroso e t a l . , 1942. C l a r k , 1934. Heuser and S t r e e t e r , 1929. O x e n r e i d e r and Day, 1966.  C SE EE /  coitus start of estrus end o f e s t r u s c o i t u s f o l l o w e d w i t h i n 6 hours  Appendix 2.  Specimen No.  The volume o f growing c o r p o r a l u t e a o f t h e second o v u l a t o r y c y c l e (CL 2 ) ; t h e volume o f d e g e n e r a t i n g c o r p o r a l u t e a o f t h e f i r s t o v u l a t o r y c y c l e (CL l ) ; and t h e r a t i o o f t h e l a r g e s t CL 1 ( i n t h e same p a i r o f o v a r i e s ) ; a t i n t e r v a l s f o l l o w i n g second o v u l a t i o n  Estim Age CL 2a (days)  Z24 V19 V21  1 1 1  X61 K73 Z23 V20 Vll V30 V28  2 2 2  Z26  V27 a b  -  5  2.5  45 5+  118, 107  3 3  V24  -A  (mm-) 7.1 7.2, 6.3 12, 18, 21, 29, 38,  1.5  Vol CL 1 (mm3)b  Vol C L  6.4-  11 8.0 17 27 30 8 5 / 58 66 77, 68  82  27 14., 12 6.7, 5.412, 5 8.4-, 4.1 6.3, 5.9 4-.8, 4-.-45.5, 5.3 5.7, 4.2 3.5 3.3, 1.2 2.8 0.8  Ratio Vol L g s t CL 2 to the v o l L g s t CL 1 0.3 0.5 0.9 1.0 2.1 3.3 6.5 6.9 14.9 18.8 23.3 29.3 14.8  E s t i m a t e d by t h e c l e a v a g e s t a g e o f t h e ovum. I f two p r e s e n t t h e volumes o f b o t h a r e g i v e n . Only i f volume i s > 10 mm i s i t r e c o r d e d t o n e a r e s t d e c i 3  mal.  Appendix 3. The t i m i n g and v a r i a b i l i t y o f t h e b r e e d i n g seasons f r o m 1963 t o 1967 based on s t a t i s t i c s d e r i v e d from frequency d i s t r i b u t i o n s of dates of ovulation.  Dates o f Second 1963  196-4  1965  1966  1967  X  25.2 3.15 0.72 19  21.3 3.19 1.02 10  20.1 3.80 1.43 7  26.7  3^56 0.80 20  x n  23.0 3.20 0.78 17  2-4.7 2.95 0.93 10  21.3 3.19 1.03 10  19.5 3.73 1.52 6  27.4 4.00 0.97 16  X  All Does  s s_ n  Adults  O v u l a t i o n from 1963 t o 1967  X  s s  4.00  0.97 17  The d a t e s o f f i r s t o v u l a t i o n advanced 8 days and added t o t h e d a t e s o f second o v u l a t i o n . 1963  1964  1965  1966  1967  24.4 5.88 0.82 51  23.9 5.42 1.01 29  20.9 5.07 1.06 23  22.2 5.09 1.17 19  26.9 3.66 0.80 21  X s  23.8  x n  0„80 40  23.1 4.72 1.14 17  21.1 5.10 1.14 20  21.5 4.73 1.15 17  26.4 3.83 0.96  Adults  .All Does  X  s x n s  s  5.06  16  Appendix 4.  A g e - s p e c i f i c pregnancy r a t e and f e t u s - p r o d u c t i o n  Concept Age ( y r )  0.5  1.5  -4  1  c°>  vO ON  2.5 3.5  r-H  6  6  8.5  2.5  4.5 1.5 2.5  5.5  13.5  T o t a l or Avg All years combined  8  6 2  6.5  1.5  2.5 3.5 A.5  5.5  6.5  8.5  9.5 12.5 13.5  T o t a l or Avg  No. Preg.  0 8 7  8 5  T o t a l or Avg  vO vO 1 UA vO OA  8 10  4.5 5.5  9.5 12.5 -<f UA vO sO O 1 rH  No.  8 5 5 2  1 1  5 1 47 1 1  3 3  3 3  5 1 51  1 1 8 13 12 6 9 6 6 2  5 1  1  61  1 1 8 11 11 6 9 6  5  2  5 1 1 57  % Preg.  00.0 80.0  87.5 100.0 100.0 100.0  83.3  100.0 100.0  100.0 92.2 100.0 100.0 100.0 100.0 100.0 100.0 100.0 84.6 91.7  100.0 100.0 100.0 83.3 100.0 100.0  100.0 100.0 93.4  r a t e o f N o r t h w e s t Bay f e m a l e s , 1963-66.  Healthy Fetuses  Moribund Fetuses  Fetuses p e r doe  0 . 9 11 8  0 0  00.00 0.90  13  10 11 4 8 2 76 2 2 3 3 2 1 9 12 16 8 15 12  11 4 ' 8 2 1 89  0  2 0 0 0 0 1  0 3 0 0 0 0 0 0 0 0 0 2 0 0 0 0 1 0 0 3'  Fetuses per p r e g . doe  00.00 1.13  Healthy Fetuses per Doe  00.00 0.90 1.38 1.33 1.63 2.00  2.00  1.57 1.67 1.63 2.00 2.20 2.00  2.00  2.00  1.80  2.00 1.60 2.00  2.00 2.00 1.00 1.00 2.00 1.00  2.00 2.00 1.00 1.00 2.00 • 1.00  2.00 2.00 1.00 1.00 2.00 1.00  0.92 1.33 1.67 1.67 2.00  1.09 1.45 1.33 1.67 2.00 2.20 2.00  0.92 1.33 1.33 1.67 2.00  1.38  1.67 1.63 2.00  1.83  1.80  1.83  2.00  1.80  2.00 1.00  1.80  2.00 1.00  '  1.83  1.83  2.00 1.60 2.00 1.00  Healthy Fetuses per preg. doe  00.00 1.13  1.57 1.33 1.63 2.00 2.20 2.00 1.60 2.00 2.00  2.00  1.00 1.00 2.00 1.00  1.09 1.45 1.33 1.67 2.00 • 2.20 2.00 1.60 2.00 1.00 ro o  208  Appendix  Year  5.  The r e l a t i o n s h i p between t h e number o f c o r p o r a l u t e a (CL) o f t h r e e s i z e s and t h e number o f f e t u s e s i n 55 p r e g n a n t does.  Concept. No. No. o f C L Age ( y r ) >5mm3 >l0mm3 >30mm3  Total Fetuses  Fetuses Per CL >30 mm  Moribund Fetuses  Healthy F e t u s e s Per CL>30mm3.  0 0 2 0 0 0 0 1 0  1 .00 1.14 0.73 1 .00 1 .00 1 .00 1 .00 0.89 1 .00  3  9 5 6 7 5 5 2 5 1  8 8 11 11 10 11 4 9 2  7 8 11 10 10 11 4 9 2  7 7 11 10 10 11 4 9 2  7 8 10 10 10 11 9 2  1 .00 1 .14 0.91 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00  45  74  72  71  71  1 .00  3  0.96  1 1  2 2  2 2  2 2  2 2  1 .00 1 .00  0 0  1 .00 1 .00  2  4  4  4  4  1 .00  0  1 .00  1.5 2.5 5-5 13.5  3 3 1 1  3 3 2 1  3 3 2 1  3 3 2 1  3 3 2 1  1 .00 1 .00 1 .00 1 .00  0 0 0 0  1 .00 .1 .00 1 .00 1 .00  or Avg  8  9  9  9  9  1 .00  0  1.00  1.5 2.5 3.5 4.5 5.5 6.5 8.5 9.5  12 9  11 13 11 13 12 11  10 13 11 12 12 11  10 12 11 12 12 11  10 13 10 12 12 11  9 2 1  1.00 1.08 0.91 1 .00 1 .00 1.00 1.00 1.00 1 .00 1.00  0 0 2 0 0 0 0 1 0 0  1.00 1 .08 0.73 1.00 1.00 1.00 1 .00 0.89 1 .00 1 .00  84  1.00  3  0.96  1.5 2.5 3.5 4.5 5.5 6.5 8.5 9.5 12.5 T o t a l s or Avg i  -4- ir\ vO NO ON \—  2.5 4-5  T o t a l s or Avg  vO  I  ON  O O  w  12.5 13.5 T o t a l s o r Avg  6  8  6 5  2  A  5  4  4  1 1  9 2 1  9 2 1  9 2 1  55  87  85  84  A  • A  Appendix  6.  The a g e - s p e c i f i c o v u l a t i o n r a t e (based on c o u n t s o f c o r p o r a l u t e a ) o f f e m a l e s t h a t had o v u l a t e d i n November and early-December. (The sample s i z e i s i n parentheses) O v u l a t i o n Rate I n t h e Year  Indicated  Age ( y r )  1963  1964  1965  1966  1967  1963-67  1-5  1.08 (13) 1.65 (17) 1.20 (5) 2.00 (1) 2.00 (8) 1.75  1.17 (12) 1.40 - (5) 1.50 (2) 1.75  I.4O  1.00 (6) 1.33 (3) 1.67 (3)  1.00 (5) 2.00 (2) 2.00 (1) 1.00 (2) 2.00 (1) 2.00 (2) 1.67 (3) 1.50 (2)  1.12 (41) 1.51 (35) 1-54 (13) 1.50 (12) 1.91 (11) 1.82 (11) 1.85 (13) 1.90  2.5 3.5 4.5  5-5 6.5 7.5-10.5 11.5 & +  ti)  2.00 (3) 2.00 (6)  ti)  2.00 (1) 1.67 (3)  (5) 1.25 (8) 2.00 (2) 1.33 (3) 1.00 (1) 1.00 (1) 2.00 (3) 2.00 (1)  1.50 (2) 2.00 (1) 2.00 (3) 2.00 (2) 2.00 (1)  (10)  o  Appendix 7.  Temperature and p r e c i p i t a t i o n d e v i a t i o n s from t h e normal f o r each month f r o m 1963 t o 1967 a t t h e Nanaimo a i r p o r t , B r i t i s h Columbia (Annual r e p o r t s o f t h e Dept. o f A g r i c u l t u r e , P r o v i n c e o f B r i t i s h C o l u m b i a ) .  Temperature D e v i a t i o n (F) from t h e I n d i c a t e d Mean f o r each Month Month  63 64 65 6667  J  F  M  A  M  J  J  A  S  0  N  D  35  38  40  46  53  58  63  62  57  49  41  38  48  -3 +4 0 +1 +3  +5 +1 0 +1 +1  +2  0 -1 +1 +1  0  +1 0 +1  -3  +1  +3 -1  +2  0  0  +1  +1  -2  0 0  -2  -2  +2  +2  -2 +2  +5  +1  +5  +1 +3  -1 +1  +1 +1  +1 -5 -1 +3 0  +1  -2 -2  +1 +1  +2  -2  -1  -2  Annual  0 +1 +1  +2  P r e c i p i t a t i o n D e v i a t i o n ( i n c h e s ) f r o m t h e I n d i c a t e d Mean f o r each Month Month  J  F  M  A  M  J  J  A  S  0  N  D  Annual  X*  6.7  4.9  3.6  2.3  1.5  1.5  1.0  1.2  1-5  4.2  6.4  7.3  42.1  • 63  -4.6 +4-1 -1-5 +2.5 +1.8  +0.9  +0.3 -1.1  +0.7 -1.9 -0.4 -1.1 +0.9  +1.4 -1.0 -0.2 -0.5 -0.2  +0.3 +0.3 -0.8 +0.2 -1.1  +0.7 +0.4 -0.2 +0.2 -0.2  -0.5 -0.3 +1.4 -1.0 -1.2  -0.8 -H0.3 -1.0 +0.1 +0.1  +4.4  -0.4 -2.0 +0.1 +1.1 -4-0  +3o0 -1.2 +4-0 +1.3 +1.9  +5.6 -8.2 -0.3 -3.4 +3.0  64 65 66 ' 67 *  -3.1 +0.1 -2.6  -2.2  -2.9  +4.0 +3.8  l o n g - t e r m average.  -2.7 +1.3 -0.7 +3.4  Appendix 8 .  The l o s s o f ova a t t h r e e s t a g e s o f t h e r e p r o d u c t i v e c y c l e as r e v e a l e d by t h e r a t i o between t h e number o f f e t u s e s and the number o f c o r p o r a l u t e a .  Females Pregnant at Collection Age Class  Total Fetuses: Corpora L u t e a  G a i n (+) or- L o s s (-) o f Ova  A l l Females That Became P r e gnant Viable fetuses: Corpora L u t e a  (%)  A l l Females That Produced C o r p o r a Lutea  G a i n (+) or l o s s (-) o f ova (%)  Loss o f Ova  Viable Fetuses: Corpora L u t e a  1.5  12:12 (100:100)'  0 0  12:13 (92:100)  -7.7  12:14 (86:100)  14-3  2.5  17:16 (106:100)  +6.2  17:16 (106:100)  +6.2  17:17 (100:100)  0.0  3.5  9:10 (90:100)  -10.0,  7:10 .(70:100)  -30.0  7:10 (70:100)  30.0  54:54 (100:100)  -1.9  53:54 (98:100)  -1.9  53:56 (95:100)  92:92 (100:100)  0 0  89:93 (96:100)  4.3  89:97 (92:100)  8.3  2.5&TT  80:80 (100:100)  0 0.  77:80 (96:100)  3.8  77:83 (93:100)  7.2  3.5&+-  63:64 (98:100)  60:64 (94:100)  6.3  60:66 (91:100)  9.1  4-5&+  .'  -1.6  '  5-4  

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