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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., Un i v e r s i t y of 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 accept t h i s thesis as conforming to the required standard THE ••JNIVER5ITY OF BRITISH CQLUMBL "FEBRUARY, 1970 In presenting t h i s thesis in p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make i t f r e e l y available for reference and study. I further agree tha permission for extensive copying of th i s thesis for s c h olarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of t h i s thesis f o r f i n a n c i a l gain shall not be allowed without my written permission. Department of ZOOLOGY  The University of B r i t i s h Columbia Vancouver 8, Canada Date FEBRUARY, 1970 i i ABSTRACT Ovarian changes, patterns of reproduction, and age-specific produc-t i v i t y of female Columbian black-tailed deer (Odocoileus hemionus columbianus) on Vancouver Island were elucidated l a r g e l y by examination of serial,stained sections of ovaries from UUU females. Well-defined, 8 or 9-day, f o l l i c u l a r cycles occur during the breed-ing season i n November and early December. Many f o l l i c l e s rupturing at f i r s t ovulation are asynchronous and of extreme sizes. R e l a t i v e l y small (4-5 mm ), short-lived (8-day) corpora lutea develop after f i r s t ovulation; these rapid-l y regress after second ovulation, while the corpora lutea of pregnancy grow 3 to 100 mm , within 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 and corpora lutea were dated by the cleavage stage of f e r t i l i z e d ova. These provided c r i t e r i a for estimating ovulation dates i n other females. In each cycle, about 50% of the adult females ovulated within an 8-day period. The mean date of f i r s t ovulation i n each of the 5 years was approximately November 16; the second ovulation followed after a remarkably short period of 8-9 days. About 96% of the females conceived at second ovulation and U% on subsequent ovulations. A high proportion of ' s i l e n t heats' accompany f i r s t ovulation, as indicated by lack of sperm on four of s i x ova. Growth curves, based on fetuses conceived at second ovulation, provided the conception dates of two l a t e conceivers—those containing more than one generation of regressed corpora lutea of non-pregnancy. Females may cycle at l e a s t f i v e times i f pregnancy does not occur. Accessory corpora lutea develop i n large and small unruptured f o l -l i c l e s , i n small ruptured f o l l i c l e s , and i n regressing corpora lutea. They i i i occurred i n 4-7% of females between f i r s t and second o v u l a t i o n ^ and i n 36% of pregnant females. Minimum l o s s of ova was 8 . 3 % i n a l l females t h a t produced corpora l u t e a and A-3% i n females t h a t became pregnant, of which 3.1,% was moribund f e t u s e s . Corpora l u t e a of pregnancy, corresponding to the number of f e t u s e s , develop i n t o d i s t i n c t i v e s c a r s , which 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 previous season, as w e l l as the lo 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 estimated from these. The average number of v i a b l e f e t u s e s per doe increased progres-s i v e l y from 0.91 i n y e a r l i n g s to 1.81 i n the 5-5 to 6.5 age-classes, and t h e r e a f t e r decreased. These changes i n f e r t i l i t y w i t h age were mirrored by changes i n weight and g i r t h . The r e l a t i v e c o n t r i b u t i o n of fawns by each age-class 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 decreased w i t h age. One hundred females of r e p r o d u c t i v e age produced about 137 fawns. i v TABLE OF CONTENTS Page 1. INTRODUCTION > 1 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. 1 C o l l e c t i o n Of The Material - 7 5. 2 Weights And Measurements 8 5. 3 Age Determination 9 5. A H i s t o l o g i c a l Technique 10 5. 5 Recovery Of Ova 13 5. 6 Microscopy 1-4 6. RESULTS 15 SECTION 1. STRUCTURE OF THE FETAL OVARY 15 1. 1 Cortico-Medullary Relationships ' 17 1.2 The Surface Epithelium 18 1.3 The Germ C e l l 18 1 . 4 F o l l i c u l a r Development 20 SECTION 2. 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 . . . . . . . . . 27 3.2 The Preovulatory F o l l i c l e 30 .3.3 Normal Atresia Of F o l l i c l e s . . 36 3. A Atypical Atresia Of F o l l i c l e s 4-6 3. 5 Polyovular F o l l i c l e s 4-7 3. 6 F o l l i c u l a r Cysts 4-8 3. 7 Primordial F o l l i c l e s . 4-9 3. 8 Tertiary F o l l i c l e s .• • • 51 CHAPTER A- THE FORMATION AND DEGENERATION OF CORPORA LUTEA. . 73 4...1 The Problem Of Mechanically-Ruptured F o l l i c l e s 73 "A- 2 The Growth Rate Of Corpora Lutea 75 4-. 3 Temporal Changes In The Ratio Between The Volumes Of Corpora Lutea Of The F i r s t And Second Generation. . . . 77 V Page 4. 4 Estimating The Age Of Corpora Lutea 77 4. 5 Histology Of Young Corpora Lutea 81 4- 6 The Early-Degeneration Of Corpora. Lutea From the F i r s t Ovulatory Cycle 91 4- 7 The Fate Of First-Cycle Corpora Lutea 94 4. 8 Corpora Lutea In The Pregnant Doe 96 4. 9 Accessory Corpora Lutea 101 4-10 Scars Derived From Corpora Lutea 104-4-. 11 Corpora Lutea And Scars In Macroscopic Sections 112 SECTION 3. THE BREEDING SEASON AND THE LENGTH OF THE ESTROUS CYCLE . 116 3. 1 The Frequency Of Ruptured F o l l i c l e s And Corpora Lutea In Periodic Samples 117 3. 2 Dates Of Ovulation Estimated From Ovarian Structures . . . 124-3. 3 The Estimated Dates Of Conception Determined From Fetal Growth Curves 127 3. 4- The Occurrence Of Corpora Lutea Of Non-Pregnancy In Late Breeders , 130 3. 5 Dates Of P a r t u r i t i o n 132 SECTION 4-. THE EFFECT OF AGE ON FERTILITY AND PRODUCTIVITY 134 4.. 1 Numbers Of Embryos And Fetuses 134 4. 2 Numbers Of Corpora Lutea 134 4- 3 Numbers Of Scars Derived From Corpora Lutea Of Pregnancy . 138 4. 4 The Productivity Of The Population 14.0 SECTION 5- THE EFFECT OF SIZE ON FERTILITY 144 7. DISCUSSION AND CONCLUSIONS . 148 7. T General Comments 148 7. 2 Development Of The ;Fetal Ovary 152 7. 3 Estimating The Age Of Ovarian Structures By Use Of Segmentation Stages Of Ova And Fetal Growth Curves. . . . 153 7. 4 The Weight Of Ovaries And Ovarian Function 155 7. 5 The Structure And Function Of F o l l i c l e s 156 7. 6 The Corpus Luteum 166 7. 7 Ovarian Scars 172 7. 8 The Breeding Season 175 7. 9 The Length Of The Estrous Cycle In Deer 178 7.10 F e r t i l i t y And Productivity Of Northwest Bay Deer Relative To Other Populations 182 7.11 The Ef f e c t Of Age On F e r t i l i t y 186 7.12 The Effect Of Size On F e r t i l i t y 187 8. SUMMARY . . . . 190 LITERATURE CITED • 194 APPENDIX 204 v i LIST OF TABLES Table Page 1. The number, age,and c o l l e c t i o n period of deer whose ovaries were studied 8 2. Measurements of deer fetuses and chara c t e r i s t i c s of their ovaries at selected periods during gestation 15 3. 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 year-l 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,con-t 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 to f i r s t ovulation 56 5. The number and condition of 25 ovulated ova recovered from the oviducts and u t e r i of 19 does, 1964.-67 66 6. Stages i n the advanced degeneration of f i r s t - c y c l e corpora lutea (CL 1) 95 7. Characteristics of structures 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. . . 123 9. The age-specific pregnancy rate and fetus production rate of females i n the 1963-64 season 135 10. The age-specific ovulation rate, based on corpora lutea, of a l l females collected from 1963 to 1967 137 11. The ovulation rate of age-classes of females i n combined samples from 1963 to 1967. 137 « 12. The age-specific average number of scars of 5 months derived from corpora lutea of pregnancy i n the reproductive seasons from 1962 to 1967 139 13. 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 fetuses, corpora lutea, and scars.. 14-3 14-. The t o t a l and r e l a t i v e productivity of each age-class at Northwest Bay 14-3 15. A comparison between the ovulation rate i n females from above and below 2000 feet at Northwest Bay from 1963 to 1966 . 150 v i i LIST OF FIGURES Figure Page 1. The age-specific weight of paired ovaries from deer collected i n November and early-December from 1963 to 1966 24 2. Changes i n the weight of ovaries from four age-classes of deer collected between October and June, 1963-64 . . . . 25 3. The age-specific number of primordial f o l l i c l e s , p e r millimeter of outer cortex,in 10 p. sections from the ovaries of deer collected i n November and early-December, 1963-67 50 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 collected i n November and early-December, 1963-67 53 5. The frequency of the largest f o l l i c l e , and i t s state of maturity, i n fawns, yearlings, and adults prior to f i r s t ovulation of the current breeding season. . . . . 55 6. The r e l a t i o n s h i p between the largest active f o l l i c l e and the largest a t r e t i c f o l l i c l e i n pairs of ovaries from pre-ovulatory females 59 7. The r e l a t i o n s h i p between the largest active, second-cycle f o l l i c l e and the largest a t r e t i c f o l l i c l e of previous cycles. 60 8. The r e l a t i o n s h i p between the volume of the largest active second-cycle f o l l i c l e and the volume of the largest f i r s t -cycle corpus luteum (CL 1) i n yearling and adult females . . 63 9. The regressions of the volumes of the largest third-cycle f o l l i c l e on the largest second-cycle corpus luteum (CL 2) i n yearling and adult females . 65 10. The growth rate of the largest third-cycle 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 11. Seasonal changes i n the volume of the largest f o l l i c l e i n pregnant females 74 12. - The growth rate of second-generation corpora lutea (CL 2) whose ages were estimated from the cleavage stage of ova . . 76 13. Temporal changes i n the r a t i o between the volume of the largest second-generation corpus luteum (CL 2) and the volume of the largest shrinking, f i r s t - g e n e r a t i o n corpus luteum (CL 1) 78 v l i i F i g u r e Page 1-4. The volume of degenerating f i r s t - c y c l e corpora l u t e a (CL 1) accompanying second-cycle corpora l u t e a (CL 2) whose ages were estimated by 1) the cleavage stage of ova and 2) s e v e r a l c r i t e r i a . 93 15- The volumes of corpora l u t e a of pregnancy (CLP) a t f i v e stages of g e s t a t i o n 97 16. The a g e - s p e c i f i c volumes of scars 5 and 17 months old de r i v e d from corpora l u t e a of pregnancy 108 17. Regression of the average number of corpora l u t e a of pregnancy scars on the age of females at the previous breeding season 113 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 119 19- 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 obtained d u r i n g November and early-December from 1963 to 1967. . . . 121 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 o v u l a t i o n s i n combined samples from the breeding seasons of 1963 to 1966 126 21 . 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 date 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 of f e t u s 1 2 8 22. The r e g r e s s i o n of f e t a l forehead-rump l e n g t h on 1) the date 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 23. The frequency 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 dates on which fawns were tagged from 1960 to 196-4 133 24-. The age s t r u c t u r e of females (fawns excluded) taken from the Northwest Bay p o p u l a t i o n between 1960 and 1966 14-1 25. 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 , chest girth,and dressed weight of e i g h t age-classes of females 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 of f o l l i c l e growth and a t r e s i a . . . 163 27. Schematic r e p r e s e n t a t i o n of r e p r o d u c t i v e c y c l e s i n deer. . . 181 i x LIST OF PLATES Plate Page 1. Development of the Fetal Ovary 16 • 2. Histology of the Fetal Ovary 19 3. The Components of F o l l i c l e s • 29 4- . The Pre-and Post-ovulatory Oocyte 34-5- Preovulatory and Degenerate Oocytes 38 6. Meiotic 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. Atypical Atresia of F o l l i c l e s 68 10. The Ovulated Ovum • 69 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. Early Development of Corpora Lutea From the Second Ovulatory Cycle 86 14-. Changes i n Corpora Lutea During Pregnancy 99 .15- Scars Derived From Corpora Lutea 110 16. Color Photomicrographs of Ovarian Structures 116 X LIST OF APPENDICES Appendix Page 1. The e a r l y cleavage of f e r t i l i z e d ova i n s e l e c t e d mammalian species 204 2. The volume of growing corpora 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 degenerating corpora 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 -gest 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 3. The t i m i n g and v a r i a b i l i t y of the breeding seasons from 1963 to 1967,based on s t a t i s t i c s d erived from frequency d i s t r i b u t i o n s of dates of o v u l a t i o n 206 4. A g e - s p e c i f i c pregnancy r a t e and fetus-product!on r a t e of Northwest Bay females, 1963 - 66 207 5. The r e l a t i o n s h i p between the number of corpora l u t e a (CL) of three s i z e s and the number of f e t u s e s i n 55 preg-nant does 208 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 of females t h a t had ovulated i n November and early-December 209 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 the normal f o r each month from 1963 to 1967 at the Nanaimo a i r -p o r t , B r i t i s h Columbia 210 8. The l o s s of ova a t three stages of the r e p r o d u c t i v e c y c l e , as revealed by the r a t i o between the number of f e t u s e s and the number of corpora l u t e a 211 x i ACKNOWLEDGEMENTS I am indebted to my research supervisor,Dr. Ian McTaggart-Cowan, Dean of Graduate Studies, who i n i t i a t e d t h i s study, provided guidance, stimulation, and research funds. Special thanks go to the other members of my. research committee: Drs. D. Chitty, V/. S. Hoar, and Ii. D. Fisher, a l l of the Department of Zoology; Dr. W. D. K i t t s , Department of Animal Science; and Dr. P. J. Bandy, Animal Resource Ecology and the B r i t i s h Columbia F i s h and W i l d l i f e Branch. Dr. Bandy was e s p e c i a l l y h e l p f u l i n the wr i t i n g phase of t h i s study. Dr. J. F. Bendall, Department of Zoology, k i n d l y agreed to serve as pro tern Supervisor i n the absence of 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 permitted me to c o l l e c t female deer and, thanks to Dr. Bandy, to use valuable equipment i n t h e i r research laboratory. I am g r a t e f u l f o r funds received from Canadian Industries Ltd., the Canadian W i l d l i f e Service, and the Un i v e r s i t y of B r i t i s h Columbia. Many people assi s t e d i n the c o l l e c t i o n of 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 father, 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 herein and Mr. W. A. Low f o r h e l p f u l suggestions.. I appreciate most of a l l the help contributed by my wife A l i c e , who typed t h i s t h e s i s and provided a propitious environment f o r the conception and long gestation of t h i s study. 1 1. Introduction. Population size i s mediated or regulated through the r e l a t i v e action of two a n t i t h e t i c a l processes, reproduction and mortality. The part that each of these inextr i c a b l y - r e l a t e d processes plays i n determining animal numbers i s imperfectly known for a l l species, but most population regulation theorists (Lack, 195-4; P i t e l k a et a l . , 1955; Wynne-Edwards, 1962; Ghitty, 196-4; Ch 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 less important than changes i n mortality. The accu-mulated information gained from studies of reproductive function w i l l even-t u a l l y elucidate the r o l e of reproduction i n the regulation of animal numbers. This i s a study of reproduction i n Columbian black-tailed deer, a species whose numbers fluctuate remarkably i n response to habitat 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 wild ungulates. Knowledge of reproductive functions has come la r g e l y from studies of common laboratory and domestic species, yet a r t i f i c i a l selection and unnatural habitat have undoubtedly modified or dampened the natural re-productive rhythms of these species. In spite of adaptive specializations i n the reproductive processes of many mammalian species, the basic neuro-endocrine system i s thought to be similar i n a l l . Insights i n t o the mecha-nism of reproductive function i s obtained through comparative studies of many species. " I t i s evident that the elucidation of the diverse pattern of mammalian reproduction i s dependent upon research from a comparative viewpoint" (Anderson and Melampy, 1966). In. t h i s study, the pattern 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 analysis of the i r ovaries, 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 natural 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 preg-nant females, a method cost l y to both the population and to the investigator. Since the i n i t i a l discovery by Cheatum (194-9) that estimates of future and past reproductive performance could be obtained by examining the ovaries of deer, the method has been applied to a variety of species with varying suc-cess. Notable among these are studies on white-tailed deer (Cheatum and Severinghaus, 1950; Gibson, 1957; Ransom, 1967), black-tailed deer (Taber, 1953; Golley, 1954), mule deer (Robinette et a l . , 1955), elk (Halazon and Buechner, 1956; Morrison, 1960), moose (Pimlott, 1959; Simkin, 1967), and caribou (McEwan, 1962). In most of the above studies the ovaries \7ere ex-amined macroscopically and i n s u f f i c i e n t information was obtained on the origins and variations of the various ovarian structures. I t i s imperative to find quantitative relationships between corpora lutea and fetuses, and between corpora lutea and corpora lutea scars. In t h i s study, the r e l a t i o n -ship between morphology of the ovary and reproductive function i s ascertained. Age-specific reproductive function i s l i t t l e known i n natural populations because of the d i f f i c u l t y of accurately determining age. Pre-vious studies of deer reproduction have related function to age estimated by the r e l a t i v e l y inaccurate tooth wear and eruption method. In t h i s study, many aspects of reproductive function are related to age determined accur-a t e l y from sections of teeth (Low and Cowan, 1963; Ransom, 1966; Gi l b e r t , 1966; Erickson and Seliger, 1969). In addition, t h i s study i s f a c i l i t a t e d by two recent research projects on the relationships between deer and the envi-ronment of the Northwest Bay study area. One researcher examined the vege-ta t i o n of a series of age-classes following logging and estimated the use by deer of each serai stage (Gates, 1968). Another investigator examined the ef f e c t of hunting and changes i n vegetation on the quantity and q u a l i t y of the 3 population (Smith, 1968). Their data enabled me to examine the reproductive performance of individuals and groups of individuals i n terms of known changes i n the population and i n terms of certain aspects of the environ-ment. The s p e c i f i c objectives of t h i s study are: 1. To determine, at various stages of the annual reproductive cycle, 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 lutea. 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 cycle, mortality rate of ova, and f e t a l growth patterns. 3. To determine age-specific reproductive rates of female Columbian black-tailed 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 effect of age on intra-uterine mortality. 4-. To evaluate the methods of gross and microscopic ovarian analysis as a means of determining past, present and future n a t a l i t y of a population of deer. 5. To evaluate some of the factors that affect the f e r t i l i t y of the population.. 4 2. The Study Area. Reproduction i s i n f l u e n c e d by the ha b i t a t , w h i c h i n t u r n 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 . A b r i e f 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 the v e g e t a t i o n , may be found i n Gates (1968) and Smith (1968). Northwest Bay i s an uninhabited area of about 135 square m i l e s i n the Englishman R i v e r watershed on the eastern slope of Vancouver I s l a n d , B. C. E l e v a t i o n s range from 4-00 f e e t i n the east, to n e a r l y 6000 f e e t (Mt. Arrowsmith) i n the west, although most of the area 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 are inc l u d e d i n the Vancouver I s l a n d Land form,while those below 2000 f e e t , forming p a r t of the Coa s t a l Trough, are c l a s s i f i e d as Nanaimo Lowlands (Holland, 1964). The e n t i r e r e g i o n d i p s toward the northeast,but 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 the r e g i o n . There i s a remarkable p r e c i p i t a t i o n g r a d i e n t from the high e l e v a t i o n of the western border to the low e l e v a t i o n of the east e r n s i d e . The western p o r t i o n ( i n the Coast C l i m a t i c Region) r e c e i v e s more than 100 inches y e a r l y , whereas the eastern edge ( i n the Southwest Coast C l i m a t i c Region) r e c e i v e s only 30 to 4-0 inc h e s . Most of the r e g i o n , however, r e c e i v e s from 4-0 to 60 inches a n n u a l l y . In win t e r , the higher e l e v a t i o n s are blanketed i n deep snow,but a t low e l e v a t i o n s snow cover i s sporadic 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 of the general study area i n c l u d e s two reg i o n s subdivided i n t o three zones: a C o a s t a l Douglas F i r Zone at e l e v a t i o n s up to 1500 f e e t ; a Co 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 Mountain Hemlock Zone a t e l e v a t i o n s between 3700 and 6000 f e e t . Subzones and t h e i r p l a n t a s s o c i -a t i o n s have been described by K r a j i n a (1964). Douglas F i r (Pseudotsuga  m e n z i e s i i var. m e n z i e s i i ) i s the most common climax species f o l l o w e d by 5 (Tsuga heterophylla), western red cedar (Thuja p l i c a t a ) , balsam f i r (Abies  grandis) and lodgepole pine (Pinus contorta) (Gates, 1968). Clear-cut patch-logging of Northwest Bay commenced i n 1939 and has continued to the present, thereby creating a great number of serai 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 factors, the age and composition of the vegetation varies considerably within the study area. Smith (1968) divided the area i n four zones and calculated the percentage of various s e r a i stages i n each zone. Characteristics of certain serai 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 calculate the serai stage composition that was optimal for 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 black-tailed deer i s the only wild ungulate present. Mountain l i o n s are the i r chief natural predator, with black bears and eagles constitu-t i n g a minor threat to fawns. Each autumn a more important predator, man, takes several hundred deer from the population. Hunting i s allowed within the logging claim only on weekends and holidays because loggers are present during the week. The hunting season begins i n early September and runs to the end of November. Antlerless 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 related to the stage of population growth or decline. During t h i s study the Northwest Bay deer population was declining primarily because of a de t e r i o r -ation i n habitat and, secondarily,because of hunting (Smith, 1 9 6 8 ) . The number of deer removed steadily increased from a low of 120 i n 1 9 5 6 ,to 6 approximately 700 per year i n 1962 and 1963. Included i n the t o t a l f o r the l a t t e r year were 78 females k i l l e d e x p r e s s l y f o r t h i s study. I f these are excluded, the peak k i l l by hunters was 1962. I n 1961-62, Gates (1968), on the b a s i s of p e l l e t - g r o u p counts, estimated winter and summer d e n s i t i e s of 50 and 67 deer per square mile w i t h i n a 10 square mile p o r t i o n of Northwest Bay. Since 1962-63, the number of deer k i l l e d has s t e a d i l y d e c l i n e d to a low of 142 i n 1968. Although some of these changes r e f l e c t changes i n hunting pressure, they 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 years. Smith (1968) estimated t h a t , i n each year from i960 to 1962, hunting m o r t a l i t y accounted f o r 20% of the 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%. He concluded t h a t p r i o r to and d u r i n g the period of t h i s study there was no o v e r a l l s i g n i f i c a n t change i n the age s t r u c t u r e of the po p u l a t i o n . There was, however, a gradual d e c l i n e i n average weight of deer w i t h i n sex and age c l a s s e s . The above f i n d i n g s and others 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 , both w i t h r e s p e c t to numbers and p h y s i c a l c o n d i t i o n , from which the samples f o r t h i s study were drawn. The d e c l i n e i n the popula-t i o n may have been abetted by two w i n t e r s (1964.-65 and 1965-66) of 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 Reproductive Cycle Of Deer. Some f e a t u r e s of the r e p r o d u c t i v e c y c l e i n the Columbian black-t a i l e d deer have been summarized by Cowan (1956), Brown (1961), Ommundsen (1967), and West (1968). Deer are polytocous (may r e l e a s e more than one ovum i n an estrous period) and m u l t i o v u l a r (may pass through s e v e r a l estrous c y c l e s i f conception does not occur). E s t r u s l a s t s 24- to 36 hours and the 7 l e n g t h of the estrous 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 deer i s 22 to 28 days. Breeding u s u a l l y occurs i n November and December but may extend i n t o March. Fawns of 0. hemfonus columbianus r a r e l y conceive except i n c a p t i v i t y . The m a j o r i t y of y e a r l i n g s produce o n l y one faun,whereas older does produce one, two, or o c c a s i o n a l l y j t h r e e fawns. Implantation of embryos occurs 27 to 30 days a f t e r conception (Cheatum and Morton, 194-2; Hudson and Browman, 1959)-G e s t a t i o n averages about 203 days i n confined b l a c k - t a i l e d does,but v a r i e s between 183 and 212 days (Cowan, 1956; Brown, 1 9 6 l ) . Most fawns are born i n June. 5. Methods. 5. 1 C o l l e c t i o n Of Tissues. Most of the t i s s u e s used i n t h i s study were obtained from female deer taken by hunters d u r i n g the r e g u l a r f a l l h unting seasons from 1963 to 1967. Reproductive t r a c t s , weights, body measurements, and the lower jaw were obtained from each deer at a hunter-check s t a t i o n operated by the B r i t i s h Columbia F i s h and W i l d l i f e Branch. Because the s t a t i o n was lo c a t e d on the on l y open access road i n t o the study area, a l l hunters were stopped and provided w i t h diagrams, and i n 1 9 6 3 ,with photographs, so they could i d e n t i f y the female r e p r o d u c t i v e t r a c t and leave i t i n t a c t i n the deer f o r l a t e r r e -moval a t the s t a t i o n . When a r e p r o d u c t i v e organ was removed, the appearance of the mammary gland, u t e r u s , and o v a r i e s was noted. I f surface o v u l a t i o n s i t e s were present, 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 small drawing of the ovary. The f r e s h weight of each u t e r u s , severed a t mid - c e r v i x , was a l s o obtained. From October 1963 to June 1964, an a d d i t i o n a l 77 female 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 to 8 post-partum). These co 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 addition, f i v e animals raised at the University 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 tre a t -ed with e s t r a d i o l i n an experiment involving the histology of the p i t u i t a r y gland, two were used to obtain known-age fetuses for a study by Ommundsen (1966), and one was an injured animal of 9 months. A t o t a l of 4-61 females were available for study.(Table l ) . In addition, 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 Period Fawns Yrlg. Adults Total 1963 Nov-Dec 24- 26 73 123 1963-64 Dec-June 9 16 62 77 1964- Nov-Dec 9 28 4-0 87 1965 Jan 0 0 2 2 1965 Nov 15 21 51 87 1965 Apr-May 0 .3 5 8 1966 Nov 0 8 26 34-1967 Nov-Dec 1 12 20 33 Other s 1 2 7 10 Subtotals 59 . 116 286 4-61 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 for loss of blood. Weight of the animal,with a l l viscera removed (dressed weight^ was also measured to the nearest pound on a plajtform or beam scale. I f the l i v e r remained i n the carcass , 2 pounds were subtracted, and i f the heart remained, the measured weight was reduced by 1 pound. The fresh weight 9 of the 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 gland, l i v e r , h e a r t , kidneys, and spleen were measured to the nearest 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 gland, adrenal glands, and t h y r o i d glands were measured on e l e c t r i c balances to a degree of p r e c i s i o n a p p r o p r i a t e to the weight of the 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). Hind-foot l e n g t h I s the d i s t a n c e between the t i p of the hoof and the t i p of the calcaneous 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 the animal j u s t p o s t e r i o r to the f o r e l e g s , measured to the nearest 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 , of 10 pounds c a p a c i t y , attached to the r u l e ) . Crown-rump and forehead-rump lengths are the d i s t a n c e i n a s t r a i g h t l i n e from the crown (embryo) or forehead ( f e t u s ) to the rump, measured to the nearest 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 of deer were estimated by the wear and e r u p t i o n method when the lower jaw was removed from the deer at the c h e c k - s t a t i o n . At a l a t e r date, the assigned ages were rechecked by the same method, except t h a t known-age jaws were used comparatively to i d e n t i f y age c l a s s e s . Be^ cause the above method was not accurate f o r deer older than two years, the t o o t h - s e c t i o n method (Low and Cowan, 1963) was used to determine the age of. 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 study. The ac-curacy of the t o o t h - s e c t i o n method was confirmed 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 sec-tioned, and ethyl alcohol-formalin-acetic acid (AFA) was used to harden the tissues for macroscopic analysis i n 1967. The l a t t e r , a colorless f i x a t i v e , did not mask any color inherent i n the tissue. In contrast, the p i c r i c acid i n Bouin's f l u i d colored the tissue a bright 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 structures. After a few days i n either f i x a t i v e , t h e ovaries were transferred to 70% ethanol for storage. Gross sections of ovaries were obtained i n a manner described by Cheatum (194-9). A sharp scalpel was used to make a series of p a r a l l e l i n -cisions about 1 mm apart throughout the ovary. These in c i s i o n s did not sever the h i l a r portion,so that a book of sections 1 mm thick was produced. A l l structures were measured and described as the ovary was s l i c e d . Some ovaries, cut transversely i n the above manner, were subsequently dehydrated, cleared, and embedded i n paraplast for the preparation of th i n micro-scopic sections. 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 tissue removed from the thick sections. Small blocks of ovarian tissue,containing pertinent or perplexing structures, were e a s i l y processed using routine h i s t o l o g i c a l methods. Several such structures i n a pair of ovaries were processed together,and a l l placed on one or two s l i d e s . Blocks of tissue were cut into various sizes and shapes to i d e n t i f y each structure. Prior to 1967, whole ovaries were sectioned s e r i a l l y i n their 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 not d i s r u p t e d . T r i a l runs 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 could not be obtained u s i n g r o u t i n e h i s t o l o g i c a l procedures. I t was necessary to f o l l o w dehydration w i t h two immersions i n methyl berizoate f o r a t o t a l of 4-0 hours,before f i n a l c l e a r i n g i n benzene. This technique i s recommended by Humason (196.2) f o r tough, f i b r o u s t i s s u e . The embedding medium was p a r a p l a s t . Embedded o v a r i e s were sectioned a t 8 or 10 u on a standard r o t a r y microtome. I n i t i a l l y , o v a r i e s were cut at 10 u, and i n consecutive order, every t e n t h s e c t i o n was placed on l a b e l l e d albuminized s l i d e s . Be-cause the diameter of the embedded oocyte was s l i g h t l y greater than 100 u, every oocyte was sampled. Subsequently, the i n t e r v a l between s e c t i o n s was doubled (every 20th s e c t i o n was taken) to reduce the number of s e c t i o n s . S e r i a l s e c t i o n s of oocytes from medium and l a r g e f o l l i c l e s were obtained because the appearance of the oocyte i s of d i a g n o s t i c value i n i n t e r p r e t i n g the stage of f o l l i c l e maturation and the h e a l t h of the oocyte. A few e x t r a s e r i a l s e c t i o n s of each ovary were taken f o r s p e c i a l s t a i n i n g methods. Corpora l u t e a from a l l c o l l e c t i o n s were stained i n the same s o l u t i o n s and processed i n a few hours, so th a t t i n c t o r i a l d i f f e r e n c e s between corpora l u t e a could be a t t r i b u t e d to d i f f e r e n c e s i n the t i s s u e and not to d i f f e r e n t batches of s t a i n . S l i d e s were stained w i t h Masson's trichrome (Humason, 1962) or a modified Massons trichrome c o n t a i n i n g orange G. Regaud 1 s or Weigert's hematoxylin was used. Selected s l i d e s were . stained 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 trichrome, c a r b o l f u c h s i n , methylene bl u e , t o l u i d i n e blue,and al d e h y d e t h i o n i n . 5. 4-. 2 U t e r i . U t e r i were f i x e d i n Baker's f o r m a l i n mixture (Humason, 1962), then t r a n s f e r r e d to AFA. Small r e c t a n g u l a r 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 caruncles were removed from each uterus and placed i n 70% ethanol. D u p l i c a t e h i s t o l o g i c a l s e c t i o n s , 12 8 u i n thickness, were prepared and stained, 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 ovulation, or had recently ovulated, were sectioned and checked for the presence of sperm and ova. Oviducts were dehydrated and embedded i n a f l a t , coiled state so that a minimum number of sections were required to permit a search for the ovum. Sections were cut at 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 pellucida of the ovum stained red, making the search for ova r e l a t i v e l y easy. When an ovum was found, additional sections of i t were obtained from stored oviduct sections. Few ova were found i n the 196-4 c o l l e c t i o n , probably because they were accidentally washed out when the reproductive tracts were immersed i n the f i x a t i v e . In 1965 and 1966,the oviduct termini were tied 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 rate of ova. 5. -4. 4- Vaginal smears. Vaginal smears were obtained to re l a t e changes i n the surface of the vagina to ovarian function and to determine, from the presence of sperm, i f the doe had recently copulated. Smears were obtained by passing a s l i d e over the surface of the vaginal epithelium 2 cm from the cervix. Early i n t h i s study,smears were air - d r i e d or fixed for a few minutes i n absolute ethanol and l a t e r stained with Weigert's hematoxylin and ponceau 2R - acid fuchsin, but they were not sa 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. In 1967,smears were fixed 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 with Harris' 13 hematoxylin and the Schoor I I I . s t a i n (deNeff, 1965). 5. 4-. 5 Teeth. Roots of t e e t h were d e c a l c i f i e d f o r 2 to 4- days i n a mixture of 25% formic a c i d and 5% f o r m a l i n . A f t e r n e u t r a l i z a t i o n i n aqueous l i t h i u m carbonate the r o o t s were dehydrated, c l e a r e d , imbedded i n p a r a p l a s t , and sectioned a t 10 or 12 u. Every 25th s e c t i o n was placed on s l i d e s and l a t e r s t a i n e d f o r 30 to 4-0 minutes i n H a r r i s ' haematoxylin. Some s l i d e s were stained i n t h i o n i n , methylene blue, t o l u o d i n e blue, and 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 su p e r i o r to those obtained w i t h hematoxylin s t a i n i n g . 5. 5. Recovery Of Ova. . The r e p r o d u c t i v e t r a c t s of r e c e n t o v u l a t o r s were kept c o o l , transported to the l a b o r a t o r y , and f l u s h e d a f t e r r e s e c t i o n of the tubes from the u t e r u s . The o s t i a l end of the tube was secured around a number 22 needle, attached to a 10 ml syringe c o n t a i n i n g a 0.9% NaCl s o l u t i o n . The contents of the tube, 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 glass 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 l i g h t a t 25X. I f the f i r s t f l u s h i n g was u n s u c c e s s f u l , s e v e r a l more were t r i e d w i t h increased f l u s h i n g pressure. The u t e r i n e horns were fl u s h e d w i t h a greater volume of the s o l u -t i o n . The connective t i s s u e septa between horns were cut so t h a t the horn could be l i g a t u r e d near the u t e r i n e body. The t i p of the u t e r i n e horn was severed, and the syringe needle was i n s e r t e d through the w a l l of the horn a t the l i g a t u r e . Segmenting ova sank to the bottom of the g l a s s , whereas most of 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 horns f l o a t e d higher i n the 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 encountered, 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 for 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 features, afteruhich they were transferred i n saline In a pipette to a well s l i d e . Then the ova were viewed under higher magnification, stained with d i l u t e solutions of toluidine blue, ponceau 2R - acid fuchsin, a l c i a n blue and hematoxylin, and then temporarily mounted i n physiological saline on a glass s l i d e . 5. 6 Microscopy. A magnification of 25 was sa t i s f a c t o r y for most i d e n t i f i c a t i o n procedures,including the loca t i o n of oocytes and ova. Linear and area measurements were made at a l l magnifications with a 20 x 20 u n i t ocular grid. The volumes of nearly-spherical objects were calculated using the formula V = 4/3 If (r^ x X r^) where r j _ , T^, and r ^ represent the three-dimension r a d i i . The volumes of non-spheroid objects were obtained by the grid method, where V = A x d, where A (area) was determined at i n t e r v a l s throughout the structure by counting grid intersections, and d (depth) was the i n t e r v a l between sections i n which A was determined. Usually,every section on the s l i d e was used i n determining volumes,but for large structures, A was determined from every 2nd, 4-th, or 8th section on the s l i d e . Suf-f i c i e n t precision i n the measurement of volume was achieved i f A was determined i n at l e a s t 10 sections,and at lea s t 40 to 50 grids covered the structure i n the section i n which i t s area was greatest. In practice,a magnification and section i n t e r v a l was chosen to meet the above require-ments, grids were counted, t o t a l l e d , then multiplied by a factor dependent on the magnification and the section i n t e r v a l . Compound binocular microscopes equipped with ocular scales were used for detailed h i s t o l o g i c a l evaluation and measurement of structures. 15 6. RESULTS. SECTION 1. STRUCTURE OF THE FETAL OVARY.• Development of the f e t a l ovary and oogenesis were studied i n a sam-pl e of 14- f e t u s e s and one p o s t n a t a l fawn. Measurements of the fetuses, and 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 recorded i n Table 2. The l e a s t v a r i a b l e of the three f e t a l measurements i n the t a b l e , h i n d - f o o t l e n g t h , was used to estimate f e t a l age. One known-age f e t u s , U34, was obtained from a doe r a i s e d i n confinement at the U n i v e r s i t y of B r i t i s h 0 Columbia. Although the f e t u s of 102 days was s l i g h t l y l a r g e r than a f e t u s of s i m i l a r age from a w i l d doe, the weight and degree of development of i t s o v a r i e s were e q u i v a l e n t to t h a t of f e t u s e s 122 to 14-4- days old from w i l d deer (Table 2 ) . Table 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 t h e i r o v a r i e s at s e l e c t e d periods d u r i n g g e s t a t i o n . Fetus Ovaries (both) Specimen Age FR HF Wt Wt Diam (u) Diam Largest No. (Days) (mm) (mm) (g) (mg) Largest F o l l i c l e (u) Sex C e l l and type T32 64- 79 20 20 4.8 14 T27 68 89 24 26 8.0 12 T36 73 112 31 53 16.5 11 • T37 79 126 37 63 12.1 12 • H 98 166 60 151 15.4 18 U34- 102 187 71 257 29.2 29 32P T43 122 225 92 417 18.0 25 31P T52 123 222 93 442 25-0 20 28P T84 14-4- 301 127 858 29.5 28 35Py T65 170 350 170 1395 29.2 35 60S T66 177 366 182 1674 42.4 30 43 S T76 194 4-03 211 2103 31.0 65 125T T77 202 4-60 225 2852 33 . 9 33 104T T70 204- 445 227 2670 42.7 72 220 T BG 1-2PP 239 2200 28.0 60 150T FR - Forehead-rump len£ Py - Primary HF - Hind-foot l e n gth S - Secondary P - P r i m o r d i a l T - T e r t i a r y PP - Post-par turn F a c i n g page 1 6 P l a t e 1 Development of the F e t a l Ovary ( A l l cross s e c t i o n s of o v a r i e s a t 10X) 1. Two abnormally-fused 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 white r e g i o n i s the mesonephric rudiment th a t gives r i s e to the r e t e apparatus. 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 ovary i n a f e t u s 102 days old from a doe r a i s e d i n c a p t i v i t y . The r e t e o v a r i ( c e n t r a l elongate s t r u c t u r e ) has condensed and a small medulla i s evident (white r e g i o n ) . 3. An ovary 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 contains l a r g e vessels.. Growth of the c o r t e x produces a mushroom-l i k e form. U. An ovary i n a f e t u s about \L\L, days o l d . The width of the c o r t e x , r e l a t i v e to the medulla, i s l e s s than i n younger o v a r i e s . 5. An ovary i n a f e t u s about 170 days o l d . The medulla, 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 (white r e g i o n s ) . F o l l i c l e s , to the secondary stage, are abundant i n the c o r t e x . 6 . An ovary of a post-partum fawn. The c o r t e x i s h y a l i n i z e d and appears dark i n the photograph. The c o r t i c o - m e d u l l a r y border 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 general there was a p r o g r e s s i v e i n c r e a s e , w i t h age,in the 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 , and s i z e of the l a r g e s t f o l l i c l e . The two ovaries of the 98-day f e t u s were p a r t i a l l y fused ( P l a t e l . l ) , b u t they may have separated l a t e r i f death had not intervened. The i n -f e r r e d sequence of o v a r i a n development, from the e a r l i e s t stage of the s e r i e s , to the term f e t u s , i s presented below. 1. 1 C o r t i c o - M e d u l l a r y R e l a t i o n s h i p s . 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 of 6 4 days, the c o r t e x was dominant and s t r u c t u r a l l y uniform throughout. A loose network of connective t i s s u e f i b e r s supported clumps and whorls of e p i t h e l i a l c e l l s , i n c l u d i n g sex c e l l s . The medulla was 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 age of 98 days, when i t was composed l a r g e l y of a d i f f u s e r e t e o v a r i i ( P l a t e 1. 1). The growth of the medulla was p r o g r e s s i v e up to a f e t a l age of 177 days, when i t reached i t s maximum s i z e r e l a t i v e to the c o r t e x ( P l a t e 1.1-5). Coincident w i t h growth of the medulla, was a l o s s of e p i t h e l -i a l c e l l s from the zone and 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 of connective t i s s u e . There was a marked i n c r e a s e i n the number and h y a l i n i -z a t i o n of 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 . During e a r l y growth, the o v a r i a n medulla merged i n t o the outer cortex,but at 170 days, when the c o r t e x was r e l a t i v e l y t h i n n e s t , the de-marcation was sharp ( P l a t e 1. 5). From 73 to I 4 4 days of age,there was a marked l o b u l a t i o n of the l o o s e l y k n i t outer c o r t e x ( P l a t e 2. 2, 7, and 8 ) . I n older f e t u s e s (170 days to term), the c o r t e x became f i b r o u s and a l a y e r of f i b e r s , the t u n i c a albuginea, formed under the surface 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 ovary w i t h c o r t e x and medulla p r o p o r t i o n a t e to t h a t of a fawn of 5 months. 18 1. 2 The Surface E p i t h e l i u m . The surface 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 to the i n t e r i o r f o r the e n t i r e period of development,but p r o l i f e r a t i o n was g r e a t e s t from 98 to 14-4- days a f t e r conception,and had v i r t u a l l y ceased at term ( P l a t e 2.2 and 6). I n the f e t u s of 177 days, 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 to l o c a l i z e d areas of the o v a r i a n surface. 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 only a small number entered the c o r t e x at one l o c a t i o n . U s u a l l y , t h e e p i t h e l i u m was a s i n g l e l a y e r of c e l l s , but i n some areas l a y e r i n g occurred 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 albuginea was w e l l e s t a b l i s h e d (170 and 177 days),the surface e p i t h e l i a l c e l l s commonly entered the u n d e r l y i n g t i s s u e at a tangent. I n mid-gestation o v a r i e s , 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 g r e a t e s t , c e l l s o c c a s i o n a l l y entered m e i o t i c prophase i n the surface l a y e r . From 14-4- to 177 days of f e t a l development,the e p i t h e l i u m was a smooth l a y e r of c u b o i d a l c e l l s , b u t the e p i t h e l i u m became stre t c h e d and 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 . 1. 3 The Germ C e l l . The 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 i n Table 2. Only 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 from other 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- to 79 days o l d . Most of these a c i d o p h i l i c c e l l s measured 5 to 8 u i n diameter,but some a t t a i n e d diameters of 14- P-. I n the 98-day f e t u s , oogenic c e l l s were more numerous i n the outer cortex,where most ranged between 7 and 15 u. By t h i s stage, some germ c e l l s had reached the r e s t i n g phase of meiosis 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 oocytes,but 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 from m i t o t i c d i v i s i o n of only the nucleus,or from coalescence of F a c i n g page 19 P l a t e 2 H i s t o l o g y of the F e t a l Ovary. 1. The cor t e x of the ovary 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 dark areas) occur i n l o o s e l y organized connective 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 outer c o r t e x of an ovary i n a f e t u s 102 days old taken from a ca p t i v e doe. Two i n v a g i n a t i o n s of the germinal e p i t h e l i u m are evident. Note the cords of c e l l s (X4OO). 3. The same ovary as 2.2 to show n u c l e i of oogonia i n prophase of meiosis (X800). 4. Same as 2. 3. Note the telephase ( l e f t center) of m i t o s i s and other n u c l e i i n prophase of meiosis (X4OO). 5. The deep c o r t e x of the same ovary to i n d i c a t e the increased amounts of connective t i s s u e and to i l l u s t r a t e t hat many oogonia n u c l e i have reached the d i c t y a t e stage of meiosis (X400). 6. The surface e p i t h e l i u m and subjacent c o r t e x of an ovary 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 contains many oogonia i n prophase of meiosis. Thickening of the germinal e p i t h e l i u m (top l e f t ) i s the f i r s t stage i n the i n v a g i n a t i o n of c e l l s i n t o the c o r t e x (X600). 7. The outer c o r t e x , i n c l u d i n g the germinal e p i t h e l i u m , of a f e t a l ovary about 122 days o l d . Clumps (or cords) of c e l l s , i n c l u d i n g m u l t i -n ucleate oogonia w i t h condensed n u c l e i , are numerous. The p o o r l y defined dark grey area (lower center) i s a blood v e s s e l (X4OO). 8. The same s e c t i o n as 2. 7 but deep i n the c o r t e x where oogonia and primary oocytes are l o o s e l y e n c i r c l e d by elongate pre-granulosa c e l l s (X800). 9. The cor t e x of an ovary about 144 days o l d . There i s a marked i n c r e a s e i n the amount of connective t i s s u e between the sex c e l l s t h a t range i n development from oogonia to primary oocytes (X400). 10. An ovary from a p o s t n a t a l fawn to 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 increased amount of connective t i s s u e (X400). 20 cytoplasm ( P l a t e 2. 1, 7 and 9 ) . 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 i n older f e t a l o v a r i e s . I n f e t a l o v a r i e s 102, 122,and 123 days ol d , t h e r e were a l l stages of s e x - c e l l development, from oogonia to the primary oocyte stage ( P l a t e 2.2-8). The germ c e l l s occurred i n clumps or cords immediately below the e p i t h e l i u m but they were more sc a t t e r e d deep i n the c o r t e x . A great number of sex c e l l s i n the outer c o r t e x were i n the m e i o t i c prophase of 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 the primary oocyte stage and were enclosed i n p r i m o r d i a l f o l l i c l e s . Many oogonia and primary oocytes were a t r e t i c . In the 1 4 4 - d a y f e t a l o v a r i e s there 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 , e s p e c i a l l y around the p e r i p h e r y , but a greater p r o p o r t i o n of the germ c e l l s had en-larged to the primary oocyte stage 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 of the sex c e l l s i n o v a r i e s 170 and 177 days old 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 . Most primary oocytes were ensheathed i n e p i t h e l i a l c e l l s and some were contained i n secondary 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 there was c o n s i d e r a b l e growth of the p r i -mary oocyte and increased numbers of a t r e t i c oocytes. The m a j o r i t y of oocytes,located i n p r i m o r d i a l or primary f o l l i c l e s , r a n g e d between 15 and 20 u i n diameter. Large oocytes i n secondary and t e r t i a r y f o l l i c l e s were en-veloped i n a zona p e l l u c i d a , w h i c h formed around oocytes as small as 22 u , i f the oocyte was ringed w i t h f o l l i c u l a r c e l l s . 1. A F o l l i c u l a r Development. The s i z e and type of the 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 sum-marized 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 of one continuous l a y e r of f l a t t e n e d e p i t h e l i a l c e l l s around the sex c e l l . E p i t h e l i a l c e l l s formed an incomplete r i n g around primary oocytes as e a r l y as 102 days i n a f e t u s from a doe reared 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 old from w i l d deer. At t h i s stage the e p i t h e l i a l or granulosa c e l l s were t h i n l y d i s t r i b u t e d over the 20 to 25 u oocyte, which had reached the 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 . At 144- days, many f o l l i c l e s were surrounded by c u b o i d a l granulosa c e l l s and thereby had reached the p r i m a r y - f o l l i c l e stage. 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 of granulosa c e l l s , but no lumen, occurred i n f e t a l o v a r i e s older than 170 to 177 days. These f o l l i c l e s a t t a i n e d diameters of 43 to 60 u, contained oocytes of 30 to 35 P-, and were s t i l l a t the p r i m o r d i a l or primary stage of development. A s i m i l a r p i c t u r e of 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 , except t h a t some f o l l i c l e s , d e e p i n the c o r t e x , had reached the 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 stage. These a t t a i n e d diameters of 220 u, contained oocytes up to 72 u, and were enveloped i n t h i c k l a y e r s of granulosa and t h e c a l c e l l s ( P l a t e 1. 6). Many of the l a r g e f o l l i c l e s were degenerate and some were replaced w i t h h y a l i n connective t i s s u e ( P l a t e 2. 10). 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 uterine horns. Except for 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 cavity because there i s no peritoneal capsule or ovarian bursa. Ovaries of prepubertal deer are flattened ovoid structures with a smooth surface. At puberty, ovaries become much enlarged and flattened i f many small f o l l i c l e s but no large f o l l i c l e s have developed. At the other extreme, the development of one large f o l l i c l e i n a small ovary r e s u l t s i n a spherical form. Large f o l l i c l e s protrude s l i g h t l y from the normal outline -of the ovary. Extrusive or herniated f o l l i c l e s , and the corpora lutea and corpora lutea scars .that develop from them, create protuberances of various shapes, including spherical, bulbular, and mushroom. In addition to extrusive f o l l i c l e s and large protrusive corpora lutea, there are smaller surface features that vary considerably i n form, size,and color. Often, they form small protuberances less 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. Black ones represent newly-ruptured f o l l i c l e s or the surface ovulation scars of former corpora lutea of pregnancy. A l l of the structures indicate the point of an ovulation, recent or old, and their color i s i n d i c a t i v e of the i r age. Young active corpora lutea are represented by red protuberances and older corpora lutea by cream, yellow, or orange bulbs of tissue on the ovarian surface. Pale orange or yellow protuberances represent degenerate corpora lutea or their scars. Occasionally, p i t s are found on the surface of the ovary that indicate former' ruptures of small, partly-extrusive 23 f o l l i c l e s . The weight of deer o v a r i e s was r e l a t e d to the season and age of the doe. W i t h i n age-classes, the weights of o v a r i e s d u r i n g the breeding season changed i n s i g n i f i c a n t l y from 1963 to 1967. Consequently, o v a r i a n weights from a l l years were combined to show more a c c u r a t e l y the r e l a t i o n -s h i p between age and weight. There was a general i n c r e a s e i n s i z e , w i t h age, up to the com-bined 7.5 to 9-5 a g e - c l a s s , although a s i g n i f i c a n t d i f f e r e n c e between adjacent means only occurred between ages 1.5 and 2.5 ( F i g - 1). There was v i r t u a l l y no d i f f e r e n c e i n the weight of l e f t and r i g h t o v a r i e s from any a g e - c l a s s . In breeding-season c o l l e c t i o n s , mean weights f o r 278 p a i r s of o v a r i e s from deer of a l l ages were 62.3 g ( i t ) and 62.9 g ( r t ) . F i g . 2 shows seasonal changes i n the weight of ovaries from f o u r age-classes of deer. P l o t t e d values are averaged f o r each age-class per c o l l e c t i n g p e r i o d . The data were not 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 trends were apparent. I n fawns there was a decrease i n o v a r i a n weight from f a l l to mid-winter. Ovaries of y e a r l i n g s i n c r e a s e d i n weight from October to mid-winter and then s t a b i l i z e d . A c o n s i s t e n t f e a t u r e of a d u l t o v a r i e s was the marked i n c r e a s e i n weight be-tween e a r l y May and mid-June. This i s a t t r i b u t e d to 1) an i n c r e a s e i n the volume of corpora l u t e a and 2) 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 . I n June there was no i n c r e a s e i n o v a r i a n weight of females 2 years o l d , because two of the three i n the sample had given b i r t h and contained shrunken corpora l u t e a . Chapter 2. The Surface E p i t h e l i u m . The surface 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 source of 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 of germ c e l l s 36 28 26 24 22 20 18 16 14 12 10 8 6 4 2 F i g . 1. The age-specific weight of paired ovaries from deer collected i n November and early-December from 1963 to 1966. (Mean, S^-t^s* s, range, and sample s i z e ) . * 95% confidence l i m i t |41 0.5 1 77 1 1.5 53 '2.5 28 3.5 22 15 4-5 5-5 '. AGE (YEARS) 16 6.5 7.5-9.5 I 12 10.5S+ 1 — j 1 1 : 1 1 ; i ~ i 7 0 N . D J J ' M A M J M O N T H ( i f any) may be related to the estrous cycle (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 usually flattened and contained elongate, f l a t -tened n u c l e i . In some areas, the epithelium was stretched u n t i l i t constituted l i t t l e more than a membrane with sparse, t h i n nuclei. Occasionally, no epithelium was found. At dips i n the surface, at invaginations, and between lobes, the c e l l s were cuboidal or even columnar. Obviously, th 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 generally i n a stretched state. 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, espe c i a l l y when i n a stretched condition. There were few invaginations of surface epithelium i n t o the cortex of fawn ovaries. Invaginations were more numerous i n ovaries with large numbers of primordial 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 at p i t s i n the surface and at invaginations. 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 into the stroma below the tunica albuginea. Some of the.invaginated c e l l s seemed to develop into large c e l l s resembling oogonia, but i f oogenesis occurred after 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 involved. 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 tunica 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 of tu b u l e s , i n or below the t u n i c a , termed sub-surface c r y p t s by H a r r i s o n and Mathews (1951). Some fawn o v a r i e s contained l o n g tubules below the t u n i c a , s i m i l a r to those noted i n the badger (Neal and H a r r i s o n , 1958). The appearance of the surface e p i t h e l i u m of y e a r l i n g o v a r i e s was s i m i l a r to t h a t of fawn o v a r i e s , except 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 of 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 the c o r t e x . Dur-i n g the breeding season,the surface e p i t h e l i u m of 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 of the surface of many o v a r i e s / t h e r e was a t h i c k zone of 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 does,although 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 female 5-5 years o l d . Chapter 3. The Ovarian F o l l i c l e . 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. 1. 1 The Antrum. In the antrum of the deer f o l l i c l e , there i s a f i n e i n t e r c o n n e c t i n g network of membranes or f i b e r s c o n t a i n i n g s p h e r i c a l a c i d o p h i l i c bodies up to 3 u i n diameter. These membranes or f i b e r s are continuous w i t h , and may o r i g i n a t e from, the outer granulosa c e l l membranes. Because the a c i d o p h i l i c spherules seem to o r i g i n a t e at the antro-granulosa boundary, they may be degradation products of granulosa c e l l s . I n s m a l l f o l l i c l e s the network f i l l s the antrum but 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 occupies l e s s than one-half the antrum. 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 , i t s f l u i d becomes p r o g r e s s i v e l y more blue when s e c t i o n s are st a i n e d i n a n i l i n e blue. 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 or glycogen are present i n the f l u i d . 3. 1. 2 The Oocyte. In fawns, a l l or almost a l l sex c e l l s have reached the primary oocyte stage and are i n p r i m o r d i a l f o l l i c l e s . As the granulosa c e l l s i n c r e a s e i n number, the oocyte 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 ceases. The zona p e l l u c i d a , 4 to 7 u t h i c k , i s traversed a t r i g h t angles by processes t h a t presumably provide communication between the granulosa c e l l s and the oocyte ( P l a t e 3:4 and 8 ) . Except f o r these almost sub-microscopic processes, the zona p e l l u c i d a i s a homogenous a n i l i n e blue and PAS-positive l a y e r i n some p r e p a r a t i o n s . I n other m a t e r i a l the membrane appears to be laminated ( 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 next to the nuclear membrane of the oocyte and an outer a n i l i n e - p o s i t i v e l a y e r . 3. 1.3 The Membrana Granulosa. 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 from the germinal e p i t h e l i u m (Brambell, 1956), but i n deer they o f t e n appear to develop from surrounding stromal c e l l s . Whatever t h e i r o r i g i n , the 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 the contained oocyte enlarges. F o l l i c u l a r 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 diameter of about 200 u and forms pools i n 300 u f o l l i c l e s . At one p o i n t on the membrana granulosa of v e s i c u l a t e f o l l i c l e s , a mound of c e l l s , the cumulus oophorus, p r o j e c t s i n t o the antrum. Although u s u a l l y l o c a t e d opposite the outermost surface of the f o l l i c l e , the 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 the si d e of the f o l l i c l e or even adjacent to the f o l l i c u l a r and ov a r i a n s u r f a c e . The v e s i c u l a t e n u c l e i of granulosa c e l l s are g e n e r a l l y 5 to 7 u Facing page 29 P l a t e 3. The Components of 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 ) , primary and secondary f o l l i c l e s . The nuc l e o l u s i n the nucleus of 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 oocyte 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 occurred i n most f o l l i c l e s between the primary and small t e r t i a r y stages (X200). 3. The oocyte w i t h i n t h i s primary f o l l i c l e contains one s p h e r i c a l b a s o p h i l i c i n c l u s i o n (X800). U- A layered zona p e l l u c i d a c o n t a i n i n g granulosa c e l l processes and a discon t i n u o u s chromophilic 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 occurs between the 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 the ooplasm (X2000). 5. Two oocytes i n a t e r t i a r y f o l l i c l e . Such p o l y o v u l a r f o l l i c l e s occurred i n many deer (X80). 6. A Call-Exner body on the inner surface of the membrana granulosa. These s t r u c t u r e s , of unknown s i g n i f i c a n c e , were numerous i n some females (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 the verge of f i r s t o v u l a t i o n . The oocyte and surrounding r i n g of c e l l s i s v i r t u a l l y f r e e from the cumulus oophorus (X200). 8. A p o r t i o n of an oocyte to i n d i c a t e l a y e r i n g of the zona p e l l u c i d a and the appearance of the ooplasm. The outer, ooplasm was f i n e l y granular i n some oocytes of f o l l i c l e s near rupture whereas the c e n t r a l ooplasm (lower l e f t ) was c o a r s e l y granular (X2000). 30 i n diameter, average just under 6u, and large nuclei adjacent to the lumen may excede 8 u. There i s a gradient of increasing nuclear sizes from the basement membrane to the lumen, with those i n the cumulus oophorus being the largest. In mature f o l l i c l e s some of the c e l l s near the lumen have basophilic cytoplasm, whereas most others have aci d o p h i l i c cytoplasm. T i n c t o r i a l differences 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. Circular groupings of granulosa c e l l s around a small central region 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 their significance could not be ascertained. Granulosa c e l l s were d i v i d i n g i n a l l active f o l l i c l e s , except those near ovulation. The thickness of the membrana granulosa was maximal (50 to 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 cys 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 usually quite uniform around the f o l l i c l e , i t was thicker i n a region where the f o l l i c l e was unable to stretch, such as adjacent to another f o l l i c l e . 3 . 2 The Preovulatory F o l l i c l e . To f u l l y i nterpret 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 physiological rupture from those undergoing early degeneration. By physiological rupture, I mean normal ovulation, presumably mediated by the sequential secretion of hormones from the p i t u i t a r y gland, as opposed to the' "mechanical" or necrotic 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 ovulation, i t was possible to 31 d i s t i n g u i s h those near ovulation, although the exact i n t e r v a l to rupture could not be estimated. Because ovaries of 367 females were collected throughout the breeding season, November 6 to December 6, i t followed that, at the time of death,several females were within one day of ovulation. One doe was undoubtedly on the verge of ovulation, as indicated 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 following 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 ovulation contained a f l u i d that was positive for 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 c e l l s . In 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 properties 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 ovulation con=-tained an accumulation of a c i d o p h i l i c droplets along the surface of the stratum granulosum. The o r i g i n and chemistry of these droplets i s not known but similar spherules are commonly associated with thecal blood vessels that loop into the granulosa below the cumulus oophorus. 3. 2. 2 The Oocyte. Including the zona pel 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 size as oocytes i n small t e r t i a r y f o l l i c l e s . Con-sequently, size was of no diagnostic value i n determining the degree, of f o l l i c u l a r maturation. The zona pellucida was 4- to 5 }X thick i n most healthy f o l l i c l e s , b u t was thicker around a t r e t i c oocytes. In sections stained with Masson's trichrome containing ponceau 2R, an outer layer of the zona was positive' for a n i l i n e blue,but the inner layer was a c i d o p h i l i c . Often the inner layer was discontinuous 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 usually appeared uniform and f i n e l y granular. Occasionally,there were splotchy regions i n the cytoplasm and the central portion was more coarsely granular (Plate 3. 8 ) . S e r i a l sections revealed that, prior to ovulation, most nuclei were large, vesiculate, and i n the r e s t i n g phase of meiosis. This was the state of the nuclei i n oocytes of large f o l l i c l e s i n a doe already inseminated (Plate 4. 4) • Nuclei reached a maximum size 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 v i r t u a l l y clear. Prior to the alignment of chromosomes at metaphase, the chromatin became condensed into one or two larger clumps that were often associated with i n f o l d i n g of the nuclear membrane next to the zona pellucida. 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 either free from the mural granulosa or were held i n tenuous bond (Plate 3. 7 ) . Loosening of the cumulus was accomplished through autolysis of granulosa c e l l s at i t s base. C e l l autolysis usually created several elongate spaces p a r a l l e l to the granulosa layer. Fusion of these resulted i n a f r e e - f l o a t i n g outer portion which contained the oocyte, or the degen-erating 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 ultimately a space, was commonly found a few c e l l s d i stant from the oocyte. Cells r e - ' maining around the oocyte contained larger nuclei (to 8 u) and greater amounts of finely-granular, a c i d o p h i l i c cytoplasm. In some f o l l i c l e s j u s t prior to ovulation, the c e l l s remaining around the oocyte radiated out on tapered stalks of cytoplasm (corona radiata formation). Oocytes trapped i n 33 ruptured f o l l i c l e s were s t i l l encircled by attached granulosa c e l l s (Plate 4. 7-9),and free 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 after i t had v i r t u a l l y ceased i n the remainder of the granulosa. Cessation of mitosis then proceeded from the periphery of the cumulus towards the oocyte, where i t occurred u n t i l , and during, corona radiata formation. The l a t t e r process precedes ovulation 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 prior to the ovulation 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 thick-ness from several ranks of c e l l s , to a single layer of c e l l s ( i n very large f o l l i c l e s ) . Generally, the granulosa layer was thicker and more active 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 pseudostratified layer of columnar c e l l s . 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 stalks of cytoplasm (Plate 7. 5). Nuclei i n these columnar c e l l s were opposed to the membrana propria,thereby creating 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 basophilic cytoplasm. In preovulatory f o l l i c l e s , numerous ac i d o p h i l i c granules or droplets were produced at the surface of the granulosa. These droplets or granules were secreted by the granulosa c e l l s ; resulted 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, clear spaces the size of granulosa c e l l s usually occurred. These spaces indicated the autolysis of surface c e l l s . In 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 usually some indications of c e l l death, t y p i c a l of early 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 ovulation of the season. Note the r i n g of c e l l s around the oocyte and the vesiculate nucleus within 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 radiata. 3. An oocyte i n a growing fourth-cycle f o l l i c l e an estimated 12 days after second ovulation. 4. An oocyte i n a f o l l i c l e near rupture. The female was within hours of f i r s t ovulation of the season. Note the lamellar nature of the zona pellucida. 5. An oocyte i n a f o l l i c l e within hours of rupture. The female, approaching second ovulation, contained sperm i n i t s oviducts. The cumulus oophorus was detached from the mural granulosa. 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 cycle. 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 viable. The zona pellucida i s thick; the ooplasm i r r e g u l a r i n outline 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 cycle. The nucleus of the oocyte i s large and vesiculate. There i s no i n d i c a t i o n of nuclear maturation. 9 . A section 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 cycle. The nuclear membrane i s i n d i s t i n c t but the chromatin has not clumped into one mass. The nuclei of the granulosa c e l l s surrounding the oocyte are vesiculate but those of the mural granulosa are condensed. 3 5 included cytoplasmic vacuolation and autolysis of c e l l s below the surface, formation of pyknotic nuclei 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 f i n e f i b e r s . The l a t t e r c e l l s produced a fibrous 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 f o l l i c l e s (Plate 7.-4). More intense staining was associated with cyto-plasmic shrinkage, increased granulation, and nuclear condensation. Some nuclei changed from the round, vesicular type of growing f o l l i c l e s to oblate, almost-solid structures. In newly-ruptured f o l l i c l e s , c e l l and nuclear condensation (resulting i n hyperchromasia) commonly occurred during the metaplasia of granulosa c e l l s to l u t e a l c e l l s . 3 . 2. 4 The Theca. In a l l large f o l l i c l e s the theca interna 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 large f o l l i c l e s of the f i r s t estrous cycle. I t was often thickest i n the area below the cumulus oophorus. Most of the oblate or fusiform c e l l s contained irregularly-shaped, oblate nu c l e i . The vacuolated cyto-plasm of thecal c e l l s i n growing f o l l i c l e s became more dense and acido-p h i l i c i n some f o l l i c l e s j u st before ovulation. C e l l d i v i s i o n s were rare i n the thecal c e l l s of preovulatory f o l l i c l e s . . As ovulation neared, the vessels i n the theca interna became large and distended with blood, causing them to push into the granulosa layer (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 highly a c i d o p h i l i c spherules ranging from 1 to 8 u i n diameter. These spherules were also located within the 36 i vessels, where they presumably originated. Their significance 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 vessels that served the f o l l i c l e . 3. 3 Normal Atresia Of F o l l i c l e s . 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 character-i 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 primordial stage of development. Atresia of primordial f o l l i c l e s was indicated by cytoplasmic changes i n the oocytes, including: hypochromasia of the ooplasm, formation of large vacuoles, enlargement or shrinkage, and inclusions. 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 mem-brane. Cytolysis of the f o l l i c u l a r c e l l s around the oocyte occurred at an early stage of degeneration. In some instances the f o l l i c u l a r c e l l s were resorbed leaving the oocyte surrounded only by stromal c e l l s . A thick zona pellucida usually developed around the oocyte. Oocytes incorporated i n a zona pellucida degenerated slowly. I f a t r e s i a occurred before zona pellucida formation, the oocyte autolysed, leaving a space surrounded by a few flattened 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 ositive f l u i d coagulum, and eventually by stromal connective tissue. Early indications of a t r e s i a i n primary and secondary f o l l i c l e s included, the formation of spherical a c i d o p h i l i c inclusions i n the ooplasm (Plate 3. 2 and 3), vacuolation of the ooplasm, and the absence of mitosis i n the granulosa. As a t r e s i a progressed, the oocyte was replaced by hyalin material and u l t i m a t e l y by stromal connective tissue. 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 a r b i -t r a r i l y d i v i d e d i n t o e i g h t stages so t h a t r e f e r e n c e could be made to a p a r t i -c u l a r stage 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 of the degenerating f o l l i c l e could be i n t e r r e l a t e d . Apparently, f o l l i c l e s q u i c k l y passed through the f i r s t few stages of t h i s c l a s s i f i c a t i o n , but l a t e r stages occupied a c o n s i d e r a b l e time. 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 are described as f o l l o w s : 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 pressure, were misshapen by other a c t i v e f o l l i c l e s or corpora l u t e a . In c o n t r a s t to 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 of 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 g r a n u l a r , " d i r t y " (from c e l l d e t r i t u s ) , coagulated, and achromatic. As the f o l l i c l e became more degenerate,the 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 t i n c t o r i a l 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 evident as e a r l y as the t r a n s i t i o n a l stage. By the t h i r d stage of a t r e s i a , t h e f l u i d was e s s e n t i a l l y achromatic. Coagulation of the f l u i d created f i s s u r e s as e a r l y as stage one of a t r e s i a . By stage 5,there were whorls of grey d e t r i t u s i n the shrunken lumen. 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 degenerate oocytes of l a r g e f o l l i c l e s s t ained i n t e n s e l y and the endoplasmic r e t i c u l u m i n the ooplasm became more prominent. The appearance of vacuoles 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 . As a t r e s i a progressed, these enlarged to give the cytoplasm a c o a r s e l y vacu-olated appearance ( P l a t e s 5 and 6 ) . Huge vacuoles or spaces developed i n a F a c i n g page 38 P l a t e 5 P r e o v u l a t o r y and Degenerate Oocytes. 1. The cumulus oophorus and contained oocyte i n a 59 mm-5 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 approaching f i r s t o v u l a t i o n ( X 2 0 0 ) . 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 of the corona 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 to the oocyte. The c e l l s of the corona 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 degenerate oocyte i n an a t r e t i c 41 mm f o l l i c l e . The f o l l i c l e , having f a i l e d to rupture at f i r s t o v u l a t i o n 1 to 2 days p r e v i o u s l y , was destined to l u t e i n i z e and become an accessory 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 chromatin i n the oocyte of a 43 mm a c t i v e f o l l i c l e . The o v a r i e s of t h i s doe contained a newly-ruptured 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 to rupture (X800). 5. A b a s o p h i l i c zona, of unknown s i g n i f i c a n c e , adjacent to the nucleus of an oocyte. The 33 mm^  f o l l i c l e c o n t a i n i n g the oocyte was i n the o v a r i e s of a y e a r l i n g approaching i t s f i r s t o v u l a t i o n (X800). 6. A degenerate oocyte, c o n t a i n i n g two p o l a r bodies, i n an a t r e t i c f o l l i c l e (stage 3 to 4 of a t r e s i a ) ( X 2 0 0 ) . 7. Nuclear fragmentation i n a oocyte l o c a t e d i n a 119 mm^  l u t e i n i z e d f o l l i c l e . F i r s t o v u l a t i o n occurred about 6 days p r e v i o u s l y ( X 4 0 0 ) . 3 8. Large vacuoles and the degenerate nucleus i n an oocyte of a 33 mm growing f o l l i c l e i n a doe 2.5 years old p r i o r to f i r s t o v u l a t i o n of the season (X4OO). 9. An enlarged, fragmented, and vacuolated oocyte i n a small (1.5 mm diam) f o l l i c l e . C e l l d i v i s i o n s s t i l l occurred i n the surrounding granulosa ( X 4 0 0 ) . 1 0 . Two-celled oocyte 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 (stage 5 or 6 of a t r e s i a ) i n a doe approaching f i r s t o v u l a t i o n of the season ( X 4 0 0 ) . 11 . I n v a sion of the oocyte by phagocytic c e l l s i n an a t r e t i c f o l l i c l e ( X 4 0 0 ) . P l a t e 6 Fac i n g page 39 M e i o t i c D i v i s i o n In the Oocyte. 1 . Nuclear c o n f i g u r a t i o n i n the oocyte of a f o l l i c l e estimated to be w i t h i n a fe\^ hours of o v u l a t i o n ( X 4 O O ) . 2 . Metaphase of meiosis I i n a 6 7 mm^  f o l l i c l e on the verge of rupture ( X 4 0 0 ) . 3. E a r l y telophase of meiosis 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 Stage of maturation (X800). 4. Late telophase of meiosis I i n a. 51 mnP a t r e t i c f o l l i c l e (Stage 2 of a t r e s i a (X400). 5. Higher m a g n i f i c a t i o n of 6 . 4 (X2000). 6. The f i r s t p o l a r body and chromatin of the secondary oocyte i n a Stage-2 a t r e t i c f o l l i c l e ( X 2 0 0 0 ) . 7 . E a r l y metaphase of meiosis I I i n a Stage 3 a t r e t i c f o l l i c l e . A p o r t i o n of the f i r s t p o l a r body i s evident (X2000). 8. Late metaphase of meiosis I I i n a small a t r e t i c f o l l i c l e (Stage 5)• The f i r s t p o l a r body i s degenerate (X400). 9- Higher m a g n i f i c a t i o n of . 6 . 8. I n the o r i g i n a l photomicrograph the metaphase s p i n d l e was c l e a r l y evident as were c e l l processes through the zona p e l l u c i d a (X2000). 3 1 0 . Late anaphase or e a r l y telophase of meiosis I I i n a 30 mm f o l l i c l e a t Stage 4 of a t r e s i a ( X 4 0 0 ) . 1 1 . A two-celled oocyte i n a small f o l l i c l e at Stage 6 of a t r e s i a ( X 4 0 0 ) . few oocytes. Nuclear changes occurred i n the degenerate oocyte including: abnormal enlargement of the nucleus, abnormal clumping of chromatin, breakdown of the nuclear membrane, and loss 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 divided.(Plate 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 pellucida. 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 just inside the zona pellucida. In some oocytes the remaining chromosomes i n the ooplasm divided again by the 4-th or 5th stage of at r e s i a . A few divided into two c e l l s , a process termed pseudocleavage rather than parthenogenesis (Ingram, 1962). In a t r e t i c oocytes, the borders of the zona pellucida often became i n d i s t i n c t and pitted with f i s s u r e s . The membrane, 3 to 5 p. thick around active 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 indications 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 granulosa-cell processes from the zona pellucida. 3.3. 3 The Granulosa. The physiological 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. In the 1 to 8 scale of a t r e s i a , the early stages were defined l a r g e l y by the appearance of the granulosa. Three ch 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 early a t r e s i a . These were: l ) Absence of mitosis i n a l l granulosa c e l l s , including those of the cumulus oophorus 41 (and the corona rad i a t a ) . 2) Shrinkage of the cytoplasm and nuclei v/ith concomittant hyperchromasia. 3) The appearance of new c e l l types on the surface of the granulosa layer. 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 portion of the cumulus oophorus. In 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 , mitosis were found only i n c e l l s of the corona radiata. Thereafter c e l l d i v i s i o n s ceased. Ce l l s i n the cumulus oophorus dissapeared r a p i d l y during the early stages of at r e s i a . Large lacunae occurred at 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 also occurred a few c e l l s distant from the oocyte. By the f i r s t stage of at r e s i a , the cumulus- oophorus was detached from the mural granulosa, or was nearly detached, and many c e l l s were undergoing autolysis. By the second stage of a t r e s i a , 20 to 50% of the c e l l s were undergoing autolysis and many c e l l s contained pyknotic n u c l e i . In 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 active c e l l s around the oocyte. Only 10 to 4-0% of the c e l l s remained by the thi r d stage of at r e s i a . The remain-ing c e l l s , except those of the corona radiata ( i f present), were.degenerate and enclosed i n a peripheral network of fin e f i b e r s . By the fourth 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 . A t h i n layer of c e l l s , occasionally i n r a d i a l arrangement (Plate 5. 1 - 3 ) , s t i l l persisted around the oocyte. Only scattered,small clumps of degenerate c e l l s remained at the f i f t h stage of . at r e s i a , and only one layer of c e l l s encircled 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 clusters of c e l l s i n f o l l i c l e s i n the si x t h stage of a t r e s i a . Changes i n the mural granulosa at the various stages of at r e s i a U2 are i l l u s t r a t e d i n P l a t e 7. Shrinkage and hyperchromasia of c e l l s and n u c l e i were f i r s t evident 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 stage. The l a y e r of 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 to the basement membrane and to one another. C e l l death proceeded from the antrum towards the basement membrane and the 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 of a t r e s i a , occurred along the l u m i n a l border of the granulosa. These c e l l types i n c l u d e d a f i b e r - p r o d u c i n g c e l l , c e l l s w i t h pyknotic n u c l e i , c e l l s e x i b i t i n g 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 other c e l l s w i t h f e a t u r e s which u s u a l l y s i g n i f y c e l l death (Nieburgs, 1967). A s m a l l number of these abnormal c e l l types, and increased i n d i c a t i o n s of c e l l pathology i n the remainder of the membrana granulosa, were t y p i c a l of the stage of 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". F i b e r p r o d u c t i o n continued u n t i l a cont 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 surface 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 stage,the i r r e g u l a r l y - s h a p e d n u c l e i were concentrated c l o s e to the basement membrane. In 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 c e l l s continued u n t i l o n l y one l a y e r of c e l l s remained c l o s e l y attached to the thickened basement membrane. At t h i s t h i r d stage of a t r e s i a , t h e f i b r o u s network was t h i c k , contained 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 several-ranked degenerate c e l l s appeared to develop i n t o f i b r o b l a s t - l i k e c e l l s a l i g n e d p a r a l l e l to the f o l l i c u l a r w a l l . 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 from, or r e p l a c e d , the granulosa c e l l s a t the f o u r t h stage of a t r e s i a . The t h i c k n e s s of these c e l l s v a r i e d from one to s e v e r a l . They produced a l a y e r of amorphous h y a l i n t i s s u e , the thi c k n e s s of which was p r o p o r t i o n a l to the th i c k n e s s of the l a y e r of f i b r o b l a s t i c c e l l s . These d e p o s i t i o n s , along w i t h f u r t h e r h y a l i n i z a t i o n of the former theca i n t e r n a and shrinkage of the f o l l i c l e , caused the w a l l of the s t r u c t u r e to become p r o g r e s s i v e l y t h i c k e r (stages Facing page 43 P l a t e 7. The F o l l i c u l a r W a l l . 1. The membrana granulosa, theca i n t e r n a , and a p o r t i o n of the theca externa of 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 the granulosa (two i n the photograph) and the t h i n l a y e r of p o o r l y defined theca i n t e r n a c e l l s are c h a r a c t e r i s t i c of growing f o l l i c l e s ( X 4 O O ) . . 3 2. The w a l l of 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 of development. The enlarged blood v e s s e l s i n the theca i n t e r n a , a l a y e r of unattached granulosa, and the columnar and po l a r c e l l s i n the mural granulosa are t y p i c a l of " t r a n s i t i o n a l " f o l l i c l e s (X800). 3. Another view of the above f o l l i c l e . The blood v e s s e l s of the theca i n t e r n a have invaginated i n t o the b a s a l r e g i o n of the degenerate cumulus oophorus. P o l a r , columnar c e l l s r a d i a t e out from the v e s s e l loops ( X 2 0 0 ) . 4. The w a l l of a 50 rnrrP f o l l i c l e between the t r a n s i t i o n a l stage and stage one of a t r e s i a . Many n u c l e i of both the membrana granulosa and the theca i n t e r n a are condensed i n t h i s f o l l i c l e which was estimated to be on the verge of rupture ( X 4 O O ) . 5 . The w a l l of a f o l l i c l e i n the t r a n s i t i o n a l phase (between growth and a t r e s i a ) of development. Note the " s t a l k e d " columnar granulosa 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 the l a r g e blood'vessel i n the theca i n t e r n a (X800). 6. The w a l l of a 62 mnr f o l l i c l e i n the f i r s t stage of a t r e s i a . Forma-t i o n of the r e t i c u l a r network on the surface of the membrana granulosa t y p l i f i e s t h i s stage. Such f o l l i c l e s may rupture at f i r s t e s t r u s or l u t e i n i z e , and become an accessory corpus luteum. 3 7. The w a l l of a 4 4 mm f o l l i c l e i n the second stage of a t r e s i a . There i s a continuous zone of f i b e r s on the degenerate membrana granulosa ( X 4 0 0 ) . 3 8. The w a l l of a 22 mm . f o l l i c l e i n the t h i r d stage of a t r e s i a . Death of granulosa c e l l s has r e s u l t e d i n a wide zone of f i b e r s and degenerate c e l l s ( X 4 0 0 ) . 3 9. The w a l l of a 7 mm f o l l i c l e i n the f o u r t h stage of a t r e s i a . Auto-l y s i s of the granulosa c e l l s i s n e a r l y complete. The bas a l l a y e r of 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 of a f o l l i c l e i n the f o u r t h to f i f t h stage of a t r e s i a . Note the m i t o s i s ( r i g h t center) i n the c e l l s a l i g n e d p a r a l l e l to the thickened 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). 3 11. The w a l l of a 1.8 mnr f o l l i c l e i n the f i f t h stage of a t r e s i a . A c l u s t e r of degenerate granulosa c e l l s p e r s i s t i n the lumen. The former t h e c a l l a y e r s ( l e f t t h i r d of the 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 one-quarter) i s composed of 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 secreted by the f i b r o b l a s t s (X800). 12. The w a l l of a f o l l i c l e i n the s i x t h stage of a t r e s i a . A t h i c k l a y e r of connective t i s s u e ( l e f t t w o - t h i r d s of photograph) surrounds a small lumen. F i b r o b l a s t s are s t i l l s e c r e t i n g amorphous t i s s u e (X400). s i x and seven of a t r e s i a ) . The t r a n s f o r m a t i o n of degenerate granulosa c e l l s i n t o a c t i v e , s p i n d l e - s h a p e d c e l l s resembling f i b r o b l a s t s ( A t r e s i a 4) was retarded i n l a r g e f o l l i c l e s i n which a very t h i n (one to three c e l l s ) granulosa l a y e r was present. F o l l i c l e s w i t h only a s i n g l e l a y e r of granulosa c e l l s p e r s i s t e d f o r l o n g p e r i o d s . Such f o l l i c l e s , u s u a l l y i n d i -c a t i v e of abnormal endocrine f u n c t i o n (Moulton, 1961), are termed " c y s t i c " because of t h e i r slow degeneration (Brambell, 1956). The 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 at the f o u r t h stage 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 enlarged i n t o c e l l s r e -sembling young l u t e a l c e l l s ( P l a t e 7. 10 and P l a t e 8. 1). This hypertropy and h y p e r p l a s i a of t i s s u e u s u a l l y occurred i n only a p o r t i o n of the degenerate f o l l i c l e . Yellow pigment, apparently a product of l i p o i d degeneration, formed on degeneration of the n e o p l a s t i c t i s s u e . About the f i f t h stage 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 s e m i s p h e r i c a l , s t r u c t u r e of a c t i v e and dead c e l l s developed i n some f o l l i c l e s ( P l a t e 8. 3-6). Apparently i t o r i g i n a t e d from a r e t r a c t i o n of most of the former granulosa to one l o c a t i o n on the w a l l of the f o l l i c l e . S p i n d l e - s h a p e d , f i b r o b l a s t i c 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 and, to a l e s s e r extent, the i n n e r surface of the s t r u c t u r e . Most of the r e -maining i n t e r n a l c e l l s were degenerate. P e c u l i a r b a s o p h i l i c , f i b r o u s , and amorphous m a t e r i a l was produced i n the middle of the t i s s u e l a y e r . 3. 3. 4 The Theca. C e l l s of the theca i n t e r n a began degenerating l a t e r than those of the granulosa. I n d i c a t i o n s of a t r e s i a i n the theca i n c l u d e d 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 , 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 ressure, 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) increased amounts of amorphous h y a l i n t i s s u e . I n small 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 " g l a s s y " Facing page 45 P l a t e 8 Hyper p l a s i a and M e t a p l a s i a of Tissue i n A t r e t i c F o l l i c l e s . 1. E a r l y p r o l i f e r a t i o n of 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 f o l l i c l e ( X 4 O O ) . 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 from the degeneration of a small neo-plasm such as p i c t u r e d i n 8. 1. Some yellow pigment i s secreted around the degenerate, vacuolated 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 the f i f t h stage of a t r e s i a ( X 4 O ) . 4. A p o r t i o n of the s t r u c t u r e i n 8.3 at higher m a g n i f i c a t i o n . The outer c e l l s are f i b r o b l a s t i c - l i k e ( l e f t s i d e of photograph) and the in n e r c e l l s are degenerate, g r a n u l o s a - l i k e c e l l s . Between the' aforementioned l a y e r s i s p e c u l i a r , s t r u c t u r e l e s s , b l a c k - s t a i n e d m a t e r i a l ( X 4 O O ) . 5. A s i m i l a r but l a r g e r and more degenerate s t r u c t u r e than the one shown i n 8.3 above ( X 4 O ) . r 6. A p o r t i o n of the above s t r u c t u r e at higher m a g n i f i c a t i o n ( X 4 O O ) . 3 7. E a r l y l u t e i n ! z a t i o n of a 16 mm f o l l i c l e about one day a f t e r f i r s t o v u l a t i o n . A c t i v e c e l l s occur i n both the membrana granulosa (to r i g h t of dashes) and the theca 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 evident i n the theca 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 of a 12 mm f o l l i c l e 1 to 2 days f o l l o w i n g f i r s t o v u l a t i o n . A metaphase of m i t o s i s i s present i n the theca i n t e r n a (to l e f t of dashes) (X800). 3 9. L u t e i n i z e d w a l l of 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 o v u l a t i o n . The l o c a t i o n of the 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 granulosa and the theca 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 of the l u t e a l t i s s u e o r i g i n a t e d from granulosa c e l l s (X400). 46 membrane, formed around the lumen. Small a t r e t i c f o l l i c l e s i n which the g l a s s y membrane has become f o l d e d i n t o v a r i o u s shapes are termed "ribbon a t r e t i c " 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 (stages f i v e to.seven) u n t i l a s o l i d , t h i c k , amorphous band of a n i l i n e -p o s i t i v e t i s s u e surrounded the s h r i n k i n g lumen. Complete r e s o r p t i o n of the h y a l i n t i s s u e g r a d u a l l y ensued, l e a v i n g no t r a c e of the former f o l l i c l e . 3. 4 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% of 312 female deer underwent an a t y p i c a l form of a t r e s i a . I t occurred i n females of a l l ages and at any stage of the breed-i n g season,but was most extensive i n a fawn of 5 months. I n some females , on l y two or three f o l l i c l e s i n a p a i r of ova r i e s were i n v o l v e d but i n others 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 of degenerate t i s s u e . O c c a s i o n a l l y , t h i s c e n t r a l area, corresponding to the former antrum, was f i b r i n o u s , which suggests t h a t i t was der i v e d at l e a s t i n p a r t from the blood ( P l a t e 9. 3 and 4-). The p e r i p h e r a l 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 contained many leucocytes arranged i n c i r c u l a r p a t t e r n by c o l l a g e n i c f i b e r s of the 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 elements. The l a t t e r were pro-l i f e r a t i n g and, i n p l a c e s , extended a short d i s t a n c e i n t o the c e n t r a l n e c r o t i c r e g i o n ( P l a t e 9. l ) . Scattered around and ahead of these i n v a d i n g elements,were mobile phagocytic c e l l s t h a t invaded the 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 m a t e r i a l i n the c e n t r a l zone continued u n t i l o n l y the phagocytes remained and then the r e g i o n was chromophobic,except f o r the evenly 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 c h a r a c t e r i s t i c s of neutrophils, each with two or three spherical chromatin masses (Plate 9). Concurrently, the inflammation spread outward to involve a l l of the former theca and portions of the surrounding stroma. Portions of the thecal connective tissue were replaced by hyperplastic c e l l s and multinucleate giant c e l l s ; then by achromatic tissue with numerous, vacuo-lated and polymorphonuclear c e l l s ; and f i n a l l y by fibrous connective tissue. In some f o l l i c l e s , most of the peripheral portion was l a r g e l y acromatic and contained bizarre vacuolated c e l l s amongst the f i b e r s , d e t r i t u s , and active connective tissue c e l l s (Plate 9- 7). As the whole structure 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 highly variable amounts of hyalinized or collagenated connective tissue f i b e r s . In hyalinized structures, the c e l l spaces were smaller and contained yellow or orange-brown c e l l remnants. Cells i n loose non-encapsulated structures were large; the contents achromatic and degenerate.. Later,the connective tissue capsules were resorbed and eventually the lacunae was replaced by connective tissue stroma. Some of the above-mentioned features are i n d i -cative of a chronic inflammatory condition (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 early primordial stage, and probably did not pro-ceed past that stage. Nevertheless, several f o l l i c l e s containing two or r a r e l y three or four, oocytes had reached'the small t e r t i a r y f o l l i c l e stage (Plate 3.5). The largest polyovular f o l l i c l e , containing four oocytes, was 1.3 mm i n diameter. The greatest number of primary or large polyovular f o l l i c l e s , 18, occurred i n the ovaries of a doe 2.5 years old. This doe had the largest 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 collected. I t s ovaries contained innumerable polynuclear oocytes and polyovular primordial f o l l i c l e s . Tubules containing as many as 10 oocytes were numerous. The second largest number of polyovular f o l l i c l e s , eight, occurred i n the ovaries of a yearling. 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 routine examination of every tenth section, because the pr o b a b i l i t y of sectioning two or more oocytes with one s l i c e decreases as the size 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 section through each of hundreds of f o l l i c l e s . At least one oocyte i n each active polyovular f o l l i c l e was normal i n appearance ,but most supernumary oocytes were small and retarded, i n devel-opment. They were often located i n the mural granulosa or i n a poorly de-veloped cumulus oophorus. In many instances,the f o l l i c l e s were s t i l l grow-ing, as indicated by mitosis. 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 f o l l i c l e s . 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 cysts 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 lined by a single layer of granulosa c e l l s , was rare i n deer of Northwest Bay. One occurred i n an adult doe collected on October 30, prior to the breeding season. Another occurred i n a doe on the verge of i t s f i r s t ovulation of the current season. The lumena of both f o l l i c l e s were clear and achromatic. The second type of c y s t was f o l l i c u l a r i n form, l i n e d w i t h c i l i a t e d columnar e p i t h e l i u m , contained f l u i d , and occurred i n the h i l a r r e g i o n or i n the mesovarium. These s t r u c t u r e s , termed serous cystadenomas, v a r i e d great-l y i n s i z e and represented metaplasia or n e o p l a s i a of coelomic e p i t h e l i u m (Sternberg, 1963). The neoplasms, which d i d not appear to 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% (7/398) of a l l does. The l a r g e s t , c o n t a i n i n g about 200 ml of f l u i d , occurred i n a pregnant doe c o l l e c t e d on May 7. 3. 7 P r i m o r d i a l F o l l i c l e s 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 albuginea, 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 of each ovary. The circumference of the zone c o n t a i n i n g the primor-d i a l f o l l i c l e s was c a l c u l a t e d from the diameter of the s e c t i o n ( i f spheroid) 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 ocular s c a l e , i f the s e c t i o n had a r e c t a n g u l a r or i r r e g u l a r shape. The l e n g t h of the zone ( i n mm) c o n t a i n i n g the p r i m o r d i a l f o l l i c l e s was d i v i d e d i n t o the f o l l i c u l a r count to o b t a i n an index of f o l l i c u l a r occurrence per l i n e a r mm of the f o l l i c u l a r zone. Values 'obtained f o r the two o v a r i e s were averaged. G e n e r a l l y there was a p r o g r e s s i v e d e c l i n e i n the number of primor-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 ( F i g . 3). There was a . s i g n i f i c a n t de-crease (P < 0.05) i n the number of f o l l i c l e s between the 1.5 and the 2.5 age c l a s s e s , and between the l a t t e r and the 3.5 age c l a s s . Other d i f f e r e n c e s were not s i g n i f i c a n t . The index of p r i m o r d i a l - f o l l i c l e numbers was h i g h l y v a r i a b l e , e s p e c i a l l y i n young animals. The index v a r i e d from 1.2 to 17.5 i n fawns and from 2.8 to 27.4- i n y e a r l i n g s . I t i s d i f f i c u l t to c o n v e r t , a c c u r a t e l y , t h e s e two-dimension i n d i c e s of f o l l i c u l a r numbers to absolute numbers f o r each ovary s i n c e the t h i r d dimension must be considered. C a l c u l a t i o n s i n d i c a t e an Fig.. 3. 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 outer c o r t e x i n 10 u sections from the ovaries of deer c o l l e c t e d i n November and early-December, 1963-67. (Mean, 95% confidence l i m i t s , standard d e v i a t i o n and sample s i z e ) . 11 10 9 30 32 28 PH c o & q o. 1—t o !—I n § O c 2 24 20 1 17 T 17 I 4 1 • 4 "oTF — 1 — 2.5 4.5 6.5 8.5 • 10.5 AGE (YEARS) L2.5 14. 5 16. 18.5 o average number of about 50,000,and a range of from 8,000 to 200,000 prim-o r d i a l f o l l i c l e i n each ovary 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 every tenth s e c t i o n of one ovary from the o l d e s t deer, aged 19.5 years. With due c o n s i d e r a t i o n to 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 and i n t e r v a l , i t was c a l c u l a t e d t h a t the ovary contained 535 p r i m o r d i a l f o l l i c l e s and 15 secondary f o l l i c l e s . Assuming equal numbers of f o l l i c l e s i n the other ovary, the doe contained j u s t over 1000 oocytes. I t was estimated e a r l i e r t h a t each fawn ovary contained a s t o r e of from 10,000 to 200,000 oocytes, w i t h a mean of 50,000. Based on the above s t a t i s t i c s the average fawn had 100 times more oocytes than the o l d e s t doe. The average s i z e and appearance of p r i m o r d i a l f o l l i c l e s v a r i e d c o n s i d e r a b l y between i n d i v i d u a l s of a l l age-classes,but 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 to stages of the estrous c y c l e . However, as the breeding season progressed there appeared to be a greater number of 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 oocytes were enclosed i n a t h i c k zona p e l l u c i d a . •• 3. 8 T e r t i a r y F o l l i c l e s . 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 f o l l i c l e s , they 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 0.5- 4.1 mm3 Class I I 4-2-20 mrn^  Class I I I 21 -30 mmo Class IV > 30 mm 3. 8. 1 Class I T e r t i a r y F o l l i c l e s . Class I f o l l i c l e s were numerous i n breeding-season o v a r i e s , but t h e i r importance to the r e p r o d u c t i v e process i s not known. U n l i k e smaller f o l l i c l e s , growth and a t r e s i a of Class I f o l -l i c l e s was c y c l i c . 52 The r e l a t i o n s h i p between the numbers of Class I f o l l i c l e s and the age of the female was i n v e s t i g a t e d i n 101 p a i r s of ova r i e s from the breeding season of 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 Class I f o l l i c l e s were most numerous i n deer 2.5 years o l d , and l e a s t numerous i n does older than 9 years. The number of Cl 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 lower than i n any age-class except.the o l d e s t group (P < 0.05). No Class I f o l l i c l e s were present i n the o l d e s t doe (19-5 y e a r s ) , and another old doe contained only two such f o l l i c l e s . A general decrease i n the number of Cla s s I f o l l i c l e s occurred a f t e r the breeding season. I n fawns, the number of Cla s s I f o l l i c l e s de-creased s i g n i f i c a n t l y from a mean of 50 i n November (N = 23), to a mean of 16, i n e i g h t fawns c o l l e c t e d from December to June. I n y e a r l i n g s , t h e average number decreased from 38 i n November (N = 25), to 23 (N = 13), i n pooled samples from December to June. Class I f o l l i c l e s were not counted i n ov a r i e s of a d u l t s c o l l e c t e d from December to May, but a p r o g r e s s i v e decrease i n f o l l i c u l a r numbers was apparent. A f t e r n o t i n g the r e l a t i o n s h i p between s i z e of 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 hypothesized t h a t the s i z e of the ovary was l a r g e l y d i c t a t e d by the number of Class I f o l l i c l e s and not by the number or s i z e of l a r g e r f o l l i c l e s . This hypothesis was teste d i n a sample of 24-p a i r s of o v a r i e s from 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 the t o t a l number of s m a l l f o l l i c l e s i n a p a i r of o v a r i e s and the weight of the two o v a r i e s . Thus, ovary s i z e d i f f e r e n c e s between fawns, as' measured by weight, was l a r g e l y due to d i f f e r e n c e s i n the number of Cl a s s 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 a p p l i e d to the ova r i e s of old e r deer, but to a l e s s e r extent. Fig. -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 collected i n November and early December, 1963-67. 100 • .24 CO w O I—I CO co < 1-1 o o 90 80 70 60 -I 50 40 -I 30 o s 20 10 -23 25 13 10 Sample size Standard deviation 1 95% confidence.limit (Sx-t.os) • Mean 6 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 Prior 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 largest f o l l i c l e i n pairs of ovaries collected during the breeding season,revealed differences i n f o l l i c u l a r development between fawns, yearlings, and adults (Fig. 5). The histograms for fawns and yearlings include- animals collected from November 6 to December 5. The histogram for adults includes animals collected between November 6 and November 23,when most were approaching their f i r s t ovulation of the season. The mode class 3 3 3 was 11-20 mm i n fawns, 31-4-0 mm i n yearlings, and 4-1-50 mm 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 yearlings 3 and adults attained volumes up to 100 mm . The largest f o l l i c l e i n some year-l i n g s did 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 . larger than 20 mm i n each cycle. Many of the large f o l l i c l e s i n the ovaries had stopped growing, as indicated by cessation 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 ("early-atretic" and " t r a n s i t i o n " f o l l i c l e s i n Fig. 5). Two fawns with large f o l l i c l e s exibited unusual ovarian a c t i v i t y . 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 (Atresia 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 de-veloped i n fawns, two Class IV f o l l i c l e s were found i n 13% of yearlings i n ''. 1963, and i n 11% of a l l yearlings collected from 1963 to 1966 (Table 3). From 1963 to 1964, the frequency of yearlings with 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. Prior to f i r s t ovulation, 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 with age up to 4«5 years,and then 55 F i g . The frequency of the l a r g e s t f o l l i c l e , and i t s s t a t e of ma t u r i t y , i n fauns, y e a r l i n g s , and a d u l t s p r i o r to f i r s t • o v u l a t i o n of the cu r r e n t breeding season. 40 35 30 25 20 15 10 5 Fawns n = 45 FOLLICULAR STATE [ | E a r l y - A t r e t i c T r a n s i t i o n a l A c t i v e 35 -I 30 25 20 15 10 5 Adults n = 95 HL 0-10 11-20 VOLUME 21-30 (MM3) J l -40 4'i-50 60 b l -70 71-80 81-90 91-100 OF THE LARGEST FOLLICLE IN EACH FEMALE 56 Table 3. Per cent frequency of one Class IV, two Class IV, and one Class IV plus one Class 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 cent Frequency Year Sample S i z e One Class IV f o l l i c l e ( >30.mm3) Two Class IV f o l l i c l e s One Class IV & One Class I I I f o l l i c l e (> 30mm3+20-30mm3) 1963 16 69 13 13 1964 1 5 73 07 00 1965 14 71 07 00 1966 2 100 5 0 00 1963-66 47 72 11 4 decreased to a lower l e v e l i n older animals (Table 4 ) . Table 4 . The per cent frequency of a d u l t does,in s i x age c l a s s e s , c o n t a i n i n g only one or two Class IV f o l l i c l e s , p r i o r to f i r s t o v u l a t i o n . Per cent frequency i n age-- c l a s s Age-class (yr) 2.5 3.5 4.5 5.5 6.5-8.5 9.5&^ One Class IV F o l l i c l e * 52 42 ' 15 33 33 25 Two Class IV F o l l i c l e s 4 8 58 85 . 67 67 75 Sample S i z e 29 19 13 12 15 4 > 30mm3 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 ve r y old p r e o v u l a t o r y ; females, the two Class IV f o l l i c l e s were out of phase. Only i n one of f i v e y e a r l i n g s were both f o l l i c l e s r a t e d i n the same stage of development. In three of the othe r s , the l a r g e r f o l l i c l e of the two was more advanced,- and one was d e f i n i t e l y a t r e t i c (Stage 2 of a t r e s i a ) . Two y e a r l i n g s each had one Class IV f o l l i c l e and one Class I I I f o l l i c l e . I n both i n s t a n c e s the f o l l i c l e s were only s l i g h t l y out of phase. About one-half ( 2 1 / 4 O ) of the two Class IV 57 f o l l i c l e s i n a d u l t s were a t the same stage of maturation but others were out of phase by as much as four stages i n the a c t i v e , t r a n s i t i o n a l , a t r e s i a 1, a t r e s i a 2, etc. c l a s s i f i c a t i o n . U s u a l l y , however, the out-of-phase f o l -l i c l e s 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 i . e . one or two stages out of phase. Class 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 occurred i n 39% of a d u l t o v a r i e s (37 of 95). U s u a l l y the e a r l y - a t r e t i c f o l l i c l e was the l a r g e s t one i n the o v a r i e s . When f o l l i c l e s were out of phase, o f t e n only one ruptured to form a corpus luteum and the other developed i n t o an accessory corpus luteum. This phenomenon was more common i n young females (1.5 to 3.5 years) than i n older does. O c c a s i o n a l l y , a t r e t i c f o l l i c l e s ruptured a t the f i r s t o v u l a t i o n . There were notable d i f f e r e n c e s between the 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 the volume of the l a r g e s t f o l l i c l e i n each doe j u s t p r i o r to o v u l a t i o n . F o l l i c l e s i n females on the verge of second o v u l a t i o n a t t a i n e d smaller but l e s s v a r i a b l e volumes than f o l l i c l e s i n does approaching f i r s t o v u l a t i o n . F o l l i c l e s near f i r s t r upture ranged i n volume 3 from about 20 to 100 mm , whereas f o l l i c l e s i n seven females on the verge 3 3 of 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 the l a t t e r group, 35 mm , was i n the ovar i e s of an inseminated y e a r l i n g i n e s t r u s . Females approaching second o v u l a t i o n , l i k e those approaching f i r s t o v u l a t i o n , contained v a r i o u s combinations of Class I I , I I I , and IV f o l l i c l e s . The most frequent combination i n a d u l t s was two Class IV f o l l i c l e s . A lso of frequent occurrence was a s i n g l e Class IV f o l l i c l e , and one Class IV f o l l i c l e p lus one Class I I or I I I f o l l i c l e . 58 3. 8. 3 The Relationship Between Active and A t r e t i c F o l l i c l e s i n Preovulatory Female Deer. The occurrence and nature of c y c l i c f o l l i c u l a r development was investigated by comparing the volume of the largest active f o l l i c l e i n each female to the volume of the largest a t r e t i c f o l l i c l e (presumably from the previous cycle). Cyclic development would be indicated 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 largest active and largest a t r e t i c f o l l i c l e s i n a pair of ovaries. In addition, the a t r e t i c f o l l i c l e s should become progressively more a t r e t i c as they decreased i n size,while the successive,active f o l l i c l e became larger. Both of the above relationships were found i n adults and yearlings collected during October, and i n a l l females collected during the breeding season (Fig. 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 largest active and the largest a t r e t i c f o l l i c l e was most evident i n adults, including those collected i n October, and l e a s t evident i n fawns and some yearlings. The progressive a t r e s i a of the shrinking f o l l i c l e was also most evident i n adults. Some fawns and a few yearlings did not produce f o l l i c l e s larger 3 than 10 to 20 mm , which resulted i n a group of intercepts near the o r i g i n i n Fig. 6. 3. 8. 4 The Atresia of F o l l i c l e s After F i r s t and Second Ovulation. The volume of the largest a t r e t i c f o l l i c l e plotted against the volume of the largest second-cycle (growing) f o l l i c l e i n the same pair of ovaries i s presented i n F i g . 7. The volume of the largest active f o l l i c l e i s a measure of time because one, two, or three f o l l i c l e s begin growth soon after ovu-l a t i o n and continue to grow for several days u n t i l second ovulation. I t i s 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 larger than about 20 mm3, 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 largest second-cycle f o l l i c l e had reached about 10 mm (about 2 59 Fig. 6. The rel a t i o n s h i p between the largest active f o l l i c l e and the largest a t r e t i c f o l l i c l e i n pairs of ovaries, from pre-ovulatory females. The degree of a t r e s i a from 1 (early atresia) to 8 (late 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 indicated (L = l u t e i n i z e d f o l l i c l e ) . 50 H AO 30 -| 20 10 o •H r H r H O P H o -H •P CD < • -p M (D U0 • . 1 1 - 2 .t 3 2 •t 3 3 4 2-3 5-6 Adults Nov. 6-23 10 — i — 20 5 - 6 30 40 50 60 50 -4.0 30 20 10 • 1-2 2 -1 t .3 t '•. 2.2-3 '5 Fawns Nov. 6-Dec. 5 10 20 — i r— 30 40 50 90 75< 604 50-4.0-30-20-1-2 2 - 3 • 1 t •» « t . 4 1 .4-5 3-4 Yearlings Nov. 6-Dec. 5 * . 2 - 5 * • 4 . • \ • " 5 - 6 6 6 . . .5 ' .5 * 7 -7 " T " I I 1 30 40 50 60 3< 10 20 LARGEST ACTIVE FOLLICLE (MMJ) 50 AO 30 20 10 1-2 Adults and Yearlings ( a ) Oct. 1963 1-2 2-3 " lO 20 ~~30 4~0 50" F i g . 7. 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-c 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 of previous 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 ) . 60 50 1 4.0 30 20 10 o 3 L D L u t e i n i z e d ° N o n - l u t e i n i z e d O L L 0 L O Y e a r l i n g s ® L 0 oo o o o — I — — I 1 1 1 1 — 1 — 10 20 30 -40 50 60 70 80 70 J (185 mm3) B L (120 mm3) ^ 6 0 o M 5 0 o ^ AO o H E-i § 30 EH 20 O 43 10 3L L |L L 0 ° o a L o L oo Adults 10 20 30 4.0 50 60 70 LARGEST ACTIVE SECOND-CYCLE FOLLICLE (MM3) 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 undoubtedly would have l u t e i n i z e d i n another day or two. The i n c i d e n c e of Class I I I and Class IV 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 corpora l u t e a was 31% ( 5 / l 6 ) . I n a l l o v a r i e s 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 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 d u l t s , there 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 increased i n volume a f t e r l u t e i n i z a t i o n began,because two a t t a i n e d volumes of 185 and 120 mm ( F i g . 7 ). They were l a r g e r than 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 occurred i n 4-2% (5/12) of 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 to be older than 2 days. Volumes 3 of the l u t e i n i z e d f o l l i c l e s were 58, 31, 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 occurred i n only one of the y e a r l i n g s and i t was a sma l l f o l l i c l e . The 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 (accessory corpora l u t e a ) 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). Be cause l u t e i n i z a t i o n was not evident f o r a day 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 corpora l u t e a l a r g e r than 10 mm , and second-cycle f o l l i c l e s l a r g e r than 5 mm , were included i n the sample. The i n c i d e n c e was h i g h e s t 62.5% (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 of 17 a d u l t females w i t h l u t e i n i z e d f o l l i c l e s . F i v e of the females contained two t o fou r l u t e i n i z e d f o l l i c l e s and the f i f t h female had I 4 accessory corpora l u t e a i n i t s o v a r i e s . This e x c e p t i o n a l female was the 3 only one w i t h two Class IV ( >30 mm ) l u t e i n i z e d f o l l i c l e s . 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 females 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 than i n those o v u l a t i n g two or three 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 yearlings and adults, a wave of atr 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 larger than 10 mm and soon shrank to an i n s i g n i f i c a n t size. Often,the highly a t r e t i c f o l l i c l e s of previous cycles were larger than those orig i n a t i n g from second-cycle f o l l i c u l a r development. In adults, only one Class IV f o l l i c l e (75 mm3) f a i l e d to rupture 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 at second ovulation). One other large f o l l i c l e f a i l e d to l u t e i n i z e . After the second ovulation, luteinized f o l l i c l e s occurred i n only 11% (5/44-) of the adults. Most of these were small (Class I) f o l l i c l e s . After second ovulation, no a t r e t i c f o l l i c l e s larger than 11 mrrr 3 occurred i n 15 yearlings,and the mean size was less than 5 mm . 3.8. 5 The Growth of Second-Cycle F o l l i c l e s Relative to the Growth  of First -C y c l e Corpora Lutea. After the f i r s t ovulation, growth of corpora lutea was contemporaneous with growth of the f o l l i c l e s that were destined to ovulate at the next estrus. The re l a t i o n s h i p between the volumes of the largest f i r s t - c y c l e corpus luteum and the largest second-cycle f o l l i c l e was similar i n yearlings and i n adults,but v a r i a b i l i t y was greater i n yearlings and the two curves diff e r e d s l i g h t l y (Fig.. 8). In t h i s relationship,active corpora lutea were distinguished from early-degenerate ones,for the l a t t e r were vacuolate and shrinking i n volume prior to second estrus. The curves i n Fig. 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 for f o l l i c l e s and one for corpora lutea. Early growth of the corpus luteum was faster than that of the f o l l i c l e , b u t after 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 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 -c y c l e corpus luteum (CL 1) i n y e a r l i n g and a d u l t females. . (Curves f i t by eye). ® A c t i v e CL 1 64 whereas the f o l l i c l e continued to grow. Most f o l l i c l e s l a r g e r than 30 mm were near second o v u l a t i o n and u s u a l l y they accompanied degenerate corpora l u t e a ( F i g . 8). I n the l a s t 1 to 3 days p r i o r to second o v u l a t i o n , many 3 3 of these corpora l u t e a shrank i n volume from 4-0-50 mm to 25-4-0 mm . For t h i s reason, the curve i s f i t t e d only to coordinates represented by a c t i v e corpora l u t e a . I n y e a r l i n g females, the r a t e of f o l l i c u l a r growth exceded t h a t of the corpus luteum a t an e a r l i e r stage than i n a d u l t s . I a t t r i b u t e t h i s d i f f e r e n c e to l e s s e r corpus luteum 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 not be r e a l because of the sm a l l sample s i z e f o r y e a r l i n g s . 3. 8. 6 The Growth of Third-Cycle F o l l i c l e s R e l a t i v e to the Growth of Second-Cycle Corpora Lutea. The r e l a t i o n s h i p between the volumes of corpora 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 of the cu r r e n t season, c o n t r a s t s remarkable w i t h the r e l a t i o n s h i p between the s t r u c t u r e s a f t e r f i r s t o v u l a t i o n . The l a r g e s t second-cycle corpus luteum grew a t about twice the 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 ( F i g . 9). Moreover, the 3 corpora l u t e a continued to grow u n t i l many a t t a i n e d volumes of 100 to l60mm . By t h i s stage, some of the f o l l i c l e s were i n e a r l y stages of a t r e s i a and were succeeded by a f o u r t h generation of f o l l i c l e s . . The growth of the l a r g e s t f o l l i c l e , r e l a t i v e to the growth of the l a r g e s t corpus luteum, was greater i n y e a r l i n g s females than i n a d u l t s ( F i g . 9). 3. 8. 7 Determining the Growth Rate of F o l l i c l e s and Corpora Lutea. In past s e c t i o n s ,the growth of f o l l i c l e s r e l a t i v e to 'the growth of the corpora l u t e a was presented but no data were obtained on the growth r a t e of f o l l i c l e s and corpora l u t e a . T h i s . i n f o r m a t i o n was procured by an 65 The r e g r e s s i o n s 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 a d u l t females. (Regressions f i t "by l e a s t - s q u a r e s method to a c t i v e -f o l l i c l e s o n l y ) . -E A c t i v e F o l l i c l e © A t r e t i c F o l l i c l e Y e a r l i n g s n = 15 0.35x - 0.15 0.98 "I 1 1 1 1 1 1 1 I 1 1 I I 1 1 1 ' 32 64 9 6 128 1 The F i r s t Stage Of F o l l i c u l a r A t r e s i a 2 A Late r Stage Of F o l l i c u l a r A t r e s i a 1+ © y = O . 4 I 6 X + 0.91 r = 0.91 I 1 1 1 1 1—1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 r-32 64 96 128 160 L A R G E S T C L 2 ( M M 3 ) ' 66 i n d i r e c t method of e s t i m a t i n g the age of ova recovered from the oviducts and u t e r i . The o v u l a t o r y c y c l e from which the ova were d e r i v e d , the number of ova found i n each r e p r o d u c t i v e t r a c t , the c e l l stage, the presence or absence i of sperm on the ovum, the l o c a t i o n of the ovum, and the method of recovery are presented i n Table 5. Table 5. 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. Specimen Ovulatory No. Ova C e l l Recovery No. Cycle Recovered L o c a t i o n Stage Sperm Method X62 F i r s t 1 Oviduct 1 N i l OS K9 ii 1 II 1 11 11 Z l l II 1 II 1 n 11 Z24 Second 1 n 1 Few 11 V21 II 2 1! 1 . Many FL V19 II 2 11 1 11 ti Z26 II 2* II 1-2 Few OS Z28 F i r s t 1 II 2 N i l 11 X61 Second 1 II 4 Few 11 Z23 II 2 II 4 Many 11 K73 it 1 II 4-8 11 n Z15 F i r s t 1 II 5 11 11 Z22 n 1 II 6 11 n V20 Second 2 II 8 11 • F l V l l II 2 II 8-16 Many 11 V30 II 1 11 16 ti n V28 it 1 Uterus 32-64 11 11 V24 ti 1 ii 64-128 11 11 V27 n 1 II B i a s t u l a 11 11 OS Oviduct Sec t i o n s F l F l u s h i n g of Oviduct or Uterus * One Fragmented P e r t i n e n t observations r e l a t i n g to the above ova are 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 found, both were a t the same stage of development. 67 2. Ova from four of s i x deer, which ovulated at f i r s t estrus, had no sperm attached to the zona pellucida (Plate 10. 2-4). Nevertheless, oviducts of one of the four contained a two-celled ovum with fragmented nuclei'(Plate 10. 4). Many-sperm were attached to the zona pellucida of the other two ova from the f i r s t ovulatory cycle and they had divided into f i v e and s i x c e l l s . In contrast, abundant sperm was attached to the zona pelluci 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 sectioning oviducts than by flushi 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 rates of cleavage i n the ova of other mammals because the rate has not been determined i n deer. Ova of a d i v e r s i t y of mammalian species segment at about the same rate (See Appendix 1 for cleavage rates, and Hamilton and Laing, 1946, f o r a review). The average of the rates of cleavage i n the goat, sheep, and cow probably provide the best estimate for deer because these three domestic species most cl o s e l y resemble deer i n the length of the estrous cycle, time of implantation, and gestation period. The approximate average rate i n these species i s as follows: Facing page 68 Plate- 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 3 1 . A t y p i c a l a t r e s i a of a 1 2 mm f o l l i c l e . A zone of elongate 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 area (black m a t e r i a l on r i g h t s ide of photograph) and the theca externa (X800). 2 . The 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 . 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 ) contains l e u c o c y t e s . The outer zone ( f a r l e f t ) i s the theca externa. The r e g i o n corresponding I n l o c a t i o n to the former stratum granulosum and theca i n t e r n a contains 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 leucocytes ( 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 leucocytes 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-. Leucocytes 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 of a f o l l i c l e 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. Phagocytic c e l l s are d e s t r o y i n g an oocyte. A p o r t i o n of 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 of the t h i n zona p e l l u c i d a i s evident (X800). 6. Leucocytes 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 stage of a t y p i c a l a t r e s i a (X800). 7. An advanced stages of degeneration. Connective t i s s u e c e l l s occur between the degenerate c e l l s which c o n t a i n pale cream or y e l l o w pigment ( X 4 O O ) . 8. A scar 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 are l o c a t e d i n a network of connective t i s s u e c e l l s and f i b e r s (X4OO). 9. A small f o l l i c l e i n advanced stages of abnormal a t r e s i a . N e c r o t i c t i s s u e (black) s t i l l occurs i n the center 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 white 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. Several f o l l i c l e s (appear as white areas 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 ovary (X5). F a c i n g page 69 P l a t e 10 The Ovulated Ovum. 1 . A one-celled ovum f l u s h e d from the o v i d u c t , f i x e d i n 5% f o r m a l i n , and stained w i t h a d i l u t e s o l u t i o n of t o l u i d i n e blue ( X 4 O O ) . 2. S e c t i o n of a one-celled ovum derived from the f i r s t o v u l a t o r y c y c l e . Note the l a c k of sperm and the discon t i n u o u s chromophilic band i n the zona p e l l u c i d a (X800). 3. A degenerate one-celled ovum from the f i r s t o v u l a t o r y c y c l e . No sperm was found on the zona p e l l u c i d a ( X 4 O O ) . 4 . A two-celled ovum from the f i r s t o v u l a t o r y c y c l e . No sperm was found on the zona p e l l u c i d a and development had probably ceased. Two n u c l e i are present i n each c e l l ( X 4 O O ) . 5. A s i x - c e l l e d ovum from the f i r s t o v u l a t o r y c y c l e . Note the chromo-p h i l i c l a y e r i n the zona p e l l u c i d a and the numerous sperm on the surface of the zona p e l l u c i d a (X800). 6. A one-celled ovum from the second o v u l a t o r y c y c l e . The black object i n the ooplasm i s the sperm head. Only a few sperm were attached to 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 from the second o v u l a t o r y c y c l e . Sperm are abundant on the zona p e l l u c i d a ( X 4 O O ) . 8. A f i v e - c e l l e d ovum from the second o v u l a t o r y c y c l e . Note the abundant sperm and the columnar epthelium i n the oviduct ( X 4 O O ) . 70 No. of c e l l s Time si n c e o v u l a t i o n (days) 1 0-1 2 1.5 4 - 8 2 9- 16 3 17- 64 4 65-128 5 The above r a t e s were ap p l i e d o n l y to f e r t i l i z e d ova shed at the second es t r u s of the season. 3. 8. 8 The Rate of F o l l i c u l a r Growth. 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 , from 1 to 5 days a f t e r second-cycle o v u l a t i o n , was determined i n 12 does ( F i g . 10). I n these, the elapsed time since o v u l a t i o n was estimated from the cleavage stage of recovered ova. Growth was apparently r a p i d and l i n e a r from 1 to 5 days p o s t - o v u l a t i o n . As e a r l y as 4 to 5 days, f o l l i c l e s grew to a volume of 30 mm (near o v u l a t o r y s i z e i n some females). The growth r a t e of second-cycle f o l l i c l e s was not determined because only two f e r t i l i z e d ova were obtained from f i r s t - c y c l e o v u l a t i o n s , and these ova were thought to be retarded i n development. I t i s hypothe-s i z e d , t h a t the r a t e of f o l l i c u l a r growth was s i m i l a r 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 obtained f o r t h i r d - c y c l e f o l l i c l e s would apply to the f i r s t and second c y c l e s . F o l l i c u l a r growth d u r i n g the breeding season seemed to be independent of corpora 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 second-generation corpora l u t e a d e r i v e d from second-c y c l e f o l l i c l e s . Although most of the females conceived a t second e s t r u s , f o l l i c u l a r growth and development continued. . As the t h i r d - c y c l e f o l l i c l e s became a t r e t i c , a new ( 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 pregnancy and l a r g e a c t i v e corpora l u t e a . 71 F i g . 10. 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 o v u l a t i o n was estimated from the cleavage stage of ova ( r e g r e s s i o n f i t t e d by l e a s t squares). O Y e a r l i n g l 1 1 1 r 1 . 2 3 A 5 DAYS SINCE SECOND OVULATION 72 3. 8. 9 Seasonal Changes i n F o l l i c l e s • Seasonal changes occurred i n the 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 of fawns. I n a l a r g e sample (N = 4-6) of o v a r i e s c o l l e c t e d i n November, the 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 5 mm3 (N = 3), and by January to March, i t was 1 mm3 (N - A) • However, a female 1 year o l d , c o l l e c t e d i n June, contained a 30 mm3 a c t i v e f o l l i c l e 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 of 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 as 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 (Class I I I or IV) occurred i n y e a r l i n g and a d u l t s i n October. Thick-walled a t r e t i c f o l l i c l e s 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 IV ruptured f o l l i c l e , which s t i l l contained the attached oocyte, was present i n a doe c o l l e c t e d October A- A Class IV, e a r l y - a t r e t i c f o l l i c l e was near rupture i n another 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 the f i r s t three c y c l e s of the breeding season was described p r e v i o u s l y . Because most females conceived at second e s t r u s and few deer were obtained a f t e r December 6, l i t t l e i n f o r -mation was procured on the nature of c y c l e s a f t e r the t h i r d . Only one doe ( c o l l e c t e d November 28, 1965) contained two generations of corpora l u t e a and a newly-ruptured f o l l i c l e . I n t h i s doe, the second generation corpus luteum was more degenerate (by 1 to 2 days) than were f i r s t - c y c l e corpora 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 ruptured i n a doe c o l l e c t e d on November 27, 1965. Perhaps t h i s was a mechanical • rupture because the second-generation corpora l u t e a were s i m i l a r i n appearance to normal corpora l u t e a of the same age i n pregnant females. The u t e r i n e h i s t o l o g y of the doe was c h a r a c t e r i s t i c of a pregnant doe , r a t h e r than one which had j u s t ovulated. Reduced f o l l i c u l a r development d u r i n g mid-pregnancy i s a l s o e v i d e n t i n the graph d e p i c t i n g the volume of 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 at f i v e periods during gestation (Fig. 11). There were s i g n i f i -cant differences i n the sizes of the largest 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 largest 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 collected between mid-December and mid-June, there was usually a recipr o c a l r e l a t i o n s h i p between the volumes of the largest active and the largest a t r e t i c f o l l i c l e i n a pair of ovaries. This form of c y c l i c a l development was less evident from January to March than i n December, May, and June. Many of the large f o l l i c l e s i n females carry-ing 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 for longer periods i n pregnant females during the winter months than during the breeding season. Four females collected during the winter and spring of 1963-64 were not v i s i b l y pregnant. The ovaries of only one of these, a doe aged 7 years,collected on May 14, contained no corpora lutea and exibited n e g l i g i b l e f o l l i c u l a r a c t i v i t y . The other three females,collected i n February and March,were not v i s i b l y pregnant but they were either i n early stages of pregnancy or were s t i l l cycling. A l l three contained large f o l l i c l e s at various stages of development and degeneration. Chapter 4- The Formation And Degeneration Of Corpora Lutea. 4..1 The Problem Of Mechanically-Ruptured F o l l i c l e s . In deer ovaries i t was often d i f f i c u l t to di 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 at other times. F o l l i c l e s may rupture at any time of the year and at any stage of the f o l l i c u l a r cycle. These "mechanical" ruptures were espe c i a l l y common between ovulations and were a potential source of error i n the inter p r e t a t i o n of ovarian functions. F i g . 11. Seasonal changes i n the volume of the largest f o l l i c l e i n pregnant females. 80 70' S 6o1 E-i I 50' 40 o 30 20J 1-3 g 10' T Dec. 13 20 V — I Fe D. 2 70 1 Mar. 22 119 14 1 May 10 168 8 Sample size Range • Standard deviation A 95% confidence l i m i t Mean June 12 DATE OF COLLECTION 200 AVG. 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. Pertinent observations were as follows: 1 . Commonly, the granulosa was a t r e t i c i n mechanically-ruptured f o l l i c l e s but i t was also a t r e t i c i n some f o l l i c l e s ruptured during physiological ovulation, es p e c i a l l y those of the f i r s t cycle. 2. C e l l d i v i s i o n i n the theca, and to a lesser extent i n the granulosa ( i f a c t i v e ) , was cha 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 occurring i n pregnant does. 3 . During the breeding season the size of ruptured f o l l i c l e s was one of the best c r i t e r i a for separating ruptures at estrus from ruptures at other times. From the size of the collapsed structure, and the degree of fo l d i n g , i t was possible 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 large. During the breeding season, most f o l l i c l e s ruptured at estrus were large. 4. Non-extrusion of the oocyte was cha 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 also occurred In f o l l i c l e s ruptured at ovulation. In summary, f o l l i c l e s ruptured outside of estrus could often, but not always, be distinguished from those ovulated at estrus. 4. 2 The Growth Rate Of Corpora Lutea. The growth rate of second-generation corpora lutea was determined by the cleavage stage of the ova i n 12 females (data i n Appendix 2 ) . Early growth of corpora lutea was rapid and the maximum growth rate was attained i n about 2 . 5 to 3 days (Fig. 1 2 ) . The volumes of corpora lutea 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 of second-generation corpora lutea -"" (GL 2),whose ages were estimated from the cleavage stage of ova. The largest CL 2 i n a pair of ovaries. Considered a t y p i c a l and omitted from the curve f i t t i n g . 77 they were a t y p i c a l l y l a r g e corpora l u t e a . A growth curve f o r f i r s t - g e n e r a t i o n corpora l u t e a was not obtained but t h e i r growth,during the f i r s t 2 to 3 days, was expected to f o l l o w t h a t of the next generation. A f t e r 2 to 3 days, the growth of f i r s t - g e n e r a t i o n corpora l u t e a ceased and a f t e r another 2 to 3 days they s t a r t e d to degenerate. 4. 3 Temporal Changes I n The R a t i o Between The Volumes Of Successive  Corpora Lutea. Because second-generation corpora l u t e a are 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 corpora l u t e a are 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 of the e a r l y age of second-generation corpora l u t e a . The r a t i o between the volumes of the l a r g e s t corpus luteum of each generation was e s t a b l i s h e d f o r each of 12 females from which segmenting ova were recovered ( F i g . 13 and Appendix 2 f o r d a t a ) . The time elapsed si n c e o v u l a t i o n was estimated from the developmental stage of the ova. The r a t i o i n the volumes of the two generations of corpora l u t e a changes r a p i d l y i n the f i r s t 3 days a f t e r o v u l a t i o n . The r a t e of change begins to d e c e l l e r a t e a f t e r 3 days,but o b v i o u s l y the r a t i o continues to i n c r e a s e past 5 days (the r a t i o o f t e n exceeded 50 a t an estimated 10 days a f t e r o v u l a t i o n ) . The r a t i o method i s undoubtedly a more accurate measure of elapsed time since second o v u l a t i o n than are the growth curves f o r corpora l u t e a and f o l l i c l e s , because there i s l e s s v a r i a t i o n between females than w i t h i n females i n the s i z e of f o l l i c l e s and corpora l u t e a . 4. U E s t i m a t i n g The Age Of Corpora Lutea. F i v e c r i t e r i a were used to estimate the age of corpora l u t e a 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 second-generation corpus luteum (GL 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) (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 cycle. 1. The cleavage rate of ova. 2. The volume of the corpus luteum. 3. The volume and degree of maturation of f o l l i c l e s of succeeding 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 cycle. 5. The r a t i o betv/een the volumes of successive corpora lutea. Of the f i v e c r i t e r i a , the cleavage rate of ova vas the standard and provided a base for the quantitative components of the other methods. Corpora lut e a , whose ages were estimated by the cleavage rate of ova,will henceforth be termed "known-age" corpora lutea. The ages of second-cycle corpora lutea,to 5 days of age,were e s t i -mated p a r t l y by objective methods (the four curves for growth and degeneration of corpora lutea and f o l l i c l e s ) and p a r t l y by subjective methods (the h i s t o -l o g i c a l comparison of corpora of unknown age to corpora lutea of known age). The ages of corpora lutea older than 5 days were estimated subjectively 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 lutea. The usefulness of f o l -l i c l e s i n determining the stage of the estrous cycle after 4 to 5 days was de-pendent on f i n d i n g the length of the estrous cycle. Data presented i n the next section indicate that the average i n t e r v a l between f i r s t and second ovulation was 8 days. I assumed that.successive f o l l i c u l a r cycles were the same length and that f o l l i c u l a r growth and maturation rates were equivalent i n the consecutive cycles. I f t h i s assumption i s v a l i d , i t follows that the average i n t e r v a l be-tween f i r s t and second ovulation, 8 days, was also the length of the f o l l i c u l a r cycles preceeding f i r s t ovulation and succeeding second ovulation. Thus, the stage of a f o l l i c u l a r cycle, and thereby the age of corpora lutea, was estimated from the size of f o l l i c l e s and their degree of maturation i n r e l a t i o n to an 80 8-day cycle. During the f i r s t 3 days,the age of corpora lutea derived from the f i r s t ovulation was estimated by: l ) comparing the volumes of the corpora lutea to the growth curve for second-cycle corpora lutea, 2) com-paring the volumes of second-cycle f o l l i c l e s to the growth curve for t h i r d -cycle 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 lutea to known-age corpora lutea of the second cycle. The ages of older corpora lutea from the f i r s t ovulation were estimated by comparing the volumes of second-cycle f o l l i c l e s to the growth curve of third-cycle 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 lutea. Blastocysts recovered from f i r s t - c y c l e ovulations were not v a l i d indicators of the age of corpora lutea. Corpora lutea were often older .than indicated by the ova. 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 lutea with those derived from the second cycle, whose ages were estimated by blastocyst development, revealed that one was about one day old and the other two were about 1.5 days old. 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 after ovulation, the corpus luteum associated with t h i s two-celled ovum was comparable i n development to an older corpus luteum. Development of f e r t i l i z e d ova from the f i r s t cycle was also retarded. On the basis of h i s t o l o g i c a l character-i s t i c s ,the corpora lutea corresponding to the ova containing 5 and 6 c e l l s were estimated to be 6 and 3 to A days old, respectively,rather than 2 days old as indicated by the ova. 81 4- 5 H i s t o l o g y Of Young Corpora Lutea. 4. 5. 1 The Recently-Ruptured F o l l i c l e . Three morphological c h a r a c t e r i s t i c s were evident i n s e c t i o n s of 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 low m a g n i f i c a t i o n ; a ruptured o v a r i a n surface not y e t bridged by new connective 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 which had j u s t ruptured, the w a l l was moderately f o l d e d around a small 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 granulosa, f i r m l y attached to the membrana p r o p r i a , resembled p r e o v u l a t o r y granulosa. But i n most ruptured f o l l i c l e s , the attached granulo-sa c e l l s contained elongate condensed n u c l e i and hyperchromatic cytoplasm ( P l a t e 13. 2 ) . Often,the granulosa c e l l s around f r e e oocytes trapped i n ruptured f o l l i c l e s were l e s s chromatic, l e s s g r anular, and the n u c l e i l e s s condensed, than the c e l l s i n the mural granulosa ( P l a t e 4. 9 ) . C e l l d i v i s i o n s occurred i n f r e q u e n t l y i n the membrana granulosa and the theca 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 time,the membrana granulosa, c o n t a i n i n g both 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 from the membrana p r o p r i a . At t h i s stage, c e l l d i v i s i o n occurred more f r e q u e n t l y i n the c e l l s , which 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 granulosa c e l l s ) . In most ruptured f o l l i c l e s , the appearance of the t h e c a l c e l l s was unchanged,but i n a few the cytoplasm became granular and hyperchromatic, some n u c l e i changed from oblate v e s i c u l a t e to the s p h e r i c a l condensed type, and m i t o s i s occurred. , In most ruptured f o l l i c l e s , engorged blood v e s s e l s occurred a l l along the i n n e r border of the theca i n t e r n a to form a d i s t i n c t i v e boun-dary between i t and the membrana granulosa ( 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 the f o l d s of the 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 into the central c a v i t y i f the granulosa layer was de-tached from the membrana propria. 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 central c l o t or coagulum i n these f o l l i c l e s . The above description 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 at f i r s t estrus were a t r e t i c and therefore exibited d i f f e r -ent c h a r a c t e r i s t i c s . In females ovulating two f o l l i c l e s at f i r s t estrus, often one was active and the other was i n early stages of a t r e s i a . The major differences encountered 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 wall of a t r e t i c f o l l i c l e s did not always fold but remained quite rigid,although 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 after rupture was highly variable. In 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 at ovulation. Presumably,the granulosa layer was a t r e t i c and free from the membrana propria at the time of rupture.' 3. Many of the granulosa and thecal 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 did not form following rupture. 4-. 5. 2 One Day After Ovulation. Corpora lutea estimated to be one day old s t i l l had l ) an ir r e g u l a r shape, 2) a small lumen containing a blood and f l u i d coagulum, 3) prominent fo l d i n g of the former membrana 83 granulosa, and 4) a l o o s e l y - k n i t appearance.- F i v e known-age corpora l u t e a from the second o v u l a t o r y c y c l e averaged 6 mm3 i n volume. Commonly, the s t r u c t u r e was pear or flask-shaped. The i n c r e a s e i n s i z e r e s u l t e d p a r t l y from an i n c r e a s e i n the number and s i z e of c e l l s , and p a r t l y , f r o m an accumulation of f l u i d , i n c l u d i n g blood. The l a t t e r occurred because the surface rupture was bridged by a t h i n l a y e r of connective t i s s u e . E x tensive 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 a 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 of these corpora l u t e a . Large s i n u s o i d s , l o c a t e d i n the apices of 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 to the center of the c o l l a p s e d s t r u c t u r e . Rupture of one or more of these v e s s e l s r e s u l t e d i n massive blood p e r f u s i o n throughout the c e n t r a l r e g i o n of the young corpus luteum. The r e s u l t a n t c l o t contained l a r g e numbers of leucocytes ( P l a t e 13. 3 ) . Pronounced f o l d i n g of the membrana granulosa occurred 1 day a f t e r o v u l a t i o n . The granulosa c e l l s a t t a i n e d diameters of 13-14 u and were 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 11.2). The number of l u t e a l c e l l s per f i e l d of view a t 1250X was about 50, and there was about one d i v i d i n g c e l l i n each f i e l d of view a t 500X. N u c l e i of the granulosa c e l l s were small and most were v e s i c u l a t e . Condensed n u c l e i were u s u a l l y confined to the more c e n t r a l r e g ions of-the corpus luteum, where elongate and hyperchromatic c e l l s - o c c u r r e d . The general boundary between the granulosa and the theca was s t i l l e v i d ent ( 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 granulosa c e l l s to create a d i f f u s e boundary. Many t h e c a l c e l l s contained 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 sparse cytoplasm. C e l l -d i v i s i o n was more frequent i n the theca than i n the membrana granulosa. Many of-the 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 elongate and hyperchromatic ( P l a t e l l . - ' l ) . I n two corpora l u t e a , abundant F a c i n g page 84 P l a t e 11 Development of Corpora Lutea From the F i r s t Ovulatory Cycle. ( A l l photomicrographs X800). 1. A corpus luteum one day 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), contains elongate, 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 are derived from the granulosa. 2. The edge of a corpus luteum about one day o l d . The c e l l s of both the granulosa (to r i g h t of dashes) and the theca are undergoing l u t e i n ! z a t i o n . C e l l d i v i s i o n s are beginning i n both zones (note anaphase i n theca). 3. C e n t r a l r e g i o n of a corpus luteum about one day o l d . Folds of granulosa c e l l s are separated by connective t i s s u e membranes ( v i s i b l e i n center of photograph). The cytoplasm of the c e l l s i s sparse. 4. A corpus luteum ( f i r s t c y c l e ) estimated to be 1.5 days o l d . There i s e x tensive p r o l i f e r a t i o n of blood v e s s e l s ( l a r g e one along l e f t border of photograph). The c e l l i n prophase i s probably an e n d o t h e l i a l c e l l . Note the l a r g e r and more granular l u t e a l c e l l s . 5. A corpus luteum 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 are l a r g e r and t h e i r borders are more d i s t i n c t . Connective t i s s u e c e l l s are forming between the l u t e a l c e l l s . 6. A corpus luteum 2.5 to 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 (lower 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 the l u t e a l c e l l s . 7. A corpora luteum 2.5 to 3 days a f t e r o v u l a t i o n . M i t o s i s (telophase upper'center) has v i r t u a l l y ceased. 8 . A corpus luteum estimated to be 3 days o l d . Many l u t e a l c e l l s have vacuolated cytoplasm, and a few are degenerate. H i g h l y chromophilic c e l l s w i t h condensed n u c l e i occur (lower l e f t . ) . 9. A corpus luteum 4 to 5 days o l d . There i s extensive v a c u o l a t i o n and shrinkage of c e l l s . 10. A corpus luteum about 6 to 7 days o l d . Note the extensive vacuola-t i o n and shrinkage of c e l l s . C e l l s and n u c l e i are p o o r l y d e f i n e d . 11. A corpus luteum i n an inseminated y e a r l i n g doe on the verge of second o v u l a t i o n of the season. Although some c e l l s are vacuolated and shrunken and leucocytes occur (lower 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 occurs (telophase upper c e n t e r ) . 12. A f i r s t - c y c l e corpus luteum i n a y e a r l i n g female having j u s t ovulated a t second e s t r u s . Many c e l l s and n u c l e i are p o o r l y defined because they are i n e a r l y stages of a u t o l y s i s . P l a t e 12 F a c i n g page 85 Degeneration of the Corpus Luteum From the F i r s t Ovulatory Cycle. • 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 ontains 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 degenerate corpus luteum one day a f t e r second o v u l a t i o n . Few c e l l s remain (X800). 3 3. A p o r t i o n of a 5-5 mm degenerate corpus luteum two days a f t e r second o v u l a t i o n . H y a l i n connective t i s s u e i s forming between c e l l s undergoing a u t o l y s i s (X800). 4 . A p o r t i o n of a 4 . 7 mm3 degenerate corpus luteum 3 days a f t e r secont 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 center of the s t r u c t u r e ( r i g h t ) . The normal s t a t e of degeneration i s p i c t u r e d to the l e f t (X80). 3 5. A p o r t i o n of a 2.8 mm degenerate corpus luteum 4 days a f t e r second o v u l a t i o n . H y a l i n connective 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 degenerate corpus luteum 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 vacuoles 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 degenerate corpus luteum about 10 days a f t e r second o v u l a t i o n . Small,angular c e l l s and l e u c o c y t e s occur i n the vacuolated, h y a l i n i z e d s t r u c t u r e (X800). 3 8. A s e c t i o n of a 0.3 mm scar of a corpus luteum of non-pregnancy i n a non-pregnant doe c o l l e c t e d May I 4 . This female contained nine s i m i l a r s t r u c t u r e s i n i t s o v a r i e s . The p i c t u r e d scar was estimated to be from the second-last o v u l a t o r y c y c l e of the past breeding season (X800). 3 .9- A p o r t i o n of the 0.5 ronr scar o r i g i n a t i n g from the youngest corpus luteum of non-pregnancy i n the doe described i n 12.8. Yellow pigment o f t e n forms around the s p e r i c a l vacuoles evident i n the scar (X800). 3 10. A 0.07 mm scar i n a doe k i l l e d May 13- The scar was derived from a corpus luteum of non-pregnancy of the previous 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 vacuoles (X800). 3 11. A 0.05 mm corpus luteum of non-pregnancy scar about 11 months o l d . A few s m a l l , t h i c k - w a l l e d blood 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 these s c a r s . The c e l l spaces are 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 luteum of non-pregnancy scar about 12 months o l d . This one has a h y a l i n capsule ( r i g h t and lower r i g h t ) around a p o r t i o n of the c e n t r a l r e g i o n (X200). Facing page 86 Plate 13 ' Early Development of Corpora Lutea From the Second Ovulatory Cycle. ( A l l photographs X800). 1. The central region of a corpus luteum about one day old. Nuclei of the granulosa c e l l s are becoming vesiculate. Many leucocytes are scattered amongst the l u t e a l c e l l s . 2. The wall 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 nuclei. A large blood vessel and a mitotic figure occur i n the theca interna (to r i g h t of dashes). 3. The central region of a corpus luteum one day after ovulation. Most nuclei of l u t e a l c e l l s are now vesiculate. Large numbers of leucocytes (right side of photograph) are present. -4. The peripheral region 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 tissue 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 ) . Cells 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 central region of a corpus luteum about 2 days old. Connective tissue, associated with the endothelial 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 after ovulation. Many of the l u t e a l c e l l s have peripherally-vacuolated cytoplasm. 7. A corpus luteum 5 to 6 days after ovulation. The cytoplasm of the l u t e a l c e l l s i s extensively vacuolated. A central zone of f i n e granular cytoplasm contains a golgi 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 old. 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 old. Angular hyper-chromatic c e l l s with condensed nuclei occur at t h i s stage. 10. A corpus luteum estimated to be 11 to 12 days old. Portions of two hyperchromatic c e l l s containing condensed nuclei are present along the r i g h t edge of the photograph. 87 l e u c o c y t e s occurred 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 from the theca externa formed a t r i a n g u l a r wedge of t i s s u e at the base of each t h e c a l f o l d . The c e n t r a l p o r t i o n of each f o l d developed i n t o f i b r o u s tongues of t i s s u e termed trabeculae. These t r a b e c u l a , bearing l a r g e blood v e s s e l s , extended to the center of the corpus luteum and became.the source of the connective t i s s u e framework of the gland. 4. 5. 3 One And One-Half Days A f t e r O v u l a t i o n . Corpora l u t e a 1.5 days old a t t a i n e d volumes of 12 mm , were more rounded, and contained a c e n t r a l amorphous coagulum. The d e n s i t y of l u t e a l c e l l s was approximately 4-0 per f i e l d of view a t 1250X, and the frequency o f - ' d i v i s i o n s was about two per f i e l d of view a t 500X. Many c e l l d i v i s i o n s occurred along the edges of the widened tr 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 the granulosa c e l l zone from the trabeculae. The rounder granulosa c e l l s , c o n t a i n i n g v e s i c u l a t e n u c l e i , a t t a i n e d diameters up to 15 p.. There was extensive p r o l i f e r a t i o n of 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. 4). 4. 5. 4 Two Days A f t e r O v u l a t i o n . Corpora l u t e a estimated to be 2 days old were compact s t r u c t u r e s w i t h uniform round or oblate shapes. The connective t i s s u e bridge over the surface rupture area was t h i c k e r . E i g h t 3 3 known-age corpora l u t e a averaged 23 mm , and ranged from 8 to 38 mm . L u t e a l t i s s u e protruded from the o v a r i a n surface of some o v a r i e s . The f o l d i n g of t i s s u e s was l e s s obvious on the second day than on the f i r s t . The s m a l l , c e n t r a l lumena were bounded by young connective t i s s u e c e l l s and contained f l u i d . • ' The l u t e a l c e l l s a t t a i n e d diameters of 20 p., which reduced the average number of c e l l s per f i e l d of view (a t 1250X) to about 20. 88 P r o l i f e r a t i o n of l u t e a l c e l l s reached a maximum i n corpora l u t e a approaching 2 days ( f o u r - c e l l e d ovum). In these, an average of s i x m i t o t i c f i g u r e s per f i e l d of view was common,but by about 2.5 days ( 8 - c e l l e d ovum), the number of c e l l d i v i s i o n s decreased to about two. The l a r g e s t l u t e a l 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 of the s t r u c t u r e , were vacuolated. 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 of the theca, i n c l u d i n g f i b r o b l a s t s , were found through-out the corpus luteum. Many of the t h e c a l c e l l s were s m a l l , elongate, and hyper chroma t i c ( P l a t e 13. 4-) • Some of them were probably a s s o c i a t e d w i t h the 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 sinuses were fewer i n number but they were s t i l l a s s o c i a t e d w i t h blood pigment. Medium-sized v e s s e l s entered the gland v i a the trabeculae and d i v i d e d i n t o smaller v e s s e l s t h a t i n s i n u a t e d between groups of l u t e a l c e l l s . 4-. 5- 5 Three Days A f t e r O v u l a t i o n . Three known-age corpora l u t e a of 3 the second c y c l e averaged 70 mm , but undoubtedly they were e x c e p t i o n a l l y 3 l a r g e and the true mean volume was probably between 4-0 and 50 mm . Enlarged l u t e a l c e l l s occurred throughout 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 -form appearance. The d e n s i t y of l u t e a l c e l l s was reduced to about 14- c e l l s i n a f i e l d of view a t 1250X, and an average of two d i v i s i o n s occured i n a f i e l d of view a t 500X. 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 diameters of 24- contained small vacuoles. The c e n t r a l lumen, which contained f i b r o b l a s t s , was smoothly bounded by young connective t i s s u e . The corpus luteum was u n i f o r m l y smooth around the p e r i p h e r y , the trabeculae having thinned c o n s i d e r a b l y . Thin-walled blood v e s s e l s of medium s i z e occurred around the per i p h e r y . F i b r o b l a s t s and amorphous connective t i s s u e replaced many of the 89 l a r g e s i n u s o i d s found i n younger corpora l u t e a . Other zones of loose connective t i s s u e developed a t the expense of l u t e a l c e l l s . Increased numbers of connective t i s s u e c e l l s created a p r i m i t i v e network amongst the l u t e a l c e l l s ( P l a t e 11. 6). The c h a r a c t e r i s t i c s of corpora l u t e a of the same age from the f i r s t o v u l a t o r y c y c l e were comparable to the above, except t h a t almost a l l c e l l d i v i s i o n had ceased and angular 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 , vacuolated cytoplasm occurred f o r the f i r s t time ( P l a t e 11. 8 ) . 4. 5. 6 Four Days A f t e r O v u l a t i o n . Corpora l u t e a of the f i r s t and 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 from t h i s stage onward. Corpora l u t e a from 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 corpora l u t e a , supporting a m u l t i - c e l l e d (16 to 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 occurred i n one of these.' The increased 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 f i e l d of view at 1250X) of 10. But,the d i s t i n g u i s h i n g c h a r a c t e r i s t i c 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 the h i g h degree of c e l l v a c u o l a t i o n and even c e l l death, 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). A few of the c e l l s contained 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 deeply chromatic but vacuolated cytoplasm. A new 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 , granular zone and a vacuolated outer zone, made i t s f i r s t appear-ance. This 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 uterus and h e r e a f t e r I w i l l term them 'conception c e l l s ' . " The.bands of connective t i s s u e were wider, more f i b r o u s and c o l l a g e n i c . 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 i n diameter. Small v e s s e l s and c a p i l l a r i e s occurred throughout the s t r u c t u r e . 90 Most corpora l u t e a d e r i v e d from the f i r s t c y c l e ranged between 30 and 50 mm3, and contained wide bands of connective t i s s u e between the vacuolated c e l l s ( P l a t e 11. 9)- Considerable a u t o l y s i s of l u t e a l c e l l s i n the r e g i o n of the connective t i s s u e bands, r e s u l t e d i n c e l l u l a r d e b r i s . k. 5- 8 S i x to 8 Days A f t e r O v u l a t i o n . Corpora l u t e a i n ova r i e s c o n t a i n i n g f o l l i c l e s of the succeeding c y c l e i n the t r a n s i t i o n a l stage of development, were estimated to be 6 to 8 days o l d . Although lumena i n 3 these were g e n e r a l l y s m a l l , one contained a 39 mm , f l u i d - f i l l e d c e n t r a l c a v i t y . The c y t o l o g y of most second-generation corpora l u t e a of t h i s age was s i m i l a r to corpora l u t e a 5 days o l d . Some l a r g e , vacuolated, l u t e a l c e l l s were much l a r g e r , a t t a i n i n g diameters of 30 ; i ( P l a t e 13. 8). About one-half of the c e l l s were of the conception type. From 6 to 8 days a f t e r f i r s t o v u l a t i o n , the corpora l u t e a became p r o g r e s s i v e l y more degenerate. Sections of corpora l u t e a a t low m a g n i f i c a t i o n appeared hypochromatic because of c e l l v a c u o l a t i o n , shrinkage, and a u t o l y s i s ( P l a t e 11. 10). B a s o p h i l i c , 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 leuc o c y t e s occurred. Nine corpora l u t e a i n deer on the verge of second o v u l a t i o n 3 averaged 32.6 mm . Obviously some r e d u c t i o n i n volume occurred a f t e r maximum development a t about U to 5 days a f t e r o v u l a t i o n . Hemorrhagic corpora l u t e a occurred i n two does, i n c l u d i n g one i n e s t r u s . The ov a r i e s of the l a t t e r were hyperemic and edematous and blood had r e c e n t l y escaped i n t o the c e n t r a l lumen of the corpus luteum. Young, a c t i v e , and d i v i d i n g c e l l s occurred throughout the s t r u c t u r e ( P l a t e 11. 11), 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 by the t h i n - w a l l e d f o l l i c l e s w i t h f r e e oocytes and by the presence of sperm i n the o v i d u c t s . 91 4. 5. 9 Nine to 15 Days A f t e r O v u l a t i o n . Corpora l u t e a of the second o v u l a t o r y 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 estimated to be 9 to 12 days o l d . C h a r a c t e r i s t i c s of these corpora l u t e a were as f o l l o w s : 1. 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 diameters of 30 _u and a few reached 35 P-. 2. About one-half of the l u t e a l c e l l s were of the conception type and most others were l a r g e and vacuolated. The ch r o m o p h i l i c , angular 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 occurred i n many of the l u t e a l c e l l s . 3. The thinner and more-fibrous trabeculae contained elongate spaces. 4. A t h i c k l a y e r of c o l l a g e n i c f i b e r s bordered the c e n t r a l lumen ( i f p r e s e n t ) . F i b r o b l a s t s and amorphous connective t i s s u e p a r t i a l l y f i l l e d the c e n t r a l lumen. 5. The i n t e r c e l l u l a r network of connective t i s s u e , i n c l u d i n g c a p i l l a r i e s , more d e f i n i t e l y separated n e a r l y every l u t e a l c e l l . " 4". 6. The E a r l y Degeneration Of Corpora Lutea From The F i r s t Ovulatory  Cycle. Corpora l u t e a d e r i v e d from the f i r s t o v u l a t o r y c y c l e degenerated r a p i d l y a f t e r the rupture of second-cycle f o l l i c l e s . I n f i v e females w i t h newly-ruptured f o l l i c l e s , t h e average volume of e i g h t corpora l u t e a 3 3 was 28.7 mm , as compared to 32.6 mm i n females j u s t p r i o r to second o v u l a t i o n . But,one day a f t e r second o v u l a t i o n , degenerating corpora l u t e a 3 3 averaged only about 15 mm and were as small as 5-4- mm . Two days a f t e r 92 ovulation the corpora lutea were shrunken to an average volume of about 5 mm but thereafter shrinkage was slower (Fig. 14- and Appendix 2). Two curves depicting the volume of degenerate corpora lutea are presented i n F i g . 14. One i s based on known-age corpora lutea and the other i s based on corpora lutea whose ages were estimated by the c r i t e r i a previously outlined i n 4. 4. Included i n one of the above methods was a comparison of degenerate corpora lutea of unknown age to degenerate corpora lutea dated by the cleavage stage of the ova. Both curves i n Fig. 14 indicate an extremely rapid shrinkage during the f i r s t 2 to 3 days after second ovulation. Reasons for the f a s t i n i t i a l shrinkage and slower shrink-age after 3 days was evident i n h i s t o l o g i c a l sections. Immediately after 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 early stages of autolysis. One day after second ovulation, c y t o l y s i s of c e l l s was extensive and only a limited number of r e l a t i v e l y i n t a c t c e l l s remained. Cells i n the central portion of the structure were le a s t affected. The connective tissue of the trabeculae formed wide bands of tissue between groups of c e l l s . Two days after second ovulation, the connective tissue formed large a c e l l u l a r zones i n the structure and a network of hyalinized tissue started to develop. Remains of degenerate c e l l s were present i n most corpora lutea, but i n some more-degenerate structures, only c e l l spaces containing c e l l u l a r debris remained. Active c e l l s persisted i n the center of a few scars that were 2 days old. Three days after second ovulation, the bands of hy a l i n tissue between the c e l l remnants were wider and by the fourth to f i f t h day only a reduced number of shrunken c e l l lacunae persisted. Five days after the i n i t i a l rapid degeneration, almost the entire structure was hyalinized. Less degenerate c e l l s were scattered throughout these and older scars. C e l l s bordering the central lumen were 93 . F i g . 14. The volume of degenerating f i r s t - c y c l e corpora l u t e a (CL 1) accompanying second-cycle corpora l u t e a (CL 2) whose ages were estimated by 1) the cleavage 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 ) . 601 94 s t i l l active i n a few corpora lutea of non-pregnancy. The above changes are i l l u s t r a t e d i n Plate 12. 4.. 7 The Fate Of First-C y c l e Corpora Lutea. Stages i n the advanced degeneration of corpora lutea derived from the f i r s t ovulatory cycle 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-ti o n of the f i r s t - c y c l e corpora lutea begins at second ovulation. Shrinkage of the scars was gradual but continuous from November to March, after which the average volumes of those measured was about constant 3 at 0.04 to 0.05 mm . Apparently,the scars occasionally persisted for about 2 years,but some were barely discernable after 12 months. Exluded from the above samples,were degenerate f i r s t - c y c l e corpora lutea that underwent secondary l u t e i n i z a t i o n . These,of course,were much larger. General changes i n the histology of the degenerating corpus luteum included: 1) a reduction i n the amount and s t a i n a h i l i t y of the hya l i n tissue, 2) a reduction 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 4) a reduction i n the stromal response to the scar. Some scars older than 120 days contained vacuoles and c e l l spaces bordered by b r i l l i a n t yellow pigment. Others, with less pigment, appeared as white spaces i n a.pale hyalin structure. 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 tissue. Although most surface scars were small plugs of hyalin tissue, some were larger 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 lutea are i l l u s t r a t e d i n Plate 12. Degenerate corpora lutea that developed into accessory corpora Table 6. Stages i n the advanced degeneration of f i r s t - c y c l e corpora l u t e a (CL l ) and t h e i r 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 . Avg CL 1 ' Age Scar Mean of CL 1 (mm^) C o l l . scars (x and Sample Date (days) range) S i z e 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 30 3.38 H y a l i n i z e d network of f i b e r s around c e l l lacunae c o n t a i n i n g n a t u r a l y e l l o w -Nov. 5 (1.2- 14 orange pigment and c e l l remnants. A few vacuolated c e l l s remain trapped i n 4.0) amorphous a n i l i n e - p o s i t i v e connective t i s s u e . 5 1.95 C e l l s and lacunae fewer. C e l l a u t o l y s i s n e a r l y complete. Amorphus bands of Dec. 10 (1.1- 12 connective t i s s u e more extensive but there are patches of no n - h y a l i n i z e d 3.5) . connective t i s s u e c e l l s and f i b e r s . 12 1.02 C e l l lacunae scattered throughout a n i l i n e - p o s i t i v e , h y a l i n e connective t i s s u e . Dec. 10 (0.14- 10 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 capsule of f i b e r s 2.3) reduced. Two have a c t i v e l u t e a l c e l l s i n center. 2 0.07 Less a n i l i n e - p o s i t i v e c e n t r a l core of amorphous connective t i s s u e which con-Feb. 69 •(.03- 18 t a i n s lacunae and some yellow m a t e r i a l . I n some there i s a p a l e , more h y a l i m 0.09) band around the c e n t r a l core of lacuna. Scar i s surrounded by compressed a c i d o p h i l i c stromal c e l l s . 21 0.04 As above, but whole s t r u c t u r e l e s s chromatic, except f o r lacunae, x^hich may Mar, 118 (0.01- 17 be b r i l l i a n t yellow. Less stromal response. Scar i s l o c a t e d near surface of 0.09) ovary and may be traced to the surface where a rupture scar i s u s u a l l y presen' 10 0.04 Hyaline core of scar wasting or being resorbed by stromal c e l l s . A group May 167 • .. • (0.01- 37 of yellow pigmented c e l l s or lacunae occurs i n , or adjacent t o , degenerate 0.08) h y a l i n e t i s s u e . 13 0.05 June 201 (0.03- 8 As i n preceeding stage. 0.07) 20 0.05 St r u c t u r e can u s u a l l y be traced to the surface of the ovary where a h y a l i n e Nov. 360 (0.02- 10 scar may be present. Some have a c e n t r a l core of h y a l i n e t i s s u e , w i t h y e l l o w 0.10) . c e l l s i n a r i n g around i t . I n others,almost a l l amorphous c o n n e c t i v e • t i s s u e has been resorbed and a group of y e l l o w c e l l s l i e s i n the stroma near the surface of the ovary.  96 lutea (Type 2) did not follow the above pattern of resorption. Upon degeneration,they developed i n t o miniature corpora lutea of pregnancy scars (Chapter 4-. 9 ) . 4. 8 Corpora Lutea In The Pregnant Doe. Corpora lutea at f i v e stages of pregnancy were obtained from periodic 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 adult females i n the sample were assumed to be pregnant because their u t e r i were enlarged, convoluted, and distended with f l u i d . 4. 8. 1 Size. Corpora lutea increased i n size during gestation, except for a period near mid-gestation when the mean volume was less than at any other period (Fig. 15). The apparent reduction i n size could be attributed 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 for the December and January-February c o l l e c t i o n s . 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 size of corpora l u t e a , i n comparison to the three e a r l i e r sampling periods. Three does collected i n February,which were not v i s i b l y pregnant, contained smaller than average corpora lutea. These corpora lutea received separate designations i n Fig. 15. In addition, the rapidly-regressing corpora lutea i n two post-parturient does were not included i n the s t a t i s t i c s for the June c o l l e c t i o n period. Primary corpora lutea i n pregnant females ranged i n volume from 3 13 to 156 mm . The smallest one was undoubtedly exceptional because the second smallest was 46 mm^ . F i g . 15.' The volumes of corpora l u t e a of pregnancy (CLP) at f i v e stages of g e s t a t i o n . 150 -j UO 130 120 110 100 1 PH O o S 1-1 o > 90 80 i 70. 60 50 AO 30 • 20 • • No v i s i b l e conceptual O Post-Parturant o •rH P P, CD O C O O A © Range Standard d e v i a t i o n 35% confidence l i m i t Mean "i5 Sample s i z e 26 20 10 Xi p o 20 30 NOVEMBER "75 20 IT DECEMBER 20 10 <;u ^ JANUARY 10 20 29 FEBRUARY 10 20 31 MARCH 10 20 • 30 • APRIL 10 20 MAY 10 20 JUNE 98 4. 8. 2 Histology. In the following account of the h i s t o l o g i c a l and c y t o l o g i c a l changes i n corpora lutea during gestation, the stage of gestation i s the i n t e r v a l between the mean collection-period date and November 25, the mean date of conceptions. Corpora lutea i n two does with r e l a t i v e l y small fetuses were excluded from the following descriptions. Reference should be made to Plate 14 before reading the descriptions of corpora lutea. Corpora lutea from the December 12 c o l l e c t i o n period were, on the average, about 17 days old. 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 vesiculate n u c l e i . A f a i r number of conception c e l l s , w i t h central 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 nuclei and golgi apparatus,occurred throughout the gland. Also present,were small numbers of angular, deeply chromatic c e l l s with condensed nuclei (Plate I4. l ) . Older corpora lutea i n the group contained variably sized, l i g h t l y a c i d o p h i l i c c e l l s including larger (to 35 p), more-vacuolated c e l l s . Some of these contained small amounts of yellow l i p o i d pigment. Up to 40% of the l u t e a l c e l l s i n these older corpora lutea were va r i a b l y sized, angular or s t e l l a t e , hyperchromatic c e l l s with peripheral vacuoles. About 9 or 10 l u t e a l c e l l s , including n u c l e i , occurred i n a f i e l d of view at 1250X. P r a c t i c a l l y every c e l l was bordered by connective tissue c e l l s , including those of the vascular system. Corpora lutea i n deer from the February 2 c o l l e c t i o n period were about 69 days old. The average l u t e a l c e l l was much larger 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 , including n u c l e i , occurred i n a f i e l d of view at 1250X. However, the largest c e l l s were only s l i g h t l y larger (39 p). Most c e l l s were large, spheroid or oblate, l i g h t l y stained and contained small peripheral vacuoles and semi-vesiculate n u c l e i . Only a small proportion 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 Lutea During Pregnancy ( a l l photomicrographs X800). 1. A s e c t i o n of a corpus luteum about 25 days a f t e r conception. Most l u t e a l c e l l s c o n t a i n s m a l l vacuoles throughout. Angular chromo-p h i l i c c e l l s (lower l e f t ) are common. The l a r g e c e l l (lower center) contains some yel l o w pigment. 2. A s e c t i o n of a corpus luteum 62 days a f t e r conception. Note the 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 are condensed. 3. A s e c t i o n of a corpus luteum about 72 days a f t e r conception. Note the angular c e l l s c o n t a i n i n g condensed n u c l e i . 4. A s e c t i o n of a corpus luteum about 119 days a f t e r conception. There i s a r i n g of extensive 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 spheroid c e l l s (upper r i g h t ) . Some c e l l s have d i e d . 5. A s e c t i o n of a corpus luteum about 159 days a f t e r conception. Some enlarged c e l l s c o n t a i n granules around the n u c l e i . 6. A s e c t i o n of a corpus luteum about 171 days a f t e r conception. A few c e l l s are 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 r i g h t ) . 7. A p o r t i o n of a corpus luteum about 200 days a f t e r conception (near term). In a d d i t i o n to the l a r g e angular c e l l s there are sma l l c e l l s (one at the r i g h t border and two at the top, j u s t l e f t of c e n t e r ) . 8. A p o r t i o n of a corpus luteum i n a doe near p a r t u r i t i o n . A l l c e l l s are i n v a r i o u s stages of degeneration. 3 9. A s e c t i o n of a degenerate,22 mm corpus luteum i n a doe tha t had given b i r t h a few days previous to c o l l e c t i o n . The c e l l s are com-p l e t e l y vacuolated. A p o r t i o n of 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 of a degenerate 12 mm corpus luteum i n a second doe which had given b i r t h a few days previous to being k i l l e d . This s t r u c t u r e i s more degenerate than the one above. Only 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 cut i n cross 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.' Angular, chromophilic c e l l s w i t h condensed n u c l e i were not numerous except i n zones undergoing c e l l a u t o l y s i s . The connective t i s s u e network was more ex t e n s i v e . In p l a c e s , two or three connective t i s s u e c e l l s separated adjacent l u t e a l c e l l s . A t h i c k l a y e r of c o l l a g e n i c f i b e r s surrounded the l a r g e blood v e s s e l s . Corpora l u t e a from the c o l l e c t i o n period centered on March 21 were about 117 days o l d . As i n the previous group,most of the c e l l s were l a r g e , contained v e s i c u l a t e n u c l e i and numerous vacuoles. Many c e l l s contained a d i s t i n c t p e r i p h e r a l r i n g of vacuoles and others contained r e l a t i v e l y l a r g e vacuoles, yellow pigment, and a c i d o p h i l i c granules. From one-tenth to o n e - t h i r d of the c e l l s contained deeply chromophilic cytoplasm, condensed n u c l e i , and p e r i p h e r a l vacuoles. A small p r o p o r t i o n of c e l l s were s m a l l , angular, and chromophilic. Corpora l u t e a i n does c o l l e c t e d around May 10 were about 167 days o l d . A u t o l y s i s of c e l l s was widespread i n these, e s p e c i a l l y i n the center of the gland. The r e l a t i v e p r o p o r t i o n s of the c e l l types 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 vacuolated cytoplasm, comprised 20 to 80% of the l u t e a l c e l l p o p u l a t i o n . These c e l l s appeared to be most numerous i n regions undergoing c e l l a u t o l y s i s . 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 vesi c u l a t e - n u c l e i were e x t e n s i v e l y vacuolated. Scattered amongst the l a r g e c e l l s were small c e l l s w i t h l a r g e vacuoles. In r e g i o n s of c e l l a u t o l y s i s , spherules of cytoplasm were common. The w a l l s of the major blood v e s s e l s were t h i c k . Corpora l u t e a from the c o l l e c t i o n p eriod centered on June 13 were, about 200 days o l d . Two does had given b i r t h and most of the other does were w i t h i n a few days of p a r t u r i t i o n . Corpora l u t e a i n does approaching par-t u r i t i o n contained c e l l s up to 4-7 i n diameter,whereas c e l l s i n e a r l i e r c o l l e c t i o n s d i d not exceed about 39 _u. The m a j o r i t y of 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 contained 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 . A few c e l l s around the p e r i p h e r y of the gland contained yellow pigment. Up t o one-tenth 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 , chromophilic c e l l s i n which l a r g e vacuoles apparently developed as the c e l l aged. The exceedingly degenerate corpora l u t e a i n the two p o s t - p a r t u r i e n t females contained only a few shrunken, vacuolated c e l l s . Consequently,the 3 3 corpora l u t e a were shrunken to volumes of 22 mm and 12 mm . F i n e , y e l l o w , pigment was present i n the degenerate, vacuolated c e l l s t h a t p e r s i s t e d , e s p e c i a l l y those trapped i n h y a l i n connective t i s s u e . I n the smaller more degenerate corpus luteum, c o n s i d e r a b l e granular pigment was present along the i n s i d e of a t h i c k , f i b r o u s , outer capsule. Also present I n the c o l l a g e n i c capsule, were t h i c k - w a l l e d blood v e s s e l s which f o r m e r l y served the 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 of 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 con-t r i b u t e v i a b l e ova are termed accessory or secondary corpora l u t e a . They occur only i n o v a r i e s c o n t a i n i n g primary corpora l u t e a . (Primary corpora l u t e a are d e r i v e d from f o l l i c l e s 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 , ova). I c l a s s i f i e d accessory corpora l u t e a i n deer i n t o three types, based on t h e i r source. Type 1 accessory corpora l u t e a o r i g i n a t e d i n unruptured f o l l i c l e s , e s p e c i a l l y i n l a r g e f o l l i c l e s t h a t f a i l e d to rupture at o v u l a t i o n . L u t e i n i -z a t i o n of the f o l l i c u l a r w a l l began 1 to 2 days a f t e r o v u l a t i o n . Unless the f o l l i c l e was s m a l l , only the w a l l or a p o r t i o n of the w a l l was composed of 102 l u t e a l t i s s u e . These s t r u c t u r e s were p r e v i o u s l y described i n 3. 8. L\. Type 2 accessory corpora l u t e a developed i n degenerate, f i r s t -c y c l e corpora l u t e a from c e l l s t h a t f a i l e d to degenerate, or from c e l l s t h a t became h y p e r p l a s t i c d u r i n g the e a r l y growth of second-cycle corpora l u t e 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 the c e n t r a l regions of some degenerat-i n g corpora l u t e a . There was no sharp demarcation 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 early-degenerate corpora l u t e a , but a f t e r 8 to 12 days, 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 the scar. The s m a l l , compact, s p h e r i c a l s t r u c t u r e s were surrounded by scar t i s s u e t h a t could be traced to the surface of the ovary. The amount of l u t e a l t i s s u e i n the 29 scars v a r i e d from j u s t a few c e l l s , to a volume of 0.87 mm3. Type 3 accessory corpora 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 t h a t ruptured about the same time as the primary f o l l i c l e s . These small 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 c o n t r i b u t e d v i a b l e ova. Type 3 accessory corpora 3 l u t e a , w i t h volumes of l u t e a l t i s s u e to 16 mm , were the l a r g e s t of the three types. Because they were i n d i s t i n g u i s h a b l e from, and r a r e l y overlapped i n s i z e w i t h s m a l l , primary corpora l u t e a , an a r b i t r a r y d e c i s i o n on the demarcation s i z e was made. I f a corpus luteum w i t h an o v u l a t i o n scar was l e s s than one-quarter 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 , i t was considered accessory. I n pregnant animals,the s m a l l e s t primary corpus luteum was about one-quarter of the s i z e of the l a r g e r corpus luteum i n the same female. The i n c i d e n c e of the three types of accessory corpora l u t e a was r e l a t e d to the age of the doe and to the chronology of the estrous c y c l e s . F o l l o w i n g f i r s t - c y c l e o v u l a t i o n , the i n c i d e n c e of Type 1 accessory corpora l u t e a i n y e a r l i n g s and a d u l t s was UU% and 4-8%, r e s p e c t i v e l y . I f accessory l u t e a l t i s s u e developed, i t was always i n the l a r g e s t a t r e t i c f o l l i c l e , 103 although smaller f o l l i c l e s i n the same p a i r of ov a r i e s l u t e i n i z e d o c c a s i o n a l l y . A f t e r f i r s t - c y c l e o v u l a t i o n there was no Type 3 accessory corpora l u t e a i n 50 females, although some corpora l u t e a were between one- . quarter and one-half the s i z e of the l a r g e r corpora l u t e a i n the same p a i r of o v a r i e s . A f t e r second o v u l a t i o n , TyP e 1 accessory corpora l u t e a were not 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 they occurred mostly i n s m a l l f o l l i c l e s (see 3. 8. 4). I n the f i r s t 2 weeks f o l l o w i n g second o v u l a t i o n , Type 2 secondary corpora l u t e a occurred i n a bout 41% (7/17) of the does w i t h degenerate f i r s t - c y c l e corpora l u t e a . The i n c i d e n c e of a c t i v e l u t e a l t i s s u e , i n otherwise degenerate corpora lutea,was 31% (9/29)- U s u a l l y , o n l y one of two corpora l u t e a of non-pregnancy i n a p a i r of o v a r i e s contained a c t i v e l u t e a l t i s s u e . Type 3 accessory corpora l u t e a occurred i n only 3.2% (2/62) of the females w i t h second-cycle corpora l u t e a estimated to be between 2 and 14 days o l d . Both accessory corpora l u t e a were i n the o v a r i e s of females 2.5 years o l d . Four other deer contained corpora l u t e a between one-quarter and one-half the volume of the l a r g e r corpora l u t e a i n the same p a i r of o v a r i e s . The i n c i d e n c e of a l l types of accessory corpora l u t e a i n pregnant does was 35.9% (19/53). They occurred i n females of a l l ages, but the 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 old does. Of 3 the f i v e l a r g e s t accessory corpora l u t e a , one was 16 mm i n volume and the 3 other f o u r measured between 2.4 and 8.4 mm . Most accessory corpora l u t e a 3 were.less than 1 mm i n volume. The l a r g e s t one occurred i n a non-pregnant female c o l l e c t e d on March 24. Apparently, a l l 3 types of accessory corpora l u t e a survived to the t e r m i n a t i o n of pregnancy. As pregnancy proceeded, the source (and t h e r e f o r e type) of the accessory corpora 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 to 104 determine. Ovulation stigmas healed and the corpora lutea became modified i n form. For example, a few months after i n i t i a l development of Type 2 accessory corpora lutea, hypertropy of the l u t e a l c e l l s and atrophy of the surrounding scar tissue made diagnosis d i f f i c u l t . Only a b a l l of l u t e a l tissue remained i n the stroma, not unlike Type 1 accessory corpora lutea after the f o l l i c l e was resorbed. I f a surface rupture scar was present, i t was d i f f i c u l t to dis t i n g u i s h some Type 2 structures from Type 3 accessory corpora lutea. In a l l three types,the l u t e a l c e l l s resembled those of the primary corpora lutea. 4. 10 Scars Derived From Corpora Lutea. The term corpus albicans was o r i g i n a l l y used to 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 structure i s hyalinized and appears colorless i n fresh, f i x e d , and ro u t i n e l y stained (hematoxylin and eosin) sections. In many species, the term corpus albicans has been applied to a l l types of scars derived from corpora lutea and also 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 . In 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 ori g i n a t i n g from corpora lutea may be c l a s s i f i e d as follows: 1. Scars derived from corpora lutea of non-pregnancy. 1. 1 Scars derived from degenerate, f i r s t cycle corpora lutea. 1. 2 Scars derived from degenerate corpora lutea of the second, t h i r d , etc. cycle i f pregnancy doesn't occur. 1. 3 Scars derived from accessory corpora lutea of the f i r s t ovulatory cycle (and subsequent cycles i f pregnancy 105 does not occur). 2. Scars derived from corpora lutea of pregnancy. 2. 1 Scars derived from normal corpora lutea of pregnancy. 2. 2 Scars derived from aberrant corpora lutea (often associated with the intra-uterine mortality of zygotes). 2. 3 Scars derived from accessory corpora lutea. The ch a r a c t e r i s t i c s of each type are presented i n the following pages. 4. 10. 1 Scars not Derived From Luteal Structures. The most numerous ovarian scars r e s u l t from the normal at r e s i a of f o l l i c l e s . They are charac-terized by large amounts of amorphous, hyalin connective tissue, which gives them a white appearance i n fresh and fixed material. However, they are a n i l i n e -positive when stained with trichrome methods. In microscopic sections, or upon hasty examination of stained sections, these scars can be confused with scars derived from corpora lutea 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. Occasionally, a few of the major blood vessels, which formerly served large f o l l i c l e s , p e r s i s t for long periods. Abnormal or necrotic a t r e s i a of f o l l i c l e s resulted i n scars con-taining variable amounts of connective tissue associated with degenerate c e l l s and thei r products. As the necrotic structure regressed, peculiar creamy-white material was produced i n spaces between the collagenic 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 lutea, were described previously in.4. 6 and 4- 7. They may be confused with si m i l a r h y a l i n struc-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 lutea always contained remnants of l u t e a l c e l l s and the structure often extended to the surface of the ovary, where some scar tissue was evident. In addition, corpora lutea scars were usually s o l i d structures, whereas scars replacing f o l l i c l e s contained a central lumen , which gradually decreased i n size. Scars r e s u l t i n g from the second and subsequent ovulations, which did not r e s u l t i n conception, were not common i n the ovaries because most does conceived at second estrus. The scars were similar 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 lutea, except they were larger at equivalent stages of regression. The ovaries of one non-pregnant doe, collected May 14, contained ten 0.1 to 0.8 mm3 scars from the previous breeding season. Scars r e s u l t i n g from accessory corpora lutea of the f i r s t cycle were small and soon disappeared. They were usually crescent shaped and had no connection to the surface of the ovary. Apparently,the size of the scar was proportional to the size of the accessory corpus luteum from which i t was derived. The largest active accessory corpus luteum (Type l ) contained 3 11.3 mm of l u t e a l tissue. L\. 10. 3 Corpora Lutea of Pregnancy Scars. Corpora lutea that supported a pregnancy for an extended period of time, degenerated into scars with 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 within a few days i t was one- f i f t h to one-tenth of i t s pre-parturition volume. At 3.5 months post-partum, the structure had a volume of about 3.4- mm (one t h i r t i e t h of i t s • former size), and a l l l u t e a l c e l l s had been resorbed. The structure of the scars 5 to 6 months after 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 early December. 107 Because corpora l u t e a never or r a r e l y developed i n w i l d fawn deer of the study area, l a r g e scars found i n females between 2.0 and 2.4 years of age were de r i v e d from corpora l u t e a of pregnancy of the previous November to 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 of scars was from females 2.4 to 2^5 years old at the time of c o l l e c t i o n i n November or e a r l y December. The mean volume of 49 scars was 2.24 +.0.10 mm ( F i g . 16). 3 Excluded from the above sample were a l l scars l e s s than 1.0 mm , a l l of which are beli e v e d to be scars derived from accessory corpora l u t e a . 3 Included i n the above sample were s i x l a r g e scars of 3.5 to 4>8mm , which may have a r i s e n from l a r g e corpora l u t e a or from a l a t e p a r t u r i t i o n . Both are 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 corpora l u t e a of 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 scars lowered the mean scar volume to 2.08 + 0.06 mm (N = 43). There was a gradual i n c r e a s e i n the s i z e of scars estimated to be 5 to 6 months old,as the age of the females increased ( F i g . 16). A s i g -n i f i c a n t d i f f e r e n c e occurred between the volume of scars i n young does (2.5 and 3.5 years old) and old does (ol d e r than 7 y e a r s ) ( A n a l y s i s of var i a n c e F = 7.35, P < 0.01 w i t h 6 and 213 d f ) . A m a c r o s c o p i c a l l y v i s i b l e surface p r o t r u s i o n , u s u a l l y l o c a t e d i n the center of a small depressed area, marked the l o c a t i o n of most scars of 5 months. The p r o t r u s i o n , composed of a s o l i d h y a l i n mass of connective t i s s u e , was connective to the main body of the scar l o c a t e d deeper i n the stroma. The m a j o r i t y of these s c a r s , l o c a t e d i n the outer c o r t e x , were oblate spheroid i n form; others were d i t o r t e d by f o l l i c l e s and corpora l u t e a . * Unless otherwise stated the plus and minus f i g u r e a f t e r a mean represents the standard e r r o r . ^ 4' B £ 3' o 00 F i g . 16. The a g e - s p e c i f i c volumes of scars 5 and 17 months old d e r i v e d from corpora l u t e a of pregnancy. Scars 5 months old are i n the upper row. (Mean, 95% confidence l i m i t s , standard d e v i a t i o n , range, and sample s i z e ) . 49 37 24 5 months 1y months V 2 3 1 24 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 scar of 5 months was ensheathed i n an a n i l i n e - p o s i t i v e , outer connective t i s s u e capsule of c o l l a g e n i c f i b e r s . The capsule was t h i c k around' some scars but was b a r e l y d i s c e r n i b l e around others. 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 which passed through the capsule along one side of the scar and extended a short 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. 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 of the corpus luteum. The c e n t r a l p o r t i o n of the scar was composed of h y a l i n connective 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 l i p o g e n i c pigment. The l a r g e r , outer 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 , but 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 to s t a i n . Most of the l a r g e v e s s e l s s t i l l contained blood c e l l s but u s u a l l y the lumena of the smaller 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 occluded. I n s e c t i o n s stained w i t h PAS, 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 pigment, a l l stained r e d . In s e c t i o n s stained i n combined PAS and Massons's trichrome, the red scars contrasted markedly a g a i n s t the blue stroma ( P l a t e 16). A l i p o g e n i c pigment, ov 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 scars of 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 not s p e c i f i c to the pigment. I n s e c t i o n s stained w i t h Masson's trichrome, 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 16). • The o v a r i e s of does 3.5 years old contained scars f i t t i n g the above d e s c r i p t i o n and other s i m i l a r but more degenerate scars t h a t were o b v i o u s l y 17 months o l d . These represent corpora l u t e a of pregnancy I n the does when they were between 1.5 and 2.0 years o l d . I n females 3.5 years o l d , the s i z e of 21 scars estimated to be 17 months old was 1.17+0.07 mm3. This i s s i g n i f i c a n t l y smaller (P < 0.001) than the scars of 5 months i n females aged 2.5 years, although the ranges overlap. The s i z e of scars Facing page 110 P l a t e 15 Scars Derived From Corpora Lutea 1 . A scax derived from a corpus luteum of pregnancy. About 5 months have elapsed s i n c e the corpus luteum began to degenerate ( X 2 0 ) . 2 . P o r t i o n s of adjacent scars derived from corpora l u t e a of pregnancy. Note the chromophobic h y a l i n i z e d regions around the p e r i p h e r a l blood v e s s e l s of the scar 17 months old (on the l e f t ) and the small amount of amorphous connective t i s s u e w i t h i n the scar. The t i s s u e between the scars 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 scar of 5 months (on the r i g h t ) . The l a t t e r scar contains amorphous connective t i s s u e between the blood v e s s e l s ( X 4 I ) . 3. A scar aged 5 months de r i v e d from a p a r t l y - h e r n i a t e d corpus luteum. A t h i c k band of h y a l i n i z e d connective t i s s u e marks the former rupture s i t e on the surface of the ovary ( X 4 I ) . 4 . A scar of 5 months derived from a completely-herniated corpus luteum of pregnancy ( X 4 I ) . 5. Four old scars derived from corpora l u t e a of pregnancy. The three scars,composed almost e n t i r e l y of a chromatic h y a l i n t i s s u e ^ a r e probably between 1 0 and 17 years old ( X 2 0 ) . 6. The c e n t r a l r e g i o n of a scar of 5 months to demonstrate the t h i c k -walled 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 connective t i s s u e c o n t a i n i n g n u c l e i , pigment and vacules (X800). 7. A s e c t i o n of a degenerate corpus luteum i n the ovaries of a doe k i l l e d November 9- The 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 laden w i t h granules. The slow and abnormal form of degeneration i s a t t r i b u t e d to f e t a l mummification (X800). 8. A 0.9 mm3, h y a l i n - t y p e scar b e l i e v e d to represent a corpus luteum th 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 -walled blood v e s s e l s occur around the p e r i p h e r y of a c e n t r a l mass of amorphous h y a l i n t i s s u e (X80). 111 estimated to be 17 months old, i n does older than 3.5 years, i s presented i n F i g . 16. There was a gradual i n c r e a s e i n the s i z e of these scars w i t h the age of does. I n s p e c t i o n of F i g . 16 r e v e a l s t h a t , f o r each age-group 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 scars of the two ages. However, there was c o n s i d e r a b l e overlap i n s i z e s between scars w i t h i n an age-class, and t h e r e f o r e not a l l scars 5 months old can be d i s t i n g u i s h e d , o n the basis of s i z e alone,from o l d e r s c a r s . Surface scars marking the rupture 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 5 months of age, but they v/ere g e n e r a l l y smaller and l e s s s t a i n a b l e . Most scars aged 17 months were d i s t o r t e d by f o l l i c l e s or corpora l u t e a , e s p e c i a l l y i n young animals because of t h e i r greater f o l l i c u l a r a c t i v i t y . Compared to the younger s c a r s , the demarcation of the 17-month scar from the surrounding stroma was more d e f i n i t e . There was no i n d i c a t i o n of an outer c o l l a g e n i c capsule. The outer blood v e s s e l s were s m a l l e r , more occluded, and 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 o l d . The 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 continuous w i t h the major o v a r i a n v e s s e l s i n the hilum, were more c o i l e d and r e t r a c t e d . The h y a l i n c e n t r a l p o r t i o n of the scar was much reduced, l e s s chromatic, and . commonly contained f i s s u r e s . This r e d u c t i o n of t i s s u e placed the s m a l l , t h i c k - w a l l e d , c e n t r a l v e s s e l s i n c l o s e p r o x i m i t y . Only a small amount of more deeply stained pigment was present i n the scar. The pigment was of d i v e r s e shapes, whereas pigment i n scars aged 5 months scars were o f t e n s h e r o i d . I n females aged 4-5 years, which contained f i v e or s i x scars i n t h e i r o v a r i e s , the scar t h a t had degenerated most and appeared older than t y p i c a l s c ars of 17 months,was assumed to be 29 months o l d . G e n e r a l l y these scars were s m a l l e r , contained l i t t l e or no h y a l i n connective t i s s u e or pigment, were more vacuolated and l e s s chromatic than younger s c a r s , and 112 the peripheral vessels were usually achromatic. I t was apparent that scars 27 months old could not alx-zays be distinguished from those of 17 months. In older does,there were scars 4I, 53, 65, etc. months old. Since 3 most of these scars exceeded 0.5 mm i n volume,, i t was apparent that degenera-t i o n and absorption of the scar proceeded at a slow rate after the second year. From the progression of changes i n the scars, i t appeared that a l l scars derived from corpora lutea of pregnancy persisted i n the ovaries for the l i f e of the doe. I f t h i s was true, and the pregnancy rate of does did not change appreciably with age, then a regression of the average number of scars per age-class on age should reveal a strong correlation. Such was the case (r = 0.998)'('Fig. 17). 4. 11 Corpora Lutea And Scars In Macroscopic Sections. At certain periods of the reproductive cycle, each major type of ovarian structure (corpora lutea, 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 color and size c h a r a c t e r i s t i c s (Table 7). At other times during the breeding season, mistakes i n i d e n t i t y were made,as' revealed by examination of the same structures i n thin,stained sections. The ages of structures i n Table 7 were estimated from their volumes and their h i s t o l o g i c a l appearance i n t h i n sections. Active corpora lutea were d i s t i n c t i v e structures but at least one small newly-ruptured f o l l i c l e was missed i n the macroscopic sections. F i r s t -cycle corpora lutea 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 similar i n color (orange) and size to pregnancy scars of 5 months. As a r e s u l t , three degenerate,first-cycle corpora lutea F i g . 17 Regression of .the-, average number of corpora l u t e a of pregnancy scars on the age of females at the previous breeding season. (Sample s i z e by each p o i n t ) . 28 1 © l © 26 • I © 24' 22 1 I I 1 I 1 1 1 1 I I « 1 1 1 I I 1 !— 2.5 4.5 6.5 8.5 10.5 12.5 14.5 16.5 18.5 AGE OF DOE AT THE PREVIOUS BREEDING SEASON Table 7. 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 s e c t i o n s of preserved o v a r i e s . S t r u c t u r e i F i r s t - C y c l e Corpus Luteum ]De; generating F i r s t - C y c l e Corpus Luteum Accessory Corpus Luteum Age(days)j 0-1 2-3 6-8 .j 1-2 3-4 5-8 9+ Various Color j B zones & st r e a k s . ¥ pe r i p h . t i s s . W C j C PaY DeC, CY, C. Y, PaO CO, PaO PaO, PaOY YO Same as corresp. CLl or CL2. Size(two ] diam mm) j 3-5x1-3 5x4 6-7x j 4-6 j 3-4x2-3 2.5-3x 1.5-2 1.5-2x 1-2 2x1-2 Various Bulb-shaped or f l a t t e n e d . Folded w a l l . Cent, lumen Oblate spher. or flask-shaped. F o l d i n g i n d i s -t i n c t . Sm. lumen S o l i d j oblate 1 Spher i Oblate spheroid or s p h e r i c a l Oblate spheroid or s p h e r i c a l Same as previous stage Same as •previous stage Type 1 hollow. Spher. to c r e s c . Shaped. Type 2 & 3 oblate or spher. S t r u c t u r e j Second-Cycle Corpus Luteum Scar Of Pregnancy JAt r e t i c F o l l i c l e Age(days)j 0-1 1-2 3-4 5-8 9+ 5-6 17-18 29-30 ! Various •months months months \ See • W W C w c w c DeO | P e r i p h e r y W. Color j Corresp. abund ant few B 0 0 j Center normally CL 1 B str e a k s streaks PaO PaO PaO ! c l e a r . Center ] of some PaY or | Y PaO S i ze(two ] 3 - 4 X 4-7x 5-8x 7-8.5x 7-10x 1.5-2.5 l - 2 x Avg 1 i Various diam mm) j 1-3 2-5 3-7 5.5-7 7-8 • x l - 2 0.5-1 j See Oblate, Reduced S o l i d , Same Spher., o b l a t e , U s u a l l y F l a t t e n e d Large lumen Corresp. spher., or lumen. oblate as flask-shaped. f l a t t e n e d and i surrounded by a CL 1 f l a s k - s h a p - Compact spheroid prev. R a d i a l pat- o f t e n i white capsule Form | ed . Lumen stage t e r n of blood curved 1 to a s o l i d mass u s u a l l y sm. 3 v e s s e l s & ! of white t i s s u e . occas. l a r g e pigment. ! R=red, 0=orange, Y=yellow, C= cream, W^white, B=Black, De^deep, Pa=pal e. Spher. = spheroid, sm. = s m a l l . 115 were i n i t i a l l y mistaken for pregnancy scars. Four other structures i n gross sections were i n d i s t i n c t but were estimated to be corpora lutea of non-pregnancy. This i d e n t i f i c a t i o n was confirmed i n thin sections. In addition, two unidentified,colored structures noted i n thick sections were i d e n t i f i e d i n thin sections as degenerate corpora lutea of non-pregnancy. At le a s t one small,degenerate corpus luteum of non-pregnancy,found i n thin sections,was missed i n macroscopic sections. Many pregnancy scars were not v i s i b l e i n gross sections. Accessory corpora lutea exibited the same texture and color as primary corpora lutea i n the same pair of ovaries. Because of limited development of l u t e a l tissue, they were not confused with primary corpora lute a , nevertheless, some small ones would c e r t a i n l y be missed In macro-scopic sections. I t was not always possible to estimate the age of pigmented corpora lutea of pregnancy scars i n macroscopic sections. The ovaries of does older than 4- years usually contained three to s i x pigmented scars. The larger, more rounded and deeply pigmented scars were assumed to be 5 months old, but they c l e a r l y graded into older scars. One scar of 5 months was barely v i s i b l e i n the ovaries of a female aged 2.5 years. Prior to macroscopic sectioning, 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, spherical, 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 variable 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 (ide n t i f i e d i n thin sections as necrotic 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 tresia). Facing Page 116 P l a t e 16 Color Photomicrographs Of Ovarian S t r u c t u r e s 1 . Two generations of corpora l u t e a . The most re 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 corpus luteum was c y s t i c and f i l l e d w i t h blood. (Masson's trichrome, X16). 2. A corpus luteum of pregnancy about 7 days a f t e r o v u l a t i o n and a shrunk-en corpus luteum from the f i r s t ovulation.(Masson 1s trichrome, X16). 3. A t i n y scar from the corpus luteum 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 scars u s u a l l y c o n t a i n more h y a l i n connective t i s s u e than i s present i n t h i s scar.(Masson's trichrome, X160). 4. Corpora l u t e a of pregnancy scars aged 5 months (On the l e f t ) and 17 months (on the r i g h t ) . Note the capsule of c o l l a g e n i c f i b e r s around the younger scar. 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 present. (Masson's trichrome, X16). 5- A corpus luteum of pregnancy scar 5 months o l d . The c e n t r a l p o r t i o n of the scar i s magenta when stained i n a combination of p e r i o d i c a c i d -S c h i f f and Masson's trichrome. (X16). 6. Pregnancy scars of 5 months (bottom) and 17 months (to 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 evident. The younger scar c o n t a i n s greater amounts of h y a l i n connective tissue.(Masson's trichrome, X32). 7. Ovarian f u s c i n pigment i n a pregnancy scar aged 5 months. The s e c t i o n was stained i n c a r b o l f u s c h i n which i s s p e c i f i c f o r l i p o g e n i c pigment ( X I 6 0 0 ) . 8. Pigment i n a pregnancy scar of 5 months stained i n p e r i o d i c a c i d -. S c h i f f . The pigment i s mostly i n the form of spherules. 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 achromatic. 1 0 . A t h i c k s e c t i o n through an ovary f i x e d i n A.F.A. The pigmented structure- below the l a r g e f o l l i c l e i s a pregnancy scar 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 evident 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 to define,accurately,the breeding season for two main reasons: 1. To determine the i n t e r v a l between f i r s t and second ovulation. 2. To determine the mean date of conceptions so that the age of embryos, corpora lutea, 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 ovulations, so that successive corpora lutea and f o l l i c l e s could be fixed 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 for the f i r s t and second time i n samples obtained i n November and early December. 2) The dates of f i r s t and second ovulation estimated from c h a r a c t e r i s t i c s of corpora lutea and f o l l i c l e s . 3) The dates of conception estimated from f e t a l growth curves. A) The occurrence of degenerate corpora lutea i n l a t e breeders. 5) The dates of p a r t u r i t i o n estimated from records of tagged fawns. I t was possible to estimate the length 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 ovulation was never successful. Because large numbers of deer were obtained throughout the breeding season, ovulation-frequency curves were constructed for f i r s t and second ovulations and they were compared s t a t i s t i c a l l y . Fortunately, most of the females ovulated for the f i r s t time within a span of about 2 weeks and for the second time within 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 for 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 F o l l i c l e s And Corpora Lutea In  P e r i o d i c Samples. Most of the o v a r i e s came from deer k i l l e d by hunters 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 given year 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 the two week-end days was used. Even then the sample s i z e was inadequate f o r some week-ends of some years. O v u l a t i o n i n c i d e n c e curves 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 older) 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 small and v a r i a b l e percentage of l a t e o v u l a t o r s and non-breeders. I n a d d i -t i o n , the l e n g t h of the estrous c y c l e may be more v a r i a b l e i n y e a r l i n g s than i n older females. Separate graphs were constructed f o r does w i t h d e f i n i t e corpora l u t e a and f o r does w i t h e i t h e r corpora l u t e a or newly-ruptured 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 could be mechanical r u p t u r e s . F i g . 18 shows the percentage of does having ovulated f o r the f i r s t and second time a t f o u r periods (weekends) d u r i n g November of 1963. The curve was smoothed through the p o i n t s because of the small sample s i z e f o r the c o l l e c t i o n periods November 16-17 and 23-24-The period of f i r s t o v u l a t i o n extended from the f i r s t few days of November,until about November 29, when a l l does i n a sample of 21 had ovulated. By November 9-10, only about 12% had ovulated but the i n c i d e n c e increased to about 60% by the f o l l o w i n g weekend. I t i s deduced from the.graph t h a t 50% of a l l does ovulated f o r the f i r s t time between November 9 and November 23. About 50%. of the a d u l t does ovulated 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 of the 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 ) . N O V E M B E R 120 presumably,extended i n t o the f i r s t week of December. I t i s deduced from the graph ( F i g . 18) t h a t about 50% of a l l does ovulated f o r the second time between November 17 and November 27. Second o v u l a t i o n s began i n a d u l t females about November 17, and reached the 50% i n c i d e n c e l e v e l by November 25. About 50% of the a d u l t does ovulated w i t h i n a span of 9 days. I n the years 1964- to 1967, the dates of f i r s t and second o v u l a t i o n s were s i m i l a r to those of 1963. Curves f i t t e d to the a v a i l a b l e samples i n d i c a t e . t h a t the date on which the 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 the 4- year period from 1963 to 1966. Because the breeding season v a r i e d o n ly s l i g h t l y from year to year, the r e s u l t s f o r a l l 5 years were combined to i n c r e a s e the sample s i z e . From 1963 to 1967 samples were a v a i l a b l e f o r each day (except December 4-) from November 6 to December 6. The o v u l a t i o n i n c i d e n c e curves f o r a l l does, based on a sample of 298 females, were s i m i l a r to those f o r the 1963 sample. The curves i n d i c a t e that 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 the 50% i n c i d e n c e l e v e l by November 16, and terminated about November 28 a t the 96 to 97% i n c i d e n c e l e v e l ( F i g . 19). The remaining 3 to 4-% represents l a t e o v u l a t o r s and non-ovulators (mostly y e a r l i n g s ) . The data 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, followed by a c e s s a t i o n of a c t i v i t y u n t i l November 12. Because there was l i t t l e i n d i c a t i o n of a s i m i l a r bimodal d i s t r i b u t i o n of o v u l a t i o n dates i n the curve f o r second-cycle o v u l a t i o n s , the 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 through the p o i n t s . About 50% of a l l does ovulated f o r the f i r s t time between November 13 and November 21,and 75% ovulated between November 12 and November 27. Ad u l t does-completed 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 began about November 17 and terminated e a r l y i n December. About 50% of a l l F i g . 19- The ovulation incidence based on d a i l y samples obtained during November and early December from 1963 to 1967. — i — A —r-6 10 12 U N O V E M B E R 16 20 22 24 26 30 2 4 DECEMBER 122 does ovulated f o r the second time between November 18 and November 26,and 75% ovulated between November 18 and December 1. Adult does completed second o v u l a t i o n around December 2. Because the 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 values f o r periods of 2, 3, and 4 days were c a l c u l a t e d , and the mean dates f o r each period were weighted according to the sample s i z e f o r each day of the p e r i o d . The e f f e c t of these running averages was a r e d u c t i o n i n v a r i a t i o n about the curve f i t t e d through the p o i n t s . Emphasis was placed on o b t a i n i n g an accurate estimate of the mean dates of f i r s t and second o v u l a t i o n because these dates were necessary to date ov a r i a n s t r u c t u r e s and f e t u s e s . Curves f o r the frequency of d e f i n i t e corpora l u t e a per u n i t of time g e n e r a l l y p a r a l l e l e d , but on the average lagged about one day behind, the o v u l a t i o n i n c i d e n c e curves f o r females w i t h corpora l u t e a and newly-ruptured f o l l i c l e s . The gap between the curves, which are not i l l u s t r a t e d , was g r e a t e s t (up to 2 days) i n the mid p o r t i o n of the curves — at the peak of f i r s t and second o v u l a t i o n . The l e n g t h of the estrous c y c l e , 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 s , was deduced, from the o v u l a t i o n i n c i d e n c e curves based on combined samples from 1963 to 1967 . I n t e r v a l s based on the i n c i d e n c e of does w i t h corpora l u t e a and ruptured f o l l i c l e s and does w i t h corpora l u t e a only, are tabulated f o r a l l deer,and a d u l t does,at o v u l a t i o n i n c i d e n c e l e v e l s of 25, 50, and 75%, u s i n g running averages f o r time groupings of 1, 2, 3, and U days (Table 8 ) . I t i s deduced from the curves t h a t the 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 or 9 days . The reasons f o r accurately* 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 the 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. J Interval Between Ovulations | Interval Between Ovulations Ovulation ] Based on CL & NRF** Ovulation | Basec on CL only. Incidence ! Time grouping (days)-"-I--X- Incidence | Time groupin g (days) Level (%)* j 1 2 3 4 Level (%) ! 1 2 3 4 A l l does (1.5 25 1 10.0 10.0 10.0 9.0 25 | 7.0 9.0 9-5 8.5 years old and 50 j 8.0 9.0 9-0 9.0 50 j 8.0 9.0 8.5 9.0 old er) 75 j 8.0 9.0 • 7.0 10.0 75 i 7 - 5 8.0 6.5 8.5 Means ! 8.7 9.3 8.7 9.3 Means j 7.5 8.7 8.2 8.7 1 Adult does 25 ! 8 - 5 10.0 9-5 9.0 25 ] 7.0 8.5 8.0 7.5 (2.5 years old 50 | 7.0 9.0 8.5 8.0 50 i 7.0 8.0 7.5 7.5 and old er) 75 j 7.0 9-0 8.5 8.5 75 ] 6.5 8.0 7.0 7.5 Means i 7.5 9-3 8.8 8.5 Means ! 6.8 8.2 7.5 7.5 The i n t e r v a l between the curves was determined at these three arbitrarily-chosen l e v e l s . Ovulation incidence curves were established for corpora lutea plus newly-ruptured f o l l i c l e s (CL & NRF) and for corpora lutea (CL) alone. The curves were f i t t e d to weighted running averages of ovulation 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 Estimated From Ovarian S t r u c t u r e s . C e r t a i n aspects of the breeding 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 dates 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 , as described 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 of the breeding seasons from 1963 to 1967 were p l o t t e d i n histogram form. The frequency d i s t r i b u t i o n s revealed the f o l l o w i n g p o i n t s : 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 6 and November I 4 to 16, r e s p e c t i v e l y . 2. The dates of f i r s t o v u l a t i o n s spanned a greater time period than the dates of second o v u l a t i o n s . 3. On the average, y e a r l i n g s ovulated l a t e r i n the season than a d u l t s and t h e i r dates of o v u l a t i o n were more v a r i a b l e . 4. The breeding season occurred at about the same time i n each of the f i v e years except i n 1967, when i t was l a t e r . 5. There was a tendency f o r o v u l a t i o n s to decrease i n number d u r i n g , and f o r a day or two a f t e r , the weekend hunting p e r i o d s . Means, standard deviations,and standard e r r o r s were c a l c u l a t e d from the frequency 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 ) . I n 1963 the mean date of f i r s t o v u l a t i o n was November 1 7 + 1 f o r a l l 32 does and November 16 + 1 f o r 24 a d u l t does. The mean date of second o v u l a t i o n was November 16 + 1 f o r 24 a d u l t does. The mean date of 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 of the nature of the sampling d u r i n g November, the above mean dates of f i r s t and second o v u l a t i o n s are considered to 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 analysed s t a t i s t i c a l l y to determine, i f , i n some years, the breeding season was e a r l i e r or l a t e r than i n other 125 years. Tests between the means derived from the d i s t r i b u t i o n s for f i r s t ovulation were not made because of the small sample size for most years, the obviously 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 ovulation were compared, but the comparisons were not v a l i d i n a l l cases because a l l the does (especially yearlings) had not completed second ovulation 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 difference between the mean ovulation dates of 1963 and 1964- (P < 0.05), but the difference between the means for adults was not s i g n i f i c a n t . Because a few adults had not completed second ovulation 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 difference existed between ovulation dates i n 1963 and 1964. Further comparisons between years were made after the d i s t r i b u t i o n of dates of f i r s t ovulation was shifted 8 days forward and added to the d i s t r i b u t i o n of dates for second ovulation 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 size for each year and p a r t i a l l y compensated for 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. In 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 ovulation from 1963 to 1966 were combined because there was no s t a t i s t i c a l difference between them. S i m i l a r l y , d i s t r i b u t i o n s for second ovulation were combined (Fig. 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 indicate that, over the four year period, the mean dates of f i r s t and second ovulations were November 14 to 15 and November 23 respectively. 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. Yearlings ovulated s l i g h t l y l a t e r than adults. The standard deviation of the dates of f i r s t ovulation was nearly twice that of the dates of second-ovulation. The mean date for the second ovulation was s l i g h t l y early because a few does had not completed second ovulation at the termination of the sampling period. For th i s reason, the actual 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 ovulation 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 for second ovulation. The resultant 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 la t e dates. The difference between the 1967 mean date of second ovulations for a l l does, and the corresponding mean for combined samples from the other four years, was highly s i g n i f i c a n t (P < 0.001). Similarly,the mean for 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 for adults from the combined samples from the other four years (P< 0.01). 3. 3 The Estimated Dates Of Conception Determined From Fetal Growth Curves. Some information on the breeding season and length of the estrous cycle 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 collected for t h i s study, found that hind-fcot length correlated best with mean developmental ranks based on numerous morphological c r i t e r i a . The curv i l i n e a r r e l a t i o n s h i p between hind-foot length (post-fixation) 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 Fig. 21. Because the mean date of second ovulation i n 1963 was November 25, the growth of the hind foot was also expressed i n terms of the mean gestation age. In 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 raised i n F i g . 21. The relationship 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 fetus. The l a t t e r was calculated from November 25, the mean date of conceptions i n 1963. Also included are two known-age fetuses and samples from 1965 and 1966.(Eye-fit curve). 230 210 190-170 w 150 EH O S3 S 130i rH g 110 I a 90-70 50-30-10 n Known-age Fetus O Fetus from a yearling A Conceived on a cycle subsequent to Cycle 2. 54 Specimen no. 50 60 70 _> , i_ MEAN GESTATION AGE 80 90 100 110 120 .130 1-40 150 160 170 M a 3 o - p u •H ra 190 200 210 220 10 20 30 JANUARY 10 20 29 FEBRUARY iO 20 30 MARCH DATE 10 20 APRIL 30 10 20 MAY 10 . 20 JUNE 30 ro oa 129 c a p t i v i t y . Only fetuses conceived at the second ovulation i n 1963, as determined by ovarian analysis, were included i n the regression. ThuSj two does (T54 and T56),that conceived 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 for fetuses from wild deer. The hind-foot lengths of the fetuses from two does collected i n 1965 were also i n close agreement with the extrapolated growth curve for the 1963-64 samples. This was expected- because the•breeding seasons of 1963 and 1964 occurred at about the same time each November. That the majority of points for 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 for forehead-rump length was established to deter-mine i f the relationship between the points for the various samples and the curve was similar to that for hind-foot length. The pattern was similar i n a l l respects (Fig. 22). 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 regression. 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 gestation age. Forehead-rump length was more variable than hind-foot length i n spite of the measurements for twins being averaged and represented by a single point. Based on the growth curve for the hind foot,a yearling doe (T54) conceived January 10, 46 days after the average date of conceptions i n 1963. Because i t s ovaries contained one corpus luteum and three corpora lutea of non-pregnancy, the doe probably conceived on i t s fourth cycle. I f the doe ovulated for the second time on November 25, then i t underwent two more cycles i n a period of 46 days. I t i s possible that t h i s doe started to cycle l a t e i n the breeding season as do many yearlings 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 adult 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 lutea and seven corpora lutea of non-pregnancy, i t i s l i k e l y that t h i s doe conceived on either the fourth or f i f t h cycle, with the l a t t e r being more probable. I f the doe conceived for the second time on November 25, then i t underwent two or probably three cycles i n a period of 36 days. Both females (T54 and T56) are important for they provided an i n d i c a t i o n of the length of estrous cycles 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 cycle 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 lutea of non-pregnancy were located i n the ovaries of a doe 2 . 5 years of age collected on February 3 . Because the corpora lutea of non-pregnancy were very small (0.08, 0.06 and 0.02 mm3), either the embryo had been resorbed or the female had stopped cy c l i n g several weeks previously, and the corpus luteum was persistent. The doe passed through three or probably four cycles before becoming temporarily pregnant or ceasing to cycle. The ovaries of a yearling taken on February 4 contained two 3 r e l a t i v e l y small corpora lutea (69 and 32 mm ), a very small corpus luteum 3 of non-pregnancy, and a large (5-5 mm ), hyalinized corpus luteum of non- • pregnancy. I t was deduced that t h i s animal became pregnant on cycle two, but there was zygote mortality, corpus luteum degeneration, and subsequent development of the two existent corpora lutea. 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) indicated 131 Fig. 22. The regression 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 fetus. (See Fig. 21) . 4 2 0 ' 4.00-380 -3 6 0 ' 340 3 2 0 ' 3 0 0 ' 2 8 0 2 6 0 ' 240 2 2 0 ' 2 0 0 ' 1 s o -i l 6o -' 1 4 0 : 1 2 0 H : 1 0 0 80' :-6o EH O Pi I - j - Known-age fetus O Fetus from a yearling Conceived on a cycle subsequent to cycle 2 5 4 specimen no. 4°" 50 60 7 0 MEAN GESTATION AGE (DAYS) 8 0 "90 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 T " 0 20 30 JANUARY 1 0 2 0 2 8 FEBRUARY 10 2 0 3 0 MARCH 10 20 30 APRIL 1 0 2 0 . MAY 3 0 1 0 JUNE DATE 132 t h a t o v u l a t i o n s had ceased or d i d not occur w i t h each f o l l i c u l a r c y c l e . 3 A y e a r l i n g , c o l l e c t e d on March 24, contained a 114 mm corpus 3 luteum, an accessory corpus luteum of 16 mm , and four corpora l u t e a of non-pregnancy. The doe had probably c y c l e d f i v e times. F o l l i c u l a r 3 development i n c l u d e d a waning Stage 3 f o l l i c l e of 19 mm , and a growing 3 f o l l i c l e of 19 mm . A non-pregnant doe,approaching 7 years of age,taken on May 14, contained no corpora l u t e a and ten corpora l u t e a of non-3 pregnancy,ranging i n volume from 0.1 to 0.8 mm . At an average of two corpora l u t e a per c y c l e , t h i s doe had passed through f i v e c y c l e s before o v u l a t i o n s ceased many weeks p r i o r to 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 to 1964, crews of the B r i t i s h Columbia F i s h and W i l d l i f e Branch tagged newly-born fawns a t Northwest Bay. The frequency d i s t r i b u t i o n and derived s t a t i s t i c s of the tagging dates f o r a l l seasons but the f i r s t , when the search f o r fawn began l a t e , are shown i n F i g . 23. During 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 occurred between the d i s t r i b u t i o n s f o r each year. The mean date of the combined d i s t r i b u t i o n , June 1 4 , i s 203 days l a t e r than the mean date of second-cycle o v u l a t i o n s f o r the p e r i o d 1963-66. I f the average fawn was tagged w i t h i n three days of b i r t h , the average g e s t a t i o n period under n a t u r a l c o n d i t i o n s i s 200 to 203 days,or equal to th a t f o r c a p t i v e deer. The standard d e v i a t i o n s of fawn-tagging dates (5 days) was s i m i l a r to the standard d e v i a t i o n of 6 days f o r the dates of second o v u l a t i o n s from 1963 to 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 which fawns were tagged from 1960 to 1964-. 4 6 8 10 12 14 16 18 20 22 24 26 28 30 JUNE x = 14 ±0.37 (SE) s = 5 n = 207 134 SECTION 4. THE EFFECT OF AGE ON FERTILITY AND PRODUCTIVITY. Information 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 from counts of f e t u s e s , corpora l u t e a , and scars derived from corpora l u t e a of pregnancy. Corpora l u t e a r a r e l y develop i n w i l d fawns, although l a r g e and ruptured f o l l i c l e s are common d u r i n g the breeding season. Only one s m a l l scar i n d i c a t i v e of a former corpus luteum of non-pregnancy, was noted i n 70 p a i r s of o v a r i e s from y e a r l i n g s . None of the u t e r i from fawns or short 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 of a previous pregnancy, nor were a c t i v e corpora l u t e a ever encountered. Thus, fawns are excluded from the f o l l o w i n g r e s u l t s . 4. 1 Numbers Of Embryos And Fetuses. Fetuses were obtained from 61 females c o l l e c t e d at p e r i o d i c i n t e r -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 . The m a j o r i t y of samples came from the 1963-64 r e p r o d u c t i v e season, but a few were taken i n the f o l l o w i n g two seasons. C o l l e c t i o n s f o r each period were made w i t h i n a period of 10 days. R e s u l t s of these counts 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 females 5.5 to 8.5 years o l d . A l -though sample s i z e s were small f o r each a g e - c l a s s , there was a general i n c r e a s e i n the pregnancy r a t e w i t h age, up to 3.5 years, 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 to 5.5 years (Appendix 4.). I n samples from a l l years,the pregnancy r a t e was 84.6% i n y e a r l i n g s , 91.7% i n females 2.5 years o l d , and 97.2% i n females older than 3 years. I n samples from 1963-64, the pregnancy r a t e s were s i m i l a r to the above. Only one female, older, than 3 years, was not pregnant i n a sample of 33 does (Table 9). 135 Table 9- The a g e - s p e c i f i c pregnancy r a t e and f e t a l r a t e of females i n 1963-64-Conception Age (yr) Sample S i z e Per Cent Pregnant Fetuses Per Doe 0.5 8 0.0 0.00 1.5 10 80.0 0.90 2.5 8 87.5 1.38 3.5 6 100.0 1.67 4.5 8 100.0 1.60 5.5-6.5 11 90.9 1.91 8.5 & + 8 100.0 1.75 In samples from 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 from the r a t e s i n the other age-classes. However, there 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 the mean f e t a l r a t e f o r does 2.5 to 4.5 years of age (I.46 + 0.12, N = 24) and the r a t e f o r does 5.5 to 8.5 years o l d (1.93 + 0.16, N = 14). I n samples from 1963-64, there was a gradual i n c r e a s e i n the f e t u s r a t e , w i t h age (Table 12). The maximum r a t e was a t t a i n e d i n the 5.5 to 6.5 age-group. The only s et of t r i p l e t f e t u s e s occurred i n a female 6.7 years o l d . Only three of 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 fetus,and a second l a r g e f e t u s attached to the 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 cord,occurred i n the uterus of a doe 3.6 years o l d . I t i s d o u b t f u l i f the l a t t e r f e t u s would have been born i n a v i a b l e con-d i t i o n . One member of a twin p a i r , i n a doe of 9.7 years, was i n an advanced stage of r e s o r p t i o n . 4. 2 Numbers Of Corpora Lutea. I n a sample of 56 females,the number of corpora l u t e a l a r g e r 3 than 30 mm corresponded e x a c t l y to the number of f e t u s e s . One doe of 3.5 136 years contained two l a r g e corpora l u t e a i n i t s ovaries and only one f e t u s i n i t s u t e r u s , but another doe (2.5 years old) contained a second 3 f e t u s t h a t was represented by an e x c e p t i o n a l l y small (12.8 mm ) corpus 3 luteum. The next s m a l l e s t corpus luteum represented by a f e t u s was 49 mm i n volume (the smaller of two), and the s m a l l e s t s i n g l e corpus luteum was 73 3 mm . In other does, small corpora l u t e a w i t h volumes of 16, 13, 8, 6,and 3 4 mm were not represented by f e t u s e s . These, and many other smaller l u t e a l bodies, were accessory corpora l u t e a and were excluded from comparisons between the number of corpora 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 probably would have d i e d , each corpus luteum l a r g e r than 30 mm was rep-resented by O.96 h e a l t h y f e t u s e s . This f i g u r e i s c o n s e r v a t i v e because many of the c o l l e c t i o n s were taken e a r l y i n the period of g e s t a t i o n . P r i o r to 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 estimated the o v u l a t i o n r a t e from counts of corpora l u t e a . Accessory corpora l u t e a , those l e s s than one-quarter 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 females of Northwest Bay conceived at second e s t r u s , i t was important to determine i f f i r s t - c y c l e corpora l u t e a could be used to estimate 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 corpora l u t e a from both c y c l e s , there were 93 f i r s t - c y c l e corpora l u t e a and 97 second-cycle, corpora 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 = 0.8, X =0.08 w i t h one d f ) . Unequal numbers of corpora l u t e a from the two c y c l e s were most common i n young and old does. Thus, the number of corpora l u t e a i n females having o n l y ovulated once,was added to those having ovulated t w i c e , t o i n c r e a s e the sample s i z e and place more confidence i n the mean o v u l a t i o n r a t e s f o r each age c l a s s (Table 10). 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 corpora l u t e a , of a l l females c o l l e c t e d from 1963 to 1967. Age Sample Corpora Lutea (years) Size per doe SE 1.5 41 1.12 0.05 2.5 35 1.51 0.10 3.5 13 1.54. 0.15 -4.5 12 1.50 0.15 5-5 11 1.91 0.16 6.5 11 1.82 0.12 7.5-10.5 13 1.85 0.10 11.5 & + 10 1.90 0.18 In a l l the samples from 1963 to 1967, the o v u l a t i o n r a t e was 1.12 per year-l i n g doe, about 1.52 per doe 2.5 to 4-.5 years old and 1.82 to 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 not d e c l i n e i n females over 11.5 years of age. Because of the small sample s i z e s only the mean r a t e f o r y e a r l i n g s d i f f e r e d s i g n i f i c a n t l y from the means f o r other females. The means f o r samples from each of the breeding seasons i s i n Appendix 6. When the sample s i z e s were increased by grouping age-classes, the e f f e c t of age on the o v u l a t i o n r a t e was more c l e a r l y demonstrated (Table 11). Table 11. The o v u l a t i o n r a t e of age-classes of females i n combined samples from 1963 tp 1967. Age of doe (yr) 1.5 2.5-4.5 5.5-6.5 7.5-10.5 11.5&+ 5.5&t-N 41 60 22 13 10 45 X 1.12 1.52 1.86 1.85 1.90 1.87 s 0.33 0.57 0.47 0.38 0.57 0.46 SX 0.05 0.07 0.10 0.10 0.18 0.07 S55«t.05 " 0.10 0.14 0.21 0.22 0.41 0.14 * 95% confidence l i m i t s of the means 138 I t i s obvious i n the above Table t h a t the o v u l a t i o n r a t e of y e a r l i n g " f e m a l e s d i f f e r e d s i g n i f i c a n t l y from older does; t h a t the 2.5 to 4.5 group d i f f e r e d s i g n i f i c a n t l y from other groups; and that groups of does older than 5 years d i f f e r e d i n s i g n i f i c a n t l y from one another. The above counts 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 having ovulated 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 of a l l females i n each age c l a s s . For example, about 20% of the y e a r l i n g s d i d not ovulate. 4. 3 Number Of Scars Derived From Corpora Lutea Of Pregnancy. A measure of past r e p r o d u c t i v e performance was obtained by count-i n g scars 5 months old t h a t r e s u l t e d from corpora l u t e a of pregnancy. U n t i l they were about 8 months o l d , scars from the preceeding 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 from older scars 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 study were c o l l e c t e d 5 to 6 months a f t e r p a r t u r i t i o n , the counts of scars are b e l i e v e d to be accurate. Scars r e s u l t i n g from corpora l u t e a of non-pregnancy, accessory corpora l u t e a , and aberrant corpora l u t e a were exluded. The last-mentioned a t y p i c a l s c a r s , c o n t a i n i n g l i t t l e or no pigment and few blood v e s s e l s , were be l i e v e d to be associated 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 scars l e s s than one-half the s i z e of the l a r g e s t s c a r , i n the same p a i r of o v a r i e s , were excluded because probably they developed from 3 accessory corpora l u t e a . A l l those excluded were l e s s than 1.0 mm i n volume. I n combined samples from the breeding seasons of 1963 to 1966, the average number of scars 5 months old Increased s t e a d i l y w i t h age up to 4-5 years (age a t the previous breeding season), and t h e r e a f t e r remained near 2.00 scars per female (Table 12). In combined samples from a l l years,there 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 age-classes up Table 12. The age-specific average number of scars of 5 months d e r i v e d from corpora l u t e a of pregnancy • i n the rep r o d u c t i v e seasons from 1962 to 1966.(Sample s i z e s are i n parentheses and standard e r r o r s are presented where a p p l i c a b l e ) . The Average Number of Scars per Female i n the Season I n d i c a t e d Age* 1962-63 1963-64 1964-65 1965-66 1962 to 1966 1 1.5 1.09+0.10 0.94 0.74 0.33 0.97+0.06 (33) ' (17) (17) (3) (70) 2.5 1.46+0.14 1.43 0.86 1.20 1.28+0.09 (13) (7) (7) (5) (32) 3-5 1.86+0.14 1.73 1.40 1.50 1.68+0.10 (7) (11) :(5) (2) (25) 4.5 . 1.94+0.06 2.00 2.00 1.50 1.90+0.07 (16) (1) (2) (2) (21) 5.5 2.00+0.00 1.40 1.50 2.00 1.82+0.08 (10) (5) (2) (5) (22) 6.5 2.00 2.00 2.00 2.00+0.00 (2) (2) (2) (6) ' 7.5-10.5 2.00 1.75 1.60 2.00 1.89+0.08 (6) • (4) (5) (3) - (18) 11.5-18.5 1.60 1.50 2.00 1.63+0.18 (5) (2) (1) (8) * The age of the females at the time of the conceptions that e v e n t u a l l y r e s u l t e d i n the sc a r s . UJ UO to L\,5 years. Over the 4-year period there was an o v e r a l l general decline i n the average number of scars per doe i n each age class up to 4.5 years. The decline i n scar production was most evident i n yearlings. Differences i n the means between years within an age-class were 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 , except between 1962 and 1964 i n the 1.5 age-group. However, the sample sizes for 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 lutea of pregnancy scars per doe, because the scars persisted 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 at the previous breeding season. 4. 4 The Productivity Of The Population. The productivity or b i r t h rate of the population was estimated by r e l a t i n g age-specific rates of reproduction to the age structure of the population. In t h i s paper, productivity i s defined as the average number of young (at birth) produced by each female of reproductive age. As defined above, productivity i s equivalent to the "General F e r t i l i t y Rate" used by demographers to measure human reproductive rates, except that the base i s 100 females instead of 1000. The age structure of the population was estimated from the age structure of deer k i l l e d by hunters (Fig. 24). The ages of these deer were determined by the tooth section method. I t was necessary to pool samples from several years to obtain an adequate sample. Young females, e s p e c i a l l y Fig. 24. The age structure of females (fawns excluded) taken from the Northwest Bay population between 1960 and 1966. 40 30 EH o 20 o cy 10 1. 1963-66 Hunter Sample n = 336 n n n E H 20 10 2. 1963-65 Personal Sample 78 n n _a C L 1.5 2.5 3.5 4.5 5.56.5 7.5 8.5 9.510.5 12.5 AGE-CLASS (YEARS) U.5 16.5 19.5 U 2 y e a r l i n g s , are probably over-represented i n the hunter sample, because they are l e s s wary than older does. This 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 females k i l l e d s p e c i f i c a l l y f o r t h i s study ( F i g . 24)• Most of these were taken at n i g h t and outside of the hunting season, when females were l e s s wary. Nevertheless, t h i s d i s t r i b u t i o n may be biased towards o l d e r , more p r o d u c t i v e , females because i n many i n s t a n c e s a doe was i d e n t i f i e d by the presence of a fawn. An estimate of the mean number of v i a b l e fawns produced by each age-class was obtained 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 production (Table 13). P r i o r to t h e i r amalgamation, each of the r a t e s was reduced by 5% to account f o r post-i m p l a n t a t i o n f e t a l m o r t a l i t y . The observed f e t a l m o r t a l i t y of 3.3% was increased to 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 age-class i s the product of the per cent frequency of the age-class i n the p o p u l a t i o n and the mean number of l i v e fawns born to each female i n the age-class (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 of v i a b l e f e t u s e s produced by 100 females of breeding age. The r e l a t i v e c o n t r i b u t i o n of each age-class to the t o t a l annual production 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 age-class i s expressed as a percentage of t o t a l p r o d u c t i o n (Table 14-). I t i s apparent 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 young to the p o p u l a t i o n and t h a t the c o n t r i b u t i o n of i n d i v i d u a l age-classes decreases w i t h age t h e r e a f t e r . Although i t i s not evident i n the Table, the 4-.5 and 5.5 age-classes c o n t r i b u t e d about equal numbers of fawns. 143 Table 13 . 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,(Sample s i z e i n parentheses). Age Class (yr) Adjusted F e t a l R a t e 1 Adjusted-O v u l a t i o n R a t e 2 Adjusted Scar R a t e 3 Weighted Average Rate 4 1.5 2.5 3.5 4.5 5.5-6.5 0.87 (13) 1.26 (12) 1.59 ( 6) 1.59 ( 9) 1.82 (12) 0.90 (41) 1.31 (35) 1.42 (13) 1.39 (12) 1.86 (22) 0.92 (70) 1.22 (32) 1.60 (25) 1.80 (21) 1.77 (28) 0.91 (124) 1.27 ( 79) 1.55 ( 44) 1.64 ( 42) 1.81 ( 42) 1. The t o t a l number of fe t u s e s (both v i a b l e and moribund) d i v i d e d by the t o t a l number of females, and the r e s u l t reduced 5% to 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 the o v u l a t i o n r a t e (of those o v u l a t i n g i n the f a l l ) times the per cent pregnant (84.6% of y e a r l i n g s , 91.7% of 2.5 years, and 97.2% of 3.5 years and older females, (Ap-pendix 4) times a 5% r e d u c t i o n to account f o r f e t a l m o r t a l i t y . 3. The pregnancy-scar r a t e (scars 5 months old)reduced by 5% to account f o r f e t a l m o r t a l i t y . .4. Weighted according to the sample s i z e . Table 14. 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. Age Per cent Freq of . L i v e Fawns " P r o d u c t i v i t y R e l a t i v e Class the Age-Class Born per Doe per Age-Class P r o d u c t i v i t y (yr) i n the P o p u l a t i o n per 100 Does per Age-Class (%) 1.5 30 0.91 27.3 20.0: 2.5 21 1.27 26.7 19.5 3.5 15 1.55 23.2 17.0 4.5 9 1.64 14.8 10.8 5.5-6.5 14 1.81 25.3 18.5 7.5 & + 11 1.76 19.4 14.2 T o t a l s 100 136.7 100.0 144-SECTION 5- THE EFFECT OF SIZE ON FERTILITY. I hypothesized that within an age-class, larger does would have a higher reproductive rate than smaller ones. The size of females was evaluated from measurements of the hind foot, chest.girth, and dressed weight. 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 thereafter re-mained almost constant (Fig. 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 differences occurred only to age 2.5 years. The r e l a t i o n s h i p between size of doe and ovulation rate, i n 1963, was explored. In these test s , dressed weight, g i r t h , and length of hind foot, were used to separate each age-class into' two groups of equal size. I f , for any doe, at least 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 "large" category. I f a doe had ovulated twice and the number of f i r s t - c y c l e corpora lutea dif f e r e d from the number of second-cycle corpora lutea (this r a r e l y occurred), then the average of the two was used. A l l eight yearlings that ovulated contained just one current corpus luteum, however, s i x of the eight does were larger than the average of a l l yearlings collected during the 1963 breeding season. A greater percent-age of the smaller yearlings had not ovulated by the termination of the hunting season on December 1.. The f e r t i l i t y of yearlings weighing less than 55 pounds (field-dressed weight) i n November was low; those weighing less than 50 pounds seldom produced corpora lutea. In contrast, some yearlings with dressed weights over 70 pounds produced two corpora lutea. I f only one corpus luteum developed, there was usually a second large f o l l i c l e present, i n d i c a t i n g that the yearling was nearly capable of ovulating 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 , chest g i r t h and dressed weight of e i g h t age-classes of females • c o l l e c t e d d u r i n g November and early-December, 1963-66. (Mean, 95% confidence l i m i t s , standard d e v i a t i o n , range, and sample s i z e ) . AO CK5 1.5 2*. 5 3T5 Z?5 5?5 6.5-7.5 8.5&f AGE U 6 the two yearlings 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 rate of yearlings had an important bearing on the i r f e r t i l i -ty. Twelve females, 2.5 years old, were divided into equal-sized large and small groups. •The mean ovulation rates for the respective large and small groups were 2.08 + 0.20 and 1.58 + 0.33 (NS). An in v e s t i g a t i o n of the re l a t i o n s h i p between the size of females 2.5 years old and the number of scars derived from corpora lutea 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 into two almost numerically-equal groups on the basis of the three size 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. In the aggregated r e s u l t s for a l l years,the mean for 25 large females was 1.00 + 0.08. scars as compared to 0.85 + 0.10 scars for 27 small does (NS). In older does, differences between large and small females were generally 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 sizes, nevertheless, some trends were apparent. For example, i n 1963, s i x large 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 largest 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 large 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 f e r t i l i t y . 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 , but i n s i g n i f i c a n t , d e c l i n e i n the-weight of female fawns and y e a r l i n g s . During the period 1962 to 1966, there was a decrease i n f e r t i l i t y , which was most evident i n young females. For i n s t a n c e , from 1963-64 to 1966-67 the mean number of pregnancy s c a r s , i n females 2.5 years o l d , decreased as f o l l o w s : 1.09, 0.94, 0.74 and 0.33. These scars r e p r e -sented corpora l u t e a of pregnancy i n y e a r l i n g s i n the previous r e p r o d u c t i v e season. S i m i l a r , but l e s s dramatic r e d u c t i o n s a l s o occurred i n the next two age-classes. I n females 4-5 years of age and o l d e r , the mean number of scars 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 i n d i c a t e t h a t , w i t h i n young age-classes of female deer, 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 or growth r a t e . 148 7. DISCUSSION AND CONCLUSIONS. 7. 1 General Comments. In t h i s study, the descriptive phase was an essential prelude to l a t e r sections i n which the pattern 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 cycle 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 function 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) also suffer because of inadequate sample sizes, undated structures, 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 defining, within narrow l i m i t s , the period of ovulations and conceptions; and by determining the age of the females from tooth sections. In previous investigations of the deer ovary, only a few dozen pairs of ovaries were sectioned i n t h e i r entirety. 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 pairs of ovaries, and both gross and t h i n sections of 33 pairs. Descriptions of structures are v i r t u a l l y useless unless they can be related temporally to v i t a l reproductive events, such as ovulation or p a r t u r i t i o n . The chronology of events i n the ovaries, including the quanti-t a t i v e changes i n the size of structures, 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 at the f i e l d s tation — f a i l e d because of inexperience and poor working conditions. Next, the oviducts were sectioned (a laborious 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 laboratory, up to 2 days after they were collected at the f i e l d station. The ova provided the best information available on the stage of the cycle i n the natural population, although there are undefined sources of error inherent i n the method (discussed l a t e r ) . These ova also provided valuable information on the frequency of f e r t i l i z a t i o n at the two ovulations, and concrete evidence for ' 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 ovulation, because they defined the length of the f i r s t estrous cycle and provided a basis for f e t a l growth curves. I was able to ascertain, within narrow l i m i t s , the period of f i r s t and second ovulations, primarily because large numbers of ovaries were obtained from throughout the breeding season, and because I was able to estimate dates of ovulation using c r i t e r i a e s t a b l i s h -ed from structures dated by means of ova. The timing of conceptions corres-ponded cl o s e l y to that of second ovulations, because over 95%> of the females conceived at second ovulation. 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 for curves depicting the growth of fetuses. Fortunately, I was able to exclude fetuses conceived on cycles subsequent to the-second. The ovaries of females hosting these fetuses contained more than one generation of scars from corpora lutea of non-pregnancy. The f e t a l growth curves, the f i r s t for the species and for a natural population of deer, were used to estimate the conception dates of two l a t e breeders — females that had cycled several times before becoming pregnant. The curves, i n conjunction with counts of scars from corpora lutea of non-pregnancy, provided a means of estimating the length of estrous cycles subsequent to the second. Periodic sampling of the population during the gestation period pro-vided information on the rel a t i o n s h i p between corpora lutea and fetuses, and 150 on the amount of prenatal mortality. Estimates of both are required before corpora lutea and t h e i r scars can be used to estimate fetus and fawn production. Fortunately, accessory corpora lutea can be excluded from primary ones by size 3 alone. Remarkably, i n pregnant females, the number of corpora lutea over 30 mm equalled the number of fetuses. Included i n the sample,was a female with two fetuses and only one 3 corpus luteum larger 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 just over 25% of the volume of the larger one. Thus, each primary corpus luteum was represented by 0.99 fetuses (85:84-). Ovarian analysis, i n conjunction with counts of ova, embryos, and fetuses, i s the only means of determining the rate and timing.of prenatal mortality. The observed percentage of moribund fetuses, 3.4%, was a r b i t r a r i l y increased to 5%,because coll e c t i o n s were made throughout gestation. The ovary may be likened to a kymograph, which records past repro-ductive events. Thus, the number of corpora lutea 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 for the l i f e of the female. The number of cycles experienced by a doe prior to conception or reproductive f a i l -ure was estimated from the number of scars derived from corpora lutea of non-pregnancy. Usually, only scars from the previous reproductive season are present, because of the i r rapid degeneration. In contrast, scars derived from corpora lutea 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 certainty from older scars. I t soon became evident that accurate measures of future, present,and past productivity could be obtained by microscopic examination of ovaries. Along with means of determining age of the females, the method i s a useful t o o l i n studies of comparative productivity. One advantage i s that 151 reproductive performance can be examined i n r e t r o s p e c t — after some change i n the population or the environment has occurred. In addition, the long term reproductive output of individuals and the population can be compared quan-t i t a t i v e l y to a th e o r e t i c a l or ecological standard. Perhaps, information ob-tained from ovarian analysis i s one of the best means of evaluating the s u i t a b i l i t y of a pa r t i c u l a r environment for a population. Gross analysis of ovaries, although much quicker than microscopic analysis, provides considerably less information. Productivity of the current year may be obtained from counts of corpora lutea but only a crude estimate of productivity i n the immediate past season i s obtained from counts of pigmented scars. The l a t t e r usually exceed counts of corpora lutea 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 i n d i s t i n c t . In addition, the regressing corpora lutea of non-pregnancy are s i m i l a r , for a b r i e f period, to scars of pregnancy. Both structures 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 t h e i r color. L i t t l e information on the extent of repeat breeding can be obtained by gross analysis. After reviewing the l i t e r a t u r e , Teer et a l . (1965) con-cluded that corpora lutea of ovulation, accessory corpora lutea, and other l u t e i n i z e d structures were r e l a t i v e l y uncommon i n most cervids. More detailed investigations may show, as I have, that these structures are common. In the following pages, certain 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 knowledge t h i s i s the f i r s t study of f e t a l deer o v a r i e s . The sequence of development corresponds c l o s e l y to t h a t of man, the monkey (Macaca mulatta) (van Wagenen and Simpson, 1965), and the domestic cow (van Tienhoven, 1968). In deer, f o l l i c u l a r development begins e a r l i e r but proceeds more s l o w l y to the t e r t i a r y f o l l i c l e stage than i t does i n the primates. T e r t i a r y f o l l i c l e s do not exceed 0.25 mm i n diameter i n ' p e r i n a t a l deer o v a r i e s but they a t t a i n diameters of 1 to 4 mm i n man. In deer, as i n other 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 a t r e t i c . Development of the c o r t e x and medulla 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 described f o r man and the monkey,except t h a t connective t i s s u e investment appears e a r l i e r and i s more extensive i n deer. F e t a l o v a r i e s are p a r t i t i o n e d by a connective t i s s u e network a t 64 days and the t u n i c a albuginea I s w e l l developed by 170 days. I n c o n t r a s t , connective t i s s u e p r o l i f e r a t i o n i n the human ovary i s not r a p i d u n t i l a f t e r 180 days,and the 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 (van Wagenen and Simpson, 1965). 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 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 surface 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 time. This p a t t e r n i s s i m i l a r to t h a t of man, but i n the macaque monkey,ovogenesis continues i n t o the f i r s t few post-partum months. In deer, as i n the monkey, there 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 . They are most.abundant i n deer o v a r i e s 98 to 123 days a f t e r conception. I t i s concluded t h a t the germinal e p i t h e l i u m c o n t r i b u t e s c e l l s t h a t develop i n t o sex c e l l s . However, one cannot make f i n a l c o nclusions .153 about detailed 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 fetus from a captive doe were larger, further developed, and contained greater numbers of sex c e l l s than f e t a l ovaries of equal age i n wild does, i s worth further investigation. There i s l i t t l e doubt that the penned doe was better nourished than i t s wild counterparts. The f e t a l ovary, which undergoes progressive developmental changes, may be a more sensitive indicator of the maternal environment, and ultimately the physical 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 for 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 histology and function of the post-natal ovary, but i n some instances, provides information concerning endocrine changes i n the female host. For example, the 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-crine 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 Fetal Growth  Curves. Descriptions of structures are of l i t t l e value unless they can be related to a reference point, such as ovulation 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 directed into means of estimating the ages of corpora lutea and f o l l i c l e s . The ages of ovarian structures 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 lutea and f o l l i c l e s i n females from which ova were recovered. I assumed that the rate of cleavage of deer ova i s equivalent to about the average of the rates 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 occur i n the r a t (Schlafke and Enders, (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 rates because i t i s d i f f i c u l t to determine the moment of ovulation. Austin (1957) observed considerable v a r i a t i o n after the 1 - c e l l stage i n the f i e l d vole (Mlcrotus  a g r e s t i s ) . 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 differences among ova and asynchronous rupture of f o l l i c l e s . 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 large considering the source of v a r i a b i l i t y between ovulation 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. In most domestic species of ungulates,the ova enter the uterus 3 to 4 days after ovulation at about the 8-celled stage (Hartmann, 1962).. More recent studies indicate that some ova containing only 3 or 4 c e l l s enter the uterus as early as 2 days after ovulation (Austin, 1957; Oxenreider and Day, 1966). Three ova containing 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 for ova. The ages of ovarian structures, after 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 for black-tailed deer. Most i n -vestigators r e l i e d on the f e t a l growth curve for white-tailed 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 ovulation i n formulation of the curves,i.e. females with more than one generation of corpora lutea of non-pregnancy were excluded. Sample sizes were large, 155 consequently,the mean date of second ovulations and the mean sizes of fetuses from second ovulation 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 fetuses. The curve based on hind-foot length i s preferred to that for g i r t h or weight because the hind foot i s the le a s t variable of the three measurements (weight i s the most variable). Fetal growth curves are not r e l i a b l e indicators of f e t a l age i n the l a t t e r stages of gestation because of differences i n growth rate, differences between singletons and twins, and differences between sexes (Ommundsen, 1967). For example, there was a s i g n i f i c a n t difference i n weight between male (heavier) and female fetuses i n the sample taken just prior 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, with age, up to 7.5 to 9-5 years. The ovaries of old females were s l i g h t l y l i g h t e r . Excluding fawns, the curve depicting age-specific changes i n ovarian weight clos e l y p a r a l l e l s the corresponding curve for the b i r t h rate. 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 la 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 lesser extent by enlargement of corpora lutea. 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 difference between the weights of 156 the l e f t and r i g h t o v a r i e s , which i s i n keeping w i t h the o b s e r v a t i o n 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 sp e c i e s , one ovary 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 than the other ( A s d e l l , 1964). 7. 5 The S t r u c t u r e And F u n c t i o n Of F o l l i c l e s . P r i o r to t h i s study, v i r t u a l l y nothing was known about the f o l l i c u l a r c y c l e i n any w i l d ungulate. I n deer, a 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 occurs i n which f o l l i c l e s develop from an i n s i g n i f i c a n t s i z e to m a t u r i t y I n about 8 days. This r a p i d growth of f o l l i c l e s , s t a r t i n g immediately a f t e r o v u l a t i o n , has been reported i n few s p e c i e s . Corner (1921) and Hammond (1927) beli e v e d t h a t f o l l i c l e s i n the p i g and cow grew to o v u l a t o r y s i z e over a period of two or three estrous c y c l e s (about 6 weeks). More r e c e n t l y , i t was discovered t h a t two f o l l i c u l a r c y c l e s occur w i t h i n each 21-day estrous c y c l e i n c a t t l e (Bane and R a j a k o s k i , 1961). The 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 a f t e r e s t r u s and the second s t a r t s about 12 to 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 to 7 days a f t e r o v u l a t i o n . 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 days, because o v u l a t i o n occurs on day 2 of the estrous c y c l e . 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 guinea p i g . Estrous c y c l e s as short as 10 days have been recorded f o r the horse, although the average l e n g t h of the c y c l e i s 22 days ( B e r l i n e r , 1959). A l l of these observations suggest t h a t two w e l l -defined f o l l i c u l a r c y c l e s may occur w i t h i n the estrous c y c l e of some spec i e s . The short 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 , s h o r t -l i v e d corpus luteum and an 8-day f o l l i c u l a r c y c l e , w i t h f o l l i c u l a r growth beginning immediately a f t e r o v u l a t i o n . 157 Several weeks prior to the breeding season, single large 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 breed-ing season two large f o l l i c l e s usually 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 size 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 prior to the breeding season but no corpora lutea develop. Cyclic f o l l i c u l a r development continues throughout gestation, but i t i s dampened during the mid-portion of pregnancy. The length of the f o l l i c u l a r cycle may be extended during t h i s period. Invariably, only one large active f o l l i c l e i s present at a time i n females contain-ing 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. Cyclic production of f o l l i c l e s , at in t e r v a l s corresponding to the length of the estrous cycles, occur i n pregnant rats and guinea pigs (Perry and Rowlands, 1962). .'Precocious' ripening 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). In the mare, the c y c l i c production of f o l l i c l e s and corpora lutea i s most prevalent i n the second and thi r d month of pregnancy (Amoroso et a l . , 194-8). Post-conception ovulations, which r e s u l t i n corpora lutea, occur i n the African elephant (Perry, 1953), elk (Halazon and Buechner, 1956; Morrison, 1960), and the mare (Berliner, 1959). Some of the large f o l l i c l e s rupture i n pregnant deer but corpora lutea do not develop. F o l l i c l e s i n deer ovaries grow to ovulatory size during the l a s t 6 weeks of pregnancy, and the presence of large regressing f o l l i -cles indicates 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 cycles 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 gestation 158 has not been reported i n ar t i o d a c t y l s , although i t occurs i n the porcupine (Mossman and Judas, 194-9) • In elk, f o l l i c l e s decrease i n average size during gestation (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 histology and cytology of growing f o l l i c l e s i n deer are quite similar to the descriptions for other large 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 prior to ovulation were examined closely. The preovulatory f o l l i c l e i n deer ovaries has many of the cha 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. Characteristics s i g -n i f y i n g f o l l i c u l a r maturation were: 1) loosening of the cumulus oophorus, 2) increased vascularization, 3) cessation of mitosis i n granulosa c e l l s , except those immediately around the oocyte, and 4-) thinning of the ovarian and f o l l i c u l a r w a l l . Probably the most universal char a c t e r i s t i c i s c e l l autolysis i n the cumulus oophorus. This loosen-ing of the oocyte occurs just prior to ovulation i n guinea pigs (Myers et a l . , 1936; Hartman, 1962), rats (Odor, 1955; Hartman, 1962), swine (Spalding et a l . , 1955), c a t t l e (Corner, 1920), rabb i t s , and man (Brambell^, 1956). Autolysis of the c e l l s of the cumulus oophorus also occurs i n a t r e t i c f o l l i c l e s of deer, including those that l u t e i n i z e . Blandau (1966a) reported that dispersion of c e l l s did not occur i n large, 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-with cumulus oophorus dispersion i n some preovulatory f o l l i c l e s of deer, i s formation of the corona radiata. Apparently, t h i s s t r i k i n g r a d i a l 159 arrangement of granulosa c e l l s around the oocyte occurs i n most mammals (Brambell, 1956). The e n t i r e process of cumulus oophorus a u t o l y s i s and corona r a d i a t a formation may be induced by l u t e i n i z i n g hormone from the a n t e r i o r p i t u i t a r y (Blandau, 1965b). P r o l i f e r a t i o n of v a s c u l a r loops i n t o the membrana granulosa below the cumulus oophorus occurs i n most l a r g e f o l l i c l e s of deer,but the loops enlarge as o v u l a t i o n nears. T a l l , columnar c e l l s w i t h l e s s dense cytoplasm r a d i a t e out from these loops, as i n the cow (Corner, 1920). 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 mural granulosa, 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 ncreased v a s c u l a r i t y of the f o l l i c l e i s one of the best i n d i c a t o r s of impending o v u l a t i o n i n man. According to Burr and Davis (1951), there 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 of f o l l i c l e s and the thinness of f o l l i c u l a r and ova 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 . F o l l i c l e s t h a t have extended through the -fibers of the t u n i c a albuginea are destined to rupture e i t h e r p h y s i o l o g i c a l l y or mechanically. Enlargement or l u t e i n i -z a t i o n of theca c e l l s does not occur i n deer p r i o r to ov u l a t i o n , a l t h o u g h i t does occur i n some other species ( 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 are present d u r i n g the breeding season, one of them i s o f t e n out of phase w i t h the other. E v i d e n t l y , both do not always rupture a t the same time. Although there may have been an i n t e r v a l of a day or two between some of the r u p t u r e s , n e v e r t h e l e s s , both 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 of a 1 to 2 day i n t e r v a l between ruptures i n w h i t e - t a i l e d deer. McEwan (1962) b e l i e v e s t h a t the accessory corpora l u t e a i n c a r i b o u .are produced from asynchronous rupture of f o l l i c l e s . However, there are many i n s t a n c e s i n which 16o one of the large f o l l i c l e s does not rupture at f i r s t ovulation. Such f o l l i c l e s i n v a r i a b l y develop into accessory corpora lutea. 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 tissue. The f o l l i c l e s which rupture at second ovulation are mature and at the same stage of development presumably because the st a r t of thei r growth i s synchronized by the f i r s t ovulation. Thus, most of the ova shed are v i a b l e , whereas many of the ova shed at f i r s t ovulation are probably incapable of producing embryos,even i f provided with a suitable environment. The selective advantage of the second ovulation i s at once apparent. In deer,the atresia 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 primarily on the stage of development. Detection of early 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 prior to rupture. The e a r l i e s t stages of atr e s i a are i d e n t i f i e d by multiple c h a r a c t e r i s t i c s , involving a l l of the f o l l i c u l a r components. However, the most diagnostic tissues are the membrana granulosa, the vascular system i n the theca interna, and the zona pellucida and i t s attachments to 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 at r e s i a . This v a r i a b i l i t y probably accounts for 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 character-i s t i c s of atr e s i a (Ingram, 1962). In deer f o l l i c l e s , t h e granulosa usually s i g n i f i e d a t r e s i a f i r s t . Oocytes i n large 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 disassociates 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 ova, although most showed one or more signs of degeneration. Hypertropy and hyperplasia of c e l l s occur inside 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 similar i n appearance to l u t e a l c e l l s but they are smaller, hence I term them "semi-luteinized" 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 degenera-t i o n . The peculiar 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 may occur i n other mammals besides deer. Mossman and Judas (194-9) described concentric layers of young i n t e r s t i t i a l tissue 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 structures as one type of accessory corpora lutea. Such structures have been termed"corpora lutea atretica"(Brambell,1956), a term also applied to f u l l y l uteinized f o l l i c l e s . I think the term i s misleading and should be abandoned. I consider only structures containing t y p i c a l l u t e a l c e l l s , a s corpora lutea or accessory corpora lutea. The material i n the center of the walls of these c i r c u l a r structures has not been described previously,and remains unidentified. I t i s naturally black or i s stained black by Masson's trichrome. I t was shown that atresia of f o l l i c l e s took two forms i n deer. The a t y p i c a l form of degeneration involving leucocytes, i s unlike any described i n the l i t e r a t u r e , 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 rate 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 dating events. I was able to obtain information on t h i s topic by establishing the r e l a t i o n s h i p 162 betv/een the degeneration of l a r g e f o l l i c l e s and the growth of the next c y c l e 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 growth was estimated from segmentation stages of o v a , i t was p o s s i b l e to estimate the r a t e of f o l l i c u l a r a t r e s i a . The general r e l a t i o n s h i p , s i m p l i f i e d and diagrammed i n F i g . 26, i s e x i b i t e d by does of a l l ages from 6 weeks p r i o r to the breeding season to the end of g e s t a t i o n , although dampened f o l l i c u l a r a c t i v i t y occurs d u r i n g the middle h a l f of g e s t a t i o n . For the purposes of the f i g u r e , i t i s assumed that f o l l i c l e s grow a t the same r a t e throughout d i f f e r e n t c y c l e s of the season. I t i s evident that the f o l l i c l e passes through the f i r s t three stages of a t r e s i a i n one or two days, 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. Although i t i s not evident i n the f i g u r e , the l a s t two stages (seven and 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 , u n l i k e e a r l i e r stages, i s l a r g e l y dependent on the s i z e of the degenerating f o l l i c l e . S t u r g i s (194-9) has conducted one of the few s a t i s f a c t o r y s t u d i e s on the r a t e of f o l l i c u l a r a t r e s i a . The temporal changes i n the r e g r e s s i n g f o l l i c l e of the deer are s i m i l a r to those he d e s c r i b e s i n the monkey. The general p r o g r e s s i v e d e c l i n e i n the numbers of p r i m o r d i a l f o l l i c l e s w i t h age (in deer) i s comparable to the s i t u a t i o n i n other s p e c i e s . The d e c l i n e i n numbers i s not as r a p i d i n deer as i n small mammals,but the l a t t e r have short continuous cycles,whereas the deer i s a seasonal breeder. There i s a continuous d e c l i n e i n the number of oocytes, w i t h time, i n man (Block, 1952), the r a t • (Mandl and Shelton, 1959), and the mouse (Jones and Krohn, 1961). In the l a t t e r two species,the d e c l i n e i n numbers of p r i m o r d i a l f o l l i c l e s i s e x p o n e n t i a l . I n deer, as i n other s p e c i e s , there i s great i n d i v i d u a l v a r i a t i o n i n the number of oocytes. For example, i t was c a l c u l a t e d t h a t fawns have from 8,000 to 200,000 p r i m o r d i a l f o l l i c l e s i n each ovary. Hafez (1961) estimated t h a t h e i f e r s of 3 months c o n t a i n 100,000 to 300,000 oocytes i n t h e i r o v a r i e s . Neoformation of oocytes 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 of 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 given f o r one. 'The other curves r e p r e -sent stages of a t r e s i a and a t r a n s i t i o n a l (T) stage. 7- 1 : » 1 1 1— 10 20 30 40 50 60 VOLUME LARGEST ACTIVE FOLLICLE 164 i f i t occurs. There i s no i n d i c a t i o n t h a t the stock of oocytes i s ever exhausted i n deer as i t i s i n man (Block, 1952). The o l d e s t doe, 19.5 years, s t i l l harbored about 2,000 oocytes. The number of small 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 but they have no bearing on the f e r t i l i t y of the doe. Fawns and females aged 2.5 years contained the grea t e s t numbers of sm a l l f o l l i c l e s and there was a d e f i n i t e r e d u c t i o n i n small f o l l i c l e numbers i n o ld does. Perhaps,the number of small f o l l i c l e s may i n d i c a t e the type or amount of gonadotropic hormone s t i m u l a t i o n . The f a c t t h a t fawns and females aged 2.5 years c o n t a i n great numbers of small f o l l i c l e s , but fewer l a r g e f o l l i c l e s , could be a t t r i b u t e d to an imbalance of 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 (LH). I t was noted t h a t i f an i n d i v i d u a l contained a l a r g e number of s m a l l f o l l i c l e s , the l a r g e s t f o l l i c l e s were r e l a t i v e l y s m a l l . I n con t r a s t , some y e a r l i n g s and old does w i t h very few small f o l l i c l e s contained one or two r e l a t i v e l y l a r g e f o l l i c l e s . Seemingly p e c u l i a r to deer, i s the formation of abundant chromophilic bodies i n oocytes l o c a t e d i n small f o l l i c l e s . These i n c l u s i o n s , which seem to o r i g i n a t e i n the nucleus and spread outward, are u n l i k e s t r u c t u r e s i n oocytes of the mouse (Gresson, 1933), oppossum (Martinez-Esteve, 1942), and monkey ( P o l l a k , 1926). Because the I n c l u s i o n s occur i n f o l l i c l e s w i t h r a p i d l y - p r o l i f e r a t i n g granulosa, they may not s i g n a l the onset of a t r e s i a as suggested by Ingram (1962). The i n c l u s i o n s are s i m l l i a r to the 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 the r a t (Gresson, 1933),but i n deer they are many times l a r g e r . The i n c l u s i o n s may be associated w i t h the s h i f t i n g of the 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 diameter,the nucleus seems to disappear 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 diameter when the s h i f t of the nucleus occurs. Brambell (1956) described f a t t y y o l k spheres i n the 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 significance of these structures, which occur i n the rabbit, p r a i r i e dog, cat, 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 f o l l i c l e s . Possibly, they s i g n i f y a r e l a t i v e l y high secretion of f o l l i c l e -stimulating 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 black-tailed deer of Northwest Bay. The largest polyovular f o l l i c l e encountered was 1.2 mm i n diameter, f a r below ovulatory size. Hundreds of large f o l l i c l e s were examined for multiple oocytes and none was found. Even i f polyovular f o l l i c l e s reached ovulatory 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 viable, because, i n most small f o l l i c l e s , only one oocyte i s of normal size. Nevertheless, embryos i n excess of corpora lutea have been attributed to polyovular f o l l i c l e s i n the wild Norway r a t (Davis and H a l l , 1950), and rabbits (Allen, Brambell and M i l l s , 1947). Three instances of polyovulation have been reported i n white-tailed deer by Hesselton (1967). Other examples of single corpora lutea 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 lutea had fused. F o l l i c u l a r cysts are rare i n the ovaries of Northwest Bay deer. As noted by Ingram (1962), the cysts are lined with a single layer of epithelium and the 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 hor-mone. 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 cycle i n which the membrana granulosa thins to one layer 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 either ovulate or become accessory corpora lutea. The second type of cyst (derived from coelomic epithelium) i s more common. One exceptionally large cyst was recovered from a pregnant doe. 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 white-t a i l e d deer were undoubtedly scar tissue that formed after the regression of partly-extrusive corpora lutea. I concur with the opinion of Morrison (i960), who examined elk ovaries, that information on the reproductive status of herds can be obtained from analysis of f o l l i c u l a r development prior to the breeding season. The 3 number, size, and appearance of f o l l i c l e s over about 10 mm provides a crude estimate of the number that w i l l rupture. 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 at 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 lutea of short dura-t i o n occur i n ungulates. Buechner et a l . (1966) suggested that corpora lutea of short duration might occur i n the Uganda kob,and Perry (1953) be-lieved that " r e l a t i v e l y short-lived" corpora lutea occurred i n the African "elephant (not an ungulate). Interestingly, Short and Buss (1965) were unable to detect progesterone i n corpora lutea of the elephant. According to Perry and Rowlands (1962), i t i s well established that corpora lutea of non-pregnancy are r e l a t i v e l y inactive i n the r a t , mouse, and possibly i n many other small rodents. The short-lived corpora lutea derived from the f i r s t ovulation 3 grow to a volume of about 50 mm i n a span of 2 or 3 days and they begin to shrink after another 2 or 3 days. The functional 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 th i r d day and the structure shows h i s t o l o g i c a l indications of 167 degeneration 1 to 2 days before second o v u l a t i o n . U s u a l l y , sheep do not ovulate f o r 2 to 7 days a f t e r exogenous progesterone i s withdrawn (Robinson, 1959), and the l e v e l of progesterone i n the blood f a l l s r a p i d l y 2 days before e s t r u s i n c y c l i n g sheep (Robinson, 1966). S i m i l a r l y , e n u c l e a t i o n of the bovine corpus luteum r e s u l t s i n e s t r u s 2 to 5 days l a t e r (Hammond, 1927; Hansel, 1966). The extremely r a p i d degeneration of the f i r s t - c y c l e corpus luteum a f t e r second o v u l a t i o n i s an outstanding 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 than i n the cow (McNutt, 1924.) or p i g (Corner, 1919). B o l i n g (1942) noted a r a p i d decrease i n the volume of the previous corpus luteum 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 r a p i d r e g e n e r a t i o n of p e r s i s t e n t corpora l u t e a i n hypophysectorn!zed r a t s . S i m i -l a r l y , the r e g r e s s i o n of corpora l u t e a of pseudopregnancy i n the r a b b i t was f a c i l i t a t e d by very s m a l l doses of exogenous l u t e i n i z i n g hormone (Stormshak and Casida, 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 f o r the sudden decrease i n the volume of corpora l u t e a i n deer. W i t h i n three days a l l but a few c e l l s degenerate completely. The curves e s t a b l i s h e d f o r growing and degenerating f o l l i c l e s and corpora l u t e a provide 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 of s t r u c -t u r e s and the stage of the estrous c y c l e . The curve based on the r a t i o of the volumes of the a c t i v e and r e g r e s s i n g corpora l u t e a unquestionably provides the best estimate of the e a r l y age of second-cycle corpora l u t e a . Of the four curves developed, t h i s one i s l e a s t s u b j e c t to v a r i a t i o n s between animals i n the s i z e of f o l l i c l e s and corpora 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 of s t r u c t u r e s w i t h i n females than between them. I n e s t a b l i s h i n g temporal r e l a t i o n s h i p s between the f i r s t and second c y c l e s , the growth r a t e s of f o l -l i c l e s and corpora l u t e a were assumed to be e q u i v a l e n t . There i s general agreement t h a t the e a r l y growth of corpora 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 the same w i t h i n species t h a t range i n s i z e from the mouse to the elephant (Perry and Rowlands, 1962). G e n e r a l l y , the e a r l y development of the corpus luteum of pregnancy i n deer i s s i m i l a r , i n both r a t e of growth and t i s s u e changes, to the c y c l i c corpora l u t e a of the p i g (Corner, 1921), goat ( H a r r i s o n , 194.8b), sheep (Robinson, 1959), and the cow (Hammond, 1927). In deer,the corpus luteum of pregnancy grows r a p i d l y i n the f i r s t 4- or 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 to 20 days. The above p a t t e r n of growth i s t y p i c a l of the corpora l u t e a of. pregnancy and non-pregnancy i n many species. 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 mid-pregnancy, and e s p e c i a l l y the sudden increase i n s i z e i n the l a t e p r e n a t a l months. The corpora l u t e a of most a r t i o d a c t y l species a t t a i n maximum s i z e w i t h i n the f i r s t month and remain 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 occurs (Brambell, 1956; H a r r i s o n , 1962). However, c e l l shrinkage (and perhaps cor-pus luteum shrinkage) has been reported by the 24-th day i n the monkey (Corner, 194-5), and between the 35th and the 4-5th day i n the goat ( H a r r i s o n , 194-8a). Corpora l u t e a remain a t about the same s i z e throughout g e s t a t i o n i n the c a r i b o u (McEwan, 1962), cow (Corner, 1921; Hammond, 1927), and the A f r i c a n elephant (Buss and Smith, 1966). Trauger and Haugen (1965) stated t h a t "the average s i z e of the corpora l u t e a apparently i n c r e a s e d d u r i n g pregnancy" i n the w h i t e - t a i l e d deer. I n the sp e c i e s , the l u t e a l c e l l s en-larged 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 to p a r t u r i t i o n (Gibson, 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. - Tissue changes d u r i n g e a r l y development of the corpus luteum were remarkably s i m i l a r to d e s c r i p t i o n f o r the p i g (Corner, 1919), monkey (Corner, 169 194-5), horse (Harrison, 194-6), man (Corner, 1955) and, especially, the goat (Harrison, 194-8). My observation that hemorrhage of large amounts of blood occurs several hours after ovulation coincides with Corner's (194-6) inte 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 at f i r s t ovulation do not develop into corpora hemorrhagica. In th i s respect they are similar to most ruptured f o l l i c l e s i n man (White et a l . , 1951). The amount of blood associated with the young corpus luteum i s highly variably among mammals (Harrison, 194-8a, b). The r e l a t i v e contributions of the thecal and granulosal layers to the l u t e a l tissue, i s also variable among mammals. In deer, both layers contribute c e l l s but the granulosa layer appears to contribute the bulk of the l u t e a l tissue, as i t does i n most artiodactyls (Harrison, 194-8a). Nevertheless, i n deer, the thecal layer apparently contributes a l l of the l u t e a l tissue i n f i r s t cycle f o l l i c l e s i n which the granulosa i s degen-erate and i s extruded at rupture. Also, considerably mitotic a c t i v i t y was noted i n the c e l l s of the theca interna located along the edges of the invaginated folds of thecal tissue. In addition, c e l l s located i n the theca externa were converted into 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 fate of the c e l l s from the respective layers could not be followed with certainty past the second or t h i r d day. In contrast to most descriptions, mitotic d i v i s i o n s occurred frequently i n developing corpora lutea. They occurred from ovulation u n t i l about the fourth day, although occasionally they were observed at l a t e r stages. Changes i n the number of d i v i s i o n s and the size of the l u t e a l 170 c e l l s provide a quantitative means of evaluating the age of corpora lutea. 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 better i n d i c a t i o n of c e l l size 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 lutea progressively decreased with time,whereas i n deer i t increased to a peak at about 1.5 days after ovulation and decreased sharply thereafter. The corpora lutea of deer are usually of the compact type, as i n the sheep, pig,and goat (Harrison, 194-Sa). However, some corpora lutea with large central lumena, similar to those of the cow (Hammond, 1927), monkey (Corner, 194-5) and man (Corner, 1956), develop after the f i r s t ovulation. These 'cystic' corpora lutea form i n f o l l i c l e s which were abnormally large at rupture. In these f o l l i c l e s , the t h i n granulosa and theca interna layers produce only a band of l u t e a l tissue around a large cavity before development ceases. Many of the very large f o l l i c l e s that produce these corpora lutea are a t r e t i c prior to rupture. The cytology of the corpus luteum of pregnancy gradually changed during gestation. Of significance, are the "conception c e l l s " which seem to s i g n i f y presence of the blastocyst i n the uterus. These c e l l s , which are most prominent 5 to 10 days after conception, have been noted i n young corpora lutea i n the pregnant cat (Dawson, 194-6), goat (Harrison, 1948b) and the pig (Corner, 1919). As noted by Gibson (1957) i n the white-tailed deer, there was a gradual increase i n maximum c e l l size during gestation. She also noted the large vacuoles i n the c e l l s , l a t e In gestation. Why there i s c e l l u l a r enlargement just prior to b i r t h , i s not known. The significance 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 nuclei may be thecal c e l l s . Hyper-chromasia i n large c e l l s seemed to be associated with c e l l death. Accessory corpora lutea occur i n most species of mammals (Brambell, 171 1956; Harrison, 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 ovulation. Because they r a r e l y overlap i n size with primary corpora lutea, they are a potential source of error i n the determination of ovulation rate from counts of primary corpora lutea. In deer, few errors w i l l be made i f corpora lutea smaller than one-quarter the size of the largest corpus luteum i n the same pair of ovaries are c l a s s i f i e d as accessory. Accessory corpora lutea i n many species are e a s i l y confused with primary ones. In elk, they originate i n f o l l i c l e s which rupture i n the pregnant female (Halazon and Buechner, 1957; Morrison, i960). Pimlott (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 less than ha l f the size of the larger corpus luteum i n the same pair of ovaries. However, his c r i t e r i o n of size was diameter, whereas I used volume. The accessory corpora lutea which occur i n red deer (Cervus elaphus) are also much smaller than the primary ones (Douglas, 1966). McEwan (1962) suggested that, i n caribou, accessory corpora lutea developed i n f o l l i c l e s which ruptured out of phase. His description bf ovulation s i t e s suggests that some accessory corpora lutea may develop i n the regressing corpus luteum of the f i r s t ovulatory cycle. I have termed these Type 3 accessory corpora lutea. Gibson (1957) termed them "corpora lutea aberrans" i n white-tailed deer. This term was used o r i g i n a l l y by Corner (1945) to describe a persistent corpus luteum which formed from a corpus luteum of estrus In the monkey. Although represented by a rupture s i t e on the surface of the ovary, Type 3 accessory corpora lutea are small and cannot be confused with primary corpora lutea i n black-ta 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 after the f i r s t ovulation than after the second, and forms i n f o l l i c l e s that are out of phase or are too small to rupture. 172 When formed i n large f o l l i c l e s these accessory corpora lutea 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 lutea of pregnancy can be used to estimate, accurately,the b i r t h rate 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 rates obtained from counts of scars derived from corpora lutea of pregnancy must be reduced by the amount of intrauterine mortality. 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 gestational period. This technique i s valuable because the hunting season of many deer herds occurs prior to or during the breeding season, and i t i s during the hunting season that large 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 for at le a s t two successive years. That i s , the number of active corpora lutea 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 for the l i f e of the doe. I t i s therefore possible to estimate the t o t a l past fawn production of any f e -male. An estimate of viable fawn production i s obtained by adjusting the rate by the amount of f e t a l mortality. In addition, the v a r i a b i l i t y i n f e r t i l i t y between ind i v i d u a l s can be r e a d i l y ascertained. Furthermore, a regression of scar numbers with age produces a curve that describes the long term fetus 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 at the previous conception. The slope, 1.885, i s the rate at which scars are produced. 173 Counts of pregnancy scars 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 long-term, e c o l o g i c a l p r o d u c t i v i t y of a popu-l a t i o n to a t h e o r e t i c a l curve 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 species. I t a l s o has a p p l i c a t i o n i n comparisons of the l o n g 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 only technique a p p l i c a -b l e to deer by which past 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 deterimed. P r i o r to t h i s study, i t was not known t h a t scars derived from corpora l u t e a of 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 female deer. Although Gibson counted as many as 18 pigmented scars i n the o v a r i e s of w h i t e - t a i l e d deer, some old females contained few s c a r s , suggesting t h a t pregnancy scars d i d not p e r s i s t i n these. Simkin (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 the number of pigmented scars i n s t a i n e d s e c t i o n s of moose o v a r i e s . Pregnancy scars p e r s i s t f o r many years i n the cetacea (Mackintosh, 194-6) and the cow (McNutt, 1924). Scars may p e r s i s t f o r many years i n numerous other species but few people 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 Gibson (1957) t h a t , i n deer, only the pregnancy scars of the previous breeding season are d i s t i n c t from older s c a r s . I n some females there i s a d e f i n i t e sequence of scars of d i f f e r e n t ages,but i n others the penultimate s e t of scars cannot be d i s t i n g u i s h e d from older s c a r s . With age there i s a p r o g r e s s i v e decrease i n : 1) the e x t e r n a l capsule of connective t i s s u e f i b e r s , 2) the amount of pigment, 3) the amount of h y a l i n m a t e r i a l , 4-) the number of s m a l l , c e n t r a l l y - l o c a t e d blood vessels,-5) the s t a i n a b i l i t y of the blood v e s s e l s which p e r s i s t . The only t i s s u e s t h a t remain i n old scars are the t h i n - w a l l e d blood v e s s e l s . I t i s probably e l a s t i n i n the w a l l s of these v e s s e l s which makes them so p e r s i s t a n t . E l a s t i n i s probably the most r e s i s t a n t of a l l the body p r o t e i n s to chemical change 174 (Ham and Leeson, 1961). There appear to be some bas 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 deer and b l a c k - t a i l e d deer i n the r a t e of corpus luteum degeneration. Gibson (1957) noted t h a t pigment-bearing 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 o u t l i n e s and n u c l e i f o r a t l e a s t one and one-half years a f t e r p a r t u r i t i o n i n white-t a i l e d deer. I n a d d i t i o n , l a r g e vacuolated c e l l s were present in. the corpora l u t e a 2 weeks a f t e r p a r t u r i t i o n , and scars aged 6 months were composed l a r g e l y of pigmented l u t e a l c e l l s . 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 deer. Few l u t e a l c e l l s remain a few days a f t e r b i r t h and none are present 5 months post-partum. G o l l e y (1954-) reported t h a t i n b l a c k - t a i l e d deer a l l l u t e a l c e l l s had disappeared from the pregnancy scars w i t h i n 2 months. In t h i s study the corpora l u t e a of only one doe contained l a r g e , granulated c e l l s . The cause of t h i s retarded r e g r e s s i o n was probably some m a t e r i a l i n the u t e r u s , perhaps remnants of a r e s o r t i n g f e t u s . Corpora l u t e a p e r s i s t i n mule deer which have resorbed embryos or have aborted (Robinette et a l . , 1955). There are a l s o d i f f e r e n c e s between the two species i n the r a t e of r e g r e s s i o n of corpora l u t e a of non-pregnancy. One 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 i n b l a c k - t a i l e d deer, the s t r u c t u r e s are composed of a small 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 a few s m a l l , t h i c k - w a l l e d blood v e s s e l s . I n w h i t e - t a i l e d deer, s t r u c t u r e s of the 3 same age are 10 times l a r g e r (O.4O mm ) and c o n t a i n pigmented l u t e a l c e l l s s c a t t e r e d throughout h y a l i n connective t i s s u e . I cannot agree w i t h Brambell (1956) t h a t the r e t r o g r e s s i v e processes are e s s e n t i a l l y s i m i l a r whether the corpora l u t e a are of o v u l a t i o n or pregnancy. He noted, however, th a t d e t a i l e d systematic accounts of corpus luteum r e g r e s s i o n are not numerous. The corpora l u t e a of o v u l a t i o n or non-pregnancy re g r e s s r a p i d l y i n deer because no outer connective t i s s u e capsule i s produced and the blood v e s s e l s are few and t h i n - w a l l e d . The 175 dominant h y a l i n connective t i s s u e i s resorbed q u i t e r a p i d l y . Some y e l l o w pigment i s produced around the p e r i p h e r y of degenerating c e l l s but i t i s not i n the form of granules as i s the pigment produced i n degenerating corpora l u t e a of pregnancy. C l e a r l y , the type of scar which 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 of the corpus luteum. The l a r g e r the s t r u c t u r e , the greater the number, s i z e , and t h i c k n e s s of the supporting blood v e s s e l s , and the greater the d e p o s i t i o n of pigment upon r e g r e s s i o n . Thus, scars b e l i e v e d to be as s o c i a t e d w i t h embryonic m o r t a l i t y are in 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 scars derived from corpora l u t e a of non-pregnancy and those of pregnancy. Scars d e r i v e d from accessory corpora l u t e a have a l l the c h a r a c t e r -i s t i c s of the corresponding scars from the primary corpora l u t e a w i t h which they were a s s o c i a t e d . Thus, t h e i r age i s estimated s o l e l y on the b a s i s of 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 they vary markedly i n s i z e . Most of them are of an i n s i g n i f i c a n t s i z e and could never be confused w i t h scars derived from primary corpora l u t e a . However, scars are probably r o u g h l y propor-t i o n a t e i n s i z e to the l u t e a l mass from which they are derived and i t was noted e a r l i e r t h a t Type 3 accessory corpora l u t e a o c c a s i o n a l l y overlapped i n s i z e w i t h primary corpora l u t e a . Any scar l e s s than o n e - t h i r d the s i z e of the l a r g e r scar of the same age i n the same p a i r of ov a r i e s was considered to be der i v e d from an accessory corpus luteum. 7 . 8 The Breeding Season. The t i m i n g and v a r i a b i l i t y of conceptions and b i r t h s i s important, f o r i t r e l a t e s to n a t u r a l s e l e c t i o n and to 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, seasonal breeders produce t h e i r young a t a time of year d u r i n g which environmental f a c t o r s are optimal f o r the s u r v i v a l of the young and, to a l e s s e r e x tent, the a d u l t s . As pointed out by S a d l e i r (1969), there i s 176 l i t t l e information on the r e l a t i v e effects of environmental stresses on sur-v i v a l at various stages of the reproductive cycle. On the average, the breeding season of white-tailed and mule deer i s e a r l i e r and more condensed at higher latitudes than at lower latitudes (Einarson, 1956; Severinghaus and Cheaturn, 1956). These observations are v a l i d for the Columbian black-tailed deer. Most conceptions occur i n the l a s t 2 weeks of November at Northwest Bay (49° 10' N latitude) 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 for 5 populations of the subspecies varied from November 15 to December 25 (Bischoff, 1957). Ultimately, the timing of the breeding season i s linked unquestionably to bioclimatic factors,and l a t i t u d e i s only a crude measure of regional climatic differences. The l a t e r and more variable breeding season i n southern deer, usually can be explained by these bioclimatic factors. Usually, the fawns are produced shortly after the s t a r t of the season of accelerated plant growth, which occurs as early a s . A p r i l i n some locations and as l a t e as July i n others. In northern l a t i t u d e s , there i s , or has been, considerable selec-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. In the present study only two of 49 does conceived l a t e (early i n January), although three others were s t i l l c y cling i n February and March. I t i s u n l i k e l y that they conceived. 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 within a period of 15 days (Brown, 1961). Breeding seasons of similar length occur i n mule deer (Chattin, 1948; Robinette and Gashwiler, 1950; Lassen et a l . , 1952) and white-tailed deer (Cheatum and Morton, 1946; Shaw and McLaughlin, 1951; Ransom, 1966b). The fawns of l a t e breeders probably do not survive except i n years when environmental conditions are nearly optimal. A fawn born i n 177 mid-July i n the Cedar Creek enclosure i n Oregon soon died (Hines, 1968). S i m i l a r l y , a fawn estimated to have been born i n August a t Northwest Bay was abandoned and near death when found i n November. The s m a l l amount of v a r i a b i l i t y i n the t i m i n g of f i v e successive breeding season (from 1963 to 1967) at Northwest Bay i n d i c a t e s t h a t the proximal cause of the breeding season i s some environmental f a c t o r t h a t i s s e a s o n a l l y constant (such as photoperiod), or the season i s c o n t r o l l e d by endogenous rhythms w i t h which environmental f a c t o r s may i n t e r a c t . The s y n c h r o n i z a t i o n of most r e p r o d u c t i v e c y c l e s can be explained by e x t e r n a l f a c t o r s . Although the t i m i n g of the breeding season may be c o n t r o l l e d l a r g e l y by photoperiod, b i o c l i m a t i c f a c t o r s may modify the s y n c h r o n i z a t i o n . I n 1967 the breeding season was l a t e . The e a r l y p o r t i o n of the s p r i n g (March and A p r i l ) of 1967 was colder and wetter than normal, whereas the summer (June to September) was abnormally warm and dry. The c l i m a t e may have a f f e c t e d the animals d i r e c t l y , or i n d i r e c t l y through changes i n the ve g e t a t i o n . Captive, female w h i t e - t a i l e d deer on a low l e v e l of n u t r i t i o n con-ceived about 10 days l a t e r than wel l - n o u r i s h e d females (Verme, 1965)-Some y e a r l i n g s are l a t e breeders because they are j u s t . a t t a i n i n g puberty and presumably have marginal endocrine st i m u l u s . Y e a r l i n g s express a l l degrees of s e x u a l i t y , from a nonovulatory s t a t e , to f e r t i l i t y equal to th a t of a d u l t s . G e n e r a l l y , s m a l l y e a r l i n g s are anovular, l a t e breeders, or f a i l to conceive, even although they ovulate. P o s s i b l y , some y e a r l i n g s undergo s e v e r a l o v u l a t o r y c y c l e s without e x p e r i e n c i n g a true e s t r u s . Year-l i n g s i n an enclosed herd of b l a c k - t a i l e d deer i n Oregon were the l a s t to bear fawns each year ( 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 varies from 24-to 29 days (Cheatum and Morton, 194-2; Cowan, 1956; Haugen, 1959). In 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 free-ranging deer. Therefore, i t was s t a r t l i n g to find that the i n t e r v a l between f i r s t and second ovulation was only 8 or 9 days. I have a tentative explana-t i o n for the discrepancy between this new f i n d i n g , and the r e s u l t s of pre-vious workers. . Female deer i n the study area apparently never conceive on thei r f i r s t ovulatory cycle but most conceive at second ovulation. Prio r to f i r s t ovulation, sperm could not be detected i n the vaginal smears of females whose f o l l i c l e s exibited 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 ovulation, i t i s possible that a large percentage of females may not come in t o f u l l estrus at 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, i960), goats (Fraser, 1968), and horses (Berliner, 1959). There i s also some evidence that s i l e n t heats occur i n free-ranging Uganda kob (Buechner et a l . , 1966), elk (Morrison, i960), moose (Simkin, 1965), African elephants (Perry, 1953), hedgehogs,and bankvoles (Perry and Rowlands, 1962). In sheep, s i l e n t heats are common at the beginning and at the end of the breeding season (Asdell, 1964). In c a t t l e , s i l e n t heats are common i n the early 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 cycle i n deer do not develop into viable 179 embryos. I n animals w i t h a short breeding season, such as deer, each season may be l i k e n e d to the reattainment of puberty, i n t h a t each female g r a d u a l l y comes i n t o f u l l r e p r o d u c t i v e performance. Studies on the seasonal changes i n the t o t a l gonadotropic content of p i t u i t a r y glands from mule deer i n d i c a t e there i s a gradual i n c r e a s e i n hormone l e v e l s i n the gland d u r i n g the summer and f a l l ( G r e i s e r and Browman, 1956). In animals e x p e r i e n c i n g s i l e n t heat, a p p a r e n t l y there i s s u f f i c i e n t gonadotropic s t i m u l a t i o n to evoke o v u l a t i o n and growth of the corpus luteum but i n s u f f i c i e n t s t i m u l a t i o n to e l i c i t estrous behavior. However, some of the w i l d deer experience e s t r u s at f i r s t o v u l a t i o n and t h e i r ova are f e r t i l i z e d . N e vertheless, these soon f a i l and the female undergoes a second o v u l a t i o n , which 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 estrous c y c l e s of two or more le n g t h s . The short 8 to 9 day f i r s t ( o v u l a t o r y ) c y c l e may be f o l l o w e d by c y c l e s of 24 to 28 days. Short (8 to 9-day) f o l l i c u l a r 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 of 8 to 9 days i s p o s s i b l e . I f conception does not occur a t second o v u l a t i o n , the female may continue i n 8 to 9-day f o l l i c u l a r c y c l e s w i t h e s t r u s o c c u r r i n g every second, t h i r d , or f o u r t h f o l l i c u l a r c y c l e . O v u l a t i o n may, or may not, occur w i t h each of these f o l l i c u l a r c y c l e s . This hypothesis e x p l a i n s c y c l e s of 8, and 24 to 28 days. One would a l s o expect c y c l e s of 16 days. Some other species of mammals experience short c y c l e s and c y c l e s of d i f f e r e n t l e n g t h s . I n the Uganda kob, Buechner et a l . (1966) concluded t h a t the l e n g t h of the estrous c y c l e was 20 to 26 days. However, t h e i r data c l e a r l y I n d i c a t e some estrous 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 ) every 12 to 13 days, and s h o r t e r c y c l e s of 5 to 7 days, i n which some estrous ac-t i v i t y was present ( v i s i t s to the t e r r i t o r i a l breeding grounds). Most of these s h o r t e r , sub-estrous c y c l e s were confined to females a t t a i n i n g puberty. 180 Remarkably, a few short cycles 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 functional l i f e of the corpus luteum. I t i s well documented that pro-gesterone prevents ovulation (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 cycle" of 8 days, which i s modi-f i e d by the functional l i f e of the corpus luteum (Fig. 27). Short cycles occur i f the functional l i f e - s p a n of the corpus luteum i s less than that of the f o l l i c l e . Long cycles apparently occur i f the corpus luteum i s active for a period s u f f i c i e n t to block the ovulation of two cycles of f o l l i c l e s . Presumably, ovulation and estrous occur at the end of the t h i r d f o l l i c u l a r cycle i n captive females because the corpus luteum i s no longer functional. Theoretically, intermediate-length cycles should occur i f the corpus luteum i s functional for more than 8 days but less than perhaps 13 to 15 days. Con-ceivably, a deer could experience cycles of a l l three lengths. I t has been shown that, i n seasonal breeders, the i n t e n s i t y and duration of estrus i n -creases with each cycle as the animal comes into f u l l reproductive condi-t i o n (West, 1968; Fraser, 1 9 6 9 ) -The i n t e r v a l between the f i r s t and second ovulatory cycle i n wild 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 ovulation and these animals, e s p e c i a l l y the three yearlings, may have started to cycle l a t e i n the season. Neverthe-l e s s , one adult apparently underwent two or probably three cycles (subsequent to the second) i n 36 days. A yearling doe probably cycled twice (3rd and 4-th cycle) i n a minimum period of 4-6 days. These scanty observations indicate that the length of the t h i r d and fourth cycles i n deer probably l i e s somewhere between 12 and 23 days. Fig. 27. Schematic representation of reproductive cycles i n deer. The bars represent the functional l i f e span of corpora lutea (CL) and f o l l i c l e s ( F o i l ) . 1. Short Cycle ( f i r s t cycle i n wild; subsequent cycle i n wild?; f i r s t cycle i n some captive deer?) j 5-6 days | 8 days 0 0 E or SE E NP P CL F o i l 0 E P Long Cycle (captive deer; second and subsequent cycle i n wild deer?) U - 1i 0 E P CL F o i l Intermediate-Length Cycle (the o r e t i c a l ; second and subsequent cycles i n wild?) 0 NP 8 8 CL F o i l 0 E P "0 = Ovulation SE - Si l e n t estrus E = Estrus NP = Pregnancy does not occur P = Pregnancy may 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 prior to the f i r s t d e f i n i t e estrus i n a captive yearling doe. This a c t i v i t y may have indicated 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 lutea 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 factor. Conceptions i n penned white-tailed deer on a high l e v e l of n u t r i t i o n were distr i b u t e d bimodally with an i n t e r v a l of about 30 days between the modes. Most con-ceptions i n females on low-level n u t r i t i o n occurred between the mode dates of the high-plane females (Verme, 1965). Possibly 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 pseudo-pregnant cycles of small mammals. In confined deer, some stimulus may cause production of large, long-lived corpora lutea. I t i s well known that female mice housed i n groups can experience long pseudopregnant cycles, even i n the absence of mating (the LeeBoot eff e c t , Parkes, 1962). 7. 10 F e r t i l i t y And Productivity Of Northwest Bay Deer Relative To Other  Populations. The Northwest Bay population has the highest f e r t i l i t y of a l l investigated populations of 0. h. columbianus. The pregnancy rate was 85% • i n yearlings, 92% i n females 2.5 to 3.0 years old, and 97% i n females older than 3 years. The above rate i n adults was duplicated by herds i n C a l i f o r n i a (Bischoff, 1958) but lower rates were recorded i n Washington State (Brown, 1961). Fetus production by females of Northwest Bay was higher than i n the 183 other p o p u l a t i o n s . For example, i n 1963-64, y e a r l i n g s averaged 0.90 f e t u s e s per doe. The corresponding r a t e s i n C a l i f o r n i a and Washington were 0.36 and O.45 r e s p e c t i v e l y (Bischoff,.1958; Brown, 1961). The f e t a l r a t e i n a l l a d u l t s of Northwest Bay, 1.68 per female, was a l s o higher than the 1.62 recorded i n C a l i f o r n i a herds ( B i s c h o f f , 1958) and the 1.32 l i s t e d f o r the Washington p o p u l a t i o n (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 herds 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 production of f e t u s e s under the e x i s t i n g environmental c o n d i t i o n s . Adult Columbian b l a c k - t a i l e d deer i n the study r e g i o n , i n Washington S t a t e , and 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 order, the r a t e s were 1.67, 1.57 (Brown, 1961) and 1.70 ( B i s c h o f f , 1958). The o v u l a t i o n r a t e i n 45 a d u l t females taken throughout g e s t a t i o n (1.67 corpora l u t e a per female) was equal to 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 breeding season. Pregnancy, f e t a l , and o v u l a t i o n r a t e s of some herds of mule and w h i t e - t a i l e d deer are s i m i l a r to the r a t e s i n the study herd, but i n others the r a t e s are much higher (Robinette et a l . , 1955; Julander et a l . , 1961 ; Banasiak, 1964; Hesselton et a l . , 1965; Ransom, 1967b). I n mule deer, and 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 deer, puberty and f u l l r e p r o d u c t i v e output i s a t t a i n e d much e a r l i e r than i n Columbian b l a c k - t a i l e d deer. 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 process i s one measure of the f e r -t i l i t y of a p o p u l a t i o n . In deer, the e f f i c i e n c y of the p o s t - i m p l a n t a t i o n 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 . Only 3.3% of the f e t u s e s examined i n t h i s study were moribund, which i s s i m i l a r to the 2 .6% recorded by Brown (1961). These estimates are c o n s e r v a t i v e because 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 throughout g e s t a t i o n . However, ob-s e r v a t i o n s on the extent of i n t r a u t e r i n e m o r t a l i t y i n deer have shown th a t 184 only a small p r o p o r t i o n of i t occurs i n the l a s t t h r ee-quarters of the ges-t a t i o n a l period (Taber, 1953; Robinette et a l . , 1955; Brown, 1961; Plesselton et a l . , 1965; Ransom, 1967). None of the 243 f e t u s e s examined by Taber (1953) and 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 1000 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 , 1958; Robinette et a l . , 1955; N e l l i s , 1968). 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 p o p u l a t i o n s of w h i t e - t a i l e d deer (Teer et a l . , 1965; Ransom, 1967). I n the above d i s c u s s i o n , only 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 considered. I n most species,the g r e a t e s t l o s s of ova occurs p r i o r to the stage a t which embryos are m a c r o s c o p i c a l l y v i s i b l e . In deer, t h i s l o s s may be estimated by comparing the number of f e t u s e s to the number of corpora l u t e a . 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 are r a r e , monozygotic twinning i s r a r e , and t h a t a l l primary corpora l u t e a present i n the o v a r i e s are 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 at the o v u l a t i o n i n which conception occurred. From my observations, and the observations of other i n v e s t i g a t o r s of deer reproduction, i t i s concluded t h a t the above assumptions are v a l i d . No l a r g e p o l y o v u l a r f o l l i c l e s occurred i n more than 4OO p a i r s of o v a r i e s t h a t I examined. Ne i t h e r was there any evidence of monozygotic twinning. G o l l e y 0 954) reported one i n s t a n c e i n which one corpus luteum was a s s o c i a t e d w i t h 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. I n a sample of 774 v i a b l e f e t u s e s i n Utah mule deer, monozygotic twinning was suspected i n f o u r i n s t a n c e s (Robinette et a l . , 1955). I n w h i t e - t a i l e d deer, Hesselton (1967) has recorded three cases i n which the number of corpora l u t e a was one l e s s than the number of f e t u s e s . Because the f e t u s e s were of d i f f e r e n t sex, he concluded t h a t the supernumary f e t u s e s arose from p o l y o v u l a r f o l l i c l e s . Teer et a l . , (1965) observed fewer corpora l u t e a than embryos i n 2.5% of 525 pregnant females. N e v e r t h e l e s s , both events are unquestionably r a r e and may be ignored i n comparisons between the numbers of corpora l u t e a and f e t u s e s . 185 The t h i r d assumption i s v a l i d i f accessory corpora lutea are excluded from the counts of corpora lutea. In deer, this separation can be made on the basis of size. Remarkably, i n t h i s study, the number of corpora lutea larger than 30 mm3 (92), i n females pregnant when collected, equalled the number of fetuses. Because three of the fetuses were moribund, each corpus luteum was represented on the average by 0.97 viable fetuses. In addition, one of the non-pregnant females (a yearling) had been temporarily pregnant e a r l i e r i n the season, as revealed by a large fibrous scar i n i t s ovaries. Therefore, the minimum loss of embryos, after approximately the implantation stage,was 4.3% (4-/93). In late November or December c o l l e c t i o n s , the three females that didn't become pregnant would have contained four corpora lutea. 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 viable fetuses. Therefore, the minimum t o t a l loss of ova ivras 8.3%. The above calculations, and others s p e c i f i c to age-classes, are summarized i n Appendix 8. The ov e r a l l loss of ova i n females of Northwest Bay i s lower than the loss i n other populations of deer. In pregnant females,, the number of corpora lutea usually exceeds the t o t a l number of fetuses by 3% to 12% (Taber, 1953; Robinette et a l . , 1955; Bischoff, 1958; Taber and Dasmann, 1958; Brown, 1961; Teer et a l . , 1965; N e l l i s , 1968). The loss of embryos after 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 calculated difference between the ovulation rate i n samples from the breeding season and the maximum production of viable 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 implanta-t i o n stage) to the number of corpora lutea. The pre-implantation loss i n t h i s study was about 4-%. Some of i t represents loss of ova that were never f e r -t i l i z e d . The minimum pre-implantation loss 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 . , 1955). 7. 11 The Effect 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 at Northwest Bay gradually increase with age from 1.5 years to 5-5-6.5- years and thereafter decreased s l i g h t l y . A similar trend was noted i n white-tailed deer by Severinghaus and Cheatum (1956). Nevertheless, females apparently reproduce to f u l l capacity almost u n t i l they become decrepit from old age. Semi-tame deer aged 17 to 21 years have been known to produce at or near capacity 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 rates, fetus production, and ovulation rates i n old females have been noted i n Columbian black-tailed 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 loss 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 un-productive on poor range. The age composition of the female component of the population has an important bearing on productivity. The rate of increase of a population (excluding mortality and dispersion) i s profoundly influenced by the time at which puberty and f u l l reproductive capacity i s attained. In t h i s r e s -pect, the ecological or realized n a t a l i t y of the Northwest Bay population i s considerably less than the p a r t i a l reproductive potential of the species, as exibited by females kept i n c a p t i v i t y under somewhat less than optimum cond-tions. 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 black-tailed deer are capable of producing one fawn at about one year of age (Rampont, 1926; Shantz, 194-3, quoted i n Brown, 1961; Cowan and Wood, 1955) and thereafter twins are usually produced. Excluding fawns, the above rate of fetus production would y i e l d a productivity 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 productivity was attained even less 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; Bischoff, 1958). Puberty, and f u l l reproductive capacity (for a p a r t i c u l a r environment) i s reached more quickly 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 white-tailed deer (Morton and Cheatum, 194-6, 194-6, G i l l , 1956; Robb, 1959; Haugen and Trauger, 1962; Banasiak, 1964; Hesselton et a l . , 1965; Nixon, 1965; Ransom, 1967). Studies of both natural and confined populations of deer have shown that n u t r i t i o n i s a key factor i n the rate at which females a t t a i n puberty and f u l l reproductive capacity (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 at which puberty and f u l l reproductive output i s attained i s undoubtedly dependent on the rate of growth, the subject of the next topic. 7. 12 The Effect Of Size On F e r t i l i t y . 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 effects of s i z e , within age classes, 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 indicate that within an age class at Northwest Bay, large 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 old, e s p e c i a l l y i n yearlings. Those less than about 2/3 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 rate of adults. Several investigators have shown that the f e r t i l i t y of deer i n difference herds, or within one herd over a period of time, correlates w e l l with differences i n physical 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 rate i n yearling mule deer dying from malnutrition 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 size and productivity of white-tailed deer i n several r e -gions of West V i r g i n i a . The per cent difference i n weight between two herds of mule deer was exceeded by the per cent difference i n the ovulation rate (Julander et a l . , 1961). Bansiak (1964) ranked white-tailed deer from f i v e regions i n Maine on the basis of three size measurements and found that the ovulation rate correlated quite w e l l with the ranks. As the size of deer i n the Seneca Army Depot increased, the productivity of the herd also i n -creased (Hesselton et a l . , 1965). Nixon (1956) also found a good c o r r e l a t i o n between productivity and the size 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) after a study of the small white-tailed deer of the Central Mineral Basin i n Texas. The productivity of subspecies of deer that are not genetically isolated apparently correlates well with multiple physical varients over extensive geographical regions. In a l l of the above studies on natural populations, differences i n size were attributed to n u t r i t i o n a l differences. Growth rate differences between and within species, or even within a population, have an important bearing on productivity. Bandy (1965) showed that, within a subspecies, the rate of growth (as measured by t o t a l weight) was retarded on a low plane of n u t r i t i o n , thereby increasing the age 189 at which the ultimate maximum size was attained. Murphy and Coates (1966) noted that growth of fawns was retarded when they were fed low protein di e t s . When fawns of a r e l a t i v e l y small and unproductive white-tailed deer herd ( i n the Central Adirondacks, New York) were transferred to corrals i n a less harsh environment, they grew larger and became much more productive than thei 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 size and productivity of young-deer during the course of t h i s study i s i n accord with the above r e s u l t s . I t i s hypothesized that the productivity of a deer population could be e s t i -mated quite accurately from size measurements taken i n November - i f the exact age of each female was determined. I t would be necessary to establish the r e l a t i o n s h i p between the size variable and the reproductive rate, within each age-class (older age-classes could be grouped). Large samples would be required to establish accurate re l a t i o n s h i p s , however, a sta r t could be made from information obtained i n t h i s study. 190 8. SUMMARY Ovarian changes, p a t t e r n s of r e p r o d u c t i o n , and a g e - s p e c i f i c pro-d u c t 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 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 examination of o v a r i e s . The o v a r i e s of 444 females, r a n g i n g i n age from 64 days post-conception to 19-5 y e a r s , were sectioned a t i n t e r v a l s of 90 p. or 190 p; another 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 sub-sequently i n h i s t o l o g i c a l s e c t i o n s . Reproductive f u n c t i o n was r e l a t e d to s p e c i f i c ages as determined from stained s e c t i o n s of 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 important f i n d i n g s . 1. Oogenesis v i r t u a l l y ceases i n the f e t a l ovary 30 days before b i r t h . Ovaries i n a f e t u s from a c a p t i v e doe were l a r g e r and f u r t h e r developed than those i n f e t u s e s of the same age from w i l d females. 2. Well-defined f o l l i c u l a r c y c l e s occur throughout most of the year, i n c l u d -i n g the period of g e s t a t i o n . They occur at 8-day i n t e r v a l s d u r i n g the breeding season and t h e i r growth seems to be independent of l u t e a l func-t i o n . The average s i z e of the l a r g e s t f o l l i c l e i n pregnant females i n -creased s i g n i f i c a n t l y from 21 mm3 i n February and March to 45 mm3 i n May and June. 3 . 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 stage of maturation; 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 t h a t 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 ; these and smaller ones occurred i n 47% of the females a f t e r f i r s t o v u l a t i o n . 4 . Apparently, pregnancy never ensues from the f i r s t o v u l a t i o n of the season i n November. Even f e r t i l i z e d ova soon cease development. The. presence of sperm on only two of s i x ova suggests.that ' s i l e n t heats' occur frequent-l y a t f i r s t o v u l a t i o n . 191 5. R e l a t i v e l y s m all corpora l u t e a (45 mm ) develop a f t e r the f i r s t o v u l a t i o n , but they cease growth a f t e r 2-3 days, begin to degenerate a f t e r about 5 or 6 days, and r a p i d l y r e gress a f t e r second o v u l a t i o n . 6. Corpora 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 days, r a p i d l y grow to 100 mm . N i n e t y - s i x per cent of the females con-ceived a t second o v u l a t i o n . 7. The breeding season occurred at about the same time i n each of the 5 years, except a f t e r the hot, dry summer of 1967, when i t was 3-4- days l a t e . 8. 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%, of the a d u l t does ovulated f o r the f i r s t time between November 10 and November 18, and f o r the second time be-tween November 17 and November 25. On the average, y e a r l i n g s ovulated l a t e r than a d u l t s and t h e i r dates of o v u l a t i o n were more v a r i a b l e . 9. Scars of corpora l u t e a of non-pregnancy i n l a t e and non-coneeivers, i n d i c a t e t h a t females c y c l e a t l e a s t f i v e times i f pregnancy does not occur. 10. ' There were 93 f i r s t - c y c l e corpora l u t e a and 97 corpora l u t e a from the second c y c l e i n females t h a t had ovulated twice at the time of c o l l e c t i o n . Thus, f u t u r e p r oduction of f e t u s e s can be estimated i n females having ovulated only once a t the time 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 breeding season were a s c e r t a i n e d i n 12 females whose f o l l i c l e s and corpora l u t e a were dated by the cleavage stage of f e r t i l i z e d ova (shed a t second o v u l a t i o n ) . I t was assumed t h a t deer ova segment at about the same r a t e as the ova of domestic ungulates. Dates of o v u l a t i o n f o r a l l females c o l l e c t e d I n November and e a r l y December were estimated from c r i t e r i a formulated from the 12 females. 192 12. Corpora l u t e a of pregnancy contained 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 to p a r t u r i t i o n ; increased i n s i z e d u r i n g the l a s t few 3 weeks of the 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 than 30 mm corresponded i n number to the f e t u s e s i n pregnant females. 13. Only three of 92 f e t u s e s (3.4%) were d e f e c t i v e , but t h i s estimate i s cons e r v a t i v e because samples were obtained throughout g e s t a t i o n . The minimum l o s s of ova i n females t h a t became pregnant was 4.3%; i t was 8.3% i n a l l females t h a t produced corpora l u t e a d u r i n g the breeding season. 14. Three types of accessory corpora l u t e a occur i n deer: Type 1 develops i n unruptured f o l l i c l e s , Type 2 forms i n r e g r e s s i n g corpora l u t e a of non-pregnancy, and Type 3 develops i n small f o l l i c l e s t h a t r u p t u r e at or about the time of o v u l a t i o n . Any corpora l u t e a l e s s than one-quarter the volume of the l a r g e s t corpus luteum i n a p a i r of o v a r i e s , were defined as accessory corpora l u t e a . They occurred i n 36% i n pregnant females. 15. F e t a l growth curves 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 nly f e t u s e s conceived at second o v u l a t i o n s , the mean date of which was known. Then the curves were used .to determine the conception dates of two females t h a t conceived 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 estrous c y c l e s subsequent to the second, was between 12 and 23 days.. 16. Corpora l u t e a of 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 rs f o r the l i f e of the female. Thus, the long-term r e p r o d u c t i v e performance of the p o p u l a t i o n was revealed by the l i n e a r r e l a t i o n s h i p between the age of the doe and the average number of scars per doe. However, only scars up to about 8 months of age are 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 of scar 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 to the age of the doe. 193 17. Some of 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 corpora l u t e a on non-pregnancy, accessory corpora l u t e a , and scars of corpora l u t e a of pregnancy, could not 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 s e c t i o n s . 18. Reproductive r a t e s were obtained from counts of f e t u s e s , corpora l u t e a , and corpora l u t e a s c a r s . These were used, i n c o n j u n c t i o n w i t h the age d i s t r i b u t i o n , to estimate the p r o d u c t i v i t y of the p o p u l a t i o n . 19. The average number of v i a b l e f e t u s e s per doe increased p r o g r e s s i v e l y , w i t h age, from 0.91 i n y e a r l i n g s to 1.81 i n the 5-5 and 6.5 age-classes, and t h e r e a f t e r i t decreased. 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 from each age-class decreased w i t h age. One hundred females of r e p r o d u c t i v e age produce about 137 fawns. 20. 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Thesis, Univ. of B r i t i s h Columbia, Vancouver. Winters, L.. M., W. W. Green and R. E. Comstock. 1942. P r e n a t a l development of the bovine. Minnesota Agr. Exp. Sta. Tech. B u l l . No. 151. White, 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 observations on the corpus luteum of human pregnancy w i t h s p e c i a l r e f e r e n c e to corpora 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.34:55-74-White, M. 1966. P o p u l a t i o n ecology of some w h i t e - t a i l e d d e e r i n South T e x a s . Ph.D. Thesis, Purdue Univ. Wynne-Edwards, V. C. 1962. Animal d i s p e r s i o n i n r e l a t i o n to s o c i a l be-haviour. Hafner Pub. Co. Appendix 1. The e a r l y cleavage of 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 P o i n t Elapsed time (hr) from reference p o i n t to recovery a t va r i o u s c e l l stages-.-* 1 - c e l l 2 - c e l l 3-to 4 - c e l l 5-to 8 - c e l l 9-to 16-cell Morula C a t t l e * C 33 50-62 62-64 110 134 ' C a t t l e ^ EE 23-52 40-56 44-66 46-96 71-141 144 Goat 3 SheepT: C 30 30-48 60 85 98 .- 120-140 C 0-38 38-39 42 44 65-77 96 Swine? SE^ 0-51 51-66 66-72 90-110 110-114 Swine 36-54 60-69 60-90 75-96 96+ Ov u l a t i o n occurs about 24-30 hr a f t e r the s t a r t of estrus and about 10-15 hr po s t - e s t r u s i n the cow (Ref 2); about 30-36 hr a f t e r the s t a r t of estrus i n the goat ( S a l i s b u r y , 1961); about 24 hrs a f t e r the s t a r t of esfcrus i n the sheep ( A s d e l l , 1964); and about 30 hr a f t e r the s t a r t of es t r u s i n the p i g (Ref 6). Reference. 1. Winters e t a l . , 1942. 2. Hamilton and La i n g , 1964. 3. Amoroso e t a l . , 1942. 4. C l a r k , 1934. 5. Heuser and S t r e e t e r , 1929. 6. Oxenreider and Day, 1966. C c o i t u s SE s t a r t of e s t r u s EE end of 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. The volume of growing corpora l u t e a of the second o v u l a t o r y c y c l e (CL 2); the volume of degenerating corpora 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 l ) ; 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 ) ; at 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 Specimen No. Estim Age CL 2 (days) a Vol C L - A (mm-5) Vol CL 1 (mm3)b R a t i o V ol Lg s t CL 2 to the v o l Lg s t CL 1 Z24 1 7.1 27 0.3 V19 1 7.2, 6.4- 14., 12 0.5 V21 1 6.3 6.7, 5.4- 0.9 Z26 1.5 12, 11 12, 5 1.0 X61 2 18, 8.0 8.4-, 4.1 2.1 K73 2 21, 17 6.3, 5.9 3.3 Z23 2 29, 27 4-.8, 4-.-4- 6.5 V20 2.5 38, 30 5.5, 5.3 6.9 V l l 3 85/ 58 5.7, 4.2 14.9 V30 3 66 3.5 18.8 V28 4- 77, 68 3.3, 1.2 23.3 V24 5 82 2.8 29.3 V27 5+ 118, 107 0.8 14.8 a - Estimated by the cleavage stage of the ovum. b I f two present the volumes of both are given. Only i f volume i s > 10 mm3 i s i t recorded to nearest d e c i mal. Appendix 3. The t i m i n g and v a r i a b i l i t y of the breeding seasons from 1963 to 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 o v u l a t i o n . Dates of Second O v u l a t i o n from 1963 to 1967 1963 196-4 1965 1966 1967 All Does X s s_ X n 3^56 0.80 20 25.2 3.15 0.72 19 21.3 3.19 1.02 10 20.1 3.80 1.43 7 26.7 4.00 0.97 17 Adults X s s 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 The dates of f i r s t o v u l a t i o n advanced 8 days and added to the dates of second o v u l a t i o n . 1963 1964 1965 1966 1967 .All Does X s s x n 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 Adults X s s x n 23.8 5 .06 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 16 Appendix 4. A g e - s p e c i f i c pregnancy r a t e and fet u s - p r o d u c t i o n r a t e of Northwest Bay females, 1963-66. Concept Age (yr) No. No. Preg. % Preg. Healthy Fetuses Moribund Fetuses Fetuses per doe Fetuses per preg. doe Healthy Fetuses per Doe Healthy Fetuses per preg. doe -4 1 c°> vO ON 0.5 1.5 2.5 3.5 4.5 5.5 6.5 8.5 9.5 12.5 8 10 8 6 8 5 6 2 5 1 0 8 7 6 8 5 5 2 5 1 00.0 80.0 87.5 100.0 100.0 100.0 83.3 100.0 100.0 100.0 0 . 9 11 8 13 10 11 4 8 2 0 0 0 2 0 0 0 0 1 0 00.00 0.90 1.38 1.67 1.63 2.00 1.83 2.00 1.80 2.00 00.00 1.13 1.57 1.67 ' 1.63 2.00 2.20 2.00 1.80 2.00 00.00 0.90 1.38 1.33 1.63 2.00 1.83 2.00 1.60 2.00 00.00 1.13 1.57 1.33 1.63 2.00 2.20 2.00 1.60 2.00 T o t a l or Avg 51 47 92.2 76 3 -<f UA vO sO O 1 rH 2.5 4.5 1 1 1 1 100.0 100.0 2 2 0 0 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 vO vO 1 UA vO OA r-H 1.5 2.5 5.5 13.5 3 3 1 1 3 3 1 1 100.0 100.0 100.0 100.0 3 3 2 1 0 0 0 0 1.00 1.00 2.00 1.00 1.00 1.00 2.00 • 1.00 1.00 1.00 2.00 1.00 1.00 1.00 2.00 1.00 T o t a l or Avg 8 8 100.0 9 0 All years combined 1.5 2.5 3.5 A.5 5.5 6.5 8.5 9.5 12.5 13.5 13 12 6 9 6 6 2 5 1 1 11 11 6 9 6 5 2 5 1 1 84.6 91.7 100.0 100.0 100.0 83.3 100.0 100.0 100.0 100.0 12 16 8 15 12 11 4 ' 8 2 1 0 0 2 0 0 0 0 1 0 0 0.92 1.33 1.67 1.67 2.00 1.83 2.00 1.80 2.00 1.00 1.09 1.45 1.33 1.67 2.00 2.20 2.00 1.80 2.00 1.00 0.92 1.33 1.33 1.67 2.00 1.83 2.00 1.60 2.00 1.00 1.09 1.45 1.33 1.67 2.00 • 2.20 2.00 1.60 2.00 1.00 T o t a l or Avg 61 57 93.4 89 3' ro o 208 Appendix 5. The r e l a t i o n s h i p between the number of corpora l u t e a (CL) of three s i z e s and the number of f e t u s e s i n 55 pregnant does. Year Concept. No. No. of C L T o t a l Fetuses Moribund Healthy Age (yr) >5mm3 >l0mm3 >30mm3 Fetuses Per CL Fetuses Fetuses Per >30 mm3 CL>30mm3. 1.5 9 8 7 7 7 1 .00 0 1 .00 2.5 5 8 8 7 8 1 .14 0 1.14 3.5 6 11 11 11 10 0.91 2 0.73 4.5 7 11 10 10 10 1 .00 0 1 .00 5.5 5 10 10 10 10 1 .00 0 1 .00 6.5 5 11 11 11 11 1 .00 0 1 .00 8.5 2 4 4 4 A 1 .00 0 1 .00 9.5 5 9 9 9 9 1 .00 1 0.89 12.5 1 2 2 2 2 1 .00 0 1 .00 To t a l s or Avg 45 74 72 71 71 1 .00 3 0.96 i -4- ir\ vO NO ON \— 2.5 4-5 1 1 2 2 2 2 2 2 2 2 1 .00 1 .00 0 0 1 .00 1 .00 Tot a l s or Avg 2 4 4 4 4 1 .00 0 1 .00 vO I ON 1.5 3 3 3 3 3 1 .00 0 1 .00 2.5 3 3 3 3 3 1 .00 0 .1 .00 5-5 1 2 2 2 2 1 .00 0 1 .00 13.5 1 1 1 1 1 1 .00 0 1 .00 or Avg 8 9 9 9 9 1 .00 0 1.00 O O w 1.5 12 11 10 10 10 1.00 0 1.00 2.5 9 13 13 12 13 1.08 0 1 .08 3.5 6 11 11 11 10 0.91 2 0.73 4.5 8 13 12 12 12 1 .00 0 1.00 5.5 6 12 12 12 12 1 .00 0 1.00 6.5 5 11 11 11 11 1.00 0 1.00 8.5 2 4 4 A • A 1.00 0 1 .00 9.5 5 9 9 9 9 1.00 1 0.89 12.5 1 2 2 2 2 1 .00 0 1 .00 13.5 1 1 1 1 1 1.00 0 1 .00 To t a l s or Avg 55 87 85 84 84 1.00 3 0.96 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 counts of corpora l u t e a ) o f females that had ovulated i n November and early-December. (The sample s i z e i s i n parentheses) Ovulation Rate I n the Year I n d i c a t e d Age (yr) 1963 1964 1965 1966 1967 1963-67 1-5 1.08 1.17 I.4O 1.00 1.00 1.12 (13) (12) (5) 1.25 (6) (5) (41) 2.5 1.65 1.40 1.33 2.00 1.51 (17) - (5) (8) (3) (2) (35) 3.5 1.20 1.50 2.00 1.67 2.00 1-54 (5) (2) (2) (3) (1) (13) 4.5 2.00 1.75 1.33 1.50 1.00 1.50 (1) ti) (3) (2) (2) (12) 5-5 2.00 1.00 2.00 2.00 1.91 (8) (1) (1) (1) (11) 6.5 1.75 2.00 1.00 2.00 2.00 1.82 ti) (1) (1) (3) (2) (11) 7.5-10.5 2.00 1.67 2.00 2.00 1.67 1.85 (3) (3) (3) (2) (3) (13) 11.5 & + 2.00 2.00 2.00 1.50 1.90 (6) (1) (1) (2) (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 the normal f o r each month from 1963 to 1967 at the Nanaimo a i r p o r t , B r i t i s h Columbia (Annual r e p o r t s of the Dept. of A g r i c u l t u r e , Province of B r i t i s h Columbia). Temperature D e v i a t i o n (F) from the Indicated Mean f o r each Month Month J F M A M J J A S 0 N D Annual 35 38 40 46 53 58 63 62 57 49 41 38 48 63 -3 +5 +2 0 0 +1 -3 +1 +3 +2 0 +1 +1 64 +4 +1 +1 -1 -2 0 -2 -1 0 -2 -5 0 65 0 0 +1 +1 -2 +1 +1 +1 -2 +2 +2 -1 +1 66- +1 +1 +2 +1 0 -2 -2 +2 +1 -1 +1 +3 +1 67 +3 +1 -1 -2 0 +5 +1 +5 +3 +1 +1 0 +2 P r e c i p i t a t i o n D e v i a t i o n (inches) from the In 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 +0.9 +0.3 +0.7 +1.4 +0.3 +0.7 -0.5 -0.8 +4.4 -0.4 +3o0 +5.6 64 +4-1 -3.1 -1.1 -1.9 -1.0 +0.3 +0.4 -0.3 -H0.3 -2.7 -2.0 -1.2 -8.2 65 -1-5 +0.1 -2.9 -0.4 -0.2 -0.8 -0.2 +1.4 -1.0 +1.3 +0.1 +4-0 -0.3 66 ' +2.5 -2.6 +4.0 -1.1 -0.5 +0.2 +0.2 -1.0 +0.1 -0.7 +1.1 +1.3 -3.4 67 +1.8 -2.2 +3.8 +0.9 -0.2 -1.1 -0.2 -1.2 +0.1 +3.4 -4-0 +1.9 +3.0 * long-term average. Appendix 8 . The l o s s of ova a t three stages of the r e p r o d u c t i v e c y c l e as revealed by the r a t i o between the number of fetuses and the number of corpora l u t e a . Females Pregnant at C o l l e c t i o n A l l Females Became Preg That nant A l l Females That Produced Corpora Lutea Age Clas s T o t a l Fetuses: Corpora Lutea Gain (+) or- Loss (-) of Ova (%) V i a b l e f e t u s e s : Corpora Lutea Gain (+) or l o s s (-) of ova (%) V i a b l e Fetuses: Corpora Lutea Loss of Ova 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 4-5&+ .' 54:54 (100:100) -1.9 53:54 (98:100) -1.9 53:56 (95:100) ' 5-4 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) -1.6 60:64 (94:100) 6.3 60:66 (91:100) 9.1 

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