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Control of annual reproduction in the female harbor seal, Phoca vitulina Bigg, Michael Andrew 1972

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1 I Z U ^ Control of annual reproduction i n the female harbor s e a l , Phoca v i t u l i n a . by Michael Andrew Bigg M.Sc., Univ e r s i t y of B r i t i s h Columbia, 1966 A t h e s i s submitted i n p a r t i a l f u l f i l m e n t of the requirements f o r the degree of Ph.D. i n the Department of Zoology We accept t h i s t h e s i s as conforming to the required standard The U n i v e r s i t y of B r i t i s h Columbia January, 1972 In presenting t h i s thesis i n 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 s h a l l make i t f r e e l y available for reference and study. I further agree that permission fo r extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT I t was noted recently that the annual timing of the b i r t h season varies r e g i o n a l l y i n the harbor s e a l Phoca v i t u l i n a . In any one area b i r t h s occur once a year during a 1 - 2 1/2 month perio d but between areas vary by up to 5 or 6 months with c l i n a l v a r i a t i o n s over much of the species d i s t r i b u t i o n . To explain these v a r i a t i o n s , t h i s study describes the reproduc-t i v e cycle of female harbor seals and i n v e s t i g a t e s the factors which c o n t r o l reproductive timing. The study i s divided i n t o one f i e l d p r o j e c t and two laboratory experiments. The f i e l d p r o j e c t decribes the annual pregnancy and nonpregnancy cycles and discusses the r o l e of negative s t e r o i d feedbacks from the corpus luteum and/or placenta i n annual reproductive timing. I t i s based on a macroscopic and microscopic examination of 135 adult g e n i t a l organs c o l l e c t e d i n southeastern Vancouver Island. The f i r s t laboratory experiment describes the estrous cycle of 15 captive adults based on vaginal smears and vaginal and uterine b i o p s i e s . These females were representatives of three regions, southeastern.and northeastern Vancouver Island and northern C a l i f o r n i a , and were kept under the same outdoor conditions f o r up to 4 years. From t h i s the r o l e s of n u t r i t i o n , temperature and genetic v a r i a t i o n i n reproductive timing are discussed. In the second experiment, the e f f e c t of photoperiod on the timing of estrus i s tested.and the existence of an endogenous cycle i s discussed f o r females from southeastern Vancouver Island and northern C a l i f o r n i a . . The r e s u l t s show that females i n a l l populations have a i i i monestrous c y c l e . In southeastern Vancouver Island the pregnancy cycle consists of 2 - 2 1/2 months delay of b l a s t o c y s t implantation, 8 - 8 1/2 months placentation and 6 weeks of l a c t a t i o n . The f o l l i c u l a r phase extends from s h o r t l y a f t e r p a r t u r i t i o n to the end of l a c t a t i o n and the l u t e a l phase from conception to term. A nonpregnancy cycle occurs when: 1) no ovulation takes place, which r e s u l t s i n 10 months of anestrus followed by 3-4 weeks of proestrus; 2) ovulation but no f e r t i l i z a t i o n occurs, which r e s u l t s i n 2-3 months pseudopregnancy and 7 months of anestrus; and 3) ovulation and f e r t i l i z a t i o n are followed by a prematurely terminated pregnancy, which r e s u l t s i n anestrus. The main reproductive d i f f e r e n c e between populations i s that the whole cycle i s s h i f t e d e i t h e r e a r l i e r or l a t e r and i s i n phase with a d i f f e r e n t time of the year. Within each population, photoperiod and an endogenous cycle are the main proximate controls f o r the annual timing of estrus. Photoperiod probably sets the annual timing of the endogenous cycle so that estrus occurs at a p a r t i c u l a r time of the year. Between populations, genetic v a r i a t i o n maintains d i f f e r e n c e s i n the timing of estrus. Differences i n timing may be maintained by p o p u l a t i o n - s p e c i f i c responses to the annual l i g h t c y c l e . In t h i s way each population would set the timing of the endogenous cycle d i f f e r e n t l y so that estrus occurs at a d i f f e r e n t time of the year. The adaptive value of a p o p u l a t i o n - s p e c i f i c response to photoperiod could be to permit each population to set the timing i v of the entire cycle so that estrus, conception, implantation, birth and weaning occur at the optimum times for survival. The ultimate factor in determining the timing of the whole cycle may be food (shrimp, Crangon) for the newly weaned pups. V TABLE OF CONTENTS Page Abstract • i i Table of contents v L i s t of tables v i i L i s t of figures .... v i i i L i s t of p l a t e s '• • x I. Reproductive c y c l e i n southeastern Vancouver Island .. 1 Abstract 2 Introduction 4 Materials and methods 7 Results • 11 1. Pregnancy cycle 11 A. Conception , 11 B. Delay of implantation 16 C. Placentation ' • 17 D. L a c t a t i o n ' 20 2. Nonpregnancy cycle 22 Discussion v. .' 25 I I . Estrous cycle i n c a p t i v i t y 43 Abstract 44 v i Page Introduction 46 Materials and methods 48 Results 56 1. Captive environment 56 2. Estrous cycle 56 A. Estrus 62 B. Pseudopregnancy 63 C. Anestrus .... 65 D. Proestrus .... 66 3. Time of estrus 67 Discussion 74 I I I . E f f e c t of photoperiod on estrus 90 Abstract 91 Introduction ...92 Materials and methods .. 93 Results 95 Discussion 99 Acknowledgments L i t e r a t u r e c i t e d 105 106 v i i LIST OF TABLES Table Page I. Summary of the number and s i z e of graafian f o l l i c l e s i n each ovary, s i z e and type of corpus luteum and type of uterine and vaginal epithelium during each stage of pregnancy and during nonpregnancy . .. 15 I I . Summary of the contents of the vaginal smear and the type of vaginal and uterine epithelium during each stage of the estrous cycle 59 v i i i LIST OF. FIGURES Figure Page 1. Diagrammatic representation of the female reproductive t r a c t showing the most frequently, discussed regions 8 2. Macroscopic and microscopic changes i n the o v a r i e s , uterus and vagina during 9 stages of pregnancy 13 3. Frequencies of newborn pups observed ( t o t a l , 46), at 4-day i n t e r v a l s 18 4. Pregnancy cycle of Phoca.vitulina i n d i f f e r e n t regions .. 26 5. Equipment to r e s t r a i n seals .... 49 6. Equipment to sample vaginal f l u i d 52 7. Comparison of the average monthly v a r i a t i o n i n water temperature and daylength i n one holding tank and i n three f i e l d regions 57 8. Annual v a r i a t i o n i n the percent of parabasal intermediate and s u p e r f i c i a l type e p i t h e l i a l c e l l s i n the vaginal f l u i d and i n the s t r e t c h a b i l i t y of the vaginal f l u i d during the estrous cycle 60 i x Figure Page 9. The seasonal timing of estrus i n i n d i v i d u a l females from southeastern Vancouver Island, northeastern Vancouver Island and Humboldt Bay during 1-4 years of c a p t i v i t y 68 10. Comparison of standard length i n 32 fetuses from southeastern Vancouver Island and 16 from northeastern Vancouver Island c o l l e c t e d during e a r l y and middle p l a c e n t a t i o n times 7 1 11. E f f e c t of 18 hr and 6 hr daylengths on the onset time of estrus i n i n d i v i d u a l females from southeastern Vancouver Island and Humboldt Bay .................................................. 96 X LIST OF PLATES Plate Page I. Mature f o l l i c l e and v a r i a t i o n i n the corpus luteum during pregnancy 35 I I . Uterine endometrium from conception to e a r l y p l a c e n t a t i o n .. 37 I I I . Uterine endometrium from term to f i r s t h a l f of l a c t a t i o n 39 IV. Vaginal mucosa during pregnancy • • • 41 V. Vaginal biopsies at d i f f e r e n t stages of the estrous cycle 82 VI. C e l l s i n the vaginal f l u i d during the estrous cycle .. 84 VII. Uterine biopsies between estrus and anestrus 86 VIII. Uterine biopsies between anestrus and proestrus ...... 88 Reproductive cycle i n southeastern Vancouver Island. 2 ABSTRACT In the f i e l d , the harbor s e a l has a monestrous, monovulatory reproductive c y c l e . B i r t h s occur between l a t e June and e a r l y September with a peak i n l a t e J u l y . Mating occurs mainly during the f i r s t h a l f of September, at the end or s h o r t l y a f t e r a 6 week period of l a c t a t i o n . Pregnancy l a s t s 10 1/2 months and includes a 2 - 2 1/2 month delay of b l a s t o c y s t implantation and 8 - 8 1/2 month of p l a c e n t a t i o n . I f pregnancy i s missed or prematurely terminated, estrus does not occur again u n t i l about the same time as i n l a c t a t i n g females. In other populations the cycle i s about the same as i n southeastern Vancouver Island except that i t i s phase s h i f t e d e i t h e r e a r l i e r or l a t e r over a range of 5 or 6 months. The f o l l i c u l a r phase of a pregnancy cy c l e begins s h o r t l y a f t e r p a r t u r i t i o n and l a s t s u n t i l ovulation. During t h i s time the f o l l i c l e number and s i z e increase, estrogen production increases and uterine and vaginal e p i t h e l i a p r o l i f e r a t e . The l u t e a l phase follows conception and l a s t s u n t i l p a r t u r i t i o n . At t h i s time the f o l l i c l e s i z e and number are u s u a l l y small, the corpus luteum secretes progesterone and the reproductive e p i t h e l i a are mainly secretory. The reduction i n s t e r o i d l e v e l s after the l o s s of the placenta and the degeneration of the corpus luteum following p a r t u r i t i o n probably t r i g g e r s the onset of the f o l l i c u l a r phase. Thus, negative s t e r o i d feedbacks probably modify the annual timing of estrus during pregnancy. However, because the low s t e r o i d l e v e l s which follow a missed or prematurely terminated pregnancy 3 do not trigger f o l l i c u l a r development, negative steroid feedbacks play only a minpr role i n the timing of annual breeding. 4 INTRODUCTION The harbor s e a l , Phoca v i t u l i n a , i s widely d i s t r i b u t e d along the c o a s t a l regions of the north A t l a n t i c and north P a c i f i c . Scheffer (1958) divided t h i s s e a l i n t o 5 geographical subspecies which include v i t u l i n a , found i n the eastern A t l a n t i c , concolor, i n the western A t l a n t i c , mellonae, i n the Seal Lakes, Quebec, r i c a r d i , i n the eastern P a c i f i c and largha, i n the western P a c i f i c . Whether the form largha i s a subspecies or a separate species i s i n dispute (McLaren, 1966). In a recent review of the l i t e r a t u r e and personal correspondence on the reproductive cycle of t h i s species, I concluded that there are large r e g i o n a l v a r i a t i o n s i n the seasonal timing of the b i r t h p e r i o d (Bigg, 1969a). In any one region b i r t h s occur over a 1 - 2 1/2 month period. Between regions, however, the seasonal timing v a r i e s by up to 5 or 6 months with v a r i a t i o n s grading i n t o one another to form c l i n e s over much of the species d i s t r i b u t i o n . Of p a r t i c u l a r i n t e r e s t i s the v a r i a t i o n shown by P_.v. r i c a r d i . In northern Alaska the b i r t h season occurs during March and A p r i l and p r o g r e s s i v e l y l a t e r southeastward to southern Puget Sound, Washington, where i t occurs during August and September. In Willapa Bay, on the outer coast of Washington, young are born i n May and June (Newby, 1966) and p r o g r e s s i v e l y e a r l i e r southward to Mexico, where b i r t h s are i n March. Such large r e g i o n a l v a r i a t i o n s and extensive c l i n e s are rare among marine and t e r r e s t r i a l mammals. Reasons f o r the v a r i a t i o n s are not known since adequate information on the seasonal timing of reproductive events i n the f i e l d and under known environmental conditions i n c a p t i v i t y are 1 lacking. The most pe r t i n e n t studies to date on female reproduction c o n s i s t of a d e s c r i p t i o n of the reproductive organs (Harrison, et a l , 1952) , four f i e l d studies which o u t l i n e the main reproductive events (Fisher, 1954; Harrison, 1963; Bishop, MS, 1968; Bigg, 1969b), and a few observations on annual changes i n reproductive organs (Harrison, et a l , 1952; Harrison, 1960, 1963; Amoroso, et a l , 1965). Information on reproduction i n c a p t i v i t y i s l i m i t e d to one small experimental i n v e s t i g a t i o n (Harrison, 1963). The consensus i s that the cycle i s annual and consists of a short b i r t h season followed by l a c t a t i o n , breeding, delay of implantation and pla c e n t a t i o n . I t i s unclear, however, whether estrus and ovulation occur at other times of the c y c l e , p a r t i c u l a r l y following a missed or prematurely terminated pregnancy. V i r t u a l l y nothing i s known about the annual v a r i a t i o n i n f o l l i c u l a r development or microscopic changes i n the reproductive organs. This i s the f i r s t of three studies i n v e s t i g a t i n g the factors which c o n t r o l reproductive timing of the adult female harbor s e a l i n d i f f e r e n t regions. Generally, the main c o n t r o l l i n g f a c t ors i n vertebrates are considered to be s t e r o i d feedbacks, n u t r i t i o n , temperature, genetic v a r i a t i o n , photoperiod and an endogenous cycle (van Tienhoven, 1968; Sadleir,1969; Kovacic, 1970). The purpose of t h i s study i s to f i r s t describe the reproductive cycle during pregnancy and nonpregnancy i n one region, southeastern Vancouver Island, B r i t i s h Columbia. P a r t i c u l a r emphasis i s placed on determining when estrus occurs, whether the species i s monestrous or polyestrous and whether pregnancy a f f e c t s the timing of estrus . 6 Secondly, the possible r o l e of estrogen and progesterone feedbacks i n reproductive timing i s discussed. The study i s based on a mac-roscopic and microscopic examination of the ovaries, uterus and vagina of adult females c o l l e c t e d from a l l months of the year. The macroscopic examination includes a d e s c r i p t i o n of the number and s i z e of f o l l i c l e s and the s i z e of the corpus luteum and conceptus and the microscopic examination, the type and s i z e of c e l l s i n the uterine and vaginal e p i t h e l i a and i n the corpus luteum and f o l l i c l e s . In most mammals these reproductive t i s s u e s change during the cycle and are good i n d i c a t o r s as to whether an i n d i v i d u a l i s under f o l l i c u l a r (estrogenic) or l u t e a l (progesteronic) stimulation (Turner, 1963; Nalbandov, 1964). Usually, estrogen secreted by mature f o l l i c l e s causes p r o l i f e r a t i o n (hypertrophy and hyperplasia) of the uterine and vaginal e p i t h e l i a , whereas progesterone from the corpus luteum produces d i f f e r e n t i a t i o n (secretion) of these e p i t h e l i a . Laws (1956) and Craig (1964) used b a s i c a l l y the same indices i n describing the cycles of the southern elephant s e a l (Mirounga leonina) and northern f u r s e a l (Callorhinus u r s i n u s ) . Since t h i s study i s a d e s c r i p t i o n of the reproductive cycle under natural environmental conditions, i t i s used as a base-line against which the two l a t e r studies, on captive females held under c o n t r o l l e d environmental conditions, are i n t e r p r e t e d and compared. 7 MATERIALS AND METHODS Between 1964 and 1969, I c o l l e c t e d 112 pregnant or l a c t a t i n g females and 16 nonpregnant adults i n southeastern Vancouver Island and the adjacent mainland, B r i t i s h Columbia. The 21 females taken during the delay of b l a s t o c y s t implantation were considered to be pregnant since a l l possessed a recently formed corpus luteum. I t i s l i k e l y that no more than one or two were not pregnant because reproductive f a i l u r e f o r the e n t i r e cycle averages only about 12% (Bigg, 1969b). Females were considered nonpregnant adults i f they were c o l l e c t e d during the normal placentation p e r i o d and di d not possess a healthy conceptus 'but had a corpus luteum or corpus albicans i n one ovary. The number of adults c o l l e c t e d during each month was as follows: J F M A M J J A S O N D Pregnant or . 1 0 10 9" 14 10 2 7 11 9 6 15 9 l a c t a t i n g Nonpregnant 2 2 5 4 2 1 0 0 0 0 0 0 An a d d i t i o n a l 7 females which were s a c r i f i c e d during estrus i n the two experimental studies reported l a t e r , are included i n the study to supplement the shortage of specimens representing t h i s stage. Figure 1 diagrammatically shows the reproductive t r a c t of the adult. The ovaries, uterine horns and vaginae of a l l were f i x e d . i n 10% formalin. When present, the standard length of the conceptus was noted to a i d i n de s c r i b i n g the maternal stage of pregnancy or l a c t a t i o n . Each ovary was transversely sectioned i n t o s l i c e s 2 mm th i c k with a razor blade. The diameters 8 Figure 1. Diagrammatic representation of the female reproductive t r a c t showing the most frequently discussed regions. 9 Ovary Uterine horn Uterine lumen Cervical canal Cervix Vagina Hymen Urethral papilla.; Vestible C l i t o r i s Surface epithelium Gland Stroma Mus cle 1 "H I-I 4-> O) S o xs r-UJ Epithelium Surface pit Lamina propria] — f Muscle 10 width + depth ( 2 ) of the l a r g e s t f o l l i c l e and the corpus luteum and the number of f o l l i c l e s >2 mm diameter were noted. Tissue samples were taken from the corpus luteum, f o l l i c l e s , middle region of the uterine horns and the upper vaginae. Tissues were p o s t f i x e d i n Zenker's a c e t i c a c i d s o l u t i o n , imbedded i n p a r a f f i n wax, sectioned at 7 u and stained with Crossmon's modification of Mallory's connective ti s s u e s t a i n (Crossmon, 1937). Measurements of c e l l height were taken from the surface and glandular (subsurface region) e p i t h e l i a of the uterine endometrium of the horn without a conceptus. From the vaginal mucosa, the height of the outer c e l l l ayer was taken from the epithelium, between the surface p i t s . A t o t a l of 20 measurements of each epithelium was taken per female with f i v e females representing each stage of pregnancy. The e p i t h e l i a i n a l l nonpregnant females were measured. Females were considered to be i n estrus when the vaginal epithelium was s t r a t i f i e d , squamous and a c t i v e l y e x f o l i a t i n g . This con d i t i o n occurs at estrus i n many other mammals (Eckstein and Zuckerman, 1956, p. 43). 11 RESULTS 1. Pregnancy Cycle A. Estrus and conception Estrus and conception took place at the end of l a c t a t i o n or s h o r t l y afterwards, mainly during the f i r s t h a l f of September. This conclusion was suggested from the following observations. None of the s i x l a c t a t i n g females which were taken near weaning time (as in d i c a t e d by large suckling pups and lean mothers), on August 24 (1 specimen), 25 (1) and 29 (2) and September 14 (1) and 17 (1), were i n estrus or had ovulated. Two females netted with t h e i r pups and kept i n c a p t i v i t y during the suckling p e r i o d came i n t o estrus at the end of l a c t a t i o n , one during the f i r s t week of September and the other during ..the.. second week. pf..-.Se&tember. Three, post-partum females taken.in the f i e l d on. September 5, 17 and 20 had completed l a c t a t i o n and were i n estrus. Recent ovulations, as i n d i c a t e d by the presence of a newly formed corpus luteum, were recorded i n females which had completed l a c t a t i o n by September 20 (1) and October 9 (3) and 10.(1) and in,females ovulating f o r the f i r s t time by August 28 (1), September 4 (2) and 12 (1) and October 3 (2). Two of the newly mature females from August 28 and September 4 had j u s t completed estrus because the vaginal epithelium had only p a r t i a l l y d e s t r a t i f i e d . Since l a c t a t i o n l a s t e d 6 weeks (p.20), and b i r t h s occurred from l a t e June to e a r l y September (Fig. 3) , the onset of estrus and conception i s expected to extend from e a r l y August to middle October. During estrus a'/, s i n g l e ovulation occurred i n the opposite 1 2 ovary from the previous year. A l l 1 6 multiparous females c o l l e c t e d during the delay of b l a s t o c y s t implantation, when the degenerating corpus luteum of the previous year was s t i l l i d e n t i f i a b l e , had the most re c e n t l y formed corpus luteum i n the opposite ovary. Thus, there i s an annual a l t e r n a t i o n of ovarian function. The macroscopic and microscopic changes which occurred i n the reproductive organs during each stage of pregnancy are summar-iz e d i n F i g . 2 and Table 1 . At estrus f o l l i c u l a r development occurred i n one ovary and consisted of the marked enlargement of one f o l l i c l e . Other f o l l i c l e s i n both ovaries remained small i n s i z e and number. The preovulatory f o l l i c l e was characterized by a folded granulosa l a y e r , u s u a l l y 6 - 1 0 c e l l s t h i ck (Plate I , A ) . Small, healthy f o l l i c l e s had unfolded granulosa l a y e r s , 4 - 6 c e l l s t h i c k . The e p i t h e l i a of the uterine endometrium and vaginal mucosa were p r o l i f e r a t i n g since m i t o t i c f i g u r e s were common. The c e l l s of the surface-epithelium of the endometrium were t a l l pseudo-s t r a t i f i e d columnar many of which possessed c i l i a (Plate I I , A, B, C). Uterine glands were generally abundant and composed of low columnar c e l l s . A small amount of e p i t h e l i a l s e c r e t i o n i n the endometrium was indicated by a p i c a l buds on a few surface c e l l s and a t h i n mucus i n the lumina of a few glands. The endometrial stroma was edematous and l i n e d i n the lumen evenly. The vaginal epithelium was composed of s t r a t i f i e d squamous c e l l s , 1 0 - 1 5 t h i c k (Plate I V , A, B). 13 i Figure 2. Macroscopic and microscopic changes i n the ovaries, uterus and vagina during 9 stages o f pregnancy. A. V a r i a t i o n i n diameter of the corpus luteum (hatched box) and the l a r g e s t f o l l i c l e i n the ovary with the corpus luteum (black box) and without the corpus luteum (open box); note that the corpus luteum forms i n the opposite ovary i n consecutive years. B. V a r i a t i o n i n number of graafian f o l l i c l e s i n the ovary with the corpus luteum (black box) and without the corpus luteum (open box). C. V a r i a t i o n i n height of the epithelium l i n i n g the surface (hatched box) and glands (open box) of the empty uterine horn and the outer c e l l l a y e r l i n i n g the vaginal lumen (black box). Symbols: C, conception; D, delay of implantation; P, placentation; L, l a c t a t i o n ; e, e a r l y ; m, middle; 1, l a t e ; 1st, f i r s t h a l f ; 2nd, second h a l f ; box, 95% confidence l i m i t s ; v e r t i c a l l i n e , range; h o r i z o n t a l l i n e , mean; parentheses, enclose sample s i z e . 15 Table I. Summary of the number and s i z e of Graafia n f o l l i c l e s i n each ovary, s i t e and type of corpus luteum and the type of uterine'and v a g i n a l e p i t h e l i u m d u r i n g each stage of pregnancy and during nonpregnancy. Reproductiv stage No. and s i z e of f o l l i c l e s Ovary w i t h corpus luteum Ovary without corpus luteum Size and type of corpus luteum Glands Type of u t e r i n e e p i t h e l i u m Type of vagina 1 e p l t h e l l m Pregnancy Conception Delay of Implan-t a t i o n - e a r l y -middle many, small many, small few, small few, large many, small many, sma11 sm a l l , s e c r e t o r y s m a l l , reduced s e c r e t i o n s m a l l , s e c r e t o r y p r o l i f e r a t i v e s e c r e t o r y s e c r e t o r y p r o l i f e r a t i v e s e c r e t o r y s e c r e t o r y p r o l i f e r a t i v e d e s t r a t 1 f y l n g d e s t r a t i f y i n g P l e c e n t a t l o n - e a r l y -middle - l a t e few, small few, small few, small i n c r e a s i n g , small I n c r e a s i n g , small i n c r e a s i n g , secre- s e c r e t o r y t o r y i n c r e a s i n g , secre- s e c r e t o r y t o r y l a r g e , s e c r e t o r y s e c r e t o r y quiescent quiescent quiescent. quiescent s e c r e t o r y s e c r e t o r y L a c t a t i o n -1st h a l f many, small decreasing, de-generating few, i n c r e a s i n g many, i n c r e a s i n g s m a l l , degenera-ted quiescent p r o l i f e r a t i v e quiescent p r o l i f e r a t i v e quiescent p r o l i f e r a t i v e Nonpregnanc'v g e n e r a l l y few, g e n e r a l l y few, s m a l l , s e c r e t o r y s e c r e t o r y t o quiescent quiescent small small t o degenerated quiescent 16 B. Delay of implantation Following conception, the b l a s t o c y s t remained unimplanted f o r 2 - 2 1/2 months u n t i l about middle November. No implantations were found i n i n d i v i d u a l s c o l l e c t e d on November 2, 7, 17, 20, 24 and 25, but new implantations were found i n those taken on November 17, 18, 23, 26 and 29 i n a l l 11 taken between December 5 and 31. Delay of implantation was d i v i d e d i n t o e arly (August and September specimens), middle (October) and l a t e periods (November). F o l l i c u l a r s t i m u l a t i o n , as" i n d i c a t e d by the number of f o l l i c l e s , occurred i n both ovaries during e a r l y and middle delay of implanta-t i o n but decreased i n l a t e delay. The corpus luteum during t h i s time remained small although appeared to change i n secretory a c t i v i t y , since most l u t e a l c e l l s were unvacuolated during e a r l y delay and l a t e delay and vacuolated during middle delay (Plate I, B, C). The corpus contained small (largest 20-25u), c l o s e l y packed l u t e a l c e l l s andjiad l i t t l e connective ti s s u e and a few i n t e r c e l l u l a r spaces. The e p i t h e l i a i n the endometrium were secretory, and i n the vagina, quiescent. Secretion i n the uterus was i n d i c a t e d by many a p i c a l buds on the surface epithelium, and abundant t h i n secretions i n most glandular lumina (Plate I I , D, E, F ) . The surface epithelium increased i n height during e a r l y and middle delay but decreased i n l a t e delay to low columnar with only some areas of p s e u d o s t r a t i f i c a t i o n . C i l i a t e d c e l l s were common only during the early delay and mitoses were rare throughout the delay. The stroma was compact and thrown i n t o regular shallow f o l d s around the lumen. 17 The glandular epithelium increased i n height throughout the delay pe r i o d to t a l l columnar and gland c o i l i n g increased. During e a r l y delay subnuclear vacuoles formed i n the glandular epithelium and i n middle delay supra-nuclear vacuoles were evident. The vacuoles gradually increased i n s i z e and number u n t i l by l a t e delay they comprised most of the c e l l volume. In the vagina the epithelium d e s t r a t i f i e d to 2-4 f l a t t e n e d c e l l layers over a basal cuboidal l a y e r by e a r l y delay. In middle or l a t e delay i t became a t r a n s i t i o n a l type. This consisted of two c e l l l a y e r s , a basal cuboidal layer covered by an outer cuboidal layer except i n the mucus-secreting surface p i t s where the outer layer was e i t h e r low or t a l l columnar (Plate IV, C, D). C. Placentation Placentation, the p e r i o d from implantation to b i r t h , l a s t e d 8 - 8 l/2~months since most b i r t h s occurred i n l a t e J u l y (Fig. 3). This stage was d i v i d e d i n t o e a r l y placentation (females with fetus <^  249 mm; average period, middle November - middle February, based on Bigg, 1969b), middle pl a c e n t a t i o n (250-599 mm; l a t e February -middle May) and l a t e p l a c e n t a t i o n (>_ 600 mm; l a t e May - l a t e J u l y ) . During p l a c e n t a t i o n , slow f o l l i c u l a r stimulation occurred i n the ovary without the corpus luteum, whereas gradual f o l l i c u l a r suppression took place i n the other. The corpus luteum was f u n c t i o n a l throughout pl a c e n t a t i o n and gradually increased i n s i z e to occupy about the ovarian volume by term. Growth r e s u l t e d from an increase i n l u t e a l c e l l s i z e (30-35u), v a s c u l a r i t y , connective 1 8 Figure 3. Frequencies of newborn pups observed ( t o t a l , 46), at 4-day i n t e r v a l s . NUMBER 20 t i s s u e and i n t e r c e l l u l a r spaces (Plate I, D and E). Central implantation of the b l a s t o c y s t occurred i n the uterine horn associated with the corpus luteum. The developing fetus and membranes eventually f i l l e d most of t h i s horn leaving the other horn empty. The following d e s c r i p t i o n r e f e r s only to the empty horn. During the period of placentation the e p i t h e l i a of the endometrium became les s secretory, whereas those of the vagina increased i n s e c r e t i o n . The.surface epithelium i n the endometrium was not as secretory as i t was during the delay since the a p i c a l buds were r a r e l y present and the epithelium gradually decreased i n height to cuboidal by term (Plate I I I , A, B, C). In the glands a thick rather than t h i n secretion was produced through-out placentation. The amount of s e c r e t i o n gradually decreased as in d i c a t e d by fewer lumina with secretions and a decrease i n the height of the epithelium to cuboidal by term. The number of cytoplasmic vacuoles also decreased s u b s t a n t i a l l y . From middle placentation u n t i l term, the stroma became more edematous forming t a l l i r r e g u l a r f o l d s which projected i n t o the uterine lumen. In the vagina the epithelium was t r a n s i t i o n a l during e a r l y p l a c e n t a t i o n but was secretory during middle and l a t e placentation (Plate IV, E, F ) . By term, the outer c e l l l ayer i n a l l regions was t a l l columnar and the secretory p i t s were replaced by branching f i n g e r -l i k e p r o j e c t i o n s of the lamina p r o p r i a . D. L a c t a t i o n L a c t a t i o n l a s t e d about six weeks, since most b i r t h s occurred 21 i n l a t e July and most pups were deserted by t h e i r mothers during the f i r s t week i n September. This duration was also suggested from two females which were kept i n c a p t i v i t y with t h e i r pups. One female, captured at term, suckled i t s pup f o r 6 weeks and the other, captured with i t s pup which I estimated to be at l e a s t one week o l d , weaned i t a f t e r an a d d i t i o n a l 4 weeks. Lactating specimens were divided i n t o those c o l l e c t e d during the f i r s t and second halves of l a c t a t i o n , on the ba s i s of the weight of the su c k l i n g pup. Because pups weigh an average of 10 kg at b i r t h and 24 kg at weaning (Bigg, 1969b), the f i r s t h a l f of l a c t a t i o n included those with pups weighing <^  17 kg and the second h a l f , those with pups >_ 18 kg. During l a c t a t i o n , r a p i d f o l l i c u l a r s t i m u l a t i o n , as i n d i c a t e d by an increase i n f o l l i c l e s i z e and number, occurred i n the ovary without the corpus luteum. In the other ovary, v'f6llicular-development was generally suppressed although an' increase' i n f o l l i c u l a r s i z e occurred i n the second h a l f of l a c t a t i o n . At the end of l a c t a t i o n , the f o l l i c l e destined to ovulate, u s u a l l y i n the ovary without the corpus luteum, increased markedly i n s i z e . The number and s i z e of other f o l l i c l e s i n both ovaries decreased. Within a few days a f t e r b i r t h the corpus luteum began to degenerate r a p i d l y . By the second h a l f of l a c t a t i o n most corpora were about h a l f the s i z e of those at term and contained mainly thick h y a l i n i z e d blood vessels and dense connective t i s s u e plus a few degenerating l u t e a l c e l l s (Plate I, F ) . The e p i t h e l i a of the endometrium and vagina were quiescent or regressive i n most of the f i r s t h a l f of l a c t a t i o n and p r o l i f e r a t i v e 22 i n the second h a l f . In the f i r s t h a l f , the glandular and surface e p i t h e l i a of the endometrium were nonsecretory and composed of cuboidal c e l l s (Plate I I I , D-I). The stroma of the empty horn became compact but i n the post-parturn horn i t remained edematous. Many large h y a l i n i z e d blood v e s s e l s , which r e s u l t e d from pregnancy, bulged along the surface of the post-partum endometrium. During the second h a l f of l a c t a t i o n , the epithelium of both horns under-went hyperplasia and hypertrophy. Some regions of the surface epithelium were p s e u d o s t r a t i f i e d and possessed a few c i l i a but few c e l l s were secretory. The glands grew i n s i z e and number and few contained vacuoles. The stroma was edematous i n both horns. During the f i r s t h a l f of l a c t a t i o n , the vaginal epithelium regressed almost to a t r a n s i t i o n a l state (Plate IV, G, H). In the second h a l f , however, the basal l a y e r began to p r o l i f e r a t e a c t i v e l y which r e s u l t e d i n a s t r a t i f i e d squamous epithelium of about eight c e l l s t h i c k by the end of l a c t a t i o n . The'outermost l a y e r consisted of degenerating mucous c e l l s which were sloughed by the time estrus a r r i v e d . 2. Nonpregnancy cycle A l l 16 nonpregnant adults c o l l e c t e d between January 11 and June 8 , the usual placentation time, were i n reproductive stages between a normal recent pregnancy and anestrus. None were i n proestrus, estrus or delay of implantation as judged by a comparison with the t y p i c a l appearance of the ovaries, u t e r i and vaginae during the pregnancy c y c l e . L i t t l e f o l l i c u l a r s timulation was 23 i n d i c a t e d i n nonpregnant females since the f o l l i c l e s were small i n s i z e (average 4 mm, range 1-7 mm) and number (average 15, range 0-29). A l l females possessed e i t h e r a corpus luteum or a recently formed corpus albicans. The corpora v a r i e d i n s i z e (5-14 mm) and appearance between those already described f o r e a r l y placentation and those i n the second h a l f of l a c t a t i o n . The l a r g e s t corpora were f i l l e d mainly with healthy c l o s e l y packed and unvacuolated l u t e a l c e l l s with t h i n l y d i s t r i b u t e d connective t i s s u e . As the corpus regressed l u t e a l c e l l s were l e s s abundant and the connective t i s s u e content increased u n t i l i n the smallest corpus only a small connective t i s s u e knot remained. The uterine e p i t h e l i a were e i t h e r secretory as described during placentation, or were i n regressive stages which v a r i e d to quiescence. The surface epithelium i n a l l was nonsecretory and composed of e i t h e r cuboidal or low columnar c e l l s (6-14u). In most females (14) the uterine glands secreted a thick f l u i d and the lumina were l i n e d with cuboidal epithelium (7-12y) which frequently contained vacuoles. Two females appeared to be i n anestrus since the glandular lumina contained no secretions, the epithelium contained few vacuoles and both specimens had the smallest corpora of a l l nonpregnant females c o l l e c t e d . In a l l females the uterine stroma was compact and the vaginal epithelium was of the t r a n s i t i o n a l type. Since there was no evidence of recent or imminent ovulations over a 5 month peYIdd between normal breeding seasons, i t i s u n l i k e l y that out-of-season ovulations occur. Females, therefore, appear to 2 4 be monestrous in both a pregnancy and nonpregnancy cycle. This indicates that the cause of nonpregnancy i s either missed f e r t i l i z a -tion or resorption or abortion of the conceptus rather than failure to ovulate because a l l nonpregnant females had a corpus luteum or a recently formed corpus albicans. 25 DISCUSSION The duration of reproductive events i n the pregnancy cycle of the harbor s e a l i n southeastern Vancouver Island was approximately the same as i n other populations. In d i f f e r e n t populations of P. v. r i c a r d i , P_. v. concolor and P_. v. v i t u l i n a b i r t h s occur once a year, l a c t a t i o n l a s t s 3-6 weeks, estrus and ovulation occur within a few weeks a f t e r l a c t a t i o n and implantation occurs 1 1/2 -3 months l a t e r (Bigg, 1969b). These data i n d i c a t e that Phoca v i t u l i n a i s monestrous during a pregnancy cy c l e . The r e l a t i v e timing of the f i r s t ovulation at puberty may d i f f e r between populations i n the United Kingdom and i n the eastern P a c i f i c . Venables and Venables (1959, p. 666, 668) observed mating behaviour i n young females about.4 months early i n Shetland, Scot-land and Harrison (1963, p. 103) noted that two young females ovulated f o r the f i r s t time about two months e a r l y i n The Wash, England. In southeastern Vancouver Island and i n Alaska (Bishop, MS, 1968), however, f i r s t ovulations occurred at the same time as i n parous females. When the data obtained here are integrated with those of other populations described i n the e a r l i e r l i t e r a t u r e , i t i s c l e a r that the main d i f f e r e n c e between populations i s i n the seasonal timing of reproductive events. The seasonal timing of the b i r t h season, f o r example, varies r e g i o n a l l y by up to 5 or 6 months (Bigg, 1969a). For a comparison of the reproductive events i n d i f f e r e n t regions, F i g . 4 summarizes the most r e l i a b l e data on the annual timing of b i r t h , weaning, ovulation and implantation. i 26 Figure 4. Pregnancy cycle of Phoca v i t u l i n a i n d i f f e r e n t regions. The most r e l i a b l e times are given f o r e i t h e r the peak or middle of b i r t h (0), weaning (•), mating (A) and implantation "(A) . Regions and sources: 1. Southern Puget Sound, Washington; Hart, e t a l , 1965. 2. Southeastern Vancouver Island, B r i t i s h Columbia; present study. 3. Holland; Havinga, 1933. 4. Shetland Islands, Scotland; Venables and Venables, 1955. 5. The Wash, England; Harrison, 1963. 6. Tugidak Island, Alaska; Bishop, MS, 1968. 7. Nova S c o t i a and New Brunswick; F i s h e r , 1954. 8. Skeena River, B r i t i s h Columbia; Fisher, 1952. 9. Willapa Bay, Washington; Scheffer and S l i p p , 1944; Newby, 1966. 10. Humboldt Bay, C a l i f o r n i a ; Bigg, 1969a; Finch, MS, 1966. 11. Baja, C a l i f o r n i a , Mexico; Bigg, 1969a. REGION J — I I I I I I l I I l o \ \ \ \ \ b \ \ \ \ \ \ ex •*o—o„ \ •• \ ° \ \ J \ \ \ \ \ \ J 1 1 1 1 • • 28 These data suggest that the other reproductive events also vary r e g i o n a l l y by several months, although a l l keep about the same r e l a t i v e timing to the b i r t h season. Thus each population has s h i f t e d the e n t i r e cycle to be i n phase with a d i f f e r e n t time of the year. L i t t l e data are a v a i l a b l e as to whether females are mon-estrous during a nonpregnancy cycle i n regions other than south-eastern Vancouver Island. Bishop (MS, 1968) found no evidence of out-of-season ovulations i n two nonpregnant adults c o l l e c t e d i n Alaska, one and eight months p r i o r to the usual mating season. The lack of records on unusually e a r l y or l a t e embryos, fetuses and b i r t h s implies that a monestrous cycle e x i s t s i n t h i s species dur-i n g nonpregnancy. Most out-of-season ovulations would be expected to be f e r t i l i z e d since males are i n breeding condition f o r about 9 months of the year (Bigg, 1969b). The reproductive cycle of the harbor s e a l i s s i m i l a r to that of other species of Pinnipedia. In most species the cycle i s also d i s t i n c t l y annual although there are species d i f f e r e n c e s i n the duration of reproductive events (Harrison, 1969). For example, l a c t a t i o n may l a s t from a few weeks to several months, mating may occur from a few days to several months a f t e r p a r t u r i t i o n , implanta-t i o n from immediately a f t e r f e r t i l i z a t i o n to 5 months l a t e r and p l a c e n t a t i o n may l a s t 7-9 months. Unlike the harbor s e a l , however, most species e x h i b i t l i t t l e population v a r i a t i o n i n reproductive timing. The grey s e a l (Halichoerus grypus) i s the main exception with r e g i o n a l v a r i a t i o n s of about 6 months (Davies, 1957)., 29 I t i s unclear whether other seals are monestrous or poly-estrous. The extensive data of C r a i g (1964) indi c a t e that the northern f ur se a l (Callorhinus ursinus) i s monestrous. Hamilton (1939), Laws (1956) and McLaren (1958), however, suggest that the southern sea l i o n (Otaria bryonia), southern elephant seal (Mirounga  leonina) and the ringed s e a l (Pusa hispida) are polyestrous. But t h e i r evidence i s not completely convincing since no females were c o l l e c t e d i n estrus during out-of-season periods and the recently formed corpora l u t e a which they describe may, i n f a c t , be from recently terminated normal pregnancies. The question of whether a species has one estrous p e r i o d a year or several i s of considerable importance i n determining which factors c o n t r o l reproductive timing. For example, how does a polyestrous species maintain a s p e c i f i c a l l y timed annual cycle when ovulations can take place at any time of the year? The usual explanation f o r t h i s i s that the seasonal timing of male breeding i s so short that out-of-season ovulations would not be f e r t i l i z e d (Laws, 1956, p. 58; McLaren, 1958, p. 80). I f , on the other hand, a species i s monestrous, what mechanism keeps estrus r e s t r i c t e d to a short p e r i o d each year? In t h i s case the c o n t r o l i s complex and i s p h y s i o l o g i c a l l y rather than e c o l o g i c a l l y imposed. In many mammals the duration between consecutive estrous periods i s c o n t r o l l e d by negative s t e r o i d feedbacks which operate during pregnancy (Turner, 1963; Nalbandov, 1964). High l e v e l s of progesterone from the corpus luteum and progesterone and estrogen ' from the placenta u s u a l l y suppress ovulation through 30 a negative feedback ac t i n g on the hypothalamus - a n t e r i o r p i t u i t a r y - gonad axis. Thus, the timing of the next estrus i s determined by when the l e v e l s are low enough, such as when pregnancy i s missed or the corpus luteum and placenta degenerate and are l o s t due to termination of pregnancy... The ro l e of. t h i s negative feedback i n the annual timing of estrus i n the harbor s e a l i s now discussed from a consideration of the r e l a t i v e l e v e l s of these steroi d s as in d i c a t e d by microscopic and macroscopic changes i n the reproduc-t i v e organs. Although the actions of estrogen and progesterone cannot always be separated i n mammals i t i s generally believed that the former causes uterine and vaginal e p i t h e l i a to p r o l i f e r a t e (hyperplasia and hypertrophy) and that the l a t t e r encourages these t i s s u e s to d i f f e r e n t i a t e (secrete). The period of the cycle when estrogen predominates and leads to estrus i s usually c a l l e d the f o l l i c u l a r phase, when progesterone predominates, the l u t e a l phase, and when neither are high, the anestrous phase. The main reproduc-t i v e events i n the pregnancy and nonpregnancy cycle of the harbor s e a l f a l l i n t o these categories. Although sparse, the data given by Harrison, e t a l (1952), Harrison (I960, 1963) and Amoroso, e t a l (1965), support t h i s . With few exceptions, the ba s i c changes i n the reproductive organs of the harbor s e a l also agree with those reported by Laws (1956) and Craig (1954) f o r the southern elephant s e a l and the northern .fur s e a l and by Harrison et; a l (1952) f o r a v a r i e t y of s e a l species. The f o l l i c u l a r phase of .the pregnancy cycle i n the harbor s e a l began s h o r t l y a f t e r p a r t u r i t i o n and terminated with ovulation 31 s h o r t l y a f t e r l a c t a t i o n with ovulation. During t h i s time the increased s i z e and number of f o l l i c l e s apparently produced large q u a n t i t i e s of estrogen since the uterine and vaginal e p i t h e l i a p r o l i f e r a t e d . At the same time the corpus luteum degenerated. I found no evidence f o r a post-partum rejuvenation of the corpus luteum as Harrison (1960) postulated to occur i n the E n g l i s h harbor s e a l . The f o l l i c u l a r phase of the northern fur s e a l d i f f e r s from that of the harbor s e a l since i n the former species i t begins i n l a t e pregnancy and estrus occurs within a few days a f t e r p a r t u r i -t i o n (Craig, 1964). This study, along with that of Bishop (MS, 1968), confirms that the harbor s e a l has an annual a l t e r n a t i o n of ovarian function l i k e many other species of s e a l (Harrison, 1969). The reason for the a l t e r n a t i o n probably stems from the f a c t that slow f o l l i c u l a r development which began i n middle placentation was suppressed i n the ovary with the corpus luteum. This suppression during placenta-t i o n probably d i d not permit enough time f o r f o l l i c u l a r development to occur i n t h i s ovary between p a r t u r i t i o n and the end of l a c t a t i o n . Why f o l l i c u l a r development d i d not occur i n t h i s ovary during placentation i s unclear although i t may have r e s u l t e d , as Enders, et a l (1946) suggested f o r the northern f u r s e a l , from the mechan-i c a l pressure of the growing corpus luteum which by term comprised about h a l f the ovarian volume. The l u t e a l phase began with the formation of the corpus luteum and l a s t e d u n t i l term. During t h i s time the reproductive organs were p r i m a r i l y progesteronic although some estrogenic changes 32 occurred i n the delay of implantation. The progesteronic features included a f u n c t i o n a l (healthy appearing) corpus luteum and associated secretory e p i t h e l i a i n the uterus throughout pregnancy and i n the vagina during middle and l a t e placentation. Glandular vacuoles, which are not reported i n other s e a l s , are common during t h i s phase and may be evidence f o r glandular s e c r e t i o n or presecretion as they are i n humans a f t e r ovulation (Witt, 1963, p. 41). Progester-one s e c r e t i o n was apparently reduced during middle delay of implanta-t i o n since many l u t e a l c e l l s became vacuolated at t h i s time. Vacuolation of the corpus luteum i s common among seals during the delay and i s thought to i n d i c a t e reduced progesterone s e c r e t i o n (Harrison, 1969). The vacuolation disappeared and the corpus luteum was thus f u l l y f u n c t i o n a l again by implantation. From implantation u n t i l term the corpus increased i n s i z e and presumably progesterone s e c r e t i o n (Nalbandov, 1964). Since Harrison (1960) found progesterone i n the corpus luteum and placenta of term harbor seals i n England i t i s l i k e l y that both organs secrete t h i s s t e r o i d throughout p l a c e n t a t i o n . The corpus luteum d i d not begin r a p i d degeneration u n t i l a f t e r p a r t u r i t i o n . This d i f f e r s from the corpus of the northern f u r se a l which becomes nonfunctional a f t e r e a r l y p l a c e n t a t i o n (Craig, 1964). The estrogenic features which occurred during the l u t e a l phase include a b r i e f estrogen surge i n ea r l y and middle delay of implantation. This was in d i c a t e d by a marked increase i n the number of f o l l i c l e s and an hypertrophied uterine epithelium. The uterine e p i t h e l i a appeared essentially, the same as i n the pre-33 implantation p e r i o d of the cat (Dawson and Kosters, 1944) and i n many other mammals during the delay of implantation (Wimsott, 1963). In the mink t h i s estrogen surge may lead to ovulation and s t r a t i f i c a t i o n of the vaginal epithelium (Enders and Enders, 1963). No preovulatory f o l l i c l e s or vaginal s t r a t i f i c a t i o n s occurred i n the harbor s e a l at t h i s time suggesting a r e l a t i v e l y lower estrogen surge f o r t h i s s e a l . By implantation time the f o l l i c l e number was low and the epithelium of the uterus was secretory i n d i c a t i n g that the estrogen l e v e l s had dropped. The hormonal cause of implantation i s s t i l l unknown f o r any species with obligate delay of implantation (not l a c t a t i o n induced) since estrogen and/or progesterone administration cannot hasten implantation time (Daniel, 1970). I t i s not known whether estrogen i s produced i n the placenta of the harbor s e a l . Harrison et a l , (1952) and Laws (1956) suggested that some .species of s e a l have f o l l i c u l a r stimulation s h o r t l y a f t e r implantation. This was not reported by Craig (1964) i n the northern fur s e a l and was not found i n the harbor s e a l . These data suggest that the onset of the f o l l i c u l a r phase'is influenced by negative s t e r o i d feedbacks during pregnancy since marked f o l l i c u l a r development coincided with the probable r a p i d reduction i n s t e r o i d l e v e l s associated with the l o s s . o f the placenta and degeneration of the corpus luteum following p a r t u r i t i o n . Thus, a female which, gives b i r t h to a pup e a r l y or l a t e i n the b i r t h season would be expected to have a correspondingly hastened or delayed onset of r a p i d f o l l i c u l a r , development. 34 Between the annual.periods of estrus, nonpregnant females went from the normal l u t e a l phase of pregnancy to an anestrous phase. The f a c t that the anestrous phase remained despite the l o s s of the placenta.and. degeneration of the corpus.luteum.indicates that the high l e v e l s of. s t e r o i d d i d not r e s t r i c t estrus to a short time each year. Had .they, been the main f a c t o r then the low levels, of s t e r o i d s following.a missed or. prematurely terminated pregnancy would have r e s u l t e d i n r a p i d . f o l l i c u l a r development s h o r t l y a f t e r reproductive f a i l u r e . . These, data, coupled .with those, of .the preg-. nancy cycle i n d i c a t e that negative steroid-feedbacks play only a r e l a t i v e l y minor r o l e i n the annual timing of.estrus i n the harbor s e a l . Some other.endogenous and/or exogenous fa c t o r s must be involved. 35 i P l a t e I. Mature f o l l i c l e and v a r i a t i o n i n the corpus luteum during pregnancy. Scale =50 u A Ovulatory f o l l i c l e with folded granulosa l a y e r . B Corpus luteum during e a r l y delay of implantation containing small, unvacuolated l u t e a l c e l l s . C Corpus luteum during middle delay of implantation with small vacuolated (arrow) and dead (d). l u t e a l c e l l s . D Corpus luteum during e a r l y placentation with small unvacuolated c e l l s . E Corpus luteum at term with large l u t e a l c e l l s i n a loose a s s o c i a t i o n with each other. F Degenerating corpus luteum during the f i r s t h a l f of l a c t a t i o n with small l u t e a l c e l l s and large h y a l i n i z e d blood vessels (arrow). 36 37 Plate I I . Uterine endometrium from conception to e a r l y placentation. Scale = 5 0 ^ A. Uterine endometrium at conception; glands abundant and moderately c o i l e d ; stroma edematous and l i n e s the lumen evenly. B. Surface epithelium of "A" showing p s e u d o s t r a t i f i e d columnar c e l l s with c i l i a (arrow). C. Glandular epithelium of "A" showing low columnar c e l l s and t h i n s e c r e t i o n (tn). D. Uterine endometrium during middle delay of implantation; glands enlarged and t i g h t l y c o i l e d ; stroma compact and l i n e s the lumen unevenly. E. Surface epithelium of "D" showing enlarged p s e u d o s t r a t i f i e d c e l l s . F. Glandular epithelium of "D" showing enlarged c e l l height and subnuclear vacuoles (arrow). G. Uterine endometrium during early p l a c e n t a t i o n ; glands smaller; stroma compact and l i n e s lumen more evenly. H. Surface epithelium of "G" showing low c e l l height. I. Glandular epithelium of "G" showing cuboidal c e l l s with many subnuclear vacuoles and a thick s e c r e t i o n i n the lumen (tk). 39 Plate I I I . Uterine endometrium from term to f i r s t h a l f of l a c t a t i o n . Scale = 50 y A. Uterine endometrium of empty horn at term; glands sparce and loo s e l y c o i l e d ; stroma edematous and l i n e s the lumen unevenly. B. Surface epithelium of "A" showing cuboidal c e l l s . C. Glandular epithelium of "A" showing cuboidal c e l l s and thick luminal s e c r e t i o n . D. Uterine endometrium of empty horn during f i r s t h a l f of l a c t a t i o n ; glands sparce and l o o s e l y c o i l e d ; stroma compact and l i n e s lumen evenly. E. Surface epithelium of "D" showing cuboidal c e l l s . F. Glandular epithelium of "D" showing enlarging c e l l s and scant luminal s e c r e t i o n . G. Uterine endometrium of post parturn horn during f i r s t h a l f of l a c t a t i o n ; glands sparce and l o o s e l y c o i l e d ; stroma edematous and has large h y a l i n i z e d blood vessels (arrow). H. Surface epithelium of "G" i n d i c a t i n g cuboidal c e l l s over h y a l i n i z e d blood v e s s e l . I. .Glands of "G" with cuboidal epithelium and some luminal s e c r e t i o n . 41 Plate IV. Vaginal mucosa during pregnancy. Scale = 50 ^ A. Vaginal mucosa at conception showing surface p i t s with squamous c e l l s . B. S t r a t i f i e d squamous epithelium of "A". C. Vaginal mucosa i n ea r l y placentation showing t r a n s i t i o n a l epithelium on the surface and i n the surface p i t s . D. T r a n s i t i o n a l epithelium of "C" i n d i c a t i n g that the bottom of the surface p i t s has mucous-secreting c e l l s . E. Vaginal mucosa at term i l l u s t r a t i n g the absence of surface . p i t s and presence of f i n g e r - l i k e p rojections of stromal t i s s u e . F. Simple t a l l columnar epithelium of "F" which secretes mucus. G. Vaginal mucosa during f i r s t h a l f of l a c t a t i o n i n d i c a t i n g the reformed surface p i t s . : H. T r a n s i t i o n a l epithelium of "G". 42 43 I I . Estrous cycle i n c a p t i v i t y . i 44 ABSTRACT The estrous cycle of captive females from southeastern and northeastern Vancouver Island and Humboldt Bay was the same, i n duration and timing, as expected i n the respective f i e l d populations. Also, as i n the f i e l d , the cycle was monestrous and monovulatory. A few females had a second estrus s h o r t l y a f t e r the f i r s t . Estrus usually l a s t e d 3-5 weeks but ranged from 1-9 weeks. Ovulation occurred i n about h a l f the females. Because estrus was prolonged and ovulation d i d not always occur, females may be induced ovulators. When ovulati o n occurred estrus was followed by 2-3 months of pseudopregnancy, 7 months of anestrus and 3-4 weeks of proestrus. When no ovulation occurred anestrus l a s t e d about 10 months. The changes which occurred i n the e p i t h e l i a of the vaginal mucosa and uterine endometrium and i n the contents of the vaginal f l u i d are described f o r each stage of the c y c l e . The vagi n a l smear was a us e f u l index., to detect estrus. N u t r i t i o n was not an important proximate c o n t r o l i n the annual timing of estrus and temperature was u n l i k e l y to be. Genetic d i f f e r e n c e s appeared to cause v a r i a t i o n s i n the breeding seasons between populations. The annual timing of the reproductive cycle i n each population may have evolved from the f a c t that t h i s seal i s nonmigratory and has an extensive narrow c o a s t a l d i s t r i b u t i o n which permitted the formation of many d i s c r e t e breeding populations. A r e g i o n a l l y v a r i a b l e s e l e c t i v e f a c t o r which operated 45 on the newly weaned pups, such as food abundance (shrimp, Crangon), may have established the differences in reproductive timing for each population. Selection within each population was probably for a genotype with adaptations to u t i l i z e exogenous and endogenous proximate cues in a specific manner to produce the appropriate weaning time. 46 INTRODUCTION In the previous study I c l a r i f i e d three points about the control of reproductive timing within and between populations of harbor seals under natural environmental conditions. F i r s t , a l l populations examined appear.to have essentially.the same monestrous reproductive cycle. Second, .the main difference between populations is that each has shifted ..the phasing of the entire.cycle to a different time of-the year.. Third, negative steroid feedbacks from the corpus luteum and/or placenta are of only.minor.importance in governing the annual, timing of estrus.. Nothing.is known of the role of other possible controls of estrus i n this species, such as nutrition, temperature, genetic variation, photoperiod, and endogenous, cycles. The purpose of this study i s to experimentally determine the role of nutrition and.genetic variation and to discuss the role of temperature i n the .annual timing of estrus in different populations of harbor seals. . For this, I brought into captivity adult females from three f i e l d populations and kept them under the same outdoor conditions for.up to 4 years. The estrous cycles of captive females are described for. the f i r s t , time and compared with those under natural conditions. If the timing of estrus in the f i e l d i s main-s-tained by seasonal variations in nutrition, a regular food.supply in captivity should.change the seasonal timing.of estrus. Also, since physiological variations between populations may be due to either phenotypic or.genotypic responses to different, environments (Prosser, 1955), population.differences i n the timing of estrus 47 may be due to either acclimatization or genetic adaptation to each region. Thus, i f the differences in timing variations are phenotypic responses to different environments, they should disappear in a common environment. However, i f they are genotypic responses differences should.remain. The annual variation of water tempera-ture i n captivity.was also recorded. The estrous cycle was described from vaginal smears and vaginal and uterine hiopsies. These procedures are commonly used in reproductive studies of-terrestrial.mammals (Eckstein and Zuckerman, 1956), but to date have not been employed on any marine mammal. It i s clear from the previous study that changes i n the epithelia of the uterus and vagina.are good,indices of ovarian act i v i t y . i n this species..A.routine.method for restraining each seal i s described. 48 . . MATERIALS AND.METHODS . . . Between 1966 and 1969, 8 adult females were obtained from southeastern Vancouver Island, British.Columbia (49°N, 123°W), 3 from northeastern Vancouver, Island (50°N, 125°W) and 4 from Humboldt Bay, C a l i f o r n i a (41°N, 124°W). A l l seals were captured i n nets and brought d i r e c t l y to the holding tanks at the Vivarium compound, Un i v e r s i t y of B r i t i s h Columbia, Vancouver. Seals were housed i n p a i r s for. up, to 4 breeding seasons i n 6 outdoor tanks with females fr o m - C a l i f o r n i a and B r i t i s h Columbia kept separately. The holding tanks, were wood-stave water storage tanks,.8 1/2' i n diameter by 3 1/2' - 4 1/2' deep. Each tank was supplied with fresh running water (8 gal/min) to a depth of 1.1/2' - 2 1/2', a drain and a haulout platform. I n d i v i d u a l seals were fed 5-6 l b s of h e r r i n g per day. Herring were purchased from l o c a l f i s h i n g companies, which netted and.froze them.in mid-winter. The same l o t of herring was fed to.the seals over the remainder of the year. Each s e a l was given a supplement of 15 milligrams thiamine (Beminal; Ayerst Laboratories) intramuscularly every 2. weeks. Maximum and minimum water temperatures i n one tank were taken d a i l y during 1968 and 1969. The. average monthly temperature was c a l c u l a t e d by averaging a l l d a i l y temperatures ^ m a x + m"'-n^ recorded each month. To determine reproductive condition, each female was p h y s i c a l l y r e s t rained, sampling instruments were in s e r t e d into, the vagina and uterus and the epithelia.were checked f o r evidence of p r o l i f e r a t i o n , . secr e t i o n or quiescence... During r e s t r a i n t , the holding .tank was., drained and the s e a l captured with a purse net (Fig. 5A). This 4 9 Figure 5 . Equipment to r e s t r a i n s e a l s . A, purse net; B, net clamp; C, portable examination c a r t with only one of the ten straps shown and with rear brackets f o r binding the hind f l i p p e r apart removed; D, portable A-frame with,overhead t r o l l y , block and t a c k l e and s e a l tank. 50 51 apparatus consisted of 2 boards (2" x 4" x 5') and a piece of polypropylene seine n e t t i n g (5' x 5') stapled together to form a s l i n g i n which the.lower three-quarters of each end was.closed with twine. The s e a l was worked i n t o the purse by p o s i t i o n i n g the apparatus so that a board lay along each side, of the animal and the net covered i t s back. The animal was r o l l e d to one side over a board and the two boards p u l l e d together and l i f t e d so.that the s e a l s l i d to the bottom of the purse. A s e l f - l o c k i n g rope h o i s t (No. 12, Durbin Durco, St.. Louis, Mo.)., connected to an. overhead t r o l l y and a metal A-frame support (Fig. 5b ) » were used to l i f t the net and s e a l over the side of the tank onto an examination c a r t . At t h i s p o i n t a c t i v e seals often - tended to turn i n the net and so were fu r t h e r r e s t r a i n e d with a net clamp (Fig. 5B).. This was made from two pieces of wood (2" x 1" x 5') loo s e l y bolted at one end and was secured around the net j u s t over the seal's back. The examination c a r t (Fig.. 5C) consisted of a wooden trough with 10 nylon straps, a wooden frame and a p a i r of wheels. The s e a l was strapped i n t o the trough.and the back end of the purse net unbound . so that the boards could be hung over the sides of the c a r t and the head, back and rear of the animal exposed f o r examination. For the reproductive examination, the seal's hind legs were strapped apart to brackets on the back of the c a r t . Tissue sampling was done i n a room at the Vivarium. The sampling instruments included: an elongated glass proctoscope (Fig. 6A and B). to.enter-the vagina; several glass rods with rubber t i p s (Fig. 6C) to c o l l e c t vaginal, mucus; r e c t a l biopsy ..forceps. 52 Figure 6. Equipment to sample vaginal f l u i d . A and B, glass ' proctoscope; C, glass sampling rod. A l l . glass i s i • pyrex tubing; measurements are i n mm. 53 5 4 (16" stem length) f o r b i o p s i e s ; and a narrow beam l i g h t to locate the cervix. Instruments were s t e r i l i z e d by autoclaving. Samples of vaginal f l u i d were taken at 2-14 day i n t e r v a l s and vaginal biopsies at i n t e r v a l s of 2-40 days. Uterine biopsies were taken at 2-14 day i n t e r v a l s during proestrus and estrus and i r r e g u l a r l y at other times. They were taken mainly around estrus as a precaution against i n f e c t i o n since a greater natural immunity i n mammals e x i s t s at t h i s time (Jubb. and Kennedy, 1963, p. 409). A n t i b i o t i c s . ( t o t a l of 2000 mg a m p i c i l l i n , 1,600,000 IU p e n i c i l l i n G procaine, 2 mg dihydrostreptomycin) were given intramuscularly f o r 2-3 days a f t e r each uterine biopsy.. Part of the vaginal f l u i d c o l l e c t e d from the upper vagina was f i x e d and stained in.the manner suggested by Hartman (1944) f o r r a t vaginal smears. The c e l l s found i n the f l u i d were c l a s s i f i e d on the basis of Wied's (1958).standardized terminology f o r human vaginal cytology and the frequencies of each.type of e p i t h e l i a l c e l l was calculated from, a sample of 400 e x f o l i a t e d c e l l s . A frequency value c a l c u l a t e d from .400 c e l l s i s within +5% of the true value 95% of the time. As e p i t h e l i a l c e l l s were e x f o l i a t e d f o r only a short . time during the c y c l e , the r a t i o s were a r b i t r a r i l y determined only., when they made up more than .20% of a l l c e l l types i n a sample of 400 randomly viewed c e l l s , excluding red blood c e l l s . Red blood c e l l s were not normally found i n the f l u i d except f o r . a few days a f t e r a biopsy, while healing occurred. ..The remaining, vaginal f l u i d was. checked, f o r " s t r e t c h a b i l i t y " (spinnbarkeit).by. touching. . a drop of f l u i d with a glass s l i d e and.determining the height of 55 the f l u i d column.before breaking. Usually 3-5 t r i a l s were needed to get a good estimate. This i s a u s e f u l procedure f o r detecting ovulation time i n humans (Cohen, et a l , 1952). Vaginal and uterine b i o p s i e s , usually -3-5 mm.in diameter, were f i x e d i n 10% formalin, Bouin's f l u i d or Zenker's a c e t i c a c i d s o l u t i o n . Tissues were . . imbedded i n p a r a f f i n wax, sectioned at 7 y and stained with Crossmon's modification of Mallory's connective t i s s u e s t a i n (Crossmon, 1937.)... Changes were recorded i n the type of epithelium l i n i n g the surface .. and glands of the .uterine endometrium and the luminal surface of the vaginal mucosa- Females, were, considered to be i n estrus when the vaginal epithelium was s t r a t i f i e d squamous, and a c t i v e l y e x f o l i a t i n g as noted p r e v i o u s l y (p. 12 ). Each examination.for t i s s u e samples required about 20 . ... minutes from the time the.seal was captured u n t i l i t was released. A t o t a l of 980 examinations were made. To confirm the c o r r e l a t i o n of uterine and -vaginal changes with ovarian a c t i v i t y the 15. females were s a c r i f i c e d at.-different stages of the cycle. - Of these, 4 from southeastern Vancouver Island and 4 .from Humboldt Bay were f i r s t used i n a study on the e f f e c t of photoperiod (p. 90 ) on.reproductive timing. Euthanasia of a l l specimens was by sodium pentabarbitol (Barb-Euthol, Haver-Lockhart Laboratories) i n j e c t e d i n t o the extradural vein at 5 grains/10 l b s body weight. 56 RESULTS 1. Captive Environment Figure 7A gives the average monthly v a r i a t i o n of water tempera-tures i n c a p t i v i t y . Since changes i n temperature and photoperiod may a f f e c t reproductive timing when-females are brought i n t o c a p t i v i t y , Figures 7, B-D summarize, f o r comparative purposes, the average monthly v a r i a t i o n i n sea water temperature and photoperiod i n the three regions from which females were taken. In c a p t i v i t y the water temperature va r i e d from 3-4°C i n winter to 12-14°C i n summer. In southeastern .Vancouver Island, depending on the l o c a l i t y , i t ranged from 7. to 11°C and 6 to-18°C and i n northeastern Vancouver Island,.7 to 14°C ( H o l l i s t e r , MS, 1964). Near Humboldt Bay i t va r i e d fromlO-14°C (Anon., 1962). Photoperiod i n the holding tanks was considered to be the same as i n southeastern Vancouver Island since both areas are the same l a t i t u d e ( 4 9°N). Here the day length v a r i e d from 8 hours i n the winter to 16 hours... i n the summer, i n the northeastern Vancouver Island (50°N).the range, was only about 1/2 hr longer and i n Humboldt Bay.it was 2 hr l e s s (Anon., 1966). I t i s c l e a r from these comparisons that water temperature and photoperiod v a r i e d i n about the same pattern . i n c a p t i v i t y as i n the f i e l d . Thus, i f . photoperiod and temperature - -had an e f f e c t i n the ..field i t was not markedly changed by c a p t i v i t y . 2. Estrous Cycle  Table II and Figure 8 summarize the changes i n e p i t h e l i a of ... the vagina and uterus.and i n the c e l l u l a r , and mucus content of the vaginal f l u i d during the estrous c y c l e . Data from the.three groups 57 Figure 7. Comparison of the average monthly v a r i a t i o n i n water temperature arid daylength i n one holding tank and i n three f i e l d regions. A. Water temperature i n one tank during 1968 (-rr-) and 1969 (•••). B. Surface water temperature at Departure Bay (—) and Race Rocks (•••), southeastern Vancouver Island. C. Surface water temperature at Cape Mudge (•••), northeastern Vancouver Island and at Crescent C i t y ( ) , near Humboldt Bay, C a l i f o r n i a . D. Daylength i n southeastern Vancouver Island (•••), northeastern Vancouver Island ( -) and Humboldt Bay (—"—), C a l i f o r n i a . o TEMPERATURE (°C) , 1 , , 1 . 1 I 1 1 1 1 1 •n 1 \ \ \ \ \ \ \ \ > \ \ \ » \ \ \ \ \\ - \ \ \ \ \ * \ \ \ \ \ \ \ \ \ 1 \ 1 V > / / ' I / / / / / / / / / / O / / / / / / / / / z / / / — / / o / 1 1 .11,1 1 1 1 1 CD TEMPERATURE (°C) > TEMPERATURE (°C) 00 I 59 Table II. Summary of the contents of the vaginal smear and the type of vaginal and uterjrflne epithelium during each stage of' the estrous cycle. Reproductive stage Vaginal smear Type of epithelium Fluid Epithelial cells Leucocytes Vagina Uterine gland Uterine surface Estrus 3-5 weeks Fseudopregnancy 8-15 weeks Anestrus 7>-10 months Proestrus 3-4 weeks Watery, no mucus Mucus Mucus Watery mucus Many Rare Rare Gradual increase Few Many Many Gradual decrease Proliferative Proliferative Proliferative Quiescent Secretory Secretory Quiescent Quiescent Quiescent Proliferative Proliferative Proliferative 60 Figure 8. " Annual variation i n the percent of parabasal ( ), intermediate (•••) and superficial (— —) type epithelial cells in the vaginal f l u i d and in the stretchability ( ) of the vaginal f l u i d during the estrous cycle. - -. Symbols: horizontal bar, mean; vertical bar, 9 5 % confidence limits; numbers in parentheses, sample size; - and .+ numbers, months before and after estrus. 62 of seals were combined since no consistent d i f f e r e n c e s were found between them. A. Estrus On the basis of 16 complete estrous cycles i n 11.females, estrus occurred once a year i n 9 females. In 2 females estrus occurred a second time 3-5 weeks a f t e r the f i r s t . From 18 periods of estrus recorded, the duration was. usually 3-5 weeks with, a range of 1-9 weeks as i s i n d i c a t e d i n the following summarized data: Duration (weeks) 1 2 3 4 5 6 7 8 9 Frequency 1 3 4 4 4 0 0 1 1 When a second estrus occurred i t l a s t e d 2-3 weeks and was about 2-3 weeks shorter.than the f i r s t . Throughout estrus the va g i n a l and uterine e p i t h e l i a were p r o l i f e r a t i n g . The v a g i n a l epithelium was 10-15 c e l l s t h i c k and the outer layer was sloughing i n a l l areas (Plate V, A-B). E x f o l i a t e d . e p i t h e l i a l c e l l s were the most obvious c e l l s i n the vaginal smear (Plate VI, A-E). Intermediate type c e l l s ( c e l l s with l i v i n g nucleus and much cytoplasm) were the most common (74%). Other c e l l types included parabasal (14%) (immature, l i v i n g nucleus, l i t t l e cytoplasm) and s u p e r f i c i a l (13%) (mature, pyknotic nucleus, much cytoplasm). Occasionally parabasal and intermediate c e l l s were f i l l e d with large vacuoles g i v i n g the cytoplasm a foamy appearance. Anucleate squamous c e l l s ( keratinized or c o r n i f i e d ) were generally not present because the va g i n a l epithelium does not c o r n i f y . .. .. However, since the v e s t i b u l a r epithelium does, some c e l l s from t h i s region were o c c a s i o n a l l y c a r r i e d i n t o the vagina'during 63 sampling and thus.appeared i n the.smear. Other types of c e l l s found in|the smear.. during estrus included leucocytes (mostly polymorphs) and h i s t i o c y t e s . . Usually the vaginal fluid.was watery and contained l i t t l e or.no mucus and thus had a s t r e t c h a b i l i t y of 0 mm. This was because most mucus-secreting c e l l s i n the vagina and cervix were e x f o l i a t e d . The f l u i d originated.from the uterus. By the end of the f i r s t week of estrus the epithelium of the uterine endometrium reached maximum p r o l i f e r a t i o n . . During t h i s time mitoses were common, i n both the.surface and glandular e p i t h e l i a . In the. former, the c e l l s were t a l l p s e u d o s t r a t i f i e d columnar and i n the l a t t e r , low.columnar (Plate VII, A-C). C i l i a . occurred on many, c e l l s i n both.regions.. The surface epithelium was not secretory, as i n d i c a t e d by the lack of a p i c a l buds, but the glands were s l i g h t l y secretory since, a t h i n s e c r e t i o n occurred i n . -some lumina. The.stroma was edematous. When estrus l a s t e d more than one week, c e l l s i z e s and types remained the same. On the basis of 6 females, which were s a c r i f i c e d a f t e r being i n estrus f o r about.2, 4, 12, 22, 45, 60 days, females were ready to. ovulate throughout.estrus, but d i d not, a t l e a s t not u n t i l the end of estrus. Each female contained a large (14 mm, range 10-^20 mm) healthy, f o l l i c l e , i n one ovary. There was no. evidence of recently formed corpus albicans suggesting that females were not polyovulatory during extended periods of estrus. . B. Pseudopregnancy  At the end of estrus about h a l f the females ovulated as determined from uterine biopsies and by s a c r i f i c e d i n d i v i d u a l s . 64 Whether ovulation occurred or not the vaginal epithelium became quiescent ( t r a n s i t i o n a l ) by 5-8 weeks (Plate, C-D). By about one week a f t e r estrus the epithelium decreased i n thickness to 4-6 . . squamous c e l l s and by .2 weeks,..to 2T4 c e l l s . Over the next few weeks, the basal and.surface layers became cuboidal. In the sur-face p i t s , the outer, l a y e r gradually increased i n s i z e to a t a l l columnar and became secretory. These tis s u e changes were r e f l e c t e d i n the c e l l u l a r and mucus content of the vaginal f l u i d . E p i t h e l i a l c e l l s gradually, decreased in.number as d e s t r a t i f i c a t i o n proceeded and the amount of mucus increased as the surface p i t s became . secretory.. For. 1^2 weeks a f t e r estrus e p i t h e l i a l c e l l s were common and. composed mainly of parabasal c e l l s . (58%) .plus some intermediate types (39%) and a few s u p e r f i c i a l c e l l s (.3%). A f t e r 2 weeks, a l l types of e p i t h e l i a l c e l l s r a p i d l y decreased in. number, whereas, leucocytes r a p i d l y increased, by migrating through the thinning surface epithelium. The amount of mucus which occurred during the f i r s t two weeks was small and d i d not change the f l u i d ' s s t r e t c h a b i l i t y from 0 mm.. ..By 3-8 weeks, however, . s u f f i c i e n t mucus was secreted to increase s t r e t c h a b i l i t y t o . 4 mm. • By the time the epithelium was t r a n s i t i o n a l the vaginal f l u i d contained much thick mucus. Females which ovulated.experienced a 3-month perio d of pseudopregnancy with the uterine epithelium and corpus luteum undergoing essentially.. the same secretory changes as those pre-v i o u s l y described i n pregnant females during the delay of implanta-t i o n (p. 19. By 2 weeks a f t e r o v u l a t i o n the surface epithelium 65 developed many a p i c a l buds, increased i n height, l o s t most of. the c i l i a and was thrown.into shallow f o l d s . .. The glands increased . i n c o i l i n g and sec r e t i o n , the epithelium increased s l i g h t l y i n height and subnuclear vacuoles formed i n the epithelium (Plate VII, D—F). The endometrial stroma became, compact. Over the next 6 weeks the e p i t h e l i a of the surface and glands remained enlarged and secretory and subnuclear vacuoles became more abundant. A f t e r 8 weeks, the e p i t h e l i a of. both areas decreased i n s i z e and secretory a c t i v i t y and the number of subnuclear vacuoles decreased markedly. By 15 weeks a l l e p i t h e l i a were.cuboidal.or low columnar.and only s l i g h t l y s e c r e t o r y . i n d i c a t i n g an anestrous state. Five females, s a c r i f i c e d 2-3 days and 3,6,8 and.15 weeks a f t e r ovulation, had large (12-15.mm) healthy-appearing corpora lut e a . Those k i l l e d at 3, 6 and 8 weeks had many vacuolated l u t e a l c e l l s as found i n w i l d females during the delay of implanta-t i o n . The corpus of the. female, k i l l e d a t 15 weeks was beginning to degenerate since, connective t i s s u e was abundant, although-healthy l u t e a l c e l l s s t i l l made, up .most of the corpus. The f o l l i c l e s i z e was small (3^7mm).in a l l - females. C. Anestrus When ovulation d i d not occur, the e p i t h e l i a of the uterine , endometrium regressed.-to a quiescent state within 3^ .4 weeks. ..By t h i s time, the surface and glandular e p i t h e l i a changed to a nonsecretory cuboidal.type and.no subnuclear vacuoles formed.in the glands (Plate VII,. G-I).. ..Two unovulated females k i l l e d 7.and 13 weeks a f t e r estrus had only small f o l l i c l e s (5 mm) i n each ovary. 66 The uterine and vaginal e p i t h e l i a remained quiescent f o r u s u a l l y 7-10 months (Plate V, E-F; VIII, A-C). During t h i s time, the vaginal f l u i d was composed p r i m a r i l y of thick mucus, which stretched .7-1.1 mm, containing many leucocytes, but r a r e l y e p i t h e l i a l c e l l s (Plate VI, F ) . D. Proestrus The f i r s t marked signs of p r o l i f e r a t i o n i n the vaginal and uterine e p i t h e l i a occurred about 3-4 weeks before estrus. At t h i s time, c e l l s i n the b a s a l . l a y e r of the vaginal epithelium and i n the surface and glandular epithelium of the uterus began to d i v i d e and . increase i n s i z e . By about 2 weeks p r i o r to estrus the vaginal epithelium was 3-5 c e l l s t h i c k , the surface epithelium of the-uterus was composed of low or t a l l p s e u d o s t r a t i f i e d c e l l s and the uterine glands were more.abundant and were l i n e d with cuboidal or low columnar c e l l s . (Plate V, G-H; VIII, D-F). Usually the only change i n the vaginal f l u i d by t h i s time was a decrease i n v i s c o s i t y and an increase i n s t r e t c h a b i l i t y to an average of 1.4 mm. During the week before estrus, the vaginal epithelium increased i n thickness to 6-8 squamous c e l l s , and i n a few areas began to e x f o l i a t e . These were mainly parabasal (55%).and i n t e r -mediate (43%) c e l l s plus a few s u p e r f i c i a l c e l l s (6%). Leucocytes were common but reduced i n number as a r e s u l t of the thickened e p i t h e l i a l b a r r i e r which prevented c e l l u l a r , migration i n t o the v a g i n a l f l u i d . Because the mucus-secreting c e l l s of the surface p i t s were a l s o gradually sloughed, the amount of mucus i n the vaginal f l u i d was reduced which lowered the s t r e t c h a b i l i t y to .7mm. 67 The e p i t h e l i a of the endometrium increased i n height and some c e l l s possessed c i l i a . The endometrial stroma was edematous. Two females s a c r i f i c e d during the week before estrus had 9mm - f o l l i c l e s which .. were probably not yet of ovulatory s i z e , since females i n estrus have f o l l i c l e s 10-:20mm .in diameter (av. 14mm). 3. Time, of estrus . Figure 9 summarizes the onset time, of estrus i n i n d i v i d u a l females from each of the three populations kept.for.1T4 years. Only the times f o r :the f i r s t estrus which occur, i n a female, each year are given. These are accurate to +_ 3 days. The f i r s t females to come i n t o estrus were the four from Humboldt Bay which ranged from May 11 to June 22. The three females from northeastern. Vancouver Island began l a t e r , between Ju l y 10 and September 7, and. the 8 from southeastern.Vancouver Island began the l a t e s t from August 6 to September. 25. Within each population,... there was no tendency.for estrus to occur, e a r l i e r or l a t e r with increasing time i n c a p t i v i t y . A check on the cycles of four seals examined during two consecutive, years, i n d i c a t e that the timing may change . by up to 2 weeks e a r l i e r and .5. weeks l a t e r than the previous year. Thus, the timing of estrus in.the 3 groups d i f f e r e d . The onset of estrus i n females, from -Humboldt Bay did.not overlap those of seals from Vancouver Island.. Estrus i n females, from northeastern Vancouver-Island was s l i g h t l y e a r l i e r than i n the southeastern population \ r although much overlap .occurred. The timing of.estrus i n each captive group coincided with the expected range i n onset times of estrus in the respective 6 8 Figure 9. The seasonal timing of estrus i n i n d i v i d u a l females from southeastern Vancouver Island (0), northeastern Vancouver Island (•) and Humboldt Bay (A) during 1-4 years of c a p t i v i t y . The expected range ( ) i n onset times of estrus i n the respective f i e l d populations is given below the captive times. Symbol - — , connects consecutive .times of estrus in the same individual. 6 9 v 4 . h 1 3 " o - 9 n d co £-< A A \ v - v - \ — r N \ \ \ A * St O O OO^ O 00 00 o FIELD A -~\— o--o M J A MONTH 70 f i e l d populations.. The expected range i s the range of b i r t h times plus 6 weeks since the cycle i s monestrous and estrus begins about 6 weeks a f t e r the usual p a r t u r i t i o n time (p. 20 ). Thus, because b i r t h s occurred i n southeastern Vancouver Island from June 24 to September 6 (Fig. 3), the expected range of estrus was from e a r l y August to middle October. In c a p t i v i t y , the range i n females from t h i s area was early. August to l a t e September or within the expected f i e l d .range. Although no ..pupping data are a v a i l a b l e from northeastern Vancouver Island, b i r t h and estrus probably began s l i g h t l y e a r l i e r than the southeastern populations because pupping generally occurs e a r l i e r as one goes northward along the B r i t i s h Columbia coast (Bigg, 1969a). : Figure 10.confirms t h i s conclusion by a comparison of f e t a l lengths from the two areas. The slopes of the growth curves i n each population are not d i f f e r e n t (P. <.05).but the time i n t e r c e p t of the northern group does d i f f e r (P. <.01) by about 15 days e a r l i e r than the southern group. Thus, assuming equal growth rates f o r the r e s t of gestation, the northern group should give b i r t h and come i n t o estrus about two weeks e a r l i e r , during middle . Jul y to l a t e September. In c a p t i v i t y the range f o r t h i s group was from middle J u l y to ea r l y September and thus was also within the -expected time. As i n c a p t i v i t y the expected range of the north- . eastern and southeastern groups i n the f i e l d d i f f e r e d s l i g h t l y i n timing with much overlap. The best estimate .for the: b i r t h time of seals i n Humboldt Bay came from J. Turk, the aquarist who netted the Humboldt Bay 71 Figure 10. Comparison of standard length of 32 fetuses from southeastern Vancouver Island (0) and 16 from northeastern Vancouver Island (•) c o l l e c t e d during e a r l y and middle pl a c e n t a t i o n times. Equations are based on 16 January as day 1. 72 I 73 seals used i n t h i s experiment. He observed that pupping took place from March 25 to May 7. This time agrees generally with Rosenthal (1968) who noted that the f i r s t pups were born i n t h i s Bay on A p r i l 2. Also, I v i s i t e d .the region on May 19, 1967 and observed that there were many pups, most of which were nearly weaned making the peak b i r t h season i n . middle t o . l a t e A p r i l . Thus, the expected. . . range i n estrus.onset for. t h i s region.was e a r l y May to middle June, as i n the.captive group. As i n c a p t i v i t y , the. expected onset times of estrus i n f i e l d females.from Humboldt Bay were much e a r l i e r than those from Vancouver Island, with no overlap i n times. 74 DISCUSSION This study confirms the conclusion of the previous study that females are monestrous. .. A few i n d i v i d u a l s (2 out. of 11), however, have a second estrus s h o r t l y a f t e r the f i r s t . . The f a c t that estrus was prolonged and ovulation d i d not occur i n a l l seals are strong i n d i c a t i o n s that females are induced ovulators (Turner, 1963). Normally, i n induced ovulators, c e r v i c a l stimulation from mating causes ovulation, whereas,.in spontaneous ovulators, ovula-. .. t i o n occurs regardless of mating. Some female harbor s e a l s , however, ovulated despite the.absence of males..._ This may be explained by . . the f a c t that e i t h e r some in d i v u a l s eventually ovulatedwithout mating/ as with some other induced ovulators (Nalbandov, 1964, p.163) or some were stimulated to ovulate ..during the vaginal examinations. . Other data i n t h e . l i t e r a t u r e on.induced ovulation i n seals are inconclusive since.the.duration of estrus preceding ovulation i s not reported.. Harrison. (1963) noted that two captive harbor seals •' ovulated spontaneously and Craig (1964, .MS .1967, p, 26), found-that 5 out of 9 northern f u r seals had not ovulated a f t e r p o s s i b l y 10 days of estrus; 4 had ovulated. , . Pseudopregnancy i s shown to occur i n the harbor s e a l , the f i r s t time t h i s has been i n d i c a t e d f o r any s e a l . I n . t e r r e s t r i a l mammals pseudopregnancy follows an u n f e r t i l i z e d o v u l a t i o n and occurs when the corpus luteum.from t h i s ovulation secretes progesterone f o r a prolonged period,...usually f o r about-half the normal duration, of a pregnancy (Turner, 1963). This r e s u l t s i n v i r t u a l l y the same . progesteronic changes taking place i n the u t e r i n e e p i t h e l i a as 75 during a pregnancy. In the harbor seal pseudopregnancy lasted for 2-3 months or about the same duration as the delay of implantation ( 2 - 2 1/2 months) in pregnant females. The histological appearance of the ovaries, uterus and vagina during the other reproductive events in captivity, such as anestrus, proestrus and estrus were identical with those described previously in females from the f i e l d for anestrus,(nonpregnant), second half of lactation and estrus respectively. The vaginal smear of the harbor seal was shown to be a good indicator of estrus. The vaginal smear cycle has not been previously described for any other species of marine mammal. The cycle was basically the same as that described for domesticated mammals in that the number and type of epithelial cells exfoliated vary during the cycle (Cole and Cupps, 1959; Hafez, 1970). The harbor seal, however, has a much more clearly defined cycle than most mammals since epithelial cells are exfoliated only around the time of estrus wheres, in many other mammals some epithelial cells are exfoliated at a l l times. The main difference between the vaginal smear of the harbor seal and the domestic species during estrus i s that intermediate type epithelial c e l l s are the most common in the former and superficial cells in the latter. This difference probably only indicates a greater rate of exfoliation i n the seal which would prevent complete maturation of cells before exfoliation. The fact that the duration and timing of the cycle in; the*three captive populations was the same as in the respective f i e l d populations i s significant with respect to the control of reproductive timing i n 76 each population.- I t ind i c a t e s that the main factors which c o n t r o l the annual timing of estrus i n the f i e l d were also present e i t h e r within the captive environment or within the animals. Wild mammals do not always keep the same reproductive timing when they are brought i n t o c a p t i v i t y . Marshall (1942, p. 69, 70) and A s d e l l (1965, p. 12) report that the r a b b i t , cat, dog and bison are seasonal breeders, i n the w i l d but continuous breeders i n captivity,. The present study i n d i c a t e s that an annual v a r i a t i o n i n n u t r i t i o n d i d not. maintain the p r e c i s e l y timed annual cycle since the food t y p e l o t . a n d quantity remained the same throughout the . year i n c a p t i v i t y . . Photoperiod and temperature could be environ-mental cues since the annual v a r i a t i o n i n c a p t i v i t y was about the same as i n the f i e l d . However., i t i s u n l i k e l y that water tempera-ture was an important f a c t o r . Although i t a f f e c t s reproductive timing i n some t e r r e s t r i a l animals, where the annual a i r temperature flu c t u a t e s markedly, seals l i v e i n an environment where these f l u c t u a t i o n s a r e - r e l a t i v e l y small, and thus would l e s s l i k e l y to be an important cue. Also, because water temperatures d i f f e r between c l o s e l y located areas (Fig. 7B) , within the probable home range of a population, i t i s u n l i k e l y to be a us e f u l cue f o r accurately timing the cyc l e . Hafez- (1968) suggests that, even i n t e r r e s t r i a l mammals, .temperature i s r e l a t i v e l y unimportant because of marked r e g i o n a l d i f f e r e n c e s . From the present data, nothing can be s a i d about the roles, of photoperiod or endogenous c y c l e s . The data suggest^.however, that genetic v a r i a t i o n i s important, i n maintaining d i f f e r e n c e s in...the timing of estrus 77 between populations. . This was i n d i c a t e d by. the. f a c t that.the tim-. ing of estrus remained d i f f e r e n t i n the three populations despite being kept under the same environmental.conditions f o r up to 4 years Genetic d i f f e r e n c e s .in the c o n t r o l of reproductive timing between populations.of the. same species have also been shown, i n the same . manner, i n mink (Hannson,... 1947), w h i t e - t a i l e d deer (Adams, .1960) and sheep. (Smith, 1967). This i n d i c a t e s that v a r i a t i o n i n reproduce t i v e timing of the harbor.seal r e s u l t s from population evolution. . . rather than population a c c l i m a t i z a t i o n i n d i f f e r e n t environments.. Thus, each population.with a diffe r e n t , b i r t h season i s a separate . . genotype. This agrees with the suggestion of Mayr (1966) that geographical v a r i a t i o n s i n animal characters are.usually adaptive. The evolution of breeding seasons i s generally thought, to involve ultimate and proximate factors (Baker, 1938; Lack, 1954). The ultimate f a c t o r .establishes the timing of the c y c l e through natural s e l e c t i o n and proximate f a c t o r s maintain the timing, through p h y s i o l o g i c a l means. Ultimate f a c t o r s , such as food supply, predation o r weather,, s e l e c t f o r a genotype with adaptations which produce the optimum reproductive timing f o r s u r v i v a l of the population. Proximate f a c t o r s , such.as photoperiod or temperature. act as cues f o r these.adaptations to maintain the optimum timing .. f o r the genotype. In the case of the harbor s e a l , the ultimate f a c t o r seems to have v a r i e d .regionally and selected, f o r l o c a l genotypes with adaptations .to u t i l i z e the proximate factors, i n a s p e c i f i c manner so as to maintain a unique timing f o r each.geno-type. ... 78 Stutz (1967) also suggested that the harbor s e a l i n the eastern P a c i f i c may be divided i n t o g e n e t i c a l l y d i s t i n c t populations. He showed that seals from B r i t i s h Columbia and Alaska could be. d i v i d e d i n t o three large populations on the basis of pelage pattern f r e -quencies. He hypothesized that these d i f f e r e n c e s may have evolved because t h i s s e a l i s nonmigratory and inhabits a narrow l i t t o r a l zone along the coast. This probably r e s u l t e d i n a low genetic interchange between adjacent populations with the subsequent forma-t i o n o f d i s c r e t e breeding stocks. The same i s o l a t i n g p r i n c i p l e also most reasonably explains how regional d i f f e r e n c e s i n reproduc-t i v e timing have evolved, given a r e g i o n a l l y v a r i a b l e s e l e c t i v e f a c t o r . That a s e l e c t i v e f a c t o r does e x i s t i n the harbor, seal,, p a r t i c u l a r l y where c l i n e s occur, i s supported by Mayr's (1966) working hypothesis that c l i n a l v a r i a t i o n i n d i c a t e s a geographically v a r i a b l e s e l e c t i v e f a c t o r . Some v a r i a t i o n s i n timing, however, may not be.adaptive, but rather may be determined by gene flow, p a r t i c u l a r l y within c l i n e s . C l i nes are the product of two forces s e l e c t i o n , which tends to make each population uniquely adapted to the l o c a l environment, and gene flow which, tends to make a l l populations of a species the same (Mayr, 1966, p. 361). Thus, s e l e c t i o n may have, operated -only i n c e r t a i n regions but because of gene flow between these populations, intermediately located populations would have intermediate c h a r a c t e r i s t i c s which produces the character gradient. What the s e l e c t i v e f a c t o r . i s . i n the harbor.seal, i s not completely obvious. One problem i n i d e n t i f y i n g the ultimate 79 f a c t o r , as noted by S a d l e i r (1969, p. 48), i s to determine the stage of the cycle which i s the most susceptible to environmental influences. He suggests that i n mammals t h i s time i s from l a t e term u n t i l the end of l a c t a t i o n . Lack (1954) argued that i n b i r d s the hatching time i s the most important. In general, the l i t e r a t u r e on seals has stressed the importance of the b i r t h season i n e s t a b l i s h i n g reproductive timing. A v a r i e t y of ultimate factors have been suggested, Davies (1957), f o r example, suggested that the b i r t h season of the grey s e a l (Halichoerus grypus) may be determined by the presence or absence of seasonally forming i c e . Ice formation, however, would not be responsible f o r regional v a r i a t i o n i n the b i r t h season of the harbor s e a l i n the eastern P a c i f i c since it.forms seasonally only i n p a r t of Alaska. Ling (1969) proposed that the timing of the annual cycle ( b i r t h s , molt, movements) of s e v e r a l species i n h a b i t i n g the same small i s l a n d s may be staggered to reduce competition f o r s u i t a b l e food and space. Although the cycle of the harbor s e a l may be c o n t r o l l e d i n some i n s u l a r populations i n t h i s way, most populations i n h a b i t mainland areas or i s l a n d s without competition with other s e a l s . F i s h e r (MS, 1954), working on the harp s e a l (Pagophilus groenlandicus) and Ca r r i c k ejt al_ (1962) , working on the southern elephant s e a l (Mirounga leonina), postulated that the b i r t h season i s determined by the seasonal a v a i l a b i l i t y of invertebrate food f o r the newly weaned pups. In t h i s case, pups would be born and weaned i n time to take advantage of a short food abundance.... A review of the l i t e r a t u r e on the food of newly weaned harbor 80 seals i n d i c a t e s that the food type i s s p e c i f i c f o r a short time and thus may also be of s e l e c t i v e value. For the f i r s t few weeks a f t e r weaning, pups feed mainly on the shrimp Crangon (Crago) before changing to a f i s h d i e t i n a l l areas recorded of the P a c i f i c and A t l a n t i c . These shrimps were abundant i n the. stomachs of newly weaned pups i n Holland (Havinga, .1933), England . (Sergeant, 1951), Maine, United States (Fisher, MS, 1949; personal communication), Skeena River and southeastern Vancouver.Island, Canada (Fisher, 1952, personal communication; Bigg, unpublished data). Also Scheffer (1928) noted that shrimps occurred i n the stomachs of very small seals (presumably pups) i n Washington State,. United States. The f a c t that Crangon is.widely d i s t r i b u t e d i n the c o a s t a l regions of the A t l a n t i c and P a c i f i c (DeMan, 1920), and has seasonal spawning migrations from shallow to deep water ( I s r a e l , 1936; Lloyd and Yonge, 1947; P r i c e , 1962) add to the p o s s i b i l i t y of i t being the ultimate f a c t o r f o r the species. I t i s not p o s s i b l e yet, how-ever, to c o r r e l a t e reproductive timing of the s e a l with shrimp movements since better data are needed on the species of shrimp eaten and on the timing of shrimp migrations. One would expect that the seasonal migrations (seasonal a v a i l a b i l i t y ) of Crangon would vary r e g i o n a l l y by up to 5-6 months i n the same pattern of v a r i a t i o n as the b i r t h season of harbor s e a l s . Although no data on t h i s p o i n t are a v a i l a b l e f o r Crangon, the p o s s i b i l i t y e x i s t s because the northern shrimp (Pandalus b o r e a l i s ) has. r e g i o n a l . . d i f f e r e n c e s i n the timing of the spawning season of up to 4-5 months (Haynes and Wigley, 1969). 81 Presumably, the s e l e c t i v e e f f e c t of Crangon i s about the same throughout the d i s t r i b u t i o n of the s e a l since the duration of the b i r t h season i s about the same (1 T 2 1/2 months) i n a l l populations. In other vertebrates the ultimate f a c t o r does not n e c e s s a r i l y act i n the same degree i n a l l populations. One species of sparrow, . . f o r example, tends to have longer breeding seasons toward the equator ( M i l l e r , 1960) and d i f f e r e n t races of sheep have varying lengths of breeding seasons (Robinson, 1951). On the basis of the data c o l l e c t e d i n t h i s study and.the pre-vious study I suggest the following explanation f o r the evolution of the seasonal reproduction i n d i f f e r e n t populations of harbor s e a l s . Within each population the timing of the cycle was deter-mined by a s e l e c t i v e f a c t o r , p o s s i b l y Crangon. S e l e c t i o n was against i n d i v i d u a l s which were weaned too e a r l y or too l a t e to feed on the b r i e f abundance of food. Since i n many regions the seasonal timing of food abundance d i f f e r e d , the optimum times to wean the young d i f f e r e d between populations. S e l e c t i o n i n each population was f o r a genotype with adaptations to u t i l i z e proximate factors i n a s p e c i f i c manner so that mating, p a r t u r i t i o n and weaning occurred at the appropriate time. Nothing can be s a i d at t h i s stage about . . the adaptations because nothing i s known of the proximate f a c t o r s which c o n t r o l estrus w i t h i n d i f f e r e n t populations. 82 Plate V. Vaginal biopsies at d i f f e r e n t stages of the estrous c y c l e . Scale = 50 u A Estrus; surface epithelium i s s t r a t i f i e d squamous; surface p i t s are widely separated. B Surface epithelium of A showing outer squamous c e l l s . C Anestrus, 6 weeks a f t e r estrus, subsurface p i t s are small and close together; surface epithelium i s t r a n s i t i o n a l . D Surface epithelium of C showing the cuboidal outer and inner layers E Anestrus, 6 weeks before estrus, surface p i t s small, surface epithelium i s t r a n s i t i o n a l . F Surface epithelium of E i n d i c a t i n g the presence of mucus s e c r e t i n g c e l l s at the base of the surface p i t . G Proestrus, 2 1/2 weeks before estrus, subsurface p i t s enlarged and surface epithelium p r o l i f e r a t i n g . H Surface epithelium of G showing the formation of a t h i r d c e l l l a y e r . 8 4 Plate VI. C e l l s i n the vaginal f l u i d during the. estrous c y c l e . Scale = 20 A. Parabasal e p i t h e l i a l c e l l . B. Intermediate e p i t h e l i a l c e l l . C. S u p e r f i c i a l e p i t h e l i a l c e l l . D. Intermediate e p i t h e l i a l foam c e l l . E. T y p i c a l smear during estrus which contains mainly intermediate c e l l s ( i ) , some parabasal (b) and s u p e r f i c i a l (s) c e l l s , few leucocytes (1) and no mucus. F. T y p i c a l smear during anestrus which contains mainly leucocytes and mucus and no e p i t h e l i a l c e l l s . 85 86 Plate VII. Uterine biopsies between estrus and anestrus. Scale = 50 y A Estrus, glands enlarged and stroma i s edematous and l i n e s uterine lumen evenly. B Surface epithelium of A showing t a l l columnar e p i t h e l i a l c e l l s , some with c i l i a . C Uterine gland of A showing low columnar c e l l s with t h i n s e c r e t i o n i n lumen. D Pseudopregnancy, 8 weeks a f t e r ovulation; note i r r e g u l a r surface l i n i n g and densely c o i l e d glands. E Surface epithelium of D showing p s e u d o s t r a t i f i e d epithelium. F Uterine gland of D showing subnuclear vacuoles (arrow) and luminal section. G Anestrus, 7 weeks a f t e r estrus, with no ovulation, surface epithelium i s even and glands small. H Surface epithelium of G showing cuboidal c e l l s . I Uterine gland of G showing back o f luminal s e c r e t i o n and cuboidal c e l l s and no subnuclear vacuoles. 88 Plate VIII. Uterine biopsies between anestrus and proestrus. Scale - 50 y A Anestrus, 6 weeks before estrus, surface epithelium i s even, glands small and sparce. B Surface epithelium of A showing cuboidal c e l l s . C Uterine gland of A showing cuboidal c e l l s D Proestrus, 2 1/2 weeks before estrus, surface epithelium enlarged and even, glands more abundant and enlarged. E Surface epithelium of D showing, p r o l i f e r a t i o n . F Uterine gland of D showing low columnar c e l l s . 89 90 III. Effect of photoperiod on estrus. 91 ABSTRACT Long (18 hr) and short (6 hr) daylengths r e s p e c t i v e l y hasten and delay the onset time of estrus i n some females from southeastern Vancouver Island and Humboldt Bay. These daylengths extend the perio d over which estrus occurs from an expected 2 1/2 months to 7 1/2 months i n females from southeastern Vancouver Island and from 1 1/2 months to 4 1/2 months i n those from Humboldt Bay. Some i n d i v i d u a l s from both populations do not respond to these daylengths. I n d i v i d u a l d i f f e r e n c e s i n response may be due to v a r i a t i o n s i n the strength of an endogenous reproductive c y c l e . The r o l e of photoperiod i n the two populations i s probably to set the timing of the endogenous cycle so that estrus keeps i n phase with a p a r t i c u l a r time of the year. Population d i f f e r e n c e s i n the timing of estrus may be due to p o p u l a t i o n - s p e c i f i c responses to photoperiod. 92 INTRODUCTION In the previous study I suggested that r e g i o n a l v a r i a t i o n s i n reproductive timing of the harbor s e a l r e s u l t e d from population-s p e c i f i c adaptations which u t i l i z e proximate factors in. a s p e c i f i c manner to maintain d i f f e r e n t breeding seasons. The types of adaptations were not discussed because l i t t l e is.known o f the proximate factors which c o n t r o l the-annual timing of estrus except that n u t r i t i o n , temperature and negative s t e r o i d feedbacks were u n l i k e l y to be normally important. Since photoperiod i s generally considered to be the most important environmental proximate f a c t o r i n mammals (Sadleir, 1968), the purpose .of t h i s study i s to t e s t the e f f e c t of t h i s f a c t o r on the seasonal timing of estrus i n two populations of harbor seals and to discuss the r e s u l t s as they r e l a t e to adaptations i n each population. The. p o s s i b l e existence of an endogenous cycle i s al s o discussed. The seals used i n t h i s study came from southeastern Vancouver Island and Humboldt Bay and were chosen because t h e i r reproductive timing d i f f e r e d and the estrous cycles were already described i n c a p t i v i t y under n a t u r a l photoperiods. Seals from each p o p u l a t i o n -were put under e i t h e r continuous long (18 hr) or short (6 hr) daylengths to determine whether the next estrus was hastened or. delayed. Most photoperiodic experiments on reproductive timing . i n mammals use f i x e d l i g h t and dark r a t i o s to simulate e i t h e r constant winter or summer l i g h t i n g ( S a d l e i r , ,1969, p. 87). 93 MATERIALS AND METHODS. . . Four nonpregnant adults from southeastern Vancouver Island, B r i t i s h Columbia and 4 from Humboldt Bay, C a l i f o r n i a , were used i n t h i s study. Each female had previously experienced from 1 to 3 breeding seasons i n c a p t i v i t y under n a t u r a l photoperiods before being placed under a r t i f i c i a l daylengths. On October 1 and December 20, 1969 r e s p e c t i v e l y , about 5-17 weeks a f t e r the l a s t estrus, females from each population were s p l i t i n t o p a i r s and placed i n t o 4 tanks, each covered by a l i g h t -proof hut. Each hut was about 12* by 16', 8' - 10' high and was il l u m i n a t e d by f i v e 300 watt filament lamps and two 40 watt fluorescent lamps (4' long) mounted 3-6? over the water surface. L i g h t i n t e n s i t y at the water surface i n the centre of the tank was 160 foot candles and at the edge, 20. These i n t e n s i t i e s were considered adequate to t e s t photoperiod since Yeates (1949), Bissonette (1932) and Hart (1951) recorded photoperiodic e f f e c t s on reproductive timing i n sheep and f e r r e t s with as low as 3-24 foot candles. E l e c t r i c fans v e n t i l a t e d the huts by c i r c u l a t i n g fresh a i r through l i g h t traps. Pairs of seals.from each population were given e i t h e r 6 hr (0900-1500) or 18 hr (0300-2100) daylengths u n t i l the next estrus occurred. Maximum and minimum water tempera-tures were taken d a i l y i n one tank to determine whether the presence of the hut a l t e r e d the temperature compared to the outside tanks. The holding tanks, husbandry and v a g i n a l examinations f o r reproductive condition was.the same as described in.the previous study. Females were considered to be i n estrus when the v a g i n a l 94 smear contained primarily intermediate type epithelial c e l l s , few leucocytes and watery .vaginal.fluid as described earlier.for this reproductive stage. 95 RESULTS The average monthly v a r i a t i o n i n water temperature i n the enclosed tank remained with.+_ 1/2C of outside tank tested i n d i c a t -ing that the presence of the huts had a n e g l i g i b l e e f f e c t on water temperature. Therefore, by the same arguments given i n the previous study (p. 76 ), temperature remained unimportant i n the annual c o n t r o l . Figure 11 shows the e f f e c t of long and short daylengths on the onset time of estrus i n females from.both populations by com-paring the time of estrus of i n d i v i d u a l s under a r t i f i c i a l daylengths with those under n a t u r a l photoperiods i n c a p t i v i t y during the per i o d immediately preceding l i g h t treatments and .with the expected onset times i n the respective f i e l d populations.• Changes i n the timing of estrus under a r t i f i c i a l daylengths are considered meaning-f u l only i f they occur outside the expected time i n the f i e l d . The previous study showed that when females were brought i n t o c a p t i v i t y and kept under n a t u r a l photoperiods, they came i n t o estrus within the same range of time as expected i n the f i e l d . Both populations of seals reacted to long and short daylengths i n about the same manner. Under 18 hr of l i g h t , one female from southeastern Vancouver Island both from Humboldt Bay came i n t o estrus 4-8 weeks e a r l i e r than the e a r l i e s t expected time i n the f i e l d . The remaining females from southeastern Vancouver Island came i n t o estrus i n the same week.as the previous year and thus wit h i n the expected range. Under .6 hr photoperiods, ..one female from each region came i n t o estrus 4^16 weeks l a t e r than expected 96 Figure : 11. E f f e c t of 18 hr and 6 hr daylengths on the onset time .of estrus i n i n d i v i d u a l females from southeastern Vancouver Island and Humboldt Bay. Symbols: , 18 hr daylength; , 6 hr daylength; 0, onset time of estrus under natural daylength i n c a p t i v i t y ; • , onset time of estrus under a r t i f i c i a l daylength i n c a p t i v i t y ; h o r i z o n t a l bar, expected onset time of estrus i n the f i e l d . 9 8 i n the f i e l d . The remaining females from Vancouver Island and Humboldt Bay came i n t o estrus with 2 weeks o f the previous year and wi t h i n the expected f i e l d range of times. The data i n d i c a t e that long daylengths hastened and short daylengths delayed estrus i n some i n d i v i d u a l s from both populations. The combined e f f e c t of long and short daylengths increased the range of onset times of estrus i n females from southeastern Vancouver Island from the expected 2 1/2 months to 7 1/2 months.and i n females from Humboldt Bay from 11/2 to 4 1/2 months. : However, photoperiod had no e f f e c t on some i n d i v i d u a l s from both populations. 99 DISCUSSION Normally i n ph o t o p e r i o d i c a l l y s e n s i t i v e mammals, long day-lengths hasten the onset time of estrus i n species which.breed i n the spring and summer and short daylengths delay estrus; whereas i n species which breed i n the f a l l , the reverse i s true (Nalbandov, 1964). Since the two populations of harbor seals examined breed during the spring and summer i n the f i e l d they responded to long and short daylengths i n the same manner as other mammals which breed at t h i s time. The f a c t that some seals d i d not respond.to e i t h e r long or short daylengths in d i c a t e s that i n d i v i d u a l v a r i a t i o n s i n response e x i s t . However, such v a r i a t i o n are not unusual, i n ph o t o p e r i o d i c a l l y s e n s i t i v e mammals. Hart (1951) and Thomson (1954), f o r example, gave f e r r e t s extra l i g h t during the winter which induced some to come i n t o estrus e a r l i e r than controls but d i d not a f f e c t others. Donovan (1967), s i m i l a r l y reported that some female f e r r e t s kept under 8 hr daylengths from b i r t h came i n t o estrus at the same time as expected under outdoor l i g h t i n g , whereas, others d i d not come i n t o estrus even a f t e r s e v e r a l a d d i t i o n a l months. He noted further that some females kept under 16 hr daylengths came i n t o estrus within a few months a f t e r the s t a r t of the treatment and others d i d not come i n t o estrus even a f t e r several years. Radford (1961, p. 153) found that some Merino sheep kept under 12 hr daylengths were seasonal breeders and others were continuous breeders. The reasons f o r i n d i v i d u a l v a r i a t i o n s i n response to photo-i i i . • 100 perio d i n mammals are generally poorly understood. Donovan (1967) a t t r i b u t e d v a r i a t i o n s i n the f e r r e t to seasonal di f f e r e n c e s i n response to the same treatment and to i n d i v i d u a l d i f f e r e n c e s i n the strength of an endogenous sexual rhythm. Although l i m i t e d , data from the current study i n d i c a t e that v a r i a t i o n s i n response of the harbor s e a l occurred within rather than between treatments. There were no marked dif f e r e n c e s i n response to photoperiod between t r e a t -ments begun on October 1. compared to December 20, but there were dif f e r e n c e s w i t h i n the long and short day treatments at both times. Thus, i n d i v i d u a l d i f f e r e n c e s i n response to photoperiod i n the harbor s e a l may be due to i n d i v i d u a l d i f f e r e n c e s i n the strength of an endogenous cy c l e . The question as to whether a true annual sexual rhythm e x i s t s i n mammals i s c o n t r o v e r s i a l . In a recent review of the l i t e r a t u r e on sheep and f e r r e t reproduction, S a d l e i r (1969) argued that there i s no convincing evidence f o r the existence of an annual sexual rhythm only a seasonal peri o d of re f r a c t i v e n e s s which does not maintain accurate timing over successive years. He concluded that the seasonal nature of breeding i n t h i s group i s e n t i r e l y dependent upon external environmental s t i m u l i . On the other hand, Pengelley and Asmundsen (1970) recently gave conclusive evidence f o r an i n t e r n a l annual rhythm i n hibernating mammals and migratory b i r d s . Whether the harbor s e a l has e i t h e r a true annual rhythm or some other endogenous mechanism which produces an annual p e r i o d i c i t y f o r one or. two years must be determined by. f u r t h e r experimentation. In e i t h e r case the f a c t o r i s accurate i n some i n d i v i d u a l s since 101 they came i n t o estrus within 2 weeks of. the previous year regard-les s of whether they received long or short daylengths. To cover both p o s s i b i l i t i e s I w i l l r e f e r to t h i s f a c t o r as an endogenous cycle f o r the remainder of the c y c l e . The degree to which the annual cycle of animals i s dependent on photoperiod f o r r e g u l a r i t y v a r i e s between species. Wolfson (1964, p. 4) described three categories of dependence. In the f i r s t category photoperiod merely acts to phase a strong endogenous cycle w i t h i n the seasons. In the second category i t controls the seasonal phasing and frequency of an endogenous cycle and i n the t h i r d category i t completely controls the phasing, frequency and existence of the c y c l e . The harbor s e a l p r o b a b l y . f a l l s i n the f i r s t category since a strong annual endogenous.cycle seems to e x i s t which, depending on the i n d i v i d u a l , i s modified by photo-period. This conclusion agrees with Farner (1965, p. 361) who stated that photoperiodic controls are not r i g i d i n mammals, although they do provide important p r e c i s e timing f o r events that would occur anyway, p o s s i b l y because of crude endogenous quasi-annual timers. Although the endogenous cycle of the harbor s e a l was unaffected by photoperiod i n some i n d i v i d u a l s over a one year period, i t seems l i k e l y that photoperiod would have a modifying e f f e c t over several years. This i s suggested by the f a c t that other endogenous cycles, such as : c i r c a d i an. rhythms., i n other species, are seldom completely accurate to 24 hrs and .thus eventually get out of phase with night and day. and need.resetting usually by 102 photoperiod (Menaker and Eskin,. 1968). A r e l a t i v e strong endogenous reproductive cycle i n the harbor s e a l may have evolved to prevent wasteful sexual a c t i v i t y throughout, most: of the year when breeding attempts would not lead to successful r a i s i n g of the pups. The mechanism by which photoperiod sets the annual timing of O n breeding i n mammals i s v i r t u a l l y vknown although the photosexual pathway seems to involve the o p t i c nerves, c e r v i c a l ganglia and p i n e a l gland (van Tienhoven, 1968). During dark periods the p i n e a l gland secretes melatonin, which depresses ovarian a c t i v i t y , whereas during the day melatonin i s not secreted and .ovarian a c t i v i t y increases. Whether mammals measure photoperiod by a c i r c a d i a n rhythm of p h o t o s e n s i t i v i t y as i n b i r d s i s not known. Whatever the mechanism i s by which photoperiod sets the endogenous c y c l e , i t i s presumably g e n e t i c a l l y f i x e d f o r each species. This f a c t i s shown by the v a r i e t y of breeding times which d i f f e r e n t p h o t o p e r i o d i c a l l y s e n s i t i v e species of mammals are w e l l known to have. I suggest that, within the harbor s e a l , t h i s g e n e t i c a l l y f i x e d response to photoperiod i s an important adaptive f a c t o r i n the c o n t r o l of di f f e r e n c e s i n reproductive timing between popula-t i o n s . While most species of p h o t o p e r i o d i c a l l y s e n s i t i v e mammals are divided i n t o e i t h e r long-day (spring-summer) or short-day ( f a l l ) breeders (Wolfson, 1964, p. 35), the harbor s e a l breeds during both long- and short-days depending on the population. This suggests that the population s p e c i f i c adaptation f o r maintaining r e g i o n a l d i f f e r e n c e s i n d i c a t e d e a r l i e r (p. 81 ) i s a population-103 s p e c i f i c response to photoperiod.. Thus, each.population of harbor seals which has a d i f f e r e n t timing f o r reproduction may.use photoperiod i n a unique manner, so.that estrus w i l l occur at a p a r t i c u l a r time of the year. There i s good evidence from other studies that population-s p e c i f i c responses to photoperiod. occur within the same species. This i s w e l l known f o r reproductive: growth i n plants (Meyer, et a l 1960, p. 474) and pupation rates i n i n s e c t s ( D a n i l e v s k i i , 1965). Data are l e s s abundant i n vertebrates but nonetheless imply t h i s i n the breeding season of the sparrow ( M i l l e r , 1960) and sheep (Robinson, 1951; S a d l e i r , 1969) and the metabolic responses of the beaver (Aleksiuk and Cowan, 1969). Just how each population of harbor s e a l would respond d i f f e r e n t l y to photoperiod must be determined by further, experi-mentation. However, on the basi s . o f studies on other species two ways are p o s s i b l e . F i r s t , as suggested by D a n i l e v s k i i (1965) f o r d i f f e r e n t populations of i n s e c t s , each population could respond d i f f e r e n t l y to the same number, of hours of. l i g h t per.day. Here, fo r example, one population may be stimulated maximally to breeding condition by 16 hr of l i g h t and another ..population by . 18 hrs. Second, as suggested by Wolfson (1964), f o r d i f f e r e n t species of mammals, each population could respond to a s p e c i f i c phase of the inc r e a s i n g or decreasing daylength. In t h i s case, . one population may be stimulated.to breed by an e a r l y p a r t of . the i n c r e a s i n g . l i g h t regime i n spring,, another by. a. l a t e r p a r t . and s t i l l another by a p a r t of the decreasing l i g h t c y c l e . 104 The adaptive s i g n i f i c a n c e of a p o p u l a t i o n - s p e c i f i c response to photoperiod i n the harbor s e a l seems c l e a r . In a l o c a l l y adapted population, the annual l i g h t cycle would set the timing of estrus to a p a r t i c u l a r time of the year. This time would, be important to the s u r v i v a l of the population because i t would also set the time of conception, implantation, b i r t h and weaning. As discussed i n the previous study (p. 80 ) the seasonal timing of weaning may be the s e l e c t i v e (ultimate) f a c t o r i n e s t a b l i s h i n g reproductive timing. Since the optimum time to. wean the young probably v a r i e s between populations the proximate mechanisms must also change to adjust the time of estrus. A genetic adjustment i n the mechanism by which photoperiod sets the.annual timing of the endogenous cycle would be of obvious s e l e c t i v e value. The conclusion that d i f f e r e n t populations of harbor s e a l are adapted to l o c a l environments i s of considerably p r a c t i c a l import-ance. Mayr (1966, p. 318) stated that w i l d l i f e managers frequently give l i t t l e a t t e n t i o n to the s p e c i f i c p h y s i o l o g i c a l adaptations of l o c a l populations. This has r e s u l t e d i n the l o s s of m i l l i o n s of. d o l l a r s from unsuccessful transplant of animals to unsuitable environments. The harbor s e a l would seem to be a good.example of where a transplanted population, would qui c k l y :die o f f i f ' . . c onsideration was not given to the o r i g i n . o f the stock and to the transplant environment. i 105 ACKNOWLEDGMENTS This research was d i r e c t e d and funded by Dr. H. D. F i s h e r , Department of Zoology. I appreciated h i s help and encouragement during the research and i n the preparation of the manuscript. The following people also c r i t i c a l l y read the manuscript: Drs. N. Auersperg, M. J . Taylor, A. M. Perks and H. C. Nordan, Department of Zoology and Dr. R. M. T a i t , Department of Animal Science. Dr. H. C. Nordan kin d l y constructed the holding tanks f o r captive seals and provided working space at the Vivarium. 106 LITERATURE CITED Adams, W. H. 1960. Population ecology of white-tailed deer in northeastern Alabama. Ecol. 41: 706-715. Aleksiuk, M., and I. McT. Cowan. 1969. 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