Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The reproductive cycle of the river otter, Lutra canadensis, in the marine environment of southwestern… Stenson, Garry B. 1985

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1985_A1 S72.pdf [ 14.14MB ]
Metadata
JSON: 831-1.0096748.json
JSON-LD: 831-1.0096748-ld.json
RDF/XML (Pretty): 831-1.0096748-rdf.xml
RDF/JSON: 831-1.0096748-rdf.json
Turtle: 831-1.0096748-turtle.txt
N-Triples: 831-1.0096748-rdf-ntriples.txt
Original Record: 831-1.0096748-source.json
Full Text
831-1.0096748-fulltext.txt
Citation
831-1.0096748.ris

Full Text

THE REPRODUCTIVE CYCLE OF THE RIVER OTTER, LUTRA CANADENSIS, IN THE MARINE ENVIRONMENT OF SOUTHWESTERN BRITISH COLUMBIA by GARRY B. STENSON B. Sc., U n i v e r s i t y of A l b e r t a , 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September 1985 © Garry B. Stenson, 1985 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 for 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 or her representatives. I t 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 QQ I c><^  ^  The University of B r i t i s h Columbia 1956 Main Ma l l Vancouver, Canada V6T 1Y3 Date DE-6 (3/81) i i ABSTRACT Knowledge of r e p r o d u c t i o n i n the r i v e r o t t e r , L u t r a  c a n a d e n s i s , and of the f a c t o r s which c o n t r o l the r e p r o d u c t i v e c y c l e , i s l i m i t e d . The r e p r o d u c t i v e b i o l o g y of r i v e r o t t e r s i n the marine environment of B r i t i s h Columbia, and some of the f a c t o r s which may i n f l u e n c e t h e i r r e p r o d u c t i v e c y c l e , i n p a r t i c u l a r , the e f f e c t of p h o t o p e r i o d , were examined. The changes t h a t o c c u r r e d i n the anatomy, h i s t o l o g y and e n d o c r i n o l o g y of the annual r e p r o d u c t i v e c y c l e were determined i n w i l d (226 male, 146 female) and c a p t i v e (9 male, 5 female) o t t e r s . R e p r o d u c t i v e c y c l e s were s i m i l a r i n both groups. As i n the f r e s h - w a t e r environment, c o a s t a l o t t e r s a r e s e a s o n a l b r e e d e r s w i t h a p r o l o n g e d , o b l i g a t o r y p e r i o d of embryonic di a p a u s e p r i o r t o i m p l a n t a t i o n . Changes were s i m i l a r t o those r e p o r t e d i n o t h e r m u s t e l i d s . In males, development of the s e m i n i f e r o u s t u b u l e s was d i v i d e d i n t o a s e r i e s of n i n e d i s t i n c t phases used t o q u a n t i f y the degree of r e p r o d u c t i v e a c t i v i t y p r e s e n t . T e s t o s t e r o n e c o n c e n t r a t i o n s were h i g h l y c o r r e l a t e d t o changes i n t e s t i c u l a r a c t i v i t y , and f e l l i n t o the low end of the range of c o n c e n t r a t i o n s seen i n m u s t e l i d s . V a g i n a l smear ty p e s were found t o i d e n t i f y c o r r e c t l y the r e p r o d u c t i v e s t a t e of female o t t e r s . E s t r a d i o l c o n c e n t r a t i o n s of c a p t i v e o t t e r s were h i g h l y v a r i a b l e , but the v a l u e s o b t a i n e d and the p a t t e r n of s e c r e t i o n were s i m i l a r t o tho s e of o t h e r c a r n i v o r e s . A l t h o u g h e s t r a d i o l l e v e l s of c a p t i v e o t t e r s were reduced i n the f i n a l two y e a r s of the s t u d y , normal o v a r i a n development o c c u r r e d . D u r i n g pregnancy, p r o g e s t e r o n e c o n c e n t r a t i o n s c o r r e s p o n d e d t o changes obse r v e d i n l u t e a l c e l l c y t o l o g y . I p r e s e n t e v i d e n c e s u g g e s t i n g t h a t female o t t e r s a r e monestrous, induced o v u l a t o r s . The t i m i n g of r e p r o d u c t i v e e v e n t s of o t t e r s i n the marine environment was s i m i l a r t o those of o t t e r s i n h a b i t i n g f r e s h water a t comparable l a t i t u d e s . R e p r o d u c t i v e a c t i v i t y of males began i n the l a t e autumn and a d u l t s were c a p a b l e of b r e e d i n g from l a t e J a n uary through mid-May. Females e x h i b i t e d e s t r u s from l a t e F e b r u a r y through mid-May, w i t h the m a j o r i t y of matings p r o b a b l y o c c u r r i n g d u r i n g March and A p r i l . A f t e r a n i n e t o e l e v e n month p e r i o d of embryonic d i a p a u s e , i m p l a n t a t i o n o c c u r r e d the subsequent F e b r u a r y . B i r t h s o c c u r r e d a f t e r an e s t i m a t e d two month g e s t a t i o n and p a r t u r i e n t 1 o t t e r s mated d u r i n g l a c t a t i o n . Male o t t e r s were s e x u a l l y mature a t the end of t h e i r second y e a r . Only 55% of females bred a t age c l a s s 2 and s e x u a l m a t u r i t y was d e l a y e d u n t i l the f o l l o w i n g year i n the remainder of the f e m a l e s . Pregnancy r a t e s were h i g h among females over 3 y e a r s of age and o v u l a t i o n r a t e s were s i m i l a r t o those of o t h e r s t u d i e s . To d e t e r m i n e the i n f l u e n c e of p h o t o p e r i o d on the t i m i n g of r e p r o d u c t i v e e v e n t s , c a p t i v e o t t e r s were exposed t o i n c r e a s e d p h o t o p e r i o d s . W ith one e x c e p t i o n , s p ermatogenesis and e s t r u s o c c u r r e d e a r l i e r under e x p e r i m e n t a l c o n d i t i o n s . My r e s u l t s suggest t h a t the t e r m i n a t i o n of embryonic d i a p a u s e i s a l s o under p h o t o p e r i o d i c c o n t r o l . V a r i a t i o n s i n t emperature or food were not a s s o c i a t e d w i t h changes i n r e p r o d u c t i v e c o n d i t i o n . T h i s study s u g g e s t s t h a t p h o t o p e r i o d p r o b a b l y c o n t r o l s the t i m i n g of the r e p r o d u c t i v e c y c l e of o t t e r s i n B r i t i s h C olumbia. i v TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES ix LIST OF FIGURES xi ACKNOWLEDGEMENTS xiv Chapter I: General Introduction 1 Chapter I I : Male Reproductive Cycle 8 Introduction 9 Materials and Methods 12 I. H i s t o l o g i c a l Examination of the Reproductive Cycle .. 12 A. Source of Material 12 B. Age Determination 13 C. Examination of H i s t o l o g i c a l Material 14 D. Phase Analysis Procedure 15 II. Captive animals 16 A. Examination and Sampling of otters 16 B. Blood Analysis 18 III. S t a t i s t i c a l Analysis 19 Results 21 I. H i s t o l o g i c a l Examination of the Seminiferous Tubules 21 A. C e l l Types Present 21 B. Phase Analysis 23 II. The Annual Cycle and Sexual Maturity 29 A. The Testis 29 1. The Seminiferous tubules 29 2 . The I n t e r t u b u l a r T i s s u e 35 B. The E p i d i d y m i s 37 C. T e s t e s S i z e 44 D. Summary 47 E. S e x u a l M a t u r i t y 48 I I I . E n d o c r i n o l o g y of the Male R e p r o d u c t i v e C y c l e 49 A. R e p r o d u c t i v e c y c l e of c a p t i v e males 49 B. T e s t o s t e r o n e C o n c e n t r a t i o n s 52 D i s c u s s i o n 56 A. H i s t o l o g y 56 B. Phase a n a l y s i s 57 C. S e x u a l M a t u r i t y 59 D. The a n n u a l c y c l e i n B r i t i s h Columbia 61 E. T e s t o s t e r o n e c o n c e n t r a t i o n s 64 Chapter I I I : Female R e p r o d u c t i v e C y c l e 68 I n t r o d u c t i o n 69 M a t e r i a l s and Methods 72 I . Necropsy E x a m i n a t i o n s 72 A. Source of M a t e r i a l 72 B. Age D e t e r m i n a t i o n 73 C. E x a m i n a t i o n of H i s t o l o g i c a l M a t e r i a l 73 I I . C a p t i v e A n i m a l s 75 A. E x a m i n a t i o n and Sampling of o t t e r s 75 B. B l o o d A n a l y s i s 77 I I I . S t a t i s t i c a l A n a l y s i s 78 R e s u l t s 79 I . H i s t o l o g i c a l E x a m i n a t i o n of the R e p r o d u c t i v e Organs 79 A. Ovarian Cycle 79 1. General Description 79 2. F o l l i c u l a r growth 83 3. Pregnancy 85 B. Uterus and Placenta 91 C. Blastocysts 97 D. Vaginal Cycle 97 II . The Annual Cycle, Reproductive Rates and Sexual Maturity 104 A. Annual reproductive cycle 104 B. Pregnancy rates 111 C. Ovulation rates 111 D. Blastocysts 113 E. Implanted embryos 114 F. Sexual maturity 114 I I I . Endocrinology of the Female Reproductive Cycle ....115 A. Reproductive cycle of captive females 115 1 . Estrus 116 2. Mating 119 3. P a r t i a l estrus 120 B. E s t r a d i o l concentrations 121 1. Nonpregnant adults 121 2. Pregnant otters 126 3. Immature otters 131 4 . Wild otters 131 C. Progesterone concentrations 133 1. Immature and nonpregnant adults 133 v i i 2. Pregnant o t t e r s 136 3. W i l d o t t e r s 137 D i s c u s s i o n 138 A. H i s t o l o g y 138 B. V a g i n a l smears 140 C. Induced o v u l a t i o n 142 D. E s t r o u s p e r i o d s 146 E. The a n n u a l c y c l e i n B r i t i s h Columbia 146 F. S e x u a l M a t u r i t y 152 G. E s t r a d i o l c o n c e n t r a t i o n s 155 1. Nonpregnant females 155 2. Pregnant females 162 3. P a r t i a l e s t r u s 164 H. P r o g e s t e r o n e c o n c e n t r a t i o n s 166 1. Nonpregnant and immature o t t e r s 166 2. Pregnancy 166 3. Pseudopregnancy 170 Chapter IV: The e f f e c t s of i n c r e a s e d p h o t o p e r i o d on r e p r o d u c t i v e a c t i v i t y 173 I n t r o d u c t i o n 174 M a t e r i a l s and Methods 177 R e s u l t s 180 I . The e f f e c t of i n c r e a s e d p h o t o p e r i o d on female o t t e r s 180 A. E s t r u s 180 1 . Experiment 1 180 2. Experiment 2 183 3. Experiment 3 186 v i i i B. Implantation .. 187 II . The e f f e c t of increased photoperiod on male otters .188 1 . Experiment 1 188 2. Experiment 2 191 3. Experiment 3 194 4. A l l experiments 201 Discussion 205 A. Environmental Cues 205 B. Females 206 1 . Estrus 206 2. Implantation 208 C. Males 209 D. Photoperiod as a Cue 211 Chapter V: General Discussion 216 Summary 223 References Cited 226 Appendix 1: Testicular Abnormalities 248 Appendix 2: Testosterone Concentrations 253 Appendix 3: E s t r a d i o l Concentrations 255 Appendix 4: Progesterone Concentrations 258 LIST OF TABLES T a b l e I . O r i g i n of male specimens 12 T a b l e I I . C a p t i v e male o t t e r s 17 T a b l e I I I . D e s c r i p t i o n of phases of spermatogenesis 24 T a b l e IV. R e l a t i v e volumes of i n t e r l o b u l a r t i s s u e 36 T a b l e V. C o r r e l a t i o n between i n d i c a t o r s of r e p r o d u c t i v e a c t i v i t y 48 T a b l e V I . O r i g i n of female necropsy samples 72 Tab l e V I I . C a p t i v e female o t t e r s 76 T a b l e V I I I . O v a r i a n d i m e n s i o n s of d i f f e r e n t age c l a s s e s ... 80 Tab l e IX. O v a r i a n and u t e r i n e d i m e n s i o n s of a d u l t o t t e r s .. 82 Tab l e X. Co r p o r a l u t e a and l u t e a l c e l l d i a m e t e r s 86 T a b l e X I . U t e r i n e d i m e n s i o n s of d i f f e r e n t age c l a s s 92 T a b l e X I I . C e l l t y p e s p r e s e n t i n the v a g i n a l smears 98 T a b l e X I I I . Pregnancy, o v u l a t i o n and f e r t i l i t y r a t e s of a d u l t o t t e r s 112 T a b l e XIV. E s t r a d i o l c o n c e n t r a t i o n s 127 T a b l e XV. E s t r a d i o l c o n c e n t r a t i o n s i n a pregnant o t t e r ....130 T a b l e XVI. P r o g e s t e r o n e and e s t r a d i o l c o n c e n t r a t i o n i n w i l d o t t e r s 132 T a b l e X V I I . Mean p r o g e s t e r o n e c o n c e n t r a t i o n s d u r i n g pregnancy 132 T a b l e X V I I I . R e p r o d u c t i v e c y c l e s of r i v e r o t t e r s i n N o r t h America 149 T a b l e XIX. E x p e r i m e n t a l p r o t o c o l 178 Table XX. Phase analysis of the seminiferous tubules xi LIST OF FIGURES Plate I 25 Figure 1. Immature seminiferous tubule 25 Figure 2. Phase 1 seminiferous tubule 25 Figure 3. Phase 2 seminiferous tubule 25 Figure 4. Phase 3 seminiferous tubule 25 Plate II 27 Figure 5. Phase 4 seminiferous tubule 27 Figure 6. Phase 5 seminiferous tubule 27 Figure 7. Phase 6 seminiferous tubule 27 Figure 8. Phase 7 seminiferous tubule 27 Figure 9. Phase 8 seminiferous tubule 27 Figure 10. Phase Analysis 30 Figure 11. Seminiferous tubule diameters 33 Figure 12. Leydig c e l l nucleus diameters 33 Figure 13. Ductus epididymis diameter 38 Plate III 41 Figure 14. Immature epididymis 41 Figure 15. Inactive adult epididymis 41 Figure 16. Active adult epididymis 41 Figure 17. I n t r a - e p i t h e l i a l lumen 41 Figure 18. T e s t i s weight 45 Figure 19. T e s t i s volume 45 Figure 20. T e s t i s volume of wild and captive otters 50 Figure 21. Serum testosterone concentration and t e s t i s volume 53 P l a t e IV 87 F i g u r e 22. Corpora l u t e a of e a r l y o v u l a t i o n 87 F i g u r e 23. L u t e a l c e l l s of e a r l y o v u l a t i o n 87 F i g u r e 24. Corpora l u t e a of p r e i m p l a n t a t i o n 87 F i g u r e 25. L u t e a l c e l l s of p r e i m p l a n t a t i o n 87 F i g u r e 26. Corpora l u t e a of p o s t i m p l a n t a t i o n 87 F i g u r e 27. L u t e a l c e l l s of p o s t i m p l a n t a t i o n 87 P l a t e V 93 F i g u r e 28. A n e s t r o u s u t e r u s 93 F i g u r e 29. E s t r u s u t e r u s 93 F i g u r e 30. Pregnant u t e r u s : p r e i m p l a n t a t i o n p e r i o d 93 F i g u r e 31. Pregnant u t e r u s : p o s t i m p l a n t a t i o n p e r i o d .... 93 P l a t e VI 99 F i g u r e 32. A n e s t r o u s v a g i n a 99 F i g u r e 33. E s t r o u s v a g i n a 99 F i g u r e 34. A n e s t r o u s v a g i n a l smear 99 F i g u r e 35. P r o e s t r o u s v a g i n a l smear 99 F i g u r e 36. E s t r o u s v a g i n a l smear 99 F i g u r e 37. M e t e s t r o u s v a g i n a l smear 99 F i g u r e 38. F o l l i c u l a r s t a g e s p r e s e n t i n a d u l t o v a r i e s 106 F i g u r e 39. Annual changes i n maximum f o l l i c u l a r s i z e 108 F i g u r e 40. E s t r u s i n c a p t i v e female o t t e r s 117 F i g u r e 41. I n d i v i d u a l e s t r a d i o l p r o f i l e s 122 F i g u r e 42. M o n t h l y e s t r a d i o l c o n c e n t r a t i o n s 124 F i g u r e 43. E s t r a d i o l c o n c e n t r a t i o n s d u r i n g pregnancy 128 F i g u r e 44. P r o g e s t e r o n e c o n c e n t r a t i o n s i n immature and x i i i nonpregnant o t t e r s 134 F i g u r e 45. P r o g e s t e r o n e c o n c e n t r a t i o n s d u r i n g pregnancy ...134 F i g u r e 46. E s t r a d i o l c o n c e n t r a t i o n s and e s t r u s p e r i o d s of females d u r i n g . E x p e r i m e n t 1 181 F i g u r e 47. E s t r u s p e r i o d s o b s e r v e d d u r i n g Experiment 2 ....184 F i g u r e 48. E s t r u s p e r i o d s o b s e r v e d d u r i n g Experiment 3 ....184 F i g u r e 49. P r o g e s t e r o n e c o n c e n t r a t i o n s of an e x p e r i m e n t a l female d u r i n g pregnancy 184 F i g u r e 50. T e s t i s volumes d u r i n g Experiment 1 189 F i g u r e 51. T e s t o s t e r o n e c o n c e n t r a t i o n s d u r i n g Experiment 1 189 F i g u r e 52. T e s t i s volumes d u r i n g Experiment 2 192 F i g u r e 53. T e s t o s t e r o n e c o n c e n t r a t i o n s d u r i n g Experiment 2 192 F i g u r e 54. T e s t i s volumes d u r i n g Experiment 3 195 F i g u r e 55. T e s t o s t e r o n e c o n c e n t r a t i o n s d u r i n g Experiment 3 195 F i g u r e 56. S e m i n i f e r o u s t u b u l e d i a m e t e r s 199 F i g u r e 57. T e s t i s volumes of e x p e r i m e n t a l o t t e r s 202 F i g u r e 58. T e s t o s t e r o n e c o n c e n t r a t i o n s of e x p e r i m e n t a l o t t e r s 202 P l a t e V I I 249 F i g u r e 59. Abnormal t e s t i s #505 249 F i g u r e 60. Abnormal e p i d i d y m i s 249 F i g u r e 61. Abnormal t e s t i s #303 249 x i v ACKNOWLEDGMENTS I would l i k e t o acknowledge the h e l p of a l l of the people who have c o n t r i b u t e d i n v a r i o u s ways t o the s u c c e s s of t h i s s t u d y . My s i n c e r e thanks t o my s u p e r v i s o r , Dr. H.D. F i s h e r , f o r h i s support and a d v i c e . I would a l s o l i k e t o thank the members of my r e s e a r c h committee, Drs. H.C. Nordan, A.M. P e r k s , D.M. S h a c k l e t o n , and A.R.E. S i n c l a i r f o r t h e i r i n t e r e s t , a d v i c e , and c o n s t r u c t i v e c r i t i c i s m i n r e v i e w i n g t h i s t h e s i s . To Dr. L.V. Moore, I e x t e n d a s p e c i a l thanks f o r a l l of the work she d i d h e l p i n g me s o r t out my v a r i o u s i d e a s and g e t t i n g them on paper i n a manner t h a t would make sense t o o t h e r s . I would l i k e t o thank the many t r a p p e r s , and F i s h and W i l d l i f e Branch p e r s o n n e l , who h e l p e d me c o l l e c t samples, i n p a r t i c u l a r Guy A n t i l l a and the crew a t Camp Keough. I am a l s o g r a t e f u l t o the p e o p l e who h e l p e d me c a r e f o r my o t t e r s and c o l l e c t b l o o d samples. The a d v i c e and a s s i s t a n c e of Dr. B.D. Murphy and the members of h i s l a b c o n c e r n i n g the i n s and o u t s of radioimmunoassays, i s g r a t e f u l l y acknowledged. To my f a m i l y and f r i e n d s , I a p p r e c i a t e a l l of the h e l p and the support I have r e c e i v e d . In p a r t i c u l a r , thanks t o a l l of the p a s t and p r e s e n t r e s i d e n t s of the penthouse, l i f e w i l l never be the same. A l s o t o Grant Badgero, thanks f o r a l l the rowing. A v e r y s p e c i a l t h a n k s t o my w i f e , J a n e t , f o r her l o v e , ° s u p p o r t , and p a t i e n c e t h r o u g h a v e r y t r y i n g t i m e . T h i s work was s u p p o r t e d by N a t u r a l S c i e n c e s and E n g i n e e r i n g X V Research Council grants to Dr. H.D. Fisher and by National Research Council, Canadian W i l d l i f e Service, and University of B r i t i s h Columbia Scholarships to G.B. Stenson. 1 CHAPTER I GENERAL INTRODUCTION The reproductive cycle of most wild mammals is poorly understood, usually because of l o g i s t i c constraints. Reproduction and the factors which influence i t have been studied extensively in only a few mammals; our knowledge i s based primarily on laboratory and domestic animals which are readily available and tractable. From these studies, we have attempted to generalize to wild species which have not been studied in d e t a i l , relying heavily upon the limited information available and taxonomic relationships. However, given the variation known to exist within taxa, such generalizations are often inadequate, p a r t i c u l a r l y when dealing with a species such as the river otter which i s wide-ranging and l i v e s in a variety of habitats. River otters, Lutra canadensis (Schreber), inhabit aquatic environments throughout North America from approximately 70°N to 25°S (Hall 1981). They are common in many fresh-water lakes and streams, and are abundant in the marine environment of both the A t l a n t i c and P a c i f i c coasts (Toweill and Tabor 1982; Larsen 1983; Woolington 1984). Our knowledge of their reproduction i s lim i t e d . The purpose of my study was to describe the reproductive cycle of L. canadensis and to examine some of the factors which may influence t h e i r reproductive cycle. U n t i l recently, information about reproduction in otters was primarily anecdotal. Hamilton and Eadie (1964) were the f i r s t to examine the reproductive organs of L. canadensis and 2 found that otters in New York State are seasonal breeders which mate in the spring and give b i r t h approximately 12 months l a t e r . This prolonged gestation i s the result of a dormant period p r i o r to implantation; a condition referred to as 'embryonic diapause' or 'delayed implantation'. Since their study, various authors have examined otter reproductive tracts in an attempt to determine the timing of reproductive events and to estimate population parameters (Tabor and Wight 1977; Mowbray e_t a l . 1979; H i l l and Lauhachinda 1981; Lancia and Hair 1983). However, none of these studies described the entire reproductive cycle of female otters in d e t a i l and v i r t u a l l y nothing was known about reproduction in males. Also, changes in the plasma concentrations of gonadal hormones have not been examined previously. The f i r s t objective of my study was to examine in d e t a i l the changes which occur in the reproductive organs of both male and female otters during the annual reproductive cycle. This information w i l l provide basic information necessary to understand much of otter biology. Such data are of p a r t i c u l a r importance at this time because of growing interest in the species as a furbearer and the placing of L. canadensis on Appendix II of CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) in 1977 (Greenwalt 1977). This study w i l l also increase our knowledge of reproduction among the Mustelidae and seasonal breeders in general, and provide a description of embryonic diapause that should increase our understanding of the variety and 3 significance of th i s form of reproduction. Although the reproductive biology of ri v e r otters i s not well known, other members of the Mustelidae have been examined extensively. The reproductive cycles of the mink, Mustela vison (Hansson 1947; Enders 1952; Moller 1973, 1974; Pilbeam et a l . 1979; Boissin-Agasse et a l . 1982), the f e r r e t , Mustela  putorius (Allanson 1932; Hamilton and Gould 1940; Neal et-a l . 1977), the stoat, Mutela erminea (Deanesly 1935; Gulamhusein and Tarn 1974; King and Moody 1982), the spotted skunk, Spilogale  putorius (Mead I968a,b; Mead and Eik-Nes I969a,b; Mead 1981), and the European badger, Meles meles (Neal and Harrison 1958; Canivenc 1966; Ahnlund 1980; Audy et a l . 1982) have been described in d e t a i l and thus provide a good basis for comparison. Reproductive cycles among mustelids are fundamentally similar owing to a common phylogeny, but vary greatly in d e t a i l . One reason for this v a r i a t i o n i s the presence of embryonic diapause in many members of t h i s family. Species exhibiting t h i s t r a i t have a period during which the blastocyst i s r e l a t i v e l y inactive and remains unattached in the uterine lumen. In mustelids, t h i s period i s usually accompanied by reduced l u t e a l and uterine a c t i v i t y and diapause is" considered obligatory in these species (see reviews in Wright 1963; Mead 1981; Mead and Wright 1983). Embryonic diapause occurs in a variety of other mammals and i s common among members of the Macropodidae, Ursidae, and Pinnipedia (Renfree and Calaby 1981). It appears to be important in extending the reproductive cycle so that reproductive events occur at favourable times of 4 the year (Sadlier 1969a; Renfree 1978; Enders 1981). However, the occurrence of diapause in mammals appears to be species s p e c i f i c and many d i f f e r e n t forms are present (Weir and Rowlands 1973; Renfree 1978). Therefore, the mechanisms involved i n , as well as the factors c o n t r o l l i n g , embryonic diapause must also vary greatly between species. Among otter species, embryonic diapause i s not common; i t occurs in the North American rive r otter, L. canadensis, and the sea otter, Enhydra l u t r a , but i s not known to occur in other members of the subfamily Lutrinae. (Sinha et a l . 1966; Sinha and Conaway 1968; Harris 1968; Duplaix-Hal.l 1975). Reproduction in mammals is a complicated process that must be adapted in some manner to existing environmental conditions. Such adaptations have resulted in a variety of reproductive patterns and have made i t d i f f i c u l t to determine how the reproductive cycle i s affected by various environmental factors (Sadleir 1969a). To understand the importance of environmental factors, i t i s necessary to establish a basic understanding of the reproductive cycle of wild mammals, preferably by examining a species under d i f f e r e n t ecological conditions. The second objective of t h i s study therefore, was to determine the timing of reproductive events of river otters in the marine environment of southwestern B r i t i s h Columbia. The presence of otters in the marine environment provided an int e r e s t i n g opportunity to examine the influence of l o c a l environmental conditions ( i . e . coastal vs freshwater) on reproduction. A l l animals examined inhabited the marine 5 environment and were c l a s s i f i e d as L. c. mira (Hall 1981). Previous studies on the reproductive cycle of r i v e r otters have concentrated upon fresh-water environments, although a few included some coastal individuals (e.g. Tabor and Wight 1977). The timing of reproductive events, together with the pregnancy rates and age of sexual maturity, were a l l found to vary among populations (Wilson 1959; McDaniel 1963; Hamilton and Eadie 1964; Tabor and Wight 1977; Mowbray et a l . 1979; H i l l and Lauhachinda 1981; Lancia and Hair 1983). Generally, in the southern regions, breeding, implantation, and births occur e a r l i e r than in northern areas. Reproductive events in the south extend over a minimum three to four month period, whereas they are confined to a r e l a t i v e l y short period in northern populations. In addition, sexual maturity may be advanced and births may occur only every second year in some southern populations. These differences may r e f l e c t adaptations to the d i f f e r e n t environments inhabited, or may represent basic genetic differences between populations. In B r i t i s h Columbia, l o c a l environmental conditions are similar to those of freshwater areas further south; temperatures are moderate and seasonal fluctuations are minimal (Anon. 1982). Thus i t was of interest to examine i f the reproductive cycle coincides with more southernly populations, or with those of a comparable la t i t u d e . In the marine environment food, primarily i n t e r t i d a l f i s h (Stenson e_t a l . 1984; Larsen 1984), appears abundant throughout the year. Bronson (1985) suggests that in areas with a constant food supply mammals w i l l breed as often as possible. For 6 example, the breeding season of wild rodents has been extended by the presence of ad d i t i o n a l food during the winter (Fordham 1971; T a i t t 1981; T a i t t and Krebs 1981). It i s possible, therefore, that under favourable environmental conditions coastal otters in B r i t i s h Columbia may also have an extended breeding season. Given the l a t i t u d i n a l differences in reproductive parameters observed among populations of L. canadensis, the th i r d objective of t h i s study was to investigate the influence of photoperiod on the timing of reproductive events. For a seasonal breeder, i t should be advantageous to use environmental cues to anticipate the subsequent seasonal changes by inducing the metabolic preparation necessary for reproduction. Baker (1938) distinguished these 'proximal' factors, which maintain the timing of the reproductive events through physiological means, from 'ultimate' factors, which es t a b l i s h the timing of the cycle through natural s e l e c t i o n . Photoperiod i s the most commonly used predictor of annual events in temperate mammals (Sadleir 1969a, Herbert 1977; Bronson 1985), and altered photoperiod induces reproductive a c t i v i t y in a number of mustelids (Bissonnette 1932; Hammond 1951; Mead 1971; Thorpe and j Herbert 1976; Canivenc and Bonnin 1979; Boissin-Agasse et a l . 1982). In Chapters II and I I I , I describe in d e t a i l the histology and endocrinology of the reproductive cycle of L. canadensis and present the f i r s t account of the reproductive cycle of otters in a marine environment. I also present techniques to determine 7 the reproductive state of l i v e otters which can be used to standardize future studies on reproductive biology. In Chapter IV, I examine the influence of increased photoperiod on the timing of spermatogenesis, estrus, and implantation in rive r o tters. 8 C H A P T E R I I R E P R O D U C T I V E C Y C L E O F T H E M A L E R I V E R O T T E R 9 INTRODUCTION The reproductive cycle of the male ri v e r otter, Lutra  canadensi s, i s poorly understood. E a r l i e r studies of reproduction in otters have consisted almost e n t i r e l y of an examination of female reproductive organs (Liers 1951,1958; Hamilton and Eadie 1964; Tabor and Wight 1977; Mowbray e_t a l . 1979; H i l l and Lauhachinda 1980) while the reproductive cycle of the male has been ignored or dealt with s u p e r f i c i a l l y . The changes that occur in the morphology and histology of the reproductive organs and the endocrinology of the annual reproductive cycle of the male are unknown. This study had three objectives: 1) to describe in d e t a i l the histology and endocrinology of the annual reproductive cycle of the male river otter, 2) to develop a technique to standardize the degree of spermatogenesis observed, and 3) to determine the annual reproductive cycle of male otters in B r i t i s h Columbia. The c y c l i c a l changes that occur in the anatomy and histology of the reproductive organs of juvenile and adult otters were described. In addition, variations in serum testosterone concentrations throughout the year were determined and correlated with the changes observed in the reproductive organs. The c y c l i c a l changes which occur in reproductive organs and androgen concentrations during the annual reproductive cycle have been described for a variety of mustelids such as the mink, Mustela vison (Onstad 1967; Bassur and Ramos 1973; Pilbeam et a l . 1979; Boissin-Agasse et a_l. 1982), the stoat, Mustela  erminea (Deanesly 1935; Gulamhusein and Tarn 1974), the fe r r e t , 10 Mustela putorius (Allanson 1932; Neal et a l . 1977), and the European badger, Meles meles (Ahnlund 1980; Audy et a l . 1982). This study provides the f i r s t comparable investigation in the otter and as such w i l l increase our knowledge of reproductive development in the Mustelidae and other mammalian seasonal breeders. The second objective of t h i s investigation was to develop a technique to determine the timing of spermatogenesis in otters. Although stages of seminiferous epithelium development have been c l a s s i f i e d by .various authors (Roosen-Runge and Giesel 1950; Clermont and Leblond 1955; Berndtson 1977), these techniques were developed for animals which undergo continuous spermatogenesis and are based primarily upon the changes which occur in the spermatids. They do not consider the detailed c e l l u l a r changes which occur in the seminiferous tubules during the onset and cessation of spermatogenesis that are essential in understanding the reproductive cycle of seasonal breeders such as the ot t e r . The reproductive development of seasonal breeders has been successfully i l l u s t r a t e d using a technique modified from these e a r l i e r methods, based upon the t o t a l c e l l u l a r associations present in the seminiferous tubules throughout the year (Marsh et a l . 1984; Johnston and Buss 1967). Using t h i s modified technique, I i d e n t i f i e d a series of phases which describe the process of spermatogenesis in the otter. These phases are e a s i l y i d e n t i f i e d and provide a method of standardizing the study of reproduction in the r i v e r otter. The t h i r d objective of t h i s investigation was to determine 11 the annual reproductive cycle of male r i v e r otters in the coastal environment of southwestern B r i t i s h Columbia. L i t t l e i s known concerning the timing of reproductive events in otters; a c t i v i t y i s believed to begin in late autumn (Liers 1951; Hamilton and Eadie 1964) and mating can occur between January and May depending upon the location (Liers 1951; McDaniel 1963; Mowbray e_t a_l. 1979; Melquist and Hornocker 1983; Woolington 1984). This investigation i s the f i r s t to examine the timing of spermatogenesis among male otters and w i l l provide a basis for comparison with future studies. 12 MATERIALS AND METHODS J_. H i s t o l o q i c a l Examination of the Reproductive Cycle A. Source of Material Specimen material consisted of 226 reproductive tracts c o l l e c t e d from coastal areas of southern B r i t i s h Columbia during 1975-81 (Table I ) . The majority (69.5%) came from the Barkley Sound region of Vancouver Island (49° N 125° W); professional trappers provided 94.7% of a l l specimens. Other sources of material included federal and p r o v i n c i a l w i l d l i f e o f f i c e r s and my own trapping e f f o r t s . Table I. Origin of male river otter necropsy samples. Total number of samples is 226. Vancouver Island Barkley Sound 157 East Coast 56 Mainland Coast Rivers Inlet 10 Howe Sound 3 Reproductive t r a c t s , bacula and skulls were removed in the f i e l d and placed in 10% seawater formalin. When th i s was not possible, carcasses were frozen whole and the reproductive t r a c t s were removed and fixed in formalin when brought to the laboratory. 13 B. Age D e t e r m i n a t i o n The age of each a n i m a l sampled was determined and i t was a s s i g n e d t o an age c l a s s . A p r i l 1 was a r b i t r a r i l y assumed as the d a t e of b i r t h f o r a l l samples. A l t h o u g h b i r t h may have o c c u r r e d e a r l i e r the a p p r o x i m a t i o n d i d not a f f e c t the i n t e r p r e t a t i o n of the r e s u l t s . O t t e r s e s t i m a t e d t o be 0 t h r o u g h 12 months o l d were p l a c e d i n age c l a s s 0, 13 - 24 months i n age c l a s s 1, 25 -36 months i n age c l a s s 2, e t c . . Age c l a s s e s were grouped i n t o t h r e e g e n e r a l c a t e g o r i e s f o r d i s c u s s i o n : j u v e n i l e (age c l a s s 0 ) , y e a r l i n g (age c l a s s 1), and a d u l t (age c l a s s e s 2 and a b o v e ) . Samples ranged i n age from e i g h t months t o 14 y e a r s . Ages were determined by the use of cementum growth l a y e r s (Stephenson 1977) and c o n f i r m e d w i t h baculum and s k u l l c h a r a c t e r i s t i c s as d e s c r i b e d by F r i l e y (1949), Hooper and Ostenson (1949), and Lauhachinda (1978). B a c u l a and s k u l l s were c l e a n e d by b o i l i n g and the lower l e f t c a n i n e was removed f o r s e c t i o n i n g . The t e e t h were d e c a l c i f i e d i n 5% n i t r i c a c i d f o r 24-48 h o u r s , washed i n t a p water f o r a minimum of f i v e minutes and s e c t i o n e d i m m e d i a t e l y . L o n g i t u d i n a l s e c t i o n s 12-15 jim t h i c k were removed from the c e n t r e of the t o o t h u s i n g a f r e e z i n g microtome. S e c t i o n s were f l o a t e d onto a l b u m i z e d s l i d e s and a i r d r i e d . T e e t h were s t a i n e d w i t h P a p a n i c o l a o u ' s h a e m a t o x y l i n ( 3 -5 m i n u t e s ) , a i r d r i e d and mounted w i t h permount. To d e t e r m i n e the best t o o t h t o be examined, f i v e s k u l l s of v a r i o u s ages were s e l e c t e d . Two c a n i n e s ( C 1 , C , ) , one premolar ( P , ) , one molar ( M 3 ) , and one i n c i s o r ( I 3 ) were removed and 14 pr e p a r e d as d e s c r i b e d . In each a n i m a l , the age i n d i c a t e d by each t o o t h was i d e n t i c a l . The lower c a n i n e was chosen because of the ease of h a n d l i n g and the l a r g e r amount of cementum p r e s e n t . S k u l l s from 15 r i v e r o t t e r s whose ages were known or c o u l d be approximated were examined t o c o n f i r m the a c c u r a c y of t h i s age d e t e r m i n a t i o n t e c h n i q u e . Of t h e s e , s i x were young of the y e a r , s i x were y e a r l i n g s , and f o u r were a d u l t s . With the e x c e p t i o n of one y e a r l i n g , a l l were were a s s i g n e d t o the proper age c l a s s . Some d i f f i c u l t y was encountered due t o s p l i t t i n g of a n n u l i i n the b a s a l p o r t i o n of the cementum, p a r t i c u l a r l y among the young a n i m a l s . The best r e a d i n g s were found t o occur a l o n g the s i d e of the r o o t . C. E x a m i n a t i o n of H i s t o l o g i c a l M a t e r i a l Each f i x e d t e s t i s was trimmed of f a t and the e p i d i d y m i s and weighed t o 0.01 g. T e s t i s l e n g t h ( L ) , w i d t h a t m i d - l e n g t h (W) and d o r s o - v e n t r a l t h i c k n e s s a t m i d - l e n g t h (T) were measured u s i n g v e r n i e r c a l i p e r s and the t e s t i c u l a r volume was e s t i m a t e d from the form u l a of S e t c h e l l and Waites (1964): TV = 4/37T • L / 2 • W/2 • T/2 C r o s s s e c t i o n s of the r i g h t t e s t i s and e p i d i d y m i s were removed f o r h i s t o l o g i c a l s t u d y . To determine i f a l l r e g i o n s of the t e s t i s were a t the same stage of r e p r o d u c t i v e a c t i v i t y , I examined s e c t i o n s from f i v e d i f f e r e n t a r e a s of s e l e c t e d t e s t e s . T i s s u e samples were washed i n water, d e h y d r a t e d i n e t h a n o l , 1 5 c l e a r e d i n x y l e n e and embedded i n p a r a f f i n , u s i n g s t a n d a r d h i s t o l o g i c a l t e c h n i q u e s . B l o c k s were s e c t i o n e d a t 10 ym, mounted, s t a i n e d w i t h h e m a t o x y l i n and e o s i n . The d iameter of twenty (20) s e m i n i f e r o u s t u b u l e s a t r i g h t a n g l e s t o t h e i r l o n g a x i s was d e t e r m i n e d u s i n g a c a l i b r a t e d o c u l a r micrometer a t X200 m a g n i f i c a t i o n . I f the m a t e r i a l was s u f f i c i e n t l y p r e s e r v e d t o a l l o w o b j e c t i v e h i s t o l o g i c a l e x a m i n a t i o n , the c e l l t y p e s i n the t e s t i s were i d e n t i f i e d . At l e a s t ten c e l l s of each type were measured u s i n g the o c u l a r micrometer at X1000. The t h i c k n e s s of the muscle and e p i t h e l i a l l a y e r s and the t o t a l d i a m e t e r , i n c l u d i n g muscle l a y e r s , of ten t r a n s v e r s e s e c t i o n s of each d u c t u s e p i d i d y m i s was a l s o measured. The r e l a t i v e volumes of the i n t r a t u b u l a r and e x t r a t u b u l a r t i s s u e were examined i n a subsample of w e l l p r e s e r v e d t e s t e s u s i n g a W e i b e l g r a t i c u l e . T h i s i n v o l v e s r e c o r d i n g the c e l l type a p p e a r i n g a t 42 p o i n t s i n each of a s e r i e s of ten random f i e l d s at X400. Counts beyond t h i s l e v e l produce o n l y s l i g h t v a r i a t i o n s (Marsh et a l . 1984). D. Phase A n a l y s i s Procedure The c e l l u l a r a s s o c i a t i o n s o b s e r v e d i n the s e m i n i f e r o u s t u b u l e s were d i v i d e d i n t o a s e r i e s of n i n e phases. The phase p r e s e n t i n each of twenty (20) t r a n s v e r s e s e c t i o n s of t u b u l e s i n one s e c t i o n from each o t t e r was i d e n t i f i e d a t a m a g n i f i c a t i o n of X400. The annual changes i n t e s t i c u l a r a c t i v i t y were q u a n t i f i e d , based on the o c c u r r e n c e of t h e s e phases i n the t e s t e s . 1 6 I I . C a p t i v e a n i m a l s A. E x a m i n a t i o n and Sampling of o t t e r s To p r o v i d e i n f o r m a t i o n f o r a l l p e r i o d s of the annual c y c l e and t o determine t e s t o s t e r o n e c o n c e n t r a t i o n s f o r c o r r e l a t i o n w i t h the h i s t o l o g i c a l e x a m i n a t i o n of w i l d specimens, n i n e male r i v e r o t t e r s were l i v e - t r a p p e d and h e l d a t the Animal Care C e n t r e , U n i v e r s i t y of B r i t i s h C o l u m b i a , f o r p e r i o d s r a n g i n g from f o u r t o 41 months (Table I I ) . H o l d i n g f a c i l i t i e s c o n s i s t e d of pens (3.7 X 3.7 m) surrounded by c h a i n l i n k and s u b j e c t t o n a t u r a l e n v i r o n m e n t a l c o n d i t i o n s . Den boxes and p o o l s w i t h f r e s h , r u n n i n g water were a v a i l a b l e i n a l l pens. The o t t e r s were m a i n t a i n e d on a d i e t of f i s h ( h e r r r i n g and/or salmon) supplemented w i t h B-complex v i t a m i n s . Each a n i m a l r e c e i v e d 1-1 1/2 kg of f i s h per day and body weig h t s were r e c o r d e d each week. Except f o r e x p e r i m e n t a l p e r i o d s , a l l a n i m a l s were housed t o g e t h e r and were f r e e t o move between t h r e e a d j a c e n t pens. A l t h o u g h the I 3 was removed from each o t t e r , ages c o u l d o n l y be d e t e r m i n e d from some a n i m a l s by c o u n t i n g the cementum a n n u l i . The age c a t e g o r y of the o t h e r o t t e r s were e s t i m a t e d based on body s i z e . O t t e r s were examined at one t o two week i n t e r v a l s f o l l o w i n g ketamine h y d r o c h l o r i d e ( K e t a s e t , Rogar, D o r v a l , Quebec, 10 mg/kg) a n a e s t h e s i a . O c c a s i o n a l l y e x a m i n a t i o n s o c c u r r e d w i t h g r e a t e r ( d a i l y ) or l e s s e r (monthly) f r e q u e n c y . Body weigh t s and t e s t i c u l a r d i m e n s i o n s , measured through the scrotum, were r e c o r d e d and a 5-7 ml b l o o d sample was o b t a i n e d by j u g u l a r 17 T a b l e I I . M a l e river o t t e r s l i v e - t r a p p e d and h e l d i n c a p t i v i t y , 1 9 7 7 - 8 1 . A n i m a l C a p t u r e D u r a t i o n o f c a p t i v i t y D a t e L o c a t i o n Age ( y r s ) (months ) Ml 1 3 / 0 2 / 7 7 B a r k l e y S d . 7 3 6 . 0 M2 1 4 / 0 3 / 7 8 P o r t Ha rdy a d u l t 4 1 . 0 M3 0 5 / 0 4 / 7 8 P o r t Hardy a d u l t 4 0 . 5 M4 1 9 / 0 3 / 7 9 P o r t Hardy 9 1 8 . 0 M5 0 5 / 0 5 / 7 9 P o r t Hardy 4 2 6 . 0 M6 0 1 / 0 3 / 8 0 Bowen I s . a d u l t 1 6 . 0 M9 2 4 / 1 1 / 8 0 V i c t o r i a 1 9 . 0 M10 1 1 / 0 4 / 8 1 P o r t Hardy a d u l t 4 . 0 M i l 2 2 / 0 4 / 8 1 P o r t Ha rdy a d u l t 4 . 0 18 v e n i p u n c t u r e . To s t a n d a r d i z e the p r o c e d u r e s , a n i m a l s were s u b j e c t e d t o the minimum p o s s i b l e s t r e s s b e f o r e a n a e s t h e t i z i n g and b l o o d samples were u s u a l l y c o l l e c t e d between 9 am and noon. A l l a n i m a l s were b l e d as soon as they c o u l d be h a n d l e d , u s u a l l y 5-10 minutes a f t e r the ketamine i n j e c t i o n . B l o o d samples were c e n t r i f u g e d and the s e r a f r o z e n (-40°C) u n t i l a n a l y z e d . T e s t i c u l a r b i o p s i e s were performed monthly from November 1980 t h r o u g h J u l y 1981. A s c a l p e l was used t o c u t through the s c r o t a l sac and t u n i c a a l b u g i n e a of the l e f t t e s t i s and a sample of the s e m i n i f e r o u s t u b u l e s was removed w i t h a b i o p s y i n s t r u m e n t . B i o p s i e s were f i x e d i n f o r m a l i n and examined h i s t o l o g i c a l l y as d e s c r i b e d above. Surgery was performed under a s e p t i c c o n d i t i o n s ; i n c i s i o n s h e a l e d r a p i d l y and no l a s t i n g e f f e c t s were n o t i c e d . B. B l o o d A n a l y s i s Serum t e s t o s t e r o n e c o n c e n t r a t i o n s were determined by radioimmunoassays which I performed i n the l a b o r a t o r y of Dr. B. Murphy, U n i v e r s i t y of Saskatchewan, Saskatoon. T e s t o s t e r o n e was e x t r a c t e d from 0.5 ml a l i g u o t s of serum a c c o r d i n g t o the method of Moger and Armstrong (1974). Radioimmunoassays were preformed on d u p l i c a t e 200 ul a l i q u o t s of e x t r a c t e d t e s t o s t e r o n e , r e c o n s t i t u t e d i n 1 ml assay b u f f e r , i n s t a n d a r d assay t u b e s . S t a n d a r d s c o n t a i n i n g 23-1400 pg t e s t o s t e r o n e i n 200 utl b u f f e r were a l s o assayed i n d u p l i c a t e . A p p r o x i m a t e l y 1.5-2.0 X 10" dpm 3H t e s t o s t e r o n e i n 50 ul b u f f e r was added, f o l l o w e d by 100 u l of a 1:4000 d i l u t i o n of s p e c i f i c 19 a n t i s e r u m (NCR-E98, b l e e d i n g of 18-4-78). F o l l o w i n g o v e r n i g h t i n c u b a t i o n a t 4°C, unbound s t e r o i d s were removed by add i n g 100 j i l human gamma g l o b u l i n (10%) and 450 ^1 p o l y e t h y l e n e g l y c o l ( 3 0 % ) . Assay tubes were i n c u b a t e d on i c e f o r t e n minutes and then c e n t r i f u g e d f o r 35 mi n u t e s . The s u p e r n a t a n t s o l u t i o n was d i s c a r d e d and the p r e c i p i t a t e d i s s o l v e d i n 200 ul 0.5N NaOH, n e u t r a l i z e d w i t h 0.5N HC1, and t r a n s f e r r e d t o s c i n t i l l a t i o n v i a l s . A t o l u e n e - b a s e d s c i n t i l l a t i o n f l u i d was added (4.5 ml) and a l l o w e d t o e q u i l i b r a t e o v e r n i g h t b e f o r e c o u n t i n g . C r o s s r e a c t i o n s and v a l i d a t i o n of the a n t i s e r u m a r e o u t l i n e i n Cook et a l . (1982) . The serum samples were not chromatographed b e f o r e assay and the r e s u l t s r e p o r t e d a re the sum of 5 a - d i h y d r o t e s t o s t e r o n e and t e s t o s t e r o n e c o n c e n t r a t i o n s . These r e s u l t s a r e r e f e r r e d t o as t e s t o s t e r o n e or androgen l e v e l s t h r o u g h o u t t h i s paper. T e s t o s t e r o n e e x t r a c t i o n v a r i e d from 54-100%. The i n t r a a s s a y c o e f f i c i e n t s of v a r i a n c e were 0.3-7.9%; the i n t e r a s s a y c o e f f i c i e n t of v a r i a n c e ranged from 15-25%. I I I . S t a t i s t i c a l A n a l y s i s A l l samples were p l a c e d i n t o monthly groups and s u b j e c t e d t o a n a l y s i s of v a r i a n c e . In the presence of a s i g n i f i c a n t F v a l u e , S c h e f f e ' s t e s t was c a r r i e d out on matched p a i r s of r e s u l t s t o determine s i g n i f i c a n t d i f f e r e n c e s . I n d i v i d u a l comparisons between means were made by S t u d e n t ' s t t e s t . In s i t u a t i o n s i n v o l v i n g e x t r e m e l y s m a l l samples s i z e s K r u s k a l l -W a l l i s and Mann-Whitney U t e s t s were used t o determine 2 0 s i g n i f i c a n t differences. A l l values are expressed as monthly means ± standard error of the mean (SEM). 21 RESULTS I_. H i s t o l o g i c a l E x a m i n a t i o n of the S e m i n i f e r o u s T u b u l e s A. C e l l Types P r e s e n t S e r t o l i c e l l s ; S e r t o l i c e l l s were common a l o n g the basement membrane of s e m i n i f e r o u s t u b u l e s of immature o t t e r s ( P l a t e I , F i g . 1 ) . The n u c l e i were e l o n g a t e d , a v e r a g i n g 8.07 Mm i n l e n g t h and u s u a l l y a l i g n e d w i t h t h e i r l o n g a x i s p e r p e n d i c u l a r t o the basement membrane. The c y t o p l a s m was i n d i s t i n c t . In a d u l t o t t e r s the S e r t o l i c e l l s ( P l a t e I , F i g . 2) were m o r p h o l o g i c a l l y s i m i l a r t o those i n immature a n i m a l s . However, they were uncommon and d i f f i c u l t t o observe once sp e r m a t o g e n e s i s began. Spermatogonia: Three t y p e s of spermatogonia (Type A, Type B, and 'w i n t e r spermatogonia') were p r e s e n t i n the t u b u l e s of j u v e n i l e and a d u l t o t t e r s . Both Type A and Type B spermatogonia were v i s i b l e around the p e r i p h e r y of a l l t u b u l e s ( P l a t e I , F i g . 1, F i g . 2 ) . Type A spermatogonia were l a r g e c e l l s w i t h l i g h t c y t o p l a s m and s p e r i c a l or o v a l n u c l e i a v e r a g i n g 6.5 Mm i n diam e t e r (range = 5-10 um). The n u c l e u s c o n t a i n e d e v e n l y d i s t r i b u t e d c h r o m a t i n g r a n u l e s , a w e l l - s t a i n e d n u c l e o l u s and a d i s t i n c t membrane. Type B spermatogonia had round n u c l e i w i t h a d e f i n i t e membrane and c o a r s e r , more abundant c h r o m a t i n . They were s m a l l e r than Type A, r a n g i n g from 5-7 Mm i n di a m e t e r and a v e r a g i n g 5.3 Mm. E n l a r g e d spermatogonia termed ' w i n t e r spermatogonia' ( C o u r r i e r 1927) were common i n the c e n t r a l area 22 of immature t e s t e s . They were a l s o p r e s e n t i n the t u b u l e s of a s p e r m a t i c mature a n i m a l s , but a t lower d e n s i t i e s . These ' w i n t e r spermatogonia' were l a r g e r than e i t h e r Types A or B and were o c c a s i o n a l l y m u l t i n u c l e a t e d . The n u c l e i were p a l e s t a i n i n g , averaged 8.1 urn i n d i a m e t e r , and ranged from 6.25-12.5 um. The n u c l e a r membrane was w e l l d e f i n e d and the c y t o p l a s m c o n s p i c u o u s . T h e i r g e n e r a l appearance was t h a t of d e g e n e r a t i n g spermatogonia which have been sloughed o f f . Spermatocytes: P r i m a r y spermatocytes o c c u r r e d away from the basement membrane of the t u b u l e . They c o n t a i n e d rounded n u c l e i a v e r a g i n g 6.7 urn i n diameter (range = 6.25-7.5 um) i n which the v a r i o u s s t a g e s of m e i o s i s were c l e a r l y v i s i b l e ( P l a t e I , F i g . 3 ) . Secondary spermatocytes were l e s s common due t o the speed of m e i o t i c d i v i s i o n s . The n u c l e i were s p h e r i c a l and i n t e r m e d i a t e i n s i z e between p r i m a r y spermatocytes and s p e r m a t i d s . The mean n u c l e a r diameter was 5 um (range = 3.75-5.5 M m ) . U n l i k e p r i m a r y spermatocytes the n u c l e a r membrane was d i s t i n c t . The c y t o p l a s m was c o n s p i c u o u s i n a l l s p e r m a t o c y t e s . Spermatids and Spermatozoa: Newly formed s p e r m a t i d s ( P l a t e I I , F i g . 6) p o s s e s s e d s m a l l , round n u c l e i which ranged from 3.75-5 Mm and averaged 3.8 Mm i n d i a m e t e r . The n u c l e a r membrane was c l e a r l y o u t l i n e d and the c y t o p l a s m was e a s i l y seen. As s p e r m i o g e n e s i s proceeded the shape of the n u c l e i changed; they became o v a l , f l a t t e n e d and t h e i r a f f i n i t y f o r h a e m a t o x y l i n i n c r e a s e d ( P l a t e I I , F i g . 7 ) . The c y t o p l a s m e l o n g a t e d and e v e n t u a l l y a d i s t i n c t t a i l became v i s i b l e ( P l a t e I I , F i g . 8 ) . Mature spermatozoa had heads 5-6 Mm i n l e n g t h . 23 B. Phase A n a l y s i s The development of the s e m i n i f e r o u s e p i t h e l i u m was d i v i d e d i n t o n i n e e a s i l y r e c o g n i z a b l e phases, based upon the assemblages of c e l l t y p e s p r e s e n t (Table I I I ) . The immature s t a t e was r e p r e s e n t e d by Phase 0 t u b u l e s ( P l a t e I , F i g . 1 ) ; no s i g n s of a c t i v i t y were p r e s e n t and development d i d not proceed beyond t h e o c c a s i o n a l p r i m a r y spermatocyte. S e x u a l l y mature o t t e r s were c h a r a c t e r i z e d by the p r e s e n c e of s e m i n i f e r o u s t u b u l e s i n the st a g e s of development r e p r e s e n t e d by Phases 1 - 8. Phase 1 ( P l a t e I , F i g . 2) t u b u l e s were i n a c t i v e and appeared s i m i l a r t o the immature s t a t e . T h i s phase was seen d u r i n g the summer months and r e p r e s e n t s the u s u a l r e s t i n g s t a t e . Phase 2 ( P l a t e I , F i g . 3) was an i n t e r m e d i a t e phase w h i l e Phases 3 ( P l a t e I I , F i g . 4) and 4 ( P l a t e I I , F i g . 5) were p r e s e n t d u r i n g the i n c r e a s e d a c t i v i t y p r e c e d i n g s p e r m i o g e n e s i s . Phases 1 - 4 were c h a r a c t e r i s t i c of a s p e r m a t i c o t t e r s . Phases 5 through 7 ( P l a t e I I , F i g s . 6,7,8) were t y p i c a l of o t t e r s u ndergoing a c t i v e s p e r m i o g e n e s i s and were c h a r a c t e r i z e d by round s p e r m a t i d s (Phase 5) which dev e l o p e d i n t o mature sperm (Phase 7 ) . A h i g h p r o p o r t i o n of Phase 7 t u b u l e s i n a t e s t i s was an i n d i c a t i o n of maximum r e p r o d u c t i v e a c t i v i t y . Phase 8 t u b u l e s ( P l a t e I I , F i g . 9) were i n d i c a t i v e of t e s t e s a t the end of sperm p r o d u c t i o n . D e g e n e r a t i n g spermatozoa and s p e r m a t o c y t e s were p r e s e n t i n the lumen of some t u b u l e s and a n i m a l s w i t h the m a j o r i t y of t u b u l e s a t t h i s stage o f t e n c o n t a i n e d spermatozoa i n the e p i d i d y m i s . T h i s appeared t o be a 24 T a b l e I I I . D e s c r i p t i o n s o f p h a s e s used i n a n a l y s i s o f s e m i n i f e r o u s t u b u l e s o f r i v e r o t t e r s . P h a s e 0 i s c h a r a c t e r i s t i c o f immature o t t e r s w h i l e P h a s e s 1-8 a r e p r e s e n t i n s e x u a l l y ma tu re m a l e s . Phase 0 : Phase 1: P h a s e 2 : P h a s e 3 : P h a s e 4 : Phase 5 : P h a s e 6 : P h a s e 7 : Immature t e s t i s . S m a l l , c l o s e d s e m i n i f e r o u s t u b u l e s l i n e d w i t h a s i n g l e l a y e r o f s p e r m a t o g o n i a and S e r t o l i c e l l n u c l e i . ' W i n t e r s p e r m a t o g o n i a ' and t h e o c c a s i o n a l s p e r m a t o c y t e o c c u r i n t h e c e n t r a l a r e a o f t h e t u b u l e s . T y p e s A and B s p e r m a t o g o n i a and S e r t o l i c e l l n u c l e i l i n e t h e p e r i p h e r y o f s o l i d t u b u l e s . ' W i n t e r s p e r m a t o g o n i a ' o f t e n p r e s e n t , b u t a r e n o t commmon. The o c c a s i o n a l s p e r m a t o g o n i a may be u n d e r g o i n g m i t o s i s . A p p e a r a n c e s i m i l a r t o immature t e s t i s . S i m i l a r t o P h a s e 1 b u t w i t h a l ow number o f p r i m a r y s p e r m a t o c y t e s p r e s e n t . Numerous p r i m a r y s p e r m a t o c y t e s p r e s e n t . S e c o n d a r y s p e r m a t o c y t e s may be s e e n . P r i m a r y and s e c o n d a r y s p e r m a t o c y t e s o r g a n i z e d i n t o l a y e r s . M a t u r a t i o n d i v i s i o n s o c c u r i n g be tween s p e r m a t o c y t e g e n e r a t i o n s . Some s p e r m a t i d s may be p r e s e n t . Round s p e r m a t i d s numerous . Some u n d e r g o i n g s p e r m i o g e n e s i s and e l o n g a t i n g . Two g e n e r a t i o n s o f s p e r m a t i d s p r e s e n t . E l o n g a t e d s p e r m a t i d s a s s o c i a t e d i n w e l l o r g a n i z e d b u n d l e s be tween round s p e r m a t i d s . T a i l s v i s i b l e and o r i e n t a t e d t o w a r d s t h e l u m e n . Sperm l i n e t u b u l e l u m e n . Round s p e r m a t i d s a l s o p r e s e n t . P h a s e o f h i g h e s t a c t i v i t y . P h a s e 8 : S p e n t t u b u l e a t end o f p e r i o d o f a c t i v i t y . I n c o m p l e t e a s s e m b l a g e o f c e l l l a y e r s p r e s e n t . I f lumen i s p r e s e n t i t may be l a r g e and c o n t a i n a s s o r t e d c e l l t y p e s . 25 P l a t e I . C e l l u l a r a s s o c i a t i o n s c h a r a c t e r i s t i c of Phase 0 t h r o u g h Phase 3 s e m i n i f e r o u s t u b u l e s of L. c a n a d e n s i s . A l l s e c t i o n s s t a i n e d w i t h H and E. A l l m a g n i f i c a t i o n s a p p r o x i m a t e l y 525X. F i g u r e 1. Phase 0 s e m i n i f e r o u s t u b u l e s c h a r a c t e r i s t i c of s e x u a l l y immature o t t e r s . T ubules a r e narrow, c l o s e d and l i n e d w i t h spermatogonia (S) and S e r t o l i c e l l n u c l e i . 'Winter spermatogonia' (W) and the o c c a s i o n a l s p e r m a t o c y t e may occur i n the c e n t r e of the t u b u l e . F i g u r e 2. Phase 1 s e m i n i f e r o u s t u b u l e s p r e s e n t i n the t e s t e s of a mature r i v e r o t t e r . Appearance i s s i m i l a r t o immature t u b u l e s w i t h a p e r i p h e r a l l a y e r of type A and type B spermatogonia and S e r t o l i c e l l n u c l e i . Phase 1 i s t h e l e a s t a c t i v e phase i n mature o t t e r s . F i g u r e 3. Phase 2 s e m i n i f e r o u s t u b u l e s . S i m i l a r t o Phase 1 t u b u l e s but w i t h a low number of p r i m a r y spermatocytes ( P s ) . Note the i n a c t i v e L e y d i g c e l l s ( L ) . F i g u r e 4. Phase 3 s e m i n i f e r o u s t u b u l e . Numerous p r i m a r y s p e r m a t o c y t e s and the o c c a s i o n a l secondary spermatocyte are p r e s e n t . 2 6 27 P l a t e I I . C e l l u l a r a s s o c i a t i o n s c h a r a c t e r i s t i c of Phase 4 t h r o u g h Phase 8 s e m i n i f e r o u s t u b u l e s of s e x u a l l y mature L. c a n a d e n s i s . F i g u r e 5. Phase 4 s e m i n i f e r o u s t u b u l e c o n s i s t i n g of a basement membrane l i n e d w i t h spermatogonia and S e r t o l i c e l l n u c l e i . P r i m a r y and secondary s p e r m a t o c y t e s p r e s e n t and o r g a n i z e d i n t o l a y e r s . S p e r m a t i d s (arrow) may be p r e s e n t . M a g n i f i c a t i o n a p p r o x i m a t e l y 320X. F i g u r e 6. Phase 5 s e m i n i f e r o u s t u b u l e . Round s p e r m a t i d s numerous. Some s p e r m a t i d n u c l e i becoming e l o n g a t e d . Note a c t i v e L e y d i g c e l l s . M a g n i f i c a t i o n a p p r o x i m a t e l y 260X. F i g u r e 7. Phase 6 s e m i n i f e r o u s t u b u l e . E l o n g a t e d s p e r m a t i d s a s s o c i a t e d w i t h S e r t o l i c e l l s and o r g a n i z e d i n t o b u n d l e s . T a i l s p r e s e n t and o r i e n t a t e d towards the lumen. M a g n i f i c a t i o n a p p r o x i m a t e l y 400X. F i g u r e 8. Phase 7 s e m i n i f e r o u s t u b u l e s . Abundant spermatozoa l i n e t u b u l e lumen. Phase 7 i s the most a c t i v e phase. M a g n i f i c a t i o n a p p r o x i m a t e l y 260X. F i g u r e 9. Phase 8 s e m i n i f e r o u s t u b u l e c h a r a c t e r i s t i c of t u b u l e s at the end of s e x u a l a c t i v i t y . Note i n c o m p l e t e assemblage of c e l l t y p e s and d e c r e a s e d t u b u l e d i a m e t e r . M a g n i f i c a t i o n a p p r o x i m a t e l y 400X. 2 9 t r a n s i t o r y phase and was o f t e n d i f f i c u l t t o r e c o g n i z e . I I . The Annual C y c l e and Se x u a l M a t u r i t y A. The T e s t i s J_. The S e m i n i f e r o u s t u b u l e s On the b a s i s of the c y c l i c changes which o c c u r r e d i n t h e i r t e s t i s , o t t e r s two y e a r s of age and o l d e r were c o n s i d e r e d s e x u a l l y mature i n t h i s s t u d y . No s i g n i f i c a n t d i f f e r e n c e s were found among o t t e r s o l d e r than age c l a s s 1 and s p e r m a t o g e n e s i s appeared t o be p r o c e e d i n g n o r m a l l y even i n the o l d e s t a n i m a l (14 y e a r s ) . W i t h few e x c e p t i o n s , no d i f f e r e n c e s were seen between s e c t i o n s of the same t e s t i s or between c o n t r a l a t e r a l t e s t e s . Two a n i m a l s e x h i b i t e d u n i l a t e r a l d e g e n e r a t i o n of the t e s t e s and are d e s c r i b e d i n Appendix 1. J u v e n i l e o t t e r s were s e x u a l l y immature and a l l of the t e s t e s examined c o n t a i n e d Phase 0 t u b u l e s e x c l u s i v e l y . Y e a r l i n g a n i m a l s were found t o be m a t u r i n g s e x u a l l y and the phases p r e s e n t a r e d i s c u s s e d i n d e t a i l l a t e r (see s e x u a l m a t u r i t y ) . I n a c t i v e t u b u l e s (Phase 1) were predominate i n the t e s t e s of mature o t t e r s d u r i n g the months of J u l y and August ( F i g . 10). The lumen was f i l l e d w i t h S e r t o l i c e l l c y t o p l a s m and an o c c a s i o n a l ' w i n t e r s p e r m a t o g o n i a ' . The f i r s t s i g n s of spe r m a t o g e n e s i s were seen i n September when Phase 2 t u b u l e s , i n d i c a t i n g low l e v e l s of a c t i v i t y , were found i n some t e s t e s . P r o l i f e r a t i o n of the s e m i n i f e r o u s e p i t h e l i u m o c c u r r e d d u r i n g the 30 F i g u r e 10. Phase A n a l y s i s of mature o t t e r t e s t e s . See T a b l e I I I f o r e x p l a n a t i o n of phases. Phase 7 i n d i c a t e s f u l l y a c t i v e s p e r m a t o g e n e s i s . T o t a l number of t u b u l e s examined i s 3500. 31 O c 0 cr CD c CD O i _ CD 0_ 100 75 50 2 5 i 100 75-i 50 25 1001 75 50 ] 25 100 75 50 25 H 100 75 5 0 -2 5 -100 75 50 25 July n=120 - i r i Aug n--2o T 1 1 1 1 1— Sept sn--20 -i 1 1 1 r-Oct n--0 Nov rr-400 Dec n=tl80 1 2 3 4 5 6 7 8 - i — i — i i i Jan n=840 Feb n=500 March n=120 April n=140 May n=120 June n=40 i i — i — i — i — 1 2 3 4 5 6 7 8 P h a s e 32 f a l l and by November most of the t u b u l e s c o n t a i n e d p r i m a r y s p e r m a t o c y t e s u n d e r g o i n g m e i o s i s (Phase 3 ) . Spermatogenic a c t i v i t y i n c r e a s e d throughout December and January as i n d i c a t e d by the i n c r e a s e i n the p r o p o r t i o n of t u b u l e s c o n t a i n i n g secondary s p e r m a t o c y t e s and s p e r m a t i d s (Phases 4 - 6 ) . A l t h o u g h Phase 7 t u b u l e s were f i r s t seen i n December, they d i d not make up the m a j o r i t y of the t u b u l e s i n any t e s t i s u n t i l F e b r u a r y . Based on the h i g h p r e c e n t a g e of Phase 7 t u b u l e s p r e s e n t , maximum spermatogenic a c t i v i t y o c c u r r e d from F e b r u a r y through A p r i l . Phase 8 t u b u l e s , i n d i c a t i n g the end of s p e r m a t o g e n e s i s , were f i r s t o b s e r v e d i n l a t e A p r i l and were common i n t e s t e s c o l l e c t e d d u r i n g May and June. The s e m i n i f e r o u s t u b u l e s were s m a l l i n j u v e n i l e o t t e r s and d i d not v a r y s i g n i f i c a n t l y throughout the year ( F i g . 11), Among a d u l t s however, the mean monthly diameter of the s e m i n i f e r o u s t u b u l e s v a r i e d s e a s o n a l l y , r e f l e c t i n g the degree of r e p r o d u c t i v e a c t i v i t y p r e s e n t . D u r i n g the summer months t u b u l e d i a m e t e r s were s i m i l a r t o tho s e of immature a n i m a l s . As the t u b u l e e p i t h e l i u m p r o l i f e r a t e d , however, the diameter of the s e m i n i f e r o u s t u b u l e s i n c r e a s e d . S i g n i f i c a n t i n c r e a s e s i n the mean monthly d i a m e t e r s o c c u r r e d i n December, January and F e b r u a r y (p<.00l) and a maximum dia m e t e r of 181.1 ± 16.3 /xm o c c u r r e d i n March ( F i g . 11). As spermatogenesis ended i n l a t e A p r i l , mean monthly t u b u l e d i a m e t e r s a l s o began t o decrease t o i n a c t i v e l e v e l s . The changes which o c c u r r e d i n the d i a m e t e r s of the s e m i n i f e r o u s t u b u l e s among y e a r l i n g o t t e r s appeared t o r e f l e c t 3 3 F i g u r e 11. S e m i n i f e r o u s t u b u l e d i a m e t e r s of immature (0 O) , y e a r l i n g (• •) and a d u l t (• D o t t e r s . V a l u e s e x p r e s s e d as mean diameter ± SEM. Sample s i z e i n d i c a t e d above b a r s . F i g u r e 12. L e y d i g c e l l n u c l e u s d i a m e t e r s i n immature (O O) and a d u l t (• •) o t t e r s . Y e a r l i n g s a r e grouped w i t h a d u l t s . V a l u e s e x p r e s s e d as mean dia m e t e r ± SEM. Sample s i z e i n d i c a t e d above b a r s . 3 5 the approach of s e x u a l m a t u r i t y ( F i g . 11). Tubule d i a m e t e r s c o u l d not be d i s t i n g u i s h e d from those of immatures d u r i n g November but were s i g n i f i c a n t l y d i f f e r e n t i n December (P<0.05) and J a n u a r y (P<0.001). A l t h o u g h mean d i a m e t e r s were s i g n i f i c a n t l y d i f f e r e n t from a d u l t s i n the t h r e e months f o r which d a t a are a v a i l a b l e , a s i g n i f i c a n t i n c r e a s e (p<0.00l) s i m i l a r t o t h a t seen i n the a d u l t s o c c u r r e d between December and January i n j u v e n i l e s . 2. The I n t e r t u b u l a r T i s s u e I n t e r t u b u l a r t i s s u e was m o d e r a t e l y abundant i n immature t e s t e s , c o m p r i s i n g a p p r o x i m a t e l y 23% of the t o t a l volume (Table I V ) . L e y d i g c e l l s accounted f o r t w o - t h i r d s of the i n t e r t u b u l a r t i s s u e and c o n t a i n e d s p h e r i c a l or o v a l n u c l e i w i t h one or two n u c l e o l i , g r a n u l a r c h r o m a t i n and a prominent n u c l e a r membrane. The n u c l e a r diameter averaged 4.4 /im ( F i g . 12) t h roughout the s a m p l i n g p e r i o d . Very l i t t l e c y t o p l a s m was p r e s e n t and no v a c u o l e s were observed. In mature o t t e r s the r e l a t i v e volume of t e s t i c u l a r i n t e r t u b u l a r t i s s u e was found t o d e c r e a s e w i t h i n c r e a s i n g r e p r o d u c t i v e a c t i v i t y (Table I V ) ; p r o b a b l y a r e s u l t of the p r o p o r t i o n a t e l y g r e a t e r i n c r e a s e t h a t o c c u r r e d i n t u b u l a r d i a m e t e r . D u r i n g peak spermatogenesis i n March, i n t e r t u b u l a r t i s s u e a c c o u n t e d f o r o n l y 14% of the t o t a l t u b u l a r volume compared w i t h 40% d u r i n g J u l y and August when the t e s t e s were i n a c t i v e . When the r e l a t i v e volume of the L e y d i g c e l l s was m u l t i p l i e d by the average t e s t i s weight t o e s t i m a t e the t o t a l T a b l e I V . R e l a t i v e vo lumes o f i n t e r - and e x t r a - t u b u l a r t i s s u e i n t h e i n t e r l o b u l a r a r e a o f t h e t e s t e s o f o t t e r s f o r d i f f e r e n t r e p r o d u c t i v e s t a t e s . A l l v a l u e s e x p r e s s e d as means + S . E . R e p r o d u c t i v e N a I n t e r - t u b u l a r E x t r a - t u b u l a r Volume {%) E s t i m a t e d W e i g h t C a t e g o r y Volume L e y d i g o f L e y d i g C e l l s b {%) C e l l s O t h e r (gm) Immature : 5 7 7 . 3 + 1.3 1 4 . 8 + 1.6 7 . 8 + 1.7 0 . 1 1 M a t u r e : November 4 7 5 . 4 + 2 . 5 1 5 . 3 + 1.1 9 . 3 + 1.7 0 . 7 8 December 4 7 6 . 2 + 2 . 6 1 5 . 8 + 1.1 8 . 1 + 1 .7 0 . 9 8 J a n u a r y 6 8 0 . 3 + 1.7 1 4 . 4 + 1.6 5 . 3 + 0 . 8 1.44 F e b r u a r y 4 8 2 . 6 + 1.4 1 3 . 0 + 1 .5 4 . 4 + 0 . 4 1 .59 March 3 8 6 . 0 + 2 . 6 1 1 . 0 + 2 . 5 3 . 0 + 0 . 1 1 .43 A p r i l 4 7 2 . 3 + 4 . 3 2 0 . 9 + 2 . 2 6 . 7 + 2 . 3 2 . 1 1 May 2 7 8 . 7 + 1.9 1 2 . 6 + 3 . 6 8 . 7 + 1.7 0 . 8 9 J u l y 2 5 5 . 9 + 1.2 2 6 . 9 + 1.1 17 .1 + 2 . 3 1 .03 A u g u s t 1 5 8 . 7 3 0 . 5 1 0 . 7 1.56 Sep tember 1 7 6 . 3 1 6 . 3 7 . 4 0 . 5 5 a Each sample c o n s i s t s o f 42 p o i n t s f rom t e n f i e l d s o f v i e w p e r t e s t i s , b W e i g h t e s t i m a t e d by m u l t i p l y i n g % v o l X a v e r a g e w e i g h t o f t e s t i s . LO CTV 3 7 L e y d i g c e l l w e i g h t , t h e l a t t e r tended t o i n c r e a s e d u r i n g r e p r o d u c t i v e a c t i v i t y . An e x c e p t i o n t o t h i s t r e n d o c c u r r e d i n August when an i n c r e a s e i n e s t i m a t e d L e y d i g c e l l w e i g h t s o c c u r r e d . However, t h i s may be an a r t i f a c t of the s m a l l sample s i z e . The changes t h a t o c c u r r e d i n the d i a m e t e r of the n u c l e i were a l s o used t o i n d i c a t e the a c t i v i t y of the L e y d i g c e l l s . From May t h r o u g h August, the mean monthly d i a m e t e r s of the L e y d i g c e l l n u c l e i were s i m i l a r t o tho s e i n immature t e s t e s ( F i g . 12). N u c l e a r s i z e i n c r e a s e d d u r i n g the f a l l and w i n t e r , and peaked d u r i n g the b r e e d i n g season i n F e b r u a r y and March. As sperm a t o g e n e s i s d e c r e a s e d the diameter of the L e y d i g c e l l s d e c l i n e d . These r e s u l t s i n d i c a t e an i n c r e a s e i n an d r o g e n i c a c t i v i t y of the L e y d i g c e l l s i n synchrony w i t h the development of the s e m i n i f e r o u s e p i t h e l i u m . Changes i n serum t e s t o s t e r o n e l e v e l s i n c a p t i v e o t t e r s , d e s c r i b e d l a t e r , q u a n t i f y the a c t u a l s e c r e t o r y output of the L e y d i g c e l l s . L e y d i g c e l l n u c l e a r d i a m e t e r s of y e a r l i n g o t t e r s were not s i g n i f i c a n t l y d i f f e r e n t from a d u l t s i n the t h r e e months (November - Ja n u a r y ) f o r which da t a i s a v a i l a b l e . B. The E p i d i d y m i s The e p i d i d y m i d e s of j u v e n i l e o t t e r s d i d not v a r y s i g n i f i c a n t l y t hroughout t h e y e a r . They c o n s i s t e d of narrow t u b u l e s ( F i g . 13), l i n e d w i t h a l a y e r of p s e u d o s t r a t i f i e d e p i t h e l i a l c e l l s and s u r r o u n d e d by a t h i c k band of c i r c u l a r m uscle. The p r i n c i p a l e p i t h e l i a l c e l l s were c u b o i d a l and no 38 Figure 13. Ductus epididymis diameter in immature (O O), yearling (• •) and mature (• •) otters. Values expressed as mean diameter ± SEM. Sample size indicated above bars. Ductus Epididymis Diameter (um) 4 0 s t e r i o c i l i a were v i s i b l e ( P l a t e I I I , F i g . 14). The e p i t h e l i a and muscle l a y e r s d i d not v a r y s i g n i f i c a n t l y i n t h i c k n e s s throughout the y e a r , a v e r a g i n g 16.6 ± 1.6 and 18.8 ± 0.8 um r e s p e c t i v e l y . C h a r a c t e r i s t i c a l l y , i n immature o t t e r s the muscle l a y e r was a p p r o x i m a t e l y the same t h i c k n e s s as the e p i t h e l i a l l a y e r . In s e x u a l l y mature o t t e r s the e p i d i d y m i d e s were found t o undergo c y c l i c changes which c o r r e s p o n d e d t o those of the t e s t e s . D u r i n g months when the t e s t i s was i n a c t i v e , the d u c t u s e p i d i d y m i s c o n s i s t e d of a t h i c k , c i r c u l a r smooth muscle l a y e r s u r r o u n d i n g a p s e u d o s t r a t i f i e d e p i t h e l i a l l a y e r ( P l a t e I I I , F i g . 15). T h i s e p i t h e l i a l l a y e r was composed of b a s a l c e l l s and low columnar p r i n c i p a l c e l l s . The lumen was o f t e n absent and no s t e r i o c i l i a were seen. As i n immature o t t e r s , the muscle and e p i t h e l i a l l a y e r s were a p p r o x i m a t e l y the same t h i c k n e s s (14-16 Mm) a l t h o u g h the t o t a l d i a m e t e r was s i g n i f i c a n t l y l a r g e r (p<.05). As s p e r m a t o g e n e s i s proceeded i n the t e s t i s , the du c t u s e p i d i d y m i s d i a m e t e r s i n c r e a s e d ( F i g . 13) and the p r i n c i p a l c e l l s e l o n g a t e d . N u c l e i of the p r i n c i p a l c e l l s became o v o i d and c o n f i n e d t o the b a s a l p o r t i o n of the c e l l ( P l a t e I I I , F i g . 16). A lumen d e v e l o p e d and s t e r i o c i l i a 6-10 Mm l o n g were v i s i b l e . By November the h e i g h t of the e p i t h e l i a l l a y e r i n c r e a s e d t o an average of 27.2 ± 1.5 Mm w h i l e the mean muscle l a y e r t h i c k n e s s d e c r e a s e d t o 12.7 ± 0.7 Mm. Sperm were f i r s t seen i n l a t e December and by mid Ja n u a r y a l l of the males c o n t a i n e d sperm i n t h e i r e p i d i d y m i d e s . D u r i n g the peak of r e p r o d u c t i v e a c t i v i t y i n 41 P l a t e I I I . Photomicrographs of d u c t u s e p i d i d y m i d e s of L. c a n a d e n s i s . A l l s e c t i o n s s t a i n e d w i t h H and E. F i g u r e 14. Ductus e p i d i d y m i d e s of an immature r i v e r o t t e r . Note the t h i n c u b o i d a l e p i t h e l i a l l a y e r and t h i c k band of c i r c u l a r muscle. M a g n i f i c a t i o n a p p r o x i m a t e l y 400X. F i g u r e 15. Ductus e p i d i d y m i d e s of a mature r i v e r o t t e r d u r i n g a p e r i o d of s e x u a l i n a c t i v i t y . S i m i l a r t o those of immature o t t e r s . M a g n i f i c a t i o n a p p r o x i m a t e l y 340X. F i g u r e 16. Ductus e p i d i d y m i s of a s e x u a l l y a c t i v e o t t e r c o n t a i n i n g mature spermatozoa. Note the columnar e p i t h e l i a l c e l l s , t h i n c i r c u l a r muscle l a y e r and the presence of s t e r e o c i l i a . M a g n i f i c a t i o n a p p r o x i m a t e l y 260X. F i g u r e 17. I n t r a - e p i t h e l i a l lumen ( I ) p r e s e n t i n the d u c t u s e p i d i d y m i s of a mature r i v e r o t t e r . Lumina were most common i n samples c o n t a i n i n g e a r l y s t a g e s of spermatogenesis i n the t e s t e s . M a g n i f i c a t i o n a p p r o x i m a t e l y 280X. 42 4 3 March, the e p i t h e l i a l l a y e r reached a maximum h e i g h t of 41.7 ± 2.0 * i m and the muscle l a y e r d e c r e a s e d t o a minimum t h i c k n e s s of 6.1 ± 0.16 M m . The s i g n i f i c a n t change (F = 16.12, df = 9,69, P<.001) i n t u b u l a r d i a m e t e r from a minimum of 108 ± 6.5 Mm i n J u l y and August t o a maximum of 233.4 ± 14.3 Mm i n March appears t o be due t o an i n c r e a s e i n lumen s i z e as w e l l as the i n c r e a s e d h e i g h t of the e p i t h e l i a l l a y e r . T h i s growth of the e p i t h e l i a l c e l l s may r e f l e c t i n c r e a s e d s e c r e t o r y output d u r i n g a c t i v i t y . Sperm were not p r e s e n t i n the e p i d i d y m i d e s of specimens c o l l e c t e d a f t e r A p r i l and d u r i n g May the lumen of some ductus c o n t a i n e d l a r g e amounts of c e l l u l a r d e b r i s . As spermatogenesis ended the t u b u l e s took on an i n a c t i v e appearance. The e p i d i d y m i d e s of j u v e n i l e o t t e r s were s i m i l a r i n appearance t o those of a d u l t s . The mean d i a m e t e r of the ductus e p i d i d y m i s of y e a r l i n g s was s i g n i f i c a n t l y d i f f e r e n t from j u v e n i l e s i n December and January (p<0.0l) but c o u l d not be d i f f e r e n t i a t e d from a d u l t s a t any t i m e . L a r g e , round c a v i t i e s were found i n the l u m i n a l e p i t h e l i a of the e p i d i d y m i d e s of some males ( P l a t e I I I , F i g . 17). These ' i n t r a - e p i t h e l i a l l u m i n a ' (Onstad 1967) were common i n animals c o l l e c t e d d u r i n g November and December and were o c c a s i o n a l l y p r e s e n t i n J a n u a r y and F e b r u a r y samples. Some lumina c o n t a i n e d l a r g e c e l l s w i t h s m a l l round n u c l e i and c o n s p i c u o u s c y t o p l a s m . A l t h o u g h t h e i r f u n c t i o n i s unknown t h e i r appearance and the f a c t t h a t they a r e p r e s e n t when the e p i d i d y m i s i s becoming a c t i v e s u g g ests t h a t they may be s e c r e t o r y i n n a t u r e . 4 4 C . T e s t e s S i z e T h e m e a n t e s t i s w e i g h t o f i m m a t u r e o t t e r s i n c r e a s e d s i g n i f i c a n t l y ( F = 8 . 1 3 , d f = 5 , 5 0 , p < . 0 0 l ) f r o m 0 . 7 ± 0 . 0 3 g m a t e i g h t m o n t h s t o 1 .1 ± 0 . 0 6 g m b y o n e y e a r o f a g e ( F i g . 1 8 ) . T h i s g r a d u a l i n c r e a s e i n w e i g h t l i k e l y r e f l e c t e d t h e o v e r a l l i n c r e a s e i n s i z e o f t h e a n i m a l s b u t d u e t o t h e m a n n e r i n w h i c h s a m p l e s w e r e c o l l e c t e d , i t w a s n o t p o s s i b l e t o e x p r e s s t e s t i s w e i g h t a s a f u n c t i o n o f t o t a l b o d y w e i g h t . T h e m e a n m o n t h l y t e s t i s v o l u m e o f i m m a t u r e o t t e r s w e r e s m a l l a n d d i d n o t v a r y s i g n i f i c a n t l y t h r o u g h o u t t h e y e a r ( F i g . 1 9 ) . A l t h o u g h t h e m e a n t e s t i s w e i g h t s a n d v o l u m e s o f y e a r l i n g o t t e r s w e r e g r e a t e r t h a n j u v e n i l e s , t h e d i f f e r e n c e s w e r e s i g n i f i c a n t ( p < 0 . 0 0 l ) i n J a n u a r y o n l y . Y e a r l i n g s s h o w e d s i g n i f i c a n t i n c r e a s e s i n b o t h w e i g h t s a n d v o l u m e s b e t w e e n D e c e m b e r a n d J a n u a r y ( p < 0 . 0 0 l ) b u t w e r e s m a l l e r t h a n a d u l t s i n a l l t h r e e m o n t h s ( p < 0 . 0 0 l ) . A m o n g a d u l t m a l e s , t h e t e s t e s w e i g h t s ( F i g . 1 8 ) a n d v o l u m e s ( F i g . 1 9 ) s h o w e d c y c l i c a l c h a n g e s w h i c h r e f l e c t e d t h e a n n u a l c y c l e o f t e s t i c u l a r a c t i v i t y . A s s p e r m a t o g e n e s i s b e g a n i n t h e f a l l t h e m e a n t e s t i s w e i g h t i n c r e a s e d f r o m t h e m i n i m u m s i z e s s e e n d u r i n g t h e s u m m e r m o n t h s ( F i g . 1 8 ) . C o i n c i d i n g w i t h t h e p r o l i f e r a t i o n o f t h e s e m i n i f e r o u s e p i t h e l i u m i n D e c e m b e r t h r o u g h F e b r u a r y , t h e w e i g h t i n c r e a s e d r a p i d l y ( p < . 0 0 l ) t o p e a k d u r i n g t h e b r e e d i n g s e a s o n . A s s p e r m a t o g e n e s i s d e c l i n e d t h e t e s t i c u l a r s i z e q u i c k l y r e t u r n e d t o r e s t i n g l e v e l s . T h e t e s t i s w e i g h t s o f m a t u r e o t t e r s w e r e l a r g e r t h a n t h o s e o f i m m a t u r e s a t a l l t i m e s o f t h e y e a r ( p < . 0 0 l ) . 4 5 Figure 18. Testis weight in immature (O O), yearling (• •) and mature (• •) ott e r s . Values expressed as mean weight ± SEM. Sample size indicated above bars. Figure 19. Testis volume in immature (O O), yearling (• •) and mature (• •) ott e r s . Values expressed as mean volume ± SEM. Sample size indicated above bars. 13 12 11 10 9 8 7 6. 5. 4 . 3 . 2 . 1 10-. 9 -8 . E 7. o 6 . CO E 5 . 4. CO +-> 3 -CO |2 2 . 1 _ - i 1 r N D J F M A M J Month Figure 18 —i 1 1 A S O Figure 19 N D - i 1 1 1 1 r F M A M J J A S O Month 47 Changes i n t e s t i c u l a r volumes among a d u l t o t t e r s f o l l o w e d a p a t t e r n s i m i l a r t o t h a t of t e s t i c u l a r w e i g h t s ( F i g u r e 19); d u r i n g J u l y and August volumes were s i g n i f i c a n t l y s m a l l e r (p<0.00l) than those a t o t h e r t i m e s of the year and r a p i d growth o c c u r r e d i n December through F e b r u a r y . A l t h o u g h peak t e s t i s volume was reached i n F e b r u a r y , i t was not s i g n i f i c a n t l y d i f f e r e n t from t h a t of March when o t h e r i n d i c a t o r s of r e p r o d u c t i v e a c t i v i t y r e a c h t h e i r maximum. L i t t l e d i f f e r e n c e i n s i z e was found t o occur between c o n t r a l a t e r a l t e s t e s i n e i t h e r j u v e n i l e or a d u l t o t t e r s . Only f i v e a d u l t o t t e r s had t e s t e s i n which one was l e s s than 40% of the volume of the o t h e r . Phase a n a l y s i s and measurements of the t u b u l a r d i a m e t e r s i n d i c a t e d t h a t i n t h r e e a n i m a l s both t e s t e s were a t the same stage of r e p r o d u c t i v e a c t i v i t y . The two o t h e r e x c e p t i o n s i n v o l v e d u n i l a t e r a l d e g e n e r a t i o n of the t e s t e s and are d e s c r i b e d i n Appendix 1. D. Summary of the E x a m i n a t i o n of R e p r o d u c t i v e Organs Among the a d u l t o t t e r s , the v a r i o u s i n d i c a t i o n s of r e p r o d u c t i v e a c t i v i t y were h i g h l y c o r r e l a t e d ( T a ble V) and showed the same annual c y c l e of a c t i v i t y . The t e s t e s were i n a c t i v e d u r i n g the summer months ( J u l y , August) and low l e v e l s of a c t i v i t y began i n the f a l l . From December th r o u g h F e b r u a r y t e s t i s volume, t e s t i s w e i g h t , s e m i n i f e r o u s t u b u l e d i a m e t e r and duc t u s e p i d i d y m i s d i a m e t e r a l l i n c r e a s e d r a p i d l y . Peak a c t i v i t y o c c u r r e d d u r i n g the months of F e b r u a r y , March and A p r i l , 48 T a b l e V. C o r r e l a t i o n s ( r v a l u e s ) between i n d i c a t o r s of r e p r o d u c t i v e a c t i v i t y i n o t t e r s g r e a t e r than one y e a r of age. In a l l i n s t a n c e s p<.00l. T e s t i s Weight T e s t i s Volume .94 Sem. Tubule D i a . .71 Ductus E p i d . D i a . .75 T e s t i s Sem. Tubule Volume Diameter .69 .71 .75 i d e n t i f y i n g t h i s as the c e n t e r of the b r e e d i n g season. By l a t e May a c t i v i t y had d e c l i n e d t o r e s t i n g l e v e l s . E. S e x u a l M a t u r i t y The r e s u l t s of my study i n d i c a t e t h a t i n B r i t i s h Columbia, male r i v e r o t t e r s a t t a i n s e x u a l m a t u r i t y d u r i n g t h e i r second y e a r . Phase a n a l y s i s of the s e m i n i f e r o u s e p i t h e l i u m showed sp e r m a t o g e n e s i s f i r s t o c c u r r e d among y e a r l i n g o t t e r s . However, peak a c t i v i t y appeared t o occur l a t e r i n y e a r l i n g s than i n a d u l t s . In b o t h age groups Phase 2 and 3 t u b u l e s were the most common d u r i n g November. However, by January spermatogenesis had p r o g r e s s e d as f a r as Phase 7 i n a d u l t s ( F i g . 10) w h i l e i n j u v e n i l e s , Phases 3 and 4 predominated (61.7% and 28.3% r e s p e c t i v e l y ) . Only 5% of the t u b u l e s of y e a r l i n g s c a p t u r e d i n January c o n t a i n e d s p e r m a t i d s (Phase 5) and none were e l o n g a t e d . A l t h o u g h no samples were c o l l e c t e d from y e a r l i n g o t t e r s d u r i n g the b r e e d i n g season, a specimen c o l l e c t e d i n May c o n s i s t e d of Phase 8 t u b u l e s . These t u b u l e s a r e c h a r a c t e r i s t i c of 'spent' 49 t e s t e s , i m p l y i n g t h a t f u l l s p e r m a t o g e n e s i s had o c c u r r e d . Other i n d i c a t o r s of r e p r o d u c t i v e a c t i v i t y support the c o n t e n t i o n t h a t o t t e r s are s e x u a l l y mature by 24 months of age. B e g i n n i n g a t l e v e l s i n t e r m e d i a t e between a d u l t s and j u v e n i l e s , s e m i n i f e r o u s t u b u l e d i a m e t e r s ( F i g . 11), t e s t e s volume ( F i g . 18), t e s t i s weight ( F i g . 19), and du c t u s e p i d i d y m i s d i a m e t e r s ( F i g . 13) a l l i n c r e a s e d i n a manner s i m i l a r t o t h a t of a d u l t s . Among y e a r l i n g o t t e r s , s i g n i f i c a n t i n c r e a s e s (p<.OU1) o c c u r r e d i n t e s t i s volume, weight and s e m i n i f e r o u s t u b u l e d i a m e t e r s between November and December. A l s o , L e y d i g c e l l n u c l e i d i a m e t e r s , an i n d i c a t o r of a n d r o g e n i c a c t i v i t y , were not s i g n i f i c a n t l y d i f f e r e n t from a d u l t s i n a l l of the months f o r which d a t a a re a v a i l a b l e . These s i m i l a r i t i e s i n d i c a t e t h a t y e a r l i n g o t t e r s were becoming l e s s l i k e j u v e n i l e s and more s i m i l a r t o a d u l t s as the season p r o g r e s s e d . I I I . E n d o c r i n o l o g y of the Male R e p r o d u c t i v e C y c l e A. Reproduct i v e c y c l e of capt i v e males Based on the n i n e a n i m a l s examined i n my s t u d y , male o t t e r s e x h i b i t a normal c y c l e of r e p r o d u c t i v e a c t i v i t y w h i l e i n c a p t i v i t y . A l t h o u g h t e s t i s volumes of c a p t i v e o t t e r s were l a r g e r due t o the t h i c k n e s s of the s c r o t a l s a c , the annual changes i n volume were s i m i l a r t o those of w i l d a d u l t s ( F i g . 2 0 ) . As i n w i l d o t t e r s , the t e s t i s volumes of c a p t i v e a n i m a l s i n c r e a s e d r a p i d l y d u r i n g t h e l a t e f a l l and w i n t e r t o peak d u r i n g the b r e e d i n g season. A l t h o u g h the maximum t e s t i s 50 F i g u r e 20. T e s t i s volume of w i l d (• • ) and c a p t i v e (• •) o t t e r s . V a l u e s e x p r e s s e d as monthly mean volume ± SEM. Sample s i z e i n d i c a t e d next t o b a r s . I 1 1 1 1 1 1 1 1 1 1 I N D J F M A M J J A S O Month 52 volume was reached i n March compared t o F e b r u a r y f o r w i l d o t t e r s , t h i s d i d not appear t o i n d i c a t e a d i f f e r e n c e i n development. Both w i l d and c a p t i v e o t t e r s were a t s i m i l a r s t a g e s of spermatogenesis and t h e r e were no s i g n i f i c a n t d i f f e r e n c e s between mean t e s t i s volumes i n F e b r u a r y , March or A p r i l i n e i t h e r groups. T h i s d i f f e r e n c e i n t e s t i c u l a r volumes may be due t o the number of w i l d samples (n = 4 ) . Other measures of r e p r o d u c t i v e a c t i v i t y a l s o i n d i c a t e d t h a t a normal c y c l e of spermatogenesis o c c u r r e d i n c a p t i v e males. A n a l y s i s of the monthly t e s t i c u l a r b i o p s i e s showed t h a t the degree of spermatogenesis p r e s e n t and the s e m i n i f e r o u s t u b u l e d i a m e t e r s were s i m i l a r t o those of w i l d specimens a t the same time of the y e a r . B. T e s t o s t e r o n e C o n c e n t r a t i o n s Serum t e s t o s t e r o n e c o n c e n t r a t i o n s were found t o vary s i g n i f i c a n t l y (F = 6.27, df = 11,185, p < . 0 0 l ) . D u r i n g the annual r e p r o d u c t i v e c y c l e ( F i g u r e 2 1 ) . From the end of A p r i l t o the mi d d l e of October the serum c o n c e n t r a t i o n s were e x t r e m e l y low; 85% of the samples c o n t a i n e d l e s s than 2.5 ng/ml of t e s t o s t e r o n e . With the onset of s p e r m a t o g e n e s i s , c o n c e n t r a t i o n s rose and reached a p l a t e a u i n F e b r u a r y . From mid F e b r u a r y t o mid A p r i l the mean t e s t o s t e r o n e c o n c e n t r a t i o n s were always g r e a t e r than 3.5 ng/ml and i n d i v i d u a l peaks were as h i g h as 8.7 ng/ml. The maximum monthly mean l e v e l , which o c c u r r e d i n March, was s i g n i f i c a n t l y d i f f e r e n t than the l e v e l s seen i n May and June ( p < . 0 0 l ) . At the end of the b r e e d i n g season t e s t o s t e r o n e 5 3 F i g u r e 2 1 . Serum t e s t o s t e r o n e c o n c e n t r a t i o n (• •) and t e s t i s volume (O O) t a k e n a t two week i n t e r v a l s from c a p t i v e a d u l t r i v e r o t t e r s . V a l u e s e x p r e s s e d as mean SEM. Sample s i z e i n d i c a t e d next t o b a r s . Testosterone Concentration (ng/ml) 55 c o n c e n t r a t i o n s r e t u r n e d t o b a s e l i n e l e v e l s (1.5 ± 0.34 - 2.5 ± 0.8 ng/ml). I n d i v i d u a l t e s t o s t e r o n e c o n c e n t r a t i o n s a r e p r e s e n t e d i n Appendix 2. The mean t e s t i s volumes f o r two week s a m p l i n g p e r i o d s i s a l s o shown i n F i g u r e 21. There i s a s i g n i f i c a n t c o r r e l a t i o n ( r = 0.62, p<.00l) between t e s t i s volume and serum t e s t o s t e r o n e c o n c e n t r a t i o n s throughout the annual r e p r o d u c t i v e c y c l e . 56 DISCUSSION A. H i s t o l o g y The p a t t e r n of development of the p r i m a r y and secondary r e p r o d u c t i v e organs i n d i c a t e s t h a t the o t t e r i s a t y p i c a l mammalian s e a s o n a l b r e e d e r . The h i s t o l o g i c a l appearance of the organs d u r i n g the r e p r o d u c t i v e c y c l e i s s i m i l a r t o t h a t of o t h e r m u s t e l i d s such as the mink (Onstad 1967; B a s r u r and Ramos 1973; N i e s c h l a g and B i e n i e k 1975; P i l b e a m e t a_l. 1979), the s t o a t (Deanesly 1935), and the f e r r e t ( A l l a n s o n 1932; N e a l et a l . 1977). The w i n t e r spermatogonia which were found i n the c e n t r a l a r e a of immature and i n a c t i v e a d u l t t u b u l e s , a l s o occur i n f e r r e t s ( A l l a n s o n 1932) and s t o a t s (Deanesly 1935; Gulamhusein and Tarn 1974). T h e i r o c c a s i o n a l appearance a l o n g the basement membrane of the t u b u l e s i n o t t e r s and t h e i r degenerate appearance s u p p o r t s the c o n t e n t i o n t h a t they may a r i s e by the h y p e r t r o p h y of p e r i p h e r a l spermatogonia which then m i g r a t e i n t o the c e n t r a l a rea (Gulamhusein and Tarn 1974). Spermatogonia may d e v e l o p i n t o Type A or B spermatogonia when the t e s t i s i s a c t i v e but degenerate i n t o w i n t e r spermatogonia when a c t i v i t y i s low. There a r e no r e p o r t e d d e s c r i p t i o n s of the s e m i n i f e r o u s c y c l e of o t t e r s t o compare my r e s u l t s w i t h . However, the changes seen i n t e s t i s volumes of o t t e r s caught i n B r i t i s h Columbia were comparable t o those of o t t e r s i n New York when I e s t i m a t e d t e s t i c u l a r volumes from the average t e s t i s s i z e s r e p o r t e d by H a m i l t o n and E a d i e (1964). S i m i l a r age s p e c i f i c 57 t e s t i c u l a r d i mensions and w e i g h t s were r e p o r t e d by Tabor (1974) and L a n c i a and H a i r (1983) but t h e s e were not c o r r e l a t e d t o changes i n t e s t i c u l a r a c t i v i t y . B. Phase a n a l y s i s The d i v i s i o n of the annual c y c l e of the s e m i n i f e r o u s e p i t h e l i u m i n t o e a s i l y r e c o g n i z a b l e component phases a l l o w s the r e p r o d u c t i v e s t a t e of specimens t o be q u i c k l y i d e n t i f i e d . I t i s a l s o a p r e r e q u i s i t e f o r the q u a n t i t a t i v e i n v e s t i g a t i o n of spermatogenesis i n any a n i m a l . In the p a s t , two b a s i c approaches have been used i n the c h o i c e of c r i t e r i a f o r the i d e n t i f i c a t i o n of s t a g e s . The f i r s t was based p r i m a r i l y upon changes i n the acrosomic system and morphology of d e v e l o p i n g s p e r m a t i d s ( L e b l o n d and Clermont 1952); the second approach r e l i e d upon changes i n the shape of the s p e r m a t i d n u c l e i , the o c c u r r e n c e of m e i o t i c d i v i s i o n s and the arrangement of s p e r m a t i d s w i t h i n the g e r m i n a l e p i t h e l i u m (Roosen-Runge and G e i s e l 1950). A l t h o u g h both were developed f o r use i n c o n t i n u o u s b r e e d e r s such as the r a t , i t was the concept of c e l l u l a r a s s o c i a t i o n s found i n the l a t t e r approach which has been m o d i f i e d f o r use i n s e a s o n a l b r e e d e r s (Johnston and Buss 1967; Marsh et. a l . 1984). T h i s m o d i f i e d t e c h n i q u e has the advantage of i d e n t i f y i n g the onset and d e c l i n e of s p e r m a t o g e n e s i s which i s i m p o r t a n t when d e a l i n g w i t h s e a s o n a l b r e e d e r s . I t can be used t o i d e n t i f y q u a n t i t a t i v e l y the t i m i n g of r e p r o d u c t i v e e v e n t s and t o t e s t f o r p o s s i b l e d i f f e r e n c e s between sam p l i n g a r e a s and age groups. I t can a l s o be used t o 58 i d e n t i f y j u v e n i l e s and a n i m a l s t h a t a r e m a t u r i n g , a problem the o r i g i n a l approaches d i d not d e a l w i t h . I t was on t h i s concept of a s s o c i a t i o n s of c e l l t y p e s seen i n the t u b u l e s throughout s p e r m a t o g e n e s i s t h a t the n i n e phases I i d e n t i f i e d i n the r i v e r o t t e r were based. Phase 0 was c h a r a c t e r i s t i c of immature a n i m a l s w h i l e Phases 1 - 4 r e p r e s e n t e d a s p e r m a t i c o t t e r s . These l a t t e r phases a r e e q u i v a l e n t t o the Phases - 4 - 0 i n the dugong, Dugong duqon (Marsh e t a l . 1984). Phases 5 - 7 were found i n o t t e r s u n d e r g o i n g s p e r m i o g e n e s i s and a r e a s i m p l i f i e d v e r s i o n of the e l a b o r a t e s e r i e s based on changes i n the s p e r m a t i d s which have been d e v e l o p e d f o r c o n t i n u o u s b r e e d e r s (Clermont 1972). They ar e a l s o e q u i v a l e n t t o Phases 1 - 5 of Marsh et a_l. (1984). The f i n a l phase, Phase 8, was seen a f t e r s p e r m a t o g e n e s i s ended and i d e n t i f i e d the end of the b r e e d i n g season. In t h i s s t u d y , phase a n a l y s i s was a l s o i m p o r t a n t i n a s s e s s i n g the r e p r o d u c t i v e s t a t e of c a p t i v e a n i m a l s through the use of t e s t i c u l a r b i o p s i e s . S m a l l samples of the t e s t e s were removed and t h e r e p r o d u c t i v e s t a t e of the a n i m a l s was determined by the c l a s s i f i c a t i o n of the t u b u l e s . The degree of r e p r o d u c t i v e development i n d i c a t e d by phase a n a l y s i s of b i o p s i e s was c o n f i r m e d by the e x a m i n a t i o n of the e n t i r e t e s t e s i n t h r e e c a s e s . T h i s t e c h n i q u e was q u i c k and the a n i m a l s r e c o v e r e d r a p i d l y , t h u s a l l o w i n g the r e p r o d u c t i v e s t a t e of l i v e a n i m a l s t o be e a s i l y d e t e r m i n e d . Once the c y c l e of the s e m i n i f e r o u s e p i t h e l i u m i n an a n i m a l has been d e s c r i b e d and c l a s s i f i e d i n t o e a s i l y r e c o g n i z a b l e 59 phases, f u t u r e i n v e s t i g a t o r s can use the same t e c h n i q u e t o d i r e c t l y compare r e s u l t s . By d o i n g t h i s f o r the r i v e r o t t e r i t i s hoped t h a t f u t u r e s t u d i e s on the r e p r o d u c t i v e c y c l e of male o t t e r s w i l l be s t a n d a r d i z e d . C. S e x u a l M a t u r i t y My r e s u l t s , i n d i c a t i n g t h a t male r i v e r o t t e r s i n B r i t i s h Columbia a t t a i n s e x u a l m a t u r i t y between 20-24 months of age, are c o n s i s t e n t w i t h those of o t h e r s t u d i e s ( L i e r s 1951; H a m i l t o n and E a d i e 1964). I n d i c a t o r s of r e p r o d u c t i v e a c t i v i t y such as changes i n the t e s t i s s i z e , L e y d i g c e l l n u c l e a r d i a m e t e r s and e p i d i d y m i s development, suggest t h a t p u b e r t a l o t t e r s a r e c a p a b l e of b r e e d i n g i n the s p r i n g of t h e i r t h i r d y e a r . A l t h o u g h phase a n a l y s i s s u g g e s t s t h a t spermatogenesis may occur s l i g h t l y l a t e r than i n a d u l t s , t h e r e was no i n d i c a t i o n t h a t m a t u r i t y would have been d e l a y e d f o r another year i n any of the y e a r l i n g s sampled p r i o r t o the b r e e d i n g season. The o n l y a v a i l a b l e sample of a 25-26 month o l d o t t e r f o l l o w i n g the b r e e d i n g season c o n t a i n e d Phase 8 t u b u l e s , i n d i c a t i n g t h a t spermatogenesis had o c c u r r e d a t the end of i t s second y e a r . P h y s i o l o g i c a l and b e h a v i o u r a l m a t u r i t y has been r e p o r t e d t o occur a t d i f f e r e n t ages i n o t t e r s . L i e r s (1951) r e p o r t e d t h a t c a p t i v e male o t t e r s would not mate u n t i l they a t t a i n e d b e h a v i o u r a l m a t u r i t y a t f i v e or s i x y e a r s of age a l t h o u g h they are p h y s i o l o g i c a l l y c a p a b l e of i t e a r l i e r . However, i t i s not known i f w i l d o t t e r s show a s i m i l a r d e l a y and i t i s p o s s i b l e t h a t t h i s i s an e f f e c t of c a p t i v i t y . W i l d males t e n d t o be 6 0 s o l i t a r y ( M e l q u i s t and Hornocker 1983; W oolington 1984) w h i l e c a p t i v e a n i m a l s are u s u a l l y grouped. The p r e s ence of males i n c l o s e v i c i n i t y i s known t o i n h i b i t e f f e c t i v e p u b e r t y i n a v a r i e t y of mammals ( S a d l e i r 1969a). Both of the male o t t e r s which mated i n my study were e s t i m a t e d t o have been a t l e a s t s i x y e a r s o l d , w e l l above the p h y s i o l o g i c a l age of m a t u r i t y . T h i s does not n e c e s s a r i l y support L i e r s ' c o n t e n t i o n of a b e h a v i o u r a l m a t u r i t y i n r i v e r o t t e r s however, s i n c e o l d e r , as w e l l as younger, males had the o p p o r t u n i t y t o breed but d i d not do so. More i n f o r m a t i o n on the f a c t o r s i n f l u e n c i n g b r e e d i n g among w i l d and c a p t i v e o t t e r s i s n e c e s s a r y t o determine i f the presence of o t h e r males l i m i t e d the amount of b r e e d i n g t h a t o c c u r r e d among my c a p t i v e a n i m a l s . Many mammals advance s e x u a l m a t u r i t y under f a v o r a b l e c o n d i t i o n s and ' d e l a y i t under u n f a v o r a b l e c o n d i t i o n s ( S a d l e i r 1969a). A l t h o u g h the age of s e x u a l m a t u r i t y was advanced i n c o n d i t i o n s of good n u t r i t i o n i n female weasels ( K i n g 1980), t h e r e was no such change observed among the male o t t e r s i n t h i s s t u d y . A l l of the a n i m a l s examined i n t h i s study were c o l l e c t e d from c o a s t a l a r e a s of B r i t i s h Columbia which q u a l i t a t i v e l y appears t o be v e r y f a v o u r a b l e . The temperature i n t h i s r e g i o n i s m i l d and denning s i t e s a r e r e a d i l y a v a i l a b l e ; food appears t o be abundant and prey s p e c i e s eaten do not v a r y w i t h season as i n f r e s h w a t e r h a b i t a t s (Stenson e t a l . 1984). I f age of s e x u a l m a t u r i t y v a r i e s w i t h l o c a l e n v i r o n m e n t a l c o n d i t i o n s i n r i v e r o t t e r s , m a t u r i t y may be e x p e c t e d t o occur e a r l i e r i n t h i s h a b i t a t . However, the age of s e x u a l m a t u r i t y appears t o be 61 c o n s i s t e n t throughout the range of L. c a n a d e n s i s ( L i e r s 1951; H a m i l t o n and E a d i e 1964; M e l q u i s t and Hornocker 1983). Thus i t appears t h a t e i t h e r the age of s e x u a l m a t u r i t y among males does not v a r y , or a l l a r e a s s t u d i e d t o date are c o n s i s t e n t i n the q u a l i t y of h a b i t a t p r e s e n t . D. The annual c y c l e i n B r i t i s h Columbia E x a m i n a t i o n of the male r e p r o d u c t i v e organs i n d i c a t e s t h a t a c t i v i t y b e g i n s i n the l a t e autumn i n B r i t i s h Columbia. A r a p i d i n c r e a s e i n spermatogenesis and t e s t i s development o c c u r s near the w i n t e r s o l s t i c e and spermatozoa, which are f i r s t seen i n l a t e December, are p r e s e n t i n the e p i d i d y m i d e s of a l l mature o t t e r s by mid J a n u a r y . Peak t e s t i c u l a r a c t i v i t y o c c u r s from l a t e F e b r u a r y t h r o u g h A p r i l and d u r i n g May spermatogenesis d e c l i n e s . S i m i l a r c y c l e s of r e p r o d u c t i v e a c t i v i t y were seen i n both w i l d and c a p t i v e o t t e r s . The p e r i o d d u r i n g which b r e e d i n g c o u l d occur was e s t i m a t e d by the presence of sperm i n the e p i d i d y m i d e s . Spermatozoa were f i r s t seen i n l a t e December but were not p l e n t i f u l u n t i l l a t e J anuary or e a r l y F e b r u a r y . In l a t e A p r i l the e p i d i d y m i d e s were s t i l l f i l l e d w i t h mature sperm and b r e e d i n g was p o s s i b l e a l t h o u g h Phase 8 t u b u l e s , i n d i c a t i n g the end of s p e r m a t o g e n e s i s , were common i n most t e s t e s . Samples of e p i d i d y m i d e s were not a v a i l a b l e from e a r l y and mid-May, but the t u b u l e d i a m e t e r s remained r e l a t i v e l y l a r g e d u r i n g t h i s p e r i o d , s u g g e s t i n g t h a t spermatozoa may s t i l l be a v a i l a b l e f o r b r e e d i n g . By l a t e May both t e s t e s and e p i d i d y m i d e s l a c k e d spermatozoa i n d i c a t i n g the 62 end of the b r e e d i n g season. Male o t t e r s , l i k e most mammals ( S a d l e i r 1969a; L i n c o l n 1981), a r e c a p a b l e of b r e e d i n g f o r l o n g e r p e r i o d s than females and t h e r e f o r e , the a c t u a l p e r i o d of b r e e d i n g i s c o n t r o l l e d by the t i m i n g of e s t r u s i n the female. In B r i t i s h Columbia e s t r u s o c c u r s i n female o t t e r s between l a t e F e b r u a r y and e a r l y May (Chapter I I I ) . T h i s p e r i o d extends p a s t the peak of sp e r m a t o g e n e s i s but i s c o n s i s t e n t w i t h the presence of sperm i n the e p i d i d y m i s . Because the m a j o r i t y of s t u d i e s o n l y r e p o r t b r e e d i n g d a t e s which a re d e t e r m i n e d by the female, i t i s d i f f i c u l t t o compare the t i m i n g of r e p r o d u c t i v e a c t i v i t y among male o t t e r s i n d i f f e r e n t l o c a l e s . G e n e r a l l y , the b r e e d i n g season of o t t e r s i n sout h e r n B r i t i s h Columbia i s s i m i l a r t o t h a t r e p o r t e d i n the n o r t h e r n U n i t e d S t a t e s (Best 1962; H a m i l t o n and Ea d i e 1964; Mowbray e t a l . 1979; M e l q u i s t and Hornocker 1983). However, t h e r e i s an i n d i c a t i o n t h a t the t i m i n g of spermatogenesis may change w i t h l a t i t u d e . In the s o u t h e r n U n i t e d S t a t e s o t t e r s have been r e p o r t e d t o breed as e a r l y as January (McDaniel 1963; H i l l and Lauhachinda 1980). In c o n t r a s t , January b r e e d i n g has not been r e p o r t e d among w i l d o t t e r s i n n o r t h e r n a r e a s a l t h o u g h i t has o c c u r r e d i n c a p t i v i t y ( L i e r s 1951). In New York sperm were found i n the e p i d i d y m i d e s of a l l a d u l t o t t e r s (n = 7) c o l l e c t e d i n November and December ( H a m i l t o n and Ea d i e 1964) whereas i n my st u d y , o n l y s l i g h t l y f u r t h e r n o r t h , sperm were not p r e s e n t u n t i l l a t e December. There i s a l s o e v i d e n c e s u g g e s t i n g t h a t spermatogenesis o c c u r s l a t e r a t h i g h e r l a t i t u d e s . Samples from e i g h t a d u l t male 63 o t t e r s (not i n c l u d e d i n t h e s e r e s u l t s ) were o b t a i n e d from the Queen C h a r l o t t e I s l a n d s d u r i n g J a n u a r y 1977. Development of the s e m i n i f e r o u s t u b u l e s was l e s s advanced i n t h e s e specimens than i n samples c o l l e c t e d i n the s o u t h e r n p a r t of the p r o v i n c e . In t e s t e s from the Queen C h a r l o t t e I s l a n d s Phase 3 and 4 t u b u l e s were the most common (43.3% and 26.7% r e s p e c t i v e l y ) and no sperm were p r e s e n t (Phases 6 and 7 ) . In c o n t r a s t , over 95% of the t e s t i s samples c o l l e c t e d i n s o u t h e r n B r i t i s h Columbia d u r i n g January c o n t a i n e d Phase 7 t u b u l e s , i n d i c a t i n g g r e a t e r r e p r o d u c t i v e a c t i v i t y . A l t h o u g h the samples s i z e i s s m a l l , t h i s s u p p o r t s the i d e a t h a t s p e r m a t o g e n e s i s , and l i k e l y b r e e d i n g , occur l a t e r as the l a t i t u d e i n c r e a s e s . The use of phase a n a l y s i s e n a b l e s such minor changes t o be d e t e c t e d and the a p p l i c a t i o n of t h i s t e c h n i q u e t h r o u g h o u t the range of the o t t e r may a l l o w a t e s t of t h i s h y p o t h e s i s . A r e c e n t study i n s o u t h e a s t A l a s k a a l s o i n d i c a t e s t h a t b r e e d i n g may occur l a t e r i n h i g h e r l a t i t u d e s . W o o l i n g t o n (1984) observed mating o c c u r r i n g as l a t e as May 31 on Baranof I s l a n d . In c o n t r a s t , o t t e r s i n s o u t h e r n B r i t i s h Columbia have completed b r e e d i n g by t h i s time and have ceased s p e r m a t o g e n e s i s . The o b s e r v a t i o n t h a t b r e e d i n g season v a r i e s w i t h l a t i t u d e s u g g e s t s t h a t r e p r o d u c t i v e a c t i v i t y may be a f f e c t e d by e x t e r n a l f a c t o r s . P h o t o p e r i o d has been found t o c o n t r o l development of the r e p r o d u c t i v e organs i n a v a r i e t y of m u s t e l i d s ( B i s s o n n e t t e 1932; Harvey and M a c f a r l a n e 1958; Rust and S h a c k e l f o r d 1969; Duby and T r a v i s 1972; B o i s s i n - A g a s s e e t a l . 1982) and may a l s o be i m p o r t a n t i n i n f l u e n c i n g the r e p r o d u c t i v e c y c l e of r i v e r 64 o t t e r s . In B r i t i s h Columbia s e a s o n a l t e s t i s a c t i v i t y i s i n i t i a t e d d u r i n g the d e c l i n i n g p h o t o p e r i o d p r i o r t o the w i n t e r s o l s t i c e w h i l e a c t i v i t y r i s e s s h a r p l y and peaks when d a y l e n g t h i s on the i n c r e a s e . The e f f e c t of p h o t o p e r i o d on the r e p r o d u c t i v e c y c l e i s examined i n Chapter IV. E. T e s t o s t e r o n e c o n c e n t r a t i o n s The c y c l i c a l changes which o c c u r r e d i n the serum t e s t o s t e r o n e c o n c e n t r a t i o n s were found t o r e f l e c t the development of the p r i m a r y and secondary r e p r o d u c t i v e organs. A h i g h c o r r e l a t i o n e x i s t e d between t e s t o s t e r o n e l e v e l s and t e s t i s volume, both showing s i g n i f i c a n t i n c r e a s e s between December and F e b r u a r y and near peak l e v e l s from l a t e F e b r u a r y through e a r l y A p r i l . T e s t i s volume and serum t e s t o s t e r o n e c o n c e n t r a t i o n s d e c r e a s e d d u r i n g A p r i l and were low from May t h rough October when r e p r o d u c t i v e a c t i v i t y was a t a minimum. The development of the s e m i n i f e r o u s t u b u l e s , ductus e p i d i d y m i s and L e y d i g c e l l s f o l l o w e d a s i m i l a r p a t t e r n . Gonadal a c t i v i t y i s a l s o c o r r e l a t e d w i t h t e s t o s t e r o n e l e v e l s i n a number of m u s t e l i d s i n c l u d i n g the mink ( P i l b e a m e t a l . 1979; S u n d q v i s t e t a l . 1984), the s t o a t (Gulamhusein and Tarn 1974); the f e r r e t (Neal e t a l . 1977), the stone marten (Audy 1978), and the European badger (Audy. et a l . 1982). T e s t o s t e r o n e l e v e l s were observed t o r i s e s l i g h t l y i n l a t e O c t o b e r , c o i n c i d i n g w i t h the b e g i n n i n g of s p e r m a t o g e n e s i s . C o n c e n t r a t i o n s then d e c l i n e d b r i e f l y b e f o r e a subsequent r i s e to peak l e v e l s i n F e b r u a r y . A l t h o u g h not s t a t i s t i c a l l y 65 s i g n i f i c a n t , t h i s i n i t i a l r i s e o c c u r r e d a t a time when r e d u c t i o n d i v i s i o n was b e g i n n i n g i n the p r i m a r y s p e r m a t o c y t e s and Phase 3 and 4 t u b u l e s were f i r s t seen i n the t e s t e s . A minor r i s e i n t e s t o s t e r o n e a s s o c i a t e d w i t h the b e g i n n i n g of spermatogenesis has been observed i n o t h e r mammals such as the s t o a t (Gulamhusein and Tarn 1974), the b l a c k bear ( M c M i l l i m e t a l . 1976), and the r e d s t a g ( L i n c o l n 1971), and may be n e c e s s a r y f o r the i n i t i a t i o n of sper m a t o g e n e s i s ( S t e i n b e r g e r 1971). High t e s t o s t e r o n e l e v e l s do not appear t o be ne c e s s a r y f o r mating t o occur' i n o t t e r s . I n t h i s s t u d y , maximum c o n c e n t r a t i o n s of serum t e s t o s t e r o n e c o i n c i d e d w i t h peak spermatogenesis r a t h e r than the p e r i o d of maximum b r e e d i n g . A l t h o u g h b r e e d i n g may oc c u r i n March when t e s t o s t e r o n e l e v e l s were a t t h e i r h i g h e s t , most appears t o occur d u r i n g A p r i l and e a r l y May (Chapter I I I ) when t e s t o s t e r o n e l e v e l s were d e c l i n i n g . T h i s p a t t e r n d i f f e r s from t h a t of the m a j o r i t y of m u s t e l i d s where t e s t o s t e r o n e l e v e l s remain h i g h d u r i n g the e n t i r e b r e e d i n g season (Gulamhusein and Tarn 1974; N e a l et a l . 1977; Ma u r e l et a l . 1981; Audy et a l . 1 982) and from mink where t e s t o s t e r o n e d e c l i n e s b e f o r e e i t h e r maximum t e s t i s volume or b r e e d i n g occur ( P i l b e a m et a l . 1979; S u n d q v i s t et a_l 1984). Even i n mammals i n which peak t e s t o s t e r o n e l e v e l s , maximum t e s t i s s i z e and b r e e d i n g season a l l c o i n c i d e , androgen l e v e l s and l i b i d o are p o o r l y c o r r e l a t e d ; t e s t o s t e r o n e l e v e l s i n the b l o o d a r e c o r r e l a t e d w i t h the development of the s e m i n i f e r o u s t u b u l e s r a t h e r than b r e e d i n g i t s e l f ( S e t c h e l l 1978). The v a l u e s o b t a i n e d f o r t e s t o s t e r o n e c o n c e n t r a t i o n s i n the 66 serum of r i v e r o t t e r s i n B r i t i s h Columbia a r e much lower than t h o s e found i n the mink ( N i e s c h l a g and B i e n i e k 1975; P i l b e a m e t a l . 1979), the s t o a t (Gulamhusein and Tarn 1974) and the f e r r e t (Neal e t a l . 1977). They a r e however, comparable t o tho s e of the European badger (Maurel e t a l . 1981; Maure l et B o i s s i n 1982; Audy e t a l . 1982). The s i g n i f i c a n c e of t h e s e d i f f e r e n c e s i n t e s t o s t e r o n e l e v e l s i s not u n d e r s t o o d s i n c e androgen c o n c e n t r a t i o n s appear t o v a r y g r e a t l y among d i f f e r e n t mammals and even among members of the same f a m i l y ( G u s t a f s o n and Shemesh 1976). Androgen l e v e l s i n w i l d o t t e r s a r e u n l i k e l y t o be v e r y d i f f e r e n t from those d e t e r m i n e d i n t h i s study s i n c e serum c o n c e n t r a t i o n s i n r e c e n t l y caught a n i m a l s d i d not v a r y s i g n i f i c a n t l y from those h e l d i n c a p t i v i t y f o r over t h r e e y e a r s . In s t u d i e s on European badgers (Audy e_t a_l. 1982) and w h i t e -t a i l e d deer ( M i r a r c h i et a l . 1978) no d i f f e r e n c e s were found i n the t e s t o s t e r o n e l e v e l s of c a p t i v e and w i l d i n d i v i d u a l s . Because of the d i f f i c u l t y i n o b t a i n i n g b l o o d samples from o t t e r s , I was unable t o d e t e r m i n e i f d i u r n a l or s h o r t - t e r m f l u c t u a t i o n s of serum t e s t o s t e r o n e c o n c e n t r a t i o n s were p r e s e n t . A d i u r n a l rhythm or p u l s a t i l e r e l e a s e of t e s t o s t e r o n e has been demonstrated i n a number of mammals i n c l u d i n g men (Southern and Gorden 1975), r a t s ( H o s t e t l e r and P i a c s e k 1977), rams ( L i n c o l n 1976) and m u s t e l i d s such as the f e r r e t ( R e i g e r and Murphy 1977), the badger (Maurel e t a l . 1981) and the mink ( B o i s s i n - A g a s s e et a l . 1982). The e p i s o d i c r e l e a s e of t e s t o s t e r o n e i s l i k e l y t o occur i n o t t e r as w e l l and c o u l d e x p l a i n the l a r g e v a r i a b i l i t y 67 observed between individual samples that i s reflected in the SEM. In summary, thi s study provides the f i r s t detailed description of the changes that occur in the reproductive organs and serum testosterone concentrations during the annual reproductive cycle of male otters. The development of the seminiferous tubules in otters is similar to other members of the family Mustelidae and the values obtained for testosterone f a l l into the low end of the range of concentrations seen in mustelids. The changes observed in the seminiferous epithelium throughout the year were divided into e a s i l y recognizable phases which were used to analyze the degree of reproductive a c t i v i t y of each specimen. In the coastal environment of B r i t i s h Columbia, male otters were found to be seasonal breeders and to become sexually mature at the end of their second year. Reproductive a c t i v i t y begins in the late autumn and adults are capable of breeding from late January through mid May although actual breeding may not begin u n t i l late February or March. CHAPTER I I I REPRODUCTIVE CYCLE OF THE FEMALE RIVER OTTER 69 INTRODUCTION The r e p r o d u c t i v e c y c l e of the female r i v e r o t t e r , L u t r a  c a n a d e n s i s , has not p r e v i o u s l y been s t u d i e d i n d e t a i l , and, as f o r most w i l d mammals, i n f o r m a t i o n i s v e r y l i m i t e d . H a m i l t o n and E a d i e (1964) were the f i r s t t o d e s c r i b e u n i m p l a n t e d embryos and t h e g e n e r a l appearance of the c o r p o r a l u t e a and l u t e a l c e l l s b e f o r e and a f t e r i m p l a n t a t i o n . To a l a r g e e x t e n t l a t e r s t u d i e s have been concerned e i t h e r w i t h the t i m i n g of i m p l a n t a t i o n ( H i l l and Lauhachinda 1981), or g e n e r a l p o p u l a t i o n parameters (Tabor and Wight 1977; Mowbray et a_l. 1979). They i n d i c a t e t h a t female o t t e r s a r e s e a s o n a l b r e e d e r s and e x p e r i e n c e an o b l i g a t o r y , p r o l o n g e d p e r i o d of embryonic d i a p a u s e p r i o r t o i m p l a n t a t i o n . However, h i s t o l o g i c a l changes which accompany t h i s form of pregnancy are, p o o r l y u n d e r s t o o d and the g e n e r a l h i s t o l o g y of r e p r o d u c t i v e organs and o v a r i a n development have not been d e s c r i b e d . In a d d i t i o n , the e n d o c r i n o l o g y of the annual r e p r o d u c t i v e c y c l e of the female o t t e r i s unknown. My s t u d y had t h r e e o b j e c t i v e s : 1) t o d e s c r i b e i n d e t a i l the h i s t o l o g y and e n d o c r i n o l o g y of the a n n u a l r e p r o d u c t i v e c y c l e of t h e female o t t e r , 2) t o i d e n t i f y the changes which occur i n the v a g i n a l smears and t o r e l a t e them t o the r e p r o d u c t i v e s t a t e of the f e m a l e , and 3) t o d e t e r m i n e the a n n u a l r e p r o d u c t i v e c y c l e of female o t t e r s i n B r i t i s h Columbia. Annual changes i n the r e p r o d u c t i v e organs and gonadal s t e r o i d hormone c o n c e n t r a t i o n s were d e t e r m i n e d i n j u v e n i l e s , nonpregnant a d u l t s and a pregnant a d u l t o t t e r . The a n n u a l changes i n e s t r a d i o l c o n c e n t r a t i o n were d e t e r m i n e d and c o r r e l a t e d w i t h the b r e e d i n g c y c l e and changes i n 70 the r e p r o d u c t i v e organs. P r o g e s t e r o n e l e v e l s were determined and r e l a t e d t o the changes obse r v e d i n l u t e a l c y t o l o g y d u r i n g the p r e i m p l a n t a t i o n and p o s t i m p l a n t a t i o n p e r i o d s of pregnancy. The d a t a p r o v i d e d i n t h i s study a r e p a r t i c u l a r l y i m p o r t a n t because e s t r o g e n l e v e l s among m u s t e l i d s a r e l a r g e l y unknown ( P i l b e a m e t a l . 1979; Mondain-Monval 1980; R a v i n d r a and Mead 1984) and because embryonic d i a p a u s e i s p o o r l y u n d e r s t o o d . A d e l a y i n i m p l a n t a t i o n has d e v e l o p e d s e p a r a t e l y i n many of the m u s t e l i d s and can va r y c o n s i d e r a b l y i n form (Renfree 1978; Mead and Wright 1983). D e t a i l e d s t u d i e s of the r e p r o d u c t i v e c y c l e and p r o g e s t e r o n e c o n c e n t r a t i o n s a r e a v a i l a b l e f o r a number of o t h e r m u s t e l i d s such as the mink, M u s t e l a v i s o n (Hansson 1947; Enders 1952; P i l b e a m e_t a_l. 1 979), the s p o t t e d skunk, S p i l o g a l e  p u t o r i u s (Mead I968a,b; Mead and Ei k - N e s I969a,b; Mead 1981), the s t r i p e d skunk, M e p h i t i s mephitus (Wade-Smith and Richmond 1978) and the European badger, Meles meles (Neal and H a r r i s o n 1958; Canivenc 1966; Ahnlund 1980). T h i s study p r o v i d e s the f i r s t comparable i n v e s t i g a t i o n i n the r i v e r o t t e r . As such, i t w i l l i n c r e a s e our knowledge of r e p r o d u c t i o n among the M u s t e l i d a e and among mammals which e x h i b i t embryonic d i a p a u s e . The second o b j e c t i v e of t h i s study was t o i d e n t i f y the p a t t e r n of changes which o c c u r i n v a g i n a l smears of the o t t e r and t o determine i f smear type c o u l d be used s u c c e s s f u l l y t o i d e n t i f y r e p r o d u c t i v e s t a t e , thus p r o v i d i n g a n o n - i n v a s i v e method of f o l l o w i n g the r e p r o d u c t i v e c y c l e of r i v e r o t t e r s . Smears have been s u c c e s s f u l l y used t o i d e n t i f y the r e p r o d u c t i v e s t a t e of a v a r i e t y of mammals (D'Souza 1978) i n c l u d i n g two 71 s p e c i e s of m u s t e l i d s , the f e r r e t , M u s t e l a p u t o r i u s ( H a m i l t o n and Gould 1940) and the mink (Hansson 1947; Enders 1952; T r a v i s e_t a l . 1978). Because smear t y p e s and the r e l a t i o n s h i p between the e s t r o u s v a g i n a l smear and the o c c u r r e n c e of b r e e d i n g a r e s p e c i e s s p e c i f i c (D'Souza 1978; T r a v i s e t a_l. 1979), i t was n e c e s s a r y f o r me t o determine the s p e c i f i c smear t y p e s p r e s e n t i n the female r i v e r o t t e r . The a n e s t r o u s and e s t r o u s smears were e a s i l y i d e n t i f i e d , and the comparison of o v a r i a n h i s t o l o g y t o the v a g i n a l smear t y p e s p r e s e n t i n t h i s study i n d i c a t e d t h a t v a g i n a l smears c o u l d be used t o c o r r e c t l y i d e n t i f y the r e p r o d u c t i v e s t a t e of both c a p t i v e and w i l d o t t e r s . The t h i r d o b j e c t i v e of t h i s s e c t i o n of my i n v e s t i g a t i o n was to compare the annual r e p r o d u c t i v e c y c l e of the female r i v e r o t t e r s i n B r i t i s h Columbia t o those of o t t e r s throughout N o r t h A m e r i c a . P r e v i o u s s t u d i e s i n d i c a t e t h a t d i f f e r e n c e s occur between the b r e e d i n g p a t t e r n s of o t t e r s throughout t h e i r range. Pregnancy r a t e s and age of s e x u a l m a t u r i t y v a r y g r e a t l y and b i r t h s can occur between l a t e December and May depending upon the l o c a t i o n ( L i e r s 1951; H a m i l t o n and Eadie 1964; Mowbray et a l . 1979; H i l l and Lauhachinda 1981; L a n c i a and H a i r 1983; Wo o l i n g t o n 1984). T h i s study i s the f i r s t t o examine the r e p r o d u c t i v e c y c l e of r i v e r o t t e r s i n c o a s t a l B r i t i s h Columbia and as such, p r o v i d e s an i n d i c a t i o n of the i n f l u e n c e l o c a l h a b i t a t has upon r e p o d u c t i v e c y c l e s i n o t t e r s . 72 MATERIALS AND METHODS I_. Necropsy E x a m i n a t i o n s A. Source of M a t e r i a l A t o t a l of 146 female r e p r o d u c t i v e t r a c t s were c o l l e c t e d i n sout h w e s t e r n B r i t i s h Columbia d u r i n g 1975 -81 (Table VI) from the sou r c e s l i s t e d i n Chapter I I . A l l samples were c o l l e c t e d from c o a s t a l a r e a s and the m a j o r i t y (83.6%) were from the B a r k l e y Sound r e g i o n . P r o f e s s i o n a l t r a p p e r s accounted f o r 75.3% of a l l samples. Table V I . O r i g i n of female r i v e r o t t e r necropsy samples. T o t a l number of samples i s 146. The t r e a t m e n t of samples c o l l e c t e d by t r a p p e r s i s o u t l i n e d i n Chapter I I . In samples which I c o l l e c t e d , the u t e r i were removed and f l u s h e d w i t h p h y s i o l o g i c a l s a l i n e ( 0.9% NaCl) t o r e c o v e r u n i m p l a n t e d b l a s t o c y s t s b e f o r e f i x a t i o n . B l o o d samples were c o l l e c t e d from f i v e o t t e r s i n the f i e l d and the serum was s e p a r a t e d u s i n g v a c u t a i n e r serum s e p a r a t i o n t u b e s . The f r o z e n Vancouver I s l a n d B a r k l e y Sound E a s t Coast 1 22 1 4 M a i n l a n d Coast R i v e r s I n l e t Howe Sound 6 4 73 serum samples were l a t e r thawed, c e n t r i f u g e d and t r e a t e d as o u t l i n e d f o r the b l o o d samples c o l l e c t e d from c a p t i v e a n i m a l s . B . Age D e t e r m i n a t i o n A l l a n i m a l s were aged u s i n g cementum a n n u l i and s k u l l c h a r a c t e r i s t i c s as o u t l i n e d i n Chapter I I . Each a n i m a l was p l a c e d i n t o an age c l a s s based on a t h e o r e t i c a l b i r t h d a t e of A p r i l 1 and a s s i g n e d t o one of t h r e e c a t e g o r i e s : j u v e n i l e (age c l a s s 0 ) , y e a r l i n g (age c l a s s 1 ) , and a d u l t (age c l a s s e s 2 and above) (Chapter I I ) . O t t e r s ranged i n age from e i g h t months t o 11 y e a r s . C. E x a m i n a t i o n of H i s t o l o g i c a l M a t e r i a l A f t e r f i x a t i o n i n 10% b u f f e r e d f o r m a l i n , the o v a r i e s were removed from the b u r s a , weighed t o 0.01 g and t h e i r d i m e n s i o n s d e t e r m i n e d u s i n g v e r n i e r c a l i p e r s . O v a r i a n volumes were e s t i m a t e d from the f o r m u l a of S e t c h e l l and Waites (1964) as i n Chapter I I : OV = 4/3TT • L/2 • W/2 • T/2 The u t e r i were opened and f l u s h e d w i t h s a l i n e t o r e c o v e r u n i m p l a n t e d b l a s t o c y s t s . The presence of b l a s t o c y s t s and/or c o r p o r a l u t e a were used t o d e f i n e pregnancy. A f t e r c l e a r i n g i n b e n z y l benzoate ( O r s i n i 1962), the u t e r i were examined f o r p l a c e n t a l s c a r s and i m p l a n t a t i o n s i t e s . Samples of u t e r i , p l a c e n t a and v a g i n a were removed and p r e p a r e d f o r l i g h t m i c r o s c o p y as d e s c r i b e d i n Chapter I I . A subsample of f i v e 74 u t e r i from a n i m a l s c l a s s i f i e d as b e i n g immature, a n e s t r o u s , e s t r o u s ( o n l y t h r e e a v a i l a b l e ) , and i n the p r e i m p l a n t a t i o n and p o s t i m p l a n t a t i o n p e r i o d s of pregnancy were examined i n d e t a i l and the h e i g h t of twenty l u m i n a l and g l a n d u l a r e p i t h e l i a l c e l l s were measured u s i n g a c a l i b r a t e d o c u l a r micrometer. U s i n g s t a n d a r d h i s t o l o g i c a l t e c h n i q u e s , the o v a r i e s were s e c t i o n e d s e r i a l l y a t 6 um, s t a i n e d w i t h h a e m a t o x y l i n and e o s i n , and e v e r y t e n t h s e c t i o n was removed f o r d e t a i l e d s t u d y . An o c u l a r micrometer was used t o d e t e r m i n e the d i a m e t e r s of the c o r p o r a l u t e a ( C L ) , the c o r p o r a a l b i c a n t i a (CA), a minimum of twenty l u t e a l c e l l s from each CL, and a n t r a l f o l l i c l e s over 400 um i n d i a m e t e r . The d i a m e t e r used was the mean of two p e r p e n d i c u l a r d i a m e t e r s measured i n the s l i d e c o n t a i n i n g the l a r g e s t c r o s s s e c t i o n a l a r e a . F o l l i c l e s were d e s c r i b e d as p r i m a r y ( f o l l i c l e s w i t h a s i n g l e l a y e r of g r a n u l o s a c e l l s ) , p r e a n t r a l ( f o l l i c l e s w i t h a membrana g r a n u l o s a of v a r y i n g t h i c k n e s s w i t h o u t a n t r a ) , a n t r a l ( f o l l i c l e s of v a r i o u s s i z e w i t h a n t r a ) , or p r e o v u l a t o r y ( l a r g e f o l l i c l e s c h a r a c t e r i z e d by l a r g e s i z e ) . The were f u r t h e r c l a s s i f i e d a c c o r d i n g t o t h e i r d i ameter i n t o s i x s t a g e s : Stage I (<250 um), Stage I I (250-599 M m ) , Stage I I I (600-799 M m ) , Stage IV (800-999 M m ) , Stage V (1.00-1.49 mm), and Stage VI (>1.49 mm). Corpora l u t e a were c l a s s i f i e d d epending on the l e v e l of a c t i v i t y of t h e i r l u t e a l c e l l s , and on the p r e s e n c e or absence of i m p l a n t e d embryos i n the accompanying u t e r u s . The r e l a t i v e volumes of o v a r i a n components such as CL, CA, f o l l i c l e s , c o n n e c t i v e t i s s u e and i n t e r s t i t i a l c e l l s were d e t e r m i n e d u s i n g a W e i b e l g r a t i c u l e i n the manner d e s c r i b e d i n 75 Chapter I I . The pregnancy r a t e was d e f i n e d as the p e r c e n t a g e of mature females which c o n t a i n e d c o r p o r a l u t e a a f t e r the b r e e d i n g season ceased. S i n c e few samples were c o l l e c t e d between b i r t h and mating, t h i s d e f i n i t i o n a l s o i n c l u d e d the m a j o r i t y of females which were r e p r o d u c t i v e l y a c t i v e . The pregnancy r a t e s t h e r e f o r e g i v e a good i n d i c a t i o n of the percentage of r e p r o d u c t i v e l y a c t i v e f e m a l e s . The date of i m p l a n t a t i o n of embryos was e s t i m a t e d from crown-rump l e n g t h s ( H i l l and Lauhachinda 1981). 11. C a p t i v e Animals A. E x a m i n a t i o n and Sampling of o t t e r s S i x female r i v e r o t t e r s were l i v e - t r a p p e d and h e l d a t the U n i v e r s i t y of B r i t i s h Columbia f o r p e r i o d s r a n g i n g from e i g h t t o 42.5 months (Table V I I ) . At one t o two week i n t e r v a l s body w e i g h t s , b l o o d samples, and v a g i n a l smears were taken f o l l o w i n g the regime o u t l i n e d i n Chapter I I . B l o o d samples were c e n t r i f u g e d and s t o r e d a t -40°C u n t i l a n a l y z e d . V a g i n a l smears were c o l l e c t e d u s i n g a c o t t o n swab, f i x e d i n e q u a l p a r t s of 100% e t h a n o l and e t h e r and s t a i n e d w i t h S c h o r r ' s s t a i n . The c e l l s found i n the smear were c l a s s i f i e d on the b a s i s of Wied's (1958) s t a n d a r d i z e d t e r m i n o l o g y f o r human v a g i n a l c y t o l o g y . However, the term ' c o r n i f i e d c e l l ' was used i n t h i s s tudy t o a l l o w comparisons w i t h o t h e r s t u d i e s . D u r i n g the 1981 e s t r o u s c y c l e smears were taken d a i l y , and a l s o h o u r l y over two 24 hour p e r i o d s , from F3 and F6 t o determine w i t h i n a n i m a l v a r i a b i l i t y . 7 6 T a b l e V I I . Fema le r i v e r o t t e r s l i v e - t r a p p e d and h e l d i n c a p t i v i t y , 1 9 7 7 - 8 1 . A n i m a l C a p t u r e D u r a t i o n o f c a p t i v i t y Da te L o c a t i o n Age ( y r s ) (months) F2 1 0 / 0 2 / 7 8 G u l f I s l a n d s 1 4 2 . 5 F3 0 5 / 0 4 / 7 8 P o r t Hardy a d u l t 4 0 . 5 F5 0 5 / 0 5 / 7 8 P o r t Ha rdy 7 3 9 . 0 F6 0 4 / 0 3 / 8 0 Bowen I s l a n d 2 1 8 . 5 F7 1 1 / 0 9 / 8 0 P o r t Hardy 1 1 0 . 0 77 In May 1981 the r e p r o d u c t i v e t r a c t s of two o t t e r s were removed t o d e t e r m i n e i f the smear type c o r r e s p o n d e d w i t h the h i s t o l o g y of the ovary and r e p r o d u c t i v e t r a c t . The f i r s t day of e s t r u s was e s t i m a t e d t o occur mid-way between the day of the f i r s t f u l l y c o r n i f i e d smear and the p r e c e d i n g s a m p l i n g d a t e . The l a s t day of e s t r u s was s i m i l a r l y e s t i m a t e d t o occur mid-way between the date of the l a s t e s t r o u s and the f i r s t m e t e s t r o u s smear. S l i g h t m o d i f i c a t i o n s o c c u r r e d i n c a ses when the date c o u l d be d e t e r m i n e d more p r e c i s e l y , based on the n a t u r e of the smears. Thus on aver a g e , the dates r e p o r t e d f o r the onset and end of e s t r u s are c o r r e c t t o w i t h i n a week. Females were c o n s i d e r e d t o be s e x u a l l y mature at the b e g i n n i n g of t h e i r f i r s t f u l l e s t r u s . A n i m a l s e x h i b i t i n g p a r t i a l e s t r u s were c o n s i d e r e d t o be immature. B. B l o o d A n a l y s i s E s t r o g e n and p r o g e s t e r o n e c o n c e n t r a t i o n s were determined on the s t o r e d s e r a samples u s i n g radioimmunoassay t e c h n i q u e s . P r o g e s t e r o n e c o n c e n t r a t i o n s were determined u s i n g p r e v i o u s l y d e s c r i b e d p r o c e d u r e s ( M c K i b b i n e t a l . 1984; Moger and Armstrong 1974). A l i q u o t s of serum (0.5 ml) were e x t r a c t e d w i t h ten volumes of r e d i s t i l l e d hexane, d r i e d under f i l t e r e d a i r and d i s s o l v e d i n assay b u f f e r . The mean p r o g e s t e r o n e r e c o v e r y was 86.9% and the s e n s i t i v i t y of the a s s a y was 25 pg/ml. A s i n g l e assay was performed and the i n t r a - a s s a y c o e f f i c i e n t of v a r i a t i o n c a l c u l a t e d between d u p l i c a t e s ranged from 0.1 t o 9.9%. 78 Serum c o n c e n t r a t i o n s of e s t r a d i o l 17/3 were det e r m i n e d by the R e p r o d u c t i v e E n d o c r i n e L a b o r a t o r i e s , Department of V e t e r i n a r y P h y s i o l o g i c a l S c i e n c e s , C o l l e g e of V e t e r i n a r y M e d i c i n e , U n i v e r s i t y of Saskatchewan, Saskatoon, Saskatchewan. The p r o c e d u r e s f o r t h i s a ssay a r e o u t l i n e d i n R a w l i n g s et a l . (1984). A l i q u o t s of 1 ml of serum were e x t r a c t e d and the s e n s i t i v i t y of t h i s a s s a y was 3 pg/ml e s t r a d i o l . C r o s s r e a c t i o n s w i t h e s t r o n e , t e s t o s t e r o n e , and p r o g e s t e r o n e were 2.7%, 0.02% and <0.01% r e s p e c t i v e l y . The i n t r a - and i n t e r a s s a y c o e f f i c i e n t s of v a r i a t i o n were 11% and 17% r e s p e c t i v e l y . The term e s t r o g e n may be used t o r e f e r t o the r e s u l t s of t h i s assay a l t h o u g h e s t r a d i o l 17/3 i s the p r i m a r y component. Due t o the l a r g e amounts of serum n e c e s s a r y , some samples were i n s u f f i c i e n t f o r both s t e r o i d a s s a y s . In t h e s e samples e s t r a d i o l c o n c e n t r a t i o n s were not de t e r m i n e d . I I I . S t a t i s t i c a l A n a l y s i s Samples were grouped and s u b j e c t e d t o the s t a t i s t i c a l a n a l y s i s o u t l i n e d i n Chapter I I . A l l v a l u e s a r e ex p r e s s e d as mean ± s t a n d a r d e r r o r of t h e mean (SEM). 7 9 RESULTS I_. H i s t o l o g i c a l Examinat i o n of the Reproduct i v e Organs A. O v a r i a n C y c l e K G e n e r a l D e s c r i p t i o n The 146 o t t e r o v a r i e s I examined were e n c l o s e d i n an o v a r i a n bursa and were l e n t i c u l a t e or o v a l i n shape. The o u t e r s u r f a c e was g e n e r a l l y smooth or s l i g h t l y l o b u l a t e d and the s u r f a c e e p i t h e l i u m c o n s i s t e d of a s i n g l e l a y e r of c u b o i d a l or low columnar c e l l s w i t h e i t h e r rounded or s l i g h t l y e l o n g a t e d n u c l e i . The t u n i c a a l b u g i n e a formed a t h i n l a y e r of c o n n e c t i v e t i s s u e beneath the s u r f a c e e p i t h e l i u m . S u b s u r f a c e f i s s u r e s , s h a l l o w i n v a g i n a t i o n of the o v a r i a n s u r f a c e w i t h l i t t l e or no b r a n c h i n g , were p r e s e n t i n some o v a r i e s . They were l i n e d w i t h a c o n t i n u a t i o n of the s u r f a c e e p i t h e l i u m and t u n i c a a l b u g i n e a . The anatomy and h i s t o l o g y of the r e p r o d u c t i v e organs changed w i t h both age and r e p r o d u c t i v e s t a t e . The o v a r i e s of j u v e n i l e o t t e r s were s m a l l (Table V I I I ) and t h e r e was no sharp d e m a r c a t i o n between the c o r t e x and m e d u l l a . The c o r t e x was c h a r a c t e r i z e d by a l a r g e number of p r i m a r y f o l l i c l e s r a n g i n g from 28-44 Mm i n di a m e t e r and Stage I p r e a n t r a l f o l l i c l e s . F o l l i c l e s r a r e l y exceeded 150-200 um i n diameter b e f o r e a t r e s i a o c c u r r e d and a t r e t i c f o l l i c l e s were numerous. No a n t r a l f o l l i c l e s were p r e s e n t and p o l y o v u l a r f o l l i c l e s were r a r e . C o n n e c t i v e t i s s u e was abundant, c o m p r i s i n g 62.8 ± 7.4% of the T a b l e V I I I . Mean o v a r i a n w e i g h t s (mg) and vo lumes (rnm^) o f r i v e r o t t e r s i n d i f f e r e n t age c l a s s e s . A l l v a l u e s a r e mean + SEM. Age Sample W e i g h t Sample Volume C l a s s S i z e S i z e 0 35 6 4 . 0 + 5 . 7 a 45 5 4 . 2 + 4 . 5 d 1 9 1 2 5 . 8 + 6 . 2 b 12 1 1 2 . 4 + 1 5 . 5 e 2 11 1 7 8 . 2 + 1 4 . 7 C 20 2 1 1 . 0 + 2 4 . 0 f >3 56 2 1 7 . 3 + 1 2 . l c 68 1 9 9 . 0 + 1 5 . 0 f I 2 67 2 1 0 . 9 + 1 0 . 5 C 88 2 0 2 . 0 + 1 2 . 0 f a , b , c , d , e , f - D i f f e r e n t s u p e r s c r i p t s w i t h i n a co lumn i n d i c a t e s i g n i f i c a n t l y d i f f e r e n t means , p < 0 . 0 1 . 81 o v a r i a n volume, w h i l e i n t e r s t i t i a l t i s s u e was i n a c t i v e and ac c o u n t e d f o r o n l y 30.8 ± 7.5% of the o v a r y . A l t h o u g h l a r g e r , the o v a r i e s of y e a r l i n g and a d u l t o t t e r s were s i m i l a r i n g e n e r a l appearance t o those of j u v e n i l e s . The o v a r i e s of y e a r l i n g s were s i g n i f i c a n t l y d i f f e r e n t i n s i z e (p<.0l) from those of both j u v e n i l e s and a d u l t s (Table V I I I ) and are d e s c r i b e d i n d e t a i l l a t e r (see s e x u a l m a t u r i t y ) . Mean o v a r i a n volumes and w e i g h t s d i d not d i f f e r s i g n i f i c a n t l y among a d u l t s a l t h o u g h w e i g h t s were s l i g h t l y lower i n age c l a s s 2 than i n o l d e r a n i m a l s . S i g n i f i c a n t changes i n o v a r i a n weight were observed throughout the r e p r o d u c t i v e c y c l e of a d u l t o t t e r s . O v a r i a n w e i g h t s were l a r g e r i n r e p r o d u c t i v e l y a c t i v e o t t e r s (Table IX) w i t h a n e s t r o u s o v a r i e s b e i n g s i g n i f i c a n t l y s m a l l e r than those of pregnancy ( p < . 0 l ) . The mean we i g h t s of p a i r e d o v a r i e s c o l l e c t e d from o t t e r s d u r i n g e s t r u s v a r i e d g r e a t l y ; two females c o n t a i n e d s m a l l o v a r i e s (207.5 and 227.8 mg), whereas the o v a r i e s from a t h i r d were much l a r g e r (599 mg). However, the l a t t e r o t t e r had r e c e n t l y g i v e n b i r t h and the presence of c o r p o r a a l b i c a n t i a may account f o r i t s g r e a t e r w e i g h t. In the a d u l t ovary the me d u l l a was c o n c e n t r a t e d i n the r e g i o n of the h i l u s and c o n s i s t e d p r i m a r i l y of c o n n e c t i v e t i s s u e and b l o o d v e s s e l s . The c o r t e x c o m p r i s e d most of the ovary and c o n t a i n e d a v a s t a r r a y of p r i m a r y , p r e a n t r a l and s m a l l (Stages I and I I ) a n t r a l f o l l i c l e s t hroughout the e s t r o u s c y c l e . I n t e r s t i t i a l c e l l s were abunda'nt and were l o c a t e d i n d i s c r e t e clumps, u s u a l l y around the a t r e t i c f o l l i c l e from which they were T a b l e I X . O v a r i a n w e i g h t (mg) , vo lume (mm 3) and u t e r i n e d i a m e t e r (mm) o f a d u l t r i v e r o t t e r s (age c l a s s e s 2+) i n v a r i o u s r e p r o d u c t i v e s t a t e s . A l l v a l u e s a r e mean +_ SEM. n = sample s i z e . R e p r o d u c t i v e O v a r i a n O v a r i a n U t e r i n e S t a t e We igh t (n) Volume (n) D i a m e t e r (n) A n e s t r u s 1 6 7 . 9 + 1 0 . 0 ( l l ) b 1 6 0 . 3 + 2 4 . 0 ( 1 5 ) d 3 . 3 + 0 . 2 ( 7 ) e E s t r u s 9 : n u l l i p a r o u s 2 1 6 . 9 + 1 0 . 4 (2) 1 4 0 . 2 + 2 2 . 3 (2 ) 4 . 8 + 0 . 8 (2) p o s t p a r t u m 599 .0 (1) 2 2 . 5 (1 ) P r e i m p l a n t a t i o n 2 0 9 . 9 + 11 .1 ( 4 9 ) c 2 1 2 . 0 + 1 5 . 5 ( 6 5 ) d 4 . 2 + 0 . 1 ( 1 9 ) f P o s t i m p l a n t a t i o n 2 4 1 . 8 + 2 9 . 0 ( 4 ) c 2 1 3 . 7 + 3 0 . 4 ( 6 ) d 5 . 3 + 0 . 1 ( 6 ) 9 a - E s t r u s was d i v i d e d i n t o a n i m a l s w h i c h had g i v e n b i r t h t h e y e a r b e f o r e and t h o s e t h a t d i d n o t b e a r y o u n g . See t e x t f o r d e t a i l s . Due t o s m a l l samp le s i z e no c o m p a r i s o n s were made w i t h o t h e r means. b , c , d , e , f , g - D i f f e r e n t s u p e r s c r i p t s w i t h i n a co lumn i n d i c a t e s i g n i f i c a n t l y d i f f e r e n t means , p < 0 . 0 1 . 83 d e r i v e d . Based upon the c l a s s i f i c a t i o n s of Mossman (1937), t h r e e d i s t i n c t t y p e s of i n t e r s t i t i a l c e l l s were i d e n t i f i e d : type I or g r a n u l a r i n t e r s t i t i a l c e l l s were s m a l l , i r r e g u l a r , and c o n t a i n e d s m a l l q u a n t i t i e s of c y t o p l a s m , type I I (mature) were l a r g e r , rounded, had an i n c r e a s e d amount of c y t o p l a s m and appeared t o be s e c r e t o r y , t y p e I I I ( i r r e g u l a r ) were l a r g e , v a c u o l a t e d c e l l s w i t h s m a l l p y k n o t i c n u c l e i . The l a t t e r type appeared t o be d e g e n e r a t i n g c e l l s and were p r o b a b l y d e r i v e d from Type I I i n t e r s t i t i a l c e l l s . The r e l a t i v e abundance and ty p e s of i n t e r s t i t i a l t i s s u e s p r e s e n t v a r i e d w i t h r e p r o d u c t i v e a c t i v i t y . With the e x c e p t i o n of a g r e a t e r number of d e v e l o p i n g f o l l i c l e s , a d u l t o v a r i e s d u r i n g a n e s t r u s were s i m i l a r t o those of j u v e n i l e s . They d i f f e r e d from more a c t i v e o v a r i e s i n b e i n g l e s s v a s c u l a r and w i t h o u t a n t r a l f o l l i c l e s l a r g e r than Stage I I . C o n n e c t i v e t i s s u e and i n t e r s t i t i a l c e l l s , p r e d o m i n a t e l y Type I , were abundant a c c o u n t i n g f o r 48.2 ± 3.3% and 44.4 ± 3.2% of the t o t a l o v a r i a n volume r e s p e c t i v e l y . 2. F o l l i c u l a r growth A n t r a were f i r s t o b s e r v e d when f o l l i c l e s were 1 40-200 j i m i n d i a m e t e r . The ovum reached maximum dia m e t e r (100.0 ± 3.34 M m , n = 100) w h i l e the f o l l i c l e s were s t i l l i n Stage I . As f u r t h e r growth o c c u r r e d , t h e c a i n t e r n a c e l l s h y p e r t r o p h i e d and formed a moderately t h i c k l a y e r around the e n t i r e f o l l i c l e . A c t i v e t h e c a c e l l s were l a r g e , rounded and had abundant, p a l e s t a i n i n g c y t o p l a s m . F o l l i c u l a r a t r e s i a was observed a t a l l s t a g e s of 84 development a l t h o u g h i t was most common among Stage I I f o l l i c l e s . In the e a r l y s t a g e s of a t r e s i a , f o l l i c l e s c o n t a i n e d a l a r g e number of p y k n o t i c g r a n u l o s a c e l l s , c e l l d e b r i s i n the antrum, and a degenerated o o c y t e . More advanced a t r e s i a r e s u l t e d i n f o l l i c l e s c h a r a c t e r i z e d by the absence of a g r a n u l o s a c e l l l a y e r and by the h y p e r t r o p h y of the t h e c a i n t e r n a c e l l s . I f a t r e s i a o c c u r r e d i n s m a l l i n a c t i v e f o l l i c l e s , t he t h e c a l c e l l s resembled Type I i n t e r s t i t i a l t i s s u e . However, i f the f o l l i c l e s were l a r g e r and more a c t i v e b e f o r e a t r e s i a o c c u r r e d , the t h e c a i n t e r n a c e l l s underwent h y p e r t r o p h y t o become rounded w i t h l a r g e amounts of p a l e s t a i n i n g c y t o p l a s m . Large a t r e t i c f o l l i c l e s formed d i s t i n c t n o n - l u t e a l s t r u c t u r e s termed c o r p o r a a t r e t i c a (Mossman and Duke 1973). D u r i n g e s t r u s the ovary was h i g h l y v a s c u l a r i z e d and maximum development of the g r a n u l o s a and t h e c a i n t e r n a l a y e r s was observed. P r e o v u l a t o r y f o l l i c l e s averaged 2.31 ± 0.13 mm i n diameter (range = 2.0-2.8 mm, n = 7) and the oocyte and a d j o i n i n g c o r o n a l c e l l s were f r e e i n the a n t r a p r i o r t o o v u l a t i o n . The o v a r i e s a l s o c o n t a i n e d numerous Stage I I and one or two Stage V f o l l i c l e s which may r e p r e s e n t a second, s m a l l e r wave of f o l l i c l e s . The presence of numerous c o r p o r a a t r e t i c a i n d i c a t e d t h a t the m a j o r i t y of f o l l i c l e s underwent a t r e s i a p r i o r t o o v u l a t i o n . The amount of i n t e r s t i t i a l t i s s u e i n c r e a s e d throughout f o l l i c u l a r growth and reached a maximum of 41.4 ± 5.2% of the t o t a l o v a r i a n volume a t e s t r u s . C o n n e c t i v e t i s s u e d e c r e a s e d t o 29.4 ± 7.3% w h i l e e n l a r g e d f o l l i c l e s a c c o u n t e d f o r the remainder. The p e r c e n t a g e of Type I i n t e r s t i t i a l c e l l s 8 5 d e c r e a s e d as f o l l i c u l a r growth proceeded and Type I I c e l l s o n l y were p r e s e n t at e s t r u s . No l a r g e f o l l i c l e s or l u t e a l b o d i e s were p r e s e n t i n the o v a r i e s of nonpregnant, a d u l t o t t e r s f o l l o w i n g the b r e e d i n g season. The o v a r i e s of a mature c a p t i v e o t t e r which had completed e s t r u s , as i n d i c a t e d by the changes i n her v a g i n a l smears, but had not b r e d , were a l s o examined. O v u l a t i o n had not o c c u r r e d and t h r e e l a r g e c o r p o r a a t r e t i c a (700-900 um) were v i s i b l e . F o l l i c l e s l a r g e r than Stage I I were not p r e s e n t and Type I I I i n t e r s t i t i a l c e l l s were common. 3_. Pregnancy Three t y p e s of c o r p o r a l u t e a were obser v e d d u r i n g pregnancy i n the r i v e r o t t e r : the c o r p o r a l u t e a of o v u l a t i o n , c o r p o r a l u t e a of p r e i m p l a n t a t i o n pregnancy and the c o r p o r a l u t e a of i m p l a n t a t i o n . The c o r p o r a l u t e a of o v u l a t i o n were s i m i l a r i n s i z e t o the p r e o v u l a t o r y f o l l i c l e s ( T a b l e X, range = 2.04-2.43 mm) and c o n t a i n e d a c e n t r a l antrum ( P l a t e IV, F i g . 22). The l u t e a l c e l l s were s m a l l (Table X ) , rounded or e l o n g a t e d i n shape and had l i t t l e c y t o p l a s m ( P l a t e IV, F i g . 23). A c o n n e c t i v e t i s s u e network and a v a s c u l a r t r a b e c u l a e were p r e s e n t between ,the l u t e a l c e l l s . A l t h o u g h Type I I i n t e r s t i t i a l c e l l s were abundant i n o v a r i e s which had r e c e n t l y o v u l a t e d , some Type I I I c e l l s were p r e s e n t which i n d i c a t e d t h a t d e g e n e r a t i o n was o c c u r r i n g . The c o r p o r a l u t e a of p r e i m p l a n t a t i o n pregnancy ( P l a t e IV, F i g . 24) a r e s m a l l and r e l a t i v e l y i n a c t i v e . The c o r p o r a l u t e a 8 6 T a b l e X . D i a m e t e r s o f c o r p o r a l u t e a and l u t e a l c e l l s d u r i n g p r e g n a n c y i n r i v e r o t t e r s . Twenty l u t e a l c e l l s were measu red i n each f e m a l e . Month C o r p o r a l u t e a Number Mean + SEM (mm) L u t e a l c e l l s Number o f Mean + SEM F e m a l e s (um) P r e i m p l a n t a t i o n : May 3 a November December J a n u a r y T o t a l 56 115 183 2 . 2 2 + 0 . 1 1 2 . 0 8 + 0.10& 1.99 + 0 . 0 4 b 2 . 0 6 + 0 .04& 2 . 0 3 + 0 . 0 3 b 1* 2 a 7 21 31 1 5 . 6 1 8 . 8 3 + 2 . 1 7 1 7 . 6 6 + 0 . 6 7 d 1 8 . 4 3 + 0 . 5 0 d 1 8 . 4 3 + 0 . 4 0 d P o s t i m p l a n t a t i o n : F e b r u a r y 19 3 . 6 3 + 0.22C 3 7 . 8 0 + 0 . 4 0 e a - S t a t i s t i c a l c o m p a r i s o n s were n o t made due t o s m a l l samp le s i z e . b , c , d , e - D i f f e r e n t s u b s c r i p t s w i t h i n a co l um i n d i c a t e s i g n i f i c a n t l y d i f f e r e n t means , p < 0 . 0 1 . 87 P l a t e IV. Photomicrographs of c o r p o r a l u t e a and l u t e a l c e l l s d u r i n g pregnancy i n L. c a n a d e n s i s . A l l s e c t i o n s a r e s t a i n e d w i t h H and E. F i g u r e 22. Corpora l u t e a of e a r l y o v u l a t i o n . Note the c e n t r a l antrum and the c o n n e c t i v e t i s s u e network. M a g n i f i c a t i o n a p p r o x i m a t e l y 40X. F i g u r e 23. Enlargement of l u t e a l c e l l s i n F i g u r e 22. C e l l s g e n e r a l l y s m a l l and rounded, or e l o n g a t e d i n shape. M a g n i f i c a t i o n a p p r o x i m a t e l y 400X. F i g u r e 24. Corpora l u t e a of p r e i m p l a n t a t i o n . Note the s m a l l , r e l a t i v e l y i n a c t i v e appearance and low degree of v a s c u l a r i z a t i o n . Compare w i t h F i g u r e 26. M a g n i f i c a t i o n a p p r o x i m a t e l y 40X. F i g u r e 25. Enlargement of l u t e a l c e l l s i n F i g u r e 24. C e l l s s m a l l and no v a c u o l e s p r e s e n t . Compare w i t h F i g u r e 27. M a g n i f i c a t i o n a p p r o x i m a t e l y 400X. F i g u r e 26. Corpora l u t e a of p o s t i m p l a n t a t i o n . Note the a c t i v e appearance and i n c r e a s e d v a s c u l a r i z a t i o n i n the o v a r y . M a g n i f i c a t i o n a p p r o x i m a t e l y 25X. F i g u r e 27. Enlargement of l u t e a l c e l l s i n F i g u r e 26. C e l l s l a r g e , a c t i v e l y s e c r e t i n g and surrounded by abundant b l o o d c e l l s ( B ) . M a g n i f i c a t i o n a p p r o x i m a t e l y 400X. 88 89 were s i m i l a r i n diameter t o those of o v u l a t i o n (range = 0.95-3.3 mm) and d i d not vary s i g n i f i c a n t l y d u r i n g the p e r i o d of d e l a y i n i m p l a n t a t i o n (Table X ) . A n t r a were observed i n some of the c o r p o r a l u t e a c o l l e c t e d d u r i n g the e a r l y and m i d d l e p r e i m p l a n t a t i o n p e r i o d s but not a t the end of the d e l a y p e r i o d . D u r i n g the p r e i m p l a n t a t i o n p e r i o d the l u t e a l c e l l s were n o n v a c u o l a t e d , round or p o l y g o n a l i n shape and ranged i n s i z e from 10-25 Mm ( P l a t e IV, F i g . 2 5 ) . Only s m a l l l u t e a l c e l l s were p r e s e n t and the mean s i z e d i d not v a r y d u r i n g the p e r i o d the b l a s t o c y s t s were f r e e i n the u t e r u s (Table X ) . By t h e end of the d e l a y p e r i o d l u t e a l c e l l s had a g r a n u l a r appearance a l t h o u g h no v a c u o l e s were observed. F o l l i c u l a r growth o c c u r r e d throughout p r e i m p l a n t a t i o n and Stage V f o l l i c l e s were o b s e r v e d by the end of d e l a y . The a c t i v i t y of the i n t e r s t i t i a l t i s s u e appeared t o v a r y w i t h f o l l i c u l a r development. Type I c e l l s were dominant when f o l l i c l e s were s m a l l w h i l e Type I I c e l l s were more abundant near the end of the p r e i m p l a n t a t i o n p e r i o d when f o l l i c l e s were l a r g e r . Some Type I I i n t e r s t i t i a l c e l l s were p r e s e n t throughout the d e l a y p e r i o d and were c o n f i n e d t o the p e r i p h e r y of the c o r p o r a l u t e a . At the end of the d e l a y p e r i o d the c o r p o r a l u t e a i n c r e a s e d i n s i z e and a c t i v i t y t o form the c o r p o r a l u t e a of p o s t i m p l a n t a t i o n pregnancy ( P l a t e IV, F i g . 26). These c o r p o r a were h i g h l y v a s c u l a r i z e d and were s i g n i f i c a n t l y l a r g e r i n dia m e t e r (p<.00l) than those observed d u r i n g the p r e i m p l a n t a t i o n p e r i o d ( Table X, range = 1.44-4.5 mm). The l u t e a l c e l l s had a l s o i n c r e a s e d s i g n i f i c a n t l y i n s i z e ( T a ble X, range = 30-47 Mm, 90 P<.001), and were p o l y g o n a l i n shape w i t h l a r g e amounts of v a c u o l a t e d c y t o p l a s m ( P l a t e IV, F i g . 27). Corpora l u t e a of p o s t i m p l a n t a t i o n were p r e s e n t i n the o v a r i e s of o t t e r s which c o n t a i n e d u t e r i n e s w e l l i n g s and b l a s t o c y s t s which had shed t h e i r zona p e l l u c i d a , but were s t i l l u n i m p l a n t e d , which i n d i c a t e s t h a t i n c r e a s e d a c t i v i t y of the l u t e a l c e l l s began b e f o r e a c t u a l i m p l a n t a t i o n o c c u r r e d . Samples from the l a t e p o s t i m p l a n t a t i o n p e r i o d were not a v a i l a b l e f o r e x a m i n a t i o n and i t i s unknown i f the c o r p o r a l u t e a are m a i n t a i n e d t h r o u g h o u t the e n t i r e g e s t a t i o n p e r i o d or i f l u t e a l d e g e n e r a t i o n o c c u r s p r i o r t o p a r t u r i t i o n . D u r i n g the p o s t i m p l a n t a t i o n p e r i o d o v a r i e s c o n t a i n e d f o l l i c l e s which d i d not appear t o be a c t i v e l y growing and were s i m i l a r i n s i z e t o those of l a t e d e l a y . The i n t e r s t i t i a l t i s s u e was p r e d o m i n a t e l y i n a c t i v e (Type I ) which may r e f l e c t the reduced f o l l i c u l a r a c t i v i t y p r e s e n t . Along w i t h normal c o r p o r a l u t e a , u n u s u a l l y s m a l l ones were observed i n the o v a r i e s of f o u r o t t e r s ; t h r e e c o l l e c t e d d u r i n g the d e l a y p e r i o d and one w i t h i m p l a n t e d embryos. These c o r p o r a l u t e a appeared normal w i t h the e x c e p t i o n of t h e i r s i g n i f i c a n t l y s m a l l e r s i z e ( d i a m e t e r s = 0.94, 1.01, 1.14 and 1.44 mm r e s p e c t i v e l y , p < . 0 0 l ) . A l l appeared t o be a t the same stage of development as the o t h e r c o r p o r a i n the same ovary and t h e r e was no e v i d e n c e t h a t they had been formed as a r e s u l t of secondary o v u l a t i o n s . D e g e n e r a t i n g ova or remnants of the zona p e l l u c i d a were not v i s i b l e i n these c o r p o r a which suggests t h a t they were not a c c e s s o r y c o r p o r a l u t e a formed from the l u t e i n i z a t i o n of a t r e t i c f o l l i c l e s or from f o l l i c l e s which f a i l e d t o o v u l a t e . 91 The l u t e i n i z a t i o n of a t r e t i c f o l l i c l e s was o b s e r v e d i n a s i n g l e o v a r y . A 400 urn l u t e a l body was p r e s e n t which had formed from some of the t h e c a l c e l l s s u r r o u n d i n g an a t r e t i c Stage I I f o l l i c l e . F o l l i c u l a r growth proceeded r a p i d l y i n o t t e r s which had r e c e n t l y g i v e n b i r t h . The o v a r i e s of p a r t u r i e n t females c o n t a i n e d p r e o v u l a t o r y f o l l i c l e s and abundant Type I I i n t e r s t i t i a l t i s s u e . Corpora a l b i c a n t i a which a v e r a g e d 2.4 ± 0.09 mm (n = 3) i n diameter were a l s o p r e s e n t and c o n s i s t e d of d e g e n e r a t i n g l u t e a l c e l l s , b l o o d v e s s e l s and c o n n e c t i v e t i s s u e . Corpora a l b i c a n t i a d i d not p e r s i s t and were not v i s i b l e i n o v a r i e s c o l l e c t e d s i x months a f t e r the b i r t h p e r i o d . B. U t e r u s and P l a c e n t a The endometrium of the b i c o r n u a t e u t e r u s was composed of f o u r t o s i x l o n g i t u d i n a l f o l d s and c o n t a i n e d g l a n d s which c o n s i s t e d of a s t r a i g h t neck r e g i o n and a c o i l e d b a s a l p o r t i o n . The myometrium was comprised of an i n n e r c i r c u l a r , middle o b l i q u e , and an o u t e r l o n g i t u d i n a l l a y e r of smooth muscle. The mi d d l e o b l i q u e l a y e r a l s o c o n t a i n e d numerous b l o o d v e s s e l s which p e n e t r a t e d the i n n e r m y o m e t r i a l l a y e r t o e n t e r t h e e n d o m e t r i a l f o l d s . J u v e n i l e o t t e r s had s m a l l , s l e n d e r u t e r i ( T a b l e X I ) ; the endometrium was t h i n and the u t e r i n e g l a n d s simple* s t r a i g h t t u b e s . The l u m i n a l e p i t h e l i u m c o n s i s t e d of a s i n g l e l a y e r of c u b o i d a l c e l l s r a n g i n g i n h e i g h t from 2.5-10 um (mean = 5.7 ± 0.5 um). G l a n d u l a r e p i t h e l i a l c e l l s were a l s o c u b o i d a l or low T a b l e X I . U t e r i n e d i a m e t e r s (mm) and l e n g t h s (mm) o f c o r n u a o f r i v e r o t t e r s i n d i f f e r e n t age c l a s s e s . A l l v a l u e s a r e mean + SEM. Age Sample D i a m e t e r Sample L e n g t h C l a s s S i z e ( r a n g e ) S i z e ( r a n g e ) 0 14 1 . 6 0 + 0 . 1 0 3 14 4 9 . 8 + 2 . 6 d ( 1 . 2 - 2 . 6 ) ( 2 6 . 3 - 6 6 . 2 ) 1 7 2 . 6 2 + 0 . 1 8 b 7 5 4 . 6 + 2 . 0 d ( 2 . 0 - 3 . 2 ) ( 4 4 . 2 - 6 1 . 2 ) 2 5 3 . 7 5 + 0 . 5 4 b c 5 6 2 . 5 + 1 . 8 e ( 2 . 5 - 5 . 6 ) ( 5 8 . 4 - 6 8 . 5 ) >3 27 4 . 8 3 + 0 . 6 9 c 27 7 5 . 3 + 3 . 3 f ( 2 . 6 - 2 2 . 5 ) ( 4 8 . 3 - 1 2 3 . 4 ) >2 32 4 . 6 6 + 0 . 5 9 c 31 7 3 . 2 + 2 . 9 f ( 2 . 5 - 2 2 . 5 ) ( 4 8 . 3 - 1 2 3 . 4 ) a , b , c , d , e , f - D i f f e r e n t s u p e r s c r i p t s w i t h i n a co lumn i n d i c a t e s i g n i f i c a n t l y d i f f e r e n t means , p < 0 . 0 1 . 93 P l a t e V. P h o t o m i c r o g r a p h s of u t e r i from L. c a n a d e n s i s . A l l s e c t i o n s s t a i n e d w i t h H and E. A l l m a g n i f i c a t i o n s a p p r o x i m a t e l y 340X. F i g u r e 28. T r a n s v e r s e s e c t i o n of an u t e r u s d u r i n g a n e s t r u s . Note the t h i n , c u b o i d a l e p i t h e l i a l l a y e r and the s h o r t , s t r a i g h t u t e r i n e g l a n d s . F i g u r e 29. T r a n s v e r s e s e c t i o n of an u t e r u s d u r i n g e s t r u s . S i m i l a r t o t h a t of a n e s t r u s but w i t h a h i g h e r e p i t h e l i a l l a y e r and more abundant, deeper u t e r i n e g l a n d s . F i g u r e 30. T r a n s v e r s e s e c t i o n of an u t e r u s d u r i n g the p r e i m p l a n t a t i o n p e r i o d of pregnancy. Note the columnar e p i t h e l i a l c e l l s and abundant, a c t i v e l y s e c r e t i n g u t e r i n e g l a n d s . F i g u r e 31. T r a n s v e r s e s e c t i o n from a n o n - p l a c e n t a l p o r t i o n of an u t e r u s w i t h i m p l a n t e d embryos. L u m i n a l e p i t h e l i a l c e l l s a r e t a l l , columnar and c o n t a i n b a s a l n u c l e i . U t e r i n e g l a n d s a r e a c t i v e and l i n e d by v a c u o l a t e d e p i t h e l i a l c e l l s ( V ) . 94 95 columnar and were 4-10 j i m (mean = 5.69 ± 0.8 Aim) h i g h . The myometrium was t h i n (130-190 Mm a c r o s s ) and had l i t t l e v a s c u l a r i z a t i o n . The u t e r i of a d u l t s were l a r g e r than those of immatures ( T a b l e XI) and e x h i b i t e d c y c l i c a l changes i n development and d i a m e t e r a s s o c i a t e d w i t h the annua l r e p r o d u c t i v e c y c l e . A n e s t r o u s u t e r i were a v a s c u l a r and had s m a l l d i a m e t e r s (Table IX) r a n g i n g from 2.5-4.2 mm w i t h t h o s e of parous i n d i v i d u a l s b e i n g l a r g e r than those of n u l l i p a r o u s ones. The lumen was l i n e d by c u b o i d a l e p i t h e l i a l c e l l s 2-5 Mm i n h e i g h t and the u t e r i n e glands were s h o r t and s t r a i g h t ( P l a t e V, F i g . 28). The stroma was nonedematous and the myometrium was r e l a t i v e l y t h i n (250-400 M m ) . D u r i n g e s t r u s the u t e r i had g r e a t e r v a s c u l a r i z a t i o n and l a r g e r d i a m e t e r s (Table I X ) . C u b o i d a l or low columnar l u m i n a l e p i t h e l i a l c e l l s i n c r e a s e d i n h e i g h t t o 7-14 Mm and the u t e r i n e g l a n d s were more abundant, deeper and showed s i g n s of a c t i v i t y ( P l a t e V, F i g . 29). The g l a n d s were t u b u l a r , s l i g h t l y c o i l e d and l i n e d w i t h low columnar c e l l s 9-11 Mm h i g h . The t h i c k n e s s of the myometrium d i d not i n c r e a s e d a l t h o u g h the v a s c u l a r l a y e r was more pronounced. The u t e r u s of an o t t e r i n e s t r u s which had r e c e n t l y g i v e n b i r t h was much l a r g e r i n diameter (22.5 mm) and had edematous stroma w i t h d e g e n e r a t i n g u t e r i n e g l a n d s . D u r i n g the p r e i m p l a n t a t i o n p e r i o d of pregnancy the endometrium was congested w i t h deep, c o i l e d g l a n d s which were i n an a c t i v e s e c r e t o r y s t a t e ( P l a t e V, F i g . 30). A p i c a l buds were v i s i b l e on some of the s u r f a c e c e l l s and the lumen of many 96 g l a n d s c o n t a i n e d s e c r e t o r y p r o d u c t s . These g l a n d s were l i n e d w i t h columnar e p i t h e l i a l c e l l s w i t h round, b a s a l n u c l e i and ranged from 9-12 um i n h e i g h t . The l u m i n a l e p i t h e l i u m was t a l l e r than i n nonpregnant a n i m a l s (14-19 jum) and was c o m p r i s e d of columnar c e l l s w i t h e l o n g a t e d , c e n t r a l l y l o c a t e d n u c l e i w h i l e the myometrium was s i m i l a r t o t h a t p r e s e n t d u r i n g e s t r u s . The u t e r i had i n c r e a s e d v a s c u l a r i z a t i o n and s i g n i f i c a n t l y l a r g e r d i a m e t e r s r a n g i n g from 3.1-5.38 mm (Table IX, p < . 0 l ) . U t e r i c o l l e c t e d e a r l y i n the p r e i m p l a n t a t i o n p e r i o d appeared l e s s a c t i v e than those c o l l e c t e d l a t e r . The r e s u m p t i o n of l u t e a l a c t i v i t y and the i n i t i a t i o n of i m p l a n t a t i o n were a s s o c i a t e d w i t h a marked i n c r e a s e i n e n d o m e t r i a l development ( P l a t e V, F i g . 31). The d i a m e t e r of those p o r t i o n s of t h e u t e r i not i n v o l v e d i n p l a c e n t a t i o n i n c r e a s e d s i g n i f i c a n t l y t o 5.3-5.6 mm (Table IX, p<.0l) and v a s c u l a r i z a t i o n of t h e endometrium was e x t e n s i v e . The l u m i n a l e p i t h e l i u m v a r i e d i n h e i g h t from 20-29 Mm and the u t e r i n e g l a n d s were numerous, h i g h l y c o i l e d and had g r e a t l y d i l a t e d necks. The myometrium was w e l l d e v e l o p e d and ranged from 500 t o 900 Mm i n t h i c k n e s s . The e n d o t h e l i o c h o r i a l p l a c e n t a was c o m p l e t e l y zonary and the u t e r i n e g l a n d s were g r e a t l y d i l a t e d and extended deep i n t o the endometrium. S m a l l (5.5-6.7 mm) haemophagous organs (Creed and B i g g e r s 1964) were suspended i n the a l l a n t o i c c a v i t i e s of a u t e r u s c o n t a i n i n g two 20mm embryos a l t h o u g h none were obse r v e d i n u t e r i w i t h more r e c e n t i m p l a n t a t i o n s . 97 C. B l a s t o c y s t s The b l a s t o c y s t s c o n s i s t e d of a t r o p h o b l a s t l a y e r , the i n n e r c e l l mass and a zona p e l l u c i d a a p p r o x i m a t e l y 9 Aim t h i c k . In f r e s h l y r e c o v e r e d b l a s t o c y s t s the t r o p h o b l a s t was c l o s e l y apposed t o the zona but q u i c k l y c o l l a p s e d when p l a c e d i n f i x a t i v e . The di a m e t e r of the zona p e l l u c i d a d i d not v a r y d u r i n g the d e l a y p e r i o d and ranged from 1.5-2.0 mm. D. V a g i n a l C y c l e Vaginae of o t t e r s were l o n g , muscular and underwent c y c l i c a l changes i n the v a g i n a l e p i t h e l i u m a s s o c i a t e d w i t h the annual r e p r o d u c t i v e c y c l e . E a s i l y i d e n t i f i e d changes were observed i n the v a g i n a l c y c l e which c o r r e l a t e d w i t h the a n n u a l r e p r o d u c t i v e c y c l e . These changes which a r e summarized i n T a b l e X I I , i n d i c a t e t h a t the v a g i n a l smears c o r r e c t l y i d e n t i f y the r e p r o d u c t i v e s t a t e of the female r i v e r o t t e r . The v a g i n a e of immature o t t e r s were undeveloped and appeared s i m i l a r t o those of a n e s t r o u s a d u l t s d e s c r i b e d below. The v a g i n a l smear was v e r y s c a n t y and c o n s i s t e d p r i m a r i l y of p a r a b a s a l (62.8%, range = 32.6-85.2%) and i n t e r m e d i a t e e p i t h e l i a l c e l l s (23.3%, range = 12.0-48.9%). The v a g i n a l lumen of a n e s t r o u s a d u l t s were l i n e d by t h r e e t o f i v e l a y e r s of c u b o i d a l e p i t h e l i u m which gave r i s e t o the 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 t y p i c a l of the a n e s t r o u s v a g i n a l smear ( P l a t e V I , F i g . 3 4 ) . I n t e r m e d i a t e e p i t h e l i a l c e l l s were the most common c e l l type i d e n t i f i e d and acc o u n t e d f o r 56.6% 9 8 T a b l e X I I . Summary o f t h e c e l l t y p e s p r e s e n t i n t h e v a g i n a l smears o f r i v e r o t t e r s R e p r o d u c t i v e E p i t h e l i a l C e l l s L e u k o c y t s S t a t e P a r a b a s a l I n t e r m e d i a t e C o r n i f i e d A n e s t r u s ++ +++ + P r o e s t r u s + +++ + + E s t r u s + +++ + M e t e s t r u s + ++ ++ +++ P r e g n a n t ++ +++ + Immature +++ ++ + + few (<10%) ++ m o d e r a t e (10 - 50%) +++ many (>50%) 99 P l a t e V I . P h o t o m i c r o g r a p h s of the v a g i n a and v a g i n a l smears of L. c a n a d e n s i s • V a g i n a l s e c t i o n s a re s t a i n e d w i t h H and E. V a g i n a l smears a r e s t a i n e d w i t h S c h o r r ' s t r i p l e s t a i n . A l l m a g n i f i c a t i o n s a p p r o x i m a t e l y 400X. F i g u r e 32. L o n g i t u d i n a l s e c t i o n t h r o u g h an a n e s t r o u s v a g i n a . Note the l a r g e , rounded e p i t h e l i a l c e l l s which g i v e r i s e t o the i n t e r m e d i a t e c e l l s of the v a g i n a l smear. F i g u r e 33. L o n g i t u d i n a l s e c t i o n t h r o u g h an e s t r o u s v a g i n a . E p i t h e l i a l l a y e r c o n s i s t s of numerous l a y e r s of f l a t t e n e d , c o r n i f i e d c e l l s . F i g u r e 34. An a n e s t r o u s v a g i n a l smear. L a r g e , rounded i n t e r m e d i a t e c e l l s ( I ) and p a r a b a s a l c e l l s (P) p r e s e n t . L e k o c y t e s common i n some smears. F i g u r e 35. A p r o e s t r o u s v a g i n a l smear c o n t a i n i n g i n t e r m e d i a t e c e l l s ( I ) , s u p e r i f i c a l c e l l s w i t h p y k n o t i c n u c l e i ( S ) , and the o c c a s i o n a l c o r n i f i e d c e l l ( C ) . Note the a n g u l a r shape of many i n t e r m e d i a t e c e l l s . F i g u r e 36. An e s t r o u s v a g i n a l smear. N u c l e a t e d and non-n u c l e a t e d c o r n i f i e d e p i t h e l i a l c e l l s p r e s e n t . F i g u r e 37. A m e t e s t r o u s v a g i n a l smear. L e u k o c y t e s (L) and mucus s t r a n d s common. C o r n i f i e d c e l l s g r a d u a l l y r e p l a c e d by i n t e r m e d i a t e c e l l s . ' 100 101 (range = 24.5-79%) of the t o t a l c e l l s p r e s e n t . These c e l l s were l a r g e (19-38 ym) w i t h abundant, p a l e s t a i n i n g c y t o p l a s m and round or o v a l n u c l e i a v e r a g i n g 10.39 ± 0.36 um i n d i a m e t e r . P a r a b a s a l c e l l s , s m a l l (8-15 um), round immature e p i t h e l i a l c e l l s w i t h l i t t l e c y t o p l a s m , were a l s o common and comprised a p p r o x i m a t e l y 16% (range = 1.5-43.5%) of the c e l l s p r e s e n t . L e u k o c y t e s and mucus v a r i e d g r e a t l y i n abundance throughout the a n e s t r o u s p e r i o d but were u s u a l l y p r e s e n t i n s m a l l amounts o n l y . Minor changes o c c u r r e d i n the v a g i n a l smear d u r i n g p r o e s t r u s ( P l a t e V I , F i g . 3 5 ) . I n t e r m e d i a t e c e l l s i n c r e a s e d i n number and were more a n g u l a r i n shape w h i l e p a r a b a s a l c e l l s d e c r e a s e d i n abundance and l e u k o c y t e s became r a r e . As the v a g i n a l e p i t h e l i u m p r o l i f e r a t e d the number of l a y e r s l i n i n g the lumen i n c r e a s e d and the c e l l s became f l a t t e n e d . In the l a t e r s t a g e s of p r o e s t r u s , p o r t i o n s of the v a g i n a l e p i t h e l i u m were c o r n i f i e d and the m a j o r i t y of the e p i t h e l i a l c e l l s p r e s e n t i n the smears had a p a r t i a l l y c o r n i f i e d , squamous appearance. S u p e r f i c i a l c e l l s , s i m i l a r i n appearance t o i n t e r m e d i a t e c e l l s but w i t h p y k n o t i c n u c l e i , o c c u r r e d o c c a s i o n a l l y and the number of e o s i n o p h i l i c c e l l s g r a d u a l l y i n c r e a s e d . As e s t r u s approached, the number of squamous e p i t h e l i a l c e l l s i n c r e a s e d w h i l e o t h e r c e l l t y p e s d e c l i n e d . Changes between a n e s t r u s and e s t r u s o c c u r r e d s l o w l y and no smear d e f i n i t i v e of the p r o e s t r o u s p e r i o d c o u l d be i d e n t i f i e d . D u r i n g e s t r u s the v a g i n a l e p i t h e l i u m c o n s i s t e d of 10-20 l a y e r s of f l a t t e n e d , c o r n i f i e d c e l l s which were sloughed i n t o the lumen ( P l a t e V I , F i g . 3 3 ) . The v a g i n a l smear was dominated 1 02 by t h e s e c o r n i f i e d c e l l s which were l a r g e (31-38 A i m ) , a n g u l a r and c o m p r i s e d 90-100% of the c e l l s p r e s e n t ( P l a t e V I , F i g . 36). A l t h o u g h most of the c o r n i f i e d c e l l s examined e a r l y i n e s t r u s c o n t a i n e d p a l e s t a i n i n g or p y k n o t i c n u c l e i 8.4 ± 0.3 Aim i n d i a m e t e r , c e l l s w i t h o u t n u c l e i were abundant d u r i n g l a t e e s t r u s i n most f e m a l e s . However, the presence of n u c l e a t e d and non-n u c l e a t e d c o r n i f i e d c e l l s v a r i e d g r e a t l y among d i f f e r e n t c y c l e s . D u r i n g the e a r l y s t a g e s of e s t r u s the v a g i n a l smears c o n t a i n e d c o r n i f i e d c e l l s o n l y . However, as e s t r u s proceeded the smears were l e s s c o n s i s t e n t and low numbers of l e u k o c y t e s were p r e s e n t i n many specimens. The presence of l e u k o c y t e s a l s o v a r i e d among smears taken h o u r l y , or d a i l y , from the same o t t e r d u r i n g e s t r u s . Low numbers of l e u k o c y t e s appeared t o be normal d u r i n g the e s t r o u s p e r i o d of the r i v e r o t t e r and were not c o n f i n e d t o the end of e s t r u s . V a g i n a l smears c o n t a i n i n g l a r g e q u a n t i t i e s of l e u k o c y t e s , mucus and the reappearance of i n t e r m e d i a t e c e l l s o c c u r r e d d u r i n g m e t e s t r u s ( P l a t e V I , F i g . 37). L e u k o c y t e s were the dominant c e l l type p r e s e n t (mean = 70%, range = 38.5-83.9%) and the number of i n t e r m e d i a t e c e l l s i n c r e a s e d as the c o r n i f i e d c e l l s d e c r e a s e d i n abundance. The m e t e s t r o u s p e r i o d c o n t i n u e d f o r t h r e e t o s i x weeks a f t e r which a t y p i c a l a n e s t r o u s smear rea p p e a r e d . I f mating o c c u r r e d , t h e r e was an i n f l u x of c o r n i f i e d c e l l s i n t o the v a g i n a l smear and a f t e r two t o t h r e e weeks the c o r n i f i e d v a g i n a l e p i t h e l i u m was c o m p l e t e l y sloughed o f f . For the remainder of pregnancy, the e p i t h e l i u m appeared s i m i l a r t o 1 03 t h a t d u r i n g a n e s t r u s . S i m i l a r l y , a f t e r the i n i t i a l p e r i o d of c o r n i f i e d smears, the v a g i n a l smears of pregnant o t t e r s c o u l d not be d i s t i n g u i s h e d from th o s e of a n e s t r o u s f e m a l e s . They c o n s i s t e d p r i m a r i l y of i n t e r m e d i a t e c e l l s , t h e o c c a s i o n a l p a r a b a s a l c e l l , low numbers of l e u k o c y t e s and v a r i a b l e amounts of mucus. To determine i f the smears a c c u r a t e l y r e f l e c t the degree of o v a r i a n a c t i v i t y p r e s e n t , the o v a r i e s were removed from c a p t i v e o t t e r s and compared w i t h t h e i r v a g i n a l smears. Due t o the l i m i t e d number of females a v a i l a b l e , s i n g l e specimens from each of the f o u r s t a g e s of the e s t r o u s c y c l e were examined. P r e o v u l a t o r y f o l l i c l e s were p r e s e n t o n l y i n the o v a r i e s of the a n i m a l s e x h i b i t i n g the e s t r o u s smears, w h i l e l a r g e c o r p o r a a t r e t i c a were found i n the female c l a s s i f i e d as m e t e s t r u s . The o v a r i e s of the a n i m a l s showing a n e s t r u s and p r o e s t r u s smears c o n t a i n e d Stage I I and Stage V f o l l i c l e s r e s p e c t i v e l y i n d i c a t i n g t h a t the r e p r o d u c t i v e s t a t e of each female was c o r r e c t l y i d e n t i f i e d by the v a g i n a l smear. The amount of mucus i n the v a g i n a l smears, the s w e l l i n g of the v u l v a and the p r e s ence of b l o o d i n the v a g i n a were a l s o m o n i t o r e d d u r i n g the e s t r o u s c y c l e . A l l t h r e e c o n d i t i o n s were found t o be l o o s e l y c o r r e l a t e d w i t h the a n n u a l c y c l e but were too v a r i a b l e t o be used w i t h any c o n f i d e n c e as i n d i c a t o r s of e s t r u s . The v a g i n a l smears were u s u a l l y d r y d u r i n g a n e s t r u s and m o i s t d u r i n g e s t r u s or m e t e s t r u s . However, the amount of mucus p r e s e n t d u r i n g e s t r u s v a r i e d g r e a t l y and smears c o n t a i n i n g l a r g e amounts of mucus o c c u r r e d throughout the c y c l e . The appearance 104 of b l o o d i n the v a g i n a was s i m i l a r l y not c o n f i n e d t o the e s t r o u s p e r i o d . The s i z e of the v u l v a v a r i e d d u r i n g the e s t r o u s c y c l e and was u s u a l l y e n l a r g e d d u r i n g e s t r u s . However, the amount and d u r a t i o n of the s w e l l i n g v a r i e d g r e a t l y and no attempt was made t o q u a n t i f y the amount of s w e l l i n g o b s e r v e d . I I . The Annual C y c l e , R e p r o d u c t i v e Rates and S e x u a l M a t u r i t y A. Annual r e p r o d u c t i v e c y c l e E x a m i n a t i o n of the r e p r o d u c t i v e organs of w i l d and c a p t i v e o t t e r s i n d i c a t e d t h a t i n B r i t i s h Columbia, e s t r u s o c c u r r e d from e a r l y March through mid-May, i m p l a n t a t i o n was the f o l l o w i n g F e b r u a r y and b i r t h s o c c u r r e d l a t e March and A p r i l . A l l of the a d u l t females (age c l a s s 2 and above) were s e x u a l l y mature w i t h age c l a s s 2 females b e i n g e i t h e r pregnant or e n t e r i n g t h e i r f i r s t b r e e d i n g season. No s i g n i f i c a n t d i f f e r e n c e s i n the anatomy or h i s t o l o g y of the r e p r o d u c t i v e organs were found among a d u l t age c l a s s e s or between parous and n u l l i p a r o u s a n i m a l s . O v a r i a n development appeared t o be p r o c e e d i n g n o r m a l l y i n a l l of the a d u l t s examined and no i n d i c a t i o n s of s e n i l i t y were obs e r v e d . R e p r o d u c t i v e a c t i v i t y was e x t r e m e l y low d u r i n g August; the o v a r i e s c o n t a i n e d few a n t r a l f o l l i c l e s ( F i g . 38) and the o v a r i a n i n t e r s t i t i a l t i s s u e and u t e r u s were i n a c t i v e . By e a r l y w i n t e r (November and December) f o l l i c u l a r growth had o c c u r r e d and a c t i v e i n t e r s t i t i a l c e l l s and numerous Stage I I and I I I f o l l i c l e s were p r e s e n t i n the o v a r i e s . F o l l i c u l a r development 105 had proceeded as f a r as Stage V i n a few a n i m a l s and the mean diameter of the l a r g e s t f o l l i c l e per ovary had r i s e n from 250 um i n August t o 571 ± 31.2 um i n November and December ( F i g . 3 9 ) . A c t i v i t y i n c r e a s e d r a p i d l y d u r i n g January and by February a l l of the o v a r i e s c o n t a i n e d f o l l i c l e s l a r g e r than 600 /xm. U t e r i n e a c t i v i t y had a l s o i n c r e a s e d d u r i n g the w i n t e r and e x t e n s i v e development of the endometrium and the u t e r i n e g l a n d s was p r e s e n t by F e b r u a r y . P r e o v u l a t o r y f o l l i c l e s were p r e s e n t i n the o v a r i e s of a n u l l i p a r o u s female c o l l e c t e d on March 5 and l a r g e f o l l i c l e s were v i s i b l e i n a l l of the o v a r i e s sampled through e a r l y May ( F i g . 3 8). The presence of f r e s h c o r p o r a l u t e a i n a specimen t r a p p e d on May 17 i n d i c a t e d t h a t i t had r e c e n t l y bred w h i l e f o l l i c l e s l a r g e r than Stage 2 were not found i n o v a r i e s c o l l e c t e d i n l a t e May. A l t h o u g h the sample s i z e was s m a l l d u r i n g the s p r i n g , t h i s i n d i c a t e s t h a t the b r e e d i n g season of o t t e r s i n B r i t i s h Columbia began i n e a r l y March and c o n t i n u e d through u n t i l e a r l y or mid May. E s t r o u s specimens c o l l e c t e d l a t e i n the b r e e d i n g season c o n t a i n e d f r e s h c o r p o r a a l b i c a n t i a w h i l e no i n d i c a t i o n s of r e c e n t p r e g n a n c i e s were seen i n samples c o l l e c t e d e a r l i e r i n the b r e e d i n g season. T h i s suggests t h a t p a r t u r i e n t o t t e r s may e n t e r e s t r u s l a t e r than females which were not pregnant the p r e v i o u s y e a r . The e s t r o u s p e r i o d s o b s e r v e d among c a p t i v e o t t e r s agree w i t h the e s t i m a t e of the b r e e d i n g season based on samples from w i l d o t t e r s and are d e s c r i b e d i n d e t a i l l a t e r . The o v a r i e s of one female (F6) which had e n t e r e d e s t r u s i n e a r l y March, 106 F i g u r e 38. Annual changes i n the p e r c e n t a g e of a d u l t o v a r i e s c o n t a i n i n g maximum f o l l i c u l a r d i a m e t e r of v a r i o u s s t a g e s . F o l l i c u l a r d i a m e t e r s of each s t a g e a r e : Stage I <250 / x m , Stage I I 250-599 M m , Stage I I I 600-799 M m , Stage IV 800-999 M m , Stage V 1000-1499 M m , Stage VI >1500 M m . P r e o v u l a t o r y f o l l i c l e s a r e i n Stage V I . n = number of p a i r s of o v a r i e s examined. 107 100-75. 50 . 2 5 . 100. 75. O CD D CT Q) 5 0 . 25J 100.. 75 . 5 0 . 25 . 1 II August n--1 n i 1 1 November December n--27 ~i 1 January n=44 iv v V S t a g e February n--Tl i J r March April n=2 May n=3 1 1 1 IV V VI 108 F i g u r e 3 9 . Monthly v a r i a t i o n s i n the mean maximum f o l l i c u l a r d i a m eter of r i v e r o t t e r s . The samples i n May a r e d i v i d e d i n t o e a r l y and l a t e p e r i o d s t o i n d i c a t e the d i f f e r e n c e s which occur as the b r e e d i n g season ends. V a l u e s e x p r e s s e d as mean + SEM. Sample s i z e i n d i c a t e d above the b a r s . 1 10 contained preovulatory f o l l i c l e s when examined on May 5 and with one exception, estrus in the captive otters began in early or mid March and continued u n t i l mid May. After the breeding season, the ovaries of wild otters quickly became inactive i f ovulation did not occur. If mating had occurred, however, the oocyte was ovulated, f e r t i l i z e d , formed a blastocyst and entered a period of delayed implantation which lasted approximately nine to 11 months, depending upon when mating occurred. During t h i s period, the blastocysts were unattached in the upper portion of the cornua and the corpora lutea and uteri showed signs of low levels of a c t i v i t y . The preimplantation period continued u n t i l early February when the corpora lutea enlarged and the u t e r i and l u t e a l c e l l s increased in a c t i v i t y . Corpora lutea of postimplantation were present in the ovaries of a l l of the samples col l e c t e d a f t e r the f i r s t week in February although they were absent before t h i s period. The mean date of implantation of four samples with implanted embryos was February 15 ± 2 days. A somewhat later date of implantation was indicated in a f i f t h specimen c o l l e c t e d on February 20 in which postimplantation corpora lutea were present although implanted embryos and uterine swellings were absent. In this animal activation of the corpora lutea probably occurred quite recently and implantation would have occurred near the end of February. Thus, these data indicate that implantations occur over a r e l a t i v e l y short period of time during the month of February and possibly into early March in B r i t i s h Columbia, although the t o t a l extent of the implantation period cannot be 111 d e t e r m i n e d due t o the s m a l l number of samples c o l l e c t e d . B i r t h s a r e l i k e l y t o occur d u r i n g A p r i l i n B r i t i s h Columbia a l t h o u g h no specimens were a v a i l a b l e from the l a t e p o s t i m p l a n t a t i o n p e r i o d of pregnancy. However, a female which had a p p a r e n t l y g i v e n b i r t h r e c e n t l y was c o l l e c t e d on A p r i l 12. She had f r e s h c o r p o r a a l b i c a n t i a , a s w o l l e n u t e r u s , p l a c e n t a l s c a r s , e n l a r g e d t e a t s and w e l l d e v e l o p e d mammary g l a n d s . T h i s female was the o n l y a d u l t sampled d u r i n g A p r i l and no i n d i c a t i o n s of r e c e n t b i r t h s were found i n any o t h e r samples. B. Pregnancy r a t e s The pregnancy r a t e of o t t e r s i n my sample was h i g h , as i n d i c a t e d by the presence of c o r p o r a l u t e a i n the o v a r i e s of 82.5% of a l l a d u l t females (Table X I I I ) . The pregnancy r a t e was o b s e r v e d t o v a r y w i t h age, however; o n l y 55% of the age c l a s s 2 females were pregnant w h i l e 90.9% of the o l d e r females had bred (Table X I I I ) . There were no d i f f e r e n c e s between age c l a s s e s above two and the p e r c e n t a g e of females pregnant d i d not v a r y among the d i f f e r e n t y e a r s of the s t u d y . C. O v u l a t i o n r a t e s Based on the number of c o r p o r a l u t e a p r e s e n t , the average number of f o l l i c l e s o v u l a t e d per female o t t e r was 2.9 ± 0.06 (Table X I I I ) . The mean number of c o r p o r a l u t e a i n c r e a s e d s l i g h t l y w i t h age but the d i f f e r e n c e s were not s i g n i f i c a n t . There was no e v i d e n c e of o v u l a t i o n s d u r i n g the p r e i m p l a n t a t i o n T a b l e X I I I . P r e g n a n c y r a t e s and mean numbers o f c o r p o r a l u t e a , b l a s t o c y s t s and i m p l a n t e d embryos o f a d u l t r i v e r o t t e r s i n B r i t i s h C o l u m b i a 1 9 7 5 - 1 9 8 1 . n = number o f f e m a l e s . Age C l a s s Number o f F e m a l e s Number P r e g n a n t P e r c e n t P r e g n a n t C o r p o r a l u t e a mean + SEM (n) B l a s t o c y s t s mean + SEM (n) I m p l a n t e d Embryos mean +_ SEM (n ) 2 20 11 55 2 . 8 0 + 0 . 1 2 (11) 2 . 0 (1) 3 21 19 9 0 . 5 2 . 8 8 + 0 . 1 8 (16) 3 . 0 + 0 . 0 (3 ) 2 . 0 (1 ) 4 18 16 8 8 . 9 2 . 9 4 + 0 .11 (16) 3 . 0 (1 ) 2 . 5 + 0 . 5 (2 ) 5 12 10 8 3 . 3 2 . 8 0 + 0 . 1 3 (10) 2 . 0 + 0 . 0 (3 ) 6 7 7 100 2 . 8 6 + 0 . 1 4 (7) 3 . 0 (1) 7 4 4 100 3 . 0 0 + 0 . 0 0 (4 ) 3 . 0 (1 ) 8 3 3 100 3 . 3 0 + 0 . 3 3 (3) 11 1 1 100 3 . 0 (1) T o t a l 3 86 71 8 2 . 5 2 . 9 0 + 0 . 6 8 (68) 2 . 6 + 0 . 2 (9) 2 . 5 + 0 . 2 9 (4) T o t a l b 66 60 9 0 . 9 2 .91 + 0 . 0 7 (57) a - Age c l a s s e s 2 and a b o v e . b - Age c l a s s e s 3 and a b o v e . 1 13 p e r i o d and the average number of c o r p o r a d i d not v a r y d u r i n g pregnancy. A l t h o u g h no ob v i o u s d i f f e r e n c e s i n f o l l i c u l a r a c t i v i t y were noted between c o n t r a l a t e r a l o v a r i e s , the number of c o r p o r a l u t e a p r e s e n t i n the r i g h t o v a r i e s were s i g n i f i c a n t l y g r e a t e r than i n the l e f t (1.71 ± 0.1 vs 1.19 ± 0.1, p < . 0 l ) . Of the 15 females i n which c o r p o r a l u t e a were c o n f i n e d t o a s i n g l e o v a r y , 80% had c o r p o r a p r e s e n t i n the r i g h t ovary o n l y . I t i s unknown i f t h i s asymmetry has any s i g n i f i c a n c e . D. B l a s t o c y s t s The mean number of b l a s t o c y s t s r e c o v e r e d per pregnant o t t e r was 2.56 (Table X I I I ) which c o r r e s p o n d s c l o s e l y t o the mean number of c o r p o r a l u t e a i n the o t t e r s from which they were r e c o v e r e d ( 2 . 8 9 ) . T h i s e s t i m a t e was based on n i n e a n i m a l s i n which I was r e a s o n a b l y c o n f i d e n t of r e c o v e r i n g a l l of the b l a s t o c y s t s p r e s e n t . In s i x , the number of b l a s t o c y s t s r e c o v e r e d agreed w i t h the number of c o r p o r a l u t e a w h i l e i n the o t h e r t h r e e b l a s t o c y s t c o u n t s were l o w e r , p o s s i b l y due t o i n t r a -u t e r i n e m o r t a l i t y , d e g e n e r a t i o n of the b l a s t o c y s t s b e f o r e c o l l e c t i o n or l o s s d u r i n g c o l l e c t i o n . B l a s t o c y s t s were always found i n f r e s h l y caught females c o n t a i n i n g c o r p o r a l u t e a a l t h o u g h they were r a r e l y seen i n the t r a p p e r caught samples due t o the poor s t a t e of p r e s e r v a t i o n . No d i f f e r e n c e s were observed between the r e p r o d u c t i v e organs of o t t e r s w i t h r e c o v e r e d and assumed b l a s t o c y s t s . 1 14 E. Implanted embryos I m p l a n t a t i o n s i t e s were p r e s e n t i n f o u r o t t e r s and based on thes e a n i m a l s , the mean l i t t e r s i z e was 2.5 ± 0.29 (Table X I I I ) . In t h r e e o t t e r s the number of i m p l a n t a t i o n s i t e s agreed w i t h the number of c o r p o r a l u t e a p r e s e n t i n the o v a r i e s . In the f o u r t h , two i m p l a n t e d embryos were p r e s e n t w h i l e the o v a r i e s c o n t a i n e d two c o r p o r a l u t e a of average s i z e and a t h i r d , s m a l l e r c o r p u s . These s m a l l c o r p o r a may be an a c c e s s o r y c o r p o r a l u t e a a l t h o u g h no d e g e n e r a t i n g oocyte or zona p e l l u c i d a were v i s i b l e . The c l o s e c o r r e l a t i o n between the number of c o r p o r a l u t e a and the number of i m p l a n t e d embryos i n these a n i m a l s i n d i c a t e t h a t i n t r a - u t e r i n e m o r t a l i t y may be low i n B r i t i s h Columbia. F. S e x u a l m a t u r i t y My r e s u l t s show t h a t i n B r i t i s h Columbia, the age of s e x u a l m a t u r i t y v a r i e d among i n d i v i d u a l s . Of the 12 a n i m a l s i n age c l a s s 1, the o v a r i e s of two o t t e r s were i n a c t i v e and l a c k e d a n t r a l f o l l i c l e s , w h i l e t h r e e o t h e r s c o n t a i n e d l a r g e f o l l i c l e s , a c t i v e (Type I I ) i n t e r s t i t i a l c e l l s , and w e l l d e v e l o p e d u t e r i . These t h r e e o t t e r s , and p o s s i b l y seven o t h e r s which showed r e l a t i v e l y a c t i v e o v a r i e s , appeared t o be r e p r o d u c t i v e l y a c t i v e and would have been c a p a b l e of b r e e d i n g a t the end of age c l a s s 1. A l l of the o t t e r s examined i n t h i s study had a t t a i n e d s e x u a l m a t u r i t y by the end age c l a s s 2. The o v a r i e s c o u l d not be d i s t i n g u i s h e d from o l d e r a d u l t s on the b a s i s of f o l l i c u l a r 115 development or s i z e ( T a b l e V I I ) . No r e p r o d u c t i v e a c t i v i t y was o b s e r v e d among j u v e n i l e o t t e r s (age c l a s s 0 ) . V a r i a t i o n s i n the age of s e x u a l m a t u r i t y was a l s o o b s e r v e d among t h e c a p t i v e o t t e r s . A l t h o u g h the t h r e e immature o t t e r s d i d not show c o m p l e t e l y c o r n i f i e d v a g i n a l smears d u r i n g e s t r u s u n t i l age c l a s s 3, two (F2, F7) d i d e x h i b i t p a r t i a l e s t r u s d u r i n g age c l a s s 2. The degree of f o l l i c u l a r development which o c c u r r e d d u r i n g these p e r i o d s i s unknown a l t h o u g h i n F2 p a r t i a l e s t r u s was accompanied by a r i s e i n e s t r a d i o l c o n c e n t r a t i o n s ( d e s c r i b e d below) which may i n d i c a t e e x t e n s i v e o v a r i a n a c t i v i t y . A s i m i l a r r i s e i n e s t r a d i o l was not observed i n F7 d u r i n g p a r t i a l e s t r u s and the t h i r d immature (F6) showed no e v i d e n c e of s e x u a l m a t u r i t y u n t i l she was a year o l d e r . I I I . E n d o c r i n o l o g y of the Female R e p r o d u c t i v e C y c l e A. R e p r o d u c t i v e c y c l e of c a p t i v e females Of t h e f i v e c a p t i v e o t t e r s examined, one (F7) was immature and two o t h e r s (F2, F6) a t t a i n e d s e x u a l m a t u r i t y d u r i n g t h e i r second y e a r i n c a p t i v i t y . A t o t a l of n i n e complete e s t r o u s c y c l e s were m o n i t o r e d i n f o u r a d u l t s . Three of these c y c l e s o c c u r r e d under e x p e r i m e n t a l c o n d i t i o n s and w i l l be d i s c u s s e d i n Chapter I V . H i s t o l o g i c a l e x a m i n a t i o n of r e p r o d u c t i v e organs and changes obser v e d i n v a g i n a l smears i n d i c a t e t h a t the a n n u a l c y c l e of r e p r o d u c t i v e a c t i v i t y i n c a p t i v e a n i m a l s was s i m i l a r t o t h a t of w i l d o t t e r s . The o v a r i e s and u t e r i of c a p t i v e o t t e r s appeared 1 16 normal i n a l l r e s p e c t s and were s i m i l a r t o t h o s e d e s c r i b e d i n w i l d f e m a l e s . Stages of f o l l i c u l a r development o b s e r v e d i n the o v a r i e s were c o n s i s t e n t w i t h w i l d o t t e r s a t the same time of year and the m a j o r i t y of e s t r o u s p e r i o d s o c c u r r e d between e a r l y March and mid-May which agrees w i t h the b r e e d i n g season e s t i m a t e d from w i l d specimens. J_. E s t r u s W ith the e x c e p t i o n of one c y c l e which began i n e a r l y F e b r u a r y (F5, 1980), e s t r o u s smears of o t t e r s under n a t u r a l p h o t o p e r i o d s were f i r s t observed between F e b r u a r y 25 and March 13 ( F i g . 4 0 ) . The mean f i r s t date of e s t r u s based on these s i x c y c l e s was F e b r u a r y 26 ± 6 days. E s t r u s ended i n one a n i m a l (F2, 1980) on A p r i l 11 a l t h o u g h i n most i t c o n t i n u e d u n t i l e a r l y or mid May. Three of the females (F2, F3, F6) e x h i b i t e d a s i n g l e e s t r u s o n l y w h i l e F5 had a second p e r i o d of e s t r u s i n each of the t h r e e y e a r s she was m o n i t o r e d . Based on the n i n e p e r i o d s of e s t r u s r e c o r d e d , the d u r a t i o n averaged 5.5 ± 0.7 weeks (range = 3-8 weeks). When a second e s t r u s o c c u r r e d , i t f o l l o w e d a 2-3 week p e r i o d of m e t e s t r u s and l a s t e d f o r 3-5 weeks. In each case the second e s t r o u s p e r i o d was 2-3 weeks s h o r t e r than the f i r s t and was c h a r a c t e r i z e d by v a g i n a l smears c o n t a i n i n g a g r e a t e r number of l e u k o c y t e s than i n the smear of the f i r s t or s i n g l e e s t r u s . 1 17 F i g u r e 40. P e r i o d s d u r i n g which e s t r o u s ( i — i ) or p a r t i a l e s t r o u s 0 0 v a g i n a l smears were o b t a i n e d from c a p t i v e r i v e r o t t e r s under n a t u r a l p h o t o p e r i o d s . M e t e s t r o u s smears a r e i n d i c a t e d by • . The o v a r i e c t o m y of F6 i s i n d i c a t e d by X. See t e x t f o r d e s c r i p t i o n of smears. 118 F2 1979 F6 1981 F3 1981 F2 1980 F5 1980 F5 1980 F5 1979 H • H H • H ~i 1 1 1 r J F AA A M Month 119 2. M a t i n g A l t h o u g h a l l of the a d u l t females appeared t o e n t e r e s t r u s n o r m a l l y i n each year of the s t u d y , mating o c c u r r e d i n the f i r s t y ear of the study o n l y . Both of the a d u l t females p r e s e n t i n 1979 were i n v o l v e d i n mating a t t e m p t s d u r i n g t h e i r r e s p e c t i v e e s t r o u s p e r i o d s . In l a t e March, and a g a i n from l a t e A p r i l t h r o ugh e a r l y May, a male was r e p e a t e d l y observed a t t e m p t i n g t o mount F5. I n t r o m i s s i o n was not observed and t h e r e was no e v i d e n c e t h a t o v u l a t i o n or pregnancy o c c u r r e d (see p r o g e s t e r o n e b e l o w ) . The attempted matings o c c u r r e d d u r i n g both of the p e r i o d s i n which c o r n i f i e d v a g i n a l smears were observed and i n the i n t e r v e n i n g m e t e s t r o u s p e r i o d she was not approached by any male. M a t i n g s c a r s were obser v e d on the neck of the second female (F3) when examined on A p r i l 29. She was i n e s t r u s a t the time and a l t h o u g h no mating was o b s e r v e d , she had not been under c l o s e o b s e r v a t i o n . F o l l o w i n g the o b s e r v a t i o n of mating s c a r s , t h i s female e x h i b i t e d hormonal changes c h a r a c t e r i s t i c of pregnancy i n m u s t e l i d s ( d e s c r i b e d b e l o w ) . However, she f a i l e d t o g i v e b i r t h the f o l l o w i n g s p r i n g a l t h o u g h she had s w o l l e n t e a t s , and an e n l a r g e d v a g i n a l opening i n e a r l y March 1980. No mating a t t e m p t s were obser v e d i n subsequent y e a r s a l t h o u g h t h e r e i s e v i d e n c e t h a t F3 became pregnant i n 1981. In A p r i l p r o g e s t e r o n e l e v e l s rose and an i n c r e a s e o c c u r r e d i n the number of c o r n i f i e d c e l l s i n the v a g i n a l smears s i m i l a r t o t h a t observed f o l l o w i n g m a t i n g . M a t i n g may have o c c u r r e d u n n o t i c e d or o v u l a t i o n may have been caused by the t a k i n g of v a g i n a l smears. D u r i n g t h i s p e r i o d a s e r i e s of d a i l y and h o u r l y smears 120 were taken from F3 w h i l e she was l i g h t l y a n e s t h e t i z e d w i t h carbon d i o x i d e but not ketamine as on p r e v i o u s o c c a s i o n s . The female escaped the f o l l o w i n g summer and the f a t e of t h i s pregnancy i s not known. 3. P a r t i a l e s t r u s Of the t h r e e immature females examined, changes were observed i n the v a g i n a l smears of two (F7, F2) d u r i n g the b r e e d i n g season. In March 1979 the v a g i n a l smears of F2 changed from t h a t of a t y p i c a l immature a n i m a l t o become s i m i l a r t o those observed from a d u l t s d u r i n g p r o e s t r u s ( F i g . 40). I n t e r m e d i a t e c e l l s were abundant and the o c c a s i o n a l c o r n i f i e d c e l l was p r e s e n t . A f u l l e s t r o u s smear was never obse r v e d and i n mid-May the 'immature' smear r e t u r n e d . T h i s smear c o n t i n u e d u n t i l the next s p r i n g when a t y p i c a l a d u l t e s t r o u s c y c l e o c c u r r e d . The p e r i o d of m o d i f i e d smear, r e f e r r e d t o as p a r t i a l e s t r u s , l a s t e d seven weeks and was accompanied by changes i n serum e s t r a d i o l c o n c e n t r a t i o n s d e s c r i b e d below. F7 underwent a t h r e e week p e r i o d of p a r t i a l e s t r u s i n January 1981 w h i l e i n v o l v e d i n an e x p e r i m e n t a l l y i n c r e a s e d p h o t o p e r i o d (Chapter I V ) , Both p e r i o d s of m o d i f i e d v a g i n a l smears o c c u r r e d w h i l e accompanying a d u l t s were i n e s t r u s a l t h o u g h the amount of f o l l i c u l a r growth which o c c u r r e d i n the immature o t t e r s d u r i n g p a r t i a l e s t r u s i s not known. A l l t h r e e immature females were e n t e r i n g age c l a s s 2, an age a t which s e x u a l m a t u r i t y i s a t t a i n e d i n some w i l d o t t e r s . 121 B. E s t r a d i o l c o n c e n t r a t i o n s K Nonpregnant a d u l t s I n d i v i d u a l e s t r a d i o l c o n c e n t r a t i o n s were h i g h l y v a r i a b l e t hroughout the study ( F i g . 41) and e s t r a d i o l p r o f i l e s were s i g n i f i c a n t l y d i f f e r e n t (p<0.00l) i n the f i r s t year of the study (June 1978 - May 1979) than i n the l a s t two (June 1979 - August 1981). L e v e l s ranged from 3-120 pg/ml d u r i n g the f i r s t y e a r , but d i d not exceed 33 pg/ml i n subsequent y e a r s . In the f i r s t y e a r , e s t r a d i o l c o n c e n t r a t i o n s of a d u l t females (F3, F5) were low d u r i n g the f a l l and i n c r e a s e d d r a m a t i c a l l y d u r i n g November and December ( F i g s . 41 and 42 ) . F o l l o w i n g a b r i e f d e c l i n e , second r i s e o c c u r r e d i m m e d i a t e l y p r i o r t o , or d u r i n g , e s t r u s . A t h i r d , l o w e r , r i s e i n e s t r a d i o l o c c u r r e d l a t e r i n the e s t r o u s p e r i o d i n both a d u l t s ( F i g . 4 1 ) . E s t r u s e s t r a d i o l c o n c e n t r a t i o n s r e t u r n e d t o b a s e l i n e l e v e l s at the end of e s t r u s . A l t h o u g h l a r g e i n c r e a s e s i n e s t r a d i o l were not observed i n the subsequent y e a r s , the p a t t e r n of change i n the l e v e l s was s i m i l a r t o t h a t of the f i r s t ( F i g s . 41 and 42 ) . E s t r a d i o l c o n c e n t r a t i o n s were low d u r i n g summer and f a l l (3-18 pg/ml) and rose s l i g h t l y i n November and December. L e v e l s rose a g a i n s l i g h t l y d u r i n g the b r e e d i n g season. Over the t h r e e y e a r s of the s t u d y , the mean e s t r a d i o l c o n c e n t r a t i o n i n December (30.3 ± 6.2 pg/ml) was s i g n i f i c a n t l y h i g h e r (p<0.05) than those of a l l othe r months w i t h the e x c e p t i o n of November and February ( F i g . 4 2 ) . D u r i n g the peak of r e p r o d u c t i v e a c t i v i t y i n A p r i l , the e s t r a d i o l l e v e l s (15.5 ± 2.1 pg/ml) were a l s o s i g n i f i c a n t l y 122 F i g u r e 41. I n d i v i d u a l e s t r a d i o l p r o f i l e s of e x p e r i m e n t a l and n o n e x p e r i m e n t a l immature (• •) and a d u l t (• •) o t t e r s from September 1978 t h r o u g h August 1981. S t i p p l i n g i n d i c a t e s p e r i o d s of e s t r u s and p a r t i a l e s t r u s . F3 was s u b j e c t e d t o i n c r e a s e d p h o t o p e r i o d s from Dec. 21 1978 - May 11 1979 and from Dec. 9 1979 - Aug. 26 1980. F2 and F7 were s u b j e c t e d t o i n c r e a s e d p h o t o p e r i o d s from Nov. 4 1980 - June 17 1981. E x p e r i m e n t a l c o n d i t i o n s a r e d e s c r i b e d i n Chapter IV. Arrows i n d i c a t e (1) when mating o c c u r r e d and (2) when the o v a r i e s were removed from F2 and F6. F2 1 I I *1 'I 1 1 1 1 "T— i ' - f ' j ' I 1 1 T I I F 3 "I ' i — i " i — i — r • i i r i i"T i r*i i r -100- F5 1 1 1 1 1 i i "i 1 1 1 i F6 1 1 1 1 1 ' ' i • • i 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 F7 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I S J M S J M S J M A 1 9 7 8 1 9 7 9 1 9 8 0 1 9 8 1 M o n t h 124 F i g u r e 42. Mean monthly c o n c e n t r a t i o n s of e s t r a d i o l f o r r i v e r o t t e r s under n a t u r a l p h o t o p e r i o d d u r i n g A) 1978-79, B) 19 7 9 - 8 1 , and C) a l l y e a r s p o o l e d . V a l u e s e x p r e s s e d as mean + SEM. Sample s i z e i n d i c a t e d below the b a r s . Month 1 26 g r e a t e r ( p < . 0 5 ) than th o s e i n J u l y and September. Mean e s t r a d i o l l e v e l s were g r e a t e r d u r i n g e s t r u s than d u r i n g a n e s t r u s i n each y e a r of the stu d y (Table X I V ) . A l t h o u g h no surges i n e s t r a d i o l were observed d u r i n g the l a s t two y e a r s , f o l l i c u l a r growth d i d oc c u r among c a p t i v e f e m a l e s . P r e o v u l a t o r y f o l l i c l e s were p r e s e n t i n the o v a r i e s of F6 d u r i n g e s t r u s 1981 a l t h o u g h peak e s t r a d i o l l e v e l s (20-29 pg/ml) were o n l y s l i g h t l y g r e a t e r than t h a t observed d u r i n g the r e s t of the year ( F i g . 4 1 ) . The o v a r i e s of F2, removed a f t e r e s t r u s 1981, a l s o appeared t o have undergone normal f o l l i c u l a r growth. Large c o r p o r a a t r e t i c a (>750 urn) and s m a l l a n t r a l f o l l i c l e s were p r e s e n t even though e s t r a d i o l c o n c e n t r a t i o n s d i d not exceed 25 pg/ml d u r i n g development and e s t r u s ( F i g . 4 1 ) . E s t r a d i o l c o n c e n t r a t i o n s of o v a r i e c t o m i z e d females were s i m i l a r t o those d u r i n g a n e s t r u s (Table X I V ) . 2. Pregnant o t t e r s E s t r a d i o l c o n c e n t r a t i o n s were low throughout most of the p r e i m p l a n t a t i o n p e r i o d a l t h o u g h a s l i g h t , but s t a t i s t i c a l l y i n s i g n i f i c a n t , r i s e o c c u r r e d d u r i n g J u l y and August ( F i g . 43, Ta b l e X V ). - Serum l e v e l s a l s o i n c r e a s e d s l i g h t l y i n December a f t e r the p h o t o p e r i o d had been e x p e r i m e n t a l l y i n c r e a s e d , but b e f o r e a r i s e i n p r o g e s t e r o n e a s s o c i a t e d w i t h i m p l a n t a t i o n o c c u r r e d . F o l l o w i n g i m p l a n t a t i o n , mean e s t r a d i o l l e v e l s were s i m i l a r t o those seen d u r i n g the p r e i m p l a n t a t i o n p e r i o d ( F i g . 43, T a b l e XV). T a b l e X I V . Serum e s t r a d i o l c o n c e n t r a t i o n s ( p g / m l ) o f a l l c a p t i v e r i v e r o t t e r s i n c l u d i n g e x p e r i m e n t a l a n i m a l s . A l l v a l u e s a r e means + SEM. R e p r o d u c t i v e P o o l e d Y e a r s 1978 -79 1979 -81 S t a t e N Mean N Mean N Mean A n e s t r u s 109 1 3 . 3 + 1.3»>c 16 3 1 . 4 + 7 . 0 e 93 1 0 . 2 + 0 . 5 9 E s t r u s 46 1 8 . 4 + 2.7C 12 3 4 . 8 + 8 . 5 e 34 1 2 . 7 + 1 . 2 h Immature 64 9 . 9 + 0 . 8 d 5 6 . 4 + i.zf 59 1 0 . 2 + 0 . 8 9 h P a r i t a l e s t r u s 3 5 3 6 . 8 + 1 8 . 0 3 5 5 . 3 + 4 3 . 0 2 9 . 0 + 1.0 O v a r i e c t o m i z e d 10 1 0 . 4 + 1 .7bd 10 1 0 . 4 + 1 . 7 9 " a - No c o m p a r i s i o n s were made w i t h t h i s d a t a due t o t h e s m a l l samp le s i z e and ex t reme v a r i a t i o n . b , c , d , e , f , g , h - D i f f e r e n t s u p e r s c r i p t s w i t h i n a co lumn i n d i c a t e s i g n i f i c a n t l y d i f f e r e n t means , p < 0 . 0 5 . 128 F i g u r e 4 3 . E s t r a d i o l c o n c e n t r a t i o n s d u r i n g pregnancy i n a c a p t i v e r i v e r o t t e r . Arrow i n d i c a t e s when the p h o t o p e r i o d was e x p e r i m e n t a l l y i n c r e a s e d . I m p l a n t a t i o n was e s t i m a t e d t o occur near the end of December. D e t a i l s of e x p e r i m e n t a l c o n d i t i o n s a r e d e s c r i b e d i n Chapter IV. V a l u e s e x p r e s s e d as mean + SEM. Sample s i z e i n d i c a t e d next t o the b a r s . 129 20i N 1 • 1 1 1 — — , , — — , , N A M J J A S O N D J F Month 130 T a b l e X V . Serum e s t r a d i o l c o n c e n t r a t i o n s ( p g / m l ) o f a p r e g n a n t r i v e r o t t e r (F3 ) w h i c h f a i l e d t o g i v e b i r t h . A l l v a l u e s a r e means + SEM. Means a r e n o t s i g n i f i c a n t l y d i f f e r e n t . R e p r o d u c t i v e P o o l e d Y e a r s 1979-81 S t a t e N Mean N Mean A n e s t r u s E s t r u s P r e i m p l a n t a t i o n P o s t i m p l a n t a t i o n 31 1 1 . 6 + 1 . 8 12 2 4 . 0 + 7 . 0 18 9 . 3 + 0 . 6 7 9 . 3 + 1.0 23 7 . 8 + 0 . 5 5 1 1 . 0 + 2 . 0 18 9 . 3 + 0 . 6 7 9 . 3 + 1.0 131 3. Immature o t t e r s B a s a l e s t r a d i o l l e v e l s of immature o t t e r s were lower t h a n , or s i m i l a r t o those of a n e s t r o u s a d u l t s (Table XIV, F i g . 41). I n c r e a s e s i n e s t r a d i o l c o n c e n t r a t i o n s o c c u r r e d i n one female (F2) d u r i n g the f i r s t year of the study and peak l e v e l s of 85 pg/ml were observed d u r i n g p a r t i a l e s t r u s ( F i g . 4 1 ) . T h i s i n c r e a s e was s i m i l a r t o t h a t o b s e r v e d i n a d u l t s d u r i n g 1979 a l t h o u g h i t o c c u r r e d l a t e r i n the e s t r o u s p e r i o d and was not preceded by a r i s e the p r e v i o u s w i n t e r . An i n c r e a s e i n e s t r a d i o l l e v e l s was not a s s o c i a t e d w i t h p a r t i a l e s t r u s i n F7 ( F i g . 41). However, e s t r a d i o l c o n c e n t r a t i o n s were low i n a l l of the a n i m a l s at t h a t time and i n c r e a s e s a s s o c i a t e d w i t h f o l l i c u l a r growth were d i f f i c u l t t o d e t e c t . 4. W i l d o t t e r s As i n the c a p t i v e a n i m a l s , serum e s t r a d i o l c o n c e n t r a t i o n s of f i v e w i l d o t t e r s were low and v a r i e d g r e a t l y between i n d i v i d u a l s (Table X V I ) . A l l of the c o n c e n t r a t i o n s observed were w i t h i n the range of v a l u e s o b t a i n e d f o r c a p t i v e o t t e r s over the t h r e e y e a r s of the s t u d y . 132 T a b l e X V I . Serum e s t r a d i o l and p r o g e s t e r o n e c o n c e n t r a t i o n s o f w i l d r i v e r o t t e r s . Da te R e p r o d u c t i v e E s t r o g e n P r o g e s t e r o n e S t a t e ( p g / m l ) ( n g / m l ) 5 / 7 / 8 1 Immature (<1 y r ) 10 0 . 2 6 / 1 / 7 9 Immature (<1 y r ) 16 0 . 9 6 / 1 / 7 9 Immature (1 y r ) 19 0 . 9 6 / 1 / 7 9 P r e i m p l a n t a t i o n 13 6 . 2 6 / 1 / 7 9 P r e i m p l a n t a t i o n 33 4 . 7 T a b l e X V I I . Serum p r o g e s t e r o n e c o n c e n t r a t i o n s d u r i n g 1978 -79 o f a p r e g n a n t o t t e r (F3 ) w h i c h f a i l e d t o g i v e b i r t h . A l l v a l u e s e x p r e s s e d as mean + SEM. R e p r o d u c t i v e N P r o g e s t e r o n e S t a t e ( n g / m l ) Nonp regnan t 13 1.0 + 0 . 5 * P r e i m p l a n t a t i o n 8 2 . 4 + 0 . 3 0 b P o s t i m p l a n t a t i o n 8 1 3 . 2 4 + 2.7C a , b , c , , - D i f f e r e n t s u p e r s c r i p t s w i t h i n a co lumn i n d i c a t e s i g n i f i c a n t l y d i f f e r e n t means , p < 0 . 0 1 . 133 C. P r o g e s t e r o n e c o n c e n t r a t i o n s _1_. Immature and nonpregnant a d u l t s P r o g e s t e r o n e c o n c e n t r a t i o n s were determined d u r i n g one pregnant, f i v e nonpregnant a d u l t , and t h r e e immature r e p r o d u c t i v e c y c l e s . These r e s u l t s i n d i c a t e t h a t p r o g e s t e r o n e l e v e l s i n c r e a s e s i g n i f i c a n t l y d u r i n g the p r e i m p l a n t a t i o n and p o s t i m p l a n t a t i o n p e r i o d s of pregnancy whereas i n nonpregnant a d u l t s and immature o t t e r s p r o g e s t e r o n e l e v e l s remain r e l a t i v e l y c o n s t a n t . P r o g e s t e r o n e c o n c e n t r a t i o n s were low i n both nonpregnant and immature o t t e r s throughout the y e a r ( F i g . 44). S i g n i f i c a n t changes were not o b s e r v e d , but a s l i g h t i n c r e a s e i n the p r o g e s t e r o n e l e v e l s of nonpregnant a d u l t s d i d occur i n January and F e b r u a r y . Mean p r o g e s t e r o n e l e v e l s of nonpregnant a d u l t s (0.77 ± 0.09 ng/ml, n = 129) were h i g h e r and more v a r i a b l e than those of immatures (0.47 ± 0.04 ng/ml, n = 71, p<.01). The p r o g e s t e r o n e c o n c e n t r a t i o n s of F5 d i d not i n c r e a s e f o l l o w i n g r e p e a t e d mating a t t e m p t s i n 1979, i n d i c a t i n g t h a t o v u l a t i o n had not o c c u r r e d and she d i d not become p r e g n a n t . There was a l s o no s i g n i f i c a n t change i n mean p r o g e s t e r o n e c o n c e n t r a t i o n s (0.53 ± 0.13 ng/ml, n = 10) f o l l o w i n g the removal of the o v a r i e s and u t e r i from two females i n d i c a t i n g t h a t the r e p r o d u c t i v e organs were not the source of the b a s a l p r o g e s t e r o n e l e v e l s . The p r o g e s t e r o n e l e v e l s f o r i n d i v i d u a l o t t e r s are p r e s e n t e d i n Appendix 4. 134 F i g u r e 44. Mean p r o g e s t e r o n e c o n c e n t r a t i o n s of immature (O O) and nonpregnant (• •) o t t e r s under n a t u r a l p h o t o p e r i o d . V a l u e s e x p r e s s e d as means + SEM. Sample s i z e i n d i c a t e d next t o the b a r s . F i g u r e 45. P r o g e s t e r o n e c o n c e n t r a t i o n s i n F5 d u r i n g pregnancy. Arrows i n d i c a t e (1) when mating s c a r s were observed and (2) when the female was f i r s t s u b j e c t e d t o an i n c r e a s e d d a y l e n g t h . D e t a i l s of e x p e r i m e n t a l c o n d i t i o n s a r e d e s c r i b e d i n Chapter IV. E 1 0, 15. 2 to. CD CL Figure 44 i _ i _ i i i i — i — i — i — i — i — i — i — i — - i — , F M A M J J A S O N D J F M A Month Figure 45 F MAM'J'J'A'S' O'N'D'J' F' ' ' 1979 M A 1980 Month 136 2. Pregnant o t t e r s The serum p r o g e s t e r o n e c o n c e n t r a t i o n s of a s i n g l e pregnant o t t e r (F3) which f a i l e d t o g i v e b i r t h a re shown i n F i g . 45. P r i o r t o mating i n l a t e A p r i l 1979, p r o g e s t e r o n e l e v e l s were s i m i l a r t o those of nonpregnant o t t e r s (Table X V I I ) . From May t h r o u g h November however, p r o g e s t e r o n e c o n c e n t r a t i o n s i n c r e a s e d s i g n i f i c a n t l y (p<.0l) and ranged from 1.16 t o 3.90 ng/ml d u r i n g the e s t i m a t e d p r e i m p l a n t a t i o n p e r i o d . On December 9 F3 exposed t o an e x p e r i m e n t a l l y i n c r e a s e d p h o t o p e r i o d (16L:8D) d e s i g n e d t o h a s t e n i m p l a n t a t i o n (see Chapter IV) and, f o l l o w i n g a b r i e f time l a g , p r o g e s t e r o n e c o n c e n t r a t i o n s rose s h a r p l y t o a peak of 21 ng/ml i n J a n u a r y . Serum p r o g e s t e r o n e then d e c l i n e d and r e t u r n e d t o nonpregnant l e v e l s by the end of F e b r u a r y . A l t h o u g h b i r t h was not o b s e r v e d , the t o t a l time p e r i o d from the . a c t i v a t i o n of the c o r p o r a l u t e a t o the r e t u r n t o i n a c t i v e l e v e l s of p r o g e s t e r o n e was a p p r o x i m a t e l y 70 days. Based on these d a t a i t i s e s t i m a t e d t h a t f o l l o w i n g mating i n l a t e A p r i l the c o r p o r a l u t e a of p r e i m p l a n t a t i o n formed and s e c r e t e d low l e v e l s of p r o g e s t e r o n e u n t i l l a t e December when the c o r p o r a l u t e a of p o s t i m p l a n t a t i o n d e v e l o p e d . The g r e a t e r a c t i v i t y of the l u t e a l c e l l s produced i n c r e a s e d l e v e l s of p r o g e s t e r o n e which r e t u r n e d t o b a s a l l e v e l s near the e s t i m a t e d time of b i r t h i n l a t e F e b r u a r y or e a r l y March. P r o g e s t e r o n e c o n c e n t r a t i o n s c h a r a c t e r i s t i c of the p r e i m p l a n t a t i o n p e r i o d of pregnancy i n F3 d u r i n g 1979 were a l s o o b s e r v e d i n F3 i n 1981. B e g i n n i n g i n l a t e A p r i l , p r o g e s t e r o n e rose t o l e v e l s s i m i l a r t o t h e e a r l i e r p e r i o d of d e l a y e d 137 i m p l a n t a t i o n (mean =3.18 ± 0.4, n = 9, p>.05) and remained e l e v a t e d (1.95-6.23 ng/ml) u n t i l August. U n f o r t u n a t e l y , i n e a r l y September F3 escaped ending t h i s p a r t of the s t u d y . 3. W i l d o t t e r s The serum p r o g e s t e r o n e c o n c e n t r a t i o n s of f i v e w i l d o t t e r s (Table XVI) a r e c o n s i s t e n t w i t h those observed among the c a p t i v e a n i m a l s i n the same r e p r o d u c t i v e s t a t e . P r o g e s t e r o n e l e v e l s of immature o t t e r s were a l l below 1 ng/ml w h i l e those of a d u l t s w i t h u n i m p l a n t e d b l a s t o c y s t s were w i t h i n the range of v a l u e s o b t a i n e d i n F3 d u r i n g the p r e i m p l a n t a t i o n p e r i o d of pregnancy. 138 DISCUSSION A. Hi s t o l o q y T h i s study has shown t h a t i n B r i t i s h C o lumbia, o t t e r s a re s e a s o n a l b r e e d e r s which e x h i b i t a g e s t a t i o n p e r i o d c h a r a c t e r i z e d by a p r o l o n g e d p e r i o d of embryonic d i a p a u s e p r i o r t o i m p l a n t a t i o n . The r e p r o d u c t i v e organs of r i v e r o t t e r s were found t o undergo annual c y c l e s of a c t i v i t y which are a n a t o m i c a l l y and h i s t o l o g i c a l l y s i m i l a r t o those of ot h e r m u s t e l i d s such as the mink (Hansson 1947; Enders 1952; Enders and Given 1977), the f e r r e t , M u s t e l a p u t o r i u s (Buchanan 1966; Deanesly 1967; Rowlands 1974), the s t o a t , M u s t e l a erminea (Deanesly 1935; K i n g and Moody 1982), the European badger (Neal and H a r r i s o n 1958; Canivenc 1966; Ahnlund 1980), the sea o t t e r Enhydra l u t r a ( S inha et a l . 1966; Si n h a and Conaway 1968) and the s p o t t e d skunk (Mead I968a,b; G r e e n s i d e s and Mead 1973; Sinha and Mead 1976). The d i s t i n c t i v e l u t e a l and u t e r i n e c y c l e observed d u r i n g pregnancy i n the o t t e r had not been d e s c r i b e d i n d e t a i l p r e v i o u s l y , but i s s i m i l a r t o t h a t of o t h e r m u s t e l i d s which e x h i b i t a p e r i o d of embryonic d i a p a u s e (see r e v i e w s i n Enders and G i v e n 1977; S c h l a f k e e t a l . 1981; Mead 1981; Mead and Wright 1983). In the o t t e r , the f o l l i c u l a r g r a n u l o s a c e l l s undergo i n c o m p l e t e l u t e i n i z a t i o n f o l l o w i n g o v u l a t i o n t o form a corpus luteum of p r e i m p l a n t a t i o n which i s s m a l l and r e l a t i v e l y i n a c t i v e . T h i s c o r pus luteum remains throughout the n i n e t o e l e v e n month d i a p a u s e p e r i o d , d u r i n g which the the b l a s t o c y s t i s 139 f r e e i n the upper p o r t i o n of the u t e r u s . I t i s unknown i f c e l l d i v i s i o n o c c u r s i n the b l a s t o c y s t s of o t t e r s d u r i n g t h i s p e r i o d as i t does i n o t h e r m u s t e l i d s , where i t has been observed t o p r o c e e d s l o w l y and t o be r e s t r i c t e d p r i m a r i l y t o the t r o p h o b l a s t (Mead and Wright 1983). D u r i n g t h i s p r e i m p l a n t a t i o n p e r i o d , the u t e r i n e e p i t h e l i u m of o t t e r s i s s e c r e t o r y and the l u t e a l c e l l s a r e s m a l l , n o n - v a c u o l a t e d and r e l a t i v e i n a c t i v e . S h o r t l y b e f o r e i m p l a n t a t i o n o c c u r s , the b l a s t o c y s t resumes embryonic development, the l u t e a l c e l l s i n c r e a s e a c t i v i t y , and the corpus luteum doubles i n s i z e . S i m i l a r v a r i a t i o n s i n s i z e between the c o r p o r a l u t e a of p r e i m p l a n t a t i o n and p o s t i m p l a n t a t i o n were r e p o r t e d by H a m i l t o n and Eadi e (1964), Tabor (1973) and Lauhachinda (1978). The u t e r i n e endometrium a l s o i n c r e a s e s i n t h i c k n e s s p r i o r t o i m p l a n t a t i o n and the numerous u t e r i n e g l a n d s become h i g h l y s e c r e t o r y . A l t h o u g h the n a t u r e of the u t e r i n e s e c r e t i o n i s unknown, changes i n the amount and the c o m p o s i t i o n of the u t e r i n e f l u i d have been found t o occur s h o r t l y b e f o r e i m p l a n t a t i o n i n s e v e r a l s p e c i e s of m u s t e l i d s ( D a n i e l 1970; Enders and G i v e n 1977; Mead e_t a_l. 1979; Bonnin and Canivenc 1980). The r e l a t i o n s h i p between age groups and o v a r i a n s i z e found i n t h i s study was a l s o r e p o r t e d by Tabor (1973), e xcept t h a t the mean w e i g h t s he r e p o r t e d were an o r d e r of magnitude lower than I o b s e r v e d (8.0-25.4 mg vs 64.0-210.9 mg). However, the o v a r i a n d i m e n s i o n s I r e c o r d e d were s l i g h t l y s m a l l e r than those Tabor o b s e r v e d so c o n c e i v a b l y a d e c i m a l p o i n t may have been m i s p l a c e d i n h i s r e p o r t e d w e i g h t s . Minor v a r i a t i o n s i n s i z e may be due t o 140 the amount of m o i s t u r e i n the o v a r i e s b e f o r e w e i g h i n g or the i n c l u s i o n of c i r c u m o v a r i a n t i s s u e . B. V a g i n a l smears Changes i n the v a g i n a l smears were found t o be c o r r e l a t e d t o o v a r i a n development i n a l l of the a n i m a l s examined and t h u s , i d e n t i f y c o r r e c t l y the r e p r o d u c t i v e s t a t e of the female. The v a g i n a l h i s t o l o g y and c e l l t y p e s o b s e r v e d a r e s i m i l a r t o those of o t h e r m u s t e l i d s i n which v a g i n a l smears have been used t o determine the r e p r o d u c t i v e c y c l e s . As i n o t h e r m u s t e l i d s (Hamilton and Gould 1940; Hansson 1947; Enders 1952), the a n e s t r o u s and e s t r o u s p e r i o d s c o u l d be c l e a r l y i d e n t i f i e d on the b a s i s of the c h a r a c t e r i s t i c smears p r e s e n t w h i l e the changes which occur d u r i n g p r o e s t r u s were g r a d u a l and i t was d i f f i c u l t t o determine t h e i r d u r a t i o n . The p r e s ence of l e u k o c y t e s d u r i n g e s t r u s suggests t h a t the v a g i n a l smears of o t t e r s are more s i m i l a r t o the f e r r e t ( H a m i l t o n and G ould 1940) than t o the mink (Hansson 1947; Enders 1952; T r a v i s e t a l . 1978) or the s p o t t e d skunk (Mead 1968a,b; G r e e n s i d e s and Mead 1973) which do not show l e u k o c y t e s u n t i l e s t r u s i s c o m p l e t e d . However, due t o the v a r i a b l e presence of n u c l e a t e d c o r n i f i e d c e l l s , the e s t r u s p e r i o d of o t t e r s c o u l d not be d i v i d e d i n t o two d i s t i n c t s t a g e s , based on the presence or absence of n u c l e i as i n the f e r r e t ( H a m i l t o n and Gould 1940). The presence of a v a g i n a l smear dominated by c o r n i f i e d c e l l s i s a good i n d i c a t o r of e s t r u s i n the o t t e r . P r e o v u l a t o r y f o l l i c l e s were obser v e d i n the o v a r i e s of o t t e r s which had a 141 c o r n i f i e d v a g i n a o n l y and a l l of the mating a t t e m p t s i n t h i s s t udy o c c u r r e d when the female had a c o r n i f i e d e s t r o u s smear. O v u l a t i o n s a l s o occur d u r i n g the p e r i o d of maximum v a g i n a l c o r n i f i c a t i o n i n the s p o t t e d skunk ( G r e e n s i d e s and Mead 1973) a l t h o u g h mink w i l l a c c e p t a male b e f o r e the c o r n i f i e d smear i s p r e s e n t ( T r a v i s et a_l. 1979). Female o t t e r s may not be a b l e t o o v u l a t e d u r i n g the l a t t e r s t a g e s of a p r o l o n g e d p e r i o d of c o r n i f i e d v a g i n a l smears, however, and the end of e s t r u s may not be i n d i c a t e d by the presence of a mete s t r o u s smear. As i n the mink (Hansson 1947; Enders and Enders 1963) and the s t r i p e d skunk (Smith 1974), a f t e r mating t h e r e i s an i n f l u x of c o r n i f i e d c e l l s i n t o the v a g i n a l smear of o t t e r s which remained d u r i n g the e a r l y s t a g e s of pregnancy. A l s o , c o r n i f i e d c e l l s were p r e s e n t a f t e r the o v a r i e s and u t e r i were removed from an e s t r o u s female ( F 6 ) . C o r n i f i e d smears a l s o c o n t i n u e a f t e r o v u l a t i o n i n the f e r r e t ( H a m i l t o n and Gould 1940) and the c a t ( F o s t e r and Hisaw 1935) a l t h o u g h i n most domestic mammals e s t r o u s smears end s h o r t l y a f t e r mating or o v u l a t i o n ( S t a b e n e l d t 1974). My data i n d i c a t e t h a t i n the o t t e r , e s t r u s o c c u r s d u r i n g the p e r i o d i n which a c o r n i f i e d v a g i n a l smear i s p r e s e n t and may end b e f o r e the m e t e s t r o u s smear i s f i r s t o bserved. Other i n d i c a t o r s of o v a r i a n a c t i v i t y such as the presence of b l o o d i n the v a g i n a , s w e l l i n g of the v u l v a and the amount and n a t u r e of mucus p r e s e n t , were found t o v a r y w i t h the r e p r o d u c t i v e c y c l e but were c o n s i d e r e d u n r e l i a b l e i n d i c a t o r s of e s t r u s . The presence of b l o o d and mucus i n the v u l v a and v a g i n a l smear may have been a f f e c t e d by h a n d l i n g the a n i m a l s . 1 42 The amount p r e s e n t appeared t o be g r e a t e r when d i f f i c u l t i e s were en c o u n t e r e d c a p t u r i n g the female f o r e x a m i n a t i o n . S i m i l a r l y , t h e s e i n d i c a t o r s a r e not u s e f u l i n the f e r r e t ( H a m i l t o n and Gould 1940) a l t h o u g h v u l v a r s w e l l i n g has been used s u c c e s s f u l l y i n the mink ( T r a v i s et a_l. 1978). The a b i l i t y t o use n o n - d e s t r u c t i v e methods such as v a g i n a l smears i s i m p o r t a n t when d e t e r m i n i n g the r e p r o d u c t i v e c y c l e of v a l u a b l e a n i m a l s such as r i v e r o t t e r s . O t t e r s are common d i s p l a y a n i m a l s i n zoos and a q u a r i a and have r e c e n t l y become the o b j e c t of a number of r e s e a r c h p r o j e c t s i n v o l v i n g r a d i o -t e l e m e t r y . I t i s d i f f i c u l t t o o b t a i n l a r g e numbers of a n i m a l s f o r s tudy and a n o n - i n v a s i v e method of d e t e r m i n i n g t h e r e p r o d u c t i v e s t a t e a l l o w s the maximum amount of i n f o r m a t i o n t o be o b t a i n e d w i t h o u t harm. Because the o c c u r r e n c e of v a g i n a l c o r n i f i c a t i o n i s s p e c i e s s p e c i f i c and many a n i m a l s do not conform t o the c l a s s i c rodent c y c l e (D'Souza 1978). Thus, i t i s i m p o r t a n t f o r s t u d i e s such as t h i s t o i d e n t i f y the a n n u a l changes which occur i n the v a g i n a l smears of w i l d a n i m a l s and t o c o r r e l a t e the smear types w i t h the h i s t o l o g i c a l e x a m i n a t i o n of r e p r o d u c t i v e t i s s u e . C. Induced o v u l a t i o n R i v e r o t t e r s have been s u s p e c t e d of b e i n g i n d u c e d o v u l a t o r s (Mead and W r i g h t 1983) d e s p i t e a l a c k of e v i d e n c e . There a r e t h r e e s e p a r a t e o b s e r v a t i o n s from my study which p r o v i d e i n d i r e c t e v i d e n c e s u p p o r t i n g t h i s c o n t e n t i o n . The f i r s t o b s e r v a t i o n i s the l a c k of c o r p o r a l u t e a or c o r p o r a a l b i c a n t i a i n the o v a r i e s 143 of females which e x h i b i t e d e s t r u s , but were not mated. When examined, the o v a r i e s showed no i n d i c a t i o n t h a t o v u l a t i o n had o c c u r r e d and c o n t a i n e d c o r p o r a a t r e t i c a or s m a l l f o l l i c l e s o n l y . The second i s the low l e v e l s of p r o g e s t e r o n e which remained f o l l o w i n g e s t r u s i n nonmated fe m a l e s . No r i s e i n p r o g e s t e r o n e a s s o c i a t e d w i t h o v u l a t i o n and c o r p o r a l u t e a f o r m a t i o n was seen which i n d i c a t e s t h a t o v u l a t i o n had not o c c u r r e d . The t h i r d o b s e r v a t i o n i s the p r o l o n g e d e s t r u s and the i r r e g u l a r p a t t e r n of v a g i n a l smears e x h i b i t e d by the f e m a l e s . S i m i l a r o b s e r v a t i o n s have been r e p o r t e d f o r a v a r i e t y of induced o v u l a t o r s and appear t o be a c h a r a c t e r i s t i c of a n i m a l s which do not o v u l a t e s p o n t a n e o u s l y (Enders 1952; MacFarlane and T a y l o r 1982). My d a t a i n d i c a t e t h a t the r i v e r o t t e r i s a l s o an induced o v u l a t o r a l t h o u g h t o o b t a i n d i r e c t e v i d e n c e i t would be n e c e s s a r y t o d i v i d e females i n t o p a i r e d and unmated groups and m o n i t o r f o r s i g n s of o v u l a t i o n . The amount of s t i m u l a t i o n n e c e s s a r y f o r o v u l a t i o n i n the o t t e r i s unknown. M u l t i p l e c o p u l a t i o n s a r e n e c e s s a r y t o induce o v u l a t i o n i n the mink and s h o r t - t a i l e d weasel w h i l e a s i n g l e p r o t r a c t e d mating i s s u f f i c i e n t i n the f e r r e t ( H e r b e r t 1977). In t h i s s t u d y , r e p e a t e d mounting a t t e m p t s , but w i t h o u t apparent i n t r o m i s s i o n , were o b s e r v e d i n one o t t e r (F5) who f a i l e d t o o v u l a t e . Mounting a l o n e may be i n s u f f i c i e n t s t i m u l u s f o r o v u l a t i o n , or a l t e r n a t i v e l y , mating may have o c c u r r e d and she s i m p l y f a i l e d t o o v u l a t e . The f a i l u r e t o o v u l a t e among mated females has been r e p o r t e d i n o t h e r m u s t e l i d s w i t h induced o v u l a t i o n (Enders 1952; Wade-Smith and Richmond 1978; H a m i l t o n 144 and Gould 1940). Data from t h i s s tudy a l s o s u g g e s t s t h a t o v u l a t i o n may be in d u c e d a r t i f i c i a l l y by the r e p e a t e d p r o c e s s of c o l l e c t i n g v a g i n a l smears. A l t h o u g h mating was not o b s e r v e d , F3 showed r i s e s i n p r o g e s t e r o n e c o n c e n t r a t i o n s c h a r a c t e r i s t i c of pregnancy d u r i n g 1981. The female had l i m i t e d a c c e s s t o a male d u r i n g the b r e e d i n g p e r i o d and mating s c a r s were not p r e s e n t . B r e e d i n g may have o c c u r r e d unobserved or o v u l a t i o n may have been induced by the t a k i n g of v a g i n a l smears. Smears were taken f r e q u e n t l y w h i l e the female was not f u l l y a n e s t h e t i z e d and t h i s r e p e a t e d s t i m u l u s may have been s u f f i c i e n t t o induce o v u l a t i o n and caused F3 t o e n t e r pseudopregnancy. A l t e r n a t i v e l y , the p r o g e s t e r o n e changes seen i n F3 may have been the r e s u l t of spontaneous o v u l a t i o n . O v u l a t i o n s among unmated females have been observed i n i n d u ced o v u l a t o r s such as mink ( M o l l e r 1974) and s t r i p e d skunks (Wade-Smith et. a l . 1980). U n f o r t u n a t e l y , i t i s not known i f t h i s female was pregnant or pseudopregnant because she escaped b e f o r e b i r t h s h o u l d have o c c u r r e d . However, the l a c k of o v u l a t i o n s among most of my c a p t i v e o t t e r s i n d i c a t e s t h a t spontaneous o v u l a t i o n s a r e not common i n o t t e r s , and t h a t the amount of s t i m u l u s n e c e s s a r y f o r o v u l a t i o n t o oc c u r i s g r e a t e r than t h a t n o r m a l l y p r o v i d e d by v a g i n a l smear c o l l e c t i o n s from a n e s t h e t i z e d a n i m a l s a t w i d e l y spaced i n t e r v a l s . An e x c e p t i o n may oc c u r when an a n i m a l i s l i g h t l y a n a e s t h e t i z e d or when the e x a m i n a t i o n s a re r e p e a t e d over a s h o r t p e r i o d of t i m e . Knowledge of the pr e s e n c e of spontaneous or induced o v u l a t i o n i s i m p o r t a n t when i n t e r p r e t i n g the d a t a c o l l e c t e d from 145 t r a p p e d a n i m a l s . R e s e a r c h e r s have c o l l e c t e d r e p r o d u c t i v e t r a c t s and used the i n f o r m a t i o n o b t a i n e d from c o u n t s of c o r p o r a l u t e a t o e s t i m a t e p o p u l a t i o n parameters such as f e r t i l i t y r a t e s and r e c r u i t m e n t (Tabor and Wight 1977; Mowbray e t a l . 1979). Due t o the the poor s t a t e of p r e s e r v a t i o n of many of these samples, b l a s t o c y s t s c o u n t s a r e o f t e n not a v a i l a b l e (as i n t h i s s t udy) and t h e r e f o r e i t i s im p o r t a n t t o know i f c o r p o r a l u t e a g i v e a t r u e i n d i c a t i o n of mating and assumed f e r t i l i z a t i o n s or merely of o v u l a t i o n s . T h i s study i n d i c a t e s t h a t under normal c i r c u m s t a n c e s the s t i m u l u s of mating i s n e c e s s a r y f o r o v u l a t i o n t o occur i n o t t e r s and because the v a s t m a j o r i t y of males are f e r t i l e (Chapter I I ) , the presence of c o r p o r a l u t e a s h o u l d be a good i n d i c a t o r of pregnancy. R e p r o d u c t i v e a t t r i b u t e s , such as induced o v u l a t i o n and p r o l o n g e d e s t r u s , may i n c r e a s e b r e e d i n g s u c c e s s among o t t e r s . Induced o v u l a t i o n has been observed i n a number of mammals, the m a j o r i t y of which te n d t o be s o l i t a r y (Zarrow and C l a r k 1968). O t t e r s a r e a l s o s o l i t a r y a n i m a l s and do not have a l o n g c o u r t s h i p p e r i o d ( M e l q u i s t and Hornocker 1983; Woo l i n g t o n 1984; p e r s o n a l o b s e r v a t i o n ) . I t may be advantageous f o r female o t t e r s t o be in d u c e d o v u l a t o r s so t h a t r e s o u r c e s were not wasted by o v u l a t i n g when a male i s not p r e s e n t . A l o n g e s t r o u s p e r i o d would a l l o w the female t o be r e c e p t i v e u n t i l she i s l o c a t e d by the male and f o r mating t o occur a l m o s t i m m e d i a t e l y . 146 D. E s t r o u s p e r i o d s T h i s study i n d i c a t e s t h a t r i v e r o t t e r s a r e g e n e r a l l y monestrous w i t h a s i n g l e p r o l o n g e d e s t r o u s p e r i o d . One c a p t i v e o t t e r d i d e x h i b i t two e s t r o u s p e r i o d s per year however, s u g g e s t i n g t h a t a second, s h o r t e r p e r i o d may occur i n some females i f mating does not o c c u r . A second e s t r u s has a l s o been observed i n o t h e r monestrous, induced o v u l a t o r s such as the harbour s e a l , Phoca v i t u l i n a ( B i g g 1973) and may r e p r e s e n t a subsequent wave of f o l l i c u l a r development (Nalbandov 1976). The Stage V f o l l i c l e s o b s e r v e d i n the o v a r i e s of some e s t r o u s o t t e r s may a l s o r e p r e s e n t a second, s m a l l e r wave of f o l l i c l e s which can d e v e l o p i f mating does not o c c u r e a r l y i n the e s t r o u s p e r i o d . The l e n g t h of the e s t r o u s p e r i o d , i d e n t i f i e d by a c o r n i f i e d v a g i n a l smear, v a r i e d i n t h i s s t u d y from 20-56 days and averaged a p p r o x i m a t e l y 40 d a y s . A l t h o u g h more v a r i a b l e , t h i s i s comparable t o the o b s e r v a t i o n s of L i e r s (1951) who r e p o r t e d a s i n g l e e s t r o u s p e r i o d of 42-46 days i n o t t e r s w i t h maximum r e c e p t i v i t y o c c u r r i n g e v e r y s i x days. E. The a n n u a l c y c l e i n B r i t i s h Columbia The r e s u l t s of my s t u d y i n d i c a t e t h a t e s t r u s and mating a r e c o n f i n e d t o a r e l a t i v e l y s h o r t s p r i n g b r e e d i n g season and t h a t a n i n e t o e l e v e n month p e r i o d of embryonic d i a p a u s e i s o b l i g a t o r y i n o t t e r s . F o l l i c u l a r a c t i v i t y was low o u t s i d e of the b r e e d i n g season and t h e r e a r e no r e c o r d s of u n u s u a l l y e a r l y or l a t e embryos, f e t u s e s or b i r t h s . There was no e v i d e n c e of out of 147 season o v u l a t i o n s and i f they d i d occur i t would be u n l i k e l y t h a t f e r t i l e males would be p r e s e n t (Chapter I I ) . A l s o , t h e r e was no e v i d e n c e of secondary o v u l a t i o n s o c c u r r i n g i n o t t e r s which had mated as has been observed i n the mink (Enders 1952; Hansson 1947) and the European badger (Ahnlund 1980). H i l l and Lauhachinda (1981) suggest t h a t some r i v e r o t t e r s may breed i n the l a t e w i n t e r or e a r l y s p r i n g and then g i v e b i r t h w i t h o u t e x h i b i t i n g d e l a y e d i m p l a n t a t i o n . A l t h o u g h males do produce sperm as e a r l y as l a t e December or January i n B r i t i s h Columbia (Chapter I I ) , none of the w i l d females were c a p a b l e of o v u l a t i n g b e f o r e March. One c a p t i v e female (F5) however, e n t e r e d e s t r u s e a r l y i n F e b r u a r y (1980) but mating d i d not o c c u r . There i s no reason t o s u s p e c t t h a t she would not have had a normal pregnancy and g i v e n b i r t h the f o l l o w i n g s p r i n g . T h i s e a r l y e s t r u s may have been r e l a t e d t o the u n s u c c e s s f u l mating a t t e m p t s which o c c u r r e d the p r e v i o u s y e a r , or may be an i n d i c a t i o n t h a t non-p a r t u r i e n t o t t e r s breed e a r l i e r than those which have r e c e n t l y g i v e n b i r t h . O b v i o u s l y , f u r t h e r r e s e a r c h i s n e c e s s a r y t o determine the f u l l e x t e n t of i n d i v i d u a l v a r i a t i o n p r e s e n t among the e s t r o u s p e r i o d s and b r e e d i n g c y c l e s of r i v e r o t t e r s . I m p l a n t a t i o n a l s o appears t o be c o n f i n e d t o a r e l a t i v e s h o r t time p e r i o d . My r e s u l t s i n d i c a t e t h a t i m p l a n t a t i o n s b e g i n a f t e r the f i r s t week i n F e b r u a r y and are completed by l a t e F e b r u a r y or e a r l y March. Assuming a p o s t i m p l a n t a t i o n p e r i o d of two months (see b e l o w ) , i m p l a n t a t i o n l a t e r than e a r l y March would r e s u l t i n b i r t h i n mid or l a t e May. E s t r u s would occur two t o t h r e e weeks l a t e r , which i s a f t e r male o t t e r s have ceased 148 r e p r o d u c t i v e a c t i v i t y (Chapter I I ) . Thus, the h i g h pregnancy r a t e s of o t t e r s i n B r i t i s h Columbia i n d i c a t e t h a t i m p l a n t a t i o n s a r e u n l i k e l y t o occur l a t e r than e a r l y March, a l t h o u g h the t o t a l e x t e n t of the i m p l a n t a t i o n p e r i o d c o u l d not be de t e r m i n e d due t o the the l a c k of samples. The r e p r o d u c t i v e c y c l e of L. c a n a d e n s i s i n southwestern B r i t i s h Columbia i s s i m i l a r t o t h a t observed i n o t h e r a r e a s of i t s range, a t s i m i l a r l a t i t u d e s ( H a m i l t o n and E a d i e 1964; Tabor and Wight 1977; M e l q u i s t and Hornocker 1983) and i s i n agreement w i t h the b i r t h of a c a p t i v e o t t e r i n Vancouver i n l a t e March (Best 1962). An e x a m i n a t i o n of a v a i l a b l e i n f o r m a t i o n on r i v e r o t t e r s (Table X V I I I ) i n d i c a t e s t h a t these d a t a a r e c o n s i s t e n t w i t h a t r e n d f o r r e p r o d u c t i v e e v e n t s i n o t t e r s t o oc c u r e a r l i e r i n the y e a r , and f o r p e r i o d s of i m p l a n t a t i o n , p a r t u r i t i o n and b r e e d i n g t o become l o n g e r , as l a t i t u d e d e c r e a s e s . In southern r e g i o n s b r e e d i n g , i m p l a n t a t i o n and b i r t h a r e spread over extended p e r i o d s of up t o f o u r months ( H i l l and Lauhachinda 1981; L a n c i a and H a i r 1983, M c D a n i e l 1963; Mowbray e t a l . 1979) w h i l e i n n o r t h e r n a r e a s r e p r o d u c t i v e e v e n t s occur l a t e r and are c o n f i n e d t o r e l a t i v e l y s h o r t p e r i o d s of t i m e . S i m i l a r t r e n d s have been obse r v e d i n a v a r i e t y of mammals ( S a d l e i r 1969a; Lee 1970; Bronson 1985). T h i s v a r i a t i o n among o t t e r p o p u l a t i o n s i n d i c a t e s t h a t an e x t e r n a l f a c t o r may be a c t i n g as a p r o x i m a l c o n t r o l on t h e t i m i n g of r e p r o d u c t i v e a c t i v i t y . S i m i l a r i t i e s between r e p r o d u c t i v e c y c l e s of o t t e r s l i v i n g i n the marine environment of B r i t i s h Columbia and those l i v i n g under much d i f f e r e n t c l i m a t i c c o n d i t i o n s i n f r e s h water l o c a l e s 149 Table XVIII. Sumnary of available Information on the reproductive cycle of lutra canadensis. Location Mating Implantation Birth Reference Florida S.E. Texas Alabama S Georgia North Carolina Maryland Missouri (captive) S. Minnesota (captive) Michigan New York Idaho Oregon British Columbia Vancouver (captive) Calgary (captive) Northern Alberta Southern Alaska January - April February December -February December - February January -early April March - April April - early May March - mid May November - January* mid November -late February mid - late January15 late January -early February February February May early March late January -•1d March January -February January - March8 February -April March0 March 1 - 15 late December -late March March - April* March - April late March -early April April late March -April late March late March late April early May McDaMel 1963 Foy 1984 Lauhachinda 1978, Hill and Lauhachinda 1978 Wilson 1959 Land a and Hair 1983 Mowbray et a l . 1979 Best 1962 L1ers 1951, 1966 Hooper and Ostenson 1949 Hamilton and Eadie 1964 Mel quist and Hornocker 1983 Tabor and Wight 1977 this study Best 1962 Best 1962 Reid et al 1985 Woolington 1984 Larson 1983 a - peak periods. Implantation and births may occur earlier or later. b - estimated based on crown - rump lengths of implanted embryos (Hill and Lauhachinda 1981) 150 a t s i m i l a r l a t i t u d e s , s u g g e s t s t h a t t h i s f a c t o r i s not c o n f i n e d t o l o c a l h a b i t a t . S e a s o n a l changes i n d a y l e n g t h vary w i t h r e s p e c t t o l a t i t u d e o n l y and a r e not a f f e c t e d by l o c a l c o n d i t i o n s . P h o t o p e r i o d i s known t o induce e a r l y i m p l a n t a t i o n i n a number of m u s t e l i d s (Pearson and Enders 1944; Hansson 1947; W r i g h t 1963; Murphy and James 1974; Mead 1971) and w i l l a l t e r l u t e a l f u n c t i o n i n s p o t t e d skunks (Mead 1971) and mink ( A l l a i s and M a r t i n e t 1978). In B r i t i s h C olumbia, r e p r o d u c t i v e a c t i v i t y i n o t t e r s may be s t i m u l a t e d by i n c r e a s e d p h o t o p e r i o d s i n c e f o l l i c u l a r growth i n c r e a s e s s i g n i f i c a n t l y a f t e r the w i n t e r s o l s t i c e , and i m p l a n t a t i o n o c c u r s d u r i n g F e b r u a r y when the d a y l e n g t h i s i n c r e a s i n g . The same p a t t e r n appears t o occur t hroughout most of the o t t e r ' s range a l t h o u g h i n the southern U n i t e d S t a t e s i m p l a n t a t i o n o c c u r s between November and February ( H i l l and Lauhachinda 1981; L a n c i a and H a i r 1983). T h i s i n d i c a t e s t h a t i m p l a n t a t i o n may not be induced by i n c r e a s i n g p h o t o p e r i o d s i n a l l o t t e r p o p u l a t i o n s and the p r o x i m a l f a c t o r ( s ) which c o n t r o l r e p r o d u c t i v e e v e n t s may v a r y i n d i f f e r e n t a r e a s . The e f f e c t of p h o t o p e r i o d on the r e p r o d u c t i v e c y c l e of o t t e r s i n B r i t i s h Columbia w i l l be examined i n g r e a t e r d e t a i l i n Chapter IV. O v u l a t i o n , i m p l a n t a t i o n and pregnancy r a t e s of o t t e r s i n B r i t i s h Columbia were w i t h i n the ranges of those observed i n o t h e r l o c a l e s . No s i g n i f i c a n t d i f f e r e n c e s were observed i n the number of c o r p o r a l u t e a or i m p l a n t e d embryos i n v a r i o u s s t u d i e s a l t h o u g h pregnancy r a t e s were found t o v a r y g r e a t l y . The h i g h r a t e of pregnancy among o t t e r s i n t h i s s tudy i n d i c a t e s t h a t 151 o t t e r s b reed a n n u a l l y i n B r i t i s h Columbia. Annual b r e e d i n g a l s o o c c u r s i n New York ( H a m i l t o n and E a d i e 1964) and Oregon (Tabor and Wight 1977) a l t h o u g h low pregnancy r a t e s suggest t h a t some o t t e r s may not bear young e v e r y year i n M a r y l a n d (Mowbray et a l . 1979) and Alabama and G e o r g i a ( H i l l and Lauhachinda 1981). A l t h o u g h f u r t h e r study i s n e c e s s a r y t o determine the r e l a t i o n s h i p between h a b i t a t and f r e q u e n c y of b r e e d i n g , these v a r i a t i o n s i n b r e e d i n g a r e a f u r t h e r s u g g e s t i o n t h a t o t t e r p o p u l a t i o n s a l t e r t h e i r r e p r o d u c t i v e p a t t e r n s i n response t o t h e i r environment. A l t h o u g h t h i s s tudy was a b l e t o p r o v i d e o n l y c i r c u m s t a n t i a l e v i d e n c e f o r the l e n g t h of the p o s t i m p l a n t a t i o n p e r i o d i n r i v e r o t t e r s , a two month p o s t i m p l a n t a t i o n p e r i o d was i n d i c a t e d by the a v a i l a b l e d a t a . In w i l d o t t e r s i m p l a n t a t i o n s were found t o begin i n F e b r u a r y and b i r t h s o c c u r r e d i n l a t e March or A p r i l . A c a p t i v e female was e s t i m a t e d t o complete her pregnancy i n l a t e F e b r u a r y or e a r l y March, s l i g h t l y over two months a f t e r the s i g n i f i c a n t r i s e i n p r o g e s t e r o n e c o n c e n t r a t i o n s a s s o c i a t e d w i t h the end of the p r e i m p l a n t a t i o n p e r i o d . A l t h o u g h young were not o b s e r v e d , t h i s e s t i m a t e was based on the presence of s w o l l e n t e a t s , an e n l a r g e d v a g i n a l opening and the d e c l i n e of p r o g e s t e r o n e c o n c e n t r a t i o n s t o nonpregnant l e v e l s (1 ng/ml). The d e c l i n e of p r o g e s t e r o n e l e v e l s t o nonpregnant c o n c e n t r a t i o n s i s a s s o c i a t e d w i t h the end of pregnancy i n most of the m u s t e l i d s (mink, M o l l e r 1973; P i l b e a m et a l . 1979; s p o t t e d skunk, Mead and Eik-Nes 1969b; f e r r e t , Heap and Hammond 1974; European badger, Canivenc and Bonnin 1981). A l l o w i n g f o r the p e r i o d between 152 resumption of development and i m p l a n t a t i o n , i t would suggest a p o s t i m p l a n t a t i o n p e r i o d of 60-65 days. A l t h o u g h a s h o r t e r g e s t a t i o n p e r i o d i s p o s s i b l e , t h i s e s t i m a t e i s s i m i l a r t o 60-63 days e s t i m a t e d by H a m i l t o n and E a d i e (1964) and Lauhachinda (1978) and t o the t o t a l g e s t a t i o n p e r i o d of o t t e r s t h a t do not e x h i b i t embryonic d i a p a u s e such as Amblonyx, L u t r o g a l e , and L u t r a l u t r a ( D u p l a i x - H a l l 1975). F u r t h e r s t u d y i n v o l v i n g the ex a m i n a t i o n of o t t e r s d u r i n g a l l s t a g e s of pregnancy i s ne c e s s a r y t o determine the d u r a t i o n of the p o s t i m p l a n t a t i o n p e r i o d . F. S e x u a l M a t u r i t y T h i s study i n d i c a t e s t h a t i n B r i t i s h C o lumbia, some female o t t e r s a t t a i n s e x u a l m a t u r i t y near the end of t h e i r second year (age c l a s s 1) w h i l e the remainder do so a t the end of t h e i r t h i r d (age c l a s s 2 ) . The p r o p o r t i o n of y e a r l i n g s t h a t become mature i s unknown, but the pregnancy r a t e s of age c l a s s 2 a n i m a l s i n d i c a t e s t h a t s l i g h t l y over h a l f of the females breed at the end of t h e i r second y e a r . D u r i n g the e n t i r e study a p p r o x i m a t e l y 55% of the age c l a s s 2 females c o n t a i n e d c o r p o r a l u t e a as compared t o 90.9% f o r o l d e r a n i m a l s . In the f o u r y e a r s i n which s u f f i c i e n t sample s i z e s were a v a i l a b l e f o r comparison (1976-79), the p e r c e n t a g e s were s i m i l a r which s u g g e s t s t h a t 50-60% of the females a r e mature by the b e g i n n i n g of age c l a s s 2. By age c l a s s 3 a l l , or n e a r l y a l l , have a t t a i n e d s e x u a l m a t u r i t y and have b r e d . P h y s i o l o g i c a l , b e h a v i o u r a l , or s o c i a l f a c t o r s may have r e s u l t e d i n t h e lower pregnancy r a t e s I o b s e r v e d among 153 younger a n i m a l s . The a t t a i n m e n t of s e x u a l m a t u r i t y among the c a p t i v e o t t e r s was s i m i l a r t o t h a t observed among w i l d f e m a l e s . The presence of p a r t i a l e s t r u s and the i n c r e a s e d e s t r a d i o l c o n c e n t r a t i o n o b s e r v e d i n F2 i n d i c a t e t h a t one, and p o s s i b l y two, of these o t t e r s may have been c a p a b l e of b r e e d i n g d u r i n g age c l a s s 2 a l t h o u g h e s t r u s was d e l a y e d u n t i l age c l a s s 3 i n the t h i r d . S e x u a l m a t u r i t y among the c a p t i v e o t t e r s i s d i s c u s s e d f u r t h e r i n the d i s c u s s i o n of p a r t i a l e s t r u s . The p o s s i b i l i t y t h a t s e x u a l m a t u r i t y may be d e l a y e d u n t i l a f t e r age c l a s s 2 i n some o t t e r s has been suggested by H i l l and Lauhachinda (1981) t o e x p l a i n the low pregnancy r a t e s observed i n t h e i r s t u d y . My study s u p p o r t s t h e i r s u g g e s t i o n and showed t h a t f i r s t b r e e d i n g o c c u r s i n some o t t e r s d u r i n g age c l a s s 3. In c o n t r a s t t o the s e f i n d i n g s , p r e v i o u s s t u d i e s have found t h a t a l l female o t t e r s were mature by the end of t h e i r second year ( L i e r s 1951; H a m i l t o n and E a d i e 1964; Tabor and Wight 1977). A l t h o u g h no pregnant y e a r l i n g s were observed i n t h i s s t u d y , they have been r e p o r t e d among c a p t i v e ( L i e r s 1958) and w i l d (Mowbray et a l . 1979) o t t e r s . - Thus i t appears t h a t the age of s e x u a l m a t u r i t y i s v a r i a b l e among and w i t h i n o t t e r p o p u l a t i o n s . Such v a r i a t i o n s among i n d i v i d u a l s have a l s o been r e p o r t e d f o r a v a r i e t y of m u s t e l i d s such as the w e a s e l , M u s t e l a n i v a l i s ( K i n g 1980), the European badger (Ahnlund 1980), the American badger, T a x i d e a t a x u s (Wright 1966), the w o l v e r i n e , Gulo q u l o (Rausch and Pearson 1972; L i s k o p et §_1. 1981) and the American marten, Martus americana (Markley and B a s s e t 1942). 154 In mammals, v a r i a t i o n s i n the age of s e x u a l m a t u r i t y may be due t o endogenous d i f f e r e n c e s or exogenous c o n d i t i o n s such as n u t r i t i o n , l i g h t , temperature or p o p u l a t i o n d e n s i t y ( S a d l e i r 1969a). Among the m u s t e l i d s , female weasels show advanced s e x u a l m a t u r i t y i n y e a r s of abundant food ( K i n g 1980), but f a i l t o breed i n y e a r s of low v o l e numbers (Tapper 1979). In sea o t t e r s , poor p h y s i c a l h e a l t h may d e l a y e s t r u s or i n c r e a s e the l e n g t h of the r e p r o d u c t i v e c y c l e ( L o u g h l i n e t a_l. 1981; G a r s h e l i s e t a l . 1985). I t i s unknown i f t h e observed d i f f e r e n c e s among r i v e r o t t e r p o p u l a t i o n s r e f l e c t g e n e t i c or e n v i r o n m e n t a l v a r i a t i o n s i n t h e i r h a b i t a t s . I n c r e a s e d n u t r i t i o n may e x p l a i n the b r e e d i n g of a y e a r l i n g o t t e r i n c a p t i v i t y ( L i e r s 1958). I f f a v o r a b l e e n v i r o n m e n t a l c o n d i t i o n s and good n u t r i t i o n can advance s e x u a l m a t u r i t y i t would be p r e d i c t e d t h a t the r e l a t i v e l y m i l d c l i m a t e and a p p a r e n t l y abundant f o o d p r e s e n t i n the c o a s t a l environment of B r i t i s h Columbia (Stenson e_t a l . 1984) s h o u l d r e s u l t i n s e x u a l l y mature y e a r l i n g s . However, t h i s was not observed which s u g g e s t s t h a t e i t h e r the amount of food p r e s e n t w i l l not advance m a t u r i t y , or t h a t o t h e r f a c t o r s may be p r e s e n t which d e l a y the date of f i r s t b r e e d i n g . A t t a i n m e n t of s e x u a l m a t u r i t y i s i n v e r s e l y r e l a t e d t o p o p u l a t i o n d e n s i t y i n a number of mammals ( S a d l e i r 1969a) or can be s u p pressed by the presence of a d u l t females i n r o d e n t s (Drickman 1979; Vandenbergh 1983). P o p u l a t i o n e s t i m a t e s f o r o t t e r s sampled i n t h i s study are not a v a i l a b l e a l t h o u g h c o a s t a l a r e a s appear t o support g r e a t e r d e n s i t i e s than f r e s h water h a b i t a t s ( Woolington 1984; p e r s o n a l o b s e r v a t i o n ) . S e x u a l m a t u r i t y may be 1 55 d e l a y e d among female o t t e r s i n c o a s t a l B r i t i s h Columbia due t o h i g h p o p u l a t i o n d e n s i t i e s r e s u l t i n g i n l i m i t e d food s u p p l y or s o c i a l p r e s s u r e s . G. E s t r a d i o l c o n c e n t r a t i o n s J_. Nonpregnant females T h i s study i s the f i r s t i n v e s t i g a t i o n of the changes which occur i n e s t r a d i o l c o n c e n t r a t i o n s d u r i n g the an n u a l r e p r o d u c t i v e c y c l e of r i v e r o t t e r s . I t i s a l s o one of the few s t u d i e s t o determine e s t r o g e n l e v e l s among the M u s t e l i d a e ; p r e v i o u s l y the annual c y c l e of e s t r o g e n s e c r e t i o n had been de t e r m i n e d i n the mink ( T r a v i s et a l . 1978; P i l b e a m et a l . 1979) and the s p o t t e d skunk (Mead and Eik-Nes 1969a). A l t h o u g h t h e c o n c l u s i o n s drawn from my study of the e s t r o g e n c y c l e a r e l i m i t e d due t o the v a r i a b l e e s t r a d i o l c o n c e n t r a t i o n s o b s e r v e d , they p r o v i d e an i n d i c a t i o n of the p a t t e r n s of e s t r o g e n s e c r e t i o n among m u s t e l i d s and i n d i c a t e a r e a s i n which f u r t h e r r e s e a r c h i s n e c e s s a r y . D u r i n g the f i r s t year of the study (1978-1979) serum e s t r a d i o l l e v e l s of a d u l t s were found t o v a r y s i g n i f i c a n t l y d u r i n g the annual r e p r o d u c t i v e c y c l e . E s t r a d i o l c o n c e n t r a t i o n s were low throughout the summer and autumn when o v a r i a n a c t i v i t y was reduced and a s i g n i f i c a n t i n c r e a s e o c c u r r e d i n November and December when f o l l i c u l a r development was i n c r e a s i n g . A second i n c r e a s e i n e s t r a d i o l was r e c o r d e d near the b e g i n n i n g of e s t r u s , a l t h o u g h the r e l a t i o n s h i p between the e s t r a d i o l s u rges and the onset of e s t r u s , as ^ i n d i c a t e d by the v a g i n a l smears, was not 1 56 c o n s i s t e n t between the two a d u l t s . The subsequent i n c r e a s e i n e s t r a d i o l l e v e l s seen d u r i n g e s t r u s may be a s s o c i a t e d w i t h a second wave of f o l l i c u l a r development. The r i s e i n e s t r a d i o l seen d u r i n g p a r t i a l e s t r u s i n one a n i m a l (F2) w i l l be d i s c u s s e d i n d e t a i l l a t e r . An i n c r e a s e i n e s t r a d i o l c o n c e n t r a t i o n s d u r i n g the e a r l y w i n t e r has a l s o been o b s e r v e d i n the mink ( T r a v i s e t a l . 1978; P i l b e a m e t a_l. 1979). The s i g n i f i c a n c e of t h i s i n i t i a l r i s e i s unknown a l t h o u g h i t may be a s s o c i a t e d w i t h the i n i t i a t i o n of o v a r i a n a c t i v i t y i n the o t t e r . U n l i k e the mink however, e s t r a d i o l l e v e l s i n o t t e r s were found t o i n c r e a s e near, or d u r i n g , e s t r u s . In t h i s r e s p e c t the p a t t e r n of e s t r a d i o l s e c r e t i o n i n the o t t e r i s s i m i l a r t o those of dogs (Concannon et a l . 1975) and c a t s (Verhage et a_l. 1 976) where peak l e v e l s occur i m m e d i a t e l y p r i o r t o b r e e d i n g . The l e v e l s of e s t r a d i o l o bserved i n t h i s s t u d y are s i m i l a r t o those of the mink ( T r a v i s et_ a l . 1978; P i l b e a m et, a_l. 1979) and o t h e r c a r n i v o r e s ( c a t , Verhage et a l . 1976; dog, Concannon et a l . 1975; Feder 1981). A l t h o u g h the l e v e l s were s i g n i f i c a n t l y l o w e r , the p a t t e r n of e s t r a d i o l s e c r e t i o n d u r i n g the f i n a l two y e a r s of my study was s i m i l a r t o t h a t of the f i r s t . Throughout most of the year l e v e l s were low, but v a r i a b l e , and s l i g h t i n c r e a s e s o c c u r r e d d u r i n g the e a r l y w i n t e r and d u r i n g e s t r u s . The c y c l i c a l changes seen i n the v a g i n a l smears and the p r e s ence of f o l l i c u l a r growth i n o v a r i e s i n d i c a t e s t h a t r e p r o d u c t i v e a c t i v i t y o c c u r r e d n o r m a l l y i n my c a p t i v e f e m a l e s . T h i s a c t i v i t y s u g g e s t s t h a t development of the p r i m a r y and secondary r e p r o d u c t i v e organs can 157 occur i n the absence of l a r g e serum e s t r a d i o l l e v e l s . Sex s t e r o i d t a r g e t t i s s u e s have been shown t o be v e r y s e n s i t i v e t o s m a l l changes i n e s t r o g e n l e v e l s i n the dog (Concannon e t a l . 1975) and the l a b o r a t o r y r a t (Feder 1981), and the s m a l l i n c r e a s e s I observed d u r i n g e s t r u s may have been s u f f i c i e n t t o s t i m u l a t e r e p r o d u c t i v e development i n my o t t e r s . An a l t e r n a t i v e p o s s i b i l i t y i s t h a t the r e p r o d u c t i v e a c t i v i t y o bserved was due t o i n c r e a s e s i n e s t r a d i o l which I d i d not d e t e c t . The sampling f r e q u e n c y used may have missed s h o r t d u r a t i o n surges of e s t r a d i o l . The presence of e s t r o g e n surges c o u l d a l s o e x p l a i n the poor c o r r e l a t i o n between e s t r o g e n v a l u e s and e s t r o u s smears observed i n the f i r s t year of the s t u d y . I n v e s t i g a t i o n s u t i l i z i n g l a r g e sample s i z e s and more f r e q u e n t sampling a r e n e c e s s a r y t o de t e r m i n e the p a t t e r n of e s t r a d i o l s e c r e t i o n d u r i n g the annual r e p r o d u c t i v e c y c l e i n o t t e r s . A t h i r d p o s s i b l e e x p l a n a t i o n f o r the presence of r e p r o d u c t i v e development i n the absence of h i g h l e v e l s of e s t r a d i o l , i s t h a t the a c t i v i t y was i n response t o an e s t r o g e n which was not assayed f o r , such as e s t r o n e . E s t r a d i o l 17/3 and e s t r o n e a r e the major e s t r o g e n s produced i n the mammalian ovary (Turner and Bagnara 1971; P e t e r s and McNatty 1980) and e s t r a d i o l has been i d e n t i f i e d as the p r i m a r y r e g u l a t o r of f o l l i c u l a r a c t i v i t y i n most mammals examined (see review i n Verhage 1976; Feder 1981). Among the m u s t e l i d s , ' Mondain-Monval e t a l . ( 1980) have shown t h a t w h i l e e s t r o g e n c o n c e n t r a t i o n s were low d u r i n g pregnancy i n the European badger, e s t r o n e l e v e l s exceeded those of e s t r a d i o l . These a u t h o r s f e l t however, t h a t much of t h i s 158 i n c r e a s e d e s t r o n e was due t o an e x t r a - o v a r i a n s o u r c e , p o s s i b l y the a d r e n a l s , and so i t d i d not r e f l e c t the r e l a t i v e e s t r o g e n c o n c e n t r a t i o n s i n the ovar y . Mead and Eik-Nes (1968a) a l s o found t h a t e s t r o n e l e v e l s were h i g h e r than e s t r a d i o l f o r much of the year a l t h o u g h e s t r a d i o l c o n c e n t r a t i o n s i n c r e a s e d d u r i n g e s t r u s t o exceed those of e s t r o n e which d i d not v a r y s i g n i f i c a n t l y t h r o u g h o u t the y e a r . I t i s r e a d i l y apparent t h a t f u r t h e r study i s n e c e s s a r y t o dete r m i n e the r e l a t i v e importance of d i f f e r e n t e s t r o g e n s on o v a r i a n a c t i v i t y i n the r i v e r o t t e r and among m u s t e l i d s as a whole. Due t o our l a c k of knowledge of the v a r i e t y of f a c t o r s t h a t can a f f e c t hormonal l e v e l s i n c a p t i v e a n i m a l s , i t i s d i f f i c u l t t o d etermine t h e cause of the d i f f e r e n c e s i n e s t r a d i o l s e c r e t i o n observed between the f i r s t and subsequent y e a r s . The absence of e s t r a d i o l s u r g e s i n the f i n a l two y e a r s of the study may have been i n re s p o n s e a c u m u l a t i v e e f f e c t or t o a change which o c c u r r e d a t one s p e c i f i c p o i n t i n t i m e . However, i f the a l t e r e d s e c r e t i o n was due a s i n g l e f a c t o r , or an i n t e r a c t i o n of m u l t i p l e f a c t o r s , which changed a f t e r the 1979 b r e e d i n g season, the n a t u r e of t h i s change i s not o b v i o u s . The c a r e and h a n d l i n g of the o t t e r s was not a l t e r e d d u r i n g the cour s e of the study and a l l b l o o d samples were t r e a t e d i n the same manner. A l l serum samples were a s s a y e d a t the same time and the assay p r o c e d u r e s were c o n s i s t e n t . The o n l y change which d i d occur was the s u b s t i t u t i o n of salmon f o r h e r r i n g as food , b e g i n n i n g i n the f a l l of 1979. A l t h o u g h n u t r i t i o n can have an i m p o r t a n t e f f e c t upon r e p r o d u c t i o n (see re v i e w s i n S a d l e i r I969a,b), both of 159 t h e s e s p e c i e s are eaten by w i l d o t t e r s i n t h i s a r e a (Stenson et a l . 1984) and appeared t o be of e q u a l n u t r i t i o n a l v a l u e . A l s o , mink f e e d on P a c i f i c salmon showed no s i g n s of i m p a i r e d f e r t i l i t y ( A u l e r i c h e t ' a_l. 1973). The r e p e a t e d use of an a n e s t h e t i c may have a f f e c t e d e s t r a d i o l s e c r e t i o n a l t h o u g h p r o l o n g e d d a i l y use of ketamine h y d r o c h l o r i d e d i d not cause s i g n i f i c a n t a l t e r a t i o n s i n e s t r o g e n and p r o g e s t e r o n e c o n c e n t r a t i o n s i n rhesus monkeys (Channing e_t a_l. 1977). The most l i k e l y cause of the v a r i a b l e e s t r o g e n c o n c e n t r a t i o n s and the absence of e s t r a d i o l surges d u r i n g 1980 and 1981 i s the v a r i o u s forms of s t r e s s which a c t upon c a p t i v e o t t e r s . Under c o n d i t i o n s of s t r e s s , a d r e n a l output i s i n c r e a s e d and can a l t e r normal g o n a d o t r o p h i n s e c r e t i o n t h r o u g h a d i r e c t e f f e c t of c o r t i c o s t e r o n e or as a r e s u l t of o t h e r a d r e n a l s t e r o i d s ( E c k s t e i n 1977; Ramaley 1981). When w h i t e - t a i l e d d e e r , O d o c o i l e u s v i r g i n i a n u s , were s u b j e c t e d t o the s t r e s s of c a p t i v i t y , e s t r o g e n p r o f i l e s were e r r a t i c and u n i n f o r m a t i v e (Woolfe and Harper 1977). S t r e s s has been shown t o d e l a y or i n h i b i t s u c c e s s f u l r e p r o d u c t i o n i n a v a r i e t y of mammals (see r e v i e w s i n S a d l e i r 1969a; C h r i s t i a n 1980; Ramaley 1981) and among the m u s t e l i d s , s t r e s s a s s o c i a t e d w i t h i s o l a t i o n d e l a y e d e s t r u s and i n h i b i t e d s e x u a l development i n mink ( G i l b e r t and B a i l e y 1967) and p o s s i b l y s p o t t e d skunks ( G r e e n s i d e s and Mead 1973). A l s o , the low f e r t i l i t y r a t e s observed among mink i n a number of s t u d i e s may be due t o the s t r e s s of f r e q u e n t h a n d l i n g (Papke e t a l . 1980; A l l a i s and M a r t i n e t 1978; D a n i e l 1971). The e f f e c t s of s t r e s s induced by c a p t i v i t y may a l s o account f o r the 160 l a c k of b r e e d i n g among L. c a n a d e n s i s i n zoos and a q u a r i a . A l t h o u g h b r e e d i n g has o c c u r r e d i n c a p t i v i t y , i t i s r e s t r i c t e d p r i m a r i l y t o p r i v a t e c o l l e c t i o n s where t h e r e were few d i s t r a c t i o n s and where dens were i s o l a t e d from the p u b l i c both v i s u a l l y and a c o u s t i c a l l y ( D u p l a i x - H a l l 1975). I t would be w o r t h w h i l e t o determine i f o t t e r s i n zoos a l s o show reduced e s t r a d i o l p r o f i l e s s i m i l a r t o those I obs e r v e d . My c a p t i v e o t t e r s may have been s u b j e c t e d t o two typ e s of s t r e s s : 1) the a c c u m u l a t i v e e f f e c t s of c a p t i v i t y , h a n d l i n g and human c o n t a c t i n the h o l d i n g f a c i l i t i e s , and 2) s t r e s s r e s u l t i n g from the presence of o t h e r o t t e r s . The presence of a d d i t i o n a l o t t e r s may have caused s o c i a l t e n s i o n s which i n f l u e n c e d the r e p r o d u c t i v e c y c l e s of the females i n t h i s s t u d y . S o c i a l f a c t o r s have been shown t o a l t e r r e p r o d u c t i v e a c t i v i t y i n a v a r i e t y of mammals t h r o u g h the r o l e of pheromones or as a r e s u l t of the s t r e s s i n v o l v e d i n i n t e r a c t i n g w i t h c o n s p e c i f i c s (see re v i e w s i n S a d l e i r 1969a; Drickamer 1981; M c C l i n t o c k 1983). The number of o t t e r s i n the h o l d i n g a r e a i n c r e a s e d as the study p r o g r e s s e d and f o r most of the y e a r , c a p t i v e o t t e r s c o u l d move f r e e l y between h o l d i n g pens. O t t e r s were never i s o l a t e d from a u d i t o r y or o l f a c t o r y communication w i t h each o t h e r , though p h y s i c a l i n t e r a c t i o n s were l i m i t e d t o p a i r s or s m a l l groups d u r i n g the b r e e d i n g season and d u r i n g e x p e r i m e n t a l p r o c e d u r e s (Chapter I V ) . In c o n t r a s t , w i l d o t t e r s a r e u s u a l l y s o l i t a r y w i t h l a r g e groups o c c u r r i n g o c c a s i o n a l l y ( M e l q u i s t and Hornocker 1983; W o o l i n g t o n 1984; H a t l e r 1972; p e r s o n a l o b s e r v a t i o n ) . The c a p t i v e o t t e r s d i d not e x h i b i t s i g n s of a g g r e s s i o n and appeared 161 t o a c t i v e l y s o l i c i t s o c i a l i n t e r a c t i o n s , but the e f f e c t of t h i s a r t i f i c i a l s o c i a l s t r u c t u r e upon t h e i r normal r e p r o d u c t i v e a c t i v i t y i s unknown. The l a c k of c l e a r i n c r e a s e s i n e s t r a d i o l a f t e r the f i r s t y ear of t h i s study may account f o r the absence of mating a c t i v i t y among the c a p t i v e a n i m a l s d u r i n g the f i n a l two y e a r s . Both a d u l t females were i n v o l v e d i n mating or attempted mating i n the s p r i n g of 1979 and a l l a t t e m p t s o c c u r r e d near, or d u r i n g , p e r i o d s of i n c r e a s e d e s t r a d i o l c o n c e n t r a t i o n s observed i n t h i s year o n l y . None of t h e s e a d u l t s were observed t o mate i n f o l l o w i n g y e a r s , s u g g e s t i n g t h a t i n c r e a s e d e s t r a d i o l l e v e l s a re n e c e s s a r y f o r e s t r o u s b e h a v i o u r t o o c c u r . E s t r a d i o l l e v e l s i n the c a p t i v e o t t e r s d u r i n g the l a s t two y e a r s of the study may have been s u f f i c i e n t f o r o v a r i a n a c t i v i t y t o occur but i n s u f f i c i e n t t o t r i g g e r e s t r o u s b e h a v i o u r . E s t r o u s b e h a v i o u r c o i n c i d e s w i t h peak e s t r o g e n c o n c e n t r a t i o n s i n most mammals (see r e v i e w s i n Concannon et a l . 1975; Verhage et a_l. 1976; E a y r s ejt a l . 1977; Feder 1981) and i n the r a t l o r d o s i s behaviour r e q u i r e s h i g h e r l e v e l s of e s t r a d i o l than t h a t n e c e s s a r y f o r v a g i n a l c o r n i f i c a t i o n , or g o n a d o t r o p h i n r e l e a s e (Feder 1981). The importance of e s t r o g e n s f o r mating b e h a v i o u r among m u s t e l i d s i s unknown and the o n l y s p e c i e s examined i n d e t a i l i s the mink. As mentioned p r e v i o u s l y , the p a t t e r n of e s t r o g e n s e c r e t i o n i n r i v e r o t t e r s seems t o be d i f f e r e n t from t h a t observed i n mink whose e s t r a d i o l c o n c e n t r a t i o n s d e c l i n e s i g n i f i c a n t l y b e f o r e b r e e d i n g o c c u r s ( T r a v i s e t a l . 1976; P i l b e a m e t a l . 1979). The presence of o v a r i a n development t y p i c a l of e s t r u s 162 w i t h o u t the accompanying e s t r o u s b e h a v i o u r , known as s i l e n t or q u i e t h e a t , has a l s o been obs e r v e d i n domestic mammals (mare, H i l l m a n and Loy 1975; cow, Robinson 1977; ewe, R o b e r t s o n 1977) and may be a c c e l e r a t e d by s t r e s s ( Robertson 1977). Such a n i m a l s may not be r e c e i v i n g s u f f i c i e n t e s t r o g e n or t h e r e may be an improper b a l a n c e between s t e r o i d l e v e l s needed t o t r i g g e r p s y c h o l o g i c e s t r u s (Turner and Bagnara 1971). A s i m i l a r s i t u a t i o n may have o c c u r r e d i n the c a p t i v e o t t e r s i n my s t u d y . 2. Pregnant females Serum e s t r a d i o l c o n c e n t r a t i o n s were low d u r i n g pregnancy i n the o t t e r . V a l u e s f l u c t u a t e d between 5 and 16 pg/ml, s i m i l a r t o t h o s e o b s e r v e d i n pregnant mink ( P i l b e a m e_t a l . 1979), European badgers (Mondain-Monval e t a_l. 1980) and s p o t t e d skunks ( R a v i n d r a and Mead 1984). D u r i n g the p r e i m p l a n t a t i o n p e r i o d , e s t r a d i o l c o n c e n t r a t i o n s i n pregnant o t t e r s v a r i e d s l i g h t l y w i t h peak l e v e l s o c c u r r i n g i n the m i d d l e of the p r e i m p l a n t a t i o n p e r i o d ( J u l y and A u g u s t ) . Mondain-Monval e t a l . (1980) a l s o r e p o r t e d i n c r e a s e d s e c r e t i o n of e s t r o g e n s d u r i n g the m i d d l e of the p r e i m p l a n t a t i o n p e r i o d i n the European badger which were c o r r e l a t e d w i t h p e r i o d i c waves of f o l l i c u l a r development and r e o c c u r r e d a t 7 t o 8 week i n t e r v a l s . S i m i l a r l y , the f l u c t u a t i n g e s t r o g e n l e v e l s o b s e r v e d i n t h i s s t u d y may be due t o f o l l i c u l a r development and a t r e s i a d u r i n g embryonic d i a p a u s e i n the o t t e r . E s t r a d i o l may be i m p o r t a n t i n p r o v i d i n g the p r o p e r hormonal environment f o r the s u r v i v a l of the b l a s t o c y s t s d u r i n g the p r e i m p l a n t a t i o n (Canivenc and Bonnin 1981). 163 No s i g n i f i c a n t i n c r e a s e i n e s t r a d i o l was obser v e d p r i o r t o i m p l a n t a t i o n i n t h i s study and e s t r a d i o l c o n c e n t r a t i o n s were s i m i l a r d u r i n g the p r e i m p l a n t a t i o n and p o s t i m p l a n t a t i o n p e r i o d s of pregnancy. T h i s suggest t h a t o t t e r s a re s i m i l a r t o o t h e r m u s t e l i d s i n t h a t i m p l a n t a t i o n does not appear t o be i n i t i a t e d by an i n c r e a s e i n e s t r a d i o l l e v e l s ( P i l b e a m e_t a_l. 1979; Mondain-Monval e_t a l . 1980; R a v i n d r a and Mead 1984). A l t h o u g h I found a s l i g h t i n c r e a s e i n a s i n g l e sample p r i o r t o i m p l a n t a t i o n , h i g h e r e s t r a d i o l l e v e l s o c c u r r e d e a r l i e r i n the p r e i m p l a n t a t i o n p e r i o d w i t h o u t i n d u c i n g i m p l a n t a t i o n . There i s no e v i d e n c e t h a t e s t r o g e n i s r e q u i r e d t o induce i m p l a n t a t i o n i n any o t h e r c a r n i v o r e and e s t r o g e n a d m i n i s t e r e d a l o n e or i n c o m b i n a t i o n w i t h p r o g e s t e r o n e has f a i l e d t o induce i m p l a n t a t i o n i n any m u s t e l i d (Mead and Wright 1981; R a v i n d r a and Mead 1984). In the s p o t t e d skunk, e s t r o g e n l e v e l s d e c r e a s e d as the diameter of the b l a s t o c y s t i n c r e a s e d p r i o r t o i m p l a n t a t i o n , s u g g e s t i n g t h a t e s t r o g e n may i n f a c t i n h i b i t l u t e a l f u n c t i o n and i m p l a n t a t i o n ( R a v i n d r a and Mead 1984). I t was not p o s s i b l e t o determine i f a s i m i l a r r e l a t i o n s h i p o c c u r s between e s t r a d i o l l e v e l s and i m p l a n t a t i o n i n the r i v e r o t t e r . Changes i n e s t r o g e n c o n c e n t r a t i o n s , n e c e s s a r y f o r i n i t i a t i n g i m p l a n t a t i o n , may have o c c u r r e d but were not r e c o g n i z e d due t o the s m a l l sample s i z e s , the l e n g t h of time between samples or the o v e r a l l v a r i a b i l i t y of the e s t r a d i o l c o n c e n t r a t i o n s . 164 3. P a r t i a l e s t r u s A p e r i o d of v a g i n a l p r o l i f e r a t i o n termed ' p a r t i a l e s t r u s ' was observed i n two of the t h r e e immature c a p t i v e o t t e r s w h i l e accompanying a d u l t s were i n e s t r u s . In mammals, development of the v a g i n a l e p i t h e l i u m r e f l e c t s i n c r e a s e s i n e s t r o g e n c o n c e n t r a t i o n s which a r e a s s o c i a t e d w i t h o v a r i a n development. Growing f o l l i c l e s a r e the p r i m a r y source of e s t r o g e n s and t h u s , v a g i n a l e p i t h e l i u m development i s r o u t i n e l y used as a b i o a s s a y f o r e s t r o g e n l e v e l s and an i n d i c a t o r of o v a r i a n a c t i v i t y (Turner and Bagnara 1971; P e t e r s and McNatty 1980). T h e r e f o r e , the changes i n the v a g i n a l smears observed i n these o t t e r s i n d i c a t e t h a t o v a r i a n development was o c c u r r i n g a l t h o u g h i t i s unknown i f the l e v e l of a c t i v i t y was g r e a t enough f o r o v u l a t i o n t o o c c u r . Both of the o t t e r s showing p a r t i a l e s t r u s were i n age c l a s s 2, an age when some w i l d females mate f o r the f i r s t time ( d e s c r i b e d e a r l i e r ) . Thus, these c a p t i v e females may have been e x h i b i t i n g a normal e s t r u s w i t h s l i g h t l y reduced v a g i n a l c o r n i f i c a t i o n as has been seen i n c a t s (D'Souza 1978). Complete c o r n i f i c a t i o n of the v a g i n a l e p i t h e l i u m may not d e v e l o p d u r i n g the f i r s t b r e e d i n g season a l t h o u g h i t was observed i n subsequent y e a r s i n F2. P a r t i a l e s t r u s d i d not occur i n any a d u l t female. T h i s s u g g e s t s t h a t p a r t i a l e s t r u s may be a s s o c i a t e d w i t h the f i r s t b r e e d i n g season of some o t t e r s . An a l t e r n a t i v e p o s s i b i l i t y i s t h a t r e p r o d u c t i v e development d i d o c c u r but was i n s u f f i c i e n t f o r o v u l a t i o n t o o c c u r . S t i m u l a t i o n of the o v a r i e s and r e p r o d u c t i v e t r a c t may have o c c u r r e d i n these o t t e r s but no p r e o v u l a t o r y f o l l i c l e s 1 65 dev e l o p e d . Wright (1966) found t h a t w h i l e the r e p r o d u c t i v e organs of j u v e n i l e American badgers a l l showed marked s t i m u l a t i o n d u r i n g the b r e e d i n g season, o n l y a m i n o r i t y would mate and c o n c e i v e . The f a i l u r e of t h e s e o t t e r s t o rea c h f u l l m a t u r i t y may e x p l a i n why the v a g i n a l smear r e t u r n e d t o the immature type i n both f e m a l e s . The r e s u l t s of the e s t r a d i o l a s s a y s d u r i n g p a r t i a l e s t r u s were not i n f o r m a t i v e due t o the h i g h v a r i a b i l i t y p r e s e n t . In 1979, p a r t i a l e s t r u s i n F2 was accompanied by s i g n i f i c a n t i n c r e a s e s i n e s t r a d i o l l e v e l s and p r e o v u l a t o r y f o l l i c l e s may have been p r e s e n t . However, a l t h o u g h b o t h of the a d u l t o t t e r s were mounted i n 1979, F2 was not mated and i t i s unknown i f she was f u l l y mature. E s t r a d i o l c o n c e n t r a t i o n s d i d not vary d u r i n g p a r t i a l e s t r u s i n F7 and l e v e l s were s i m i l a r t o those o b s e r v e d throughout the y e a r . The p a r t i a l e s t r o u s p e r i o d was of s h o r t d u r a t i o n and f o l l i c u l a r growth may have been l e s s than i n F2. S i g n i f i c a n t i n c r e a s e s i n e s t r a d i o l c o n c e n t r a t i o n s were not observed among the a d u l t o t t e r s i n 1981 and t h u s , no c o n c l u s i o n s c o n c e r n i n g the degree of o v a r i a n a c t i v i t y and e s t r o g e n l e v e l s d u r i n g t h i s p e r i o d i n F7 can be made. Al t h o u g h the degree of s e x u a l development among i n d i v i d u a l s of the same age may be a f f e c t e d . b y body s i z e ( S a d l e i r 1969a), no s i g n i f i c a n t v a r i a t i o n s i n weight were o b s e r v e d among the t h r e e immature o t t e r s . Because the t h i r d female (F6) d i d not show e s t r u s u n t i l age c l a s s 3, t h i s i n d i c a t e s t h a t the d i f f e r e n c e s i n the degree of s e x u a l a c t i v i t y o b s e r v e d among t h e s e t h r e e females were f o r reasons o t h e r than d i f f e r e n c e s i n body we i g h t . 166 H. P r o g e s t e r o n e c o n c e n t r a t i o n s J_. Nonpregnant and immature o t t e r s P r o g e s t e r o n e c o n c e n t r a t i o n s among c a p t i v e j u v e n i l e and nonpregnant a d u l t o t t e r s remained low (<1 ng/ml) throughout the year and were s i m i l a r t o tho s e o b t a i n e d from w i l d j u v e n i l e o t t e r s . The s l i g h t , but s t a t i s t i c a l l y i n s i g n i f i c a n t , i n c r e a s e observed among the a d u l t s d u r i n g J a n u a r y and F e b r u a r y o c c u r r e d near the p e r i o d of r a p i d f o l l i c u l a r development p r i o r t o e s t r u s and was s h o r t l y a f t e r a s i m i l a r r i s e i n e s t r a d i o l c o n c e n t r a t i o n s . S l i g h t i n c r e a s e s i n p r o g e s t e r o n e l e v e l s have a l s o been observed i n mink near t h e i n i t i a t i o n of f o l l i c u l a r development ( P i l b e a m e t a l . 1979) and i n s p o t t e d skunks where they c o i n c i d e d w i t h e s t r u s (Mead and Eik-Nes 1969b). However, the s i g n i f i c a n c e of these i n c r e a s e s a r e unknown and the importance, i f any, of t h i s s l i g h t r i s e t o f o l l i c u l a r development i n the o t t e r c o u l d not be de t e r m i n e d . F o l l o w i n g e s t r u s , p r o g e s t e r o n e c o n c e n t r a t i o n s of unmated o t t e r s d i d not i n c r e a s e s u g g e s t i n g t h a t o v u l a t i o n and the f o r m a t i o n of c o r p o r a l u t e a does not occur s p o n t a n e o u s l y i n the o t t e r . 2. Pregnancy The serum p r o g e s t e r o n e c o n c e n t r a t i o n s observed d u r i n g pregnancy i n a c a p t i v e o t t e r a r e c o n s i s t e n t w i t h the h i s t o l o g i c a l changes which o c c u r i n the l u t e a l c e l l c y t o l o g y of w i l d o t t e r s . The r i s e i n p r o g e s t e r o n e c o n c e n t r a t i o n s observed 167 f o l l o w i n g mating c o r r e s p o n d s t o the f o r m a t i o n of the r e l a t i v e l y i n a c t i v e c o r p o r a l u t e a of p r e i m p l a n t a t i o n found i n w i l d o t t e r s . S i m i l a r p r o g e s t e r o n e l e v e l s were a l s o seen i n w i l d females d u r i n g the d e l a y p e r i o d . At the time when i m p l a n t a t i o n i s assumed t o have o c c u r r e d (see Chapter I V ) , p r o g e s t e r o n e l e v e l s r o s e d r a m a t i c a l l y , r e f l e c t i n g the i n c r e a s e d l u t e a l a c t i v i t y seen d u r i n g the p o s t i m p l a n t a t i o n p e r i o d i n w i l d o t t e r s . S i m i l a r p r o g e s t e r o n e p r o f i l e s and c o r r e l a t i o n s w i t h l u t e a l c y t o l o g y have been r e p o r t e d f o r o t h e r m u s t e l i d s which e x h i b i t a p e r i o d of d e l a y i n i m p l a n t a t i o n (see r e v i e w s i n Mead 1981; Mead and Wright 1983). Thus, the o t t e r i s s i m i l a r t o o t h e r m u s t e l i d s i n t h a t l u t e a l a c t i v i t y and p r o g e s t e r o n e c o n c e n t r a t i o n s a r e low d u r i n g the p e r i o d of embryonic d i a p a u s e and t h a t i m p l a n t a t i o n i s a s s o c i a t e d w i t h a s i g n i f i c a n t i n c r e a s e i n a c t i v i t y of the l u t e a l c e l l s and p r o g e s t e r o n e l e v e l s . A l t h o u g h h i s t o l o g i c a l e x a m i n a t i o n s of c o r p o r a l u t e a d u r i n g l a t e pregnancy a re n e c e s s a r y t o determine when l u t e o l y s i s o c c u r s , they were not a v a i l a b l e i n t h i s s t u d y . However, s i n c e serum p r o g e s t e r o n e l e v e l s d e c l i n e d d u r i n g the l a t t e r h a l f of pregnancy i n the o t t e r and the ovary has been i d e n t i f i e d as the p r i m a r y s o u r c e of p r o g e s t e r o n e d u r i n g pregnancy i n m u s t e l i d s such as the mink and the s p o t t e d skunk ( M o l l e r 1974; Mead and Swannack 1978), i t appears t h a t l u t e a l d e g e n e r a t i o n may occur p r i o r t o p a r t u r i t i o n i n the o t t e r . I f p a r t u r i t i o n i s assumed t o occur i n l a t e F ebruary or e a r l y March, as i n d i c a t e d by the s w e l l i n g of the t e a t s and e n l a r g e d v u l v a a t t h i s t i m e , then p r o g e s t e r o n e l e v e l s d e c l i n e d a p p r o x i m a t e l y h a l f way through the 168 60-65 day p o s t i m p l a n t a t i o n p e r i o d ( H a m i lton and E a d i e 1964; Lauhachinda 1978; t h i s s t u d y ) . A d e c l i n e i n t h e p r o g e s t e r o n e c o n c e n t r a t i o n s d u r i n g the l a t t e r h a l f of pregnancy has been obser v e d i n a number of pregnant and/or pseudopregnant m u s t e l i d s (mink, M o l l e r 1973, P i l b e a m e t a l . 1979; f e r r e t s , Heap and Hammond 1974; s t r i p e d skunks, Wade-Smith et a_l. 1 980; s p o t t e d skunks, Mead and Eik-Nes 1969b). In some mammals, the d e c l i n e i n p r o g e s t e r o n e l e v e l s may f a c i l i t a t e normal p a r t u r i t i o n (Concannon et a l . 1975). My study i n d i c a t e s t h a t the p a t t e r n of p r o g e s t e r o n e s e c r e t i o n d u r i n g pregnancy i s v e r y s i m i l a r i n the r i v e r o t t e r t o those of o t h e r m u s t e l i d s . A l t h o u g h changes i n the c o r p o r a l u t e a and p r o g e s t e r o n e c o n c e n t r a t i o n s are t e m p o r a l l y c o r r e l a t e d w i t h changes i n the development of the embryo, the f a c t o r s which c o n t r o l embryonic diapause and i m p l a n t a t i o n i n the o t t e r are unknown. The c o n t r o l of d i a p a u s e and n i d a t i o n has been examined i n a v a r i e t y of m u s t e l i d s (see r e v i e w s i n Renfree 1978; Mead 1981; Mead and Wright 1983) and i n a l l , the ovary i s r e q u i r e d f o r b l a s t o c y s t s u r v i v a l d u r i n g d i a p a u s e and f o r the i n d u c t i o n of i m p l a n t a t i o n . However, a l l a t t e m p t s t o induce i m p l a n t a t i o n by a d m i n i s t e r i n g p r o g e s t e r o n e t o i n t a c t or o v a r i e c t o m i z e d m u s t e l i d s have f a i l e d , i n d i c a t i n g t h a t p r o g e s t e r o n e a l o n e i s not s u f f i c i e n t t o induce i m p l a n t a t i o n (Mead 1981; R a v i n d r a and Mead 1984). An a d d i t i o n a l , but unknown, f a c t o r ( s ) of l u t e a l o r i g i n i s / a r e n e c e s s a r y f o r i m p l a n t a t i o n t o o c c u r i n the the f e r r e t and mink (Foresman and Mead 1978; Murphy et a l . 1983). The d r a m a t i c changes which occur i n the l u t e a l c e l l s and p r o g e s t e r o n e 169 concentrations of otters at or shortly before renewed development and implantation suggest that similar l u t e a l factors may control embryonic diapause in the rive r otter. The synchronous nature of the timing of implantation among in d i v i d u a l otters suggests that an environmental factor may t r i g g e r the end of embryonic diapause (see Chapter IV). This factor may act upon the endocrinological axes in some manner which i s yet unknown to affect hormonal changes which result in the increased l u t e a l a c t i v i t y observed prior to implantation. In a l l of the mustelids which have been examined, this increase in l u t e a l function i s dependent exclusively upon hormonal cues from the p i t u i t a r y and does not involve hormones of embryonic or placental o r i g i n (Mead 1975; Murphy and Moger 1977; Mead and Swannack 1978; Duby e_t a_l. 1972). Prolactin has been found to maintain l u t e a l function and i n i t i a t e implantation in mink (Papke et a l . 1980; Martinet et a l . 1981; Murphy et a l . 1981; Murphy 1983) and fe r r e t s (Donovan 1967; Murphy 1979; McKibbin et a l . 1984) although i t s involvement in terminating embryonic diapause in other mustelids such as the river otter i s questionable. Otters breed during the postpartum period and embryonic diapause begins while the female i s l a c t a t i n g . If suckling elevates p r o l a c t i n levels in the otter as in other mammals (Turner and Bagnara 1971; Tepperman 1980), th i s would indicate that increased pr o l a c t i n does not induce l u t e a l function and implantation early in the delay period. The cessation of lactation also has no e f f e c t on diapause .in the otter although i t does end f a c u l t a t i v e diapause in some 170 m a r s u p i a l s (Renfree 1981). The c o u r s e of pregnancy i s s i m i l a r l y u n a f f e c t e d by l a c t a t i o n i n the European badger (Canivenc and Bonnin 1981) and o v i n e p r o l a c t i n d i d not a l t e r c o r p o r a l u t e a f u n c t i o n i n the s p o t t e d skunk (Mead 1981). I t appears t h a t the p i t u i t a r y c o n t r o l of embryonic d i a p a u s e can v a r y g r e a t l y among d i f f e r e n t s p e c i e s even w i t h i n one f a m i l y . 3. Pseudopregnancy One female (F3) underwent an a p p a r e n t l y normal p e r i o d of pregnancy but f a i l e d t o g i v e b i r t h . T h i s may have been because she re a b s o r b e d the embryos e a r l y i n pregnancy, a b o r t e d , d e s t r o y e d her pups, or o v u l a t e d w i t h , or w i t h o u t , mating and e n t e r e d pseudopregnancy. There was no e v i d e n c e of a b o r t i o n or p a r t u r i t i o n and the .female appeared t o be ready t o g i v e b i r t h the s p r i n g f o l l o w i n g m a t i n g . She had an e n l a r g e d v u l v a and s w o l l e n t e a t s a l t h o u g h no young were found and b l o o d was not p r e s e n t i n her v u l v a or i n the nest box straw t o i n d i c a t e t h a t b i r t h had o c c u r r e d . S t r e s s has been shown t o i n h i b i t r e p r o d u c t i o n i n mink ( G i l b e r t and B a i l e y 1967; D a n i e l s 1971) and the s t r e s s of c a p t i v i t y and the f r e q u e n t e x a m i n a t i o n s may have t r i g g e r e d embryo r e s o r p t i o n i n F3. An i n c r e a s e i n c o r p o r a l u t e a a c t i v i t y and p r o g e s t e r o n e c o n c e n t r a t i o n s a s s o c i a t e d w i t h the normal p e r i o d of i m p l a n t a t i o n was a l s o o b s e r v e d i n s t r i p e d skunks which f a i l e d t o g i v e b i r t h (Wade-Smith e_t a_l 1980) and i n unmated mink which had o v u l a t e d s p o n t a n e o u s l y ( M o l l e r 1974). I t i s p o s s i b l e t h a t t h i s c a p t i v e female underwent pseudopregnancy. Pseudopregnancy has not been d e s c r i b e d i n the 171 river otter although i t has been reported or suggested in other mustelids with embryonic diapause (Enders 1952; Moller 1974; Wade-Smith et a l . 1980). If F3 was pseudopregnant, i t suggests that pseudopregnancy is of approximately the same duration as pregnancy in otters, as i t i s in ferrets (Heap and Hammond 1974), cats (Verhage et a l 1976) and possibly striped skunks (Wade-Smith et a_l. 1980). In these animals, true pregnancy and pseudopregnancy can only be distinguished by the lack of implanted embryos during the postimplantation period. Although one otter with corpora lutea of postimplantation, but no embryos or uterine swellings, was observed in this study, i t i s not known i f the lack of embryos was due to pseudopregnancy. Blastocysts which may have implanted in the near future may have been present, but were not found due to the poor state of preservation. The occurrence of psuedopregnancy may also explain the increased progesterone concentrations observed in F3 during 1981 when she was not observed to mate and may have been induced to ovulate by repeated vaginal smears. In summary, this study provides the f i r s t detailed description of the histology and endocrinology of the annual reproductive cycle of female river otters and describes the reproductive chronology of otters in coastal B r i t i s h Columbia. There, otters are monestrous, induced ovulators, exhibiting a seasonally regulated reproductive cycle which includes a prolonged obligatory period of embryonic diapause prior to implantation. Ovarian a c t i v i t y began in the late autumn or early winter and estrus usually occurred between early March and 1 72 mid May. F o l l o w i n g f e r t i l i z a t i o n the b l a s t o c y s t entered a nine to eleven month dormant p e r i o d , d u r i n g which the l u t e a l c e l l s and the u t e r i n e glands showed low l e v e l s of a c t i v i t y . Implantations occurred i n February and f o l l o w i n g a g e s t a t i o n p e r i o d of approximately two months, b i r t h s were from l a t e March through A p r i l . Mating oc c u r r e d d u r i n g l a c t a t i o n i n p a r t u r i e n t o t t e r s . Some o t t e r s a t t a i n e d sexual maturity at the end of t h e i r second year while i t was delayed u n t i l the f o l l o w i n g year in the remainder of the p o p u l a t i o n . The changes which occurred i n the r e p r o d u c t i v e organs and the progesterone c o n c e n t r a t i o n s during the annual r e p r o d u c t i v e c y c l e were s i m i l a r to those o c c u r r i n g in other mustelids with embryonic diapause. The v a g i n a l smear types were found to i d e n t i f i e d c o r r e c t l y the r e p r o d u c t i v e s t a t e of o t t e r s and, although e s t r a d i o l c o n c e n t r a t i o n s were h i g h l y v a r i a b l e i n t h i s study, the values obtained and the p a t t e r n of s e c r e t i o n are s i m i l a r to those of other c a r n i v o r e s . E s t r a d i o l surges were observed i n the f i r s t year of the study only although e s t r a d i o l l e v e l s i n c r e a s e d d u r i n g e s t r u s i n a l l years. The reduced estrous behaviour observed among c a p t i v e animals may have been a consequence of the lower e s t r a d i o l c o n c e n t r a t i o n s which occurred d u r i n g the f i n a l years of the study. 173 CHAPTER IV THE EFFECTS OF INCREASED PHOTOPERIOD ON REPRODUCTIVE ACTIVITY 174 INTRODUCTION In p r e v i o u s c h a p t e r s I have shown t h a t i n B r i t i s h Columbia, c o a s t a l r i v e r o t t e r s a re s e a s o n a l b r e e d e r s and t h a t t i m i n g of r e p r o d u c t i v e e v e n t s i s s i m i l a r t o t h a t of o t t e r s l i v i n g a t the same l a t i t u d e , but i n f r e s h w a t e r . As s e a s o n a l b r e e d e r s , o t t e r s presumably have adapted t h e i r l i f e c y c l e s t o ensure t h a t r e p r o d u c t i v e e v e n t s (and b i r t h i n p a r t i c u l a r ) occur a t a time when food a v a i l a b i l i t y and c l i m a t i c c o n d i t i o n s combine t o m i n i m i z e the p r o b a b i l i t y of r e p r o d u c t i v e f a i l u r e . In a s e a s o n a l environment, such as t h a t of B r i t i s h Columbia, s e a s o n a l c o n d i t i o n s a r e p r e d i c t a b l e and an e n v i r o n m e n t a l cue may be used t o induce the m e t a b o l i c p r e p a r a t i o n n e c e s s a r y f o r r e p r o d u c t i o n . A v a r i e t y of e n v i r o n m e n t a l f a c t o r s a r e known t o s t i m u l a t e r e p r o d u c t i o n i n mammals; they i n c l u d e food a v a i l a b i l i t y , s o c i a l cues, t e m p e r a t u r e , r a i n f a l l and d a i l y p h o t o p e r i o d . Because of embryonic d i a p a u s e , the t i m i n g of b i r t h i n o t t e r s i s d e t e r m i n e d by the date of i m p l a n t a t i o n , r a t h e r than mating i t s e l f . T h e r e f o r e , a f a c t o r , or c o m b i n a t i o n of f a c t o r s , may s e r v e as a s t i m u l u s f o r mating and i m p l a n t a t i o n t o o c c u r , r e s u l t i n g i n b i r t h a t an a p p r o p r i a t e t i m e . In B r i t i s h Columbia, female r i v e r o t t e r s e x h i b i t e s t r u s from March t h r o u g h mid-May. F o l l o w i n g mating t h e r e i s a p r o l o n g e d p e r i o d of embryonic d i a p a u s e d u r i n g which l u t e a l a c t i v i t y i s low (Chapter I I I ) . The subsequent F e b r u a r y , l u t e a l a c t i v i t y i n c r e a s e s s i g n i f i c a n t l y , embryos i m p l a n t , and b i r t h s o c cur from l a t e March t h r o u g h A p r i l . In male o t t e r s , r e p r o d u c t i v e a c t i v i t y b e g i n s i n the l a t e autumn, w i t h a r a p i d 175 i n c r e a s e i n sper m a t o g e n e s i s and t e s t i s development o c c u r r i n g around the w i n t e r s o l s t i c e (Chapter I I ) . T e s t o s t e r o n e c o n c e n t r a t i o n s i n c r e a s e s i g n i f i c a n t l y between December and Feb r u a r y and peak r e p r o d u c t i v e a c t i v i t y o c c u r s from l a t e F e b r u a r y t h r o u g h A p r i l . In the p r e v i o u s c h a p t e r I suggested t h a t r e g i o n a l v a r i a t i o n s i n r e p r o d u c t i v e t i m i n g a r e c o r r e l a t e d w i t h changes i n l a t i t u d e , r a t h e r than w i t h l o c a l e n v i r o n m e n t a l c o n d i t i o n s . In a d d i t i o n , c a p t i v e o t t e r s w i t h abundant food have s i m i l a r r e p r o d u c t i v e c y c l e s t o those of w i l d o t t e r s . T h i s suggests t h a t l o c a l c o n d i t i o n s of temperature and food a v a i l a b i l i t y may not be important as p r o x i m a l f a c t o r s i n c o n t r o l l i n g t he r e p r o d u c t i v e c y c l e of o t t e r s . I n s t e a d , r e p r o d u c t i o n may be i n f l u e n c e d by a f a c t o r , such as p h o t o p e r i o d , which i s c o n s i s t e n t between s i m i l a r l a t i t u d e s and i s not r e l a t e d t o l o c a l c o n d i t i o n s . A l t h o u g h the i n f l u e n c e of e n v i r o n m e n t a l f a c t o r s on the r e p r o d u c t i v e c y c l e of o t t e r s has not been s t u d i e d b e f o r e , p h o t o p e r i o d i s a common p r e d i c t o r of annual e v e n t s i n temperate mammals ( S a d l e i r 1969a; Hoffman 1973; H e r b e r t 1977; Turek and Campbell 1979; Bronson 1985). I t has a l s o been shown t o induce r e p r o d u c t i v e a c t i v i t y i n a number of m u s t e l i d s , i n c l u d i n g the f e r r e t , M u s t e l a p u t o r i u s ( B i s s o n n e t t e 1932; Donovan 1967; H e r b e r t 1969; Thorpe and He r b e r t 1976), the mink, M u s t e l a v i son (Hammond 1951; Duby and T r a v i s 1972; Murphy and James 1974; B o i s s i n - A g a s s e e t a_l. 1982), the European badger, Meles meles (Canivenc and Bonnin 1979; Canivenc e_t a l . 1981 ), the western s p o t t e d skunk, S p i l o g a l e  p u t o r i u s l a t i f r o n s (Mead 1971), and the l o n g - t a i l e d w e a sel, 176 M u s t e l a f r e n a t a (Wright 1963). In B r i t i s h Columbian o t t e r s , i m p l a n t a t i o n , e s t r u s and peak spermatogenesis o c c u r d u r i n g s p r i n g when p h o t o p e r i o d i s i n c r e a s i n g . The o b j e c t i v e of t h i s s e c t i o n of the study was t o det e r m i n e the i n f l u e n c e of i n c r e a s e d p h o t o p e r i o d on the t i m i n g of the r e p r o d u c t i v e c y c l e . Because of t h e r e s t r i c t e d n a t u r e of the b r e e d i n g season and the l i m i t e d a v a i l a b i l i t y of a n i m a l s , a s e r i e s of t h r e e e x p e r i m e n t s was performed. In each, e x p e r i m e n t a l a n i m a l s were exposed t o a long p h o t o p e r i o d (16 hr) t o determine i f t h i s exposure would h a s t e n the onset of e s t r u s or the peak of s p e r m a t o g e n e s i s . A l t h o u g h the number of pregnant o t t e r s was l i m i t e d , the a b i l i t y of i n c r e a s e d d a y l e n g t h t o induce i m p l a n t a t i o n was a l s o examined. 1 77 MATERIALS AND METHODS A s e r i e s of t h r e e e x p e r i m e n t s was conducted a t the U n i v e r s i t y of B r i t i s h Columbia ( l a t i t u d e 49° 15' N, l o n g i t u d e 123° 15' W) t o d e t e r m i n e the e f f e c t s of i n c r e a s e d p h o t o p e r i o d on the r e p r o d u c t i v e c y c l e of r i v e r o t t e r s . W i l d o t t e r s were c a p t u r e d i n c o a s t a l r e g i o n s of southwestern B r i t i s h Columbia and h e l d a t the Animal Care C e n t r e as d e s c r i b e d i n C h a p t e r s I I and I I I . In male o t t e r s , t e s t i s volumes, which have been c l o s e l y c o r r e l a t e d w i t h o t h e r i n d i c a t o r s of r e p r o d u c t i v e a c t i v i t y (Chapter I I ) , and serum t e s t o s t e r o n e c o n c e n t r a t i o n s were measured t o determine the degree of r e p r o d u c t i v e a c t i v i t y . In a d d i t i o n , t e s t i c u l a r b i o p s i e s were performed monthly d u r i n g the f i n a l experiment as d e s c r i b e d i n Chapter I I . Tubule d i a m e t e r s were measured and phase a n a l y s i s was used t o determine the degree of s p e r m a t o g e n e s i s p r e s e n t . V a g i n a l smear t y p e s and gonadal s t e r o i d c o n c e n t r a t i o n s ( e s t r o g e n and p r o g e s t e r o n e ) were used as i n d i c a t o r s of r e p r o d u c t i v e a c t i v i t y i n f e m a l e s . Females were c o n s i d e r e d t o be i n e s t r u s when the v a g i n a l smear c o n s i s t e d p r i m a r i l y of c o r n i f i e d e p i t h e l i a l c e l l s as d e s c r i b e d i n Chapter I I I . E x a m i n a t i o n s c h e d u l e s and radioimmunoassay t e c h n i q u e s have been d e s c r i b e d i n C h a p t e r s I I and I I I . The e x p e r i m e n t a l p r o t o c o l s f o r the t h r e e e x p e r i m e n t s are summarized i n T a b l e XIX. In each e x p e r i m e n t , a n i m a l s were exposed t o e i t h e r an i n c r e a s e d p h o t o p e r i o d of 16 hours of d a y l i g h t (16L:8D) or a n a t u r a l l i g h t regime which ranged between 8.2L:15.8D and 16.2L:7.8D (Anon. 1982). To c o n t r o l f o r the p o s s i b l e t r i g g e r i n g e f f e c t of s h o r t p h o t o p e r i o d upon T a b l e X V I X . Summary o f p r o t o c o l s o f t h r e e e x p e r i m e n t s t o t e s t t h e e f f e c t o f i n c r e a s e d p h o t o p e r i o d on t h e t i m i n g o f r e p r o d u c t i v e c y c l e s o f r i v e r o t t e r s . E x p e r i m e n t D a t e Group A n i m a l s P h o t o p e r i o d T e m p e r a t u r e D e c . 2 0 , 1978 -May 1 1 , 1979 D e c . 9 , 1979 -J u n e 3 0 , 1980 N o v . 4 , 1980 -J u n e 3 0 , 1981 C o n t r o l E x p e r i m e n t a l C o n t r o l E x p e r i m e n t a l C o n t r o l 1 C o n t r o l 2 b E x p e r i m e n t a l D Ml M2 F 2 * F5 M3 F3 Ml M2 F2 F5 M2 M5 F3 M3 M5 F3 F6 M6 F5 M2 F2 F 7 * N a t u r a l 16L :8D N a t u r a l 16L :8D N a t u r a l N a t u r a l 0 1 6 L : 8 D N a t u r a l N a t u r a l N a t u r a l N a t u r a l N a t u r a l 10°C 10°C a - Age c l a s s 2 f e m a l e s underwent p a r t i a l e s t r u s o n l y . b - E x p o s e d t o a p h o t o p e r i o d o f 8L :16D f rom O c t o b e r 2 - November 4 , 1980 . c - N a t u r a l p h o t o p e r i o d a p p r o x i m a t e d i n an e n v i r o n m e n t a l chambe r . 179 spermatogenesis i n experiment 3, both groups i n the e x p e r i m e n t a l chambers ( c o n t r o l 2 and e x p e r i m e n t a l ) were s u b j e c t e d t o a p h o t o p e r i o d of 8L:16D from October 2 u n t i l November 4 1980. E x p e r i m e n t a l a n i m a l s were housed i n v e n t i l a t e d , l i g h t p roof rooms (3.4 X 3.4 m or 3.4 X 4.0 m), each c o n t a i n i n g two den boxes and a p o o l w i t h f r e s h , r u n n i n g water. The f l u o r e s c e n t and i n c a n d e s c e n t l i g h t s i l l u m i n a t i n g the e x p e r i m e n t a l chambers p r o v i d e d a l i g h t i n t e n s i t y of 2000 l u x (3.0 W/m2) a t ground l e v e l i n the c e n t r e of the room. In the f i r s t two experiments the temperature of the room was u n c o n t r o l l e d and approximated t h a t of the c o n t r o l pens, a l t h o u g h i t was s l i g h t l y h i g h e r (2-8°C) on c o l d e r days. In the t h i r d e x p e r i m e n t , e x p e r i m e n t a l a n i m a l s were housed i n a room i n which the temperature was c o n t r o l l e d at 10°C. C o n t r o l a n i m a l s were h e l d i n pens exposed t o n a t u r a l d a y l i g h t and temperature as d e s c r i b e d i n Chapter I I , w i t h the e x c e p t i o n of the o t t e r s i n c o n t r o l group 2, experiment 3. These a n i m a l s were housed i n e x p e r i m e n t a l chambers and f o l l o w i n g a p e r i o d of 8L:16D, the l i g h t regime was a l t e r e d weekly t o mimic the n a t u r a l p h o t o p e r i o d . To c o n t r o l f o r the e f f e c t s of i n c r e a s e d t emperature i n the e x p e r i m e n t a l chambers, t h i s room was a l s o m a i n t a i n e d a t 10°C. 180 RESULTS I_. The e f f e c t of i n c r e a s e d p h o t o p e r i o d on female o t t e r s A. E s t r u s J_. Experiment _1_ The r e s u l t s of the f i r s t e xperiment suggests t h a t i n c r e a s e d p h o t o p e r i o d i n d u c e s e a r l y e s t r u s among e x p e r i m e n t a l females. E s t r o u s v a g i n a l smears were obser v e d i n the e x p e r i m e n t a l female at the b e g i n n i n g of F e b r u a r y , a p p r o x i m a t e l y s i x weeks a f t e r the b e g i n n i n g of the e x p e r i m e n t , whereas the a d u l t female i n the c o n t r o l group d i d not e n t e r e s t r u s u n t i l the b e g i n n i n g of March ( F i g . 4 6 ) . The age c l a s s 2 f e m a l e , which was a l s o i n the c o n t r o l group, d i d not e x h i b i t a f u l l e s t r u s but i n s t e a d showed a p a r t i a l e s t r u s (Chapter I I I ) which c o i n c i d e d w i t h e s t r u s i n the c o n t r o l a d u l t . The e s t r o u s p e r i o d s of both c o n t r o l a n i m a l s were s i m i l a r t o those of w i l d o t t e r s i n B r i t i s h Columbia (Chapter I I I ) and e s t r u s c o n t i n u e d u n t i l May i n a l l of the females i n t h i s e x p e r i m e n t . I t s d u r a t i o n i n the e x p e r i m e n t a l female (13 weeks) was g r e a t e r than t h a t observed i n e i t h e r of the c o n t r o l f e m a l e s . A l t h o u g h e s t r u s d i d occur e a r l i e r i n the e x p e r i m e n t a l a d u l t than i n the c o n t r o l a n i m a l s , the e f f e c t of the i n c r e a s e d p h o t o p e r i o d i s unknown, because i n a subsequent experiment d e s c r i b e d below, e s t r u s was observed t o occur i n e a r l y F e b r u a r y i n a female under n a t u r a l p h o t o p e r i o d . E s t r a d i o l c o n c e n t r a t i o n s of i n d i v i d u a l o t t e r s d u r i n g the 181 F i g u r e 46. Experiment 1. E s t r a d i o l c o n c e n t r a t i o n s and e s t r o u s p e r i o d s ( s t i p p l e d ) of female o t t e r s exposed t o a p h o t o p e r i o d of 16L:8D (F3) or under n a t u r a l l i g h t regimes (F5, F 2 ) . Arrows i n d i c a t e (1) when the p h o t o p e r i o d was f i r s t i n c r e a s e d and (2) when mating at t e m p t s o c c u r r e d . F2 underwent p a r t i a l e s t r u s o n l y (see Chapter I I I ) . 182 O ' N ' D ' J ' F ' M ' A ' M ' J Month 183 f i r s t e xperiment a r e shown i n F i g . 4 6 . As mentioned i n Chapter I I I , changes i n the v a g i n a l smears were a b e t t e r i n d i c a t o r of r e p r o d u c t i v e a c t i v i t y than e s t r a d i o l , because l e v e l s were h i g h l y v a r i a b l e and showed s i g n i f i c a n t changes i n the f i r s t year of the s t u d y o n l y . No r e l a t i o n c o u l d be d e t e r m i n e d between the t i m i n g of e s t r u s and the e s t r a d i o l s u r g e s , a l t h o u g h e s t r a d i o l c o n c e n t r a t i o n s were s l i g h t l y h i g h e r d u r i n g e s t r u s than d u r i n g a n e s t r u s (see Chapter I I I ) . Peak e s t r a d i o l c o n c e n t r a t i o n s o c c u r r e d s l i g h t l y e a r l i e r i n the e x p e r i m e n t a l female, however, the s i g n i f i c a n c e of t h i s d i f f e r e n c e i s unknown due t o the h i g h v a r i a b i l i t y of the l e v e l s . Both the e x p e r i m e n t a l female (F3) and the a d u l t c o n t r o l (F5) were i n v o l v e d i n mating a t t e m p t s d u r i n g t h i s e x p e r i m e n t , a l t h o u g h matings o c c u r r e d a t s i m i l a r t i m e s i n b o t h a n i m a l s . 2. Experiment 2 The e s t r o u s p e r i o d s of females d u r i n g experiment 2 are summarized i n F i g . 47. The e x p e r i m e n t a l female e x h i b i t e d a very s h o r t e s t r u s which began i n the second week of March, a p p r o x i m a t e l y two t o t h r e e weeks a f t e r the e s t i m a t e d end of pregnancy. Among the c o n t r o l a n i m a l s e s t r o u s p e r i o d s v a r i e d g r e a t l y ; one female (F5) had c o r n i f i e d v a g i n a l smears a t the b e g i n n i n g of F e b r u a r y w h i l e the second (F2) d i d not e n t e r e s t r u s u n t i l e a r l y March. The e s t r o u s p e r i o d of the former was u n u s u a l l y e a r l y because i t d i d not b e g i n b e f o r e l a t e F e b r u a r y i n any of my o t h e r c o n t r o l f e m a l e s . Due t o the low and v a r i a b l e n a t u r e of the e s t r a d i o l l e v e l s 184 F i g u r e 47. Experiment 2. E s t r o u s p e r i o d s of e x p e r i m e n t a l ( s t i p p l e d ) and c o n t r o l female o t t e r s ( c r o s s - h a t c h e d ) The e x p e r i m e n t a l o t t e r was f i r s t exposed t o a p h o t o p e r i o d of 16L:8D on December 9. F i g u r e 48. Experiment 3. E s t r o u s p e r i o d s of c o n t r o l , Groups 1 ( c r o s s - h a t c h e d ) and 2 (open b o x ) , and e x p e r i m e n t a l , Group 3 ( s t i p p l e d ) , female o t t e r s . F7 underwent a p e r i o d of p a r t i a l e s t r u s o n l y (see Chapter I I I ) . Groups 2 and 3 were exposed t o a s h o r t p h o t o p e r i o d (8L:16D) on October 2. On November 4 group 3 was exposed t o a l o n g p h o t o p e r i o d (16L:8D) and group 2 was r e t u r n e d t o a n a t u r a l l i g h t regime. F i g u r e 49. P r o g e s t e r o n e c o n c e n t r a t i o n s of an o t t e r which f a i l e d t o g i v e b i r t h w h i l e exposed t o a p h o t o p e r i o d of 16L:8D d u r i n g experiment 2. Arrow i n d i c a t e s when p h o t o p e r i o d was f i r s t i n c r e a s e d . F i g u r e 47 F 3 F 5 F 2 F M A Month M F 7 F 2 ^ ^ ^ ^ ^ ^ f f l F 5 [—: F 6 F 3 F igu re 4 8 F M A Month M 2 5 i E 2 0 CD C o CD 1 5 -10 CO CD CO O F i g u r e 4 9 V ~ ~ I 1 1 1 1 1 O N D J F M Month 186 o b s e r v e d , i t c o u l d not be d e t e r m i n e d i f c o n c e n t r a t i o n s v a r i e d between the e x p e r i m e n t a l and c o n t r o l groups. Surges i n e s t r a d i o l were not obser v e d d u r i n g E x p e r i m e n t s I I and I I I , and no r e l a t i o n s h i p c o u l d be ob s e r v e d between serum l e v e l s and the t i m i n g of e s t r u s . T h e r e f o r e , e s t r a d i o l c o n c e n t r a t i o n s o b s e r v e d d u r i n g the f i n a l two e x p e r i m e n t s have not been p r e s e n t e d i n t h i s s t u d y . They have been d e s c r i b e d i n Chapter I I I and a r e p r e s e n t e d i n Appendix 3. 3. Experiment 3 The b e s t e v i d e n c e f o r the advancement of e s t r u s by i n c r e a s e d p h o t o p e r i o d was p r o v i d e d by the t h i r d e x p e r i m e n t . The i n c r e a s e d d a y l e n g t h i n d u c e d gonadal a c t i v i t y , which r e s u l t e d i n the o c c u r r e n c e of e s t r u s i n e x p e r i m e n t a l females w e l l b e f o r e t h a t of e i t h e r c o n t r o l group 1 or 2 ( F i g . 4 8 ) . E s t r o u s v a g i n a l smears were observed among the Group 3 females a p p r o x i m a t e l y e i g h t weeks a f t e r the p h o t o p e r i o d was i n c r e a s e d . Note, however, t h a t a l t h o u g h one of the females (F7) underwent a b r i e f p e r i o d of p a r t i a l e s t r u s o n l y , i t s onset d i d c o i n c i d e w i t h the f u l l e s t r u s observed i n the o t h e r e x p e r i m e n t a l o t t e r . The date of f i r s t e s t r u s d i d not v a r y s i g n i f i c a n t l y among females i n the two c o n t r o l groups and t h i s i n d i c a t e s exposure t o a s h o r t p h o t o p e r i o d and h i g h e r t e m p e r a t u r e s d i d not induce the e a r l y e s t r u s o b s e r v e d i n the e x p e r i m e n t a l a n i m a l s . 187 B. I m p l a n t a t i o n The s i g n i f i c a n t i n c r e a s e i n p r o g e s t e r o n e c o n c e n t r a t i o n s observed i n the e x p e r i m e n t a l o t t e r d u r i n g experiment 2 ( F i g . 49) s u g g e s t s t h a t t e r m i n a t i o n of embryonic diapause was induced by a l t e r e d p h o t o p e r i o d . P r o g e s t e r o n e c o n c e n t r a t i o n s r a n g i n g from 1.10 t o 3.90 ng/ml (mean = 2.4 + 0.30, n = 8) s u g g e s t s t h a t the e x p e r i m e n t a l female was i n the p r e i m p l a n t a t i o n p e r i o d of pregnancy p r i o r to the experiment (Chapter I I I ) . F o l l o w i n g the i n c r e a s e i n p h o t o p e r i o d , however, p r o g e s t e r o n e l e v e l s i n c r e a s e d s i g n i f i c a n t l y t o a peak of 21 ng/ml i n January ( F i g . 4 9 ) . These h i g h l e v e l s , i n d i c a t i v e of i n c r e a s e d l u t e a l a c t i v i t y a s s o c i a t e d w i t h the end of embryonic diapause (Chapter I I I ) , remained u n t i l F e b r u a r y when they began t o d e c l i n e . By the b e g i n n i n g of March, p r o g e s t e r o n e c o n c e n t r a t i o n s had r e t u r n e d t o nonpregnant l e v e l s (Chapter I I I ) . From the b e g i n n i n g of the e x p e r i m e n t a l p e r i o d u n t i l the end of F e b r u a r y p r o g e s t e r o n e c o n c e n t r a t i o n s were s i g n i f i c a n t l y h i g h e r than d u r i n g the p r e - e x p e r i m e n t a l p e r i o d (mean = 12.74 + 2.45, p < 0 . 0 l ) . A l t h o u g h t h i s female f a i l e d t o g i v e b i r t h , the presence of s w o l l e n t e a t s , an e n l a r g e d v a g i n a l opening and the low l e v e l s of p r o g e s t e r o n e a l l suggest t h a t b i r t h would have o c c u r r e d near the end of F e b r u a r y or i n e a r l y March. T h i s compares t o l a t e March or A p r i l i n w i l d o t t e r s (Chapter I I I ) . U n f o r t u n a t e l y , no pregnant c o n t r o l female was a v a i l a b l e . A l t h o u g h one of the females i n the c o n t r o l group (F5) was a l s o i n v o l v e d i n mating a t t e m p t s the p r e v i o u s s p r i n g , she d i d not -e x h i b i t changes i n p r o g e s t e r o n e a s s o c i a t e d w i t h pregnancy. 188 P r o g e s t e r o n e l e v e l s remained low (0.31-1.23 ng/ml) and were w i t h i n the range of v a l u e s observed among nonpregnant o t t e r s (Chapter I I I ) . P r o g e s t e r o n e c o n c e n t r a t i o n s a r e p r e s e n t e d i n Appendix 4. Based on the h i g h pregnancy r a t e among w i l d o t t e r s (Chapter I I I ) , the a d u l t females chosen f o r e x p e r i m e n t a l 1 were o r i g i n a l l y assumed t o be pregnant. The low p r o g e s t e r o n e c o n c e n t r a t i o n s observed (0.43-1.10 ng/ml) i n d i c a t e d t h a t they were n o t . 11. The ef f e c t of i n c r e a s e d p h o t o p e r i o d on male o t t e r s j _ . Experiment j _ The r e s u l t s of a l l t h r e e e x p e r i m e n t s show t h a t t e s t i c u l a r a c t i v i t y i n male o t t e r s i s p r o b a b l y s t i m u l a t e d by exposure t o i n c r e a s e d p h o t o p e r i o d s , and t h a t d u r a t i o n of t e s t i c u l a r development i s not a f f e c t e d by the e x p e r i m e n t a l c o n d i t i o n s . In the f i r s t e x p e r i m e n t , the t e s t i s volume of the e x p e r i m e n t a l male (M3) i n c r e a s e d r a p i d l y i mmediately f o l l o w i n g the i n c r e a s e i n t h e a r t i f i c i a l p h o t o p e r i o d ( F i g . 50). Maximum volume o c c u r r e d on March 1 and by e a r l y A p r i l , the t e s t i s had begun t o d e c l i n e i n s i z e . In c o n t r a s t , the t e s t i s of males under n a t u r a l p h o t o p e r i o d d i d not i n c r e a s e i n volume u n t i l the end of J a n u a r y and peak v a l u e s o c c u r r e d i n m i d - A p r i l , s i x weeks l a t e r than those of the e x p e r i m e n t a l a n i m a l . The p a t t e r n of- t e s t o s t e r o n e s e c r e t i o n v a r i e d g r e a t l y between i n d i v i d u a l s i n t h i s experiment ( F i g . 51). T e s t o s t e r o n e 189 F i g u r e 50. Experiment 1. T e s t i s volumes of male o t t e r s under n a t u r a l l i g h t regimes ( M i l 1 , M2 • •) or exposed t o a p h o t o p e r i o d of 16L:8D (M3 O O ) . Arrow i n d i c a t e s when p h o t o p e r i o d was f i r s t i n c r e a s e d . F i g u r e 51. Experiment 1. T e s t o s t e r o n e c o n c e n t r a t i o n s of male o t t e r s . See F i g u r e 50 f o r key t o the symbols. 190 20-E o 0 E CO "to 5 H . 0 15H 10-F i g u r e 5 0 -i r-O N D J F M Month M 0 c o l _ 0 co o CO 0 p-5" 4 3 2 1 F igure 51 O N D J F M A M J Month 191 c o n c e n t r a t i o n s i n the e x p e r i m e n t a l male r o s e o n l y s l i g h t l y f o l l o w i n g the i n c r e a s e i n p h o t o p e r i o d and remained r e l a t i v e l y c o n s t a n t t hroughout the ex p e r i m e n t . A s i m i l a r p a t t e r n was observed among one of the c o n t r o l males (M1) a l t h o u g h the second c o n t r o l (M2) showed a more s u b s t a n t i a l i n c r e a s e i n mid-February. The p a t t e r n of i n c r e a s e s i n t e s t o s t e r o n e i n the l a t t e r male was s i m i l a r t o t h a t observed among c o n t r o l a n i m a l s i n subsequent y e a r s . Both of the males l a c k i n g o b v i o u s peaks i n t e s t o s t e r o n e showed normal t e s t i c u l a r growth and were i n v o l v e d i n mating d u r i n g t h i s p e r i o d . T h i s would suggest t h a t normal r e p r o d u c t i v e development had o c c u r r e d . E p i s o d i c r e l e a s e of t e s t o s t e r o n e , or the t e c h n i q u e s of sa m p l i n g and s t o r a g e used i n the f i r s t y e a r of the s t u d y , may have r e s u l t e d i n the low t e s t o s t e r o n e c o n c e n t r a t i o n s measured a l t h o u g h s i m i l a r v a l u e s were obse r v e d o c c a s i o n a l l y i n subsequent y e a r s . 2. Experiment 2 The r e s u l t s of the second experiment f u r t h e r i n d i c a t e d t h a t t e s t i c u l a r development and r e g r e s s i o n i n o t t e r s can be advanced by exposure t o an i n c r e a s e d p h o t o p e r i o d . I n c r e a s e s i n t e s t i s volume ( F i g . 52) and t e s t o s t e r o n e c o n c e n t r a t i o n s ( F i g . 53) were obser v e d i n the e x p e r i m e n t a l a n i m a l s b e f o r e s i m i l a r i n c r e a s e s o c c u r r e d among the c o n t r o l group. S i m i l a r l y , the t e s t e s of the e x p e r i m e n t a l o t t e r s underwent r e g r e s s i o n w e l l b e f o r e those of males under n a t u r a l l i g h t c o n d i t i o n s . As i n the p r e v i o u s e x p e r i m e n t , t h e r e was c o n s i d e r a b l e v a r i a t i o n between i n d i v i d u a l s i n b o t h groups and among the c o n t r o l a n i m a l s . T e s t o s t e r o n e 192 F i g u r e 52. E x p e r i m e n t 2. T e s t i s volumes of male o t t e r s under n a t u r a l l i g h t regimes (M1 • •, M2 • •, M 6 A • ) o r exposed t o a p h o t o p e r i o d of 16L:8D (M30 O, M5 • • ) . Arrow i n d i c a t e s when the p h o t o p e r i o d was f i r s t i n c r e a s e d . M1 d i e d on F e b r u a r y 19. F i g u r e 53. E x p e r i m e n t 2. T e s t o s t e r o n e c o n c e n t r a t i o n s of male o t t e r s . See F i g u r e 52 f o r key t o the symbols. 2 5 F igure 52 cT 20 E o © 1 5 E 1 5 o > CO 10 CO 51 O N J F M Month M O ' N ' D J F ' M - 1 A ' M"1 J ' Month 1 94 c o n c e n t r a t i o n s were o b s e r v e d t o i n c r e a s e e a r l i e r i n one c o n t r o l o t t e r (M1) than i n the o t h e r two. The p a t t e r n of t e s t i c u l a r growth was s i m i l a r among thes e t h r e e o t t e r s , however, and the s i g n i f i c a n c e of t h i s e a r l y r i s e i n t e s t o s t e r o n e l e v e l s i n M1 i s unknown. T h i s a n i m a l d i e d of undetermined causes on Feb r u a r y 19 and i t i s not known i f serum t e s t o s t e r o n e c o n c e n t r a t i o n s were a f f e c t e d by h i s poor h e a l t h . 3. Experintent 3 As i n the p r e c e d i n g e x p e r i m e n t s , t e s t i c u l a r a c t i v i t y was advanced i n males exposed t o an i n c r e a s e d p h o t o p e r i o d . I n d i c a t o r s of t e s t i c u l a r a c t i v i t y were s i m i l a r among a l l t h r e e groups d u r i n g O c t o b e r , when two groups ( c o n t r o l 2 and e x p e r i m e n t a l ) were exposed t o a s h o r t d a y l e n g t h . However, a c t i v i t y of the male i n group 3 (M2) i n c r e a s e d r a p i d l y f o l l o w i n g t r a n s f e r t o 16 hours of d a y l i g h t . Maximum t e s t o s t e r o n e c o n c e n t r a t i o n ( F i g . 55) and t e s t i s volume ( F i g . 54) o c c u r r e d d u r i n g December and l a t e J a n uary r e s p e c t i v e l y i n the e x p e r i m e n t a l male, w e l l b e f o r e males i n e i t h e r c o n t r o l group. A l t h o u g h v a l u e s s u b s e q u e n t l y d e c l i n e d , a second, s m a l l e r r i s e i n t e s t o s t e r o n e , s i m i l a r t o t h a t seen d u r i n g J u l y and August i n c o n t r o l a n i m a l s , o c c u r r e d i n l a t e A p r i l ( F i g . 5 5 ) . E x a m i n a t i o n of the s e m i n i f e r o u s t u b u l e s c o n f i r m e d the d i f f e r e n c e s i n r e p r o d u c t i v e a c t i v i t y o b s e r v e d between the e x p e r i m e n t a l and c o n t r o l groups. A n a l y s i s of the phases p r e s e n t i n the t e s t e s i n d i c a t e d t h a t r e p r o d u c t i v e a c t i v i t y and subsequent r e g r e s s i o n was advanced i n group 3 (Table XX). 195 F i g u r e 54. Experiment 3 . T e s t i s volumes of male o t t e r s i n Group 1 (M3» •, M5 • • ) , Group 2 (M6D •) and Group 3 (M20 O). Arrows i n d i c a t e (1) when groups 2 and 3 were p l a c e d i n an e n v i r o n m e n t a l chamber ( p h o t o p e r i o d 8L:16D, temperature 10°C) and (2) when the p h o t o p e r i o d of the e n v i r o n m e n t a l chambers was changed t o t h a t of the n a t u r a l l i g h t regime (Group 2) or i n c r e a s e d t o 16L:8D (Group 3 ) . Group 1 was exposed t o n a t u r a l d a y l i g h t . See t e x t f o r e x p l a n a t i o n of e x p e r i m e n t a l p r o t o c o l . F i g u r e 55. Experiment 3. T e s t o s t e r o n e c o n c e n t r a t i o n s of male o t t e r s . See F i g u r e 54 f o r key t o the symbols. 1 9 6 Month 197 T a b l e X X . S e m i n i f e r o u s t u b u l e p h a s e s p r e s e n t 1n b i o p s i e s o f c o n t r o l ( G r o u p 1 a n d 2 ) a n d e x p e r i m e n t a l ( G r o u p 3 ) r i v e r o t t e r s f r o m November 1980 t h r o u g h J u n e 1 9 8 1 . B o l d t y p e I n d i c a t e s m o s t a b u n d a n t p h a s e s . P h a s e 7 t u b u l e s a r e t h e m o s t a c t i v e . See T a b l e I I I f o r e x p l a n a t i o n o f p h a s e t y p e s . T e n t u b u l e s w e r e e x a m i n e d i n e a c h s a m p l e . P h a s e s P r e s e n t ( J t u b u l e s ) A n i m a l s N o v . 17 D e c . 17 F e b . 4 M a r c h 4 A p r i l 9 May 17 J u n e 15 G r o u p 1 : M3 ( 4 0 ) (60) 3 ( 1 0 ) 4 ( 4 0 ) 5 (50) 6 ( 4 0 ) 7 (60) 6 ( 2 0 ) 7 (80) 6 ( 2 0 ) 7 (80) 7 ( 3 5 ) 8 (65) 8 (70) 1 ( 3 0 ) M5 4 ( 1 5 ) 5 (65) 6 ( 2 0 ) ( 1 5 ) (85) 7 (100) 7 (100) 7 ( 2 5 ) 8 (75) 8 (65) 1 ( 3 5 ) G r o u p 2 : M6 3 ( 1 0 ) 4 (60) 5 ( 3 0 ) ( 2 0 ) (80) 6 ( 1 0 ) 7 (90) 8 (100) 8 ( 3 0 ) 1 (70) G r o u p 3 : M2 1 ( 1 5 ) 5 (60) 7 (100) 6 ( 5 ) 8 (95) 8 (100) 8 ( 3 0 ) 2 (50) 6 ( 3 0 ) 7 (95) 1 ( 5 ) 1 (70) 3 ( 3 5 ) 7 ( 1 0 ) 198 D e s p i t e the s i m i l a r t e s t i c u l a r phases p r e s e n t i n the c o n t r o l and e x p e r i m e n t a l a n i m a l s i n November, by December phase 7 t u b u l e s were p r e s e n t i n the t e s t i s of the e x p e r i m e n t a l male (M2). In c o n t r a s t , the t e s t e s of males exposed t o n a t u r a l l i g h t regimes were not as a c t i v e and spermatogenesis had p r o g r e s s e d as f a r as phase 6 i n o n l y one a n i m a l . Phase 7 t u b u l e s were not observed i n e i t h e r group 1 or group 2 males u n t i l the f o l l o w i n g sampling p e r i o d . The presence of phase 8 t u b u l e s i m p l i e s t h a t s p e r m a t o g e n e s i s had ceased i n the group 2 male by e a r l y A p r i l a l t h o u g h i t c o n t i n u e d f o r a t l e a s t one month i n the o t h e r males. The p e r i o d d u r i n g which phase 7 t u b u l e s were p r e s e n t was s i m i l a r i n a l l of the males which i n d i c a t e s t h a t the i n c r e a s e d p h o t o p e r i o d d i d not a f f e c t the d u r a t i o n of r e p r o d u c t i v e a c t i v i t y . A l t h o u g h the advanced a c t i v i t y o bserved i n t e s t i s volume, t e s t o s t e r o n e c o n c e n t r a t i o n s and phase a n a l y s i s i n the group 3 male was not i n d i c a t e d by changes i n the s e m i n i f e r o u s t u b u l e d i a m e t e r s , the e a r l y d e c l i n e i n r e p r o d u c t i v e a c t i v i t y was ob v i o u s ( F i g . 56). The e a r l y development appears normal a l t h o u g h maximum di a m e t e r o c c u r r e d i n Fe b r u a r y and v a l u e s s u b s e q u e n t l y d e c l i n e d . Among the o t h e r males, t u b u l a r d i a m e t e r s c o n t i n u e d t o i n c r e a s e and maximum v a l u e s o c c u r r e d i n March. However, i t i s p o s s i b l e t h a t the maximum diameter of M2 was not measured. The maximum diameter o b s e r v e d was lower i n the e x p e r i m e n t a l male than i n the o t h e r s even though peak t e s t i s s i z e was s i m i l a r . Tubule d i a m e t e r s a r e c l o s e l y c o r r e l a t e d w i t h t e s t i s volume (Chapter I I ) and measurements were not a v a i l a b l e 199 F i g u r e 56. Experiment 3. S e m i n i f e r o u s t u b u l e d i a m e t e r s of male o t t e r s . Group 1 ( M 3 » — • , M51 •) was h e l d under n a t u r a l c o n d i t i o n s . Group 2 (M6D • ) and Group 3 (M2 0 O) were exposed t o a p h o t o p e r i o d of 8L:16D f o r one month from October 2 t h r o u g h November 4. Arrow i n d i c a t e s when Group 2 was exposed t o a n a t u r a l l i g h t regime and Group 3 was s u b j e c t e d t o a p h o t o p e r i o d of 16L:8D. Groups 2 and 3 were m a i n t a i n e d a t 10°C. Month 201 from l a t e January which i s when maximum t e s t i s volume o c c u r r e d i n t h i s a n i m a l . T h e r e f o r e , i t i s p o s s i b l e t h a t the s e m i n i f e r o u s t u b u l e s reached s i m i l a r s i z e s i n a l l of the o t t e r s i n t h i s s t u d y . R e p r o d u c t i v e a c t i v i t y was not s i g n i f i c a n t l y d i f f e r e n t i n the two c o n t r o l groups. A l t h o u g h t h e r e were minor v a r i a t i o n s between i n d i v i d u a l s , b o t h groups were s i m i l a r w i t h r e s p e c t t o t e s t i s s i z e , t e s t o s t e r o n e c o n c e n t r a t i o n s , t u b u l e d i a m e t e r s or phases of s p e r m a t o g e n e s i s p r e s e n t i n the s e m i n i f e r o u s t u b u l e s . T h i s i n d i c a t e s t h a t t h e p e r i o d of s h o r t p h o t o p e r i o d and i n c r e a s e d t e m p e r a t u r e t h a t group 2 was s u b j e c t e d t o , d i d not a f f e c t the t i m i n g of r e p r o d u c t i v e development i n t h i s i n d i v i d u a l . The r e p r o d u c t i v e development of t h r e e o t h e r males caught d u r i n g the e x p e r i m e n t a l p e r i o d , was s i m i l a r t o t h a t of the c o n t r o l groups a t the time of c a p t u r e . S u b s e q u e n t l y , w h i l e i n c a p t i v i t y , a l l of the males f o l l o w e d s i m i l a r p a t t e r n s of a c t i v i t y and t e s t i c u l a r r e g r e s s i o n . R e s u l t s of a l l f o u r measurements of r e p r o d u c t i v e a c t i v i t y used i n t h i s experiment i n d i c a t e d t h a t r e p r o d u c t i v e a c t i v i t y of c a p t i v e male o t t e r s under a n a t u r a l l i g h t regime was s i m i l a r t o t h a t of w i l d o t t e r s i n s o u t h w e s t e r n B r i t i s h C olumbia. 4 . A l l e x p e r i m e n t s A s i m i l a r p a t t e r n of response of male o t t e r s t o i n c r e a s e d p h o t o p e r i o d was o b s e r v e d i n a l l t h r e e of the e x p e r i m e n t s . T e s t i c u l a r growth o c c u r r e d e a r l i e r i n the e x p e r i m e n t a l o t t e r s 202 F i g u r e 57. C o m p a r i s i o n of t e s t i s volumes of a l l e x p e r i m e n t a l male o t t e r s , as a f u n c t i o n of the time from the b e g i n n i n g of exposure t o the i n c r e a s e d (16L:8D) • p h o t o p e r i o d . Experiment 1 (M30 O ) , Experiment 2 (M3» • , M51 •) and Experiment 3 (M2D • ) . See t e x t f o r d e t a i l s of e x p e r i m e n t s . F i g u r e 58. C o m p a r i s i o n of t e s t o s t e r o n e c o n c e n t r a t i o n s of a l l e x p e r i m e n t a l male o t t e r s , as a f u n c t i o n of the time from the b e g i n n i n g of exposure t o i n c r e a s e d (16L:8D) p h o t o p e r i o d . See F i g u r e 57 f o r key t o the symbols. Figure 57 i i : i 1 1 1 0 5 10 15 20 2 5 30 Weeks j 1 1 1 1 1 1 0 5 10 15 2 0 2 5 3 0 Weeks 204 than i n the c o n t r o l groups, and i n each e x p e r i m e n t , maximum volumes o c c u r r e d between 10 and 12 weeks a f t e r the s t a r t of the a r t i f i c i a l l y i n c r e a s e d p h o t o p e r i o d ( F i g . 57). F u r t h e r , i n each e x p e r i m e n t , the t e s t e s of the e x p e r i m e n t a l males underwent a normal p e r i o d of a c t i v i t y b e f o r e d e c l i n i n g s p o n t a n e o u s l y . T h i s spontaneous d e g e n e r a t i o n r e s u l t e d i n e x p e r i m e n t a l males b e i n g r e p r o d u c t i v e l y i n a c t i v e a t ti m e s when o t t e r s under n a t u r a l p h o t o p e r i o d s were s t i l l u n dergoing s p e r m a t o g e n e s i s . A l t h o u g h t e s t o s t e r o n e v a l u e s showed g r e a t i n d i v i d u a l v a r i a t i o n , a s i m i l a r p a t t e r n of e a r l y s e c r e t i o n and r e g r e s s i o n was observed i n e x p e r i m e n t a l a n i m a l s , w i t h peak v a l u e s o c c u r r i n g between s i x and ten weeks a f t e r the b e g i n n i n g of each p e r i o d of a r t i f i c i a l l y i n c r e a s e d d a y l e n g t h ( F i g . 5 8 ) . 205 DISCUSSION A. E n v i r o n m e n t a l Cues The r e s u l t s of my study i n d i c a t e t h a t e s t r u s and s p e r m a t o g e n e s i s were advanced i n r i v e r o t t e r s exposed t o a r t i f i c i a l l y i n c r e a s e d d a y l e n g t h . There was a l s o e v i d e n c e t h a t i n c r e a s e d p h o t o p e r i o d induced the t e r m i n a t i o n of embryonic d i a p a u s e . In c o n t r a s t , i n c r e a s e d temperature d i d not advance spermatogenesis or e s t r u s s u g g e s t i n g t h a t r e p r o d u c t i v e t i m i n g i n o t t e r s i s not c o n t r o l l e d by t h i s s t i m u l u s . The l a c k of a temperature e f f e c t i s perhaps u n d e r s t a n d a b l e because the annual changes i n mean ambient temperature i n southwestern B r i t i s h Columbia a r e r e l a t i v e l y minor when compared t o the d a i l y v a r i a t i o n s . Between. November and March, when the a b i l i t y t o determine season i s c r u c i a l , the mean monthly temperatures v a r y by o n l y 3.3°C, but d a i l y t e m p e r a t u r e s may v a r y by as much as 24° C (Anon. 1982). The s i m i l a r i t y between the r e p r o d u c t i v e c y c l e s of w i l d o t t e r s ( C h a p t e r s I I and I I I ) and those of c a p t i v e o t t e r s f e d on a c o n s t a n t d i e t would a l s o argue f o r changes i n n u t r i t i o n h a v i n g l i t t l e e f f e c t on r e p r o d u c t i v e a c t i v i t y . While s e a s o n a l changes i n temperature and food a v a i l a b i l i t y can a f f e c t s u r v i v a l of the a d u l t s and young and thus may be u l t i m a t e l y r e s p o n s i b l e f o r d i c t a t i n g the t i m i n g of b i r t h , they a r e not n e c e s s a r i l y the f a c t o r s used as cues by o t t e r s t o r e g u l a t e t h e i r r e p r o d u c t i v e a c t i v i t y . A l t h o u g h the number of a n i m a l s a v a i l a b l e f o r study was s m a l l and f a c t o r s such as temperature or n u t r i t i o n may have mo d e r a t i n g i n f l u e n c e s ( S a d l e i r I969a,b; D e s j a r d i n s and Lopez 206 1983), my r e s u l t s suggest t h a t the p r i m a r y e n v i r o n m e n t a l f a c t o r c o n t r o l l i n g the t i m i n g of the r e p r o d u c t i v e c y c l e of o t t e r s i n B r i t i s h Columbia i s the annual c y c l e of d a i l y p h o t o p e r i o d . B. Females J_. E s t r u s C o n t r o l of r e p r o d u c t i v e a c t i v i t y of females by p h o t o p e r i o d was i m p l i c a t e d by advancement of e s t r u s demonstrated i n two of the t h r e e experiments (1 and 3 ) . In the t h i r d (experiment 2 ) , the t i m i n g of e s t r u s r e f l e c t e d the pregnancy of the e x p e r i m e n t a l female and as i n w i l d o t t e r s (Chapter I I I ) , e s t r u s o c c u r r e d s h o r t l y a f t e r the e s t i m a t e d time of b i r t h . In o n l y a s i n g l e c o n t r o l a n i m a l (F5, experiment 2) d i d e s t r u s occur as e a r l y as observ e d among the e x p e r i m e n t a l f e m a l e s . T h i s may r e f l e c t e i t h e r a normal degree of v a r i a b i l i t y , commonly seen among the r e p r o d u c t i v e c y c l e s of female mammals ( S a d l e i r 1969a), a moder a t i n g e f f e c t by o t h e r e n v i r o n m e n t a l cues such as n u t r i t i o n , or the e f f e c t s of the c o n d i t i o n s of c a p t i v i t y . P h o t o p e r i o d has been found t o c o n t r o l the t i m i n g of e s t r u s i n o t h e r m u s t e l i d s such as the f e r r e t ( B i s s o n n e t t e 1932; Hammond 1951; Donovan 1967) and the mink (Hammond 1951; Duby and T r a v i s 1972). F u r t h e r s t u d i e s a r e n e c e s s a r y t o determine the amount of i n d i v i d u a l v a r i a t i o n i n e s t r u s among o t t e r s , but the l a c k of advanced e s t r u s among the m a j o r i t y of the c o n t r o l a n i m a l s , and the u n e q u i v o c a l r e s u l t s of experiment 3, support the importance of p h o t o p e r i o d ' s r o l e i n c o n t r o l l i n g e s t r u s i n r i v e r o t t e r s . 207 The l a c k of any change i n the t i m i n g of e s t r u s by c o n t r o l group 2 (experiment 3) a f t e r exposure t o a s h o r t p h o t o p e r i o d (8L:16D) i n d i c a t e s t h a t s h o r t p h o t o p e r i o d a l o n e does not advance e s t r u s i n the e x p e r i m e n t a l group as i t does i n the mink (Hammond 1953; Duby and T r a v i s 1972). However, s h o r t p h o t o p e r i o d s may a f f e c t r e p r o d u c t i v e a c t i v i t y i n female o t t e r s by i n i t i a t i n g f o l l i c u l a r a c t i v i t y , as i n the mink (Hammond 1951; Duby and T r a v i s 1972), or by t e r m i n a t i n g a p h o t o r e f r a c t o r y p e r i o d , as i n the f e r r e t (Donovan 1967; Thorpe and H e r b e r t 1976). The low l e v e l of f o l l i c u l a r development o b s e r v e d among o t t e r s c o l l e c t e d b e f o r e the w i n t e r s o l s t i c e (Chapter I I I ) , p o i n t s t o i n c r e a s i n g p h o t o p e r i o d b e i n g r e s p o n s i b l e f o r i n d u c i n g both f o l l i c u l a r growth and i m p l a n t a t i o n i n a n i m a l s from B r i t i s h Columbia. Though the l e n g t h of e s t r u s i s v a r i a b l e i n o t t e r s due t o the p r e sence of induced o v u l a t i o n , two e x p e r i m e n t a l a n i m a l s (F3, 1979; F2, 1981) showed p r o l o n g e d e s t r u s w h i l e the o t h e r two (F3, 1980; F7, 1981) had e x t r e m e l y s h o r t p e r i o d s of e s t r u s and p a r t i a l e s t r u s . Lengthy e s t r o u s p e r i o d s have a l s o been r e p o r t e d i n e x p e r i m e n t a l f e r r e t s ( H e r b e r t and K l i n o w s k a 1978) and may be due t o the p r o l o n g e d s t i m u l a t i o n by the l o n g p h o t o p e r i o d . In c o n t r a s t , the s h o r t e s t r o u s p e r i o d s may be r e l a t e d t o reduced f o l l i c u l a r growth p r i o r t o the e x p e r i m e n t a l p e r i o d . F7 showed p a r t i a l e s t r u s o n l y and f o l l i c u l a r growth may not have been complete i n t h i s i n d i v i d u a l (see Chapter I I I ) . E s t r u s i n F3 (1980) may have been s h o r t e n e d by her r e c e n t l y completed pregnancy. An i n c r e a s e i n f o l l i c u l a r growth u s u a l l y o c c u r s i n pregnant o t t e r s i n J a n u a r y , j u s t p r i o r t o the end of the 208 p r e i m p l a n t a t i o n p e r i o d . S i n c e embryonic d i a p a u s e appears t o have been t e r m i n a t e d i n December (see b e l o w ) , t h i s p e r i o d of f o l l i c u l a r growth may not have o c c u r r e d . F o l l i c u l a r growth does not o c c u r d u r i n g the p o s t i m p l a n t a t i o n p e r i o d of pregnancy (Chapter I I I ) , which would a l s o r e s u l t i n reduced o v a r i a n a c t i v i t y . 2. I m p l a n t a t i o n My study s u g g e s t s t h a t the t e r m i n a t i o n of embryonic d i a p a u s e i n the o t t e r i s a f f e c t e d by p h o t o p e r i o d . In l a t e December, a f t e r an e x p e r i m e n t a l female was exposed t o an i n c r e a s e d p h o t o p e r i o d , p r o g e s t e r o n e c o n c e n t r a t i o n s i n c r e a s e d s i g n i f i c a n t l y , i n d i c a t i n g i n c r e a s e d l u t e a l a c t i v i t y . T h i s i s n o r m a l l y a s s o c i a t e d w i t h the end of embryonic d i a p a u s e and i m p l a n t a t i o n i n the o t t e r (Chapter I I I ) and o t h e r m u s t e l i d s ( r e v i e w s i n Mead 1981, Mead and Wright 1983). A l t h o u g h I had no pregnant c o n t r o l s f o r c o m p a r i s o n , I d i d not f i n d s i g n i f i c a n t i n c r e a s e s i n l u t e a l c e l l s i z e and a c t i v i t y b e f o r e F e b r u a r y i n w i l d o t t e r s from s o u t h w e s t e r n B r i t i s h Columbia (Chapter I I I ) . Thus, I f e e l t h e s e r e s u l t s i n d i c a t e t h a t the o t t e r i s s i m i l a r t o o t h e r m u s t e l i d s such as the s p o t t e d skunk, S p i l o g a l e p u t o r i u s  l a t i f r o n s (Mead 1971) and the mink (Murphy and James 1974; M a r t i n e t e t a_l. 1981) i n t h a t i n c r e a s e d l u t e a l a c t i v i t y , and t h e r e f o r e i n c r e a s e d p r o g e s t e r o n e c o n c e n t r a t i o n s , may be induced by i n c r e a s e d p h o t o p e r i o d . Changes i n d a y l e n g t h have a l s o been shown t o t e r m i n a t e d i a p a u s e and induce e a r l y i m p l a n t a t i o n i n a number of o t h e r m u s t e l i d s (American marten, M a r t e s americana, 209 Pearson and Enders 1944; l o n g t a i l e d w e a s e l , M u s t e l a f r e n a t a , W right 1963; European badger, Meles meles, Canivenc and Bonnin 1979, Canivenc e_t a l . 1981; European marten, Martes martes, Bonnin and Canivenc 1980). A l t h o u g h temperature was not s t r i c t l y c o n t r o l l e d i n t h i s e x p e r i m e n t , I f e e l t h a t i t was u n l i k e l y t o have induced l u t e a l a c t i v i t y . A s l i g h t r i s e was o b s e r v e d i n the temperature of the e x p e r i m e n t a l chamber but t h i s i n c r e a s e was m i n i m a l and w i t h i n the range i n d a i l y t e m p e r a t u r e s observed i n p r e c e d i n g and f o l l o w i n g months (Anon. 1982). S i m i l a r l y , l u t e a l a c t i v i t y was not induced by n u t r i t i o n a l changes s i n c e food s u p p l y d i d not v a r y d u r i n g the e x p e r i m e n t a l and n o n - e x p e r i m e n t a l p e r i o d s . S i m i l a r l y , , t h e s e p o t e n t i a l cues have not been found t o a f f e c t the t i m i n g of embryonic d i a p a u s e i n o t h e r m u s t e l i d s ( T r a v i s and P i l b e a m 1980; Canivenc e t a l . 1981; Mead and Wright 1983). C. Males The changes observed i n t e s t i c u l a r a c t i v i t y i n my e x p e r i m e n t s i n d i c a t e t h a t r e p r o d u c t i v e a c t i v i t y of male o t t e r s was a l s o a f f e c t e d by exposure t o i n c r e a s e d p h o t o p e r i o d s . E x p e r i m e n t a l o t t e r s c o n s i s t e n t l y e x h i b i t e d s i g n s of .advanced r e p r o d u c t i v e development and r e g r e s s i o n , and peak a c t i v i t y o c c u r r e d e a r l i e r i n a l l of the e x p e r i m e n t a l o t t e r s than i n the c o n t r o l a n i m a l s . P h o t o p e r i o d i s known t o i n f l u e n c e the t i m i n g of r e p r o d u c t i v e a c t i v i t y i n m u s t e l i d s such as the mink (Duby and T r a v i s 1972; Klochkov 1980; B o i s s i n - A g a s s e e t a l . 1982) and the f e r r e t ( B i s s o n n e t t e 1932; Hammond 1951; Rust and S h a c k e l f o r d 210 1969; V i n c e n t 1970) and a l s o i n o t h e r male mammals (see reviews i n S a d l e i r 1969a; Turek and Campbell 1979; E l l i o t and Goldman 1981). A l t h o u g h i n c r e a s e s i n t e s t o s t e r o n e l e v e l s g e n e r a l l y agreeded w i t h o t h e r parameters of male r e p r o d u c t i v e a c t i v i t y , t h e r e was c o n s i d e r a b l e v a r i a t i o n i n t i m i n g and amount of the i n c r e a s e s . A h i g h degree of i n d i v i d u a l v a r i a b i l i t y i n t e s t o s t e r o n e c o n c e n t r a t i o n s i s r e p o r t e d i n mink ( B o i s s i n - A g a s s e et a l . 1982) and i t may be due t o an e p i s o d i c r e l e a s e of t e s t o s t e r o n e . Such p u l s a t e d r e l e a s e i s c o r r e l a t e d w i t h r e p r o d u c t i v e a c t i v i t y and has been obser v e d i n a v a r i e t y of mammals i n c l u d i n g m u s t e l i d s ( R e i g e r and Murphy 1977; Maurel e t a l . 1981; B o i s s i n - A g a s s e e t a l . 1982; Southern and Gorden 1975; L i n c o l n 1976). The presence of e a r l y s t a g e s of s p e r m a t o g e n e s i s i n s e m i n i f e r o u s t u b u l e s p r i o r t o the w i n t e r s o l s t i c e observed i n c a p t i v e (experiment 3) and i n w i l d o t t e r s ( C h a p t e r I I ) l e a d s me to c o n c l u d e t h a t an i n c r e a s e i n the p h o t o p e r i o d i s not n e c e s s a r y f o r s permatogenesis t o be i n i t i a t e d . I n c r e a s i n g l i g h t does, however, appear t o have the a b i l i t y t o h a s t e n m a t u r a t i o n . In t h i s r e s p e c t the o t t e r i s s i m i l a r t o t h e mink which i s s t i m u l a t e d by s h o r t p h o t o p e r i o d s , but whi c h breeds under i n c r e a s i n g p h o t o p e r i o d i n the e a r l y s p r i n g (Hammond 1951; Duby and T r a v i s 1972). In my s t u d y , the t i m i n g of r e p r o d u c t i v e a c t i v i t y was not a f f e c t e d by exposure t o a s h o r t p h o t o p e r i o d i n October ( c o n t r o l 2, experiment 3) a l t h o u g h a l o n g e r p e r i o d of exposure may be n e c e s s a r y f o r a response t o o c c u r (Klochkov 21 1 1980). F u r t h e r study i s n e c e s s a r y t o d e t e r m i n e the e f f e c t of s h o r t p h o t o p e r i o d upon spe r m a t o g e n e s i s i n the r i v e r o t t e r . D. P h o t o p e r i o d as a Cue In a p r e d i c t a b l e environment such as B r i t i s h Columbia, i t i s advantageous f o r a n i m a l s t o respond t o a cue which would a l l o w m e t a b o l i c p r e p a r a t i o n f o r f a v o u r a b l e c o n d i t i o n s and t o ensure t h a t r e p r o d u c t i v e e v e n t s o c c u r a t a time when food a v a i l a b i l i t y and c l i m a t e ensure minimum r e p r o d u c t i v e f a i l u r e . P h o t o p e r i o d i s the most a c c u r a t e p r e d i c t o r of s e a s o n a l e v e n t s i n B r i t i s h Columbia and t h e r e f o r e , i t i s not unexpected t h a t r i v e r o t t e r s u t i l i z e changes i n the d a y : n i g h t c y c l e t o determine the t i m i n g of s e a s o n a l r e p r o d u c t i v e a c t i v i t y . The h i g h degree of c o n s i s t e n c y from year t o year makes p h o t o p e r i o d the most " n o i s e f r e e " (Turek and Campbell 1979) a n n u a l p r e d i c t o r , and t r a n s i t o r y changes i n l i g h t c o n d i t i o n s may be b u f f e r e d by a b r i e f d e l a y i n response t o p h o t o p e r i o d i c s t i m u l i . T h i s d e l a y appeared t o occur i n many of the a n i m a l s i n t h i s s t u d y and i t i s common among a n i m a l s which respond t o p h o t o p e r i o d i c a d j u s t m e n t s ( B i s s o n n e t t e 1931; H e r b e r t 1977; T r a v i s and P i l b e a m 1980; K l o c h k o v 1980). Owing t o i t s h i g h degree of p r e d i c t a b i l i t y , p h o t o p e r i o d i s the most commonly used p r e d i c t o r of s e a s o n a l e v e n t s among temperate mammals (see r e v i e w s i n S a d l e i r 1969a; Hoffman 1973; Turek and Campbell 1979; Bronson 1985). Changes i n p h o t o p e r i o d may induce r e p r o d u c t i v e development d i r e c t l y o r may s i m p l y h e l p s y n c h r o n i z e an endogenous c y c l e w i t h e x t e r n a l e n v i r o n m e n t a l c o n d i t i o n s . The e x i s t e n c e of endogenous c y c l e s has y e t t o be examined i n o t t e r s 212 but among the m u s t e l i d s t h e r e i s e v i d e n c e of endogenous s t i m u l i , independent of l i g h t , i n d u c i n g r e p r o d u c t i v e a c t i v i t y i n mink ( K i r k 1962; Murphy and James 1974) and f e r r e t s (Rust and S h a c k e l f o r d 1969; H e r b e r t et a l . 1978). In both s e x e s , r e p r o d u c t i v e a c t i v i t y ceased w i t h o u t a change i n p h o t o p e r i o d . T h i s s u g g e s t s t h a t e i t h e r the d u r a t i o n of s p e r m a t o g e n e s i s and e s t r u s i s independent of l i g h t once i t i s i n i t i a t e d , or t h a t the a n i m a l s become r e f r a c t o r y t o c o n t i n u e d s t i m u l a t i o n by l o n g d a y l e n g t h s , as seen i n the f e r r e t (Donovan 1967; Thorpe and H e r b e r t 1976). There i s no i n d i c a t i o n t h a t exposure of the o t t e r s t o l o n g p h o t o p e r i o d (16L) was i n h i b i t o r y , as i n the mink (Duby and T r a v i s 1972; B o i s s i n - A g a s s e e_t a l . 1982). The d u r a t i o n of sper m a t o g e n e s i s was s i m i l a r i n both e x p e r i m e n t a l and c o n t r o l a n i m a l s and a l t h o u g h the l e n g t h of e s t r u s v a r i e d g r e a t l y , the wide range i n d i c a t e s t h a t l o n g p h o t o p e r i o d d i d not i n h i b i t i t . Thus, a l t h o u g h c o n t r o l o t t e r s a l s o t e r m i n a t e r e p r o d u c t i v e a c t i v i t y b e f o r e June when a 16 hour d a y l e n g t h o c c u r s (Anon. 1982), t h e r e i s no i n d i c a t i o n t h a t the end of the b r e e d i n g season i s determined by i n c r e a s i n g p h o t o p e r i o d i n the o t t e r . The v a r i a t i o n s observed i n the responses of the c a p t i v e o t t e r s t o changes i n p h o t o p e r i o d suggest t h a t p h o t o p e r i o d i c s t i m u l i a f f e c t i n d i v i d u a l s d i f f e r e n t l y . Such d i f f e r e n c e s may account f o r the v a r i a b i l i t y o b s e r v e d among w i l d o t t e r s c o l l e c t e d on the same d a t e , p a r t i c u l a r l y i n the degree of spermatogenesis among males c o l l e c t e d i n l a t e December (Chapter I I ) . I n d i v i d u a l v a r i a t i o n i n response t o changes i n p h o t o p e r i o d i s seen i n a 213 number of mammals ( d e e r , Goss and Rosen 1973; ha r b o r s e a l s , Phoca v i t u l i n a , B i g g and F i s h e r 1975; golden hamsters, M e s o c t r i e t u s a u r a t u s , S t e t s o n et. a_l. 1975; deer mice, Peromyscus sp., W h i t s e t t and M i l l e r 1982, D e s j a r d i n s and Lopez 1983) and among the m u s t e l i d s , i n d i v i d u a l v a r i a t i o n o c c u r s i n f e r r e t s ( B i s s o n n e t t e 1932; Hammond 1951) and mink ( M a r t i n e t e t a l . 1981; B o i s s i n - A g a s s e et. a l . 1982). The r e p r o d u c t i v e s t r a t e g y of male o t t e r s a l l o w s f o r g r e a t e r v a r i a b i l i t y i n response of i n d i v i d u a l s t o changes i n p h o t o p e r i o d . The f u n c t i o n of p h o t o p e r i o d i c responses i n males i s t o ensure t h a t r e p r o d u c t i v e a c t i v i t y i s s y n c h r o n i z e d between males and fe m a l e s . However, because males are u s u a l l y ready t o breed w e l l b e f o r e e s t r u s b e g i n s (Chapter I I ) , c l o s e l y c o n t r o l l e d responses t o p h o t o p e r i o d a r e not n e c e s s a r y . T h i s c o u l d e x p l a i n the h i g h degree of v a r i a t i o n seen among c a p t i v e and w i l d males, p a r t i c u l a r l y i n l a t e December when sperm are f i r s t found i n some an i m a l s (experiment 3, Chapter I I ) . In c o n t r a s t , a s t r o n g s e l e c t i v e p r e s s u r e f o r b i r t h t o occur a t a f a v o u r a b l e time would r e s u l t i n a g r e a t e r degree of synchrony among females i n t h e i r response t o p h o t o p e r i o d i c s t i m u l i . Owing t o the presence of embryonic d i a p a u s e i n r i v e r o t t e r s , the t i m i n g of b i r t h i s determined by the date of i m p l a n t a t i o n . T h e r e f o r e , a l t h o u g h the t i m i n g of e s t r u s may v a r y , the p h o t o p e r i o d i c c o n t r o l of i m p l a n t a t i o n would not be e x p e c t e d t o v a r y g r e a t l y . A h i g h degree of synchrony i n the t i m i n g of i m p l a n t a t i o n was seen among w i l d o t t e r s (Chapter I I I ) , and i t appears t h a t such a s e l e c t i v e p r e s s u r e c o u l d e x i s t i n B r i t i s h Columbian o t t e r s . 214 The t e r m i n a t i o n of embryonic di a p a u s e as a response t o i n c r e a s i n g p h o t o p e r i o d , e x p l a i n s the t i m i n g of i m p l a n t a t i o n and b i r t h of o t t e r s i n B r i t i s h Columbia and o t h e r n o r t h e r n a r e a s (see T a b l e X V I I I , Chapter I I I ) . However, i t does not account f o r i m p l a n t a t i o n p r i o r t o the w i n t e r s o l s t i c e which has been observed i n the s o u t h e a s t e r n U n i t e d S t a t e s ( M c D a n i e l 1963; H i l l and L a u h a c h i n d a 1981; L a n c i a and H a i r 1983). I suggest t h a t t h e r e may be d i f f e r e n c e s among p o p u l a t i o n s i n t h e i r response t o p h o t o p e r i o d i c changes; s o u t h e r n p o p u l a t i o n s of o t t e r s may be r e s p o n d i n g t o d i f f e r e n t c r i t i c a l p h o t o p e r i o d s or they may not respond t o p h o t o p e r i o d i c s t i m u l i a t a l l . G e n e t i c d i f f e r e n c e s between p o p u l a t i o n s occur i n a number of mammals (mink, Hansson 1947, H a t l e r 1976; harbour s e a l , B i g g 1973; d e e r , Goss and Rosen 1973; sheep, Bowman 1973, K a r s c h and F o s t e r 1981; mice, Peromyscus sp. , D e s j a r d i n s and Lopez 1980, Lynch e_t a l . 1981) and t h e r e appears t o be a g e n e r a l t r e n d among mammals f o r a p o s i t i v e r e l a t i o n s h i p between l a t i t u d e and the degree of p h o t o p e r i o d i c response (Bronson 1985; D e s j a r d i n s and Lopez 1980). In s o u t h e r n a r e a s o t t e r s may respond t o o t h e r e n v i r o n m e n t a l f a c t o r s such as r a i n f a l l or n u t r i t i o n t o s y n c h r o n i z e t h e i r r e p r o d u c t i v e c y c l e s w i t h f a v o u r a b l e c o n d i t i o n s . Lauhachinda (1978) was u n s u c c e s s f u l i n h i s attempt t o c o r r e l a t e i m p l a n t a t i o n d a t e s w i t h d a i l y changes i n t e m p e r a t u r e , but i t may be a more g e n e r a l r e s p o n s e t h a t he examined. Such d i f f e r e n c e s between p o p u l a t i o n s a r e not s u r p r i s i n g s i n c e p h o t o p e r i o d i s not the " u l t i m a t e f a c t o r " d i r e c t l y d e t e r m i n i n g the most advantageous 215 time f o r r e p r o d u c t i o n . I n s t e a d , i t i s o n l y a "proximate f a c t o r " t h a t a l l o w s the organism t o p r e p a r e f o r the c o n d i t i o n s t o come (Baker 1938). The e a r l i e r and l e s s r e s t r i c t e d i m p l a n t a t i o n d a t e s o b s e r v e d i n s o u t h e r n a r e a s l e a d s t o the h y p o t h e s i s t h a t o p t i m a l c o n d i t i o n s f o r r e p r o d u c t i v e s u c c e s s i n o t t e r s may l a s t l o n g e r , and occur a t d i f f e r e n t t imes of the y e a r s , than i n n o r t h e r n p o p u l a t i o n s . The e x a m i n a t i o n of the f a c t o r s which c o n t r o l the r e p r o d u c t i v e c y c l e i n v a r i o u s l o c a t i o n s i s an i n t e r e s t i n g t o p i c f o r f u r t h e r r e s e a r c h which i s n e c e s s a r y b e f o r e we w i l l b e g i n t o u n d e r s t a n d the d i f f e r e n c e s which occur between p o p u l a t i o n s . 216 CHAPTER V GENERAL DISCUSSION The r e s u l t s of t h i s study on c o a s t a l o t t e r s i n B r i t i s h Columbia r a i s e some i n t e r e s t i n g q u e s t i o n s c o n c e r n i n g the f a c t o r s which u l t i m a t e l y c o n t r o l the r e p r o d u c t i v e c y c l e of o t t e r s , and mammals i n g e n e r a l . A l t h o u g h o t t e r s may be e x p e c ted t o a l t e r t h e i r r e p r o d u c t i v e c y c l e t o one of a l e s s s e a s o n a l n a t u r e because of the r e l a t i v e l y s t a b l e environment a l o n g the c o a s t , the r e s u l t s of my study i n d i c a t e t h a t the r e p r o d u c t i v e c y c l e of r i v e r o t t e r s i s s i m i l a r i n f r e s h and marine h a b i t a t s a t s i m i l a r l a t i t u d e s . In the marine environment o t t e r s are s t r i c t l y s e a s o n a l b r e e d e r s w i t h a p r o l o n g e d , o b l i g a t o r y p e r i o d of embryonic d i a p a u s e . Based on my d a t a , I c o n c l u d e t h a t p h o t o p e r i o d p r o b a b l y c o n t r o l s the t i m i n g of the r e p r o d u c t i v e c y c l e i n o t t e r s i n B r i t i s h Columbia. Thus, the s i m i l a r i t i e s between the r e p r o d u c t i v e t i m i n g among o t t e r s l i v i n g a t s i m i l a r l a t i t u d e s i s l i k e l y the r e s u l t responses t o s i m i l a r p h o t o p e r i o d s . However, I was unable t o address the q u e s t i o n of which f a c t o r s a r e u l t i m a t e l y r e s p o n s i b l e f o r the t i m i n g of the r e p r o d u c t i v e c y c l e of c o a s t a l o t t e r s . I f the c o a s t a l environment i s as m i l d as i t appears t o be, why a r e o t t e r s s t r i c t l y s e a s o n a l b r e e d e r s ? Our l i m i t e d knowledge of the f a c t o r s which u l t i m a t e l y c o n t r o l b r e e d i n g i n mammmals, and o t t e r s i n p a r t i c u l a r , means t h a t d i s c u s s i o n i s n e c e s s a r i l y s p e c u l a t i v e . However, by p r e s e n t i n g some t h e o r i e s c o n c e r n i n g the mechanisms i n v o l v e d , f u r t h e r r e s e a r c h may be s t i m u l a t e d . The presence of s e a s o n a l b r e e d i n g suggests t h a t an o p t i m a l 217 p e r i o d f o r r e p r o d u c t i o n e x i s t s . A l t h o u g h the presence of s e a s o n a l b r e e d i n g i n o t t e r s may be due s o l e l y t o t h e i r p a s t e v o l u t i o n a r y h i s t o r y , the h i g h degree of synchrony I obser v e d among i m p l a n t a t i o n d a t e s s u g g e s t s t h a t t h e r e i s p r o b a b l y a c o n t i n u i n g s e l e c t i v e p r e s s u r e f o r s e a s o n a l b r e e d i n g among c o a s t a l o t t e r s . T h i s synchrony i s l e s s pronounced i n s o u t h e r n p o p u l a t i o n s where b i r t h s may be spread over two t o t h r e e months (McDaniel 1963; H i l l and Lauhachinda 1981; L a n c i a and H a i r 1983). F a c t o r s which may c o n t r o l the b r e e d i n g season have been d i s c u s s e d e x t e n s i v e l y (Baker 1938; S a d l e i r 1969a; Immelmann 1971; Weir and Rowlands 1973; Bronson 1985). G e n e r a l l y , any f a c t o r ( s ) which d e c r e a s e s r e p r o d u c t i v e s u c c e s s w i l l f a v o u r s e a s o n a l b r e e d i n g . An adequate food supply i s c o n s i d e r e d by most a u t h o r s t o be the p r i m a r y f a c t o r c o n t r o l l i n g of s e a s o n a l b r e e d i n g , a l t h o u g h c l i m a t i c c o n d i t i o n s and c o m p e t i t i o n may a l s o be i m p o r t a n t (Baker 1938; S a d l e i r 1969b; Immelmann 1971; M i l l a r 1977). Bronson (1985) s u g g e s t s t h a t mammals may be ex p e c t e d t o breed as o f t e n as p o s s i b l e whenever they d w e l l i n a r e a s w i t h a c o n s t a n t food s u p p l y . Even though a t f i r s t g l a n c e , food appears to be abundant f o r o t t e r s i n the c o a s t a l environment, i t i s p o s s i b l e t h a t t h e r e i s some s e a s o n a l f l u c t u a t i o n i n i t s a v a i l a b i l i t y . I n t e r t i d a l f i s h a r e p r e s e n t throughout the y e a r , but may v a r y i n abundance s e a s o n a l l y ; some f i s h s p e c i e s move i n t o deeper water d u r i n g the w i n t e r and most spawn d u r i n g l a t e s p r i n g (Hart 1973; Green 1971; Wourms and Evans 1974; Grossman 1982). T h e r e f o r e , the amount of energy expended by o t t e r s i n o b t a i n i n g food may v a r y c o n s i d e r a b l y w i t h season. 218 Kruuk and Hewson (1978) found t h a t i n S c o t l a n d c o a s t a l d w e l l i n g L u t r a l u t r a , which f e e d upon s i m i l a r prey s p e c i e s t o L. c a n a d e n s i s , swam g r e a t d i s t a n c e s t o f e e d i n g a r e a s and spent c o n s i d e r a b l e amounts of time f o r a g i n g . L ess than 20% of the d i v e s they o b s e r v e d (n = 120) were s u c c e s s f u l and they c o n c l u d e d t h a t o t t e r s had d i f f i c u l t i e s o b t a i n i n g food d u r i n g some p e r i o d s of the y e a r . Kruuk and Hewson (1978) a l s o found t h a t u n l i k e s o u t h e r n p o p u l a t i o n s where b i r t h s occur throughout the year (Stephens 1957; J e n k i n s and Harper 1982), b i r t h s were r e s t r i c t e d t o the s p r i n g i n S c o t l a n d . I t i s p o s s i b l e t h a t t h i s r e s t r i c t e d b i r t h p e r i o d may be r e l a t e d t o the d i f f i c u l t i e s i n o b t a i n i n g food they o b s e r v e d among o t t e r s . In A l a s k a , Woolington (1984) found t h a t c o a s t a l d w e l l i n g L. c a n a d e n s i s spent more time a c t i v e and i n the water d u r i n g the w i n t e r than d u r i n g o t h e r t i m e s . T h i s s u g g e s t s t h a t ' A l a s k a n o t t e r s may expend more energy o b t a i n i n g food i n the w i n t e r than a t o t h e r times of the y e a r . Young o t t e r s may be more a f f e c t e d by l i m i t e d food s u p p l i e s than a d u l t s . In l a r g e mammals a v a i l a b i l i t y of adequate food f o r growing o f f s p r i n g may be more i m p o r t a n t i n d e t e r m i n i n g the presence of s e a s o n a l b r e e d i n g than l a c t a t i o n a l demands on the mother (Bronson 1985). O t t e r s born i n A p r i l are weaned a p p r o x i m a t e l y two months l a t e r i n June or J u l y ( L i e r s 1951; Johnstone 1978), a time when young f i s h a r e e x t r e m e l y abundant (Green 1971; Grossman 1982). Developmental r a t e s of young m u s t e l i d s a r e not known a l t h o u g h , Watson (1978) found t h a t the f o r a g i n g e f f i c i e n c y of a t e n month o l d European o t t e r was c o n s i d e r a b l y lower than t h a t of an a d u l t . E s t e s e_t a_l. (1981) 219 a l s o r e p o r t e d t h a t j u v e n i l e sea o t t e r s were more o f t e n u n s u c c e s s f u l f o r a g i n g than a d u l t s . Thus, b i r t h s may be l i m i t e d t o A p r i l or May i n B r i t i s h Columbia by the energy n e c e s s a r y f o r f o r a g i n g d u r i n g the w i n t e r , e s p e c i a l l y among j u v e n i l e s . E a r l y s p r i n g b i r t h s a l l o w the young of the year t o be l a r g e enough and e x p e r i e n c e d enough t o s u r v i v e t h e i r f i r s t w i n t e r . In a d d i t i o n , the i n f l u x of f i s h i n t o s h a l l o w water t o spawn i n the s p r i n g r e s u l t s i n an abundant food s o u r c e f o r both a d u l t s d u r i n g l a c t a t i o n and young f o l l o w i n g weaning. C l i m a t i c c o n d i t i o n s may a l s o a c t t o c o n t r o l the t i m i n g and l e n g t h of the b r e e d i n g season i n some s p e c i e s . However, i n the c o a s t a l environment of s o u t h e r n B r i t i s h Columbia, a b s o l u t e changes i n t emperature are r e l a t i v e l y minor (Anon. 1982) and t e m p e r a t u r e s a r e comparable t o m i l d e r c l i m a t e s f u r t h e r s o u t h , i n c o n t r a s t , the t i m i n g of the r e p r o d u c t i v e c y c l e i s s i m i l a r t o a r e a s a t the same l a t i t u d e , r e g a r d l e s s of the a n n u a l changes i n t e m p e r a t u r e . T h e r e f o r e , ambient t e m p e r a t u r e s a l o n e p r o b a b l y have l i t t l e e f f e c t on s e a s o n a l b r e e d i n g i n my study a r e a . B e f o r e the f o r e g o i n g h y p o t h e s i s can be e v a l u a t e d , d e t a i l e d s t u d i e s on the energy r e q u i r e m e n t s of b oth a d u l t s and j u v e n i l e s a r e needed. The amount of energy expended d u r i n g f o r a g i n g and the energy n e c e s s a r y f o r r e p r o d u c t i o n i n d i f f e r e n t seasons must be d e t e r m i n e d . A l s o , the importance of the s e a s o n a l i t y of a h a b i t a t may be i n d i c a t e d by s t u d i e s i n an a s e a s o n a l environment such as c a p t i v i t y . S i n c e the degree of s e a s o n a l i t y p r e s e n t i n the marine environment has y e t t o be q u a n t i f i e d , i n f o r m a t i o n about how o t t e r s i n t e r a c t w i t h t h e i r environment and s t u d i e s on 2 2 0 the abundance of prey s p e c i e s a r e needed t o d e t e r m i n e i f food i s i n f a c t l i m i t i n g a t some p o i n t . Answers t o q u e s t i o n s such as t h e s e w i l l e nable us t o determine i f changes i n a v a i l a b l e energy can account f o r the r e s t r i c t e d b r e e d i n g season I o b s e r v e d . As w i t h s e a s o n a l b r e e d i n g , i t i s p o s s i b l e t h a t the o c c u r r e n c e of embryonic d i a p a u s e i n c o a s t a l o t t e r s i s a s p e c i e s s p e c i f i c c h a r a c t e r i s t i c t h a t was s e l e c t e d f o r under d i f f e r e n t e c o l o g i c a l c o n d i t i o n s and now i s a n e u t r a l t r a i t . L i t t l e i s known c o n c e r n i n g the time n e c e s s a r y f o r e v o l u t i o n a r y change or the l e n g t h of time i n which o t t e r s have been p r e s e n t i n the c o a s t a l environment. However, t h i s e x p l a n a t i o n does not e x p l a i n why ' c h e a t e r s ' do not p r o s p e r by b r e e d i n g a t o t h e r t i m e s of the year or why t h e r e i s not g r e a t e r v a r i a t i o n i n d i a p a u s e as seen i n the sea o t t e r ( L o u g h l i n e t a l . 1981). The presence of s t r i c t l y s e a s o n a l b r e e d i n g and an o b l i g a t o r y p e r i o d of embryonic diapause i n d i c a t e s t h a t e i t h e r g e n e t i c v a r i a t i o n i s minimal i n r i v e r o t t e r s or t h a t s e l e c t i v e f o r c e s a r e p r e s e n t which favour t h e i r r e t e n t i o n . At l e a s t t h r e e p o s s i b l e advantages can be suggested f o r the e x i s t e n c e of embryonic d i a p a u s e and a change i n the b r e e d i n g season. One advantage i s t h a t the t i m i n g of b i r t h no l o n g e r depends upon the t i m i n g of m a t i n g . I n s t e a d i t becomes dependent upon the t i m i n g of i m p l a n t a t i o n which can be i n d uced by an e n v i r o n m e n t a l cue such as p h o t o p e r i o d . T h i s a l l o w s b i r t h s t o be s y n c h r o n i z e d w i t h o u t a s i m i l a r s y n c h r o n i z a t i o n i n mating which can occur over a r e l a t i v e l y l a r g e time p e r i o d . T h i s i s p a r t i c u l a r l y e v i d e n t i n the mink where the l e n g t h of the d e l a y 221 i s i n v e r s e l y p r o p o r t i o n a l t o the d a t e of mating (Hansson 1947; Enders 1952). The a b i l i t y t o mate o v e r a wide time p e r i o d would be advantageous t o s o l i t a r y a n i m a l s such as the o t t e r ( M e l q u i s t and Hornocker 1983; L a r s e n 1983; W o o l i n g t o n 1984) and may a c t i n c o n j u n c t i o n w i t h i n d uced o v u l a t i o n and a p r o l o n g e d e s t r u s , t o extend the b r e e d i n g season and a l l o w m a t i n g whenever a female i s l o c a t e d by the male. Embryonic d i a p a u s e i s common among the m u s t e l i d s (Mead and Wright 1983), most of which are s o l i t a r y a n i m a l s ( P o w e l l 1979; Hornocker e t a l . 1983). A l a c k of a v a i l a b l e energy d u r i n g the l a t e w i n t e r may a l s o s e l e c t f o r an adjustment i n the b r e e d i n g season. I f energy i s l i m i t e d e a r l y i n the y e a r , owing t o a r e d u c t i o n i n the prey base (see above), i t would be advantageous t o d e l a y mating u n t i l food i s abundant. Large amounts of energy a r e n e c e s s a r y t o f i n d a mate, e s p e c i a l l y i n s p e c i e s w i t h d i s p e r s e d , s o l i t a r y i n d i v i d u a l s , and o t t e r s o f t e n t r a v e l l o n g d i s t a n c e s d u r i n g the b r e e d i n g season ( M e l q u i s t and Hornocker 1983; Woolington 1984). Hence, i n B r i t i s h Columbia mating i n A p r i l may be f a v o u r e d due t o the abundant food p r e s e n t . I n the c o a s t a l environment the same s e l e c t i v e p r e s s u r e s which may be a c t i n g t o m a i n t a i n s e a s o n a l b r e e d i n g can a l s o r e s u l t i n a l a t e r b r e e d i n g season. S t u d i e s t o d e t e r m i n e i f food i s l i m i t e d d u r i n g the l a t e w i n t e r are n e c e s s a r y t o d e t e r m i n e i f t h i s h y p o t h e s i s has m e r i t . A t h i r d p o s s i b l e e x p l a n a t i o n f o r the presence of embryonic diapause l i e s i n the f a c t t h a t m a t i n g c o i n c i d e s w i t h the l a c t a t i o n p e r i o d . A l t h o u g h the most e n e r g e t i c a l l y s t r e s s f u l p e r i o d s f o r both males ( f i n d i n g a mate) and females ( l a c t a t i o n ) 2 2 2 c o i n c i d e , t h e r e may be an advantage due t o a change i n beh a v i o u r of females d u r i n g the denning p e r i o d . D u r i n g l a c t a t i o n , a c t i v i t y of the females d e c r e a s e s and they remain near the den f o r much of the day ( M e l q u i s t and Hornocker 1983). Because o t t e r s can have r e l a t i v e l y l a r g e (1-40 km), l i n e a r home ranges ( M e l q u i s t and Hornocker 1983; L a r s e n 1983; Wo o l i n g t o n 1984), the i n c r e a s e d p r e d i c t a b i l i t y of f i n d i n g a female i n a g i v e n area may p r o v i d e a s l i g h t advantage. A l t h o u g h the importance of s o c i a l f a c t o r s such as t h i s can never be f u l l y d e t e r m i n e d , some i n d i c a t i o n may be p r o v i d e d by more d e t a i l e d s t u d i e s on the be h a v i o u r of o t t e r s . L i t t l e i n f o r m a t i o n i s a v a i l a b l e on the a c t i v i t y p a t t e r n s of o t t e r s d u r i n g the b r e e d i n g season and on the methods males use t o l o c a t e f e m a l e s . I t i s a l s o not known where mating o c c u r s i n r e l a t i o n t o the den and i f females a r e bred by t r a n s i e n t s or males on a d j a c e n t home ranges. These i d e a s a re n e c e s s a r i l y s p e c u l a t i v e owing t o the i m p r e c i s e n a t u r e of our knowledge of the s o c i a l s t r u c t u r e of o t t e r s and the s e l e c t i v e f a c t o r s o p e r a t i n g . In a d d i t i o n , t h e r e i s the l i k e l i h o o d t h a t m u l t i p l e causes have o p e r a t e d on the l i f e h i s t o r y s t r a t e g y and t h a t they may have been i n f l u e n c e d by chance h i s t o r i c a l e v e n ts ( S t e a r n s 1977). F u r t h e r r e s e a r c h i n be h a v i o u r and e c o l o g y s h o u l d c o n s i d e r some of the q u e s t i o n s posed here and i n t u r n , may suggest new p o s s i b i l i t i e s . 223 Summary The f i r s t o b j e c t i v e of t h i s study was t o d e s c r i b e the a n n u a l changes which occur i n the r e p r o d u c t i v e organs of r i v e r o t t e r s . In Chapters I I and I I I , I p r o v i d e the f i r s t d e t a i l e d d e s c r i p t i o n of the anatomy, h i s t o l o g y and e n d o c r i n o l o g y of the male and female r e p r o d u c t i v e organs d u r i n g the a n n u a l c y c l e . G e n e r a l l y , my r e s u l t s s u pport and expand upon the few d e s c r i p t i o n s a v a i l a b l e f o r o t t e r s ( H a m i lton and E a d i e 1964; Tabor 1974; Lauhachinda 1978), and i n d i c a t e t h a t the e n d o c r i n o l o g y and the changes t h a t occur i n the r e p r o d u c t i v e organs, are s i m i l a r t o those of o t h e r m u s t e l i d s (Hansson 1947; Enders 1952; Pilbeam e t a l . 1979; Mead 1968a,b; Mead 1981; N e a l and H a r r i s o n 1958; Ahnlund 1980; S i n h a et. a_l. 1966; Enders and Given 1977). In t h i s s t u d y , I p r e s e n t e d a method, phase a n a l y s i s , t h a t can be used t o s t a n d a r d i z e the d e s c r i p t i o n of spermatogenesis and t o q u a n t i f y the degree of r e p r o d u c t i v e a c t i v i t y among males. I a l s o d e s c r i b e d the v a g i n a l smear c y c l e i n o t t e r s , which p r o v i d e s a n o n - i n v a s i v e method of d e t e r m i n i n g the r e p r o d u c t i v e s t a t e of f e m a l e s . The second o b j e c t i v e of my study was t o determine i f the l o c a l environment would a f f e c t the t i m i n g or n a t u r e of the r e p r o d u c t i v e c y c l e . My r e s u l t s show t h a t t h e r e , as i n o t h e r a r e a s , o t t e r s a r e s e a s o n a l b r e e d e r s w i t h a p r o l o n g e d (9-11 months), o b l i g a t o r y p e r i o d of embryonic d i a p a u s e p r i o r to i m p l a n t a t i o n . M a t i n g , i m p l a n t a t i o n and b i r t h o c c u r r e d a t times comparable t o those of L. c a n a d e n s i s l i v i n g a t s i m i l a r 224 l a t i t u d e s , but i n f r e s h water. In males, r e p r o d u c t i v e a c t i v i t y began i n the l a t e autumn and a d u l t s were c a p a b l e of b r e e d i n g from l a t e January through mid-May. Females e x h i b i t e d e s t r u s from l a t e F e b r u a r y t h r o u g h mid-May a l t h o u g h the m a j o r i t y of m ating appeared t o occur d u r i n g March and A p r i l . A f t e r a n i n e t o e l e v e n month p e r i o d of embryonic d i a p a u s e , the c o r p o r a l u t e a and b l a s t o c y s t s i n c r e a s e d a c t i v i t y and i m p l a n t a t i o n o c c u r r e d the subsequent F e b r u a r y . The o v u l a t i o n r a t e s , number of young and age of s e x u a l m a t u r i t y of males were s i m i l a r t o those r e p o r t e d i n p r e v i o u s s t u d i e s ( H a m i l t o n and E a d i e 1946; Tabor and Wight 1977; Mowbray et a l . 1979; H i l l and Lauhachinda 1981). Some females were found t o mature l a t e r i n t h i s s t u d y , which may be r e l a t e d t o the h i g h e r p o p u l a t i o n d e n s i t i e s observed among c o a s t a l p o p u l a t i o n s ( L a r s e n 1983; W o o l i n g t o n 1984; p e r s o n a l o b s e r v a t i o n ) . The t h i r d o b j e c t i v e was t o examine the r o l e of p h o t o p e r i o d i n c o n t r o l l i n g the t i m i n g of r e p r o d u c t i v e e v e n t s . In Chapter I I I , I p r e s e n t e v i d e n c e t h a t changes i n p h o t o p e r i o d i n f l u e n c e the t i m i n g of s p e r m a t o g e n e s i s , e s t r u s and p o s s i b l y i m p l a n t a t i o n i n my c a p t i v e o t t e r s . My r e s u l t s i n d i c a t e t h a t the t i m i n g of the annual r e p r o d u c t i v e c y c l e i n o t t e r s i n B r i t i s h Columbia i s i n f l u e n c e d by p h o t o p e r i o d i c s t i m u l i r a t h e r than the l o c a l e n v i r o n m e n t a l c o n d i t i o n s . T h i s study p r o v i d e s a b a s i s f o r f u r t h e r r e s e a r c h on the r e p r o d u c t i v e c y c l e of r i v e r o t t e r s . I t i s hoped t h a t f u r t h e r i n v e s t i g a t i o n s w i l l use the t e c h n i q u e s describe'd here t o s t a n d a r d i z e the i n f o r m a t i o n o b t a i n e d . Q u e s t i o n s c o n c e r n i n g the 2 2 5 f a c t o r s which c o n t r o l the r e p r o d u c t i v e c y c l e can be answered b e s t w i t h s t a n d a r d i z e d d a t a . 226 REFERENCES CITED Ahnlund, H. 1980. S e x u a l m a t u r i t y and b r e e d i n g season of the badger, Meles meles i n Sweden. J . Z o o l . , London. 190:77-95. A l l a i s , C. and L. M a r t i n e t . 1978. R e l a t i o n between d a y l i g h t r a t i o , plasma p r o g e s t e r o n e l e v e l s and t i m i n g of n i d a t i o n i n mink ( M u s t e l a v i s o n ) . J . Reprod. F e r t . 54:133-136. A l l a n s o n , M. 1932. The r e p r o d u c t i v e p r o c e s s e s of c e r t a i n mammals. I l l The r e p r o d u c t i v e c y c l e of the male f e r r e t . P r o c . Roy. Soc. London. S e r . B 110:295-312. Anonymous. 1982. Canadian C l i m a t e Normals 1951-1980. Temperature and P r e c i p i t a t i o n . B r i t i s h C olumbia. Atmospheric Environment S e r v i c e . Environment Canada, Ottawa, O n t a r i o . Audy, M.C. 1978. Etude u l t r a s t r u c t u r o l e c e l l u l e s de L e y d i g e t de S e r t o l i au c o u r s du c y c l e s e c u e l s a i s o n n i e r de l a fourne (Martes f o i n a E r x ) . Gen. Comp. E n d o c r i n o l . 36:462-476. Audy, M.C, B. M a r t i n , G. Charron and M. Bonnin. 1982. S t e r o i d b i n d i n g p r o t e i n s and t e s t o s t e r o n e l e v e l i n the badger plasma d u r i n g the annu a l c y c l e . Gen. Comp. Endocr. 48:239-246. A u l e r i c h , R.J., R.K. R i n g e r , and S. Iwamoto. 1973. R e p r o d u c t i v e f a i l u r e and m o r t a i l i t y i n mink f e d on Great Lakes f i s h . J . Reprod. F e r t . , S u p p l . 19:365-376. Baker, J.R. 1938. The e v o l u t i o n of b r e e d i n g season. I n : de Beer, G.R. ( e d . ) . E v o l u t i o n : E s s a y s on A s p e c t s of E v o l u t i o n a r y B i o l o g y . O x f o r d U n i v . P r e s s . London, pp. 161-177. B a r s u r , P.K. and A.S. Ramos. 1973. S e a s o n a l changes i n the a c c e s s o r y sex g l a n d s and gonaducts of male mink. Can. J . Z o o l . 51:1125-1132. 227 B e r n d t s o n , W.E. 1977. Methods f o r q u a n t i f y i n g mammalian sp e r m a t o g e n e s i s : a r e v i e w . J . Anim. S c i . 44:818-833. B e s t , A. 1962. The Canadian o t t e r , L u t r a c a n a d e n s i s i n c a p t i v i t y . I n t . Zoo Yb. 4:42-44. B i g g , M.A. 1973. A d a p t a t i o n s i n the b r e e d i n g of the harbor s e a l , Phoc v i t u l i n a . J . Reprod. F e r t . , S u p p l . 19:131-142. B i g g , M.A. and H.D. F i s h e r . 1975 E f f e c t of p h o t o p e r i o d on annual r e p r o d u c t i o n i n female harbour s e a l s . Rapp. P.-v. Reun. Cons. i n t . E x p l o r . Mer. 169:141-144. B i s s o n n e t t e , T.H. 1932. M o d i f i c a t i o n of mammalian s e x u a l c y c l e s . 1. R e a c t i o n s of f e r r e t s ( P u t o r i u s v u l g a r i s ) of both sexes t o e l e c t r i c l i g h t added a f t e r dark i n November and December. P r o c . Roy. Soc. London. S e r . B. 110:322-336. B o i s s i n - A g a s s e , L., J . B o i s s i n and R. O r t a v a n t . 1982. C i r c a d i a n p h o t o s e n s i t i v e phase and p h o t o p e r i o d i c c o n t r o l of t e s t i s a c t i v i t y i n the mink ( M u s t e l a v i s o n P e a l e and B e a u v o i s ) , a s h o r t - d a y mammal. B i o l . Reprod. 26:110-119. Bonnin, M. and R. C a n i v e n c . 1980. E n v i r o n m e n t a l f a c t o r s i n v o l v e d i n d e l a y e d i m p l a n t a t i o n . P r o g r e s s i n Reprod. B i o l . 7:173-188. Bowman, J . C. 1973. P h o t o p e r i o d i c i t y i n Mammals. J . Reprod. F e r t . , S u p p l . 19:85-88. Bronson, F.H. 1985. Mammalian r e p r o d u c t i o n : an e c o l o g i c a l p e r s p e c t i v e . B i o l . Reprod. 32:1-26. Buchanan, G.D, 1966. R e p r o d u c t i o n i n the F e r r e t ( M u s t e l a f u r o ) . I . U t e r i n e h i s t o l o g y and h i s t o c h e m i s t r y d u r i n g pregnancy and pseudopregnancy. Am. J . Anat. 118:195-216. C a n i v e n c , R. 1966. A study of p r o g e s t a t i o n i n the European badger (Meles m e l e s ) . I n : Rowland, J.W. ( e d . ) . Comparative B i o l o g y of R e p r o d u c t i o n i n Mammals. Academic P r e s s . London, pp 15-26. 2 2 8 C a n i v e n c , R. and M. Bonnin. 1979. Delayed i m p l a n t a t i o n i s under e n v i r o n m e n t a l c o n t r o l i n the badger (Meles meles L . ) . Nature 278:849-850. C a n i v e n c , R. and M. Bonnin. 1981. E n v i r o n m e n t a l c o n t r o l of d e l a y e d i m p l a n t a t i o n i n the European badger (Meles m e l e s ) . J . Reprod. F e r t . , S u p p l . 29:25-33. C a n i v e n c , R. , M. Bonnin and C. R i b e s . 1981. T r i g g e r i n g of egg-i m p l a n t i n g by p r o l o n g i n g the dark phase of p h o t o p e r i o d i n the European badger Meles meles L. C. R. Acad. S c i . P a r i s . S e r . I I I . 292:1009-1012. C a n i v e n c , R. , C. Mauget, M. Bonnin and R.J. A i t k e n . 1981. D e l a y e d I m p l a n t a t i o n i n the Beech Marten (Martes f o i n a ) . J . Z o o l . , London. 193:325-332. Channing, C P . , S. F o w l e r , B. Engel and K. V i t e k . 1977. F a i l u r e of d a i l y i n j e c t i o n s of ketamine HCL t o a d v e r s e l y a l t e r m e n s t r a l c y c l e l e n g t h , b l o o d e s t r o g e n and p r o g e s t e r o n e l e v e l s i n the rhesus monkey. P r o c . Soc. Exp. B i o l . Med. 155:615-619. C h r i s t i a n , J . J . 1980. E n d o c r i n e f a c t o r s i n p o p u l a t i o n r e g u l a t i o n . I n : Cohen, M., R. Malpass and H. K l e i n ( e d s . ) . B i o s o c i a l Mechanisms of P o p u l a t i o n R e g u l a t i o n . Y a l e U n i v . P r e s s . New Haven, pp. 55-116. Concannon,P., T. P i l b e a m and H. T r a v i s . 1980. Advanced i m p l a n t a t i o n i n mink (M u s t e l a v i s o n ) t r e a t e d w i t h medroxyprogesterone a c e t a t e d u r i n g e a r t l y embryonic d i a p a u s e . J . Reprod. F e r t . 58:1-6. Cook, S. J . , N.C. Rawlings and R.I. Kennedy. 1982. Q u a n t i t a t i o n of s i x androgens by combined h i g h performance l i q u i d chromatography and radioimmunoassay. S t e r o i d s 40:369-380. C o u r r i e r , R. 1927. Etude sur l e determinisme des c a r a c t e r e s s e x u e l s s e c o n d a i r e s chez quelques mammiferes a l ' a c t i v i t e t e s t i c u l a i r e p e r i o d i q u e . Archs B i o l , . P a r i s , 37:173. 229 D'Souza, F. 1978. The d e t e c t i o n of o e s t r u s . I n : Watson, P.F. ( e d . ) . A r t i f i c i a l b r e e d i n g of non-domestic a n i m a l s . Academic P r e s s . London, pp. 175-193. D a n i e l , J.C. 1970. C o i n c i d e n c e of embryonic growth and u t e r i n e p r o t e i n i n the f e r r e t . J . Embryol. Exp. Morph. 24:305-312. D a n i e l , J.C. 1971. T e r m i n a t i o n of pregnancy i n mink caused by r e p e a t e d i n j e c t i o n s d u r i n g the p e r i o d p r e c e d i n g i m p l a n t a t i o n . J . Anim. S c i . 33:659-661. D e a n e s l y , R. 1935. The r e p r o d u c t i v e p r o c e s s e s of c e r t a i n mammals. P a r t IX : Growth and R e p r o d u c t i o n i n the s t o a t . P h i l . T r a n s . Roy. Soc. London. 225:460-492. D e a n e s l y , R. 1944. The r e p r o d u c t i v e c y c l e of the female weasel ( M u s t e l a n i v a l i s ) . P r o c . Z o o l . S o c , P a r t I I I . 114:339-349. D e a n e s l y , R. 1967. E x p e r i m e n t a l o b s e r v a t i o n s on the f e r r e t c o r p u s luteum of pregnancy. J . Reprod. F e r t . 13:183-186. D e s j a r d i n s , C. and M.J. Lopez. 1980. Sensory and nonsensory m o d u l a t i o n of t e s t i s f u n c t i o n . I n : S t e i n b e r g e r , A. and E. S t e i n b e r g e r . ( e d s . ) . T e s t i c u l a r Development, S t r u c t u r e and F u n c t i o n . Raven P r e s s . New York. pp. 381-388. D e s j a r d i n s , C. and M.J. Lopez. 1983. E n v i r o n m e n t a l cues evoke d i f f e r e n t i a l response i n p i t u i t a r y t e s t i c u l a r f u n c t i o n i n deer mice. E n d o c r i n o l o g y . 112:1398-1406. Donovan, B.T. 1967. The c o n t r o l of corpus luteum f u n c t i o n i n the f e r r e t . A r c h . Anat. M i c r o . , 56, S u p p l . 3-4:315-325. D r i c k a m e r , L.C. 1979. A c c e l e r a t i o n and d e l a y of f i r s t e s t r u s i n w i l d Mus musculus. J . Mammal. 60:215-216. Duby, R.T. and H.F. T r a v i s . 1972. P h o t o p e r i o d i c c o n t r o l of f u r growth and r e p r o d u c t i o n i n the mink ( M u s t e l a v i s o n ) . J . Exp. Z o o l . 182:217-226. 230 Duby, R.T., T. P i l b e a m and H.F. T r a v i s . 1972. The i n f l u e n c e of m e l a t o n i n and h y s t e r e c t o m y on plasma p r o g e s t e r o n e l e v e l s of the mink ( M u s t e l a v i s o n ) . B i o l . Reprod. 7:125. D u p l a i x - H a l l , N. 1975. R i v e r o t t e r s i n c a p t i v i t y : a r e v i e w . I n : M a r t i n , R.D. ( e d . ) . B r e e d i n g endangered s p e c i e s i n c a p t i v i t y . Academic P r e s s . New York. pp. 315-327. E a y r s , J.T., A. G l a s s and H.H. Swanson. 1977. The ovary and nervous system i n r e l a t i o n t o b e h a v i o u r . I n : Zuckerman, S. and B.J. Weir ( e d s . ) . The Ovary. Volume I I , P h y s i o l o g y . Academic P r e s s . New York. pp. 399-455. E c k s t e i n , P. 1977. E n d o c r i n e a c t i v i t y of the o v a r y . I n : Zuckerman, S. and B.J. Weir ( e d s . ) . The Ovary. Volume I I , P h y s i o l o g y . Academic P r e s s . New York. pp. 275-313. E l l i o t t , J.A. and B.D. Goldman. 1981 . S e a s o n a l r e p r o d u c t i o n : p h o t o p e r i o d and b i o l o g i c a l c l o c k s . I n : A d l e r , N.T. ( e d . ) . N e u r o e n d o c r i n o l o g y of R e p r o d u c t i o n . Plenum P u b l . New York, pp. 377-423. En d e r s , A.C. 1981. Embryonic d i a p a u s e - p e r s e c t i v e s . J . Reprod. F e r t . , S u p p l . 29:229-241. En d e r s , A.C. and R.L. G i v e n . 1977. The endometrium of d e l a y e d and e a r l y i m p l a n t a t i o n . I n : Wynn, R.M. ( e d . ) . B i o l o g y of the U t e r u s . Plenum P r e s s . New York. pp. 203-243. En d e r s , R.K. 1952. R e p r o d u c t i o n i n the mink ( M u s t e l a v i s o n ) . P r o c . Am. P h i l . Soc. 96:691-755. E n d e r s , R.K. and A.C. Enders . 1963. Morphology of the female r e p r o d u c t i v e t r a c t d u r i n g d e l a y e d i m p l a n t a t i o n i n the mink. I n : Enders, A.C. ( e d . ) . Delayed I m p l a n t a t i o n . U n i v . C h i c a g o P r e s s . C h i c a g o , pp. 129-140. E s t e s , J.A., R.J. Jameson and A.M. Johnson. 1981. Food s e l e c t i o n and some f o r a g i n g t a c t i c s of sea o t t e r s . P r o c . Worldwide F u r b . Conf. F r o s t b u r g S t a t e C o l l e g e , MD. pp. 606-641. 231 Feder, H.H. 1981. E s t r o u s c y c l i c i t y i n mammals. I n : A d l e r , N.T. ( e d . ) . N e u r o e n d o c r i n o l o g y and R e p r o d u c t i o n . Plenum P r e s s . New York. pp. 279-348. Fordham, R.A. 1971. F i e l d p o p u l a t i o n s of deer mice w i t h s u p p l e m e n t a l f o o d . E c o l o g y . 52:138-146. Foresman, K.R. and R.A. Mead. 1978. L u t e a l c o n t r o l of n i d a t i o n i n t he f e r r e t ( M u s t e l a p u t r i o u s ) . B i o l . Reprod. 18:490-496. F o s t e r , M.A. and F.L. Hisaw. 1935. E x p e r i m e n t a l o v u l a t i o n and r e s u l t i n g pseudopregnancy i n a n e s t r o u s c a t s . Anat. Rec. 62:75-93. Foy, M.K. 1984. Seasonal movement, home range, and h a b i t a t use of r i v e r o t t e r i n s o u t h e a s t e r n Texas. M.S. T h e s i s . Texas A&M U n i v . C o l l e g e S t a t i o n . 102 pp. F r i l e y , C.E. 1949. Age d e t e r m i n a t i o n , by use of the baculum, i n the r i v e r o t t e r , L u t r a £. c a n a d e n s i s S c h r e b e r . J . Mammal. 30:102-110. G a r s h e l i s , D.L., A.M. Johnson and J.A. G a r s h e l i s . 1985. S o c i a l o r g a n i z a t i o n of sea o t t e r s i n P r i n c e W i l l i a m Sound, A l a s k a . Can. J . Z o o l . 62:2648-2658. G i l b e r t , F.F. and E.D. B a i l e y . 1967. The e f f e c t of v i s u a l i s o l a t i o n on r e p r o d u c t i o n i n the female ranch mink. J . Mammal. 48:113-118. Goss, R.J. and J.K. Rosen. 1973. The e f f e c t of l a t i t u d e and p h o t o p e r i o d i n the growth of a n t l e r s . J . Reprod. F e r t . , S u p p l . 19:111-118. Green, J.M. 1971. L o c a l d i s t r i b u t i o n of 0 1 i q o c o t t u s maculosus G i r a r d and oth e r t i d e p o o l c o t t i d s of the west c o a s t of Vancouver I s l a n d , B r i t i s h Columbia. Can. J . Z o o l . 49:1111-1 1 28. 232 G r e e n s i d e s , R.T. and R.A. Mead. 1973. O v u l a t i o n i n the S p o t t e d Skunk ( S p i l o q a l e p u t o r i u s l a t i f r o n s ) . B i o l . Reprod. 8:576-584. G r e e n w a l t , L.A. 1977. I n t e r n a t i o n a l t r a d e i n endangered s p e c i e s of w i l d fauna and f l o r a . Fed. R e g i s t . 42:10462-10488. Grossman, G.D. 1982. Dynamics and o r g a n i z a t i o n of a rocky i n t e r t i d a l f i s h assemblage: the p e r i s t e n c e and r e s i l i e n c e of taxocene s t r u c t u r e . Am. Nat. 119:611-637. Gulamhusien, A.P. and W.H. Tarn. 1974. R e p r o d u c t i o n i n the male s t o a t , M u s t e l s e r m i n e a . J . Reprod. F e r t . 41:303-312. G u s t a f s o n , A.W. and M. Shemesh. 1976. Changes i n plasma t e s t o s t e r o n e l e v e l s d u r i n g the annual r e p r o d u c t i v e c y c l e of the h i b e r n a t i n g b a t , M y o t i s l u c i f u g u s , w i t h a survey of plasma t e s t o s t e r o n e l e v e l s i n a d u l t male v e r t e b r a t e s . B i o l . Reprod. 15:9-24. H a l l , E.R. 1981. The Mammals of N o r t h A merica. John W i l e y and Sons, New York. pp. 1029-1031. H a m i l t o n , W.J. and W.R. E a d i e . 1964. R e p r o d u c t i o n i n the o t t e r , L u t r a c a n a d e n s i s . J . Mammal. 45:242-252. H a m i l t o n , W.J. and J.H. G o u l d . 1940. The normal o e s t r u s c y c l e of the F e r r e t : the c o r r e l a t i o n of the v a g i n a l smear and the h i s t o l o g y of the g e n i t a l t r a c t , w i t h notes on the d i s t r i b u t i o n of g l y c o g e n , the i n c i d e n c e of growth, and the r e a c t i o n t o i n t r a - v i t a m s t a i n i n g by t r y p a n b l u e . T r a n s . Roy. Soc. E d i n . 60:87-109. Hammond, J . 1951. C o n t r o l by l i g h t of r e p r o d u c t i o n i n f e r r e t s and mink. Nature 167:150-151. Hansson, A. 1947. The p h y s i o l o g y of r e p r o d u c t i o n i n mink ( M u s t e l a v i s o n ) w i t h s p e c i a l r e f e r e n c e t o d e l a y e d i m p l a n t a t i o n . A c t a Z o o l . 28:1-136. 233 H a r r i s , C.J. 1968. O t t e r s : a Study of Recent L u t r i n a e . W e i d e n f i e l d & N i c o l s o n , London. H a r t , J .L. 1973. P a c i f i c f i s h e s of Canada. B u l l . F i s h . Res. Board Can. No. 180. Harvey, N.E. and W.V. M a c f a r l a n e . 1958. The e f f e c t s of day l e n g t h on the c o a t - s h e d d i n g c y c l e s , body-weight, and r e p r o d u c t i o n of the f e r r e t . A u s t . J . B i o l . S c i . 11:189-199. H a t l e r , D.F. 1972. Mammals of P a c i f i c Rim N a t i o n a l Park. The r i v e r o t t e r . U n p u b l i s h e d r e p o r t t o the N a t i o n a l and H i s t o r i c P a r k s Branch. Dept. of I n d i a n A f f a i r s and N o r t h e r n Development, pp. 122-132. H a t l e r , D.F. 1976. The c o a s t a l mink on Vancouver I s l a n d , B r i t i s h Columbia. Ph.D. T h e s i s . U n i v . B r i t i s h Columbia. Vancouver, B r i t i s h C o lumbia. 360 pp. Heap, R.B. and J . Hammond. 1974. Plasma p r o g e s t e r o n e l e v e l s i n pregnant and pseudopregnant f e r r e t s . J . Reprod. F e r t . 39:149-152. H e r b e r t , J . 1977. E x t e r n a l f a c t o r s and o v a r i a n a c t i v i t y i n mammals. I n : Zuckerman, S. and B.J. Weir ( e d s . ) . The Ovary. Volume I I , P h y s i o l o g y . Academic P r e s s . London, pp. 457-505. H e r b e r t , J . and M. K l i n o w s k a . 1978. Day l e n g t h and the annual r e p r o d u c t i v e c y c l e i n the f e r r e t ( M u s t e l a f u r o ) : the r o l e of the p i n e a l body. I n : Assenmacher, I and D.S. F a r n e r ( e d s . ) . E n v i r o n m e n t a l E n d o c r i n o l o g y . S p r i n g e r - V e r l a g e . New York. pp. 87-93. H e r b e r t , J . , P.M. S t a c e y and D.H. Thorpe. 1978. R e c u r r e n t b r e e d i n g seasons i n p i n e a l e c t o m i z e d or o p t i c -n e r v e s e c t i o n e d f e r r e t s . J . E n d o c r i n o l . 78:389-397. H i l l , E.P. and V. L a u h a c h i n d a . 1981. R e p r o d u c t i o n i n r i v e r o t t e r s from Alabama and G e o r g i a . P r o c . Worldwide Fu r b . Conf. F r o s t b u r g S t a t e C o l l e g e , MD. pp. 478-486. 234 H i l l m a n , R.B. and R.G. Loy. 1975. Oestrogen e x c r e t i o n i n mares i n r e l a t i o n t o v a r i o u s r e p r o d u c t i v e s t a t e s . J . Reprod. F e r t . , S u p p l . 23:223-230. Hoffmann, J . 1973. The i n f l u e n c e of p h o t o p e r i o d s on r e p r o d u c t i v e f u n c t i o n i n female mammals. I n : Greep, R.O. and E.B. Astwood ( e d s . ) . Handbook of P h y s i o l o g y . S e c t i o n V I I , V o l . 2, P a r t 1. American P h y s i o l o g i c a l S o c i e t y . Washington, D.C. pp. 57-77. Hooper, E.T. and B.T. Ostenson. 1949. Age groups i n M i c h i g a n o t t e r . Occas. Pap. Mus. Z o o l . U n i v . M i c h . 518:1-22. Hornocker, M.G., J.P. M e s s i c k and W.E. M e l q u i s t . 1983. S p a t i a l s t r a t e g i e s i n t h r e e s p e c i e s of M u s t e l i d a e . A c t a Z o o l . F e n n i c a . 174:185-188. H o s t e t l e r , M. W. and B.E. P i a c s e k . 1977. P a t t e r n s of p i t u i t a r y gonadal hormone s e c r e t i o n d u r i n g a 24 hour p e r i o d i n the male r a t . B i o l . Reprod. 16:495-498. Immelmann, K. 1971. E c o l o g i c a l a s p e c t s of p e r i o d i c r e p r o d u c t i o n . I n : F a r n e r r , D.S. and J.R. K i n g ( e d s . ) . A v i a n B i o l o g y . V o l . I . Academic P r e s s , New York. pp.341-389. J e n k i n s , D. and R.J. H a r p e r . 1982. F e r t i l i t y i n European o t t e r s ( L u t r a l u t r a ) . J . Z o o l . 197:299-300. Johnson, O.W. and I.O. Buss. 1967. The t e s t e s of the A f r i c a n e l e p h a n t s (Loxodonto A f r i c a n a ) . I . H i s t o l o g i c a l f e a t u r e s . J . Reprod. F e r t . 13:11-21. J o h n s t o n e , P. 1978. B r e e d i n g and r e a r i n g the Canadian o t t e r L u t r a c a n a d e n s i s a t Mole H a l l W i l d l i f e Park 1966-1977. I n t . Zoo Yb. 18:143-147. K a r s c h , F . J . and D.L. F o s t e r . 1981. E n v i r o n m e n t a l c o n t r o l of s e a s o n a l b r e e d i n g : a common f i n a l mechanism g o v e r n i n g s e a s o n a l b r e e d i n g and s e x u a l m a t u r a t i o n . I n : G i l m o r e , D. and B. Cook ( e d s . ) . E n v i r o n m e n t a l F a c t o r s i n Mammal R e p r o d u c t i o n . U n i v . Park P r e s s . B a l t i m o r e , M a r y l a n d , pp. 30-53. 235 K i n g , C M . 1980. P o p u l a t i o n b i o l o g y of the weasel M u s t e l a n i v a l i s on B r i t i s h game e s t a t e s . H o l a r c t i c E c o l . 3:160-168. K i n g , C M . 1981. The r e p r o d u c t i v e t a c t i c s of the s t o a t ( M u s t e l a  erminea) i n New Zealand f o r e s t s . P r o c . Worldwide Fu r b . Conf. F r o s t b u r g S t a t e C o l l e g e , MD. pp. 443-468. K i n g , C M . and J.E. Moody. 1982. The b i o l o g y of the s t o a t M u s t e l s erminea i n the N a t i o n a l P a r k s of New Zealand. 4. R e p r o d u c t i o n . New Zealand J . Z o o l . 9:103-118. K i r k , R.J. 1962. The e f f e c t of d a r k n e s s on the mink r e p r o d u c t i v e c y c l e . Fur Trade J . Can. 40:8-9. K l o c h k o v , D.V. 1980. I n f l u e n c e of s h o r t e n e d d a y l i g h t on s p e r m a t o g e n e s i s of young minks. Ontogenez. 11:91-95. Kruuk, H. and R. Hewson. 1978. S p a c i n g and f o r a g i n g of o t t e r s ( L u t r a l u t r a ) i n a marine h a b i t a t . J . Z o o l . 185:205-212. L a n c i a , R.A. and J.D. H a i r . 1983. P o p u l a t i o n s t a t u s of bobcat ( F e l i s r e f u s ) and r i v e r o t t e r ( L u t r a c a n a d e n s i s ) i n N o r t h C a r o l i n a . U n p u b l i s h e d c o m p l e t i o n r e p o r t , P r o j e c t E-1, N o r t h C a r o l i n a S t a t e U n i v e r s i t y . 65 pp. L a r s e n , D.N. 1983. H a b i t a t s , movements, and foods of r i v e r o t t e r s i n c o a s t a l s o u t h e a s t e r n A l a s k a . M. Sc. T h e s i s . U n i v . of A l a s k a . F a i r b a n k s , A l a s k a . 149 pp. L a u h a c h i n d a , V. 1978. L i f e h i s t o r y of the r i v e r o t t e r i n Alabama w i t h emphasis of f o o d h a b i t s . Ph.D. T h e s i s . Auburn U n i v . , Auburn, Alabama. 169 pp. L e b l o n d , C P . and Y. Clermont. 1952. D e f i n i t i o n of the s t a g e s of the c y c l e of the s e m i n i f e r o u s e p i t h e l i u m i n the r a t . Ann. N.Y. Acad. S c i . 55:548-573. Lee, R. 1970. L a t i t u d e and p h o t o p e r i o d i s m . A r c h i v . f u r M e t e o r o l . Geophys. B i o k l i m a t o l . S e r . B. 18:325-332. 236 L i e r s , E.E. 1951. My f r i e n d s the l a n d o t t e r . N a t u r a l H i s t o r y . 60:320-326. L i e r s , E.E. 1958. E a r l y b r e e d i n g i n the r i v e r o t t e r . J . Mammal. 39:438-439. L i e r s , E.E. 1966. Notes on b r e e d i n g the Canadian o t t e r , L u t r a c a n a d e n s i s , on c a p t i v i t y and l o n g e v i t y r e c o r d s of b e a v e r s , C a s t o r c a n a d e n s i s . I n t . Zoo Yb. 6:171-172. L i n c o l n , G.A. 1971. The s e a s o n a l r e p r o d u c t i v e changes i n the r e d deer s t a g (Cervus e l a p h u s ) . J . Z o o l . , London. 163:105. L i n c o l n , G.A. 1976. Se a s o n a l v a r i a t i o n i n the e p i s o d i c s e c r e t i o n of l u t e i n i z i n g hormone and t e s t o s t e r o n e i n the ram. J . E n d o c r i n o l . 69:213-226. L i n c o l n , G.A. 1981. Se a s o n a l a s p e c t s of t e s t i c u l a r f u n c t i o n . I n : B u r g e r , H., and D. de K r e t s e r ( e d s . ) . Raven P r e s s . New York. pp. 255-302. L i s k o p , K.S., R.M.F.S. S a d l e i r and B.P. Saunders. 1981. R e p r o d u c t i o n and h a r v e s t of w o l v e r i n e (Gulo g u l o ) i n B r i t i s h Columbia. P r o c . Worldwide Fur b . Conf. F r o s t b u r g S t a t e C o l l e g e , MD. pp.469-477. L o u g h l i n , T.R., J.A. Ames and J.E. Vandevere. 1981. Annual r e p r o d u c t i o n dependency p e r i o d and apparent g e s t a t i o n p e r i o d i n two C a l i f o r n i a n U.S.A. sea o t t e r s Enhydra l u t r i s . U.S. N a t l . Mar. F i s h S e r v . F i s h B u l l . 79:347-349. Lynch, G.R., H.W. Heath and C M . J o h n s t o n . 1981. E f f e c t of g e o g r a p h i c a l o r i g i n on the p h o t o p e r i o d i c c o n t r o l of r e p r o d u c t i o n i n the w h i t e - f o o t e d mouse, Peromyscus  l e u c o p u s . B i o l . Reprod. 25:475-480. M a c F a r l a n e , J.D. and J.M. T a y l o r . 1982. Nature of e s t r o u s and o v u l a t i o n i n M i c r o t u s t o w n s e n d i i (Bachman). J . Mammal. 63:104-109. 237 M a r k l e y , M.H. and C.F. B a s s e t 1942. H a b i t s of c a p t i v e marten. Am. M i d i . Nat. 28:604-616. Marsh, H., G.E. Heinsohn, and T.D. G l o v e r . 1984. Changes i n the male r e p r o d u c t i v e organs of the dugong, Dugong dugon ( S i r e n i a : Dugondidae), w i t h age and r e p r o d u c t i v e a c t i v i t y . A u s t . J . Z o o l . 32:721-742. M a r t i n e t , L., C. A l l a i s and D. A l l a i n . 1981. The r o l e of p r o l a c t i n and LH i n l u t e a l f u n c t i o n and b l a s t o c y s t growth i n the mink ( M u s t e l a v i s o n ) . J . Reprod. F e r t . , S u p p l . 29:119-130. M a u r e l , D. and J . B o i s s i n . 1982. Metabolisme p e r i p h e r i q u e de l a t e s t o s t e r o n e en r e l a t i o n avec l e c y c l e a n n u e l de l a t e s t o s t e r o n e e t de l a 5 a l p h a d i h y d r o t e s t o s t e r o n e p l a s m a t i q u e s chez l e B l a i s e a u europeen (Meles meles) et l e Renard noux (Vulpes v u l p e s ) . Can. J . Z o o l . 60:406-416. M a u r e l , D., A.M. L a u r e n t and J . B o i s s i n . 1981. S h o r t - t e r m v a r i a t i o n s of plasma t e s t o s t e r o n e l e v e l s i n the European badger (Meles m e l e s ) . J . Reprod. F e r t . 61:53-58. M c C l i n t o c k , M.K. 1983. Pheromonal r e g u l a t i o n of the o v a r i a n c y c l e : enhancement, s u p p r e s s i o n and synchrony. I n : Vandenbergh, J.G. ( e d . ) . Pheromones and R e p r o d u c t i o n i n Mammals. Academic P r e s s . New Y ork. pp. 113-149. M c D a n i e l , J.C. 1963. O t t e r p o p u l a t i o n s t u d y . P r o c . Southeast A s s o c . F i s h and Game Comm. 17:163-168. M c K i b b i n , P.E., K. Rajkumar and B.D Murphy. 1984. R o l e of l i p o p r o t e i n s and r p o l a c t i n i n l u t e a l f u n c t i o n i n the f e r r e t . B i o l . Reprod. 30:1160-1166. M c M i l l i n , J.M., U.S. S e a l , L. Rogers and A.W. E r i c k s o n . 1976. Annual t e s t o s t e r o n e rhythm i n the b l a c k bear (Ursus  a m e r i c a n u s ) . B i o l . Reprod. 15:163-167. Mead, R.A. 1968a. R e p r o d u c t i o n i n e a s t e r n forms of the s p o t t e d skunk (genus S p i l o g a l e ) . J . Z o o l . , London. 156:119-136. 238 Mead, R.A. 1 9 6 8 b . R e p r o d u c t i o n i n w e s t e r n forms of the s p o t t e d skunk (genus S p i l o g a l e ) . J . Mammal. 4 9 : 3 7 3 - 3 9 0 . Mead, R.A. 1971. E f f e c t s of l i g h t and b l i n d i n g upon d e l a y e d i m p l a n t a t i o n i n the s p o t t e d skunk. B i o l . Reprod. 5:214-220. Mead, R.A. 1975. E f f e c t s of hypophysectomy on b l a s t o c y s t s u r v i v a l , p r o g e s t e r o n e s e c r e t i o n and n i d a t i o n i n the s p o t t e d skunk. B i o l . Reprod. 12:526-533. Mead, R.A. 1981. Delayed i m p l a n t a t i o n i n m u s t e l i d s w i t h s p e c i a l emphasis on the s p o t t e d skunk. J . Reprod. F e r t . , S u p p l . 29:11-24. Mead, R.A. and K.B. E i k - N e s . 1969a. Oestrogen l e v e l s i n p e r i p h e r a l b l o o d plasma of the s p o t t e d skunk. J . Reprod. F e r t . 18:351-353. Mead, R.A. and K.B. E i k - N e s . 1969b. S e a s o n a l v a r i a t i o n s on plasma l e v e l s of p r o g e s t e r o n e i n western forms of the s p o t t e d skunk. J . Reprod. F e r t . , S u p p l . 6:397-403. Mead, R.A. and P.L. W r i g h t . 1983. R e p r o d u c t i v e c y c l e s of M u s t e l i d a e . A c t a Z o o l . F e n n i c a 174:169-172. Mead, R.A., A.W. Rourke and A. Swannack. 1979. Changes i n u t e r i n e p r o t e i n s y n t h e s i s d u r i n g d e l a y e d i m p l a n t a t i o n i n the western s p o t t e d skunk and i t s r e g u l a t i o n by hormones. B i o l . Reprod. 21:39-46. Mead, R.A. and A. Swannack. 1978. E f f e c t s of hysterectomy on l u t e a l f u n c t i o n i n the western s p o t t e d skunk ( S p i l o g a l e  p u t o r i u s l a t i f r o n s ) . B i o l . Reprod. 18:379-383. M e l q u i s t , W.E. and M.G. Hornocker. 1983. E c o l o g y of r i v e r o t t e r s i n west c e n t r a l Idaho. W i l d l . Monogr. No. 83. 6 0 pp. M i l l a r , J.S. 1977. A d a p t i v e f e a t u r e s of mammalian r e p r o d u c t i o n . E v o l u t i o n . 31:370-386. 239 M i r a r c h i , R.E., B.E. Howland, P.F. S c a n l o n , R.L. K i r k p a t r i c k and L.M. S a n f o r d . 1978. S e a s o n a l v a r i a t i o n i n plasma LH, FSH, p r o l a c t i n and t e s t o s t e r o n e c o n c e n t r a t i o n s i n a d u l t male w h i t e - t a i l e d d e e r . Can. J . Z o o l . 56:121-127 Moger, W.H. and D.T. Armstrong. 1974. Changes i n serum t e s t o s t e r o n e l e v e l s f o l l o w i n g a c u t e LH t r e a t m e n t i n immature and mature r a t s . B i o l . Reprod. 11:1-6 . M o l l e r , O.M. 1973. The p r o g e s t e r o n e c o n c e n t r a t i o n s i n the p e r i p h e r a l plasma of the mink ( M u s t e l a v i s o n ) d u r i n g pregnancy. J . E n d o c r i n o l . 56:121-132. M o l l e r , O.M. 1974. Plasma p r o g e s t e r o n e b e f o r e and a f t e r o v a r i e c t o m y i n unmated and pregnant mink, M u s t e l s v i s o n . J . Reprod. F e r t . 37:367-372. Mondain-Monval, M., M. Bonnin. R. Canvienc and R. S c h o l l e r . 1980. Plasma e s t r o g e n l e v e l s d u r i n g d e l a y e d i m p l a n t a t i o n i n the European badger (Meles mele L ) . Gen. Comp. E n d o c r i n o l . 41:143-149. Mossman, G.W. 1937. The t h e c a l g l a n d and i t s r e l a t i o n t o the r e p r o d u c t i v e c y c l e . A study of the c y c l i c changes i n the ovary of the pocket gopher, Geomys b a r s r i u s (Shaw). Am. J . Anat. 61:289-319. Mossman, H.W. and K.L. Duke. 1973. Comparative Morphology of the Mammalian Ovary. U n i v . Wisconson P r e s s . M a d i s o n , Wi s c o n s i n . Mowbray, E.E., D. P u r s l e y and J.A. Chapman. 1979. The s t a t u s , p o p u l a t i o n c h a r a c t e r i s i c s and h a r v e s t of the r i v e r o t t e r i n M a r y l a n d . Maryland W i l d l i f e A d m i n i s t r a t i o n . W i l d l i f e E c o l o g y No. 2. 16 pp. Mowbray, E.E., J.A. Chapman and J.R. G o l d s b e r r y . 1976. P r e l i m i n a r y o b s e r v a t i o n s on o t t e r d i s t r i b u t i o n and h a b i t a t p r e f e r e n c e s i n M aryland w i t h d e s c r i p t i o n s of o t t e r f i e l d s i g n . N o r t h e a s t F i s h W i l d l . Conf. 33:125-131. 240 Murphy, B.D. 1983. P r e c o c i o u s i n d u c t i o n of l u t e a l a c t i v a t i o n and t e r m i n a t i o n of d e l a y e d i m p l a n t a t i o n i n mink w i t h the dopamine a n t a g o n i s t p i m o z i d e . B i o l . Reprod. 29:658-662. Murphy, B.D. and D.A. James. 1974. The e f f e c t s of l i g h t and s y m p a t h e t i c i n n e r v a t i o n t o the head on n i d a t i o n i n mink. J . Exp. Z o o l . 187:267-276. Murphy, B.D. and W.H.Moger. 1977. P r o g e s t i n s of mink g e s t a t i o n : the e f f e c t s of hypophysectomy. E n d o c r i n o l . Res. Comm. 4:45-60. Murphy, B.D., R.A. Mead and P. E. M c K i b b i n . 1983. L u t e a l c o n t r i b u t i o n t o the t e r m i n a t i o n on p r e i m p l a n t a t i o n d e l a y i n mink. B i o l . Reprod. 28:497-503. Murphy, B.D., P.W. Concannon, H.F. T r a v i s and W. H a n s e l . 1981. P r o l a c t i n : the hypophyseal f a c t o r t h a t t e r m i n a t e s embryonic d i a p a u s e i n mink. B i o l . Reprod. 25:487-491. Nalbandov, A.V. 1976. R e p r o d u c t i v e P h y s i o l o g y of Mammals and B i r d s . W.H. Freeman and Company. San F r a n c i s c o . N e a l , E.G. and R.J. H a r r i s o n . 1958. R e p r o d u c t i o n i n the European badger. T r a n s . Z o o l . Soc. Lond. 29:67-130. N e a l , J . , B.D. Murphy, W.H. Moger and L.W. O l i p h a n t . 1977. R e p r o d u c t i o n i n the male f e r r e t . Gonadal a c t i v i t y d u r i n g the a n n u a l c y c l e : r e c r u d e s c e n c e and m a t u r a t i o n . B i o l . Reprod. 17:380-385. N e s c h l a y , E. and H. B i e n i e k . 1975. E n d o c r i n e t e s t i c u l a r f u n c t i o n i n the mink d u r i n g the f i r s t year of l i f e . A c t a E n d o c r i n o l . 79:375-379. Onstad, O. 1967. S t u d i e s on p o s t n a t a l t e s t i c u l a r changes, semen q u a l i t y and anomalies of r e p r o d u c t i v e organs i n the mink. A c t a E n d o c r i n o l . , S u p p l . 117:1-134. O r s i n i , M.W. 1962. Technique of p r e p a r a t i o n , s t u d y and photography of b e n z y l - b e n z o a t e c l e a r e d m a t e r i a l f o r e m b r y o l o g i c a l s t u d i e s . J . Reprod. F e r t . 3:283-287. 241 Papke, R.L., P.W. Concannon, H.F. T r a v i s and W. H a n s e l . 1980. C o n t r o l of l u t e a l f u n c t i o n and i m p l a n t a t i o n i n the mink by p r o l a c t i n . J . Anim. S c i . 50:1102-1107. P e a r s o n , O.P. and R.K. Enders. 1944. D u r a t i o n of pregnancy i n c e r t a i n m u s t e l i d s . J . Exp. Z o o l . 95:21-35. P e t e r s , H. and K.P. McNatty. 1980. The Ovary. U n i v e r s i t y of C a l i f o r n i a P r e s s . B e r k e l e y , C a l i f o r n i a . P i l b e a m , T.E., P.W. Concannon and H.F. T r a v i s . 1979. The annual r e p r o d u c t i v e c y c l e of mink ( M u s t e l s v i s o n ) . J . Anim. S c i . 48:578-584. P o w e l l , R.A. 1979. M u s t e l i d s p a i n g p a t t e r n : v a r i a t i o n s on a theme by M u s t e l a . Z. T i e r p s y c h o l . 50:153-165. Ramaley, J.A. 1981. S t r e s s and f e r t i l i t y . I n : G i l m o r e , D. and B.Cook ( e d s . ) . E n v i r o n m e n t a l F a c t o r s i n Mammal R e p r o d u c t i o n . U n i v e r s i t y Park P r e s s . B a l t i m o r e , pp. 125-141 . Rausch, R.A. and A.M. P e a r s o n . 1972. Notes on the w o l v e r i n e i n A l a s k a and the Yukon T e r r i t o r y . J . W i l d l . Manage. 36:249-268. R a v i n d r a , R. and R.A. Mead. 1984. Plasma e s t r o g e n l e v e l s d u r i n g pregnancy i n the western s p o t t e d skunk. B i o l . Reprod. 30:1153-1159. R a w l i n g s , N.C., J.A. J e f f c o a t e and D.L. R i e g e r . 1984. The i n f l u e n c e of e s t r a d i o l - 1 7 B and p r o g e s t e r o n e on p e r i p h e r a l serum c o n c e n t r a t i o n s of l u t e i n i z i n g hormone and f o l l i c l e s t i m u l a t i o n hormone i n the o v a r e c t o m i z e d ewe. T h e r i o g e n o l o g y 22:473-488. R e i d , D.G., W.E. M e l q u i s t , J.D. W o o l i n g t o n and J.M. N o l l . 1985. The e f f e c t s of i n t r a p e r i t o n e a l t r a n s m i t t e r i m p l a n t s on r e p r o d u c t i o n i n r i v e r o t t e r s . J . W i l d l . Manage. In p r e s s . 242 R e i g e r , D. and B.D. Murphy. 1977. E p i s o d i c f l u c t u a t i o n i n plasma t e s t o s t e r o n e and d i h y d r o t e s t o s t e r o n e i n male f e r r e t s d u r i n g the b r e e d i n g season. J . Reprod. F e r t . 51:511-514. R e n f r e e , M.B. 1978. Embryonic d i a p a u s e i n mammals - a develo p m e n t a l s t r a t e g y . I n : C l u t t e r , M.E.(ed.). Dormancy and Developmental A r r e s t . Academic P r e s s . New York. pp. 1-46. R e n f r e e , M.B. 1981. Embryonic d i a p u a s e i n m a r s u p i a l s . J . Reprod. F e r t . , S u p p l . 29:67-78. R e n f r e e , M.B. and J.H. C a l a b y . 1981. Background t o d e l a y e d i m p l a n a t i o n and embryonic d i a p a u s e . J . Reprod. F e r t . , S u p p l . 29:1-9. R o b e r t s o n , H.A. 1977. R e p r o d u c t i o n i n the ewe and the goat. I n : C o l e , H.H. and P.T. Cupps ( e d s . ) . R e p r o d u c t i o n i n Domestic A n i m a l s . Academic P r e s s . New York. pp. 475-498. Robin s o n , T .J. 1977. R e p r o d u c t i o n i n c a t t l e . I n : C o l e , H.H. and P.T. Cupps ( e d s . ) . R e p r o d u c t i o n i n Domestic A n i m a l s . Academic P r e s s . New York. pp. 433-454. Roosen-Runge, E.C. and L.O. G i e s e l . 1950. Q u a n t i t a t i v e s t u d i e s on spermatogenesis i n the a l b i n o r a t . Am. J . Anat. 87:1-30. Rowlands, I.W. 1974. R e p r o d u c t i v e s t u d i e s i n M u s t e l i d a e . J . Z o o l . 173:116-118. R u s t , C C , and R.M. S h a c k e l f o r d . 1969. E f f e c t of b l i n d i n g on r e p r o d u c t i v e and pe l a g e c y c l e s i n the f e r r e t . J . Exp. Z o o l . 171:443-450. S a d l e i r , R.M.F.S. 1969a. The E c o l o g y of R e p r o d u c t i o n i n W i l d and Domestic Mammals. Methuen, London. S a d l e i r , R.M.F.S. 1969b. The r o l e of n u t r i t i o n i n the r e p r o d u c t i o n of w i l d mammals. J . Reprod. F e r t . , S u p p l . 6:39-48. 243 S c h l a f k e , S., A.C. Enders and R.L. G i v e n . 1981. C y t o l o g y of the endometrium of d e l a y e d and e a r l y i m p l a n t a t i o n w i t h s p e c i a l r e f e r e n c e t o mice and m u s t e l i d s . J . Reprod. F e r t . , S u p p l . 29: 1 35-141 . S e t c h e l l , B.P. 1978. The Mammalian T e s t i s . E l e k Books L i m i t e d . London. S e t c h e l l , B.P. and G.M.H. W a i t e s . 1964. B l o o d f l o w and the uptake of g l u c o s e and oxygen i n the t e s t e s and e p i d i d y m i s of the ram. J . P h y s i o l . , London. 171:411-425. S i n h a , A.A. and C.H. Conaway. 1968. The ovary of the sea o t t e r . Anat. Rec. 160:795-806. S i n h a , A.A. and R.A. Mead. 1976. M o r p h o l o g i c a l changes i n the t r o p h o b l a s t u t e r u s and c o r p u s luteum d u r i n g d e l a y e d i m p l a n t a t i o n and i m p l a n t a t i o n i n the western s p o t t e d skunk. Am. J . Anat. 145:331-356. S i n h a , A.A., C.H. Conaway and K.W. Kenyon. 1966. R e p r o d u c t i o n i n the female sea o t t e r . J . W i l d l . Manage. 30(1) : 121 -130. S o u t h e r n , A.L. and G.G. Gordon. 1975. Rhythms and t e s t o s t e r o n e m e t a b o l i s m . J . S t e r o i d . Biochem. 6:809-813. S t a b e n f e l d t , G.H. 1974. P h y s i o l o g i c , p a t h o l o g i c and t h e r a p e u t i c r o l e s of p r o g e s t i n s i n d o m e s t i c a n i m a l s . J . Am. V e t . Med. A s s o c . 164:311-317. S t e a r n s , S.C. 1977. The e v o l u t i o n of l i f e - h i s t o r y t r a i t s : a c r i t i q u e of the t h e o r y and a r e v i e w of the d a t a . Ann. Rev. E c o l . 8:145-171. S t e i n b e r g e r , E. 1971. Hormonal c o n t r o l of mammalian s p e r m a t o g e n e s i s . P h y s i o l . Rev. 51:1-22. Stenson, G.B., G.A. Badgero and H.D. F i s h e r . 1984. Food h a b i t s of t h e r i v e r o t t e r L u t r a c a n a d e n s i s i n the marine environment of B r i t i s h C o lumbia. Can. J . Z o o l . 62:88-91. 244 Stephens, M.N. 1957. The o t t e r r e p o r t . U n i v . Fed. Anim. W e l f a r e , London. 88 pp. Stephenson, A.B. 1977. Age d e t e r m i n a t i o n and m o r p h o l o g i c a l v a r i a t i o n of O n t a r i o o t t e r s . Can. J . Z o o l . 55:1577-1583. S t e t s o n , M.H., J.A. E l l i o t t and M. Menaker. 1975. P h o t o p e r i o d i c r e g u l a t i o n of hamster t e s t i s : c i r c a d i a n s e n s i t i v i t y t o the e f f e c t s of l i g h t . B i o l . Reprod. 13:329-339. S u n d q v i s t , C , A. L u k o l a and M. V a l t o n e n . 1984. R e l a t i o n s h i p between serum t e s t o s t e r o n e c o n c e n t r a t i o n s and f e r t i l i t y i n male mink ( M u s t e l a v i s o n ) . J . Reprod. F e r t . 70:409-412. Tabor, J.E. 1973. P r o d u c t i v i t y and p o p u l a t i o n s t a t u s of r i v e r o t t e r s i n western Oregon. M. Sc. T h e s i s . Oregon S t a t e U n i v . C o r v a l i s , Oregon. 62 pp. Tabor, J.E. and H.M. Wight. 1977. P o p u l a t i o n s t a t u s of r i v e r o t t e r i n western Oregon. J . W i l d l . Manage. 41:692-699. T a i t t , M.J. 1981. The e f f e c t of e x t r a food on s m a l l rodent p o p u l a t i o n s : I . Deermice (Peromyscus m a n i c u l a t u s ) • J . Anim. E c o l . 50:111-124. T a i t t , M.J. and C.J. K r e b s . 1981. The e f f e c t of e x t r a food on s m a l l rodent p o p u l a t i o n s . I I . V o l e s ( M i c r o t u s townsendi i ) J . Anim. E c o l . 50:125-137. Tapper, S. 1979. The e f f e c t of f l u c t u a t i n g v o l e numbers ( M i c r o t u s a g r e s t i s ) on a p o p u l a t i o n of weasels ( M u s t e l a n i v a l i s ) on f a r m l a n d . J . Anim. E c o l . 48:603-617. Tepperman, J . 1980. M e t a b o l i c and E n d o c r i n e P h y s i o l o g y . Year Book M e d i c a l P u b l i s h e r s , I n c . C h i c a g o . Thorpe, P.A. and J . H e r b e r t . 1976. S t u d i e s on the d u r a t i o n of the b r e e d i n g season and p h o t o r e f r a c t o r i n e s s on female f e r r e t s p i n e a l e c t o m i z e d or t r e a t e d w i t h m e l a t o n i n . J . E n d o c r i n o l . 70:255-262. 245 T o w e i l l , D.E. and J.E. Tabor. 1982. R i v e r o t t e r . I n : Chapman, J.A. and G.A. F e l d h a n e r ( e d s . ) . W i l d Mammals of N o r t h A m e r i c a . John Hopkins U n i v . P r e s s . B a l t i m o r e , pp. 688-703. T r a v i s , H.F. and T.E. P i l b e a m . 1980. Use of a r t i f i c i a l l i g h t and day l e n g t h t o a l t e r the l i f e c y c l e s of mink. J . Anim. S c i . 50:1108-1112. T r a v i s , H.F., T.E. P i l b e a m , W.J. Gardner and R.S. C o l e . 1978. R e l a t i o n s h i p of v u l v a r s w e l l i n g t o e s t r u s i n mink. J . Anim. S c i . 46:219-224. Turek, F.W. and C.S. Campbell. 1979. P h o t o p e r i o d i c r e g u l a t i o n of n e u r o e n d o c r i n e - g o n a d a l a c t i v i t y . B i o l . Reprod. 20:32-50. T u r n e r , C D . and J.T. Bagnara. 1971. G e n e r a l E n d o c r i n o l o g y . W.B. Saunders Company. P h i l a d e l p h i a . Vandenbergh, J . C 1983. Pheromonal r e g u l a t i o n of p u b e r t y . I n : Vandenbergh, J . C ( e d . ) . Pheromones and R e p r o d u c t i o n i n Mammals. Academic P r e s s . New York. pp. 95-112. Verhage, H.G., N.B. Beamer and R.M. Brenner. 1976. Plasma l e v e l s of e s t r a d i o l and p r o g e s t e r o n e i n the c a t d u r i n g p o l y e s t r u s , pregnancy and pseudopregnancy. B i o l . Reprod. 14:579-585. V i n c e n t , D.S. 1970. M o d i f i c a t i o n of the annual o e s t r o u s c y c l e of the f e r r e t by v a r i o u s regimes of a r t i f i c i a l l i g h t . J . E n d o c r i n o l . 4 8 : i i i . Wade-Smith, J . , M.E. Richmond, R.A. Mead and H. T a y l o r . 1980. Hormonal and g e s t a t i o n a l e v i d e n c e f o r d e l a y e d i m p l a n t a t i o n i n the s t r i p p e d skunk, M e p h i t i s m e p h i t i s . Gen. Comp. E n d o c r i n o l . 42:509-515. Wade-Smith, J.eand M.E. Richmond. 1978. Induced o v u l a t i o n , development of the co r p u s luteum and t u b a l t r a n s p o r t i n the s t r i p e d skunk ( M e p h i t i s m e p h i t i s i s ) . Am. J . Anat. 153:123-142. 246 Watson, H. 1978. C o a s t a l o t t e r s i n S h e t l a n d . U n p u b l i s h e d r e p o r t t o the V i n c e n t W i l d l i f e T r u s t . Earsham, S u f f o l k . 92 pp. Webb, D.R. and J.R. K i n g 1984. E f f e c t s of w e t t i n g on i n s u l a t i o n of b i r d and mammal c o a t s . J . Therm. B i o l . 9:189-191. Weir, B . J . and I.W. Rowlands. 1973. R e p r o d u c t i v e s t r a t e g i e s of mammals. Ann. Rev. E c o l . S y s t . 4:139-163. W h i t s e t t , J.M. and L.L. M i l l e r . 1982. P h o t o p e r i o d and r e p r o d u c t i o n i n female deer mice. B i o l . Reprod. 26:296-304. W i l s o n , K.A. 1959. The o t t e r i n N o r t h C a r o l i n a . P r o c . S.E. Assoc. F i s h Game Comm. 13:267-277. Woolf, A. and J.D. Har p e r . 1979. P o p u l a t i o n dynamics of a c a p t i v e w h i t e - t a i l e d deer herd w i t h emphasis on r e p r o d u c t i o n and m o r t a l i t y . W i l d l . Mong. No. 67. W o o l i n g t o n , J.D. 1984. H a b i t a t use and movements of r i v e r o t t e r s a t K e l p Bay, Baranof I s l a n d , A l a s k a . M. Sc. T h e s i s . U n i v . of A l a s k a . F a i r b a n k s , A l a s k a . 147 pp. Wourms, J.P. and D. Evans. 1974. The annual r e p r o d u c t i v e c y c l e of the b l a c k p r i c k l e b a c k , X i p h i s t e r a t r o p u r e u s , a P a c i f i c c o a s t b l e n n i o d f i s h . Can. J . Z o o l . 52:795-802. W r i g h t , P.L. 1942. De l a y e d i m p l a n t a t i o n i n the l o n g - t a i l e d weasel ( M u s t e l a f r e n a t a ) , the s h o r t - t a i l e d weasel ( M u s t e l a  c i c o g n a n T l and the marten (Mates a m e r i c a n a ) . Anat. Rec. 83:341-354. W r i g h t , P.L. 1963. V a r i a t i o n s i n r e p r o d u c t i v e c y c l e s i n N o r t h A m e r i c a . I n : E n d e r s , A.C. ( e d . ) . Delayed I m p l a n t a t i o n . U n i v . Chicago P r e s s . C h i c a g o , pp. 77-98. W r i g h t , P.L. 1966. O b s e r v a t i o n s on the r e p r o d u c t i v e c y c l e of the American badger ( T a x i d e a t a x u s ) . I n : Rowlands, I.W. ( e d . ) . Comparative B i o l o g y of R e p r o d u c t i o n i n Mammals, pp. 27-46. 247 W r i g h t , P.L. 1969. The r e p r o d u c t i v e c y c l e of the male American badger, T a x i d e a t a x u s . J . Reprod. F e r t . , S u p p l . 6:435-446. Zarrow, M.X. and J.H. C l a r k . 1968. O v u l a t i o n f o l l o w i n g v a g i n a l s t i m u l a t i o n i n a spontaneous o v u l a t o r and i t s i m p l i c a t i o n . J . E n d o c r i n o l . 40:343-353. 248 APPENDIX 1 TESTICULAR ABNORMALITIES A b n o r m a l i t i e s were ob s e r v e d i n the t e s t e s of f i v e o t t e r s . Of t h e s e , t h r e e (#175, #400, #505) showed b i l a t e r a l h y p o p l a s i a of the spermatogenic e p i t h e l i u m ; the m a j o r i t y of the s e m i n i f e r o u s t u b u l e s were degenerate a l t h o u g h the i n t e r s t i t i a l t i s s u e and a few t u b u l e s appeared normal ( P l a t e V I I , F i g . 59). In some of the abnormal t u b u l e s , the e p i t h e l i u m was not complete and o n l y s c a t t e r e d S e r t o l i c e l l s and spermatogonia were p r e s e n t . In o t h e r s , the g e r m i n a l e p i t h e l i u m appeared v a c u o l a t e d . P r i m a r y s p e r m a t o c y t e s were p r e s e n t i n many of the t u b u l e s but had an abnormal appearance; the c y t o p l a s m was e n l a r g e d and the n u c l e i p y c n o t i c . G i a n t m u l t i - n u c l e a t e d c e l l s were a l s o p r e s e n t i n the c e n t r a l a r e a of some t u b u l e s . These t h r e e o t t e r s , c o l l e c t e d i n January of d i f f e r e n t y e a r s , were from two t o fo u r y e a r s of age. T e s t i c u l a r volumes (average of both t e s t e s = 3.04, 2.65 and 3.24 cm 3) were lower than normal f o r the time of year (7.04 ± 0.19 c m 3 ) . The r e m a i n i n g two o t t e r s showed u n i l a t e r a l a b n o r m a l i t i e s of the t e s t e s . One male (#64) was a 14 year o l d c o l l e c t e d on December 10, 1975. One t e s t i s was absent and t h e r e was no ev i d e n c e i t had been p r e s e n t . The e p i d i d y m i s a s s o c i a t e d w i t h t h i s m i s s i n g t e s t i s was w e l l v a s c u l a r i z e d and e n l a r g e d due t o p r o l i f e r a t i o n of the c o n n e c t i v e t i s s u e stroma ( P l a t e V I I , F i g . 6 0 ) . The few t u b u l e s p r e s e n t i n the e p i d i d y m i s were s m a l l and i n a c t i v e . They resembled th o s e of immature a n i m a l s , 249 P l a t e V I I . P h o t omicrographs from mature L. c a n a d e n s i s w i t h abnormal t e s t e s . A l l s e c t i o n s s t a i n e d w i t h H and E. F i g u r e 59. Abnormal t e s t i s from o t t e r #505. Note the v a c u o l a t e d e p i t h e l i a l l a y e r and i n c o m p l e t e c e l l u l a r assemblage. C o n t r a l a t e r a l t e s t i s had s i m i l a r a b n o r m a l i t i e s . M a g n i f i c a t i o n a p p r o x i m a t e l y 340X. F i g u r e 60. Abnormal ductus e p i d i d y m i s from o t t e r #64 l a c k i n g one t e s t i s . E p i d i d y m i s was e n l a r g e d due to h y p e r t r o p h y of the stroma and the i n c r e a s e d v a s c u l a r i z a t i o n . C o n t r a l a t e r a l t e s t i s and e p i d i d y m i s was normal. M a g n i f i c a t i o n a p p r o x i m a t e l y 260X. F i g u r e 61. S e c t i o n from the abnormal t e s t i s of o t t e r #303. Note e n l a r g e d L e y d i g c e l l s (L) and degenerate s e m i n i f e r o u s t u b u l e s ( T ) . C o n t r a l a t e r a l t e s t i s was h y p e r t r o p h i e d but showed normal s p e r m a t o g e n e s i s . M a g n i f i c a t i o n a p p r o x i m a t e l y 260X. 2 5 0 251 c o n s i s t i n g of a t h i c k c i r c u l a r muscle l a y e r s u r r o u n d i n g low p s u e d o s t r a t i f i e d e p i t h e l i u m . The c o n t r a l a t e r a l t e s t i s appeared normal; t e s t i s volume and development of the spermatogenic e p i t h e l i u m were c o n s i s t e n t w i t h o t h e r a n i m a l s sampled i n December. The second o t t e r w i t h a u n i l a t e r a l a b n o r m a l i t y of the t e s t e s was caught on December 8, 1976. Both t e s t e s were p r e s e n t i n t h i s t h r e e year o l d (#303) a l t h o u g h one was dege n e r a t e . The abnormal t e s t i s was v e r y s m a l l (0.68 cm 3) and dark i n appearance. The t u n i c a a l b u g i n e a was t h i c k (470 urn) and most of the t e s t i s l a c k e d s e m i n i f e r o u s t u b u l e s . I t c o n s i s t e d p r i m a r i l y of c o n n e c t i v e t i s s u e s t r a n d s w i t h many round, u n i d e n t i f i e d c e l l s s c a t t e r e d throughout ( P l a t e V I I , F i g . 6 1 ) . These c e l l s were l a r g e , r a n g i n g i n s i z e from 6-30 urn and had abundant g r a n u l a r c y t o p l a s m which s t a i n e d yellow-brown w i t h H a r r i s ' h a e m a t o x y l i n and e o s i n . One or more i r r e g u l a r l y p l a c e d , p y c n o t i c n u c l e i were o c c a s i o n a l l y p r e s e n t . The c e l l s were found i n l a r g e clumps, between c o n n e c t i v e t i s s u e s t r a n d s and w i t h i n the c o l l a p s e d remnants of the s e m i n i f e r o u s t u b u l e s . The lumen and a l l e p i t h e l i a l c e l l s were absent from the t u b u l e s which were reduced t o a c i r c u l a r band of t h i c k e n e d basement membrane. The t e s t i s was w e l l v a s c u l a r i z e d w i t h many l a r g e c a p i l l a r i e s p r e s e n t . In a s m a l l s e c t i o n of t h i s t e s t i s , the s e m i n i f e r o u s t u b u l e s were not c o m p l e t e l y degenerate and v a r y i n g numbers of abnormal S e r t o l i c e l l s , spermatogonia and p r i m a r y spermatocytes were p r e s e n t . These c e l l s c o n t a i n e d e n l a r g e d c y t o p l a s m , p y c n o t i c 252 n u c l e i , and u s u a l l y d i d not form a c o m p l e t e g e r m i n a l l a y e r . These c e l l s were not undergoing normal s p e r m a t o g e n e s i s . Between these t u b u l e s were numerous L e y d i g c e l l s ; some were normal i n appearance w h i l e o t h e r s had g r a n u l a r c y t o p l a s m . T h i s s u g g e s t s t h a t the l a r g e , u n i d e n t i f i e d c e l l s which were abundant throughout the r e s t of the t e s t i s may be abnormal L e y d i g c e l l s . Spermatogenesis appeared t o be p r o c e e d i n g n o r m a l l y i n the c o n t r a l a t e r a l t e s t i s a l t h o u g h the t e s t i c u l a r volume (7.37 cm 3) was l a r g e r than normal f o r o t t e r s i n December (4.58 ± 0.2 c m 3 ) . T h i s t e s t i s may have h y p e r t r o p h i e d t o compenstate f o r the i n a c t i v i t y of the abnormal t e s t i s . 253 A p p e n d i x 2 Serum t e s t o s t e r o n e c o n c e n t r a t i o n s ( n g / m l ) o f n i n e c a p t i v e r i v e r o t t e r s f r om O c t o b e r 1978 - A u g u s t 1 9 8 1 . D a t e M l M2 M3 M4 M5 M6 M9 M10 M i l 1978 O c t . 6 0 . 1 3 1 .70 1 1 ___ __ O c t . 30 2 . 8 8 3 . 1 9 N o v . 13 3 . 2 2 ___ D e c . 9 1 .74 1.57 D e c . 21 1.87 0 . 6 1 2 . 7 4 3 1979 J a n . 19 3 . 3 2 1.26 3 . 9 0 J a n . 26 3 . 2 6 F e b . 3 2 . 2 3 ___ F e b . 8 3 . 4 6 1 .08 3 . 2 5 —— F e b . 19 4 . 1 3 F e b . Mav< 28 o 1 .64 4 . 8 0 3 . 9 3 roar. M a r . 21 4 . 2 0 3 . 4 0 O . DO 3 . 7 7 A p r . 2 2 . 9 8 4 . 2 5 _____ A p r . 12 3 . 3 2 2 . 2 0 3 . 1 4 5 . 3 4 — ___ A p r . 29 , 1.78 2 . 5 2 1.58 May 11 0 . 9 9 1.19 3 . 1 1 ° 1.56 0 . 9 1 J u n e 7 3 . 6 0 0 . 8 6 2 . 8 1 1 .22 0 . 6 5 J u n e 27 1 .83 0 . 7 1 2 . 2 4 1.14 J u l y 15 1 .90 1.91 3 . 5 4 1 .45 0 . 6 7 ___ J u l y 28 3 . 0 1 1.38 2 . 9 8 2 . 4 1 0 . 8 2 —-O c t . 4 0 . 9 0 O c t . 21 _ _ , 1.84 1.61 2 .27 N o v . 9 3 . 8 1 1.11 2 . 2 7 1.17 1.24 N o v . 23 2 . 1 0 2 . 0 1 2 . 1 7 1.52 1 .58 _ _ _ _ __ D e c . 9 1.91 1.26 2 . 9 2 a 1.21 1 .409 ___ ___ D e c . 20 1 .45 1 .89 1.97 1.50 — — — — — 1980 J a n . 5 2 . 0 1 1 .88 2 . 7 5 1 .43 1.88 J a n . 18 3 . 1 4 2 . 8 1 1 .29 F e b . 2 , . 5 . 7 3 1.74 F e b . 8 7 . 8 3 6 . 4 9 F e b . 15 5 . 8 2 3 . 4 3 8 . 7 6 1 .93 3 . 7 8 —__ F e b . 19 _ 4 . 9 3 F e b . 23 7 . 0 2 F e b . 28 3 . 9 6 8 . 6 8 2 . 9 0 4 . 4 2 ( c o n t i n u e d n e x t page) 254 A p p e n d i x 2 . ( c o n t i n u e d ) Da te M l M2 M3 M4 M5 M6 M9 MIO M i l 1980 M a r . 9 8 . 9 3 2.71 M a r . 14 —_ 2 . 9 2 4 . 8 8 4 . 2 0 M a r . 23 , 4 . 3 1 , „ A p r . 4 3 . 6 9 2 . 9 4 5 . 6 5 A p r . 18 2 . 7 7 2 . 5 7 4 . 0 4 A p r . 29 1 .62 May 8 2.74 3 . 2 1 1.25 May 19 1 .62 0 . 8 2 1.67 May 31 1.37 1.78 1.38 J u n e 15 2 . 0 8 1 . 1 5 b 1.27 J u l y 5 2 . 0 1 1.57 1 .33 J u l y 29 1 .22 1 .95 1.74 A u g . 26 3 .71 2 . 2 6 1.67 S e p . 11 1.67 3 . 0 2 1 .52 O c t . 2 1.94C 1.71 1. O c t . 16 1 .43 3 . 9 6 N o v . 1 1.813 3 . 6 5 N o v . 17 2 . 3 7 2 . 9 0 D e c . 5 4 . 9 0 1 .22 D e c . 17 — 6 . 6 5 2 . 0 8 — 1981 J a n . 7 5 . 9 4 3 . 3 4 J a n . 23 5 . 0 9 2 . 7 5 F e b . 4 2 . 7 6 F e b . 10 3 . 3 9 F e b . 19 2 . 0 5 7 . 5 3 M a r . 4 1 .42 8 . 7 0 M a r . 21 2 . 0 6 __ A p r . 9 2 . 5 7 6 . 3 4 A p r . 20 4 . 4 9 1.77 A p r . 27 — 2 . 8 0 2 . 0 8 May 15 1.66 2 . 4 9 May 26 3 . 5 4 __ J u n e 15 1 .55b 1 .39 J u l y 7 1 .40 J u l y 28 — 1.86 — A u g . 18 — — — 4 . 6 8 4 . 2 8 4 . 3 0 1 .80 3 . 1 8 1.57 1 .32 2 . 3 3 —— 1.12 3 . 7 9 1 .99 2 . 5 5 0 . 9 3 2 . 3 1 1 . 5 0 b 2 . 0 5 1.86 . 1.66 1.51 2 . 9 1 2 . 2 9 0 . 9 4 3 . 6 7 1.67 2.24C 1 .73 3 .71 3 . 8 0 d 2 . 3 9 4 . 2 5 1 .82 2 . 5 9 1.06 1 .88 0 . 6 6 2 . 6 0 2 . 3 6 4 . 9 3 1.82 2 . 9 0 — 1 .48 — — 2 . 3 6 2 . 6 4 5 . 0 8 5 . 3 0 1 .48 —— 4 . 4 6 1.87 4 . 2 8 1 .03 1 .80 2 . 6 8 1.87 1 .16 1.57 2 . 1 8 1 .69 1.27 0 . 7 3 1 .68 0 . 5 0 _ _ „ —— 1.44 1.02 1.32 0 . 9 1 0 . 6 6 1.82 0 . 7 7 2 . 5 0 1.97 2 . 2 5 1 .82 1 .03 0 . 6 2 1 .62 2 . 2 6 . 1 .29 1.62 1 .73 1 .80 1.57 a - E x p o s e d t o i n c r e a s e d p h o t o p e r i o d . b - Removed f r om i n c r e a s e d p h o t o p e r i o d . c - E x p o s e d t o s h o r t e d ( 8 L : 1 6 D ) p h o t o p e r i o d and 10°C. d - E x p o s e d t o n a t u r a l l i g h t r e g i m e and 10°C. 255 A p p e n d i x 3 . Serum e s t r a d i o l c o n c e n t r a t i o n s ( p g / m l ) o f f i v e c a p t i v e r i v e r o t t e r s f r om A u g u s t 1978 - A u g u s t 1 9 8 1 . D a t e F23 F3 F5 F 6 D F 7 C 1978 A u g . 28 7 12 13 O c t . 6 14 22 O c t . 30 13 , , N o v . 13 22 120 D e c . 9 „ i. 33 47 —— ____ D e c . 21 9 5 7 f 52 — 1979 J a n . 19 9 12 11 J a n . 26 15 F e b . 8 4 1 0 d 37 F e b . 17 , 8 3 d „ F e b . 28 3 i g d 96 M a r . 9 1 6 d 2 7 d M a r . 21 6 2 4 d 2 2 d A p r . 2 75e 1 5 d A p r . 12 6 6 d 23 A p r . 29 . 10dh 2 9 d 1, May 11 85e _ 9 —_ J u n e 7 13 8 19 J u n e 27 8 11 19 J u l y 15 8 10 10 — J u l y 28 10 10 16 O c t . 21 6 13 N o v . 9 18 5 24 N o v . 23 6 8 8 D e c . 9 8 f D e c . 20 — 10 20 1980 J a n . 5 6 12 10 J a n . 18 8 7 9 J a n . 25 5 F e b . 2 3 F e b . 8 . 8 F e b . 15 7 11 1 7 d F e b . 23 12 „ F e b . 28 8 1 4 d ( c o n t i n u e d n e x t page) 256 A p p e n d i x 3 . ( c o n t i n u e d ) Da te F 2 a F3 F5 F6b F 7 C 1980 M a r . 2 10 M a r . 9 6 18 M a r . 14 I d 7d lid 20 M a r . 23 10d 7d lid 11 A p r . 4 4 d 8 18 2 A p r . 18 8 8 33d 13 A p r . 29 9 5 17d 15 May 8 10 7 19d 19 May 19 8 8 14d 12 May 31 11 7 8 16 J u n e 15 13 69 15 19 J u l y 5 4 9 7 11 6 J u l y 29 8 11 17 10 A u g . 26 8 10 13 17 5 S e p . 11 6 9 7 6 O c t . 2 9 11 14 17 5 O c t . 16 6 11 12 24 3 O c t . 31 1* 8 20 N o v . 4 17 , ~ 6 f N o v . 17 6 4 10 8 3 N o v . 24 i, , , ______ 3 D e c . 5 10 5 25 4 8 D e c . 17 5 8 26 3 7 1981 J a n . 7 10d 12 11 9 8e J a n . 15 8d 3 23 1 0 e J a n . 23 1 2 d 10 ____ 5 F e b . 4 , 7 16 F e b . 10 3d _____ 6 F e b . 19 lid 8 11 26 8 M a r . 4 3d 17d 8d 19 8 M a r . 21 7 14d 1 3 d 1 9 d 8 A p r . 9 3 1 0 d i lid 2 9 d 6 A p r . 20 5 9d 15 2 i d 4 A p r . 27 13 9d ____ 1 7 d J 8 May 15 9 1 4 d 12 _____ May 22 5J 10 May 26 — 6 14 11 ~7 ( c o n t i n u e d n e x t page) A p p e n d i x 3 . ( c o n t i n u e d ) Da te F 2 a F3 F5 F 6 b F 7 C 1981 J u n e 15 39 6 _ 16 49 J u l y 6 4 12 17 17 4 J u l y 28 7 12 14 18 _ A u g . 11 3 _ _ 13 _ A u g . 18 — 16 — — — a - Immature a t b e g i n n i n g o f s t u d y . P a r t i a l e s t r u s o b s e r v e d f rom March 21 1979 - May 11 1 9 7 9 . F i r s t f u l l e s t r u s e n t e r e d on March 14 1 9 8 0 . b - Immature a t b e g i n n i n g o f s t u d y . F i r s t f u l l e s t r u s e n t e r e d on March 21 1 9 8 1 . c - Immature a t b e g i n n i n g o f s t u d y . P a r t i a l e s t r u s o b s e r v e d f r om J a n u a r y 6 1981 - J a n u a r y 15 1 9 8 1 . d - E s t r u s v a g i n a l smear p r e s e n t , e - P a r t i a l e s t r u s v a g i n a l smear p r e s e n t , f - S u b j e c t e d t o i n c r e a s e d p h o t o p e r i o d . g - Removed f r om i n c r e a s e d p h o t o p e r i o d . h - M a t i n g s c a r s p r e s e n t on A p r i l 29 1 9 7 9 . i - P o s s i b l e m a t i n g on A p r i l 12 1 9 8 1 . j - O v a r i e s and u t e r i r e m o v e d . A p p e n d i x 4 . Serum p r o g e s t e r o n e c o n c e n t r a t i o n s ( n g / m l ) o f f i v e c a p t i v e r i v e r o t t e r s f r om A u g u s t 1978 - A u g u s t 1 9 8 1 . D a t e F2a F3 F5 F6b F 7 C 1978 A u g . 28 0 . 7 3 0 . 6 0 O c t . 6 0 . 5 8 0 . 6 1 O c t . 30 , . 0 . 5 7 ___ N o v . 13 1.1 0 . 7 9 0 . 3 8 D e c . 9 1 .37 0 . 7 4 0 . 3 8 D e c . 21 0 . 4 6 2 . 2 7 0 . 6 2 — 1979 J a n . 19 0 . 6 6 0 . 9 5 0 . 7 9 J a n . 26 ___ 0 . 7 3 —_ F e b . 8 1 .10 0 . 4 0 0 . 4 8 ___ F e b . 17 ___ 1.10 ___ F e b . 28 0 . 9 8 0 . 9 6 2 . 2 0 ___ M a r . 9 ___ 1.60 0 . 9 0 M a r . 21 0 . 6 8 1.41 0 . 4 3 ___ A p r . 2 0 . 5 3 1.34 A p r . 12 1.26 0 . 7 6 0 . 4 8 _— A p r . 29 . 2 . 9 1 d 0 . 3 0 _ May 11 0 . 7 0 J u n e 7 1 .20 3 . 9 0 . 3 8 , , J u n e 27 0 . 8 2 3 . 0 3 0 . 2 8 J u l y 15 0 . 7 3 1.80 0 . 3 8 ___ J u l y 28 2 . 0 8 1.57 0 . 6 1 O c t . 21 1.16 0 . 8 4 N o v . 9 0 . 5 7 1 .54 0 . 9 1 N o v . 23 0 . 7 8 3 . 2 3 e 0 . 4 9 D e c . 20 — 3 . 9 3 0 . 3 1 1980 J a n . 5 1.17 1 9 . 0 0 0 . 3 8 _ J a n . 18 0 . 6 6 2 1 . 0 0 0 . 3 8 t — J a n . 25 2 1 . 0 0 — F e b . 2 0 . 9 6 — F e b . 8 i, 1 4 . 0 0 F e b . 15 0 . 8 1 9 . 2 0 0 . 6 5 F e b . 23 _ 6 . 7 ___ F e b . 28 0 . 6 4 7 .1 0 . 8 4 — ( c o n t i n u e d n e x t page) 2 5 9 A p p e n d i x 4 . ( c o n t i n u e d ) D a t e F 2 * F3 F5 F6b F 7 C 1980 M a r . 2 1 .73 M a r . 9 1 .22 ______ 0 . 4 2 M a r . 14 0 . 4 8 0 . 8 0 0 . 3 6 0 . 3 5 M a r . 23 0 . 3 8 1 .40 0 . 4 3 0 . 2 7 A p r . 4 0 . 7 6 1.04 0 . 3 8 0 . 2 7 A p r . 18 0 . 5 1 0 . 5 8 0 . 1 9 0 . 2 8 A p r . 29 0 . 5 9 0 .41 1 .23 0 . 3 7 May 8 1 .19 0 . 5 2 0 . 3 0 0 . 2 1 May 19 0 . 2 0 _____ 0 . 2 7 0 . 3 0 May 31 0 . 2 0 0 . 6 9 0 . 2 2 0 . 2 9 J u n e 15 0 . 5 8 0 . 3 9 0 . 2 2 M J u l y 5 0 . 4 9 0 . 5 4 0 . 3 7 0 . 3 4 0 . 2 2 J u l y 29 0 . 8 4 0 . 2 0 _ _ _ 0 . 2 2 0 . 2 7 A u g . 26 0 . 2 0 1 .04 1 .02 0 . 2 4 0 . 3 4 S e p . 11 0 . 5 9 0 . 5 6 0 . 3 6 0 . 2 9 O c t . 2 0 . 5 7 0 . 4 5 0 . 3 9 0 . 3 4 0 . 2 3 O c t . 16 0 . 5 7 0 . 4 3 0 . 4 8 0 . 5 2 0 . 2 1 O c t . 31 . , 0 . 4 2 _____ 0 . 2 7 N o v . 4 0 . 8 2 0 . 4 2 , 0 . 4 6 N o v . 17 0 . 5 4 0 . 5 4 0 . 3 6 0 . 2 8 0 . 2 8 N o v . 24 . 0 . 3 4 D e c . 5 0 . 5 4 0 . 3 0 0 . 2 9 0 . 3 7 D e c . 17 2 . 7 0 0 . 4 8 0 . 3 0 0 . 2 3 0 . 2 4 1981 J a n . 7 0 . 4 0 0 . 4 7 0 . 3 8 0 . 2 3 J a n . 15 0 . 5 1 0 . 3 9 0 . 3 0 J a n . 23 5 . 9 0 0 . 7 0 0 . 3 0 F e b . 4 _____ 0 . 7 4 0 . 3 3 I, F e b . 10 0 . 3 8 —— ______ _______ 0 . 2 3 F e b . 19 0 . 5 8 0 . 3 0 0 . 6 1 0 . 3 5 0 . 1 6 M a r . 4 0 . 4 4 0 . 6 0 0 . 6 0 0 . 2 6 0 . 1 5 M a r . 21 0 . 5 8 0 . 2 0 0 . 6 9 0 . 2 7 0 . 1 8 A p r . 9 0 . 4 7 0 . 4 2 f 0 . 4 0 0 . 3 8 0 . 3 9 A p r . 20 0 . 2 0 „ 0 . 3 4 0 . 3 4 0 . 1 4 A p r . 27 0 . 8 0 1 .95 0 . 2 4 9 0 . 2 5 May 15 . | 4 . 0 0 0 . 3 4 0 . 2 7 0 . 2 3 May 22 1 .679 2 . 9 0 i. May 26 — 3 . 5 0 0 . 2 3 0 . 2 4 0 . 2 3 ( c o n t i n u e d n e x t page) A p p e n d i x 4 . ( c o n t i n u e d ) D a t e F 2 a F3 F5 F 6 b F 7 C 1981 J u n e 15 1 .22 2 . 4 1 0 . 3 3 0 . 3 6 0 . 2 0 J u l y 6 0 . 4 6 1 .82 0 . 4 2 0 . 2 1 0 . 2 0 J u l y 28 0 . 5 9 6 . 2 3 0 . 3 4 0 . 2 2 ________ A u g . 11 1 .39 , ______ 0 . 3 7 —— A u g . 18 — 0 . 3 0 _ a - Immature a t b e g i n n i n g o f s t u d y . P a r t i a l e s t r u s o b s e r v e d f r om March 21 1979 - May 11 1 9 7 9 . F i r s t f u l l e s t r u s e n t e r e d on Ma rch 14 1 9 8 0 . b - Immature a t b e g i n n i n g o f s t u d y . F i r s t f u l l e s t r u s e n t e r e d on M a r c h 21 1 9 8 1 . c - Immature a t b e g i n n i n g o f s t u d y . P a r t i a l e s t r u s o b s e r v e d f rom J a n u a r y 6 1981 - J a n u a r y 15 1 9 8 1 . d - M a t i n g s c a r s p r e s e n t on A p r i l 29 1 9 7 9 . e - S u b j e c t e d t o i n c r e a s e d p h o t o p e r i o d on December 9 1 9 7 9 . f - P o s s i b l e m a t i n g on A p r i l 12 1 9 8 1 . g - o v a r i e s and u t e r i r e m o v e d . 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0096748/manifest

Comment

Related Items