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The role of photoperiod and endocrine factors in the control of nest-building and courtship in the male… McDonald, Pam 1983

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THE ROLE OF PHOTOPERIOD AND ENDOCRINE FACTORS IN THE CONTROL OF NEST-BUILDING AND COURTSHIP IN THE MALE RING DOVE ( STREPTOPELIA RISORIA ). By PAM MCDONALD B . S c , U n i v e r s i t y Of Toronto, 1973 M.Sc, U n i v e r s i t y Of B r i t i s h Columbia, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n 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 J u l y 1983 © Pam McDonald, 1983 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of p\ o <-^ s^   The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6 (3/81) i i ABSTRACT Male r i n g doves ( S t r e p t o p e l i a r i s o r i a ) h e l d on long days perform more n e s t - b u i l d i n g than b i r d s h e l d on short days. T h i s d i f f e r e n c e p e r s i s t s even i f c i r c u l a t i n g androgen i s maintained at s i m i l a r l e v e l s i n both groups by c a s t r a t i o n and treatment with exogenous t e s t o s t e r o n e . I t t h e r e f o r e appears that daylength can a l t e r n e s t - b u i l d i n g through some means other than changes i n gonadal androgen p r o d u c t i o n . The experiments d e s c r i b e d i n t h i s t h e s i s examined three mechanisms which might be r e s p o n s i b l e f o r the e f f e c t of daylength on male nest-b u i l d i n g . The f i r s t s e r i e s of experiments t e s t e d the h y p o t h e s i s that n e s t - b u i l d i n g a c t i v i t y i s g r e a t e r under long days as a r e s u l t of i n c r e a s e d a r o m a t i z a t i o n of t e s t o s t e r o n e i n t o estrogen. C a s t r a t e d male doves were implanted with e s t r a d i o l benzoate, t e s t o s t e r o n e propionate (TP), or a combination of TP and an aromatase i n h i b i t o r , ATD, and exposed to e i t h e r long or short photoperiods. Males h e l d under long days continued to b u i l d more a c t i v e l y , r e g a r d l e s s of the type of hormone being used. The r e s u l t s thus i n d i c a t e that d i f f e r e n c e s i n the r a t e of a r o m a t i z a t i o n cannot account f o r the e f f e c t of p h o t o p e r i o d . In a second s e r i e s of experiments an attempt was made to determine whether an i n c r e a s e i n the p r o d u c t i o n of hypothalamic l u t e i n i z i n g hormone r e l e a s i n g hormone (LHRH) under long daylengths s t i m u l a t e s n e s t - b u i l d i n g , as a r e s u l t of a synergism with t e s t o s t e r o n e . Long- and short-day c a s t r a t e d males were t r e a t e d with exogenous TP or TP and LHRH to see i f the i n f l u e n c e of short daylengths c o u l d be rev e r s e d by LHRH. LHRH treatment had no e f f e c t on the n e s t i n g a c t i v i t y of e i t h e r s h o r t - or long-day b i r d s . F i n a l l y , s e v e r a l t e s t s were performed which focussed on the r o l e of the p i n e a l gland. Removal of the p i n e a l caused a decrease i n n e s t i n g a c t i v i t y i n long-day b i r d s , but not i n short-day b i r d s . The e f f e c t of pinealectomy does not depend on the presence of the gonads, s i n c e the b i r d s d i s p l a y e d s i m i l a r responses f o l l o w i n g c a s t r a t i o n and t e s t o s t e r o n e treatment. In the second t e s t , the a b i l i t y , of pinealectomy t o n u l l i f y the s t i m u l a t o r y i n f l u e n c e of long daylengths was confirmed. In a d d i t i o n , i n j e c t i o n s of chic k e n p i n e a l e x t r a c t i n c r e a s e d n e s t -b u i l d i n g i n p i n e a l e c t o m i z e d and i n short-day p i n e a l - i n t a c t b i r d s , suggesting that the p i n e a l produces a substance which i s capable of s t i m u l a t i n g n e s t i n g a c t i v i t y . The i d e n t i t y of t h i s substance i s unknown. P i l o t t e s t s using melatonin f a i l e d to show any s t i m u l a t o r y e f f e c t of t h i s compound on n e s t i n g behaviour. However, s e r o t o n i n , which i s a l s o present i n the p i n e a l i n hig h c o n c e n t r a t i o n , d i d i n c r e a s e n e s t - b u i l d i n g i n short-day p i n e a l e c t o m i z e d males. These r e s u l t s i n d i c a t e that i n response to long daylengths the p i n e a l gland of male doves r e l e a s e s a compound, p o s s i b l y s e r o t o n i n , which s t i m u l a t e s n e s t -b u i l d i n g a c t i v i t y . i v TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v i i i LIST OF FIGURES x ACKNOWLEDGEMENTS x i i I . I n t r o d u c t i o n 1 I I . G e n e r a l Methods 9 A. Experimental Animals 9 B. S u r g i c a l Procedures and Hormone A d m i n i s t r a t i o n 11 C. T e s t i n g Procedure 13 1. Stimulus Females 13 2. Recording Technique 14 3. B e h a v i o u r a l Recordings 15 D. A n a l y s i s of Data 20 I I I . Androgen metabolism and daylength-dependent changes i n n e s t - b u i l d i n g behaviour i n male doves 21 A. I n t r o d u c t i o n 21 B. The E f f e c t i v e n e s s of V a r i o u s Androgen M e t a b o l i t e s i n R e s t o r i n g Male Reproductive Behaviour 30 1. I n t r o d u c t i o n 30 2. M a t e r i a l s and Methods 31 3. R e s u l t s '. 32 4. D i s c u s s i o n 37 C. A r o m a t i z a t i o n and the E f f e c t of Daylength on the N e s t - b u i l d i n g Behaviour of C a s t r a t e d Doves 40 V 1. M a t e r i a l s and Methods 41 A) EB, TP, and TP+ATD implants 41 B) TP and TP+ATD i n j e c t i o n s 41 C) Androgenic e f f e c t s of ATD 42 2. R e s u l t s 42 A) EB, TP, and TP+ATD implants 42 B) TP and TP+ATD i n j e c t i o n s 43 C) Androgenic e f f e c t s of ATD 54 3. D i s c u s s i o n 54 4. Summary 58 IV. The E f f e c t of L u t e i n i z i n g Hormone R e l e a s i n g Hormone and Photoperiod on N e s t - B u i l d i n g i n Male Doves 60 A. I n t r o d u c t i o n 60 B. M a t e r i a l s and Methods 61 C. R e s u l t s 62 D. D i s c u s s i o n 68 E. Summary 72 V. The r o l e of the p i n e a l i n mediating the b e h a v i o u r a l response to photoperiod 73 A. I n t r o d u c t i o n 73 1. The P i n e a l and Reproduction i n Mammals 75 2. The P i n e a l and Reproduction i n B i r d s 78 3. The P i n e a l and Reproductive Behaviour . 82 B. The E f f e c t of Pinealectomy on the Behaviour of Male Doves 83 1. M a t e r i a l s and Methods 84 2. R e s u l t s 85 v i 3. D i s c u s s i o n 92 4. Summary 100 C. The B e h a v i o u r a l E f f e c t of Exogenous Me l a t o n i n Treatment 101 1. M a t e r i a l s and Methods 102 2. R e s u l t s 103 3. D i s c u s s i o n 108 4. Summary 113 D. The E f f e c t of Chicken P i n e a l E x t r a c t Treatment on N e s t - b u i l d i n g i n Male Doves 113 1. I n t r o d u c t i o n 113 2. M a t e r i a l s and Methods 114 A) Experimental Subj e c t s 114 B) P r e p a r a t i o n of e x t r a c t s 115 3. R e s u l t s 116 4. D i s c u s s i o n 131 5. Summary 136 E. The E f f e c t of Exogenous S e r o t o n i n on Nesting A c t i v i t y i n P i n e a l e c t o m i z e d , Short-day B i r d s 136 1. I n t r o d u c t i o n 136 2. M a t e r i a l s and methods 141 3. R e s u l t s 141 4. D i s c u s s i o n 144 5. Summary 149 . General D i s c u s s i o n 151 A. C a t e g o r i e s of male r e p r o d u c t i v e a c t i v i t y i n the r i n g dove 151 v i i B. Photoperiod and the Breeding C y c l e i n Ring Doves ....159 C. Photoperiod and Male Behaviour 161 D. The Mechanism Mediating the E f f e c t of Photoperiod on Male N e s t - B u i l d i n g i n Doves 163 E. G e n e r a l i t y of Photoperiod-induced Changes i n the Response to S t e r o i d s 171 F. Why Should N e s t - b u i l d i n g Be Under P h o t o p e r i o d i c C o n t r o l ? A F u n c t i o n a l E x p l a n a t i o n 173 V I I . C o n c l u s i o n 180 XVII. References C i t e d 181 XVIII. Appendix A. A b b r e v i a t i o n s 222 v i i i LIST OF TABLES Table I. The e f f e c t of photoperiod on androgen metabolism . 28 Table I I . Copulatory a c t i v i t y and female behaviour f o l l o w i n g treatment of the males with v a r i o u s androgen m e t a b o l i t e s 37 Table I I I . Female behaviour and c o p u l a t o r y a c t a i v i t y f o l l o w i n g treatment of the males with TP, EB, or TP + ATD 46 Table IV. Male . c o u r t s h i p f o l l o w i n g treatment with i n j e c t i o n s of TP or TP + ATD 49 Table V. Copulatory behaviour and female a c t i v i t y f o l l o w i n g treatment of the males with i n j e c t i o n s of TP or TP + ATD 50 Table VI. The e f f e c t of ATD alone on male behaviour 55 Table V I I . The e f f e c t of LHRH on c o p u l a t o r y a c t i v i t y and female behaviour 63 Table V I I I . The e f f e c t of LHRH on male c o u r t s h i p behaviour 65 Table IX. The e f f e c t of pinealectomy and melatonin treatment on gonadal development i n b i r d s 79 Table X. Male c o u r t s h i p f o l l o w i n g pinealectomy 86 Table XI. Copulatory behaviour and female a c t i v i t y f o l l o w i n g pinealectomy of males 87 Table X I I . Pinealectomy and t e s t i s s i z e 91 Table X I I I . The e f f e c t of pinealectomy on the c o u r t s h i p behaviour of T P - t r e a t e d c a s t r a t e s 95 Table XIV. Copulatory behaviour and female a c t i v i t y f o l l o w i n g pinealectomy of T P - t r e a t e d c a s t r a t e s 96 Table XV. C o u r t s h i p behaviour, c o p u l a t o r y a c t i v i t y , and female n e s t - s o l i c i t i n g f o l l o w i n g l a t e - d a y melatonin i n j e c t i o n of the males 104 Table XVI. The p r e - l a y i n g behaviour of p a i r s f o l l o w i n g treatment of the males with e a r l y morning i n j e c t i o n s or implants of melatonin 109 Table XVII. Male c o u r t s h i p f o l l o w i n g pinealectomy 117 Table XVIII. Copulatory behaviour and female a c t i v i t y f o l l o w i n g pinealectomy of males 118 Table XIX. The e f f e c t of c h i c k e n p i n e a l e x t r a c t on male c o u r t s h i p 125 Table XX. Copulatory behaviour and female a c t i v i t y f o l l o w i n g treatment of the males with p i n e a l e x t r a c t ...126 Table XXI. Other changes i n behaviour f o l l o w i n g treatment of the males with p i n e a l e x t r a c t 130 Table XXII. M o u l t i n g i n P i n e a l e c t o m i z e d B i r d s 132 Table XXIII. S e r o t o n i n and male c o u r t s h i p behaviour 145 Table XXIV. Copulatory behaviour and female a c t i v i t y f o l l o w i n g s e r o t o n i n treatment of males ....146 Table XXV. C o r r e l a t i o n s among the v a r i o u s r e p r o d u c t i v e d i s p l a y s , 0 157 Table XXVI. P o s s i b l e mechanisms mediating the e f f e c t of ph o t o p e r i o d on n e s t - b u i l d i n g 165 X LIST OF FIGURES Fi g u r e 1. Routes of t e s t o s t e r o n e metabolism i n c e n t r a l t i s s u e s 22 F i g u r e 2. Male behaviour f o l l o w i n g treatment with v a r i o u s androgen m e t a b o l i t e s 33 F i g u r e 3. Male c o u r t s h i p f o l l o w i n g treatment with TP, EB, or TP + ATD implants 44 F i g u r e 4. The e f f e c t of implants of TP, EB, or TP + ATD on male n e s t - b u i l d i n g 47 F i g u r e 5. The e f f e c t of i n j e c t i o n s of TP or TP + ATD on male n e s t - b u i l d i n g 52 F i g u r e 6. The e f f e c t of LHRH on male n e s t - b u i l d i n g behaviour 66 F i g u r e 7. Melatonin s y n t h e s i s *. 74 F i g u r e 8. The e f f e c t of pinealectomy on male n e s t - b u i l d i n g 89 F i g u r e 9. The e f f e c t of pinealectomy on n e s t - b u i l d i n g i n T P - t r e a t e d c a s t r a t e s 93 F i g u r e 10. The e f f e c t of l a t e - d a y melatonin i n j e c t i o n s on male n e s t - b u i l d i n g .106 F i g u r e 11. N e s t - b u i l d i n g behaviour of males f o l l o w i n g early-morning i n j e c t i o n s or implants of melatonin ......111 F i g u r e 12. The e f f e c t of pinealectomy on male n e s t - b u i l d i n g 120 F i g u r e 13. The e f f e c t of c h i c k e n p i n e a l e x t r a c t on male n e s t - b u i l d i n g ......122 F i g u r e 14. Changes i n male n e s t - b u i l d i n g as a r e s u l t of p i n e a l e x t r a c t treatment 128 F i g u r e 15. The e f f e c t of s e r o t o n i n on male n e s t - b u i l d i n g ..142 F i g u r e 16. The s h i f t s i n behaviour seen d u r i n g the r e p r o d u c t i v e c y c l e 153 x i i X IX. ACKNOWLEDGEMENTS I w i s h t o thank my s u p e r v i s o r , Dr. N. R. L i l e y , f o r h i s guid a n c e and s u p p o r t t h r o u g h o u t the s t u d y , and f o r h i s h e l p f u l d i s c u s s i o n and c a r e f u l c r i t i c i s m o f t h i s t h e s i s . P a r t o r a l l of t h e m a n u s c r i p t has b e n e f i t t e d from c r i t i c a l r e a d i n g by D r s . B. G o r z a l k a , A. P e r k s , A. P h i l l i p s and J.N.M. S m i t h . I am g r a t e f u l t o Dr. Mei Cheng of R u t g e r s U n i v e r s i t y f o r the doves which formed the n u c l e u s o f our c o l o n y ; Dr. D. J . M a r s h a l l o f A y e r s t L a b o r a t o r i e s f o r t h e g i f t of s y n t h e t i c LHRH; Dr. T. M. John f o r h i s a d v i c e on the p i n e a l e c t o m y p r o c e d u r e ; and J a c k i e of H a l l m a r k P o u l t r y , Vancouver, f o r the c h i c k e n heads used t o make the p i n e a l e x t r a c t . I a l s o want t o thank my husband, Stephen B e t t , f o r h i s a s s i s t a n c e d u r i n g numerous "emergencies", and f o r p r o o f i n g t h i s t h e s i s . T h i s s t u d y was s u p p o r t e d by the N a t i o n a l S c i e n c e and E n g i n e e r i n g C o u n c i l of Canada t h r o u g h an o p e r a t i n g g r a n t t o Dr. N. R. L i l e y , and by a N a t i o n a l S c i e n c e and E n g i n e e r i n g C o u n c i l s c h o l a r s h i p , U n i v e r s i t y of B r i t i s h Columbia Open S c h o l a r s h i p s , and an H. R. M a c M i l l a n F a m i l y F e l l o w s h i p g r a n t e d t o me. 1 I. INTRODUCTION The r e l a t i o n s h i p between daylength and gonadal development in b i r d s was f i r s t noted i n the e a r l y p a r t of t h i s century (Rowan, 1926; M a r s h a l l , 1936; R i l e y & W i t s c h i , 1938; B i s s o n n e t t e , 1939) and annual changes i n photoperiod are now r e c o g n i z e d as a major environmental f a c t o r determining the t i m i n g of a v i a n breeding seasons (see reviews by Farner & F o l l e t t , 1979; W i n g f i e l d & Farner, 1980; F o l l e t t & Robinson, 1980) Information from the photoperiod i s t r a n s l a t e d i n t o changes i n endocrine a c t i v i t y , which i n t u r n r e g u l a t e both 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 aspects of r e p r o d u c t i o n . In b i r d s , as i n mammals, breeding i s c o n t r o l l e d by hormones from the b r a i n , p i t u i t a r y and gonads. F o l l o w i n g exposure of b i r d s to long daylengths there i s a marked i n c r e a s e i n both p i t u i t a r y and plasma l e v e l s of l u t e i n i z i n g hormone (LH) and f o l l i c l e s t i m u l a t i n g hormone (FSH) ( W i n g f i e l d et a l . , 1980; F o l l e t t & Robinson, 1980). These changes i n the p r o d u c t i o n and r e l e a s e of p i t u i t a r y gonadotropins are l i k e l y a r e s u l t of i n c r e a s e d s t i m u l a t i o n by the hypothalamus, s i n c e they are a b o l i s h e d by l e s i o n s i n the t u b e r o - i n f u n d i b u l a r region (Assenmacher & J a l l a g e a s , 1978; S t e t s o n , 1969). The a v i a n gonads r e l e a s e a number of s t e r o i d s i n response to gonadotropic s t i m u l a t i o n : both the ovary and t e s t i s produce estrogen, androgen and progesterone (Hohn & Cheng, 1967; Huang et a l . , 1979). The s t e r o i d s , i n t u r n , are r e s p o n s i b l e f o r the development and maintenance of accessory r e p r o d u c t i v e s t r u c t u r e s and secondary sex c h a r a c t e r i s t i c s such as plumage and b i l l 2 c o l o u r a t i o n (see reviews by L o f t s & Murton, 1973; Murton & Westwood, 1977). They are a l s o r e s p o n s i b l e f o r the c o n t r o l of r e p r o d u c t i v e behaviour ( S i l v e r et a l . , 1979). The i d e n t i t y of n e u r a l s t r u c t u r e s c o n t r o l l i n g hypothalamic a c t i v i t y , i f i n f a c t these e x i s t , i s unknown. Many b i r d s p e c i e s have been shown to respond to l i g h t even a f t e r b l i n d i n g ( F o l l e t t , 1973; O l i v e r et a l . , 1979; H a r r i s o n & Becker, 1969). L e s i o n s i n the p r e - o p t i c area (POA) w i l l block photoresponses i n Japanese q u a i l (Davies & F o l l e t t , 1975) and pigeons ( B o u i l l e & Bayle, 1973),, but a n t e r i o r d e a f f e r e n t a t i o n of the POA has no e f f e c t , suggesting that input from higher c e n t e r s may not be e s s e n t i a l (Davies & F o l l e t t , 1975). I t i s p o s s i b l e that the hypothalamus i s i t s e l f r esponsive to l i g h t p e n e t r a t i n g the s k u l l . D i r e c t i l l u m i n a t i o n of the hypothalamus using o p t i c f i b e r s or luminescent p a i n t has been shown to s t i m u l a t e t e s t i s growth i n some b i r d s ( O l i v e r et a l . , 1979; Homma et a l . , 1980). I t i s a l s o p o s s i b l e , however, that the POA i s c o n t r o l l e d by substances t r a n s p o r t e d i n the c i r c u l a t o r y or v e n t r i c u l a r systems and that these may act alone or i n c o n j u n c t i o n with n e u r a l i n p u t . I t i s thus c l e a r that annual c y c l e s of development i n the r e p r o d u c t i v e t r a c t s of photoresponsive b i r d s are due to l i g h t -induced changes i n the hormonal output of the hypothalamus and p i t u i t a r y . C o n s i d e r a t i o n of the p h y s i o l o g i c a l system i s f r e q u e n t l y accompanied by the t a c i t assumption that annual .cycles of behaviour are a d i r e c t r e f l e c t i o n of the seasonal changes i n c i r c u l a t i n g l e v e l s of gonadal s t e r o i d s (see diagrams 3 i n Murton & Westwood, 1977; W i n g f i e l d & Farner, 1980). In other words, as daylength i n c r e a s e s i n the s p r i n g , s t i m u l a t i o n of the hypothalamus and p i t u i t a r y r e s u l t s i n a dramatic r i s e i n gonadal s t e r o i d o g e n e s i s . As l e v e l s of t e s t o s t e r o n e i n c r e a s e i n males, or estrogen and progesterone i n females, a p p r o p r i a t e p a t t e r n s of r e p r o d u c t i v e behaviour begin to emerge. S e v e r a l l i n e s of evidence support such a scheme i n b i r d s . I t i s c e r t a i n l y t rue that recrudescence of the gonads i s accompanied by l a r g e i n c r e a s e s i n the p r o d u c t i o n of s t e r o i d s by both the o v a r i e s and t e s t e s ( W i n g f i e l d & Farner, 1980; Farner & F o l l e t t , 1979). I t i s a l s o t r u e that i n v i r t u a l l y a l l b i r d s p e c i e s which have been examined the occurrence of r e p r o d u c t i v e behaviour, be i t t e r r i t o r i a l defense and song, c o u r t s h i p and mating, or p a r e n t a l c a r e , i s dependent upon the presence of gonadal s t e r o i d s ( S i l v e r et a l . , 1979). Gonadectomy v i r t u a l l y e l i m i n a t e s sexual a c t i v i t y i n both male and female b i r d s of numerous s p e c i e s , while replacement therapy u s i n g exogenous s t e r o i d s u s u a l l y r e i n s t a t e s the l o s t behaviour p a t t e r n s . Although the occurrence of sexual behaviour i s dependent upon hormone p r o d u c t i o n , i t does not n e c e s s a r i l y f o l l o w that the l e v e l of a c t i v i t y i s a simple r e f l e c t i o n of l i g h t - i n d u c e d changes i n s t e r o i d c o n c e n t r a t i o n . The p o s s i b i l i t y that p hotoperiod may a l t e r the b e h a v i o u r a l response to s t e r o i d s , r a t h e r than simply a c t i n g on hormone l e v e l s , was f i r s t proposed by Robert Hinde and h i s a s s o c i a t e E l i z a b e t h S t e e l . They found that female c a n a r i e s , which normally b u i l d t h e i r nests i n the s p r i n g , c o u l d be induced to b u i l d i n the winter i f they were 4 exposed to long daylengths ( S t e e l & Hinde, 1966). E s t r a d i o l i n j e c t i o n s a l s o s t i m u l a t e d n e s t - b u i l d i n g i n o v a r i e c t o m i z e d females, but only i f they were exposed to long days; e s t r a d i o l had l i t t l e e f f e c t under short days ( S t e e l & Hinde, 1972b; F o l l e t t et a l . , 1973). Since the d i f f e r e n c e s i n b u i l d i n g r a t e s d i d not appear to depend on e i t h e r i n c r e a s e d gonadotropin l e v e l s (Hinde & S t e e l , 1975; Hinde et a l . , 1974) or d i f f e r e n c e s i n the amount of time a v a i l a b l e f o r b u i l d i n g ( S t e e l & Hinde, 1976a) the authors concluded that photoperiod a l t e r s responsiveness to hormonal s t i m u l a t i o n . These r e s u l t s were p a r a l l e l e d by l a t e r work on females of two other domestic s p e c i e s : the budgerigar and the r i n g dove. Ovariectomized, e s t r a d i o l - t r e a t e d female budgerigars exposed to e i t h e r s o l i d (14L:10D) or s k e l e t o n (6L:6D:2L:1OD) long days enter the nest box sooner, and spend longer i n the nest box than do females h e l d on short (8L:16D) days (Gosney & Hinde, 1975). I n t a c t female r i n g doves engage i n more n e s t - s o l i c i t i n g and n e s t - b u i l d i n g a c t i v i t y when h e l d under long days ( L i l e y , 1976a), even i f both l o n g - and short-day b i r d s are t r e a t e d with h i g h l e v e l s of exogenous estrogen and/or progesterone ( L i l e y , 1976b). In Japanese q u a i l , on the other hand, photoperiod does not i n f l u e n c e the s t e r o i d i n d u c t i o n of behaviour. In t h i s s p e c i e s both males and females respond to short daylengths with a drop i n sexual a c t i v i t y (Adkins, 1973; Adkins & Nock, 1976). Androgen treatment of e i t h e r long-day c a s t r a t e s or short-day i n t a c t males causes an i n c r e a s e i n sexual behaviour to the l e v e l seen i n i n t a c t long-day b i r d s (Beach & Inman, 1967; Sachs, 5 1969), as does estrogen treatment of o v a r i e c t o m i z e d long-day or i n t a c t short-day females (Adkins & A d l e r , 1972; Noble, 1972; Adkins & Nock, 1976). These r e s u l t s have been i n t e r p r e t e d as evidence that long daylengths and s t e r o i d treatment have the same e f f e c t s and should t h e r e f o r e be c o n s i d e r e d e q u i v a l e n t (Sachs, 1969; Adkins & Nock, 1976). The male r i n g dove p r o v i d e s a u s e f u l model f o r examining the i n t e r a c t i o n between daylength, e n d o c r i n o l o g y and a v i a n breeding behaviour. The hormonal c o n t r o l of i t s r e p r o d u c t i v e behaviour has been thoroughly examined by Lehrman and h i s co-workers (see review by Cheng, 1979); i t c o u r t s r e a d i l y i n the l a b o r a t o r y s i t u a t i o n , and males, u n l i k e females, are e a s i l y gonadectomized. C a s t r a t i o n of male doves a b o l i s h e s a l l a s p e c t s of t h e i r r e p r o d u c t i v e behaviour ( E r i c k s o n & Lehrman, 1964) while i n t r a m u s c u l a r i n j e c t i o n or i n t r a c r a n i a l implants of exogenous t e s t o s t e r o n e r e s t o r e c o u r t s h i p and n e s t - b u i l d i n g (Hutchison, 1967; B a r f i e l d , 1971; Martinez-Vargas,1974). E s t r a d i o l r e -i n s t a t e s n e s t - o r i e n t e d behaviour, but not the more a g g r e s s i v e d i s p l a y s (Martinez-Vargas, 1974, Cheng & Lehrman, 1975; Hutchison, 1976). Since radioimmunoassays have f a i l e d to d e t e c t e s t r a d i o l i n the plasma of male doves (Korenbrot et a l . , 1974), androgen appears to be the gonadal hormone r e s p o n s i b l e f o r the c o n t r o l of r e p r o d u c t i v e behaviour. T h i s i s supported by the presence of i n c r e a s e d androgen l e v e l s i n breeding males (Feder et a l . , 1977; Hutchison & Katongole, 1975) and by the o b s e r v a t i o n that i n t r a c r a n i a l androgen implants s t i m u l a t e c o u r t s h i p a c t i v i t y i n the same b r a i n r e g i ons that c o n c e n t r a t e 6 r a d i o a c t i v e t e s t o s t e r o n e (Hutchison, 1971; Martinez-Vargas et a l . , 1974). In e a r l i e r experiments examining the e f f e c t of daylength on the r e p r o d u c t i v e behaviour of male doves (McDonald, 1976), I found that the l e v e l s of both c o u r t s h i p and n e s t - b u i l d i n g a c t i v i t y were i n c r e a s e d by exposure to long (16L:8D) photoperiods. Exogenous t e s t o s t e r o n e treatment r e s u l t e d i n i n c r e a s e d n e s t - s o l i c i t i n g and n e s t - b u i l d i n g i n short-day (8L:16D) b i r d s , suggesting that the daylength might a c t through changes i n t e s t i c u l a r androgen p r o d u c t i o n . In order to t e s t t h i s p o s s i b i l i t y , c a s t r a t e d males were i n j e c t e d with t e s t o s t e r o n e propionate while being h e l d on one of the two experimental l i g h t regimes. No d i f f e r e n c e s i n c o u r t s h i p were seen between the two groups, but n e s t i n g a c t i v i t y was s t i l l g r e a t e r i n those b i r d s exposed to long days. These r e s u l t s i n d i c a t e that photoperiod may a l t e r c o u r t s h i p through changes i n endogenous androgen p r o d u c t i o n . The e f f e c t on n e s t - b u i l d i n g p e r s i s t e d , however, even when androgen l e v e l s were b e l i e v e d to be maintained at s i m i l a r l e v e l s . T h i s suggests that i n the case of n e s t - b u i l d i n g , daylength a f f e c t s b e h a v i o u r a l s e n s i t i v i t y to the hormonal s t i m u l a t i o n . The p o s s i b i l i t y that environmental f a c t o r s can a l t e r behaviour through means other than changes i n gonadal s t e r o i d p r o d u c t i o n has f u n c t i o n a l importance, i n that i t pr o v i d e s a mechanism f o r temporal s e p a r a t i o n of the v a r i o u s responses which depend on the same hormone. The r e s u l t s from experiments on c a n a r i e s , b u d g e r i g a r s , and r i n g doves c l e a r l y suggest that 7 photoperiod causes changes i n responsiveness to hormones. However, in none of these i n s t a n c e s have the mechanisms u n d e r l y i n g the a l t e r a t i o n i n responsiveness been determined. Thus, our understanding of how daylength c o n t r o l s behaviour i s s t i l l at the 'black-box' s t a g e — l i t t l e i s known of the events which l i n k a l t e r e d photoperiod with the r e s u l t i n g s h i f t s i n a c t i v i t y . P r i o r assumptions that f l u c t u a t i o n s i n s t e r o i d l e v e l are important, may prove to be a c c u r a t e i n some i n s t a n c e s , but other changes o b v i o u s l y occur as w e l l . The experiments o u t l i n e d i n t h i s t h e s i s were designed to examine how photoperiod b r i n g s about changes in the n e s t i n g behaviour of male doves. Three d i f f e r e n t mechanisms were c o n s i d e r e d . A l l three mechanisms have been i m p l i c a t e d i n 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 v e p h y s i o l o g y and i n the c o n t r o l of sexual behaviour i n a v a r i e t y of s p e c i e s . In the f i r s t s e r i e s of experiments I examined the p o s s i b i l i t y that daylength a l t e r s the a r o m a t i z a t i o n of androgen i n t o estrogen by c e n t r a l t i s s u e s . T e s t o s t e r o n e i s converted by the a v i a n b r a i n i n t o s e v e r a l m e t a b o l i t e s which d i f f e r i n t h e i r a b i l i t y to induce sexual behaviour (Adkins-Regan, 1981). One of the most a c t i v e of these m e t a b o l i t e s i s estrogen (see below). Because the r a t e of metabolism of androgen i n some t i s s u e s has been shown to be a f f e c t e d by photoperiod, i t seemed p o s s i b l e that long daylengths may l e a d to i n c r e a s e d l e v e l s of estrogen formation. I f estrogen i s the hormone r e s p o n s i b l e f o r c o n t r o l l i n g n e s t i n g , then daylength-dependent changes i n a r o m a t i z a t i o n c o u l d account f o r the observed d i f f e r e n c e s i n n e s t - b u i l d i n g . I t e s t e d t h i s 8 suggestion by comparing the n e s t - b u i l d i n g a c t i v i t y of short-day and long-day c a s t r a t e d doves t r e a t e d with estrogen, t e s t o s t e r o n e , or t e s t o s t e r o n e and an a r o m a t i z a t i o n i n h i b i t o r . Daylength i s known to i n f l u e n c e endogenous l e v e l s of those hormones which are r e s p o n s i b l e f o r p r o d u c t i o n of gonadal s t e r o i d s , s p e c i f i c a l l y , hypothalamic l u t e i n i z i n g hormone r e l e a s i n g hormone (LHRH) and p i t u i t a r y gonadotropins. Because LHRH s t i m u l a t e s sexual a c t i v i t y i n female doves (Cheng, 1977), i n a second s e r i e s of experiments an attempt was made to determine whether i t i s the i n c r e a s e d p r o d u c t i o n of the hypothalamic hormone under long daylengths that s t i m u l a t e s nest-b u i l d i n g , as a r e s u l t of a synergism with t e s t o s t e r o n e . Long-and short-day c a s t r a t e d males were t r e a t e d with exogenous t e s t o s t e r o n e or t e s t o s t e r o n e and LHRH to see i f the i n f l u e n c e of s h o r t daylengths c o u l d be r e v e r s e d by LHRH. F i n a l l y , s e v e r a l t e s t s were performed which focussed on the r o l e of the p i n e a l gland. In some mammals the p i n e a l gland mediates photoperiod-induced changes i n r e p r o d u c t i v e development, and i t seemed reasonable to suggest that i t might p l a y a s i m i l a r r o l e i n c o n t r o l l i n g n e s t i n g behaviour i n b i r d s . T h i s hypothesis was t e s t e d using the c l a s s i c a l e n d o c r i n o l o g i c a l method of removal and replacement. F i r s t , the e f f e c t of p i n e a l removal on n e s t - b u i l d i n g was examined, then attempts were made to r e v e r s e the e f f e c t u sing exogenous p i n e a l p r o d u c t s . 9 I I . GENERAL METHODS A. Experimental Animals The dove colony at the U n i v e r s i t y of B r i t i s h Columbia c o n s i s t s of the descendants of ten breeding p a i r s obtained from Rutgers U n i v e r s i t y i n 1974. P a i r s of doves were bred i n the l a b o r a t o r y i n 120x45x65 cm aluminium cages. The young were l e f t with t h e i r parents u n t i l they were four weeks o l d and were then t r a n s f e r r e d to l a r g e a v i a r i e s , each c o n s i s t i n g of an i n s i d e room (6.2x3.6x2.4 m) connected to an e n c l o s e d outer area (3.8x3.9x2.4 m). The b i r d s were h e l d i n mixed groups of 60 to 80 i n d i v i d u a l s f o r 8 to 10 months. They were then sexed by e x p l o r a t o r y laporotomy, banded and ret u r n e d t o the a v i a r i e s u n t i l needed. Approximately 540 b i r d s were r a i s e d , i n t o t a l , f o r t h i s study. The b i r d s were p r o v i d e d with food (Purina pigeon chow) and water ad l i b i t u m . Vitamin supplements ( A v i t r o n ) were added to the water once a week. Breeding p a i r s r e c e i v e d v i t a m i n s and ground o y s t e r s h e l l s twice a week. While being used f o r experiments, the b i r d s were h e l d i n i s o l a t i o n cages made of aluminium and wire mesh. Each cage measured 43x43x43 cm and was equipped with a perch, water tube and feeder. The s i d e s of the cages were s o l i d and the racks were p o s i t i o n e d so that the animals were i n constant v i s u a l i s o l a t i o n . Each h o l d i n g room c o n t a i n e d two racks of 12 cages. B e h a v i o u r a l o b s e r v a t i o n s were made in 120x45x65 cm breeding cages. Each was d i v i d e d i n h a l f by a removable opaque p a r t i t i o n . Both s i d e s of the cages were f i t t e d with a perch, 10 water tube and feeder, and a g l a s s n e s t i n g bowl was suspended 10 cm above the f l o o r on the l e f t s i d e . Nine o b s e r v a t i o n cages were p o s i t i o n e d along one w a l l of the o b s e r v a t i o n room. The b i r d s c o u l d not see i n t o other cages and were separated from the observer by a black p l a s t i c b l i n d . Both the o b s e r v a t i o n and the h o l d i n g rooms were l i g h t e d by e i g h t 40 watt f l u o r e s c e n t tubes mounted on the c e i l i n g and c o n t r o l l e d by an automatic timer. The average l i g h t i n t e n s i t y near the f r o n t of the cages was 161 +22 l u x . Temperatures were h e l d at 20 + 1 C. Unless otherwise s t a t e d , the experimental b i r d s were h e l d on long days of 16 hours l i g h t : 8 hours dark (LD = 16L:8D) or short days of 8 hours l i g h t : 16 hours dark (SD = 8L:16D), with the l i g h t s t u r n i n g on at 8:30am. B i r d s h e l d f o r laporotomy, c a s t r a t i o n or pretreatment were g e n e r a l l y exposed to a 12L:12D, while stimulus females were kept on 14L:10D. The r e p r o d u c t i v e experience of i n d i v i d u a l b i r d s was unknown; undoubtedly most mature b i r d s had engaged i n some c o u r t s h i p while being h e l d i n the a v i a r i e s , but the absence of a p p r o p r i a t e nest s i t e s and n e s t i n g m a t e r i a l prevented the s u c c e s s f u l h a t c h i n g of eggs and subsequent p a r t i c i p a t i o n i n p a r e n t a l a c t i v i t i e s . I n a b i l i t y to complete the breeding c y c l e , coupled with the r e l a t i v e l y high d e n s i t y of b i r d s , probably d i s r u p t e d the formation of s t a b l e p a i r bonds. However, s i n c e the ages of the b i r d s v a r i e d - - a n d p o s s i b l y t h e i r s t a t e of r e p r o d u c t i v e development as w e l l — a l l b i r d s were bred i n the l a b o r a t o r y before being used. P a i r s were p l a c e d i n 120x45x65 cm cages c o n t a i n i n g n e s t i n g m a t e r i a l (12 cm le n g t h s of s t r i n g ) and 11 a nest bowl, and remained together u n t i l they had b u i l t a nest and l a i d two f e r t i l e eggs. F o l l o w i n g the i n i t i a l breeding experience the b i r d s were again returned to the a v i a r i e s . At any given time, about 5% of the male doves are s e x u a l l y unresponsive. Because of the small sample s i z e s employed in the experiments, I decided not to use these b i r d s . Immediately before being used i n any experiment, a l l males were p a i r e d with a female f o r two 10-minute p e r i o d s . Any b i r d which f a i l e d to d i s p l a y c o u r t s h i p (bow-cooing and/or n e s t - s o l i c i t i n g ) was not used. B. S u r g i c a l Procedures and Hormone A d m i n i s t r a t i o n C a s t r a t i o n was performed under sodium p e n t a b a r b i t o l (•Nembutal) a n a e s t h e s i a (3.0-3.5 mg p e n t a b a r b i t o l / b i r d ) i n a two-stage o p e r a t i o n , the r i g h t t e s t i s being removed one week a f t e r the l e f t one. U s u a l l y the e n t i r e t e s t i s was e x c i s e d i n one p i e c e . A f t e r a recovery p e r i o d of two weeks each male was t e s t e d with a female f o r two 10-minute p e r i o d s i n order to ensure that the c a s t r a t i o n was complete. Any males that e i t h e r bowed or s o l i c i t e d ( l e s s than 8%) were d i s c a r d e d . The procedure f o r pinealectomy was based on the one used by John e t . a l . (1978) f o r pigeons. B i r d s were a n a e s t h e t i z e d with sodium p e n t a b a r b i t o l , then p l a c e d under a b i n o c u l a r microscope. The s k i n on the top of the head was cut with s c i s s o r s , and the spongy bone removed with f o r c e p s . The hard bone over the p i n e a l r e g i o n was then p r i e d o f f , t a k i n g care not to rupture the u n d e r l y i n g blood s i n u s . Once the p i n e a l area was 12 c l e a r the meninges were cut and a slow, steady stream of s a l i n e was run over the area to wash away the blood and enable l o c a t i o n of the p i n e a l body. The p i n e a l was grasped with f i n e f o rceps and p u l l e d l o o s e , along with the c h o r o i d plexus. The area was then covered with Gelfoam (Upjohn) and sutured. Sham o p e r a t i o n s on c o n t r o l b i r d s f o l l o w e d the same procedure to the p o i n t of r u p t u r i n g the sinus and l o c a t i n g the p i n e a l . Hormone i n j e c t i o n s were ad m i n i s t e r e d e i t h e r subcutaneously or i n t r a m u s c u l a r l y i n the p e c t o r a l r e g i o n , a l t e r n a t i n g between the r i g h t and l e f t s i d e s . Unless otherwise s t a t e d , a l l i n j e c t i o n s were given at approximately 9:00 am. Hormone implants were made f o l l o w i n g the procedure of Smith e t . a l . (1977), with the exception of the implants used i n the LHRH experiment. One end of a short l e n g t h of s i l a s t i c t ubing (1.98mm i d , 3.17mm od; Dow Corning) was s e a l e d with a wooden plug and coated with s i l a s t i c medical s i l i c o n e adhesive type A (Dow C o r n i n g ) . C r y s t a l l i n e hormone was then packed i n t o the tubing u n t i l i t f i l l e d a s p e c i f i e d l e n g t h and the second end s e a l e d . Implants used i n the LHRH experiment were prepared i n the same way, except that the wooden plugs were not used. The cap s u l e s were soaked i n .9% s a l i n e o vernight and then p l a c e d under the s k i n at the back of the neck, f o l l o w i n g a d m i n i s t r a t i o n of a l o c a l a n a e s t h e t i c (*Xylocaine). 13 C. T e s t i n g Procedure J_. Stimulus Females Stimulus female b i r d s were brought i n from the a v i a r i e s and h e l d i n p a i r s f o r one week. They were then moved i n t o i s o l a t i o n cages f o r a second week, and two days before use were p l a c e d i n the r i g h t hand s i d e of an o b s e r v a t i o n cage. The females remained i n the o b s e r v a t i o n cages throughout the t e s t p e r i o d , on a l i g h t c y c l e of 14L:10D. The sexual a c t i v i t y of a female dove i s dependent, i n p a r t , on the r e p r o d u c t i v e behaviour of her mate (Martinez-Vargas, 1971). C i r c u l a t i n g l e v e l s of both estrogen and progesterone i n c r e a s e i n females f o l l o w i n g p a i r i n g (Korenbrot e t . a l . , 1974; S i l v e r e t . a l . , 1974), as a r e s u l t of exposure to male c o u r t s h i p . While e s t r a d i o l alone can support many of the b e h a v i o u r a l p a t t e r n s a s s o c i a t e d with female r e p r o d u c t i o n (Cheng, 1973b), both estrogen and progesterone are necessary f o r the d i s p l a y of n e s t - b u i l d i n g (Cheng & S i l v e r , 1975). By t r e a t i n g females with a combination of the two hormones, i t i s p o s s i b l e to mimic the endocrine s i t u a t i o n normally experienced j u s t p r i o r t o l a y i n g , l e a d i n g to h i g h l e v e l s of n e s t - o r i e n t e d a c t i v i t y immediately a f t e r p a i r i n g . In order to ensure that each of the males used i n the experiments was exposed to a s e x u a l l y a c t i v e female, r e g a r d l e s s of the male's own l e v e l of a c t i v i t y , s t i m u l u s females were t r e a t e d with exogenous estrogen and progesterone, beginning one week p r i o r to the o b s e r v a t i o n p e r i o d and c o n t i n u i n g through to 14 the end of o b s e r v a t i o n s . In the f i r s t two experiments females r e c e i v e d d a i l y i n j e c t i o n s of .2mg e s t r a d i o l benzoate (EB) + ,1mg progesterone (P) (Sigma) i n 0.2 ml s a l i n e . In the remaining experiments females were implanted with 5mm s i l a s t i c c apsules c o n t a i n i n g a 4:1 r a t i o of E:P one week before o b s e r v a t i o n s began. 2. Recording Technique Because only four p a i r s of b i r d s c o u l d be observed at a given time, the i n d i v i d u a l s used f o r an experiment were d i v i d e d i n t o s e t s of 8-10, c o n t a i n i n g approximately equal numbers of b i r d s from each treatment group being examined. These s e t s were staggered at one week i n t e r v a l s - - i n other words, p r o c e d u r a l week 3 f o r set 1 = week 2 f o r set 2 = week 1 f o r set 3. Beh a v i o u r a l r e c o r d i n g s were made over a 5-day t e s t p e r i o d . Males were moved from t h e i r i s o l a t i o n cages i n t o the l e f t s i d e of an o b s e r v a t i o n cage at 9:00 am. The p a r t i t i o n s were removed from 4 cages at 9:30 am and the a c t i v i t y of the 4 p a i r s recorded s i m u l t a n e o u s l y f o r 15 min. us i n g check sheets d i v i d e d i n t o 15-second i n t e r v a l s . T h i s procedure was then repeated f o r the second group of 4 p a i r s . Three more s e t s of r e c o r d i n g s were made at 11:30, 13:30 and 15:30. A f t e r the l a s t o b s e r v a t i o n p a r t i t i o n s were r e p l a c e d . The males were r e t u r n e d to the i s o l a t i o n cages on the d i f f e r e n t photoperiods and the females remained i n the r i g h t h a l f of the o b s e r v a t i o n cages. Thus, each of the males was p a i r e d with a female f o r 6.5 hours every day but c o ntinued to experience the experimental photoregimes. A l l 1 5 of the females, on the other hand, were he l d on the same 14L:10D l i g h t c y c l e . During the day each p a i r had f r e e access to 12cm lengths of s t r i n g spread on the f l o o r of the cage on the s i d e o pposite to the nest bowl. S t r i n g was added as necessary, keeping approximately 24 p i e c e s on the f l o o r . In order to minimize d i f f e r e n c e s i n the s t i m u l i r e c e i v e d from the nest, the s t r i n g i n the nest bowls was removed and counted at the end of each day. Any p i e c e s l y i n g d i r e c t l y beneath the nest had presumably been c a r r i e d over the 1" high p a r t i t i o n holder which l a y a c r o s s the c e n t r e of the cage, and were t h e r e f o r e a l s o counted. 3^ B e h a v i o u r a l Recordings D e s c r i p t i o n s of dove c o u r t s h i p behaviour have been p u b l i s h e d by s e v e r a l authors (see L o v a r i & Hutchison, 1975; L i l e y , 1976a; M i l l e r & M i l l e r , 1958; Hutchison, 1970a). The f o l l o w i n g b r i e f summary of the a c t i v i t i e s recorded i n t h i s study i s taken from McDonald (1976). 1) Chasing and pecking When a p a i r of r i n g doves i s f i r s t p l a c e d together the male u s u a l l y rushes towards the female, h i s head h e l d low and h i s rump f e a t h e r s r u f f l e d . I f she f l e e s he c o n t i n u e s to chase her, f r e q u e n t l y pecking her on the head and neck. T h i s d i s p l a y i s o f t e n accompanied by a 'kah' or laughing c a l l . 16 2) Bow-cooing Chasing r a p i d l y g i v e s way to the 'bow-coo' i n which the male s t r e t c h e s h i s neck upward, then bows forward with h i s b i l l p o i n t i n g towards the ground, u t t e r i n g a c h a r a c t e r i s t i c cooing sound. The u n d e r l y i n g m o t i v a t i o n of the bow-coo has been the s u b j e c t of c o n s i d e r a b l e s p e c u l a t i o n (see L o v a r i & Hutchison, 1975; Davies, 1974b; Goodwin, 1956a; L o f t s & Murton, 1973), but i t i s g e n e r a l l y agreed to be i n d i c a t i v e of a mixture of a g g r e s s i v e and sexual t e n d e n c i e s . 3) Male n e s t - s o l i c i t i n g F o l l o w i n g repeated bow-coos the male u s u a l l y moves to a corner of the cage or to the nest bowl and begins nest-s o l i c i t i n g ; s t a nding or s q u a t t i n g i n an o b l i q u e p o s i t i o n , h i s head c l o s e to the ground, he v i b r a t e s both wings si m u l t a n e o u s l y , i n t e r m i t t e n t l y u t t e r i n g 'nest-coos'. V a r i o u s forms of t h i s behaviour occur, i n c l u d i n g o b l i q u e p o s t u r i n g i n the absence of e i t h e r cooing or w i n g - f l i p p i n g , or both. In the f o l l o w i n g t e s t s n e s t - s o l i c i t i n g was d e f i n e d by the presence of both the o b l i q u e stance and w i n g - f l i p p i n g . 4) Female n e s t - s o l i c i t i n g S o l i c i t i n g by the male appears to a t t r a c t the f e m a l e — s h e e v e n t u a l l y approaches him and begins to spend more and more time i n h i s v i c i n i t y , o f t e n engaging i n a l l o p r e e n i n g . A f t e r a p e r i o d which may vary from a few minutes to s e v e r a l days the female a l s o begins to n e s t - s o l i c i t . I n i t i a l l y t h i s e l i c i t s an a g g r e s s i v e response from the male, who 17 pecks at her head and neck. N e s t - s o l i c i t i n g by the female i s a l s o v a r i a b l e - - w i n g - f l i p s and/or nest-coos i n the absence of the o b l i q u e posture being common. For t h i s reason i t was i d e n t i f i e d simply by the occurrence of wing-f l i p p i n g at the n e s t . 5) N e s t - b u i l d i n g As the female becomes f i r m l y a t t a c h e d to the nest s i t e male s o l i c i t i n g i s r e p l a c e d by n e s t - b u i l d i n g . B u i l d i n g i s a co-o p e r a t i v e e f f o r t : f o l l o w i n g v a r y i n g amounts of time spent p i c k i n g up and dropping n e s t i n g m a t e r i a l ( h a n d l i n g ) , the male c a r r i e s a p i e c e to the female, who remains at the n e s t . She takes the m a t e r i a l from the male and tucks i t beneath her. However, t h i s d i v i s i o n of labour i s not s t r i c t (see White, 1975a) and females may o c c a s i o n a l l y p i c k up m a t e r i a l and c a r r y i t to the nest. 6) Copulatory behaviour Behaviours a s s o c i a t e d with c o p u l a t i o n u s u a l l y occur d u r i n g the a f t e r n o o n . The female approaches the male and pecks g e n t l y at the base of h i s b i l l , while r h y t h m i c a l l y f l i p p i n g her wings (begging). The male then takes her b i l l i n h i s and appears to r e g u r g i t a t e food, which she swallows ( b i l l i n g ) . A f t e r one or s e v e r a l bouts of begging and b i l l i n g the female crouches, her wings extended. T h i s i s f o l l o w e d e i t h e r by a r e p e t i t i o n of the above sequence, or by mounting and c o p u l a t i o n . 18 7) Appeasement crouch Crouching may a l s o occur s h o r t l y a f t e r a p a i r i s p l a c e d together, i n response to chasing and bowing by the male. T h i s appears to f u n c t i o n as an appeasement d i s p l a y , and so has been l a b e l l e d appeasement c r o u c h i n g . Most of the b e h a v i o u r a l a c t i v i t e s recorded were scored i f they were observed w i t h i n a 15 sec. i n t e r v a l . Some a c t i v i t i e s which have very short d u r a t i o n s , ( c a r r y i n g , c o p u l a t o r y d i s p l a y s ) were noted as f r e q u e n c i e s , s i n c e these d i s p l a y s c o u l d occur s e v e r a l times w i t h i n one 15 sec. i n t e r v a l . The d i s p l a y s recorded were as f o l l o w s : 1) chase/peck (no. i n t e r v a l s ) - Because chasing and pecking occur i n c l o s e temporal a s s o c i a t i o n , bouts of one r a p i d l y g i v i n g way to the ot h e r , and because the u n d e r l y i n g m o t i v a t i o n of both appears to have a strong a g g r e s s i v e component, these two behaviours have been grouped t o g e t h e r . 2) bow-coos (no. i n t e r v a l s ) 3) male n e s t - s o l i c i t i n g (no. i n t e r v a l s ) 4) male-at-nest (no. i n t e r v a l s ) - T h i s i s a measure of the t o t a l amount of time spent by the male at the nest s i t e , and i n c l u d e s s i t t i n g , a l l o p r e e n i n g , and s o l i c i t i n g at the nest. 5) male c a r r y i n g (no. events) 19 6) male n e s t - b u i l d i n g (no. i n t e r v a l s ) - T h i s i s a measure of the t o t a l amount of time spent n e s t - b u i l d i n g . I t i n c l u d e s time spent h a n d l i n g or c a r r y i n g s t r i n g . In order to account f o r short breaks between these two behaviours d u r i n g which the male r e t u r n e d from the nest to the f l o o r , or appeared to search among the p i e c e s of s t r i n g before a c t u a l l y h a n d l i n g them, any i n t e r v a l s of l e s s than 30 seconds o c c u r r i n g between han d l i n g and/or c a r r y i n g are a l s o i n c l u d e d . 7) female n e s t - s o l i c i t i n g (no. i n t e r v a l s ) 8) female-at-nest (no. i n t e r v a l s ) - T h i s was recorded i n the same manner as male-at-nest. 9) female c a r r y i n g (no. events) 10) female n e s t - b u i l d i n g (no. i n t e r v a l s ) - T h i s i s a measure of the amount of time which the female spent performing 'male-type' b u i l d i n g , and was measured i n the same way as male n e s t - b u i l d i n g . I t does not i n c l u d e s i t t i n g at the nest and t u c k i n g m a t e r i a l brought by the male. 11) begging (no. events) 12) b i l l i n g (no. events) 13) sexual crouch (no. events) - T h i s category i n c l u d e s only those crouches preceded by begging and b i l l i n g . 14) mount/copulation (no. events) 20 15) appeasement crouch (no. events) - T h i s i n c l u d e s crouches which o c c u r r e d w i t h i n the f i r s t few minutes a f t e r the p a r t i t i o n was removed and were preceded by chasing and/or bowing. D. A n a l y s i s of Data Data from the b e h a v i o u r a l r e c o r d i n g s were t e s t e d with a K r u s k a l - W a l l i s one-way a n a l y s i s of v a r i a n c e , a = .05 ( S i e g e l , 1956). If o v e r a l l d i f f e r e n c e s were s i g n i f i c a n t then s e l e c t e d p a i r s were compared ( K o l s t o e , 1975) with Mann-Whitney U - t e s t s , a = .05 ( S i e g e l , 1956). Non-parametric c o r r e l a t i o n c o e f f i c i e n t s (Spearman rank t e s t ) and p a i r e d t e s t s (Wilcoxon t e s t ) were a l s o employed i n some s i t u a t i o n s . Unless otherwise s t a t e d , a l l t e s t s were based on the t o t a l a c t i v i t y recorded over the f i v e - d a y o b s e r v a t i o n p e r i o d (=20 15-minute o b s e r v a t i o n s ) . 21 I I I . ANDROGEN METABOLISM AND DAYLENGTH-DEPENDENT CHANGES IN NEST-BUILDING BEHAVIOUR IN MALE DOVES A. I n t r o d u c t i o n Over the past few years much a t t e n t i o n has been focussed on the p o s s i b i l i t y that t e s t o s t e r o n e i s a c t u a l l y a 'prehormone' with no b i o l o g i c a l a c t i v i t y i t s e l f , but having i t s e f f e c t s through conve r s i o n to i t s v a r i o u s m e t a b o l i t e s (Zigmond, 1975). Tes t o s t e r o n e metabolism has now been i m p l i c a t e d i n most androgen-induced responses, i n c l u d i n g the c o n t r o l of a d u l t sexual a c t i v i t y ( L u t tge, 1979; L o f t s & Massa, 1980; Davies et a l . , 1980; Feder, 1981a; Feder, 1981b; Adkins-Regan, 1981; M a r t i n i , 1982). I was i n t e r e s t e d i n the p o s s i b i l i t y that daylength a l t e r s the con v e r s i o n of t e s t o s t e r o n e to one of i t s more a c t i v e m e t a b o l i t e s , estrogen, i n doves, thus l e a d i n g to d i f f e r e n c e s i n the ' c e n t r a l a c t i v a t i o n ' of n e s t - b u i l d i n g . Photoperiod-dependent changes i n androgen metabolism might, then, account f o r the observed v a r i a t i o n s i n n e s t - b u i l d i n g seen i n b i r d s whose androgen l e v e l s are h e l d c o n s t a n t . F i g u r e 1 i n d i c a t e s the routes of t e s t o s t e r o n e metabolism which are found i n most v e r t e b r a t e s . In b i r d s , u n l i k e mammals, hypothalamic 5a-reductase a c t i v i t y i s e i t h e r very s l i g h t ( s t a r l i n g , B o t t o n i & Massa, 1981; Japanese q u a i l , Massa et a l . , 1979; dove, Steimer & Hutchison, 1981a; turkey, Massa & Sharp, 1981) or absent (Japanese q u a i l , B a l t h a z a r t et a l . , 1979; c o c k e r e l , Nakamura & Tanabe, 1974). The major m e t a b o l i t e i d e n t i f i e d i n Japanese q u a i l i s androstenedione (AE), f o l l o w e d F i g u r e 1. The major r o u t e s o f androgen m e t a b o l i s m i n c e n t r a l and p e r i p h e r a l t a r g e t t i s s u e s . From L u t t g e e t al„, 1975„ 23 by 5/3-metabolites (5/3dihyrotestosterone = 50DHT, 5/33adiol) ( B a l t h a z a r t et a l . , 1979; Massa et a l . , 1979) while i n other a v i a n s p e c i e s p r o d u c t i o n of AE i n the hypothalamus i s l e s s s i g n i f i c a n t and the major products are 5j3DHT and 5/3androstanediol (5j3DIOL) (dove, Steimer & Hutchison, 1981b; s t a r l i n g , Massa et a l . , 1977; B o t t o n i & Massa, 1980; c o c k e r e l , Massa £ Sharp, 1981). Low r a t e s of a r o m a t i z a t i o n to e s t r a d i o l have a l s o been noted ( c h i c k e n , C a l l a r d et a l . , 1978; dove, Steimer & Hutchison, 1980). Although i t i s by no means the most abundant m e t a b o l i t e , e s t r a d i o l has r e c e i v e d c o n s i d e r a b l e a t t e n t i o n with regard to the c o n t r o l of male sexual a c t i v i t y . Because of the h i g h potency of estrogen i n r e l a t i o n to the a c t i v a t i o n of r e p r o d u c t i v e responses i n males, numerous recent papers have suggested that a r o m a t i z a t i o n of t e s t o s t e r o n e i n t o estrogen i s a necessary step f o r the i n d u c t i o n of male sexual behaviour i n some mammals (Luttge, 1979; Beyer et a l . , 1976a). The major l i n e s of evidence s u p p o r t i n g t h i s h y p othesis a r e : 1) T e s t o s t e r o n e i s conve r t e d i n t o estrogen i n b i o l o g i c a l l y s i g n i f i c a n t q u a n t i t i e s i n the b r a i n ( L i e b e r b e r g & McEwen, 1977; Rezek, 1977; S h o l l & Goy, 1981). 2) Estrogen i s pi c k e d up by r e c e p t o r s i n the male CNS (Ciacco et a l . , 1979; Muldoon, 1980; Whalen, 1980). 24 3) Estrogen and aromatizable androgens w i l l induce male behaviour, nonaromatizable androgens w i l l not ( C h r i s t e n s e n & Clemens, 1974; Sodersten, 1975; W a l l i s & L u t t g e , 1975; but see a l s o Gorzalka et a l . , 1975; Cochran & Perachio, 1977; and reviews by Beyer, 1976a; Luttge et a l . , 1976). 4) A n t i - e s t r o g e n s and aromatase b l o c k e r s decrease b e h a v i o u r a l responsiveness to t e s t o s t e r o n e (Beyer et a l . , 1976b; L u t t g e , 1975; M o r a l i et a l . , 1977). There i s evidence that a r o m a t i z a t i o n i s important f o r the c o n t r o l of masculine sexual behaviour i n b i r d s as w e l l as mammals. The p r e o p t i c - a n t e r i o r hypothalamic r e g i o n (POA-AH) i s probably a major s i t e of androgen a c t i o n i n the avian b r a i n (Gardner & F i s h e r , 1968; B a r f i e l d , 1969; Meyer & Salgen, 1970; B a r f i e l d , 1971; Hutchison, 1967). T h i s area c o n t a i n s aromatase ( C a l l a r d et a l . , 1978) and has been shown to convert t e s t o s t e r o n e (T) to estrogen (E) i n male doves (Steimer & Hutchison, 1980). Estrogen i s c o n c e n t r a t e d i n the POA, b a s a l hypothalamus and amygdala (Kim et a l . , 1978; Martinez-Vargas et a l . , 1976) where i t s t i m u l a t e s t r a n s l o c a t i o n of the r e c e p t o r to the nucleus (Steimer & Hutchison, 1981a). I t i s thus c l e a r t h a t aromatase a c t i v i t y i s h i g h i n those c e n t r e s c o n t r o l l i n g male c o u r t s h i p . Information on the a b i l i t y of v a r i o u s T m e t a b o l i t e s to induce r e p r o d u c t i v e behaviour i s a v a i l a b l e f o r only a few b i r d s p e c i e s . Estrogen promotes mounting and nest-box o r i e n t a t i o n i n male budger i g a r s (Brockway, 1974) and c o p u l a t i o n i n Japanese q u a i l (Adkins & A d l e r , 1972). Copulatory a c t i v i t y i n Japanese 25 q u a i l i s s t i m u l a t e d by aromatizable androgens (T, T propio n a t e , AE) but not by nonaromatizable ones (5aDHT, 5aDHT benzoate, 5/3DHT, androsterone) (Adkins, 1977; Wada, 1982). In a d d i t i o n , the b e h a v i o u r a l response to T i s decreased by concurrent treatment with a n t i - e s t r o g e n s (Adkins-Nock, 1976) or with the aromatase b l o c k e r ATD (Adkins et a l . , 1980). Deviche et a l . (1982) r e c e n t l y r e p o r t e d that i n a d u l t Japanese q u a i l 5/3DHT given i n c o n j u n c t i o n with .5 mg T i n c r e a s e s male neck-grabs, suggesting that i t may be weakly a n d r o g e n i c — b u t s i n c e only 3 of 80 p o s s i b l e measures of the e f f e c t of 50DHT on sexual behaviour were s i g n i f i c a n t , the r e s u l t s appear to co n f i r m , r a t h e r than deny a lack of response to t h i s androgen. S e v e r a l workers have found t h a t e s t r a d i o l i s i n e f f e c t i v e i n r e i n s t a t i n g e a r l y p a t t e r n s of a g g r e s s i v e c o u r t s h i p i n c a s t r a t e d male doves, while i t i s more e f f e c t i v e , (Hutchison, 1970a; Hutchison, 1971), l e s s e f f e c t i v e (Cheng & Lehrman, 1975) or e q u a l l y as e f f e c t i v e (Martinez-Vargas, 1974) as t e s t o s t e r o n e i n terms of n e s t - s o l i c i t i n g . One paper rep o r t e d that e s t r a d i o l benzoate (EB) w i l l a l s o induce n e s t - b u i l d i n g and i n c u b a t i o n i n c a s t r a t e s (Martinez-Vargas, 1974). The a b i l i t y of 5a and 50-reduced m e t a b o l i t e s t o r e s t o r e e a r l y c o u r t s h i p a c t i v i t y i n c a s t r a t e d doves i s s l i g h t compared with the e f f e c t of T. Hypothalamic implants of d i h y d r o t e s t o s t e r o n e (DHT), DHT a c e t a t e , or DHT propionate f a i l to s t i m u l a t e bow-cooing and induce only low l e v e l s of n e s t - s o l i c i t i n g (unpublished r e s u l t s c i t e d i n Hutchison, 1976 & 1978). Systemic DHTP, but not DHT, causes a s i g n i f i c a n t i n c r e a s e i n bow-cooing and chasing, but not i n nest-26 s o l i c i t i n g , while p l a i n DHT only induces chasing (Adkins-Regan, 1981). The c o n t r a d i c t i o n s i n these r e s u l t s can be accounted f o r , i n p a r t , by d i f f e r e n c e s i n the form of the hormone ad m i n i s t e r e d and i n the l e n g t h of the o b s e r v a t i o n p e r i o d s . Steimer & Hutchison (1981) have r e c e n t l y t e s t e d hypothalamic implants of 5/3DHT and found them to be t o t a l l y i n e f f e c t i v e . Androstenedione, the other major m e t a b o l i t e of T, has r e c e i v e d l e s s a t t e n t i o n . Saad & S i l v e r ( c i t e d i n S i l v e r et a l . , 1979) r e p o r t e d that subcutaneous implants of AE are as e f f e c t i v e as those of TP i n r e s t o r i n g both a g g r e s s i v e (bow-cooing, chasing) and n e s t - o r i e n t e d ( n e s t - s o l i c i t i n g ) c o u r t s h i p . P i e t r a s and Wenzel (1974) t r e a t e d c a s t r a t e d pigeons with AE and DHT and only observed bow-cooing i n the A E - t r e a t e d group. Cohen and Cheng (1982) have r e c e n t l y examined the c o u r t s h i p behaviour of male doves i n response to implants of T, 5aDHT, 5/3DHT, and E i n the nucleus i n t e r c o l l i c u l l a r i s ( I C o). T h i s r e g i o n has been shown to mediate v o c a l i z a t i o n s i n doves (Cohen, 1981) and to concentrate both E and T. The authors found that male nest - c o o i n g was s p e c i f i c a l l y induced by ICo implants. There was l i t t l e d i f f e r e n c e i n the responses to T, 5aDHT or E (5/3DHT was i n e f f e c t i v e ) , i n d i c a t i n g t h at a r o m a t i z a t i o n i s not e s s e n t i a l f o r ICo a c t i v a t e d n e s t - c o o i n g . The c o n t r o l of sexual behaviour i n male c h i c k s appears to be l e s s hormone-specific than i n the preceeding two s p e c i e s . J u v e n i l e c o p u l a t i o n has been r e p o r t e d i n c h i c k s t r e a t e d with AE, 5ametabolites (Young & Rogers, 1978) and 5/3metabolites ( B a l t h a z a r t & H i r s c h b e r g , 1979b). I t i s p o s s i b l e , of course, 27 that these s t e r o i d s s t i m u l a t e the r e l e a s e of endogenous androgen i n the c h i c k . I f , as suggested, metabolism p l a y s an important r o l e i n r e g u l a t i n g androgenic e f f e c t s , then the response of any t a r g e t t i s s u e w i l l be determined not only by the amount of T re a c h i n g i t , but a l s o by the metabolic a c t i v i t y of the t i s s u e i t s e l f . Of major importance to the q u e s t i o n s being addressed i n t h i s paper i s the p o s s i b i l i t y that androgen metabolism can be a l t e r e d by p h o t o p e r i o d . The number of s t u d i e s which have examined t h i s problem i s small and the r e s u l t s are o f t e n d i f f i c u l t to i n t e r p r e t due to d i f f e r e n c e s i n methodology. Table I summarizes c u r r e n t knowledge of the e f f e c t s of daylength on androgen c o n v e r s i o n by v a r i o u s t i s s u e s . A quick glance at the t a b l e i s enough to show that photoperiod i s , i n f a c t , an e f f e c t i v e r e g u l a t o r , and that i n most cases longer daylengths are a s s o c i a t e d with higher r a t e s of metabolism. In hamsters hypothalamic a r o m a t i z a t i o n i s g r e a t e r under long days (Horst, 1979). U n f o r t u n a t e l y , the e f f e c t of photoperiod on hypothalamic a r o m a t i z a t i o n i n b i r d s has not yet ..been examined. I t should a l s o be kept i n mind that most of the experiments l i s t e d i n v o l v e d g o n a d a l l y i n t a c t animals, and a r o m a t i z a t i o n i s known to be a f f e c t e d by f l u c t u a t i o n s i n endogenous s t e r o i d l e v e l s ( M a r t i n i , 1982). In summary, a r o m a t i z a t i o n has been shown to occur i n the a v i a n b r a i n , and most a v a i l a b l e evidence i n d i c a t e s that i t may be important i n the c o n t r o l of male sexual behaviour. In a d d i t i o n , although the i n f o r m a t i o n i s somewhat d i v e r s e and 28 Table I. The effect of photoperiod on androgen metabolism in various tissues in the male. Unless otherwise indicated, a l l animals were gonadally intact. SPECIES TISSUE METABOLITE EFFECT a r a t 'dwarf hypothalamus pituitary prostrate 'hypothalamus hamster 'amygdala 'hypothalamus 'amygdala chamster prostate plasma l i v e r ^ s t a r l i n g hypothalamus pituitary hyperstriaturn 5«mets "duck 9Japanese quail Japanese quail testes testes hypothalamus pituitary hyperstriaturn cloacal gland hypothalamus pituitary hyper s tr ia turn cloacal gland 5*mets AE 5«mets AE 5«mets 5*mets 5Bmets AE 5*mets 5Bmets AE 5«imets 5Bmets AE 5*mets T/AE 5«mets AE 5*mets 56mets AE 5«mets 5Bmets AE 5*mets 5fJmets AE 5«mets 5Bmets AE 5Bmets AE 5*mets 5Bmets AE 5Bmets AE 5«mets 5Bmets AE LI?«14L:10D>DD LL>14L:10D>DD 14L:10D>6L:10D 14L:10D>6L:10D 14L:10D>6L:10D 16L:8D>8L:16D 16L:8D>8L:16D 16L:8D>8L:16D 16L:8D>8L:16D 14L:10D>1L:23D May>Nov May>Nov May<Nov May<Nov May<Nov spring<fall May<Oct 16L>8L 16L>8L 16L<8L 16L>PL 20L>8L 20L>8L 20L<8L 20L>8L 20L< 8L Sources; a Shapiro et a l . , 1977 k Horst, 1979 - animals were castrated 6 wk before measurements intact c Callard & Solomon, 1980 - prostate and plasma - castrated 6 hr before measurements Frehn et a l . , 1974 - li v e r d Bottoni & Massa, 1980;. Bottoni & Massa, 1981 e Cardinali et a l . , 1971 f Horst & Paulke, 1977 9 Balthazart et a l . , 1979 h Massa et a l . , 1979 30 o c c a s i o n a l l y c o n t r a d i c t o r y , a general p a t t e r n emerges which suggests that androgen s y n t h e s i s and c o n v e r s i o n i s s t i m u l a t e d by exposure to long photoperiods. B. The E f f e c t i v e n e s s of V a r i o u s Androgen M e t a b o l i t e s i n  R e s t o r i n g Male Reproductive Behaviour j_. I n t r o d u c t i o n F l u c t u a t i o n s i n the r a t e of t e s t o s t e r o n e metabolism, such as those d e s c r i b e d above, o f f e r a p o s s i b l e e x p l a n a t i o n of the e f f e c t s of photoperiod on the responsiveness of male doves to t e s t o s t e r o n e treatment. If long photoperiods can a l t e r hypothalamic c o n v e r s i o n of t e s t o s t e r o n e , e i t h e r by i n c r e a s i n g the p r o d u c t i o n of e f f e c t i v e m e t a b o l i t e s such as E and AE, or d e c r e a s i n g the c o n v e r s i o n to i n e f f e c t i v e 5a and 5/3-reduced androgens, t h i s would l e a d to higher l e v e l s of " a c t i v e " hormone i n the hypothalamus and thus, p o s s i b l y , to g r e a t e r b e h a v i o u r a l a c t i v i t y . The involvement of t e s t o s t e r o n e metabolism i n the c o n t r o l of n e s t i n g a c t i v i t y i n doves presupposes that androgen m e t a b o l i t e s d i f f e r i n t h e i r a b i l i t y to s t i m u l a t e n e s t - b u i l d i n g . At the time t h i s experiment was designed, no data were a v a i l a b l e on the e f f e c t s of p e r i p h e r a l implants of the v a r i o u s m e t a b o l i t e s on dove c o u r t s h i p . In a d d i t i o n , most of the p r e v i o u s s t u d i e s were based on 3 to 30 minute o b s e r v a t i o n p e r i o d s and focussed e x c l u s i v e l y on e a r l y c o u r t s h i p a c t i v i t y . In the one study i n which behaviour was recorded over a p e r i o d of s e v e r a l days, i t was found that EB i s as e f f e c t i v e as TP f o r the i n d u c t i o n of 31 n e s t - b u i l d i n g (Martinez-Vargas,1974). The f o l l o w i n g p i l o t study was designed to determine the a b i l i t y of implants or i n j e c t i o n s of v a r i o u s T m e t a b o l i t e s to induce c o u r t s h i p and n e s t i n g a c t i v i t y over a 5-day p e r i o d . 2. M a t e r i a l s and Methods Twenty-one c a s t r a t e d male doves which had been used i n pre v i o u s experiments, but had r e c e i v e d no hormone treatments f o r at l e a s t 6 months, were f i r s t t e s t e d to ensure that they were r e p r o d u c t i v e l y i n a c t i v e , and were then p l a c e d i n i s o l a t i o n cages on 8L:16D f o r 4 weeks. Hormone treatment began one week p r i o r to t e s t i n g and conti n u e d throughout the t e s t p e r i o d . The hormones used were: TP ( t e s t o s t e r o n e p r o p i o n a t e ) , EB ( e s t r a d i o l benzoate), 5aDHT ( 5a-androstan-1 7/3-ol-3-one) , 5/3DHT (etiocholan-1 7/3-ol-3-one), and AE ( 4~androstene-3,17-dione). The b i r d s were d i v i d e d i n t o 8 groups as f o l l o w s : TPim 2.5mm implant n=2 EBim 5.0mm implant n=3 EBin j .2mg i n .2ml s a l i n e / d n=3 5aDHTim 5.0mm implant n=3 5aDHTinj .2mg i n .2ml s a l i n e / d n=2 5j3DHTim 5.0mm implant n=3 5|3DHTin j .2mg i n ,2ml s a l i n e / d n=3 AEin j .2mg i n .2ml s a l i n e / d n=2 B e h a v i o u r a l r e cords were made f o l l o w i n g the procedure 32 o u t l i n e d e a r l i e r , except that i n t h i s case only two 10-minute o b s e r v a t i o n p e r i o d s , at 9:30 and 11:30, were used. The males continued to be h e l d on an 8L:16D l i g h t c y c l e throughout the t e s t i n g p e r i o d . 3 i R e s u l t s F i g u r e 2 i l l u s t r a t e s the occurrence of male preening, c o u r t s h i p and n e s t - b u i l d i n g a c t i v i t y . Both of the b i r d s t r e a t e d with TP e x h i b i t e d c o u r t s h i p and n e s t - b u i l d i n g . EB f a i l e d to s t i m u l a t e bow-cooing, but was as e f f e c t i v e as t e s t o s t e r o n e i n terms of n e s t - s o l i c i t i n g , time spe n t - a t - n e s t , and a l l three measures of b u i l d i n g . 5aDHT-treatment r e s u l t e d i n low l e v e l s of n e s t - s o l i c i t i n g , while both 5/JDHT and AE were almost t o t a l l y i n e f f e c t i v e . 50DHT d i d induce n e s t - s o l i c i t i n g i n 3 males, but comparison with the 'male-at-nest' graph i n d i c a t e s that the d i s p l a y was never performed at the n e s t - s i t e . None of the three androgen m e t a b o l i t e s supported bow-cooing, and o n l y 1 of 6 5/3DHT, 1 of 5 5aDHT and 0 of 2 AE males e x h i b i t e d n e s t - b u i l d i n g . Table II i n d i c a t e s the number of p a i r s which d i s p l a y e d c o p u l a t o r y behaviour, female c o u r t s h i p , and female nest-b u i l d i n g . The high a g g r e s s i v e a c t i v i t y (based on chasing) and low l e v e l of c o u r t s h i p d i s p l a y e d by the 50DHT and AE males i s r e f l e c t e d i n the low l e v e l s of c o u r t s h i p seen i n t h e i r mates (2 of the 4 5/3DHT females and the 1 AE female which d i d c o u r t were only a c t i v e f o r a few minutes). 33 F i g u r e 2. The p r e - i n c u b a t i o n b e h a v i o u r of c a s t r a t e d doves t r e a t e d w i t h i n j e c t i o n s (.2 mg/d) or i m p l a n t s of e s t r a d i o l benzoate ( E B ) , 5 a d i h y d r o t e s t o s t e r o n e (5aDHT), 5 / 3 d i h y d r o t e s t o s t e r o n e (50DHT), a n d r o s t e n e d i o n e (Andro) or t e s t o s t e r o n e p r o p i o n a t e ( T P ) . Each l i n e r e p r e s e n t s t h e t o t a l number of e v e n t s or 15-second i n t e r v a l s i n w h i c h the a c t i v i t y o c c u r r e d , based on t e n 10-minute o b s e r v a t i o n p e r i o d s . 34 40-, 20-^ .2mg 5n .2mg 5mm .2 mg 5mm •2mg Z5rr EB 5« DH T 5B DHT ANDRO T P 2 5 0 -2 0 0 -150-O 100-Z Z 5 0 -LU ^ o-l—LU lLI III II I I I III U I • 2m g 5 mm >2mg 5mm .2 mg 5 mm -2 mg 2,5 m m EB 5«DHT 5 B DHT ANDRO TP M A L E AT NEST NEST SOLICITING BOW-COO in 5» O I in CD o 10 3 IO 3 to 3 ID in 3 3 to 3 ID i n 3 > z ro o 3 TO (O o tO —1 D 3 3 in O _1_ o o _JL_ In O in $» I to 3 to In 3 3 to 3 In 3 3 In O O O _ L _ i n O _ J _ o to 3 to 3 3 to 3 to i n 3 3 to Jj in 3 to Ol 3 to C5 o O O I in I m —i 3 —i 3 3 3 > > z to Z to 3 o 3 to to O O to to —1 i n — • In T3 3 3 3 3 i n _ l _ O -J STRING C A R R Y I N G NEST BUILDING 3 3 3 tO 3 ID Ul 3 Jo 3 • to O a X Cn —i 3 3 > z to o 3 TO to o to C» TJ 3 3 o o I o o to o o Cn 5» O I to 3 to in 3 3 K> 3 to o _1_ o _JL_ cn 3 3 tO Cn 3 to O o I Cn —1 3 3 > Z Jo o 3 to o JO —( In ,T3 3 3 in J» O I Cn tO 3 to In 3 3 ro 3 to In 3 3 X Cn 3 3 > z to o 3 to o {O —< Cn 3 3 to cn _ L _ 37 Table I I . Copulatory a c t i v i t y and female behaviour. Data represent the number of p a i r s d i s p l a y i n g each behaviour p a t t e r n at l e a s t once d u r i n g the 5-day t e s t p e r i o d . A l l b i r d s r e c e i v i n g the same hormone are grouped, r e g a r d l e s s of mode of a d m i n i s t r a t i o n . TP EB 5«(DHT 5j?DHT AE n= 2 6 5 6 ' 2 Copulatory a c t i v i t y begging b i l l i n g c r o u c h i n g mounting 1 0 1 1 1 0 0 0 4 2 2 1 2 1 0 0 2 1 1 1 Female behaviour n e s t - s o l i c i t i n g a t - n e s t n e s t - b u i l d i n g c a r r y i n g 2 2 0 0 6 6 3 1 5 5 3 1 4 4 1 0 1 1 1 0 4. D i s c u s s i o n The r e s u l t s seen here concur with those of other workers. Estrogen i s h i g h l y e f f e c t i v e when a d m i n i s t e r e d e i t h e r as implants or i n j e c t i o n s , and r e s u l t s i n a c y c l e of n e s t - o r i e n t e d behaviour which i s i n d i s t i n g u i s h a b l e from that seen u s i n g t e s t o s t e r o n e . The i n a b i l i t y of 5a and 50DHT to s t i m u l a t e bow-cooing and t h e i r low e f f i c a c y i n terms of n e s t - s o l i c i t i n g are i n agreement with the r e s u l t s of Hutchison (1978) and Saad et a l . ( c i t e d i n 3 8 Adkins-Regan, 1981). S e v e r a l c o m p l i c a t i n g f a c t o r s may be r e s p o n s i b l e f o r the lack of response to DHT. F i r s t , i t i s p o s s i b l e that slow r e l e a s e from implants or r a p i d removal from the c i r c u l a t i o n r e s u l t i n l i t t l e hormone reachin g the c e n t r a l c o n t r o l systems. B a l t h a z a r t & H i r s c h b e r g (1979a) measured the r e l e a s e of v a r i o u s androgens from s i l a s t i c implants both i n  v i t r o (15 days i n s a l i n e ) and in v i v o (18 days i n c h i c k s ) . They found that 50DHT, T and AE are r e l e a s e d q u i t e r a p i d l y , 5aDHT somewhat l e s s q u i c k l y and TP very s l o w l y . A s i m i l a r order of i n  v i v o r e l e a s e r a t e s was found i n Japanese q u a i l (Wada, 1982). However, Saad et a l . ( c i t e d i n Adkins-Regan, 1981) rep o r t e d h i g h plasma androgen c o n c e n t r a t i o n s i n 5aDHT-implanted doves, so low c i r c u l a t i n g l e v e l s are u n l i k e l y to account f o r the observed r e s u l t s . A f a i l u r e of DHT to r e s t o r e androgen receptor c o n c e n t r a t i o n s c o u l d a l s o c o n c e i v a b l y be p e r t i n e n t i n the present t e s t , which u t i l i z e d long-term c a s t r a t e s . Adkins-Regan' (1981) study, however, used c a s t r a t e s which had r e c e i v e d d a i l y i n j e c t i o n s of 100 ug TP f o r 2 weeks p r i o r to t e s t i n g and she s t i l l observed low responsiveness to 5aDHT. I n t e r p r e t i v e problems may a l s o a r i s e due to the comparison of n a t i v e androgens such as 5aDHT and 5/3DHT with e s t e r i f i e d TP and EB. E s t e r i f i c a t i o n g e n e r a l l y slows down the metabolism of a hormone, lengthening i t s b i o l o g i c a l a c t i v i t y (Feder, 1981c). The a b i l i t y of hypothalamic t e s t o s t e r o n e implants to induce c o u r t s h i p i n c a s t r a t e d male doves i s i n c r e a s e d i f TP i s used (Hutchison, 1971). However, the low b e h a v i o u r a l response to the 39 DHTs seen here, p a r t i c u l a r l y i n terms of n e s t - o r i e n t e d a c t i v i t y , i s u n l i k e l y to be the r e s u l t of using the f r e e a l c o h o l form f o r s e v e r a l reasons. F i r s t , as a l r e a d y mentioned, DHT-implanted b i r d s have high c i r c u l a t i n g androgen l e v e l s . Moreover, although i n j e c t i o n s of propionated 5aDHT are more potent than n a t i v e 5aDHT f o r s t i m u l a t i o n of e a r l y a g g r e s s i v e c o u r t s h i p (chasing and bow-cooing) they do not induce n e s t - s o l i c i t i n g (Adkins, 1981). And f i n a l l y , there i s evidence suggesting that e s t e r i f i c a t i o n may a c t u a l l y decrease the e f f i c a c y of DHT i n r a t s (Yahr, 1979) and doves (Hutchison, 1976). The observed l a c k of response to AE i s i n agreement with p r e v i o u s r e s u l t s based on the treatment of c a s t r a t e d pigeons ( P i e t r a s & Wenzel, 1974) but i n d i r e c t c o n f l i c t with the o b s e r v a t i o n s of Saad & S i l v e r (unpublished r e s u l t s c i t e d i n S i l v e r et a l . , 1979) who found implants of AE to be as e f f e c t i v e as TP i n c a s t r a t e d doves. T h i s hormone w i l l i n c r e a s e c o p u l a t i o n i n i n t a c t male q u a i l h e l d on short days i f a d m i n i s t e r e d as implants (Adkins et a l . , 1980) but not i f given as i n j e c t i o n s (Adkins, 1977), and i s r e l a t i v e l y i n e f f e c t i v e i n rodents i f given a f t e r a long p o s t - c a s t r a t i o n p e r i o d (Larsson, 1979) as was the case here. These r e s u l t s do not support the hypothesis that a r o m a t i z a t i o n i s an e s s e n t i a l step i n the c o n t r o l of behaviour i n doves, but the hig h l e v e l of response to EB, coupled with the low potency of the nonaromatizable DHTs, does i n d i c a t e that known androgen m e t a b o l i t e s d i f f e r i n t h e i r a b i l i t y to induce n e s t i n g behaviour and that changes i n r a t e s of androgen 40 co n v e r s i o n c o u l d thus c o n c e i v a b l y l e a d to d i f f e r e n c e s i n nest-b u i l d i n g . C. A r o m a t i z a t i o n and the E f f e c t of Daylength on the Nest- b u i l d i n g Behaviour of C a s t r a t e d Doves The i n t e r a c t i o n between daylength and m e t a b o l i t e - i n d u c t i o n of behaviour was examined by t e s t i n g the e f f e c t s of subcutaneous implants of TP, EB or combined TP + ATD (an aromatase i n h i b i t o r ) on n e s t i n g behaviour i n c a s t r a t e d males h e l d under d i f f e r e n t p h o t o p e r i o d s . The experiment was designed to t e s t the hyp o t h e s i s that the high l e v e l s of n e s t - b u i l d i n g which occur under long daylengths are the r e s u l t of i n c r e a s e d r a t e s of a r o m a t i z a t i o n . I f short-day b i r d s are l e s s a c t i v e due to low r a t e s of a r o m a t i z a t i o n , then I a n t i c i p a t e d t h a t c a s t r a t e d b i r d s t r e a t e d with EB would have high l e v e l s of c i r c u l a t i n g E and would e x h i b i t high l e v e l s of n e s t - b u i l d i n g on e i t h e r short or long days. In the same v e i n , i n t e r f e r e n c e with a r o m a t i z a t i o n by use of an aromatase b l o c k e r , would be expected to decrease b u i l d i n g , p a r t i c u l a r l y on long days. An a d d i t i o n a l t e s t was performed u t i l i z i n g i n j e c t i o n s of TP or TP + ATD to circumvent the p o s s i b i l i t y t h a t ATD i s not r e l e a s e d from s i l a s t i c implants r a p i d l y enough to e l i m i n a t e a l l a r o m a t i z a t i o n . S t u d i e s of the a n t a g o n i s t i c e f f e c t s of ATD implants on m a s c u l i n i z a t i o n i n neonatal mice and f e r r e t s (McEwen et a l . , 1979; Baum et a l . , 1983) and t e s t o s t e r o n e i n d u c t i o n of maternal behaviour i n female r a t s (Bridges & R u s s e l , 1982) i n d i c a t e that the aromatase b l o c k e r w i l l pass through s i l a s t i c , 41 but no i n f o r m a t i o n i s a v a i l a b l e on the r a t e of r e l e a s e . A f i n a l p i l o t study was performed using i n j e c t i o n s of ATD alone. ATD i s a s y n t h e t i c androgen, and i t might act as an androgen a g o n i s t i n b i r d s . j_. M a t e r i a l s and Methods a) EB, TP, and TP+ATD implants F i f t y - s i x e x p e r i m e n t a l l y naive male doves which had been sexed and given i n i t i a l breeding experience were brought i n weekly from the a v i a r i e s i n groups of seven and p l a c e d on a 16L:8D l i g h t c y c l e . They were then p r e t e s t e d , c a s t r a t e d and p o s t - t e s t e d . A f t e r 6 weeks indoors, h a l f were put on an 8L:16D c y c l e . Seven weeks l a t e r each b i r d was given a s i l a s t i c implant c o n t a i n i n g e i t h e r 5mm e s t r a d i o l benzoate (EB, Sigma) alone, 2.5mm t e s t o s t e r o n e propionate (TP, Sigma) alone, or 2.5 mm TP + 5 mm 1,4,6-androstatriene-3,17-dione (ATD, S t e r a l o i d s ) separated by a small wooden p l u g . F i v e d a i l y s e t s of four 15-minute b e h a v i o u r a l o b s e r v a t i o n s were made du r i n g the f o l l o w i n g week, a f t e r which the hormone implants were removed. b) TP and TP+ATD i n j e c t i o n s The b i r d s used i n the p r e v i o u s experiment (a, above) were kept on the same l i g h t c y c l e s f o r another 14 weeks. S i x t e e n b i r d s on each daylength were then randomly d i v i d e d i n t o two groups: TP - b i r d s were i n j e c t e d d a i l y with 0.2mg TP i n 0.05 ml 42 25% EtOH at 9:00 and with 0.1 ml EtOH at 9:00 and 16:00, and TP + ATD - b i r d s were i n j e c t e d d a i l y with 0.2 mg TP i n 0.05 ml 25% EtOH at 9:00 and with 0.25 mg ATD i n 0.1 ml EtOH at 9:00 and 16:00. The i n j e c t i o n s continued f o r two weeks. Be h a v i o u r a l r e c o r d i n g s were made durin g the second week. c) Androgenic e f f e c t s of ATD Ten c a s t r a t e s which had not been used i n any experiments f o r at l e a s t s i x months were h e l d on 16L:8D f o r 3 weeks, then p a i r e d with hormone-treated females over a 5-day t e s t p e r i o d . Two 10-minute r e c o r d i n g s were made at 9:30 and 11:30. F i v e b i r d s were i n j e c t e d d a i l y at 9:00 with 0.5 mg ATD i n 0.1 ml 25% EtOH and f i v e with the v e h i c l e alone, beginning one week before the t e s t p e r i o d and c o n t i n u i n g to the 5th day of o b s e r v a t i o n s . 2. R e s u l t s a) EB, TP, and TP+ATD implants I n d i v i d u a l l e v e l s of a c t i v i t y i n t h i s t e s t v a r i e d so g r e a t l y that few s i g n i f i c a n t d i f f e r e n c e s were found between hormone or photoperiod groups, although s e v e r a l trends were apparent. K r u s k a l - W a l l i s t e s t s i n d i c a t e d s i g n i f i c a n t d i f f e r e n c e s among groups f o r c h a s i n g , bow-cooing, n e s t - b u i l d i n g , b i l l i n g , and mounting. Mann-Whitney U - t e s t s were used to compare long and short-day groups given the same hormone treatment, and TP vs EB or TP + ATD groups h e l d on the same 43 l i g h t c y c l e s . As expected on the b a s i s of e a r l i e r r e s u l t s , both TP and EB- t r e a t e d males e x h i b i t e d h i g h l e v e l s of n e s t - o r i e n t e d behaviour, while chasing and bow-cooing were only d i s p l a y e d by TP b i r d s ( f i g . 3). The aromatase i n h i b i t o r ATD d i d not have an a n t a g o n i s t i c e f f e c t on the male a c t i v i t i e s recorded, and i n f a c t was a s s o c i a t e d with mean l e v e l s of n e s t - s o l i c i t i n g which were c o n s i d e r a b l y higher than those d i s p l a y e d i n males t r e a t e d with TP alone. L e v e l s of female behaviour and c o p u l a t o r y a c t i v i t y were s i m i l a r i n a l l groups ( t a b l e I I I ) . Short daylengths were a s s o c i a t e d with i n c r e a s e d bow-cooing i n the TP and TP+ATD t r e a t e d groups and s l i g h t l y decreased nest-s o l i c i t i n g i n the EB and TP+ATD groups. In terms of nest-b u i l d i n g , the mean l e v e l s of a c t i v i t y seen i n b i r d s exposed to long photoperiods were twice as high as those of short-day b i r d s , f o r a l l three hormone treatments ( f i g . 4). The average r e l e a s e r a t e of TP was 0.59 mg/d, based on the weight before and a f t e r use. EB was r e l e a s e d more slowly (0.40 mg/d), d e s p i t e the f a c t t h a t the implant was twice as l a r g e . The combined TP + ATD implants l o s t an average of 0.77 mg/d, i n d i c a t i n g r e l e a s e of approximately 0.18 mg/d of the aromatase b l o c k e r . b) TP and TP+ATD i n j e c t i o n s R e s u l t s from males i n j e c t e d with TP or TP + ATD are shown i n t a b l e s IV and V and i n f i g . 5. The observed l e v e l s of r e p r o d u c t i v e d i s p l a y s were f a r below those seen e a r l i e r i n the 44 F i g u r e 3. The e f f e c t of subcutaneous i m p l a n t s of t e s t o s t e r o n e p r o p i o n a t e ( T P ) , e s t r a d i o l benzoate ( E B ) , or TP + t h e aromatase i n h i b i t o r ATD (ATD) on t h e c o u r t s h i p b e h a v i o u r of l o n g - and s h o r t - d a y c a s t r a t e s . The v a l u e s r e p r e s e n t t h e mean l e v e l of a c t i v i t y based on t o t a l s f o r the 5-day t e s t p e r i o d . N=8 f o r a l l g r o u p s . L o n g - d a y b i r d s Short-day b i r d s a = s i g n i f i c a n t d i f f e r e n c e from s h o r t - d a y TP group based on Mann-Whitney U - t e s t , 2 - t a i l e d , p<.05 N E S T - S O L I C I T BOW - C O O C H A S E assays* + > to I _l O -L ^ ^ ^ ^ 4 6 T a b l e I I I . C o p u l a t o r y a c t i v i t y and female b e h a v i o u r f o r p a i r s i n which the males were c a s t r a t e d and i m p l a n t e d w i t h t e s t o s t e r o n e p r o p i o n a t e ( T P ) , e s t r a d i o l b e n z o a t e ( E B ) , or TP + aromatase i n h i b i t o r (TP + ATD), w h i l e b e i n g h e l d on l o n g (LD) or s h o r t (SD) days. The v a l u e s r e p r e s e n t the mean and r a n g e , based on the t o t a l number o f i n t e r v a l s o r o f e v e n t s o v e r t he 5-day t e s t p e r i o d . n=8 f o r a l l g r o u p s . TP EB TP + ATD LD SD LD SD LD SD c o p u l a t i on beg 1 . 8 ( 0 - 5 ) 1 0 . 8 ( 0 - 3 0 ) ( 0 - 3 2 ) 1 0 . 6 ( 0 - 3 1 * ) 12 . 1 ( 0 - 3 0 ) 12 . 0 ( 1 -31 ) b i l l a 0.I4 ( 0 - 2 ) 2 . 5 ( 0 - 1 0 ) 0 . 6 ( 0 - 1 . ) 1 . 6 ( 0 - 1 * ) 6 . 3 * ( 0 - 1 3 ) 5 . 3 ( 0 - 2 0 ) c r o u c h 0 . 5 ( 0 - 2 ) 3 . 1 ( 0 - 9 ) 2 . 0 ( 0 - 1 3 ) 3 . 8 ( 0 - 1 1 ) 3 . 8 ( 0 - 1 2 ) 1. .1* ( 0 - 1 1 ) mount* 0 . 1 ( 0 - 1 ) 1 . 9 * ( 0 - l t ) 1 . 0 ( 0 - 8 ) 1 . 6 ( 0 - 5 ) 0 . 6 ( 0 - 1 * ) 1 . 6 ( o-i*) o b e h a v i o u r n e s t - s o l i c i t 6 l l * U 0 2 - 8 3 1 * ) 6 0 6 ( 2 3 0 - 1 0 9 2 ) 5 5 5 ( 2 9 3 - 7 7 0 ) 1*70 ( 2 5 3 - 7 8 1 * ) 5 2 2 ( 2 9 7 - 6 1 9 ) 3 9 5 ( 1 7 7 - 5 6 7 ) a t - n e s t 1 0 0 5 ( 7 5 0 - 1 1 7 2 ) 7 6 7 ( 2 1 2 - 1 1 1 4 6 ) 8 8 6 ( 2 8 6 - 1 1 6 1 ) 801* ( 5 1 3 - 1 0 8 9 ) 8 3 7 ( 7 1 * 6 - 9 5 1 ) 6U 9 ( 1 * 1 5 - 1 1 2 7 ) b u i l d i n g 6 . 0 ( 0 - 1 3 ) 2 . 3 ( 0 - 8 ) 7 . 8 (0-1*1*) 3 . 9 ( 0 - 1 1 * ) 6 . 6 ( 0 - 2 5 ) 6 . 5 ( 1 - 2 2 ) c a r r y i n g 1 . 8 ( 0 - 7 ) 1.1* ( 0 - 6 ) 2 . 6 ( 0 - 1 2 ) 1 . 0 ( 0 - 1 . ) 1 . 2 ( 0 - 1 . ) 2 . 6 ( 0 - 1 5 ) a. c r o u c h 2 . 3 ( 0 - 1 1 ) 1 . 8 ( 0 - 6 ) 0.1* ( 0 - 2 ) 0 . 1 ( 0 - 1 ) 2 . 9 ( 0 - 8 ) 3 . 0 ( 0 - 1 1 ) a p . 0 5 K r u s k a l - W a l l i s AN OVA * s i g n i f i c a n t l y d i f f e r e n t from TP-LD, based on 2 - t a i l e d U - t e s t , p .05 47 F i g u r e 4. The e f f e c t of subcutaneous i m p l a n t s of t e s t o s t e r o n e p r o p i o n a t e ( T P ) , e s t r a d i o l benzoate ( E B ) , or TP + t h e aromatase i n h i b i t o r ATD (ATD) on the n e s t -b u i l d i n g b e h a v i o u r of l o n g - and s h o r t - d a y c a s t r a t e s . The v a l u e s r e p r e s e n t t h e mean l e v e l of a c t i v i t y based on t o t a l s f o r t h e 5-day t e s t p e r i o d . N=8 f o r a l l g r o u p s . Long-day S h o r t - d a y S T R I N G ( pieces/ C A R RYING \even ts) N E S T - BU I LD I NG (intervals) 49 Table IV. The courtship a c t i v i t y of castrated male doves injected with testosterone propionate (TP) or TP + ATD (an aromatase in h i b i t o r ) while being held on long or short photoperiods. The values represent the means and ranges based on the t o t a l number of 15-second in t e r v a l s , or events over the 5-day test period. LONG DAY SHORT DAY TP n=8 ATD n=8 TP n=8 ATD n=8 Male courtship chase 6.3 (0-15) 7.3 (0-33) 6.6 (1-14) 4.4 (0-15) bow-coo 0.4 (0-2) 0.3 (0-1) 0.4 (0-3) 0.0 (0-0) s o l i c i t 33 (0-82) 19 (0-91) 39 (0-195) 49 (0-287) at-nest 37 (0-80) 18 (0-62) 44 (0-228) 44 (0-289) '50 T a b l e V. The e f f e c t of t e s t o s t e r o n e p r o p i o n a t e (TP) or TP + ATD (ATD) i n j e c t i o n s on c o p u l a t o r y a c t i v i t y of c a s t r a t e s and on the b e h a v i o u r of t h e i r mates. The v a l u e s r e p r e s e n t the means and ranges based on t h e t o t a l number of 15-second i n t e r v a l s or the t o t a l number of e v e n t s over t h e 5-day t e s t p e r i o d . LONG DAY SHORT DAY TP n=8 ATD n=8 TP n=8 ATD n=8 Copulatory a c t i v i t y beg 4.0 (0-17) 4.1 (0-13) 6.6 (0-37) 7.6 (0-18) b i l l 0.3 (0-2) 0.8 (0-4) 2.1 (0-15) 2.2 (0-9) crouch 1.0 (0-7) 1.0 (0-3) 3.1 (0-21) 2.6 (0-6) mount 0.4 (0-3) 0.3 (0-1) 0.3 (0-1) 0.9 (0-3) Female behaviour a.crouch (0-8) (0-9) (0-8) (0-4) s o l i c i t 457 (117-933) 291 (0-850) 506 430 (9-1116) (154-786) at-nest 656 (160-1138) 289 (0-886) 587 (2-1112) 493 (89-1170) nest-build 7.6 (0-33) 1.6 (0-11) 1.4 (0-5) 2.7 (0-12) carry 3.1 (0-12) 0.9 (0-6) 0.5 (0-2) 0.6 (0-5) 52 F i g u r e 5. The e f f e c t of d a i l y i n j e c t i o n s of t e s t o s t e r o n e p r o p i o n a t e ( T P ) , or TP + t h e aromatase i n h i b i t o r ATD (ATD) on the n e s t - b u i l d i n g b e h a v i o u r of l o n g - and s h o r t -day c a s t r a t e s . The v a l u e s r e p r e s e n t the mean l e v e l of a c t i v i t y based on t o t a l s f o r the 5-day t e s t p e r i o d . N=8 f o r a l l groups. Long-day t e s t o s t e r o n e t r e a t e d b i r d s S h o r t - d a y t e s t o s t e r o n e t r e a t e d b i r d s Long-day t e s t o s t e r o n e + ATD t r e a t e d b i r d s S h o r t - d a y t e s t o s t e r o n e + ATD t r e a t e d b i r d s S T R ING C A R R Y N E S T - B U I L D I N G 54 hormone-implanted b i r d s . As b e f o r e , there was a t r e n d (not s i g n i f i c a n t ) towards decreased n e s t - b u i l d i n g on short days, but in a d d i t i o n , i n t h i s case ATD-treatment seemed to i n t e r f e r e with the n e s t i n g response i n b i r d s h e l d on long daylengths. c) Androgenic e f f e c t s of ATD ATD alone d i d not induce a g g r e s s i v e c o u r t s h i p ( c h a s i n g , bow-cooing) or n e s t - b u i l d i n g , even though the females p a i r e d with the ATD-males were r e l a t i v e l y a c t i v e ( t a b l e V I ) . The aromatase i n h i b i t o r d i d cause an i n c r e a s e i n n e s t - s o l i c i t i n g and in the time spent at the nest, although the r e s u l t i n g values were very low. 3. D i s c u s s i o n E s t r a d i o l benzoate i s c l e a r l y as potent a s t i m u l a t o r of n e s t - s o l i c i t i n g and n e s t - b u i l d i n g as t e s t o s t e r o n e p r o p i o n a t e . In f a c t , i t i s l i k e l y t h a t e s t r a d i o l i s the more e f f e c t i v e of the two, s i n c e i t was being r e l e a s e d from the implants at a slower r a t e . There are a number of ways of i n t e r p r e t i n g the male dove's responsiveness to estrogen. I t i s p o s s i b l e that the response to e s t r a d i o l i s b i o l o g i c a l l y unimportant, that i t i s simply a pha r m a c o l o g i c a l e f f e c t (Gorzalka et a l . , 1975). Estrogen may be a c t i n g v i a the r e l e a s e of a d r e n a l androgen, although t h i s seems q u e s t i o n a b l e i n view of the f a c t t h a t there i s no evidence of androgen p r o d u c t i o n by the h e a l t h y avian a d r e n a l (Ljunngren, Table VI. The courtship and nesting a c t i v i t y of castrated male doves injected with 0.5 mg/d ATD, or with the vehicle (control), and the behaviour of th e i r mates. The values represent the mean, median and range, based on the t o t a l s for f i v e 10-minute observations. n=5 for both groups. CONTROL ADT 0) tn • C c IX a) 10 IX IB male courtship e H B U chase/peck 1.4 0 (0-4) 0.8 0 (0-3) bow-coo 0.2 0 (0-1) 0 0 (0) n e s t - s o l i c i t i n g 0.8 0 (0-2) 32.6 10 (0-83) at-nest 4.2 0 (0-21) 35.2 29 (0-72) male nest-building nest-building 1.2 0 (0-5) 1.4 0 (0-5) carrying 0 0 (0) 0.2 0 (0-1) s t r i n g 5.4 1 (0-24) 9.8 10 (0-25) female a c t i v i t y n e s t - s o l i c i t i n g 132 153 (32-181) 175 168 (44-35 at-nest 98 83 (0-198) 146 162 (6-321 ne s t-bu i l d ing 1.6 2 (0-3) 3.6 0 (0-16) carrying 0.2 0 (0-1) 1.2 0 (0-6) 56 1969). Estrogen may a l s o i n t e r a c t with c e n t r a l androgen r e c e p t o r s , but again t h i s i s probably not a v a l i d e x p l a n a t i o n s i n c e e s t r o g e n - s p e c i f i c r e c e p t o r s have been found i n the c e n t r a l t i s s u e s of s e v e r a l b i r d s p e c i e s , i n c l u d i n g doves (Martinez-Vargas, et a l . , 1976; Steimer & Hutchison, 1981). I t seems probable, then, that estrogen p l a y s a r o l e i n the normal c o n t r o l of male a c t i v i t y , p a r t i c u l a r l y i n l i g h t of the known uptake of E by the dove b r a i n (Martinez-Vargas et a l . , 1976). The presence of estrogen c o u l d be due e i t h e r to p e r i p h e r a l p r o d u c t i o n , or to a r o m a t i z a t i o n i n c e n t r a l t i s s u e s . Korenbrot et a l . (1974) f a i l e d to d e t e c t e s t r a d i o l i n the plasma of male doves, u s i n g a lower l i m i t of 25pg/ml. Other r e s e a r c h e r s have r e p o r t e d the presence of estrone (89pg/ml) and e s t r a d i o l (85pg/ml) i n the plasma of the c l o s e l y r e l a t e d c o l l a r e d dove, S t r e p t o p e l i a decaocto (Peczeley & Pethos, 1979) and i n the pigeon (16pg e s t a d i o l / m l , Haase et a l . , 1976) but the s i g n i f i c a n c e of such low l e v e l s of immunoreceptive substances i s not c l e a r . Observations of c e n t r a l aromatase a c t i v i t y (Steimer & Hutchison, 1980) i m p l i c a t e c e n t r a l androgen metabolism as the most l i k e l y source of e s t r o g e n . The f a i l u r e of ATD implants to i n t e r f e r e with the e f f e c t of exogenous t e s t o s t e r o n e c o u l d be the r e s u l t of inadequate l e v e l s of i n h i b i t o r reaching the b r a i n . At f i r s t g lance, r e s u l t s based on ATD i n j e c t i o n s support t h i s i d e a . However, the males i n t h i s t e s t were unresponsive compared with t h e i r e a r l i e r a c t i v i t y f o l l o w i n g i m p l a n t a t i o n of TP. The most obvious reason a c c o u n t i n g f o r the l a c k of response i s the i n a b i l i t y of TP 57 i n j e c t i o n s to r e s t o r e r e p r o d u c t i v e a c t i v i t y i n long-term c a s t r a t e s . The responsiveness of male doves to t e s t o s t e r o n e d e c l i n e s with i n c r e a s i n g time s i n c e c a s t r a t i o n , and priming with high l e v e l s of t e s t o s t e r o n e i s needed to r e i n s t a t e f u l l a c t i v i t y (Hutchison, 1974). Implants which provide continuous r e l e a s e of the hormone are more e f f e c t i v e than i n j e c t i o n s , and I have observed long-term c a s t r a t e s court only two days a f t e r i m p l a n t a t i o n . I t i s l i k e l y t h a t 7 days of T i n j e c t i o n s were incap a b l e of r e s t o r i n g f u l l responsiveness i n the present s i t u a t i o n . Of p a r t i c u l a r i n t e r e s t here i s the f a c t t h a t u n l i k e the androgen r e c e p t o r , estrogen r e c e p t o r s ( i n females, at l e a s t ) do not r a p i d l y disappear f o l l o w i n g gonadectomy (Muldoon, 1980a). If t e s t o s t e r o n e a c t s as a r e s u l t of uptake by both androgen and estrogen ( f o l l o w i n g a r o m a t i z a t i o n ) r e c e p t o r s , then perhaps the e f f e c t of ATD i s masked i n the implant s i t u a t i o n due to h i g h androgen uptake, but becomes apparent when androgen receptor c o n c e n t r a t i o n i s low and most uptake i s v i a a r o m a t i z a t i o n and estrogen r e c e p t o r uptake. The f a c t that ATD, which i s a s y n t h e t i c androgen, can induce c o p u l a t o r y a c t i v i t y i n c a s t r a t e d male r a t s (Landau, 1980) and causes s l i g h t i n c r e a s e s i n n e s t - s o l i c i t i n g i n c a s t r a t e d doves suggests that i t i s probably weakly androgenic. I f t h i s i s the case then i t i s d i f f i c u l t to i n t e r p r e t apparent i n h i b i t o r y e f f e c t s of ATD s i n c e these may be due to c o m p e t i t i v e i n h i b i t i o n of TP at the r e c e p t o r l e v e l (Landau, 1980) r a t h e r than to decreased a r o m a t i z a t i o n . The r e s u l t s of the major experiment d i d not support the 58 hypothesis that photoperiod a l t e r s male n e s t - b u i l d i n g a c t i v i t y as a r e s u l t of changes i n t e s t o s t e r o n e a r o m a t i z a t i o n . Mean values f o r a l l three measures of n e s t - b u i l d i n g were twice as great under long days as under short ones i n a l l three hormone-treatment groups. N e i t h e r EB nor concurrent TP and ATD treatment, both of which would be expected to e l i m i n a t e d i f f e r e n c e s i n r a t e s of a r o m a t i z a t i o n , were capable of overcoming the e f f e c t of photoperiod. In a d d i t i o n , the decreased responsiveness to EB as w e l l as to TP under short daylengths suggests that photoperiod does not a c t through i n c r e a s e d metabolic i n a c t i v a t i o n of t e s t o s t e r o n e , s i n c e i t seems u n l i k e l y t h a t EB would be s i m i l a r l y a f f e c t e d . Despite the lack of s t a t i s t i c a l s i g n i f i c a n c e the data support the c o n c l u s i o n that long photoperiods a f f e c t responsiveness to estrogen as w e l l as androgen, i n d i c a t i n g that a l t e r e d a r o m a t i z a t i o n i s not an e s s e n t i a l f a c t o r , and that daylength a c t s independently of the type of hormone s t i m u l a t i o n being used. 4. Summary 1) In the i n i t i a l p i l o t study i t was shown that e s t r a d i o l induces male c o u r t s h i p and n e s t - b u i l d i n g i n c a s t r a t e d males, but u n l i k e t e s t o s t e r o n e does not s t i m u l a t e bow-c o o i n g . A few of the b i r d s t r e a t e d with 5a and 50 DHT performed n e s t - s o l i c i t i n g > but the response was much l e s s than t h a t seen with EB or TP. 59 2) B i r d s implanted with EB f a i l e d to e x h i b i t a g g r e s s i v e c o u r t s h i p , but were as a c t i v e as TP-implanted males i n terms of n e s t - o r i e n t e d c o u r t s h i p and n e s t - b u i l d i n g . Concurrent TP and ATD treatment was a s s o c i a t e d with higher l e v e l s of c o u r t s h i p , but had no e f f e c t on n e s t - b u i l d i n g . L e v e l s of male n e s t i n g were somewhat higher under long days than under short ones i n b i r d s i n j e c t e d with TP and TP + ATD, but o v e r a l l a c t i v i t y was very low. 3) ATD alone r e s u l t s i n low l e v e l s of n e s t - s o l i c i t i n g , and may be weakly androgenic. 4) Measures of c o p u l a t o r y a c t i v i t y and female behaviour showed l i t t l e v a r i a t i o n among the experimental groups. 5) There were no c o n s i s t e n t e f f e c t s of photoperiod on any a c t i v i t i e s other than n e s t - b u i l d i n g . Long-day b i r d s performed more n e s t - b u i l d i n g than t h e i r short-day c o u n t e r p a r t s , r e g a r d l e s s of the type of hormone treatment, i n d i c a t i n g t h at d i f f e r e n c e s i n r a t e s of a r o m a t i z a t i o n are not r e s p o n s i b l e f o r the i n f l u e n c e of photoperiod on n e s t -b u i l d i n g . 60 IV. THE EFFECT OF LUTEINIZING HORMONE RELEASING HORMONE AND PHOTOPERIOD ON NEST-BUILDING IN MALE DOVES A. I n t r o d u c t i o n The s t i m u l a t o r y e f f e c t of long daylengths on the avian p i t u i t a r y - g o n a d a x i s i s g e n e r a l l y assumed to r e f l e c t an inc r e a s e i n the pr o d u c t i o n and r e l e a s e of l u t e i n i z i n g hormone r e l e a s i n g hormone (LHRH) by the hypothalamus. D i r e c t evidence of such changes i s sparse: gonadotropic- r e l e a s i n g a c t i v i t y of q u a i l hypothalami i s in c r e a s e d by exposure to long days ( B i c k n e l l & F o l l e t t , 1977) but c i r c u l a t i n g l e v e l s have not yet been measured (Jackson, 1979). N e v e r t h e l e s s , the l a r g e i n c r e a s e s i n LH and FSH c o n c e n t r a t i o n s seen f o l l o w i n g p h o t o s t i m u l a t i o n are probably i n d i c a t i v e of i n c r e a s e d LHRH r a t h e r than an i n c r e a s e i n p i t u i t a r y responsiveness to the hypothalamic hormone, s i n c e p h o t o p e r i o d has l i t t l e e f f e c t on the a b i l i t y of the decapeptide t o s t i m u l a t e gonadotropin r e l e a s e ( F o l l e t t & Robinson, 1980). LHRH i s found i n s e v e r a l b r a i n regions o u t s i d e of the hypothalamus (Jackson, 1979; Weindl et a l . , 1982), suggesting t h a t i t may serve f u n c t i o n s other than the c o n t r o l of p i t u i t a r y hormone p r o d u c t i o n . Hypophysectomy i n c r e a s e s the l o r d o s i s response to EB treatment i n ov a r i e c t o m i z e d female r a t s , and i t has been proposed that the change i n behaviour i s the r e s u l t of in c r e a s e d l e v e l s of LHRH (Crowley et a l . , 1976). S e v e r a l s t u d i e s have now shown that subcutaneous i n j e c t i o n s or i n t r a c r a n i a l i n f u s i o n of s y n t h e t i c LHRH w i l l i n c r e a s e l o r d o s i s i n estrogen-primed o v a r i e c t o m i z e d female r a t s (Moss & McCann, 61 1973; Moss & McCann, 1975; Dudley & Moss, 1976; Foreman & Moss, 1977). The response i s not due to changes i n gonadotropin r e l e a s e , s i n c e i t cannot be mimicked with LH or FSH treatment (Moss & McCann, 1975) and i t i s not e l i m i n a t e d by hypophysectomy ( P f a f f , 1973). The g e n e r a l i t y of t h i s e f f e c t has yet to be e s t a b l i s h e d . Although systemic LHRH i s e f f e c t i v e i n EB-primed female mice (Luttge & Sheets, 1977) and doves (Cheng, 1977) i t has only a s l i g h t e f f e c t i n the guinea p i g , and i s not a c t i v e i n the female hamster (unpublished r e s u l t s c i t e d i n C a r t e r & Davis, 1977). The present study was undertaken i n order to determine whether changes i n LHRH l e v e l s a r i s i n g through exposure to d i f f e r e n t daylengths c o u l d account f o r the p h o t o p e r i o d i c e f f e c t s on male n e s t - b u i l d i n g i n doves observed i n the e a r l i e r experiments. B. M a t e r i a l s and Methods E x p e r i m e n t a l l y naive mature male r i n g doves were h e l d i n v i s u a l i s o l a t i o n on a photoperiod of 6L:18D f o r 8 weeks. They were c a s t r a t e d as d e s c r i b e d e a r l i e r and 2 weeks l a t e r given subcutaneous androgen implants." The implants c o n t a i n e d approximately 8mg of t e s t o s t e r o n e p r o p i o n a t e (TP) (Sigma) which was r e l e a s e d at an average r a t e of 0.4 mg/day, based on weights before and a f t e r use. The males were then p l a c e d on long (16L:8D) or short (8L:16D) photoperiods f o r 7 weeks, at which time the implants, now empty, were r e p l a c e d with f r e s h ones. The f o l l o w i n g week the males were p a i r e d with females and t h e i r 62 behaviour recorded. H a l f of the males on each photoperiod r e c e i v e d d a i l y subcutaneous i n j e c t i o n s at 9:00am of 20 ug s y n t h e t i c LHRH (Ayerst L a b o r a t o r i e s ) i n 0.1ml s a l i n e and h a l f r e c e i v e d 0.1 ml s a l i n e o n l y , beginning the day before the f i r s t o b s e r v a t i o n . C. R e s u l t s There were 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 female c o u r t s h i p a c t i v i t y (female n e s t - s o l i c i t i n g ; female-at-nest) or i n l e v e l s of c o p u l a t o r y d i s p l a y s (begging; b i l l i n g ; female crouch; mounting) between e i t h e r photoperiod or hormone groups, whether these were examined as d a i l y scores or t o t a l s f o r the 5-day t e s t p e r i o d ( t a b l e V I I ) . Treatment with LHRH had no s i g n i f i c a n t e f f e c t on male c o u r t s h i p or n e s t - b u i l d i n g under e i t h e r p hotoperiod, based on the 5-day t o t a l s ( t a b l e V I I I ) , comparisons at 4 d i f f e r e n t times of day, or d a i l y s c o r e s . LD males performed more n e s t - b u i l d i n g than SD males i n both hormone and s a l i n e - t r e a t e d groups ( f i g , 6). In a d d i t i o n , long-day LHRH-treated b i r d s d i s p l a y e d higher l e v e l s of chasing and bow-cooing ( t a b l e V I I I ) . Although the t o t a l amount of time spent by males n e s t - s o l i c i t i n g and s i t t i n g at the nest over the 5-day p e r i o d d i d not d i f f e r , LD males d i d d i s p l a y s i g n i f i c a n t l y higher l e v e l s of both these a c t i v i t i e s on days 1 and 2 i n the s a l i n e - t r e a t e d groups (p<.05, Mann-Whitney U - t e s t , 2 - t a i l e d ) . 63 T a b l e V I I . The e f f e c t of LHRH i n j e c t i o n s on t h e c o p u l a t o r y a c t i v i t y of T P - i m p l a n t e d l o n g - and s h o r t - d a y c a s t r a t e s and t h e b e h a v i o u r o f t h e i r mates. The v a l u e s r e p r e s e n t the means and ranges based on the t o t a l number of 15-second i n t e r v a l s o r the t o t a l number of e v e n t s over t h e 5-day t e s t p e r i o d . 64 LONG DAY SHORT DAY Control n=10 LHRH n=10 Control n=10 LHRH n=9 Copulatory a c t i v i t y beg 3.8 (0-22) 6.5 (0-12) 7.7 (0-19) 4.6 (0-30) b i l l 2.6 (0-18) 1.1 (0-6) 3.7 (0-14) 2.1 (0-12) crouch 2.7 (0-16) 2.3 (0-9) 4.1 (0-15) 2.1 (0-11) mount 0.3 (0-1) 0.7 (0-3) 1.2 (0-4) 0.6 (0-2) Female behaviour a.crouch 3.2 (0-8) 2.9 (0-9) 2.0 (0-8) 1.8 (0-4) s o l i c i t 361 242 (133-961) (48-431) 354 248 (156-590) (43-671) at-nest 620 568 (214-1098) (136-961) 596 (37-1042) 369 (20-930) nest-bu i l d 3.4 (0-14) 5.8 (0-26) 3.0 (0-10) 2.6 (0-14) carry 0.3 (0-2) 2.2 (0-14) 0.0 (0-0) 1.3 (0-4) 65 T a b l e V I I I . The e f f e c t o f LHRH i n j e c t i o n s on the c o u r t s h i p a c t i v i t y o f l o n g - and s h o r t - d a y T P - i m p l a n t e d c a s t r a t e s . * — p<.02 long-day v s s h o r t - d a y , U - t e s t , 2 - t a i l e d LONG DAY SHORT DAY C o n t r o l n=10 LHRH n=10 C o n t r o l n=10 LHRH n=9 Male c o u r t s h i p chase 11.0 (3-36) 13.1 (3-23) 6.5 (2-18) *5.0 (1-13) bow-coo 7.4 (0-22) 12.0 (0-27) 3.5 (0-13) *0.8 (0-3) s o l i c i t 437 (267-689) 448 (249-707) 338 (169-622) 383 (127-805) a t - n e s t 455 (202-711) 405 (232-601) 330 (115-571) 283 (21-796) 66 F i g u r e 6. T h e e f f e c t o f d a y l e n g t h a n d LHRH i n j e c t i o n s on t h e n e s t - b u i l d i n g b e h a v i o u r o f T P - i m p l a n t e d c a s t r a t e d d o v e s . T h e v a l u e s r e p r e s e n t t h e mean l e v e l o f a c t i v i t y , b a s e d on t h e t o t a l s f o r t h e 5 - d a y t e s t p e r i o d . L C l o n g - d a y c o n t r o l LRH l o n g - d a y LHRH • SC s h o r t - d a y c o n t r o l SS= SRH s h o r t - d a y LHRH a p < . 0 5 l o n g - d a y v s s h o r t - d a y , 2 - t a i l e d U - t e s t S T R I N G ( pieces/ o o _ l _ O O o o _ l •. o o !L J3 o I M CO o O llllllllllllllllil C A R R Y I N G (events) N E S T - B U I L D ING (intervals) • o u o U t o o o o o o o o 1 1 1 1 1 1 1 lllllllllllll 6 8 D. D i s c u s s i o n The present data c o n f i r m e a r l i e r s t u d i e s (McDonald & L i l e y , 1978) which suggested that long photoperiods s t i m u l a t e male n e s t - b u i l d i n g v i a some nongonadal mechanism. In a d d i t i o n , they i n d i c a t e t hat i n i t i a l l e v e l s of male c o u r t s h i p are s i m i l a r l y a l t e r e d , an e f f e c t suggested by pr e v i o u s r e s u l t s (McDonald & L i l e y , 1978), but not s t a t i s t i c a l l y confirmed. R e l e a s i n g f a c t o r d i d not in c r e a s e the n e s t - b u i l d i n g response of short-day b i r d s . Hinde & S t e e l a l s o f a i l e d to incr e a s e n e s t - b u i l d i n g i n e s t r a d i o l - t r e a t e d SD female c a n a r i e s using d a i l y i n j e c t i o n s of 1ug LHRH (unpublished r e s u l t s c i t e d i n Hinde & S t e e l , 1977). The f a i l u r e of LHRH treatment to a l t e r any of the observed a c t i v i t i e s can be e x p l a i n e d i n s e v e r a l ways. I t i s p o s s i b l e that the dose used (20 ug/day) was too low to have an e f f e c t . However t h i s seems u n l i k e l y as the same dose s t i m u l a t e d sexual behaviour i n female doves (Cheng, 1977) and i n j e c t i o n s of as l i t t l e as 0.15ug are e f f e c t i v e i n female r a t s (Moss & McCann, 1975). Only 1ug LHRH i s r e q u i r e d to i n c r e a s e plasma LH i n c a n a r i e s (unpublished r e s u l t s c i t e d i n Hinde & S t e e l , 1977). The extremely short h a l f - l i f e of LHRH i n the plasma ( C a r t e r & Davies, 1977; S c h a l l e y et a l . , 1978) c o u l d c o n c e i v a b l y mean that the treatment regime used i n t h i s experiment r e s u l t e d i n i n c r e a s e d LHRH l e v e l s over too short a p e r i o d to cause a response. Jackson (1979) s t a t e s that the h a l f - l i f e of the hormone v a r i e s from 15 minutes to 3 hours. I t would thus be expected that c i r c u l a t i n g l e v e l s would be hig h d u r i n g at l e a s t one of the o b s e r v a t i o n s e s s i o n s used. In 69 a d d i t i o n , the b e h a v i o u r a l response observed by others extends from 1.5 to 48 hours a f t e r i n j e c t i o n s i n female r a t s ( P f a f f , 1973; Dudley & Moss, 1976) and from 2 to 18 hours i n female doves (Cheng, 1977), suggesting that the o b s e r v a t i o n p e r i o d s used here should cover the p e r i o d of responsiveness. A more s e r i o u s problem a r i s e s from the s p e c i f i c i t y of the response to LHRH. E a r l y o b s e r v a t i o n s of enhanced gonadotropic a c t i v i t y i n pigeons and chi c k e n s f o l l o w i n g treatment with s y n t h e t i c mammalian hormone i n d i c a t e d that LHRH i s not s p e c i e s -s p e c i f i c ( S c h a l l e y et a l . , 1978). T h i s c o n c l u s i o n was supported by immunochemical and chromatographic s i m i l a r i t i e s between chicken and mammalian LHRH, but recent accounts of low c r o s s -r e a c t i v i t y between r a t and q u a i l hypothalamic e x t r a c t s ( H a t t o r i , 1980) and d i f f e r e n c e s i n chromatographic p r o p e r t i e s on i o n -exchange columns (Jackson, 1980) suggest that the avian and mammalian p e p t i d e s are not i d e n t i c a l (Miyamato et a l . , 1982). The responsiveness of female doves to s y n t h e t i c LHRH ( a l s o from Ayerst L t d . ) , however, i n d i c a t e s t hat the hormone used here should be e f f e c t i v e (Cheng, 1977). A l t e r n a t i v e l y , i t may be that males, u n l i k e females, are b e h a v i o u r a l l y unresponsive to LHRH. Some i n f o r m a t i o n i s now a v a i l a b l e on the r e a c t i o n of males to r e l e a s i n g hormone. In both i n t a c t and c a s t r a t e d , TP-primed male r a t s LHRH decreases the l a t e n c y of mounts or i n t r o m i s s i o n s , but does not change t h e i r frequency (Moss et a l . , 1978). Ryan & F r a n k e l (1978) f a i l e d to observe any a l t e r a t i o n i n the sexual a c t i v i t y of i n t a c t males i n response to e i t h e r subcutaneous or i n t r a c r a n i a l 70 treatment. Using a s l i g h t l y d i f f e r e n t approach, Dorsa et a l . (1981) found that a h i g h l y potent LHRH analog (which presumably decreases endogenous LHRH a c t i v i t y , but i s i t s e l f b e h a v i o u r a l l y i n e f f e c t i v e ) i n h i b i t e d sexual behaviour i n i n t a c t males and i n c a s t r a t e s with small t e s t o s t e r o n e implants, but had no e f f e c t when given i n c o n j u n c t i o n with much l a r g e r doses of t e s t o s t e r o n e . Hopes that hypothalamic hormone would a i d i n treatment of impotency i n humans r e c e i v e d some support from e a r l y t e s t s i n males with hormone d e f i c i e n c i e s , but more recent s t u d i e s on p h y s i o l o g i c a l l y normal men have y i e l d e d negative r e s u l t s (Mauk et a l . , 1980). Another p o s s i b l e reason f o r the lack of response to LHRH i n male doves i s that the hig h d a i l y dose of TP (0.4mg/day) may have masked any e f f e c t of the hypothalamic hormone. In previous s t u d i e s i n which LHRH was found to s t i m u l a t e sexual behaviour i t was used i n c o n j u c t i o n with s u b - t h r e s h o l d doses of s t e r o i d hormones. When the peptide has been a d m i n i s t e r e d with b e h a v i o u r a l l y - a c t i v e doses of s t e r o i d (female r a t , Modianos & P f a f f , 1977; female dove, Cheng, 1977) or to i n t a c t animals, (male r a t s , Ryan & F r a n k e l , 1978; man, Mauk et a l . , 1980; female r a b b i t , Baldwin et a l . , 1974) i t i s i n e f f e c t i v e . Moore et a l . (1982) have a l s o r e p o r t e d L H R H - f a c i l i t a t i o n of male sexual a c t i v i t y i n an amphibian, but the response i s only seen at the beginning of the breeding season (when androgen l e v e l s are low?), and not at the peak. The r e s u l t s presented here do not e l i m i n a t e the p o s s i b i l i t y that LHRH can modify the behaviour of male doves i n some 71 s i t u a t i o n s , f o r example, i n the presence of very low l e v e l s of c i r c u l a t i n g t e s t o s t e r o n e . They do, however, i n d i c a t e that the i n f l u e n c e of photoperiod on n e s t - b u i l d i n g and e a r l y c o u r t s h i p behaviour observed i n male doves r e c e i v i n g h i g h l e v e l s of exogenous androgen cannot be accounted f o r by d i f f e r e n c e s i n pr o d u c t i o n of LHRH. LHRH c o u l d act i n d i r e c t l y through changes i n gonadotropin p r o d u c t i o n . LH l e v e l s are ap p a r e n t l y high d u r i n g the nest-b u i l d i n g phase i n pigeons (Murton & Westwood, 1975), s t a r l i n g s (Dawson & Goldsmith, 1982) and both female and male doves (Cheng & F o l l e t t , 1976; Cheng & B a l t h a z a r t , 1982). However, the involvement of gonadotropins i n c o n t r o l of n e s t i n g behaviour i s u n l i k e l y . Cheng & B a l t h a z a r t (1982) measured changes i n plasma FSH and LH l e v e l s i n doves completing an e n t i r e c o u r t s h i p c y c l e . Hormone l e v e l s were s i m i l a r i n four groups of males exposed to v a r y i n g n e s t i n g c o n d i t i o n s , d e s p i t e l a r g e d i f f e r e n c e s i n the b u i l d i n g a c t i v i t y e x h i b i t e d by the groups. A l s o , although i t has been suggested that LH i n f l u e n c e s avian a g o n i s t i c behaviour (see McDonald, 1976 f o r review), i t has no e f f e c t on nest-b u i l d i n g i n ov a r i e c t o m i z e d , e s t r o g e n - t r e a t e d female c a n a r i e s (Hinde & S t e e l , 1975) and doves (Cheng, 1977) or i n male red-b i l l e d quelea(Crook £ B u t t e r f i e l d , 1968). In a d d i t i o n , exogenous LHRH treatment s t i m u l a t e s gonadotropin r e l e a s e i n b i r d s (pigeon and c h i c k e n , S c h a l l e y et a l . , 1978; sparrow, W i n g f i e l d & Farner, 1980) but does not a l t e r the n e s t i n g response i n doves, i n d i c a t i n g that the behaviour i s not a f f e c t e d by the assumed i n c r e a s e i n endogenous gonadotropin r e s u l t i n g 72 from LHRH treatment. E. Summary 1) Exposure to long daylengths was a s s o c i a t e d with g r e a t e r n e s t i n g a c t i v i t y i n TP-implanted c a s t r a t e s t r e a t e d with s a l i n e or LHRH. Long-day LHRH b i r d s a l s o d i s p l a y e d more ag g r e s s i v e c o u r t s h i p than short-day LHRH males. 2) LHRH treatment d i d not a l t e r any of the measures of b e h a v i o u r a l a c t i v i t y i n e i t h e r SD or LD b i r d s , i n d i c a t i n g that i n c r e a s e d LHRH r e l e a s e i s not r e s p o n s i b l e f o r long-day induced changes i n n e s t i n g . 73 V. THE ROLE OF THE PINEAL IN MEDIATING THE BEHAVIOURAL RESPONSE TO PHOTOPERIOD A. I n t r o d u c t i o n During the past few years the p i n e a l gland, a small organ a r i s i n g from an e v a g i n a t i o n of the t h i r d v e n t r i c l e , has gained prominence as a s t r u c t u r e which may be i n v o l v e d i n mediating the e f f e c t of photoperiod on r e p r o d u c t i o n . Although the vast m a j o r i t y of s t u d i e s which l i n k the p i n e a l with seasonal r e p r o d u c t i v e c y c l e s are based on the hamster, i t seems worthwhile to explore the p o s s i b i l i t y t h a t i t i s a l s o i n v o l v e d i n t r a n s d u c i n g i n f o r m a t i o n about daylength i n doves. Both the mammalian and the avian p i n e a l organs are c o n s i d e r e d to be endocrine glands. The p i n e a l produces a number of substances, among which i s melatonin, c o n s i d e r e d by most r e s e a r c h e r s to be the major p i n e a l hormone ( R e i t e r , 1980; C a r d i n a l i , 1981). Melatonin i s an indoleamine which i s d e r i v e d from s e r o t o n i n (5-hydroxytryptamine = 5HT) v i a a metabolic pathway i n v o l v i n g two enzymes: NAT and hydroxyindole-o-methyl t r a n s f e r a s e (HIOMT) (Balemans, 1979; see f i g . 7). The hormone was f i r s t i s o l a t e d from bovine p i n e a l s i n 1958 by Lerner and h i s c o l l e a g u e s (Wurtman, 1971). Soon afterwards i t was re p o r t e d that HIOMT i s present e x c l u s i v e l y i n the p i n e a l (Wurtman, 1971). I t i s now known that melatonin s y n t h e s i s i s not r e s t r i c t e d to the p i n e a l , but a l s o occurs i n the r e t i n a s of f i s h , amphibians, b i r d s , and mammals, as w e l l as i n the Harderian gland and the gut of r a t s (see review by Ralph, 1981). Aside from melatonin, Figure 7. Routes of indole metabolism i n the pineal gland. From Balemans, 1975. oca -xcr^xxri Tryptophan dry) tryptophan 5*HydroxytryptopHan <5-HTP) aVfarboayloM 5 - MetKoH y U yptopho n <5-MTP 5-Hydroxytryptomine <5-HT> o*<cto»t <*AO> 5-Hydroxyindole oceetaldertyde <5-HIAt> Akghel d^ tfdr 09.1*01. S-Memoxytryptamine <s-MT, henittfoit •xxr- nocn. "Can. 5-Hydroxyindole ocelk ocid 5-HydroxytryptopHol <5-HIAA>. <5-HTL> H r d . o . r " « i o * » - 0 -<MIOMT> H T d,e»,.~*ol.-0-WMtKyt t r o n t l . r O M (HIOMT) N-Acetyl 5-Hydroxytryptomi <N-Ac-5-HT) M r d. M r . tx»oW-0-m.ihr{ t 'on t f r ro te <HIOMl> "°XXJ^™ "TJCO ""Ton M M M 5-Methoxyindole ocetic ocid S-MetKoxylryptophol Melatonin <5-MIAA> <5-MTl> (Met) 75 the p i n e a l c o n t a i n s at l e a s t nine other i n d o l e compounds, i n c l u d i n g s e r o t o n i n , 5-hydroxytryptophol, and 5-methoxytryptophol (Pevet, 1981). S e v e r a l p e p t i d e s have a l s o been found (see reviews by Ebels & Benson, 1978; Vaughan, 1981). One of the most d i s t i n c t i v e f e a t u r e s of the p i n e a l gland i s i t s s t r o n g d i u r n a l rhythm of enzyme a c t i v i t y and melatonin r e l e a s e . In a l l animals s t u d i e d so f a r , p i n e a l melatonin content peaks d u r i n g the dark phase of the l i g h t c y c l e ( R e i t e r , 1981). Although p i n e a l f u n c t i o n appears to be based on true c i r c a d i a n rhythms which p e r s i s t i n constant dark (Ralph et a l . , 1975; B i n k l e y , 1980), i t i s h i g h l y responsive to modulation by l i g h t s t i m u l i . A one-minute l i g h t f l a s h d u r i n g the dark phase r e s u l t s i n an immediate drop i n NAT a c t i v i t y ( I l l n e r o v a & Vanecek, 1979) and melatonin l e v e l s ( I l l n e r o v a et a l . , 1978). Darkness d u r i n g the daytime i s i n e f f e c t i v e (Hoffmann, 1981). The most common e f f e c t of the l i g h t c y c l e on the p i n e a l i s the entrainment of the enzyme and melatonin rhythms. In male hamsters exposure to l i g h t c y c l e s of 10L:14D or 14L:10D does not a l t e r the amplitude or d u r a t i o n of the p i n e a l melatonin c r e s t but the c y c l e i s e n t r a i n e d so that the peak f a l l s four hours b e f o r e dawn on both photoperiods (Tamarkin et a l . , 1979). _1_. The P i n e a l and Reproduction i n Mammals The f i r s t experimental i n d i c a t i o n of the l i n k between the p i n e a l and r e p r o d u c t i v e p h y s i o l o g y was d i s c o v e r e d by Wurtman'and h i s co-workers, who showed that o v a r i a n weight i n female r a t s i s i n c r e a s e d by pinealectomy (Wurtman et a l . , 1961) and decreased 76 by melatonin treatment (Wurtman et a l . , 1963). Although much work was done examining p i n e a l - n e u r o e n d o c r i n e i n t e r a c t i o n s i n the r a t (see C a r d i n a l i , 1981, f o r review), i t soon became apparent that photoperiod p l a y s a major r o l e i n c o n t r o l l i n g the system, l e a d i n g to i n c r e a s e d emphasis on other, more photoresponsive s p e c i e s . Most of the evidence i n d i c a t i n g the importance of the p i n e a l as a photoperiod transducer i s based on o b s e r v a t i o n s of the golden or S y r i a n hamster ( M e s o c r i c e t u s auratus ). The r e p r o d u c t i v e c y c l e i n hamsters i s dependent mainly upon the i n h i b i t o r y e f f e c t of short daylengths d u r i n g the autumn, r e s u l t i n g i n a s e x u a l l y q u i e s c e n t p e r i o d which i s f o l l o w e d by spontaneous recrudescence i n the s p r i n g ( R e i t e r , 1975). Pinealectomy blocks the i n h i b i t o r y e f f e c t of short days (Tamarakin et a l . , 1976), and l a t e - a f t e r n o o n i n j e c t i o n s of melatonin r e s u l t i n gonadal atrophy i n p h o t o s t i m u l a t e d animals ( R e i t e r et a l . , 1978). Although these r e s u l t s suggest t h a t melatonin i s an a n t i g o n a d a l hormone s e c r e t e d by the p i n e a l gland in response to short daylengths, the s i t u a t i o n i s a c t u a l l y much more complex. I t now appears that the a n t i g o n a d a l e f f e c t of melatonin i s dependent upon the rhythm of the hormone (as a r e s u l t of the combined e f f e c t s of both endogenous and exogenous l e v e l s ) and the a b i l i t y of melatonin to down-regulate i t s own r e c e p t o r s (Vacas & C a r d i n a l i , 1979; see R e i t e r , 1978; R e i t e r , 1980 f o r d e t a i l s ) . However, other s p e c i e s such as the f e r r e t do respond to the presence of melatonin r a t h e r than to the hormone rhythm (C a r t e r et a l . , 1982). 77 Gonadal r e g r e s s i o n has been l i n k e d to the p i n e a l i n a number of other mammalian s p e c i e s . Pinealectomy prevents s h o r t -day i n v o l u t i o n i n d j u n g a r i a n hamsters ( Phodopus sunqorus ; Hoffmann, 1978a) and i n v o l e s ( M i c r o t u s a g r e s t i s ; F a r r a r & C l a r k , 1976) while exogenous melatonin i n h i b i t s long-day induced recrudescence i n the a r t i c hare ( K u d e r l i n g et a l . , 1979), d j u n g a r i a n hamster (C a r t e r & Goldman, 1981), weasel (Rust & Meyer, 1969) and s e v e r a l mouse sp e c i e s (Turek et a l . , 1976; G l a s s & Lynch, 1981). The p i n e a l appears to be i n v o l v e d i n the response to long days as w e l l . U n l i k e the golden hamster, most p h o t o s e n s i t i v e animals a c t i v e l y respond to long p h o t o p e r i o d s - - i n c r e a s e d daylengths s t i m u l a t e development of the r e p r o d u c t i v e t r a c t . In some of these animals, such as the d j u n g a r i a n hamster ( C a r t e r et a l . , 1982), Mesocricetus b r a n d t i (Hoffmann, 1981b), and the f e r r e t (Herbert, 1981), pinealectomy delays recrudescence under long days, and i n the hamster melatonin prevents short-day induced r e g r e s s i o n (Goldman,1982). Observations such as these have l e d s e v e r a l authors to q u e s t i o n the s t r i c t l y a n t i g o n a d a l r o l e of the p i n e a l / m e l a t o n i n o u t l i n e d above, proposing i n s t e a d that the p i n e a l gland serves to convey p h o t o p e r i o d i c i n f o r m a t i o n , whether i t i s pro- or a n t i - g o n a d a l (Hoffmann, 1978b; Brown et a l . , 1981). 78 2. The P i n e a l and Reproduction i n B i r d s The involvement of the avian p i n e a l gland i n the c o n t r o l of breeding i s q u e s t i o n a b l e (see t a b l e I X ). Many s t u d i e s of b i r d s have f a i l e d to f i n d any changes i n the r e p r o d u c t i v e t r a c t f o l l o w i n g pinealectomy (Japanese q u a i l , Homma et a l . , 1972, Siopes & Wilson, 1974, O i s h i & Lauber, 1974a; H a r r i s ' sparrow, Donham & Wilson, 1969; white-crowned sparrow, Oksche et a l . , 1972), l e s i o n i n g of the p i n e a l (house f i n c h , Hamner & B a r f i e l d , 1970) or melatonin treatment (Japanese q u a i l , A r r i n g t o n , 1967, O i s h i & Lauber, 1974b; w h i t e - t h r o a t e d sparrow, Turek & Wolfson, 1978). S i m i l a r l y , n e i t h e r pinealectomy (Donham & Wilson, 1970) nor exogenous melatonin (Storey & N i c h o l l s , 1978) a l t e r the onset or t e r m i n a t i o n of p h o t o r e f r a c t o r i n e s s i n b i r d s . In those s i t u a t i o n s i n which the p i n e a l does i n f l u e n c e gonadal development, r e s u l t s are o f t e n i n c o n s i s t e n t , v a r y i n g with the age and sex of the t e s t animals. Thus, S a y l e r & Wolfson (1967) observed a t r a n s i t o r y delay i n the maturation of the ovary i n j u v e n i l e female q u a i l f o l l o w i n g p i n e a l removal, but saw no e f f e c t i n males. A r r i n g t o n (1967), on the other hand, recorded a decrease i n short-day induced r e g r e s s i o n i n pi n e a l e c t o m i z e d male q u a i l , but not i n females. Melatonin treatment has been r e p o r t e d to d e l a y ( A r r i n g t o n , 1967; Homma et a l . , 1967), s t i m u l a t e (Wright & P r e s l o c k , 1975),or have no e f f e c t ( O i s h i & Lauber, 1974b) on gonadal development i n Japanese q u a i l - - a l t h o u g h these d i s c r e p a n c i e s can probably be accounted f o r , i n l a r g e p a r t , by v a r i a t i o n i n the age and sex of the b i r d s and i n the l e v e l and route of melatonin 7 9 Table IX. The e f f e c t of pinealectomy and melatonin treatment on gonadal development i n b i r d s . *=based on lesions. sex gonad l i g h t reference PINEALECTOMY chicken M 4 ? Ralph, 1970 P LD, SD Godkhindi et a l . , 1971 Japanese q u a i l F 4. LD Sayler & Wolfson, 1967 M LD F DD, b l i n d F LL, SD, LD Homma et a l . , 1967 M.F - SD, LD Arrington, 1967 M,F — SD, LD Oi s h i & Lauber, 1974a H,F - SD, LD Siopes & Wilson, 1974 Harr i s ' sparrow M,F - SD Donham & Wilson, 1979 *house f i n c h M - SD, LD Hamner & B a r f i e l d , 1970 white-crowned M — SD, LD Oksche et a l . , 1972 sparrow duck M 4- LD C a r d i n a l i et a l . , 1971 M 4- DD,blind Hisano et a l . , 1972 F 4- LL Cuello et a l . , 1971 weaverbird M t SD, LD Balasubramanian & Saxena, 1973 Saxena et a l . , 1979 house sparrow M _ LD Menaker et a l . , 1970 M — DD,LD.13L Takahashi et a l . , 1978 M 4- LD skeleton it M 4- 3L.-23D Gwinner et a l . , 1981 M t 3L:21D n s t a r l i n g M _ LD,SD,LL Gwinner et a l . , 1981 M 4- LL(dim) M 12L:12D Gwinner & Dittami, 1980 MELATONIN white-throated F _ SD, LD Turek & Wolfson, 1978 sparrow M — LD •i chicken M, young * ? Balemans, 1972 M ? •t Japanese q u a i l M*F(y) a. ? Arrington, 1969 M,F — ? •i M - SD, LD Oishi & Lauber, 1974b M,F(y) 4- ? Homma et a l . , 1967 F t ? Wright & Preslock, 1975 junco M,F - ? c i t e d i n Turek & Wolfon, foxsparrow M.F — ? •i 80 a d m i n i s t r a t i o n . C l e a r e f f e c t s of the p i n e a l gland have, however, been seen i n two b i r d s p e c i e s . In ducks, i l l u m i n a t i o n of the p i n e a l region of b l i n d males h e l d i n constant dark (DD) r e s u l t s i n t e s t i c u l a r growth. T h i s response to l i g h t i s p a r t i a l l y i n h i b i t e d by pinealectomy (Hisano et a l . , 1972). Removal of the gland i n i n t a c t ducks h e l d on n a t u r a l l i g h t c y c l e s causes a temporary d e c l i n e i n t e s t i s s i z e ( C a r d i n a l i et a l . , 1971). Female ducks exposed to constant l i g h t undergo a decrease i n ovary weight f o l l o w i n g pinealectomy ( C u e l l o et a l . , 1972). The t r o p i c a l weaverbird ( Ploceus p h i l l i p p i n u s ) e x h i b i t s the o p p o s i t e response: pinealectomy leads to p r e c o c i o u s development i n j u v e n i l e s , and to i n c r e a s e d t e s t i s s i z e i n both l o n g - and short-day a d u l t s , and prevents r e g r e s s i o n at the end of the breeding c y c l e (Balasubramanian & Saxena, 1973; Saxena et a l . , 1 979) . D e s p i t e the p a r a d o x i c a l nature of the i n f o r m a t i o n c u r r e n t l y a v a i l a b l e , the obvious responsiveness of the avian p i n e a l to p h o t i c s t i m u l i ( B i n k l e y , 1980), and the frequent, a l b e i t c o n t r a d i c t o r y , i n d i c a t i o n s of i t s e f f e c t s on gonadal f u n c t i o n have l e d reviewers to conclude that i t i s probably i n v o l v e d i n the c o n t r o l of r e p r o d u c t i v e c y c l e s i n b i r d s , although perhaps a c t i n g i n a more g e n e r a l , i n t e g r a t i v e manner ra t h e r than i n the d i r e c t a n t i g o n a d a l r o l e u s u a l l y a s s i g n e d to the mammalian gland (see reviews by Ralph, 1970; Menaker & Oksche, 1974; Ralph, 1978). There i s now c o n s i d e r a b l e support f o r the h y p othesis that 81 the p i n e a l i s i n v o l v e d i n the c o n t r o l of c i r c a d i a n rhythms i n b i r d s . Pinealectomy a b o l i s h e s c i r c a d i a n rhythms of a c t i v i t y i n house sparrows (Gaston & Menaker, 1968; B i n k l e y et a l . , 1971; B i n k l e y et a l . , 1972), J a v a sparrows (Ebihara & Kawamura, 1980) and w h i t e - t h r o a t e d sparrows (McMillan, 1972) h e l d on DD, but has no i n f l u e n c e on b i r d s h e l d on l i g h t : d a r k c y c l e s . T h i s e f f e c t of pinealectomy i s presumably mediated by hormonal, r a t h e r than n e u r a l outputs, s i n c e no d i s r u p t i o n i s seen f o l l o w i n g s e c t i o n i n g of the p i n e a l s t a l k or t r a n s p l a n t a t i o n of the p i n e a l (Zimmerman & Menaker, 1975). M e l a t o n i n i n j e c t i o n s are r e p o r t e d to synchronize a c t i v i t y c y c l e s i n p i n e a l e c t o m i z e d s t a r l i n g s (Gwinner & Benzinger, 1978), while melatonin implants a b o l i s h or shorten the rhythm i n DD sparrows (Turek et a l . , 1976); however these e f f e c t s c o u l d be due to melatonin-induced suppression of a c t i v i t y (Hendel & Turek 1978; Datta & King, 1980). Because pinealectomy f a i l s to i n f l u e n c e a c t i v i t y i n b i r d s exposed to l i g h t c y c l e s , the gland may serve to couple and/or d r i v e other, e x t r a p i n e a l o s c i l l a t o r s which are a l s o s e n s i t i v e to l i g h t (Gwinner, 1978; Takahashi & Menaker, 1980). The importance of the p i n e a l v a r i e s among s p e c i e s ; pinealectomy produces arrhythmia i n some, but not a l l , s t a r l i n g s (Gwinner, 1978; Rutledge & Angle, 1977) and has no e f f e c t i n chickens and Japanese q u a i l (Takahashi & Menaker, 1980). The response i s not l i m i t e d to a c t i v i t y c y c l e s , as pinealectomy a l s o i n t e r f e r e s with the d i u r n a l rhythm of t h e rmoregulation i n c h i c k e n s (Cogburn et a l . , 1976) and sparrows ( B i n k l e y et a l . , 1971). P h o t o p e r i o d i c c o n t r o l of breeding c y c l e s i s dependent upon 82 a c i r c a d i a n rhythm of s e n s i t i v i t y to l i g h t s t i m l u l a t i o n ( F o l l e t t et a l . , 1981), and i t i s p o s s i b l e that the p i n e a l c o u l d a f f e c t r e p r o d u c t i o n through changes i n the mechanism c o n t r o l l i n g s e n s i t i v i t y . Takahashi et a l . (1978) observed i n h i b i t i o n of t e s t i c u l a r growth i n p i n e a l e c t o m i z e d house sparrows h e l d on a 1L:11D:1L:11D photoperiod, but not i n b i r d s h e l d on a constant 13L:11D c y c l e , suggesting involvement of the c i r c a d i a n system. Gwinner and h i s c o l l e a g u e s have s t u d i e d the r e l a t i o n s h i p between t e s t i c u l a r growth and the p i n e a l i n s t a r l i n g s h e l d on v a r i o u s d i f f e r e n t l i g h t regimes. Pinealectomy had no e f f e c t on b i r d s exposed to 8L:16D; 12L:12D; 16L:8D or LL of h i g h i n t e n s i t y , although gonadal i n h i b i t i o n d i d occur i n one group exposed to LL of.low i n t e n s i t y (Gwinner et a l . , 1981). Removal of the p i n e a l a l s o r e s u l t e d i n the l o s s of a second c y c l e of t e s t i s growth on 12L:12D (Gwinner & D i t t a m i , 1980). Attempts to r e l a t e changes i n a c t i v i t y rhythms f o l l o w i n g pinealectomy with expected changes i n gonadal development were u n s u c c e s s f u l (Gwinner et a l . , 1981; Gwinner & D i t t a m i , 1982). _3 „ The P i n e a l and Reproductive Behaviour Once again , d e s p i t e a wide body of knowledge about r e p r o d u c t i v e p h y s i o l o g y , l i t t l e a t t e n t i o n has been focussed on the accompanying behaviour. Only two s t u d i e s have been done examining the e f f e c t of pinealectomy on r e p r o d u c t i v e behaviour. Baum (1968) p i n e a l e c t o m i z e d neonatal male r a t s , then reared them i n constant dark. He observed a t r a n s i e n t i n c r e a s e i n the sexual a c t i v i t y of j u v e n i l e s , but no e f f e c t i n a d u l t s . T h i s 83 r e s u l t i s not unexpected i n view of the f a c t that p i n e a l removal and l i g h t d e p r i v a t i o n have only modest and t r a n s i t o r y e f f e c t s on the r e p r o d u c t i v e organs of r a t s ( R e i t e r , 1980). Sampson (1975) examined the e f f e c t of neonatal pinealectomy on r a t maternal behaviour. P i n e a l e c t o m i z e d females proved to be poorer mothers: they b u i l t s m a l l e r nests and spent more time away from the nest on the f i r s t day postpartum. They a l s o r e ared 11% fewer pups to weaning than t h e i r i n t a c t c o u n t e r p a r t s . In a d d i t i o n , i t was found that v i r g i n females which had been p i n e a l e c t o m i z e d d i s p l a y e d l e s s pup-induced maternal behaviour. D a i l y i n j e c t i o n s of melatonin w i l l decrease accessory sexual organ weight i n a d u l t male r a t s , but have no e f f e c t on sexual behaviour (Mas et a l . , 1979; Mas & Oakiu, 1978), although another p i n e a l i n d o l e , 5-methoxytryptophol, does reduce c o p u l a t o r y a c t i v i t y (Mas & Oakiu, 1978). In female mice t i n y implants of melatonin i n the a n t e r i o r hypothalamus and SCN i n c r e a s e n e s t i n g behaviour, as measured by the amount of c o t t o n c o l l e c t e d each day (Glass & Lynch, 1981). B. The E f f e c t of Pinealectomy on the Behaviour of Male Doves In s p i t e of c o n t r a d i c t i o n s between d i f f e r e n t s p e c i e s , the p i n e a l i s c l e a r l y i m p l i c a t e d i n the p h o t o p e r i o d i c c o n t r o l of mammalian r e p r o d u c t i v e p h y s i o l o g y . The connection i s l e s s obvious i n b i r d s , but there i s growing evidence of p i n e a l -induced changes i n r e p r o d u c t i v e c a p a c i t y here as w e l l . The f o l l o w i n g experiments were designed to examine whether the p i n e a l gland might somehow be a s s o c i a t e d with the modulating 84 i n f l u e n c e of daylength on the behaviour of male r i n g doves. In the i n i t i a l t e s t the e f f e c t of p i n e a l removal was examined i n male doves h e l d on long or short daylengths. Work based on mammals suggests that the primary r o l e of the p i n e a l i s to mediate the i n h i b i t o r y e f f e c t of short p h otoperiods. I t h e r e f o r e a n t i c i p a t e d t h a t pinealectomy of the doves would r e s u l t i n i n c r e a s e d n e s t i n g a c t i v i t y under short days. Because of the i n d i c a t i o n s that the p i n e a l i s progonadal i n some b i r d s I p i n e a l e c t o m i z e d long-day i n d i v i d u a l s as w e l l . A f t e r the f i r s t set of b e h a v i o u r a l o b s e r v a t i o n s were completed, the b i r d s were c a s t r a t e d and implanted with TP c a p s u l e s i n order to e l i m i n a t e p o s s i b l e d i f f e r e n c e s i n c i r c u l a t i n g t e s t o s t e r o n e l e v e l s r e s u l t i n g from the pinealectomy. They were then r e t e s t e d . J _ . M a t e r i a l s and Methods F i f t y e x p e r i m e n t a l l y naive a d u l t male r i n g doves were sexed, g i v e n i n i t i a l b reeding experience and housed as d e s c r i b e d e a r l i e r . I n t a c t male doves were brought i n from the a v i a r i e s i n February and h e l d on a 8L;16D l i g h t c y c l e . The b i r d s were sham-operated or p i n e a l e c t o m i z e d and one week l a t e r moved i n t o i s o l a t i o n cages on e i t h e r long (16L:8D) or short (8L;16D) daylengths. Thus there were four experimental groups: long-day c o n t r o l (LC) and p i n e a l e c t o m i z e d (LP), and short-day c o n t r o l (SC) and p i n e a l e c t o m i z e d (SP) b i r d s . A f t e r 8 weeks each male was p a i r e d with a female which had r e c e i v e d 7 d a i l y i n j e c t i o n s of EB + P. Behavioural r e c o r d i n g s were taken 4 times a day over the 5-day t e s t p e r i o d . 85 Four weeks a f t e r the end of the o b s e r v a t i o n p e r i o d the males were c a s t r a t e d . The l e f t t e s t i s was weighed, measured by volume displacement of s a l i n e i n a 3 cc s y r i n g e , and f i x e d i n Bouins s o l u t i o n . S e c t i o n s were s t a i n e d with haematoxylin + e o s i n and examined f o r the stage of spermatogenesis, as d e f i n e d by L o f t s et a l . , 1966. F o l l o w i n g c a s t r a t i o n , the b i r d s from the above experiment were h e l d f o r a f u r t h e r 7 weeks on the long and short p h o t o p e r i o d s . They were then implanted with 5 mm long c a p s u l e s c o n t a i n i n g TP and r e t e s t e d with new females f o l l o w i n g the procedure o u t l i n e d above. 2. R e s u l t s R e s u l t s from c o n t r o l b i r d s agree with those seen i n e a r l i e r experiments. There was no s i g n i f i c a n t e f f e c t of photoperiod on l e v e l s of male c o u r t s h i p , c o p u l a t o r y behaviour, or on the a c t i v i t y of the females p a i r e d with the males ( t a b l e s X & X I ) . As i n d i c a t e d i n f i g . 8, however, a l l three measures of male n e s t - b u i l d i n g were g r e a t e r f o r LC than f o r SC b i r d s . T h i s d i f f e r e n c e was e l i m i n a t e d by pinealectomy: LP males b u i l t at l e v e l s s i m i l a r to those d i s p l a y e d by both short-day groups, and were s i g n i f i c a n t l y l e s s a c t i v e than LC b i r d s . Pinealectomy had no e f f e c t on those a c t i v i t i e s which are not a l t e r e d by p h o t o p e r i o d — i . e . , male c o u r t s h i p and c o p u l a t o r y behaviour and the behaviour of the s t i m u l u s females. Long daylengths were a s s o c i a t e d with higher t e s t i s volume 8 6 T a b l e X. The e f f e c t o f p i n e a l e c t o m y on the c o u r t s h i p a c t i v i t y o f males h e l d on l o n g and s h o r t d a y s . The v a l u e s r e p r e s e n t the means and ranges based on the t o t a l number o f 15-second i n t e r v a l s or the t o t a l number o f e v e n t s over the 5-day t e s t p e r i o d . LONG DAY SHORT DAY Sham P i n e a l x Sham P i n e a l x n=9 n = l l n=8 n=12 Male c o u r t s h i p chase 16.8 (2-36) 15.0 (3-48) 14.5 (6-27) 18.7 (4-42) bow-coo 14.5 (3-32) 13.5 (0-39) 13.0 (4-41) 14.2 (2-34) s o l i c i t 385 448 (169-492) (315-628) 385 331 (135-613) (200-462) a t - n e s t 375 408 (181-502) (209-628) 367 313 (141-663) (85-502) 87 Table XI. The e f f e c t of p h otoperiod and pinealectomy on the c o p u l a t o r y behaviour of i n t a c t males, and on the r e p r o d u c t i v e behaviour of t h e i r mates. The v a l u e s re p r e s e n t the mean and range based on the t o t a l number of 15-second i n t e r v a l s or the t o t a l number of observed events over the 5-day t e s t p e r i o d . LONG DAY SHORT DAY Sham n=9 P i n e a l x n = l l Sham n=8 P i n e a l x n=12 C o p u l a t o r y a c t i v i t y beg 7.3 (0-26) 7.2 (0-21) 6.4 (0-12) 5.9 (0-16) b i l l 3.7 (0-11) 5.8 (0-18) 4.5 (0-9) 2.7 (0-11) c r o u c h 3.5 (0-9) 3.7 (0-13) 2.9 (0-5) 3.2 (0-9) mount 2.6 (0-5) 2.7 (0-7) 1.9 (0-4) 1.8 (0-5) Female be h a v i o u r a.crouch 1.6 (0-5) 2.2 (0-7) 2.6 (0-5) 1.9 (0-11) s o l i c i t 383 (214-662) 333 (0-603) 372 382 (119-786) (226-586) a t - n e s t 612 (385-838) 495 (0-804) 571 (80-1002) 525 (91-817) n e s t - b u i l d 4.9 (0-36) 3.9 (0-16) 2.0 (0-7) 2.7 (0-16) c a r r y 2.1 (0-17) 0.8 (0-3) 0.6 (0-2) 0.8 (0-4) 89 F i g u r e 8. The e f f e c t of d a y l e n g t h and p i n e a l e c t o m y on the n e s t - b u i l d i n g b e h a v i o u r of g o n a d a l l y - i n t a c t male doves. The v a l u e s r e p r e s e n t the mean l e v e l of a c t i v i t y , based on the t o t a l s f o r the 5-day t e s t p e r i o d . long-day c o n t r o l long-day p i n e a l e c t o m i z e d s h o r t - d a y c o n t r o l s h o r t - d a y p i n e a l e c t o m i z e d a p<.02 s i g n i f i c a n t l y d i f f e r e n t from LC, based on 2 - t a i l e d U - t e s t b p<.05 s i g n i f i c a n t l y d i f f e r e n t from LC, based on 2 - t a i l e d U - t e s t S T R I N G (pieces) C A R R Y I N G (events) N E S T B U I L D I N G (interval*) 91 and t e s t i s weight i n c o n t r o l b i r d s ( t a b l e X I I ) . No e f f e c t of Table X I I . The e f f e c t of pinealectomy and daylength on t e s t i s s i z e and volume. Values represent mean ± SE. Long days Short days C o n t r o l P i n e a l x C o n t r o l P i n e a l x n=9 n=11 n=8 n=12 t e s t i s 476±31 435±35 383±32* 432±27 weight (mg) t e s t i s 0.611.06 0.54±.03 0.47±.04* 0.55±.03 volume (ml) * = p<.05 SC < LC t - t e s t , 1 - t a i l e d d aylength was seen i n p i n e a l e c t o m i z e d b i r d s , and pinealectomy d i d not cause s i g n i f i c a n t changes i n t e s t i s weight or volume under e i t h e r daylength, although there d i d appear to be a t r e n d towards i n c r e a s e d t e s t i s s i z e under short days and decreased s i z e under long days with pinealectomy. A l l of the b i r d s e x h i b i t e d asynchronous se m i n i f e r o u s t u b u l e s and a l l were presumably i n breeding c o n d i t i o n as evidenced by the presence of spermatozoa i n the lumen of some t u b u l e s . C a s t r a t e d , t e s t o s t e r o n e - i m p l a n t e d b i r d s behaved i n a manner s i m i l a r to that of i n t a c t b i r d s , although l e v e l s of a g g r e s s i v e c o u r t s h i p and n e s t - b u i l d i n g were lower f o l l o w i n g c a s t r a t i o n and 92 TP-treatment. Male n e s t i n g behaviour was again the only a c t i v i t y to show s i g n i f i c a n t changes i n response to photoperiod or pinealectomy ( f i g . 9, t a b l e s XIII & XIV). A l l three measures of n e s t i n g a c t i v i t y were s i g n i f i c a n t l y h igher i n LC than i n SC b i r d s . Pinealectomy had no e f f e c t under sho r t days, but caused a d e c l i n e i n n e s t i n g behaviour under long days. 3. D i s c u s s i o n In g e n e r a l , pinealectomy has been shown to have l i t t l e e f f e c t on gonad s i z e i n b i r d s . Most of the evidence s u p p o r t i n g a r e l a t i o n s h i p between the p i n e a l and r e p r o d u c t i v e development comes from animals which were not f u l l y s e n s i t i v e to the photoperiod, e i t h e r because they were p r e p u b e r t a l (Japanese q u a i l , A r r i n g t o n , 1967; S a y l e r & Wolfson, 1967), had been b l i n d e d (domestic ducks, Hisano et a l . , 1972 - see Bayle, 1980 and Homma et a l . , 1980 f o r d i s c u s s i o n of the r o l e s of eyes versus e x t r a r e t i n a l r e c e p t o r s ) , were exposed to low l i g h t i n t e n s i t i e s ( s t a r l i n g s , Gwinner et a l . , 1981), or were t e s t e d e a r l y i n the breeding season (domestic ducks, C a r d i n a l i et a l . , 1971; sparrows, Takahashi et a l . , 1978). The only s p e c i e s i n which f u l l y photoresponsive a d u l t s have been shown to r e a c t to pinealectomy i s the t r o p i c a l weaver b i r d , which i s a l s o the only s p e c i e s to d i s p l a y s t i m u l a t i o n of t e s t i s growth f o l l o w i n g removal of the p i n e a l (Balasubramanian & Saxena, 1973). I t i s p o s s i b l e that i n most cases any e f f e c t of the p i n e a l gland on gonadal development i s masked by a much stronger c o n t r o l exerted by the photoperiod independently of the p i n e a l , and that 93 F i g u r e 9. The e f f e c t of daylength and pinealectomy on the n e s t - b u i l d i n g behaviour of TP-implanted c a s t r a t e s . The val u e s represent the mean l e v e l of a c t i v i t y , based on the t o t a l s f o r the 5-day t e s t p e r i o d . • LC long-day c o n t r o l LP long-day p i n e a l e c t o m i z e d SC short-day c o n t r o l SP short-day p i n e a l e c t o m i z e d a p<.02 s i g n i f i c a n t l y d i f f e r e n t from LC, based on 2 - t a i l e d U -test b p<.05 s i g n i f i c a n t l y d i f f e r e n t from LC, based on 2 - t a i l e d U - t e s t STR I N G (p l e c e s ) C A R R Y I N G (eve n t$) N E S T B U I L D I N G ( in terva l s ) vo 95 T a b l e X I I I . The e f f e c t o f p i n e a l e c t o m y on the c o u r t s h i p a c t i v i t y o f T P - i m p l a n t e d c a s t r a t e s h e l d on l o n g or s h o r t d a y s . The v a l u e s r e p r e s e n t the means and ranges based on the t o t a l number o f 15-second i n t e r v a l s or the t o t a l number o f e v e n t s over the 5-day t e s t p e r i o d . LONG DAY SHORT DAY Sham n=9 P i n e a l x n = l l Sham n=5 P i n e a l x n=9 Male c o u r t s h i p chase 9.9 (1-30) 7 . 6 (1-20) 5.4 (1-9) 7.7 (0-22) bow-coo 7.0 (0-17) 3.7 (0-12) 3.8 (2-7) 9.9 (0-38) s o l i c i t 359 397 (209-586) (144-665) 312 285 (35-523) (134-437) a t - n e s t 357 401 (67-649) (89-786) 335 318 (32-606) (96-718) 96 Table XIV. The e f f e c t of photoperiod and pinealectomy on the c o p u l a t o r y behaviour of TP-implanted c a s t r a t e s , and on the r e p r o d u c t i v e behaviour of t h e i r mates. The valu e s re p r e s e n t the means and ranges based on the t o t a l number of 15-second i n t e r v a l s or the t o t a l number of events over the 5-day t e s t p e r i o d . LONG DAY SHORT DAY Sham n=9 Pinealx n=ll Sham n=5 Pinealx n=9 Copulatory a c t i v i t y beg 11 (0-54) 10 (0-20) 12 (3-22) 14 (0-41) b i l l 4.7 (0-20) 3.7 (0-13) 8.4 (0-18) 5.0 (0-14) crouch 5.8 (0-18) 4.4 (0-13) 6.2 (0-12) 4.7 (1-11) mount 2.6 (0-7) 1.5 (0-5) 4.0 (0-6) 2.0 (0-4) Female behaviour a.crouch 4.6 (0-11) 2.6 (0-9) 1.6 (0-4) 3.0 (0-6) s o l i c i t 291 (124-612) 395 (139-673) 326 316 (18-509) (55-753) at-nest 584 516 (295-842) (67-937) 435 743 (15-649) (149-1047) nest-build 1.6 (0-5) 4.1 (0-35) 6.8 (0-19) 3.4 (0-10) carry 0.2 (0-1) 0.1 (0-1) 2.8 (0-7) 0.7 (0-5) 98 r e s p o n s e s t o p i n e a l e c t o m y a r e g e n e r a l l y s e e n i n s i t u a t i o n s w h e r e t h e r e i s a weak p h o t o r e s p o n s e . C e r t a i n l y t h e r i n g d o v e c a n be c l a s s i f i e d a s " w e a k l y p h o t o r e s p o n s i v e " , s i n c e p h o t o p e r i o d c a u s e s o n l y s l i g h t ( 1 . 5 X ) c h a n g e s i n t e s t i s s i z e c o m p a r e d w i t h t h o s e s e e n i n many s e a s o n a l b r e e d e r s . B a l t h a z a r t e t a l . ( 1 9 8 1 a & b ) o b s e r v e d a d e c r e a s e i n t e s t i s w e i g h t f o l l o w i n g t h e e x p o s u r e o f d o v e s t o r e d u c e d d a y l e n g t h s f o r 5 w e e k s . R e g r e s s i o n was r a p i d l y f o l l o w e d by s p o n t a n e o u s r e c r u d e s c e n c e , s o t h a t by 15 w e e k s t h e t e s t e s w e r e a s l a r g e a s t h o s e o f l o n g - d a y b i r d s . I n t h i s e x p e r i m e n t t h e s h o r t - d a y b i r d s w e r e k e p t on 8 L : 1 6 D f o r 16 w e e k s p r i o r t o c a s t r a t i o n . The s m a l l d i f f e r e n c e i n t e s t i s w e i g h t may i n d i c a t e t h a t s p o n t a n e o u s r e c r u d e s c e n c e h a d o c c u r r e d . The e f f e c t o f p i n e a l e c t o m y on g o n a d a l d e v e l o p m e n t i n d o v e s i s u n c l e a r - - a l t h o u g h r e m o v a l o f t h e p i n e a l d i d n o t c a u s e a s i g n i f i c a n t c h a n g e i n t e s t i s w e i g h t o r v o l u m e u n d e r e i t h e r d a y l e n g t h , i t d i d a p p e a r t o d i m i n i s h t h e d i f f e r e n c e s e e n b e t w e e n c o n t r o l b i r d s h e l d on t h e t w o p h o t o p e r i o d s . The b e h a v i o u r o f t h e d o v e s was more c l e a r l y a f f e c t e d by p h o t o p e r i o d a n d p i n e a l e c t o m y . The r e s u l t s c o n f i r m e a r l i e r t e s t s i n d i c a t i n g t h a t m a l e n e s t - b u i l d i n g i s s t i m u l a t e d by e x p o s u r e t o l o n g d a y l e n g t h s . The r e s p o n s e t o t h e d a y l e n g t h i s d e p e n d e n t u p o n t h e p r e s e n c e o f t h e p i n e a l g l a n d , b u t c o n t r a r y t o t h e s i t u a t i o n i n r o d e n t s , i n d o v e s t h e p i n e a l m e d i a t e s t h e s t i m u l a t o r y e f f e c t o f l o n g d a y s , r a t h e r t h a n t h e i n h i b i t o r y e f f e c t o f s h o r t o n e s . T h u s , i f t h e p i n e a l i s r e m o v e d , l o n g - d a y b i r d s e x h i b i t t h e l o w l e v e l s o f n e s t i n g a c t i v i t y n o r m a l l y a s s o c i a t e d w i t h s h o r t p h o t o p e r i o d s . The p o s i t i v e e f f e c t o f t h e 99 p i n e a l on dove behaviour i s not t o t a l l y unexpected, given e a r l i e r i n d i c a t i o n s of i t s progonadal r o l e i n s e v e r a l avian s p e c i e s , and i t s importance i n conveying long-day s t i m u l a t i o n i n some mammals (Hoffmann, 1981a; Herbert, 1981). Since p i n e a l e c t o m i z e d doves, u n l i k e sham-operated ones, d i s p l a y e d no d i f f e r e n c e s i n gonad s i z e under the two photoregimes, i t i s p o s s i b l e that the d e c l i n e i n n e s t i n g seen i n the long-day p i n e a l e c t o m i z e d b i r d s arose from a decrease i n t e s t i c u l a r androgen. R e s u l t s from the second experiment c l e a r l y i n d i c a t e that t h i s i s not the case. In the c a s t r a t e d , t e s t o s t e r o n e - i m p l a n t e d males, a l l of whom presumably had e q u a l l y h i g h l e v e l s of c i r c u l a t i n g androgen, pinealectomy s t i l l a b o l i s h e d the i n c r e a s e i n n e s t - b u i l d i n g normally seen under long d a y l e n g t h s . I t thus seems l i k e l y that the p i n e a l i s i n v o l v e d i n conveying photodependent changes i n responsiveness to androgens. The data presented here p r o v i d e no i n d i c a t i o n of a d d i t i v e e f f e c t s of short p h o t o s t i m u l a t i o n and pinealectomy. The f a c t t h a t pinealectomy a l t e r s only n e s t i n g b e h a v i o u r — t h e one a c t i v i t y which i s c o n s i s t e n t l y i n f l u e n c e d by ph o t o p e r i o d - - g i v e s f u r t h e r evidence that the p i n e a l i s a c t i n g as a mediator of pho t o p e r i o d e f f e c t s r a t h e r than through an independent mechanism. I f pinealectomy was having a d e b i l i t a t i n g e f f e c t (Ralph, 1970) or was ca u s i n g a general change i n behaviour separate from the photoperiod response, f o r example by changing the c i r c a d i a n rhythm of o v e r a l l a c t i v i t y , then i t seems probable t h a t other measures of behaviour would a l s o be a f f e c t e d . However, the only c l e a r i n f l u e n c e of pinealectomy was the 100 r e v e r s a l of long-day induced i n c r e a s e s in n e s t - b u i l d i n g ; other p h o t o p e r i o d - r e s i s t a n t a c t i v i t i e s were u n a l t e r e d . 4. Summary 1) I n t a c t , sham-operated male doves e x h i b i t e d higher l e v e l s of n e s t - b u i l d i n g on long days than on short ones. Photoperiod had no e f f e c t on male c o u r t s h i p , c o p u l a t o r y a c t i v i t y , or female behaviour. 2) Pinealectomy n u l l i f i e d the s t i m u l a t o r y e f f e c t s of long daylengths, reducing n e s t i n g a c t i v i t y i n long-day b i r d s but not i n the short-day group. P i n e a l removal had no e f f e c t on any other b e h a v i o u r a l a c t i v i t i e s . 3) T e s t i s weights were higher under long daylengths i n sham-operated males, but not i n p i n e a l e c t o m i z e d ones. 4) S t a n d a r d i z a t i o n of c i r c u l a t i n g androgen l e v e l s by c a s t r a t i o n and t e s t o s t e r o n e i m p l a n t a t i o n had no e f f e c t on the response to photoperiod or pinealectomy. As b e f o r e , n e s t i n g a c t i v i t y was the only behaviour which v a r i e d among the groups. Long-days s t i m u l a t e d n e s t i n g i n sham-operated b i r d s , but had no e f f e c t f o l l o w i n g pinealectomy. 101 C. The Be h a v i o u r a l E f f e c t of Exogenous Melatonin Treatment Since melatonin i s g e n e r a l l y thought to be the major p i n e a l hormone, demonstration of i t s a b i l i t y to reverse the e f f e c t of pinealectomy would provide f u r t h e r evidence of the p i n e a l ' s r o l e i n c o n t r o l l i n g n e s t - b u i l d i n g . The gonadal response of hamsters ( M e s o c r i c e t u s auratus ) to exogenous melatonin treatment has been found to vary a c c o r d i n g to both the mode and timing of the hormone treatment. Late a f t e r n o o n i n j e c t i o n s of melatonin cause gonadal atrophy i n i n t a c t animals ( R e i t e r et a l . , 1978a), but not i n p i n e a l e c t o m i z e d ones ( R e i t e r et a l . , 1976). I f the melatonin i s given e i t h e r as an e a r l y morning i n j e c t i o n or i n the form of a continuous r e l e a s e implant, i t i s i n e f f e c t i v e (Tamarkin et a l . , 1976), or even progonadal ( R e i t e r et a l . , 1977; R e i t e r et a l . , 1978b). In the f i r s t p i l o t t e s t I chose to use l a t e - d a y i n j e c t i o n s , s i n c e they were most s u c c e s s f u l i n r e v e r s i n g p i n e a l - m e d i a t e d changes i n hamsters. Late-day i n j e c t i o n s presumably mimic the n a t u r a l a f t e r n o o n r i s e i n melatonin which occurs i n b i r d s and mammals exposed to short daylengths ( B i n k l e y , 1980; R o l l a g et a l . , 1979). Since I d i d not know whether the e f f e c t of melatonin r e q u i r e s the presence of the p i n e a l i n b i r d s , as i n mammals, both i n t a c t and p i n e a l e c t o m i z e d males were used. I expected that the melatonin would have the opp o s i t e e f f e c t of pinealectomy and of exposure to short photoperiods: that i s , that i t would i n c r e a s e n e s t i n g both i n long-day p i n e a l e c t o m i z e d and short-day b i r d s . Because of the la c k of success of the aft e r n o o n melatonin 1 02 i n j e c t i o n s (see below), I dec i d e d to t r y other modes of a d m i n i s t r a t i o n . Melatonin i s r a p i d l y removed from the system ( C a r d i n a l i , 1981), and perhaps has only very short term e f f e c t s on behaviour. To t e s t t h i s , b i r d s were i n j e c t e d with l a r g e (.5mg) doses of melatonin j u s t before the b e h a v i o u r a l o b s e r v a t i o n s began. A l s o , as I was l o o k i n g f o r a s t i m u l a t o r y , r a t h e r than i n h i b i t o r y , e f f e c t of melatonin and s i n c e melatonin implants are progonadal i n hamsters ( R e i t e r et a l . , 1977) I a l s o t e s t e d t h i s route of a d m i n i s t r a t i o n . Again, I was a n t i c i p a t i n g an i n c r e a s e i n the n e s t i n g a c t i v i t y of short-day b i r d s . j_. M a t e r i a l s and Methods In the f i r s t p i l o t t e s t , the b i r d s used i n the preceding pinealectomy experiments were l e f t on a l i g h t c y c l e of 6L:18D f o r 4 months i n order to s t a n d a r d i z e t h e i r photoresponsiveness. The b i r d s were then p l a c e d on long (16L:8D, l i g h t s on 23:30-15:30) or short (7L:17D, l i g h t s on 8:30-15:30) days f o r 8 weeks. A l l of the males r e c e i v e d a 2.5 mm TP implant at the s t a r t of the 7th week. In a d d i t i o n , h a l f of each group was i n j e c t e d with 25 ug of melatonin (Sigma) i n .1 ml peanut o i l or with the v e h i c l e alone, each day one hour before the l i g h t s were turned o f f , beginning 7 days p r i o r to the o b s e r v a t i o n p e r i o d and c o n t i n u i n g to the 5th day of o b s e r v a t i o n s . Two 15-minute b e h a v i o u r a l r e c o r d i n g s were made each day at 9:30 and 11:30. In the second p i l o t study 21 p i n e a l - i n t a c t , c a s t r a t e d b i r d s which had not been used i n any experiments f o r at l e a s t 2 months were h e l d on 8L:16D f o r 4 weeks. A l l of the b i r d s were then 103 implanted with a 2.5 mm capsule of TP. Seven males a l s o r e c e i v e d a 5 mm melatonin implant (melatonin implants = Mim group). Beginning the same day and c o n t i n u i n g through the o b s e r v a t i o n p e r i o d , every b i r d was given e i t h e r a subcutaneous i n j e c t i o n of 0.5 mg melatonin i n .1 ml peanut o i l (melatonin i n j e c t i o n s = Minj group) or .1 ml of the v e h i c l e (Mim group, and c o n t r o l = C group) once a day at 9:00. Two 10-minute b e h a v i o u r a l o b s e r v a t i o n s were made a t 9:30 and 11:30. The power ( l i g h t ) was o f f f o r 3 hours on one a f t e r n o o n , r e q u i r i n g e l i m i n a t i o n of s t r i n g counts f o r a l l groups on the f o u r t h o b s e r v a t i o n day. 2. R e s u l t s The data based on b i r d s r e c e i v i n g a f t e r n o o n melatonin i n j e c t i o n s are given i n t a b l e XV and f i g . 10. The sample s i z e s were very s m a l l , but i t i s c l e a r t h a t melatonin d i d not have any marked e f f e c t on male c o u r t s h i p or n e s t - b u i l d i n g . The a c t i v i t y of long-day males was unexpectedly low: there was no d i s c e r n a b l e e f f e c t of e i t h e r photoperiod or p i n e a l removal i n the o i l -i n j e c t e d groups. T h i s r e s u l t may be due to the a l t e r e d l i g h t c y c l e s used i n t h i s experiment. The l i g h t s came on i n the lo n g -day room at 23:30, thus the b i r d s were not p a i r e d u n t i l 10 hours a f t e r 'dawn', whereas short-day b i r d s and long-day groups i n other experiments were p a i r e d o n l y one hour a f t e r ' l i g h t s - o n ' . The n e s t - b u i l d i n g a c t i v i t y of doves drops o f f i n the l a t e a f t e r n o o n (Martinez-Vargas, 1974; McDonald, 1976) and I suspect that t h i s d e c l i n e r e s u l t e d i n u n u s u a l l y low a c t i v i t y i n long-day 104 Table XV. The e f f e c t of l a t e - d a y i n j e c t i o n s of melatonin on the behaviour of TP-implanted c a s t r a t e s and t h e i r mates. The v a l u e s represent the means and ranges based on the t o t a l number of 15-second i n t e r v a l s or the t o t a l number of events over ten 10-minute o b s e r v a t i o n p e r i o d s . 105 _ * o < t>-f>i «>MM * v i o<<J xx. i S £ • I • O CD I s i F v i I • C V \ N M — » « t - ~ ~ «M J . * CD ^—• i ~ ~ CM^ • I • I C I M I fNvO MN M I MGL D M O- — — « — _ _ _ «D <w < r \ «g V\ O O » C U M T S t w f s j M M f t O - M A 0 K 3 > ^CD S £ V- ~ i SA ti, - i - i IU i w ~ 2 £ ~ ~ 2. £ ma. ON it) * *« - 1 • 1 O CS) ON — *V •* O «M 1 r\ E> i M — CD r*. IV O r^f — fw ON 1 M 1 K- 1 rf rf W -ON r - \ - < K > — fN- WN-t © N r f — rv •£ ^ • I • I ©I N I ON I Ml CI © I •N«-< o o **ON « * r f t r s > rf r > f*\ OD cc >^ ^-O 0D fs rf CD |N-IS) « I ?w ^ • i • i — i ^ i r\ i j f-rf ON i r > o e o N ON eve o rf «v> « — rf rf ONI tr\rg ©>c \C «- — H • I .1 N N «— I M | O l c c rf o «Mo ~*c- «MON «M e «a w v * K r ^ rv l w% — MI C- I » 106 F i g u r e 10. The e f f e c t of i n j e c t i o n s of 25 ug melatonin l a t e i n the day on the n e s t - b u i l d i n g behaviour of l o n g - and short-day males which had been p i n e a l i c t o m i z e d (PINX) or sham-operated (SHAM). A l l b i r d s were c a s t r a t e d and implanted with TP. The v a l u e s are based on the t o t a l s of t en 15-minute o b s e r v a t i o n p e r i o d s . i n d i v i d u a l score T mean score N E S T - BUILDING S T R I N G w I O D H D > o Z Q D > -< 2 m — r -z X o ,r-» to m > g r-•° El o o o Ul o to o 13 H D > Z X to n: > O Z o D > IS o o 8 o to o o _1_ o o - 1 Z X w > .2 m z X to > 2 m o 108 b i r d s i n the c u r r e n t experiment. In the second t e s t melatonin implants and e a r l y morning i n j e c t i o n s again f a i l e d to cause any n o t i c e a b l e changes i n male r e p r o d u c t i v e behaviour ( t a b l e XVI, f i g . 11). Melatonin implants were a s s o c i a t e d with higher mean l e v e l s of both a g g r e s s i v e and n e s t - o r i e n t e d c o u r t s h i p than seen i n the other groups, 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 were 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 n e s t - b u i l d i n g e i t h e r : melatonin may have s l i g h t l y suppressed n e s t i n g a c t i v i t y , but t o t a l s t r i n g counts were q u i t e c l o s e . 3_. D i s c u s s i o n The r e s u l t s obtained from t e s t s u s ing exogenous melatonin i n d i c a t e d that i t i s not an e f f e c t i v e s t i m u l a t o r of nest-b u i l d i n g a c t i v i t y . Research with hamsters has p l a i n l y shown that responsiveness to melatonin i s dependent upon the presence of the p i n e a l , and the ti m i n g and route of melatonin a d m i n i s t r a t i o n . The negative r e s u l t s u s i n g doves might represent use of an i n a p p r o p r i a t e experimental d e s i g n . In doves, u n l i k e hamsters, the p i n e a l appears to have a p o s i t i v e e f f e c t . Thus, long-day b i r d s c o u l d a l r e a d y be d i s p l a y i n g a maximal l e v e l of response, which c o u l d not be f u r t h e r augmented by melatonin treatment. However, r e s u l t s from p a r t of the afte r n o o n i n j e c t i o n t e s t , and from the second p i l o t study are based on short-day animals which performed low l e v e l s of nest-b u i l d i n g and s t i l l had the p o t e n t i a l f o r an i n c r e a s e i n the response. In a d d i t i o n , three d i f f e r e n t modes of a d m i n i s t r a t i o n 109 Table XVI. The c o u r t s h i p and c o p u l a t o r y a c t i v i t y of TP-implanted c a s t r a t e s t r e a t e d with a 5mm implant of melatonin (Mim), d a i l y i n j e c t i o n s of 0.5 mg melatonin ( M i n j ) , or v e h i c l e i n j e c t i o n s (C). The v a l u e s represent the mean and range, based on t o t a l s from ten 10-minute o b s e r v a t i o n p e r i o d s . C o n t r o l Mim M i n j n=7 n=7 n=7 Male c o u r t s h i p chase 5.3 7.0 3.9 (0-18) (4-14) (0-13) bow-coo 2.3 7.3 3.9 (0-13) (0-14) (0-16) s o l i c i t 87 152 90 (27-185) (58-247) (14-170) a t - n e s t 94 148 89 (39-225) (66-225) (21-174) C o p u l a t o r y a c t i v i t y beg 6.4 3.9 9.6 (0-16) (0-17) (0-19) b i l l 2.7 1.3 3.4 (0-13) (0-9) (0-12) c r o u c h 2.1 1.4 3.0 (0-6) (0-6) (0-6) mount 0.4 0.0 0.6 (0-2) (0-0) (0-2) Female b e h a v i o u r s o l i c i t 239 191 208 (110-304) (91-265) (129-356) a t - n e s t 299 241 258 (172-393) (119-361) (98-393) n e s t - b u i l d 2.1 1.1 1.4 (0-11) (0-5) (0-3) c a r r y 1.0 (0-5) 0.4 (0-2) 0.6 (0-3) 111 Figure 11. The e f f e c t of early morning melatonin injectsions or subcutaneous melatonin implants on the nest-building behaviour of short-day TP-implanted castrates. The values represent t o t a l s based on ten 10-minute observation periods. C control Min j early melatonin inj e c t i o n s 0 . 5 mg/d Mim melatonin implants 5 mm individual score mean score S T R I N G (p ieces ) N E S T - B U I L D I N G (intervals) o o o Ul o ro o o I r o Ui O ro Ul Ul o i Ul o o ro Ul 1 13 were used: e a r l y and l a t e - d a y i n j e c t i o n s and c o n t i n u o u s - r e l e a s e implants. In no case c o u l d any change i n n e s t - b u i l d i n g be determined. I t t h e r e f o r e appears that i f the p i n e a l i s a c t i n g through r e l e a s e of a hormone, that hormone i s not melatonin. 4. Summary 1) The n e s t - b u i l d i n g a c t i v i t y of sham-operated and p i n e a l e c t o m i z e d male doves h e l d on long or short days was not a f f e c t e d by l a t e - a f t e r n o o n i n j e c t i o n s of melatonin. S i m i l a r l y , both early-morning i n j e c t i o n s and constant r e l e a s e implants f a i l e d to simulate n e s t i n g i n short-day b i r d s . D. The E f f e c t of Chicken P i n e a l E x t r a c t Treatment on Nest- b u i l d i n g i n Male Doves _K I n t r o d u c t i o n The apparent i n a b i l i t y of melatonin to a f f e c t n e s t - b u i l d i n g a c t i v i t y , as found e a r l i e r , might suggest that p i n e a l messages are t r a n s m i t t e d by n e u r a l , r a t h e r than hormonal pathways. The f u n c t i o n of the p i n e a l - e f f e r e n t nerve f i b e r s i n b i r d s has not been determined; even t h e i r s i t e of t e r m i n a t i o n i s unknown (Ueck, 1979). There i s , however, no i n d i c a t i o n that the p i n e a l ' s s t a l k need be i n t a c t f o r the occurrence of any of i t s r e c o g n i z e d e f f e c t s . Thus, t r a n s p l a n t a t i o n of a p i n e a l i n t o the 1 14 eye chamber of a p r e v i o u s l y p i n e a l e c t o m i z e d , arrhythmic s t a r l i n g w i l l r e s t o r e an a c t i v i t y rhythm which matches that of the donor (Gwinner, 1978). Observation of a n t i g o n a d o t r o p i c a c t i v i t y i n p i n e a l e x t r a c t s from weaverbirds, the potency of which corresponds to the r e p r o d u c t i v e c o n d i t i o n of the b i r d (Saxena et a l . , 1979), suggests that gonadal responses are a l s o independent of n e u r a l output. The a l t e r n a t i v e e x p l a n a t i o n f o r the f a i l u r e of melatonin to reverse the e f f e c t of pinealectomy i s that the hormone treatment used was i n a p p r o p r i a t e , e i t h e r through the c h o i c e of the wrong mode of a d m i n i s t r a t i o n or the wrong substance. The f i r s t of these e x p l a n a t i o n s seems u n l i k e l y because s e v e r a l routes of melatonin a d m i n i s t r a t i o n were t e s t e d , none of which had a n o t i c e a b l e i n f l u e n c e on male behaviour. I d e c i d e d to use p i n e a l e x t r a c t s i n order to determine i f any p i n e a l substance c o u l d reverse the e f f e c t of gland removal. G o n a d a l l y - i n t a c t b i r d s were used, r a t h e r than t e s t o s t e r o n e -t r e a t e d c a s t r a t e s , s i n c e i n the p r e v i o u s experiment no marked changes i n gonadal development were seen as a r e s u l t of pinealectomy. 2. M a t e r i a l s and Methods a) Experimental S u b j e c t s The f o l l o w i n g procedure, from pinealectomy to the end of the e x t r a c t experiment, was performed on f i v e s e t s of 8-10 b i r d s , staggered at one-week i n t e r v a l s . Every set c o n s i s t e d of 2-3 i n d i v i d u a l s from each treatment group (short-day sham and 115 p i n e a l e c t o m i z e d = SC, SP; long-day sham and p i n e a l e c t o m i z e d =LC, LP). Seventy e x p e r i m e n t a l l y naive male doves which had been sexed and given p r i o r breeding experience were p r e t e s t e d and p i n e a l e c t o m i z e d or sham-operated. F o r t y - e i g h t of these were p l a c e d on the long or short photoperiods f o r 8 weeks, then b e h a v i o u r a l r e c o r d i n g s were made over a 5-day p e r i o d . A l l b i r d s r e c e i v e d a subcutaneous i n j e c t i o n of ,2ml .9% s a l i n e between 9:00- 9:30 am beginning seven days before t e s t i n g and c o n t i n u i n g to the end of the r e c o r d i n g s e s s i o n . The b i r d s were r e t a i n e d on the same l i g h t c y c l e s f o r an a d d i t i o n a l 4 weeks, then r e t e s t e d . In t h i s case, each i n d i v i d u a l r e c e i v e d an i n j e c t i o n of chi c k e n p i n e a l e x t r a c t every morning f o r the week preceding the t e s t and d u r i n g the t e s t p e r i o d i t s e l f . b) P r e p a r a t i o n of e x t r a c t s Two-hundred and f i f t y chicken heads were obtained from Hallmark P o u l t r y Co. (Vancouver, Canada) i n three batches. The c h i c k e n s , r e p r e s e n t i n g approximately equal numbers of males and females, were about 7 weeks o l d . They were d e c a p i t a t e d i n the morning and the heads were kept r e f r i g e r a t e d u n t i l used, no more than 4 hours l a t e r . The p i n e a l and c h o r o i d plexus were removed from each head. These were ground up i n s a l i n e , spun down i n a c e n t r i f u g e , and the supernatant d i l u t e d with .9% s a l i n e to a c o n c e n t r a t i o n of 50 glands/30 ml. The e x t r a c t was frozen i n 2 ml q u a n t i t e s f o r a maximum p e r i o d of 3 weeks, and thawed each 116 day j u s t before being i n j e c t e d . The b i r d s r e c e i v e d e x t r a c t from 1/3 gland i n .2 ml sc once a day f o r 12 days (= a t o t a l of 4 g l a n d s / b i r d ) . 3. R e s u l t s As b e f o r e , n e i t h e r pinealectomy nor daylength a l t e r e d any measures of male c o u r t s h i p , c o p u l a t o r y a c t i v i t y or female behaviour ( t a b l e s XVII and X V I I I ) . Long daylengths were a s s o c i a t e d with s i g n i f i c a n t l y higher l e v e l s of n e s t - b u i l d i n g than short days i n sham-operated c o n t r o l s ( f i g . 12). Once more, pinealectomy r e s u l t e d i n a s i g n i f i c a n t decrease of l o n g -day scores to a l e v e l comparable to that seen i n short-day b i r d s . U n l i k e the p r e v i o u s t e s t , however, there appeared to be an a d d i t i v e e f f e c t of p h o t o p e r i o d - and pinealectomy-induced i n h i b i t i o n . Pinealectomy caused a s i g n i f i c a n t d e c l i n e i n the amount of s t r i n g c o l l e c t e d by short-day b i r d s , although d i f f e r e n c e s i n c a r r y i n g and n e s t - b u i l d i n g were not s i g n i f i c a n t . In a d d i t i o n , exposure to short daylengths was a l s o capable of c a u s i n g a drop i n a l l measures of n e s t i n g even i n the absence of the p i n e a l gland (LP vs SP, f i g . 12). When a l l i n d i v i d u a l s were t r e a t e d d a i l y with i n j e c t i o n s of c h i c k e n p i n e a l e x t r a c t , there were no longer any d i f f e r e n c e s i n n e s t i n g a c t i v i t y between groups of sham-operated b i r d s h e l d on the two photoregimes ( f i g . 13). In a d d i t i o n , LP b i r d s were as a c t i v e as LC ones. The e x t r a c t treatment d i d not, however, a b o l i s h the d i f f e r e n c e i n n e s t i n g between long and short-day p i n e a l e c t o m i z e d b i r d s . 117 T a b l e X V I I . The e f f e c t o f p i n e a l e c t o m y and d a y l e n g t h on the c o u r t s h i p a c t i v i t y o f g o n a d a l l y - i n t a c t m a les. The v a l u e s r e p r e s e n t the means and ranges based on the t o t a l number o f 15-second i n t e r v a l s or the t o t a l number o f ev e n t s over the 5-day t e s t p e r i o d . LONG DAY SHORT DAY Sham n=12 P i n e a l x n=12 Sham n=12 P i n e a l x n=12 Mal e c o u r t s h i p chase 7.1 (1-20) 9.7 (3-20) 13.2 (2-26) 6.9 (1-17) bow-coo 14.9 (0-42) 13.4 (0-53) 21.0 (0-60) 7.8 (0-15) s o l i c i t 355 361 (124-604) (124-618) 259 (72-650) 308 (1-743) a t - n e s t 357 337 (162-606) (132-696) 260 (90-601) 308 (59-728) 118 Table XVIII. The effect of photoperiod and pinealectomy on the copulatory behaviour of intact males, and on the reproductive behaviour of th e i r mates. The values represent the mean and range based on the t o t a l number of 15-second inter v a l s or the t o t a l number of observed events over the 5-day test period. 119 LONG DAY SHORT DAY Sham n=12 P i n e a l x n=12 Sham n=12 P i n e a l x n=12 C o p u l a t o r y a c t i v i t y beg 10.0 (0-44) 8.3 (0-28) 11.7 (1-26) 10.5 (0-26) b i l l 5.5 (0-19) 2.9 (0-14) 5.9 (0-18) 5.3 (0-16) c r o u c h 3.3 (0-19) 1.9 (0-7) 3.8 (0-8) 3.8 (0-8) mount 1.6 (0-6) 1.0 (0-2) 2.1 (0-3) 2.0 (0-5) Female b e h a v i o u r a.crouch 2.3 (0-9) 2.1 (0-8) 3.7 (1-9) 3.3 (1-10) s o l i c i t 687 614 (259-1033) (293-944) 668 513 (372-956) (195-912) a t - n e s t 745 754 (331-1016) (503-1125) 761 (328-1129) 687 (232-1143) n e s t - b u i l d 7.4 (0-71) 8.0 (0-57) 4.0 (0-21) 2.3 (0-15) c a r r y 1.8 (0-17) 1.5 (0-14) 1.4 (0-7) 0.8 (0-6) 120 F i g u r e 12. The e f f e c t of d a y l e n g t h and p i n e a l e c t o m y on the n e s t - b u i l d i n g b e h a v i o u r of g o n a d a l l y - i n t a c t male doves. The v a l u e s r e p r e s e n t the mean l e v e l of a c t i v i t y , based on the t o t a l s f o r the 5-day t e s t p e r i o d . LC long-day c o n t r o l LP long-day p i n e a l e c t o m i z e d SC s h o r t - d a y c o n t r o l SZZ SP s h o r t - d a y p i n e a l e c t o m i z e d groups w i t h d i f f e r e n t s u b s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t , based on 2 - t a i l e d U - t e s t s , p < .05 S T R I N G (pieces) C A R R Y I N G (events) N E S T - B U I L D I N G (intervals) III rt cr rt 122 F i g u r e 1 3 . T h e c o m b i n e d e f f e c t o f d a i l y i n j e c t s i o n s o f c h i c k e n p i n e a l e x t r a c t , p i n e a l e c t o m y a n d d a y l e n g t h o n t h e n e s t - b u i l d i n g b e h a v i o u r o f g o n a d a l l y - i n t a c t m a l e d o v e s . T h e v a l u e s r e p r e s e n t t h e mean l e v e l o f a c t i v i t y , b a s e d on t h e t o t a l s f o r t h e 5 - d a y t e s t p e r i o d . LC l o n g - d a y c o n t r o l LP l o n g - d a y p i n e a l e c t o m i z e d SC s h o r t - d a y c o n t r o l SP s h o r t - d a y p i n e a l e c t o m i z e d g r o u p s w i t h d i f f e r e n t s u b s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t , b a s e d on 2 - t a i l e d U - t e s t s , p < . 0 5 S T R I N G (pieces) _ KJ U o o o o o o J I I C A R R Y I N G (events) N E S T - B U I L D I N G (intervols) w *. m o o o o o 1 1 1 1 1 o o o Ul o _J 124 Male n e s t - o r i e n t e d c o u r t s h i p appeared to be g r e a t e r on long-days than on short-days ( t a b l e XIX). The d i f f e r e n c e between sham-operated groups was, i n f a c t , s i g n i f i c a n t f o r the time spent by the male at the nest, but these r e s u l t s can only be c o n s i d e r e d t e n t a t i v e , s i n c e the p r i o r a n a l y s i s of v a r i a n c e f a i l e d to reach s t a t i s t i c a l s i g n i f i c a n c e . Other measures of male c o u r t s h i p , c o p u l a t o r y a c t i v i t y , and female behaviour d i d not d i f f e r among the v a r i o u s groups ( t a b l e XX). F i g u r e 14 i n d i c a t e s the d i r e c t i o n and mean magnitude of the change i n n e s t - b u i l d i n g between the f i r s t ( v e h i c l e - t r e a t e d ) and second ( e x t r a c t - t r e a t e d ) t e s t s . In the long-day sham-operated b i r d s n e s t i n g a c t i v i t y was lower f o l l o w i n g e x t r a c t treatment. In the other three groups there were s i g n i f i c a n t i n c r e a s e s i n two (LP, SP) or three (SC) of the measures examined. Although the magnitude of the change appeared s l i g h t i n some cases, i n every i n s t a n c e i t represented a mean i n c r e a s e or decrease of at l e a s t 25% of the l e v e l d i s p l a y e d i n the f i r s t ( v e h i c l e ) experiment, and i n some s i t u a t i o n s was as high as 100%. Wilcoxon t e s t s ( 2 - t a i l e d ) were performed on data f o r other a c t i v i t i e s as w e l l . Of these remaining 52 comparisons (13 a c t i v i t y measures x 4 groups) only the 8 l i s t e d i n t a b l e XXI showed a s i g n i f i c a n t change. E x t r a c t - t r e a t m e n t was a s s o c i a t e d with an i n c r e a s e i n bow-cooing i n SP b i r d s and a decrease i n cha s i n g i n LP and SC b i r d s . Copulatory a c t i v i t y (begging and b i l l i n g ) was lower i n the SC group. The l a r g e s t and most c o n s i s t e n t change was seen i n female n e s t - s o l i c i t i n g ; there was a s i g n i f i c a n t d e c l i n e i n groups of females p a i r e d with both LP 125 T a b l e XIX. The e f f e c t o f c h i c k e n p i n e a l e x t r a c t t r e a t m e n t on the c o u r t s h i p a c t i v i t y o f p i n e a l e c t o m i z e d and sham-o p e r a t e d male doves h e l d on l o n g or s h o r t d a y l e n g t h s . The v a l u e s r e p r e s e n t the means and ranges based on the t o t a l number o f 15-second i n t e r v a l s or the t o t a l number o f e v e n t s over the 5-day t e s t p e r i o d . LONG DAY SHORT DAY Sham n=12 P i n e a l x n=12 Sham n=12 P i n e a l x n=12 Male c o u r t s h i p chase 5.8 (0-16) 5.3 (0-24) 6.7 (1-19) 5.9 (2-17) bow-coo 16.8 (0-56) 14.6 (0-97) 20.9 (2-79) 13.9 (0-45) s o l i c i t 366 (131-608) 322 (114-688) 247 (97-409) 261 (2-494) a t - n e s t 396 337 (145-728) (128-645) 224 (110-447) 254 (90-505) 126 Table XX. The c o p u l a t o r y a c t i v i t y and female behaviour of p a i r s i n which the males were exposed to long or short days a f t e r being sham-operated or p i n e a l e c t o m i z e d . A l l males were t r e a t e d with p i n e a l e x t r a c t . The v a l u e s r e p r e s e n t the means and ranges based on the t o t a l number of 15-second i n t e r v a l s or the t o t a l number of events over the 5-day t e s t p e r i o d . 127 LONG DAY Sham n=12 P i n e a l x n«12 SHORT DAY Sham n=12 P i n e a l x n=12 C o p u l a t o r y a c t i v i t y beg 5.6 (0-17) 4.3 (0-20) 4.8 (0-12) 7.4 (0-28) b i l l 2.8 (0-16) 1.8 (0-8) 2.6 (0-9) 2.8 (0-6) c r o u c h 2.2 (0-5) 2.6 (0-8) 2.3 (0-6) 2.3 (0-6) mount 1.4 (0-3) 1.3 (0-4) 1.8 (0-3) 1.8 (0-5) Female b e h a v i o u r a.crouch 1.5 (0-4) 3.0 (0-23) 1.5 (0-6) 1.1 (0-3) s o l i c i t 381 457 (163-634) (273-757) 468 465 (104-717) (225-638) a t - n e s t 207 328 (441-1057) (359-1182) 348 (377-1139) 294 (514-1128) n e s t - b u i l d 3.8 (0-12) 3.8 (0-29) 6.0 (0-31) 5.0 (0-22) c a r r y 0.9 (0-6) 1.4 (0-10) 2.4 (0-6) 4.3 (0-3) 128 F i g u r e 14. The change i n the n e s t - b u i l d i n g b e h a v i o u r of male doves as a r e s u l t of t r e a t m e n t w i t h c h i c k e n p i n e a l e x t r a c t . LC long-day c o n t r o l LP long-day p i n e a l e c t o m i z e d SC s h o r t - d a y c o n t r o l SP s h o r t - d a y p i n e a l e c t o m i z e d a p<.05 b p<.05 based on W i l c o x o n t e s t , 1 - t a i l e d based on W i l c o x o n t e s t , 2 - t a i l e d 130 T a b l e X X I . Changes i n a c t i v i t i e s o t h e r than n e s t - b u i l d i n g , as a r e s u l t o f p i n e a l - e x t r a c t t r e a t m e n t o f the males. The v a l u e s r e p r e s e n t the mean change i n a c t i v i t y and the mean o f the change as a p e r c e n t o f the v e h i c l e - t r e a t m e n t l e v e l s . A l l d i f f e r e n c e s are s i g n i f i c a n t at*A=.05, W i l c o x o n m a t c h e d - p a i r s s i g n e d - r a n k t e s t , 2 - t a i l e d . A c t i v i t y Group Mean Change Mean % Change Male C o u r t s h i p chase LP -4.4 -45% SC -6.5 -49% bow-coo SP +6.2 +80% C o p u l a t o r y a c t i v i t y beg SC -6.8 -58% b i l l SC -3.3 -56% Female b e h a v i o u r a.crouch SP -2.2 -67% s o l i c i t LC -306 -45% LP -157 -26% 131 and LC males, as w e l l as a n o n s i g n i f i c a n t change i n the remaining two groups (SP, -49; SC, -200). I n i t i a l l y , I had no i n t e n t i o n of measuring the i n c i d e n c e of moulting i n the v a r i o u s groups. However, soon a f t e r the f i r s t set of b i r d s were p l a c e d on the experimental l i g h t regimes i t was noted that the SP b i r d s l o s t n e a r l y a l l of the f e a t h e r s on t h e i r heads, as w e l l as many on the wings. I decided to keep t r a c k of the occurrence of moulting f o r the f i r s t 6 weeks a f t e r the b i r d s were put on the two l i g h t c y c l e s (= f i r s t 9 weeks a f t e r p i nealectomy). Table XXII shows that moulting i s a f f e c t e d by daylength and p i n e a l removal (p<.00l, Chi-square t e s t ) . A l l but one of the SP b i r d s moulted w i t h i n 3 weeks of being p l a c e d on the short daylength. A few of the sham-operated b i r d s a l s o moulted, but none of the long-day ones d i d . At the end of the e x t r a c t experiment the LP b i r d s were a l s o p l a c e d on 8L;16D. Wit h i n 3 weeks 9 of 12 had l o s t most of t h e i r head f e a t h e r s and 2 d i s p l a y e d l i g h t moulting. 4. D i s c u s s i o n The e f f e c t of pinealectomy on moulting was not a n t i c i p a t e d , but i t lends f u r t h e r support to the s u p p o s i t i o n that the p i n e a l i s i n v o l v e d i n responsiveness to daylength. The t i m i n g of moulting i n b i r d s v a r i e s from s p e c i e s to s p e c i e s , and seems to be r e l a t e d to p a t t e r n s of m i g r a t i o n and r e p r o d u c t i o n (see review by Payne, 1972). In many s p e c i e s , moulting, which i s e n e r g e t i c a l l y c o s t l y , occurs at the end of the breeding season (Murton & Westwood, 1977) i n response to the shortened 1 32 Table XXII. The e f f e c t of photoperiod and pinealectomy on moulting i n male doves. Values represent the number of b i r d s which f i r s t e x h i b i t e d heavy moult d u r i n g the s p e c i f i e d p e r i o d of exposure to the experimental l i g h t c y c l e s . Latency to moult 1-3 weeks 3-6 weeks no moult Long day Short day C o n t r o l n=12 0 0 1 2 P i n e a l x n=1 2 0 0 12 C o n t r o l n=1 2 2 3 7 P i n e a l x n=1 2 1 1 0 1 photoperiod ( O i s h i & Lauber, 1974a). Although fe a t h e r replacement i s maintained by h i g h t h y r o x i n e l e v e l s , i t appears that o t h e r , d i f f e r e n t mechanisms are e s s e n t i a l f o r the i n d u c t i o n of moulting ( W i n g f i e l d & Farner, 1980). T h i s second mechanism might i n v o l v e a d e c l i n e i n s t e r o i d p r o d u c t i o n (Payne, 1972). Pigeons and doves moult s t e a d i l y throughout the breeding season, but the r a t e can be a c c e l e r a t e d by t h y r o x i n e i n j e c t i o n s (Tanabe, 1980) or by exposure to short photoperiods, and can be delayed with androgen or estrogen treatment (Murton & Westwood, 1977; Kobayashi, 1954). Pinealectomy i n c r e a s e d f e a t h e r l o s s soon a f t e r exposure to short daylengths, and i t i s d u r i n g t h i s p e r i o d that decreased photoperiod i s r e p o r t e d to cause t e s t i c u l a r r e g r e s s i o n and lowered plasma t e s t o s t e r o n e l e v e l s i n doves ( B a l t h a z a r t et a l . , 1981). Removal of the p i n e a l might augment the i n h i b i t o r y e f f e c t of the shor t days on t e s t i s f u n c t i o n , or i t may s t i m u l a t e moulting through i n c r e a s e d t h y r o i d f u n c t i o n or even through an a l t e r e d s e n s i t i v i t y to androgen s t i m u l a t i o n . Pinealectomy i s a l s o r e p o r t e d to advance the c o l l a p s e of the 1 33 t e s t e s and the onset of moulting i n s t a r l i n g s h e l d on 12L:12D (Gwinner & D i t t a m i , 1980). The r e s u l t s of t h i s experiment c o n f i r m the e a r l i e r o b s e r v a t i o n of decreased n e s t i n g behaviour i n long-day b i r d s f o l l o w i n g pinealectomy. Again, long-day sham-operated c o n t r o l s were much more a c t i v e than long-day p i n e a l e c t o m i z e d males or than short-day shams. The interdependance of the two f a c t o r s was a l s o r e a f f i r m e d : only n e s t - b u i l d i n g i s a l t e r e d by e i t h e r p h o t o p eriod or p i n e a l removal. P i n e a l e x t r a c t treatment i n c r e a s e d n e s t - b u i l d i n g i n both short-day groups, and i n long-day p i n e a l e c t o m i z e d b i r d s . I t might be argued that the chicken e x t r a c t c o n t a i n e d a s t i m u l a t o r y substance, the p r o d u c t i o n of which has no r e l e v a n c e to the p h o t o p e r i o d i c c o n t r o l of n e s t i n g i n doves. If t h i s were the case, one would assume that the e x t r a c t should i n c r e a s e a c t i v i t y i n a l l groups, with the p o s s i b l e e x c e p t i o n of LC males which may a l r e a d y be d i s p l a y i n g a maximum response. In f a c t , the e x t r a c t not only f a i l e d to i n c r e a s e n e s t - b u i l d i n g i n the LC b i r d s , but the a c t i v i t y l e v e l s of t h i s group a c t u a l l y showed a s i g n i f i c a n t decrease from t h e i r pre-treatment l e v e l s . T h i s observed d e c l i n e i n long-day c o n t r o l s thus e l i m i n a t e s the p o s s i b i l i t y of a non-s p e c i f i c i n c r e a s e between the two experiments which was t o t a l l y independent of the e x t r a c t treatment, and a l s o p o i n t s to a f u n c t i o n a l r e l a t i o n s h i p between the p r o d u c t i o n or presence of some p i n e a l substance, and the e f f e c t of the p i n e a l and p h o t o p eriod on behaviour. Although n e s t - b u i l d i n g was the only category of behaviour 1 34 showing a c o n s i s t e n t change, the l e v e l s of s e v e r a l other a c t i v i t i e s were s i g n i f i c a n t l y a l t e r e d between the f i r s t and second t e s t s . Of these, a l l but one (bow-cooing) were reduced i n the second experiment. Again, there appears to be a marked d i s t i n c t i o n between n e s t - b u i l d i n g and the other components of r e p r o d u c t i v e a c t i v i t y . The d e c l i n e i n female n e s t - s o l i c i t i n g i s i n t e r e s t i n g . I t appears to represent a decrease i n the a c t i v e d i s p l a y of c o u r t s h i p - - t h e p a s s i v e measure of time spent a t - t h e -nest was not a f f e c t e d . In a d d i t i o n , the change seems u n r e l a t e d to the treatment of the males, s i n c e i t was seen i n almost a l l females. There i s some evidence of an annual s h i f t i n the responsiveness of female doves. L i l e y (1976b) found a s t i m u l a t o r y e f f e c t of long photoperiods on the behaviour of female doves dur i n g the s p r i n g , but not i n the w i n t e r . Perhaps a v a r i a t i o n i n responsiveness o c c u r r e d here, although only a one-month p e r i o d separated the two t e s t s . A l t e r n a t i v e l y , the females may have been a f f e c t e d by the experience of being p a i r e d f o r 5 days and then separated only one month before being r e -p a i r e d with a new male. The present r e s u l t s d i f f e r from those of the pre v i o u s pinealectomy experiment i n one important regard: i n a d d i t i o n to a c o n s i d e r a b l e r e d u c t i o n i n the n e s t - b u i l d i n g of long-day b i r d s , pinealectomy a l s o r e s u l t e d i n a s i m i l a r , but much s m a l l e r , decrease i n short-day males. The n e s t i n g a c t i v i t y of LP b i r d s i s g r e a t e r than that of SP b i r d s , and t h i s d i f f e r e n c e p e r s i s t s f o l l o w i n g e x t r a c t treatment. The most l o g i c a l e x p l a n a t i o n f o r these d i f f e r e n c e s i s that daylength i s i n f l u e n c i n g behaviour 135 through two d i s t i n c t , a d d i t i v e mechanisms: the major e f f e c t of photoperiod depends on the presence of the p i n e a l gland, but long daylengths a l s o s t i m u l a t e n e s t - b u i l d i n g , to a much l e s s e r e xtent, independently of the p i n e a l . Although the b i r d s were go n a d a l l y i n t a c t , i t i s u n l i k e l y t h a t v a r i a t i o n i n t e s t i c u l a r androgen p r o d u c t i o n was r e s p o n s i b l e f o r the pineal-independant e f f e c t as: 1) i n the e a r l i e r experiment no d i f f e r e n c e s were observed i n t e s t i s weight or volume i n p i n e a l e c t o m i z e d b i r d s h e l d on long and short days 2) although daylength i n f l u e n c e s c i r c u l a t i n g androgen l e v e l s i n male doves, t h i s e f f e c t d i s a p p e a r s a f t e r 13 weeks ( B a l t h a z a r t et a l . , 1981) 3) i f photoperiod does a l t e r androgen l e v e l s and i f these are causing d i f f e r e n c e s i n behaviour, one would expect to f i n d s i m i l a r changes in other testosterone-dependent a c t i v i t i e s , such as c o u r t s h i p F u r t h e r t e s t s are needed i n order to determine the nature of t h i s second photoperiod mechanism. Since i t was not observed i n the f i r s t pinealectomy experiment, i t may be r e l a t e d to a seasonal s h i f t i n responsiveness to l i g h t s t i m u l i (see F o l l e t t & Robinson, 1980). V 136 5. Summary 1 ) Pinealectomy decreases the s t i m u l a t o r y e f f e c t of long ' daylengths on n e s t - b u i l d i n g behaviour. 2) Treatment with chicken p i n e a l e x t r a c t c o u n t e r a c t s both short day- and pinealectomy-induced i n h i b i t i o n of n e s t - b u i l d i n g . 3) The e f f e c t of daylength on n e s t - b u i l d i n g p e r s i s t s to some extent f o l l o w i n g pinealectomy, i n d i c a t i n g the e x i s t e n c e of a second, pineal-independent mechanism mediating photoresponsiveness. E. The E f f e c t of Exogenous S e r o t o n i n on Nesting A c t i v i t y i n  P i n e a l e c t o m i z e d, Short-day B i r d s J _ . I n t r o d u c t i o n The r e s u l t s from the p i n e a l e x t r a c t experiment show t h a t the a v i a n p i n e a l gland c o n t a i n s some substance which i s capable of r e v e r s i n g the low l e v e l s of n e s t - b u i l d i n g seen i n p i n e a l e c t o m i z e d and i n short-day exposed b i r d s . The i d e n t i t y of t h i s substance i s unknown, but i t does not appear to be melatonin. Numerous authors have p r e v i o u s l y argued t h a t melatonin i s not the only a c t i v e p i n e a l c o n s t i t u e n t a f f e c t i n g the r e p r o d u c t i v e system, s i n c e m e l a t o n i n - f r e e bovine p i n e a l e x t r a c t s have a n t i - g o n a d o t r o p i c a c t i v i t y i n r a t s and mice ( O r t s , 1977; Damian et a l . , 1978; Damian et a l . , 1981). The importance of melatonin has a l s o been qu e s t i o n e d on the b a s i s of 1 37 immunization experiments. Hamsters immunized a g a i n s t melatonin or NAS, which i s an e s s e n t i a l i ntermediate i n melatonin s y n t h e s i s , d i s p l a y the same decrease i n gonad weight under s h o r t days as c o n t r o l s (Brown et a l . , 1976). In r a t s , immunization a g a i n s t melatonin a c t u a l l y causes a decrease i n plasma t e s t o s t e r o n e , suggesting that melatonin i s pro- rather than a n t i - g o n a d a l (Brown et a l . , 1981). Many other p i n e a l compounds have been proposed as the 'true' p i n e a l hormone. Pavel and h i s c o l l e g u e s maintain t h a t a r g i n i n e v a s o t o c i n (AVT) i s the a c t u a l p i n e a l hormone, and t h a t melatonin a c t s as a r e l e a s e r f o r v a s o t o c i n (Pavel & G o l d s t e i n , 1979). However, there i s now s e r i o u s doubt concerning both the i d e n t i t y of t h i s p i n e a l peptide ( R e i t e r , 1981) and the a c t i v i t y of AVT ( R e i t e r , 1980). Larger p e p t i d e s with r e p o r t e d gonad-i n h i b i t i n g q u a l i t i e s have a l s o been r e p o r t e d (Ebels & Benson, 1978; Vaughan, 1981). There i s another p o s s i b i l i t y , however, which stands out from the r e s t . S e r o t o n i n (5HT), the p r e c u r s o r of melatonin, i s present i n the p i n e a l , and has been i m p l i c a t e d i n the c o n t r o l of r e p r o d u c t i v e behaviour i n mammals (see below). In a d d i t i o n , 5HT treatment and pinealectomy have both been shown to have p a r a l l e l e f f e c t s on a c t i v i t y l e v e l s i n j u v e n i l e sockeye salmon (Byrne, 1968). The e x t r a c t used i n the c u r r e n t study c o n t a i n e d e v e r y t h i n g except membrane-bound substances and t h e r e f o r e can give no i n d i c a t i o n of the a c t i v e component. However, the f a c t that the e x t r a c t , which was produced from daytime k i l l e d b i r d s , was so e f f e c t i v e suggests that melatonin was not the important 138 c o n s t i t u e n t . P i n e a l melatonin content d e c l i n e s s h a r p l y i n b i r d s f o l l o w i n g the onset of l i g h t ( B i n k l e y , 1980). The p i n e a l glands of 8-week o l d chickens have daytime melatonin c o n c e n t r a t i o n s of 3.8ng/gland (Ralph et a l . , 1975; Pang et a l . , 1977). Thus, my doves were probably r e c e i v i n g l e s s than 1.5ng melatonin/day, most of which would be c l e a r e d from the system before r e a c h i n g the b r a i n ( C a r d i n a l i , 1981). Since the i n j e c t i o n of 25ug or 500 ug melatonin had no d i s c e r n a b l e e f f e c t when examined e a r l i e r , i t i s l i k e l y that some compound other than melatonin i s r e p o n s i b l e f o r the e f f e c t i v e n e s s of the e x t r a c t . L e v e l s of-5HT in the p i n e a l are higher than those of other i n d o l e s (Quay,1974). Quay, (1966) r e p o r t e d an e a r l y morning peak c o n c e n t r a t i o n of s e r o t o n i n i n the pigeon p i n e a l . In the c h i c k e n , 5HT reaches a peak c o n c e n t r a t i o n of 27ng/gland e a r l y i n the day (Brammer & B i n k l e y , 1979). The amount c o n t a i n e d i n one dose of e x t r a c t would thus be q u i t e low (9ng), but would s t i l l be much higher than melatonin. The exact r o l e of the high c o n c e n t r a t i o n of s e r o t o n i n i n the p i n e a l gland i s unknown. Some i s turned i n t o melatonin by the a c t i o n of the enzymes HIOMT and NAT (see f i g . 7), but c o n v e r s i o n to melatonin can only account f o r a small p o r t i o n of the 5HT present (Pevet, 1981). Some s e r o t o n i n i s metabolised by monoamine oxiadase (MAO), and some i s taken up by nerve t e r m i n a l s (Brammer & B i n k l e y , 1979). Wurtman, i n h i s summary of a symposium on the p i n e a l g l a n d h e l d i n 1970, posed s e v e r a l q u e s t i o n s f o r f u t u r e p i n e a l r e s e a r c h (Wolstenholme & Knight, 1971), one of which was: "Does [the p i n e a l ] s e c r e t e s e r o t o n i n ? " . 1 39 L i t t l e a t t e n t i o n has been focussed on the p o s s i b i l i t y that s e r o t o n i n i s r e l e a s e d from the p i n e a l , and today we are no c l o s e r to answering h i s q u e s t i o n . There appears to be a c l o s e a s s o c i a t i o n between p i n e a l f u n c t i o n and hypothalamic s e r o t o n i n l e v e l s . Pinealectomy and melatonin treatment have been shown to a l t e r l e v e l s of hypothalamic s e r o t o n i n i n r a t s (Moszkowska et a l . , 1971; Anton-Tay, 1971) and f e r r e t s (Yates & Herbert, 1979). 5HT treatment can block pinealectomy-induced o v u l a t i o n i n r a t s (Mess et a l . , 1981) and can a l s o mimic some of the e f f e c t s of melatonin treatment on gonadotropic a c t i v i t y (Kamberi et a l . , 1970; M a r t i n et a l . , 1977). These r e s u l t s have l e d s e v e r a l authors to propose that the p i n e a l a c t s on the r e p r o d u c t i v e system through melatonin-induced a c t i v a t i o n of s e r o t o n e r g i c pathways i n the hypothalamus (Moszkowska et a l . , 1971; T r e n t e n i et a l . , 1979; Mess et a l . , 1981). T h i s suggestion i s supported by the o b s e r v a t i o n that the rhythm of 5HT i n the hypothalamus of female f e r r e t s i s s i m i l a r l y a f f e c t e d by exposure to short photoperiods and by treatment with melatonin (Yates & Herbert, 1979). S e r o t o n i n has been shown to i n f l u e n c e r e p r o d u c t i v e behaviour i n rodents. In r a t s , d e p l e t i o n of s e r o t o n i n p o t e n t i a t e s female r e c e p t i v i t y (Ward et a l . , 1975; E v e r i t t et a l . , 1975b; Meyerson & Malmnas, 1977; E v e r i t t , 1977; Crowley & Zemlan, 1981a), and male c o p u l a t o r y a c t i v i t y (Dewsbury, 1975; S o u l a i r a c & S o u l a i r a c , 1975; Gessa & Tagliamonte, 1975; A h l e n i u s et a l . , 1980). These r e s u l t s are commonly i n t e r p r e t e d as evidence that s t e r o i d hormones a c t i v a t e sexual behaviour by 140 d e p r e s s i n g b r a i n s e r o t o n e r g i c mechanisms ( E v e r i t t et a l . , 1975; Ward et a l . , 1975), but the f a c t that monoamine i n h i b i t o r s are i n e f f e c t i v e i n the absence of s t e r o i d s t i m u l a t i o n suggests i n s t e a d that n e u r o t r a n s m i t t e r s f u n c t i o n by a l t e r i n g r esponsiveness to hormones (see Nock & Feder, 1981). S e v e r a l p o i n t s can be drawn from the s t u d i e s c i t e d here which i n d i c a t e an a s s o c i a t i o n of photoperiod, the p i n e a l , s e r o t o n i n l e v e l s and behaviour: 1) B r a i n 5HT a c t i v i t y i s r e l a t e d to p i n e a l a c t i v i t y s i n c e - s e r o t o n i n f r e q u e n t l y has the same e f f e c t as melatonin on r e p r o d u c t i v e endocrinology -melatonin treatment can a l t e r hypothalamic 5HT rhythms and content 2) Photoperiod can a f f e c t hypothalamic and p i n e a l 5HT rhythms 3) S e r o t o n i n i n h i b i t s s t e r o i d - i n d u c e d sexual behaviour. 4) 5HT treatment mimics the e f f e c t of pinealectomy on a c t i v i t y l e v e l s i n young salmon. Taken together, the evidence suggests that p i n e a l s e r o t o n i n c o u l d be r e s p o n s i b l e f o r the gland's i n f l u e n c e on n e s t - b u i l d i n g i n doves. Very l i t t l e i s known of the importance of monoamines i n c o n t r o l of avian behaviour. One experiment which d i d analyse the e f f e c t s of v a r i o u s drugs on bow-cooing and n e s t - s o l i c i t i n g i n doves showed a decrease i n these a c t i v i t i e s f o l l o w i n g POA-AH implants of e i t h e r 5HT or PCPA (a 5HT s y n t h e s i s i n h i b i t o r ) (Cascione, 1978). If the a c t i o n of the chicken p i n e a l e x t r a c t was due to the presence of 5HT, then exogenous s e r o t o n i n , l i k e 141 the e x t r a c t , should be capable of s t i m u l a t i n g n e s t - b u i l d i n g i n short-day b i r d s . I dec i d e d to t e s t t h i s p o s s i b i l i t y u s ing p i n e a l e c t o m i z e d b i r d s i n order to e l i m i n a t e any d i r e c t a c t i o n of s e r o t o n i n on the p i n e a l . 2. M a t e r i a l s and methods T h i s t e s t was performed r e - u s i n g the p i n e a l e c t o m i z e d b i r d s from the e x t r a c t experiment. At the c o n c l u s i o n of the e x t r a c t study both the long- and short-day p i n e a l e c t o m i z e d males were h e l d on 8L:16D f o r another 8 weeks, then d i v i d e d i n t o f i v e new set s of 8-10 each. Half of the b i r d s i n each set r e c e i v e d d a i l y sc i n j e c t i o n s of 150 ug of s e r o t o n i n (5-hydroxytryptamine c r e a t i n e s u l f a t e , Sigma) d i s s o l v e d i n .2ml of .9% s a l i n e (= approximately 1mg/kg/d), and h a l f r e c e i v e d s a l i n e alone, from the beginning of the week p r i o r to t e s t i n g to the end of the t e s t i n g p e r i o d . Two of the 5HT b i r d s were e l i m i n a t e d due to adverse r e a c t i o n s to the i n j e c t i o n s — b o t h e x h i b i t e d c o n v u l s i o n s and one d i e d soon a f t e r w a r d s . 3_o R e s u l t s Short-day p i n e a l e c t o m i z e d b i r d s i n j e c t e d with o n l y s a l i n e continued to e x h i b i t very low l e v e l s of n e s t i n g a c t i v i t y i n comparison to those d i s p l a y e d by p i n e a l - i n t a c t b i r d s i n previous experiments ( f i g . 15). The d a i l y i n j e c t i o n s of s e r o t o n i n caused a marked and s i g n i f i c a n t i n c r e a s e i n a l l t h r e e measures of n e s t - b u i l d i n g , but had no e f f e c t on any of the other 142 F i g u r e 15. The e f f e c t of d a i l y i n j e c t i o n s of s e r o t o n i n on the n e s t - b u i l d i n g b e h a v i o u r of s h o r t - d a y p i n e a l e c t o m i z e d male doves. The v a l u e s r e p r e s e n t means, based on t o t a l s f o r the 5-day t e s t p e r i o d . C c o n t r o l 5HT d a i l y i n j e c t i o n s of 0.15 mg s e r o t o n i n a p<.05 based on 2 - t a i l e d U - t e s t 01 O Oi O O O ' ' I CARRYING N E S T - BUILDING iiiiiimiiii immimii 1 44 b e h a v i o u r a l parameters recorded ( t a b l e s XXIII and XXIV). 4. Di s c u s s i o n S e r o t o n i n i n j e c t i o n s , l i k e the p i n e a l e x t r a c t treatment, caused a s i g n i f i c a n t , s p e c i f i c i n c r e a s e i n the n e s t i n g a c t i v i t y of short-day b i r d s . C u r i o u s l y , the b i r d s again showed a response o p p o s i t e to that found i n mammals. In the l a t t e r both the p i n e a l and s e r o t o n i n i n h i b i t r e p r o d u c t i o n , while i n the doves both s t i m u l a t e n e s t - b u i l d i n g , a r e p r o d u c t i v e a c t i v i t y . The way i n which 5HT might b r i n g about t h i s change i s unknown, but there are s e v e r a l f e a s i b l e e x p l a n a t i o n s . Exogenous s e r o t o n i n treatment might i n f l u e n c e n e s t - b u i l d i n g i n doves by s t i m u l a t i n g t e s t o s t e r o n e s e c r e t i o n . Systemic 5HT can d i r e c t l y i n h i b i t gonadal f u n c t i o n i n g i n r a t s , r e s u l t i n g i n decreased c i r c u l a t i n g t e s t o s t e r o n e l e v e l s (see review by Kinson, 1976). In c o t u r n i x q u a i l 5HT i s repo r t e d to i n h i b i t gonad growth under long daylengths ( E l Halawani et a l . , 1977) and to s t i m u l a t e growth under short days ( M i l l e r , 1979). I n j e c t i o n s of 5HTP (a s e r o t o n i n p r e c u r s o r ) w i l l i n c r e a s e t e s t i s s i z e i n house sparrows or q u a i l h e l d on nonstimulatory photoperiods, but only i f given at s p e c i f i c times i n r e l a t i o n to the l i g h t c y c l e ( M i l l e r , 1979). I t i s u n l i k e l y that t h i s e x p l a n a t i o n can account f o r the e f f e c t of s e r o t o n i n i n doves, as 5HT treatment d i d not a f f e c t the d i s p l a y of the other androgen-dependent male a c t i v i t i e s . S e r o t o n i n has a f a i r l y u b i q u i t o u s d i s t r i b u t i o n i n the b r a i n (Crowley & Zemlan, 1981; Fuxe & Ljunngren, 1965; Ikeda & Gotoh, 145 Table XXIII. The courtship a c t i v i t y of short-day pinealectomized males treated with saline or 0.15 mg 5HT/d. The values represent the means and ranges based on the t o t a l number of 15-second in t e r v a l s or the t o t a l number of events over the 5-day test period. Control 5HT n=12 n=10 Male courtship chase 14.5 10.4 (4-71) (0-23) bow-coo 8.9 13.5 (0-22) (0-42) s o l i c i t 207 179 (58-378) (43-372) at-nest 212 169 (38-382) (39-366) 146 Table XXIV. The c o p u l a t o r y a c t i v i t y and female behaviour of p a i r s i n which the p i n e a l e c t o m i z e d males were i n j e c t e d with s a l i n e or 0.15 mg/d 5HT while being h e l d on short days. The values represent the means and ranges based on the t o t a l number of 15-second i n t e r v a l s or the t o t a l number of events over the 5-day t e s t p e r i o d . C o n t r o l n=12 5HT n=10 C o p u l a t o r y a c t i v i t y beg b i l l c r o u c h mount Female be h a v i o u r a.crouch s o l i c i t a t - n e s t n e s t - b u i l d c a r r y 4.4 (0-25) 2.1 (0-13) 1.7 (0-7) 1.1 (0-6) 2.6 (0-13) 614 (265-913) 929 (226-1185) 5.4 (0-30) 2.4 (0-13) 11.3 (0-23) 7.2 (0-16) 4.9 (0-21) 2.8 (0-6) 1.2 (0-8) 487 (176-744) 870 (430-1073) 4.7 (0-18) 2.4 (0-10) 1 48 1971; Sharp & F o l l e t t , 1968). I t i s t h e r e f o r e p o s s i b l e that hypothalamic s e r o t o n i n may be i n f l u e n c i n g n e s t - b u i l d i n g behaviour q u i t e independently of any p i n e a l e f f e c t s , and that the r e s u l t s of 5HT i n j e c t i o n s bear no relevance to the p i n e a l or p h o t o p e r i o d . However, in doves, s e r o t o n i n a c t s only on nest-b u i l d i n g a c t i v i t y . Photoperiod and the p i n e a l a l s o s p e c i f i c a l l y a f f e c t n e s t - b u i l d i n g . I t t h e r e f o r e seems l i k e l y that s e r o t o n e r g i c a c t i v i t y r e p r e s e n t s an important l i n k i n the c h a i n of events connecting daylength, the p i n e a l , and n e s t - b u i l d i n g behaviour. No c o n c l u s i o n about the source of endogenous 5HT can be drawn from the r e s u l t s given here, but the apparent lac k of response to melatonin i n j e c t i o n s may i n d i c a t e that s e r o t o n i n i t s e l f passes from the p i n e a l to the hypothalamus. Although i t has been t r a d i t i o n a l l y accepted that the b l o o d - b r a i n b a r r i e r i s impermeable to monoamines, i n c l u d i n g s e r o t o n i n (Rapoport, 1976), there are i n d i c a t i o n s that the b a r r i e r breaks down i n c e r t a i n r e g i o n s , p a r t i c u l a r y i n the c i r c u m v e n t r i c u l a r organs (Weindl & Schinko, 1975; Larsson & Beyer, 1981). The b a r r i e r has been shown to be incomplete i n the v e n t r a l hypothalamus, a l l o w i n g e n t r y of catecholamines ( L i c h t e n s t e i g e r & Langemann, 1966; W e i l -Malhorbe et a l . , 1959) and 5HT (Knigge et a l . , 1975; Bulat & Sopek, 1968). Although s e r o t o n i n i s r e p o r t e d not to enter the b r a i n i n a d u l t ducks ( c i t e d i n Genst et a l . , 1981) i t i s unclear whether s p e c i f i c b r a i n r e g i o n s were examined in t h i s case. S y s t e m i c a l l y a d m i n i s t e r e d 5HT i s accumulated i n the b r a i n s of e e l s and neonate c h i c k s (Genst et a l . , 1981). Thus, there i s 149 evidence suggesting that some of the 5HT i n j e c t e d i n t o male doves in the c u r r e n t experiment may enter the hypothalamus. Although s e r o t o n i n i s g e n e r a l l y c o n s i d e r e d to be a n e u r o t r a n s m i t t e r , i t may a c t as a neurohormone as w e l l . The d i s t i n c t i o n s between these c l a s s e s of n e u r o s e c r e t i o n are not as c l e a r as they were once thought to be: neurohormones, such as LHRH and TRH are widely d i s t r i b u t e d i n the b r a i n ( S c o t t & Kobisch-Dudley, 1975; Silverman & Zimmerman, 1978), and have been shown to a l t e r both behaviour and c e n t r a l n e u r a l a c t i v i t y (Moss et a l . , 1978), suggesting that they act both as hormones and n e u r o t r a n s m i t t e r s (Silverman & Zimmerman, 1978; Moss et a l . , 1978). In the case of 5HT, u l t r a s t r u c t u r a l c h a r a c t e r i s t i c s of s e r o t o n e r g i c t e r m i n a l s i n the c i r c u m v e n t r i c u l a r organs i n d i c a t e that s e r o t o n i n , as w e l l as a c t i n g as a s y n a p t i c t r a n s m i t t e r , may be s e c r e t e d i n t o the bloodstream or CSF as a neurohormone (Calas et a l . , 1978; B u i j s , 1982). I t has been proposed that s e r o t o n i n r e a c h i n g the b r a i n through the v e n t r i c u l a r system a c t s on r e c e p t o r s l o c a t e d near the v e n t r i c u l a r s u r f a c e r a t h e r than as a n e u r o t r a n s m i t t e r (Datta & King, 1980). The i n f o r m a t i o n a v a i l a b l e i n d i c a t e s that 5HT a l t e r s n e s t i n g behaviour i n doves through c e n t r a l e f f e c t s r a t h e r than changes i n gonadal or p i n e a l a c t i v i t y . The s i m i l a r i t y of the responses to s e r o t o n i n and to p i n e a l e x t r a c t suggests t h a t the p i n e a l may act v i a s e r o t o n e r g i c mechanisms. I t i s not c l e a r whether the e x t r a c t c o n t a i n s some substance which s t i m u l a t e s hypothalamic 5HT mechanisms, or whether 5HT i t s e l f passes from the p i n e a l to the b r a i n , e i t h e r i n the c i r c u l a t o r y or the v e n t r i c u l a r system. 150 On the b a s i s of the accumulating evidence that s e r o t o n i n may serve as a neurohormone, and the demonstration of high c o n c e n t r a t i o n s of 5HT i n the p i n e a l , I propose that p i n e a l 5HT i s r e l e a s e d i n response to long daylengths, and passes to the hypothalamus, where i t s t i m u l a t e s c e n t r e s c o n t r o l l i n g n e s t i n g behaviour. 5. Summary 1) D a i l y i n j e c t i o n s of exogenous s e r o t o n i n i n c r e a s e n e s t -b u i l d i n g i n p i n e a l e c t o m i z e d , short-day males, but have no e f f e c t on any of the other a c t i v i t i e s recorded. S e r o t o n i n may be the p i n e a l hormone r e s p o n s i b l e f o r p h o t o p e r i o d i c c o n t r o l of n e s t i n g . 151 VI. GENERAL DISCUSSION A. C a t e g o r i e s of male r e p r o d u c t i v e a c t i v i t y i n the r i n g dove F o l l o w i n g p a i r i n g , r i n g doves g e n e r a l l y proceed through a p r e d i c t a b l e sequence of b e h a v i o u r a l a c t i v i t i e s . Information from the temporal sequencing and c o r r e l a t i o n s among the v a r i o u s behaviour p a t t e r n s , along with experimental evidence of t h e i r r e l a t i o n s h i p to i n t e r n a l and e x t e r n a l s t i m u l i , a l l suggest that the d i s p l a y s which occur d u r i n g the p r e - l a y i n g p e r i o d can be d i v i d e d i n t o s e v e r a l b a s i c c a t e g o r i e s , each c o n t r o l l e d by s l i g h t l y d i f f e r e n t f a c t o r s . L o v a r i and Hutchison (1975) d i d a thorough a n a l y s i s of the b e h a v i o u r a l t r a n s i t i o n s o c c u r r i n g e a r l y i n the male dove r e p r o d u c t i v e c y c l e . They d i s t i n g u i s h e d between a g g r e s s i v e and n e s t - o r i e n t e d c o u r t s h i p , and proposed that the c a u s a l mechanisms c o n t r o l l i n g these two c a t e g o r i e s d i f f e r . A g g r e s s i v e c o u r t s h i p c o n s i s t s of c h a s i n g and bow-cooing. The u n d e r l y i n g m o t i v a t i o n of bow-cooing i n doves and pigeons has been d i s c u s s e d at l e n g t h by s e v e r a l authors (Goodwin, 1956a; F a b r i c i u s & Jansson, 1963; Davies, 1970; L o v a r i & Hutchison, 1975), and most conclude on the b a s i s of temporal sequences and p o s t u r a l components that i t r e p r e s e n t s a combination of a g g r e s s i v e , sexual, and p o s s i b l y f e a r t e n d e n c i e s . Chasing i s u s u a l l y c o n s i d e r e d to be o v e r t l y a g g r e s s i v e , but d i f f e r e n c e s between ch a s i n g d i r e c t e d at females and c h a s i n g d i r e c t e d at other males suggest that t h i s behaviour, l i k e d r i v i n g i n the pigeon (Goodwin, 1956b) a l s o r e f l e c t s combined sexual and a g g r e s s i v e m o t i v a t i o n (McDonald, 1976). 152 N e s t - s o l i c i t i n g seems to be d i s t i n c t from the preceding two d i s p l a y s . Although a l l three a c t i v i t i e s emerge soon a f t e r p a i r i n g and peak w i t h i n one day, both bowing and c h a s i n g disappear r a p i d l y , while n e s t - s o l i c i t i n g remains high f o r at l e a s t four days ( E r i c k s o n , 1973; L o v a r i & Hutchison, 1975). Both a g g r e s s i v e and n e s t - o r i e n t e d c o u r t s h i p depend upon the presence of gonadal s t e r o i d s ( E r i c k s o n , 1970; Martinez-Vargas, 1974; Cheng & Lehrman, 1975; McDonald, 1976), and i t has been suggested that the t r a n s i t i o n to n e s t - s o l i c i t i n g may r e s u l t from a decrease i n t e s t o s t e r o n e l e v e l s ( E r i c k s o n et a l . , 1967; Hutchison, 1970b). However, i t has a l s o been shown that a male w i l l bow-coo towards a strange female, r e g a r d l e s s of the stage of r e p r o d u c t i o n he i s engaged i n with h i s own mate ( E r i c k s o n , 1973; S i l v e r & B a r b i e r e , 1977). I t thus appears that the male's s h i f t from a g g r e s s i v e to n e s t - o r i e n t e d behaviour i s not the d i r e c t r e s u l t of changes in androgen s t i m u l a t i o n , but i s a l s o determined by s t i m u l i from the female. Because of t h e i r common focus on the nest s i t e , nest-s o l i c i t i n g and n e s t - b u i l d i n g are o f t e n grouped together as ' n e s t - o r i e n t e d ' behaviour ( F a b r i c i u s & Jansson, 1963). However, examination of the a c t i v i t y of p a i r s of doves proceeding through a r e p r o d u c t i v e c y c l e suggests an i n v e r s e r e l a t i o n s h i p between these two behaviour p a t t e r n s : as the female becomes a t t a c h e d to the nest s i t e , n e s t - s o l i c i t i n g by the male wanes and nest-b u i l d i n g i n c r e a s e s s h a r p l y (see f i g . 16 from McDonald, 1976; a l s o L o v a r i & Hutchison, 1975). Long-term c a s t r a t e d male doves do not engage i n n e s t - b u i l d i n g (Martinez-Vargas, 1974; McDonald, 153 F i g u r e 16. Changes i n the p r e - i n c u b a t i o n b e h a v i o u r of male doves i n r e l a t i o n t o the date when the f i r s t egg was l a i d by t h e female (day 0 ) . The v a l u e s r e p r e s e n t d a i l y means. CH/PK = chase/peck BC = bow-coo NS = n e s t - s o l i c i t i n g NB = n e s t - b u i l d i n g 154 DAYS IN RELATION TO FIRST EGG 155 1976). L i k e n e s t - s o l i c i t i n g , n e s t - b u i l d i n g i s r e a d i l y r e s t o r e d by systemic i n j e c t i o n s (Martinez-Vargas, 1974), capsules (McDonald, 1979) or i n t r a h y p o t h a l a m i c implants ( E r i c k s o n & Hutchison, 1977) of t e s t o s t e r o n e . E r i c k s o n & Hutchison (1977) r e p o r t e d that n e s t - b u i l d i n g d e c l i n e s very slowly f o l l o w i n g removal of the t e s t e s , and p e r s i s t s i n a few b i r d s f o r as long as 6-8 weeks a f t e r gonadectomy. The r e l a t i v e independence of n e s t - b u i l d i n g from immediate androgen l e v e l s i s supported by o b s e r v a t i o n s t h a t male doves which have been c a s t r a t e d a few days p r i o r to p a i r i n g w i l l s t i l l perform both n e s t - s o l i c i t i n g and n e s t - b u i l d i n g (unpublished r e s u l t s c i t e d i n S i l v e r , 1978). The p e r s i s t e n c e of b e h a v i o u r a l responses to hormones f o r a short p e r i o d a f t e r the removal of s t e r o i d s has been noted i n other animals, and suggests that processes subsequent to receptor uptake continue f o r some time (Beyer et a l . , 1978), d e l a y i n g the t e r m i n a t i o n of the response. The s h i f t i n behaviour from male c o u r t s h i p to n e s t - b u i l d i n g may depend p r i m a r i l y upon the behaviour of the female. Males p a i r e d with females which spend much of t h e i r time at the n e s t - s i t e gather more n e s t i n g m a t e r i a l than males p a i r e d with unresponsive females (Martinez-Vargas & E r i c k s o n , 1973). The c o n d i t i o n of the nest i s a l s o c r u c i a l , s i n c e b i r d s given f u l l nests b u i l d l e s s than those given an empty nest bowl (White, 1975b). Another major category of r e p r o d u c t i v e behaviour recognized by F a b r i c i u s & Jansson (1963) i s that of s e x u a l , or c o p u l a t o r y a c t i v i t y . C o p u l a t i o n occurs throughout the r e p r o d u c t i v e c y c l e , and u n l i k e other p r e - i n c u b a t i o n a c t i v i t i e s , i s most frequent 156 l a t e i n the day (Martinez-Vargas & E r i c k s o n , 1973; Cheng et a l . , 1981). Hormonal c o n t r o l of sexual d i s p l a y s appears d i s t i n c t from that of c o u r t s h i p and n e s t - b u i l d i n g . Begging, b i l l i n g , c r o u c h i n g and mounting are re p o r t e d to p e r s i s t a f t e r the disappearance of c o u r t s h i p a c t i v i t y i n male and female gonadectomized doves (Cheng, 1973a) and pigeons (Carpenter, 1933a & 1933b). The occurrence of c o p u l a t o r y behaviour bears no c l e a r r e l a t i o n s h i p to the endocrine s t a t e i n female doves, as assessed by ovary and o v i d u c t development ( L i l e y , 1976a) and i s not i n f l u e n c e d by exogenous hormone treatment ( L i l e y , 1976b). I f a i l e d to observe any c o p u l a t i o n s by long-term c a s t r a t e s , but that may have been because the females p a i r e d with c a s t r a t e s d i d not s o l i c i t c o p u l a t i o n s (McDonald and L i l e y , 1978). Table XXV i l l u s t r a t e s the degree of c o r r e l a t i o n among the v a r i o u s d i s p l a y s performed by s a l i n e - t r e a t e d , sham-operated males and t h e i r females i n the f i n a l pinealectomy experiment. The r e s u l t s tend t o support the d i v i s i o n s proposed above: a g g r e s s i v e d i s p l a y s (chasing/pecking and bow-cooing) are p o s i t i v e l y c o r r e l a t e d , as are the v a r i o u s measures of nest-b u i l d i n g and the d i f f e r e n t components of c o p u l a t o r y behaviour. There i s a str o n g c o r r e l a t i o n between a g g r e s s i v e c o u r t s h i p and appeasement crouches, s u p p o r t i n g the n o t i o n that the female uses t h i s d i s p l a y i n response to a t t a c k s by males. No p o s i t i v e c o r r e l a t i o n s were found among the major groups of male behaviour, with the e x c e p t i o n of b i l l i n g vs chasing and bow-coo i n g . The c o r r e l a t i o n data thus support the d i v i s i o n of male a c t i v i t y i n t o four d i s t i n c t c a t e g o r i e s : a g g r e s s i v e c o u r t s h i p , 157 Table XXV. C o r r e l a t i o n s among the v a r i o u s c o u r t s h i p a c t i v i t i e s d i s p l a y e d by the s a l i n e - t r e a t e d c o n t r o l b i r d s i n the second pinealectomy experiment. Spearman rank c o r r e l a t i o n c o e f f i c i e n t s are gi v e n o n l y i f s i g n i f i c a n t (p<.05, 2 - t a i l e d ) . 0 -J o bow—coo 3 3 co CB n e s t - s o l i c i t 10 3 3 £ a tn m a l e - a t - n e s t 3 3 3 3 CD CD CO CO vO 3 3 3 3 f o 00 CO CO CO U l «> 00 3 3 3 3 O t o CO CO CO 00 VO 03 n e s t - b u i l d c a r r y s t r i n g 3 3 3 on U l 3 3 ^ , • • ^ tn co cn .c C J coco 2 n e s t — s o l i c i t CO ui * 3 3 3 3 3 3 3 3 _ . . . co co to m coco coco j - a t - n e s t I 3 O l 3 3 3 on CT> 3 3 „ v , • . j to H cococo on v-» coco Q n e s t - D u i l a in 3 3 3 3 3 3 3 3 3 _ „ = o-icocn cococo coco cocn 9 c a r r y 3 3 In 3 3 3 3 3 *«k *k KJ co co NJ cococo co co r->cs beo I • • • VO 3 3 3 On 3 3 3 3 3 3 on i-i co m cn ro cn tn cn coco K H b i l l On J> U l I CT. -J 3 3 3 on 3 3 3 3 3 3 3 £ > C J CO CO to O cococo co co to co crouch O N ] O J 0 • • • 00 CT> 0> 3 3 3 m 3 3 3 3 3 3 3 CD vo vo co <g co to co co co to co tn co mount On 0"> t o O SSS2 s s s s s s s s s ** aPP. crouch oSS'ST ^040^^° » O 3 3 3 Vn ^ i J I C rr O III CO o 6" CO CO C r t O t i n . O K' CO H* a 9 rt r t o 8ST 159 n e s t - o r i e n t e d c o u r t s h i p , n e s t - b u i l d i n g and c o p u l a t o r y d i s p l a y s . The s e p a r a t i o n of the p r e - i n c u b a t i o n behaviour of males i n t o these four c a t e g o r i e s i s s i g n i f i c a n t i n terms of the r e s u l t s from the present study. Only n e s t - b u i l d i n g a c t i v i t y was a f f e c t e d by photoperiod, pinealectomy and e x t r a c t or s e r o t o n i n treatment. The f a c t t h at daylength can a l t e r n e s t - b u i l d i n g i n the absence of changes i n c o u r t s h i p and c o p u l a t o r y a c t i v i t y i s not unexpected i n view of the f a c t t h at each of these types of behaviour i s i n f l u e n c e d by a d i s t i n c t c o n t r o l mechanism. B. Photoperiod and the Breeding C y c l e i n Ring Doves Because of i t s t r o p i c a l o r i g i n s and long d o m e s t i c a t i o n i t has been suggested t h a t the r i n g dove, S t r e p t o p e l i a r i s o r i a , i s unresponsive to photoperiod (van Tienhoven, 1968). The r i n g dove i s a domesticated form which i s now thought to be d e r i v e d from the A f r i c a n c o l l a r e d dove, S. r o s e o g r i s e a ( V a u r i e , 1961; Goodwin, 1967; Parkes, 1973). The a n c e s t r a l A f r i c a n c o l l a r e d dove i s found i n e q u a t o r i a l r e g i o ns where there are onl y s l i g h t annual v a r i a t i o n s i n daylength. L i t t l e i s known of i t s r e p r o d u c t i v e c y c l e s , although i t r e p o r t e d l y has an extended breeding season l a s t i n g from January to June, and w i l l breed c o n t i n u o u s l y i n some re g i o n s (MacWorth-Praed & Grant, 1970). Domestication i s known to be a s s o c i a t e d with lengthened breeding c y c l e s i n b i r d s , and may a l s o r e s u l t i n a l o s s of p h o t o r e f r a c t o r i n e s s (Haase, 1980; Tanabe, 1980), but even i n s p e c i e s i n which there has been s e l e c t i o n f o r i n c r e a s e d breeding, as i n c h i c k e n s , the a b i l i t y to respond to photoperiod 160 p e r s i s t s (Wilson & Cunningham, 1980; Casey et a l . , 1973), although i t i s perhaps somewhat dampened. I t i s a l s o o f t e n assumed that photoperiod i s not a major f a c t o r c o n t r o l l i n g r e p r o d u c t i o n i n t r o p i c a l s p e c i e s , p a r t l y because of the small degree of change i n daylength throughout the year, and p a r t l y because of the extended breeding p e r i o d s o f t e n seen i n t r o p i c a l s p e c i e s (Murton & Westwood, 1977; Gwinner, 1981). S e v e r a l authors have r e c e n t l y q uestioned t h i s g e n e r a l i z a t i o n (Singh & Chandola, 1981; W i n g f i e l d & Farner,1980). I t has been shown that b i r d s l i k e the budgerigar, whose sexual a c t i v i t y i s normally c o n t r o l l e d by other proximate f a c t o r s ( i n t h i s case r a i n f a l l ) , s t i l l respond to photoperiod ( S h e l l s w e l l et a l . , 1975). In f a c t , a c c o r d i n g to Singh & Chandola (1981) " a l l s u b t r o p i c a l / t r o p i c a l s p e c i e s i n v e s t i g a t e d so f a r have been found to be p h o t o s e n s i t i v e " (p. 293). E a r l y o b s e r v a t i o n s of c a p t i v e r i n g doves suggested that they w i l l r e s t r i c t t h e i r breeding c y c l e s i f h e l d under n a t u r a l temperate c o n d i t i o n s . Whitman (1919) re p o r t e d an i n c r e a s e i n the i n t e r v a l between c l u t c h e s d u r i n g the l a t e f a l l and w i n t e r . C r a i g (1909) a l s o noted a d e c l i n e i n breeding a c t i v i t y i n the autumn. Doves he l d at s e m i - l i b e r t y i n England u s u a l l y bred from l a t e February or March to September (Goodwin, 1952), an i n t e r v a l which c l o s e l y corresponds to the c y c l e of S. decaocto in England, and of the r i n g doves h e l d under n a t u r a l l i g h t and temperature c y c l e s here at UBC (personal o b s e r v a t i o n ) . Ring doves kept i n outdoor a v i a r i e s i n A u s t r a l i a are a l s o a c t i v e from l a t e winter ( J u l y ) to m i d - f a l l ( A p r i l ) (Davies, 1974a). The 161 d e c l i n e i n breeding a c t i v i t y d u r i n g the winter months suggests that doves w i l l respond to photoperiod when h e l d under the a p p r o p r i a t e c o n d i t i o n s , although concurrent f l u c t u a t i o n s i n temperature may a l s o be important. Taken together, these s t u d i e s suggest that photoperiod-induced changes i n the behaviour of male and female doves, such as those seen i n the l a b o r a t o r y , can l e a d to a seasonal 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 under n a t u r a l c o n d i t i o n s . C. Photoperiod and Male Behaviour Hutchison (1974) compared the f i r s t three minutes of c o u r t s h i p a c t i v i t y of l o n g - and short-day c a s t r a t e s which had r e c e i v e d b r a i n implants of TP. He found that there were no d i f f e r e n c e s i n bow-cooing or n e s t - s o l i c i t i n g l e v e l s as a r e s u l t of exposure to d i f f e r e n t photoperiods, but the short-day b i r d s d i d perform l e s s c h a s i n g . Although the s h o r t - and long-day groups were t e s t e d many years a p a r t , the r e s u l t s suggested that "implants of t e s t o s t e r o n e propionate are...more e f f e c t i v e i n i n i t i a t i n g c o u r t s h i p i n . . . c a s t r a t e s maintained on a long photoperiod (I3h/day) than i n c a s t r a t e s maintained on a short photoperiod (8.5h/day)" (Hutchison, 1975b, p.132). Hutchison a l s o r e f e r s to an unpublished experiment conducted on groups of doves h e l d on 14L or 6L per day. I n t a c t males d i s p l a y e d more cha s i n g under long days. The same males were then c a s t r a t e d and given i n t r a h y p o t h a l a m i c t e s t o s t e r o n e implants. There were no d i f f e r e n c e s i n the peak d u r a t i o n or l a t e n c y of c h a s i n g , l e a d i n g to the c o n c l u s i o n that "photoperiod [has] l i t t l e i n f l u e n c e on 162 the l e v e l of a g g r e s s i v e c o u r t s h i p behaviour induced by t e s t o s t e r o n e " (Hutchison, 1975a, p.183; 1975b, p.132). Hutchison's r e s u l t s seem to i n d i c a t e a l a c k of e f f e c t of p h otoperiod on t e s t o s t e r o n e - a c t i v a t e d a g g r e s s i v e c o u r t s h i p . A s i m i l a r l a c k of e f f e c t was found i n my p r e v i o u s study examining the i n f l u e n c e of photoperiod on r e p r o d u c t i v e a c t i v i t y over a two-week p e r i o d (McDonald, 1976). T h i s i s i n agreement with most of the experiments d e s c r i b e d i n t h i s paper. In s i x of the seven s e t s of data I found no s i g n i f i c a n t d i f f e r e n c e s between l o n g - and short-day b i r d s f o r e i t h e r chasing or bow-cooing. The t o t a l amount of n e s t - s o l i c i t i n g d i s p l a y e d over the f i v e - d a y t e s t p e r i o d d i d not show any s i g n i f i c a n t d i f f e r e n c e s with photoperiod, although there was a c o n s i s t e n t tendency f o r mean l e v e l s to be g r e a t e r under long days. I n i t i a l l e v e l s of n e s t - s o l i c i t i n g (day 1) were higher i n LD than SD b i r d s i n the LHRH experiment, but t h i s e f f e c t was not seen i n any of the subsequent t e s t s . Taken together the data suggest that n e s t -s o l i c i t i n g i s not s t r o n g l y a f f e c t e d by daylength. Copulatory a c t i v i t y was not a l t e r e d by the l i g h t regime under which the males were h e l d . L i l e y (1976a) a l s o found that female c o p u l a t o r y behaviour i s independent of daylength. The f i n a l c ategory of behaviour to be c o n s i d e r e d i s n e s t -b u i l d i n g . N e s t i n g a c t i v i t y was s i g n i f i c a n t l y g r e a t e r i n l o n g -day exposed males i n n e a r l y a l l of the t e s t s d e s c r i b e d here, The l a c k of d i f f e r e n c e s i n female behaviour, and the general absence of a c o r r e l a t i o n between n e s t - b u i l d i n g and the female's n e s t - o r i e n t e d a c t i v i t y ( t a b l e XXV) e l i m i n a t e s the l i k e l i h o o d 163 that s u b t l e changes i n the male's e a r l y c o u r t s h i p were a f f e c t i n g n e s t - b u i l d i n g by a l t e r i n g the female's response. I t i s of i n t e r e s t t h at the few other examples of photoperiod-induced changes i n b e h a v i o u r a l responses to s t e r o i d s i n b i r d s have a l s o c e n t e r e d on n e s t - b u i l d i n g . Thus, i n female c a n a r i e s ( S t e e l & Hinde, 1972a & b), budgerigars (Gosney & Hinde, 1975) and r i n g doves ( L i l e y , 1976a & b) i t i s n e s t - o r i e n t e d a c t i v i t y which changes with the photoregime. D. The Mechanism M e d i a t i n g the E f f e c t of Photoperiod on Male N e s t - B u i l d i n g i n Doves There are a number of d i f f e r e n t ways i n which photoperiod c o u l d a l t e r n e s t i n g i n male doves. Daylength c o u l d be a c t i n g on c e n t r a l mechanisms c o n t r o l l i n g n e s t i n g behaviour through d i r e c t n e u r a l i n p u t . Although i t would be very d i f f i c u l t to e l i m i n a t e such a p o s s i b i l i t y , the known e f f e c t of photoperiod on the endocrine system i n d i c a t e s that the most u s e f u l approach to the q u e s t i o n of how daylength a l t e r s n e s t - b u i l d i n g behaviour probably l i e s i n examination of the v a r i o u s a s p e c t s of the hormonal system which might a f f e c t the d i s p l a y of such an androgen-dependent behaviour. As more s o p h i s t i c a t e d means of s t u d y i n g the endocrine system are developed, i t becomes apparent that hormone a c t i v i t y cannot be adequately e x p l a i n e d simply on the b a s i s of changes i n hormone p r o d u c t i o n , but must be looked at i n terms of the e n t i r e complex of events a s s o c i a t e d with the c o n t r o l of hormone c o n c e n t r a t i o n , uptake and metabolism. Any of these aspects of 1 64 hormonal responsiveness might be a l t e r e d by photoperiod, r e s u l t i n g i n the observed s t i m u l a t i o n of male n e s t - b u i l d i n g under long days (see t a b l e XXVI). J _ . A c t i v e hormone l e v e l s Changes i n p e r i p h e r a l l e v e l s of t e s t o s t e r o n e are u n l i k e l y to be r e s p o n s i b l e f o r the r e a c t i o n to photoperiod. T e s t o s t e r o n e - i m p l a n t e d c a s t r a t e s continue to e x h i b i t i n c r e a s e d n e s t i n g a c t i v i t y under long daylengths, even though they are r e c e i v i n g the same amount of androgen as are short-day b i r d s . Increased t e s t o s t e r o n e p r o d u c t i o n by the ad r e n a l s under long days c o u l d r e s u l t i n higher blood l e v e l s . I t has been r e p o r t e d that there i s no evidence of androgen p r o d u c t i o n by the h e a l t h y a v i a n a d r e n a l c o r t e x (Ljunggren, 1969; Hohn, 1961). However, measurements of plasma androgen l e v e l s c a s t some doubt on t h i s . Feder et a l . (1977) found r e l a t i v e l y high c o n c e n t r a t i o n s of both t e s t o s t e r o n e and DHT i n male doves which had been c a s t r a t e d f o r 2-3 weeks. On the other hand, McCreery & Farner (1979) f a i l e d to observe any plasma androgen i n c a s t r a t e d sparrows. The r e l a t i v e r a t e s of c l e a r a n c e and metabolism of plasma t e s t o s t e r o n e haven't been examined i n doves, but i n T-implanted gonadectomized hamsters photoperiod has no e f f e c t on serum androgen l e v e l s (Campbell et a l . , 1978). D e f i n i t e c o n c l u s i o n s c o n c e r n i n g the r e l a t i v e l e v e l s of c i r c u l a t i n g androgen i n doves exposed to d i f f e r e n t daylengths w i l l have to wait u n t i l blood samples from the b i r d s can be analysed, but i t seems extremely u n l i k e l y that the s l i g h t d i f f e r e n c e s which might a r i s e from the XXVI. P o s s i b l e s i t e s o f a c t i o n o f p h o t o p e r i o d r e s u l t i n g i n changes i n n e s t - b u i l d i n g b e h a v i o u r I . D i r e c t e f f e c t s on the CNS I I . Changes i n the l e v e l o f a c t i v e hormone 1) c i r c u l a t i n g l e v e l s - t e s t i s p r o d u c t i o n - a d r e n a l p r o d u c t i o n - r a t e o f c l e a r a n c e / m e t a b o l i s m 2) b r a i n l e v e l s - uptake -metabolism to a c t i v e or i n a c t i v e forms I I I . C i r c a d i a n system IV. Synergism w i t h o t h e r hormones 1) LHRH, g o n a d o t r o p i n s 2) p r o l a c t i n 3) t h y r o i d hormones 4) a d r e n a l hormones V. P i n e a l g l a n d 166 above f a c t o r s would be capable of a l t e r i n g the behaviour of the b i r d s , c o n s i d e r i n g the extremely high doses of exogenous hormone which were being used. The exact r e l a t i o n s h i p between c i r c u l a t i n g hormone l e v e l s and the d i s p l a y of behaviour i s u n c e r t a i n . S e v e r a l e a r l y papers showed a strong c o r r e l a t i o n between t e s t o s t e r o n e dosage and c o p u l a t o r y behaviour i n c a s t r a t e d r a t s (Bermant & Davidson, 1974). Many other s t u d i e s , however, have f a i l e d to f i n d any dose-dependant changes i n male b e h a v i o u r a l a c t i v i t y (mouse, Champlin et a l . , 1963; r a t , Damassa et a l . , 1977). Gorman (1977) attempted to c o r r e l a t e d a i l y p a t t e r n s of behaviour i n e i d e r ducks with d i u r n a l f l u c t u a t i o n s i n plasma androgen, but c o u l d f i n d no a s s o c i a t i o n between the two. There i s no c o r r e l a t i o n between the sexual a c t i v i t y of male doves d u r i n g the f i r s t 15 minutes a f t e r p a i r i n g and plasma T l e v e l s 24 hours l a t e r (O'Connell et a l . , 1981a). Feder et a l . (1977) repo r t e d that t e s t o s t e r o n e l e v e l s i n male doves were p o s i t i v e l y c o r r e l a t e d with w i n g - f l i p p i n g , but not with a g g r e s s i v e c o u r t s h i p or n e s t - b u i l d i n g . A s i m i l a r d i s s o c i a t i o n of p h y s i o l o g i c a l a s p e c t s of r e p r o d u c t i o n ( i e . endocrine l e v e l s ) and r e p r o d u c t i v e behaviour has been found i n a number of d i f f e r e n t circumstances (see review by Schwartz, 1982). In g e n e r a l , the p e r i o d of peak r e p r o d u c t i v e c a p a c i t y c o - i n c i d e s with the mating season, a l l o w i n g s t e r o i d s to play the dual r o l e of c o n t r o l l i n g both 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 a c t i v i t y . Crews (1983) has proposed that the p h y s i o l o g i c a l r o l e of the hormonal system arose p r i o r to i t s involvement i n the c o n t r o l of behaviour. I f 167 t h i s i s t r u e , i t would not be s u r p r i s i n g that the l i n k between hormone l e v e l s and behaviour should be loose, thus a l l o w i n g enough f l e x i b i l i t y to accommodate minor d i v e r g e n c e s between 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 responses. Changes i n hormone l e v e l s may s t i l l p l a y a v i t a l r o l e i n the p a t t e r n i n g of behaviour, i f i t i s assumed that d i f f e r e n t d i s p l a y s have d i f f e r e n t t h r e s h o l d s f o r a c t i v a t i o n . However, l e v e l s of .2-.4 mg TP/day would undoubtedly surpass the t h r e s h o l d f o r r e p r o d u c t i v e d i s p l a y s i n male doves, making i t u n l i k e l y that c i r c u l a t i n g c o n c e n t r a t i o n s are an important f a c t o r i n the c u r r e n t study. Even assuming that c i r c u l a t i n g l e v e l s of t e s t o s t e r o n e under d i f f e r e n t photoperiods are equal, i t i s s t i l l p o s s i b l e that those c e n t e r s c o n t r o l l i n g n e s t i n g behaviour r e c e i v e g r e a t e r hormonal s t i m u l a t i o n as a r e s u l t of d i f f e r e n c e s i n uptake, or c o n v e r s i o n to a c t i v e / i n a c t i v e forms under the two l i g h t c y c l e s . Only one of these p o s s i b i l i t i e s was examined i n t h i s study: the a r o m a t i z a t i o n of t e s t o s t e r o n e to estrogen. The e f f e c t of p h o t o period p e r s i s t e d f o l l o w i n g estrogen i m p l a n t a t i o n , i n d i c a t i n g that d i f f e r e n c e s i n the r a t e of a r o m a t i z a t i o n are not e s s e n t i a l . I n a c t i v a t i o n to i n e f f e c t i v e m e t a b o l i t e s such as 5a or 5/3DHT, or d i f f e r e n c e s i n r e c e p t o r c o n c e n t r a t i o n c o u l d a l s o a l t e r e f f e c t i v e t e s t o s t e r o n e l e v e l s i n the b r a i n . Female c a n a r i e s , which e x h i b i t g r e a t e r n e s t i n g a c t i v i t y on long days than on short days, do not show any d i f f e r e n c e s i n hypothalamic uptake of r a d i o a c t i v e estrogen under d i f f e r e n t l i g h t c y c l e s (unpublished r e s u l t s c i t e d i n Hinde & S t e e l , 1977), suggesting 168 that p h o t operiod has l i t t l e e f f e c t on c e n t r a l s t e r o i d r e c e p t o r s . A l s o , the f a c t that daylength had the same e f f e c t on the behaviour of EB doves as on that of T P - t r e a t e d ones s t r o n g l y suggests t h a t photoperiod a c t s at a p o i n t which i s independent of the s t e r o i d i n v o l v e d , and i s probably t h e r e f o r e u n a f f e c t e d by the r a t e s of uptake and metabolism of s p e c i f i c hormones. 2. C i r c a d i a n system The a b i l i t y of daylength both to e n t r a i n and set the phase angle of endogenous rhythms (Bunning, 1973) suggests that the c i r c a d i a n system may p r o v i d e a l i n k between the photoperiod and i t s i n f l u e n c e on n e s t - b u i l d i n g . Meier (1972, 1975) proposed that seasonal changes i n the r e p r o d u c t i v e f u n c t i o n of b i r d s stem from d i f f e r e n t p a t t e r n s of r e l e a s e of p r o l a c t i n and c o r t i c o s t e r o n e . However, attempts to r e p l i c a t e h i s experiments have f a i l e d (Farner & Gwinner, 1980). A l s o , i t appears that p r o l a c t i n , i n doves at l e a s t , has no d i u r n a l rhythm (Goldsmith et a l . , 1981). The involvement of rhythms of t e s t o s t e r o n e r e l e a s e i s a l s o u n l i k e l y i n view of the continued response of TP-implanted c a s t r a t e d doves to d a y l e n g t h . And f i n a l l y , o v e r a l l p a t t e r n s of d a i l y a c t i v i t y are s i m i l a r i n l o n g - and short-day b i r d s , i n d i c a t i n g that the decrease i n n e s t - b u i l d i n g seen i n SD males i s not due to a s h i f t i n the peak n e s t i n g a c t i v i t y o u t s i d e of the o b s e r v a t i o n p e r i o d . 3. Synergism with other hormones Another p o s s i b l e e x p l a n a t i o n f o r the d i f f e r e n c e s i n 169 b u i l d i n g a c t i v i t y seen under the two photoregimes i s that nongonadal hormones are i n v o l v e d - - e i t h e r d i r e c t l y , v i a synergism with t e s t o s t e r o n e , or i n d i r e c t l y , through a l t e r e d hypothalamic s e n s i t i v i t y to androgens. L e v e l s of hypothalamic LHRH and p i t u i t a r y gonadotropins are i n c r e a s e d by long daylengths, as a l r e a d y d e s c r i b e d . Although LHRH s t i m u l a t e s sexual a c t i v i t y i n o v a r i e c t o m i z e d female r a t s (Moss & McCann, 1973) and doves (Cheng, 1977) t r e a t e d with low doses of EB or EB + P, I f a i l e d to reverse the i n h i b i t o r y e f f e c t of s h o r t days on male n e s t i n g using exogenous LHRH i n j e c t i o n s . Since the treatment would a l s o be expected to i n c r e a s e LH and FSH l e v e l s , i t appears that n e i t h e r gonadotropins nor r e l e a s i n g hormone are r e s p o n s i b l e f o r the e f f e c t of day l e n g t h . Photoperiod has been i m p l i c a t e d i n the c o n t r o l of s e v e r a l other nongonadal hormones, i n c l u d i n g p r o l a c t i n (Burke & Dennison, 1980), t h y r o i d hormones (Assenmacher & J a l l a g a e a s , 1980) and adren a l hormones (Assenmacher, 1973). However, none of these hormones has been shown to i n f l u e n c e n e s t - b u i l d i n g i n male b i r d s . P r o l a c t i n l e v e l s are low du r i n g the n e s t - b u i l d i n g phase i n male and female r i n g doves (Goldsmith et a l . , 1981), c a n a r i e s (Goldsmith, 1982) and s t a r l i n g s (Dawson & Goldsmith, 1982). In a d d i t i o n , f o r c e d r e - b u i l d i n g f o l l o w i n g removal of the nest i s normally accompanied by a d e c l i n e i n p r o l a c t i n i n c a n a r i e s (Goldsmith, 1982). There i s l i t t l e evidence suggesting that t h y r o i d hormones can a l t e r r e p r o d u c t i v e behaviour i n b i r d s . Cheng (1977) f a i l e d to observe any e f f e c t of t h y r o i d r e l e a s i n g hormone on the sexual behaviour of o v a r i e c t o m i z e d female doves. 170 Progesterone, which may be produced p r i m a r i l y by the a d r e n a l s i n b i r d s (McCreery & Farner, 1979), a c t s s y n e r g i s t i c a l l y with e s t r a d i o l to induce n e s t - b u i l d i n g i n female doves (Cheng & S i l v e r , 1975). U n l i k e females, male doves show no v a r i a t i o n i n plasma progesterone l e v e l s d u r i n g the breeding c y c l e ( S i l v e r et a l . , 1974), and i n the pigeon progesterone a c t u a l l y d e c l i n e s f o l l o w i n g p a i r i n g (Haase et a l . , 1976). McCreery and Farner (1979) found no change i n plasma progesterone l e v e l s of white-crowned sparrows h e l d on d i f f e r e n t p h o t o p e r i o d s . F u r t h e r evidence a g a i n s t the involvement of adr e n a l progesterone i s p r o v i d e d by o b s e r v a t i o n s that dexamethasone (an ACTH i n h i b i t o r ) has l i t t l e e f f e c t on the c o u r t s h i p behaviour of male doves ( S i l v e r & Buntin, 1973) and that exogenous progesterone decreases n e s t - b u i l d i n g i n i n t a c t male pigeons (Murton et a l . , 1969). 4. P r o d u c t i o n of a p i n e a l hormone Two l i n e s of evidence g i v e c o n v i n c i n g support to the p r o p o s a l that daylength a f f e c t s male n e s t - b u i l d i n g , i n p a r t , through changes i n p i n e a l hormone p r o d u c t i o n . F i r s t , only n e s t -b u i l d i n g behaviour, the one a c t i v i t y which i s c o n s i s t e n t l y a f f e c t e d by photoperiod, was a l t e r e d by p i n e a l removal, Secondly, treatment with c h i c k e n p i n e a l e x t r a c t was capable of s e l e c t i v e l y i n c r e a s i n g n e s t i n g i n those groups which presumably have low p i n e a l a c t i v i t y — s h o r t - day i n t a c t and p i n e a l e c t o m i z e d b i r d s and long-day p i n e a l e c t o m i z e d b i r d s — b u t not i n the one group which would be expected to have high endogenous a c t i v i t y , 171 long-day i n t a c t b i r d s . The f a c t that s e r o t o n i n i n j e c t i o n s a l s o i n c r e a s e d only n e s t - b u i l d i n g suggests that 5HT may be the p i n e a l substance r e s p o n s i b l e f o r mediating the e f f e c t of p h o t p e r i o d on d a y length. E. G e n e r a l i t y of Photoperiod-induced Changes i n the Response to S t e r o i d s In b i r d s the major e f f e c t of photoperiod on hormone-responsiveness i n v o l v e s n e s t - b u i l d i n g behaviour. T h i s might suggest that a l i g h t - i n d u c e d a l t e r a t i o n i n the a c t i o n of s t e r o i d s i s a phenomenon with l i t t l e g e n e r a l a p p l i c a b i l i t y . In a c t u a l f a c t , t h i s i s not the case. The f i r s t r e p o r t of d i f f e r e n c e s i n the b e h a v i o u r a l response of c a s t r a t e d males to androgen s t i m u l a t i o n i n v o l v e d the breeding a c t i v i t y of male s t i c k l e b a c k . Hoar (1962) observed that n e i t h e r m e t h y l t e s t o s t e r o n e nor mammalian gonadotropins c o u l d induce l e v e l s of a g o n i s t i c and n e s t - b u i l d i n g behaviour i n short-day males comparable to those seen under long days. Over the past few years more and more evidence has accumulated suggesting that daylength can a f f e c t numerous types of hormone-response r e l a t i o n s h i p s . A number of s t u d i e s have i n d i c a t e d a r e l a t i o n s h i p between the time of year and hormonal c o n t r o l of behaviour i n a v a r i e t y of s p e c i e s (ewes, Goodman et a l . , 1979 & 1981, F l e t c h e r & Lindsay, 1971, Raeside & McDonald, 1959, Reardon & Robinson, 1961; rams, Mattner, 1977; male and female deer, L i n c o l n et a l . , 1972, F l e t c h e r & Short, 1974, Short, 1976; female l i z a r d s , Crews, 1981) or between photoperiod and 1 72 behaviour (rams, Howies et a l . , 1980; male hamsters, G a r r e t t & Campbell, 1980). While i n t e r p r e t a t i o n of these r e s u l t s i s complicated by a f a i l u r e to c o n t r o l other environmental f a c t o r s , such as seasonal changes i n temperature and the behaviour of mates, or l e v e l s of endogenous hormone p r o d u c t i o n , they do suggest a r o l e f o r photoperiod i n the c o n t r o l of the b e h a v i o u r a l responsiveness to hormones. The a b i l i t y of s t e r o i d s to e l i c i t s exual behaviour i n one mammalian s p e c i e s under c o n d i t i o n s of c o n t r o l l e d l i g h t and hormone l e v e l s has now been s t u d i e d . T e s t o s t e r o n e - t r e a t e d c a s t r a t e d male hamsters perform more i n t r o m i s s i o n s and e j a c u l a t i o n s when h e l d under long days than when exposed to short days (Morin & Zucker, 1978; Campbell et a l . , 1978). Long daylengths i n c r e a s e d the d u r a t i o n and decreased the l a t e n c y of the l o r d o s i s response i n hormone-t r e a t e d o v a r i e c t o m i z e d female hamsters i n one t e s t (Schwartz & G e r a l l , 1978), although no s i g n i f i c a n t e f f e c t of photoperiod was found i n another study (unpublished r e s u l t s c i t e d i n Zucker et a l . , 1980). In a d d i t i o n to i t s e f f e c t on behaviour, daylength may a l s o i n f l u e n c e p h y s i o l o g i c a l responses to hormones. Photoperiod a l t e r s hypothalamic s e n s i t i v i t y to s t e r o i d feedback i n i n t a c t male q u a i l (Davies et a l . , 1976), sheep (Moss, 1979), grouse (Sharp & Moss, 1977), and c a s t r a t e d male hamsters ( E l l i s & Turek, 1980), and a f f e c t s p i t u i t a r y responsiveness to LHRH s t i m u l a t i o n i n i n t a c t female r a t s (Steger et a l . , 1976), i n t a c t male q u a i l (Davies & F o l l e t t , 1980), c a s t r a t e d male turkeys ( E l Halawani et a l . , 1980) and sheep ( L i n c o l n , 1977), and i n 1 73 ov a r i e c t o m i z e d ewes (Land et a l . , 1979). I t i s p o s s i b l e that photoperiod may prove to play a major r o l e i n the c o n t r o l of re p r o d u c t i o n both through changes i n p h y s i o l o g i c a l and i n b e h a v i o u r a l responsiveness to hormones. F. Why Should N e s t - b u i l d i n g Be Under P h o t o p e r i o d i c C o n t r o l ? A F u n c t i o n a l E x p l a n a t i o n U l t i m a t e l y , one i s l e f t with the q u e s t i o n of why nest-b u i l d i n g behaviour i s so much more s e n s i t i v e to p h o t o p e r i o d i c c o n t r o l than are other r e p r o d u c t i v e a c t i v i t i e s . Why should daylength a c t on a response which normally begins s e v e r a l days a f t e r c o u r t s h i p i s i n i t i a t e d ? The presence of an adequate nest i s e s s e n t i a l f o r m a i n t a i n i n g i n c u b a t i o n , and f o r s u c c e s s f u l h a t c h i n g of the eggs (White, 1975c; Cheng & B a l t h a z a r t , 1982). If completion of the breeding c y c l e i s blocked under short days as a r e s u l t of i n h i b i t i o n of n e s t - b u i l d i n g , then the energy i n v e s t e d i n the performance of e a r l i e r d i s p l a y s which are not a f f e c t e d by the photoperiod (bow-cooing, n e s t - s o l i c i t i n g , c o p u l a t o r y a c t i v i t y ) c o u l d be viewed as wasted. A number of f a c t o r s need to be c o n s i d e r e d i n t r y i n g to understand such seemingly maladaptive behaviour. I t i s p o s s i b l e that the apparent waste of energy a s s o c i a t e d with the performance of high l e v e l s of c o u r t s h i p by short-day b i r d s stems from the experimental design used. In t h i s study, as i n the vast m a j o r i t y of experiments d e a l i n g with r i n g dove behaviour, u n n a t u r a l l y h i g h l e v e l s of c o u r t s h i p may have been d i s p l a y e d as a r e s u l t of the p r a c t i s e of studying only newly 174 p a i r e d animals. Ring doves can l i v e f o r many years and they form permanent, monogamous p a i r s . I t i s t h e r e f o r e p o s s i b l e that c o u r t s h i p i s important only i n the r e l a t i v e l y r a r e s i t u a t i o n s i n which new p a i r bonds are being formed, and that low l e v e l s of c o u r t s h i p g e n e r a l l y occur a f t e r the f i r s t breeding c y c l e and a c t p r i m a r i l y i n p a i r maintenance. The concept of the p a i r bond i s a ra t h e r vague one, o f t e n used to r e f e r to widely d i f f e r e n t forms and degrees of attachment. E r i c k s o n (1978) has d i s c u s s e d many of the problems surrounding the d e f i n i t i o n and measurement of p a i r bonds. With t h i s i n mind, I propose to d e f i n e the " p a i r bond", as I w i l l use i t here, as the tendency of two doves to remain together and to d i r e c t t h e i r r e p r o d u c t i v e e f f o r t s to each o t h e r . I have a l s o made the assumption that the i n t e r a c t i o n s o c c u r r i n g soon a f t e r p a i r i n g are e s s e n t i a l f o r the formation of such a bond. I have argued that the d i f f e r e n t stages of male behaviour are a s s o c i a t e d with d i f f e r e n t c o n t r o l l i n g f a c t o r s . One reason f o r t h i s may be the s y n c h r o n i z a t i o n of the two p a r t n e r s — e a c h i n d i v i d u a l proceeds to the next step i n the c y c l e only a f t e r r e c e i v i n g the a p p r o p r i a t e s t i m u l i from the mate, nest, e t c . In a d d i t i o n , divergence of c o n t r o l i s undoubtedly l i n k e d to d i f f e r e n c e s i n the f u n c t i o n of each stage. I would l i k e to suggest that i t i s n e s t - b u i l d i n g which i s the key determinant of the onset of a c t u a l breeding (as opposed to p a i r formation) i n e s t a b l i s h e d p a i r s . Although c o u r t s h i p may s t i m u l a t e p a r t i a l development of the female's r e p r o d u c t i v e t r a c t , i t s major f u n c t i o n may be to e s t a b l i s h and maintain the p a i r bond between 175 the male and female. In experiments i n v o l v i n g newly p a i r e d b i r d s the two processes are o c c u r r i n g s i m u l t a n e o u s l y : the i n t e r a c t i o n s t a k i n g p l a c e a c t both i n formation of a p a i r bond and i n i n d u c t i o n of a new l a y i n g c y c l e . In e s t a b l i s h e d p a i r s , d i s p l a y s a s s o c i a t e d with bond formation would be expected to be l e s s f r e q u e n t . There i s l i t t l e sexual dimorphism i n d o v e s — m a l e s tend to be somewhat heavier than females, but otherwise they are i n d i s t i n g u i s h a b l e (to the human eye, at l e a s t ) . The sexes can, however, be e a s i l y i d e n t i f i e d on the b a s i s of t h e i r behaviour. Chasing, bow-cooing and n e s t - s o l i c i t i n g a l l appear soon a f t e r a p a i r of r e p r o d u c t i v e l y a c t i v e b i r d s come i n t o c o n t a c t , and ag g r e s s i v e c o u r t s h i p , which i s only performed by males, i s a c l e a r i n d i c a t i o n of gender. E r i c k s o n (1978) has proposed' that the bowing d i s p l a y i s important f o r s p e c i e s r e c o g n i t i o n , and may a l s o be used by females to i d e n t i f y r e p r o d u c t i v e l y a c t i v e males. Very l i t t l e i s known about the s i m i l a r i t i e s or d i f f e r e n c e s i n p r e - i n c u b a t i o n behaviour between the i n i t a l and subsequent breeding c y c l e s . S i l v e r & B a r b i e r e (1977) found that males bow-coo more towards strange females than towards t h e i r own mates. They conclude that the behaviour of the female e l i c i t s a g g r e s s i v e c o u r t s h i p . The d i f f e r e n c e i n male behaviour may a l s o be a r e f l e c t i o n of f a m i l i a r i t y with the female. Males which have been i s o l a t e d f o r two weeks bow-coo more i f p a i r e d with a strange female than with t h e i r former mate ( E r i c k s o n & M o r r i s , 1972). In the only d e t a i l e d study f o l l o w i n g the b i r d s ' behaviour through c o n s e c u t i v e c y c l e s , E r i c k s o n (1973) found that 176 males perform roughly the same amount of chasing and bowing towards t h e i r own mates as towards strange females d u r i n g the e a r l y stages of the f i r s t breeding c y c l e . T h i s soon changes, however, and the male subsequently d i r e c t s v i r t u a l l y a l l of h i s a g g r e s s i v e c o u r t s h i p towards the strange females. There was a s l i g h t resurgence of bowing towards the mate at the onset of the second c y c l e , but t h i s was l e s s than that seen i n the i n i t i a l c y c l e . I t thus appears that a g g r e s s i v e c o u r t s h i p has l i t t l e importance in t i m i n g the appearance of the eggs, but i s used as a means of a d v e r t i s i n g the sex of the performing b i r d , and i n d i r e c t l y a s c e r t a i n i n g the sex of the second i n d i v i d u a l , through i t s response. Observations of p o s i t i v e c o r r e l a t i o n s between the d i s p l a y of c h a s i n g and bowing and the l a t e n c y to o v i p o s i t i o n support t h i s idea ( L o v a r i & Hutchison, 1975; Hutchison & L o v a r i , 1976; McDonald, 1976; Zenone, 1979). F a b r i c i u s and Jansson (1963) proposed that n e s t - s o l i c i t i n g serves to e s t a b l i s h the n e s t - s i t e . The d i s p l a y of nest-s o l i c i t i n g i s a t t r a c t i v e to female doves. In the e a r l y stages of c o u r t s h i p the female only s o l i c i t s i n the v i c i n i t y of the male; i f he leaves she u s u a l l y stops d i s p l a y i n g , and i f he changes l o c a t i o n she g e n e r a l l y f o l l o w s . Males d i s p l a y high l e v e l s of n e s t - s o l i c i t i n g f o r the f i r s t few days a f t e r p a i r i n g , then r e s t r i c t t h e i r a c t i v i t y to short p e r i o d s e a r l y i n the day. A f t e r 4 weeks of s e p a r a t i o n from the mate n e s t - s o l i c i t i n g i n c r e a s e s a g a i n , but d e c l i n e s more r a p i d l y than i n the f i r s t c y c l e ( E r i c k s o n , 1973). N e s t - s o l i c i t i n g i s almost c e r t a i n l y i n v o l v e d i n pair-bond formation ( F a b r i c i u s & Jansson, 1963) as 177 i t i s extremely u n l i k e l y that a female would remain with a male which only d i s p l a y e d a g g r e s s i v e c o u r t s h i p towards her. Nest-s o l i c i t i n g i s a l s o important f o r inducing o v u l a t i o n i n the female ( E r i c k s o n & Lehrman, 1964; Cheng, 1974; L i l e y , 1976a). In order f o r n e s t - b u i l d i n g to p l a y an important r o l e i n ti m i n g r e p r o d u c t i o n , i t i s necessary that i t (1) reoccur i n each breeding c y c l e , and (2) s t i m u l a t e o v u l a t i o n i n females. Nest-b u i l d i n g by e s t a b l i s h e d p a i r s has not been examined, but two l i n e s of evidence s t r o n g l y suggest that i t does occur: b i r d s which are s u p p l i e d with a completed nest s t i l l engage i n some b u i l d i n g a c t i v i t y (White, 1976b; Cheng & B a l t h a z a r t , 1982), and the c l o s e l y r e l a t e d Indian c o l l a r e d dove ( S. decaocto ) i s re p o r t e d to b u i l d a new nest or to renovate the o l d one at the commencement of each c y c l e i n the w i l d (Rana, 1975). Other w i l d doves w i l l a l s o add n e s t i n g m a t e r i a l to o l d nests (White, 1975b). A c l o s e c o r r e l a t i o n between n e s t - b u i l d i n g and o v u l a t i o n has been observed i n c a n a r i e s (White & Hinde, 1968) and Bengalese f i n c h e s ( S l a t e r , 1970) as w e l l as i n r i n g doves (White, 1975b; McDonald, 1976). O v i p o s i t i o n can be delayed by d e p r i v i n g the b i r d s of n e s t i n g m a t e r i a l (Hinde & Warren, 1959; Lehrman et a l . , 1961), suggesting that e i t h e r p a r t i c i p a t i o n i n n e s t - b u i l d i n g or t a c t i l e s t i m u l i a r i s i n g from the nest i s important f o r f o l l i c u l a r development. In order to separate these two f a c t o r s White (1975b) presented p a i r s of doves with a completed nest or an empty nest bowl, and n e s t i n g m a t e r i a l . The b i r d s given a f u l l nest b u i l t l e s s , but a l s o o v u l a t e d l a t e r than those which had to b u i l d t h e i r own nest. Cheng & B a l t h a z a r t 178 (1982), i n a s i m i l a r study, found that b i r d s which engage i n more n e s t - b u i l d i n g a c t i v i t y have lower FSH l e v e l s and ovulate e a r l i e r than l e s s a c t i v e b i r d s . The authors conclude that p a r t i c i p a t i o n i n n e s t - b u i l d i n g s t i m u l a t e s female o v u l a t i o n . I t seems that both n e s t - s o l i c i t i n g and n e s t - b u i l d i n g by the male may a f f e c t the t i m i n g of e g g - l a y i n g . In a d d i t i o n , other f a c t o r s such as the time of year (Cheng et a l . , 1980) and the female's own n e s t - c o o i n g behaviour (Cohen & Chen, 1979; Cohen & Cheng, 1981) can i n f l u e n c e f o l l i c u l a r development. Thus, i t i s l i k e l y t h a t the t i m i n g of o v u l a t i o n depends on the combined e f f e c t of these f a c t o r s , and that each p l a y s a c o n t r i b u t i n g but not an i n d i s p e n s i b l e r o l e . N e s t - s o l i c i t i n g was not a f f e c t e d by d a y length i n these experiments, but i t i s p o s s i b l e that s o l i c i t i n g alone would be inadequate s t i m u l a t i o n f o r o v a r i a n development i n short-day females. The female's response to the male depends upon the s i z e of the l a r g e s t f o l l i c l e (Cheng, 1974), and i t has been shown that f o l l i c l e s i z e i s reduced by exposure to short daylengths ( L i l e y , 1976a). That photoperiod, through changes i n n e s t i n g , c o u l d i n f l u e n c e the p r e c i s e timing of breeding w i t h i n a much broader p e r i o d of r e p r o d u c t i v e a c t i v i t y , now seems more comprehensible. Male doves would b e n e f i t from f i n d i n g a mate or c o p u l a t i n g with a strange female r e g a r d l e s s of the time of year, so those d i s p l a y s a s s o c i a t e d with p a i r formation ( c h a s i n g , bow-cooing, n e s t - s o l i c i t i n g ) or i n s e m i n a t i o n ( b i l l i n g , mounting) are u n a f f e c t e d by p hotoperiod. Although n e s t - s o l i c i t i n g can s t i m u l a t e o v u l a t i o n i n the females, i t might be an inadequate st i m u l u s f o r short-day females in the 1 79 a b s e n c e o f n e s t i n g a c t i v i t y . T h e r e c o u l d be no a d v a n t a g e t o t h e e x p e n d i t u r e o f l a r g e a m o u n t s o f e n e r g y on n e s t - b u i l d i n g , o v u l a t i o n a n d i n c u b a t i o n a t t i m e s o f t h e y e a r when t h e c h a n c e o f s u r v i v a l o f t h e s q u a b s i s l o w . T h e r e f o r e , t h e o n s e t o f r e p r o d u c t i v e a c t i v i t y c o u l d be t i m e d by t h e a b i l i t y o f d a y l e n g t h t o r e g u l a t e n e s t - b u i l d i n g a n d , c o n s e q u e n t l y , f e m a l e o v u l a t i o n . 180 V I I . CONCLUSION The r e s u l t s presented here are of p a r t i c u l a r importance s i n c e they not only i n d i c a t e a p o s s i b l e mechanism through which an e x t e r n a l f a c t o r (photoperiod) a l t e r s b e h a v i o u r a l responsiveness to androgenic s t i m u l a t i o n , but they a l s o p rovide the f i r s t example of an e f f e c t of pinealectomy on a d u l t r e p r o d u c t i v e behaviour. In a d d i t i o n , they supply one of the few c l e a r i n d i c a t i o n s of p i n e a l involvement i n a v i a n breeding c y c l e s . 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ABBREVIATIONS androstenedione 1,4,6-androstatriene-3,17-dione estrogen e s t r a d i o l benzoate 5<x»dihydrotestosterone 56 d i h y d r o t e s t o s t e r o n e 5o^dihydrotestosterone propionate f o l l i c l e - s t i m u l a t i n g hormone 5-hydroxytryptamine = s e r o t o n i n hydroxyindole-O-methyltransferase l u t e i n i z i n g hormone l u t e i n i z i n g hormone r e l e a s i n g hormone N - a c e t y l t r a n s f e r a s e p r e o p t i c area t e s t o s t e r o n e t e s t o s t e r o n e propionate 

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