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A study on the function of foam from the proctodeal gland of the male Japanese quail (Coturnix coturnix… Hickman, Andrew Richard 1984

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A STUDY ON THE FUNCTION OF FOAM FROM THE PROCTODEAL GLAND OF THE MALE JAPANESE QUAIL (COTURNIX COTURNIX JAPONICA) WITH RESPECT TO ITS EFFECTS ON SPERM COMPETITION BY ( ? ) ANDREW RICHARD HICKMAN B.A. (Zoology), DePauw University, 1981 A THESIS SUBMITTED IN.PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Poultry Science) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1984 @ Andrew Richard Hickman, 1984 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 o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f 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 of 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 o f my department o r 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 g a i n 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 o f The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) ABSTRACT The e f f e c t of foam f r o m t h e male J a p a n e s e q u a i l on sperm c o m p e t i t i o n was i n v e s t i g a t e d by comparing f e r t i l i t y and mating behaviour of foam-producing (sham-operated) and f o a m l e s s (foam g l a n d c a u t e r i z e d ) males i n two male / f i v e female mating group s i t u a t i o n s . In v i t r o examinations of sperm b e h a v i o u r i n foam were a l s o c a r r i e d out. Over an eight-week e x p e r i m e n t a l p e r i o d , foam-producing males s i r e d more than 90% of the progeny produced when i n competition with a f o a m l e s s male. Pens i n which two f o a m l e s s males were competing had s i g n i f i c a n t l y lowered f e r t i l i t y and h a t c h a b i l i t y of eggs, w h i l e pens w i t h two foam-producing males had the h i g h e s t f e r t i l i t y . Foamless males attempted and completed c o p u l a t i o n s as o f t e n or more o f t e n than foam-producing males and examinations of females post-copula revealed that sperm t r a n s f e r was o c c u r r i n g during c o p u l a t i o n s by both t y p e s of males. R e s u l t s i n d i c a t e t h a t foam a l l o w s a male t o c o p u l a t e l e s s f r e q u e n t l y and yet maintain good f e r t i l i t y l e v e l s . Foam s i g n i f i c a n t l y h i n d e r e d the p e n e t r a t i n g a b i l i t y of q u a i l sperm i n t h i n a l b u m i n - f i l l e d c a p i l l a r y tubes (75 mm l o n g x 1.2 i n n e r diameter). On the other hand, foam s i g n i f i c a n t l y prolonged q u a i l sperm m o t i l i t y . The behaviour of q u a i l sperm when mixed with foam from the same or from a d i f f e r e n t male, was i n d i s t i n g u i s h a b l e when examined on microscope s l i d e s at 400x m a g n i f i c a t i o n . I found that during c o p u l a t i o n by q u a i l , sperm and foam are mixed and deposited i n t o the female's coprodeum, rather than i n the o v i d u c t as with most other birds examined. In conclusion, because the temporal p a t t e r n of egg l a y i n g i n the J a p a n e s e q u a i l ( l a t e i n t h e day) p r e c l u d e s a peak i n copulations at the optimum time for f e r t i l i z a t i o n (within an hour p o s t - o v i p o s i t i o n ) , foam may aid a male's r e p r o d u c t i v e s u c c e s s by s u s p e n d i n g and s u s t a i n i n g h i s sperm i n the female's coprodeum, thus a v o i d i n g e x c e s s i v e l o s s of sperm v i a o v i p o s i t i o n . These experiments a l s o suggest a hypothesis about a r o l e for foam i n sperm c o m p e t i t i o n : i t s p h y s i c a l presence f i l l s s p a c e which may p r e c l u d e normal foam and semen deposition by subsequent copulations and thus increase the chances of the foam/sperm mass from subsequent copulations being excreted by the female. i v TABLE OF CONTENTS Page ABSTRACT • i i TABLE OF CONTENTS i v LIST OF TABLES v i i LIST OF FIGURES x ACKNOWLEDGEMENTS x i INTRODUCTION 1 GENERAL MATERIALS AND METHODS 9 A) Experimental B i r d s 9 B) Incubation and Rearing 10 C) Operations 11 EXPERIMENT 1 14 A) H i s t o r y and Rearing of Experimental B i r d s 14 B) Experimental Procedure 15 C) Data Analyses 18 •D) Re s u l t s 21 1. E f f e c t of q u a i l foam on f e r t i l i t y and h a t c h a b i l i t y under n a t u r a l mating c o n d i t i o n s 21 2. E f f e c t of q u a i l foam on sperm competition 21 3. E f f e c t of c o n d i t i o n of the foam gland on the male's mating behaviour 22 4. D i f f e r e n c e s between genotypes i n homosexual c o p u l a t i o n s . 29 EXPERIMENT 2 32 A) M a t e r i a l s and Methods 32 V TABLE OF CONTENTS (Continued) EXPERIMENT 3 38 Experiment 3A 39 A) Materials and Methods 39 B) Analyses 43 C) Results 45 1. E f f e c t of foam on sperm penetration 45 2. E f f e c t of incubation temperature on sperm penetration... 45 3. E f f e c t of time of examination on sperm penetration 45 4. E f f e c t of observers on sperm penetration 46 5. E f f e c t of r e p l i c a t i o n on sperm penetration 46 6. E f f e c t s of the s i g n i f i c a n t i n t e r a c t i o n s involving the main e f f e c t s on sperm penetration 48 7. E f f e c t of foam on chicken sperm m o t i l i t y 53 Experiment 3B 53 A) Materials and Methods 54 1. Semen c o l l e c t i o n from male Japanese q u a i l 54 2. Experimental procedure 55 a) Sperm penetration and m o t i l i t y i n c a p i l l a r y tubes.... 55 b) Sperm m o t i l i t y on microscope s l i d e s 56 B) Analysis 57 C) Results 57 1. E f f e c t of foam on quail sperm penetration 57 2. E f f e c t of foam on qu a i l sperm m o t i l i t y 57 DISCUSSION 59 v i TABLE OF CONTENTS (Continued) BIBLIOGRAPHY 66 APPENDICES 75 Experiment 4.... 76 ANOVA T a b l e s 85 v i i LIST OF TABLES Table Page 1. F e r t i l i t y , h a t c h a b i l i t y , and phenotypic r a t i o of progeny i n the four treatments (summed over three r e p l i c a t i o n s ) 23 2. Phenotypes of progeny from females i n the four mating groups 24 3. Mean number of mating attempts (MA) and completed copulations (CC) by UBC-W males per observation period 26 4. S i g n i f i c a n t 3-way i n t e r a c t i o n between foam gland operation, operation of competing male, and time of day; mean number of mating attempts (MA) by UBC-W males per observation 27 5. Mean number of mating attempts (MA) and completed copulations (CC) by UBC-A males per observation period 28 6. Time of day x operation of foam gland i n t e r a c t i o n ; mean number of mating attempts (MA) by UBC-A males per observation 30 7. A comparison of the t o t a l number of mating attempts (MA) and completed copulations (CC) by UBC-W and UBC-A males 31 8. The presence of sperm and foam i n d i f f e r e n t parts of the female's cloaca immediately a f t e r copulation 35 9. Mean distance (mm) t r a v e l l e d by rooster sperm through c a p i l l a r y tubes per examination 47 10. S i g n i f i c a n t 2-way i n t e r a c t i o n between time and contents of tube; mean distance (mm) t r a v e l l e d by rooster sperm through c a p i l l a r y tubes per examination 49 LIST OF TABLES (Continued) v i i i 11. S i g n i f i c a n t 2-way i n t e r a c t i o n between time and temperature; mean distance (mm) t r a v e l l e d by rooster sperm through c a p i l l a r y tubes per examination 50 12. S i g n i f i c a n t 2-way i n t e r a c t i o n between time and observer; mean distance (mm) t r a v e l l e d by rooster sperm through c a p i l l a r y tubes per examination 51 13. S i g n i f i c a n t 2-way i n t e r a c t i o n between time and contents of tube; mean m o t i l i t y ranks for rooster sperm i n c a p i l l a r y tubes 52 14. Duration of q u a i l sperm m o t i l i t y on microscope s l i d e s 58 15. Number of completed copulations and number of females producing f e r t i l e eggs by treatment 80 16. T o t a l f e r t i l i t y and duration of f e r t i l i t y by females that l a i d at le a s t one f e r t i l e egg 81 17. Occurrences of sperm transf e r during copulations by foamless and foam-producing males 83 18. Analysis of variance for t o t a l f e r t i l i t y 1 of a l l incubated eggs i n Experiment 1 85 19. Analysis of variance for percent h a t c h a b i l i t y 1 of a l l f e r t i l e eggs i n Experiment 1 86 20. Analysis of variance for number of white progeny typed out of a l l progeny typed 1 i n Experiment 1 87 21. Analysis of variance for percent h a t c h a b i l i t y 1 of white progeny out of a l l f e r t i l e eggs i n Experiment 1 88 LIST OF TABLES (Continued) i x 22. A n a l y s i s of variance f o r number of mating attempts per observation-^ by UBC-W males i n Experiment 1 89 23. A n a l y s i s of variance f o r number of completed c o p u l a t i o n s per observation^ by UBC-W males i n Experiment 1 90 24. A n a l y s i s of variance f o r number of mating attempts per observation^ by UBC-A males i n Experiment 1 91 25. A n a l y s i s of variance f o r number of completed c o p u l a t i o n s per observation^ by UBC-A males i n Experiment 1 92 26. A n a l y s i s of variance f o r r o o s t e r sperm penetration through c a p i l l a r y tubes 93 LIST OF FIGURES x Figure Page 1. S a g i t t a l s e c t i o n of cloaca of a d u l t male Japanese q u a i l . . . . 2 2. Method of r e s t r a i n t f o r c a u t e r i z a t i o n of foam gland 13 3. C a u t e r i z a t i o n of foam gland 13 A. Floor" pen as used i n Experiment 1 16 5. Foam being e x p e l l e d from the female's coprodeum 36 6. Cloaca of a female chicken opened along the d o r s a l m i d l i n e and l a i d f l a t 37 7. Diagram of a prepared c a p i l l a r y tube A l x i ACKNOWLEDGEMENTS I would l i k e to express my a p p r e c i a t i o n and i n d e b t e d n e s s t o Dr. K.M. Cheng, t h e s i s a d v i s o r and f r i e n d , f o r h i s guiding i n s i g h t s and i n t e r e s t i n my research. My l e a r n i n g e x p e r i e n c e s have been g r e a t l y enhanced by h i s knowledge. A p p r e c i a t i o n i s a l s o extended t o the members of my Advisory Committee, P r o f e s s o r B.E. March, Dr. J.N.M. Sm i t h , and Dr. R.C. F i t z s i m m o n s f o r t h e i r a d v i c e and suggestions. Graduate students and s t a f f of the P o u l t r y Science Department a t UBC, p r o v i d e d both an i n t e l l e c t u a l l y s t i m u l a t i n g and congenial atmosphere i n which to conduct research. S p e c i a l t h anks i s due t o the s t a f f of the Q u a i l G e n e t i c Stock Centre, UBC, e s p e c i a l l y Cathleen N i c h o l s f o r her sympathetic ear and moral s u p p o r t when i t was needed most. I am g r e a t f u l f o r being twice awarded the Jacob B i e l y S c h o l a r s h i p which p r o v i d e d n e c e s s a r y a s s i s t a n c e d u r i n g my r e s e a r c h . This research was supported by A g r i c u l t u r e Canada Operating grant # 1027 and U n i v e r s i t y of B r i t i s h Columbia N a t u r a l , Applied and H e a l t h Sciences research grant # N81-184. [ 1 ] INTRODUCTION The male Japanese q u a i l (Coturnix c o t u r n i x j a p o n i c a ) produces a t h i c k foam from the proctodeal gland i n the d o r s a l w a l l of the cl o a c a ( C o i l and Wetherbee, 1959; Ikeda and T a j i , 1954; Nagra et a l . , 1959) ( F i g 1 ) . A l t h o u g h t h e p r o d u c t i o n of foam i s unique t o the genus Co t u r n i x , homologous v e s t i g i a l g l a n d u l a r t i s s u e has been found i n c h i c k e n s and t u r k e y s (King, 1975, 1981; Komarek, 1970), i n ducks and geese (Komarek, 1971), and p o s s i b l y a l s o i n Northern Fulmars ( H a t c h , 1983) . Such f i n d i n g s may i n d i c a t e a much wider occurrance of these glands i n the Cla s s Aves. The production of foam has r e s u l t e d i n the common name "foam g l a n d " f o r the p r o c t o d e a l g l a n d of the Japanese q u a i l . The term " c l o a c a l gland" had a l s o been used i n the l i t e r a t u r e . In t h i s t h e s i s I s h a l l use the common name foam gland i n r e f e r r i n g to the proctodeal gland of the male Japanese q u a i l . The p r o c t o d e a l gland occurs i n both sexes of the afore-mentioned domestic b i r d s i n c l u d i n g the Japanese q u a i l ( K i n g , 1975; Komarek, 1 9 7 1 ) . I n the female q u a i l , a s m a l l amount of foam i s sometimes produced, e s p e c i a l l y i n the o l d e r b i r d s ( M c F a r l a n d e t a l . , 1968). Females, immature males, and c a s t r a t e s can be stim u l a t e d to produce foam by the a d m i n i s t r a t i o n of t e s t o s t e r o n e ( A d k i n s and A d l e r , 1972; H u t c h i s o n , 1978; M c F a r l a n d e t a l . , 1968; Nagra e t a l . , 1959; Schumacher and B a l t h a z a r t , 1983). The development (Schafersman and Klemm, 1977; Siopes and Wilson, [ 2 ] FIGURE 1: S a g i t t a l section of cloaca of adult male Japanese q u a i l . Figure taken from Klemm ejt a]_., 1973. [3] 1975) and the morphology (Klemm et a l . , 1973; McFarland et a l . , 1968; Tamura and F u j i i , 1967) of the male's foam gland have been s t u d i e d . The foam gland i s a c t u a l l y an "aggregate gland" of discre t e glandular units united by connective tissue (Klemm et a l . , 1973). Hypertrophy o f t h e g l a n d u l a r t i s s u e i s s e a s o n a l l y ( p h o t o p e r i o d i c a l l y ) c o n t r o l l e d ; the glands are androgen-dependent with the s i z e of the r e s u l t i n g protuberance highly correlated with sexual behavior (Sachs, 1967, 1969). Wilson et a l . (1962) found that the foam glands s t a r t e d producing foam only a f t e r the t e s t e s weight had reached 0.75 gm. Thus, the c l o a c a l protuberance of the male Japanese q u a i l i s a good i n d i c a t o r of the male's sexual condition and androgen l e v e l s (Sachs, 1967, 1969) and can be used to measure t e s t i c u l a r a c t i v i t y over time without having to k i l l the male to obtain t e s t i c u l a r weight (Siopes and Wilson, 1975). In t h i s context, there has been much re s e a r c h done r e c e n t l y on t e s t o s t e r o n e metabolism i n the foam gland, i n comparison with metabolism i n the b r a i n of the male Japanese q u a i l , and i t s e f f e c t s on behaviour and sexual development (e.g. Adkins and Pniewski, 1978; Adkins et a l . , 1980; Balthazart and Schumacher, 1984; Balthazart et a l . , 1979, 1980, 1984; Cohen-Parsons et a l . , 1983; Davies et a l . , 1980; Massa et a l . , 1979, 1980; for review, see Adkins-Regan, 1981; Ottinger, 1983; M a r t i n i , 1982). The s e c r e t i o n of the foam gland i s a viscous mucoid—referred to as a g l y c o m u c o p r o t e i n by M c F a r l a n d e t a 1 . ( 1 9 6 8 ) and as a m u c o p o l y s a c c h a r i d e by F u j i i and Tamura (1967) and Klemm et a l . (1973)—that apparently foams with CO2 and H2 produced from c l o a c a l [A] bacteria (Escherichia c o l i and Proteus m i r a b i l i s ) metabolizing glucose (McFarland et a l . , 1968). Pellerano-Domini and Renzoni (1969) found the s e c r e t i o n to be AA % p r o t e i n , A.A % polysaccharides, and 5.7 % uronic a c i d . Uronic acid i s involved with the oxidative breakdown of glucose (Martin et a l . , 1983). Secretions from a c t i v e foam glands are sulfated while secretions from the non-stimulated female and immature and c a s t r a t e d male foam glands are non-sulfated ( F u j i i and Tamura, 1967). No c o n c l u s i v e evidence has been presented on the function of the foam but because i t i s d e p o s i t e d along with semen i n t o the female d u r i n g c o p u l a t i o n and i t s p r o d u c t i o n i s androgen-dependent (Sachs, 1967, 1969), i t i s most commonly t h o u g h t t o be c o n c e r n e d w i t h r e p r o d u c t i o n (Ikeda and T a j i , 1954; Klemm et a l . , 1973; McFarland et a l . , 1968; Ogawa et a l . , 197A; Wetherbee, 1961). Other suggestions as to the foam's f u n c t i o n have been: ( i ) i t prevents post-copulatory sperm leakage from the female (Perez and Juarez, 1966), ( i i ) i t a c t s as a l u b r i c a n t f o r the male's p h a l l u s (Renzoni, 1968), and ( i i i ) because i t i s also occasionally excreted with feces, i t might function as a t e r r i t o r i a l marker (Schleidt and Shalter, 1972). Although accummulated evidence suggests that foam from the male Japanese q u a i l p l a y s a ro l e i n reproduction, Marks and Lepore (1965) and Lepore and Marks (1966) obtained f a i r f e r t i l i t y u s i n g a r t i f i c i a l insemination (Al) without mixing foam with the inseminated semen. The Al may have bypassed a f a c t o r i n the process of normal mating, but foam i s a p p a r e n t l y not necessary for achieving f e r t i l i z a t i o n i n the Japanese q u a i l . [5] A comparison of d a i l y o v i p o s i t i o n and peak c o p u l a t i o n times between Japanese q u a i l and other domestic birds provides some i n s i g h t s for the development of a hypothesis on the function of foam i n natural mating. Chickens and ducks lay most of t h e i r eggs early i n the l i g h t p o r t i o n of the day (Tanabe and Nakamura, 1980; Wilson, 1964), turkeys lay mostly during l a t e morning or early afternoon (Wilson, 1964), and Japanese q u a i l l a y d u r i n g the l a s t 2-4 hours of the day (Konishi, 1980; Wilson, 1964). In a l l of these birds, ovulation normally follows o v i p o s i t i o n of the p r e v i o u s egg by 15-75 minutes (Sturkie, 1976). F e r t i l i z a t i o n of an ovulated ovum occurs i n the in f u n d i b u l u m ( A l l e n and G r i g g , 1957; Howarth, 1974). A second v i t e l l i n e membrane formation ( B e l l a i r s e_t_ al_., 1963) and albumin d e p o s i t i o n around the yolk s u f f i c i e n t to preclude f e r t i l i z a t i o n occurs within 15-30 minutes a f t e r ovulation i n these b i r d s ( G i l b e r t , 1971; Woodard and Mather, 1964). Sperm normally takes an hour to traverse the oviduct (Allen and Grigg, 1957) but near the time of o v u l a t i o n , sperm can t r a v e r s e the oviduct within 10-15 minutes (Bobr et a l . , 1964b; Howarth, 1971). On t h e o t h e r hand, an egg i n t h e o v i d u c t , e s p e c i a l l y a h a r d - s h e l l e d egg, i s an e f f e c t i v e block to the upward t r a v e l of sperm and apparently lowers the concentrations of sperm subsequently s t o r e d i n the u t e r o v a g i n a l (UV) sperm host glands (Bobr et a l . , 1964a). A r t i f i c i a l insemination at times when a h a r d - s h e l l e d egg i s i n the o v i d u c t a l s o r e s u l t s i n lowered f e r t i l i t y (Moore and Byerly, 1942; Parker, 1945; Wyne et a_l., 1959). Thus, a male should copulate within an hour p o s t - o v i p o s i t i o n to o p t i m i z e h i s chance of f e r t i l i z i n g an ovum. However, i n a fl o c k s i t u a t i o n i t w i l l be d i f f i c u l t f o r a male [6] to d e t e r m i n e the egg l a y i n g time f o r each female or to have the opportunity to copulate with a female at the a p p r o p r i a t e time; the male would do best to copulate when most or a l l of the females i n the flock have l a i d to maximize h i s chance of c o p u l a t i n g with a female without a hard-shelled egg i n her oviduct. Under these circumstances, the male's evolutionary s t r a t e g y of t i m i n g c o p u l a t i o n f o r the best chance of f e r t i l i z a t i o n would be a compromise between two opposing time f a c t o r s . The male should time copulation so that: 1) i t ' s l a t e enough i n the day that i f copulating with a female c h o s e n a t random from th e f l o c k , t h e r e would be no h a r d - s h e l l e d egg i n the female's o v i d u c t , but i t ' s early enough so that 2) c o p u l a t i o n would occur near the time of o v u l a t i o n so h i s sperm would have the best chance of f e r t i l i z i n g the ovulated ovum. Such p r e d i c t i o n i s t r u e i n chickens (Wood-Gush, 1971), turkeys (Smyth and L e i g h t o n , 1953), and ducks ( R a i t a s u o , 1964); i n these s p e c i e s , copulation frequencies peak d a i l y near the end of the normal time curve for egg l a y i n g . However, i n Japanese q u a i l , the p r e d i c t e d peak for copulations would be a f t e r dark because egg l a y i n g peaks just 2-4 hours before dark with some females laying c o n s i s t e n t l y a f t e r dark (Opel, 1966; Wilson and Huang, 1962). As i t i s , there's no clear peak i n copulation frequency i n Japanese q u a i l , with only a s i g n i f i c a n t low c o r r e s p o n d i n g to the peak of egg laying a c t i v i t i e s (Ottinger et a l . , 1982). Thus, perhaps, the foam gland developed i n the q u a i l because i t s exudate played a r o l e i n compensating for the lack of a convenient [7] "insemination window" (Cheng e_t al_., 1983) as occurs i n other domestic b i r d s . The r o l e of the foam might be to a i d sperm i n r e a c h i n g the oviduct and to also block sperm from subsequent copulations. F u r t h e r evidence to argue f o r a r o l e by the male's foam i n n a t u r a l mating can be seen from a comparison of w i l d and domestic Japanese q u a i l . In the wild, Japanese q u a i l are mostly monogamous but polygyny o f t e n occurs with a s u r p l u s of females (Moreau and Wayre, 1968; Wetherbee, 1961); under about 600 years of domestication (Howes, 196A; Yamashina, 1961), Japanese q u a i l have become promiscuous (Wetherbee, 1961). T h i s c o n d i t i o n has d r a s t i c a l l y i n c r e a s e d the i n t e n s i t y of sperm competition within the female. At the same time, the s i z e of the foam g l a n d has i n c r e a s e d s i g n i f i c a n t l y under d o m e s t i c a t i o n ( S c h l e i d t and S h a l t e r , 1972). The p h y s i c a l c h a r a c t e r i s t i c s of foam make i t an i d e a l v e h i c l e f o r s p e r m i c i d e s i n human v a g i n a l c o n t r a c e p t i v e s ( B e r n s t e i n , 1971; Dingle and Tietz e , 1963; G r e e n s t e i n , 1965; Hafez, 1980; Sobrero, 1970); perhaps the qua i l ' s foamy exudate also evolved i n response to i t s e f f e c t s on sperm competition within the female. I n t r a s e x u a l s e l e c t i o n w i l l be e s p e c i a l l y intense for males to reduce competition from sperm of another male (Parker, 1970; T r i v e r s , 1972). Sperm competition occurs i n insects (Parker, 1970), r e p t i l e s (Devine, 1975; Gibson and F a l l s , 1975), mammals (Dewsbury, 1984), f i s h (Van Tienhoven, 1983), as well as i n birds (Allen and Champion, 1955; Cheng et a l . , 1983; McKinney et_ a_l. , 1984; Payne and Kahrs, 1961). T h e r e have evolved numerous s t r a t e g i e s by males to reduce sperm competition i n t h e i r favor, e.g. prolonged or repeated c o p u l a t i o n s [8] ( i n s e c t s : P a r k e r , 1970; Smith, 1979; mammals: Ogelsby et al_., 1981; bird s . : W e l t y , 1979), mate g u a r d i n g ( i n s e c t s : P a r k e r , 1970; salamanders: Arnold, 1976; birds: McKinney e_t al^., 1984), sperm blocks or copulatory plugs ( i n s e c t s : Parker, 1970; r e p t i l e s : Devine, 1975, 1977; Ross and Crews, 1977; mammals: Hartung and Dewsbury, 1978; Voss, 1979). There has been no reported example of a sperm block o c c u r i n g i n birds (McKinney et a l . , 1984). T h i s t h e s i s r e s e a r c h was conducted to determine the function of the foam from the foam glands of the male Japanese q u a i l and i t s r e l a t i o n s h i p to sperm competition. Two hypotheses were tested: 1) The foam gland exudate of the male Japanese q u a i l p l a y s a rol e i n the male's natural mating, and 2) The foam of the male Japanese q u a i l i n c r e a s e s t h a t male's r e p r o d u c t i v e s u c c e s s by e n h a n c i n g i t s own f e r t i l i z i n g capacity and/or by hindering sperm of competing males from achieving f e r t i l i z a t i o n . GENERAL MATERIALS AND METHODS [9] A) EXPERIMENTAL BIRDS The b i r d s used i n a l l the experiments were e i t h e r w i l d - t y p e (UBC-A) or white (UBC-W) plumage Japanese q u a i l . Both s t r a i n s are maintained as randombred l i n e s at the UBC Quail Genetic Stock Centre. The wild-type plumage UBC-A l i n e was acquired from the U n i v e r s i t y of Washington (Pullman) i n 1964. The l i n e has been closed since 1968 (Somes, 1981) and maintained with a population si z e of 100 females and 50 males each g e n e r a t i o n f o r at le a s t 40 generations at the time of th i s experiment. B i r d s from the UBC-W l i n e carry an autosomal recessive mutation producing white feathers with occasional patches of darker f e a t h e r s (Roberts et a l . , 1978). This l i n e originated i n 1976 from a pair of white birds acquired from a l o c a l breeder. The o r i g i n a l b i r d s were ou t c r o s s e d to the UBC-A l i n e and the F-^'g were intermated to obtain the white progeny. UBC-W has s i n c e been maintained as a randombred l i n e of 66 females and 33 males. This l i n e had gone through at lea s t 12 g e n e r a t i o n s of c l o s e d random breeding at t h e s t a r t of t h e s e experiments. These two l i n e s were chosen f o r the experiments because the recessive white plumage gene of the UBC-W b i r d s c o u l d be used as a ge n e t i c marker. Using only UBC-W females, unequivocal determination of paternity was p o s s i b l e — t h e homozygous dominant UBC-A males would [10] produce only heterozygous wild-type o f f s p r i n g and the recessive UBC-W males would produce only homozygous white o f f s p r i n g . B) INCUBATION AND REARING B i r d s to be used i n the experiments were raised i n groups of 70 with approximately equal proportions of UBC-A and UBC-W b i r d s of both sexes. T h i s was done to minimize b i a s due to p r e f e r e n c e f o r own phenotype i n mating (Immelmann, 1972; Gallagher, 1976, 1977; Bateson, 1978; Truax and Si e g e l , 1982). Groups were reared i n "Marsh Farm" game ' brooders (71 cm x 92 cm x 15 cm) (Marsh Mfg., Garden Grove, Ca.) u n t i l a b o u t 5 weeks of age. UBC-A females were then.removed from the experiment and the remaining e x p e r i m e n t a l b i r d s were moved to and maintained i n community holding cages u n t i l the s t a r t of experiments. Males of both l i n e s were mixed but were kept separate from the UBC-W f e m a l e s . A l l e x p e r i m e n t a l b i r d s were wingbanded f o r i n d i v i d u a l i d e n t i f i c a t i o n and debeaked at about 4 weeks of age t o r e d u c e feather-pecking and cannibalism (Mahn and Blackwell, 1967). Feed (26% protein turkey s t a r t e r r a t i o n ) and water were a v a i l a b l e ad_ l i b i t u m during the rearing and experimental periods. In experiments where f e r t i l i t y , h a t c h a b i l i t y , and phenotype of the o f f s p r i n g were to be determined, eggs were c o l l e c t e d d a i l y and i d e n t i f i e d by pen or cage number and date. When more than one female was kept per pen, eggs from a given female could be matched by the [11] s p e c i f i c patterns, c o l o r s , shapes, and s i z e s of the egg s h e l l s (Jones et a l . , 1964). Eggs were stored i n a cooler room at about 11°C f o r a maximum of 6 days and then set for incubation i n batches once a week. These eggs were a r t i f i c i a l l y incubated and transferred on the 14th day of i n c u b a t i o n f o r h a t c h i n g i n i n d i v i d u a l pedigree baskets i n two Jamesway Model 252 incubators. A l l eggs not hatched a f t e r 18 days of i n c u b a t i o n were stored at l l o C and were broken out l a t e r to determine i f they were i n f e r t i l e ( K o s i n , 1944) or were e m b r y o n i c d e a t h s . Embryonic deaths were c l a s s i f i e d as early deads (ED; embryos that died before 8 days of i n c u b a t i o n ) , l a t e deads (LD; embryos t h a t d i e d between 8 days of i n c u b a t i o n and p i p p i n g ) , and p i p s (embryos that pipped but were unable to emerge from the s h e l l ) . Plumage phenotypes could be determined during the LD stage (Padgett and Ivey, 1960). C) OPERATIONS In two of the experiments, some treatments required males not producing foam and yet m a i n t a i n i n g s p e r m a t o g e n e s i s and s e x u a l behaviour. Therefore, i t was necessary to operate on the foam glands of these males. As the foam gland i s h y p e r t r o p h i e d a f t e r sexual maturity, the best time for the operation would be before the gland i s f u l l y developed. Rather than t r y i n g to remove the gland s u r g i c a l l y , i t was destroyed by cautery. This was done to minimize d i s t o r t i o n of the c l o a c a l a r e a . A s i m i l a r p r o c e d u r e has been s u c c e s s f u l i n [12] r e n d e r i n g other glands non-functional (e.g. avian s a l t gland, Hughes, 1977) A preliminary study was conducted u s i n g 3, 4, and 5-week o l d non-experimental c h i c k s . Both destroying the gland by using dry i c e applied l o c a l l y and by e l e c t r i c cautery ( H y f r e c a t o r ) were t r i e d . I found that c a u t e r y was most e f f e c t i v e between 4 and 5 weeks of age; the best r e s u l t s were obtained with approximately 90 v o l t s . A l l b i r d s to be operated on were trimmed of f e a t h e r s i n the cl o a c a l region. They were r e s t r a i n e d by wrapping with a pi e c e of f o l d e d "Kimwipes" t i s s u e and then s e c u r i n g with masking tape ( F i g . 2). Approximately 0.15 to 0.20 ml of l o c a l anesthetic (Xylocaine) was i n j e c t e d i n t o the d o r s a l l i p of the cloaca and 60 to 90 seconds were allowed for the anesthetic to take e f f e c t . A s i n g l e 1 cm cut with a number 3 s u r g i c a l s c a l p e l was made l o n g i t u d i n a l l y from the centre of the cloaca's d o r s a l l i p to expose the i n n e r c l o a c a l s u r f a c e . One p e r s o n w i t h i n s u l a t e d f o r c e p s kept the s u r f a c e to be c a u t e r i z e d exposed w h i l e a second person operated the H y f r e c a t o r ( F i g . 3 ) . Cotton swabs (Q-Tips) were used to cl e a r the area when needed. After the glandular ti s s u e was destroyed, t a l c powder was sprinkled over the area to he l p stop any b l e e d i n g . The b i r d was then released into a holding box with water a v a i l a b l e and was p e r i o d i c a l l y observed to ensure t h a t bleeding had stopped, and that the bird was moving around before being r e p l a c e d i n i t s o r i g i n a l cage. The time r e q u i r e d to operate on one b i r d was l e s s than 10 minutes. Sham-operated birds used as co n t r o l s were t r e a t e d i d e n t i c a l l y to the c a u t e r i z e d b i r d s except that the c a u t e r i z a t i o n i t s e l f was not performed. [13] [14] EXPERIMENT 1 There i s no empirical data i n the l i t e r a t u r e about the r o l e of the male Japanese q u a i l ' s foam gland s e c r e t i o n i n natural matings. This experiment was designed to determine whether the foam p l a y s a r o l e i n n a t u r a l mating by examining i t s e f f e c t s i n a 2-male/5-female mating group s i t u a t i o n using sexually mature males e i t h e r producing foam or with foam glands c a u t e r i z e d . One male can induce above 61% f e r t i l i t y when kept with 5 females (Woodard and Abplanalp, 1967). Two males were used i n order to study the e f f e c t s of foam on sperm competition within the female. A) HISTORY AND REARING OF EXPERIMENTAL BIRDS The birds used i n t h i s experiment (UBC-A and UBC-W s t r a i n s ) were h a t c h e d on 20 August, 1982, and were reared with the. sexes and genotypes mixed. F o r t y - f i v e males from each l i n e and a l l 66 UBC-W females were used i n the experiment. Sexes were separated at four [15] weeks of age and the UBC-W females were then maintained i n community holding cages u n t i l the beginning of the experiment. After eight weeks of age, 60 UBC-W females were placed i n t h e i r experimental f l o o r pens for an ac c l i m a t i z a t i o n period of 10 days. A l l experimental males were operated on before they were 6 weeks of age (30 males from each l i n e were cauterized and 15 males from each were sham-operated). Males were separated by genotype and placed i n community holding cages two days a f t e r a l l operations were completed. P r i o r to the experiment, a " f e r t i l i t y t e s t " was performed with 20 cauterized males (10 males from each l i n e ) . Males were i n d i v i d u a l l y p a i r e d w i t h f e m a l e s i n c a g e s f o r 6 days; eggs were c o l l e c t e d , incubated for 5 days, and broken out to determine f e r t i l i t y . Males from p a i r s where the female was c o n s i s t e n t l y l a y i n g i n f e r t i l e eggs were e l i m i n a t e d from the experiment. Because o f time and cage a v a i l a b i l i t y , not a l l the cauterized males used i n the experiment were tested for f e r t i l i t y . B) EXPERIMENTAL PROCEDURE Six chicken-wire f l o o r pens with wood shavings l i t t e r (each pen = 122cm x 92cm x 46cm high) were placed i n each of two rooms with the same dimentions (3.45 m x 3.65 m). Each pen contained f i v e mature UBC-W females and two mature males (one UBC-A and one UBC-W) ( F i g . 4). FIGURE A: Floor pen as used i n Experiment [17] The s i d e s of the pens were covered with b l a c k p l a s t i c sheets to provide v i s u a l i s o l a t i o n between pens. The f l o o r pens were used f o r three r e p l i c a t i o n s of these four treatments: 1) UBC-W male sham-operated; UBC-A male cauterized, 2) UBC-W male cauterized; UBC-A male sham-operated, 3) both males cauterized, and 4) both males sham-operated. During the experiment, eggs were c o l l e c t e d from the experimental pens, i d e n t i f i e d , and a r t i f i c i a l l y i n c u b a t e d . Phenotypes of progeny were r e c o r d e d and t h e p e r c e n t progeny s i r e d by foam-producing (sham-operated) and foamless ( c a u t e r i z e d ) males was d e t e r m i n e d . F e r t i l i t y , h a t c h a b i l i t y , and % embryonic death were calculated for each female. B e h a v i o u r a l o b s e r v a t i o n s were c a r r i e d o ut on two of the r e p l i c a t e s to monitor any changes i n mating performance due to the o p e r a t i o n s and to d e t e c t the occu r r a n c e of any mate preference by females. Eighteen 20-minute o b s e r v a t i o n s were done on each of the e i g h t pens — s i x d u r i n g each of the three time periods spanning the l i g h t portion of the day (morning: 0700-1100 hrs; afternoon: 1100-1700 hrs; evening: 1700-2100 h r s ) . A l l observations were made by the same observer s i t t i n g on a st o o l on a raised platform next to the pens. A c l i p b o a r d with data s h e e t s , pen and a watch were used for recording observations. Only reproductive behaviours and aggressive behaviours i n v o l v i n g at l e a s t one male were recorded. The observer sat qu i e t l y for 5 minutes before b e g i n n i n g to r e c o r d o b s e r v a t i o n s . The UBC-W females i n each pen were i n d i v i d u a l l y i d e n t i f i a b l e from s p e c i f i c [18] plumage p a t t e r n s on t h e i r heads and backs; UBC-W males were marked with a spot of paint on t h e i r backs for quick i d e n t i f i c a t i o n . The experiment lasted eight weeks. In order to minimize bias due to any p a r t i c u l a r male, two sets of males were used and r o t a t e d every two weeks so that the f i r s t set of males was used on weeks 1,2,5, and 6, while the second set was used on weeks 3,4,7, and 8. W i t h i n each set, the males were never placed with the same group of females during r o t a t i o n , but each female treatment group always r e c e i v e d males with the same o p e r a t i o n s as b e f o r e . Eggs were c o l l e c t e d and hatched for another two weeks a f t e r the removal of males at the end of the e i g h t h week. E i g h t f e m a l e s needed r e p l a c i n g d u r i n g the 10 weeks of egg c o l l e c t i o n — three due to death and a treatment group of f i v e due to e x c e s s i v e aggression" towards males. Eight males also needed replacing during the experiment due to excessive aggression from the females. C) DATA ANALYSES Data analyzed included developmental condition of eggs c o l l e c t e d from the experimental pens a f t e r 18 days of i n c u b a t i o n ( i n f e r t i l e , embryonic death, f e r t i l e , hatch), phenotypes of progeny (wild-type or white f e a t h e r s ) , and c o p u l a t o r y behaviour ("mount" i n d i c a t i n g a copulation attempt and "t a i l - b e n d " i n d i c a t i n g a completed copulation). Analysis of variance (ANOVA) was applied for the t h r e e f e r t i l i t y and [19] h a t c h a b i l i t y t r a i t s . Arcsine transformation (Steel and T o r r i e , 1980, p. 236) was a p p l i e d t o a l l p e r c e n t a g e s b e f o r e a n a l y s e s . The s t a t i s t i c a l model used was: Yijk = H + Ri + T j + E i j k ' and i=l,2,3; j=l,2,3,4; k=l,2 5; where Y ^ = o n e G f t h e dependent variables (% f e r t i l i t y , % hatch, or % embryonic d e a t h ) . Y i j k i s t * i e condition of the incubated eggs of the kth female i n the j t h treatment of the i 1 - * 1 r e p l i c a t i o n , u = the t h e o r e t i c a l p o p u l a t i o n mean, = e f f e c t of the i t h r e p l i c a t i o n , T.- = e f f e c t of the j t h treatment, and E±jk = r a n ^ o ' n e r r o r . Percent white progeny phenotyped (number of white i d e n t i f i e d / number of chicks phenotyped) and percent white progeny hatched (number of white hatched / number f e r t i l e eggs) were also analysed by ANOVA. Percentages were again transformed using the a r c s i n e t r a n s f o r m a t i o n . A s i m i l a r s t a t i s t i c a l model was used: Y i j k l = P + Ri + 0j + C k + OC-jk + E i j k l ' and i = l , 2 , 3 ; j = l , 2 ; k=l,2; 1=1,2,...,5; where Y ± j k x = one of the dependent v a r i a b l e s (% white c h i c k s phenotyped, % w h i t e c h i c k s h a t c h e d ) . ^ i j k l i s the percent white progeny produced by the 1^ female from the i t h r e p l i c a t i o n s i r e d by the UBC-W male with the j t n type of operation i n competition with the UBC-A male with the k t n type of operation, u = the t h e o r e t i c a l population mean, R^ = e f f e c t of the i t h r e p l i c a t i o n , 0j = e f f e c t of whether the UBC-W male was cauterized or sham-operated , C k = e f f e c t of whether the competing UBC-A male was c a u t e r i z e d or sham-operated, 0 C j k _ e f f e c t of the 2-way i n t e r a c t i o n involving the main e f f e c t s , and Ej.jkl = r a n ^ o m e r r o r . [20] A n a l y s e s were p e r f o r m e d on the males of both genotypes to determine f r e q u e n c i e s of attempted c o p u l a t i o n s ("mounting") and completed c o p u l a t i o n s ("tail-bending"). Because of the small number of copulations involved, a l l values were transformed using the square root t r a n s f o r m a t i o n ((Y + 0.5).5) ( S t e e l and T o r r i e , 1980, p. 234) before a n a l y s i s . The s t a t i s t i c a l model used was: Y i j k l m = P + R i + °j + C k + 0 C j k + E l i j k + T i + TOij + T C l k + TOCijk + E2ijklm' and i = l , 2 ; j = 1,2; k=l,2; 1=1,2,3; m=l,2,...,6; where Y i j k l m i s the number of copulations by the UBC-W male with the j t h type of operation (1 = cauterized; 2 = sham-operated) i n competition with the UBC-A male with the k t n type of operation (1 = sham-operated; 2 = c a u t e r i z e d ) d u r i n g the mth o b s e r v a t i o n o f the 1 t h time p e r i o d i n the i t h r e p l i c a t i o n , u = the t h e o r e t i c a l population mean, = e f f e c t of the i t n r e p l i c a t i o n , 0j = e f f e c t of whether the UBC-W male was cauterized or sham-operated, C k = e f f e c t of whether the competing UBC-A male was c a u t e r i z e d or sham-operated, 0 C j k = e f f e c t of the i n t e r a c t i o n of the main e f f e c t s , E l i j k = error term for t e s t i n g the main e f f e c t s , T i = e f f e c t of the 1th time p e r i o d , T O ^ and T C l k = e f f e c t s of the 2-way in t e r a c t i o n s i n v o l v i n g time, TOCijk = e f f e c t o f t n e 3-way i n t e r a c t i o n , and E2j_j k-^ m = error term for t e s t i n g the sub-plot e f f e c t s . The mean s e p a r a t i o n s between t r e a t m e n t s were t e s t e d u s i n g Duncan's New M u l t i p l e Range Test (Steel and T o r r i e , 1980, p.187) i n a l l of the above ANOVAs. [21] D) RESULTS 1. E f f e c t of q u a i l foam on f e r t i l i t y and h a t c h a b i l i t y under natural mating conditions: Male Japanese q u a i l that were producing foam f e r t i l i z e d more eggs than males t h a t were f o a m l e s s . F e m a l e s i n p e n s w i t h two foam-producing males had the highest f e r t i l i t y (97.5%) while females in pens with two foamless males had the lowest f e r t i l i t y (26.4%) (T a b l e 1). In between these extremes was the f e r t i l i t y of females i n pens with one foam-producing and one foamless male (75.6% and 73.2%, Table 1). There was no s i g n i f i c a n t d i f f e r e n c e i n h a t c h a b i l i t y of f e r t i l e eggs from pens with two foam-producing males (70.2%) as compared with h a t c h a b i l i t y from pens with one foam-producing and one foamless male (73.6% and 75.9%); h a t c h a b i l i t y of f e r t i l e eggs from pens with two foamless males, however, was s i g n i f i c a n t l y lower (53.4%, Table 1). 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 t h e t h r e e r e p l i c a t i o n s i n these f e r t i l i t y and h a t c h a b i l i t y data. 2. E f f e c t of q u a i l foam on sperm competition: In competition for f e r t i l i z i n g the females' eggs, UBC-W and UBC-A males were about equally as e f f e c t i v e (51.5% and 48.5%, r e s p e c t i v e l y ; Table 1) when both males were foam-producing. In s i t u a t i o n s where one of the males was foam-producing and the other male was foamless, the [22] foam-producing male (whether UBC-W or UBC-A) had a d i s t i n c t advantage and s i r e d almost a l l of the progeny produced by the females (98.7% for UBC-W males and 99.4% for UBC-A males, Table 1). On the other hand, when two foamless males were competing the UBC-A males had an apparent advantage and s i r e d 74.5% of the o f f s p r i n g while UBC-W males s i r e d only 25.5% of the of f s p r i n g (X 2 = 25.77, df = 1, p < 0.001). Another i n t e r e s t i n g o b s e r v a t i o n i s shown i n Table 2. In pens where both males were foam-producing, a l l of the females had progeny from both males. In pens where a foam-producing male was competing with a foamless male, females either produced progeny s i r e d by both males or produced progeny s i r e d e x c l u s i v e l y by the foam-producing male. However, i n pens where both males were foamless, some females produced progeny s i r e d e x c l u s i v e l y by UBC-W males, some produced progeny s i r e d e x c l u s i v e l y by UBC-A males, and o t h e r s produced both types of progeny. 3. E f f e c t of c o n d i t i o n of the foam gland on the male's mating behaviour: There was no s i g n i f i c a n t difference i n the frequency of attempted or completed copulations between foam-producing and foamless UBC-W males (Table 3) (However, see s i g n i f i c a n t 3-way i n t e r a c t i o n described on page 25). There was a l s o no s i g n i f i c a n t d i f f e r e n c e i n t h e s e f r e q u e n c i e s whether the UBC-W male (foam-producing or foamless) was competing with a foam-producing or foamless male, i . e . the c o n d i t i o n TABLE 1: F e r t i l i t y , h a t c h a b i 1 i t y , and phenotypic r a t i o of progeny i n the four treatments (summed over three r e p l i c a t i o n s ) . ^ CONDITION CONDITION NUMBER NUMBER OF % WHITE*** % WHITE5** OF UBC-W OF UBC-A OF PERCENT** PERCENT3* CHICKS PROGENY CHICKS TREATMENT MALE MALE 2 EGGS SET FERTILITY HATCHABILITY TYPED TYPED HATCHED 1 Foam Foamless 663 75.6^ 73.6 b 418 98.7 d 70.7 d 2 Foamless Foam 660 73.2 b 75.9 b 424 0.6 a 0.4 a 3 Foamless Foamless 696 26.4a 53.4 a 109 25.5 b 9.5 b 4 Foam Foam 666 97.5 C 70.2 b 540 51.5 C 32.3 C 1 Comparisons only w i t h i n a column by Duncan's M u l t i p l e Range Test; means followed by d i f f e r e n t l e t t e r 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 . 2 UBC-A males were used as markers f o r proportion of progeny s i r e d by UBC-W males. 3 % h a t c h a b i l i t y of f e r t i l e eggs. 4 (number of white o f f s p r i n g typed / number of o f f s p r i n g typed) x 100 ro 5 (number of white c h i c k s hatched / t o t a l number of f e r t i l e eggs) x 100 w * p<0.01 p<0.005 [24] TABLE 2: Phenotypes of progeny from females i n the f o u r treatment groups*. Female UBC-W: Foam UBC-W: Foamless UBC-W-.Foamless UBC-W: Foam Rep. ID UBC-A : F o a m l e s s UBC-A:Foam UBC-A:Foamless UBC-A:Foam 1 both both UBC-W both 2 both both both both 3 UBC-W UBC-A UBC-A both 4 UBC-W both both both 5 both UBC-A UBC-W both 6 both UBC-A both both 7 UBC-W UBC-A UBC-A both 8 UBC-W both both both 9 both both UBC-A both 10 UBC-W UBC-A both both 11 UBC-W UBC-A both both 12 UBC-W UBC-A both both 13 UBC-W UBC-A both both 14 UBC-W UBC-A UBC-A both 15 both UBC-A UBC-A both * Each treatment had 15 females (5 females / pen, with 3 r e p l i c a t i o n s ) . [25] of the competing male's foam gland had no s i g n i f i c a n t e f f e c t on the UBC-W males' mating frequencies (Table 3). Furthermore, 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 the f r e q u e n c i e s of both attempted and completed copulations between the three time periods i n a day f o r the UBC-W males ( T a b l e 3). None of the 2-way i n t e r a c t i o n s involving the main e f f e c t s were s i g n i f i c a n t . There was, however, a s i g n i f i c a n t 3-way i n t e r a c t i o n i n v o l v i n g the main e f f e c t s (condition of the UBC-W male's foam'gland, condition of the competing male's foam gland, and time of day) i n f r e q u e n c y of attempted c o p u l a t i o n s (Table 4 ) . Although the separation was not c l e a r cut by Duncan's New M u l t i p l e Range T e s t , t h e t r e n d was t h a t foamless UBC-W males attempted copulation more often when i n competition with foam-producing males than when competing with foamless males (0.286 and 0.092 re s p e c t i v e l y , Table 3). The s i g n i f i c a n t 3-way i n t e r a c t i o n may have masked t h i s e f f e c t . Again, there were no s i g n i f i c a n t r e p l i c a t i o n e f f e c t s involved. Since i n a l l the treatment pens a UBC-W male was competing with a UBC-A male f o r t h e f i v e females, the mating f r e q u e n c i e s of the competing males were not independent and could not be compared i n the same a n a l y s i s . The frequencies of attempted and completed copulations by UBC-A males were t h e r e f o r e examined i n separat e a n a l y s e s . The f r e q u e n c i e s of both attempted and completed c o p u l a t i o n s d i f f e r e d s i g n i f i c a n t l y between foam-producing and foamless UBC-A males (Table 5 ) . F o a m l e s s UBC-A males attempted and completed s i g n i f i c a n t l y (p<0.01 and p<0.05, resp e c t i v e l y ) more copulations than foam-producing UBC-A males. Neither the condition of the competing male nor the time of day had a s i g n i f i c a n t e f f e c t on the mating frequencies (Table 5 ) . [26] TABLE 3: Mean number of mating attempts (MA) and completed copulations (CC) by UBC-W males per observation period (20 minutes)!. TREATMENT MA se CC se Condition of UBC-W males: FOAM .184 + .027 .148 ± - 0 2 5 FOAMLESS .188 + .029 .118 + .026 Condition of competing UBC-A males: FOAM .286 + .032 .212 .+ .030 FOAMLESS .092 + .021 .059 + .018 Time of Day: 0700-1100 h .231 + .036 .124 + .030 1100-1700 h .158 + .032 .124 + .030 1700-2100 h .171 + .035 .153 + .034 1 None of the treatment e f f e c t s was s i g n i f i c a n t l y d i f f e r e n t . [27] TABLE 4: S i g n i f i c a n t 3-way i n t e r a c t i o n between foam gland operation, operation of competing male, and time of day; mean number of mating attempts (MA) by UBC-W males per observation!. TIME TREATMENT 1 TREATMENT 2 TREATMENT 3 TREATMENT 4 OF UBC-W:Foam UBC-W:Foamless UBC-W:Foamless UBC-W:Foam DAY UBC-A:Foamless UBC-A:Foam UBC-A:Foamless UBC-A:Foam se se se se 0700-1100 .200 +.068ab .407 +.092a .129 +.058ab .200 +.068ab 1100-1700 .063 +.043ab .327 +.090ab .000 b .224 +.074ab 1700-2100 .177 +.08iab .327 +.090ab .000 b .200 +.068ab Means followed by d i f f e r e n t l e t t e r superscripts are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) by Duncan's Mul t i p l e Range Test. [28] TABLE 5: Mean number of mating attempts (MA) and completed copulations (CC) by UBC-A males per observation period (20 minutes). TREATMENTS MA se CC se Condition of UBC-A males: Foam .294 + .034* .157 +_ .027' Foamless .540 + .051 .365 .036 Condition of competing UBC-W males: Foam .384 .043 .265 + .034 Foamless .442 .044 .249 + .031 Time of Day: 0700-1100 h .413 + .056 .232 + .041 1100-1700'h .423 + .054 .330 +_ .043 1700-2100 h .403 + .050 .212 + .030 * p<0.05 [29] On the other hand, there was a s i g n i f i c a n t 2-way i n t e r a c t i o n involving the condition of the male and time of day i n the frequency of attempted copulations (Table 6 )—UBC-A foamless males attempted s i g n i f i c a n t l y more copulations than the UBC-A foam-producing males only during the 11:00-17:00 hrs time period of each day (0.678 by foamless males and 0.200 by foam-producing males). Mating i s usually infrequent during t h i s part of the day i n Japanese q u a i l (Ottinger, e_t a l . , 1982). There were no s i g n i f i c a n t r e p l i c a t i o n e f f e c t s . Although there was no d i r e c t comparison, UBC-A males attempted and completed copulation twice as often as UBC-W males (Table 7). While there was no difference i n the mating frequencies between foam-producing and foamless UBC-W males (Table 3), foamless UBC-A males mated s i g n i f i c a n t l y more often than foam-producing UBC-A males (Table 5) . This may be the reason why i n pens where two foamless males were competing, the UBC-A males were able to s i r e more progeny than the foamless UBC-W males (Table 1). A. Differences between genotypes i n homosexual copulations: During the observation periods, homosexual copulations between the experimental males were recorded. UBC-W males mounted UBC-A males 33 times r e s u l t i n g i n nine apparently completed copulations. UBC-A males, however, were observed mounting UBC-W males only once during the experiment. Foamless UBC-W males attempted homosexual copulation 21 times while foam-producing UBC-W males attempted only 12 times. [30] TABLE 6: S i g n i f i c a n t time of day x condition of foam gland i n t e r a c t i o n ; mean number of mating attempts (MA) by UBC-A males per observation!. TIME OF DAY FOAM FOAMLESS se se 0700-1100 h .379 + .059ab .448 + ,098ab 1100-1700 h .200 + .047b .678 + .093a 1700-2100 h .310 + .068ab .502 + .073ab 1 Means followed by d i f f e r e n t l e t t e r superscripts are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) by Duncan's Mul t i p l e Range Test. [31] TABLE 7: A comparison of the t o t a l number of mating attempts (MA) and completed copulations (CC) by UBC-W and UBC-A males. MALES MA CC UBC-W 35 26 UBC-A 79 48 [32] EXPERIMENT 2 Results from Experiment 1 showed that, i n n a t u r a l matings, foam from the c l o a c a l foam glands aids the male i n f e r t i l i z i n g more eggs when i n competition with another male. However, i t was not conclusive as to: 1) how the foam aids the male i n f e r t i l i z a t i o n , or 2) whether the foam can act as a block to sperm from subsequent matings. Experiments 2 and 3 address these two questions. In t h i s experiment, the second question was examined by attempting to determine the order of transfer of foam and semen to the female during an e j a c u l a t i o n . I f foam was transferred following insemination or mixed with the semen, then I expect that foam would act as a blocking agent to hinder sperm t r a v e l from any subsequent inseminations. On the other hand, i f foam t r a n s f e r was found to preceed semen t r a n s f e r , then the sperm block hypothesis would have to be reconsidered. A) MATERIALS AND METHODS P a i r c o p u l a t i o n s were staged and f l u i d and foam samples were scraped from t h r e e d i f f e r e n t c l o a c a l r e g i o n s of t h e f e m a l e s [33] immediately post-copula and examined microscopically. These three regions were: 1) just inside the l i p of the cloaca, 2) foam expelled from the female's cloaca with l i g h t squeezing, and 3) scrape of the everted oviduct opening. The slides were examined under 400x magnification to determine where the greatest concentration of sperm occurred. Mature UBC-A wild-type Japanese quail were used and copulations were staged after oviposition in the la t e afternoon so there would be no hard-shelled egg in the uterus to hinder everting the females' cloacae. A male was placed i n the female's cage and observed until copulation with cloacal contact had occurred. If no copulation occurred within 5 minutes (Schein e_t a l . , 1972's "rule of thumb" for whether a male w i l l perform sexually), a different male was used with the same female. After copulation, the female was removed and held ventral-side up with the head downwards in the experimenter's l e f t hand. Without squeezing, the cloaca was s l i g h t l y opened and a sample taken with a small v i n y l spatula (a straightened paper c l i p with electrical tape trimmed to a bulb shape at one end) and transferred directly to pre-labelled Slide 1. After squeezing out some foam, a second sample was taken with a new spatula and transferred to Slide 2. After f u l l y everting the female, the t h i r d sample (obtained with a t h i r d spatula) was taken from the everted oviduct opening and placed on Slide 3. Slides were examined microscopically and scored for the presence of foam and relative density of sperm. A score of 1 was given for the [34] presence of sperm, a 2 was given f o r higher sperm c o n c e n t r a t i o n s compared to a 1, and a 3 was given f o r the presence of the h i g h c o n c e n t r a t i o n sperm mass. Absence of sperm was given the score 0. Only the presence or absence of foam was recorded. B) RESULTS As r e f l e c t e d i n the f l u i d and foam samples from the c l o a c a l opening and the p a r t i a l l y squeezed c l o a c a , sperm were always found mixed with foam; sperm were a l s o found i n the oviduct, but with no foam preceding them (Table 8 ) . Thus, sperm are a p p a r e n t l y always mixed with foam at i n s e m i n a t i o n , and enter the oviduct without foam occurring between sperm and the infundibulum ( s i t e of f e r t i l i z a t i o n ; Olsen and Neher, 1948; Burke et a l . , 1969). Sperm from subsequent matings may have the p r e c e e d i n g male's foam t o t r a v e r s e b e f o r e entering the oviduct. In c h i c k e n s (Lake, 1966), turkeys (Hale and Schein, 1962), and most waterfowl (Guaryahu et a l . , 1984; Van Tienhoven, 1983), the semen i s d e p o s i t e d d i r e c t l y i n t o the female's o v i d u c t d u r i n g n a t u r a l matings. However, during t h i s experiment, I found t h a t the foam / semen mass of the male Japanese q u a i l i s deposited into the coprodeum of the female rather than her oviduct as had been expected ( F i g 5 ) . The coprodeum i s the most f r o n t a l of three c l o a c a l sections ( with the urodeum and proctodeum, r e s p e c t i v e l y , more caudal) and s t o r e s f e c a l [35] TABLE 8: The presence of sperm and foam i n d i f f e r e n t parts of the female's cloaca immediately a f t e r copulation. SAMPLE CLOACAL OPENING PARTIALLY SQUEEZED OVIDUCT OPENINC NUMBER Sperm Foam Sperm Foam Sperm Foam 1 1 1 1 1 1 0 2 1 1 2 1 1 0 3 1 1 3 1 1 0 4 1 1 3 1 1 0 5 1 1 2 1 1 0 6 1 1 - 0 0 7 2 1 1 1 1 0 8 - 1 1 1 0 9 0 1 0 1 0 0 10 1 1 1 1 0 = absence of sperm / foam 1 = presence of sperm / foam 2 = higher sperm concentrations compared to 1 3 = presence of high concentration sperm mass - = no observation [36] FIGURE 5: Foam being expelled from the female's coprodeum. [37] FIGURE 6: Cloaca of a female chicken, opened along the dorsal midline and l a i d f l a t . P proctodeum U urodeum C coprodeum R rectum RO right oviduct LO le f t oviduct UR ureter Figure taken from Komarek, 1971. [38] m a t e r i a l from the rectum f o r defecation (King, 1981) (see F i g 6 for the c l o a c a l anatomy of a female c h i c k e n ) . Chicken and q u a i l are r e l a t e d s p e c i e s w i t h i n Family P h a s i a n i d a e (Welty, 1979) and have a s i m i l a r c l o a c a l anatomy. The implications of t h i s discovery for sperm competition w i l l be discussed. EXPERIMENT 3 R e s u l t s from Experiment 1 i n d i c a t e d t h a t i n a c o m p e t i t i v e s i t u a t i o n , foam-producing males f e r t i l i z e d more eggs than foamless males. T h i s p r o v i d e s e v i d e n c e t h a t h i s foam a i d s t h e male i n f e r t i l i z a t i o n i n a c o m p e t i t i v e s i t u a t i o n . However, I o b t a i n e d no information on how f e r t i l i t y was enhanced or i f foam may hinder sperm from subsequent matings i n traversing the oviduct. In Experiment 2, I found that: i ) foam and sperm are mixed at insemination and sperm can enter the oviduct ahead of the foam, and i i ) foam and sperm are deposited i n t o the coprodeum (rather than the o v i d u c t i t s e l f ) and sperm and foam from s u b s e q u e n t matings are deposited posterior to the f i r s t male's foam. These r e s u l t s i n d i c a t e that the foam i s placed i n the female's c l o a c a [39] where i t could play a r o l e i n sperm competition. Experiment 3 was designed to study the a b i l i t y of foam to block sperm p e n e t r a t i o n i n v i t r o and a t the same time examine the p o s s i b i l i t y that foam aids f e r t i l i z a t i o n by enhancing or p r o l o n g i n g sperm m o t i l i t y . Because semen from domestic chickens can be obtained e a s i l y and i n large quantity, t h i s in v i t r o study was f i r s t conducted us i n g c h i c k e n sperm (Experiment 3A) and then l a t e r repeated using q u a i l sperm (Experiment 3B). EXPERIMENT 3A A) MATERIALS AND METHODS The technique used to measure sperm penetration through foam was based on a method described by Birrenkott et a l . (1977) for evaluating the q u a l i t y of a v i a n semen. G l a s s c a p i l l a r y tubes measuring 75 mm long and 1.2 mm inner diameter were marked with i n d e l i b l e i n k every 5 mm along t h e i r length. Foam from mature male quail was c o l l e c t e d by a gentle squeeze of the c l o a c a l protuberence immediately before the experiment i n 2 ml p l a s t i c auto-analyzer cups. Foam a c c i d e n t a l l y mixed with feces was not used. E i g h t marked c a p i l l a r y tubes were f i l l e d [40] with foam up to the 10 mm mark by suctioning and then further f i l l e d with thin albumin (at room temperature, 210-22° C) from chicken eggs u n t i l only 5 mm was l e f t empty at the end of the c a p i l l a r y tube. The foam was c l o s e s t to the open end ( F i g 7). The o p p o s i t e end was then s e a l e d with a s i l i c o n gel sealer. For the c o n t r o l , eight more marked c a p i l l a r y tubes were f i l l e d with only t h i n albumin (again leaving 5 mm empty a t one end and s e a l i n g the o p p o s i t e end). Each prepared c a p i l l a r y tube was embedded v e r t i c a l l y with the open end uppermost i n one of two styrofoam h o l d e r s ( s t y r o f o a m b l o c k s , each with h o l e s punched for eight c a p i l l a r y tubes) so that each holder contained four e x p e r i m e n t a l - c o n t r o l p a i r s of c a p i l l a r y tubes. Semen was c o l l e c t e d from a rooster by the massage method (Burrows and Quinn, 1935,1937; Lake, 1957) and t r a n s f e r r e d to a 1 cc t u b e r c u l i n s y r i n g e . Equal volumes (about 5 j j l ) of the semen were g e n t l y d e p o s i t e d w i t h a 23-guage needle into each of the 16 c a p i l l a r y tubes, making sure that the semen did not pass the 5 mm mark at the tubes' open ends and t h a t no a i r bubbles o c c u r r e d between the semen and the contents of the tube. The order of semen deposition was randomized between holders and between p a i r s . Each c a p i l l a r y tube was s e a l e d with s i l i c o n g e l immediately a f t e r semen deposition. When a l l the tubes were s e a l e d , one of the styrofoam holders was placed i n an incubating oven at 41° C (the bird's body temperature) and the time was r e c o r d e d . The other styrofoam h o l d e r was l e f t at room temperature (2lo_22oC) and was l o o s e l y covered with a paper towel to reduce the amount of l i g h t reaching the incubating sperm. A f t e r 15 m i n u t e s of i n c u b a t i o n , e i g h t c a p i l l a r y tubes were FIGURE 7: Diagram of a prepared c a p i l l a r y tube. -T r- -i 1 i 1 1 1 r-foam v thin albumin semen eal seal [42] examined i n sequence' by each of two observers under microscopes at 400x magnification for about 2 minutes. The order of examination was randomized both by p a i r s and by h o l d e r s (temperature) with the two observers examining c a p i l l a r y tubes i n an o p p o s i t e o r d e r , i . e . i f Observer A s t a r t e d with an " e x p e r i m e n t a l " c a p i l l a r y tube at "room temperature", then Observer B started with a " c o n t r o l " c a p i l l a r y tube at "body temperature"... Each c a p i l l a r y tube was replaced into i t s o r i g i n a l p o s i t i o n a f t e r examination. The c a p i l l a r y tubes were again examined 30 minutes a f t e r the f i r s t examination. For each examination, both c o n c e n t r a t i o n and m o t i l i t y of sperm w i t h i n each 5 mm s e c t i o n of the c a p i l l a r y tubes were examined. The s c a l e used f o r s c o r i n g the sperm c o n c e n t r a t i o n per s e c t i o n o f c a p i l l a r y tube was: A: sperm present en masse, B: more than 10 sperm present, but not en masse, C: between 5 and 10 sperm present, and D: fewer than 5 sperm present. The scoring scale for m o t i l i t y was m o d i f i e d from the 5 - c l a s s s c a l e used by Wilcox (1959): A: active movement of a l l sperm, B: moderate movement of most sperm, C: sluggish movement of sperm at best, and D: no movement of sperm. Semen from f i v e r o o s t e r s was used; each rooster was sampled twice. A l l semen samples from one rooster constituted a r e p l i c a t i o n . [A3] B) ANALYSES The maximum distance t r a v e l l e d by sperm i n the c a p i l l a r y tube was determined by the 5 mm section furthest away from the s t a r t i n g p o i n t that scored a "C" or above for concentration. This dependent variable was analyzed by ANOVA with repeated measures. The s t a t i s t i c a l model used was: Y i j k l m n = P + R i + C j + T k + °1 +CTj k + CO^ + T 0 k l + CTO j k l + E l i j k l + S m + S C m j + S T m k + S O m i + S C T m j k + S C O m j i +  S T O m k l + S C T O m j k l + E 2 i j k l m n , and i=l,2,3,4,5; j=l,2; k=l,2; 1=1,2; m=l,2; n=l,2,3,4; where Y-jj k;[ mn i s the maximum d i s t a n c e t r a v e l l e d ( i n mm) by the sperm from the i t h male i n the tube with the j t h contents (l=foam; 2=no foam) incubated at the k t h temperature examined by the 1th observer during the mzn time of o b s e r v a t i o n from t h e n t h sample, p. = t h e t h e o r e t i c a l p o p u l a t i o n mean, R ± = e f f e c t of the i t h r e p l i c a t i o n , Cj = e f f e c t of whether there was foam in.the tube, T k = e f f e c t of whether i n c u b a t i o n was at room or body temperature, 0-^  = e f f e c t of examination by the 1 t h observer, S m = e f f e c t of time of examination, C T j k , COj^, T0 k]^, SC mj> [44] ^ m k » a n d SOra]_ = e f f e c t s of the 2-way i n t e r a c t i o n s , CTOj^i, S C T m j k , S C O m j i , and STO mkl = e f f e c t s of the 3-way i n t e r a c t i o n s , S C T O m j k l = e f f e c t of the 4-way i n t e r a c t i o n , E 1 i j k l = error term f o r tes t i n g the e f f e c t s of the main plo t , and E2ijklmn = t h e s u b - P l o t error term. The mean s e p a r a t i o n s between treatments were tested using Duncan's New Mul t i p l e Range Test. Sperm m o t i l i t y was analyzed by the Kruskal-Wallis k-Sample Signed Rank Test ( S t e e l and T o r r i e , 1980, p. 544). The average m o t i l i t y s c o r e f o r each c a p i l l a r y tube was c a l c u l a t e d by, f i r s t summing the scores for each section and then d i v i d i n g by the number of s e c t i o n s s c o r e d . These averages were then ranked and the Kruskal-Wallis test was applied. P a i r w i s e comparisons of sperm m o t i l i t y i n c a p i l l a r y tubes were made between each of these treatments: 1) tubes with foam at the 15 minute examination, 2) tubes with foam at the 45 minute examination, 3) tubes without foam at the 15 minute examination, and 4) tubes without foam at the 45 minute examination. [45] C) RESULTS 1. E f f e c t of foam on sperm penetration. The presence of q u a i l foam s i g n i f i c a n t l y hindered the t r a v e l of c h i c k e n sperm through c a p i l l a r y tubes (Table 9, p<0.005). Sperm t r a v e l l i n g through c a p i l l a r y tubes without foam averaged 20.0 mm per o b s e r v a t i o n a v e r a g e d a c r o s s temperature, time of o b s e r v a t i o n , o b s e r v e r , and r e p l i c a t i o n , w h i l e sperm w i t h foam t o p e n e t r a t e t r a v e l l e d an average of only 11.6 mm per observation. 2. E f f e c t of incubation temperature on sperm penetration. Sperm were a b l e t o t r a v e l f u r t h e r when incubated at body temperature (41°C) than when inc u b a t e d at a room te m p e r a t u r e of 210-220C (Table 9, p<0.005). At room temperature, sperm t r a v e l l e d an average of 14.4 mm per observation averaged across tube contents, time of o b s e r v a t i o n , observer, and r e p l i c a t i o n , but t r a v e l l e d 17.2 mm per observation at body temperature. 3. E f f e c t of time of examination on sperm penetration. As expected, sperm had pe n e t r a t e d s i g n i f i c a n t l y f u r t h e r when examined at the second observation (45 minutes) than when examined at the f i r s t observation (15 minutes) of the c a p i l l a r y tubes (Table 9, [46] p<0.005). After 15 minutes of incubation, sperm had averaged 12.4 mm of t r a v e l while a f t e r 45 minutes of incubation, sperm had penetrated an average of 19.3 mm through the tube. 4. E f f e c t of observers on measurements of sperm penetration. Sperm was r e c o r d e d as t r a v e l l i n g f u r t h e r through tubes by Observer B than by Observer A ( T a b l e 9, p<0.05); however, the d i f f e r e n c e was c o n s i s t e n t — b o t h o b s e r v e r s recorded f u r t h e r sperm penetration through tubes without foam, at body temperature, and at the second examination. Observer A recorded an average penetration of 15.0 mm per observation averaged across a l l f a c t o r s while Observer B recorded an average penetration of 16.7 mm per observation. 5. E f f e c t of r e p l i c a t i o n on sperm penetration. A r e p l i c a t i o n was represented by semen c o l l e c t e d from one of the f i v e r o o s t e r s . There was a s i g n i f i c a n t r e p l i c a t i o n e f f e c t ; the average penetration of sperm for each r e p l i c a t i o n varied from a low of 13.2 mm to a high of 18.6 mm (Table 9, p<0.005). These r e p l i c a t i o n differences r e f l e c t i n d i v i d u a l v a r i a t i o n i n sperm m o t i l i t y between the d i f f e r e n t males. [47] TABLE 9: Mean distance (mm) t r a v e l l e d by rooster sperm through c a p i l l a r y tubes per examination. TREATMENT DISTANCE TRAVELLED E f f e c t of contents of tubes5'" se Foam 11.66 + .05 Foamless 20.05 + .06 E f f e c t of incubation temperature**: 21°-22° C (Room) 14.45 + .06 41° (Body) 17.25 + .07 E f f e c t of observer*: A 14.98 + .06 B 16.72 + .07 E f f e c t of time of examination**: 15 minutes 12.39 + .04 45 minutes 19.31 + .07 E f f e c t of replication''"'': 1 18.55 + .09 2 15.70 + .10 3 17.11 + ..10 4 14.65 + .10 5 13.24 + .08 p<0.05 ** p<0.005 [48] 6. E f f e c t s of the s i g n i f i c a n t i n t e r a c t i o n s involving the main e f f e c t s on sperm penetration. Of the 2-way i n t e r a c t i o n s examined, only those inv o l v i n g time were s i g n i f i c a n t . The Time of O b s e r v a t i o n x C o n t e n t s o f Tube i n t e r a c t i o n shows that not only had the sperm i n tubes without foam already penetrated s i g n i f i c a n t l y further by 15 minutes than sperm i n tubes with foam, but also the sperm's increase of penetration between 15 and 45 minutes was s i g n i f i c a n t l y greater i n the tubes without foam (Table 10, p<0.005). I t was a l s o observed t h a t by 15 minutes the sperm i n tubes without foam had a l r e a d y t r a v e l l e d s i g n i f i c a n t l y further than sperm i n tubes with foam would t r a v e l by 45 minutes. The Time x Temperature i n t e r a c t i o n was a l s o s i g n i f i c a n t (Table 11, p<0.005). Although sperm penetrated further at body temperature than at room temperature, the differe n c e between the two temperatures i n distance penetrated was larger at 45 minutes than at 15 minutes. The s i g n i f i c a n c e i n the Time x Observer i n t e r a c t i o n was due to the f a c t t h a t although t h e r e was no s i g n i f i c a n t d i f f e r e n c e i n the distance of penetration recorded by the two observers at the 15-minute e x a m i n a t i o n , O b s e r v e r B r e c o r d e d a g r e a t e r i n c r e a s e i n sperm penetration than Observer A at the 45 minute examination (Table 12, p<0.005). None of the 3-way i n t e r a c t i o n s examined were s i g n i f i c a n t . The 4-way i n t e r a c t i o n was highly s i g n i f i c a n t (p<0.005), but no clear trend could be interpreted. [49] TABLE 10: S i g n i f i c a n t 2-way i n t e r a c t i o n between time and contents of tube; mean distance (mm) t r a v e l l e d by rooster sperm through c a p i l l a r y tubes per examination!. CONTENTS TIME OF EXAMINATION OF TUBE 15 MIN 45 MIN se se Foam 9.12 + .04a 14.19 + .08" Foamless 15.66 + .05c 24.44 + .07d A l l means are s i g n i f i c a n t l y d i f f e r e n t (p<0.01) by Duncan's Multi p l e Range Test. [50] TABLE 11: S i g n i f i c a n t 2-way i n t e r a c t i o n between time and temperature; mean distance (mm) t r a v e l l e d by rooster sperm through c a p i l l a r y tubes per examination!. TEMPERATURE TIME OF EXAMINATION 15 MIN 45 MIN se se 21°-22° C (Room T.) 11.59 + .05 a 17.31 + ,08c 41° C (Body T.) 13.19 + .06 b 21.31 + .10 d A l l means are s i g n i f i c a n t l y d i f f e r e n t (p<0.01) by Duncan's Multi p l e Range Test. O [51] TABLE 12: S i g n i f i c a n t 2-way i n t e r a c t i o n between time and observer; mean distance (mm) t r a v e l l e d by rooster sperm through c a p i l l a r y tubes per examination^. OBSERVER TIME OF EXAMINATION 15 MIN 45 MIN se se A 11 .91 + .05 a 18.06 + .09 b B 12 .88 + .06 a 20.56 + .10 c Means followed by d i f f e r e n t l e t t e r superscripts are s i g n i f i c a n t l y d i f f e r e n t (p<0.01) by Duncan's Multiple Range Test. [52] TABLE 13: S i g n i f i c a n t 2-way i n t e r a c t i o n between time and contents of tube; mean m o t i l i t y ranks for rooster sperm i n c a p i l l a r y tubes!. CONTENT OF TUBES TIME OF EXAMINATION 15 MIN 45 MIN Foam 45 .72 b 24.00 a Foamless 51 .82° 40.45 b Means followed by d i f f e r e n t l e t t e r superscript are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) by Kruskal-Wallis k-Sample Signed Rank Test. [53] 7. E f f e c t of foam on chicken sperm m o t i l i t y . Japanese q u a i l foam caused a s i g n i f i c a n t decrease i n c h i c k e n sperm m o t i l i t y over time. There was no s i g n i f i c a n t difference i n m o t i l i t y of sperm i n tubes with or without foam a t 15 m i n u t e s . However, at 45 minutes, sperm m o t i l i t y i n c a p i l l a r y tubes with foam was s i g n i f i c a n t l y lower than the m o t i l i t y of sperm i n tubes without foam at the same time ( T a b l e 13). Moreover, i n tubes without foam, sperm m o t i l i t y at 45 minutes was not s i g n i f i c a n t l y d i f f e r e n t from m o t i l i t y i n tubes with foam at 15 minutes. EXPERIMENT 3B R e s u l t s from E x p e r i m e n t 3A showed t h a t foam from the male Japanese q u a i l hinders the t r a v e l of chicken sperm in. v i t r o . In order to determine i f the foam would have a s i m i l a r e f f e c t on Japanese q u a i l sperm, the experiment was repeated using q u a i l sperm and t h i n albumin from quail eggs. However, due to the inconsistency of obtaining semen from any given male q u a i l , a balanced experimental d e s i g n s i m i l a r to the one used for examining chicken sperm was not possible. [54] A) MATERIALS AND METHODS 1. Semen c o l l e c t i o n from male Japanese q u a i l . Semen was c o l l e c t e d using a modification of the method by Marks and L e p o r e (1965) a f t e r s u g g e s t i o n s by H. P. VanKrey ( p e r s o n n a l communication). The male, with f e a t h e r s near the c l o a c a l opening c l i p p e d , was held i n the l e f t hand with the bird's breast i n the palm and the c o l l e c t o r ' s thumb was placed just below the ventral l i p of the c l o a c a f o r support. The foam was f i r s t removed by two or three squeezes to the c l o a c a l protuberance with the thumb and f o r e f i n g e r of the r i g h t hand, and was discarded. Gentle but firm massage "strokes" were then applied by the thumb and for e f i n g e r of the r i g h t hand to the caudal area of the b i r d with p a r a l l e l movements di r e c t e d down the sides of the c l o a c a . A f t e r t h r e e to f i v e s t r o k e s , a f i n a l f i r m e r squeeze to the s i d e s of the cloaca n a t u r a l l y everted the phallus and caused an e j a c u l a t i o n . I f the creamy white semen d i d not appear i n the groove of the p h a l l u s , the process was repeated; i f no semen appeared a f t e r t h i s second attempt, the male was r e p l a c e d and a d i f f e r e n t male was obtained for semen c o l l e c t i o n . The semen obtained was removed from the male with a m i c r o p i p e t t e (Drummond D i g i t a l M i c r o d i s p e n s e r ) f o r immediate transfer i n t o c a p i l l a r y tubes. For a microscope s l i d e study of q u a i l sperm m o t i l i t y (see page 41), the semen was removed with a small v i n y l spatula. [55] 2. Experimental procedure. a) Sperm penetration and m o t i l i t y i n c a p i l l a r y tubes. The volume of semen per ejaculate i n q u a i l i s low compared with t h e volume of c h i c k e n semen (3.87-6.90 u l vs 0.41-2.25 m l , r e s p e c t i v e l y , Buxton and O r c u t t , 1975). An ejaculate from one male furnished enough semen for at most two e x p e r i m e n t a l - c o n t r o l p a i r s of c a p i l l a r y tubes. Because of the longer time needed to t r a i n q u a i l males to respond to semen c o l l e c t i o n , only f i v e males were f u l l y t r a i n e d and used. Not a l l males were used to the same extent because o f i n c o n s i s t e n c y i n s e m e n p r o d u c t i o n . A t o t a l o f 12 e x p e r i m e n t a l - c o n t r o l pairs of c a p i l l a r y tubes were examined between 1 January and 17 February, 1983. Because q u a i l sperm lose m o t i l i t y quickly in_ v i t r o (Ogasawara and Huang, 1963; Schein e_t_ £l_. , 1972), the i n c u b a t i o n t i m e f o r the c a p i l l a r y tubes a f t e r semen d e p o s i t i o n was reduced to f i v e minutes a f t e r which the maximum distance t r a v e l l e d by sperm was s c o r e d . The c a p i l l a r y tubes were incubated only at room temperature (21-22°C) to reduce v a r i a t i o n s i n temperature by s h o r t p e r i o d s i n an i n c u b a t i n g oven. M o t i l i t y of sperm was also scored at the 5 minute observation and t h e r e a f t e r checked p e r i o d i c a l l y to determine i n which t u b e s ( e x p e r i m e n t a l or c o n t r o l ) sperm m o t i l i t y lasted longest. The same c r i t e r i a as i n Experiment 3A were used for scoring sperm concentration and m o t i l i t y . [56] b) Sperm m o t i l i t y on microscope s l i d e s . In addition to examining penetration and m o t i l i t y of q u a i l sperm i n c a p i l l a r y tubes, I compared sperm m o t i l i t y with and without foam on microscope s l i d e s . Four comparisons were made. In two of these, semen from a male was mixed with i t s own foam and i n the remaining two comparisons, semen from a cauterized male was mixed with foam from a d i f f e r e n t male. I mixed semen with foam from the same and from d i f f e r e n t males to check i f the foam had an immune or suppressant response a g a i n s t sperm from other males. Thus, comparisons of sperm m o t i l i t y could be made both between foam vs no foam and between foam and semen from the same male _vs_ foam and semen from d i f f e r e n t males. Sl i d e s were prepared just p r i o r to semen c o l l e c t i o n : approximately 20-25 u l of t h i n albumin from a f r e s h q u a i l egg was placed on both s l i d e s and approximately 20-25 u l of f r e s h l y obtained foam was added to one s l i d e . A small quantity (around 5 Jul) of f r e s h l y obtained q u a i l semen was mixed with the c o n t e n t s on each s l i d e , a cover s l i p was a p p l i e d , and the s l i d e s were placed on separate microscopes at AOOx magnification. The s l i d e s were p e r i o d i c a l l y checked f o r up to 55 minutes a f t e r the addition of semen and the m o t i l i t y of the sperm was observed and scored. [57] B) ANALYSIS The same c r i t e r i a used to determine maximum distance t r a v e l l e d by chicken sperm i n Experiment 3A were applied to.determine q u a i l sperm penetration i n c a p i l l a r y tubes. However, due to the smaller number of samples observed, only a comparison of sperm t r a v e l i n tubes with or without foam was made u s i n g the Student's t - t e s t for paired samples ( S t e e l and T o r r i e , 1980, p. 102). M o t i l i t y d a t a from both the c a p i l l a r y tube and microscope s l i d e studies were summarized, but no analyses were performed. C) RESULTS 1. E f f e c t of foam on q u a i l sperm penetration. As with chicken sperm, q u a i l sperm were able to penetrate further through the c a p i l l a r y tubes when there was no foam to contend with. Without foam, sperm t r a v e l l e d an average of 7.90 mm while with foam, sperm only averaged 2.80 mm (t = 4.64, df = 11, p<0.01). 2. E f f e c t of foam on q u a i l sperm m o t i l i t y . The e f f e c t of foam on q u a i l sperm m o t i l i t y was observed both i n c a p i l l a r y tubes and on microscope s l i d e s . In the c a p i l l a r y tube e x p e r i m e n t , sperm m o t i l i t y was more vi g o r o u s i n tubes with foam [58] TABLE 14: Duration of q u a i l sperm m o t i l i t y on microscope slides-^. TREATMENT TIME OF FINAL EXAMINATION MOTILITY2 Foam and sperm from same male: Sample 1 Sample 2 No foam: Sample 1 Sample 2 Foam and sperm from d i f f e r e n t males: Sample 3 Sample 4 No foam: Sample 3 Sample 4 (minutes) 16 55 4 10 45 45 45 11 vigorous vigorous ceased poor vigorous good poor poor 1 2 Samples with the same number in d i c a t e sperm from a sing l e male. On s l i d e s without foam, sperm remained motile only when associated with a i r bubbles. [59] compared to sperm m o t i l i t y i n tubes without foam at the 5 minute observation. In c a p i l l a r y tubes without foam, sperm m o t i l i t y was poor or had ceased w i t h i n an average of 8.5 minutes of incubation whereas i n c a p i l l a r y tubes with foam, sperm m o t i l i t y was often s t i l l v i g o r o u s a f t e r 10.6 minutes. The a b i l i t y of foam to prolong sperm m o t i l i t y i n v i t r o became apparent when observed on the microscope s l i d e s .(Table 14). Sperm a s s o c i a t e d with foam remained motile for more than 55 minutes while sperm not associated with foam usually ceased m o t i l i t y within 4 to 11 minutes. Occasionally, where a i r bubbles were trapped under the cover s l i p s on microscope s l i d e s without foam, sperm a s s o c i a t e d with these a i r bubbles remained motile for much longer than sperm not associated with bubbles on the same s l i d e s . Whether or not foam was from the same male whose semen was used on the microscope s l i d e apparently did not d i f f e r e n t i a l l y a f f e c t the duration of sperm m o t i l i t y . DISCUSSION T h i s r e s e a r c h was conducted to s t u d y t h e f u n c t i o n of foam s e c r e t e d by the foam gland of the male Japanese q u a i l . The f i r s t question asked was, "Does foam play a r o l e i n a f f e c t i n g the male's [60] r e p r o d u c t i v e success during natural matings?" Results from Experiment 1 c l e a r l y i n d i c a t e that i t does. Sham-operated (foam-producing) males s i r e d s i g n i f i c a n t l y more progeny than c a u t e r i z e d (foamless) males ( T a b l e s 1 and 2 ) . B e h a v i o u r a l o b s e r v a t i o n s d u r i n g E x p e r i m e n t 1 indicated that cauterized males attempted and completed copulations at l e a s t as often as sham-operated males (Tables 4 and 6). The difference i n f e r t i l i t y , t h e r e f o r e , was not due to d i f f e r e n c e s i n mating frequencies between the foamless and foam-producing males. Ogawa e t a l . '(1974) n o t e d t h a t the volume of a r t i f i c i a l l y ejaculated semen from males whose foam glands (along with part of the c l o a c a ) were s u r g i c a l l y removed was only'about a t h i r d of t h a t obtained from normal males, although semen from the g l a n d l e s s males had a higher sperm concentration. Never-the-less, the t o t a l number of sperm per ejaculate was 40 % higher i n normal males. In the present e x p e r i m e n t s , t h e p r o c t o d e a l g l a n d of some males was rendered n o n - f u n c t i o n a 1 by c a u t e r y and was not s u r g i c a l l y r e m o v e d . M a c r o s c o p i c a l l y , no r e d u c t i o n i n semen volume per e j a c u l a t e was noticed i n these males compared to sham-operated or normal b i r d s . However, I d i d not measure semen volume from the males. A l s o , I n o t i c e d no gross d i f f e r e n c e s i n sperm m o t i l i t y when semen was c o l l e c t e d from c a u t e r i z e d m a l e s , mixed w i t h foam, examined m i c r o s c o p i c a l l y , and compared with semen of normal males. In a s e p a r a t e experiment (see Appendix, p. 80), examinations of c l o a c a l samples and expelled foam from females p o s t - c o p u l a r e v e a l e d t h a t 45 out of 99 (45.5%) samples c o n t a i n e d sperm a f t e r c o p u l a t i o n with cauterized males, while 16 out of 27 (61.5%) samples contained sperm [61] a f t e r c o p u l a t i o n with normal males. The d i f f e r e n c e i n f e r t i l i t y between males with and without foam i n Experiment 1, t h e r e f o r e , was not due to semen quality or quantity, although further experiments are needed to confirm t h i s observation. I then asked, "How did foam increase the reproductive success of males?" Two p o s s i b l e answers to t h i s q u e s t i o n a r e : 1) foam could i n c r e a s e a male's r e p r o d u c t i v e success by a i d i n g the f e r t i l i z i n g a b i l i t y of i t s own sperm. 2) foam c o u l d a l s o i n c r e a s e a male's r e p r o d u c t i v e success by h i n d e r i n g the f e r t i l i z i n g a b i l i t y of sperm from any other males copulating with the same female. In the I n t r o d u c t i o n , a h y p o t h e s i s on the function of foam was developed based on differences i n o v i p o s i t i o n time between the q u a i l and other domestic birds such as chickens and ducks (see page A). In t h i s hypothesis, foam i s suspected to be an agent which suspends sperm near the o v i d u c t opening f o r slow r e l e a s e of sperm into the vagina while blocking the t r a v e l of sperm from any subsequent c o p u l a t i o n s . Results from the present experiments not only provide support but also a refinement for t h i s hypothesis. Experiment 2 revealed that a f t e r a successful copulation, foam i s deposited into the coprodeum of the female. When examined immediately a f t e r a s u c c e s s f u l copulation, sperm was well mixed with foam. Sperm was also always found i n the oviduct; foam was found only once i n the oviduct, and then only as a few stray bubbles. Experiment 3 indicated that i n v i t r o , foam prolonged the m o t i l i t y of q u a i l sperm to a great e x t e n t ; sperm were e i t h e r a t t r a c t e d to or trapped i n the bubbles of the foam. The above o b s e r v a t i o n s are c o n s i s t e n t with the "sperm [62] block" hypothesis. The f a c t t h a t foam was found i n the coprodeum of the female and not i n the oviduct provides an excellent demonstration that sperm can be suspended i n a "pocket" out 'of the way of an egg as i t moves down the o v i d u c t . Foam may even b u f f e r sperm a g a i n s t t h e a d v e r s e e n v i r o n m e n t o f t h e c l o a c a as f e c a l m a t e r i a l i s harmful to the v i a b i l i t y of sperm (Boone and Hughes, 1970). Foam i s o c c a s i o n a l l y found on f e c e s e x c r e t e d by the female and on the s h e l l s of f r e s h l y l a i d eggs (personal observation), but never i n the amount o r i g i n a l l y d e p o s i t e d i n t o the female. T h e r e f o r e , even i f c o p u l a t i o n occurs before o v i p o s i t i o n , enough sperm (suspended i n foam) w i l l remain i n the female a f t e r the egg i s l a i d to f e r t i l i z e the next egg, or to be stored i n her u t e r o v a g i n a l sperm-host g l a n d s . Unless c o p u l a t i o n o c c u r s l a t e i n t h e evening ( a f t e r o v i p o s i t i o n ) , without such a mechanism the e j a c u l a t e of the male, being s m a l l i n q u a n t i t y and v i s c o u s i n c o n s t i t u t i o n , c o u l d e a s i l y be e l i m i n a t e d by the female along with the egg (Bobr e_t ad., 1964b) without l e a v i n g enough sperm to maintain good f e r t i l i t y . In n a t ure, t h i s hypothesized function of foam may not be c r u c i a l as w i l d Japanese q u a i l are mostly monogamous (Whetherbee, 1961; Moreau, 1951). Wild males m a i n t a i n t e r r i t o r i e s and form pair-bonds with one female. Males can copulate with t h e i r mate more f r e q u e n t l y and/or at appropriate times to optimize f e r t i l i z a t i o n . The foam gland of the wild q u a i l i s not w e l l developed compared with the gland of domestic q u a i l and volume of foam produced i s only a f r a c t i o n of that from domestic q u a i l ( S c h l e i d t and S h a l t e r , 1972). Under domestic [ 6 3 ] conditions, Japanese q u a i l are promiscuous; a larger number of females than males are usually maintained i n a given cage. Competition f o r f e r t i l i z a t i o n among males under these conditions i s intense and would exert a strong s e l e c t i o n pressure for development of the males' foam gland i f the foam aided the males' reproductive success. The Chinese Painted Quail, ( E x c a l f a c t o r i a c h i n e n s i s ) i s c l o s e l y r e l a t e d to the Japanese q u a i l . Females of t h i s species also lay eggs l a t e i n the evening ( N i c h o l s , unpublished d a t a ) . T h i s s p e c i e s i s domesticated but has seldom been r a i s e d i n high d e n s i t i e s as with Japanese q u a i l . Chinese Painted q u a i l have remained monogamous even a f t e r d o m e s t i c a t i o n and t h e males of t h i s s p e c i e s have only rudimentary glandular ti s s u e i n t h e i r proctodeum. In 2-male mating groups s i m i l a r to the ones i n Experiment 1, Cheng (unpublished data) has observed that unless one male completely dominates the other male, each w i l l form a s m a l l t e r r i t o r y i n an opposite corner of the pen and a s s o c i a t e c l o s e l y with one or two females, while some females w i l l " f l o a t " f r e e l y between the two males. Occasionally, a male i n one corner w i l l rush into the t e r r i t o r y of the other male and s t e a l c o p u l a t i o n s with females associated with that male. Experiment 1 showed t h a t when both males were foam-producing, these occasional copulations were successful in f e r t i l i z a t i o n as a l l females i n the pen had progeny from both males. On. the other hand, foamless males may not be a b l e to a c h i e v e f e r t i l i z a t i o n even when s u c c e s s f u l i n s t e a l i n g c o p u l a t i o n s . In pens where one male was foamless and one male foam-producing, females either produced progeny [64] from b o t h males or p r o d u c e d progeny s i r e d e x c l u s i v e l y by the foam-producing male. Thus, the foam-producing males- were s u c c e s s f u l i n f e r t i l i z i n g eggs l a i d by females associated with foamless males, but the reverse did not occur. This becomes apparent i n the r e s u l t s from pens where both males were foamless. In these pens, some females produced progeny s i r e d e x c l u s i v e l y by one male, while some females produced progeny s i r e d e x c l u s i v e l y by the other male. Some females produced progeny s i r e d by both males; presumably, these were the females which " f l o a t e d " between the two males. Behavioural observations i n Experiment 1 showed that foamless UBC-A m a l e s a t t e m p t e d and c o m p l e t e d more c o p u l a t i o n s t h a n foam-producing UBC-A males. The even proportion of progeny found when two foam-producing males were competing (Table 1), however, indicates a s i m i l a r frequency of copulation between the foam-producing UBC-W and UBC-A males. T h i s i m p l i e s that foamless UBC-A males were copulating more often than the foamless UBC-W males, and may be an e x p l a n a t i o n why a higher p r o p o r t i o n of progeny from Treatment 3 (Experiment 1) were si r e d by the UBC-A males. Furthermore, i n pens where both males were foamless, not only t o t a l f e r t i l i t y but a l s o h a t c h a b i l i t y of f e r t i l e eggs was s i g n i f i c a n t l y lowered. In chickens and other domestic b i r d s i t has been shown t h a t aged sperm (from the UV sperm-host glands) w i l l s i g n i f i c a n t l y lower egg h a t c h a b i l i t y (Nalbandov and Card, 1943). A l l these r e s u l t s i n d i c a t e that without foam, not enough sperm from the male survive to be stored i n the sperm-host glands. This may be due to the harsh c o n d i t i o n s of the cloaca or to d i f f i c u l t i e s i n sperm reaching the oviduct. With small amounts of s t o r e d sperm being [65] o n l y p e r i o d i c a l l y sparcely replenished, s t a l e sperm w i l l account for more f e r t i l i z a t i o n s . The question of whether foam can also block sperm from subsequent copulations, however, remains unsettled. Experiment 3 shows that foam does i n t e r f e r e with sperm t r a v e l i n v i t r o , but since foam i s deposited i n t o the coprodeum and not i n the o v i d u c t , the s i g n i f i c a n c e of t h i s r e s u l t i s not c l e a r . However, the s t a b i l i t y of the foam (Fujihara and Nishiyama, 1984), and the d i f f i c u l t y sperm has t r a v e l l i n g through foam, may i n d i c a t e t h a t sperm from two copulations w i l l not become mixed. Females often evacuate foam with d e f e c a t i o n and o v i p o s i t i o n . These observations lead Cheng (personal communication) to hypothesize t h a t although foam does not d i r e c t l y b l o c k t h e e n t r a n c e t o the o v i d u c t , i t o c c u p i e s s p a c e i n the coprodeum so that foam from subsequent copulations i s deposited closer to the c l o a c a l opening and thus has a better chance of being eliminated by the female before any foam from the f i r s t male. Thus, sperm from the male that mates f i r s t has a c o m p e t i t i v e edge over sperm from any subsequent copulations. The t e s t i n g of t h i s hypothesis, however, i s beyond the scope of t h i s t h e s i s . 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[75] APPENDICES [76] EXPERIMENT 4 Results from Experiments 2 and 3 i n d i c a t e that: 1) d u r i n g n a t u r a l c o p u l a t i o n s , sperm and foam are mixed yet sperm can precede the foam from the coprodeum i n t o the oviduct, and 2) foam from the male Japanese q u a i l h i n d e r s sperm t r a v e l i n v i t r o . T h i s experiment was an attempt to determine whether rn vivo, foam can act as a blocking agent to sperm from subsequent i n s e m i n a t i o n s , by s t u d y i n g i t s e f f e c t s on the onset of f e r t i l i t y . I f foam acts as a blocking agent to sperm t r a v e l , then i t s presence i n the female's c l o a c a b e f o r e i n s e m i n a t i o n should increase the latency of the f i r s t f e r t i l e egg l a i d a f t e r the insemination. The experiment a l s o allowed f o r d e t e r m i n a t i o n of the e f f e c t of foam on f e r t i l i t y and duration of f e r t i l i t y i n i n d i v i d u a l matings. However, due to poor f e r t i l i t y among ex p e r i m e n t a l females, i n s u f f i c i e n t data were c o l l e c t e d to allow for analyses and meaningful i n t e r p r e t a t i o n . The experimental procedure, however, may be of i n t e r e s t to r e s e a r c h e r s i n t h i s area of study. Thus, the experiment i s described here with a b r i e f discussion. A) MATERIALS AND METHODS The b i r d s used i n t h i s experiment were from the same.two s t r a i n s (UBC-A "wild-type" and UBC-W "white phenotype") as b i r d s used i n the other experiments, and were reared under s i m i l a r conditions. Again, [77] UBC-W females and males from both s t r a i n s were used because of the recessive white plumage genetic marker. Twenty males of each s t r a i n underwent cauterizations of t h e i r foam glands (see page 10) to provide sexually mature, foamless males. A f e r t i l i t y test was again performed with 12 cauterized males from each s t r a i n ; a t o t a l of nine males were thus excluded from the experiment. Six foam-producing males from both s t r a i n s were also maintained for the experiment. Thirty-two " v i r g i n " females were placed i n i n d i v i d u a l cages (30cm x 50cm x 25cm) four days p r i o r to the s t a r t of the experiment. E x p e r i m e n t a l males were a l s o i n d i v i d u a l l y caged as were 20 non-experimental males (10 UBC-W and 10 UBC-A) for the use of t h e i r foam. Males were kept s e p a r a t e because of r e p o r t s t h a t male-male c o p u l a t i o n s o f t e n include sperm tr a n s f e r (Adkins, 1974). During the experiment, females were s u b j e c t e d to one of the f o l l o w i n g f o u r treatments within 25 minutes a f t e r o v i p o s i t i o n : 1) The female was allowed to copulate with a foamless male and then foam was a r t i f i c i a l l y deposited into her cloaca 2) Foam was a r t i f i c i a l l y deposited into the female's c l o a c a and then she was allowed to copulate with a foamless male. 3) The female was allowed to copulate with a foamless male. 4) The female was allowed to copulate with a normal male. During copulations, the male was plac e d i n t o the female's cage f o r 15 m i n u t e s and o b s e r v a t i o n s were made to d e t e c t completed copulations using the same c r i t e r i a as i n Experiment 1. Foam used i n the experiment (0.30-0.45 ml foam deposited with a l c c tuberculin syringe) was always f r e s h l y c o l l e c t e d from the non-experimental males [78] p h e n o t y p i c a l l y d i f f e r e n t from the c o p u l a t i n g male. O f f s p r i n g phenotypes were l a t e r checked for paternity i n case any sperm had been mixed with the foam. A f t e r a completed copulation, a c l o a c a l sample from the p a r t i a l l y e v e r t e d female was obt a i n e d with a s m a l l v i n y l s p a t u l a , t r a n s f e r r e d to a microscope s l i d e , and examined (400x) to determine whether sperm transf e r had occurred. Poor f e r t i l i t y was n o t i c e d i n the f i r s t three hatches which I thought might be due to the p h y s i c a l m a n i p u l a t i o n i n v o l v e d i n o b t a i n i n g the p o s t - c o p u l a c l o a c a l samples from the females. Thus, s t a r t i n g with the f i f t h r e p l i c a t i o n , c l o a c a l samples were no longer taken. Instead, heavy cardboard "foam catchers" were suspended under the female's cage for 15 to 30 minutes a f t e r removal of the male i n the t r e a t m e n t s i n v o l v i n g foam (1,2 and 4 ) . Foam found on the suspended board was examined m i c r o s c o p i c a l l y f o r the presence of sperm. R e s u l t s would r e f l e c t the frequency of p o s t - c o p u l a foam excretion by the female as well as provide a conservative e s t i m a t e of the frequency of sperm t r a n s f e r . Because of occasional non-laying days for i n d i v i d u a l females and periods where females would lay within a few minutes of each o t h e r , t h r e e or four evenings per r e p l i c a t i o n were r e q u i r e d to stage a minimum of 20 c o p u l a t i o n s - - f i v e f e m a l e s f o r each of t h e f o u r t r e a t m e n t s . Eggs from these females were then c o l l e c t e d for at le a s t 10 days, i d e n t i f i e d by female and date, and a r t i f i c i a l l y incubated and h a t c h e d as i n t h e o t h e r experiments to determine f e r t i l i t y and paternity of o f f s p r i n g . Females were reused, but were allowed at least 10 days between copulations and the genotype of the male partner [79] was changed i n each r e p l i c a t i o n to d e t e c t any f e r t i l i z a t i o n from previous matings. There were a t o t a l of eight r e p l i c a t i o n s i n v o l v i n g 190 staged c o p u l a t i o n s — 5 0 with Treatment 1, 48 with Treatment 2, 47 with Treatment 3, and 45 with Treatment 4. B) RESULTS AND DISCUSSION The i n s t a n c e s of females l a y i n g f e r t i l e eggs a f t e r a s i n g l e staged copulation were so low that s t a t i s t i c a l a n a l y s e s f o r t e s t i n g the d i f f e r e n c e s between treatments i n the latency of l a y i n g the f i r s t f e r t i l e egg were not a p p l i e d ( T a b l e 15). I n s t e a d , the d a t a a r e presented i n tables and the trends are discussed. Data r e s u l t i n g from examinations of both the post-copula female c l o a c a l samples and the "foam catcher" foam drops are also presented and discussed. 1. E f f e c t of foam oh f e r t i l i t y . The f i r s t three treatments involved copulations with cauterized, foamless males. When no foam was added (Treatment 3 ) , the t o t a l f e r t i l i t y (among females t h a t l a i d at l e a s t one f e r t i l e egg) for a 10-day period a f t e r the copulation was 18.52% (5 f e r t i l e eggs out of 27 i n c u b a t e d ) . The addition of foam, e i t h e r before or a f t e r allowing copulation, resulted i n 34.69% f e r t i l i t y (34 f e r t i l e out of 98), which i s comparable to the f e r t i l i t y l e v e l a c h i e v e d with copulations by normal males (37.59% ; 53 f e r t i l e out of 141) (Table 16). Thus, these [80] TABLE 15: Number of completed c o p u l a t i o n s and number of females producing f e r t i l e eggs by treatment. FACTORS Number of completed copulations Number of females laying at lea s t one f e r t i l e egg Number of females^ where onset could be determined Totals 155 34 1 copula, then foam 40 TREATMENTS 2 3 foam, then copula 40 copula by foamless male 40 copula by normal male 35 18 25 13 Females that did not have a cracked egg or non-laying day between copulation and f i r s t f e r t i l e egg. o [81] TABLE 16: T o t a l f e r t i l i t y and duration of f e r t i l i t y by females that l a i d at least one f e r t i l e egg''\ TREATMENTS 1 2 3 A DAYS AFTER COPULATION copula, then foam foam, then copula copula by foamless male copula by normal male 1 A/7 1/A 0/4 3/14 2 4/5 A/A 3/3 12/14 3 2/6 3/4 1/2 11/15 4 3/6 2/4 0/2 8/14 5 2/7 0/5 1/4 10/16 6 2/6 3/5 0/4 3/17 7 2/6 1/5 0/2 3/14 8 1/5 0/4 0/2 3/13 9 0/5 0/3 0/2 0/11 10 0/5 0/2 0/2 0/13 TOTALS: 20/58 1A/A0 5/27 53/141 P E R C E N T A G E S : 3 A . 4 8 % 3 5 . 0 0 % 1 8 . 5 2 % 3 7 . 5 9 % Number of f e r t i l e eggs / number of eggs l a i d (by females that l a i d at l e a s t one f e r t i l e egg). [82] r e s u l t s p r o v i d e a d d i t i o n a l support for a conclusion of Experiment 1, that foam aids a male in f e r t i l i z a t i o n . I was hoping to show i n t h i s experiment t h a t i n Treatment 2, where foam was added before allowing c o p u l a t i o n , o v i p o s i t i o n of the f i r s t f e r t i l e egg would be delayed compared to o v i p o s i t i o n of the f i r s t f e r t i l e eggs from Treatment 1, where foam was added a f t e r c o p u l a t i o n , and i n c o n t r o l Treatment 4. Examining data from Table 2 f o r f e r t i l i t y on the f i r s t day a f t e r c o p u l a t i o n shows the t r e n d e x p e c t e d , but t h e r e a r e t o o few f e r t i l e eggs f o r s t a t i s t i c a l c e r t a i n t y . However, the data show that lack of foam caused a decrease not only i n t o t a l f e r t i l i t y , but also i n duration of f e r t i l i t y with respect to a l l other treatments. 2. Frequency of sperm transfer by cauterized and normal males. The occurrence of sperm t r a n s f e r d u r i n g a p p a r e n t l y s u c c e s s f u l c o p u l a t i o n s was recorded both by examining samples from the female's c l o a c a p o s t - c o p u l a and by examining foam e x c r e t e d by the f e m a l e p o s t - c o p u l a . Foamless males f a c i l i t a t e d sperm transfer i n 45.5% of the copulations checked (45 out of 99), while 61.5% (16 out of 26) of the copulations by normal foam-producing males involved sperm transfer (Table 17). These r e s u l t s are consistent with Adkins (1974), who found the frequency of ejaculatory and non-ejaculatory copulations to be not s i g n i f i c a n t l y d i f f e r e n t . Foam was f o u n d e x c r e t e d by t h e f e m a l e w i t h i n 30 minutes post-copula i n 43 out of 89 (48 %) c o p u l a t i o n s where a foam c a t c h e r [83] TABLE 17: Occurrences of sperm transfer during copulations by foamless and foam-producing males. METHOD OF SAMPLING SPERM NO SPERM 1. Microscope s l i d e of c l o a c a l sample from female post-copula: Copula, then foam (TR 1) 8 11 Foam, then copula (TR 2) 8 IA Copula, no foam (TR 3) 11 12 Normal copula (TR A) 9 9 2. Examination of foam excreted by female post-copula: Copula, then foam (TR 1) 8 12 Foam, then copula (TR 2) 10 5 Normal copula (TR A) 7 1 TOTALS: Foamless males Normal males A5 16 5A 10 [84] was a p p l i e d . Sperm was found mixed with the excreted foam i n 58% of the examined foam droppings. The poor f e r t i l i t y seen i n t h i s experiment was unexpected. Sperm transfer was known to be occurring enough to provide better f e r t i l i t y . In s i m i l a r staged matings where the major difference was in placing the female i n t o the male's cage f o r c o p u l a t i o n , a much b e t t e r f e r t i l i t y l e v e l was achieved (Cheng, unpublished d a t a ) . Indeed, females were sometimes reluctant to copulate, whereas, i n the study by Cheng (personal communication), copulations were normally immediate. An important c o n c l u s i o n of t h i s t h e s i s was given a d d i t i o n a l support by r e s u l t s o b t a i n e d i n t h i s experiment: foam can apparently aid a male i n f e r t i l i z a t i o n even when not i n a competitive s i t u a t i o n . [85] TABLE 18: Analysis of Variance for t o t a l f e r t i l i t y l of a l l incubated eggs i n Experiment 1. Source df SS MS F Replication 2 74.70 37.35 0.217 Treatment 3 18,891.71 6297.24 36.550* Error 54 9303.48 172.29 Total 59 28,269.89 1 Arcsine transformation applied to data before a n a l y s i s . *** p<0.005 [86] TABLE 19: A n a l y s i s of V a r i a n c e f o r percent h a t c h a b i l i t y * of a l l f e r t i l e eggs i n Experiment 1. Source df SS MS F Rep l i c a t i o n 2 355.47 177.74 1.40 Treatment 3 1702.17 567.39 4.48** Error 54 6833.05 126.54 Tot a l 59 8890.69 i Arcsine transformation applied to data before a n a l y s i s . ** p<0.01 [87] TABLE 20: Analysis of Variance for number of white progeny typed out of a l l progeny typed! i n Experiment 1. Source df SS MS F R e p l i c a t i o n 2 908.36 454.18 1.587' Operation (0) 1 33,569.07 33,569.07 117.329'' ' Co n d i t i o n 2 (C) 1 15,152.71 15,152.71 52.961'' f 0 x C 1 512.99 512.99 1.793 Error 54 15,449.99 286.11 Tot a l 59 65,593.12 Arcsine transformation applied to data before a n a l y s i s . ^ , Condition of foam gland of the competing UBC-A male. *** p<0.005 [88] TABLE 21: A n a l y s i s of V a r i a n c e f o r percent h a t c h a b i l i t y 1 of white pro§ ieny out of a l l f e r t i l e eggs i n Experiment 1. Source df SS MS F Repl i c a t i o n 2 570.61 285.30 1.655 Operation (0) 1 18,403.71 18,403.71 106.756""'"" Condition^ (C) 1 5,039.46 5,039.46 29.233"""'" 0 x C 1 270.09 270.09 1.567 Error 54 9,309.16 172.39 ' Total 59 33,593.03 Arcsine transformation applied to data before a n a l y s i s . 1 Condition of foam gland of the competing UBC-A male. o~ *t. """'" p<0.005 [89] TABLE 22: A n a l y s i s of V a r i a n c e f o r number of mating attempts per observation^ by UBC-W males i n Experiment 1. Source df SS MS F R e p l i c a t i o n 1 0.091 0.091 1.083 Operation (0) 1 0.001 0.001 0.005 Co n d i t i o n 2 (C) 1 0.400 0.400 4.762 0 x C 1 0.277 0.277 3.298 Error 1 3 0.252 0.084 Time (T) 2 0.051 0.026 0.513 T x 0 2 0.022 0.011 0.217 T x C 2 0.146 0.073 1.440 T x 0 x C 2' 0.451 0.226 4.459* Error 2 128 6.487 0.051 Tota l 143 8.177 1 Square root transformation applied to data before a n a l y s i s . 2 Condition of foam gland of the competing UBC-A male. * p<0.05 [90] TABLE 23: A n a l y s i s of V a r i a n c e f o r number of completed copulations per observation-^ by UBC-W males i n Experiment 1. Source df SS MS F Repli c a t i o n 1 0.008 0.008 0.10 Operation (0) 1 0.013 0.013 0.17 Co n d i t i o n 2 (C) 1 0.333 0.333 4.27 0 x C 1 0.1A3 0.143 1.83 Error 1 3 0.23A 0.078 Time (T) 2 0.011 - 0.006 0.13 T x 0 2 0.010 0.005 0.11 T x C 2 0.013 0.006 0.13 T x 0 x C 2 0.179 0.090 2.00 Error 2 128 5.799 0.045 Tota l 1A3 6.743 ! Square root transformation applied to data before ana l y s i s . 2 Condition of foam gland of the competin g UBC-A male. [91] TABLE 2A: Analysis of Variance for number of mating attempts per observation^ by UBC-A mal es i n Experiment 1. Source df SS MS F Repli c a t i o n 1 0.018 0.018 0.38 Operation (0) 1 0.595 0.595 12.39* Co n d i t i o n 2 (C) 1 0.033 • 0.033 0.69 0 x C 1 0.025 0.025 0.52 Error 1 3 0.1 AA 0.0A8 Time (T) 2 0.002 0.001 0.01 T x 0 2 0.883 0.AA2 3.69* T x C 2 0.37A 0.187 1.56 T x 0 x C 2 0.769 0.38A 3.21* Error 2 128 15.325 0.120 Total 1A3 18.168 Square root transformation applied to data before a n a l y s i s . 2 Condition of foam gland of the competing UBC-W male. * p<0.05 [92] TABLE 25: Analysis of Variance for number of completed copulations per observation^ by UBC-A males i n Experiment 1. Source df SS MS F Re p l i c a t i o n 1 0.082 0.082 2.03 Operation (0) 1 0.515 0.515 12.77* C o n d i t i o n 2 (C) 1 0.003 0.003 0.07 0 x C 1 0.001 0.001 0.01 Error 1 3 0.121 0.040 Time (T) 2 0.125 0.062 0.85 T x 0 2 • 0.120 0.060 0.83 T x C 2 0.242 0.121 1.67 T x 0 x C 2 0.460 0.230 3.17* Error 2 128 9.288 0.072 Tota l 143 10.957 Square root transformation applied to data before a n a l y s i s . Condition of foam gland of the competing UBC-W male. * p<0.05 [93] TABLE 26: Analysis of Variance for rooster sperm penetration through c a p i l l a r y tubes. Source df SS MS F Repl i c a t i o n 4 10.99 2.75 5.62"" Observer (0) 1 2.41 2.41 4.93* Contents (C) 1 56.32 56.32 115.19* Temperature (T) 1 6.26 6.26 12.80' 0 x C 1 1.10 1.10 2.45 0 x T 1 0.36 0.36 0.74 C x T 1 0.04 0.04 0.08 0 x C x T 1 0.12 0.12 0.24 Error 1 120 58.67 0.49 Time (S) 1 38.33 38.33 465.45' S x 0 1 0.46 0.46 5.65* S x C 1 2.77 2.77 33.64-S x T 1 1.16 1.16 14.09"" S x 0 x C 1 0.03 0.03 0.36 S x 0 x T 1 0.07 0.07 0.82 S x C x T 1 0.00 0.00 0.00 S x 0 x C x T 1 1.81 1.81 22.03* Error 2 180 14.82 0.08 T o t a l 319 195.72 * p < 0 . 0 5 ** p < 0 . 0 0 5 

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