Science, Faculty of
Zoology, Department of
DSpace
UBCV
Grove, Bryon Dennis
Master of Science - MSc
University of British Columbia
Although guppy females may store sperm in the ovary for several months, they become sexually receptive for a short time every 22-30 days. Females may remate during these receptive periods and consequently, a potential competition between stored and fresh sperm for oocytes may occur. Earlier studies indicated that sperm from the most recent insemination dominates fertilization of new broods. However, these studies provide no clues to how this apparent sperm precedence occurs. There are three possible explanations. First, sperm from new inseminations may outnumber stored sperm and thus, have a statistical advantage. Second, since fertilization in guppies occurs within a few days of insemination, this timing of insemination (relative to egg maturity) may give fresh sperm an advantage. Finally, the location of fresh sperm in the ovary may give them greater access to mature eggs. In this study, females were artificially inseminated with known numbers of spermatophores to see how the proportion of one male's sperm in a mixed insemination determines its contribution to new broods; 2.) the number of spermatophores in inseminations affects the degree of precedence shown by sperm from re-inseminations; and 3.) the timing of re-insemination relative to the female reproductive cycle affects the degree of precedence shown by re-inseminations. An autosomal, recessive allele was used as a genetic marker to distinguish between different males' sperm. In addition, ovaries of females artificially inseminated twice were examined histologically to locate sperm from each insemination. Sperm from one of the inseminations was radioactively labelled. These experiments indicated that sperm precedence does not necessarily result from a statistical advantage to sperm from a re-insemination. Also sperm from re-inseminations occurring up to a week before a new batch of oocytes is mature have strong precedence over stored sperm, although the degree of precedence may be reduced. Autoradiographical analysis of sections of ovaries, however, indicated that the location of sperm storage sites may contribute to sperm precedence. Sperm clusters found in tubules closely associated with oocytes come predominantly from re-inseminations. A model is proposed suggesting that movement of sperm into these tubules immediately after insemination accounts for the dominance of new inseminations during fertilization. The possible functional significance of sperm storage and sperm precedence in poeciliids is discussed.
Ail ANALYSIS OF IHTBAOVARIAS SPEEM INTERACTIONS IN THE GUPPX, PQECILIA RETICULATA by BBYON DENNIS GBOVE B . S c , The U n i v e r s i t y of B r i t i s h Columbia A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIBEHENTS FOB THE DEGBEE OF BASTES OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Zoology) Be accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA Hay 1980 (c) Byron Dennis Grove, 1980 I n 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 t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e 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 a n d s t u d y . I f u r t h e r a g r e e 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 c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f 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 n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f ^ n i n g y T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 D a t e Q^A^2£^ln Abstract Although guppy females may s t o r e sperm i n the ovary f o r s e v e r a l months, they become s e x u a l l y r e c e p t i v e f o r a s h o r t time every 22-30 days., Females may remate during these r e c e p t i v e p e r i o d s and consequently, a p o t e n t i a l c o m p e t i t i o n between s t o r e d and f r e s h sperm f o r oocytes may occur. E a r l i e r s t u d i e s i n d i c a t e d t h a t sperm from the most r e c e n t i n s e m i n a t i o n dominates f e r t i l i z a t i o n of new broods. However, these s t u d i e s p r o v i d e no c l u e s t o how t h i s apparent sperm precedence occurs. There are three p o s s i b l e e x p l a n a t i o n s . F i r s t , sperm from new i n s e m i n a t i o n s may outnumber s t o r e d sperm and thus, have a s t a t i s t i c a l advantage.„ Second, s i n c e f e r t i l i z a t i o n i n guppies occurs u i t h i n a few days of i n s e m i n a t i o n , t h i s t i m i n g of i n s e m i n a t i o n { r e l a t i v e t o egg maturity ) may g i v e f r e s h sperm an advantage. F i n a l l y , the l o c a t i o n of f r e s h sperm i n the ovary may g i v e them g r e a t e r access to mature eggs. In t h i s study, females were a r t i f i c i a l l y inseminated with known numbers of spermatophores to see how the p r o p o r t i o n of one male's sperm i n a mixed i n s e m i n a t i o n determines i t s c o n t r i b u t i o n to new broods; 2.) the number of spermatophores i n i n s e m i n a t i o n s a f f e c t s the degree of precedence shown by sperm from r e - i n s e m i n a t i o n s ; and 3.), the t i m i n g of r e - i n s e m i n a t i o n r e l a t i v e t o the female r e p r o d u c t i v e c y c l e a f f e c t s the degree of precedence shown by i i i r e - i n s e m i n a t i o n s . An autosomal, r e c e s s i v e a l l e l e was used as a g e n e t i c marker to d i s t i n g u i s h between d i f f e r e n t males' sperm. In a d d i t i o n , o v a r i e s of females a r t i f i c i a l l y inseminated twice were examined h i s t o l o g i c a l l y to l o c a t e sperm from each i n s e m i n a t i o n . Sperm from one of the i n s e m i n a t i o n s was r a d i o a c t i v e l y l a b e l l e d . These experiments i n d i c a t e d t h a t sperm precedence does not n e c e s s a r i l y r e s u l t from a s t a t i s t i c a l advantage to sperm from a r e - i n s e m i n a t i o n . A l s o sperm from r e - i n s e m i n a t i o n s o c c u r r i n g up to a week before a new batch of oocytes i s mature have s t r o n g precedence over s t o r e d sperm, although the degree of precedence may be reduced. A u t o r a d i o g r a p h i c a l a n a l y s i s of s e c t i o n s of o v a r i e s , however, i n d i c a t e d that the l o c a t i o n of sperm s t o r a g e s i t e s may c o n t r i b u t e t o sperm precedence. Sperm c l u s t e r s found i n t u b u l e s c l o s e l y a s s o c i a t e d with oocytes come predominantly from r e -i n s e m i n a t i o n s . A model i s proposed suggesting t h a t movement of sperm i n t o these t u b u l e s immediately a f t e r i n s e m i n a t i o n accounts f o r the dominance of new i n s e m i n a t i o n s d u r i n g f e r t i l i z a t i o n . The p o s s i b l e f u n c t i o n a l s i g n i f i c a n c e of sperm storage and sperm precedence i n p o e c i l i i d s i s d i s c u s s e d . i v l§:klS. of Contents Abstr a c t .......................... . . . . . . . . . . . . . . . . . . . . . i i L i s t of T a b l e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v i L i s t of . F i g u r e s . . ...... - ......... ... . . . . ..... ..... . . ... . v i i i L i s t of P l a t e s .. . . . •. • .* . . .-,. . .................,*........ i x Acknowledgements .... ... • • .. ...... • • .. ........ ... • • .... . . x I n t r o d u c t i o n ............................. ............ ... 1 General Methods and M a t e r i a l s ..........................7 Stocks used i n t h i s study ......................... 7 A r t i f i c i a l i n s e m i n a t i o n procedures 9 A. ) o b t a i n i n g spermatophores from males ....... 10 B. ) i n s e m i n a t i o n of the female ................11 Section I The r e l a t i o n s h i p between the p r o p o r t i o n of g e n e t i c a l l y d i s t i n c t sperm i n mixed in s e m i n a t i o n s and the r e l a t i v e c o n t r i b u t i o n of those sperm to broods ..... 16 I n t r o d u c t i o n ......• ...• Methods and M a t e r i a l s .. Be s u i t s ' * :•••:•#'*••-•••••«! D i s c u s s i o n ... 16 ... 17 ... 18 ...24 Se c t i o n I I The e f f e c t of spermatcphore number i n ins e m i n a t i o n s on the success of r e -i n s e m i n a t i o n s .•........................... 29 I n t r o d u c t i o n ............. .. Methods and M a t e r i a l s ...... R e s u l t s .. ................. .............•...... 4 Di s c u s s i o n ..... ....... ..... ...29 ...30 • • 32 . ..48 Se c t i o n I I I The e f f e c t o f t i m i n g of r e - i n s e m i n a t i o n on the success of sperm from r e - i n s e m i n a t i o n s 56 In t r o d u c t i o n .......... ............................56 Methods and M a t e r i a l s ............................. 57 Resul t s ......... 59 D i s c u s s i o n . ............. ............... . .... .... . ..71 Sec t i o n IV A u t o r a d i o g r a p h i c a l a n a l y s i s of the l o c a t i o n of sperm from separate i n s e m i n a t i o n s i n the ovary ................ 75 I n t r o d u c t i o n ............... .... ...................75 V Methods and M a t e r i a l s .............................76 a. ) production of r a d i o a c t i v e l y l a b e l l e d sperm 76 b. ) a r t i f i c i a l i n s e m i n a t i o n of females ........77 c. ) h i s t o l o g i c a l procedures ...................79 d. ) a p p l i c a t i o n and development of the nu c l e a r t r a c k emulsion . ... ........ ..... ............ 80 Re s u l t s ........81 D i s c u s s i o n . ........... . . ....... ... .......... ...... . 90 General D i s c u s s i o n .............. ...........•........... 97 B i b l i o g r a p h y ............ ........ .... .......................108 v i L i s t o f Tables Table 1 The number of progeny of each genotype i n the f i r s t broods of females inseminated with th r e e d i f f e r e n t p r o p o r t i o n s of cjd sperm ......20 Table 2 A comparison between the gd/gd freguency i n the broods and the cjd a l l e l e frequency i n the i n s e m i n a t i o n s f o r each treatment group ....... 23 Table 3 The number of females inseminated at the beginning of the experiment, the number of females r e - i n s e m i n a t e d , and the number of r e -inseminated females which gave b i r t h . Values f o r the two d i f f e r e n t i n s e m i n a t i o n seguences i n each treatment group are separated ........ 34 Table 4 The percentage of s u c c e s s f u l i n s e m i n a t i o n s and r e - i n s e m i n a t i o n s f o r each i n s e m i n a t i o n sequence i n three treatment groups ........... 37 Table 5 The number of • /gd and qd/gd progeny i n f i r s t and second broods produced ty females i n the t h r e e treatment groups .......................39 Table 6 The mean s i z e s of f i r s t and second broods r e s u l t i n g from i n s e m i n a t i o n s with e i t h e r .gd or + sperm. The mean s i z e s of pooled broods f o r each treatment group are a l s o r e p o r t e d ...42 Table 7 An a n a l y s i s of variance comparing brood s i z e s among i n s e m i n a t i o n sequences, f i r s t and second broods, and treatment groups ..........47 Table 8 Contingency t a b l e f o r a c h i - s q u a r e t e s t comparing the c o n t r i b u t i o n of f i r s t and second i n s e m i n a t i o n s to second broods between . the three treatment groups ...*.«....*....,...50 Table 9 The number of +/gd and qd/gd progeny i n f i r s t and second broods produced by females a f t e r pre- and post-parturn r e - i n s e m i n a t i o n s ........61 v i i Table 10 The mean s i z e s of f i r s t and second broods r e s u l t i n g from i n s e m i n a t i o n s with e i t h e r c[d or • sperm. , The mean s i z e s of pooled broods f o r each treatment group are a l s o presented ..63 Table 11 An a n a l y s i s of v a r i a n c e comparing mean brood s i z e s among i n s e m i n a t i o n sequences, f i r s t and second broods, and treatment groups ..........66 Table 12 Contingency t a b l e s f o r c h i - s g u a r e t e s t s comparing the c o n t r i b u t i o n of f i r s t and second i n s e m i n a t i o n s t o second broods between i n s e m i n a t i o n sequences f o r A.) ,pre-partum r e -i n s e m i n a t i o n s and B.) post-partum r e -ins e m i n a t i o n s ..... ...........................68 Table 13 Contingency t a b l e f o r c h i - s q u a r e t e s t comparing the c o n t r i b u t i o n c f f i r s t and second i n s e m i n a t i o n s to second broods between treatment groups • .... ... . ..... . . .«•... ......... .70 v i i i l i s t o f F i g u r e s F i g u r e 1 Mean s i z e of A.) f i r s t broods and B.) second broods a f t e r i n s e m i n a t i o n s with 40, 100, and 200 spermatophores ................ ... ........ 44 F i g u r e 2 Diagram . o f a l o n g i t u d i n a l s e c t i o n of a guppy ovary .... . .......... ... ........ ......... .... . . 84 i x L i s t of P l a t e s P l a t e 1 A.) Male guppy with gonopodium p a r t i a l l y e r e c t e d . B. )„ S y r i n g e , p o l y e t h y l e n e t u b i n g , and modified micro-hematocrit tube f o r counting spermatophores. C. ) Mo d i f i e d m i c r o - c a p i l l a r y tube with tapered end. D. ) I n t r o d u c t i o n of m i c r o - c a p i l l a r y tube i n t o the female r e p r o d u c t i v e duct ......15 P l a t e 2 a.) a t u b u l e c o n t a c t i n g an oocyte. B. ) An immature oocyte an e a r l y stage of development. C. ) An immature oocyte at an i n t e r m e d i a t e stage of development. D. ) M a g n i f i c a t i o n of P l a t e 2a showing spermatozoa and exposed emulsion at the d i s t a l end of a t u b u l e . E. ) M a g n i f i c a t i o n of P l a t e 2C showing i n t e n s e l y exposed area of the emulsion. •P.), The tubule i n P l a t e 2E one s e c t i o n l a t e r . 86 X Acknowledgements I wish to thank my s u p e r v i s o r . Dr. J.D. McPhail, f o r s u p p o r t i n g t h i s work and f o r h i s c o n s t r u c t i v e c r i t i c i s m of the manuscript a t i t s v a r i o u s stages of development. Drs. L i l e y , Gass, and Acton a l s o reviewed the manuscript and I wish to thank them f o r t h e i r c r i t i c i s m s . S e v e r a l others were i n s t r u m e n t a l to the s u c c e s s f u l completion of t h i s work. I thank Henke Luyten who a s s i s t e d g r e a t l y with the a u t o r a d i o g r a p h i c a l procedures used i n t h i s study. I wish a l s o t o express my a p p r e c i a t i o n to Suzin Crosby f o r her l e s s o n s i n h i s t o l o g i c a l t e c h n i g u e s , f o r use of equipment and s u p p l i e s , and f o r photographing some of the f i g u r e s . F i n a l l y , I am indebted t o Margaret McDonald f o r her time and a d v i c e i n the p r o d u c t i o n of p l a t e s and f i g u r e s as w e l l as f o r her a s s i s t a n c e i n e d i t i n g the manuscript. Without her help t h i s t h e s i s c o u l d not have been completed. I wish a l s o to acknowledge NSEBC f o r a postgraduate s c h o l a r s h i p and the Department of Zoology f o r p r o v i d i n g me with a graduate a s s i s t a n t s h i p . 1 I n t r o d u c t i o n Prolonged sperm r e t e n t i o n w i t h i n female r e p r o d u c t i v e systems i s a s u r p r i s i n g l y common phenomenon throughout the animal kingdom. In keeping with the d i v e r s i t y of r e p r o d u c t i v e modes in animals, t h i s sperm s t o r a g e i s h i g h l y v a r i a b l e . Both the degree and means of s t o r i n g sperm vary between, and w i t h i n , major taxoncmic groups. Despite t h i s v a r i a b i l i t y , two b a s i c problems are common to sperm st o r a g e i n animals. F i r s t , s i n c e sperm have l i m i t e d metabolic machinery, they have been c o n s i d e r e d i n c a p a b l e of long-term maintenance. The f a c t t h a t sperm are known t o s u r v i v e i n the female r e p r o d u c t i v e system f o r months or even years i m p l i e s s o p h i s t i c a t e d p h y s i o l o g i c a l mechanisms f o r sperm s t o r a g e . These mechanisms have been the s u b j e c t of c o n s i d e r a b l e i n q u i r y (Forbes, 1961; Parker, 1970; Parkes, 1960; T h i b a u l t and l e v a s s e u r , 1973), The second, and perhaps more important problem, i s the s i g n i f i c a n c e of sperm storage as a r e p r o d u c t i v e s t r a t e g y . The p a r t t h a t sperm storage p l a y s i n r e p r o d u c t i o n , how i t enhances r e p r o d u c t i v e output, and the e f f e c t i t has upon other aspects of r e p r o d u c t i o n are important f o r understanding the e v o l u t i o n a r y and e c o l o g i c a l consequences of sperm s t o r a g e . I n c o n t r a s t to s t u d i e s on the p h y s i o l o g y of sperm s t o r a g e , the e v o l u t i o n a r y s i g n i f i c a n c e of sperm storage i s exceedingly d i f f i c u l t to determine, and so has r e c e i v e d l i t t l e a t t e n t i o n . 2 Hith the exce p t i o n of the i n s e c t s , sperm storage i n the i n v e r t e b r a t e s i s not as s e l l known as i t i s i n the v e r t e b r a t e s . In many i n v e r t e b r a t e s (e.g. opis t o b r a n c h gastropods, t u r b e l l a r i a n s , a r t h r o p o d s ) r females possess s t r u c t u r e s t o hold sperm before f e r t i l i z a t i o n (Barnes, 197 4) and these may be capable of m a i n t a i n i n g sperm f o r prolonged p e r i o d s . O n f o r t u n a t e l y , the d u r a t i o n of sperm v i a b i l i t y w i t h i n these s t r u c t u r e s i s unknown. More i s known about sperm st o r a g e i n i n s e c t s . Sperm storage i s documented i n f i v e i n s e c t o r d e r s and i s p a r t i c u l a r l y w e l l known i n D r o s o p h i l a . , Parker (1970) reviewed the mechanisms employed by i n s e c t s known to s t o r e sperm and considered the consequences and b e h a v i o u r a l phenomena l i n k e d with sperm s t o r a g e . For the v e r t e b r a t e s a more e x t e n s i v e survey of female sperm s t o r a g e i s a v a i l a b l e . Every major v e r t e b r a t e group c o n t a i n s members i n which - females s t o r e sperm and the d i v e r s i t y of adapt a t i o n s i s high. The v i a b i l i t y of s t o r e d sperm ranges from a few days t o years and, depending upon the group, storage s i t e s can be f o l d s , t u b u l e s , or d i v e r t i c u l a e anywhere along the female r e p r o d u c t i v e t r a c t . The r e l a t i o n s h i p between sperm and - female t i s s u e s ranges from a very simple a s s o c i a t i o n to one i n v o l v i n g complex m o d i f i c a t i o n s of the storage s i t e e p i t h e l i u m {Dent, 1970; Hoffmann and Wimsatt, 1972; Simsatt e t a l , 1966). . For a few groups, the anatomical d e t a i l s are w e l l s t u d i e d , but l i t t l e i s known about the phys i o l o g y of sperm s u r v i v a l and a c t i v i t y d uring s t o r a g e . Forbes (1961), Parkes (1960), and T h i b a u l t 3 and Levasseur {197 3) reviewed sperm storage i n .vertebrates, but provided only a curs o r y treatment of the phenomenon i n f i s h . Gardiner (1S76) p r e s e n t s a more d e t a i l e d review of f i s h . V a r i a b i l i t y i n the modes of s t o r i n g sperm among the v e r t e b r a t e s suggests t h a t sperm s t o r a g e has d i f f e r e n t r o l e s and consequences i n v e r t e b r a t e r e p r o d u c t i o n . E s s e n t i a l l y two b a s i c p a t t e r n s of sperm storage occur i n the v e r t e b r a t e s . S e v e r a l groups delay f e r t i l i z a t i o n f o r a prolonged p e r i o d a f t e r c o p u l a t i o n {bats, Himsatt e t a l , 1966; seme l i z a r d s and snakes. Fox, 1956, 1963; embiotocid f i s h e s , G a r d i n e r , 1976; blue shark, P r a t t , 1979). In these animals, sperm storage i s e s s e n t i a l t o r e p r o d u c t i o n . Presumably d e l a y i n g f e r t i l i z a t i o n ensures t h a t c o p u l a t i o n , g e s t a t i o n , and r e a r i n g occur only a t optimal times of the year. In other groups f e r t i l i z a t i o n occurs soon a f t e r c o p u l a t i o n , but spermatozoa remain v i a b l e i n the female r e p r o d u c t i v e system f o r c o n s i d e r a b l e l e n g t h s of time. The purpose of sperm storage i n such s i t u a t i o n i s not obvious and may vary from animal t o anim a l . Perhaps i t se r v e s to maintain female r e p r o d u c t i v e output when male a v a i l a b i l i t y i s low, As a r e s u l t of prolonged sperm st o r a g e i n some v e r t e b r a t e s , v i a b l e sperm may be present i n , the female r e p r o d u c t i v e system when another i n s e m i n a t i o n occurs. I f t h i s happens, t h e r e i s a p o t e n t i a l f o r com p e t i t i o n f o r mature oocytes between the sperm of two males. The consequences of t h i s are t w o f o l d . Depending on the nature 4 of the c o m p e t i t i o n , female r e p r o d u c t i v e output may be a f f e c t e d i n seme way. For example, genetic v a r i a b i l i t y w i t h i n and between broods w i l l depend upon the outcome of any i n t r a s p e c i f i c sperm i n t e r a c t i o n s . In a d d i t i o n , an i m p l i c i t c o m p e t i t i o n between males may e x i s t . Again, depending upon the outcome of sperm i n t e r a c t i o n s , male success may be a f f e c t e d by a female's r e p r o d u c t i v e h i s t o r y . Of the v e r t e b r a t e s , the p o e c i l i i d s , a f a m i l y of t e l e o s t f i s h , best exemplify the s u p e r i m p o s i t i o n of m u l t i p l e i n s e m i n a t i o n s on prolonged sperm s t o r a g e . In t h i s group, r e p r o d u c t i o n i s extremely s p e c i a l i z e d and i n c l u d e s : i n t e r n a l f e r t i l i z a t i o n ; e i t h e r v i v i p a r i t y or o v o v i v i p a r i t y ; sometimes s u p e r f o e t a t i o n ; and a well developed a b i l i t y t o s t o r e sperm i n the ovary. A s h o r t and r e g u l a r r e p r o d u c t i v e c y c l e a l s o p r e v a i l s i n the group and i s what makes sperm s t o r a g e such a c u r i o u s phenomenon i n the p o e c i l i i d s . Females are r e c e p t i v e t o males f o r a s h o r t time i n each c y c l e . T h i s r e c e p t i v e p e r i o d corresponds to the completion of oogenesis and f e r t i l i z a t i o n . V i a b l e sperm remain i n the ovary through s e v e r a l c y c l e s {van Oordt, 1928; Hinge, 1937), and consequently females commonly r e t a i n the sperm of more than one male {Borowsky and Kallman 1976; Hildemann and flagner, 1954; i i n g e , 1937)., P r e l i m i n a r y assessments of the success of sperm from d i f f e r e n t i n s e m i n a t i o n s have been made. Breeding experiments using genetic markers i n d i c a t e that i n the guppy, P o e c i l i a r e t i c u l a t a , an i n s e m i n a t i o n d i r e c t l y f o l l o w i n g the b i r t h of a brood has precedence i n f e r t i l i z i n g 5 the next brood (Hildemann and Wagner, 1954; Hinge, 1937). Since Hinge (19 37) does not support h i s c l a i m s with experimental r e s u l t s , i t i s i m p o s s i b l e to assess h i s c o n c l u s i o n s , but Hildemann and Wagner (1954) give the mating records of ten females, . In e i g h t , the r e - i n s e m i n a t i o n c o n t r i b u t e d almost e n t i r e l y t o the subseguent brood. The s i g n i f i c a n c e of sperm storage i n the r e p r o d u c t i v e b i o l o g y of P o e c i l i a r e t i c u l a t a cannot be f u l l y understood u n t i l the f a c t o r s governing sperm precedence are known. From the above s t u d i e s i t i s c l e a r t h a t a second i n s e m i n a t i o n predominates over the f i r s t i n a subseguent brood, but s i n c e the g u a l i t y and g u a n t i t y of spermatozoa i n these i n s e m i n a t i o n s i s unknown, l i t t l e can be i n f e r r e d about how a recent i n s e m i n a t i o n gains precedence. .. The s i m p l e s t e x p l a n a t i o n of sperm precedence i s a d i s p a r i t y i n the number of a v a i l a b l e spermatozoa between the two i n s e m i n a t i o n s . I t i s expected that l o s s e s through f e r t i l i z a t i o n and m o r t a l i t i e s before and during sperm storage w i l l reduce the number of s t o r e d sperm t o l e v e l s below t h a t of a new i n s e m i n a t i o n , as a consequence, a male's su c c e s s a f t e r a mating would depend upon the number of sperm i n h i s ins e m i n a t i o n and the number of in s e m i n a t i o n s preceding h i s . Many other hypotheses are a l s o p o s s i b l e . . Since r e -in s e m i n a t i o n occurs when oocytes are mature (Rosenthal, 1952; T h i b a u l t and S c h u l t z , 1978; Turner, 1937), sperm from a r e - i n s e m i n a t i o n may be i n a p o s i t i o n t o f e r t i l i z e oocytes b e f o r e s t o r e d spermatozoa can. A l t e r n a t i v e l y , a c o m p e t i t i v e i n t e r a c t i o n between spermatozoa from d i f f e r e n t males or 6 d i f f e r e n c e s i n sperm v i a b i l i t y , c ould e x p l a i n the phenomenon. C l e a r l y , an e x p l a n a t i o n of the mechanism of sperm precedence w i l l help i n understanding the importance of sperm storage i n guppy r e p r o d u c t i o n , and a l s o g i v e i n s i g h t s i n t o the p h y s i o l o g y of sperm s t o r a g e . In the present study p r e d i c t i o n s based on two hypotheses about the f a c t o r s governing sperm precedence are t e s t e d . I propose t h a t i f sperm precedence by the second i n s e m i n a t i o n r e s u l t s from a d i s p a r i t y i n the number of spermatozoa from the f i r s t and second i n s e m i n a t i o n s , then the s i z e of the i n s e m i n a t i o n s should determine the d i s p a r i t y and hence t h e degree of sperm precedence. I f the precedence gained by the second i n s e m i n a t i o n r e s u l t s from i n s e m i n a t i o n o c c u r r i n g a t the same time eggs mature, changing the t i m i n g of the second i n s e m i n a t i o n with r e s p e c t to the female r e p r o d u c t i v e c y c l e should change the success of the second i n s e m i n a t i o n . In a d d i t i o n I have employed autoradiography to determine the a c t i v i t y of sperm from two d i f f e r e n t i n s e m i n a t i o n s w i t h i n the ovary and t h e i r a f f e c t upon each o t h e r . P o s s i b l y , the l o c a t i o n of s t o r e d sperm from two i n s e m i n a t i o n s determines the amount of sperm precedence gained by r e - i n s e m i n a t i o n s . , 7 General Methods and M a t e r i a l s The b a s i c technique used i n t h i s study was to a r t i f i c i a l l y inseminate female guppies twice with known amounts of sperm, and then assess the i n t e r a c t i o n between the sperm from the two i n s e m i n a t i o n s . • To separate the r e s u l t s of the two i n s e m i n a t i o n s , i t was necessary t h a t sperm frem one i n s e m i n a t i o n c a r r i e d a marker. Where the success of an i n s e m i n a t i o n was assessed through o f f s p r i n g p a t e r n i t y , a g e n e t i c marker was used. To i d e n t i f y the sperm of two d i f f e r e n t i n s e m i n a t i o n s w i t h i n the ovary, a r a d i o i s o t o p e was used. Stocks used i n t h i s study The g e n e t i c s of a l a r g e number of t r a i t s i n P o e c i l i a r e t i c u l a t a are known and so a v a r i e t y of g e n e t i c markers are a v a i l a b l e . O nfortunately most are not e a s i l y o b t a i n e d from aguarium supply o u t l e t s . f o r t h i s study, I chose the xanthic mutant phenotype, "Gold", as a g e n e t i c marker. I t i s c o n t r o l l e d by a s i n g l e r e c e s s i v e autosomal a l l e l e ( c[d ) (Goodrich et a l , 1944; Singe, 1927) and g i v e s the homozygote ( gd/gd ) a g e l d body c o l o u r . Using the qd a l l e l e as a g e n e t i c marker has s e v e r a l advantages., Stocks are r e a d i l y obtained from aquarium supply d e a l e r s . . A l s o , newborn qd / j d i n d i v i d u a l s are e a s i l y 8 d i s t i n g u i s h e d from both + /qd and +/+ young. T h i s removes the need t o r a i s e the broods before s c o r i n g t h e i r phenotypes. F i n a l l y , the g e n e t i c s of the t r a i t are simple enough that phenotype f r e q u e n c i e s d i r e c t l y i n d i c a t e the f r e q u e n c i e s of the two a l l e l e s i n the broods. Brood s t o c k s of female and male qd/qd and •/* guppies were i n i t i a l l y obtained from Hartz Mountain Pet S u p p l i e s L i m i t e d . The two s t r a i n s were maintained s e p a r a t e l y i n 84 l i t r e g l a s s a q u a r i a . Pregnant females were i s o l a t e d i n 20 l i t r e g l a s s aquaria u n t i l they had given b i r t h and then were placed back i n t o the a p p r o p r i a t e brood stock tank.. O f f s p r i n g of the two s t r a i n s were kept separate i n 40 and 84 l i t r e g l a s s a q u a r i a . Since a s t o c k of v i r g i n females was r e q u i r e d f o r the experiments, males were removed from the tanks as soon as t h e i r sex c o u l d be determined. They were kept i n 40 l i t r e g l a s s a q u a r i a and used as a sperm supply. V i r g i n females were net used i n the experiments u n t i l they were at l e a s t four months o l d . For the d u r a t i o n of the experiments, a l l f i s h were kept i n an environment chamber at a constant temperature of 25 C and a constant photoperiod of 16 hours c f l i g h t and 8 hours of darkness. A i l 20 l i t r e a q u a r ia were f i t t e d with undergravel f i l t e r s and a sand s u b s t r a t e . F i l t r a t i o n i n the 40 and 84 l i t r e aquaria was accomplished e i t h e r with undergravel f i l t e r s and a sand s u b s t r a t e or s m a l l c o rner f i l t e r s . 9 a r t i f i c i a l i n s e m i n a t i o n procedure. To ensure t h a t females were inseminated with known amounts of sperm, a l l i n s e m i n a t i o n s were a r t i f i c i a l . In essence, a r t i f i c i a l i n s e m i n a t i o n i n v o l v e s e x t r a c t i n g spermatophores from a male, suspending these spermatophores i n a s a l i n e s o l u t i o n , drawing the suspension i n t o a micro-p i p e t t e , and i n j e c t i n g i t i n t o the female r e p r o d u c t i v e t r a c t . The use of a r t i f i c i a l i n s e m i n a t i o n i n s t u d i e s of p o e c i l i i d g e n e t i c s and r e p r o d u c t i o n i s not new. , C l a r k (1950) f i r s t d e s c r i b e d a simple procedure f o r i n s e m i n a t i n g Xiphophorus faelleri. In C l a r k ' s technigue, spermatophores were simply suspended i n a 0.8 % NaCl s o l u t i o n , and then, i n j e c t e d i n t o the female. B i l l a r d (1966) d e s c r i b e d a s i m i l a r t e c h i n i g u e f o r a r t i f i c i a l l y i n s e m i n a t i n g P o e c i l i a r e t i c u l a t a . However, s i n c e B i l l a r d inseminated female guppies with known amounts of sperm, h i s technigue was more complex than C l a r k ' s . B i l l a r d suspended spermatophores i n F l i c k i n g e r ' s s o l u t i o n t o preserve spermatophore i n t e g r i t y and measured the number of spermatophores i n h i s i n s e m i n a t i o n s v o l u m e t r i c a l l y with a s p e c i a l l y designed p i p e t t e . , My study a l s o r e g u i r e d known numbers of spermatophores i n i n s e m i n a t i o n s . However, r a t h e r than measure q u a n t i t i e s of spermatophores v o l u m e t r i c a l l y , I counted spermatophores. Not only d i d counting spermatophores g i v e a more accurate measure of spermatophore number, but i t a l s o made i t e a s i e r to mix spermatophores from d i f f e r e n t males. 10 a. ) Obtaining s | e r j from males. Before s t r i p p i n g spermatophores from a male gappy, the male was a n a e s t h e t i z e d i n a 300 mg/1 s o l u t i o n of MS 222, The time i t took f o r the a n a e s t h e t i c t o have e f f e c t v a r i e d , but I u s u a l l y waited j u s t u n t i l the male stopped b r e a t h i n g . Guppies are extremely hardy and o n l y a few males died from t h i s treatment. Once a n a e s t h e t i z e d , the male was placed on a w e l l - s l i d e so that the base of the gonopodium was j u s t i n s i d e the w e l l . I then viewed the gonopodium under a d i s s e c t i n g microscope. The w e l l of the s e l l - s l i d e c o n tained a 0.8% NaCl s o l u t i o n and served as a c o l l e c t i n g v e s s e l f o r spermatophores. Holding the a n t e r i o r h a l f of the male with thumb and f o r e -f i n g e r of one hand, i t s gonopodium was swung forward ( P l a t e 1A) and h e l d i n p l a c e by s l i g h t l y r o t a t i n g the male so that the gonopodium was pressed a g a i n s t the s l i d e . With a b l u n t d i s s e c t i n g needle, the t e s t i c u l a r r e g i o n , j u s t a n t e r i o r to the anus, was r e p e a t e d l y stroked p o s t e r i o r l y u n t i l spermatophores stopped f l o w i n g i n t o the w e l l of the w e l l - s l i d e . When no more spermatophores were a v a i l a b l e , the male was c a r e f u l l y removed from the s l i d e and placed i n f r e s h aguarium water to recover from the a n a e s t h e t i c . The w e l l of the w e l l - s l i d e was then topped up with the 0.8% NaCl s o l u t i o n . Since males that recovered from the a n a e s t h e t i c before the s t r i p p i n g was complete became , i m p o s s i b l e to handle, t h i s s t r i p p i n g procedure had to be done g u i c k i y . Although B i l l a r d (196 6) found t h a t spermatophores 11 d i s s o c i a t e d i n a 0.8? NaCl s o l u t i o n , i n my a r t i f i c a l i n s e m i n a t i o n s , spermatophores u s u a l l y remained i n t a c t i n 0. 8% NaCl. P o s s i b l y , sperm s u r v i v e i n some s a l i n e s b e t t e r than i n o t h e r s , but u n f o r t u n a t e l y , t h i s has not been i n v e s t i g a t e d f o r guppy sperm. I used the. NaCl s o l u t i o n s i n c e i t was easy t o prepare. b.) Insemination of the female. , I t was e s s e n t i a l t o work through t h i s p o r t i o n of the procedure as q u i c k l y as p o s s i b l e . , although spermatophores g e n e r a l l y remained i n t a c t i n ,0*9% NaCl s o l u t i o n f o r up to ten minutes, sometimes they began d i s s o l v i n g soon a f t e r being removed from the male. When t h i s o c c u r r e d , the spermatophores e i t h e r d i s s o l v e d before they c o u l d be counted or they stuck t o the glassware. Once spermatophores were c o l l e c t e d i n the well of the w e l l - s l i d e , the d e s i r e d number of spermatophores was counted. F i r s t , a m i c r o - p i p e t t e was f a s h i o n e d by h e a t i n g and drawing out a F i s h e r b r a n d micro-hematocrit tube {1.2 mm 1. D.) and breaking the t a p e r e d end t c produce an opening approximately 0.5 ma i n diameter. t h i s m i c r o - p i p e t t e was then a t t a c h e d with a pi e c e o f Intramedic p o l y e t h y l e n e t u b i n g (0. 965 mm I.D.) to a -1.ee p l a s t i c s y r i n g e ( P l a t e 1B) . By sl o w l y withdrawing the plunger of the s y r i n g e , spermatophores were drawn i n t o the m i c r o - p i p e t t e and counted one at a time. 12 Hhen the r e q u i r e d number o f spermatophores were drawn i n t o t h e m i c r o - p i p e t t e , the m i c r o - p i p e t t e was detached from the s y r i n g e and held v e r t i c a l l y so t h a t the spermatophores would s e t t l e i n t o the tapered end. O c c a s i o n a l l y , l i g h t tapping of the m i c r o - p i p e t t e was necessary before the spermatophores would s e t t l e . Once the spermatophores had s e t t l e d , they were t r a n s f e r r e d t o a modified m i c r o - c a p i l l a r y tube. This tube was f a s h i o n e d by heating and drawing out one end of a 10 u l Drummond m i c r o - c a p i l l a r y tube, breaking the t i p of the tapered end, and then, f i r e p o l i s h i n g the broken t i p . The diameter of the opening at the tapered end of the tube was approximately 0.35 mm (Plate 1C). Spermatophores i n s a l i n e were t r a n s f e r r e d by c a p i l l a r y a c t i o n t o the m i c r o - c a p i l l a r y tube by g u i c k i y touching the tapered ends of the mi c r o - p i p e t t e and the m i c r o - c a p i l l a r y tube. With p r a c t i c e i t was p o s s i b l e t o t r a n s f e r the spermatophores i n 2 to 3 p i of s a l i n e s o l u t i o n . Next, the m i c r c - c a p i l l a r y tube, c o n t a i n i n g spermatophores, was held v e r t i c a l l y so t h a t the spermatophores again s e t t l e d t o the tapered end., The tube was then l i g h t l y tapped to d i s l o d g e spermatophores s t i c k i n g to the s i d e . As soon as the spermatophores were s e t t l e d the tube was s e t a s i d e u n t i l a female was ready to be inseminated. Females were a n a e s t h e t i z e d the same way males were and then placed on t h e i r backs onto a p i e c e o f damp cheese c l o t h . Again i t was necessary to do the i n s e m i n a t i o n with the a i d of a d i s s e c t i n g microscope. The m i c r o - c a p i l l a r y 13 tube was attached to the rubber d i s p e n s o r bulb s u p p l i e d with the tubes and i n s e r t e d , t a pered end f i r s t , i n t o the female r e p r o d u c t i v e duct ( P l a t e 1D). The spermatophores and s a l i n e were s l o w l y e j e c t e d from the m i c r o - c a p i l l a r y tube by squeezing the bulb. As soon as the tube was evacuated, i t was q u i c k l y withdrawn and the female was p l a c e d i n f r e s h aguarium water to recover from the a n a e s t h e t i c . Aside from the obvious advantage of the m i c r o - c a p i l l a r y being s m a l l enough to f i t the r e p r o d u c t i v e t r a c t of female guppies, i t o f f e r e d s e v e r a l other advantages...„ Since the volume of the tube was very s m a l l , the volume of f l u i d drawn i n t o the m i c r o - c a p i l l a r y tube, and the i n j e c t i o n of spermatophores i n t o the female, c o u l d be p r e c i s e l y c o n t r o l l e d . Drawing the spermatophores i n t o the micro-c a p i l l a r y tube by c a p i l l a r y a c t i o n r a t h e r than by s u c t i o n a l s o meant more p r e c i s e c o n t r o l i n t r a n s f e r r i n g spermatophores from the m i c r o - p i p e t t e to the m i c r o - c a p i l l a r y tube. I attempted to keep the volume of spermatophore suspension to a minimum to prevent damage t o the female and to a v o i d washing the spermatophores back out of the r e p r o d u c t i v e t r a c t , but 2 p i was the s m a l l e s t volume t h a t c o u l d be achieved., O c c a s i o n a l l y , females were inseminated with . spermatophores i n approximately 5 p i of s a l i n e . However, t h i s d i d not appear to a f f e c t female s u r v i v a l nor the success of the i n s e m i n a t i o n . 14 P l a t e 1: A) Male guppy with gonopodium p a r t i a l l y e r e c t e d . B) S y r i n g e , p o l y e t h y l e n e t u b i n g , and modified micrc-hematocrit tube f o r counting spermatophores. C) Mo d i f i e d m i c r o - c a p i l l a r y tube with tapered end. The white clump at the t i p of the tube i s a clump of spermatophores. D) I n t r o d u c t i o n of the m i c r o - c a p i l l a r y tube i n t o the female r e p r o d u c t i v e duct. 16 S e c t i o n I The r e l a t i o n s h i p between the p r o p o r t i o n of g e n e t i c a l l y d i s t i n c t sperm i n mixed i n s e m i n a t i o n s and the r e l a t i v e c o n t r i b u t i o n of those sperm to broods. I n t r o d u c t i o n Hildemann and Sagner {1951) and Binge (1937) i n v e s t i g a t e d i n t r a s p e c i f i c sperm c o m p e t i t i o n i n P o e c i l i a r e t i c u l a t a . In t h e i r s t u d i e s females were mated and then, a f t e r the f i r s t brood, remated and the p a t e r n i t y of subseguent broods determined. Binge (1937) di d not d e s c r i b e h i s mating procedure, but i n a l l Hildemann*.s and Wagner's t r i a l s , females homozygous f o r x a n t h i c , mutant, autosomal, r e c e s s i v e a l l e l e s were f i r s t mated with males c a r r y i n g the same a l l e l e s and then remated with males homozygous f o r a dominant a l l e l e ( u s u a l l y the • a l l e l e ) . . As a conseguence of t h e i r e x p e r i m e n t a l design, i t i s not p o s s i b l e t o determine i f the precedence of the second i n s e m i n a t i o n i s due t o the sequence of i n s e m i n a t i o n s c a r r y i n g the two a l l e l e s or t o the f a c t t h a t the second i n s e m i n a t i o n was made with sperm c a r r y i n g a dominant a l l e l e a t the marker l o c u s . Perhaps the genotype of the second i n s e m i n a t i o n determined i t s s u c c e s s . One of the aims of my study i s to e s t a b l i s h whether or not a l l e l i c d i f f e r e n c e s i n sperm a f f e c t f e r t i l i z a t i o n s uccess. A second aim of t h i s p o r t i o n of the study i s to e s t a b l i s h the r e l a t i o n s h i p between the p r o p o r t i o n of a 17 male's sperm i n the t o t a l ovary sperm p o o l and h i s c o n t r i b u t i o n to a brood, My f i r s t h ypothesis s t a t e s t h a t the r e d u c t i o n i n the number of sperm from the f i r s t i n s e m i n a t i o n which occurs between i n s e m i n a t i o n s g i v e s sperm from a l a t e r i n s e m i n a t i o n precedence. A necessary c o r o l l a r y of t h i s h y p o t h e s i s i s t h a t when sperm from two males are used i n the same i n s e m i n a t i o n , the r e l a t i v e success of the two males' sperm i s a f u n c t i o n of the numbers of each male's sperm. The experiments i n t h i s p o r t i o n of the study are designed to answer both q u e s t i o n s . By a r t i f i c i a l l y i n s e m i n a t i n g females with varying p r o p o r t i o n s of qd and * sperm, not only have I s i m u l a t e d simultaneous i n s e m i n a t i o n s by two males, but I can a l s o assess the e f f e c t of male genotype on sperm s u c c e s s . Methods and M a t e r i a l s A l l i n s e m i n a t i o n s i n t h i s s e c t i o n were a r t i f i c i a l . Each i n s e m i n a t i o n contained the spermatophores of both qd/qd and males. , Spermatophores were obtained from each male and kept separate i n w e l l - s l i d e s . Using a s y r i n g e , the a p p r o p r i a t e number of spermatophores from each male were drawn i n t o a m i c r o - p i p e t t e {produced from a hematocrit tube) and the e n t i r e i n s e m i n a t i o n t r a n s f e r r e d t o a 10 u l micro-c a p i l l a r y tube. Each c o n t a i n e d a t o t a l of 100 spermatophores, but with mixtures of 25:75, 50:50, and 75:25 of 3 d : + spermatophores. 18 V i r g i n a^d/qd females, were inseminated once with one of the sperm mixtures. Twenty s i x i n s e m i n a t i o n s were mixed 25:75; 26 i n s e m i n a t i o n s were mixed 50:50; and 18 i n s e m i n a t i o n s were mixed 75:25. These females were then i s o l a t e d i n 20 l i t r e g l a s s a q u a r i a . The young from c o n s e c u t i v e broods were c o l l e c t e d as scon a f t e r b i r t h as p o s s i b l e and counted and scored f o r phenotype. Since females normally g i v e b i r t h a t n i g h t , the young were not removed from the f e m a l e 1 s tank u n t i l morning. I t i s p o s s i b l e t h a t the females ate some of t h e i r young, but un l e s s t h i s p r e d a t i o n i s s e l e c t i v e , i t i s u n l i k e l y that t h i s e f f e c t e d the r e s u l t s . The young born d u r i n g the day appeared a l e r t with a w e l l developed escape response, and at no time d i d I observe a female eat any of her o f f s p r i n g . In an attempt t o prevent females c a n n i b a l i z i n g t h e i r o f f s p r i n g , I t r i e d using breeding t r a p s , but females d i d not do w e l l i n them. Using p l a n t s i n the tank to provide the young with cover o n l y proved to be a hindrance when removing young. Once the young were s c o r e d , the two phenotypes were kept s e p a r a t e l y i n 40 l i t r e g l a s s a q u a r i a and used f o r f u t u r e experiments. Res u l t s Table 1 summarizes the r e s u l t s c f the t h r e e s e t s of a r t i f i c i a l i n s e m i n a t i o n s . I have presented the number of young of each phenotype i n a l l the f i r s t broods and then 19 Table 1 : The number c f progeny of each genotype i n the f i r s t broods of females inseminated with three d i f f e r e n t p r o p o r t i o n s of gd sperm. F r e q u e n c y o f gd_ a l l e l e N o . o f f e m a l e s F e m a l e i n t h e i n s e m i n a t i o n i n s e m i n a t e d 1 . 2 . 3 . 0 . 2 5 26 4 . 5 . 6 . t o t a l 1 . 2 . 3 . 4 . 5 . 6 . 7 . 8 . 9 . t o t a l 0 . 5 0 26 1 . 2 . 3 . 4 . 0 . 7 5 18 5 . 6 . 7 . 8 . t o t a l . o f g d / g d p r o g e n y N o . o f + / g ^ p r o g e n y % o f f e m a l e s g i v i n g b i r t h 9 7 1 5 2 2 5 19 10 41 8 20 3 5 9 4 16 3 4 3 8 20 7 27 5 8 10 9 11 11 20 3 4 3 1 100 100 42 11 3 0 23 20 4 1 13 1 12 3 1 4 8 0 106 40 35 21 pooled o f f s p r i n g numbers t c produce a t o t a l f o r each treatment group. Table 1 i n d i c a t e s c o n s i d e r a b l e v a r i a b i l i t y i n phenotype f r e q u e n c i e s f o r a l l broods w i t h i n each treatment group. T h i s v a r i a b i l i t y was highest when i n s e m i n a t i o n s c o n t a i n e d equal numbers of spermatophores from qd/qd and •/* males. Females inseminated with other p r o p o r t i o n s of the two sperm types a l s o produced broods with h i g h l y v a r i a b l e phenotype f r e q u e n c i e s , but there was a c l e a r tendency f o r the a l l e l e of the most abundant sperm i n an i n s e m i n a t i o n t o predominate i n each brood. A measure of h e t e r o g e n e i t y i n these data can be c a l c u l a t e d u sing a G - s t a t i s t i c , but s i n c e brood s i z e s are s m a l l , i t s value i s q u e s t i o n a b l e . , As a r e s u l t cf s m a l l brood s i z e s , data i n each treatment group were pooled and then compared to the expected phenotype f r e q u e n c i e s ( i . e . the three a l l e l i c f r e q u e n c i e s i n the i n s e m i n a t i o n s ) . The +/qd frequency i n broods from i n s e m i n a t i o n s c o n t a i n i n g jgd a l l e l e f r e q u e n c i e s of .25, .50, and .75 are .27, .50, and .73. Table 2 p r e s e n t s a s t a t i s t i c a l comparison c f the qd a l l e l e f r e q u e n c i e s i n the i n s e m i n a t i o n s with the +/gd f r e q u e n c i e s i n the broods. The value of G f o r each treatment qroup i s not s i g n i f i c a n t at the 0.05 l e v e l . although 70 females were inseminated i n t h i s p o r t i o n of the study, only 23 gave b i r t h . Table 1 r e p o r t s the percentage of s u c c e s s f u l a r t i f i c i a l i n s e m i n a t i o n s i n each treatment group. . These data suggest that i n s e m i n a t i o n s with predominantly <jd sperm were the most s u c c e s s f u l . . D e s p i t e 22 Table 2 : A comparison between the gd/gd frequency i n the broods and the qd a l l e l e frequency i n the i n s e m i n a t i o n s f o r each treatment group. P r o p o r t i o n o f g_d/gd_ P r o p o r t i o n o f gd a l l e l e p r o g e n y i n p o o l e d i n t h e i n s e m i n a t i o n s b r o o d s 0 . 2 7 0 . 5 0 0 . 7 3 V a l u e s o f G n o t 0 . 2 5 0 . 5 0 0 . 7 5 s i g n i f i c a n t a t p - ^ 0 . 0 5 24 t h i s d i f f e r e n c e i n a r t i f i c i a l i n s e m i n a t i o n success between treatment groups, peeled values i n a l l treatment group are sm a l l e r than expected. U n f o r t u n a t e l y , time and space d i d not permit me to re-do the experiment. D i s c u s s i o n The 1:1 r e l a t i o n s h i p observed between the freguency of gd a l l e l e i n the i n s e m i n a t i o n s and the " g o l d " phenotype freguency i n the broods i n d i c a t e s t h a t : 1.) a l l e l i c d i f f e r e n c e s i n sperm d i d not i n f l u e n c e the r e s u l t s , and 2.) when the e f f e c t s of a time l a g between two i n s e m i n a t i o n s were removed, male success was determined by the p r o p o r t i o n of h i s sperm i n the sperm pool. Both c o n c l u s i o n s are important because they s a t i s f y the c o n d i t i o n s necessary f o r using the 3 d a l l e l e as a g e n e t i c marker and f o r proposing f u r t h e r hypotheses. although the r e l a t i o n s h i p i s c l e a r when broods w i t h i n treatment groups are pooled, the phenotype f r e q u e n c i e s i n each brood are h i g h l y v a r i a b l e . T h i s i s probably i n p a r t due t o the s m a l l s i z e of most broods. Small d i f f e r e n c e s i n the number of each phenotype between s m a l l broods t r a n s l a t e i n t o s u b s t a n t i a l d i f f e r e n c e s i n the p r o p o r t i o n of each phenotype. U n f o r t u n a t e l y i t i s u n l i k e l y t h a t l a r g e r broods can be obtained. Binge (1927) noted t h a t with v i r g i n females the f i r s t brood tended to be s m a l l and t h i s appears to be the case i n t h i s , and my o t h e r , experiments. Undoubtedly the a r t i f i c i a l i n s e m i n a t i o n technique i s 25 another source of v a r i a b i l i t y i n brood phenotype f r e q u e n c i e s , Spermatophores c o u l d not be o b t a i n e d from the males s i m u l t a n e o u s l y , and as a r e s u l t , spermatophores from one male sere suspended i n s a l i n e s o l u t i o n l o n g e r than t h o s e of the other male. T h i s may have produced d i f f e r e n c e s i n the v i a b i l i t y of sperm from the two males. In a d d i t i o n , i t was t e c h n i c a l l y d i f f i c u l t t o mix the spermatophores from the two males before an i n s e m i n a t i o n . Because of these two f a c t o r s , t h e males 1 sperm may not have been d i s t r i b u t e d randomly i n the i n s e m i n a t i o n , and thus f e r t i l i z a t i o n by the d i f f e r e n t sperm may not have been random. / S t u d i e s on the g e n e t i c s of other a l l e l e s i n P o e c i l i a r e t i c u l a t a show phenotype freguency v a r i a b i l i t y between i n d i v i d u a l c r o s s e s (Hinge, 1927). U n f o r t u n a t e l y , pooled data were used to e s t a b l i s h the g e n e t i c s of the jgd a l l e l e (Goodrich e t a l , 1944) and t h e r e f o r e : an independant measure of phenotype freguency v a r i a b i l i t y between i n d i v i d u a l broods a f t e r n a t u r a l i n s e m i n a t i o n s i s u n a v a i l a b l e . , Since the problem of s m a l l brood s i z e s i s compounded by e r r o r s i n t r o d u c e d by a r t i f i c i a l i n s e m i n a t i o n , a n a l y s i s of d i f f e r e n c e s between broods would be d i f f i c u l t t o i n t e r p r e t . The importance of e s t a b l i s h i n g the e f f e c t of sperm genotype on sperm success i n t h i s study cannot be overlooked. I t i s e n t i r e l y p o s s i b l e that i n c o m p e t i t i v e s i t u a t i o n s , s m a l l a l l e l i c d i f f e r e n c e s may c o n f e r d i f f e r e n t i a l s u c c e s s upon sperm. An example i s the x a n t h i c mutant phenotype, "Cream", i n P o e c i l i a r e t i c u l a t a . "Cream" i s c o n t r o l l e d by r e c e s s i v e , autosomal a l l e l e s a t two l o c i 26 and has been used as a g e n e t i c marker i n mating s t u d i e s (Haskins and Haskins, 1950; Hildemann and Hagner, 1954). T h i s phenotype, however, c h a r a c t e r i s t i c a l l y shows reduced v i a b i l i t y and consequently the sperm c a r r y i n g the a l l e l e or the r e s u l t i n g h y b r i d zygotes may be l e s s v i a b l e than normal genotypes. Prout and Bungaard (1S77) have shown that simple g e n e t i c d i f f e r e n c e s i n D r o s o p h i l a melanoqaster can a f f e c t sperm s u c c e s s i n c o m p e t i t i v e s i t u a t i o n s . Of the 70 females inseminated, only 23 gave b i r t h . T h i s low success i n d i c a t e s t h a t a r t i f i c i a l i n s e m i n a t i o n i s not completely e f f e c t i v e . Other workers ( B i l l a r d , 1966; C l a r k , 1950; L i l e y , pers. comm.) have a l s o r e p o r t e d low success with a r t i f i c i a l i n s e m i n a t i o n s i n p o e c i l i i d s . Low success with a r t i f i c i a l i n s e m i n a t i o n may be the r e s u l t of two f a c t o r s . Since spermatophores were t r a n s m i t t e d i n a p h y s i o l o g i c a l s a l i n e s o l u t i o n , the composition cf the s o l u t i o n may have a f f e c t e d sperm v i a b i l i t y . ; C l a r k (1950) used a 0.8% H a d s o l u t i o n and r e p o r t e d 60% success with Xiphcphorus, another genus of p o e c i l i i d . To maintain the i n t e g r i t y of spermatophores while i n s e m i n a t i n g guppies, B i l l a r d (1966) used F l i c k i n g e r p h y s i o l o g i c a l s a l i n e and r e p o r t e d l e v e l s of success comparable to C l a r k ' s . G ardiner (1976) used a b u f f e r e d s a l i n e t o study sperm storage i n Cymatoqaster aqgregata. He i n d i c a t e d t h a t pH was c r i t i c a l to sperm s u r v i v a l . The a c t u a l i n s e m i n a t i o n may a l s o have c o n t r i b u t e d to the low s u c c e s s . I n s e r t i o n of a m i c r o - p i p e t t e i n t o the female r e p r o d u c t i v e system may damage the system or i n some other 27 way upset the female's r e p r o d u c t i v e p h y s i o l o g y . Ia a d d i t i o n t c these p o s s i b l e disadvantages i n a r t i f i c i a l i n s e m i n a t i o n , the r e p r o d u c t i v e c o n d i t i o n of the females may be r e s p o n s i b l e f o r some of the l a c k of i n s e m i n a t i o n s u c c e s s . L i l e y (1968) p o s t u l a t e d an endogenous r e p r o d u c t i v e c y c l e c o n t r o l l i n g egg maturation and egg degeneration i n v i r g i n females which had a p e r i o d of approximately 30 days ( s i m i l a r to the p e r i o d of the non-v i r g i n female brood c y c l e ) . I f such a r e p r o d u c t i v e c y c l e occurs i n v i r g i n females, i n s e m i n a t i o n s when mature eggs are beginning to degenerate and the next batch of oocytes are beginning to mature would mean delayed f e r t i l i z a t i o n f o r one r e p r o d u c t i v e c y c l e ( i . e . i f these females were capable of s t o r i n g sperm) and subsequent delay i n the production of a brood. Thus, I may have d i s c a r d e d some females before they gave b i r t h . However, I cannot exclude the p o s s i b i l i t y t h a t some females i n t h i s experiment were i n f e r t i l e . The d i f f e r e n c e i n i n s e m i n a t i o n s u c c e s s between treatment groups suggests t h a t gd and + sperm may d i f f e r i n v i a b i l i t y or v i g o r . T h i s s u g g e s t i o n c o n t r a d i c t s the brood phenotype freguency data from each treatment group, but the f a c t t h at i n s e m i n a t i o n success was g r e a t e s t when i n s e m i n a t i o n s contained predominantly gd spermatophores appears t c i n d i c a t e t hat gd sperm are more v i g o r o u s than + sperm. The r e l a t i o n between i n s e m i n a t i o n success and the p r o p o r t i o n of gd sperm i n i n s e m i n a t i o n s a l s o c o n t r a d i c t s data i n the experiments i n the f o l l o w i n g s e c t i o n s . The data i n these experiments suggest that + . sperm may be more 28 vigorous than qd sperm. T h i s i n c o n s i s t e n c y i n the r e l a t i v e successes of and + sperm i s d i s c u s s e d i n the next s e c t i o n . Although refinement of the a r t i f i c i a l i n s e m i n a t i o n technique may i n c r e a s e i t s e f f e c t i v e n e s s , most l i k e l y , l a r g e numbers of i n s e m i n a t i o n s would have to be performed to generate moderate sample s i z e s . . I n t h i s study, doubling or t r i p l i n g the number of i n s e m i n a t i o n s would have r e q u i r e d much more time and space than was a v a i l a b l e . .,. 2 9 Section I I The e f f e c t of spermatophore number i n i n s e m i n a t i o n s on the success of r e -i n s e m i n a t i o n s . I n t r o d u c t i o n L i t t l e can be i n f e r r e d from p r e v i o u s s t u d i e s of i n t r a s p e c i f i c sperm co m p e t i t i o n i n guppies {Hildemann and Hagner, 1954; Hinge, 1937) about the nature of i n t e r a c t i o n s between sperm from d i f f e r e n t i n s e m i n a t i o n s or t h e phenomenon sperm precedence. In these s t u d i e s , males and females were kept together f o r up to twenty days. , Dnder such circumstances i t i s i m p o s s i b l e to estimate both the amount of sperm i n t r o d u c e d a t each i n s e m i n a t i o n and the number of i n s e m i n a t i o n s performed by each male. Consequently the i n f l u e n c e of the amount of sperm i n t r o d u c e d by a male on that male's success i s not known. The o b s e r v a t i o n that a second i n s e m i n a t i o n has precedence i n f e r t i l i z i n g eggs over the f i r s t i n s e m i n a t i o n can be e x p l a i n e d by a d i s p a r i t y i n the number of sperm a v a i l a b l e f o r f e r t i l i z i n g egqs from each i n s e m i n a t i o n . Sperm from the second i n s e m i n a t i o n probably outnumber sperm from the f i r s t i n s e m i n a t i o n f o r a number of reasons, but the most obvious i s a r e d u c t i o n through e i t h e r f e r t i l i z a t i o n of an e a r l i e r brood or storage of o n l y a p o r t i o n of the sperm from the f i r s t i n s e m i n a t i o n . The d i r e c t r e l a t i o n s h i p observed i n the p r e v i o u s s e c t i o n < between the p r o p o r t i o n a l 30 r e p r e s e n t a t i o n of a male's sperm i n the t o t a l sperm pool and h i s success i n c o n t r i b u t i n g to a brood makes the sperm number hypothesis appear reasonable, k number of a r t i f i c i a l i n s e m i n a t i o n experiments sere designed to t e s t the h y p o t h e s i s . The hypothesis p r e d i c t s that when the s i z e of the f i r s t and second i n s e m i n a t i o n s are e q u a l , any d i s p a r i t y between the number of sperm from each, i n s e m i n a t i o n a v a i l a b l e t o f e r t i l i z e the second brood w i l l be due t c a r e d u c t i o n i n the number of sperm from the f i r s t i n s e m i n a t i o n . Note, however, that t h e r e may be t h r e s h o l d e f f e c t s . I f the s i z e of i n s e m i n a t i o n s are so l a r g e t h a t the ovary i s , , s a t u r a t e d , , with, sperm, the second i n s e m i n a t i o n may not have precedence simply because enouqh sperm s t i l l remain from the f i r s t i n s e m i n a t i o n t o do an e f f e c t i v e job* , The purpose of the present s e c t i o n i s to t e s t t h i s p r e d i c t i o n . The procedure i s s i m i l a r t o t h a t of Hildemann and Hagner (1954), but s i n c e the i n s e m i n a t i o n s are a r t i f i c i a l the amount of sperm i s known* Methods and M a t e r i a l s Three groups of v i r g i n g o l d females were a r t i f i c i a l l y inseminated with 40, 100, and 200 spermatophores. These q u a n t i t i e s were chosen t o produce a l a r g e range i n the s i z e of the ovary sperm p o o l . , B i l l a r d <1966) demonstrated t h a t the q u a n t i t y of sperm i n an i n s e m i n a t i o n a f f e c t s the number of o f f s p r i n g per brood and the number of broods a female 31 produces. I n h i s study females inseminated with 40 or fewer spermatophores produced s u c c e s s i v e l y s m a l l e r broods. Successive broods produced a f t e r i n s e m i n a t i o n s with 100 spermatophores, on the other hand, were l a r g e r and d i d not s i g n i f i c a n t l y change i n s i z e . These o b s e r v a t i o n s i n d i c a t e that the amount of s t o r e d sperm from i n s e m i n a t i o n s c o n t a i n i n g 100 spermatophores does not d i m i n i s h as n o t i c e a b l y as i t does f o r s m a l l e r i n s e m i n a t i o n s . A f t e r d o ubling the s i z e of E i l l a r d ' s l a r g e s t i n s e m i n a t i o n , I assumed sperm l e v e l s i n the ovary were s u f f i c i e n t l y high t o reduce the d i f f e r e n c e i n the number of f i r s t and second i n s e m i n a t i o n sperm a v a i l a b l e f o r f e r t i l i z a t i o n o f the second brood. . Each female was kept i n a 20 l i t r e g l a s s aquarium u n t i l she gave b i r t h to her f i r s t brood. The young were c o l l e c t e d from the aguarium as soon a f t e r b i r t h as p o s s i b l e , counted, and then e i t h e r d i s c a r d e d , or i f they were the gold phenotype, saved f o r f u t u r e use. The female was then immediately re-inseminated (the second insemination) with the same amount of sperm as used i n the f i r s t i n s e m i n a t i o n . . Since females u s u a l l y give b i r t h a t n i g h t , r e -i n s e m i n a t i o n u s u a l l y d i d not immediately f o l l o w b i r t h . Although t h i s time l a p s e between b i r t h and r e - i n s e m i n a t i o n c o u l d not be a c c u r a t e l y known, i t never exceeded 24 hours. The p e r i o d of time between b i r t h and the f e r t i l i z a t i o n of the next brood has been r e p o r t e d t o be from s i x to twelve days ( D i l d i n e , 1936; Rosenthal, 1952; Turner, 1937)., I f t h i s i s so, r e - i n s e m i n a t i o n i n my experiments occurred w e l l 32 before completion of f e r t i l i z a t i o n of the next brood. Females were returned to t h e i r tanks a f t e r r e - i n s e m i n a t i o n and the o f f s p r i n g of the second broods were counted and t h e i r p a t e r n i t y determined. To determine how much each i n s e m i n a t i o n c o n t r i b u t e d to the second brood, one of the i n s e m i n a t i o n s c a r r i e d the ad a l l e l e . , H i t h i n the three treatment groups, some of the females r e c e i v e d the g e n e t i c a l l y marked sperm i n the f i r s t i n s e m i n a t i o n and others r e c e i v e d the marked sperm i n the second i n s e m i n a t i o n . T h i s was done to c o n t r o l f o r the p o s s i b i l i t y t h a t the sperm genotype a f f e c t s sperm s u c c e s s . The schedule of i n s e m i n a t i o n s and number of i n s e m i n a t i o n s are presented i n Table 3. Not a l l females were inseminated a second time. Some die d before p a r t u r i t i o n , and others ( e i t h e r because of i n f e r t i l i t y or a poor insemination) d i d not give b i r t h . Since the average time between the f i r s t a r t i f i c i a l i n s e m i n a t i o n and the f i r s t brood was 36 days, females t h a t d i d not give b i r t h within 50 days were d i s c a r d e d from the experiment. Res u l t s Again I experienced d i f f i c u l t y o b t a i n i n g an adequate sample s i z e by using a r t i f i c i a l i n s e m i n a t i o n . Table 3 presents the number of females i n i t i a l l y inseminated, the 3 3 Table 3 ; The number of females inseminated at the beginning of the experiment, the number of females re-inseminated, and the number re-inseminated females that gave b i r t h a second time.,. Values for the two insemination sequences i n each treatment group are separated. T r e a t m e n t g r o u p I n s e m i n a t i o n s e q u e n c e N o . o f f e m a l e s i n s e m i n a t e d A gd_;+ 26 ( i n s e m . w i t h 40 s p e r m a t o p h o r e s ) +;gd_ 21 B gd_;+ 27 ( i n s e m . w i t h 100 s p e r m a t o p h o r e s ) +;gd_ 17 C gd_;+ 24 ( i n s e m . w i t h 200 s p e r m a t o p h o r e s ) +;g_d_ 24 N o . o f f e m a l e s N o . o f r e - i n s e m i n a t e d f e m a l r e - i n s e m i n a t e d g i v i n g b i r t h 5 9 7 8 3 6 4 5 9 13 6 6 35 number of females which gave b i r t h and were re-inseminated, and the number of females which gave b i r t h a f t e r the second i n s e m i n a t i o n . Table 1 giv e s the percentage of s u c c e s s f u l i n s e m i n a t i o n s . The o v e r a l l success of a r t i f i c i a l i n s e m i n a t i o n was low, but success v a r i e d between some groups of i n s e m i n a t i o n s . Insemination of v i r g i n females with gd sperm tended t o be l e s s s u c c e s s f u l than i n s e m i n a t i o n with + sperm. However, the success of these i n s e m i n a t i o n s with: .gd sperm d i d i n c r e a s e as the number of spermatophores i n i n s e m i n a t i o n s i n c r e a s e d . The success of the f i r s t i n s e m i n a t i o n with • sperm a l s o i n c r e a s e d s l i g h t l y as the number of spermatophores i n i n s e m i n a t i o n s i n c r e a s e d . The success of second i n s e m i n a t i o n s was g r e a t e r than the success o f f i r s t i n s e m i n a t i o n s . In a d d i t i o n , the success of i n s e m i n a t i o n s with .gd or + sperm, was s i m i l a r . T h i s d i f f e r e n c e i n success between f i r s t and second i n s e m i n a t i o n s may simply be the r e s u l t of s e l e c t i o n f o r f e r t i l e females. The v i r g i n females used i n t h i s experiment may not a l l have been f e r t i l e , but s i n c e only females which had given b i r t h were re-i n s e m i n a t e d , only f e r t i l e females r e c e i v e d the second i n s e m i n a t i o n . Table 5 presents the number of gd/gd and »/qd o f f s p r i n g i n f i r s t and second broods of a l l females s u c c e s s f u l l y inseminated t w i c e . The order of i n s e m i n a t i o n with g_d and + sperm, as w e l l as the number of spermatophores used i n i n s e m i n a t i o n s , are a l s o i n d i c a t e d . In a l l c a s e s , second i n s e m i n a t i o n s had precedence over f i r s t i n s e m i n a t i o n s i n c o n t r i b u t i n g to the second brood. The degree of precedence 36 Table 4 : The percentage of s u c c e s s f u l i n s e m i n a t i o n s and r e - i n s e m i n a t i o n s f o r each i n s e m i n a t i o n sequence i n the t h r e e treatment groups. T r e a t m e n t g r o u p I n s e m i n a t i o n % o f t o t a l f e m a l e s s e q u e n c e r e - i n s e m i n a t e d A g d j + 1 9 . 2 ( i n s e m . w i t h 40 s p e r m a t o p h o r e s ) +;gd_ 4 2 . 8 B g d ; + 2 5 . 9 ( i n s e m . w i t h 100 s p e r m a t o p h o r e s ) +;cjd_ 4 7 . 1 C 3d_;+ 3 7 . 5 ( i n s e m . w i t h 200 s p e r m a t o p h o r e s ) + ;gd_ 5 4 . 2 % o f r e - i n s e m i n a t i o n s % o f t o t a l f e m a l e s g i v i n g r e s u l t i n g i n b i r t h b i r t h a f t e r r e - i n s e m i n a t i o n 6 0 . 0 6 6 . 7 5 7 . 1 6 2 . 5 6 6 . 7 4 6 . 2 1 1 . 5 2 8 . 6 1 4 . 8 2 9 . 4 2 5 . 0 2 5 . 0 38 Table 5: The number of +/3<l and gd/qd progeny i n f i r s t and second broods produced by females i n the three treatment groups. T r e a t m e n t g r o u p A ( i n s e m . w i t h 40 s p e r m a t o p h o r e s ) B ( i n s e m . w i t h 100 s p e r m a t o p h o r e s ) C ( i n s e m . w i t h 200 s p e r m a t o p h o r e s ) F e m a l e 1 . 2 . 3 . 4 . 5 . 6 . 7 . 8 . 9 . 1 . 2 . 3 . 4 . 5 . 6 . 7 . 8 . 9 . 1 . 2 . 3 . 4 . 5 . 6 . 7 . 8 . 9 . 1 0 . 1 1 . 1 2 . F i r s t B r o o d S e c o n d B r o o d I n s e m i n a t i o n N o . o f gd/gdi N o . o f +/g_d_ N o . o f g d / g d N o . o f +/. s e q u e n c e p r o g e n y p r o g e n y p r o g e n y p r o g e n y 2 i ; + 2 _ _ 43 g d ; + 25 - 4 4 5 g d ; + 12 - 2 6 + ; g d - 17 36 1 + ; g d - 33 36 + ;gd~ - 15 36 +;gd~ - 12 39 _ + ; g d - 26 8 3 + ;gd_ - 32 2 1 g d ; + 11 _ _ ' 27 g d ; + 2 - _ 43 g d ; + 12 - 30 gd~;+ 12 - _ 49 + ; g d 8 30 _ + ; g d 26 14 2 +;gd" - 20 10 6 + ; g d - 44 9 6 + ;g<[ *• 1 10 g d ; + 43 — • 2 47 gdV 21 - 9 22 g d ; + 14 - 5 36 gd_;+ 59 - 6 62 g d ; + 32 - _ 60 g d ; + 41 - _ 61 + ; g d - 31 4 2 ^ +;gd" - 25 3 8 2 + ; g d - - 25 14 1 + ; g d 31 24 1 + ;<£[ 13 1 5 -+ ; g d . - 5 31 -40 v a r i e d c o n s i d e r a b l y , but i n h a l f of the cases sperm from second i n s e m i n a t i o n s c o n t r i b u t e d to 100% of the second brood, Table 6 i n d i c a t e s t«o tren d s i n the mean s i z e of f i r s t and second broods i n a l l treatments. F i r s t , second broods i n a l l treatments tended to be l a r g e r than f i r s t broods. Second, the mean s i z e o f f i r s t and second broods tended t o i n c r e a s e as the number of spermatophores i n the i n s e m i n a t i o n s i n c r e a s e d . , a c o n s i s t e n t t r e n d i n brood s i z e s d i d not occur between treatments A and B (inseminations with 40 and 100 spermatophores), but a c l e a r i n c r e a s e i n the mean s i z e of f i r s t and second broods occurred a f t e r i n s e m i n a t i o n s with 20 0 spermatophores. In a d d i t i o n t o the d i f f e r e n c e s i n mean brood s i z e between treatment groups and f i r s t and second broods, broods produced a f t e r i n s e m i n a t i o n s with jgd sperm appeared to d i f f e r i n s i z e from broods produced a f t e r i n s e m i n a t i o n s with * sperm. Table 6 a l s o g i v e s the mean s i z e of broods produced a f t e r i n s e m i n a t i o n s with cjd and * sperm. , When comparing mean s i z e of f i r s t and second broods, note t h a t females inseminated f i r s t with gd sperm were re-i n s e m i n a t e d with + sperm and v i c e versa. The mean brood s i z e s r e p o r t e d i n t h i s t a b l e i n d i c a t e t h a t broods produced a f t e r i n s e m i n a t i o n s with 3d sperm tended to be sm a l l e r than broods produced a f t e r i n s e m i n a t i o n s with + sperm. F i g u r e 1a and 1B i l l u s t r a t e how mean brood s i z e v a r i e d between sperm genotype and treatment group i n f i r s t and second broods.. In F i g u r e 1 a , the mean 4 1 Table .6: The mean s i z e s o f f i r s t and second broods r e s u l t i n g from i n s e m i n a t i o n s with e i t h e r gd or + sperm. The mean s i z e s of pooled broods f o r each treatment group a r e a l s o r e p o r t e d . The value i n parentheses i s the standard d e v i a t i o n and n i s the number of broods. N o . o f s p e r m a t o p h o r e s I n s e m . w i t h i n t h e i n s e m i n a t i o n s cjd_ s p e r m F i r s t B r o o d s I n s e m . w i t h + s p e r m 40 1 3 . 0 ( 1 1 . 5 ) 2 2 . 5 ( 9 . 1 ) n = 3 n = 6 100 9 . 2 ( 4 . 9 ) n = 4 1 9 . 8 ( 1 6 . 7 ) n = 5 200 3 5 . 0 ( 1 6 . 3 ) n = 6 2 1 . 7 ( 1 4 . 8 ) n = 6 S e c o n d B r o o d s P o o l e d I n s e m . w i t h I n s e m . w i t h P o o l e d b r o o d s gd^ s p e r m + s p e r m b r o o d s 1 9 , 3 ( 1 0 . 3 ) 3 3 . 3 ( 2 2 . 1 ) 2 7 . 0 ( 1 5 . 8 ) 2 9 . 1 ( 1 7 . 0 ) n = 9 n = 6 n = 3 n = 9 1 5 . 1 ( 1 3 . 4 ) 1 7 . 4 ( 7 . 5 ) 3 7 . 3 ( 1 0 . 5 ) 2 6 . 2 ( 1 3 . 4 ) n = 9 n = 5 n = 4 n = 9 2 8 . 3 ( 1 4 . 8 ) 2 8 . 0 ( 1 2 . 6 ) 5 1 . 7 ( 1 3 . 9 ) 3 9 . 8 ( 1 7 . 5 ) n = 12 n = 6 n = 6 n = 12 43 F i g u r e 1S: fleao s i z e of f i r s t broods a f t e r i n s e m i n a t i o n s with 40, 100, and 200 spermatophores. Bars r e p r e s e n t 1 standard d e v i a t i o n on e i t h e r s i d e of the mean. F i g u r e 1B: Wean s i z e o f second broods a f t e r i n s e m i n a t i o n s with 40, 100, and 200 spermatophores. Bars re p r e s e n t 1 standard d e v i a t i o n on e i t h e r s i d e of the mean, 45 s i z e of f i r s t broods produced a f t e r i n s e m i n a t i o n s with + sperm remained constant as the number c f spermatophores i n the i n s e m i n a t i o n s i n c r e a s e d . In c o n t r a s t , mean brood s i z e remained lew a f t e r i n s e m i n a t i o n s with 40 and 100 gd spermatophores, but a f t e r i n s e m i n a t i o n s with 200 cjd spermatophores, the mean, brood s i z e i n c r e a s e d markedly. F i g u r e IB i n d i c a t e s t h a t the mean s i z e of second broods a l s o tended to be l a r g e s t a f t e r i n s e m i n a t i o n s with • sperm. Note t h a t , i n a d d i t i o n to t h i s , the mean s i z e of second broods i n c r e a s e d as the number of spermatophores i n the in s e m i n a t i o n s i n c r e a s e d . I t appears from these mean brood s i z e data t h a t qd sperm are not as vigorous or v i a b l e as • sperm. However, the sudden i n c r e a s e i n the mean s i z e o f f i r s t broods f o l l o w i n g i n s e m i n a t i o n with 200 .gd spermatophores ( F i g . :. 1A) suggests t h a t the number of cjd sperm i n an i n s e m i n a t i o n may have to reach a t h r e s h o l d l e v e l b efore they are as e f f e c t i v e as • s p e r m . I t i s d i f f i c u l t to s p e c u l a t e f u r t h e r with these few data* Since a s i m i l a r t rend i n the mean s i z e of second broods ( F i g . 1B) d i d not occur a f t e r i n s e m i n a t i o n s with £d sperm, p o s s i b l y the trend seen i n F i g u r e H i s an a r t i f a c t of v a r i a b l e data and s m a l l sample s i z e . , T a b l e 7 presents r e s u l t s of an a n a l y s i s of v a r i a n c e comparing brood s i z e v a r i a b i l i t y among treatments, o r d e r of i n s e m i n a t i o n with ajl and + sperm, and f i r s t and second broods. among these c a t e g o r i e s , the d i f f e r e n c e s were s i g n i f i c a n t at the 0.05 l e v e l . To t e s t the n u l l h y p o t h e s i s t h a t the number of 4 6 Table 7: An a n a l y s i s of v a r i a n c e comparing brood s i z e s among i n s e m i n a t i o n sequence, f i r s t and second broods,., and treatment groups. S o u r c e d f T o t a l 59 Among i n s e m i n a t i o n 1 s e q u e n c e s Among b r o o d s 1 Among t r e a t m e n t 2 g r o u p s I n t e r a c t i o n 7 W i t h i n 4 8 * s i g n i f i c a n t a t t h e 0 . 0 5 l e v e l SS MS F t e s t 1 5 6 1 3 . 4 1 3 3 7 . 9 1 3 3 7 . 9 7 . 8 6 * 1 7 7 1 . 3 1 7 7 1 . 3 1 0 . 4 0 * 2 0 6 4 . 4 1 0 3 2 . 2 6 . 0 6 * 2 2 6 7 . 5 3 2 3 . 9 1 . 9 0 8 1 7 2 . 3 1 7 0 . 3 48 spermatophores i n i n s e m i n a t i o n s does not a f f e c t the success of sperm i n r e - i n s e m i n a t i o n s , I compared the r e l a t i v e f r e q u e n c i e s of each phenotype i n the second brood between treatment groups. Si n c e , the second i n s e m i n a t i o n was always the most s u c c e s s f u l and the number of females i n each of the groups was s m a l l , I pooled the data f o r each treatment group before making the comparison. The r e s u l t s of the comparison using a c h i - s g u a r e d contingency t e s t f o r three independaat samples are presented i n Table 8. 8 i t h a c h i - s q u a r e equal to 0.852, df=2, no s i g n i f i c a n t d i f f e r e n c e i n phenotype frequency e x i s t s between the second broods of each of the t h r e e treatment groups, Conseguently the n u l l h y p o thesis cannot be r e j e c t e d and I conclude t h a t the amount of sperm i n i n s e m i n a t i o n s does not e f f e c t the success of the second i n s e m i n a t i o n . D i s c u s s i o n The s i m p l e s t hypothesis e x p l a i n i n g the apparent advantage t h a t second i n s e m i n a t i o n s have over f i r s t i n s e m i n a t i o n s i s a s t a t i s t i c a l one. In a l l f e r t i l i z a t i o n s a p r o p o r t i o n of the a v a i l a b l e sperm w i l l be used. I f f e r t i l i z a t i o n e f f i c i e n c y i s not h i g h , the number of sperm l o s t i n t h i s way may be c o n s i d e r a b l e . As a r e s u l t , the number of sperm l e f t i n the ovary a f t e r f e r t i l i z a t i o n w i l l be l e s s than i n the o r i g i n a l i n s e m i n a t i o n . I f the next 49 T a b l e 8: C o n t i n g e n c y t a b l e f o r a c h i - s q u a r e t e s t comparing t h e c o n t r i b u t i o n of f i r s t and second i n s e m i n a t i o n s t o second broods between the t h r e e t r e a t m e n t groups. , V a l u e s i n p a r e n t h e s e s are c a l c u l a t e d e x p e c t e d v a l u e s . T r e a t m e n t T r e a t m e n t T r e a t m e n t g r o u p g r o u p g r o u p A B C T o t a l number o f p r o g e n y p r o d u c e d f r o m t h e f i r s t i n s e m i n a t i o n . 11 ( 1 3 . 7 ) 14 ( 1 2 . 3 ) 26 ( 2 5 . 0 ) T o t a l number o f p r o g e n y p r o d u c e d f r o m t h e s e c o n d i n s e m i n a t i o n . 251 ( 2 4 8 . 3 ) 222 ( 2 2 3 . 7 ) 452 ( 4 5 3 . 0 ) c h i - s q u a r e = 0 . 8 5 2 : n o t s i g n i f i c a n t a t p * = : 0 . 0 5 , 2 d f 51 i n s e m i n a t i o n i s as l a r g e as the f i r s t , and i f f e r t i l i z a t i o n i s a s t o c h a s t i c p r o c e s s , then the second i n s e m i n a t i o n w i l l be at a s t a t i s t i c a l advantage i n f e r t i l i z i n g the next brood of eggs. Depending upon the number of sperm i n each i n s e m i n a t i o n and the f e r t i l i z a t i o n e f f i c i e n c y of each i n s e m i n a t i o n , the success of the second i n s e m i n a t i o n may vary. I t i s c l e a r from the r e s u l t s cf my a r t i f i c i a l i n s e m i n a t i o n experiments t h a t the p r e d i c t i o n i s not borne out. There i s no d i f f e r e n c e i n the pooled phenotype f r e q u e n c i e s of the broods a c r o s s treatment groups. In a l l broods, the genotype of the second i n s e m i n a t i o n predominates. T h i s i n d i c a t e s that a d i f f e r e n c e i n the number of sperm from two in s e m i n a t i o n s i s not n e c e s s a r i l y a cause of the apparent advantage of the second i n s e m i n a t i o n . P o s s i b l y 200 spermatophores were not enough f o r any d i s p a r i t y between the number of s t o r e d sperm (from the f i r s t i n s emination) and the number of f r e s h sperm (from the second insemination) to be markedly d i f f e r e n t from a d i s p a r i t y i n sperm number r e s u l t i n g from i n s e m i n a t i o n s with HO spermatophores. T h i s , however, i s u n l i k e l y . B i l l a r d ' s (1966) o b s e r v a t i o n that the s i z e of c o n s e c u t i v e broods dwindles a f t e r one i n s e m i n a t i o n with 50 spermatophores, but remains constant a f t e r one i n s e m i n a t i o n with 100 spermatophores, suggests t h a t l o s s of sperm due t o f e r t i l i z a t i o n and storage i s more n o t i c e a b l e a f t e r i n s e m i n a t i o n s with l e s s than 50 spermatophores than a f t e r i n s e m i n a t i o n s with mere than 100 spermatophores. T h e r e f o r e , 52 i f sperm from the second i n s e m i n a t i o n had precedence d a r i n g f e r t i l i z a t i o n simply because they outnumbered sperm from the f i r s t i n s e m i n a t i o n , r e - i n s e m i n a t i o n s with 200 spermatophores should have had the l e a s t success i n my experiments. T h i s was not the case. The amount of precedence shown by second i n s e m i n a t i o n s i n t h i s experiment f u r t h e r s trengthens the argument t h a t t h i s precedence cannot be accounted f o r by a p u r e l y s t a t i s t i c a l h y p o t h e s i s . I f sperm from r e - i n s e m i n a t i o n s gained precedence because they outnumbered s t o r e d sperm, the s t r o n g dominance of second i n s e m i n a t i o n s i n f e r t i l i z i n g second broods would have meant t h a t 95% of the sperm from f i r s t i n s e m i n a t i o n s were l o s t . B i l l a r d 1 s (1966) data, however, suggest t h a t much l e s s sperm i s l o s t a f t e r an i n s e m i n a t i o n . I f 95% of the sperm i n an i n s e m i n a t i o n were l o s t , c o n s e c u t i v e broods produced a f t e r i n s e m i n a t i o n s with 100 spermatophores would dwindle i n s i z e as r a p i d l y as broods produced by females inseminated with 40 spermatophores r a t h e r than remaining c o n s t a n t . On the b a s i s of d i f f e r e n c e s i n brood s i z e between f i r s t and second broods, one c o u l d argue that sperm from second i n s e m i n a t i o n s predominate i n f e r t i l i z a t i o n of second broods because these sperm are more v i g o r o u s . Brood s i z e data i n t h i s experiment i n d i c a t e t hat females produced l a r g e r second broods than f i r s t broods and t h i s may suggest g r e a t e r v i g o r or v i a b i l i t y i n sperm from second i n s e m i n a t i o n s . , However, Rosenthal (1952), Turner (1937), and Winge (1922) noted t h a t f i r s t broods produced by females inseminated once were a l s o 53 the s m a l l e s t . Since f e c u n d i t y i s p o s i t i v e l y c o r r e l a t e d with female s i z e i n P o e c i l i a r e t i c u l a t a (Purser, 1938; T h i b a u l t and S c h u l t z , 1978; Turner, 1937), they suggested that t h i s d i f f e r e n c e between the s i z e s of f i r s t and second broods r e s u l t e d from an i n c r e a s e i n f e c u n d i t y with growth, although i n my experiment, I d i d not measure a female's l e n g t h ; each time she gave b i r t h , most females were n o t i c e a b l y s m a l l e r a t the b i r t h of t h e i r f i r s t brood than they were a t the completion of the experiment. T h e r e f o r e , the d i f f e r e n c e i n mean brood s i z e between ; f i r s t and second broods was probably due to f e c u n d i t y i n c r e a s i n g with age and growth. Although sperm from the second i n s e m i n a t i o n dominated i n f e r t i l i z i n g the second brood, i n a l l c a s e s , a r t i f i c i a l i n s e m i n a t i o n with gd sperm tended to be l e s s s u c c e s s f u l than a r t i f i c i a l i n s e m i n a t i o n with + sperm. Both the number of s u c c e s s f u l i n s e m i n a t i o n s and the s i z e of r e s u l t i n g broods were l e s s when gd sperm were used. These data suggest that gd sperm may be l e s s v i a b l e or vig o r o u s than + sperm. However, as I mentioned i n S e c t i o n I , d i f f e r e n c e s i n a r t i f i c i a l i n s e m i n a t i o n success, and brood s i z e , a s s o c i a t e d with d i f f e r e n c e s i n sperm genotype are net c o n s i s t e n t . The brood phenotype f r e q u e n c i e s obtained i n the experiment i n Sec t i o n I i n d i c a t e there i s no d i f f e r e n c e i n v i g o r between * and gd sperm, hut d i f f e r e n c e s i n the success of a r t i f i c i a l i n s e m i n a t i o n between d i f f e r e n t treatments i n the experiment suggest that gd r a t h e r than + sperm may be the most v i g o r o u s . 54 F a c t o r s such as f e c u n d i t y and female f e r t i l i t y a l s o a f f e c t brood s i z e and success of i n s e m i n a t i o n . Since the number of females i n each treatment group of my experiments was s m a l l , any f a c t o r s (other than sperm genotype) which could a f f e c t brood s i z e and i n s e m i n a t i o n success may not have v a r i e d randomly among the d i f f e r e n t groups of i n s e m i n a t i o n s . T h e r e f o r e , although these d i f f e r e n c e s appear to be r e l a t e d to sperm genotype d i f f e r e n c e s , they may be f o r t u i t o u s . On the ether hand, phenotype f r e g u e n c i e s w i t h i n broods d i r e c t l y r e f l e c t d i f f e r e n t sperm v i a b i l i t i e s . S i n c e mean phenotype f r e g u e n c i e s i n the experiments of t h i s s e c t i o n d i d not d i f f e r between i n s e m i n a t i o n s with qd sperm and i n s e m i n a t i o n s with + sperm, .gd and * sperm are probably e q u a l l y v i g o r o u s . Although a d i f f e r e n c e i n the number of sperm a v a i l a b l e from f i r s t and second i n s e m i n a t i o n s i s not necessary to g i v e second i n s e m i n a t i o n s precedence, t h i s does not mean t h a t d i f f e r e n c e s i n the s i z e of two i n s e m i n a t i o n s do not a f f e c t f e r t i l i z a t i o n success i n subsequent broods. The s i z e of i n s e m i n a t i o n s probably v a r i e s from male to male, and i f a female was re-inseminated with a s m a l l e r amount of sperm than i n the preceding i n s e m i n a t i o n , the second i n s e m i n a t i o n may not f a r e w e l l . A s i m i l a r s i t u a t i o n may e x i s t i f a female has been inseminated s e v e r a l times., The l a s t i n s e m i n a t i o n may be so s m a l l compared t o the amount of sperm s t o r e d i n the ovary that i t s success i s reduced. The outcome of such s i t u a t i o n s cannot be determined from the r e s u l t s of t h i s part of my study; however, i f two unequal 5 5 i n s e m i n a t i o n s were performed i n a s e t of i n s e m i n a t i o n s s i m i l a r to the ones i n t h i s p a r t of the study, or the success of a t h i r d i n s e m i n a t i o n was monitored, f u r t h e r i n s i g h t i n t o the problem may be gained. S e v e r a l other hypothesis can account f o r second i n s e m i n a t i o n s g a i n i n g precedence over f i r s t i n s e m i n a t i o n s . Stored sperm, e i t h e r because of t h e i r age or because of a l a c k of some v i t a l substance present during i n s e m i n a t i o n , may be l e s s v i a b l e than sperm from f r e s h i n s e m i n a t i o n s . A l t e r n a t i v e l y , s t o r e d sperm may be i n r e g i o n s of the ovary that g i v e them l e s s access t o mature eggs than sperm from a new i n s e m i n a t i o n or they may be d i s p l a c e d from storage s i t e s by f r e s h sperm. Some of these h y p o t h e s i s are not as d i f f i c u l t as others to t e s t experimently and they should be i n v e s t i g a t e d f i r s t . The next s e c t i o n looks a t the e f f e c t of time of r e - i n s e m i n a t i o n r e l a t i v e to the female brood c y c l e on the degree of precedence gained by the second i n s e m i n a t i o n . I f some c h a r a c t e r i s t i c of s t o r a g e makes s t o r e d sperm l e s s s u c c e s s f u l than f r e s h sperm, a new in s e m i n a t i o n w e l l b e f o r e eggs are mature (hence sperm from both i n s e m i n a t i o n s are stored) may not have precedence over the f i r s t i n s e m i n a t i o n d u r i n g f e r t i l i z a t i o n of f r e s h l y matured eggs. , 56 S e c t i o n I I I The e f f e c t of t i m i n g of r e - i n s e m i n a t i o n on the success of sperm from r e - i n s e m i n a t i o n s . I n t r o d u c t ion Under n a t u r a l c o n d i t i o n s the t i m i n g of i n s e m i n a t i o n i s r e s t r i c t e d i n p o e c i l i i d s . Breeding i s continuous i n many po p u l a t i o n s , but i n d i v i d u a l females are r e c e p t i v e f o r only a few days a f t e r p a r t u r i t i o n ( C a r l s o n , 1963; L i l e y , 1966). In most p o e c i l i i d s , t h i s p e r i o d of r e c e p t i v i t y corresponds to the f i n a l maturation of the next batch of eggs. Consequently, new i n s e m i n a t i o n s i n t r o d u c e f r e s h sperm i n t o the ovary j u s t as a new s e t of eggs become a v a i l a b l e f o r f e r t i l i z a t i o n . In a l l pr e v i o u s s t u d i e s of i n t r a s p e c i f i c sperm c o m p e t i t i o n i n P o e c i l i a r e t i c u l a t a , i n s e m i n a t i o n s were n a t u r a l (Bowden, 1970; Hildemann and Hagner, 1954; Winge,1 937). „ Since t h i s occurs only when females are r e c e p t i v e and c a r r y i n g mature eggs, i t has been i m p o s s i b l e t o determine the i n f l u e n c e t h a t the t i m i n g of an i n s e m i n a t i o n , r e l a t i v e t o egg maturation, has on f e r t i l i z a t i o n . Since mature eggs are a v a i l a b l e a t the time a new i n s e m i n a t i o n , or j u s t a f t e r i n s e m i n a t i o n , f r e s h l y i n t r o d u c e d sperm may have g r e a t e r access to eggs than s t o r e d sperm. Sperm a c t i v i t y b e f o r e f e r t i l i z a t i o n has not been d e s c r i b e d , so i t i s not p o s s i b l e t o compare the r e l a t i v e success of competing s t o r e d sperm and f r e s h sperm. 57 In t h i s s e c t i o n , I use a r t i f i c i a l i n s e m i n a t i o n to examine the a f f e c t s of the time of r e - i n s e m i n a t i o n on success. Females were re-in s e m i n a t e d d u r i n g g e s t a t i o n and the success of t h i s i n s e m i n a t i o n i s compared to that of r e -i n s e m i n a t i o n s o c c u r r i n g d i r e c t l y a f t e r p a r t u r i t i o n {the n a t u r a l time of r e - i n s e m i n a t i o n ) . . Methods and M a t e r i a l s 51 v i r g i n gd/qd females were a r t i f i c i a l l y inseminated with 100 spermatophores. 27 of the i n s e m i n a t i o n s were with gd sperm and 2U with • sperm. , approximately 7 t o 12 days l a t e r a l l females were re-inseminated with TOO spermatophores, but of the genotype opposite to t h a t used i n the f i r s t i n s e m i n a t i o n . Females were then kept s e p a r a t e l y i n 20 l i t r e g l a s s a g u a r i a . , The time between . the two i n s e m i n a t i o n s was chosen so t h a t the r e - i n s e m i n a t i o n s occurred a f t e r f e r t i l i z a t i o n of the f i r s t batch of eggs, but before development of the second brood proceded f a r . I n t h i s way, only the f i r s t i n s e m i n a t i o n c o u l d c o n t r i b u t e to the f i r s t brood, and the p o s s i b i l i t y of p h y s i c a l damage to the developing embryos from the r e - i n s e m i n a t i o n was minimized.,. Although .Stolk (1950) r e p o r t e d that an o c c l u s i o n apparatus c l o s e s the gonoduct opening during brood development, I was a b l e to i n s e r t the m i c r o - c a p i l l a r y tube c o n t a i n i n g spermatophores i n t o the female t r a c t with l i t t l e d i f f i c u l t y and no p h y s i c a l 58 damage to the female was apparent. De s p i t e the l a p s e of 7 to 12 days between the i n i t i a l i n s e m i n a t i o n and r e - i n s e m i n a t i o n , the second i n s e m i n a t i o n c o n t r i b u t e d t o the f i r s t brood of some females. undoubtedly i n v i r g i n females the p e r i o d between i n s e m i n a t i o n and complete f e r t i l i z a t i o n of the f i r s t brood i s v a r i a b l e . An endogenous r e p r o d u c t i v e c y c l e c o n t r o l l i n g egg maturation has been p o s t u l a t e d f o r v i r g i n females ( l i l e y , 1968)., P o s s i b l y eggs were at v a r i o u s stages of maturity when 1 inseminated the v i r g i n females i n my experiments. I used only those females whose f i r s t brood was f e r t i l i z e d e n t i r e l y by sperm from the f i r s t i n s e m i n a t i o n i n the a n a l y s i s , I f the f i r s t brood was f e r t i l i z e d by a mixture of sperm from the two i n s e m i n a t i o n s , t h i s meant t h a t r e - i n s e m i n a t i o n occurred p r i o r t o the beginning of embryo development and was not c o n s i s t e n t with my experimental design.. In a d d i t i o n , I c o u l d not be c e r t a i n of the v i a b i l i t y of sperm from the f i r s t i n s e m i n a t i o n , i f the brood was f e r t i l i z e d predominantly by sperm from the second i n s e m i n a t i o n . The. success of the second i n s e m i n a t i o n i n t h i s s e c t i o n i s compared to t h a t of treatment B of the p r e v i o u s s e c t i o n . T h i s p r o v i d e s the a p p r o p r i a t e comparison f o r determining how the t i m i n g of a r e - i n s e m i n a t i o n a f f e c t s i t s s u c c e s s . The only d i f f e r e n c e between the two treatments i s the time of r e - i n s e m i n a t i o n r e l a t i v e to the female r e p r o d u c t i v e c y c l e . 59 Results Table 9 presents the number of • /gd and gd/gd progeny i n the f i r s t and second broods of females i n both treatments. The data f o r females re-inseminated post-partum (Treatment B) are from Table 5, Treatment B., I n a l l c a s e s , the second i n s e m i n a t i o n c o n t r i b u t e d the most to the second brood. However, the amount of dominance gained by the second insemination d i f f e r e d between treatments and i n s e m i n a t i o n s with d i f f e r e n t sperm genotypes. , The mean s i z e c f broods f e r t i l i z e d a f t e r i n s e m i n a t i o n s with e i t h e r g d or + sperm are given s e p a r a t e l y f o r each treatment group i n Table 10., These data i n d i c a t e two c l e a r trends. F i r s t , second broods were l a r g e r than f i r s t broods i n both treatments. Second, females i n Treatment A produced l a r g e r broods ( f i r s t and second broods} than females i n Treatment B. An a n a l y s i s o f v a r i a n c e of brood s i z e s (Table 11) i n d i c a t e s t h a t both of these brood s i z e d i f f e r e n c e s are s i g n i f i c a n t f o r p < 0.05. In c o n t r a s t , d i f f e r e n c e s i n brood s i z e between broods f e r t i l i z e d a f t e r i n s e m i n a t i o n s with gd sperm and broods f e r t i l i z e d a f t e r i n s e m i n a t i o n s with + sperm do not f o l l o w a c o n s i s t e n t t r e n d . ;« Females i n Treatment E tended t o produce the l a r g e s t broods a f t e r i n s e m i n a t i o n s w i t h s p e r m . In Treatment A, broods f e r t i l i z e d a f t e r i n s e m i n a t i o n s with gd sperm tended to be s l i g h t l y l a r g e r than than those broods f e r t i l i z e d a f t e r i n s e m i n a t i o n s with + sperm. The a n a l y s i s of v a r i a n c e c a l c u l a t i o n i n Table 11 i n d i c a t e s that the 60 Table 9 ; The number of +/£d and gd/gd progeny, i n f i r s t and second broods produced by females a f t e r pre- and post-partum r e -i n s e m i n a t i o n s . T r e a t m e n t g r o u p F e m a l e 1 . 2 . A 3 ' / 4 ( p r e - p a r t u m , - ' r e - i n s e m i n a t i o n ) g ' l\ 8 . 1 . 2 . 3 . B 4 . ( p o s t - p a r t u m 5 . r e - i n s e m i n a t i o n ) 6 . 7 . 8 . 9 . F i r s t B r o o d S e c o n d B r o o d I n s e m i n a t i o n N o . o f g d / g d N o . o f +/gd N o . o f g d / g d N o . o f +/' s e q u e n c e p r o g e n y p r o g e n y p r o g e n y p r o g e n y gd;+ 31 4 62 g d V 13 - 3 39 gd";+ 4 - - 39 +;gd - 2 46 15 +;gd" - 20 17 2 +;gd" - 23 60 13 + -M 3 3 4 16 + ; g l - 14 45 -gd-,+ 11 27 gd";+ 2 - 43 gdV 12 - - 30 gd";+ 12 - - 49 + ;gd - 8 30 -+ ;gd" 26 14 2 + ;gd" 20 10 6 + ;gd" - 44 9 6 + ;gd" 1 10 -62 Table 10: The mean s i z e s of f i r s t and second broods r e s u l t i n g from i n s e m i n a t i o n s with e i t h e r <jd or • sperm. The mean s i z e s of pooled broods f o r each, treatment group are a l s o r e p o r t e d . Values i n parentheses are the standard d e v i a t i o n s and n i s the number of broods. I n s e m . w i t h gd s p e r m I n s e m . w i t h + s p e r m P r e - p a r t u m 1 6 . 0 ( 1 3 . 8 ) 1 2 . 4 ( 9 . 6 ) r e - i n s e m i n a t i o n s n = 3 n = 5 P o s t - p a r t u m 9 . 3 ( 4 . 9 ) 1 9 . 8 ( 1 6 . 7 ) r e - i n s e m i n a t i o n s n = 4 n = 5 P o o l e d b r o o d s I n s e m . w i t h gd s p e r m I n s e m . w i t h + s p e r m P o o l e d b r o o d s 1 3 . 8 ( 1 0 . 5 ) n = 8 4 9 . 6 ( 2 0 . 2 ) n = 5 4 7 . 7 ( 1 2 . 5 ) n = 3 4 9 . 4 ( 1 7 . 2 ) n = 8 1 5 . 1 ( 1 3 . 4 ) n = 9 1 7 . 4 ( 7 . 5 ) n = 5 3 7 . 3 ( 1 0 . 5 ) n = 4 2 6 . 2 n ( 1 3 . 4 ) = 9 CO 64 sequence of i n s e m i n a t i o n s with gd and + sperm ( i . e . gd -* + o r + gd ) did not a f f e c t brood s i z e t r e n d s between f i r s t and second broods and treatment groups. T h i s means t h a t brood s i z e d i f f e r e n c e s i n t h i s experiment are not r e l a t e d t o sperm genotype d i f f e r e n c e s . Although brood s i z e d i f f e r e n c e s between treatment groups and f i r s t and second broods were not r e l a t e d to the sequence of inseminations with gd and • sperm, the degree of dominance shown by the second i n s e m i n a t i o n appears to d i f f e r between the two i n s e m i n a t i o n sequences. , As seen i n Table 9, there was a tendency f o r the second i n s e m i n a t i o n to be more dominant when + r a t h e r than gd sperm were used i n the i n s e m i n a t i o n . Chi-sguare a n a l y s i s of the a v a i l a b l e data (Tables 12A and 12B) shows t h i s d i f f e r e n c e i n dominance between inseminations with gd or + sperm to be s i g n i f i c a n t (p < 0.05) i n both Treatments A and B. U n f o r t u n a t e l y the number of broods r e s u l t i n g from these i n s e m i n a t i o n s may be too s m a l l to overcome the a f f e c t s of f a c t o r s other than sperm genotype which c o u l d b i a s the d a t a . Table 13 p r e s e n t s a c h i - s q u a r e contingency t a b l e of second brood phenotype f r e q u e n c i e s i n females subjected to pre- and post-partum r e - i n s e m i n a t i o n s . Despite the apparent frequency d i f f e r e n c e s between r e - i n s e m i n a t i o n s with gd and + sperm, broods i n each treatment group were pooled. At p < 0.05, df=1, the c h i - s g u a r e v a l u e , 8.67, i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e i n the degree of precedence gained between pre- and post-partum r e - i n s e m i n a t i o n s . . T h e r e f o r e the h u l l h y p o t h e s i s (that t h e r e i s no d i f f e r e n c e i n the 6 5 Table 1 1 : An a n a l y s i s of va r i a n c e comparing mean brood s i z e s among i n s e m i n a t i o n seguence, f i r s t and second broods, and treatment groups. . S o u r c e d f T o t a l 33 Among i n s e m i n a t i o n 1 s e q u e n c e s Among b r o o d s 1 Among t r e a t m e n t 1 g r o u p s I n t e r a c t i o n 4 W i t h i n 26 * s i g n i f i c a n t a t t h e 0 . 0 5 l e v e l SS 1 2 3 4 5 . 6 4 7 . 8 4 3 5 9 . 6 1 5 1 0 . 6 1 8 6 8 . 3 4 5 6 0 . 0 MS 4 7 . 8 4 3 5 9 . 6 1 5 1 0 . 6 4 6 7 . 1 1 7 5 . 4 F t e s t 0 . 2 7 3 2 4 . 8 6 * 8 . 6 1 2 . 6 6 67 Table 12 : C o n t i n g e n c y t a b l e s f o r c h i - s g u a r e t e s t s comparing the c o n t r i b u t i o n of f i r s t and second i n s e m i n a t i o n s to second broods between i n s e m i n a t i o n sequences f o r A) p r e -partum r e - i n s e m i n a t i o n s and E) p o s t - p a r t u m r e - i n s e m i n a t i o n s . V a l u e s i n p a r e n t h e s e s are c a l c u l a t e d e x p e c t e d v a l u e s . A ) I n s e m i n a t i o n s e q u e n c e T o t a l number o f p r o g e n y 7 p r o d u c e d f r o m t h e f i r s t i n s e m i n a t i o n . ( 1 4 . 7 ) T o t a l number o f p r o g e n y 140 p r o d u c e d f r o m t h e s e c o n d i n s e m i n a t i o n . ( 1 3 2 . 3 ) c h i - s q u a r e = 1 8 . 0 1 : s i g n i f i c a n t a t p < 0 . 0 5 , l d f B) I n s e m i n a t i o n s e q u e n c e T o t a l number o f p r o g e n y 0 p r o d u c e d f r o m t h e f i r s t i n s e m i n a t i o n . ( 8 . 8 ) T o t a l number o f p r o g e n y 149 p r o d u c e d f r o m t h e s e c o n d i n s e m i n a t i o n . ( 1 4 0 . 2 ) c h i - s q u a r e = 2 5 . 1 9 : s i g n i f i c a n t a t p < 0 . 0 5 , 1 d f 6 9 Table 1.3: Contingency t a b l e f o r a c h i - s g u a r e t e s t comparing the c o n t r i b u t i o n of f i r s t and second i n s e m i n a t i o n s to second broods between treatment • groups. Values i n parentheses are c a l c u l a t e d expected values. 70 T r e a t m e n t g r o u p A T o t a l number o f p r o g e n y 53 p r o d u c e d f r o m t h e f i r s t i n s e m i n a t i o n . ( 4 2 ) T o t a l number o f p r o g e n y 3 4 2 p r o d u c e d f r o m t h e s e c o n d i n s e m i n a t i o n . ( 3 5 3 ) T r e a t m e n t g r o u p B 14 ( 2 5 ) 222 ( 2 1 0 ) c h i - s q u a r e = 8 . 6 7 : s i g n i f i c a n t a t p < 0 . 0 5 , l d f 71 success of pre- and post-partum r e - i n s e m i n a t i o n s ) must be r e j e c t e d . Since I do not know whether the v i a b i l i t y o f sperm from f i r s t i n s e m i n a t i o n s or the time of r e - i n s e m i n a t i o n r e l a t i v e to egg maturation a f f e c t e d c o n t r i b u t i o n of r e - i n s e m i n a t i o n s t o f i r s t broods i n some females i n Treatment A, i t i s im p o s s i b l e to compare the r e l a t i v e success of i n s e m i n a t i o n and r e - i n s e m i n a t i o n between Treatments A and B, , In a d d i t i o n , I was unable t o determine the d i f f e r e n c e s i n success between a r t i f i c i a l i n s e m i n a t i o n s with gd and a r t i f i c i a l i n s e m i n a t i o n s with + sperm i n Treatment A of t h i s experiment. D i s c u s s i o n In the pre v i o u s s e c t i o n s of t h i s study, only d i f f e r e n c e s i n brood s i z e , or a r t i f i c i a l : i n s e m i n a t i o n s u c c e s s , appeared r e l a t e d t o d i f f e r e n c e s i n sperm genotype. The c o m p e t i t i v e a b i l i t i e s of .gd and + sperm were egual. In t h i s experiment, however, d i f f e r e n c e s i n a b i l i t y of sperm from r e - i n s e m i n a t i o n s t o dominate during f e r t i l i z a t i o n are c o r r e l a t e d with sperm genotype, but d i f f e r e n c e s i n mean brood s i z e are not a s s o c i a t e d with sperm genotype. T h i s i n c o n s i s t e n c y i n the a s s o c i a t i o n of brood d i f f e r e n c e s and sperm s u c c e s s with sperm genotype suggests t h a t .gd and • sperm may be approximately equal i n v i g o r and t h a t s t o c h a s t i c processes and e r r o r s a s s o c i a t e d with technigue may have biased the s m a l l samples c f breeds.. 7 2 R e j e c t i o n of the n u l l hypothesis must be i n t e r p r e t e d with c a r e . D i f f e r e n t degrees of sperm precedence a s s o c i a t e d with sperm genotype d i f f e r e n c e mean that a f t e r p o o l i n g broods, the amount t h a t sperm from r e - i n s e m i n a t i o n s dominate f e r t i l i z a t i o n w i l l depend on how broods f o l l o w i n g r e -i n s e m i n a t i o n with gd and «• sperm are r e p r e s e n t e d i n the pooled brood s i z e . I n Treatment B, broods f e r t i l i z e d a f t e r r e - i n s e m i n a t i o n s with > sperm were the l a r g e s t and hence c o n t r i b u t e d the most to the pooled brood s i z e . In Treatment a , more second broods were f e r t i l i z e d a f t e r r e - i n s e m i n a t i o n s with gd sperm and consequently they c o n t r i b u t e d the most to the peeled brcod s i z e i n that treatment group. The r e s u l t of t h i s d i f f e r e n c e between Treatments a and B i n the weighting of second broods f e r t i l i z e d a f t e r r e - i n s e m i n a t i o n s with the two sperm genotypes i s that r e - i n s e m i n a t i o n s i n Treatment A appear l e s s a b l e t o dominate i n f e r t i l i z a t i o n than r e - i n s e m i n a t i o n s i n Treatment B. Since there i s doubt about whether or not sperm genotype a f f e c t s the success of sperm i n c o m p e t i t i o n , the apparent d i f f e r e n c e i n success between pre- and post-partum r e - i n s e m i n a t i o n s may be f o r t u i t o u s . In a d d i t i o n , i f f a c t o r s other than sperm genotype a f f e c t e d brood s i z e , or a r t i f i c i a l i n s e m i n a t i o n s u c c e s s , i n a non-random way, the s m a l l sample s i z e may have allowed these b i a s e s to be emphasized. I f sperm from pre-partum r e - i n s e m i n a t i o n s are indeed l e s s capable than sperm from post-partum r e - i n s e m i n a t i o n s of dominating f e r t i l i z a t i o n of second broods, i t i s not c l e a r why t h i s d i f f e r e n c e occurs. P o s s i b l y , developing embryos 73 occlude p o r t i o n s of the o v a r i a n lumen and prevent sperm from r e a c h i n g some of the storage s i t e s . _ a l s o , the ovary's c a p a c i t y to s t o r e sperm may be l e s s during g e s t a t i o n . U n f o r t u n a t e l y , anatomical evidence f o r these p o s s i b i l i t i e s i s l a c k i n g . . Despite d i f f e r e n c e s i n the degree of precedence gained by r e - i n s e m i n a t i o n s between treatment groups and sperm genotype i n t h i s experiment, sperm from a l l r e - i n s e m i n a t i o n s had precedence over sperm from the f i r s t i n s e m i n a t i o n . Thus, although the time of r e - i n s e m i n a t i o n r e l a t i v e to the female r e p r o d u c t i v e c y c l e and sperm genotype.may a f f e c t the success of r e - i n s e m i n a t i o n s , the second i n s e m i n a t i o n s t i l l m aintains precedence over the f i r s t i n s e m i n a t i o n . The r e s u l t s of my experiments up to t h i s p o i n t i n d i c a t e t h a t : •1 .,).-• the t o t a l sperm p o o l i n the ovary a f t e r a female i s r e -inseminated does not work as a complete u n i t d u r i n g f e r t i l i z a t i o n . I n s t e a d sperm from r e c e n t i n s e m i n a t i o n s are favoured. , 2 . ) dominance of sperm from second i n s e m i n a t i o n s oyer sperm from f i r s t i n s e m i n a t i o n s i s e s t a b l i s h e d even be f o r e eggs are mature. The r e s u l t s of a few i n s e m i n a t i o n s not reported here suggest t h a t t h i s dominance may be e s t a b l i s h e d immediately a f t e r r e - i n s e m i n a t i o n . 3. ) dominance i s not always complete and the degree of dominance shown by sperm from r e - i n s e m i n a t i o n s may vary depending on circumstances. I t i s , n e v e r t h e l e s s , i m p o s s i b l e t o get a c l e a r p i c t u r e of 74 how sperm from r e - i n s e m i n a t i o n s dominate over sperm from f i r s t i n s e m i n a t i o n s with t h i s i n f o r m a t i o n a l o n e . D i r e c t i n d i c a t i o n of how sperm from d i f f e r e n t i n s e m i n a t i o n s are d i s t r i b u t e d throughout the ovary i s r e q u i r e d as w e l l . The purpose of t h e f i n a l part of t h i s study was t o qather some i n f o r m a t i o n about sperm d i s t r i b u t i o n i n the ovary and the r e s u l t s of t h a t i n v e s t i q a t i o n are r e p o r t e d i n the next s e c t i o n . 75 S e c t i o n IV. a u t o r a d i o g r a p h i c a l a n a l y s i s of the l o c a t i o n of sperm from separate i n s e m i n a t i o n s i n the ovary. I n t r o d u c t i o n My e a r l i e r experiments i n t h i s study demonstrated t h a t sperm st o r a g e i n the ovary must be organized s i n c e sperm from the most recent i n s e m i n a t i o n are more l i k e l y to f e r t i l i z e mature eggs than sperm from e a r l i e r i n s e m i n a t i o n s . The d i s p o s i t i o n of sperm i n the ovary may c o n t r i b u t e t o t h i s advantage of new sperm over o l d e r sperm. In t h i s s e c t i o n , I i n v e s t i g a t e the anatomy of sperm storage by using sperm from two i n s e m i n a t i o n s . a p o s s i b l e e x p l a n a t i o n f o r sperm precedence i s t h a t sperm from the most r e c e n t i n s e m i n a t i o n are s t o r e d at l o c a t i o n s that give c l e a r access to mature eggs, a l t e r n a t i v e l y , i f sperm move i n t o a l l sperm storage s i t e s a f t e r i n s e m i n a t i o n s , sperm from new i n s e m i n a t i o n s may d i s p l a c e o l d e r sperm from storage s i t e s . P r e v i o u s workers ( B a i l e y , 1933; J a l a b e r t and B i l l a r d , 1969; Kadow, 1954; Ryder, 1885; Stuhlman, 1887; Hinge, 1922) rep o r t e d pockets and d i v e r t i c u l a e of the o v a r i a n lumen of guppies. Many of these d i v e r t i c u l a e c o n t a c t eggs and some workers suggested t h a t these might be the s i t e of f e r t i l i z a t i o n ( B i l l a r d , 1966; J a l a b e r t and B i l l a r d , 1969). I f sperm are normally s t o r e d i n those d i v e r t i c u l a e c o n t a c t i n g eggs, t h i s may be where dominance of sperm from new i n s e m i n a t i o n s ever sperm from e a r l i e r i n s e m i n a t i o n s i s 76 e s t a b l i s h e d . By using a r a d i o i s o t o p e to mark sperm from d i f f e r e n t i n s e m i n a t i o n s , i t should be p o s s i b l e to determine i f e i t h e r of the two e x p l a n a t i o n s of sperm precedence are t e n a b l e . Methods and M a t e r i a l s a » ) Production of r a d i o a c t i v e l y l a b e l l e d sperm. The a u t o r a d i o g r a p h i c a l procedure used i n t h i s study was developed t o l a r g e extent by Luytens (unpubl. MS), 250 u C i of thymidine (raethy1- 3H) ( s p e c i f i c a c t i v i t y approximately 50 UCi/mmole) d i s s o l v e d i n 250 u l of s t e r i l e water was obtained from Hew England t h i s c o n c e n t r a t i o n was used throughout the experiment. L a b e l l e d thymidine was administered by i n t r a p e r i t o n e a l i n j e c t i o n with a 50 u l Hamilton s y r i n g e f i t t e d with a 30 gauge n e e d l e , k male was a n a e s t h e t i z e d with MS 222 and then placed cn i t s s i d e on a piece of dampened paper towel. The procedure was viewed with a d i s s e c t i n g microscope, Spermatophores were f i r s t s t r i p p e d from the male i n the manner d e s c r i b e d e a r l i e r . The needle was then pushed a n t e r i o r l y under the integument d o r s a l t o the base o f the gonopodium and 2 u l of the u n d i l u t e d thymidine s o l u t i o n were i n j e c t e d i n t o the p e r i t o n e a l space. The needle was g u i c k l y , but g e n t l y , withdrawn and the male was placed i n f r e s h aguarium water to r e c o v e r . 77 F i v e days a f t e r the f i r s t i n j e c t i o n , males were r e -i n j e c t e d on the opposite s i d e of t h e i r bodies with 2 u l of the thymidine s o l u t i o n . Thus the t o t a l r a d i o a c t i v e dose admi n i s t e r e d to males was t u C i . Luyten (unpubl. MS) found t h i s dosage to r e s u l t i n the same amount of l a b e l l i n g as l a r g e r doses, but the volume of f l u i d used was l e s s l i k e l y t o cause harm to the male. A f t e r the i n j e c t i o n s , males were kept i n 20 l i t r e g l a s s aguaria u n t i l the 28th day a f t e r the f i r s t i n j e c t i o n . T e s t s by Luytens (unpubl. MS) i n d i c a t e d t h a t l a b e l l i n g reaches a maximum (approximately 80%) 28 days a f t e r the f i r s t i n j e c t i o n and then drops s h a r p l y a few days l a t e r . . b.) A r t i f i c i a l i n s e m ination of females. The p a t t e r n of a r t i f i c i a l i n s e m i n a t i o n s was s i m i l a r to t h a t i n the p r e v i o u s s e c t i o n : v i r g i n females were inseminated twice with egual amounts of sperm and the time between i n s e m i n a t i o n s was approximately a t h i r d of the r e p r o d u c t i v e c y c l e . To d i s t i n g u i s h sperm from the two i n s e m i n a t i o n s , sperm from one of the i n s e m i n a t i o n s were l a b e l l e d with t r i t i a t e d thymidine. To reduce problems i n t e r p r e t i n g the a u t o r a d i o g r a p h i c d a t a , i n s e m i n a t i o n s were c a r r i e d out i n the f o l l o w i n g manner. Three v i r g i n females were i n i t i a l l y inseminated a r t i f i c i a l l y with 100 spermatophores. Ten days l a t e r they 78 sere again inseminated, but t h i s time with 100 spermatcphores c o n t a i n i n g l a b e l l e d sperm. , In a d d i t i o n , two other v i r g i n females sere s i m i l a r i l y inseminated, but the order o f i n s e m i n a t i o n with l a b e l l e d and u n l a b e l l e d sperm was r e v e r s e d . a l l females were maintained i n 20 l i t r e g l a s s aquaria u n t i l the tenth day a f t e r r e - i n s e m i n a t i o n . R e c i p r o c a l i n s e m i n a t i o n s were important f o r two reasons. F i r s t , they allowed me t o assess d i f f e r e n c e s i n the a c t i v i t y of r a d i o a c t i v e l y l a b e l l e d and. u n l a b e l l e d sperm w i t h i n the ovary. Secondly, t h i s was the only means of determining i f sperm from both i n s e m i n a t i o n s occur together or s e p a r a t e l y i n storage s i t e s . Since r a d i o a c t i v e sperm are detected i n d i r e c t l y on a photographic emulsion* r a d i o a c t i v e sperm c o u l d mask t h e presence of u n l a b e l l e d sperm i n sperm c l u s t e r s . Comparing the r e s u l t s of a r e v e r s e s e t of r e c i p r o c a l i n s e m i n a t i o n s would r e v e a l whether t h i s was o c c u r r i n g . Io check the r a d i o a c t i v i t y of l a b e l l e d sperm, a s e t of v i r q i n females was inseminated with 100 spermatophores from a l l of the r a d i o a c t i v e l y t r e a t e d males. Thus f o r every female inseminated with both l a b e l l e d and u n l a b e l l e d sperm, there was a c o n t r o l female, inseminated with the same batch of l a b e l l e d sperm. These c o n t r o l females were maintained i n 20 l i t r e g l a s s aquaria and removed a t the same time as the corresponding treatment female. 7 9 c « ) H i s t o l o g i c a l procedures. Ten days a f t e r the second i n s e m i n a t i o n , females {and corresponding c o n t r o l females) were k i l l e d f o r h i s t o l o g i c a l examination of the o v a r i e s . , Females were k i l l e d i n Bouin's f i x a t i v e and a f t e r approximately one hour, they were d e c a p i t a t e d . T h i s allowed p e n e t r a t i o n of the f i x a t i v e i n t o the i n t e r n a l organs. , F i x a t i o n continued o v e r n i g h t . A f t e r f i x a t i o n the o v a r i e s were e x c i s e d from the bodies and washed i n s e v e r a l changes of 70% e t h a n o l . T h i s part of the p r e p a r a t i o n was important f o r subseguent s e c t i o n i n g . Because of t h e i r high y o l k c o n t e n t , f i s h eggs become hard from f i x a t i o n . I f any f i x a t i v e remains, the eggs tend to s h a t t e r d u r i n g s e c t i o n i n g , and thus destroy surrounding t i s s u e s . Once the o v a r i e s were washed, the t i s s u e s were dehydrated and i n f i l t r a t e d i n a F i s h e r H i s t o m a t i c T i s s u e P r o c e s s o r , Model 166. The o v a r i e s were t r e a t e d with two changes of 9535 e t h a n o l a t two hours per change, and then t r e a t e d with two changes of 100% e t h a n o l , a l s o at two hours per change. F i n a l dehydration of t i s s u e s b e f o r e P a r a p l a s t i n f i l t r a t i o n was done i n two changes of xylene, the f i r s t change f o r twenty minutes, the second change f o r one hour. Shorter treatment times i n xylene l e f t the o v a r i e s too hard f o r s a t i s f a c t o r y s e c t i o n i n g . I n f i l t r a t i o n o f t i s s u e s with P a r a p l a s t was done i n two changes of P a r a p l a s t at two hours per change. I n f i l t r a t e d o v a r i e s were f i n a l l y embedded i n P a r a p l a s t so that t r a n s v e r s e s e c t i o n s c o u l d be made., 8 0 The o v a r i e s were s e r i a l l y s e c t i o n e d at 10 am t h i c k n e s s . Although care was taken to preserve as many of the s e c t i o n s as p o s s i b l e , d i f f i c u l t i e s i n s e c t i o n i n g as a r e s u l t of egg hardness made i t imp o s s i b l e t c preserve a l l o f the s e c t i o n s . Once mounted upon s l i d e s , s e c t i o n s were washed i n xylene to remove the P a r a p l a s t and coated with a nu c l e a r t r a c k emulsion. d.) A p p l i c a t i o n and development of the n u c l e a r t r a c k emulsion. Kodak NTB nuclear t r a c t emulsion was used to d e t e c t r a d i o a c t i v e l y l a b e l l e d sperm i n t i s s u e s e c t i o n s of o v a r i e s . L i k e a l l n u c l e a r t r a c t emulsions, t h i s i s very s e n s i t i v e to l i g h t and t h e r e f o r e , the emulsion was a p p l i e d t o the s l i d e s and developed a f t e r exposure, i n a darkroom, e i t h e r i n the dark or, i f i t was necessary t o see, with a s a f e t y l i g h t . The s t o c k emulsion, normally s t o r e d a t 4 C, was melted i n a water bath a t 45 C. T h i s took approximately 30 t o 40 minutes. Once melted, a s m a l l amount of the emulsion was d i l u t e d 1:1 with d i s t i l l e d water and placed i n a c l e a n , 50 ml g l a s s v i a l . One by one, the g l a s s s l i d e s c o n t a i n i n g t i s s u e s e c t i o n s were held i n the d i l u t e d emulsion f o r 2 seconds, p u l l e d out, and the excess emulsion allowed to run back i n t o the v i a l . Coated s l i d e s were stood upright and l e f t to a i r dry i n the dark f o r two hours. 8 1 Once d r i e d , the s l i d e s were placed i n s l i d e boxes along with a package of D r y e r i t e . The boxes were se a l e d with black e l e c t r i c a l tape, wrapped i n a heavy, b l a c k , p l a s t i c bag, and s t o r e d i n the r e f r i g e r a t o r at 4 C f o r 17 days. A f t e r 17 days of exposure, t h e s l i d e boxes were opened i n the darkroom using a s a f e t y l i g h t and the coated s l i d e s were processed i n the f o l l o w i n g manner., 1.1 4 minutes i n D-19 Developer (at 20 C). 2. ) 30 seconds i n water (at 20 C ) . 3. ) 8 minutes i n Kodak f i x e r (at 20 C ) . , 4. ) 1 minute i n Edwal 4 and 1 h y p o e l i m i n a t o r (at 20 C) . 5. ) Hashed i n water f o r 20 minutes. 6. ) A i r d r i e d . When the emulsion on the s l i d e s was dry, the t i s s u e s e c t i o n s were s t a i n e d with E h r l i c h * s hematoxylin and e o s i n s t a i n and then permanently covered. S e c t i o n s were examined under both b r i g h t f i e l d i l l u m i n a t i o n and phase c o n t r a s t with a Z e i s s GFL microscope. , Results The gross anatomy of the guppy ovary was d e s c r i b e d by Turner (1937). The ovary i n P o e c i l i a r e t i c u l a t a i s a s i n g l e , hollow organ which occupies a major p o r t i o n of the body c a v i t y . The o v a r i a n lumen occupies mainly the p o s t e r o -d c r s a l part of the ovary, and connects to the e x t e r i o r v i a a 82 shor t duct t h a t e x i t s j u s t p o s t e r i o r to the anus. In the c a u d a l p o r t i o n of the ovary the oocytes are l a t e r a l and v e n t r a l to the lumen, but more a n t e r i o r l y they r e s t r i c t , the lumen. F i g u r e 2 presents a diagram of a l o n g i t u d i n a l s e c t i o n of a guppy ovary. The c r o s s - s e c t i o n a l shape of the lumen v a r i e s between females, and can be anywhere from d o r s o - v e n t r a l l y f l a t t e n e d t o l a t e r a l l y f l a t t e n e d . I n approximately the a n t e r i o r two-t h i r d s of the ovary, p r o j e c t i o n s of the lumen extend amongst the o o c y t e s . , There i s no g e n e r a l p a t t e r n to the d i s t r i b u t i o n of these p r o j e c t i o n s , but they a r e i n c l o s e p r o x i m i t y to a l l oocytes.. The e p i t h e l i u m e n c l o s i n g the lumen i s composed of a s i n g l e l a y e r of columnar c e l l s . , apparently oocyte d i s t r i b u t i o n w i t h i n the ovary i s c o r r e l a t e d with oocyte m a t u r i t y . Three l e v e l s of oocyte development, as w e l l as d e v e l o p i n g embryos, were evident i n females inseminated twenty days before f i x a t i o n . The s m a l l e s t oocytes occurred c e n t r a l l y i n the ovary, surrounding the lumen and i t s p r o j e c t i o n s . These were d i s t i n g u i s h e d from the other oocytes by a dense, homogeneous appearance ( P l a t e 2B). D s u a l l y a s m a l l nucleus occurred i n the center of each, but o c c a s i o n a l l y only a hole was p r e s e n t . Successive stages of egg development oc c u r r e d more d i s t a l t o the lumenal p r o j e c t i o n s and embryos tended to occur towards the o u t s i d e of the ovary.,, Oocytes a t an i n t e r m e d i a t e stage of development possessed an obvious n u c l e u s , surrounded by a v e s i c u l a r - l i k e matrix (most l i k e l y y o l k ) . A c u b o i d a l c e l l u l a r l a y e r surrounded the e n t i r e 83 F i g u r e 2: Diagram of a l o n g i t u d i n a l s e c t i o n of a guppy ovary. D e t a i l s of i n t e r n a l s t r u c t u r e s are not to s c a l e . . 85 P l a t e 2: A) A tubule c o n t a c t i n g an oocyte. Exposure of the emulsion can be seen at the d i s t a l end of the t u b u l e . B) An immature oocyte at an e a r l y stage of development. Nucleus i s ev i d e n t and i s surrounded by a homogeneous matrix. C) An immature oocyte at an i n t e r m e d i a t e stage of development. Note the i n t e n s e l y exposed area of the emulsion (lb) a t the d i s t a l end of a t u b u l e c o n t a c t i n g the oocyte. . D) M a g n i f i c a t i o n of P l a t e 2A showing spermatozoa and exposed emulsion a t the d i s t a l end of the t u b u l e . E) M a g n i f i c a t i o n of P l a t e 2C showing the i n t e n s e l y exposed area of the emulsion ( l b ) . . Spermatozoa are j u s t v i s i b l e at the edge of the exposed a r e a s . E) Same tubule as i n P l a t e 2E, but one s e c t i o n l a t e r . The spermatozoa appear t o be o r i e n t e d with the tubule e p i t h e l i u m . Symbols eg - oocyte l b - exposed emulsion lm - o v a r i a n lumen sp - spermatozoa tb - tubule 87 oocyte {Plate 2C)» I co u l d not see a nucleus i n the l a r g e s t oocytes, and although the ' egg c o n s i s t e d of a dense, homogeneous matrix {yolk), t h i s matrix tended t o be broken up. A l a y e r o f squamous c e l l s surrounded oocytes at t h i s stage o f development. , A s s o c i a t e d with each oocyte was a s i n g l e t u b u l e of varying l e n g t h . These t u b u l e s a p p a r e n t l y r e p r e s e n t i n v a g i n a t i o n s of the columnar l a y e r of the lumenal e p i t h e l i u m . Mostly, the t u b u l e s end b l i n d l y , and separate the oocyte or f o l l i c u l a r membrane of o l d e r oocytes from the lumen by a s i n g l e columnar e p i t h e l i a l l a y e r . O c c a s i o n a l l y however, t u b u l e s appear to open onto the l a r g e r , y o l k y oocytes.. The t u b u l e s a l s o p e r s i s t where embryos are dev e l o p i n g , but here these t u b u l e s are completely open. Undoubtedly, young pass through these channels a t b i r t h . P l a t e 2A i l l u s t r a t e s a t u b u l e c o n t a c t i n g an oocyte. A p p a r e n t l y , sperm are s t o r e d i n the t u b u l e s . In some cases, I found sperm a s s o c i a t e d with the e p i t h e l i u m of the main p o r t i o n of the o v a r i a n lumen, p a r t i c u l a r l y with the d o r s a l e p i t h e l i u m : however, such sperm o c c u r r e d i n low numbers and without any c o n s i s t e n t o r i e n t a t i o n t o the e p i t h e l i u m . In c o n t r a s t , sperm abounded i n the tub u l e s near the d i s t a l end. Such sperm were g e n e r a l l y o r i e n t e d i n a r e g u l a r p a t t e r n with the e p i t h e l i u m {Plate 2F). U n f o r t u n a t e l y , the q u a l i t y of t i s s u e p r e p a r a t i o n d i d not allow a f i n e r examination and I was unable to assess the a c t u a l a s s o c i a t i o n between the tu b u l e e p i t h e l i u m and the sperm. Most f r e q u e n t l y , sperm were i n t u b u l e s a s s o c i a t e d 88 with the o l d e s t oocytes, hat sperm c o u l d be found i n the tubu l e s a s s o c i a t e d with oocytes of a l l ages. although the a u t o r a d i o g r a p h i c a n a l y s i s of sperm a c t i v i t y was r e l a t i v e l y s u c c e s s f u l , i t was not as p r e c i s e a d i a g n o s t i c t o o l as I had hoped. „ I had hoped that r a d i o a c t i v e l e v e l s i n sperm would be high enough to i d e n t i f y i n d i v i d u a l , l a b e l l e d sperm. T h i s was not so. R a d i o a c t i v e sperm c o u l d only be detected when sperm were clumped and c l o s e to the s u r f a c e cf the t i s s u e s e c t i o n . Since sperm g e n e r a l l y occurred at high d e n s i t i e s i n t u b u l e s , f i n d i n g l a b e l l e d sperm t h e r e presented no problem, but d i s t i n g u i s h i n g between l a b e l l e d and u n l a b e l l e d sperm was i m p o s s i b l e . I d e a l l y , a comparison of the r e l a t i v e p r o p o r t i o n o f marked sperm i n the c o n t r o l and treatment females would g i v e the most i n f o r m a t i o n ^ , U n f o r t u n a t e l y , high sperm d e n s i t i e s and the i n d i r e c t way o f d e t e c t i n g r a d i o a c t i v i t y made sperm counts u n r e l i a b l e . When females were inseminated with u n l a b e l l e d sperm and then r e - i n s e m i n a t e d with l a b e l l e d sperm, r a d i o a c t i v i t y was de t e c t e d i n a l l t u b u l e s t h a t c o n t a i n e d sperm. In t u b u l e s with high d e n s i t i e s of sperm the l a b e l was p a r t i c u l a r l y e v i d e n t . At lower d e n s i t i e s , s i g n s of r a d i o a c t i v i t y were s c a t t e r e d . U n f o r t u n a t e l y , I c o u l d not t e l l i f a l l of these sperm were l a b e l l e d . In the r e c i p r o c a l s et of i n s e m i n a t i o n s r a d i o a c t i v i t y c o u l d not be d e t e c t e d , although c o n t r o l females contained r a d i o a c t i v e sperm. Again, I c o u l d not t e l l i f a l l sperm were from the second i n s e m i n a t i o n s i n c e l a b e l l e d sperm c o u l d have been present i n low numbers, but 89 the i n d i c a t i o n from the two s e t s of i n s e m i n a t i o n s i s that sperm from the second i n s e m i n a t i o n predominated i n the t u b u l e s . The o v a r i e s of treatment females were not examined i f r a d i o a c t i v e sperm c o u l d not be found i n the o v a r i e s of the c o r r e s p o n d i n g c o n t r o l females, For t h i s reason, one female from each of the r e c i p r o c a l i n s e m i n a t i o n s was d i s c a r d e d . P l a t e s 2D, 2E, and 2F show l e v e l s of r a d i o a c t i v i t y i n c l u s t e r s of sperm f o r two d i f f e r e n t i n s e m i n a t i o n s . In P l a t e 2D, the female was inseminated i n i t i a l l y with u n l a b e l l e d sperm and then re-inseminated with l a b e l l e d sperm. R a d i o a c t i v e l a b e l l i n g does not appear heavy i n t h i s photomicrograph, but a t lower m a g n i f i c a t i o n (Plate 2A) great e r exposure of the emulsion i s ev i d e n t . , In P l a t e s 2E and 2F, the female was re-inseminated with r a d i o a c t i v e l y l a b e l l e d sperm only. Note t h a t the c o r r e l a t i o n between exposed emulsion and sperm i s net p e r f e c t and t h a t the extent of exposed emulsion may vary between s e c t i o n s . P o s s i b l y t h i s d i f f e r e n c e i n d e t e c t i o n of l a b e l between s e c t i o n s r e s u l t s from sperm o c c u r r i n g a t d i f f e r e n t depths i n the s e c t i o n . J a l a b e r t and B i l l a r d (1969) and Kadow (1954) r e p o r t e d sperm i n a n t e r o - d o r s a l d i v e r t i c u l a e of the o v a r i a n lumen and they c o n s i d e r e d these d i v e r t i c u l a e to be the major sperm storage s i t e . , Although i n my s e c t i o n s , the lumen of some o v a r i e s ended a n t e r i o r l y i n b l i n d pockets, I d i d not f i n d sperm c l u s t e r s i n them. O c c a s i o n a l l y sperm were present i n these p o c k e t s , but never i n l a r g e numbers. Thus, even i f 90 some of these sperm were l a b e l l e d , I probably would not have detected r a d i o a c t i v i t y . D i s c u s s i o n The ovary of the guppy i s an extremely complex organ t h a t probably serves s e v e r a l f u n c t i o n s . Hot only i s i t the s i t e of oogenesis, but a l s o i t i s the s i t e of f e r t i l i z a t i o n . In a d d i t i o n , i t provides the necessary requirements f o r i n t e r n a l embryo development., In many p o e c i l i i d s , only qas exchange and p r o t e c t i o n are provided by the ovary, but i n other s p e c i e s the female p r o v i d e s some, or a l l , of the n u t r i t i o n a l requirements (in a d d i t i o n to yolk) of the embryo (Scrimshaw, 1945; T h i b a u l t and S c h u l t z , 1978; Turner, 1940). In c o n t r a s t to other o v o v i v i p a r o u s or v i v i p a r o u s f i s h , the ova r i a n f o l l i c l e p l a y s an important r o l e i n ontoqenesis i n p o e c i l i i d s . T h i s s t r u c t u r e remains i n t a c t u n t i l p a r t u r i t i o n (Turner, 1937) . . T h i s p a r t i c u l a r mode of o v o v i v i p a r i t y (and v i v i p a r i t y ) can occur only i n c o n j u n c t i o n with s p e c i a l m o d i f i c a t i o n s f o r f e r t i l i z a t i o n . I f the oocyte does not e n t e r the lumen before f e r t i l i z a t i o n , t here must be some way f o r sperm t o penet r a t e the b a r r i e r s between sperm and egg. In P o e c i l i a r e t i c u l a t a , t h i s problem apparently i s s o l v e d by the c o n t a c t between t u b u l e s and ooc y t e s . The c o n s i s t e n c y and i n t i m a c y of the tubule and oocyte a s s o c i a t i o n suggests the t u b u l e s 91 are the s i t e of f e r t i l i z a t i o n . Other workers a l s o noted the e x i s t e n c e of t u b a l e s . Eyder (1885), Stuhlman (1887), and B a i l e y (1933) r e f e r r e d to them as " d e l l e s " . , Hinge (1927) and Kadow (1954) both r e p o r t e d sperm i n the t u b u l e s , and B i l l a r d (1966) r e f e r r e d to them as "entonnoir de f e c o n d a t i o n " , implying that they f u n c t i o n as a route f o r spermatozoa during f e r t i l i z a t i o n . The development of t h i s a s s o c i a t i o n between t u b u l e s and oocytes has not been d e s c r i b e d . . Apparently the a s s o c i a t i o n develops e a r l y , s i n c e t u b u l e s are found a s s o c i a t e d with the s m a l l e s t oocytes i n the ovary. Sperm do not appear to d i s c r i m i n a t e between t u b u l e s , and are found i n t u b u l e s a s s o c i a t e d with oocytes of any age. , Hinge (1927) presents a photomicrograph of a c l u s t e r of sperm i n a t u b u l e c o n t a c t i n g a s m a l l , immature oocyte. There i s some q u e s t i o n , however, as to t h e exact s i t e of sperm sto r a g e . Sperm commonly occur i n the t u b u l e s f o r up to t s e n t y days a f t e r i n s e m i n a t i o n , but the d u r a t i o n of v i a b i l i t y i n these s t r u c t u r e s has not been assessed (Kadow, 1954; Hinge, 1927). , The presence of spermatozoa i n a n t e r o -d o r s a l pockets of the lumen a l s o i s known ( J a l a b e r t and B i l l a r d , 1969; Kadow, 1954).. C u r i o u s l y , J a l a b e r t and B i l l a r d (1969) do not mention sperm storage i n the t u b a l e s . They c o n s i d e r the s i t e of sperm storage to be i n the a l v e o l a ted d i v e r t i c u l a e of the a n t e r o - d o r s a l p o r t i o n of the ovary, U l t r a s t r u c t u r a l l y , sperm i n t h i s region are embedded i n f o l d s of the e p i t h e l i a l c e l l plasma membranes, a s i t u a t i o n common i n v e r t e b r a t e s that s t o r e sperm. 92 Although i n some females I found pockets a t the antero-d o r s a l end of the o v a r i a n lumen, these pockets contained few sperm. In a d d i t i o n , I o c c a s i o n a l l y found sperm i n the d o r s a l e p i t h e l i u m .of the lumen. However, due to low sperm density and i r r e g u l a r sperm o r i e n t a t i o n i n these s t r u c t u r e s , I c o u l d not c o n f i r m the e a r l i e r workers* o b s e r v a t i o n s that these a r e the major sperm s t o r a g e s i t e s . In t h i s study the use of a r a d i o a c t i v e l a b e l t o i d e n t i f y the sperm of d i f f e r e n t i n s e m i n a t i o n s proved to be only p a r t i a l l y s a t i s f a c t o r y . In autoradiography, the r a d i o a c t i v e m a t e r i a l i s i d e n t i f i e d by c o r r e l a t i n g an exposed r e g i o n i n the emulsion with some s t r u c t u r e immediately beneath the exposed r e g i o n . I f an i s o t o p e with a low beta p a r t i c l e energy, such as t r i t i u m , i s used, a number of problems are encountered. The most s i g n i f i c a n t i s s e l f -a b s o r p t i o n by the t i s s u e s e c t i o n . . as s e c t i o n t h i c k n e s s i n c r e a s e s , the e f f i c i e n c y of r e c o r d i n g the r a d i o i s o t o p e i n the emulsion drops r a p i d l y (Palk and King, 1963). In t h i s study I had to compromise on s e c t i o n t h i c k n e s s to o b t a i n a good s e t of s e r i a l s e c t i o n s , and as a consequence, I s a c r i f i c e d e f f i c i e n c y . T h i s i s undoubtedly t h e reason why r a d i o a c t i v i t y c o u l d be detected only when spermatozoa were near the s u r f a c e of the s e c t i o n * . In a d d i t i o n to s e l f -a b s o r p t i o n , exposure to background r a d i a t i o n and contamination of s e c t i o n s , p a r t i c u l a r l y by spreading the c o n t e n t s of ruptured c e l l s a c r o s s the s e c t i o n s , must be coped with i n autoradiography. I d e a l l y , the advantage of low energy r a d i o i s o t o p e s i s high r e s o l u t i o n . However, i n my 93 study, t h i s advantage was negated by t h i c k s e c t i o n s and continuous t e a r i n g of t i s s u e s d u r i n g s e c t i o n i n g . Since r a d i o a c t i v i t y was present i n tubule sperm c l u s t e r s when the second i n s e m i n a t i o n c o n t a i n e d l a b e l l e d sperm, but was not present i n sperm c l u s t e r s when the f i r s t i n s e m i n a t i o n c o n t a i n e d l a b e l l e d sperm, the sperm i n the tub u l e s of the treatment females must have come mostly from the second i n s e m i n a t i o n . How these sperm come to predominate i n the tub u l e s i s a matter of s p e c u l a t i o n . Since sperm are found i n t u b u l e s a s s o c i a t e d with a l l stages of oocytes, i t i s p o s s i b l e t h a t sperm p e r s i s t i n a t u b u l e u n t i l the oocyte matures. I f t h i s occurs, a second i n s e m i n a t i o n can only dominate i n . these t u b u l e s by d i s p l a c i n g the sperm alr e a d y present. . A l t e r n a t i v e l y , i f the tubules are f r e e of sperm at the time of the second i n s e m i n a t i o n , sperm from t h i s new i n s e m i n a t i o n can take up the a v a i l a b l e space. . It i s not known how long sperm p e r s i s t i n the t u b u l e s . , In c o n t r o l females k i l l e d 20 days a f t e r i n s e m i n a t i o n , sperm s t i l l p e r s i s t e d i n the t u b u l e s . These females c o n t a i n e d w e l l developed embryos and t h e r e f o r e the oocytes a s s o c i a t e d with the t u b u l e s probably were not mature., However, although sperm may p e r s i s t i n the tubules f o r up t o 20 days, I am not c e r t a i n how many t u b u l e s i n treatment females contained sperm from the f i r s t i n s e m i n a t i o n when the females were re - i n s e m i n a t e d . , In these treatment females, not a l l tubules c o n t a i n e d sperm. Conseguently I cannot t e l l i f sperm from r e - i n s e m i n a t i o n s d i s p l a c e d sperm a l r e a d y present 94 or i f sperm were s t o r e d i n unoccupied t u b u l e s . Begardless of whether or not sperm from the second i n s e m i n a t i o n gain access to t u b u l e s by d i s p l a c i n g pre-e x i s t i n g sperm, the o b s e r v a t i o n t h a t sperm from the second i n s e m i n a t i o n dominate before eggs are ready to be f e r t i l i z e d e x p l a i n s the r e s u l t s of the pr e v i o u s two s e c t i o n s . ahen an i n s e m i n a t i o n occurs during g e s t a t i o n , the sperm gain a c c e s s t o the t u b u l e s and p e r s i s t i n them u n t i l the a s s o c i a t e d oocytes are ready f o r f e r t i l i z a t i o n . Since eggs are f e r t i l i z e d a few days a f t e r p a r t u r i t i o n (Rosenthal, 1954; T h i b a u l t and S c h u l t z , 1978; Turner, 1937) , sperm from r e -i n s e m i n a t i o n s immediately f o l l o w i n g p a r t u r i t i o n can a l s o gain access to the t u b u l e s before mature eggs are f e r t i l i z e d , and thus may dominate. These sperm have precedence. I f f e r t i l i z a t i o n of a new brood of eggs begins before r e - i n s e m i n a t i o n , the r e - i n s e m i n a t i o n w i l l not c o n t r i b u t e as h e a v i l y t o the next brood. Rosenthal (1952) has shown t h a t the success of r e - i n s e m i n a t i o n s decreases as the time between p a r t u r i t i o n and r e - i n s e m i n a t i o n i n c r e a s e s . Indeed, i f sperm precedence i s achieved i n such a manner, the number sperm i n a r e - i n s e m i n a t i o n w i l l only have an a f f e c t i f t h e r e are not enough sperm to gain access to a l l tub u l e s a s s o c i a t e d with maturing oocytes. I t i s u n l i k e l y that sperm are only s t o r e d i n t u b u l e s a s s o c i a t e d with oocytes. Kadow (1954) and J a l a b e r t and B i l l a r d (1969) i d e n t i f i e d d i v e r t i c u l a e i n the a n t e r o - d o r s a l p o r t i o n of the ovary t h a t can c o n t a i n l a r g e numbers of sperm. The a s s o c i a t i o n between the spermatozoa and the 9 5 plasma membrane of the lumenal e p i t h e l i a l c e l l s i n t h i s r e g i o n i n d i c a t e s these are a s i t e of long-term s t o r a g e . , In a d d i t i o n to t h i s anatomical i n f o r m a t i o n , many commonly re c o g n i z e d r e p r o d u c t i v e phenomema i n guppies suggest t h a t sperm must p e r s i s t i n a g e n e r a l s t o r a g e area. I f sperm were s t o r e d s o l e l y i n the t u b u l e s , the eggs of s e v e r a l broods would have to be present i n the ovary when an i n s e m i n a t i o n o c c u r r e d . . He know t h a t up to e i g h t broods can be f e r t i l i z e d from a s i n g l e i n s e m i n a t i o n (Hinge, 1937). However, I have found only t h r e e groups of oocytes ( r e p r e s e n t i n g d i f f e r e n t stages of development) i n the ovary. Turner (1937) and S t o l k (1951) a l s o reported only t h r e e stages and t h i s makes i t u n l i k e l y t h a t the eggs of e i g h t broods would be developed enough to possess tubules. A l s o , i n s t u d i e s examining i n t r a s p e c i f i c sperm c o m p e t i t i o n , the f i r s t i n s e m i n a t i o n i s , o c c a s i o n a l l y , r e - i n s t a t e d a f t e r an i n i t i a l dominance by sperm from the second: i n s e m i n a t i o n (Hildemann and Wagner, 1954). Again, i f the t u b u l e s were the s o l e sperm st o r a g e s i t e s , t h i s change i n sperm dominance c o u l d be accounted f o r only i f sperm from r e - i n s e m i n a t i o n s were s e l e c t i v e a g a i n s t t u b u l e s a s s o c i a t e d with the l e a s t mature oocytes. T h i s does not appear to be the case ( l i n g e , 19 22; pers obs.). The most t e n a b l e model at t h i s time i s t h a t a more gene r a l sperm storage r e c e p t a c l e e x i s t s i n a d d i t i o n to the t u b u l e s . At c o p u l a t i o n , a p o r t i o n of the sperm move to the t u b u l e s and a p o r t i o n move to a r e c e p t a c l e i n the a n t e r o -d o r s a l p o r t i o n of the ovary. Sperm i n the t u b u l e s f e r t i l i z e oocytes as they mature. A f t e r p a r t u r i t i o n , another 9 6 c o p u l a t i o n again sends sperm t o both r e g i o n s of the ovary. Those going to the t u b u l e s g a i n access before the oocytes mature, and as the oocytes mature, these sperm f e r t i l i z e them before sperm from the previous i n s e m i n a t i o n can gain access to the oocytes. Those sperm from the r e - i n s e m i n a t i o n which enter the sperm s t o r a g e r e c e p t a c l e are s t o r e d i n a p o s i t i o n t h a t g i v e s them a c o m p e t i t i v e edge over a l r e a d y e x i s t i n g sperm, and thus give them precedence i f a new i n s e m i n a t i o n does not occur. As the sperm from the most re c e n t i n s e m i n a t i o n are used up, sperm from previous i n s e m i n a t i o n s can again i n c r e a s e t h e i r success i n f e r t i l i z i n g eggs.. An examination c f how sperm from d i f f e r e n t i n s e m i n a t i o n s are d i s t r i b u t e d i n the a n t e r o - d o r s a l o v a r i a n storage s i t e would g i v e some i n s i g h t i n t o t h i s problem. 3 97 General D i s c u s s i o n ft wide v a r i e t y of female sperm storage systems occur i n f i s h e s . . Although i n t e r n a l f e r t i l i z a t i o n i s t y p i c a l of s e v e r a l f a m i l i e s , sperm st o r a g e does not f o l l o w a c o n s i s t e n t taxonomic p a t t e r n . . Sperm sto r a g e occurs i n . s h a r k s , s u r f -perches {Embictocidae), and p o e c i l i i d s . Other groups probably a l s o s t o r e sperm, but to date, t h e r e has been no attempt t o survey the phenomenon i n a l l f i s h e s . I n t e r n a l f e r t i l i z a t i o n (a p r e - r e g u i s i t e f o r sperm storage] i s rep o r t e d i n some genera of c o t t i d s { i . e . C l i n o c o t t u s , O l i q o c o t t u s . and Orthonopias - B o l i n , 1941; M o r r i s , 1952, 1956). Morris {1952) re p o r t e d a case of p o s s i b l e sperm storage i n C l i n o c o t t u s r e c a l v u s . , In Sebastes, i n t e r n a l f e r t i l i z a t i o n and o v o v i v i p a r i t y a l s o o c c u r s , but too l i t t l e i s known about r e p r o d u c t i o n i n t h i s group to comment on the p o s s i b i l i t y of sperm storage {Breder and Bosen, 1966).. A l l three f i s h groups that are known to s t o r e sperm possess s t r i k i n g l y d i f f e r e n t modes of r e p r o d u c t i o n . There are d i f f e r e n c e s i n t h e i r female r e p r o d u c t i v e systems, and conseguently i n t h e i r modes of sperm s t o r a g e . In a d d i t i o n , the r e p r o d u c t i v e ecology of each group i s s u f f i c i e n t l y d i f f e r e n t t o suggest that sperm st o r a g e may ser v e d i f f e r e n t f u n c t i o n s i n each group. The shark r e p r o d u c t i v e system has a t y p i c a l v e r t e b r a t e o r g a n i z a t i o n . As i n a l l ether v e r t e b r a t e s t h a t s t o r e sperm, sperm a r e s t o r e d , and f e r t i l i z a t i o n takes p l a c e , i n the 98 r e p r o d u c t i v e t r a c t . , Hetten (1911) r e p o r t s sperm i n the tub u l e s of the o v i d u c a l gland i n S c y l i o r h i n u s c a n i c u l a . In the b l u e shark, Prionace g l a u c a , spermatozoa occur i n the tubules i n the p o s t e r i o r s h e l l - s e c r e t i n g p o r t i o n of the o v i d u c a l gland ( P r a t t , 1979). , Since a f u n c t i o n a l s h e l l i s net produced i n t h i s s p e c i e s , P r a t t suggests the s h e l l -s e c r e t i n g t u b u l e s have taken on a new f u n c t i o n - sperm s t o r a g e , The u l t r a s t r u c t u r a l d e t a i l s of sperm storage i n sharks are unknown. The d u r a t i o n c f sperm r e t e n t i o n i n females i s unknown i n most s p e c i e s , but i n female blue sharks, sperm may p e r s i s t f o r one t c two years ( P r a t t , 1 9 7 9 ) . In t h i s s p e c i e s , f i v e year o l d s mate i n l a t e s p r i n g , but f e r t i l i z a t i o n does not occur u n t i l the f o l l o w i n g s p r i n g . G e s t a t i o n takes 9 - 1 2 months and another mating i s probably r e q u i r e d t o r e p l e n i s h the sperm supply ( P r a t t , 1979). Since f e r t i l i z a t i o n i s delayed f o r a year, sperm s t o r a g e i s an e s s e n t i a l p a r t of blue shark r e p r o d u c t i o n . U n f o r t u n a t e l y , the f u n c t i o n a l s i g n i f i c a n c e of delayed f e r t i l i z a t i o n i n t h i s s p e c i e s i s not apparent, Embiotocids and p o e c i l i i d s , although p h y l o g e n e t i c a l l y u n r e l a t e d , have s i m i l a r modes of r e p r o d u c t i o n . T h e i r r e p r o d u c t i v e systems are t y p i c a l of t e l e o s t s . Females have a s i n g l e , hollow ovary (fused p a i r e d o v a r i e s ) which connects t o the e x t e r i o r v i a a sh o r t duct., Both groups are l i v e -bearers and s t o r e sperm i n t u b u l e s or d i v e r t i c u l a e i n the ovary. T h e i r r e p r o d u c t i v e c y c l e s , however, d i f f e r d r a s t i c a l l y and as a consequence, sperm s t o r a g e appears t o 99 serve d i f f e r e n t r o l e s i n ,the two f a m i l i e s . Embiotocids, or s u r f - p e r c h e s , are i n s h o r e , marine p e r c o i d s . They are endemic t o the north P a c i f i c Ocean. Eigenmann (1892) was the f i r s t to note that male and female r e p r o d u c t i v e c y c l e s are s i x months out of phase. , He s t u d i e d Cymatogaster aggreqata and d i s c o v e r e d t h a t sperm sere s t o r e d f o r prolonged p e r i o d s . . Apparently, females are inseminated i n t h e summer and f e r t i l i z a t i o n occurs i n the winter when eggs a r e mature. G e s t a t i o n takes approximately s i x months {Turner, 1952; Biebe, 1968)., Sperm are s t o r e d i n pockets i n the lumenal e p i t h e l i u m and are i n t i m a t e l y a s s o c i a t e d with the e p i t h e l i a l c e l l s (Gardiner, 1976). U l t r a s t r u c t u r a l i n v e s t i g a t i o n s r e v e a l t h a t sperm are embedded i n i n v a g i n a t i o n s of the c e l l plasma membrane, much as occurs i n other sperm s t o r i n g v e r t e b r a t e s ( C u e l l a r , 1966; Hoffmann and Himsatt, 1972; Simsatt e t a l , 1966)., As i n s h a r k s , sperm s t o r a g e a p p a r e n t l y p l a y s an e s s e n t i a l p a r t of embiotocid r e p r o d u c t i o n . Given the long g e s t a t i o n p e r i o d (Turner, 1952; Wiebe, 1968), delayed f e r t i l i z a t i o n i s l i k e l y a s t r a t e g y to ensure t h a t c r i t i c a l stages i n embryo development do not occur d u r i n g the winter and t h a t young are born i n l a t e s p r i n g or e a r l y summer when food may be more a v a i l a b l e . I t i s not known i f sperm p e r s i s t i n the ovary beyond f e r t i l i z a t i o n . , In c o n t r a s t to embiotocids, the female r e p r o d u c t i v e c y c l e i n p o e c i l i i d s i s s h o r t , ranging from about 22 days to 40 days (Colson, 1969; Turner, 1937; van Oordt, 1928; Winge, 1937). Eor s h o r t p e r i o d s i n each c y c l e , females are 100 r e c e p t i v e and f e r t i l i z a t i o n g e n e r a l l y occurs a few days a f t e r i n s e m i n a t i o n ( D i l d i n e , 19.31; Rosenthal, 1952; Turner, 1937). Sperm can remain v i a b l e i n the ovary f o r s e v e r a l months and f e r t i l i z e each new batch o f eggs as they mature (van Oordt, 1928; Hinge, 1937). T h i s s i t u a t i o n i s unique i n the v e r t b r a t e s . Females g e n e r a l l y mate with more than one male (Borowsky and Kallman, 1976; Haskins et a l , 1961) even thouqh sperm from each i n s e m i n a t i o n p e r s i s t s i n the ovary. Consequently the r o l e of sperm storage i n p o e c i l i i d r e p r o d u c t i o n i s not c l e a r . The f a c t t h at female p o e c i l i i d s are a b l e to maintain r e p r o d u c t i v e output when sperm a v a i l a b i l i t y i s low suqqests t h a t sperm storaqe may be an a d a p t a t i o n t o e c o l o q i c a l f a c t o r s t hat a f f e c t the a v a i l a b i l i t y of males (e.g. s e l e c t i v e m o r t a l i t y on . males, drought and f l o o d i n g which might l e a v e females i s o l a t e d from males).. Sperm s t o r a g e would allow females, i n the absence of males, t o c o l o n i z e new h a b i t a t s or r e c o l o n i z e areas s u b j e c t t o e c o l o g i c a l c a t a s t r o p h e . . However, evidence to suggest that female p o e c i l i i d s commonly f a c e shortages i n sperm supply i s c i r c u m s t a n t i a l . P o e c i l i i d s l i v e i n a v a r i e t y of h a b i t a t s ranging from fast-moving, deep streams t o s h a l l o w , lowland streams and ponds. In fast-moving streams, females may be washed downstream, but there i s no evidence t h a t females swept t o another area can e s t a b l i s h a new p o p u l a t i o n or t h a t t h i s i s a frequent occurrence. I n lowland ponds and streams, f l o o d s may a l s o sweep females i n t o new areas, but again the freguency of t h i s o c c u r r i n g i s unknown. F l o o d i n g 101 may allow c o l o n i z a t i o n of new areas or r e c o l o n i z a t i o n of p r e v i o u s l y populated areas. However, i t seems u n l i k e l y that only females move i n t o these newly f l o o d e d r e g i o n s . In T r i n i d a d , ponds c o n t a i n i n g guppies dry up i n the dry season and a r e fl o o d e d again i n the wet season ( L i l e y , p e rs. comm.). L i l e y observed that males and females r e c o l o n i z e d these f l o o d e d areas t o g e t h e r . Sex r a t i o s skewed i n favour of females have been r e p o r t e d i n p o p u l a t i o n s of some p o e c i l i i d s (Krumholtz, 1963; Seghers, 197 3). I f t h i s skewing of sex r a t i o s i s common i n p o e c i l i i d p o p u l a t i o n s and c r e a t e s a sperm supply shortage, sperm s t o r a g e may be an a d a p t a t i o n t o low male a v a i l a b i l i t y . U n f o r t u n a t e l y , i t i s not known how skewed the sex r a t i o must be to produce a sperm supply shortage. Since females are r e c e p t i v e f o r only p a r t of t h e i r r e p r o d u c t i v e c y c l e , the e f f e c t i v e sex r a t i o at any gi v e n moment i s probably d i f f e r e n t from the observed sex r a t i o . , Thus, f o r a shortage i n sperm supply t o e x i s t , the p r o p o r t i o n of males i n p o p u l a t i o n s may have to be much lower than normally observed. Since p o e c i l i i d females are capable of s t o r i n g sperm f o r r e l a t i v e l y long periods, i t i s c u r i o u s t h a t females remain s u s c e p t a b l e to f u r t h e r i n s e m i n a t i o n s . A s i n g l e mating p r o v i d e s enough sperm f o r more than one brood and only when the sperm supply i n the ovary dwindles do females r e g u i r e another i n s e m i n a t i o n . The f a c t that females of some p o e c i l i i d s { Xiphophcr us macnlatus and P o e c i i i a r e t i c u l a t a ) are inseminated by more than one male suggests t h a t m u l t i p l e 102 i n s e m i n a t i o n combined with sperm storage has some ad a p t i v e value. M u l t i p l e i n s e m i n a t i o n i n p o e c i l i i d s means t h a t females can accumulate sperm i n t h e i r o v a r i e s . , B i l l a r d (1966) and Zander (1962 - c i t e d by Borowsky and Kallman, 1976) r e p o r t e d t h a t i n P o e c i l i a r e t i c u l a t a and Xiphophorus brood s i z e i n c r e a s e s as the number of spermatophores (and hence the amount of sperm) i n i n s e m i n a t i o n s i n c r e a s e s . These r e s u l t s suggest that i f the number of spermatophores t r a n s f e r r e d d u r i n g i n s e m i n a t i o n i s s m a l l , s t o r i n g sperm from these i n s e m i n a t i o n s may r e s u l t i n i n c r e a s e d brood s i z e s . O n f o r t u n a t e l y , the number of spermatophores t r a n s f e r r e d d u r i n g i n s e m i n a t i o n i s unknown., B i l l a r d ' s (1966) data i n d i c a t e d t h a t i n P o e c i l i a r e t i c u l a t a , broods r e s u l t i n g from a r t i f i c i a l i n s e m i n a t i o n s with 100 spermatophores were s l i g h t l y l a r g e r than broods produced a f t e r n a t u r a l i n s e m i n a t i o n s . T h i s would suggest t h a t i f sperm from a r t i f i c i a l i n s e m i n a t i o n s are as e f f e c t i v e as sperm i n n a t u r a l i n s e m i n a t i o n s , l e s s than 100 spermatophores are t r a n s f e r r e d i n n a t u r a l , i n s e m i n a t i o n s . I f sperm from a r t i f i c i a l i n s e m i n a t i o n s are l e s s e f f e c t i v e than sperm i n n a t u r a l i n s e m i n a t i o n s , n a t u r a l i n s e m i n a t i o n s may c o n t a i n c o n s i d e r a b l y fewer than 100 spermatophores. S i n c e the mean brood s i z e o b tained from a l l i n s e m i n a t i o n s by B i l l a r d (1966) was much l e s s than brood s i z e s r e p o r t e d by other workers (Rosenthal, 1952; T h i h a u l t and S c h u i t z , 1978; pers. obs.),, p o s s i b l y the amount of sperm i n one of B i l l a r d ' s i n s e m i n a t i o n s was not enough t o produce the maximum brood s i z e . B e s u l t s from guppy mate s e l e c t i o n experiments by 103 G a n d o l f i (1971) provide f u r t h e r evidence t h a t the number of sperm i n a s i n g l e i n s e m i n a t i o n i s i n s u f f i c i e n t , to produce the l a r g e s t brood. Females inseminated by two males produced broods almost twice as l a r g e as broods produced by females inseminated by a s i n g l e male. Thus, i f accumulation of sperm i n the ovary has the same e f f e c t as i n c r e a s i n g the number of sperm i n i n s e m i n a t i o n s , sperm storage may be a means f o r females to maximize r e p r o d u c t i v e output. „ Since female guppies expend more i n r e p r o d u c t i o n than males and hence would probably l o s e the most from mistakes i n mate s e l e c t i o n , females are expected t o be more s e l e c t i v e than males when chosing a mate. Indeed, female guppies do appear to be s e l e c t i v e i n t h e i r c h o i c e of males to mate with ( G a n d o l f i , 1971; P a r r , 1976, 1977).,. Although female guppies may s t o r e the sperm from one male, i f a more f i t male comes along , and the female can p e r c e i v e male f i t n e s s , she would b e n e f i t from mating with the male and thus, she would be expected t o mate with the s u p e r i o r male.. S i n c e sperm from the most r e c e n t i n s e m i n a t i o n f e r t i l i z e most or a l l of the f o l l o w i n g broods u n t i l i t i s g r a d u a l l y r e p l a c e d by sperm s t o r e d from e a r l i e r i n s e m i n a t i o n s , the phenomenon of sperm precedence may maximize the b e n e f i t of matings with more f i t males. The anatomical model t h a t I proposed i n S e c t i o n IV to e x p l a i n sperm precedence does not n e c e s s a r i l y c o n f l i c t with the p r o p o s a l t h a t accumulation of sperm i n the ovary may i n c r e a s e brood s i z e . I f i n s e m i n a t i o n s are so s m a l l that sperm does not reach a l l t u b u l e s a s s o c i a t e d with mature 104 oocytes, sperm from e a r l i e r i n s e m i n a t i o n s , s t o r e d i n the a n t e r o - d o r s a l d i v e r t i c u l a e of the o v a r i a n lumen, may move i n t o the remaining t u b u l e s . T h i s means that i n s e m i n a t i o n s c o n t a i n i n g a very s m a l l number of spermatophores may not show sperm precedence. The number of spermatophores i n my a r t i f i c i a l i n s e m i n a t i o n s were probably l a r g e enough f o r sperm t o reach most tubules a s s o c i a t e d with mature oocytes and hence i n my experiments, sperm from r e - i n s e m i n a t i o n s f e r t i l i z e d most of the mature oocytes. One of the b a s i c components of sperm storage i n v e r t e b r a t e s i s maintenance o f spermatozoa. . Presumably sperm remain i n a c t i v e d u r i n g storage (Himsatt e t a l , 1966) and then are r e a c t i v a t e d when mature oocytes become a v a i l a b l e . The p h y s i o l o g y of sperm storage maintenance d u r i n g s t o r a g e has r e c e i v e d some a t t e n t i o n (Gardiner, 1976; Hoffmann and Simsatt, 1972; Bacey, 1975). Since i n most v e r t e b r a t e s t h a t s t o r e sperm, sperm a r e c l o s e l y a s s o c i a t e d with the plasma membrane of the e p i t h e l i a l c e l l s of storage s i t e s , i t i s p o s t u l a t e d t h a t these e p i t h e l i a l c e l l s p r o v i d e n u t r i t i o n f o r sperm. In b a t s , fiacey (1975) found an abundance of glycogen, enzymes a s s o c i a t e d with c o n v e r s i o n of glycogen to monosaccharides, and f r u c t o s e present i n the u t e r i n e f l u i d (uterus i s sperm storage s i t e i n b a t s ) . Since mammalian sperm are capable of a e r o b i c r e s p i r a t i o n and f r u c t o l y s i s , p o s s i b l y the substances i n the u t e r i n e f l u i d of bats s u s t a i n the s t o r e d sperm. The pr o d u c t i o n of p o t e n t i a l sperm me t a b o l i t e s by c e l l s of sperm storage s i t e s o f sperm s t o r i n g t e l e o s t s has not been . i n v e s t i g a t e d . However, sperm from 105 Cymatoqaster aqqceqata and P o e c i l i a r e t i c u l a t a are capable of m e t a b o l i z i n g e x t r a c e l l u l a r glucose (in f i s h with e x t e r n a l f e r t i l i z a t i o n , sperm must r e l y on endogenous glucose) and Gardiner (1976) p o s t u l a t e d t h a t sperm from these s p e c i e s o b t a i n n u t r i t i o n from o v a r i a n lumenal e p i t h e l i a l c e l l s at storage s i t e s . In c o n t r a s t , v i r t u a l l y nothing i s known of the mechanisms c o n t r o l l i n g sperm d e a c t i v a t i o n and r e a c t i v a t i o n or r e l e a s e from sto r a g e . Dent (1970) noted that a m y o e p i t h e l i a l l a y e r around the spermatheca of the r e d -spotted newt and suggested t h a t c o n t r a c t i o n of t h i s l a y e r expels sperm i n t o the r e p r o d u c t i v e t r a c t . Fox (1956) p o s t u l a t e d t h a t i n g a r t e r snakes, eggs moving down the r e p r o d u c t i v e t r a c t sgueeze sperm from the s t o r a g e tubules i n the r e p r o d u c t i v e t r a c t . More r e c e n t l y , however, Hoffmann and wimsatt (1972) found that the e p i t h e l i a l c e l l s of sperm storage s i t e s i n g a r t e r snakes become vacuolated and suggested that t h i s may be r e l a t e d to r e l e a s e of s t o r e d sperm. In P o e c i l i a r e t i c u l a t a , c e l l s of the a n t e r o - d o r s a l d i v e r t i c u l a e a l s o appear to become vacuolated and J a l a b e r t and B i l l a r d (1969) suggest t h a t t h i s v a c u o l a t i o n may be r e l a t e d t o sperm r e l e a s e from storage s i t e s . U n f o r t u n a t e l y , these a r e merely c a s u a l o b s e r v a t i o n s from anatomical s t u d i e s and no attempt has been made to t e s t these s p e c u l a t i o n s . In P o e c i l i a r e t i c u l a t a , f e r t i l i z a t i o n of a new batch of eggs by s t o r e d sperm occurs a few days a f t e r p a r t u r i t i o n (Bosenthal, 1952; T h i b a u l t and S c h u l t z , 1978; Turner, 1937). T h i s time lapse between p a r t u r i t i o n of one brood and 106 f e r t i l i z a t i o n of the next brood may r e s u l t from a l a p s e between p a r t u r i t i o n and sperm r e l e a s e or a l a p s e between p a r t u r i t i o n and egg maturation. However, fiosenthal*s (1952) study suggests t h a t r e l e a s e of sperm from s t o r a g e s i t e s i s delayed. Sperm from new i n s e m i n a t i o n s immediately a f t e r p a r t u r i t i o n f e r t i l i z e most of the next batch of eggs. I f s t o r e d sperm were r e l e a s e d immediately a f t e r p a r t u r i t i o n , i t i s u n l i k e l y t h a t sperm from the new i n s e m i n a t i o n would dominate f e r t i l i z a t i o n . F a c t o r s c o n t r o l l i n g r e l e a s e of s t o r e d sperm from storage s i t e s , v i t e l l o g e n e s i s , and p a r t u r i t i o n may be l i n k e d i n some way to produce a c o o r d i n a t e d system. / S e c r e t i o n of gonadotropic and o v a r i a n s t e r o i d s are a s s o c i a t e d with v i t e l l o g e n e s i s , i n c r e a s e d s e x u a l behaviour ( L i l e y , 1972), and perhaps, s e c r e t i o n of a s e x u a l phermcne (Crow and L i l e y , 1979) i n the guppy, p o s s i b l y these hormones a l s o c o n t r o l , d i r e c t l y , or i n d i r e c t l y , the r e l e a s e cf s t o r e d sperm from storage s i t e s . However, at present there i s no d i r e c t evidence a v a i l a b l e . In c o n c l u s i o n , although the f u n c t i o n a l s i g n i f i c a n c e of sperm storage i n guppy females i s not c l e a r , the o r g a n i z a t i o n ; of sperm storage g i v e s gappy females c o n s i d e r a b l e f l e x i b i l i t y i n r e p r o d u c t i o n . I f sperm a v a i l a b i l i t y i s low. a t times, females c a n , maintain r e p r o d u c t i v e output. In a d d i t i o n , accumulation of sperm from more than one i n s e m i n a t i o n may enhance brood s i z e s , but at the same time, sperm from new i n s e m i n a t i o n s can dominate over s t o r e d sperm during f e r t i l i z a t i o n . Thus, although 107 sperm s t o r a g e may maximize r e p r o d u c t i v e output, females can b e n e f i t immediately from matings with s u p e r i o r males, a n a t o m i c a l l y , the guppy ovary appears to be s t r u c t u r e d so th a t s t o r e d sperm can f e r t i l i z e s e v e r a l broods, but a l s o , so th a t f r e s h sperm can have precedence over s t o r e d sperm. U n f o r t u n a t e l y , some of the d e t a i l s of sperm a c t i v i t y i n the ovary a r e l a c k i n g . The time of r e a c t i v a t i o n of s t o r e d sperm r e l a t i v e to p a r t u r i t i o n and whether f r e s h sperm dominate i n the oocyte t u b u l e s by e n t e r i n g unoccupied t u b u l e s or by d i s p l a c i n g o l d e r sperm that are present i n t u b u l e s are f a c t o r s a f f e c t i n g sperm precedence. 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Zoology
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An analysis of intraovarian sperm interaction in the guppy, Poecilia reticulata
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