Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Effects of gonadectomy and methyl testosterone on the reproductive behavior of the blue gourami (Trichogaster… Johns, Laurence Sipprell 1966

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

Item Metadata

Download

Media
831-UBC_1966_A6_7 J6.pdf [ 4.5MB ]
Metadata
JSON: 831-1.0104653.json
JSON-LD: 831-1.0104653-ld.json
RDF/XML (Pretty): 831-1.0104653-rdf.xml
RDF/JSON: 831-1.0104653-rdf.json
Turtle: 831-1.0104653-turtle.txt
N-Triples: 831-1.0104653-rdf-ntriples.txt
Original Record: 831-1.0104653-source.json
Full Text
831-1.0104653-fulltext.txt
Citation
831-1.0104653.ris

Full Text

THE EFFECTS OF GONADECTOMY AND METHYL TESTOSTERONE ON THE REPRODUCTIVE BEHAVIOR OF THE BLUE GOURAMI (TRICHOGASTER TRICHOPTERUS) by LAURENCE SIPPRELL JOHNS B.A., U n i v e r s i t y o f B r i t i s h Columbia, 1959 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In The Department of Z o o l o g y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA August, 1966 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y aval]able for reference and study, I further agree that permission-for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. I t i s understood that copying or publication of* t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my wri t t e n permission. Department of The Uni v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT The blue gourami (Trichogaster trichopterus) , does not appear to go through a r i t u a l i z e d prespawning behavior sequence. The female i s apparently brought into a state of sexual a c t i v i t y as a r e s u l t of the presence of a dark, nest building male. Spawning is i n i t i a t e d by the female and i t consists of a stereotyped cycle of behavioral events. In the majority of cases, male castration r e s u l t s in the cessation of nest building, a reduction of colour change, a p a r t i a l atrophy of the morphological secondary sexual c h a r a c t e r i s t i c s (S.S.C.), and the absence of spawning. Treatment with methyl testosterone brings back a l l of these c h a r a c t e r i s t i c s . In a few cases, castration resulted in only a p a r t i a l reduction of nest-building, colour change and morphological secondary sexual c h a r a c t e r i s t i c s and the retention of spawning. It i s t e n t a t i v e l y suggested that there may be an extragonadal source of androgen. It seems the physical act of spawning i s i i necessary to trigger f u l l parental behavior regardless of gonadal condition. Agonistic behavior i s not apparently affected by castration. Methyl testosterone given to unoperated females resulted i n male-like agonistic behavior, coloration, secondary sexual c h a r a c t e r i s t i c s and some evidence of nest building. i i i TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i i i LIST OF TABLES V LIST OF FIGURES v i ACKNOWLEDGEMENTS v i i i Chapter 1. Introduction 1 Chapter 2. Materials and Methods 6 Chapter 3. The Reproductive Behavior of the 14 Blue Gourami Chapter 4. An Analysis of the Effects of Gonadectomy on Male Reproductive Behavior 2 7 Chapter 5. An Analysis of the Effects of Methyl Testosterone on Male Reproductive Behavior 53 Chapter 6. Some Morphological Effects of Gonadectomy and Methyl Testos-terone Treatment on the Secondary Sexual Characteristics of the Male and Female 69 Chapter 7. Parental Behavior seen i n Sham Operated, Gonadectomized and Testosterone Treated Gonadec-tomized Males 76 iv Page Chapter 8. An Analysis of the Agonistic Behavior between Gonadectomized and Sham Operated Males 85 Chapter 9 . General Discussion and Conclusions. 93 LITERATURE CITED I l l APPENDIX . . . 113 V LIST OF TABLES TABLE Page I Results of reoperation at the conclusion of a l l experiments 49 II Results of egg transfers 78 III Fish combinations used to test agonistic behavior 86 IV Sh and unoperated male f i n r a t i o s 114 V Unoperated female f i n r a t i o s 114 VI Gonadectomized (Gil and G i l l ) male f i n r a t i o s 115 VII Gonadectomized hormone treated male f i n r a t i o s 116 VIII Gonadectomized Control (GII and GUI) male f i n r a t i o s 116 IX Hormone treated unoperated female f i n r a t i o s 117 v i LIST OF FIGURES FIGURE Page 1. A q u a l i t a t i v e record of spawning times and nest building in sham operated and gonadectomized males 30 2. Approach 34 3. Display . 34 4. Butt „ 37 5. Male Chase, Female Flee 37 6. Nest Building 40 7. Hiding 40 8. The percentage of male approaches r e s u l t i n g i n female f l e e i n g to Hiding 41 9. The spawning cycle 44 10. Mean spawning cycles and nest building in t e r v a l s for Sh and unoperated males . . . . 46 11. Mean spawning sequences and nest building intervals for G i l males 47 12. A q u a l i t a t i v e record of spawning times and nest building i n G-H treated and G control males 55 13. Approach 58 14. Display 58 15. Butt 60 v i i FIGURE * Page 16. Chase, F l e e 60 17. Nest B u i l d i n g . 62 18. H i d i n g 62 19. The percentage of male Approaches r e s u l t i n g i n female F l i g h t i n t o H i d i n g . . 64 20a. Mean spawning c y c l e s and nest b u i l d i n g i n t e r v a l s f o r G—H males showing o v e r a l l mean and Sh o v e r a l l mean 66 20b. E f f e c t s of Gonadectomy and Methyl T e s t o s t e r o n e on d o r s a l f i n r a t i o s . . . . 71 21. Mean f r e q u e n c i e s of behavior p a t t e r n s recorded f o r Sh-Sh, Sh-G, and G-G male encounters 89 22. Average bout l e n g t h f o r each c l a s s . . . . 89 v i i i ACKNOWLEDGEMENTS I am p a r t i c u l a r l y grateful to my supervisor Dr. N.R. L i l e y who made t h i s study possible. He obtained f i n a n c i a l assistance, was constantly a v a i l -able for discussion and guidance and devoted hours of his time to reading and he l p f u l c r i t i c i s m of the thesi s . I am grate f u l also to the other members of my committee. Dr. W.S. Hoar, Dr. C.C. Lindsey and Dr. P. Ford for their guidance throughout the year and their h e l p f u l c r i t i c i s m of the thesis. I say thank you also to my fellow student^ Mr. J. Wiebe for many useful suggestions and to Mr. S. Borden who programmed and computed the s t a t i s t i c s . To my wife and children, for their continuous support during my return to university, I am eter n a l l y g r a t e f u l . This work was supported by a grant from the National Research Council of Canada and by the Graduate Scholarship Fund. CHAPTER 1 INTRODUCTION During the past two decades much research has been directed towards the r o l e of hormones i n reproductive behavior among the vertebrates. The majority of this work however has involved the mammals. U n t i l recently, the r e s u l t s seemed rather clear cut, i . e . , gonadectomy resulted i n a loss of sexual behavior and replacement therapy of exogenous hormones brought back sexual behavior to pre-operative l e v e l s . Recently however, as research has spread to include a wider range of species the s i t u -ation has become less simple than was once thought. In the higher mammals, experience appears to play an increasing r o l e . For example, copulation may p e r s i s t for months or years in castrated male cats depending on the l e v e l of previous experience (RosenbJ^atz^and Aronson, 1958) . Harlow (1962) showed that monkeys raised on dummy mothers and deprived of play contacts with peers were unable to mate in adulthood. The recent work involving prenatal or postnatal hormonal sex determination of the brain has opened a whole new area of research. The r e s u l t s of these recent mammalian findings may help to explain the apparently contradictory r e s u l t s being -2-reported from the lower vertebrates, f i s h i n p a r t i c u l a r . No one has investigated the r o l e of experience or develop-mental factors i n f i s h thus f a r . Aronson (1959) reviewed the work involving f i s h . He reports that among females, spayed jewel f i s h (Hemichromis bimaculatus.) and Siamese f i g h t i n g f i s h (Betta  splendens) exhibited n« sexual a c t i v i t i e s when placed with r i p e males. Similar r e s u l t s were obtained from the female West African mouth breeding c i c h l i d ( T i l a p i a macrocephala). Spayed swordtails (Xiphophorus h e l l e r i ) o n the other hand remained sexually a t t r a c t i v e to males. Among males he noted that several authors have found nearly a complete cessation of nest building behavior following castration of the three spined stickleback (Gosterosteus aculeatus). Castrated Salmo salar showed no interest i n females. Castrated male jewel f i s h and f i g h t i n g f i s h were reported to show t y p i c a l courtship, spawning, f e r t i l i z a t i o n and brooding. There i s some doubt as to how f e r t i l i z a t i o n could occur i f castration was com-plete! One and a half months from castration, the male blue acara ^(Aq^idens l a t i f r o n s ) s t i l l displayed spawning behavior with l i t t l e change i n the frequency of occurrence or duration from preoperative l e v e l s . The only difference -3-noted was an increase i n nest passing behavior and a decrease i n nest building. In male p l a t y f i s h following gonadectomy, the only change i n sexual behavior was a decrease i n gonapodial thrusting. In goby males (Bathygobius soporator) spawning behavior i s not affected by castration but operated males became indiscriminate and courted males and females i n the same manner. Aronson drew the following conclusions: 1) Removal of testes or ovary causes eventual decline in some or a l l sexual behavior-2) In a l l species studied, the decline i s greater i n the female than i n the male. 3) In a l l species, c e r t a i n elements are affected more r a p i d l y than others. 4) In males, parts of the sexual pattern remain for long periods after castration. Pickford <1952, 54) and Aronson (1957) state there i s some evidence showing that c e r t a i n components of re-productive behavior may be controlled d i r e c t l y by p i t u i t a r y action. Aronson suggested t h i s may be a p r i m i t i v e con-d i t i o n i n vertebrates. Wai and Hoar (1962) confirmed that the testes was necessary for nest building i n sticklebacks but that a high l e v e l of p i t u i t a r y gonadatrophin was essen— t i a l for the normal expression of the behavior. L i l e y (1965) -4-concluded that gonads were not essential for sexual behavior i n the female guppy. On the other hand the gonad appears to exert a regulatory influence superimposed on a more d i r e c t neural and/or p i t u i t a r y control of receptive behavior. The present research was i n i t i a t e d on the basis of th i s rather confusing and c o n f l i c t i n g background of findings. The choice of an Anabantid f i s h , the blue gourami, (Tr ichogaster tr ichopterus) as an experimental animal was made largely as a r e s u l t of reading an excellent description of i t s reproductive behavior by M i l l e r (1964). It was r e a l i z e d from M i l l e r ' s work that t h i s was a species with an elaborate set of behavior patterns, e a s i l y bred and hardy under laboratory conditions and about which very l i t t l e was known i n terms of behavioral regulatory mechanisms. It was thought that i f some preliminary studies along this l i n e were undertaken and proved successful this would be an excellent experimental animal for future research. The Anabantids are a group of highly s p e c i a l i z e d fishes native to much of South East Asia. There are some 16 genera containing 50 species. Forselius (1959) i n a large monograph, gave a comprehensive review of the -5-l i t e r a t u r e dealing with the systematica, d i s t r i b u t i o n and biology of these f i s h e s . However, his observations were mainly concerned with the genus C o l i s a . Hodges and Behre ^1953) were the f i r s t to publish an abbreviated description of the spawning behavior of _T. trichopterus. The present work i s an exploratory study of the r o l e of hormones in the reproductive behavior of the blue gourami. A " c l a s s i c a l approach" has been adopted. This involved observing the effects of gonadectomy and replace-ment therapy. It was assumed that the gonads are the s i t e of hormone production. As far as possible, a l l aspects of reproductive behavior were considered, i . e . , sexual, agonistic and parental behavior. I was interested to learn whether the various components have a common causal factor or control mechanism or whether they are controlled independently. -6-CHAPTER 2 MATERIALS AND METHODS A. Materials A l l observations were carried out i n twenty-four, glass and st a i n l e s s s t e e l aquaria measuring 51 cm x 27 cm x 31 cm and each holding approximately 43 l i t e r s . Numerous other tanks were u t i l i z e d however, as recovery, breeding, stock and holding tanks. These ranged i n s i z e from 12 to 200 l i t e r s . The observation tanks were mounted on two stands each holding twelve tanks i n two rows of s i x . Each was equipped with a sub-sand f i l t e r which was covered by a layer of course Del Monte (E.I. 16) white sand to a depth of about one inch. The temperature i n a l l tanks o ° varied between 25 and 29 C. The mean temperature i n a l l tanks maintained throughout was 27°C. (80°F). The tanks were f i l l e d with tap water which was allowed to stand at least two days before f i s h were placed i n i t . About 3 gms of sea s a l t were added to each tank. Each tank had a layer of f l o a t i n g plants which were thinned about every 2 weeks. One plant was anchored i n the sand at each end of every tank to provide cover. The tanks were illuminated by soft white fluorescent tubes mounted 23 cm above the centre l i n e of t h e t a n k s . A l l f i s h were k e p t on a t w e l v e hour p h o t o p e r i o d from t h e t i m e t h e y were h a t c h e d . O r i g i n a l l y t w e l v e p a i r s o f B l u e Gouramis were purch a s e d from l o c a l aquarium d e a l e r s i n Vancouver. These f i s h were u t i l i z e d i n p r e l i m i n a r y f a m i l i a r i z a t i o n o b s e r v a t i o n s and e x p e r i m e n t s . However, a l l f i s h used i n t h e a c t u a l e x p e r i m e n t s were bred i n t h e l a b from t h e s e o r i g i n a l s . I n t h i s way t h e age and h i s t o r y o f a l l f i s h c o u l d be s t a n d a r d i z e d . T h i s a l s o c u t down on p o t e n t i a l s o u r c e s o f d i s e a s e . The a d u l t f i s h were f e d once a day w i t h f r o z e n b r i n e shrimp (Artemia) as t h e i r s t a p l e d i e t . T h i s was s u p p l e -mented w i t h v a r i o u s forms of d r i e d foods and l i v e r and pablum m i x t u r e s . The f r y were r a i s e d i n i t i a l l y w i t h "Wardley's S m a l l P r y " s u s p e n s i o n u n t i l t h e y were about 1 cm i n l e n g t h . They were t h e n g i v e n f r e s h h a t c h e d l i v i n g A r t e m i a u n t i l t h e y were l a r g e enough t o h a n d l e t h e f r o z e n form. R e c o r d i n g s were made u s i n g an E s t e r l i n e Angus, 20 c h a n n e l , i n k r e c o r d e r w h i c h gave measures o f d u r a t i o n and f r e q u e n c y . T h i s was o p e r a t e d by remote c o n t r o l from a p o r t a b l e keyboard w h i c h c o u l d be t a k e n t o any tank d e s i r e d . -8-Different codes were used on this keyboard depending on the type of sequence being recorded. Operations were performed under an Olympus binocular dissecting microscope. B. Procedures 1) Behavioral testing During the course of preliminary observations of the animal's normal reproductive behavior the following testing method gradually evolved. It was found that i f a male and female were placed in a tank and separated by a loose f i t t i n g opaque p a r t i t i o n , they became highly sexually stimulated after two or three days. Generally a breeding nest was constructed during this period. Upon removing the p a r t i t i o n , courtship would begin and i n about 70% of the cases proceed to spawning on the morning or afternoon of the second day. Behavior was recorded on. the days following release u n t i l spawning occurred. The following i s an outline of the testing procedure in a l l the experiments to be described l a t e r : Day 1 Male and female placed i n a tank separated by a loose f i t t i n g black p l e x i g l a s s p a r t i t i o n . Day 2 No change. -9-Day 3 No change. Day 4 A.M. P a r t i t i o n removed. Record 1. Behavioral recording made immediately following release for 15 minutes. P.M. Record I I . 15 minute behavioral recording. Day 5 A.M. Record I I I . 15 minute behavioral recording. P.M. Record IV. Generally a continuous behavioral recording throughout spawning, (2-4 hours) . Day 6-10 Qualitative observations made as to nest size, state of eggs and/or f r y . Day 10 Female removed, test terminated. Except for minor variations to be described l a t e r , this method was employed throughout to test normal males, gonadectomized males and gonadectomized hormone treated males. 2), Gonadectomy A great^.deal of time and e f f o r t was expended in perfecting the following technique which may be broken down into 4 steps. -10-a) Anaesthetic The f i s h was placed in 500 ml of a sol'n. of It 2500 strength Tricaine Methanesulphonate (M.S. 222) in water. This sol'n was maintained at a temperature of o 16-17 C giving the f i s h a ten degree temperature shock along with the anaesthetic. The f i s h was removed as soon as i t began to lose balance and l i e over and while i t was s t i l l breathing. This varied between 2 to 3 minutes. b) Operation The p a r t i a l l y anaesthetized f i s h was placed on i t s r i g h t side on a sponge pad i n a wax f i l l e d operating tray designed for the f i s h ; i . e . s l i g h t l y concave to f i t the contour of the f i s h . When in place on the pad i t was t o t a l l y immersed to a depth of 5 mm i n a standard o fresh water teleost s a l i n e at a temperature of 20"C. The f i s h was then pinned down by means of 3 sponge rubber s t r i p s . One was placed across the mid region from just anterior to the dorsal f i n to below the middle of the anal f i n . The second crossed over the caudal peduncle. The t h i r d passed over the snout and eye region but did not cover the operculum. This l a t t e r s t r i p was placed so the g i l l s were free to move as the f i s h breathed slowly throughout the operations. It i s believed -11-many f i s h were l o s t due to this head s t r i p being too t i g h t and thus causing death by suffocation. A small glass nozzle was inserted into the mouth di r e c t i n g a flow of fresh aerated aquarium water over the g i l l s . A curved i n c i s i o n was made s l i g h t l y anterior to and p a r a l l e l with the natural posterior curve of the body cavity. On cutting two r i b s d o r s a l l y i n the i n c i s i o n the curved f l a p was folded back and held using a special clamp designed from a "bobby pin". This gave ample access to the body cavity. Following the removal of the testes, the f l a p was returned and held with one suture using s u r g i c a l s i l k . c) Revival Often upon removal of the holding s t r i p s , the f i s h began swimming almost immediately. Usually, however, i t was assisted with a r t i f i c i a l r e s p i r a t i o n by means of sq u i r t i n g water into the g i l l chamber, on and off i n time with i t s g i l l movements. d) Recovery Following i n i t i a l r e v i v a l , the f i s h was placed in one of four, a,ll glass, three gallon recovery tanks -12-containing a 50% sol'n of the f i s h s a l i n e and maintained at 27 degrees C. These tanks were equipped with outside f i l t e r s . A l l f i s h were kept i n these tanks for three or four days before being replaced i n their regular aquarium tanks where they remained for complete recovery. After 4 weeks they were retested. Sham operated control f i s h were given the exact treatment described above except the testes were not removed. 3) Hormone Administration A l l f i s h were anaesthetized under the same conditions described. They were then held i n moist cotton wool for i n j e c t i o n . Each f i s h received a series of 4 injections^one every two days. Each i n j e c t i o n contained 1 mg of methyl testosterone suspension in 0.05 ml of d i s t i l l e d water and Tween 80 (one drop/25 ml). These were administered i n the peritoneal cavity using a No. 26 hypodermic needle. On the day following the 4th i n j e c t i o n , (8 days from the i n i t i a l treatment) p a r t i t i o n s were i n s t a l l e d and females added. This led d i r e c t l y to the behavioral testing procedure described above. -13-4) Fish Measurements Fish were placed on a moistened sponge pad and measurements were made of their length and f i n s using a pair of d r a f t i n g d i v i d e r s . Each f i s h was measured twice i . e . once following gonadectomy and once following hormone treatment. Two measurements were taken. a) the standard length (snout to caudal peduncle). b) the distance from the snout to the d i s t a l t i p of the dorsal f i n . The two r e s u l t i n g lengths gave a r a t i o as shown below. Snout to Dorsal Fin Length = Fin to Length Ratio Standard Length 5) Egg Removal, Transfers and Incubation Eggs were removed from tanks by means of lowering a small p e t r i e dish (submerged beneath the nest) and slowly l i f t i n g . Any plants caught i n the dish were trimmed off with scissors as the dish was l i f t e d . The eggs and bubbles were placed from the dish into other tanks by means of a teaspoon. Eggs to be incubated were l e f t i n the p e t r i e dish and the whole dish plus eggs floated in a tank containing water at the desired temperature. -14-CHAPTER 3 THE REPRODUCTIVE BEHAVIOR OF THE BLUE GOURAMI Introduction A long series of preliminary studies enabled the observer to become fa m i l i a r with the various behavior patterns involved i n the t o t a l reproductive behavior. The i n d i v i d u a l behavior patterns recorded were described in d e t a i l by M i l l e r (1964) and w i l l be presented only b r i e f l y here i n the f i r s t section. A general q u a l i t a t i v e account of the reproductive and agonistic behavior i s included i n the second section to provide a better under-standing of the experimental work to follow. A. L i s t of behavior patterns recorded The following i s a b r i e f description of the behavior patterns recorded during t h i s research. The records were taken i n terms of frequency or duration of behavior or both. The symbols given beside the names indicate the behavior pattern code l e t t e r s and w i l l appear again i n this thesis. -15-I. Male Behavior 1) Approach (A) The act of purposely swimming towards the female in any way from any d i r e c t i o n . 2) Pelvic Fin Ray Contact (P) The act of reaching out and contacting the body of the female at any point with the p e l v i c f i n rays. 3) P a r t i a l Display (Fin Erection) (E) The act of erecting the f i n s in what i s regarded as a low i n t e n s i t y display. This means a display lacking sigmoidal body t o r t i o n . 4) F u l l Display (D) A high i n t e n s i t y display which involves f u l l f i n erection plus l a t e r a l sigmoidal body t o r t i o n . This display may take place i n front of the other f i s h (frontaL), or p a r a l l e l to i t (lateral). No d i s t i n c t i o n was made since one phase may lead into the other and back again depending on the motor component. 5) Butting or Biting (B) The act of butting or b i t i n g at the female generally at the anal f i n or caudal peduncle area. 6) Chasing (Ch) The act of pursuing the female around the tank r a p i d l y . It i s generally accompanied by Butting. -16-7) Colour Change (Co) The darkening of the male through the reproductive period was recorded i n terms of i n t e n s i t y on a scale of three: 1 = blanched; 2 = v e r t i c a l dark bars giving a grey mottled appearance; 3 = inky black a l l over. 8) Nest Building (N) The act of gulping a i r at the surface and spewing out bubbles into and around the nest, c o l l e c t i n g , mouthing and placement of eggs. 9) Rubbing (R) The act of moving back and f o r t h under the female rubbing her ventral surface just p r i o r to spawning. 10) Spawning (S) This includes f i v e separate behavior patterns and only the i n c l u s i o n of a l l f i v e i n succession on a record indicates a complete spawning act. a) E n c i r c l i n g : - The act of curving around the female u n t i l the clasp i s established. b) Clasping:- The actual r i g i d grip of the female by the male. c) Rollover:- The act of inverting the female at the end of the clasp. d) Oviposition:- The release of eggs and presumably spermatazoa. e) Swimming Inh i b i t i o n : - The period of time from the breakup after r o l l o v e r u n t i l the female recovers and the male begins to chase her away. II. Female Behavior Certain behavior patterns of the female were recorded simultaneously. Many of these are i d e n t i c a l to the male patterns and therefore only new patterns w i l l be elaborated on. 11) Approach (A) 12) Pelvic Contact (P) 13) P a r t i a l Display (E) 14) F u l l Display (D) 15) B i t i n g or Butting (B) 16) Fleeing (F) The act of rapid retreat from the male, usually simultaneously with the male Chasing. 17) Colour Change (Co) 18) Hiding (H) The act of remaining inconspicuous behind or below substrate plants or occasionally among the surface plants. 19) Appeasement (Ap) The act of r o l l i n g over l a t e r a l l y when the male approaches exposing to him the flanks and b e l l y . This act appears to i n h i b i t male attack. -18-20) F in Tugging (Ft) The act of grasping a f i n (usually the dorsal or caudal) i n the mouth and rocking backwards and forwards or sideways. This appears to be the most aggressive act employed by thi s species and usually occurs following a long series of Butts. The behavior patterns described w i l l be referred to throughout t h i s thesis. To c l a r i f y , they w i l l be "c a p i t a l i z e d " whenever used. B. A Qualitative Description of Reproductive Behavior  Trichogaster trichopterus, the Blue Gourami, i s a bubble nest breeder. Like the other Anabantids, i t posses-ses a labyrinth chamber dorsal to the pharynx which enables i t to store a i r and u t i l i z e a e r i a l oxygen to some extent. Indeed, some members of the group can remain out of water for long periods of time using t h i s apparatus. With a secretion of mucus from within the pharyngeal chamber, the male gourami can, on gulping i n a i r , issue a stream of bubbles at the air-water interface. By continuous gulping and blowing, the animal builds a mound of bubbles which may reach 15 cm in diameter and 1 cm deep. I t i s generally anchored to the f l o a t i n g plants or i n their -19-absence, spreads out along the sides of the tank. This nest becomes the f o c a l point of the reproductive behavior. The nest i s generally begun during the i n i t i a l phase under the conditions of the tes t i n g procedure outlined i n Chapter I i . e . during the time when the male and female are separated by the p a r t i t i o n . In f a c t the presence of a nest at t h i s time i s a good in d i c a t i o n that spawning w i l l take place shortly after release. Sexual dimorphism i n T. trichopterus i s less d i s t i n c t than that found i n other c l o s e l y r e l a t e d species. In the non-reproductive phase, i t i s limited mainly to two c h a r a c t e r i s t i c s . 1) The normal male possesses a large, elongated and pointed dorsal f i n , whereas the female has a much shorter, rounded dorsal f i n . 2) The abdominal region of the male i s f l a t or smoothly curved l a t e r a l l y whereas the female possesses a d i s t i n c t abdominal bulge l a t e r a l l y caused by pressure from the ovaries. This abdominal bulge was used as a rough in d i c a t i o n of the phy s i o l o g i c a l state of readiness to spawn i n the female. When i n the reproductive phase of behavior the male -20-becomes increasingly darker u n t i l , during spawning, he i s inky black. The female r a r e l y darkens as much, but i s more of a grey-black at this time. When not spawning both sexes are a s i l v e r y powder blue with very l i t t l e d i s t i n c t i o n between them. The actual reproductive behavior can be sub-divided into three phases. I Prespawning Courtship II Spawning III Post Spawning Parental I Prespawning Courtship I n i t i a l l y , upon release the two f i s h Approach each other extending the Pelvic f i n rays by which they contact each other. Then follows a series of l a t e r a l or f r o n t a l Displays with or without Colour darkening. The Display consists of a f u l l erection of the median f i n s plus a r i g i d body t o r t i o n which i s best described as sigmoid i n shape. The extent of Display and darkening appear to depend on the r e l a t i v e size of the f i s h . The larger the female i s , the more intense the male Displays. The Display sequence i s usually terminated by a Butt or Bite by the male aimed at the region of the caudal preduncle or anal f i n t i p of the female. She may return the Display and Butt to the male and i f so the bout may continue. Usually, the male asserts his dominance after one or two exchanges, p a r t i c u l a r l y i f he has a nest i n the tank. The male Butt i s usually followed by the female either Appeasing or Fleeing with the male pursuing i n a Chase. The female then seeks refuge behind one of the bottom plants. However, she soon w i l l emerge and the whole sequence i s repeated. Gradually over the course of t h i s f i r s t day the male becomes more aggressive. His Butts and Chases increase i n vigour u n t i l the female i s nearly completely confined to Hiding. Between the i n t e r -vals of Chasing the male spends more and more time Nest Building. During the morning of the second day he i s occupied at Nest Building almost constantly, except to defend the nest from the female. During t h i s period of intense Nest Building the colour of the male darkens and remains dark unless he i s disturbed. A sudden noise or movement w i l l cause blanching i n about 3 seconds. In about 70% of the t r i a l s spawning began around noon on the second day. -22-II Spawning The spawning sequence appears to be a stereotyped reaction chain. It may begin i n one of two ways: 1) The male may Approach the Hiding female, Display to her and lead her to the nest. This behavior was, however, r a r e l y observed. 2) The female leaves her spot of refuge on her own i n i t i a t i v e and r a p i d l y Approaches the Black, Nest Building male and Butts him smartly two or three times i n rapid succession on the side just posterior to the central l a t e r a l spot. This gesture appears to i n h i b i t the male's aggression, but i n extreme cases he w i l l turn on the female and a v i o l e n t Chase r e s u l t s following which the female seeks refuge and the male returns to Nest Building. Normally, however, the Butt e l i c i t s a male Display which proceeds to Rubbing. The Rubbing behavior i s unique to T. trichopterus (Miller 1964). I t consists of the following actions. The male orients himself d i r e c t l y under the nest at an angle of 30 degrees to the water surface, head up. The female approaches from above and behind the male. She moves into a p o s i t i o n -23-p a r a l l e l to the surface so that the male's dorsum contacts her b e l l y and genital areas. Then the male moves backwards and forwards about once a second and may continue from 20 to 120 seconds. He then draws forward i n a curving Display. The female moves into the curve and places her head above his dorsum. The male continues to tighten the curve u n t i l he wraps com-p l e t e l y around the female who i s now oriented almost v e r t i c a l l y to the water surface. If both the f i s h are oriented properly, t h i s r e s u l t s i n the Clasp by which the male grips the female t i g h t l y and begins to vibrate. Sexual ducts are brought very close during the Clasp. The Clasp l a s t s about 35 seconds and i s terminated by R o l l Over. The caudal f i n s of both f i s h cause t h i s which r e s u l t s i n a complete inversion of the female so that her genital opening i s directed upward. It i s at this point that the gametes of both sexes are released. The eggs and sperm f l o a t to the surface to become caught up in the sti c k y bubbles. The male now releases the female and both begin to sink slowly with no other move-ment. The male recovers f i r s t and gently begins to nudge the female down and away from the nest s i t e . The female -24-recovers almost immediately and begins to swim with the male i n rapid pursuit. The Chase i s of short duration. The female Hides and the male returns to the nest. He now begins c o l l e c t i n g any stray eggs and spews them into the bubble nest. He works continuously, Nest Building and egg placing and mouthing. Generally he clumps the eggs in the center of the nest. The whole spawning cycle may be repeated many times over a period of up to four hours. Quite often the i n i t i a l spawning attempts do not r e s u l t in ov i p o s i t i o n . The same occurs near the end of the spawning when the l a s t clasp produces only three or four eggs or none after which the male w i l l not tolerate the female under the nest again. During the course of the spawning up to 1000 eggs may be l a i d . I l l Parental Behavior The f r y hatch i n about twenty four hours. The male shows parental behavior which consists of c o l l e c t i n g , mouthing and spewing the f r y into the bubbles. After about two days, however, the f r y become active and spread out through the tank. After t h i s , the time spent nest tending gradually diminishes over a period of about f i v e -25-to six days. Males have been l e f t with growing f r y for upwards of four weeks during which time they were apparently completely i n h i b i t e d from eating them. Fry or eggs placed with a male not recently spawned are immediately eaten. No accurate measure of the time length between i n d i v i d u a l spawning cycles was obtained since the female was removed one week after spawning. Indications are that a new cycle might s t a r t i n about ten days to two weeks. However, on three occasions t h i s observer noted pairs spawn on successive days. C. A Description of Agonistic Behavior When two males are released together the behavior which r e s u l t s i s quite d i f f e r e n t from that which occurs between a male and female. The males Approach each o t h e r - i n i t i a l l y and exchange a number of Pelvic Ray Contacts and then begin to c i r c l e each other with a series of F u l l Lateral Displays. Sooner or l a t e r one of the males w i l l turn i n and begin Butting the other on the flank and caudal peduncle regions. The f i s h r e c eiving the Butts remains i n the Display flexure. Then i t w i l l suddenly turn and begin Butting. The o r i g i n a l Butting f i s h now assumes the Display posture. -26-Th us a series of mutual,' alternate Display and Butting sequences begin. During t h i s time both f i s h become inky black i n colour. As Displaying and Butting pro-gresses alternate Fin Tugging begins to appear following long Butting sessions. This behavior continues u n t i l one of the f i s h suddenly stops Butting, blanches and either swims away or Flees. The v i c t o r r a r e l y pursues the loser, instead both simply part company and return to their o r i g i n a l ends of the tank. Once dominance has been established there i s l i t t l e further recourse to such h o s t i l i t i e s . If the dominant f i s h happens to have a nest i n his half of the tank, he w i l l defend i t by a rushing Approach, a Display or a Chase. -27-CHAPTER 4 AN ANALYSIS OF THE EFFECTS OF GONADECTOMY ON MALE REPRODUCTIVE BEHAVIOR Introduction A comparison was made between the reproductive behavior recorded from a group of sham-operated, control males and a group of gonadectomized males. An analysis of the r e s u l t s also provides quantitative date to supplement the descriptive account given previously. Procedure Young males were i s o l a t e d from a large growing tank as soon as sexing was possible and raised to maturity i n a l l male tanks. V i r g i n males were then selected from these tanks as desired and paired with v i r g i n females of the same age. The pairs were then put through the tes t i n g procedure described i n Chapter 1 but no records were taken. A l l the f i s h , male or female, which spawned on the second or t h i r d day after release were selected for the experiment. A l l others were rejected. Thus a l l experimental f i s h used were capable of spawning -28-r e a d i l y under the test s i t u a t i o n and had had one spawning experience p r i o r to t e s t i n g . As mentioned e a r l i e r , the number of males responding i n this way amounted to about seventy percent of those tested. The selected females were placed together u n t i l required. The selected males were either gonadectomized or sham-operated as controls and allowed to recover for one month. At t h i s time they were again paired with the selected females and put through the t e s t and recorded. A l -together over a period of two months 6 sham operated controls and 16 gonadectomized experimental males were tested. Results A Time to Spawning Fi g . 1 shows the day and time at which spawning occurred following the removal of the p a r t i t i o n on Day 4. The "G" and "Sh" numbers indicate either gonadectomized or sham-operated and were assigned to the f i s h following the operation. From th i s chart (Fig. 1), i t can be seen that 100% -29-of the Sh males spawned under the test s i t u a t i o n , although Sh~j did not spawn u n t i l Day 7. The duration of the spawning varies between two to four hours a l -though th i s chart i s not intended to be accurate i n this respect. It can be seen also that spawning may take place at any time of day but there i s a tendency for i t to occur during the f i r s t half of the day. A l l spawning f i s h had previously b u i l t a bubble nest during Days 1-3 and i n a l l cases this was maintained following spawning u n t i l after Day, 10. Only once during a year's observations was a spawning seen without the presence of a nest. It may be noted that no spawning has ever been known to take place at night. Further, when observed for short periods at night the f i s h were usually s i t t i n g q u i e t l y on the sand or among the plants and so i t has been assumed that most a c t i v i t y takes place during daylight hours and therefore the dark hours have been eliminated from a l l charts and graphs. The gonadectomized f i s h f a l l into three categories. Category I:- Those G f i s h which spawned within seven days from release and produced viable or f e r t i l i z e d eggs (4 f i s h : - G, _ ,^ , f t . „ ) . These f i s h were Sh 2 m 0k ft w o n 3 O Q h . W ». ^> r M•*-• c e h . A ^ . o S > ' 1 5 w V c u ^ • ^ " w «t o n 7 - • W « 11 w * 1 1 " 12 " 14 ^ " 15 " 16 II 17 V 9 W 18 w 19 (A •»~«-W W w W V Oi 20 II (/, 22 « • .1 ' 24 25 t i 26 2 / 1 1 1 .1 10 12 2 4 Day 4 F I G U R E 1. 1 r r 1 1 1 1 10 12 2 4 Day 5 \ q u a l i t a t i v e l e s t b u i l d i n c n a l e s . 1 1 1 1 10 12 2 4 Day 6 D A 2 r e c o r d o f s j i n sham-ope I I I ! 10 12 2 4 Day 7 Y and T 1 IV s p a w n i n g t i m e ; r a t e d a n d gc 10 12 2 4 Day 8 IE (hours ) 2 S a n d m a d e c t o m i z ec 10 12 2 4 Day 9 KEY J 10 12 2 4 Day 10 ' Spawning • Nest -31-reoperated and found to have regenerated t e s t i c u l a r tissue, t h i s was removed. During t h i s process died. The remaining three were allowed a month's recovery and retested from whence they f e l l into either category II or I I I . Category I I : - Those f i s h which spawned within seven days of release but f a i l e d to f e r t i l i z e any eggs (5 f i s h : - G.. .. _ . Q ) . After a spawning of a G i i , u / , i"» i y f i s h , about 50% of the eggs were removed and incubated separately to check for v i a b i l i t y . F i g . 1 also shows a much greater v a r i a b i l i t y i n the spawning time from release among the Cat. II f i s h than among the controls. Only two f i f t h s spawned within the f i r s t three days compared to f i v e sixths of the controls. Category I I I : - Those which f a i l e d to show any sexual behavior (11 f i s h ) . The categories II and III w i l l be referred to throughout. From th i s point on they w i l l be abbreviated as GII and GUI. B. Prespawning Behavior The three 15 minute records for each pair sampled the behavior from the time of release (a.m. Day 4) to -32 the middle of Day 5 when the majority of Sh spawnings began. The r e s u l t s have been presented as a series of mean frequencies, durations or percentages. Although th i s research i s pri m a r i l y concerned with male behavior, a good deal of female behavior was recorded as we l l . Some of t h i s has been included as i t r e f l e c t s the male behavior i n that i t i s a response to male actions and thereby helps to i l l u s t r a t e male behavior. A l l data presented i n Figs. 2-8 were programmed through the University of B r i t i s h Columbia^ computing center. A c a l c u l a t i o n of the mean and standard error for every graph point was made. In addition, the following comparisons between the f i s h groups were made at each of the three Record points. Sh males compared with GII males Sh males compared with GUI males These comparisons were made using the Welch te s t . This i s a " t t e s t " which assumes independence and normality but does not assume equality of variances. It i s a conservative t e s t . Values for p were obtained i n each case. Those values s i g n i f i c a n t at or below the 5% l e v e l have been included i n the text i n every case. In - 3 3 -addition, larger values have been included to the 20% l e v e l to i l l u s t r a t e trends. For a l l points of com-parison where no "p" value appears i t may be assumed there i s very l i t t l e s i g n i f i c a n t difference. Approach F i g . 2 The mean frequencies of male and female Approach are given i n F i g . 2. Rl i n each case i s marked by a high frequency of Approach as thi s i s the i n i t i a l "meeting period" recorded immediately after removal of the p a r t i t i o n . The males and females generally Approach each other and frequently contact each other with the pe l v i c f i n rays during any i n i t i a l encounter regardless of their p h y s i o l o g i c a l condition or sex. As time pro-gresses, the frequency of Approach decreases s t e a d i l y i n both sexes. It i s generally always higher i n the males, however. G i l and III males follow a very similar pattern to the Sh males during Rl and 2 but in R3 the frequency of Approach tends to l e v e l off for G U I males (R3: Sh to G U I p = 0.0540). Correspondingly, the frequency of Approach for G U I females i s s l i g h t l y higher than the Sh females during a l l three Records (Rl: Sh to GUI females p = 0.0494)? (R3: Sh to G U I female p = 0.1278). b) ? A pproach - 3 5 -It w i l l be seen l a t e r that t h i s i s a r e s u l t of a lower aggressive l e v e l on the part of the GUI males. Displaying and Butting (Figs. 3-4) These figures indicate trends i n the male and female Displaying and Butting. Examining the Sh males f i r s t i t can be seen that i n i t i a l l y there i s an equal amount of Display between males and females and that with time, the frequency of D f a l l s to a very low l e v e l i n the female. In the male, i t i s maintained and may even r i s e i n R3 since spawning has either started or i s imminant. G i l males and females show p a r a l l e l trends but at a lower l e v e l . Only once did a G i l male spawn at this time, hence the very low l e v e l of Display. In R3, the p value for the difference between the Sh and G i l males i s 0.1070. In R2 and R3 for female Display, comparing Sh and G i l females p values are 0.1747 and 0.1073 respe c t i v e l y . A very d i f f e r e n t picture was seen i n the case of the G U I males and their females. The difference between the Sh and GUI males for Rl of Displaying and Butting i s highly s i g n i f i c a n t (p = 0.0037 and 0.0212 -36-r e s p e c t i v e l y ) . S i m i l a r l y a s i g n i f i c a n t difference appears between the Sh and G U I females i n Rl for Butting (p = 0.0280). Mutual Displaying and Butting and some Fin Tugging was regu l a r l y observed i n the majority of cases. During these observations i t became very obvious to t h i s observer that these f i s h were behaving i n an agonistic manner almost i d e n t i c a l to that of two normal males released together (Chapter 7) or for that matter two females. A casual observation made about 3-4 weeks after the operation began now to c l a r i f y i t s e l f . Many of the G U I males had considerably shortened and more rounded female-like dorsal f i n s . Indeed i t was d i f f i c u l t to d i s t i n g u i s h them from the females. This observer suggests the G U I males were i n i t i a l l y mistaken for females by the females. It may be further noted that female Butting of the male was never observed under normal heterosexual contact except during spawning. Chasing and Fleeing (Fig. 5a-b) The trend i n male Chasing i s i l l u s t r a t e d i n F i g . 5a. The amount of actual Chasing by Sh males i s very s l i g h t i n i t i a l l y but tends to increase as spawning time nears. G i l males show a similar trend i n i t i a l l y which levels FIGURE 4 a) (f Butt KEY • Sh A Cat II O Cat I I I T r 10T FIGURE 5 a) ( ? Chase 20 10 R1 12 4 R 2 6 Time (Hours ) 8 ~ 1 — 10 R 3 10 b) ? B u t t -38-off i n R3 as spawning i s not imminant. G U I males follow a similar trend but at a lower l e v e l . F i g . 5b indicates the trend i n female Fleeing. Once again there i s a close p a r a l l e l between the Sh and G i l females. Both show a high frequency of Fleeing i n i t i a l l y which indicates male dominance has been established immediately. G U I females i n i t i a l l y Flee much less since the males at t h i s time have not estab-lish e d dominance. In one case, the female remained dominant throughout the test period and Butted and Chased the male into Hiding which i s a complete reversal of the normal trend. The difference between Rl Sh female Fleeing and GUI female Fleeing i s indicated by p= 0.1664. The general decrease i n Fleeing frequency noted in R2 and R3 i s perhaps misleading. I t does not indicate a lowering of male aggressive tendencies but simply that the females Flee less frequently because they are spending more and more time Hiding. Nest Building (N) and Hiding (H) (Fig. 6-7) In Sh males, as spawning time nears there i s a dramatic increase i n the amount of N. F i g . 6a shows -39-th e mean number of actual nest building t r i p s made to the nest. It can be e a s i l y seen that G U I males did not Nest Build. GII males show no N at R2 and a mean frequency of one t r i p per 15 minutes i n R3. The differences i n R3 between Sh and GII males and between Sh and G U I males are s i g n i f i c a n t (p = 0.0215 and 0.0106 re s p e c t i v e l y ) . The average time spent i n N per Nest Building t r i p i s i l l u s t r a t e d i n F i g . 6b (Total N duration divided by frequency of N). It appears as though GII males are spending more time Nest Building i n R3 but this figure r e s u l t s since they average only one t r i p . In actual fact, only two of the f i v e GII f i s h were Nest Building at th i s time since the majority did not spawn u n t i l l a ter (Fig. 1). The frequency and mean duration per t r i p of female Hiding behavior i s indicated i n Fi g . 7a-b. In the Sh females, the frequency of H drops i n R3 but the mean time per t r i p increases correspondingly, i n d i c a t i n g a much larger t o t a l duration. Once again a similar trend i s set by the GII females but at a lower l e v e l . The occurrence of H appears to be a d i r e c t r e s u l t of male aggressiveness and therefore these graphs are a -41-FIGURE 8. The percentage of male Approaches r e s u l t i n g i n female Fleeing to Hiding. -42-good in d i c a t i o n of the male aggression lev e l s at the time. As might be expected therefore, the GUI females show the lowest frequency of H and furthermore this varies l i t t l e throughout. The difference between G i l and Sh females i n R2 i s s i g n i f i c a n t (p = 0.0385). In F i g . 7b another i n d i c a t i o n of the lack of female intimidation by the GUI males may be seen by the f a c t that the mean time spent i n H per t r i p a c t ually drops i n R3 for G U I females. In summary of the prespawning behavior trends observed, i t appears that the Sh males tend to establish dominance over the females very soon after meeting and continue to exert t h i s dominance by being increasingly aggressive as spawning time nears. This r e s u l t s i n females Fleeing into Hiding. This aggressive tendency was evident i n a l l males tested but at d i f f e r e n t l e v e l s . This i s c l e a r l y i l l u s t r a t e d in F i g . 8. Here the percentage of male Approaches which r e s u l t i n female F l i g h t into Hiding has been plotted for each category. Note once again the G i l males achieve a l e v e l between the Sh and GUI males. Values of p were not obtainable -43-for t h i s graph due to the manner of i t s makeup i . e . percentages computed from means. Therefore i t can only be taken as an ind i c a t i o n of trends. C. The Spawning Cycle The behavior involved i n the spawning cycle was described q u a l i t a t i v e l y i n Chapter 2 and w i l l there-fore not be elaborated here. The names of the events and their order of occurrence are indicated i n Pig. 9. Also shown are points where the cycle may be broken or shortened. Incomplete cycles generally occur at the beginning and at the end of a spawning. The actual number of cycles performed by any mating pair varies tremendously. This observer over a period of one year recorded as few as four cycles and as many as t h i r t y two. The l a t t e r includes a l l incomplete attempts i . e . , a breakup before successful termination i n o v i p o s i t i o n . Actually, i n this case, only fourteen of the t h i r t y two attempts were carried to successful termination i n o v i -p o s i t i o n . For each complete spawning cycle a record of the duration of each event comprising i t was obtained. After recording a number of such cycles, the durations -44-C l a s p i n g E nci rc l ing \ \ \ Ro l l over FIGURE 9. The Spawning Cycle. Cross arrows indicate points where cycle may be shortened by breakup after which i t must begin again. Once ro l l o v e r s t a r t s , the cycle completes, but eggs may not always be released. -4£~ of each repeating event were averaged to give a mean spawning cycle and nest building i n t e r v a l for one pair of f i s h . Figure 10 indicates the mean cycles for Sh^ f Sh 5, and Sh-^  plus previous records of f i v e other normal spawnings to give more complete picture. The l a t t e r were not involved i n th i s experiment. The eight mean spawning cycles were averaged among themselves to give an o v e r a l l mean spawning cycle and nest building i n t e r v a l . A similar treatment of the GII spawnings was made (Fig. 11). It was only possible to record three of these spawnings. In comparing Figs. 10 and 11, i t can be seen that there i s p r a c t i c a l l y no measurable difference. From the two o v e r a l l average readings the only difference i s in a s l i g h t l y longer average Rubbing period and a shorter average Nest Building i n t e r v a l for the GII males. The l a t t e r would indicate a s l i g h t l y higher frequency of spawning cycles. An in t e r e s t i n g point to note i s that i n a l l cases (Sh and G males) the duration of the actual Clasp varies the l e a s t . A successful FIGURE 10 Mean spawning cycles and nest building intervals for Sh and unoperated males. FIGURE 11 Mean Spawning Sequences and Nestbuilding Intervals for G i l males. a  Clasp i . e . one leading to Oviposition, was never observed to be less than twenty four seconds or more than f o r t y f i v e seconds. Further, the length of Clasp remained f a i r l y constant for each f i s h . If the average clasp time was e.g. 26 seconds for Sh^, then the time for a l l Sh^ Clasps varied l i t t l e from 26 seconds. A l l the behavior patterns occurred i n the GII males that occurred i n the controls; further, they occurred i n the same order and at the same in t e n s i t y and for similar duration so as to coordinate p e r f e c t l y with the females and achieve o v i p o s i t i o n . In no case did any egg hatch. The eggs l e f t i n the nest with the GII males i n attendance had invari a b l y disappeared by the next morning. These were either eaten by the male or disintegrated i n the water. The former was assumed to be the case since the incubated eggs were s t i l l present the next morning though white and bloated. Evidence of Regeneration Nearly a month following the termination of experiments a l l the gonadectomized males were reoperated to determine i f i n fac t any t e s t i c u l a r regeneration had ORIGINAL DAYS TO CATEGORY FISH OPERATION FINAL EVIDENCE OF REGENERATION AND DATE OPERATION DORSAL FIN DESCRIPTION III CONTROLS G 15 16/3/66 109 Yes-2-3 mm Testis Tissue Fin long G 16 16/3/66 109 Yes- 1 mm Testis Tissue Fin long G 23 1/4/66 94 No trace Fin shor G 26 26/4/66 68 No trace Fin shor II CONTROLS G 11 11/3/66 114 No trace Fin long G 17 24/3/66 100 No trace Fin long G 19 3/5/66 62 Yes-2-3 mm Testis Tissue Fin long III HORMONE G 20 24/3/66 115 No trace Fin med. G 21 1/4/66 95 No trace Fin med. G 22 1/4/66 95 No trace Fin med. G 25 16/4/66 79 No trace Fin long G 12 15/4/66 80 No trace Fin long II HORMONE G 14 16/3/66 110 No trace Fin long 1 Id 5 TABLE I A summary of reoperation findings at conclusion of a l l experiments. taken place during the three to four months since the o r i g i n a l operations. The findings have been summarized i n Table I along with a q u a l i t a t i v e description of the dorsal f i n at the time. Discussion On examination of the r e s u l t s i t can be r e a d i l y seen that a l l Sh males spawned under the test s i t u a t i o n . Further, these males and a l l G males had previously spawned under the same conditions p r i o r to operation. Therefore i t may be assumed that the differences noted for the G males on the second testing are due to the effects of gonadectomy and presumably the lack of gonadal hormones. It was noted that four G males did spawn and f e r t i l i z e eggs. On reoperating a large e a s i l y distinguishable regrowth of t e s t i c u l a r material was seen in the ventral region of the abdominal cavity immediately posterior to the rectum. This was presumably caused from an incomplete removal of the duct during the o r i g i n a l operation. Since the majority of G males i . e . , GUI males, showed very l i t t l e reproductive behavior i n an organized -51'-form i t would appear that gonadectomy was complete and the r e s u l t i n g lack of hormones was responsible. It i s i n t e r e s t i n g to note however, that the G U I males s t i l l showed a l l the behavioral elements of the Sh males but at a much lower i n t e n s i t y l e v e l . There was no b u i l d up of aggressions to a l e v e l necessary to intimidate the female into F l i g h t and 'Hiding for increasing periods. Neither was there Nest Building. However, f i v e G males (Gil) did spawn. These f i s h displayed a l l the normal behavior but at a l e v e l which f e l l between the controls and G U I (Fig. 9). This resulted i n a delayed spawning and one i n which no eggs were f e r t i l i z e d . It was suspected that regeneration had occurred. If so, i t must have been of a nature or i n a p o s i t i o n not allowing the release of sperm. It appears also that once the male agression l e v e l reaches a cert a i n point whether r a p i d l y (Sh) or over a longer period ( G i l ) , spawning i s possible providing the female i s ready and cooperative. Once spawning started, the behavior shown by the G i l males was v i r t u a l l y indistinguishable from the Sh males. -sa-l t i s suggested that the main r o l e of gonadal hormone i s i n the prespawning organizational period and not in the actual spawning i t s e l f . The r e s u l t s of reoperation show that i n f a c t by the end of three to four months some f i s h had regenerated small amounts of t e s t i c u l a r t i s s u e . However, at the time of the present experiment, these res u l t s were not known. It i s the author's intent, therefore, to reserve further discussion of these r e s u l t s to Chapter 9 after a l l data have been presented. -53-CHAPTER 5 AN ANALYSIS OF THE EFFECTS OF METHYL TESTOSTERONE ON THE MALE REPRODUCTIVE BEHAVIOR I n t r o d u c t i o n A comparison was made between t h e r e -p r o d u c t i v e b e h a v i o r r e c o r d e d from a group o f gonadectomized c o n t r o l males and a group o f gonadectomized males t r e a t e d w i t h m e t h y l t e s t o s t e r o n e . The r e s u l t s a r e a l s o com-p a r e d w i t h t h e b e h a v i o r d i s p l a y e d by a group of sham o p e r a t e d males d e s c r i b e d i n Chapter 4. Pro c e d u r e G i l and G U I from t h e p r e v i o u s e xperiment were u t i l i z e d h e r e . These were d i v i d e d r o u g h l y i n h a l f t o p e r m i t one group o f seven t o r e c e i v e t e s t o s t e r o n e and one group of F o u r t e e n gonadectomized males made up of seven t o a c t as c o n t r o l s . The groups were composed o f : Group I Hormone t r e a t e d : -G i l G 14 G 1 8 G U I G 20 G G G G 21 12 22 25 -54-Group II Controls:-GII G G G, n 11 17 19 GUI G_, G G G, , 26 23 15 1 6 Group I males were treated with methyl testosterone by the method described i n Chapter 2. The control group received exactly the same treatment but with no hormone added, i . e . they received injections of d i s t i l l e d water containing "Tween80" only. Following one week's treatment a l l f i s h were paired with females and tested i n the same manner as before. From this point on, gonadectomized f i s h receiving hormone treatment w i l l be referred to as G-H males. Results A. Time to Spawning A record of the number, the time and the day of spawning for each f i s h has been summarized on F i g . 12. Looking f i r s t at Group I i t can be seen that f i v e sixths out of the f i s h tested, spawned within the te s t i n g period. Unfortunately the second GII male, G^g, died just p r i o r to 1 Hormone Treated 2 Con t ro l " 18 "20" " 2 2 " 2 5 "21 L '• 12 " 2 6 "15' m died pr-/or to M v — ^ A V w V W w jfm^amm^ V v 10 12 2 4 Day 4 10 12 2 4 Day 5 10 12 2 4 Day 6 10 12 2 4 Day 7 10 12 2 4 Day 8 » » » f 10 12 2 4 Day 9 10 12 2 4 Day 10 FIGURE 12. D A Y and T I M E (hours) A q u a l i t a t i v e record of spawning times and nest building i n G-H treated and G control males. KEY *~-H S p a w n i n g • Nest testing, probably as a r e s u l t of injury through i n j e c t i o n . Therefore, of the f i v e G U I males given the hormone treatment, four spawned with G22 spawning twice (Day 8 and 10). G 2 i H b u i l t a large nest i n i t i a l l y but i t tapered off during the week and no spawning occurred. None of these f i s h spawned p r i o r to hormone treatment (Fig. 1). Quite a variable picture as to spawning day can be seen which i s quite reminiscent of G i l males previously (Fig. 1). The Group II controls show an even more in t e r e s t i n g pattern. F i r s t l y the GUI males did not spawn nor did they undertake nest b u i l d i n g . The three G i l males a l l spawned as before but th i s time, a l l on Day 5. This behavior follows c l o s e l y that of the Sh males (Fig. 1). It might also be noted that nearly a month had passed i n most cases since the previous te s t i n g and two months since the o r i g i n a l operation. B. Pre Spawning Behavior The histograms (Figs*. 13-19) indicate the pre-spawning a c t i v i t i e s of the G-H treated males and the GII and III controls i n the same manner as was presented i n Chapter 4. In addition the o r i g i n a l Sh male trends have been included for comparison. Once again the data appearing i n Figs.13-19 have been treated s t a t i s t i c a l l y i n the same manner as those presented i n Chapter 4. Here also the "p" values have been included where applicable for the following comparisons: H treated G to Cat. II Controls H treated G to Cat. I l l Controls H treated G to Or i g i n a l Sh Controls Approach (Fig. 13) The mean frequencies of male and female Approach are given i n F i g . 13a-b. Once again male A i s highest in Rl and diminishes through R3 except the G U I males who show a l e v e l i n g off at R2 (G-H males to G U I males, p = 0.1153). There i s however, a s i g n i f i c a n t difference between GII males and G-H males i n Rl (p = 0.0396). The G-H and Sh males follow a p a r a l l e l trend i n Approach. GII males are similar but at a lower l e v e l . The GUI females and G-H females show a p a r a l l e l trend i n Approach. FIGURE 13 a) o* Approach KEY 9 G - H treated A Cat II O Cat I I I — Sh T r FIGURE 14 a) D isp lay — i — 10 — i — 12 4 R. 6 8 Time (Hours ) 10 b) ^ Approach i-Display and Butting F i g . 14-15 ' In both male and female Displaying D and Butting B there i s a higher frequency i n Rl for G U I than for any of the others. However, the differences here are not as s i g n i f i c a n t as they were i n the previous experi-ment. The G-H males and females are a l l very close to G U I i n R l . GII males p a r a l l e l the Sh males for D but at a lower l e v e l . In R2 and R3 there i s a similar trend between GII, G-H and Sh males. Only GUI males continue to drop. For male B GII and G-H males drop to nearly zero i n R2. There i s no tendency to p a r a l l e l the Sh males i n B. None of these differences, however are s i g n i f i c a n t . Female B did not occur for any f i s h after R2 except for GUI. Here GII and Sh females showed no B at a l l . Chasing and Fleeing F i g . 16a-b. The frequency of Chasing drops o f f for G-H males whereas i t s t e a d i l y increases for Sh males. GUI males show quite an upswing for Chasing after an i n i t i a l drop off during Day 4. GII males showed very l i t t l e chasing throughout. ~6X~ There i s a p a r a l l e l trend between the G—H male Chasing and Butting and G U I female Fleeing. G i l females p a r a l l e l Sh females for Fleeing tendency. In these two graphs trends can be seen, but differences between the groups are not s i g n i f i c a n t . Nest Building F i g . 17 Turning to the trends for male Nest Building i t is i n t e r e s t i n g to note the close p a r a l l e l between G i l males and Sh males for both frequency and mean duration per t r i p . The G-H males on the other hand show a much lower frequency of N but a correspondingly higher mean time per t r i p . When these values are computed in terms of t o t a l duration of N for a 15 minute observation i t becomes apparent the G-H males and G i l males are spending s l i g h t l y less time than the Sh males. G U I males did not nest b u i l d (Comp. G U I males and G-H males: p = 0.0948). Hiding F i g . 18 i The trends i n female Hiding appear to fluctuate greatly but actually each female i s showing the same trend on a t o t a l duration basis i . e . that of an increase KEY • G - H t rea ted A Cat II 63 •» i n the amount of time spent i n Hiding. The only females not showing a net increase are those of GUI. If the two graphs are multiplied together i t can be seen that Sh females are spending the most time i n Hiding by R3, the G-H females are next, GII females next with G U I females the l e a s t . In summary of the prespawning behavior. F i g . 19 i l l u s t r a t e s the percentage of male Approaches r e s u l t i n g i n female Fleeing into Hiding. Here can be seen a steady increase for GII males in d i c a t i n g a s t e a d i l y increasing aggression l e v e l which might be expected since a l l three spawned on Day 5 (Fig. 12). However, the f i n a l l e v e l did not reach the height found for Sh males. The G-H males showed a p a r a l l e l trend but at a much lower l e v e l . This i s surprising since three (fifths^pf these f i s h also spawned on Day 5 (Fig. 12), yet there i s a large difference between these and the Sh males. The G U I then their dominance f e l l or aggression dropped r i g h t off to a very low l e v e l i n R3. This drop i s r e f l e c t e d i n F i g . 13a-b by an upswing of A for both G U I males and males followed the G-H males exactly through Day 4 but — 1 1 1 1 1 1 > 1 0 12 2 4 6 8 10 R l R2 R 3 Time (Hours) FIGURE 19. The percentage of male Approaches r e s u l t i n g i n female F l i g h t into Hiding. -65-their females. Fi g . 16a-b for G III show an increase i n Chasing and Fleeing at R3 but t h i s f'emale Fleeing i s not terminating in Hiding (Fig. 18). Thus the GUI females as i n the previous experiment are not being intimidated into Hiding by dominant and increasingly aggressive, nest building males, and as before they did not spawn. C. The Spawning sequence Each G-H male which spawned was recorded for upwards of two hours. During t h i s time approximately eight spawning cycles and nest building i n t e r v a l s were recorded for each p a i r . The behavior patterns involved followed exactly the same cycle as outlined i n F i g . 9. A mean spawning cycle and nest building i n t e r v a l for each G-H pair was calculated (Fig. 204. Then an o v e r a l l mean calculated from the i n d i v i d u a l means was set up. Also i n F i g . 20athe o v e r a l l mean from the eight Sh and unoperated males (Fig. 10) was included for comparison. Noteworthy here are the facts that i n the case of the GUI males, four out of f i v e did spawn with KEY G 1 4 H G 2 0 H G 2 2 H G 2 5 H G 1 2 H jfRubbing E n c i r c l i n g §§§Clasping H R O I l o v e r 1 • I n h i b i t i o n Nest B u i l d i n g Mean C y c l e G-H males Mean C y c l e Sh males — i — 10 — I — 20 — r -40 — i — 60 80 100 T ime — i — 120 140 160 — I — 180 200 (Sec) Mean Spawning Cycles T i m e ( M i n ) Mean N e s t b u i l d i n g I n t e r v a l s 15 FIGURE 20a Mean Spawning Cycles and N e s t b u i l d i n g I n t e r v a l s f o r G-H males showing o v e r a l l mean and Sh o v e r a l l mean. hormone and the spawning pattern was completely organized i n every respect. Further i t can be seen there was very l i t t l e v a r i a t i o n of the means for each f i s h except for the nest b u i l d i n g i n t e r v a l . The over-a l l mean i s p r a c t i c a l l y i d e n t i c a l to that for the Sh males. F i g . 12 shows far more v a r i a t i o n of the means among the Sh and unoperated male controls. Discussion The r e s u l t s presented indicate quite conclusively that methyl testosterone treatment restored much of the reproductive behavior up to and including spawning among the GUI males. Indeed, the spawning cycles recorded were far more regular than those recorded for Sh males. It seems highly probable that the lone GII male, G i 4 H * which received hormone treatment would have spawned without i t - a s did the three GII controls. In t h i s experiment G U I controls showed much the same behavior as they did previously (Chapter 4) in that they f a i l e d to intimidate the female into Hiding, did not Nest b u i l d and did not spawn. Yet on re-operating, two of the four showed some regeneration. -6§-The GII controls on the other hand spawned again showing similar behavior to the previous t e s t i n g . However, i n th i s experiment they a l l spawned on Day 5 whicli i s reminiscent of the o r i g i n a l Sh control l e v e l . -69-CHAPTER 6 SOME MORPHOLOGICAL EFFECTS OF GONADECTOMY AND METHYL TESTOSTERONE TREATMENT ON THE SECONDARY SEXUAL CHARACTERISTICS OF THE MALE AND FEMALE Introduction About three weeks to one month following gonadectomy a pronounced shortening and rounding of the male dorsal f i n was observed. Measurements were made of these and of unoperated males and females for comparison. Later, following hormone administra-t i o n to ce r t a i n males, further measurements were made to provide further evidence of the hormonal e f f e c t s . Procedure The f i r s t set of measurements was made on May 31, 1966 or approximately f i v e weeks following gonadectomy. The f i n a l set of measurements was made during the f i r s t week of July, 1966 or about ten days following the termination of hormone treat-ment. - 70-Th e following groups of f i s h were measured: I Unoperated and Sh males II Unoperated females III Gonadectomized males (a) GII (b) GUI IV Hormone treated Gonadectomized males (a) GII (b) GUI V Hormone treated females Results The raw data (cm) and their r e s u l t i n g r a t i o s together with the mean r a t i o s calculated for each group have been included as a series of tables i n Appendix I. The meaniratios and th e i r standard errors for each group have been plotted with respect to an approximate time scale (Fig. 20^. This scale i s approximate since a l l f i n a l measurements were not i n f a c t made the same day but spread over a week or more depending on t e s t i n g time, day or period of certa i n groups. F i g . 20bshows the range of f i n to length r a t i o s on the v e r t i c a l axis. At an a r b i t r a r y point before (3 0£ at c 4) in O Q 1^>50| 1-000-0-950-0-900-0-850-0.800 r Gonadectomy 1st dVlVleasure (? H. Treatment L. A r b i t r a r y In i t i t i a l Measure 9 H. . Treatment Final 9. Measure ••Final tf Measure —A KEY • Sh » V a Cat II G <?<? O Cat III G d V SB Cat l l - G - H 0 C a t III G - H A Normal 99 A H - 99 A p r i l M a y J u n e Ju l y FIGURE 20b. Effects of gonadectomy and methyl testosterone on dorsal f i n r a t i o s . - 7 2 -gonadectomy a group of Sh males and normal females were measured and their mean r a t i o s plotted to give a standard or base l i n e for control males and females. It was assumed that these two values would not deviate much and hence they were car r i e d across the time scale. The male value of 1.035 indicates the distance from snout to dorsal f i n t i p was 1.035 times the standard length. The mean female r a t i o was 0.891. Following gonadectomy, males were c l a s s i f i e d according to their behavior into two groups (GII and G i l l ) . On May 31, 1966, these two Categories of males were measured separately and the mean r a t i o s for each group plotted. From th i s measure two things became apparent. a) A l l males measured f i v e to eight weeks after gonadectomy revealed a lower f i n to length r a t i o than the control males. b) There was a d i s t i n c t difference i n the amount of f i n loss between the GII and G U I males. The GII males which displayed reproductive behavior -73-including spawning but at a lower l e v e l than Sh males had a correspondingly lower f i n r a t i o of 1.017. The G U I males on the other hand which displayed much less reproductive behavior and did not spawn had a s t i l l lower r a t i o , i . e . , 0.957 which was about mid way between control males and females. Methyl testosterone was administered to 50% of the GII and G U I males and ten days following cessation of this treatment, a l l were remeasured including the 50% receiving control i n j e c t i o n s . The plotted r e s u l t s show a s p l i t i n both G categories. It can be seen that testosterone treatment produced an increase i n the f i n r a t i o i n both groups,. Since there was only one GII-H male, the reading of 1.050 may not be r e l i a b l e , however, hormone appears to cause i t to become more male-like than the controls. The f i v e G U I males receiving hormone showed a dramatic mean increase from 0.957 to 1.020. This measure correlates very c l o s e l y with the pre-hormone measure of the GII males (1.017). Of the G males receiving control injections the GUI group showed a further mean drop to 0.946. - 7 4 The GII group showed a s l i g h t increase (1.017-1.028). F i n a l l y , after treating a group of six unoperated females with methyl testosterone for an extended period (these were o r i g i n a l l y used to test dose levels and i n j e c t i o n methods) they were measured i n mid June. Here can be seen a dramatic increase i n their f i n to length r a t i o s over their controls (0.891 - 0.975). Discussion The shape and si z e of the dorsal f i n i s the most obvious morphological difference between the male and female blue gourami. Apparently the larger, pointed shape of the male f i n i s under the control of gonadal secretions. In some cases following gonadectomy there was a d r a s t i c reduction i n f i n si z e to the point where i t became almost: indistinguishable from the female f i n (GUI males). In other cases gonadectomy resulted i n only a s l i g h t f i n atrophy (GII males). Exogenous replacement by testosterone resulted i n a regrowth of the f i n to some extent i n a l l cases. On i n j e c t i n g i n t a c t -75-females with testosterone th e i r external morphology began to take on a male appearance. Therefore the dorsal f i n has been classed as a morphological secondary sexual c h a r a c t e r i s t i c ( s . S.C.) whose s i z e and shape i s controlled by androgens. « -76-CHAPTER 7 PARENTAL BEHAVIOR SEEN IN SHAM OPERATED, GONADECTOMIZED AND TESTOSTERONE TREATED GONADECTOMIZED MALES Introduction Parental behavior as displayed by the male blue gourami consists of accepting the eggs at the time of spawning and maintaining a nest around them u n t i l they hatch. For two or three days following hatching, stray f r y are c o l l e c t e d i n the mouth and spewed back into the nest. After t h i s time the f r y are too active and the male gives up c o l l e c t i n g , but the nest may be maintained for another week to ten days. Certain aspects of t h i s behavior were tested on Sh, GII and G-H treated males. Procedure F e r t i l i z e d eggs were removed from the nests of normal and sham operated males by the method outlined i n Chapter 2. These eggs were given to other Sh males as well as G males and G-H treated -77-males i n various stages of the reproductive cycle for the purpose of determing when i n the cycle the parental phase began. Eggs were given at the following times. a) Before courtship i . e . before adding the female. , b) During courtship but before spawning. c) Immediately after spawning i . e . same or next day. d) One week after spawning. e) Two weeks after spawning. Eggs present i n the tanks of the f i s h to be tested as a r e s u l t of their own spawning, were removed p r i o r to the addition of the test eggs. Results Table II summarizes the number of times the males of each group either accepted and cared for the eggs given, or rejected and ate them, at the points during the reproductive cycle described above. -78-No. of T r i a l s Time of Addition Accepted Rejected 7 Before courtship. 0 7 5 During courtship -before spawning. 1 4 7 Immediately after spawning. 7 0 4 1 week post spawning. 4 0 3 2 weeks post spawning. 0 3 EGGS GIVEN TO Sh MALES 7 Before courtship. 0 7 4 During courtship -before spawning. 0 4 3 Immediately after spawning. 3 0 1 1 week post spawning 1 0 EGGS GIVEN TO G MALES Immediately after spawning. EGGS GIVEN TO G-H MALES TABLE I I . SUMMARY OF RESULTS OF EGG TRANSFERS -79-From th i s table i t may be seen that Sh males would not accept eggs before the addition of the females i . e . , before courtship, nor would they accept eggs during courtship. There was one case where a male which was dark and nest building and apparently close to spawning did at least i n i t i a l l y , accept a number of eggs that were added. But usually a male i n th i s condition would eat eggs immediately. A l l G males tested showed i d e n t i c a l behavior. However, once spawning has occurred, i . e . clasping and oviposition, both Sh and G males appear to accept the addition of f e r t i l i z e d eggs. Sh eggs were transferred back and f o r t h when several spawnings occurred on the same day and each accepted the other 1s eggs. A rough idea of how long following spawning th i s parental acceptance phase l a s t s was obtained by giving males eggs at one week post spawning and at two weeks post spawning. The Sh males accepted eggs after one week i n a l l cases and rejected them after two weeks. This indicates an egg acceptance period from immediately after or during spawning u n t i l somewhere between one and two -80-week s l a t e r . I t was in t e r e s t i n g to note that i n both the one and the two week tests, some tanks contained l i v i n g f r y and others did not (eggs having been removed in t o t a l before hatching). Therefore, the presence or absence of f r y did not a f f e c t egg acceptance at one week nor did i t e f f e c t egg re j e c t i o n at two weeks. The G males which spawned (GII) accepted f e r t i l i z e d eggs i n place of their own i n f e r t i l e eggs i n a l l cases. One such male accepted eggs after one week»s duration. An opportunity for tes t i n g at two weeks duration did not present i t s e l f but i t may be assumed they would r e j e c t as did the Sh males. The G-H treated males were given f e r t i l i z e d eggs immediately following spawning and accepted i n a l l cases. No other tests were made on these f i s h . Some in t e r e s t i n g observations were made on the parental behavior displayed by the GII males and the G-H males regarding their own eggs. In a l l these cases, the eggs produced were checked and found to be i n f e r t i l e . However, 30-40 percent of the eggs were always l e f t with the f i s h . Here i t was noted -81-that i n i t i a l l y (during and immediately following spawning) the males co l l e c t e d and concentrated the eggs in the nest i n the usual manner. However, as time passed, the eggs began to disappear and by the next morning in. a l l cases there was hardly an egg to be found. It was assumed that when the eggs began to disintegrate as a r e s u l t of b a c t e r i a l and fungal attack, they were weeded out and eaten by the male so that by morning, a l l had disappeared. This assumption i s based on the fa c t that the eggs previously removed were s t i l l i n t a c t the next morning but were .white with fungus.' Discussion The experiments described above were ca r r i e d out as part of an examination of the gonadal r o l e i n a l l aspects of reproductive behavior i . e . , i s parental behavior under separate control from sexual behavior? An examination of the r e s u l t s would appear to indicate that something to do with the act of spawning i t s e l f , or the sight of eggs i n the nest, triggers the mechanism of parental behavior. V i r t u a l l y no eggs were accepted by any male who had not performed -82-the spawning cycle at least once. The usual or natural reaction of any male which i s not i n the parental phase i s to eat the eggs immediately on discovery. Therefore some mechanism set o f f by the spawning act i n h i b i t s t h i s usual tendency. Apparently t h i s i n h i b i t i o n only la s t s for approximately ten days after which time eggs are eaten again. The int e r e s t i n g point here i s that f r y are not eaten after ten days. Indeed, males have been l e f t with their f r y for up to a month with no apparent decrease i n the number of f r y . Yet, i f f r y are placed in a tank with an unspawned male, they are quickly eaten as are the eggs. One male which had resided with several dozen fr.y for about two weeks was removed and castrated then given two days i n a recovery tank after which i t was returned to the tank containing the f r y . Apparently no f r y were eaten on i t s return. It may be postulated then, that the spawning act triggers a neural or endocrine mechanism which causes the i n h i b i t i o n of egg eating and probably stimulates the c h a r a c t e r i s t i c parental behavior. As time -83-progresses, the e f f e c t of t h i s mechanism slowly decreases, but long before i t has ceased to be e f f e c t i v e , the eggs have hatched into f r y which the male also i s i n h i b i t e d from eating. Possibly by a process of learning the male avoids eating the f r y or i n other words becomes used to their presence. This learning process l a s t s long after cessation of the i n i t i a l i n h i b i t o r y mechanism. This seems even more probable when one remembers that f e r t i l e eggs only l a s t about twenty four hours; thus the f i s h has very l i t t l e time i n which to "learn" to avoid them whereas the f r y may be present for many weeks. Thus i t i s possible that a c e r t a i n time after spawning, fresh f e r t i l e eggs may be offered to a male and be immediately eaten, while at the same time hundreds of f r y are m i l l i n g about the feeding male. The trigger for egg acceptance i s l i k e l y associated with the actual physical or t a c t i l e sensations produced by the spawning act and not by any chemical nature of the f e r t i l i z e d eggs themselves. This seems l i k e l y since G males and G-H males on spawning i n i t i a l l y cared for their i n f e r t i l e eggs i n the same manner as the Sh -84-males. The i n f e r t i l e eggs were only eaten after they began to decompose. The eating i n t h i s case was l i k e l y a hygenic response which any male, regard-less of gonadal state would perform, since l i k e l y , not every single egg i n a f e r t i l e spawning i s viable. The f i n a l point which comes from these r e s u l t s i s simply that the egg acceptance mechanism triggered during spawning i s not mediated by androgen from the testes. This i s demonstrated by the f a c t that GII males spawned and accepted eggs i n the usual way. When given f e r t i l e eggs, these were cared for as well. As was mentioned e a r l i e r (Chapter 4) on examining the Cat II G males at the conclusion of a l l experiments, no trace of t e s t i c u l a r tissue could be found i n two of the three. Therefore, the presence or absence of testes apparently has no e f f e c t on egg acceptance once spawning i s achieved. However, this evidence does not r u l e out the p o s s i b i l i t y that testosterone may s t i l l be important, i . e . , i f i t was being made available from some other source. -85-CHAPTER 8 AN ANALYSIS OF THE AGONISTIC BEHAVIOR BET-WEEN GONADECTOMIZED AND SHAM OPERATED MALES Introduction Agonistic behavior displayed between males was tested i n a series male-male encounters. Different combinations of males were used. Sh males were paired with each other and with G males. Also G males were paired with each other. Any differences noted could be attributed to the effects of gonadectomy. Procedure The t e s t i n g procedure was i d e n t i c a l to that used for male-female interactions. In thi s case the two males to be tested were placed i n a tank and separated by a black p a r t i t i o n . On the morning of Day 4 the p a r t i t i o n was removed and one behavioral recording was made which was terminated when one male established i t s e l f as dominant. Three classes of male interactions -86-were tested with four t r i a l s i n each c l a s s . Table III indicates the classes and their t r i a l s . A l l of these records were made pri o r to the commencement of hormone treatment. TABLE III FISH COMBINATIONS USED TO TEST AGONISTIC BEHAVIOR Class T r i a l Combination 1. Sh male vs Sh male 1 Sh & - Sh 4 2 Sh 6 - Sh 5 3 Sh 7 - Sh 3 4 Sh ? - Sh 4 2. Sh male vs G male 1 Sh 5 - G 2 3 2 Sh 6 - G 1 5 3 Sh 5 - G 1 2 4 Sh 4 - G 2 5 3. G male vs G male 1 G 1 6 - G 2 1 2 G12 - G 2 3 3 G26 " G25 4 G12 " G26 -87-Results During the recording of the three groups tested i t became apparent to this observer that there was very l i t t l e difference between them. Sh males paired with each other or G males, or G males with each other a l l behaved i n a similar fashion to that described i n Chapter 3. One observed difference was that the G males did not become as dramatically black as the Sh males although they were very dark. The bout lengths varied considerably within each c l a s s . It appeared that the lengths of the disputes varied with s i z e differences between the two p a r t i c i p a t i n g f i s h rather than with their gonadal state. The closer the two were to each other i n siz e , the longer the bout. The frequencies of four behavior patterns (Approach, Display, Butting and Fin Tugging) were taken for each f i s h i n each bout. Since a l l bout lengths varied, the raw frequencies were computed as rates i . e . , number per 10 minutes. In each bout the winner and loser were computed separately. A mean rate for each behavior pattern recorded for the winner and the loser was compiled from the four t r i a l s i n each c l a s s . F i g . 21 shows the r e s u l t i n g mean -88-rates presented as a series of winner and loser bars for each class and each behavior pattern used. F i g . 22 indicates the mean bout length for the three classes. From th i s graph i t can be seen that the o v e r a l l average bout length was about f i f t e e n min. The longest dispute observed was twenty seven minutes, while the shortest was three minutes. From the bar graph the following observations were made: 1. There i s l i t t l e difference in the mean rates for Approach, Display or Fin Tugging between the three classes or between the i n d i v i d u a l winners and losers. 2. A considerable amount of Butting occurred for the three classes. 3. There i s a tendency for the winner to Butt less than the loser i n a l l three classes. This Tendency i s apparent only for Butting. 4. The frequency of Fin Tugging i s very low. This may be misleading since each Fin Tugging episode may l a s t several seconds whereas a single Butt i s more or less instantaneous. 80 Ul c 0) 3 o GO c i 60-o - 4 0 u c V 3 a « 41 KEY I W inner D Loser Sh | Sh-G I G A p p r o a c h Sh I S h - G | G D i s p l a y Sh Sh-G B u t t W i l l G Sh | Sh JLmTL G | F in Tug FIGURE 21. Mean frequencies of behavior patterns recorded for Sh-Sh, Sh-G, and G-G male encounters. 20-15-c .E « 10-E 5-Sh Sh-G FIGURE 22. Aver-age bout length for each c l a s s . -90-5. There appears to be a c o r r e l a t i o n between the mean rates of Display and Approach i n a l l three Classes. Discussion From the re s u l t s presented i t would appear that gonadectomy has l i t t l e e f f e c t on the i n i t i a l agonistic behavior. Both the Sh and G males follow the same sequences of Approach, Display, Butting and Fin Tugging in alternate fashion with accompanying darkening. G males however, were not observed to be as dark as Sh males. It i s suggested that colour darkening may be under the influence of androgens since a l l the G males used were of the GUI group which had not spawned (Chapter 4) nor had they exhibited colour darkening when paired with females. A l l GII and GUI males under hormone treatment showed intense colour'change when spawning. Certain G U I males when paired with each other or Sh males were i n i t i a l l y treated as females since they were highly female-like i n appearance (Chapter 6). The male-female behavior was of short duration, since the female-like males did not turn and Flee but remained and -91-returned the Butting. The rates for Approach and Display are very similar since just about every Approach resulted i n Display. The lower rate for Butting i n G—G p a i r i n g i s probably a r e s u l t of the fac t that two of the G-G t r i a l s were of very short duration (three-four min.) Since the f i r s t 30 seconds of any encounter i s taken up with Approach, Pelvic Contact and Display, the high rate of Butting does not become apparent with such a short bout. The winning f i s h i n every case was s l i g h t l y larger than i t s oponent. Of the four G—Sh t r i a l s , three were won by Sh f i s h and one by a G f i s h . Here again the winning G male was the largest. A f a i r l y d e f i n i t e trend appears i n Butting where the los i n g f i s h tends to Butt more than the winner. This again probably r e s u l t s from s i z e difference. It would seem that from a s t r i c t l y physical basis, a greater number of Butts from a smaller i n d i v i d u a l would be necessary to achieve the same r e s u l t as a smaller number of Butts from a larger i n d i v i d u a l . The moment of decision was quite dramatic. The los i n g f i s h for no apparent reason, suddenly blanched, ceased a l l attacks and swam away. Several of the Sh f i s h had bubble nests at the time of release. There was no apparent attempt to defend t h i s nest on the part of i t s owner. The dispute ranged throughout the tank equally. However, the test was terminated following completion of the dispute. Other observations, suggest that nest or t e r r i t o r i a l defence would develop sometime following the i n i t i a l encounter. -93-CHAPTER 9 GENERAL DISCUSSION AND CONCLUSIONS I General Discussion a) Normal Reproductive Behavior It seems worth mentioning at the beginning of th i s discussion that a l l of the behavior observed, des-cribed and recorded i n this thesis occurred i n a laboratory, i . e . aquarium s i t u a t i o n . This was also true of M i l l e r ' s (1964) work. A l l r e s u l t s therefore must be interpreted with t h i s i n mind.. This observer f e e l s that a l l of the behavior patterns described would be seen i n the f i e l d , but not necessarily at the same frequencies or durations since the natural environment would be less confining. Interesting also i s the point that a very reliable'method for predicting spawning evolved from th i s study. In 70 percent of the t r i a l s involving i n -experienced f i s h of both sexes, spawning occurred within two or three day's after the single opaque p a r t i t i o n was removed. M i l l e r describes a nonreproductive or s o c i a l phase occurring between reproductive periods. This -94-aspect of behavior was never c l e a r l y observed i n the present study. The animals would enter into a re-productive phase whenever the environment was manipulated i n the manner described. Those f a i l i n g to spawn in t h i s s i t u a t i o n always showed a c e r t a i n amount of sexual behavior. After working with the blue gourami for over a year and observing many spawnings, this observer has not been able to define c l e a r l y any p a r t i c u l a r behavior patterns as "courtship". From the analysis of Sh prespawning behavior (Chapter 4) a number of trends were described. I n i t i a l l y , i t appears that Approach, Pelvic f i n ray contacting and alternate Displaying are important. These give way to male Butting and Chasing and female Fleeing and Hiding. The Sh male asserts his dominance over the female very soon after release. Male aggressive behavior towards the female increases s t e a d i l y with the r e s u l t that she i s intimidated into Hiding more frequently and for longer periods. The increased aggression i s accompanied by gradual darkening and an increase i n the frequency and duration of Nest Building. U n t i l actual spawning begins there i s no r i t u a l i z e d courtship behavior -95-such as i s found i n other species, e.g. stickleback, b i t t e r l i n g and guppy. Furthermore, the s t a r t of the spawning cycle, which i s the only part of the behavior that could be described as at a l l r i t u a l i z e d , appears to be completely dependent on the female. Here i s the most in t e r e s t i n g question of t h i s study. What causes a female which has been Butted and Chased, often having the whole d i s t a l portion of her anal f i n torn away, to suddenly swim forth, apparently of her own v o l i t i o n , and i n i t i a t e spawning? A possible explanation i s that during the time from release u n t i l spawning and as a r e s u l t of seeing the male i n the act of nest building, a f i n a l maturation of the female gonad i s triggered. This might involve a readying of a c e r t a i n number of eggs for release plus an increase i n gonadal secretions. When these secretions, presumably steroids, reach a c e r t a i n l e v e l as a r e s u l t of constant v i s u a l stimulation, sexual behavior i s i n i t i a t e d . This could occur through d i r e c t action on the brain by the steroids or by r e f e r r a l , through the p i t u i t a r y . Lehrman (1965), describes mechanism of t h i s type -in the female r i n g dove. He claims i t i s the sight of the male i n the -96-act of nest building which brings the female into reproductive condition. This i s accomplished through a neural-endocrine l i n k i . e . from the eye to the brain to the p i t u i t a r y to the gonad. Due to the lack of complete understanding of the contribution of the female i n terms of prespawning behavior, i t has had to be assumed during t h i s study that a l l females would react i n the same manner to a presented male. Thus any large differences i n the time from the release u n t i l spawning, or the lack of spawning altogether, were attributed tto some change or f a i l u r e i n male behavior as a r e s u l t of gonadectomy. In summary, a mature male when i n the presence of a mature female w i l l usually commence nest building. Nest building i s accompanied by colour change and t e r r i t o r i a l behavior. This leads to the following hypothesis: there i s no r i t u a l i z e d prespawning behavior in t h i s species. It i s the sight of the Black, Nest building male which brings the female into reproductive condition. The discussion to follow attempts to show that nest building, colour change and the morphological secondar sexual c h a r a c t e r i s t i c s i n the male are the important factor -97- i i n bringing the female into condition and that a l l three are mediated by androgen. b) Effects of Gonadectomy The performance of the Sham operated males following one month's recovery from operation indicates that the differences observed on the part of gonadectomized males on r e t e s t i n g .could not be attributed to any physical effects of the operation i t s e l f . The behavioral evidence presented i n Chapter 4 showed that eleven out of sixteen castrated males (GUI) did not succeed i n e l i c i t i n g a spawning response i n the females. P a r a l l e l i n g the change i n behavior was a dramatic reduction i n the s i z e and shape of their dorsal f i n s (Chapter 6). As described i n Chapter 3, the s i z e and shape of the dorsal f i n i s the only gross morphological difference between the sexes. In the case of the f i v e G males which did spawn (GII), t h e i r behavioral records indicated that a lower l e v e l of aggression was reached than by the Sh males. But s t i l l t h i s was above the aggression l e v e l of the GUI males and included nest b u i l d i n g . Correspondingly there was a s l i g h t reduction i n the dorsal f i n s i z e for these -98-f i v e over the Sh males but this was well above that of the majority or G III males. It has been mentioned e a r l i e r that the GUI males were extremely female-like in appearance and furthermore i n i t i a l l y were displaying t y p i c a l agonistic behavior with the females. This observer suggests that the females could hot d i s t i n g u i s h the GUI males from females and treated them accordingly. P i c c i o l o (1964) conducted a number of tests involving v i s u a l , chemical, auditory, l a t e r a l l i n e and p e l v i c f i n ray s t i m u l i . He concluded that sexual discrimination in the blue gourami i s s t r i c t l y v i s u a l . He placed males with dorsal f i n s clipped to look l i k e females i n glass containers f l o a t i n g i n aquaria. Undipped males were placed i n other glass containers. When females were placed i n the aquaria he noted they approached the containers holding undipped males far more often than those containing clipped males. This evidence backs the present findings regarding the f a i l u r e of the females to discriminate and their r e s u l t i n g agonistic behavior. The author feels that apart from the morphological -99-changes r e s u l t i n g from gonadectomy, the most important behavioral change was the f a i l u r e on the part of the males to b u i l d nests. As mentioned e a r l i e r i t seems l i k e l y that the sight of a f u l l y mature male i n the act of nest building with t y p i c a l dark coloration brings the female into reproductive condition. The high l e v e l of aggression accompanying nest building i s undoubtedly a function of i t , i . e . s t r i c t l y t e r r i t o r i a l i n nature, with l i t t l e or no d i r e c t connection with courtship. An observation made pr i o r to the experiments may help to i l l u s t r a t e t h i s . Two unoperated males and females were l e f t together i n a 100 l i t e r tank. The larger of the two males b u i l t a nest at one end and defended i t vigorously against both females and the other male. These females were not driven into hiding but merely to the other end of the tank which was much larger than the regular tanks used. Then after a day or so both females were observed spawning with the large male alternately. In fa c t one female was observed r a p i d l y Approaching and Butting the male who was already engaged in Clasping the other female. The Clasp was broken and the male commenced to Rub the intruder^ Here then, both -100-females had come into reproductive condition at the same time as a r e s u l t of the sight of the dark, nest building male. If the hypothesis i s correct, then the f a i l u r e of spawning among the GUI f i s h can be explained i n two ways. F i r s t , as a r e s u l t of gonadectomy and presumably the lack of gonadal hormones, these males had l o s t t h e i r secondary distinguishing features and f a i l e d to nest b u i l d with accompanying colour change and t e r r i t o r i a l aggression. Secondly, the females i n i t i a l l y f a i l e d to dis t i n g u i s h them as males, and behaved towards them as they would to normal females. On receiving no further s t i m u l i from the males, the females f a i l e d to come into reproductive condition. From the evidence presented i n Chapter 8 i t can be seen that gonadectomy had v i r t u a l l y no ef f e c t on male agonistic behavior. Here, a l l G males tested were of Category I I I . The only,observed difference was the fact that these G males did not undergo as dramatic a colour change as the Sh males. Therefore i t i s suggested that colour change i s p a r t l y under gonadal control since these f i s h showed very l i t t l e darkening when paired with -101-females. However, gonadectomized males are quite capable of agonistic behavior and even of establishing dominance over an Sh r i v a l . Further, the G U I males behaved towards normal females i n the same manner as they did toward the Sh males i . e . a high rate of Display, Butting and Fin Tugging. Females also behave i n i t i a l l y towards each other i n the same manner but for a shorter duration and without colour darkening. Thus both males and females are capable of agonistic behavior but the female form lacks the i n t e n s i t y and accompanying colour change. It i s assumed the females would not normally be secreting androgens. With regards to parental behavior (Chapter 7), i t was shown that no males either operated or unoperated would accept eggs or f r y u n t i l they had gone through the physical process of spawning at least once. G U I males never achieved spawning so i t was impossible to test whether they were capable of parental behavior. GII males diq^io^t^spawn however and entered into a parental phase i n the same manner as the controls. These males i n i t i a l l y cared for the inviable eggs and only ate them when they began to decompose. When given f e r t i l e eggs they were looked after i n the usual way. -102-These observations suggest that some neural or endocrine mechanism triggers parental behavior and associated egg eating i n h i b i t i o n as a r e s u l t of spawning and that t h i s mechanism i s not necessarily associated with the testes. Further this i n h i b i t o r y mechanism seems to l a s t only a short while (approximately ten days) after which the males w i l l no longer accept eggs but they w i l l continue to avoid f r y . Perhaps during the period of i n h i b i t i o n the males become used to the f r y through constant association i . e . by a learning process. When a l l G males were reopened following com-pl e t i o n of the hormone experiment i t was noted that two of the^fouryGIII males and one of the three GII males showed some testes regeneration. The four GUI con-t r o l s on retest (Chapter 5) showed no more i n c l i n a t i o n to Nest build than they did previously. Their dorsal f i n s i z e was on the average s l i g h t l y smaller than before and yet regeneration was discovered i n two. On the basis of the r e s u l t s of the hormone treatments, i t was f e l t the only plausible explanation could be that this regeneration was either non existent or too small at the time of testing to have much eff e c t , i . e . to produce a hormone l e v e l s u f f i c i e n t to promote Nest building. -103-In support of the above suggestion, i t may be pointed out that reoperation took place a month after termination of the hormone experiment. At thi s time, two instances of s l i g h t regeneration were found. The dorsal f i n s of these two were noticeably longer at the time of reoperation than when previously measured. It was f e l t therefore that this re-generation must have occurred to a large extent following the second testing. The GII controls present a more in t e r e s t i n g picture s t i l l . These f i s h when tested f i r s t showed only s l i g h t dorsal f i n atrophy and their behavior was of a s u f f i c i e n t i n t e n s i t y to cause spawning but they f a i l e d to f e r t i l i z e eggs. At the time of their second testing their dorsal f i n s were p r a c t i c a l l y i d e n t i c a l to the Sh males and further the time from release to spawning was also similar to Sh males. Their aggression l e v e l was closer to the Sh males than was that of the G-H males. A l l of these observations suggested re-generation of testes; yet on reoperating, none could be found i n two of the three. Two possible explanations suggest themselves. Either their behavior was being mediated by some other endocrine or neural factor, or by androgen from some other source. If either of -104-these mechanisms were f u n c t i o n i n g , then the most i n t e r e s t i n g q u e s t i o n of a l l a r i s e s i . e . how d i d such, a s u b s t i t u t e system become e s t a b l i s h e d i n some f i s h , o b v i o u s l y from very soon a f t e r gonadectomy, and not i n o t h e r s ? F u r t h e r , was the behavior of the s i n g l e GII regenerate being mediated by i t s t e s t e s a l l along, or was i t too under some other c o n t r o l and t h e . r e -g e n e r a t i o n o n l y a r e c e n t occurrence? The l a t t e r was thought to be the case s i n c e the amount of r e g e n e r a t i o n was very s i m i l a r to the amount found i n the two G U I males. A f u r t h e r check (not p o s s i b l e here) w i l l be to c a r r y out a h i s t o l o g i c a l examination of the p i t u i t a r i e s to see i f they show the hypertrophy c h a r a c t e r i s t i c of c a s t r a t e d animals. c) The E f f e c t s of Methyl T e s t o s t e r o n e Replace  ment Methyl t e s t o s t e r o n e a d m i n i s t r a t e d to G U I males brought back nest b u i l d i n g , t e r r i t o r i a l aggression, c o l o u r darkening, e l o n g a t i o n and p o i n t i n g of t h e i r d o r s a l f i n s and i n four out of f i v e cases, spawning r e s u l t e d . T h i s evidence i n d i c a t e s almost beyond doubt t h a t androgen i s i n f a c t very much concerned w i t h mediating sexual -105-behavior and i n regulating the secondary sexual c h a r a c t e r i s t i c s i n the males. The effects of testosterone on the morphological secondary sexual c h a r a c t e r i s t i c s of f i s h i s well docu-mented. Wai and Hoar (1962) found the effects of testosterone the same regardless of genetic sex. They measured the height of kidney tubule c e l l s of the three-spine stickleback. They found testosterone caused an increase i n the siz e of these c e l l s in juveniles and adults of both sexes. Gorbman (1962) says "sex secondaries are almost u n i v e r s a l l y regulated by sex steroids". He reports that i n gobies, dorsal f i n spines elongate with testosterone treatment, as does the sword of the swordtail (Xiphophorus) and the gonadapod of Platypoecilus. He further states that male secondaries w i l l develop in females with androgen treatment. In the present work testosterone caused an increase i n the dorsal f i n length in GII and GUI males and in unoperated females. Not only did the dorsal f i n s of the treated females increase but their behavior was modified as well. -106-Among the six treated females an increased amount of t y p i c a l l y male agonistic behavior was observed. The important aspects here were the increase i n i n t e n s i t y and duration of Displaying, Butting and Fin Tugging plus the fact that extreme colour darkening accompanied the behavior. The most in t e r e s t i n g behavior of a l l was the f a c t that these females engaged i n a lim i t e d amount of Nest building. On opening one of these females, a very much degenerate ovary was discovered but no indi c a t i o n of any other gross changes. These r e s u l t s add further strength to the suggestion that androgen i s in fac t the missing i n -gredient following gonadectomy of the male and that i t is responsible for nest building in the male. It seems l i k e l y i t i s also responsible for colour darkening which together with nest building form the v i s u a l s t i m u l i for female reproductive behavior. Colour change was the only thing missing i n the G male agonistic behavior. This fa c t raises a question. Why did testosterone bring on t y p i c a l male agonistic behavior among the females whereas i t s lack, did not appear to reduce the in t e n s i t y of the -107-behavior i n the G males? A possible explanation here might be the r o l e of experience. A l l of the males had had plenty of experience with male contacts before gonadectomy, whereas each had had only one spawning experience before the operation. There has been much discussion recently as to the r o l e of the p i t u i t a r y i n reproductive behavior. Wai and Hoar (1962) have suggested that gonadotropin i s e s s e n t i a l for the normal expression of behavior. Aronson (1957) said there i s some evidence for the d i r e c t action of the p i t u i t a r y on c e r t a i n aspects of sexual behavior. L i l e y (1965) suggested the gonad appears to exert a regulatory influence superimposed on a more d i r e c t neural and/or p i t u i t a r y control of r e c e p t i v i t y i n the female guppy. Clemens, et a l (1966) treated guppies for 60 days after b i r t h with testosterone. They obtained a sex r a t i o of nine males to one female. These males a l l showed male secondary coloration and produced copious amounts of sperm, yet only a few s i r e d young. They suggest that the endocrine control of the p i t u i t a r y was involved i n the s i r i n g f a i l u r e . They quotei. Hoar (1962) as f i n d i n g endocrine controls from J - 108-th e gonads and the p i t u i t a r y are responsible for coordination and timing of reproductive a c t i v i t i e s . In the present experiment i n view of the above findings one might suspect a p i t u i t a r y or neural mechanism was regulating the behavior of the GII male controls without testes. Yet the effects of testosterone on the GUI males i n bringing back kthe morphological S.S.C. and sexual behavior, neither of which were l o s t i n the GII males, suggest an extra-gonadal source of" testosterone } perhaps the interrenals. II Summary of Conclusions 1. Blue gouramis may be e a s i l y and predictably induced to spawn by manipulating the environment. 2. Male prespawning behavior in t h i s species seems to be confined to the act of nest building, with accompanying t e r r i t o r i a l behavior and colour darkening. Spawning i t s e l f involves a stereotyped behavioral cycle. 3. It appears that the presence of the dark nest building male provides the stimulus s i t u a t i o n necessary to bring the female into a sexually receptive condition. After a cer t a i n period of exposure to this stimulus she i n i t i a t e s spawning. -109-4. Sexual recognition or discrimination by the female appears to be a s t r i c t l y v i s u a l process. 5. Castration eliminates nest building, t e r r i t o r i a l behavior and the spawning cycle, l i m i t s colour change, and causes atrophy of the. morphological secondary sexual c h a r a c t e r i s t i c s i n the majority of males, however i t does not reduce agonistic behavior in s o c i a l l y raised males. It was impossible to say from th i s experiment whetherkspawning was eliminated as a r e s u l t of a lack of androgen or from the f a i l u r e of prespawning behavior. 6. Parental behavior i s apparently triggered by the act of spawning regardless of gonadal condition. The male i s i n i t i a l l y i n h i b i t e d from eating the eggs by a chemical or neural mechanism of r e l a t i v e l y short duration, during which time the f r y hatch. Continued i n h i b i t i o n appears to be the r e s u l t of a learning process-. 7. Testosterone replacement causes a regrowth of the morphological S.S.C., restores nest building, colour change and spawning in gonadectomized males. 8. Testosterone given to non-operated females caused a growth of male morphological S.S.C., and brought -110-on male-like agonistic behavior, colour change and some nest building. 9. A few males, following gonadectomy, retained their morphological S.S.C., continued to nest bui l d , to show t e r r i t o r i a l ..behavior and colour change and to spawn. It i s suggested that testosterone from an extragonadal source was being made available since no testes regeneration was found. -111-LITERATURE CITED ARONSON, L.R. 1957. Reproductive and Parental Behavior. Physiology of Fishes Vol. I I . Ed. by Brown, M.E. Academic Press. ' . 1959. Hormones and Reproductive Behavior: Some Phylogenetic Considerations. Comparative Endocrinology. Ed. by Gorbman, A Wiley. New York. •• 1965. Environmental Stimuli A l t e r i n g the Physiological Conditions of the Individual Among Lower Vertebrates. Sex and Behavior. Ed. by Beach, F.A. BROWNLEE, K.A. 1965. S t a t i s t i c a l Theory and Methodology  In Science and Engineering 2nd Ed. Wiley, New York. CLEMENS, H.P., McDERMITT, C., INSLEE, T. 1966. The Effects of Feeding Methyl Testosterone to Guppies for 60 Days After B i r t h . Copeia. No. 2: 280-284. FORSELIUS, S. 1957. Studies of Anabantid Fishes I-III. Zoologiska Bidrag Fran Uppsala 32: 53-597. GORBMAN, A. 1962. A Textbook In Comparative Endocrinology. Wiley & Sons. New York. HODGES, W.R., and Behre, E.H. 1953. Breeding Behavior, Early Embryology, and Melanophore Develop-ment i n the, Anabantid Fish (Trichogaster  trichopterus). Copeia 1953: 100-107. LEHRMAN, D.S. 1965. Interaction Between Internal and External Environments in the Regulation of the Reproductive Cycle of the Ring Dove.. Sex and Behavior. Ed. by Beach, F.A. -112-LILEY, N.R. 1965. The Role of the Gonad in the Control of Sexual Behavior in the Female Guppy (Poecilia r e t i c u l a t a ) . American Zoologist  Vol. 5 No. 4: 278. MILLER, R.J. 1964. Studies on the Social Behavior of the Blue Gourami; (Trichoqaster  trichopterus). Copeia 3: 469-496. PICCIOLO, A.R. 1964. Sexual and Nest Discrimination i n Anabantid Fishes of the Genera Colisa and Trichogaster. Ecological Monographs 34 53-77. r \ ROSENBLATZJJ.S. and ARONSON, L.R. 1958. The Influence of Experience and Behavior and the Effects of Androgen on Prepubertal Castrated Male Cats. Animal Behavior 6: 171-172. WAI, E.H., and HOAR, W.S. 1962. The Secondary Sex Characteristics and Reproductive Behavior of Gonadectomized Sticklebacks Treated with Methyl Testosterone. Canadian  Journal of Zoology. 41: 611-628. -113-APPENDIX Introduction This appendix contains the raw data concerning the f i s h measurements made i n connection with Chapter 5 . The following tables contain the snout to f i n length, standard length and r e s u l t i n g r a t i o for Sh males, unoperated females, GII and GUI males following gonadectomy, G males following hormone treatment, GII and GUI males following control treatment, and unoperated hormone treated females. -114-Fish Snout-fin (cm) Snout length (cm) Ratio Sh 3 5.3 5.2 1.02 s h 4 5.65 5.4 1.045 s h 5 5.7 5.6 1.02 s h 6 6.0 5.85 1.025 Sh ? 5.3 5.0 1.06 Norm 5.2 5.0 1.04 Norm 5.0 4.9 1.02 Norm 4.85 4.6 1.055 Mean Ratio 1.035 TABLE IV Sh and Unoperated Male f i n ra t i o s F i sh Snout-fin (cm) Snout length (cm) Ratio 1 5.2 5.9 0.882 2 4.75 5.4 0.880 3 4.95 5.45 0.908 4 4.55 5.1 0.893 5 4.5 5.0 0.900 6 4.5 5.1 0.882 Mean Ratio 0.891 TABLE V Unoperated female f i n r a t i o s -115-Pish Snout-fin Snout length Ratio (cm) (cm)  G 1 ; L 6.40 6.15 1.04 G 1 4 6.15 6.15 1.00 G 1 ? 5.95 5.95 1.00 G 1 8 5.80 5.70 " 1.02 G 1 9 5.60 5.45 1.027 Mean r a t i o GII males 1.017 G 1 2 5.75 5.65 1.022 G 5.57 5.55 1.027 G 1 6 5.37 5.70 0.924 G 2 Q 5.50 5.70 0.965 G 2 1 5.10 5.50 0.927 G 2 2 4.95 5.10 0.972 G 2 3 5.00 5.44 0.927 G 2 5 4.80 5.15 - 0.932 G o c 5.20 5.70 0.912 Z D Mean r a t i o G III males 0.957 TABLE VI Gonadectomized (GII and GUI) male f i n r a t i o s -116-Fish Snout-fin (cm) Snout length (cm) Ratio G14 6. 20 5. 90 1.050 G20 5.70 5.65 1.008 G22 • 5.25 5.10 1.030 G25 5.40 5.20 1.039 G21 5.40 5.50 0.983 G12 6.10 5.85 1.040 Mean r a t i o GII 1 f i s h = 1.050 Mean r a t i o G UI 5 f i s h = 1.020 TABLE VII Gonadectomized, Hormone treated male f i n r a t i o s Fish Snout-fin Snout length (cm) Ratio Cat.II G l l G 1 ? G19 Cat. I l l G26 G23 G15 G16 6.35 6.20 5.90 5.40 5.20 5.80 5.50 6.25 6.00 5.70 5.90 5.50 5.75 1.015 1.033 1.035 0.915 0.945 1.008 6.00 0.917 Mean r a t i o GII 3 f i s h = 1.028 Mean r a t i o G UI 4 f i s h = 0.946 TABLE VIII Gonadectomized control (GII & GUI) male f i n r a t i o s -117-Fish Snout-fin (cm) Snout length (cm) Ratio 1 4.4 4.6 0.958 2 4.5 4.8 0.938 3 4.55 4.65 0.980 4 4.60 4.55 1.010 5 4.55 4.75 0.958 6 5.00 5.00 1.000 Mean r a t i o = 0.975 TABLE IX Hormone treated unoperated f i n r a t i o s female 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0104653/manifest

Comment

Related Items