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Some metabolic effects of synthetic sodium thyroxine, thiourea and methyl testosterone on the commoon… Gardner, Robert Renwick 1953

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SOME METABOLIC EFFECTS OF SYNTHETIC SODIUM THYROXINE, THIOUREA AND METHYL TESTOSTERONE ON THE COMMON GOLDFISH by ROBERT RENWICK GARDNER A Thesis Submitted i n P a r t i a l Fulfilment of the Requirements for the Degree of MASTER OF ARTS i n the Department of ZOOLOGY We accept t h i s thesis as conforming to the standard required from candidates f o r the degree of MASTER OF ARTS Members of the Department of Zoology October 1953 The University of B r i t i s h Columbia V ABSTRACT A group of f i v e g o l d f i s h , Carassius auratus, treated ten and sixteen days with a 1 : 3>980,000 concentra-t i o n of synthetic sodium thyroxine during the winter, had t h e i r oxygen consumption s i g n i f i c a n t l y raised. Five goldfish treated with .03 per cent thiourea f or the same lengths of time during the same period had t h e i r oxygen consumption s i g n i f i c a n t l y depressed. Two series of s i m i l a r treatments of three, seven, fourteen and twenty-one days duration throughout the summer did not s t a t i s t i c a l l y a l t e r the oxygen consumption and ammonia excretion. Treatments with a 1 : 1500,000 concentration of methyl testosterone for three, seven, fourteen and twenty-one days s t a t i s t i c a l l y elevated the oxygen consumption and ammonia excretion. The thiourea, synthetic sodium thyroxine and methyl testosterone treatments for three, seven, fourteen and twenty-one days did not appreciably a l t e r the t o t a l l i p i d and moisture content of the goldfish tissue. These treatments do not appear to a l t e r the oxygen carbon dioxide tension at death nor the length of time to asphyxiate the goldfish. ACKNOWLEDGMENT I wish to acknowledge my debt to Dr. W. S. Hoar fo r his encouragement, advice and invaluable d i r e c t i o n throughout t h i s study. I wish also to thank Dr. W. A. Clemens for his interest and helpful c r i t i c i s m of the manu-s c r i p t . Gratitude i s due to the National Research Council and the President's Fund for f i n a n c i a l assistance without which t h i s i n v e s t i g a t i o n would not have been possible. In addition the cooperation and assistance of my fellow-students, e s p e c i a l l y Aline Redlich, has been of great value. i i TABLE OF CONTENTS Page INTRODUCTION 1 MATERIALS AND METHODS 6 Oxygen Consumption Experiments 7 Ammonia Excretion Experiments 14 Ether Soluble Experiments 1 5 Moisture Determinations 16 Carbon Dioxide, Oxygen Tension at Death and Time to Asphyxiate 16 RESULTS 18 Oxygen Consumption 18 (Series 1) (Series 2) 20 (Series 3) 21 (Series 4) 23 Ammonia Excretion Experiments 25 Moisture and Ether Soluble Experiments 25 Asphyxiation Experiments 29 DISCUSSION 30 CONCLUSION . 44 LITERATURE CITED 4$ APPENDIX 52 S t a t i s t i c s on Series 2 and 3 Oxygen Consumption data. i i i e L I S T OF T A B L E S P a g e T A B L E I . . S T A T I S T I C A L SUMMARY OF THE S E R I E S I OXYGEN CONSUMPTION RESULTS 19 T A B L E I I SUMMARY OF THE TREATMENT AND S T A T I S T I C A L D E T A I L S OF S E R I E S 4 RESP IRAT ION EXPERIMENTS 24 T A B L E I I I SUMMARY OF AMMONIA E X C R E T I O N RESULTS 26 T A B L E IV E T H E R SOLUBLES EXPERIMENT - PER CENT OF ETHER SOLUBLES IN WET MACERATED G O L D F I S H T I S S U E 27 T A B L E IV PART I I . MOISTURE CONTENT EXPERIMENTS - PER CENT MOISTURE IN WET MACERATED GOLDFISH T I S S U E 28 i v L I S T OF F I G U R E S P a g e F i g u r e 1 R e s p i r a t i o n A p p a r a t u s 10 F i g u r e 2 A p p a r a t u s f o r t h e c o l l e c t i o n o f ammonia e x c r e t i o n 1 4 v t o f o l l o w P a g e F i g u r e 3 S e r i e s 1 o x y g e n c o n s u m p t i o n r e s u l t s 18 F i g u r e 4 E x a m p l e o f S e r i e s 2 o x y g e n c o n s u m p t i o n r e s u l t s 20 F i g u r e 5 Summary o f means and p l u s and m i n u s one s t a n d a r d d e v i a t i o n f o r S e r i e s 2 o x y g e n c o n s u m p t i o n r e s u l t s 20 F i g u r e 6 Summary o f means and p l u s and m i n u s one s t a n d a r d d e v i a t i o n f o r S e r i e s 3 o x y g e n c o n s u m p t i o n r e s u l t s 21 F i g u r e 7 Summary o f means and p l u s and m i n u s one s t a n d a r d d e v i a t i o n f o r u s i n g s i n g l e g o l d -f i s h and u s i n g f i v e g o l d f i s h t r e a t e d w i t h d o u b l e and t r i p l e t h e u s u a l c o n c e n t r a t i o n o f t h y r o x i n e 24 F i g u r e 8 P e r c e n t d e v i a t i o n f r o m t h e c o n t r o l s f o r t h e ammonia e x c r e t i o n r e s u l t s 26 F i g u r e 9 C a r b o n d i o x i d e o x y g e n t e n s i o n s a t d e a t h o f t h e g o l d f i s h ^ 29 F i g u r e 10 T i m e t o a s p h y x i a t e and t h e c a r b o n d i o x i d e t e n s i o n a t d e a t h o f t h e g o l d f i s h 29 SOME METABOLIC E F F E C T S OF SYNTHETIC SODIUM T H Y R O X I N E , THIOUREA AND METHYL TESTOSTERONE ON THE COMMON GOLDFISH INTRODUCTION In higher vertebrates, e s p e c i a l l y mammals, thyroxine elevates oxygen consumption, increases excretion of nitrogen and potassium, affects the excretion of sodium chloride and therefore a l t e r s the water metabolism, reduces l i v e r glyco-gen, increases the e x c i t a b i l i t y of the organism and affects growth and maturation (Turner, 1948; Gaunt, et a l , 1944). Clear cut results i n mammals lead to the theory that thyroxine acts i n some way as a catalyst i n oxidative processes. Thio-urea i n h i b i t s the function of the thyroid gland and tends to have an opposite e f f e c t to thyroxine. (Astwood, 1949; Means, 1949; Turner, 1948). Among lower vertebrates the picture i s not so cl e a r . Some species of amphibians show increases i n oxygen consumption only at certain stages i n t h e i r metamorphosis when they are treated with thyroxine (Prosser, et a l 1950). Frogs treated with thyroxine have raised nitrogen excretions with no subsequent change i n the oxygen consumption (Mansfeld, 1949). Feeding mammalian thyroid gland to amphibians hastens metamor-phosis (Prosser, et a l , 1950). Thiourea, on the other hand, retards amphibian metamorphosis (Gordon, et a l , 1943). 2 With fish, numerous workers failed to show a con-sistent elevated oxygen consumption, and i t was generally believed that thyroid materials had no effect on this aspect of fish metabolism until the work of Smith and Matthews, (194-8). However, these workers could obtain their results only from fish of a certain size. Other workers such as Etkin, et al. (194-0) and Smith and Everett, (1943) were able to show sporadic elevations of the oxygen consumption in fish, but statistics indicated that a consistent significant increase did not exist. The investigation of the nitrogen output of fish treated with thyroxine seems to have been carried out first by Redlich, (195#, unpublished). Although she obtained increases of the ammonia excretion for goldfish, the effect varied and the results are rather confused. Redlich also reports that thyroxine treated gold-fish withstood asphyxiation longer than untreated ones. Mansfeld, (1949) reports that the oxygen deficiency of the thyroid glands of dogs and rabbits caused an increase in the activity of the gland and a subsequent elevation in the amount of nitrogen eliminated. He also puts forth strong arguments to prove that thyroxine brings about an acceleration of combustion only when i t has an opportunity to act on anaerobic phases of'metabolism or on enzymes. Mansfeld's and Redlich's works promoted much speculation on the effects of thyroxine on fish anaerobic metab-olism in our laboratory. Therefore, experiments were undertaken 3 i n the present study to determine the oxygen, carbon dioxide tensions of the water media at death of thyroxine treated gol d f i s h asphyxiated i n water containing varying concentrations of oxygen and carbon dioxide. The l i p i d or ether soluble content of thyroxine treated g o l d f i s h was also investigated since Smith (1929) found that when the ammonia excretion was raised i n his experiment by i n a n i t i o n , ( i . e . catabolism of body proteins) then the other metabolites such as l i p i d s ( fats) were much reduced. Thyroxine often produces di u r e s i s i n mammals (Gaunt, et a l . 194-4) and Redlich reports a reduced moisture content of thyroxine treated go l d f i s h , therefore t h i s aspect of metabolism was re-investigated. Synthetic sodium thyroxine produces marked metabolic effects i n young salmon as evidenced by guanine deposition, (Hoar, 1951 and Robertson, 1949), abnormal growth (Dales, 1953), unpublished) and increased a c t i v i t y (Hoar, et a l . 1952). This compound was therefore chosen for the re-examination of the calorigenic ( i . e . r e s p i r a t i o n and nitrogen excretion) effect of thyroxine as well as for the l i p i d , moisture and asphyxiation studies. Considerable confusion exists i n the l i t e r a t u r e on thiourea's effects on f i s h metabolism; therefore, experiments p a r a l l e l to those f o r the thyroxine treatment were undertaken on thiourea treated goldfish. 4 Many f i s h d e m o n s t r a t e a c l o s e r e l a t i o n s h i p b e t w e e n s e x u a l and t h y r o i d a c t i v i t y a s e v i d e n c e d b y c h a n g e d t h y r o i d h i s t o l o g y ( H o a r , 1951) and i n c r e a s e d r a t e o f s e x u a l m a t u r a t i o n o f P l a t y p o e c l l u s when f e d t h y r o i d powier ( G r o b s t e i n and B e l l a m y , 1939)• S i n c e m e t h y l t e s t o s t e r o n e p r o d u c e d b e h a v i o r s i m i l a r t o t h y r o x i n e i n chum and c o h o f r y ( H o a r , e t a l . 1952), i t was c h o s e n f o r e x p e r i m e n t s p a r a l l e l t o t h o s e f o r t h y r o x i n e i n t h e p r e s e n t s t u d y . A g a i n i n mammals , m e t h y l t e s t o s t e r o n e e l e v a t e s o x y g e n c o n s u m p t i o n , r a i s e s n i t r o g e n , s o d i u m and p o t a s s i u m r e t e n t i o n , b u t p r o m o t e s c r e a t i n u r e a ( P i n c u s and T h i m a n n , 1950). M a n n , (1939) seems t o h a v e b e e n t h e o n l y p e r s o n t o i n v e s t i g a t e t h e e f f e c t s o f g o n a d a l h o r m o n e s o n f i s h r e s p i r a t i o n . He f o u n d t h a t t h e o x y g e n c o n s u m p t i o n o f f i s h c o u l d be e l e v a t e d u s i n g t h e s e h o r m o n e s . T h e n i t r o g e n e x c r e t i o n o f f i s h t r e a t e d w i t h t e s t o s t e r o n e was n o t p r e v i o u s l y i n v e s t i g a t e d . T h e t h e s i s p r o b l e m i s , t h e n , t o r e - e x a m i n e t h e e f f e c t s o f s y n t h e t i c s o d i u m t h y r o x i n e o n f i s h r e s p i r a t i o n , n i t r o g e n e x c r e t i o n , l i p i d and m o i s t u r e c o n t e n t s and t o d e t e r -m i n e what e f f e c t s m e t h y l t e s t o s t e r o n e h a s i n p a r a l l e l e x p e r i -m e n t s . A n o t h e r p a r t o f t h e p r o b l e m i s t o a s c e r t a i n what e f f e c t t h e a s p h y x i a t i o n o f s i n g l e g o l d f i s h t r e a t e d r e s p e c t i v e l y w i t h t h e t h r e e compounds had o n t h e g a s c o n t e n t o f t h e w a t e r m e d i u m , w h i c h c o n t a i n e d v a r y i n g amounts o f o x y g e n and c a r b o n d i o x i d e . P r e v i o u s s t u d i e s h a v e n o t b e e n made o n t h e e f f e c t s 5 o f t h e s e h o r m o n e s o n f i s h a s p h y x i a t i o n e x p e r i m e n t s and o n t h e e f f e c t s o f m e t h y l t e s t o s t e r o n e o n r e s p i r a t i o n , ammonia e x c r e -t i o n and t h e l i p i d and m o i s t u r e c o n t e n t o f g o l d f i s h . 6 MATERIALS AND METHODS A p p r o x i m a t e l y e i g h t h u n d r e d g o l d f i s h , C a r a s s i u s  a u r a t u s , r a n g i n g i n s i z e f r o m f i v e t o t w e n t y - t h r e e grams w e r e u s e d i n t h i s s t u d y . T h e g o l d f i s h were o b t a i n e d f r o m t h e G o l d -f i s h S u p p l y C o m p a n y , S t o u f f v i l l e , O n t a r i o , i n o n e - h u n d r e d t o t w o - h u n d r e d q u a n t i t i e s , and w e r e h e l d a week t o a month i n l a r g e a q u a r i a . When new f i s h w e r e p l a c e d i n a q u a r i a t h e w a t e r t e m p e r a t u r e wsts r a i s e d 1° C . p e r d a y ( F r y , e t a l . 1948) u n t i l a t e m p e r a t u r e o f 20-21° C . was r e a c h e d . A l l f i s h were m a i n -t a i n e d a t 20-21° C . s i n c e H u x l e y (1929) p o s t u l a t e d t h a t t h e t h y r o i d g l a n d a c t e d a s a t e m p e r a t u r e r e g u l a t o r i n c o l d b l o o d e d v e r t e b r a t e s b e l o w 20° C . and B e t t s (1949) s u g g e s t s t h a t t h i s t e m p e r a t u r e i s o p t i m u m f o r g o l d f i s h . A f t e r t h e minimum h o l d i n g p e r i o d o f a w e e k , l o t s o f t w e n t y - t w o t o t h i r t y - e i g h t g o l d f i s h were t r a n s f e r r e d t o t h r e e s m a l l e r ( t h i r t y l i t r e ) a q u a r i a and t r e a t e d w i t h h o r m o n e s a s f o l l o w s : 1 : 3,980,000 c o n c e n t r a t i o n o f s y n t h e t i c s o d i u m t h y r o x -i n e B . D . H . , .03 p e r c e n t t h i o u r e a , and 1 : 1,500,000 c o n c e n -t r a t e o f m e t h y l t e s t o s t e r o n e B . D . H . T h e s e c o n c e n t r a t i o n s w e r e b a s e d o n p r e v i o u s s t u d i e s o f R e d l i c h (1951> u n p u b l i s h e d ) and H o a r , e t a l . (1952). T h e w a t e r i n e a c h o f t h e t h r e e a q u a r i a was c h a n g e d d a i l y f o r t w e n t y - o n e d a y s . A f t e r e a c h c h a n g e t h e h o r m o n e s 7 were dissolved i n separate small volumes of water and poured into the respective aquaria. A l l goldfish, whether under treatment or i n the supply tanks were fed twice to three times d a i l y with a commercially prepared goldfish food, except before an experiment when food was withheld for a period of twenty-four hours. Goldfish were usually withdrawn from the three hormone treatment aquaria for experiments at interv a l s of three, seven, fourteen and twenty-one days. A l l g o l d f i s h f o r controls were taken from the supply tanks as they were needed. Oxygen Consumption Experiments The apparatus used for these experiments i s a modifi-cation of that used by Fry and Hart (1948). It combines features of the continuous flow method of r e s p i r a t i o n study of Adkins ( 1 9 3 0 ) , Clausen ( 1 9 3 6 ) , H a l l ( 1 9 2 9 ) , Haugard and Irving ( 1 9 4 3 ) , Keys ( 1 9 3 0 ) , Spoor (1946), and others; and the closed system of S c h l a i f e r ( 1 9 3 9 ) , Powers ( 1 9 2 2 ) , and others i n which no water flows through the re s p i r a t o r s . The modification to the Fry and Hart equipment was the provision of hemostats to shut o f f the "B" hoses (figure 1 ) when the water flow was stopped and when samples were taken. Paired water samples with comparablevoxygen contents were d i f f i c u l t to obtain with the unmodified method, since Fry and Hart assumes that water samples could be obtained from the re s p i r a t i o n flasks before the incoming water from the "B" hoses mixed with the' water already i n the f l a s k s . 8 L i g h t was e x c l u d e d f r o m t h e a p p a r a t u s w i t h a t i g h t f i t t i n g c o v e r and d i s t u r b a n c e s w e r e k e p t t o a min imum d u r i n g a n e x p e r i m e n t . S p o o r (194-6) f o u n d t h a t t h e s e f a c t o r s a f f e c t e d t h e o x y g e n c o n s u m p t i o n o f g o l d f i s h . A c c o r d i n g t o S p o o r ( 1 9 4 6 ) , S c h l a i f e r ( 1 9 3 9 ) ? E s c o b a r ( 1 9 3 6 ) , and S c h u e t t (1933)> c r o w d i n g and n u m b e r s o f f i s h a f f e c t t h e o x y g e n c o n s u m p t i o n . F i v e f i s h i n e a c h r e s p i r a -t i o n f l a s k was f o u n d t o be t h e o p t i m u m n u m b e r . G o l d f i s h must be i n a b a s a l r e s t i n g c o n d i t i o n i f r e s u l t s f r o m s u c c e s s i v e e x p e r i m e n t s a r e t o be c o m p a r e d . T h i s was a c c o m p l i s h e d b y c o n d i t i o n i n g t h e f i s h t o t h e r e s p i r a t i o n f l a s k s f o r t w e n t y - f o u r h o u r s p r i o r t o e x p e r i m e n t a t i o n , w i t h -h o l d i n g f o o d t w e n t y - f o u r h o u r s b e f o r e t h e c o n d i t i o n i n g , r e d u c i n g l i g h t and d i s t u r b a n c e s d u r i n g e x p e r i m e n t s . T w e n t y - f o u r h o u r s was c h o s e n a s t h e l e n g t h o f t h e c o n d i t i o n i n g p e r i o d s i n c e W e l l s (1932) and F r y and H a r t ( 1 9 4 8 ) o b s e r v e d t h a t t h e m e t a b o l i s m o f f i s h t r a n s f e r r e d t o r e s p i r a t i o n c h a m b e r s r i s e s r a p i d l y , b u t d i m i n i s h e s a f t e r f i v e t o s i x h o u r s , and d o e s n o t become b a s a l f o r a l m o s t t w e n t y - f o u r h o u r s . W a t e r a t a r a t e o f 150 c . c . p e r m i n u t e was a l l o w e d t o f l o w t h r o u g h t h e f l a s k s d u r i n g a l l c o n d i t i o n i n g p e r i o d s . B a t t e r i e s o f t h r e e o r f o u r r e s p i r a t i o n f l a s k s e a c h c o n t a i n i n g a d i f f e r e n t g r o u p o f w e i g h e d g o l d f i s h were u s e d s i m u l t a n e o u s l y . A l l f l a s k s were i m m e r s e d i n t h e w a t e r -f i l l e d t u b and t h e " A " h o s e s ( f i g u r e 1) were a t t a c h e d t o t h e i r 9 respective nipples which passed through the wa l l of the tub. The HD" hoses are attached to the nipples on the outside of the tub and the screw clamps are adjusted to allow a water flow of 1 5 0 c c . per minute through the f l a s k s . This rate was the lowest that could be maintained and i s s l i g h t l y higher than that used by Matthews and Smith (1947), 43 - 128 c c . per minute fo r parrot f i s h , or by Spoor (1946), 70-100 c c . per minute fo r goldfish. The "B" hoses are l e f t open to the water i n the tub. The "C" hoses protrude through holes i n the cover to the outside, where they are clamped with spring clamps, o Water (20-21 C.) flowed into the tub at a s u f f i c i e n t rate to maintain a constant volume. A twenty-four hour experimental period followed the twenty-four hour conditioning. Five tests usually were carried out at s i x hour intervals during the twenty-four hours. According to Fry and Hart (1948), and Spoor (1946), the goldfish have a low oxygen consumption between lat e evening and dawn, therefore the selection of the time int e r v a l s i n the present study allowed at least one test to be done i n t h i s period. Each test during the twenty-four hours consisted of taking duplicate preliminary water samples from each of the res p i r a t i o n f l a s k s and duplicate samples again af t e r a one or a four-hour period during which no water flowed through the f l a s k s . Oxygen determinations were made on each water sample and the results f o r each duplicate were averaged. The oxygen content of the members of a duplicate sample were within a range of two per cent of each other. The differences i n the oxygen content of 10 t h e p r e l i m i n a r y and f i n a l w a t e r s a m p l e s i n d i c a t e t h e amount o f o x y g e n u s e d b y t h e f i s h e x p r e s s e d a s c . c . o f o x y g e n u s e d p e r g ram o f g o l d f i s h p e r h o u r . S a m p l i n g was s t a g g e r e d s l i g h t l y t o a l l o w t h e same l e n g t h o f t i m e f o r t h e e x p e r i m e n t i n e a c h r e s p i r a t o r . F i g u r e 1 A p p a r a t u s u s e d t o d e t e r m i n e t h e o x y g e n c o n s u m p t i o n o f t h e g o l d f i s h A , B , C and D , v a r i o u s h o s e s ; H , h e m o s t a t ; C O , c o v e r i n g o v e r t h e a p p a r a t u s t o e l i m i n a t e l i g h t and o t h e r d i s t u r b a n c e s ; 0, o v e r f l o w ; R F , r e s p i r a t i o n f l a s k ; S C , s c r e w c l a m p ; S P C , s p r i n g c l a m p ; WS, w a t e r s u p p l y ; WB, w a t e r b a t h . 11 The detailed procedure to obtain water samples was as follows: 1. Close spring clamp (not shown) on "D" hoses and remove screw clamps (SPC) from the "C" hoses. 2. Clamp hemostats on the "B" hoses. 3. Remove the spring clamps (SPC) from the^C" hoses. 4. Collect paired water samples in 66 c.c. sample bottles by releasing the spring clamps on the " D " hoses. 5 . Release the hemostats on the "B" hoses to allow the flasks to r e f i l l with water. 6 . Replace the spring clamps (SPC) -on "C". 7. Replace the hemostats at "B" for one or four hours. 8. After one or four hours repeat step 3? allowing about 25 c.c. of water to pass before repeating step 4. 9 . Repeat steps 5 and 6 . 1 0 . Replace the screw clamps (SC) on MD" hoses and remove the spring clamps. The screw clamps are re-adjusted to give a flow of 1 5 0 c.c. per minute through the flasks. A recovery period of two or five hours was allowed between tests in which the water flowed through the respirators. A modification of the standard Winkler method reported by E l l i s , et al. (1948), was used for a l l oxygen determinations. Fifty c.c. alloquots of the 66 c.c. water samples treated with . 5 c.c. of each reagent were titrated with .0.25N sodium thiosulfate. In experiments similar to the present study Adkins (1930) and Graham (1949) found that the collection of nitrites had a neglig-ible effect on the oxygen determinations. Therefore in our ex-periments the permanganate modification of the Winkler method outlined by Allee and Oesting (1934), and Wilder (1937), to eliminate the nitrite effect was not used. 1 2 F o u r s e r i e s o f r e s p i r a t i o n e x p e r i m e n t s w e r e u n d e r t a k e n i n t h i s t h e s i s . T h e f o l l o w i n g o u t l i n e s u m m a r i z e s t h e d e t a i l s o f e a c h s e r i e s . S e r i e s D a t e G r o u p s o f L e n g t h o f L e n g t h o f N o . o f N o . o f F i s h u s e d s h u t - o f f r e c o v e r y t e s t s d o n e f i s h u s e d p e r i o d p e r i o d d u r i n g 24 i n e a c h h o u r s r e s p i r a t o r 2 9 / 1 / 5 2 1 0 and 1 6 4 h r s 2 h r s 5 5 d a y T h y r o x -1 t o i n , T h i o -u r e a t r e a t -6 / 2 / 5 2 m e n t s and c o n t r o l s 3 0 / 6 / 5 2 3 , 7 , 14 and 21 d a y 1 h o u r 5 h r s t o t r e a t m e n t s w i t h t h y -2 1 / 7 / 5 2 r o x i n , t h i o -u r e a , t e s t o -s t e r o n e and c o n t r o l s 3 1 / 7 / 5 2 Same a s 2 1 h o u r 5 h r s 5 5 3 t o 1 8 / 8 / 5 2 Same a s 3 , 7 , 1 4 , 3 . 21 d a y t r e a t m e n t s 2 h r s 4 h r s 5 1 w i t h t h y r o x -i n and c o n t r o l s 2 8 / 8 / 5 2 7 d a y t r e a t - 1 h o u r 5 h r s 3 T ment w i t h t h y r o x i n and c o n t r o l 13 Goldfish f o r series 1 were treated ten days with the hormones before a r e s p i r a t i o n study was undertaken, a f t e r which the f i s h were returned to t h e i r respective aquaria f o r another s i x days of treatment to make a t o t a l of sixteen days treatment before the second experiment was done. Four hour shut-off periods were used, since t h i s was the procedure used by Fry and Hart (1948). These workers do not mention the time allowed for recovery but Graham ( 1 9 4 9 ) used an hour i n his work on trout. Two hours was a r b i t r a r i l y used i n series 1 of the present study. Another series of experiments was done before series 2, i n which a l l groups of gold f i s h used the oxygen down to the same low l e v e l , i ndicating that the shut-off period was too long. Preliminary studies p r i o r to the commencement of series 2 indicated that i n an hour shut-off period the goldfish used 50 - 75 per cent of the available oxygen, therefore t h i s i n t e r v a l was used i n subsequent experiments. A f i v e hour recovery period between tests i n the remaining series allowed a reasonable time for t i t r a t i o n and re s t . The need f o r a shorter shut-off period i s thought to be due to seasonal differences (series 1, i n winter, and series 2 and 3 i n summer) and size differences. The t o t a l weights of the goldfish i n series 2 and 3 were nearly double that of series 1, therefore they would use the available oxygen quicker without necessarily increasing the amount of oxygen consumed per gram per hour. 1 4 T h e r e a r e two p a r t s t o t h e m i s c e l l a n e o u s e x p e r i m e n t s o f s e r i e s 4 . P a r t one was d o n e w i t h s e r i e s 3 ( o u t l i n e p . 1 2 ) P a r t two was d o n e a t a l a t e r d a t e u s i n g two g r o u p s o f f i s h t r e a t e d s e v e n d a y s w i t h c o n c e n t r a t i o n s o f 1 : 1 , 9 9 0 , 0 0 0 and 1 : 1 , 3 2 6 , 6 6 7 s y n t h e t i c s o d i u m t h y r o x i n e B . D . H . r e s p e c t i v e l y . Ammonia E x c r e t i o n E x p e r i m e n t s T h e a p p a r a t u s ( f i g u r e 2 ) c o n s i s t s o f a b a t t e r y o f f o u r s p e c i a l 2 5 0 0 w i d e m o u t h E r l e n m e y e r c o l l e c t i n g f l a s k s a s s e m b l e d i n a c o n s t a n t t e m p e r a t u r e b a t h , e a c h w i t h i t s own a i r s u p p l y . F i g u r e 2 A p p a r a t u s u s e d t o c o l l e c t e x c r e t e d ammonia A S , a i r s u p p l y ; C , c o v e r i n g o v e r t h e a p p a r a t u s ; C F , c o l l e c t -i n g f l a s k s ; 0 , o v e r f l o w p i p e s ; R, r e s e r v o i r ; WB, w a t e r b a t h ; W S , w a t e r s u p p l y . 1 5 G r o u p s o f f i v e g o l d f i s h f r o m e a c h o f t h e t h r e e h o r -mone t r e a t m e n t s w i t h c o n t r o l s w e r e w e i g h e d and p l a c e d i n t h e i r r e s p e c t i v e f l a s k s . W a t e r a t a t e m p e r a t u r e o f 20 - 2 1 ° C . was a l l o w e d t o f l o w t h r o u g h t h e f l a s k s a t a r a t e o f 1 5 0 c c . p e r m i n u t e d u r i n g a t w e n t y - f o u r h o u r c o n d i t i o n i n g p e r i o d . T h i s r a t e was f o u n d t o be s u f f i c i e n t t o p r e v e n t t h e c o l l e c t i o n o f a m m o n i a . T h e a i r s u p p l y was r e d u c e d t o a f i n e s t r e a m o f b u b b l e s t o p r e v e n t d i s t u r b i n g t h e f i s h . A t w e n t y - f o u r h o u r ammonia c o l l e c t i n g p e r i o d i n w h i c h no w a t e r f l o w e d t h r o u g h t h e f l a s k s f o l l o w e d t h e c o n d i t i o n i n g . D u p l i c a t e 50 c . c . s a m p l e s o f w a t e r were p i p e t t e d f r o m e a c h f l a s k a f t e r t h e c o l l e c t i n g p e r i o d . F r o m e a c h , two 10 c . c . s a m p l e s w e r e p i p e t t e d i n t o c o l o r i m e t r i c t u b e s and t h e ammonia d e t e r m i n e d b y t h e d i r e c t n e s s l e r i z a t i o n method o f V a n S l y k e (Hawk, e t a l . 1948 ) u s i n g a C e n c o C o l o r i m e t e r w i t h a r e d f i l t e r , and ammonia f r e e d i s t i l l e d w a t e r a s a b l a n k . ' T h e a m o u n t s o f ammonia i n t h e f o u r 10 c . c . s a m p l e s d i d n o t v a r y more t h a n t h r e e p e r c e n t f r o m e a c h o t h e r , and were t h e r e f o r e a v e r a g e d . B y k n o w i n g t h e v o l u m e o f t h e c o l l e c t i n g f l a s k up t o t h e t o p n i p p l e (2000 c . c ) , and t h e s i z e o f t h e s a m p l e (10 c c ) , and t h e a v e r a g e d amount o f ammonia p r e s e n t i n t h e f o u r 10 c . c . s a m p l e s , t h e t o t a l ammonia e x c r e t i o n c a n be e x p r e s s e d a s mg. o f ammonia p e r g ram o f g o l d f i s h p e r t w e n t y - f o u r h o u r s . Two s e t s o f e x p e r i m e n t s were c a r r i e d o u t a t t h r e e , s e v e n , f o u r t e e n and t w e n t y - o n e d a y s o f hormone t r e a t m e n t . E t h e r S o l u b l e E x p e r i m e n t s E i g h t t o t w e l v e g o l d f i s h f r o m e a c h o f t h e f o u r g r o u p s 16 a t t h r e e , s e v e n , f o u r t e e n and t w e n t y - o n e d a y s of t r e a t m e n t w e r e m a c e r a t e d i n a W a r i n g B l e n d e r . T r i p l i c a t e s a m p l e s o f t w e l v e t o f i f t e e n grams w e r e p l a c e d i n p r e - w e i g h a 3 1 2 5 c c . F l o r e n c e F l a s k s and t h e e t h e r s o l u b l e s o r t o t a l l i p i d s w e r e e x t r a c t e d u s i n g t h e method d e s c r i b e d b y H o a r and C o t t l e ( 1 9 5 2 ) . M o i s t u r e D e t e r m i n a t i o n s W e i g h e d d u p l i c a t e s a m p l e s o f s i x t o t w e l v e grams w e r e t a k e n f r o m t h e r e m a i n i n g g o l d f i s h h o m o g e n a t e p r e p a r e d f o r t h e e t h e r s o l u b l e e x p e r i m e n t s . T h e s e s a m p l e s were d r i e d t o c o n s t a n t w e i g h t i n a d r y i n g o v e n a t a t e m p e r a t u r e o f 9 0 - 9 5 C . T h e t i m e r e q u i r e d t o do t h i s was s i x t o e i g h t d a y s . C a r b o n D i o x i d e , O x y g e n T e n s i o n a t D e a t h and T i m e t o A s p h y x i a t e . T h e method o f F r y , e t a l . ( 1948 ) was u s e d . S i n g l e g o l d f i s h f r o m t h e f o u r g r o u p s w e r e s e a l e d i n p i n t j a r s c o n t a i n -i n g v a r y i n g c o n c e n t r a t i o n s o f c a r b o n a t e d w a t e r and s u f f i c i e n t o o x y g e n a t a t e m p e r a t u r e o f 20 - 21 C . D i s t u r b a n c e s were r e d u c e d b y p l a c i n g t h e s e a l e d j a r s i n c o v e r e d b o x e s . O b s e r v a -t i o n s w e r e made e v e r y f i f t e e n m i n u t e s u n t i l t h e f i s h d i e d , a s e v i d e n c e d b y t h e a b s e n c e o f g i l l movements ( F r y , e t a l . 1 9 4 8 ) . U s u a l l y , t e n o r more g o l d f i s h were u s e d f o r a s p h y x i a t i o n s a t e a c h i n t e r v a l o f hormone t r e a t m e n t ( i . e . a t t h r e e , s e v e n , f o u r t e e n and t w e n t y - o n e d a y s ) . F o u r w a t e r s a m p l e s ( 66 c c . ) w e r e t a k e n a s s o o n a f t e r 1 7 d e a t h a s p o s s i b l e . B e c a u s e o f t h e number o f s a m p l e s i n v o l v e d a t l e a s t two J a r s , i n some c a s e s , w e r e n o t s a m p l e d u n t i l f o u r o r f i v e h o u r s a f t e r t h e d e a t h o f t h e f i s h . T h e W i n k l e r method p r e v i o u s l y d e s c r i b e d was u s e d t o d e t e r m i n e t h e o x y g e n c o n t e n t o f t h e w a t e r i n one p a i r o f s a m p l e s , t h e r e s u l t s o f w h i c h w e r e a v e r a g e d . C a r b o n d i o x i d e c o n t e n t o f 2 5 c . c . a l l o q u o t s f r o m t h e s e c o n d p a i r o f w a t e r s a m p l e s were d e t e r m i n e d b y t i t r a t i n g w i t h N/44 s o d i u m h y d r o x i d e u s i n g p h e n o l p h t h a l e i n a s a n i n d i c a t o r , ( W e l c h , 1948). T h e t i t r a t i o n s were c o n t i n u e d u n t i l a f a i n t p i n k c o l o r r e m a i n e d two m i n u t e s . O x y g e n and c a r b o n d i o x i d e t e n s i o n s w e r e c a l c u l a t e d b y r e l a t i n g t h e q u a n t i t y o f g a s e s p r e s e n t t o t h e s o l u b i l i t y a t t h e e x p e r i m e n t a l t e m p e r a t u r e and m u l t i p l y i n g t h i s f r a c t i o n b y 7 6 0 . 1 8 R E S U L T S O x y g e n C o n s u m p t i o n I n t h e p r e l i m i n a r y e x p e r i m e n t s , s e r i e s 1 , ( f i g u r e 3 and T a b l e I ) t h e o x y g e n c o n s u m p t i o n f o r t h e t h y r o x i n t r e a t e d f i s h was h i g h e r t h a n t h e c o n t r o l s f o r b o t h t h e t e n and s i x t e e n d a y s o f hormone t r e a t m e n t ( t e n d a y s P = < 0 . 1 ; s i x t e e n d a y s P = > . 0 1 ) . S i n c e b o t h e x p e r i m e n t s w e r e p e r f o r m e d o n t h e same f i s h t h e r e s u l t s were c o m b i n e d f o r f u r t h e r s t a t i s t i c a l t r e a t -ment t o i n d i c a t e t h a t t h e o x y g e n c o n s u m p t i o n o f t h e t h y r o x i n t r e a t e d f i s h was s i g n i f i c a n t l y h i g h e r t h a n t h a t o f t h e u n t r e a t e d c o n t r o l f i s h (P = > . 0 1 ) . . T h e two e x p e r i m e n t s a r e s i m i l a r . I n e a c h c a s e t h e h i g h p o i n t s o f t h e o x y g e n c o n s u m p t i o n d u r i n g t h e t w e n t y - f o u r h o u r s t u d y o c c u r i n t h e same " s h u t - o f f " p e r i o d s , 8 - 1 2 p . m . f o r t h e t h y r o x i n t r e a t e d f i s h and t h e u n t r e a t e d c o n t r o l f i s h . A l s o i n b o t h e x p e r i m e n t s t h e o x y g e n c o n s u m p t i o n f o r t h y r o x i n a n d t h i o u r e a t r e a t e d f i s h and t h e c o n t r o l s r e m a i n e d i n t h e same g e n e r a l r a n g e ( f i g u r e 3 ) . T h e o x y g e n c o n s u m p t i o n f o r t h e t h i o u r e a t r e a t e d f i s h was l o w e r t h a n t h e c o n t r o l s i n b o t h e x p e r i m e n t s ( t e n d a y P = . 0 5 " , G 1 , s i x t e e n d a y P = / - . O l ) . T h e r e s u l t s i n t h i s f i r s t s e r i e s p r o m p t e d t h e c o n d u c t i o n o f t h r e e f u r t h e r s e r i e s . t o f a c e F i g u r e 3 F i g u r e 3 - S e r i e s 1 C o m p a r i s o n o f t h e o x y g e n c o n s u m p t i o n a t s i x h o u r i n t e r v a l s d u r i n g t w e n t y - f o u r h o u r p e r i o d s o f g o l d f i s h t r e a t e d t e n d a y s w i t h t h y r o x i n and t h i o u r e a and a n u n t r e a t e d c o n t r o l g r o u p w i t h t h e same f i s h r e t u r n e d t o t h e i r r e s p e c t i v e a q u a r i a t o r e c e i v e a n a d d i t i o n a l s i x d a y s t r e a t m e n t , o r a t o t a l o f s i x t e e n d a y s t r e a t m e n t . G = A = X = T h y r o x i n t r e a t e d g o l d f i s h . T h i o u r e a t r e a t e d g o l d f i s h . C o n t r o l o r u n t r e a t e d g o l d f i s h . 1 ' - 1 , J 1 — """"7""""" I O D A Y - J A N U A R Y 2 9 \ \ / ' \ ' - — X ' A a 1 1 1 1 16 D A Y F E B R U A R Y 5 I O A M - 2 P M 3-7PM 8-I2PM U 5 A M 6 - I O A M W A T E R S H U T - O F F P E R I O D S 19 T A B L E I S T A T I S T I C A L SUMMARY OF THE S E R I E S I OXYGEN CONSUMPTION R E S U L T S S t a t i s t i c T h y r o x i n C o n t r o l T h i o u r e a C o n t r o l 10 d a y * Means . .113 .1 .080 .1 t r e a t m e n t I-P < .1 .1 .05 - .02 Means .116 .093 .080 .093 16 d a y ] t r e a t m e n t P > .01 > .01 10 and 16 d a y Means .115 .096 .081 .096 c o m b i n e d P > .01 > .01 t r e a t m e n t s , * Mean amount o f o x y g e n u s e d p e r gram o f f i s h p e r h o u r d u r i n g t h e t w e n t y - f o u r h o u r e x p e r i m e n t . 20 T h e r e s u l t s f o r s e r i e s 2 d i f f e r e d f r o m t h o s e f o r s e r i e s 1. I n o n l y one c a s e ( ? t h d a y , J u n e 31) t h e o x y g e n c o n s u m p t i o n o f t h e t h y r o x i n t r e a t e d g o l d f i s h was h i g h e r t h a n t h e c o n t r o l s . A s i g n i f i c a n t s t a t i s t i c a l d i f f e r e n c e was n o t p r e s e n t (P = s l i g h t l y l e s s t h a n . 1 ) . T h e o x y g e n c o n s u m p t i o n f o r t h e t h r e e , f o u r t e e n and t w e n t y - o n e d a y t r e a t m e n t s was l o w e r t h a n t h e c o n t r o l s b u t i n no c a s e was t h e r e a s i g n i f i c a n t d i f f e r e n c e ( S e e f i g u r e 5 and a p p e n d i x ) . T h i o u r e a t r e a t e d g o l d f i s h showed l i t t l e v a r i a b i l i t y i n t h e i r o x y g e n c o n s u m p t i o n s . T h e s e v e n , f o u r t e e n and t w e n t y -one d a y t r e a t m e n t s had o x y g e n c o n s u m p t i o n s s i g n i f i c a n t l y h i g h e r t h a n t h e c o n t r o l s , b u t t h e f i s h i n t h e t h r e e d a y t r e a t m e n t had a n o x y g e n c o n s u m p t i o n l e s s t h a n t h e c o n t r o l s b u t n o t s i g n i f i -c a n t l y l e s s (P = s l i g h t l y l e s s t h a n . 1 ) . T e s t o s t e r o n e t r e a t e d g o l d f i s h had a n o x y g e n c o n s u m p t i o n h i g h e r t h a n t h e c o n t r o l s i n e a c h e x p e r i m e n t . O n l y t h e s e v e n , f o u r t e e n and t w e n t y - o n e d a y t r e a t m e n t s were s t a t i s t i c a l l y s i g n i f -i c a n t l y d i f f e r e n t t h a n t h e c o n t r o l s . When t h e r e s u l t s o f t h e t h r e e hormone t r e a t m e n t s a r e c o m p a r e d i n e a c h t r e a t m e n t p e r i o d , c o n s i d e r a b l e v a r i a b i l i t y i s p r e s e n t . T h e r e a r e s i g n i f i c a n t d i f f e r e n c e s b e t w e e n t h e t h r e e d a y t h y r o x i n and t e s t o s t e r o n e r e s u l t s , t h r e e d a y t h i o u r e a and t e s t o s t e r o n e , f o u r t e e n d a y t h y r o x i n and t h i o u r e a , t w e n t y - o n e d a y t h y r o x i n and t h i o u r e a , t w e n t y - o n e d a y t h i o u r e a and t e s t o s t e r o n e and t h e t w e n t y - o n e d a y t h i o u r e a and t e s t o s t e r o n e . t o f a c e F i g u r e 4 F i g u r e 4 E x a m p l e s o f s e r i e s 2 o x y g e n c o n s u m p t i o n a t i n t e r v a l s o f s i x h o u r s d u r i n g t w e n t y - f o u r h o u r p e r i o d s f o r two g r o u p s o f g o l d f i s h t r e a t e d s e v e n and f o u r t e e n d a y s w i t h c o n c e n -t r a t i o n s o f 1 : 3 , 9 8 0 , 0 0 0 s y n t h e t i c s o d i u m t h y r o x i n B . D . H . , . 0 3 p e r c e n t t h i o u r e a and 1 : 1 , 5 0 0 , 0 0 0 m e t h y l t e s t o s t e r o n e B . D . H . 0 = T h y r o x i n t r e a t e d g o l d f i s h . A = T h i o u r e a t r e a t e d g o l d f i s h . • = T e s t o s t e r o n e t r e a t e d g o l d f i s h . X = U n t r e a t e d c o n t r o l g o l d f i s h . .150 g .125 o x • 2 g .IO ^^ ^^ ^ U u z - 0 7 5 O H CL .05 3 f> .1 Z o u Z -125 UJ . 0 7 5 . 0 5 7 DAY J U N E 30 14 DAY JULY 8 - 1 2 A M 5 - 6 P M 11- 12PM 5 - 6 A M 11-12AM WATER S H U T - O F F PERIODS t o f a c e F i g u r e 5 F i g u r e 5 Summary o f t h e means o f f i v e o x y g e n c o n s u m p t i o n d e t e r m i n -a t i o n s d u r i n g t w e n t y - f o u r h o u r s and p l u s and m i n u s one s t a n d a r d d e v i a t i o n f o r s e r i e s 2 . 0 = T h y r o x i n t r e a t e d g o l d f i s h . A = T h i o u r e a t r e a t e d g o l d f i s h . O = T e s t o s t e r o n e t r e a t e d : g o l d f i s h . X = U n t r e a t e d c o n t r o l g o l d f i s h . .160 OC O X "f.140 U u z g a .120 5 z> CO z O u Z HI >• x o .10 .08 3 DAY JULY 21 7 DAY JUNE 30 14 DAY JULY 8 21 DAY JULY 15 2 1 S e r i e s 3 r e s u l t s w e r e s i m i l a r t o t h o s e o f s e r i e s 2 . A g a i n t h e mean o x y g e n c o n s u m p t i o n f o r one o f t h e t h y r o x i n t r e a t m e n t s ( t h r e e d a y , J u l y 3 1 ) was h i g h e r t h a n t h e c o n t r o l , b u t no s i g n i f i c a n t d i f f e r e n c e was p r e s e n t (P = < 0 . 5 ) . ( S e e f i g u r e 6 , and a p p e n d i x ) . I n t h e s e v e n , f o u r t e e n and t w e n t y -one d a y t h y r o x i n t r e a t m e n t s t h e g o l d f i s h had l o w e r a v e r a g e c o n s u m p t i o n s t h a n t h e i r c o n t r o l s , b u t no s i g n i f i c a n t d i f f e r e n c e e x i s t e d . T h i o u r e a t r e a t e d f i s h show g r e a t v a r i a b i l i t y i n t h e i r a v e r a g e o x y g e n c o n s u m p t i o n . T h e t h r e e - d a y ( J u l y 3 1 ) and t h e t w e n t y - o n e d a y ( A u g u s t 1 8 ) t h i o u r e a t r e a t m e n t s h a v e o x y g e n c o n s u m p t i o n h i g h e r t h a n t h e c o n t r o l s , b u t a s i g n i f i c a n t d i f f e r e n c e i s p r e s e n t o n l y i n t h e t w e n t y - o n e d a y t r e a t m e n t . O x y g e n c o n s u m p t i o n s f o r t h e s e v e n and f o u r t e e n d a y t r e a t m e n t s a r e s i g n i f i c a n t l y l o w e r t h a n t h e c o n t r o l s . T h e t e s t o s t e r o n e t r e a t e d f i s h had o x y g e n c o n s u m p t i o n s h i g h e r t h a n t h e c o n t r o l s i n t h e t h r e e , s e v e n , and t w e n t y - o n e d a y t r e a t m e n t s , b u t a s i g n i f i c a n t d i f f e r e n c e was p r e s e n t o n l y a t t w e n t y - o n e d a y s . When t h e r e s u l t s o f t h e t h r e e hormone t r e a t m e n t s a r e c o m p a r e d c o n s i d e r a b l e v a r i a b i l i t y i s p r e s e n t . T h e r e a r e s i g -n i f i c a n t d i f f e r e n c e s b e t w e e n t h e s e v e n d a y t h i o u r e a and t e s t o -s t e r o n e r e s u l t s , t w e n t y - o n e d a y t h y r o x i n and t e s t o s t e r o n e , and t w e n t y - o n e d a y t h y r o x i n and t h i o u r e a ( s e e a p p e n d i x ) . S t a t i s t i c s o n t h e t o t a l d a t a f o r s e r i e s 2 and 3 show 22 t h a t t e s t o s t e r o n e t r e a t e d g o l d f i s h u s e s i g n i f i c a n t l y more o x y g e n t h a n t h e c o n t r o l s (P =>0.01) ( s e e a p p e n d i x ) . T h e t h i o u r e a t r e a t e d f i s h h a v e a n o x y g e n c o n s u m p t i o n h i g h e r t h a n t h e i r : c o n t r o l s b o r d e r i n g o n s i g n i f i c a n c e (P = 0.1 - 0.05). T h y r o x i n t r e a t e d f i s h h a v e a n o x y g e n c o n s u m p t i o n l o w e r t h a n t h e c o n t r o l s b u t n o t s i g n i f i c a n t l y l o w e r (P =>0.5). T h e r e a r e s i g n i f i c a n t d i f f e r e n c e s b e t w e e n t h e t h y r o x i n and t h i o u r e a t r e a t m e n t s , t h y r o x i n and t e s t o s t e r o n e , and t h i o u r e a and t e s t o s t e r o n e t r e a t m e n t s . t o f a c e F i g u r e 6 F i g u r e 6 Summary o f t h e means o f f i v e o x y g e n c o n s u m p t i o n d e t e r m i n a -t i o n s d u r i n g t w e n t y - f o u r h o u r s and p l u s and m i n u s one s t a n d a r d d e v i a t i o n f o r s e r i e s 3 . 0 = T h y r o x i n t r e a t e d g o l d f i s h . A = T h i o u r e a t r e a t e d g o l d f i s h . P = T e s t o s t e r o n e t r e a t e d g o l d f i s h . X = U n t r e a t e d c o n t r o l g o l d f i s h . /8o .160 2 J . I 40 .120 £ . I O O 3 DAY JULY 31 7 DAY AUGUST 4 14 DAY A U G U S T II 21 DAY AUGUST 18 . 0 8 21 S e r i e s 4 r e s u l t s a r e f o r m i s c e l l a n e o u s e x p e r i m e n t s . Two o f t h e f o u r e x p e r i m e n t s ( t h r e e and s e v e n d a y t r e a t m e n t s ) w i t h s i n g l e g o l d f i s h t r e a t e d w i t h 1 : 3 , 9 8 0 , 0 0 0 t h y r o x i n had a v e r a g e o x y g e n c o n s u m p t i o n s l o w e r t h a n t h e i r c o n t r o l s , h u t n o t s i g n i f i c a n t l y l o w e r ( f i g u r e 7 and T a b l e I I ) . T h e f o u r t e e n and t w e n t y - o n e d a y t r e a t e d f i s h had a v e r a g e o x y g e n c o n s u m p t i o n s h i g h e r t h a n t h e i r c o n t r o l s , t h e r e s u l t s f o r t h e t w e n t y - o n e d a y t r e a t m e n t b e i n g s i g n i f i c a n t l y h i g h e r . Two g r o u p s o f f i v e g o l d f i s h t r e a t e d w i t h d o u b l e and t r i p l e t h e u s u a l t h y r o x i n c o n c e n t r a t i o n f o r s e v e n d a y s ( A u g u s t 2 8 ) had o x y g e n c o n s u m p t i o n s l o w e r t h a n t h e i r c o n t r o l s b u t n o t s i g n i f i c a n t l y l o w e r . P e r h a p s t h e s e r e s u l t s w o u l d h a v e b e e n s i g n i f i c a n t l y l o w e r t h a n t h e c o n t r o l s i f t h e u s u a l f i v e i n s t e a d o f t h r e e d e t e r m i n a t i o n s had b e e n t a k e n d u r i n g t h e t w e n t y - f o u r h o u r e x p e r i m e n t a l p e r i o d . T h e r e a p p e a r s t o be l i t t l e d i f f e r -e n c e i n t h e o x y g e n c o n s u m p t i o n s b e t w e e n g r o u p s o f g o l d f i s h t r e a t e d w i t h d o u b l e and t r i p l e t h e u s u a l c o n c e n t r a t i o n o f s y n t h e t i c s o d i u m t h y r o x i n . - T h e a v e r a g e o x y g e n c o n s u m p t i o n s o f s i n g l e t h y r o x i n and c o n t r o l g o l d f i s h were h i g h e r t h a n a n y o f t h e g r o u p s o f f i v e u n t r e a t e d c o n t r o l f i s h o r t h o s e t r e a t e d w i t h t h y r o x i n e and t h i o u r e a i n s e r i e s 1 , 2 and 3 ' T h e s e r e s u l t s a r e i n a g r e e m e n t w i t h S c h u e t t ( 1 9 3 3 ) who f o u n d t h a t g r o u p s o f g o l d f i s h u s e d l e s s o x y g e n p e r w e i g h t t h a n s i n g l e f i s h . 2 4 T A B L E I I . SUMMARY OF THE TREATMENT AND S T A T I S T I C A L D E T A I L S OF S E R I E S 4 R E S P I R A T I O N EXPERIMENTS N o . o f d a y s N o . o f N o . o f c o n c e n t r a - S e x o f G o l d f i s h t r e a t m e n t g o l d f i s h d e t e r m i n - t i o n o f T r e a t e d U n t r e a t e d i n e x p e r i - a t i o n s t h y r o x i n G o l d - C o n t r o l s m e n t a l t a k e n u s e d f o r f i s h g r o u p s . d u r i n g 24 t r e a t e d h o u r s g r o u p s 1 3 1 5 1 : 3 , 9 8 0 , 0 0 0 2 7 1 5 «• ? </ 3 14 1 5 / ? 4 2 1 1 5 ? J 5 7A 5 3 1 : 1 , 9 9 0 , 0 0 0 6 7B 5 3 1 : 1 , 3 2 6 , 6 6 7 S T A T I S T I C S FOR S E R I E S 4 RESP IRAT ION EXPERIMENTS Mean 0 2 C o n -s u m p t i o n d u r i n g 24 h o u r s f o r t h y r o x i n t r e a t e d G o l d f i s h c c / g m / h r Mean 0 2 C o n -s u m p t i o n d u r i n g 24 h o u r s f o r u n t r e a t e d c o n t r o l s c c / g m / h r t P 1 . 1 4 2 . 1 4 9 . 2 7 3 4 2 . 1 5 6 . 2 0 6 1 . 1 6 7 9 . 5 - . 1 3 . 1 7 2 . 1 5 9 ' " . 5 9 7 1 <-5 4 . 3 3 4 . 2 1 1 4 . 7 9 1 5 > . 0 1 5 . 1 1 8 . 1 7 4 1 . 1 3 7 1 . 5 - 1 6 . 1 2 0 1 . 0 7 7 1 • 5 - . 1 to face Figure 7 Figure 7 Summary of the means of five results obtained during twenty-four hour periods and plus and minus one standard deviation for the miscellaneous experiments i n series 4 for single goldfish treated three, seven, fourteen and twenty-one days with 1 : 3 , 9 8 0 , 0 0 0 concentration of thyroxin and two groups of five f i s h treated seven days with thyroxin concentrations of 1 s 1 , 9 9 0 , 0 0 0 (A) and 1 : 1 , 3 2 6 , 6 6 7 (B). Thyroxin treated goldfish Untreated control goldfish . 3 7 0 3 DAY J U L Y 31 .330 a. o i 5 < J3 U U 290 z 0 . 2 5 0 a OJ z O u 2IO UJ S .170 .130 . 0 9 0 7 DAY AUGUST 4 14 DAY AUGUST II 21 DAY AUGUST 18 7 DAY AUGUST 28 . 0 5 0 2 5 Ammonia E x c r e t i o n E x p e r i m e n t s . T h e ammonia e x c r e t i o n o f t h e t h y r o x i n t r e a t e d g o l d f i s h d i m i n i s h e d w i t h t i m e ( f i g u r e 8 ) . P a i r e d r e s u l t s were u n o b t a i n -a b l e f o r t h e t w e n t y - o n e d a y t h i o u r e a and f o u r t e e n d a y t e s t o s t e r o n e t r e a t m e n t s a s some f i s h d i e d d u r i n g t h e e x p e r i m e n t s . S t a t i s t i c s c o m p u t e d o n t h e t o t a l d a t a do n o t i n d i c a t e s i g n i f i c a n t d i f f e r -e n c e s i n t h e ammonia e x c r e t i o n o f t h e t h y r o x i n and t h i o u r e a t r e a t e d g o l d f i s h and t h e i r r e s p e c t i v e c o n t r o l s ( T a b l e I I I ) . T e s t o s t e r o n e t r e a t e d g o l d f i s h e x c r e t e d s i g n i f i c a n t l y more ammonia t h a n t h e i r c o n t r o l s (P = . 0 5 - . 0 1 ) . A g a i n when t h e t o t a l d a t a a r e c o n s i d e r e d t h e r e i s a s i g n i f i c a n t d i f f e r e n c e o n l y b e t w e e n t h e t h y r o x i n and t e s t o s t e r o n e t r e a t m e n t s . M o i s t u r e and E t h e r S o l u b l e E x p e r i m e n t s . T h e r e s u l t s f o r t h e s e e x p e r i m e n t s ( T a b l e I V ) i n d i c a t e t h a t hormone t r e a t m e n t s d i d n o t a p p r e c i a b l y a l t e r t h e m o i s t u r e and e t h e r s o l u b l e c o n t e n t o f t h e g o l d f i s h . T A B L E I I I SUMMARY OF AMMONIA EXCRET ION RESULTS D a t e N o . o f d a y s N o . o f mg N H 3 e x c r e t e d p e r gm. o f f i s h p e r 24 h o u r s t r e a t m e n t f i s h T h y r o x i n T h i o u r e a T e s t o s t e r o n e C o n t r o l 18 / 5 / 5 2 3 5 . 7 2 7 . 8 2 0 . 9 3 3 . 7 0 7 2 9 / 8 / 5 2 3 5 . 6 6 1 . 7 2 2 . 8 6 4 . 6 2 3 2 1 / 5 / 5 2 7 5 . 8 3 8 . 7 0 3 .184 . 9 1 1 3 0 / 6 / 5 2 7 5 1 . 0 8 1 . 1 7 1 . 4 7 . 8 1 6 2 9 / 5 / 5 2 14 5 . 5 1 3 . 6 8 5 1 . 3 0 . 9 4 7 8 / 7 / 5 2 14 5 1.24 1.24 1.048 1 2 / 6 / 5 2 2 1 3 . 6 4 7 1 . 1 8 5 1 . 8 9 1 . 5 8 1 5 / 7 / 5 2 2 1 T h y r o x i n . 9 8 9 — 1 . 1 2 6 3 C o n t r o l s 5 2 5 / 8 / 5 2 2 1 7 — — 1 . 3 6 0 • 9 1 7 S T A T I S T I C S ON TOTAL AMMONIA EXCRETION DATA Mean o f m g . NH3 e x c r e t e d p e r gm. o f f i s h i n 24 h r s T h y r o x i n ' T h i o u r e a T e s t o s t e r o n e c o n t r o l c o n t r o l c o n t r o l . . 8 3 7 .970 .932 970' 1 . 3 8 .970 T h y r o x i n T h y r o x i n T h i o u r e a t h i o u r e a t e s t o s t e r o n e t e s t o s t e r o n e .837 .932 . 8 3 7 1 . 3 8 .932 1 . 3 8 . 7 6 8 8 6 .14902 2.1739 . 3 9 4 6 8 3 . 7 7 8 7 1 . 6 3 8 0 D e g r e e s o f f r e e d o m ' 14 13 13 1 3 •13 14 P r o b a b i l i t y L e v e l .5-1 < - 5 . 0 5 - . 0 1 <-5 >'.oi < . 1 t o f a c e F i g u r e 8 F i g u r e 8 P e r c e n t d i f f e r e n c e s f r o m t h e c o n t r o l s i n t h e amount o f ammonia e x c r e t e d ( e x p r e s s e d a s m g . ammonia p e r g ram o f f i s h p e r t w e n t y - f o u r h o u r s ) b y t h e hormone t r e a t e d f i s h a t e a c h t r e a t m e n t i n t e r v a l . O p e n b a r = t h y r o x i n t r e a t e d g o l d f i s h . S t i p p l e d b a r = t h i o u r e a t r e a t e d g o l d f i s h , s o l i d b a r = t e s t o s t e r o n e t r e a t e d g o l d f i s h . +100 -1 1 2 7 T A B L E IV ETHER SOLUBLES EXPERIMENT - PER CENT OF ETHER S O L U B L E S IN WET MACERATED GOLDFISH T I S S U E . D a y s o f T h y r o x i n T h i o u r e a T e s t C o n t r o l T r e a t m e n t % e t h e r Mean % e t h e r Mean % e t h e r Mean % e t h e r Mean s o l u b l e s s o l u b l e s s o l u b l e s s o l u b l e s 3 1 . 6 4 6 1 . 6 2 8 1 . 1 1 1 2 . 3 6 3 1 . 9 5 1 1 . 7 5 1 1 . 6 7 3 1 . 4 7 6 1 . 4 3 9 1 . 8 4 3 1 . 6 4 0 1 . 7 3 0 7 . 8 8 6 1 . 5 4 3 . 9 9 8 1 . 9 8 9 . 8 9 3 . 8 4 4 . 7 3 6 1 . 0 6 1 . 0 1 4 . 9 5 2 1 . 6 1 0 1 . 7 3 4 . 7 5 4 . 9 0 1 . 8 4 4 1 . 7 1 3 14 2 . 3 8 3 1 . 6 8 1 1 . 3 8 4 . 8 7 9 4 1 . 3 7 4 1 . 7 7 5 1 . 4 4 5 1 . 5 4 3 1 . 1 8 0 1 . 3 1 0 1 . 1 6 0 1 . 1 0 1 1 . 5 7 0 1 . 5 0 5 1 . 3 6 8 1 . 2 6 6 2 1 2 . 3 8 4 1 . 7 6 7 2 . 4 5 8 I . 6 7 8 I . 8 6 7 1 . 5 3 6 1 . 8 6 1 2 . 0 2 7 2 . 4 5 6 1 . 5 3 9 2 . 2 8 0 2 . 8 8 5 A v e r a g e I . 6 0 9 1 - 5 3 4 1 . 5 3 9 1 . 4 1 8 T a b l e IV c o n t i n u e d n e x t p a g e 28 T A B L E IY MOISTURE CONTENT EXPERIMENTS - PER CENT C o n t i n u e d MOISTURE IN WET MACERATED GOLDFISH T I S S U E D a y s o f T h y r o x i n T h i o u r e a T e s t o s t e r o n e C o n t r o l T r e a t m e n t % M o i s t u r e % M o i s t u r e % M o i s t u r e % M o i s t u r e 3 80.43 79.36 79-18 80.98 80.34 79.13 79.64 78.96 7 80.07 77-71 79-67 78.56 80.64 77.91 79.35 79.23 14 79.49 79.06 78.29 79-12 77.37 79.08 77.81 79.19 21 77.45 7 8 . 14 77.39 78.03 A v e r a g e 79-15 78.55 78.99 79-34 29 A s p h y x i a t i o n E x p e r i m e n t s T h e p l o t s o f o x y g e n and c a r b o n d i o x i d e t e n s i o n s ( f i g u r e 9) a r e s i m i l a r f o r e a c h o f t h e f o u r t r e a t m e n t p e r i o d s . I f c u r v e s were d r a w n t h r o u g h t h e a g g r e g a t e r e s u l t s f o r e a c h hormone t r e a t m e n t a t e a c h p e r i o d t h e y w o u l d a p p r o x i m a t e e a c h o t h e r up t o t h e 40 mm. o x y g e n t e n s i o n i n a l l e x c e p t t h e t h r e e d a y t r e a t m e n t . A s e c o n d s e t o f l i k e c u r v e s c o u l d be d r a w n i n a n o p p o s i t e d i r e c t i o n t h r o u g h t h e r e s u l t s g r o u p e d t o w a r d t h e u p p e r r i g h t o f e a c h g r a p h . T h e r e s u l t s i n t h i s c a s e w e r e f o u n d t o be d u e t o a d i s s o c i a t i o n b e t w e e n t h e o x y g e n and c a r b o n d i o x i d e i n t h e w a t e r . T h e z e r o o x y g e n r e s u l t s p l o t t e d o n t h e a b s c i s s a a r e p r e s u m e d t o be a r t i f a c t s s i n c e t h e g a s d e t e r m i n a t i o n s w e r e c o n -d u c t e d i n some c a s e s f i v e h o u r s a f t e r d e a t h o f t h e g o l d f i s h . S i m i l a r r e s u l t s c o u l d n o t a l w a y s be o b t a i n e d when t h e d e t e r m i n -a t i o n s were made i m m e d i a t e l y f o l l o w i n g d e a t h . F i g u r e 10 shows a r e l a t i o n s h i p b e t w e e n t h e c a r b o n d i o x i d e t e n s i o n s and t h e t i m e t o a s p h y x i a t e e a c h g o l d f i s h . H i g h c a r b o n d i o x i d e t e n s i o n s (100 mm. o f Hg o r g r e a t e r ) p r o d u c e d t h e most r a p i d a s p h y x i a t i o n s . L i t t l e d i f f e r e n c e i n t h e r e l a t i o n s h i p i s s e e n f o r t h e f o u r t r e a t m e n t p e r i o d s and t h e f o u r g r o u p s o f f i s h r e p r e s e n t e d . I f s t r a i g h t l i n e s were d r a w n t h r o u g h t h e r e s u l t s f o r e a c h o f t h e f o u r d i f f e r e n t g r o u p s o f f i s h l i t t l e d i f f e r e n c e w o u l d be s e e n b e t w e e n them i n a n y o f t h e t r e a t m e n t p e r i o d s o r b e t w e e n t h e f o u r p e r i o d s . t o f a c e F i g u r e 9 F i g u r e 9 C a r b o n d i o x i d e and o x y g e n t e n s i o n s a t d e a t h f o r e a c h g o l d f i s h a s p h y x i a t e d . 0 = T h y r o x i n t r e a t e d g o l d f i s h . A = T h i o u r e a t r e a t e d g o l d f i s h . • = T e s t o s t e r o n e t r e a t e d g o l d f i s h . X = U n t r e a t e d c o n t r o l g o l d f i s h . t 80 X 5 z 6 0 O 00 z UJ I-Z 40 LU o > x O 20 3 DAY • x o x 7 DAY • A 80 I 5 60 O 00 Z LU 4 0 UJ O >- ' x O 20 ^P 0. 14 DAY • x 6x n 21 DAY • o ^ o X AO o O A O X >PO O ^ O ^ 0 ^ ffirSMn^QlA , A A X 8 f X D I Q_ O IOO 200 300 CARBON DIOXIDE TENSION MM. Hg O IOO 200 300 CARBON DIOXIDE TENSION MM.Hg t o f a c e F i g u r e 10 F i g u r e 10 C a r b o n d i o x i d e t e n s i o n a t d e a t h and t i m e t o a s p h y x i a t e I n m i n u t e s f o r e a c h g o l d f i s h . 0 = T h y r o x i n t r e a t e d g o l d f i s h . A = T h i o u r e a t r e a t e d g o l d f i s h . D = T e s t o s t e r o n e t r e a t e d g o l d f i s h . X = U n t r e a t e d c o n t r o l g o l d f i s h . cn 2 0 0 X 2 2 Z O co Z U J hx-A O I50K-LU Q Q IOO Q z o CQ or < u 5 0 h -i — r OK 3 DAY X ° X X T J OA 7 DAY O AA° A X-X A O • X X X 6 X 2 2 0 0 O 00 Lo 150 LU I-LU Q X O Q  IOO o CD or < U 5 0 hx-hn-14 DAY 21 DAY o° o »' ,o • x • • • ox v ° X o • AA AR • A • A q^< O A D A DA AA X X X o o x a Dx • o O O 2 0 0 4 0 0 T I M E 6 0 0 O 2 0 0 4 0 0 T I M E 6 0 0 3 0 D I S C U S S I O N G o l d s m i t h (194-9) , and P r o s s e r , e t a l . (1950) h a v e r e v i e w e d t h e m a j o r w o r k s o n t h e e f f e c t s o f t h y r o i d m a t e r i a l s o n f i s h . T h e y r e p o r t t h a t t h e s e m a t e r i a l s g e n e r a l l y p r o m o t e g r o w t h i n f i s h and p o s s i b l y c o n t r o l f i s h m e t a m o r p h o s i s . I n a d d i t i o n t o t h i s H o a r (1951) s t a t e s : M a m m a l i a n t h y r o i d p r e p a r a t i o n s b r i n g a b o u t c h a r a c t e r i s t i c c h a n g e s i n t h e h i s t o l o g i c a l p i c t u r e o f t h e t h y r o i d o f s a l m o n , a l t e r b o d y p r o p o r t i o n s o f f i s h , t h e c h l o r i d e m e t a b o l i s m and t h e n i t r o g e n m e t a b o l i s m a s i n d i c a t e d b y d e p o s i t i o n o f g u a n i n e i n t h e s k i n . T h e a b o v e r e v i e w e r s a g r e e t h a t t h e t h y r o i d i s i n some way i n v o l v e d i n t h e r e g u l a t i o n o f f i s h m e t a b o l i s m , b u t c o n s i d e r a b l e c o n t r o v e r s y e x i s t s a s t o w h e t h e r i t r e g u l a t e s t h e c a l o r i g e n i c a s p e c t s o f m e t a b o l i s m . E t k i n , e t a l . (194-0) f o u n d t h a t g o l d f i s h f e d d e s i c c a t e d t h y r o i d t a b l e t s had o x y g e n c o n s u m p t i o n w h i c h d i f f e r e d s i g n i f i c a n t l y f r o m t h e i r c o n t r o l s i n t e n o u t o f t w e n t y - s i x e x p e r i m e n t s . S i x o f t h e e x p e r i m e n t s p r o d u c e d r e s u l t s s i g n i f i c a n t l y h i g h e r and f o u r w e r e s i g n i f i -c a n t l y l o w e r t h a n t h e i r c o n t r o l s , b u t i n t h e s t a t i s t i c a l c o n -s i d e r a t i o n o f a l l o f t h e r e s u l t s t h e r e was no s i g n i f i c a n t d i f f e r e n c e e i t h e r w a y . S m i t h and E v e r e t t (194-3) r e p o r t t h a t t h e o x y g e n c o n s u m p t i o n o f g u p p i e s c o u l d be r a i s e d i n some c a s e s a s much a s 1 2 . 5 p e r c e n t when t h e y were f e d d e s i c c a t e d 31 t h y r o i d t a b l e t s . A g a i n , t h o u g h , s t a t i s t i c s i n d i c a t e no s i g -n i f i c a n t i n c r e a s e . Two R u s s i a n w o r k e r s , S a k s and Zamkwa (1952) m e n t i o n t h a t t h y r o x i n e i n a c o n c e n t r a t i o n o f 1 : 2,000,000 a d m i n i s t e r e d t o t h i o u r e a t r e a t e d s a l m o n and s t u r g e o n l a r v a e d e s t r o y e d t h e o x y g e n l o w e r i n g e f f e c t o f t h e t h i o u r e a . S m i t h and M a t t h e w s ( 1 9 4 8 ) a p p e a r t o be t h e o n l y w o r k e r s t h a t h a v e r e p o r t e d c o n s i s t e n t i n c r e a s e s i n t h e o x y g e n c o n s u m p t i o n o f f i s h u s i n g B a t h y s t o m a o v e r 15 grams i n j e c t e d w i t h s e a w a t e r e x t r a c t s o f m a c e r a t e d t h y r o i d g l a n d s o f p a r r o t f i s h . T h i s e x p e r i m e n t i n d i c a t e s t h r e e p o s s i b i l i t i e s : t h e i n c r e a s e d o x y g e n c o n s u m p t i o n was c a u s e d b y some u n i d e n t i f i e d m a t e r i a l s i n t h e e x t r a c t , o r t h e e f f e c t s o f t h y r o i d m a t e r i a l s o n t h e r e s p i r a t i o n o f some f i s h i s r e l a t e d t o s i z e , o r t h e r e may be s p e c i e s o r c l a s s s p e c i f i c i t i e s o f t h y r o i d ' s e f f e c t o n f i s h r e s p i r a t i o n . G o l d s m i t h (1949) i s c a u t i o u s a b o u t p l a c i n g t o o much e m p h a s i s o n t h e S m i t h and M a t t h e w s s t u d y a s o n l y one g r o u p o f e x p e r i m e n t s was d o n e . I n t h e p r e s e n t s t u d y t h e r e s p i r a t i o n o f t h e t h y r o x i n e t r e a t e d g o l d f i s h was n o t s i g n i f i c a n t l y a l t e r e d i n s e r i e s 2 and 3 « T h e s e r e s u l t s a r e c o m p a t i b l e w i t h t h e p r e v i o u s l y d e s c r i b e d f i n d i n g s o f E t k i n , e t a l . ( 1 9 4 0 ) , and S m i t h and E v e r e t t ( 1 9 4 3 ) . S e r i e s 1 r e s u l t s show a s i g n i f i c a n t i n c r e a s e i n t h e o x y g e n c o n s u m p t i o n o f t h y r o x i n e t r e a t e d f i s h . S i n c e s e r i e s 1 was d o n e d u r i n g t h e w i n t e r , s e a s o n a l v a r i a b i l i t y may be t h e e x p l a n a -t i o n f o r t h e d i s c r e p a n c y b e t w e e n t h e r e s u l t s o f t h i s and t h e o t h e r s e r i e s . Some e v i d e n c e s o f s e a s o n a l v a r i a b i l i t y d o e s 32 e x i s t f o r f i s h . H o a r (1951) r e p o r t s a s e a s o n a l a l t e r a t i o n i n t h e h i s t o l o g y o f s a l m o n t h y r o i d t i s s u e . H o a r , e t a l . (1952) a l s o m e n t i o n s t h a t u n t r e a t e d g u p p i e s h a v e r a i s e d o x y g e n c o n -s u m p t i o n a t s e x u a l m a t u r i t y . S e x u a l m a t u r i t y o f g o l d f i s h t a k e s p l a c e i n l a t e s p r i n g and e a r l y summer , t h e r e f o r e t h e h i g h e s t o x y g e n c o n s u m p t i o n s h o u l d o c c u r a t t h i s t i m e . I n many c a s e s t h e a v e r a g e o x y g e n c o n s u m p t i o n o f t h e summer c o n t r o l f i s h was h i g h e r t h a n t h a t f o r t h e w i n t e r o n e s ( f i g u r e s 3> 4 , 5 and 6). T h e r e a r e o t h e r s e a s o n a l v a r i a t i o n s i n f i s h r e s p i r a -t i o n , w h i c h a r e u n e x p l a i n e d , t h a t a r e o p p o s i t e t o t h o s e a s s o c i a t e d w i t h r e p r o d u c t i o n . F u n d u l u s had a s l i g h t l y h i g h e r m e t a b o l i s m i n w i n t e r t h a n i n summer a t t e m p e r a t u r e s o f 15° G . o r b e l o w ( W e l l s , 1935). T h e m e t a b o l i s m o f e u n n e r , T a u t o g l a b r u s , was a f f e c t e d s i m i l a r l y a t t h e same t e m p e r a t u r e ( H a u g a r d and I r v i n g , 1 9 4 3 ) . I t i s u n c e r t a i n w h e t h e r t h i s i s t r u e a b o v e 15° C . a s t h e s e w o r k e r s d o n o t a p p e a r t o h a v e c o n t i n u e d t h e i r s t u d i e s b e y o n d t h i s t e m p e r a t u r e . No l i t e r a t u r e o f t h i s n a t u r e seems t o be p r e s e n t f o r g o l d f i s h . I n a l l r e s p i r a t i o n e x p e r i m e n t s t h e r e a r e many f a c t o r s s u c h a s s e x d i f f e r e n c e s , i n d i v i d u a l and g r o u p v a r i a b i l i t y and p r e v i o u s t e m p e r a t u r e h i s t o r y o v e r w h i c h t h e r e s e a r c h e r h a s no c o n t r o l . No l i t e r a t u r e seems t o e x i s t o n t h e s e x d i f f e r e n c e s o f f i s h r e s p i r a t i o n , b u t T a b l e I I shows t h a t s u c h a p h e n o m e n o n c o u l d e x i s t . I n d i v i d u a l v a r i a b i l i t y i n t h e o x y g e n c o n s u m p t i o n 33 o f r e a r e d g u p p i e s o f t h e same s e x and w e i g h t was s e e n by-S m i t h and E v e r e t t (1943). T h e s i n g l e g o l d f i s h o f s e r i e s 4 ( f i g u r e 7) shows t h i s n i c e l y . T h e c o n t r o l s o f s e r i e s 1, 2 and 3 ( f i g u r e s 3» 4 , 5 and 6) d e m o n s t r a t e t h e i n t e r o r i n t r a g r o u p v a r i a b i l i t y i n r e s p i r a t i o n t h a t c a n o c c u r . I n t h e p r e s e n t s t u d y , t h e t e m p e r a t u r e h i s t o r y o f t h e g o l d f i s h was n o t known p r i o r t o t h e i r a r r i v a l a t o u r l a b o r a t o r y . T h e r e -f o r e t h e f i s h w e r e c o n d i t i o n e d t o 20° C . a t t h e r a t e o f 1 ° C . p e r d a y and w i t h h e l d f r o m e x p e r i m e n t a t i o n a min imum o f a w e e k , t h e p e r i o d recommended b y F r e e m a n (1950). G a r d n e r , e t a l . (1922) f o u n d t h a t e n v i r o n m e n t a l c o n d i t i o n s two weeks p r e v i o u s t o e x p e r i m e n t a t i o n i n f l u e n c e d t h e o x y g e n c o n s u m p t i o n o f g o l d f i s h . T h e p r e v i o u s d i s c u s s i o n s c a n a l s o be u s e d t o e x p l a i n t h e v a r i a b i l i t i e s e x i s t i n g i n s t u d i e s o t h e r t h a n t h o s e o n r e s p i r a t i o n i n t h i s t h e s i s , e s p e c i a l l y f o r t h e t e n d e n c y f o r t h e amount o f ammonia e x c r e t e d b y t h e c o n t r o l s t o i n c r e a s e a s t h e i n v e s t i g a t i o n p r o g r e s s e d ( T a b l e I I I ) . I f h o r m o n e s a f f e c t t h e r e s p i r a t i o n , ammonia e x c r e t i o n and o t h e r a s p e c t s o f g o l d f i s h m e t a b o l i s m , t h e n t h e e f f e c t s h o u l d be g r e a t e n o u g h t o o v e r r i d e t h e u n c o n t r o l l e d ' v a r i a b i l i t i e s . S e r i e s 1, ( f i g u r e 3)? t h e e f f e c t o f m e t h y l t e s t o s t e r o n e o n r e s p i r a t i o n ( f i g u r e s 4 , 5 and 6) and t h e e f f e c t o f m e t h y l t e s t o s t e r o n e o n ammonia e x c r e t i o n ( T a b l e I I I and f i g u r e 8) a r e a l l i n s t a n c e s i n w h i c h t h e u n c o n t r o l l e d v a r i a b i l i t i e s h a v e b e e n s u r m o u n t e d . ' R e t u r n i n g a g a i n t o t h e e f f e c t s o f t h y r o i d m a t e r i a l s o n f i s h r e s p i r a t i o n , i t i s n o t e w o r t h y t h a t a l l p r e v i o u s s t u d i e s 34 as well as the present one were done on f i s h i n a resting state. Sporadic increases i n the oxygen consumption exist i n a l l of the experiments. This may mean that thyroid materials have t h e i r influence on active rather than resting metabolism. Mansfeld (1949) indicates that thyroid materials stimulate active rather than resting metabolism i n mammals. Another topic that may be s i g n i f i c a n t i s the concentration of the thy-roid materials used. In series 4 (figure 7? August 28) doubling and t r i p l i n g the usual concentrations of thyroxine produced an average oxygen consumption lower than the controls. Perhaps i f f i v e instead of the three tests were done during the twenty-four hours, s t a t i s t i c a l s i g n i f i c a n t differences would e x i s t . Similar oxygen consumptions lower than the controls are seen i n series 2 and 3« This could mean that the concentration of thyroxine was too high. Dahlstrom and Sjostrand (1948) demonstrated the importance of thyroxine concentration i n humans when they de-pressed oxygen consumptions with low concentrations of thyroxine; with larger doses they were able to produce increased r e s p i r a -tions and with s t i l l larger doses an unexplained depression took place again. Hydrogen ion concentration of the thyroxine medium i s another matter that could be important. Unfortunately i n the present studies no readings of thi s nature were taken. Marzulli :(1942) points out the importance of the hydrogen ion concentration of the thyroxine media i n his studies on tadpole metamorphosis. He found that a s l i g h t l y a c i d i c media greatly increased the accelerating e f f e c t of thyroxine on metamorphosis and r e s p i r a t i o n , whereas a s l i g h t l y basic media retarded 35 t h y r o x i n e ' s e f f e c t . P e r h a p s h y d r o g e n i o n c o n c e n t r a t i o n s i m i l a r l y a f f e c t s t h y r o x i n e ' s a c t i o n o n f i s h . T h y r o i d e c t o m i z e d r a t s h a v e a n i n c r e a s e d o u t p u t o f u r i n a r y n i t r o g e n w h i c h c a n be r e d u c e d b y s m a l l p h y s i o l o g i c a l d o s e s o f t h y r o x i n e ( R u p p , e t a l . 194-9) . T h e s e w o r k e r s f o u n d t h a t t h e same amount o f t h y r o x i n e had no e f f e c t o n t h e e x c r e t i o n o f i n t a c t a n i m a l s , b u t a l a r g e r d o s a g e d i d i n c r e a s e t h e u r i n a r y n i t r o g e n . T h e s i z e o f t h e t h y r o x i n e d o s e , t h e n , i s a n i m p o r t a n t c o n s i d e r a t i o n i n p r o d u c i n g a n e l e v a t e d n i t r o g e n e x c r e t i o n i n r a t s . S y n t h e t i c s o d i u m t h y r o x i n e d i d n o t i n c r e a s e t h e ammonia e x c r e t i o n o f g o l d f i s h ( T a b l e 3 and f i g u r e 8) as was r e p o r t e d b y R e d l i c h (195f? u n p u b l i s h e d ) . T h e p r e s e n t s t u d y u s e d a n i m p r o v e d t e c h n i q u e i n w h i c h d i s t u r b a n c e s w e r e r e d u c e d and a t w e n t y - f o u r h o u r c o n d i t i o n i n g o f t h e f i s h t o o k p l a c e p r i o r t o e x p e r i m e n t a t i o n . R e d l i c h t r e a t e d t h e g o l d f i s h i n h e r a p p a r a -t u s and c r e a t e d c o n s i d e r a b l e d i s t u r b a n c e b y r e m o v i n g t h e f i s h f r o m t h e w a t e r when t h e w a t e r was c h a n g e d . T h e r e i s a s t r o n g s u g g e s t i o n h e r e t o o t h a t t h y r o x i n e a c t s o n t h e a c t i v e r a t h e r t h a n t h e r e s t i n g m e t a b o l i s m t o i n c r e a s e t h e ammonia e x c r e t i o n . B y n o t u s i n g a c o n d i t i o n i n g p e r i o d R e d l i c h was a b l e t o i m m e d -i a t e l y b e g i n c o l l e c t i n g t h e ammonia e x c r e t e d b y t h e f i s h a t t h e c o m p l e t i o n o f t h e v a r i o u s t r e a t m e n t s . T h e p r e s e n t i n v e s t i g a -t i o n may h a v e m i s s e d a r i s e i n t h e ammonia e x c r e t i o n b y u s i n g t h e t w e n t y - f o u r h o u r c o n d i t i o n i n g p e r i o d . 36 Turner (194-8) reports that goitrogenic substances such as thiourea or thiouradl i n t e r f e r e d i r e c t l y with the thyroid gland to i n h i b i t i t s action and cause metabolic responses i n mammals opposite to what occurs when thyroid substances are administered. Astwood (1949) suggests that thiourea's or thiouradl's mode of action i s to compete with other materials such as tyrosine i n the synthesis of thyro-globulin or thyroxine which have part of t h e i r structure s i m i l a r to that of these goitrogens. Thiourea produces a growth retardation i n f i s h i n contrast to the growth stimulating effect associated with administration of thyroid substances. Vivien, et a l . (1952) showed t h i s using guppies, and Goldsmith et a l . (1944) also demonstrate s i m i l a r effects on hybrids of a Platypoecllus  maculatus and Xiphophorus h e l l e r i i cross. Both authors report a retardation of sexual maturity, absence of secondary sex c h a r a c t e r i s t i c s and a marked hyperplasia of the thyroid tissues i n thiourea treated f i s h . Hatley (1950) describes the increased l i v e r glycogen and blood sugar l e v e l of carp immersed i n a 1 : 1000 concentration of thiourea. This effect i s the opposite to that reported i n mammals treated with thyroxine. The available l i t e r a t u r e indicates that the adminis-t r a t i o n of antithyroid substances depressed the oxygen consumpi-t i o n of f i s h i n most cases. Osborn (1951) successfully 37 l o w e r e d t h e o x y g e n c o n s u m p t i o n o f minnows b y 20 p e r c e n t i n p r e p a r a t i o n f o r t r a n s p o r t a t i o n b y i m m e r s i n g them i n s o l u t i o n s c o n t a i n i n g up t o 388 p . p . m . t h i o u r a c i l . S a k s and Zamkwa (1952) f o u n d t h a t t h i o u r e a d e p r e s s e d t h e o x y g e n c o n s u m p t i o n o f s a l m o n and s t u r g e o n l a r v a e . M a t t h e w s and S m i t h ( 1 9 4 7 ) ? t h o u g h , were u n a b l e t o d e p r e s s t h e r e s p i r a t i o n o f F u n d u l u s b y i n j e c t i n g t h i o u r e a . T h e r e s u l t s o f S e r i e s 1 ( f i g u r e 3) r e s u l t s a r e i n a c c o r d w i t h t h e r e s p i r a t i o n d e p r e s s i n g e f f e c t o f a n t i t h y r o i d compounds m e n t i o n e d i n t h e l i t e r a t u r e a v a i l a b l e . G r e a t v a r i -a b i l i t y e x i s t s i n t h e S e r i e s 2 and 3 t h i o u r e a r e s u l t s ( f i g u r e 4 , 5 and 6) . I n some c a s e s t h e o x y g e n c o n s u m p t i o n i s l o w e r t h a n t h e c o n t r o l s and i n o t h e r i n s t a n c e s i t i s h i g h e r . I n most c a s e s w h e r e i t i s h i g h e r t h e r e a r e s t a t i s t i c a l s i g n i f i c a n t d i f f e r e n c e s ( a p p e n d i x ) . I t a p p e a r s t h e n t h a t t h i o u r e a ' s e f f e c t may be s e a s o n a l a s was p o s t u l a t e d p r e v i o u s l y f o r t h e t h y r o x i n e r e s u l t s . T h e i n c o n s i s t e n c y o f t h e s e r i e s 2 and 3 r e s u l t s may be e x p l a i n e d b y t h e i n i t i a l s t i m u l a t i n g a c t i o n o f t h i o u r e a o n t h e t h y r o i d g l a n d r e p o r t e d b y T u r n e r ( 1948 ) and b y L e v e r , e t a l . ( 1 9 4 9 ) f o r L e b i s t e s r e t i c u l a t u s . T u r n e r e x p l a i n s t h a t t h e e f f e c t o f t h i o u r e a i s t o i n h i b i t t h e p r o d u c t i o n o f i o d o t h y r o g l o b u l i n b y t h e t h y r o i d g l a n d and t h e r e f o r e i n c r e a s e t h e s e c r e t i o n o f t h y r o t r o p i n b y t h e a n t e r i o r p i t u i t a r y t h u s e n l a r g i n g t h e s e c r e t o r y e p i t h e l i u m o f t h e t h y r o i d t i s s u e . T h e m o b i l i z a t i o n o f t h y r o g l o b u l i n i s r a i s e d t o e l e v a t e o r m a i n t a i n 38 t h e n o r m a l m e t a b o l i c r a t e f o r a f e w d a y s u n t i l a l l o f t h e c o l l o i d a l s t o r e o f t h y r o g l o b u l i n i s e x h a u s t e d . A t t h i s t i m e t h e p r o d u c t i o n o f t h y r o g l o b u l i n i s s u b n o r m a l and t h e m e t a b o l i c r a t e d e c r e a s e s . L e v e r , e t a l . (1949) c o r r o b o r a t e s T u r n e r ' s s t a t e m e n t s o n t h e i n i t i a l s t i m u l a t i o n o f t h i o u r e a o n t h e t h y -r o i d g l a n d i n h i s s t u d y o n f i s h . He o b s e r v e d a n i n c r e a s e i n t h e t h y r o i d a c t i v i t y shown b y t h e i n c r e a s e d s i z e o f t h e t h y r o i d g l a n d f o l l i c l e s a f t e r i m m e r s i n g t h e L e b l s t e s f o r a week i n s o l u t i o n o f t h i o u r e a up t o . 6 p e r c e n t . F u r t h e r t r e a t m e n t i n a c t i v a t e d t h e g l a n d a s shown b y d e c r e a s e d f o l l i c l e s i z e . C a r e f u l e x a m i n a t i o n s o f t h e h i s t o l o g i c a l p r e p a r a t i o n s o f t h e g o l d f i s h t h y r o i d t i s s u e o b t a i n e d a t t h e t i m e o f e x p e r i m e n t a t i o n a r e n e e d e d t o s u b s t a n t i a t e t h e p r e v i o u s d i s c u s s i o n i n o u r s t u d y . E x p e r i m e n t s l o n g e r t h a n t w e n t y - o n e d a y s were n o t c a r r i e d o u t . No e v i d e n c e e x i s t s t o i n d i c a t e w h e t h e r l o n g e r t r e a t m e n t w o u l d p r o d u c e a c o n s i s t e n t l o w e r i n g o f t h e o x y g e n c o n s u m p t i o n . G o l d f i s h t r e a t e d w i t h t h i o u r e a had ammonia e x c r e t i o n s s o m e t i m e s l e s s and s o m e t i m e s g r e a t e r t h a n t h e c o n t r o l s ( f i g u r e 8 and t a b l e I I I ) . A s t a t i s t i c a l c o n s i d e r a t i o n o n t h e t o t a l d a t a d i d n o t i n d i c a t e a s i g n i f i c a n t d i f f e r e n c e f r o m t h e c o n t r o l ( t a b l e I I I ) . T h e p r e v i o u s d i s c u s s i o n u s e d t o e x p l a i n t h i o u r e a ' s v a r i a b l e e f f e c t o n f i s h r e s p i r a t i o n c a n a l s o be u s e d h e r e t o e x p l a i n t h e v a r i a b i l i t y o f t h e ammonia e x c r e t i o n . Mann (1939) showed t h a t p e r c h , b i t t e r l i n g and r o a c h i n j e c t e d i n t e r p e r i t o n e a l l y w i t h s e x h o r m o n e s o f t h e same s e x a s t h e f i s h , i n c r e a s e d t h e i r o x y g e n c o n s u m p t i o n s . H a s l e r and 39 M e y e r ( 1 9 4 2 ) f o u n d t h a t t h e o x y g e n c o n s u m p t i o n o f g o l d f i s h c o u l d be r a i s e d 36 p e r c e n t b y t h e i n j e c t i o n o f c a r p p i t u i t a r y e x t r a c t s . When t h e g o l d f i s h were c a s t r a t e d t h i s i n c r e a s e was a b o l i s h e d , i n d i c a t i n g t h a t t h e e x t r a c t s t i m u l a t e d t h e g o n a d s w h i c h I n t u r n i n c r e a s e d t h e o x y g e n c o n s u m p t i o n b y some m e c h a n i s m . T h e e l e v a t e d r e s p i r a t i o n o f t h e g o l d f i s h 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 ( f i g u r e s 3> 4 , 5? 6 and a p p e n d i x ) i s i n k e e p i n g w i t h t h e l i t e r a t u r e c i t e d a b o v e . T h e r e s u l t s a r e a l s o s i m i l a r t o t h o s e r e p o r t e d b y P i n c u s and T h i m a n n (1950) f o r mammals 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 . M e t h y l t e s t o s t e r o n e h a s a n i t r o g e n r e t e n t i o n e f f e c t o n mammals b u t i n c r e a s e s r a t h e r t h a n d e c r e a s e s c r e a t i n u r e a ( P i n c u s and T h i m a n n (1950). G o l d f i s h 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 h a v e t h e i r ammonia e x c r e t i o n s s i g n i f i c a n t l y i n c r e a s e d ( T a b l e I I I and f i g u r e 8). G o l d f i s h do n o t e x c r e t e c r e a t i n e o r c r e a t i n i n e ( P r o s s e r , e t a l . 1950) b u t m e t h y l t e s t o s t e r o n e may e n h a n c e t h e c a t a b o l i s m o f o t h e r p r o t e i n s u b s t a n c e s t o e l e v a t e t h e ammonia e x c r e t i o n . T h e i n c r e a s e d ammonia e x c r e t i o n o f g o l d f i s h 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 c o r r e s p o n d s t o a n i n c r e a s e d o x y g e n c o n s u m p t i o n . T h e r e a r e c o r r e s p o n d i n g r e l a t i o n -s h i p s e x i s t i n g b e t w e e n t h e o x y g e n c o n s u m p t i o n and ammonia e x c r e t i o n f o r t h e o t h e r two t r e a t m e n t s . No s t a t i s t i c a l s i g n i f i -c a n t c h a n g e s i n t h e o x y g e n c o n s u m p t i o n a s a r e s u l t o f t h i o u r e a o r t h y r o x i n e t r e a t m e n t s o c c u r r e d and t h e c o r r e s p o n d i n g ammonia e x c r e t i o n d a t a r e m a i n e d s t a t i s t i c a l l y u n c h a n g e d . 40 No a p p r e c i a b l e c h a n g e t o o k p l a c e i n e t h e r s o l u b l e s o r m o i s t u r e o f t h e g o l d f i s h when t h e y were t r e a t e d w i t h t h e t h r e e h o r m o n e s ( T a b l e I V ) . I n g e n e r a l e t h e r s o l u b l e s r a n g e f r o m ;.5 - 1 . 5 p e r c e n t and a r e s i m i l a r t o t h e v a l u e s f o u n d b y H o a r and C o t t l e (1952) f o r u n t r e a t e d g o l d f i s h a c c l i m a t e d t o 2 0 ° C . M o i s t u r e r e s u l t s f o r t h e t h y r o x i n e t r e a t e d g o l d f i s h do n o t a g r e e w i t h t h e r e d u c e d m o i s t u r e c o n t e n t o f g o l d f i s h t r e a t e d s i m i l a r l y b y R e d l i c h (195'/-? u n p u b l i s h e d ) . T h e d i f f e r -e n c e c a n p e r h a p s be a t t r i b u t e d t o t e c h n i q u e . R e d l i c h u s e d d e a d g o l d f i s h f r o m h e r a s p h y x i a t i o n e x p e r i m e n t s f o r m o i s t u r e d e t e r m i n a t i o n s and t h e y may h a v e t a k e n o n c o n s i d e r a b l e w a t e r a f t e r d e a t h . F r o m t h e r e s u l t s o b t a i n e d i t a p p e a r s t h a t t h e f i s h t r e a t e d w i t h t h e t h r e e h o r m o n e s were i n good c o n d i t i o n w i t h r e s p e c t t o f a t c o n t e n t and m o i s t u r e c o n t e n t . R a t s w e r e more r e s i s t a n t t o a n o x i a when t h e y had 1 p e r c e n t t h i o u r e a a d d e d t o t h e i r d r i n k i n g w a t e r f o r t h r e e m o n t h s , b u t d a i l y t r e a t m e n t s o f 2 m g . t h y r o x i n e f o r t h r e e weeks d e c r e a s e d t h e r e s i s t a n c e o f a d i f f e r e n t g r o u p ( L e b l o n d , 1 9 4 4 ) . H u g h e s and A s t w o o d (1944 ) f o u n d t h a t r a t s t r e a t e d w i t h t h i o -u r a c i l had i n c r e a s e d r e s i s t a n c e t o l o w b a r o m e t r i c p r e s s u r e , l o w o x y g e n t e n s i o n a t a t m o s p h e r i c p r e s s u r e and c a r b o n m o n o x i d e p o i s o n i n g . S y n t h e t i c s o d i u m t h y r o x i n e , t h i o u r e a and m e t h y l t e s t o s t e r o n e d i d n o t a l t e r t h e n o r m a l a s p h y x i a t i o n p i c t u r e f o r f i s h i n o u r s t u d y ( f i g u r e s 9 and 1 0 ) . T h e u s e o f c a r b o n d i o x i d e t o v a r y t h e o x y g e n c o n t e n t 41 o f t h e w a t e r f o r f i s h a s p h y x i a t i o n s h a s c e r t a i n d i s a d v a n t a g e s . C a r b o n d i o x i d e i s t o x i c t o f i s h ( W e l l s , 1918) and a c t s a l s o a s a n a n e s t h e t i c . F i s h ( 1942 ) u s e d c a r b o n d i o x i d e e x t e n s i v e l y i n c o n c e n t r a t i o n s up t o 200 p . p . m . t o a n e s t h e t i z e a d u l t s a l m o n and t r o u t i n p r e p a r a t i o n f o r t r a n s p o r t a t i o n . I n t h e p r e s e n t s t u d y i t was d i f f i c u l t t o d e t e r m i n e when d e a t h t o o k p l a c e . I n t h e l a t e r s t a g e s o f a n e s t h e s i a t h e g i l l movements were s e v e r -a l m i n u t e s a p a r t , b u t u n t i l j u s t b e f o r e d e a t h p a r t i a l r e c o v e r i e s o c c u r r e d f r e q u e n t l y when g i l l movements c o u l d be r e a d i l y s e e n . C a r b o n i c a c i d i s f o r m e d when c a r b o n d i o x i d e i s a d d e d t o w a t e r t h u s a l t e r i n g t h e h y d r o g e n i o n c o n c e n t r a t i o n o f t h e w a t e r m e d i a . T h e h y d r o g e n i o n c o n c e n t r a t i o n a f f e c t s t h e o x y g e n t e n s i o n a t d e a t h o f f i s h and s u b s e q u e n t l y t h e r a t e o f a s p h y x i a t i o n , s i n c e i t l i m i t s t h e f i s h e s ' a b i l i t y t o t a k e up o x y g e n a t l o w t e n s i o n s . W i e b e , e t a l . ( 1 9 3 4 ) f o u n d t h i s t o be t h e c a s e f o r f r e s h w a t e r f i s h , a l t h o u g h g o l d f i s h were u n u s u a l l y t o l e r a n t t o c h a n g e s i n h y d r o g e n i o n c o n c e n t r a t i o n s s i n c e t h e y were a b l e t o r e d u c e t h e o x y g e n down t o .15 p . p . m . o v e r a h y d r o g e n i o n r a n g e o f 5.2 -8 . 5 and b e y o n d . P o w e r s ( 1922 ) a l s o f o u n d t h a t t h e h y d r o g e n i o n c o n c e n t r a t i o n a f f e c t e d t h e u p t a k e o f o x y g e n b y m a r i n e f i s h . A s p h y x i a t i o n s t u d i e s u s i n g a n i n e r t gas s u c h a s h e l i u m o r e v e n n i t r o g e n i n p l a c e o f c a r b o n d i o x i d e s h o u l d be a t t e m p t e d . Z e r o o x y g e n d e t e r m i n a t i o n s o b t a i n e d when t h e t i t r a -t i o n s w e r e d e l a y e d s e v e r a l h o u r s c o u l d be a t t r i b u t e d t o t h e e x t r a c o l l e c t i o n o f n i t r o g e n o u s m a t e r i a l s w h i c h a r e u n d o u b t e d l y 42 produced following death. These materials, especially n i t r i t e s , could interfere with the standard Winkler method used, and would produce lower results than those which would be obtained i f the permanganate modification were used (Allee and Qesting, 1934). / Some goldfish took as long as eight hours to die, during which time considerable ammonia and nitrogenous wastes would collect. The ammonia could aid i n the asphyxiation, since trout l i v i n g i n water containing more than .3 p.p.m. ammonia had the oxygen content of their blood reduced to one seventh and the carbon dioxide content raised to 15 per cent (Brockway, 1950). There are great v a r i a b i l i t i e s i n the oxygen tensions of comparable f i s h at death for the same carbon dioxide ten-sions (Fry et a l . 1948). Also there are individual v a r i a b i l i -ties i n the asphyxiation times of comparable goldfish from the same and different shipments receiving the same hormone and the same length of treatment (figure 10). In future, studies on the respiration effects of thyroxine should be undertaken on active f i s h metabolism and resting winter metabolism. The effects of varying concentra-tions and hydrogen ion concentrations of thyroxine solutions should also be f u l l y investigated. Further studies on the ammonia excretion of hormone treated f i s h should be attempted using f i s h i n both the active and resting states. For future 43 a s p h y x i a t i o n s t u d i e s some r e l a t i v e l y i n e r t g a s s h o u l d be u s e d i n s t e a d o f c a r b o n d i o x i d e , t h e p e r m a n g a n a t e m o d i f i c a t i o n o f t h e W i n k l e r m e t h o d f o r o x y g e n d e t e r m i n a t i o n s s h o u l d be u s e d , and many more a s p h y x i a t i o n ® s h o u l d be d o n e f o r e a c h hormone t r e a t m e n t , b u t w i t h f e w e r e x p e r i m e n t s d o n e s i m u l t a n -e o u s l y . O t h e r t e c h n i q u e s u s i n g t h e W a r b u r g a p p a r a t u s s h o u l d a l s o be u s e d t o d e t e r m i n e w h e t h e r h o r m o n e s s u e h a s t h y r o x i n e a f f e c t t h e a n a e r o b i c m e t a b o l i s m o f f i s h . 44 CONCLUSIONS 1. Treating the same group of g o l d f i s h i n Series 1 for ten and sixteen days with a 1 : 3,980,000 concentration of synthetic sodium thyroxine during the winter, elevated t h e i r oxygen consumption. Separate groups of f i s h treated f o r three, seven, fourteen and twenty-one days with the same concentra-t i o n of synthetic sodium thyroxine during the summer did not have raised oxygen consumptions or an increased ammonia exeretion. 2. The group of f i s h treated ten and sixteen days with .03 per cent thiourea during the winter had t h e i r oxygen consumption depressed. Treatments of three, seven, fourteen and twenty-one days duration with the same concentration of thiourea on separate groups of f i s h during the summer did not s i g n i f i c a n t -l y a f f e c t the oxygen consumption and ammonia excretion. 3. Summer treatments f o r three, seven, fourteen and twenty-one days with a concentration of 1 : 1,500,000 methyl testosterone increased the oxygen consumption and ammonia excretion. 4. Summer treatments with the three compounds did not a l t e r the t o t a l l i p i d and moisture content nor the gas tensions at death and the asphyxiation times. 4$ LITERATURE CITED Adkins, M. A.,"A method fo r determining the basal metabolism of fishes," Proc. Soc. E x p t l . B i o l . Med, 28 : 259-6 3 , 1930. A l l e e , W. C , and R. Oesting,"A c r i t i c a l examination of Winkler's method for determining dissolved oxygen i n r e s p i r a t i o n studies with aquatic animals," Phvsiol. Zool. 7 : 509-41, 1934. Astwood, E. B.,"Mechanisms of action of various antithyroid compounds," Ann. N. Y. Acad. S c i . 50 : 419-443, 1949. Betts, C. L.,"The gold f i s h . V a r i e t i e s , maintenance and breed-ing," Wajtex_Llfe,'' series 8, Marshall Press Ltd. London, 1949. Brockway, D. R.,"Metabolic products (of f i s h ) and t h e i r effects," Prog. F i s h C u l t u r i s t , 12 : 127 - 29 , 1950. Clausen, R. G.,"Oxygen consumption i n fresh water f i s h e s . " Ecology, 17 : 216, 1936. Dahlstrom, H. and T. Sjostrand, "Relation between the dosage of desiccated thyroid and i t s effect on oxygen consump-t i o n i n healthy individuals," Acta. Med. Scand. 132 : 353-63, 1948. Dales, S.,"Some effects of thyroxine on developing chum salmon, Oncorhynchus keta, and the comparative action of thiourea, the halides and adrenaline," Thesis, Univ. of B r i t i s h Columbia, 1953. 46 E l l i s , M. M., B. A. Westfall and M. D. E l l i s , "Determination of water quality," Fish and Wild Life Service, U. S. Dept. of the Interior Research Report 9, 1948. Escobar, A. R., "Motility factors in mass physiology; Loco-motor activity of fish under conditions of isolation, homotypic grouping and heterotypic grouping," Physiol,  Zool. 9 : 66-78, 1936. Etkin, W., R. W. Root and B. P. Mofshin, "The effect of thyroid on oxygen consumption of goldfish," Physiol. Zool. 13 : 415 - 20, 1940. Fish, F. T., "Anesthesia of fish by high carbon dioxide con-centrations," Trans. Amer. Fish. Soc. 72 : 25-29, 1942. Freeman, J. A., "Oxygen consumption, brain metabolism and respiratory movements during temperature acclimation with special reference to lowered temperature," Biol. Bull. 99 : 416 - 24, 1950. Fry, F. E. J . , E. C. Black and V. S. Black. "Influence of temperature on the asphyxiation of young goldfish Carassius auratus L. under various tensions of oxygen and carbon dioxide," Biol. Bull. 92 : 217 -24, 1948. Fry, F. E. J. and J.-S. Hart, "The relation of temperature to oxygen consumption in the goldfish," Biol. Bull. 94 : 66 - 77, 1948. Gardner, J. A., G. King and E. B. Powers, "The respiratory exchange in fresh water fish, III Goldfish," Biochem: Jour. 16 : 523 - 29, 1922. Gaunt, R., M. Cordsen and M. Lillng, "Water intoxication in relation to thyroid and adrenal function," Endocrin-ology 35 : 105, 1944. Goldsmith, E. D., "Phylogeny of the thyroid, descriptive and experimental," Ann. N. Y. Acad. Sci. 50 : 2$3-3l6, 1949. 47 G o l d s m i t h , E . D., R . F . N i g r e l l i , A . S . G o r d o n , H. A . C h a r i p p e r and M . G o r d o n , " E f f e c t o f t h i o u r e a o n f i s h d e v e l o p m e n t , " E n d o c r i n o l o g y , 35 s 132 - 3 4 , 1 9 4 4 . G o r d o n , A . S . , E . D . G o l d s m i t h and H . A . C h a r i p p e r , " E f f e c t o f t h i o u r e a o n t h e d e v e l o p m e n t o f t h e a m p h i b i a n , " N a t u r e , 152 ( 3 8 6 1 ) : 504 - 5 0 5 , 1 9 4 3 . G r a h a m , G . M . , " S o m e e f f e c t s o f t e m p e r a t u r e and o x y g e n p r e s s u r e o n t h e m e t a b o l i s m and a c t i v i t y o f S a l v e l i n u s  f o n t i n a l i s S p e c k l e d t r o u t , " C a n . J o u r n a l o f R e s e a r c h , D, 27, P P . 2 7 0 - 2 8 8 , 1 9 4 9 . G r o b s t e i n , C , and A . W. B e l l a m y , " S o m e e f f e c t s o f f e e d i n g t h y r o i d t o i m m a t u r e f i s h e s , " P r o c . S o c . E x p t l . B i o l . and M e d . 41 : 363 - 6 5 , 1 9 3 9 . H a l l , F . G . , " T h e i n f l u e n c e o f v a r y i n g o x y g e n t e n s i o n s u p o n t h e r a t e o f o x y g e n c o n s u m p t i o n i n m a r i n e f i s h e s , " Am. J o u r . P h y s i o l . 88 : 2 1 2 , 1 9 2 9 . H a s l e r , A . D . , and R. K . M e y e r , " R e s p i r a t o r y r e s p o n s e s o f n o r m a l and c a s t r a t e d g o l d f i s h t o t e l e o s t and mammal ian h o r m o n e s , " J o u r . E x p t l . Z o o l , 91 : 3 9 1 - 4 0 4 , 1 9 4 2 . H a t l e y , J . , " A c t i o n de l a t h i o u r e e s u r l a m e t a b o l i s m e g l u c i d i q u e de l a c a r p e ( C r y p i n u s C a r p o L ) , " C o m p t . R e n d . S o c . B i o l . P a r i s , 1 4 4 , ( 1 3 - 1 4 ) : 955-57, 1950. H a u g a r d , N . , and L . I r v i n g , " T h e i n f l u e n c e o f t e m p e r a t u r e u p o n t h e o x y g e n c o n s u m p t i o n o f t h e c u n n e r ( T a u t o g l a b r u s a d s p e r s u s Walbaum) i n summer and w i n t e r , J o u r . C e l l . C o m p . P h y s i o l . 21 : 1 9 - 2 6 , 1 9 4 3 . Hawk, P . B . , B . L . O s e r and ¥ . H . S u m m e r s o n , P r a c t i c a l P h y s i o -l o g i c a l C h e m i s t r y , T h e B l a k i s t o n C o . , P h i l a d e l p h i a , 1 9 4 8 . H o a r , W. S . , "Hormones i n f i s h , " U n i v . T o r o n t o . S t u d . B i o l . , 59, P u b . O n t . F i s h . R e s . L a b . 71 : 1 - 51, 1951. 48 H o a r , ¥ . S . , and M . K . C o t t l e , " S o m e e f f e c t o f t e m p e r a t u r e a c c l i m a t i z a t i o n o n t h e c h e m i c a l c o n s t i t u t i o n o f g o l d -f i s h t i s s u e s , " C a n . J o u r . Z o o l . 30 : 4 9 - 5 4 , 1 9 5 2 . H o a r , ¥ . S . , D . M a c K i n n o n and A . B. R e d l i c h , " E f f e c t s o f some h o r m o n e s o n t h e b e h a v i o r o f s a l m o n f r y , " C a n . J o u r . Z o o l . 3 0 t 273 - 2 8 6 , 1 9 5 2 . H u g h e s , A . M., and E. B . A s two o d , ' T h y r o i d f u n c t i o n and r e s i s t a n c e t o a n o x i a i n the r a t " J o u r . A v i a t i o n Med. 1 5 : 1 5 2 -5 7 , 1 9 4 4 . H u x l e y , J . S . , ' T h y r o i d i n c o l d - b l o o d e d v e r t e b r a t e s , " N a t u r e , 1 2 3 : 7 1 2 , 1 9 2 9 . K e y s , A . B . , " T h e m e a s u r e m e n t o f t h e r e s p i r a t o r y e x c h a n g e o f a q u a t i c a n i m a l s " B i o l . B u l l . 59 s 1 8 7 - 9 8 , 1 9 3 0 . L e b l o n d , C . P., " I n c r e a s e d r e s i s t a n c e t o a n o x i a a f t e r t h y r o i d -e c t o m y and a f t e r t r e a t m e n t w i t h t h i o u r e a , " P r o c . S o c . E x p t l . B i o l , and M e d . 55 : 1 1 4 - 1 1 6 , 1 9 4 4 . L e v e r , J . , J . M i l t e n b u r g and G. J . von O o r d t , " E f f e c t o f a s h o r t t r e a t m e n t w i t h t h i o u r e a o n t h e f i s h t h y r o i d g l a n d , ' P r o c . K o n i n k l . N e d e r l a n d . A k a d . W e t e n s c h a p . 52 : 296 -3 0 0 , 1 9 4 9 . Mann, H . , " T h e e f f e c t o f c o l c h i c i n e and s e x h o r m o n e s o n t h e o x y g e n c o n s u m p t i o n o f f i s h , " Z o o l . Ariz. 127 * 3 1 5 - 1 8 , 1939* M a r z u l l i , F . N . , " E f f e c t s o f h y d r o g e n - i o n c o n c e n t r a t i o n on t h e m e t a m o r p h i c p a t t e r n o f t h y r o x i n * and i o d i n e t r e a t e d t a d p o l e s " J o u r . G e n . P h y s i o l . 25 : 6 2 3 - 4 7 , 1 9 4 2 . M a n s f e l d , G., T h e t h y r o i d h o r m o n e s and t h e i r a c t i o n , F r e d e r i c k M u l l e r , L o n d o n , 1 9 4 9 . M a t t h e w s , S . A . , and D . C . S m i t h , " T h e e f f e c t s o f t h i o u r e a o n t h e o x y g e n c o n s u m p t i o n o f F u n d u l u s " P h y s i o l . Z o o l . 20 s 161 - 6 4 , 1 9 4 7 . 49 M e a n s , J . H . , T h e f u n c t i o n o f t h e t h y r o i d g l a n d , C . C . T h o m a s , S p r i n g f i e l d , 1 1 1 . , 1 9 4 9 . O s b o r n , P . E . , " T h i o u r a d l as a n a i d i n h o l d i n g and t r a n s p o r t i n g f i s h , " P r o g . F i s h C u l t u r i s t 13 s No. 2 : 75 - 78, 1951. P i n c u s , G . , and K. V . T h i m a n n , T h e H o r m o n e s , v o l . 2, A c a d e m i c P r e s s I n c . , New Y o r k , 1950. P o w e r s , E . B . , " T h e p h y s i o l o g y o f r e s p i r a t i o n o f f i s h e s i n r e l a t i o n t o h y d r o g e n ion c o n c e n t r a t i o n o f t h e m e d i u m , " J o u r . G e n . P h y s i o l . 4 : 305, 1922. P r o s s e r , C . L . , e t a l . , C o m p a r a t i v e a n i m a l p h y s i o l o g y , W. B . S a u n d e r s C o m p a n y , P h i l a d e l p h i a , L o n d o n , 1950. R e d l i c h , A . B . , " i n f l u e n c e o f t h y r o i d hormones o n t h e n i t r o g e n e x c r e t i o n and t h e r e s i s t a n c e o f g o l d f i s h , C a r a s s i u s a u r a t u s , t o a n o x i a | T h e s i s , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1951. R o b e r t s o n , 0 . H . , " T h e o c c u r r e n c e o f i n c r e a s e d a c t i v i t y o f t h e t h y r o i d g l a n d i n r a i n b o w t r o u t a t t h e t i m e o f t r a n s -f o r m a t i o n f r o m p a r r t o s i l v e r y s m o l t , " P h y s i o l . Z o o l . , 21 : 282-295, 1949. R u p p , Y., K . E . P a s c h k i s , and A . K a n t a r o w , " T h e I n f l u e n c e o f T h y r o x i n o n P r o t e i n m e t a b o l i s m , " E n d o c r i n o l o _ g y , 44*. 449, 1 9 4 9 . * S a k s , M . G . , and M. A . Zamkwa, " E f f e c t o f t h i o u r e a o n gas m e t a b o l i s m o f l a r v a e s a l m o n and s t u r g e o n , " D o k l a d y A k a d . N a u k , S . S . S . R . , 84 •. 1101 - 3 , 1952. S c h f c t t , F . , " S t u d i e s i n mass p h y s i o l o g y . T h e e f f e c t s o f n u m b e r s o n t h e o x y g e n c o n s u m p t i o n o f f i s h e s E c o l o g y 14 : 106 - 22 , 1933• S c h l a i f e r , A . , " A n a l y s i s o f t h e e f f e c t o f numbers upon t h e o x y g e n c o n s u m p t i o n o f C a r a s s i u s a u r a t u s , " P h y s i o l . Z o o l . 12 : 381 - 92, 1939. 50 S m i t h , H . ¥ . , " T h e e x c r e t i o n o f ammonia and u r e a b y t h e g i l l s o f f i s h , " J . B i o l . C h e m . , 81 : 727 - 4 2 , 1929. S m i t h , D . C , and G . M . E v e r e t t , " T h e e f f e c t o f t h y r o i d hormone o n g r o w t h r a t e , t i m e o f s e x u a l d i f f e r e n t i a t i o n and o x y g e n c o n s u m p t i o n i n t h e f i s h - L e b i s t e s r e t i c u l a t u s , J o u r . E x p t l . Z o o l . , 94 : 229 - 4 0 , 1 9 4 3 . S m i t h , D . C , and S . A . M a t t h e w s , " P a r r o t f i s h t h y r o i d e x t r a c t and i t s e f f e c t s u p o n t h e o x y g e n c o n s u m p t i o n i n t h e f i s h B a t h y s t o m a , Am. J o u r . P h y s i o l . 153 * 215-25, 1 9 4 8 . S p o o r , W. A . , " A q u a n t i t a t i v e s t u d y o f t h e r e l a t i o n b e t w e e n t h e a c t i v i t y and o x y g e n c o n s u m p t i o n o f g o l d f i s h and i t s a p p l i c a t i o n t o t h e m e a s u r e m e n t o f r e s p i r a t o r y m e t a b o l i s m i n f i s h e s , " B i o l . B u l l . 91 : 312 - 25 , 1946. T u r n e r , C . D . , G e n e r a l E n d o c r i n o l o g y , W. B . S a u n d e r s C o . , P h i l a d e l p h i a , 1 9 4 8 . V i v i e n , J . , and. M . L . G a i s e r , " A c t i o n o f p r o l o n g e d t r e a t m e n t w i t h t h i o u r e a o n t h e s t r u c t u r e and f u n c t i o n o f t h e t h y r o i d and p i t u i t a r y o f L e b i s t e s r e t i c u l a t u s . M o d i f i c a t i o n o f t h e s t a g e s o f g r o w t h i n h e i g h t and g e n i t a l d i f f e r e n t i a t i o n , " "Compt. R e n d . S o c . B i o l . P a r i s , 234 s 1643 - 5, 1952. W e l c h , P . S . , L i m n o l o g i c a l M e t h o d s , T h e B l a k i s t o n C o . , P h i l -a d e l p h i a , 1 9 4 8 . W e l l s , M. M . , " T h e r e a c t i o n s and r e s i s t a n c e o f f i s h e s t o c a r b o n d i o x i d e and c a r b o n m o n o x i d e , " B u l l . , I l l i n o i s  S t a t e L a b . N a t . H i s t . 11 : 557 - 73, 1 9 1 8 . W e l l s , N . A . , " V a r i a t i o n s i n r e s p i r a t o r y m e t a b o l i s m o f P a c i f i c k i l l i f i s h , F u n d u l u s p a r v i p l n n i s due t o s i z e , s e a s o n and c o n t i n u e d c o n s t a n t t e m p e r a t u r e , " P h y s i o l . Z o o l . 8 : 318, 1935. 51 W e l l s , N . A . , " T h e i m p o r t a n c e o f t h e t i m e e l e m e n t i n t h e d e t e r m i n a t i o n o f t h e r e s p i r a t o r y m e t a b o l i s m o f ' f i s h e s , " P r o c . N a t l . A c a d . S c i . , W a s h . , 1 8 : 5 8 0 - 5 8 5 , 1 9 3 2 . -W i e b e , A . H . , A . M . M c G a v i c k , A . C . F u l l e r and H . C . M a r k u s , " T h e a b i l i t y o f f r e s h w a t e r f i s h t o e x t r a c t o x y g e n a t d i f f e r e n t h y d r o g e n i o n c o n c e n t r a t i o n s , " P h y s i o l . Z o o l . 7 s 435 - 48, 1934. W i l d e r , J . , "A c o r r e l a t i o n o f r e s u l t s o n o x y g e n c o n s u m p t i o n o b t a i n e d b y t h e W i n k l e r m e t h o d and b y r e s p i r o m e t e r s u s i n g a s a s t a n d a r d t h e m e t h o d s o f V a n S l y k e , " P h y s i o l . Z o o l . , 1 0 : 4 6 5 - 7 2 , 1 9 3 7 -o * O r i g i n a l p a p e r s n o t c o n s u l t e d . APPENDIX S T A T I S T I C S ON S E R I E S 2 AND 3 OXYGEN CONSUMPTION DATA D a t e J u l y 7, 1952 ?°* 0 ^ c t f -M T h y r o x i n T h i o u r e a T e s t o s t e r o n e T h y r o x i n T h y r o x i n T h i o u r e a d a y s b t a t i s t i c c o n t r o l c o n t r o l c o n t r o l T h i o u r e a T e s t o s t e r o n e T e s t o s t e r o n e t r e a t -ment J u l y 31 , 1952 J u n e 3 0 , 1952 A u g . 1952 3 X Mean .112 .125 .108 .125 . 1 3 4 .125 .112 .108 .112 .134 .108 . 1 3 4 * t 1 . 0442 1.0976 .60811 .42998 2.6683 2.0569 P r o b a -b i l i t y .5 - .1 .5 - .1 .5 - .1 < . 5 .05 - .02 .1 - .05 3 Mean .116 .111 .130 .111 . 1 4 9 .111 .116 .130 .116 . 1 4 9 .130 . 1 4 9 t .3786 1.5397 1.8423 1.31528 1.6780 1.0182 P r o b a -b i l i t y <-5 .5 +-.1 .1 .5 - . 1 .5 - .1 7 Mean .102 .087 .119 .087 .125 .087 .102 .119 .102 .125 .119 .125 t 1 . 3 6 7 8 11.0407 3.0079 1 . 6340 1.6838 .4568 P r o b a -b i l i t y .5 - .1 >.01 > .02 .5 - . 1 .5 - .1 7 Mean .129 . 1 3 4 .119 . 1 3 4 . 1 4 4 . 1 3 4 .129 .119 .129 . 1 4 4 .119 . 1 4 4 t 1.3927 1.00568 .7077 1 . 0 9 4 4 2.82309 P r o b a -b i l i t y <-5 .5 - .1 .5 - .1 - < . 5 . 5 - . 1 .05 - .02 X Mean number o f c c . o f o x y g e n consumed p e r gram o f f i s h d u r i n g 24 h o u r s * 8 d e g r e e s o f f r e e d o m . c o n t i n u e d n e x t p a g e S T A T I S T I C S O H SERIES 2 and 3 OXYGEN C O N S U M P T I O N DATA c o n t i n u e d ETo. o f T h y r o x i n T h i o u r e a T e s t o s t e r o n e T h y r o x i n T h y r o x i n T h i o u r e a D a t e d a y s S t a t i s t i c c o n t r o l c o n t r o l c o n t r o l T h i o u r e a T e s t o s t e r o n e T e s t o s t e r o n e t r e a t -ment J u l y 8 14 M e a n .093 .096 .125 .096 .116 .096 .093 .125 .093 .116 .125 .116 1952 t .3758 3.9847 2.0699 3.4297 2.0535 .90403 P r o b a -b i l i t y < . 5 > . 0 1 .1 - . 0 5 >.01 .1 - . 0 5 > . 5 A u g . 11 1952 14 M e a n .117 .139 .126 .139 .135 .139 .117 .126 .117 .135 .126 .135 t 2.09006 1.2946 .53819 .8857 2.2359 1 . 2049 P r o b a -b i l i t y .1 - .05 .5 - . 1 .5 .5 1- .05 .5 - .1 J u l y 15 1952 21 M e a n .089 . 0 9 4 .110 .094 .133 .094 .089 .110 .089 .133 .110 .133 t . .7666 3 - 5 8 4 7 6.8817 3.4913 6.3289 4 . 1 8 1 2 P r o b a -b i l i t y > . 5 y .01 > . Q 1 > . 0 1 > .01 >.01 A u g . 18 1952 21 Mean .116 .122 .145 .122 .160 .122 .116 . 1 4 5 .116 .160 . 1 4 5 .160 t .63032 3.5272 4.1726 2.7324 3.5656 1.5012 P r o b a -b i l i t y < .5 >.01 > .01 . 0 5 - . 0 2 y .01 9 ^) ™ • 1L c o n t i n u e d n e x t p a g e APPENDIX S T A T I S T I C S ON TOTAL OXYGEN CONSUMPTION DATA - c o n t . N o . o f T h y r o x i n T h i o u r e a T e s t o s t e r o n e T h y r o x i n T h y r o x i n T h i o u r e a D a t e d a y s S t a t i s t i c c o n t r o l c o n t r o l c o n t r o l T h i o u r e a T e s t o s t e r o n e T e s t o s t e r o n e t r e a t -ment Mean .109 .114- .123 . 1 1 4 .137 . 1 1 4 .109 .123 .109 .137 .123 .137 * t .§948 1.9808 3 . 4 2 0 1 3.2547 5.7190 3.1015 P r o b a -b i l i t y .> .5 . 1 - .05 > .01 > . 0 1 ^ . 0 1 ^ . 0 1 * 38 d e g r e e s o f f r e e d o m . 

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