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The change in the degree of unsaturation of body fats during acclimation of goldfish (Carassius auratus)… Hunter, John Gerald 1948

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3 7  /?*-8- ftp Cop - / .  The Change I n The Degree  Of U n s a t u r a t i o n  Of Body F a t s D u r i n g A c c l i m a t i o n 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 ) To H i g h Temperature,  by John G e r a l d  A Thesis  Hunter  submitted i n P a r t i a l  Fulfillment  o f t h e R e q u i r e m e n t s f o r t h e Degree o f  ' MASTER in  OF ARTS  t h e Department of  Zoology  The U n i v e r s i t y o f B r i t i s h  April,  1948  Columbia  The  C h e m i c a l Change I n The  Unsaturation  Of Body F a t s  A c c l i m a t i o n Of G o l d f i s h auratus)  To H i g h  Degree  Of  During  (Carassius  Temperature.  by  J.G.  Hunter  Abstract An  a t t e m p t has  metabolic  changes o c c u r i n g  (Carassius auratus) acclimated to  b e e n made t o  to temperature.  temperatures.  o f t h e i r b o d y f a t s was determinations.  general  The  The  the  then  fully  have been f o l l o w e d .  i n temperature there unsaturation.  unsaturation  I t has  value  acclimated  iodine values.  f a t s a r e more s a t u r a t e d a t h i g h e r amount o f u n s a t u r a t i o n  were  reacclimated  found u s i n g Wij's i o d i n e  f a t s of g o l d f i s h  goldfish  goldfish  change i n t h e  t e m p e r a t u r e s show d i f f e r e n t  Changes i n t h e process  The  the  during.acclimation of  t o c e r t a i n t e m p e r a t u r e s and  different  different  f o l l o w some o f  during  In  temperatures. the  acclimation  been found upon  i s a decrease i n the  to  amount  of  increase  2.  Table o f  Introduction Materials Chemical  Contents  .  Page  and M e t h o d s . . . . Reactions  3 8  and E q u a t i o n s  14  Reaction o f Wij's S o l u t i o n . . . .  14  R e a c t i o n o f IC1 w i t h u n s a t u r a t e d o l e i c Standardization  acid..  14  o f sodium thiosulphate.-...... •  15  C a l c u l a t i o n o f the i o d i n e value Results.......  15 18  Discussion.....  •  28  C o n c l u s i o n s and Summary  32  Acknowledgements  33  Appendix  34  References...  ........•..*.............•...»*.•.•  46  The Change In  The  Degree of Unsaturation  Of Body Fats During Acclimation of Goldfish (Carassius auratus)  to High  Temperature  Introduction  The melting point of f a t s i n poikilothermic animals and higher plants varies with the temperature of deposition, (Terroine, et a l . 1930}  Heilbrunn, 1943).  Fat formed at a higher  temperature i s found to be more s o l i d than f a t formed at a lower temperature.  Since the melting point of a f a t depends l a r g e l y on  i t s degree of unsaturation, the iodine value of a f a t as a measure of double bonds i n the f a t t y acid chains i s a good measure f o r i t s melting point.  I t has been suggested that the e s s e n t i a l  feature of temperature acclimation i s a change i n the melting point of the f a t s but no one seems to have attempted to follow these changes during acclimation.  This experiment i s so designed  to show that during and a f t e r acclimation there i s a change i n the number of unsaturated  double bonds i n the o i l s of the g o l d f i s h  (Carassius auratus) with the higher temperatures producing smaller number of unsaturated  a  bonds.  Examples of temperature adaption are found i n the work of Loeb and Wastenays (1912), Hathaway (1927), Wells (1935), Sumner and Doudoroff (1938), Fry, Brett and Clawson (1942), and  Brett (1946), a l l r e l a t i n g to f i s h .  These works, however, make  no reference to the metabolic changes involved i n the process of acclimation.  These seem to be very imperfectly understood.  No one has attempted to follow the chemical changes which occur during temperature acclimation and the resultant changes a f t e r temperature acclimation has been completed. Acclimation i s the a b i l i t y of an organism to make some adjustment to enable i t to meet unfavorable conditions of i t s physical and chemical environment (Heilbrunn, 1943)* Various theories have been put f o r t h i n an e f f o r t to explain temperature acclimation.  Davenport (1897), suggested that increase  in  tolerance might be caused by a lowering of the water content of protoplasm, with a consequent r a i s i n g of the temperature necessary to cause coagulation.  Loeb and Wastenays (1912) compare  acclimation with the annealing of g l a s s . the work of Miss Behre (1918) who  Hathaway (1927) c i t e s  f e e l s that acclimation i s an  adjustment i n metabolic rate but that continued  exposure to high  or low temperature causes a gradual return to the o r i g i n a l s t a t e . Heilbrunn  (1924-), explains the temperature c o e f f i c i e n t of the  process of coagulation of protoplasm i s of the same order of magnitude as that which i s found i n the heat coagulation of proteins.  He also states that heat coagulation of protoplasm  d i f f e r s from heat coagulation of proteins i n being r e v e r s i b l e .  Further, acclimation i s apparently associated with some change i n the state of f a t t y materials of the c e l l (Heilbrunn, 1943).  Such  a change i s indicated by v i s i b l e differences i n the f a t s of heat treated protoplasm. The l i t e r a t u r e on the nature of f a t s i n r e l a t i o n to the environmental temperature of the poikilothermic animals and plants i s rather extensive. Terroine, Batterer, and Roehrig (1930) shoved that the nature of the reserve f a t s , body f a t s and phosphatides was correlated with the temperature at which the animal or plant was living.  B u l l (1937) states that animals attempt to store the high-  est melting point f a t that can safely be stored.  Fraenkel and Hoff  (1940) show i n the breeding of two c l o s e l y a l l i e d species of f r u i t f l i e s at d i f f e r e n t temperatures that the degree of unsaturation of the phosphatides was d i r e c t l y dependent upon the temperature of breeding.  Pearson and Raper (1927) found s i m i l a r r e s u l t s i n t h e i r  work on the t o t a l f a t of two species of fungus.  They showed the  mean iodine value of Aspergillus niger to be 149 i f bred at 18^C, 129 at 25°G, and 95 i f the culture had been kept at 35°C.  In  Rhizopus nigricans the corresponding figures were 88 f o r 12°C and 75 f o r 2 5 ° C  Dean and H i l d i t c h (1933) working on sow f a t found  that i t becomes more saturated with a r i s e i n temperature, with a close r e l a t i o n e x i s t i n g between the saturation change and the temperature change.  Brockelsby and Bailey (1932) reported that the o i l  of each species of salmon taken from more Northern l o c a l i t i e s showed greater unsaturation than salmon taken i n more Southern l o c a l i t i e s .  This might, of course, be related to the u t i l i z a t i o n of the o i l s i n the spawning migration. Lovern (1938) found i n h i s experiments with eels (Anguilla v u l g a r i s ) that lower temperatures lead to more unsaturated f a t s while higher temperatures lead to increased saturat i o n of the f a t s .  Temperature,  i t appears, plays i t s r o l e i n  a f f e c t i n g the degree of unsaturation i n the animal f a t s . Diet as pointed out by Lovern (1938) when providing a source of f a t may or may not change the type of f a t being deposited. An animal usually deposits f a t only when the food eaten exceeds the amount u t i l i z e d by the body "for energy requirements.  The nature of  the deposited f a t s depends to a c e r t a i n extent upon the nature of the food eaten.  By changing the d i e t of an animal the nature of  the deposited f a t s can be changed.  Corn fed hogs, f o r example,  produce soft l a r d s due t o the high o i l centent of the d i e t . S i m i l a r l y c a t t l e fed on oil-cake d i e t y i e l d softer tallows than those fed on a grass (largely carbohydrate) d i e t (Brocklesby, 1941). When the energy content of the d i e t i s i n excess of that required by the animal and the d i e t also contains an excess of f a t , the f a t deposited i n the body w i l l tend to resemble that ingested. the other hand, an active animal fed on a balanced diet w i l l  On produce  a f a t t y p i c a l of the species i r r e s p e c t i v e of the nature of the f a t ingested.  Guha, H i l d i t c h , and Lovern (1930) report the unsaturated  f a t t y acids of the CgQ and Cg£ series were known at one time as c h a r a c t e r i s t i c only of f i s h e s and marine mammals, but now have been found i n algae.  7. The proportion found i n algae i s much lower than i n f i s h l i v e r o i l s . I t was  reported by G r i i n and Halden (1929) that the f a t s of b i r d s  whose d i e t i s composed of f i s h also have these f a t s present.  Lovern  (1938) studied the r e l a t i o n s between d i e t a r y and body f a t s i n eels (Anguilla  ^ulg,aiasj.  He found that d i e t s low i n f a t content had  no  appreciable e f f e c t on the f a t deposited by eels but d i e t r i c h i n f a t content, although l a i d down with the r e l a t i v e proportions  of  the acids i n the various carbon series unchanged, altered the degree of unsaturation.  The action of t h i s mechanism i n the eel  i s accelerated by a r i s e i n temperature.  The f a c t that d i e t i s a  determining f a c t o r i n the degree of unsaturation i s i l l u s t r a t e d i n the feeding experiments of Y u i l l and Craig (1937) on the larvae of L u c i l i a s e r i c a t a . Reared on d i f f e r e n t d i e t s they showed a difference i n iodine value of t h e i r f a t s according to the food offered them. Temperature a f f e c t s the rate of metabolism (Wells 1935). The rate of metabolism i n turn a f f e c t s the rate at which f i s h become sexually mature.  Lovern (1934) has shown i n salmon (Salmo  salar) that there i s a high degree of s e l e c t i v i t y of o i l s from the depot f a t i n developing  I  The  work o f  Lovern,  ovaries.  t h e s e men  1930.  is cited  by  Guha, H i l d i t c h  and  8.  Materials and Methods  Goldfish (Carassius auratus) were procured from two d i f f e r e n t sources.  One hundred and ten (110) were obtained from  a breeder i n S t o u f f v i l l e , Ontario, while another eighty f i v e  (85)  were bought from a l o c a l breeder on Lulu Island, near Vancouver, B r i t i s h Columbia.  In both cases these f i s h had been reared i n  outdoor ponds subject to c l i m a t i c changes of temperature and l i v e d on food caught i n the ponds. The experiment was carried out i n three sections. Section I the goldfish from Lulu Island were used.  For  Although kept  i n an outdoor pond, these f i s h had experienced a f a i r l y constant temperature of about 12°C f o r the previous two weeks.  Within two  days of t h e i r a r r i v a l they were divided i n t o three groups and placed i n aquaria at temperatures of 12°C, 20°C and 33°C.  They  were given time enough t o acclimate to these temperatures (Brett, 194-6) before iodine value determinations were made.  In a l l experi-  ments the work of Brett (194-6) was used as a basis f o r determining the time required f o r acclimation of the g o l d f i s h . Section I I was comprised of f o r t y f i v e g o l d f i s h from Ontario which had been acclimated to 12°C.  They, too, were grouped  i n three aquaria with temperatures of 12°C, 25°C, and 33°C, and given time to acclimate to these temperatures before determinations were conducted.  9 In Section I I I s i x t y of the Ontario bred g o l d f i s h were kept a t 5°C f o r sixteen days. to that temperature  In t h i s way they became acclimated  (Brett, 1946). When acclimation was complete  ( i . e . i n sixteen days) they were divided i n t o three groups and placed i n aquaria with temperatures of 5°C, 15°C, and 25°C. Twelve iodine value determinations were made a t i n t e r v a l s during the acclimation process.  The same number of g o l d f i s h were taken f o r  iodine value determination from each temperature grouping a t each of the time i n t e r v a l s (see tables i n Appendix). The oxygen supply i n a l l the aquaria was maintained a t saturation l e v e l by f o r c i n g compressed a i r through porous b a l l s placed i n each aquarium.  Determinations of the amount of oxygen  present were made from time t o time by the Winkler method. The g o l d f i s h i n a l l experiments were fed one h a l f gram per f i s h of commercially prepared g o l d f i s h food twice a week during t h e i r time i n the laboratory. with each group of f i s h .  The consumption of t h i s food varied  No accurate quantitative measure of the  amount used was possible. The sampling method used i n setting up the d i f f e r e n t i  aquaria i n each section and i n taking samples f o r f a t analyses was a simple one.  The g o l d f i s h nearest t o the f a r l e f t hand corner of  the aquarium was taken.  In t h i s way a random d i s t r i b u t i o n of the  g o l d f i s h was insured. There was no s e l e c t i v i t y of sampling as shown on examination of the r e s u l t s .  10  The tanks used i n t h i s experiment consisted of one outside concrete tank (2* x 3* x 12').  This tank was aeriated by a stream of  splashing water from the laboratory's plumbing system.  I t provided  a body of water large enough to withstand the d a i l y fluctuations i n temperature and was used only f o r retaining f i s h p r i o r to experimentation.  A refrigerated tank (4  f  x 2' x 3') with a sensitive thermo  regulator was available f o r low temperature work.  There were a l s o a  series of glass aquaria (18" x 12" x 12") with "Lo-Lag Immersion" heaters and "Fenwal Bimetal" thermo regulators.  In one instance a  glass aquarium was kept i n a household r e f r i g e r a t o r t o maintain a 5°C temperature.  In no case d i d the temperature vary more than 2°C.  A l l aquaria, with the exception of the previously mentioned concrete tank, were kept saturated with oxygen by means of compressed a i r . In a resume of the properties of unsaturated o i l s and f a t t y acids Bailey (Brockelsby, 1941) states: "Unsaturated f a t t y acids contain one or more double bonds, so that they and the o i l s containing them have the property of taking up various other substances by chemical addition.  Among the substances which can take  place i n such addition reactions are oxygen, hydrogen (under the influence of a c a t a l y s t ) , and the halogens, — c h l o r i n e , bromine and iodine.  The addition of oxygen enters i n t o both oxidative r a n c i d i t y  and the formation of paint f i l m s * the addition of hydrogen produces a s o l i d f a t from a l i q u i d o i l j the addition of the halogens i s used mainly i n the quantitative measurement of unsaturation.  The u t i l i t y  of halogen addition i n the measurement of unsaturation l i e s i n the  11.  f a c t s that, by careful control of conditions the reaction can be brought quantitatively to completion,and  that the excess of unused  halogen can be r e a d i l y measured." The iodine value determined by the W i j s method 1  (Brockelsby, 1941) was used as a measure of the degree of unsaturation of the g o l d f i s h o i l s .  I t s determination i s based on the reaction of  an unstable compound of two halogens  (IC1) with the unsaturated  f a t t y acids, whereby the unstable IC1 breaks up with i t s twp components adding on instantaneously a t the double bonds of the unsaturated m a t e r i a l . In t h i s procedure an excess of halogenating reagent was added to a small amount of the o i l dissolved i n a suitable solvent such as carbon t e t r a c h l o r i d e .  A f t e r allowing the  mixture to stand i n the dark u n t i l the reaction i s complete, the amount of unabsorbed halogen reagent i s determined by t i t r a t i o n . The iodine value i s the percentage of iodine with which the material under t e s t i s capable of combining under the prescribed conditions. The g o l d f i s h , a f t e r length, weight, colour, and sex had been recorded, were cut i n t o small pieces with a pair of s c i s s o r s . One and a h a l f times as much by weight of anhydrous sodium sulphate was added to the cut pieces of goldfish and the two were ground together i n a mortar and p e s t l e .  The anhydrous sodium sulphate  takes up much of the water from the goldfish t i s s u e .  The mixture  of ground g o l d f i s h and sodium sulphate was then placed i n a porous f i l t e r thimble and set up i n a soxhlet extractor with peroxide-free  '  Were  idtlcd  ethyl ether. ?ud Two drops of 1% a l c o h o l i c hydroquinoneHo prevent  oxidation of f a t t y acids.  Tests f o r peroxide i n the ether were  carried out r e g u l a r l y . The o i l and other ether soluble products were then extracted f o r s i x hours i n a water bath at A2°C.  The f a c t that ether  soluble constituents other than o i l s were extracted means that the iodine values reported i n t h i s experiment are f o r t o t a l ether soluble and not only the f a t t y a c i d s .  Hence s i m i l a r iodine values  do not necessarily mean that the same f a t t y acids are present but give r e l a t i v e measurements of the nature of the f a t s as f a r as saturation goes.  The b o i l i n g point of ethyl ether i s 34.5°C and  the temperature of the extraction process would not exceed t h i s even i n the 42°C water bath. •42°C polymerize.  Unsaturated o i l s above the temperature of  At the end of s i x hours the ether was a l l but  d i s t i l l e d o f f the o i l products. remain a t 34.5°C.  In t h i s way the temperature would  The remaining ether was separated from the o i l s  by a vacuum dessicator.  This treatment also eliminated any water  which may have escaped the anhydrous sodium sulphate. The o i l was then weighed i n t o 2 ml. v i a l s i n o.lOOO gram aliquots.  The v i a l containing the o i l was c a r e f u l l y placed i n a 250  ml. iodine f l a s k and 25 ml. of the neutral solvent carbon t e t r a chloride added.  This was followed by 25 ml. of Wlj's s o l u t i o n .  The  Wij's solution was prepared by dissolving 12 grams of C. P. d i c h l o r a mine T i n 700 ml. of C. P. g l a c i a l a c e t i c a c i d .  Sixteen decimal s i x  (16.6) grams of C. P. potassium iodide were then added.  When a l l was  dissolved, the solution was made up t o 1000 ml. with a c e t i c a c i d .  13 The Wij's solution needed i n t h i s experiment was a l l prepared at the one time and stored i n amber coloured bottles which were kept i n a dark cupboard.  There has been considerable controversy over the  s t a b i l i t y of Wij's solution but Child (1945) reports that Wij's solution kept at 81°F f o r ten months showed no change i n the iodine values obtained* A f t e r the addition of the W i j s solution to the f l a s k and 1  the stopper of the f l a s k has been moistened with 15$ potassium iodide solution to prevent evaporation of the halogenating agent, i t i s stored i n a dark cupboard f o r t h i r t y minutes to allow complete addition t o take place. products are formed.  In the presence of l i g h t substitution  At the end of t h i r t y minutes 20 ml. of 15%  potassium iodide and 100 ml. of d i s t i l l e d water were added.  This  solution i s now t i t r a t e d against a tenth normal solution of sodium thiosulphate.  The sodium thiosulphate solution has been standardized  against a c a r e f u l l y prepared tenth normal solution of potassium dichromate.  A few drops of a stable starch solution are added when  the solution turns a straw yellow colour.  This causes the solution  to turn blue (reacts with the remaining iodine) and a very sensitive end point can be reached. A similar t i t r a t i o n without the g o l d f i s h o i l present i s carried out with each determination.  This i s c a l l e d a blank t i t r a t i o n .  Usually two blanks are done, one before and one a f t e r the o i l t i t r a tion.  The c o e f f i c i e n t of expansion f o r the sodium thiosulphate i s  great enough to cause an appreciable difference i n the volume  14 required f o r only a s l i g h t change i n temperature.  The difference of  the volumes of sodium thiosulphate required by the o i l t i t r a t i o n and the average of the blank t i t r a t i o n s gives the amount of iodine absorbed by the o i l . CHEMICAL REACTIONS & EQUATIONS i Reaction of the Wi.j's Solution: CH3COOH -r KI  >  CH C00K  CHj 2HI  2  HI  CH^  ' + j^j) S0 NC1  +  3  >  S0 NH  2  +  j^jj 2  2IC1  2  Reaction of I C 1 vrlth unsaturated o l e i c acid (found i n a l l f i s h o i l s )  CH3(CH )TCH 2  =  CHtCH^TCOOH  +•  IC1—>  I CI I \ CH3(CH ) CH-CH(CH ) C00H 2  7  2  7  The end product i s monoiodomonochloroleic a c i d , formed by the addition of iodine quantitatively with the o l e i c a c i d . The addition of the 2 0 ml. of 15$ potassium iodide t o the solutions a f t e r coming from the dark cupboard i s t o complete the formation of I C 1 from the dichloramine T.  Any excess of potassium  iodide i s not important since the hydrogen iodide (Hi) formed from i t s action of a c e t i c acid i s not t i t r a t a b l e .  15. In these experiments duplicate o i l extractions were carried out on the same f i s h as well as duplicate t i t r a t i o n s being made on the same o i l s . Standardization of the sOdlum thiosulphate:  K Cr 0 2  2  7  + 6 KI  3I  2  + 14HC1  > 8KC1  > 6NaI  + 6Na S 03 2  2  +• 2CrCl3  + 3I  + 7H 0  2  2  +• 3Na S^0£, 2  When the standardization i s complete the Na^S^O^ i s colourless. A stable starch solution was used as an i n d i c a t o r . The Wij's solution when t i t r a t e d with sodium thiosulphate gave the same end product.  3IC1  +  6Na S 0 2  2  3  *  3NaI  + 3NaCl  + 3Na S^05 2  Calculation of the iodine value: In the standardization of the sodium thiosulphate i t was shown that one molecular weight of potassium dichromate i s equivalent to six molecular weights of sodium thiosulphate.  16.  294.22 gram3 of K C r 0 y 2  -  2  6 X 158.12 grams of Na-jSgO^ A ml. of Na S 03  25 ml. of tenth normal (1/60 molar) K Cr 0<7 ° 2  Then A ml. of N a ^ O ^  =  2  2  25 ml. of 0.1 N K C r 0 2  2  2  7  25 1000  X  _1 60  -  25  X  294.22  grams o f . K C r o 0  Z  25 IS00  X  294.22 60  X 6 x 158.12 grams of 294.22 Na S2°3  =  mols. of K Cg 0 2  ?  2  7  2  (The molecular weight of iodine i s 126.93 grams)  . . 1 ml. of Na S 0 * * d  =  25 X 6 X 158.12 X 126.93 grams of I 1000 60 158.12  =  25 X 1000  = 25 1000  12.693  grams of I  12.693 A  grams of I  X  2  2  2  Now l e t "M" represent the number of milligrams of iodine equivalent to one ml. of sodium thiosulphate.  Then  M  =  25 x 12.693 A  The number of milligrams of I  mg.  2  which combine with the o i l i s J  | (blank) - ( t i t r a t i o n ) ) The iodine value i s Wt  of I  2  of I ,  X  M  mg.  combined with o i l x 100  Wt. of o i l  Iodine value i s  ( (blank) - ( t i t r a t i o n ) j  $4 x 100  1000 x Wt of o i l ( i n grams)  Iodine value i s  j (blank) - ( t i t r a t i o n ) j 10 x Wt of o i l  /  M  Results In Sections I and I I the g o l d f i s h were acclimated to d e f i n i t e temperatures before the iodine values .were determined. In Section I the g o l d f i s h , which had been acclimated to temperatures of 1 2 ° , 2 0 ° , and 33°C showed mean iodine values of 1 2 2 . 9 5 , 1 0 9 . 9 9 , and 1 0 5 . 3 3 respectively (Table I ) . The r e s u l t s are shown graphically i n Figure 1 .  Using Snedecor's 0-94-6)* s t a t i s t i c a l  method of variance, an F value of 4.08 was calculated.  This means  that there i s a s i g n i f i c a n t difference between these groups of f i s h . The chances are l e s s than 5 i n 100 that such differences could a r i s e through chance alone.  Indications are that temperature was respon-  s i b l e f o r the difference i n iodine values.  Variance does not show  trend, but i t does v e r i f y the trend r e s u l t i n g from the work, which i s i n accordance with the hypothesis of t h i s experiment.  Table I .  Values obtained from g o l d f i s h used i n Section I .  Temperature °C  Number of Samples  Mean Iodine Value  Standard Error  12°  9  122.95  20°  12  109.90  4.03  33°  5  105.32  8.13  it  .513  Footnote - A l l s t a t i s t i c a l s t a t i s t i c a l methods, formulae and symbols used i n t h i s thesis are from Snedecor (1946).  130  120 -  110  100  12°  20°  33  c  Temperature °C Figure 1.  Mean iodine values of g o l d f i s h acclimated to above temperatures.  F. Value  =  4.08  20,  In Section I I the goldfish had been acclimated to temperatures of 12°, 25°, and 33°C,  Included with these c a l c u l a t i o n s  are data from Section I I I of the g o l d f i s h which had become f u l l y acclimated to 5°, 15° and 2 5 ° C The mean iodine values f o r the temperature groupings of 5°, 12°, 15°, 25°, and 33°C were 128.73, ,127.64, 117.37, 120.00, and 114.00 respectively (Table I I ) . Table I I . Values obtained from g o l d f i s h used i n Section I I .  Temperature °C  Number of Samples  Mean iodine Value  Standard Error  5  19  128.73  3.4  12 '  13  127.64  8.2  15  8  117.37  5.8  25  8  120.00  3.5  33  10  114.00  2.8  The r e s u l t s are shown graphically i n Figure 2.  These  iodine values were a l s o compared by the variance method and an F value of 8.36  resulted. This again means a s i g n i f i c a n t difference  e x i s t s between the groups of g o l d f i s h .  The trend shown by the mean  iodine values, however, does not f i t the hypothesis of t h i s experiment as p r e c i s e l y as the iodine values of the g o l d f i s h i n Section I . The general regression of these values, nevertheless, i s confirmatory.  130  120 -  110 -  100 5°  12°  15°  Temperature Figure 2.  25°  33°  °C  Mean iodine values of g o l d f i s h acclimated to above temperatures.  F. Value = 8.37  For the 25°C temperature group there may have been other factors influencing the degree of unsaturation of the o i l s .  The r e l a t i o n  of diet and metabolic rate could very possibly have caused t h i s difference at t h i s p a r t i c u l a r temperature. In Section I I I goldfisb from Ontario were sampled during and a f t e r t h e i r temperature acclimation.  Iodine values  were determined f o r f i s h from each temperature at twelve d i f f e r e n t time i n t e r v a l s (See Appendix). iodine values i n figure 3  The graphs of the  of the g o l d f i s h kept at 5°, 15°, and  25°C show the trend to be i n agreement with the hypothesis of t h i s experiment.  A regression l i n e has been f i t t e d to the data  of each temperature group.  We might expect l i t t l e change i n  iodine value during the experiment with the g o l d f i s h represented i n figure 3 which were acclimated and kept at 5°C. had a regression c o e f f i c i e n t of -.24.. expected r e s u l t .  These g o l d f i s h  This i s very close to the  The g o l d f i s h at 15°C and 25°C had regression  c o e f f i c i e n t s of -1.53 and -1.75.  These regression c o e f f i c i e n t s  are i n l i n e with the hypothesis on which the experiments were performed.  They indicate increased saturation i n the o i l s of the  g o l d f i s h during acclimation to higher temperature. the change i s greater at higher temperatures.  In addition  Temperature i s  apparently a f a c t o r causing a change i n the unsaturation of the goldfish o i l s .  Time Figure 3e  Goldfish acclimated t o 5°C and set up i n three temperature groups 5°,  1 5 ° and 25°C.  24.  I t must be noted, however, that the v a r i a t i o n i n values i s rather great (figure 3, tables i n Appendix) and that the regression c o e f f i c i e n t s do not show a highly s i g n i f i c a n t s t a t i s t i c a l trend  (TableJtfl).  Table I I I . Values obtained from g o l d f i s h used i n Section I I I .  Temperature °C  Number of Determinations  b  t  5  12  -.24  .17  15  12  -1.53  1.21  .25  128  25  9  -1.75  .936  .38  135  P  Fiducial Limits 127.92  %M -  For t h i s analysis the 95$ l e v e l of s i g n i f i c a n c e has been chosen. The order i s apparent but the v a r i a b i l i t y i s great enough to invalidate the trend at t h i s l e v e l of s i g n i f i c a n c e .  This may mean  that the t o t a l ether extract contains a v a r i e t y of compounds some of which are not affected by temperature or that various other factors (sex, s i z e , heredity) a f f e c t the iodine value more d e f i n i t e l y than temperature.  The same order, however, i s apparent i n  a l l experiments and the temperature r e l a t i o n s h i p seems unquestionable. In an attempt to improve the picture the data f o r the d i f f e r e n t sexes and s i z e s have been considered separately. The amount of unsaturation of o i l s i n the male and female g o l d f i s h was very s i m i l a r and could not be shown to d i f f e r by the variance  25. analysis (Table 17). Table IV. Values obtained when sexes of the g o l d f i s h were compared f o r differences i n iodine value.  Sex  Number of Determinations  Mean Iodine Values  Standard Error  Male  H  131  3.6  Female  14  127  3.4  Likewise a t e s t f o r a s i g n i f i c a n t difference e x i s t i n g between the sexes when they were treated as paired samples of the same length group showed no difference (Table V ) .  Table V.  Values obtained when sexes of the same length groups of g o l d f i s h were compared f o r differences i n iodine value.  Sex  Number of Determinations  Mean Iodine Value  Standard Error  Male  9  129  1.35  Female  9  125  2.17  A ponderal index (Thompson,1942) was used t o determine the r e l a t i v e condition of the entire group of g o l d f i s h used.  The  r e s u l t s , as shown i n figure 4, proved to be an almost normal frequency d i s t r i b u t i o n curve, and showed a very small difference i n general  condition.  I t was not f e a s i b l e to attempt a c o r r e l a t i o n  between iodine value and condition when the l a t t e r was uniform i n the groups.  Likewise the data d i d not  c o r r e l a t i o n of iodine value with colour of f i s h .  so  permit I t may  be  noted i n passing that the colour of the g o l d f i s h was about 90$ red and 10$ black, s i l v e r and varying degrees of combinations of the three colours.  There was an apparent l o s s of melanin  pigment at 25°C and an even greater l o s s at 33°C.  We have no  way of knowing whether t h i s had an e f f e c t on the r e s u l t s .  27.  25  r  24  Zl Zl  21  IS  Z+" Z-7 Zf Z<f 5.0 1.1 31  3.t 3-7 3 / 3-9  3-3 3-*  f-0 */  Condition Factor Figure U-  Frequency d i s t r i b u t i o n curve of condition of  g o l d f i s h from a l l groups.  R  =  W  E  L  G  H  T  X  IOQ.OOQ  Length^  28, Discussion,  The g o l d f i s h i n Section I show a d e f i n i t e decrease i n the amount of unsaturation with an increase i n temperature.  In  Section I I there was a general decrease i n unsaturation f o r the • groups but i n c e r t a i n groups i r r e g u l a r i t i e s appeared.  The gold-  f i s h acclimated to 25°C had an iodine value s l i g h t l y greater than those acclimated to 15°C.  Apparently  some f a c t o r other than  temperature has been i n f l u e n t i a l or more i n f l u e n t i a l i n the  one  temperature group than i n another. I t i s noticed i n a comparison of the iodine values from Sections I and I I that the o i l s from Section II are more unsaturated than the o i l s from Section I ,  This possibly could be  due to the breeding or d i e t a r y h i s t o r y of the two groups. reported by Brockelsby  It i s  (1941) that the average unsaturation of  o i l s from f i s h of one species may d i f f e r from the average unsaturation of f i s h of the same species caught i n d i f f e r e n t waters.  This i s i l l u s t r a t e d by the d i s t r i b u t i o n curve of the  A t l a n t i c and P a c i f i c h a l i b u t l i v e r o i l s . l e s s unsaturated than the l a t t e r .  The former are i n general  The a b i l i t y of f i s h to produce  depot f a t s with c h a r a c t e r i s t i c s peculiar to t h e i r species must mean that they can e i t h e r modify t h e i r ingested f a t s or select their diet.  Since a l l the g o l d f i s h were fed the same food they  must have been able to modify t h e i r ingested f a t s or b u i l d f a t s from carbohydrates istics oils. thynnus  new  and proteins peculiar to t h e i r character-  Lovern (1936) i n his work on the tunny (Thynnus  ) found that the high s t e a r i c acid present in- the  29.  f i s h was not a r e s u l t of d i e t since the stearic acid content from the p y l o r i c caeca (consisting l a r g e l y of food f a t ) has a lower stearic acid content.  To some extent, therefore, f i s h can control  the saturated f a t t y acids i n t h e i r depot f a t s .  I t i s reported i n  B u l l e t i n 59 (Brockelsby, 1941) of the F i s h e r i e s Research Board that i t i s possible t o c l a s s i f y f i s h to t h e i r correct genus and even species by t h e i r iodine value.  The g o l d f i s h from both  were at some advanced stage of sexual maturity.  Lovern  sources  (1934)  found i n salmon a f i s h which does not eat during i t s l a t e sexual development, a selective mobilization of the depot f a t s , and the developing ovaries and testes u t i l i z e d a greater percentage of unsaturated than saturated o i l s .  Differences i n maturity of the  g o l d f i s h may have caused i r r e g u l a r i t i e s i n r e s u l t s . In Section I I I , although the r e s u l t s do not f a l l within 5% p r o b a b i l i t y tables, they conform to the general hypothesis of t h i s paper.  The g o l d f i s h i n t h i s section had f i r s t been acclima-  ted to 5°C before being set up i n three temperature groups.  A  regression c o e f f i c i e n t of 0.0 was the t h e o r e t i c a l value f o r the 5°C group since they had already been acclimated to t h i s temperature and were going to continue l i v i n g at the same temperature.  The r e s u l t  of - . 2 4 shows the regression c o e f f i c i e n t i s not f a r from the but i t suggests that the o i l s became more saturated.  expected,  Since, a t t h i s  temperature, the consumption of food was n e g l i g i b l e , there may have been a drain over the long period of time on the unsaturated which were already present.  Lovern  fats  (1934) states that i n starving  30. salmon there i s s e l e c t i v i t y i n the o i l s f o r developing ovaries. These g o l d f i s h were developing sexually. Both groups 15°G  and 25°C showed a decrease i n  unsaturation, the l a t t e r group showing more change than the former. Although the average r e s u l t s follow the expected order throughout i t must be admitted i t i e s i n d e t a i l appear.  that many i r r e g u l a r -  As stated i n the introduction food, species  (heredity), and sexual maturity as well as temperature may expected to modify the f a t s .  be  Diet during the course of the experi-  ment was the same f o r a l l f i s h .  Unfortunately the data do not  permit the evaluation of sexual maturity and heredity e f f e c t . may  have been responsible f o r c e r t a i n i r r e g u l a r i t i e s .  The  This  goldfish,  apart from being procured from two separate sources, are u s u a l l y subjected to breeding programs i n the desire f o r attainment more marketable commercial form.  of a  Aida (1921) reports a breeding  experiment dealing with colour inheritance on a f r e s h water f i s h , (Aplocheilus l a t i p e s ) .  In dealing with four separate colours Aida  found the r e s u l t i n g colour pattern to follow the Mendelian p r i n c i p l e . The g o l d f i s h i n t h i s experiment were of d i f f e r e n t colour patterns. The colour i s due to carotenoid and melanin*. unsaturated the o i l s .  compounds and may  Carotenoids  are  possibly a l t e r the iodine value of  A phenomenon reported i n t h i s work and reported by Wells  (1935) i n connection with h i s work on the P a c i f i c K i l l i f i s h (Fundulus parvepinnis). and which i s i n d i c a t i v e of a p h y s i o l o g i c a l change i n the f i s h i s an apparent l o s s of melanin pigment at high temperatures.  \  31. In addition i t seems worthwhile noting that the goldfish had d i f f e r e n t rates of metabolism at the d i f f e r e n t tures.  Wells  tempera-  (1935) reports that fishes subjected to a lower  temperature have a lower rate of metabolism than those already l i v i n g at those temperatures or below the given temperature.  I t was observed  i n t h i s experiment that amounts of food eaten, a c t i v i t y , rate and extent of opercular movement varied with the temperature. measurements were not tabulated.  Counts and  The d i f f e r e n t rates of metabolism  would be instrumental i n determining the amount of food consumed by the g o l d f i s h at each temperature. Mendel  (1928) may be pertinent.  The findings of Anderson and  They note that the change from one  type of s p e c i a l l y produced depot f a t to another can be brought about f a r more r a p i d l y by depleting the f a t reserves through b r i e f periods of f a s t i n g p r i o r to the change i n d i e t .  This might possibly be a  determining f a c t o r i n changing the o i l s i n the g o l d f i s h .  Certainly  while the acclimation process was f i r s t i n progress the consumption of food dropped considerably returning to a l e v e l of equal standing with t h e i r rate of metabolism a f t e r they were f u l l y or p a r t l y acclimated.  32. Conclusions and Summary The r e s u l t s conform to the theory that temperature modifies the composition  of the f a t s of the g o l d f i s h . An increase i n  temperature decreases the number of unsaturated  bonds r e s u l t i n g i n a  higher melting point of the body f a t s . The study of the change i n the amount of unsaturat i o n during acclimation suggests there i s a chemical  change i n the o i l s  over the period of time necessary f o r acclimation as found by Brett (194-6).  The r e s u l t s also show, as i n the 5°C group of Section I I I ,  there was a decrease i n unsaturation a f t e r acclimation was  complete.  This could not have been a d i r e c t r e s u l t of temperature and must be attributed to some other f a c t o r probably i n t e r r e l a t e d with temperature. A l l of the foregoing findings are not s i g n i f i c a n t when based on 5% p r o b a b i l i t y tables.' The  statistically  different  temperature groupings as i n Sections I and I I , however, are s i g n i f i c a n t and the o v e r a l l trend i s i n accordance with other research on temperature acclimation.  I r r e g u l a r i t i e s are attributed to dietary h i s t o r y ,  heredity or differences i n degree of sexual maturity.  Acknowledgements  The suggestion of t h i s problem came from Dr. W. S. Hoar f o r whose a i d , opinions, and c r i t i c i s m s I am g r e a t l y indebted. The assistance from Dr. R. H. Wright of the B. C. Research Council, Dr. B. E. B a i l e y and Dr. L. A. Swain of the P a c i f i c F i s h e r i e s Station and Dr. W. Chalmers of Western Chemical Industries Limited have been s i n c e r e l y appreciated.  34.  Appendix  Table VI. Goldfish used i n Section I which were acclimated to 12°C.  Number 1  2  3  4  5  6  7  8  9  Footnote. and  Length  Weight  6.8  8.2  M  6.7  7.9  F  6.9  8.1.  F  6.1  6.5  F  6.5  6.3  F  7.3  8.6  F  7.7  11.1  F  6.4-  9.2  F  6.8  7.2  M  127.00  5.5  5.3.  M•  127.00  6.8  9.0  M-"  122.55  6.4  6.7  F  6.6  7.4  M"  6.6  7.8  M  7.5  10.6  F  6.5  6.9  M  6.6  7.1  M  6.7  8.2  M  A l l lengths  Sex  Iodine Value 116.58  129.65  120.40  120.91  121.28  119.87  128.34  a r e measured i n c e n t i m e t e r s ,  a l l w e i g h t s m e a s u r e d i n grams.  Table V I I . Goldfish used i n Section I which were acclimated to 20°C.  Number  Length  1  6.8  8.0  M  7.2  8.6  F  6.1  5.7  F  6.8  8.5  F  8.8  13.2  M  5.7  5.3  F  7.1  7.8  F  7.0  8.3  M  6.0  6.1  M  6.7  8.1  F  7.2  11.4  F  6.6  8.7  F  6.9  9.8  F  7.3  9.7  F  6.6  8.8  M  7.1  11.4  F  7.3  10.6  M  7.4  11.4  M  6.8  8.0  M  7.5  10.4  F  6.9  8.1  M  6.6  7.6  F  2  3  4  5  6  7  8  9  10  Weight  Sex  Iodine Value  88.18  105.54  120.09  96.10  126.77  124.23  116.56  124.50  123.24  102.40  37. Table V I I .  Goldfish used i n Section I which were acclimated to 20°C.  Number  11  12  Length  Weight  Sex  7.3  10.0  F  6.7  9.4  F  7.0  9.4  M  7.3  10.8  M  Iodine  Value  94.60  97.65  Table V I I I .  Goldfish used i n Section I which were acclimated to 33°C.  Number  Length  Weight  Sex  1  6.2  9.7  M  6.6  8.1  F  6.5  8.1  F  6.3  7.2  F  3  5.7  5.4  ?  107.11  4  6.3  6.8  F  74.11  6.4  6.8  ?  7.7  9.5  F  7.4  8.9  •>  7.0  8.6  «  2  5  Iodine Value  110.63  120.76  114.02  Table IX, Goldfish used i n Section I I which were acclimated t o 12°C,  Number  Length  Weight  Sex  Iodine Value  1  7.4  12.0  F  126.86  2  7.5  11.3  M  131.50  3  7.5  11.6  M  163.37  duplicate  164.43  4  7.8  12.7  M  128.85  5  8.1  13.6  M  128.27  6  8.9  18.5  F  154.65  duplicate 7  8.5  18.1  F duplicate  154.02 155.59 149.49  8  7.0  9.1  M  123.62  9  6.6  10.6  M  123.44  10  7.0  9.9  F  119.78  11  7.4  11.4  F  125.31  12  8.7  13.9  F  132.30  13  8.2  15.4  F  130.07  40. Table X.  Goldfish used i n Section I I which were acclimated to 25°C.  Number  Length  Weight  Sex  Iodine Value  1  7.9  10.6  F  123.92  2  7.1  10.2  F  129.26  3  7.7  13.0  M  132.86  4  8.7  16.8  M  159.30  5  8.5  18.0  F  128.85  6  8.8  17.2  F  123.32  7  6.3  7.0  ?  116.49  8  7.5  10.3  F  113.66  9  7.7  10.9  ?  118.47  10  7.3  11.0  F  119.46  11  8.4  16.4  M  115.61  12  8.1  16.2  F  135.09  Table XI.  Goldfish used i n Section II which were acclimated t o 33°C,  Number  Length  Weight  1  6.8  8.7  2  6.3  6.5  ?  108.85  3  8.1  14.3  F  117.89  4  7.2  11.0  M  128.26  5  7.5  12.5  F  104.55  6  8.9  23.6  F  103.56  7  8.6  H.9  ?  104.95  8  7.1  9.5  F  120.86  9  6.8  10.8  ?  127.40  10  7.4  11.3  F  112.11  Sex  Iodine Value  110.67  Table X I I .  G o l d f i s h used acclimated while over  Time P e r i o d  1  i n S e c t i o n I I I which had been  t o 5°C and k e p t  at t h i s  temperature  i o d i n e v a l u e d e t e r m i n a t i o n s were made a p e r i o d o f time.  Length  8,0  Weight  Sex  14.4  M duplicate  Iodine  Value  142.54. 142.19  2  7.3  13.0  M  126.77  3  7.3  12.1  M  112.91  4  9.2  22.8  M  165.29  duplicate:  163.05  9.2  22.8  M  164.69  5  7.5  10.6  M  129.58  6  7.1  11.1 "  F  124.91  7  6.9  12.1  F  112.88  7.5  11.3  M  123.96  7.4  11.4  M  125.92  7.2  11.5  M  119.44  7.5  12.3  M  139.58  7.2  10.0  F  125.44  8  9  43. Table X i i .  Goldfish used i n Section I I I which had been acclimated to 5°C and kept at t h i s  temperature  while iodine value determinations were made over a period of time.  Time Period  Length  Weight  Sex  Iodine Value  7.3  10.7  M  134.61  7.5  11.2  M  110.98  6.9  9.6  M  123.02  8.3  15.8  F  123.71  7.1  9 .5  M  125.44  7.6  H.8  F  116.74  duplicate 9.3  22.2  M duplicate  116.58 162.80 162.85  Table X I I I .  Goldfish used i n Section I I I which had been acclimated to 5°C. and then placed i n a 15°C. aquarium during the time iodine value determinations were being made.  Time Period  Length  Weight  Sex  Iodine Value  1  7.5  12.5  M  122.35  2  7.5  13.0  F  121.25  3  8.6  18.1  4  7.4  9.2  5  8.5  16.4  6  7.0  9.0  8.6  16.1  7.4  10.4  F  122.50  7.3  13.4  M  133.19  8.9  20.4  M  145.63  7.8  15.6  F  114.18  7.2  9.9  M  131.50  7.3  12.6  F  115.o7  8.2  15.6  F  141.41  9.0  17.0  M  111.90  7.7  11.4  M  108.26  9.3  22.5  F  109.66  9.4  21.3  M  106.85  8  9  10  11  12  .  M duplicate M M duplicate M M duplicate  127.24 135.09 129.42 152.60 157.75 129.41 165.59 164.99  Table XIV. Goldfish used i n Section I I I which had been acclimated to 5°C. and then placed i n a 25°C. aquarium during the time iodine value determinations were made.  Time Period  1  Length  8.6  Weight  20..1  Sex  Iodine Value  F  159.88  duplicate  162.56  2  7.2  12.0  F  118.83  3  7.4  11.4  F  127.30  4  7.9  12.4  F  129.13  5  7.8  12.2  M  162.93  6,9  9,3  M  123.18  7.8  12.4  M  164.49  6  duplicate 7  8  9  164.24  7.5  12.6  M  129.73  7.3  11.7  F  126.16  7.1  9.9  M  120.97  7.1  7.8  F  128.69  7.5  10.7  M  134.74  8.0  12.5  M  106.84  References Aida, Tatuo. 1921.  On the inheritance of colour i n f r e s h water f i s h : Aplocheilus l a t i p e s , Temmick and Schlegel, with special references to sex-linked inheritance. Genetics, 6: 554 - 5 7 3 .  Anderson, W. E. and L. B. Mendel. 1928.  The r e l a t i o n of the d i e t to the q u a l i t y of f a t produced i n the animal body. Jour. B i o l . Chem. 76: 729 - 7 4 7 .  Brett, J . R. 1946.  Rate of gain of heat tolerance i n goldfish (Carassius auratus) Ontario F i s h e r i e s Research Board, No. 5 3 .  Brockelsby, H. N. 1941.  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Lovern.  o f the mixed f a t t y  acids  present  i n the g l y e e r i d e s o f cod l i v e r  certain  fish  liver  B i o c h e m . J o u r . 24: Hathaway,  50  and  oils. 266  -  290.  E.S. 1927.  Q u a n t i t a t i v e changes produced ization by  on  fishes  B u l l . U.S.  the and  by  tolerance of high  acclimattemperatures  amphibians.  Bureau F i s h e r i e s , 43:  169  -  192.  48. Heilbrunn, L. V.  -  1924. The c o l l o i d I V The h e a t  •  chemistry  of proloplasm.  coagulation of  protoplasm.  Amer. Jour. Physiol. 6 2 : 190* ~ 1 9 9 . Heilbrunn, L. V. 1943. An outline of general physiology. 2nd Ed. 1947. W. B. Saunders Company, Philadelphia and London. Loeb, J . and H. Wastenays. 1912. On the adaption of f i s h to higher  (Fundulus)  temperatures.  Jour. Exp. Zool. 12:  543 - 557.  Lovern, J . A. 1934' CCLV IV  Fat metabolism i n fishes  Mobilisation of depot f a t i n the salmon  Biochem. Jour. 28: 1955 - 1961. Lovern, J . A. 1936.  CCLXXXV F a t M e t a l o b i s m X Hydrogenation 50:  i n fishes.  i n the f a t depots  o f t h e tunny,  2023 - 2026. -B-<-t">* •J*"'*  Lovern, J . A. 1938. Fat metabolism i n fishes  XIII  Factors influencing the composition of the  depot f a t of f i s h e s . Biochem. Jour. 2£i  1214 - 1 2 2 4 .  49. P e a r s o n , L.K. and H.S',. R a p e r . 1927. The i n f l u e n c e of  o f temperature  t h e f a t formed  Biochem.  hy l i v i n g  on t h e n a t u r e organisms.  J o u r . 2 1 : 875 - 8 7 9 .  Snedecor, G.W. 1946.  S t a t i s t i c a l M e t h o d s , F o u r t h E d . Iowa S t a t e College Press.  Ames, Iowa.  Sumner, F.B. and P. D o u d o r o f f , 193,8. Some e x p e r i m e n t s upon  temperature  a c c l i m a t i z a t i o n and r e s p i r a t o r y in  fishes.  Biol. T e r r o i n e , E.F., 1930.  metabolism  Bull.^  7 4 : 403 - 4 2 9 .  C H . H a t t e r e r , and P. R o e h r i g . L e s a c i d e s g r a s des p h o s p h a t i d e s chez l e s animaux p o i k i l o t h e r m e s , l e s v e g e t a u x superieurs e t l e s microorganismes. B u l l .  Chi. j W Biol. Thompson, D * a r c y  a-  Jf.  6/2-7°*-  12: 682 - 7 0 2 .  W.  * 1942. Growth and f o r m ,  Cambridge  University  Press,  London. Wells,  N.A. 1935.  Variations  i n the r e s p i r a t o r y metabolism o f  the  Pacific  due  to size,  killifish  (Fundulus p a r v e p i n n i s )  season, and continued constant  temperature. P h y s i o l . Z o o l . * 8:  318.  50. Yulll,  J . S . and R o d e r i c k 1937. The  Craig.  nutrition  Serieata I I The  of f l e s h  f l y larvae,  (Meig.)  development  J o u r . Exp.  Zool.  of f a t .  75: 169  -  178.  Lucilia  

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