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The effect of dietary fat on the heat tolerance of goldfish (Carassius auratus) Dorchester, John E. C. 1948

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££3 -3? -Z>  The E f f e c t o f Dietary Fat on the Heat Tolerance o f Goldfish (Carassius auratus)  ty John E. C. Dorchester  A Thesis submitted i n P a r t i a l F u l f i l l m e n t  of the Requirements f o r the Degree o f  . MASTER OF ARTS  i n the Department o f  ZOOLOGY  The University o f B r i t i s h Columbia August, 19A8.  7  - i -  The E f f e c t of Dietary Fat oh the Heat Tolerance of Goldfish (Carassius auratus)  by  J . E. C.  Dorchester  Abstract An attempt has been made to a l t e r the degree of unsaturation of the body f a t s of g o l d f i s h (Carassius auratus) and correlate these changes with any modifications of heat tolerance subsequently fish.  exhibited by the  The g o l d f i s h were fed three d i f f e r e n t d i e t s each containing a f a t  o f d i f f e r e n t degree of unsaturation.  The f a t s used were p i l c h a r d o i l  (iodine value of 181.7), herring o i l (iodine value of 128.4.) and l a r d (iodine value of 66.2).  Heat'resistance was tested by holding the f i s h  at a constant high temperature and observing the time to death.  Var-  i a t i o n s i n the a b i l i t y of the groups to withstand high temperature were then compared to differences i n the degree of unsaturation of t h e i r extracted f a t s .  I t was found that while d i e t could e f f e c t changes i n the  degree of unsaturation of the g o l d f i s h f a t s to approximately  5A% of the  t h e o r e t i c a l l e v e l , and that these changes i n turn modified the heat r e sistance of the g o l d f i s h , no quantitative r e l a t i o n s h i p was established.  - i i-  TABLE OF CONTENTS Page Introduction  •  •  1  Materials and Methods  5  Preparation o f Diets and Feeding  5  Environmental Control  7  Determination o f Heat Resistance  8  Fat Extraction and Analysis  9  Results  12  The E f f e c t o f Diet on the Unsaturation o f the Extracted Fats  12  E f f e c t o f Dietary Fat on the a b i l i t y o f Goldfish to withstand High Temperature  16  E f f e c t o f Diet on Behaviour i n respect to Temperature ... 21 Discussion  26  Summary  35  Acknowledgements  36  Appendix  37  References  •  *  •  ••••••  55  INTRODUCTION  L i f e i s maintained within a r e l a t i v e l y narrow range of temperatures.  This has attracted the attention of a great many workers,  with the r e s u l t that there i s voluminous l i t e r a t u r e on the subject of temperature i n r e l a t i o n to l i f e .  The observation that an extreme  temperature f o r one organism may be an optimum or even the opposite extreme f o r another, has l e d to much work on upper and lower l e t h a l l i m i t s f o r numerous plants and animals.  Other work has dealt with  optimum and preferred temperatures f o r plants and animals and v i t a l processes.  By comparison the attempts to investigate the physiological  processes involved have been r e l a t i v e l y few. of heat death have evolved'.  However, several theories  No attempt w i l l be made to l i s t them a l l ,  nor w i l l the author presume to argue t h e i r various merits.  A few w i l l  be mentioned along with the generally expressed c r i t i c i s m s of them, i n an e f f o r t to demonstrate the d i v e r s i t y and scope of the problem. The various theories f a l l into three main classes.  The  first  of these classes deals with interference with metabolic processes. Thus, i n 1905, Winterstein suggested that when animals are subjected to high temperatures, the metabolic rate i s increased so greatly that the organism i s unable to supply enough oxygen to the c e l l s and tissues. Therefore death i s due to asphyxiation.  Harvey (1911) working with  nerve conduction i n medusae observed that there i s an optimum temperature f o r conduction, the rate f a l l i n g o f f above and below t h i s l e v e l . Since enzyme reactions i n common with a l l v i t a l processes, behave i n a  - 2 -  s i m i l a r manner, he ascribed the phenomenon of heat death to the breakdown of the enzymes.  Mayer (1917) experimenting with a number of species  of corals showed that t h e i r a b i l i t y to stand high temperatures  was  roughly the same as t h e i r a b i l i t y to withstand high concentrations of carbonic a c i d .  He therefore proposed that i t i s the accumulation of  t h i s acid due to the increased metabolism r e s u l t i n g from high temperatures that causes heat death.  He disagreed with the asphyxia theory on  the grounds that by t e s t , the corals can survive i n the dark i n the absence of oxygen f o r long periods.  He c r i t i c i z e d the enzyme theory  because heat death i s , to a l i m i t e d extent, reversible and t h i s could not be so were the i n a c t i v a t i o n of enzymes responsible. The second c l a s s of theories developed from the of Loeb and Wastehays, (1912) on the f i s h (Fundulua).  experiments  According to  these workers, the maximum temperatures the f i s h could stand varied with the concentration of the sea water or Ringer's solution of the surrounding medium.  However, experiments with sucrose solutions  demonstrated that i t was not a case of simple osmosis.  They therefore  postulated that a r i s e i n temperature brings about c e r t a i n changes i n the permeability of the surface c e l l s of the body which r e s u l t i n death.  They further postulated that these changes may be overcome or  modified, i f the temperature change i s slow enough, by the s a l t s i n the blood or surrounding medium.  In t h i s manner they explain the  adaptation of an organism to higher  temperatures.  A t h i r d c l a s s of theories deals with the coagulation and  p r e c i p i t a t i o n o f the protoplasm.  Thus Brodie and Richardson (1899) •  explained heat death on the basis o f the coagulation o f the protein matter o f the l i v i n g c e l l .  This idea i s s t i l l popular, though many-  workers disagree on the grounds that protein usually does not coagulate much under 50°C. while many plants and animals suffer heat death at much lower temperatures than t h i s .  Heilbrunn (1924) endorsed the view  that i t i s a coagulation o f the c e l l contents, but from h i s experiments on the coagulation'of the protoplasm o f the eggs o f sea-urchins (Arbacia) and clams (Cuminga). f e l t that i t i s the l i q u i f a c t i o n o f the l i p o i d s i n the c e l l which allows the p r e c i p i t a t i o n o f the protoplasm.  He based  h i s assumption on the f a c t that, i n general, organisms i n a cooler habitat have more l i q u i d f a t s than those i n a warmer habitat.  In l a t e r  work he connected h i s calcium release theory o f anaesthetic action with the l i q u i f a c t i o n o f the protoplasmic f a t a t high temperature. According to t h i s composite idea when an animal i s exposed to l e t h a l heat the l i p o i d s of the c e l l membrane melt, releasing, the c o r t i c l e calcium, t h i s calcium i n turn causing changes i n the protoplasmic viscosity. Fraenkel and Hopf (1940) i n an attempt to show the r e l a t i o n ship between the degree o f unsaturation o f the phosphatides and l e t h a l temperature, bred two species o f blow-fly larvae a t two d i f f e r e n t temperatures and then subjected the larvae to l e t h a l temperatures. C o n f l i c t i n g r e s u l t s were obtained.  On the one hand a l l the larvae o f  both species raised a t the higher temperature, not only exhibited a  gain i n heat tolerance o f 1°C. over those raised at the lover temperature, but also the phosphatides extracted from the former larvae had iodine values 26 u n i t s higher than the l a t t e r group.  On the other hand larvae  of Phormia t e r r a nova and Calliphora erythrocephala while producing phosphatides of i d e n t i c a l iodine number, exhibited a difference i n l e t h a l temperature o f 7°C,  The f a c t that they were o f d i f f e r e n t species,  however c l o s e l y related, might explain the discrepancy. In any case, i t can be seen that the problem i s very complex. The answer probably l i e s i n the i n t e r a c t i o n o f a number of factors.  The  purpose of t h i s thesis i s to explore further one of these f a c t o r s , namely, the r e l a t i o n s h i p between the degree of unsaturation of the protoplasmic f a t s and temperature tolerance. Accordingly, goldfish (Carassius auratus) were fed d i e t s cont a i n i n g f a t s of d i f f e r e n t degree of unsaturation, and the e f f e c t of the dietary f a t on the body f a t s noted and correlated with any changes i n the response of the animals to high temperature.  MATERIALS AND METHODS  1. Preparation of the Diets and Feeding Three d i e t s were developed.  These were high i n f a t content  and contained f a t s o f d i f f e r e n t degree o f unsaturation.  The f a t s  selected were p i l c h a r d o i l (iodine value o f 180), herring o i l (iodine value o f 128.4.) and l a r d (iodine value of 66.2).  Pablum, manufactured  by the Mead Johnson Company o f Canada Ltd., constituted the base f o r a l l the d i e t s .  The f a t s were bound i n with l e c i t h i n i n the following  proportions: Pablum  - 7056 by weight  Fat  - Z% by weight  Lecithin -  5% by weight.  The f a t and l e c i t h i n were f i r s t dissolved separately i n peroxide f r e e ether and then mixed.  The solution o f f a t and l e c i t h i n  was then poured into the Pablum, mixed thoroughly and the ether extracted i n vacuo a t 37°C.  The resultant d i e t s were very serviceable, the o i l  remaining i n them f o r considerable time even i n contact with water. To ascertain the degree o f unsaturation o f the f a t s consumed by the f i s h , samples o f the d i e t s were taken immediately  a f t e r they  were prepared and iodine determinations were made upon the extracted fats.  To determine whether any considerable changes i n the f a t s occurred  a f t e r the d i e t s were made up the process was repeated i n 7 days and again i n 14 days.  The r e s u l t s are given i n table I .  - 6 -  TABLE I Iodine values of f a t s extracted from the d i e t s Iodine Values 0 Days  7 Days  Pilchard O i l Diet  159  159 - 170  Herring O i l Diet  115  115  S o l i d Fat Diet  64  65  14 Days 159 11^.3 64.1  Although no appreciable changes occurred i n two weeks, i t was deemed advisable to make up fresh d i e t s each week.  In order to reduce  the p o s s i b i l i t y of oxidation of the f a t s , the d i e t s were kept i n the f r e e z i n g compartment of the r e f r i g e r a t o r , except f o r the very few minutes during actual feeding. The Herring and Pilchard o i l s were supplied by Western Chemicals Ltd., Vancouver, while the Lard was obtained from Burns Ltd., Vancouver.  They were kept i n a frozen condition i n the cold storage  plant of the Dominion F i s h e r i e s Experimental  Station i n Vancouver.  reduce the p o s s i b i l i t y of oxidation through frequent freezing and  To un-  freezing of the p i l c h a r d and herring o i l s , a number of 50 ml. Erlenmeyer f l a s k s were f i l l e d at one time and kept i n cold storage at the station. When required, a small f l a s k of each o i l , and only as much of the frozen l a r d as was necessary was taken out.  The small f l a s k s of o i l were  subsequently unfrozen at the University, the appropriate amount of o i l weighed out and the remainder put i n t o the freezing compartment of the r e f r i g e r a t o r at the University laboratory.  As i t was impractical to feed each f i s h separately, an amount of d i e t equivalent to .3 grams per f i s h per day was used. divided into three feedings, morning, noon and night. took approximately  This was  Each feeding  t h i r t y minutes, only a small portion of food Toeing  put into each aquarium at a time, so that the animals could eat as much as possible before i t sank to the bottom and disintegrated.  I t was  necessary to change the water and clean out the aquaria once a week. 2. Environmental  Control  The experiments were carried out i n two parts, a preliminary group i n v o l v i n g 97 g o l d f i s h (Carassius auratus) procured from the Goldfish Supply Company, S t o u f f v i l l e ,  Ontario, i n November 1947,  and a  main group i n v o l v i n g 167 g o l d f i s h procured from the same source i n May 1948. The f i r s t of the preliminary experiments were performed during the winter of 1947 - 1948.  T h i r t y g o l d f i s h were divided i n t o three  groups of ten f i s h , and the groups retained i n aquaria 19" x 11.5" x  10.5"  Compressed a i r forced through a i r breakers provided adequate aeration. The temperature varied with the environment from 10 - 18°C.  The  fish  were fed the three standard d i e t s already described. For the l a s t of the preliminary experiments (May and June  1948)  sixty f i s h were divided into three groups of twenty, each group being contained i n two Turtox aquaria (19  w  x 11.5" x 10.5"), provided with  Lolag immersion heaters and Fenwal 10 amp., controls.  115 v o l t , - .5°C.  The thermostats were set f o r 20°C.  thermostat  Since the summer temper-  - 8 -  ature was capable o f elevating the water beyond the 20°C. mark, a cooling system was introduced.  This consisted of lengths of Pyrex glass tubing  (0.5" diameter) with the two ends bent at r i g h t angles forming a r e c t angular "U".  One of these "U's" was placed diagonally i n each aquarium  and connected by rubber tubing to the U*s" i n the adjacent aquaria. n  Cold water from the tap was run through the series and back into the sink.  In t h i s way a constant temperature o f 20°C. -.5°C. was maintained. Since not a l l the thermostats exhibited the same degree of  s e n s i t i v i t y , keeping each group i n two aquaria allowed s l i g h t v a r i a t i o n s i n the thermal h i s t o r y within the groups and between groups.  This  variable was eliminated from the main group o f experiments by d i v i d i n g 162 f i s h into three groups of 54, each being contained i n one large aquarium, (32" x 1 6  W  x 20").  The thermostats selected a l l exhibited the  same degree of s e n s i t i v i t y and a temperature of 20°C. -.5°C. was maintained i n each aquarium. 3. Determination of Heat Resistance In the early experiments two methods of ascertaining the resistance of the f i s h to high temperatures were t r i e d .  In the f i r s t  of these the f i s h were placed i n a small aquarium and the temperature raised at a constant rate u n t i l the l e t h a l l i m i t was reached.  This  method was discarded since i t was f e l t that there was no way to ensure i d e n t i c a l rate of change of temperature i n a l l cases, and any variations i n t h i s rate would almost c e r t a i n l y be r e f l e c t e d i n the r e s u l t s obtained. The method adopted, consisted of holding the f i s h a t a constant  high temperature and comparing the length o f survival time o f the three groups, using the 50/& l e v e l of mortality as the c r i t e r i o n .  Any f i s h  l i v i n g f o r 14 hours a t a given temperature was considered acclimatized, (Fry  et a l 1941).  The temperature selected was i n conformity with the  work o f Brett (1946).  As i n the holding tanks, compressed a i r was  bubbled into the t e s t i n g aquarium to ensure adequate oxygen content. Winkler t e s t s were made before and a f t e r the experiments. Using a small aquarium as a l e t h a l chamber necessitated t e s t i n g a sample from one group a t a time and since the thermostats used were at best only sensitive to -.5°C. therefore there would be s l i g h t v a r i a t i o n s i n the i n d i v i d u a l t e s t s .  This variable was removed i n the  main group of experiments (June - August 1948) by using a large tank (4' x 2' x 3')> and d i v i d i n g i t into three compartments by means o f two f i n e mesh p l a s t i c window screen p a r t i t i o n s .  Two heaters connected i n  series to one thermostat served to keep the water i n the tank at any desired temperature while two j e t s of compressed a i r bubbled i n vigorously provided adequate oxygen and c i r c u l a t i o n so that the temperature remained i d e n t i c a l i n a l l parts of the tank.  Samples of a l l three  groups of f i s h were tested simultaneously and therefore the f i s h f o r any one t e s t were a l l subjected to the same temperature f l u c t u a t i o n s . 4. Fat Extraction and Analysis After the f i s h had been k i l l e d by subjecting them to high temperature, the f a t was extracted.  The method was the same as that  used by Hunter (unpublished) with the exception that i n most o f the  - 10 -  experiments anhydrous sodium sulphate was not used to dry the macerated f l e s h , instead the t i s s u e , a f t e r being chopped with s c i s s o r s o r a Waring blender, was dried i n vacuo a t about U5 - 55°C.  The dried f i s h was  then placed i n the ether extraction thimbles and extracted f o r two hours with ether at 38°C.  The advantage o f t h i s system was a reduction o f the  actual extraction time thus minimizing the p o s s i b i l i t y o f oxidation due to any peroxides i n the ether.  The ether was tested f o r peroxides  before extraction was begun but. changes may have occurred during the process.  Also the o i l s thus obtained were free from any impurities  such as the anhydrous sodium sulphate which runs down the syphon carrying water with i t . Unfortunately the only means o f obtaining a vacuum was from the running water i n the laboratory and as the summer progressed the water pressure dropped to such a l e v e l that i t became impossible to obtain s u f f i c i e n t vacuum to dry the f i s h tissues.  For t h i s reason the  anhydrous sodium sulphate method was used f o r some o f the l a s t experiments (see appendix). In both cases, however, each thimble f u l l o f the tissue was extracted with ether f o r two hours when the thimble was emptied and fresh tissue put i n . This process was repeated u n t i l a l l the f i s h had been used. The mixture o f o i l and ether thus obtained was placed i n a round bottomed f l a s k and the ether drawn o f f i n vacuo a t 3 7 ° C , the f i n a l o i l was transferred to two-inch watch glasses and put into a  11 -  vacuum dessicator to remove any l a s t traces of ether and water. Iodine determinations on the o i l s were carried out i n the manner described by B a i l e y (unpublished) using Wij's solution.  - 12 -  RESULTS The E f f e c t o f Diet on the Unsaturation o f the Extracted Fats 1. Preliminary Experiments: Iodine determinations were not made f o r the winter preliminary experiments but were made f o r a l l l a t e r groups. Before feeding was commenced i n the second group o f preliminary experiments (May and June 1948), s i x pre-diet f i s h were subjected to a l e t h a l temperature (see Appendix I, Part B) and iodine determinations made on the extracted f a t s .  The average value obtained was 114*05.  Thereafter determinations were made on the f i s h as they were k i l l e d .  A  progressive desaturation o f the p i l c h a r d o i l group, and a saturation o f the s o l i d f a t group resulted. change.  The herring o i l group showed l i t t l e  The r e s u l t s are tabulated i n table I I below and shown graph-  i c a l l y i n f i g u r e 1. TABLE I I Iodine values o f the extracted f a t s from g o l d f i s h fed on the three standard d i e t s during the preliminary experiments o f May and June Iodine Values Pilchard O i l Diet (I.V.159)  Herring O i l Diet (I.V.115)  S o l i d Fat Diet (I.V.64)  114.05  114.05  114.05  12  124  112.9  90.4  21  126  115.6  34  136  111.4  Number o f Days Fed 0  -  89.2  Figure 1.  Graphic representation of the changes i n iodine values l i s t e d i n table II Pilchard o i l d i e t f i s h Herring o i l d i e t f i s h Solid f a t diet f i s h  <  -u2. Main Group of Experiments: As i n the preceding experiments, pre-diet g o l d f i s h were k i l l e d (see Appendix I, Fart C) and iodine determinations made. value being 112.78.  The  average  The  e f f e c t of the d i e t s on the extracted f a t s •a almost i d e n t i c a l with that shown above, (table I I I and figure 2).  was  TABLE III Iodine values of the extracted f a t s from goldfish fed on the three standard d i e t s during the main group of experiments. Iodine Values Pilchard O i l Diet (I.V.159)  Herring O i l Diet (I.V.115)  S o l i d Fat Diet (I.V.64)  0  112.78  112.78  112.78  6  122.3  115.5  93.4  29  128.2  112.6  88.4  32  133.4  112.9  86.5  37  136.1  113.1  86.4  46  137.1  113.2  91.38  55  137  110  85.8  Number of Days Fed  For figure 2 regression l i n e s ( f i r s t 37 days) have been f i t t e d to these data using the methods of Snedecor (1946). that a f t e r the thirty-seventh  These points show  day no p a r t i c u l a r change took place i n the  extracted f a t s of the f i s h fed on p i l c h a r d o i l and  solid fat diets.  the case of the herring o i l d i e t , no d e f i n i t e change took place at time.  .  In any  Iodine values f o r pilchard o i l d i e t Regression l i n e f o r pilchard o i l d i e t Iodine values f o r herring o i l d i e t Regression l i n e f o r herring o i l d i e t Iodine values f o r s o l i d f a t d i e t Regression l i n e f o r s o l i d f a t d i e t  b = 0.52 - 0.278  b * -.032  -  .088  b = -0.72  -  0.95  - 16 -  The E f f e c t of the Dietary Fats on the a b i l i t y of the Goldfish to withstand High Temperatures 1.  Preliminary Experiments: The r e s u l t s o f the early preliminary experiments (summarized  i n Appendix I, Part A) showed that the resistance to heat had been modified by d i e t .  However, thermal h i s t o r i e s were poorly controlled and  the number of f i s h not great enough to e s t a b l i s h the r e l a t i o n s h i p . Both these defects were remedied i n l a t e r experiments. In presenting the r e s u l t s of these l a t e r experiments, i t was f e l t that, due to the small numbers o f f i s h used i n each t e s t and the great v a r i a t i o n s among i n d i v i d u a l s o f the same group to withstand heat, the r e s u l t s o f comparable experiments could be shown to t h e i r best advantage by tabulating them c o l l e c t i v e l y (see table IV). 50$ mortality i s shown graphically i n f i g u r e 3. are summarized  The time f o r  The i n d i v i d u a l t e s t s  i n Appendix I, Parts B and C.  To t e s t further the observed differences i n heat resistance, the rate of dying i n each group f o r the f i r s t 75 minutes has been p l o t t e d i n f i g u r e 4-. This shows a d i f f e r e n t rate o f dying f o r the three groups, though a consideration of the f i d u c i a l l i m i t s does 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. 2. Main Experiments: The immediate e f f e c t of the d i e t on the f i s h used i n t h i s section, was to increase the heat tolerance o f a l l groups so that by the end o f 29 days a temperature of 35°C. had very l i t t l e e f f e c t on them.  - 17 -  TABLE IV  Results o f Preliminary experiments carried out i n May and June on the resistance to a temperature o f 35°C. F i s h fed f o r 12 to 34 days. The 50% l e v e l i s indicated by /  Pilchard O i l Diet  Herring O i l Diet  S o l i d Fat Diet  Time i n Minutes  Number Dead  Time i n Minutes  Number Dead  Time i n Minutes  Number Dead  11  1  1  1  22  1  25  2  13  2  25  3  37  3  17  3  27  4  40  4  20  4  30  5  42  5  27  6  40  7  43  6  30  7  57  8.  50  7  32  8  58  9  53/  9  33 /  9  62/  10  58  10  35  10  67  11  67  11  38  11  80  12  70  12  43  12  97  13  75  13  45  13  151  14  80  14  50  14  185  15  190  15  60  16  190  16  230  16  62  17  320  17  65  18  Acclimatized 1  Acclimatized 0  Acclimatized 3  - 18 -  60  SO  3C  Zo  to  N  •D Figure  3.  50?  mortality i n goldfish at  35°0.  - 19 -  Figure 4. Regression l i n e s f o r the f i r s t 75 minutes o f the preliminary heat resistance tests Pilchard o i l d i e t f i s h Herring o i l d i e t f i s h  b = 5.86 * 1.16 b s 7.86 - 0.808  Solid f a t diet f i s h  b = 4.4I  * 0.599  »  - 20 -  In tables V and VI are given the r e s u l t s o f t e s t s carried out a f t e r s i x days feeding and 29 days feeding respectively, shoving t h i s increase i n heat resistance. TABLE V Results o f s i x days feeding o f standard d i e t s on the resistance o f the goldfish used i n the Main Experiments to a temperature o f 35°C. S i x f i s h per group. Pilchard O i l Diet Time i n Minutes  Number Dead  Herring O i l Diet Time i n Minutes  Number Dead  S o l i d F a t Diet Time i n Minutes  Number Dead  52  1  28  1  74  1  67  2  55  2  85  2  106  3  75  3  195  4  170  4  Acclimatized 2  Acclimatized 2  Acclimatized 4  TABLE VI Results o f 29 days feeding o f standard d i e t s on the resistance o f the g o l d f i s h used i n the Main Experiments to a temperature o f 35°C. Eight f i s h per group Pilchard O i l DietTime i n Minutes  Number Dead  Acclimatized 8  Herring O i l Diet Time i n Minutes  Number Dead  1  2  70  3  Acclimatized 5  S o l i d Fat Diet Time i n . Number Minutes Dead  Acclimatized 8  21 -  Therefore a number o f experiments were performed (summarized i n Appendix I, Part C) to select a suitable temperature f o r the remainder of the experiments.  The temperature f i n a l l y adopted was 36°C.  As i n the l a s t section the r e s u l t s o f a l l the experiments a t 36°C. are presented c o l l e c t i v e l y i n table VII.  I t i s to be noted a t  t h i s point, however, that a l l these t e s t s were made a f t e r feeding had been continued f o r 46 days or more.  This i s s i g n i f i c a n t since, as has  already been pointed out, there i s a l e v e l i n g o f f o f the iodine values a f t e r the thirty-seventh day.  For t h i s reason i t i s f e l t that these  r e s u l t s are more s i g n i f i c a n t than the preceding ones. mortality i s presented i n figure 5«  The time f o r 50%  Figure 6 shows the rate o f dying  f o r the f i r s t 75 minutes, using the same method as i n the previous series. As i n the f i r s t experiments the r e s u l t s show a difference i n the a b i l i t y o f the three groups to withstand high temperature.  While  a consideration o f the f i d u c i a l l i m i t s does not show a s i g n i f i c a n t difference i n the case o f the p i l c h a r d and herring o i l d i e t s , i t does show a s i g n i f i c a n t difference i n the case o f the s o l i d f a t d i e t . The E f f e c t o f Diet on Behaviour i n respect to Temperature Although i t i s d i f f i c u l t to assess variations i n behaviour, i t was observed that i n most of the t e s t s performed the i n i t i a l reaction 1  to heat was more v i o l e n t i n the case o f the f i s h fed herring o i l than i n the f i s h fed p i l c h a r d o i l .  Both these groups displayed more i n i t i a l  d i s t r e s s than the f i s h fed on the s o l i d d i e t .  On four occasions herring  - 22  TABLE VII  Results o f the main group o f experiments on the resistance to a temperature o f 36°C. F i s h fed 46 days or more. 50% l e v e l indicated by /  Pilchard O i l Diet  Herring O i l Diet  S o l i d Fat Diet  Time i n Minutes  Number Dead  Time i n Minutes  Number Dead  Time i n Minutes  Number Dead  8 11 16 18 20 25 27 30 33.5 35 37 42.5 45 46 60 65 77 82 137  1 2 3 4 7 11 13 H 15 18 20 22 24 25 26 27 28 29 30  1 7 8 10 12 14 15 20 21 22 24 33 35/ 37 39 40 48 49 57 80  1 2 3 4 6 7 9 10 11 12 13 14 16 18 20 21 24 27 28 30  8 9 18 27 30 33.5 36 38 42.5 50 55 58 60 / 62 72 77 80 85 110 190 195 420 540  2 3 4 5 6 8 9 10 11 12 13 14 16 17 18 19 20 21 22 23 24 25 27  /  Acclimatized 0  Acclimatized 0  1  Acclimatized 3  - 23 -  So  30 Hi  20 s /o  0  Q  0  .5  VI  >i  — Figure 5.  5  Dur  —  50% mortality i n goldfish at 36°C  Pilchard o i l d i e t f i s h Herring o i l d i e t f i s h Solid f a t diet f i s h  b « 7.34 b • 6.82 b s 4.42  * 1.32 - 1.08 - 0.293  -  25  -  o i l d i e t f i s h died o f shock immediately, an occurrence which was not r e corded f o r the other f i s h . Even a t room temperature, the more unsaturated f a t d i e t s produced nervous symptoms i n the f i s h .  On one occasion a t l e a s t , the  herring o i l group became so nervous a t a temperature o f 18°C. as to a t t r a c t the attention o f strangers entering the laboratory.  Subsequently  two o f these f i s h died a t t h i s temperature i n a manner s t r i k i n g l y r e sembling heat death. The reason f o r t h i s behaviour i s unknown but Brown (1931) noticed s i m i l a r symptoms amongst pigs fed on f i s h o i l s .  - 26 -  DISCUSSION Relation o f Dietary to Body Fats Four f a c t o r s , temperature, sexual maturity, species and d i e t have been shown to influence the composition of the f a t s i n plants and animals* Pearson and Raper (1927) showed that the iodine values o f the f a t t y acids produced by two species o f fungi varied with the temperature at which these organisms were grown.  Dean and H i l d i t c h (1933) demon-  strated that the body temperature influenced the composition of the depot f a t s o f pigs, the outermost layers of f a t being more unsaturated than the innermost.  Lovern (1936) found that the tunny (Thymus thynnus)  which has a body temperature some three degrees higher than the water i n which i t l i v e s , had more saturated f a t t y acids than other marine f i s h . F i n a l l y , Bailey (1936) showed that Sockeye salmon (Oncorhynchus nerka) and Pink salmon (Oncorhynchus gorbuscha) caught i n the northern P a c i f i c coast waters had more unsaturated f a t s than f i s h of the same species caught i n the more southerly waters o f f the Fraser River. Greene (1913) showed the progressive l o s s o f stored f a t i n the Spring salmon (Oncorhynchus tshawytscha) during i t s spawning migration while Lovern (1934) found that i n the case o f the male salmon (Salmo salar) some s e l e c t i v e mobilization of the depot f a t s took place during the spawning season. Undoubtedly, species and d i e t are two very important factors. I t i s d i f f i c u l t to determine where the influence of one ends and the  27 -  other begins. "  Brockelsby (1941) discusses these factors and points out:  Since d i e t and l i f e habits have a profound e f f e c t on the  nature of the deposited f a t s , animals i n general tend to form s p e c i f i c f a t s only insofar as t h e i r d i e t and habits are specific.  However, any species l i v e s on a more or l e s s character-  i s t i c d i e t and the f a t s formed are, within c e r t a i n l i m i t s , c h a r a c t e r i s t i c o f that species.  "  I t seems d e f i n i t e that a l l animals possess, to a greater o r l e s s e r degree, some mechanism f o r modifying the ingested f a t s to s u i t the s p e c i f i c requirements.  However, i f the f a t consumed varies too  greatly from the normal requirements of the animal, or i f the rate of ingestion i s too high, the mechanism cannot successfully modify i t a l l , and part at l e a s t of the foreign f a t w i l l be deposited r e l a t i v e l y unchanged. Although many controlled feeding experiments have been performed on various farm animals, with a view to a l t e r i n g the f a t s f o r marketing purposes, very l i t t l e has been done on the controlled feeding of  fish.  To the author's knowledge, the work of Lovern (1938) and the  experiments contained herein, constitute the only two recorded experiments i n t h i s f i e l d .  For t h i s reason the r e s u l t s obtained are o f  particular interest. Using eels (Anguilla vulgaris) Lovern (1938) experimented with two d i f f e r e n t d i e t s , mussels and herrings.  He found that there was no  appreciable modification o f the e e l f a t toward mussel, probably because  - 28 -  i t was not fed at a s u f f i c i e n t l y high l e v e l . a t i o n toward herring f a t however.  He observed some modific-  This l a t t e r food contained a much  higher percentage of f a t . The f i n a l products obtained corresponded very well to d e f i n i t e mixtures of e e l and herring o i l , but a quantitive turnover of ingested and depot f a t was not demonstrated. The present experimental r e s u l t s agree with those of Lovern i n that d i e t produced modifications i n the degree of unsaturation of the g o l d f i s h f a t s , when the ingested f a t s d i f f e r e d s u f f i c i e n t l y from the s p e c i f i c v a r i e t i e s normally present i n the f i s h (see tables I and I I ) . Further, the amount of change i n the case of the pilchard o i l and l a r d d i e t was only approximately 54$ of the t h e o r e t i c a l .  Herring o i l , as has  been demonstrated (figure 2), produced very l i t t l e e f f e c t . There are two possible explanations f o r t h i s phenomenon.  One  i s that since the f a t s were fed to the f i s h at too high a l e v e l f o r them to be converted to the s p e c i f i c f a t s , they were stored r e l a t i v e l y unchanged i n the adipose tissues, the f i s h meanwhile l i v i n g on t h e i r s p e c i f i c f a t s previously stored.  A f t e r the l a t t e r were exhausted,  the  f a t s obtained through the d i e t s would then be converted to the s p e c i f i c ' type required f o r the metabolic processes.  This, then, would mean that  the d i e t a r y f a t s bear no r e l a t i o n to the p h y s i o l o g i c a l f a t s .  This view,  while conforming to the views of Lovern, i s contrary to those of Rittenberg and Schoenheimer (1937) whose experiments with deuterium indicate a constant turnover between depot and body or p h y s i o l o g i c a l f a t . The other explanation i s that, because of the nature of the  - 29 -  ingested f a t s and the rate at which they were consumed, the regulating mechanism was only capable of modifying the f a t s to a c e r t a i n degree and i t was,in t h i s form that they were stored.  When the s p e c i f i c f a t s were  exhausted, the ingested f a t s were then used i n the form i n which they were stored, replacing a l l the f a t s i n the animal body, both depot and physiological. This l a t t e r view follows more c l o s e l y the experimental r e s u l t s i n that the regression c o e f f i c i e n t of the f i s h fed on the herring o i l d i e t (figure 2) i s -.032, i n d i c a t i n g that there was an immediate and constant desaturation o f the ingested f a t s to the s p e c i f i c type.  I t i s true i n  t h i s case that the ingested f a t s (iodine value 115) were very l i t t l e d i f f e r e n t from the s p e c i f i c v a r i e t y (iodine value 112 - LL4) but the l e v e l of feeding was equally as high as i n the other two cases.  Again  the r e s u l t s of the experiments on heat resistance show a r e l a t i o n s h i p between the degree o f unsaturation of the dietary f a t s and the temperature tolerance of the organism.  Since i t i s the protoplasmic or body  f a t s that are assumed to be involved here, no such r e s u l t could be obtained were the dietary f a t s converted  to s p e c i f i c f a t s before use by  the f i s h .  Relation of Fats to Heat Tolerance Figure 4 i l l u s t r a t e s that i n the Preliminary Group o f experiments the order of increase of heat tolerance imparted by the d i e t s was herring o i l , p i l c h a r d o i l and s o l i d f a t .  The f i d u c i a l l i m i t s i n t h i s  graph are l i s t e d i n table 8, from the formulae:  - 30 -  1-^  =  b + t.sb  ±2 - b - t.sb where  b = the regression c o e f f i c i e n t t = p r o b a b i l i t y a t the p a r t i c u l a r l e v e l desired sb = standard error o f b TABLE VIII  F i d u c i a l l i m i t s o f b values f o r g o l d f i s h used i n the preliminary experiments Diet  F i d u c i a l Limits o f b Values 99$  level  95$  level  Herring  8.98 6.74  8.66 7.05  Pilchard  7.47 4.25  7.02 4.70  Solid  5.24 3.58  5.00 3.82  A study o f the table reveals an overlapping o f the b values f o r herring and p i l c h a r d , and p i l c h a r d and s o l i d a t the 99$ l e v e l .  This  means that 1$ o f the f i s h i n the herring o i l and p i l c h a r d o i l groups could have shown the same degree o f heat tolerance. f o r the p i l c h a r d and s o l i d f a t groups.  The same i s true  However, i t i s s i g n i f i c a n t that  overlapping does not occur between the herring and s o l i d d i e t groups, i n d i c a t i n g that the chances f o r a f i s h fed on herring o i l e x h i b i t i n g the same response to heat as a f i s h f e d on l a r d are l e s s than 1 i n 100. A consideration o f the 95$ l e v e l shows that the pilchard and s o l i d overlap but the pilchard and herring do not.  Thus the chances f o r herring  o i l d i e t f i s h and p i l c h a r d o i l d i e t f i s h dying a t the same rate are l e s s  - 31 -  than 5 i n 100. A study of f i g u r e 6 reveals that i n the main group of experiments the order of dying changed i n the case of the f i s h fed p i l c h a r d and herring o i l d i e t s . herring and l a r d .  The order i n these experiments was - p i l c h a r d ,  This i s the order expected i f the hypothesis upon  which the experiments were based i s correct.  An examination of the  f i d u c i a l l i m i t s indicates that the difference between the e f f e c t s of the p i l c h a r d and herring o i l d i e t s i s not s t a t i s t i c a l l y s i g n i f i c a n t since at both the 95% l e v e l and the 99% l e v e l considerable overlapping occurs. However, i n these experiments the behaviour of the f i s h maintained on the s o l i d f a t (lard) d i e t i s s i g n i f i c a n t l y d i f f e r e n t from both the p i l c h a r d and herring o i l d i e t f i s h .  This i s true even f o r the 99% l e v e l .  Thus, there i s a discrepancy between the r e s u l t s obtained i n the preliminary experiments and those of the main group as regards to the pilchard o i l and herring o i l d i e t s .  There are three f a c t o r s that  may be of importance i n explaining t h i s contradiction. 1.  I t w i l l be r e c a l l e d that during the preliminary experiments  each group was maintained i n two separate aquaria and that due to the v a r i a t i o n i n s e n s i t i v i t y o f the thermostats, s l i g h t differences i n thermal history were unavoidable.  Loeb and Wastenays (1912) have pointed  out that immunity to a higher temperature i s l o s t very slowly when once acquired and that short d a i l y exposures to a higher temperature a marked change i n immunity.  produces  Therefore i t i s possible that even s l i g h t  v a r i a t i o n s i n thermal h i s t o r y might be s i g n i f i c a n t .  - 32 -  2.  This same reasoning applies to a c r i t i c i s m of the method  of k i l l i n g the f i s h i n the preliminary experiments. v  Here i t w i l l be re-  c a l l e d a small aquarium served as a l e t h a l tank, and each sample was tested separately.  This l e d to v a r i a t i o n s i n thermal h i s t o r i e s i n the  t e s t s themselves, which might possibly become apparent i n the f i n a l results. 3.  F i n a l l y , the preliminary experiments were a l l c a r r i e d out  within 34- days of the commencement of feeding.  I t has been shown that  during t h i s time the degree of unsaturation of the extracted f a t s  was  constantly changing which may have some bearing on the r e s u l t s obtained. This seems to be the most l i k e l y  explanation.  In the main group of experiments the sources of v a r i a t i o n j u s t described were eliminated.  The f a c t that the r e s u l t s of the herring o i l  and p i l c h a r d o i l t e s t s are not s i g n i f i c a n t l y d i f f e r e n t may  either be  attributed to the f a c t that the numbers of f i s h a v a i l a b l e f o r experimentation were not large enough or that while the degree of saturation of the protoplasmic  fatsplays a part i n heat tolerance, some other f a c t o r ,  or f a c t o r s , are also involved.  Fraenkel and Hopf (1940) concluded that  an a d d i t i o n a l f a c t o r must be involved a f t e r t h e i r experiments on blowf l y larvae.  Working alone Hopf (1940) showed that exposure to high  temperature caused an increase i n the l i p o i d phosphorus, inorganic phosphorous and adenyl pyro phosphate P of the Haemolymph o f blow-fly larvae. Lovern (1932-1934) analyzed the composition  of the f a t t y acids  - 33  o f a number of marine and fresh water f i s h and found that there were d i s t i n c t differences.  In 1935  the same author examined three species of  fresh water Crustacea and one species of marine Crustacea and found the same class d i s t i n c t i o n s .  This may have some bearing on the r e s u l t s ob-  tained i n these experiments since the goldfish,which are a fresh water species, were fed d i e t s containing the o i l s of marine f i s h e s .  The f a c t  that the iodine values changed considerably i n the case of the pilchard o i l d i e t and s o l i d f a t d i e t , indicates an a l t e r a t i o n i n the composition of the depot f a t .  However, the f a c t that the iodine values of the  herring o i l group varied very l i t t l e does not necessarily imply that there was no change i n the composition of the depot f a t s .  I t has been  suggested that the f a t s were stored i n a somewhat modified form i n the case of the l a r d and p i l c h a r d o i l d i e t s .  This could equally well apply  i n the case of the herring o i l d i e t without causing the iodine values to change appreciably.  I f t h i s were so, i t may  be s i g n i f i c a n t i n view of  the r e s u l t s obtained i n the heat resistance experiments, that the p i l c h a r d (Sardinops caerulea Girard) generally inhabits more southerly waters than the herring (Clupea p a l l a i s i i  Valenciennes).  Another point of i n t e r e s t i s the increased tolerance to a temperature of 35°C. exhibited by the f i s h used i n the main experiments a f t e r a short period of feeding.  This may  be attributed to the better  condition of these f i s h , since those used i n the preliminary group had already been maintained i n the laboratory f o r a number of months and t h e i r feeding had been unavoidably i r r e g u l a r . The generally improved  - 34 -  n u t r i t i o n seems to a f f e c t the heat tolerance. A review o f the r e s u l t s obtained i n these experiments shows that d i e t can be a f a c t o r i n determining the type o f f a t produced within the f i s h .  I t also indicates the v e r a c i t y o f the general hypothesis that  greater immunity from the e f f e c t s o f heat i s derived from more s o l i d dietary f a t s .  However, these relationships are not quantitative and the  degree o f unsaturation o f these f a t s i s probably not the only mechanism involved i n heat tolerance.  - 35 -  SUMMARY  1.  The composition of the depot f a t s of g o l d f i s h may  be  altered by feeding f a t s of a d i f f e r e n t degree of saturation at a sufficiently  2.  high l e v e l ,  This process i s rapid at f i r s t but a f t e r a change o f approximately 54% of the t h e o r e t i c a l i s reached very l i t t l e further change occurs.  3.  The degree of saturation of the dietary f a t s influences the heat tolerance of the f i s h i n conformity with the general theory that the higher the melting point of the f a t the the heat resistance of the f i s h .  greater  However, since some a d d i t i o n a l  f a c t o r i s involved which may modify t h i s resistance, the r e s u l t s are not  4.  quantitative.  The f i s h o i l d i e t s modify both the normal behaviour and the i n i t i a l reaction to heat.  There i s a general tendency  f o r the f i s h o i l s to produce nervous symptoms.  - 36 -  ACKNOWLEDGEMENTS  I wish to acknowledge the Interest taken i n t h i s thesis by Dr. W. A. Clemens, Head o f the Department o f Zoology of the University of B r i t i s h Columbia, and to thank Dr. W. S. Hoar, by whom t h i s problem was suggested, f o r h i s continuous assistance and h e l p f u l c r i t i c i s m s .  I also wish to thank Dr.  B. E. Bailey o f the Dominion F i s h e r i e s Experimental Station i n Vancouver. The assistance o f fellow students, e s p e c i a l l y that given by I. Barrett, L. Robertson and G. Potter, has been sincerely appreciated.  - 37 -  APPENDIX I PART A - D e t a i l s and Results of the Winter Preliminary Experiments. Feeding was commenced on December 18, 19-47. December 30. 1947.  Experiment 1(a);  Four g o l d f i s h , one from each d i e t group and one control which had not been fed any s p e c i a l d i e t , were placed i n an aquarium and the temperature raised 4° per hour.  Results are presented i n table IX below.  TABLE IX Results of preliminary experiment 1(a)  Diet Group  Lethal Temperature  Herring  34°C  Control  35.5°C  Solid  36°C.  Pilchard  36.2°C.  - 38 -  January 2. 1948.  Experiment 2(a);  Four g o l d f i s h , one from each d i e t group and one c o n t r o l , were placed i n an aquarium at 34°C., l e f t f o r 30 minutes and then the temperature was immediately raised to 36°C.  The time of death i s given i n  table X.  TABLE X Results of preliminary experiment 2(a)  Diet Group  Time o f Death  Herring  1 minute, 15 seconds  Solid  1 minute, 30 seconds  Control  1 minute, 30 seconds  Pilchard *  2 minutes , 50 seconds  -  January 13. 1948.  39  -  Experiment 3 ( a ) i  One goldfish from each d i e t group was placed i n an aquarium at 9.2°C.  The temperature was raised 1°C. every 90 seconds.  temperature was noted and the r e s u l t s are given i n table XI.  TABLE XI Results of preliminary Diet Group  experiment 3(a)  Lethal  Temperature  Herring  35.5°C  Pilchard  36.1°C.  Solid  36.8°C.  The l e t h a l  - 40  February 16. 1948.  Experiment 4(a);  The remaining f i s h i n the three groups were tested i n water at 34°C.  Time to death i s noted i n table XII.  TABLE XII Results of preliminary experiment 4(a) Diet Group  Pilchard  Time to Death  5 minutes'  Herring  50 minutes  Solid  80 minutes  -a FART B - Results of the Second Section of the Preliminary Experiments carried out i n May and June, 1948  TABLE XIII Results o f experiment 1(b) on s i x pre-diet f i s h tested a t 35°C.  Number Dead  Time i n Minutes  •1  27  4  47  6  60  - 42 -  TABLE X I V Results o f experiment 2(b). group subjected to 35°C.  Pilchard O i l Diet F i s h  S i x f i s h per d i e t  Feeding time 12 days.  Herring O i l Diet F i s h  S o l i d Fat Diet F i s h  Time i n Minutes  Number Dead  Time i n Minutes  Number Dead  Time i n Minutes  Number Dead  25  1  38  1  22  1  37  2  43  2  151  2  43  3  45  3  185  3  50  4  60  4  320  4  75  5  62  5  80  6  65  6 ...  Acclimatized  2  - 43  TABLE XV Results of experiment 3(b). subjected to 35°C.  Pilchard O i l Diet F i s h  S i x f i s h per group  Feeding time 21 days.  Herring O i l Diet F i s h  S o l i d Fat Diet F i s h  Time i n Minutes  Number Dead  Time i n Minutes  Number Dead  Time i n Minutes  Number Dead  40  1  13  1  25  1  42  2  17  2  27  2  53  4  27  4  40  3  67  5  50  5  62  4  70  6  60  6  67  5  97  6  - 44  TABLE XVI Results o f experiment 4(b).  The remaining f i s h i n  each group, 5 p i l c h a r d , 6 herring and 8 s o l i d subjected to 35°C.  Pilchard O i l Diet F i s h  Feeding time 34 days.  S o l i d Fat Diet F i s h  Herring O i l Diet F i s h  Number Dead  Time i n Minutes  Number Dead  Time i n Minutes  Number Dead  11  1  1  1  25  1  58  2  20  2  30  2  190  3  30  3  40  3  230  4  32  4  57  4  33  5  58  5  35  6  80  6  190  7  Time i n Minutes  Acclimatized  1  Acclimatized  1  - 45 -  PART C - Results o f Main Group o f Experiments. Feeding was commenced on June 20, 1948.  TABLE XVII Results o f experiment 1 ( c ) .  Five pre-diet f i s h  subjected to a temperature o f 35°C.  Number Dead  Time i n Minutes  1  15  2  21  3  29  4  50  5  110  - 46 -  TABLE XVIII Results o f experiment 2 ( c ) . S i x f i s h per d i e t group subjected to 35°C.  Pilchard O i l Diet F i s h  Feeding time 6 days.  S o l i d Fat Diet F i s h  Herring O i l Diet F i s h  Time i n Minutes  Number Dead  Time i n Minutes  Number Dead  Time i n Minutes  Number Dead  52  1  28  1  74  1  67  2  55  2  85  2  106  3  75  3  195  4  170  4  Acclimatized 2  Acclimatized 2  Acclimatized 4  - 47 -  TABLE XIX Results o f experiment 3(c). subjected to 35°C.  Pilchard O i l Diet F i s h All became acclimatized  Eight f i s h per d i e t group Feeding time 29 days.  Herring O i l Diet F i s h  2 died o f shock  S o l i d Fat Diet F i s h  All  1 died i n 70 minutes  became  5 became acclimatized  acclimatized  - 48 -  TABLE XX Results o f experiment 4(c). Four f i s h per d i e t group placed i n the heating tank.  Temperature raised 1°C. per  hour u n t i l a l l the f i s h died.  Temperature  Number Dead Pilchard O i l  Feeding time 32 days.  Number Dead Herring O i l  Number Dead S o l i d Fat  38.3  1  1  38.8  4  2  1  4  2  39. 39.2  3  39.5  4  - 49 -  TABLE XXI Results o f experiment 5(c). S i x f i s h per d i e t group subjected to 37°C.  Pilchard O i l Diet F i s h  Feeding time 37 days.  Herring O i l Diet F i s h  S o l i d Fat Diet F i s h  Time i n Minutes  Number Dead  Time i n Minutes  25  1  25  3  25  2  45  6  45  6  45  4  70  6  Number Dead  Time i n Minutes  Number Dead  50 -  TABLE XXII Results of experiment 6(c). subjected to 36°C.  Pilchard O i l Diet F i s h Time i n Minutes  Number Dead  S i x f i s h per d i e t group  Feeding time 46 days.  Herring O i l Diet F i s h Time i n Minutes  S o l i d Fat Diet F i s h  Number Dead  Time i n Minutes  Number Dead  16  1  1  1  33.5  2  30  2  14  2  42.5  3  33.5  3  40  3  60  5  42.5  5  49  6  195.  6  46  6  - 51 -  TABLE XXIII Results o f experiment 7(c)., subjected to 36oC.  Pilchard O i l Diet F i s h  Eight f i s h per d i e t group  Feeding time 55 days.  S o l i d Fat Diet F i s h  Herring O i l Diet F i s h  Time i n Minutes  Number Dead  Time i n Minutes  Number Dead  Time i n Minutes  Number Dead  11  1  7  1  58  1  20  2  10  2  77  2  27  3  12  3  85  3  35  4  15  4  190  4  37  5  33  5  540  6  65  6  39  7  77  7  80  8  137  8  Acclimatized  2  - 52 -  TABLE XXIV Results o f experiment 8 ( c ) . Sixteen f i s h per d i e t group subjected to 36°C.  Pilchard O i l Diet F i s h  Feeding time 59 days.  Herring O i l Diet F i s h  S o l i d Fat Diet F i s h  Time i n Minutes  Number Dead  8  1  8  1  8  2  18  2  12  2  9  3  20  4  15  3  18  U  25  8  20  4  27  5  27  9  21  5  30  6.  35  11  22  6  36  7  37  12.  24  7  38  8  45  14  35  9  50  9  60  15  37  11  55  10  82  16  48  14  62  11  57  15  72  12  80  16  80  13  110  14  420  15  Time i n Minutes  Number Dead  Time i n Minutes  Number Dead  Acclimatized  1  tt  r 53 -.  APPENDIX I I Average Lengths, Weights and Iodine Values of F i s h Used. TABLE XXV F i s h used i n second section o f the preliminary  experiments.  Date Killed  Experiment Number  Diet Fed  Av. Length i n cm.  Feb. 17, '48  Kb)  Pilchard  6.78  13.3  Herring  6.74  11.06  Solid  7.0  13.9  Av. Weight i n grams  Average Iodine Value  mt  May 26, «48  2(b)  Pre-diet  6.8  June 8, '48  3(b)  Pilchard  6.6  10  124.  Herring  6.7  10.1  113.7  Solid  7.'5  10.3  90.4  Pilchard  6.8  9.6  126.6  Herring  6.8  11.5  115.5  Solid  7.0  11.6  Pilchard  7.1  12.0  136.  Herring  6.6  11.6  111.4  Solid  7.1  11.6  88.9  June 17, '48  June 30, "48  4(b)  5(b)  9.25  114.045  -  TABLE XXVI F i s h used i n the main group of experiments  Date Killed  Av. Weight i n grams  Average Iodine Value  Experiment Number  Diet Fed  Av. Length i n cm.  Pre-diet  7.1  11.1  112.78  Pilchard / Pilchard Herring / Herring Solid / Solid  7.25 7. 7.25 7.2 7.3 7.4  9.5 11. 11.5 11. 12.2 11.2  119.25 124.9 118.2 113.47 95.77 91.14  June 18,  '48  Kc)  June 26,  '48  2(c)  J u l y 19,  •48  3(c)  Pilchard Herring / Herring Solid  7.2 6.7 6.8 6.9  12. 9.4 9.4 11.  128.25 116.35 109. 88. a  J u l y 22,  •48  4(c)  Pilchard Herring Solid  7. 7.2 7.1  10. 12.5 12.2  133.45 112.9 86.5  J u l y 27,  '48  5(c)  Pilchard Herring Solid / Solid  7.2 7.3 7. 7.2  13.1 13.3 11. 14.  136.1 113.1 87.75 85.3  .  Aug.  5, •48  6(a)  Pilchard Herring Solid  7.6 7.1 7.3  16. 13. 15.5  137.1 113.2 91.38  Aug.  14, •48  7(c)  Pilchard Herring Solid / Solid  7.36 7.26 7.5 7.5  14. 13.75 14.2 14.2  137. 110.86 83.14 88.5  Aug.  18,•48  8(c)  Pilchard Herring Solid  7.7 7.6 7.5  17. 16.5 16.  -  /  F i s h that became acclimatized or showed greater resistance to heat than others i n the same group sample.  - 55 -  REFERENCES Bailey, B. E. 1936.  The N u t r i t i v e value o f marine animal products, VII  The vitamin A and D potency o f the o i l s  from B r i t i s h Columbia canned salmon. B i o l . Bd. Can. 2 (5) : 431 - 437. Bailey, B. E. 1946.  Unsaturation. Unpublished.  1 Brett, J . R. I946.  Rate o f gain of heat tolerance i n g o l d f i s h (Carassius auratus) Publ. Ont. F i s h . Res. Lab. 64  Brockelsby, H. N. 1941.  The chemistry and technology of marine animal o i l s with p a r t i c u l a r reference to those i n Canada. F i s h . Res. Bd. Can. B u l l 59.  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