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The cold-resistance of goldfish (Carassius auratus) fed certain lipid diets Irvine, Donald Grant 1954

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THE GOLD-RESISTANCE OF GOLDFISH (Carassius auratus) FED CERTAIN LIPID DIETS by Donald Grant Irvine A thesis submitted i n partial fulfilment of the requirements for the Degree of Master of Arts in the Department of Zoology We accept this thesis as conforming to the standard required from candidates for the degree of Master of Arts Members of the Department of Zoology The University of British Columbia June 1954 ABSTRACT The effects of dietary phospholipid and cholesterol upon the body lipids and cold resistance of the goldfish (Carassius auratus) were studied. Two series of diets were used to compare the effects of these supplements in the presence and absence of a large o i l supple-ment. It was found that dietary phospholipid and cholesterol both tend to increase cold-resistance of the goldfish, the relative effectiveness of these treatments varying with season and with duration of dietary treatment prior to chilling. The increased cold resistance afforded by dietary cholesterol or phospholipids was closely correlated with the increased cholesterol content of the tissues, but only weakly correlated with the less modified phospholipid content of the tissues. Although feeding these diets also resulted in definite changes in moisture con-tent, total lipi d content, unsaturation of total lipids and cholesterol/ phospholipid ratio, no obvious correlation between these factors and cold resistance of the goldfish was noted. In addition to diet, i t was found that cold narcosis, season, and size, as well as sex of the goldfish tested, modified the observed cold resistance. ACKNOWLEDGMENTS I wish to extend my sincere thanks to Dr.W.S.Hoar, for the suggestion of this problem, for his continuous active interest in this study, and for his most valuable assistance and helpful criticisms. For advice upon chemical methods, I am particularly indebted to Drs. D.S.Idler and L.A.Swain, Pacific Fisheries Experimental Station, and to Mrs.Merva K.Cottle, University of Washington. For the translation of a pertinent Italian paper I wish to thank E.Merler, Department of Chemistry, University of British Columbia, and for the translation of the Japanese article, my sincere thanks go to the Japanese Consulate i n Vancouver. I have also greatly appreciated the assistance of fellow stu-dents, especially that given by J.G.Robertson, M.H.A.Keenleyside, F.G.Hess, and W.H.Lewis. The generosity of the National Research Council in financing this project is most gratefully acknowledged. TABLE OF CONTENTS PAGE ACKNOWLEDGMENTS INTRODUCTION 1 MATERIALS AND METHODS General 3 Biological 6 Chemical 9 RESULTS General Observations 15 Influence of Non-Dietary Factors upon Cold Resistance: Size ,. . 17 Sex 18 Season 19 Effects of Diets apon Cold Resistance . . . 21 Physiological Results of Experiments with Diets A,B,C,D 22 Physiological Results, Diets P,E,I... . 23 Biochemical Results, Diets P,E,F. ... * 27 Summary of Results 32 DISCUSSION 34 Non-Dietary Factors . 36 Size 36 Sex 37 Season 37 Assessing Non-Dietary Factors . . . . . 38 Diets in Relation to Theories of Cold Death . 39 Melting-Point Theory 39 Calcium Release and Fat Release Theories 40 Permeability and Osmoregulation Theories 41 Body Insulation Theory • • 42 Biological Antioxidant Hypothesis . . . 48 Cold-Metabolism Theory 56 SUMMARY 61 LITERATURE CITED LIST OF TABLES PAGE TABLE I. Composition of Diets . . . . . . . . . 4 II. Dietary Experiments 5 HI. Size Effects 17 IV. Sex Effects , . . 19 V. Narcosis and Cholesterol . . . . . . 26 LIST OF FIGURES FIGURE 1. Length Effect 2. Weight Effect 3. Sex Effect 4. Sex Effect 5. Season Effect 6. Gold Test Response 7. Cold Test Response 8. Cold Test Response 9. Cold Test Response 10. "Initial Protection" 11. Feeding Effect/Time 12. Feeding Effect/Time 13. Total Lipid 14. Moistures 15. Lipid-Water Relation 16. Iodine Numbers 17. Cholesterol 18. Phospholipids 19. Cholesterol/Phospholipid 20. Cholesterol/Total Lipid 21. Relative Cholesterol 22. Relative Phospholipid INTRODUCTION The ability to withstand heat or cold has long been associated both scientifically and popularly with the "fats 1 1. It has been known for many years that acclimatization to cold increases the unsaturation of the tissue lipids, whereas acclimatization to heat causes the lipids to become more saturated, and hence more solid. It i s somewhat surprising, therefore, to find that diet-evoked changes in unsaturation of total body lipids of the goldfish bring about neither consistently Increased cold resistance with increased unsaturation nor regularly enhanced heat resistance with increased saturation (Hoar and Dorchester, 1949)• Nevertheless, the work supporting these conclusions has been repeated and expanded (Hoar and Cottle, 1952), and i t i s now well established that there i s no close correlation between total lipi d unsaturation and thermal resistance of the goldfish, even though the l i p i d diets fed were found to influence markedly the fishes' thermal resistance. Clearly the lipids had an effect, but not one based upon the level of l i p i d unsaturation induced in the tissues. In view of this apparent discrepancy between degree of l i p i d unsaturation and thermal resistance, and following the line of reasoning employed by Mayer et Schaeffer (1914), Sundstroem (1927), and Wilbur and Del Porno (1949)> Cottle of this laboratory investigated changes in the cholesterol, phospholipid, and fatty acid fractions of goldfish acclima-tized to different temperatures. This work showed that acclimatization to cold decreased tissue phospholipids, but had no significant effect upon 2 tissue cholesterol. The total l i p i d increased while the water content of the tissues decreased in cold acclimatization. Other experiments i n this "laboratory, however, showed that diet- or hormone-induced high phospholipid content of the tissues was beneficial in cold-resistance. The present series of experiments was undertaken to investigate further the validity of the unsaturation-cold resistance anomaly, and to cast some light upon the effect of dietary l i p i d supplements upon tissue lipids and thermal resistance of the goldfish. In order to investigate and evaluate non-dietary nidifying factors operating upon observed c h i l l resis-tance, the effects of length, weight, and sex of the fish, proportion of the fish deeply narcotized, the season, and lipid level in the control diet were also studied as an integral part of the whole dietary program. MATERIALS AND METHODS A. General To reduce the number of li p i d factors to be considered in in-terpreting the data, a new group of diets was prepared, designated as P, E, and F. These diets differ from the diets A, B, C, and D previously studied in this laboratory in that they differ in only one l i p i d fraction at a time, the control containing neither supplements of o i l nor appre-ciable lecithin (fuller description of a l l diets used appears i n Table I). Since, however, continued study of diets A, B, G, and D was likely to pro-vide useful comparative data in con-junction with the P, E, F series, and since this earlier series of diets had proved successful in this labora-tory, and had already provided preliminary data on thermal resistance in relation to dietary complexes of lipids, i t was also decided to continue studying the effects of diets A, B, G, and D upon the cold-resistance of goldfish. The eighth diet (Q) was used in order to test the validity of conclusions based upon comparisons between diets A and F, since these two diets differ not only in o i l content, but also in the nature of the phospholipid supplement. To determine the effect of season and any un-controllable factors upon the cold-resistance and chemical composition of the goldfish, the group of diets, A, B, C, D was fed to two different shipments of goldfish, and the group of diets P, E, F was fed to three shipments. A l l the experimental animals were obtained from the Goldfish Supply House, Stouffville, Ontario. 4 TABLE I COMPOSITION OF DIETS Diet Pablum*'' Pilchard O i l 2 Cholesterol^ Phospholipids A 70g 25g - 5g Lecithin^ B 70 18.75 6.25g 5 II C 70 20 - 10 u D 70 15 5 10 II E •50 - 50 -F 50 - 50 lecithin, cephalin, and inositol phosphatide.-* P 100 - - -Q 70 - - 5 Lecithin2*-1. Mixed cereals, Mead Johnson & Co. 2. Kindly supplied by Dr.Sven Lassen, Van Camp Sea Foods, Terminal Island, California. 3. Fisher Laboratory Chemical. 4. Animal lecithin (practical), Eastman Kodak. 5. This mixture of phospholipids was conveniently available as "Granulestin", generously supplied by J.ELchberg, President of the American Lecithin Company. TABLE II DIETARY EXPERIMENTS Lot # of Goldfish Season Cold Test Diets Days Feedings Date Number of Fish I Winter 152 A,B,C,D. 40A19 Nov.24/52 40 I W 252 A,B,C,D. 42/126 Nov.26/52 40 IIA Summer 153 P,E,F. 45/130 June 19/53 51 IIA S 253 P,E,F. 25/73 June 22/53 51 IIB s 4 P,E,F. 65/186 July 9/53 51 IIB s 5 P,E,F. 46/130 July 12/53 51 III s 6 A,B,C,D,Q. 24/68 Aug.14/53 S5 III s 7 A,B,C,D,Q. 38/110 Aug.29/53 85 IV Winter 8 P,E,F. 7/21 Nov.4/53 51 IV W 9 P,E,F. 14/41 Nov.11/53 51 IV •W 11 P>E,F. 21/62 Nov.18/53 51 607 The experiments were so arranged that the seven main diets were evaluated in terms of modified cold resistance of both "Summer" and "Winter" fish. Details of the experimental program are presented in Table II. B. Biological The group of experiments reported here used approximately 1,800 goldfish procured in two winter shipments and two summer shipments from Stouffville, Ontario, and further treated as described below. AH goldfish used were immediately transferred to aquaria containing aerators and dechlorinated water at the same temperature as that in the shipping cans on arrival at this laboratory. The method of Fry et al (1942) was followed in bringing these fish to an acclimatization temperature of 20° C, maintained in the aquaria by means of a Fenwal thermoregulator and Lo-lag immersion heater. The goldfish were always maintained at 20° C. for at least one week prior to the first feeding of the special diets, their nutritional requirements in the meantime being met by single daily feedings of Pablum (or the prepared goldfish food "Pallerina" in the preliminary experiments). In addition to thermal standardization, steps were taken toward randomization of the fish among the aquaria used, allow-ing eighty five fish to each large (153 litre) aquarium. Diets were administered to the fish three times per day, seven days a week, each feeding being the largest amount that could be consumed without giving any undue turbidity to the water. To assure active maximum consumption of the diets, the fish were conditioned to a feeding sign made by the experimenter upon approaching the aquaria. Following the methods already 7 worked out in this laboratory, the diets were prepared fresh each week, and stored under refrigeration in the case of oil-containing diets, while the fish were carefully maintained at 20° G ± ^° throughout the weeks of feeding. Aquarium water was changed at least once per week, care being taken to mix the hot and cold streams of dechlorinated water to assure i t s discharge at 20° C. into the aquaria. Having, then, relatively large stocks of fish completely accli-matized to 20° C,, but fed different diets, methods of sampling these fish for cold-resistance tests, and methods of measuring cold-resistance had to be chosen. An approach to randomization was made in selecting the optimum-size sample of 17 fish from each diet, for each cold-test, immediately prior to the test in question. Directly from their 20° G. feeding aquaria, the "selected" fish were transferred into a single, screen-partitioned approximately 2° C, thermoregulated and aerated chilling-tank. The chilling-tank compartments limited by the vertically-placed screens, were designated as A,B,C, or 1,2,3,k, into which compartments the selected goldfish from diets P,E, and F, or A,B,C, and D, were placed by some careful worker having no knowledge of the theoretical expectations. Only he knew which dietary treatment corresponded to each compartment of the chilling-tank, and only after the data had been recorded by compartments and the test completed did this investigator learn the identity of the fish groups. This technique is believed to have removed virtually a l l bias from the results. Such removal of bias from the cold test observations i s essential, for i t has been long recognized that there i s great difficulty in deter-8 niining when a chilled fish has actually died (Fujita, 1906; Sumner and Doudoroff, 1938). F.D.Smith, (1950, unpub.) realizing this difficulty, has proposed an electrical shock method of determining an arbitrary end point approximating the death-point of chilled goldfish. However, since this method may well be damaging to the tissues i f used routinely at 250 volts, and would be too time-consuming to apply in the most refined form, and also in view of the fact that the Japanese worker, M.Fujita, found as early as 1906 that dead fish also responded to electrical stimuli similar to those which el i c i t responses in living chilled fish, i t was decided instead to use a reproducible series of mechanical stimu-lations. This method included a very brief removal from the water, onto a cold wet surface, i f the fish did not respond at an earlier phase. The method i s an adaptation of that of Burridge and Hoar (unpub. notes) with emphasis upon mechanical stimulation of the caudal peduncle, in view of the established fact that the caudal peduncle is the most sensitive re-gion of the body in many chilled fishes, including the carp (Fujita, 1906). However, this criterion of c h i l l death, while giving reproducible results, is not a quantitative measure of irreversible c h i l l damage, or truly lethal effect, since several fish from preliminary tests, apparently dead by this mechanical stimulus sequence test, recovered completely upon being returned to 16° C. water for one hour. This preliminary finding resulted in adoption of an additional test to determine the degree of chill-damage more accurately - that is, a l l fish apparently dead by the mechanical test were placed in buckets of continuously aerated, dechlorinated water main-tained at 16° - 20° C. and examined one hour later. Any goldfish not showing complete recovery at this time were termed "truly dead", as i t is almost 9 certain that they would not survive at 20 C. for more than a few days at mostj any goldfish recovering fully, on the other hand, was termed a "recovery". The term "removals" was used to refer to a l l those fish which failed to respond to the mechanical stimulus sequence test. Thus, a "removal" is a "recovery" i f i t "revives" completely on rewarming, or a "truly dead" fish i f i t fails to recover completely. Clearly, the "recoveries" are a measure of the tendency toward very deep cold-narcosis. Since each chilled fish was treated separately in recording pertinent data, much additional information was afforded. The pertinent data referred to above includes not only the time at which each fish was removed from the chill-tank, the temperature, com-partment, and the reaction of the fish to warming for one hour, but also the weight, length, and sex of the fish. Smith (1950, unpub.) had found length and weight of the goldfish to influence the chill-mortality rate. Once these data had been obtained, the goldfish were surface-dried with paper towels to a characteristic "tackiness", and, sealed in labelled polyethylene bags, were quickly frozen and subsequently maintained in cold-storage lockers until time permitted their chemical analyses, C, Chemical The methods previously used in this laboratory for the extraction and determination of moisture, total lipid, phospholipid, cholesterol, and li p i d unsaturation were a l l subjected to tests evaluating their accuracy, precision, percent recovery, and their efficiency. The results of these tests led to the adoption of two new techniques, a significant change in a third technique, and adoption with only minor adjustments of the other techniques already used in this laboratory for l i p i d analyses. The data from these tests of chemical methods, together with recommendations regard-ing their use, the avoidance of common difficulties encountered in their use, and apparatus used for greatest efficiency, plus a detailed flow-sheet of the determinations, are on fi l e with Dr.W.S.Hoar, Department of Zoology, University of British Columbia. A brief survey of the techniques found satisfactory for following the changes in moisture and various lipid fractions of the goldfish i s given below. 1. Moisture and volatile constituents (new method) For this determination, the Genco Moisture Balance was used. This standard apparatus consists basically of an infrared lamp placed above the pan of a torsion balance precalibrated to read 100 percent with wet tissue. The heat from the infrared lamp dries the tissue, and the torsion balance reads percent moisture directly. It was found that averaged t r i -plicate determinations using the Moisture Balance and conventional oven drying method checked within a maximum of 0.28 percent, when using a Power-stat in the current supply to the Balance, to keep the Blendored tissue at approximately 80° C. At this temperature, the determination i s completed in 25 minutes, as compared to six or seven days required for the oven method. It is true that the oven method is slightly more precise than the Moisture Balance, but i t is very doubtful whether the slight gain in pre-cision afforded by the more tedious, time-consuming, oven method warrants the continued use of the latter in studies of this type. 2. Phospholipids (new method) After many partially or totally unsatisfactory t r i a l determina-tions of phospholipids using the microoxidative method of Bloor, as modified by Wilbur, (personal communication to Mrs, Merva K,Cottle), and finally employing a nitrogen atmosphere to increase reproducibil-ity of this method, i t was decided to modify the procedure slightly by determining the phospholipids directly instead of through the interven-tion of an excess of oxidizing agent. It had been noted that the amount of material centrifuged down in the acetone-precipitation step was a l -ways proportional to the known phospholipid content of the lipid solu-tions being studied; yet the colours produced upon the most carefully controlled oxidation of duplicates often were very dissimilar, and only occasionally were the determinations fully satisfactory. Since Lovern (1953) has recently pointed out the great difficulties in obtaining repro-ducible results with the long "established" method of Bloor, especially with regard to the use of magnesium chloride, and on the advice of Dr, D.R,Idler, Chemist at the Pacific Fisheries Experimental Station, Van-couver, British Columbia, i t was decided to precipitate the phospholipids in the absence of magnesium chloride by using three volumes of chilled ace-tone to one volume of ether extract of the lipids and chilling this mixture for one-half hour. Difficulties, however, were s t i l l present, and weighing of the washed chilled-acetone precipitate in the centrifuge tube was tried. The method was most successful, quite readily reproducible, checked well with previous multiple determinations using the microoxidative method, and was simple and rapid, especially where ether solutions of lipids were to be assayed, as in this work. In addition, i t was found that the precipitate weighed contained less than 0.060 milligram cholesterol, which i s within the limit of accuracy of this new phospholipid method. Additional evidence suggests that this precipitate contains under 5 micrograms of cholesterol. The supernate, which can be used for cholesterol determinations, was found to contain less than 0.06 milligrams of phospholipid. Thus the phospholipids precipitated appear to be of sufficient purity to indicate the usefulness of the method in following changes in the phospholipid content of goldfish tissues. 3. Total lipids (method greatly modified) Since the anhydrous sodium sulphate - dry ether extraction of goldfish tissue yielded surprisingly l i t t l e l i p i d material, and since i t has often been pointed out that ether extraction of tissues fails to break some lipids from their protein binding, while hot alcohol extraction overcomes this difficulty (Leathes, 1925; McArthur, 1952; personal communi-cation to Dr. W.Hoar; Fisheries Research Board of Canada, Bull. 89, 1952) extraction of tissue dried with anhydrous sodium sulphate was carried out with hot Bloor's mixture (3 parts ethyl alcohol to 1 part anhydrous peroxide-free diethyl ether). It was soon realized that the materials extracted in this manner were not a l l ether-soluble. To obtain results that could be validly compared with the ones already obtained in this laboratory, the ether-solubles were exhaustively extracted from the weighed Bloor's solubles, leaving a residue which was ether-insoluble, probably gangliosides or other carbohydrate-containing compounds. This ether-insoluble residue was weighed, giving by differences the weight of total ether-solubles in the extracted tissue. This method was adopted for extraction and determination of total lipids. However, this method is not entirely suitable for further quantitative analysis of the ex-tracted lipids as the residue must be dried for an extended period, until i t comes to constant weight. During this time i t is very likely that cer-tain labile lipids, especially phospholipids, and highly unsaturated oils, could break down to a considerable extent, despite the fact that the dry-ing i s done in vacuo. For this reason, lipids for determination of the cholesterol content, iodine number, and phospholipid content, were ex-tracted from the tissue mince mixed with anhydrous sodium sulphate, in a Waring Blendor, using anhydrous peroxide-free ether as solvent. The ex-tracted lipids in ether solution were placed in sealed glass-stoppered flasks stored in the dark. Total lipid determinations were made on these ether solutions for aliquots of fixed size (4 mis.) as a basis for calculation of percen-tages of cholesterol and phospholipids in the total lipi d extract, and in order to estimate the volume of the aliquots that should be taken for determination of the iodine numbers. In view of the excellent results ob-tained using the new semimicrogravimetric method for phospholipids, the total lipids were determined from these solutions i n an analogous way. The measured aliquot (4 mis.) was placed in a weighed 15 ml. centrifuge tube, the tube tightly stoppered with a cork previously pierced with a knitting needle, and placed in a vacuum chamber adjusted so that the vacuum slowly increased. This arrangement eliminates "bumping" and maintains an ether atmosphere above the residue until the vacuum i s highest and the tube ready to be reweighed. This semimicrogravimetric method, using a sensitive chainomatic balance, proved to be rapid, simple, precise, and accurate. 14 G. Cholesterol Cholesterol was determined by the routine Liebermann-Burchard method, as used by Cottle (1951* unpub. notes). Great care was exercised to keep the reagents anhydrous. Automatic pipettes were used to eliminate pipetting hazards, to speed the determinations, and to increase precision. A cholesterol standard was run with each series of unknowns. Apart from these minor ones, no changes were necessary. D. Iodine Number The routine method of Wij was employed. Cottiers finding, that the iodine number determined was partially dependent upon the weight of the lipids used i n the determination, was confirmed, and care was exer-cised to keep the weight of the lipids used for determination of iodine values to 100 mg. * 10 mg. Precision and accuracy of the unmodified method were excellent. 15 RESULTS A. General Observations Data obtained from duplicate and triplicate dietary experiments, together with data upon the feeding responses, general laboratory history and condition of the goldfish stocks point to suitability of the data for detailed analysis. However, due to the mass of data assembled, graphic analyses will be used in place of complex statistical treatments, in or-der to present the salient findings in readily appraisable form. Consid-erable data were taken during the thermal standardization and dietary admin-istration phases of the project, and these are on f i l e with Dr.W.S.Hoar of this laboratory. It suffices here to point out that a l l goldfish were in apparently good condition throughout the dietary treatments, and only once was disease noted in a few fish, and these were quickly discarded. It was noticed several times during the feeding portion of the experiments that goldfish on Diet F were most prone to come to the surface of the well-aerated aquaria, and gulp air, while goldfish on a l l other treatments, tinder identical conditions other than diet, rarely, i f ever, did this. When-ever fish were found at the surface, the aquarium water was changed, and no increase in the very low mortality rate occurred at these times. It i s necessary also, to observe that while the effect of feeding diets A,B,C, and D for 38 to 42 days was not found to be quan-titatively reproducible in terms of cold-resistance, determined for three separate stocks of fish, the trends are very clearly shown (Figures 7, 8, 9). Using less complicated diets (P,E, and F), i t i s seen that both thermal resistance (Figures 11 and 12) and chemical determinations (Figures 13,18,20, and 21) were considerably more reproducible, at 130 feedings (45-46 days). These findings in themselves are insufficient to prove the validity of comparisons made, however, without consideration of other factors found to influence the cold resistance as measured in these experiments. These influencing factors were found to be the size and sex of the fish, the season, and the proportions of fish exhibiting shock, narcosis, and true death in response to chilling. Of these, the shock effect has been largely eliminated by waiting one hour or more before removing torpid fish from the c h i l l tank; the seasons during which the tests were run have been purposely selected; and the other factors have proved largely uncontrollable. Instead of attempting partial control of this latter group of factors, sufficient numbers of goldfish have been used and careful recording of these factors (narcosis or true death in the cold; weight, length, and sex) has been carried out. Data assembled in this way have been analyzed graphically for a l l factors listed. TABLE III AVERAGE SIZE OF GOLDFISH REMOVED EARLY, INTERMEDIATE, AND LATE IN COLD TESTS Diet Early Period Av.Leng. Av. Wt. Middle Av.Leng. Period Av.Wt. Late Period Av.Leng. Av.Wt. A 72.3 8.75 75.8 10.62 82.2 11.76 B 70.6 8.01 76.0 9.56 79.9 10.39 C 71.8 8.09 80.5 13.48 81.6 11.12 D 74.4 9.22 78.8 11.74 82.1 12.22 P 78.9 10.5 79.9 10.9 82.0 13.3 E 76.6 9.09 80.9 11.7 85.2 13.2 F 80.2 10.7 80.8 11.3 90.0 15.7 B. Influence of Non-Dietary Factors upon Cold-Resistance 1. Size Larger goldfish tend to die off last in chilling tests. This is very apparent in Table III and less apparent but s t i l l noticeable in the relationship between median lethal time for 50 percent true death (MLT50) and mean length of the goldfish chilled, especially for diet P fish (Figure 1), and also in the relationship between MLT50 and mean weight of the goldfish, especially for diet E fish (Figure 2). Thus the To follow page' 17 SUMMER WINTER a o .1 o 16 / p - / F—F x \ \ > — > 1 1 1 ! 1 I 1 1 1 73 7.9 AVERAGE LENGTH 91 CM. Figure 1. TRUE RESISTANCE TO CHILLING AS RELATED TO BODY (FORK) LENGTH Letters on the lines identify the diets fed to the goldfish prior to chilling. MLT50 i s the median lethal time for 50$ of the sample. To follow page 17 SUMMER WINTER 1 " I AVERAGE WEIGHT ;ure 2. TRUE RESISTANCE TO CHILLING. AS RELATED TO BODY WEIGHT Letters on the lines identify the diets fed to the goldfish. MLT50 i s the median lethal time for 50% of the sample. 18 "effect" of size i s apparent both within a given cold-test and between cold-tests, though not between diets or seasons. More minute preliminary analyses of the data give evidence for a positive correlation between weight/leng"th or condition coefficients, and MLT50. This confirms and expands upon the findings of Smith (unpub.), and Keys (1931). 2. Sex Female goldfish tend to die off faster than the males, under identical conditions of chilling following identical dietary and thermal history. The tendency is quite pronounced, in terms of true death rate. However, the males tend to enter a deep cold narcosis more often than female goldfish do, and consequently, since deep narcosis i s far more common in the early portion of each cold-test, the greater tendency of the females to die off in this period i s considerably obscured when only the conventional "removals" data are considered. Furthermore, i t was noted that the stocks of fish used had consistently an overall sex ratio of one male to 0 . 8 4 female. Hence even a one male to 0 .90 female sex ratio of dying fish in the early portion of a cold-test represents a considerable differential mortality rate tending to eliminate the females. In the last third of the average cold test, the sex-ratio of dying fish i s in the neighbourhood of one male to 0.7 female, since most of the originally smaller number of females in the sample have already died. The opposing effects of sex upon the mortality rates of goldfish are evident in Table IV and in Figures 3 and 4 . The dome-shaped curves resulting from plotting mean time for 50 percent removed (MRT50) against percent of males in the sample is thought to be a reflection of the opposing factors, males tending To follow page IS Figure 3. APPARENT RESISTANCE TO CHILLING AS RELATED TO SEX Letters on the lines identify the diets fed to the goldfish. MRT50 i s the median removal time for 50$ of the sample -this value includes deep cold-narcosis. To follow page 18 _ J I I I L t I L IO 30 50 70 PERCENT MALES Figure 4-TRUE RESISTANCE TO CHILLING AS RELATED TO SEX Letters on the lines identify the dietary pretreatments; letters with subscript 1 are Winter fishj those without subscripts are Summer fish. MLT50 i s the median lethal time for 50% of the sample. 19 TABLE IV SEX-DIFFERENGES IN GOLDFISH KILLED OR NARCOTIZED EARLY, INTERMEDIATE, AND LATE IN COLD TESTS Mortality Early Period Middle Period Late Period Overall Measure Males Females Males Females Males Females Males Females Removal 39/45 52/44 44/25 135/114 1:1.15 1:0.85 1:0.57 1:0.84 Deep Narcosis 15/12 8/5 1/0 24A7 1:0.80 1:0.63 1:0.00 1:0.71 True Death 23/32 38/30 45/26 106/88 1:1.39 1:0.79 1:0.58 1:0.83 to decrease the removal time by entering deep narcosis; females also de-creasing the removal time by actually dying off. Conceivably, the combined effects would be at a minimum where there i s an approximately one-to-one sex ratio, as shown in the data. Further analysis of the data has shown that females do not differ significantly from the males in average length or in average weight, and therefore the effect of sex i s distinct from that of size. 3. Season There appears to be a seasonal effect of considerable importance, both in cold resistance and in bio->chemical response to diets. This effect was noticeable in the results for hydrogenated pilchard o i l diets (Hoar and Cottle, 1952), as well as in the present data for diet series P,E,F, and also for series A,B,C,D. The difference i n cold resistance of fish fed a given diet for a certain number of days while maintained at a con-stant temperature during the winter is characteristically greater than that of fish treated in an identical manner in the summer (Figure 5). This phenomenon was responsible for the failure of one cold-test in which fully standardized and acclimatized goldfish, following identical dietary treatments, were chilled at the same temperature as used for their summer "analogues". There was complete survival. It is tempting, of course, to state unequivocally that these effects are truly due to season. However, i t is felt that while i t is very probable that these great differences in cold resistance are indeed seasonal per se, further work must be done to test the validity of this contention. Thus i t was found in the present work that the Winter fish tested with Diets P,E, and F, were significantly larger than the Summer fish tested with the same diets. In addition, chemical evidence points to a higher total lipid content, and lower moisture content in Winter fish than in Summer fish, but this may also be interpreted as a greater rate of modification of tissues by dietary means in Winter fish. Similar to these findings is the discovery of much higher concentrations of lecithin in Winter haddock than in Summer ones (Bahr and Wille, 1931). Tending to support the concept of seasonal effects, also, is the observation that trend lines on the graphs rarely tend to encompass both Winter and Summer points, while the points tend to become grouped by seasons, as well as by To follow page 20 4 0 30 — O 5 2 0 — IO — jnnL IVI52 IV92 IV63 nniu jnn Figure 5. APPARENT RESISTANCE TO CHILL BIG AS RELATED TO SEASON Open bars, Winter fish Striped bars, Summer f i s h . Letters identify dietary pretreatment. IV series from data of Cottle for partially hydrogenated pilchard o i l diets. diets. In addition, repeated dietary experiments check well during the same season, but not well for different seasons. Thus, comparing a l l the data, but especially Diets C and D, there has been found evidence suggestive of greater importance of dietary cholesterol in the summer, but dietary phospholipid in the winter, in promoting cold-resistance of the goldfish. Lending added support to this view is the finding that the only diet regularly providing markedly increased cold resistance in both seasons is heavily supplemented with both cholesterol and phospho-lipid (Diet D). See Figures 6,7,8,9. It is clear that the seasonal factor warrants further attention. G. Effects of Diets upon Cold Resistance As has been noted in the preceding section, the measure of c h i l l -mortality usually employed gives estimates which are the resultant of the two factors: deep cold narcosis, and true death due to chilling. In the terminology of this report, both "true deaths" and "recoveries" together constitute the "removals". The "removals" are known to be slightly farther on in the process of cold-induced necrobiosis than goldfish just failing to respond to prodding with a glass rod at the caudal peduncle. Nevertheless, the relative picture obtained by the prodding method will most probably correspond very closely with that obtained by the removal method. Certain-ly there is a closer relationship between "prodding" data and "removal" data than there is between prodding data and true death records. Thus the . work done previously in this laboratory may be analyzed in terms of "removals". To follow page 21 I ' 1 I L I _ L 3 4 5 6 PROBITS i Figure 6. RESULTS OF CHILL-TEST 6 Diet lQ • Diet (EL Diet .k. Diet B 11 111 111 i t i i i To follow page 21 SO IO O * 5 [ = — r PERCENT DEAD I0 20 40 60 80 90 " i — i — i i i i i i i i i i i i i — i — r - I • _ i j- _ 5 PROBITS Figure 7. RESULTS OF CHILL-TEST 7 Diet D Diet £. Diet A. i » 1 1 1 » i i 11 Viet I To follow page 21 Figure 8. RESULTS OF CHILL-TEST lUl Diet D) Diet C Diet Af\ Diet B 1 1 1 1 1 1 1 1 1 To follow page 21 J 1 I I I L 3 4 5 6 PROBITS Figure 9. RESULTS OF CHILL-TEST 252 Diet IP Diet C Diet A 22 It is crucial to distinguish between removals and true deaths, not only because sex differences have been found to exert different effects upon the two measurements, but also because certain diets have analogous differential effects. In both cases this means that there is an effect upon frequency at which deep narcosis is found in the sample chilled. The tendency for deep narcosis to develop drops off rapidly about one-third of the way through each cold-test. However, i t must not be assumed that the tendency for removal curves for different diets to come permanently closer together by the mid portion of cold tests i s due to disappearance of the deep narcosis factor. This is not always the correct interpretation, for the cold-tank temperature was regularly allowed to drop slowly from the i n i t i a l approxi-mate 2.8° C. to near 0.4° C. in 48 hours, and therefore later in a cold test is also colder in that test. This technique ensures a constant severe cold-stress upon a l l specimens but renders difficult the interpretation of time-effects within a chill-test. YJIth these points in mind, a survey of the dietary effects should be more meaningful. The physiological and biochemical effects of the diets administer-ed will be shown graphically and description will be largely limited to references to the graphs illustrating the features mentioned. 1. Physiological Results of Experiments with Diets A,B,C, and D Despite the complexity of these diets, several features are evi-dent in the cold-resistance of fish consuming them. Thus Diet A fish, consuming the most o i l but l i t t l e lecithin and virtually no cholesterol, showed the poorest cold resistance after 38 days of feeding. (Figures 7> 8,9). Diet B fish, consuming second most o i l , l i t t l e lecithin, but most 23 cholesterol, exhibited about the same degree of cold resistance i n the summer as did diet A fish, but much greater resistance than Diet A fish when tested in winter. This protection against cold was particularly-evident in the first half of both cold tests, and may be termed " i n i t i a l protection" (Figure 10), This i n i t i a l protection offered by Diet B i s evident in both "removal" and "true death" c h i l l mortality data. Consid-ering next Diet C (less o i l , more lecithin, no appreciable cholesterol), i t i s seen (Figures 8 and 9) that this diet offered goldfish increased resistance to cold, especially in the winter. Diet C afforded more pro-tection with longer feeding period, at least up to 42 days, and afforded the greatest protection of any of the ABCD series of diets i n the winter. Diet D, on the other hand, containing least o i l , more lecithin, as well as much cholesterol, gave greatest cold resistance of the series, during the summer, and second-greatest cold resistance i n the winter. Diet D was the only diet of the ABCD series which was consistently "beneficial" (relative to Diet A) in both halves of the year. In brief, then, the ABCD experiments show increased c h i l l resistance associated with cholesterol supplements i n the summer, and with lecithin supplements in the winter. Supplements of both together provided increased cold resistance in both seasons. The lecithin effect was spread over the whole duration of each cold-test, whereas the cholesterol effect tended to be present only in the i n i t i a l period of c h i l l . 2. Physiological Results of Experiments with Diets PtE, and F No great changes in true chill-death rate were produced by Diets E and F as compared with Diet P as control. When noticeable effects i n To follow page 23 Figure 10. DIETARY CHOLESTEROL AND COLD RESISTANCE c Circles in order from the centre show 20, 40, 60 and 80$ mortality. The seven axes show the effect of two high cholesterol diets, fed for varying periods. Solid lines joining the axes are controls; broken lines, chol-esterol-fed fish. Inner stippled band shows protective effect at 2|r hours' exposure; outer band shows variable but usually slight or detrimental effect (V marks) during prolonged exposure (15 hours). true chill-death rate were produced by the two lipid-supplemented diets, these effects were usually somewhat detrimental. However, quite a differ-ent picture was found for those diets in terms of "removals", and since most work in the field of chill-resistance has been done using such a criterion of death, and also since the degree of "torpor" of a fish at chilling temperatures is also some measure of its cold-resistance, the data obtained appear here in considerable detail. Consequently, unless otherwise stated, the data regarding fish on Diets P,E, and F are for "removals". To simplify assimilation of the data obtained, the time required for 25 percent removal of Diet P (Pablum, = control) fish was determined for each cold-test, and the conversion factors necessary to convert each of these numbers to "one" (arbitrary unit of physiological control time), were calculated. The appropriate conversion factor was then multiplied by the actual time required for the corresponding Diet E, and the corres-ponding Diet F fish to reach 25 percent removal. This gave the ratio of resistance time for the treatments (E,F) to the resistance time of the controls (P). These values are relative median removal times (RMRT), and since these were calculated not only for the 25 percent level of removals, but also for 50 percent and 75 percent removals, subscripts are used to designate the level of removals to which the relative time values corres-pond. For example, RMRT50 means "the relative median removal time for 50 percent of the sample", and indicates the ratio of the time i t took 50 percent of the experimental sample to become so torpid as to be or appear dead in the cold-tank, to the time i t took 50 percent of the control sample to reach the same degree of torpor under identical conditions. The original resistance times were taken from smoothed plots of cumulative percent dead versus log time using log-probit paper> according to accept-ed methods of graphic analysis of quantal response. (Miller, 1950). Curves obtained by this same method were compared as such, without further treat-ment in the case of cold-test responses of fish on diets A,B,C,D, and Q. The complexity of the physiological responses to Diets E and F makes i t highly desirable to consider Diet P responses to cold merely as the reference level for evaluating the effects of Diets E and F. Therefore no separate discussion of the cold resistance of Diet P fish w i l l be given. Diet E Diet E (half Pablum, half cholesterol) was.found to increase the thermal resistance of goldfish very greatly, the effect becoming greater with increasing numbers of feedings. This effect was found to be most marked i f 25 percent removal was taken as the comparison level; less marked at 50 percent removal level, and unimportant at 75 percent level. Further-more, the picture of increasing resistance with increasing numbers of days on diets (number of feedings) found at RMRT25 ^ ad changed to a clearly peaked curve of the form (Figure 11) by RMRT50. These observa-tions, plus the fact that Diet E has either l i t t l e effect or a detrimental effect in terms of true death rate due to chilling, indicated that the protection offered by Diet E was really protection from deep cold narcosis. Further analysis of the data has proved this point (Table V). In summary, then, Diet E fish have been found to have increased cold resistance over Diet P fish, the increase being proportional, up to a To follow page 25 Figure 11. THE TIME EFFECT IN DIETARY MODIFICATION OF CHILL-RESISTANCE The horizontal line at 1 represents Diet P gold-fish. Letters on the respective lines identify the other goldfish by diet. RMRT50 i s the median removal time for 50$ of the sample compared as a ratio, to the median removal time for 50$ of the controls. TABLE V ANALYSIS OF "INITIAL PROTECTION" DUE TO DIET E: NUMBERS OF GOLDFISH SHOWING DEEP COLD NARCOSIS EARLY, INTERMEDIATE, AND LATE IN COLD TESTS Days Fed Early Middle Late Overall Diet P Diet E Diet P Diet E Diet P Diet E Diet P Diet E 7 I/O O/O 1/0 2/0 21 3 A OA 0/0 3/2 25 6/5 4/3 1/0 11/8 46 5A 1/0 0/0 6 A Totals 15/7 5/4 2/0 22/11 certain point, to the duration of dietary treatment, and being due to a decreased tendency of these cholesterol-supplemented fish to enter deep cold—narcosi s• Diet F The data obtained for Diet F fish subjected to the same cold-tests exhibits a pattern remarkably similar to that obtained for Diet E fish, in comparison with those of the control diet (P). However, for Diet F, the RMRT50 and also the RMRT25 curves, while of the same general shape as that sketched above for Diet E fish, differ from the E curve in their timing. The Diet F fish pass through the stages of the curve more rapidly than Diet E fish do. Also, the Diet F fish follows the sketched curve not only for 50 percent removals, but also, unlike Diet E fish, for 27 25 percent removals as well. That i s to say, the "Granulestin" (phospho-lipid) supplemented goldfish during the course of continual dieting gain cold resistance sooner and lose i t 3D oner than do cholesterol-supplemented goldfish. This i s clearly seen in Figures 11 and 12. Finally, i t should he observed that the relative cold-resistance of Diet E fish (cholesterol-supplemented) i s on the average higher than that for Diet F fish (phospho-lipid supplemented) in the summer, but not in the winter. Diet Q was designed to test the effects of a diet chemically midway between Diets A and F. (Table 1). Feeding Diet Q (Pablum supple-mented with animal lecithin only) resulted in cold-resistance changes very closely paralleling those found with Diet C fish undergoing identical tests at the same time. The "removals" picture for the two Diets Q and C was the same, while the true death picture showed the Q fish to be slightly less resistant than G fish, when those receiving the same number of feed-ings were compared. It appears from these preliminary tests that the amount of pilchard o i l in the diet had l i t t l e influence upon the results obtained, at least in the case of lecithin supplements. It is also note-worthy that without the highly unsaturated o i l supplement, only one-half the amount of lecithin was required for equal changes in cold resistance, 3. Biochemical Results of Experiments with Diets P,E, and F Progressive changes in tissue lipids occurred in control fish (Diet P) as well as in experimentals (Diets E and F). It was therefore decided to record the biochemical data in two ways: fi r s t , absolute values of each fraction determined and for each diet, plotted against number of days fed, to show something of the dynamics of the processes occurring in To follow page 27 4 6 WEEKS ON DIETS Figure 12. THE TIME EFFECT BI DIETARY MODIFICATION OF CHILL-RESISTANCE The horizontal l i n e at 1 represents Diet P goldfish. Letters on the respective lines identify the other goldfish by diet. RMRT25 i s the time required for 25p removal on a specified"diet, referred back to the unit of time i t took for 25$ removal of the respective controls. dietary modification of the goldfish; and second, relative values for these fractions, calculated analogously to the relative mortality rates described in the preceding section. The relative values for the various quantitative analyses of the goldfish tissue are a l l referrable to the value for these respective analyses conducted upon Diet P fish of the same cold-test, the values for Diet P fish being reduced to unity. For this reason chemical values for only Diet E and Diet F goldfish appear on the relative value graphs, Diet P values having been adjusted to "one unit" hence forming the straight line parallel to the X-axis, and cutting the Y-axis at one unit. The results, both absolute and relative, arranged by diet, are presented descriptively, with reference to the pertinent graphs and tables, in the following sections. Chemical Effects common to Diets P.E. and F All these diets caused a progressive increase in the total l i p i d content of the tissues (Figure 13), and a concomitant progressive decrease in the moisture content of the tissues (Figure 14). Apparently the winter fish either contained more lipid and less water upon arrival "at this l a -boratory, or they responded more rapidly to the diets. This matter i s being investigated. Examination of the inverse relationship between water and total lipi d content of goldfish tissue was extended to data gathered for Diet P fish which had been used as controls in preliminary hormone ex-periments, and previous experiments conducted by the author, and also to data collected by Merva K. Cottle for Pablum-fed fish acclimatized to different temperatures. It was found that there is a precise inverse relationship between the log percent ether-extractable lipids and percent To follow page 28 Figure 13. THE RATE OF DIETARY MODIFICATION OF LIPID CONTENT OF WET GOLDFISH TISSUE Letters on the graph identify the dietary treatments To follow page 28 Figure 14. THE RATE OF DIETARY MODIFICATION OF WATER CONTENT OF GOLDFISH TISSUE Letters on the graph identify the dietary treatments. Extrapolation to zero time is hypothetical. moisture (Figure 15), and a less precise relationship between the log percent ether-soluble Bloor's-extractable lipids and percent moisture. (All percentages here are calculated on the basis of wet tissue weight). This inverse relationship between percent lipids and percent water is found to be independent of goldfish stock, season, acclimatization tempera-tures, death temperature, and in the present study, diet. These findings confirm and extend the finding of Hoar and Cottle (1952) that there is an inverse relationship between water content and lipid content of goldfish acclimatized to different temperatures. Chemical Effects of Diet P As noted above, feeding Diet P (or E or F) resulted in a large increase in total lipi d content of the tissues. Diet P may be regarded as a high-carbohydrate ration, and while i t is true that there is 3.0 percent lipids in the diet, i t is probable that much of the fattening resulted, as in mammals, from biosynthesis of fat from excess carbohy-drate in the body. If this were so one would expect an increased satur-ation of the extracted "fats". This was the case upon prolonged feeding (Figure 16). Goldfish fed Diet P also showed a slow but constant de-crease in the ratio of cholesterol to total lipids, and a slow constant decrease in cholesterol to phospholipid ratio. The phospholipid content of the tissues rose slowly throughout the 65 days, while cholesterol con-tent rose slowly for 45 days and then f e l l slowly for at least twenty days. Such unavoidable changes in the composition of the control fish made i t imperative to express the experimental data ultimately in rela-tive units, based upon the constitution of the controls for each stage To follow page 29 J I I I I I L_ 74 76 82 86 PERCENT WATER Figure 15. THE INVERSE RELATIONSHIP BETWEEN TISSUE WATER AND ETHER-EXTRACTABLES IN THE GOLDFISH A l l points plotted are for goldfish fed Pablum only, but at different seasons and at acclimatiza-tion temperatures from 5° to 35° C. To follow page 29 Figure 16. THE CHANGES IN TOTAL LIPID UNSATURATION PRODUCED BY CONTINUED FEEDING OF DIETS P, E, and F. Letters on the graph identify the dietary treatments 30 at which comparisons were to be made. Comparison of the absolute-unit graph Figure 18 with the corresponding relative-unit graph Figure 22 re-veals the simplification afforded by plotting relative values. Chemical Effects of Diet E Diet E had spectacular effects. The water content of summer goldfish receiving this diet was consistently much higher, and the lipid content much lower than these values for goldfish on Diets P and F. (Figures 13 and. 14). Also, the cholesterol content of the Diet E fish was increased at least fourfold, reaching its highest concentration in 46 days, and then dropping slightly (Figure 17). The phospholipid con-tent of these fish, however, reached a peak in 25 days, and then f e l l for at least 21 days (Figure 18). The methods used for determining phospho-lipids measured only those which were ether-soluble, and this may explain why Diet E (cholesterol-supplemented Pablum) resulted in the highest con-centration of phospholipids in the tissue, while phospholipid supplements (Diet F) did not alter their tissue level. The ratio of cholesterol to phospholipid and cholesterol to total lipid were rather irregular, but of the same trend. (Figures 19 and 20). The data available show a progressive saturation of the total body lipids with continued administration of Diet E. It was noted that by the forty-fifth day of feeding, the extracted lipids were quite solid at room temperature. Upon evaporating the Bloor's extracts of these fish to dryness under vacuum at 50° C, i t was found that the nearly solvent-free residues of only Diet E fish extract spattered ex-tensively. This phenomenon may possibly be related to the greater satura-tion of these lipidsj at least, i t shows a distinct difference between the To follow page 30 Figure 17. THE CHANGES IN TISSUE CHOLESTEROL LEVEL PRODUCED BY CONTINUED FEEDING OF DIETS P, E, and F. Letters on the graph identify*the dietary treatments. To follow page 30 Figure 18 THE CHANGES IN TISSUE PHOSPHOLIPID LEVEL PRODUCED BY CONTINUED FEEDING OF DIETS P, E, and F. Letters on the graph identify the dietary treatments. To follow page 30 O 3 6 9 WEEKS ON DIETS Figure 19. PROGRESSIVE CHANGES IN CHOLESTEROL/PHOSPHOLIPID RATIO PRODUCED BY PROLONGED FEEDING OF DIETS P, E, and F. Letters on the graph identify the dietary treatments. Figure 20. PROGRESSIVE CHANGES IN CHOLESTEROL/TOTAL LIPID RATIO PRODUCED BY PROLONGED FEEDING OF DIETS P, E, and F. Letters on the graph identify the dietary treatments. body lipids of these fish and those on Diets P and F, which never ex-hibited spattering. « A brief look at the relative picture shows (l) that the percent cholesterol in the body lipids of Diet E fish increases over the same value for Diet P fish, at a constant logarithmic rate for the first 46 days in the summer experiments, but (2) the relative percent cholesterol in the body lipids then decreases slightly. (Figure 21). It is also apparent that for diet E fish the relative percent cholesterol in body lipids is somewhat correlated with the RMRT25 or RMRT50 (relative median removal times), Figures 11 and 12. It should also be noticed at the same time that the correlation between relative percent cholesterol in body lipids of Diet F fish and their cold-resistance, is very weak indeed). Chemical Effects of Diet F Chemical effects of this diet were less pronounced than might be expected. There was no consistent difference from the control in water content of the tissues, and while i t appears that the Granulestin supple-ment increased the rate of lipid uptake or synthesis, but later tended to keep the lipid content regulated at a moderately high level (as could be expected - West and Todd,01951)> conclusive evidence is not yet avail-able. However, feeding this diet did produce an i n i t i a l "crash" in the cholesterol content of the goldfish. (Figure 17). Following this, the cholesterol content slowly and apparently erratically increased. The cholesterol to phospholipid and cholesterol to total lipid ratios both f e l l i n i t i a l l y in Diet F goldfish, and then from 25th to the 65th day, To follow page 31 z o 9 Q. o 10.0 o o: U J O I O 4 6 WEEKS ON DIETS Figure 21. PROGRESSIVE CHANGES IN CHOLESTEROL/TOTAL LIPID RATIO PRODUCED BY PROLONGED FEEDING OF DIETS P, E, and F. The concentrations of cholesterol found are expressed in terms of control units, the respective control cholesterol content being taken as unity. Diet P fish are represented by the horizontal line at 1; letters on the graph identify the other fish by diets. virtually coincided with Diet P values, tending to l i e very slightly above the latter. (Figures 19 and 20). Administration of Diet F caused a pro-gressive increase in Iodine number. This may be an zntioxidant effect. Rather unexpectedly, the phospholipid content of the goldfish on Diet F, receiving 50 percent of their diet as phospholipids, was no higher than control values on the average. This may or may not be due to the fact that only ether-extractable phospholipids were measured in these studies. The values for phospholipid content of the tissues of Diet F fish were found to follow a dome-shaped curve, when plotted against "days fed", with a very slight crest near the 25th day of feeding. (Figure 18). Finally, the rela-tive phospholipid content curve (Figure 22) i s of a rather unusaal form, but this same form is also quite noticeable in the curve depicting relative cold resistance of Diet F fish. (Figure 11). During this comparison i t is also noticeable that there is a much weaker correlation between rela-tive phospholipid content of the tissues of Diet E fish and their rela-tive cold resistance. D. Summary of Results Although length, weight, and sex of the goldfish used were found to modify observed cold resistance, repeated attempts to explain the ob-served cold resistances, using these three factors, without regard to diet or season, failed entirely. This strengthens the argument that the observed cold resistance i s predominantly a function of season and diet. Tests of biochemical and physiological reproducibility and continuity of these ex-periments also showed that diet tended to override the other factors to a large extent, but suggested that seasonal effects were more important than To follow page 32 IO.O 4 6 WEEKS ON DIETS Figure 22. PROGRESSIVE CHANGES IN PHOSPHOLIPID CONTENT OF GOLDFISH, AS PRODUCED BY CONTINUED FEEDING OF DIETS P, E, and F. The phospholipid concentrations are plotted in terms of control units of phospholipid. The percent phos-pholipids in wet tissue of the respective control is reduced to unity. Diet P fish are represented by the horizontal line at 1; letters on the graph identify the other fish by diets. 33 heretofore believed. Physiological and biochemical data and comparisons (as delimited by the purpose of this research), have been graphed for ready appraisal. Physiologically, the graphs show that cholesterol supple-ments as well as phospholipid supplements increase cold-resistance, the relative effectiveness of either depending upon season, concentration in the diet, and number of days on the diet. Biochemically the graphs show that while Diets P,E, and F a l l cause progressive fattening of the gold-fish, coupled with proportional water loss, there are specific biochemical changes produced by each diet, viz,, P produced only small specific ef-fects, whereas E caused a great decrease i n Iodine number and total l i p i d content as well as a great increase in cholesterol and total water content of the tissues, while F produced an increase in iodine number and a sharp i n i t i a l decrease in cholesterol. Also significant was the finding of a characteristic relationship between relative cold resistance and number of days on diet. This relationship differed in detail and timing between Diets E and F in relation to P. The relative time-resistance relation was closely correlated with relative body cholesterol content for Diet E fish, but with the relative body phospholipid content for Diet F fish. 34 DISCUSSION Several aspects of the relationship between lipids and physio-logical thermal resistance have recently been investigated using the gold» fish. Lipid unsaturation in relation to temperature was srtudied by Hunter (1948) by varying the environmental temperature, and by Dorchester (1948), by feeding saturated and unsaturated fats, then subjecting the fish to thermal stress, Cottle (1951)• studied the relationship from both these approaches, and also determined the effects of acclimatization tempera-ture upon other li p i d fractions, notably phospholipids and cholesterol. In the same period, Smith (1950) worked upon a detailed analysis of the whole c h i l l reaction in the goldfish. The value of such a study to workers in the field of lipid/thermal stress interrelationships is obvious. How-ever, the discussions and bibliographies of these papers are of only limit-ed interest here, for they are not directly concerned with dietary choles-terol and phospholipids. Duplication of this previous discussion and bibliography will be avoided here. By way of summary, however, the salient features follow. While body lipids are less saturated at lower acclimatization temperatures, and lipids may be made less saturated by diet, such diet-evoked unsatura-tion i s not consistently beneficial to chilled goldfish. In fact, there is no consistent relationship between degree of diet-evoked saturation and heat-resistance either, even though a direct proportion exists between acclimatization temperature and saturation of the body lipids for a stand-ard diet. Similarly, while i t had been shown that high phospholipid levels 35 in the tissues were associated with high acclimatization temperatures, analysis of assembled data showed dietary supplements of phospholipid in-creased cold-resistance, and diet-or hormone-induced high phospholipid levels in the tissues were also associated with enhanced cold - not heat -resistance of these fish (Hoar, 1952, M.S.report). Clearly, there are anomalies in these results as outlined above. Without considering, for the moment, the complexity of the actual situation as given under Results, i t should be pointed out that there may be a simple explanation for the phospholipid anomaly, and a partial explanation for the variability of chemical results obtained by different workers in this field. First, the high phospholipid content of wara-acclimatized gold-fish may not have anything to do with primary physiological adjustment to heat, but may well be merely the result of increased activity of these fish at high temperatures. It is well known that much-used muscles, especially skeletal muscles of active organisms have greatly increased phospholipid content in comparison to little-used skeletal muscles or smooth muscles generally. (Bloor, 1936, Bloor and Snider, 1934). Secondly, as pointed out by Corran (1946), lecithin i s likely to hydrolyze during cold storage to a certain extent, unlike cholesterol which (p.181), "is relatively stable and i s not likely to change appreciably on keeping." The chief uninvestigated possible source of error for the various lipid (or other chemical) determinations i s the effect of the cold-test itself upon the chemical constitution of the goldfish. Thus virtually a l l values so far determined in these studies have been the resultant of the chemical state immediately prior to the test, together with the metabol-ic changes occurring during the test. It is virtually impossible to treat sufficient numbers of goldfish to determine pre-chill and post-chi l l chemical values when different diets, weights, lengths, sex ratios and seasons must be considered. In fact, the reasonable concordance of the data obtained in these studies is encouraging in view of the opinion held by the eminent lipid nutritionist G.O.Burr (1946), that dietary effects of fats give very irregular pictures, inconsistent results on repetition of experiments being usual. Non-Dietary Factors The physiological precision of the methods used, though not extreme, is clearly sufficient, especially i f the modifying factors of size, sex, and season are taken into consideration. Size Size effects (weight, length, surface area, condition co-efficients, etc.) have been demonstrated by several previous workers. Thus Colbert et al (1946) found that small alligators c h i l l more quickly; Keys (1931) found that larger fish (Fundulus parvipinnis) have greater chill-tolerance than smaller fish of the same species. This is related, apparently, to the greater g i l l area relative to body size in these small-er K i l l i f i s h . Finally, F.D.Smith (1950, unpub.) has found a positive correlation between weight over length of the goldfish and the survival times, as well as a negative correlation between the surface area over the weight of the goldfish and the survival times. It has been definitely established that the smaller goldfish are less resistant to chilling. Sex It i s likely that the tendency for female goldfish to die off more rapidly than similarly treated males in the experiments reported here is not an artefact. Further work is required, however, to establish this point, and to test the observed greater tendency of male goldfish to become deeply cold-narcotized. It should be mentioned that no effect upon chil l removal rate was found with testosterone treatment in preliminary experiments carried out in this laboratory. However, in view of the atten-tion afforded to lipids in this study, i t is worth noting that sex differ-ences in lipid metabolish are well-established. Thus Tsuruta (1931), and also Kim and Ivy (1952), have found male-female .differential susceptibil-ity to bile salts, held to be a reflection of differences in lipid meta-bolism between the sexes. Also, Deuel (1952) found that male rats need ten times as much of the essential fatty acid, linoleic, as do female rats. Using estrogens, Stamler (1954) induced a marked hyperphospholipemia in the fowl, and estrogen pellets are currently being implanted in fowl regardless of sex, to increase the muscle lipids. Thus the sex effect found in chilling goldfish may have as a basis, sex-differences in lipid metabolism. Season The seasonal effect apparent in this study should not be too exhaustively speculated upon, until the long-term research necessary to test thoroughly i t s validity and probe its gross mechanism is completed. However, i t should be noted that since Hippocrates, some measure of cor-relation between physiological state (often incidence of disease) and the seasons has been suggested or demonstrated, even taken for granted. That 38 frogs are physiologically very different in the two halves of the year is evident from the habitually used terms of the physiologist, "Winter frog" and "Summer frog". However, the goldfish i s quite active, even in 4° C. water, i f acclimatized, and thus differs from the Winter frog and the even more striking examples of animals showing seasonal physiological differences. The finding of D.J.Smith (1953), that there are seasonal differences in pigs at the pharmacological level, and the finding of Oka (1952) that there is a great seasonal difference in the redox potential of the rat liver in situ, both suggest the reality of the seasonal dif-ference in the goldfish. In view of the constancy of the temperature main-tained for the fish in this laboratory, and in view of the great importance of photoperiodic effects in the physiology of so many organisms, work assessing the effect of photoperiod upon cold resistance of the goldfish may be indicated. Assessment of Non-Dietary Factors It is believed that a l l goldfish used in this study were of approximately the same age. However, without raising our own goldfish, there i s no practicable way of learning much of the past history, or age, or sex ratio of the living stocks of fish. In addition, i t has not been practicable to use large numbers of fish in each cold test. There-fore, instead of calculating the age- and sex-specific mortality rates according to methods of Pearl (1922), the influence of size and sex, as well as other factors, is assessed by graphic analysis of extensive data with suitable replication. In calculating the true death rate, a further precept of Pearl (1922) should be (and was) followed: that a correction be made so that the sample size considered be not the original one chilled, but the original minus those which have proven to be alive, though re-moved to another tank because of their earlier apparent mortality. That i s , a meaningful mortality rate considers the actual fraction of the population exposed to risk at that particular time. These foregoing points were considered first not because they are most important, but because they should be borne in mind for proper evaluation of the rest of the discussion and for the proper assessment of the data in relation to the discussion. Diets in Relation to Theories of Cold Death The dietary factors studied can be related to several theories of cold-death. Among these theories are the: li p i d melting-point theory; calcium-release and fat-release theories; body insulation theory; per-meability and osmoregulation theory; viscosity theory; Selye's stress theory; the anoxia theory; the cold metabolism theory, and the "biologi-cal antioxidant hypothesis." The last-named i s proposed by the author to augment the preliminary; metabolic chart relating biochemically, sub-stances found physiologically to have beneficial effects when administer-ed to animals prior to chilling, or found to drop in concentration signi-ficantly during c h i l l . Melting-Point Theory Although this was not the prime purpose of the s tudy, dramatic evidence was secured showing that the melting point of the total body  lipids bears definitely no necessary relationship to cold-resistance of the goldfish. The most resistant fish had the hardest lipids - exactly 40 contrary to theoretical expectations. It i s clear that some other li p i d fraction of the tissues or some function of the tissues modified by the diet is more important than total lipi d unsaturation. This finding i s not new, and i t has been suggested that i t is the melting point of the phospholipids that i s crucial - not the phospholipids of the blood, liver, and intestinal mucosa, but of the muscles, nerves, and brain. (Terroine et Hatterer, 1930; Sinclair, 1935). Of course, under the conditions of the experiments carried out to date in this laboratory, the relatively very large amount of quite changeable depot fat, and transport phospholipid, would mask any constancy of iodine number (unsaturation) of the "element constant" (cellular phospholipids), and a thorough investi-gation of this possible relationship of m.p. of cellular phosphatides to thermal acclimatization and resistance should be made. Such a study might well embrace a physical study of the sphingomyelins of the goldfish, for Sano (1922) found changes in optical rotation and solubility near 40° C. for cat brain sphingomyelin, possibly related to the upper lethal temperature of this mammal. Calcium Release and Fat Release Theories If the unsaturation of the total body lipids is acceptable as a measure of the unsaturation of the cell membrane lipids, and i f too, the degree of unsaturation of these lipids i s crucial to the calcium release theory, as appears to be the case, then this theory as i t applies to cold- death i s untenable in its present form. The discussion of the melting-point theory above i s pertinent also to this theory. The calcium release theory of Heilbrunn i s paralleled by his less-known "fat-release theory" (Heilbrunn and Daugherty, 1938). It i s interesting to note that there is in this 1938 paper on fat release, a suggestion of a relationship between water content and free lipid in Amoeba, as well as a seasonal effect upon magnitude of the fat release. While i t is true that the fat release was studied using X-radia-tion as the stimulus, Heilbrunn himself points out that any stimulus, and particularly local increases in temperature would also be expected to re-lease fat from the centre of the cells. Presumably chilling would be a satisfactory stimulus for fat-release. In this case, the water l i p i d re-? lationship and the greater "release" of fat in the summer are of consider-able interest, since they parallel two of the findings for goldfish i n the present study. However, these analogies are admittedly weak, and i t will take at least several years of work to test the validity of these parallel theories of Heilbrunn, that of calcium release, and that of fat release, in relation to c h i l l death of the goldfish. Since the work in this laboratory has repeatedly demonstrated the falseness of the oversimplified forms of the melting-point and calcium release theories of cold-resistance, there has been a shift of emphasis to the body insulation, permeability and osmoregulation theories of cold death. Permeability and Osmoregulation Theories Despite the fact that the phospholipid:cholesterol ratio has been found (Hoar and Cottle, 1952) to vary directly with the water content, and acclimatization temperature of the goldfish, no definite correlation be-tween this ratio and either water content of the tissues or thermal resis-42 tance was found in the present study. This means that the present data do not support the theroies of Mayer et Schaeffer (1914), and of Wilbur and Del Porno (1949), which link the cholesterolrphospholipid ratio with water content, and this in turn with cold resistance. However, fair agreement of both cold resistance and water content with the cholesterol: fatty acid ratio of the goldfish tissues is not ruled out. Generally speaking, high cholesterol content in the present study was associated with high percent water, and also with enhanced cold-resistance. High water content was not closely correlated with high cold resistance i n phospholipid-supplemented fish, however. The remarks so far have referred to the permeability theory, but these can be used to apply to the osmoregulation theory as well. More specifically belonging to a discussion of the osmoregulation theory is the finding that phospholipid feeding apparently caused lipoid deposition in the g i l l s . This would almost certainly change the permeability and func-tional efficiency of the g i l l , not only for oxygen, but also for water and possibly chlorides in addition. The magnitude of this effect is com-pletely unknown. Apart from this i t was noted that a definite inverse relationship between total lipids and water content obtained i n a l l the goldfish. This clearly wouldnH be significant for modified chill-resis** tance due to specific diets, however. Body Insulation Theory Recently, some attention has been paid to the idea of thermal insulation, due in part to the-finding of Smith (unpub.) that large gold-fish, (with relatively small surface area per unit volume, and relatively small g i l l area for their total surface) are more chill-resistant than smaller goldfish of approximately the same age. He also found that the decline of body temperature in larger goldfish was slightly slower than in the smaller ones. This size effect has been completely confirmed qualitatively as a result of the present study. The data do not, however, support the idea that cold resistance i s simply a function of body size, or even dominantly so, because diet modified the observed cold resistance much more profoundly than did size of the goldfish used. In addition to mere size, thermal insulation may be due to increased body lipid concen-tration. In the present study, no correlation was apparent between total l i p i d concentration i n the body, and cold-resistance. Viscosity Theory The lipid melting-point, calcium release, body insulation, permeability, and osmoregulatory theories have not afforded satisfactory explanation of the present results. A theory closely allied with the permeability, melting-point, and calcium release theories, namely the viscosity theory, offers a slightly better picture. The principal viscos-ity theory of temperature effects upon protoplasm is that of Belehradek (1935), which proposes that cold increases the viscosity of cells so that processes involving diffusion particularly are slowed to such an extent that l i f e can no longer exist. Due to the partial dependence of the protoplasmic viscosity upon the melting point of the lipids, and the cholesterol:phospholipid ratio, we cannot regard this theory as one likely to explain the present data. It must be stated, however, that the protoplasmic viscosity is dependent upon far more factors than the two mentioned here. It should be made clear that two oils of the same iodine number may have grossly different viscosities, especially as a function of their lecithin content (Fisheries Research Board of Canada, Bull, 87j 1952). It is also noteworthy that lecithin with cephalin promotes dual emulsions, and the nature of the predominant or exclusive phase i s controlled by cholesterol:phospholipid ratio. Very high ratios (9 cholesterol: 1 lecithin) stabilize the emulsion in the o i l in water state (Chem.Pub.Co., 1946). If this happened to occur intracellularly, a very rapid exchange of water and water-solubles across the cell boundary might be expected. The cholesterolphospholipid ratio for whole fish was ob-served to be forced to values this high by feeding Diet E. Associated with Diet E was high tissue water concentration. Beyond stating this parallelism, i t i s unwise to go further at this time. Further discussion of the "fluidizing" function of certain lipids may be found in Kartha (1951). The " f i t " of the experimental data to the theories so far dis-cussed is poor. A much better agreement, requiring no weak analogies or unwarranted extrapolation from theory or data, is found upon examining the present results from Selye's point of view. Selye's Stress Theory Selye's general adaptation syndrome or G.A.S. i s a stereotyped series of histological and biochemical changes in an organism undergoing prolonged stress. The kind of stress matters l i t t l e , and although the outward signs of the G.A.S. are of many different kinds, the internal mechanism of response to stress is remarkably consistent. The onset of stress is marked by shock, during which specific resistance (to the stress-or) and general resistance are both lowered; this is followed by counter shock, where the specific resistance starts to rise above normal, and during 45 which the general resistance (also called "crossed resistance") reaches its supranormal peak; the stressed organism now enters the "stage of resistance", where the specific resistance plateaus at a high level, but the general resistance falls gradually to a very low level. Finally, i f the stressor has not been eliminated by this time, the stage of exhaustion ensues, the specific resistance dropping progressively, through normal, to zero, with the general resistance also reaching n i l . This i s death due to stress. Generally, a specific stressor builds up a specific resis-tance to that stressor only, in the body, depressing the general resistance to other stressors, but occasionally, examples are found where a certain stressor results in increased resistance to a certain group of stressors. This is the less usual, crossed resistance phenomenon. This positive crossed resistance phenomenon i s briefly discussed by Selye in his journal "Stress" (Selye, 1950). If we regard the diets E (half Pablum, half chol-esterol) and F (half Pablum, half Soyabean phosphotides) themselves as stressors, or the conditions they produce in the goldfish consuming them as stressors, then the reason for the observed curves of cold-resistance, and cholesterol versus time, is quite apparent on the basis of crossed resistance developing as protection against not only the real diet-ary lipid stress, but also the non-existent c h i l l (See Selye, 1949, PP. 838;841). Since the temperature of the feeding tanks i s kept constant at the moderate temperature 20° C, the goldfish cannot have experienced ther-mal stress prior to the actual c h i l l test. Because the feeding of phos-pholipids or cholesterol results in the development of high crossed-resis-tance to cold, whereas the specific stressor usually decreases the crossed resistance, i t seems most likely that cold-stress acts upon the metabolism in the same way excessive amounts of cholesterol or phospholipids do. Although the effect of excess dietary phospholipids is not well known, one major effect of excess cholesterol is the selective using-up of highly unsaturated fatty acids to form cholesteryl esters. (West and Todd, 1951, p.917). This explanation (crossed resistance to dietary stressors) i f correct, certainly implicates more than ever, cholesterol and phospholipids in cold-resistance of the goldfish. Once again, the similarity of the relative resistance curves obtained in this research (Figures 11 and 12) to the "classical" curve of Selye's G.A.S. (Selye 1949, p.838) is emphasized. There i s considerable further support for this hypothesis. It is certainly easy to regard the 50 percent cholesterol diet (E) as a stressor, although the goldfish very definitely appeared healthy after consuming 186 feedings of i t in 65 days. In regarding the phospholipid diet (F) as a stressor, i t may be necessary to think of i t as causing partial anoxia of the tissues, resulting in stress in the indirect manner. On the other hand, Deuel points out (1952) that dietary fat can counteract stress, Selye correlates loss of fats and cholesterol with specific phases of the G.A.S. (Selye, 1949, p.843)* The changes given for adrenal cholesterol in the G.A.S. were found for the whole body cholesterol of "Granulestin"-fed goldfish. Further work is certainly indicated, for i t i s difficult to reconcile this hypothesis of dietary stressors with the observation that the fish appeared in excellent health. There was no indication whatever of any deleterious effects of the diets at room temperature, even after two months of feeding. Anoxia Theory Although partial anoxia has been discussed already to a certain extent, the anoxia theory of cold-death has not been. This theory holds that cold-death i s due to failure of some part or parts of the respira-tory system resulting in prolonged severe anoxia of the tissues. Evident-ly, however, i t takes extreme anoxia to k i l l a poikilotherm at chilling temperatures. Goldfish removed from the chilling tank after an absence of breathing rhythm or even any observable isolated breathing movements for more than 20 hours have often fully recovered. The work of Storer and Hempelmann (1952) with chilled infant mice (poikilothermic at this stage) provides a parallel (p.346), "Although the heart stops beating, the metabolism falls to zero, and a l l signs of l i f e are absent in animals chilled below 8° C.j recovery i s usually prompt once the animals are re-warmed." However, tissue anoxia i s a significant factor in cold resis-tance, for Adolph (1948) has shown that animals live longer i n oxygen than in nitrogen even when respiratory movements have stopped. Also, fish can acclimatize to lowered environmental oxygen levels, actually needing less oxygen after a period of acclimatization to shortage (Shepard, 1953* unpub.). It may be surmised that the antioxidant acti-vity could cause a continuous very slight shortage of oxygen in the tissues. (Diet F fish are much more prone to surface under standard aquarium conditions). Together these points show that phospholipids may possibly increase cold-resistance of goldfish by some form of acclimatization to partial anoxia prior to chill-testing. Possibly, dietary phospholipids fed over a certain pre-test period results in the ability of the goldfish to live with less oxygen. This brings us to the question of biological antioxidants. Biological Antioxidant Hypothesis Biological antioxidants, for the purposes of this discussion, are compounds used in very low concentrations to prevent the autooxida-tion of labile compounds in industry, but also of such low toxicity that they may be safely administered to living organisms. Fortunately, a study has been done upon the toxicity to goldfish of many industrial antioxi-dants. (Sollmann, 1949). This worker found that the immersion toxicity was much greater than the injection toxicity for the antioxidants tested. This suggests the action of antioxidants in immersion i s upon the g i l l s . Certainly, this could be related to the effects of lecithin upon the g i l l s of goldfish. However, before further consideration of specific antioxi-dants i s undertaken, i t i s necessary to mention something of their nature and mode of action. Antioxidants may be of a wide variety of basic structural formulae. They rarely stand alone i f from a biological source, and i t is common for two antioxidants to work synergistically, or for a non-antioxidant to be synergistic with a given antioxidant. (Golumbic, 1946). Antioxidants are frequently but not always readily oxidizable substances. They are associated with less-known inhibitols in the plants, substances which are absent from animals. Hence, i t takes much less antioxidant to stabilize the animal fats than i t does to increase the stability of the already-stabilized plant fats. An ordinary o i l (biological) will exhibit a period of latency before rancidification occurs. This is the induc-tion period. Since the induction period is totally removed by refining, i t is believed due to ill-defined mixtures of natural antioxidants in the o i l as i t existed in the organism. Thus the feeding of unrefined plant 49 fats or seed oils is quite different, in terms of biological antioxidant dosage, from feeding animal fats and oils* It has been found that the antioxidants responsible for the induction period of natural fats are actually being destroyed during induction, through the action, of peroxi-dases. (Boehm and Williams, 1945). Theoretically at least, this permits the association of biological antioxidants with the metabolism of tyro-sine, adrenalin, and possibly tryptophane. It should be emphasized that the effect of a given antioxidant is very dependent upon i t s nature and concentration, the nature of the substance to be "protected", the presence or absence of water, and espe-cially the other antioxidants or synergists which may be present. It is often found that an antioxidant for one compound i s not an antioxidant for another, while i t is generally true that excess of an antioxidant will reverse its effect, resulting in a higher rate of oxidation than when i t is absent from the system. It i s apparent that the effect of an addition of some antioxidant to a complex system is not at present predictable from the known principles of antioxidant action. The effect can only be deter-mined empirically. Since 1922 interest of some biologists has been turned toward the possible applications of the antioxidant concept to the biological sciences. Pioneers in this field were Moureau et Dufraisse (1922), who suggested that the larger concentration of antioxidant phenols in plants than i n animals was associated with the lower level of metabolic activity in,plants. Since this early suggestion of biological antioxidant activity was made, numerous papers have appeared on aspects of the subject* De Caro (1933) showed thyroxine to be an antioxidant for lipids. Numerous re« search workers showed that dietary antioxidants, especially vitamin E, protected vitamin A, and decreased the dietary requirement for A, Other workers have partially controlled the oxygen consumption of dystrophic muscles with vitamin E; others have shown reduction of abnormal pigment formation on administration of biological antioxidants; s t i l l others have changed the stability of body fat by altering antioxidant intake in the diet. There are many more papers in this general field (See: Macy Fdn,, Biological Antioxidants, 1946-50), Evidence has been gained for the i n vivo action of several biological antioxidants, especially alpha tocopherol and thiourea. That the action of many compounds is truly due to their antioxidant activity was demonstrated by Fieser (1948), who found such a close correlation between the in vitro antirespiratory activity of hydroxyalkylnaphtho-quinones and their antimalarial activity that the in vitro anti-oxidant test was used to screen new preparations, and even to evaluate one com-pound against another. The well-known lipid nutritionist, G.O.Burr, in 1946 pointed out that the deleterious effects of feeding slightly per-oxidized fats resulted not from their (low) toxicity, but from their de-pletion of the body's natural antioxidants, thus reducing the stability of the body fat, increasing the need for certain B vitamins, and decreas-ing the tissue linoleic acid. There are two schools of thought with regard to the action of vitamin E as a biological antioxidant, one de-cidedly against this explanation of vitamin E activity. Despite this, the general evidence for biological antioxidants bringing about physiolor-gical effects other than toxicity reactions i s convincing of their importance in biology. A recent textbook of biochemistry devotes several sections to the concept.(West and Todd, "Textbook of Biochemistry", 1st ed., pp. 729,793,868,464-465.) Continuing this trend, Ferrando (1953) recently proposed the name "antioxidant vitamin" for alpha tocopherol. Feeding crude lecithin to goldfish i n the present series of experiments increased their cold resistance. Moreover, thyroxine immer-sion and thiourea immersion both increase the cold resistance of gold-fish (Hoar, unpublished data). It seemed, therefore, that i t was not merely a coincidence that these three substances offering increased cold resistance to fish, were also biological antioxidants. Soon i t was noted in this series of experiments that the feed-ing of phospholipids did not produce a steady rise in cold-resistance, but a delayed rise to a peak, and then a progressive decline, ending well below the control level. At the same time i t was recognized that such a curve of cold resistance plotted against duration of feeding period, i n conditions of limited oxygen, would be expected upon the basis of Hickman1s (1949) theoretical curve for biological antioxidant activity (p,120). Thus, increased body antioxidant concentration with increased time of feeding (not necessarily the same antioxidant) would result according to Hickman,s hypothesis, i n a decrease of "unwanted" oxidations down to a low level, followed by a slow rise through the normal level to high levels of unwanted reactions (quinoid effect). This sequence, where oxygen i s non-limiting, would be paralleled by no change at a l l in the level of "wanted" reactions, until the antioxidant level had risen very high. At very high and excessive antioxidant levels the "wanted" reaction would drop gradually i n intensity. In the case of limited oxygen this 52 means that with low concentration (short feeding) of antioxidant, avail-able oxygen would be used to support considerable "useless" oxidation, and normal metabolism would suffer somewhat. With moderate concentrations (intermediate duration of feeding) of antioxidant, most of the oxygen avail** able would go to the "normal" metabolism, due to severe curtailment of un-desirable oxidations. With high concentrations (prolonged feeding) of antioxidant, the available oxygen would be used more for non-metabolic oxidation with increasing concentration of antioxidant once this range had been entered. From the beginning this would mean an i n i t i a l decrease in resistance to anoxia, followed by considerable increase in resistance to anoxia to supranormal values, followed again by decreasing resistance, this time to very low levels. Clearly, Hickman,s theory of biological antioxidant dynamics explains the observed results of feeding Diet F in these experiments, (Figure 12) , and under conditions of limiting oxygen, the curve obtained by plotting the proportion of "useful" oxidation to "useless" oxidation according to the relationship of Hickman i s a curve very similar to that found i n the General Adaptation Syndrome of Selye, At present i t i s not certain whether antioxidant activity can give the i n i t i a l low portion of the G.A.S, curve or not. If small additions of an antioxidant increased the rate of unwanted oxidation while having no ef-fect upon the wanted oxidations under conditions of ample, oxygen, such a "dip" i n the curve of anoxic resistance (and hence probably cold-resistance) would be obtained from purely theoretical considerations on conditions of limiting oxygen. It i s curious that similar curves have been given by Leopold and Thimann (1949) for the effect of auxin concentration upon the development of plant parts, while the antioxidant thyroxine has been shown by Davis (1934) to act in plants as an auxin, inhibiting root and vege-tative shoot growth while providing development of flowering parts. Per* haps these similarities of curves are not accidental. Since the biological antioxidant hypothesis seemed to be somewhat productive, a preliminary review of the literature was made to determine just what compounds had (1) been found to increase cold resistance in ani-mals, or (2) been found to f a l l in concentration during cold exposure of various types. This was done with a view to assessing the biological antioxidant hypothesis on the basis of numbers of biological antioxidants found to alter or be altered in cold resistance. At the same time, as many compounds as possible i n these two classes were recorded whether anti-oxidants or not with the hope that a preliminary metabolic chart might be drawn up to show the general biochemical relationships among the compounds known to be concerned in cold stress or i t s amelioration. This search for other instances of cold resistance associated with compounds that are biological antioxidants resulted in the discovery of five instances. Working without knowledge of the preliminary findings of this laboratory with regard to thiourea-immersed fish, P.Y.Fortune of Leeds University found the same results; that i s , thiourea markedly increas-es cold resistance of fishes. Dugal and Fortier (1952) have found that the antioxidant ascorbic acid enhances cold-resistance of those mammals incap-able of synthesizing this vitamin. Also, Monier and Weiss (1953) have shown an increased excretion of metabolic derivatives of ascorbic acid following temperature shock. The less potent biological antioxidant vita-mins belonging to the B-complex have also been implicated in cold stress. Finally, the non-protein sulfhydryl compounds of blood and liver have been 54 found to decline with cold stress (Bartlett and Register, 1953). The importance of maintaining the SH groups of enzymes intact i s well known. It may well be that biological antioxidants are largely concerned with maintaining these groups in the reduced form. This concept of biological antioxidants acting to control the "background" redox poten-t i a l in tissues thus allowing maximal or optimal operation of the enzymes of those tissues, is voiced by West and Todd (1951> "Textbook of Biochem-istry" 1st ed., p .465), and by Guzman Barron (1950), the latter referring to the SH groups as the most primitive regulators of cell metabolism. It is not surprising then that such sulfhydryl compounds should prove impor-tant in ch i l l resistance, for during chilling the nervous system i s vir-tually inactivated, the hormone-carrying blood is static, and the tissue viscosity is quite high. Any consideration of the sulfhydryl group leads to speculation regarding the redox potential of the tissues. In this regard, Oka (1952) has found the seasonal shift of redox potentials of rat liver as previously mentioned. In addition, a recent paper (Maye, et al., 1953) contains a discussion of the possible role of Lipids in (bacterial) metabolism as important oxidation-reduction systems. During this search of the literature, i t was also found that inositol, chemically pure lecithin, cholesterol, and naturally-occurring sterols were completely devoid of antioxidant activity. (Fisheries Re-search Board of Canada, Bull. 87, 1952; and Mattill, 1931). However, cephalins were also present in a l l phospholipid diets fed in the present study, and these compounds are good antioxidants. (Fisheries Research Board of Canada, Bull. 87, 1952). Thus the antioxidant effect of phospho-lipid diets may be used as an explanation of their effectiveness in afford-ing cold resistance to the goldfish, whereas this explanation cannot be used in any direct form, to explain enhanced cold resistance due to chol-esterol feeding. There remains the task of testing this biological antioxidant hypothesis, using many biological antioxidants in different concentrations. This work i s to commence immediately. It is hoped to study the mechanism of the protection offered by thiourea, distinguishing between its SH effects (Gyorgy, 1946), i t s lipid oxidation effect (Sumiki et al , 1951), effect upon phospholipid synthesis (Cornatzer et al, 1953), and its effect upon the thyroid (Smith, Sladek, and Kellner, 1953). Similarly, ascorbic acid will be tried as a biological antioxidant also providing ch i l l resistance. Here i t will be necessary to follow up the association of ascorbic acid with very adtively oxygen-transporting tissues (Day, 1949). This reminds one of the phospholipid effect upon goldfish g i l l s , and Bloor's findings of in-creased phospholipid in the most active muscles. Also to be borne in mind i s the effect of ascorbic acid upon the autooxidation of lipids (Lehmann and Watts, 1951). Finally,. none of these biological antioxidants should be divorced from considerations of usual intermediary metabolism, even though i t i s true that virtually none of them has been assigned any very specific place in the metabolic scheme. The recent discovery of glutathione in a carbohydrate intermediary metabolism mechanism, and the implication of a group of biological antioxidants as such, in the mechanism of oxida-tion of parahydroxyphenylpyruvic acid (La Du and Greenberg, 1953) 56 (tyrosine metabolism) indicates the likelihood of further discoveries of biological antioxidant action directly in intermediary metabolism mechanisms. Gold-Metabolism Theory It has long been considered that metabolism during chilling i s shifted away from the "normal" metabolism that takes place in the usual thermal environment. This question is intimately linked, though not identical, with the problem of chemical heat regulation. One of the ear-l i e r theories of control of heat production in the body was that of Sajous (1928), who suggested that control was effected by means of the adrenal medulla, and the compound cholesterol (through i t s control on lecithin, which " i s " the actual substance doing the ultimate controlling). Working upon heat stress, Sundstroem (1927) concluded that low phospholipid levels, especially in relation to cholesterol levels, were beneficial in tropical areas, while much later, Milch, et.al. (1953), working with low tempera-tures, concluded that cholesterol is important in the repair of cold-damage. Wilbur and Del Porno (1949) found a higher phospholipid:cholester-ol ratio in Arctic fish than i n Temperate Zone fish, and concluded the rate of fat turnover was greater in the Arctic fish. Thus, several pre-liminary attempts have been made to relate heat- and cold-resistance to specific sections of the metabolism. In the case of phospholipids and cholesterol, the picture i s made very complex by some definite but variable correlations between the metabolism of the two. In the (developing) fish, Glover, Morton and Rosen (1952) have suggested from experimental evidence, that the choles-terol i s synthesized largely, i f not wholly, from the phospholipids. Quite clearly, too, the thyroid gland plays a large part in determining the phospholipid and cholesterol levels in the tissues. (Kim and Ivy, 1952; Stamler, 1954; Pasternak and Page, 1934; Onizawa, 1929; Irvine, unpub.) So do the ovaries (Stamler, 1954)* Cholesterol has also been called one of the most potent anti-thyroid substances, as well as one of the greatest anti-calorigenic compounds. The complex interrelation-ships between cholesterol and phospholipid metabolism are far from being elucidated. There i s , of course, a close relationship between phospholipid, cholesterol, and fat metabolism. It has been shown in several cases that high fat diets are beneficial in resisting cold stress (Dugal, LeBlond, and Therien, 1945), while the decrease in R.Q. in the cold, indicating the combustion of fats to the virtual exclusion of carbohydrates and pro-teins, is well known. However, in the cold the R.Q. often descends to extremely low levels which have not been adequately explained (Gardner et a l , 1922). In addition to this evidence for the "switching over" to fat-catabolism in the cold, Tannenbaum and Silverstone (1953) point out that "Increasing the fat content of a diet results in an increased efficiency of energy utilization" and " ... this "saving" of net body energy might be regarded as equivalent to an increase in caloric intake." In conclusion, a metabolic chart related to cold stress i s presented, covering the lipids chiefly, but including a l l the compounds commonly associated with cold stress, as revealed by the literature, togeth-er with a few additional compounds essential for continuity of the scheme. It is surprising that a l l the known factors f i t into such a close-knit scheme. It must, however, be emphasized that this chart can not be re garded as more than a preliminary guide to study of the metabolism res ponsible for cold resistance. *9 GOLD-RESISTANCE METABOLISM increased metabolic ef: methionine \ mercaptans* \ \ \ \ .-choline adrenalin* B complex* / / iciency Krebs Cycle l e c i t h i i j i I •fatty acids I essential f.*.* ! A \ ^ ^ v e r _ function^ /" I 4 I .pyridoxine fats*. repair of cold-damage increased resistance to stressor adrenal function acetoacetate ascorbic acid* 1 : 1 : » ifolic acid* 1 tyrosine 4< other biological antioxidants •*' Acety:U-pantothenic acid* Co A 3 £2EL metabolic conversion - _ _ cofactors sparing action, or synergesis. * substances directly implicated in cold-resistance metabolism. COLD RESISTANCE METABOLISM References: 1. Dr.J.Biely, Department of Poultry Husbandry, U.B.C., 1954, (personal interview) 2 . Kielley, W.W., and Louise B.Bradley, 1954, Glutathione thio-lesterase, J.B.C. 206:327-333. 3 . Ochoa, S., and F.Lynen, 1953. Biochemica et Biophysics Acta 12:299. 4 . LaDu, B.N.Jr., and D.M.Greenberg, 1953. Science 117:111. 5. Milch, L.J., R.F.Redmond, W.W.Calhoun, and the Cardiovascular Research Group, 1953. Plasma lipoprotein changes induced by acute local cold injury, Amer. Jour. Med. Sci. 225:416-420. 6. Burr, G.O., 1946. The role of fat i n the diet, Chap.5, pp.62-82, in ed. Wohl, Dietotherapy: Clinical Application of Modern Nutrition. Phila., Saunders Co. 7. Tannenbaum, A., and H.Silverstone, 1953. Nutrition in Relation to Cancer, in ed. J.P.Green'stein and A.Haddan, Advances in Cancer Research 1:451-501. A l l unnumbered relationships are to be found in West, E.S. and W.R.Todd, Textbook of Biochemistry, 1st Ed., New York, The Macmillan Company, x i i + 1,345 pp. 61 SUMMARY 1. Dietary supplements of phospholipids or cholesterol generally-increased the cold resistance of the goldfish receiving them. The relative effectiveness of these two supplements varied with season and number of feedings. Correlation between body content of the supplemented lipid and cold resistance was good. 2. There was no correlation between cold resistance and : total body lipid; total lipid unsaturation; cholesterol:phospholipid ratio; or water content of the goldfish. 3 . Non-dietary factors modifying the chill-mortality rates were found to include length, weight, and sex of the fish, as well as season, and cold narcosis. Longer and heavier fish died off later in a given chill-test. Female goldfish were less cold-resistant than corresponding males, but their tendency to die first was partly masked by the greater tendency of male fish to enter deep narcosis. Winter goldfish were much more cold-resistant than Summer goldfish. 4. Cholesterol feeding prevented or delayed the i n i t i a l mortality in each chilling. This effect was partly due to failure of the cholesterol-treated fish to enter deep narcosis. Cholesterol had no significant effect upon mortality rate after the i n i t i a l period of each cold-test, however. 5. A characteristic curve of changing relative cold resistance with increasing number of feedings was obtained for goldfish on a cholesterol-supplemented diet, and for those on a phos-pholipid-supplemented one. The curve has been explained in terms of the biological antioxidant theory of Hickman, and also in terms of the General Adaptation Syndrome of Selye, postulating a positive crossed resistance phenomenon. 6. Feeding the Diets P,E, and F to goldfish resulted in a pro-gressive increase in total lipi d but a progressive decrease in water content in a l l cases. Specific effects produced were: a) Diet P (100$ Pablum) - slight saturation of body lipids. b) Diet E (50$ Pablum, 50$ cholesterol) - high water and choles-terol content; low li p i d and iodine number (marked hardening of lipids). c) Diet F (50$ Pablum, 50$ soya phosphatides) - slight unsatura-tion of body lipids; otherwise, not different in gross lipoid nature from Diet P goldfish. 7. There was a precise inverse relationship between the log of the total ether-extractable li p i d , and water content of a l l goldfish, regardless of their acclimatization or death temperature. 8. A tentative metabolic chart has been prepared to link together those compounds already known to be involved in the mechanism of cold-resistance of some animals. It centres around the lipid metabolism. BIBLIOGRAPHY ADOLPH, E.F. Tolerance to cold and anoxia in infant rats. Amer. J.  Physiol. 15^:366-377, 1948. BAHR, 0., and WILLE, 0. article in Fischwirtschaft 7:129, 1931, cited in LOVERN, J.A., and OLLEY, JUNE. The lipids of fish. 2. The acetone-soluble lipids of the flesh of the haddock. Biochem. J. 5AJ12S-137, 1953. BARRON, E.S.G. Effect of ionizing radiation on sulfhydryl systems, in Mackenzie, C.G. ed., Biological Antioxidants: Transactions  of the Fifth Conference. Josiah Macy, Jr. Fdn. 1950:81-115. 1950. BARTLETT, R.G., and REGISTER, U.D. Effect of cold and restraint on blood and liver non-protein sulfhydryl compounds. Proc. Soc. Exptl. Biol. 83_:708-709, 1953. BELEHRADEK, J. Temperature and living matter, Protoplasma-monographien 8, Berlin, 1935. Cited in HEILBRUNN, L.V. An outline of general  physiology. 2nd ed., Chapt. XXXI, p.419-432. W.B.Saunders Com-pany, Phila. and London, 1943. BLOGR, W.R. The cholesterol content of muscle, J.Biol. Chem. 114:639-648, 1936. BLOOR, W.R., and SNIDER, RUTH H. Phospholipid content and activity in muscle, J.Biol.Chem. 107:459-470, 1934. BOEHM, E., and WILLIAMS, R. Article in Pharm. J.. 1945, cited in SOLLMANN, T., Correlation of the aquarium goldfish toxicities of some phenols, quinones, and other benzene derivatives with their inhibition of auto oxidation. J. Gen. Physiol. 3_2:671-680, 1949. BRETT, J.R. Rate of gain of heat tolerance in goldfish (Carassius auratus). Pub.'n. Ontario Fisheries Research Board, No. 53. 1946. • Some principles in the thermal requirements of fishes. Unpub-lished paper presented at a symposium on The Ecological Require-ments of Cold Water Fishes, sponsored by the Western Society , of the Ecological Society of America (1953). BURR, G.O. The role of fat i n the diet. Chapt.V in WDHL, G. ed., Dietotherapy:Clinical application of modern nutrition, W.B. Saunders Company, Phila. and London, 1946. CAHN, T., HOUJET, J., and AGH), R. Validity of calculations of the content of phospholipids in tissue. Compt. rend. 228:275-277, 1949. i i CHEMICAL PUBLISHING COMPANY. Emulsion technology theoretical and applied. Including the Symposium on Technical Aspects of Emulsions. 2nd enlarged ed. Brooklyn, 1946. COLBERT, E.H., COWLES, R.B., and BOGERT, CM. Temperature tolerance in the North American alligator, Bull. Amer. Mus. Nat. Hist. 8 6 : 327-375, 1946. CORNATZER, W.E., GALLO, D.G., and DAVISON, J.P. Some hormonal effects on phosphorylation in the liver of rats. Proc. Soc. Exptl. Biol. Med. 84_:103-105, 1953. CORRAN, J.W. Section 9 in Emulsion technology, theoretical and applied; including the symposium on technical aspects of emulsions. 2nd enlarged ed. Chemical Publishing Company, Brooklyn, 1946. COTTLE, MERVA K. Unpublished notes in f i l e of DR.W.S.HOAR, Department of Zoology, U.B.C., 1951. ___________ . Temperature tolerance of the goldfish (Carassius auratus) in relation to the degree of unsaturation of body lipids, the cholesterol, phospholipid, fatty acid, and water content of the tissues. Masters Thesis, University of British Columbia, (unpub.), 1951. DAVIS, E.E. Influence of thyroxine on the growth of plants. Plant  Physiol. 9:377-384, 1934. DAY, M.F. The distribution of ascorbic acid in tissues of insects. Austral. J. Scient. Res., B. 2 : 1 9 - 3 0 , 1949. DE CARO, L. Azione della tiroxina suH' autoossidazione degli acidi grassi non saturi. Boll, soc. biol. sper. 8:160-161, 1933. DEUEL, H.J.Jr. The lipids: their chemistry and biochemistry, vol . 1 , Chemistry, Interscience Publishers, New York, 1951. ' . Recent advances in relation to fat metabolism, Nutritional Observatory 13_: 6 4 - 6 8 , 1952. DORCHESTER, J.E.C. The effect of dietary fat on the heat tolerance of goldfish (Carassius auratus). Masters Thesis, University of British Columbia, (unpub.), 1948. D0UD0R0FF, P. The resistance and acclimatization of marine fishes to temperature change. I. Experiments with Girella nigricans (Ayres), Biol. Bull. 83:219-244, 1942. DUGAL, L.P., and FORTIER, G. Ascorbic acid and acclimatization to cold in monkeys, J.Appl. Physiol. 143-146, 1952. 11X DUGAL, L.P., LE BLOND, CP., and THERIEN, M. Resistance to extreme temperatures in connection with different diets, Can.J. Research, E. 2_3:244-258, 1945. DIMM, MARY E., and RALLI, ELAINE P. Factors influencing adrenalectomized rats to stress. Metabolism 2:153-164, 1953. FERRANDO, R. Vitamin E, antivitamins E, et phenomenes de reproduction, in Nutrition et fonctions de reproduction III. Editions du Centre National de la Recherche Scientifique, Paris, 1953. FIESER, L.F. Naphthoquinones as antimalarials and inhibitors of res-piration, in MACKENZIE, CG. ed., Biological antioxidants:  transactions of the third conference, Josiah Macy, Jr. Fdn. 1948:24-28, 1948. FISHERIES RESEARCH BOARD OF CANADA. Marine oils with particular refer-ence to those of Canada, 2nd ed. Fish. Research Bd. Can. Bull. 89, 1952. FORTUNE, PAMELA Y. Personal communication to DR. W.S.HOAR, Department of Zoology, University of British Columbia. FRAZER, A.C Differentiation in the absorption of olive o i l and oleic acid in the rat. J.Physiol. ___2:306-312, 1943. FRY, F.E.J. Effects of the environment on animal activity. Univ. Toronto Studies, Biol. Series. 55. Publ. Ontario Fish.  Res. Lab. 68:1-62, 1947. FRY, F.E.J., BRETT, J.R., and CLAWSON, G.H. Lethal limits of tempera-ture for young goldfish, Rev. Can, de biol. 1:50-56. FUJITA, M. Kwanden-Denki no Gyorui ni oyobosu hanwo Jikken. (Experi-ments on the reactions of fishes both living and dead, towards induction currents of electricity.) Dobuts Zasshi, (Tokyo), 18:153-154, 1906. GARDNER, J.A., KING, G., and POWERS, E.B. The respiratory exchange in fresh water fish. III. Goldfish. Biochem. J. 16:523-529, 1922. GLOVER, MARY, MORTON, R.A., and ROSEN, D.G. Astaxanthin, cholesterol, and lipins in developing salmon eggs. Biochem. J. 50:425-429, 1952. GOLDSMITH, Grace A. Clinical research on the newer vitamins. Nutritional Observatory 13_:76-84, 1952. i v GOLUMBIC, C. Kinetic studies on the antioxygenic synergism between tocopherol and ascorbic acid. Biological antioxidants; transactions of the first conference. Josiah Macy Jr. Fdn. 1946:42-48, 1946. ~ GYORGY, P. in published discussion following the paper of GOLUMBIC, C. Kinetic Studies on the antioxygenic synergism between tocopherol and ascorbic acid. Biological antioxidants: transactions of the fi r s t conference. Josiah Macy Jr. Fdn. 1946:42-48, 1946. HEILBRUNN, L.V. An outline of general physiology. 2nd ed. revised. W.B.Saunders Company, Phila. 1948. HEILBRUNN, L.V., and DAUGHERTY, KATHRYN. Fat release in amoeba after irradiation. Physiol. Zool. _:383-387. 1938. HOAR, W.S. Unpublished notes. 1949. Studies in the biochemistry of temperature acclimatization in fishes. National Research Council MS Report. (Unpub.) 1952. HOAR, W.S., and DORCHESTER, J.E.C. The effect of dietary fat on the heat tolerance of goldfish (Carassius auratus). Can.J.Research, D. 22,:85-91, 1949. HOAR, W.S., and COTTLE, MERVA K. Dietary fat and temperature tolerance of goldfish. Can.J.Zool. 3_0:41-48. 1952. Some effects of temperature acclimatiza-tion on the chemical constitution of goldfish tissues, Can. J. Zool. 3.0:49-54. 1952. HUNTER, J.G. The change in the degree of unsaturation of body fats during acclimation of goldfish (Carassius auratus) to high tempera-ture. Masters Thesis, University of British Columbia, (Unpub.) 1948. HICKMAN, K.C.D. Practical Application of Physiological Antioxidants, in MACKENZIE, C.G., ed. Biological antioxidantstransactions  of the fourth conference, Josiah Macy Jr. Foundation, 1949: 114-123, 1949. HAWK, P.B., OSER, B.L., and SUMMERSON, W.H. Practical physiological  chemistry. 12th ed. Blakiston Company, New York, 1951. IRVINE, D.G. Unpublished data on thyroxine/lipids relationships, 1953 V JONES, J.H. Vitamin E in health and disease. Chapter 14 of WOHL, M.G., ed., Dietotherapy:clinical application of modern  nutrition, W.B.Saunders Company, Philadelphia, 1946. KARTHA, A.R.S. Hypothesis of fluidizing function of unsaturated acids  in natural fats. A separate published by K.Maharaja's College, Ernakulam, India, 1:110-128. 1951. KTFiLLEY, W.W., and BRADLEY, LOUISE B. Glutathione thiolesterase, J. Biol. Chem. 206:327-333, 1954. KIM, K.S., and IVY, A.C. Factors influencing cholesterol absorption. Amer. J. Physiol. 171:302-318. 1952. LA DU, B.N.Jr., and GREENBERG, D.M. Ascorbic acid and the oxidation of tyrosine. Science 117:111-112, 1953. LEHMANN, BARBARA T., and WATTS, BETTY M. Antioxidants in aqueous fat systems. J. Amer. Oil Chemists' Soc. 28:475-477, 1951. LEOPOLD, A.C, and THIMANN, K.V. The effect of auxin on flower i n i t i a -tion, 1949. Cited in WARDLAW, C.W., Phylogeny and Morpho- genesis. Macmillan, London, 1952. (pp. 212-213). LOVERN, J.A., and OLLEY, JUNE. The lipids of fish. 2. The acetone-soluble lipids of the flesh of the haddock. Biochem. J. 5_4_:128-137, 1953. LUNDBERG, W.O., GYORGY, P., TAYLOR, H.S., BAXTER, J.G., and SWIFT, C.E. in (published) discussion following C.E.Swift's paper "Some properties and reactions of methyl hydroperoxide oleate," in MACKENZIE, C.G. ed., Biological antioxidants:transactions  third conference. Josiah Macy Jr. Fdn. 1946:16^25^ 1946. LUYET, B.J., and GEHENIO, P.M. Life and death at low temperatures. No.l in a series of monographs ed. by Luyet, B.J., Biodynamica, Normandy, Missouri. 1940. LYNEN, F., and OCHOA, S. Enzymes of fatty acid metabolism. Biochemica  et Biophysica Acta 12:299-314. 1953. MATTILL, H.A. Antioxidants and the autooxidation of fats, J. Biol. Chem. 20:141-151. 1931. MAYER, A., et SCHAEFFER, G. Recherches sur les constantes cellulaires teneur des cellules en eau. I. Discussion theorique. L*eau, constante cellulaire. J.physiol. et path, gen. 16:1-16, 1914. v i MAYER, A., et SCHAEFFER, G. Recherches sur les constantes cellulaires teneur des cellviles en eau. II Rapport entre la teneur des cellules en lipoides en leur teneur en eau. J. physiol. et  path, gen. 16:23-38, 1914. MAYR, R.L., GRIMM, M., JACONIA, D., and KULL, F.G. Role of lipids in formation of yellow pigment from p-amino-benzoic acid and p-amino-salicylic acids. Proo. Soc. Exptl. Biol. Med. 8_: 378-383, 1953. MUCH, L.J., REDMOND, R.F., CALHOUN, ¥.¥., and the CARDIOVASCULAR RESEARCH GROUP. Plasma lipoprotein changes induced by acute local cold injury. Amer. J.-Med. Sci. 225:416-420. 1953. MILLER, L.C. "Biological assays involving quantal responses," in the place of statistical methods in biological and chemical experimentation. Ann. N.Y. Acad. Sci., _52:903-919, 1950. MONIER, MARY M., and WEISS, ROSLYN J. Increased excretion of dehydroascorbic and diketogulonic acids in urine of rats after standardized temperature shock. Proc. Soc. Exptl. Biol. Med. __:93-94. 1953. MOORE, R.N., and BICKFORD, W.G. Comparative evaluation of several antioxidants in edible fats. J. Amer. Oil Chemists' Soc. 2_:l-4, 1952. MOUREAU, C, and DUFRAISSE, C. Anti-oxidases. Compt. rendu, soc. biol. 86:321-323, 1922. NATIONAL RESEARCH COUNCIL (CANADA). Effects of low temperatures on small mammals, N.R.C. News. 2(3)si, 1954. OKA, Y. On the seasonal variation of the redox potential of the liver in situ, (in Japanese). Bull. Exptl. Biol. 2:179-180, 1952. ONIZAWA, J. Article in J. Biochem. (Japan) 10:425, 1929, cited in BLOOR, W.R., The cholesterol content of muscle, J. Biol. Chem. _•_: 639-648, 1936. PASTERNAK, L., and PAGE, E.H. Article in Biochem. Zeitschr. 274:122, 1934, cited in BLOOR, W.R., The cholesterol content of muscle, J. Biol. Chem. _J_:639-648, 1936. PEARL, R. The biology of death. Monographs on experimental biology, J.B.Lippincott and Company, Phila. and London, 1922. SAJOUS, C.E. de M. Tissue respiration as the function of the internal secretions which science has sanctioned. Proc. Am. Phil. Soc. 6J:307-318, 1928. " v i i SANO, M. Article in J.Biochem. (Japan) 1:17-20, 1922. (Chemical  Abstracts 16:15966, 1922). SELYE, H. The general-adaptation-syndrome and the diseases of adapta-tion, from SELYE, H., Textbook of Endocrinology, Acta Incorporated, Montreal, 1949, (pp. 836-867). __________ Stress (The physiology and pathology of exposure to stress: a treatise based on the concepts of the general-adaptation-syndrome and the diseases of adaptation). Acta Incorporated, Montreal, 1950. SHEPARD, M. Doctoral Thesis, University of Toronto, (unpub.) 1953. SINCLAIR, R.G. The metabolism of phospholipids VIII. The passage of elaidic acid into tissue phospholipids. Evidence of the intermediate role of liver phospholipid in fat metabolism, J. Biol. Chem. 111:515-526, 1935. SMITH, D.C., SLADEK, S.A., and KELLNER, A.W. The effect of mammalian thyroid extract on the growth rate and sexual differentiation i n the fish, Lebistes reticulatus. treated with thiourea. Physiol. Zool. 26:117-123, 1953. SMITH, D.J. Variations in vascular reactivity produced by season, cold stress and immaturity; role of thyroid and adrenal cortex. Amer. J. Physiol. 172:118-128, 1953. SMITH, F.D. Mechanisms involved in the injury and death of fish by chilling temperatures. Masters Thesis, University of British Columbia, (unpub.) 1950. SOLLMANN, T. Correlation of the aquarium goldfish toxicities of some phenols, quinones, and other benzene derivatives with their inhibition of autooxidative reactions. J. Gen. Physiol. 32: 671-680, 1949. STAMLER, J. Endocrine influences on lipid metabolism and atherosclero-sis, Proc. Inst. Med. Chicago 20:17-18, 1954. STORER, J.B., and HEMPELMANN, L.B. Hypothermia and increased survival rate of infant mice irradiated with X-rays. Amer. J. Physiol. 121:341-348, 1952. SUMIKI, Y., TAMURA, S., and SAKATE, K. Inhibition of the autooxidation of oils and fats. I. The influences of sulfur-containing compounds on the autooxidation of methyl oleate. J. Agric.  Chem. Soc. Japan 2_>:237-240, 1951. v i i i SUMNER, F.B., and DOUDOROFF, P. Some experiments on temperature acclimatization and respiratory metabolism in fishes, Biol. Bull. 7_±:403-429, 1938. SUNDSTROEM, E.S. The physiological effects of tropical climate. Physiol. Rev. 2*320-362, 1927. TAKAHASHI, K. On the nutritive value of fats and lipoids. J. Chem. Soc. Japan 4_: 201-242, 1922. TANNENBAUM, A., and SHVERSTONE, H. Nutrition in relation to cancer, in GREENSTEBI, J.P., and HADDOW, A., eds. Advances in cancer  research, vol . 1 . Academic Press, New York, 1953. TERROINE, E.F., et HATTERER, G. Les acides gras des phosphatides des tissues d'homoeothemes sont-ils independants de la nature de 1»alimentation? Bull, soc. chim. biol. 12:674-681, 1930. TSURUTA, T. Article in Med. Bull. Univ. Cincinnati 6:100, 1931, cited in KIM, K.S., and IVY, A.C., Factors influencing cholesterol absorption, Amer. J. Physiol. 12:302-318, 1952. WEST, E.S., and TODD, W.R. Textbook of biochemistry. Macmillan, New York, 1951. WILBUR, C.G. Personal communication to Mrs. Merva K. Cottle; on f i l e in the Department of Zoology, University of British Columbia. WILBUR, C.G., and DEL P0M0, M. Comparative study of lipids in whole carcasses of Arctic and non-Arctic fish. Proc. Soc. Exptl. Biol. Med. 22:418-420, 1949. 


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