temperature tolerance of goldfish (carassiu auratuss ) …
TRANSCRIPT
TEMPERATURE TOLERANCE OF GOLDFISH (Carassius auratus)
IN RELATION TO THE DEGREE OF UNSATURATION OF
BODY LIPIDS, THE CHOLESTEROL, PHOSPHOLIPID,
FATTY ACID, AND WATER CONTENT
OF THE TISSUES
by
Merva Kathryn Cottle
A Thesis submitted i n p a r t i a l fulfilment
of the requirements for the Degree of
Master of Arts
i n the Department of
Zoology
We accept t h i s thesis as conforming to the ^standard required for candidates f o r the
degree of Master of Arts .
Members of the Department of Zoology
The Univ e r s i t y of B r i t i s h Columbia
A p r i l , 1951
TEMPERATURE TOLERANCE OF GOLDFISH (Carassius auratus)
IN RELATION TO "THE DEGREE OF UNSATURATION OF
BODY LIPIDS, THE CHOLESTEROL, PHOSPHOLIPID,
FATTY ACID, AND WATER CONTENT
OF THE TISSUES.
. Abstract
Ten d i f f e r e n t f a t s (three i n duplicate)-including
natural and hydrogenated fat s were arranged i n four series of
diets to allow a range i n degree of saturation of diet i n each
s e r i e s . These were fed to 1300 go l d f i s h (100 f i s h per f a t
d i e t ) . The degree of unsaturation of f i s h l i p i d altered i n
accordance with the degree of unsaturation of the diet and the
tolerance of the f i s h to high and low temperatures was modi
f i e d . In none of the four series was there a precise c o r r e l a
t i o n between melting point of t o t a l l i p i d s and degree of
tolerance to high and low temperatures. Since t o t a l l i p i d i s
not necessarily protoplasmic l i p i d i t i s possible that the
degree of unsaturation of protoplasmic l i p i d s (phosphatides)
and temperature tolerance of the f i s h would show a more
precise c o r r e l a t i o n .
Changes occuring i n water content, l i p i d content,
and l i p i d constituents of f i s h tissues and iodine values of
t o t a l l i p i d were studied i n gol d f i s h acclimatized to 5°, 15°,
i i
o o 20 , and 35'.' C. Water content increased while l i p i d content
decreased with a r i s e i n the temperature of acclimatization of
f i s h . The cholesterol .: phospholipid r a t i o varied inversely
and the cholesterol : f a t t y acid r a t i o varied d i r e c t l y as both
the water content and temperature of acclimatization. Iodine
values of the t o t a l f i s h l i p i d increased with a decrease i n
temperature of acclimatization.
I t i s concluded that i n the process of acclimat
i z a t i o n to d i f f e r e n t temperatures, changes i n the degree of
unsaturation of t o t a l l i p i d as well as possible changes i n
permeability to water occur and that both factors may be of
importance i n resistance of the f i s h to further thermal
changes.
i i i
Acknowledgements
I am indebted to Dr. W. S. Hoar f o r h i s valuable
guidance and under whom i t has been my p r i v i l e g e to work. I
also wish to thank Dr. W. A. Clemens fo r h i s assistance;
Dr. B. E. Bailey, P a c i f i c F i s h e r i e s Experimental Station and
Dr. C. Wilber, St. Louis University, both f o r advice i n
chemical procedures; and my husband for aid i n many phases of
the study. My thanks also go to the National Research Council
whose f i n a n c i a l assistance made the project possible.
Table of Contents
page Introduction . • • • . • • • • • • • • • » * . . . • . . . 1
Review of Literature . • • • . . . . . * . . « • • . . * . 2
Materials and Methods . * » • • • « . . . . . . 7
Feeding Experiments . . . . . . . . . . . 8
Acclimatization Experiments . « • • • • • • • • • • . 1 4
Results . . . . . . . . . . . . . . . . . » 19
Feeding Experiments . . . . . . . .19
E f f e c t of Diet on Uhsaturation of Fi s h L i p i d o • 19
Condition of Fi s h during Feeding Period . . . »_19 E f f e c t of Dietary Fat on High and Low Temperature Tolerance of Goldfish • . • • . . . . . . • 20
E f f e c t of Dietary Fat on Moisture and L i p i d ' Contents of Tissue . . . . . . . . . . . . . . . 22
Acclimatization Experiments • 24
Temperature Acclimation and Temperature Tolerance 24 Temperature Acclimation and Moisture Content, L i p i d Content, L i p i d Constituents of F i s h Tissue and Iodine Values of T o t a l L i p i d s . . . . . . . 24
Discussion . . . . . . . . . . . 26
Summary and Conclusions . . . . . . . . . . . . . . . . . • 31
Appendix . . . . . . . . . . . . . . . 33
Lite r a t u r e Cited 40
L i s t of Tables
page Table I . 10
Table I I 15
Table I I I . . , . . . . . . . . . . . . . . . . 23
Table IV to follow 24
Table V 33
Table VI . . . . . . . . . . 34 .
Table VII 35
Table VIII 36
Table IX • 37
Table X 38
Table XI 39
L i s t of Figures o
. . . - . page Figure 1 • • to follow 8
Figure 2 M " 20
Figure 3 . . . 20
Figure 4 . . . " " 21
Figure 5 ". n 21
Figure 6 . " " 21
Figure 7 ". 22
Figure 8 • . . " n. 22 .
Figure 9 ." " .24
Figure 10 " " 24
Introduction
A number of studies (Henriques and Hansen, 1901;
Belehradek, 1935; and Hunter, unpub.) have suggested that pro
toplasmic l i p i d s , either d i r e c t l y or i n d i r e c t l y , play an
important role i n temperature acclimatization. Heilbrunn
(1943) postulated that the melting point of body l i p i d s was
related to the temperature tolerance such that increased melt
ing point of the l i p i d s resulted i n increased tolerance to
high temperatures. Hoar and Dorchester.(1949) altered the
body f a t s of g o l d f i s h by feeding diets containing a high pro
portion of d i f f e r e n t f a t s and found only a very general
rela t i o n s h i p between degree of unsaturation of body f a t s and
tolerance to high temperatures. Lower l e t h a l temperatures
were not investigated i n these experiments.
In the present study an attempt has been made to
test more r i g i d l y the r e l a t i o n s h i p between melting point of
body l i p i d s and temperature tolerance (both upper and lower
l e t h a l temperatures) and to investigate the e f f e c t s of temp-
erature acclimatization on the nature of the body l i p i d s .
Since a r e l a t i o n s h i p between moisture content of tissues and
the r e l a t i v e amounts of tissu e cholesterol and phospholipid
was recognized by Mayer and Schaeffer (1914) these factors
have been studied most c a r e f u l l y i n the acclimatized f i s h .
z
I t was hoped that these experiments would show
whether or not melting point of body f a t s i s involved i n temp
erature tolerance and e s t a b l i s h changes that occur i n l i p i d
constituents and water content of tissues during temperature
acclimatization.
Review of Literature
Melting'Point of L i p i d s and Temperature Acclimatization.
Data on the r e l a t i o n s h i p between melting point of
lipids,and temperature acclimatization have been recorded for
both plants and animals. The following i s a b r i e f summary of
the more important contributions. Pearson and Raper (1927)
reported that fungus grown at d i f f e r e n t temperatures possessed
f a t t y acids of iodine values which varied inversely with temp
erature. Likewise, McNair (192,9) studied the r e l a t i o n s h i p of
plant o i l s to climate and found s i m i l a r r e s u l t s , He showed
that with few exceptions, plants of warmer regions contained
more saturated o i l s than those of cooler regions. The s o l i d i
f i c a t i o n point of f a t t y globules i n aphids was found" by
Ackerman (1929) to vary with temperature of habitat. A lower
temperature was associated with a f a t of lower melting point.
Studies on acclimatization of f i s h to d i f f e r e n t temperatures
carried out by Lovern (1938) and Hunter (unpub.) showed a
si m i l a r trend of desaturation of l i p i d s at lower temperatures.
3
Dean and H i l d i t c h (1933) found the degree of unsaturation of
depot f a t of hogs was c l o s e l y correlated to the temperature of
the s i t e .
Temperature;; of acclimatization not only a f f e c t s
t o t a l body l i p i d (composed pr i m a r i l y of depot fat) but also
protoplasmic l i p i d s , the phosphatides. This was shown for
poikilothermic animals by Terroine and co-workers (1930). The
r e s u l t s of Fraenkel and Hopf (1940) on temperature tolerance
and degree of saturation of phosphatides of blow-fly larvae
also conformed to the general hypothesis.
According to Heilbrunn (1943) degree of unsaturation
of protoplasmic l i p i d s i s of importance i n a b i l i t y of c e l l s to
r e s i s t high temperature. This author postulated that heat
death involved l i b e r a t i o n of calcium from cortex to c e l l
i n t e r i o r and l i q u e f a c t i o n of c o r t i c a l region. Normally c a l
cium; of the cortex i s held i n a loosely combined calcium-
p r o t e i n - l i p i d complex and when heated the l i p i d releases
calcium to the c e l l i n t e r i o r . The e f f e c t of heat i s more
pronounced when f a t s are more unsaturated. -Hence more s o l i d
f a t s confer upon c e l l s increased tolerance to heat. Heilbrunn
also pointed out that cold acted s i m i l a r l y i n causing calcium
release and l i q u e f a c t i o n of c e l l cortex.
4
Relation between Dietary and Body Fat.
Since the present investigation i s based i n part on
dietary experiments i t i s important to understand tne e f f e c t
of dietary f a t on body f a t s . Anderson and Mendel (1928) i n
t h e i r c l a s s i c a l study showed that a diet high i n f a t altered
the iodine value of body f a t i n accordance with the iodine
value of dietary f a t . Similar r e s u l t s were reported f o r pigs
by Brown (1931) and E l l i s and I s b e l l (1926). Lovern (1938)
experimenting oni eels and Hoar and Dorchester (1948) on gold
f i s h also found that the melting point of body l i p i d s of f i s h
resembled that of dietary f a t when the l a t t e r was fed at high
l e v e l s .
S i n c l a i r , i n a series of experiments (1929, 1930, and
1931), showed that f a t t y acids of phospholipids, as well as
those of depot f a t , resembled the f a t t y acids of the dietary
f a t . However, contrary to t h i s Terroine and Hatterer (1930)
concluded that the phospholipids (element constant) did not
change qu a n t i t a t i v e l y or q u a l i t a t i v e l y with d i e t . In a more
recent study of S i n c l a i r ' s (1935) the two viewpoints may be
p a r t l y reconciled. In t h i s work S i n c l a i r found that f a t t y
acids of phospholipids present i n l i v e r , i n t e s t i n a l mucosa,
and blood changed quite r a p i d l y to resemble f a t t y acids of the
d i e t , whereas phospholipids present i n nerve and muscle tissue
were slower to respond and indicated a s e l e c t i o n of the more
unsaturated f a t t y acids. The f i r s t group of phospholipids,
a c c o r d i n g to S i n c l a i r , f u n c t i o n e d i n the t r a n s p o r t a t i o n of
f a t t y a c i d s while the second group were important i n c e l l u l a r
s t r u c t u r e and p r e s e r v e d to some degree from wear and t e a r .
T h i s l a t t e r group would compare to T e r r o i n e ' s element
constant.
T i s s u e Water and Temperature T o l e r a n c e .
The s u g g e s t i o n t h a t water content was a l i m i t i n g
f a c t o r i n t o l e r a n c e to h i g h temperature was made by Davenport
(1897). He s t a t e d t h a t the lower the water content the h i g h e r
the temperature r e q u i r e d to cause c o a g u l a t i o n of c e l l contents.
Dehydration was a l s o shown t o i n c r e a s e c o l d h a r d i n e s s i n i n s e c t s
(Payne,1921). Loeb and Wasteneys (1912) found t h a t Fundulus
t o l e r a t e d maximum temperatures i n h y p e r t o n i c s o l u t i o n s . They
p o s t u l a t e d t h a t a r i s e i n temperature brought about changes i n
p e r m e a b i l i t y of the s u r f a c e body c e l l s which r e s u l t e d i n death.
R e c e n t l y Doudoroff (1945) r e p o r t e d a 23% l o s s i n water f o r o
Fundulus, a c c l i m a t i z e d t o 14 C. and p l a c e d f o r two days i n un-o
d i l u t e d sea water at 1 C. I f lowever, Fundulus was p l a c e d i n
45% sea water at 1° C. no l o s s of water o c c u r r e d and the f i s h
t o l e r a t e d lowered temperature f o r a longer p e r i o d . Doudoroff
concluded t h a t the e f f e c t of c o l d on the osmoregulative t i s s u e s
was r e s p o n s i b l e f o r h a s t e n i n g death but t h a t i t was not the
o n l y f a c t o r i n v o l v e d .
A c c o r d i n g t o Mayer and S c h a e f f e r (1914) water content
of t i s s u e s v a r i e d d i r e c t l y w i t h the l i p o c y t i q u e
6
c o e f f i c i e n t s , which are r a t i o s of cholesterol : phospholipid
and cholesterol : f a t t y a c i d . Their work was substantiated
by extensive studies on the various relationships i n tissues
of homiothermes, and also included some data on poikilothermes.
Sundstroem (1927) postulated a possible change i n r a t i o of
cholesterol : phospholipid which affected the permeability of
the red c e l l s of humans and r a t s i n t r o p i c a l climate and
accounted f o r t h e i r increased water content. The l i p i d con
stituents of '••Arctic stickleback (Pygosteus pungitius) as com-s
pared to guppies (Giardinus guppyi) were determined by Wilber
and Del Porno (1949), however, moisture determinations were not
included i n t h e i r analyses-.
Other Metabolic Changes i n Temperature Acclimatization of
Poikilotherms.
.The e f f e c t of temperature acclimatization on oxygen
consumption i n f i s h has been investigated by a number of
workers (Gardner and co-workers, 1922; and Fry and Hart, 1948).
Wells (1935) found oxygen consumption of Fundulus parvipinnis
increased with temperature. The same author (1935 a) acclima
t i z e d f i s h to high and low temperatures. When oxygen con
sumption of both groups was tested at an intermediate
temperature f i s h acclimatized to low temperature gave highest
r e s u l t s . Fox (1939) recorded a f a s t e r heart beat f o r f i s h
acclimatized to a low temperature and tested at a higher
7
temperature. Freeman (1950) found oxygen consumption, brain
metabolism,and resp i r a t o r y movements of go l d f i s h increased
with the temperature of acclimatization up to a temperature of
30° 0. However, f i s h acclimated to a high temperature i f
placed i n a low temperature showed considerable i n i t i a l de
crease i n metabolism followed by gradual increase u n t i l the
metabolism resembled that of a f i s h acclimated to that temp
erature. Likewise Peiss and F i e l d (1950) measured metabolism
of brain and l i v e r tissue of Polar cod (Boreogadus saida) and
Golden orfe (Idus melanotus) at a temperature intermediate
between temperatures of acclimation of the cod and orfe and
found the polar cod showed a higher rate of metabolism.
Materials and Methods -
Two groups of experiments have been carried out:
Group I involved feeding f i s h diets containing a high l e v e l of
fat of d i f f e r e n t types f o r a 30 - 60 day feeding period and
subsequently testing temperature tolerance of the f i s h , de
termining water and l i p i d content of the t i s s u e s , and degree
of unsaturation of the l i p i d s . Group II involved acclima
t i z i n g f i s h to d i f f e r e n t temperatures, and l a t e r determining
water content, l i p i d content and l i p i d constituents of the
tissues and degree of unsaturation of the l i p i d s . Approx
imately 2,000 go l d f i s h were used i n the experiments. These
were procured from the Goldfish Supply Company, S t o u f f v i l l e ,
Ontario.
8
Feeding Experiments.
Ten d i f f e r e n t f a t s , three of which were used i n dup
l i c a t e , were selected f o r the d i e t s . These may be grouped
into four r e l a t e d series which provide a range i n degree of
unsaturation f o r each s e r i e s .
P i l c h a r d o i l , Herring; o i l , and Lard (Natural f a t series) •
The pilchard and herring o i l s used i n the diets were
obtained from Western Chemical Co. and the l a r d from Burns Co.
Iodine values of the o r i g i n a l o i l s were: pilchard oil—-172,
herring o i l — 1 3 4 , and lard—<65, and iodine values of o i l s
extracted from the diets were 147, 114, and 64 as shown i n
figure 1, graph A.
Hydrogenated P i l c h a r d o i l s (HPO Series 1)•
Four hydrogenated pilchard o i l s of d i f f e r e n t degree
of saturation were prepared by Dr. Swain of the P a c i f i c
F i s h e r i e s Experimental Station* The o i l s were hydrogenated at
25 l b s . pressure of hydrogen and 180° C. i n the presence of
0«26% n i c k e l as found i n "Sele c t o l A." This material, a
hardened f a t , contained a suspension of 16.44% n i c k e l . The
n i c k e l was removed af t e r hydrogenation by f i l t r a t i o n followed
by washing with d i l u t e s u l f u r i c acid and water. The f i n a l
product contained 1.4% of the hardened f a t and les s than 0.05
mgms. Ni/gm. f a t . Iodine values of the o i l s when f i r s t pre
pared were as follows: 182, 154, 92 and 64. When checked
to follow page 8 ISO
I20
90
Ui us
5
z E o
A 60
140
I20-
KX>-
Bao NATURAL FATS HPO SERIES Id a a. cso a CRISCO
F i g . l . Graph A - Iodine values of 13 f a t diets used i n the feeding experiments.
Graph B - Iodine values o f . l i p i d extracted from f i s h fed the 13 f a t d i e t s . Natural f a t s from l e f t to r i g h t i n order: pilchard o i l , h e r r i n g . o i l , and l a r d .
9
2 months l a t e r , at the commencement of the experiment, iodine
values of the two most l i q u i d o i l s were s l i g h t l y a l t e r e d : 150
and 138. Iodine values of the o i l extracted from the diets
were 91.5, 103, 84, and 64 and are shown i n figure 1, graph A.
Hydrogenated Pilchard o i l s (HPO Series 2)
compared with Lard.
Three hydrogenated pilchard o i l s prepared by Dr»
Swain and l a r d were used i n these d i e t s . Iodine values for
o r i g i n a l o i l s were 150, 92, 65, and l a r d 64, and f o r o i l s ex
tracted from diets 132, 85, 65, and 64, as shown i n figure 1,
graph A.
Cottonseed o i l and Crisco (CSO and Crisoo S e r i e s ) •
Cottonseed o i l procured from Western Wholesale Drug
Co. and commercial " c r i s c o " (hydrogenated cottonseed o i l ) were
also used. Iodine values f o r o r i g i n a l o i l s were cottonseed
oil-*106 and c r i s c o — 7 7 and for o i l s extracted from the diets
were 100 and 78 as shown i n figure 1, graph A.
The diets were f r e s h l y prepared every seven to ten
days, according to the dir e c t i o n s of Hoar and Dorchester
(1949) and stored i n the r e f r i g e r a t o r * The f i s h were fed nine
times d a i l y . At each feeding one-^half teaspoon of the diet
was fed each 100 f i s h . Individual feedings were small i n
order to minimize the amount of food which f e l l to the bottom
of the aquaria and disintegrated. Glass aquaria contained the
10
f i s h i n each case. The aquarium water was aerated continu
ously and changed once or twice a week. The water fouled
quickly i n the aquarium containing f i s h on the cottonseed diet
and i t was found necessary to change the water approximately
every two days. The temperature i n a l l the aquaria was main
tained at 20° + 1.5° C.
The four series were fed over d i f f e r e n t periods.
Table I shows duration of feeding period f o r each s e r i e s .
Table I.. Duration of feeding period p r i o r to temperature
tolerance t e s t s .
Series Days fed before low temperature tolerance test
Days fed before high temperature tolerance t e s t
Natural Fats 48 52
HPO Series 1 60 65
HPO Series 2 50 49
CSO & Crisco No test made 32
Samples of four to f i v e f i s h from each aquarium were
taken at three or four i n t e r v a l s during the feeding periods
and iodine values of body l i p i d s determined i n order to follow
progress i n the changes of t h e i r unsaturation.
Afte r completion of the feeding period f i s h were
k i l l e d during temperature tolerance t e s t s .
11
Temperature Tolerance Tests*
Low Temperature Test.
A thermostatically controlled r e f r i g e r a t o r tank was
divided into separate compartments by p l a s t i c netting so- that
the temperature throughout the tank was uniform during t e s t i n g
of a s e r i e s . Approximately half the f i s h from each diet were
plaoed i n separate compartments i n the tank. Temperatures f o r
the four tests were 2.2° * 0.3° C. and were checked at f r e
quent i n t e r v a l s . While t e s t i n g the natural f a t series the
thermoregulation f a i l e d to function f o r several hours and
allowed water to go down to a temperature of 0.7° C. I t i s
emphasized, however, that a l l f i s h i n that series were sub
jected to i d e n t i c a l thermal conditions. Checks to remove the
dead f i s h were carried out at frequent i n t e r v a l s during the
f i r s t twelve hours of the t e s t , after which a check every
three or four hours s u f f i c e d * D i f f i c u l t y arose i n d i s t i n
guishing dead f i s h from l i v i n g ones, since many of the f i s h
l a y motionless on the bottom of the tank. The most r e l i a b l e
test consisted i n prodding the f i n s with a glass rod. Those
which exhibited a negative response were considered dead. The
test was completed i n 48 hours and any survivors removed.
High Temperature Test.
The remaining f i s h from each diet i n a series were
placed i n separate compartments of the same tank as used above
12
i n the low temperature tolerance t e s t . In t h i s case the water
was heated by thermostatically controlled immersion heaters to
a temperature of 3 6 ° * 1° C The f i s h were again tested by
prodding the f i n s with a glass rod and dead f i s h removed.
After 24 hours the test was completed and any survivors
removed*
Approximately h a l f the f i s h which died i n the low
temperature test were pooled into l o t s A, B, C, and D depend
ing upon the time period i n which they died: i . e . l o t A —
f i r s t 12 hour period, l o t B—second 12 hour period and so on;
and o i l extracted. The remaining half of f i s h which died at
the low temperature were used for moisture determinations*
Fi s h which died at the high temperature were discarded except
for those of CSO and 1 c r i s c o ' series which were used for o i l
extraction since no low temperature tests were made on t h i s
s e r i e s .
Moisture Determinations•
After wiping o f f excess water from the surface the
whole f i s h was placed i n a test tube and weighed. The f i s h
were dried to constant weight- (usually about four days) i n an
open oven of 90° C. The f i s h of HPO series 2 were pooled into
l o t s A, B, C and D, depending upon the time period i n which
they died and marcerated i n a Waring blendor. Samples of
tissue from each l o t were weighed i n weighing bottles and
13
dried to constant weight i n an oven as above.
O i l Extraction
The f i s h were macerated i n a Waring blendor and f o r
a quantitative t e s t the weight of a small sample of wet tissue
was recorded* The tiss u e was dried i n a suction f l a s k on a
water bath of 50°-60° 0. The dry, cri s p y tissue was then
transferred to a thimble and o i l extracted by the A. S. T. M.
apparatus* Extraction time was two hours i n a water bath of
3 7 ° C. The ether was then removed by suction i n a water bath
of 3 7 ° C. and the o i l placed i n a dessioator for f i n a l drying
and weighing*
Iodine Determination
Iodine values, commonly used as a measure of the
degree of unsaturation of o i l s , were determined according to
W i j i f s Method. A known amount of IC1 was allowed to react
with a sample of o i l and the excess IC1 was then t i t r a t e d with
sodium t h i o s u l f a t e . The difference between the t i t r a t i o n of
the blank and that of the sample gave the amount of IC1 r e
quired to saturate the double bonds of the sample. This was
expressed as an iodine value by converting to the grams of
iodine which would saturate a one-hundred-gram sample of o i l .
14
Acclimatization Experiments
Three acolimatization experiments were carried out.
A preliminary experiment consisted of acclimatizing 50 f i s h to
15° and 50 to 35° C. The p r i n c i p a l experiment consisted of
acclimatizing 100 f i s h to each of three temperatures: 5°, 20°
and 35° G. A repeat on the p r i n c i p a l experiment was made and
involved the same number of f i s h acclimatized to the same
temperatures.
The f i s h were' retained i n glass aquaria heated by
means of immersion heaters, except f o r those acclimatized to
5° C. which were held i n the r e f r i g e r a t o r tank. Temperatures
were raised or lowered, as required, one degree per day u n t i l
the temperature of acclimatization was attained. The time r e
quired to raise or lower the temperature of the aquaria to that
desired and time allowed at that temperature are shown i n
Table I I .
Diets were similar f o r a l l f i s h , regardless of temp
eratures. Three times d a i l y one-half teaspoon of.Pablum
(Meads Company) was fed to each 100 f i s h . Once a week t h i s
was supplemented by one teaspoon of dog meal.(Gaines).
The aquarium water was aerated continuously and
changed once or twice a week.
15
Table II.- Time allowed i n the three experiments for f i s h to
aoolimatize to the d i f f e r e n t temperatures.
Experiment Temperature
of acclimatization
Days required Days at to r a i s e or desired
lower temperature temperature
15° 0 33
,35° se 12
5° l l 14 P r i n c i p a l
30° 0 25
35° 16 9
5° 15 12 Repeat
20° 0 22
35° 16 12
Temperature Tolerance.
To determine whether the time f o r acclimatization
was s u f f i c i e n t , temperature tolerance of the f i s h of the p r i n
c i p a l experiment was determined. One glass aquarium, held at
38° C. and the r e f r i g e r a t o r tank at 2° C. were each divided by
p l a s t i c netting into three compartments. Half of the acclima
t i z e d f i s h from each of the three temperatures were placed i n
compartments i n the 2° tank and the remainder of the f i s h were
plaoed i n the 36° tank. The t e s t s were sim i l a r to those de
scribed above for the diet f i s h . After completion of the
te s t s the dead f i s h were ground, moisture content determined,
16
and o i l extracted.
Moisture Determinations.
Moisture content of f i s h i n the preliminary experi
ment was measured as described i n feeding experiments, f o r
natural f a t series and HPO series 1, exeept 'that a vacuum oven
at 90° C. and 5 l b s . pressure replaced the e l e c t r i c oven. In
the p r i n c i p a l experiments and repeat experiments moisture con
tent was determined as described i n feeding experiments for
HPO series 2.
O i l Extraction.
In the preliminary experiments o i l was extracted
as described i n the feeding experiments. A simpler and more
e f f i c i e n t method of f a t extraction was suggested by Dr. Swain
and used i n the p r i n c i p a l and repeat experiments. This con
si s t e d of shaking the macerated tissue with cold peroxide-free
ether and anhydrous sodium s u l f a t e . Ether was d i s t i l l e d o f f
and o i l placed i n a dessicator f o r f i n a l drying. Quantitative
measurements were made by weighing the sample of tissue to be
extracted and l a t e r weighing the o i l product.
Chemical Procedures.
Iodine values were determined by Wiji's method as
described f o r feeding experiments.
17
Phospholipid content of g o l d f i s h o i l was determined
oxidatively by the method of Bloor (1929), Acetone and mag
nesium chloride p r e c i p i t a t e d phospholipids from an ethereal
solution of a 2-10 mgm. sample of o i l . The supernatant l i q u i d
was poured o f f and retained f o r cholesterol determination.
The residue was then dissolved i n moist ether, and the ether
evaporated on a hot water bath. S i l v e r reagent, a s u l f u r i c
acid solution of s i l v e r n i t r a t e and potassium dichromate o x i
dized the organic portion of the phospholipid. The potassium
dichromate was reduced i n an amount proportional to phosphol
i p i d present* The unreduced potassium dichromate l i b e r a t e d :
free iodine which was t i t r a t e d by standardized sodium t h i o -
sulphate. The difference between the sample t i t r a t i o n and
the t o t a l t i t r a t i o n ( t i t r a t i o n of blank) gave the number of
cc. of potassium dichromate required to oxidize phospholipid.
The phospholipid content of the o i l could then be calculated
since the number of cc. of potassium dichromate required to
oxidize a known amount of phospholipid was given.
Equations:
K 2 C r 2 0 7 > 8H 2S0 4-* 2K 2S0 4 + 2 C r 2 ( S 0 4 ) 3 * 8H20 +• 30.2
Reaction of the potassium dichromate and s u l f u r i c
acid of the s i l v e r reagent. The s i l v e r present acts as a
cata l y s t , shortens the time and makes the reaction certain-
18
Phospholipid uses 0 2 freed from above reaction as
follows:
2C 4 2H 4 80 gNP 12102-^ 84C0 2 1- HgP0 4 -r- N a. (Gleopalmityl l e c i t h i n )
Unreduced potassium dichromate l i b e r a t e d I g from K l .
6KI •*• K 2 C r 2 0 7 -r 7H 2S0 4—^ 3 I 2 4K 2S0 4 -f- C r 2 ( S 0 4 ) 3 -r 7H 20.
Free iodine formed i s t i t r a t e d by standardized
sodium t h i o s u l f a t e .
3I2-r- 6Na 2S 20 3 —> 6NaI + 3Na 2S 40g.
Cholesterol content of o i l s was measured according
to Bloor's methods (1916, 1936). The acetone f i l t r a t e from
the phospholipid determination was conveniently employed for
t h i s . The acetone was evaporated and the residue dissolved i n
chloroform. Acetic anhydride and concentrated s u l f u r i c acid
were added and caused acetylation of the carbonyl group, f o l
lowed by condensation to produce a color compound which dev
eloped on standing. The i n t e n s i t y of the color compound was
read on a Cenco photolometer and compared with a standard
curve 0
Fatty acid content, according to Wilber ( l e t t e r ) and
Hawke and Bergeim (1931), i s the difference between t o t a l l i p i d
and c h o l e s t e r o l . Since cholesterol was calculated i n per cent
of t o t a l l i p i d the remaining per cent was f a t t y acids.
19
Results
Fee ding? Experiments.
Effeot of Diet on Unsaturation of F i s h L i p i d .
Changes i n iodine value of f i s h t o t a l l i p i d s i n r e
sponse to the feeding of high f a t diets f o r a 40 day period
are shown i n figure 1, graph B. O r i g i n a l iodine value of f i s h
l i p i d before feeding f a t diets was 114. The diet containing
pilchard o i l of natural f a t series produced a much higher
degree of unsaturation of f i s h l i p i d than any other d i e t .
Diets of iodine values 132, 103, and 92 a l l of HPO se r i e s ,
iodine value of 100 of CSO s e r i e s , and iodine value of 114,
herring o i l of natural f a t series a l l produced i n the f i s h
l i p i d s s i m i l a r degrees of unsaturation. The l a r d d iets of
iodine value 63 produced i n the f i s h l i p i d a comparable degree
of saturation as that produced by HPO series 1 diet of iodine
value 64. -However the diet of iodine value of 65 i n HPO
series 2 was not as e f f e c t i v e i n producing a saturated f i s h
l i p i d .
Condition of Fish during Feeding Period.
The g o l d f i s h fed herring o i l i n the diet suffered a
mortality of 17$ during the f i r s t week. However, for the r e
mainder of the feeding period the survivors appeared to be i n
good condition. The f i s h on the pi l c h a r d o i l d i e t , l a r d d i e t ,
and those on HPO series 1 and 2 fed avidly and appeared i n
20
good condition during the feeding period* The CSO and crisco
d i e t s produced marked lethargic symptoms i n the f i s h * Mortal
i t y was high during the 32-day feeding period* Twenty-six
per cent of f i s h on cottonseed o i l d i e t and 19$ of crisco-fed
f i s h succumbed. E l l i s and I s b e l l (1926) noted peculiar be
haviour of pigs fed cottonseed o i l .
E f f e c t of Dietary Fat on High and Low Temperature
Tolerance of Goldfish.
Data fo r the low and high temperature tests appear
i n figures 2, 4, 5, and 6. A thermograph i s included i n f i g
ure 2, graph A to show the temperature variance caused by
f a i l u r e of the thermoregulation apparatus. Results of natural
f a t series indicate that the f i s h on the herring o i l diet were
most r e s i s t a n t to cold, followed by f i s h on l a r d diet and p i l
chard o i l d i e t i n order of decreasing resistance* The r e s u l t s
of Burridge (unpub.) shown i n figure 3 f o r a group of g o l d f i s h
fed s i m i l a r d i e t s and subjected to low temperatures showed the
same order of resistance* However, i n h i s r e s u l t s f i s h on
l a r d diet and those on herring o i l diet showed increased r e
sistance as compared with r e s u l t s of present study. F i s h on
the l a r d diet showed highest degree of resistance to high
temperatures, while tolerance of those f i s h on the pilchard
o i l diet was approximately equal to resistance of those on
herring o i l d i e t . These findings are substantiated by those
of Hoar and Dorchester (1949) f o r a group of g o l d f i s h fed the
to f o l l o w page 20
l O O r
HOURS F i g . 2 . Temperature t o l e r a n c e of g o l d f i s h f e d n a t u r a l f a t s i n
the- d i e t t s . P i l c h a r d o i l ; h e r r i n g o i l ; and l a r d — ? G r a p h A - t o l e r a n c e to low temperatures (see thermograph) and graph B - t o l e r a n c e t o h i g h t emperature, 3 5.1 - 3 5 .
to f o l l o w page 20
i o o r
7 5
• I HOURS
F i g . 3 . R e s u l t s of Burr i d g e (unpub.) to show t o l e r a n c e of g o l d f i s h ..fed n a t u r a l f a t s i n the d i e t s to low temperature (see thermograph). P i l c h a r d o i l ; h e r r i n g o i l — — ; and l a r d .
21
same diets*
Data f o r HPO series 1, plotted i n figure 4, indicate
that f i s h fed softer hydrogenated pilchard o i l s diets of
iodine value 103 and 92, were much more re s i s t a n t to high and
s l i g h t l y more r e s i s t a n t to low temperatures than f i s h on the
more s o l i d hydrogenated o i l diets of iodine values 84 and 63*
As a group, f i s h of HPO series 2 were more re s i s t a n t
to low temperatures but l e s s resistance to high temperatures
(figure 5) compared with HPO series 1* In both low and high
temperature tests f o r HPO series 2 f i s h fed lea s t hydrogenated
o i l s were more to l e r a n t , as was the case i n HPO series 1. The
f i s h on the l a r d diet showed thermal tolerance similar to those
fed on HPO series diet of iodine value 10-12 points higher
than the iodine value of the l a r d diet*
High temperature tolerance for f i s h of 0S0 and
cri s c o series i s shown i n figure 6. Mo r t a l i t y was exceeding
l y low f o r t h i s series compared to mortality of the other
three s e r i e s . F i s h fed hydrogenated o i l were les s r e s i s t a n t
to heat than f i s h on the l i q u i d o i l diet* Because of the high
mortality during the feeding period i t i s questionable whether
the r e s u l t s have any si g n i f i c a n c e .
To f a c i l i t a t e comparison of high and low temperature
tolerances with the iodine value of f i s h total, l i p i d the time
required f o r 50$ mortality i s shown i n histograms,
HOURS
F i g . 4 . Temperatur.e t o l e r a n c e of g o l d f i s h fed HPO s e r i e s 1 d i e t of I.V. 103 ; I.V. 92 ; I.V. 84 ; and I.V. 64 ••• Q Gragh A shows t o l e r a n c e to low temperature (2.1 -2.4 C) and graph B shows t o l e r a n c e to h i g h temperature (34.1 -35°C).
HOURS
F i g , 5 . Temperature t o l e r a n c e o f g o l d f i s h f e d HPO s e r i e s ' 2 d i e t of I.V. 132- ; I.V. 85 ; I.V. 65 ; and I.V. 64 ( l a r d ) . . . . Ggaph A shows t o l e r a n c e to low temperature (2.2 -2.4 C) and graph B shows t o l erance to hi g h temperature (34.9 -35.7 C ) .
t o f o l l o w page £1
lOOr
6 12 18 24
HOURS
F i g . 6 . Temperature t o l e r a n c e o f g o l d f i s h f e d c o t t o n seed o i l ; and c r i s c o . Temperature of the w a t e r d u r i n g t e s t i n g was 3 3 . 9 - 3 5 . 8 C.
22
figures 7 and 8. Because of uncontrollable temperature d i f
ferences during t e s t i n g of the four s e r i e s , i t i s not possible
to compare i n d i v i d u a l iodine values of f i s h l i p i d from one
series with an i n d i v i d u a l of another s e r i e s . However, the
trends of each series as a whole are comparable.
E f f e c t of Dietary Fat on Moisture and L i p i d Contents of
Tissues and Iodine Values of T o t a l L i p i d ,
Moisture contents of f i s h of the various feeding
experiments are shown i n Table I I I , Comparing natural f a t
series and HPO series 1 and 2, f i s h on the natural f a t s con
tained s l i g h t l y l e s s water than those of HPO series 1 and 2,
F i s h on i n d i v i d u a l diets of p i l c h a r d o i l , herring o i l , and
l a r d of natural f a t series showed inappreciable differences
i n water content compared to variations i n i n d i v i d u a l deter
minations (Appendix, Table V). F i s h of HPO series 1 showed
increases i n water content as diet f a t became more f l u i d . The
same trend did not appear i n HPO series2. Since determina
tions i n series 2 (Appendix, Table VII) were based on samples
of pooled f i s h b r ie instead of i n d i v i d u a l f i s h as i n series 1
(Appendix, Table VI) the r e s u l t s of series 2 are probably more
r e l i a b l e . Water contents of f i s h from CSO and crisco series
were not determined.
Fish of the natural f a t series contained s l i g h t l y
more l i p i d than f i s h from the other series (Table I I I ) .
to f o l l o w page 22
H P O S E R W 2
8 8 55o/* SURVIVAL 1
.21 1
IOI %2afi> SURVIVAL 1 IOS SURVIVAL
H P O
SERIES I
NATURAL PATS
SO.
113.
-fl2_
IO 20 30 40 4 8
HOURS
F i g . 7 . Time f o r 50% m o r t a l i t y i n low temperature t o l e r a n c e t e s t s f o r f i s h f e d d i f f e r e n t f a t d i e t s . Iodine v a l u e s of l i p i d e x t r a c t e d from.the f i s h are shown i n the bars,,
to follow page 22
CSO * CRISCO
94 60^> SURVIVAL 109 80o/» SURVIVAL
HPO SERIES 2 i o n
HPO SERIES I
NATURAL FATS
J B L A .
9 8
IQ5
96
87 109 135
60 120
MINUTES
ISO
106 40</> SURVIVAL H3 48 </>SURVIVAL
1440
Fig.8. Time f o r 50% mortality i n high temperature tolerance Seats for f i s h fed d i f f e r e n t fa't d i e t s . Iodine values of l i p i d extracted from the f i s h are shown i n the bars.
23
Table I I I . Moisture and l i p i d oontents of f i s h tissue and
iodine value of f i s h l i p i d extracted from f i s h fed
the various d i e t s .
Iodine value Series of di e t i Moisture % L i p i d $ Iodine value
147 74.59 5,08 135 Natural (pilchard o i l ) Fats - 114 74.96 4.96 109
(herring o i l ) 64 74.84 5.43 87
(lard)
HPO 103 79.61 1.95 106 Series 1 92' 77.33 2.87 113
84 76.76 3.28; 95 64 76.59 3.08 W
HPO 132 77.52 4.26 105 Series 2 85 77.18 3.49 101
65 77.55 4.24 98 64 77.30 4.25 88
(lard)
CSO & 100 1.53 109 Crisco (CSO)
.76 1.86 94 (crisco)
The low f a t content of f i s h of the CSO and crisco series may
be attributed to the shorter feeding period and the poor con
d i t i o n of the f i s h . That variati o n s i n l i p i d content of f i s h
of pilchard o i l , herring o i l , and l a r d diets denotes d i s p a r i t y
i s doubtful since differences between the three diets i s small
compared with variations between i n d i v i d u a l determinations of
any one diet (Appendix, Table 7111). This would also apply
to the HPO series and CSO and crisco series, the i n d i v i d u a l
24
determinations of which are shown i n the Appendix
'('Tables IX, X, XI).
Iodine values of t o t a l l i p i d extracted from f i s h
which died during the temperature tolerance tests are shown
i n Table I I I . These values are means of eight i n d i v i d u a l
determinations on l i p i d of f i s h of l o t s A, B, C and D which
died during the four 12-hour periods of the low temperature
tes t and are shown i n the Appendix (Tables VIII, IX, X, XI).
Very l i t t l e change occurred i n iodine value of l i p i d s of f i s h
from any of the diets during the 48-hour t e s t .
Acclimatization Experiments.
Temperature- Acclimatization and Temperature Tolerance.
Data for the, temperature tolerance t e s t s of the
p r i n c i p a l experiment are shown i n figure 9. From the r e s u l t s
i t i s evident that f i s h had been acclimatized.
Temperature Acclimatization and Moisture Content, L i p i d
Content, L i p i d Constituents of F i s h Tissue and Iodine
. Values of T o t a l L i p i d s .
Results are summarized i n Table IV and shown graph
i c a l l y i n figure 10. Water content of f i s h tissue f o r the
three experiments showed a s l i g h t increase with a r i s e i n
temperature except for .fish acclimatized to 35° C. i n the
repeat experiment. This was possibly an error since
to follow page 24
6 12 l g 24 H O U R S
Fig.9. Temperature 0tolerance of goldfish acclimated to 5 C ; 2CTC- ; and 35 C - rf Gragh A shows tolerance to - low temperature (1.9 -2,3 C) and graph B shows tolerance to high temperature (35.9° 36.2°C).
Table IV. Determinations of some constituents of tissues of go l d f i s h acclimatized
to d i f f e r e n t temperatures.
Acclimation Moisture T o t a l Iodine Phospholipids Cholesterol temperature % l i p i d % value % of fo of wet % of fo of wet
l i p i d tissue l i p i d t issue
Preliminary
15° 80.23 (6) 0.92 (3) 101.1 (2) — — — —
0 35 83.07 (8) 0.54 (5) 91.2 (2) — — — —
P r i n c i p a l 0 5 81.42 (2) 1.35 (6) 123.9 (2) 0.92 (3) 0.012 12.75 (4) 0.172 o
20 82.98 U) 1.07 (5) 111.2 (5) 2.82 (6) 0.030 17.15 (6) 0.183
0 35 84.61 (2) 0.63 (6) 105.8 (5) 11.81 (5) > 0.074 27.7 U) 0.173
Repeat -
5° 80.94 (4) 1.54 (4) 114.1 (2) 6.4 (6) 0.099 13.2 (6) 0.203 20° 81.61 (4) 1.30 (4) 110.9 (2) 6.6 (6) 0.086 13.8 (6) 0.179 35° 81.06 (4) 1.38 (4) 102.8 (2) 9.84 (6) 0.136 15.67 (6) 0.216
Table IV. continued.
Acclimation temperature
Fatty acids fo of fo of-wet l i p i d tissue
Cholesterol Phospholipid
Cholesterol Fatty acid
P r i n c i p a l
5° 87.25 1.17 14.33 0.15 0
20 82.85 0.88 6.10 0.21 o
35 72.30 0.45 2.34 0.38
Repeat
5° 86.8 1.33 2.05 0.15
20° 86.2 1.12 2.08 0.16 0
35 84.33 1.16 1.58 0.19
Number i n brackets indicates number of determinations.
F i g . 10. Histograms to show some constituents (expressed i n percent of wet tissue) of goldf i s h acclimated to d i f f e r e n t temperatures. Values for the preliminary experiment are shown i n cross-hatch, for the p r i n c i p a l experiment i n black, and for the repeat experiment i n white.
to f o l l o w page 24
MOSTUflE
CHOLESTEROL 5° 2CP 35°
• 0 2
• 2 0 CHOLESTEROL PHOSPHOLIPID
-IO
LIPID
FATTY ACID
S° 2 0 ° 35° 15 •
OS -
CHOLESTEROL 3 5 © Q . 4 " FATTY ACID
O 2 -
{ 2 5
Black (unpub.) also found water content of g o l d f i s h to i n
crease with a r i s e i n temperature of acclimatization. In
her experiments the water content of f i s h acclimatized to
2 - 11° C. was 80$ and of f i s h acclimatized to 11 - 26° C.
was 82$. Her r e s u l t s were the average of 8 and 13 samples
re s p e c t i v e l y .
~ L i p i d content of g o l d f i s h tissue decreased s l i g h t l y -
as the temperature increased, with the exception again of
the f i s h maintained at 35° G. i n repeat experiments. I t
would appear that these f i s h are not comparable.
Iodine values of the t o t a l l i p i d s decreased as the
temperature of acclimation of g o l d f i s h increased. A r i s e of
15° C. decreased the iodine value an average of 7.5 points or
change i n iodine value per degree was 0.51 points. Hunter
(unpub.) reported si m i l a r changes i n iodine value with temp
erature. His r e s u l t s showed an average change per degree of
0.5 points.
Phospholipid contents of the f i s h tissue from the
p r i n c i p a l experiment and the repeat experiment were not the
same but showed a similar trendy that i s , with an increase i n
temperature of acclimation the phospholipid content increased.
A l l the r e s u l t s were low compared with those of Wilber and
Del Pomo (1949). Cholesterol content of the f i s h from the
p r i n c i p a l experiment showed very s l i g h t variations with
changes i n temperature acclimation as shown i n figure 10.
86
Cholesterol content of the f i s h employed i n the repeat ex
periment was s l i g h t l y higher and more variable for the d i f
ferent temperatures than the cholesterol content of those
employed i n the p r i n c i p a l experiment. Fatty acid values f o r
f i s h of the p r i n c i p a l experiment decreased with a r i s e i n
temperature. Results f o r the f i s h of the repeat experiment
showed smaller changes with a change i n temperature of acclim
a t i z a t i o n . However, the'values followed a trend similar to
that of the p r i n c i p a l experiments except f o r f a t t y acid con
tent of f i s h acclimated to 35° C. as shown i n figure 10.
Cholesterol : phospholipid r a t i o of the f i s h from
the p r i n c i p a l experiment decreased with a r i s e i n temperature
of acclimation. Although the r a t i o s i n the repeat experiment
showed the same trend as the p r i n c i p a l experiment the de
crease i n the r a t i o with a r i s e i n temperature was s l i g h t as
shown i n figure 10. Cholesterol : f a t t y acid r a t i o of the
tissues increased with the temperature. This increase was
more marked i n the f i s h of the p r i n c i p a l experiment than i n
those of the repeat experiment.
Discussion.
In accordance with the general hypothesis, that the
degree of saturation of l i p i d s of plants and aniiaals i s de
pendent upon the temperature of the environment, Lovern (1938)
and Hunter (1948) showed that the melting points of f i s h
27
l i p i d s were raised when fish, were acclimatized to higher
temperatures. Melting points of f i s h l i p i d s were also raised
by feeding a high l e v e l of saturated f a t i n the d i e t , as
shown by Lovern (1938) and Hoar and Dorchester (1948)• In
the present investigation the temperature tolerances of f i s h
whose body l i p i d s had been altered by diet were determined
and i t was found that although temperature tolerance was
modified by changes i n melting points of the f i s h l i p i d s ,
degree of unsaturation of body l i p i d s of f i s h was not d i r
e c t l y related to temperature tolerance.
In neither natural f a t series, HPO series,nor GSO
and crisco series was an increased melting point of body
l i p i d s r e l a t e d to increased tolerance to high temperatures
or a decreased melting point of body l i p i d s r e l a t e d to i n
creased tolerance to low temperatures, figures 7 and 8* In
natural f a t series f i s h on herring o i l diet were most r e s i s
tant to low temperature followed by f i s h on l a r d diet and
pil c h a r d o i l diet respectively* F i s h on l a r d d i e t showed
greatest tolerance to high temperatures followed by f i s h on
pilchard o i l diet and herring o i l diet (figure 2). In HPO
series 1 and 2 f i s h on the more l i q u i d f a t diets were most
res i s t a n t to both low and high temperatures (figures 4 and 5).
The higher l i p i d content of f i s h on HPO series 2 over those
of HPO series 1 may account f o r increased resistance of a l l
f i s h to low temperatures and decreased resistance of f i s h to
high temperatures as compared with f i s h of HPO series 1.
28
F i s h of the CSO and crisco series were comparable to HPO
series 1 and 2 i n that f i s h on diet containing more l i q u i d
f a t were more r e s i s t a n t to high temperature than f i s h on diet
containing harder f a t .
Though temperature tolerance of f i s h with t o t a l
body l i p i d s of d i f f e r e n t melting point show marked variati o n s
there i s no orderly r e l a t i o n between tolerance to high temp
erature and high melting point of t o t a l body l i p i d or t o l e r
ance to low temperature and low melting point of t o t a l body
l i p i d s . According to S i n c l a i r (1935) the degree of unsat-
uration of the group of phospholipids which pa r t i c i p a t e i n
oxidation and formation of c e l l u l a r membranes do not neces
s a r i l y resemble degree of unsaturation of dietary l i p i d or
t o t a l body l i p i d . This group of phospholipids i s most pre
dominant i n nerve and muscle t i s s u e . I t i s possible that the
degree of unsaturation of the phospholipid f a t t y acids of
these tissues i s related to temperature tolerance of the
f i s h , a r e l a t i o n s h i p which would merit further i n v e s t i g a t i o n .
The role of water content and water balance i n r e
l a t i o n to temperature resistance was studied recently by
Doudoroff (1945). He concluded that a breakdown of osmo
regulatory tissues hastened death at low temperatures* In
the present study water content of g o l d f i s h acclimatized to
d i f f e r e n t temperatures increased with the temperature of
acclimation. T o t a l l i p i d s of g o l d f i s h were also determined
29
and found to vary inversely with the water content.
Two p o s s i b i l i t i e s may account for increased water
i n f i s h acclimatized to higher temperatures: f i r s t l y in-?
creased metabolic water as a r e s u l t of higher metabolic rate
and secondly increased permeability to water. An increased
metabolic rate at higher temperatures was shown f o r go l d f i s h
by Fry and Hart (1948) and Freeman (1950). This would r e s u l t
i n greater u t i l i z a t i o n of food, less f a t stored i n depots 5and
more metabolic water formed i n oxidation. This would also
explain the higher l i p i d content of the f i s h acclimated to
low temperatures. Page and Babineau (1950) recorded a r e
placement of water by gain i n f a t f o r rats kept at low
temperatures.
According to Brooks and Brooks (1941) permeability
i s the rate of movement of a substance through the permeable
layer under a given d r i v i n g force. There are thus two con
cepts involved—the d r i v i n g force and permeability as a
property of a membrane• Generally water enters c e l l s more
r a p i d l y at a higher temperature due i n part to a speeding up
of d i f f u s i o n or a change i n dr i v i n g force. In experiments on
Fundulus i n sea water, Doudoroff (1945) found that controls
at 14° C. contained more-water than those at 20° C. In t h i s
case the hypotonic f i s h l o s t water possibly due to increased
d r i v i n g force at the higher temperature. In the hypertonic
g o l d f i s h the opposite occurred and water inflow increased
30
(results of present study). Experiments on red blood c e l l s
carried out by.Lucke and McCutcheon (1932) snowed that 2 - 3
times as much water entered c e l l s f o r a 10° r i s e i n
temperature.
The change i n permeability as a property of the
membrane would also appear to be involved i n temperature
changes. Mayer and Schaeffer (1914) established the l i p o -
cytique c o e f f i c i e n t s , cholesterol : phospholipid and
cholesterol : f a t t y acid and found them to vary d i r e c t l y with
water content. In the present investigation cholesterol :
phospholipid varied inversely whereas cholesterol : f a t t y
acid varied d i r e c t l y as both water content and temperature
• of acclimation. The discrepancy may be p a r t i a l l y explained
i n that Mayer and Schaeffer*s r e s u l t s were based on i n d i v i
dual organs of homeothermes with only scattered data on
poikilothermes. Since the r a t i o s , s as found i n the present,
study, varied with the temperature and with water content
i t i s possible they indicate some change i n permeability of
the membrane. To study further the p o s s i b i l i t i e s of changes
i n permeability to water by a change i n the temperature of
acclimatization i t would be int e r e s t i n g to study hemolysis of
blood c e l l s of f i s h acclimatized to d i f f e r e n t temperatures
and also determine water content and l i p i d constituents of
the f i s h t i s s u e .
:, .:. 31
The q u e s t i o n remains whether the change i n water
content and p e r m e a b i l i t y to water with a c c l i m a t i z a t i o n confer
upon f i s h i n c r e a s e d t o l e r a n c e t o c o l d or hot water. I t would
seem t h a t i f , as Doudoroff p o i n t e d out, osmotic r e g u l a t i o n
was a f a c t o r i n b r i n g i n g about death then f i s h which are
a c c l i m a t i z e d must have been able t o e s t a b l i s h osmotic r e g
u l a t i o n , p o s s i b l y by changes i n l i p o c y t i q u e r a t i o s and
thus changes i n p e r m e a b i l i t y of the ' c e l l s t o water. As
changes i n the degree of u n s a t u r a t i o n of l i p i d s a l s o accom-
pany.fi 5 a c c l i m a t i z a t i o n p e r m e a b i l i t y of the c e l l s t o water
and m e l t i n g p o i n t of the l i p i d s are p o s s i b l y i n t e r r e l a t e d
f a c t o r s f u n c t i o n i n g i n t o l e r a n c e t o h i g h or low temperatures.
Summary and Con c l u s i o n s
1. In g e n e r a l the degree of u n s a t u r a t i o n of g o l d
f i s h t o t a l l i p i d s was a l t e r e d i n accordance w i t h the g e n e r a l
t h e o r y t h a t the m e l t i n g p o i n t of l i p i d s tends t o resemble the
m e l t i n g p o i n t of d i e t a r y f a t when the l a t t e r I s f e d at a
hi g h l e v e l .
these.
2. T o l e r a n c e o f * f i s h t o h i g h and low temperatures
was m o d i f i e d by the change i n the u n s a t u r a t i o n of t o t a l l i p i d s
but no c o r r e l a t i o n s between t o l e r a n c e to h i g h temperatures
and h i g h e r m e l t i n g p o i n t of l i p i d s or t o l e r a n c e t o low temp
e r a t u r e s and lower m e l t i n g p o i n t of l i p i d s were found.
32 i
3# The water content of f i s h acclimatized to 5°,
15°, 20°, and 35° C. increased s l i g h t l y with the increase i n
acclimatization temperature. The water content of f i s h i n
the feeding experiments was r e l a t i v e l y constant.
4. The l i p i d content of acclimatized f i s h varied
inversely with the temperature of acclimation.
5. The cholesterol : phospholipid r a t i o varied
inversely and cholesterol ; f a t t y acid r a t i o varied d i r e c t l y
as the water content and the temperature of a c c l i m a t i z a t i o n .
6. The iodine values of acclimatized f i s h changed
with temperatures, an average of 7 points per .15° C.
7. The changes i n water content, lipocytique co
e f f i c i e n t s and degree of unsaturation with change i n tempera
ture indicate possible adaptions which occur and which
increase resistance of f i s h to further changes i n temperature.
i
to follow page
Appendix
33
T a b l e V. M o i s t u r e d e t e r m i n a t i o n s o f g o l d f i s h o f n a t u r a l
f a t s e r i e s .
P i l c h a r d o i l H e r r i n g o i l L a r d
74.16 75.36 75.22
77.69 74.00 75.00
76.87 74.70 72.23
71.60 75.31 76.01
73.69 71.77 74. 39
73.44 76.97 73.9?
76.18 77.49 74.30
73.90 76.43 73.43
74.78 73.95 77.22
74.36 74.69 75.12
73.82 73.85 75.15
Means 74.59 74.95 74.84
34
Table VI . M o i s t u r e determinations of g o l d f i s h of HPO
s e r i e s r .
D i e t of i o d i n e value 103
D i e t of i o d i n e value 92
D i e t of i o d i n e value 84
D i e t of i o d i n e value 64
77.35 75.91 74.19 75.03
79.17 76.88 77.59 76.44
78.48 77.11 76.30 75.61
83.0£ 79.47 76.89 77.73
77.20 73.40
78.17 76.36
77.01
Means 79.61 77.33 76.76 76.59
Table V I I . Moisture determinations of g o l d f i s h of HPO
series 2.
Diet of iodine value 132
Diet of iodine value 85
Diet of iodine value 65.
Diet of iodine value 64 (lard)
78.80 76.70 77.78 77.57
76.22 79.59 79.55 78.61
76.60 75.25 78.73 75.99
74.54 74.51
77.14
78.79
Means 77.52 77.18 77.55 77.30
36
Table VIII. L i p i d oontent and iodine values of o i l ex
tracted from f i s h which died during the low
temperature tolerance t e s t .
Lots P i l c h a r d o i l Herring o i l Lard
L i p i d '-/ b Iodine L i p i d °/o Iodine L i p i d % Iodine value value value
A 5.48 133.2 5.11 103.6 6.25 87.6
134.0 104.1 87.4
B 5.40 132.3 6.10 106.8 5.97 84.56
134.8 106.8 84.6
C 5.57 121.4 4.39 109.9 4.55 93.6
124.5 110.1 91.7
D 3.83 137.6 4.23 114.1 4.97 83.8
137.3 115.0 83.6
Means 5.08 134.8 4.96 108.7 5.43 87.1
37
Table IX. L i p i d oontent and iodine values of o i l ex
tracted from f i s h of HPO series 1 which died
during the low temperature tolerance t e s t .
Lots Diet of I.V. 103
Diet of I.V. 92
Diet l of I.V.
34 Diet of I.V.
64
L i p i d % I.V. L i p i d fo I.V. L i p i d % I.V. L i p i d % I.V.
A 2.22 109.8
110.3
3.51 114.6
113.2
3.98 96.9
96.4
3.62 90.0
89.2
B 2*05 107.1
107.1
2.43 111.5
111.5
94.8
95.3
2.52 89;4
90.3
C 1.59 100.1
100.7
2.94
_ _ _ _
1.70 94.1
93.7
3.12 91.5
90.0
D 1.94 107.1
108.3
2.62 114.0
113.6
4.17 97.8
98.0
- - —
Means 1.95 106.3 2.87 113.0 3.28 95.9 3.08 90.1
38
Table X. L i p i d oontent and iodine values of o i l extracted
from f i s h of HPO series 2 which died during the
low temperature tolerance t e s t .
Lots Diet of I.V. 132
Diet of I.V. 85
Diet of I.V. 65
Diet 64
of I.V. (lard)
L i p i d # I.V. L i p i d % I.V. L i p i d % I.V. L i p i d % I.V.
A 2.79 107.4
103.0
2.79 100.7
101.3
3.33 98.6
98.3
90.1
B — 4.52 99.2
99.6 4.04 89.3
89.9
C 6.52 106.2 4.65 93.6 5.38 99.7 4.24 84.4
106.2 92.7 99.8 83.2
D 4.23 101.1
101.7
99.6 100.0
94.9 — 83.8
3.50 110.2 3.02 102.5 3.73 96.3 4.47 90.1 Survivors
110.2 96.5 90.9
Means 4.26 105.0 3.49 100.8 4.24 98.1 4.25 88.3
39
Table X I . L i p i d content and iodine values of o i l extracted
from f i s h of CSO and crisco series which died
during the high temperature tolerance t e s t .
Lots Cottonseed o i l Crisco
L i p i d % Iodine value L i p i d % Iodine value
A 1.28 106.8 1.61 91.4
106.7 91.4
B 1.79 108.2 2.11 96.7
113.3 96.4
Means 1.53 108.8 . 1.86 94.4
40-
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