Lipid Content of Freshwater Fish

Prog. Lipid Res. Vol. 26, pp. 281-347, 1987 0163-7827/87/$0.00+ 0.
50
Printed in Great Britain. All rights reserved © 1987 Pergamon Journals Ltd
THE LIPID COMPOSITION A N D BIOCHEMISTRY

OF FRESHWATER FISH
R. JAMESHENDERSON and DOUGLAS R. TOCHER

N.E.R.C. Unit of Aquatic Biochemistry, Department of Biological Science,
University of Stifling, Stifling FK9 4LA, Scotland, U.K.
CONTENTS
I. INTRODUCTION 283
II. LIPm CONTENTS AND CLASS COMPOSITIONS 283
A. General aspects 283
B. Whole fish 284
C. Muscle 284
D. Liver 288
E. Adipose tissue and viscera 289
F. Brains and eyes 289
G. Gills 289
H. Intestinal tissue 290
I. Gonads 290
1. General aspects 290
2. Ovary 290
3. Testes 290
J. Blood 291
K. Skin 291
L. Bone 291
III. FATTY A a o COMPOSmONS 291
B. Total lipid 292
C. Neutral lipids 294
1. Triacyiglycerols 294
2. Wax esters 295
3. Cholesteryl esters 295
4. Others 295
D. Polar lipids 296
I. Total phospholipid 296
2. Phospholipid classes 297
(a) Phosphatidylcholine and phosphatidylethanolamine 297
(b) Others 297
IV. LIPID Svwrrmsxs 299
A. Fatty acid synthesis 299
1. Carbon source 299
2. Tissue sites 300
3. Products of fatty acid synthetase 301
B. Fatty acid esterification 301
1. General pathways 301
2. Glycerogenesis and glyceroneogenesis 302
C. Desaturation and elongation of fatty acids 303
D. Reduction of fatty acids 306
E. Cholesterol synthesis 306
V. METABOLISMOF DIETARY LIPIDS 307
B. Digestion 307
I. Emulsification 307
(a) Role of bile 307
(b) Bile composition 307
2. Lipolytic enzymes 307
(a) Triacylglycerol hydrolases (lipases) 308
(b) Wax ester hydrolases 308
(c) Other lipolytic enzymes 309
3. Products of digestion 309
C. Absorption 309
I. Location 309
2. Digestibility and absorption 310
3. Fates of digestion products 310
D. Transport of plasma lipids 310
281
282 R.J. Henderson and D. R. Tocher
V1. ESSENTIALFATTY ACIDS 312

B. Deficiency pathologies 313
C. Qualitative and quantitative requirements 313
D. Autoxidation and protective mechanisms 314
I. Mechanisms of autoxidation 315
2. Protective mechanisms 315
(a) ~-Tocopherol 315
(b) Catalase 315
(c) Glutathione (GSH) peroxidase 315
(d) Superoxide dismutase (SOD) 316
E. Roles of essential fatty acids 316
1. Biomembranes 316
2. Eicosanoid metabolism 316
(a) Occurrence and synthesis 316
(b) Substrate fatty acids 317
(c) Functions of eicosanoids 318
Vll. LIPID CATABOLISM 318
A. Lipid mobilization 318
2. Starvation 318
3. Gonad maturation 319
4. Hormone-sensitive lipase 320
5. Transport of mobilized lipid 320
B. Fatty acid oxidation 321
2. Mitochondrial oxidation 322
3. Peroxisomal oxidation 323
VIII. EMBRYONICAND EARLY LARVAL DEVELOPMENT 324
A. Eggs 324
1. Lipid content and class composition 324
2. Fatty acid composition 324
B. Lipid catabolism during development 325
IX. DIETARY AND ENVIRONMENTAL INFLUENCES 327
A. Dietary influences 327
I. Fatty acid composition 327
(a) Natural diets 327
(b) Synthetic diets 327
2. Lipogenic enzymes 328
(a) Dietary lipid 328
(b) Starvation 329
B. Temperature 329
1. Lipid content 329
2. Lipid classes 330
(a) Phospholipids 330
(b) Neutral lipids 330
ta) Total lipid 330
(b) Neutral lipids 331
Ic) Phospholipids 331
4. Lipid synthesis 332
C. Salinity 334
1. Lipid content 334
2. Lipid classes 335
4. Lipid synthesis 335
X. ANADROMOUSFISH 335
A. Life cycle 335
B. Lipid content 336
I. Parr smolt transformation 336
2. Spawning migration 336
C. Lipid classes 337
I. Parr-smolt transformation 337
2. Spawning migration 337
D. Fatty acid composition 337
1. Parr and smolts 337
2. Aduhs 338
XI. CONCLUDING REMARKS 339
ACKNOWLEDGEMENTS 340
REFERENCES 340
Biochemistry of freshwater fish 283
I. INTRODUCTION
Fish constitute almost one-half of the total number of recognized vertebrate species. Of
these fish species, about 39%, some 8,400, are found in, or almost always in, fresh water. ~
It is impossible to classify some species as being purely fresh water or marine since some
marine species ascend rivers for short distances, and several fresh water and marine species
are also common in brackish water estuaries. In addition, species may be purely fresh water
in some areas but anadromous in others. Anadromous fish (i.e. fish which spawn in fresh
water but spend much of their time in the sea) are included in this article since lipids
play an important role in their life cycle. The vast majority of species of freshwater fish
are tropical, and the number of species declines towards the polar regions. However, the
number of individuals of temperate species can be large. The range of habitats occupied
by freshwater fish is wide, especially in terms of environmental temperature (equatorial to
polar), light intensity (dark caves to high altitude lakes) and water movement (torrential
streams to stagnant lakes). In addition, the type of diet consumed ranges from purely
herbivorous to strictly carnivorous.
Fish have always been important as a part of the human diet. In addition to fish caught
by traditional methods in the wild, fish farmed commercially now contribute significantly
to the total volume of fish consumed by humans. Salmonids have long been used
successfully in aquaculture in temperature areas and the farming of tilapia and carp is well
established i~ the more tropical regions. The economic importance of aquaculture has
meant that cbnsiderable research has been carried out on the lipid metabolism of several
freshwater species in relation to their growth and development under culture conditions.
For this reason, much of the data reviewed here is derived from studies with commercially
cultured species.
Most of the world's fisheries are based on marine species and, consequently, the vast
bulk of commercial fish oils are of marine origin. The beneficial use of such oils in the
treatment and prevention of cardiovascular and other diseases is well established and is
currently an area of active research, j69"227Although these beneficial effects are generally
accredited to oils from marine species, the lipids from freshwater species have been shown
to exert similar effects on the lipid profiles of plasma in humans) s°
Although an occasional reference is made in this article to marine fish, we have
concentrated on freshwater species and have generally not attempted to compare the two
types. Detailed reviews concerning the lipids of marine fish species can be found
elsewhere. 3"35°The common names ascribed to species in the literature is confusing and one
species can have more than one common name. When required, we have called fish by the
common name used in the original work.
II. LIPID CONTENTS AND CLASS COMPOSITIONS
A. General Aspects
Early observations on the distribution of lipid reserves among the tissues of fish in
general are reviewed elsewhere. 431 In Table 1, the range of values reported by different
workers for the lipid contents of whole fish or fish tissues are presented for individual
species together with the major lipid class component, where known, and references to the
original published data. The values quoted in Table 1 for lipid content were almost entirely
obtained by workers using conventional techniques of extraction with organic solvents. A
mathematical approach recently employed to determine the chemical composition of one
of the Characidiidae family (Oligosarcusjenynsi) demonstrated that, while it was directly
related to the length of the fish, the lipid content of the whole fish showed the poorest
correlation with length in comparison with protein, ash and water contents, due to
variations with spawning period and nutritional state. 189 In view of such variations, the
most accurate method of determining the lipid content of fish is likely to remain that of
direct solvent extraction with subsequent gravimetric quantitation after the removal of
solvent.
284 R.J. Henderson and D. R. Tother
B. Whole Fish
In general, the lipid content of whole fish of a given species is related to size. Whole
European eels (Anguilla anguilla) weighing less than 50 g contained 39% of their dry
weight as lipid, whereas larger individuals weighing in excess of 200 g had lipid contents
of 55% of dry weight. 98 Similarly, the lipid content of the blunt nose minnow (Pimephales
notatus) increased with increasing fish size from about 6% of wet weight in 1 g fish to
almost 20% in fish weighing 4 g.~3~
Tilapia nilotica of body weight 44 g and reared on commercial diets contained 8.3% of
their weight as lipid, of which some 89% were triacylglycerols, the remainder being mostly
polar lipids. "8 On a whole fish basis, the lipid contents of sheepshead (Aplodinotus
grunniens), tullibee (Coregonus artedii), maria (Lota Iota) and alewife (Alosa pseudo-
harengus) were 11.9%, 8.0%, 3.7% and 9.6%, respectively. 2 The bodies of Atlantic salmon
(Salmo salar) smolts caught in the wild were recently shown to contain 1.7% lipid, of
which phospholipids and triacylglycerols accounted for 59% and 24%, respectively. 7 The
proportion of sterols in the lipid was half that of triacylglycerols.
C. Muscle
For ease of discussion, published data on fillets, flesh and eviscerated carcasses have all
been assembled under the heading of muscle, since on a wet weight basis this is the major
component of these preparations. It should be noted, however, that depending on the
preparation and the techniques employed in its production, other tissues such as skin,
nerves and bones may also have been present. Detailed studies of the lipid contents of fillets
from North American freshwater fish have been reported previously ~38.2t7'42zand only the
overall range of values obtained in those and other studies is presented for individual
species in Table 1.
Fillets from the following fish have been shown to have very low lipid contents of less
than 2% of the wet weight: burbot (Lota Iota), bream (Abramis brama), rock bass
(Ambloplites rupestris), crappie (Pomoxis annularis), black crappie (Pomoxis nigro-
maculatus), yellow perch (Perca flavescens), northern pike (Esox lucius), walleye pike
(Stizostedion vitreum ), pike perch (Lucioperca lucioperca), roach (Rutilus rutilus ), suckers
(Catostomus sp.), sunfish (Lepanis gibbosus), snakehead (Channa argus) and vendace
(Coregonus albula ).
A study on six species of subtropical cyprinodontids (killifishes or toothcarps) estab-
lished that the lipid contents of these fish were low and ranged from 2% to 17% of the
dry weight of the body remaining after removal of the gonads, the exact values depending
on species, sex and season. 96 Likewise, in snakeheads from India and Korea, lipid
accounted for only 0.8% to 1.2% of the wet weight of the flesh. 215'36s
For most of the fish species listed above, the lipid class composition has not been
reported. However, lipid from snakehead fillets comprised 72% neutral lipid, 25%
phospholipid and 3% glycolipid with triacylglycerols accounting for 92% of the neutral
lipid. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) comprised 65% and
28%, respectively, of the phospholipids. 2~5 The lipid content (2% wet weight) of muscle
from the parr stage of cherry salmon (Oncorhynchus masou) was twice that of the fish's
smolt stage. 3°5 In the parr, the neutral and polar lipids were present in almost equal
proportions, whereas polar lipid predominated in the smolt lipid.
Lipid contents in the range 0.7% to 5% have been reported for the muscles, fillets or
gutted carcasses of white bass (Morone chrysops), bullhead (Anneiurus melas), brown
bullhead (Ictalurus nebulosus), drum (Aplodinotus grunniens), lake herring/whitefish
(Leucichthys or Coregonus artedi), white perch (Morone americanus), brook trout
(Salvelinus fontinalis), guppy (Poecilia reticulata), Tilapia nilotica, American smolt
(Osmerus mordax), surf smolt (Hypornesus japonicus), ayu or sweet smelt (Plecoglossus
ahioelis), crucian carp (Carassius carassius) and coho salmon (Oncorhynchus kisutch).
Again, no details are available for the lipid class compositions of most of the above species.
B i o c h e m i s t r y o f f r e s h w a t e r fish 285
TASLE 1. L i p i d C o n t e n t s o f F r e s h w a t e r F i s h
Lipid Principal Fatty
content lipid acid
Species Tissue (% wet wt) class composition Ref.
Abramis brama Fillet 1.8 - + 328

(bream)
Alosa pseudoharengus W h o l e fish 9.6 - + 2, 4
(alewife)
Ambloplites rupestris Fillet 0.7 - + 217
(rock bass)
Ameiurus melas melas Fillet 0.8--4.4 - 421
(bullhead)
Anguilla anguilla (C) Whole body 39-55* - - 98
( E u r o p e a n eel)
Anguilla australis Carcass 2-22 - + 459
( s h o r t fin eel)
Anguilla japonica Muscle 22. I TAG + 215,339
( J a p a n e s e eel)
Aplodinotus grunniens Fillet 3.2 - + 217
(drum/sheepshead) W h o l e fish 11.9 - + 2,4
Blicca bjoerkna Liver 4.3-23.8 - - 228
(white b r e a m ) Muscle 3.9-7.3 - - 228
Gonads 3.7-8.5 - - 228
Carassius auratus (C) Brain 7.5--9.2 PL + 335
(goldfish)
Carassius carassius Flesh 1.1 - + 471
(Crucian carp) Viscera 4.8 - - 471
Catostomus commersonni Fillet 1.2-1.9 - - 217,421
(white s u c k e r )
Channa argus Muscle 0.9 TAG + 215
(snakehead)
Channa gachua Liver 2.4 - + 368
(brown snakehead) Viscera 1.0 - + 368
Body 0.S - + 368
Channa marulius Liver 2.2 - + 368
(giant snakehead) Viscera 1.0 - + 368
Body 0.8 - + 368
Channa punctata Liver 3.0 - + 368
(green s n a k e h e a d ) Viscera 1.0 - + 368
Body 0.8 - + 368
Channa striatus Liver 2.0 - + 368
(stripped snakehead) Viscera 2.5 - + 368
Body 1.2 - + 368
Coregonus albula Fillet 1.7 - + 328
(vendace) Roe 9.8 TAG + 193
Coregonus artedii Fillet 2.5 - + 138
(lakeherring/tullibee) W h o l e fish 8.0 -- + 2,4
Coregonus clupeaformis Fillet 2.2-18.8 - + 138,421
(whitefish)
Coregonus lavaretus Liver M 6.6 - + 139
(powan) F 5.3 - + 139
Cyprinus carpio Fillet 1.5-12.5 TAG + 215,217
(carp) 421
Esox lucius Fillet 0.6-1.7 - + 328,421
( n o r t h e r n pike) Liver M 4.0--10.7 - + 139,274
F 3.1-6.9 - - 274
Muscle M , F 0.6--0.8 - - 274
Testes 3.1-5.4 - - 274
Ovary 1.5-6.2 - - 274
Etroplus maculatus Muscle I. 16 - + 285
(orange chromide)
Etroplus suratensis Muscle 2.3 - + 285
(pearl s p o t )
Gestria australis Liver M 6.2-38.0 - - 273
(lamprey) F 4.7-39.5 - - 273
Muscle M 3.8-25.2 - - 273
F 2.9-25.8 - - 273
Testes 2.4-8.3 - - 273
Ovary 8.4-15.2 - - 273
Heteropneustes fossilis Muscle 0.86 - + 285
( s t i n g i n g catfish)
286 R.J. H e n d e r s o n a n d D . R. T o c h e r
TABLE 1--continued
content lipid acid
Species Tissue ( % w e t wt) class composition Ref.
Hypomesus olidus Whole body 4.3 - + 471

( p o n d smelt)
Ictalurus nebulosus Fillet 2.7 - + 217
(brown bullhead)
Ictalurus punctatus (C) Carcass 23.8-43.6* - - 15
( c h a n n e l catfish) (C) Gills 1.6 PL + 296
lctiobus sp. Fillet 1.6-6. I - - 421
( b u f f a l o fish)
Lepomis gibbosus Fillet 0.7 - + 217
( p u m p k i n s e e d sunfish)
Leucisus rutilis Liver M 1.0 - + 139
(roach) F 2.5 PL + 139
Roe 3.7 PL + 193,194
Leucisus reighardi Fillet 13. I - - 421
(chub)
Lota Iota Fillet 0.7-1.2 - + 217,421
(maria/burbot) Whole body 3.7 - + 2, 4
Roe 7.0 SE/WE + 193, 194
Lucioperca lucioperca Fillet 0.8 - + 328
(pike p e r c h )
Mastecembelus armatus Muscle 0.56 - + 285
( s p i n y eel)
Morone chrysops Fillet 2.2-3.8 - + 217, 421
(white b a s s )
Morone americanus Fillet 2.5 - + 217
(white p e r c h )
Oncorhynchus keta Muscle M 1.3 TAG - 400
(chum salmon) F 1.6 TAG - 400
(C) Liver 5.4-9.5 NL + 411,412
(C) Larvae 4.0-12.5 NL - 342
Oncorhynchus kisutch (C) Muscle 2.4-2.6 - - 254
(coho salmon) (C) Liver 3.9-5.6 NL - 411
Oncorhynchus masou Flesh P 2.2-4.6 TAG + 313,315
(cherry salmon) (C) Whole body S 2.0 PL - 305
(C) P 3.5 NL - 305
(C) Liver S 4.2 PL - 305
(C) P 5.0 PL - 305
Oncorhynchus nerka Flesh 6.1-15.0 - + 471
(sockeye salmon) Head 20.7 - + 471
Ophiocephalus punctatus Muscle 0.8 - + 285
(green s n a k e h e a d )
Osmerus mordax Fillet 1.5-3.4 - + 217, 421
( A m e r i c a n smelt)
Perca flavescens W h o l e fish M 2.6-18.4" - - 297
(yellow p e r c h ) W h o l e fish F 2.0-17.1" - - 297
Fillet 0.8 - + 217
Perca fluviatilis Liver F 3.1 - + 139
(perch) Roe 4.1 SE/WE + 193, 194
Pimelodus clarias Muscle M 9.3-20.5 - + 304
(mahdi) F 9,2-20.1 - + 304
Plecoglossus altivelis Flesh M 1.0-5.4 - + 312
(sweet smelt) F 1.0-4.6 TAG + 312
Liver M 3.1-9.4 - - 312
F 3.0-9.0 -- - 312
Testes 2.2-4.4 - - 312
Ovary 4.1~.9 TAG + 312
Poecilia reticulata (C) Whole body 5.0 TAG + 92
(guppy) Liver 6.3 TAG + 92
Pornoxis annularis Fillet 0.8-1.6 - - 421
(crappie)
Pomoxis nigromaculatus Fillet 1.5 - + 217
(black crappie)
Ptychocheilus oregonensis Fillet 2.1-8.6 - - 421
(squawfish)
Puntius filamentosus Muscle 1.14 -- + 285
(filamented barb)
T^at$ I--continued
content lipid acid
Species Tissue (% wet wt) class composition Ref.
Salmo gairdneri Fillet 2.5-3.1 - + 138, 217
(rainbow trout) (C) Gills 2.2 - - 296
(C) Liver 3.6-5.6 TAG + 163, 242
((2) Adipose tissue 82 TAG + 163, 242
(C) Eggs/Roe 9.2-I 1.3 TAG + 193,452
((2) Sperm 0.5-1.3 - + 452
(C) Ovary 5.5 TAG - 457
Salmo salar Fillet 4.0 - + 217
(Atlantic salmon) Whole body 1.7 PL + 7
Liver 10.0 TAG + 139
Salmo trutta
(brown trout) Fillet 4 - + 328
Liver M 2.5 - + 139
F 2.7 - + 139
Salvelinus fontinalis Fillet 3.4 - + 217
(brook trout) ((2) Liver 3--6 - - 289
Salvelinus namaycush Fillet 1.9-19.4 - + 2 1 7 , 421
(lake trout)
Scardinius
erythrophthalmus Body 3.2 - + 359
(rudd) Skin 0.3 - + 359
Spirinchus lanceolatus Flesh 9.9 - + 471
(longfin smelt)
Stizostedion vitreum Fillet 0.7-2.0 - + 217, 421
(walleye pike)
Thaleichthys pacificus Fillet 4.6--9.0 - - 421
(fiver smelt)
Tilapia mossambica (C) Muscle 11.5 - + 284
Tilapia nilotica (C) Whole body 8.3 TAG + 358
Muscle M 0.8--2.2 TAG/FFA - 107
F 1.5-2.0 TAG/FFA - 107
Liver M 4.3-21.7 TAG/FFA - 107
F 12.5-20.2 FFA/PL - 107
Testes 5.4-13.4 FFA/PL - 107
Ovary 12.2-25.5 FFA/TAG - 107
(C), reared in captivity; M, male; F, female; S, smolt, P, parr; NL, neutral lipid; PL, polar lipid; TAG,
triacylglycerols; SE, steryl esters; WE, wax esters; FFA, free fatty acids; *, dry weight basis; +, fatty acid
compositions given in reference.
Triacylglycerols and steryl esters, however, made up 52% and 29%, respectively, o f the
neutral lipid, which itself accounted for 7 4 0 o f the total lipid in the eviscerated b o d y o f
the guppy. 92 P C was the principal c o m p o n e n t o f the phospholipid fraction. T h e lipid (1.5%
o f wet weight) in the muscle o f c o m m o n carp from K o r e a comprised 77% neutral lipids,
21% phospholipids and 2% glycolipids. 2~ Triacylglycerols accounted for 89% o f the
neutral lipids and P C comprised 84% o f the phospholipids.
Fish species f r o m which fillets containing up to 10% (wet weight) lipid have been
obtained include buffalo (Ictiobus, sp.), smelt (Thaleichthys pacificus), Atlantic salmon
(Salmo salar) and squawfish (Ptychocheilus oregonensis). 5.7% o f the wet weight o f muscle
in r a i n b o w trout (Salmo gairdneri) maintained on a commercial diet was lipid o f which
neutral lipid c o m p o s e d 82%. 242 The flesh o f the longfin smolt (Spirinchus lanceolatus) had
a lipid content o f 9.9%. 47~ Muscle f r o m c h u m salmon (Onchorhynchus keta) contained 0.8
to 2.9 g lipid per 100 g tissue in the study o f A n d o et aL, ~2"~3who f o u n d that the relative
p r o p o r t i o n s o f the constituent lipid classes changed during spawning migration. The lipid
content o f muscle from white bream (Blicca bjoerkna) was also influenced by spawning
cycle and varied f r o m 3.9% to 7.3%. z28 Changes in lipid content and composition in
relation to the spawning migrations o f a n a d r o m o u s fish are discussed in Section X.
The lipid content o f fillets f r o m some freshwater fish species can be in excess o f 10%
o f the wet tissue weight as reported for c o m m o n carp (Cyprinus carpio), c h u b (Leucichthys
reighardi), lake trout (Salvelinus mamaycush ) and a whitefish (Coregonus clupeaformis).
288 R.J. Hendersonand D. R. Tocher
Eels (Anguilla japonica) of body weight 210-285 g contained 22% of their muscle wet
weight as lipid, of which triacylglycerols comprised 88%. 215 The lipid content of mandi
(Pimelodus elaria), a Brazilian fish, varied from 8.3% to 20.5% depending on season, but
the constituent lipid classes were not quantified. 3°4
The foregoing values for the lipid contents of fillets represent those of the whole fillet,
but within individual fillets from some species, the lipid may not be uniformly distributed.
For example, within an individual fillet from lake trout, the lipid content decreased from
15.7% at the anterior to 5.2% at the posterior. Furthermore, the ventral areas contained
more lipid than the dorsal area, especially in the anterior region. 217 Similarly, in sockeye
salmon (Oncorhynehus nerka), anterior and posterior regions of the flesh contained 8.1 and
6.1% lipid, respectively, and lipid accounted for 15.1% of the tissue weight in ventral
flesh.47~ The distribution pattern of lipid within the fillets is related to the overall lipid
content since variation in lipid content with region was not so obvious in the salmon fillet
which contained only 4% lipid overall. 217 In contrast to the distribution of lipid in lake
trout and salmon fillets, the lipid content of gutted sections of yellow and silver eels
(Anguilla australis) increased markedly from the pectoral to tail regions. 459
At the subcellular level, the phospholipids of mitochondrial membranes from the lateral
line muscle of goldfish (Carassius auratus) were present at the level of 1.96 mg/g muscle
at 20°C? 33 PC accounted for 48%, PE 34% and cardiolipin (CL) 11% of the total
phospholipids. All other phospholipids were present in proportions of less than 3%.
D. Liver
The values obtained for the lipid content (6.7% to 10.7%) of the liver of northern
pike 139-z74are much greater than those reported for the muscle of the same species. 217.274.328,421
Similarly, livers from Atlantic salmon caught in Scottish coastal waters contained 10%
lipid) 39 whereas fillets of the same species from North American lakes had a lipid content
of only 4°/o. 139'217 The lipid content of liver in Tilapia nilotica maintained on various
synthetic diets ranged from 10.7% to 21.9%. 358'4°3 Similar values have been reported for
the livers of this species caught in the wild by EI-Sayed et a/., t°7 who suggested that the
liver was the main lipid storage organ. Although some freshwater fish do possess livers
with relatively high lipid contents, no species has yet been documented as having a liver
lipid content as high as that of some benthic marine species of fish. For example, the liver
of cod (Gadus morhua) can contain up to 67% of wet weight as lipid whereas the muscle
has a very low lipid content. 183 Under normal nutritional and environmental conditions,
lipid-rich livers are not common in freshwater fish. Cells containing lipid droplets have
been observed by microscopy in the liver of cultivated ayu, but these cells are not
considered to store lipid reserves in wild ayu. a°8
Gunstone et aL t39 reported that the livers of perch (Percafluviatilis), brown trout (Salmo
trutta), roach (Leucisous rutilus) and sea trout (Salmo trutta) all contained less than 5%
lipid and those of male and female powan (Coregonus lavaretus) had lipid contents of 6.6%
and 5.3%, respectively. However, no quantitative data on the lipid class compositions were
provided. Although the livers of snakeheads only contained 2 to 3% lipid, this organ was
still more lipid-rich than either flesh or viscera. 36s
The hepatic lipid content of rainbow trout held in aquaria and fed artificial diets has
been reported to be in the range 3.6% to 5.6% of tissue wet weight. Is3,~63'242Values of 25%
to 50% have been reported for the proportion of neutral lipid in trout hepatic
lipid. ~53'ts4~42'452Phospholipids constituted 44% of the total lipid in livers of trout at 20°C. 153
The neutral lipid was made up of 71% triacylglycerols, 10% cholesteryl esters, 9%
cholesterol, 3% diacylglycerols, 3% free fatty acids, 2% wax esters and less than 1% each
of hydrocarbon and an unidentified component.~s4 In the phospholipids, PC accounted for
62.8%, PE 16.0%, phosphatidylinositol (PI) 7.6%, sphingomyelin (SM) 5.1%, CL 3.0%,
phosphatidylserine (PS) 1.9% and lysophosphatidylcholine (LPC) 1.0%/53
Of the liver weight in the guppy, 6.3% was lipid, in which the percentage of neutral lipid
exceeded that of phospholipids 3-fold. Triacylglycerols made up 39% of the neutral lipid,
the remainder being almost equal proportions of steryl esters and cholesterol. The
phospholipids consisted of 37% PC, 30% SM, 19% PE and an unusually high 14% LPC. 92
Phospholipids accounted for 1.5% of the weight of goldfish liver and were composed
of 57% PC, 22% PE, 7% PI, 6% SM, 4% PS and 4% was an unidentified component? 49
Depending on diet, the lipid content of the livers of chum salmon maintained in
freshwater varied from 5.4% to 9.5% of the tissue wet weight and neutral lipid consistently
comprised over 60% of the total lipid. 4"'4~2 Under similar conditions, the livers of coho
salmon contained 3.9% to 5.6% lipid of which neutral lipid accounted for over 70%. 4j~
The livers of parr and smolt stages of cherry salmon contained 5.0% and 4.2% lipid,
respectively, and polar lipids predominated in both stages. 3°5
Large seasonal variations were also observed in the lipid content (4.2 to 2.3%) of the
liver in white bream. 22s
E. Adipose Tissue and Viscera
Distinct adipose tissue is known to occur in several species of freshwater fish including
rainbow trout,~63'242 coho salmon TM and pike perch. HaIn fish, as in mammals, adipose tissue
is considered to function as a store of reserve lipid. The most obvious adipose tissues in
salmonids occur within the visceral cavity in association with the mesentery of the gut and
gonads. For this reason, viscera are included in this section along with adipose tissue since
the latter can contribute considerably to the total lipid content of the viscera. The contents
of the stomach in some instances may also contribute to measurements of visceral lipid.
In the studies referred to below, the viscera excluded gonads, kidney and liver.
Adipose tissue removed from the viscera of rainbow trout fed synthetic diets was
lipid-rich, containing 82% lipid on a wet weight basis) 63 A similar value of 85% has been
reported for the lipid contents of the adipose tissues associated with the intestines and
gonads of the white bream, us
Lipid accounted for 13°/0 to 31% of the weight of viscera taken from rainbow trout
maintained on synthetic diets ~s6 and for about 35% of the visceral weight in brook trout
fed commercial trout pellets. 2s9 Considering the high lipid content of adipose tissue, it is
surprising how few analyses of lipid class composition have been carried out. The visceral
lipid of rainbow trout was composed primarily of triacylglycerols with much smaller
proportions of cholesteryl esters, free fatty acids, cholesterol and phospholipids. 4°~
In contrast to the lipid-rich viscera of salmonids, the viscera of 4 species of snakeheads
have been shown to have low lipid contents of only 1.0% to 2.5% of wet weight. ~
F. Brains and Eyes

The brain of the goldfish was shown by Roots 33~to contain 9.3 mg total lipid and 4.4 mg
phospholipids per 100rag tissue at 15°C. The phospholipids consisted of 50% PC, 32%
PE and 6% PS, and all three classes were further shown to contain plasmalogens) ~
Another study of goldfish brain phospholipids (3.9% of tissue weight) reported their
composition to be 47% PC, 35% PE, 8% PS, 3% PI, 2% SM, 3% phosphatidic acid (PA)
and 2% unknowns. 249 Driedzic et aL ~°4 determined that, in the brain of goldfish at 30°C,
60% of the total PE and 7% of the PC on a molar basis was in the form of plasmalogens
which together only accounted for 53.5% of the total alk-l-enyl ether-containing lipids of
the tissue. The remaining unidentified plasmalogens were suggested to be plasmalogen
forms of neutral lipids.
The eyes of the guppy contained 3.5% lipid, 72% of which was neutral lipid comprised
of 44% steryl esters, 38% triacylglycerols and 18% cholesterol. PC and PE constituted
42% and 31%, respectively, of the phospholipids which also contained high proportions
(21%) of LPC. 92
G. Gills
The lipid composition of gills in freshwater species had not been extensively surveyed.
Neutral lipids were shown to be present in the lipid extracted from trout gills but were
J.P.L.R. 26/4---C
not quantitated by Hazel, 157 who reported the gill phospholipids of trout acclimated to
20°C to be composed of 56% PC, 20% PE, 6% PI, 5% PS, 5% SM and 4% CL. Half
of the phospholipids in mitochondria from the gills of goldfish reared at 30°C consisted
of PC plus PI. 67 The remainder was made up of 25% PE, 14% SM, 6% CL and 4%
unknowns.
In a recent examination of the gill lipids of aquatic animals, the lipid contents of gills
from rainbow trout and channel catfish (Ictalurus punctatus) were found to be 2.2% and
1.6% of the wet weight, respectivelyfl6 Although the constituent lipid classes were not
examined in detail, the content of polar plasmalogens of catfish gills was estimated to be
22% of the phospholipids and that of neutral plasmalogens to be 1.5% of the neutral lipid.
Trout gill lipid had a much lower plasmalogen content of only 3.6% of the total lipid.
H. Intestinal Tissue
The lipid content of whole intestinal tissue of fish has rarely been reported. However,
the intestinal mucosa of goldfish reared at 30°C was reported to contain 3.6 mg lipid per
g mucosa. :75 The lipid of microsomes prepared from the mucosa comprised 58%
phospholipid and 42% neutral lipid (including 16% cholesterol). The phospholipids were
composed of 4 8 0 PC, 23% PS + PI + SM, 18% PE and 10% unknowns.
PC was also the major component (47%) of phospholipids isolated from the membranes
of the intestinal brush border of rainbow trout. 2.5 Other constituents were PE 22%,
PS 9%, CL 8.5%, PI 6.3%, SM 4.5% and LPC 3.0%.
I. Gonads
1. General Aspects
In this section, only the lipids of whole gonads are described. The lipid content of fish
ovaries is highly dependent upon the stage of the sexual cycle, but many studies do not
provide details of the fish's sexual maturity. A considerable amount of information is
available for the lipid content and composition of the eggs or roe produced by the ovaries
of fish, and the data pertaining to freshwater species are considered in Section VIII in
relation to embryonic development. The influence of spawning migrations on the lipid
contents of gonads is discussed in Section X.
2. Ovary
Ovaries removed from sockeye salmon captured in river water had a lipid content of
10.7% 177 and lipid accounted for 3.7% to 8.5% of the wet weight of the ovary in white
bream. 22s Commercially reared rainbow trout had ovaries which contained 5.5% lipid
composed of 59.4% triacylglycerols, 32.2% polar lipids, 4.3% sterols, 3.1% steryi esters
and 1.0% partial acyiglycerols?55
The ovaries from wild Tilapia nilotica were rich in lipid, the content ranging from 12.2%
to 25.5% of wet weight, t°7 More than half of this lipid was reportedly in the form of free
fatty acids. Such high levels of free fatty acids are not usually encountered in the lipids
of animals. Triacylglyeerols accounted for about one third of the total lipid and cholesterol
and phospholipids together made up less than 20%.
3. Testes
For a given species, the lipid content of the testes is always less than that of the ovary.
Testes taken from Tilapia nilotica caught in their natural habitat in Egypt contained 5.4%
to 13.4% lipid, depending on the season.~°7 Averaged over the year, free fatty acids were
the major component (37.2%) of this testicular lipid as in ovarian lipid. These unnaturally
high levels of free fatty acids may have arisen from lipolysis of acylglycerols before
extraction of the lipidS. Other constituents were phospholipids, cholesterol and

triacylglycerols.
The lipid content of testes from sockeye salmon was only 2%, m and that of testes from
northern pike ranged from 3.1 to 5.4% according to the month of sampling. TM
J. Blood
The lipid content and composition of fish blood is dependent tO a large extent on the
nutritional history of the animal. The transport of dietary lipids in fish is considered in
Section V, and the influence of starvation and gonad maturation in Section VII. A recent
study on trout erythrocytes has shown that, in the membranes of these cells, the
cholesterol-phospholipid ratio was 0.6 for fish fed a commercial diet. 2~
K. Sk/n
In some marine species of fish such as mackerel (Scomber scombrus) and capelin
(Mallotus villosua), deposits of lipid between the skin and muscle can account for a large
proportion of the fish's total lipid reserves. 3 Such subdermal lipid stores also exist in
rainbow trout; 20% to 40% of the dry weight of the skin was reportedly lipid in
comparison with muscle, which contained only 12% to 25% of the dry weight as lipid, a°
In contrast, the skin of the rudd (Scardinius erythrophthalmus) contained only 0.3% of its
fresh weight as lipid) 59 The lipid content of the skin of the common carp has been
determined as being 8% of the wet weight, 5.7% 'free' lipid and 2.3% 'bound' lipid. 79The
'free' lipid was 80% neutral lipid of which triacylglycerols were the major component and
over 50% of the 'bound" lipid was phospholipids, in which PC pedominated and PS and
PE were present in lesser but equal proportions. Ackman and Takeuchi 7 have recently
reported the skins of smolts of Atlantic salmon to contain 4.7% lipid, of which
triacylgiycerols comprised 83%. In another recent study of the phospholipid components
present in the skin secretion of four Indian freshwater fish (Catla catla, Rim rita, Clarias
batrachus and Channa striatus), Mittal and Nigam 276found that PC was always the major
component and, together with PE, made up over 60% of the total phospholipid. SM
accounted for 11.9% to 14.8% and the other components were each less than 10% of the
total with the proportions decreasing in the order PS, PA, LPC, phosphatidylglycerol (PG)
and lysophosphatidylethanolamine (LPE).
L. Bone
The occurrence of lipid in the bones of marine fish has been well documented. 3'232
Although examination of histological sections has revealed the presence of lipid in the skull
and other bones of sticklebacks and minnows, :14 to our knowledge, no compositional data
are available for the lipids of bones in freshwater fish. The head of sockeye salmon was
found to contain 20.7% lipid and to be richer in lipid than any regions of flesh. 471Although
other tissues such as brain and muscle were still present, the high lipid content of the head
may have been due, at least in part, to lipid deposits within the skull.
III. FATTY ACID COMPOSITIONS
A. General Aspects
An abundance of data exists for the fatty acid compositions of lipids from freshwater
fish reared in captivity. However, the fatty acid compositions offish caught in their natural
habitats are described here rather than those fed artificial diets, since the fatty acid com-
position of a fish's body lipid is markedly influenced by that of its dietary lipid (Section IX).
Early analyses performed before the routine use of gas chromatography revealed a
general difference in fatty acid composition between the total lipid from freshwater and
marine fish; lipid from freshwater fish contained higher proportions of saturated fatty acids
and C18 polyunsaturated fatty acids (PUFA), but lower levels of C20 and C22 unsaturates.
than lipid from marine fish. 171 Ackman 2 discussed these differences in detail pointing out
that, for fish from northerly latitudes, it is the high proportions of CI 8 PUFA, rather than
the low levels of C20 and C22 fatty acids, that are typical of freshwater fish. The
substantially lower ratio of (n - 3) to (n - 6) PUFA in the total lipid of freshwater fish
than marine fish was also commented on. Other analytical studies have confirmed these
basic differences in fatty acid compositions between freshwater and marine fish
lipid. 139,285,470,471
In Table 1, fish species for which details of the fatty acid composition are available in
the original publications are indicated. Only the general occurrence of fatty acids is
described here.
B. Total Lipid
The fatty acid composition of total lipid is obviously influenced by that of its constituent
lipid classes. Whereas lipid-rich tissues are likely to have triacylglycerols as their principal
lipid, phospholipids generally predominate in those of low lipid content. Consequently,
comparisons between different fish and tissues of the fatty acid compositions of their total
lipid are of limited value since differences in lipid content and lipid class composition will
influence the observed fatty acid pattern. In addition, seasonal fluctuations in lipid content
are observed in many freshwater fish, especially those in temperate waters, and the fatty
acid composition of the total lipid will vary considerably with season. Temperature also
exerts influences on fatty acid composition. This effect accounts, in part, for the observed
differences between tropical and temperate species and is discussed further in Section IX.
Saturated fatty acids can range from 9% to 36% of the fatty acids in the total lipid of
whole body and tissues of freshwater fish from temperate waters. 2'139In tropical fish, the
content of saturates tends to be slightly higher and can reach 45*/. (Table 2). 171'285Within
the saturates, 16:0 predominates. Smaller proportions of 18:0 and 14:0 are always present
with 18:0 usually exceeding 14:0; 12:0 also occurs, but in proportions of less than 2%.
The saturate 20:0 has been reported as a minor component of lipid from several
sp~ies, 2,s'13,215,3°4,4sg'471 but more recently it has been shown to account for 6.5% of the total
lipid fatty acids in the dorsal muscle of wild carp. 215 Fewer analyses have found 22:0,
although it has been shown to occur in the total muscle lipids of eels from New Zealand 459
and Korea, 215 snakeheads, 215 carp 2~ and chum salmon. 13 The odd numbered saturates
(13:0, 15:0, 17:0 and 19:0) generally account for less than 2.4% of the fatty acids in fish
total lipid, with 15:0 and 17:0 being the principal ones. 215"2s5"368 Anadromous smelts,
however, can contain up to 5% of these fatty acids in their total lipid s'471 and 15:0 and
17:0 can make up 2.1% and 6.0%, respectively, of the fatty acids in the Brazilian mandi. 3°4
The monoene content of total lipid can range from 15.3 % in perch liver ~39to about 51%
in the muscle of eel.215 The monoene 18:1 predominates, followed by 16:1 and then 20: 1.
although in chum salmon caught after spawning in fresh water the proportion of 20:1 in
muscle liver exceeded that of 16:1.13 Small proportions (less than 2%) have been reported
in different studies for each of 14: 1, 2'139"215"304'459 15: 1, 215'471 17: 1,13"215"368'472 22:12'139'215'328"459
and 24:12'138'139
The major dienoic fatty acid found in the total lipid tissues of freshwater fish caught
in their natural habitat is 18: 2 (n - 6). Of the fatty acids in the visceral lipid of the tropical
green snakehead 18.5% were 18:2 (n - 6), 368but the proportion is usually less in other fish.
The diene 16:2 (n - 6) occurs in proportions of less than 2 % 2'139as do 20:2 (n - 9), 2 20:2
(n - - 6 ) , 2'1°5'139 and 22:2 (n - 6 ) ; 2,368 22:2 (n - 9 ) has also been reported to occur in some
tropical murrels. 3~ Dienes in total usually account for some 3 to 6% of the total lipid fatty
acids in temperate species, but the level can be higher in tropical species :m and may vary
with season. ~
Trienoic fatty acids, of which 18:3 (n - 3) is usually the major constituent, generally
comprise less than 10% of the fatty acids in total lipid. The analyses of Puustinen et al. 328
TAn~ 2. Fatty Acid Composition (wt %) of Total Lipid from Four Fish Caught in the
Wild
Ophiocephalus Cyprinus Mallotus
Salmo salar punctatus carpio villosus
Species (Atlantic salmon) (green snakehead) (carp) (capelin)
Tissue Whole body Muscle Muscle Muscle
Location FW FW FW SW
Canada India Korea Norway
Ref. 7 285 215 167
14:0 1.5 0.7 2.3 4.8
16:0 14.2 24.0 19.6 22.1
16:1 (n - 7) 5.5 5.8 9.4 8.5
17:0 - 3.0 1.6 -
18:0 5.3 13.9 4.5 1.8
18:1 (n - 9) 12.6 14.0 23.4 26.0
18:2 (n - 6) 3.1 3.8 3.9 1.4
18:3 (n - 3) 2.2 0.5 6.0 -
18:4 (n - 3) - 1.8 0.2 1.4
20:1 (n - 9) 0.8 0.7 0.8 2.3
20:4 (n - 6) 8.0 6.1 3.5 -
20:5 (n - 3) 4.6 6.0 6.0 13.8
22:1 (n - 11/13) 0.2 0.3 - 2.0
22:4 (n - 6) 1.3 1.4 2.0 -
22:4 (n - 3) - 1.0 - -
22:5 (n - 6) 2.0 - - -
22:5 (n - 3) 3.3 2.2 1.2 -
22:6 (n - 3) 15.4 6.7 5.1 11.3
Total saturates 24.4 45.2 36.3 28.7
Total monoenes 26.5 24.5 35.6 38.8
Total PUFA 45.3 29.9 27.9 26.5
(n - 3) 27.2 18.2 18.5 26.5
(n - 6) 15.8 11.3 9.4 1.4
(n - 3)/(n - 6) 1.7 i.6 2.0 18.9
The values quoted for total saturates, monoenes and PUFA are those reported in the
original studies and include some fatty acids not presented here. FW, fresh water; SW,
sea water.
s h o w e d 20:3 (n - 6) to be the p r i n c i p a l triene in the lipid o f several f r e s h w a t e r species being

u p to 7 % o f the total fatty acids. H o w e v e r , in the s a m e s t u d y 2 1 % o f the fatty acids in
herring (Clupea harengus) lipid were also r e p o r t e d l y 20:3 (n - 6 ) . This fatty a c i d is n o t
usually f o u n d in the lipid f r o m h e r r i n g a n d o t h e r m a r i n e species 3'35° a n d was p r o b a b l y
misidentified in the s t u d y o f P u u s t i n e n et al. 32s M o r e typically, 20:3 (n - 6) a c c o u n t s for
less t h a n 1.5% o f total lipid fatty acids, as d o 16:3 ( n - 4 ) , 18:3 ( n - 6 ) a n d 20:3
(n - 3). :a39'36s
T e t r a e n e s can a m o u n t to up to 27.6% o f the total lipid fatty acids in t r o p i c a l murrels. 3u
R e g a r d l e s s o f species a n d g e o g r a p h i c a l location, 20:4 (n - 6) is a l w a y s the m a j o r t e t r a e n o i c
fatty acid. :'7"23'285 Lesser p r o p o r t i o n s o f 18:4 ( n - 3 ) , :'139':jS'36s 2 0 : 4 ( n - 3 ) 2 a n d 22:4
(n - 6) 2:39'2~5'36s are found. T r a c e a m o u n t s o f 16:4 (n - 1), 21:4 (n - 5?) a n d 2 2 : 4 (n - 3)
have also been r e p o r t e d . :
D e p e n d i n g o n tissue a n d species, p e n t a e n o i c fatty acids can be the p r i n c i p a l g r o u p o f
P U F A in t o t a l lipid :'139'2~5"2'7 a n d usually c o m p r i s e 1.5% to 16.3% o f total lipid fatty
acids. 2a39':lT'Ss5T h e p e n t a e n e 20:5 (n - 3) p r e d o m i n a t e s , b u t 22:5 (n - 6) has been r e p o r t e d
in the r a n g e 2 . 7 - 5 . 1 % in the b o d y lipid o f murrels, a g r o u p o f fish also rich in 2 0 : 4
(n - 6), 36s b u t 22:5 (n - 6) is generally a m i n o r c o m p o n e n t o f total lipid. T h e p e n t : e r i e
22:5 ( n - 3) can o c c u r at the level o f a r o u n d 2 % in n o n l i p i d - r i c h tissuefl a39,368 T r a c e
a m o u n t s o f 21:5 (n - 2 ? ) have also been r e p o r t e d . 2
W i t h o u t exception, 2 2 : 6 (n - 3) is the principal h e x a e n o i c fatty acid in t o t a l lipid in b o t h
t e m p e r a t e 2:3s'~39 a n d t r o p i c a l 2a'SsS'~ species. It occurs at levels o f between 0.30/0285 a n d
30% :17 o f the t o t a l fatty acids.
T h e r a t i o o f (n - 3) to (n - 6) P U F A in the total lipid o f freshwater fish is typically in
the r a n g e 0.5 to 3.82"23"139'2t7'3~c o m p a r e d to 4.7 to 14.4 in m a r i n e fish. 3"139T h e a c t u a l values
depend on tissue and species with lowest values generally being found in tropical
species. 23,368
Analysis of hydrogenated fatty acid methyl esters of oils from four North American
freshwater species showed the existence of branched chain saturated fatty acids (mainly
15:0 and 17:0) in the range 1.6 to 4.0%. 5 The total proportion of iso was always
greater than that of ante iso. Small percentages of 4,8,12-trimethyltridecanoic,
2,6,10,14-tetramethylpentadecanoic (pristanic) and 3,7,11,15-tetramethylhexadecanoic
(phytanic) acids were also found at a combined level of less than 0.6%. These poly-
branched acids have also been reported in total lipid of European fish. 139 Although the
formation of these derivatives from phytol is likely to be commonplace in the herbivorous
invertebrates of the freshwater food chains, their occurrence in the lipids of freshwater fish
is not well documented, presumably due to the low levels in which they occur.
From 74% to 85% of the 18:1 present in commercial oils from four freshwater species
was the (n - 9 ) isomer, the remainder being (n - 7 ) . 6 The 16:1 was always the (n - 7 )
isomer. In the same oils, 20:1 ( n - 9) was the predominant eicosaenoic acid, but in
sheepshead 35% of the total was the (n - 1 1) isomer, whereas in the tullibee 46% was the
(n - 7) isomer. The proportions of (n - 11), (n - 9) and (n - 7) isomers varied with species
for 22: 1, but this fatty acid itself never exceeded 0.2% of the total fatty acids.
A series of fatty acids containing a furan ring, which may be substituted with one or
two methyl groups, has been shown to account for 30% and 27% of the fatty acids in the
total lipid of liver and testes, respectively, of northern pike. ~a2"~33Other North American
freshwater fish have been reported to contain large proportions (20% to 50%) of these
fatty acids in their liver lipid, t3z Analyses of hepatic total lipid from several European fish,
including pike, showed furanoid fatty acids to constitute less than 2.9% of the total fatty
acids.~39
C. Neutral Lipids
1. Triacylglycerols
For fish caught in the wild, the relative abundance of groups of fatty acids within
triacylglycerols decreases in the order monoenes > saturates > PUFA. t39,2ts.3]l,314`459Excep-
tions to this general rule are the triacylglycerols of roach liver ~39and the body of Atlantic
salmon smolts. 7 In both these instances, the level of P U F A exceeded that of saturates. In
the anadromous sweet smelt in fresh water, the proportion of saturates exceeds monoenes
in triacylglycerols) ~:
As with total lipid, 18:1 (n - 9 ) is the predominant monoene and is usually the major
fatty acid in triacylglycerols. Then 16:0 is generally the next most abundant followed by
16:1 ( n - 7), although these two fatty acids can also occur in approximately equal
proportions within triacylglycerols.7'2~5
The level of P U F A in the fish triacylglycerols of sweet smelt caught in fresh water was
31.1%,3~2 but P U F A more typically account for lower proportions of triacylglycerol fatty
acids. 139'314Within the published values of triacylglycerol PUFA, the (n - 3) exceeds the
(n - 6) series. The ratio of (n - 3) to (n - 6) ranges from about 1.08, as in cherry salmon
parr, 3~4to 3.3 observed in the same species in the smolt stage. TM Values for other freshwater
fish mainly fall within this range. 7't39'4~9This ratio is low in comparison with that of 8.3
to 11.4 observed with triacylglycerols of marine herring and menhaden (Brevoortia
tyrannis). 3 Most of the P U F A which occur in total lipid are also found in triacylglycerois
to varying degrees. 24:4 (n - 6) and 24:6 (n - 3) have been reported to be present in very
low levels (less than 0.2%) in muscle triacylglycerols of eels from New Zealand. 459
In the liver and testes of northern pike, up to 67.8% and 23.4%, respectively, of
triacylglycerol fatty acids have been recorded as furanoid fatty acids.~2 Much lower levels
(0.7%) of these fatty acids occur in the hepatic triacylglycerols of the roach. ~39
Fatty acids are distributed assymetrically within triacylglycerol molecules of fish caught
in the wild. 64 For burbot and sheepshead, position 1 contained mostly monoenoic and
saturated fatty acids with only 12% to 20% of the total P U F A residing in this position.
In both fish, position 2 contained about 50% and 25% of the total saturates and
monoenes, respectively. Approximately 43% of the total PUFA was located in this
position of triaeylglyccrols from both fish, but PUFA was still the least abundant group
of fatty acids in terms of mass in this position. Position 3 was poor in saturates but rich
in monoenes---about 70mo1%. PUFA comprised about 17mo1% of the fatty acids in
position 3, corresponding to 36 to 45% of the total PUFA present in triacylglycerols.
Goldfish reared on commercial diets also display a similar stereospecific distribution of
fatty acids within triacylglycerols, with 16:0 predominating in position 2 and PUFA being
distributed mainly between positions 2 and 3. In contrast, rainbow trout fed laboratory
diets synthesize triacylglycerols in which saturated fatty acids are esterified chiefly in
positions 1 and 3 and unsaturated (both mono- and poly-) fatty acids in position 2. 64,239
Despite the strong influence of dietary fatty acids (Section IX), triacylglycerols of fish
fed synthetic diets conform to the above general fatty acid pattern with monoenes and
saturates predominating. 92'~3'~'45~
2. Wax Esters
Only the wax esters present in freshwater fish reared in captivity have been analyzed.
In the wax esters of the roe from the gourami, Trichogaster cosby, 16:0 and 18:1 (n - 9 )
accounted for 45% to 50% and 25% to 30%, respectively, of the fatty alcohols, us'~ The
14:0, 18:0, 16:1 and 20:1 fatty alcohols occurred in proportions within the range 2% to
10%, and 18:2 (n - 6 ) and 18:3 (n - 3 ) at levels of less than 2%. About 50% of the fatty
acid moieties were 18: 1 (n - 9). The acids 18:2 (n - 6), 18: 3 (n - 3), 20:4 (n - 6) and 22: 6
(n - 3) accounted for 14%, 50,20/o and 7% of the total fatty acids. The roe wax esters
of the related species Trichogaster trichopterus were of similar composition. 337 The body
wax esters of gourami species resembled those of the roe. ~
The fatty acid composition of the small amount of wax ester present in the liver of
rainbow trout was found to be dependent on environmental temperature. I~ The 16: 0, 18: 4
(n - 3), 18:1 (n - 9) and 14:0 accounted for 24.9%, 12.0%, 10.8% and 10.7% of the fatty
acid moieties at 20°C. The fatty alcohol components were not reported.
3. Cholesteryl Esters
The fatty acid compositions of cholesteryl esters in freshwater fish have not been
extensively analyzed.
Furanoid fatty acids were specifically associated with cholesteryl esters in the testes of
the pike, where they accounted for 92% of the total fatty acids in this lipid class. 132In the
liver of the roach, cholesteryl esters contained 36.2% furanoid fatty acids, 16.4% (n - 6 )
PUFA, 9.9% (n - 3) PUFA and 16.5% monoenoic fatty acids. ~39No saturated fatty acids
were present.
The steryl esters of cherry salmon parr in the wild were richer overall in PUFA (24.1%
vs. 19.8%) and saturates (36.9% vs. 31.3%) than were the triacylglycerois. 3j4 However,
18:2 (n - 6 ) and 18:3 (n - 6 ) were present in higher proportions in the latter lipid.
No general pattern is apparent for the fatty acid composition of cholesteryl esters in fish
reared in captivity. In the livers of parr and smolt stages of steelhead trout, cholesteryl
esters were richer in saturates and poorer in PUFA than were the triacylglycerols. 372The
monoene contents were similar. However, in the liver of rainbow trout, cholesteryl esters
contained 35.7%, and triacylglycerols only 11.1% of PUFA at 20°C, with the levels of
saturates and monoenes being lower in the cholesteryl esters than in the triacylglycerols. ~
Of the cholesteryl ester fatty acids; 2.2% was 24:4 (n - 6), but only a trace of this fatty
acid was present in triacylglycerols.
4. Others
Little is known of the fatty acid composition of partial acylglycerols and nonesterified
fatty acids in fish lipids, presumably because these lipid classes are usually only minor
components. No definite pattern is obvious from the little information that is available.
In the flesh of cherry salmon parr, diacylglycerols had the same total monoene content
(48%) as triacylglycerols, but contained slightly higher levels (24.9% vs. 19.8%) of PUFA
and correspondingly lower proportions of saturates. 314 In the same species, 47.8% of the
nonesterified fatty acids were saturated, the highest proportion in any lipid class examined.
Monoenoic and polyunsaturated fatty acids comprised 31.6% and 20.6% of the total
nonesterified fatty acids.
The hepatic diacylglycerols of rainbow trout reared on commercial diets at 20°C
contained higher levels of saturates and PUFA than triacylglycerois.~54Correspondingly,
the level of monoenoic fatty acids was only 32.5% in diacylglycerols but 56.2% in
triacylglycerols. Nonesterified fatty acids from these fish comprised almost equal propor-
tions of saturated, monounsaturated and polyunsaturated fatty acids.
D. Polar Lipids
1. Total Phospholipid
Total phospholipids from tissues of wild fish are characteristically rich in PUFA which
can account for up to 58% of the fatty acids present in this lipid fraction. 139In comparison
with triacylglycerols, total phospholipid contains much higher and lower proportions of
PUFA and monoenes, respectively, but similar levels of saturates. 7'13'139'215'312 The PUFA
of phospholipids are of longer chain length than those in neutral lipids with the ratio of
C20 + C22 PUFA to C18 PUFA in phospholipid being generally 4.3 to 9.8 times greater
than that observed in neutral lipid. 7'13"139'312'314
In most cases, 22:6 (n - 3 ) is the major PUFA of total phospholipid, 7:3"132:39.312,314
although, in the liver of the tropical Channa punctatus, 20:4 (n - 6 ) slightly exceeds 22:6
(n - 3 ) ) o5 In temperate species, 20:5 (n - 3 ) and 20:4 (n - 6 ) are both common constit-
uents of total phospholipid with their relative proportions varying with species. Thus, the
proportion of 20:4 (n - 6) exceeds that of 20:5 (n - 3) in total phospholipid from roach 139
and pike livers m32and the bodies of Atlantic salmon smolts,7 whereas the reverse situation
exists in muscle of cherry salmon 13"314and sweet smelt31: caught in fresh water.
The C18 PUFA in total phospholipid are dominated by 18:2 (n - 6 ) and 18:3 (n - 3).
Small percentages (less than 1%) of 18:4 (n - 3) have been reported in phospholipid from
cherry salmon parr and sweet smelt. 312"3j4 The proportion of 18:2 (n - 6 ) is frequently
greater than that of 18:3 (n - 3 ) . 7"1°5"132"139"312 In addition to 20:4 (n - 6) and 20:5 (n - 3),
other C20 PUFA reported in total phospholipid of wild fish include 20:2 (n - 6),~°5'31420:3
( n - - 6 ) 105'139'314 and 20:4 (n - 3). 312 These fatty acids never total more than 2% of the fatty
acids.
22:3 (n - 6), I°s 22:4 (n - - 6 ) , 7"I05"139"312 22:5 (n - - 6 ) 7"105'132'139'312 and 22:5
( n - - 3) 7"13'105'132'139'312can all occur in total phospholipid up to individual levels of about 3%
of total fatty acids. The ratio of (n - 3) to (n - 6) PUFA in the phospholipid fraction of
wild freshwater fish is about 1.6 to 2.0 for published analyses. 7'~9 The corresponding value
in marine fish is in the range 7.8 to 1 8 . 5 . 3
Unlike neutral lipids, phospholipids do not contain furanoid fatty a c i d s . 132'~39
In fish reared in captivity, the phospholipid fraction conforms to the general pattern of
high unsaturation seen with wild fish. However, the pattern of fatty acids, particularly
PUFA, reflects that of the dietary lipid. Thus, the ratio of ( n - 3) to ( n - 6) PUFA
observed in tissue phospholipid is greatly influenced by whether (n - 3) or (n - 6) PUFA
predominate in the diet (Section IX). The environmental water temperature also influences
the fatty acid composition of phospholipids. This feature has been well studied in cultured
fish (Section IX).
Much information is available for the fatty acid composition of total phospholipid from
the whole body, 7'473-47s muscle,215"372"448adipose tissue 372 and liver 14a'163"372"411'~8"451 of salm-
onids reared on synthetic diets. The phospholipid fatty acid composition of peripheral
blood cells from channel catfish has also recently been described. 53At the subcellular level,
the phospholipid fractions of mitochondrial membranes from goldfish gills67 and lateral
line muscle433 have been analyzed for fatty acid Composition. The muscle mitochondrial
phospholipid contained about 50% PUFA. 1so and ante iso methyl branched fatty acids
accounted for over 2% of the total fatty acids.
2, Phospholipid Classes
(a) Phosphatidylcholine and phosphatidylethanolamine. The fatty acid compositions of
individual phospholipid classes in wild freshwater fish have not been studied. For this
reason, the data presented here originate mostly from fish reared in captivity.
Of all the phospholipids isolated from trout liver and examined by Hazel) 53 PE and PC
had the highest contents of 22:6 (n - 3), both being about 32% for fish maintained at
20°C. The two classes differed, however, in their contents of other fatty acids. Most
notably, the percentage of 16:0 in PC was 27.5 but only 11.1 in PE. Similarly, in liver
mitochondrial phospholipids from carp reared at 26°C, PC and PE resembled each other
in 22:6 ( n - 3) content (29%), but PC was again richer than PE in 16:0 (30.1% vs.
11.7%). '~3
In the intestinal phospholipid of guppy 92 and trout, 245 the differences between the fatty
acid compositions of PC and PE were small and both had lower proportions of 22:6
(n - 3) than found elsewhere for the hepatic PC and PE of trout m and carp. ~3 In the
microsomal fraction prepared from the intestine of goldfish acclimated to 30°C, PC and
PE had low contents of 22:6 (n - 3 ) (6.4% and 12.1%, respectively). 275 The major fatty
acid of PC was 16:0 (33%) and 18:0 (39.7%) that of PE; 20:4 (n - 6) was the most
abundant PUFA after 22:6 (n - 3) in both phospholipid classes. From the above studies
and that of Takahashi et al. 399in which the composition of PC from several species of fish,
both marine and freshwater, were compared, emerges the general pattern in which ca. 30%
of the fatty acids in PC of freshwater fish are 16:0.
High performance liquid chromatography (HPLC) analysis 399established that 16:0/22:6
(n - 3 ) and 16:0/20:5 (n - 3 ) were the principal molecular species of PC present in the
muscle of rainbow trout and carp. Highly unsaturated species such as 20: 5 (n - 3)/20: 5
(n - 3), 20: 5 (n - 3)/22: 6 (n - 3) and 22: 6 (n - 3)/22:6 (n - 3) were present only in small
proportions. However, it is likely that 18: 1 (n - 9)/22: 6 (n - 3) and 18: 1 (n - 9)/20: 5
(n - 3) were also present in the 16: 0/20: 5 (n - 3) or 16: 0/22: 6 (n - 3) component peaks,
since 18 : l (n - 9) accounted for substantial proportions of the total fatty acids in PC. A
species in which 16:0 and 22:6 (n - 3 ) were the major fatty acids (28.2% and 40.7%,
respectively) was also found to be the most abundant form of PC present in microsomal
phospholipids of goldfish adapted to 6°C. 275
Within PC and PE molecules, PUFA are typically concentrated in position 2 and
saturated fatty acids in position 1. For microsomal phospholipids of goldfish reared at
30°C, 16:0 and 18:0 together comprised over 90% of the fatty acids in position l while
C20 and C22 PUFA were confined to position 2. 275Environmental temperature influences
this stereospecific distribution (Section IX). This definite preference of PC and PE for
unsaturated fatty acids in position 2 is also seen in trout liver.~53 PE contains virtually all
the constituent PUFA in position 2 whereas almost all the saturates and monoenes are
located in position 1. PC shows the same distinct trend but differs slightly by having
monoenes in both positions.
Recently Hazel and Zerba j6' have shown 16:0/22:6, 16:0/18:1, 16:0/20:3 and
! 6: 0/22: 5 to be the principal molecular species of PC present in the phospholipids of trout
mitochondrial and microsomal membranes (Table 3). In the same membranes, the most
abundant species of PE were 18:1/20:4, 14:0/16:0, 18:0/22:6 and 18:1/22:6. It was
apparent that PE partly balances its high content of C40 polyunsaturated species with high
levels of shorter chain C30 saturated and monoenoic species. It was also notable that the
principal component species of PC was absent from PE and vice versa.
(b) Others. PI of trout liver is characterized by a high content of 20:4 (n - 6) and 18:0 jSa
and, in this respect, resembles PI of terrestrial mammals. The presence of 20:4 (n - 6 ) ,
298 R. J. Henderson and D. R. Tocher
TAaLE 3. Molecular Species Compositions of Phosphatidylcholine and Phosphatidyl-

ethanolamine from Trout Liver Membrane Fractions ~6~
Heavy Light
Mitochondria microsomes microsomes
Molecular
species Temp" 5' 20 5 20' 5 20'-
Phosphatidylcholine
14:0/16:2 b 0.6 2.5 - - 0.4 2.4
16:0/16:0 1.9 5.3 3.2 1.4 2.4 4.3
16:0/18:1 10.3 12.9 12.7 13.0 10.7 17.4
16: 1/18:1 2.0 13.7 2.7 4.9 1.9 5.3
18:0/18:1 0.9 2.0 -- -- 1.0 2.1
18:0/18:2 2.6 3.6 2.4 4.7 3.0 3.2
18:1/18:1 . . . . 0.8 2.4
16:0/20:3 8.5 7.6 10.0 9.7 9.5 7.1
16:0/20:5 6.2 2.1 6.6 2.8 6.0 3.4
16:0/22:6 39.6 24.2 34.3 46.3 37.6 27.8
16:0/22:5 3.7 4.6 5.6 3.7 4.7 4.5
16:1/22:6 3.4 3.0 3.3 2.1 3.8 1.8
18:2/20:4 3.8 2.5 4.0 2.5 3.5 2.3
18:2/20:5 2.9 2.0 - - 2.6 1.8
18:0/22:6 2.7 3.0 - - 2.5 3.0
Phosphatidylethanolamine
14:0/16:0 14.0 6.7 13.1 19.9 14.6 16.0
14:0/16:1 1.2 12.9 1.5 4.2 4.9 13.9
14:0/16:2 1.0 3.0 1.9 2.4 1.0 3.7
16:0/16:1 . . . . 0.5 2.9
16:0/18:1 1.9 3.7 2.1 1.9 1.5 3.0
16:1/18:1 - - 1.2 2.1 1.1 3.6
18:0/18:2 - - 1.4 3.6 1.2 2.7
18:1/18:1 4.3 6.0 4.0 2.3 4.1 3.5
16:0/20:3 3.2 3.0 4.3 3.3 3.6 3.6
18:2/18:3 0.7 3.2 1.3 2.8 1.0 2.9
18:0/20:3 1.3 3.2 2.3 3.7 1.7 3.3
18:1/20:4 23.9 18.1 16.6 16.7 15.1 8.1
18:0/20:5 5.4 5.0 7.6 5.4 7.8 3.9
18:2/20:4 2.2 2.7 2.7 1.3 - -
16:1/22:6 4.1 2.8 5.9 3.7 6.3 2.4
18:2/20:5 7.2 6.9 5.0 5.5 5.7 3.8
18:0/22:6 9.3 7.3 10.5 6.4 9.8 3.8
18:0/22:5 5.6 1.8 4.8 2.1 4.7 1.3
18:1/22:6 7.8 5.7 8.1 6.1 9.6 2.4
Values are percentages.
a Temperature to which trout were acclimated;
b The first listed fatty acid of each pair is not necessarily in position 1.
specifically in PI of aquatic organisms, has been attributed to its possible role in the
provision of 20: 4 (n - 6) for eicosanoid formation, and PI in fish may play an important
role in the transduction of hormone signals through the biomembrane as in mammals (see
Ref. 34). PS of trout liver resembles PI in terms of 18:0 and 16:0 contents. 153 The 22:6
( n - 3) accounts for about 25% of the total fatty acids in PS and the proportion of 20:4
( n - 6) exceeds that of 20:5 (n - 3).
In livers of trout maintained at 20°C, 16:0 was the major fatty acid (26.3%) of SM and
the proportion of 22:4 (n - 6) was slightly greater than that of 22:6 (n - 3) 0 0 . 9 % vs.
9.0%); 16:0 was also the principal fatty acid of CL (23.9%). The 16:l (n - 7) was present
in this phospholipid in higher proportion than in any other liver phospholipid class of fish
at 20°C. In comparison with PC, LPC had a lower 22:6 (n - 3), but higher 20:5 (n - 3)
content. The 16:0 and 18:l ( n - - 9 ) levels were similar.
The principal fatty aldehydes of PE plasmalogens from the brains of goldfish reared at
30°C were reported to be 18:0 (30% to 35%) and 18:l (28% to 30%). 336 Lesser
proportions of 14:0~ 14: l, 16:0 and 16:1 fatty aldehydes were also found. In contrast,
16:0 was the major (30% to 40%) fatty aldehyde of PC plasmalogen in the same tissue,
and 16: l, 18:0 and 18: l were present at lower levels.
IV. LIPID SYNTHESIS
A. Fatty Acid Synthesis
1. Carbon Source
Although the enzymes have not been fully characterized, it is generally assumed that
fatty acid biosynthesis in freshwater fish proceeds via pathways qualitatively similar to
those which operate in mammals. Thus, it can be expected that the two carbon acetyI-CoA
unit is carboxylated to malonyl-CoA, which is subsequently converted to fatty acids by
the fatty acid synthetase complex via a series of condensation and reduction reactions
involving the utilization of N A D P H . 437
In omnivorous mammals, the acetyl-CoA used for fatty acid synthesis originates within
the mitochondrion mainly from carbohydrate metabolism. The natural diets of carnivo-
rous fish such as salmonids and the majority of fish species are rich in protein which
consequently supplies a large part of the fishes' dietary energy along with lipid (see Ref.
~A.). Although salmonids and carp 255'256'4~3are able to utilize dietary carbohydrate as a
source of energy, the rate of glucose oxidation in rainbow trout 257 and carp 2s2 has been
shown to be considerably less than that of amino acid oxidation. Studies mainly with
salmonids have established that, in carnivorous fish, the enzymes involved in the
conversion of amino acids to pyruvate and tricarboxylic acid cycle intermediates are active
(see review by Walton and Cowey, Ref. 444). Consequently, carbon derived from amino
acids can be incorporated into citrate which can then leave the mitochondrion and become
a substrate for ATP citrate lyase in the cytosol. The action of this enzyme generates
cytos01ic acetyl-CoA as a substrate for acetyl-CoA carboxylase.
The importance of ATP citrate lyase in fatty acid synthesis is obvious and its presence
in tissues active in the production of fatty acids would seem obligatory. However, the
enzyme does not appear to be ubiquitous in the lipogenic tissues of fish. The liver of coho
salmon has been shown to contain an active ATP citrate lyaseTM but no detectable activity
was found in the livers of rainbow trout 22 or channel catfish. 253 Only very low activities
of ATP citrate lyase were found in tissues of the American eel (Anguilla rostrata), 17whereas
substantial activity of the enzyme was reported in the liver of the European eel) The
absence of ATP citrate lyase activity and the lack of incorporation of radioactivity from
~4C-aspartate into fatty acids by hepatocytes from the American eel 3~1 led Aster and
Moon ~7to conclude that the carbon source for fatty acid synthesis in this species was one
which did not require citrate as an intermediate, and to suggest that acetate originating
from intestinal processes could act as a precursor for fatty acid synthesis in this species.
Although radioactivity from 14C-acetate has been shown to be actively incorporated into
fatty acids by liver slices of the European eel, l this species also contains an active ATP
citrate lyase, as stated above.
Slices of liver from rainbow trout incorporated more radioactivity from 14C-alanine than
from laC-glucose into fatty acids) 63 In addition to providing evidence for the operation
of ATP citrate lyase in trout liver, this observation also suggested that amino acids were
the preferred carbon source for fatty acid biosynthesis in this tissue.
As in mammals, the N A D P H required for fatty acid synthesis in fish is generated in the
cytosol by glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase
(both enzymes of the hexose monophosphate shunt), NADP-malate dehydrogenase (malic
enzyme) and NADP-isocitrate dehydrogenase. These four enzymes have all been shown
to be active in the lipogenic tissues of rainbow trout, 22coho salmon, 255'256eeP'~7 and channel
catfish. 253
Overall, although protein would seem to be the major natural source of carbon for fatty
acid synthesis in fsh, the relative contributions of carbohydrate and protein remain to be
established. The use of isolated fish hepatocytes as suggested by Moon et aL 277 may provide
a means of studying this aspect of fatty acid synthesis.
2. T i s s u e S i t e s
Comparisons of the capacities of fish tissues to synthesize fatty acids de n o v o have

involved the measurement of both the rates of lipogenic enzymes in v i t r o and the rates of
incorporation of radioactivity from low molecular weight precursors into fatty acids.
In coho salmon, the activities of ATP citrate lyase, malic enzyme, glucose 6-phosphate
dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase and fatty
acid synthetase were all higher in homogenates of liver than of adipose tissue. 254'2~5The
same enzymes were also found to be more active in the liver of channel catfish than in
its adipose tissue.253 Similarly, when expressed in terms of activity per g wet tissue weight,
the cytoplasmic NADPH-producing enzymes and malate dehydrogenase in American eel
were all more active in liver than in visceral adipose tissue or muscle, leading to the
conclusion that liver was the primary organ involved in fatty acid synthesis de n o v o in this
fish. ~7
The importance of liver as a site of fatty acid synthesis in coho salmon was confirmed
by Lin et al., 256 who reported that liver slices incorporated l172nmoles tritium from
tritiated water into fatty acids per 2 hr per 100 mg tissue, whereas, on the same basis, only
40 nmoles were incorporated by adipose tissue. By maintaining fish in tanks containing
tritiated water, the same workers observed that the incorporation of tritium into fatty acids
increased linearly in liver but not in adipose tissue. They calculated that the rate of fatty
acid synthesis in vivo in the liver was 6 times greater than that of adipose tissue. In rainbow
trout, the rates of incorporation of t4C-glucose, ~4C-alanine, ~4C-palmitic acid and tritiated
water into triacylglycerols were all reported by Henderson and SargenP 63 to be higher in
liver than adipose tissue when expressed in terms of tissue weight. However, when the rates
were presented in terms of DNA, only the rate of ~4C-alanine incorporation was higher
in liver slices than in adipose tissue fragments. Since DNA is an index of cell number, this
observation suggested that individual liver and adipose tissue cells could synthesize fatty
acids at similar rates. However, the trout liver contained about 26 times as much DNA
per unit weight than adipose tissue and, on a whole body basis, the liver of the trout was
considered to be more productive in the synthesis of fatty acids than the adipose tissue.
In the same study, the rate of esterification of ~4C-palmitic acid into triacylglycerols was
higher in adipose tissue than in liver on a DNA basis, and a higher proportion of
incorporated ~4C-glucose carbon was recovered in the glycerol moiety of triacylglycerols
in adipose tissue than in liver. It was suggested, therefore, that adipose tissue was adapted
for the uptake and storage, in triacylglycerols, of fatty acids originating from the diet or
from synthesis d e n o v o in the liver. ~63 This suggestion was supported by the results of a
more recent study which demonstrated that isolated trout adipocytes could take up glucose
and amino acids but that the rates of uptake were less than 25% of those observed with
rat adipocytes at the same temperature. 82
It has frequently been stated that fish have only a limited capacity for fatty acid synthesis
d e novo. ~4"445 A recent study435 estimated the total rate of fatty acid synthesis in trout
hepatocytes using tritium incorporation from tritiated water and found the rate to be
similar to that usually observed with hepatocytes from normal fed rats. The rate of fatty
acid synthesis was also found to be related to the amount of glycogen in the trout's liver,
with hepatocytes rich in glycogen exhibiting the highest rates. The rates of tritium
incorporation observed in other studies have also suggested that fish livers can exhibit
substantial rates of fatty acid synthesis. ~63'~8°
Fatty acid synthesis has also been demonstrated in the ovary of rainbow trout in vitro. 455
In fish with small ovaries, ~4C-acetate was incorporated by fragments of ovarian tissue
mainly into polar lipids, but with tissue from large ovaries, most of the incorporated
radioactivity was recovered in triacylglycerol fatty acids. This finding suggests that, while
the main source of ovarian lipid is likely to be that of vitellogenin and other serum
lipoproteins (Section V), fatty acid synthesis de n o v o by the ovary itself might also
contribute to the overall accumulation of lipid in the ovary during recrudescence, at least
in the trout.
Biochemistryof freshwaterfish 301
As discussed earlier, substantial amounts of lipid can occur in subdermal depots within
fish. In the rudd, skin lipids were heavily labeled by ~4C-acetate injected into the fish. 359
It remains, however, to be established whether skin lipid originates entirely from the diet
or hepatic synthesis, or whether skin tissues are themselves capable of fatty acid synthesis
de novo.
3. Products of Fatty Acid Synthetase

In comparison with that of mammals and microorganisms:37 the fatty acid synthetase
enzyme complex has not been well studied in fish, particularly freshwater species. A
comparative study of the products of fatty acid synthetase in animals ~°6 found that the
radioactivity from ~4C-acetate incorporated into fatty acids by homogenates of trout liver
was distributed 57% in 16:0, 29% in 14:0, 4% each in 12:0 and 18:0, 3% in 8:0 plus 10:0
and 1% in 20:0. For carp liver, the distribution pattern was 41% 16:0, 38% 14:0, 12%
12:0, 6% 10:0, 2% 18:0, and 1% in both 8:0 and 20:0.
Other studies on the products of fatty acid synthetase in fish have used marine species
including plaice ( Pleuronectes platessa ), ~ catfish ( Arius felis ), 445and flounder ( Platichithys
flesus). 146 In all of these studies, 16:0 and 18:0 contained most of the radioactivity from
labeled acetate or acetyl-CoA precursors.
On the basis of this evidence, it appears that the products of fatty acid synthetase in
freshwater fish generally resemble those of mammals.
B. Fatty Acid Esterification
1. General Pathways
Studies both in vivo and in vitro with radioactively labeled substrates such as acetate and
various fatty acids have established that fatty acids formed de novo and those originating
from dietary lipid are readily esterified into neutral lipids and phospholipids by fish
tissues. 1'23']45"163'2°°'219'359'435By analogy with the mammalian pathways ofesterification, these
fatty acids can be expected to be esterified to yield triacylglycerols and phospholipids via
the glycerol-3-phosphate pathway, the enzymes involved being located in the microsomal
membrane fraction of the cells.
The enzyme necessary for the synthesis de novo of PC, CDP-choline-l,2-diacylglycerol
choline phosphotransferase, was shown to be active in the microsomes of trout liver,
goldfish liver and goldfish brain, t74,:49 The microsomes of trout liver were also shown to
be capable of the reacylation of LPC to PC by the presence of acyl-CoA:
1-acyl-sn-glycero-3-phosphorylcholine acyltransferase, ~75an enzyme involved in the turn-
over of PC. The enzymes utilized for the biosynthesis of the different phospholipid classes
have not been examined in detail in fish.
Dietary fatty acids are known to be re-esterified into triacylglycerols in the intestinal
epithelium of teleost fish) 7s Both monoacylglycerols and glycerol are likely to be products
of triacylglyceroi digestion in freshwater fish (Section V). Consequently, both the glycerol
3-phosphate and monoacylglycerol pathways of triacylglycerol synthesis can be expected
to operate in fish intestinal tissues to an extent which depends on species.
As discussed below in Section V, lipids are transported from the liver to extrahepatic
storage tissues as components of lipoproteins. Since the triacylglycerols of lipoproteins are
hydrolyzed to their constituent fatty acids and glycerol moieties prior to their uptake into
the cells of extrahepatic tissues, such as adipose tissue and muscle, these tissues must
possess enzymes for the re-esterification of fatty acids to form storage triacylglycerols. The
pathways have not been studied in fish tissues, but the ability of trout adipose tissue to
incorporate a large proportion of glucose carbon into triacyiglycerol suggests that the
glycerol 3-phosphate pathway can operate in this tissue./63
Cholesteryl esters are another neutral lipid into which fatty acids can be incorporated
by their esterification with cholesterol. In studies with carp,"6 rudd359 and northern pike, 2~9
a small portion of the radioactivity recovered in tissue lipids after the injection of
t4C-acetate was present in steryl esters. With the exception of the liver from warm-adapted
carp, the steryl ester was always poorly labeled in comparison with triacylglycerols and
had very low specific activity. Similarly, slices of liver from eel incorporated small amounts
of 14C-acetate into steryl esters) None of these studies with taC-acetate provided data on
the distribution of incorporated radioactivity between the sterol and fatty acid moieties.
Although consistently small amounts (less than 6% of total radioactivity) were recovered
in cholesteryl esters of intestinal epithelial cells during the 18 hr after administration of
~4C-palmitic acid directly to the stomach of trout, the cholesteryl esters of the plasma
contained only traces of radioactivity, the vast majority being present in triacylglycerols)75
In keeping with the low amounts of cholesteryl esters observed in fish lipids, esterification
into this class of neutral lipid does not appear to be a major fate of dietary or newly
synthesized fatty acids.
Some fish species are capable of esterifying fatty acids into wax esters, although this lipid
class is not generally abundant in freshwater species. Sand et al. 347 demonstrated that roe
of the gourami were capable of esterifying oleic acid into wax esters, the principal lipid
of that tissue. The intestine, ovary, adipose tissue, kidney and particularly the hepato-
pancreas of the carp all esterified 16:0 acid to oleyl alcohol to form the wax ester oleyl
palmitate in vitro. 2°8 More recently, when ~4C-16:0 was incubated with carp plasma in the
presence of soy bean lecithin, about 17% of the radioactivity was recovered in both wax
esters and phospholipids. No other lipid class containing esterified fatty acids was
labeled. 268 Incubation of ~4C-cholesterol in the same system produced extensive incorpo-
ration of radioactivity into cholesteryl esters. When soy bean lecithin was omitted from
the incubation medium, the formation of wax ester from t4C-16:0 was markedly decreased,
suggesting that a lecithin: alcohol acyltransferase similar to the plasma lecithin: cholesterol
acyltransferase which transfers fatty acids from PC to the 3-~-hydroxyl group of
cholesterol, was involved in the synthesis of wax esters in plasma.
The amounts of taC-acetate incorporated into wax esters of eel gills in vivo have been
reported to be greater than that incorporated into triacylglycerols,~45 although the
significance of this fact is not obvious.
2. Glycerogenesis and Glyceroneogenesis
The formation of glycerol in the form of its phosphorylated derivatives from glucose
directly via glycolysis is termed glycerogenesis, while its formation from noncarbohydrates
is known as glyceroneogenesis. The formation of glycerol for use in triacylglycerol
synthesis becomes critical in fish consuming lipids such as wax esters which contain no
glycerol. This occurs in those salmonids which have a sea-going phase in their life history
and consume calanoid copepods, an abundant form of marine zooplankton which can
have one-third of their dry weight as neutral lipid in the form of wax ester. 3s2 Rainbow
trout fed calanoid copepods in freshwater aquaria had, as a consequence of this diet, a
large input of wax esters, but neither wax esters nor fatty alcohols occurred to any great
extent in their blood and only a small percentage of the dietary wax ester was excreted
in the fish's faeces. 3~ Furthermore, the principal body lipid of trout which consumed
calanoid copepods was triacylglycerols. Bauermeister and Sargent 2a have shown that the
conversion of dietary wax ester to triacylglycerols occurs in the intestinal tissue of rainbow
trout fed calanoid copepods. A similar situation exists in the freshwater gourami, the roe
of which is rich in wax ester. Unfertilized eggs are eaten by the female gourami and the
constituent wax ester converted to triacylglycerols in the intestine.329
In these two instances, both the fatty acid and fatty alcohol (after dehydrogenation to
fatty acid) moieties of the wax ester are incorporated into triacylglycerols. NADH is
generated during the dehydrogenation of the fatty alcohol by the intestinal caeca in both
of the above species. 28'42~ Of the tritium from [1-31-l]oleyl alcohol fed to gouramis and
recovered as intestinal triacylglycerols, 72% was present in position 2 of the glycerol
moiety with the remainder residing in the 1- and 3-positions. ~7 This predominant labeling
Biochemistryof freshwaterfish 303
of position 2 was attributed to the reduction of dihydroxyacetone phosphate with the

tritium in the other positions arising from the reduction of phosphoglyceric acid or
glyceraldehyde 3-phosphate. The formation of glycerol 3-phosphate from glucose only
involves the consumption of NADH at the glycerol 3-phosphate dehydrogenase stage,
whereas the conversion of a glycogenic amino acid such as aspartate to glycerol
3-phosphate involves NADH utilization at the glyceraldehyde 3-phosphate dehydrogenase
stage in addition to the glycerol 3-phosphate dehydrogenase step. Thus, twice as much
NADH is theoretically used in glyceroneogenesis as in glycerogenesis. The ratio of the
number of tritium atoms from the C, of [l-3H]hexadecanol incorporated in triacylglycerol
glycerol to the number of [1-'4C]hexadecanol molecules incorporated into triacylglycerol
fatty acids in intestinal preparations of rainbow trout was 1.6: 3. zg Had the triacylglycerol
glycerol originated from glyceroneogenesis, the theoretical ratio would have been 4:3
whereas 2:3 is the expected ratio for glycerogenesis.2~ '4C-Glucose was also incorporated
by the trout intestine into triacylglycerol glycerol at a faster rate than ~4C-aspartate. 28This
experimental evidence suggests that the glycerol moiety of triacylglycerols formed during
wax ester assimilation is apparently derived from carbohydrate metabolism via glycolysis
rather than from the metabolism of amino acids.
C. Desaturation and Elongation of Fatty Acids

The lipids of fish are characteristically rich in unsaturated fatty acids, whereas the
products of the fatty acid synthetase are saturated fatty acids. While fish incorporate
unsaturated fatty acids from the diet directly into body lipids (Section V), they are also
capable of modifying both dietary and endogenously synthesized fatty acids by the
processes of desaturation and elongation. The pathways of desaturation and elongation
which could operate in the tissues of freshwater fish are presented in Fig. 1. In common
with all other vertebrates, fish do not possess the A12 and AI5 desaturase enzymes
necessary for the synthesis of 18: 2 (n - 6) and 18: 3 (n - 3), respectively, and consequently,
must obtain these fatty acids or their longer chain derivatives in the diet.
The use of two carbon units for elongation reactions as well as fatty acid synthesis de
novo has been demonstrated by the recovery of radioactivity in fatty acids other than 16:0
and 18:0 when carp T M is.~59and eels ~45have been injected with ~4C-acetate. Similar results
have been obtained in vitro in studies with trout hepatocytes ~59and slices of eel liver. ~45In
all these studies, the presence of radioactivity in 16:1 and 18:1 (n - 9) indicated that fatty
acids synthesized endogenously had been subjected to desaturation reactions. The recovery
of radioactivity in the C20 and C22 PUFA of both the (n - 3) and (n - 6) series also
demonstrated the use of ~4C-acetate units in elongation steps. The pattern of distribution
of radioactivity from ~4C-acetate in fatty acids of body lipids is notably dependent on the
fish's diet and the environmental temperature. Feeding a low-fat diet containing 87% of
its dry weight as carbohydrate to carp increased the proportions of radioactivity from
injected ~4C-acetate recovered in 16:0 and 18:1 in comparison with fish fed a control diet
of 13.6% lipid and 28.7% carbohydrate, t~7 Conversely, the levels of radioactivity from
injected ~4C-acetate recovered in the long chain PUFA was highest in tissue phospholipids
of carp fed dietary lipid containing 30% each of 18:2 ( n - 6) and 18:3 (n - 3 ) . "8
Experiments involving the administration of 14C-18:3 ( n - 3) to fish by injection or
feeding have firmly established the ability of rainbow trout and the general inability of
marine species to desaturate and elongate 18:3 ( n - 3 ) to C20 and C22 poly-
unsaturates. 2°°'3~6.~9Not all freshwater species may be as active in this conversion as trout.
The ayu and eel both converted ~4C-18:3 (n - 3) to pentaenes and hexaenes at rates of only
36% and 20%, respectively, of those observed with trout, although the rates were still
greater than those observed with marine species. ~ The general inability of marine fish to
desaturate and elongate C18 PUFA dictates the requirements of these fish for preformed
C20 and C22 PUFA as essential fatty acids in their diet.
Further evidence for the desaturation and elongation of dietary C 18,PUFA comes from
nutritional studies with salmon and trout in which feeding 18:2 ( n - 6) or 18:3 (n - 3 )
~o
Fat ty acid Diet Diet

~ ~ ~ s y n t h e t a s e " -. -. 18:2 ( n - 6) 18:3 (n - 3)
Desaturase ~.t / ..,// ~ -.,....,.~.. I I
I I
14:0 ~ 16:0 18:0 ! i
A9
1 1 +' ;'
14:1 16:1 = 18:1 ~20:1 18:1 ~ 20:1----~22:1 ~ 24:1 18:2 = 20:2 " 22:2 18:3 20:3
~6
18:2 ---20:2 ~ 22:2 18:3 ~ 20:3 ~ 22:3 18:4 = 2 0 :4 ~ 22:4
e~
~5
1
2(1:3 ~ 22:3
1 1
20: 4 ~ 22:4
1 D 24:4 20:5 ~ 22:5
~4 1
22:6 gle
22:4 22:5
(n-5) (n-7) (n- 9) ( n - 6) (n- 3)
FIG. 1. Possible pathways of desaturation and elongation of fatty acids in freshwater fish. Only fatty acids which have been reported at levels of greater than 0.5% in lipids
are included. Vertical and horizontal arrows represent desaturation and elongation steps, respectively.
Biochemistryof freshwater fish 305
as the only P U F A in the diet corresponded with the appearance of (n - 6) and (n - 3) C20
and C22 PUFA in the polar lipids, a~°'4j2'~s'*49 Analytical evidence for the conversion of
dietary 20:5 (n - 3 ) to 22:6 (n - 3 ) was provided by an early study of Kayama et al., TM
who reared guppies from young solely on Artemia, the lipid of which contained only 20:5
(n - 3 ) , and found the lipid of adult fish to contain 17% 22:6 (n - 3 ) .
In mammals, it is generally accepted that the substrate preference of the A6 desaturase
is 18:3 (n - 3) > 18:2 (n - 6) > 18:1 (n - 9) and that competition exists between these
three possible substrates. 6° The situation appears to be similar in freshwater fish. The
anounts of 18:3 (n - 3), 18:2 (n - 6) and 18:1 (n - 9) desaturated by liver were 1.34, 0.56
and 0.04 nmol/min/mg protein, respectively, in Pimelodus maculatus in vitro. 298 Studies in
which diets containing various ratios of 18:3 (n - 3) to 18:2 (n - 6) in triacyiglycerols were
fed to salmonids, with subsequent analysis of the body lipids, demonstrated that 18:3
(n - 3) is a more effective inhibitor of the desaturation and elongation of 18: 2 (n - 6) than
18:2 (n - 6) is of 18:3 (n - 3). 473'474Furthermore, the longer chain members of the (n - 3)
series of PUFA, such as 22:6 (n - 3), can exert feedback inhibition on the A6 desaturase
in rainbow trout and so prevent the desaturation of both 18:2 (n - 6) and 18:3 (n - 3). 245
As a consequence of the substrate specificity of the A6 desaturase, 18:1 (n - 9 ) only
becomes a substrate for desaturation in the absence of 18: 3 (n - 3) and 18: 2 (n - 6). This
is discussed later in relation to dietary influences.
Apart from establishing that the A9, A6 and A5 desaturases are contained in the
microsomal fractions of cells, 94'14°'2°6'298the enzymes of the fatty acid desaturating systems
of fish have not been characterized. It can only be assumed that the system comprises three
proteins (cyt b5, cyt b5 reductase and the desaturase) and that overall the system utilizes
molecular oxygen as the oxidant and N A D H as a coreductant, as in mammals. 59'~85 On
the basis of differences in responses to changes in environmental temperature, the presence
of dieldrin and cycloheximide and heat inactivation, Ninno et al. 298 concluded that A9, A6
and A5 desaturations were achieved in P. maculatus by three different enzymes, each with
different properties. The pH optima of these three desaturases in trout liver microsomes
have been shown to be in the range 6.8-7.3, in keeping with that in mammalian tissues, j'*
The rate of desaturation of 18:0-CoA by trout liver microsomes was five times more than
that of 18: 0 as the free acid, suggesting that the formation of the CoA derivative may be
rate-limiting for A9 desaturation in vitro, j4o However, in the same study, 18:2 (n - 6)-CoA
and 18:2 (n - 6) free acid were desaturated by the A6 desaturase at equal rates, indicating
that the activation of fatty acids by microsomes is not rate-determining for the further
desaturation of PUFA in vitro.
A recent comparative study using hepatocytes isolated from trout and rat livers
demonstrated that, when the trout hepatocytes were incubated at 12°C and those from rat
at 37°C, the A6 desaturase activities were similar in both species and that 18:3 (n - 3) was
a better substrate than 18:2 (n - 6).~4~ In contrast, the A5 desaturation measured with 20:3
(n - 6 ) as substrate was ca. 4 times higher in rat than trout hepatocytes. The difference
between the species was less obvious with 18: 2 (n - 6) and 18: 3 (n - 3) as initial substrates
for A5 desaturation, but trout were consistently less active than rat. The trout hepatoeytes
possessed almost twice the A5 desaturase activity of rat hepatocytes with 20:5 (n - 3) as
substrate, but 20:3 (n - 6 ) was not a substrate for A4 desaturation in cells from either
species. The elongations of 18:3 (n - 6) to 20:3 (n - 6), 18:4 (n - 3) to 20:4 (n - 3) and
20:5 (n - 3 ) to 22:5 (n - 3 ) were carried out equally well by the trout and rat, although
hepatocytes from the former species were more efficient in the elongation of 20:4 (n - 6 )
to 22:4 (n - 6 ) . Trout hepatocytes incorporated considerably more of the radioactivity
from substrate fatty acids into 20:2 ( n - 6 ) , 20:3 ( n - 3) and 22:3 ( n - 6 ) than rat
hepatocytes. These fatty acids, considered to be dead end-products which would not be
further desaturated or elongated, were more obvious in triacylglycerols, although they are
not normal components of trout liver lipids. It has been suggested that these dead
end-products are stored in triacylglycerols until they are subsequently released and
retroconverted to form substrates for the more active pathways of desaturation and
elongation, j4j'365 However, although retroconversion of 22:6 (n - 3 ) to 22:5 (n - 3 ) and
J.P.L.R, 26/4--D
20:5 (n - 3) has been demonstrated in the rat, TM analytical evidence suggests that rainbow
trout are incapable of retroconverting 22:6 (n - 3 ) to shorter chain PUFA. 472
Experiments in vivo have established that, although furanoid fatty acids are not
synthesized de nov, by freshwater fish, they are elongated and chain-shortened. ~ Both of
these processes occur on the portion of carbon chain nearest the carboxyl group. The
presence of the furan ring does apparently prevent the desaturation of these fatty acids.
D. Reduction of Fatty Acids

The extensive recovery of radioactivity from ~4C-labeled fatty acids and ~4C-acetate in
the fatty alcohol moieties of wax esters formed by gourami roe 33°'345 demonstrated the
ability of this tissue to reduce fatty acids to fatty alcohols. The production of fatty alcohol
from fatty acid is likely to be the rate-limiting step in wax ester formation since fatty
alcohols are rapidly incorporated into wax esters and free fatty alcohols never occur in
high levels in the total lipid. In roe from the gourami species, Trichogaster cosby, the fatty
acid reduction activity was associated with the microsomal fraction and the CoA derivative
of the fatty acid was the required substrate. 137 NADPH rather than NADH was utilized
as the reducing cofactor and very little fatty aldehyde intermediate was observed. In
keeping with hexadecanol being the major fatty alcohol of the gourami roe wax esters, 346
the highest rates of reduction by microsomes from this tissue were obtained with palmitic
acid as substrate. ~37The rates of reduction of 18:1 (n - 9) and 18:2 (n - 6) fatty acids were
both about half that observed with 16:0, although polyunsaturated fatty alcohols are only
minor components of the roe wax ester. The NADPH-fatty acyI-CoA oxidoreductase
responsible for the fatty alcohol formation in the gourami has a very strong association
with the subcellular membranes. Recent attempts in our laboratory to purify the enzyme
from the roe of Trichogaster trichopterus have failed to dislodge it from the particulate
fraction, although a 10-fold purification was achieved by treatment of this fraction with
3 M NaCI. 337
Little is known of the fatty alcohol forming enzyme in those tissues of carp which can
synthesize wax esters. It is notable from the results of Kayama et al. z°8 that the
incorporation of ~4C-palmitic acid into wax ester by a fraction purified from carp
hepatopancreas was greatest in the presence of NADH, ATP and CoA. This suggests that,
although the enzyme also utilizes fatty acyl-CoA, the reducing cofactor requirement of the
enzyme differs from that of the gourami roe.
Furanoid fatty acids can also be reduced in gourami roe in vivo to the corresponding
fatty alcohols. ~ Of the radioactivity originating from a ~4C-labeled furanoid fatty acid and
recovered in wax ester, 40% was present in the fatty alcohol moiety.
E. Cholesterol Synthesis
The ability of freshwater fish to synthesize cholesterol has been demonstrated by several
studies. In general, cholesterol was not heavily labeled with radioactive acetate in studies
in vivo with carp tl6 and rudd. 359 In northern pike injected with 14C-acetate, cholesterol had
the lowest specific activity of all the lipid classes examined.2t9
Various estimates have been made of the rate of cholesterol synthesis in relation to that
of fatty acid synthesis. In trout hepatocytes, the rate of fatty acid synthesis from acetate
exceeded that of sterol synthesis by 2- to 4-fold, depending on temperature. ~59This is in
good agreement with the study in which 17"/o to 35*/, of the total incorporation of tritium
from tritiated water into lipid was located in sterols with the exact percentage being related
to the assay temperature and the thermal history of the fish. ~6°
More recently, it has been shown that the rate of cholesterol synthesis in trout
hepatocytes is related to the glycogen content of the liver from which they were prepared.435
The rate of cholesterol formation at 20°C in hepatocytes of low glycogen content was
0.24 nmol/mg protein/hr compared with 0.16 nmol/mg protein/hr in cells of high glycogen
content. These rates corresponded to 74% and 7*/0 of the total lipid synthesis in
hepatocytes of low and high glycogen content, respectively. Since glycogen content is
related to nutritional status, it can be concluded that during starvation the formation of
cholesterol accounts for a large proportion of the lipid synthesis which is occurring.
However, the nutritional state did not affect the incorporation of ~4C-acetate into sterols
by liver slices from the European eel; 10 to 44% of the total radioactivity in neutral lipid
was located in free sterolsJ
Although the pathway of cholesterol synthesis in fish has not been examined in great
detail, it can be assumed that the conventional mammalian route via hydroxy
methylglutaryl-CoA operates.
v. METABOLISM OF DIETARY LIPIDS
A. General Aspects
The following is a brief account of the major aspects and the most recent work on lipid
digestion, absorption and transport in freshwater fish. More detailed reviews on various
specific areas in this field are already available. 37.m,:~.235,24L335
B. Digestion
1. Emulsification
(a) Role of bile. The physico-chemical properties of lipids and their interactions with
water dictate that their digestion in the intestine is dependent upon the interaction of the
dietary fat with bile and lipolytic enzymes. 56 Bile is produced in livers of all fishes, is stored
in the gallbladder and is delivered to the anterior intestine or the pyloric caeca via the bile
duct. H3 The mechanisms of emulsification are largely unstudied in fish but would appear
to be similar to mammalian systems. Similarly, the enterohepatic circulation of bile is
relatively unstudied in fish m but, presumably, bile salts are absorbed in the terminal ileum
by an active transport mechanism as exists in mammals and birds. ~°3
(b) Bile composition. Fish bile is primarily a solution of bile salts but can also contain
bile pigments, mucin, other lipids (e.g. phospholipids, cholesterol and acylglycerols,
proteins including digestive enzymes, urea and inorganic ions including HCO~-, and is thus
alkaline, ts~
Haslewood ~ 5 2 has reviewed the bile acid and alcohol compositions of many fish
species. Generally, fish groups show an evolutionary trend from the more primitive C27,
5~-alcohol sulphates to the more advanced C24, 5fl-acids with taurine as the major
conjugate.~5° Salmonids have primarily cholic acid with lesser amounts of chenodeoxycho-
lic acid) 32 Rainbow trout bile contained cholate and chenodeoxycholate at 85*/0 and 14%,
respectively, of the total bile salts with over 92% conjugated with taurine. 99 About
one-third of the cholic acid was conjugated with glycine and a further 9% was sulphated. 99
In the Indian freshwater species, rohu and mrigal, mainly taurine derivatives of lithocholic
acid were found. TM Bile phospholipids are primarily PC jSI and are important for the
formation of a stable emulsion in most fish species. 235
Bile is also an excretion route for certain substances not suitable for elimination via the
kidneys, a83 In this respect, the products of haem catabolism, the bile pigments biliverdin
and bilirubin, are also found in fish bile. Whereas biliverdin is generally found uncon-
jugated, bilirubin in several species of freshwater fish is found as the mono-
glucuronide. ~°.~
2. Lipolytic Enzymes
The study of lipolytic enzymes in fish, in general, has been complicated by the lack of
a discrete pancreas in most teleost species. "3 As a result, a variety of different tissue and
enzyme preparations from various species have been studied using several different assay
methods making comparisons often rather difficult.234'353 However, the more recent
biochemical studies have provided some interesting results.
(a) Triacylglycerol hydrolases (lipases). Triacylglycerol is generally the major neutral
lipid available in the diets of freshwater fish s8 and, therefore, triacylglycerol hydrolases
have been the most studied lipolytic enzymes.
Early work showed lipolytic activity was found in extracts of intestinal mucosa of the
killifish (Fundulus), 2~ mixed liver and pancreas extracts and intestinal contents in
goldfish,s49 tilapia stomach extracts 283 and carp intestine] 8 Histochemical studies in teleosts
revealed lipase activity in stomach, pyloric caeca, liver, intestine, pancreas and hepa-
topancreas (when present). 2°4"355Previously it had been shown in tilapia and perch that
areas of the digestive tract with strong iipase activity also had high esterase activity. ~
Esterase activity was also found in the liver, spleen, intestine, pyloric caeca and stomach
of rainbow trout] TM A rather nonspecific carboxylic esterase activity was also found
throughout the digestive tract of the perch. ~72 In an anadromous sturgeon, the pancreas
was the main digestive enzyme and lipase secreting organ with lipase activity also detected
in stomach, intestine and pyloric caeca. 326 The high level of lipase activity found in the
pyloric caeca of many fish, 325'355 including rainbow trout 426 and marine fish, 26~ indicates a
major and perhaps general role for this organ in lipid digestion in fish. 3~s,426
Precise characterization of fish lipases and their specificities have been difficult to
determine consistently because of the crude extracts and poorly defined enzyme prepara-
tions used. Leger and coworkers have studied a lipase prepared from intercaecal
adipo-connective tissue from the rainbow trout. 238 The enzyme activity was sensitive to
organic solvents, but was purified 36-fold with a recovery of 20%.233 The optimum pH was
ca. 8.5 and the Km for substrate was 1.3 x 1 0 - 6 M (interface concentration)? 35 Optimum
hydrolysis of triolein required Ca 2÷ and the presence of bile salts, although the activity
was not dependent upon bile salts. 237 A colipase for this enzyme was implied by the
response of the activity to bile salt concentration, 237 and porcine colipase activated the
rainbow trout lipase. 24° A colipase with a molecular wt of 9000-9400 was purified and
characterized from the dogfish, Squalus acanthias. 39° Furthermore, colipase structure and
function are known to be remarkably conserved evolutionarily through the vertebrate
groupsfl 1 Together, this evidence suggests the existence of the lipase-colipase system in
freshwater fish.
Tocher and Sargent 426 studied lipolytic activities in caecal content extracts from rainbow
trout. The triacylglycerol lipase activity was not dependent upon Ca 2+ but was stimulated
greatly by bile salts, particularly taurocholate and taurochenodeoxycholate in a ratio
similar to that found in trout gallbladder bile. 426 The activity was optimum at pH 8 and
30°C. In this study, two phases of activation due to bile salt were observed, one at
concentrations up to the critical micellar concentration (cmc) of the bile salts used due to
the action of "true" lipase (see Ref. 235), and the second beyond the cmc possibly due
to the action of so-called nonspecific lipase. The physiological bile salt concentration in
trout caeca was around the cmc of the bile salts at 5 - 1 0 m M . 426 Bile salt-dependent
nonspecific lipase has been purified and partially characterized from shark pancreas 317'322
and its presence has been suggested in other marine and anadromous fish. 32t The results
with rainbow trout 236'426 suggest the existence of both these enzymes in freshwater fish] 35
Partially purified trout pancreatic lipase showed some fatty acid specificity and
hydrolyzed triacylglycerols containing 18:1 (n - 9) in preference to 16: 0. 236Intestinal fluids
from anadromous Pacific pink salmon (Oncorhynchus gorbuscha) and striped bass (Morone
saxatilis) taken from the sea were able to hydrolyze the methyl esters of 20:4 (n - 6) and
20:5 ( n - 3) acids, which are more resistant to hydrolysis by mammalian pancreatic
lipase, m
(b) Wax ester hydrolases. Wax esters are not such an important dietary lipid input in
the freshwater environment as they are in the marine environment. 3s'36However, they will
form a major component in the diets of the sea-going forms of anadromous, zooplankton-
feeding fish. 27'35~ Freshwater gouramis incorporated label into their tissue lipids when fed
radiolabeled wax ester. 329The intestinal fluid of sea-caught striped bass and pink salmon, 321
carp hepatopancreas 2°8 and pyloric caecal extracts from rainbow trout 426 all hydrolyzed
wax esters. The rates of hydrolysis varied considerably in the studies but were from 4
times 3~ to 50 times 426 slower than triacylglycerol hydrolysis. Feeding rainbow trout diets
rich in wax esters increased the ability of the caecal extracts to hydrolyze wax esters,
whereas the hydrolysis of triacylglycerols was decreased. 426 However, fish normally
consuming wax ester-rich diets did not appear to have a superior ability to hydrolyze
them. 318 Increased retention time in the gut, particularly caeca, was postulated as an
important feature of wax ester hydrolysis in these fish. 3is Conversely, Mankura et al. 269
found increased wax ester hydrolase activity in the pyloric caeca of marine fish that
consume wax esters. Bile salts, particularly taurocholate and taurochenodeoxycholate, in
a physiological ratio and range were required for full activity of the wax ester hydrolase
from rainbow trout caecal extracts. 426
The specificity of the wax ester hydrolases of freshwater fish for fatty acid and alcohols
is not known, but Lie and Lambertsen ~52found that wax ester hydrolase in cod digestive
juices showed a preference for polyenoic fatty acids but had no specificity for the alcohol
groups.
(c) Other lipolytic enzymes. Little information is available on other lipolytic activities
in freshwater fish. Tother and Sargent 426 found steryl ester hydrolase activity in pyloric
caecai extracts from rainbow trout. The activity was dependent upon primary bile salts
and was optimal at 20°C, similar to the wax ester hydrolase activity. Furthermore, it was
stimulated in parallel with wax ester hydrolase activity upon wax ester feeding. 426 These
results suggest that the steryl ester and wax ester hydrolase activities may reside in the same
enzyme protein. Similarly, the pH optima for both enzyme activities were in the range
7.4-7.9 when endogenous bile was used. 426However, cholesteryl oleate was hydrolyzed 2-5
times faster than palmityloleate and 10-20 times slower than triolein.
As major components of biological membranes, phospholipids can be a major and
consistent source of lipid to fish. However, although intracellular tissue phospholipases
have been described in rainbow trout,188'29° little is known about intestinal phospholipases.
3. Products of Digestion
Experiments in which trout were fed with labeled triacylglycerols showed that they
retained, or partly retained, the fatty acid distribution in position 2. 63'65 Hydrolysis of
triacylglycerols primarily to 2-monoacylglycerols and fatty acids has been demonstrated
in rainbow trout caecal extracts. 426 Leger and Bauchart 236 found that partially purified
pancreatic lipase from trout could also hydrolyze the fatty acid at position 2 if it was
unsaturated. Hydrolysis of oleic acid from position 2 of triacyiglycerols was found with
sea-caught pink salmon. 32~
Therefore, it appears that products of triacylglycerol digestion in fish will include
glycerol as well as monoacylglycerol and free fatty acids. Steryl and wax esters are
hydrolyzed to fatty acids, sterol and fatty alcohol, respectively, all of which are
absorbed. ~5'~s'347'4z6Phospholipid digestion has not been studied in fish, but presumably
results in the formation of free fatty acid and lysophospholipids, which are the products
absorbed in mammals. 51'52
C. Absorption
1. Location
The absorption of lipid in fish occurs primarily in the anterior intestine and the pyloric
caeca. H'37"23s'2~ However, absorption may occur further down the intestine, particularly
after a very high dietary lipid input. 356 Furthermore, different regions of the gut may be
of more importance in some species such as mid-gut in half-beak and goldfish,'Sua~ whereas
pyloric caeca may be the primary region for lipid absorption in rainbow trout. '~'
2. Digestibility and Absorption

Digestibility of fats increased with the degree of unsaturation, j9'2° and with water
temperature. 14,~sThe digestibility of individual fatty acids decreased with increasing chain
length up to C1 8 and then inceased with further increases in chain length to C22 in rainbow
trout, s° Furthermore, high fat diets and small fish size are associated with lower
digestibility of lipids) a'46~ Absorption of lipids in fish is slow and can taken many
hours. 32°'375 However, the water temperature does not seem to play a major role in the
specific absorption processes, z35
3. Fates of Digestion Products

The major absorbed products of lipid digestion, free fatty acids, glycerol,
2-monoacylglycerol, sterols, fatty alcohols and lysophospholipids are further metabolized
in the enterocytes of the intestinal mucosa. Greater than 90% of absorbed lyso-
phospholipids are re-esterified with fatty acids to phospholipids in mammals 244'477and this
probably occurs also in fish. Whereas most free sterol may be re-esterified with fatty acid
to steryl esters, Sand et al. 347 showed that only a small proportion of fatty alcohol was
re-esterified to wax ester in the gourami, the majority of fatty alcohol being oxidized within
the enterocyte to the corresponding fatty acid. 26'2s'347Due to the presence of both glycerol
and 2-monoacylglycerol, re-esterification of free fatty acids into triacylglycerols will occur
through both the glycerol-3-phosphate and monoacylglycerol pathways.
The reacylation reactions occur primarily in the endoplasmic reticulum leading to the
production of chylomicron-like and very low density lipoprotein (VLDL)-Iike particles in
the lumen as observed in carp, 299 tench (Tinca tinca) ~ and t r o u t . 25'376"377'434 Feeding trials
in trout revealed that lipid load and degree of unsaturation affected the relative production
of chylomicrons and VLDL. 377'434High lipid levels and PUFA gave large chylomicrons
whereas high saturated fatty acids led to the production of smaller VLDL particles.
Cytoplasmic fat droplets have also been o b s e r v e d 39'299'3°°'377 and these may be a storage form
that occurs during lipid overloading, including wax ester feeding, 25 and saturation of the
lipoprotein synthesis pathways.
In carp, lipoprotein may be transported to the liver directly and solely via the portal
system. 299 Although this transport pathway may also occur in other fish such as trout and
tench, it appears that the majority of intestinal lipoprotein in these fish is transported via
the lymphatic s y s t e m 37`3s'3°°'37s before appearing in the circulatory s y s t e m 77"373"378 as in
mammals. Some evidence exists for the transport of some free fatty acid in albumin
complexes via the portal blood in trout. 2°7"333 It is postulated that this route is only
significant in certain situations such as refeeding after starvation, 37 or when glycerol or
monoacylglycerol are limiting, as in wax ester feeding. 235
D. Transport of Plasma Lipids

In general, lipids are insoluble in aqueous solutions and so are transported in the plasma
in complexes with proteins, called lipoproteins which include chylomicrons, VLDL, low
density lipoprotein (LDL) and high density lipoprotein (HDL). As well as density, these
vary in size, lipid:protein ratio, lipid class composition, apoprotein composition and
molecular organization. 25°'36°Although the precise density ranges, relative proportions and
detailed chemical compositions of the lipoprotein classes can vary between species,
lipoprotein structure and composition are comparable throughout the vertebrates, includ-
ing fish. 76
In fish, chylomicrons and some VLDL are synthesized in the intestine from dietary lipid
and are transported into the circulation via the lymphatic system as described previously.
However, most VLDL is synthesized in the liver, in rainbow trout at least) 34'376Most work
on lipoprotein metabolism in freshwater fish has been performed on rainbow
t r o u t 47'77'120"324"334"378 and a few other species, including pink salmon, 293channel catfish 272and
carp. ~78'287
Biochemistry o f freshwater fish 311
Total plasma lipid can be very high in fish, reaching almost 2 g/dl in trout m which is
far higher than in man. 76 Most of the lipid was contained in the lipoproteins, but 5-10%
was present as frc¢ fatty acids probably bound to albumin, m~°,323Albumin may also
transport ca. 50% of the csterified lipids in the carp, 28s the rest being lipoprotein bound. ~6
In rainbow trout, H D L is the predominant class ranging from 0.5-2.3 g/dl, followed by
LDL (0.2-1.1 g/dl) and then VLDL (0.1-0.7g/dl). 77'121'123'376'37sH D L was also the main
lipoprotein class in pink salmon 293and channel catfish. 272The relative amounts of VLDL,
LDL and H D L vary with species, age, nutrition and sexual cycle of the fish. m2°
The chemical compositions of rainbow trout lipoproteins in comparison with the human
ones are shown in Table 4. Due to the much higher serum total lipid, most fish tend to
have higher serum total cholesterol than mammals. 12° In relation to this, the ratio of
esterified cholesterol to free cholesterol is higher in the more advanced fish species such
as salmonids.m2° PC is the predominant phospholipid class in fish lipoproteins, similar to
most vertebrates. 76Another difference, in comparison with mammalian lipoprotcins, is the
very high level of PUFA, particularly 22:6 (n - 3) and to a lesser extent 20:5 (n - 3),
found in fish plasma lipoproteins.Z~°'243'293'37sThe fatty acid compositions of the major lipid
classes in the various lipoproteins from rainbow trout are shown in Table 5. The
apoprotein compositions of fish lipoproteins are similar to mammalian lipoproteins with
primarily apoprotein A (I and II) in HDL, apoprotein B in LDL and a mixture of
apoproteins B, C and E in VLDL and apoproteins A, B and C in
chylomicrons. 76'77'2~2'293'3~'378Although direct studies in fish are lacking, it is possible that
TASLE 4. Protein and Lipid Class Composition of Rainbow

TrouP and Human b Lipoprotein Classes (wt %)
P TL PL T A G CHOL CE
Chylomicrons:
Trout 5 95 8 84 l 2
Human 2 98 8 84 l 4
VLDL:
Trout l0 90 21 45 8 16
Human l0 90 18 50 6 16
LDL:
Trout 29 71 25 21 7 15
Human 20 80 22 8 1 40
HDL:
Trout 45 55 29 11 3 12
Human 50 50 25 7 3 15
*Averaged figures from various studies. 77''2L'23,37s
Waken from Faegerman. H2
P = p r o t e i n ; T L = t o t a l lipid; P L = p h o s p h o l i p i d ; T A G =
triacylglyccrol; CHOL = cholesterol; CE = cholesteryl ester.
TABLE 5. Composition of the Major Fatty Acids in the Predominant

Lipid Classes of Rainbow Trout Lipoproteins (wt %)
VLDL LDL HDL
Fatty Acid PL TAG PL TAG CE PL CE
16:0 29 15 28 13 17 26 19
16:1 4 8 4 7 3 4 4
18:0 I0 4 7 4 2 6 3
18: l (n - 9) 16 31 14 33 14 13 14
18:2 (n --6) 4 8 4 8 5 5 5
18:3 (n --3) l 2 l 2 0.5 l 0.3
20:4 (n -- 6) 2 1 3 l 2 4 2
20:5 (n --3) 2 3 3 3 6 3 6
22:6 (n - 3) 22 !l 26 15 33 30 32
(n -- 6) P U F A 6 l0 8 12 I1 l0 l0
(n -- 3) P U F A 25 19 31 24 41 36 40
Averaged data from various studies. 77,'2Lt23"235
PL ffi phospholipid; TAG = triacylglycerol; CE = cholesteryl ester;
P U F A = polyunsaturated fatty acids.
the apoproteins will have the same metabolic functions such as enzyme activators (AI and
C) and receptor binding (B and E), as in the much-studied mammalian systems. :65
The major enzymes of lipoprotein catabolism and remodelling have been identified in
rainbow trout. Lipoprotein lipase (LPL) activity and salt-resistant lipase (hepatic lipase)
were found in red and white muscle, adipose tissue, liver, heart and brain, 4548 whereas
lecithin:cholesterol acyltransferase (LCAT) activity was found in the plasma? 6 The
presence of these enzymes and studies on lipid transport and uptake in rainbow trout 47
suggested that the processes and mechanisms involved were similar to those in mammals.
Therefore, triacylglycerols in chylomicrons and VLDL are hydrolyzed at tissue sites by
LPL with resultant uptake of the hydrolysis products. Excess surface constituents of
chylomicrons and VLDL "bud off" as nascent H D L particles (similar to HDL3) , which
can also be secreted by the liver. HDL3 can take up free cholesterol from peripheral tissues
which is then esterified by the action of plasma LCAT resulting in mature HDL2.
Remnants of chylomicron and VLDL hydrolysis may be taken up by liver, but further
action of LPL leads to the formation of intermediate density lipoprotein (IDL) and
subsequently LDL. Changes in the apoprotein content of the lipoproteins occur along with
these lipid changes.
One further lipoprotein class, vitellogenin, is found specifically in mature oviparous
female fish or estrogen-injected fish. 70'90'303'385'439Therefore, it is not concerned directly with
transport of dietary lipid. Vitellogenin is synthesized in the liver and is transported to the
ovary during the first stage of oogenesis (oocyte growth) termed vitellogenesis. 438.439.454The
transcription of the vitellogenin protein genes is hormonally regulated by estrogens, 4~4'4~5
with further regulation of the overall vitellogenic process by pituitary gonado-
trophins. 6s'69'456Vitellogenin is a higher density lipoprotein than HDL, containing ca. 80%
protein and 20% lipid, which is predominantly phospholipid 3°3 rich in ( n - 3 )
PUFA. 122'148"243Vitellogenin leaves the circulation and passes through the ovarian follicle
by intercellular routes, 367 binds to the oocyte membrane via a specific protein subunit, 468
and is taken up by the developing egg by micropinocytosis. 367"442The endocytic vesicles
undergo fusion and condensation, during which time vitellogenin is cleaved into
phosphate-rich phosvitin 398'~°'.4] and lipid-rich lipovitellin, 184 before final fusion with the
developing yolk spheres. 439 Lipovitellin in trout eggs was composed of 77% protein and
23% lipid with a lipid composition similar to HDL. 379 During the early stages of
vitellogenesis, VLDL levels in the plasma may also be increased in response to es-
trogen, 122'439and VLDL is probably also taken up by developing oocytes by a receptor-
mediated endocytotic process, 439at least in eggs with high triacylglycerol content and lipid
droplets. Endogenous synthesis of lipid in the oocyte may only occur in yolk vesicles which
may be the precursors of cortical alveoli 443 and, hence, do not represent yolk lipid.
VI. ESSENTIAL FATTY ACIDS
A. General Aspects
PUFA cannot be synthesized de novo by fish but are required for normal growth and
development and the maintenance of cellular functions. Therefore, they must be obtained
in the diet. Linoleic acid, 18:2 ( n - 6 ) , is the principal essential fatty acid (EFA) for
terrestrial animals, whereas the essentiality of ct-linolenic acid, 18:3 ( n - 3), although
generally accepted, has been more difficult to establish quantitatively? 23However, extreme
carnivores appear to be deficient in chain elongation and desaturation mechanisms and
may also require longer chain ( n - 6) and ( n - 3) PUFA. Early fish nutrition studies
demonstrated that the 18:2 (n -6)-rich vegetable oils alone give relatively poor results. 72
Later work established the absolute requirement for (n - 3) PUFA for fish, whereas the
degree of (n - 6) PUFA requirement is still unclear. The importance of EFA requirements
in fish nutrition is emphasized by the volume of work performed on the subject over the
last decade, and which has been reviewed on several o c c a s i o n s . 72"ss'89"196"197"~
Bioeh~aistry of freshwater fish 313
B. Deficiency Pathologies
Poor growth rate and feed conversion have been found in EFA deficiency in rainbow
trout, T M carp, ~ chum salmon, 4~2eel4°2and tilapia. ~°3Interestingly, similar symptoms were
also reported for rainbow trout fed a large excess of (n - 3) PUFA. ~ Most of the studies
on the detailed pathologies of EFA deficiency have been performed on the rainbow trout.
An early symptom of EFA deficiency is an increased hepatosomatic index. The livers of
EFA-deficient trout 75"*~sand salmon 4~2 were pale and swollen and contained more lipid,
primarily neutral lipid. 4~2'449This was probably due to impaired lipoprotein biosynthesis,
preventing normal lipid transport from the liver, as found in EFA-deficient rats. ~25
Deprivation of P U F A over several months leads to shock syndrome in rainbow trout
where the fish appear to lose consciousness in response to sudden shocks. 75"45° Other
long-term pathologies include fin erosion) 7° wh.,;h may be associated with bacterial
infections,75 enlarged hearts with possibly lipid protrusions of blood vessels, decreased
haemoglobin in blood and increased water content in muscle and viscera. 75'45°At a more
cellular level, the livers of EFA-deficient trout had an increased respiration rate with more
fragile mitochondria that exhibited a higher swelling rate. TM EFA deficiency in broodstock
fish affects the number and quality of eggs produced, leading to decreased fecundity and
hatch rates. 452 Furthermore, increased mortalities of eggs from ( n - 3) PUFA-deficient
trout were noted at days 8 and 22 after fertilization?47
C. Qualitative and Quantitative Requirements

The most complete data on dietary EFA requirements for freshwater fish are for
rainbow trout. Rainbow trout require ca. 1% 18:3 ( n - 3) in their diet for optimum
growth, 75'45°more specifically within a range of 0.8-1.7% when the diet contains up to 5%
lipid? 4s When the amount of lipid in the diet is increased up to 10-15%, the 18:3 (n - 3)
requirement also increases but remains at ca. 20% of the total lipid. 4°7 Rainbow trout
appear to have a lower requirement for ( n - 6) PUFA. Addition of 18:2 ( n - 6) did
improve growth compared to totally PUFA-deficient diets, but no mixture of 18: (n - 6)
and 18:3 (n - 3) totalling 1% was better than 1% 18:3 (n - 3) alone. 75 In an early study,
18:3 ( n - 3 ) and 22:6 ( n - 3) were found to be equally efficient EFA's for troutY 2
However, it was later reported that the long chain (n - 3) PUFA, 20:5 and 22:6, were
more efficient than 18:3 (n - 3 ) and also had additive effects on growth with only 0.25%
of each P U F A required by trout. 4°8 A combined total of 0.5% or over of these P U F A can
be easily supplied by supplementing diets with small amounts of marine fish oil (see
Refs 3, 405). The lower requirement for the longer chain ( n - 3) PUFA is perhaps
indicative of less efficient chain elongation and desaturation mechanisms consistent with
the carnivorous lifestyle of the rainbow trout. Furthermore, the elongation and desatur-
ation mechanisms were less efficient with the (n - 6) series than with the (n - 3) series. 2~
With coho salmon, the optimum level of 18:3 (n - 3 ) in the diet was 1-2.5% whereas
18:2 (n - 6 ) , in the form of trilinolein in excess of 1% in the diet, depressed growthY 3.474
However, with chum salmon, optimum growth was obtained with diets containing 1%
18:2 (n - 6) and also 1% 18:3 (n - 3) or 0.5-1.0% of the longer chain (n - 3) PUFA. a~2
The (n - 6 ) EFA requirement for salmon is, therefore, unclear.
The requirements are also not fully understood for channel catfish. Whereas they grow
well on diets supplemented with beef tallow or menhaden oil, high dietary 18:2 (n - 6 )
and/or 18:3 (n - 3 ) levels depress growth. 392 Stickney et al. 394 found better growth rates
of channel catfish using diets containing 18:2 (n - 6) or 18:1 (n - 9) than a diet with 18:3
(n - 3). Recently diets containing purified individual fatty acids at a level of 6% of the
diet were investigated. Feed conversion ratio was poor with all diets, especially 16:0 and
18:3 ( n - 3) and muscle myopathies were most prominent with diets containing 18:2
(n - 6) and 18:3 (n - 3). TM The 18:3 (n - 3) requirement of this species appears to be very
low. In general, channel catfish have a lower EFA requirement than salmonids and this
may be related to environmental temperature as they inhabit warmer waters than the
salmonids. 72
314 R.J. Henderson and D. R. Tother
Optimal growth of carp was obtained with 1% 18:2 ( n - 6 ) and 1% 18:3 ( n - 3 )

together. 4°6 As with trout, 20:5 (n - 3) and 22:6 (n - 3) at a combined level of 0.5% was
as efficient as 1% 18:3 (n - 3). A requirement for both 18:2 (n - 6) and 18:3 (n - 3) each
at a level o f 0.5% was reported for optimal growth and feed conversion of eels at 25°C. 4°2
Tilapia zillii grew better at 27°C when fed diets containing 1% 18:2 (n - 6 ) or 20:4
(n - 6 ) whereas 18:3 (n - 3 ) and 20:5 (n - 3 ) were not as efficient EFA's as the (n - 6 )
PUFAfl °3 With Tilapia nilotica, a minimum level of 0.5% 18:2 (n - 6 ) was required for
optimum growth whereas 18:3 ( n - 3) was less efficient and longer chain ( n - 3) and
(n - 6 ) P U F A had no EFA value for this species. 4°3-4°4 Tilapia aurea grew well on diets
with up to 2% 18:2 (n - 6 ) , but growth was depressed when levels of 18:3 (n - 3 ) were
at or above 1%.393 However, in a recent study, the same species grew well on diets that
contained differing levels of fish oils, with optimal growth on diets with 10% menhaden
oil. 395 The level of 18:3 (n - 3 ) never reached 1% in any of these diets and the authors
concluded that the growth reductions previously observed were probably due specifically
to 18:3 (n - 3 ) rather than to combined (n - 3 ) PUFA.
Diets optimized for growth may not necessarily result in optimal fish quality. When
lipids of Atlantic salmon smolts were recently compared in hatchery and wild fish, the
hatchery fish contained 4 times as much lipid in total with almost twice as much lipid in
the skin. 7 Wild fish had higher levels of 20:4 (n - 6) in their lipids whereas hatchery smolts
were richer in 18:2 ( n - 6), indicating a possible deficiency in salmon elongation and
desaturation mechanisms. Significantly, this excess of 18:2 ( n - 6) was implicated in
dermal lesions such as fin rot found in the hatchery smolts. 7 Similarly, dorsal muscle lipid
in cultured rainbow trout and carp had higher levels of 18:2 ( n - 6) than their wild
counterparts) 97 Conversely, cultured eels had a higher (n - 3)/(n - 6) ratio in the dorsal
muscle lipids than the wild eels. 397
Most studies on EFA requirements have been performed on young fish, but not on very
small newly-hatched larvae. 446 It is entirely possible that the EFA requirements of early
larval fish are yet to be fully appreciated. The consistent occurrence of high levels of
PUFA, in particular longer chain PUFA, in fish eggs (see Section VIII) suggests that
emergent larvae may have a higher requirement for long chain ( n - 3) P U F A than is
indicated by experiments on older fish. Certainly 20:5 ( n - 3 ) and 22:6 ( n - 3 ) were
required for normal growth of larval ayu. :°L~7 Incorporation studies of dietary radioactive
20:5 (n - 3) showed high incorporation into many tissues, and the authors concluded that
this fatty acid is likely to be utilized as a constituent of cellular membranes rather than
to be oxidized for energy) ~8 Based on all this data, Kanazawa 196postulated that relatively
large amounts of exogenous 20:5 (n - 3) may be necessary for larval fish that are growing
at a rapid rate.
Work using purified microparticulate diets showed that larval ayu also required
phospholipids in their diet to prevent the incidence of malformations such as scoliosis. 199
PC or PC plus PI gave good growth and survival rates, whereas PE was much less
effective. 195 A saturated species of PC, dipalmitoyl, was ineffective whereas unsaturated
species were effective. ~95'2°2 Similarly, bonito egg PC was more effective than soybean or
chicken egg PC's. ~99"2°2 Therefore, larval ayu require intact phospholipid molecules
containing unsaturated fatty acids with either inositol or choline headgroups. The exact
relationship of this requirement to EFA requirement is not known but it is believed they
are required in addition to (n - 3 ) PUFA. ~97 Although rapidly growing larval fish will
require large amounts of phospholipid for new cellular membranes, the precise mech-
anisms behind the requirement are unclear.
D. Autoxidation and Protective Mechanisms

Fish iipids are characterized by high levels of P U F A that are highly susceptible to
oxidation. The oxidation of P U F A in biomembranes would have profound effects on
membrane structure and function and so several mechanisms exist in vivo to prevent this
from occurring. The following is only a brief account of both the mechanisms of oxidation
and prevention. The reader is referred to other recent reviews for more detailed accounts
of this subject in relation to fish. 3°'j49
1. Mechanisms of Autoxidation
PUFA oxidation, referred to as autoxidation because the rate increases as the reaction
proceeds, occurs via free radical chain reactions. The free radicals are usually metabolites
of oxygen and are produced by normal metabolic processes. Hydrogen peroxide, H:O2,
produced by peroxisomal oxidases and mitochondrial electron transport, is a potential
source of the hydroxyl radical, "OH. The hydroxyl radical is itself produced along with
HO2 in the electron transport of oxidative phosphorylation. In the cytosol, the superoxide
anion radical, O~-, generated by several oxidases, is converted to the more reactive "OH
by an iron-catalyzed reaction:
Fe 3+ + 02- --~ Fe 2+ + 02
F e 2+ + H202 ---*Fe 3+ + "OH + O H -
Initiation of PUFA autoxidation occurs when "OH abstracts a hydrogen atom from a
methylene group in a PUFA leaving a carbon radical which rearranges to form a
conjugated diene. The diene can react easily with oxygen to form a peroxy radical, R-OO',
which can abstract a hydrogen atom from another PUFA to yield a lipid hydroperoxide
and a carbon radical on the second PUFA. These are the propagation reactions that
proceed in an accelerating manner due to the introduction of new radicals from the
decomposition of lipid hydroperoxides to alkoxy and hydroxyl radicals. Harmful alde-
hydes, ketones and alcohols can be produced in the cell from the alkoxy radicals.
The chain reactions can be terminated when two radicals interact to form a nonradical
product or by the donation of a hydrogen atom to a peroxyradical by an antioxidant
compound.
2. Protective Mechanisms
(a) ~-Tocopherol. The inverse relationship between vitamin E status and lipid per-
oxidation in animals has been known for many years. 4° Fish have a dietary requirement
for the lipophilic molecule ot-tocopherol (vitamin E), t43 and the requirement increased with
increasing level of dietary PUFA in rainbow trout. 453 It has also been suggested that
vitamin E requirements of fish may be higher at lower water temperatures due to increased
membrane PUFA associated with homeoviscous adaptation. 3°'87
Vitamin E is a radical scavenger and acts as a terminator of lipid peroxidation by
donating a hydrogen atom to lipid peroxyradicals. The vitamin E radical so formed is not
sufficiently reactive to abstract hydrogen atoms from PUFA due to the distribution of the
unpaired electron around the aromatic ring. The effectiveness of vitamin E is largely a
result of its inclusion in the interior of the lipid membrane bilayer. 176'382Approximately
92-96% of total liver vitamin E partitioned into the membranous nuclear, mitochondrial
and microsomal fractions in rainbow trout. 3°
(b) Catalase. Peroxisomal catalase prevents PUFA autoxidation by removing hydrogen

peroxide produced by oxidases in the organelle via the reaction mechanism shown below:
2H20 2--, 2H20 + 02
However, peroxisomal catalase has a high Km for its substrate and cannot remove low
levels of H20: generated elsewhere in the cell.
(c) Glutathione (GSH) peroxidase. The selenium-containing enzyme GSH peroxidase

has a low Km for H2Oe and degrades it via the following reaction:
2GSH + H202 ~ 2H20 + GSSG (oxidized glutathione).
However, this enzyme will also reduce hydroperoxides of PUFA. ~42 Bell et al) 2 purified
GSH peroxidase from trout liver and showed that the molecular weight and Km for H202
was similar to that of the mammalian enzyme, confirming its role as a H202 scavenger in
fish tissues. The selenium dependency of the enzyme was demonstrated by its reduced
activity in the livers of trout maintained on low Se diets. 33 However, the level of dietary
vitamin E did not affect the activity of GSH peroxidase. 3~
Glutathione S-transferase, a nonSe-dependent enzyme, has some GSH peroxidase
activity in mammals, 23° and some Se-independent peroxidase activity was reported in black
bullhead. 162 However, no such activity was found in rainbow trout liver. 29'31"33
(d) Superoxide dismutase (SOD). This enzyme removes the superoxide anion radical by
the following reaction mechanism:
O~-+H*~HO~
HO~ + O~- + H ÷ ---,n202 + 02
Overall 20~- + 2H + ~ H202 + 02
SOD activity has been demonstrated in various tissues of the rainbow trout. ~°~ In
mitochondria, SOD is a manganese-containing enzyme whereas in cytosol it contains
copper and zinc. In rainbow trout, SOD activity in various tissues was related to the
dietary levels of these minerals, especially manganese. 222'223 This enzyme may be particu-
larly important in protecting against membrane autoxidation in Antarctic fishes existing
in waters of low temperature but relatively high oxygen concentration: 62
E. Roles of Essential Fatty Acids
1. Biomembranes
Biological membranes play the same fundamental roles in the structure and function of
fish cells and tissues as they do in all animals. Therefore, fish biomembranes contain ca.
25% up to 80% lipid, predominantly phospholipid and varying amounts of cholesterol,
as do mammalian membranes. Similarly, the maintenance of the correct lipid composition
is critical for the functioning of fish biomembranes, and factors that cause alterations in
the lipid structure can profoundly influence cellular metabolism. Obviously, PUFA play
a major role in biomembrane function as principal components of the structural
phospholipids. As phospholipids are primarily membrane associated, their detailed class
and fatty acid composition reflects that of biomembranes in general. These compositions
and the factors that affect them and the effect this has on the metabolism and physiology
of the fish are discussed in more detail elsewhere in this review.
2. Eicosanoid Metabolism
(a) Occurrence and synthesis. Prostaglandins have been found and studied in a large
range of freshwater fish, including carp, 24'8L2~°'3°1'3°6tench, 8t brown trout, 8~ brook trout) ~4
rainbow trout, -~°9 tilapia (T. mossambica), 24 Asian catfish (Heteropneustes fossilis and
Clarius batrachus), 24 pond loach (Misgurnus anguillicaudatus), 3°7 eel 386 and sheat-fish
(Parasilurus asotus). 3°1 Prostaglandins can be synthesized by virtually every fish tissue so
far studied, including gills, kidney, spleen, intestine, stomach, liver, heart, skeletal muscle,
brain, fin, skin, air sac, ovaries and ovarian fluid, testes and milt and blood throm-
bocytes. 81"2°921°'3°1"3°2'3°6'3°7In many instances, an exogenous fatty acid precursor was used
in the studies. However, prostaglandin synthesis from endogenous fatty acid substrate has
been shown in ovarian tissues of pond loach 3°7 and brook trout, TM carp, tench and trout
gills8~ and a variety of other tissues including testes, gastrointestinal tract, liver, heart,
brain, air sac and kidney of carp and shear-fish) °~ Gills were found to be more active than
intestine, heart, kidney, spleen and milt in carp, tench and trout in synthesizing
prostaglandins from exogenously added substrate. 8L3°6 However, gastrointestinal tract,
heart, air sac, skin and fins in carp and stomach, air sac and heart in sheat-fish all
synthesized more prostaglandin from endogenous substrates than gills. 3°1
In recent years, it has been shown that lipoxygenase enzyme products, including
leukotrienes (LT) are also found in freshwater fish tissues including trout gill, ~26-~2s'~3°
American eel gill 32s and trout skin. t29 Several different tissue preparations have been used
in studies on eicosanoid metabolism in fish, ranging from whole gill filaments 325and intact
cells 2o9'2~° to whole tissue homogenates, 8''3°6 subcellular fractions ~zs''3° and microsomes? 4
However, detailed biochemical characterization of the pathways involved have not been
performed. There is no evidence to suggest 34that the pathways involved differ substantially
from. those well characterized in mammalian systems 55'343
(b) S u b s t r a t e f a t t y acids. Dihomo-7-1inolenic acid (20:3 n - 6 ) has been used as an

exogenous substrate for prostaglandin production in tissue from carp, tenth and trout,
leading to the production of prostaglandin E1 (PGE0 and prostaglandin F, (PGF~). sl'~
Production of 1-series prostaglandins from endogenous 20:3 (n - 6) was not detected in
these tissues, whereas PGE2 from endogenous arachidonic acid (20:4 n - 6) was detected, s~
The subsequent addition of 20:3 ( n - 6) inhibited the production of PGE2 from endo-
genous substrate but PGEI production was still less than PGE2 in tenth and trout gills but
not carp gills, s~ Similarly, PGF2 produced from endogenous acid was detected and
measured in ovarian tissue of pond loach. 3°7 Exogenous 20:4 (n - 6 ) was metabolized to
PGD2, PGE2 and PGF2 by microsomes from liver, kidney and intestinal tissue from carp,
tilapia and Asian catfish. 24 When the 20:4 ( n - 6) metabolism intermediate PGH2 was
added to the microsomes, there was a 3- to 4-fold increase in the production of PGEz but
not PGD2 or PGF2. 24 Thrombocytes from carp also produced primarily PGE2, PGF2 and
PGDz from exogenously added 20:4 (n - 6 ) with, surprisingly, little or no thromboxane
B2 (TxB2) detected. 2~° This was in contrast to rainbow trout thrombocytes in which TxB2
was the main product, with lesser amounts of PGE2 and PGF2, produced from added 20:4
(n - 6). 21°
Rainbow trout thrombocytes also produced TxB3 from exogenously added eicosapenta-
enoic acid (20:5 n - 3), whereas no labeled cyclooxygenase products were produced after
the addition of labeled docosahexaenoic acid ( 2 2 : 6 n - 3 ) . 2o9 PGE3 produced from
endogenous 20:5 (n - 3) was measured in many tissues from both carp and sheat-fish, 3°1
although the amounts reported were generally lower than those reported for endogenous
PGE2 production in carp, tenth and trout gill homogenates. ~ Mai et al. 2ss reported the
finding of PGE4 produced from 22:6 (n - 3 ) in trout gill, but this was later retracted. ~27
The available data suggest that, in spite of the preponderance of (n - 3 ) PUFA and
specifically 20:5 (n - 3) and 22:6 (n - 3) in their tissue lipids, 20:4 (n - 6) is the preferred
substrate in vivo for the cyclooxygenase pathways in freshwater fish.
German et al. 127 first reported the presence of endogenously-produced lipoxygenase
products in fish tissue, in this case, a trihydroxy derivative of 22:6 (n - 3 ) in trout gills.
Subsequently, it was shown that lipoxygenase activity in trout gill metabolized exogenous
20:4 ( n - 6 ) , 20:5 ( n - 3 ) and 22:6 ( n - 3 ) to the corresponding 12- and 14-
monohydroxylated derivatives, namely 12-hydroxyeicosatetraenoic acid (12-HETE),
12-hydroxyeicosapentaenoic acid and 14-hydroxydocosahexaenoic acid, respectively. ~2s
Further metabolism of the monohydroxylated derivatives of 20:4 (n - 6) and 22:6 (n - 3)
to various trihydroxy compounds was observed in response to the exogenously added
acids. ~3° However, further metabolism of 20:5 ( n - 3) was not studied. Therefore, the
major lipoxygenase activity in the rainbow trout gill appears to be a 12-1ipoxygenase,
perhaps more accurately termed an (n - 9)-lipoxygenase, as found in mammalian plate-
lets. TM Similarly, in trout skin, 20:4 ( n - 6 ) and 22:6 ( n - 3 ) were converted into
lipoxygenase products with 12-HETE identified as the major monohydroxy product of
20:4 (n - 6 ) metabolism. ~29Conversely, the presence of a 5- [or (n - 16)]-lipoxygenase in
American eel gill tissue was indicated by the metabolism of exogenously added 20:4 (n - 6)
to LTC4, D4 and E4.325When the reaction products were partially purified and bioassayed
in a guinea pig lung strip preparation, they gave LTC4-1ike activity. 32s
There is little in the data available on freshwater fish to indicate which fatty acid, 20:4
( n - 6 ) , 20:5 ( n - 3 ) or 22:6 ( n - 3 ) , is the preferred substrate for the lipoxygenase
enzyme. However, comparative studies of exogenously added 20:4 (n - 6) and 20:5 (n - 3)
metabolism in peripheral blood neutrophils from a marine teleost, the plaice, showed that
20:4 (n - 6) was the preferred substrate for the 5-1ipoxygenase pathway in these cells. 4z8'42~
(c) Functions of eicosanoids. The data suggest that prostaglandins perform similar
functions in fish as they do in mammals. 34 The identification of prostaglandins in testes
and ovaries of fish 134'30|'302 implied a role for them in fish reproduction. This subject has
been comprehensively reviewed) s8 Briefly, intraperitoneal injection of PGE~, or PGF2 into
females induced ovulation in catfish, 374 whereas the cyclooxygenase inhibitor, in-
domethacin, blocked ovulation in carp. 2°s Clomiphene citrate, which induces gonado-
trophin release in carp, 61 restored ovulation in indomethacin-treated carp, 2°5 suggesting
that indomethacin blocks preovulatory gonadotrophin release. Prostaglandins are proba-
bly also required more directly for ovulation. 3s8 In addition, prostaglandins may act on
the brain to elicit behavioral changes 389 such as spawning activities. 387
The identification of TxB 2 in rainbow trout thrombocytes2°9 suggested that the control
of thrombocyte aggregation and blood clotting in fish may involve a TxB2/PGI2 balance
as in mammals. Piomelli325 demonstrated that leukotrienes from eel gills had the same
biological effects on mammalian lung tissue as mammalian LTC4 and it is possible they
may play a role in modulating gill function at a local level. A LTB extract from plaice
neutrophils stimulated with the calcium ionophore A23187 was found to have chemotactic
activity for plaice leukocytes,353"43°suggesting similar functions for this derivative in fish.
If. LIPID CATABOLISM
A. Lipid Mobilization
1. General Aspects
Fish can survive for much longer periods without food than homeothermic animals and,
for many fish species, a period of fasting during the winter months is part of the natural
life cycle. The long spawning migrations undertaken by anadromous fish are usually
undertaken without feeding, the energy supply being mainly from mobilized lipid reserves.
In addition, for many fish species, gonadal maturation occurs during a period of
nonfeeding such as the upstream migration of anadromous fish and, to a large extent, the
development of the gonads, particularly in the female, is dependent on mobilized lipid
reserves. Although the mobilization of lipid during starvation and gonadal development
will have many features in common, the two situations are discussed separately since
starvation is not always accompanied by the maturation of gonads.
2. Starvation
Changes in the proximste compositions of both marine and freshwater fish during
starvation have been described elsewhere. 26~'~62Although the glycogen content of the tissues
in some species may fall sharply at the outset of starvation, it can be replaced by
gluconeogenesis from amino acids, whereas the level of lipid shows a more gradual but
definite decline as starvation continues?62 An inverse relationship is apparent between the
lipid and water contents of muscle tissues in fish. 262'358
The mobilization of lipid in response to starvation has been demonstrated in tilapia, 3ss
rainbow trout, 47'~86"4°~carp, 2s°'2s: European eel93and northern pike. 179Some anatomical lipid
depots appear to be more readily mobilized than others in response to food deprivation.
The lateral line (dark) muscle of rainbow trout lost 31% of its original lipid during
starvation, whereas almost half of the lipid of the dorsal (light) muscle was mobilized. 333
Rainbow trout fed for 50 days accumulated lipid mainly in visceral deposits with smaller
increments in the lipid contents of muscles and liver. Subsequent starvation of the fish led
to an extensive depletion of lipid from the visceral deposits with only the loss of small
amounts of lipid from muscle and liver. Whereas visceral lipid declined almost immediately
in response to the cessation of feeding, the lipid stored in muscle was not mobilized until
after 27 days of starvation? 86 This suggests that in salmonids visceral adipose tissue is the
most mobile store of lipid. Likewise, the lipid depots associated with the intestine were
depleted before the hepatic lipid stores during starvation in northern pikef179
In keeping with their role as the storage form of lipid, triacylglycerols are always
mobilized before phospholipids from lipid depots during starvation. Within the overall
lipid mobilization, some selectivity exists in the pattern of fatty acids released from the
storage lipid and differences may even exist in this respect between the lipid depots present
in an individual fish. Starvation of rainbow trout resulted in the preferential mobilization
of saturated fatty acids from visceral lipid stores but monounsaturated acids 16: 1, 18:1
(n - 9 ) and 20:1 (n - 9 ) from muscle and liver lipid/s6 (n - 3 ) PUFA were apparently
specifically retained in the lipid of all these depots during starvation. Whereas (n - 9) and
(n - 6) unsaturates were also retained in visceral lipid, both these families of fatty acids
were mobilized from muscle, but only the (n - 9) from the liver. The specific decrease in
18:1 (n - 9 ) from the body and liver lipids of rainbow trout in response to starvation
leading to increases in the proportion of 22:6 (n - 3) in the remaining triacylglycerols and
polar lipids has also been noted by other workers. 4t°
The lipid content of the muscle decreased from 11.5% to 2.3% and that of the intestinal
tissues from 30.7% to 4.4% in Tilapia mossambica starved for one month. 2~ C20 and C22
PUFA accounted for higher proportions of the lipid lost from muscle reserves than from
the intestinal depots. Satoh et al) 5s found that no specific changes related to starvation
occurred in the triacylglycerols of Tilapia nilotica at 25°C, but that a slight preference for
the mobilization of 18:2 (n - 6 ) was apparent in fish starved at 15°C. The starvation of
carp has also been shown to result in a specific retention of 22: 6 (n - 3) in both polar and
neutral lipids, with 18:1 ( n - 9 ) , 18:2 ( n - 6) and 16:0 all being mobilized preferen-
tially. 2~°'41°Trienoic fatty acids were neither specifically mobilized nor retained.
In general, 22:6 (n - 3 ) is preferentially retained by fish subjected to food deprivation,
while monounsaturated fatty acids are readily mobilized for use in energy production.
3. Gonad Maturation
Most studies on the mobilization of lipid during periods of gonadal maturation have
been carried out on marine j67'~69and anadromous fish species. Mobilization of lipid in the
latter group is considered below (Section X) in relation to spawning migrations. In cultured
rainbow trout, the lipid content of the viscera is known to be inversely related to the
gonadosomatic index (GI). Fish of GI 0.2 contained 4.16g lipid in visceral deposits
whereas only 0.13 g lipid was present in the viscera of fish of similar body weight but
having a GI of l l.9. 4°~ A prolonged starvation imposed upon rainbow trout during a
period prior to gonad development leads to reduced fecundity as a result of the deposition
of insufficient reserves, including lipid, for future use in egg production. 363
In laboratory-controlled experiments with yellow perch, Newsome and Leduc 297showed
that death ensued when the lipid content of the fish, both males and females, fell below
the critical level of 2.2% of the total dry weight of the body. In the same study, it was
demonstrated that, in mature females of the same species taken in the field, most of the
lipid was located in the ovary for at least 4 months before spawning in spring. The lipid
content of the body (excluding the ovary) of these females came close to or even below
the critical lipid level of 2.2% during winter or early spring. In contrast, mature males
managed to maintain a level of ca. 5% lipid in their bodies, excluding testes. This difference
between the sexes was used to explain the selective mortality of mature females during
winter. In winter, the utilization of lipid stores for gonad production was implicated by
the observation that the lipid content of immature perch of both sexes was consistently
higher than that of mature fish.
The body lipid content declined in perch during the period of actual spawning, and
within the male the amount of visceral lipid decreased during spermatogenesis, 267'297
suggesting that mobilized lipid provided energy for this process. The apparent development
of ovaries in female pike over winter, without any extensive depletion of body lipid, may
be related to the predatory feeding habit of this species ensuring a dietary input during
this timefl TM Nevertheless, pike do not feed during the spawning period and a depletion of
reserves occurs at this time for energy provision associated with spawning activity and final
egg maturation. Although the visceral lipid content does not decline over this period and
protein is quantitatively the major energy source, a decrease of some 17% occurs in the
muscle lipid, suggesting lipid mobilization specifically from this tissue.
4. Hormone-Sensitive Lipase
Little is known of the mobilizing lipases in fish. A triacylglycerol lipase has been shown
to exist in the mesenteric adipose tissue of the steelhead trout. 37° Located in the cytosol,
this lipase had an apparent molecular weight of 48,000 and was distinct from lipoprotein
lipase. It may have a similar role to the hormone-sensitive lipase of mammalian adipose
tissue, and may be involved in the mobilization of the intracellular triacylglycerol reserves.
Both intra- and extracellular lipids have been shown by electron microscopy to be
present in the red and white muscles of rainbow trout. 28t The mechanism by which such
lipids are mobilized from muscle and subcutaneous depots is still ill-defined. The red
muscle of rainbow trout contains a triacylglycerol lipase, which has a pH optimum of 7
and is inhibited by NaF, both features of mammalian hormone-sensitive lipase. *~However,
the activity of the enzyme is not stimulated by adrenalin, and its role in starvation-induced
lipid mobilization is uncertain. A lipase which can hydrolyze triacylglycerols has also been
reported in the lysosomes of trout red muscle. 43 The authors suggested that this enzyme
may be more involved in the breakdown of lipids post mortem than in the release of fatty
acids from lipid reserves during starvation. 43 A physiological role in vivo is, therefore,
unclear.
In contrast to the situation in mammals, adrenalin, noradrenalin, glucagon and
adrenocorticotrophic hormone do not increase the lipolysis of triacylglycerols in the
adipose tissue of several species of freshwater fish. TM Lipase activity in rainbow trout
adipose tissue is, however, influenced by thyroid hormones. Fish treated with physiological
doses of thyroxine had higher levels of free fatty acids in this tissue than did control fish. 289
Muscle lipid was not affected by the administration of thyroxine. -'8~
Wiegand and Peter 457'458measured the plasma level of lipids in goldfish injected with sex
steroid hormones and provided evidence that the mobilization of lipid in female fish with
small ovaries is stimulated by estrogen. Testosterone had no apparent effect on the level
of plasma lipids. In the staghorn sculpin (Leptocothus armatus), the amount of lipid
associated with the viscera has been shown to be inversely related to the concentration of
estradiol in the serum, 95 implying activation of the mobilizing lipase by the hormone.
Jezierska et al.186 have suggested that the tissue-specific pattern of fatty acid mobilization
observed in rainbow trout during starvation may result from differences in the
stereospecific structure of triacylglycerols between tissues. PUFA predominate in position
2 and saturates in positions I and 3 of triacylglycerols in trout adipose tissue. 239 Faster
rates of hydrolysis of the 1 and 3 positions could lead to a preferential mobilization of
the saturated fatty acids in these positions. When coupled with a lower rate of hydrolysis
of fatty acids from position 2 by the lipase, the preferential location of saturated and
monounsaturated fatty acids at positions 1 and 3 of triacylglycerols and of PUFA at
position 2 may explain the general pattern of fatty acid mobilization during starvation.
5. Transport of Mobilized Lipid

In rainbow trout, the concentration of free fatty acids in plasma increased upon
starvation, at least in the Short term. Although the increase was significant 5 days after
cessation of feeding, the concentration of free fatty acids gradually declined until after 28
days the concentration was not significantly different from that in the plasma of fish
starved for only one day. Prolonged starvation up to 10 weeks had no further effect on
the plasma free fatty acid concentration. 4~ More recently, Black and Skinner 47 found the
plasma concentration of triacylglycerols and cholesterol to be higher in fed trout than in
those starved for 8 weeks. No differences were observed in the plasma concentration of
free fatty acids. When eels were starved, the first storage lipid to be depleted was hepatic
triacylglycerol and only after a long period of starvation, e.g. 95 days93 or 145 days, 229did
the plasma free fatty acid concentration increase, indicating the mobilization of extra-
hepatic lipids.
The seasonal peaks of free fatty acid concentration observed in the plasma of coho
salmon from North American lakes have been related to the mobilization of reserve lipids
for use in the development of gonads. TM In fish which undergo ovarian maturation during
a period of nonfeeding, some of the free fatty acids from mobilized lipid taken up by the
liver can be expected to be incorporated into vitellogenin for transport to the developing
gonad.
In mammals, free fatty acids mobilized from lipid reserves during starvation are
transported in the plasma bound to albumin. An albumin-like protein capable of binding
and transporting free fatty acids is considered to exist in the plasma of teleost fish but its
occurrence has not been widely documented. ~2°'2s7 The ketone bodies, acetoacetate and
3-hydroxybutyrate are formed from acetyl-CoA in the livers of starving mammals when
the rate of fatty acid oxidation exceeds the capacity of the TCA cycle to oxidize the
acetyl-CoA produced. These ketone bodies are transported in the plasma to those
extrahepatic tissues which can utilize them as an energy source. No detectable activity of
3-hydroxybutyrate dehydrogenase, the enzyme which synthesizes 3-hydroxybutyrate from
acetoacetate, was found in the liver of rainbow trout in a study which compared the rates
of the enzymes involved in ketogenesis in the livers of mammals and fish (all marine with
the exception of rainbow trout). 476 The activity of 3-0xo-acid-CoA transferase was more
than 10-fold greater in trout than mammalian liver. The high activity of this enzyme was
considered to reduce the rate of formation of ketone bodies in the trout by ensuring that
any acetoacetate formed is converted immediately back to acetoacetyl-CoA, which is
broken down to acetyl-CoA for complete oxidation. 476 This mechanism for reducing
ketone body formation is probably common to teleost fish in general, both freshwater and
marine, and can account for the observed absence of ketone bodies in the plasma of these
fish during starvation.
B. Fatty Acid Oxidation
1. General Aspects
Although the oxidation of protein provides the bulk of the energy used in sustained
swimming in salmonids, fatty acid oxidation also contributes significantly. This was
demonstrated in a recent study by van den Thillart, 432 who injected 14C-labeled glucose,
palmitate or several amino acids individually into rainbow trout and measured the rates
of oxygen consumption and 14CO2 production during rest and swimming at 80% of the
maximum speed. Glucose oxidation was extremely low in both resting and actively
swimming fish, and carbohydrate was, therefore, assumed to contribute little to the energy
utilized during swimming. Protein and lipid oxidation were estimated to provide 80% and
20%, respectively, of the total substrates oxidized in the whole fish at rest and 90*/0 and
10% during sustained swimming. Lipid oxidation has also been shown to supply energy
for sustained swimming in coho salmon. 224 The muscle employed in slow swimming, the
red lateral line muscle, is known in trout to have a high capacity for the oxidation of fatty
acids. 42'97This is also true of the heart. By comparison, the liver, kidney and particularly
white muscle have only a limited capacity to oxidize fatty acids. 42 Much of the energy to
LP.L.R. 2614---E
322 R . J . Henderson and D. R. Tocher
fuel the slow rhythm contractions which are characteristic of both lateral line and cardiac
muscle presumably comes from fatty acid oxidation.
As indicated above, the oxidation of fatty acids mobilized from reserve lipids is a major
source of energy during starvation.
2. Mitochondrial Oxidation
The overall mechanism of mitochondrial fatty acid oxidation in fish can be assumed to
proceed via a B-oxidation system similar to that operating in mammals. The oxidation of
z4C-18:1 and 14C-16:0 tO 14CO2 by mitochondria isolated from various trout tissues is
known to be greatly stimulated by the simultaneous presence of carnitine and C o m . 42 Such
observations provide evidence for the involvement of fatty acyl-CoA derivatives and a
carnitine-dependent transport of fatty acids in mitochondrial fatty acid oxidation.
In general, mitochondrial fl-oxidation in mammals displays a preference in vitro for the
carnitine derivatives of shorter chain (C10 to C14) fatty acids, with the derivatives of C20
and C22 monoenes being particularly poor substrates. ]°,~ In our own study of the
chain-length specificity of B-oxidation in trout liver mitochondria in vitro, a distinct trend
was also found for acylcarnities of increasing chain length to be oxidized at decreasing
rates j66 (Table 6). However, the difference between the highest and lowest rates [with 12:0
and 22:6 ( n - 3) derivatives, respectively] was only 2-fold. The trout from which the
mitochondria were isolated had been maintained on a diet of which 22:1 ( n - 11)
comprised 7.6% of the lipid component and it was clearly noticeable that 22:1 (n - 11)
carnitine was oxidized at a similar rate as 16:0 carnitine by the mitochondria. With 18:!
(n - 9 ) and 22:1 (n - 9 ) derivatives, no differences were noticeable between the cis and
trans isomers. Our results suggested that the mitochondrial fatty acid B-oxidation in trout,
and perhaps fish in general, can utilize a broader spectrum of fatty acyl derivatives than
that of mammals.
Although there is a general trend during starvation for the preferential mobilization of
monoenoic and saturated fatty acids, lesser but substantial amounts of PUFA are also
released from the reserve lipids. Mitochondria must be capable of the oxidation of the wide
range of fatty acids present in fish depot and dietary lipids.
Using carp, Murata and Higashi 279 estimated the rats of fatty acid oxidation by
mitochondria from dark muscle in vitro by measuring the rates of decrease of fatty acids
TABLE 6. Chain length Spccificities of Mitochondrial and Per-

oxisomal B-Oxidation o f Fatty Acids in Liver of Rainbow
Trout in vitro ~
Acyl-CoA/carnitinea Mitochondria b Peroxisomal b
12:0 120 272
14:0 107 159
16:0 100 100
16:1 (n - 7) 86 109
18:0 1|0 53
18:1 (n - 9) 107 57
18:1 (n - 9) trans 107 57
18:2 (n -6) 106 83
18:3(n - 3) 92 33
20:0 83 5
20:1 (n -9) 90 28
20:4 (n -6) 71 0
22:1 (n -11) 101 4
22:1(n -9) 76 8
22:1 (n - 9 ) trans 75 0
22:6 (n --3) 63 0
IAcylearnitines and acyl-CoAs were used for assay of mito-
chondrial and peroxisoraal fl-oxidation, respectively.
bRates are exprcssed as a percentage o f that obtained with 16:0.
For mitochondria, 100% = 89 ngatoms 0/hr/mg protein and for
peroxisomes 100% ==2.86 nmoles N A D + reduced/min/mg
protein.
originally supplied as a mixture to the incubation system. On this basis, 18:1 and 16:1 were
oxidized at much higher rates than 16:0, 18:0, 18:2, 18:3, 20:4 and 22:6. Murata 27s
subsequently showed by measuring oxygen uptake that the rate of fl-oxidation of 22:6
(n - 3) by mitochondria prepared from carp hepatopancreas was lower than that observed
with dark muscle mitochondria. Similarly, the oxidation rates of 18: l and 22:1 were higher
with mitochondria from dark muscle than from hepatopancreas, both being considerably
higher than the oxidation rate of 22:6 (n - 3 ) . In the same study, very little oxidation of
22:6 (n - 3 ) was observed with mitochondria from the red muscle of rainbow trout and
none at all was detected with mitochondria from the same tissue of Tilapia zillia.
The limited rate of oxidation of 22:6 (n - 3) coupled with its apparent retention during
lipid mobilization might ensure that this PUFA is specifically retained to fulfill its role as
a major constituent of biomembrane phospholipids. The monoenes which are readily
mobilized from reserve lipid are also good substrates for fl-oxidation and, consequently,
may be the primary source of lipid-derived energy.
3. Peroxisomal Oxidation
The characteristics of peroxisomal p-oxidation and its induction by diets rich in 22:1
have been described elsewhere. 5s'3'° The existence of peroxisomes has been demonstrated
in many tissues of carp '36 and goldfish. 84'3s~The ability of these organdies in fish to oxidize
fatty acids was first noted by Small and Connock, 3sl who observed that several tissues in
goldfish contained an active peroxisomal palmitoyl-CoA oxidase. Fish feeding on lipid-rich
calanoid copepods experience a large dietary input of 22:1 (n - 11) by virtue of the
composition of the copepod wax ester. By analogy with the situation in rats, such fish can
be expected to possess an active peroxisomal//-oxidation system. Although peroxisomes
were detectable by electron microscopy in the livers and intestines of trout fed calanoid
copepods, the activity of peroxisoal palmitoyl-CoA oxidase was no higher in fish fed this
diet than in those maintained on a control diet of low 22: I content? ~ Diets of natural
food stuffs high in lipid and 22:1 (n - 11) do not, therefore, induce peroxisomal oxidation
in trout. Furthermore, peroxisomal palmitoyi-CoA oxidation activity was not induced in
the livers of rainbow trout fed calanoid copepods together with di-(2-ethylhexyl)phthalate,
a plasticizer known to be a potent inducer of peroxisomal//-oxidation in rats. ~ However,
the total activity of peroxisomal palmitoyl-CoA oxidation in the livers of rainbow trout
fed a diet containing 11.5% partially hydrogenated fish oil and 3.5% capelin oil was double
that in livers of fish fed 15% capelin oil.'65 Since the ratio of 20:1 and 22:1 to PUFA was
6:1 in the diet containing the partially hydrogenated fish oil but only 1:1 in capelin oil,
it was concluded that the induction of peroxisomal oxidation resulted from an imbalance
of long chain monoenes to PUFA in the diet. Within copepod lipid, the ratio of 22:1
to PUFA is ca. 2:1 'rs and higher ratios are unlikely to occur naturally. Consequently,
peroxisomal//-oxidation need not be induced to catabolize the fatty acids in a typical fish
diet.
Peroxisomes isolated from the livers of rainbow trout fed partially hydrogenated fish oil
showed a definite preference for the CoA derivatives of saturated fatty acids, particularly
12:0 and 14:0, while 22: I-CoA and long chain PUFA-CoA were poorly oxidized (Table
6). ~ This further suggested that the oxidation of 22: l(n - 11) did not occur in the
peroxisomes of fish cells. A definite oxidation of 22:1 (n - I 1) did, however, occur in trout
fed on calanoid copepods for at least 7 weeks, since the 22:1 (n - 11) level of the fish's
body lipid was only about one-third that of the copepod lipid. ~'1~ The apparent ability
of trout mitoehondria to oxidize a wide spectrum of fatty acylcarnitines offers a route for
the oxidation of 22:1 (n - 11).
The rate of peroxisomal paimitoyl-CoA oxidation in trout liver was found to be
substantially lower than that of mitochondrial carnitine palmitoyltransferase. '~ Per-
oxisomal/~-oxidation, therefore, appears to make only a small contribution to the overall
oxidation of fatty acids in fish cells, as in the case in mammals under .normal physiological
324 R.J. Henderson and D. R, Tocher
conditions. Consequently, most of the oxidation of fatty acids mobilized for energy
provision during starvation takes place in the mitochondria.
viii. EMBRYONIC AND EARLY LARVAL DEVELOPMENT
A. Eggs
1. Lipid Content and Class Composition
Lipids are a major constituent of all fish eggs, along with water and protein, although
the relative amount of lipid and the class composition vary considerably with spe-
cies.193'258'291'436Consequently, lipids play an important role in the energy metabolism of fish
eggs after fertilizationfl'26t The lipids of eggs from some commercially important species
such as salmon and trout have been under study for up to 50 yr. 226"263'384However, the range
of freshwater fish species in which detailed lipid analyses of the eggs have been performed
is still far from extensive.
The majority of freshwater fish eggs contain from 2.5% to 10% lipid as a percentage
of the wet weight. Lower lipid levels up to ca. 5% were found in roach and perch, ~93
northern pike, TM tilapia ~°7 and many marine species. 427 Lipid levels between 5% and 10%
were found in burbot (Lota Iota), rainbow trout, ~93 and whitefish or cisco (Coregonus
albula)) 91"26°The eggs with higher lipid contents such as trout and salmon usually have
distinct oil globules as well as yolk lipid. 226"263"384In striped bass eggs, over 50% of the total
weight of the egg is in a single large oil droplet. ~°s The amount of lipid in the egg can also
vary within species. Thus, rainbow trout eggs have been reported as having low (ca. 4%
of wet wt) 71"357or high (ca. 10%) 193 lipid contents. Similarly, a study over 4 yr showed that
the lipid content of roach roe decreased from 6.4% to 2.3%. 225 It seems likely, therefore,
that egg lipid content can vary with the nutritional status of the mature adult fish.
Relatively high levels of polar lipids (predominantly phospholipids) are generally
associated with eggs containing lower total lipid content, such as roach, 193 pike TM and
various other freshwater 258 and marine fish.427 There are little detailed analyses of the
phospholipid composition in roe although, in one study, PC accounted for 69-82% of the
total phospholipids of several freshwater species. 2s8 Similarly, PC was the major phos-
pholipid class in rainbow trout and whitefish roe, comprising ca. 90% and 61% of the total
phospholipids, respectively. ~92The remaining phospholipid was mainly PE with all other
components comprising less than 2%. m Higher lipid contents in eggs are associated with
increased levels of neutral lipid classes, and storage in the form of distinct oil globules
rather than as the phospholipid-rich yolk proteins, lipovitellin) °8 Rainbow trout and
whitefish egg lipid contain ca. 50% and 68% neutral lipid, respectively, which is
predominantly triacylglycerol. ~9~'~93The lipid droplets in embryonated eggs of the annual
fish (Nothobranchius guentheri) contained almost 75% triacylglycerol.62 However, in
gourami roe, 85% of the total lipids are wax esters. 348 Similarly, wax esters and/or steryl
esters account for over 80% of the total lipid in perch and burbot roe ~93 and over 90%
of the oil globule lipid in striped bass eggs. ~°8
Lipids are utilized as energy sources throughout embryonic development in fish, 57'418and
consistent with this the amount of egg lipid appears to correlate with the time interval
between spawning and hatching or first fo~d. 193 Hence, freshwater spawners such as the
salmonids with long incubation periods (ca. 20 weeks compared with 20 days for many
marine fish49) have higher egg lipid levels.
As with egg lipid contents, the food supply and quality to the adult female have also
been shown to affect the lipid class composition. In particular, the concentration of neutral
lipid classes, triacylglycerol and cholesteryl esters, were found to vary in whitefish roe. 259
2. Fatty Acid Composition

Roe and eggs are generally richer in P U F A than other fish tissues. 3'191't94'243 In a range
offish including roach, perch, burbot, rainbow trout and whitefish, the total PUFA ranged
from almost 42% to over 53% of the total fatty acids.t91'~94The PUFA were predominantly
of the (n - 3) series, particularly 22:6 followed by 20: 5, 22:5 and 18:3, totalling from 30%
to 42% of total PUFA.~9~'~94In these species, (n - 6) PUFA, predominantly 20:4 and 18:2,
ranged from over 3% to almost 13% of total fatty acids, 191'194significantly higher than the
levels found in marine fish roe in general. 427 There were significant differences between
species in the (n - 3)/(n - 6) ratios of the egg lipid fatty acids, varying from 11.8 in roach
to 2.4 in rainbow trout) 94 Similarly, Bolgova et aL s4 found that carp egg lipid had higher
levels of (n - 6 ) PUFA, especially 20:4 and 18:2, in comparison with salmon egg lipid
which was richer in ( n - 3) PUFA. The saturated and monounsaturated fatty acids
generally found in significant amounts in freshwater fish roe include 16:0, 18:1 (n - 9 ) ,
16:1 (n - 7), 18:1 (n - 7) and 18:0) 94
Surprisingly, roach, which had the highest percentage (78%) of phospholipids in the egg
lipid, had the lowest percentage of PUFA (41.8 %), ~93.,94whereas whitefish, with the highest
percentage of triacylglycerol (65%), had the highest PUFA content (53.2%)) 9~a~ This is
opposite to the situation generally found in fish tissues and to that found in marine fish
eggs where phospholipid was more PUFA-rich. 427The fatty acids of the wax esters in perch
and burbot were highly unsaturated. 9 Gourami wax ester fatty acids contained ca. 34%
PUFA, a third of which was 18:2 (n - 6) with the rest primarily 22:6 (n - 3), 22:5 (n - 3)
and 18:3 (n - 3 ) . ~s In the steryl/wax ester-rich oil globule of striped bass eggs, PUFA
accounted for only 25% of the total fatty acids, whereas the phospholipid-rich yolk lipid
contained 36% P U F A ) °s The fatty alcohol compositions of the wax esters in gourami,
perch and burbot were all predominantly saturated and monounsaturated C16 and C18
moieties. 9,3~
To some extent, the fatty acid composition of fish roe tends to be conserved in
comparison with body lipids) 94'353 For instance, the fatty acid compositions of rainbow
trout roe were very similar in two studies separated both temporally and geographi-
cally. 7U~ This may also reflect some constancy in the culture diets of these fanned fish.
However, the fatty acid compositions of wild striped bass eggs from different aged fish
from different geographical locations were basically similar. I°s Kaitaranta and Linko 194
concluded that the component fatty acids were the same for all the species they studied
and that the same fatty acids were either predominant or minor regardless of the species.
Natural diets of the female fish in the wild may only affect the absolute amount of some
individual fatty acids, without affecting the overall fatty acid profile of the roe. 353
B. Lipid Catabolism During Development

Upon hatching, the larvae of many fish species are unable to feed as the intestinal tract
and mouth parts are not fully developed.5° Therefore, throughout embryogenesis in the egg
and then after hatching up to the first feed, the larvae gain nutrition from the endogenous
energy reserves of the yolk and, when present, lipid droplets.
The different possible substrates for energy production during development of teleost
fish were regarded as being utilized sequentially beginning with carbohydrate, then protein
and finally lipid. 1°2 Later it was proposed that lipid was increasingly utilized as devel-
opment proceeds. 49 Certainly, during the whole developmental period, the lipid content of
rainbow trout and Atlantic salmon eggs decreased greatly, sr'3~s Similarly, the total lipid
content in chum salmon decreased after hatching up to yolk sac absorption. 242
Biochemical studies in developing rainbow trout eggs investigating the ability of various
externally added substrates to stimulate respiration were reviewed by Terner. 4Is Respira-
tion was stimulated by the major product of fatty acid metabolism, acetate, to a greater
extent than by glycolytic substrates, including glucose. 41~ However, acetate was also
incorporated into polar and subsequently neutral lipids. 419 The combined rates of the
gluconeogenic and glycogenolytic pathways in developing trout eggs could not account for
all the endogenous respiration. 4~7 These studies suggested that lipids were the most
important energy source for developing rainbow trout eggs.
Boulekbache 57 reported that the major energy stores in the eggs of trout and loach were
glycogen and lipid. The lipid was utilized throughout embryogenesis and increasingly so
in the later stages. However, protein was the main energy source during the development
of two coregonid species (C. lavaretus and C. albula), and lipid was only significantly
utilized in the later stages when hatching was temperature-delayed. 9~ A similar situation
occurred in the annual fish where the developing eggs only utilized the lipid droplets as
an emergency energy source if hatching was delayed. 62In contrast to these studies, Eldridge
et al) °9'n° found that striped bass embryos utilized 14% of the oil globule up to hatching
and a further 14% was consumed by the larvae in the time period up to first feed. During
this period, the yolk was progressively and completely utilized with almost 60% consumed
between hatching and first feed. In calorific terms, the yolk supplied 55% and 60% of the
energy during embryonic and early larval development, respectively. 1°9:~°
During the development of rainbow trout, phospholipid was slowly but continuously
metabolized, whereas triacylglycerol was not utilized until after hatching. 384In the Atlantic
salmon, triacylglycerol was catabolized throughout development but PC, which originally
accounted for over 94% of the total phospholipids in the egg, was also continuously
metabolized so that by the swim-up fry stage, a PC: PE ratio approaching that of muscle
was achieved, s6 In the whitefish, triacylglycerol decreased immediately post fertilization,
but then phospholipid decreased slightly with PC decreasing from 76% to 61% of the total
phospholipids by hatching. 292 Kim 2~6 reported that during development of the grass carp
(Ctenopharyngodon) phospholipids were utilized continuously, partly for cell division and
organogenesis, but also for energy. However, during early embryogenesis in loach
(Misgurnus fossilis) eggs, the composition of the major phospholipids was constant with
some variability in the minor classes, t35 Interestingly, continual utilization of phospholipid,
specifically PC, was observed during embryonic development of a marine teleost, Atlantic
herring, whereas net consumption of triacylglycerol only occurred after hatching. 424In the
striped bass, neutral lipids, mainly steryl/wax esters but also triacylglycerol, were the
principal lipids utilized during development.~°° After fertilization of gourami eggs, the wax
esters are rapidly hydrolyzed so that they are only minor components of the fry. ~37"33°
Overall, though it appears that phospholipid may often be a significant energy source
during embryonic development of freshwater fish, the major portion must be retained,
along with cholesterol, as constituents of the developing larval body) °°'2~6
A possible and potentially undesirable consequence of phospholipid catabolism for
energy could be a greater loss of PUFA originally concentrated in the eggs. During salmon
development, where both triacylglycerol and PC were utilized, there was no significant
alteration of fatty acid composition in either triacylglycerol or phospholipid: 6 although
PUFA levels may have been slightly increased in swim-up fry. Selective retention of 18:2
( n - 6 ) and 18:3 ( n - 3) in comparison with 18:1 ( n - 9 ) was observed during the
development of the gourami) 3° Female gourami were fed radiolabeled acids and fertilized
eggs were later collected from 3 to 4 days onwards. Analysis of the eggs and fry showed
that 18: 2 and ! 8:3 were catabolized more slowly than ! 8: 1, perhaps as a consequence of
their incorporation into structural phospholipids in the rapidly developing embryo.
Selective retention of PUFA was also observed during embryonic development of herring
where PUFA produced by the hydrolysis of PC were selectively retained in the neutral lipid
pool at the expense of monoenes. 425Fatty alcohols produced by the hydrolysis of wax esters
during gourami development were presumably oxidized to fatty acids and catabolized, as
they were only minor components of the fry. 137'33°
Clearly, the literature contains some conflicting data indicating that there are significant
differences between the various species studied regarding lipid metabolism during em-
bryonic and early larval development. The extent and exact timing of lipid utilization as
an energy source, along with the specific lipid classes and fatty acids consumed, have all
shown distinct inter-specific variations. No clear pattern for these variations has yet
emerged, suggesting that they may be a consequence of the specific life cycles and
individual physiologies of the species so far studied rather than an evolutionary
trend.
IX. DIETARY AND ENVIRONMENTAL INFLUENCES
A. Dietary Influences
(a) Natural diets. The fatty acid composition of tissue lipids in freshwater fish are
markedly influenced by the patterns of fatty acids in their dietary lipid. The fatty acid
profile of lipids in wild fish reflects the availability of fatty acids in the aquatic food chain.
Algae feature prominently in the diet of the early life stages of some freshwater fish. In
general, the lipids of freshwater algae contain higher levels of C18 than C20 or C22
PUFA. '~7 Although the relative proportions of 18:2 (n - 6 ) and 18:3 (n - 3 ) are species
dependent, both usually occur. The 20:5 (n - 3 ) acid can account for a large proportion
of the fatty acids in some algal species, particularly diatoms, but 22:6 ( n - 3) is rarely
found. ~ A limited amount of lipid from terrestrial plants, containing predominantly 18: 3
(n - 3 ) and lesser amounts of 18:2 (n - 6 ) , is also likely to find its way into the aquatic
food chain.
Aquatic insects, either as adults, larvae or nymphs, are a major food source for
freshwater fish, particularly salmonidsfl 5'33a Although 18:2 ( n - 6 ) and 18:3 ( n - 3 )
predominate in the PUFA of these insects, 20:4 (n - 6 ) and 20:5 (n - 3 ) can constitute
up to 7% and 25%, respectively, of the total fatty acids. 147The lipids of predatory fish
preying on small fish can be expected to be influenced by the fatty acid composition of
their prey. Overall, the lipids at the different trophic levels in the freshwater food chain
are characterized by 18:2 (n - 6 ) , 18:3 (n - 3 ) and 20:5 (n - 3 ) . On the other hand, the
phytoplankton and zooplankton of the marine food chain contain lipids in which 18:3
(n - 3), 20:5 (n - 3) and 22:6 (n - 3) predominate and which have low levels of (n - 6)
P U F A ) 5° Consequently, the basic differences between freshwater and marine fish in terms
of PUFA composition stem largely from differences in the fatty acid composition of their
diets.
(b) Synthetic diets. When freshwater fish are deprived of dietary lipid and then fed an
oil derived from a marine fish species, the fatty acid composition of the body lipid in the
freshwater fish gradually resembles that of the marine oil by containing significant levels
of C20 and C22 PUFA. m In the wild, the presence of 22:1 (n - 11) in the neutral lipids
of anadromous salmon feeding in the sea 13reflects their diet of marine calanoid copepods.
The level of 22:1 (n - 11) has also been shown to increase substantially in the total lipid
of rainbow trout maintained in freshwater and fed marine calanoid copepods) 64,~6sIt is
notable from the data of Ackman and Takeuchi 7 that smolting salmon caught in fresh
water in the wild contained only 0.2% of their total lipid fatty acids as 22:1 (n - 11/13)
whereas, in those obtained from a commercial hatchery, 22: l accounted for about 8% of
the fatty acids in total lipid. The latter group of salmon had been reared on a fish oil of
which 14.5% of the fatty acids were 22:1 and which was obviously of marine origin.
Analytical evidence also suggests that trans fatty acids may also be deposited in the tissue
lipids of trout fed partially hydrogenated fish oils) 65
Many studies on the EFA requirements of salmonids have revealed that the PUFA
composition of the tissue lipids reflects the dietary fatty acid pattern. 73.411,44s:72,473From
nutrition studies using pure fatty acids or their methyl esters, it is known that feeding
increasing levels of 18:3 (n - 3) to trout previously maintained on a fat-free diet leads to
the deposition of 18:3 (n - 3) in polar and, particularly, neutral lipids, while the level of
its conversion product, 22:6 (n - 3), increases in phospholipids: 72The percentage of 22:6
(n - 3) in the phospholipids and neutral lipids in rainbow trout is also directly dependent
on its level in the dietfl°7'41]'475
The fatty acid 18:2 (n - 6) is deposited in the lipids of fish fed diets containing this
PUFA. 163'24:'2"~'2~'41°'473-475The presence of high levels of 18:2 (n - 6) in commercial fish oils
such as catfish oil 395 stems from the fish being reared on diets rich in vegetable oil. When
fed to salmonids, especially in the absence of (n - 3 ) PUFA, 18:2 (n - 6 ) along with the
20:4 (n - 6 ) and 22:5 (n - 6 ) arising from it are extensively incorporated into the lipids,
particularly the phospholipids. This dietary influence of 18:2 ( n - 6 ) can be seen in
salmonids at the level of the whole body, 473-47s m u s c l e , 242.411 liver, 163"242'248'411 intestine, 242,248
brain, 24a adipose tissue, 163"242erythrocytes246'248 and gametes?48
In the absence of 18:3 (n - 3 ) and 18:2 (n - 6 ) , 18:1 (n - 9 ) becomes a substrate for
desaturation and elongation leading to the formation of 20:3 (n - 9) which is incorporated
into phospholipids. Consequently, the phospholipids of ErA-deficient fish are character-
ized by their content of 20:3 (n - 9 ) . Increased levels of 20:3 (n - 9 ) have been reported
mainly in the phospholipids of the liver or whole body of salmonids fed ErA-deficient
diets.'~9"473'474 In a study of ErA-deficiency in relation to reproduction in rainbow trout,
Watanabe et al. 45~ found that only the sperm phospholipids of fish fed an EFA-deficient
diet for 3 months contained any 20:3 (n - 9 ) . None was present in liver or eggs.
It is noteworthy that recent studies in our own laboratory have shown that cell lines
derived from salmonid species, such as rainbow trout, Atlantic salmon and chinook
salmon, have very low PUFA contents, especially (n - 3) PUFA. 43° The fatty acid profile
of these isolated cells strongly reflects that of the lipid available to the cells in the medium,
usually foetal calf serum.
2. Lipogenic Enzymes
(a) Dietary lipid. Experimental evidence suggests that the rate of fatty acid synthesis de
novo in fish is modulated by the level of lipid in the diet. For example, the activity of
acetyl-CoA carboxylase in the liver of brook trout has been shown to be almost halved
by changing the fish from a fat-free diet to one containing 9% lipid. 3"~7When coho salmon
were maintained on diets containing equal amounts of protein but levels of lipid ranging
from 11.5% to 46% of the total calories, the hepatic activities of fatty acid synthetase and
the NADH-producing enzymes, with the exception of isocitrate dehydrogenase, were
lowest in the group of fish fed the diet of highest lipid content, z54 Changing coho salmon
from a high carbohydrate to a high lipid diet resulted in a depression of the above lipogenic
enzymes, but several weeks were required before these changes in enzyme activities were
observed? 55 Further experiments with coho salmon demonstrated that the rates of fatty
acid synthesis measured in vivo are influenced by dietary lipid levels. Fish fed a high
carbohydrate diet for three weeks, starved for two days and then re-fed a diet containing
20% lipid incorporated considerably less tritium from tritiated water (present in the
aquarium water) into hepatic fatty acids than fish r.,e-fed a diet containing only 5% lipid. 2~6
The lipogenic enzymes in the adipose tissue of coho salmon were found to be less
influenced by changes in dietary lipid content than those of the liver. TM Similarly, although
fatty acid synthetase and the NADPH-generating enzymes were all stimulated by a high
carbohydrate (low lipid) diet in both the liver and adipose tissue of the channel catfish,
only the hepatic enzymes were depressed by a high lipid diet. 253
In rainbow trout, liver glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehy-
drogenase and isocitrate dehydrogenase activities were found to decrease as the level of
lipid in the diet increased from 5.2% to 19.6%. tg° The change in the lipid content of the
diet was achieved, however, by corresponding alterations in the proportion of protein. No
significant changes in the rates of incorporation of m4C-glucose, ~4C-alanine or tritiated
water into triacylglycerol fatty acids by liver slices from rainbow trout were noticeable in
response to levels of dietary lipid between 2% and 100/0.163 In the same study the rate of
esterification of 14C-palmitic acid into triacylglycerols by adipose tissue slices increased
directly with the level of dietary lipid, suggesting that the esterification enzymes of fish
adipose tissue may respond to the amount of dietary lipid available for deposition in lipid
reserves.
Carp fed diets which differed in 18:3 ( n - 3) content but had similar levels of 18:2
(n--6) incorporated ~4C-acetate into hepatic lipid at different rates. *.7 The pattern of
distribution of radioactivity between the constituent fatty acids also depended on diet. The
16:0 and 18:1 were the most labeled when corn oil containing 50% 18:2 (n - 6) and 1.5%
18: 3) was the dietary lipid. From their experiments, the authors concluded that 18:3
(n - 3) rather than 18:2 (n - 6) specifically suppresses fatty acid synthesis de novo in carp
and that a minimum of 1% 18:3 (n - 3) in the total diet is required for this inhibition to
be effective.
The weight of evidence available suggests that the rate of fatty acid biosynthesis de novo
in fish is influenced by the level of fatty acids derived from the diet and that a balance
exists between the intake of dietary lipid and endogenous fatty acid synthesis. The exact
mechanisms of the control exerted by dietary fatty acids on lipogenic enzymes in fish have
not been examined in detail.
(b) Starvation. As stated above, fish can withstand very long periods of starvation, but
it can be expected that, during the period of food deprivation, the rate of lipogenesis de
novo will decrease in response to the fall in the amount of substrates available for
conversion to fatty acids.
In the livers of coho salmon, a reduction in the rate of fatty acid synthesis, as measured
by tritium incorporation from tritiated water, was observed both in vivo and in vitro within
two days of withholding food. 2~6Assays in vitro of hepatic lipogenic enzymes revealed that
fatty acid synthetase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehy-
drogenase were still active after two days fasting but that their activities had decreased
significantly after 23 days starvation. 255 Glucose 6-phosphate dehydrogenase activity in
rainbow trout has also been shown to be greatly reduced by starvation. 66
From the activities of individual NADPH-producing enzymes, Aster and M o o n 17
calculated that the liver of a fed American eel could potentially produce 17.62/~mol
NADPH/min for use in fatty acid synthesis whereas that of an eel fasted for 4 to 6 months
was only capable of generating 9.03/~mol on the same basis. It was notable that, whereas
the activity of glucose 6-phosphate dehydrogenase was markedly depressed by starvation,
that of isocitrate dehydrogenase was unaffected. In the liver of the European eel, the
activities of several NADPH-generating enzymes and ATP citrate lyase have been shown
to remain unchanged after a fast of 1 to 3 weeks, whereas the activities of the specific fatty
acid forming enzymes, acetyl-CoA carboxylase and fatty acid synthetase, decreased 2-fold
and 5-fold, respectively, during this period of starvation.~ Decreased fatty acid synthesis
during starvation has also been demonstrated in the European eel by the observed 6- to
20-fold decrease in the rate of incorporation of ~4C-acetate into fatty acids by liver slices
from fish starved for up to 39 weeks, j'j45 The activity of lipogenic enzymes is not lost
completely in eels during prolonged starvation. Even after 95 weeks without food, eel livers
were still capable of incorporating small amounts of ~4C-acetate into fatty acids.
B. Temperature
1. Lipid Content
Although the effect of environmental temperature on the fatty acid composition of fish
lipid has been well studied, few published values exist for the lipid contents of fish and
their tissues in direct relation to temperature. Seasonal variations in the amounts of lipid
present in temperature fish coincide generally with changes in environmental temperature
but can also be related to changes in the availability of food. An increase in the
consumption of diet with increasing temperature is true of fish in general and can explain
the almost linear increase in the lipid content of the carcasses of channel catfish on a dry
weight basis from 23.8% at a water temperature of 18°C to 43.6% at 34°C) 5
The amounts of lipid present in whole goldfish m and guppies 2H have been reported as
showing slight increases with decreasing water temperature. At the tissue level, the brains
of goldfish acclimated to 5°C contained 9.2% lipid whereas those offish acclimated to 30°C
had only 7.3%. 187"335
Rainbow trout, a species more associated with cold waters, had larger livers when
adapted to 5°C than 20°C. The hepatic lipid content of each group was similar, however. ~53
2. Lipid Classes
(a) Phospholipids. Phospholipids are a major component of biomembranes. In keeping
with other poikilothermic organisms, freshwater fish can alter the composition of their
biomembrane lipids in response to changes in environmental temperature. The term
homeoviscous adaptation is used to describe the phenomenon by which poikilotherms
maintain their biomembranes in a constant fluid state independent of their environmental
temperature. During homeoviscous adaptation, lipids are the only structural components
of biomembranes known to alter in composition. The temperature-induced changes
associated with the iipids of the cell membranes of fish, particularly rainbow trout, have
been reviewed in detail elsewhere. 156
Although the amount of total phospholipid in liver does not change during thermal
adaptation in rainbow trout, m significant alterations occur in the relative proportions of
the individual phospholipid classes. The proportion of PE, in particular, is subject to
change. In rainbow trout, PE constituted 20.2% of the phospholipids in livers from fish
acclimated to 5°C but only 16% of the hepatic phospholipids of fish maintained at 20°C. ~53
In the gills of the same species, a similar increase in the proportion of PE has been noted
in response to cold acclimation) 57'15s Increased proportions of PE in association with
adaptation to low temperatures have also been reported for goldfish brain ~°4 and
intestine,275 the liver of the green sunfish (Leponis cyanellus), 83 goldfish gill67 and muscle433
mitochondria and carp liver mitochondrial membranes. 464 It has been suggested that this
specific increase in PE favors a fluid membrane structure by virtue of the difficulty with
which this phospholipid forms compact lamellae, because of its small polar head group. 156
Cold acclimation did not result in any significant changes in the amount of PC in the
liver of rainbow trout, m In the gills of this species, the proportion of PC decreased from
55.6% to 51.5% of the total phospholipids within 3 days of transfer from 20°C to 5°C. ~5s
PC has also been shown to decrease in microsomal membranes of the liver of the green
sunfish during cold acclimation. 83 The ratio of PC to PE has been suggested as providing
a sensitive index to changes in phospholipid composition in response to changes in
environmental temperature in fish. ~58
The relative amounts of CL and SM both decreased significantly in the livers of rainbow
trout exposed to low water temperatures.~3 However, in gill tissue of the same species, the
proportion of CL increased and that of SM did not vary during cold acclimation. 157.15sOnly
in the liver of the green sunfish has the proportion of PI in phospholipids been shown to
change (increase) in relation to cold environmental temperatures. 83
The plasmalogen content of phospholipids from the brains of goldfish acclimated to 5°C
was significantly less than that of brain phospholipids from fish maintained at 3 0 ° C . 336
More specifically, the proportion of alk-l-enyl acylglycerophosphatidylethanolamine has
been shown to decrease in the brain of the goldfish during cold acclimation/°4 This change,
together with an increased proportion of diacyl-PE, generated at 5~C a molar ratio of PE
to its plasmalogen form of approximately double that observed at 30°C.~°4 The proportions
of PC and its plasmaiogen form remained constant with acclimation temperature.
Decreases in the plasmalogen content of phospholipids in relation to cold acclimation have
been noted in goldfish nervous tissue27° and carp muscle.464
(b) Neutral lipids. In fish, environmental temperature influences the relative abundance
of neutral lipid classes to a lesser extent than that of phospholipids. Although the amount
of neutral lipid in trout liver decreased with decreasing acclimation temperature, tri-
acylglycerols were always the predominant component of the neutral lipid, m'154 Also, in
trout liver, the cholesterol content, and consequently the molar ratio of phospholipid to
cholesterol, did not change with environmental temperature, t54 In carp, however, liver
mitochondrial membranes had a lower cholesterol content and ratio of cholesterol to
phospholipid when prepared from fish adapted to 10°C than 32°C. 463

(a) Total lipid. It is well established that the fatty acid composition of a fish's lipid is
influenced by the temperature of the water in which it lives. Early studies demonstrated
that for a given species the degree of unsaturation of total lipid is usually directly related
to the fish's environmental temperature with the proportion of unsaturated fatty acids
increasing as the temperature decreases (see Ref. 171). The extent to which this trend
occurs depends on species and tissue. The fatty acid compositions of total body lipid from
guppies 2"a2° and mosquito fish (Gambusia affinis) 22° typically display this increased
unsaturation at lower temperatures.
The fatty acid composition of total lipid from the carcass of goldfish was not found to
be influenced by changes in environmental temperature, whereas in the fish's intestinal lipid
the proportions of 20:4 ( n - 6) and 22:6 ( n - 3 ) increased markedly with decreasing
acclimation temperature. 213In contrast, the fatty acid pattern of total lipid from the carcass
of Tilapia mossambica was influenced more than that of intestinal lipid by water
temperature. TM Total lipid extracted from the livers of carp adapted to 5°C contained more
20:4 ( n - 6) and 22:6 ( n - 3) than lipid from fish adapted to 20°C. H6 Farkas et al. I~s
demonstrated that in carp the increased unsaturation of total lipid in response to a decrease
in environmental temperature can occur within a few hours. Hepatocytes isolated from the
livers of rainbow trout acclimated to 5°C had higher proportions of PUFA, particularly
the (n - 3 ) series, in their total lipid than cells from warm (20°C) acclimated fish. 159'366
Monounsaturated, and to a lesser extent saturated, fatty acids decreased in the total lipid
of the hepatocytes during this cold adaptation.
The lipid class composition of total lipid obviously varies with tissue and species, and
changes in the fatty acid composition of total lipid do not always reflect the specific
changes which occur in individual constituent lipid classes in relation to environmental
temperature.
(b) Neutral lipids. The fatty acid compositions of neutral lipid classes are influenced by
environmental temperature, but these classes have not been well studied in this respect.
During acclimation to low temperature, the levels of all PUFA, both (n - 3) and (n - 6)
series, increased in the triacylglycerols of trout liver. 1~ A small increase in the mono-
unsaturates and a reduction in the level of saturates within the triacylglycerols also
occurred. Although wax ester was only a minor lipid component, an increased degree of
unsaturation in its fatty acids was observed in the same study. The other neutral lipids
examined--free fatty acids, diacylglycerols and cholesteryl esters--all displayed decreases
in overall unsaturation with lowering of environmental temperature, due mainly to
reductions in their contents of monounsaturates.
(c) Phospholipids. Phospholipids are characteristically richer in PUFA than neutral

lipids and it is within phospholipids that the most obvious changes occur during
temperature adaptation by fish. The extent to which such changes take place in
phospholipids varies with the phospholipid class and the type of membrane but an increase
in fatty acid unsaturation in response to decreased environmental temperature is invari-
able. 53,67.s5.~s3,187,213.335.420,433.464
Exposure to cold temperatures for only a few hours has been shown to be sufficient to
increase the PUFA content of the total phospholipid fraction of livers from carp and
sheatfish (Silurus glanis).~lS The increased unsaturation of phospholipids is primarily due
to higher levels of ( n - 3) PUFA, I1s'153'275'42°'433although ( n - 6) PUFA rather than the
(n - 3) series have been reported to increase in mitochondrial membranes from carp liver
during cold acclimation. 463
A trend towards higher unsaturation with lower temperature has also been noted for
the fatty aldehyde moieties of plasmalogens in goldfish nervous tissue. 2~°'336"364
Differences occur between phospholipid classes in the manner by which they achieve a
higher degree of unsaturation during cold adaptation. In the liver of cold-acclimated
rainbow trout, a large increase in the proportion of22:6 (n - 3) occurred in PC, the major
phospholipid, whereas in PE, SM and PS 20:5 (n - 3 ) but not 22:6 (n - 3 ) increased. ~53
In PI, the proportion of 20:4 ( n - 6), the predominant PUFA in this phospholipid,
increased. A decrease in 16:0 content was common to all phospholipid classes, but the
proportions of monounsaturated fatty acids were not greatly affected in any phospholipid
by decreased environmental temperatures) *°'153 In the phospholipids of goldfish intestine,
the 22:6 (n - 3 ) contents of both PC and PE at 5°C were double those of fish raised at
30oC. 275
Temperature-induced changes in the levels of 22:6 (n - 3) and 20:5 (n - 3) in PC and
PE in rainbow trout occur specifically at position 2. The 22:6 (n - 3) increased from 45.9%
at 20°C to 72.2% at 5°C of the fatty acids in position 2 of PC. m In the phospholipids
of goldfish intestinal membranes, only position 2 of PE showed enrichment in 22:6 (n - 3)
at low temperatures whereas, in PC positions 2 and especially l, both increased their
contents of this fatty acid. 275 Decreased saturate and increased monounsaturate contents
of position 1 of both PE and PC were also noted. 275
Within a given membrane, not all phospholipid classes are influenced to the same extent
by changes in environmental temperature. Adaptation to cold environmental temperature
(5°C) has recently been shown to correspond with changes in the molecular species of PC
and PE present within the membranes of trout hepatocytes.~6~ In general, the proportions
of long chain polyunsaturated molecular species such as 16:0/22:6 increased with
decreasing temperature, whereas those of shorter chain mono- and dienoic species such as
16: 1/18:1 and 14:0/16:2 decreased (Table 3). These changes were most evident in the PC
and PE of the light microsomal fraction. In mitochondrial membranes, the alterations were
more noticeable in PC than PE. Although the reverse situation was true of the heavy
microsomal fraction, this fraction was the least influenced overall.
The general pattern of changes observed in the fatty acid composition of lipids in fish
during thermal acclimation under laboratory conditions also extends to freshwater fish
undergoing seasonal adaptations to environmental temperature in the wild. Carp collected
from their natural habitat during late summer when the water temperature was 22°C had
lower levels of PUFA in their hepatic phospholipids than fish taken from water of
temperature 5°C during winter, tt6 Similarly, in the livers of a tropical air-breathing teleost
(Channa punctatus), the proportions of C20 and C22 PUFA in phospholipids were higher
in winter (16°C) than summer (32°C). ~°5
The shift in fish towards more unsaturated phospholipids with decreasing environmental
temperature is generally accepted as a means of maintaining the biomembranes in a fluid
and hence functional, state, since the increased degree of unsaturation in phospholipids
is considered to confer a loose structure upon the membranes. 156 However, data obtained
from a computer modelling study suggested that 22:6 ( n - 3) can exist in helical and
hairpin forms within phospholipid molecules and that, consequently, phospholipids rich
in this PUFA may actually pack more tightly within biomembranes than phospolipids
containing monoenes. 16 In the same study, it was also pointed out that 1-stearoyl,
2-1inoleoyl PC had a melting point of - 16°C whereas species with more unsaturated fatty
acids (up to 20:4) in position 2 actually melted at the higher temperature of -12°C.
However, both these temperatures are still well below normal physiological levels.
Accordingly, the precise role of long chain PUFA during homeoviscous adaptation in fish
is not fully understood.
4. Lipid Synthesis
Conflicting evidence exists for the effect of environmental temperature on the rate of
fatty acid synthesis in fish. Several studies with trout 97,j6°and goldfish22t have indicated that
the rate of fatty acid synthesis increased during cold acclimation, whereas no differences
were found in the rates of fatty acid synthesis in hepatocytes isolated from rainbow trout
acclimated to different temperatures. ~59'435 A definite decrease in the rate of fatty acid
synthesis in the liver, but not the gills, of eels in fresh water was observed at 20°C in
comparison with 30°C but, when measured in vivo, temperature-associated differences were
less obvious in both tissues, j45 Sterol synthesis, as measured by the incorporation of
radioactivity from ~4C-acetate, proceeded at a higher rate in hepatocytes from cold-

acclimated than warm-acclimated rainbow trout. 1~9
Regardless of whether the actual rate of fatty acid synthesis is dependent on acclimation
temperature, a definite change in the pattern of fatty acids produced by fish occurs in
response to changes in environmental temperature. When carp were injected with
14C-acetate, a higher percentage of the radioactivity incorporated into hepatic total lipid
was recovered in long chain PUFA in fish maintained at 5°C than those maintained at
22°C. "6 The distribution of radioactivity among the fatty acids was dependent on the
experimental temperature rather than the temperature to which the fish were adapted,
suggesting that the fish were capable of rapid adjustments in the overall pattern of fatty
acids produced. This suggestion was confirmed by studies in vivo in which slices of carp
liver, preincubated at 5°C for 4 hr with ~4C-acetate and then incubated in the absence of
labeled substrate at 25°C for 2 hr, increased the labeling of 16:0 and 18:0 while decreasing
that mainly of the C22 PUFA. H5 Conversely, with slices preincubated at 25°C and then
transferred to 5°C, the recovery of radioactivity in C20 and C22 PUFA increased.
Hepatocytes from trout acclimated to 5°C incorporated a greater percentage of
~4C-acetate into unsaturated fatty acids and less into saturated fatty acids, when assayed
at 5°C than when the assay temperature was 20°C) 59 However, unlike the carp, the
acclimation temperature did influence the type of fatty acids produced by the trout
hepatocytes. Hepatocytes from 20°C-acclimated trout did not display an increased labeling
of unsaturated fatty acids in response to acute lowering of incubation temperature.
Increased activities of desaturase and elongation enzymes can account, at least in part,
for the increased proportions of long chain PUFA observed in lipids of fish upon lowering
of environmental temperature. Although under normal dietary situations, A9 desaturase
activity does not lead to the production of PUFA in fish, the specific activity of this enzyme
as measured by the conversion of 16:0 to 16:1 was 1.68 times higher in the hepatopancreas
of carp raised at 15-18°C than in that from fish maintained at 28-30°C. 2°9 The activities
of the A9 and A6 desaturases associated with the endoplasmic reticulum of carp liver have
also been shown to increase markedly during the adaptation of the fish to cold
temperatures. 362'465This induction of desaturases activity was complete within 48 to 72 hr
of exposure to cold and was attributed to a synthesis of additional desaturase enzyme
protein. 362Farkas H5incubated carp liver slices with ~4C-18: 0, ~4C-18:2 (n - 6) and 14C-18: 3
(n - 3) at 5°C in the absence or presence of cycloheximide. The presence of cycloheximide
corresponded with a reduced desaturation of all three substrate fatty acids and suggested
that an actual increase in the amount of desaturates occurs in response to cold exposure.
The activities of the A9, A6 and A5 desaturases were all higher in liver microsomes
prepared from Pimelodus maculatus acclimated at 15°C than in those from 30°-acclimated
fish .94,298
Hepatocytes isolated from rainbow trout acclimated to 5°C converted a higher
proportion of 18:3 (n - 3 ) to desaturation and elongation products than cells obtained
from 20°C-acclimated trout when assayed at their acclimation temperatures. 365.3ssA similar
relationship to temperature was found for the conversion of 18:2 (n - 6 ) to longer chain
PUFA but this percentage of substrate converted was much less than with 18:3 (n - 3 ) .
Although 18:1 (n - 9) also displayed higher desaturation and elongation rates at the lower
acclimation temperature, the overall percentage change was small. Such studies provide
evidence that desaturation and elongation reaction rates can compensate for changes in
environmental temperature and enhance the production of P U F A , particularly those of
the (n - 3 ) series, at low temperatures.
Coupled with the synthesis of increased proportions of long chain PUFA during cold
acclimation, adaptations occur in the pattern of fatty acids incorporated into phos-
pholipids and it is likely that the cold-induced changes in phospholipid fatty acid
composition are brought about by several mechanisms acting in conjunction. Evidence has
been obtained for the preferential utilization of diacylglycerols containing PUFA by
choline phosphotransferase during the synthesis of PC de novo at low temperatures in
goldfish 249and rainbow trout. 47s Furthermore, it has been shown that C20 rather than CI 8
PUFA are incorporated into phospholipids of trout tissues in vitro and that this specificity
is enhanced by both acute and chronic exposure to cold. t~3"~55'36sThus, trout hepatocytes
incubated with ~4C-18:2 (n - 6 ) or ~4C-18:3 (n - 3 ) showed a distinct preference for the
incorporation of the C20 PUFA derived from these substrates, particularly from 18:3
(n - 3) into phospholipids, and this preference was somewhat higher with cells from cold
(5°C) than from warm (20°C)-acclimated fish. The 22: 6 (n - 3) originating from exogenous
18:3 (n - 3) was incorporated into phospholipids to a lesser extent than 20:5 (n - 3) but
greater than 22:5 (n - 6). The incorporation of 22:6 (n - 3) was slightly enhanced in cells
from cold-acclimated fish.
Although the incorporation of PUFA into phospholipids synthesized de novo during
membrane turnover might produce highly unsaturated phospholipids in biomembranes
during homeoviscous adaptation, it has been shown that the actual rates of incorporation
of 18:1 ( n - 9 ) , 18:2 ( n - 6 ) and 18:3 ( n - 3 ) into total phospholipid are higher in
hepatocytes from warm- than cold-acclimated rainbow trout? 55 This implies that general
membrane turnover may proceed at a lower rate in fish adapted to cold temperatures.
Evidence has been obtained, however, for the increased involvement at lower temperatures
of a deacylation-reacylation cycle in the phospholipids of trout hepatocytes. ~55 The
preferential reacylation of the lysophospholipid generated in this cycle with long chain
PUFA would permit a rapid change in membrane unsaturation in response to lowering
of environmental temperature. The substrate specificity of phospholipase A 2 in trout liver
microsomes has been studied with respect to acclimation temperature. 29° When assayed at
5°C, the enzyme from cold-acclimated trout showed a preference order of 18:1 > 18:2
( n - 6)> 18:0 for the fatty acid composition of position 2 whereas the enzyme from
warm-acclimated fish preferred 18:0 > 18:2 > 18: 1, 16:0 being present in position 1 in
each case. The substrate preference of the enzyme from warm-acclimated fish was more
dependent on assay temperature and the presence of microsomal lipids than that from
cold-acclimated fish. The specificity of the phospholipase from cold-acclimated trout might
explain why monounsaturated fatty acids do not accumulate in phospholipids during
homeoviscous adaptation. It would seem feasible that the preferential reacylation of
position 2 with a PUFA could give rise to the phospholipids enriched in long chain PUFA
typical of cold-adapted membranes. Although the acyl-CoA:l-acyl-sn-glycero-3-
phosphorylcholine acyltransferase has been shown to utilize 18: I-CoA at a greater rate
than 16:CoA, ~75its complete substrate preference at low temperatures has not been fully
examined.
As noted above, the level of PE in phospholipid is generally elevated in fish by exposure
to cold. This additional PE may arise from the decarboxylation of PS as suggested by
Hazel, '55 who demonstrated that PS had a higher specific activity in trout hepatocytes
incubated with t4C-18:2 (n - 6 ) and ~4C-18:3 (n - 3 ) at 5°C than at 20°C.
C. Salinity
1. Lipid Content
The most obvious difference between fresh water and sea water is the high concentration
of inorganic salts in the latter, and for this reason changes observed in the lipids of fish
transferred from fresh water to sea water under laboratory conditions can be considered
as responses to change in environmental salinity. Changes from fresh water to sea water
in the wild are usually associated with changes in diet, and are discussed in relation to
anadromous fish (Section X).
When guppies were adapted from fresh water to sea water, the lipid content of the whole
body decreased from 5% to 3.6% of wet weight, with the eyes, liver, gonads and muscles
all containing reduced amounts of lipid. 92 Increases were observed, however, in the lipid
contents of the gills, digestive tract and kidney; all organs involved in osmoregulation.
Rainbow trout maintained either in fresh water, or water having salinities of 8%0 or 20%0,
but fed the same diet all had similar lipid contents in their gutted carcasses, jg°
2. Lipid Classes
Few published data are available for the class composition of lipid in fish subjected to
changes in environmental salinity.
With the exception of muscle, the proportion of phospholipids to neutral lipids increased
in all tissues of the guppy during sea water adaptation. 92 Tissue specific changes were
notable in the proportions of the individual phospholipid classes. In the natural lipids, the
proportion of cholesteryl esters increased while that of triacylglycerols decreased in all
tissues examined.
The intestine of rainbow trout did not display any changes in the relative proportions
of phospholipid classes during sea water adaptation. 245 PC always accounted for about
45% of the total phospholipids.

Although the ratio of (n - 3) to (n - 6) PUFA was 0.6 in fresh water and 2.1 in sea
water for eels caught in the wild, no changes occurred in the fatty acid composition of gill
lipid in freshwater eels when they were maintained in sea water for 3 months without
feeding. 42° Changes associated with sea water adaptation have, however, been demon-
strated in the fatty acid patterns of tissue lipids in other fish species. The most significant
alteration observed in the fatty acid composition of intestinal membranes of trout
transferred from fresh to sea water occurred in PC, the major phospholipid, in which the
proportion of total ( n - 3) PUFA increased from 17.7% to 33.4% within one day of
transfer. 24s Correspondingly, the level of (n - 6 ) PUFA fell from 8.3% to 6.0% and that
of saturated fatty acids from 58.3% to 46.3%. The increase in the (n - 3 ) PUFA in PC
was almost entirely accounted for by the increased proportion of 22:6 (n - 3 ) .
The percentage of 22:6 (n - 3 ) in total lipid increased, and that of 16:0 decreased, in
all tissues of the guppy during sea water adaptation. 9: In PC and particularly PE of the
digestive tract, the levels of 22:6 ( n - 3 ) and 20:4 ( n - 6 ) increased and decreased,
respectively. Consequently, the ratio of ( n - 3) to ( n - 6) PUFA increased in both
phospholipids. The fish were fed the same diet throughout the period of adaptation.
4. Lipid Synthesis
The influence of salinity on the activity of lipogenic enzymes in fish has not been studied
to any great extent. In rainbow trout, the activities of glucose 6-phosphate dehydrogenase,
isocitrate dehydrogenase and 6-phosphogluconate dehydrogenase, enzymes which generate
N A D P H for use in fatty acid synthesis, were all independent of the salinity of the water
in which the fish were kept. 29° The activity of glucose 6-phosphate dehydrogenase has,
however, been reported to be higher in the livers of Xiphopharous maculatus maintained
in one-third sea water than in fresh water. ~24Sulpholipids apparently exhibited an increased
turnover in the gills of eels during adaptation to sea water although there was no increase
in phospholipid synthesis. 479
X. A N A D R O M O U S FISH
A. Life Cycle
Anadromous fish typically develop into adults in the ocean but breed and spend the early
part of their lives in fresh water. Many salmonid species are anadromous. Full details of
the salmon's life cycle can be found elsewhere. ~95 Briefly, the eggs of the Atlantic salmon
hatch in fresh water into alevins which, after absorption of the yolk sac, become fry. The
fry eventually develop into parr which remain in fresh water for 1 to 4 yr. Each year, some
of the parr assume the silver color of adults and are then termed smolts. The smolts migrate
downstream and right out to sea where they develop into adult fish over another 1 to 4 yr.
As spawning time approaches, the adult fish re-enter freshwater rivers and migrate
upstream to the spawning area where eggs are laid among the gravel of riverbeds. Fish
which have spawned are known as kelts. Almost all the males perish at this stage but many
of the females survive until they reach the sea again where most die. A few females may
survive and return the following year to spawn a second time.
B. Lipid Content
1. Parr-Smolt Transformation
The parr-smolt transformation of salmonid fish is accompanied by a decrease in the
total body lipid. The lipid content of the flesh of parr stage cherry salmon caught in the
wild varied from 4.9% in summer to 1.0% in winter. 3~3In Spring, the lipid content of those
remaining as parr increased rapidly with an increased availability of food, reaching 4.2%
in April. Parr undergoing smolting and seaward migration in the same month, however,
contained only 1.9% lipid. Similarly, in coho salmon, the liver and muscle contained 3%
and 5% lipid, respectively, in parr but only 1.5% and 0.8% in smolts. 466During parr-smolt
transformation of steelhead trout from a commercial hatchery, lipid was substantially
depleted from dark muscle, light muscle and liver but mesenteric lipid showed no change
in amount? 7~ Overall, the depletion of lipid accompanying parr-smolt transformation is
likely to reflect its utilization as a source of energy for use in the various morphological,
behavioral and physiological changes associated with preparing the fish for adaptation to
life in sea water.
A requirement for dietary lipid in parr-smolt transformation has recently been
demonstrated by feeding studies with cherry salmon?°5 The rate of smolt production was
higher in a group of fish fed a diet in which the protein to lipid ratio was 41:16 than in
a group receiving dietary protein and lipid in the ratio of 41:4. Ackman and Takeuchi 7
have reported that healthy smolts of Atlantic salmon caught in the wild contained less lipid
in their whole bodies and skin than hatchery-reared smolts that had a high incidence of
fin erosion. Their results also suggested that the quality as well as the amount of lipid is
critical for the development of healthy smolts.
2. Spawning Migration
The depletion of total body lipid and the utilization of mobilized lipid as the principal
substrate for energy provision during the spawning migration of salmon are well known
(see Refs 171, 261). Over a migration of l135km in North America, the average male
sockeye salmon lost 95 g and, the female 116 g, of lipid from their flesh representing 69.3%
and 82.3 %, respectively, of the lipid present in that tissue at the outset of migration.tVv The
lipid contents of the combined head, skin, bones and tail (trimmings) decreased by 127 g
in males and 134 g in females, and were the main source of mobilized lipid during spawning
migration. Of the internal organs, the alimentary tract experienced the greatest loss of lipid,
5.3 g and 4.0 g in males and females, respectively, representing over 90% of the initial lipid
in each case. It was also notable that most of the lipid depleted from the flesh and
alimentary canal occurred within the first 400 km of migration, whereas the trimmings lost
the bulk of their lipids during the remainder of the migration. The female sockeye salmon
utilized more reserve lipid than males during spawning migration. In addition to utilizing
lipid as an energy source, females channel mobilized lipid into maturing gonads. The testes
consumed only 0.33% of the lipid lost from the body reserves in males whereas 8.2% of
that depleted from females' body lipid was deposited in the ovaries.
Chum salmon with very immature gonads were found to have dorsal muscles containing
2.9% to 10.4% lipid in males and 3.6% to 12.4% in females when caught in the
sea. ~2"1~-399,a°°Two months later, during spawning migration towards the coast, the lipid
content of male muscle had decreased to I. 1% lipid and in females to 1.0-1.4%; both sexes
Biochemistryof fi~shwaterfish 337
now had high gonadosomatic indices. During subsequent upstream migration in fresh
water, a slight increase in muscle lipid content was apparent (1.2% to 1.3% in males, 1.3%
to 1.6% in females). After spawning, only 0.8% to 1.1% of male and 0.8% to 0.9% of
the female muscle was lipid, and gonads were hardly present. The overall depletion of lipids
by females was greater than that in males, again implying the utilization of mobilized lipid
for the development of ova.
Analysis of blood sampled from pink salmon taken on the coast and upstream at the
spawning site showed that the lipid concentration was 1,968 mg/dl blood in the former
group of fish but only 640 mg/dl in the spawning fish. 319 The cholesterol concentration was
similar in both groups of fish. The concentrations of triacylglycerols and phospholipids,
however, had fallen substantially between the fish entering fresh water and spawning 60
miles upstream. The results confirmed that fish undertaking spawning migrations do not
feed and are effectively starving.
Nonfeeding during spawning migration is also a feature of the anadromous sea
lampreys. In these fish, the liver can be a major reserve of lipid. The duration of the
spawning migration depends on the actual species and may be related to the amount of
total lipid reserves.273 Although lipid depletion occurs during spawning migration, the
mobilized lipid is not utilized in gonad maturation to the extent that it is in salmonids.
C. Lipid Classes
1. Parr-Smolt Transformation
With the exception of liver, triacylglycerols were the principal constituent of the lipid
from serum and those tissues of the steelhead trout parr examined by Sheridan et al. 371
Decreases in lipid content associated with smolt transformation in this species were due
mainly to losses of triacylglycerols. The phospholipid content of tissues and serum did not
change significantly with smolting. Similarly, triacylglycerols accounted for 75% of the
total flesh lipid in the freshwater parr of the cherry salmon in the wild but only 59°/, of
the lipid in parr undergoing smolting during seaward migration, a14 It was considered that
the proportion of triacylglycerols declined as the seaward migration progressed.
2. Spawning Migration
Active feeding in the sea allows the fish to accumulate lipid in the muscle and visceral
depots as triacylglycerols. The lipid depleted from body stores during the spawning
migration of salmonids is almost entirely triacylglycerols. The amount of triacylglycerols
in the dorsal muscle of chum salmon decreased from 2.1 g/100g tissue in males and
3 g/100 g in females, to less than 0.2 g/100 g in both sexes, between feeding in the sea and
spawning) 2"~3The phospholipid content of the muscle remained constant. Changes in the
proportions of individual phospholipids have not been examined.
D. Fatty Acid Composition
1. Parr and Smolts

The fatty acid composition of the lipids changes with life stages in anadromous
salmonids. Early analyses of the different life stages of Atlantic salmon showed that the
fatty acid patterns of lipid of parr are typical of freshwater fish, whereas those of the adults
caught in the sea resembled marine fish (see Ref. 171). Smolt lipids are more akin to sea-
water than freshwater fish.
Differences in diet have been assumed to be an important factor in producing the
observed differences in fatty acid compositions between juvenile salmon in fresh water and
adults in the sea. 33sHowever, Sheridan et aL 372 have reported that, in fresh water, the lipids
of smolts of steelhead trout held in captivity had higher levels of long chain PUFA than
J~.L.R. 26/4--F
the parr from which they had developed. They concluded that the smolt assumed a
marine-type pattern in their lipids while still in fresh water and before they entered the
sea. Furthermore, since the fish were fed the same diet throughout the period of
smoltification, they concluded that the changes in fatty acid profiles were not solely
dictated by changes in the diet.
In wild cherry salmon, the proportion of PUFA in steryl esters, triacyl- and di-
acylglycerols, free fatty acids and phospholipids was higher in muscle lipid of parr
migrating, presumably as smolts, towards the sea than of nonmigrating parr remaining in
fresh water. 314 The proportions of 20:5 ( n - 3) and 22:6 ( n - 3 ) showed the largest
increases. Conversely, the levels of monoenes, particularly 16:1 and 18: 1, were lower in
all lipids except the free fatty acids of seaward migrating fish. The proportion of saturated
fatty acids did not change greatly in any lipid class. Similarly, the increased proportions
of PUFA observed in the triacylglycerols and phospholipids of hatchery-reared steelhead
trout during smolting were mostly attributable to specific increases in 20:5 (n - 3 ) , 22:5
(n - 3 ) and 22:6 (n - 3 ) , although the levels of 20:5 (n - 3 ) and 22:6 (n - 3 ) were still
less than are routinely found in purely marine fish. 372The lipid depletion associated with
parr-smolt transformation in salmonids involves, therefore, a relatively greater decrease
in saturated and monounsaturated fatty acids, than PUFA. Unlike the cherry salmon,
saturated fatty acids declined in all lipid classes, particularly triacylglycerols during
smolting in the steelhead trout, whereas the proportion of monoenes remained constant.
The resulting enrichment of smolt lipids in (n - 3) PUFA may be a prerequisite for their
adaptation to life in sea water.
The (n - 6) PUFA have been shown to constitute 15.8% of the fatty acids in the body
total lipids of smolting Atlantic salmon taken in the wild in Canada. 7 In the phospholipids,
10.4% of the fatty acids were 20:4 (n - 6). Hatchery-reared fish of the same life stage
contained 10% to 12% (n - 6 ) PUFA in their total lipid but only 1.6% to 1.8% 20:4
(n - 6) in their total phospholipid. Most of the (n - 6) PUFA was present instead as 18:2
( n - 6) in triacylglycerols. This difference in fatty acid composition was considered to
result from a selective accumulation of 20:4 (n - 6) by the wild parr. The hatchery-reared
fish were provided with dietary lipid containing 9.1% 18:2 (n - 6 ) but only 0.3% 20:4
(n - 6), and appeared unable to convert 18:2 (n - 6) to 20:4 (n - 6). Since hatchery fish
noticeably suffered from a higher incidence of fin erosion, the inclusion of higher levels
of 20:4 (n - 6) in their diet might alleviate these symptoms. However, suitable dietary oils
rich in 20:4 (n - 6) are rare. The requirement for 20:4 (n - 6) might vary with species since
the phospholipids of the parr of other anadromous salmonid species have been shown to
contain less than 5% of this P U F A . 314'372
2. Adults
The influence of diet on the fatty acid composition of the lipid of adult salmonids caught
at sea is evident from the presence of 22:1 (n - 11) originating from the wax esters of
marine calanoid copepods. ]3't39
The percentage of monoenoic fatty acids present in total lipid of the muscle declined
during spawning migration in the chum salmon with a corresponding increase occurring
in the proportion of PUFA. j3 The proportion of saturated fatty acids remained fairly
constant. Although these changes might at first sight indicate a specific utilization of
monoenes during the migration, they can largely be explained by the decreasing ratio of
triacylglycerols to phospholipids. Triacylglycerols greatly outweighed phospholipids be-
fore spawning migration, whereas the reverse situation was true of lipid from the fish post
spawning. 13"4°°Thus, the unsaturated nature of the phospholipids dictated the fatty acid
composition of the total lipid after spawning. In general, the pattern of fatty acids utilized
during spawning migration resembled the fatty acid composition of the total lipid present
at the outset of the migration although a slight preference for the mobilization of
monoenes and retention of PUFA is obvious (Table 7).
Analyses have shown that the overall fatty acid composition of total phospholipid in
TABLE 7. Consumption of Total Lipid from Muscle during Spawning
Migration and Spawning of Female Churn Salmon13
Outset of Lipid
migration Post spawning consumed
Fatty acids
(g/100g muscle) 3.115 0.599 2.516
Fatty acid
composition (wt %)
14:0 8.3 4.6 9.2
15:0 1.6 - 2.0
16:0 15.7 18.9 15.0
16: I 6.4 2.3 7.4
17:0 1.6 2.2 1.5
17:1 1.0 -- 1.2
18:0 3.3 5.0 2.9
18:1 21.0 17.4 21.8
18:2 2.3 2.2 2.4
19:1 1.5 1.I 1.6
20:0 2.7 I.i 3.1
20:1 8. I 6.0 8.6
20:4 0.4 1.1 0.2
20:5 5.3 9.5 4.3
22:0 1.3 - 1.6
22:1 7.7 3.8 8.6
22:5 0.7 1.5 0.5
22:6 11.1 23.3 8.2
Total saturates 34.5 31.8 35.3
Total monoenes 45.7 30.6 49.2
Total PUFA 19.8 37.6 15.5
chum salmon muscle does not change greatly during spawning migration.13 However, using
HPLC, Takahashi e t al. ~ ° have shown that 16:0/22:6 ( n - 3) is the most abundant
molecular species of PC in the muscle lipid of coho salmon caught in the sea. As the fish
migrate upstream, the proportion of 16:0/22:6 ( n - 3) was found to decrease, being
replaced largely by 22: 6 (n - 3)/22: 6 (n - 3) and 20: 5 (n - 3)/22: 6 (n - 3) species. The
main molecular species of PC in fish after spawning was again 16:0/22:6 (n - 3). The
significance of these changes is not clear.
XI. CONCLUDING REMARKS

Compared with the large number of freshwater fish species which exist, the information
reviewed in this article stems from relatively few species. Nevertheless, the wide variety of
lipid classes and fatty acids present in these fish is obvious. Although the basic
biochemistry is likely to be similar in all freshwater fish, further studies of fish from
different habitats may reveal specific adaptations o f lipid composition and biochemistry
to environment and life style. It is clear from studies to date that P U F A play a central
role in the physiology o f fish. Consequently, the diets employed in the culture of fish should
provide the fish with the correct levels and ratios o f ( n - 3) and ( n - 6) PUFA. In
particular, attention should be focused on the P U F A requirements of broodstock fish to
ensure that the ova produced contain the optimum levels and type o f P U F A for the growth
and development o f the early fish stages.
Evidence suggests that (n - 6) P U F A are specifically converted to eicosanoid derivatives
in fish despite the presence of high levels of (n - 3) PUFA. In view o f the current interest
in the use o f long chain (n - 3) P U F A in human nutrition and therapeutics, freshwater
fish may provide a useful model for the study of the basic interactions between (n - 3) and
(n - 6) P U F A in a system which is adapted to the simultaneous presence o f both series
of PUFA.
The elucidation o f fundamental biochemical pathways and mechanisms by further
studies of lipid biochemistry in freshwater fish should contribute substantially to im-
provements in aquaculture leading to the production o f more fish o f higher quality and
optimum lipid composition for human nutrition.
Acknowledgements--We wish to express our thanks to Professor John R. Sargent, Director of the N.E.R.C., Unit
of Aquatic Biochemistry, for permission to write this review.
( R e c e i v e d 2 2 A p r i l 1987)
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Lipid Content of Freshwater Fish

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Prog. Lipid Res. Vol. 26, pp. 281-347, 1987 0163-7827/87/$0.00+ 0.

THE LIPID COMPOSITION A N D BIOCHEMISTRY

R. JAMESHENDERSON and DOUGLAS R. TOCHER

V1. ESSENTIALFATTY ACIDS 312

II. LIPID CONTENTS AND CLASS COMPOSITIONS

Abramis brama Fillet 1.8 - + 328

Hypomesus olidus Whole body 4.3 - + 471

F. Brains and Eyes

extraction of the lipidS. Other constituents were phospholipids, cholesterol and

III. FATTY ACID COMPOSITIONS

s h o w e d 20:3 (n - 6) to be the p r i n c i p a l triene in the lipid o f several f r e s h w a t e r species being

TAaLE 3. Molecular Species Compositions of Phosphatidylcholine and Phosphatidyl-

IV. LIPID SYNTHESIS

A. Fatty Acid Synthesis

Comparisons of the capacities of fish tissues to synthesize fatty acids de n o v o have

3. Products of Fatty Acid Synthetase

B. Fatty Acid Esterification

2. Glycerogenesis and Glyceroneogenesis

of position 2 was attributed to the reduction of dihydroxyacetone phosphate with the

C. Desaturation and Elongation of Fatty Acids

Fat ty acid Diet Diet

18:2 ---20:2 ~ 22:2 18:3 ~ 20:3 ~ 22:3 18:4 = 2 0 :4 ~ 22:4

(n-5) (n-7) (n- 9) ( n - 6) (n- 3)

D. Reduction of Fatty Acids

v. METABOLISM OF DIETARY LIPIDS

2. Digestibility and Absorption

3. Fates of Digestion Products

D. Transport of Plasma Lipids

TASLE 4. Protein and Lipid Class Composition of Rainbow

TABLE 5. Composition of the Major Fatty Acids in the Predominant

VI. ESSENTIAL FATTY ACIDS

C. Qualitative and Quantitative Requirements

Optimal growth of carp was obtained with 1% 18:2 ( n - 6 ) and 1% 18:3 ( n - 3 )

D. Autoxidation and Protective Mechanisms

(b) Catalase. Peroxisomal catalase prevents PUFA autoxidation by removing hydrogen

(c) Glutathione (GSH) peroxidase. The selenium-containing enzyme GSH peroxidase

E. Roles of Essential Fatty Acids

(b) S u b s t r a t e f a t t y acids. Dihomo-7-1inolenic acid (20:3 n - 6 ) has been used as an

If. LIPID CATABOLISM

5. Transport of Mobilized Lipid

B. Fatty Acid Oxidation

TABLE 6. Chain length Spccificities of Mitochondrial and Per-

viii. EMBRYONIC AND EARLY LARVAL DEVELOPMENT

2. Fatty Acid Composition

B. Lipid Catabolism During Development

IX. DIETARY AND ENVIRONMENTAL INFLUENCES

3. Fatty Acid Composition

(c) Phospholipids. Phospholipids are characteristically richer in PUFA than neutral

radioactivity from ~4C-acetate, proceeded at a higher rate in hepatocytes from cold-

3. Fatty Acid Composition

D. Fatty Acid Composition

1. Parr and Smolts

XI. CONCLUDING REMARKS

244. LEKIM, D. and BETZING, H. Z. Physiol. Chem. 357, 1321-1331 (1976).

310. Osu~, T. and HAStflMOTO,T. Trends Bioehem. Sci. 9, 317-319 (1984).

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