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Lipid digestibility in fish: a review

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 Olsen and Ringø: Lipid digestibility in fish: a review 1

LIPID DIGESTIBILITY IN FISH: A REVIEW

Rolf Erik Olsen1* and Einar Ringø2

1 Finnmark College, Follumsvei, Alta, Norway. Present address: Institute of Marine Research,
Matre Aquaculture Research Station, N-5984 Matredal, Norway.
2 Department of Foof Safety and Infection Biology, Norwegian School of Veterinary Medicine,
Tromsø, Norway.

Key words: Review, lipid digestibility, fish

* Corresponding author: Tel.: +47 56367530; fax: +47 56367585 [E-mail address: rolf.erik.olsen@imr.no]

Recent Research Developments in Lipid Research Vol 1, pp 199-264. 1997

CONTENTS
ABSTRACT……………………………………………………………….……… 2
1. INTRODUCTION………………………………………………...…………… 2
2. DIGESTIVE PHYSIOLOGY………………………………………….……… 2
3. LIPID DIGESTION…………………………………………………....……… 7
3.1. Lipid digestion in mammals………………………………………....……. 7
3.2. Lipid digestion in fish………………………………………………...…… 8
3.2.1. Site of digestion………………………………………………..…… 8
3.2.2. Origin of lipases……………………………………………….…… 9
3.2.3. Nature of fish hydrolases/lipases…………………………………… 10
3.2.4. Fatty acid specificity…………………………………………..…… 14
3.2.5. Phospholipases…………………………………………………...… 14
3.2.6.Wax ester hydrolase………………………………………………… 15
3.2.7. Other lipases/hydrolases……………………………….…………… 16
3.2.8. Level of lipid on lipase activity………..…………………………… 16
4. LIPID ABSORPTION………………………………………………………… 18
4.1. Absorption in mammals…………………………………………………… 18
4.2. Absorption in fish………………………………………………….……… 18
5. FACTORS AFFECTING LIPID DIGESTIBILITY……………………...… 21
5.1. Determination of digestibility……………………………………..……… 23
5.2. Dietary lipid content………………………………………………….…… 24
5.3. Influence of lipid composition (melting point) …………………………… 25
5.4. Fatty acid digestibility…………………………………………..………… 27
5.5. Other lipids……………………………………………………………….. 29
5.6. Pyloric caecae……………………………………………………………. 29
5.7. Size of fish……………………………………………………………….. 30
5.8. Feed intake……………………………………………………………….. 33
5.9. Temperature……………………………………………………………… 34
5.10. Salinity………………………………………………………………….. 35
5.11. Starvation and seasonal variation………………………………………. 36
5.12.Dietary short organic acids……………………………………………… 37
5.13. Antibiotics…………………………………………………………….… 38
5.14. Bacterial contribution…………………………………………………… 38
5.15. Other factors…………………………………………………………….. 39
6. REFERENCES……………………………………………………………..… 41
 Olsen and Ringø: Lipid digestibility in fish: a review
ABSTRACT
The present paper reviews lipid
digestibility in fish. Lipid digestibility
depends on numerous factors such as a)
the rate and extent to which the food is
susceptible to digestive enzymes, b) the
amount and activity of the digestive
enzymes, c) the length of time the food
is exposed to the diet, and d) the rate
and mechanism of absorption of the
various nutrients. Each of these main
factors may furthermore be influenced
by numerous secondary factors. These
include other dietary components,
dietary formulation, and extrinsic
factors such as age and temperature.
The results presented in the current
review also include several earlier
manuscripts and some classical
experiments with fish from the
beginning of this century. References
cited include works published in well
known journals, and to some extent
also minimally circulated journals. In
order to give the readers satisfactory
information on lipid digestibility we
have also included some basic
information on digestive physiology in
fish.
1. INTRODUCTION
The utilisation of dietary lipids
involves several steps in which the
lipid components are made available to
the various cells of the body. Sheridan
(1) defined the biochemical processing
of dietary lipid into 11 steps that give a
good review over the process. These
steps included digestion, absorption,
reconstitution into triacylgycerols
(TAG) and lipoproteins, transport to
storage and utilising sites and
eventually incorporation into cells,
membranes, lipid depots, export lipids
(eggs etc) or utilisation for energy
2
through β-oxidation. Various aspects
regarding these general processes of
lipid utilisation have been the subject
of several reviews (1-9). However,
these authors have mainly focused on
the biochemical processes of lipid
utilisation, while the process of
digestibility of lipids has not recieved
much attention. Lipid digestibility
refers to the total availability of dietary
lipids, and may be influenced by
numerous factors including dietary
composition, size, salinity etc. This
review will focus on the lipid
digestibility, defined as digestion
(enzymatic degradation of dietary
lipid) and absorption in fish, and the
various factors influencing this
process. Although the knowledge of
these processes in fish is increasing,
some aspects are still unclear. In some
cases some lines will therefore be
drawn to what is known from
mammalian systems.
2. DIGESTIVE PHYSIOLOGY
There are many variations in the
morphology of the gastrointestinal
tract between various fish species. The
design is obviously to ensure optimum
utilisation of dietary nutrients, which
in many cases means efficient primary
digestion and a large intestinal
absorptive surface area. Different fish
species seem to have adapted different
approaches to accomodate this
objective. Of particular interest to fish
nutritionalists is the comparison of
morphological features in relation to
natural diets. In order to compare data
obtained from one fish species with
other species, it is essential to make
divisions into a broad line of common
morphological features. It is generally
accepted to divide fish into carnivorous
(eating fish and larger invertebrates),
 Olsen and Ringø: Lipid digestibility in fish: a review
herbivorous (comsuming mainly plant
material), omnivorous (mixed diet
eaters) and detritivorous (feeding
largely on detritus). However,
controversy exists as several authors
suggest that such divisions may not
always be correct since most species
consume mixed diets or their feeding
habits may change through the life-
cycle.
In the following we will give a brief
outline of some physiological
characteristics of the digestive system
of some fish species mentioned in this
paper. Information regarding the gross
intestinal morphology of some fish
have been compiled in Table 1. The
intention is to provide the reader with
some basic background information in
order to aid the interpretation of
possible differences in digestive
strategies between various species of
fish. Readers with special interest in
the digestive physiology of fish in
general are referred to comprehensive
reviews elsewhere (2-3,12,19,33-34)
and the references therein. In the
present paper we have used common
names of fish and their latin
equivalents given by Love (35), and
systematic classification according to
Nelson (36).
One commonly used classification of
fish is whether they possess a stomach
or not. Balistiae, Cyprinidae,
Cyprinodontidae and Labridae have no
stomach. In these fish the characteristic
gastric glands are lacking and the
esophagus empties directly into the
intestine. Cyprinidae have well
developed pharyngeal teeth. In
addition, stomachs may be absent
within families (Blennidae) and even
the same genus (Gobius). A stomach is
defined as a portion of the digestive
tube with distinctive cell lining, where
Table 1. Gross intestinal characteristics of some of the fishes mentioned in this article.
3
acid is secreted, usually along with
some digestive enzymes like pepsin.
Within this group of fish there are
several families that are important in
aquaculture (Ictaluridae, Plecoglos-
sidae, Salmonidae, Serranidae,
Carangidae and Sparidae). The shape,
size and structure of the stomach varies
significantly between species, and is to
a large extent related to the nature of
their diet (9,12). According to the early
work of Suyehiro (12), stomachs are
classified into five catergories
according to their appearance; I-shape
(Pleuronectidae, Esox), U-shape
(Salmonids), V-shape (Plecoglossidae,
Mugilidae, Salmonidae, Sparidae), Y-
shape (Mugilidae, Clupeidae) and -I
shape (Carangidae, Gadidae,
Scombridae, Serranidae). In
omnivorous species like catfish and
tilapia (3,9) the stomach is often found
to be sac shaped similar to that in
mammals. A common feature of a
carnivorous stomach is that it is large.
Several members of Clupeidae,
Channidae, Mugilidae, Acipenseridae,
Coregoninae (Coregonus spp.),
Chanidae (milkfish, Chanos chanos)
(3,5,10) have gizzard like
modifications of the pyloric stomach.
This development is often attributed to
microphagous, detrivorous and
herbivorous feeding (3). In grey-mullet
(Mugil cephalus), mineral particles
ingested with plant detrius and
microalgae act as grinding paste (3). It
has also been suggested that the
gizzard partly compensates for poor
dentitation (5). In some cases the
stomach can have extreme functions
such as in the Vieja (Loricaria parva)
where the thin-walled stomach serves
as a respiratory organ.
 Olsen and Ringø: Lipid digestibility in fish: a review 4

__________________________________________________________________________________
FAMILY S PC(N) RIL Remarks References
__________________________________________________________________________________
ACIPENSERIDAE (anadromous and freshwater; northern hemisphere)
Acipenser transmontanus + +(many) 0.9 Carnivorous 10,11
CLUPEIDAE (primary marine, some freshwater and anadromous; worldwide)
Clupea pallasii + +(25-27) 0.5 Carnivorous 12
ENGRAULIDIDAE (marine, occasionally freshwater; Atlantic, Indian and Pacific)
Engraulis mordax + +(21) 2.0 Filter feeder 13
CHANIDAE (marine and brackisk (occasionally freshwater); Indian and tropical Pacific)
Chanos chanos + + 3.0 Omnivore 14
CYPRINIDAE ((freshwater; North America, Africa and Eurasia)
Hypophthalmichthys molotrix - - 4.5-7/13 Omni/Herb 15/5
Labeo calbasu - - 4-10 Herb/Omni 5/15
Labeo horie - - 15-21 Detrivorous 5, 16
Cyprinus carpio - - 1.8-2.5 Omnivorous 12, 17
Ctenopharyngodon idella - - 2-2.5 Herbivorous 5, 18
Rutilus rutilus - - 0.9-1.1 Herb/Omni ?/19
Catla catla - - 4.5 Omnivorous 5
Cirrhina mrigala - - 8 Detrivorous 5
Gobio gobio - - 0,7-0,9 Carn/Omni 17
ICTALURIDAE (freshwater; North America (southern Canada to Guatemala))
Ictalurus punctatus + - 1.5 Omnivorous 20
ESOCIDAE (freshwater; northern hemisphere)
Esox lucius + - Carnivorous 16
PLECOGLOSSIDAE (anadromous; Japan, Korea and China)
Plecoglossus altivelis + +(350-400) 1.3 Carnivorous 12
SALMONIDAE (freshwater and anadromous; northern hemisphere)
Salvelinus alpinus + +(20-75) Carnivorous 21
S. malma + +815-50) Carnivorous 21
S. fontinalis + +(25-55) Carnivorous 21
S. namayash + +(95-220) Carnivorous 21
Salmo salar + +(40-80) 0.7-0.8 Carnivorous 21
S. trutta + +(32-78) Carnivorous 22
Oncorhynchus mykiss + +(25-80) 0.5 Carnivorous 21
O. gorbuscha + +(100-250) Carnivorous 21
O. nerka + +(40-110) 1 Carnivorous 21, 12
O. keta + +(150-200) 1.7 Carnivorous 12
GADIDAE (marine with one holarctic freshwater species; Arctic, Atlantic, and Pacific)
Gadus morhua + +(140-591) 1-1.5 Carnivorous 23
CYPRINODONTIDAE (fresh- and brackish water, rarely costal marine; southern Canada to South
America, Africa, Madagaskar, southern Europe, and southern Asia)
Fundulus heteroclitus - - 1.0-1.1 24, 25
HOLOCENTRIDAE (tropical marine; Atlantic, Indian and Pacific)
Myripristus berndi + +(10-13) 1.2 13
Holocentrus ensifer + +(10-13) 1.2 13
PERCICHTHYIDAE (marine, brackish and freshwater; tropical and temperate regions)
Morone saxatilis + +(6) 0.5 20
SERRANIDAE (marine (a few freshwater); tropical and temperate seas)
Dicentrarchus labrax + +(5) 3.5 26
Lates calcarifer + 3.5 12
 Olsen and Ringø: Lipid digestibility in fish: a review 5

CENTRARCHIDAE (freshwater; North America)

Micropterus salmoides + +(24-28) 0.7-0.9 Carnivorous 27
Lepomis gibbosus + +(7-8) Carnivorous 27
CARANGIDAE (marine (rarely brackish), Atlantic, Indian and Pacific)
Seriola quinqueradiata + +(250-300) 3.0 Carnivorous 12
Trachurus trachurus + +(15) 3.0 Carnivorous 12
LUTJANIDAE (marine (rarely in estuaries); Atlantic, Indian and Pacific)
Lutjanus kasmira + +(5) 1.0 13
SPARIDAE (marine (very rarely brackish and freshwater); Atlantic, Indian and Pacific)
Sparus auratus + +(4) 3
Chrysophrys major + +(4) 3.2 12
MULLIDAE (marine (rearly brackish water); Atlantic, Indian and Pacific)
Parupens multifasciatus + +(reduced) 1.0 13
CICHLIDAE (fresh- and brackish water; Central and South America; West Indies; Africa, Madagascar,
Syria and coastal India)
Sarotherodon mossambicus + - 6.3 Omnivorous 18, 28
MUGILIDAE (coastal marine and brackish water (some are freshwater); all tropical and temperate
seas)
Mugil auratus + +(7-12) Detritivorous
M. cephalus + +(2) 4-5 Detritivorous 29, 30
LABRIDAE (marine; Atlantic, Indian and Pacific)
Thalossoma duperreyi - - 1.0 13
Chelinus rhodacrous - - 1.0 13
ACANTHURIDAE (marine; all tropical seas) s.359
Haso hexacanthus + +(5) 4.4 13
SCOMBRIDAE (marine; tropical and subtropical seas)
Scomber japonicus + +(150) 3 Carnivorous 12
BOTHIDAE (marine; Atlantic, Indian and Pacific)
Paralichthys olivaceus + +(4) 2.5 Carnivorous 12
Scophthalmus maximus + +(2) 0.5 Carnivorous 31
PLEURONECTIDAE (marine, occasionally in brackish water, rearly in fresh water, Arctic, Atlantic
and Pacific)
Hippoglossus hippoglossus + +(4) Carnivorous 32
Limanda herzensteini + +(4) Carnivorous
BALISTIDAE (marine; Atlantic, Indian and Pacific)
Sufflamen brusa - - 2.0 13
__________________________________________________________________________________
S = Stomach, PC= Pyloric caecae, N = Number of pyloric caecae, RIL = relative intestinal length to
body length
 Olsen and Ringø: Lipid digestibility in fish: a review
According to Kapoor et al. (5) many
microphagous and herbivorous fish
consume large amounts of indigestible
ballast (sand, mud, cellulose etc) which
is of no use to the fish, and the use of
stomach as reservoir would be of little
significance. Consequently, they argue
that the reduction of stomach size seen
in tilapias (Cichlidae), and even
disappearance may be considered a
consequence of eating food containing
high proportions of ballast. Whatever
reason, the lack of stomach does not
indicate that these fish have no primary
digestion. Many stomachless fish have
developed pharyngeal teeth or gizzards
for grinding food as substitute for
stomachs (3, 12).
The intestine of fish is the main
digestive/absorptive organ. Some fish
have a relative intestinal length (RIL =
length of intestine/length of body) less
than one (less than the length of their
body) while others have intestinal
lengths ten to twenty times their body
lengths. The highest RIL generally
occurs in species that feed on detritus
and microalgae, while the shortest is
found in strict carnivores. The RIL of
carnivorous fish is usually 0.5-2.4, for
omnivorous (intermediate intestine
length) 0.8-5 and for herbivorous 2-21.
In Mugil cephalus, the intestine is
closely coiled, in the stoneroller
(Campostoma anomalum) (Cyprinidae)
it is wound around the swimbladder
(35). There are also differences in RIL
within the same species. To some
extent this may be due to changes in
feeding habits. As reported by Sinha &
Moitra (37) for the Indian rohu-carp
(Labeo rohita), and Lassuy (38) for
damselfish (Stegastes lividus), the RIL
of the herbivorous adult fish is higher
than that of fry feeding on
zooplankton. De Silva and co-authors
(39) found differences in RIL between
populations of the Asian ciclid
6
Etroplus suratensis feeding in a lagoon
where diets were primarily molluscs,
and a reservoir where diets were
predominantly macrophytes. De Silva
& Anderson (40) suggested that the
domestication of common carp
(Cyprinus carpio) has increased its
RIL, and that carbohydrate-rich diets
also could induce changes. Major
differences in intestinal lenght can also
be seen by comparing literature values
published on the same fish species.
While primarily herbivorous silver
carp (Hypophthalmichthys molotrix)
(Cyprinidae), were repported to have a
RIL of 13 (5), omnivorous fish had a
RIL of 4.5-7 (15).
From these observations it may be
assumed that longer intestinal length of
herbivorous compared to carnivorous
fish is due to the requirement for a
larger absorptive surface. However, it
is also possible that herbovorous (or
detrivorous) fish consuming plant
fibers and detrious rely on extended
intestines in order to increase the
utilization efficiency which is not
directly related to surface area. Some
fishes, mainly carnivorous fish seem to
have come up with alternative
solutions for increasing the absorptive
capacity without increasing the length
of the intestine. Sturgeons
(Acipenseridae), some other primitive
bony fishes, as well as sharks and
related cartilagous fish possess a
curious internal structure known as the
spiral valve (3,10). The absorptive
surface is increased by the corkscrew
course of a fold of tissue down the
length of the intestine. At the pyloric
end of the stomach, many fish have
finger-like pouches or blind-sacs (the
pyloric caecae from the Greek pyloros,
a gatekeeper, and the Latin caecus,
blind). The caecae of different species
vary considerably in size, state of
branching and connection to the gut,
 Olsen and Ringø: Lipid digestibility in fish: a review
and the number varies from 1 to more
than more than 1000 (12). These may
be very numerous as in Plecoglossidae,
Scombridae and Salmonidae, few as in
Percidae and flatfishes (Bothidae and
Pleuronectidae), or completely absent
as in Cyprinidae, Ictaluridae and
Labridae. The structure is similar to
that of the anterior intestine (3,5,21),
and is believed increase the surface
area for food absorption
(2,11,13,20,41-42). In rainbow trout
(Oncorhynchus mykiss) and Atlantic
cod (Gadus morhua), pyloric caecae
contribute to 70 % of the postgastric
surface area (11,20). These fish are
mostly carnivorous or omvinorous, and
the development may simply be a more
convenient arrangement for increasing
the surface area without increasing the
length of the intestine. Another
alternative to long intestines may be
seen in fish with relatively few pyloric
caecae where this arrangement is not
sufficient to impose a major increase in
intestinal surface area. In largemouth
bass (Micropterus salmoides)(25
pyloric caecae), and striped-bass
(Morone saxatilis) (6 pyloric caecae),
the caecae contribute to only 42% and
16% respectively of the postgastric
surface area. However, according to
Buddington & Diamond (11,20), these
fish may compensate by a thickening
of the gut. The authors also found that
gut weight in all fish species studied by
them (eigth) agreed by a factor of 2
although relative gut length varied 14-
fold, gut thickness 6-fold and the
number of caecae from 0 to 222.
3. LIPID DIGESTION
3.1. Lipid digestion in mammals
The main bulk of dietary lipid in most
mammals is triacylglycerol (TAG),
with smaller amounts of phospholipids
(PL) and sterol esters (SE) (43). In
mammals lipid digestion is initiated in
7
the stomach by the action of lingual or
gastric lipase (44-48). The hydrolysis
takes place in the absence of bile salts
and produces partial glycerides and
free fatty acids (FFA). Although the
quantitative contribution to lipid
digestion is low, the lipase is believed
to play a significant role in modifying
the lipid-water interface thereby
promoting the activity of pancreatic
lipases in the intestine (44-48). Some
emulsification may also take place in
the stomach (49). When the digesta
reaches the anterior intestine, it is
mixed with bile and pancreatic juice.
The pancreatic bicarbonate increase
the intestinal pH to values optimal for
digestive enzymes, while the detergent
action of bile salts emulsifies the lipid.
In mammals the typical droplet size is
reduced from 50 000 to 2 000 Å (46).
Mammalian pancreatic juice contains
several lipolytic enzymes. The
predominating appears to be pancreatic
lipase (EC 3.1.1.3) that hydrolyses
TAG. It is positional 1,3-specific
meaning that the end products would
be mainly 2-monoacylglycerols (2-
MAG) and FFA (50-51). There are
however certain fatty acids that are
more resistant to hydrolysis
irrespective of position on the glycerol
molecule. This is especially true for
long chain polyunsaturated fatty acids
(PUFA) of marine origin such as
20:5n-3 and 22:6n-3 (42,46,52). As
these fatty acids are distributed both in
1,3 and 2 position of TAG, this lead to
the accumulation of diacylglycerols
(DAG) that are not efficiently
assimilated. One peculiar property of
the pancreatic lipase is that it is
inhibited by surface-active agents like
bile salts (53-54). To overcome this
inhibition nature has come up with
another pancreatic protein, colipase,
that restores the lipase activity (53-55).
 Olsen and Ringø: Lipid digestibility in fish: a review
Pancreatic juice also contains lipolytic
proteins that hydrolyse PL, SE etc.
They all seem to require bile salts for
activity. Luminal phospholipids are
digested by phospholipase A2 to 1-
lysophospolipids and FFA (56-57).
Carboxylester lipase (EC 3.1.1.1) or
bile salt-activated lipase as we have
chosen to refer to throughout this text
is an interesting enzyme with a number
of hydrolase activities. Since the
names of enzymes often refer to the
substrate used in the documentation of
activity, it is possible that many of the
previously described enzymes from
pancreatic juice including non-specific
lipase, cholesterol esterase,
carboxylester hydrolase, lysophospho-
lipase, carboxylesterase, bile-salt
stimulated lipase, bile salt-dependent
lipase etc. (for review see 58) all refer
to this enzyme which consequently
may be central in lipid digestion. The
major difference from pancreatic lipase
(EC 3.1.1.3) is that bile salt-activated
lipase is capable to hydrolyse water-
soluble esters (esterase activity) and
that the activity on water insoluble
substrates is dependent on the presence
bile salts. No colipase is required. Bile
salt-activated lipase is believed to play
a central role in the hydrolysis of SE,
and other carboxylic esters such as
vitamin A palmitate (59). Furthermore,
the lipolytic activity does not seem to
be inhibited by long chain PUFA as is
the case for pancreatic lipase (60-61).
The lipase has been suggested to aid
the digestion of diets containing long
chain PUFA that would otherwise
cause a build-up of DAG (60-61). The
activity towards TAG is low however
compared to pancreatic lipase. The
enzyme is basically 1,3-specific, and
although some hydrolysis of 2-esters
have been observed (62-64), the rate is
low and probably of little physiological
importance (58).
8
3.2. Lipid digestion in fish
3.2.1 Site of digestion
It is generally believed that the
oesophagus functions as a passageway
for food into the stomach. However in
some fish, like Chanos chanos, the
oesophagus is relatively well
developed with several spiral folds that
increase the surface area and contains
numerous mucus glands (14, 65).
Findings of both protease- (66) and
lipase-activity (67) in Chanos chanos
shows that the oesophagus may
contribute significantly to nutrient
digestion. The possibility of lipoclastic
activity in the stomach of teleost fish
was suggested already in 1912 by
Polimanti (68). Later studies have
shown evidence of lipolytic activity in
stomach of several fishes including
herring, (Clupea harengus) (69),
tilapia (Tilapia mossambica) (70),
murrel (Ophiocephalus (Channa)
punctatus) (71), catfish (Clarias
batrachus) (71-72), white sturgeon
(Acipenser transmontanus) (73-74),
lake sturgeon (Acipenser fulvescens)
(10), Gadus morhua (75), Chanos
chanos (67), Zoarces anguillaris (76),
and turbot (Scophthalmus maximus)
(77). The activity is generally found to
be much lower than in the intestine.
Although the significance of the gastric
lipolytic activity in fish is not known,
it seems reasonable to assume that it
may contribute to primary digestion in
ways similar to that of mammals.
Possible correlation with food habits
was suggested when Patankar (78)
found higher lipase activity in the
stomach of the carnivore Osteoletus
ruber compared to herbivorous and
omnivor-ous fish species. However it
should be noted that studies on the
composition of dietary lipids in the
stomach of both Arctic char
 Olsen and Ringø: Lipid digestibility in fish: a review
(Salvelinus alpinus) (Olsen
unpublished data, 79) and Gadus
morhua (75), have failed to find
significant contribution of stomach
lipases to lipid digestion.
Lipase activity has been found in gut
or gut contents of most fish species
studied (e.g. 2,5,13,72,77,80-86). It
seems like a general rule that most of
the intestinal lipase activity is located
in the proximal part of the intestine,
and in the pyloric caecae if present
(17,25,67, 69,71,73,75, 87-92). The
lipolytic activity is also known to
extend into the lower parts of the
intestine. But for most fishes like
Gadus morhua (75,89), Chanos chanos
(67), Acipenser transmontanus (73)
and the cyprinid Tor Khudree (87), the
activity declines from anterior to
posterior parts of the intestine.
However one should notice that these
fishes all have long digestive tracts
and/or well developed pyloric caecae.
The situation may be different in fish
with no or few pyloric caecae and short
digestive tracts. In Scophthalmus
maximus, Koven et al. (77) found a
progressive increase in the lipolytic
activity from foregut to rectum, and
Munilla-Moran & Stark (31) reported a
similar tendency for esterase being
highest in stomach/pyloric caecae
region, lowest in the foregut and
increasing progressively towards the
rectum. This may be an adaptation
caused by the short digestive tract
(50% of body length) and few pyloric
caecae (see Table 1). In plaice
(Pleuronectes platessa) however,
Bayliss (93) also found higher activity
in the mucous layer of the distal part of
the intestine compared to proximal
segments despite the fact that this fish
have numerous pyloric caecae.
3.2.2 Origin of lipases
9
There are several possible sources for
digestive lipases in fish including
pancreas, intestinal wall, allochthonous
and autochthonous bacteria in the
intestine (chapter 5.14), and the diet.
As in mammals, the prime source of
digestive lipases in fish is assumed to
be the pancreas (2-3,5). Barnard (94)
even suggested that pancreatic lipase is
the major enzyme involved in the
digestion of TAG in all vertebrates.
Furthermore in anchovy (Engraulis
mordax), Patton et al. (42) found no
lipase activity in empty intestines, only
in a green fluid, which was found in
the intestine post-absorption. Similarly,
Olatunde & Ogunbiyi (95) found no
lipase activity in the intestines of three
tropical Schilbeidae after they had
been starved for 72 hours. These
observat-ions demonstrate the possible
predomi-nance of pancreas as origin of
digestive lipases.
It is however well recognised, that
digestive lipases may be secreted by
the intestinal mucosa in teleost fish
(3,5). This is particularly obvious for
stomachs where lipase activity has
been found in the stomach of fishes
like Clupea harengus (69), Clarias
batrachus, Channa punctatus (71) and
Chanos chanos (67). The origin of
lipases found in fish intestines is less
certain. Several studies have found
high lipase activity in mucous layers or
whole intestinal segments (washed) of
fishes like Clupea harengus (69),
Clarias batrachus (71,84), Channa
punctatus (71), the cyprinids Tor
Khudree (87) and Ctenopharyngodon
idella (80), killifish, Fundulus
heteroclitus (24- 25) and Pleuronectes
platessa, (93) indicate the possible
synthesis of lipase in the intestinal
segments. These data should however
be interpreted with caution, as lipase
activity particularly in fish during
feeding may be due to the possible
 Olsen and Ringø: Lipid digestibility in fish: a review
absorption of the pancreatic enzyme
into the intestinal mucosa. This option
was forwarded already in 1937 by
Vonk, (96) a view supported in fairly
recent reviews e.g. by Smith (9) and
Fänge & Grove (3), pointing out that
enzymes isolated from the intestinal
extracts may still be pancreatic, and the
two sources being extremely difficult
to separate.
There are however some reports
strengthening the possible existence of
intestinal and/or bacterial produced
lipases. In both Clarias batrachus (72),
and grass carp, Ctenopharyngodon
idella (80), strong lipase activity was
found in the intestinal segments even
after being starved for 48 and 24 hours
respectively. In Scophthalmus
maximus, Koven et al. (77) found two
different substrate specificities in
digesta from different sections of the
intestinal tract. Based on the enzyme
characteristics it seems likely that the
pancreatic enzyme may have been
excreted into the anterior part of the
digestive tract, while an intestinal
excretion was predominant in the
hindgut. The possibility of bacterial
lipases cannot be excluded however.
From the pattern of immuno-
histochemical staining in red sea bream
(Pagrus major), Uematsu et al. (86)
suggested that PLA2 was produced and
secreted by cells in the pyloric caecae
and anterior intestine. In Chanos
chanos, Borlongan (67) reported of
different pH optima of lipases from
pancreatic and intestinal extracts. It
should be pointed out however, that it
different pH optima may be due to
accompanying substances and not
necessarily different enzymes (96).
3.2.3. Nature of fish hydrolases/
lipases
10
Lipid nutrition in fish may vary
considerably between environmental
habitats and species, making it hard to
make generalisations. For example
marine flatfish consume substantial
amounts of benthic invertebrates,
containing mainly phospholipids,
whereas capelin (Mallosus villosus)
and sea going salmonids to a large
extent rely on diets containing wax
ester (WE) rich copepods (8,97-98).
Such differences could obviously
cause genetic differences in the
digestive capacities in addition to
variations in the design of the intestinal
tract. In freshwater environments,
TAG is the most abundant lipid class
in prey organisms as insects (4,8,99)
and is thus the principal metabolic
energy source. The fatty acid
compositions may contain relatively
high amounts of long chain PUFA like
20:4n-6 and 20:5n-3. Marine lipids
may deviate even more from human
diets being particularly rich in long
chain PUFA like 20:5n-3 and 22:6n-3
(8,98).
In general, one would expect to find
fish digestive lipases to have high
activities towards TAG as well as WE
(and SE) and PL. Furthermore, the
TAG lipases should be capable to
hydrolyse long chain PUFA at high
rates. If these requirements are held up
against the predominating mammalian
lipases (chapter 3.1), the classical
pancreatic lipase-colipase system (EC
3.1.1.3) would seem to be excluded.
Rather, one would expect a non-
specific lipase similar to the
mammalian bile salt-activated lipase
(EC 3.1.1.1) to predominate in fish
lipid digestion. As opposed to
mammals, many fish species have a
diffuse pancreas that may be scattered
in the mesenteries, liver or around bile
ducts (3,100). This has hampered the
identification of digestive lipases in
 Olsen and Ringø: Lipid digestibility in fish: a review
fish. Exceptions are the Chondricht-
hyes (Elasmobranchi and Holocephali)
and a few bony fishes, Osteichthyes,
like lungfishes (Dipnoi) (100), and
some teleost fish like the northern pike
(Esox lucius) and anguillid eels (9).
There seem to be some differences in
the lipases from cartilaginous and bony
fish, and in the following section, a
distinction will therefore be made
between Osteichthyes and Chondricht-
hyes.
Since a pancreatic lipase-colipase
system was found to be the
predominating lipase in mammals,
earlier works set out to characterise
this enzyme from fish. However, the
existence of the colipase-pancreatic
lipase system in bony fish
(Osteichthyes) proved very difficult to
verify. The property of a lipase from
Oncorhynchus mykiss was studied by
Leger and co-workers (101-103). The
presence of a colipase, and thereby,
pancreatic lipase was implied by the
response of the activity to bile salt
concentration (102) and by the
stimulation of the enzyme by porcine
colipase and a “colipase” preparation
separated by ultra-centrifugation (103).
However as pointed out by Gjellesvik
(104), the increase in activity these
authors found by the colipase preparat-
ions were marginal (from 80% to
100% of initial activity), and the
specific activity of the preparations
was 15 U/mg which was less than what
would be expected from a pancreatic
lipase. Furthermore optimum activity
required bile salts (9.6mM Na-
taurocholate) although not being
dependent on it (102). Furthermore,
the enzyme also hydrolysed fatty acids
from the 2-position of TAG if it
contained at least one double bond
(101). Later Tocher & Sargent (105)
found that most of the lipolytic activity
disappeared from crude caecal extracts
11
of Oncorhynchus mykiss upon removal
of bile by dialysis, and was returned by
addition of pure bile salts. They also
found that porcine colipase was unable
to prevent the inhibition of lipolytic
activities by cholate. Consequently, in
Oncorhynchus mykiss, a bile salt-
activated lipase is most likely the
predominant TAG lipase, possibly
accompanied by a pancreatic lipase-
colipase system.
In Gadus morhua, indications of lipase
activity had been found in both gut
contents (106) and crude extracts from
pyloric caecae (107). Feeding studies
indicated that the enzyme was 1,3-
specific (106), but analysis of depot
lipids showed that the 2-position was
only partially retained (108) indicating
the possibility of a complete
hydrolysis. Later, Lie & Lambertsen
(109) showed that the lipolytic activity
was stimulated by bile salts in Gadus
morhua intestinal juice. Lie and co-
workers (75,109) also found
indications of an apparently complete
hydrolysis of TAG to FFA and
glycerol along with the preferential
hydrolysis of PUFA esterified to TAG.
Attempts to isolate the lipase activity
from Gadus morhua using acetone
extracted gut powder (106), or bile salt
free olive oil emulsions from pyloric
caecal extracts both had failed (110).
Gjellesvik et al. (111) partly purified a
lipase from Gadus morhua pyloric
caecae, and in a series of publications
this enzyme was further purified and
characterised (104,112-113). The
lipase was dependent on bile salt for
activity on insoluble substrates.
Furthermore it possessed 1,3-specifici-
ty towards TAG, and was also able to
hydrolyse SE and 4-nitrophenyl esters.
Possible similarities towards the
human enzyme were indicated when
Gjellesvik (113) compared human and
Gadus morhua bile salt-activated
 Olsen and Ringø: Lipid digestibility in fish: a review
lipases finding only minor differences
in fatty acid specificity.
A lipase has also been purified from
sardine (Sardinella longiceps)
hepatopancreas (114). The enzyme
appears to be 1,3-specific and has
relatively high activity towards
tributyrine (940 U/mg) and triolein
(200 U/mg). Bile salt inhibition or
colipase stimulation was, however, not
studied, but the enzyme did hydrolyse
triolein in the absence of bile salts.
Consequently a classical or modified
pancreatic lipase system may be
expected.
In Atlantic salmon, (Salmo salar)
Gjellesvik and co-workers (115)
isolated a cDNA sequence from
pancreas that encoded for bile salt-
activated lipase (EC 3.1.1.1). The
primary structure showed 58% identity
with mammalian bile salt-activated
lipase. The lipolytic activity was
further studied in pyloric caecae by
Celius (116). The enzyme had a
specific activity of 91 U/mg towards
triolein in the presence of 20 mM
taurocholate, and was also able to
hydrolyse 4-nitrophenyl esters.
Furthermore, the activity towards
water insoluble substrates of both
crude extracts and purified enzyme
was very low, although quite
noticeable in the absence of bile salts.
Accordingly, it seems reasonable to
assume that the main TAG lipase in
Salmo salar is a bile salt-activated
lipase. The positional specificity was
not studied, but it is known from in
vivo feeding studies that complete
hydrolysis to FFA and glycerol
probably takes place (117).
Celius (116) isolated a lipase from
Scophthalmus maximus pancreatic
tissue. The catalytic properties were
very similar to that of Salmo salar bile
salt-activated lipase, and she concluded
that this probably is the only TAG
12
lipase in Scophthalmus maximus.
However, crude extracts had 30% of
maximum activity towards triolein in
the absence of bile salts while no
activity was found in purified extracts.
This could indicate the presence of a
lipase-colipase system in
Scophthalmus maximus, or simply a
contamination of bile salts in the
pancreatic extracts. Koven et al.
(77,83) on the other hand found
indications of both positional and non-
specific lipase activity in crude
extracts from gut contents.
Using acetone powder from the
mesenteric fat of top minnow
(Triportheus sp.), Patton et al. (118)
found indications of a true pancreatic
lipase (EC 3.1.1.3). The enzyme did
not require bile salts for activity. Due
to its relatively high activity the
authors also ruled out misidentification
of adipose tissue lipase.
Few other fish species have been
analysed with regard to digestive lipase
specificity. Furthermore, various
studies are difficult to compare since a
wide range of substrates and
experimental conditions have been
applied. However, the stimulation of
lipolytic activity of intestinal extracts
by bile salts have been known to occur
for a long time in fish like Fundulus
heteroclitus (24), Clupea harengus
(69) and Pleuronectes platessa (93).
Also the lipase activity from homogen-
ised larvae of Morone saxatilis (119)
was stimulated by bile acid. Conse-
quently many assays have been
performed using bile acids in the
reaction medium of fishes like Clarias
batrachus (72,84), Ctenopharyngodon
idella (80), Acipenser transmontanus
and Acipenser fulvescens (10,73-74).
Other studies have been performed on
various fish without the addition of
bile acids to the reaction medium
(42,67,81-82,85,87,120). These studies
 Olsen and Ringø: Lipid digestibility in fish: a review
however were carried out using crude
extracts of chyme or mucous that most
likely contained some bile salts.
One possible exception is Cyprinus
carpio, where a lipase from
hepatopancreas was purified tenfold
and the activity assayed using Ediol
without the need of bile salts (120).
The controversy as to the nature of the
digestive lipases in fish still remains
unsolved. With a possible exception of
Sardinella longiceps and Triportheus
sp., most information at present argues
in favour of a bile salt-activated lipase
as the main digestive enzyme in teleost
fish, while the existence of pancreatic
lipase-colipase is less certain. Also in
Cyprinus carpio, Kayama et al. (120)
were not able to separate the activities
of wax ester hydrolase, lipase and
esterase from hepatopancreas despite a
10 fold purification using ammonium
sulphate fractionation and gel
filtration. This could indicate that the
activities were due to the same
enzyme. The predominance of a bile
salt-activated lipase that is capable to
hydrolyse TAG, WE as well as SE
containing long chain PUFA is
however essential to the fish. The
presence of two major lipases
hydrolysing TAG is logic to assume
however, and has been suggested by
several researches, although on various
grounds. For example Leger (6)
suggested the existence of two
enzymes in fish mainly based on their
line of publications on Oncorhynchus
mykiss lipases. Tocher & Sargent (105)
came to a similar conclusion
suggesting one enzyme hydrolysing
TAG, and another predominantly WE
and SE. In Scophthalmus maximus,
Koven et al. (83,77) suggested the
presence of two lipolytic enzymes that
acted on different parts of the intestinal
tract. In the foregut, 1,3-specific
activity predominated producing
13
mainly FFA and MAG. In the hindgut
and particularly rectum, they found a
non-specific lipase producing mainly
FFA and glycerol.
A puzzling observation in both
Oncorhynchus mykiss, Sardinella
longiceps and Gadus morhua (and
perhaps Salmo salar and Scophthalmus
maximus), is that although 1,3-
specificity is found upon purification
of the enzyme, one of the most
prominent features of these fish
(Oncorhynchus mykiss, Gadus morhua
and Scophthalmus maximus) as well as
species like Salvelinus alpinus (121),
Engraulis mordax, (42,85), pink
salmon, Oncorhynchus gorbuscha,
(42), Morone saxatilis (42,85) and
speckled char (Salvelinus fontinalis)
(108), is the seemingly compete
hydrolysis to FFA in crude extracts or
in vivo studies. This obvious mismatch
between purified enzymes and crude
extracts could indicate that the
experimental conditions influence the
hydrolytic activity, or that there are
other lipases in these fish yet to be
purified.
A colipase from dogfish (Squalus
acanthius), an elasmobranch,
(Chondrichthyes), pancreas on the
other hand capable to activate porcine
lipase (122), and restored the lipase
activity of both human and porcine
pancreatic lipase inhibited by bile salts
(123). The colipase was eventually
purified and sequenced (124) showing
the existence of a colipase-pancreatic
lipase in Squalus acanthius. However,
a partly purified pancreatic lipase
preparation from this species (125) had
very low specific activity 1,27 U/mg,
was inhibited by p-mercuricbenzoate,
and had relatively high activity
towards WE, thus showing several
similarities with the bile salt-activated
lipase. The existence of a colipase has
also been shown in hagfish, Myxine
 Olsen and Ringø: Lipid digestibility in fish: a review
glutinosa, (Cephalaspidomorphi),
ratfish Chimera monstrosa, rayfish
Raja radiata and Greenland shark
Somnius microcephalus (123). In
skates, Raja erinacea and Raja radiata
(126-127), lipase activity was detected
in pancreatic acetone extracts.
Furthermore, the activity was 1,3-
specific for TAG, and the specific
activity was comparable to mammalian
pancreatic lipase. Furthermore, long
chain PUFA were poor substrates for
the lipase (127). In dogfish, Squalus
suckleyi, blue shark, Proniace glauca,
and sting ray, Urolophus halleri,
Patton (128) suggested that pancreatic
lipase was the predominant lipolytic
enzyme. This assumption was,
however, based on the observation that
only 5% of unsaturated MAG (non-
specific lipase) was hydrolysed.
A possible exception from the
tendency is leopard shark (Triakus
semifasciata). In the absence of bile
salts, the pancreatic extracts behaved
like hog pancreatic lipase being
specific for primary esters in the
absence of bile acids. However, when
bile was added, TAG hydrolysis
increased and there was a loss of
positional specificity (128-129), as
well as an increase in WE hydrolysis
(85). Moreover, the enzyme
hydrolysed methyl esters of C20 PUFA
that resisted hydrolysis by pancreatic
lipase (129). These results indicate the
presence of two lipases, a pancreatic
lipase, and a bile salt-activated lipase
in leopard shark. Thus in cartilaginous
fish, the classical pancreatic lipase
system may be of more significance
than in bony fish. Furthermore, there
seem to be major differences between
different fish species and further
research is needed in this field.
3.2.4. Fatty acid specificity
14
Regardless of nature and origin of
digestive lipases in fish, the main
objective is to ensure optimum
utilisation of dietary lipids. When data
for various teleost fish species are
compared, the specificity of teleost fish
digestive lipases do seem to have some
uniform specificity. In general PUFA
appear to be released at higher rates
than saturated fatty acids or monoenes
(75,83,109,112,130). Monoenes are
again better substrates for lipases than
saturated fatty acids at equivalent chain
lengths (42,75,109,113). Chain length
may also influence lipolysis. Longer
chain fatty acids seem to be hydrolysed
at decreasing rates with increasing
chain length (109,112-113). In fish
such as Oncorhynchus gorbuscha,
Engraulis mordax and Morone
saxatilis, digestive lipases released
52/33%, 42/28% and 41/30%
respectively of 16:0/18:0 when these
were located in 1,3 position of TAG
(42). This suggests the following
specificity for lipolysis of dietary
TAG: PUFA > MONO > SAT, and
short chain > longer chain.
3.2.5. Phospholipases
The lipolysis of phospholipids and the
hydrolysis of various carboxylic acid
esters such as wax esters and sterol
esters is less studied in fish. Fish do
not seem to possess significant
amounts of intestinal phospholipase A2
activity as described in mammals for
the removal of sn-2 fatty acids from
phospholipids (4,104,113). This is
rather surprising since phospholipids
are a significant part of the dietary
lipid. However, some hydrolysis of
dietary phosphatidylcholine in the
intestinal lumen has been indicated in
Cyprinus carpio (81). In Scophthalmus
maximus (77), digesta from rectum did
hydrolyse phosphatidylcholine, but at
rates much slower than that of TAG
 Olsen and Ringø: Lipid digestibility in fish: a review
and sterol esters. Using immuno-
histochemical staining, Uematsu and
co-workers (86) found phospholipase
A2 activity in intestinal cells of Pagrus
major. Whether this enzyme was
secreted into the lumen or only
associated with the cell membrane is
uncertain however. Phospholipase A2
activity has also been shown in
Morone saxatilis larvae (119). This
study was however analysed in whole
fish making it difficult to differentiate
phospholipase A2 activity from
intestine and body mass. These data
may indicate that phospholipase A2
does not exist in appreciable amounts
in the fish lumen. Consequently it can
be assumed that other enzymes
hydrolyse dietary phospholipids such
as bile-salt activated lipase, or that
phospholipids are hydrolysed at the
surface of the mucous, or by the
intestinal microflora (chapter 5.14).
Possible absorption without hydrolysis
is discussed in chapter 4.2.
3.2.6. Wax ester hydrolase
Wax ester hydrolase activity has been
shown to occur in many fish species
including Engraulis mordax (85,131),
Gadus morhua (109), Morone saxatilis
(42,85,119), Oncorhynchus gorbuscha
(42), chum salmon (Oncorhynchus
keta) (131), Cyprinus carpio (120),
jack mackerel (Trachurus
symmetricus) (85,131), Oncorhynchus
mykiss (105), chub mackerel (Scomber
japonicus), blue drum (Nibea
mitsukurii) brown sole (Limanda
herzensteini) bastard halibut,
Paralichthys olivaceus, yellowtail,
Seriola quinqueradiata, Pagrus major,
and black seabream, Acanthopagrus
schlegeli (90). Indirect evidence of WE
hydrolysis have also been shown in
Clupea harengus (132), Oncorhynchus
mykiss (132) and gourami Trichogaster
cosby (133) due to WE digestion and
15
the occurrence of free fatty
acids/alcohol from wax in the digesta.
Wax esters are generally hydrolysed
much slower than TAG by fish lipases
(13,42,75,85,90,105,109,119,130). As
with TAG, there appear to be some
selectivity in the hydrolysis of wax
esters. Studies with spot goby
Chaparrudo flavescens, scad
Trachurus trachurus (134), Clupea
harengus and Oncorhynchus mykiss
(132) have all shown that PUFA from
zooplankton wax esters are more
extensively absorbed than 20:1n-9 and
22:1n-11. It is also known that the
digestive juice from adult Gadus
morhua displays no specificity for the
hydrolysis of wax esters with respect
to the fatty alcohol composition (109).
There is some controversy as to
whether there is a specific wax ester
hydrolase in fish. Patton (128) and
Patton et al. (42) suggested that the
bile salt-activated lipase found in both
shark and teleost fish was much better
adapted for hydrolysis of WE than the
pancreatic lipase, an argument
supported by Gjellesvik and co-
workers (104,111-113). Also, the lack
of success in separating wax ester
hydrolase from TAG hydrolase in
Cyprinus carpio hepatopancreas
supports this possibility (120). In a
comparative study using intestinal
fluid, Patton & Nevenzel (85) on the
other hand found great variations in the
rate of hydrolysis of TAG versus WE
ranging from 4.4 in Trachurus
symmetricus through 2.5 for Morone
saxatilis to 1.2 for Engraulis mordax.
In any event, current information on
TAG lipases in fish clearly indicate
that this enzyme would be sufficient
for the digestion of wax esters.
3.2.7. Other lipases/hydrolases
Esterase activities have been found in
the gastrointestinal tract of fish like
 Olsen and Ringø: Lipid digestibility in fish: a review
Scophthalmus maximus (31,135),
Chanos chanos (14), Cyprinus carpio
(120,136), Oncorhynchus mykiss (136-
137), Scomber japonicus, Nibea
mitsukurii, Limanda herzensteini,
Paralichthys olivaceus, Pagrus major,
(90), Seriola quinqueradiata,
Acanthopagrus schlegeli (136), eel,
Anguilla japonica and ayu,
Plecoglossus altivelis (136) and many
more (9,15,88, see also 138 and
references therein). Carboxylic
esterase have been observed in perch,
Perca fluviatilis, (138), cholesterol
ester hydrolase in Oncorhynchus
mykiss (105) and Scophthalmus
maximus (77). The lipase is generally
found throughout the gastrointestinal
tract varying somewhat from species to
species although no particular
tendency can be seen.
The physiological role of the esterase
in lipid digestion is not yet clear, and
the definitions and distinction between
lipase and esterase has not been
defined with absolute clarity. Esterases
are generally defined as enzymes
hydrolysing water soluble carboxylic
esters while lipases act on water
insoluble neutral esters. However, on
basis of the recent developments in the
understanding of both mammalian and
fish lipases, it seems evident that the
bile salt-activated lipase (EC 3.1.1.1),
which is suggested to predominate in
fish, could be defined both as a lipase
and esterase depending on substrate
used. In this regard it is interesting to
note that the single bile salt-activated
lipase found in Gadus morhua
hydrolysed triacylglycerols, 4-nitro-
phenyl esters and cholesterol esters
(111-112). A partly purified enzyme
from Cyprinus carpio hydrolysed
ediol, metylbutyrate and oleylpalmitate
(120).
Furthermore, the definition of
lipase/esterase activity varies with the
16
authors. In Clarias batrachus, for
example Mukhopadhyay (84) defined
the lipase activity as the difference
between the hydrolysis of α-napthyl
laurate with taurocholate and the assay
run without taurocholate, which was
defined as esterase activity, while Das
& Tripathi (80) defined lipase activity
as the hydrolysis of α-naphtyl laurate
with taurocholate. Studying various
cyprinids Dhage (88) defined the
hydrolysis of tributyrine as lipase
activity, while the same substrate was
named esterase by Morishita et al.
(136). Consequently it is important to
define international standards for
assays of esterase, lipase and other
hydrolase activities.
3.2.8 Level of lipid on lipase activity
In rats increased dietary lipid load is
known to increase both pancreatic
lipase activity and mRNA levels (139).
From an evolutionary point of view,
one would expect to find similar
adaptations in fish. Indeed increased
lipolytic activity has been suggested as
a response to increased dietary lipid
content and composition by several
authors (17,67,72,87,140). In adult
Ctenopharyngodon idella which
ingested a mixed diet (artificial and
natural) in a pond had higher lipase
activity than fingerlings fed either leaf
protein concentrate or Lemna minor
diets (80). In this study however,
juveniles were fed in indoor tanks,
while adults were fed in ponds, and the
higher lipase activity in intestine of
adult fish may be due to factors other
than diet. In Oncorhynchus mykiss,
Tocher & Sargent (105) found
indications of increased WE and SE
hydrolase activities in caecal extracts
compared to TAG when fed WE rich
diets. This was only significant at 20
mM cholate while no difference was
 Olsen and Ringø: Lipid digestibility in fish: a review
found at lower and higher levels. The
ability to metabolise fatty alcohols to
TAG was however the same whether
the fish were fed diets rich in TAG or
wax esters (141). Mankura et al. (90)
compared WE hydrolase, esterase and
lipase activities between seven species
of fish, and found major differences in
activity. They suggested that fish
feeding on diets containing wax ester
compensate by increasing wax ester
hydrolase activity. The study was
however based on few fish that were
obtained from a local fish dealer.
Consequently, the dietary condition
and history of these fish was not
known.
Other studies have suggested that
changing the enzyme activity may not
be the major strategy to compensate
for various lipid content in diet. In
Clarias batrachus, Mukhopadhyay
(84) found no difference in the
intestinal lipase activity when the fish
were fed on either a natural diet, or
artificial diets containing 3-4% lipid of
different origin. Similarly, Chesley
(142) and Benson and co-workers
(131) found only slight variability in
the concentration of digestive lipases
in marine fishes. In one interesting
study, Patton & Benson (13) fed wax
esters to various fish species some of
which would be accustomed to wax
esters in their diets, while others were
not. They were not able to find any
major difference in wax ester digesting
enzymes (and perhaps fat digesting
enzymes in general). Rather they
suggested that fish have adapted
another strategy in which the food is
retained for a longer time in the
intestinal segments, allowing more
time for digestion. In for example
Engraulis mordax, 87% of a dietary
dose of WE was recovered (mainly in
gut contents) 24h after administration,
compared to only 9-10% in the
17
stomachless wrasse (Thalossoma
duperreyi) indicating that the content
was voided before assimilation could
take place.
Thus studies within species and
between species seem to yield
contradictory results. However, minor
adjustments to a change of dietary
source must be within range of most
fish. On the other hand adaptations to
fundamentally different diets, is left to
evolution. In the case of WE, the
emulsification rather than lipolysis
may be the rate limiting factor for
digestion. Using various fish species,
Patton & Benson (13) showed that the
assimilation of radioactively labelled
wax esters was significantly improved
when the wax ester carrier was
substituted with TAG. Lie and
Lambertsen (109) found no such effect
in Gadus morhua when the rate of
hydrolysis was compared using
substrates containing either pure WE,
TAG, or a 1:1 mixture of both
compounds. However, in an in vivo
study, they did find increased digestion
of wax esters when Gadus morhua
were fed diets supplemented with
TAG.
4. LIPID ABSORPTION
4.1. Absorption in mammals
In mammals, the main hydrolytic
products, 2-MAG and FFA, become
solubilised in bile salt micelles, diffuse
through the “unstirred water layer” to
the intestinal mucosa where they are
absorbed as monomers (43,46,143).
The emulsification is thought to be rate
limiting for absorption (144). The
transfer of lipolytic products from the
oil phase to the micellar phase appears
to be rate limiting for absorption, and
depends in part on pH, temperature,
ionisation (soap) and the solubility of
the individual fatty acid. In a
 Olsen and Ringø: Lipid digestibility in fish: a review
physiological system, the solubility of
the fatty acids is particularly dependent
on chain length and degree of
unsaturation (46,144). For saturated
fatty acids, the solubility in the
micelles increase logarithmically with
decreasing chain length. Unsaturated
fatty acids are more soluble than
saturated fatty acids with the solubility
increasing with unsaturation, and
decreasing with high chain length. The
transport across the “unstirred water
layer“ is assumed to proceed by
diffusion (143) and to a large extent
being a physical process. Similarly, the
uptake into the enterocyte has been
assumed to proceed by means of
passive diffusion (46,143). However,
recent studies from mammals indicate
that cellular uptake of some long chain
fatty acids may be carrier mediated
(145-147). The absorption of medium
chain fatty acids (C6 to C10 or C12)
deviates from the traditional pathway.
They are not widely distributed in
normal dietary lipids except from milk
and some other vegetable products like
coconut oil. Due to their high solubility
in water, they can solubilise without
the aid of bile salts, and can diffuse
directly through the “unstirred water
layer” (144,148). Furthermore, the
absorption into the enterocyte is very
fast compared to long chain fatty acids,
and a major part are transported with
the portal vein as free fatty acids (148-
149).
4.2. Absorption in fish
The process of absorption of individual
fatty acids has not been extensively
studied in fish. As for mammals, most
research has focused on the absorption
of the quantitatively major neutral lipid
classes, primarily TAG and to some
extent WE, while relatively little
information is available regarding the
absorption of other lipid classes such
18
as PL. There is also little information
regarding the physical processes
occurring in the gut lumen during lipid
digestion. However, since bile aided
emulgation and transport of the free
fatty acids and partial glycerides to a
large extent is a physical process, one
would not expect to find many
significant differences between fish
and mammals in these processes.
However, since fish live in different
temperature regimes compared to
mammals, one would expect to find
some variations with regard to
substrate specificity and efficiency,
and e.g. bile and bile acid composition.
In general however, available data
indicate that the process of lipid
absorption in fish is essentially similar
to that of mammals (91,150-151). The
rate of absorption is however
generally assumed to proceed much
slower than that of mammals and may
take hours (25,81-82,122,150-151).
Much of this difference is doubtless
associated with the lower maintenance
temperature, which has a marked
influence on the rate of nutrient uptake
(2,5). The abosrption of lipid in fish is
clearly comparmentalised to certain
regions of the gut (Table 2).
Although frequently stated in early
histological based studies (e.g. 155-
156), the present view is that the
absorption of lipid in stomach is likely
to be insignificant. Austreng (152)
found that 8,5% of dietary lipid was
assimilated in the stomach of
Oncorhynchus mykiss while Strømsnes
(153) found a negative assimilation of
lipid (-15%) in Atlantic halibut
(Hippoglossus hippoglossus). These
differences are probably more due to
methodological problems than actual
data. Negative digestibility may imply
that other nutrients are absorbed in the
stomach such as amino acids. It is also
possible that chromic oxide may be
 Olsen and Ringø: Lipid digestibility in fish: a review 19

transported further down the -160), which to a large extent coincides

alimentary tract at a rate different from
that of the digesta (see chapter 5.1). In
Salvelinus alpinus and Gadus morhua,
Olsen (unpublished data) and Lie et al.
(75) respectively found no assimilation
of lipid in the stomach.
As in mammals, the proximal part of
the intestine appears to be the main
area involved in lipid absorption in fish
(17,25,28,67,71,89,91,109,121,154,157
with the predominating lipase activity.
Although several studies, covering the
range from carnivorous to herbivorous
fish, have shown that lipid is also
absorbed along the lower part of the
intestine (17,28,67,75,89,121,153-
154,159), the digestive/absorptive
functions generally diminish as the
feaces progresses towards the rectum
(9,160), see also Table 2.

Table 2. Lipid digestibility in various parts of the gastrointestinal tract of various fish
species*.
_________________________________________________________________________
Fish S PC AN PS R/F REF
_________________________________________________________________________
Salvelinus alpinus 0 70 81 88 - 121
Oncorhynchus mykiss 9 48 76 86 90 152
Gadus morhua - 46 83 85 88 27
Hippoglossus hippoglossus -15 - 72 88 96 153
Heterotis niloticus - 43** 68 78 - 154***
_________________________________________________________________________
S=stomach; PC=pyloric caecae; AN=anterior intestine; PS=posterior intestine; R/F=rectum/faeces.*
The intestinal segments were not divided into sections in the same manners by the various authors.
Consequently the naming of various sections are approximations and may not be completely correct.
** No pylorus caecae present, data are from fore-gut, mid-gut and hind-gut. *** Digestibility was
calculated on basis of the chemical composition of stomach content

There does however seem to be certain fossilis) compared to Ophiocephalus

interesting differences between various
fish species. Although more
comparative studies are warranted, to
avoid differences due to analytical
errors, some of the differences are
outlined in the following text. In
Scophthalmus maximus, a species
having a short digestive tract and
lacking a functional pylorus, Koven et
al. (77,83), suggested that the hind-
gut/rectum region was a major site for
lipid absorption. In a comparative
study, Sastry and Garg (159) observed
that the posterior intestine was more
actively absorbing dietary lipid in
stinging catfish (Heteropneustes
(Channa) punctatus where most of the
lipid was assimilated in the proximal
region. In other species like Chanos
chanos that have both pyloric caecae
and a long digestive tract (67) or Gadus
morhua (75,89), Oncorhynchus mykiss
(152) and Salvelinus alpinus (121) that
have well developed pylorus caecae,
lipid absorption is always lower at the
posterior end of the intestine.
Lipid absorption in the lower segments
of the intestine may be important under
certain dietary conditions such as after
high dietary lipid input (159). In Gadus
morhua, Lied & Lambertsen (89) found
that lipid digestion in the lower part of
 Olsen and Ringø: Lipid digestibility in fish: a review
the intestine was significant only when
the fish were fed whole fish pointing to
a possible difference between feeding
studies using easily digestible
dry/moist pelleted diets and natural
conditions where the rate of digestion
may be highly different.
Although the rate of hydrolysis of WE
are slower than TAG, it is generally
assumed that they are extensively
assimilated (13,97,132,141). This may
be due to an evolutionary strategy
where diets are retained in the intestine
allowing more time for hydrolysis
thereby increasing total absorption
efficiency. For example in Engraulis
mordax, 80% of a dietary dose of WE
was recovered in the gut 24 hours after
feeding, compared to only 0,2% in the
stomachless Thalossoma duperreyi,
indicating that most of the WE was
voided before assimilation could take
place (13). Sargent et al. (132) found
very high rates of assimilation (>95%)
of dietary WE when Clupea harengus
and Oncorhynchus mykiss were fed
frozen zooplankton containing WE.
Assimilation of both WE and free fatty
alcohols were much higher in Clupea
harengus however. Fatty alcohols
released during the hydrolysis of wax
esters are oxidised to fatty acids
(13,97,133,141,161-163) prior to
incorporation into fish lipids. The
accumulation of free fatty alcohols in
faeces of fish species like Gadus
morhua (164), sprat, Chaparrudo
flavescens (134) and Oncorhynchus
mykiss (132) however indicate that
absorption or oxidation may not be as
efficient as for fatty acids. In Clupea
harengus however, Sargent et al. (132)
found a very low free fatty alcohol/free
fatty acid ratio, indicating an efficient
rate of assimilation in this species.
Phospholipids are extensively digested
and utilised in fish and have been
reported to improve growth in larval
20
fish (165-166). In Cyprinus carpio,
Iijima et al. (81) found that
phosphatidylcholine was more readily
absorbed than TAG. In Salvelinus
alpinus, lipid digestibility was 87% and
98% when the fish were fed a
commercial diet and a Mallosus
villosus roe diet respectively (167).
Although these diets are not directly
comparable, it is interesting to note that
Mallosus villosus roe contained 45%
polar lipids compared to 7% for the
commercial diet, and virtually no polar
lipid was found in the gut contents.
This obviously occurs despite the lack
of, or low activity of, appropriate
luminal phospholipases. This has led to
speculations towards the existence of
an absorptive pathway without the
requirement for hydrolysis (possibly
pinocytosis) of the more easily
emulsified phospholipids in larvae of
Scophthalmus maximus (168) and
Gadus morhua (164). Direct absorption
of lipid, e.g. through pinocytosis was
debated in the 1960-70’ties with some
enthusiasm in both mammals (169-170)
and fish (28,159) mostly through
histological approaches. These
questions were never completely
resolved however, and although the
possibility of a direct absorption of
phospholipids is still an intriguing idea
(157,160) it is nonetheless generally
accepted that hydrolysis prior to
absorption is the major pathway in fish.
It is however interesting to note that
that phospholipids with polar head
groups may cross the enteric brush
border in mammals (171) in certain
circumstances, and that the
supranuclear inclusions found in some
fish species are the result of pinocytosis
of proteins that also contain lipid (172).
These observations could indicate at
least some phospholipids may be
absorbed intact, possibly through some
 Olsen and Ringø: Lipid digestibility in fish: a review 21

kind of interaction between lipid and is already completed (mechanical

protein.
5. FACTORS AFFECTING LIPID
DIGESTIBILITY
It is generally accepted that the design
of the gastrointestinal tract of fish is to
optimise the utilisation of nutrients in
relation to the feeding habitat (see
chapter 2). Of particular importance
appears to be the primary digestion or
grinding by the stomach or a gizzard
etc. Feeding non-optimal diets may
accordingly influence nutrient
digestibility. For example using the
Asian cichlid Etroplus suratensis, De
Silva & Perera (173) found lower
digestibility of protein (59-75%) and
perhaps also lipid (65-70%) than
expected when the fish were fed the
aquatic macrophyte Hydrilla
verticellata. The authors explained this
by the fact that the Etroplus suratensis
used was predominantly a molluscan
feeder with the characteristic
pharyngeal teeth, and a short intestinal
length compared to herbivores.
Consequently short intestinal length
and incomplete rupture of diet could
have caused the observed effects. Most
feeding studies however have been
performed using artificial diets where
the primary decomposition of the diet
blending, rupture, extruding etc.). This
does in many ways partially substitute
for the primary digestion, and one
would expect that nutrient utilisation
would increase, or that species
differences would not be as pronounced
as they are in nature. However, feeding
large amounts of small pelleted diets
may paradoxically overload digestive
capacity of the fish. This theory termed
gastrointestinal overload (174) is
explained by a very rapid emptying of
the stomach leading to intestinal
overload and reduced nutrient
absorption. The theory was based on
fish with stomachs, but could certainly
apply to stomachless fish as well.
Unfortunately, most feeding studies
have been concerned with the
digestibility of protein, energy or dry
matter that only indirectly at best
describes lipid digestibility.
Consequently, although we in the
present section have tried to keep
stringent to factors affecting lipid
digestibility, some references are also
given to results obtained with other
dietary nutrients. Some of the lipid
digestibility data presented in this
chapter is given in Table 3.

Table 3. Lipid digestibility, fish weight, temperature and variable (if any) of some of
the studies mentioned in this article.
__________________________________________________________________________________
Weight Temp. Lipid Signif-
Species (grams) (oC) Variable digest(%) icance Ref
Fresh water
Oncorhynchus mykiss 135-162 17 protein 96-98 no 175
500-900 11 - 91 158
80 3 lipid 46-91 yes 176
80 11 lipid 49-89 yes 176
150-170 14 - 75 - 177
54-78 18 lipid 14-96 yes 178
 Olsen and Ringø: Lipid digestibility in fish: a review 22

16 11-14 lipid 94-98 no 179

150 15 lipid 90 no 180
45-70 14-16 protein 89-93 yes 181
200 7-15 - 85-89 - 182
586 7-15 - 91-93 - 182
170-190 8 carbohydrate 85-88 no 183
170-190 18 carbohydrate 94-95 no 183
Salvelinus alpinus 50-150 7 lipid 50-97 yes 184
400 8 - 87 - 167
400 8 - 98 - 167
210 8 lipid 78-82 yes 185
150 10 lipid 87-90 no 121
Salmo salar 75 9 - 76-90 yes 186
O. niloticus/O. mossambicus 10-35 26 lipid 32-84 yes 187
10-35 26 protein 32-84 yes 187
O. niloticus/ O. aureus 500 26 carbohydrate 92-98 no 188
O. aureus 4.6-6 26 protein 93-95 no 189
4.6-6 26 carbohydrate 92-95 no 189
Pagrus major 44-46 18-20 lipid* 77-91 yes 190
Cyprinus carpio 32-36 12 lipid 34-91 yes 178
32-36 24 lipid 31-94 yes 178
15-17 28 lipid 40-89 yes 178
Acipenser baeri 116-118 18 lipid 68-90 yes 191
Ictalurus punctatus 140-160 23 lipid 72-64 no 192
140-160 28 lipid 94-76 yes 192

Sea water
Oncorhynchus mykiss 440 8-11 carbohydrate 78-82 no 193
Salmo salar 300 15 lipid 77-86 yes 117
Salvelinus alpinus 400 8 - 81 - 167
400 8 - 94 - 167
Gadus morhua 48-52 - lipid 67-96 yes 194
367-450 8 carbohydrate 85 no 195
1100-1800 - - 85-87 - 89
Hippoglossus hippoglossus 600-1500 7-9 lipid 85-94 no 196
__________________________________________________________________________________
 Olsen and Ringø: Lipid digestibility in fish: a review
5.1. Determination of digestibility
Digestibility of nutrients is the
quantitative sum of the digestive
processes and can be determined
directly (the quantity ingested and
faecal matter voided are determined) or
indirectly (by use of an internal
indigestible marker). Unlike
comparable investigations on
terrestical animals, those in fish is
hampered by the difficulty of
collecting feces from the environment
in which they live. Several workers
have developed methods for the
collection of faecal matter for fish
digestion studies. Nose (197) dissected
the digestive tract into several parts,
Inaba and colleagues (198) stripped the
contents of the large intestine using
slight pressure until feces was naturally
expelled by the fish, Forster & Gabbott
(199) used a false bottom of plastic
mesh in the tank, Cho et al. (181) used
a group of caged fish in a feeding tank
during the day which were transferred
to a feces collection tank at night. In
contrast to these investigations, Smith
(200) employed a metabolism chamber
for fish, a direct method for total feces
cellection. However, as the collection
of fish feces alway presented specific
problems, Choubert et al. (201)
developed a sophisticated automatic
continous fecal collector.
Advantages and disadvantages
involved in the use of the direct - and
indirect methods have been the subject
of comprehensive reviews over the last
two decades, and the method of choice
is often a matter of opinion (7,9,202-
206). Among the internal and external
markers used in digestibility studies
(Table 4), there is no agreement as to
what is the better. Although the most
commonly used marker in fish and
animals is the indirect chromic oxide
(Cr2O3) method developed in 1918 by
Edin (223), some investigations have
23
raised serious doubts about its use
(213-218,224). A marker has to be
indigestible, should not interfere with
the physiology of digestion, should
move along the gut at the same rates as
the rest of the food material, should not
be absorbed into the fish, and should
not be toxic. However, these conditions
are not always met. Firstly, there is
some controversy as to whether the
time of collection of feces affects
digestibility estimations. According to
Knapka et al. (213) the excretion of
Cr2O3 in burros (Equus asinus), was
not always complete and varied
throughout the day. On the other hand,
De Silva et al. (203) did not find any
differences between digestibility
estimations based on fecal material
voided during the day or during the
night in Oreochromis aureus. Another
aspect which may affect nutrient
digestibility is the transit time of the
indicator through the digestive tract. In
an early study, Bowen (214) found
indications that Cr2O3 may move along
the gut of Cyprinus carpio at a rate
different from the rest of the food
material due to its higher density. This
may cause errors in digestibility
estimations. Later, De Silva &
Owoyemi (215) confirmed this finding
in Sarotherodon mossambicus, fry.
Using Sarotherodon niloticus fry at
three salinities and four artificial diets,
De Silva & Perera (224) clearly
showed that digestibility had daily
variations. The fish appeared to have
one or two days of high digestibility,
followed by a day of low digestibility.
Tacon & Rodrigues (216) compared
the apparent digestibility coefficients
of nutrients in Oncorhynchus mykiss
using Cr2O3 at three dietary
concentrations (0.5%, 1% and 2%), and
observed that nutrient digestibility
 Olsen and Ringø: Lipid digestibility in fish: a review 24

Table 4. Digestibility markers used in fish studies.

___________________________________________________________________
Marker References

Internal
Acid-insoluble ash 207
Ash 208
Cellulose 209
Crude fibre 210
Hydrolysis-resistant
organic matter 211
Long chain fatty acids 212
n-alkanes 184

External
Chromic oxide Numerous, e.g. 198,213-218
Metallic powder 219
Polyethylene 216
Radioisotopic 220-221
Titanium (IV) oxide 222
_________________________________________________________

was significantly higher at the 2% (Oreochromis niloticus*Oreochromis

inclusion level. With respect to prawns
and shrimps, Leavitt (217) raised
serious doubts about the use of Cr2O3
as marker, based on the same reasons
as that of earlier investigations. In a
comparative study using Salvelinus
alpinus, the nutrient digestion was
measured using n-alkanes, Cr2O3 and
celite as markers, but only small
differences in digestibility for a few
feedstuffs was estimated between the
different markers (184,225-226). On
the other hand, Ringø (218), working
with Salvelinus alpinus in fresh- and
seawater demonstrated that a where a = amount of nutrient in feces
supplement of 1% Cr2O3 significantly
affected dietary lipid during passage
through the digestive tract, and the
allochtonous and autochtonous
microbiota of the fish. Based on these
results the author concluded that the
use of Cr2O3 may led to errorous
results in nutrition studies of Salvelinus
alpinus. Similarly, in hybrid tilapia
aureus), increasing dietary chromic
oxide from 0,5% to 2% significantly
lowered nutrient and lipid
digestibilities (188). Inclusion time had
no influence on digestibility however.
The apparent digestibility coeffcient
(ADC) of a nutrient determined by a
external indicator is expressed by the
equation:
% Cr2O3 in feed a
ADC = 100 - 100 ⎯⎯⎯⎯⎯⎯ X ⎯
% Cr2O3 in feces b
and b = amount of nutrient in feed.
5.2. Dietary lipid content
Increasing the lipid content may
eventually overload the fish’s digestive
capacity. On the other hand, there is
evidence showing that high dietary
lipid may delay gastric evacuation
(227-228, see also 229), which would
 Olsen and Ringø: Lipid digestibility in fish: a review
give more time for the utilisation of
dietary lipid. Consequently, the fish are
met with two opposite effects when
dietary lipid level varies. There are also
seemingly species differences in the
tolerance for lipid contentns in diet. In
Gadus morhua, increasing dietary lipid
from 15% to 25% by dry weight had no
influence on lipid digestibility, while a
further increase to 39% reduced
digestibility from 96% to 67% (194).
This was probably due to overload of
the digestive system since undigested
TAG was found in gut lipids of fish fed
high lipid levels whereas only lipolytic
products were found in fish maintained
on lower lipid levels. Protein
digestibility was also reduced in this
group, which could indicate diarrhoea.
In catfish, Ictalurus punctatus,
Andrews et al. (192) found that
increasing the dietary lipid content
from 2.5% to 17.5% gradually
decreased digestibility. This study
however used mainly animal fat to
increase the dietary lipid content. This
may have influenced the results as
animal fat contain high amounts of
long chain saturated fatty acids that are
known to have relatively poor
digestibility. In Siberian sturgeon
(Acipenser baeri), lipid digestibility
was reduced from 90% to 68% when
dietary lipid was increased from 13%
and 23% (191). In contrast to these
results, Oncorhynchus mykiss and
Hippoglossus hippoglossus seem to
tolerate high levels of dietary lipid.
When Oncorhynchus mykiss were fed
marine fish oils, lipid digestibility was
94-98% at dietary lipid levels of 5-25%
of diet by dry weight (179). Choubert
et al. (180) and Refstie & Austreng
(230) came to a similar conclusion
when Oncorhynchus mykiss were fed
from 4 to 15% and 5 to 17% lipid by
dry weight respectively. Berge and
Storebakken (196) reported that lipid
25
digestion by Hippoglossus
hippoglossus was not significantly
affected by dietary level ranging from
18 to 38% of dry weight. There was
however a tendency towards increased
digestion (84.9% to 94.1%) when
dietary lipid increased from 18% to
31%. Protein digestibility was
approximately 85% regardless of diet.
In young red tilapia, which is a hybrid
between Oreochromis niloticus and
Oreochromis mossambicus, lipid
digestibility appeared to increase when
dietary lipid increased from 6% to 24%
(187). Lipid digestibility was generally
low however, in many cases below
50%, particularly at low dietary lipid
levels (187). A further interpretation of
the data is also difficult, as data on
dietary lipid source was missing as was
the method of lipid extraction. In eel,
Anguilla anguilla, Spannhof & Kühne
(231) found good correlation between
lipid digestibility and dietary lipid
content. At low lipid levels (7%), lipid
digestibility was 72%, while being
around 90% or more when lipid
content increased to between 20 and
30% of diet. The experimental diets did
involve the use of different fishmeal
mixtures, and could involve other
factors than lipid content such as fatty
acid composition or even peroxidation.
5.3. Influence of lipid composition
(melting point)
Some studies on the energy
requirements in fish have indicated that
animal fat, such as lard and tallow or
hydrogenated beef tallow can be used
as an energy source in fish diets once
the EFA requirements are met
(178,232-234). An early study from
1935 by McCay and Tunison (quoted
by 235), raised the question of possible
effect of the melting point on lipid
digestibility. They showed that lipid
digestibility increased when Salvelinus
 Olsen and Ringø: Lipid digestibility in fish: a review
fontinalis were fed good quality oils
(84%-94%) with low melting points
compared to hydrogenated oils (56%-
78%). Later Takeuchi et al. (178)
evaluated the lipid digestibility in
Cyprinus carpio and Oncorhynchus
mykiss fed various lipids with different
melting points. In general for both fish
species, lipid digestibility increased as
the melting points decreased (Table 5).
Hydrogenated oils with melting point
of 53oC generally had less than 35%
lipid digestibility. On the other hand,
beef tallow and hydrogenated fish oil
with melting points of 38oC were
efficiently utilised (> 70%) indicating
that they could be used as energy
source without having any adverse
effect. Hydrogenated fish oils of
melting point 53oC had 48% saturated
fatty acids of chain length C18 or more
compared to 15% and 19% for
hydrogenated fish oil of melting point
38 and beef tallow respectively. Good
quality fish oils were digested by
around 90% or more. These data
basically agree with that of Austreng et
al. (176) who fed hydrogenated capelin
oil with various melting points to both
mink and Oncorhynchus mykiss. They
also observed a progressive decrease in
lipid digestibility from 85% to 49%
with melting points increasing from
26
21oC to 41oC (Table 5). Furthermore,
the depression in lipid digestibility
became apparent at about the same
time in mink fed similar oils. Based on
these results, Austreng and colleges
suggested that the fatty acid
composition rather than melting point
itself was the main factor governing
lipid digestibility (see chapter 5.4).
Unsaturated fish- and vegetable-oils
had high digestibility coefficients 85-
91%. This also seems to agree with
data of Olsen et al. (121) who found
that lipid digestibility of Salvelinus
alpinus was very high (87-90%) when
they were fed hydrogenated coconut oil
that was rich in medium chain fatty
acids. In Salvelinus alpinus, (184), and
Oncorhynchus mykiss (236-237), lipid
digestibility varied from 50% to 97%
and 61% to 97% respectively when the
fish were fed various meal products.
The lowest digestibility was always
observed with meat or plant meals and
highest with fishmeal. The fatty acid
compositions and melting point of the
diets were not given in these studies
however, limiting interpretation of the
data. However, it seems reasonable to
assume that both meat and plant meals
contained large amounts of saturated
fatty acids.
 Olsen and Ringø: Lipid digestibility in fish: a review 27

Table 5. Influence of lipid source on lipid digestibility (by dry weight) in some fish
species. Oils used before and after hydrogenation to various melting points are
indicated above the melting point.
⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯
Dietary Cyprinus Oncorhynchus Salvelinus
lipid carpioa mykissa mykissb fontinalisc
⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯
Regular lipid
Beef tallow 72
Soybean oil 89
Cod liver oil 87
Salmon oil 94
Cuttlefish liver oil 93

Fresh and hydrogenated oils

Cottonseed oil 90
Capelin oil 85
Pollock liver oil 91 96
Hydrogenated oil m. p. 21oC 75
33oC 70
38oC 72 72
41oC 49
o
43 C 49 78(56)*
45oC 37 53
53oC 34 15
⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯
m.p - melting point,
a
(178), Cyprinus carpio fed at 12oC and 32-36g and Oncorhynchus mykiss at 17.5oC and 54-
78g.
b
(176), fed at 11oC and 80g,
c
McCay and Tunison (1935) as quoted by (235).
* The authors found 78% lipid digestibility in fish fed 25% lipid of diet, and 56% in those fed
7% lipid of diet.saturated fatty acids.

5.4. Fatty acid digestibility (75,79,83,89,117,130, 238). When

The digestibility of the individual fatty
acids in fish is highly dependent on
both chain length and unsaturation
(Table 6). In general, fatty acid
digestibility decreases with increasing
chain length, and increases with
unsaturation (89,117,153,167,176-
177,185), sometimes causing long
chain saturated fatty acids (SAT) and
monounsaturated fatty acids (MONO),
especially 22:1n-11 to accumulate in
the undigested fecal lipid fraction
administered as FFA, the digestibility
is known to be: PUFA > MONO >
SAT, and short chain > long chain
(117,121). In many cases PUFA may
be virtually eliminated from faeces
lipids at such rates that selective
absorption has been suggested
(83,121,238). Lower absorption of long
chain MONO and in particular SAT
may be explained by lower polarity of
these fatty acids (46). As mentioned
above (Chapter 5.3), there also appears
 Olsen and Ringø: Lipid digestibility in fish: a review 28

to be some relationship between > 20:1n-9(24oC) > 22:1n-9(34oC).

melting point of the individual fatty
acids and their digestibility, apparently
decreasing with increasing melting
point (89,117,176). In one study, Olsen
et al. (121) found that lipid digestibility
was very high in Salvelinus alpinus
when fed partially hydrogenated
coconut oil supplemented with certain
fatty acids. The rate of utilisation of the
supplemented fatty acids could be
explained by their melting points
being; 18:3n-3(-11oC) ≥ 18:2n-6(-5oC)
However, the digestibility of 12:0 was
very high (> 90%) despite having a
melting point of 44oC. This clearly
shows that melting point is only one of
many factors influencing lipid
digestibility. For example, high
digestibility of 12:0 may be due to the
fact that it is a medium chain fatty acid
(Chapter 4.1) that is well soluble in
water, and can diffuse through the
“unstirred water layer” without the aid
of bile salts.

Table 6. Digestibility of individual fatty acids (% of diet by dry weight) in diets for
various fish species.
⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯
Oncorhynchus Salmo Salvelinus
a b c d
mykiss mykiss mykiss salar alpinus alpinusf apinusg
e

⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯
Total lipid 87 75 86 86 87 90
Fatty acids
10:0 98
12:0 96 967
14:0 90 92 87 78 86 86 88
16:0 80 87 78 65 81 80 84
18:0 74 78 66 52 71 77 76
20:0 59* 69
22:0 100**
16:1 88 97 89 94 96 89 96
18:1 86 93 93 91.5 91 87 92
20:1 93 96 85 85 88 88
22:1 98 95 89 68 77 70
18:2n-6 97 95 91 82 87 98
18:3n-3 94 91 94 90 93 99
20:5n-3 100 97 93 97 97 95
22:6n-3 100 97 88 91 94 92
⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯
a
(178), Fish (80g) fed cod liver oil.
b
(177) Fish (150-170g) fed commercial diet.
c
From Nose (1967) as quoted by (249).
d
(117). Fish (300g) fed menhaden oil.
e
(185) Fish (210g) fed commercial diet.
f
(167) Fish 400g) fed commercial diet
g
Data are calculated from (121) not presented in the original paper.
*, coeluted with 18:2n-6, ** probably misidentification with 18:4n-3 (see 75)
Consequently, the digestibility of these very high. This is indeed the case in
fatty acids would be expected to be mammals like pig where the
 Olsen and Ringø: Lipid digestibility in fish: a review
digestibility of coconut oil, which
contains high amounts of 12:0 was
found to be better than beef tallow and
corn oil which contained mostly C16-
C18 fatty acid (239). It would not be
unlikely if a similar situation applies to
fish.
5.5. Other lipids
Wax esters and free alcohols
containing particularly 22:1 seem to be
relatively poorly utilised compared to
fatty acids and WE containing e.g. 16:0
(132). This may indicate that such lipid
accumulate in faeces and eventually
precipitate in sediments. Indeed WE
high in 22:1 alcohols have been
identified in a North Sea Oil slick
(240) and floating faecal pellets in an
enclosure with Gadus morhua
juveniles fed marine zooplankton
(164). In Salmo salar, digestibility of
ethyl esters was significantly lower
than that of free fatty acids and TAG
(117) which could indicate lower
specificity of lipolytic enzymes.
A recent study with Salvelinus alpinus
demonstrated that inclusion of 2.5%
18:2n-6 methyl ester into a commercial
diet significantly lowered both growth
rate and total lipid digestibility. This
was due to decreased feed intake and
reduced total lipid digestibility.
Decreases in the digestibility of 16:0,
18:0, 20:1 and 22:1 was also observed.
However, no effect was seen on the
utilisation of the n-3 PUFA (18:3, 18:4,
20:5 and 22:6) (185). The higher
digestibility coefficient for 18:2n-6
when the char were fed the commercial
diet supplemented with 18:2n-6 methyl
ester compared to control, may be due
to higher absorption rate for 18:2n-6
methyl ester compared to 18:2n-6 of
TAG. Alternatively, the differences
may be due to the small amounts of
lipid which remain in the faeces when
the char were fed the control diet. The
29
digestibility coefficient found for
18:2n-6 when the char were fed the
18:2n-6 diet may therefore represent
the "true" value. The depressed
digestion of some dietary fatty acids
may be due to differences in intestinal
microbiota, as demonstrated by Ringø
(241).
5.6. Pylorus caceae
The function of the pyloric caecae in
fish remained a mystery for a long
time. The general assumption was that
they carried out fermentation as in
birds and mammals, a view originally
presented by Aristotele “and where to
putrify and concoct it”. This view was
challenged only in this century. In his
classic work form 1913, Greene (156)
stated that fat absorption takes place
abundantly in the pyloric cæca in King
salmon (Oncorhynchus tschawytscha)
indicating the function of these
structures in lipid digestibility. Later
studies showed that the pyloric caecae
were rich sources of several digestive
enzymes (20,69,71,242-248) indicating
that they could function in the
digestion of nutrients. In 1975, Patton
and coworkers (13,42) suggested that
the pyloric caecae also functioned as to
insure retention and provide an
increased surface area for food
absorption, particularly high lipid diets.
The same year Bergot et al. (41)
showed that this was indeed the case in
Oncorhynchus mykiss, where the
pyloric caecae increased the serosal
surface area of the small intestine by
more than 3.2 times, and over 2 times
the total intestine. Twelve years later,
in 1987, Buddington & Diamind (20)
confirmed this study on Oncorhynchus
mykiss, and showed that similar
situations applied to other fish species
as well, including Gadus morhua.
A natural consequence of these
observations, is that increased number
 Olsen and Ringø: Lipid digestibility in fish: a review
of pyloric caecae would increase
nutrient digestibility and consequently
fish growth. An excellent experimental
fish for studying this hypothesis is
Oncorhynchus mykiss. As opposed to
fish like Salvelinus malma (250),
Salvelinus namaychus (251) and
Salvelinus alpinus (252) where the
number of pyloric caecae increases
throughout the whole life, the number
of pyloric caecae in Oncorhynchus
mykiss appears to be precociously fixed
(253) although they may vary from 28
to 145 between various families (41).
The hypothesis was forwarded already
in 1975 by Bergot and coworkers
(41,254), but evidence for such an
effect have been scarce and
inconsistent. Using families of
Oncorhynchus mykiss with varying
number of pyloric caecae, Bergot et al.
(255) and later de la Noüe et al. (256)
found some effects on food conversion
and the absorption of amino acids and
carbohydrates. However, no
differences were oberved in protein-
(256-257), lipid- (180,257) and energy-
digestibilities (256). This lack of effect
may be attributed to the observation
that the pylorus cacea size change with
their numbers, if they are short they are
numerous, if they are long they are few
(41,254,257). As a natural
consequence, the total surface area is
likely to be similar.
5.7. Size of the fish
In general, the digestive tract of newly
hatched fish larvae deviates
significantly from that of adult fish
being a straight tube considerably
shorter and structurally less developed
(9,14,258-265). The first time post
hatch, the larvae rely exclusively on
yolk sac for nutrients. The transition to
exogenous feeding following yolk sac
absorption is considered critical for
30
survival of the fish larvae. According
to Smith (9), there appear to be two
major strategies to deal with these
start-up problems. Fish with sinking
eggs and large amounts of yolk
material produce large and well
developed larvae with relatively
complete and functional digestive
tracts capable of feeding on relatively
large particles. Fish with small,
floating eggs and little yolk material,
however, start feeding with minimal
digestive capability. Fish with less
developed digestive tracts often lack a
fully functional stomach at absorption
of the yolk sac (14,258,261,263-266).
Until fully developed these fish rely
more on mechanical digestion, if
possible, and digestive enzymes for the
assimilation of nutrients. Furthermore,
the gastric evacuation rate may be
significantly shorter compared to adults
(258,267) which further limit the
assimilation efficiency. Consequently,
direct feeding on artificial diets to fish
larvae with less developed digestive
system have, except for some
freshwater species (268-271), been met
with limited success (259). For
example, Kanazawa et al. (272) fed
nylon-protein microcapsules to newly
hatched larvae of red sea bream
(Chrysophrys (Pagrus) major), and
Plecoglossus altivelis, but found them
to be poorly digested and scarcely
supported growth. Similarly, all protein
microcapsules fed to larvae of Lates
calcarifer were not broken down (273).
Live food however, such as rotifer
(Brachionus plicatilis) and arterial
nauplii (273-275) have all shown to
support growth of several fish species.
These and similar observations have
led to the suggestion that lack of
growth and survival in fish with poorly
developed digestive tracts can be due
to deficiencies in digestive capacities
(135,259,276).
 Olsen and Ringø: Lipid digestibility in fish: a review
As noted earlier, the rate of lipid
digestibility in fish is slow. In larvae,
the time allowed for lipid digestion is
likely to be further reduced.
Furthermore, there are indications that
the activities of several digestive
enzymes may be low at first feeding
(258,277-279), which may further
restrict nutrient assimilation. Lack of
intestinal lipase activity has been noted
in early larvae of both Scophthalmus
maximus (280), and Esox lucius (281).
In Morone saxatilis, Ozkizilcik et al.
(119) found that the lipase activity of
whole larvae was low until initial
feeding when the lipase activity
increased 18-fold during the next 13
days. It was however notable that the
increase in lipase activity became
notable only after 5 days of feeding,
leaving this critical period with low
lipolytic activity. In Chrysophrys
(Pagrus) major, phospholipase A2
activity appeared in the epithelial
crypts of the pyloric caecae only after
85 days post hatch (86). Furthermore,
although esterase was found in
intestinal segments of Chanos chanos
larvae at first feeding (14), activity in
the stomach was only found after
metamorphosis (60 days). This could
indicate that these larvae do not have
the digestive capacity to digest large
amounts of dietary lipid. The success
of live prey may in this aspect be due
to the additional supplementation of
prey enzymes aiding the digestion
(135,168,266,277,282). The poor
growth of Morone saxatilis larvae
(271), and Menidia menidia (see 283)
fed freeze-dried brine shrimp compared
to live shrimp, and Clarias gariepinus
(284) fed frozen brine shrimp
compared to decapsulated cysts may be
explained by damage to the digestive
enzymes. Furthermore, in larvae of
Plecoglossus altivelis, Chrysophrys
(Pagrus) major) and Paralichthys
31
olivaceus, (272,285-286), micro-bound
diets have been successfully applied on
10 day old larvae, particularly when
fed in combination with smaller
amounts of live food. In Sparus
auratus (285) and Sciaenops ocellata
(287), successful combinations have
been obtained from first feeding. In
Dicentrarchus labrax, histological
examination showed that all-protein
bound microcapsules were broken
down in the intestine when rotifers
were present (273). On the other hand,
feeding diets supplemented with
digestive enzymes have generally been
met with little or no success
(168,259,276).
The existence of some unknown
substances is an alternative theory and
cannot be excluded (259,288-291).
Various «unknown» substances or
factors supplied by live prey have been
suggested, including stimulation of
endogenous protease secretion (292-
294), and the requirement of free
amino acids for energy (295-296). One
major and often overlooked difference
between artificial diets and live food
however, is the lipid class composition.
In most artificial diets, TAG is the
main lipid source, while live diets, like
the commonly used brine shrimp
contain significantly amounts of polar
lipids as well (297). In nature similar
situations may occur. When Gadus
morhua juveniles were fed in an
enclosure system, their fatty acid
composition closely resembled that of
the polar lipids of the predominant prey
organism, Calanus finmarchicus (164).
Furthermore, the development of
zooplankton from nauplii to copepod
stages closely followed that of Gadus
morhua larval development. Prior to
metamorphosis, more than 60% of
zooplankton lipids were composed of
polar lipid classes. By the completion
of the alimentary tract at approximately
 Olsen and Ringø: Lipid digestibility in fish: a review
50 days or 40 mm (261), a good
digestive capacity is expected. At this
time higher copepodit stages (>IV)
were predominating, and the total
content of polar lipids were reduced to
42%. Furthermore, prior to
metamorphosis, large amounts of
faeces containing undigested wax
esters were found floating in the
enclosure. This faeces was devoid of
polar lipids. The faeces disappeared
around metamorphosis. Requirements
for phospholipids for growth have also
been reported for several larval fish
species such as Chrysophrys (Pagrus)
major (272,298), Plecoglossus altivelis
(299-300) and knife jaw (Oplegnathus
fasciatus) (298). The exact
mechanisms for these effects remains
to be elucidated. Limited ability of
synthesising phospholipids by fast
growing larvae has been suggested
(166). Considering the amphiatic
nature of phospholipids, Olsen et al.
(164) suggested that polar lipids could
emulsify even in the absence of bile
acids, and subsequently be absorbed by
pinocytosis, a major pathway for
absorption of other macromolecules in
fish larvae (258,264,301). This
mechanism would also bypass low
lipase activity. Koven et al. (168)
observed that absorption of
radiolabelled oleic acid (18:1n-9) as
free fatty acid improved upon feeding
with soybean lecithin to Sparus aurata
larvae, and seems to support the
emulsifying theory. See also Chapter
4.2.
Fish larvae with reasonably well
developed digestive tract are known to
accept and survive on artificial feeds
composed of the same or similar
ingredients as adults, immediately after
yolk-sac absorption (74,302-304). This
shows that the digestive capacity may
not deviate to any major extent from
that of larger fish. However, low levels
32
of intestinal lipases have been observed
in Acipenser transmontanus, followed
by an increase towards transformation
and then decrease again (10,73-74). It
is also known that the supplementation
of phospholipids may have a major
influence on growth rate of small fish.
In Salmo salar and Oncorhynchus
mykiss fry, Poston (305-306) showed
that supplementation of 4%
phosphatidylcholine increased growth
and feed conversion. In larger fish
(1,7g or larger), the effect was not
apparent which could indicate that the
digestive system was fully capable of
utilising the diets.
The influence of size on lipid
digestibility in young and adult fish is
less certain. Assays on lipolytic
activity have been met with
contradictory results. In Cyprinus
carpio, the esterase activity increased
in the intestine between 35g and 382g
while the activity decreased in the
pyloric caecae of Seriola
quinqueradiata with size (22-938g),
while it changed little in intestine of
Oncorhynchus mykiss and Anguilla
japonica when analysed at sizes 12-
908g and 26-187g respectively (136).
In Ctenopharyngodon idella, Das &
Tripathi (80) found higher lipase
activity in adult fish than in fingerlings.
However, experimental conditions
were different so a direct comparison is
not possible. Similarly, the influence of
size on lipid digestibility is
inconclusive. In their study on
Cyprinus carpio and Oncorhynchus
mykiss fed, Takeuchi et al. (178) found
that digestibility of hydrogenated oils
with high melting points (53oC) was
significantly lower in fish weighing
less than 10 grams. Cho & Slinger
(236) found no difference in lipid
digestibility in Oncorhynchus mykiss
ranging from 37g to 113g. In Heterotis
niloticus, fed natural diets, lipid
 Olsen and Ringø: Lipid digestibility in fish: a review
digestibility increased progressively
from 72% in fish of around 100-300g
to 87% in fish between 1000-2000g of
weight (154). Further increases to
3000g in weight seemed to decrease
digestibility somewhat (78%). These
data were however obtained from fish
fed natural diets, and could also reflect
differences in diet and its chemical
composition. Furthermore, the lipid
digestibility was calculated as the
difference between faeces and stomach
contents which could include major
analytical errors.
5.8. Feed intake
From the point of view of applied fish
nutrition, is essential whether or not the
amount of feed ingested affects
nutrient digestibility. Data on this
matter are often inconclusive and
somethimes contradictory however. In
an early work, Gerking (307) showed
that meal size of Tenebrio molitor
larvae (meal worms), had little or no
effect on the assimilation efficiency in
Lepomis macrochirus weighing around
30g at 24.6oC. In accordance with this
observation, Bondi et al. (308)
demonstrated that nutrient digestibility
by Cyprinus carpio, did not
significantly change with increases in
the amount of food eaten (from 2 to 6
g/fish/day). There was however a
tendency towards increased
digestibility coefficients of protein,
lipids and fibres at the highest feeding
level. Likewise, in 1972 Beamish (309)
showed that meal size had no influence
on the digestibility coefficients in
largemouth bass. Other investigations
have, however, showed that increased
food intake in Oncorhynchus mykiss is
correlated with decreases in the
apparent digestibility coefficients
(ADC) (310-316). These results may
be explained by gastrointestinal
overload leading to reduced nutrient
33
absorption (Chapter 5). However,
reductions in ADC does not necessarily
include reductions in the digestibility
of all nutrients. Using Oncorhynchus
mykiss, Windell and colleagues (182)
found that increases in meal sizes from
0.4% to 1.6% of body weight /day
decreased the digestibilities of dry
matter and carbohydrate, but had no
effect on the digestibility of lipid and
protein. Henken et al. (312) on the
other hand found that the apparent
digestibilities of protein and dry matter
by Clarias gairiepinus was negatively
correlated with feeding level. The
digestibility of lipid was not studied
however. Information regarding the
influence of feeding frequency on
nutrient digestibility is scarce. Hudon
& de la Noüe (317) found no
difference in apparent digestibility of
dry matter, protein and energy when
feeding frequency of Oncorhynchus
mykiss was increased from two to six
times per day. Similarly, in
Hippoglossus hippoglossus, Strømsnes
(153) found no influence on lipid
digestibility when the fish were fed 1
or 3 times a week.
5.9. Temperature
Contrary to endotherms, where
enzymes are active in a more or less
constant temperature, the digestive
enzymes of poikilotherms organisms
(ectotherms) such as fishes are affected
by external temperature (318-321).
Increasing the temperature may also
increase enzyme secretions (137,322),
influence the rate of absorption of
nutrients through the intestinal wall
(323-325), or improve the enzyme-
substrate affinity (326). However, the
studies conducted on the influence of
temperature (acclimatized to different
temperatures or a sudden rise in
 Olsen and Ringø: Lipid digestibility in fish: a review
temperature) on nutrient digestibility
have showed conflicting results.
Cyprinus carpio. Shcherbina &
Kazlauskene (327) reported that the
digestibility of lipid by increased from
79% to 86% when the temperature
increased from 16 to 26oC. In contrast
they found a decrease in the
digestibilities of both protein and crude
cellulose. Takeuchi and coauthors
(178) also found that lipid digestibility
increased with temperature in when the
fish were fed beef tallow or
hydrogenated fish oils (Table 5).
Digestibility of pollock liver oil on the
other hand was high and similar (c.
90%) at all temperatures. This shows
the importance of melting point on the
lipid digestibility by Cyprinus carpio.
Oncorhynchus mykiss. In
Oncorhynchus mykiss, Atherton &
Aitken (328) showed that fat
digestibility increased with increasing
water temperature. Windell and
colleagues (182) on the other hand
found little influence on nutrient
utilisation when digestibilities were
compared using three water
temperatures (7oC, 11oC, 15oC) and
three fish sizes (18g, 207g, 586g). The
only effect seen, was that small fish
(18g) had reduced digestibilities of
protein, lipid and carbohydrate when
acclimated to 7oC compared to the
other fish. Brauge et al. (183) also
observed that the digestibility
coefficients of all major nutrients,
including lipid and energy, increased
when the fish were reared at 18oC
compared to 8oC. Oliva-Teles &
Rodrigues (329) demonstrated
significant improvement of the ADC of
dry matter, protein and energy by
increasing water temperature from
14.9oC to 21.8oC. In contrast to these
results, Cho & Slinger (236) showed
that the nutrients digestibility did not
vary within the temperature range 9oC
34
to 15oC. There was however a marginal
increase (ca. 2.5 %), in the digestibility
of dry matter, energy, crude fat and
crude protein when the temperature
increased to 18oC. This increase was
not significant however. Choubert et
al. (201,330) also demonstrated that
nutrient digestibility was somewhat
higher at 18oC than at 15oC, but no
statistical differences were observed in
their study. Austreng et al. (176)
showed that the digestibility of fat and
fatty acids was essentially the same
within the temperature range 3oC to
11oC. Similarly, Hudon (313) found no
effect of temperature on the lipid
digestibility following a drop in
temperature from 9.0oC to 6.3oC.
Ictalurus punctatus. Andrews et al.
(192) conducted a study on fish reared
at two environmental temperatures
(23oC and 28oC) and fed three various
lipid levels. They clearly demonstrated
a significant higher digestible energy
and apparent lipid absorbability at
28oC.
Dicentrarchus labrax. Santulli and
coworkers (331) examined the effect of
temperature on gastric evacuation rate
and the digestibility of lipid and found
an increase in gastric evacuation and a
reduction in lipid absorption at higher
temperatures (20oC and 25oC).
However, by lowering the rearing
temperature to 15oC led to a decrease
in the rate of gastric emptying with a
prolonged lipid absorption.
The rate of gastric emptying depends
on the species and probably many other
factors such as temperature, food type,
meal size, fish size and even method
and frequency of feeding. It is well-
known that gastric evacuation rate and
total transit rate are increased by
increasing temperature in several fish
species (3) including Oncorhynchus
mykiss (332) which in turn shorten the
 Olsen and Ringø: Lipid digestibility in fish: a review
exposure time of feed to digestive
enzymes at higher temperatures. On the
other hand, some studies have
demonstrated that enzyme activities
(proteases and amylase) are increased
with increasing temperature in Rutilus
rutilus (333-334). These two effects of
changes in water temperature are
opposed; the first might decrease
wheras the second might increase the
digestibility of food at high
temperature. Which factor that
predominates is probably dependent on
species, genetic stock, melting point
and fatty acid composition of dietary
lipid and several other factors not yet
established. For example in
Dicentrarchus labrax, gastric
evacuation does not seem to be
compensated by increased efficiency of
nutrient utilisation, while this seems to
be the case for Ictalurus punctatus.
However, relatively few studies have
been performed to date and more
information is warranted. Of particular
interest would be more comparative
studies.
An interestin hypothesis regarding the
influence of temperature on nutrient
digestibility was forwarded by Brett
(335) as an attempt to explain the
diurnal migration made by juveniles of
Oncorhynchus nerka in Lake Babine
(British Columbia, Canada) during the
summer. When this oligotrophic lake
has a strong termocline, fishes fed at
the surface in warm (12-15oC) water at
dawn and dusk, but descend into cooler
(4-6oC) water to digest their meal.
Brett had shown earlier that sockeye
salmon juveniles have increased
digestion efficiency at lower
temperatures and lower rations and
thus hypothesized that they were
maximizing their digestion
(assimilation efficiency) and growth by
making the vertical migration. Later,
Biette & Geen (336) tested this
35
hypothesis by feding under-yearling
sockeye salmon 14C-labeled Daphnia at
5.5oC and 15.8oC, and a cyclic
combination of the two temperatures.
The results of this study confirmed the
hypothesis of Brett as fish grew better
under cyclic than undet constant
temperature.
5.10. Salinity
The use of migratory fish in
comparative studies in order to
determine apparent digestibility of lipid
and dietary fatty acids in fish held in
freshwater and seawater is interesting,
because the intestine must function
differently in these two environments.
Fish drink little or no water in fresh
water (337-338), and water diffuses
inward across the gills. In contrast, in
sea water the situation is the reverse of
that in freshwater, as fish loose water
and ions by diffusion and, therefore,
the fish would gradually become more
dehydrated. To compensate for this, the
fish drink seawater (337-339).
Determination of apparent digestibility
in fish held in seawater might therefore
be complicated due to increased
drinking, as the ingested water may
increase the water content of the
digesta and alter its mechanical
processing by the gut.
Ringø (167) using Salvelinus alpinus
fed two different diets in freshwater (S
= 0 o/oo) and seawater (S = 35 o/oo ),
clearly demonstrated that apparent
digestibility of lipid and some of the
dietary fatty acids (16:0, 18:0, 16:1,
18:1, 18:2n-6, 20:5n-3 and 22:6n-3)
was significant lower in seawater-
adapted fish compared with freshwater-
adapted charr. One possible
explanation is lower absorption of
especially long chain saturated fatty
acids (SAT) and monounsaturated fatty
acids (MONO) due to the presence of
divalent cations in feces. Long chain
 Olsen and Ringø: Lipid digestibility in fish: a review
fatty acids are known to form insoluble
soaps (340), with the solubility being
inversely related to chain length and
degree of saturation. Freshwater
contains only 1% and 0.1% the amount
of calcium and magnesium respectively
of the concentrations found in
seawater. Consequently, relatively low
amounts would be expected to form in
the gastrointestinal tract of fish
maintained in freshwater. In Gadus
morhua, Lied & Lambertsen (89)
found high contents of long chain SAT
and MONO, particularly 14:0, 16:0,
18:0 and 22:1 in the soap fraction of
faeces which certainly would reduce
digestibility. Lie and coworkers (75)
also suggested that salts of SAT and
MONO form micelles which solubilize
and transport FFA and MAG to
absorption sites, and that these may not
be absorbed but redused repeatedly
until accumulating in feces. Lower
digestibility of SAT and MONO
compared to PUFA of the present study
is most likely due to lower polarity as
described above, or to lack of specific
transport mechanisms.
5.11. Starvation and seasonal
variations
It is well known that fish species such
as salmonids are exposed to differences
in food availability during the year, and
that only small amounts of dietary
components are available to maintain
growth and metabolism especially
during the cold winter months. This
implicate that a long period of
starvation may affect enzyme activity
and nutrient digestibility. It is well-
known that starvation generally
reduces the hydrolytic capacity of the
intestine (341) by reducing the activity
of the digestive enzymes (110,342). In
addition, a period of starvation may
lead to morphological changes of the
digestive tract. This may of cource
36
affect nutrient digestibility. According
to Windell (343) striking
morphological changes were especially
prominent in the pyloric caecae of
starved Lepomis macochirus after 7
days. The noticeable shrunken
condition became progressively
advanced in fish starved for 25 days,
and at that time a drop in the
digestibility of approximately 50%
occurred. To maximise digestive
efficiency following a period of
starvation, it has been proposed that
rate of gastric evacuation may be an
adaptive mechanism (344). Food
remaining in the digestive tract for a
longer period of time may allow for
more complete enzymatic degradation
resulting in more efficient digestion
and absorption.
In a recent study, Jobling and
coworkers (345) examined whether
apparent nutrient digestibilities of
various diets fed to Gadus morhua
showed seasonal changes. Digestibility
of both protein and energy were
significantly higher in June than
September, but no significant
differences in lipid digestibility were
found between sampling times.
However, to what extent the
differences in digestibility of protein
and energy were influenced by
differences in methods of faecal
collection, variable water temperature,
fish size or the use of ash as marker is
not known. On the other hand, some
differences in hydrolysis of wax esters
and triglyceride by Engraulis mordax
intestinal juice was found between fish
caught in February and October (42).
As less research is available on how
starvation and seasonal variations may
affect lipid digestibility this topic
should be further investigated as it may
be of importance to the commercial
aquaculture industry.
 Olsen and Ringø: Lipid digestibility in fish: a review
5.12. Dietary short organic acids
Short chain fatty acids (SCFA) are
defined as carboxylic acids having
from 1 to 5 karbonatoms (C1-C5). In
mammals several acids such as acetic-,
lactic-, propionic- and butyric-acid are
produced during fermentation of
carbohydrate by micro-organisms in
the colon. Under normal conditions,
these SCFA are rapidly absorbed by
the mucosa by passive diffusion (346-
347). Carbohydrate malabsorption
frequently results in increased net
production of organic acids and
possible acidification of colonic
contents. Acetic acid for example is
known to degenerate enterocytes and
increase ion permeability (348).
Dependent on the nature of acid, some
SCFA like acetate and butyrate may
increase calcium absorption in the
colon, while butyrate are known to
enhance sodium and water absorption
in rats (349). Dietary inclusions of
SCFA are known to influence the
intestinal microflora generally without
having any adverse effect on the
animal and have been used with some
success in reduction of the number of
Enterobacteriaceae in store diets and
the incidence of Salmonella infections
in pigs (350). This is controversial
however as Izat et al. (351) found no
clear tendency in broilers.
Little information is available from
studies in fish. Although nutritional
studies have been performed using
silages, these diets are not controlled
with regard to the content of SCFA. In
Salvelinus alpinus, Ringø (352)
demonstrated that 1% acetate improved
growth rate, and enhanced the
digestibility of crude lipid and in
particular the fatty acids 14:0, 16:0,
18:0, 18:1, 18:2n-6, 20:1 and 22:1.
Curiously, there were no improvement
of the digestibility of n-3
37
polyunsaturated fatty acids. In contrast,
substituting acetate for formate had no
influence on digestibility whatsoever.
In an earlier study, Ringø (79) found
that the supplementation of 1% Na-
lactate significantly increased growth
rate of Salvelinus alpinus. He also
found indications of improved water
absorption (diarrhoea preventing
effect) and increases in lipid
digestibility. On the other hand, using
1% Na-propionate had the opposite
effect, reducing growth rate and
probably depressed lipid digestibility.
In contrast to these results, it is
interesting to note that dietary lactate
does not increase growth rate and lipid
digestibility of Salmo salar (353-354)
which may be due to different rates of
gastric emptying. Based on these
studies, there is no clear single effect of
SCFA observed per se. Since the
influence is so markedly different, it
seems likely that a major part of the
effect of dietary SCFA such as lactate
in Salvelinus alpinus is due to their
influence on the intestinal microflora.
The hypothesis is controversial
however, and more research is required
on this topic.
5.13. Antibiotics
Antibiotics are frequently used in fish
culture, and attempts have been made
to determine whether antibiotics have
any detectable effect on nutrition and
organisms of the intestinal tract, in
addition to the supression of those
causing disease. If this is the case
antibiotics may well be a valuable tool
in obtaining information on the
nutritional significance of the intestinal
microbiota. Thus it has been shown in
endotherms that addition to the diet of
small doses of antimicrobial agents
affect the absorption of carbohydrates
(355), proteins (356) and lipid (357-
358). With respect to improved lipid
 Olsen and Ringø: Lipid digestibility in fish: a review
absorption in mammals, the effects
observed were attributed to the action
of antibiotics on the intestinal
microflora (357-358). It has been
suggested that the characteristics of
animals receiving antibiotics are
similar to those of germ-free animals,
particularly with regard to the
reduction in thickness of the small
intestine and the increase in the size of
the caecum (359). However, in a early
work of Scrimshaw et al. (360) the
authors suggest that antibiotics reduce
the wall thickness of the intestines and
facilitate the absorption of food.
Cravedi and coworkers (177)
demonstrated that administration of
chlor-amphenicol (CP), oxolinic acid
(OA) and oxytetracycline (OT),
effective against bacterial diseases, had
no significant influence on the lipid
digestibility in Oncorhynchus mykiss.
However, the authors showed
enhanced digestion of 20:1 and 22:1
when CP was given at 0.5% level, and
of 22:1 at a 5% level of OA. Moreover,
significantly improved digestibility of
18:2 (n-6) and 18:3 (n-3) was observed
when CP, OA and OT were given at
0.5% dose. Choubert and colleagues
(180) have reported the effect of
dietary fat level and antibiotics on the
apparent lipid digestibility in
Oncorhynchus mykiss. In their study, a
combination of two antibiotics,
Gentamycin (3.45 g/kg) a broad
spectrum antibiotics, and Flumequine
(0.8 g/kg) which is commonly used
against fish bacterial diseases were
used. Antibiotic supplementation of the
lipid rich diet (15%) enhanched the
apparent digestibility of lipid from
90% to 94%, but no such effect was
observed by feeding the fish the lipid
poor diet (4%). This effect may be
related to the intestinal microbiota, as
Lesel et al. (361) observed differences
in the microbial community between
38
the the rearing groups. This is a
controverdsial hypothesis which calls
for further investigations.
5.14. Bacterial contribution
Digestion involves enzymatic
hydrolysis of organic macro-molecules,
however it is easy to visualize that
slow passage of food through the
gastrointestinal tract rather slowly will
maximize digestion and absorption,
and perhaps even allow time for
bacterial activity to assist in the
digestion. It is well established that
microbial digestion plays an important
role in the digestive processes of
terrestrial animals (362-365). Fish, on
the other hand, have been inadequatly
studied in this respect, and the
controversial nature of this field have
lead to such statements as; as
salmonids are carnivorous fish,
microorganisms do not play an
important role in the digestion, the
small bacterial population in the trout
digestive tract have no effect on
digestion and microbial degradation of
nutrient in the digestive tract of
Oncorhynchus mykiss is not very
active. However, during the last two
decades, several studies have suggested
that intestinal microorganisms may
have a beneficial effect in the
nutritional processes in fish (366-376)
making the nutrients more available for
absorption.When discussing whether or
not intestinal bacteria can be an
alternative source of lipolytic enzymes,
it is surprising to notice that only some
information is available concerning
bacterial lipases. This may be of some
interest as the heterotrophic micro-
organisms in the large intestine to some
extent may utilize dietary lipid.
Digestive tract flora with respect to
lipolysis can theoretically act in two
different ways in the gastrointestinal
tract: (1) by contribution to triglyceride
 Olsen and Ringø: Lipid digestibility in fish: a review
lipolysis through bacterial action, and
(2) by changing pancreatic lipase
secretion or inactivating it by bacterial
proteases. Many bacterial lipases are
extracellular enzymes, and are
classified into three types according to
their specificity ; non-specific lipases,
1,3-specific lipases and fatty acid
specific lipases (377). The production
of bacterial lipases is influenced by
temperature, ratio of nitrogen to
carbon, inorganic salts and oxygen. In
general, bacterial lipase synthesis is
stimulated by lipids such as lard,
butter, olive oil and fatty acids (378).
Bacterial lipase activity is assayed
using 1% Tween 80 or 0.75%
tributyrin resulting in zones of clearing
in solid media (379-381). Ringø and
colleagues (382) demonstrated that
some bacterial genera recovered from
the epithelical mucosa in the foregut,
midgut and the hindgut regions of
Salvelinus alpinus degraded tributyrin.
Consequently, the authors suggested
that these bacterial genera
(Agrobacterium, Pseudomonas,
Brevibacterium, Microbacterium and
Staphylococcus) may contribute to
nutritional processes in Salvelinus
alpinus. Lipase activity has also been
reported in bacterial isolates from the
gastrointestinal tract of
Ctenopharyngodon idella (381).
However, the authors did not present
any data in which bacterial
genera/species lipase activity was
found.
An other aspect when discussing
whether or not the indigenous
microflora may contribute to the
nutritional processes in fish, especially
in fish larvae which undergoes
metamorphosis, is the phospholipase
activity in intestinal bacteria.
Hydrolysis of phospholipids is well
documented for several bacteria e.g.
Bacillus cerus (383), but description on
39
phospholipase activity in fish bacteria
is less studied. In a preliminary
investigation, several bacterial species
isolated from Scophthalmus maximus
larvae and adult salmonids were tested
for the presence of PLC-type enzyme
(Guddal & Ringø unpublished data).
PLC activities were detected in the
bacterial species (Vibrio campellii,
Vibrio alginolyticus and Vibrio
pelagius) isolated from Scophthalmus
maximus larvae, and the activity was
especially high in V. campellii. Further
experiments will be performed to
reveal if bacterial phospholipase
activity has any effect on early
development and health of fish larvae.
5.15. Other factors
In 1983, Luquet and (384) collegues
showed that the reduced growth rate of
Oncorhynchus mykiss by ingestion of
saturated hydrocarbons (n-paraffins,
pristane and dodecyclo-hexane) could
not be fully explained by the decrease
in food consumption. In addition, the
authors suggested that the significant
decrease of dry matter and lipid
digestibilities also contributed to the
reduced growth rate.
It is well known that binders are useful
in reducing the waste from wet and
moist pelleted fish feeds. In salmonid
diets, alginate and guar gum most
commonly used. However, both
binders are known to reduce protein
and lipid digestibility in Oncorhynchus
mykiss (385-386). The greatest
reduction in lipid digestibility (from
98.4% to 56.1%) was noted when the
Oncorhynchus mykiss were fed 8%
guar gum (385). The effect of alginate
on protein and lipid digestibility has
also been investigated in Dicentrarchus
labrax (387). This binder did not
significantly affect protein and lipid
digestibility when included in the diet
at 8% level. However, at 15%, it
 Olsen and Ringø: Lipid digestibility in fish: a review
caused a small but significant drop in
protein digestion and a highly
significant drop in lipid digestion. Agar
si a gum derived from seaweeds, and is
used as a gelling agent, and in aquatic
feeds as a binder. It is indigestible in
animals, and may be considered as a
dietary fiber. Feeding up to 10% of
dietary agar to hybrid tilapia, (O.
niloticus*O. aureus), had no influence
on lipid digestibility (189).
Interactions between carbohydrate and
lipid in nutritional studies have been
studied in some investigations,
however, variyng the dietary
carbohydrate content had no influence
on lipid digestibility in Oncorhynchus
mykiss neither in seawater (193) nor
freshwater (183,230). The same appear
to occurr in Gadus morhua, where lipid
digestibility was constant at 85% (6-
8% lipid dry/wt) with digestible
carbohydrate varying from 0-35%
(195). In Salmo salar, Arnesen and co-
workers (388) reported of a marginal
but significant increase in lipid
digestibility from 90% to 95% when
fish meal was partially exchanged with
untreated oat starch. The effect was
especially notable with inclusions from
0 to 15% oat starch (90% to 94%),
while no further improvement (95%)
was observed at higher levels up to
30% oat starch. No influence was
observed when 30% oat starch was
exchaged with heat treated maize meal
with lipid digestibility being around
92% to 96%. Using various oat
preparations (whole, groats, meal and
bran) of around 23% of diet for
Oncorhynchus mykiss, Arnesen &
Krogdahl (389) could not find any
influence on lipid digestibility, always
being high at around 95%. In hybrid
tilapia, Shiau & Liang (188) found no
major influence on lipid digestion
when glucose was substituted for
starch in the diet although there was a
40
tendency towards decreases in
digestibility with inclusion of glucose.
In eel, Anguilla anguilla, Spannhof &
Kühne (231) found no influence on
lipid digestibility when starch was
partially substituted for cellulose.
Increasing the starch content from from
0 to 35% caused a marginal and
nonsignificant decrease in lipid
digestibility from 92 to 87%. However
increasing starch content further to
50% of diet decreased lipid
digestibility to 75%.
Relatively few studies have evaluated
the influence of dietary protein on lipid
digestibility. In a recent study, it was
demonstrated that varying the dietary
protein content (15%, 20% and 30%)
had little influence on lipid digestibility
in red tilapia, (O.mossambicus * O.
niloticus) (187). There was, however, a
tendency towards increased lipid
digestibility at increasing protein
content. Partly replacement of fish
meal protein with soybean protein had
no influence on lipid digestibility
(390). In Oncorhynchus mykiss, Gomes
and co-workers (175) found that lipid
digestibility remained constant at 96-
98% when graded levels (0-45%) of
colzapro, a co-extruded product of
rapeseed and peas was used as partial
replacement of fishmeal. Similary, Cho
et al (181,390) found little difference
in lipid digestibilities when Clupea
harengus meal was partly replaced
with soybean meal for Oncorhynchus
mykiss. The authors did however find
indications that inclusion of small
amounts of fermentation by products
could marginally reduce lipid
digestibility (from 93% to 90%) (181).
In hybrid tilapia increasing the dietary
protein level from 24% to 35% had no
influence on lipid digestibility (189).
Lipid peroxidation may constitute a
significant problem in fish nutrition as
PUFA are very susceptible to lipid
 Olsen and Ringø: Lipid digestibility in fish: a review
peroxidation. In red sea bream
(Chrysophtrys major), Sakaguchi &
Hamaguchi (190) found a significant
decrease in lipid digestibility (from 91
% to 77 %) when the fish were fed
highly peroxidized pollock liver oil.
Oxidized lipid is very toxic, and can
influence numerous processes involved
in lipid digestion. For example,
peroxidized lipid is known to form
lipid complex that would not be readily
absorbed. Moreover, free radicals are
highly toxic compound that may attack
and damage membrane proteins,
enzymes, lipid etc.
During the last decades, some attention
has been focused on antinutritional
factors and their effect on nutrient
digestibility in fish. Inclusion of
purified soybean trypsin inhibitors in
diets for Oncorhynchus mykiss reduced
protein digestibility from 93 to 70%,
but had no influence on lipid
digestibility (96-99%) (391).
Furthermore, several soybean products
have been elvaluated on their effect on
nutrient digestibility (186), and nutrient
excretion (392-393). Feeding Salmo
salar varying levels of commercially
available soybean molasses, it was
shown that inclusion levels of 10% or
more depressed the digestibility of fat
and in particular the fatty acids 16:0,
20:1 and 22:1 (186). However, no
significant effect was observed on the
digestibility of 20:5n-3 and 22:6n-3.
According to van den Ingh et al. (394)
morphological changes occcured in the
distal intestine when Salmo salar were
fed soybean meal heat treated to
differing degrees and containing
varying levels of soybean lectin and
trypsin inhibitor. However, as lipid
digestion and absorption occur in the
pyloric caecae and this region was not
investigated with regard to
morphological changes, no conclusion
41
can be drawn and further investigations
are needed.
In addition to the factors described
above, there are several others that may
influence lipid digestibility, and
amongst them are day length
(photoperiod), stock density, parasitic
infection, and reptroductive syclus.
These are aspects that have been little
studied, probably because they have
been viewd as having a marginal
importance. However, this stratergy
may be a wrong assessment of the
situation.
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