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Morphological and histochemical study of intestine in wildand reared European eel (Anguilla anguilla L.)
S. Kuzir • E. Gjurcevic • S. Nejedli • B. Bazdaric •
Z. Kozaric
Received: 7 February 2011 / Accepted: 26 July 2011 / Published online: 5 August 2011
� Springer Science+Business Media B.V. 2011
Abstract Diet-related differences between the ratio
of intestine length to body size and the enzymatic
activity in the intestinal tract of wild and reared
European eel (Anguilla anguilla L.) were studied.
Compared with reared eel, wild eel showed signifi-
cantly shorter relative intestine length. For the
purpose of histochemical examination, different parts
(anterior, middle and posterior) of intestine proper
were used. Activities of non-specific esterase, alka-
line and acid phosphatase, and aminopeptidase were
examined in each segment. All enzymes were present
in the intestines of both wild and reared European eel.
Fish from both groups showed similar enzyme
distribution within the enterocytes, but distribution
and intensity of enzyme activity along the intestine
vary depending on the group. Generally, reared
European eel showed highest enzymatic activity and
wider distribution of enzymes throughout all parts of
the intestine. These results suggest that different diets
could be one of the reasons for observed changes.
Keywords European eel � Relative intestine length �Intestine enzymes � Enzyme histochemistry
Introduction
Gastrointestinal tract of fishes shows a marked
diversity in its morphology and function, and this is
in correlation with taxonomy and different feeding
habits (Clarke and Witcomb 1980; Buddington et al.
1997; Nachi et al. 1998; Hellberg and Bjerkas 2000).
As in other vertebrates, the ability of fish to utilize
ingested nutrients mainly depends on the presence of
appropriate enzymes in appropriate locations along the
lumen of the gastrointestinal tract. Diverse enzymes
involved in digestive and absorptive processes have
been reported in a variety of adult fish (Kuz’mina and
Gelman 1997; Cahu et al. 2000; Kozaric et al. 2004;
Kozaric et al. 2006). Distribution intensity of digestive
enzymes across the gastrointestinal tract varies with
feeding habits and intestinal morphology (Kolkovski
2001; Gawlicka et al. 2002). The final stage of
digestion in vertebrates is controlled by enterocytes
expressing the brush border enzymes such as disac-
charidase, alkaline phosphatase and transpeptidase
(Semenza 1986; Ferraris et al. 1992).
In Anguilla japonica, digestive protease activities
were studied by Chiu (2002), and in Anguilla
S. Kuzir (&) � S. Nejedli � Z. Kozaric
Department of Anatomy, Histology and Embryology,
Faculty of Veterinary Medicine, University of Zagreb,
Heinzelova 55, 10000 Zagreb, Croatia
e-mail: kuzir@vef.hr
E. Gjurcevic
Department of Biology and Pathology of Fish and Bees,
Faculty of Veterinary Medicine, University of Zagreb,
Heinzelova 55, 10000 Zagreb, Croatia
B. Bazdaric
DALMAR d. o. o. Obala kralja Petra Kresimira IV. 64,
23 211 Pakostane, Croatia
123
Fish Physiol Biochem (2012) 38:625–633
DOI 10.1007/s10695-011-9543-7
anguilla, pepsinogens and pepsins from stomach
were identified by Wu et al. (2009). Therefore, this
paper presents histochemical distribution and activity
level of four digestive enzymes (non-specific ester-
ase, alkaline and acid phosphatase, and aminopepti-
dase) in each part of the intestines of wild eel (WE)
and reared eel (RE). Furthermore, this paper
describes gut of eel and the ratio of intestine length
to total fish length.
The aim of the study is to determine the morpho-
logical and enzymatic activity differences in the
intestines of WE and RE.
Materials and methods
The study was based on two groups of eel: 14 wild
adult eels caught during June in the Zrmanja River
and 12 eels obtained from a commercial fish hatchery
in Croatia. The first group was fed on natural feed.
The second group was fed daily as followed: glass
eels were fed in the first 2 weeks with cod roe, which
was replaced gradually by commercial dry feed. After
the second week, fish were fed only with complete
(extruded) starter feed for glass eel and fingerlings,
followed by complete feed (extruded) for eel accord-
ing to the manufacturer’s recommendations (Trouw
Nutrition Nederland).
In both examined fish groups, the fish weighed
between 500 and 750 g. After sampling, the fish were
killed in a separate tank by overdose of MS 222.
Research was carried out as a part of scientific project
with ethical committee approval and was completed
in accordance with Croatian law and regulation.
Morphological measurement
Total length (TL) of each fish was measured.
Gastrointestinal tract was removed and separated
from other viscera, and the coiled intestine was
dissected out to its full length. Length of the
stretched-out intestine (IL) was measured from
pylorus to the rectum. Relative intestine length was
calculated as IL/TL 9 100 (%).
Tissue sampling and processing
Gastrointestinal tracts in all fishes were partially
filled with ingested material. After dissection, a small
fragment of each part of the intestine was excised
from the locations shown in Fig. 1. Anterior intestine
(C-1), middle intestine (C-2) and posterior intestine
(C-3) were fixed in cold (4�C) formol-calcium for
24 h and then transferred to cold (4�C) sucrose
solution. Tissues were sectioned in a cryocat into
10-lm-thick cuts, and twenty sections of each parts
of intestine were taken for histochemical detection of
enzymatic activity. Histochemical techniques of
enzymatic activity detection were followed as per
Lojda et al. (1979) for the following enzymes: non-
specific esterase substrate, 1-naphthyl-acetate (pH
6.5); alkaline phosphatase substrate, sodium b-glyc-
erolphosphate (pH 9.4); acid phosphatase substrate,
sodium b-glycerolphosphate (pH 5.5). Aminopepti-
dase was detected using L-leucyl-4-methoxy-2-
naphthylamide hydrochloride (pH 6.5) as substrate
(Sheenan and Hrapchak 1980). One set of sections
was incubated in the medium containing the above-
mentioned substrate, while control sections were
incubated in the same medium without substrate.
Non-specific esterase and alkaline phosphatase were
incubated for 30 min and acid phosphatase for
60 min, all at room temperature (i.e., 22�C). Amino-
peptidase was incubated for 60 min at 37�C. After
incubation, sections were rinsed and mounted in
glycerin jelly or Canada balsam. Enzymatic activity
was visually analyzed and further described accord-
ing to the observed intensity of color reaction, i.e., no
enzymatic reaction (-), weak (barely detectable)
reaction (?), moderate reaction (??) or strong
reaction (???).
Results
Morphological measurement
Average TL in WE was 51.9 ± 7.7 cm, ranging from
36 to 63 cm, while average TL in RE was
56.4 ± 7.0 cm, ranging from 42 to 65 cm. Average
Fig. 1 Alimentary tract of eel, schema. Anterior intestine (C-
1), middle intestine (C-2) and posterior intestine (C-3)
626 Fish Physiol Biochem (2012) 38:625–633
123
intestinal length in WE was 15.5 ± 2.5 cm, ranging
from 11 to 21 cm. Average intestinal length in RE
was 30.5 ± 5.3 cm, ranging from 22 to 39 cm
(Table 1). Compared with RE (53.9%), WE showed
significantly shorter (29.9%) relative intestine length.
Enzyme histochemistry
In both investigated eel groups, the intestine was
short and showed a uniform histological structure
throughout its entire length. Mucosa was thrown into
numerous thin folds that were sometimes packed
together in a complex reticulate pattern, increasing
the surface area of the intestine. Based on the present
histochemical study, mucosa contained only two
types of cells. Columnar epithelial cells formed the
major part of mucosa. Goblet cells were distributed
along the length of the intestine but increase in the
number toward the posterior part of the intestine.
Enzymatic activity in the intestinal epithelium was
associated exclusively with the columnar absorptive
cells (Table 2) and was never seen in the mucous-
secreting goblet cells.
Anterior intestine (I-1)
In the anterior intestine of WE, a moderate activity of
non-specific esterase (Fig. 2a) was localized in apical
part of enterocytes. The anterior intestine of RE
showed strong activity of non-specific esterase
(Fig. 2b). The brush border of the enterocytes of
both WE and RE were characterized by strong
activity of alkaline phosphatase (Fig. 3a). Supranu-
clear region of enterocytes of WE showed strong acid
phosphatase activity. Activity of acid phosphatase in
enterocytes of RE was similar to those observed in
WE ones (Fig. 4a). There was no activity of amino-
peptidase in any part of anterior intestine mucosa in
both WE and RE.
Middle intestine (I-2)
In the middle part of the WE intestine, non-specific
esterase activity was moderate and localized in apical
cytoplasm of enterocytes (Fig. 2c). In the middle
intestine of RE, the localization of non-specific
esterase was as in the WE, but its activity was strong
(Fig. 2d). In the middle intestine of WE, the activity
of alkaline phosphatase was weak in the brush border
(Fig. 3b), but in the same intestinal part of RE, its
activity was strong (Fig. 3c). Middle intestines of WE
and RE showed strong acid phosphatase activity.
Acid phosphatase is in the supranuclear parts of
enterocytes (Fig. 4b). The brush border of the
enterocyte in the middle intestine of WE showed
moderate activity of aminopeptidase. In the RE, the
Table 1 Total length (TL)
(cm) and intestine length
(IL) (cm) of examined WE
and RE
WE wild eel, RE reared eel
x = mean value, ± standard
deviation
Wild european eel (WE) Reared european eel (RE)
Total length
(cm)
Intestine length
(cm)
% Total length
(cm)
Intestine length
(cm)
%
63 17 26.9 56 35 62.5
60 16 26.6 61 30 49.1
62 16 25.8 57 27 47.3
55 18 32.7 60 32 53.3
55 16 29.0 60 37 61.6
55 16 29.0 56 31 55.3
56 21 37.5 58 33 56.8
51 17 33.3 65 39 60.0
48 14 29.1 42 22 52.3
45 13 28.8 43 23 53.4
50 14 28.0 62 31 50.0
49 15 30.6 57 26 45.6
42 13 30.9 – – –
36 11 30.5 – – –
x = 51.9 ± 7.7 x = 15.5 ± 2,5 x = 29.9 x = 56.4 ± 7.0 x = 30.5 ± 5.3 x = 53.9
Fish Physiol Biochem (2012) 38:625–633 627
123
activity of aminopeptidase was moderate in the apical
part of enterocytes and strong in the brush border
(Fig. 5a).
Posterior intestine (I-3)
Posterior intestines of both groups of investigated fish
showed similar localization of investigated enzymes
as in previous parts. Activity of non-specific esterase
in the enterocytes WE was weak and localized in
apical parts of enterocytes (Fig. 2e), but in RE, the
activity of the enzyme was strong (Fig. 2f). Alkaline
phosphatase in the posterior intestine of WE showed
weak activity, and it was localized in the brush border
of enterocytes (Fig. 3d). In the RE, activity of
alkaline phosphatase was strong and also localized
in the brush border of enterocytes (Fig. 3e). Entero-
cytes of both WE and RE acid phosphatase showed
moderate activity and localized in the supranuclear
part of enterocytes (Fig. 4c). Activity of aminopep-
tidase in the enterocytes of the posterior intestines of
WE was weak and localized in the brush border
(Fig. 5b). In the RE, the activity of aminopeptidase
was strong and localized in the apical part of the
intestine cell, as well as in the brush border (Fig. 5c).
Discussion
Dependence of the intestine length on the type of
food is known in fish (Kapoor et al. 1975; Gebruk
et al. 1997). Adult fish that feed on detritus and
vegetation have an intestine more than three times the
length of the body, whereas in carnivorous fish, this
ratio is always about one or less (Borutskii and
Verigina 1961). Serajuddin and Saleem (1994)
researching spiny eel (Mastacembelus armatus)
affirm total gut length/total fish length ratio was
0.64, while intestine length/total length ratio was
0.27–0.34. Exploring Anguilla japonica, Chiu (2002)
found out that relative gut length of adult was about
0.2. According to this, the examined eel in our
research (WE and RE) fully belong to the group of
carnivorous fish with total intestine length/total fish
length ratio 0.29 (WE) and 0.53 (RE). Discrepancy
between those two groups is not negligible. So, if the
long intestine of fish feeding on plant material
suggests that one or more essential components of
the diet are difficult to be digested (Leveque 1997), it
is reasonable to assume that RE have the same
problem with available food quality. However, it is
more reasonable to assume that the observed differ-
ences are due to the food quantity (i.e., higher food
intake in RE). Recent study of digestion in some
loricariidae (German et al. 2010) revealed that when
facing food shortages, some fish may reduce the size
and function of their gastrointestinal tract. It could be
assume that large quantities of food lead to the
extension of the digestive tract and increased resorp-
tive surface. Moreover, we cannot exclude different
ages of examined RE and WE as a cause of these
changes, since relative length of the alimentary tract
decreases with eel life-cycle stage (Kloppmann
2003).
Digestion of ingested food can be in the gut lumen,
at the apical membrane (with membrane-bounded
enzymes) and intracellular in the enterocytes.
In the RE, the strong activity of non-specific
esterases was observed in apical part of enterocytes,
in all investigated intestinal segments. In WE, the
anterior and middle segment of the intestine dis-
played moderate activity, but in the posterior part, its
activity was weak. Similar intracellular distribution
of this enzyme has been previously reported in other
fish species (Gisbert et al. 1999; Tengjaroenkul et al.
2000). According to Chakrabarti et al. (1995),
Table 2 Enzymatic activity in the examined part of the eel
intestine
Anterior Middle Posterior
Non-specific esterase
WE (AP) ?? ?? ?
RE (AP) ??? ??? ???
Alkaline phosphatase
WE (BB) ??? ? ?
RE (BB) ??? ??? ???
Acid phosphatase
WE (SN) ??? ??? ??
RE (SN) ??? ??? ??
Aminopeptidase
WE (BB) - ?? ?
RE (BB) - ??? ???
RE (AP) - ?? ???
WE wild eel, RE reared eel, AP apical part of enterocyte, BBbrush border, SN supranuclear part of enterocyte. Level of the
staining intensity: (-) absent, (?) weak, (??) moderate,
(???) strong
628 Fish Physiol Biochem (2012) 38:625–633
123
non-specific esterase is involved in the digestion of
glycerol esters of fatty acids in most vertebrates,
including fish. Cytoplasmic activity of non-specific
esterase is associated with microsomes of endoplas-
mic reticulum and with various organelles such
as Golgi apparatus, mitochondria and lysosomes
(Deimling and Bocking 1976). However, in our
investigation, intestinal enzymes showed different
activity in WE and RE. Also, in our study, the
intensity of non-specific esterase was stronger in all
intestinal segments in RE than in WE, especially in
the posterior segment of the intestine. Evidence from
ultrastructural investigations (Noaillac-Depeyre and
Gas 1979; Stroband and Debets 1978) indicates that
lipids are absorbed in the first intestinal segments of
herbivorous grass carp (Ctenopharyngodon idella)
and carp (Cyprinus Carpio).
Lipid vacuoles in the intestinal epithelium as an
indicator of luminal absorption and a mechanism of
lipid storage were observed by Sarasquete et al.
(1995) in gilthead sea bream (Sparus aurata) and
Ribeiro et al. (1999) in Solea senegalensis. Activity
of non-specific esterase suggests that particular
segments of intestines may be important in carbox-
ylic esters’ hydrolysis occurring mainly during lipid
and carbohydrate metabolism.
Alkaline phosphatase is a transmembrane enzyme
(Kuz’mina and Gelman 1997) widely distributed and
localized in the brush border and apical part of
enterocytes (Gawlicka et al. 1995). In WE, alkaline
Fig. 2 Activity and localization of non-specific esterase in the eel intestine. a: anterior part WE (??), b: anterior part RE (???),
c: middle part WE (??), d: middle part RE (???), e: posterior part WE (?), f: posterior part RE (???)
Fish Physiol Biochem (2012) 38:625–633 629
123
phosphatase activity was strong in the anterior
intestine but weak in the middle and posterior
intestinal segments. However, in RE, all investigated
intestinal segments showed strong enzymatic activity.
According to Lojda et al. (1979), this enzyme is
found primarily in cell membranes where active
transport takes place. Intestinal alkaline phosphatase
is considered to be involved in the absorption of
nutrients such as lipids, glucose, calcium and inor-
ganic phosphate (Routaby and Portmann 1988;
Dupuis et al. 1991). According to these, it could
undoubtedly be concluded that the intestinal diges-
tion and transportation of nutrients occur mainly in
the anterior intestinal segments in WE, but in RE, all
Fig. 3 Activity and localization of alkaline phosphatase in the eel intestine. a: anterior part RE (???), b: middle part WE (?),
c: middle part RE (???), d: posterior part WE (?), e: posterior part RE (???)
630 Fish Physiol Biochem (2012) 38:625–633
123
intestinal segments are the place of very intensive
intestinal absorption of nutrients. Similar distribution
and the role of alkaline phosphatase in the intestinal
absorption of various fish species have been found by
Tengjaroenkul et al. (2000), Kozaric et al. (2004) and
Kozaric et al. (2006).
Acid phosphatase is one of the marker enzymes for
lysosomes. According to Kjorsvik et al. (1991) and
Sarasquete et al. (1995), intracellular protein digestion
occurs in the enterocytes of the rectal part of fish
intestines. Ultrastructural observation on the distal
parts of fish intestines shows the presence of protein-
containing vesicles in the enterocyte cytoplasm
(Stroband and Debets 1978; Noaillac-Depeyre and
Gas 1979; Georgopoulou et al. 1986; Murray et al.
1996). Presence of protein within these vesicles in the
enterocytes may be the explanation of the strong
activity of acid phosphatase in enterocytes of this
portion of the intestine, since the activity of this
enzyme is also related to pinocytic activity and
intracellular digestion. According to these authors,
pinocytosis by the enterocytes has been suggested as an
alternative pathway of protein digestion in teleost. In
both investigated eel groups, activity of acid phospha-
tase was strong in the anterior and middle intestinal
segments and moderate in posterior segments. Accord-
ing to the above-mentioned authors, we can conclude
that the enterocytes of anterior and middle intestinal
Fig. 4 Activity and localization of acid phosphatase in the eel
intestine. a: anterior part WE (???), b: middle part WE
(???), c: posterior part WE (??)
Fig. 5 Activity and localization of aminopeptidase in the eel
intestine. a: middle part RE (???), b: posterior part WE (?),
c: posterior part RE (???)
Fish Physiol Biochem (2012) 38:625–633 631
123
segments in WE and RE play a major role in gut protein
digestion, while the enterocytes of the posterior
segment also participate in intracellular protein diges-
tion, but to a lesser extent.
There was no activity of aminopeptidase in the
anterior intestine in WE and RE. In other intestinal
segments, it was observed in various intensities. In
WE, its activity declined in the posterior intestine, but
in RE, it is strong in both the middle and posterior
intestinal segments. Although Tsen and Wang (1982)
found that pepsin, trypsin and chymotrypsin have
main roles in protein digestion in the gut of Japanese
eel, presence of aminopeptidase in middle and
posterior intestinal segments suggests that these
segments also may be responsible for the digestion
of peptides and the absorption of nutrients (Lojda
et al. 1979; Baglole et al. 1998). No activity of
aminopeptidase and strong activity of acid phospha-
tase in the anterior intestinal segment imply the
smaller role of this intestinal segment in peptide
hydrolysis but may suggest the more important role in
pinocytosis of macromolecules (Stroband et al. 1979;
Stroband and Van Der Veen 1981).
WE is a carnivorous fish, which eats various kinds
of fish, mollusks and other invertebrate animals (De
Nie 1987; Lammens et al. 1985; Kangur et al. 1999)
having a higher level of lipids and protein in its diet.
High content of protein and lipid in RE diet is
probably responsible for the higher activity of
esterase, acid phosphatase and aminopeptidase,
enzymes involved in lipid absorption and protein
ingestion by pinocytosis and hydrolysis. Therefore,
the logical question is do RE need such a high content
of proteins and lipids for their growth and develop-
ment as well as for muscle quality. In the end, we can
agree with Satoh (2002) that there is still a great need
to develop a high-performance diet for RE.
Acknowledgments The authors would like to thank brothers
Bazdaric and K. Drasner who provided fish for the research.
This study was supported by the Ministry of Science,
Education and Sport of Croatia (project No. 053-0010501-
2107).
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