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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