Aquaculture 245 (2005) 273 – 286
www.elsevier.com/locate/aqua-online
Growth and morphological changes in the digestive tract
of rainbow trout (Oncorhynchus mykiss) and pacu (Piaractus
mesopotamicus) due to casein replacement with soybean proteins
Teresa Ostaszewskaa,*, Konrad Dabrowskib,c, Maria E. Palaciosb,
Marta Olejniczaka, Mateusz Wieczoreka
a
Faculty of Animal Sciences, Warsaw Agricultural University, 02 787 Warsaw, Poland
b
School of Natural Resources, Ohio State University, Columbus, Ohio 43210, USA
c
University of Warmia and Mazury, Olsztyn, Poland
Received 16 September 2004; received in revised form 9 December 2004; accepted 10 December 2004
Abstract
The effect of diets in which 50% of casein-gelatin protein was replaced with extracted soybean meal (SBM) or soybean
protein concentrate (SPC) on first-feeding rainbow trout and juvenile South American pacu was examined following 3–6 fold
body weight gain. A casein-gelatin-based diet supplemented with essential amino acids, lipids and other ingredients was used as
control. After 4-weeks feeding, rainbow trout growth was significantly depressed in both SBM- and SPC-replacement
treatments whereas pacu, the adults of which are considered omnivorous or frugivorous, showed significantly improved weight
gain on the SBM-replacement diet. The enterocytes of posterior intestine of all control fish, and SBM-fed pacu showed regular
shapes. Their supranuclear regions contained numerous small absorptive vacuoles. In trout fed SPC and SBM diets, and in SPCfed pacu, posterior intestine enterocytes were excessively vacuolized. The highest pancreas activity (measured as the number of
proenzyme granules) occurred in control fish. The liver cells showed regular development in both species fed the control diet
and in pacu fed SBM and SPC diets. On the contrary, the hepatocytes of SBM and SPC-based diet fed rainbow trout showed
anomalies. In both species, the average hepatocyte nuclear volumes significantly differed among the feeding groups. The results
of histological analyses indicated that absorption and transport of nutrients to liver and pancreas were affected by the presence
of soybean products in experimental diets. The SBM diet was beneficial for pacu but adversely affected rainbow trout, while the
SPC diet resulted in extensive pathologies of digestive tract and most likely affected nutrient utilization in both species.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Soybean meal; Salmonid; Piaractus; Intestine histology; Pancreas
* Corresponding author. Tel.: +48 22 853 09 37; fax: +48 22 853 09 38.
E-mail address: [email protected] (T. Ostaszewska).
0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2004.12.005
274
T. Ostaszewska et al. / Aquaculture 245 (2005) 273–286
1. Introduction
Most practical dry feeds used in intensive fish
rearing contain fish meal as a main protein source.
Attempts have been made to formulate diets with
complete or partial replacement of fish meal with
other protein sources. Plant proteins are of special
interest due to their availability and lower prices than
animal proteins. In order to avoid side effects of fish
meal (attractants, hormones, minerals) we used
purified sources of proteins (casein and gelatin) in
the present study.
Protein quality depends on its digestibility and
amino acid composition. The use of plant products in
fish feeding is limited due to the content of endogenous
antinutrients and unbalanced amino acid composition
(Francis et al., 2001). Many of plant antinutrients and
toxic substances are natural insecticides and they may
also adversely affect fish, resulting in growth reduction, serious poisoning or even death. Some plant
products, such as soybean meal, corn, wheat, barley,
cottonseed or rapeseed meal are, however, routinely
used in fish feeding (Hendricks, 2003).
Soybean is one of the most promising plant protein
sources as a fish meal substitute in fish feeding as it
contains 35–40% and 45–50% of crude protein in fullfat and extracted meal, respectively (Lusas and Riaz,
1995). The use of soybean meal is justified by its high
protein quality, well balanced amino acid composition, the only limitation is its low methionine
concentration (Dabrowski et al., 1989; Storebakken
et al., 2000).
Soybean meal is routinely used in trout, catfish and
carp feed production, but its use is limited due to the
content of antinutrients including protease inhibitors
(trypsin inhibitor), lectins (hemagglutinins), protein
antigens, phenolic compounds, oligosaccharides
(about 30% of indigestible carbohydrate), phytic acid
salts, saponins, etc. These antinutrients may reduce
feed consumption and nutrient utilization by the fish,
and consequently adversely affect their growth rate.
The available data show that feeds with high soybean
meal content disturb food digestion and nutrient
absorption (Dabrowski and Dabrowska, 1981), cause
intestinal lesions and flattening of mucosa folds,
therefore reducing absorptive surface of salmonid fish
intestine (van den Ingh et al., 1991, 1996; Rumsey
et al., 1994; Bureau et al., 1998; Bakke-McKellep et
al., 2000; Refstie et al., 2000). In Atlantic salmon,
soybean meal induced inflammation of posterior
intestine (enteritis) accompanied by high water
content in feces which suggests rapid intestinal
passage of food resulting in reduced digestion and
absorption time (van den Ingh et al., 1991; Refstie
et al., 2001).
The present study was undertaken to evaluate the
effects of 50% protein substitution of casein-gelatin
with soybean meal (SBM) or soybean protein concentrate (SPC) on growth of the carnivorous rainbow trout
or omnivorous/frugivorous pacu, and morphology of
the digestive system in both fish species.
2. Material and methods
2.1. Experimental design
The experiments were carried out in the Aquaculture Laboratory, Ohio State University. Rainbow
trout (Oncorhynchus mykiss) and paku (Piaractus
mesopotamicus) of average body mass 0.13 g, and 0.4
g, respectively, were used. The fish were placed in 35
l flow-through tanks. The trout were reared at 14–16
8C, at the density of 50 individuals per tank, and pacu
at 23–26 8C, at the density of 35 fish per tank. The
fish were fed three feeds of different composition
(Table 1), formulated and produced in the laboratory
(see Lee and Dabrowski, 2003 for details). Particles
size was increased gradually from 330 Am to 1 mm.
Three replicate tanks for each experimental dietary
treatment were used.
The fish were weighed in bulk and the mean
weight calculated after 4 weeks of rearing. Six fish
from each experimental group (2 per tank) were
sampled for histological analyses. The fish were
anesthetized with MS-222 (Sigma), and fixed in
Bouin’s fluid. Histological analyses were carried out
to evaluate the following morphological features of
the digestive system: supranuclear apical area (SAA),
total enterocyte area (TEA), absorption vacuole
diameter in supranuclear areas of posterior intestine
enterocytes (AVD) (Segner et al., 1987), height of
intestinal mucosal microvilli, mucosal leukocyte cell
number, number of mucous cells (counted per 20
intestinal fold) and type of mucins, hepatocyte nuclei
size (as indicator of hepatic activity) (Segner et al.,
T. Ostaszewska et al. / Aquaculture 245 (2005) 273–286
Table 1
Composition (%) of the experimental diets
Casein-gelatin Soybean protein Soybean meal
(control diet) concentrate
(SBM diet)a
(SPC diet)a
2.2. Statistical analyses
30.00
10.00
8.00
–
–
21.25
5.00
11.00
3.00
4.00
3.00
0.05
2.00
10.00
10.00
4.00
–
32.00
13.05
5.00
11.00
3.00
4.00
3.00
0.05
2.00
10.00
10.00
4.00
44.00
–
1.05
5.00
11.00
3.00
4.00
3.00
0.05
2.00
Fish growth and feed utilization were analyzed
using a previously described statistical package (Lee
and Dabrowski, 2003). The mean values and the
standard errors (S.E.) were given throughout. The
distribution of all pancreatic parameters were asymmetric, the data were log-transformed. All parameters
for all experimental groups were analyzed using oneway ANOVA, separately for trout and pacu, according
to the model:
0.50
0.40
0.80
1.00
0.50
0.40
0.80
1.00
0.50
0.40
0.80
1.00
where: y ijkl —trait value (or logarithm in case of
pancreas) in the l-th observation of k-th individual
(fish) from j-th tank of i-th feeding group, l—general
mean, g I —the effect of i-th feeding group (i=1..3),
e ijkl —random error.
In the case of significant differences among the
groups, the post-hoc Scheffe’s test was used. Stat-
a
SPC, SBM and soybean products were obtained from Central
Soya (Bellevue, OH, USA).
b
CPSP (Soluble fish protein concentrate) Sapropeche, France.
c
Concentration of vitamins and minerals (see Lee and
Dabrowski, 2003).
Rainbow trout
1,0
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0,0
0,8
a
0,6
0,4
0,2
0,0
b
b
SPC
SBM
Initial
weight
CONTROL
Feed conversion
(feed/gain)
Paku
1,6
b
1,2
0,8
0,4
b
a
x
Initial
weight
y
y
0,0
CONTROL
WEIGHT
SPC
SBM
FEED CONVERSION
1,8
1,5
1,2
0,9
0,6
0,3
0,0
Feed conversion
(feed/gain)
1988). Three observations per fish were used for
morphometric evaluation under 400 magnification.
Metabolic activity of secretory epithelial cells of
exocrine pancreas were measured as: relative nucleolus volume (V no/V n), nucleo-cytoplasmic index (V n/
V c), cytoplasmic integrated optic density index (I D/V),
where: V n—nuclear volume, V c—cytoplasm volume,
V no—nucleolar volume, I D—cytoplasm optic density.
Morphometric evaluation of pancreatic activity was
done on 16 observations from each experimental fish.
Fish morphology was evaluated in the sections
stained according to Masson’s with trichrome, methylene green and pyronine. Mucins (carbohydrate
compounds) were identified using histochemical
methods. The sections were stained with: periodic
acid-Schiff (PAS)-glycogen, neutral mucosubstances,
then with alcian blue at pH 2.5-sialomucins (sialic
acid) or sulfamucins (sulfate ester), and alcian blue at
pH 1-sulfated glycoproteins. Staining methods proposed by Martoja and Martoja-Pierson (1970) and
Pearse (1985) were used.
Cell measurements were done using the microscope Nikon-Alphaphot-2YS2, digital camera Nikon
yijkl ¼ l þ gi þ tj þ fk þ eijkl
Mean weight (fish, g)
Casein
Casein hydrolysate
Gelatin
SBM
SPC
Dextrin
CPSPb
Fish oil (cod liver)
Lecithin
Vitamin mixturec
Mineral mixturec
Phospitan C
Carboxymethylcelulose
l-Arginine
l-Methionine
l-Lysine
Choline chloride
4300 and computer image analysis systems: MicroScan (v. 1,5) and Lucia 4,21.
Mean weight (fish, g)
Ingredients
275
Fig. 1. Fish body mass and feed conversion rate after 4 weeks of
rearing. The average values per treatment are based on triplicate
tanks weighing (n=3).
276
T. Ostaszewska et al. / Aquaculture 245 (2005) 273–286
istical analysis was done using the SPSS 11.5 package, GLM procedure (SPSS 2002).
3. Results
The rainbow trout fed with control diet showed
the largest body mass after 4 weeks of rearing and a
5.4-fold body weight increase (Fig. 1). The fish fed
diets in which casein-gelatin was substituted by
soybean products were significantly smaller. On the
contrary, the pacu fed a soybean meal (SBM)
containing feed showed larger body mass. This
was the result of improved feed conversion in fish
fed soybean meal containing diets (0.8) in comparison to fish fed with control diet (feed/gain, 1.2)
(Fig. 1). No diet related mortality was observed in
any of the species studied.
Fig. 2. The posterior intestinal epithelium in rainbow trout (A) and pacu (B) fed with control diet. Enterocytes of regular shape, supranuclear
cytoplasm filled with small absorptive vacuoles (arrow head). The posterior intestine epithelium in rainbow trout (C) and pacu (D) fed with SPC
diet. Supranuclear cytoplasm filled with absorptive vacuoles of different size (arrow head). The posterior intestine epithelium in rainbow trout
(E) and pacu (F) fed with SBM diet. (E) Supranuclear cytoplasm filled with absorptive vacuoles of different size (arrow head). (F) Cytoplasm
containing small vacuoles (arrow head) (AB/PAS staining). Scale bars=10 Am.
T. Ostaszewska et al. / Aquaculture 245 (2005) 273–286
A
B
2
*
**
30
25
20
15
10
5
0
**
CONTROL
p≤0.05
p≤0.01
2
[µm ]
**
**
SAA
SAA
[µm ]
277
SPC
SBM
*
30
25
20
15
10
5
0
CONTROL
SPC
SBM
Fig. 3. Enterocyte supranuclear apical area (SAA) for diet groups for trout (A) and pacu (B). Expressed as least square means, with standard
errors (S.E.).
3.1. Histological analyses
3.1.1. Intestine
The enterocytes of intestinal mucosa of rainbow
trout (Fig. 2A) and pacu (Fig. 2B) fed with the
control diet showed regular shapes. The cytoplasm of
enterocyte basis adjacent to the lamina propria of
mucosa was darker with dark nuclear region. Supranuclear apical part of enterocytes showed numerous
small (3.23F0.18 Am in trout, 1.74F0.18 Am in
pacu) absorptive vacuoles containing PAS positive
granulation.
The posterior intestine enterocytes of rainbow trout
fed SPC (Fig. 2C), and SBM-supplemented diet (Fig.
2E) contained absorptive vacuoles of different size
(small—1.45F0.18 Am, or large—5.26F0.39 Am).
The enterocytes of posterior intestine of SPC fed pacu
were similar to those observed in rainbow trout (Fig.
2D). Their supranuclear cytoplasm contained absorptive vacuoles of different sizes (small—1.69F0.18
Am, and large—5.48F0.39 Am) that comprised about
2/3 of enterocyte volume (Fig. 3). These vacuoles
contained PAS positive inclusions. The enterocytes of
SBM fed pacu also showed regular shape (Fig. 2F),
and their supranuclear cytoplasm contained a small
number of fine absorptive vacuoles (2.33F0.39 Am).
3.1.2. Number of mucous cells and type of mucins
The posterior intestine folds of rainbow trout fed
with the control diet showed the average number of
Table 2
Table of ANOVA for morphometric traits of trout and pacu enterocyte (SAA—supranuclear apical area, TEA—total enterocyte area, AVD—
absorption vacuole diameter in supranuclear areas of posterior intestine enterocytes)
Trait
SAA
TEA
AVD
** pb0.01.
Source of variation
Group
Tank within group
Fish within Tank
Error
Group
Tank within group
Fish within Tank
Error
Group
Tank within group
Fish within Tank
Error
Trout
Pacu
Sum of squares
Degree of
freedom
Sum of squares
Degree of
freedom
1584.413**
20.975
66.044**
145.939
2587.784**
10.854
40.353
290.271
29.914**
1.070
1.357
10.390
2
6
9
36
2
6
9
36
2
6
9
36
178.046**
104.247
106.668**
559.996
6069.619**
386.742
240.695
2603.468
41.333**
1.511
1.687
17.907
2
6
9
36
2
6
9
36
2
6
9
36
278
T. Ostaszewska et al. / Aquaculture 245 (2005) 273–286
A
B
**
**
**
50
40
40
30
20
**
**
30
20
10
10
0
0
CONTROL
*
**
[µm2]
60
50
TEA
TEA
[µm2]
60
SPC
SBM
CONTROL
SPC
SBM
p≤0.05
p≤0.01
Fig. 4. Total enterocyte area (TEA) for diet groups for trout (A) and pacu (B). Least square means (LSM) with S.E.
significant differences in the intestine epithelium
morphometric parameters were found ( pb0.05)
among the experimental groups (Table 2).
Pair comparisons revealed significant differences
of enterocyte supranuclear apical area (SAA) and total
enterocyte area (T EA) among all experimental groups
of trout (Figs. 3 and 4). For absorption vacuole
diameter (AVD) significant difference was not
observed between SPC and SBM groups (Fig. 5).
The values of all parameters were highest in the SPC
group. In pacu, the total enterocyte area (TEA)
significantly differed between SPC and SBM groups,
while the absorption vacuole diameter significantly
differed between the control and SPC group (Fig. 4).
mucous cells per fold 3.6F0.62, while fish fed with
soybean containing diets had a number twice as large
(6.8F1.12 and 6.40F0.64 SPC and SBM groups,
respectively). The mucous cells produced mainly
acidic mucins, sialomucins and sulfomucins, except
for the SBM fed trout in which mixed-acidic and
neutral secretory products were observed.
The posterior intestine epithelium of pacu, irrespectively of the diet composition, showed similar
density of mucous cells: 19F2.49, 18F1.27 and
17.1F1.27 (per 20 folds per fish) in the control,
SPC and SBM groups, respectively. The cells secreted
acidic mucins, sialomucins or sulfomucins, except for
the control group where both, acidic and neutral
secretory products occurred.
The height of intestinal mucosa microvilli was
about 2 Am and no differences among the experimental groups were observed. No leukocyte infiltration in the lamina propria of mucosa occurred.
However, for both, rainbow trout and pacu, highly
B
[µm]
6
5
4
3
2
1
0
**
**
AVD
AVD
A
CONTROL
*
**
3.1.3. Exocrine pancreas
The highest number of proenzyme granules
occurred in the pancreas of control groups in both
species (Fig. 6A, B). The results of histological
analysis of pancreas revealed that in both, trout and
SPC
SBM
[µm]
6
5
4
3
2
1
0
**
CONTROL
**
SPC
SBM
p≤0.05
p≤0.01
Fig. 5. Absorption vacuole diameters (AVD) for diet groups for trout (A) and pacu (B). Least square means (LSM) with S.E.
T. Ostaszewska et al. / Aquaculture 245 (2005) 273–286
279
Fig. 6. The cross section of exocrine pancreas of rainbow trout (A) and pacu (B) fed with control diet. Scale bars=10 Am. Cross section of
exocrine pancreas of rainbow trout (C) and pacu (D) fed with SPC diet. Cross section of exocrine pancreas of rainbow trout (E) and pacu (F) fed
with SBM diet (AB/PAS staining). Scale bars=20 Am.
pacu fed SPC diet (Fig. 6C, D) parenchymal cells
were replaced by adipose tissue (lipomatous pseudohypertrophy) in comparison with controls (A, B).
Rainbow trout fed with the SBM diet (Fig. 6E) also
showed numerous adipose cells in the pancreas, and
their pancreatic accinar secretory cells were significantly lower compared to control. This suggests
inhibitory effect of antinutrient in SBM diets on
pancreas functions.
On the contrary, the cells of exocrine pancreas in
SBM fed pacu (Fig. 6F) did not show any anomalies
and their secretory activity was similar as in the
control group. In rainbow trout, all pancreas metabolic
activity indices evaluated with morphological criteria
significantly differed among experimental groups,
while in pacu the only differences were found in the
integrated cytoplasm density (Table 3).
In the control group of rainbow trout cytoplasmic
density was significantly lower when compared to
both, SBM and SPC groups (Fig. 9). Highly
significant differences in relative nucleolus volume
resulted from the differences in the nucleus volume.
280
T. Ostaszewska et al. / Aquaculture 245 (2005) 273–286
Table 3
Table of ANOVA for trout and pacu pancreas traits (V no/V n)—relative nucleolus volume, (V n/V c)—nucleo-cytoplasmatic index, (I D/V c)—
cytoplasmatic integrated optic density index
Trait
Source of variation
V no/V n
Trout
Group
Tank within group
Fish within Tank
Error
Group
Tank within group
Fish within Tank
Error
Group
Tank within group
Fish within Tank
Error
V n/V c
I D/V c
Pacu
Sum of squares
Degree of
freedom
Sum of squares
Degree of
freedom
50.730**
32.720**
3.975
191.272
13.691**
25.695**
7.393*
115.628
18.889**
5.574**
3.207**
31.415
2
6
9
270
2
6
9
270
2
6
9
270
5.411
37.357**
7.474
276.502
1.764
19.880**
5.920
127.439
5.216**
7.020**
3.284*
41.043
2
6
9
270
2
6
9
270
2
6
9
270
* pV0.05.
** pV0.01.
The hepatocytes of SPC-based diet fed trout were
irregularly shaped, with poorly visible cell membranes
(Fig. 10C). The nuclei were pyknotic, showed
irregular shape and were located at the cell periphery.
On the contrary, the hepatocytes of SPC fed pacu (Fig.
10D) did not show any anomalies: cell and nucleus
shapes were regular. In the cytoplasm PAS positive
regions were observed indicating glycogen storage.
The hepatocytes of SBM fed rainbow trout showed
small nuclei, usually peripherally located. In the
cytoplasm mainly lipid storage areas were found
(Fig. 10E). The hepatocytes of pacu fed SBM diet
showed regular shape, and large, centrally located
nuclei. In the cytoplasm, large PAS positive areas of
glycogen storage were visible (Fig. 10E), contrary to
the trout hepatocytes storing mainly lipids.
The largest nuclei of pancreatic cells occurred in the
control fish, therefore in this group the V no/V n was the
lowest. On the contrary, SPC group showed the
smallest nuclei and their V no/V n was the highest
(Fig. 7), and V n/V c the lowest (Fig. 8). In pacu,
cytoplasmic density was significantly lower in SPC
group comparing to the control and SBM dietary
treatments (Fig. 9).
3.1.4. Liver
The hepatocytes of trout (Fig. 10A) and pacu (Fig.
10B) fed with the control diet showed large, centrally
located nuclei. The cells of regular shape, with
moderate cytoplasmic lipid content showed distinct
cell membranes. The PAS positive areas indicate
glycogen content.
A
B
**
0,10
**
0,10
**
0,08
Vno/Vn
Vno/Vn
0,08
0,06
0,04
0,04
0,02
0,02
0,00
0,00
CONTROL
*
**
0,06
SPC
SBM
CONTROL
SPC
SBM
p≤ 0.05
p≤0.01
Fig. 7. Relative nucleolus volume (V no/V n) for diet groups for trout (A) and pacu (B). Least square means (LSM) with S.E.
T. Ostaszewska et al. / Aquaculture 245 (2005) 273–286
A
B
**
0,25
0,25
**
0,20
Vn/Vc
Vn/Vc
0,20
0,15
0,10
0,15
0,10
0,05
0,05
0,00
0,00
*
**
281
CONTROL
p≤0.05
p≤0.01
SPC
CONTROL
SBM
SPC
SBM
Fig. 8. Nucleo-cytoplasmatic index (V n/V c) for diet groups for trout (A) and pacu (B). Least square means (LSM) with S.E.
dextrin or as part of soybean products might have
led to differences in available energy. However, if
present, this effect was manifested in rainbow trout
(growth depression) but not in pacu. Therefore, we
conclude that the same diet enhanced the growth
rate of pacu juveniles and may have been a result
of positive effect on protein utilization (protein
sparing effect).
The importance of posterior intestine in digestion
of macronutrients and nutrient absorption is much
lower comparing to the anterior section (Dabrowski
and Dabrowska, 1981). However, the results obtained
by Krogdahl et al. (1999) revealed that in salmon fed
high quality feed posterior intestine absorbs 5–8% of
macronutrients. Moreover, posterior intestine plays
another very important role—absorbs intact protein
(McLean et al., 1999), and reabsorbs endogenous
digestive components (e.g. taurine) (Nordrum et al.,
2000a). Therefore, pathological changes observed in
the present study indicating an increased number of
mucous cells in soybean product fed fish correspond
to decrease of many mucosal, brush-border enzymes
In both, rainbow trout and pacu highly significant
differences in hepatocyte nucleus diameter among the
experimental groups were observed (Table 4). In trout,
the largest hepatocyte nuclei occurred in the control
group, while the least, in the SBM fed fish (Fig. 11).
In pacu, there was no significant difference in
hepatocyte nucleus size between the control and SPC
group. Contrary to the trout, in pacu the largest
hepatocyte nuclei occurred in SBM group, while the
least in the control.
4. Discussion
The present study revealed that substitution of
50% of casein-gelatin protein source with soybean
meal (SBM) or soybean protein concentrate (SPC)
protein adversely affected growth rate of firstfeeding rainbow trout, and induced pathological
changes in fish alimentary system. Despite of the
fact of isonitrogenous nature of the diets, availability of carbohydrates added in the form of
A
B
**
2,00
ID/Vc
ID/Vc
1,00
0,50
1,50
1,00
0,50
0,00
0,00
*
**
**
2,00
1,50
CONTROL
p≤0.05
p≤0.01
**
2,50
**
SPC
SBM
CONTROL
SPC
SBM
Fig. 9. Cytoplasmatic integrated optic density index (I D/V c) for diet groups for trout (A) and pacu (B). Least square means (LSM) with S.E.
282
T. Ostaszewska et al. / Aquaculture 245 (2005) 273–286
Fig. 10. Cross section of liver of rainbow trout (A) and pacu (B) fed with the control diet. Hepatocytes of regular shape with centrally located
nuclei. Cross section of liver of rainbow trout (C) and pacu (D) fed SPC diet. (C) Hepatocytes of irregular shape, with small pyknotic nuclei. (D)
Hepatocytes of regular shape with centrally located nuclei. Cross section of liver of rainbow trout (E) and pacu (F) fed with SBM diet. (E)
Hepatocytes of regular shape, often with peripheral nuclei. (F) Hepatocytes of regular shape with central nuclei (AB/PAS staining). Scale
bars=10 Am.
(Krogdahl et al., 2003) and suggest a decrease in
nutrient absorption.
According to Sire and Vernier (1992), the absorption of intact protein takes place in the posterior
intestine but amino acid and peptide absorption is
limited. Absorption of protein and lipid in the rainbow
trout posterior intestine was also suggested based on
the number of vacuoles in enterocytes by Ezeasor
(1978). Histological observations of the decrease in
absorption processes in the present study may be
explained by lowered activity of digestive mucosal
enzymes.
Bakke-McKellep et al. (2000) reported reduction in
activity of intracellular (cytosolic) alkaline phosphatase around the nuclei of epithelial cells of the
posterior intestine which suggested damage to the
Golgi apparatus in the enterocytes of fish fed with
soybean products. Other studies revealed a decrease in
T. Ostaszewska et al. / Aquaculture 245 (2005) 273–286
Table 4
Table of ANOVA for hepatocyte nucleus diameter of trout and pacu
Source of
variation
Trout
Group
Tank within group
Fish within tank
Error
33.224** 2
2.209
6
3.607
9
8.853
36
Sum of
squares
Pacu
Degree of
freedom
Sum of
squares
Degree of
freedom
14.272** 2
1.990*
6
2.858*
9
4.93
36
* pV0.05.
** pV0.01.
protein digestibility when SBM was added, which was
explained with an increase in the level of soybean
protease inhibitors in the intestine, accompanied by a
decrease in activity of leucine aminopeptidase in the
posterior intestine mucosa (Dabrowski et al., 1989;
Krogdahl et al., 2003). Moreover, reduction in activity
of all enzymes in the latter studies was directly related
to an increase in soybean product levels in the diet.
The decrease in lipid and protein utilization that
resulted in growth depression of trout in the present
study may be explained by the reduction in activity of
the cytosolic enzymes and brush-border membrane
enzymes. Similar finding was reported in other studies
concerning feeding salmonid fishes SBM-containing
diets (Olli and Krogdahl, 1994, 1995; Olli et al.,
1994a; van den Ingh et al., 1996; Refstie et al., 2000).
The acidophilic, supranuclear inclusion bodies
observed in the present study in the posterior intestinal
epithelium of trout and pacu fed SPC diet, and in trout
fed SBM diet were similar to those found in fish
larvae that compensate lack of stomach and pepsin
digestion with active intracellular digestion in the
epithelial cells of the posterior intestine, preceded
with pinocytosis of macromolecules from the intestinal lumen (Bengtson et al., 1993; Gisbert et al., 1999;
Ostaszewska, 2002; Ostaszewska et al., 2003). These
inclusions disappear when the stomach becomes fully
developed in juveniles of pike-perch (Stizostedion
lucioperca L.). Absorption of SBM derived amino
acids in trout is slower compared to the fish meal
amino acids (Dabrowski and Dabrowska, 1981;
Yamamoto et al., 1998). However, partitioning of
intact protein absorption in the case of soybean—or
fish meal based diets was not examined.
Twice as many mucous cells in the epithelium of
posterior intestine of trout fed soybean containing
diets indicates the changes in the mucosa and altered
intracellular digestion processes. It is believed that the
intestinal mucous compounds participate in enzymatic
food digestion and nutrient absorption (Grau et al.,
1992). The carbohydrate compounds produced by
mucous cells play mechanic, antimicrobial and antiviral role (Zimmer et al., 1992; Scocco et al., 1998),
and even osmoregulatory functions (Smith, 1989).
The number of mucous cells increased also in the
posterior intestine of trout fed with the formula
containing full fat soybean (van den Ingh et al.,
1991). On the contrary, the number of mucous cells in
the pacu posterior intestine was not affected by
various diets.
The observed lipomatous pseudohypertrophy of
pancreas indicates impaired proenzyme production.
Lipomatous pseudohypertrophy is a pathological
change that involves replacement of normal cells of
an organ by adipose tissue (Kuroda et al., 2003). Fatty
degeneration of pancreas occurred in trout and pacu
[µm]
6
5
4
3
2
1
0
*
**
p≤0.05
p≤0.01
**
**
CONTROL
**
SPC
SBM
hepatocyte nucleus
diameter
B
hepatocyte nucleus
diameter
A
283
[µm]
6
5
4
3
2
1
0
**
**
CONTROL
SPC
SBM
Fig. 11. Hepatocyte nucleus diameters for diet groups for trout (A) and pacu (B). Least square means (LSM) with standard S.E.
284
T. Ostaszewska et al. / Aquaculture 245 (2005) 273–286
fed with SPC diet and in SBM fed trout. Krogdahl et
al. (2003) observed that fecal loss of trypsin activity
increased in fish fed with SBM containing diets up
to the level of 20–25%, but slightly decreased at
higher levels. Similar effects have been reported in
salmon (Olli et al., 1994b), trout (Krogdahl et al.,
1994) and gilthead sea bream (Robaina et al., 1995)
fed with diets containing increasing levels of
soybean trypsin inhibitors. Despite the increase in
pancreatic protease activity, soybean trypsin inhibitors reduced protein digestibility in soybean fed
trout (Dabrowski et al., 1989).
Reduction in metabolic activity of pancreatic
secretory epithelium in SPC fed trout was confirmed
also by significant differences in optical density of
cytoplasm, and in nuclear volume of pancreatic cells.
The size of hepatocyte nuclei is directly related to
metabolic activity of liver cells (Segner et al., 1988).
The largest hepatocyte nuclei were observed in trout
fed control diet, and the differences were highly
significant. In pacu, the highest volume of hepatocyte
nuclei occurred in SBM fed group, and the differences
were also highly significant.
The liver parenchyma of SPC and SBM fed trout
showed higher level of lipid accumulation comparing
to glycogen. That was accompanied by pancreas
pseudohypertophy.
The increasing lipid storage in liver and muscles
and reduced glycogen accumulation may be a sign of
phosphorus deficiency in fish (Sakamoto and Yone,
1978).
Refstie et al. (2000) reported that lipid absorption
was disturbed in salmonid fish by indigestible
soybean polysaccharides. The reduction of n-3 HUFA
concentration in diet decreases lipoprotein synthesis
(Olsen et al., 1999). That results in lipid drop
accumulation in the enterocytes. The increase in
enterocyte size accompanied by excessive vacuolization is a consequence of lipid storage and/or inhibition
of re-esterification of fatty acids. Lipid accumulation
in supranuclear enterocyte regions was observed to be
a result of reduced re-acylation and lowered rate of
lipid synthesis from lipids of plant origin (Caballero et
al., 2003). Poor lipid digestion and reduction of
assimilated energy in salmonid fishes fed soybean
meal, compared to fish fed with fish meal based diets
was related to high concentrations of indigestible
carbohydrates in soybean (Rumsey et al., 1994;
Kaushik et al., 1995; Refstie et al., 1997, 2000). High
concentration of indigestible oligosaccharides causes
an increase in osmotic tension during digestion
processes which results in the decreased intestinal
water reabsorption (Cummings et al., 1986). The
results obtained by Nordrum et al. (2000b) revealed
that SBM causes an increase in posterior intestine
permeability and a decrease in absorption efficiency in
that region. Our comparison of morphological
changes in digestive system of carnivorous and
omnivorous fish caused by soybean containing feeds
also indicates that the disturbances in digestion and
absorption were probably induced by the presence of
indigestible polysaccharides. In carnivorous trout, the
soybean containing diets (with indigestible carbohydrates) probably caused disturbances in intracellular
digestion in enterocytes, pathological changes in
pancreatic secretory epithelium (which resulted in
reduced enzymatic activity) and metabolic disturbances in liver. These disorders resulted in reduced
growth rate, and feed utilization. The omnivorous or
frugivorous pacu probably developed mechanisms
enabling this species to digest, absorb and metabolize
both, plant and animal food. That explains efficient
digestion, absorption and high pancreatic activity,
as well as high metabolic activity of liver, and
higher growth rate in SBM fed pacu. This species
probably shows enzymatic adaptation mechanism
that responds to feed quality changes with alteration of quantity and composition of proteolytic
enzymes. Such an adaptation has been recently
observed in omnivorous piracanjuba Brycon
orbignyanus, a species of the same Characidae
family as pacu (de Borba et al., 2003). The
differences in utilization of high carbohydrate diets
and protein sparing effect between the omnivorous
and carnivorous fish species were reviewed recently
by Hemre et al. (2002).
Further studies need to address specific soybean
antinutrients that are responsible for digestion and
nutrient absorption disorders in salmonid fishes.
Acknowledgments
This research was in part funded by the bAquasoyaQ
project (National Soybean Council) and US Agency
for International Development Grant No. LAG-G-00-
T. Ostaszewska et al. / Aquaculture 245 (2005) 273–286
96-90015-00 through the Aquaculture Collaborative
Research Support Program (CRSP). The Aquaculture
CRSP accession number is 1294. The opinions
expressed herein are those of the author(s) and do
not necessarily reflect the views of the USAID.
The senior author would like to thank the dean of
the Faculty of Animal Science, Warsaw Agricultural
University, Prof. J. Kulisiewicz for financing participation in the World Aquaculture Society conference
(Hawaii, 2004) at which this work was presented.
References
Bakke-McKellep, A.M., Press, C.McL., Baeverfjord, G., Krogdahl,
2., Landsverk, T., 2000. Changes in immune and enzyme
histochemical phenotypes of cells in the intestinal mucosa of
Atlantic salmon, Salmo salar L., with soybean meal-induced
enteritis. J. Fish Dis. 23, 115 – 127.
Bengtson, D.A., Borrus, D.N., Leibovitz, H.E., Simpson, K.L., 1993.
Studies on structure and function of the digestive system of
Menidia beryllina (Pisces, Atherinidae). In: Walther, B.T., Fyhn,
H.J. (Eds.), Physiological and Biochemical Aspects of Fish
Development. University of Bergen, Norway, pp. 199 – 208.
Bureau, D.P., Harris, A.M., Cho, C.Y., 1998. The effects of purified
alcohol extracts from soy products on feed intake and growth of
chinook salmon (Oncorhynchus tshawytscha) and rainbow trout
(Oncorhynchus mykiss). Aquaculture 161, 27 – 43.
Caballero, M.J., Izquierdo, M.S., Kjbrsvik, E., Montero, D.,
Socorro, J., Fernández, A.J., Rosenlund, G., 2003. Morphological aspects of intestinal cells from gilthead seabream
(Sparus aurata) fed diets containing different lipid sources.
Aquaculture 225, 325 – 340.
Cummings, J.H., Englyst, H.N., Wiggins, H.S., 1986. The role of
carbohydrates in lower gut function. Nutr. Rev. 44, 50 – 54.
Dabrowski, K., Dabrowska, H., 1981. Digestion of protein by
rainbow-trout (Salmo gairdneri Rich) and absorption of aminoacid within the alimentary-tract. Comp. Biochem. Physiol. 69A,
99 – 111.
Dabrowski, K., Poczyczynski, P., Kock, G., Berger, B., 1989. Effect
of partially or totally replacing fish meal protein by soybean
meal protein on growth, feed utilization and proteolytic enzyme
activities in rainbow trout (Salmo gardneri). New in vivo test
for exocrine pancreatic secretion. Aquaculture 77, 29 – 49.
de Borba, M.R., Fracalossi, D.M., Pezzato, L.E., Menoyo, D.,
Bautista, J.M., 2003. Growth, lipogenesis and body composition
of piracanjuba (Brycon orbignyanus) fingerlings fed different
dietary protein and lipid concentrations. Aquat. Living Resour.
16, 362 – 369.
Ezeasor, D.N., 1978. The fine structure of gastric epithelium of the
rainbow trout, Salmo gairdneri Richardson. J. Fish Biol. 19,
611 – 627.
Francis, G., Makkar, H.P.S., Becker, K., 2001. Antinutritional
factors present in plant-derived alternate fish feed ingredients
and their effects in fish. Aquaculture 199, 197 – 227.
285
Gisbert, E., Sarasquete, M.C., Williot, P., Castelló-Orvay, F.,
1999. Histochemistry of the development of the digestive
system of Siberian sturgeon during early ontogeny. J. Fish
Biol. 55, 596 – 616.
Grau, A., Crespo, S., Sarasquete, M.C., Gonzalez de Canales, M.L.,
1992. The digestive tract of the amberjack Seriola dumerili,
Risso: a light and scanning electron microscope study. J. Fish
Biol. 41, 287 – 303.
Hemre, G.-I., Mommsen, T.P., Krogdahl, 2., 2002. Carbohydrates
in fish nutrition: effects on growth, glucose metabolism and
hepatic enzymes. Aquac. Nutr. 8, 175 – 194.
Hendricks, J.D., 2003. Adventitious toxins. In: Halver, J.E., Hardy,
R.W. (Eds.), Fish Nutrition, 3rd ed. Academic Press, Elsevier
Science USA, pp. 602–641.
Kaushik, S.J., Cravedi, J.P., Lalles, J.P., Sumpter, J., Fauconneau,
B., Laroche, M., 1995. Partial or total replacement of fish meal
by soybean protein on growth, protein utilization, potential
estrogenic or antigenic effects, cholesterolemia and flesh
quality in rainbow trout, Oncorhynchus mykiss. Aquaculture
133, 257 – 274.
Krogdahl, 2., Berg Lea, T., Olli, J.J., 1994. Soybean proteinase
inhibitors affect intestinal trypsin activities and amino acid
digestibilities in rainbow trout (Oncorhynchus mykiss). Comp.
Biochem. Physiol. 107A, 215 – 219.
Krogdahl, 2., Nordrum, S., Sbrensen, M., Brudeseth, L., Rbsjb, C.,
1999. Effect of diet composition on apparent nutrient absorption
along the intestinal tract and of subsequent fasting on mucosal
disaccharidase activities and plasma nutrient concentration in
Atlantic salmon Salmo salar L. Aquac. Nutr. 5, 121 – 133.
Krogdahl, 2., Bakke-McKellep, A.M., Baeverfjord, G., 2003.
Effects of graded levels of standard soybean meal on
intestinal structure, mucosal enzyme activities, and pancreatic
response in Atlantic salmon (Salmo salar L.). Aquac. Nutr. 9,
361 – 371.
Kuroda, N., Okada, M., Toi, M., Hiroi, M., Enzan, H., 2003.
Lipomatous pseudohypertrophy of the pancreas: further evidence of advanced hepatic lesion as the pathogenesis. Pathol.
Interact. 53, 98 – 101.
Lee, K.J., Dabrowski, K., 2003. Interaction between vitamins C and
E affects their tissue concentrations, growth, lipid oxidation, and
deficiency symptoms in yellow perch (Perca flavescens). Br. J.
Nutr. 89, 589 – 596.
Lusas, E.W., Riaz, M.N., 1995. Soy protein products: processing
and use. J. Nutr. 125, 573 – 580.
Martoja, R., Martoja-Pierson, M., 1970. Técnicas de Histologia
Animal. Toray Masson S.A, Barcelona. 350 pp.
McLean, E., Rbnsholdt, B., Sten, C., Najamuddin, 1999. Gastrointestinal delivery of peptide and protein drugs to aquacultured
teleosts. Aquaculture 177, 231 – 247.
Nordrum, S., Krogdahl, 2., Rbsjb, C., Olli, J.J., Holm, H., 2000a.
Effects of methionine, cysteine and medium chain triglycerides
on nutrient digestability, absorption of amino acids along the
intestinal tract retention in Atlantic salmon (Salmo salar L.)
under pair-feeding regime. Aquaculture 186, 341 – 360.
Nordrum, S., Bakke-McKellep, A.M., Krogdahl, 2., Buddington,
R.K., 2000b. Effects of soybean meal and salinity on intestinal
transport of nutrients in Atlantic salmon (Salmo salar L.) and
286
T. Ostaszewska et al. / Aquaculture 245 (2005) 273–286
rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. 125B, 317 – 335.
Olli, J., Krogdahl, 2., 1994. Nutritive value of four soybean
products as protein sources in diets for rainbow trout (Oncorhynchus mykiss, Walbaum) reared in fresh water. Acta Agric.
Scand., A Sci. 44A, 185 – 192.
Olli, J., Krogdahl, 2., 1995. Alcohol soluble components of
soybeans seem to reduce fat digestibility in fish-meal-based diets
for Atlantic salmon, Salmo salar L. Aquac. Res. 26, 831 – 835.
Olli, J., Krogdahl, 2., van den Ingh, T.S.G.A.M., Bratt3s, L.E.,
1994a. Nutritive value of four soybean products in diets for
Atlantic salmon (Salmo salar, L.). Acta Agric. Scand., Sect. A
Sci. 44A, 50 – 60.
Olli, J., Hjelmeland, K., Krogdahl, 2., 1994b. Soybean trypsin
inhibitors in diets for Atlantic salmon (Salmo salar, L.): effects
on nutrient digestibilities and trypsin in pyloric caeca homogenate and intestinal content. Comp. Biochem. Physiol. 109A,
923 – 928.
Olsen, R.E., Myklebust, R., Kaino, T., Ringb, E., 1999. Lipid
digestibility and ultrastructural changes in the enterocytes of
Arctic charr (Salvelinus alpinus L.) fed linseed oil and soybean
lecithin. Fish Physiol. Biochem. 21, 35 – 44.
Ostaszewska, T., 2002. The morphological and histological development of digestive tract and swim bladder in early organogenesis of pike-perch larval (Stizostedion lucioperca L.) in
different rearing environments. Treatises and Monographs.
SGGW, Warsaw (in Polish).
Ostaszewska, T., Wegner, A., We˛giel, M., 2003. Development of the
digestive tract of ide, Leuciscus idus (L.) during the larval stage.
Arch. Pol. Fish. 11, 79 – 92.
Pearse, A.G.E., 1985. Histochemistry. Theoretical and Applied,
Analytic Technology, vol. 2. Churchill Livingstone, New York.
Refstie, S., Storebakken, T., Roem, A.J., 1997. Adaptation to
soybean meal in diets for rainbow trout, Oncorhynchus mykiss.
Aquaculture 153, 263 – 272.
Refstie, S., Korsben, a.J., Storebakken, T., Baeverfjord, G., Lein, I.,
Roem, A.J., 2000. Differing nutritional responses to dietary
soybean meal in rainbow trout (Oncorhynchus mykiss) and
Atlantic salmon (Salmo salar). Aquaculture 190, 49 – 63.
Refstie, S., Storebakken, T., Baeverfjord, G., Roem, A.J., 2001.
Long-term protein and lipid growth of Atlantic salmon (Salmo
salar) fed diets with partial replacement of fish meal by soy
protein products at medium or high lipid level. Aquaculture 193,
91 – 106.
Robaina, L., Izquierdo, M.S., Moyano, F.J., Socorro, J., Vergara,
J.M., Montero, D., Fernández-Palacios, H., 1995. Soybean and
lupin seed meals as protein sources in diets for gilthead
seabream (Sparus aurata): nutritional and histological implications. Aquaculture 130, 219 – 233.
Rumsey, G.L., Siwicki, A.K., Anderson, D.P., Bowser, P.R., 1994.
Effect of soybean protein on serological response, non-specific
defence mechanisms, growth, and protein utilization in rainbow
trout. Vet. Immunol. Immunopathol. 41, 323 – 339.
Sakamoto, S., Yone, Y., 1978. Effect of dietary phosphorus level on
chemical composition of red sea bream. Bull. Jpn. Soc. Sci.
Fish. 44, 227 – 229.
Scocco, P., Accili, D., Menghi, G., Ceccarelli, P., 1998. Unusual
glycoconjugates in the oesophagus of a tilapine polyhybrid. J.
Fish Biol. 53, 39 – 48.
Segner, H., Burkhardt, P., Avila, E.M., Juario, J.V., Storch, V., 1987.
Nutrition-related histopathology of the intestine of milkfish
Chanos chanos fry. Dis. Aquat. Org. 2, 99 – 107.
Segner, H., Rfsch, R., Schmidt, H., Von Poeppinghausen, K.J.,
1988. Studies on the suitability of commercial dry diets for
rearing of Coregonus lavaretus larvae from Lake Constance.
Aquat. Living Resour. 1, 231 – 238.
Sire, M.F., Vernier, J.-M., 1992. Intestinal absorption of protein in
teleost fish. Comp. Biochem. Physiol. 103A, 771 – 781.
Smith, L.S., 1989. Digestive functions in teleost fishes. In:
Halver, J.E. (Ed.), Fish Nutrition, 2nd ed. Academic Press,
London, pp. 331 – 421.
Storebakken, T., Refstie, S., Ruyter, B., 2000. Soy products as fat
and protein sources in fish feeds for intensive aquaculture. In:
Drackley, J.K. (Ed.), Soy in Animal Nutrition. Fed. Anim. Sci.
Soc, Savoy, IL, USA, pp. 127 – 170.
van den Ingh, T.S.G.A.M., Krogdahl, 2., Olli, J.J., Hendriks,
H.G.C.J.M., Koninkx, J.G.J.F., 1991. Effects of soybeancontaining diets on the proximal and distal intestine in Atlantic
salmon (Salmo salar): a morphological study. Aquaculture 94,
297 – 305.
van den Ingh, T.S.G.A.M., Olli, J.J., Krogdahl, 2., 1996. Alcoholsoluble components in soybeans cause morphological changes
in the distal intestine of Atlantic salmon, Salmo salar L. J. Fish
Dis. 19, 47 – 53.
Yamamoto, T., Unuma, T., Akiyama, T., 1998. Postprandial
changes in plasma free amino amid concentrations of rainbow
trout fed diets containing different protein sources. Fish. Sci.
64, 474 – 481.
Zimmer, G., Reuter, G., Schauer, R., 1992. Use of influenza cvirus for detection of 9-O-acetylated sialic acids on immobilized conjugates by esterase activity. Eur. J. Biochem. 204,
209 – 215.