Aquaculture research, tập 41, số 6, 2010

Aquaculture Research, 2010, 41, 793^803

doi:10.1111/j.1365-2109.2009.02356.x

Enrichment of the African catfish Clarias gariepinus
(Burchell) with functional selenium originating from
garlic: effect of enrichment period and depuration
on total selenium level and sensory properties
Edward Schram1, Rian A A M Schelvis-Smit1, Jan W van der Heul1 & Joop B Luten1,2
1

IMARES,Wageningen UR, IJmuiden,The Netherlands

2

No¢ma Marine,TromsÖ, Norway

Correspondence: E Schram, IMARES, Wageningen UR, PO box 68,1970 AB, IJmuiden,The Netherlands. E-mail:

Abstract
We wanted to optimize the procedure for the selenium enrichment of farmed African cat¢sh, using
garlic as dietary selenium source. In the ¢rst experiment we established the relation between the length
of the selenium enrichment period and the resulting
total selenium level in the ¢llet of the ¢sh. It was
found that at a dietary level of11.7 mg kg À 1 Se, a total
selenium level in the ¢llet of 0.7 mg kg À 1 was
reached in a relatively short enrichment period of 10
days before harvest. In the second experiment we studied the e¡ect of depuration on the selenium level in
the ¢llet and the sensory properties of seleniumenriched African cat¢sh. It was found that total selenium levels in the ¢llet were not a¡ected during a
7-day depuration period, while garlic odours and £avours in the raw and cooked ¢llets were signi¢cantly

reduced after 2 days of depuration.We concluded that
selenium enrichment of farmed African cat¢sh can
be obtained by selenium-enriched ¢nishing diets,
while garlic odours and £avours resulting from dietary garlic can be e¡ectively reduced in the ¢llet during a short depuration period without negatively
a¡ecting ¢llet levels of total selenium.

Keywords: African cat¢sh, functional food, selenium, garlic, depuration, sensory properties

Introduction
Dietary selenium is essential for human health (Rayman 2000; Birringer, Pilawa & Flohe 2002) and at

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Journal Compilation r 2009 Blackwell Publishing Ltd

concentrations above dietary requirement, selenium
is reported in several studies as having anti-carcinogenic e¡ects in humans (Ip 1998). However in some
European countries the average daily intake is lower
than the recommended daily intake (Rayman 2005).
The need for selenium has resulted in an increase in
selenium-rich functional foods (Dumont, Vanhaecke
& Cornelis 2006), including the development of selenium-enriched farmed ¢sh.
Functional food has been de¢ned in a European
consensus document as ‘a food can be regarded as
functional if it is satisfactorily demonstrated to a¡ect
bene¢cially one or more target functions in the body,
beyond adequate nutritional e¡ects, in a way that is
relevant to either improved stage of health and wellbeing and/or reduction of risk of disease. A functional
food must remain food and it must demonstrate its
e¡ects in amounts that can normally be expected to
be consumed in the diet: it is not a pill or a capsule,

but part of the normal food pattern’ (Diplock, Aggett,
Ashwell, Bornet, Fern & Roberfroid 1999). Aquaculture provides an excellent opportunity for the production of functional seafood products as many
factors that determine the composition of the edible
portion of ¢sh can be controlled under farming conditions.
A number of studies have demonstrated the enhancement of the selenium concentration in the
muscle tissue of ¢sh as a result of elevated levels of
dietary selenium (see Schram, Pedrero, CaŁmara, Van
der Heul & Luten 2008 for an overview). None of
these trials used garlic as source of functional selenium species while Ip and Lisk (1996) showed that

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Aquaculture Research, 2010, 41, 793–803

Selenium enrichment of African cat¢sh part II E Schram et al.

the anti-carcinogenic e⁄cacy of the selenium species
in garlic is superior to selenomethionine and selenite
(in Ip 1998).
In our previous work we took the ¢rst step towards
the development of farmed African cat¢sh enriched
with functional selenium using garlic as selenium
source (Schram et al. 2008). We established a dose^
response relation (feed to ¢sh), the growth performance in relation to dietary selenium and garlic and
a ¢rst step was taken towards establishing the retention of functional selenium in the edible portion of
the ¢sh. However, to e⁄ciently use the seleniumenriched garlic resources we also need to know the
minimally required length of the selenium enrichment period to reach target concentrations in the ¢sh
¢llet. In addition, the e¡ect of depuration, a necessary procedure to eliminate o¡-£avours (Tucker
2000) during which ¢sh are not fed, on the selenium

level in the ¢llet needs to be established to ensure targeted selenium levels are reached at harvest. The use
of garlic as a ¢sh feed ingredient demands evaluation
of the sensory properties of the ¢sh at harvest and in
relation to depuration, because the e¡ects of dietary
garlic on sensory properties of ¢sh are unknown but
likely to be present.

Feed component

Control feed

Seleniumenriched feed

Wheat
Wheat gluten
Fish meal
Fish oil
Soybean meal
Premix
Binder
Selenium-rich garlic
Total selenium (mg kg À 1)

223
100
450
60
125
17
25

0
2.1

223
100
450
60
111.5
17
25
13.5
11.7

All feed components are expressed as g kg À 1 wet weight, except
for total selenium expressed as mg kg À 1 wet weight.

garlic with a high selenium content. Garlic was supplied as a dry powder and included as such in the
feed. The control feed was the same except for the
inclusion of garlic. Experimental feeds were produced by Research Diets Services, the Netherlands,
as 4 mm steam pellets, with a proximate analysis of
46.8% crude protein,13.6% crude fat,1.1% crude ¢bre
and 10.1% crude ash. The formulation of the experimental feeds is shown in Table 1.
Experiment 1: total selenium levels and
selenium retention in relation to the length of
enrichment period

Materials and methods
Introduction
Two experiments were performed. In the ¢rst experiment ¢sh were fed a selenium-enriched feed for six
di¡erent periods before harvest to investigate the effect of the length of the enrichment period on the

total selenium level in the ¢llet at harvest. Garlic
was used as dietary selenium source. Fillet samples
were analysed for total selenium.
In the second experiment selenium enrichment
was followed by a depuration period during which
¢sh were sampled at intervals to assess the total selenium level and the sensory properties.

Experimental feeds
For both experiments two feeds were used: seleniumenriched and a control feed. Selenium enrichment
was achieved by inclusion of selenium-enriched garlic. This garlic was grown and processed by Plant
Research International,Wageningen UR, the Netherlands, with selenium forti¢cation of the soil as described in Larsen et al. (2006), which resulted in

794

Table 1 Formulation of the experimental feeds on the wet
weight basis

The six treatments consisted of six selenium enrichment periods of di¡erent length. Groups of ¢sh were
fed the selenium-enriched feed for 35, 25, 17, 10 or
5 days before harvest. The experimental period lasted
for 35 days for all treatments. Fish were fed the control feed (Table 1) before receiving the seleniumenriched feed. Table 2 provides an overview of the
treatments.
Treatments were randomly assigned to the tanks,
with triplicate tanks for each of the six treatments.
The experimental set up consisted of 18, 30 L glass
tanks placed in two rows of nine tanks. Tanks were
£own through with tap water with a selenium level
of o0.5 mg Se L À 1at a rate of 10 L h À 1. Tank e¥uents
were discharged as ¢sh are known to take up waterborne selenium across the gills (Hodson et al. 1980)
and recirculation of water combined with leaching

of selenium from the feeds and excretion by the ¢sh
could have resulted in transfer of selenium compounds between tanks.Water temperature was maintained at 25 1C throughout the experimental period.
Each tank was stocked with 14 African cat¢sh

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Aquaculture Research, 2010, 41, 793^803

Table 2 Overview of the treatments in Experiment 1
Control feed

Selenium-enriched feed

Treatment

Feeding
days

Feeding
days

Proportion of
total feed load (%)

1
2
3
4

5
6

–
1–10
1–18
1–25
1–30
1–35

1–35
11–35
19–35
26–35
31–35
–

100
76
53
34
13
0

Presented are for each treatment the experimental days during
which the control (not selenium-enriched) feed and the selenium-enriched feed were fed and the relative proportion of the
selenium-enriched feed load to the total feed load.

(Clarias gariepinus) at a sex ratio of one male to one
female. African cat¢sh were obtained from FleurenNooijen BV, the Netherlands, a commercial African

cat¢sh hatchery. After stocking, the ¢sh were allowed
to acclimatize to the experimental conditions for
5 days, during which all ¢sh were fed the control feed
(Table 1). The day after the acclimatization period is
referred to as day 1 of the experimental period. Fish
were fed by hand to visually observe satiation twice
daily (10:00 and 17:00 hours). The total feed load
was documented for each tank. Mortalities were
recorded daily and dead ¢sh were removed immediately from the tanks after detection. On days 1 and
35 (harvest), the total biomass and number of ¢sh
were determined for each tank. The mean (SD) individual weight of 230 (6.7) g at day 1 was not signi¢cantly di¡erent between tanks [analysis of variance
(ANOVA), Po0.05]. Before stocking of the experimental
tanks on day 1, three males and three females were
randomly sampled from the total stock. On day
35, three males and three females were sampled randomly from each tank. Sampled ¢sh were ¢lleted, ¢llets were pooled per tank, homogenized and stored
frozen at À 70 1C. Feed samples were taken at day 1
of the experiment and stored frozen at À 70 1C.

Selenium enrichment of African cat¢sh part II E Schram et al.

during which ¢sh were sampled at regular intervals. The total experimental period lasted for 29 days.
Treatments were randomly assigned to the tanks, with
triplicate tanks for each of the two treatments. The
experimental set up consisted of six, 400 L plastic
tanks placed in two rows of three tanks. Tank e¥uents were discharged as recirculation could have resulted in transfer of selenium compounds between
tanks. Each tank was stocked with 38 African cat¢sh
(C. gariepinus) with a mean (SD) individual weight of
796 (11) g at a sex ratio of one male to one female. The
mean initial individual weight was not signi¢cantly
di¡erent between tanks (ANOVA, Po0.05). After stocking, the ¢sh were allowed to acclimatize to the experimental conditions for 5 days, during which all ¢sh

were fed a commercial grower diet. The day after the
acclimatization period is referred to as day 1 of the
experimental period. During the ¢rst 21 days of
experimental period the tanks were £own through
with 25 1C tap water with a selenium level of
o0.5 mg Se L À 1at a rate of 35 L h À 1. At day 22, the
start of the depuration period, the £ow was increased
to 60 L h À 1. On days 1 and 22, the total biomass and
number of ¢sh was determined for each tank. Before
stocking of the experimental tanks on day 1, three
males and three females were randomly sampled
from the total stock. On days 22, 23, 24, 25 and 29,
concurring with days 0, 1, 2, 3 and day 7 of the depuration period, three males and three females were
sampled randomly from each tank. Sampled ¢shed
were ¢lleted. Per ¢sh one ¢llet was used for total selenium analysis and one ¢llet for sensory analysis.
Fillets sampled for total selenium analysis were
pooled per tank (six ¢llets per tank per sampling
point), homogenized and stored frozen at À 70 1C.

Production parameters
The speci¢c growth rate (SGR) for of the group of ¢sh
in each tank was calculated as follows:
SGR ¼ ðlnðWt Þ À lnðW0 ÞÞ Â

Experiment 2: e¡ect of depuration on total
selenium level and sensory properties of the
¢llet
Treatments consisted of two di¡erent experimental
feeds: control feed and selenium-enriched feed
(Table 1). The experimental period consisted of two

parts. The ¢rst part of the experimental period was a
rearing period of 21 days. The second part of the
experimental period was an 8-day depuration period

100
T

where SGR is the speci¢c growth rate (%BWday À 1),
BW is the body weight, Wt is the average individual
weight at harvest (g), W0 is the average individual
weight at stocking (g) and T is the number of days.
The feed conversion rate (FCR) per tank was calculated as follows:
FCR ¼

r 2009 The Authors
Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 793^803

FL
ðWt  Nt À W0  N0 Þ

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Aquaculture Research, 2010, 41, 793–803

Selenium enrichment of African cat¢sh part II E Schram et al.

where FCR is the feed conversion rate (g g À 1), FL is
the total amount of feed administered to a tank during the experiment (g), Wt is the average individual
weight at harvest (g), W0 is the average individual

weight at stocking (g), Nt is the number of ¢sh per
tank at harvest and N0 is the number of ¢sh per tank
at stocking.
The retention of selenium in the ¢sh ¢llet was calculated as follows:
À
Á À
Á
½SeŠt ÂWt  Nt  F À ½SeŠ0 ÂW0  N0  F
À
Á
R¼
½SeŠfeed ÂFL
 100%
where R is the selenium retention rate in the ¢sh ¢llet
(% of dietary intake), [Se]t is the total selenium concentration in the ¢llet at harvest (mg kg À 1),Wt is the
average individual weight at harvest (kg), Nt is the
number of ¢sh per tank at harvest, F is the ¢lleting
yield (%), [Se]0 is the total selenium concentration
in the ¢llet at stocking (mg kg À 1), W0 is the average
individual weight at stocking (kg), N0 is the number
of ¢sh per tank at stocking, [Se]feed is the total selenium concentration in feed (mg kg À 1) and FL is the
total feed load (kg).

Total selenium analysis
Homogenized samples of the muscle tissue were microwave digested in microwave-lined digestion vessels using 70% (m/v) nitric acid (5 mL) and 30%
(m/v) hydrogen peroxide (5 mL) as the oxidant mixture. After digestion, the samples were reduced in
the microwave by adding 5 mL 37% (m/v) hydrochloric acid to convert Se61 into Se41. The resulting solution was diluted to a ¢nal volume of 25 mL using 10%
hydrochloric acid. Blanks [5 mL 70% (m/v) nitric acid
and 5 mL 30% (m/v) hydrogen peroxide] were treated
in the same way. Total selenium was determined

using hydride generation Flow Injection Analysis
System^Atomic Absorption Spectrometry. NaBH4
(0.2%) in NaOH (0.05%) was used to generate the
H2Se. The accuracy of the analyses was established
by analysing the selenium content in the BCR-certi¢ed Cod-CRM422 reference sample.

Sensory analysis
The e¡ect of dietary garlic and the e¡ect of depuration on the sensory properties of the seleniumenriched African cat¢sh were assessed in a sensory

796

intensity test in Experiment 2. On days 19, 21, 22 and
26, concurring with days 0, 2, 3 and 7 of the depuration period, three males and three females were
sampled randomly from each tank, yielding 18 ¢sh
per treatment. From each ¢sh, one ¢llet was used for
sensory analysis. The ¢sh were ¢lleted on the day of
slaughter and stored in vacuum at À 25 1C. On the
day of testing six randomly selected ¢llets per treatment were thawed for 60 min in running cold tap
water, pooled per treatment and homogenized by
cooled mincing (type DRC compact 92, France). Samples (approximately 50 g) of the homogenized minced
¢llets were put in small aluminium boxes with a
three-digit random code and stored at 0 1C until preparation. Samples were prepared by placing the aluminium boxes in 1cm boiling water in a hot air oven
(Miele H 216, Miele, Germany) at 160 1C for 7 min.
Eight members of the Wageningen IMARES sensory panel participated in the sensory analysis of the
African cat¢sh samples. All panelists were trained
before sensory analysis in two1-h sessions using samples of all experimental treatments (selenium
enriched, control) and sampling days (0, 1, 3 and 7
days of depuration) in accordance with international
standards (ISO 1993). Panelists were trained to detect,
recognize, describe and scale the intensity of odours

and £avours of raw and cooked cat¢sh, using an existing sensory evaluation scheme for African cat¢sh as a
starting point. The resulting vocabulary used to describe odours and £avours (attributes) is listed inTable
3. Intensities of each attribute were scaled using a
nine-point intensity scale. For each of the four sampling days, duplicates of the selenium-enriched ¢llet
Table 3 Mean (SD, n 5 3) total selenium levels in African
cat¢sh ¢llets and selenium retention (mean1SD, n 5 3) for
di¡erent selenium enrichment periods before harvest

No.
1
2
3
4
5
6
P-value
LSD

Treatment (no.
of selenium
enrichment days)
35
25
17
10
4
0

Total selenium
level (mg kg À 1)

Selenium
retention (%)

0.99a (0.01)
0.87b (0.05)
0.80b (0.06)
0.71c (0.06)
0.41d (0.04)
0.21e (0.03)
o0.05
0.075

6.46a (0.36)
6.83ab (0.50)
8.07bc (0.46)
9.05c (0.03)
7.92bc (1.34)
4.88d (0.99)
o0.001
1.332

Di¡erent letters represent signi¢cantly di¡erent mean values
(ANOVA, Po0.05).
LSD, least square di¡erence of means at 5% signi¢cance level;
SD, standard deviation; ANOVA, analysis of variance.

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Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 793^803

Aquaculture Research, 2010, 41, 793^803

samples were evaluated in random order by each panelist in a single session. A computerized system (FIZZ,
version 2.10a 1994^2000, Biosyste¤mes, Couternon,
France) was used for data recording.

Selenium enrichment of African cat¢sh part II E Schram et al.

Table 4 Mean (standard deviation, n 5 3) ¢nal weight,
speci¢c growth rate (SGR), feed conversion rate (FCR) for
the treatments

Statistics

No.

Statistical analysis was conducted with GENSTAT 10.1.
Di¡erences in mean values of SGR, feed conversion
rate, weight gain, selenium retention and selenium
levels between treatments were analysed using oneway ANOVA. The relations between the length of the
selenium enrichment period and the response variates selenium level in the ¢llets and selenium retention were analysed with non-linear regression using
a two straight-line model. The individual mean body
weights at the sampling days in the depuration period
were tested for signi¢cant di¡erences between and
within treatments using repeated measurements
ANOVA. Mean ¢llet weights were tested for signi¢cant
di¡erences among sampling days and between treatments using repeated measurements ANOVA. In case
of signi¢cance, one-way ANOVA and repeated measurements ANOVA were followed using the least significant di¡erence (LSD) post hoc analysis to detect the
di¡erent mean values. A signi¢cance level of 5% was

used in all cases.
For the sensory analysis, the statistical analyses
were performed in SPSS version 15.0. Mean values per
sample and per attribute were calculated. Di¡erences
in the mean values were tested for signi¢cance using
one-way ANOVA, followed by the least signi¢cant difference (LSD) post hoc analysis to detect the di¡erent
mean values. A signi¢cance level of 5% was used.
Pearson’s correlation tests (two tailed) were performed to ¢nd correlations between attributes, for
which a signi¢cance level of 1% was used.

1
2
3
4
5
6
P-value

Results
Experiment 1: total selenium levels and
selenium retention in relation to the length of
enrichment period
The total selenium levels in the ¢llets were di¡erent
between treatments (ANOVA, Po0.001) and increased
with increasing length of the selenium enrichment
period (Table 4). The two straight-line regression
model accounted for 98% of the variance and resulted in an in£ection point at a selenium enrichment period of 10.3 days (Fig. 1). Selenium retention

Treatment
(Se feeding

days before Final
SGR
FCR
harvest)
weight (g) (%BW day À 1) (g g À 1)
35
25
17
10
4
0

623 (25)
566 (6)
580 (27)
565 (46)
583 (18)
589 (3)
0.14

2.86
2.57
2.64
2.57
2.65
2.66
0.17

(0.14)
(0.06)

(0.14)
(0.12)
(0.21)
(0.06)

0.79
0.84
0.83
0.89
0.86
0.90
0.56

(0.07)
(0.01)
(0.06)
(0.10)
(0.12)
(0.02)

1.2

Total selenium level in the
fillet (mg Se kg–1)

1.0

Line 2

0.8

0.6
Line 1

0.4
0.2
0.0
0

5

10 15 20 25 30 35
Days of feeding selenium
enriched feed prior to harvest

40

Figure 1 Total selenium level (mg kg À 1 Se) in African
cat¢sh ¢llets in relation to the length of the selenium enrichment period before harvest. Line 1, selenium level ¢llet 5 0.049 Â number of enrichment days10.214; line 2,
selenium level ¢llet 5 0.011 Â number of enrichment
days10.612. In£ection point (10.3, 0.72). The two straightline regression model accounts for 98% of the variance.

in the ¢llet was di¡erent between treatments (ANOVA,
Po0.001) (Table 4). The two straight-line regression
model accounted for 76% of the variance and
resulted in a sharp in£ection point at a selenium
enrichment period of 5.8 days (Fig. 2). Selenium
retention ¢rst increased with an increasing length
of the selenium enrichment period up to 5.8 days.
Beyond 5.8 days of feeding selenium-enriched feed
before harvest, selenium retention decreased (Fig. 2).

Final weight (ANOVA, P 5 0.14), SGR (ANOVA, P 5
0.17) and FCR(ANOVA, P 5 0.56) were not di¡erent
between treatments (Table 5).

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Aquaculture Research, 2010, 41, 793–803

Selenium enrichment of African cat¢sh part II E Schram et al.

10

Table 6 Sensory vocabulary for the selenium-enriched
African cat¢sh using dietary garlic as the selenium source

Line2

6

Line1

4

2

0

0

5

10 15 20 25 30 35
Days of feeding selenium
enriched feed prior to harvest

40

Figure 2 Selenium retention (%) in African cat¢sh ¢llets
expressed as the percentage of the total dietary selenium
feed load in relation to the length of the selenium enrichment period before harvest. Line 1, selenium retention 5
0.00762 Â number of enrichment days10.048; line 2, selenium level ¢llet 5 À 0.001066 Â number of enrichment
days10.099. In£ection point (5.81, 0.093). The two straightline regression model accounts for 76% of the variance.
Table 5 Mean (SD, n 5 3) total selenium levels in the ¢llets
(mg kg À 1) and mean (SD, n 5 3) ¢llet weight (g) during the
depuration period for the selenium-enriched ¢sh and the
¢sh that received the control feed
Total selenium level in
the fillet (mg kg À 1 )

Fillet weight (g)

Depuration
dayÃ

Selenium
enriched

Control

Selenium
enriched

Control

0
1
2
3
7

0.63
0.64
0.67
0.63
0.57

0.16
0.16
0.19
0.18
0.14

322
284
284
288
260

293
322
288
250
268

(0.02)
(0.04)
(0.01)
(0.03)
(0.06)

(0.02)
(0.04)
(0.01)
(0.00)
(0.03)

(7)
(31)
(32)
(3)
(51)

(41)
(12)
(23)
(37)
(18)

ÃDepuration days 0, 1, 2, 3 and 7 concur with days 22, 23, 24, 25

and 29 of the experimental period.
Total selenium levels were equal within treatments for all sampling days during the depuration period for the selenium-enriched ¢sh (ANOVA, P 5 0.10) and for the ¢sh that received the
control feed (ANOVA, P 5 0.14). Fillet weights were equal over time
within and between treatments (repeated measurements ANOVA,
Ptreatment  time 5 0.31).
SD, standard deviation; ANOVA, analysis of variance.

Experiment 2: e¡ect of depuration on total
selenium level in the ¢llet
After 21 days of ¢sh rearing, the total selenium levels
in the ¢llets were 0.62 mg kg À 1 for the selenium-

798

Sensory
attribute

Short name

Odour of the raw fillet
R_O_metal
Metallic
R_O_garlic
Garlic
R_O_sour
Sour
Odour of the cooked fillet

C_O_garlic
Garlic
C_O_cook potato
Cooked
potato
Flavour of the cooked fillet
C_T_musty
Musty
C_T_garlic
Garlic
C_T_cook potato
Cooked
potato

Total selenium concentration in
the fillet (mg Se kg–1)

Selenium retention in
the fillet (% of Se load)

8

Description of attribute

Metallic odour
Garlic odour, slight chemical
Sour odour, spoilage
Garlic odour, slight chemical
Odour of cooked potatoes

Like a wet cellar
Garlic flavour, slight chemical
Flavour of cooked potatoes

0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
0

1

2
3
4
5
6
Depuration period (Days)

7

8

Figure 3 Total selenium concentration (mg kg À 1 Se) in
the ¢llet of African cat¢sh in relation to the length of the

depuration period for the selenium () and control treatment (^). For both the selenium treatment (simple linear
regression, P 5 0.06, r2 50.25) and the control treatment
(simple linear regression, P 5 0.31, r2 50.07), the linear
relation is not signi¢cant.

enriched ¢sh and 0.16 mg kg À 1 for the ¢sh that received the control feed (Table 6). The ¢nal total mean
(SD, n 5 38) body weight after 21 days of ¢sh rearing
was 1083 (25) g for the selenium-enriched ¢sh and
1071 (19) g for the ¢sh that received the control feed.
Mean (SD, n 5 3) SGR was 1.46 (0.07)%BWday À 1 and
1.42 (0.10)%BWday À 1. Final weight (ANOVA, P 5
0.545) and SGR (ANOVA, P 5 0.614) were not di¡erent
between treatments.
The length of the depuration period was found to
have no e¡ect on the total selenium levels in the ¢llets
of both the selenium-enriched ¢sh (simple linear
regression, P 5 0.06, Fig. 3) and the ¢sh that received
the control feed (simple linear regression, P 5 0.31,

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Aquaculture Research, 2010, 41, 793^803

Selenium enrichment of African cat¢sh part II E Schram et al.

Fig. 3). Within treatments, the total selenium level
in the ¢llet was equal for all sampling days for the selenium-enriched ¢sh (ANOVA, P 5 0.10) and for the
¢sh that received the control feed (ANOVA, P 5 0.14)

(Table 6).
African cat¢sh lost weight during the depuration period (Fig. 4). Mean individual weights were
not di¡erent between treatments for each of the
sampling days during the depuration period
(repeated measurements ANOVA, P treatment  time 5
0.45). Overall mean weights were di¡erent for sampling days 0, 1 and 2 (repeated measurements ANOVA, P time o0.001, Fig. 4). By day 7 of the depuration
period, the overall mean weight loss was 8.7% of the
individual weight at the start of the depuration period. Mean individual ¢llet weights were equal for
all sampling days during the depuration period
between treatments and within treatments (repeated measurements ANOVA, P time  treatment 5
0.31,Table 6).

Experiment 2: sensory properties of seleniumenriched African cat¢sh ¢llets in relation to
depuration
Raw and cooked samples of African cat¢sh ¢llets fed
with selenium-enriched feed (with garlic as selenium
source) were found to have a rather intense garlic
odour and £avour at day 0 of the depuration period,
which had signi¢cantly decreased at day 2 of depuration period (Table 7, Fig. 5). No changes in the intensity of these attributes were observed between days 2
and 3 of the depuration period. The intensity of the
odour of both the raw and cooked samples was signi¢cantly reduced between days 3 and 7 of the depuration period, while the intensity of the £avour of the
cooked ¢llet stabilized at a minimal detectable level
in this period (Fig.5). The garlic odour correlates with
the metallic odour and with the low values of sour

Sensory score (1-9/ not
at all-very much)

9

Average weight (g)

1200
1150
1100

A
B

1050
1000

C

C

C

950

8
7
6
5
4
3
2
1
0

0

1

2
3
4
5
6
Depuration time (days)

7

1

8

Figure 4 Overall mean (n 5 6) individual weight (g) of
African cat¢sh during the depuration period. Data points
marked with di¡erent letters di¡er signi¢cantly (repeated
measurements analysisof variance, Ptimeo0.001).

2
3
4
5
6
Depuration period (Days)

7

8

Figure 5 Sensory intensity (score 1^9) in relation to
the length of the depuration period for the garlic odour
of the raw African cat¢sh (^), the odour of the cooked
African cat¢sh (&) and the £avour of the cooked African
cat¢sh ().

Table 7 Mean (standard deviation, n 5 2) intensity scores of the sensory attributes for the ¢llets during the depuration
period for the selenium-enriched African cat¢sh
Results sensory intensity score depuration day
Attributes

0

R_O_metal
R_O_garlic
R_O_sour
C_O_garlic
C_O_cook potato
C_T_musty
C_T_garlic
C_T_cook potato

4.57
6.94
3.32
4.94
5.13

3.82
5.82
5.13

2
(0.09)
(0.44)a
(0.26)a
(0.08)a
(0.53)
(0.45)
(0.80)a
(0.18)

3.13
3.75
2.82
3.07
5.57
3.19
3.76
5.75

3
(0.53)
(1.41)b
(0.92)a
(0.45)b
(0.92)
(0.27)

(0.53)b
(0.71)

2.94
3.19
2.19
2.82
5.69
3.01
3.57
5.82

P-value

7
(0.44)
(0.44)bc
(0.85)b
(0.26)bc
(0.27)
(0.18)
(0.26)b
(0.45)

2.94
1.38
1.69
2.26
5.63
3.88

2.5
5.63

(0.79)
(0.18)c
(0.27)b
(0.18)c
(0.71)
(0.35)
(0.0)c
(0.0)

0.091
0.008
0.004
0.002
0.642
0.122
0.011
0.454

Values with di¡erent letters within an attribute are signi¢cantly di¡erent (analysis of variance, Po0.05).

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Selenium enrichment of African cat¢sh part II E Schram et al.

Table 8 Correlation matrix for the intensity scores of the sensory attributes of the ¢llets during the depuration period for the
selenium-enriched African cat¢sh

Attribute

R__O__metal

R__O__garlic

R__O__sour

C__O__garlic

C__O__cook potato

C__T__musty

C__T__garlic

R_O_metal
R_O_garlic
R_O_sour
C_O_garlic
C_O_cook potato
C_T_musty
C_T_garlic
C_T_cook potato

1

0.814
1

0.766
0.894ÃÃ
1

0.879ÃÃ
0.979ÃÃ
0.888ÃÃ
1

À 0.725
À 0.467
À 0.459
À 0.545
1

0.427
0.060
À 0.023
0.216
À 0.653
1

0.821
0.949ÃÃ

0.916ÃÃ
0.958ÃÃ
À 0.354
0.005
1

C__T__cook
potato
À 0.329
À 0.396
À 0.384
À 0.516
0.392
À 0.537
À 0.416
1

ÃÃFor values marked with the correlation is signi¢cant (the two-tailed Pearson correlation test, Po0.01).

odour (Table 8). The intensity scores of the other sensory attributes were the same for all sampling days
during the depuration period (Table 7). No o¡ £avours
were detected in the ¢llet samples.

Discussion
Total selenium levels and selenium retention
in relation to the length of enrichment period
The daily selenium intake among Europeans is on
average approximately 100 mg short of the recommended daily intake of 150 mg day À 1.We aimed to ¢ll
this gap with one 150 g portion of selenium-enriched
African cat¢sh ¢llet, which demands a ¢llet selenium

concentration of approximately 0.70 mg Se kg À 1.
This target concentration was reached after an enrichment period of 10 days before harvest, using a
dietary selenium level of 11.7 mg kg À 1. This clearly
demonstrates that selenium enrichment of African
cat¢sh can be achieved by feeding selenium-enriched
feeds for a relatively short period before harvest.
We therefore consider the selenium-enriched feed a
¢nishing diet. Finishing diets for farmed ¢sh have
been studied for Atlantic salmon (e.g. Jobling, Larsen,
Andreassen, Olsen & Sigholt 2002; Bell, Henderson,
Tocher & Sargent 2004) and red seabream (Glencross
et al., 2003), all successfully aiming to boost n-3 polyunsaturated fatty acid (PUFA) levels before harvest to
compensate for initially reduced n-3 PUFA levels due
to growing ¢sh on feeds containing vegetable oils.
Finishing diets aiming to boost levels in farmed ¢sh
of nutrients important in human nutrition other
than n-3 PUFA, such as selenium, have not been
studied to date. Apart from the current study, there
are no records of studies aiming to optimize the
enrichment process in terms of the minimal required

800

feeding period for ¢nishing diets to reach target levels
at harvest.
The relation between length of the enrichment
period and selenium level in the ¢llet of African cat¢sh as established in the present study, enables the
design of a feeding programme that targets a certain
selenium level in the ¢llet. We expect, however, that
this relation is exclusive to the currently used dietary

selenium level of 13.5 mg kg À 1, but it can be used for
other ¢sh sizes by considering the proportion of selenium-enriched feed in the total feed load rather than
the number of days the selenium-enriched feed was
fed. This was shown by the selenium level in the ¢llet
reached in the second experiment, where we aimed
for a ¢nal total selenium level in the ¢llet of
0.70 mg kg À 1. This selenium level is comparable to
the level reached in treatment 4 of the ¢rst experiment, where ¢sh received 97 g of selenium-enriched
feed per ¢sh, concurring with 19% of the total estimated feed intake per ¢sh (based on the ¢nal mean
weight of 565 g and an estimated overall FCR of
0.9 g g À 1). In the second experiment we used this
result to estimate the required amount of seleniumenriched feed at 180 g per ¢sh (based on a ¢nal individual weight of1kg, an overall FCR of 0.9 g g À 1and a
proportion of selenium-enriched feed of 20% of the
total feed intake). Upon harvest, the African cat¢sh
in the second experiment had reached a mean individual weight over 1kg and a selenium level in the ¢llet
of 0.63 mg kg À 1 (Table 5), which is well in line with
the targeted 0.70 mg kg À 1 Se.
The highest selenium retention from feed to ¢llet
was reached when feeding the ¢sh the seleniumenriched feed during 10 days before harvest. The
higher retention of selenium in the treatment groups
fed selenium-enriched feeds as compared with the
control diet is most likely related to the presence of

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Aquaculture Research, 2010, 41, 793^803

selenomethionine in the selenium-enriched feeds

(Schram et al. 2008), which can substitute for methionine in protein synthesis. As a result, selenomethionine is more easily incorporated in the muscle tissue
of growing ¢sh than the inorganic selenium in the
control feed (Waschulewski & Sunde 1988). The low
retention of selenium obtained after 5 days of selenium enrichment before harvest as compared with
the other treatment groups that received seleniumenriched feed can be explained by the delay between
feed intake and actual incorporation of selenium
compounds in the ¢llet. Such a delay would have
caused that the selenium intake from the last meals
before harvest were not incorporated in the ¢llet. As
this unknown number of last meals before harvest
account for a larger proportion of the total selenium
intake for short enrichment periods, the selenium
retention is underestimated when based on the full
enrichment period of 5 days.
The decreasing selenium retention with increasing length of the enrichment period (Fig. 2) can be a
re£ection of a £attening of selenium incorporation
in the ¢llet, which is likely to occur as the amount of
selenium that can be incorporated in the ¢llet is
limited (Schram et al. 2008).

E¡ect of depuration on total selenium level in
the ¢llet
Depuration was found to have no e¡ect on the total
selenium level in the ¢llet for both the seleniumenriched ¢sh and the control treatments (Fig. 3). As
¢llet weight of the selenium-enriched African cat¢sh
did not decrease during the depuration period, despite
the considerable total body weight loss (Table 5), the
absolute amount of selenium in the ¢llet was also not
a¡ected by depuration.

Sensory properties of selenium-enriched
African cat¢sh ¢llets in relation to depuration
The intensity of the garlic odour and £avour of the
African cat¢sh samples were found to decrease signi¢cantly during the ¢rst 2 days of the depuration
period (Fig. 4), showing that depuration is probably
an e¡ective measure to eliminate the e¡ects of dietary garlic on the sensory properties of the ¢nal product, without a¡ecting other sensory characteristics
(Table 7).
As we were mainly interested in the changes of the
sensory properties of the garlic-fed African cat¢sh

Selenium enrichment of African cat¢sh part II E Schram et al.

during the depuration period and due to the absence
of information on the sensory properties of garlic-fed
¢sh, we used a sensory intensity test in this experiment and not a sensory di¡erence test nor a sensory
threshold test. Using a sensory di¡erence test would
have enabled us to detect di¡erences in the sensory
properties of the garlic-fed and the non-garlic-fed
African cat¢sh for each of the individual sampling
days during the depuration period. However, we
would not have been able to detect any changes in
the sensory properties during the course of the depuration period. Using a sensory threshold test we
would have been able to determine exactly the required length of the depuration period to eliminate
garlic odours and £avours, but for such a test, preliminary information on the changes in the sensory
intensity during the course of the depuration period
is needed.
This means that the current results give a strong
indication that it is possible to reduce the garlic £avour of the garlic-fed African cat¢sh to minimal sensory detection levels within 3 days of depuration, but
a sensory threshold test including samples of both
garlic-fed and non-garlic-fed ¢sh is needed to con¢rm

this.
During the experiment, a strong garlic-like odour
could be smelled in the experimental room, suggesting that African cat¢sh excrete compounds causing a
garlic odour after garlic consumption, but this remains to be con¢rmed by measurements of garlic
odour causing compounds. Odours resulting from
garlic consumption have been studied in humans.
Garlic odour in human breath following garlic consumption is caused by allyl mercaptan, a water soluble and indigestible sulphide compound that is taken
up in blood from the intestinal tract, followed by excretion via the skin and lungs (Takeshi, Boku, Inada,
Morita & Okazaki 1989). A similar route in ¢sh can
explain our observations in this study. Potential excretion routes of compounds causing garlic odour in
¢sh include faeces and urine, but after uptake of the
compounds from the intestinal tract in the blood, excretion (of metabolites) can also take place via the
gills.
Next to uptake from the intestinal tract, the rearing water contaminated by faeces or urine with compounds causing garlic £avours could be considered
as a source of these compounds in the ¢llets, as ¢sh
can take up contaminants from the surrounding
water via the gills (Streit 1998). Either way, uptake
in the blood of compounds causing garlic odour and
£avour is needed to explain their presence in the

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Selenium enrichment of African cat¢sh part II E Schram et al.

African cat¢sh ¢llets, not necessarily followed by incorporation from the blood in ¢llet tissues as the ¢sh
were not bled during slaughter.

All in all we consider excretion via the gills of compounds responsible for garlic odour and £avour, combined with removing their source by cessation of
dietary garlic intake, a plausible explanation for the
reduction in the intensity of these attributes in the
African cat¢sh ¢llets during depuration.
Selenium enrichment of market-sized African
cat¢sh
We previously established that African cat¢sh can be
enriched with selenium via the diet (Schram et al.
2008). However, these results were obtained in African
cat¢sh of on average 424 g at the end of the experiment
whereas market size of African cat¢sh is generally over
1kg. In Experiment 2 of the present study, the ¢nal
mean (SD) body weight was 1080 (10) g and a mean
(SD) total selenium level of 0.63 (0.02) mg kg À 1 in the
selenium-enriched ¢sh. Although a direct comparison
cannot be made as for the di¡erences in enrichment
periods and dietary selenium levels, these current ¢ndings show that selenium enrichment can also be
reached in market-sized ¢sh.
Conclusions
Based on the current study we conclude that the
length of the selenium-feeding period is an important factor a¡ecting the selenium level in the ¢sh ¢llet and that selenium enrichment during a relatively
short period before harvest is su⁄cient to reach target levels. In addition we conclude that depuration
has no e¡ect on total selenium level in the ¢sh ¢llet,
whereas o¡ £avours associated to dietary garlic are
e¡ectively removed by a short depuration period.
Acknowledgments
This study was part of SEAFOODplus, an integrated
research project ¢nancially supported by the European Commission under FP6 (food
plus.org). The authors would like to express their gratitude to Chris Kik and Olga Scholten, Plant Research
International, Wageningen UR, the Netherlands, for

the production and supply of garlic, Marion Hoek
van Nieuwenhuizen and Afke Stein for the total selenium analysis, Fleuren-Nooijen, Someren, the Netherlands, for the supply of the experimental ¢sh,
Research Diet Services, Wijk bij Duurstede, the

802

Aquaculture Research, 2010, 41, 793–803

Netherlands, for the production of the experimental
feeds, Mercedes Careche, CSIC, Madrid, Spain, for project management and the members of the sensory panel of Wageningen IMARES for the sensory analysis.
References
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Glencross B., Hawkins W. & Curnow J. (2003) Restoration of
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T. (2002) In£uence of a dietary shift on temporal changes
in fat deposition and fatty acid composition of Atlantic
salmon post-smolt during the early phase of seawater
rearing. Aquaculture Research 33, 875^889.
Larsen E.H., Lobinski R., Burger-Meyer K., Hansen M.,
Ruzik R., Mazurowska L., Rasmussen P.H., Sloth J.J.,
Scholten O. & Kik C. (2006) Uptake and speciation of selenium in garlic cultivated in soil amended with symbiotic
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Rayman M.P. (2000) The importance of selenium to human
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Schram E., Pedrero Z., CaŁmara C.,Van der Heul J.W. & Luten
J.B. (2008) Enrichment of African cat¢sh with functional

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Takeshi M., Boku T., Inada K., Morita M. & Okazaki Y.
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Aquaculture Research, 2010, 41, 804^813

doi:10.1111/j.1365-2109.2009.02357.x

Influence of the length of time after hormonal
stimulation on selected parameters of milt of
ide Leuciscus idus L.

Beata Irena Cejko1, Radosław Kajetan Kowalski1, Dariusz Kucharczyk2, Katarzyna TargonŁska2,
Sławomir Krejsze¡2, Daniel Z_ arski2 & Jan Glogowski1,3
1

Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of

Sciences, Olsztyn, Poland
2

Department of Lake and River Fisheries, Faculty of Environmental Sciences and Fisheries, University of Warmia and Mazury,

Olsztyn, Poland
3

Department of Ichthyology, Faculty of Environmental Sciences and Fisheries, University of Warmia and Mazury, Olsztyn,
Poland

Correspondence: B I Cejko, Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish
Academy of Sciences, 10-747 Olsztyn, Poland. E-mail:

Abstract

Introduction

Milt of the Leuciscus idus L. was collected from ¢ve experimental groups, and selected parameters of its
quality were analysed for 36 h (group II), 60 h (group
III), 84 h (group IV) and 108 h (group V), respectively,
after hormonal stimulation with Ovopel (1granule kg À 1 of body weight). The control (group I) ¢sh
were not subjected to hormonal stimulation. The
highest milt volume was obtained from the ¢sh in

group IV (0.70 Æ 0.55 mL), where the largest volume
of milt expressed per kilogram was also obtained
(3.03 Æ 1.94 mL kg À 1). Signi¢cant di¡erences were
also found in milt volumes obtained between group
I and groups III (Po0.01) and IV (Po0.05). The highest percentage of motile spermatozoa was found in
the milt of group IV (59%); signi¢cant di¡erences
were found between group I and groups II (Po0.01)
and III (Po0.001). The value of osmotic pressure of
seminal plasma was the highest in group IV
(203.19 Æ 37.63 mOsm kg À 1), and the lowest in
group I (118.31 Æ 41.13 mOsm kg À 1). Parameters determining milt quality and quantity indicate that the
period of 60^84 h after hormonal stimulation with
Ovopel is optimal for obtaining milt from ide.

Ide Leuciscus idus L. is a reophileous cyprinid ¢sh that
inhabits the rivers of Central and Eastern Europe as
well as Asia. The spawning period of this ¢sh in Poland occurs from April to May and lasts for up to 10
days (Tadajewska 2000). The ide is of little economic
signi¢cance but is an important natural component
of the aquatic ecosystem because it increases the production potential of rivers by occupying niches that
other ¢sh are unable to (Błachuta 1998). This ¢sh is
also important in recreational ¢sheries and in the
propagation of ornamental ¢sh.
The study of the biology of reophileous cyprinid
¢sh reproduction is mainly concerned with the production of quality stocking material in hatcheries
and developing arti¢cial spawning. This is because
river regulations and pollution have caused declines
in ide populations in Polish waters (Kruk 2007). Females and males of the genus Leuciscus are also sensitive to negative changes in antropogenic factors in
natural waters. The production of stocking material
under controlled conditions can prevent a dramatic

decline in the number of reophileous ¢sh in the ecosystem and signi¢cantly increase the number of endangered ¢sh species in open waters.
In the available literature, little attention has been
paid to the genus Leuciscus males and the rudimentary information focuses on the characteristics of the

Keywords: Leuciscus idus L., milt, hormonal stimulation, seminal plasma, motility of spermatozoa,
Ovopel

804

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Journal Compilation r 2009 Blackwell Publishing Ltd


Aquaculture Research, 2010, 41, 804^813

basic determinants of their milt quality, i.e. the volume of milt obtained, motility and concentration of
spermatozoa (Kucharczyk, Kujawa, Mamcarz,Wyszomirska & Ulikowski 1999; Kucharczyk, Borejko, TargonŁska, Roz’ ek, Chwaluczyk, Kowalski & Glogowski
2007; Krejsze¡, Kucharczyk, Kupren, TargonŁska,
Mamcarz, Kujawa, Kaczkowski & Ratajski 2008; TargonŁska, Kucharczyk, Mamcarz, Glogowski, Krejsze¡,
PrusinŁska & Kupren 2008). Only a few publications
have provided information on the cryopreservation
of milt of such ¢sh (Lahnsteiner, Berger, HorvaŁth, Urbanyi & Weismann 2000), detailed characteristics of
spermatozoa motility parameters using the computer-assisted sperm analysis (CASA) system (Kowalski,
Sarosiek, Kucharczyk,TargonŁska & Glogowski 2006),
proteolytic enzyme activity (Kowalski, Glogowski,
Kucharczyk, Goryczko, Dobosz & Ciereszko 2003) or
their inhibitors in seminal plasma (Wojtczak, Glogowski, Kołdras, Kucharczyk & Ciereszko 2003).
During spawning, all reproductive functions of
both female and male ides must be maintained. The
temperature noted during the ide spawning period

£uctuates between 8 and 15 1C and this range is also
recommended at hatcheries (Kupren 2005). Spermatozoa in males mature in spermatic ducts and are
hormonally (11-ketotestosterone and 17a, 20b-dihydroxy-4-pregnen-3-one) controlled; the entire process of spermatogenesis is conditioned by
environmental factors and is linked to the reproduction strategy of the given species. The milt produced
by the male is characterized by both individual and
seasonal variability (Kruger, Smit, Van Vuren & Ferreira 1984; Munkittrick & Moccia 1987; Aas, Refstie &
Gjerde 1991). This applies to spermatozoa morphology as well as to organic components and the biochemical parameters of seminal plasma (Glogowski,
˛
KwasŁnik, Piros, Dabrowski,
Goryczko, Dobosz, Ku$minŁski & Ciereszko 2000). Reproduction success is
in£uenced by the quality of gametes produced by
the ¢sh. Based on motility and spermatozoa concentration, we can evaluate the biological quality of milt
and the propensity of spermatozoa for fertilization.
Consequently, those parameters are characterized
¢rst and referred to as the basic ones (Kruger et al.
1984; Rurangwa, Kime, Ollevier & Nash 2004).
It is impossible to conduct controlled reproduction
of ¢sh from genus Leuciscus without hormonal stimulation (Kucharczyk 2002; Krejsze¡ et al. 2008).
Controlled reproduction of those ¢sh is conducted
on the basis of stimulation using substances of natural origin, i.e. human chorionic gonadotropin (hCG),
carp pituitary homogenate, pure gonadotropin-

Hormonal stimulation of ide males B I Cejko et al.

releasing hormone (GnRH, LH-RH) or synthetic gonadotropin releasing-hormone analogues, i.e. sGnRHa
and mGnRHa (Kucharczyk et al. 1999; Kucharczyk
2002; Jamro¤z, Kucharczyk, HakucŁ^Błaz’ owska, Krejsze¡, Kujawa, Kupren, Kwiatkowski,TargonŁska, ’arski,
Cejko & Glogowski 2008; Krejsze¡ et al. 2008; Kucharczyk, TargonŁska, ’arski, Kujawa & Mamcarz
2008).
The ¢rst synthetic analogue to the reproduction of

reophileous ¢sh was the Hungarian preparation Ovopel (HorvaŁth, Szabo¤ & Burke 1997), a mamalian analogue GnRH (D-Ala6Pro9NEt-mGnRH) with a
dopamine receptor antagonist, i.e. metoclopramide.
Ovopel has been used in ¢shery practice for over 10
years because of its wide application in the controlled
reproduction of numerous ¢sh species, high e¡ectiveness (ovulation/spermation), low purchase costs in
comparison with hCG and sGnRH in Poland and ease
of preparation (Brzuska 2001; Kucharczyk, Szczerbowski, yuczynŁski, Kujawa, Mamcarz, Wyszomirska,
˛ 2002;
Szabo¤ & Ratajski 2001; Brzuska & Białowas
Kowalski, Hliwa, Andronowska, Kro¤l, Dietrich,Wojtczak, StabinŁski & Ciereszko 2006; Krejsze¡ et al. 2008).
Dosages of Ovopel and the results of controlled reproduction of females (ovulation, latency time and
survival of the eyed-egg stage) and males (spermation, milt volume and motility of spermatozoa) belonging to genus Leuciscus were described by
Kucharczyk et al. (2008). Based on long-time studies,
it can be concluded that Ovopel also yields better results in the controlled reproduction of ide males in
natural and out of spawning seasons in comparison
with CPH and hCG (Kucharczyk et al. 1999; Kucharczyk 2002). Among GnRH analogue preparations,
Ovaprim (a salmon analogue GnRH, D-Arg6Pro9
NEt-sGnRH with a dopamine receptor antagonist
domperidone) also yields good results in ide males’
maturity stimulations (Kucharczyk et al. 2007; Jamro¤z et al. 2008). However, because of the higher cost
e¡ectiveness of Ovopel, we decided to use this preparation in the experiments presented.
The type of hormonal preparation, the dose applied and the time between the stimulation performed and obtaining the milt signi¢cantly
determine the in£uence on the e¡ects of reproduction and depend on the species that is subject to the
treatment. Spermatozoa of carp Cyprinus carpio L. are
present in the testicles all year round (Kołdras, Bieniarz & Kime 1990) while in the case of trout Oncorchynhus mykiss (Walbaum), they are found during the
reproductive season, which occurs in the spring or
autumn only (Billard 1986). Also, maturation con-

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805


Hormonal stimulation of ide males B I Cejko et al.

trolled in both species by similar hormones di¡ers
signi¢cantly in its dynamics. In salmonids, the discharge of gonadotropins occurs gradually over several days before attaining maturity (Munkittrick &
Moccia 1987), but in case of carp it takes just hours
(Billard, Cosson, Perchec & Linhart 1995). Thus, hormonal stimulation of cyprinids can be limited to several hours, while in case of salmonids it takes up to
several weeks. The temperature of water that holds
the spawners in£uences the dynamics of maturation.
Probably, as compared with carp, other cyprinids, i.e.
reophileous ¢sh spawning in colder water need
slightly more time from carp stimulation until full
spawning readiness.
Considering the poor literature resources on reophileous ¢sh, and in particular the issues related to
the biology of their milt, the goal of this study was to
determine the in£uence of the time after hormonal
stimulation on selected quality parameters of milt of
ide. Additionally, we attempted to determine the molecular weights of seminal plasma protein pro¢les for
that species.

Aquaculture Research, 2010, 41, 804^813

tains 18^20 mg D-Ala6Pro9 Net-mGnRH and 8^10 mg
metoklopramide (HorvaŁth et al. 1997). After 36 h from
the commencement of the experiment, males of
groups I and II were examined and milt was collected
using sterile syringes by delicately massaging the abdominal parts and taking care to avoid contamination

of samples with urea, faeces or blood. After 60 h (group
III) of conducting the stimulation, another group of
males was obtained from the tanks and milt was obtained as described above. Further samples were obtained after 84 h (group IV) and 108 h (group V) of
administration of Ovopel to the males. The control
(group I) consisted of ¢sh that were not treated with
any hormonal preparations. Before milt collection, individuals of each group were weighed and then the
quantity of milt obtained from them was measured.
Milt was collected after anaesthesia was administered
2-phenoxyethanol (Sigma, St Louis, MO, USA) at
0.5 mL L À 1 water. The milt obtained was transported
on ice (14 1C) to the Department of Gamete and Embryo Biology, Institute of Animal Reproduction and
Food Research of the Polish Academy of Sciences in
Olsztyn, where further analyses were carried out.

Materials and methods
Determination of the basic parameters of milt
Origin and transport of ¢sh
Male ides originated from the Knieja Fish Farm situated near Cze˛ stochowa and they belonged to a 3year-old breeding stock. In April 2008, the ¢sh
caught from the ponds were transported to the
hatchery of the Department of Lake and River Fishery of the University of Warmia and Mazury in Olsztyn, where they were placed in tanks with water at
10 1C. All males were divided into ¢ve experimental
groups, i.e. group I ^ control (n 515) and group II
(n 512), from which milt was collected after 36 h;
group III (n 511), from which milt was collected after
60 h; group IV (n 512), from which milt was collected
after 84 h; and group V (n 512), from which milt was
collected after 108 h from hormonal stimulation.
After 3 days of adaptation, the water temperature in
the tanks was increased to 12 1C and maintained at a
constant level until the end of the experiments.

Motility of ide spermatozoa was determined by a subjective method using a light microscope under
 400 magni¢cation. Spermatozoa were activated
by mixing 1 mL of milt with 30 mL of activation solution that contained 120 mM NaCl (Sigma). Motility
was determined immediately after activation by one
observer and the value of motile spermatozoa was
subjectively estimated in per cent (%). The concentration of spermatozoa was determined using the spec˛
trophotometric method (Ciereszko & Dabrowski
1993) by diluting the milt with 0.7% NaCl at 1:1000.
Absorption was measured on a Beckman DU-640
spectrophotometer (Analytical Instruments, LLS,
Golden Valley, MN, USA) at a 5 530 nm. Absorption
measurement results were then applied to the standard curve formula prepared earlier for ide using the
Bˇrker chamber (cytometric method) and the concentration values were calculated (109 mL À 1).

Hormonal stimulation and manipulations
with spawners

Determination of total protein content and
seminal plasma osmotic pressure

Hormonal stimulation was performed with an intraperitoneal injection of a single dose of Ovopel (Unictrade, Hungary) at 1granule kg À 1 (one granule con-

Seminal plasma was obtained by the centrifugation
of milt batch (10 000 g) for 10 min and next the
supernatant obtained was transferred into test tubes

806

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Aquaculture Research, 2010, 41, 804^813

Hormonal stimulation of ide males B I Cejko et al.

and stored at À 80 1C until analyses. The total protein content in seminal plasma (mg mL À 1) was determined using the method presented by Lowry,
Rosenbrough, Farr and Randall (1951), and the value
of osmotic pressure (mOsm kg À 1) was measured
using a vapor pressure osmometer 5520 (WESCOR,
Logan, UT, USA).

Separation of proteins using denaturing
electrophoresis SDS-PAGE
Electrophoresis was conducted in 12.5% polyacrylamide gels using a horizontal electrophoresis unit
Hoefer SE 250 Mighty Small II (Hoefer-Amersham
Biosciences, Piscataway, NJ, USA). Seminal plasma
samples with a known total protein content were diluted with 0.85% NaCl (Sigma) to obtain the overall
concentration of protein in the sample at 30 mg. Next,
10 mL of a twice-concentrated staining agent was
added to each sample, which were then boiled for
5 min at 100 1C. Electrophoretic separation was carried out in an electrode bu¡er (0.025 M Tris-HCl,
0.192 M glycine, 0.1% SDS) at pH: 8.3 in 200 V and
40 mA for 1.5 h (for two gels).

albumin (67.0 kDa), b-galactosidase (120 kDa) and
myosin (203 kDa), Bio-Rad, Hercules, CA, USA] using
DETERMINATION OF MOLECULAR WEIGHT software (http://
insilico.ehu.es/mini_tools/molecular_weight/).

Statistical analysis
The results obtained were characterized using the
arithmetic average and standard deviation (Æ SD).
The signi¢cance of the di¡erences between the
groups of ¢sh examined for the analysed characteristic was veri¢ed using the non-parametric Kruskal^
Wallis test (one-way ANOVA) using the GRAPHPAD PRISM
4 software (GraphPad Software Inc., USA).

Results
After the administration of Ovopel to male ides, the
milt was obtained from all individuals in each experi-

Table 2 Volume of milt, total number of spermatozoa in
milt and number of spermatozoa per kilogram body weight
from ide Leuciscus idus L. analyses group after hormonal stimulation with OvopelÃ

Fish group

Total
Volume
number of
spermatozoa
of milt
(mL kg À 1 b.w.) in milt (109)

I (n 5 15)
II (n 5 12)
III (n 5 11)
IV (n 5 12)

V (n 5 12)

0.78
1.52
2.44
3.03
2.78

Staining of gels and documentation of results
Following electrophoresis, the gels were stained
(0.025% Coomassie brilliant blue, 40% methanol and
7% acetic acid) and discoloured (discolouration agent
I: 40% methanol17% acetic acid and discolouration
agent II: 7% acetic acid15% methanol). The gels prepared were then documented using a photo camera.
The molecular weights of protein pro¢les were estimated on the basis of protein standard [aprotinin
(6.4 kDa), lysozyme (20 kDa), trypsin inhibitor (28 kDa),
carbonic anhydrose (34.1kDa), ovalbumin (43.0 kDa),

Æ
Æ
Æ
Æ
Æ

0.36
0.85
2.01
1.94
1.61

1.41
3.28
7.00
7.03
6.48

Æ
Æ
Æ
Æ
Æ

1.54
1.97
6.15
5.08
4.79

Number of
spermatozoa
in milt
(109 kg À 1 b.w.)
7.27
14.26
26.52
30.82
32.16

Æ
Æ

Æ
Æ
Æ

7.81
9.29
18.22
16.84
26.26

b.w., body weight.
ÃFish group: I, control; II, milt obtained after 36 h; III, milt obtained after 60 h; IV, milt obtained after 84 h; V, milt obtained
after 108 h after hormonal stimulation with Ovopel.

Table 1 Selected parameters of quality of ide Leuciscus idus L. milt analyses group, obtained after hormonal stimulation with
OvopelÃ

Fish group

Fish body
weight (g)

I (n 5 15)
II (n 5 12)
III (n 5 11)
IV (n 5 12)
V (n 5 12)

217.60
209.75

180.58
218.25
215.83

Æ
Æ
Æ
Æ
Æ

Volume of
milt (mL)
49.91
25.61
102.45
40.80
45.40

0.17
0.35
0.65
0.70
0.61

Æ
Æ
Æ
Æ
Æ

0.07
0.18
0.67
0.55
0.39

Percentage
of motility
spermatozoa (%)
22.80
45.83
51.82
58.75
47.50

Æ
Æ
Æ
Æ
Æ

21.39
18.81
22.72
14.79
16.03

Concentration of
spermatozoa
(109 mL À 1)

8.75
9.05
11.86
11.29
11.04

Æ
Æ
Æ
Æ
Æ

5.13
1.83
3.02
3.29
4.00

Total protein
content
(mg mL À 1)
1.97
1.84
1.88
1.82
1.98

Æ
Æ
Æ

Æ
Æ

1.10
1.08
0.91
0.82
0.80

Osmotic
pressure
(mOsm kg À 1)
118.31
171.83
191.20
203.19
176.15

Æ
Æ
Æ
Æ
Æ

41.13
50.00
39.56
37.63
35.50

ÃFish group: I, control; II, milt obtained after 36 h; III, milt obtained after 60 h; IV, milt obtained after 84 h; V, milt obtained after 108 h

after hormonal stimulation with Ovopel.

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807


Hormonal stimulation of ide males B I Cejko et al.

mental group. The largest milt volume was obtained
from ¢sh of group IV (0.70 Æ 0.55 mL), 84 h after hormonal stimulation. It was also the largest volume of
milt (3.03 Æ 1.94 mL) per kilogram of body weight
of males (Tables 1 and 2). The lowest milt volume was
obtained from males of group I, i.e. control

Aquaculture Research, 2010, 41, 804^813

(0.17 Æ 0.07 mL). Also in this case the volume of milt
per kilogram of body weight was the lowest
(0.78 Æ 0.36 mL) (Tables1and 2). A statistically significant di¡erence was found in volumes of samples obtained between group I and group III (Po0.01) as well
as between groups I and IV (Po0.05) (Fig. 1a). When

Figure 1 In£uence of the length of time after hormonal stimulation on selected parameters of milt of ide Leuciscus idus
L. analyses group. (a) Volume of milt (mL); (b) percentage of motility spermatozoa (%); (c) concentration of spermatozoa
(109 mL -1); (d) total protein content in seminal plasma (mg mL-1); (e) seminal plasma osmotic presure (mOsm kg-1);
(f) volume of milt (mL kg-1 b.w.); (g) total number of spermatozoa in obtained milt (10 9); (h) number of spermatozoa in milt
(109 kg-1 b.w.) analyses group. Data are shown as box-and-whiskers plots (lower whisker: minimum; lower box line: 25th

percentile; middle box line: median; upper box line: 75th percentile; upper whisker: maximum). Boxes labelled with di¡erent superscripts are statistically di¡erent from each other (Po0.05). Fish group: I, control; II, milt obtained after 36 h; III,
milt obtained after 60 h; IV, milt obtained after 84 h;V, milt obtained after 108 h after hormonal stimulation with Ovopel.

808

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Aquaculture Research, 2010, 41, 804^813

the volume of milt was expressed per kilogram of
body weight, a di¡erence was showed between group
I and groups IV (Po0.01) and V (Po0.01) (Fig.1f).
The percentage of motile spermatozoa after activation was low in all groups (Table 1). The highest value
was determined in group IV (59%) and the lowest in
group I (23%). In the other groups, spermatozoa motility was at the level of 46%, 52% and 48%, respectively, in groups II, III and V (Table 1). There were
signi¢cant di¡erences in the motility of spermatozoa
between groups I and II (Po0.01) and between
groups I and III (Po0. 001) (Fig. 1b). The highest milt
production expressed as a billion of spermatozoa per
kilogram body weight was found 108 h after treatment of the males with Ovopel, i.e. in group V
(32.16 Æ 26.26 Â 109 kg À 1 b.w.), while the lowest
number was recorded in case of the controls
(7.27 Æ 7.81 Â 109 kg À 1 b.w.) (Table 2). Signi¢cant
di¡erences in this parameter were found between
the control group and groups III (Po0.05), IV
(Po0.001) and V (Po0.01) (Fig. 1h).
The concentration of spermatozoa in milt of
groups III, IV and V was at a similar level (11.86 Æ

3.02; 11.29 Æ 3.29 and 11.04 Æ 4.00 Â 109 mL À 1 respectively) and slightly higher in group III. Lower
values of this parameter were recorded for groups
I (8.75 Æ 5.13 Â 109 mL À 1) and group II (9.05 Æ
1.83 Â 109 mL À 1), i.e. in the groups where milt was
collected 36 h after commencement of the experiment (Table 1). No statistically signi¢cant di¡erences

Hormonal stimulation of ide males B I Cejko et al.

(P40.05) were found between tested groups of ¢sh in
the concentration of spermatozoa in milt (Fig. 1c).
The total protein content in seminal plasma
reached very similar levels in all the groups, although
in groups II, III and IV it was slightly lower
(1.84 Æ 1.08, 1.88 Æ 0.91 and 1.82 Æ 0.82 mg mL À 1
respectively) than in the other groups (group I:
1.97 Æ 1.10 mg mL À 1 and group V: 1.98 Æ 0.88 mg
mL À 1) (Table 1). Similar to the concentration of spermatozoa, no statistically signi¢cant (P40.05) di¡erences were found between the groups (Fig. 1d).
Based on the electrophoretic images obtained, it can
be concluded that the protein pro¢les of tested groups
are characterized by an identical distribution of proteins in ide seminal plasma. The main seminal plasma proteins of that species are characterized by
molecular weights of 52, 26 and 6 kDa, while proteins with a molecular weight of 52 kDa clearly dominate (Fig. 2).
The higher seminal plasma osmotic pressure value
was determined in group IV (203.19 Æ 37.63 mOsm
kg À 1) and the lowest in group I (118.31 Æ
41.13 mOsm kg À 1). Seminal plasma osmolality of the
other groups was at similar levels (group II: 171.83 Æ
50.00 mOsm kg À 1, group III: 191.20 Æ 39.56 mOsm
kg À 1 and group V: 176.15 Æ 35.50 mOsm kg À 1, Table
1). Di¡erences in seminal plasma osmotic pressure
were found to be statistically signi¢cant between

group I and groups II (Po0.01) and III (Po0. 001)
(Fig. 1e).

Figure 2 Electrophoretic pro¢les of seminal plasma proteins of ide Leuciscus idus L. analyses group. Fish group: I, control; II, milt obtained after 36 h; III, milt obtained after 60 h; IV, milt obtained after 84 h;V, milt obtained after 108 h after
hormonal stimulation with Ovopel. Molecular mass of protein standards electrophoresed in gel are given in the column to
the right of the gel and approximate molecular mass of protein detected are given in the column to the left of the gel.

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809


Hormonal stimulation of ide males B I Cejko et al.

Discussion
In the studies by Kucharczyk et al. (1999), hormonal
stimulation of male ide during the reproductive season using CPE and Ovopel caused a double increase
in the volume of obtained milt as compared with the
control group. Signi¢cant di¡erences were also recorded in the motility of spermatozoa between the
control group (52%) and groups hormonally stimulated using hCG (77%), CPE (76%) and Ovopel (75%).
Also, in case of chub Leuciscus cephalus L., treatment
with the same substances resulted in increased milt
volume and better biological quality of ejaculate obtained as compared with the non-stimulated control
group (Krejsze¡ et al. 2008). Only in the case of dace
Leuciscus leuciscus L. did hormonal stimulation not
cause clear changes in the quantity and quality of obtained milt (Kucharczyk 2002).
In our studies, increased milt volume was found in
each of the test groups after treatment with Ovopel,
and it was the highest 84 h after injection (group IV)

(Table 1). It should also be pointed out that 36 h after
injection, the quantity of milt obtained in group II
was already twice as large as that in the control
group. This also applies to the quantity of milt obtained per kilogram of body weight of the males
(Table 2). An increase in the volume of milt possessing better spermatozoa motility parameters is also
characteristic for hormonal stimulation in case of
other ¢sh species including bream Abramis brama
(L.) (Kucharczyk, Kujawa, yuczynŁski, Glogowski, Babiak & Wyszomirska 1997), yellow perch Perca £aves˛
cens Mich. (Dabrowski,
Ciereszko, Ramseyer, Culver
& Kestemont1994) and European perch Perca £uviatilis L. (Kucharczyk, Kujawa, Mamcarz, Skrzypczak &
Wyszomirska 1998). From the obtained milt volume
[mL kg À 1 body weight (b.w.)] and number of spermatozoa per kilogram of spawner’s body weight, it can
be concluded that those values increased over time
(Table 2). This indicates that hormonal stimulation
positively in£uences the maturation of milt of that
¢sh species.
The percentage of motile spermatozoa in each
group was low and did not exceed 60% (Table 1).
Low motility values (group I: 23%, group II: 46% and
group V: 47% respectively), which correspond to low
seminal plasma osmotic pressure values (group I:
118.31 Æ 41.13, group II: 171.83 Æ 50.00 mOsm kg À 1
and group V: 176.15 Æ 35.50 mOsm kg À 1 respectively), indicate that milt was contaminated with urine during collection, which occurs frequently under
controlled conditions in many ¢sh species (Glogows-

810

Aquaculture Research, 2010, 41, 804^813

ki et al. 2000; Bokor, Mˇller, Bercse¤nyi, HorvaŁth, UrbaŁnyi & HorvaŁth 2007). It should also be pointed out
that the seminal plasma osmolality determined was
lower for reophileous ¢sh than presented in the literature (Hliwa, Kro¤l, Kowalski & Glogowski 2003; Kowalski et al. 2003; Glogowski, Kowalski & Ciereszko
2007; Cejko, Kucharczyk, TargonŁska, Kubiak, Sarosiek & Glogowski 2008).
During the peak of the reproductive season, milt
parameters such as spermatozoa concentration in
milt, total protein content, osmotic pressure and
seminal plasma ionic composition assume values optimal for the given species. The spermatozoa concentration in genus Leuciscus ¢sh is within a relatively
wide range and the values determined in our study
di¡er from those presented by Kowalski et al. (2003)
for chub (15.26 Â 109 mL À 1) and by Glogowski et al.
(2007) for ide and dace (5.70 and 4.34 Â 109 mL À 1
respectively). Our results are similar to the data presented by Kucharczyk et al. (2007), where, after treatment of males with Ovopel, the concentration of
spermatozoa in milt of wild ide and ornamental form
of these ¢sh, i.e. Leuciscus idus Auber. Orfus was 8.4
and 9.8 Â 109 mL À 1 respectively. This may be linked
(in both cases) to an application of the same spermation-inducing procedure (hormonal preparation
Ovopel, dose:1granule kg À 1 b.w.).
Proteins are the main organic component of Teleostei ¢sh seminal plasma, and their concentration in
the seminal plasma is low and does not exceed
3 mg kg À 1 (Loir, Labbe, Maisse, Pinson, Boulard,
Mourot & Chambeyron 1990). From the literature
data on the content of proteins in reophileous ¢sh
seminal plasma, it can be concluded that depending
on the ¢sh species they assume the values within a
relatively wide range of 1.25 mg mL À 1 for ide,
2.28 mg mL À 1 for chub (Kowalski et al. 2003), 1.95^
3.50 mg mL À 1 for asp Aspius aspius (L.) (Cejko et al.
2008) and 1.24^1.67 mg mL À 1 for barbel Barbus barbus L. (B. I. Cejko, pers. comm.). In this study, these
values were within a narrow range from 1.82 to

1.98 mg mL À 1.
Spermatozoa of cyprinids are maintained in an inactive state in the testes and spermatic ducts due to a
high (300 mOsm kg À 1) seminal plasma osmotic pressure (Morisawa, Suzuki, Shimizu, Morisawa & Yasuda 1983; Redondo-Mˇller, Cosson, Cosson & Billard
1991; Perchec, Jeulin, Cosson, Andre¤ & Billard 1995).
Movement initiation starts in their case when plasma
osmolality decreases below 160 mOsm kg À 1, which
occurs when milt makes contact with water or other
activating solutions (Poupard, Paxion, Cosson, Jeulin,

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Aquaculture Research, 2010, 41, 804^813

Fierville & Billard 1998). During milt collection, uncontrolled (and unwanted) spermatozoa activation
can occur through their contact with urine released
during spermation of spawners (with milt) from the
common urogenital ori¢ce (Rana, Gupta & McAndrew 1992). Most probably, such a situation occurred
in the control group where an osmotic pressure of
118 mOsm kg À 1 was recorded.
Signi¢cant di¡erences in spermatozoa motility
emerged between groups I and II and groups I and
III (Fig. 1b), i.e. the control and groups from which
milt was obtained 36 and 60 h after stimulation.
Also, in case of the seminal plasma osmotic pressure,
signi¢cant di¡erences emerged between groups I and
II and groups I and III (Fig.1e). Our observations indicate that both the value of spermatozoa motility and
seminal plasma osmolality following hormonal stimulation increased until 84 h after treatment of ¢sh
with Ovopel and then decreased gradually, reaching

the motility value of ca. 48% and osmolality value of
ca. 178 mOsm kg À 1 after 108 h from commencement
of the experiment (Table 1, Fig. 1b and e). Changes in
seminal plasma osmolality recorded in the literature
result from milt hydration that occurs during the
peak of the reproductive season and that results from
hormonal stimulation treatment of ¢sh outside the
season (Redondo-Mˇller et al. 1991). In our study,
seminal plasma osmolality increased after Ovopel injection. Statistically signi¢cant di¡erences in this
parameter were found between the control group
(118.31 Æ 41.13 mOsm kg À 1), and groups II (171.83
Æ 50.00 mOsm kg À 1, Po0.01) and III (191.20 Æ
39.56 mOsm kg À 1, Po0.001), and could have resulted from contamination of milt with urine in
group I or increased secretion of ions into seminal
plasma after hormonal stimulation.
On the basis of our studies, it can be concluded that
within 5 days (108 h) of hormonal stimulation treatment, no di¡erences were observed either in the concentration of spermatozoa in the milt or in the
content of seminal plasma total proteins. Signi¢cant
di¡erences between groups (in terms of these parameters) could indicate the presence of uncontrolled
factors or anomalies and they would indicate the beginning of milt ageing that is observed at the end of
the reproductive season. In case of European perch,
with time, changes in spermatozoa motility and
seminal plasma total proteins content were observed
at the beginning (April) and the end (May) of the reproductive season (Kro¤l, Glogowski, Demska-Zake˛ sŁ &
Hliwa 2006). Changes were also observed in the histological image of European perch gonads, where the

Hormonal stimulation of ide males B I Cejko et al.

absence of spermatides in testes in May indicated the
end of the spermatogenesis process after the completion of the reproductive season. In our studies,

changes in milt quality in the obtained sample volume, spermatozoa motility and plasma osmolality
are the e¡ect of hormonal stimulation treatment and
did not result (because of the short duration of the
study) from ageing changes.
The application of hormonal stimulation in controlled ide reproduction appears to be favourable in
the quantity of milt obtained and percentage of motile spermatozoa (Tables 1 and 2, Fig. 1a, b and f), not
causing evident changes in other determinants of its
quality (Table 1, Fig. 1c and d). The basic parameters
determining milt quality, i.e. motility and concentration of spermatozoa determined in our study (Fig. 1b
and c) indicate that the period of 60^84 h with hormonal stimulation treatment is optimal for obtaining
good-quality milt from males of that ¢sh species. This
is, as expected, a period longer than in the case of
carp, which might be related to a lower temperature
of water in which the ide reproduces. A positive in£uence of the applied hormonal stimulation procedure
also manifested through an evident increase in the
number of spermatozoa in milt of ¢sh tested over
time due to stimulation with Ovopel (Table 2).

References
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river ecosystems. Proceedings of Conference reophileous
cyprinids, Brwino¤w 1998, SGGW, pp. 17^21, (in Polish).
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doi:10.1111/j.1365-2109.2009.02358.x

Growth, survival and immune activity of scallops,

Chlamys farreri Jones et Preston, compared between
suspended and bottom culture in Haizhou Bay, China
Zonghe Yu1,2, Hongsheng Yang1, Baozhong Liu1, Qiang Xu1, Kun Xing1,2 & Libin Zhang1,2
1

Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China

2

Chinese Academy of Sciences, Graduate University, Beijing, China

Correspondence: H Yang, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China. E-mail: hshyang@ms.
qdio.ac.cn

Abstract

Introduction

We examined the growth, survival and immune response of the scallop, Chlamys farreri, during a 1-year
period in deep water of Haizhou Bay. Scallops were
cultured using two methods: (1) in lantern nets at a
5 m depth and (2) in a bottom culture system (sleeves)
on the seabed at about a 25 m depth. Shell heights,
meat dry weight and immune activities in the haemolymph (superoxide dismutase and myeloperoxidase) were measured bimonthly or quarterly from
July 2007 to June 2008. Survival was measured at
the end of the study and environmental parameters
in the experimental layers were monitored during
the experiment. The growth and immune activities
of scallops were lower when the water temperature
was high, which was consistent with the main mortality occurring in summer. The growth and immunity of scallops were higher in the suspended

culture than in the bottom culture during the experiment, with the exception of shell growth during the
last study period. Survival of scallops in the suspended culture (54.6 Æ 12.3%) was signi¢cantly lower than that in the bottom culture (86.8 Æ 3.5%) at
the end of this study. We conclude from our results
that the high mortality of C. farreri can be prevented
by culturing them in a bottom culture system before
November of the ¢rst year, and then transferring
them to a suspended culture to improve scallop production.

The scallop, Chlamys farreri (Jones et Presten), is one
of the main aquatic animals cultured in the coastal
waters of northern China. The culture operations
expanded considerably from the 1980s after the successful development of new methods of spat collection, with the total production increasing 42-fold
between 1986 and 1996 (Zhang & Yang 1999a; Guo,
Ford & Zhang 1999). After 1996, mass mortality has
occurred in the summer in most culture areas and
has drastically a¡ected the production of this species
(Xiao, Ford, Yang, Zhang, Zhang & Guo 2005). Such
mortality outbreaks have also been reported for other
bivalves like clams, mussels and oysters (Ho & Zheng
1994; Tremblay, Myrand, Sevigny, Blier & Guderley
1998; Tomaru, Kawabata & Nakano 2001; Patrick,
Faury & Goulletquer 2006). Scientists hypothesized
that the main causes of high mortality in C. farreri
are inbreeding and environmental stresses (Zhang &
Yang 1999a; Wang, Li, Qiu & Zhang 2001; Xiao et al.
2005; Ma, Liu, Mai, Deng & Liufu 2006), but the cause
of mass mortality has not been identi¢ed.
Many methods have been suggested to avoid summer mortality and enhance the production of this
scallop, including polyculture, maintaining reasonable stocking densities, improving the germ plasm,
maintaining healthy seed-stock farms and enhancing marine environmental monitoring (Zhang &

Yang 1999b; Wang et al. 2001; Ma et al. 2006). Zhang
and Yang (1999b) proposed extending the farming
areas to depths deeper than 20 m to eliminate summer mortality. The C. farreri is cultured mainly in
near shore areas o15 m in depth, which are now

Keywords: Chlamys farreri, aquaculture, seasonal
variation, water temperature, dissolved oxygen
(DO), immune activities

814

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Journal Compilation r 2009 Blackwell Publishing Ltd


Aquaculture Research, 2010, 41, 814^827

Growth, survival and immune activity of scallops Z Yu et al.

characterized by high pollution and excessive stocking densities. Extending the farm areas to deeper
water could provide the cultured scallops with more
constant environmental conditions, lower pollution
and better water exchange than shallow areas. In addition, extending culture can reduce the culture
stress on coastal areas, and, would thus contribute
towards environmental protection of the near-shore
areas.
Scallops are reared in both suspended cultures
and bottom cultures (Bergh & Strand 2001). In a suspended culture, the animals are cultured in the water
column within lantern nets, cones, cages and other
containers (Emerson, Grant, Mallet & Carver 1994;

Mendoza, Freites, Lodeiros, Lo¤pez & Himmelman
2003) or using the ear-hanging technique (Cano,
Campos & RomaŁn 2000; Hamada, Yamashita, Watanabe & Natsume 2001; Grant, Emerson, Mallet & Carver 2003). The traditional method of bottom culture
is to release scallops directly on the seabed, which
has the advantage of reducing the costs of equipment
and husbandry compared with a suspended culture (Frishman, Nooman, Naidu & Cahill 1980); however, scallops on the bottom might be a¡ected more
by benthic predators, such as crabs and seastars
(Arsenault & Himmelman 1996; Wong & Barbeau
2003). Therefore, various methods were suggested
for a bottom culture, such as corrals, pockets, sleeves,
fences and other equipment (Bergh & Strand 2001;
Freites, Himmelman, Babarro, Lodeiros & Ve¤lez
2001; Mendoza et al. 2003). The bivalves in a bottom
culture may su¡er less from bad weather and fouling
organisms than those in a suspended culture; however, growth and survival may be better in a suspended than that in a bottom culture (Emerson et al.
1994; Mendoza et al. 2003).
In China, C. farreri is cultured mainly in lantern
nets on suspended longlines in coastal areas, and
farmers have been growing scallops at increasing
densities within lantern nets and adding more longlines in the culture areas (Xiao et al. 2005). There are
few published reports on culturing C. farreri in deepwater areas or on the bottom culture of this species in
shallow water (Wang, Lan,Yang & Zhang 1992).
Bivalves always show seasonal varations in growth
and physiological conditions. Many immune parameters, such as superoxide dismutase (SOD) and
myeloperoxidase (MPO), are promising biomarkers
to evaluate the health conditions of bivalves (Holmblad & S˛derhÌll 1999; Chen, Yang, Delaporte, Zhao
& Xing 2007; Xing, Lin & Zhan 2008). All oxygenutilizing organisms have a mechanism to remove

reactive oxygen species (ROS) that are produced by
metabolism (Santovito, Piccinni, Cassini, Irato &

Albergoni 2005). Superoxide dismutase can directly
degrade ROS to defend against and repair oxidative
damage (Demple 1999) and is the most important enzyme for minimizing oxidative damage to host cells
in the immune defence of animals (Downs, Fauth &
Woodley 2001; Campa-Co¤rdova, HernaŁndez-Saavedra & Ascencio 2002). The SOD activity of bivalves
varies widely with many factors such as season, reproductive cycle, age, tissue type and environmental
stress (Lau, Wong & Garrigues 2004; Chen, Mai, Ma,
Wang, Deng, Liu, Xu, Liufu, Zhang, Tan & Ai 2007).
Myeloperoxidase is an intracellular enzyme located
mainly inside primary lysosomes (Austin & Paynter
1995); it is able to catalyse the oxidation of chloride
to hypochlorous acid (HClO), a potent bactericidal
agent and is involved in oxidative killing (Holmblad
& S˛derhÌll 1999). The SOD and MPO activities can
e⁄ciently re£ect the immune condition of bivalves.
The present study examined the growth and survival of C. farreri in suspended and bottom cultures in
an open sea area, and also evaluated their health by
measuring the immune biomarkers, SOD and MPO.

Materials and methods
We performed our experiment at the site (35108 02400
N, 119154 03000 E) in the northeast of Haizhou Bay of
the middle Yellow Sea, 48 km to the east of Rizhao
Harbor, Shandong province (Fig. 1). The study area is
at the southernmost natural distribution boundary
of C. farreri (Guo et al. 1999), with about 25 m water
depth, high-quality water, a £at seabed and muddy
sand bottom sediment.
The juveniles used in this study were obtained on1
July 2007 from Yantai, the main culture area of

C. farreri in China located on the north side of the
Shandong peninsula. The scallops (n 5 30) had an
initial mean shell height of 2.96 Æ 0.43 cm and an
initial mean dry soft tissue weight of 0.17 Æ 0.06 g.
Scallops were acclimated in the lantern nets at about
5 m depth on the longline in the study area for1week
before the experiment.
Lantern nets and a bottom culture system were
used for the experiment. Ten lantern nets (eight
layers per net; each layer was 30 cm in diameter and
20 cm in height) were used in the suspended culture,
with 30 scallops per layer (425 ind m À 2). The lantern
nets were suspended on a longline at about 5 m below the sea surface. One bottom culture system

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Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 814^827

815


Growth, survival and immune activity of scallops Z Yu et al.

Aquaculture Research, 2010, 41, 814^827

Figure 1 Study site on cultured scallops, Chlamys farreri, during 2007^2008 in Haizhou Bay, theYellow Sea, China.

(sleeves) used in the experiment, as described in Maeda-Mart|¤ nez, Ormart, Mendez, Acosta and Sicard
(2000), was 16 m in length and sectioned into 16
compartments (1m  0.2 m height). Four hundred
scallops were placed into each compartment and the

sleeves were then anchored on the seabed about 10 m
beside the loneline at about 25 m depth. Both types of
enclosures were made with a 1.5 cm mesh net.

After ¢ltration, the ¢lters were rinsed with 20 mL distilled water and frozen for later analysis. The ¢lters
were dried at 60 1C for 48 h and weighed to a constant weight. The dry weight of the retained material
at 60 1C gave the TPM. Then, the ¢lters were incinerated at 450 1C for 4 h; the weight di¡erence between
60 and 450 1C was de¢ned as the POM. The organic
fraction (fPOM) was calculated as POM/TPM.

Environmental conditions

Scallop sampling and laboratory
analysis

The environmental conditions were monitored at
5 m depth and at the near-bottom water column
every 2^3 months. Water temperature ( 1C), salinity,
dissolved oxygen (DO; mg L À 1) and chlorophyll
a (mg L À 1) were monitored using a Yellow Springs
Instruments (YSI) 650 (Yellow Springs, OH, USA).
Seawater samples were collected at these two depths
using a 5 L Niskin bottle and then pre¢ltered through
a 200 mm mesh to eliminate large particles. The total
particulate matter (TPM; mg L À 1), particulate organic matter (POM; mg L À 1) and the organic fraction
(fPOM) were determined following the study of Cranford, Armsworthy, Mikkelsen and Milligan (2005), by
¢ltering one litre of the seawater samples through
47 mm Whatman GF/C ¢lters that previously had
been ashed (450 1C for 4 h) and weighed previously.

816

The study was conducted from 9 July 2007 to 2 June
2008. After deployment, the scallops were sampled
bimonthly or quarterly. Four layers of scallops in a
suspended culture were collected randomly from
the lantern nets, and about 100 scallops were taken
arbitrarily from one compartment of the sleeves by
SCUBA divers. In order to reduce the density e¡ect,
the compartments were not sampled repeatedly at
di¡erent times. Some of the scallops (about 20 ind)
were used for haemolymph sampling, and the remaining living specimens (about 80 ind) were frozen
at À 20 1C for growth measurements. All living and
dead specimens from three di¡erent layers of lantern
nets (about 100 ind) and three di¡erent compartments of sleeves (about 1000 ind) were counted to

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Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 814^827