Induction of out-of-season spawning in Eurasian

perch Perca fluviatilis: effects of rates of cooling

and cooling durations on female gametogenesis

and spawning

Herve Migaud a, Pascal Fontaine a,*, Isdy Sulistyo a,Patrick Kestemont b, Jean-Noel Gardeur a

aLaboratoire de Sciences Animales, INPL-UHP, Nancy 1, MAN 34 rue Sainte Catherine,

54000 Nancy, FrancebUnite de Recherches en Biologie des Organismes, Facultes Universitaires Notre-Dame de la Paix,

61 rue de Bruxelles, B-5000 Namur, Belgium

Received 20 October 2000; accepted 12 May 2001

Abstract

This study was designed to determine the influence of different thermal conditions during an

out-of-season reproductive cycle on gonad recrudescence, plasma steroid levels (testosterone and

estradiol), vitellogenin (VTG) concentrations and spawning in Eurasian perch (Perca fluviatilis)

females. The experiment was performed in 450-l square polyester indoor tanks located in two

rooms equipped with controlled light and temperature devices. Four thermal regimes were tested

with two different rates of cooling from 21 to 6 �C, 3 weeks (3w) or 6 weeks (6w), and two

different durations at 6 �C, 3 months (3m) or 5 months (5m). The photoperiod was fixed at LD

12:12. A long cooling period (6w) resulted in greater gonadosomatic index (GSI) (3.6F 0.5%)

correlated with a larger oocyte diameter (787.9F 25.1 mm). The plasma levels of testosterone

(13.63F 1.18 ng ml � 1) in the former groups were higher than in the short cooling period groups

(6.82F 0.9 ng ml� 1). A longer duration at 6 �C (5m) resulted in higher GSI (14.8F 1%), plasma

testosterone levels (26.2F 0.4 ng ml � 1) and plasma protein phosphorus (PPP, 1.33F 0.3 mgml� 1). The fish from the 6w 5m batch showed the highest plasma estradiol and testosterone levels,

whereas the 3w 5m group showed the largest GSI. At the end of the experiment, several

spontaneous out-of-season spawnings were collected in batch 6w 5m. Biopsy showed that most

oocytes from groups 3w 5m and 6w 5m females were mature (migration of the germinal vesicle

0044-8486/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0044-8486 (01 )00675 -5

* Corresponding author. Tel.: +33-383-302-841; fax: +33-383-323-016.

E-mail addresses: herve.migaud@lsa-man.u-nancy.fr (H. Migaud),

pascal.fontaine@lsa-man.u-nancy.fr (P. Fontaine), patrick.kestemont@fundp.ac.be (P. Kestemont).

www.elsevier.com/locate/aqua-online

Aquaculture 205 (2002) 253–267

from central position), whereas few females showed a beginning of migration in groups 3w 3m and

6w 3m, suggesting that the gonad development and reproductive success of Eurasian perch mainly

depends on the chilling duration (long cold period) rather than on the cooling one, in order to

obtain out-of-season spawning. D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Eurasian perch; Perca fluviatilis; Female; Reproductive cycle; Out-of-season spawning; Temperature;

Steroids

1. Introduction

The Eurasian perch, Perca fluviatilis, has been identified as the first species destined for

diversification of inland aquaculture (Fontaine et al., 1993; Kestemont and Dabrowski,

1996). In Northern and Western Europe, the aquaculture development is mainly linked

with its intensive rearing in recirculating systems. In order to satisfy the market require-

ments, reproduction cycles should be controlled to obtain out-of-season spawnings and

produce fingerlings throughout the year. At present, the production of percids relies mainly

on mature breeders captured in natural habitat just prior to spawning (Malison et al., 1994;

Malison and Held, 1996). The work published focused on extending spawning periods

(Tamazouzt et al., 1994; Dabrowski et al., 1996; Ciereszko et al., 1997a,b) and spawning

synchronisation (Kucharczyk et al., 1996; Kouril et al., 1997). Moreover, the reproduction

of the Eurasian perch received little attention compared to other North American percids,

such as yellow perch (P. flavescens) (Dabrowski et al., 1996) and walleye (Stizostedion

vitreum), and there are very few data dealing with the environmental control of the

Eurasian perch reproductive cycles. Some studies documented the influence of temper-

ature in the wild (Gillet et al., 1995; Sandstrom et al., 1995, 1997; Sulistyo et al., 1998,

2000).

Among the environmental factors, photoperiod and temperature are generally consid-

ered as the most important cues in the timing of gametogenesis and spawning in temperate

fish (Scott, 1979; Bromage, 1993). In the case of yellow perch, a period of low

temperature ( < 10 �C) in the winter is required to complete gonad maturation and

reproductive success (Hokanson 1977; Heidinger and Kayes, 1986). More recently, in

the case of Percids, several studies indicated that compressed photoperiods and temper-

ature regimes favoured maturation, such as in the yellow perch(Dabrowski et al., 1996;

Ciereszko et al., 1997a), white bass(Morone chrysops)(Kohler et al., 1994), striped

bass(M. saxatilis)(Blythe et al., 1994), sunshine bass(M. chrysops�M. saxatilis)(Tate

and Helfrich, 1998) and walleye(Malison et al., 1998). These two factors affect gonad

development through the action of steroid hormones (Borg 1994; Nagahama, 1994; Peter

and Yu, 1997). Works dealing with steroid variations (mainly estradiol and testosterone),

during a reproductive cycle, were conducted in several Percid fish, such as yellow perch

(Dabrowski et al., 1996), white perch (M. americana) (Jackson and Sullivan, 1994),

walleye (Malison et al., 1994; Malison and Held, 1996), white bass (Berlinsky et al., 1995)

and striped bass (Berlinsky and Specker, 1991; Woods and Sullivan, 1993).

The present work, based on the hypothesis that temperature variations alone (fixed

photoperiod) could induce gonadal development and control the main phases of the

H. Migaud et al. / Aquaculture 205 (2002) 253–267254

Eurasian perch reproductive cycle (Sulistyo et al., 1997), was designed to (1) test the

effects of different rates of cooling and durations on the main female reproductive phases

and (2) to induce out-of-season spawnings.

2. Materials and methods

2.1. Facilities and fish

Eurasian perch broodstocks (age = 2 + , mean body weight: 364.0F 22.9 g, mean total

length: 25.1F 0.5 cm, n = 150) were reared from hatching at the laboratory in a constant

environment (T= 21 �C, photoperiod LD 12:12) and never spawned before the onset of the

experiment.

The experiment was conducted from November 1996 to August 1997. The fish were

transferred into 10, 450-l square polyester indoor tanks (14 fish tank � 1) in two rooms

equipped with controlled lights and temperature devices (six tanks in room A and four in

room B). The water-recirculated system was based on a fish-rearing process, which uses

simple aquariological techniques (Fontaine et al., 1994). Each room was lit by two

fluorescent tubes (200 lux at the water surface), at a constant photoperiod of LD 12:12.

Thermal regimes were tested, at two different cooling periods, from 21 to 6 �C, over 3(3w, room A) or 6 weeks (6w, room B), two chilling periods at 6 �C over 3 (3m) or 5

months (5m) and warming period over 2 weeks (Fig. 1). The water temperature profiles in

Fig. 1. Experiment design. The temperature variations applied in the experiment during the cooling (3w or 6w),

the chilling (3m or 5m) and the warming phases.

H. Migaud et al. / Aquaculture 205 (2002) 253–267 255

the different groups are shown in Fig. 1. The fish, first acclimated at 21 �C, were dividedinto four groups according to the different regimes. The perch were fed twice a day (at 9

a.m. and 4 p.m.) with commercial pellets (Biomar S.A., Ecolife, number 4.5), which are

composed of 46% proteins (DM), 15% fat, 8% ash and 1.7% cellulose. The feeding rate

was 0.5% of the biomass and the food excess was regularly removed.

The oxygen, the pH, the N–NH4+ and the N–NO2

� were measured twice weekly and

varied between 5 and 14, 5 and 7.9, 0.01 and 1.30, 0 and 1.78 mg l � 1, respectively.

2.2. Samplings of plasma and oocytes

At the beginning of the experiment and at the end of the cooling, chilling and warming

periods, three to five females from each group were anaesthetised in a 2-phenoxyethanol

solution (0.3 ml l� 1), then individually weighed (body weight BWF 0.1 g), measured for

standard length (SLF 1 mm), blood-sampled and dissected. A 1-ml blood sample was

taken from the heart (initial, during cooling and chilling periods and final) or the caudal

vessel (during warming period) using a heparinized syringe. The blood was then

centrifuged at 4000 rpm for 25 min and the plasma was stored in vials at � 25 �C until

assay. Biopsies were performed on each female in order to examine the germinal vesicle

position and check for ripeness. Approximately 20 oocytes were removed with a catheter,

soaked for 5 min in a clearing solution: methanol, formalin, acetic acid 1v, 1v, 1v

(Mylonas et al., 1997) and examined under a 40� magnifying glass. At the end of the

experiment, all the fish were killed for analysis.

2.3. Morphoanatomical parameters

The gonadosomatic (GSI), hepatosomatic (HSI) and viscerosomatic (VSI) indexes were

calculated as:

GSIð%Þ ¼ ðGW� 100Þ=BW,

HSIð%Þ ¼ ðLW� 100Þ=BW and VSIð%Þ ¼ ðVW� 100Þ=BW,

where BW=body weight, GW=weight of the gonads (g), LW=weight of the liver (g) and

VW=weight of the viscera (g). To determine the respective importance of the somatic and

gonad development in fish, the condition factors were determined with and without gonad

weights, as follow K1 = 100�BW/SL3 and K2 = 100� (BW�GW)/SL3, where SL= stan-

dard length (cm) (Le Cren, 1951).

2.4. Steroid assay and vitellogenin

Plasma concentrations of testosterone (T) and 17b-estradiol (E2) were determined using

RIA analysis, according to Fostier et al. (1983), after two extractions with cyclohexane/

ethylacetate (v/v). The extractions were performed on 50 ml of plasma for each steroid. The

samples were assayed in duplicate and the standards in triplicate. The vitellogenin (VTG)

was indirectly measured by the concentration of plasma protein phosphorus (PPP). The

H. Migaud et al. / Aquaculture 205 (2002) 253–267256

cross-reactivities of antisera with a variety of common steroids were described by Prat et

al. (1990).

Six samples of different concentrations were tested in each assay and were used as

quality controls for estimating intra- and inter-assay coefficients of variation (CV). As for

these two hormones, E2 and T, the sensitivity of measurement were 8 and 5 pg ml� 1,

respectively. The intra-assay CVs (n = 6) were 6.1% and 2.5%, respectively.

2.5. Statistical analysis

All quantitative data are expressed as meansF S.D. (standard deviation). As treatments

are not repeated (one experimental room/temperature regime), means data and individuals

data as well, which are not replications, but pseudoreplications (Hurlbert, 1984), cannot be

analysed by classical ANOVA. Also, the treatment effect can only be tested on individuals

data (n = 44) by a multivariate analysis. These measures correspond to three or five fish

sampled at the end of each period: initial, cooling (two levels), chilling (two levels) and

warming periods (one level).

The method used was a principal component analysis (PCA) and hierarchical

clustering with the aggregation criteria of Ward (Ward, 1963) with the SPAD 4.0

software (Lebart et al., 1996). It allows comparisons of subpopulations characterized by

a particular experimental treatment. The biometrical data (GSI, HIS, VSI, K1 and K2)

were used as active variables and the level of treatment were used as illustration

variables. Another PCA was used for hormonal data (plasma T, E2 and PPP concen-

trations) as active variables. The characteristics of the principal components and the

classes obtained by hierarchical clustering were tested with the value test. Values were

significantly different when the value test > 2, P < 0.05 (Morineau, 1984; Lebart et al.,

1996).

3. Results

3.1. Morphoanatomical parameters

At the end of the cooling period, the GSI were 2.50F 1.29% and 3.60F 0.55% in 3w

and 6w groups, respectively (Fig. 2). The period of 6 �C (3 or 5 months) induced a large

increase in the GSI. The largest GSI was found in females from groups 3w 5m and 6w

5m and reached 15.26% and 14.23%, respectively. The samples from the end of the

warming phase are not represented in Figs. 2 and 3, because perch spawned during the

increase of temperature before the sampling. The oocyte growth was affected by the

temperature profile (Fig. 3). The oocyte diameter (OD) in the initial sample averaged

358.1F 32.3 mm and increased to about 750–800 mm in all groups at the time of the

warming phase.

At the beginning of the experiment, the HIS of females was 2.40F 0.31% (n = 3). It

increased during the cooling phase and was 2.78F 0.40% and 3.32F 0.26% for group 3w

and 6w, respectively. At the end of the chilling phase, the HSI was about 2.4–2.8% in all

groups. The VSI was 8.92F 0.78% at the beginning of the experiment and decreased

H. Migaud et al. / Aquaculture 205 (2002) 253–267 257

during the cooling phase (6.80F 0.26% and 6.70F 0.40%, respectively, for group 3w and

6w). At the end of the period at 6 �C, the VSI ranged between 5% and 6% approximately,

without any group differences.

Fig. 3. Ovocyte diameter of female Eurasian perch, P. fluviatilis, during the experiment. Values represent

meanF S.D. with n= 5. Initial: initial values; Cool: end of cooling period values; Chill: end of cold chilling

period values. The different treatments tested are 3 or 6 weeks (3w or 6w) during the cooling period and the

interaction of the cooling and the chilling periods (3m or 5m): 3w 3m, 3w 5m, 6w 3m and 6w 5m.

Fig. 2. Gonadosomatic index (GSI) of female Eurasian perch, P. fluviatilis, during the experiment. GSIs values

represent meanF S.D. with n= 5. Initial: initial values; Cool: end of cooling period values; Chill: end of cold

chilling period values. The different treatments tested are 3 or 6 weeks (3w or 6w) during the cooling period and

the interaction of the cooling and the chilling periods (3m or 5m): 3w 3m, 3w 5m, 6w 3m and 6w 5m.

H. Migaud et al. / Aquaculture 205 (2002) 253–267258

The hierarchical clustering representation showed four classes drawn on the plane

defined by the two first axis on PCA, which represent 80% of global inertia (Fig. 4). The

first axis is significantly characterised by the initial and the cooling sampling periods, the

VSI, the K2 and the first class, in the left part (values test > 2). In opposition, the chilling

period, the GSI, the OD, the class 3 and the illustrative variables (the plasma T, E2 and

PPP levels), not represented in Fig. 4, characterise the right part of the axis (values

test > 2). Thus, the first axis confirms, significantly, the decrease of the VSI and the K2 and

the concomitance increase of the GSI, the OD and the plasma T, E2 and PPP levels over

the time. In class 3, as all the treatments were represented, the active variables which

characterised this class (GSI, OD, VSI and K2) were not significantly affected by the

treatment. The class 4, described by the second axis, is characterised by low HSI, K1 and

OD compared to the class 2 which is represented by higher HSI and OD.

3.2. Plasma steroid concentrations

The initial plasma T concentrations were 0.31F 0.01 ng ml� 1 (n = 4), then increased

during the cooling period to 13.23F 0.47 ng ml� 1 in group 3w and 6.82F 0.96 ng ml� 1

in group 6w (Fig. 5). It kept on increasing during the chilling phase and the plasma T

levels were 21.8F 0.6 and 27.5F 0.42 ng ml� 1 in group 3w 3m and 6w 3m, respectively,

and 24.27F 1.86 and 27.98F 0.71 ng ml� 1 in groups 3w 5m and 6w 5m, respectively.

Fig. 4. Hierarchical clustering representation for biometrical data, gonadosomatic (GSI), viscerosomatic (VSI) and

hepatosomatic (HIS) indexes, condition factors (K1 and K2) and oocyte diameter (OD). Three sampling periods

were tested (initial, cooling and chilling periods). The large symbols represent the four classes (C1, 2, 3 and 4)

and, the small one, individuals which belong to each class. The different treatments tested are 3 or 6 weeks (3w or

6w), 3 or 5 months (3m or 5m) corresponding to the cooling and the chilling periods, respectively, and the

interaction of the two periods (3w 3m, 3w 5m, 6w 3m and 6w 5m). The axis 1 and 2 represent 50% and 30% of

the global inertia, respectively.

H. Migaud et al. / Aquaculture 205 (2002) 253–267 259

After the 2-week water warming (from 6 to 14 �C), the plasma T concentrations decreased

in all four groups to about 14–19 ng ml � 1.

The initial E2 concentrations averaged 0.27F 0.01 ng ml � 1 (n= 4) (Fig. 6). At the end

of the cooling phase, the plasma E2 levels were 2.74F 0.10 and 2.06F 0.15 ng ml � 1 in

Fig. 5. Plasma concentrations of testosterone (T) in female Eurasian perch. Values represent meanF S.D. with

n= 5. Initial: initial values; Cool: end of cooling period values; Chill: end of cold chilling period values; Warm:

end of warming period values. The different treatments tested are 3 or 6 weeks (3w or 6w) during the cooling

period and the interaction of the cooling and the chilling periods (3m or 5m): 3w 3m, 3w 5m, 6w 3m and 6w 5m.

Fig. 6. Plasma concentrations of 17b-estradiol (E2) in female Eurasian perch. Values represent meanF S.D. with

n= 5. Initial: initial values; Cool: end of cooling period values; Chill: end of cold chilling period values; Warm:

end of warming period values. The different treatments tested are 3 or 6 weeks (3w or 6w) during the cooling

period and the interaction of the cooling and the chilling periods (3m or 5m): 3w 3m, 3w 5m, 6w 3m and 6w 5m.

H. Migaud et al. / Aquaculture 205 (2002) 253–267260

the females from group 3w and group 6w, respectively. At the end of the chilling period,

the plasma E2 levels averaged 4 ng ml� 1. Nevertheless, the females from group 6w 5m

achieves 5.56F 0.49 ng ml � 1. During the 2-week warming phase, the E2 concentrations

increased, particularly, in the 6w 5m group (up to 10.54F 0.45 ng ml� 1, n = 3) compared

to the others (about 6 ng ml� 1).

The initial PPP values were 0.07F 0.01 mg ml� 1 (n = 4, Fig. 7). They increased

during the cooling phase in the females from group 3w (0.52F 0.17 mg ml� 1) and

group 6w (0.49F 0.13 mg ml � 1). During the chilling period, the plasma PPP concen-

trations were 0.88 mg ml� 1 for the 3-month treatment and 1.33 mg ml� 1 for the 5-

month treatment. At the end of the warming phase, the PPP levels decreased (0.7 mgml� 1).

Through the multivariate study, different classes were identified and described with

the variables that contribute most to the drawing up of classes (Fig. 8). The initial

samples and samples done at the end of the cooling period, represented in the class 1,

are characterized by low plasma levels of T, E2 and PPP. As far as the samples

performed at the end of the chilling period are concerned, the PPP levels were

significantly higher in the class 4, mainly represented by the 5m treatment. The class

5 was characterized by significantly higher T levels, which was not linked to a treatment

effect since all the treatments were represented in this class (3w 3m, 3w 5m, 6w 3m and

6w 5m, Fig. 8). At the end of the warming phase, the class 3 confirmed that the plasma

E2 levels were significantly higher in the 6w 5m treatment compared to the other

treatments (class 2).

Fig. 7. Plasma concentrations of plasma protein phosphorus (PPP) in female Eurasian perch. Values represent

meanF S.D. with n= 5. Initial: initial values; Cool: end of cooling period values; Chill: end of cold chilling

period values; Warm: end of warming period values. The different treatments tested are 3 or 6 weeks (3w or 6w)

during the cooling period and the interaction of the cooling and the chilling periods (3m or 5m): 3w 3m, 3w 5m,

6w 3m and 6w 5m.

H. Migaud et al. / Aquaculture 205 (2002) 253–267 261

3.3. Spawning and observations

The biopsy performed 2 weeks after the warming phase showed that most of the oocytes

from the 3w 5m and 6w 5m groups were mature as the germinal vesicle migration were

observed in the sampled oocytes. The oocytes sampled from the 3w 3m and 6w 3m females

groups had still central germinal vesicle and only few of them showed a partial migration.

The eggs were taken for number estimation and transferred for incubation. Only three

females naturally spawned in the 6w 5m group in July (the 15th, 20th and 22th) and one in

the 3w 3m group in May (20th), with an absolute fecundity ranging from 35000 to 53000

eggs/fish. When the experiment ended, 15 days after the warming period, another biopsy

was performed on non-ovulated females. The sampled oocytes contained central germinal

vesicle or had moved into an atretic stage.

4. Discussion

The present study provides new data on out-of-season spawning induction of Eurasian

perch by modification of the temperature profiles and complements existing data on

morphoanatomical indexes and sex steroids changes in this species.

Fig. 8. Hierarchical clustering representation for hormonal data, testosterone (T), estradiol (E2) and plasma protein

phosphorus (PPP), with four sampling periods tested (initial, cooling, chilling and warming periods). The large

symbols represent the five classes (C1, 2, 3, 4 and 5) and, the small one, individuals which belong to each class.

The different treatments tested are 3 or 6 weeks (3w or 6w), 3 or 5 months (3m or 5m) corresponding to the

cooling and the chilling periods, respectively, and the interaction of the two periods (3w 3m, 3w 5m, 6w 3m and

6w 5m). The axis 1 and 2 represent 69% and 22% of the global inertia, respectively.

H. Migaud et al. / Aquaculture 205 (2002) 253–267262

Adequate temperature profiles (a cooling period from 20 to 6 �C, a duration at 6 �C and

a warming period till 14 �C) successfully lead to the maturation of ovaries, with an average

GSI of 21%, thus higher than in the previous studies in which the GSI only reached

10.77F 1.79% (Tamazouzt et al., 1994) or 17–19% (Sulistyo et al., 1997). During this

study, the fish were under constant photoperiod and the GSI reached almost the same

values than in natural conditions (Sulistyo et al., 1998). Eurasian perch broodstocks, reared

from hatching in an artificial environment, under steady conditions (T > 20 �C, 14L/10D),are able to keep their reproduction ability.

This experiment confirms that a chilling period (maintaining water temperature at 6 �C)should be a crucial phase in the development of fish ovaries. Studies on yellow perch

showed that oogenesis requires a winter period to induce oocyte development and

maturation (Heidinger and Kayes, 1986). As far as this species is concerned, the water

temperature must be maintained under 10 �C for at least 160 days (Hokanson, 1977).

Contrary to many species reproducing during the spring, the photoperiod is probably not

the main factor to control the oocyte development and the maturation among Eurasian

perch. Similar conclusions were reported in yellow perch by Kayes and Calbert (1979) and

Dabrowski et al. (1994). These authors showed that spawning occurred during the same 4

days in April, as did the fish in the lake, whatever the photoperiodic regime applied in the

laboratory was (LD 13.5:10.5; 10.5:13.5; 6:18; 18:6). They also demonstrated that, when

transferred from the wild in April, yellow perch spawn in captivity either under long (LD

18:6) or short (LD 6:18) photoperiod if warmed to 12 �C. Dabrowski et al. (1996)

concluded that photothermal manipulations during the period of active ova growth and

vitellogenesis, as well as during the postvitellogenic phase of ovarian development, do not

lead to faster spawning among yellow perch. Thus, shifting the entire reproductive cycle,

instead of condensing or shortening reproductive phases (Blythe et al., 1994), seems to be

a more promising tool in order to accelerate spawning (Dabrowski et al., 1996; Tate and

Helfrich, 1998). In opposition, many studies showed that in Salmonid, the photoperiod is

the most important factor among the environmental cues, for synchronisation of the

reproductive cycle (Bromage, 1993).

Although the two cooling period tested from 22 to 6 �C did not show any difference in

the GSI (from 2.5% to 3.6%), a slight increase of oocyte diameter was observed from 450

to 700 mm for 3 and 6w, respectively. These differences might be related to steroids (E2

and T) and vitellogenin levels; however, according to the results of the principal

component analysis, only the T levels were significantly higher for the 6w treatment,

while the E2 and PPP concentrations were almost steady. So, a higher amount of T may be

due to a higher use of E2 by oocytes. No cooling period effect on oocyte diameter was

observed at the end of the chilling phase (duration at 6 �C). Oocyte growth was almost

completed during the cooling period for the 6w treatment, whereas it was only completed

during the chilling period in the 3w treatment.

The relation between the plasma steroids levels and the main reproductive phases often

seem to be unclear, because of the speed conversion of these steroids into other

compounds.

The chilling period at 6 �C is very important in the reproductive cycle of Eurasian

perch and the GSI increases to approximately 11–15% when fish were exposed to a 3- or

5-month chilling period, respectively (Fig. 2). This increase of the GSI is correlated with

H. Migaud et al. / Aquaculture 205 (2002) 253–267 263

the increase of the E2 (5.5 ng ml � 1 for group 6w 5m) and the vitellogenine, which

reached 0.9 and 1.3 ng ml� 1, after a chilling period of 3 or 5 months, respectively

(Fig. 7). This GSI difference is not related to the oocyte diameter, which is approximately

the same after the chilling durations at 6 �C, but probably to an increase in the number of

yolk oocytes.

In general, the plasma sex steroid and the PPP levels observed during this experiment

were similar to those observed in natural environment (Sulistyo et al., 1998), with an

increase after the cooling period (autumnal decrease of water temperature) and during the

chilling phase, when oocytes develop. The thermophotoperiodic treatments, in closed

conditions, did not alter the endocrinological processes involved in oogenesis. Testoster-

one, which is involved mainly in estradiol production, could also play a role in oocyte

maintenance after vitellogenesis and until final maturation (Kime 1993). This could also

explain the high levels observed during the phase at 6 �C.At the end of this experiment, three spontaneous spawnings were collected from the 6w

5m group in July, whereas the natural spawning period in the east of France occurred in

April, probably in relation with the higher E2 and T levels during the reproductive cycle.

Among the three other groups, only one spontaneous spawning was observed (group 3w

3m), and the plasma E2 concentrations were significantly lower. Cierezsko et al. (1997a,b)

demonstrated that low E2 levels during vitellogenesis (winter period) led to disturbances in

ovarian development and also compromised egg quality. These results would suggest that

shifting the entire reproductive cycle is possible in Eurasian perch as it was observed in

yellow perch (Kolkovski and Dabrowski, 1998).

As for semen quality, 75% of the spermatozoa were moving for a period of only 30 s

and 60% for 35 s, after activation. This short motility duration requires males to be very

close to females at the spawning time. This result is in accordance with those observed by

Lahnsteiner et al. (1995), who showed that 80% of the spermatozoa among P. fluviatilis

are moving for only 20 s in distilled water. Different hypothesis could explain the lack of

egg fertilisation in this study. First of all, there was no enough male stimulation (female

pheromone lack). Vermeirssen et al. (1997) demonstrated that pheromone induce semen

outflow. Secondly, the storing conditions might have been unsuitable, with too few males.

Jones (1982) and Treasurer (1983) demonstrated that, in a natural habitat, at least two

males trail one female ready for spawning and are involved in egg ribbon fertilisation. The

stress induced by regular staff presence, especially at the spawning date, could be a

possible explanation, as it was observed in several studies (Kjorszvik et al., 1990). Other

factors could also explain this fertilisation problem, such as photoperiodic conditions

during the development of the male gonad. If the temperature effect on the reproductive

cycle have already been studied, the role of photoperiod on the different reproductive

stages (as gonadal development initiation, maturation or spawning) are still to be tested.

Another factor could also play an important part in successful spawning: broodstock age at

spawning time and especially the age at first maturation time, as well as the number of

previous reproductive cycle. The broodstock tested during this experiment were main-

tained under artificial conditions of temperature (22 �C) and photoperiod (12 h L/12 h D)

at the laboratory and, thus, never matured. Some studies showed that the number of fish,

which successfully reproduced during their first reproduction cycle, was lower compared

to the fish who had already spawned (Abi-Ayad, 1997).

H. Migaud et al. / Aquaculture 205 (2002) 253–267264

In conclusion, the cooling period of 6 weeks has given results with higher plasma E2

and protein–phosphorus levels. Cold chilling periods must be long. In fact, 5 months

allow normal gonad development, whereas 3 months seems to be insufficient. The present

study has shown that out-of-season spawning in Eurasian perch is possible by photo-

thermoperiodic manipulations. On the other hand, lots of atretic oocytes were observed.

When ovaries reach a mature state, the fact of maintaining females in waters at a

temperature of 14 �C for more than 2 weeks causes damage in ovaries, and oocytes

become atretic. The use of hormonal induction, such as luteinizing hormone-releasing

hormone (LHRH), could synchronize the ovulation and release the spawning in Eurasian

perch. Further researches are then necessary to determine the specific roles of the different

environmental factors during the final stages of the reproductive cycle (mainly final

maturation, ovulation and spawning), so as to better explain the atretic phenomenon.

Acknowledgements

We would like to thank all the members of the LSA laboratory staff for their active

collaboration in this study.

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