# Duration of chilling phase, but not thermal condition, influence the gonad maturation of male and female domesticated pikeperch (Sander lucioperca)

Sylvain Milla, Amine Khendek, Daniel Zarski, Yannick Ledoré, Imen Ben Ammar, Pascal Fontaine, Imen Ammar

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Sylvain Milla, Amine Khendek, Daniel Zarski, Yannick Ledoré, Imen Ben Ammar, et al.. Duration of chilling phase, but not thermal condition, influence the gonad maturation of male and female domesticated pikeperch (Sander lucioperca). Aquaculture, Fish and Fisheries, 2021, 1 (1), pp.51-59. 10.1002/aff2.21 . hal-03596479

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# Duration of chilling phase, but not thermal condition, influence the gonad maturation of male and female domesticated pikeperch (Sander lucioperca)

Sylvain Milla, Amine Khendek, Daniel Zarski, Yannick Ledoré, Imen Ammar, Pascal Fontaine

<figure><img src="image_1.png" /></figure>

## To cite this version:

Sylvain Milla, Amine Khendek, Daniel Zarski, Yannick Ledoré, Imen Ammar, et al.. Duration of chilling phase, but not thermal condition, influence the gonad maturation of male and female domesticated pikeperch (Sander lucioperca). Aquaculture, Fish and Fisheries, 2021, 1 (1), pp.51-59. 10.1002/aff2.21 . hal-03596479v2

HAL Id: hal-03596479

https://hal.science/hal-03596479v2

Submitted on 27 May 2025

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

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Distributed under a Creative Commons Attribution 4.0 International License
1

Duration of chilling phase, but not thermal condition, influence the gonad maturation
of male and female domesticated pikeperch (*Sander lucioperca*)

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Sylvain Milla*, Amine Khendek, Daniel Zarskiª, Yannick Ledoré, Imen Ben Ammarᵇ,

Pascal Fontaine

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UR AFPA, USC INRAE 340, Université de Lorraine, Boulevard des Aiguillettes,
54506, Vandoeuvre-lès-Nancy, France

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a. Present address : Institute of Animal Reproduction and Food Research | Polish
Academy of Sciences in Olsztyn | 10-748 Olsztyn POLAND

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b. Present address : URBE | University of Namur | Rue de Bruxelles 61, 5000
NAMUR, BELGIQUE

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* Corresponding author: Sylvain.Milla@univ-lorraine.fr

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Other e-mail addresses: Amine.Khendek@gmail.com; d.zarski@pan.olsztyn.pl;

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yannick.Ledoré@univ-lorraine.fr; imen.benammar@yahoo.fr; p fontaine@univ-lorraine.fr

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Data availability statement

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The datasets generated during and/or analysed during the current study are available from
the corresponding author on reasonable request.

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Funding statement

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This study was partly supported by the Eurostars project (E!9390 TRANSANDER).

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Declaration of conflict of interest
# Duration of chilling phase, but not thermal condition, influence the gonad maturation of male and female domesticated pikeperch (*Sander lucioperca*)

Sylvain Milla<fnref n="1" /> ID

Amine Khendek<fnref n="1" />

Daniel Zarski<fnref n="1" />,<fnref n="2" /> ID

Yannick Ledoré<fnref n="1" /> ID

Imen Ben Ammar<fnref n="1" />,<fnref n="3" /> ID

Pascal Fontaine<fnref n="1" /> ID

## Correspondence

Sylvain Milla, UR AFPA, USC INRAE 340, Université de Lorraine, Boulevard des Aiguillettes, 54506, Vandoeuvre-lès-Nancy, France.

Email: Sylvain.Milla@univ-lorraine.fr

## Funding information

Eurostars, Grant/Award Number: E!9390
TRANSANDER

## Abstract

This study was designed to determine the influence of the thermal level and duration of chilling period on the initiation and progress of gonadogenesis and gametogenesis under controlled conditions in pikeperch (*Sander lucioperca*). A set of 120 fish was distributed into eight tanks (15 fish per tank). The effects of two temperatures (6 or 12°C) in combination with two durations of chilling period, either short (S; 75 days at 6°C and 125 days at 12°C) or long (L; 135 days at 6°C and 185 days at 12°C), were investigated for the effect on growth, gonadosomatic index (GSI), hepatosomatic index (HSI), gamete developmental stage and sex-steroid levels (17 beta-estradiol-E2 and testosterone-T). These zootechnical and physiological parameters were selected as they reflect the progressive advancement of gametogenesis in percid including pikeperch. The results show that the temperature did not affect the reproductive parameters but down-regulated the HSI and the condition factor in females. In addition, the exposure to a longer chilling period, whatever the temperature, allowed for (1) further advancement of gonadogenesis in females (GSI = 6.5% vs. 11%), (2) further advancement of gametogenesis (0% vs. 60% of oocytes at late vitellogenesis, 15% vs. 80% of spermatozoa in males) and (3) the maintenance or increase of plasma sex steroids in both sexes (e.g. 10 ng/mL vs. 27 ng/mL of T in females). Together with already published data, these results suggest that, depending on the features of fish populations and photothermal programmes, the maintenance of temperature at either 6 or 12°C does not necessarily impact pikeperch gonad maturation. However, the duration of the chilling period is an important factor for the optimization of gonadogenesis and gametogenesis achievement before reproduction.

## KEYWORDS

gametogenesis, sander, sex steroids, temperature

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2021 The Authors. Aquaculture, Fish and Fisheries published by John Wiley & Sons Ltd.

<footnote n="1">¹ UR AFPA, USC INRAE 340, Université de Lorraine, Boulevard des Aiguillettes, Vandoeuvre-lès-Nancy, France</footnote>

<footnote n="2">² Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland</footnote>

<footnote n="3">³ URBE, University of Namur, Rue de Bruxelles 61, Namur, Belgium</footnote>
The authors declare that they have no conflict of interest.

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## Ethics approval statement

All experimental manipulations were carried out in agreement with the European and

French national legislations on animal welfare after evaluation and approval of the

experimental project (protocol number: APAFIS#5862-2016062720134545)

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## Authors's contributions

Sylvain Milla: conceptualization, acquisition of data, statistical analysis, project

administration, funding acquisition, writing

Amine Khendek: conceptualization, acquisition of data, writing, reviewing the ms

Daniel Zarski: conceptualization, project administration, funding acquisition, reviewing the

ms

Yannick Ledoré: acquisition of data, reviewing the ms

Imen Ben Ammar: acquisition of data, reviewing the ms

Pascal Fontaine: project administration, funding acquisition, reviewing the ms

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Running title: optimization of chilling period in pikeperch

Keywords: sander, temperature, gametogenesis, sex-steroids

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# 1 | INTRODUCTION

The pikeperch (*Sander lucioperca*) is among the species with the highest potential for the development and diversification of European inland aquaculture (Mylonas & Robles, 2014). The development of pikeperch aquaculture is highly limited due to, among other factors, the seasonal-ity of reproduction. This can be overcome by the environmental control of broodstock reproduction which can be reproduced at any time of the year in recirculated aquaculture systems (RAS). But until now, a very high variability of gonad maturation and reproductive performances is observed in pikeperch broodstock reared in RAS (Khendek et al., 2018; Zarski et al., 2015). In particular, lack of spawning is often observed when females are not exposed to hormonal treatments (Zarski et al., 2019). To this end, a well-established photothermal programme allow-ing for the induction of complete gonadal development is necessary.

The reproductive cycle of temperate teleosts, including percids, is strictly controlled by the fluctuation of photothermal conditions (Taranger et al., 2010; Wang et al., 2010). In Eurasian perch *Perca fluviatilis*, exposure to continuous photophase or constant photoperiod (16L/8D) from the start of the reproductive cycle inhibits or slows down the initiation of gametogenesis in both sexes (Migaud et al., 2004). In pikeperch, gonad maturation is inhibited by a continuous lighting while optimal maturation is only achieved under a naturally stimulated photoperiod (Ben Ammar et al., 2015). Investigations showed that temperature is also a major factor to modulate the game-togenesis in percids. In Eurasian perch, exposure to high temperature from the initiation of gonad development inhibits the progress of oogenesis (Abdulfatah et al., 2013; Fontaine et al., 2015). Temperature is also important to induce the spawn in walleye *Stizostedion vitreum* (Malison et al., 1998) and to induce puberty or to regulate gonadal development in domesticated pikeperch (Hermelink et al., 2011, 2013). Thus, both temperature and photoperiod seem to be crucial factors for initiation and development of gonad maturation in percids. But the data on the proper parameters of temperature and photoperiod to be applied in pikeperch broodstock are limited, and the actual photothermal programmes used are based mostly on the protocols developed for Eurasian perch (*Perca fluviatilis*). These programmes involve a long phase (around 4.5 months) of chilling period at a low temperature (6°C) with a short (8 h) photoperiod (Abdulfatah et al., 2011, 2013; Fontaine et al., 2015; Khendek et al., 2018). Another study reported that a chilling period lasting at least 3 months at 12°C should be fully capable of inducing gonadal development (Hermelink et al., 2011). This suggests that the photothermal programmes suitable for pikeperch gonadal development may include, by comparison to Eurasian perch, a much shorter chilling period (only 3 months) with a much higher thermal regime (12°C) during the chilling period. In this context, we hypothesize that contrary to Eurasian perch, the success of pikeperch gametogenesis advancement is not dependent neither on the rearing temperature nor on the duration of the chilling period.

To address this question, we compared the gonadal maturation and the sex-steroid production in domesticated pikeperch males and females submitted to different combinations of chilling durations (long-L or short-S) and temperatures (6 or 12°C). In this way, the morpho-

anatomical indexes, gonadosomatic index (GSI) and hepatosomatic index (HSI), the profile of gametogenetic stages, 17 beta-estradiol-E2 and testosterone-T plasma concentrations were measured as these physiological endpoints belong to the most widely used parameters to evaluate the gonad maturation in fish including pikeperch.

# 2 | MATERIALS AND METHODS

All experimental manipulations were carried out in agreement with the European and French national legislations on animal welfare after evaluation and approval of the experimental project (protocol number: APAFIS#5862-2016062720134545) by the local ethic committee in France (Name: CELMEA).

## 2.1 | Experimental setup

In this experiment, 120 domesticated pikeperch (F1, males and females) reared in RAS were studied. The hatching occurred at Asialor Company (Pierrevillers, France), and larvae were transferred at the same day to the Aquaculture Experimental Platform (registration number for animal experimentation C54-547-18) belonging to the URAFPA facilities of the University of Lorraine (Nancy, France). Since the juvenile stage, fish were kept in two 2 m³ RAS tanks (renewal rate of 150% per hour) supplied originally with the city water. Fish were fed daily ad libitum with commercial dry pellets (Le Gouessant pellets, proteins 47%, lipids 13%, ash 6.90%, fibre 2.2%) under lighting (16L/8D) at 22°C. At a body weight ranging between 580 and 924 g, fish (age = 25 months) were distributed into eight tanks of 2 m³ for 3 weeks at 22°C with each tank containing 15 fish. During this acclimation period, the same photoperiod of 16L/8D was applied and all environmental factors remained constant until the start of the experiment (day 0). From day 0, the four groups of fish (two tanks and 30 fish per group, statistical experimental unit = fish) were then subjected to the start of different thermal regimes (Figure 1) where long (L, 4 tanks in total) or short (S, 4 tanks in total) chilling periods were applied either at 12°C (four tanks in total) or 6°C (four tanks in total). The photoperiod kinetics followed the programme described in Fontaine et al. (2015). As no apparent sexual dimorphism exists in pikeperch, both sexes were combined in the tanks without individual knowledge of the sex at this experiment start.

In each tank, water quality was assessed three times per week. pH and dissolved oxygen were measured with a multi-parametric probe ph/Oxi 340 i (WTW, Weinheim, Germany). pH was maintained above 7.4 by addition of NaCO₃. The dissolved oxygen was maintained over 6 mg/L. The concentrations of total ammonia and nitrite-nitrogen were measured using indophenol blue colorimetric methods in which nitro-prusside was used as the catalyst and the water sample containing the ammonium ions from each tank was treated with a solution of chlorine and phenol to give indophenol blue which can be detected by the spectrophotometer (CARY I). The concentrations of total ammonia and nitrite nitrogen always remained below 1 mg/L. The temperature and
46 Abstract

This study was designed to determine the influence of the thermal level and duration of
chilling period on the initiation and progress of gonadogenesis and gametogenesis under
controlled conditions in pikeperch (*Sander lucioperca*). A set of 120 fish was distributed
into eight tanks (15 fish per tank). The effects of two temperatures (6°C or 12°C) in
combination with two durations of chilling period, either short (S; 75 days at 6°C and 125
days at 12°C) or long (L; 135 days at 6°C and 185 days at 12°C), were investigated for the
effect on growth, gonadosomatic index (GSI), hepatosomatic index (HSI), gamete
developmental stage and sex-steroid levels (17 beta-estradiol-E2 and testosterone-T).

These zootechnical and physiological parameters were selected as they reflect the
progressive advancement of gametogenesis in percids including pikeperch. The results
show that the temperature did not affect the reproductive parameters but down-regulated
the HSI and the condition factor in females. In addition, the exposure to a longer chilling
period, whatever the temperature, allowed for 1-further advancement of gonadogenesis in
females (GSI=6.5% vs 11%), 2- further advancement of gametogenesis (0% vs 60% of
oocytes at late vitellogenesis, 15% vs 80% of spermatozoa in males) and 3-the
maintenance or increase of plasma sex-steroids in both sexes (e.g. 10 ng/ml vs 27 ng/ml of
T in females). Together with already published data, these results suggest that, depending
on the features of fish populations and photothermal programs, the maintenance of
temperature at either 6°C or 12°C does not necessarily impact pikeperch gonad maturation.

However, the duration of the chilling period is an important factor for the optimization of
gonadogenesis and gametogenesis achievement before reproduction.

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<figure><img src="image_3.png" /><figcaption>**FIGURE 1** Thermal regimes applied in the groups subjected to long (L) and short (S) chilling period. Yellow targets correspond to samplings with organ and blood extractions. Red targets correspond to samplings with blood extractions only. D0 corresponds to the start of the photoperiod decrease. D30 corresponds to the start of the temperature decrease in all thermal regimes. D90 and D140 correspond to the start of the chilling period in the 12°C (red) and 6°C (blue) groups, respectively. D0, D30, D155, D185, D215 and D275 correspond to the sampling times T0, T1, T2, T3, T4 and T5, respectively</figcaption></figure>

the photoperiod were controlled automatically and continuously by a centralized technical management. Potential temperature and photoperiod changes are operated once a day and, if change, the temperature variation is fixed at 1° whatever the stage of the photothermal programme. The light intensity was fixed at 10 lx at the water surface. The water flow rate remained constant at 3 m³ per hour.

## 2.2 | Fish sampling

The first sampling of fish was performed at day 0 (T0) of the experiment, when the different photothermal programmes initiated marked by the decrease of photoperiod (Figure 1). The second sampling (only blood) occurred at day 30 (T1) before the temperature drop. The third sampling occurred at day 155 (T2) when the lowest temperature was applied in each group. The fourth sampling (only blood) was made at day 185 (T3). The fifth sampling was performed at day 215 (T4) before the resumption of the temperature increase in the two S groups. The last sampling of the experiment was performed at day 275 (T5) before the resumption of the temperature increase in the two L groups. T5 is thus the only sampling time which characterizes the long chilling period (L). At all times, fish were anaesthetised in MS-222 (150 mg/L, Sigma)

and then the blood was sampled from all groups within 5 min after netting by caudal vein puncture using a 2-mL heparinized syringe. Then, blood was centrifuged at 3000 g for 10 min to collect plasma. At times T0, T2, T4 and T5, an average of 10 males (minimum: six, maximum: 18) and six females (minimum: three, maximum: 10) were weighed and measured to calculate the condition factor (fish weight*100/length³). Next, fish were euthanized by an overdose of MS-222 (300 mg/L; Sigma). After death, gonads and liver were extracted and weighted in order to calculate the gonadosomatic (GSI) and hepatosomatic (HSI) indices (GSI = 100 × gonad weight/total fish weight, %; HSI = 100 × liver weight / total fish weight, %). At all times, sampled fish were healthy and no lesion was observed.

## 2.3 | Gonad histology

For females and males, samples of gonads were fixed in a Bouin-Holland solution for 1 week, washed once with water, twice with 70% ethanol and stored in absolute ethanol (Abdulfatah et al., 2011). Fragments of gonads were cut into 3-mm-thick slices, dehydrated/embedded in paraffin with successive baths (two baths of 1 h in alcohol 95°, two baths of 1 h in absolute ethanol, one bath of 1 h
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# 1. Introduction

The pikeperch (*Sander lucioperca*) is among the species with the highest potential for the development and diversification of European inland aquaculture (Mylonas and Robles, 2014). Development of pikeperch aquaculture is highly limited due to, among other factors, the seasonality of reproduction. This can be overcome by the environmental control of broodstock reproduction which can be reproduced at any time of the year in recirculated aquaculture systems (RAS). But until now, a very high variability of gonad maturation and reproductive performances is observed in pikeperch broodstock reared in RAS (Zarski et al. 2015; Khendek et al. 2018). In particular, lack of spawning is often observed when females are not exposed to hormonal treatments (Zarski et al. 2019). To this end, a well-established photothermal program allowing for the induction of complete gonadal development is necessary.

The reproductive cycle of temperate teleosts, including percids, is strictly controlled by the fluctuation of photothermal conditions (Taranger et al. 2010; Wang et al. 2010). In Eurasian perch *Perca fluviatilis*, exposure to continuous photophase or constant photoperiod (16L/8D) from the start of the reproductive cycle inhibits or slows down the initiation of gametogenesis in both sexes (Migaud et al. 2004). In pikeperch, gonad maturation is inhibited by a continuous lighting while optimal maturation is only achieved under a naturally stimulated photoperiod (Ben Ammar et al. 2015). Investigations showed that temperature is also a major factor to modulate the gametogenesis in percids. In Eurasian perch, exposure to high temperature from the initiation of gonad development inhibits the progress of oogenesis (Abdulfatah et al. 2013; Fontaine et al. 2015). Temperature is also important to induce the spawn in walleye *Stizostedion vitreum* (Malison et al. 1998) and to induce puberty or to regulate gonadal development in domesticated pikeperch (Hermelink et al. 2011; Hermelink et al. 2013). Thus, both

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<figure><img src="image_4.png" /></figure>

in acetone, two baths of 1 h in toluene and three baths of 1 h in a paraffin substitute dubbed Diawax). For females, sections of 6 µm were cut from the Diawax block and slides were stained with a Masson's trichrome-haematoxylin Gill III (Merck, Darmstadt, Germany), 0.5% phloxine B (VWR, California, USA) and 0.5% light green (Sigma, Saint Quentin Fallavier, France). For males, sections of 4 µm were cut from the Diawax block and slides were stained with a trichrome: Regaud iron haematoxylin (Merck), 1% Ponceau-Fushine of Masson (Sigma) and 0.1% light green (Sigma). Gonadal development was evaluated according to the following procedure.

In females, five stages were considered:

* Protoplasmic stage: protoplasmic oocytes with vacuole free cytoplasm

* Cortical alveoli stage: appearance of yolk vesicles, occupying two or three rings in the cytoplasm periphery and then oocytes are full of yolk vesicles (green colour). Follicular and cellular layers are differentiated.

* Early vitellogenesis: appearance of yolk globules in the oocytes (purple colour).

* Late vitellogenesis: oocytes accumulate yolk globules and oil droplets in all the oocytes.

* Atresia: the oocyte becomes atretic.

At each sampling time, three to six females per temperature and two slides per female were analysed. For each slide, 30 oocytes were randomly selected and assigned to the above oogenetic stage. The number of oocytes belonging to each stage was counted and was expressed relatively to the total number of oocytes.

In males, five stages were considered:

* Differentiated spermatogonia A (SPGA): the largest cells of the germ line with voluminous, irregular nucleus surrounded by voluminous cytoplasm.

* Spermatogonia B (SPGB): similar to spermatogonia A but smaller.

* Spermatocyte (SPC): smaller than spermatogonia and identified by densely staining chromatin.

* Spermatid (SPD): larger than spermatozoa with regularly spherical and dense nucleus.

* Spermatozoa (SPZ): recognized by their kidney-shaped cell with light-coloured hair-like tails.

At each sampling time, six males per temperature and two slides per male were analysed. For each slide, two pictures were taken and the areas corresponding to each spermatogenetic stage were summed and expressed relatively to the whole area of the pictures.

For both sexes, the histological analysis used an optical microscope (Leica CME) equipped with an ocular camera (Jeulin, 1.3MP) and the ImageJ software. Such analysis was also used to differentiate the fish sex for T0, T1 and T3 samples while the sex was determined visually for T2, T4 and T5 during gonad extraction.

## 2.4 | Hormone assays

The concentration of 17β-estradiol (E2, ng/mL) was assayed on 50 µL of plasma, diluted or not, using the DIAsource E2-ELISA kit (DIAsource, KAP0621). Sensitivity was 5 pg/mL. The coefficient of variation (CV) intra-assays varied between 3.1% and 2.6% and CV inter-assays varied between 4.7% and 2.4% for low and high levels, respectively. Testosterone (T, ng/mL) was assayed on 25 µL of plasma using the DIAsource Testosterone ELISA Kit (DIAsource, KAPD1559). Sensitivity was 83 pg/mL. CV intra-assays varied between 2.6% and 8.4%, and CV inter-assays varied between 9.4% and 12.1% for low and high levels, respectively.

## 2.5 | Statistical analysis

After checking the normality and homogeneity of variances (Shapiro-Wilk and Levene tests, respectively), the effects of sampling time and temperature were analysed, for each sex independently, by a two-way analysis of variance (ANOVA) followed, when significant, by Tukey (HSD) test with different N. The statistical experimental unit was the fish. The two factors were (1) the temperature (two modalities: 6 and 12°C; four tanks per modality; from 21 to 41 statistical repetitions depending on the sex and the variable) and (2) the time (six modalities: from T0 to T5; eight tanks per modality from T0 to T4 and four tanks at T5; from 8 to 44 statistical repetitions depending on the sex and the variable). Regarding the temperature*time interaction (12 modalities), there were four tanks per modality from T0 to T4 and two tanks per temperature at T5 (from 3 to 18 statistical repetitions depending on the sex and the variable). If the assumptions of the ANOVA failed, non-parametric Kruskal-Wallis followed by Mann-Whitney or bilateral

<table><caption>**TABLE 1** The effect of thermal condition (6 and 12°C) and chilling period on weight (mean ± SD, n = 3–10 in males, n = 6–18 in females; expressed in grams) of pikeperch. Sampling times T0, T2, T4 (S = short chilling period) and T5 (L = long chilling period) correspond, respectively, to days 0, 155, 215 and 275 after the beginning of the photothermal programme. For each sex, different letters indicate significant difference between the sampling times (p < 0.05)</caption><thead><tr><th>Sex</th><th>T0</th><th>Temperature</th><th>T2</th><th>T4 (S)</th><th>T5 (L)</th></tr></thead><tbody><tr><td rowspan="2">Males</td><td rowspan="2">767 ± 137 a</td><td>6°C</td><td>1538 ± 308 b</td><td>1677 ± 317 b</td><td>1243 ± 144 b</td></tr><tr><td>12°C</td><td>1481 ± 291 b</td><td>1319 ± 196 b</td><td>1466 ± 256 b</td></tr><tr><td rowspan="2">Females</td><td rowspan="2">785 ± 87 a</td><td>6°C</td><td>1467 ± 262 b</td><td>1453 ± 219 b</td><td>1484 ± 188 b</td></tr><tr><td>12°C</td><td>1531 ± 292 b</td><td>1388 ± 276 b</td><td>1757 ± 136 b</td></tr></tbody></table>
temperature and photoperiod seem to be crucial factors for initiation and development of gonad maturation in percids. But the data on the proper parameters of temperature and photoperiod to be applied in pikeperch broodstock is limited, and the actual photothermal programs used are based mostly on the protocols developed for Eurasian perch (*Perca fluviatilis*). These programs involve a long phase (around 4.5 months) of chilling period at a low temperature (6°C) with a short (8 h) photoperiod (Abdulfatah et al. 2011; Abdulfatah et al. 2013; Fontaine et al. 2015; Khendek et al. 2018). Another study reported that a chilling period lasting at least 3 months at 12°C should be fully capable of inducing gonadal development (Hermelink et al. 2011). This suggests that the photothermal programs suitable for pikeperch gonadal development may include, by comparison to Eurasian perch, a much shorter chilling period (only 3 months) with a much higher thermal regime (12°C) during the chilling period. In this context, we hypothesize that contrary to Eurasian perch, the success of pikeperch gametogenesis advancement is not dependent neither on the rearing temperature nor on the duration of the chilling period.

To address this question, we compared the gonadal maturation and the sex-steroid production in domesticated pikeperch males and females submitted to different combinations of chilling durations (long-L or short-S) and temperatures (6°C or 12°C). In this way, the morpho-anatomical indexes GSI and HSI, the profile of gametogenetic stages, 17 beta-estradiol-E2 and testosterone-T plasma concentrations were measured as these physiological endpoints belong to the most widely used parameters to evaluate the gonad maturation in fish including pikeperch.

## 2. Materials and Methods

All experimental manipulations were carried out in agreement with the European and French national legislations on animal welfare after evaluation and approval of the
<table><caption>**TABLE 2** The effect of thermal condition (6 and 12°C) and chilling period on the condition factor (mean ± SD, *n* = 3–10 in males, *n* = 6–18 in females) of pikeperch. Sampling times T0, T2, T4 (S = short chilling period) and T5 (L = long chilling period) correspond, respectively, to days 0, 155, 215 and 275 after the beginning of the photothermal programme. For each sex, different letters indicate significant differences between the sampling times (*p* < 0.05). In females, the asterisks indicate a significant temperature effect over the whole sampling period except T0 (*p* < 0.05)</caption><thead><tr><th>Sex</th><th>T0</th><th>Temperature</th><th>T2 (D155)</th><th>T4 (D215)</th><th>T5 (D275)</th></tr></thead><tbody><tr><td rowspan="2">Males</td><td rowspan="2">0.87 ± 0.13 a</td><td>6°C</td><td>1.09 ± 0.11 b</td><td>1.12 ± 0.05 b</td><td>1.06 ± 0.09 b</td></tr><tr><td>12°C</td><td>1.06 ± 0.08 b</td><td>1.02 ± 0.08 b</td><td>1.02 ± 0.16 b</td></tr><tr><td rowspan="2">Females</td><td rowspan="2">0.89 ± 0.06 a</td><td>6°C</td><td>1.11 ± 0.1 b,*</td><td>1.15 ± 0.13 b,*</td><td>1.23 ± 0.13 b,*</td></tr><tr><td>12°C</td><td>1.05 ± 0.12 b,*</td><td>1.02 ± 0.15 b,*</td><td>1.06 ± 0.12 b,*</td></tr></tbody></table>

multiple comparisons tests were used. The level of significance used in all tests was *p* < 0.05. All statistical analyses were performed using the STATISTICA software (StatSoft, Tulsa, OK, USA).

## 3 | RESULTS

No mortality occurred during the acclimation period and during the experimental time course. For both sexes, an elevation of growth and condition factor was observed between T0 and T2 (females: *p* < 10⁻³ and *p* < 10⁻², respectively; males: *p* < 10⁻³ and *p* < 10⁻³, respectively) without significant differences between the two sexes (*p* > 0.05). Then, these parameters remained steady along the experimental time course (Tables 1 and 2). In females, the temperature significantly influenced the condition factor with a significant reduction among those kept at 12°C compared to those at 6°C (*p* < 0.05).

In females, a progressive increase of GSI was observed along the experimental time course (*p* < 10⁻⁴) associated with a significant difference between T4 and T5 (*p* < 10⁻², Figure 2a). However, the temperature did not significantly influence the GSI (*p* > 0.05). The temperature (*p* < 10⁻³), time (*p* < 10⁻⁴) and the interaction between temperature and time (*p* < 0.05) significantly influenced the HSI (Figure 2b) marked by a steady and elevated level from T1 in pikeperch at 6°C contrary to those at 12°C which returned to the initial level.

Like in females, only the length of the chilling period affected the male GSI (*p* < 10⁻⁴) but no difference was noticeable between T4 and T5 (Figure 3a). The temperature (*p* < 10⁻³) and time (*p* < 10⁻⁴) significantly influenced the HSI, but the interaction between both factors was not significant (Figure 3b). The HSI was lower among males kept at 12°C compared to those kept at 6°C.

The histological analysis did not reveal any effect of temperature for either sex. However, a marked difference was observed between T4 and T5 showing a much more advanced gametogenesis when the chilling period was lengthened (Figure 4). At T4, females and males reached the early vitellogenesis stage and the spermatid stage, respectively, while at T5 they reached the late vitellogenesis stage and the spermatotozoa stage, respectively (Figure 5).

The hormonal analysis did not show any temperature effect on plasma E2 and T levels (*p* > 0.05; Figure 6) for either sex. But they revealed strong fluctuations along the experimental time course for both females and males (*p* < 10⁻⁴ for each group). In females, E2 and T plasma levels peaked at T3 and T5, respectively, with a significant

<figure><img src="image_5.png" /><figcaption>**FIGURE 2** The effect of thermal condition (6 and 12°C) and chilling period on gonadosomatic index GSI (Figure 2a, mean ± SD, *n* = 3–10) and hepatosomatic index HSI (Figure 2b, mean ± SD, *n* = 3–10) values of female pikeperch. Sampling times T0, T2, T4 (S = short chilling period) and T5 (L = long chilling period) correspond, respectively, to days 0, 155, 215, and 275 after the beginning of the photothermal programme. Different letters indicate significant differences between the sampling times or between the interactions time*temperature (*p* < 0.05)</figcaption></figure>

marked elevation of T between T4 and T5 (*P* < 0.01; Figure 6a and b). In males, the highest E2 and T levels were measured at T3 and T4 and T5, respectively (Figure 6c and d).

## 4 | DISCUSSION

Regardless of the sex of the fish, changes in temperature during the chilling period neither caused a change in the GSI level or caused any
experimental project (protocol number: APAFIS#5862-2016062720134545) by the local
ethic committee in France (Name: CELMEA).

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**2.1 Experimental setup**

In this experiment, 120 domesticated pikeperch (F1, males and females) reared in RAS
were studied. The hatching occurred at Asialor Company (Pierrevillers, France) and larvae
were transferred at the same day to the Aquaculture Experimental Platform (AEP,
registration number for animal experimentation C54-547-18) belonging to the URAFPA
facilities of the University of Lorraine (Nancy, France). Since the juvenile stage, fish were
kept in two 2 m³ RAS tanks (renewal rate of 150% per hour) supplied originally with the
city water. Fish were fed daily *ad libitum* with commercial dry pellets (Le Gouessant
pellets, proteins 47%, lipids 13%, ash 6.90%, fiber 2.2%) under lighting (16L/8D) at 22°C.

At a body weight ranging between 580 g and 924 g, fish (age=25 months) were distributed
into eight tanks of 2 m³ for 3 weeks at 22°C with each tank containing 15 fish. During this
acclimation period, the same photoperiod of 16L/8D was applied and all environmental
factors remained constant until the start of the experiment (day 0). From day 0, the four
groups of fish (two tanks and 30 fish per group, statistical experimental unit=fish) were
then subjected to the start of different thermal regimes (Figure 1) where long (L, 4 tanks in
total) or short (S, 4 tanks in total) chilling periods were applied either at 12°C (4 tanks in
total) or 6°C (4 tanks in total). The photoperiod kinetics followed the program described in
Fontaine et al. (2015). As no apparent sexual dimorphism exists in pikeperch, both sexes
were combined in the tanks without individual knowledge of the sex at this experiment
start.

In each tank, water quality was assessed three times per week. pH and dissolved oxygen
were measured with a multi-parametric probe ph/Oxi 340 i (WTW, Weilheim, Germany).

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<figure><img src="image_6.png" /></figure>

**FIGURE 3** The effect of thermal condition (6 and 12°C) and chilling period on gonadosomatic index GSI (Figure 3a, mean + SD, n = 6-18) and hepatosomatic index HSI (Figure 3b, mean + SD, n = 6-18) values of male pikeperch. Sampling times T0, T2, T4 (S = short chilling period) and T5 (L = long chilling period) correspond, respectively, to days 0, 155, 215 and 275 after the beginning of the photothermal programme. Different letters indicate significant differences between the sampling times (p < 0.05). The asterisks indicate a significant temperature effect over the whole sampling period (p < 0.05)

marked alteration of the gametogenesis stages. In contrast, in the same
species, the females reared at least 3 months at 12°C during the chill-
ing period exhibited a higher GSI than those kept at 6°C (Hermelink
et al., 2011). This apparent discrepancy may stem from five major dif-
ferences between both studies. First, the initial average weights highly
differ between both experiments (340 g vs. 775 g). Also, the absence
of weight change during the chilling period (Hermelink et al., 2011)
contrasts strikingly with the twofold weight increase observed in our
experiment, possibly due to differences in the diet composition. Sec-
ond, in the study of Hermelink et al. (2011), the rate of GSI increase
in females from the initial time was in the range of three to five fold
(initial GSI: 0.9%; final GSI: 4%). This rate is much lower than the
rate of GSI observed herein found to have increased in the range of
15-30 fold (initial GSI: 0.45%; final GSI from 7% to 12%). These dif-
ferent zootechnical performances suggest a contrasted response to
temperature between the pikeperch populations, which can be partly
explained by different features in the experimental setup. Third, the
duration of the programme was much longer in our study (9 months

<figure><img src="image_7.png" /></figure>

**FIGURE 4** The effect of thermal condition (6 and 12°C) and chilling period on oogenesis and spermatogenesis stages (n = 3-6 per histogram) of pikeperch. Sampling times T0, T2, T4 (S = short chilling period) and T5 (L = long chilling period) correspond, respectively, to days 0, 155, 215 and 275 after the beginning of the photothermal programme. Vtg, vitellogenesis. SPG, spermatogonia; SPC, spermatocytes; SPD, spermatids; SPZ, spermatozoa

vs. 6 months). This comes from the fact that, to reach the temperature
plateau during the chilling period, a slow temperature drop was applied
in our experiment (from 60 to 110 days). This kinetics is in accordance
with data on programme optimization obtained for the Eurasian perch
*Perca fluviatilis* (Abdulfatah et al., 2011) and contrary to the quick drop
applied in the study of Hermelink et al. (2011). Fourth, the level and the
variations of photoperiod applied during our photothermal programme
(Fontaine et al., 2015) contrast with the constant photoperiod main-
tained at 12 h/12 h during the studies of Hermelink et al. (2011, 2016).
Given the effect of photoperiod on oogenesis in pikeperch female mat-
uration (Ben Ammar et al., 2015), we hypothesize that natural changes
of photoperiod are necessary to optimize the GSI. Last, the contrast-
ing results may come from the features of the domesticated fish pop-
ulations, notably the fish life history. Indeed, the maturation success in
response to such a photothermal programme depends on the captive
pikeperch population used (Khendek et al., 2018).

In the present experiment, the maintenance at 12°C during the chill-
ing phase did not disrupt the gonad maturation in pikeperch. Wang
pH was maintained above 7.4 by addition of NaCO₃. The dissolved oxygen was
maintained over 6 mg/L. The concentrations of total ammonia and nitrite-nitrogen were
measured using indophenol blue colorimetric methods in which nitroprusside was used as
the catalyst and the water sample containing the ammonium ions from each tank was
treated with a solution of chlorine and phenol to give indophenol blue which can be
detected by the spectrophotometer (CARY I). The concentrations of total ammonia and
nitrite nitrogen always remained below 1 mg/L. The temperature and the photoperiod were
controlled automatically and continuously by a centralized technical management.
Potential temperature and photoperiod changes are operated once a day and, if change, the
temperature variation is fixed at 1 degree whatever the stage of the photothermal program.
The light intensity was fixed at 10 lx at the water surface. The water flow rate remained
constant at 3m³ per hour.

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## 2.2 Fish sampling

The first sampling of fish was performed at day 0 (T0) of the experiment, when the different photothermal programs initiated marked by the decrease of photoperiod (Figure 1). The second sampling (only blood) occurred at day 30 (T1) before the temperature drop. The third sampling occurred at day 155 (T2) when the lowest temperature was applied in each group. The fourth sampling (only blood) was made at day 185 (T3). The fifth sampling was performed at day 215 (T4) before the resumption of the temperature increase in the two S groups. The last sampling of the experiment was performed at day 275 (T5) before the resumption of the temperature increase in the two L groups. T5 is thus the only sampling time which characterizes the long chilling period (L). At all times, fish were anesthetized in MS-222 (150 mg/L, Sigma) and then the blood was sampled from all groups within 5 min after netting by caudal vein puncture using a 2 ml heparinized syringe.

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<figure><img src="image_8.png" /></figure>

**FIGURE 5** The effect of chilling period on the most advanced gametogenesis stage of pikeperch reached at the end of this period. Top left: early vitellogenesis stage reached at T4 (S = short chilling period). Top right: late vitellogenesis stage reached at T5 (L = long chilling period). Bottom left: spermatid stage reached at T4 (S = short chilling period). Bottom right: spermatozoa stage reached at T5 (L = long chilling period). Vtg, vitellogenesis; SPD, spermatids; SPZ, spermatozoa

et al. (2010) suggest that in percid fishes a given temperature thresh-
old exists, above which gametogenesis would be impaired. In Eurasian
perch females, a temperature in the range of 6-14° degrees does
not strongly impact the progress of gametogenesis and gonadogen-
esis (Abdulfatah et al., 2013). In yellow perch, the maintenance at
constant 23°C, instead of applying a chilling phase à 10°C, inhibits
the GSI increase and the gametogenesis advancement (Kolkovski &
Dabrowski, 1998; Shewmon et al., 2007). Our data thus support that
the achievement of percid gametogenesis in RAS conditions is not
markedly influenced by the rearing temperature below 12-14°C. At
higher temperatures, the slowdown or even blockage of gametogen-
esis is often accompanied with a decrease of sex-steroid production
(e.g. Abdulfatah et al., 2013). In our study, the absence of temperature
effect of sex-steroid levels might explain why gametogenesis was not
disrupted by the tested thermal conditions.

Despite no apparent effect on pikeperch gametogenesis, the 12°C
temperature induced the drop of HSI in both sexes associated with
a decrease in the condition factor, at least in females. This suggests
that the whole energetic metabolism was affected by such temper-
ature. These data are consistent with what observed in other fish
species for which an elevated temperature during vitellogenesis is
linked to a decrease of HSI (e.g. Im et al. 2016; Akhoundian et al. 2020).
Still, pikeperch is thought to be one of the most thermophilic per-

cid fish species studies so far (Geay & Kestemont, 2015). Thus, this
thermophilic feature does not seem to apply at such low tempera-
ture for maturing broodstock. From the perspective of the fish produc-
tion, this drop of HSI at 12°C raises questions regarding the long-term
consequences on the broodstock growth and reproductive perfor-
mance. However, the maintenance of fish at a higher temperature is
obviously beneficial for the fish farmers in terms of energetic costs.

Additionally, in both sexes, a further degree of gonadogenesis and/or
gametogenesis was observed when the chilling period was extended by
2 months (60 days). This phenomenon was not clearly demonstrated in
the experiment of Hermelink et al. (2011), and it may also be explained
by the longer duration of the programme in our study. This shows that
in our photothermal conditions, the chilling period should exceed the
length of time (135 days at 6°C or 185 days at 12°C; see Figure 1)
that is necessary for the completion of gametogenesis and gonadoge-
nesis. This further advancement may be due to the maintenance or
even increase of the plasma sex-steroids E2 and/or T during this exten-
sion of chilling period. Indeed, E2 and T are both involved in the reg-
ulation of fish hepatic vitellogenesis and spermatogenesis (Nagahama
& Yamashita, 2008; Lopes et al., 2020). In females, the vitellogenin is
incorporated into the oocytes during vitellogenesis which contribute to
the oocyte growth and the advanced gonadogenesis. Besides E2 (or T),
vitellogenin and GSI levels are often highly positively correlated with
Then, blood was centrifuged at 3000 g for 10 min to collect plasma. At times T0, T2, T4 and T5 an average of 10 males (min: 6, max: 18) and 6 females (min: 3, max: 10) were weighed and measured to calculate the condition factor (fish weight*100/length³). Next, fish were euthanized by an overdose of MS-222 (300 mg/L, Sigma). After death, gonads and liver were extracted and weighted in order to calculate the gonadosomatic (GSI) and hepatosomatic (HSI) indices (GSI = 100 × gonad weight / total fish weight, %; HSI = 100 × liver weight / total fish weight, %). At all times, sampled fish were healthy and no lesion was observed.

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## 2.3 Gonad histology

For females and males, samples of gonads were fixed in a Bouin-Holland solution for 1 week, washed once with water, twice with 70 % ethanol and stored in absolute ethanol (Abdulfatah et al. 2011). Fragments of gonads were cut into 3-mm-thick slices, dehydrated/embedded in paraffin with successive baths (2 baths of 1h in alcool 95°, 2 baths of 1 h in absolute ethanol, 1 bath of 1h in acetone, 2 baths of 1 h in toluene and 3 baths of 1h in a paraffin substitute dubbed Diawax). For females, sections of 6 µm were cut from the Diawax block and slides were stained with a Masson's trichrome- haematoxylin Gill III (Merck, Darmstadt, Germany), 0.5 % phloxine B (VWR, California, USA) and 0.5 % light green (Sigma, Saint Quentin Fallavier, France). For males, sections of 4 µm were cut from the Diawax block and slides were stained with a trichrome: Regaud iron haematoxylin (Merck), 1 % Ponceau-Fushine of Masson (Sigma) and 0.1 % light green (Sigma). Gonadal development was evaluated according to the following procedure.

In females, five stages were considered:

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* Protoplasmic stage: protoplasmic oocytes with vacuole free cytoplasm
<figure><img src="image_9.png" /><figcaption>**FIGURE 6** The effect of thermal condition (6 and 12°C) and chilling period on 17 beta-estradiol (E2) and testosterone (T) values of female (Figures 6a and 6b respectively, mean ± SD, *n* = 3–44) and male (Figures 6c and 6d respectively, mean ± SD, *n* = 6–23) pikeperch. Sampling times T0, T1, T2, T3, T4 (S = short chilling period) and T5 (L = long chilling period) correspond, respectively, to days 0, 30, 155, 185, 215 and 275 after the beginning of the photothermal programme. Different letters indicate significant differences between the sampling times (p < 0.05)</figcaption></figure>

each other during the oogenesis (Koya et al., 2003; Martyniuk et al., 2013; Khendek et al., 2017). In males, the increase of T during spermatogenesis is also often linked to the increase of GSI (Melamed et al., 2000; Khendek et al., 2018). In that study, we assume that the persistence or increase of the plasma concentrations of E2 and T from T4 to T5 was necessary to still progress in the gonadogenesis and gametogenesis processes by achieving vitellogenesis in females and the transition from the spermatid stage to the spermatozoa stage in males.

In conclusion, to optimize the advancement of pikeperch gonadal development, we recommend to apply a slow temperature decline of 2 months (60 days) after the programme start followed by temperature stabilization at 12°C for a minimum of 6 months (185 days). Still, further studies are necessary to test the consequences on gamete quality, reproductive success and broodstock growth.

## ACKNOWLEDGEMENTS

The authors acknowledge the Eurostars Programme (E!9390 TRANSANDER) that supported financially that experiment. This study was partly supported by the Eurostars project (E!9390 TRANSANDER).

## CONFLICTS OF INTEREST

The authors declare that they have no conflict of interest.

## ETHICS APPROVAL STATEMENT

All experimental manipulations were carried out in agreement with the European and French national legislations on animal welfare after evaluation and approval of the experimental project (protocol number: APAFIS#5862-2016062720134545)

## AUTHOR CONTRIBUTIONS

Sylvain Milla: conceptualization; funding acquisition; project administration; writing - review and editing. Amine Khendek: conceptualization; investigation; writing - review and editing. Daniel Zarski: conceptualization; funding acquisition; project administration; writing - review and editing. Imen Ben Ammar: investigation; writing - review and editing. Pascal Fontaine: funding acquisition; project administration; writing - review and editing.

## DATA AVAILABILITY STATEMENT

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

## PEER REVIEW

The peer review history for this article is available at https://publons.com/publon/10.1002/aff2.21.
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• Cortical alveoli stage : appearance of yolk vesicles, occupying 2 or 3 rings in the cytoplasm periphery and then oocytes are full of yolk vesicles (green colour). Follicular and cellular layers are differentiated.

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• Early vitellogenesis: appearance of yolk globules in the oocytes (purple colour).

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• Late vitellogenesis: oocytes accumulate yolk globules and old droplets in all the oocyte.

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• Atresia: the oocyte becomes atretic.

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At each sampling time, 3-6 females per temperature and 2 slides per female were analyzed.

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For each slide, 30 oocytes were randomly selected and assigned to the above oogenetic stage. The number of oocytes belonging to each stage was counted and was expressed relatively to the total number of oocytes.

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In males, five stages were considered:

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• Differentiated spermatogonia A (SPGA): the largest cells of the germ line with voluminous, irregular nucleus surrounded by voluminous cytoplasm.

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• Spermatogonia B (SPGB): similar to spermatogonia A but smaller.

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• Spermatocyte (SPC): smaller than spermatogonia and identified by densely staining chromatin.

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• Spermatid (SPD): larger than spermatozoa with regularly spherical and dense nucleus.

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• Spermatozoa (SPZ): recognized by their kidney-shaped cell with light-coloured hair-like tails.

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At each sampling time, 6 males per temperature and 2 slides per male were analyzed. For each slide, two pictures were taken and the areas corresponding to each spermatogenetic stage were summed and expressed relatively to the whole area of the pictures.

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ORCID

Sylvain Milla https://orcid.org/0000-0002-6109-1212

Daniel Zarski https://orcid.org/0000-0003-3082-9250

Yannick Ledoré https://orcid.org/0000-0002-2648-3755

Imen Ben Ammar https://orcid.org/0000-0002-8262-8349

Pascal Fontaine https://orcid.org/0000-0002-8729-1482

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Žarski, D., Fontaine, P., Roche, J., Alix, A., Blecha, M., Broquard, K.J. & Milla, S. (2019) Time of response to hormonal treatment but not the type of a spawning agent affects the reproductive effectiveness in domesticated pikeperch, *Sander lucioperca*. *Aquaculture*, 503, 527–536. Available from: https://doi.org/10.1016/j.aquaculture.2019.01.042

**How to cite this article:** Milla, S., Khendek, A., Žarski, D., Ledoré, Y., Ben Ammar, I. & Fontaine, P. (2021) Duration of chilling phase, but not thermal condition, influence the gonad maturation of male and female domesticated pikeperch (*Sander lucioperca*). *Aquaculture Fish and Fisheries*, 1, 51–59.
https://doi.org/10.1002/aff2.21
For both sexes, the histological analysis used an optical microscope (Leica CME) equipped with an ocular camera (Jeulin, 1.3MP) and the ImageJ software. Such analysis was also used to differentiate the fish sex for T0, T1 and T3 samples while the sex was determined visually for T2, T4 and T5 during gonad extraction.

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## 2.4 Hormone assays

Concentration of 17β-estradiol (E2, ng/ml) was assayed on 50 µl of plasma, diluted or not, using the DIAsource E2-ELISA kit (DIAsource, KAP0621). Sensitivity was 5 pg/ml. Coefficient of variation (CV) intra-assays varied between 3.1% and 2.6% and CV inter-assays varied between 4.7% and 2.4% for low and high levels, respectively. Testosterone (T, ng/ml) was assayed on 25 µl of plasma using the DIAsource Testosterone ELISA Kit (DIAsource, KAPD1559). Sensitivity was 83 pg/ml. CV intra-assays varied between 2.6% and 8.4% and CV inter-assays varied between 9.4% and 12.1% for low and high levels, respectively.

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## 2.5 Statistical analysis

After checking the normality and homogeneity of variances (Shapiro-Wilk and Levene tests respectively), the effects of sampling time and temperature were analyzed, for each sex independently, by a two-way analysis of variance (ANOVA) followed, when significant, by Tukey (HSD) test with different N. The statistical experimental unit was the fish. The two factors were 1-the temperature (2 modalities: 6°C and 12°C; 4 tanks per modality; from 21 to 41 statistical repetitions depending on the sex and the variable) and 2-the time (6 modalities: from T0 to T5; 8 tanks per modality from T0 to T4 and 4 tanks at T5; from 8 to 44 statistical repetitions depending on the sex and the variable). Regarding the temperature*time interaction (12 modalities), there were 4 tanks per modality from T0

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to T4 and 2 tanks per temperature at T5 (from 3 to 18 statistical repetitions depending on the sex and the variable). If the assumptions of the ANOVA failed, non-parametric Kruskal-Wallis followed by Mann-Whitney or bilateral multiple comparisons tests were used. The level of significance used in all tests was P < 0.05. All statistical analyses were performed using the STATISTICA software (StatSoft, Tulsa, OK, USA).

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### 3. Results

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No mortality occurred during the acclimation period and during the experimental time-course. For both sexes, an elevation of growth and condition factor was observed between T0 and T2 (Females: *P* < 10⁻³ and *P* < 10⁻² respectively; Males: *P* < 10⁻³ and *P* < 10⁻³ respectively) without significant differences between the two sexes (*P* > 0.05). Then, these parameters remained steady along the experimental time-course (Tables 1 and 2). In females, the temperature significantly influenced the condition factor with a significant reduction among those kept at 12°C compared to those at 6°C (*P* < 0.05).

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In females, a progressive increase of GSI was observed along the experimental time-course (*P* < 10⁻⁴) associated with a significant difference between T4 and T5 (*P* < 10⁻², Figure 2A). However, the temperature did not significantly influence the GSI (*P* > 0.05). The temperature (*P* < 10⁻³), time (*P* < 10⁻⁴) and the interaction between temperature and time (*P* < 0.05) significantly influenced the HSI (Figure 2B) marked by a steady and elevated level from T1 in pikeperch at 6°C contrary to those at 12°C which returned to the initial level.

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Like in females, only the length of the chilling period affected the male GSI (*P* < 10⁻⁴) but no difference was noticeable between T4 and T5 (Figure 3). The temperature (*P* < 10⁻³) and time (*P* < 10⁻⁴) significantly influenced the HSI, but the interaction between both

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factors was not significant (Figure 3). The HSI was lower among males kept at 12°C compared to those kept at 6°C.

The histological analysis did not reveal any effect of temperature for either sex. However, a marked difference was observed between T4 and T5 showing a much more advanced gametogenesis when the chilling period was lengthened (Figure 4). At T4, females and males reached the early vitellogenesis stage and the spermatid stage respectively while at T5, they reached the late vitellogenesis stage and the spermatozoa stage respectively (Figure 5).

The hormonal analysis did not show any temperature effect on plasma E2 and T levels (*P* > 0.05; Figure 6) for either sex. But they revealed strong fluctuations along the experimental time-course for both females and males (*P* < 10⁻⁴ for each group). In females, E2 and T plasma levels peaked at T3 and T5 respectively with a significant marked elevation of T between T4 and T5 (*P* < 0.01; Figure 6). In males, the highest E2 and T levels were measured at T3 and T4-T5 respectively (Figure 6).

## 4. Discussion

Regardless of the sex of the fish, changes of temperature during the chilling period neither caused a change in the GSI level or caused any marked alteration of the gametogenesis stages. In contrast, in the same species, the females reared at least 3 months at 12°C during the chilling period exhibited a higher GSI than those kept at 6°C (Hermelink et al. 2011). This apparent discrepancy may stem from five major differences between both studies. First, the initial average weights highly differ between both experiments (340 g vs 775 g). Also, the absence of weight change during the chilling period (Hermelink et al. 2011) contrasts strikingly with the 2-fold weight increase observed in our experiment, possibly due to differences in the diet composition. Second, in the study of Hermelink et al. (2011),
the rate of GSI increase in females from the initial time was in the range of 3-5 fold (initial GSI: 0.9%; final GSI:4%). This rate is much lower than the rate of GSI observed herein found to have increased in the range of 15-30 fold (initial GSI: 0.45%; final GSI from 7 to 12%). These different zootechnical performances suggest a contrasted response to temperature between the pikeperch populations, which can be partly explained by different features in the experimental set up. Third, the duration of the program was much longer in our study (9 months vs 6 months). This comes from the fact that, to reach the temperature plateau during the chilling period, a slow temperature drop was applied in our experiment (from 60 to 110 days). This kinetics is in accordance with data on program optimization obtained for the Eurasian perch *Perca fluviatilis* (Abdulfatah et al. 2011) and contrary to the quick drop applied in the study of Hermelink et al. (2011). Fourth, the level and the variations of photoperiod applied during our photothermal program (Fontaine et al. 2015) contrast with the constant photoperiod maintained at 12 h/12 h during the studies of Hermelink et al. (2011) and Hermelink et al. (2016). Given the effect of photoperiod on oogenesis in pikeperch female maturation (Ben Ammar et al. 2015), we hypothesize that natural changes of photoperiod are necessary to optimize the GSI. Last, the contrasting results may come from the features of the domesticated fish populations, notably the fish life-history. Indeed, the maturation success in response to such a photothermal program depends on the captive pikeperch population used (Khendek et al. 2018).

In the present experiment, the maintenance at 12°C during the chilling phase did not disrupt the gonad maturation in pikeperch. Wang et al. (2010) suggest that in percid fishes a given temperature threshold exists, above which gametogenesis would be impaired. In Eurasian perch females, a temperature in the range of 6-14° degrees does not strongly impact the progress of gametogenesis and gonadogenesis (Abdulfatah et al. 2013). In yellow perch, the maintenance at constant 23°C, instead of applying a chilling phase à
316 10°C, inhibits the GSI increase and the gametogenesis advancement (Kolkovski and 317 Dabrowski, 1998; Shewmon et al. 2007). Our data thus support that the achievement of 318 percid gametogenesis in RAS conditions is not markedly influenced by the rearing 319 temperature below 12-14°C. At higher temperatures, the slowdown or even blockage of 320 gametogenesis is often accompanied with a decrease of sex-steroid production (e.g. 321 Abdulfatah et al. 2013). In our study, the absence of temperature effect of sex-steroid 322 levels might explain why gametogenesis was not disrupted by the tested thermal 323 conditions.

324 Despite no apparent effect on pikeperch gametogenesis, the 12°C temperature induced the 325 drop of HSI in both sexes associated with a decrease in the condition factor, at least in 326 females. This suggests that the whole energetic metabolism was affected by such 327 temperature. This data is consistent with what observed in other fish species for which an 328 elevated temperature during vitellogenesis is linked to a decrease of HSI (e.g. Im et al. 329 2016; Akhoundian et al. 2020). Still, pikeperch is thought to be one of the most 330 thermophilic percid fish species studies so far (Geay and Kestemont, 2015). Thus, this 331 thermophilic feature does not seem to apply at such low temperature for maturing 332 broodstock. From the perspective of the fish production, this drop of HSI at 12°C raises 333 questions regarding the long-term consequences on the broodstock growth and 334 reproductive performance. However, the maintenance of fish at a higher temperature is 335 obviously beneficial for the fish farmers in terms of energetic costs.

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Additionally, in both sexes, a further degree of gonadogenesis and/or gametogenesis was 337 observed when the chilling period was extended by 2 months (60 days). This phenomenon 338 was not clearly demonstrated in the experiment of Hermelink et al. (2011), and it may also 339 be explained by the longer duration of the program in our study. This shows that in our 340
341 photothermal conditions, the chilling period should exceed the length of time (135 days at 6°C or 185 days at 12°C, see Figure 1) that is necessary for the completion of 342 gametogenesis and gonadogenesis. This further advancement may be due to the 343 maintenance or even increase of the plasma sex-steroids E2 and/or T during this extension 344 of chilling period. Indeed, E2 and T are both involved in the regulation of fish hepatic 345 vitellogenesis and spermatogenesis (Nagahama and Yamashita 2008; Lopes et al. 2020). In 346 females, the vitellogenin is incorporated into the oocytes during vitellogenesis which 347 contribute to the oocyte growth and the advanced gonadogenesis. Besides E2 (or T), 348 vitellogenin and GSI levels are often highly positively correlated with each other during 349 the oogenesis (Koya et al. 2003; Martyniuk et al. 2013; Khendek et al. 2017). In males, the 350 increase of T during spermatogenesis is also often linked to the increase of GSI (Melamed 351 et al. 2000; Khendek et al. 2018). In that study, we assume that the persistence or increase 352 of the plasma concentrations of E2 and T from T4 to T5 was necessary to still progress in 353 the gonadogenesis and gametogenesis processes by achieving vitellogenesis in females and 354 the transition from spermatid stage to spermatozoa stage in males. 355

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357 In conclusion, to optimize the advancement of pikeperch gonadal development, we
358 recommend to apply a slow temperature decline of 2 months (60 days) after the program
359 start followed by temperature stabilization at 12°C for a minimum of 6 months (185 days).
360
361 Still, further studies are necessary to test the consequences on gamete quality, reproductive
362 success and broodstock growth.
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5. Acknowledgments

364
365 The authors acknowledge the Eurostars Programme (E!9390 TRANSANDER) that supported financially that experiment.
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452 of response to hormonal treatment but not the type of a spawning agent affects the
453 reproductive effectiveness in domesticated pikeperch, Sander lucioperca. Aquaculture

454 503 : 527-536. https://doi.org/10.1016/j.aquaculture.2019.01.042.

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458 **Fig. 1 Thermal regimes applied in the groups subjected to long (L) and short (S) chilling period.** Yellow targets correspond to samplings with organ and blood extractions.

459 Red targets correspond to samplings with blood extractions only. D0 corresponds to the start of the photoperiod decrease. D30 corresponds to the start of the temperature decrease in all thermal regimes. D90 and D140 correspond to the start of the chilling period in the 12°C (red) and 6°C (blue) groups, respectively. D0, D30, D155, D185, D215 and D275 correspond to the sampling times T0, T1, T2, T3, T4 and T5 respectively.

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466 **Fig. 2 The effect of thermal condition (6 and 12°C) and chilling period on gonadosomatic index GSI (mean + SD, n = 3-10) and hepatosomatic index HSI (mean + SD, n = 3-10) values of female pikeperch.** Sampling times T0, T2, T4 (S=Short chilling period) and T5 (L=Long chilling period) correspond, respectively, to days 0, 155, 215 and 275 after the beginning of the photothermal program. Different letters indicate significant differences between the sampling times or between the interactions time*temperature (*P* < 0.05).

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474 **Fig. 3 The effect of thermal condition (6 and 12°C) and chilling period on gonadosomatic index GSI (mean + SD, n = 6-18) and hepatosomatic index HSI (mean + SD, n = 6-18) values of male pikeperch.** Sampling times T0, T2, T4 (S=Short chilling period) and T5 (L=Long chilling period) correspond, respectively, to days 0, 155, 215 and 275 after the beginning of the photothermal program. Different letters indicate significant differences between the sampling times (*P* < 0.05). The asterisks indicate a significant temperature effect over the whole sampling period (*P* < 0.05).

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Fig. 4 The effect of thermal condition (6 and 12°C) and chilling period on oogenesis and spermatogenesis stages (n = 3-6 per histogram) of pikeperch. Sampling times T0, T2, T4 (S=Short chilling period) and T5 (L=Long chilling period) correspond, respectively, to days 0, 155, 215 and 275 after the beginning of the photothermal program.

Vtg = vitellogenesis. SPG = spermatogonia; SPC = spermatocytes; SPD = spermatids; SPZ = spermatozoa.

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Fig. 5 The effect of chilling period on the most advanced gametogenesis stage of pikeperch reached at the end of this period. Top left: early vitellogenesis stage reached at T4 (S=Short chilling period). Top right: late vitellogenesis stage reached at T5 (L=Long chilling period). Bottom left: spermatid stage reached at T4 (S=Short chilling period). Bottom right: spermatozoa stage reached at T5 (L=Long chilling period). Vtg = vitellogenesis; SPD = spermatids; SPZ = spermatozoa.

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Fig. 6 The effect of thermal condition (6 and 12°C) and chilling period on 17 beta-estradiol (E2) and testosterone (T) values of female (mean + SD, n = 3-44) and male (mean + SD, n = 6-23) pikeperch. Sampling times T0, T1, T2, T3, T4 (S=Short chilling period) and T5 (L=Long chilling period) correspond, respectively, to days 0, 30, 155, 185, 215 and 275 after the beginning of the photothermal program. Different letters indicate significant differences between the sampling times (*P* < 0.05).

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Table 1 The effect of thermal condition (6 and 12°C) and chilling period on weight (mean ± SD, n = 3-10 in males, n = 6-18 in females; expressed in grams) of pikeperch.

Sampling times T0, T2, T4 (S=Short chilling period) and T5 (L=Long chilling period) correspond, respectively, to days 0, 155, 215 and 275 after the beginning of the

<table><thead><tr><th>Sex</th><th>T0</th><th>Temperature</th><th>T2</th><th>T4 (S)</th><th>T5 (L)</th></tr></thead><tbody><tr><td rowspan="2">Males</td><td rowspan="2">767±137 a</td><td>6°C</td><td>1538±308 b</td><td>1677±317 b</td><td>1243±144 b</td></tr><tr><td>12°C</td><td>1481±291 b</td><td>1319±196 b</td><td>1466±256 b</td></tr><tr><td rowspan="2">Females</td><td rowspan="2">785±87 a</td><td>6°C</td><td>1467±262 b</td><td>1453±219 b</td><td>1484±188 b</td></tr><tr><td>12°C</td><td>1531±292 b</td><td>1388±276 b</td><td>1757±136 b</td></tr></tbody></table>

photothermal program. For each sex, different letters indicate significant difference between the sampling times (P < 0.05).
<table><caption>Table 2 The effect of thermal condition (6 and 12°C) and chilling period on condition factor (mean ± SD, n = 3-10 in males, n = 6-18 in females) of pikeperch. Sampling times T0, T2, T4 (S=Short chilling period) and T5 (L=Long chilling period) correspond, respectively, to days 0, 155, 215 and 275 after the beginning of the photothermal program. For each sex, different letters indicate significant differences between the sampling times (*P* < 0.05). In females, the asterisks indicate a significant temperature effect over the whole</caption><thead><tr><th>Sex</th><th>T0</th><th>Temperature</th><th>T2 (D155)</th><th>T4 (D215)</th><th>T5 (D275)</th></tr></thead><tbody><tr><td rowspan="2">Males</td><td rowspan="2">0.87±0.13 a</td><td>6°C</td><td>1.09±0.11 b</td><td>1.12±0.05 b</td><td>1.06±0.09 b</td></tr><tr><td>12°C</td><td>1.06±0.08 b</td><td>1.02±0.08 b</td><td>1.02±0.16 b</td></tr><tr><td rowspan="2">Females</td><td rowspan="2">0.89±0.06 a</td><td>6°C</td><td>1.11±0.1 b,*</td><td>1.15±0.13 b,*</td><td>1.23±0.13 b,*</td></tr><tr><td>12°C</td><td>1.05±0.12 b,*</td><td>1.02±0.15 b,*</td><td>1.06±0.12 b,*</td></tr></tbody></table>

sampling period except T0 (*P* < 0.05).
<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /><figcaption>Figure 2</figcaption></figure>

<figure><img src="image_4.png" /></figure>
<caption>Figure 3</caption>

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<figure><img src="image_6.png" /></figure>
<figure><img src="image_7.png" /><figcaption>Figure 4</figcaption></figure>

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>Figure 5</figcaption></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>
<figure><img src="image_13.png" /><figcaption>Figure 6</figcaption></figure>