Temperature control of pikeperch (Sander lucioperca) maturation in recirculating aquaculture systems—induction of puberty and course of gametogenesis

Aquaculture 400–401 (2013) 36–45

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Temperature control of pikeperch (Sander lucioperca) maturation inrecirculating aquaculture systems—induction of puberty and courseof gametogenesis

B. Hermelink a,⁎, S. Wuertz a, B. Rennert a, W. Kloas a,c, C. Schulz b,d

a Department of Ecophysiology and Aquaculture, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Mueggelseedamm 310, D-12587 Berlin, Germanyb GMA–Gesellschaft für Marine Aquakultur mbH, Hafentoern, D-25761, Buesum, Germanyc Department of Endocrinology, Institute of Biology, Humboldt University Berlin, Invalidenstrasse 42, D-10099, Berlin, Germanyd Institute of Animal Breeding and Husbandry, Christian-Albrechts-University, Olshausenstraße 40, D-24098 Kiel, Germany

⁎ Corresponding author. Tel.: +49 030 64181786; faxE-mail address: [email protected] (B. Herme

0044-8486/$ – see front matter. Crown Copyright © 20http://dx.doi.org/10.1016/j.aquaculture.2013.02.026

a b s t r a c t

Article history:Received 11 July 2011Received in revised form 19 October 2012Accepted 19 February 2013Available online 13 March 2013

Keywords:ReproductionSteroidsEstradiolTestosterone11-KetotestosteroneProgestin

After temperature-induced puberty at 12 °C for 12 weeks, the progression of gametogenesis in maturingpikeperch (Sander lucioperca) was studied at 12 °C, 14 °C, 16 °C, and 18 °C, compared with a control group rearedat 23 °C. The plasma concentration of the sex steroids estradiol (E2), testosterone (T), 11-ketotestosterone(11-KT), and 17α,20β-dihydroxy-4-pregnen-3-one (17,20-P) as well as the histology of the gonad confirmedthe successful induction of puberty in all treatment groups and an advanced gonadal maturation until the end ofthe experiment in both. After 16 weeks at 14 °C, 80% of the female pikeperch examinedwere inmid vitellogenesiscompared with a slower progression at 12 °C, 16 °C, and 18 °C. After 20 weeks, all fish reared at 14 °C accom-plished mid vitellogenesis accompanied by a significant up-regulation of E2, which was also detected in the con-specifics reared at 12 °C and 16 °C, although not as advanced. In females reared at 18 °C, only a small percentagereached mid vitellogenesis accompanied by decreasing E2 concentrations, succeeding the induction of puberty atweek 12. After 20 weeks, the level of T aswell as 11-KT peaked in fish of both sexes kept between 12 °C and 16 °C.As in females, temperature influenced postpubertal maturation in males, which was indicated by significant tem-perature dependent changes of the sex steroids. E2 plasma concentrations in males exhibited a bimodal patternwith two maxima, first after 12 weeks at the onset of puberty and the second after week 26, coinciding with theend of spermatogenesis as indicated by milt production. In conclusion, postpubertal temperatures around 14 °Cpromoted a complete ripening in pikeperch of both sexes within 8 weeks, whereas higher temperaturesconstrained full gonadal maturation. Furthermore, with reference to the optimal temperature for the inductionof puberty previously discussed, slightly higher temperatures around 14 °C are optimal for postpubertal matura-tion. This clearly indicates the existence of a dynamic temperature influence for optimal maturation due topostpubertal inhibition of maturation at temperatures higher than 18 °C. Consequently, in addition to the role oflow temperatures in the induction of puberty, this is the first evidence that emphasizes the need for a closelycontrolled temperature range to be maintained during the ongoing maturation, which should be considered inyear round production of pikeperch.

Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.

1. Introduction

Undoubtedly, the reproduction of fish is a major obstacle in finfishaquaculture diversification as most species experience some degree ofreproductive dysfunction in captivity (Mylonas et al., 2010). In severalspecies, the first sexual maturation ormore precisely the onset of vitello-genesis in females or spermatogenesis in males (referred to as puberty:Wuertz et al., 2006; Hermelink et al., 2011; Taranger et al., 2010) of cap-tive fish stagnates overextended periods, as it was reported for sturgeon

: +49 030 64181682.link).

13 Published by Elsevier B.V. All rig

(Wuertz et al., 2007a), the European eel (van Ginneken et al., 2007), andtuna (Taranger et al., 2010). The duration between the onset of pubertyand final maturation has been examined in little detail, particularly inrelation to temperature or photoperiod (Taranger et al., 2010; Wang etal., 2010).

Species-specific rearing protocols, which make use of external trig-gers such as temperature or photoperiod, have contributed substantiallyin assuring reproduction in the most important aquaculture species. Inaddition, the control of reproduction is considered as a prerequisite forthe sustainable development of the aquaculture industry, particularlyin high-cost recirculation aquaculture (Martins et al., 2010). To ensurethe optimum control of reproduction, it is essential to assess species-specific triggers, not only to avoid the inhibition of maturation but

hts reserved.

37B. Hermelink et al. / Aquaculture 400–401 (2013) 36–45

only to optimise gonadal maturation. There is a broad variety of factorsregulating the reproduction cycle in teleosts. Although the availabilityof food, rain fall, or changing water levels are able to induce andsynchronize the reproduction of some species, the main trigger in thesynchronization of wild (and aquaculture) species from temperateregions is photoperiod or temperature (Taranger et al., 2010; Wanget al., 2010). Undoubtedly, various differences exist regarding thespecies-specific influence of these environmental cues in percids. In theyellow perch (Perca flavescens), the variation of temperature ratherthan photoperiod had a stronger impact on the ovarian development(Dabrowski et al., 1996);whereas for the Eurasianperch (Percafluviatilis),changing temperatures combined with a variable photoperiod are re-quired to control the reproductive cycle (Migaud et al., 2006).

The pikeperch is doubtlessly one of the most attractive percids forEuropean aquaculture commerce. The species has excellent qualityflesh, high growth performance, and a good market acceptance(Philipsen and van der Kraak, 2008; Wuertz et al., 2012), which hasincreased research focus into the species. (Hermelink et al., 2011;Lappalainen et al., 2003; Zakes andDemska-Zakes, 2009). The pikeperchis cultured for restocking measures in recreational fisheries aroundEurope (Hilge and Steffens, 1996; Kestemont et al., 2007; Lappalainenet al., 2003). At present, the increasing demand for pikeperch as foodas well as for stocking programs almost exclusively supplied from wildfish and only occasionally by outdoor farming (FAO, 2012–2013). Thus,successful breeding and full life-cycle rearing in recirculating aquaculturesystems (RAS) is a seminal alternative in order to preservewild pikeperchpopulations and secure a sustainable development of the industry. Untilrecently, hormonal therapies were applied to achieve an out-of-seasonreproduction of pikeperch (Schlumberger and Proteau, 1996; Zakes andDemska-Zakes, 2009), and only one pioneering study reported anout-of-season reproduction of adult pikeperch by a photo-thermal ma-nipulation only (Mueller-Belecke and Zienert, 2008). In this study,Mueller-Belecke and Zienert reported an accelerated maturation andspawning of mature (adult) pikeperch at 15 °C and 16 h light after acooling period lower than 10 °C combined with a photoperiod of 8-hlight (L)–16-h dark (D). Considering the results of Mueller-Belecke andZienert (2008), we set up a first experiment regarding the induction ofpuberty in virgin pikeperch of 2 years old by different low temperatureregimes (Hermelink et al., 2011). For both sexes, a temperature-relatedinduction of puberty was already observed after 12 weeks at 12 °C.However, the postpubertal influence of different temperatures on theprogressing maturation of the pikeperch remains unclear.

In vertebrates, external factors such as temperature and photoperiodare integrated at the level of the brain, modulating the secretion ofgonadotropin releasing hormones GnRH I and GnRH II, which in turncontrol the synthesis and secretion of the gonadotropins, the folliclestimulating hormone and the luteinizing hormone. In response to thecirculating gonadotropins, sex-specific steroids are in the control ofvitellogenesis and spermatogenesis. In female fish, E2 has been recog-nized as hormone regulating the synthesis of vitellogenin, whereas17,20-P regulates final maturation and induces the germinal vesiclebreakdown preceding ovulation. In males, 11-ketotestosterone (11-KT)is considered as the main androgen, although even T is important forthedifferentiation of the early stages promoting the proliferation of sper-matogonia to spermiogenesis, whereas 11-KT partly regulates the finalmaturation of the gametes (Schulz et al., 2010; Taranger et al., 2010).

In this study, the influence of temperature on the progression ofmaturation after the successful induction of puberty was studied inmale and female pikeperch to clarify if maturation can be optimized.The temperatures chosen for our investigation covered the temperaturerange met in the species distribution during gonadal ripening andspawning (Lappalainen et al., 2003; Ozyurt et al., 2011). The sex steroidsE2, 11-KT, T, and 17,20-P were assessed as crucial key players and prox-ies of the gonad maturation and as distinct time points in pikeperchreared at various temperatures postinduction of puberty. Thereby, adynamic temperature protocol for the out-of-season reproduction was

aimed at as well as a maximization of somatic growth by the inhibitionof gonadal ripening of pubescent pikeperch for on growing purposes.

2. Materials and methods

2.1. Animals, experimental setup, and sampling

Two-year-old virgin pikeperch (initial mass, 247 ± 95 g), lifetimereared under a photoperiod of 12L:12D (10000 K, 100 lx abovesurface, electronic timer without gloaming phase) at 23 °C, wererandomly stocked to fifteen 500-l tanks (turnover 2500 l/1 h, waterexchange 5% of total recirculating water volume per day), providingfive treatments, studied in triplicates arranged within one RAS. Infour experimental groups, the induction of puberty was achieved bycooling to 12 °C within 14 d and subsequent rearing at 12 °C for12 weeks as described in Hermelink et al. (2011). The last groupwas taken as a control with an unchanged water temperature of23 °C. After the cooling period, the temperature was adjusted to theexperimental set points of 12 °C, 14 °C, 16 °C, and 18 °C for another4 months to access the progress of gonad maturation. Tank effluent ofall tanks was collected in a central tube, directed through the watertreatment units and into separate cooling units (Titan 4000 coolingunits, Aqua Medic) or heaters (Titan, Aqua Medic), respectively, inorder to provide true replicates. All tanks were additionally isolatedwith insulating foil and covered with polycarbonate multiskin sheets.A commercial trout diet (DAN-EX 1750, Dana Feed, containing 16%carbohydrates, 17% fat, and 50% protein) was fed daily at 0.5% of thewhole body mass, adjusted fortnightly after each sampling point. Forexamination, fishes were anesthetized with ethyl 3-aminobenzoatemethansulfonate (MS 222 50 mg/l; Sigma Aldrich). Total length wasmeasured in a straight line from the tip of the snout to the superiorlobe of the caudal fin in 5-mm steps, and wet weight was quantifiedto the nearest 2 g. The condition factor (K) was calculated accordingto Fulton (1904) as K = (fish somatic weight × 100) / fish totallength3. Subsequently, blood was drawn from the caudal vein withheparinized syringes and centrifuged (12,000 g, 5 min), and cell-freeplasma was immediately shock frozen and stored at −80 °C until fur-ther processing. For histological analysis, gonad samples (only females)were removed by incisional biopsy from the genital porous with asterile catheter of 0.2-mm diameter according to Steffens et al. (1996),Mueller-Belecke and Zienert (2008) and Zakes and Demska-Zakes(2009). Follicle weremeasured and subsequently transferred to Bouin’ssolution for gonad histology. After sampling,fishwere allowed to recov-er in aerated tanks, before they were returned to the correspondingtreatments. Animal care and performance of experiments were incompliance with the national guidelines as approved by the LaGeSo(Landesamt für Gesundheit und Soziales, Berlin, Germany, G0095/01).

2.2. Gonad histology

Upon fixation in Bouin’s solution for 12 h, gonad samples weredehydrated in a graded series of alcohol, embedded in paraffin (Paraplast,Roth, Germany), cut to 5 μm and stained with hematoxylin–eosin.Analysis was carried out with an Olympus RX50 microscope equippedwith an Olympus XC50 digital camera. Staging of female gonads wasperformed according to Selman et al. (1993) and Lubzens et al. (2010)(Table 1).

2.3. Hormone assays

Steroid analysis was described in detail in Hermelink et al. (2011).In brief, 100 μl of plasma was extracted in 1 ml diethyl ether (Roth)after shaking for 1 min in 10 ml glass vials. After freezing of the aque-ous phase at −80 °C for 30 min, the organic phase was transferred toa new vial and the remaining aqueous phase was once more extractedas described earlier, pooled with the first extraction and allowed to

Table 1Description of gonadal stages in female pikeperch.

Previtellogenesis Early vitellogenesis Mid vitellogenesis Late vitellogenesis

● Maximum diameter,250 μm

● Maximum diameter, 400 μm ● Maximum diameter,700 μm

● Diameter, > 700 μm

● Cytoplasm is either freeof cortical alveoli

● Accumulation of lipid droplets ● Follicle filled with manysmall lipid and yolk droplets

● Follicle filled with few largeaccumulated lipid and yolk droplets

● Nascent; cortical alveoli are withinthe follicle, close to the cortical cortex

● Yolk accumulates centripetally ● Yolk accumulates centripetally

● All follicle layers developed, including jelly coat

38 B. Hermelink et al. / Aquaculture 400–401 (2013) 36–45

evaporate over night at room temperature. For analysis, the steroidfractions were redissolved in assay buffer (Cayman Chemicals). Con-centrations of E2, T, 11-KT, and 17,20-P were determined in duplicateby specific enzyme-linked immunosorbent assays using a standarddilution series according to the manufacturer’s instructions (CaymanChemicals). Absorption at 412 nm was measured with an InfiniteM200 microplate reader (Tecan). Assay characteristics are given inTable 2.

2.4. Data analysis

Data are presented asmean ± SDof n samples. For statistical analysis,the normality and the homogeneity of variances (log-transformed if nec-essary) were tested. If confirmed, one-way ANOVA and Tukey–Kramer’smultiple comparison test were carried out. If tests failed, a nonparametricDunn’s multiple comparison test was performed. Because no tank-baseddifferences were found, data from replicate tanks were combined. Differ-ences were considered to be significant if p b 0.05. Statistical analyseswere performed using the software packages SPSS 14.0 and GraphPadPrism 4.03.

3. Results

3.1. Growth parameters

3.1.1. FemalesFemale pikeperch (Table 3) revealed a significant influence of the

temperature treatment (Fig. 1a) on the growth parameters (weight,length, and K). Compared with the initial weight and length of females,higher values were observed at 23 °C, from the third month onward.Less pronounced, although congruent, the condition factor (K) steadilyincreased at 23 °C fromweek 16 onward. In all other treatments, weight,length, andKdid not increase. Just in pikeperch kept at 16 °C,weight andlength were slightly increased toward the end of the treatment phase(weeks 24 and 26).

3.1.2. MalesMale pikeperch (Table 4) showed a similar pattern as the females

regarding growth, with an increase of weight and length at males keptat 23 °C, but not in the other treatment groups (Fig. 1b). In pikeperchkept at 14 °C, weight and length were significantly increased from

Table 2Cross-reactivity, detection limits, and intra- and interassay coefficients of variability of the

Testosterone 11

Testosterone 100% 2.211-Ketotestosterone b0.01% 10Estradiol 0.01% /17,20 Progesterone / /Detection limits (pg/ml) 6 1.3Intra assay coefficient of variability 6.2% 8.6Interassay coefficient of variability 12.7% 16

week 20 onward and also less pronounced at 18 °C at weeks 24 and26. In the other treatment groups, no growth effects were observed.

3.2. Gonadal development in females

3.2.1. Follicle diameterThe follicle diameter of the temperature-treated females (Table 3)

was significantly increased after 6 weeks, whereas only minor growtheffects were detected in control females at 23 °C over the entire exper-imental period (Fig. 2). After incubation at 12 °C for 12 weeks, atemperature- and time-related increase in the follicle diameter was ob-served in the respective treatments, confirming the successful inductionof puberty. Females of the 16 °C group peaked after 18 weeks, thepikeperch reared at 14 °C at 22 weeks, and the females kept at 12 °Creached maximum follicle diameter after 26 weeks. The follicle diame-ter in the 18 °C group was highest after 18 weeks (just as the femaleskept at 16 °C), but revealed the lowest maximum follicle size of allrespective treatments. In contrast, the control fish constantly kept at23 °C did not reveal any significant increase in follicle diameter, rangingbetween mean values from 130 to 270 μm.

3.2.2. Development of gonadal stagesConcomitant with the increase of the follicle diameter at 12 °C,

females of the treatment groups revealed the successful induction ofpuberty and progressing vitellogenesis was observed in all treatmentgroups (correlated to an increase of the plasma E2 concentrations).Early and mid vitellogenic stages were already recorded from week6 onward (Fig. 3). After the induction of puberty, a clear temperature-related effect on the progressingmaturation of the gonadwas observedin both sexes. The majority of the females reached mid and late vitello-genesis shortly after the cooling, irrespective the temperature. After24 weeks, late vitellogenesis was observed in all females at 12 °C. Inthe 14 °C group, 100% of females exhibited late vitellogenic stages al-ready after 20 weeks. After 24 weeks, 100% of the females were stillin late vitellogenesis; but after 26 weeks, 30% were in early vitellogen-esis. No previtellogenic females were detected. Fishes kept at 16 °Cshowed their developmental peak after 20 weeks. In the observedpikeperch of this group, 83% were late vitellogenic. Interestingly, atthe subsequent sampling points, previtellogenic fish were identified at71% (week 24) and 73% (week 26) in the 16 °C group. At 18 °C, theabundance of late vitellogenic females was lowest in general, 33%

enzyme-linked immunosorbent assay tests used.

-Ketotestosterone Estradiol 17,20 Progesterone

% b0.01% 0.14%0% / 0.01%

100% 0.01%/ 100%19 2

% 6.5% 4.7%% 11.3% 18.7%

Table 3Number of female pikeperch sampled according to temperature and sampling point.

Temperature (°C) Sample point (weeks)

0 VI XII XVI XVIII XX XXII XXIV XXVI

23 29 6 11 9 9 3 7 8 2412 13 7 4 7 5 0 4 514 5 0 6 1 4 716 6 7 6 7 7 1118 3 6 2 5 7 8

Fig. 1. Effect of rearing temperature (12 °C, 14 °C, 16 °C, 18 °C, and 23 °C) on growth papikeperch compared with control fish kept at 23 °C, after 0, VI, XII, XVI, XVIII, XX, XXII, XXIday 0 and the dotted line the end of cooling (induction of puberty). The number of fish peprovide a better overview, data of the same sampling point are presented with an interval wand the values at day 0 are marked by letters (Tukey–Kramers’s multiple comparison test)

39B. Hermelink et al. / Aquaculture 400–401 (2013) 36–45

(week 18) and 50% (week 20). Pikeperch kept constantly at 23 °C didnot show any advanced stages at all.

3.3. Plasma sex steroids

3.3.1. FemalesIn female pikeperch examined (Table 3), the E2 level showed a

biphasic manner, with an increase during cooling, up to 1800 pg/ml, aslow decrease until week 16, followed by an significant increase for

rameters (weight, length, and condition factor) of maturing (a) female and (b) maleV, and XXVI weeks of treatment (mean ± SD). The dashed line indicates the status atr treatment and sampling point are given in Table 3 (female) and Table 4 (male). Toithin each sampling. Significant differences between the respective growth parameters.

Table 4Number of male pikeperch sampled according to temperature and sampling point.

Temperature (°C) Sample point (weeks)

0 VI XII XVI XVIII XX XXII XXIV XXVI

23 25 5 5 5 2 8 5 4 2012 11 17 8 5 7 9 8 814 7 12 7 5 8 1816 5 4 7 5 5 1018 8 9 8 7 9 11

Fig. 2. Effect of rearing temperature (12 °C, 14 °C, 16 °C, 18 °C, and 23 °C) on the folliclediameter (mean ± SD) of maturing female pikeperch compared with control femaleskept at 23 °C, after VI, XII, XVI, XVIII, XX, XXII, XXIV, and XXVI weeks of treatment(mean ± SD). The dashed line indicates the status at day 0; the dotted line the end ofcooling (induction of puberty). The number of fish per treatment and the samplingpoint are given in Table 3. To provide a better overview, data of the same samplingpoint are presented with an interval within each sampling. Significant differencesbetween the respective growth parameters and the values at day 0 are marked by letters(Tukey–Kramers’s multiple comparison test).

40 B. Hermelink et al. / Aquaculture 400–401 (2013) 36–45

females kept between 12 °C and 16 °C. Precisely, the E2 of pikeperchkept at 12 °C was significantly increased after the cooling period on-ward, ranging from 2220 to 2230 pg/ml between weeks 20 and 26(Fig. 4). In contrast, control females kept at 23 °C exhibited basic E2level between 100 and 200 pg/ml during the entire experimental peri-od. At 14 °C, elevated E2 plasma concentrations were observed untilweek 24 but dropped to 1040 pg/ml at week 26, respectively. At16 °C, maximum E2 concentrations were recorded at week 20 reaching2650 pg/ml, followed by a distinct drop to 330 and 120 pg/ml at week24 and week 26. Pikeperch kept at 18 °C, the E2 level, did not displaythis surge and decreased during postpuberty to basic level of around100 pg/ml from week 20 on. Concomitantly to E2, T increased signifi-cantly from 840 to 3100 pg/ml within the first 12 weeks of coolingbut fluctuated between 440 and 840 pg/ml in the control. Thereafter,plasma concentrations of T increased at 12 °C, 14 °C, and 16 °C. At18 °C, no increase after the cooling phase was observed. At 12 °C and14 °C, T was found at high concentrations of 28 and 100 ng/ml atweek 26. In females reared at higher temperatures, T peaks werelower in general, 16 ng/ml in the 18 °C group (week 20) and 8 ng/mlin the 16 °C group (week 22), and returned to normal values untilweek 26.

A more heterogeneous pattern was observed for 11-KT and17,20-P level (Fig. 4). In contrast to the T plasma concentrations, pro-nounced concentrations of 11-KT were also noticed in the 23 °Cgroup. Comparable with the level of T, highest concentrations weredetected in the females reared at 12 and 14 °C between weeks 22and 26. Still, significant changes were only noticed at week 18 forthe fish kept at 12 °C, 16 °C, and 18 °C. 17,20-P plasma concentrationsexhibited a discontinuous pattern. Because of high individual varia-tions between 7 and 327 pg/ml, significant elevations were onlyfound in the cold banked females at week 12 and in the 23 °C groupat week 20.

3.3.2. MalesAs in females, temperature influenced postpubertal maturation in

male pikeperch with regard to steroidogenesis (Fig. 5). E2 as well as Tand 11-KT concentrations were significantly influenced by the rearingtemperature. During the 12 weeks of cooling, E2 increased from 88to 168 pg/ml, T from 770 to 8600 pg/ml, and 11-KT from 280 to1100 pg/ml. In the control only minor differences in E2, T, and 11-KTwere detected. Thereafter, the E2 level of the 14, 16, and 18 °C groupdecreased within 4 weeks and remained low (less than 100 pg/ml)until week 24 before sharply rising to peak level of 210, 230, and210 pg/ml at week 26. In contrast, the E2 of the 12 °C males decreasedalso after the cold phase to 80 pg/ml at week 18 but wasmissing such asharp rise from week 24 to week 26; instead, only a slight increase to110 pg/ml was observed at week 24. In the conspecifics kept at 23 °CE2, concentrations varied between 75 and 137 pg/ml. At 12 °C, 14 °C,and 16 °C, T was elevated after cold banking until week 24 reaching 19,22, and 25 ng/ml, before dropping to basic values around 0.3 ng/ml atweek 26. In the 18 °C group, constantly decreasing values lower than8.5 ng/ml were determined, from week 12 onward. At 23 °C no signifi-cant increase of T concentration was detected.

11-KT peaked at week 20 with 15 ng/ml at 12 °C, 8 ng/ml at 14 °C,and 9.8 ng/ml at 16 °C. The 18 °C group peaked 2 weeks earlier at

week 18 with 2.5 ng/ml. Concomitant to T, the 11-KT values of thefemales reared at 23 °C were constantly low always around 0.3 ng/ml,except for week 22 where a significant increase was detected with3 ng/ml. For the steroid 17,20-P, no clear temperature-related effectswere observed.

4. Discussion

The present study demonstrates that variable temperature protocolsare sufficient either to provide or to preventmaturation and completionof the reproductive cycle in pikeperch kept in captivity.

We observed that different rearing temperatures had profoundeffects on somatic and gonadal growth in maturing pikeperch of bothsexes until late vitellogenesis and spermatogenesis. In addition totemperature-induced puberty (Hermelink et al., 2011), temperaturemodulates postpubertal development of the gonad. Furthermore,temperature influence reveals a temperature optima rather than a ther-modynamic acceleration of maturation at increasing temperatures.In immature fish kept at 23 °C, puberty stagnated and fish achievedtwice as much weight gain compared with temperature-induced con-specifics (Hermelink et al., 2011; Hilge and Steffens, 1996), revealingonly previtellogenic follicle. Congruently, constantly low steroid con-centrations were detected in this group. In contrast to the pikeperchkept at 23 °C, the gonadal development of their temperature-treatedconspecifics displayed progressing maturation, requiring the majorityof available nutrients and energy (Craig, 2000). Thus, temperature af-fected the endocrine regulation subsequently directing energy eitherto gonadmaturation or to somatic growth as a prerequisite of the induc-tion of puberty (Davis et al., 2007; Wuertz et al., 2007a, 2007b). Thefollowing discussion on females and males is presented separately.

4.1. Female maturation

In female pikeperch, 14 °C turned out to be the most efficienttemperature to accelerate maturation and complete vitellogenesis asevidenced by increased follicle diameter, advanced ovarianmaturation,and steroid concentrations. The individuals kept at 12 °C (which is thetemperature needed to induce puberty) also reached late vitellogenic

Fig. 3. Effect of rearing temperature (12 °C, 14 °C, 16 °C, 18 °C, and 23 °C) on ovarian development (mean ± SD) of maturing female pikeperch, after 0, VI, XXII, XIV, XVIII, XX, XXII,XXIV, and XXVI weeks of treatment (mean ± SD) showing the percentage of maturational stages observed in females sub sampled from the respective temperature treatment. Thenumber of samples is indicated above each column.

41B. Hermelink et al. / Aquaculture 400–401 (2013) 36–45

stages but compared with the 14 °C with a delay of approximately2 weeks. Surprisingly, temperatures higher than 14 °C slowed downmaturation. Inwild caught fish, mature unfertilized follicle are reportedto reach diameter between 700 and 850 μm μm (Virbickas et al., 1974),whereas fertilized ones are between 1000 and 1670 μm (Schlumbergerand Proteau, 1996; Virbickas et al., 1974) Here, at 12 °C and 14 °C,follicle grew up to 1100 μm, which is above the biggest unfertilizedfollicle reported in wild, ripe female spawners (Virbickas et al., 1974).

Therefore, it can be argued that nutrition and diet ideally met therequirements for maturation. Furthermore, temperatures and hencedynamics of energy mobilization as well as vitellogenin supportedoptimal follicle growth, resulting in maximal follicle sizes in thesetwo groups. After fertilization, the follicle absorb water, reaching di-ameters of up to 1670 μm (Lappalainen et al., 2003; Schlumbergerand Proteau, 1996). Compared with other percid species, pikeperchhas relatively small eggs and reveals substantial variation in egg sizebetween spawners (Craig, 2000; Lappalainen et al., 2003). In walleyeSander vitreus, for instance, 1.4–2.1 mm was reported, and thoseof perch eggs range from 1.0 to 1.9 mm (Marshall, 1977; Thorpe,1977). As a conclusion, with regard to the role of egg size as qualityparameter of eggs and larval nutrition (Bromage et al., 1992), opti-mizing follicle development for maximal sizes is an important goalfor aquaculture purposes. Thus, it can be argued that the nutritionand diet (DAN-EX 1750) ideally met the requirements for maturationresulting in bigger follicle size.

In the present study, sex steroid levels were assessed as proxies ofgonad maturation. E2 as main estrogen regulating vitellogenesis wasgenerally higher at 12 °C and 14 °C than that at 16 °C and 18 °C. Moreimportantly, at elevated temperatures higher than 14 °C, E2 decreasedto basic values, whereas levels supporting vitellogenesis were onlyobserved at 12 and 14 °C. At 23 °C, E2 fluctuated between 40 and500 pg/ml, and vitellogenesis was never initiated because E2maintainsvitellogenin synthesis by the liver (Lubzens et al., 2010; Wallace andSelman, 1981). For the pikeperch and his close relative the walleye(Sander vitreus), E2 concentrations around 1–2 ng/ml are observedin general during early vitellogenesis. In female Eurasian perch(P. fluviatilis), the early vitellogenesis is induced at E2 level slightlyless than 1 ng/ml (Sulistyo et al., 1998), representing the lowest con-centration during vitellogenesis in percid species. Thus, percids reveallow E2 concentrations compared with other species. In cyprinids, forinstance, it was 100 ng/ml during vitellogenesis in the Caspian Kutum

(Rutilus frisii kutum) (Heidari et al., 2010) and 20 ng/ml in carp Cyprinuscarpio (Aizen et al., 2012; Yaron et al., 2009).

The detected plasma concentrations correspond (data of regressionanalysis not presented) to the progression in follicle diameter andmaturational stages observed, which indicates an accelerated, moreeffective maturation between 12 °C and 14 °C. Large variations in plas-ma E2 at 16 °C indicate heterogenic maturation among conspecifics,confirmed by the histological analysis. This can be interpreted as firstsymptoms of reproductive dysfunction here.

In the pejerrey (Odontesthes bonariensis), short periods of warmwater fluctuations resulted in clear signs of gonadal regression (Elisioet al., 2012), including a decrease in mRNA expression of aromataseCYP19a1a. Such a reduced expression of the key enzyme convertingtestosterone to estradiol contributed to decreased E2 steroid levels.Still, high temperature may additionally affect reaction rates of suchkey enzymes outside their temperature optimum, furthermore reduc-ing enzyme activity. A temperature-related depression of aromataseconcomitant with increasing T and decreasing E2 level was also ob-served in Atlantic salmon (Salmo salar) at elevated temperatures(Anderson et al., 2012; Watts et al., 2004). Although the T level in-creased intensively in the 12 °C and 14 °C pikeperch by the completionof vitellogenesis, the T level of the 16 °C and especially 18 °C peakedalready 4 weeks earlier, indicating an inhibitory effect on aromataseexpression or activity. Still, this needs to be addressed in future investi-gations. Interestingly, we also observed a continuous slight increase ofthe second androgen 11-KT in the 12 and 14 °C treatments, whereasin the 16 °C and 18 °C treatments, the up-regulation was less intense.It has been reported that 11-KT is involved in previtellogenic folliclegrowth of short-finned eel (Anguilla australis) (Lokman et al., 2007).Furthermore, the uptake of lipids (bound as lipoproteins) into theovary was attributed to 11-KT in short-finned eel (A. australis) (Diverset al., 2010; Endo et al., 2011; Rohr et al., 2001). Some pikeperchkept at 23 °C exhibited higher concentrations of 11-KT during theprevitellogenic stage, and the role of 11-KT in female pikeperch remainsunclear and should be addressed in future investigations.

Regarding the regulating effects of 17,20-P (Barry et al., 1995)during gonadal maturation, we only found a significant change of17,20-P in the females reared at 12 °C accompanied by high variationsof the residuals especially at 23 °C. Thus, due to the low progestinconcentrations observed irrespective of the treatment and time ofsampling, we assume that 17,20-P does not play a prominent role

Fig. 4. Effect of rearing temperature (12 °C, 14 °C, 16 °C, 18 °C, and 23 °C) on plasma sex steroids (mean ± SD) of maturing female pikeperch compared with control females keptat 23 °C, after 0, VI, XXII, XIV, XVIII, XX, XXII, XXIV, and XXVI weeks of treatment (mean ± SD). The dashed lines represent hormone concentrations at day 0; the dotted line the endof cooling (induction of puberty). The number of fish per treatment and sampling point are given in Table 3. To provide a better overview, data of the same sampling point arepresented with an interval within each sampling. Significant differences between the respective concentrations and concentrations at day 0 are marked by letters (Dunn’s multiplecomparison test). E2—17ß-estradiol, T—testosterone, 11-KT—11-ketotestosterone, 17,20-P—17,20β-progesterone.

42 B. Hermelink et al. / Aquaculture 400–401 (2013) 36–45

until germinal vesicle breakdown (GVBD). In walleye, concentrationsduring previtellogenesis and vitellogenesis are low but sharply peakedand induce the GVBD during final maturation (Barry et al., 1995;Pankhurst et al., 1986). Nevertheless, we assumed that if a depressionof E2 can be observed in late vitellogenic females, this might be accom-panied with the known switch of the steroidogenic pathway to theproduction of 17,20-P (Barry et al., 1995; Lubzens et al., 2010). Thus, itbecomes visible just days before the actual GVBD, as observed in thewalleye (Barry et al., 1995). Unfortunately, we were not able to detectincreasing 17,20-P levels concomitant with decreasing E2 levels. Conse-quently, it seems that the majority of late vitellogenic pikeperch mightbe observed before synthesis of 17,20-P. However, to get a clear insightconcerning the progression of progesterone in the later gonadal matu-ration, further investigations are required.

4.2. Male maturation

Here, as a result of the minimum invasive biopsy, histologicalinvestigations were not feasible in male pikeperch. Thus, only theanalyses of the plasma steroid concentrations were available to char-acterize the progression of maturation. In this context, the establishedmajor steroids involved in the regulation of reproductive functions, Tand 11-KT (Kloas et al., 2009; Schulz et al., 2008, 2010;Weltzien et al.,2004), were observed. These androgens are known to control the

spermatogenic cycle in fish (Kloas et al., 2009; Schulz et al., 2008,2010; Weltzien et al., 2004). Although 11-KT is considered to be themost effective androgen to stimulate spermatogenesis, T (beside itsrole as the biosynthetic precursor of 11-KT) might act primarily viapositive feedback on the hypothalamus and the pituitary and there-fore on the secretion of the gonadotropins luteinizing hormone andfollicle stimulating hormone.

11-KT regulates mainly the initiation of spermatogonial proliferationtoward meiosis (Munakata et al., 2000; Schulz et al., 2010; Weltzien etal., 2004). The action of 11-KT in the different processes is mediated byfactors that are synthesized by the Sertoli cells. There aremuch evidencesgiven that these are growth factors like IGF1 and activin B (Schulz et al.,2010).

In male pikeperch, we observed increased levels of 11-KT as wellas T during the first 12 weeks of the experiment concomitant withthe induction of puberty. Both steroids peaked after 20 weeks ofincubation in the males of the 12 °C, 14 °C, and 16 °C treatments. Asimilar profile of plasma T and 11-KT level were also found in walleye(Stizostedion vitreum) (Malison et al., 1994) and Eurasian Perch(P. fluviatilis) (Sulistyo et al., 2000). Although the perch peaked at12 ng/ml (T) and 5 ng/ml 11-KT, the steroid concentrations observedin the walleye were generally lower for T (5 ng/ml) but also with aclear biphasic development (Malison et al., 1994) comparable withthe T level observed in the pikeperch. However, for 11-KT, the walleye

Fig. 5. Effects of rearing temperature (12 °C, 14 °C, 16 °C, 18 °C, and 23 °C) on plasma sex steroids (mean ± SD) of maturing male pikeperch compared with control females kept at23 °C, after 0, VI, XXII, XIV, XVIII, XX, XXII, XXIV, and XXVI weeks of treatment (mean ± SD). The dashed lines represent the hormone concentrations at day 0; the dotted line theend of cooling (induction of puberty). The number of fish per treatment and the sampling point are given in Table 4. To provide a better overview, data of the same sampling pointare presented with an interval within each sampling. Significant differences between the respective concentrations and concentrations at day 0 are marked by letters (Dunn’smultiple comparison test). E2—17ß-estradiol, T—testosterone, 11-KT—11-ketotestosterone, 17,20β-P—17,20β-progesterone.

43B. Hermelink et al. / Aquaculture 400–401 (2013) 36–45

peaked with 40 ng/ml (Malison et al., 1994) above the maximumconcentrations observed in pikeperch (15 ng/ml).

The pikeperch kept at 18 °C were generally low in their androgenlevel. Thus, it can be presumed that the increased plasma androgenconcentrations observed provided the final maturation of male gam-etes (which was indicated by milt production during the last weeksof the experiment). At the end of the experiment, correspondingwith low concentrations of androgens after peak concentrationsupon the induction of puberty, no milt production was observed atall. Regarding the regulating effects of both androgens (especially11-KT), further investigations might be focused on the expression ofthe growth factors mentioned.

Although androgens and their involvement in the spermatogenesisare investigated in detail, the role of estrogens concerning the regula-tion in proceeding gonadal maturation postpuberty is scarcely known.Increasing evidence suggests that estrogens are involved in the regula-tion of testicular functions which is supported by the expression ofat least three estrogen receptors in the testis (Schulz et al., 2010). Inmale trout, E2 application resulted in the expressions of numerousgenes, which are important for the steroidogenesis (i.e., StAR andaromatase) and spermatogenesis (Ito et al., 2007; Schulz et al., 2010).Recently, the role of estrogens in the early stages of spermatogenesiswas investigated (Amer et al., 2001;Miura andMiura, 2003). In huchen(Hucho perryi) and the Japanese eel (Anguilla japonica), estrogens are

implicated in the regulation of testicular stem cell renewal and sper-matogonial proliferation (Amer et al., 2001; Miura and Miura, 2003;Schulz et al., 2010). Nevertheless, there is still lack of data regardingthe estrogenic derived processes during the whole reproduction cycle.

In our experiment, the males kept lower than 14 °C showed a clearbimodal E2 pattern with a first peak at the end of the cooling phase(induction of puberty), followed by a second one after 26 weeks. Thisindicates the involvement of E2 in the regression of gonad developmentafter final maturation. This is supported by the observed decreasingandrogen level from week 20 onward. Comparable observations werealso made in male Gilthead seabream (Sparus aurata), where E2 levelwere accelerated at the end of spermatogenesis, spawning, and duringpostspawning events (Chaves-Pozo et al., 2007; Kadmon et al., 1985).Another newsworthy study reported that the reduction of semen vol-ume, sperm density, and sperm fertility in fully mature and spawningmale rainbow trout (Oncorynchus mykiss) appeared after the fish wereexposed to low doses of E2 (Lahnsteiner et al., 2006). Therefore, weconsider that E2 is also essential for the regulation of male gonadalmaturation.

In addition and in contrast to female pikeperch, we found elevatedE2 concentrations in males at temperatures between 12 °C, 16 °C,and 18 °C. With the current knowledge, it can only be speculated ifsex-specific differences regarding the temperature-dependentmodula-tion of enzymes like the aromatase in pikeperch might exist, or if there

44 B. Hermelink et al. / Aquaculture 400–401 (2013) 36–45

are other possible explanations. In addition, the involvement of theprogestin 17,20-P in the testicular development and maturation ofpikeperch is not yet fully understood.We foundmaximum plasma con-centrations of 17,20-P inmales kept at 23 °C, although their conspecificsin the temperature treatments peaked firstly at the end of the coolingphase and secondly just after 11-KT decreased. In our previous investi-gation (Hermelink et al., 2011), males that were steadily reared at 23 °Cnever showed any further testicular ripening but also reached compara-ble progestin level.

Furthermore, in Cottus sp. and Tinca tinca, 17,20-P peaked betweengametogenic quiescence and early spermatogenesis (Fukui et al., 2007;Pinillos et al., 2003). On this basis, it can be hypothesized that 17,20-Pis irrelevant for the regulation of puberty or early spermatogenesis. Incontrast to the warm banked pikeperch, the males of the temperaturetreatments completed spermatogenesis, which was indicated by miltproduction. According to Scott et al. (2010), the initiation of spermiationis accompanied not only by a sharp rise in plasma 17,20-P concentra-tions, influencing (among others) the sperm motility (Weltzien et al.,2004), but also by a distinct drop in plasma 11-KT concentration, compa-rable with our observations made.

4.3. Conclusion

In summary, it is evident that temperatures around 14 °C areoptimal to support vitellogenesis and spermatogenesis after the inductionof puberty at 12 °C. This is reflected in species-specific profiles of E2, T,and 11-KT. In females kept between 12 °C and 14 °C, a slight increase of11-KT indicated its role in the regulation of lipid uptake during vitellogen-esis. In the present study, the completion of vitellogenesis and spermato-genesis in 2-year-old pikeperch only by temperature was successfullyoptimized. Thereby, the basic information on temperature-related effectsand subsequent influence on plasma steroids is provided. Differentialtemperature effects on successivematurational stages suggest a potentialto optimize reproduction by establishing a dynamic temperature protocolfor the all-year production of pikeperch in RAS.

Acknowledgment

This study was supported by the German research funding DFGas part of the project “Influence of Exogenous Factors on the Endo-crine Regulation of Gonad Maturation in pikeperch Sander lucioperca”(SCHU 2308/1-1 AOBJ: 530719).

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