Reproductive performance and seasonal plasma sex

steroid and metabolite levels in a captive wild

broodstock of brill Scophthalmus rhombus L.

Ismael Hachero-Cruzado1,�, AŁ ngel Garc|¤ a-Lo¤ pez2,�, Marcelino Herrera1, LuisVargas-Chaco¡3,Gonzalo Mart|¤ nez-Rodr|¤ guez2, Juan M Mancera3 & Jose¤ I Navas1

1IFAPA (Instituto de Investigacio¤ n y Formacio¤ n Agraria y Pesquera deAndaluc|¤ a) CentroAgua del Pino. Carretera Cartaya ^

Punta Umbr|¤ a s/n, Cartaya, Huelva, Spain2Instituto de Ciencias Marinas deAndaluc|¤ a, Consejo Superior de Investigaciones Cient|¤ ¢cas, Avenida Repu¤ blica Saharaui

no. 2, Puerto Real, CaŁ diz, Spain3Departamento de Biolog|¤ a, Facultad de Ciencias del Mar yAmbientales, Universidad de CaŁ diz, Pol|¤ gono Universitario R|¤ o San

Pedro, Puerto Real, CaŁ diz, Spain

Correspondence: Ismael Hachero-Cruzado, IFAPA (Instituto de Investigacio¤ n y Formacio¤ n Agraria y Pesquera de Andaluc|¤ a) Centro

Agua del Pino. Carretera Cartaya-Punta Umbr|¤ a s/n, E-21450 Cartaya, Huelva, Spain. E-mail: ismael.hachero.ext@juntadeandalucia.es�Contributed equally in the preparation of this paper.

Abstract

This study reports egg production by captive wildbrill Scophthalmus rhombus, a potential new £at¢shspecies for Southern Europe-Mediterraneanmaricul-ture, as well as seasonal plasma levels of 17b^estra-diol, testosterone, 11^ketotestosterone, proteins,triglycerides, glucose and lactate. A mean egg pro-duction of 102800 eggs kg body weight�1 wasachieved during the 2005 spawning period (Janu-ary^March), although a continuous egg supply couldonly be obtained from some females, which had ahigher relative fecundity (261019 � 10393 eggskg�1) with12^17 eggs batches released at a mean in-terval of 3.4 days. Most eggs were obtained withwater temperatures ranging from12 to14 1C, andun-der increasing temperatures (up to 2.9 1C). Potentialegg viability (70.1 � 2.9%), fertilization (72.2 �3.4%) and hatching rates (31.9 � 3.9%) showed highvariability, with potential viability tending to de-crease as the water temperature increased (mainlybetween 16 and 17 1C) and 0% hatching above16.6 1C. The endocrine changes that brill underwentduring late gametogenesis, spawning and post-spawning periods were similar to those reported inother Pleuronectiformes. This study establishes animportant basis for further research on the biologyand physiology of brill reproduction, directed to-

wards the optimization of the breeding techniquesused currently.

Keywords: turbots, arti¢cial breeding, reproduc-tive physiology, sex hormones, metabolic parameters

Introduction

The great expansion of the marine ¢sh aquacultureindustry in Europe during the last decades can beattributed to the culture of a few species, whosemarkets already show signs of saturation. Therefore,introduction of new species (diversi¢cation of aqua-culture products), as well as solving other biological,technical and environmental problems, seems to becrucial to keep the industry economically viable.Among these new target species, £at¢shes occupya top position, because they normally have verygood market prices and present high growth rates(Person-Le Ruyet, Baudin-Laurencin, Devauchelle,Me¤ tailler, Nicolas, Robin & Guillaume 1991; Shields,Gara & Gillespie 1999; Arneri, Collella & Gianetti2001; Imsland, Foss, Conceic� a� o, Dinis, Delbare,Schram, Kamstra, Rema & White 2003; Hachero2006). One of these potential candidate £at¢shspecies for Southern Europe^Mediterranean marine

Aquaculture Research, 2007, 38, 1161^1174 doi:10.1111/j.1365-2109.2007.01782.x

r 2007 TheAuthorsJournal Compilationr 2007 Blackwell Publishing Ltd 1161

aquaculture diversi¢cation is brill Scophthalmusrhombus L., because: (1) it is well adapted to warmclimates (it inhabits coastal waters from 5 to 50mdepth along the Eastern Atlantic and the Mediterra-nean Sea; Bauchot 1987); (2) its landings are scarce;(3) its growth in the wild is rapid during the ¢rst 2years of life (Robert & Vianet 1988; Arneri et al.2001), period in which the market size (500^1000 g)can be reached in culture (I. Hachero, M. Herrera &J. I. Navas, unpubl. data); and (4) mainly due toits high market value [between 20 and 30 h kg�1;Isla Cristina (Huelva, Spain) ¢sh market]. In addition,its similarity to turbot Psetta maxima (L.), specieswhich currently dominates the £at¢sh farming sec-tor (Brown 2002), enables the application of cur-rently available breeding techniques for this species.In fact, the studies examining the biology of bothspecies and their suitability for large-scale culturebegan together in the early1970s in the United King-dom (Jones 1972). The results obtained with brill inthose preliminary studies were worse comparedwith turbot, and therefore, it was determined thatthe second species had more potential for commer-cial cultivation (Jones 1972). After these preliminarystudies and to our knowledge, no more informationhas been published on the culture of brill until thepresent day.Control of gametogenesis and reproductive events is

mediated by the brain^pituitary^gonad (BPG) axis(Weltzien, Andersson, Andersen, Shalchian-Tabrizi &Norberg 2004). It seems to be evident that other endo-crine factors linked to growth, nutritional status andmetabolism in£uence the BPG axis functions^andvice versa ^ (Luquet & Watanabe1986; Le Gac, Blaise,Fostier, Le Bail, Loir, Mourot & Weil 1993; Holloway &Leatherland1998;Weltzien et al.2004). One of themostwidely used and practical tools to obtain informationon the BPG axis and on the reproductive status of the¢sh is the assessment of plasma levels of sex steroidsduring the gonadal cycle (Merson, Casey, Martinez,So⁄entino, Chandlee & Specker 2000). Moreover, theevolution of nutritional-status-related metabolite plas-ma levels along the reproductive cycle can be useful toevaluate the health (Guijarro, Lopez-Patin� o, Pinillos,Isorna, De Pedro, Alonso-Go¤ mez, Alonso-Bedate &Delgado 2003) and physiological status of the ¢sh, be-cause gonadal maturation and spawning can drasti-cally a¡ect the internal milieu of the organism (seereferences in Svoboda, Kour› il, HamaŁ e' kovaŁ , Kalab,Savina, SvobodovaŁ & VykusobaŁ 2001).From 2002, the £at¢sh culture group at IFAPACen-

tro Agua del Pino (Cartaya, Huelva, Spain), in colla-

boration with other Institutions, has been focusingon the study of brill reproductive biology in captivity,among others aspects related to the rearing techni-que, as a necessary prerequisite for the developmentof a sustainable culture for this species. Thus, theaims of the present work, as a starting point to gaina comprehensive knowledge on the reproductive biol-ogy and physiology of brill in mariculture, are tocharacterise the annual egg production obtainedfrom a captive wild broodstock of this species and toprovide reference plasma levels of several sex steroids[17b^estradiol (E2), testosterone (T) and 11^ketotes-tosterone (11^KT)] and metabolites (proteins, trigly-cerides, glucose and lactate) during the reproductivecycle in captivity.

Material and methods

Collection and management of wild breeders

Brill breeders were caught by trawling or trammelnetting in the Gulf of CaŁ diz (SW Spain) between2002 and 2004 and thereafter transported to theIFAPACentroAgua del Pino facilities (Cartaya, Huelva,Spain) where they were acclimated to captivity andkept during the experimental period (November2004 to June 2005). At arrival, animals with serioussores in the skin and/or ¢ns were treated with oxyte-tracycline through consecutive baths during 7^10days (dose: 50 ppm) or injected with Terramicine(20% tetracycline; P¢zer Laboratories, Alcobendas,Madrid, Spain) at a dose of 0.25mL kg�1 (one singleinjection). Preventive 100 ppm formalin baths wereapplied to those ¢sh without evident wounds aftercapture. A diet of fresh or frozen European pilchardSardina pilchardus (Walbaum)was used to adapt bree-ders to feeding in captivity. Fish successfully adaptedto captivity (n524) were sexed by abdominal mas-sage in order to express oocytes or sperm, taggedwith an intramuscular passive integrated transpon-der tag (Trovan ID100A; EID Ibe¤ rica, Madrid, Spain)and stocked in four groups (Table 1). Fish were keptunder natural conditions of photoperiod (37117 0N,719 0W; Fig.1) in 4.2m2 (5m3;1.2mwater depth) rec-tangular ¢breglass tanks located indoors. No sandsubstrate was used. The tanks were supplied withrunning sea water (SW; 200% renovation per day)with 37^39 g L�1 salinity and 5^9 ppm oxygen.Water temperature £uctuated naturally (Fig. 1), ex-cept during summer when it was maintained below23 1C (titanium plates heat exchanger; Alfa LavalIberia, Alcobendas, Madrid, Spain). During the ex-

Reproductive performance in captive wild brill I Hachero-Cruzado et al. Aquaculture Research, 2007, 38, 1161^1174

r 2007 TheAuthors1162 Journal Compilationr 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 1161^1174

perimental period, ¢shwere fed (at14:00 hours) withfrozen squid Loligo spp. and bluewhitingMicromesis-tius poutassou (Risso) twice a week, and frozen mus-sels Mytilus spp., pilchard and dry pellets speci¢c forbreeders (Skretting, Cojobar, Burgas, Spain) once aweek at a maximum daily ration of 2% of body mass.

Gamete collection and arti¢cial fertilization

Because natural spawnings of brill under the currentrearing conditions had very poor potential viabilityrates during 2004 (6.7 � 1.6%), from 2005 onwards,gametes were hand-stripped frommature ¢sh and ar-ti¢cially fertilized. Arti¢cial fertilization protocolused in the present studywas based on that used cur-rently for turbot at the production scale (Omnes, Nor-mant, Suquet & Fauvel 1991; Person-Le Ruyet et al.1991), and thus it can be extrapolated to industrialhatcheries. During the spawning season (mid Janu-ary to late March), all females were periodicallychecked for expression of hyaline eggs. In order to

collect freshly ovulated eggs and thus avoid over ri-pening inside the ovary lumen (McEvoy 1984), thestripping frequency was selected individually bychecking the females daily during the beginning ofthe spawning season to establish their approximatedovulatory rhythms according to their ¢rst ovulationdate. In the majority of them, a 3-day stripping fre-quency was found to be e¡ective. However, the fe-males that could not be successfully stripped on thethird day were checked daily during the subsequentdays until the eggs were released. Tanks were alsoprovided with egg collectors and when eggs were de-tected onanunexpected ovulationday, the females inthe tank were stripped for eggs. Males maintainedspermiating over the whole reproductive season,and so they were alternatively chosen for the extrac-tion of sperm. Before handling, ¢sh were anaesthe-tized in 2^phenoxyethanol (500 ppm, maximumtime of exposure: 5min). Thereafter, eggs were hand^stripped, being collected in a dry 2 L plastic beakerand kept at room temperature (10^15 1C) until fertili-zation (ca. 20min). The total number of eggs was cal-culated volumetrically under dry conditions,counting the eggs contained in one 500 mL aliquotand extrapolating it to the total volume of eggs col-lected, which was measured in a calibrated cylinder.The same aliquot was used to calculate the potentialviability rate and to measure, after adding SW, thediameter of 10 randomly chosen potentially viableeggs. Eggs were estimated to be potentially-viablewhen they showed a perfect spherical and translu-cent aspect (McEvoy 1984; Fauvel, Omnes, Suquet &Normant 1992). Each egg batch was fertilized at 18^20 1C with freshly stripped sperm from one male,using a volume ratio eggs:sperm:sea water of

Month (2004-2005)

Dc Jn Fb Mr Ap My Jn

Tot

al d

aily

fec

undi

ty (

10−3

egg

s)

0

50

100

150

200

250 Water tem

perature (°C) or daylight (hours)8

10

12

14

16

18

20

22

Figure 1 Total daily fe-cundity during the year2005 (vertical bars) of fe-male brill held under a natu-rally £uctuating watertemperature (line plot) andphotoperiod (line and scat-ter plot). Data from the fourgroups of breeders (Tables 1and 2) have been combinedto showa single cycle.

Table 1 Composition (number of ¢sh), mean ( � SEM)weights and stocking density of the four groups of brill bree-ders used in the present study

GroupComposition(females:males)

Weight (g) Stockingdensity(kgm� 2)Females Males

A 3:3 2664 � 525 1770 � 378 3.2

B 4:2 2872 � 207 2046 � 444 3.7

C 3:3 2782 � 513 1949 � 226 3.4

D 4:2 2823 � 294 1378 � 513 3.3

Aquaculture Research, 2007, 38, 1161^1174 Reproductive performance in captive wild brill I Hachero-Cruzado et al.

r 2007 TheAuthorsJournal Compilationr 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 1161^1174 1163

100:0.2:100. Sperm density was not measured, but allsperm used for fertilization were viscous and hadhigh motility. After gentle mixing, the fertilizationmixture was left for 3min (Suquet, Billard, Cosson,Normant & Fauvel1995) before adding more SW till atotal volume of1.5 L.Thirty minutes later, the £oatingeggs were collected and one sample of 50 eggs wasincubated in 12-well culture plates ¢lled with 3mLof ¢ltered SW (two eggs per well) at temperaturesranging from 13.2 to 19.8 1C (mean temperature:15.7 1C) in order to estimate the fertilization andhatching rates. Sucha static systemwas used in orderto minimize the mechanical shocks, which can alterthe normal egg development, especiallyat themorulastage and just before hatching (Person-Le Ruyet et al.1991). Two hours after setting up incubation, thenumber of eggs with cellular division (two to four cellstage) was counted and the fertilization rate was cal-culated. Five days after fertilization, the number oflarvae in the plate was counted and the hatching ratewas calculated. All the operations were performedduring the morning.

Blood sampling and analytical techniques

From four to 14 females and from four to 10 malesstocked in the di¡erent groups were randomly bloodsampled each month from November 2004 to May2005. After being anaesthetized as above, a sample ofbloodwas collected from the caudal vessels using coldheparinized syringes.The plasma, obtained by centri-fugation (1200 g, 10min, 4 1C), was stored at � 80 1Cuntil further analyses. To reduce stress by successivehandling during the spawning season, blood extrac-tion coincided with gamete stripping manipulation.In order to obtain a reliable measurement of plasmametabolite levels, which canvarywith time after feed-ing, blood extraction was always performed between10:00 and12:00 hours (feeding at14:00 hours).Plasma levels of E2 (females), 11-KT (males) and T

were quanti¢ed by an enzyme-linked immunosor-bent assay (ELISA) according to Rodriguez, Begtashi,Zanuy and Carrillo (2000). The assay was validatedfor use with brill plasma (data not shown) by con-¢rming parallel displacement of serially dilutedpooled plasma samples to the standard curves as wellas no signi¢cant displacement of steroid-strippedplasma pools (prepared according to Barry, Lapp,Kayest & Malison1993). Steroids were extracted from5 mL plasma in1.5mL methanol. Reagents and mate-rials were the same as described in Garc|¤ a-Lo¤ pez, An-

guis, Couto, Canario, Can� avate, Sarasquete andMart|¤ nez-Rodr|¤ guez (2006). The percentages of re-covery for E2, 11^KT and T in steroid-spiked plasmapools were 80%, 85% and 90% (n54) respectively.The inter-assay coe⁄cients of variation at 50% ofbinding were 0.2% for E2 (n52), 7.0% for 11-KT(n52), and 8.0% for T (n54). The intra-assay coe⁄-cients of variation (calculated from the sample dupli-cates) were 3.8 � 0.6% for E2, 9.7 � 1.1% for 11-KTand 7.1 � 0.8% for T. The lower limit of detection forall assays was 35 pgmL�1 plasma. Main cross-reac-tivity (41%; given by the supplier; Cayman Chemi-cal Company, Ann Arbor, MI USA) was detectedwith estradiol^3^glucoronide (17%) and oestrone(4%) for the E2 antibody and with 5a^dihydrotestos-terone (27.4%), 5b-dihydrotestosterone (18.9%), an-drostenedione (3.7%) and 11^KT (2.2%) for the Tantibody. Cross-reactivity with other steroids waslower than1% for the11-KTantibody.Plasma glucose, lactate, and triglycerides were

measured using commercial microplate kits fromSpinreact (Girona, Spain). Plasma proteins concentra-tion was measured using the bicinchoninic acidmethod with a BCA protein kit (Pierce, Rockford, IL,USA) for microplates, with bovine serum albumin asstandard. These assays were run on a Bio KineticsEL-340i Automated Microplate Reader (Bio-Tek In-struments,Winoosky,VT, USA) using DELTASOFT3 soft-ware forMacintosh (BioMetallics, Princeton, NJ, USA).

Statistics

Data, presented as mean � standard error of mean(SEM), were analysed for statistical di¡erencesamong groups by one-way ANOVA or by Kruskal^Wallis one^way ANOVA on ranks if data complied ornot with normality and homogeneity of variance re-spectively. In the ¢rst case, ANOVAwas followed by Stu-dent^Newman^Keuls (SNK) while in the second byDunn’s multiple-comparison procedures, bothwith asigni¢cance level (P) of 0.05. The interaction betweenpotentially viable eggs diameter and total body mass,water temperature, date or fecundity was studied bylinear regression analysis.

Results

Egg production and quality evaluation

Captive brill breeders showed a spawning period dur-ing year 2005 lasting, taking into account the wholepopulation, from January 19th to March 21st (10.1^

Reproductive performance in captive wild brill I Hachero-Cruzado et al. Aquaculture Research, 2007, 38, 1161^1174

r 2007 TheAuthors1164 Journal Compilationr 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 1161^1174

12.0 h light per day) (Fig.1). A total of102 egg batcheswere collected during 48 days out of 61, with a totalharvest of 3654250 eggs. Daily collection rangedfrom 12432 to 205760 eggs, with a mean of 74994eggs. Although eggs were obtained from 13 femalesout of14, a continuous supplywas attained only fromfour females, accounting for 67% of the total egg har-vest (data not shown). Such females were success-fully stripped 12^17 times at a mean interval of 3.4days. The egg batches obtained from these fourfemales represented 50% of the total egg batches

collected. Although the mean relative fecundity wassigni¢cantly higher in ‘good’ females (n54) com-pared with the remaining (n59) (261019 � 10393vs. 52006 � 7414 eggs kg�1 respectively; Po0.001),batch fecundity was similar (40541 � 4185 vs.31437 � 3361eggs ovulation�1; P50.136).The diameter of potentially viable eggs ranged

from 1.28 to 1.65mm, with a mean ( � SEM) of1.42 � 0.002mm. No signi¢cant correlation wasfound between egg diameter and total body mass,water temperature at egg collection or during theprevious day of stripping, or date along the spawningseason (data not shown). However, egg diameter wasnegatively correlated with total, batch and relative fe-cundity, with the strongest correlation being withbatch fecundity (Fig. 2). Accordingly, mean diameterof potentially viable eggs obtained from the femaleswith higher fecundity (see previous paragraph) wassigni¢cantly lower (Po0.001) than that of eggs col-lected from the remaining females (1.41 � 0.003 vs.1.44 � 0.005mm respectively).The mean potential egg viability, fertilization and

hatching rates, taking into account monthly groupeddata from the four groups of breeders, did not showany signi¢cant change along the spawning season,ranging from 69% to 72%, from 66% to 82%, andfrom 22% to 39% respectively (Fig. 3). Females witha higher fecundity also showed signi¢cantly higherpotential egg viability and fertilization rates com-pared with the remaining females (76 � 3% and69 � 4% vs. 61 � 5% and 46 � 7% respectively).Hatching rate was relatively higher in the formerthan in the latter (36 � 5% vs. 23 � 6%).Table 2 summarizes the reproductive performance

characteristics obtained from the four groups of brillbreeders used in the present study.

Relationship between water temperature andegg production and quality

Although egg batches were collected withwater tem-perature ranging from10.0 to 16.7 1C in the breedingtanks (Fig.1), between 61% and 69% of the total har-vest (64^71 batches out of 102) was obtained whenthe water temperature ranged from 12 to 14 1C theprevious or the same day of collection (Fig. 4). Thetemperature of the day before egg collection was ac-counted because stripping was always performedduring the morning and therefore, it was probablethat eggs were ovulated during the day before collec-tion.Within this temperature range, 67^69% of the

Batch fecundity (10 x n eggs /ovulation)

15 20 25 30 35 40 45 50

Egg

dia

met

er (

mm

)

1.37

1.39

1.41

1.43

1.45

1.47

1.49Y = −0.002 X + 1.494r = −0.801 r = 0.641P = 0.0018

º

Figure 2 Diameter of potentially viable eggs in relationto batch fecundity per female brill during the year 2005.Data (mean � SEM) from the four groups of breeders (Ta-bles1and 2) have been combined.

Month (2005)

Jn Fb Mr

Pot

enti

al e

gg v

iabi

lity,

fer

tiliz

atio

nan

d ha

tchi

ng (

%)

0

20

40

60

80

100 (14) (55) (33)

Figure 3 Potential egg viability (black bars), fertilization(grey bars) and hatching rates (white bars) achieved bybrill breeders during the year 2005. Data (mean � SEM)from the four groups of breeders (Tables1and 2) have beencombined. Means of each parameter are not signi¢cantlydi¡erent among months (Kruskal^Wallis one-way ANOVA

on ranks; P40.05). The numbers in parenthesis indicatethe number of egg batches collected each month.

Aquaculture Research, 2007, 38, 1161^1174 Reproductive performance in captive wild brill I Hachero-Cruzado et al.

r 2007 TheAuthorsJournal Compilationr 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 1161^1174 1165

eggs were collected in February (48^50 eggs batchesout of 64^71; data not shown).The relationship between changes in water tem-

perature 4, 3 and 2 days before egg collection, andthe total fecundity is shown in Fig. 5. Between 59%and 62% of the total egg production was collectedwhen thewater temperature in the breeding tanks in-creased from 0.1 to 2.9 1C during the previous days ofstripping (59^63 batches out of102). Despite this gen-Ta

ble

2Reprodu

ctiveperforman

cech

aracteristicsob

tained

during

year

2005

from

thefourgrou

psof

brill

breedersusedin

thepresen

tstudy

Group

Total

AB

CD

No.

of

fem

ale

s(o

vula

ting)

3(2

)4

(4)

3(3

)4

(4)

14

(13)

Bio

mass

ovula

ting

fem

ale

s(k

g)

4.4

11.5

8.3

11.3

35.5

First

ovula

tion

date

15

Febru

ary

19

January

19

January

21

January

19

January

Last

ovula

tion

date

17

Marc

h16

Marc

h21

Marc

h16

Marc

h21

Marc

h

Tota

lno.

of

ovu

latio

ns

728

34

33

102

No.

of

ovula

tions

fem

ale�

12.3�

1.9

(0–6)

7.0�

2.7

(4–15)

11.3�

3.5

(5–17)

8.3�

3.2

(3–17)

7.3�

1.5

(0–17)

Ovula

tion

frequency

(days)

6.7�

3.7

(3–14)

4.2�

0.5

(1–13)

3.2�

0.1

(2–6)

3.6�

0.3

(1–8)

3.8�

0.2

(1–14)

Tota

lfe

cundity

(10�

3�

no.

of

eggs)

227.6

1,1

10.7

1,3

80.5

935.5

3,6

54.2

Batc

hfe

cundity

(10�

3�

no.

of

eggs

ovula

tion�

1)

32.5�

3.2

ab

39.7�

4.0

a40.6�

3.6

a28.3�

2.6

b35.8�

1.9

Rela

tive

fecu

ndity

(10�

3�

no

of

eggs

kg�

1)

51.5

96.7

165.4

82.8

102.8

Pote

ntia

lvia

bili

ty(%

)29.7�

11.8

c76.5�

4.3

ab

60.1�

5.6

bc

83.1�

3.0

a70.1�

2.9

Fert

iliza

tion

(%)

79.0�

15.0

a73.3�

6.6

a66.7�

5.4

a75.8�

6.2

a72.2�

3.4

Hatc

hin

g(%

)0

a38.9�

8.1

a24.4�

5.5

a36.1�

7.1

a31.9�

3.9

Dataareexpressedas

mean�

SEM.Numbers

inpa

rentheses

indicate

thedata

range.Ovu

lation

frequen

cywas

calculatedfrom

ovulation

swithpo

tentially

viable

eggs.Sa

meno

tation

deno

teshom

o-geneousgrou

ps(P40.05

).

Temperature range (°C)

10-11 11-12 12-13 13-14 14-15 15-16 16-17

Tot

al f

ecun

dity

(%

)

0

50

40

30

20

10 68

1416

45

41

26

23

3 3 35 5

6

Figure 4 Proportion of total fecundity achieved by fe-male brill breeders in relation to water temperature dur-ing the previous (black bars) and the same day (greybars) of egg collection. Data from the four groups of bree-ders (Tables1and 2) have been combined. Numbers abovebars indicate the number of egg batches.

Temperature variation (°C) before egg collection

+0.6 to +2.9 +0.1 to +0.5 0 −0.1 to −0.5 −0.6 to −2.2

Tot

al f

ecun

dity

(%

)

0

10

20

30

4040

36

30

19

27

29

8

6 8

16

17

23

19

1612

Figure 5 Proportion of total fecundity achieved by fe-male brill breeders in relation to water temperaturechanges during the four (black bars), three (grey bars)and two (white bars) days before egg collection. Data fromthe four groups of breeders (Tables 1 and 2) have beencombined. The numbers above bars indicate the numberof egg batches.

Reproductive performance in captive wild brill I Hachero-Cruzado et al. Aquaculture Research, 2007, 38, 1161^1174

r 2007 TheAuthors1166 Journal Compilationr 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 1161^1174

eral trend, the egg batches collected in January weremainlyovulated under steadyor decreasing tempera-tures (9^12-egg batches out of 14, which accountedfor12^17% of the total egg harvest, data not shown).The potential egg viability rate showed a decreas-

ing tendency among increasing water temperatureintervals, considering the temperature in the breed-ing tanks both at the same day and at the day before

egg collection (Fig. 6). No time e¡ect was observed(see Fig.3).Egg batches with potential viability and fertiliza-

tion rates equal to or higher than 70% (n541) butwith low to null hatching rates were obtained withina wide range of collection and incubation tempera-tures (from10 to17 and from13 to19 1C respectively,Fig.7). However, all of them collected and incubatedat temperatures above 13.5 and 16.6 1C (respectively)had 0% hatching (four out of 41), while those egg lotspresenting hatching rates equal to or higher than50% were always obtained at collection and incuba-tion temperatures below13.5 and16.3 1C respectively(14 out of 41; Fig.7).

Plasma sex steroid and metaboliteprofiles

Females

Plasma E2 levels remained high during the ¢rst halfof the spawning period (January 17^February 9th2005; Fig. 8a), although with elevated individualvariability as denoted by the high values of SEMThereafter, they declined progressively until baselinevalues in late April. The presence of T in the bloodstream was low both before and after the spawningperiod, while it was detected at its highest levels inthe sampling performed in the middle of the repro-ductive season (Fig. 8b). In the case of plasma totalprotein levels (Fig. 8c), they increased markedly fromDecember12004 to January172005, remained hightill March17th and subsequently decreased on April21st. Both plasma triglycerides and glucose levels(Figs. 8d and e respectively) increased gradually tillpeaking in February 2005 and thereafter, they de-creased progressively until reaching in lateApril low-er or similar values than those found in December.Plasma lactate levels remained steady at relative highvalues from December to March, decreasing signi¢-cantly in April (Fig. 8f). Available data did not allowus to correlate endocrine pro¢les with individualspawning performance, as well as to compare steroidand metabolite levels between high-and low-fecund-ity females.

Males

Both plasma11-KTandT levels (Figs.9a and b respec-tively) increased gradually till peaking in February2005 (some males showed maximum androgen con-

Temperature range (°C)10-11 11-12 12-13 13-14 14-15 15-16 16-17

Pot

enti

al e

gg v

iabi

lity

(%)

0

20

40

60

80

100 ab a a

ba a

b

bab

ab

ab

abc

ab

cb

abc

Figure 6 Potential egg viability rate achieved by brillbreeders in relation to water temperature during the pre-vious (black bars) and the same day (white bars) of egg col-lection. Data (mean � SEM) from the four groups ofbreeders (Tables1and 2) have been combined. Means withdi¡erent notations are signi¢cantly di¡erent among tem-perature ranges in the same parameter and day (Krus-kal^Wallis one-way ANOVA on ranks; Dunn’s method;Po0.05). See Fig. 4 for sample sizes.

Temperature at egg collection (°C)

15 16 1712 1310 11 14

Incu

bati

on t

empe

ratu

re (

°C)

12

13

14

15

16

17

18

19

20Hatching = 0%Hatching = 5-48%Hatching = 50-88%

Figure 7 Temperature at egg collection and incubationtemperature in relation to hatching rate obtained from ar-ti¢cial fertilization of brill gametes.Values correspond toegg batches with potential viability and fertilization ratesequal to or higher than 70% (n541). Data from the fourgroups of breeders (Tables1and 2) have been combined.

Aquaculture Research, 2007, 38, 1161^1174 Reproductive performance in captive wild brill I Hachero-Cruzado et al.

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centrations in January 2005, data not shown) andthereafter, they decreased progressively until reach-ing in lateApril signi¢cantly lower values than thosefound in December. Male plasma levels of total pro-teins (Fig. 9c), triglycerides (Fig. 9d), glucose (Fig. 9e)and lactate (Fig.9f) showed slight changes during theexperimental period, remaining at relativelyor signif-icantly lower values than those measured in females.

Discussion

Egg production and quality

Wild captive brill showed in the current study atSouthwest Iberian Peninsula a spawning period last-ing from mid-January to mid-March. In the Mediter-

ranean and Black Seas, spawning also occurs in latewinter (Bauchot 1987), although in the Adriatic Sea,the spawning season seems to be prolonged until July(Caputo, Candi, Colella & Arneri 2001). In the NorthSea, conversely, brill spawns (as turbot) from May toAugust (Jones 1972), as a consequence of the ex-pected lower temperature.Both the number of egg batches per female (up to

17; mean:7.3) and ovulation frequency (3.8 days) ob-tained for brill were similar to those reported for tur-bot, which is able to spawn up to 12^16 times perseason (Howell & Scott 1989; Person-Le Ruyet et al.1991) with a ovulatory period between 80 and 90 h(3.3^3.7 days; McEvoy1984). However, the relative fe-cundity was lower in the case of brill (102800 vs.430000 eggs kg�1 in turbot; Person-Le Ruyet et al.

Pro

tein

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abab

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(14) (5) (4) (4) (4)(a)

(c)

(e) (f)

(d)

(b)

Figure 8 Monthlychanges in plasma levels of (a) E2, (b) T, (c) proteins, (d) triglycerides, (e) glucose and (f) lactate in femalebrill.The numbers in parenthesis indicate the sample sizes for all the plasma parameters. Horizontal bars at the lower sideof each panel represent the spawning period. Means ( � SEM) with di¡erent notations are signi¢cantly di¡erent (one-wayANOVA; Student^Newman^Keuls test; Po0.05).

Reproductive performance in captive wild brill I Hachero-Cruzado et al. Aquaculture Research, 2007, 38, 1161^1174

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1991), a di¡erence possibly related to the egg size dif-ferences between both species (1.28^1.65mm for brillvs. 0.9^1.2mm for turbot, this study, Jones 1972). In-terestingly, this possibility matches the lower egg dia-meter reported for female brill showing the higherfecundities. Another (not mutually exclusive) possi-bility would be the lower weight of our breeders incomparison with turbot breeders used by Person-LeRuyet et al. (1991) (1.2^3.9 vs. 3.5^8 kg). Similar toAmerican plaice Hippoglossoides platessoides (Fabri-cius) (Nagler, Adams & Cyr 1999), but in contrast toAtlantic cod Gadus morhua L. (Kjesbu, Kryvi & Nor-berg 1996), Senegalese sole Solea senegalensis Kaup(Dinis, Ribeiro, Soares & Sarasquete 1999), turbot(Howell & Scott 1989; Mugnier, Guennoc, Lebegue,Fostier & Breton 2000) and Atlantic halibut Hippo-

glossus hippoglossus (L.) (Brown, Shields & Bromage2006) egg diameter in brill did not decrease duringthe course of the spawning season. The small num-ber of eggs per batch measured (n510) and/or thefact that only potentially viable eggs were taken intoaccount in the present study cannot be discarded asthe origin of such di¡erences. However, a circum-stance supporting our data is that the number of eggs(both potentially viable and non-viable) contained inthe 500 mL aliquot taken from each batch to calculatethe total egg number, despite £uctuating along thespawning season for each female, was similar be-tween the beginning and the end of this period.The present study obtained relatively high poten-

tial egg viability rates, similar to those reported byFauvel et al. (1992) and higher than those by Devau-

Pro

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(d)

(b)

Figure 9 Monthly changes in the plasma levels of (a) 11-KT, (b) T, (c) proteins, (d) triglycerides, (e) glucose and (f) lactatein male brill. Numbers in parenthesis indicate the sample sizes for all the plasma parameters. Horizontal bars at the lowerside of eachpanel represent the spawning period. Means ( � SEM)with di¡erent notations are signi¢cantly di¡erent (one-wayANOVA; Student^Newman^Keuls test; Po0.05).

Aquaculture Research, 2007, 38, 1161^1174 Reproductive performance in captive wild brill I Hachero-Cruzado et al.

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chelle, Alexandre, Le Corre and Letty (1988), both forturbot. However, we also found a high variabilityamong individuals and egg batches, which indicatesthat the stripping strategy used was e⁄cient to avoidegg overripening inside the ovary lumen only insome females, i.e. stripping of eggs was not well syn-chronized with ovulatory patterns in all the females.The fertilization rates obtained in the present

study were also relatively elevated, although withhigh variability. This variability could also be asso-ciated with the mismatch between the ovulatoryrhythms and the stripping frequency in some femalesleading to egg overripening. Another possibility toexplain this variability would be the use of inade-quate insemination conditions as pointed out by Su-quet et al. (1995). Therefore, we cannot rule out the‘male e¡ect’ on such variability (as well as in thatfound in the hatching rate), because we used spermfrom only one male instead of a pool of sperms to fer-tilize each egg batch and sperm density was not mea-sured (although all sperm used for fertilization wasviscous and had high motility). The fertilization va-lues obtained here were similar to those reported forturbot by Omnes et al. (1991) at the pilot scale and byChereguini, Garcia de la Banda, Rasines and FernaŁ n-dez (1999) at the experimental scale. This was trueeven though the quantity of sperm used in the pre-sent work was lower than that used by Chereguiniet al. (1999) (0.2 vs.0.5mL per100mL of eggs).Regarding hatching rates, the values obtained in

the present work with brill were similar to those re-ported by Fauvel et al. (1992), although signi¢cantlylower than those found by McEvoy (1984), both forturbot. In the latter study, such high hatching rates(up to 97%) were obtained only for ‘freshly stripped’eggs (collected within 10 h of ovulation), becauseafter being retained in the ovary lumen for1day, thehatching rate declined to 0%. Therefore, it can besuggested that, similar to turbot (McEvoy 1984) andAtlantic halibut (Norberg, Valkner, Huse, Karlsen &Ler˛y Grung1991), brill eggs overripen quickly whenretained in the ovary lumen and this is probably amajor cause of poor or variable egg quality in captivebroodstocks.Water temperature ranges for egg collection in the

present work were very similar to those reported forspawning of turbot; from 9.5^11 to 16^17 1C (Brom-ley, Sykes & Howell 1986; Person-Le Ruyet et al.1991),with the maximal production between 13 and 15 1C(Devauchelle et al. 1988; Person-Le Ruyet et al. 1991).Our data suggest that an increase in temperaturemay induce ovulation-spawning in brill, as demon-

strated previously in southern £ounder Paralichthyslethostigma Jordan & Gilbert (from14.0^15.5 to 17 1C;Smith, McVey, Jenkins, Denson, Heyward, Sullivan &Berlinsky 1999) or Senegalese sole (up to 2.5 1C; An-guis & Can� avate, 2005). It has been reported thatwater temperature is able to act on the synchroniza-tion of ¢nal phases of gamete maturation, ovulation,and spawning (Bromage, Porter & Randal 2001).Therefore, it is probable that the thermal increase sti-mulates the ¢nal maturationand ovulation of a batchof oocytes in all the species mentioned above.The potential egg viability rate in brill tended to

decrease as the water temperature in the breedingtanks increased, especially in the interval 16^17 1C.This temperature threshold coincides with that re-ported for turbot, whose eggs always showed a lowerpotential viability over 16^17 1C (Devauchelle et al.1988). This circumstance may be related to the quick-er ageing and overripening of ovulated eggs insidethe ovary lumen (Billard 1985; Howell & Scott 1989)and/or to disturbances of di¡erent endocrine-depen-dent developmental processes before ovulationunderinadequately high temperatures (van der Kraak &Pankhurst 1997; Davies & Bromage, 2002). Impairedegg viability with increasing temperatures has alsobeen reported, among others, in European seabassDicentrarchus labrax (L.) (416 1C; Carrillo, Zanuy,Prat, Serrano & Bromage 1993), Atlantic halibut(48 1C; Brown et al. 1993) and Atlantic cod (49.6 1C;van der Meeren & Ivannikov 2006).Available data suggest the existence of a thermal

threshold a¡ecting hatching, because all the eggbatches incubated at temperatures above16.6 1C had0%hatching. A similar upper limit for adequate incu-bation temperature has been reported for turbot(17 1C in the North Sea, Devauchelle et al. 1988;18 1Cin the Baltic Sea, Nissling, Johansson & Jacobsson2006). Moreover, temperatures higher than 17 1Chave been reported to induce additional deformitiesin turbot embryos (Devauchelle et al.1988).The results obtained show that an acceptable brill

eggs supply can be achieved using arti¢cial fertiliza-tion techniques similar to those currently availablefor turbot (Omnes et al. 1991; Person-Le Ruyet et al.1991). However, it is necessary to study precisely theovulatory rhythms to improve the gamete collectionmethod, the implication of water temperature varia-tions on the ¢nal stages of maturation/ovulation, thecharacteristics of brill sperm to optimize the arti¢cialfertilization protocol and the embryonic develop-ment under the current incubation system to im-prove the hatching success. Studies of these condi-

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tions that would make spontaneous natural spawn-ing possible and controllable may also be of high in-terest to brill broodstock management. In addition,the use of hormonal stimulation to overcome spawn-ing problems in some females, to synchronize ovula-tion within the broodstock and to maximize eggproduction should be evaluated in future research.

Plasma sex steroid and metabolite pro¢les

In general, the evolution of plasma sex steroid levelsin brill reported here agrees with that described pre-viously for other £at¢sh species, like turbot (Howell &Scott1989) Atlantic halibut (Methven, Crim, Norberg,Brown, Go¡ & Huse1992;Weltzien,Taranger, Karlsen& Norberg 2002), English sole Parophrys vetulus Gir-ard (Sol, Olson, Lomax & Johnson 1998), summer£ounder Paralichthys dentatus (L.) (Merson et al.2000), spotted halibutVerasper variegatus (Temminck& Schlegel) (Koya,Watanabe, Soyano, Ohta, Aritaki &Matsubara 2003) and Senegalese sole (Garc|¤ a-Lo¤ pezet al. 2006).Plasma sex hormone concentrations were highly

variable among brill individuals during the spawn-ing season, which may re£ect the asynchrony in the¢nal phases of gamete maturation and spermiation/ovulation, as reported in turbot (Howell & Scott1989). In group-synchronous multi-spawning ¢sh,such as brill (Caputo et al. 2001; this study), circulat-ing levels of steroids £uctuated in relation to the cy-cles of oocyte batch recruitment, maturation, andrelease throughout the spawning season (Howell &Scott 1989; Methven et al. 1992; Asturiano, Sorbera,Ramos, Kime, Carrillo & Zanuy 2002). A similarpattern has been observed in males, because eachincrease in the sperm productionused to be precededby increases in the synthesis of androgens(Asturiano et al. 2002).Regarding steroid pro¢les in females, it is interest-

ing to note the delayed peak of Tconcentration in re-lation to E2 surge and decline. The fact that T levelsincreased markedly or remained high whereas E2 le-vels started to decrease is characteristic of the ¢naloocyte maturation of most teleost (Scott,Witthames,Turner & Canario 1998; Sun & Pankhurst 2004). Atthis stage,T seems to be involved in the maintenanceof oocytes once vitellogenesis has been completed(Kime1993) or in the enhancement of gonadotropin-releasing hormone sensitivity of the pituitary(Trudeau, Murthy, Habibi, Sloley & Peter 1993) inpreparation for the preovulatory surge in plasma

luteinizing hormone (Mylonas, Scott & Zohar 1997).In turbot, Howell and Scott (1989) reported a closerelation between short-term peaks of T plasma levelsand maximum in egg production over the spawningseason.It is worth noting the relatively high levels of T

compared with those of 11-KT found in male brill. Inthe majority of £at¢sh studied to date (i.e. Harmin,Crim &Wiegand1995;Weltzien et al.2002; Garc|¤ a-Lo¤ -pez et al. 2006), the concentrations of plasma 11-KTduring gametogenesis and spawning are 5^10-foldhigher than those of T, as indicative of the greater re-levance of 11-KT in the control of spermatogenesisand reproductive behaviour in teleosts (Borg 1994).However, the high levels of T found in our study sug-gest a more relevant role for T in brill spermatogen-esis. To our knowledge, no information regarding theratio between the levels of these steroids is currentlyavailable for turbot to compare our results.The evolution of plasma levels of metabolites in

brill showed a clear sex-speci¢c pattern, which sug-gests the considerable di¡erences in the metabolicand energy requirements for female and male repro-ductive processes, with oogenesis requiring a higherenergy demand than spermatogenesis. Similar di¡er-ences have been reported in the plasma levels of tri-glycerides and proteins of tench Tinca tinca (L.) atprespawning (Svoboda et al. 2001; Guijarro et al.2003), although no di¡erences in serum glucose andlipid levels were found betweenmale and female Eur-opeanplaice Pleuronectes platessa L. at any time of thereproductive cycle (White & Fletcher 1985; White,Fletcher & Pope1986).The high plasma proteins levels (in conjunction

with elevated E2 concentrations) during the brillspawning season seem to be mainly associated withincreased synthesis of vitellogenin (see references inJohnson, Casillas, Myers, Rhodes & Olson 1991),whichwould be elevated during the whole reproduc-tive season, as reported in other multiple spawners(i.e. Methven et al.1992; Sun & Pankhurst 2004). Plas-ma triglyceride levels also peaked in female brill dur-ing spawning, indicating the mobilization of lipidsfrom the liver and other body tissues, both to be in-corporated by growing oocytes (Johnson et al. 1991)and to cover the great energy investment requiredfor vitellogenesis (Bon, Corraze, Kaushik & Le-Menn1997). Some ¢sh species show improved toleranceand better dietary glucose use during gonadalmaturation (Luquet & Watanabe 1986), which mayindicate the importance of carbohydrates as energysource at this stage of the life cycle (Washburn, Bruss,

Aquaculture Research, 2007, 38, 1161^1174 Reproductive performance in captive wild brill I Hachero-Cruzado et al.

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Avery & Freedland1992). Increased hepatic glycolyticactivity (Soengas, Barciela & Aldegunde 1995) andexportation of generated glucose to the ovary(Mommsen &Walsh1988) mayalso have contributedto hyperglycaemia found in female brill during pre-spawning and spawning, as reported in £ounder Pla-tichtys £esus (L.) (Petersen & Emmersen 1977) andtench (Svoboda et al. 2001). Conversely, plasma glu-cose levels declined at spawning in English sole(Johnson et al. 1991), while they were una¡ected bythe reproductive cycle in European plaice (White &Fletcher 1985). The changes in plasma lactate levelsin female brill were also in£uenced by the consider-able energy demand required for gonadal develop-ment. Lactate-derived energy seems to be moreimportant towards the end of the spawning season,when plasma levels of triglycerides and glucosestarted to decrease.The present work provides reference plasma levels

of sex steroids and metabolites for future studies onthe physiology of the reproductive events of brill.Such physiological studies will complement those re-lated to spawning performance in our goal to estab-lish a reliable breeding technique for this species.

Acknowledgments

This research has been funded by the projects DI-VERAQUA (Interreg IIIA: SP5.E36). The authorsthank J. F. Ferrer, J. M. MaŁ rquez and M. Rosano at De-sarrolloAgrario y Pesquero S.A. (Junta deAndaluc|¤ a)and A. Rodiles at IFAPACentroAgua del Pino for theiressential help with ¢sh husbandry. Thanks are alsodue to R. Osta at ICMAN for her skilful assistance insteroid measurements.

References

AnguisV. & Can� avate J.P. (2005) Spawningof captive Senegalsole (Solea senegalensis) under a naturally £uctuating tem-perature regime. Aquaculture 243,133^145.

Arneri E., Collella S. & Gianetti G. (2001) Age determinationand growth of turbot and brill in theAdriatic Sea: reversalof the seasonal pattern of otolith zone formation. Journalof Applied Ichthyology17, 256^261.

Asturiano J.F., Sorbera L.A., Ramos J., Kime D.E., Carrillo M.& Zanuy S. (2002) Group-synchronous ovarian develop-ment, ovulation and spermiation in the European seabass (Dicentrarchus labrax L.) Could be regulated by shiftsin gonadal steroidogenesis. Scientia Marina 66, 273^282.

Barry T.P., Lapp A.F., Kayest B. & Malison J.A. (1993) Valida-tion of a microtitre plate ELISA for measuring cortisol in

¢sh and comparison of stress responses of rainbow trout

(Oncorhynchus mykiss) and lake trout (Salvelinus namay-

cush). Aquaculture117,351^363.Bauchot M.L. (1987) Poissons osseux. In: Fiches FAO d’identi-

¢cation pour les besoins de la peŒ che (re¤ vision1). Me¤ diterrane¤ e

et mer Noire. Zone de peΠche 3,Vol. 2 (ed. byW. Fischer, M.L.

Bauchot & M. Schneider), pp. 891^1421. Commission des

Communaute¤ s Europe¤ ennes & FAO, Rome.Billard R. (1985) Environmental factors in salmonid culture

and the control of reproduction. In: Salmonid Reproduc-

tion (ed. by R.N. Iwamoto & S. Sower), pp.70^87.Washing-

ton Sea Grant Program, University of Washington,

Seattle,WA, USA.Bon E., Corraze G., Kaushik S. & Le-Menn F. (1997) E¡ects of

accelerated photoperiod regimes on the reproductive cy-

cle of the female rainbow trout: 1. Seasonal variations of

plasma lipids correlated with vitellogenesis. Comparative

Biochemistry and Physiology Part A118,183^190.Borg B. (1994) Androgens in teleost ¢shes. Comparative Bio-

chemistry and Physiology Part C109, 219^245.Bromage N.R., Porter M.J.R. & Randal C.F. (2001) The envir-

onmental regulation of maturation in farmed ¢n¢shwith

special reference to the role of photoperiod and melato-

nin. Aquaculture197,63^98.Bromley P.J., Sykes P.A. & Howell B.R. (1986) Egg production

of turbot (Scophthalmus maximus L.) Spawning in tank

conditions. Aquaculture 53, 287^293.Brown N. (2002) Flat¢sh farming systems in theAtlantic re-

gion. Reviews in Fisheries Science10, 403^419.Brown N.P., Shields R.J. & Bromage N.R. (2006) The in£u-

ence of water temperature on spawning patterns and

egg quality in theAtlantic halibut (Hippoglossus hippoglos-

sus L.). Aquaculture 261,993^1002.CaputoV., Candi G., Colella S. & Arneri E. (2001) Reproduc-

tive biology of turbot (Psetta maxima) and brill (Scophthal-

mus rhombus) (Teleostei, Pleuronectiformes) in the

Adriatic Sea. ItalianJournal of Zoology 68,107^113.CarrilloM., Zanuy S., Prat F., SerranoR. & BromageN. (1993)

Environmental and hormonal control of reproduction in

sea bass. In: Recent Advances in Aquaculture,Vol. IV (ed. by

F. Muir & R.J. Roberts), pp. 43^54. Blackwell Scienti¢c

Publications, Oxford, UK.Chereguini O., Garcia de la Banda I., Rasines I. & FernaŁ ndez

A. (1999) Arti¢cial fertilization in turbot, Scophthalmus

maximus (L.): di¡erent methods and determination of

the optimal sperm-egg ratio. Aquaculture Research 30,319^324.

Davies B. & Bromage N. (2002) The e¡ects of £uctuating sea-

sonal and constant water temperatures on the photoper-

iodic advancement of reproduction in female rainbow

trout, Oncorhynchus mykiss. Aquaculture 205,183^200.Devauchelle N., Alexandre J.C., Le Corre N. & LettyY. (1988)

Spawning of turbot (Scophthalmus maximus) in captivity.

Aquaculture 69,159^184.

Reproductive performance in captive wild brill I Hachero-Cruzado et al. Aquaculture Research, 2007, 38, 1161^1174

r 2007 TheAuthors1172 Journal Compilationr 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 1161^1174

Dinis M.T., Ribeiro L., Soares F. & Sarasquete C. (1999) A re-view on the cultivation of Solea senegalensis in Spain andPortugal. Aquaculture176, 27^38.

Fauvel C., Omnes M.H., Suquet M. & Normant Y. (1992) En-hancement of the production of turbot, Scophthalmusmaximus (L.), larvae by controlling overripening inmature females. Aquaculture and Fisheries Management23, 209^216.

Garc|¤ a-Lo¤ pez A., Anguis V., Couto E., Canario A.V.M., Can� a-vate J.P., Sarasquete C. & Mart|¤ nez-Rodr|¤ guez G. (2006)Non-invasive assessment of reproductive status and cycleof sex steroid levels in a captive wild broodstock ofSenegalese sole Solea senegalensis (Kaup). Aquaculture254,583^593.

Guijarro A.I., Lopez-Patin� o M.A., Pinillos M.L., Isorna E.,De Pedro N., Alonso-Go¤ mez A.L., Alonso-Bedate M. &Delgado M.J. (2003) Seasonal changes in haematologyand metabolic resources in the tench. Journal of Fish Biol-ogy 62,803^815.

Hachero I. (2006) Perspectivas del cultivo del parracho en ellitoral SuratlaŁ ntico. In: Proceedings of the III Jornadas deAcuicultura en el litoral SuratlaŁ ntico: ‘‘Innovacio¤ n en Acui-cultura: produccio¤ n y comercializacio¤ n’’ Cartaya (Huelva),Spain. pp.65^71. Consejer|¤ a deAgricultura y Pesca, JuntadeAndaluc|¤ a.

Harmin S.A., Crim L.W. & Wiegand M.D. (1995) Plasma sexsteroid pro¢les and the seasonal reproductive cycle inmale and female winter £ounder, Pleuronectes ameri-canus.Marine Biology121,601^610.

HollowayA.C. & Leatherland J.F. (1998) Neuroendocrine reg-ulationof growth hormone secretion in teleost ¢sheswithemphasis on the involvement of gonadal sex steroids. Re-views in Fish Biology and Fisheries 8, 409^429.

Howell B.R. & Scott A.P. (1989) Ovulation cycles and post-ovulatory deterioration of eggs of the turbot (Scophthal-mus maximus L.). Rapports et Proce' s-Verbaux des Re' unionsdu Conseil International pour l’Exploration de la Mer 191,21^26.

Imsland A.K., Foss A., Conceic� a� o L.E.C., Dinis M.T., DelbareD., Schram E., Kamstra A., Rema P. & White P. (2003) Areviewof the culture potential of Solea solea and S. Senega-lensis. Reviews in Fish Biology and Fisheries13,379^407.

Johnson L.L., Casillas E., Myers M.S., Rhodes L.D. & OlsonO.P. (1991) Patterns of oocyte development and relatedchanges in 17-b estradiol, vitellogenin, and plasmachemistry in English sole Parophrys vetulus Girard.Journal of Experimental Marine Biology and Ecology 152,161^185.

Jones A. (1972) Studies on egg development and larval rear-ing of turbot, Scophthalmus maximus L., and brill,Scophthalmus rhombus L., in the laboratory. Journal of theMarine Biological Association of the United Kingdom 52,965^986.

Kime D.E. (1993) ‘Classical’ and ‘non-classical’ reproductivesteroids in ¢sh. Reviews in Fish Biology and Fisheries 3,160^180.

Kjesbu O.S., Kryvi H. & Norberg B. (1996) Oocyte size andstructure in relation to blood plasma steroid hormones inindividually monitored, spawning Atlantic cod. Journal ofFish Biology 49,1197^1215.

Koya Y., Watanabe H., Soyano K., Ohta K., Aritaki M. &Matsubara T. (2003) Testicular development and serumsteroid hormone levels in captive male spotted halibutVerasper variegatus. Fishieries Science 69,792^798.

Le Gac F., Blaise O., FostierA., Le Bail P.Y., Loir M., Mourot B.&Weil C. (1993) Growth hormone (GH) and reproduction:a review. Fish Physiology and Biochemistry13, 219^232.

Luquet P. & Watanabe T. (1986) Interaction ‘‘nutrition ^ re-production’’ in ¢sh. Fish Physiology and Biochemistry 2,121^129.

McEvoy L.A. (1984) Ovulatory rhythms and over-ripeningof eggs in cultivated turbot, Scophthalmus maximus L.Journal of Fish Biology 24, 437^448.

Merson R.R., Casey C.S., Martinez C., So⁄entino B., Chan-dlee M. & Specker J.L. (2000) Oocyte development in sum-mer £ounder: seasonal changes and steroid correlates.Journal of Fish Biology 57,182^196.

Methven D.A., Crim L.W., Norberg B., Brown J.A., Go¡ G.P. &Huse I. (1992) Seasonal reproduction and plasma levels ofsex steroids and vitellogenin in Atlantic halibut (Hippo-glossus hippoglossus). Canadian Journal of Fisheries andAquatic Sciences 49,754^759.

MommsenT.P. & Walsh P.J. (1988) Vitellogenesis and oocyteassembly. In: Fish Physiology, Vol. XIA (ed. by W.S. Hoar& D.J. Randall), pp. 347^406. Academic Press, NewYork,NY, USA.

Mugnier C., Guennoc M., Lebegue E., Fostier A. & Breton B.(2000) Induction synchronisation of spawning in culti-vated turbot (Scophthalmus maximus L.) Broodstock byimplantation of a sustained-release gnrh-a pellet. Aqua-culture181, 241^255.

Mylonas C.C., Scott A.P. & ZoharY. (1997) Plasmagonadotro-pin II, sex steroids, and thyroid hormones inwild strippedbass (Morone saxatilis) during spermiation and ¢nal oo-cyte maturation. General and Comparative Endocrinology108, 223^236.

Nagler J.J., Adams B.A. & Cyr D.G. (1999) Egg production,fertility, and hatch success of American plaice held incaptivity. Transactions of the American Fisheries Society128,727^736.

Nissling A., Johansson U. & Jacobsson M. (2006) E¡ects ofsalinity and temperature on the reproductive success ofturbot (Scophthalmus maximus) in the Baltic Sea. FisheriesResearch 80, 230^238.

Norberg B.,Valkner V., Huse J., Karlsen I. & Ler˛y Grung G.(1991) Ovulatory rhythms and egg viability in theAtlantic halibut (Hippoglossus hippoglossus). Aquaculture97,365^371.

Omnes M.H., Normant Y., Suquet M. & Fauvel C. (1991)Analysis of turbot (Scophthalmusmaximus) broodstock pilotscale production. In: Proceedings of theAquaculture Europe1991 Conference: ‘‘Aquaculture and the Environment’’, Du-

Aquaculture Research, 2007, 38, 1161^1174 Reproductive performance in captive wild brill I Hachero-Cruzado et al.

r 2007 TheAuthorsJournal Compilationr 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 1161^1174 1173

blin, Ireland (ed. by N. de Pauw & J. Joyce), pp. 245^246.European Aquaculture Society. Special Publication14.

Person-Le Ruyet J., Baudin-Laurencin F., Devauchelle N.,Me¤ tailler R., Nicolas J.L., Robin J. & Guillaume J. (1991)Culture of turbot (Scophthalmus maximus). In: CRC Hand-book of Mariculture,Vol. 2 (Fin¢sh Aquaculture (ed. by J.P.McVey), pp. 21^41. CRC Press, Boston, MA, USA.

Petersen I.M. & Emmersen B.K. (1977) Changes in serumglucose and lipids, and liver glycogen and phosphorilaseduring vitellogenesis in nature in the £ounder (Platichtys£esus, L.). Comparative Biochemistry and Physiology Part B58,167^171.

Robert F. & Vianet R. (1988) Age and growth of Psetta max-ima (Linne¤ 1758) and Scophthalmus rhombus (Linne¤ 1758)in the Gulf of Lion (Mediterranean). Journal of AppliedIchthyology 4,111^120.

Rodr|¤ guez L., Begtashi I., Zanuy S. & Carrillo M. (2000)Development and validation of an enzyme immunoassayfor testosterone: e¡ects of photoperiod on plasma testos-terone levels and gonadal development in male sea bass(Dicentrarchus labrax, L.) at puberty. Fish Physiology andBiochemistry 23,141^150.

Scott A.P.,Witthames P.R.,Turner R.J. & CanarioA.V.M. (1998)Plasma concentrations of ovarian steroids in relation to oo-cyte ¢nal maturation and ovulation in female plaicesampled at sea. Journal of Fish Biology 52,128^145.

Shields R.J., Gara B. & Gillespie M.J.S. (1999) AUK perspectiveon intensive hatchery rearing methods forAtlantic halibut(Hippoglossus hippoglossus L.). Aquaculture176,15^25.

Smith T.I.J., McVey D.C., Jenkins W.E., Denson M.R., Hey-ward L.D., SullivanC.V. & Berlinsky D.L. (1999) Broodstockmanagement and spawning of southern £ounder, Para-lichthys lethostigma. Aquaculture176,87^99.

Soengas J.L., Barciela P. & AldegundeM. (1995) Variations incarbohydrate metabolism during gonad maturation infemale turbot (Scophthalmus maximus). Marine Biology123,11^18.

Sol S.Y., Olson O.P., Lomax D.P. & Johnson L.L. (1998) Gona-dal development and associated changes in plasma inplasma reproductive steroids in English sole, Pleuronectesvetulus, from Puget Sound,Washington. Fishery Bulletin96,859^870.

Sun B. & Pankhurst N.W. (2004) Patterns of oocyte growth,vitellogenin and gonadal steroid concentrations in green-back £ounder. Journal of Fish Biology 64,1399^1412.

Suquet M., Billard R., Cosson J., Normant Y. & Fauvel C.(1995) Arti¢cial insemination in turbot (Scophthalmusmaximus): determination of the optimal sperm to eggratio and time of gamete contact. Aquaculture 133,83^95.

Svoboda M., Kour› il J., HamaŁ e' kovaŁ J., Kalab P., Savina L.,SvobodovaŁ Z. & VykusobaŁ B. (2001) Biochemical pro¢le ofblood plasma of tench (Tinca tinca L.) During pre ^and postspawning period. ActaVeterinaria Brno 70, 259^268.

TrudeauV.L., Murthy C.K., Habibi H.R., Sloley B.D. & PeterR.E. (1993) E¡ects of sex steroid treatments on gonadotro-pin^releasing hormone^stimulated gonadotropin secre-tion from the gold¢sh pituitary. Biology of Reproduction48,300^307.

van der Kraak G. & Pankhurst N.W. (1997) Temperature ef-fects on the reproductive performance of ¢sh. In: GlobalWarming: Implications for Freshwater and Marine Fish,Vol. 61 (ed. by C.M.Wood & D.G. McDonald), pp. 159^176.Cambridge University Press, Cambridge.

van der Meeren T. & Ivannikov V. (2006) Seasonal shift inspawning of Alantic cod (Gadus morhua L.) By photoper-iod manipulation: egg quality in relation to temperatureand intensive larval rearing. Aquaculture Research 37,898^913.

Washburn B.S., Bruss M.L., Avery E.H. & Freedland R.A.(1992) E¡ects of estrogen onwhole animal and tissue glu-cose use in female andmale rainbow trout.AmericanJour-nal of Physiology 32,1241^1247.

Weltzien F.A.,Taranger G.L., Karlsen �. & Norberg B. (2002)Spermatogenesis and related plasma androgen levelsin Atlantic halibut (Hippoglossus hippoglossus L.).Comparative Biochemistry and Physiology Part A 132,567^575.

Weltzien F.A., Andersson E., Andersen �., Shalchian-Tabri-zi K. & Norberg B. (2004) The brain-pituitary-gonad axisin male teleost, with special emphasis on £at¢sh (Pleuro-nectiformes). Comparative Biochemistry and PhysiologyPart A137, 447^477.

White A. & Fletcher T.C. (1985) Seasonal changes in serumglucose and condition of the plaice, Pleuronectes platessaL. Journal of Fish Biology 26,755^764.

White A., Fletcher T.C. & Pope J.A. (1986) Seasonal changesin serum lipid composition of the plaice, Pleuronectes pla-tessa L. Journal of Fish Biology 28,595^606.

Reproductive performance in captive wild brill I Hachero-Cruzado et al. Aquaculture Research, 2007, 38, 1161^1174

r 2007 TheAuthors1174 Journal Compilationr 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 1161^1174