Influence of daylength on the age at first maturity and somatic growth in male sea bass (...

Ž .Aquaculture 196 2001 159–175www.elsevier.nlrlocateraqua-online

Influence of daylength on the age at first maturityand somatic growth in male sea bass

ž /Dicentrarchus labrax, L.

Lucinda Rodrıguez, Silvia Zanuy, Manuel Carrillo)´( )Consejo Superior de InÕestigaciones Cientıficas CSIC , Instituto de Acuicultura de Torre la Sal,´

12595 Ribera de Cabanes, Castellon, Spain´Received 24 May 1999; received in revised form 1 October 2000; accepted 20 October 2000

Abstract

Groups of 4-month-old sea bass were each exposed over three consecutive years to a naturalŽ . Ž . Ž . Ž .photoperiod 408N NP , constant long photoperiod 15L:9D LO and 18-month-expandedŽ . Ž .photoperiod the natural light cycle extended to 18 months EX , under natural conditions ofŽ .temperature 12.0–26.38C . During the differentiation period, a significant increase in number of

Ž . Ž .precocious males was found in the LO 26.8% and EX 17.7% groups compared to controlsŽ . Ž . Ž . Ž .5.3% P-0.05 . During the first reproductive season 20–25 months of age gonadal size GSI

Ž . Ž .in both the LO 0.82"0.16% and the EX 0.28"0.07% groups was significantly lower thanŽ .controls 1.28"0.41% . The percentage of spermiating males during the first reproductive period

Ž . Ž . Ž .was lower in LO -60% and EX -35% groups compared to controls)80% . At the secondŽ .reproductive period 32–37 months of age gonadal maturation was advanced in EX group,

whereas a significant delay was observed in LO group with respect to control. Spermiation in EXgroup was advanced with respect to control group. During the second reproductive period,

Ž .percentage of spermiating males was significantly higher in EX group;80% than in LO groupŽ . Ž-50% . LO and EX groups displayed enhanced growth 655.52"37.21 g and 376.40"6.33

. Žmm; 510.44"25.80 g and 350.14"5.27 mm, respectively compared to controls 459.93"21.20.g and 343.62"4.82 mm . The present results indicate a significant beneficial effect of constant

long and expanded photoperiods on reducing sea bass gonadal development and enhancing growthat the age of commercialisation, with a potential application in aquaculture.q2001 ElsevierScience B.V. All rights reserved.

Keywords: Photoperiod; Onset of puberty; Sea bass; Gonadal development; Growth

) Corresponding author. Tel.:q34-64-319500; fax:q34-64-319509.Ž .E-mail address: [email protected] M. Carrillo .

0044-8486r01r$ - see front matterq2001 Elsevier Science B.V. All rights reserved.Ž .PII: S0044-8486 00 00555-X

( )L. Rodrıguez et al.rAquaculture 196 2001 159–175´160

1. Introduction

Ž .The European sea bassDicentrarchus labrax, L. is an important marketablespecies. Its production by aquaculture has increased considerably as opposed to fisheries

Žthat have reached their maximal level of exploitation. Studies of their biology Pickett. Ž .and Pawson, 1994 , the photoperiodic control of reproduction Carrillo et al., 1993 ,

Ž .broodstock management and offspring quality Carrillo et al., 1995 , and recently, theŽ .implementation of sex control techniques Carrillo et al., 1999 have addressed some

problems that limit the expansion of sea bass culture. However, under culture conditionsŽ . Ž .some important problems remain including: 1 the high ratio of males:females 3:1 ,

Ž .some of which become sexually mature prior to reaching the commercial size; 2 theŽfact that puberty appears near the second year of life in males at commercialisation,

. Ž .250–500 g and 3 normally, males are smaller than females at the time of firstŽ .maturity, although there is no clear sexual dimorphism Carrillo et al., 1995 . Apart from

these problems, the increasing demand for juveniles from fish farms requires a continu-ous production of eggs and larvae outside the normal spawning season. This requiresmethods to control the reproductive cycle and to induce spawning at any time of theyear. To accomplish this goal, several studies have investigated the environmentalŽ .photoperiod, temperature and hormonal control of the reproduction in this speciesŽ .Zanuy et al., 1986, 1995; Carrillo et al., 1989, 1993; Prat et al., 1999 . Generally,spawning occurs when local climatic conditions are favourable. On the south-east coastof Spain, under natural conditions, spawning of the sea bass occurs during winter

Ž .months, under low temperatures 12–148C and short andror increasing daylengthsŽ .Barnabe 1980 . In sea bass, along with many other fish, spermiation in males lasts´

Ž .longer than spawning in females Prat et al., 1999 .There are several studies related to the effects of temperature on growth performance

Ž .Imsland et al., 1996 . However, studies on the influence of photoperiod on growth rateŽand onset of puberty are scarce Clarke and Blackburn, 1994; Amano et al., 1995;

.Imsland et al., 1997; Oppedal et al., 1997 . Among the large number of environmentalfactors considered as possible cues for pubertal development and the control ofreproduction in fish, photoperiod has been shown to be one of the most important in

Ž .salmonids and also in several marine fishes Bromage et al., 1993 , including sea bassŽ . Ž . ŽBromage et al. 1990 , Atlantic codGadus morhua Hansen et al., 1995; Norberg et

. Ž .al., 1995 and turbotScophthalmus maximus, Imsland et al. 1997 .Maturation rates and timing of spawning can be altered in sea bass by using modified

Ž .seasonally changing light cycles Zanuy et al., 1986; Prat et al., 1999 . Constant longŽ .photoperiods delay spawning Carrillo et al., 1991; Zanuy et al., 1995 and expanded

Ž . Žphotoperiods 18 months can delay spawning in both sea bass Devauchelle and Coves,. Ž .1988 and rainbow troutOncorhynchus mykiss, Bromage and Duston, 1986 . In a

Ž .similar way, it has been reported that compressed photoperiods 6, 9, 10 months canŽadvance the spawning of sea bass, gilthead sea breamSparus aurata, Girin and

. Ž .Devauchelle, 1978 and rainbow trout Bromage and Duston, 1986; Bromage, 1987 .Ž . Ž .Particularly, Rodrıguez et al. 2000 stated that compressed photoperiods 6 months´

advance the first spawning season in juvenile male sea bass. Nevertheless, there are stillno detailed studies on the effect of modified photoperiod cycles on gonadal development

( )L. Rodrıguez et al.rAquaculture 196 2001 159–175´ 161

and growth of sea bass. Moreover, there is a lack of information on the effects ofphotoperiod on initial reproductive events in sea bass, including sex differentiation andpuberty. In order to study these reproductive processes it is essential to have knowledgeof gonadal development, and how germ cells become gametes. There are a few works

Ždescribing testicular structure and stages of development in the sea bass Billard et al.,.1982; Zanuy et al., 1999 . For the first time, a morphological identification of male germ

cells during testicular recrudescence in sea bass is described in this paper.The aim of the present work is to study the long-term effects of expanded light cycles

and constant light regimes, both known to delay the natural spawning time in adult seabass, on the reproductive process of prepuberal male sea bass, including age at firstmaturity, testis development, time of spermiation and somatic growth over the first threeyears of life.

2. Materials and methods

2.1. Fish and experimental conditions

The experiment was performed at the Instituto de Acuicultura de Torre la SalŽ . ŽCastellon, Spain . Two thousand 4-month-old juvenile sea bass 0.9"0.04 g body´

. Ž .weight were purchased from GESA Gas y Electricidad, Islas Baleares, Spain . FishŽ .were distributed in four groups mixed sexes;ns500 and reared in separate identical

Ž .2000 l light-proof circular 2 m diameter fiberglass tanks provided with well aeratedŽ .running sea water salinitys37‰ . Temperature was always maintained under natural

Ž .conditions 12.0"1.08C in January 1998 to 26.3"1.08C in August 1995 for allŽ .treatments Fig. 1 . Each group was exposed to one of the following photoperiod

Ž . Ž .regimes Fig. 1 , from June 1995 when natural daylength was 15L: 9D until May1998:

Ž .Group NP: the natural light cycle 408N; 08E which served as control;Ž .Group LO: Constant long photoperiod 15L:9D ;

Group EX: Expanded photoperiod: the natural seasonal light cycle expanded over 18months.

Control group was carried out in duplicate during all the experiment to monitor tankeffect.

Ž .Fish were fed by handAad libitumB three times a day at 9:00, 11:30 and 14:00 hŽ .when they were juveniles-100 g coinciding with the first annual cycle; see Fig. 1

Žwith pelleted dry food of the appropriate size pellet size was 1 and 3 mm; 54%protein–14% fat and 47% protein–18% fat, respectively; Inve Aquaculture, Baasrode,

. Ž .Belgium; Ewos, Madrid, Spain . Coinciding with the second annual cycle under 300 g ,Žthey were fed two times a day at 10:00 and 13:30; pellet size 4.5 mm; 48%

. Žproteins–12% fat at an adequate ratio ranged from 0.5% to 5% of body weight;.feeding tables from Ewos , which was adjusted according to the seasonal changes of the

Ž . Ždaily food intake. Adult fish )300 g, third annual cycle were fed once a day at


Ž .Fig. 1. Experimental design of the temperature cycle and photoperiod regimes solid lines used in this study.Ž .The black bars represent the percentage of sea bass running males maintained under natural photoperiod NP ,

Ž . Ž .constant long photoperiod LO; 15L:9D and expanded photoperiod EX; 18 months during the first andsecond reproductive periods. Different letters represent differences between the three groups at the samemonth.

10:00, according to the established conditions for the food ratio; pellet size 7 mm; 48%.protein–12% fat . Daylength, controlled by electronic time clocks, provided 10,000 lx at

the water’s surface by using two fluorescent tubes suspended 50 cm above the tank.

2.2. Sampling protocol

Ž .Fish were anaesthetised with ethylene glycol monophenyl ether 0.5 mlrl of waterŽ . Ž .and then wet weight precision 0.01 g and fork length measured precision 1mm every


2 months during the first year when there was no gonadal maturation, except someprecocious males, and monthly during the second and third year of age. Although datawere obtained with mixed sexes, only data from males are presented in this work.

Precocity, during the sex differentiation period, was determined histologically by thepresence of spermatozoa in the lumen of the lobules. Males were gently stripped at each

Ž .sampling time normally from October to April in order to evaluate the number ofŽ .spermiating males per group. Gonadal samplesns6–30 from each treatment were

obtained from November 1995 to April 1998, except during late spring and summerŽ .months May to September . Gonads were quickly removed and weighted to the nearest

Ž .0.001g for estimation of the gonadosomatic index GSI as follows:

GSIs G rB )100,Ž .W W

whereG is the gonad weight andB is the body weight of each animal.W WŽ .Testes were fixed by immersion in gluteraldehyde 1% gluteraldehyde , embedded in

Ž .methacrylate plastic Historesin LKB , sectioned at 4–5mm, and stained with theŽ .Cleveland–Wolff technique Herlant 1960 for light microscopic analysis.

2.3. Testicular histology

ŽThe number of spermatogonia A, cysts containing the same germinal cells sper-. 2matogonia B, spermatocytes and spermatids , and spermatozoa in a total of 320 mm

Ž 2 .divided in 50 areas of 6400mm each and taken randomly along the testis of each fishwas recorded. Consequently, the relative abundance of each cellular type was calculated

Ž .according to Kestemont 1989 . Mean values of these data were obtained from fiveŽ .animals n value determined with a statistical test randomly selected per group.

2.4. Statistical analysis

Ž .The results were expressed as mean"standard error SE . Normality was ensuredŽusing the Kolgomorov–Smirnoff test after logarithmic transformation of data when

.required , and Bartlett’s test was used to establish homogeneity of variances. Data wereŽ .analyzed by two-way nested ANOVA Zar 1996 followed by Fisher’s multiple range

Ž .test P-0.05 . Percentage data were arcsin transformed prior to statistical analyses.Precocity and mortality data were analysed using a Chi-squared test. Differences wereaccepted as statistically significant whenP-0.05.

3. Results

Although data were obtained with mixed sexes, only data from males are presentedhere.

3.1. Growth

Almost three annual cycles from the beginning to the end of the experiment wereŽstudied. Each cycle was divided in a period of active somatic growth August to


November the first annual cycle, and April to November the second and third annual.cycle , and a period of reduced growth on which the reproductive events took place

Ž . Ž .November to April Fig. 2A and B . During the first annual cycle, sex differentiationŽoccurred. Males attained puberty during the second annual cycle first reproductive

.period . However, males show their full reproductive capability during the third annualŽ .cycle second reproductive period .

Ž . Ž .Fish kept under constant long LO and expanded EX photoperiods grew signifi-cantly more than controls until the beginning of the second reproductive period in

Ž . Ž .November 1997 Fig. 2A and B P-0.05 . Significant differences in somatic growthŽ .were observed between LO, EX and control LO)EX)NP groups during part of the

Ž . Ž .period of sex differentiation January 1996 to April 1996P-0.05 . During the secondŽ . Ž .growth period April 1996 to November 1996 LO and EX groups grew moreP-0.05

than control group. In all groups, growth was reduced during the first reproductiveperiod. There was an increase in weight in all groups during the third growth period,

Ž .showing significant differences between the three treatments EX)LO)NP at theŽ .middle of this period P-0.05 . In November 1997, the second reproductive period

started and growth rate in weight of EX and control groups began to decrease, whensexual maturation began. In contrast, LO photoperiod kept on growing until January

Ž .1998, reaching significantly higher values 684.7"25.2 g than control and EXŽ . Žphotoperiods 499.4"10.4 g and 523.0"19.7 g, respectively at this time Fig. 2A and

. Ž .B P-0.05 .

3.2. Gonadal deÕelopment

A certain percentage of early maturing males appeared in all groups, being signifi-Ž . Ž .cantly higher in LO and EX groups compared to controls Table 1P-0.05 . The GSI

values for the three annual cycles are represented in Fig. 3. During the period of sexdifferentiation, the gonads of the immature fish of all groups presented as thread likestructures in January, while over the following months the gonads showed an earlydifferentiated lobular structure, with GSI values less than 0.1%. In all groups the GSIvalues of precocious males were higher than those of no precocious males, at least for 2

Ž .months during the sexual differentiation period Fig. 3 . The GSI of precocious malesŽfrom LO and EX group was higher than in control group although these differences

. Žwere not significant,P)0.05 . During the first reproductive period second annual. Ž .cycle , controls had significantly higher values of GSIP-0.05 than the previous

Ž .period, with highest values in January 1997 2.33"0.14% . LO group followed theŽsame pattern as controls, but with significantly lower GSI values 1.18"0.17%, January

.1997 . The EX group exhibited lower values of GSI than the other groups during thisŽ .period, peaking in November 1996 0.49"0.15 .

Ž .During the second reproductive period third annual cycle , the control group showedŽ .higher GSI values January–February between 2.5–3.0% than at the first reproductive

Ž .period Fig. 3 . This confirms that males attain their full reproductive capability duringthis period. In LO group, GSI showed a first increment in November to peak in

Ž . Ž .December 1.84"0.25% . It decreased in January 1998 0.58"0.13% and then it


Ž . Ž . Ž .Fig. 2. Values of mean weight A and length B of male sea bass kept under natural photoperiod NP ,Ž . Ž .18-month-expanded photoperiod EX and constant long photoperiod LO; 15L:9D . Ann.Cyclesannual cycle

Ž1st annual cycle August 1995–April 1996; 2nd and 3rd annual cycles April 1996–April 1997 and April.1997–April 1998, respectively . Gr.P.ssomatic growth period; Sex D.P.ssex differentiation period;

Rep.P.s reproductive period. Discontinuous lines divide different periods. as indicate significant differencesŽ .between LO, EX and NP groupsP-0.05 ; bs indicate significant differences between LO and EX groups

Ž .with respect to NP groupP-0.05 ; cs indicate significant differences between EX and NP groups withŽ .respect to LO groupP-0.05 . Data are represented as mean"SE.


Table 1Ž .Morphological data mean values"SE of precocious males and percentage of precocity found during the sexŽ .differentiation period 9 to 11 months of age in sea bass kept under different photoperiod regimes

NP LO EX)N 152 56 62

b a aŽ .B g 60.0"3.5 80.8"5.1 78.2"6.8Wb a aŽ .B min 168.8"2.8 183.7"3.9 182.7"4.2L

Ž .GSI % 0.051"0.005 0.139"0.037 0.153"0.064Ž .Mortality % 3.2 3.9 3.4

b a aŽ .Precocity % 5.3 26.8 17.7

NPscontrol group; LOs long photoperiod; EXsexpanded photoperiod; Ns total fish examined;B sbodyW

weight; B sbody length; GSIsgonadosomatic index. Different letters indicate differences between groupsLŽ .P-0.05 . Groups without letters do not exhibit any significant differences.

)This value is the sum of the two replicates of the NP group.

Ž .showed a second higher and more consistent increment peaking in April 3.66"0.68% ,showing that in LO group the time of maturation was delayed with respect to controls.During the second reproductive period, the EX group showed a similar GSI pattern thanthe previous period of reproduction, but exhibiting higher GSI values, indicating a fullaccomplishment of gonadal development. The GSI was already high in November 1997Ž . Ž1.69"0.13% , being significantly higher than in NP and LO groups 0.69"0.20% and

. Ž .0.85"0.18%, respectively and peaking in December 1997 2.40"0.26% . All theseŽ .data indicate an advancement of the sexual maturation with respect to controls Fig. 3 .

3.3. Spermiating males

In LO and NP groups, spermiating males were first observed in November 1996,during the first reproductive period, while in EX group they appeared 1 month earlierŽ .Fig. 1 . During the first reproductive period, the proportion of spermiating males in NPwas significantly higher than in the LO group and in this higher than in EX group.During the second reproductive period, NP followed the same pattern of maturation asthe first reproductive period. Spermiation occurred from December 1997 to April 1998Ž .around 80% . The percentage of spermiating males in LO group was significantly lower

Ž .than in NP except in April 1998 . EX group showed 60% of spermiating males inOctober 1997, which were significantly higher than LO and NP groups, and peaked in

ŽNovember 1997, which again was significantly higher than the rest of the groups Fig..1 .

3.4. Testicular histology

3.4.1. Identification and characterisation of male germ cellsŽ . Ž . Ž .a Spermatogonia. Primary A and secondary spermatogonia B have been identi-

fied. Spermatogonia A: they are found as isolated cells at the periphery of the lobule,present during both the sexual resting period and the first stages of testicular develop-


Ž . Ž . Ž .Fig. 3. Gonadosomatic index GSI of male sea bass held under natural NP , constant long LO; 15L:9D andŽ .18-month-expanded EX photoperiods. During the sexual differentiation period, the GSI is represented for

Ž . Ž . Ž .precocious white bars and no precocious grey bars males left hand side scale . Black bars represent theŽ . ŽGSI for males during the first reproductive period puberty and the second reproductive period right hand

. Ž .side scale . The data are expressed as mean"SE. The asterisks indicate significant differencesP-0.05within a group at the same month between precocious and no precocious males. Different letters indicate

Ž .significant differencesP-0.05 between groups at the same month.

ment. They are characterised by its large size and rounded shape as well as by a largecentral nucleus containing a prominent nucleolus. The average nuclear area is 46.1"1.08

2 Ž .mm and the diameter ranges from 9.2 to 6.0mm. Fig. 4F . Spermatogonia B: primaryspermatogonium forms cysts that become filled with spermatogonia B following a seriesof mitosis. They are smaller than spermatogonia A, with less cytoplasm and morecondensed chromatin. The average nuclear area is 21.38"0.69mm2 and the diameter

Ž .ranges from 6.48 to 3.25mm Fig. 4F .


Ž .b Spermatocytes. Identification of primary and secondary spermatocytes was done.Primary spermatocytes: form larger cysts as a result of a meiotic division of spermatogo-nia. They have a big central and dark stained nucleus with a thin cytoplasm around.


Their average nuclear area is 11.36"0.45mm2 and their diameter ranges from 5 to 2.4Ž .mm Fig. 4D . Secondary spermatocytes: have a smaller size and are also grouped in

cysts. Central nucleus is smaller due to the higher chromatin condensation. The average2 Žnuclear area is 3.69"0.10mm and the diameter ranges between 2.9 and 1.3mm Fig.

.4D .Ž .c Spermatids. Spermatids are found in the lobule with the flagella grouped and

directed to the lumen. Nucleus can be round or long-shaped according to the maturationstage. As maturation progress, the cytoplasm becomes more scarce and the nucleussmaller. The average nuclear area ranges between 2.57"0.11 and 1.87"0.05mm2 and

Ž .the diameter size from 2.5 to 0.96mm Fig. 4D .Ž .d Spermatozoa. Found in the whole lobular area. The long-shaped nucleus is

strongly stained, and chromatin material highly condensed. Cytoplasm is indistinguish-able. The average nuclear area is 1.29"0.01mm2 and the diameter ranges from 1.85 to

Ž .1.08mm Fig. 4C,D .The relative percentages of germ cells during the first and second reproductive period

are shown in Fig. 5. Histological data during sexual differentiation are not representedŽ .because only spermatogonia A were observed in all groups data not shown , except in

the precocious animals. Early maturing males showed a clearly differentiated testis, inmaturing stages, with a relative high percentage of cysts containing spermatids andspermatozoa. Although primary and secondary spermatocytes were individually de-scribed, they were represented as only one category since no significant differences werefound between them.

Ž .During the first reproductive period, in NP only spermatogonia A 38.3% , cystsŽ .containing spermatogonia B 52.6% , and a small percentage of cysts containing

Ž .spermatocytes 8.3% were present in October 1996 in the immature differentiated testisŽ .Fig. 5 . From November 1996 to February 1997, testes contained cysts with all gametestages, with spermatocytes and spermatozoa predominating in December 1996 and

Ž .January 1997 maturing and fully mature testis . Spermatozoa increased from NovemberŽ .1996 to February 1997 Fig. 5 . The same pattern was observed during the second

reproductive period. The presence of spermatozoa increased in the same way, reachingŽ .the higher values in February 1998 and March 1998 Fig. 5 .

During the first reproductive period, the percentage of all germ cell types present inthe testes in LO group followed the same pattern as in NP, except in the percentage of

Ž .Fig. 4. Photomicrographs of sea bass male gonads at the same age 38 months held under differentŽ .photoperiod regimes: A,B testis of a control male showing a regressed testicular structure after the

Ž .reproductive period. Note in A the abundance of necrotic tissue with picnotic nucleus arrows produced by thephagocytosis of germ cells and residual sperm by macrophage or Sertoli cells. In B is shown in detail

Ž . Ž . Ž . Ž .degenerated germinal cells gc and interstitial tissue with a lot of fibroblasts arrow and vacuole va . C,DŽ .Testis of a male held under constant long photoperiod. Note the presence of spermatogonia A sgA , primary

Ž . Ž . Ž . Ž .spermatocytes sc1 , secondary spermatocytes sc2 , spermatids sm and spermatozoa sz , indicating aŽ .maturing stage of the gonad. E,F Testis of a male kept under expanded photoperiod presenting normally

Ž .developed testicular lobules in a resting stage; spermatogonia A and B sgA and sgB are predominant.Ž .Fibroblasts arrows form a discontinuous layer around the lobules. Bars20 mm for A, C and E. Bars10

mm for B, D and F.


Ž 2.Fig. 5. Relative abundance in percentage of germ cells in section of testis in a standard area of 320 mm ofŽ . Ž . Ž .male sea bass kept under natural NP , constant long LO; 15L:9D and 18-month-expanded EX photoperi-

ods during the first and second reproductive period. SGAsSpermatogonia A; SGBsSpermatogonia B;Ž .SCsSpermatocytes primaryqsecondary ; SMsSpermatids; SZsSpermatozoa. The data are represented as

the mean percentage.

presence of spermatozoa. There was only a significant percentage in December 1996,Ž . Ž .decreasing until 0% in February 1997P-0.05 Fig. 5 .

The percentage of spermatogonia A in EX photoperiod in December 1996, January1997 and February 1997 was significantly higher than in NP and LO groups in the same


months. The percentage of cysts containing spermatocytes and spermatids was very lowŽ . Ž .less than 4% in these months Fig. 5 . During the second reproductive period, both thepercentages of spermatogonia A and spermatozoa were highest in January 1998.

3.4.2. Stages of testicular deÕelopmentTesticular development in the control group is summarised in the following six

Ž . Ž .maturation stages according to Grier and Taylor 1998 and Zanuy et al. 1999 .Ž .a Undifferentiated gonads. Only isolated germinal cells and somatic elements are

present.Ž .b Early differentiated testis, containing some initially differentiated spermatogonia.

The situation of the germinal cells begins to constitute the seminiferous lobules.Ž .c Immature differentiated testis. The structure of the seminiferous lobules is

completed. Scattered spermatogonia A and spermatocysts containing spermatogonia Bbegin to appear close to the lobule periphery.

Ž .d Early maturing testis. Spermatogenesis is initiated throughout all the lobules.Spermatocysts contain cells in different stages of development.

Ž .e Maturing testis. The spermatids mature into spermatozoa and are released intolobule light. At advanced stages, the lumen of the lobules is filled with sperm that isreleased into the seminiferous duct.

Ž .f Regressed testis. Characterised by the absence of spermatocysts containingspermatocytes, spermatids and mature sperm, although residual sperm can be observedin the lobule lumen.

Histological appearance of the gonad of the three experimental groups at the end ofŽ .the experimental period April 1998 is shown in Fig. 4. Testes are regressed in NP

Ž . Ž .group Fig. 4A,B , mature-spermiating in LO group Fig. 4C,D , and in a resting stageŽ .in EX group Fig. 4E,F .

4. Discussion

The constant long photoperiod enhanced somatic growth compared to controls. Thisis thought to have been achieved by a combination of prevention of reallocation ofenergy resources towards gonadal development and a direct effect of photoperiod ongrowth performance. Moreover, the photoperiod may also affect the proportion of fish

Ž .reaching maturity, as have been reported for Atlantic salmon Berg et al., 1996 andŽ .turbot Imsland et al., 1997 . We observed that long and expanded photoperiods

exhibited a significant lower proportion of fish reaching first maturity than controls. It isknown that spawning season often depends on an interrelation between environmental

Ž .cues and endogenous rhythms, circadian andror circannual Baggerman, 1980 . In thepresent work, endogenous rhythms cannot be referred to since temperature was notconstant over the whole experiment. Temperature and photoperiod interactions areknown to control several mechanisms at different times of the year, but when there is amismatch between them, the reproductive cycle may be perturbed. During the firstreproductive cycle, gonadal maturation in EX and LO groups could be explained by adisplacement of the artificial photoperiods with respect to the natural temperature


conditions. Hence, the reduction of maturation in both groups with respect to thecontrols could be explained by the non-appropriated photoperiodic signals.

Sea bass remains sexually undifferentiated until near the end of the first year of ageŽ .Blazquez et al., 1995 . This is confirmed in the present experiment by both the low GSI´

Ž .values and the gonadal histology reported over this period. Blazquez et al. 1995´Ž .reported that sea bass males attain sexual differentiation at 11 months of age 16 cm , 2

months later than females. It is interesting to note here that during the sexual differentia-Žtion period, the LO group had a lower proportion of undifferentiated fish 6.6% vs.

. Ž .16.7%, unpublished data and a higher growth rate than control group Fig. 2 . Thesedata suggest that LO photoperiod could also have effects in accelerating the physio-logical process of sex differentiation which, in turn, may be linked to the growthenhancement. In the following months, the growth of LO group remained significantlyhigher than that of the controls, a situation that remained until the end of the experiment.The same relationship was observed with regards to precocity, which was significantly

Ž .higher in the LO group compared to controls Table 1 . In addition, early maturingmales from the LO group showed a significantly higher somatic growth than thecontrols. These results indicate a close relationship between growth and early sexual

Žmaturation. Similar studies performed in yearling chinook salmon Clarke and Black-.burn, 1994 demonstrate that precocious sexual maturation is conditional to and facili-

tated by rapid growth. These results support the hypothesis that photoperiod could beinvolved in both growth enhancement of juveniles and adult fish and the acceleration ofsex differentiation. However, it must be taken into account that data on precocity wereobtained with a low sample size out of an initial number of 500. Thus, these data need tobe interpreted with caution since the observed effects might be hardly representative.

The rate of spermiation in males, examined at the second and third annual cycle,indicated that spermiation time was affected by the photoperiodic treatments, althoughthe experiment was performed with mixed sexes and there was some evidence ofphotoperiod effects on female reproductive performance. However, the proportion offemales in the population was too low to allow any statistical treatment of the dataobtained. This is why only data from males are presented here. Under natural photope-riod, adult male sea bass spermiates from November to April. However, this long periodof spermiation is fully efficient only from December to March, probably to guarantee the

Žfertilisation of the eggs that are produced, generally, from January to March. Carrillo et.al., 1995; Prat et al., 1999 . Our results showed that the spermiation time in the controls

was shorter during the first reproductive period than during the second. During the firstreproductive period, LO photoperiod exhibited a similar pattern of variation in the

Ž .percentage of spermiating males, gonadal development GSI and proportion of germinalŽ .cells particularly in the percentage of cysts containing spermatids as the control group.

In LO group, there was not any photoperiodic signal, so testicular maturation could beŽ .induced by the right thermal conditions low temperatures , although an inhibition of

maturation was observed with respect to controls. However, during the second reproduc-tive period, the spermiation time of LO group was delayed, exhibiting a significantreduction in the proportion of spermiating males and a poor gonadal developmentcompared to control group. It suggests that in LO group this attempt of reproduction,although not completely successful, can be probably due to the absence of the appropri-


ated photoperiodic cue, since thermal conditions were suitable. In the same way, AmanoŽ .et al. 1995 demonstrated that long photoperiod delayed but did not suppress precocious

Ž .maturation in underyearling male masu salmonO. masou .During the first annual cycle, the group under expanded photoperiod also initiated

sexual differentiation. Moreover, this group showed a significant increase of thepercentage of early maturing or precocious males with respect to control group, which

Ž .was also significantly larger Table 1 . In the first reproductive period, the age at firstmaturity was advanced compared with the control group. The GSI and the percentage ofrunning males were reduced throughout this period, indicating a low degree of testicularmaturation and a rapid suppression of the reproductive function. This could be due to theinappropriate photoperiodic and thermal conditions, since in October 1996 the tempera-ture was around 208C and photoperiod was long. During the second reproductive period,testicular recrudescence and spermiation were advanced with respect to the controls.This successful maturation was probably due to the favourable increasing daylength,although the water temperature was too high, indicating that gonadal maturation mightdepend on the natural time of breeding of the fish, and the time of the year when the fish

Ž .is exposed to a specific photoperiodic signal Scott et al., 1984 . Consequently, thesudden suppression of the maturation in EX group could be due to a mismatch betweenphotoperiod and temperature, which differ from the control group during wintertime.

It is generally accepted that somatic growth is arrested during gonadal development,Ž .as shown in sea bass Zanuy and Carrillo, 1985 . Consequently, the specific alterations

of spawning time induced by environmental manipulations can also be observed in theevolution of somatic growth. From the present results and during the second reproduc-tive period, the most delayed time of spermiation was presented by the LO group andthe most advanced by the EX group.

In conclusion, we have shown that expanded photoperiod advances the onset ofpuberty in prepuberal male sea bass, although this is followed by a dramatic reduction ingonad size and the rates of spermiation. Moreover, gonadal development was signifi-cantly reduced and growth was enhanced at the age of first maturity in both LO and EXgroups, indicating a close relation between maturation and growth. In addition, theseresults confirm that environmental factors are important cues for the control of reproduc-tion in the sea bass.

Our findings could have practical applications for fish farms, which need to satisfythe increasing demands of the international market. The application of long or expandedphotoperiods would avoid complete gonadal maturation and improve the growth of fishup to the age at which they can be harvested.

Acknowledgements

This work was supported by a CICYT grant No. AGF93-0127 to Dr. S. Zanuy, aFAIR grant No. CT96-1410 to Dr. M. Carrillo and a CICYT grant No. MAR97-1883-CEto Dr. M. Carrillo. L. Rodrıguez was supported by a grant of Ministerio de Educacion y´ ´Ciencia. We would like to thank J. Monfort and C. Lopez for their help in histological´procedures; C. Marin, R. Simo, J.M. Llorens and Dr. J. Ramos for their help in hatchery´


management and sample collection and, we wish express our gratitude to A. Felip, M.A.Soubrier, M.J. Bayarri, J.M. Cerda and E. Mananos for their technical and scientific´ ˜ ´contribution.

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