Growth and gonadal development in triploid sea bass ( Dicentrarchus labrax L.) during the first two...

Ž .Aquaculture 173 1999 389–399

Growth and gonadal development in triploid seaž /bass Dicentrarchus labrax L. during the first two

years of age

Alicia Felip a, Silvia Zanuy a,), Manuel Carrillo a,Francesc Piferrer b

a ( )Consejo Superior de InÕestigaciones Cientıficas CSIC , Instituto de Acuicultura de Torre de la Sal, 12595´Ribera de Cabanes, Castellon, Spain´

b ( )Consejo Superior de InÕestigaciones Cientıficas CSIC , Institut de Ciencies del Mar, Passeig Joan de´ `Borbo, srn-08039 Barcelona, Spain´

Accepted 14 October 1998

Abstract

The aquaculture of the sea bass is now well established in Mediterranean countries. However,under culture conditions the sexual maturation is an undesirable process that reduces growth. Inaddition, spermiation around the time of marketing of fish is another inconvenience. In an attemptto solve these problems, triploidy was induced in the sea bass according to a previously publishedprotocol. A cold shock applied starting 5 min after fertilization for 10 min at 08C was used for thetriploidisation of a large volume of sea bass eggs. Control and shocked fish were reared induplicate groups under natural conditions of temperature and photoperiod using standard proce-dures for sea bass aquaculture. Triploidy was determined in a sample of fish at 3 months of age by

Ž .karyotyping 2ns48; 3ns72 , and individually checked at 15 months of age by measurement ofŽ .the major axis of erythrocytes 1.2–1.3 times larger in shocked fish than in controls; P-0.001 .

These latter analyses verified that the shocked groups were 95–97% triploid. No statisticallysignificant differences in mortality and growth performance were found between ploidies from 5

Ž .to 23 months of age 2ns172.02"2.73 g vs. 3ns158.94"2.68 g . Macroscopic observationof the gonads showed marked effects in triploid females, which had filiform ovaries. Histologicalanalysis showed that diploid females had ovaries with developing oocytes, while triploid femaleshad rudimentary gonads. In contrast, diploid and triploid males showed similar gonadal develop-ment. However, at 1 and 2 years of age testes with spermatozoa were observed in diploid males,while in triploid males this condition was never achieved. At 2 years of age, gonadosomatic index

) Corresponding author. Tel.: q34-964-319500; Fax: q34-964-319509; E-mail: [email protected]

0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0044-8486 98 00464-5

( )A. Felip et al.rAquaculture 173 1999 389–399390

Ž .GSI in triploid females was equivalent to only 16% that of diploids while that in triploid testesGSI was reduced to 40% that of diploids. The sex ratio in triploids was not different from that

Ž .usually found in cultured diploids of this species i.e., 3 males:1 female . Thus, the resultsobtained show that until 2 years of age triploids present a growth performance similar to that ofdiploids and that the triploidisation markedly affects the gonadal development of females.Nevertheless, it is required to determine the comparative growth between diploids and triploids inolder and larger fish, as well as to analyze the gonadal development of larger triploids in thisspecies. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Triploidy; Gonads; Growth; Teleost; Sea bass; Dicentrarchus labrax

1. Introduction

The sexual maturation of fish involves undesirable effects such as reduction ofgrowth rates, increased mortality during the reproductive season and inferior fleshquality. In an attempt to solve these problems, the production of sterile stocks bytriploidy induction in commercially important species has been proposed as a possiblesolution. Induction of triploidy is aimed at producing sterile fish, due to the disruption ofmeiosis during gametogenesis. As a consequence of that, triploid fish exhibit reducedgonadal development, appearing more evident in females. Several authors have reportedthat while triploid males exhibit a testicular development similar to that of diploids,

Žincluding an active spermatogenetic process Penman et al., 1987; Nakamura et al.,.1993; Hussain et al., 1995 , ovaries of triploid females, in contrast, remain rudimentary.

In this way, it is expected that triploid female fish will not divert energy into gonadalŽ .growth Utter et al., 1983; Johnson et al., 1986; Benfey, 1991 . Nevertheless, this

potential commercial advantage in triploids depends on the age of fish considered, i.e.,Ž .juveniles or adults Ihssen et al., 1990 and species.

Chromosome manipulation techniques have been mostly applied in many freshwaterŽ .species Benfey, 1989 ; however, these techniques have been also reported in marineŽ .species Sugama et al., 1992; Johnstone and Stet, 1995; Garrido-Ramos et al., 1996 .

Ž .This is the case of the European sea bass Dicentrarchus labrax L. , a marine teleost ofŽ .economic interest in the European market. According to Carrillo et al. 1995a , under

rearing conditions this species exhibits a proportion of males three times higher than thatof females, an appearance of precociously-maturing males before reaching marketablesize and a larger size in favour of females. Thus, its aquaculture might benefit from theproduction of triploid stocks to obtain functionally sterile fish preventing the sexualmaturation process and the spermiation during marketing of fish. In this regard, triploidy

Žinduction was carried out in our laboratory applying heat and pressure shocks Carrillo. Ž .et al., 1993; Zanuy et al., 1994; Carrillo et al., 1995b . Later, Colombo et al. 1995 and

Ž .Gorshkova et al. 1995 reported the use of cold shocks to avoid the extrusion of thesecond polar body. Other publications regarding chromosome set manipulation in this

Ž . Ž . Ž .species are those of Barbaro et al. 1996 , Colombo et al. 1996 , Curatolo et al. 1996 ,Ž .and Gorshkova et al. 1996 . Recently, we optimized the induction of triploidy in the sea

Žbass using cold shocks, allowing its application to a large volume of eggs Felip et al.,.1997 .

( )A. Felip et al.rAquaculture 173 1999 389–399 391

Thus, since the effects of triploidy on growth vary with species a study of the growthperformance in triploid sea bass is required to estimate the presumptive differences inthe weight gains between both ploidies, and to determine the effect of triploidy ongonadal development. The objective of this paper was to produce triploid sea bass by

Ž .cold shock using the method of Felip et al. 1997 and then to study their performancecompared to that of diploids. Specifically, in this work the effect of triploidy on bodyweight, the sex ratio and gonadal development during the first 2 years of age wereevaluated.

2. Materials and methods

2.1. Triploidy induction and ploidy determination

Ž .For this experiment, 650 ml of eggs 1 ml;650–750 eggs were fertilized. Onlyfloating eggs were used to obtain two equal groups of fertilized eggs. One was subjectedto triploidisation and the other served as the diploid control group. Triploidy wasinduced by applying a cold shock shortly after fertilization as described previouslyŽ .Felip et al., 1997 . The ploidy level from cold shock and control fish at 3 months of age

Žwas monitored by chromosomal preparations ns9 fish per ploidy; ns6 plates per. Ž .fish Kligerman and Bloom, 1977 . Ploidy was reverified in juvenile fish at 15 months

of age. Blood smears were prepared from about ;300 cold shocked fish and 20 controlŽ .fish randomly selected. The major axis of erythrocytes ns35 erythrocytes per fish

Ž .was measured Wolters et al., 1982 after staining with a solution of 20% Giemsa.

2.2. Rearing conditions and triploid performance

Ž . Ž . Ž .Duplicate diploid controls ns196; ns195 and triploid ns198; ns197Ž .groups of fish four tanks in total were reared under natural conditions of temperature

and photoperiod. Fish were first fed with rotifers and later with Artemia for 4–7 days iny1 Ž .both cases at ;1–10 individuals ml , according to Barnabe 1991 . Subsequently,´

ŽArtemia nauplii were enriched with essential nutritive components Super Selco, Artemia.Systems, Belgium . Fish were weaned around 60 DPH using a commercial diet of the

Ž .appropriate size Inve Aquaculture, Baasrode, Belgium, and Ewos, Madrid, Spain . Fishwere fed 1–4 times a day depending on age and maintained in 200-l circular fibreglasstanks until 170 DPH. Then, fish were reared in 500-l circular fibreglass tanks. Each tankwas provided with a seawater leak on the top and 1–4 oxygen leaks in the bottom toaerate seawater. At 546 DPH, fish were transferred to 2200-l tanks of similar character-

Žistics as described above range of density in the period 170–546 DPHs2.6–7.6 kgy3 .m . Weight and length measurements were performed every 2–3 months, between 5

and 23 months of age. All fish in each group were anaesthetized in a bath containingŽ y1 .2-phenoxyethanol 0.5 ml l , measured to 0.1 cm and weighed to 0.01 g. In addition,

20–27 fish per ploidy level were sacrificed at 11, 18 and 23 months of age. For eachsample, body weight and gonadal weight were determined to estimate the gonadoso-

Ž .matic index GSI . The proportion of sexes was determined by histological analysis


using conventional histology procedures to prepare the gonadal tissues. For photodocu-mentation, the weight of the selected gonads was representative of the average gonadalweight for each group.

Ž . Ž . ŽFig. 1. Metaphase spreads in diploid A and triploid B sea bass, sampled at 3 months of age ns9 fish per.ploidy; ns6 plates per fish .


2.3. Statistical analysis

Differences in the major axis of erythrocytes, body weight and GSI between diploidsand triploids were analyzed using a Student’s t-test. A x 2-test was used to determinesignificant differences in the sex ratio between both ploidies. Data are shown as

Žmean"SEM. Differences were considered significant when P-0.05 Sokal and Rohlf,.1981 .

3. Results

Karyotyping in a sample of nine control and nine cold-shocked fish at 3 months ofŽage consistently revealed metaphasic spreads with 48 and 72 chromosomes Fig. 1A and

.B, respectively . The individual examination of the major axis of erythrocytes usingGiemsa staining from the cold shocked fish and control fish at 15 months of age showed

Ž .that the major axis of erythrocytes of cold shocked sea bass 14.25"0.02 mm wereŽ .significantly larger than that of the controls 12.29"0.39 mm , i.e., 1.2–1.3 times larger

Ž .P-0.001 . Therefore, these results revealed that the control fish were all diploids, asexpected, while 95–97% of cold shocked fish were indeed triploids.

Essentially, no differences in mortality and growth performance were found betweendiploids and triploids from 5 to 23 months of age. Comparison between diploid andtriploid sea bass sampled during this period revealed a very similar evolution in forklength, on average of 23.5"0.07 cm at 2 years of age. Regarding body weight, diploids

Fig. 2. Mean body weight of diploid and triploid sea bass from 5 to 23 months of age. Data as mean from tworeplicates groups in each ploidy level, with 130–198 fish per group. SEM values were of equal or smaller sizethan the data point symbols and thus are not represented.


Fig. 3. Macroscopic appearance of diploid and triploid gonads in the sea bass at 11 months of age. Scales2cm.

Ž . Ž .attained a higher mean weight 172.02"2.73 g than triploids 158.94"2.68 g at 23months of age. Thus, triploids were about 7–8% smaller in weight than diploids. This

Ž .difference, however, was not statistically significant Fig. 2 .Macroscopic observation at 1 year of age showed that testes of triploids were

Ž .essentially similar to those of diploids Fig. 3 . Furthermore, at 11 and 18 months of ageŽ .prior to maturation , the GSI values for triploid males were similar to those for diploids.

Ž .However, at 23 months during maturation of some diploids , the GSI of triploids wasŽ .found to be lower Table 1 . At this time, the GSI in triploid males was reduced to 40%

Ž .that of diploid males 0.61"0.07 vs. 1.53"0.22 . Histological analysis showed that

Table 1Ž . Ž .Gonadosomatic index in diploid 2n and triploid 3n sea bass

Ž .Age Ploidy Gonadosomatic index %Ž .months level

Males Females

Ž . Ž .11 2n 0.04"0.00 14 0.11"0.00 6Ž . Ž .3n 0.03"0.00 12 0.04"0.00) 8Ž . Ž .18 2n 0.04"0.00 16 0.21"0.00 3Ž . Ž .3n 0.04"0.00 14 0.05"0.00) 6Ž . Ž .23 2n 1.53"0.22 21 0.55"0.01 6Ž . Ž .3n 0.61"0.07) 13 0.09"0.01) 6

Data as mean"SEM from two replicates per ploidy. The sample size is in brackets.) P -0.05


Ž . Ž . Ž . Ž .Fig. 4. Transverse section of a diploid A and a triploid B testis and a diploid C and a triploid D ovary injuvenile sea bass at one year of age. Scales50 mm. Abbreviations: sg, spermatogonia; ps, primaryspermatocyte; ss, secondary spermatocyte; o, oogonia; po, perinucleolar oocyte.


Ž . Ž .diploid Fig. 4A and triploid Fig. 4B testes at 1 year of age attained a similar degreeof development, mostly consisting of seminiferous lobules filled by spermatogonia andcysts of primary spermatocytes. Nevertheless, some cysts containing secondary sperma-tocytes were also observed. Thus, 68% of the diploid males had gonads populated withpremeiotic and meiotic germ cells but the remaining 32% had sperm when they were

Ž .microscopically analyzed ns20 fish . Since most of sea bass males normally matureduring the second year of age, fish containing sperm in the gonads were considered to bepremature males although the sperm was not released when gently pressed. In contrast,microscopic examination revealed no spermatid and no sperm formation in triploidmales at 1 year of age.

At 2 years of age, 27 randomly taken diploid fish were microscopically analyzed.Eighty-one percent of males were found to have sperm in their seminiferous lobules.

Ž .Furthermore, when checked individually ns178 , 58% of the fish in the diploidŽ .groups histological sex unknown released sperm when gently pressed. In contrast, not

a single fish in the triploid groups neither produced nor released sperm at 2 years of age.This was not due to lack of spermatic ducts in triploids since examination of the internalanatomy showed no alterations with respect to that of diploids.

Macroscopic examination showed that ovarian development was severely reduced inŽ .triploid females Fig. 3 . Histologically, diploid ovaries appeared filled with developing

Ž .oocytes in the perinucleolar stage Fig. 4C , while triploid females had rudimentaryŽ .ovaries containing mainly oogonia Fig. 4D . Nevertheless some primary oocytes were

Ž .also observed. The mean GSI was significantly higher P-0.05 for diploid femalesŽ .compared to that of triploid females Table 1 . At 23 months of age, triploid females had

Ž . Ž .a GSI 0.09"0.01 equivalent to only 16% that of diploids 0.55"0.01 .Determination of the proportion of sexes in triploids required histological examina-

tion, since the reduced development of their gonads often made it difficult to determinethe sex visually. By histological analysis 39 males were found among 59 triploid fishand 51 males in 59 diploid controls. These results were not different from the sexpopulation usually found in cultured diploids of this species, i.e., 3:1 in favour of males.

Ž 2 .Thus, triploidy did not affect the sex ratios significantly x s2.22; P)0.05 .

4. Discussion

Ž .Recently, Felip et al. 1997 optimized, at the laboratory scale, the induction oftriploidy in the sea bass using cold shocks. The present work shows that triploidy can bealso successfully obtained using a large volume of eggs without significantly affectingsurvival. Although karyotyping can accurately determine the ploidy level, it is a timeconsuming technique that requires dividing cells and good chromosome spreads. Instead,for this particular study, measurements of the major axis of erythrocytes were used to

Ž .assess ploidy in control and treated fish, as did Wolters et al. 1982 for the determina-tion of triploidy in catfish.

Differences in growth between diploid and triploid fish are age- and species-relatedŽ . ŽIhssen et al., 1990 . Thus, depending on species, triploids can grow like Galbreath and


. Ž . Ž .Thorgaard, 1997 , less Penman et al., 1987 or more Fast et al., 1995 than diploidsreared under the same conditions. The results obtained in the present work show that, atleast until 2 years of age, triploid sea bass had essentially the same growth performance

Žas their diploid counterparts although they were about 7–8% smaller in weight dif-.ferences not significant .

Triploid males usually show a progression through spermatogenesis, leading to spermŽ .production Penman et al., 1987; Nakamura et al., 1993; Hussain et al., 1995 . Because

Žof this, triploid males are usually not considered of benefit for aquaculture Benfey,.1991 . In the particular case of sea bass, it has been shown that under culture conditions

most of individuals develop as males. One of the major inconveniences of this sexbecomes evident when marketing fish during the winter months at around 2 years of age,coinciding with Christmas or Easter. Most of these fish are fully mature males thatrelease a significant amount of sperm when pressed against each other during handlingand transport to market, posing at least a problem of visual appeal. In the present study,it has been shown that triploidy did not affect the proportion of sexes and thus triploidsea bass were still mostly males. Our data showed that with an average weight of158.94"2.68 g, none of the triploid fish produced and released spermatozoa when, incontrast, 58% of the diploid fish did so at 23 months of age. These results suggest that,if triploidy induction can prevent final sexual maturation in males, it would be a goodadvantage for sea bass culture and marketing in order to solve the above mentionedproblems. The triploid females, on the other hand, at 2 years of age already revealedtheir expected inability to develop their gonads which resulted in rudimentary ovaries.

For future studies, it remains to be determined the growth pattern of triploid sea basscompared to that of diploids in adult fish of larger size, to examine other growthparameters and provide a description of the type of germ cells sequentially appearing insea bass triploid gonads in fish of different ages.

Acknowledgements

Research supported by EC grant AIR2-CT93-1543, CICYT grant AGP94-1321-CEand a contract from GESA Diversificacion to S.Z. A.F. was supported by a grant from´the Government of Valencia. The authors greatly appreciate the assistance received bythe support staff from the Instituto de Acuicultura de Torre de la Sal.

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