Induction of ovulation in captive-reared dusky grouper, Epinephelus marginatus (Lowe, 1834), with a...

Induction of ovulation in captive-reared dusky

grouper, Epinephelus marginatus (Lowe, 1834),

with a sustained-release GnRHa implant

G. Marinoa,*, E. Paninia, A. Longobardia, A. Mandichb,M.G. Finoiaa, Y. Zoharc, C.C. Mylonasd

a ICRAM Istituto Centrale per la Ricerca Scientifica e Tecnologica Applicata al Mare,

300 Via Casalotti, 00166 Rome, ItalybDipartimento di Biologia Sperimentale, Ambientale ed Applicata, Universita di Genova,

5 Viale Benedetto XV, 16132 Genoa, ItalycCenter of Marine Biotechnology, University of Maryland Biotechnology Institute,

701 E. Pratt Street, 21202 Baltimore, MD, USAd Institute of Marine Biology of Crete, P.O. Box 2214, Iraklion, Crete 71003, Greece

Received 8 July 2002; received in revised form 3 January 2003; accepted 6 January 2003

Abstract

Captive-reared dusky grouper were induced to ovulate using a sustained-release delivery system

(implant) loaded with gonadotropin-releasing hormone agonist [D-Ala6, Pro9, NEt]-GnRH (GnRHa).

Thirteen females were implanted at doses ranging from 30.5 to 68.3 Ag kg� 1 during three

experiments between late June and early September. Of the injected females, 85% responded

positively to the GnRHa implant and ovulated between 60 and 238 h after treatment, whereas none

of control fish showed any sign of maturation. No spontaneous spawning was observed, and the eggs

were manually removed from the females using gentle abdominal pressure. The mean number of

ovulations per fish was 3.8, with a maximum of nine for one female. Overall, a total of 42 ovulations

were obtained, resulting in the production of more than 5 million eggs. The average relative

fecundity was 118.3F 16.0� 103 eggs kg� 1 BW, with a maximum of 202.2� 103 eggs kg� 1 BW.

Mean percentage fertilisation and hatching were 48.2% and 52.2%, respectively. The results

demonstrate that GnRHa administration via controlled delivery systems is an effective method for

producing good quality eggs in captive dusky grouper.

D 2003 Elsevier Science B.V. All rights reserved.

Keywords: Epinephelus; Dusky grouper; Induced spawning; GnRHa; Implants

0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0044-8486(03)00036-X

* Corresponding author. Tel.: +39-06-61570495; fax: +39-06-61561906.

E-mail address: [email protected] (G. Marino).

www.elsevier.com/locate/aqua-online

Aquaculture 219 (2003) 841–858

1. Introduction

The dusky grouper (Epinephelus marginatus, Epinephelinae) is a commercially and

recreationally important species in many areas of the Mediterranean Sea, and is also

considered a potential candidate for aquaculture and sea ranching (Glamuzina et al., 2000;

Marino et al., 2000). Interest in dusky grouper reproduction has increased since 1995 when

it was listed as an endangered teleost (Berne Convention, Annex 3; Barcelona Convention,

Protocol for Mediterranean Biodiversity, Annex 3), and artificial propagation was

considered as a means for stock enhancement (Fao, 1997, Art. 9.3.5). The first attempts

at artificial reproduction were undertaken by research groups in Italy and Croatia

(Spedicato et al., 1995; Glamuzina et al., 1998a; Marino et al., 1998a,b).

Broodstocks have been formed by collecting either wild adult breeders (Spedicato and

Lembo, 1996; Marino et al., 2000) or juveniles (Glamuzina et al., 1998b). Establishment

of captive broodstock is a complex and lengthy operation because of the shortage of fish in

the nearshore waters (Di Marco and Marino, 1999; Di Marco et al., 1999) and the

protogynous hermaphroditism exhibited by this species (Marino et al., 2001). This

reproductive strategy makes it difficult to obtain a sexually balanced broodstock (Marino

et al., 2002), since sex reversal takes place at a very large size and most fish captured are

females (Marino et al., 2000). Therefore, fish must be maintained in culture for a long

period before obtaining a sexually balanced broodstock.

There are also reproductive dysfunctions that exist in wild dusky grouper maintained in

captivity. Firstly, most of the time females fail to complete vitellogenesis, and as a result

there is absence of final oocyte maturation (FOM), ovulation and spawning (Marino et al.,

2000). Furthermore, even when FOM and ovulation occur, voluntary spawning does not

follow, probably because of the complex mating behaviour required for successful

spawning (Zabala et al., 1997) and the absence of the natural environmental conditions

(e.g., substrate, hydrology and temperature). Therefore, collection of ovulated eggs must

be done by manual stripping (Glamuzina et al., 1998a; Marino and Mandich, 2000). At

present, the inadequate supply of good quality eggs is the main constraint on the artificial

propagation of the dusky grouper (Marino et al., 2000; Spedicato and Boglione, 2000),

and there is a need to develop methods for controlling reproductive processes in this

species.

Early induced spawning trials using human chorionic gonadotropin (hCG) and

synthetic gonadotropin-releasing hormone agonists (GnRHa) given in multiple injections

(Spedicato et al., 1995; Glamuzina et al., 1998a; Marino et al., 1998a,b; Spedicato and

Boglione, 2000) proved to be relatively unsuccessful. On the other hand, treatment with

GnRHa incorporated in biodegradable microspheres (designed for warm-blooded animals)

was more effective in obtaining viable and fertile eggs (Marino et al., 2000). However,

most trials resulted in only a small number of eggs, with low fertilisation and hatching

success. Such failure of the hormonal treatment has been observed in many cultured

broodstock (Mylonas and Zohar, 2001a; Zohar and Mylonas, 2001), and has been

attributed to the low levels of the GnRHa in circulation due to the rapid metabolism

and clearance of this peptide after injection (Gothilf and Zohar, 1991). Recently, GnRHa

incorporated in sustained-release delivery systems (designed for poikilothermic animals)

was proven successful in inducing FOM, ovulation and spawning in several cultured

G. Marino et al. / Aquaculture 219 (2003) 841–858842

fishes, particularly in species displaying a group-synchronous or asynchronous spawning

patterns (Mylonas and Zohar, 2001b).

The dusky grouper exhibits a multiple-batch, group-synchronous pattern of ovarian

development and spawns several times from May to August in the wild (Marino et al.,

2001). Therefore, we hypothesised that treatment of vitellogenic females with GnRHa

delivery systems could be an effective method for inducing FOM and multiple ovulations

in this species. The present study reports on the first application of sustained-release,

GnRHa-loaded Ethylene–Vinyl Acetate (EVAc) implants in captive dusky grouper

females. The effectiveness of GnRHa implants to induce ovulation was evaluated by

monitoring the percentage of ovulated fish, the kinetics of ovarian maturation and

ovulation after hormone treatment, the fecundity and egg quality (fertilisation and hatching

success).

2. Materials and methods

2.1. Experimental fish

Dusky groupers were collected off the Pelagie Islands (Southern Mediterranean Sea,

35–36jN; 10–14jE) between May 1995 and August 1997, following the procedure of Di

Marco et al. (1999). In May 1998, 40 fish ranging in weight from 1.9 to 21.0 kg were

transported to a commercial hatchery (Ittica Mediterranea, Trapani, Sicily, Italy). At that

time, fish were anaesthetised with 80 mg l� 1 MS222 (Sandoz), individually tagged by

passive integrated transponders (Fish Eagle, UK) and stocked in four outdoor tanks (20

m3) at a mean density of 2.3 kg m� 3. Male–female sex ratio was between 1:5 and 1:7.

The tanks were shaded with a drop curtain and provided with concrete or PVC tubes with a

hiding function. Fish were maintained under natural thermoperiod up to March 1999, and

under controlled thermoperiod from April 1999 to September 1999, the temperature

gradually increasing from 16 to 22 jC. The sea water renewal was 200% day� 1, and the

fish were fed ad libitum four times a week with fresh fish (pilchards and mackerel) and

cephalopods.

2.2. Induction of ovulation

Females were selected for implantation with GnRHa only if they were at an advanced

stage of gonadal development, i.e., if their ovaries had vitellogenic oocytes at diameters

>325 Am. The stage of ovarian maturity of the broodstock was inspected monthly from

April to September 1999 using a gonadal biopsy. A sterile polyethylene cannula (0.8–1.2

mm i.d.) was introduced through the ovipore of the anaesthetised female, and the oocytes

were removed from the anterior and middle part of the right ovary using gentle aspiration.

Some oocytes were immediately examined under a compound microscope in order to

select females for hormone treatment. In these females, a portion of the biopsy was fixed

in 1% buffered formalin saline solution (365 mOsm) for 24 h, after which the diameter of

at least 50 vitellogenic oocytes was measured with a stereoscope using an ocular

micrometer. Samples of ovarian tissue were also fixed in Bouin’s solution for later

G. Marino et al. / Aquaculture 219 (2003) 841–858 843

histological examination. Ovarian condition and oocyte stages were histologically deter-

mined according to Marino et al. (2001). Briefly: J, immature; F1, resting; F2, early

vitellogenesis; F3, late vitellogenesis; F4, mature; F5, partially running; F6, postspawning.

Three consecutive hormone induction trials were performed during the reproductive

season from June to September (see Table 1). For each of the three spawning experiments,

two females having an ovarian maturity stage similar to the implanted females were left

untreated and formed the control group. Experiment 1 started on the 25th of June, 1 month

before the peak of the reproductive period. Of the females examined, three individuals

with vitellogenic oocytes ranging between 325 and 400 Am in diameter were given

GnRHa implants at a mean dose of 43.4 Ag kg� 1. Two females with vitellogenic oocytes

between 300 and 400 Am in diameter were used as controls. The implants were prepared

with EVAc (Mylonas and Zohar, 2001b) and contained [D-Ala6, Pro9, NEt] GnRHa

(Bachem Bioscience, King of Prussia, PA). The cylindrical pellets (2 mm in diameter and

3 mm in length) were inserted underneath the skin in the dorsal muscle by means of a

special implanter syringe. After treatment, water temperature was increased from 20.5 to

21.5 jC, 6 h after the time of hormonal treatment.

Table 1

Individual data on induced ovulation of dusky grouper females treated with a GnRHa-loaded implant during three

experiments

Experiment Fish

I.D.

BW

(kg)

GnRHa

dose

(Ag kg� 1)

Ovulations

(n)

Total

eggs

(g)

Relative

fecundity

(� 103 kg� 1 BW)

Mean egg

buoyancy

(%)

Mean

fertilisation

(%)

Mean

hatching

(%)

1 1 3.7 40.5 2 35.0 13.5 82.0 67.6 20.9

1 2 6.1 49.2 4 248.4 58.2 66.0 56.8 88.2

1 3 3.7 40.5 3 91.7 35.4 81.2 4.1 0.0

1 4 8.0 control – – – – – –

1 5 6.9 control – – – – – –

2 1 3.7 33.8 2 217.1 83.8 70.5 17.8 49.4

2 2 6.0 41.7 1 72.5 17.2 99.4 94.9 81.7

2 3 3.7 33.8 6 305.6 118.0 81.1 30.1 28.3

2 4 8.0 31.2 – – – – – –

2 5 6.9 36.2 7 883.1 182.8 59.7 63.1 62.6

2 6 6.2 40.3 6 441.5 101.7 74.3 75.4 51.9

2 7 4.4 34.1 3 209.1 67.9 74.2 70.4 68.7

2 8 4.1 30.5 1 163.0 56.8 91.4 7.1 59.8

2 9 5.4 46.3 – – – – – –

2 10 7.5 control – – – – – –

2 11 5.0 control – – – – – –

3 7 4.4 68.3 2 147.0 47.7 46.3 14.2 87.0

3 12 4.0 62.5 1 244.5 87.3 37.2 11.0 0.0

3 13 1.97 63.4 1 234.0 169.7 29.5 56.3 58.7

3 14 1.95 64.1 1 276.0 202.2 42.0 44.8 42.7

3 15 2.00 62.5 2 80.0 57.2 70.0 22.5 0.0

3 16 3.5 control – – – – – –

3 17 4.1 control – – – – – –

Females treated twice during the reproductive season are indicated in bold style. Only ovulations of >25,000 eggs

are presented.


Experiment 2 was initiated on the 26th of July, during the peak of gonad ripeness. At

this time, we found 11 females in the broodstock with vitellogenic oocytes. Nine of them,

having oocytes from 425 to 500 Am in diameter, were implanted with GnRHa at a mean

dose of 36.4 Ag kg� 1. Three of these females were already used in Experiment 1, but since

they contained vitellogenic oocytes at the appropriate stage, they were included again in

this experiment. Control fish (n = 2) had vitellogenic oocytes between 240 and 450 Am.

Water temperature was increased from 21.5 to 22.5 jC, 6 h after the time of injection. It

was then increased gradually at an average rate of 0.05–0.1 jC every 33 h, until a

temperature of 23 jC was reached.

Experiment 3 began on the third day of September, at the end of reproductive season.

From the whole broodstock, only seven females with vitellogenic oocytes between 475

and 550 Am in diameter were found (including one individual used in Experiment 2).

Within them, five were given a mean GnRHa dose of 64.2 Ag kg� 1. Two females, having

vitellogenic oocytes between 350 and 500 Am formed the control group. Water temper-

ature was maintained constant at 23 jC. Collectively, in all three experiments, out of the

28 adult dusky grouper females examined, 17 had reached advanced stages of vitello-

genesis during the experimental period (late June–early September), making them eligible

for hormonal induction of FOM and ovulation.

After each hormone treatment, females were transferred with one or two spermiating

males (sex ratio 1:3–5 males to females) to an indoor spawning tank (15 m3) provided

with flow-through seawater and aeration. Hormonally treated and control females were

biopsied at different times after treatment in order to monitor oocyte development and

to determine the time to ovulation, since voluntary spawning was not expected. The

first post-treatment (pt) examination of ovarian maturation was done 24 h after

implantation. Time to ovulation was predicted by monitoring changes in the oocyte

diameter pattern and morphology at different times after GnRHa injection. Ripe females

were also identified by the swelling of the abdomen and the protrusion of the genital

papilla.

2.3. Artificial fertilisation and evaluation of egg quality

When females were ripe, a gentle pressure on the abdomen in a cranial–caudal

direction was applied to collect the ovulated eggs. Eggs were fertilised by the wet method

using milt obtained from natural males and hormonally sex inverted juveniles (Marino et

al., 1998b, 1999). The stripped eggs were collected in 200-ml sterile plastic containers and

were maintained at the same temperature as the tank water. Milt was collected in a 2.5-ml

microcentrifuge vials, checked for motility and density and stored in ice (4 jC).Spermatozoa were activated in seawater at a ratio of 1:1000. Eggs were transferred into

a 2-l bucket containing the seawater–milt mixture and gently stirred for about 2–5 min to

allow fertilisation and water hardening. The eggs were then rinsed to remove debris,

transferred to a decanter (10–50 l) containing filtered sea water and disinfected using

iodine at a concentration of 5 ml l� 1 for 5 min. Eggs were rinsed again and allowed to

stand without aeration until separation between sinking and floating eggs was complete

(dusky grouper eggs are hyponeustonic). The percentage of floating eggs was calculated as

the weight (g) of floating eggs divided by that of stripped eggs. The total number of


stripped eggs per spawn was estimated indirectly from their weight using the calculation:

number of eggs = dry weight� 1428.57 eggs g� 1.

Fertilisation success (%) for each strip spawn was determined from three subsamples of

eggs (n = 200) 2 h after insemination by dividing the number of fertilised eggs by the

number of floating eggs obtained. The eggs were then incubated until hatching in a 1-m3

cylindro-conical tank supplied with filtered seawater and gentle aeration. After hatching,

three subsamples of 40 ml were obtained by immersing vertically a glass pipette in the

incubator in order to estimate the number of hatched larvae. Hatching success (%) was

estimated as the number of hatched larvae divided by the number of fertilised eggs

originally placed in the incubator. Total fecundity per female (eggs kg� 1 BW) was

calculated as the sum of the eggs obtained from all the strip spawns within the

reproductive season.

2.4. Statistical analysis

Statistical analyses were conducted using SPSS Software, Advanced Statistics 7

(Chicago, IL). The existence of differences in relative fecundity and number of ovulations

per female during the three experimental periods were analysed using one-way ANOVA,

followed by the LSD multiple comparison test. Differences in quality among strip spawns

were performed by the Student’s t-test. A linear regression analysis was performed

between oocyte diameter and time to ovulation, and between the number of ovulated

eggs and body weight. All data are expressed as meansF standard errors.

3. Results

3.1. Ovarian development after GnRHa stimulation

The progression of gonadal maturation after stimulation with the GnRHa implant was

evaluated by means of oocyte diameter distribution (Fig. 1) and the histological analysis of

ovarian biopsies (Fig. 2) taken from a representative female (F3). At the time of GnRHa

treatment, vitellogenesis was not complete and the diameters of most oocytes were

between 300 and 350 Am (Fig. 1a). After 58 h from the implantation with GnRHa, part

of the oocytes completed vitellogenesis, reaching a diameter of 450–500 Am (Fig. 1b). At

137 h post-treatment, a batch of oocytes (600–700 Am) had initiated FOM, while some

bigger oocytes (750–800 Am) had almost completed the process (Fig. 1c). At the same

time, we could observe also a batch of vitellogenic oocytes ranging in diameter between

200 and 400 Am. Final oocyte maturation was completed when oocytes had a diameter of

800–850 Am at which stage ovulation occurred (Fig. 1d).

The oocyte size frequency distribution shows that cohorts of eggs at different

developmental stages matured simultaneously in the ovary of dusky grouper (Fig. 1c).

Histological analysis clearly shows how, before and after hormonal treatment, oocytes in

various developmental stages exist near each other in the ovary. At the time of injection,

vitellogenic oocytes contained lipid droplets surrounding the germinal vesicle (gv) and

small yolk granules scattered throughout the cytoplasm (Fig. 2a). At the conclusion of


vitellogenesis, the gv still occupied a central position, while some lipid droplets had started

coalescence (Fig. 2b). Final oocyte maturation was characterised by the migration of the

gv towards the periphery and the coalescence of both lipid droplets and yolk granules (Fig.

2c); another batch of vitellogenic oocytes between 250 and 500 Am in diameter were also

visible in the biopsy at the same time. During the hydrating stage, larger oocytes contained

lipid droplets and yolk granules fused in large amorphous plaques, especially in the central

Fig. 1. Oocyte size frequency distribution in a representative dusky grouper female (F3) treated with a GnRHa-

loaded implant in June (a) at the time of hormonal treatment, (b) 58 h post-treatment (c) 106 h post-treatment and

(d) 137 h post-treatment, at time of the first strip spawn.


zone of the cytoplasm. Germinal vesicle breakdown was completed within the next few

hours before oocyte hydration (Fig. 2d).


Eleven of the 13 females treated with the GnRHa implants in the three spawning

experiments responded successfully, completing vitellogenesis and undergoing FOM and

ovulation. Time to first ovulation after GnRHa treatment was found to be dependent on the

stage of ovarian development of the female at the time of GnRHa implantation. Regression

analysis showed that time to first ovulation had a significant negative correlation

(r2 = 0.764; P < 0.005) with the mode of oocyte diameter at the time of hormonal treatment

(Fig. 3).

As anticipated from previous work, spontaneous spawning did not occur and ovulated

eggs were obtained by manual stripping within 60–238 h after implantation (Fig. 4). In

Experiment 1 (Fig. 4a), the first strip spawn was performed 107 h post-treatment (pt) in

the female exhibiting the most advanced stage of ovarian development at the time of

treatment, having oocytes of 500 Am in diameter. Another female having oocytes around

400 Am gave the first ovulation at 137 h pt. The females in this experiment produced a

mean of 3.0F 0.58 ovulations each, with a mean relative fecundity of 35.7F 12.9� 103

eggs kg� 1 BW (Fig. 3a and b). Percentage fertilisation and hatching of the stripped-

spawned eggs were 50.3F 15.4% and 41.1F 20.3%, respectively (Fig. 5c and d). The

two control fish, having vitellogenic oocytes between 300 and 400 Am at the time of

Fig. 2. Photomicrographs of ovarian biopsies of dusky grouper female (F3) treated with a GnRHa implant in June.

Shown samples taken (a) at the time of hormonal treatment, (b) 58 h post-treatment, (c) 106 h post-reatment and

(d) 137 h post-treatment, at time of the first strip spawn. Previtellogenic oocytes (PVG), lipid vesicles (LV), yolk

granules stage I (Y1), yolk granules stage II (Y2), yolk granules stage III (Y3), hydrated oocytes (H).


GnRHa treatment, did not complete vitellogenesis and did not undergo FOM and

ovulation.

One month after the start of the first experiment, ovarian development proceeded in the

broodstock as indicated by the increase in the diameter of vitellogenic oocytes. At the start

of Experiment 2, nine females had vitellogenic oocytes ranging in maximum diameter

between 425 and 500 Am, but still showing no signs of FOM. Stimulation with GnRHa

was successful in 78% of the treated females (Fig. 4b) and induced a total of 26 ovulations

(mean = 3.7F 0.97 female� 1) of mean relative fecundity of 89.8F 19.8� 103 eggs kg� 1

BW (Fig. 3a and b). The first strip spawn was performed 60 h pt and the last one 238 h pt,

so the time elapsing between first and last ovulation was three times longer than in June

(Fig. 4b). Percentage fertilisation and hatching of the stripped-spawned eggs were

55.9F 5.8% and 57.4F 5.0%, respectively (Fig. 5c and d). Control fish, having oocytes

ranging from 240 to 450 Am in diameter, again showed no signs of FOM and did not

ovulate during the experimental period. Two hormone treated females (F4, F9) failed to

ovulate and they did not proceed vitellogenesis after hormone stimulation.

In Experiment 3, 100% of the females having advanced vitellogenic oocytes and given

a GnRHa implant ovulated successfully. The first strip spawn was performed 69 h pt (Fig.

4c), and proved to be delayed, with most eggs being overripe and of low fertility. Overall,

we obtained an average of 1.4F 0.24 ovulation per female with a mean relative fecundity

of 112.8F 31.0� 103 eggs kg� 1 BW (Fig. 5a and b). Percentage fertilisation and hatching

of the stripped-spawned eggs were 29.8F 3.4% and 37.7F 6.4%, respectively (Fig. 5c

and d). Similarly to the previous two experiments, control females proceeded with oocyte

maturation during the reproductive period reaching the late stage of vitellogenesis (550–

600 Am) but they did not undergo FOM or ovulation. In September (Experiment 3), signs

of follicular atresia were observed in vitellogenic oocytes of both control and hormone-

treated females, probably indicating the end of the reproductive period.

Altogether, a total number of 42 ovulations was recorded, resulting in more than 5

million eggs. Female fecundity after GnRHa implantation correlated significantly with

body weight (r2 = 0.566; P= 0.008, data not shown). The best performance in terms of

ovulation number was recorded in late July with some females ovulating up to six to seven

times (Table 1). The mean number of ovulations per female was statistically different

Fig. 3. Linear regression between the mode of oocyte diameter at the time of hormonal treatment and the time to

first ovulation in dusky grouper females. Y=� 2.0349x+ 608.14 (r2 = 0.764; P < 0.005). Only first ovulations for

which time for stripping was correctly determined are considered (n= 9).


Fig. 4. Cumulative percentage of dusky grouper females having ovulated after treatment with GnRHa-loaded

implants during (a) Experiment 1, initiated in late June; (b) Experiment 2, conducted in late July and (c)

Experiment 3, which started in early September. The ranges of maximum diameters (Am) of the biopsied oocytes

at the time of hormonal treatment in each experiment are shown. Strip spawn resulting in overripe eggs are not

taken into account in any experiment.


among the three experiments carried in June, July and August, being higher in July

(P= 0.001) (Fig. 5a). In the course of the entire reproductive season, four females ovulated

only once, one ovulated twice and six females ovulated a total of four to nine times.

Among the latter, four females ovulated either both in June and July or both in July and

September (Table 1). Within a particular experiment, the longest period between the first

and the last ovulations of an individual female was 144.5 h (F5).

Fertilisation (%) varied widely in the course of the entire reproductive season, ranging

from 0 to 98.5% with a mean value of 48.2F 4.5 for the whole study. This very large

variation was largely dependent on the latency period between the time of ovulation and

the time of stripping, which should be kept to a minimum. Especially in Experiment 3,

ovulation time was not correctly predicted and eggs were already overripe when stripped.

In other cases, biopsies happened to take place while FOM was underway, probably

influencing negatively the quality of ovulated eggs. Mean hatching rate was 52.2F 4.7%.

Fig. 5. Mean (F S.E.M.) number of ovulations (a) and relative fecundity (b), fertilisation (%) (c) and hatching

(%) (d) from three experiments (late June, late July, early September) on the induction of ovulation of dusky

grouper using a GnRHa-loaded implant. BarsF S.E.M. in (a) and (b) are calculated from data per female, while

bars in (c) and (d) are calculated considering each ovulation. There were statistically significant differences

among the mean number of ovulations and mean relative fecundity during the three experiments (ANOVA,

PV 0.001). Differences in mean fertilisation (%) and mean hatching (%) were not significantly different

(Student’s test).


4. Discussion


Broodstock management and environmental manipulation applied to dusky grouper

broodfish was effective in inducing gametogenesis without altering the periodicity of

reproduction and the mode of ovarian development. Under captive conditions, the

periodicity of reproduction is similar to that recorded in the natural environment

(Massari et al., 1999; Marino et al., 2001). Females started sexual maturation in April

and reached the final stages of vitellogenesis between late June and early September.

Histological analysis and the oocyte size frequency distribution confirmed that ovarian

development occurs according to a multiple-batch, group-synchronous pattern (Tyler and

Sumpter, 1996), as observed in naturally maturing dusky grouper females (Marino et al.,

2001). The size interval between the mean diameters of the two successive vitellogenic

oocyte batches was about 100 Am (Fig. 1). Such oocyte size frequency distribution has

been generally associated with multiple ovulations within a time interval of less than 72

h (Alvarino et al., 1992), as was observed here. As a result of GnRHa implantation, the

batch of the larger vitellogenic oocytes grew from 350–450 to 800–830 Am, and

underwent completion of vitellogenesis, FOM and ovulation within 70–80 h, while a

batch of smaller vitellogenic oocytes continued their vitellogenic growth and increased

in diameter. Females ovulated successfully after GnRHa treatment only if their ovaries

contained a batch of oocytes >350 Am in diameter at the time of implantation. This

compares favourably with other spawning induction methods with dusky grouper

(Spedicato et al., 1995; Glamuzina et al., 1998a; Marino et al., 1999), or other

Epinephelus spp. (Tucker, 1994), where minimum responsiveness has been established

at 400–500 Am. These results underscore a great advantage of using sustained-release,

GnRHa-loaded delivery systems in spawning induction protocols, namely, the ability to

apply them in females which have not completed vitellogenesis and are not yet entirely

ready to undergo FOM/ovulation.

Sustained-release GnRHa-loaded implants proved very effective in inducing FOM and

ovulation in captive-reared dusky grouper, resulting in single or multiple ovulations in 11

out of 13 hormone-treated females. Female F4 was likely to not respond because of the

presence of signs of atresia in late vitellogenic oocytes (500 Am), while female F9 did not

ovulate probably because of the clear prevalence of early vitellogenic (250 Am) over late

vitellogenic oocytes. Untreated, control females did not undergo ovulation during the

experimental period, as expected from previous work (Marino et al., 2000; Spedicato and

Boglione, 2000) and did not even exhibit any signs of FOM.

The high responsiveness resulting from hormonal treatment by means of GnRHa

implants, compares well with other studies, employing different hormonal preparations.

For example, multiple doses of hCG induced ovulation only in 16.7% of the females when

given alone, and in 14.3% of the females when it was administered simultaneously with

pituitary extracts of adult sea bass Dicentrarchus labrax (Marino et al., 1998a). In other

studies, a 100% responsiveness in inducing ovulation was achieved using two injections of

hCG spaced 24 h apart, but the treatment resulted in the production of relatively few or

overripe eggs (Glamuzina et al., 1998a; Spedicato et al., 1998). Even less successful


results, in terms of both responsiveness to hormone treatment and egg quality, were

achieved by two GnRHa injections spaced 24 h apart (Spedicato et al., 1998). GnRHa

implants also performed substantially better than GnRHa-loaded microspheres produced

for human use, which induced multiple ovulations in only a few cases (Marino et al.,

2000). GnRHa implants used here have been manufactured specifically for fish use and

release 100% of the loaded GnRHa gradually for 2 weeks after application, thus sustaining

high levels of the endogenous LH (Mylonas and Zohar, 2001b). In many fishes, GnRHa

delivery systems induce multiple ovulations/spawnings, according to the reproductive

biology of the species (Mylonas and Zohar, 2001a). The dusky grouper has a multiple-

batch, group-synchronous ovulation pattern and the results from the present experiments

indicate that sustained stimulation with GnRHa represents a good method for inducing

FOM and ovulation of many batches of eggs over the course of a few days.

Some of the females in the present study were implanted with GnRHa twice during

the reproductive season, after a ‘‘resting period’’ of 20–30 days had elapsed (Table 1). It

appears that while the first GnRHa implant application induced the maturation of the

first (i.e., largest) batches of vitellogenic oocytes, at the same time allowed the

recruitment of new batches of oocyte into vitellogenesis. These latter batches were

found to be in an advanced vitellogenic stage in the subsequent trials (Experiments 2

and 3). Females exhibiting such ‘‘rematuration’’ responded successfully in 100% of the

cases following two treatments with the GnRHa implant. Similar results have been

obtained in the white grouper Epinephelus aeneus (Hassin et al., 1997), while a

comparable spawning interval was reported for wild Epinephelus suillus cultured in

net cages (Toledo et al., 1993).

In this study, use of GnRHa implant induced an average of 3.8 and a maximum of 7

ovulations per female (Table 1) within a period of 4–5 days after treatment (Fig. 4),

allowing several batches of good quality eggs to be obtained. This is an additional,

important advantage over previous maturation-induction methods which, generally,

require multiple administrations to obtain multiple ovulations (Glamuzina et al., 1998a;

Spedicato et al., 1998; Marino et al., 2000). Multiple handling of the broodstock is labour-

intensive, time-consuming and predisposes broodfish to stress. More importantly, in

grouper, the stress due to repetitive injection is additional to the stress due to repetitive

strip spawn, which can not yet be avoided, and both can affect negatively the quality of the

produced eggs.

All hormone-treated females were manually strip-spawned, since studies by various

research groups have demonstrated sufficiently that natural spawning is extremely unlikely

in captive fish (Spedicato et al., 1995; Glamuzina et al., 1998a, Marino, 2000; Marino et

al., 2000). The dusky grouper exhibits an elaborate spawning behaviour: gamete release

occurs from a breeding pair at the conclusion of rapid 7–12 m upward ascents towards the

surface (Zabala et al., 1997). The rapid ascent of a pair seems to be a prerequisite

behaviour for the synchronisation of gamete release and egg fertilisation in other grouper

species, like Epinephelus akaara under artificial conditions (Ukawa et al., 1966; Okumura

et al., 2002) and Cephalopholis boenack in the wild (Donaldson, 1989). In our study, the

ascent behaviour was never observed and it is likely that in captive dusky grouper such

spawning behaviour was limited by the tank depth (1.5 m) and by the space, as presumed

also for other groupers (Okumura et al., 2002).


4.2. Egg production and quality

All eggs ovulated after GnRHa implantation were stripped manually from the female,

resulting in an overall mean relative fecundity of 118� 103 eggs kg� 1 BW and a

maximum of 202� 103 eggs kg� 1. Ovulation induction of dusky grouper using a single

acute GnRHa injection resulted in lower fecundity (Spedicato et al., 1998), because only

the first batch of large vitellogenic oocytes was induced to undergo FOM and ovulation.

However, total fecundity of captive females after GnRHa implantation was lower than

reported for wild dusky groupers from the natural environment, which produce up to 258–

606� 103 eggs kg� 1 BW (Brusle, 1985). This is perhaps to be expected from a fish with a

multiple-batch, group-synchronous ovarian pattern, such as the dusky grouper, which has

a 3-month spawning season (Marino et al., 2001).

The number of females with oocytes at an advanced stage of vitellogenesis increased in

July, when seven females were treated with the GnRHa implant, compared to three and

five fish, respectively, in June and September (Table 1). The mean number of ovulations

per female was also higher in July (Fig. 5), thus, suggesting that the peak of the spawning

period was in July, similarly to what was observed in nature (Marino et al., 2001). Relative

fecundity showed a weak trend towards an increase in the reproductive period. The

statistically significant higher fecundity in July and in September could have been due to

more vitellogenic oocytes reaching a stage to be responsive to the GnRHa treatment. This

was evident from the slightly higher maximum oocyte diameter observed in the gonads of

females implanted in September (Fig. 4). Both fertilisation and hatching (%) were not

significantly different during the experimental period (Fig. 5). These data suggest that in

dusky grouper oocytes undergo vitellogenesis and are viable and responsive to hormonal

stimulation throughout the 3-month-long, summer spawning period.

In the present study, GnRHa was administered at doses ranging from 30 to 68 Ag kg� 1.

Poor-quality eggs were observed in Experiment 3, when the mean dose employed was

higher than the doses used in Experiments 1 and 2, and the eggs were overripe 70 h after

hormone injection. High GnRHa dosages can result in a reduction of egg quality (Mylonas

et al., 1992; Francescon et al., 1994) or in a small number of ovulations in several cultured

fishes (Poortenaar and Pankhurst, 2000). Also, in other studies with dusky grouper, the

administration of GnRHa at high dosages (200 Ag kg� 1) by an acute treatment resulted in

low quality eggs (Glamuzina et al., 1998a). We are currently carrying out experiments to

establish such dose–response curve for the GnRHa implants in the dusky grouper, but

based on the results presented in this study we could recommend a dose not exceeding 45

Ag kg� 1 GnRHa.

Fertilisation success was variable among different batches of eggs, even when obtained

from the same individual (data not shown), being generally lower at the first stripping after

hormone treatment. This could be due to failure to predict accurately the time of first

ovulation. It is widely reported that once the eggs are ovulated into the ovarian or

abdominal cavity, they lose their nutrient and oxygen supply and eventually die. The

‘‘latency period’’ during which viability is maintained after ovulation varies greatly among

species, and also depends on parameters such as water temperature and ovulation

induction method. Therefore, the maximum allowable period between ovulation and

manual stripping varies widely among fishes. For example, in salmonids this period


extends to a few weeks (Springate et al., 1984), in Atlantic halibut (Hippoglossus

hippoglossus) it is 4–6 h (Bromage et al., 1994), whereas in the white bass it is only

30 min (Mylonas et al., 1996). In other Epinephelus spp., this period has been suggested to

be between 1 and 2 h after ovulation (Tucker, 1994). In dusky grouper, eggs stripped

immediately after ovulation had lower fertilisation (%) than eggs stripped later (data not

shown). This suggests that a maturational event takes place after ovulation and while the

eggs are in the ovarian cavity. It has been demonstrated that in some fishes the second

meiotic division in the maturing oocyte is completed after ovulation (Nagahama, 1983). If

this occurs also in the dusky grouper, then maximum fertility of the ovulated eggs can be

expected some time after ovulation is completed, as reported in other species (Hirose et al.,

1977; Springate et al., 1984).

In species such as the dusky grouper that do not spawn spontaneously in captivity, the

time of stripping is a crucial parameter in obtaining viable eggs. Apart from the

hormonal therapy, the time to ovulation after treatment depends on the oocyte devel-

opmental stage at the time of treatment or soon afterwards, and on water temperature

(Shelton, 1989). For example, a relation linking water temperature and oocyte diameter

24 h after hormonal treatment has been used to determine the optimum stripping time for

Nassau grouper (Epinephelus striatus) (Watanabe et al., 1995). Similarly, an ovulation

prediction guide has been developed for striped bass, based on the stage of FOM at the

time of hCG injection and the ambient water temperature (Watson, 1992). In dusky

grouper, we found a significant correlation between time to ovulation and the most

representative oocyte diameter class at the moment of GnRHa implantation (Fig. 3). This

relationship can be an important tool in correctly predicting the time to first ovulation

after GnRHa implantation of dusky grouper, in order to ensure efficient collection of

eggs of good quality and to minimise losses due to overripening. Very often behavioural

and external morphological changes occur in females after hormone treatment, which can

be used to enhance the accuracy of predictive methods based on oocyte stage and

temperature. It was observed that when dusky grouper females had completed ovulation,

the abdomen becomes swollen and the genital papilla becomes more visible due to its

protrusion and reddish colour. Obviously, it is impractical to visually monitor females

throughout the period after GnRHa treatment, but coupled with a coarse approximation

of expected ovulation time, such observations can assist in determining stripping times

more effectively.

In conclusion, broodstock management applied to dusky grouper was effective in

inducing gametogenesis, and hormonal treatment with a GnRHa implant was able to

induce ovulation of viable eggs for a period of two months during the natural spawning

season. Further studies are needed to investigate the environmental, social and behavioural

factors possibly responsible for hormone failure and inhibition of spontaneous spawning in

captivity.

Acknowledgements

We are grateful to Ittica Mediterranea fish farms (Trapani, Italy) for maintaining our

dusky grouper broodstock, and to the staff of Tecnologie Avanzate in Acquacoltura for


technical assistance during the experiments. We are especially grateful to Vito Balsamo,

Ernesto Azzurro and Piero De Benedetti for their technical support during hormonal

induction trials.

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