Gonadotropic hormone-releasing hormone analog (GnRH-A) induced ovulation and spawning in female winter flounder, Pseudopleuronectes americanus (Walbaum)

Aquaculture, 104 (1992) 375-390 Elsevier Science Publishers B.V., Amsterdam

375

Gonadotropic hormone-releasi (GnRH-A) induced ovulation and spawningin femalewinter flounde uronectes americanus Sharr A. Ha.miu and Laurence

W. Crinn

Ocean Sciences CentrelDcpartment of Biology, Memorial University,St. John 3, Nfra:, Canada

(Accepted 29 September I99 1)

ABSTRACT Harmin, S.A. and Crim, L.W., 1992. Gonadotropic hormone-releasing hormone analog (GnRH-A) induced ovulation and spawning in female winter flounder, Pseudopleuronectesamericznus (Walbaum). Aquaculture, 104: 375-390. Winter flounder females generally fail to ovulate spontaneously in the laboratory whether being held at wmter water temperatures ( rO”C) or after being exposed to seawater at 5 “C. Although treatment of flounder with gonadotropic hormone releasing hormone, [ D-Ala6,Pro9-NHEt] LHRH (GnRHA), resulted in ovulation of a few females at O”C, GnRH-A accelerated ovulation and spawning reliably in prespawning female flounder maintained at 5°C. While seasonal recrudescence of flounder ovaries is largely completed by December, GnRH-A was first capable of inducing spawning of some females in February, approximately 3 months prior to the normal flounder spawning season. More rapid and predictable hormone-induced ovulatory responses were observed following GnRH-A implantation of females in May. Studies of egg and larval quality, e.g., rates of egg fertilization, hatching and larval survival, demonstrated that good quality flounder offspring can be produced after accelerated spawning by GnRH-A treatment. After GnRH-A induction of spawning in February, the best flounder egg quality was observed when females had previously received brief exposure to cold winter seawater temperatures.

-1NTRODUCTION

The winter flounder is an inshore species found along the Atlantic Coast of North America from Georgia to Newfoundland and Labrador (Liem and ’ Scott, 1966). In its northern distribution spawning normally occurs in May or June as seawater temperature rise approaching 5 OC. Previous descriptions of the flounder repl*oductive cycle (Burton and Idler, Correspondence to: Dr. L.W. Crim, Ocean Sciences Centre/Department University, St. John’s, NfId., AlC 5S7, aCanada.

0044-8486/92/$05.00

of Biology, Memorial

0 1992 Elsevier Science Publishers B.V. All rights reserved.

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S.A. MARMIN

AND L.W.

CRlM

1984; Harmin, 199 1) indicate that seasonal gonadal recrudescence begins in August together with the summer feeding period. The gonadssomatic index (GSI) of females increases rapidly in the fall, continues to increase more slowly during the winter months, and then remains elevated and relatively stable until May when the spawning period begins. Just prior to the onset of spawning, an additional increase in GSI is observed, presumably reflecting a brief period of ovarian hydration. Successful induction of spawning of winter flounder held in captivity has been previously reported using human chor-ionicgonadotropin (HCG), carp pituitary extract (CPE) (Smigielski, 1975 ), and a synthetic mammalian GnRH analog (Crim, 1985; Weigand et al., 1987 ). Although GnRH-A is well known to induce spawning in many teleost fish (Crim et al., 1987), previous studies provide mixed reports of egg quality/ larval development following hormonal induction of spawning. Indeed, poor egg quality was observed following hormonal induction of spawning in the flounder (Smigielski, 1975 ), salmon (Crim and Glebe, 1984), sole (Ramos, 1986a) and the grey mullet (Lee et al., 1987). In contrast, high hatching rates and a high yield of normal-looking fry were observed in catfish (De Leeuw et al., 1985; Manickam and Joy, 1989) and the Chinese carp (Peter et al., 1988) following GnRH-A treatment. In our current studies, the effects of GnRH-A treatment on the winter and spring ovulatory responses of females and the effects of advanced spawning on ~gp,@vd c;z;a!ity v:erc iwestigab.i. Hnaddition, the eflects of cold water temperature exposure on GnRH-A induced ovulatory responses of winter flou!j+derwere in.vestigated. MATERIAd AND XETi-i&X

Experimental I;wimals Sexually matu,ring female winter flounder, ranging in weight from 300 to 700 g, were captured by SCUBA from Conception Bay, Newfoundland and transported to the laboratory where they were acclimated in 250-l tanks to ambient seawater and a simulated seasonal photoperiod (60 W incandescent light at 1 m height from fish) for 3-7 days prior to initiating experiments. For hormonal induction of spawning, the temperature was adjusted to 5°C to mimic naturally occurring spring seawater temperatures (Bigelow and Sch;n)eder, 1953). At the beginning of experiments 1 and 2, a group of 4-6 females was sacrificed to determine seasonal ovarian development including gonadosomatic index (GSI ), oocyte diameter (OCDM ) , and germinal vesicle (GV) position. Hormone administration [ D-Ala6,Prog-NHEt ] LHRH (GnRH-A ), purchased from Syndel Laboratories, Vancouver, Canada, was administered either by intramuscular pellet

GnRH-A INDUCED OVULATION AND SPAWNING IN FEMALE WINTER FLOUNDER

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implant or intraperitoneal (IP) saline injection. Blank pellets lacking hormone were implanted into implant control fish. GnRH-A ( iO0 or 120 pg/ pellet ) was incorporated into slow-release cholesterol pellets measuring 2 x 5 mm according to Crim et al. ( 1983). Fish in experiment 3 were treated with 40 pg GnRH-A contained in quick-release pellets ( 50 : 50 cholesterol : cellulose) which deliver releasing hormone more quickly (Sherwood et al., 1988 ). In experiment 1, the effects of IP GnRH-A injections (saline control or 20 ,ug/kg BW GnRH-A in saline 3 ~per week) were compared with GnRH-A application by implantation. Ovulatory response Fish were inspected daily for signs of ovulation according to abdominal swelling, softening of the body wall and finally the release of free-flowing eggs from the egg pore upon application of gentle abdominal pressure. Increases in body weight (a sign of ovarian hydration ) were also periodically monitored. In females with relatively well developed gonads, oocytes from the dorsal ovary were quickly aspirated into a 14-gauge needle without need of an anaesthetic. About 30-40 oocytes from each ovarian biopsy were cleared of yolk to determine the GV position by exposing ovarian fragments to clearing solution (ethanol: formalin : acetic acid, 6 : 3 : 1 v/v) for about 1 min. A system to stage the position of the egg germinal vesicle (GV) under the microscope was as follows: ( 1) GV in the central position, (2) GV slightly off-centre, ( 3 ) GV migrated midway to animal pole, (4) GV peripheral position against egg membranes, ( 5) germinal vesicle breakdown (GVBD), and (6 ) ovulation (GV disappeared). Mean GV position u as calculated from 3-7 females per group. Egg quality determinations Milt from 2-5 ripe males was pooled to inseminate flounder eggs in vitro. Eggs from freshly ovulated females were stripped into a precooled 250.ml beaker placed into crushed ice. Aliquots of 200-300 eggs from each female (70-80 ~1) were pipetted into 3-6 replicate petri dishes and mixed with 3050 ~1undiluted milt. Sperm activation was initiated by the addition of 0.5 ml sterilized, 5 “C seawater containing 0.1 g/l streptomycin and 0.06 g/l penicillin. After swirling the eggs and milt together, l-2 ml of clean seawater was added while the eggs began sticking to the bottom of the petri dishes. Additional washes with fresh seawater removed the excess milt. In experiment 3, males and females in equal numbers were introduced into a 250-l tank to allow spontaneous spawning. The males had been previously injected twice with 20 &kg GnRH-A to increase milt production. Tanks were inspected daily at 10.00 h for the presence of floating egg masses; the waterhardened eggs were immediately removed, washed several times, and divided into subsamples of 200-300 eggs/petri dish for subsequent incubation. After

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24 h incubation at 5 “C in the dark, egg fertilization (blastula stage) rates were determined as an index of egg viability. Opaque eggs and larvae (mortalities) were removed and seawater was renewed daily. Daily observations were recorded for determination of initiation of hatch and the duration of the hatching interval. In addition, the relative proportions of normal vs abnormal (curvature of the spine, rounded yolk sacs, or enlarged fin folds) hatched larvae were estimated. Prematurely hatched larvae (short, thickened and often curved ) were also easily recognized and considered abnormal. Experimental design GnRH-A induction of ovulation and spawning was studied at 3 different times of the year (experiments l-3) beginning in February, in March and again in May (ambient seawater temperatures 0, 0.5 and 2.0°C, respectively). Experiment 4, to study hormone-induced ovulatory responses and egg quality of flounder following exposure of females to different winter water holding temperatures, began in December 1988. Initially, two groups of females were held in either relatively warm ( 5 *C ) temperature controlled seawater (group 1) or in seasonally declining ambient seawater temperatures (group 2). In early February, after ambient seawater temperatures had fallen to O*C, females in group 2 were further subdivided into fish remai. ing at the low ambient seawater temperatures (group 2A) or females immec iately returned to 5 *C after the brief exposure to cold 0°C seawater (group ZB ) . Following GnRH-A implantation on 8 February 1989, the body weights and ovulatory responses of females were recorded for a period of 7 weeks. In this experiment, after ovulation was detected, the eggs were stripped, fertilized with sperm in vitro and eggs were incubated for 2 days until fertilization rates could be determined. Statistical analysis All data are means ?s.e.m. Ovulation data were analyzed by the Fisher’s probability exact test. Arcsine transformed GSI data were analyzed by oneway ANOVA. Analysis of body weight was performed on the untransformed percentages since changes in body weights were expressed as both positive and negative (weight loss) values. After performing the one-way ANOVA, normality of the residuals was tested using either the Shapiro-Wilk statistic (n S 5 1) or the Kolmogorov statistic (n 2 50). Significance of mean differences (P-c 0.05 ) were tested by the Duncan’s multiple range test. RESULTS

For the initial 4-week trial (experiment 1 ), oocyte maturation (GV migration), ovulatory responses and egg quality were studied following GnRH-A treatment of females in February. Autopsy of females at the beginning of this

GnRH-A INDUCED OVULATION AND SPAWNING IN FEMALE WINTER FLOUNDER

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experiment indicated that the ovaries were well developed at this time of year (GSI: 17.2 + 0.9%, n = 6) although the GV remained located in a central position (Table 1). Progressive GV analysis (Table 1) of oocytes throughout this experiment indicated that after 2 weeks, seasonal GV migration was underway (GV offcentre in oocytes, stage 2) in control females; GnRH-A treatment accelerated GV migration in some experimental females (mean GV stages 2.2 and 2.3, groups 4 and 6, respectively; maximum GV migration stage 4 in a fish in group 6). At the end of this $-week trial, while no further changes occurred in the stage 2 GV position of control female oocytes, GV migration had advanced to stage 4 or 5 (peripheral position/GVBD) in oocytes from some females receiving releasing hormone treatment. After 4 weeks no ovulatory responses had occurred in control females maintained at 5 OC beginning in February (Table 2 ); however, after GnRHA treatment, 2 females ovulated from each hormone treatment group (groups TABLE I The effect of GnRH-A treatment in February on mean germinal vesicle (GV ) stage in oocytes from prespawning female winter flounder (experiment 1) ’ Group

Treatment

Tl

72

T3

1 2 3 4 5 6

Initial Intact Sham imp. GnRH-A imp. Saline inj. GnRH-A inj.

l&O If0 l&O I+0 I+0 l&O

2&O 2fO 2.16kO.16 220 2.33kO.33

2+0 (5) 2t-0 (6) 3.4 f0.87 (5) 220 (3) 4.66+, 1.33 (3)

‘GV values are mean + s.e.m.; Tl: time zero prior to experimen:ation, all groups composed of 6 fish; 72: two weeks after hormone treatment, all groups composed of 6 fish except group 5, n= 3; and 73: final day of the 4-week experiment, (n). TABLE 2 Influence of GnRH-A treatment in Februam on gonadosomatic index (GSI) and ovulation in female winter flounder (experiment 1) ’ Group

Treatment

Number ovulated

GSI (%)

I

Initial Intact Sham imp. GnRH-A imp. Saline inj. GnRH-A inj.

0 0 0 2 0 2

17.220.8 (6) 16.7kO.8 (5) 19.0+ 1.9 (61 21.62 1.2 (4) 17.3f 1.9 (3) 24.8 (1)

2 3 4 5 6

‘GSI values are mean f s.e.m. At the beginning of the treatment each group consisted of 6 fish. ( indicate number of unovulated fish surviving on the final day of the experiment.

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4 and 6 ) by the final day of this study. Poor survival of females (Table 2 ) in the injection treatment groups (3/6 and l/6 survivors for saline control and GnRH-A injected groups, respectively) indicates that frequent handling of females greatly increases prespawning mortality ( 8/ 12 ). At the end of the study, assessment of ovarian development (GSI ) in unovulated, surviving females indicated that GnRH-A treatment increases GSI (GSI in pooled GnRH-A treated females significantly greater, PC 0.05, compared with pooled controls ) . For eggs obtained from one ovulated female treated with GnRH-A injections in February, the rate of egg fertilization was low (23%). Eighty-four percent of these embryos hatched by 1 l- 15 days and half of the hatched larvae appeared to be in normal condition. Experiment 2, beginning in mid-March, again tested the effects of GnRHA implantation on advancement of ovulation and egg quality in hormonctreated females. As before, the ovaries of females autopsied in l+vIaicHappeared relatively well developed (GSI: 14.7 +, 1.OS%,n = 4 ). After 40 days of the experimental period, just 1 of 8 sham control females ( 12.5%) had ovulated (Fig. 1). By contrast, the ovulatory response of GnRH-A imp!anted females was significantly greater (B-C0.00 1). While the first female ovulated 16 days after hormone implantation, most females ovulated within 3 weeks of the GnRH-A treatment, and by day 32 following hormone implantation all 9 females ( 100%) in this group had ovulated (mean time to ovulation=258 days). The spawning response (egg release) of ovulated females was quite variable; 5 females spontaneously deposited their ovulated eggs into the tank overnight but, without males, the e s remained unfertilized. Four females

0 ---e

4 -.--A

A----A

SHAM GnRH -A

.I .’

/ A I

II

8

16

24

32

40

DAYS OF IMPLANTATION Fig.

1. The ovulatory responses (cumulative number of females ovulated) of sham control females and females implanted with GnRH-A (II== 9) in March. Day=0 indicates beginning of experiment. (II-

81

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GnRH-A INDUCED OVULATION AND SPAWNING IN FEMALE WINTER FLOUNDER

TABLE 3 Influence of induced spawning by GnRH-A in March on fertilization rate, hatching rate and presence of normal larvae (experiment 2 ) ’ Fish no.

Fertilization rate (% )

Hatching rate (%)

Normal embryo (%)

OR105 B20 B122 B123 B124

92.2 + 1.9 66.4 + 8.9 55.7 + 1.8 49.8 f 10.8 63.2 IL7.2

14.7k4.3 86.7f4.3 54.8 + 6.9 65.92 16.1 67.0 + 2.1

64.5 f 8.8 98.2kO.l 75.2_+2.5 97.45 1.2 97.2 kO.2

‘Values are mean + s.e.m., each determined from 3 replicate petri dishes each containing 200-300 eggs; (OR) sham implant; (B) GnRH-A implant.

8642-

O(8)

1 --

I

0

5

10

15

--

20

25

DAYS OF IMPLANTATION

Fig. 2. Changes in body weight (mean percent) following GnRH-A implantation of females in May. Data in parentheses indicate the number of fish. Values arc mean + s.c.m.

retained their eggs until morning which were stripped by hand and artificially fertilized with sperm. Egg quality data as estimated by egg fertilization rates, hatching success, and larval development for individual female egg lots are shown in Table 3. Hatching of larvae, which began approximately 12 days post-fertilization, was completed within 2 weeks. For eggs collected from the only ovulating sham control female, a high fertilization rate (92%) was observed but the hatching rate was low and a relatively high proportion of abnormal larvae was observed. For females retaining eggs after being induced to ovulate with GnRH-A, the mean egg fertilization rate was 58% (range 5066%). However, mean egg survival to hatch was 68% and larval quality was very good. The third seasonal study, beginning 20 ay 1988, was carried out immediately before onset of the natural spawning period. Following GnRH-A implant treatment of females, changes in body weight were monitored as an indicator of ovarian hydration and anticipated ovulation (Fig. 2 ). In sham

S.A.HARMIN AND L.W. GRIM

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DAYS OF IMPLANTATION

Fig. 3. The ovulatory responses (cumulative number females ovulated) of sham control females ( n= 8 ) and females implanted with GnRH-A (n= I 1) in May. Day = 0 indicates beginning of experiment. TABLE 4 Evaluation of the effects of GnRH-A induced spawning in May on egg fertilization rate, hatching rate and relative amounts of normal embryos (experiment 3) ’ Fish no.

Fertilization (%)

Hatching rate (%)

Normal embryo (%)

QR7I 6177 Gl79 G182 0183 G185 Gl86

94.0~0.5 95.7 kO.6 8(f.7+ 1A 99.3+0.1 96.8 zk0.6 97.5 20.2 63.4” 3.8

58.6 k 27.4 92.7 + 1.O 93.0+ 1.2 85.42 1.8 86.3 9 5.7 94.8+ 1.0 28.2 + 2.0

3.4f_ 1.7 98.0+ I .2 98.7kO.S 97.6+0.5 94.3 k 1.3 97.5 + 0.6 68.9k2.7

‘Values arc mean ks.e.m. each determined from 5-6 replicates of petri dishes each containing 200300 eggs: (OR) sham control implant ( 1 spontaneously spawned female); (G) GnRH-A implant.

control females, body weights remained relatively stable throughout the 25 day study period although a 3.5% increase in body weight was observed for the two control females which subsequently ovulated. In contrast, within 5 days of GnRH-A implantation, significant increases in body weight, reaching a peak of 5% by day 10, occurred in the hormone-treated females. Hormone-implanted females ovulated at a significantly greater rate (9 of 11,82%) compared with control females (2 of 8,25%) (Fig. 3). The latency time, i.e. mean days to ovulation after hormone treatment, was 13days. Most ovulated females spawned within 1 day after ovulation was detected; however, the time between ovulation and spawning varied among individuals. Eggswere released from some females in a single spawn; in partially ovulated females, however, serial spawning occurred 2 or 3 times after the first spawn-

GnRH-A INDUCED OVULATION AND SPAWNING IN FEMALE WINTER FLOUNDER

383

10 8-

o-o a--a

SHAM IMP GnRH-AIMP

GROUP

1

T 6_

HIGH

O--o o-

TEMP

SHAM IMP GnRH-AIMP

AMBIENT

GROUP

2A

LOW

(8)

(8)

(8)

1

I

-

___GROUP

28

i(3 DAYS

OF

IMPLANTATION

Fig. 4. Changes in body weight (mean percent) of females exposed to different temperatureregimes prior to GnRH-A implantation in February. Prior to hormone treatment, females remained at 5°C (high temp., group 1 ), were exposed to winter ( I “C) temperatures (ambient low, group 2A1 or they were returnedto 5°C afterexposure to cold winter temperatures(iow-high, group 28). Data in parentheses indicate the number of fish. Values are mean k s.e.m.

ing. Of the 9 GnRH-A treated females induced to spawn, eggs from 6 females were fertilized spontaneously by ‘male co-occupants of the tank; two other females were partially spawned at the end of the experiment and one female spawned eggs of poor quality that were not apparently fertilized. Eggs from only 1 of 2 ovulated control females were fertilized naturally. Similar to previous results, most flounder embryos hatched within 2 weeks of egg fertilization following incubation at 5°C. Egg fertilization rates were

S.A. HARMlN AND L.W. GRIM

384 U-U

6 t 5-

a-m

SHAM IMP GnRH-A IMP

HIGH

TEMP

GROUP

/+‘-’

4-

/

3.’

2-

0-0 M 85 P 4

f 2

3

6.-. A-A

GROUP

SHAM IMP GnRH-A IMP

AMBIENT

z

1

2A

LOW

GROUP2 B

SHAM IMP GnRH-A IMP

b------A

LOW-HIGH

A.-

cl

16 DAYS

OF

L--I_1 24

32

40

48

IMPLA!JTATION

Fig. 5. The ovulatory responses (cumulative number females ovulated) of females exposed to different temperature regimes prior to GnRH-A implantation in February. Identification and number of females in groups I, 2A and 2B as indicated in Fig. 4.

high, e.g., 94% and 89%, respectively, for eggs collected from the one ovulated control fish and for eggs obtained from females induced to spawn with GnRHA (Table 4). Likewise, good quality eggs ere indicated by high hatching rates and good quality larvae obtained from females treated with GnRH-A. The results of experiment 4 show that the body weights of all groups of sham control females remained unchanged or tended to decrease towards the end of the experiment (Fig. 4). However, following GnRH-A treatment, the body weights of hormone-treated females significantly increased (up to 30 days after initiation of the experiment) in fish held at 5 “C as well as in fish

GnRH-A INDUCED OVULATION AND SPAWNING IN FEMALE WINTER FLOUNDER

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TABLE 5 influence of temperature regime and GnRH-A induced spawning in February on egg fertilization rates (experiment 4) ’ Treatment/ fish no. Group I -high W28 W26 w25 w30 W29

Fertilization rate (%I temp. 73.0f 1.0 73.0 + 3.7 23.8 + 2.4 8.7f 1.2 ND

Treatment/ fish no.

Fertilization rate (OhI

Group 2A - low temp. R17 5.5 Group 2B - low: high temp. G174 97.0 2 0.4 G172 95.9f 1.1 G171 71.7f2.1 G169 1001 mart G175 92.7 Z!I0.8

‘Valuesare mean f s.e.m.

held at low ambient temperature. Autopsy of non-ovulated females at the end of the experiment indicated that, GnRH-A treatment significantly stimulated development of the ovaries irrespective of water temperature (pooled GnRHA treated female GSI =2 1.8 ,+0.9% 2 GSI = 15.6 + 0.5% for pooled control females ) . No ovulatory responses were observed in females not receiving hormone treatment in experiment 4 (sham control female groups) irrespective of their exposure to various experimental temperature regimes (Fig, 5 ). Similarly, females responded poorly to GnRH-A treatment when they were held at low ambient seawater temperatures; just one of eight females ovulated late during the S l-day experimental period (group 2A). In contrast, GnRH-A treatment of females at 5 “C significantly stimulated ovulation (83% and 7 l%, of the different groups of females, respectively) when hormone-treated females were either briefly held at ambient seawater temperatures and returned to 5°C (group 2B) or when females were always held at a relatively warm winter temperature ( 5 OC) never experiencing the seasonally low ambient seawater temperatures (group 1) . The results of egg quality from experiment 4 (Table 5 indicate that better quality eggs were obtained from females with some exposure to low seawater temperatures (mean egg fertilization 89 and 45%, female groups 2B and 1, respectively) as opposed to females always held in 5°C seawater never experiencing low temperature conditions, DISCUSSION

These results clearly show that GnR -A treatment is highly effective for accelerating ovulation and spawning of prespawning female winter flounder, and this compares favourably with reports of Gn H-A induction in many other teleosts (Crim et al., 1987; Zohar, 1989 1. It is worth noting that non-hormone-treated female fkurder in the winter seldom ovulate early

ofspawning

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S.A.HARMIN AND L.W. CRIM

or spawn spontaneously in the laboratory even after they are exposed to elevated seawater temperatures and/or are maintained in mixed sex populations. A period of gonadal hydration occurs in female teleosts immediately prior to final egg maturation and ovulation (Clements and Grant, 1964). Indeed, increased body weight following hormone induction of spawning has been noted for a number of fish species including striped mullet (Shehadeh and Ellis, 1970)) tilapia (Babiker and Ibrahim, L 979), Japanese ayu (Hirose and Ishida, 1974; Hirose et al., 1974), northern anchovy (Leong, 197 1), and sablefish (Solar et al., 1987). The practical aspects of such findings are that ovulation of females may be accurately predicted simply by monitoring increased body weight which improves the chances of collecting freshly ovulated, high quality eggs. In female flatfish, e.g., flounder and Atlantic halibut, swelling of the abdomen above the ovary is particularly marked prior to ovulation, and in this study we have firmly established the timing of increases in body weight and subsequent ovulation of female flounder. The timing of hormone treatment is of particular importance to developing reliable hormone techniques for induction of ovulation and the spawning of good quality eggs in teieosts. This is especially true of the winter flounder where a lengthy period of time intervenes between gonad development in the fall and the onset of the normal spawning season beginning in May or June (Harmin, 1991). Although relatively well developed gonads (elevated GSI and large oocyte diameters) exist in female flounder by December, ovulation was not observed in response to GnRH-A treatment until February (Harmin, 199 1). Even in February, the response of females to GnRH-A treatment was variable ( 33% vs 7 1 and 83% in experiments 1 and 4, respectively): by March and May, a greater proportion of hormone-treated females ovulated and the time to ovulation was shortened. This suggests that GnRH-A induction of spawning in the winter flounder is more predictable as the fish approach the time of the normal spawning season. Crim and Glebe ( 1984) reported that when female Atlantic salmon were treated with GnRH-A 45 days prior to the expected time for spawning, a low rate of ovulation (30%) was recorded in hormone-implanted females. In contrast, when the hormone treatment was delayed until 1 month before spawning, 94% of the female salmon responded. Similarly, Fitzpatrick et al. ( 1987) showed that by delaying GnRH-A treatment of’who salmon 1 week (closer to spawning), the percentage of responding females increased. A reliable technique for hormonal induction of spawning depends upon short (h ) and predictable ovulatory responses of females as described for cultured freshwater fish in China (Peter et al., 1988). However, water temperatures are known to affect the timing of ovulation since hormone-induced OVUkition occurs more rapidly at warmer compared to lower water temperatures (Lam, 1982; htuny et al., 1988). At water temperatures ranging between 17 and 32OC. ovulation occurs within 24-48 h after hormone treatment in

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the common sole (Ramos, I986b), Indian carp (Kaul and Rishi, 1986) milkfish (Matte et al., 1988)) goldfish (Sokolowska et al., 1984) and spotted seatrout (Thomas and Boyd, 1988 ). Much longer latency periods to ovulation have been observed for fish held at relatively lower water temperatures I;:s 10°C), e.g. days to ovulation, as reported in the rainbow trout (Crim et al., 1983 ), Atlantic salmon (Crim and Glebe, 1984), and the sablefish (Solar et al., 1.987 ). Although flounder held at S “C ovulated within 13 days after GnRH-A treatment in May, interestingly, during the winter, a latency period of 25 days was found for flounder held at the same temperature. Development of useful techniques to control spawning in fish also requires optimization of hormone delivery methods. In February, some flounder ovulated in response to GnRH-A application both by injections and implantation but a severe increase in mortalities resulted from the handling stress associated with serial hormone injections. On the other hand, GnRH-A implantation offers the advantage of a single time hormone application and has proven effective for inducing spawning in other teleosts including the rainbow trout (Crim et al., 1983), Atlantic salmon (Crim and Glebe, 1984; Crim et al., 1986; Davies et al., 1987), sea bass (Almendras et al., 1988; Harvey et al., 1985; Garcia, 1989, 1990) and milkfish (Lee et al., 1986; Marte et al., 1987 ). Sustained release GnRH-A delivery via implantation technology proves particularly effective where long-term hormone treatment is required, e.g., stimulation of progressive gonad development and/or induction of spawning in fish with prolonged latency periods. Egg quality is a very important concern following successful induction of spawning in teleosts. Although egg fertilization and hatching rates are common indicators of egg quality, survival through the period of larval development and production of healthy fry provide the best indic ( 1985) and Manickam and Joy ( 1989) both reported GnRH-A induction of spawning and production of normal larvae in catfish. For the winter flounder, we noticed that the best egg quality, i.e., highest hatching rates and production of normal-looking larvae, resulted from fertilization of freshly ovulated eggs. Clearly, predictable control of the timing of spawning with GnRH-A treatment improves the chances collection of good quality eggs, thereby reducing potential detrimental effects on larval morphology. Sometimes, we observed low flounder egg fertilization rates but this might have occurred by testing the fertility of poor quality eggs left-over after spontaneous spawning of females. It was suggested that overripening of eggs occurs after hormonal induction of spawning in other fish including the ayu (Hirose et al., 1977) and the Japanese flounder (Hirose et al., 1979 ). In the common sole, Ramos ( 1986a) demonstrated that high doses of HCG lower egg fertilization rates. Furthermore, premature seasonal application of hormone treatment may induce precocious egg maturation and ovuMion ( Fitzpatrick et al., 1984 j causing heavy mortalities in raimbow trout eggs (Grim et

of

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S.A. HARMIN AND L.W. CRIM

al., 1983; Billard et al., 1984), a high proportion of abnormal eggs in Atlantic salmon (Crim and Globe, 1984), and lowered fertility of eggs from coho salmon (Hunter et ;?i., 19b 1; Fitzpatrick et al., 1984) and seabass (Garcia, 1989). Interestingly, the low egg fertility observed after GnRH-A treatment of the grey mullei: (Lee et al., 1987) improved when these fish were spawned with a combination of GnRH-A followed by carp pituitary homogenate. In conclusion, GnRH-A treatment is a useful technique for advancing ovulation/spawning of prespawning female winter flounder; however, the best egg quality can be expected when GnRH-A induced ovulation/spawning is performed near the time of the normal breeding season. ACKNOWLEDGEMENTS

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