Progress in Controlled Maturation and Spawning of Summer

JOURNAL OF THE WORLD AQUACULTURE SOCIETY

Vol. 29, No. 4 December, 1998

Progress in Controlled Maturation and Spawning of Summer Flounder Purulichthys dentutus Broodstock

W A D E 0. WATANABE,' EILEEN ELLIS, AND SIMON c. ELLIS Caribbean Marine Research Center, 805 East 46Ih Place, Vero Beach, Florida 32963 USA

MICHAEL W. FEELEY Marine Sciences and Technology Center, The University of Connecticut, 1084

Shennecossett Road, Groton, Connecticut 06340-6097 USA

Abstract Wild-caught, adult summer flounder Paralichthys dentatus (avg. wt. = 740 g; range = 264-

1,540 g; N = 60), collected in northeastern US coastal waters during October 1994, were transported to Vero Beach, Florida in March 1995 and held in 2.6-m3 indoor tanks through November 1995 under two artificial photothermal regimes: (1) natural regime, simulating natural habitat conditions; and (2) accelerated thermal regime, with seasonal temperature changes advanced by one month. A third group of fish was held in outdoor tanks under ambient photothermal conditions. Under all photothermal conditions, onset of vitellogenesis was associated with declining daylength and temperature, beginning in the accelerated group, then progressing to the natural and the ambient groups. From 20 September to 28 November 1995, 23 vitellogenic stage females from the accelerated and natural regimes were implanted with a cholesterol-cellulose pellet containing LHRH-a (100 pglkg body wt). Females with initial mean oocyte diameters ranging from 258-456 pm spawned voluntarily 2.5-5.5 d post- implantation, while no maturational response was obtained from females with mean diameters ranging from 165-231 pm. Two females were spawned twice during the study period by LHRH-a pellet implantation. Infrequent, natural spawning without hormone intervention was also obtained. Females released from 22.7-396.9 X 1W eggs on the first day of spawning, with fertilization and hatching rates of 0-93.470 and 0-81.1%, respectively.

The summer flounder Paralichrhys dentarus is a high-valued flatfish found in es- tuarine and shelf waters of the Atlantic coast of north America from Nova Scotia to south Florida, with greatest abundance from Cape Cod, Massachusetts to Cape Hatteras, North Carolina (Rogers and Van den Avyle 1983). Declining natural popu- lations of summer flounder (NMFS 1992) and recent advances in commercial culti- vation of similar flatfish species, including turbot Scophthalmus maximus in Europe (Person-Le Ruyet et al. 1991) and the Jap- anese flounder P. olivaceus in Asia (Matsu- oka 1995; Min 1995), has stimulated inter- est in the summer flounder as a candidate

Corresponding author's present address: The Uni- versity of North Carolina at Wilmington, Center for Marine Science Research, 7205 Wrightsville Ave., Wilmington, North Carolina 28403 USA.

for aquaculture (Bisbal and Bengston 1995a; Bengston 1995).

Considerable progress had been made in determining the environmental and feeding requirements for the early life stages of the summer flounder (Bisbal and Bengston 1995a, 1995b), and significant numbers of juveniles have been produced in the laboratory (Bisbal and Bengston 1993, 1995~). Development of cost-effective methods for large-scale production of juveniles remains a bottleneck to commercial development.

Artificial manipulation of photoperiod and temperature is indispensable to commercial production of seedstock of a number of marine fish species, including gilt- head sea bream Sparus aurata (Zohar et al. 1995), sea bass Dicentrarchus labrax (Coves et al. 1991; Carrillo et al. 1995), turbot (Person-Le Ruyet et al. 1991) and Japanese flounder (R. Murashige, Uwajima

0 Copyright by the World Aquaculture Society 1998

393

394 WATANABE ET AL.

Fisheries, personal communication). Little or no published information is available on the influence of these environmental factors on reproduction in summer flounder.

The traditional method (Smigielski 1975) for induced spawning of summer flounder involves daily injection of carp pituitary homogenate (a crude extract containing gonadotropin as well as other hormones and water-soluble compounds) and checking for ovulation before fish are manually strip- spawned, a process requiring up to 2 wk. Excessive handling can reduce spawning success and induce mortality.

Superactive analogues of mammalian gonadotropin releasing hormones, such as [D- Ala6 Des-GlylO] Luteinizing Hormone Re- leasing Hormone analog (LHRH-a), are be- ing used increasingly as spawning agents due to their high potency and low cost (Harmin and Crim 1992; Bromage 1995; Carrillo et al. 1995; Zohar et al. 1995). Acute injection is the conventional mode of LHRH-a administration, but implantation of sustained-release pellets (Crim 1985; Lee et al. 1986a; Sherwood et al. 1988), which stimulate a sustained release of endogenous gonadotropin from the pituitary, have prov- en effective in fish that spawn serially over a protracted season (Almendras et al. 1988; Kelley et al. 1994; Zohar et al. 1995) such as the summer flounder (Morse 1981). Hor- mone implantation reduces handling and can promote voluntary spawning.

The objectives of this preliminary study were to evaluate the feasibility of photothermal control of gonadal maturation of summer flounder broodstock and to deter- mine the efficacy of LHRH-a, administered in sustained-release (pellet implant) form, on voluntary spawning of photothermally- conditioned fish.

Materials and Methods Adult summer flounder (avg. wt. = 740

g; range = 264-1,540 g; N = 60) were collected by trawl during October 1994 off Milford, Connecticut and 'hckerton, New Jersey. Fish were held at these locations

through February 1995 in flow-through seawater tanks under natural photoperiod and under temperatures of 10-12 C.

In March 1995, fish were air-shipped in oxygenated plastic bags to the Caribbean Marine Research Center in Vero Beach, Florida, where this study took place between March and November 1995. Upon receipt, fish were individually tagged and held for 7 d in recirculated seawater (36 g! L) tanks at 11 C and under a photoperiod of 12 E l 2 D.

Maturation by Environmental Control The controlled-environment broodfish

tank system consisted of four circular fiberglass tanks (diam. = 1.85 m; depth = 1.2 m; vol. = 2,646 L) situated in an indoor laboratory. Tanks were divided into two groups, each comprising two tanks supported by a water-recirculating system. Each broodtank was provided with a coni- cal fiberglass cover which was fitted with a timer-controlled, fluorescent fixture containing one 15-watt daylight bulb. Average light intensity at the water surface was approximately 300 lux. Timers were adjusted weekly to simulate seasonal photoperiod changes (Fig. la). Three incandescent ceil- ing fixtures (100-watt) provided indirect il- lumination through a Plexiglas window (30 X 36 cm) on each tank. Incandescent lights were programmed to turn on and off in se- quence beginning 21 min before and after the photophase to simulate dawn and dusk, respectively. ,

Gonadal maturation of broodstock was evaluated under two different artificial photothermal regimes:

Natural regime. To obtain gonadal maturation and spawning in October and No- vember 1995, the peak reproductive period for summer flounder in southern New Eng- land and New Jersey waters (Able et al. 1989), one indoor system was stocked at a density of 12 fish per tank (3.29 kg/m3) and exposed to a photothermal regime (Fig. la) simulating conditions encountered in their natural habitat (Walford and Wicklund

SUMMER FLOUNDER MATURATION AND SPAWNING 395

15

14

13

12

11

10

9

I 0 Acralerated

2o I 350

300

250

200

150

100

50

e Accelerated 0 Natural a Ambient

.- 400 ,

0 1

P' (4)

a

b

C

8Apr &May 7-Jun 7-Jul 6-Aug 5-Sep 5-Oct 4-NOV 4-Dec

Date FIGURE 1. ( a ) Photoperiod and (b) temperature regimes used fo r study of gonadal maturation in summer

Pounder broodstock in 2.6-mJ tanks. Two artiJcial photothermal regimes were studied: ( I ) natural: simulating natural habitat conditions and (2 ) accelerated: in which seasonal temperature changes were advanced by one month. Under the natural and accelerated regimes, daylength and temperature were held constant ajier reaching autumnal levels of 10 h and 17 C, respectively. A third group of fish was held under ambient photothermal conditions in Vero Beach, Florida. (c) Average mean oocyte diameters of female broodstock over the 7-mo study period under the different photothermal regimes. Plotted points represent means ( Z S E ) while numbers in parentheses denote number of individuals sampled.

396 WATANABE ET AL

1968; Smith 1973; Rogers and Van Den Avyle 1983; US Naval Observatory 1993).

Accelerated thermal regime. To obtain early maturation and spawning in Septem- ber 1995, the other indoor system was stocked at densities of 12 and 15 fish per tank (3.29 and 4.11 kg/m3) and exposed to the same photoperiod conditions as in the natural regime, but with seasonal temperature changes advanced by one month (Fig. lb). The “accelerated thermal” regime is hereafter referred to as the “accelerated” regime for brevity. Under both artificial regimes, photoperiod and temperatures were held constant after declining daylength and temperatures reached 10 h and 17 C, respectively (Figs. la, b).

In addition to the artificial photothermal regimes, a third group of fish was maintained in two outdoor tanks at densities of 4 and 5 fish per tank (0.91 and 1.13 kg/m3) under ambient photothermal conditions (Figs. la, b). Outdoor tanks were shaded with four layers of 70% light-occluding cloth and were supplied with flow-through seawater, pumped from a near-shore location adjacent to the laboratory.

Sex ratios of fish were unknown at the time of stocking in March. Gonadal matu- rity of individual brooders was assessed at 6-wk intervals during the non-reproductive period and at approximately 2-wk intervals in-season by biopsy of anesthetized (0.5 g/ L 2-phenoxyethanol) fish, using a polyeth- ylene cannula (1.57-mm 0.d. X 1.14-mm i.d.) (Shehadeh et al. 1973). Ovarian samples were fixed in a solution of 10% for- malin in seawater. Using a compound mi- croscope fitted with an ocular micrometer, the diameters of at least 100 oocytes were measured to the nearest 50 pm, from which a mean and standard deviation were calcu- lated. General stage of oocyte development (i.e., pre-vitellogenic, cortical vesicle, vitellogenic and atretic) was determined from microscopic appearance according to Kuo et al. (1974a). Males were identified by the presence of milt when pressure was applied to the gonadal area. Fish were weighed and

measured (total length = TL) after sampling.

Fish were fed to satiation once daily (approximately 0900), a diet that alternated between Atlantic silversides Menidia menidia, squid, krill and a pelleted vitamin supplement (Zeigler Bros. Inc., Pennsylvania, USA).

LHRH-a Pellet Implantation LHRH-a pellets were implanted between

0900 and 1200 into females with mean vitellogenic oocyte diameters of at least 164 pm. Anesthetized females were implanted intramuscularly on the ocular side with a single 95% cholesterol-5% cellulose pellet (Sherwood et al. 1988) containing luteinizing hormone releasing hormone [D-Ala6 Des-GlylOl-LH-RH Ethylamide) (Sigma Chemical Co., Missouri, USA) at a nominal dose rate of 100 (range = 79.8-124) ygkg body wt. Females were returned to their broodtank after implantation and not han- dled thereafter.

Since females released eggs voluntarily, strip-spawning was not required. An egg collector, consisting of a 250-pm mesh polyester fabric (Nytex) bag situated in the sump tank, was checked five times daily for spawned eggs. Total numbers of eggs spawned (fecundity) was estimated using volumetric methods, and buoyant eggs were transferred to a 15-L incubator containing seawater at 17-20 C. Fertilization and hatching rates were expressed as percentages of total eggs and of buoyant eggs. Af- ter spawning, implanted females were sampled by cannulation to confirm the presence of fully mature (ripe), hydrated oocytes (Fig. 2c).

Water Quality Temperature, salinity and dissolved oxy-

gen were monitored daily, while pH, total ammonia-nitrogen, nitrite and nitrate were monitored twice weekly. Average daily val- ues (and ranges) over all treatments were as follows: salinity, 36.1 (32-38) g/L; dissolved oxygen, 6.89 (6.30-7.22) mgL; pH,


7.95 (7.86-8.14); total ammonia-nitrogen, 0.144 (0.042-0.185) m a ; nitrite-nitrogen, 0.135 (0.0014.201) mg/L; nitrate-nitrogen, 0.233 (0.0704.320) mgk. Water temperature in indoor tanks was maintained within 20.2 C of the prescribed level (Fig. lb).

Statistics Mean oocyte diameters of females on a

given sampling date were compared between photothermal regimes by analysis of variance. Percentages of vitellogenic females were compared by 2 X 2 G-test (So- kal and Rohlf 1981).

Results Gonadal Maturation by Environmental Control

Only pre-vitellogenic stage oocytes (< 150 pm diameter) were observed from 6 June through 28 July (Fig. lc), a period during which daylengths and temperatures were near annual maxima of 13.9-15 h and 24-30 C, respectively (Figs. la, b). Mean oocyte diameter averaged 62 pm under all photothermal regimes from June through July (Fig. lc).

Females with yolk vesicle (approximately 150-200 pm diam.) and vitellogenic- stage oocytes (approximately 200 pm diam. and above) were first observed on 30 Au- gust in both the accelerated and natural regimes, although the percentage of vitellogenic females was markedly higher (P < 0.001) in the accelerated (80%) than in the natural regime (11.8%). Mean oocyte diameter on 30 August remained low among all groups, averaging 69 pm (Fig. lc).

By 25 September, 100% of females under the accelerated and natural regimes were vitellogenic. Mean oocyte diameter among females in the accelerated group (252 pm) was higher (P < 0.01) than among those in the natural group (168 pm) (Fig. lc).

By 10 October, temperatures in both the accelerated and natural regimes were near- ing convergence at 17 C (Fig. lb). Mean oocyte diameter among females in the accelerated regime (254 pm) was not signif-

3OAug rrrly vltellogenlc

x = 63.3 pm 20 10 0

25 125 225 325 425 525 625 725 825 925

60 14Sept 6 d pre4mplant " 1 - x = 235 pm

30 b

$ i i , ,m,, , , , , , . , , , , , x o 8 + 0

25 125 225 325 425 525 625 725 825 925

x = 457 pm

10 0 : 25 125 225 325 425 525 625 725 025 925

60 Il-OCt

30 d

second lmplrntation

1.. . . . I . I

25 125 225 325 425 525 625 725 825 925

Oocyte diameter (pm)

FIGURE 2. Oocyte diameter-frequency distribution in a female summerjounder (#2) at various stages of photothermully-induced maturation- and LHRH-a induced spawning: (a) early vitellogenic, (b) 6-d pre-implantation, ( c ) post-spawning, and (d) second implantaiion. Distributions are based on samples of =I00 oocytes. In Figs. 2b, c, and d, pre-vitellogenic oocytes < I 0 0 pm, were not included.

icantly different from those in the natural regime (225 pm) (Fig. lc).

On 10 October, 75% of females held under ambient conditions were vitellogenic, and mean oocyte diameter increased to 138 pm (Fig. lc), considerably smaller (P < 0.01) than among those in the artificial re-

398 WATANABE ET AL.

gimes. Onset of vitellogenesis of fish held under ambient conditions was also correlated with declining daylength in early Octo- ber (Fig. la), but under high prevailing temperatures (27.3-28.8 C) (Fig. lb).

By 7 November, mean oocyte diameters were not significantly different between females in the accelerated and natural photothermal regimes reaching 322 and 349 pm, respectively (Fig. lc). On this date, 83.3% of females held under ambient conditions were vitellogenic, and mean oocyte diameter increased to 288 pm (Fig. lc).

Seasonal changes in oocyte diameter-frequency distribution of a representative female (#2) are shown in Fig. 2. From June through July, a unimodal frequency distribution, consisting of pre-vitellogenic stage oocytes (C150 pm), was evident among females in all groups. By 30 August, yolk vesicle and vitellogenic stage oocytes were evident among females in both the accelerated and natural regimes, although mean oocyte diameter remained low (Fig. 2a) due to a predominance of pre-vitellogenic oocytes. By 14 September, vitellogenic stage oocytes were increasing in frequency and in size (Fig 2b). and mean oocyte diameter increased to 235 pm.

Spermiation in males was first observed on 14 September in the accelerated regime and on 10 October in the natural regime. No spermiation was observed among fish held under ambient conditions.

Growth and Sex Ratios of Broodstock

Sex ratios in each photothermal regime were as follows: 23 females: 4 males (accelerated), 20 females: 4 males (natural) and 6 females: 0 males: 3 undetermined (ambient). During the period 26 April to 7 November, average TL, weight and biomass density of fish under all regimes increased from 39.1 cm, 694 g and 2.72 kg/m3, respectively, to 42.1 cm, 1,057 g and 4.28 kg/ m3. On 7 November, males averaged 37.6 (33.2-42.1) cm and 690 (472-980) g, significantly ( P < 0.01) smaller than females,

which averaged 44.4 (38.1-52.3) cm and 1,244 (628-2,070) g.

LJYRH-a Pellet Implantation During the period 20 September to 28

November 1995, 23 females from the accelerated and natural photothermal regimes were implanted with LHRH-a. Voluntary spawning (i.e., spontaneous release of hydrated eggs) following pellet implantation was obtained within 2.5-5.5 d in 13 trials from females with initial mean oocyte diameters ranging from 258-456 pm (Table 1). A majority of spawnings occured around midnight. No response to LHRH-a treatment was observed among 10 females with mean oocyte diameters ranging from 165- 231 pm. Mean oocyte diameter among these females, implanted on 20 September, did not differ significantly from that of non- implanted females through 7 November (Fig. 1).

Fecundity and Frequency of Spawning Females implanted with LHRH-a re-

leased from 27.7-396.9 X lo3 (avg. = 111.2 X lo3; 78.9 X 103/kg body wt.) eggs on the first day of spawning (Table l), then continued to spawn generally diminishing quantities of eggs daily for up to 4 wk. Fer- tilization rates of secondary spawnings were poor and seldom quantified.

The diameter-frequency distribution of oocytes sampled within 24 h of the initial spawning was typically multi-modal (Fig. 2c), generally characterized by two modes of smaller, vitellogenic stage oocytes up to 625 pm and an advanced mode of hydrated oocytes ranging from 725-975 pm (Fig. 2c). Diameter of fertilized eggs following water hardening was 973 2 1 pm (mean ? SE) and ranged from 881 to 1,034 pm. TWO females from the accelerated regime

(#1 and #2) were induced to spawn twice during the study period (Table 1). On 11 October, 16 d after their first spawning, both females were implanted a second time with a single LHRH-a pellet, with spawning obtained 5.5 d post-implantation.


For female #2, a bimodal oocyte diameter-frequency distribution with an overall mean oocyte diameter of 400 pm was observed on 11 October, the date of the second implantation (Fig. 2d). Hydrated oocytes seen on 25 September (Fig. 2c) were not evident by this date.

Egg Buoyancy, Fertilization and Hatching Fertilization rates following LHRH-a-in-

duced spawning varied from 0 to 93.4% (Table 1). l b o spawnings produced high overall fertilization rates of 81.1-93.4%, one spawning produced a moderate fertilization rate of 37.6%, while nine spawnings produced poor fertilization rates of 0- 15.3%. No correlation between fertilization rate and initial mean oocyte diameter was evident.

During incubation of eggs in full- strength seawater (36-38 g/L), the percentage of eggs that remained buoyant when aeration was suspended varied among spawnings from 0-94.3% (Table I), and was directly proportional to fertilization rate. This relationship could be described by the linear regression, y = 0.9102~ + 10.392 ($ = 0.807, N = 11, P < 0.01), where y is the buoyancy rate (%) and x is the fertilization rate (%).

Hatching rate (overall) varied among spawnings from 0-87.4%, generally increasing with higher fertilization rate (Table 1). This trend could be described by the linear regression, y = 0 . 8 1 5 ~ - 6.0651 (N = 10, ? = 0.832, P < 0.01), where y is the hatching rate (%) and x is the fertilization rate (%).

Natural Spawnings Natural spawning without hormone im-

plantation was observed on 12, 13 and 27 November in one broodtank under the natural photothermal regime (Table 1). None of the females in this tank had been treated with LHRH-a. Mean oocyte diameters of females sampled 4-5 d prior to the date of the first natural spawning ranged from 265- 413 pm (Table 1). Numbers of eggs re-

leased during natural spawning ranged from 22.7 to 69.2 X lo3, while overall fertilization and hatching rates ranged from 5.64 to 50.4% and from 5.5 to 22.7%, respectively.

Discussion Gonadal Maturation by Environmental Control

In this study, the first appearance of vitellogenic stage oocytes among female summer flounder broodstock under both artificial photothermal regimes occured in as- sociation with declining daylength and temperature (Figs. la, b) which simulated natural habitat conditions during the pre-mi- gratory period (Smith 1973). This is in accord with previous studies showing that short or decreasing photoperiod and/or low or decreasing temperature are stimulatory to gametogenesis in marine finfish species which spawn in autumn and winter, including grey mullet Mugil cephalus (Kuo et al. 1974b), red drum Sciaenops ocellutus (Ar- nold 1988), California halibut (Caddell et al. 1990) and sea bass (Canillo et al. 1995).

Under ambient photothermal conditions in Vero Beach, Florida, a location outside the ecological range for reproduction of summer flounder (Smith 1973; Able et al. 1989), vitellogenic females were also first observed under declining daylength (10 Oc- tober; Fig. la), but while high temperatures (28.4 C) prevailed (Fig. lb). This suggests that declining photoperiod may play a more dominant role than temperature in trigger- ing vitellogenesis in summer flounder. On the other hand, more rapid maturation of females in the accelerated than in the natural regime (Fig. lc) under identical photoperiod conditions (Fig. la), indicates that rate of vitellogenesis was modulated by temperature. In grey mullet, a winter spawning species, a short photoperiod (6 L: 18 D) initiated vitellogenesis, but the mag- nitude of the response was greater at 17-21 C than at 24-26 C (Kuo et al. 1974b). Stud- ies which dissociate the effects of temperature and photoperiod on gonadal recrudes-

400 WATANABE ET AL.

TABLE 1. Summarized data on hormone-induced and natural spawning of F? dentatus under accelerated and natural photothermal regimes.

Date of Initial mean implant Body wt. Photothermal oocyte

Tank No. (1995) Fish I.D. (kg) regime Treatment diameter ( p n )

3 9/20 1 1.52 Accelerated LHRH-a 301

4 3

4

3 2 1 1 1 3 2 1

9120 1011 1

1011 1

10123 10123

nae na na

11/25 11/25 I1128

2 I d

3 2d 4 5 6

nd nd nd I 8 9

I .35 1.54, I .22 1.41, 1.29 1.46 1.52 nd nd nd

1.53 1.93 1.24

Accelerated Accelerated

Accelerated

Accelerated Natural Natural Natural Natural Accelerated Natural Natural

LHRH-a LHRH-a

LHRH-a

LHRH-a LHRH-a control control control LHRH-a LHRH-a LHRH-a

258 360, 40 1 400, 311 33 1 456 265-4 13'

nd 432.5 423 362

2 11/28 10 1.24 Natural LHRH-a 353.4 3 11128 I 1 1.08 Accelerated LHRH-a 398.6

a d.p.i. = days post-implantation. For natural spawnings, date of spawning (1995) is shown. value represents time of secondary spawn. nd = no data. combined data for two females. na = not applicable.

'range of mean oocyte diameters of females in tank recorded 4-5 d before spawning.

cence in summer flounder may lead to sim- pler procedures, such as constant temperature regimes, for controlled reproduction in this species.

Patterns of ovarian maturation of summer flounder females under the different photothermal regimes (Fig. lc) were consistent with the seasonally-changing photothermal conditions, progressing from the accelerated regime to the natural and then the ambient regimes. These results demonstrate that photothermal manipulation enables precise control of gonadal recrudescence in this species.

M R H - a Pellet Implantation In this study, a single application of

LHRH-a in sustained-release form was highly effective in stimulating final maturation and ovulation in female summer

flounder, in agreement with what was reported earlier in this species (Berlinsky et al. 1997). as well as in the southern P. lethostigma (Berlinsky et al. 1996) and winter Pseudopleuronectes americanus (Harmin and Crim 1992) flounders, and in other tel- eosts (Harvey et al. 1985; Lee et al. 1986c; Almendras et al. 1988; Hodson and Sulli- van 1993). In contrast to the earlier study with summer flounder, which involved re- petitive handling and daily strip-spawning (Berlinsky et al. 1997), females in t h i s study spawned voluntarily, probably due to a longer acclimation period (12 vs. 3 mo) to captive conditions and minimal handling. The high fertilization rates (81.1-93.4%) (Table 1) attained in at least two trials in this study may well be attributable to voluntary spawning, which precludes the need to judge time of stripping in relation to ovu-

SUMMER FLOUNDER MATURATION AND SPAWNING 40 1

TABLE 1. Extended.

Fertiliz- ation Hatching Hatching

Fertiliz- rate rate rate Time of Fecundity ation Floaters Floaters (% of (% of (% of

spawn d.p.i." (XlO') (% overall) (XlW) (%) floaters) floaters) overall)

4.5 5.56 4.5 5.5

5.5

4.5 3.5

11/12 1 1/13b 1 1/27 2.5 2.5 5.5 6Sb 4.5 4.5

27.7 40.1 25.9

251.1d

1 16.9d

118 170.9 69.2 22.7 33.3

140 396.9

14.2 106.7 83.2

8.54

93.4 11.7 0 3.83

15.3

0 37.6 50.4

5.64 35.7 0 4 4 9.9

81.1 3.1

23.0 ndc 0

132.6

28.6

0 52.7 34.9 nd 11.6 0

nd 0.349

nd 96.1 nd

94.3 nd 0

52.8

38.5

0 38.8 50.4 nd

37.9 0 nd 4.1 nd

nd 90

99 nd 0 7.25

39.7

0 96.8 nd nd

94.3 0 nd

97.9 nd

90. I nd

93.6 0 0 nd

2.29

0 83.6 11 nd

63.6 0 nd nd nd

70.3 nd

87.4 0 0 nd

0.35

0 31.4 5.54 nd

22.7 0 nd nd nd

nd 57

lation, a factor critical to fertilization success (Shelton 1989).

An optimum initial mean oocyte diameter for induced spawning by LHRH-a pellet implantation was not determined, since vitellogenic females with a wide range of initial mean oocyte diameters of 258-456 pm showed a rapid ovulatory response, spawning within 2.5-5.5 d of treatment. A multi- modal egg diameter-frequency distribution (Fig. 2c), comprising ripe as well as early, mid- and late-vitellogenic stage oocytes, was evident in implanted fish for up to 4 wk after spawning, and two females were induced to spawn twice within a single season. These findings are consistent with a continuous recruitment of ripe eggs from a pool of smaller, vitellogenic oocytes and with a serial spawning characteristic of summer flounder in nature (Morse 1981; Berlinsky et al. 1997).

In this study, LHRH-a implantation of vitellogenic females with relatively small mean oocyte diameters ranging from 165- 231 km did not accelerate oocyte growth

compared to non-implanted females. This is consistent with what was reported in an earlier study (Berlinsky et al. 1997) in which LHRH-a and HCG did not stimulate oocyte growth in summer flounder females with maximum follicle diameters ranging from 180-435 pm, while carp pituitary extract was effective. These results suggest that LHRH-a alone may not be sufficient for stimulation of vitellogenesis in summer flounder. In milkiish Chanos chanos, a combination of LHRH-a pellet and liquid 17 a-methyltestosterone therapy was supe- rior to LHRH-a alone in stimulating ovarian maturation, probably due to a stimulatory effect of 17 a-methyltestosterone on gonadotropin production by the pituitary (Crim and Evans 1979; Lee et al. 1986b).

In this study, a circadian ovulatory rhythm was evident, with voluntary spawning occuring around midnight. Fertilization success of secondarily spawned egg batches was poor (Berlinsky et al. 1997), suggesting a decline in egg quality after the initial spawning. The LHRH-a pellet matrix used

402 WATANABE ET AL.

in this study (95% cholesterol-5% cellulose), which releases only 38% of the analogue after 28 d (Sherwood et al. 1988), may be inappropriate relative to the pattern of ovarian development in summer flounder. LHRH-a pellets containing higher pro- portions of cellulose, which release the analogue at faster rates (Sherwood et al. 1988), may produce a more concentrated ovulatory response, improving egg quality, and minimizing wastage of gametes.

Production of viable embryos (i.e., fecundity X overall hatching rate), is a practical measure of spawning success. In female #3 (Table I), high fertilization (93.4%) and hatching (87.4%) rates in conjunction with moderate fecundity (27.7 X lo3) produced 24,210 viable embryos. On the other hand, in female #17 (Table l), considerably lower fertilization (8 1.1 %) and hatching (57%) rates in conjunction with high fecundity (106.7 X lo3), produced substantially higher numbers of viable embryos (60,819). The results support the practical application of LHRH-a pellet implants as a spawning device in summer flounder, if predictable rates of ovulation, as well as of fertilization and hatching can be achieved. A clearer understanding of the re- lationships between brooder size, oocyte diameter-frequency distribution, pattern of LHRH-administration, and the recruitment of ripe oocytes from the vitellogenic pool is needed.

Maximum fertilization success under natural, non-induced spawning in this study was only 50.4%. Since natural spawning precludes the effects of handling stress and hormonal intervention on egg quality (Lam 1994; Lee et al. 1987). variable fertilization rates among trials may have been related in part to inconsistent performance by males, due to a lack of participation in spawning (Henderson-Arzapalo et al. 1988), inade- quate spermiation (Bengston 1995; Berlin- sky et al. 1997). or low male: female ratios. The implantation of males with sustained- release pellets containing both LHRH-a and 17 a-methyltestosterone (Lee et al. 1986 a,

1986b; Lee and Tamaru 1988) and higher ratios of male:female brooders merit eval- uation.

In this study, egg buoyancy and hatching rate were positively correlated with fertilization rate. This is consistent with the well- known observation that buoyancy in pelag- ic eggs is often better for good than for poor quality eggs (Kjorsvik et al. 1990) and sup- ports the hypothesis that variable fertilization rates may have also been related to egg quality as influenced by factors (e.g., nutri- tional) other than handling stress or hormonal intervention.

Since natural spawning without hormonal intervention was obtainable in wild- caught broodstock after one year in captivity, such spawnings are likely to become more prevalent with longer-term acclimation and with domestication. While addi- tional work is needed to improve and stan- dardize these procedures, the results of this study suggest that natural and LHRH-a-induced spawning of photothermally-conditioned fish are potentially valuable techniques to help meet increasing year-round demands for summer flounder seedstock.

Acknowledgments This work was supported by a grant from

the University of Connecticut, Marine Sci- ences and Technology Center. We thank Anthony Calabrese and David Nelson (Na- tional Marine Fisheries Service, Milford, Connecticut), Kenneth Able (Rutgers Uni- versity) and Anne Studholme (NMFS, Sandy Hook, New Jersey) for assistance in acquisition of summer flounder broodstock, the Florida Institute of Technology for fa- cilities support, Ryan Murashige (Uwajima Fisheries, Hawaii), Satoru Matsuoka and Tsuneo Morizane (Ehime Prefectural Fish- eries Experimental Station, Uwajima, Ja- pan), Bori Olla (NMFS, Newport, Oregon), David Bengston (University of Rhode Is- land), Elliot Finkel and Richard Cooper (University of Connecticut) for helpful ad- vice.

SUMMER FLOUNDER MATURATION AND SPAWNlNG 403

Literature Cited Able, K. W., R. E. Matheson, W. W. Morse, M. P.

Fahay and G. Shepherd. 1989. Patterns of summer flounder Paralichthys dentatu.s early life history in the Mid-Atlantic Bight and New Jersey estuaries. Fishery Bulletin, U.S. 88: 1-12.

Almendras, J. M., C. Duenas, J. Nacario, N. M. Sherwood and L. W. Crim. 1988. Sustained hormone release. 111. Use of gonadotropin releasing hormone analogues to induce multiple spawnings in sea bass, Lates calcarifer. Aquaculture 74:97- 1 1 1 .

Arnold, C. E. 1988. Controlled year-round spawning of red drum, Sciaenops ocellatus, in captivity. Contributions in Marine Science (Supplement to Volume 30):65-70.

Bengston, D. A. 1995. Summer flounder, Paralichthys dentatus: a potentially valuable new aquaculture species for the Northeastern United States. Book of Abstracts, Aquaculture '95: 59. World Aqua- culture Society, San Diego, California, USA.

Berlinsky, D. L., W. King V, R. G. Hodson and C. V. Sullivan. 1997. Hormone induced spawning of summer flounder Paralichthys dentatus. Journal of the World Aquaculture Society 28:79-86.

Berlinsky, D. L., W. King V., T. I. J. Smith, R. D. Hamilton 11, J. Holloway Jr. and C. V. Sulli- van. 1996. Induced ovulation of southern flounder Paralicthys lethostigma using gonadotropin releasing hormone analogue implants. Journal of the World Aquaculture Society 27: 143-152.

Bisbal, G. A. and D. A. Bengston. 1993. Reversed asymmetry in laboratory-reared summer flounder. The Progressive Fish-Culturist 5: 106-108.

Bisbal, G. A. and D. A. Bengston. 1995a. Description of the starving condition in summer flounder, Par- alichrhys dentatus, early life history stages. Fish- ery Bulletin 93:217-230.

Bisbal, G. A. and D. A. Bengston. 1995b. Effects of delayed feeding on survival and growth of summer flounder Paralichthys dentatus larvae. Marine Ecology Progress Series 121:301-306.

Bisbal, G. A. and D. A. Bengston. 199%. Develop- ment of the digestive tract in larval summer flounder. Journal of Fish Biology 47:277-291.

Bromage, N. R. 1995. Broodstock management and seed quality-general considerations. Pages 1-24 in N. R. Bromage and R. J. Roberts, editors. Broodstock management and egg and larval quality. Blackwell Science Ltd., Oxford, UK.

Caddell, S. M., D. M. Gadomski and L. R. Abbot. 1990. Induced spawning of the California halibut, Paralichthys californicus (Pisces: Paralichthydae), under artificial and natural conditions. Pages 175- 198 in C. W. Haugen, editor. The California halibut, Paralichthys californicus, resources and fisheries. Fish Bulletin 174 California Department of Fish and Game, Sacramento, California, USA.

Carrillo, M., S. Zanuy, F. Rat, J. Cerda, J. Ramos, E. Mananos and N. Bromage. 1995. Sea Bass (Dicentrarchus labrax). Pages 138-168 in N. R. Bromage and R. J. Roberts, editors. Broodstock management and egg and larval quality. Black- well Science Ltd., Oxford, UK.

Coves, D., G. Dewavrin, G. Breuil and N. Devau- chelle. 1991. Culture of sea bass (Dicentrarchus labrax L.). Pages 3-20 in J. P. McVey, editor. CRC handbook of mariculture, volume 11. Finfish aquaculture. CRC Press, Boca Raton, Florida, USA.

Crim, L. W. 1985. Methods for acute and chronic hormone administration in fish. Pages 1-13 in C.- S. Lee and 1.C. Liao, editors. Reproduction and culture of milkfish. Oceanic Institute, Hawaii and Tungkang Marine Laboratory, Taiwan.

Crim, L. W. and D. M. Evans. 1979. Stimulation of pituitary gonadotropin by testosterone in juvenile rainbow trout (Salrno gairdneri). General and Comparative Endocrinology 37: 192-196.

Harmin, S. and L. Crim. 1992. Gonadotropic hormone-releasing hormone analog (GnRH-A) induced ovulation and spawning in female winter flounder, Pseudopleuronectes americanus (Wal- baum). Aquaculture 104:375-390.

Harvey, B., J. Nacario, L. W. Crim, J. V. Juario and C. L. Marte. 1985. Induced spawning of sea bass, Lates calcarifir and rabbitfish, Siganus gut- tatus, after implantation of pelleted LHRH analogue. Aquaculture 47:53-59.

Henderson-Arzapalo, A., R. L. Colura and A. F. Maciorowski. 1988. Temperature and photoperiod induced maturation of southern flounder. Man- agement data series number 154. Texas Parks and Wildlife Department, Austin, Texas, USA.

Hodson, R. G. and C. V. Sullivan. 1993. Induced spawning of domestic and wild striped bass, Mo- rone saxatilis (Walbaum), broodstock with implanted. GnRH analogue and injected hCG. Aqua- culture and Fisheries Management 24:389-398.

Kelley, C. D., A. Moriwake, G. Miyamoto, V. Nicol and W. 0. Watanabe. 1994. The use of LHRH- a for induced spawning of five different species of marine teleost fishes. Book of Abstracts, World Aquaculture '94: 147. World Aquaculture Society, New Orleans, Louisiana, USA.

Kjorsvik, E., A. Mangor-Jensen and I. Holmefjord. 1990. Egg quality in fishes. Advances in Marine Biology 26:71-113.

Kuo, C.-M., C. E. Nash and Z. H. Shehadeh. 1974a. A procedural guide to induce spawning in grey mullet (Mugil cephalus L.). Aquaculture 3: 1-14.

Kuo, C.-M., C. E. Nash and Z. H. Shehadeh. 1974b. The effects of temperature and photoperiod on ovarian development in captive grey mullet (Mu- gil cephalus L.). Aquaculture 3:25-43.

Lam, T. J. 1994. Hormone and egg/larval quality in

404 WATANABE ET AL.

fish. Journal of the World Aquaculture Society 25: 2-12.

Lee, C.-S. and C. S. Tamaru. 1988. Advances and future prospects of controlled maturation and spawning of grey mullet (Mugil cephalus L.) in captivity. Aquaculture 74:63-73.

Lee, C.-S. and C. S. Tamaru and C. D. Kelley. 1986a. Technique for making chronic-release LHRH-a and 17 alpha-methyltestosterone pellets for intramuscular implantation in fishes. Aquacul- ture 59:161-168.

Lee, C.-S., C. S. Tamaru, J. E. Banno and C. D. Kelley. 1986b. Influence of chronic administration of LHRH-analogue and/or 17a-methyltestosterone on maturation in milkfish, Chanos chanos. Aqua- culture 59: 147-159.

Lee, C.-S., C. S. Tamaru, C. D. Kelley and J. E. Banno. 1986c. Induced spawning of milkfish, Chanos chanos, by a single application of LHRH- analogue. Aquaculture 58237-98.

Lee, C.-S., C. S. Tamaru, G. T. Miyamoto and C. D. Kelley. 1987. Induced spawning of grey mullet (Mugil cephalus) by LHRH-a. Aquaculture 62:

Matsuoka, S. 1995. A review of the nursery and growout culture techniques for flounder (Parali- chthys olivaceus) in Japan. Pages 139-145 in K. L. Main and C. Rosenfeld, editors. Culture of high-value marine fishes in Asia and the United States. The Oceanic Institute, Honolulu, Hawaii, USA.

Min, B. S. 1995. A review of the nursery and growout culture techniques for the flounder (Paralichrhys olivaceus) in Korea. Pages 147-152 in K. L. Main and C. Rosenfeld, editors. Culture of high-value marine fishes in Asia and the United States. The Oceanic Institute, Honolulu, Hawaii, USA.

Morse, W. W. 1981. Reproduction of the summer flounder, Paralichrhys dentarus (L.). Journal of Fish Biology 19:189-203.

NMFS. 1992. Status of fishery resources off the Northeastern United States for 1992. NOAA tech- nical memorandum NMFS-F/NEC-95. October 1992. Conservation and Utilization Division, Northeast Fisheries Science Center, Woods Hole, Massachussetts, USA.

Person-Le Ruyet, J., F. Baudin-Laurencin, N. De- vauchelle, R. Mebiller, J.-L. Nicolas, J. Robin

327-336.

and J. Guillaume. 1991. Culture of turbot (Scop- rhalmus maximus). Pages 21-41 in J. I? McVey, editor. CRC handbook of mariculture, volume 11. Finfish aquaculture. CRC Press, Boca Raton, Flor- ida, USA.

Rogers, S. G. and M. J. Van den Avyle. 1983. Spe- cies profiles: Life histories and environmental requirements of coastal fishes and invertebrates (South-Atlantic). Summer flounder. FWS/OBS-82/ 11.15, TR EL-82-4, October 1983.

Shehadeh, Z. H., C.-M. Kuo and K. K. Milisen. 1973. Validation of an in vivo method for moni- toring ovarian development in the grey mullet (Mugil cephalus L.). Journal of Fish Biology 5 :

Shelton, W. L. 1989. Management of finfish reproduction in aquaculture. Aquatic Science 1989: 497-535.

Sherwood, N. M., L. W. Crim, J. Carolsfeld and S. M. Walters. 1988. Sustained hormone release. I. Characteristics of in vitro release of gonadotropin- releasing hormone analogue (GnRH-A) from pellets. Aquaculture 74:75-86.

SmigielsM, A. S. 1975. Hormone-induced spawning of the summer flounder and rearing of larvae in the laboratory. Progressive Fish-Culturist 37:3-8.

Smith, W. G. 1973. The distribution of summer flounder, Paralichrhys dentatus, eggs and larvae on the continental shelf between Cape Cod and Cape Lookout, 1965-66. Fishery Bulletin US. Fish and Wildlife Service 7 1527-548.

Sokal, R. R. and F. J. Rohlf. 1981. Biometry: The principles and practice of statistics in biological research. W. H. Freeman and Company, New York, USA.

U.S. Naval Observatory. 1993. The nautical almanac for the year 1994. Nautical Almanac Office, U.S. Government Printing Office, Washington, D.C., USA.

Walford, L. A. and R. I. Wicklund. 1968. Monthly sea temperature structure from the Florida Keys to Cape Cod. Serial atlas of the marine environment, folio 15. American Geographical Society New York, New York, USA.

Zohar, Y., M. Harel, S. Hassin and A. Tandler. 1995. Gilt-head sea bream (Sparus aurafa). Pages 94-117 in N. R. Bromage and R. J. Roberts, editors. Broodstock management and egg and larval quality. Blackwell Science Ltd., Oxford, UK.

479-487.

Documents

Progress in Controlled Maturation and Spawning of Summer