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The use of luteinizing hormone releasing hormone analogue for ovulation induction in black sea bass (Centropristis striata)
Aquaculture 250 (2005) 813 – 822 www.elsevier.com/locate/aqua-online
The use of luteinizing hormone releasing hormone analogue for ovulation induction in black sea bass (Centropristis striata) David L. Berlinskya,*, William King V a, Theodore I.J. Smithb a Department of Zoology, University of New Hampshire, Durham, NH 03824, USA South Carolina Department of Natural Resources, Marine Resources Research Institute, Charleston, SC 29412, USA
b
Received 11 January 2005; received in revised form 5 April 2005; accepted 20 April 2005
Abstract Interest in commercial production of black sea bass has increased in recent years, but reliable spawning methods remain problematic. The objective of this study was to evaluate the effects of oocyte size and luteinizing hormone releasing hormone analogue (LHRHa) dosage and delivery systems on ovulatory success for in vitro fertilization. Vitellogenic females with maximum oocyte diameters of 400–625 Am were implanted with a 95% cholesterol–5% cellulose pellet containing 50 Ag of LHRHa. Fish with maximum oocyte diameters b 450 Am failed to ovulate. In contrast, 90% of fish with 500 Am oocytes spawned within 36 h and 40% of this group ovulated a second time. All of the females containing oocytes N 550 Am ovulated. In a second experiment, females with uniformly vitellogenic oocytes (N500 Am) and implanted with 50 Ag LHRHa ovulated substantial numbers of eggs (45,000–192,000 eggs/kg body weight (BW), but fertility was consistently low (0– 15%). In a third experiment, 19 of 39 females receiving implants containing 6.3–23.6 Ag LHRHa/kg BW during the spawning season ovulated, but fecundity (17,000–339,000 eggs/kg) and fertilization (0–98%) were highly variable. When fish were grouped by developmental index, calculated as the number of oocytes with diameters N 400 Am/total number of oocytes measured, there were no statistical differences among groups with respect to the number of spawns, fecundity or fertilization success. In a fourth experiment, 11 of 13 females with a clutch of fully vitellogenic oocytes that were injected with 20 or 100 Ag/kg BW LHRHa ovulated between 1 and 2 times on consecutive days. Five of seven given an implant containing 12.5 Ag LHRHa ovulated one or more times. Fish implanted with shams or injected with vehicle alone did not ovulate in any of the experiments. No differences were found in the number of spawns, fecundity or fertilization success from fish receiving different doses of injected or implanted LHRHa. Incubation of pooled eggs produced 155,000 larvae (60% hatch) and 95,000 one-gram juveniles. These results demonstrate that injected or implanted LHRHa is effective for inducing ovulation in black sea bass with maximum oocyte diameters N 500 Am. D 2005 Elsevier B.V. All rights reserved. Keywords: Black sea bass; Centropristis striata; Induced ovulation; LHRHa
* Corresponding author. Tel.: +1 603 862 0007; fax: +1 603 862 3784. E-mail address: [email protected] (D.L. Berlinsky). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.04.074
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1. Introduction Black sea bass (Centropristis striata) are found in waters along the Atlantic coast from the Gulf of Maine to northern Florida, and a subspecies inhabits the eastern Gulf of Mexico. Throughout their range this species supports important commercial and recreational fisheries (Musick and Mercer, 1977; Low, 1981). Historically, the greatest abundance along the Atlantic coast occurred in the Mid-Atlantic region, where fish were commercially harvested in traps during the summer and by otter trawls during the winter when they aggregated offshore in deeper waters (Frame and Pearce, 1973). Currently, populations are overexploited in some areas of this region and a variety of management strategies have been implemented in an attempt to maintain sustainable fisheries (Shepherd and Terceiro, 1994). Landings, however, are not expected to meet increasing consumer demand. A member of the family Serranidae, black sea bass are protogynous hermaphrodites and differentiate and spawn as females before undergoing sexual succession at 2–5 years of age (Shepherd and Idoine, 1993). Multiple spawns occur during the reproductive season, which progresses latitudinally from south to north. Along the southeastern states, spawning occurs from March to May (Wenner et al., 1986) while off the New England coast spawning usually takes place during June and July (Mercer, 1978). Fecundity varies with size and age. The lowest fecundity (17,000 eggs/ female) observed by Wenner et al. (1986) was in age 2 fish (140 mm TL) while the highest fecundity (1,050,0000 eggs/female) was observed in an older, larger female (454 mm TL). Due to a high market value and limited seasonal availability, there has been increased interest in aquaculture production of black sea bass in recent years. In response to this, several studies have been conducted to determine the environmental conditions conducive for larval and juvenile growth (Berlinsky et al., 2000, 2004; Watanabe et al., 2003). Larval growth and survival are high at temperatures between 21 and 27 8C and salinity levels N 15 g/L. (Berlinsky et al., 2004). Temperature and salinity values of 25 8C and 20–30 g/L were found to be optimal for juvenile fish (Atwood et al., 2003; Cotton et al., 2003). Information on nursery systems was provided by Stuart and Smith (2003) and this species appears suitable for culture in
recirculating systems at relatively high densities (Copeland et al., 2002; Bender et al., 2004). Substantial progress has been made in controlling reproduction in this species since Earll (1884) first produced fertilized eggs by collecting gametes from fish captured during the spawning season. Black sea bass have been induced to ovulate, and were manually spawned, following repeated administration of human chorionic gonadotropin (hCG) (Hoff, 1970; Roberts et al., 1976; Tucker, 1984). More recently, black sea bass tank spawned following administration of luteinizing hormone releasing hormone analogue (LHRHa) administered as a sustained release implant (Watanabe et al., 2003). Fish in this study spawned for up to 32 days post-implantation, but fertilization was highly variable. This paper reports the results of strip spawning trials conducted at a private aquaculture facility over two spawning seasons, using LHRHa to induce ovulation. The objectives were to: (1) investigate the influence of oocyte size on ovulatory success and (2) determine effective LHRHa doses and administration methods for ovulation in black sea bass.
2. Materials and methods 2.1. Experimental fish Two hundred adult black sea bass (0.76 F 0.1 kg) were obtained during fall 2001. Fish were captured in commercial traps off Rhode Island and held in tanks at Great Bay Aquaculture LLC (GBA, Portsmouth, NH, USA). Fish were reared and conditioned at GBA where all experiments were conducted during spring 2002 and 2003. Each fish was implanted with a passive integrated transponder (PIT) tag (Biomark, Boise, ID, USA) for individual identification. The fish were held in 2–5500 L rectangular tanks that were connected to a larger recirculating water system. Each system was equipped with biological and mechanical filtration, temperature control, ultra-violet sterilization, foam fractionation and photoperiod control. The fish were maintained under simulated natural ambient conditions (water temperature 12–22 8C, salinity 30–33 g/L). Photoperiod was controlled with timers adjusted weekly to local light/dark cycles. Half-hour dawn and dusk periods were simulated
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with rheostats and 100 W incandescent bulbs. Light intensity measured with a meter (Sper Scientific, Scottsdale, AZ) ranged from 5 lx (dawn/dusk) to 10 lx at the water surface during the daylight period. During the spawning studies, photoperiod was held at 18L:6D and water temperature was maintained between 18 and 22 8C. Fish were fed a commercial ration (11 mm pellet, 58% protein, Dana Feed A/S, Horsens, Denmark) to apparent satiation three times weekly. Water temperature and dissolved oxygen (OxyguardR, Birkerod, Denmark) were measured daily and total ammonia nitrogen and nitrite were monitored weekly using wet chemistry kits (HACHR, Loveland CO, USA). Water quality within the culture tanks remained within ranges suitable for rearing this species (Atwood et al., 2003). 2.2. Spawning protocol Females were selected for induced ovulation based on their ovarian development. Selected females were anesthetized in 100 mg/l tricaine methane sulfonate (MS-222; FinquelR; Argent Laboratories, Redmond WA) and an ovarian biopsy was obtained using a polypropylene cannula (1.49 mm o.d. 1.19 mm i.d) as described by Shehedeh et al. (1973). Candidates for induced ovulation were initially chosen based on maximum oocyte diameters measured to the nearest 25 Am using a dissecting microscope (Bausch and Lomb StereoZoom 5, Rochester NY, USA) fitted with an ocular micrometer. Oocytes were classified as vitellogenic, hydrated or atretic following Wallace and Selman (1981). To construct oocyte size-frequency distributions, biopsy samples were photographed with a digital camera (Canon PowerShot S40, Lake Success, NY) attached to a dissecting microscope (Leica model S8APO, Singapore) and oocytes with diameters N 200 Am (n = 150/ fish) were measured under a dissecting microscope using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the internet at http://rsb.info.nih.gov/ nih-image/). A developmental index (DI) was calculated as the number of oocytes with diameters N 400 Am/total number of oocytes measured. Oocytes that are 400 Am in diameter have initiated vitellogenesis in black sea bass (Cerda´ et al., 1996). After hormonal implantation, the fish were placed in a 5500 L tank
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and beginning 24 h post implantation, females were checked for evidence of ovulation by exerting gentle abdominal pressure to express eggs. If ovulation had not occurred, the developmental stage of the oocytes was determined by ovarian biopsy. This procedure was repeated after 2–8 h (for the next 48 h) depending on the proximity to ovulation as judged by the degree of oocyte development. Once ovulation had occurred, eggs were collected into a 500 ml glass beaker and their total volume recorded. A subsample of eggs (n = 200) was examined to assess quality. High quality (fertilizable) eggs from marine teleosts are generally clear, buoyant, spherical and lack a perivitelline space prior to fertilization (Kjorsvik et al., 1990; Larsson et al., 1997). An estimate of the number of eggs exhibiting these characteristics was determined. If most of the eggs appeared to be of high quality, milt was added. If eggs appeared atretic then their total volume was determined and the eggs were discarded. Males were identified based on the release of milt when slight abdominal pressure was applied. Manually expressed milt was collected into plastic transfer pipettes from 2–3 anesthetized males (1.5–2.5 ml/ fish). Milt was pooled in a 10 ml beaker and used immediately or held on ice no longer than 2 h prior to use. Sperm were activated with seawater and their motility was confirmed using a compound microscope (Olympus CH, Melville, NY, 400X). The eggs were transferred to plastic 2 L containers and fertilized by adding pooled milt (0.4–0.6 ml) and 20–40 ml of filtered seawater (34 g/L). Eggs and milt were gently mixed for 2 min. The eggs were transferred to a calibrated separatory funnel containing 700–800 ml seawater and statically incubated for 15 min to allow the buoyant (viable) and sinking (nonviable) eggs to separate. The volumes of both groups of eggs were recorded. The number of eggs/ ml was estimated (1200 eggs ml/l) based on counts of 1 ml subsamples (n = 5) of floating eggs. The percentage of fertilized eggs was determined after 2 h (4– 8 cell stage) by microscopic examination of approximately 200 eggs. The mean numbers of eggs per female, eggs per kg BW, percentage of buoyant eggs, percent of buoyant fertilized, and total eggs produced in relation to LHRHa dosage/kg BW were calculated for each spawn. No information on the treatment effects on individual hatch rates was available, as
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space limitations required the fertilized eggs to be pooled and incubated. 2.3. Experiments 1 and 2: effect of oocyte size on induced ovulation Experiment 1, conducted at the start of the spawning season (May 2002), sought to identify the minimum oocyte size necessary for LHRHa-induced ovulation. Females were biopsied and 15–20 of the largest oocytes present were measured with a microscope and an ocular micrometer. Fish (n = 18; 0.42–1.23 kg BW) with the largest clutch of oocytes N 400 Am (range = 400–625 Am) were implanted with a 95% cholesterol–5% cellulose pellet (Sherwood et al., 1988) containing 50 Ag [dAla6 Des-Gly10]-LHRH ethylamide (LHRHa; Peninsula Laboratories, Belmont CA, USA). The 2 8 mm long cylindrical pellets were implanted subcutaneously several scale-rows below the dorsal fin using a 10gauge needle attached to a modified syringe (Hodson and Sullivan, 1993). None of the biopsy samples from these fish contained atretic or hydrated oocytes which indicated that this population of females had not begun to volitionally ovulate.
Experiment 2, also conducted early in the 2002 spawning season, examined the ovulatory response to LHRHa of 6 large females (0.87–1.75 kg BW) that exhibited pre-spawning morphology (distended abdomens; Mylonas et al., 1995). Microscopic inspection of the ovarian biopsies from these 6 fish revealed that most oocytes were fully vitellogenic (N 500 Am) and this was later confirmed by analysis of the digital micrographs (DIs N 86%). As before, these fish received 50 Ag LHRHa implants (actual dose 29–57 Ag/kg). As in the previous experiment, biopsies from fish selected for this experiment showed no evidence of recent ovulation (i.e. no atretic or hydrated oocytes in the ovarian biopsies or presence of ovarian fluid). After hormonal implantation, the fish were placed in a 5500 L tank and checked for evidence of ovulation as described above. The study was terminated 96 h post-implantation. 2.4. Experiment 3: effect of developmental index on ovulation A third experiment, examining the ovulatory response of fish categorized by DI, was conducted
Fig. 1. Digital micrographs of ovarian follicles in biopsies from black sea bass prior to ovulation induction: (A) hydrated oocytes indicate previous ovulation; (B) uniformly vitellogenic oocytes in a biopsy with a high developmental index; (C) large range in oocyte diameters indicative of a biopsy with a low developmental index; (D) fully grown follicles in a biopsy taken at the time of re-implantation, 10 days following the first spawn. The fish failed to ovulate again.
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during June and July 2002. Thirty-nine females (0.43– 2.0 kg BW) were administered implants with nominal doses of 6.25 or 12.5 Ag LHRHa/implant (7.1–23.0 Ag/kg BW). Six fish served as controls and received sham implants. Fish selected for implantation had mostly fully vitellogenic oocytes (N 500 Am), but the relative proportion of fully vitellogenic oocytes present differed among fish (i.e. different DIs). The ovaries in some fish contained atretic, hydrated oocytes and relatively large volumes of ovarian fluid indicating that volitional ovulation had recently occurred (Fig. 1A). In some cases the ovarian fluid wicked through the biopsy tube by capillary action or could be expressed from the fish with slight abdominal pressure. Evidence of previous ovulation and oocyte maturational stage was recorded for each fish and ovarian biopsy samples were digitally photographed for later analysis. Post implantation, fish were sampled, rechecked and strip-spawned as described previously. Buoyant eggs were pooled for hatching after determining fertilization success. Ten days after spawning was complete, ovarian biopsies were taken from all fish, examined with a dissecting microscope, and ovarian condition was recorded. 2.5. Experiment 4: effect of LHRHa delivery method on ovulation During the subsequent spawning season (June– July 2003) a fourth experiment was conducted. Females (n = 20, 0.6–1.4 kg/BW) received LHRHa either by cholesterol–cellulose implant (12.5 Ag) or by injection of either 20 or 100 Ag/kg BW in 0.9% saline. Six fish received saline and served as controls. As in 2002, some fish in this study had volitionally ovulated previously, as determined by the presence of hydrated atretic follicles and ovarian fluid in biopsy samples. All other selection criteria and handling procedures were the same as described for earlier experiments. Two months after the spawning season ended (September), all fish were examined for evidence of sex reversal by gonadal biopsy and presence of spermiation.
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MANOVA with either developmental index or administration method and dose used as the categorical variables. All testing was performed using Systat 10.0 software (SSI, Richmond, CA, USA). Percent data were arcsine transformed prior to analysis.
3. Results 3.1. Experiments 1 and 2: effect of oocyte size on induced ovulation The ovulatory response to LHRHa was affected by oocyte size. At initiation of the experiment, the largest clutch of oocytes ranged from 400 to 625 Am prior to hormone treatment (Table 1). Neither of the fish with the largest clutch of oocytes measuring 400 Am in diameter ovulated and only 1 of 2 fish with 450 Am oocytes ovulated. In contrast, 90% of fish with 500 Am oocytes spawned within 36 h and 40% of this group spawned a second time. All females containing oocytes N550 Am spawned and one of these fish spawned a second time. Mean buoyancy of all eggs in the first spawns was 71% and ranged from 26% to 100% (Table 1). Fertilization of pooled eggs from the first spawn was 82%. Mean buoyancy of all eggs in the second spawn was 59% and ranged from 40% to 75% (Table 1). Fertilization of pooled eggs from the second spawn was 46%. In experiment 2 which examined the ovulatory success of fish exhibiting pre-spawning morphology (distended abdomens), 5 of 6 fish with uniformly, fully vitellogenic oocytes (one died from causes unrelated to the study, and was extruding eggs upon recovery) ovulated substantial numbers of eggs (45,000–192,000 eggs/kg BW), but fertility following strip spawning was consistently low (0–15%). Fertile eggs were only obtained from the 2 fish with the lowest fecundity (45,000 eggs/kg, 9% fertilization; 75,000 eggs/kg, 15% fertilization). All of the fish in this group had DIs N 86% (Fig. 1B). 3.2. Experiment 3: effect of developmental index on ovulation
2.6. Statistics Spawning data (eggs/kg, # spawns, % buoyant, % buoyant fertilized) were subjected to a one-way
In experiment 3 (2002 season), 19 implanted fish ovulated but fecundity and fertilization were highly variable (Table 2). The number of eggs/kg ovulated
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Table 1 Ovulatory response of black sea bass females implanted with LHRHaa Fish #
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 a b
Weight (kg)
0.78 0.70 0.55 1.09 0.88 0.42 0.79 0.65 1.09 0.72 0.68 0.86 0.62 0.86 1.23 0.71 0.65 0.83
LHRHa (Ag/kg BW)
Maximum oocyte diameter (Am)
Spawn 1
39.0 35.0 27.5 54.5 44.0 21.0 39.5 32.5 54.5 36.0 34.0 43.0 31.0 43.0 61.5 35.5 32.5 41.5
400 400 450 450 500 500 500 500 500 500 500 500 500 500 550 550 600 625
0 0 0 1200b 0 3600 37,200 40,800 3600 7200 18,000 15,600 18,000 13,200 52,800 180,000 12,000 55,200
Table 2 Effect of developmental index on egg production and fertilization in black sea bassa,b Developmental Spawns index (%) (#) b60 61–70 71–80 N80 a
Eggs produced
Buoyant eggs (%)
Eggs produced
Buoyant eggs (%)
30,000 63,600 80,400 10,800
48 40 75 67
72,000
63
0 67 45 62 67 100 80 62 87 91 91 73 70 26
All fish received 50 Ag LHRHa in a cholesterol–cellulose implant. Eggs discarded and not incorporated into treatment mean values.
per spawn and the fertilization rate ranged from 17,000–339,000 and 0–98%, respectively. None of the control fish ovulated. No differences were found in the number of spawns, fecundity, egg buoyancy or fertilization success among fish with different developmental indices (Table 2). Ovulation occurred from 1 to 4 days in succession and egg quality (buoyancy and fertilization) did not decrease from the first (71.6%, 43%) to the second spawn (82.8%, 52.3%). The degree of ovarian development, as determined by the DI, was not an accurate predictor of ovulatory response or egg quality. Fish with DIs as low as 45% (Fig. 1C)
1.0 F 0.0 1.7 F 0.7 2.3 F 0.4 1.6 F 0.4
Eggs/kg Buoyant Buoyant eggs BW (103) (%) fertilized (%) 63 F 11 67 F 26 86 F 21 68 F 22
98 F 2.0 54 F 25 78 F 4.3 81 F11
73 F 13 33 F 18 65 F 7.4 69 F 12
Data are mean F S.E.M. There were no statistical differences among spawning data categories when compared to the development index. b
Spawn 2
ovulated relatively high numbers of eggs, while some with indices as high as 90% failed to do so (Table 3). The one fish that was re-implanted 10 days after spawning relatively few eggs with low fertilization Table 3 Developmental indices and LHRHa doses for black sea bass females that failed to ovulate Fish #
Weight (kg)
LHRHa (Ag/kg BW)
Developmental index (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1.99 1.00 0.76 0.53 0.62 0.65 0.64 0.71 0.76 0.57 0.67 0.53 0.83 0.70 0.68 0.74
6.3 12.5 8.2 23.6 10.1 9.6 9.8 8.8 8.2 11.0 9.3 11.8 7.5 8.9 18.4 8.4
34 NA 8 96 NA 81 69 55 60 80 61 80 90 66 67 61
NA means that no data were available.
D.L. Berlinsky et al. / Aquaculture 250 (2005) 813–822 Table 4 LHRHa dose and administration method on egg production and fertilizationa,b LHRHa (Ag/kg)
Spawns (#)
Eggs/kg BW (103)
Buoyant (%)
Buoyant eggs fertilized (%)
Injection 20 100
2.0 F 0.4 2.0 F 0.3
146 F 40.1 85 F 14
65 F 8.4 69 F 10
40 F 12 38 F 9.2
Implant 12.5 Ag
1.4 F 0.2
92 F 14
80 F 8.1
38 F 10
a
Data are mean F S.E.M. b There were no statistical differences among spawning data categories when compared to administration method or dose.
(DI = 96%, Table 3) did not ovulate again, although the biopsy sample had fully grown follicles (Fig. 1D). One fish with a 90% DI that did not ovulate (Table 3) was spermiating when examined 2 months after the spawning season. Hatching rate of pooled embryos ranged from 53% to 76%. 3.3. Experiment 4: effect of LHRHa delivery method on ovulation In experiment 4 conducted in 2003, 11 of 13 LHRHa-injected fish ovulated at least once, 5 fish ovulated on 2 consecutive days and 3 fish ovulated on 3 consecutive days. Three of seven implanted fish ovulated once and 2 fish ovulated on consecutive days. No differences were found among females given implants or an injection (20 or 100 Ag/kg) with respect to the mean number of eggs/kg produced, the percentage of buoyant eggs or fertilization success. The mean number of spawns for fish injected with 20 (2.0 F 0.4) or 100 Ag (2.0 F 0.3) LHRHa was not significantly greater than that (1.4 F 0.2) for implanted fish (Table 4). Incubation of pooled eggs resulted in hatching of approximately 155,000 larvae (60% hatch) of which approximately 95,000–1.0 g juveniles were produced (61%).
4. Discussion Synthetic analogues of LHRH have been effectively used to induce ovulation and spawning in a number of commercially important finfish including many serranid species (Tucker, 1994; Mylonas and
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Zohar, 2001). Under favorable conditions, captive black sea bass will tank spawn volitionally or following administration of LHRHa, but spawning is not synchronized and fertilization success is highly variable (Cerda´ et al., 1996; Watanabe et al., 2003). Additionally, tank spawning is often prolonged over a period of weeks, which extends the labor-intensive larviculture period and the need for algal and livefeed production. In vitro fertilization is often employed to control parentage for genetic improvement and manipulation of ploidy conditions. In the present studies, commercial quantities of black sea bass larvae were produced, and the spawning period was contracted to a few days, by using in vitro fertilization following LHRHa administration. Analogues of LHRH have often been favored over gonadotropic hormone preparations (pituitary extracts and hCG) because they are non-immunogenic and provide more integrated control of final oocyte maturation (FOM) and ovulation (Zohar and Mylonas, 2001). Since LHRHa is rapidly cleared from the circulatory system (half-life 0.8 h in chinook salmon, Slater et al., 1999), multiple injections or sustainedrelease implants are often more effective than single injections for inducing multiple ovulations in batch spawning fishes with group-synchronous oocyte development (Mylonas et al., 2003). In some species, such as spotted seatrout (Cynoscion nebulosus) and red drum (Sciaenops ocellatus), with fully grown oocytes, a single injection or oral administration of LHRHa is sufficient for ovulation induction (Thomas and Boyd, 1988, 1989). Sustained release implants may be more advantageous than injections for inducing FOM and ovulation in fish with less developed ovaries, however. For instance, wild striped bass (Morone saxatilis) collected far from their spawning grounds, which have not initiated FOM, require slowrelease implanted LHRHa for successful ovulation (Hodson and Sullivan, 1993). LHRHa implants were also used to significantly advance the spawning season of winter flounder (Pleuronectes americanus; Larsson et al., 1997). In the present study, ovulation was induced in black sea bass just prior to, or during, the natural spawning season. Under these conditions, LHRHa administered as a single injection was as effective as implanted LHRHa for inducing ovulation. Human chorionic gonadotropin has also been used to induce ovulation in black sea bass with fully vitello-
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genic oocytes (Hoff, 1970; Tucker, 1984), and has been shown to be as effective as LHRHa for inducing ovulation in related Epinephelus grouper species (Watanabe et al., 1995). Considering black sea bass in spawning condition, it appears that the degree of ovarian development may be more important than the gonadotropic-inducing agent used to stimulate ovulation. Given the success obtained using hCG to induce ovulation in many serranid species (Tucker, 1994; Watanabe et al., 1995) and its recent approval by the U.S. Food and Drug Administration for use on broodstock, a direct comparison of LHRHa and hCG is warranted. The efficacy of LHRH analogues has also been shown to be influenced by dose, primary peptide structure, and water temperature (Harmin and Crim, 1992; King and Pankhurst, 2004). Previous studies of black sea bass demonstrated that a single cholesterol– cellulose pellet containing 50 Ag LHRHa (approximately 50 Ag/kg) successfully induced multiple spawning events over a prolonged period (Watanabe et al., 2003). In the present study, this dose was also administered to pre-spawning black sea bass that exhibited grossly distended abdomens and high DIs (N86%). All fish in this condition ovulated large quantities of eggs, but fertilization percentage was low. It is possible that the dose administered to these fish (mean dose, 47.2 Ag/kg) was excessive and may have prematurely stimulated FOM in underdeveloped oocytes (Taranger et al., 1992). Fertilization and hatching was adversely effected in Asian sea bass (Lates calcarifer) administered high doses of LHRHa (150–300 Ag/kg) as compared to lower dosages (4.5–75 Ag/kg; Garcia, 1989). High LHRHa doses have been shown to diminish egg quality or induce fewer ovulations in a number of other species (Mylonas et al., 1992; Poortenaar and Pankhurst, 2000; Marino et al., 2003). While the optimal dose of LHRHa has not been established for black sea bass, implants with actual doses as low as 7.1 Ag/kg were effective for inducing ovulation without decreasing fecundity or fertility. The responsiveness of black sea bass to LHRHa may change over time and for the first and subsequent spawns. Cerda´ et al. (1997) demonstrated that the maturational response of black sea bass oocytes to hCG in vitro declined as the spawning season progressed.
The frequency of spawning (inter-ovulatory cycle) has also not been identified in black sea bass. In the present study, fish were generally strip spawned at 24 h intervals following the first ovulation and no attempt was made to determine the inter-ovulatory cycle. Two fish were spawned in the morning and afternoon of the same day with high rates of fertilization. It is not known whether these represented separate or single, interrupted ovulatory events. Watanabe et al. (2003) reported that female black sea bass tank spawned for up to 4 days following implantation with LHRHa, but eggs were only collected on a daily basis. During the spawning season in the present study, the oocyte composition in the ovary included early and late vitellogenic oocytes and the DIs varied greatly among individuals. Furthermore, hydrated and atretic follicles were often observed in ovarian biopsies prior to LHRHa administration but were not always present in biopsy samples taken from fish 10 days after induced spawning. The variable responsiveness to administered LHRHa during the spawning season may reflect the number of previous spawning events completed and/or the time-point in the ovulatory cycle when the hormone was administered. Similar oocyte frequency distributions were observed in Nassau grouper (Epinephelus striata), a related protogynous hermaphrodite, in which repeated ovulatory events could be stimulated within a single season (Watanabe et al., 1995). Further research is necessary to establish the ovulatory cycle and period of over-ripening in order to minimize fish handling and preserve egg quality. Successful FOM and ovulation requires coordinated synthesis of gonadotropin and maturation-inducing steroid receptors and shifts in steroid enzymatic pathways (Nagahama et al., 1994). Typically, the efficacy of gonadotropic compounds is greatest in fish that have completed vitellogenesis and initiated FOM (Harmin and Crim, 1992); however, fertilization success is often variable and may be influenced by nutritional and environmental factors (Watanabe et al., 1995; Berlinsky et al., 1996). Black sea bass oocytes that have completed vitellogenesis are 475– 600 Am in diameter and can be induced to ovulate with hCG (Tucker, 1984, 1994) and LHRHa (Watanabe et al., 2003). In the present study, responsiveness to LHRHa was greatest in oocytes N500 Am. These results agree with in vitro studies of Cerda´ et al.
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(1996) who reported that black sea bass oocytes 490– 600 Am were more responsive to hCG than those 350– 390 Am in diameter. Watanabe et al. (2003) also found that all females that tank spawned after LHRHa implantation had maximum oocyte diameters N475 Am. The proximity to sex inversion may also influence oocyte responsiveness to LHRHa, as 1 fish with a high DI (90%), that did not spawn, was found to be a spermiating male when examined 2 months after the spawning trials. It is not known if administration of LHRH analogues can hasten sex change in black sea bass as has been reported in the gilthead seabream (Sparus aurata; Vilia and Canario, 1995) and the bluehead wrasse (Rhalassoma bifasciatum, Kramer et al., 1993). In summary, black sea bass with fully vitellogenic oocytes are responsive to LHRHa delivered in an intramuscular injection or a slow release implant. The responsiveness to the hormone appears to be influenced more by the degree of oocyte development and spawning history than dose or delivery method. Acknowledgements We thank the personnel at Great Bay Aquaculture for use of their broodstock and assistance throughout these studies. This study was supported by grants from New Hampshire Sea Grant (award number NA16RG1035) and the National Sea Grant College Program (award number NA16RG1561). This is contribution number 562 from South Carolina Marine Resources Division and number 2265 from the New Hampshire Agricultural Experiment Station. References Atwood, H.L., Young, S.P., Tomasso Jr., J.R., Smith, T.I.J., 2003. Effect of temperature and salinity on survival, growth, and condition of juvenile black sea bass Centropristis striata. J. World Aquac. Soc. 34, 398 – 402. Bender, J., Lee, R., Sheppard, M., Bulski, K., Phillips, P., Yeboah, Y., Wah, R.C., 2004. A waste effluent treatment system based on microbial mats for black sea bass Centropristis striata recycledwater mariculture. Aquac. Eng. 31, 73 – 82. Berlinsky, D.L., King V, W., Smith, T.I.J., Hamilton, R.D., Holloway, J., Sullivan, C.V., 1996. Induced ovulation of southern flounder Paralichthys lethostigma using gonadotropin releasing hormone analogue implants. J. World Aquac. Soc. 27, 143 – 152.
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Berlinsky, D., Watson, M., Nardi, G., Bradley, T., 2000. Investigations of selected parameters for growth of larval and juvenile black sea bass Centropristis striata L.. J. World Aquac. Soc. 31, 426 – 435. Berlinsky, D.L., Taylor, J., Howell, R.A., Bradley, T.M., Smith, T.I.J., 2004. The effects of temperature and salinity on early life stages of black sea bass (Centropristis striata). J. World Aquac. Soc. 35, 335 – 344. Cerda´, J., Selman, K., Wallace, R.A., 1996. Observations on oocyte maturation and hydration in vitro in the black sea bass, Centropristis striata (Serranidae). Aquat. Living Resour. 9, 325 – 335. Cerda´, J., Selman, K., Hsiao, S.-M., Wallace, R.A., 1997. Evidence for the differential regulation of ovarian follicle responsiveness to human chorionic gonadotropin in vitro in a serranid teleost, Centropristis striata. Aquaculture 159, 143 – 157. Copeland, K.A., Watanabe, W.O., Carroll, P.M., 2002. Growth and feed utilization of captive wild subadult black sea bass, Centropristis striata, fed practical diets in a recirculating system. J. World Aquac. Soc. 39, 97 – 109. Cotton, C.F., Walker, R.L., Recicar, T.C., 2003. Effects of temperature and salinity on growth of juvenile black sea bass, with implications for aquaculture. N. Am. J. Aquac. 65, 330 – 338. Earll, R.E., 1884. Hatching blackfish and Spanish mackerel. Bulletin U. S. Fish Commission, vol. 1884. Government Printing Office, Washington, DC, USA, pp. 415 – 416. Frame, D.W., Pearce, S.A., 1973. A survey of the sea bass fishery. Mar. Fish. Rev. 35, 19 – 26. Garcia, L.M.B., 1989. Dose-dependent spawning response of mature female sea bass, Lates calcarifer (Bloch), to pelleted luteinizing hormone-releasing hormone analogue (LHRHa). Aquaculture 77, 85 – 96. Harmin, S.A., Crim, L.W., 1992. Gonadotropic hormone-releasing hormone analog (GnRH-a) induced ovulation and spawning in female winter flounder, Pseudopleuronectes americanus (Walbaum). Aquaculture 104, 375 – 390. Hodson, R.G., Sullivan, C.V., 1993. Induced maturation and spawning of domestic and wild striped bass, Morone saxatilis (Walbaum), broodstock with implanted GnRH analogue and injected hCG. Aquac. Fish. Manage. 24, 389 – 398. Hoff, F.H., 1970. Artificial spawning of black sea bass, Centropristis striatus melanus, Ginsburg, aided by chorionic gonadotropic hormones. Florida Department of Natural Resources Marine Research Laboratory. Special Science Report 25, 17 pp. St. Petersburg, Florida, USA. King, H.R., Pankhurst, N.W., 2004. Effect of maintenance at elevated temperatures on ovulation and luteinizing hormone releasing hormone analogue responsiveness of female Atlantic salmon (Salmo salar) in Tasmania. Aquaculture 223, 583 – 597. Kjorsvik, E., Mangor-Jensen, A., Holmefjord, I., 1990. Egg quality in fishes. Adv. Mar. Biol. 26, 71 – 113. Kramer, C.R., Caddell, M.T., Bubenheimer-Livolsi, L., 1993. sGnRH-a [(d-Arg6, Pro9, Net) LHRH] in combination with domperidone induces gonad reversal in a protogynous fish, the bluehead wrasse, Thalassoma bifasciatum. J. Fish Biol. 42, 185 – 195. Larsson, D.G.J., Mylonas, C.C., Zohar, Y., Crim, L.W., 1997. Gonadotropin-releasing hormone analogue (GnRHa) induces
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multiple ovulations of high-quality eggs in a cold water, batchspawning teleost, the yellowtail flounder (Pleuronectes ferrugineus). Can. J. Fish. Aquat. Sci. 54, 1957 – 1964. Low Jr., R.A., 1981. Mortality rates and management strategies for black sea bass off the southeast coast of the United States. North Am. J. Fish. Manage. 1, 93 – 103. Marino, G., Panini, E., Longobardi, A., Mandich, A., Finoia, M.G., Zohar, Y., Mylonas, C.C., 2003. Induction of ovulation in captive-reared dusky grouper, Epinephelus marginatus (Lowe, 1834), with a sustained-release GnRHa implant. Aquaculture 219, 841 – 858. Mercer, L.P., 1978. The reproductive biology and population dynamics of black sea bass, Centropristis striata. PhD thesis, College of William and Mary, Williamsburg, VA. 196 pp. Musick, J.A., Mercer, L.P., 1977. Seasonal distribution of black sea bass Centropristis striata, in the Mid-Atlantic Bight with comments on ecology and fisheries of the species. Trans. Am. Fish. Soc. 106, 12 – 25. Mylonas, C.C., Zohar, Y., 2001. Use of GnRHa-delivery systems for the control of reproduction in fish. Rev. Fish Biol. Fish. 10, 463 – 491. Mylonas, C.C., Hinshaw, J.M., Sullivan, C.V., 1992. GnRHa-induced ovulation of brown trout (Salmo trutta) and its effects on egg quality. Aquaculture 106, 379 – 392. Mylonas, C.C., Zohar, Y., Richardson, B.M., Minkkinen, S.P., 1995. Induced spawning of wild American shad Alosa sapidissima using sustained administration of gonadotropin-releasing hormone analog (GnRHa). J. World Aquac. Soc. 26, 240 – 251. Mylonas, C.C., Sigelaki, I., Divanach, P., Manano˜s, E., Carrillo, M., Afonso-Polyviou, A., 2003. Multiple spawning and egg quality of individual European sea bass (Dicentrarchus labrax) females after repeated injections of GnRHa. Aquaculture 221, 605 – 620. Nagahama, Y., Yoshikuni, M., Yamashita, M., Tanaka, M., 1994. Regulation of oocyte maturation in fish. In: Hoar, W.S., Randall, D.J. (Eds.), Fish Physiology, vol. 13. Academic Press, New York, USA, pp. 393 – 439. Poortenaar, C.W., Pankhurst, N.W., 2000. Effect of luteinising hormone-releasing hormone analogue and human chorionic gonadotropin on ovulation, plasma and ovarian levels of gonadal steroids in greenback flounder Rhombosolea tapirina. J. World Aquac. Soc. 31, 175 – 185. Roberts Jr., D.E., Harpster, B.V., Havens, W.K., Halscott, K.R., 1976. Facilities and methodology for culture of the southern sea bass (Centropristis melana). Proc. World Maric. Soc. 7, 163 – 198. Shehedeh, Z.H., Kuo, C.-M., Milisen, K.K., 1973. Validation of an in vivo method for monitoring ovarian development in the grey mullet (Mugil cephalus L.). J. Fish Biol. 5, 479 – 487. Shepherd, G.R., Idoine, J.S., 1993. Length based analyses of yield and spawning biomass per recruit for black sea bass Centropristis striata, a protogynous hermaphrodite. Fish. Bull., U.S. 91, 328 – 337.
Shepherd, G.R., Terceiro, M., 1994. The summer flounder, scup and black sea bass fishery of the middle Atlantic bight and southern New England waters. NOAA Technical Report NMFS, vol. 122. US Department of Commerce, Seattle, WA. Sherwood, N.M., Crim, L.W., Carolfeld, J., Walters, S.M., 1988. Sustained hormone release: I. Characteristics of in vitro release of gonadotropin releasing hormone analogue LHRHa from pellets. Aquaculture 74, 75 – 86. Slater, C.H., Schreck, C.B., Sower, S.A., 1999. Rapid clearance of d-Ala6, Pro9Net mammalian gonadotropin releasing hormone from chinook salmon plasma. N. Am. J. Aquac. 61, 315 – 318. Stuart, K.R., Smith, T.I.J., 2003. Development of nursery systems for black sea bass (Centropristis striata). J. World Aquac. Soc. 34, 359 – 367. Taranger, G.L., Stefansson, S.O., Hansen, T., 1992. Advancement and synchronization of ovulation in Atlantic salmon (Salmo salar L.) following injections of LHRH analogue. Aquaculture 102, 169 – 175. Thomas, P., Boyd, N., 1988. Induced spawning of spotted seatrout, red drum and orangemouth corvina (Family: Sciaenidae) with luteinizing hormone analog injection. Contrib. Mar. Sci. 30, 43 – 47 (Supp. to Vol.). Thomas, P., Boyd, N.W., 1989. Dietary administration of an LHRH analogue induces spawning of spotted seatrout (Cynoscion nebulosus). Aquaculture 80, 363 – 370. Tucker Jr., J.W., 1984. Hormone-induced ovulation of black sea bass and rearing of larvae. Prog. Fish-Cult. 46, 201 – 203. Tucker Jr., J.W., 1994. Spawning by captive serranid fishes: a review. J. World Aquac. Soc. 25, 345 – 359. Vilia, P., Canario, A.V.M., 1995. Effect of LHRH on sex reversal and steroid levels in gilthead seabream (Sparus aurata). In: Goetz, F., Thomas, P. (Eds.), Proceedings of the Fifth International Symposium on the Reproductive Physiology of Fish, Fish Symp., vol. 95. The University of Texas at Austin, p. 329. Wallace, R.A., Selman, K., 1981. Cellular and dynamic aspects of oocyte growth in teleosts. Am. Zool. 21, 325 – 343. Watanabe, W.O., Ellis, S.C., Ellis, E.P., Head, W.D., Kelley, C.D., Moriwake, A., Lee, C., Bienfang, P.K., 1995. Progress in controlled breeding of Nassau grouper (Epinephelus striatus) broodstock by hormone induction. Aquaculture 138, 205 – 219. Watanabe, W.O., Smith, T.I.J., Berlinsky, D.L., Woolridge, C.A., Stuart, K.R., Copeland, K.A., Denson, M.R., 2003. Volitional spawning of black sea bass (Centropristis striata) induced with pelleted luteinizing hormone releasing hormone-analogue. J. World Aquac. Soc. 34, 319 – 331. Wenner, C.A., Roumillat, W.A., Waltz, C.W., 1986. Contributions to the life history of black sea bass, Centropristis striata, off the southeastern United States. Fish. Bull. 84, 723 – 741. Zohar, Y., Mylonas, C.C., 2001. Endocrine manipulations of spawning in cultured fish: from hormones to genes. Aquaculture 197, 99 – 136.
The use of luteinizing hormone releasing hormone analogue for ovulation induction in black sea bass (Centropristis striata) David L. Berlinskya,*, William King V a, Theodore I.J. Smithb a Department of Zoology, University of New Hampshire, Durham, NH 03824, USA South Carolina Department of Natural Resources, Marine Resources Research Institute, Charleston, SC 29412, USA
b
Received 11 January 2005; received in revised form 5 April 2005; accepted 20 April 2005
Abstract Interest in commercial production of black sea bass has increased in recent years, but reliable spawning methods remain problematic. The objective of this study was to evaluate the effects of oocyte size and luteinizing hormone releasing hormone analogue (LHRHa) dosage and delivery systems on ovulatory success for in vitro fertilization. Vitellogenic females with maximum oocyte diameters of 400–625 Am were implanted with a 95% cholesterol–5% cellulose pellet containing 50 Ag of LHRHa. Fish with maximum oocyte diameters b 450 Am failed to ovulate. In contrast, 90% of fish with 500 Am oocytes spawned within 36 h and 40% of this group ovulated a second time. All of the females containing oocytes N 550 Am ovulated. In a second experiment, females with uniformly vitellogenic oocytes (N500 Am) and implanted with 50 Ag LHRHa ovulated substantial numbers of eggs (45,000–192,000 eggs/kg body weight (BW), but fertility was consistently low (0– 15%). In a third experiment, 19 of 39 females receiving implants containing 6.3–23.6 Ag LHRHa/kg BW during the spawning season ovulated, but fecundity (17,000–339,000 eggs/kg) and fertilization (0–98%) were highly variable. When fish were grouped by developmental index, calculated as the number of oocytes with diameters N 400 Am/total number of oocytes measured, there were no statistical differences among groups with respect to the number of spawns, fecundity or fertilization success. In a fourth experiment, 11 of 13 females with a clutch of fully vitellogenic oocytes that were injected with 20 or 100 Ag/kg BW LHRHa ovulated between 1 and 2 times on consecutive days. Five of seven given an implant containing 12.5 Ag LHRHa ovulated one or more times. Fish implanted with shams or injected with vehicle alone did not ovulate in any of the experiments. No differences were found in the number of spawns, fecundity or fertilization success from fish receiving different doses of injected or implanted LHRHa. Incubation of pooled eggs produced 155,000 larvae (60% hatch) and 95,000 one-gram juveniles. These results demonstrate that injected or implanted LHRHa is effective for inducing ovulation in black sea bass with maximum oocyte diameters N 500 Am. D 2005 Elsevier B.V. All rights reserved. Keywords: Black sea bass; Centropristis striata; Induced ovulation; LHRHa
* Corresponding author. Tel.: +1 603 862 0007; fax: +1 603 862 3784. E-mail address: [email protected] (D.L. Berlinsky). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.04.074
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1. Introduction Black sea bass (Centropristis striata) are found in waters along the Atlantic coast from the Gulf of Maine to northern Florida, and a subspecies inhabits the eastern Gulf of Mexico. Throughout their range this species supports important commercial and recreational fisheries (Musick and Mercer, 1977; Low, 1981). Historically, the greatest abundance along the Atlantic coast occurred in the Mid-Atlantic region, where fish were commercially harvested in traps during the summer and by otter trawls during the winter when they aggregated offshore in deeper waters (Frame and Pearce, 1973). Currently, populations are overexploited in some areas of this region and a variety of management strategies have been implemented in an attempt to maintain sustainable fisheries (Shepherd and Terceiro, 1994). Landings, however, are not expected to meet increasing consumer demand. A member of the family Serranidae, black sea bass are protogynous hermaphrodites and differentiate and spawn as females before undergoing sexual succession at 2–5 years of age (Shepherd and Idoine, 1993). Multiple spawns occur during the reproductive season, which progresses latitudinally from south to north. Along the southeastern states, spawning occurs from March to May (Wenner et al., 1986) while off the New England coast spawning usually takes place during June and July (Mercer, 1978). Fecundity varies with size and age. The lowest fecundity (17,000 eggs/ female) observed by Wenner et al. (1986) was in age 2 fish (140 mm TL) while the highest fecundity (1,050,0000 eggs/female) was observed in an older, larger female (454 mm TL). Due to a high market value and limited seasonal availability, there has been increased interest in aquaculture production of black sea bass in recent years. In response to this, several studies have been conducted to determine the environmental conditions conducive for larval and juvenile growth (Berlinsky et al., 2000, 2004; Watanabe et al., 2003). Larval growth and survival are high at temperatures between 21 and 27 8C and salinity levels N 15 g/L. (Berlinsky et al., 2004). Temperature and salinity values of 25 8C and 20–30 g/L were found to be optimal for juvenile fish (Atwood et al., 2003; Cotton et al., 2003). Information on nursery systems was provided by Stuart and Smith (2003) and this species appears suitable for culture in
recirculating systems at relatively high densities (Copeland et al., 2002; Bender et al., 2004). Substantial progress has been made in controlling reproduction in this species since Earll (1884) first produced fertilized eggs by collecting gametes from fish captured during the spawning season. Black sea bass have been induced to ovulate, and were manually spawned, following repeated administration of human chorionic gonadotropin (hCG) (Hoff, 1970; Roberts et al., 1976; Tucker, 1984). More recently, black sea bass tank spawned following administration of luteinizing hormone releasing hormone analogue (LHRHa) administered as a sustained release implant (Watanabe et al., 2003). Fish in this study spawned for up to 32 days post-implantation, but fertilization was highly variable. This paper reports the results of strip spawning trials conducted at a private aquaculture facility over two spawning seasons, using LHRHa to induce ovulation. The objectives were to: (1) investigate the influence of oocyte size on ovulatory success and (2) determine effective LHRHa doses and administration methods for ovulation in black sea bass.
2. Materials and methods 2.1. Experimental fish Two hundred adult black sea bass (0.76 F 0.1 kg) were obtained during fall 2001. Fish were captured in commercial traps off Rhode Island and held in tanks at Great Bay Aquaculture LLC (GBA, Portsmouth, NH, USA). Fish were reared and conditioned at GBA where all experiments were conducted during spring 2002 and 2003. Each fish was implanted with a passive integrated transponder (PIT) tag (Biomark, Boise, ID, USA) for individual identification. The fish were held in 2–5500 L rectangular tanks that were connected to a larger recirculating water system. Each system was equipped with biological and mechanical filtration, temperature control, ultra-violet sterilization, foam fractionation and photoperiod control. The fish were maintained under simulated natural ambient conditions (water temperature 12–22 8C, salinity 30–33 g/L). Photoperiod was controlled with timers adjusted weekly to local light/dark cycles. Half-hour dawn and dusk periods were simulated
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with rheostats and 100 W incandescent bulbs. Light intensity measured with a meter (Sper Scientific, Scottsdale, AZ) ranged from 5 lx (dawn/dusk) to 10 lx at the water surface during the daylight period. During the spawning studies, photoperiod was held at 18L:6D and water temperature was maintained between 18 and 22 8C. Fish were fed a commercial ration (11 mm pellet, 58% protein, Dana Feed A/S, Horsens, Denmark) to apparent satiation three times weekly. Water temperature and dissolved oxygen (OxyguardR, Birkerod, Denmark) were measured daily and total ammonia nitrogen and nitrite were monitored weekly using wet chemistry kits (HACHR, Loveland CO, USA). Water quality within the culture tanks remained within ranges suitable for rearing this species (Atwood et al., 2003). 2.2. Spawning protocol Females were selected for induced ovulation based on their ovarian development. Selected females were anesthetized in 100 mg/l tricaine methane sulfonate (MS-222; FinquelR; Argent Laboratories, Redmond WA) and an ovarian biopsy was obtained using a polypropylene cannula (1.49 mm o.d. 1.19 mm i.d) as described by Shehedeh et al. (1973). Candidates for induced ovulation were initially chosen based on maximum oocyte diameters measured to the nearest 25 Am using a dissecting microscope (Bausch and Lomb StereoZoom 5, Rochester NY, USA) fitted with an ocular micrometer. Oocytes were classified as vitellogenic, hydrated or atretic following Wallace and Selman (1981). To construct oocyte size-frequency distributions, biopsy samples were photographed with a digital camera (Canon PowerShot S40, Lake Success, NY) attached to a dissecting microscope (Leica model S8APO, Singapore) and oocytes with diameters N 200 Am (n = 150/ fish) were measured under a dissecting microscope using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the internet at http://rsb.info.nih.gov/ nih-image/). A developmental index (DI) was calculated as the number of oocytes with diameters N 400 Am/total number of oocytes measured. Oocytes that are 400 Am in diameter have initiated vitellogenesis in black sea bass (Cerda´ et al., 1996). After hormonal implantation, the fish were placed in a 5500 L tank
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and beginning 24 h post implantation, females were checked for evidence of ovulation by exerting gentle abdominal pressure to express eggs. If ovulation had not occurred, the developmental stage of the oocytes was determined by ovarian biopsy. This procedure was repeated after 2–8 h (for the next 48 h) depending on the proximity to ovulation as judged by the degree of oocyte development. Once ovulation had occurred, eggs were collected into a 500 ml glass beaker and their total volume recorded. A subsample of eggs (n = 200) was examined to assess quality. High quality (fertilizable) eggs from marine teleosts are generally clear, buoyant, spherical and lack a perivitelline space prior to fertilization (Kjorsvik et al., 1990; Larsson et al., 1997). An estimate of the number of eggs exhibiting these characteristics was determined. If most of the eggs appeared to be of high quality, milt was added. If eggs appeared atretic then their total volume was determined and the eggs were discarded. Males were identified based on the release of milt when slight abdominal pressure was applied. Manually expressed milt was collected into plastic transfer pipettes from 2–3 anesthetized males (1.5–2.5 ml/ fish). Milt was pooled in a 10 ml beaker and used immediately or held on ice no longer than 2 h prior to use. Sperm were activated with seawater and their motility was confirmed using a compound microscope (Olympus CH, Melville, NY, 400X). The eggs were transferred to plastic 2 L containers and fertilized by adding pooled milt (0.4–0.6 ml) and 20–40 ml of filtered seawater (34 g/L). Eggs and milt were gently mixed for 2 min. The eggs were transferred to a calibrated separatory funnel containing 700–800 ml seawater and statically incubated for 15 min to allow the buoyant (viable) and sinking (nonviable) eggs to separate. The volumes of both groups of eggs were recorded. The number of eggs/ ml was estimated (1200 eggs ml/l) based on counts of 1 ml subsamples (n = 5) of floating eggs. The percentage of fertilized eggs was determined after 2 h (4– 8 cell stage) by microscopic examination of approximately 200 eggs. The mean numbers of eggs per female, eggs per kg BW, percentage of buoyant eggs, percent of buoyant fertilized, and total eggs produced in relation to LHRHa dosage/kg BW were calculated for each spawn. No information on the treatment effects on individual hatch rates was available, as
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space limitations required the fertilized eggs to be pooled and incubated. 2.3. Experiments 1 and 2: effect of oocyte size on induced ovulation Experiment 1, conducted at the start of the spawning season (May 2002), sought to identify the minimum oocyte size necessary for LHRHa-induced ovulation. Females were biopsied and 15–20 of the largest oocytes present were measured with a microscope and an ocular micrometer. Fish (n = 18; 0.42–1.23 kg BW) with the largest clutch of oocytes N 400 Am (range = 400–625 Am) were implanted with a 95% cholesterol–5% cellulose pellet (Sherwood et al., 1988) containing 50 Ag [dAla6 Des-Gly10]-LHRH ethylamide (LHRHa; Peninsula Laboratories, Belmont CA, USA). The 2 8 mm long cylindrical pellets were implanted subcutaneously several scale-rows below the dorsal fin using a 10gauge needle attached to a modified syringe (Hodson and Sullivan, 1993). None of the biopsy samples from these fish contained atretic or hydrated oocytes which indicated that this population of females had not begun to volitionally ovulate.
Experiment 2, also conducted early in the 2002 spawning season, examined the ovulatory response to LHRHa of 6 large females (0.87–1.75 kg BW) that exhibited pre-spawning morphology (distended abdomens; Mylonas et al., 1995). Microscopic inspection of the ovarian biopsies from these 6 fish revealed that most oocytes were fully vitellogenic (N 500 Am) and this was later confirmed by analysis of the digital micrographs (DIs N 86%). As before, these fish received 50 Ag LHRHa implants (actual dose 29–57 Ag/kg). As in the previous experiment, biopsies from fish selected for this experiment showed no evidence of recent ovulation (i.e. no atretic or hydrated oocytes in the ovarian biopsies or presence of ovarian fluid). After hormonal implantation, the fish were placed in a 5500 L tank and checked for evidence of ovulation as described above. The study was terminated 96 h post-implantation. 2.4. Experiment 3: effect of developmental index on ovulation A third experiment, examining the ovulatory response of fish categorized by DI, was conducted
Fig. 1. Digital micrographs of ovarian follicles in biopsies from black sea bass prior to ovulation induction: (A) hydrated oocytes indicate previous ovulation; (B) uniformly vitellogenic oocytes in a biopsy with a high developmental index; (C) large range in oocyte diameters indicative of a biopsy with a low developmental index; (D) fully grown follicles in a biopsy taken at the time of re-implantation, 10 days following the first spawn. The fish failed to ovulate again.
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during June and July 2002. Thirty-nine females (0.43– 2.0 kg BW) were administered implants with nominal doses of 6.25 or 12.5 Ag LHRHa/implant (7.1–23.0 Ag/kg BW). Six fish served as controls and received sham implants. Fish selected for implantation had mostly fully vitellogenic oocytes (N 500 Am), but the relative proportion of fully vitellogenic oocytes present differed among fish (i.e. different DIs). The ovaries in some fish contained atretic, hydrated oocytes and relatively large volumes of ovarian fluid indicating that volitional ovulation had recently occurred (Fig. 1A). In some cases the ovarian fluid wicked through the biopsy tube by capillary action or could be expressed from the fish with slight abdominal pressure. Evidence of previous ovulation and oocyte maturational stage was recorded for each fish and ovarian biopsy samples were digitally photographed for later analysis. Post implantation, fish were sampled, rechecked and strip-spawned as described previously. Buoyant eggs were pooled for hatching after determining fertilization success. Ten days after spawning was complete, ovarian biopsies were taken from all fish, examined with a dissecting microscope, and ovarian condition was recorded. 2.5. Experiment 4: effect of LHRHa delivery method on ovulation During the subsequent spawning season (June– July 2003) a fourth experiment was conducted. Females (n = 20, 0.6–1.4 kg/BW) received LHRHa either by cholesterol–cellulose implant (12.5 Ag) or by injection of either 20 or 100 Ag/kg BW in 0.9% saline. Six fish received saline and served as controls. As in 2002, some fish in this study had volitionally ovulated previously, as determined by the presence of hydrated atretic follicles and ovarian fluid in biopsy samples. All other selection criteria and handling procedures were the same as described for earlier experiments. Two months after the spawning season ended (September), all fish were examined for evidence of sex reversal by gonadal biopsy and presence of spermiation.
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MANOVA with either developmental index or administration method and dose used as the categorical variables. All testing was performed using Systat 10.0 software (SSI, Richmond, CA, USA). Percent data were arcsine transformed prior to analysis.
3. Results 3.1. Experiments 1 and 2: effect of oocyte size on induced ovulation The ovulatory response to LHRHa was affected by oocyte size. At initiation of the experiment, the largest clutch of oocytes ranged from 400 to 625 Am prior to hormone treatment (Table 1). Neither of the fish with the largest clutch of oocytes measuring 400 Am in diameter ovulated and only 1 of 2 fish with 450 Am oocytes ovulated. In contrast, 90% of fish with 500 Am oocytes spawned within 36 h and 40% of this group spawned a second time. All females containing oocytes N550 Am spawned and one of these fish spawned a second time. Mean buoyancy of all eggs in the first spawns was 71% and ranged from 26% to 100% (Table 1). Fertilization of pooled eggs from the first spawn was 82%. Mean buoyancy of all eggs in the second spawn was 59% and ranged from 40% to 75% (Table 1). Fertilization of pooled eggs from the second spawn was 46%. In experiment 2 which examined the ovulatory success of fish exhibiting pre-spawning morphology (distended abdomens), 5 of 6 fish with uniformly, fully vitellogenic oocytes (one died from causes unrelated to the study, and was extruding eggs upon recovery) ovulated substantial numbers of eggs (45,000–192,000 eggs/kg BW), but fertility following strip spawning was consistently low (0–15%). Fertile eggs were only obtained from the 2 fish with the lowest fecundity (45,000 eggs/kg, 9% fertilization; 75,000 eggs/kg, 15% fertilization). All of the fish in this group had DIs N 86% (Fig. 1B). 3.2. Experiment 3: effect of developmental index on ovulation
2.6. Statistics Spawning data (eggs/kg, # spawns, % buoyant, % buoyant fertilized) were subjected to a one-way
In experiment 3 (2002 season), 19 implanted fish ovulated but fecundity and fertilization were highly variable (Table 2). The number of eggs/kg ovulated
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Table 1 Ovulatory response of black sea bass females implanted with LHRHaa Fish #
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 a b
Weight (kg)
0.78 0.70 0.55 1.09 0.88 0.42 0.79 0.65 1.09 0.72 0.68 0.86 0.62 0.86 1.23 0.71 0.65 0.83
LHRHa (Ag/kg BW)
Maximum oocyte diameter (Am)
Spawn 1
39.0 35.0 27.5 54.5 44.0 21.0 39.5 32.5 54.5 36.0 34.0 43.0 31.0 43.0 61.5 35.5 32.5 41.5
400 400 450 450 500 500 500 500 500 500 500 500 500 500 550 550 600 625
0 0 0 1200b 0 3600 37,200 40,800 3600 7200 18,000 15,600 18,000 13,200 52,800 180,000 12,000 55,200
Table 2 Effect of developmental index on egg production and fertilization in black sea bassa,b Developmental Spawns index (%) (#) b60 61–70 71–80 N80 a
Eggs produced
Buoyant eggs (%)
Eggs produced
Buoyant eggs (%)
30,000 63,600 80,400 10,800
48 40 75 67
72,000
63
0 67 45 62 67 100 80 62 87 91 91 73 70 26
All fish received 50 Ag LHRHa in a cholesterol–cellulose implant. Eggs discarded and not incorporated into treatment mean values.
per spawn and the fertilization rate ranged from 17,000–339,000 and 0–98%, respectively. None of the control fish ovulated. No differences were found in the number of spawns, fecundity, egg buoyancy or fertilization success among fish with different developmental indices (Table 2). Ovulation occurred from 1 to 4 days in succession and egg quality (buoyancy and fertilization) did not decrease from the first (71.6%, 43%) to the second spawn (82.8%, 52.3%). The degree of ovarian development, as determined by the DI, was not an accurate predictor of ovulatory response or egg quality. Fish with DIs as low as 45% (Fig. 1C)
1.0 F 0.0 1.7 F 0.7 2.3 F 0.4 1.6 F 0.4
Eggs/kg Buoyant Buoyant eggs BW (103) (%) fertilized (%) 63 F 11 67 F 26 86 F 21 68 F 22
98 F 2.0 54 F 25 78 F 4.3 81 F11
73 F 13 33 F 18 65 F 7.4 69 F 12
Data are mean F S.E.M. There were no statistical differences among spawning data categories when compared to the development index. b
Spawn 2
ovulated relatively high numbers of eggs, while some with indices as high as 90% failed to do so (Table 3). The one fish that was re-implanted 10 days after spawning relatively few eggs with low fertilization Table 3 Developmental indices and LHRHa doses for black sea bass females that failed to ovulate Fish #
Weight (kg)
LHRHa (Ag/kg BW)
Developmental index (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1.99 1.00 0.76 0.53 0.62 0.65 0.64 0.71 0.76 0.57 0.67 0.53 0.83 0.70 0.68 0.74
6.3 12.5 8.2 23.6 10.1 9.6 9.8 8.8 8.2 11.0 9.3 11.8 7.5 8.9 18.4 8.4
34 NA 8 96 NA 81 69 55 60 80 61 80 90 66 67 61
NA means that no data were available.
D.L. Berlinsky et al. / Aquaculture 250 (2005) 813–822 Table 4 LHRHa dose and administration method on egg production and fertilizationa,b LHRHa (Ag/kg)
Spawns (#)
Eggs/kg BW (103)
Buoyant (%)
Buoyant eggs fertilized (%)
Injection 20 100
2.0 F 0.4 2.0 F 0.3
146 F 40.1 85 F 14
65 F 8.4 69 F 10
40 F 12 38 F 9.2
Implant 12.5 Ag
1.4 F 0.2
92 F 14
80 F 8.1
38 F 10
a
Data are mean F S.E.M. b There were no statistical differences among spawning data categories when compared to administration method or dose.
(DI = 96%, Table 3) did not ovulate again, although the biopsy sample had fully grown follicles (Fig. 1D). One fish with a 90% DI that did not ovulate (Table 3) was spermiating when examined 2 months after the spawning season. Hatching rate of pooled embryos ranged from 53% to 76%. 3.3. Experiment 4: effect of LHRHa delivery method on ovulation In experiment 4 conducted in 2003, 11 of 13 LHRHa-injected fish ovulated at least once, 5 fish ovulated on 2 consecutive days and 3 fish ovulated on 3 consecutive days. Three of seven implanted fish ovulated once and 2 fish ovulated on consecutive days. No differences were found among females given implants or an injection (20 or 100 Ag/kg) with respect to the mean number of eggs/kg produced, the percentage of buoyant eggs or fertilization success. The mean number of spawns for fish injected with 20 (2.0 F 0.4) or 100 Ag (2.0 F 0.3) LHRHa was not significantly greater than that (1.4 F 0.2) for implanted fish (Table 4). Incubation of pooled eggs resulted in hatching of approximately 155,000 larvae (60% hatch) of which approximately 95,000–1.0 g juveniles were produced (61%).
4. Discussion Synthetic analogues of LHRH have been effectively used to induce ovulation and spawning in a number of commercially important finfish including many serranid species (Tucker, 1994; Mylonas and
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Zohar, 2001). Under favorable conditions, captive black sea bass will tank spawn volitionally or following administration of LHRHa, but spawning is not synchronized and fertilization success is highly variable (Cerda´ et al., 1996; Watanabe et al., 2003). Additionally, tank spawning is often prolonged over a period of weeks, which extends the labor-intensive larviculture period and the need for algal and livefeed production. In vitro fertilization is often employed to control parentage for genetic improvement and manipulation of ploidy conditions. In the present studies, commercial quantities of black sea bass larvae were produced, and the spawning period was contracted to a few days, by using in vitro fertilization following LHRHa administration. Analogues of LHRH have often been favored over gonadotropic hormone preparations (pituitary extracts and hCG) because they are non-immunogenic and provide more integrated control of final oocyte maturation (FOM) and ovulation (Zohar and Mylonas, 2001). Since LHRHa is rapidly cleared from the circulatory system (half-life 0.8 h in chinook salmon, Slater et al., 1999), multiple injections or sustainedrelease implants are often more effective than single injections for inducing multiple ovulations in batch spawning fishes with group-synchronous oocyte development (Mylonas et al., 2003). In some species, such as spotted seatrout (Cynoscion nebulosus) and red drum (Sciaenops ocellatus), with fully grown oocytes, a single injection or oral administration of LHRHa is sufficient for ovulation induction (Thomas and Boyd, 1988, 1989). Sustained release implants may be more advantageous than injections for inducing FOM and ovulation in fish with less developed ovaries, however. For instance, wild striped bass (Morone saxatilis) collected far from their spawning grounds, which have not initiated FOM, require slowrelease implanted LHRHa for successful ovulation (Hodson and Sullivan, 1993). LHRHa implants were also used to significantly advance the spawning season of winter flounder (Pleuronectes americanus; Larsson et al., 1997). In the present study, ovulation was induced in black sea bass just prior to, or during, the natural spawning season. Under these conditions, LHRHa administered as a single injection was as effective as implanted LHRHa for inducing ovulation. Human chorionic gonadotropin has also been used to induce ovulation in black sea bass with fully vitello-
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genic oocytes (Hoff, 1970; Tucker, 1984), and has been shown to be as effective as LHRHa for inducing ovulation in related Epinephelus grouper species (Watanabe et al., 1995). Considering black sea bass in spawning condition, it appears that the degree of ovarian development may be more important than the gonadotropic-inducing agent used to stimulate ovulation. Given the success obtained using hCG to induce ovulation in many serranid species (Tucker, 1994; Watanabe et al., 1995) and its recent approval by the U.S. Food and Drug Administration for use on broodstock, a direct comparison of LHRHa and hCG is warranted. The efficacy of LHRH analogues has also been shown to be influenced by dose, primary peptide structure, and water temperature (Harmin and Crim, 1992; King and Pankhurst, 2004). Previous studies of black sea bass demonstrated that a single cholesterol– cellulose pellet containing 50 Ag LHRHa (approximately 50 Ag/kg) successfully induced multiple spawning events over a prolonged period (Watanabe et al., 2003). In the present study, this dose was also administered to pre-spawning black sea bass that exhibited grossly distended abdomens and high DIs (N86%). All fish in this condition ovulated large quantities of eggs, but fertilization percentage was low. It is possible that the dose administered to these fish (mean dose, 47.2 Ag/kg) was excessive and may have prematurely stimulated FOM in underdeveloped oocytes (Taranger et al., 1992). Fertilization and hatching was adversely effected in Asian sea bass (Lates calcarifer) administered high doses of LHRHa (150–300 Ag/kg) as compared to lower dosages (4.5–75 Ag/kg; Garcia, 1989). High LHRHa doses have been shown to diminish egg quality or induce fewer ovulations in a number of other species (Mylonas et al., 1992; Poortenaar and Pankhurst, 2000; Marino et al., 2003). While the optimal dose of LHRHa has not been established for black sea bass, implants with actual doses as low as 7.1 Ag/kg were effective for inducing ovulation without decreasing fecundity or fertility. The responsiveness of black sea bass to LHRHa may change over time and for the first and subsequent spawns. Cerda´ et al. (1997) demonstrated that the maturational response of black sea bass oocytes to hCG in vitro declined as the spawning season progressed.
The frequency of spawning (inter-ovulatory cycle) has also not been identified in black sea bass. In the present study, fish were generally strip spawned at 24 h intervals following the first ovulation and no attempt was made to determine the inter-ovulatory cycle. Two fish were spawned in the morning and afternoon of the same day with high rates of fertilization. It is not known whether these represented separate or single, interrupted ovulatory events. Watanabe et al. (2003) reported that female black sea bass tank spawned for up to 4 days following implantation with LHRHa, but eggs were only collected on a daily basis. During the spawning season in the present study, the oocyte composition in the ovary included early and late vitellogenic oocytes and the DIs varied greatly among individuals. Furthermore, hydrated and atretic follicles were often observed in ovarian biopsies prior to LHRHa administration but were not always present in biopsy samples taken from fish 10 days after induced spawning. The variable responsiveness to administered LHRHa during the spawning season may reflect the number of previous spawning events completed and/or the time-point in the ovulatory cycle when the hormone was administered. Similar oocyte frequency distributions were observed in Nassau grouper (Epinephelus striata), a related protogynous hermaphrodite, in which repeated ovulatory events could be stimulated within a single season (Watanabe et al., 1995). Further research is necessary to establish the ovulatory cycle and period of over-ripening in order to minimize fish handling and preserve egg quality. Successful FOM and ovulation requires coordinated synthesis of gonadotropin and maturation-inducing steroid receptors and shifts in steroid enzymatic pathways (Nagahama et al., 1994). Typically, the efficacy of gonadotropic compounds is greatest in fish that have completed vitellogenesis and initiated FOM (Harmin and Crim, 1992); however, fertilization success is often variable and may be influenced by nutritional and environmental factors (Watanabe et al., 1995; Berlinsky et al., 1996). Black sea bass oocytes that have completed vitellogenesis are 475– 600 Am in diameter and can be induced to ovulate with hCG (Tucker, 1984, 1994) and LHRHa (Watanabe et al., 2003). In the present study, responsiveness to LHRHa was greatest in oocytes N500 Am. These results agree with in vitro studies of Cerda´ et al.
D.L. Berlinsky et al. / Aquaculture 250 (2005) 813–822
(1996) who reported that black sea bass oocytes 490– 600 Am were more responsive to hCG than those 350– 390 Am in diameter. Watanabe et al. (2003) also found that all females that tank spawned after LHRHa implantation had maximum oocyte diameters N475 Am. The proximity to sex inversion may also influence oocyte responsiveness to LHRHa, as 1 fish with a high DI (90%), that did not spawn, was found to be a spermiating male when examined 2 months after the spawning trials. It is not known if administration of LHRH analogues can hasten sex change in black sea bass as has been reported in the gilthead seabream (Sparus aurata; Vilia and Canario, 1995) and the bluehead wrasse (Rhalassoma bifasciatum, Kramer et al., 1993). In summary, black sea bass with fully vitellogenic oocytes are responsive to LHRHa delivered in an intramuscular injection or a slow release implant. The responsiveness to the hormone appears to be influenced more by the degree of oocyte development and spawning history than dose or delivery method. Acknowledgements We thank the personnel at Great Bay Aquaculture for use of their broodstock and assistance throughout these studies. This study was supported by grants from New Hampshire Sea Grant (award number NA16RG1035) and the National Sea Grant College Program (award number NA16RG1561). This is contribution number 562 from South Carolina Marine Resources Division and number 2265 from the New Hampshire Agricultural Experiment Station. References Atwood, H.L., Young, S.P., Tomasso Jr., J.R., Smith, T.I.J., 2003. Effect of temperature and salinity on survival, growth, and condition of juvenile black sea bass Centropristis striata. J. World Aquac. Soc. 34, 398 – 402. Bender, J., Lee, R., Sheppard, M., Bulski, K., Phillips, P., Yeboah, Y., Wah, R.C., 2004. A waste effluent treatment system based on microbial mats for black sea bass Centropristis striata recycledwater mariculture. Aquac. Eng. 31, 73 – 82. Berlinsky, D.L., King V, W., Smith, T.I.J., Hamilton, R.D., Holloway, J., Sullivan, C.V., 1996. Induced ovulation of southern flounder Paralichthys lethostigma using gonadotropin releasing hormone analogue implants. J. World Aquac. Soc. 27, 143 – 152.
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Berlinsky, D., Watson, M., Nardi, G., Bradley, T., 2000. Investigations of selected parameters for growth of larval and juvenile black sea bass Centropristis striata L.. J. World Aquac. Soc. 31, 426 – 435. Berlinsky, D.L., Taylor, J., Howell, R.A., Bradley, T.M., Smith, T.I.J., 2004. The effects of temperature and salinity on early life stages of black sea bass (Centropristis striata). J. World Aquac. Soc. 35, 335 – 344. Cerda´, J., Selman, K., Wallace, R.A., 1996. Observations on oocyte maturation and hydration in vitro in the black sea bass, Centropristis striata (Serranidae). Aquat. Living Resour. 9, 325 – 335. Cerda´, J., Selman, K., Hsiao, S.-M., Wallace, R.A., 1997. Evidence for the differential regulation of ovarian follicle responsiveness to human chorionic gonadotropin in vitro in a serranid teleost, Centropristis striata. Aquaculture 159, 143 – 157. Copeland, K.A., Watanabe, W.O., Carroll, P.M., 2002. Growth and feed utilization of captive wild subadult black sea bass, Centropristis striata, fed practical diets in a recirculating system. J. World Aquac. Soc. 39, 97 – 109. Cotton, C.F., Walker, R.L., Recicar, T.C., 2003. Effects of temperature and salinity on growth of juvenile black sea bass, with implications for aquaculture. N. Am. J. Aquac. 65, 330 – 338. Earll, R.E., 1884. Hatching blackfish and Spanish mackerel. Bulletin U. S. Fish Commission, vol. 1884. Government Printing Office, Washington, DC, USA, pp. 415 – 416. Frame, D.W., Pearce, S.A., 1973. A survey of the sea bass fishery. Mar. Fish. Rev. 35, 19 – 26. Garcia, L.M.B., 1989. Dose-dependent spawning response of mature female sea bass, Lates calcarifer (Bloch), to pelleted luteinizing hormone-releasing hormone analogue (LHRHa). Aquaculture 77, 85 – 96. Harmin, S.A., Crim, L.W., 1992. Gonadotropic hormone-releasing hormone analog (GnRH-a) induced ovulation and spawning in female winter flounder, Pseudopleuronectes americanus (Walbaum). Aquaculture 104, 375 – 390. Hodson, R.G., Sullivan, C.V., 1993. Induced maturation and spawning of domestic and wild striped bass, Morone saxatilis (Walbaum), broodstock with implanted GnRH analogue and injected hCG. Aquac. Fish. Manage. 24, 389 – 398. Hoff, F.H., 1970. Artificial spawning of black sea bass, Centropristis striatus melanus, Ginsburg, aided by chorionic gonadotropic hormones. Florida Department of Natural Resources Marine Research Laboratory. Special Science Report 25, 17 pp. St. Petersburg, Florida, USA. King, H.R., Pankhurst, N.W., 2004. Effect of maintenance at elevated temperatures on ovulation and luteinizing hormone releasing hormone analogue responsiveness of female Atlantic salmon (Salmo salar) in Tasmania. Aquaculture 223, 583 – 597. Kjorsvik, E., Mangor-Jensen, A., Holmefjord, I., 1990. Egg quality in fishes. Adv. Mar. Biol. 26, 71 – 113. Kramer, C.R., Caddell, M.T., Bubenheimer-Livolsi, L., 1993. sGnRH-a [(d-Arg6, Pro9, Net) LHRH] in combination with domperidone induces gonad reversal in a protogynous fish, the bluehead wrasse, Thalassoma bifasciatum. J. Fish Biol. 42, 185 – 195. Larsson, D.G.J., Mylonas, C.C., Zohar, Y., Crim, L.W., 1997. Gonadotropin-releasing hormone analogue (GnRHa) induces
822
D.L. Berlinsky et al. / Aquaculture 250 (2005) 813–822
multiple ovulations of high-quality eggs in a cold water, batchspawning teleost, the yellowtail flounder (Pleuronectes ferrugineus). Can. J. Fish. Aquat. Sci. 54, 1957 – 1964. Low Jr., R.A., 1981. Mortality rates and management strategies for black sea bass off the southeast coast of the United States. North Am. J. Fish. Manage. 1, 93 – 103. Marino, G., Panini, E., Longobardi, A., Mandich, A., Finoia, M.G., Zohar, Y., Mylonas, C.C., 2003. Induction of ovulation in captive-reared dusky grouper, Epinephelus marginatus (Lowe, 1834), with a sustained-release GnRHa implant. Aquaculture 219, 841 – 858. Mercer, L.P., 1978. The reproductive biology and population dynamics of black sea bass, Centropristis striata. PhD thesis, College of William and Mary, Williamsburg, VA. 196 pp. Musick, J.A., Mercer, L.P., 1977. Seasonal distribution of black sea bass Centropristis striata, in the Mid-Atlantic Bight with comments on ecology and fisheries of the species. Trans. Am. Fish. Soc. 106, 12 – 25. Mylonas, C.C., Zohar, Y., 2001. Use of GnRHa-delivery systems for the control of reproduction in fish. Rev. Fish Biol. Fish. 10, 463 – 491. Mylonas, C.C., Hinshaw, J.M., Sullivan, C.V., 1992. GnRHa-induced ovulation of brown trout (Salmo trutta) and its effects on egg quality. Aquaculture 106, 379 – 392. Mylonas, C.C., Zohar, Y., Richardson, B.M., Minkkinen, S.P., 1995. Induced spawning of wild American shad Alosa sapidissima using sustained administration of gonadotropin-releasing hormone analog (GnRHa). J. World Aquac. Soc. 26, 240 – 251. Mylonas, C.C., Sigelaki, I., Divanach, P., Manano˜s, E., Carrillo, M., Afonso-Polyviou, A., 2003. Multiple spawning and egg quality of individual European sea bass (Dicentrarchus labrax) females after repeated injections of GnRHa. Aquaculture 221, 605 – 620. Nagahama, Y., Yoshikuni, M., Yamashita, M., Tanaka, M., 1994. Regulation of oocyte maturation in fish. In: Hoar, W.S., Randall, D.J. (Eds.), Fish Physiology, vol. 13. Academic Press, New York, USA, pp. 393 – 439. Poortenaar, C.W., Pankhurst, N.W., 2000. Effect of luteinising hormone-releasing hormone analogue and human chorionic gonadotropin on ovulation, plasma and ovarian levels of gonadal steroids in greenback flounder Rhombosolea tapirina. J. World Aquac. Soc. 31, 175 – 185. Roberts Jr., D.E., Harpster, B.V., Havens, W.K., Halscott, K.R., 1976. Facilities and methodology for culture of the southern sea bass (Centropristis melana). Proc. World Maric. Soc. 7, 163 – 198. Shehedeh, Z.H., Kuo, C.-M., Milisen, K.K., 1973. Validation of an in vivo method for monitoring ovarian development in the grey mullet (Mugil cephalus L.). J. Fish Biol. 5, 479 – 487. Shepherd, G.R., Idoine, J.S., 1993. Length based analyses of yield and spawning biomass per recruit for black sea bass Centropristis striata, a protogynous hermaphrodite. Fish. Bull., U.S. 91, 328 – 337.
Shepherd, G.R., Terceiro, M., 1994. The summer flounder, scup and black sea bass fishery of the middle Atlantic bight and southern New England waters. NOAA Technical Report NMFS, vol. 122. US Department of Commerce, Seattle, WA. Sherwood, N.M., Crim, L.W., Carolfeld, J., Walters, S.M., 1988. Sustained hormone release: I. Characteristics of in vitro release of gonadotropin releasing hormone analogue LHRHa from pellets. Aquaculture 74, 75 – 86. Slater, C.H., Schreck, C.B., Sower, S.A., 1999. Rapid clearance of d-Ala6, Pro9Net mammalian gonadotropin releasing hormone from chinook salmon plasma. N. Am. J. Aquac. 61, 315 – 318. Stuart, K.R., Smith, T.I.J., 2003. Development of nursery systems for black sea bass (Centropristis striata). J. World Aquac. Soc. 34, 359 – 367. Taranger, G.L., Stefansson, S.O., Hansen, T., 1992. Advancement and synchronization of ovulation in Atlantic salmon (Salmo salar L.) following injections of LHRH analogue. Aquaculture 102, 169 – 175. Thomas, P., Boyd, N., 1988. Induced spawning of spotted seatrout, red drum and orangemouth corvina (Family: Sciaenidae) with luteinizing hormone analog injection. Contrib. Mar. Sci. 30, 43 – 47 (Supp. to Vol.). Thomas, P., Boyd, N.W., 1989. Dietary administration of an LHRH analogue induces spawning of spotted seatrout (Cynoscion nebulosus). Aquaculture 80, 363 – 370. Tucker Jr., J.W., 1984. Hormone-induced ovulation of black sea bass and rearing of larvae. Prog. Fish-Cult. 46, 201 – 203. Tucker Jr., J.W., 1994. Spawning by captive serranid fishes: a review. J. World Aquac. Soc. 25, 345 – 359. Vilia, P., Canario, A.V.M., 1995. Effect of LHRH on sex reversal and steroid levels in gilthead seabream (Sparus aurata). In: Goetz, F., Thomas, P. (Eds.), Proceedings of the Fifth International Symposium on the Reproductive Physiology of Fish, Fish Symp., vol. 95. The University of Texas at Austin, p. 329. Wallace, R.A., Selman, K., 1981. Cellular and dynamic aspects of oocyte growth in teleosts. Am. Zool. 21, 325 – 343. Watanabe, W.O., Ellis, S.C., Ellis, E.P., Head, W.D., Kelley, C.D., Moriwake, A., Lee, C., Bienfang, P.K., 1995. Progress in controlled breeding of Nassau grouper (Epinephelus striatus) broodstock by hormone induction. Aquaculture 138, 205 – 219. Watanabe, W.O., Smith, T.I.J., Berlinsky, D.L., Woolridge, C.A., Stuart, K.R., Copeland, K.A., Denson, M.R., 2003. Volitional spawning of black sea bass (Centropristis striata) induced with pelleted luteinizing hormone releasing hormone-analogue. J. World Aquac. Soc. 34, 319 – 331. Wenner, C.A., Roumillat, W.A., Waltz, C.W., 1986. Contributions to the life history of black sea bass, Centropristis striata, off the southeastern United States. Fish. Bull. 84, 723 – 741. Zohar, Y., Mylonas, C.C., 2001. Endocrine manipulations of spawning in cultured fish: from hormones to genes. Aquaculture 197, 99 – 136.