B. Glamuzina, B. Skaramuca, N. Glavic, V. Kozul-1998
Aquaculture Research, 29: 769-771
Short communication:
The dusky grouper, Epinephelus marginatus (Lowe, 1834) is a popular fish for both sport and commercial fishery activities throughout the Mediterranean. The high market price (above US$ 15 in Croatia and Italy) and low standing stock of dusky grouper caused by over-exploitation of natural populations in most Mediterranean countries, combined with its fast growth and good adaptation to captivity, make this species an attractive candidate for intensive culture. Investigations on the culture of dusky grouper have been initiated in some Mediterranean countries, but published reports are scarce (Spedicato et al. 1995). The present paper gives preliminary data on sexual maturation in captivity, artificial spawning with hormonal treatments and the results of rearing trials with larvae.
Artificial spawning using broodstock raised from wild fingerlings was attempted throughout 1996 and 1997. During both years, the development of gonads was followed once a month by taking biopsy samples from the ovaries via the insertion of a 1.2-mm plastic tube in the oviduct. The fish were injected with 2000 IU/kg of human chorionic gonadotropin (HCG) in 1996 and with 200 microg/kg of analogues of mammalian luteinizing hormone releasing hormone (LHRH(a)) in 1997. Because of the limited number of females, the control groups injected with blank solution were not used. Females were induced to ovulate with two injections of hormone spaced 24 h apart. Eggs were manually stripped 16 h after the second injections and fertilized with milt from three sex-reversed males which was obtained as described previously. The fertilised eggs and hatched larvae were incubated in 300-L plastic tanks with a flow-through of ambient sea water (temperature = 23 C ; and salinity = 38 ppt). The larvae were fed with rotifers.
In the period from October to the end of May, the ovaries of the dusky grouper consisted of previtellogenic oocytes with a diameter of 30-80 microm. Two types of oocytes were observed: smaller oocytes (30-50 microm) without a differentiated nucleus and with cytoplasm darkly stained with eosin; and larger oocytes (50-80 microm) with clear cytoplasm and a visible nucleus. At the beginning of June, when sea water temperatures rose to 20 C, the larger oocytes started to grow and develop vitellogenesis. The smaller oocytes remained the same over the whole summer. The ovaries of dusky grouper in captivity are similar in appearance to those reported in wild fish with two superimposed generations of oocytes and a one-year shift between them. Vitellogenesis started at the beginning of June at the same time as the warming of the sea water to 20 C. This is 2 months later than populations in the warmer waters of Tunisia, suggesting that temperature is important in the maturation of dusky grouper.
All fish responded positively to HCG in 1996, but produced only small quantities of eggs (25-90 g). Only one fish responded positively to LHRH(a) in 1997, and only after the second treatment, producing 1080 g of eggs in two strippings. The other fish did not respond, not even after repeated treatments with LHRH(a) 15 days later, in spite of the biopsy showing the presence of mature oocytes. One reason for the poor result with LHRH(a) in 1997 may have been the unexpected cooling of the sea water near Dubrovnik from 23 C to 16-17 C over a period of 20 days in July. This rapid cooling may possibly have influenced the maturation process. However, a better understanding of the reproduction cycle of dusky grouper in captivity is needed in order to set up a final protocol for induced spawning.
Dusky grouper held in captivity under the ambient seawater conditions of Southern Adriatic can be induced to ovulate during the second half of August and early September, i.e. the same period as natural spawning in these waters, whereas these fish spawn in June and July in the warmer waters of Tunisia, and during early August in Spanish waters. These results indicate that the spawning season is temperature dependent, starting in June along the coast of Northern Africa and finishing in September in the waters of North Adriatic.
One female treated with LHRH(a) produced 580 g of eggs when stripped 16 h after second hormone injection, and 500 g of eggs the next day. The fertilization rate with mixed milt from three sex-reversed males was 95% for the first batch of eggs and 50% for the second. The diameter of fertilized eggs from dusky grouper ranged between 738 and 940 microm (mean = 846 +/- 41 microm). The eggs were transparent and spherical with a smooth chorion. Good eggs had only one oil globule, while bad eggs had two or more. The yolk was unsegmented and homogenous. At 23 C, the first cleavage occurred about 1 h 5 min after fertilization. Hatching started 30 h after fertilization and all larvae hatched within 3 h. The average ripe egg diameter was smaller than the 888 microm found in eggs obtained from fish held in captivity in Italy. Egg diameter has previously been recorded as 832 microm for wild fish spawning in the south-eastern Adriatic. The total length of newly hatched larvae in the present study was 1.52 +/- 0.066 mm, which is smaller than the larvae of all investigated world groupers and other cultivated fish in the Mediterranean area.
A period of increased eggs mortality was observed at the gastrula stage when about 10% of eggs died. The total mortality from fertilization to hatch was 27%. About 25% of hatched larvae were deformed (mostly in the spine). A steady decline in larval density in water samples indicated a constant rate of mortality from the beginning of incubation through the various phases of development. The mouths of the dusky grouper started to open 65 h after fertilization and the mouths of all the larvae were completely functional by 96 h. The maximun gape of the open mouth was between 250 and 300 microm. The functional mouth opening of dusky grouper larvae, based on assumption that it presents only 40% of maximum mouth opening, was estimated as 98-120 microm. Based on the observed mouth opening, about 70% of added rotifers had a lorica width suitable as first prey.
Rotifers were initially found in the stomachs. However, considering the massive larval mortality linked to crystal formations in the urinary bladder, the present authors cannot confirm the suitability of rotifers as a first food for dusky grouper larvae. The first larval prey is the major problem in rearing of all grouper species. Some authors have suggested that small screened rotifers with a lorica width of less than 90 microm are good prey, while others have recommended the introduction of oyster trochophores as first food either alone or in combination with small rotifers.
Crystal formation in the urinary bladder was apparent for all the larvae that had started feeding, the crystals becoming as large as the bladder itself. On the sixth day after hatching, most of the larvae had sunk to the bottom of the tanks and a massive mortality of remaining larvae occurred during the following 4 days. The present authors did not investigate the reasons for the development of crystals in the urinary bladder. The same formation was observed in the early larvae of Epinephelus fuscoguttatus (Forsskal, 1775). There are some hypotheses regarding the calculus formation in larval fish, but more research is needed in order to present an explanation for their development.
(Laboratory for Ecology and Aquaculture, Institute of Oceanography and Fisheries, Dubrovnik, Croatia)