State of Art for Larval Rearing of Grouper  

Zhenhua Ma1,2 , Huayang Guo1,2 , Nan Zhang1,2 , Zemin Bai3
1 South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China
2 Key laboratory of South China Sea Fishery Rescources Exploitation and Utilization, Ministry of Agriculature, Guangzhou, 510300, China
3 School of Applied Science, Temasek Polytechnic, 21 Tampines Avenue 1, 529757, Singapore
Author    Correspondence author
International Journal of Aquaculture, 2013, Vol. 3, No. 13   doi: 10.5376/ija.2013.03.0013
Received: 15 Apr., 2013    Accepted: 22 Apr., 2013    Published: 30 May, 2013
© 2013 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Ma et al., 2013, State of Art for Larval Rearing of Grouper, International Journal of Aquaculture, Vol.3, No.13 63-72 (doi: 10.5376/ija.2013. 03.0013)


Groupers belonging to the family of Serranidae, are widely distributed throughout sub-tropical and tropic water area. With increasing marketing demands and reduced natural resource, groupers have been considered have high aquaculture potential in tropical and subtropical waters. However, since the first attempt to culture groupers, significant problem exists in fingerling production, particular with survival rates are low and inconsistent. In this review, we will use the life cycle of grouper larvae as a framework to review internal factors regulating ontogenetic development in fish larvae and environmental factors affecting grouper larvae development. To understand the cause of high larvae mortality in early life history, we will review factors related to the ontogenetic development, and then we will focus on issues of first feeding of grouper larvae in intensive aquaculture. At the end, we will review the management strategies of using live feeds in grouper hatcheries.

Grouper; Larvae culture; Live feeds; Growth and survival rate

Groupers are species belonging to the family of Serranidae that are very important for sport and commercial fisheries (Glamuzina et al., 1998). They are widely distributed throughout sub-tropic and tropic water area. Because of the high flesh quality and elegant appearance, groupers are very attractive for human consumption. With the increasing demands in the markets, the global production of groupers reached to 198 690 mt in 2007 (Harikrishnan et al., 2011). In the live markets of China, groupers have brought high prices (up to US$70-100/kg wholesale) (McGilvray and Chan, 2001; Harikrishnan et al., 2011). Increasing marketing request and reduced natural resource made grouper being considered have high aquaculture potential in tropical and sub-tropical waters (Liao et al., 2001; Marte, 2003).

Up to present, reliable fingerling productions are still the issues hindering the development of groupers’ aquaculture industries. Since 1970s, efforts have been made to mass produce artificial grouper fingerlings (Lim, 1993). Species such as Epinephelus tauvina, E. salmoides, E. akaara, E. malabaricus, E. striatus (Chen et al., 1977; Xu et al., 1985; Huang et al., 1986; Maneewong et al., 1986; Tucker et al., 1991) have been reported for successful spawning. However, as a consequence of massive mortalities in the early development stage, grouper aquaculture remains heavily dependent on the capture and grow-out of wild-caught juvenile. According to the results published in 2003, around 70%~85% of culture groupers were grown out from wild-caught fry (Sadovy et al., 2003). Recently, as a result of successful out-door-ponds culture grouper larvae in China, China has become the biggest grouper fry supplier in Asian.Although artificial breeding has been steadily increased recently, there is a still big gap in the fry market.
Studies show that the early survival rates of groupers are very low when compared to other finfish (Duray et al., 1996; Duray et al., 1997). High mortality during the early life stages has been observed in reared grouper larvae, such as Epinephelus fuscoguttatus, Epinephelus coioides, Epinephelus suillus (Duray et al., 1997; Toledo et al., 1999; Kohno et al., 1990). Although a series of studies have been conducted to explore the optimum rearing protocol for groupers larvae, up to present most of the results are still dissatisfaction as heavy mortalities still often observed within the first two weeks after hatching (Duray and Kohno, 1988; Kohno et al., 1990; Kohno et al., 1997). Difficulties in rearing early stage larvae of groupers have become the major bottleneck hindering the development of mass fingerling production (Kohno et al., 1997). Marte (2003) summarized the difficulty of rearing grouper into three area: 1) spawned eggs and larvae are very small and the small mouth gape in early larvae limits the choice of initial live feed; 2) grouper are extremely sensitive to mechanical disturbance; 3) long duration of larval rearing (>60 days).
In this review, we will use the life cycle of grouper larvae as a framework to review internal factors regulating ontogenetic development in fish larvae and environmental factors affecting the general develoment of grouper larvae. To understand the cause of high larvae mortality in early life history, we will review factors related to the ontogenetic development, and then we focus on issues of first feeding of grouper larvae in intensive aquaculture. At the end, we will review the management strategies of using live feeds in grouper hatcheries.
1 Ontogenetic Development
1.1 Eggs and Embryo
The eggs size of groupers is generally less than one millimeter (Table 1). Like most marine teleosts, nutrition during the embryonic phase is derived from yolk reserve (Ma et al., 2012). The embryonic period starts from fertilization and ends at the commencement of exogenous feeding. It is divided into three major phase: cleavage egg, embryo, and free embryo (Moyle and Cech, 2003). Figure 1 illustrates the embryonic development of Malabar grouper Epinephelus malabaricus. Cleavage egg (A-F), and embryo (G-L) are defined according to Moyle and Cech (2003). Embryonic development is a complex process and egg quality and hatching environments directly affect embryonic development and the sizes of fish at hatching and first feeding (Robin and Gatesoupe, 2001).
Table 1 Comparison of the eggs and larvae of Epinephaline Serranids

Figure 1 Embryonic development of Epinephelus malabaricus

1.1.1Egg quality
Egg quality is generally derived from broodstock nutrition (Izquierdo et al., 2001; Mazorra et al., 2003; Sawanboonchun et al., 2008; Ma et al., 2012). Since protein, lipoprotein, glycogen, and enzymes contents in yolk reserve directly affect embryonic development (Gunasekera et al., 1995; Harrell and Woods, 1995; Sargent et al., 1999), proper controlled broodstocks nutrition is essential in breeding marine fish. For instance, Dhert et al. (1991) showed that E. tauvina broodstock given trash fish injected the emulsified enrichment diet Marila diet significantly increased oil globule diameter, total lipids, eicosapentaenoic acid, docosahexaenoic acid, and larval survival at day 7. Nutrients such as essential fatty acids (EFA) (Fernández-Palacios et al., 1995), vitamin E (Fernández-Palacios et al., 1998), carotenoids (Craik, 1985), vitamin C (Blom and Dabrowski, 1995), dietary protein, vitamin B1, and vitamin B6 (Izquierdo et al., 2001) in broodstock diets have been considered as essential for the normal development of embryos (Ma et al., 2012). Evidence indicate that the percentage of normal eggs increases with the increase of n -3 highly unsaturated fatty acids (HUFA) in broodstocks diets of gilthead seabream (Sparus aurata) (Fernández-Palacios et al., 1995). Therefore, proper control of broodstocks nutrition can improve egg quality and enhance survivorship in marine fish larvae (Izquierdo et al., 2001).

1.1.2 Temperature
Temperature can also affect embryonic development of grouper larvae. Watanabe et al. (1995) found that the variations in water temperatures within an ecological range can markedly influence development rates and survival of pre-feeding Nassau grouper (Epinephelus striatus) larvae. Table 1 shows the eggs diameter, incubation temperature, hatching time in 10 grouper species. Clearly, during the grouper embryonic development, with the increased incubation temperature, the hatching time was decreased. Similarly, studies in other species have been demonstrated that higher temperature accelerates development rate (Bermudes and Ritar, 1999; Das et al., 2006; Moran et al., 2007; Kazuyuki et al., 1988; Miranda et al., 1990). For instance, the development rate of mackerel (Scomber scombrus) eggs at 17.8 is almost three times faster than at 8.6 (Figure 2, Figure 3). A similar result has also been reported in striped trumpeter (Latris lineata) when compared with incubation temperatures between 16.2 and 8.1℃ (Bermudes and Ritar, 1999). However, high temperature over a tolerable range may lead to heat shock and fish mortality (Hopkins and Dean, 1975; Kiyono and Shinshima, 1983).

Figure 2 Functional relationship between developmental time, temperature and embryonic stage of mackerel eggs

Figure 3 Fitted relationship between developmental time and temperature at each embryonic stage (Mendiola et al., 2006)
Not like other species, grouper eggs and newly hatched larvae (such as Plectropomus leopardus) are very sensitive to stress and handling (personal conmunication with Mr Zhang, Xincun, Hainan Province, P.R. China 2013). Therfore, to minimize the handling related mortality, stocking activity are recommended to conduct at neurula-stage (after the formation of the optic vesicles) and by stocking eggs into the culture tanks 2 h before hatching so that the directly handle larvae can be avoided (Lim, 1993; Tamaru et al., 1995, Caberoy and Quninitio 1998). Furthermore,The larvae are sensitive to light during the early stages of their development and are generally kept in darkened conditions.
1.2 Larvae
Newly hatched grouper larvae are generally less than two millimeters (Table 1), with different structure, morphology and function from adult (Figure 2) (Bone et al., 1995). Similar with most fish larvae, special larval structure relevant to respiration may develop to increase the area to volume ratio for gas exchange (Houde, 2001). Grouper larvae are delicate and have a large yolk sac and an undeveloped mouth, fins, and eyes (Figure 4). By 3-4 day post hatching, the yolk and oil globule will be absorbed completed (Table 1). The larval stage starts from exogenous feeding and ends with completing metamorphosis. Like most marine fish, massive mortality normally occurs during larval stage because of vulnerability of larvae to predation, starvation, unfavorable environmental conditions and prevailing pathogens (Kamler, 1992; Moyle and Cech, 2003).

Figure 4 Development of larvae and juveniles of Epinephelus malabaricus

1.2.1 Feeding and temperature
Like most marine finfish, heavy mortalities that occur in early-stage of grouper larvae have been considered to be related to the initial feeding stage (Blaxter and Hempel, 1963a; Kohno et al., 1997; Kohno, 1998). The timing to supply feed with appropriate nutritional composition is a key consideration in marine larval fish culture (Cahu and Infante, 2001; Koven et al., 2001). After yolk sac is depleted, fish larvae rely on food from exogenous sources (Shan et al., 2008). At this point, a delay of live food supply can result in low survival, slow development and alimentary tract degeneration (Heming et al., 1982; Chen et al., 2007; Yoseda et al., 2006). Furthermore, if larvae cannot access suitable food for an extended period (defined as Point of no return by Blaxter and Hempel (1963) PNR) after yolk sac depletion, they may lose the ability for food ingestion and digestion (Blaxter and Hempel, 1963b; Kamler, 1992). During onset of exogenous feeding, fish mortality is likely to occur if the provision of first feeding is beyond the PNR (Blaxter and Hempel, 1963b). Therefore, the time at first feeding and live food provision is crucial for the growth and survival of postal larvae.
The PNR is closely related to temperature, as low temperature prolongs the time for larvae to reach the PNR and high temperature shows the opposite effect (Dou et al., 2005; Blaxter and Hempel, 1963b; Yin and Blaxter, 1987). Dou et al. (2005) suggested that the high temperature shortens the period for the first feeding larvae to learn ingesting food before the onset of irreversible starvation is a cause for mortality. Similar result also has been found by Ma et al. (unpublished), they found higher temperatures reduced the time for yellowtail kingfish larvae to reach PNR, thus fish at 25 and 27 had less time to establish their feeding capability than at 21 and 23. This may explain why massive mortality occurred earlier at high temperature than at low temperature. In grouper larvae such Malabar grouper (E. malabaricus) are very vulnerable to starvation, the feeding windows for E. malabaricus cultured at 28 are only 24 h after mouth opening (Yoseda et al., 2006). In order to increase survival and reduce starvation, it is necessary to reduce the culture temperature within the first feeding stage as evidence from previous studies indicate that lower temperature can delay exhaustion of yolk reserve and starvation in other species (Dou et al., 2005; Blaxter and Hempel, 1963b; Yin and Blaxter, 1987). After compared the hatching success and survival rate in a previous study, Watanabe et al. (1995) suggest that a lower temperature may be advantageous to higher temperature for incubating eggs and for rearing first feeding E. striatus when prey concentrations are limiting. Similarly, an operating protocol has been proposed by Ma et al. (unpublished) that use low temperature at the initial feeding stage to improve survival and increased temperature to promote feeding and growth in later stage in yellowtail kingfish larvae culture.
1.2.2 Light intensity and tank color
Light intensity and tank color in the rearing tank may affect the successful grouper larvae feeding as most of marine fish larvae are visual feeders, and light plays an important role in the foraging behavior of fish larvae (Monk et al., 2008). Previous study in E. suillus indicate that no significant preference between black and tan color regarding to the food intake and growth (Duray et al., 1996). However, more and more evidence indicates that tank color preference is more species dependent. For example research in species such as herring and turbot indicate that black wall tanks give a good contrast between food and background (Blaxter, 1968; Howell, 1979), while haddock (Melanogrammus aeglefinus) larvae did not grow and survive well in the tank coated with black wall with low light intensity (Downing and Litvak, 1999). More recent study in juvenile barramundi (Lates calcarifer) indicate that color preference changed as an effect of the ambient light environment (Ullmann et al., 2011). Therefore research should looking into both factor may improve the feeding rate of fish larvae. Surprisingly, little information can be found in literature regarding to grouper larvae preference. Therefore, future research should also towards to obtain the optimal tank color and ambient light environment.
1.2.3 Water surface death
In the larval culture of several marine teleosts, large numbers of dead larvae are often observed on the water surface around the time of first feeding (Kaji et al., 2004). The cause of surface death has been considered as a consequence of being trapped on the water surface by the water surface tension when body surface is exposed to the air (Kawabe and Kimura, 2007; 2008). Surface death is a heavy mortality factor in yolk-sac grouper larvae such as E. akaara (Kaji et al., 1995; Yamaoka et al., 2000), E. septemfasciatus (Tsuchihashi et al., 2003), E. bruneus (Sawada et al., 1999), E. fasciatus (Kawabe and Kohno, 2009). In order to overcome this problem, the addition of an oil film on the water surface is normally used to reduce water surface tension and prevent the occurrence of mass surface deaths of fish larvae (Yamaoka et al., 2000; Kawabe and Kohno, 2009). However, evidence indicates that the outcome of using oil film to prevent surface death varied among hatchery and the removal of oil film from rearing tank was difficult (Yamaoka, 2001). As an alternative substitute, egg white has been used to prevent surface death (Kaji et al., 2003).
2 Feeding Protocol and Live Feeds
2.1 Feeding Protocol
At hatching, the size and mouth are very small in groupers such as E. coiodes, E. malabricus, E. fuscoguttatus, E. suillus and C. altivelis (Table 1). The mouth gape at first feeding of E. suillus was about 150-180µm (Maneewong et al., 1986), and was about 250-300 µm in E. marginatus (Glamuzina et al., 1998). As the prey selection criteria are more by size than by taste or other senses (Yufera and Darias,
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