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groupers, snappers, cobia, pompano and tuna) is increasingly becoming popular in many parts of the world, and continues to develop rapidly in the Southeast Asian region (Rimmer, 2004). Groupers are one of the improtant species for aquaculture in both marine and brackishwater pond and off-shore cages. They are particularly popular for the live fish market, mainly in Hongkong, China, Singapore and Taiwan. Most of the grouper aquaculture production came from Asia, with China and Taiwan topping the list, followed by Indonesia and Thailand. In 2005, Asia produced a total 62,088 MT representing 24% of the total world fisheries production for groupers (FAO, 2007). The most common species for grouper culture are Orange-spotted grouper (Epinephelus coioides), Brown-marbled grouper (Epinephelus fuscoguttatus), Malabar grouper (Epinephelus. malabaricus), Humpback grouper (Cromileptes altivelus) and Giant grouper (Epinephelus lanceolatus). Giant Grouper is one of the grouper species which is has bright prospect in the market. Fast growth and high economic value are the advantages of this species compare with other grouper species. It is much sought after for the live reef fish trade with Hong Kong import statistics revealing import of around 2.4 tonnes of giant grouper in 2004 (Lau, P.F. and Parry-Jones, R. 1999). Although Taiwan has had some success in breeding, and sells giant grouper fingerlings in South East Asia, the amount of hatchery reared fish available is thought to be small, and proportion of traded individuals from
wild versus hatchery production is unknown. Indonesia and Thailand are known to be conducting research on the breeding of this species. Meanwhile, research into improving the seed production of giant grouper should be undertaken. Until now the main problem of the giant grouper culture is the lacking of natural seed stock as well as hatchery stock. The mass larval production technology for the giant grouper species is not yet established, and seed production is still not sufficient enough to meet the high demand for grow-culture. Technology cultured and wild fish populations of this species are dependent upon the production of good quality eggs. Increasing fish population through aquaculture requires increasing fingerling supply; however high mortality during early life stages remains a significant bottleneck in most of marine finfish production. Hatchery technology for an increasing number of species is continually being developed to meet with the expending interest in aquaculture and the market demand for diversification and the production of additional marine species present an enormous economic opportunity for increasing the supply of high quality, safe and wholesome aquaculture products (Lee and Ostrowski) 2001. Therefore in order to develop giant grouper culture technology, the seed production technology must be prepared. The success of seed production activity depends on the gonad maturity of broodstock, which is naturally depending on the age, size, feed, environment, season and broodstock management. A range of factors imposed by
hatchery practices has been found to influence egg quality such as stress on broodstock brought about by handling, over ripening of eggs and hormone administration in induced spawning.
One of the striking features in teleost fish is the plasticity of the sex determination with a range of gonochoristc species, where individuals are either males or females, or species displaying a change in gander during the lifetime. Research works directed to the developing the biotechnology tools to assess gender and maturational status of giant grouper is needed to be developed.
Statement of the problem
The giant grouper (Epinephelus lanceolatus) which is similar to other grouper behavior are well-known protogynous hermaphrodite teleost of the family of Serranidae (Smith, 1971). Which is changing sex from female in early life to male at older age, depending on the natural environment of the populations studied (Lasserre, 1972). The point in time at which sex-change occurs is can be interpreted on many different levels (e.g. behavioral, morphological, histological) were effects on broodstocks management of this species, which is difficult particularly concerning the prediction of natural sex inversion (age and/or size) and the artificial control of sex.
Sex determination in cultured species is a constraint to broodstock constitution. Individual identification of the breeder gender is necessary to maintain the desired sex ratio to produce the appropriate fingerling number for aquaculture production. Generally, equilibrated or determinate sex ratios are required when mature male and female pairs are necessary for induced or natural breeding. From an academic perspective, sexual
transition is a useful process in which to investigate the mechanisms controlling sex determination and differentiation.
In most species, phenotypic sex is more or less easily identified in pubertal male or female by specific external characters. In some cases, intra-ovarian biopsies or endoscopies or cannulation technique on anesthetized animals allowed gender identification, even outside the breeding season or even in immature fish. However, this technique has limitation to predict the gander of broodstock. It is necessary to develop the instrumental or methods which can easily to define the sex change technology for sake of improving the efficiency of aquaculture operations.
Currently, no reliable method for gender determination has been described in giant grouper, except may be some morphological characters observed only when fish have initiated courtship behavior. Direct observation of broodstock is not always possible due to water turbidity, and this visual method is not compatible with an efficient broodstock management. Specifically, it does not allow the constitution of male and female mating pairs before the breeding period in order to optimize fry production in this valuable species.
Throughout the years, many researchers have been developed in pursuit of obtaining reliable quantitative values of nutrient in blood plasma. Researchers were focus on developing the biochemical indicators to determine the sex and maturational status of fish in the absence of the availability of gonad tissues, generating the biological knowledge
necessary to evaluate the impacts that a shift in sex change phenomenon. In this regard, the first clues indicating a relation between the sexual maturation and the appearance of changes in the serum profiles were obtained by experiments which is demonstrated that changes in the blood component were reflect by the administration of estrogens (MacDonald and Riddle, 1945).
Non-invasive techniques based on the use of sexual steroid and /or vitellogenin (Vtg) have been reported as efficient techniques in tuna (Takemura and Oka, 1998), trout and salmon (Pottinger et al.2005) and sturgeon (Cuisset et al. 1994; Ceapa et al. 2002: Malekzadeh Viayeh et al. 2006). Vtg, a pubertal female specific glycopophosphoprotein, is correlated to oocyte development in sexually mature fish during the reproductive cycle (Kishida et al. 1992: Mananos et al. 1994; Nilsen et al.2004).
Therefore, a non-invasive sexing technique for giant grouper using both the Vtg detection in maturing females by enzyme immune assay (EIA) and the sexual steroid technique based on 17 β-estradiol (E2) plasma levels in mature and immature individuals of giant grouper were suggest to apply in this study.
Purpose and significance of the study
Determination of the sex and gonadal maturity of fish is necessary for successful commercial aquaculture operations. A numbers of techniques to assay on sex determinations have been apply. Sex linked markers allows for determinations of the genetic sex of an organism (Olmsted et al. 2011). There are several molecular techniques which generate molecular markers. Certain 5
molecular techniques have advantages over others depending on the research question being addressed. The amplified length polymorphism (AFLP) technique generates large numbers of molecular markers and provides a rapid method for scanning the genomes of different individuals for sequence variation. The AFLP technique can be used to quantify genetic variation, assist in marker assisted breeding programs.
In some cases, sex determination is an integral part of the characterization of forensic samples containing genomic DNA. PCR technique (Kobayashi et al, 2004) offers an efficient and sensitive method for sex determination by amplifying gender specific sequences, which results in different size PCR product depending on the gender of the donor. Genetic sex determination in Japanese Medaka is based on the presence or absence of the medaka male-sex determining gene, DMY, which located on the Y chromosome.
In general, determining the sex and sexual maturity of protogynous giant grouper is difficult, because both males and females are found simultaneously and there is no definite size or age at which the sex begins. In some cases, biopsies (Alam and Nakamura, 2008) or endoscopies allowed sex determination; however, most of the works above were depend on not reliable method for sex determination. marker for the onset of maturation in female fish has to discover. Easily identifiable
Since, vitellogenin (Vtg), the hepatically synthesized precursor to egg-yolk, could be an easily identifiable marker for the onset of maturation in female fish proposed (Idler et al. 1981: Le Bail and Breton 1981) as indicator in the plasma of maturing female fish. Immunoassay for VTG have been developed and validated for several species of fish
including sole, Solea vulgaris (Nunes-Rodriguez et al.1989), white spotted char, Salvelinus leucomenis, (Kwon et al. 1990), channel catfish, Ictalarus punctatus (Goodwin et al.1992), striped bass, Morone saxatilis (Tao et al. 1993; Kishida et al. 1992) tilapia, Oreochromis mossambicus (Kishida and Specker, 1993) and seabass, Dicentrachus labrax (Mannaos et al. 1994). Its present a simple and effective technique to determine the sex and sexual maturity of giant grouper were proposed in this study.
Therefore the aim of the present study was to establish a simple, efficient and nonlethal technique by using vitellogenin (Vtg) as biomarker indicator for rapid sex determination without killing or seriously injuring the sampled individuals.
Objectives of the study
The specific objective of this research were to: 1) characterized the physiological events (hormonal and histological) associated with sexual inversion from unknown to female, 2) developed the biomarker tools for identifying the sex and maturational status of individual giant grouper through biochemical assay approach to assessing gender and maturity by comparison with conventional technique based on histological examination of the gonads, and 3) precise localized of Vtg in the ovary and elucidate their functional differences in ovarian development and onset of sex change in the giant grouper.
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