Evaluating The Spawning Techniques For Bivalves

Evaluating The Spawning Techniques For BivalvesControl of reproduction is a prerequisite in modern bivalves aquculture industries due to un received quantity and flavor of natural spat as well as market demand on standardized products. Spawning is unitary critical step in bivalves hatchery, together with salad dressing and larval rearing, thus a lot of research has been done to explore prospective methods and understand the process within. Various means in spawning techniques are presented herein, i.e. gamete discovery and spawning induction by chemical or physical stimulants. Where relevant, advantage and disadvantage of each technique are discussed and compared.IntroductionBivalves are aquatic animals belong to the phylum phylum Mollusca which characterized by two calcareous shells covering a soft body, encompassing two marine and fresh-water species both marine and fresh-water species. Some well-known bivalves, such as scar, sc totallyop, mussels, and oysters has been success fully polite since decades or centuries ago (Nash, 2011). Nowadays, bivalves and other members of molluscs accounts for nearly 30% of aquaculture production with volume more than 50 million tonnes in 2006 (FAO, 2008).Intensive production is needed to bump into market demands on edible and high value bivalves (pearl oysters). One important key of intensification is control over all of the production steps, including management of reproduction which go out lead to higher productivity with lower cost in order to form best efficiencies and standardized products. Controlled spawning of molluscs is the best way to obtain reliable bring out of quality seeds and reduce dependencies upon unreliable natural spawning events to collect spat.The development of bivalves hatchery was started in 1879 when William Brooks demonstrated that oyster eggs could be spawned and developed into the larval stagecoach in laboratory (Castagna, 1983). No published report on artificial propagation of bivalv e molluscs until 1920s when William Wells successfully reared oyster larvae. These two findings appropriate important data for further development in mollus drive out aquaculture.This article will describe a number of common spawning techniques for bivalves, which divided into 2 categories gamete discovery and spawning induction. Henceforward, gamete stripping is considered not induction since the word induction refers to a persuading or stimulating action, whereas another authors may define the other way. Advantage and disadvantage considerations of each technique are included, particularly in relevance with practical farming aspects.1. Gamete findAs the name indicates, this procedure involves removal of gamete from gonad meander. Briefly, fully ripe gonad overlies the digestive tract was slash repeatedly with scalpel and washed with filtered seawater to harvest the gametes. Microscopy examination is needed to determine sex and appearance of the ova and sperms. in that respectf ore, only well-trained operators can hold back it out because of high level of difficulties.Due to its effectiveness, gamete stripping has been widely employ to obtain gametes in many Bivalvia species. Pacific oysters (Suquet et al., 2007), pearl oysters Pinctada fucata (Alagarswami et al., 1983a), and European flat oysters Crassostrea gigas (Shpigel, 1989) are some of organisms successfully spawned using gamete stripping. Furthermore, this technique obviously the only way to obtain mature gametes for species that can not be spawned by any physical or chemical stimulation.In some bivalves, viable eggs does not necessarily obtained immediately subsequently stripping because of course a series of activation process takes place in follicle prior to spawning, i. e. removal of germinal vesicles and formation of germinal spindle (Loosanof, 1953 Southgate, 2008). Loosanof and Davis (1963) treated eggs of genus Mercenaria mercenaria with 0.1N ammonium hydroxide (NH4OH), but the embryos did not develop well and leading to low percentage of normal larvae compared to their naturally spawned counterparts. So far, no reports more or less detail mechanisms on how NH4OH activates eggs, while comprehensive understanding of the process is absolutely important for optimization or probably substitute NH4OH with another potential substance.Stripping itself is a destructive and lethal method, thus it is not a sustainable way to maintain broodstock collections. Sacrificing superior broodstock must be invaluable loss that all hatcheries try to avoid it. Likewise, pearl oysters hatcheries prefer another spawning method that has no detrimental effects, considering the high value of the product and the oysters itself (Southgate, 2008).2. Spawning Induction by Physical and chemic Means2.1 Chemical stimuli2.1.1 Injections of sex steroidsThe presence of steroids in molluscs has been identified by Hagerman et al. (1957) and its fluctuations have a strong correlation with sexual matu ration, gamete release, and sex determination in bivalve (Croll and Wang, 2007). A serotonin, 5 hydroxytryptamine (5-HT), works synergistically with steroid for those functions because steroids regulate 5-HT receptor on the membrane of gametes (Kadam et al., 1991). Combinations of 5-HT and steroid intragonadal injections are the most widely used in aquaculture farming practice.Efficacy of steroid treatment must be assessed on species basis because a particular steroid may give different results in different species. Similarly, one species could give various responses to some steroids, even if they have similar chemical structure. Published reports by Wang Croll (2003 2004 2006) explain clearly about gonadal maturation as well as spawning responses in sea scallop Placopecten magellanicus towards a number of steroid injections, both in vitro and in vivo.In vitro playing area by Wang and Croll (2003) shows 10-5-10-8 M progesterone and 17-estradiol potentiated gamete release in 5-HT-i nduced gonads tissue of P. magellanicus, both female and/or male. On the other side, testosterone (10-5-10-7 M) was only effective in male and no significant effects on dehydroepiandrosterone (DHEA) and 4-androsterone injections. Application of steroid inhibitors and control group (5-HT-incubated gonads) in the experiment strongly suggest steroids and steroid receptors involve in molluscan reproduction.Those results are confirmed with in vivo larn by Wang and Croll (2006), although slightly different. Progesterone, which is able to initiate gamete release in male and female gonads tissue in vitro, demonstrated inhibitory effect in 5-HT-induced female P. magellanicus. Another interesting fact is the number of gametes released (spawning intensity) are sharply increased up to one-third hundred% and the response time after injection (spawning latency) reduced about 30%.Despite of its advantages, administration of sex steroid in bivalves farming apparently raise auxiliaryal cost sin ce serotonin must be used in parallel to obtain optimal result. In-depth economic calculations might be necessary to reach a balance between cost and benefits.2.1.2 Hydrogen peroxide (H2O2)H2O2 application to induce bivalves spawning was first reported by Morse et al. (1977). He and his colleagues found that addition of 5mM of H2O2 to alkaline seawater successfully induced spawning, in both male and female gravid abalone Haliotis rufescens, within about 3 hours after the first addition. This effect results from activation of prostaglandin endoperoxidase by peroxides, which eventually triggers the spawning response.Their paper convincingly pointed that H2O2, neither another contaminants nor substances, initiates the spawning (Table 1). Destruction of H2O2 by catalase and mercaptoethanol (reducing agent) effectively blocks spawning. Aspirin, an inhibitor of cyclooxygenase and prostaglandin peroxidase synthesis, shows endoperoxidase synthesis is required for induction of spawning by H 2O2.Table 1. Induction of spawning in gravid H. rufescens discussionTotal Number of AnimalsTestedSpawnedControls77 (47F, 30M)3 (2F, 1M)H2O18 (13 F, 5 M)0H2O231 (17 F, 14 M)30 (16 F, 14 M)Aspirin, thusly H2O226 (12 F, 14 M)2 MCatalase, then H2O25 M0Mercaptoethanol, then H2O25 M0Note F = female, M = male. Source Morse et al. (1977)Nevertheless, less success was achieved in Indian pearl oysters P. fucata with H2O2 treatment as with abalone did (Alagarswami et al., 1983b). Mild spawning only occurred with 6mM H2O2 for 5 hours immersion. Supplementation of seawater with Tris buffer at pH 9.1, which usually effective for abalone, does not works well for P. fucata. These conversely results indicate H2O2 concentration must be optimized for any cultured species, sometimes in combination with other treatments.Since peroxide method is a simple and rapid technique, an assay for spawning bivalves which previously known difficult to spawn can be developed quite easily. Morse (1984) has been abl e to identify optimal conditions for gametogenesis and spawning of green (H. fulgens), go (H. corrugata), and black abalone (H. cracherodii) using peroxide method. Spawning of these animals can be induced by H2O2 in a slightly high temperature (20-25C).2.1.3 Injections of neurotransmittersserotonin, 5-HT, is the most widely used and probably the most potent neurotransmitter for artificial spawning purpose. In vitro study shows ovarian tissue of Patinopecten yessoensis released high number of eggs after been immersed in 10-6M serotonin (Matsutani and Nomura, 1987). The study also demonstrated interaction between serotonin and its receptors in ovary, as well as on how prostaglandins regulate serotonin performance in scallop.The dexterity of serotonin in vitro was confirmed in many in vivo studies. Gibbons and Castagna (1984) injected six species of bivalves with 2mM serotonin, nearly similar to the concentration used by Matsutani and Nomura (1987), and all of them spawned as antici pate with spawning latency 15-30 minutes (Table 2). For an unknown reason, there is a tendency serotonin more likely traveled males than females.Table 2. Number of bivalves spawned by 5-HT injectionSpeciesTreatmentNumber ofTestedSpawning MalesSpawning FemalesArctica islandicaControl7000Serotonin70154Argopecten irradiansControl3521Serotonin35291Crassostrea virginicaControl3000Serotonin30210Geukensia demissaControl2010Serotonin2081Mercenaria mercenariaControl7000Serotonin32911621Spisula solidissimaControl4501Serotonin45189Source Gibbons and Castagna (1984).Positive result was also obtained in giant scrape, only Hippopus hippopus was less spiritualist to serotonin (Braley, 1985). Another tested species (Tridacna gigas, T. derasa, T. maxima, T. crocea, T. squamosa) did very well in terms of spawning latency and spawning duration, even with a low dosage of serotonin. Therefore, injection of serotonin provides reliable way to induce spawning in giant clams, besides gametes-overlay metho d.Velez et al. (1989) carried out a more comprehensive research to see effect some neurotransmitters upon spawning induction in scallop Pecten ziczac. Given the number of neurotransmitters (dopamine, epinephrine, norepinephrine), only serotonine could induce spawning process. Moreover, they noticed that females give less response to serotonine than males do similar to what Gibbons Castagna (1984) found.2.1.4 Addition of gametesTridacnid clams, such as T. maxima and T. squamosa, can be induced to spawn in captivity by introducing gonad extract from the opposite-sex into the mantel cavity (Gwyther and Munro, 1981 Munro et al., 1982). Three different gonad extracts were performed gonad biopsy, freshly blended gonad, and freeze-dried blended gonad. Unfortunately, the author did not compare the treatments and describe the relevant results more detail.This induction technique is fairly simple in principles and implementation. Spawning occurs simply because synchronized release of gametes , on which gamete act as chemical cues or pheromones (Soong et al., 2005), a natural adaptation to increase external fertilization opportunity for broadcasting-gametes organisms.2.1.5 Ultraviolet (UV)-irradiated seawaterSeawater irradiated by light was a very powerful spawning inducer. Window-pane shell Placuna placenta responded quickly after been immersed in UV-irradiated seawater (Madrones-Ladja, 1997). The number number of eggs released per female using this treatment is as high as in serotonin treatment furthermore the stimulus did not affect the viability of gametes.Besides of its reliable efficiency, this technique is very cost-effective and simpler than serotonin injection. It is also applicable for either individual or mass spawning, and does really works for both sexes of bivalves.2.2 Physical stimuli2.2.1 Temperature manipulationManipulating water temperature is the most popular methods of spawning induction in bivalve hatcheries mainly because of its ease of applicatio n and reliable effectiveness. This technique has been studying intensively and implemented in pearl oyster P. maxima (Southgate and Beer, 1997) and various edible bivalves, such as American oyster Ostrea virginica (Soria et al., 2010) and brackish water bivalve Corbicula japonica (Baba et al., 1999).Reproductive physiology of O. virginica has been discovered more than decades ago, thus artificial reproductive control method has been set up very well (Galtsoff, 1938a 1938b 1940). Sudden change in water temperature from 20 to 32-35C was found effectively stimulate spawning than long exposure to lower temperatures.In C. japonica, rising the temperature must be accompanied by salinity. Baba et al. (1999) demonstrated temperature 26C with salinity 2.3-4.5 practical salinity unit (psu) give optimum yield. Moreover, addition of gamete suspension into the water is able to increase its fecundity. These three different factors may bring some complexity in terms of hatchery practice because o ptimization of those components is more likely indispensable.2.2.2 Administration of microalgal foodImmersing razor clams Siliqua patula in high density of phytoplankton is the only method so far that scientifically effective to induced spawning process. Breese and Robinson (1981) found 2-2.5 million cells ml-1 microalgae Pseudoisochrysis paradoxa stimulates razor clams to spawn. No published report that explains this phenomenon, so the exact mechanism does not understood well. It is maybe only a basic instinct of reproduction in nature when food present abundantly in environment.This method could be a good alternative instead of gamete stripping. The number of eggs released per clam as well as hatching rate was much higher compared to those obtain by stripping. In terms of aquaculture practicality, this technique is considerably easy and fairly inexpensive.3. ConclusionsThere are numerous techniques to stimulate spawning process in bivalves. Basically, they are only mimicking natur al process involved in natural reproduction cycle. No one of those techniques is more superior to another, therefore choosing the appropriate technique should be based on species cultured. Any cost associated, for instance chemicals and equipments, must be also interpreted into consideration.