Evaluating The Spawning Techniques For Bivalves

Control of reproduction is a prerequisite in modern bivalves aquculture industries due to unreliable quantity and quality of natural spat as well as market demand on standardized products. Spawning is one critical step in bivalves hatchery, together with fertilization 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 stripping and spawning induction by chemical or physical stimulants. Where relevant, advantage and disadvantage of each technique are discussed and compared.

Introduction

Bivalves are aquatic animals belong to the phylum Mollusca which characterized by two calcareous shells covering a soft body, encompassingboth marine and fresh-water species both marine and fresh-water species. Some well-known bivalves, such as clams, scallop, mussels, and oysters; has been successfully cultured 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 meet 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 will lead to higher productivity with lower cost in order to reach best efficiencies and standardized products. Controlled spawning of molluscs is the best way to obtain reliable supply 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 stage in laboratory (Castagna, 1983). No published report on artificial propagation of bivalve molluscs until 1920’s when William Wells successfully reared oyster larvae. These two findings provide important data for further development in molluscan aquaculture.

This article will describe a number of common spawning techniques for bivalves, which divided into 2 categories: gamete stripping 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 Stripping

As the name indicates, this procedure involves removal of gamete from gonad tissue. 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. Therefore, only well-trained operators can carry it out because of high level of difficulties.

Due to its effectiveness, gamete stripping has been widely used 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 after stripping because naturally 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 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 about 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 Chemical Means

2.1 Chemical stimuli

2.1.1 Injections of sex steroids

The presence of steroids in molluscs has been identified by Hagerman et al. (1957) and its fluctuations have a strong correlation with sexual maturation, 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 study by Wang and Croll (2003) shows 10-5-10-8 M progesterone and 17β-estradiol potentiated gamete release in 5-HT-induced 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 4α-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 study 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 300% and the response time after injection (spawning latency) reduced about 30%.

Despite of its advantages, administration of sex steroid in bivalves farming apparently impose additional cost since 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 H2O2.

Table 1. Induction of spawning in gravid H. rufescens

Treatment

Total Number of Animals

Tested

Spawned

Controls

77 (47F, 30M)

3 (2F, 1M)

H2O

18 (13 F, 5 M)

H2O2

31 (17 F, 14 M)

30 (16 F, 14 M)

Aspirin, then H2O2

26 (12 F, 14 M)

2 M

Catalase, then H2O2

5 M

Mercaptoethanol, then H2O2

5 M

Note: 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 able to identify optimal conditions for gametogenesis and spawning of green (H. fulgens), pink (H. corrugata), and black abalone (H. cracherodii) using peroxide method. Spawning of these animals can be induced by H2O2 in a slightly high temperature (20-25°C).

2.1.3 Injections of neurotransmitters

Serotonin, 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 efficacy 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 expected with spawning latency 15-30 minutes (Table 2). For an unknown reason, there is a tendency serotonin more likely affected males than females.

Table 2. Number of bivalves spawned by 5-HT injection

Species

Treatment

Number of

Tested

Spawning Males

Spawning Females

Arctica islandica

Control

70

Serotonin

70

15

4

Argopecten irradians

Control

35

2

1

Serotonin

35

29

1

Crassostrea virginica

Control

30

Serotonin

30

21

Geukensia demissa

Control

20

1

Serotonin

20

8

1

Mercenaria mercenaria

Control

70

Serotonin

329

116

21

Spisula solidissima

Control

45

1

Serotonin

45

18

9

Source: Gibbons and Castagna (1984).

Positive result was also obtained in giant clams, only Hippopus hippopus was less sensitive 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 method.

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 gametes

Tridacnid 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 probability for broadcasting-gametes organisms.

2.1.5 Ultraviolet (UV)-irradiated seawater

Seawater 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 average 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 stimuli

2.2.1 Temperature manipulation

Manipulating water temperature is the most popular methods of spawning induction in bivalve hatcheries mainly because of its ease of application 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 32-35°C 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 26°C 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 optimization of those components is more likely indispensable.

2.2.2 Administration of microalgal food

Immersing 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. Conclusions

There are numerous techniques to stimulate spawning process in bivalves. Basically, they are only mimicking natural 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 taken into consideration.