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Part 4
Hatchery operation: broodstock conditioning, spawning and fertilization


4.1.1 Introduction

Conditioning broodstock is essential in the provision of larvae for culture (Figure 32).

It is the procedure by which hatcheries are able to extend their production season, removing reliance on the relatively brief period in the year when adults of the desired species are bearing mature gametes in the sea. In the case of hatcheries in marginal climates, there is distinct advantage in producing seed early in the year - often months before stock have developed and matured in nature.

Early season production in colder climates ensures that seed have a maximum growing period prior to their first over-wintering. Thereby, they are larger and more resistant to low temperature. This may be relevant in the culture of exotic species where small seed may not be as fully cold hardy at a small size as are similarly sized seed of native species. Hatchery conditioning of stock is also relevant in circumstances where an exotic species has been introduced for farming purposes but will not recruit reliably in its new habitat.

Figure 32: A typical broodstock conditioning system.

Many bivalves will mature in their first year of life as males. As they age, year by year, an increasing percentage may switch sex and become females. This is known as protandric hermaphroditism. Among the commonly cultured species in hatcheries exhibiting this form of sexual development are clams of the genus Tapes, Mercenaria, Mya and Spisula, oysters of the genus Crassostrea and the many types of mussel including Mytilus sp. and Perna sp.

Figure 33: The anatomy of a fully mature calico scallop (Argopecten gibbus): am - adductor muscle; g - gills (raised to reveal the gonad); m - mantle; o - ovary; t - testis.

Some species of bivalves are truly functional hermaphrodites. They mature both male and female gonads simultaneously (Figure 33). Gametes are spawned sequentially, usually sperm first followed by the eggs, with later reversal to sperm again within the spawning cycle. This group of monoecious species includes the northern European King scallop, Pecten maximus, the (Brazilian or Caribbean) sand scallop, Pecten (Euvola) ziczac, the bay scallop, Argopecten irradians, the calico scallop, Argopecten gibbus, the Chilean scallop, Argopecten purpuratus, and some species of Chlamys. Sexes are separate (dioecious) in other large sea scallops, e.g. Placopecten magellanicus and Patinopecten yessoensis.

Flat oysters of the genera Ostrea and Tiostrea exhibit alternate sexuality. They switch sex at the end of each reproductive cycle. A single European oyster (Ostrea edulis) can go through two or three sex reversals each spawning season when sufficient food is available and during an extended warm water period.

Conditioning case history - the Manila clam, Tapes philippinarum

Figure 34: A selection of clams commonly cultured in hatcheries. Note that the nomenclature of the genus Tapes is synonymous with Venerupis and Ruditapes in European hatcheries, thus Manila clams may be referred to as Tapes or Venerupis or Ruditapes philippinarum (with semidecussatus or semidecussata being other alternative specific names). Nomenclature is equally as confusing in some other commonly cultured bivalves).

In the Manila clam (Figure 34), as in other bivalves, egg production increases with increasing adult size. Mature females of 10 - 20 g live weight will spawn 5 - 8 million eggs on average depending on their condition and time of year they are brought into breeding condition.

Populations of 2 and 3-year-olds show close to a 50:50 sex ratio. For example, of 138 conditioned clams subjected to spawning stimuli in trials at the MAFF Fisheries Laboratory, Conwy, UK, in 1987, 54 spawned as females and 55 as males. The remaining 29 clams, that were mainly in earlier season spawning attempts, failed to liberate gametes and were probably ‘under-ripe.’

Sexual development starts in the sea when the water temperature exceeds 10°C. Gametes develop during late May or June and mature in July or August to be retained until spawning is stimulated by high temperatures (>20°C) or by a series of thermal or handling shocks. In northern European waters, where temperatures are rarely high enough to stimulate spawning, mature gametes are retained into the early winter and are then resorbed.

Maturity can be accelerated in the hatchery by holding the clams at elevated temperatures and providing them with a suitable food ration. It is possible to mature adults in the winter and early spring, before clams in the sea commence sexual development, thereby extending the period hatcheries have access to larvae. Clams in spawning condition can, therefore, be made available for most of the year. To obtain spawning in the autumn it is possible to mature juveniles from early same season spawning by conditioning them at high temperature and high food rations.

Temperate climate bivalves generally have two spawning periods within a year following spring and autumnal peaks in phytoplankton production. Tropical species exhibit less well defined spawning periods. Spawning takes place during most of the year with a low percentage of adults maturing at any point in time. This habit presents problems for hatcheries in the tropics since many of the individuals will be spent (i.e., have recently spawned) or be in the early stages of gamete development when stock is brought into the hatchery. This is wasteful in terms of time, space and food resources. However, there are ways of bringing broodstock into greater synchronicity of reproductive development (see section 4.1.3).

4.1.2 Conditioning methods Tank systems and water treatment

The basic methods for broodstock conditioning are much the same for all bivalves. It is usual for a hatchery to maintain its own stocks for production purposes in local, sea-based growout. These stocks are kept in the best possible conditions of high water flow and at low density in well maintained growout equipment. They are often the offspring of previous hatchery-reared generations, selected for desirable characteristics such as growth rate, shell shape and colouration.

Figure 35: Diagrammatic representation of A - a flow-through broodstock tank in which adults are suspended off the bottom in a mesh tray with large apertures in the base so as not to retain faeces and detritus; B - a similar tank fitted for sub-gravel filtration. Systems of type A are suitable for most species that do not require a substrate. Clams and some scallop species often condition better in tanks of type B.

Adults taken from the sea are brought into the hatchery, their shells thoroughly scrubbed and rinsed to remove epifaunal (fouling) organisms and sediment, and then placed in tanks similar to those shown in Figure 35 (see also Figure 32). Clams and also scallop species (e.g. Pecten ziczac) which are normally partially buried in the substrate in nature, feed more efficiently if they are kept in a suitable substrate. In tanks of the type illustrated, clams or scallops are allowed to bury in 10 cm deep trays filled with coarse sand or shell gravel, or in a sufficient depth of substrate over a sub-sand filter (Figure 35B). Trays are supported off the bottom of the conditioning tank when stocked with bivalves that do not require a substrate, e.g. oysters, mussels and some scallop species (Figures 35A and 36).

Figure 36: A to D - Examples of various types of flow-through tanks used for broodstock conditioning. The tray under the outflow in B - contains a mesh-based sieve, used to retain European flat oyster larvae that may otherwise be lost in the tank discharge when liberated by the adults. C - is an experimental system with each broodstock tank supplied a different diet by peristaltic pump from the reservoir tanks alongside.

Seawater used need not be filtered: the diversity of food species present in unfiltered seawater is beneficial in the conditioning process. While it is possible that the broodstock may be exposed to the infective stages of parasites or potentially pathogenic micro-organisms present in the incoming water supply, the cost benefit in not filtering the water often outweighs the risks. In most cases, conditioning takes place in flowthrough systems, which may or may not include an element of water recycling to conserve cultured algae added as feed.

It is also feasible to condition bivalves in recirculation systems where the total live weight biomass of adults (the collective weight - shells included - of all the animals in the tank) does not exceed 2 or 3 g per l. In this case, it is advisable to drain and refill the total volume of water in the system at least twice each week to prevent build-ups of bacteria and metabolites.

Both salinity and temperature should be appropriate to the species being conditioned. Most commonly cultured bivalves will undergo reproductive development and mature gametes at salinities greater than 25 PSU (practical salinity units, equivalent to parts per thousand) and temperatures of between 16 and 24°C. However, each species will have optima for both of these parameters. Manila clams and Pacific oysters for example, respond best to water temperatures between 22 and 24°C. Pacific oysters will condition at a wide range of salinities (15 to 34 PSU) while Manila clams prefer higher salinities of between 25 and 34 PSU with an optimum of around 30 PSU. The American (Virginia) oyster, Crassostrea virginica, will condition at much lower salinities. As one would expect, offshore, deeper water species require cooler conditions and near oceanic salinity.

Water flow rate through conditioning tanks should exceed 25 ml per minute per adult and no more than 5 kg live weight biomass of stock should be held in a tank of 120 to 150 l volume (Figure 37). The water should not be recycled and reused in such small tanks when heavily stocked. When bivalves from outside the immediate area are used as stock, the effluent water discharged from the tanks should be diverted to a treatment tank to prevent the transfer of pathogens and parasites to the local environment. The effluent needs to be treated with >100 mg per l free-chlorine or a similarly effective disinfecting/sterilizing agent (e.g. ozone) for a minimum 24-hour period (preferably 48 hours) before it is returned to the sea.

Figure 37: A 120 l broodstock tank stocked with 55 oysters averaging 80 g live weight. The minimum flow rate of cultured food supplemented seawater through the tank at this stock density is 1.375 l per minute.

Hatcheries usually have a separate broodstock conditioning room or locate the conditioning tanks in a quiet area of the plant where stock will not be subject to frequent disturbance. Most species respond to shadows and vibration by closing their shell valves. The less disturbance they receive, the more time they will spend feeding.

Small and medium size hatcheries usually have between 5 and 20 conditioning tanks to accommodate the various species reared and to permit the regular introduction of new stock to maintain a rotation and ensure a continuous supply of larvae. Large hatcheries may have more of the smaller tanks or fewer that are larger. When the steady production of spat of a particular species is required over an extended period of the year, new stock is brought in to start the conditioning process on a weekly or twoweekly basis. In this way, adults are available for spawning every week. Feeding broodstock

Cultured marine algal species are used most frequently as the principal food supply during conditioning. Alternative sources are natural phytoplankton bloomed extensively in outdoor tanks or ponds, or commercially available algae pastes.

Useful algal species that can be cultured intensively on a large-scale are Tetraselmis (various species, including T. chuii, T. tetrahele and T. suecica), Isochrysis galbana (and the T-Iso clone), Pavlova lutherii, Chaetoceros muelleri (previously named C. gracilis), Thalassiosira pseudonana and T. weisfloggii and Skeletonema costatum. (This list is by no means exhaustive). A mixture of these species, on a proportional basis, is more beneficial than a single species diet. Care should be taken not to feed relatively indigestible species (e.g. Chlorella sp.) or, species known to be deficient in the more highly unsaturated fatty acids (e.g. Dunaliella tertiolecta).

An example of the consequences of feeding a deficient diet is the reduced production of larvae from Ostrea edulis adults when held in filtered water and fed only Dunaliella tertiolecta (Table 8). Dunaliella is known to be lacking in the C20 and C22 highly unsaturated fatty acids considered to be nutritionally essential. In this trial, groups of 60 adults were kept in tanks provided with a through-flow of either unfiltered seawater or seawater filtered to 2 µm particle size. (The experimental tank system is shown in the bottom right photograph of Figure 36C). A daily 3% ration based on the initial dry meat weight of the oysters was provided to three of the groups as Dunaliella alone or in combination with either Tetraselmis suecica or T-Iso. Control groups were kept in both flowing filtered and unfiltered seawater without the addition of cultured algae.

Table 8. Effect of diet on the production of Ostrea edulis larvae. Key: Seawater (SW) treatment, F and UF refer to filtered and unfiltered seawater respectively; Diet, Dt - Dunaliella tertiolecta, Ts - Tetraselmis suecica, T-Iso - Isochrysis galbana (Clone T-ISO). Days - refers to the number of days from the start of conditioning until larvae were first released. Total larvae is the number of larvae produced by each group of adults in a 70-day period and when this value is divided by the number of adults in the group provides larvae/oyster. From Millican and Helm (1994). See text for further details.

SW Treatment



Total larvae






19 367





10 280


Dt + Ts



49 950


Dt + T-Iso



78 250





135 317

The elapsed time from the beginning of conditioning to the first release of larvae in each group was noted and daily counts of larval released were made during the 10- week duration of the trial. Results given in Table 8 show that the single species diet of Dunaliella delayed both the onset of larval production and reduced overall production in comparison with the alternative treatments tested. Interestingly, considerably greater numbers of larvae were released by adult oysters held in unfiltered seawater without cultured algae addition than from the other treatments. This reinforces the previously made point that there may be a cost benefit in not filtering seawater for conditioning.

The duration of the above trial encompassed the spring phytoplankton bloom when chlorophyll a in the unfiltered control seawater averaged 1.68 mg per m3 compared with 0.35 mg per m3 in the filtered control seawater. Particulate lipid averaged 62 ng per l (nanogram per l) compared with 9.7 ng per l respectively.

Methods for both intensive and extensive algal culture are described in Part 3 of this manual. Steps in the calculation of the required food ration are described below in section The calculation does not, however, apply to extensively grown phytoplankton where species diversity, abundance and overall nutritional value will vary day by day. In this case, an approximation of abundance can be made by determining the ash-free dry weight of particulate matter per unit volume, or by organic carbon analysis. Alternatively, the operator can dilute the bloomed water "by eye" to provide an adequate ration.

Algal pastes of the various nutritionally preferred species are convenient to use and suppliers provide information on the equivalent number of cells per unit volume of the product. Many of these products also bear quantitative details of nutritionally important components on the packaging. Once opened, the non-living product has a relatively long shelf life when the supplier’s instructions are closely followed. Such pastes are probably best used in flow-through conditioning and attention must be paid to the hygiene of the tanks.

Provision of a satisfactory ration of nutritionally valuable food species during conditioning has a marked beneficial effect on egg production. Calculating food ration for conditioning

The required food ration for conditioning is based on the dry meat weight of the adults. It is usually between 2 and 4% of the mean dry meat weight of the adults at the start of the conditioning period in dry weight of algae fed per day. Rations exceeding 6% are not conducive to successful conditioning. Rather, the bivalves will grow strongly in response to high feed levels and the high temperature of conditioning at the expense of reproductive development.

It is a simple procedure to determine the dry meat weight of a stock of bivalves of known live weight brought to the hatchery for conditioning. Opening a random sample of 10 or 12 individuals, removing the soft body tissues and weighing the meats after drying them to constant weight in an oven (60 to 80ºC for 48 to 72 hours) will provide data for the calculation of ration. The equation below is to determine the dry weight of algae per adult required for a 3% daily ration.

Ration g per day per adult = 3 x mean dry meat weight (g)/100

Thus, a 3% ration for an adult of 0.75 g dry meat weight amounts to 0.0225 g dry weight of algae per day. Reference to the dry weight data given for the various algae species (see Table 1 - Part 3.1) shows that 1 million Tetraselmis cells have a dry (organic) weight of about 0.2 mg.

Assuming that 50% of the 3% daily ration (= 1.5%) is to be provided to the broodstock as Tetraselmis and the total dry meat weight biomass of the stock is 50 g (converted to mg in the equation below), then:

Ration (1.5%) per day (in millions of cells)

= [(1.5x(50x1 000))/100]/0.2

= 3 750 million cells

If, for example, the harvest density of Tetraselmis on a particular day is 1.5 million cells per ml, then the volume required to feed the stock a 1.5% ration will be 3 750/1.5 = 2 500 ml, or 2.5 l. Calculation of the remainder of the ration is similar for the other component species of the diet. If instead of, or in addition to Tetraselmis, Chaetoceros muelleri is to be fed at a harvest density of 7 million cells per ml, then the volume required for a 1.5% ration will be 3.57 l. Chaetoceros muelleri has a dry weight approximating to 0.03 mg per million cells. Adjusting ration for flow-through systems

In calculating ration, account needs to be taken of the configuration of the tanks and system in which the adults are conditioned. This is not of particular concern in closed systems where algal cells, as yet uneaten, are not lost other than through sedimentation or settling on surfaces. In flow-through systems and tanks of the type shown in Figures 32, 36 and 37, however, a proportion of the algae fed will inevitably be uneaten and will be lost in the outflow. For this reason, adequately stocked tanks of 100 to 150 l with slow rates of water exchange are preferred.

From experience, a total water exchange rate in excess of 90 minutes minimizes cultured algae loss, giving the stock adequate time to filter and consume 60 to 80% of the food. For example, a tank of 150 l volume stocked with 50 oysters or scallops of 75 to 100 g live weight requires a flow of 1.25 l per minute at 25 ml per minute per adult. At this rate of flow, tank volume exchange rate is 120 minutes. Where smaller bivalves, eg. Manila clams, are stocked, numbers of adults per tank should be increased correspondingly on a live weight biomass basis.

It is also preferred that the ration is dosed continuously into the water delivery line to the tank by peristaltic pump to effect better mixing. In some hatcheries, the daily food ration is divided into several batch feeds. The seawater supply is turned off for an hour or so after each addition. This can be problematic in terms of contamination with the waste products of metabolism if the water is inadvertently not switched back on in good time.

In the absence of the means to determine percentage particle removal between the inflow and out-flow from a flow-through tank, it is recommended that food supply be calculated as a 4% ration to allow for losses discussed above. If the operator has access to an electronic particle counter and sizer (e.g. a Coulter Counter - refer to Figure 21), adjustments to ration can be made based on hard data. Two-stage early season conditioning

Conditioning can be a two-part procedure. Early in the season in temperate and cold-water climes, when adults in the wild have yet to start gamete development, it is often advantageous to provide conditions of high food availability at a temperature intermediate between the ambient and that required for conditioning. The objective is to boost the levels of food reserves in the adults that will later be mobilized in gamete development. This is more important for females than it is for males because egg development and maturation is considerably more energy intensive. Following 4 to 6 weeks of a high ration: moderate temperature regime, temperature is gradually raised (1 to 2°C per day) and food ration is somewhat reduced (from 4 to 6% to 2 to 3% per day).

Food supply for the first stage, which can be called the pre-conditioning stage, can be in the form of algal pastes, bloomed natural phytoplankton (from extensive algal culture, Part 3.4.6) or, intensively cultured algal species. It is important to bear in mind that during this stage, principally the structural lipid (phospholipid) composition of the early stage oocytes will be influenced by the diet and ration available to the broodstock. Thus, a diet deficient in highly unsaturated fatty acids (HUFAs) of known importance, including EPA (eicosapentaenoic acid, 20:5n-3) and DHA (docosahexaenoic acid, 20:6n-3), will be reflected in eggs with cell membranes with reduced content of these components. For this reason, the ration should contain nutritionally valuable diatoms (e.g. Chaetoceros muelleri or Thalassiosira sp.) and flagellates such as Pavlova lutherii or Isochrysis galbana, all of which are rich in one or other of these HUFAs.

Triacylglycerols - the neutral lipids that are laid down as reserves in maturing eggs - are accumulated during the later stages of the second, warm water phase of conditioning. These lipids are drawn upon as sources of energy during embryo and larval development. Their composition appears to be more dependent upon lipids being mobilized directly from the food ingested by the adult than it is from maternally derived reserves.

4.1.3 Conditioning bivalves in the tropics

Mention was made earlier in this chapter of the habit of many tropical species to spawn intermittently throughout most of the year. This presents problems in obtaining sufficient numbers of larvae to support production requirements of hatcheries in tropical and sub-tropical climates.

When there is little variation in seawater temperature and food availability during the year, bivalves do not to have a quiescent period - as do temperate and cold water species - that triggers synchronicity in reproductive development within a stock. This cooler period can be provided in tropical hatcheries by holding stock in water chilled to between 5 and 10ºC below ambient with an adequate food ration for a period of 4 to 6 weeks. After this period they are gradually warmed to ambient conditions when a greater percentage of adults will mature gametes synchronously. This is a similar approach in many ways to that described in section

The technique has been used with the mangrove oyster, C. rhizophorae, in Cuba. Similar methodology has also been applied successfully in conditioning Pacific oysters, C. gigas, in parts of Brazil. The problem is somewhat different in the latter case. Pacific oysters (an introduced exotic species) grow extremely well in the more southerly states of the country but they do not undergo reproductive development to the extent that they will spawn.


4.2.1 Introduction

A summary of information pertinent to conditioning and egg/larval production for a number of commonly cultured bivalves is given in Table 9.

Many temperate and cold water climate bivalves will require 4 to 8 weeks of conditioning to reach spawning readiness during late winter and early spring (Figure 38). A progressively shorter period will be required as the natural breeding season approaches. Precise timing depends on the species being conditioned, the initial condition of the broodstock, stage in gametogenesis when the bivalves begin conditioning and hatcheryrelated factors, the most important of which are temperature, diet and ration. Hatchery operators will normally use stock already undergoing gametogenesis when returned from the sea, rather than begin conditioning with sexually undifferentiated adults. Advantage can be taken during the natural spawning period of the generally better quality of eggs in terms of important reserves (particularly lipids) from adults brought into the hatchery directly from the sea. These adults may only require 7 to 12 days at conditioning temperature with an adequate food ration to mature their gametes.

When given an adequate food supply, many coastal and estuarine temperate water bivalves will require between 350 to 650 degree-days (deg d) from the start of conditioning in late winter/early spring to the time they are ready to spawn. The hatchery operator needs to know the temperature at which reproductive development starts in the sea for the species in question. This is often between 8 and 12ºC - the "biological zero" (b0) for gametogenesis - for commonly cultured species such as Crassostrea gigas, Ostrea edulis, Pecten maximus and Tapes philippinarum. Knowing what the effective b0 temperature is for reproductive development and the water temperature during the conditioning period, calculation can be made of the number of days of conditioning required. For example, if the mean conditioning temperature is 20ºC and the b0 temperature for reproductive development is 10ºC, then every day that passes the number of degree-days will increment by 20 minus 10 = 10. Thus, a 30-day conditioning period at 20ºC will accrue 300 deg d and the same period at 22ºC will amount to 360 deg d. This represents the likely minimum time period later in spring before stock will be ready to spawn. Obviously, when new stock brought back to the hatchery for conditioning have already started gametogensis, fewer degree-days will be required before the adults are spawning ready.

Table 9: Summary of information relevant to the conditioning and egg (or larval) production for a number of commonly cultured bivalves. A key to the meaning of the symbols under sex type is given at the bottom of the table. Conditioning times are for adults brought back to the hatchery early in the season (* time in days will vary considerably according to the stage in gametogenesis the adults are in when brought to the hatchery). Fecundity values are a guide only and will vary according to the size of adult spawned, its condition and other factors. The average shell lengths of fully developed, early-stage D-larvae (2 - 3 days after fertilization) are also given for comparative purposes.



Period (days*)



size (µm)


C. gigas

O - D

28 - 42

20 - 24


70 - 75

C. virginica

O - D

28 - 42

20 - 22


60 - 65

C. rhizophorae

O - D

21 - 35

20 - 22

7 - 12

55 - 60

O. edulis

L - A

28 - 56

18 - 22

1 - 3

170 - 190

T. lutaria

L - A

28 - 56

18 - 20

0.02 - 0.05

450 - 490


T. philippinarum

O - D

28 - 42

20 - 22

5 - 12

90 - 100

M. mercenaria

O - D

28 - 42

20 - 22

10 - 20

90 - 100


P. yessoensis

O - D

14 - 21

7 - 8

20 - 80

100 - 115

P. magellanicus

O - D

28 - 42

12 - 15

20 - 80

80 - 90

P. maximus

O - M

35 - 56

10 - 15

20 - 80

90 - 100

P. ziczac

O - M

14 - 28

20 - 22

7 - 15

90 - 100

A. gibbus

O - M

14 - 28

20 - 22

4 - 7

90 - 100

A. irradians

O - M

21 - 35

20 - 22

4 - 7

90 - 100


M. edulis

O - D

28 - 35

12 - 16

5 - 12

90 - 100

Key to sex-type: O - oviparous (gametes shed into the water); L - larviparous (adults brood larvae which are then shed into the water); D - Dioecious (sexes are separate); M - monoecious (hermaphroditic - both sexes in the same animal); A - alternate sexuality (sex switches in the same animal after each spawning).

Figure 38: A spawning female Manila clam (photograph courtesy Brian Edwards).

In cold water scallops, such as Pecten maximus and Placopecten magellanicus, the number of degree-days from the time adults begin conditioning to spawning readiness is within the same range. But the duration of the conditioning period for cold water bivalves can be much longer (sometimes more than 8 weeks) because the maximum temperature of conditioning is no higher than 15 or 16ºC and may be as low as 10 to 12ºC. Colder water bivalves are often gradually acclimated to the required conditioning temperature by raising temperature from the ambient at a rate of 1 or 2ºC per week. This also extends the overall conditioning period.

Spawning is the hatchery procedure by which conditioned bivalves are induced to liberate their mature gametes in response to applied stimuli. In the case of clam and scallop species, viable embryos cannot be obtained from "stripped" gametes (see the next section below for explanation of the term "stripped"). Eggs need to undergo a maturation process during passage down the oviducts before they can be successfully fertilized.

4.2.2 Gamete stripping

Fully mature gametes can be "stripped" (removed) from the Pacific oyster, Crassostrea gigas, the American (Eastern) oyster, Crassostrea virginica, the mangrove oyster, Crassostrea rhizophorae, and from other similar oviparous oyster species. This is a common and convenient way of "spawning" these species following a suitable period of conditioning.

This procedure involves sacrificing a number of ripe adults when larvae are required (Figure 39). Removing the flatter shell valve reveals the soft body tissues of the oysters. The gonad overlies the digestive tissues towards the umbone and hinge of the shell and when very ripe will extend around the adductor muscle. Either the gonad can be cut repeatedly with a scalpel and the exhuding gametes washed with filtered seawater into a part-filled beaker or bucket, or a clean Pasteur pipette can be inserted beneath the overlying gonad epithelium and the gametes removed by exerting gentle suction. The pipette contents are then transferred to a beaker or bucket containing seawater at culture temperature. In both cases, a small sample is first removed from each of the number of opened oysters. These samples are examined microscopically under x40 to x100 magnification to determine sex and the appearance of the gametes. The sperm should be motile and the eggs, which are normally pear-shaped when first removed, should round off in contact with seawater within 20 minutes. The top shell valves should be replaced on the oysters to await stripping in order to prevent dessication.

Figure 39: Stripping and transferring gametes from Pacific oysters to a beaker of filtered seawater using a Pasteur pipette.

Assuming the gametes are fully mature, the process is continued to remove gametes from the opened oysters - whose sex is now known - starting first with the females. Crassostrea oysters are extremely fecund. Seventy to 90 g females may each be carrying 80 to 120 million eggs, not all of which need to be stripped.

Care needs to be taken to prevent puncture of the digestive gland during stripping. This is to avoid contamination of the gametes with tissue and bacteria and other microorganisms of gastro-intestinal origin. Either the eggs of individual females can be collected separately in clean 2 to 5 l glass beakers or they can be pooled together in 10 to 20 l plastic buckets that are 75% filled with filtered, ultra-light disinfected seawater at the required temperature (usually 24±2ºC).

After completion of egg stripping, the males are dealt with in a similar manner. The exception is that it is more common to pool small samples of the sperm from each of the males in a 1 l glass beaker, part filled with filtered ultra-light disinfected seawater at the same temperature, making sure that the final sperm density is not too great. As a guide, one should just be able to see nearby objects through the beaker and its contents. The gametes are then ready for fertilization.

4.2.3 The special case of flat oysters

Before considering spawning in clams, scallops and mussels the special case needs to be noted concerning oysters of the genera Ostrea and Tiostrea. These, unlike other commonly cultured bivalves, do not need to be stimulated to spawn. They will spawn of their own accord during the conditioning process and will brood larvae within their mantle cavities for varying periods of time depending on species and temperature. This group of oysters, including the European flat (or "Belon") oyster, Ostrea edulis (Figure 40), the New Zealand ("Bluff" or mud) oyster, Tiostrea lutaria, and the closely related Chilean flat oyster, Tiostrea chilensis, are referred to as larviparous. The latter two species release their larvae into the surrounding water after about a 20-day brooding period when the larvae are between 450 and 490 µm shell length and are almost ready to set. In contrast, the European flat oyster releases its larvae, after a brooding period of 6 to 8 days at normal conditioning temperatures, when they are 170 to 190 µm shell length and require a further 10 to 12 days of culture before they reach maturity and are ready to set. Eggs of the New Zealand and Chilean flat oyster are 350 µm diameter compared with 150 µm in the European flat oyster.

Figure 40: Anatomy of a developing flat oyster, Ostrea edulis; am - adductor muscle; g - gonad tissue overlying the digestive gland; gl - gills; h - hinge; ic - inhalant chamber of mantle cavity. At spawning, eggs pass through the gills into the inhalant chamber of the mantle cavity where they develop to fully shelled larvae over the course of a week or more, depending on species. The parent releases larvae when they are able to ingest and digest algae. (The anatomy of oysters of the genera Tiostrea and Ostrea is essentially similar).

Figure 41: Brooding stages of the European flat oyster, Ostrea edulis. W - the "white sick" stage shortly after eggs are passed to the inhalant chamber of the mantle cavity; G - the "grey sick" stage, beyond the trochophore stage, when the shell valves are well developed but the larval organs not yet fully developed (3 to 5 days after spawning); B - the "black sick" stage at which larvae are almost fully developed and are ready to be released. White, grey and black "sick" are traditional terms applied to brooding oysters in Europe.

The above species are not mass spawners. Rather, stocks of adults produce larvae over an extended period. It is extremely rare to see mature males liberating sperm into the surrounding water and it is assumed that they do so periodically in small quantity. Adjacent female-phase oysters (these species exhibit alternate sexuality) draw in sperm in their inhalant current, in the same way as food particles, and in response, release their eggs into the exhalant chamber of the mantle cavity - as do oviparous species. However, the eggs are not expelled into the surrounding water. Instead they are passed through the gill filaments into the inhalant chamber of the mantle cavity where they are fertilized and develop over an extended period (Figure 41), to be fully motile, completely shelled veligers at the time of release (Figure 42).

Hatchery technicians experienced in rearing these species, can often identify spawning and brooding female-phase oysters from small quantities of eggs that escape mantle cavity retention and settle on the upper shell valve, adjacent to either the inhalant or exhalant mantle apertures. Brooding oysters also tend to be inactive, retaining only a minimal shell gape for long periods.

When larvae of the larviparous oysters are liberated into the water they either swim to the surface forming visible "rafts" in the case of O. edulis, or they immediately seek a surface upon which to settle and undergo metamorphosis in the Tiostrea sp. In the latter case, suitable settlement surfaces need to be added to the broodstock tanks in advance of larvae liberation. The surfaces can either be shell or plastic cultch materials or plastic mesh (see later section dealing with settlement).

Figure 42: The appearance of Ostrea edulis veliger larvae (175 µm mean shell length) at release from the adult. All larvae are normally formed except for - a - which exhibits incomplete development of one shell valve.

When the expected liberation period is reached in the case of O. edulis, tanks should be checked every 2 or 3 hours for signs of larval release. Swimming larvae can be skimmed from the water surface of the conditioning tanks with a beaker or a small 90 µm mesh sieve and transferred to a bucket of water. Alternatively, they can be allowed to flow in the tank discharge into a larger sieve of the same mesh aperture, which is partially submerged in a tray of water (Figure 43). It is always best to collect the larvae as soon after release as possible to avoid larvae becoming contaminated by adult faecal matter in the water, or being filtered out of the water by the filtration activities of the adults.

Figure 43: Experimental broodstock conditioning of Ostrea edulis. Note the green coloured sieves immersed in shallow trays to catch and retain larvae.

Once a brood has been collected they are counted (see later) and distributed between culture tanks at the appropriate density. Female-phase European flat oysters of 70 to 90 g (the size of oysters in Figure 41) will liberate broods of between 1 and 2.5 million larvae. In contrast, female-phase Tiostrea oysters, which produce considerably larger eggs, will liberate much smaller broods of 20 000 to 50 000 larvae.

Larvae can be removed from adults identified as brooding either from the conditioning tanks, or in stock brought back from growout - or from wild populations - during the natural breeding season. The steps in this procedure are illustrated in Figure 44. It is sometimes used as a method to obtain larvae before they have developed a functional gut in the later stages of brooding. This can be relevant in the summer when potentially pathogenic bacteria are prevalent. There is evidence to suggest that brooding larvae begin feeding while still in the parental mantle cavity and, thereby, may be exposed to high loads of bacteria and other micro-organisms accumulated and defaecated both by the parent and adjacent stock.

Whether larvae are liberated naturally by the stock or are removed prior to release, they are grown following the standard methodology described later in the larval culture sections of this manual. Best results are with broods that have developed to the fully shelled, motile, D-larva stage. If removed at an earlier developmental stage, food is withheld until larvae have developed a fully functional alimentary system - visible through the transparent shell valves as a darker S-shaped structure, which can be seen in Figure 42. This may take 2 or 3 days from the time of removal. Prior to this stage, the soft body tissues are a densely, granular, grey colour and the larvae only weakly motile (see Figure 41 - "grey sick" larvae).

Figure 44: A - Stripping Ostrea edulis larvae from a brooding adult. B - The top (flat) shell valve is removed, then the brooding larvae are washed through a 90 µm sieve balanced over a bucket of filtered seawater (C). D - Most of the larvae swim rapidly to the water surface where they aggregate (raft) together. They are then ready to be sampled for counting and for size determination. Photographs were taken at the Harwen Oyster Farm hatchery in Nova Scotia (courtesy John and Krista Harding).

4.2.4 Induced spawning of oviparous bivalves

Other commercial species reared in hatcheries are known as oviparous as compared with the larviparous oysters discussed above. Oviparous species shed their eggs and/or sperm into the surrounding water where fertilization takes place.

Various stimuli can be applied to induce spawning; the most successful being those that are natural and minimize stress. The description that follows is of a technique known as thermal cycling, which is the most widely used method for oviparous species. As a general rule of thumb, if stock do not respond to thermal stimuli within a reasonable period of time, the gametes they carry are most likely not fully mature.

The use of serotonin and other chemical triggers to initiate spawning is rarely beneficial. Eggs liberated using such methods are often less viable than are those produced in response to thermal cycling. Thermal cycling procedure

Mature bivalves taken from broodstock conditioning tanks are cleaned externally to remove any adhering debris and fouling organisms from their shells and then are thoroughly rinsed with filtered seawater. After cleaning they are placed in a spawning trough or tank. The preferred tank-type is a shallow, fibreglass tray of approximately 150 x 50 x 15 cm depth - 10 cm water depth (Figure 45). It needs to be of a size that can be viewed by two or more operatives who are experienced in detecting the onset of spawning by adults (an important point in the spawning of monoecious species - see later).

Figure 45: Diagram of a tray arrangement widely used for the spawning of oviparous bivalves. (After Utting and Spencer, 1991)

The trough is often fitted with a standpipe drain and two filtered seawater supplies, one heated or chilled to 12 to 15ºC and the other at 25 to 28ºC (e.g. for Crassostrea species and Manila clams). Lower temperatures apply to cooler water species. The importance is the differential between the lower and higher temperature, which will normally be about 10ºC.

The base of the trough is painted matt black or is covered by black plastic sheet to provide a dark background against which gametes being liberated can be readily seen (Figure 45).

The trough is part filled with the cooler water to a depth of about 10 cm and a small amount of cultured algae is added to stimulate the adults to open and start pumping activity. After 30 to 40 minutes the water is drained and replaced with water at the higher temperature, again with a small addition of algae. This water is drained after a similar time period and replaced with cooler water and the procedure is repeated.

The number of cool/warm cycles that are required to induce spawning depends on the state of maturity of the gametes and the readiness of the adults to spawn. In summer the adults may spawn within an hour of induction, but earlier in the season it may take 3 or 4 hours of cycling before the first animal spawns. Generally, if the adults do not respond within a 2 to 3-hour period they are returned to the conditioning tanks for a further week. Adults may start spawning on either the cool or warm part of the cycle, most commonly the warm. Although it is generally the case that males will spawn first, this cannot be guaranteed.

Additional stimuli can be provided in the form of stripped eggs, or sperm from an opened male. The gonad is located at the base of the foot in clams. In scallops it is a separate organ and can be seen when the mantle and gill tissues are lifted. If the gonad is carefully punctured with a Pasteur pipette and suction applied, quantities of gametes can be withdrawn which can then be mixed in a small volume of filtered seawater before adding to the seawater in the tray. In clams with discrete siphons, the diluted gametes are directed towards the inhalant siphon of active clams with a Pasteur pipette so that they are drawn into the mantle cavity by the pumping action of the adults. The inhalant siphon is the siphon furthest away from the hinge and has the largest diameter aperture. When spawning occurs in clams, gametes are expelled through the exhalent siphon as shown in Figure 38. The thermal shock during the second warm water cycle almost always elicits a spawning response in ripe clams and in other fully mature, oviparous bivalves within 1 to 2 hours. Spawning dioecious bivalves

In dioecious species (refer to Table 9), in which the first adults to spawn are almost invariably males, it is good practice to remove them from the trough and leave them out of water until sufficient eggs have been collected from spawning females. The reason is that sperm ages more quickly than eggs and if more than 1-hour-old at the time fertilization is made, fertilization rate may be reduced.

Figure 46: A - Pecten ziczac adults undergoing thermal cycling in a spawning tray. Note the aquarium heater used to maintain the elevated temperature. A similar tray of water is chilled with ice packs to provide the cold shock. B - Individual scallops spawning in 3 l plastic beakers immersed in a constant temperature water bath. While this species is not dioecious, the illustration applies to procedures used in spawning any species.

As each female begins to spawn it is necessary to remove it from the spawning trough and transfer it to an individual spawning dish or beaker part-filled with filtered seawater at 24-26ºC (Figure 46). The dishes/beakers are contained in a heated waterbath to maintain the temperature. The same procedure applies to spawning males, which can be identified as such by the continuous stream of milky fluid escaping from the exhalant siphon compared with the granular appearance or clumps of eggs shed by a female. Females may start spawning as much as 30 to 60 minutes after the first male begins to liberate sperm.

Time to completion of spawning for an individual is variable but gamete liberation rarely lasts for more than 40 to 60 minutes, often a shorter period in females. It may, however, be necessary to remove a spawning female from its container and place it in a fresh one when large numbers of eggs have been liberated. The presence of dense concentrations of eggs in the water inhibits pumping activity and hence the expulsion of further eggs. In addition, the female may start to filter the eggs out of suspension.

Eggs may be liberated in clumps that will eventually settle to the base of the dish or beaker. These clumps are separated when spawning is completed by carefully pouring the dish contents through a 90 µm nylon mesh sieve (eggs will not be retained by this mesh size), retaining the separated eggs on a 20 to 40 µm mesh sieve. The eggs are then gently washed into a clean glass or plastic container with filtered seawater at the required temperature. "Clumpy" eggs often do not fertilize well. The best success is most often obtained when females liberate streams of well-separated eggs that remain in suspension for longer periods than do the clumps.

When first spawned the eggs are pear-shaped but they rapidly hydrate and assume a spherical shape when in contact with seawater. Eggs from different females are collected separately to provide opportunity to visually assess quality using a microscope at about x100 magnification. Batches of eggs that do not round off after about 15 to 20 minutes in seawater should be discarded. Reproductive development in the females of oviparous bivalves is not completely synchronous so that at any point in time eggs spawned by different females will be at slightly different stages of maturation. When separation and examination of the eggs is complete, batches of eggs that appear good can be pooled in a larger volume container.

Sperm from the various males that spawn are similarly pooled. It is good practice to use eggs from at least 6 females and sperm from a similar number of males to provide larvae for a production run. This ensures satisfactory genetic variability among the offspring, the extent of which will depend on the degree of heterozygosity of the parents. Small volumes of the pooled sperm suspension are mixed with eggs during gentle agitation of the contents of the container in the proportion 1 to 2 ml per l of egg suspension. Spawning monoecious bivalves

The procedure to spawn hermaphroditic species, including many species of scallops, in which individual adults mature both eggs and sperm synchronously, is more complex. Here, the objective is to minimize chances of eggs being fertilized by sperm from the same individual (self-fertilization). It is rarely the case that an adult will spawn both eggs and sperm at the same time. More usually, sperm will be liberated first followed by the eggs. Individuals will often revert to liberating sperm once their eggs have been spawned.

There are two approaches to maximize chances of cross-fertilization. Large numbers of adults can be spawned in large-volume, deep tanks. These are fitted for flow-through so that the contribution of sperm from a particular individual is a small proportion of the total and the overall amount of sperm is continuously being diluted by water flow. When individuals switch to female production, the denser eggs are retained in the tank and chance dictates that the eggs of that individual will more likely be fertilized by the sperm of other individuals than by its own sperm. This method - applicable also to the large-scale spawning of dioecious species, where self-fertilization is not an issue - is used in mass production facilities for Argopecten purpuratus in Chile and is also used in the pond culture of bivalves in Asia.

Alternatively, permitting closer control of fertilization, each adult is transferred to a small container of filtered seawater at the required temperature once it begins to spawn (Figure 47). The container is labelled with the time and a reference number that will follow the progress of that particular adult throughout its spawning activities. As the adult spawns and clouds the water with its gametes, it is moved to a fresh, clean container after first being thoroughly rinsed with filtered water. This container is labelled with the time of transfer and the same adult-specific reference number. Careful observation is maintained on each beaker containing an adult liberating sperm in order to detect the onset of egg liberation, which is usually a sudden change. As each adult switches to egg production it is immediately removed and transferred to another container after rinsing, carrying with it the same adult-specific reference number and the time of the switch. Once sufficient eggs have been spawned, the adults are removed from the beakers before they revert back to sperm production. Thus, eggs and sperm are separately accumulated from each adult, identified as to origin by the different adult-specific reference numbers and time of production.

Mature adults with ripe gametes obtained directly from the sea can be induced to spawn in the hatchery in the same way.

Figure 47: This sequence of photographs illustrates the spawning of the monoecious calico scallop, Argopecten gibbus, at the Bermuda Biological Station for Research, Inc. (BBSR).

A - Broodstock are conditioned in the hatchery at 20-22ºC for 2 to 4 weeks during late winter, early spring. A constant flow of seawater is maintained through the tank and food is added daily.

B - The appearance of a fully mature scallop; the orange coloured ovary and white testis occupy the distal and proximal parts of the gonad respectively. The adductor muscle is centre right and the brown-coloured tissue includes the gills and mantle, which have been raised to expose the gonad.

C - Up to 20 scallops are spawned at a time in transparent plastic trays of approximately 75 x 45 x 5 cm water depth. The trays contain sufficient 1 µm filtered seawater to fully cover the scallops. One is chilled to 12°C with ice packs and the other is heated to 25 to 27ºC with a 150W aquarium heater. The scallops are cycled between the two temperatures as explained in the text.

D - Staff keep careful watch to identify scallops as they begin to spawn in the warm water tray. Spawners are rinsed with filtered seawater and transferred individually to labelled plastic beakers containing 0.5 to 1 l of seawater in other trays acting as warm water baths at the spawning temperature.

E - After liberating sperm, scallops will suddenly switch to spawning orange coloured eggs. It is important that as soon as the switch is made scallops are removed, rinsed and returned to clean beakers containing filtered seawater to continue egg liberation. If egg production is swift and prolific, sperm from other scallops will often be added at this time.

F - Eggs of good quality, determined by microscopic examination, are pooled in 10 l buckets. Note the perforated plastic plunger used to gently agitate the bucket contents to keep the fertilized eggs in suspension. The bucket may contain between 5 and 10 million eggs - judged "by eye".

4.2.5 Fertilization procedures

Before fertilization, if not already done, egg suspensions should be gently filtered through a suitable mesh-size sieve (90 µm aperture or greater) held so that the mesh is below water level in a larger volume bucket or container. This step is to remove contaminating faecal pellets from the adults prior to the addition of the sperm to reduce risk of the subsequent proliferation of bacteria and other micro-organisms during the next stage in the culture process.

Figure 48: Dividing Crassostrea gigas eggs about 50 minutes after fertilization. Most of these eggs are developing normally and are at the 2 and 4-cell stage.

The method used to fertilize eggs is essentially the same whether for monoecious or dioecious species. The one exception in hermaphroditic bivalves is to ensure that eggs are cross-fertilized with sperm from adults other than the one that provided the particular batch of eggs. For this reason, batches of eggs from the different adults are kept separate and are separately fertilized with recently shed sperm from 3 or 4 males in the ratio 2 ml of sperm per l of egg suspension. Following sperm addition, they are allowed to stand for 60 to 90 minutes before pooling - if required - with the fertilized eggs from other adults.

Figure 49: Stages in the early development of eggs; A - sperm swarming around a rounded-off egg; B - extrusion of the first polar body following fertilization; C - two-cell stage also showing the second polar body; D - four-cell stage; E - eight-cell stage. The eggs of most oviparous bivalves range in size from about 60 to 80 µm, depending on species. The time from fertilization to the various developmental stages is species and temperature dependent.

Within this time period, at the appropriate temperature for the species, the fertilized eggs will begin to divide, first almost equally into two cells and then unequally into 4 cells where one large cell will be observed capped by 3 much smaller cells. The first sign of successful fertilization, however, before cell division starts, is the extrusion from the egg of a small, transparent, dome-like structure, which is the first polar body (Figures 48 and 49). Assessment of the percentage of eggs developing normally can be made using a relatively low power microscope (x20-40 magnification). Fertilization rates almost invariably exceed 90% assuming the eggs are fully mature.

It is desirable to estimate egg numbers prior to or within 20 to 30 minutes of fertilization since development will be impaired if the density of embryos per unit volume beyond the early stages of cleavage exceeds certain specified limits. This density is specified later and the method to determine both egg and larval numbers is described in section


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