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Part 3. HATCHERY PRODUCTION PROCEDURES


3.1- BROODSTOCK MANAGEMENT

Establishing the broodstock

To work properly and with adequate safety margins, any hatchery should secure a reliable and sufficient supply of good quality fish eggs. With this aim in mind, most Mediterranean hatcheries have established their own broodstock units, where breeders of different age groups of the species involved are kept under long term stocking conditions.

However, timing, amount and quality of the eggs produced by these units do not always fit hatchery requirements, resulting sometimes in poor final outputs in terms of viable fry.

The importance of a properly dimensioned and managed broodstock unit is now fully acknowledged and its relatively high running costs are fully justified.

In the management of a fish broodstock unit the main issues to be considered refer essentially to:

Stock dimensioning

To properly determine the dimension of the broodstock unit, different parameters have to be taken into consideration. First, the seasonal production target for the hatchery has to be set. In particular the desired number of fry, their size and species and delivery timing should be planned. To this purpose, it is important to set the average survival rate from larva to weaned fry. Second, the reproductive characteristics of the species, such as sexual cycle pattern, egg productivity and latency period (see Table 3.1) play a key role in stock dimensioning. A third important aspect is represented by the possibility to introduce a year-round supply of eggs, which requires the manipulation of photoperiod and temperature to obtain out-of-season maturation and spawning.

As described in Part 2 of the manual, both seabass and gilthead seabream are seasonal breeders spawning in winter and early spring, with some differences in timing according to the specific location and the species. But whereas seabass is a synchronous gonochoric species, gilthead seabream is a proterandrous hermafroditic species where males undergo sex reversal to became females. They also differ from seabass females in being sequential spawners, i.e. they can lay 20,000-80,000 eggs every day for a period of up to four months, moreover, in captivity sex reversal is conditioned by social and hormonal factors which may lead to unfavourable sex ratio if not properly considered in establishing groups of broodstock.

For practical purposes the following table presents some conservative parameters to be considered as guidelines when dimensioning the broodstock unit.

Table 3.1 - Conservative propagation parameters under hatchery conditions for seabass and gilthead seabream

Species

Egg productivity as No. eggs per kg b.w.

Egg productivity as % of b.w.

Fertilization rate (%)

Hatching rate (%)

Viable larvae (No. Per kg per yr)1/

Latency (months)2/

Seabass

300,000

20-25

90-95

75-85

220,000

6

Gilthead seabream

800,000

50-80

90-95

70-80

560,000

12

1/Average number of viable two days old larvae that can be expected yearly per kilogram of healthy female

2/Time between the introduction of brood fish in the hatchery and their first spawning.

Although these parameters are already conservative, to face any possible egg shortage during the spawning season, a safety margin in stock size is recommended. A practical way for planning purposes is therefore to consider the following average female fecundity per season:

seabass:

120,000 2-days old larvae per kg b.w.

gilthead seabream:

350,000 2-days old larvae per kg b.w.

Table 3.2 gives further information on size at sexual maturity and optimal size for spawning to be considered in broodstock dimensioning.

Table 3.2 Size at first sexual maturity and optimal size for spawning

Species

Sex

Size at first maturity

Optimal size and age

Reform

L or W

age

wild

farmed

(years)

kg (years)

kg (years)

kg (years)

seabass

M

23-26 cm

2-31/

0.6 (2-4)

0.7 (3-4)

> 1.5 (6)

28-30 cm

22/




F

31-33 cm

4 - 51/

1-1.5 (5-8)

1.5-2 (6-8)

> 3 (9)

37-40 cm

32/




gilthead seabream

M

100-300 g

1-2

0.3-0.5 (1-2)

0.3-0.5 (2-3)


F

> 600 g

> 3

0.8-1 (3-5)

1-1.5 (4-6)

>2 (8)

1/Tunisia

2/Mediterranean France

In seabass, as fecundity and egg quality improve after the first spawning, the optimal age for female parent fish is between 5 and 8 years, whereas for males this range is lowered to 2-4 years.

When considering gilthead seabream, five age groups should be included in the broodstock, from age 1 (young males) to age 5 (old females). With this species additional precautions should be adopted. Sex reversal, which takes place at the end of the spawning season, approx. from May to September, is socially determined. In particular, the presence of young fish (potential males) during this period increases the number of older fish that become females. On the other hand, the presence of older females will inhibit sex reversal in younger fish which will remain functional males.

Furthermore, as in old fish the quality of eggs decreases, the farmer should annually replace the oldest age groups of both species with younger ones either from the wild or farmed.

A further aspect has to be considered in case hormones are extensively used to induce spawning. An improper use may induce female fish to not respond properly to repeated hormonal injections. See further for more details.

Origin, capture and handling of broodstock

Parent fish can come either from a farm or from the wild. Usually, wild fish are preferred since farmed animals could present some problems such as consanguinity or poor general conditions due to an unhealthy rearing environment. On the other hand, the use of farmed animals gives the possibility to select breeders on the basis of observed characteristics of the animals (fast growth, commercially preferred pigmentation and shape, domestication, etc.).

Wild fish have to be conditioned to captivity, which generates stress that will inhibit their sexual maturation for a certain time (latency period). It is safer to consider that new broodstock should be kept in the farm for at least six months before being used as breeders.


Fig. 20 Broodstock harvesting (photo STM Aquatrade)

The main selection criteria to identify adult fish as suitable breeders are not scientifically stated, but follow the common sense. With the advance of genetic studies, more specific criteria will probably become available related to faster growth rate and stronger disease resistance. For the time being, the following empirical selection criteria for breeders have a proven record to work for hatchery purposes:

In capturing and handling breeders, stress should always be minimised. This can be partly achieved by selecting suitable gear. The best fishing gear for wild fish is the fish barrier, a fixed trap placed at the outlet of coastal lagoons. As fish enter the trap following their migratory instinct, they can be captured without much stress. The seine net used in ponds and tanks gives also good results but it has to be handled very carefully, harvesting only a small number of fish per haul. To avoid losses of scales, the use of knotless 15-mm stretched mesh nylon nets, as well as cotton gloves, is recommended.

Trammel nets and monofilament gill nets should be avoided because they may cause unrecoverable skin damages.

Healthy adult fish can also be captured by hook and line, but care should be taken to reject all fish with major injuries in their mouth, gut or, even worse, gills, as well as those which have lost too many scales when hauled on board.

To spare additional and potentially fatal stress to recently caught fish, the temporary holding and transport containers should have the following characteristics:

Additional precautions are:

For fish transport in which oxygen is provided, consider the following:

Selection and quarantine treatment

Upon arrival at the hatchery, fish are anaesthetised and checked using the selection criteria mentioned above. Selected fish are then weighed, their sex is checked and they are immediately transferred to already prepared “quarantine tanks” to receive their first prophylactic treatment, as indicated in the quarantine protocol (Annex 1). The treatment is given as soon as fish enter the hatchery facilities: this limits the risk of introducing parasites or bacterial diseases and facilitates their recovery from handling stress. Fish not selected are discarded or sold.

The most commonly used anaesthetics for fish are: MS 222 (@20-50 ppm), 2-phenoxyethanol (@200-300 ppm) and quinaldine dissolved in acetone (@3-5 ppm). Excessive manipulation and sudden changes in water temperature and salinity have to be avoided.. Always operate gently when getting hold of brood fish, raising the fish with both hands palms up from the lower body (one below the head, the other below the anus). Never touch them with dirty or dry hands, wash them and dip them in the holding container water to have your skin well wet prior to touch the fish. The use of cotton gloves to handle fish is recommended.

The quarantine protocol (Annex 1) is designed to eliminate possible external parasites, as well as to seal wounds and abrasions caused by fishing, handling and the parasites themselves. The treatment usually followed in hatcheries is a sequence of medicated baths: first formalin (water solution with 37-40% by weight of formaldehyde, HCHO) and malachite green (zinc free oxalate or aniline green), followed by a second quick immersion in fresh water to end with a third bath in furanic antibiotics. This treatment is repeated four times every three other days. The entire procedure takes a couple of weeks. No additional specific treatment is required as fish are assumed to be healthy.

Quarantine tanks should have a flow-through water circulation, round or rounded shape, small size (4 to 6 m3) and a smooth inner surface to allow for rapid cleaning, easier harvesting and reduced use of chemicals for treatment baths. The recommended building materials are: FRP, PVC, PE or plastic-lined concrete tanks.

It is mandatory that quarantine facilities do not come into contact with other rearing units through effluents or shared equipment. They must be completely isolated from the other farm facilities to prevent possible spread of parasites and diseases, sometimes tolerated by adult fish, but often deadly for their larval stages. Thus, the effluents from quarantine tanks should be treated to remove pathogens. After the transfer of brood fish, these tanks should be drained and thoroughly disinfected with a 500 ppm hypochlorite (NaOCl) solution.

Stocking facilities

After quarantine, parent fish are moved either into larger tanks where they remain for a couple of months until full recovery, or directly into the long-term holding facilities. Lowering salinity down to 20 ppt for a few days helps the recovery of weak animals. In any case, the weight of each female fish is recorded to estimate its potential egg output (see Table 3.1). For gilthead seabream weight gives also an acceptable estimate of the broodstock sex ratio (see Table 3.2).

Long term stocking facilities exist in a variety of designs and capacities. When land area is not a constrain, earthen ponds stocked at low density (up to 0,5 kg/m3) represent a reliable and easy to manage solution. They usually measure up to 500 m2 in size with an average water depth of 1.5 m and have a rational water exchange with inlet and outlet systems through loosely screened monks to allow small fish and crustaceans, which may represent an additional source of food, to enter the pond. The outlet offers the possibility to empty the pond by gravity. During the hottest months, a shelter should cover at least 10% of the pond area to provide some protection against the sun and a quiet place to rest. If necessary, protection against fish-eating birds should also be contemplated. Another cheap solution to stock broodstock is given by floating cages, provided that suitable sheltered coves are available. Fish control is however less easy than in land-based facilities.


Fig. 21 Traditional earthen pond for broodstock long-term holding (photo STM Aquatrade)

More frequently, and depending from the design of the hatchery, stocking facilities are smaller tanks of 4-20 m3 capacity either round, or square with rounded corners,: They are made of concrete, FRP or PVC-lined, and the complete control of environmental conditions favours a higher fish density up to 2-5 kg/m3. Such tanks are also utilised to produce out-of-season spawnings (see below).

Dissolved oxygen levels in water should be maintained at near saturation with an adequate water renewal and aeration supply. Values below 50% saturation, as well as abrupt changes in other water quality parameters should be avoided. During gonadal maturation, water salinity has to be kept above 35 ppt to complete oogenesis.

Routine controls are necessary for proper management and include checking water quality parameters (salinity, temperature, dissolved oxygen, pH, etc.) and fish stock condition (general behaviour, feeding activity, diseases symptoms, prophylactic treatments, etc.). See Annex 2 for a model of the forms to be used for tank files with main daily controls and operations.

Feeding broodstock

Although studies on the effects of diets on the reproductive capacity of seabass and gilthead seabream are far from being complete, it is generally acknowledged that a diet rich in vitamins, poly-unsaturated fatty acids (n-3 PUFA) and other micro-nutrients is essential in obtaining viable eggs and larvae.

For practical purposes, two distinct feeding regimes are applied: a maintenance diet after spawning till the onset of the next ovogenesis period, some three to four months before the next spawning season, and a boosted diet thereafter to provide the essential nutritional requirements for proper gametogenesis (Annex 3).

Maintenance diet

The maintenance diet should keep spent fish or new fish breeders in good health until the onset of the gametogenesis. It should therefore be rich and varied both in quality and quantity, and should be assessed by regular controls. It should preferably be as close as possible to the fish natural diet, including fatty and lean fish, crustaceans and molluscs. According to availability from the local fishery and suppliers, trash fish may represent a cheap solution, paying due attention to the increased pollution load in the stocking facilities. To keep their original quality, trash fish should be purchased fresh and then cleaned, minced and deep frozen immediately. This process also lowers the risk of parasitic infection.

It is advisable to get broodstock used to pelletized dry feed in order to have an alternative to fresh food at hand in case of shortage. Moreover, dry feed is useful when drugs or other feed integrators have to be supplied to the fish. The use of automatic feeders is only possible with dry pellets.

Even if the choice between fresh food and dry feed depends on several factors such as their availability, cost, use of feeding equipment and management, it is advisable to use both feed types in order to benefit from a broader range of possibilities. A practical solution envisages the distribution of pellets 6 days a week supplemented with moist food twice a week, and with no feeding one day a week, typically on Sunday. This pattern reduces the workload to manageable proportions, and still provides fish with a proper diet. The daily feeding rate usually ranges between 0.7% and 1.4% of body weight in seabass and 1 to 1.5% in gilthead seabream, both adjusted in line with water temperature and physiological status of the fish (Annex 3).

From a management point of view, a feeding schedule should be prepared at regular intervals based on periodical controls of fish weight. Feeding by hand is recommended because it would be possible to prevent food leftovers, which may rapidly deteriorate water quality, and to observe the behaviour of broodfish. Food is usually given once a day, early morning or late afternoon. Water renewal in the tank is adjusted to keep DO levels at saturation and ammonium nitrogen below 1 ppm. Pollution caused by feeding fresh food can be controlled by using a flow-through water system and frequent cleaning. When broodstock is kept in a system using water recirculation, a mechanical filter to remove suspended solids is frequently added to improve the performance of the biofilter.

Breeding period diet

During gametogenesis female fish require a food richer than usual in proteins and lipids to produce the vitellogenin, which is progressively stored as yolk in the oocytes. As the sole source of food for the developing embryo and the early larval stage until feeding on live preys starts, yolk quality and quantity are key factors for a successful reproduction.

Both dry pellets and moist food are employed in this period. Dry pellets should include all the nutritional elements which are acknowledged to be essential in the development of viable larvae, such as the poly-unsaturated fatty acids (n-3 PUFA), in particular EHA (20:5 w 3) and DMA (20:6 w 3), which have to be supplied with food, as they cannot be produced by fish metabolism. In case of poor diets, the perivisceral fat of the females, rich in saturated fatty acids, is utilised for yolk production, resulting in poor egg quality and reduced larval viability.

Commercially available integrators are now widely adopted by modern hatcheries to boost the quality of pelleted feeds formulated only to grow fish. As a better alternative, specially formulated pellets or natural enrichment components such as squid oil are fed to the breeders. As a general rule, during this period the distribution of moist food should replace most of dry feed, if possible, due to its superior nutritional value.

Feed distribution follows the pattern mentioned above. A difference between the two species comes from their different spawning characteristics. As a synchronous spawner, seabass is not fed during its brief spawning period, but gilthead seabream, with its sequential emissions lasting several weeks, should always be.

Egg production

Gametogenesis

The natural process of sexual maturation in both species is briefly described below. By acting on photo and thermo-periods, it is also possible to obtain viable larvae almost throughout the year. A method to delay. spawning is described at the end of the chapter.

Under natural rearing conditions, and with variations linked to the geographical location, the sexual resting period (only oogonies present in the female gonads) lasts from the end of the spawning season till early autumn when gametogenesis starts. This process is regulated by a complex hormonal and environmental pattern, which has been better investigated in seabass.

Table 3.3 - Gametogenesis: environmental parameters and duration

Species

Period
(length)

Temperature
(°C)

Hours of light
(hours)

Duration
(days)

Seabass

Sept. to Jan.
(3 months)

20-8

14-8.5

16-95
mean: 74

Gilthead seabream

Sept. to Dec.
(3-4 months)

20-9

14-8.5

30-154
mean: 107

Both species may start ovogenesis in water with a salinity lower than 35 ppt, such as in the case of estuaries and coastal lagoons, but the final steps leading to maturation (exogenic vitellogenesis and meiotic divisions) require full seawater (= 35 ppt). Temperature has an effect on the speed of vitellogenesis and acts as a minimum/maximum threshold for the spawning.

Spermatogenesis in seabass and gilthead seabream males usually takes place under captivity conditions and even at salinity levels lower than full seawater. Spermiation starts one or two months in advance of the female spawning period and males may remain active throughout the entire spawning season.

Fig. 22.00 Ovarian development in Dicentrarchus labrax

a) Stage 1: oogonia

b) Stage 2: young oocytes

c) Stage 3: individualization of oocytes and appearance of follicular cells

d) Stage 4: differentiation of oocyte cytoplasm

e) Stage 5: separation of three areas in cytoplasm

f) Stage 6: early signs of vitellogenesis and start of membrane differentiation

Fig. 22.00 Ovarian development in Dicentrarchus labrax

g) Stage 7: organization of yolk globules in perinuclear layer

h) Stage 8: a second type of yolk globules are evident and the zona radiata appears

i) Stage 9: distribution of the two types of yolk globules

I) Stage 10: a third type of yolk globule appear

m) Stage 11: end of vitellogenesis and polar migration of the nucleus

n) Stage 12: the oocyte with oil droplet (G.h.) homogeneous yolk (V.) and cortical granules (G.c.).

Table 3.4 - Spawning: temperature range and limits (°C)

Species

Optimum range

Min. temp.

Max. temp.

Gametogenesis blocked

Seabass

13-15

9-10

18

21-22

Gilthead seabream

15-17

13-14

20

24

 

Stocking broodstock in the spawning tanks

At the onset of the spawning season it is necessary to move selected batches of breeders from their long term holding facilities to the spawning tanks, where they can be better treated and their performance can be easily monitored.

The selection of suitable fish should take into account their health status, sex and maturation stage. Sex ratio in the spawning tanks is kept at two males per female. Whereas males are choosen when they release sperm spontaneously or on stripping, the females maturation stage has to be ascertained by extracting oocytes from the ovary with the use of a catheter: only females with oocytes in the late-vitellogenic stage, i.e. with a diameter larger than 650 mm in seabass and 500 mm in gilthead seabream, are selected.

Ovarian controls start in September and last up to March in gilthead seabream and from December through February in seabass. In case of necessity, wild breeders of both species caught and stocked during the same spawning season could also be utilised, provided that their ovarian development matches the above mentioned requirements and provided also that they receive a complete quarantine treatment. However, due to fishing and handling stress, in the case of these breeders egg output can be erratic as ovarian atresia may follow.

For an ovarian biopsy, proceed as follows:

1. All equipment has to be ready, cleaned and sterilized in advance.

2. Fish are not fed 24 hours before the control. Close water inlet and lower the water level; place a net barrier to divide the tank into two compartments, keeping all fish being concentrated in one of them;

3. Add anaesthetic in sufficient quantity to obtain a light sedation (for type of drug and dosage see previous section). On a tray prepare one glass slide per female, a couple of flexible sterile catheters (1.2 mm internal diameter or 8 to 10 Berchet unit), a Pasteur pipette, cover slides and alcohol;

4. Take one female at a time from the tank (males have already been selected and released into other side of the net barrier). Introduce the sterile catheter into the urogenital papilla and the oviduct, up to the ovary for a few cm; then suck carefully a small sample of oocytes up into the catheter and place the sample on a slide;

5. After sampling, release the animal into the spawning tank, where recovery from sedation will take place. Put a few drops of sea water on the sample and a cover slide, and examine under the microscope at 5 and 10 magnifications. Using a micrometric eyepiece, measure the diameter of the bigger oocytes and record egg measurements and final destination of the fish;

6. Place the catheter in alcohol and sample the next female in the same way with the other catheter. Repeat sampling alternating catheters; Handle fish with care (use cotton gloves) and do not damage the urogenital papilla area when introducing the catheter.

7. Check oxygen levels frequently


Fig. 22.01-2-3-4-5-6-7 Follicular stages observed in ovarian biopsies of gilthead sea bream (photos A. Francescon and A. Barbaro)

Induced spawning

Egg release can be obtained either by natural spawning or inducing it by hormonal treatment. Natural spawning of both species is not uncommon to achieve in confined environments and its unpredictability in terms of both timing and output is a serious obstacle for proper planning of commercial fingerling production. Induced breeding represents therefore the almost obliged choice of most commercial hatcheries. The hormonal treatment is intended to trigger the last phases in egg maturation, i.e. a strong egg hydratation followed by their release. However, if eggs have not yet reached the late-vitellogenic (or post-vitellogenic) stage, the treatment does not work, hence the need for an ovarian biopsy. The human chorionic gonadotropin (HCG) was widely used for both species at a dosage of 800-1,000 IU per kg of body weight in seabass and 250 IU in gilthead seabream. This dosage is usually delivered in two injections in the dorsal muscles, 6 hours apart. The hormone is diluted in a 0.9% sterile saline solution as 2,000 IU per 1 ml. The hormone is easily available, and comes along with vials of saline solution. However, HCG treatment presents some serius drawbacks: not all females respond to it, egg quality may be below acceptable standards (hatching rate below 80%), being a large molecule provokes an immunization reaction, and as a result of it fish treated with this hormone cannot respond when treated again the following season. Finally it is less effective in inducing out-of-season spawning. HCG has therefore been successfully replaced by an analogue of the luteinizing hormone-releasing hormone [LH-RHa des-Gly10 (D-Ala6) LH-RH ethylamide, acetate salt]. Its small molecule (10 peptides) acts on the pituitary gland to induce the release of gonadotropins which, in turn, act on the gonads. Almost 100% of injected fish spawn eggs whose quality usually matches that of natural spawnings. No immunization effect is reported in fish treated with LH-Rha because of the small size of the decapeptide.

The native form of this hormone is not used because of its low bioactivity, due to the deactivation by specific peptidases, which cannot deactivate the analogue form. Its dosage and response are given in Table 3.5. In seabass LH-Rha is delivered with two intraperitoneal injections 4-6 hours apart at 5 and 10 mg/kg b.w. respectively. In gilthead seabream hormonal dosages are much lower. The hormone is dissolved in a 0.9% sterile saline solution as 5 mg per 1 ml.

Table 3.5 - Induced spawning. Minimum oocyte size and hormonal treatments.

Species

Min. oocyte diameter (mm)

HCG (IU/kg b.w.)

LH-RHa (mg/kg b.w.)

No. of Injections (hours)

Latency (hours)

Seabass

700

800-1,000

-

2 (6)

48-72

650

-

5-10

2 (4-6)

72

Gilthead seabream

500

100-250

1

2(4-6)

48

 

Stocking facilities for spawning

The spawning unit should be kept separated from the main hatchery building to avoid disturbance to the spawners and any possible risk of disease contamination. However, for economic reasons it is usual to keep the spawning fish inside the hatchery in a dedicated area. Spawning tanks usually have a water capacity of at least 5 m3. They should preferably be round shaped and 1 m deep. Shape and depth should be such as to provide easy access to fish and to facilitate routine operations such as cleaning, fish replacement, feeding, egg collection and controls. Very often spawning tanks are made of FRP or in epoxy-painted reinforced concrete.

As a general rule, water circulation is a flow-through system, with heated and filtered sea water to keep the desired rearing conditions. Water temperature is kept within the limits of the optimal spawning range avoiding abrupt changes when moving fish from outdoor facilities. Salinity should be above 35 ppt to improve buoyancy of fertilised eggs. Optimal stocking rate is 2 to 3 kg/m3, with a maximum acceptable load of 5 kg/m3. Sex ratio is two males per female. See annex 4 for the cleaning operations and annex 5 for the daily work plan in the spawning tanks.

Out-of-season spawning

When fertilised eggs are required outside the natural spawning period, out-of-season sexual maturation is obtained through environmental phase shifting of the gametogenesis by manipulation of photoperiod and temperature. The technique is successfully applied to both seabass and gilthead seabream and out-of-season spawning is now a current practice in many large Mediterranean hatcheries.

Different methods can be applied for out of season spawning:

The following description refers to the last phase shifting method as it gives fertilised eggs year round. The hatchery management can decide on the periods of egg production according to its marketing and/or farming needs.

The broodstock is divided in four groups including both males and females: three groups are exposed to environmental regimes that are shifted by 3,6 and 9 months respectively compared to the natural environmental regime, which is left for the fourth group. In this way, the hatchery will have a group of fish ready to spawn on each season: in winter the parent fish exposed to natural environmental conditions, in spring, summer and i autumn the other three groups. Shifting should start when fish are still in the resting phase of their sexual cycle.

If breeders are properly managed, eggs produced out of season with shifted cycles do not differ significantly in quality and quantity from the in-season eggs.

An out of phase maturation unit requires specific facilities:


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