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4 The pre-spawning process

For ease of reference, technical guidance on how to manage health and maintain biosecurity in shrimp hatcheries is arranged according to the basic hatchery production process, starting from broodstock selection through to transportation of postlarvae out of the facility. The process has been divided into two broad categories: the pre-spawning process and the post-spawning process. The pre-spawning process includes procedures for broodstock selection, maintenance, maturation, acclimatization, spawning and hatching. As these procedures require different facilities, the facility maintenance guidelines are described under the different specific facilities used in the hatchery production process. Broodstock handling, nutrition and feeding are also discussed.

4.1 Broodstock selection

Healthy broodstock that are not carriers of serious pathogens must be selected in order to achieve successful hatchery production

Some viral diseases such as Infectious Hypodermal and Haematopoietic Necrosis (IHHN) are believed to be transmitted vertically from parent to offspring (Motte et al. 2003). Such vertically transmitted diseases may be eliminated from the hatchery production system by the use of domesticated shrimp that are free from these pathogens through an appropriate Specific Pathogen Free (SPF) programme (see below).

If SPF (or "high health") shrimp free from known viruses are not available, broodstock should be tested for infection by an appropriate diagnostic test and any infected individuals destroyed. Shrimp testing negative for the disease or pathogen should still be considered a risk and placed in a quarantine facility until their health status is fully known.

Even after broodstock have been transferred from the quarantine unit, some hatcheries maintain a routine health check by monthly monitoring of the postlarvae produced. A proportion (e.g. 0.1%) of the population is sampled by PCR and haemolymph tests, and based on the results of these tests, appropriate action is taken. The number of animals to be sampled should be determined according to a sampling table that takes into consideration the size of the host population and the presumed prevalence of the pathogen (see, for example, OIE 2003).

Where possible, the animals selected as broodstock should come from a closed cycle operation, as this allows their performance history and health status to be known. Ideally, they should originate from shrimp farms located in areas with physico-chemical characteristics (salinity, temperature etc.) similar to those where the postlarvae will be stocked. Criteria used in the selection of broodstock depend on the source of the broodstock (wild or domesticated).

Wild broodstock: Because performances and growth records are not available for wild broodstock, and because there is no chance for stock improvement, there has therefore been a trend away from their acquisition and use. Wild-source broodstock were formerly preferred by hatcheries due to the belief that they produced more and stronger nauplii. In recent years, however, the high risk of introducing viral pathogens with wild broodstock has changed this preference. Additionally, it is increasingly recognised that domesticated shrimp stocks need to be developed to enable enhancement of maturation, hatchery and pond performance, which has led to a trend towards the use of broodstock reared in captivity. In the case of Penaeus vannamei, wild broodstock captured using nets from small boats are preferred, because those captured by trawlers suffer more damage. Wild females for use in a maturation facility should be 60 g body weight and with developed ovaries, and males should be approximately 40 to 50 g body weight.

Domesticated broodstock: In the past ten years, sources of domesticated shrimp stocks have become more common, with domesticated stocks of both P. vannamei and P. stylirostris now being commercially available. Closed-cycle stocks are generally supplied at a smaller size than wild animals, males being approximately 30 g and females not less than 30-35 g and usually >40 g. Females are usually supplied in a nongravid condition. Domesticated stocks may come from one of several sources. Some countries have well-established domestication programmes, whereas others rely on imported stocks. The domesticated stocks may be either genetically improved through a specific genetic improvement programme to select for desirable traits or simply selected from stocks that are free from, or suspected to be resistant or tolerant to, specific pathogens.

Several specialized types of domesticated broodstock have been developed to reduce disease risks. Specific Pathogen Free (SPF) stocks are generally maintained in highly biosecure facilities and their offspring (designated "high health" rather than SPF) are supplied to the industry. Specific Pathogen Resistant (SPR) shrimp are those that are not susceptible to infection by one or several specific pathogens, and Specific Pathogen Tolerant (SPT) shrimp are those that are intentionally bred to develop resistance to the disease caused by one, or several, specific pathogens. Lines of Penaeus stylirostris that are resistant to IHHNV, for example, are available.

When using domesticated shrimp, it essential to obtain adequate background information on the origin of the stocks and their past performance

To avoid potential genetic problems and associated poor growth and survival due to inbreeding, details of the different families or origins of the domestic stocks, whether of foreign or native origin, must be obtained.

It is also useful to have performance and development data for the candidate families or lines under a range of environmental conditions. The selection protocol used is also important, i.e. whether the stocks were selected from ponds with better performance or for survival following a disease outbreak, and the exact timing of the selection procedures. Some criteria that are used for phenotypic selection (usually done first at harvest size and later, when the females are >30 g and males are >25 g) are: relative size and general physical appearance, absence of necrosis or other (clinical or subclinical) signs of disease or ill health in muscle and exoskeleton, clean pleopods, no rostrum deformities and a translucent body.

4.2 Procedures for broodstock quarantine

Upon arrival at the hatchery, potential broodstock should be held in isolation until their disease status is ascertained

The quarantine facilities are essentially a closed holding area where shrimp are kept in individual tanks until the results of screening for viruses (and for bacteria, where applicable) are known.

The broodstock quarantine unit should be physically isolated from the rest of the hatchery facilities. If this is not possible, the hatchery design should be altered so that there is no possibility of contamination from the quarantine or holding area into the other production areas. Particular care should be taken with waste disposal and effluent treatment. Staff working in this area should not be permitted to enter other production sections and should follow sanitary protocols at all times.

The quarantine unit should have the following characteristics:

· It should be adequately isolated from all of the rearing and production areas to avoid any possible cross contamination.

· It should be in an enclosed and covered building with no direct access to the outside.

· There should be means provided for disinfection of feet (footbaths containing hypochlorite solution at >50 ppm active ingredient) and hands (bottles containing iodine-PVP (20 ppm and/or 70% alcohol) to be used upon entering and exiting the unit.

· Entrance to the quarantine area should be restricted to the personnel assigned to work exclusively in this area.

· Quarantine unit staff should enter through a dressing room, where they remove their street clothes and take a shower before going to another dressing room to put on working clothes and boots. At the end of the working shift, the sequence is reversed.

· An adequate number of plastic buckets and/or similar containers should be available in the quarantine room to facilitate effective daily routine movement of shrimp in and out of the facility.

· The quarantine facility should have an independent supply of water and air with separate treatment and disinfection systems and a system for the treatment of effluents to prevent the potential escape of pathogens into the environment.

· The seawater to be used in the facility must enter a storage tank where it will be treated with hypochlorite solution (20 ppm active ingredient for not less than 30 minutes) before inactivating with sodium thiosulfate (1 ppm for every ppm of residual chlorine) and strong aeration.

· All wastewater must be collected into another tank for chlorination (20 ppm for not less than 60 minutes) and dechlorination before release to the environment.

· All mortalities or infected animals must be incinerated or disposed of in another approved manner.

· Used plastic containers and hoses must be washed and disinfected with hypochlorite solution (20 ppm) before reuse.

· All the implements used in the quarantine unit must be clearly marked and should remain in the quarantine area. Facilities for disinfection of all equipment at the end of each day should be available.

The quarantine unit should be structured so that shrimp move from "dirty" to "clean" areas as their health status becomes clear

The individual sections of the quarantine area should be designated "dirty" or "clean" depending on whether they contain shrimp that are not yet screened for infection (pretesting) or that have been passed (posttesting). Shrimp should only move one way, from the "dirty" to the "clean" sections of the quarantine facility, and all movements should be controlled to ensure no mixing between the two areas.

On entering the quarantine area the broodstock are passed through a dip of iodine-PVP solution (20 ppm) or formalin[7] (50-100 ppm). On the third day of quarantine, a pleopod is removed from each shrimp (if held individually) or from a sample of the population (if held as a group) for analyses. If shrimp are held collectively, random samples should be taken from each container to evaluate the general condition of the population held in that container.

Groups of ten pleopods can be analysed as one sample. Any groups that give a positive result can be discarded or, in the case of a pooled sample from animals held individually, the shrimp can then be tested on an individual basis to identify and discard only the positive individuals. Infected animals should be disposed of by incineration or some other method (e.g. autoclaving and deep burial) that will prevent the potential spread of virus.

Further details on the construction and operation of a quarantine facility can be found in MAF (2001), Anon. (2002) and AQIS (2003).

Broodstock must not be released from quarantine until their health status is clearly known

The quarantine period will vary depending on the time required to complete the health screening procedure. In all cases, animals should be kept under observation in the quarantine facility until all tests are completed, and for at least a minimum of 20 days prior to transferring them to the acclimatization area. Depending on the design of the facility and the location of the quarantine unit relative to the acclimatization facility, this may involve repacking the broodstock for shipment to a distant location or their movement to a separate section of the same facility using disinfected buckets with water from the acclimatization facility.

In either case, the equipment used for the transfer should be kept separate from that used in the quarantine room and disinfected before and after transport. All equipment used in the quarantine area should remain in the quarantine area and be disinfected at the end of each day in tanks specially designated for that purpose.

Laboratory facilities and associated expertise must be determined based on the specific needs of the hatchery

Basic laboratory facilities (e.g. a microscope, some microbiological capability etc.) will be required to carry out routine inspections of shrimp health. The addition of more complex facilities to carry out PCR tests, for example, will require the construction of dedicated facilities to avoid the possibility of contamination. The design and operation of these facilities is outside the scope of this document.

4.3 Acclimatization

Shrimp that pass the initial quarantine inspection must be acclimatized to the new conditions in the maturation facility

During acclimatization, which lasts from seven days to a few weeks, the broodstock will be adjusted to the environmental conditions of the maturation facility and the types of feed that will be given. This is especially important where formulated diets will be used to supplement the natural feeds.

The acclimatisation facility must have sufficient tank space to hold the shrimp that will be introduced into the maturation facility

Such a facility also allows optimization of production of the maturation system. This is because well-acclimatized shrimp should be ready to begin producing nauplii soon after introduction to the maturation system, without excessively long periods of "down-time" (the number of days between introduction of a female into the maturation system and the first spawning) being lost.

The broodstock should spend a minimum period of seven days (and up to several weeks) in acclimatisation before being stocked in the maturation tanks

During this period any difference in temperature and/or salinity between the quarantine area and the maturation facility is gradually reduced. Feeding protocols are also adjusted so that the shrimp become accustomed to those utilized in the maturation facility. The moult stage is also observed and only females in the intermoult stage should be ablated when ready. In this way, the females to be transferred to the maturation unit will already be ablated and hence ready to begin production of nauplii almost immediately.

4.4 Maturation

The first step in larval production is the maturation and breeding of mature shrimp. The protocols to be adopted will depend to some extent on whether the hatchery operation is a component of a controlled breeding programme or if it is intended primarily for the production of postlarvae for commercial pond culture.

Depending on this distinction, the maturation system will be designed either to maximize the production of nauplii for commercial production of postlarvae or to allow for maximum control over mating and genetic crosses. Although it is possible to control mating in a conventional maturation unit, good control of individual parents requires unisex culture and artificial insemination, with larval culture and nursery systems designed for a large number of batches with relatively few larvae per batch. This presents operational challenges very different from a typical commercial hatchery or nursery system (Jahncke et al. 2002).

Appropriate infrastructure for broodstock handling consists of quarantine facilities, acclimatization facilities and the main production (maturation, spawning and hatching) facilities with their appropriate support systems.

The maturation building must be large enough to contain sufficient maturation tanks and supporting infrastructure for the requirements of the hatchery

The factors to consider in designing the facility are the level of naupliar production required, the stocking density and sex ratio of the broodstock to be used, the estimated spawning rate of the females, the estimated hatching rate, the estimated number of eggs and nauplii per female, and the production system (batch or continuous) employed.

The conditions in the maturation room must be closely controlled

The maturation room should be kept in low light, preferably with a system to control photoperiod. The photoperiod should be maintained at about 10-12 hours dark and 12-14 hours light, the light level changing between the two gradually over a period of one to two hours. Access to the maturation room should be restricted; noise (particularly loud or intermittent noise), movement and other disturbances should be kept to a minimum.

Preferably, the maturation room should have round tanks that are dark-coloured, smooth-sided, and of approximately 5 m diameter. The broodstock should be held with flow-through (new and/or recycled) water exchange of a total of 250-300% per day and a continuous, but not too vigorous air supply. Water depth is generally around 0.5-0.7 m. The shrimp are stocked at a rate of around 6-8 shrimp per sq. m. bottom surface area with a male to female ratio of 1-1.5:1. Thus, a 5 m diameter tank can accommodate 60-80 females and 60-100 males. Water temperatures are usually controlled to be maintained in the range of 28-29 °C, with a salinity of 30-35 ppt and pH of 8.0-8.2.

The feed preparation area should be adjacent to, but separated from, the maturation room

It should be equipped with all feed preparation utensils (knives, spoons, bowls/buckets, cutting surfaces, mixers, pelletisers etc.), and a fridge and a freezer to store food items.

The maturation tanks should be must be siphoned daily and cleaned regularly

Due to the high feeding rates employed, the maturation tanks require daily siphoning of uneaten food, faeces and moults. The siphon consists of two parts, a PVC tube and a hose. Each maturation tank should have its own PVC tube, but the hose may be used for all tanks. The hose should be rinsed with clean treated water before each tank is siphoned. Debris and waste siphoned from the tanks can be collected in a mesh bag placed at the end of the hose and incinerated after the cleaning operation. At the end of the working day, the hose should be washed and remain immersed inside a tank of calcium hypochlorite solution (20 ppm).

Intermittent scrubbing of tank walls and bottoms must also be undertaken if there is an excessive build-up of algae or other sedentary organisms, including protozoan fouling organisms. This can often be achieved through lowering water levels in the tank without removing the broodstock, but occasionally requires the transfer of broodstock to new tanks. It is a good idea to leave at least one tank empty for such procedures, which can then be programmed on a regular basis. Care must be taken during these cleaning exercises that the broodstock are manipulated as little as possible, as excessive disruption of mature brooders will interfere with their spawning rhythms.

The equipment used to capture the mature females should be washed before checking each tank

The hand nets used to capture mature females should be maintained in recipient(s) containing iodine-PVP and/or hypochlorite solutions (20 ppm active ingredient).

An optimal population density for natural mating should be maintained

The preferred population density for natural mating of P. vannamei broodstock is about 6-8 animals per square meter. If artificial insemination is to be done, the number can be increased up to 16 animals per square meter. It is important also to consider the biomass in weight rather than the numbers of broodstock per square meter that can be held in the tank without causing deterioration of the water quality through the feed used. A biomass/unit area of 0.2-0.3 kg/sq m is recommended.

An optimal stocking ratio for males and females should be used

Most systems will stock females and males together, usually in a 1-1.5:1 ratio. Occasionally, the sexes are kept separately. This has advantages, including reduced feeding costs for male-only tanks, because they can be reared on cheaper diets (primarily squid and enriched artificial feeds), increased sperm quality through maintaining males at lower temperatures (25-27 oC) where possible, increased stocking density of males, and facilitating artificial insemination, if this technique is employed.

However, the separation of males and females entails the capture and movement of females twice each spawning night (once to transfer to the male tank and the second time to transfer to the spawning tank), which results in excessive stress during a very vulnerable stage. In addition, mating tends to be better with mixed sexes, due to excitation of the shrimp by high hormonal concentrations in the mixed tanks. As a guide, wild broodstock usually produce spawning rates of 4-8% of females per night, while domesticated stocks tend to be more productive, producing 10-15% or more of females per night.

4.5 Spawning

A separate spawning room should be used

Spawning should take place in a separate room from the maturation area in order to keep the spawning area clean and to be able to carry out daily washing and disinfection of tanks without disturbing the broodstock. The spawning room should have sufficient and appropriate infrastructure for the level of naupliar production required.

Where possible, spawning should be carried out individually

This will reduce the risk of horizontal transfer of diseases between females. It has been shown that the tissues exuded during spawning and faeces can contain high levels of some viruses (IHHNV, HPV, BP, MBV etc.) and exposure to this can result in infection of uninfected females during collective spawning. If collective spawning must be carried out, the number of females per tank should be as low as possible to limit the number of females exposed to potential infection (i.e. one female to 200-300 litres of water).

Spawning tanks can be any size from 300 litres up to 5-8 mt, depending on the type of spawning used (individual or collective)

The tanks may be flat bottomed, but if they are slightly conical, or at least angled to the outlet, it allows easier and less damaging harvesting of all the eggs. Tanks should allow the harvest of the eggs in such a way that they can be subjected to washing or a disinfection bath after collection using formalin (100 ppm for 30 sec), or iodine PVP (50-100 ppm for 1-3 min). Treflan may also be added at 0.05-0.1 ppm to combat fungal infections. This disinfection will help to reduce the risk of disease transmission.

Spawning systems should have the best water quality possible

Water-purification steps should be taken for spawning and hatching tank water. This will typically include UV light treatment and passage through activated carbon and cartridge filtration to <1 mm. Preferably, water quality should be maintained with a temperature of 28-29 oC and salinity of 30-35 ppt, as in the maturation tanks. EDTA is often added to the spawning tank water as a chelating agent at a recommended dose depending on the heavy metal loadings of the location.

As a general principle, broodstock should be handled only when necessary to avoid unduly stressing the shrimp

Excessive chasing of individual shrimp should be avoided. When holding broodstock, grasp them firmly with the abdomen bent so that the uropods and telson are tucked between the walking legs to minimize flexing and the risk of dropping the shrimp. Avoid keeping the broodstock out of water for extended periods. For example, when transferring females to the spawning tank, they should be held as described while maintaining them underwater in beakers or buckets containing maturation water.

Sourcing of gravid females should be done in the late afternoon/early evening

Gravid females should be selected in the late afternoon or early evening (as soon as night falls), or at the most suitable time dictated by the photoperiod employed. When sourcing, use a strong, preferably waterproof, flashlight to see which of the females in the tank look gravid (those with the most highly developed, or stage IV ovaries). When a gravid female is found, use the scoop net to capture it as gently as possible and bring it to the side. The female is then inspected to see if there is a spermatophore on the thelycum. If the spermatophore is present, the female is placed in a container and transferred to the spawning room. If there is no spermatophore present, the female is placed in another container and taken elsewhere for artificial insemination (if employed) before transferral to the spawning tanks.

The fecundity, spawning rate (number of spawns per female) and length of time that the females are kept in maturation should be monitored

To avoid deterioration of the naupliar quality, ablated females should typically be retired from the maturation unit after a maximum period of three months or 15 spawns, depending on the feeding regime used and health of the spawners. Nonablated females can be spawned for up to one year. This usually requires that females be identified individually by tagging or some other method.

Egg and sperm counts should be made to determine good egg production and fertilisation

As a guide, the quantity of eggs spawned per female should be in the range of 100 000 to 140 000 eggs for females of 30 to 35 g body weight, and up to 150 000 to 200 000 eggs for 40 to 45 g females.

To ensure good fertilization, sperm should be observed and quantified regularly through sperm counts using a high powered light microscope.

A suitable system for egg collection should be employed

Spawning may be either collective, with two or more females in the spawning tank, or individual. In either case, a suitable system for harvesting the eggs, excluding broodstock faeces and ovarian tissues (using a prefilter made from 300-500 mm mesh, for example) is required.

The eggs should be collected into a receptacle with a large, mostly submerged mesh of <100 mm pore size in order to retain them without damage. Once harvested, the eggs should be washed with adequately treated seawater (filtered and sterilized) and then disinfected using iodine-PVP (50-100 ppm/10-60 sec) before rinsing again with abundant clean seawater in another recipient.

Fertilisation and hatching rates should be monitored

Following collection, the eggs are then transferred to hatching tanks in the hatching unit. A sample of the eggs harvested should be examined to determine the fertilization rate and a count made to allow an estimation of the hatching rate. The fertilization rate should be at least 50% and is more typically >75%. Where fertilization rates fall below 50%, consideration should be given to discarding the entire batch and investigations begun to determine the cause of the problem.

4.6 Hatching

Hatching should take place in an isolated and clean room

Hatching tanks (300-1 000 litre) usually have pronounced conical bottoms to allow good water circulation and aeration and easy harvesting. Tanks vary in size from tens of litres to 1 mt, and can be stocked with up to 4 million eggs/mt. Water quality should be maintained at 29-32 oC and 32-35 ppt salinity for optimal hatching. EDTA (at up to 20 ppm) and Treflan (0.05-0.1 ppm) are usually added to the water in the hatching tanks for the same reasons as with spawning.

The tank is provided with enough aeration to keep the eggs moving in suspension. The nauplii should appear approximately eight hours after stocking the eggs. After this point (typically after 12-15 hours), the aeration is stopped in order to harvest the nauplii. A dark cover or lid cover having a small hole cut in its centre is then placed over the tank and a light bulb is suspended above the hole.

The healthy nauplii are allowed to aggregate below this hole over a period of 20-30 minutes and are then collected by bucket or siphon into a separate bucket or nauplii collector, where they can be washed and disinfected. They are then held in separate tanks or buckets with aeration or sent directly to the larval rearing facilities. The unhatched eggs and weaker nauplii that remain in the hatching tank are then discarded and the tank cleaned and disinfected. The spawning and hatching tanks are washed daily with calcium (or sodium) hypochlorite solution (30 ppm active ingredient), and rinsed with abundant treated water before being refilled.

4.7 Broodstock health screening

Besides screening for general health, broodstock selected for maturation should be screened for WSSV, IHHN, TSV and YHV

Where numbers of broodstock are large, the tests may be carried out on pools of 10 individuals from different broodstock groups. A minimum sample of 150 animals for each group of 1 000 shrimp should be taken and divided into groups of 10 shrimp for each analysis. When selecting for genetic programmes, more stringent disease screening should be used to ensure freedom from pathogens. Although PCR testing should be conducted on broodstock upon arrival during their quarantine, it is worthwhile to conduct additional PCR testing (at least for WSSV) after spawning. This is because there is evidence that broodstock that tested PCR-negative for WSSV during quarantine may test positive if analysed following exposure to a stress such as spawning.

4.8 Broodstock nutrition

A good diet and feeding protocol should be essential components of the maturation programme

A good diet and feeding protocol for broodstock are key factors in the production of good quality nauplii. The appropriate quantity of feeds must be determined in relation to the biomass in the tank. In general, feeding should be at a rate of up to 20-30% of the broodstock biomass (wet weight basis or when using fresh or frozen feed). When dry feed is used, the rate will be less. The exact quantity of feed given should be adjusted frequently based on the consumption rate of each tank. The feeding should continue until only a very small amount of uneaten food remains in the tank a couple of hours after each feeding. The diet fed should be balanced, concentrating on the use of feeds high in vitamins, minerals, pigments and fatty acids (such as 20:5n3 and 22:6n3), which are essential for the production of eggs.

Cross-contamination of feed during its preparation must be avoided

Feed preparation should be carried out using good hygienic standards. Utensils (knives, tables, mixers, pelletisers etc.) must be kept clean, washed before use with iodine-PVP solution (20 ppm) and rinsed with clean water.

Fresh feeds should be really fresh, and certified free from important viruses or sterilised

When using fresh feeds such squid, polychaetes, Artemia, krill, mussels, oysters, clams etc., efforts must be made to ensure that the material is as fresh as possible. To ensure that fresh feed is not a biosecurity risk, a certificate should be requested at the time of purchase stating that the feed is free of the viruses TSV, WSSV and YHV by PCR analysis. Alternatively, the feeds may be sterilized or pasteurized (recommended) to inactivate any virus, as long as this does not affect the acceptability or nutritional quality of the feed. Ideally, different types of frozen feeds should be stored in separate freezers.

Fresh feeds should be chopped to a size suitable for ingestion

Fresh feeds need to be chopped to a size suitable for ingestion by the broodstock and washed with clean water and weighed prior to feeding. Feeding will typically be done every three to four hours during the day and night.

Artificial feeds should be enriched with nutritional additives

Artificial feed should be enriched with additives such as vitamins C and E, immunostimulants, astaxanthin, carotenoids, polyunsaturated fatty acids etc. if it is intended to complement fresh feed with dry or moist pelleted feeds. Several commercial companies produce artificial feeds to supplement the fresh feeds used in maturation, although none yet serve as full replacements. Dry or moist diets can also be economically cold-extruded (using a pelletiser or an extruder) on site using regular shrimp feeds ground to powder and incorporating the various additives mentioned above, plus a binder such as alginate or gelatine. Selection of an appropriate feed depends on the specific requirements of the maturation facility.

Dry feeds should be fed separately from fresh feeds and two to three times per day (at up to 2-3% of shrimp biomass/day) using low feeding rates each time to ensure that they are completely consumed

Domesticated Penaeus vannamei have an advantage over their wild counterparts or P. monodon because they have been raised on pelleted diets and are thus accustomed to consuming them. Wild broodstock often prove reluctant to eat dry feeds and must be acclimated to them very gradually over time. As for all management practices with broodstock, any changes made should be minimized as much as possible to limit stressing the animals. Any changes to feeding regimes, types, quantities and times should be minimized as much as possible. Hence, stocks of all feed ingredients or types used should be maintained at all times.

[7] When formalin is used, avoid using the whitish sediment at the bottom of the container (formaldehyde), as it is highly toxic.

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