NATIONAL CENTER FOR MARINE SEED RODUCTION

IN CENTRAL VIETNAM
RESEARCH INSTITUTE FOR AQUACULTURE NO.3

-
SPECIFIC PATHOGEN FREE
PROGRAMME
- (SPF) –

NGUYEN THANH VU

INTERNAL INSTITUTE DOCUMENT – DESIGNED BY THANH VU

 WHITE LEG SHRIMP
HATCHERY PRODUCTION TECHNOLOGY

 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 two broad categories:

The pre-spawning process The post-spawning process

 I/ The pre-spawning process

 Broodstock selection,
 Maintenance,
 Acclimatization,
 Maturation,
 Spawning,
 And hatching.

These procedures require different facilities, the facility

maintenance guidelines are described under the different
specific facilities used in the hatchery production process.
 I/ The pre-spawning process
1.1. Broodstock selection
 Healthy broodstock that are not
carriers of serious pathogen must
be selected in order to achieve
successful hatchery production
(OIE 2003). Shrimp broodtock’s tanks

• Employees of shrimp broodstock are produced in

Vietnam have been imported from Hawaii that have free
5 pathogens (MBV, IHHNV, TSV, WSSV, YHV).
• When using domesticated shrimp, it essential to obtain
adequate background information on the origin of the
stocks and their past performance.
I/ The pre-spawning process
1.2 Procedures for broodstock quarantine
 Upon arrival at the hatchery, potential broodstock should
be held in isolation until their disease status is ascertained.
 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.
I/ The pre-spawning process
1.2 Procedures for broodstock quarantine (cont.)
 Broodstock must not be released from quarantine until
there health status is clearly known.
 Laboratory facilities and associated expertise must be
determined based in the specific needs of the hatchery.
- Basic laboratory facilities
(e.g. a microscope, some
microbiological capability etc.)
- The addition of more
complex facilities to carry out
PCR test.
Basic laboratory facilities
 I/ The pre-spawning process
1.3 Acclimatization
 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 broodstock should spend a minimum period of seven days (and up to
several weeks) in acclimatization 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.
I/ The pre-spawning process
1.4 Maturation
 The first step in larval production is the maturation and breeding of mature
shrimp.
 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.
 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 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,
- Access to the maturation room should be restricted; noise (particularly load
or intermittent noise), movement and other disturbances should be kept to a
minimum.
 I/ The pre-spawning process
1.4 Maturation (cont.)
• 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 Maturation tanks shape
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 ration 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
28-29oC, 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
 I/ The pre-spawning process
1.4 Maturation (cont.)
 The maturation tanks should be must be siphoned daily and cleaned regularly (to
eliminate uneaten food, faeces and moults).
 The equipment (the hand nets) used to capture the mature females should be
washed before checking each tank (using iodine-PVP- 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.
 An optimal stocking ratio for males and females should be used, 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 (25-27oC) where
possible, increased stocking density of males, and facilitating artificial
insemination, if this technique is employed.
 I/ The pre-spawning process
1.5 Spawning
• A separate spawning room should be used 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.
• Where possible, spawning should be carried out
individually. This is reduce the risk of horizontal transfer of
disease between females, The spawning tank
- It has been shown that the tissues exuded during spawning and faeces can contain
high levels of some viruses (IHHNV, YHV, BP, MBV etc) and exposure to this can
result in infection of uninfected females during collective spawning.
• Spawning tanks can be any size from 300 liters 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 lease angled
to the outlet, it allows easier and less damaging harvesting of all the eggs.
- After collection the eggs, using formalin (100 ppm for 30 sec), or iodine PVP (50-
100 ppm for 1-3 min)
 I/ The pre-spawning process
1.5 Spawning (cont.)
• Spawning systems should have the best water quality possible:
- This will typically include UV light treatment and
passage through activated carbon and cartridge filtration
to < 1 μm.
- Preferably, water quality should be maintained with
a temperature of 28-29oC and salinity of 30-50 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 Broodstock with stage IV ovaries
the heavy metal loadings of the location (5-10 ppm).
• As a general principle, broodstock should be handled only when necessary to
avoid unduly stressing the shrimp. Avoid keeping the broodstock out of water
for extended period. 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.
 I/ The pre-spawning process
1.5 Spawning (cont.)
• Sourcing of gravid females should be done in the late afternoon/early
evening (as soon as night falls). 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.
Using net to capture gravid female
- 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
• Egg and sperm counts should be made to determine good egg production and
fertilization.
- The quantity of eggs spawned per female should be in the range of 100 000 to
140 000 eggs of 30-35 g body weight, and up to 150 000-200000 eggs for 40-45 g
females.
 I/ The pre-spawning process
1.5 Spawning (cont.)
• A suitable system for egg collection should be employed.
- A suitable system for harvesting the eggs, excluding broodstock faeces and ovarian
tissues (using a prefilter made from 300-500 μm mesh, for example) is required.
- The eggs should be collected into a receptacle with a large, mostly submerged
mesh of < 100 μm pore size in order to retain them without damage. Once
harvested, the eggs should be wasted with adequately treated seawater and then
disinfected using iodine-PVP (50-100 ppm/10-60 sec) before rinsing again with
abundant clean seawater in another recipient.
• Fertilization and hatching rates should be monitored.
Following the collection, the eggs are then transferred to hatching tanks in the
hatching unit. 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.
 II/ The post-spawning process
• Facility maintenance;
• Water quality management;
• Broodstock handling;
• Washing,;
• Selection;
• Holding and transport of nauplii;
• Postlarval rearing;
• Maintenance;
• Health management;
• Assessment of condition;
 II/ The post-spawning process
2.1. Facility maintenance;
• Facilities must be maintained so as to optimize the conditions for the growth,
survival and health of the shrimp broodstock, larvae and PL, minimizing the risk of
disease outbreaks.
• All tanks (used for broodstock spawning, egg hatching, and holding of nauplii and
postlarvae) and equipment should be thoroughly cleaned on a regular basis,
cleaned and disinfected after use, and cleaned and disinfected again before starting
a new production cycle.
2.2. Water quality management
• Incoming water should be cleaned and disinfected through chlorination and
filtration before being distributed to different working areas (hatchery, algal
culture, Artemia etc.).
• Incoming water should be disinfected to destroy any remaining pathogens and any
heavy metals present removed by chelation.
- Calcium (or sodium) hypochlorine (10 ppm active ingredient for not less than 30
min), and/or UV light should be used to disinfect the incoming water after initial
filtration and sedimentation.
 II/ The post-spawning process
2.2. Water quality management (cont.)
• After treatment with chlorine, the water in the
reservoir must be checked by ortho-toluidine
(3 drops in 5 ml of water sample) to ensure no
chlorine residual remains before the water in used
• Once the chlorine has dissipated or been
neutralized with sodium thiosulfate (1 ppm for every
1 ppm of chlorine ramaining), EDTA can be used
Applied to chelate any heavy metals present (quantitiesThe hatchery and reservoir tank
Depending in concentrations of heavy metals and use).
• The temperature of the water should be adjusted before it enters the production
units (generally between 28-32oC depending on area and stage).
• Sand filters must be properly maintained,
• The water to be used in the spawning and hatching tanks and pure algal culture
facilities must be the same quanlity.
 II/ The post-spawning process
2.3. Broodstock disinfection
After removing the spent females from the spawning tanks, they should be
immersed in iodine PVP (20 ppm/15 sec) before returning them to the tank
of origin.
2.4. Washing of nauplii
 Harvested nauplii at stage 4 can be treated by bath immersion in Treflan
(0.05-0.1 ppm) to prevent fugal contamination, followed by a thorough
wash in filtered and sterilized water and a dip in an iodine-PVP solution
(50-100 ppm) for 1-3 min), followed immediately by further washes with
clean seawater.
2.5. Selection of Nauplii
 Nauplii should be harvested using a light to attract them to the water
surface. Those that remain at the bottom of the tank are discarded,
reducing the percentage of weak and deformed nauplii. After harvesting,
the number of good nauplii is counted to provide the hatching rate. In good
batches, the hatching rate should be >70%.
 II/ The post-spawning process
2.5. Selection of Nauplii (cont.)
 The activity and color of the nauplii should be evaluated and the
percentage of deformities estimated.
2.6. Holding of Nauplii
 Harvested nauplii must be held under optimal condition until they are
stocked.
- The harvested nauplii can be held at a density of 20 000 – 40 000/litre,
with continuous light, clean water and aeration until they are ready to be
stocked in hatchery tanks.
- The equipment and material used to harvest the nauplii must be washed
daily with calcium hypochlorite solution (30 ppm) to prevent
contamination of subsequent batches.
 II/ The post-spawning process
2.7. Transportation of Nauplii
 The nauplii should be transported at densities of 15 000 – 30 000
nauplii/litre, depending on distance or time to the hatchery.
- Transportation is normolly done in double plastic bag containing 10-15
litres of water and filled with pure oxygen.
- The temperature of the parking and shipping water is a adjusted from
28-30o C down to between 18-25o C . Salinity
is maintained at 32-35 ppt.
- Upon arrival at the purchasing hatchery,
the nauplii should again be disinfected.
- If possible, the transport vehicle should
first be disinfected before entering the hatchery
facilities. After unpacking the nauplii, the
packing material must be incinerated. Transportation of Nauplii
 II/ The post-spawning process
2.8. Larval rearing and Maintenance
 Larval rearing should produce the best quality, high-health postlarvae
possible
- In order to achieve this, all areas involved in larval rearing must be
designed for optimal efficiency, cleanliness and productivity.
 Entrance to the larval rearing areas should be restricted to the personnel
that work in these area.
 All materials and equipment should be for the exclusive use in each room,
and should not leave the room or be used elsewhere.
 Larvae and postlarvae should be routinely checked for quality.
2.9. Larval nutrition and feed management
 High standards of feed preparation must be maintained. All feed
(Frippack, Lansy, …) preparation, especially of live feeds (algae, Artemia…)
 Entry to the algal culture and Artemia culture room must be retricted to
authorized personnel.
 II/ The post-spawning process
2.10. Larval health management
 There are many factors involved in managing larval health in the hatchery. A tight
control must be maintained on all of these factors throughout the larval rearing
cycle if good numbers of high quality larvae are to be produced.
 Stocking density.
- The density at which nauplii are stocked should not be excessive.
- In general, stocking rates for nauplii should be in the range of 100-250 nauplii/litre
(100 000 – 250 000 per mt) of water
 Water quality
- Water quality has a major impact on the health and performance of larvae batches.
Poor water quality can lead to poor growth, low survival, later moulting/staging,
increased epibiont fouling and deformities.
- The temperature should be maintained between 28 and 32o C and salinity above
30 ppt, at least until postlarval stages are reached., pH of around 8 should be
maintained.. Overfeeding is one of the major causes of water quality deterioration
and should be avoided.
 II/ The post-spawning process
2.10. Larval health management (cont.)
 Water quality (cont.)
- Hatcheries should also consider the use of probiotics and bacterial enzymes to
maintain water quanlity, prevent bacterial blooms and reduce or eliminate the
requirement for antibiotics during larval culture
 Stocking period
- Each separate unit of larvae rearing tanks within a hatchery or, preferably, the
whole hatchery should be stocked with nauplii in as short a time period as possible,
usually limited to three to four days. Prolonging this stocking period often results
in increased incidence of disease for the later-stocked larvae, presumably through
bacterial contamination from the order to the younger tanks.
- This phenomenon is often associated with the so-called “zoea 2 syndrome”,
where late zoea 1 and early 2 stage larvae refuse to eat and suffer high mortality with
associated bacterial problems. This problem maybe controlled through restricting
the time of stocking to less than four days, using probiotics and maintaining
good cleanliness in all areas of the hatchery at all times.
 II/ The post-spawning process
2.10. Larval health management (cont.)
 Nutrition and feeding
- The quantity, quality and management of feed can have an important impact on
larval health and survival. Failure to provide sufficient feed of the right quality can
lead to stress, poor growth, mortality, increased in cannibalistic behaviour, deformity
and increased levels of epobiont fouling.
- When using the formulated diets as a supplement to live feed, it is important to feed
small amounts of high quality appropriately
sized, nonpolluting diets frequently. As a guide,
particle sizes should be 10-50 μm for zoea,
100-200 μm for mysis, and 200-300 μm for early
postlarvae stages. A feeding frequency of every two
to four hours is generally regarded as sufficient.
- For majority of the larvae feed requirements ,
reliance should still be placed on high quality live
feeds, including algae and Artemia.
Algae - Chaetoceros spp
 II/ The post-spawning process
2.11. General assessment of larval condition
 Assessment of larval condition is usually done in the morning, and
decisions on water exchange, feeding and other management activities
made so that action can be taken in the afternoon.
 The larvae in each tank should be inspected two to four times each day.
Initially, a visual inspection of the larvae, the condition of the water in the
rearing tank and the feed is made.
 A sample of larvae can be taken with a beaker and inspected with the naked
eye.
 Observations are made on the larval stage, health, activity, behaviour and
abundance of feed and faeces in the water. Records may also be taken of the
water quality parameters, and the amount of food in the tank.
 The same, or a separate sample of larvae, should also be taken to the
laboratory for a more detailed microscopic examination. This will provide
information of on the stage, condition, feeding and digestion and presence
of any disease of physical deformity.
 II/ The post-spawning process
2.11. Shipping and transfer
 After postlarvae 12 stage are reached, they are sold to the farmer,
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