Hatchery standards

“COASTAL RESOURCES FOR SUSTAINABLE DEVELOPMENT PROJECT”

Shrimp Hatcheries Standards

Leonardo Galli 1

Report# 2 – Shrimp Hatcheries Standards Leonardo Galli

COASTAL RESOURCES FOR SUSTAINABLE DEVELOPMENT PROJECT”

INTERNATIONAL CONSULTANCY ON BIO-SECURITY IN SEED PRODUCTION AND AQUACULTURE

(Code: CS. 04/IAB/PCU)


Report #2

Dr. Leonardo Galli


Table of Contents

SUMMARY ............................................................................................................................................ 3

1 INTRODUCTION .......................................................................................................................... 4

2 SECTION I – HATCHERY INFRASTRUCTURE ..................................................................... 5

2.1 THE IDEAL HATCHERY ................................................................................................................ 5

3 OBJECTIVES ................................................................................................................................13

4 PRESENT DESIGN OF THE HATCHERIES ...........................................................................13

4.1 SEAWATER UPTAKE AND WATER TREATMENT ......................................................................13 4.2 PRODUCTION BUILDING ............................................................................................................13

5 PROPOSAL FOR HATCHERIES UPGRADE .........................................................................14

5.1 QUARANTINE STATION ..............................................................................................................14 5.2 HATCHERIES UPGRADE .............................................................................................................15 5.3 FENCE ..........................................................................................................................................15 5.4 SEAWATER UPTAKE AND WATER TREATMENT ......................................................................16 5.5 MATURATION .............................................................................................................................17 5.6 LARVICULTURA ..........................................................................................................................20 5.7 ALGAE ROOM ..............................................................................................................................20 5.8 ARTEMIA CYSTS HATCHING ROOM...........................................................................................22 5.9 LABORATORY FOR DAILY OBSERVATIONS ...............................................................................22 5.10 EFFLUENT TREATMENT ...........................................................................................................22 5.11 MACHINERY ROOM ..................................................................................................................23

6 SECTION II - STANDARD OPERATING PROCEDURES ...................................................24

6.1 BROODSTOCK MANAGEMENT ...................................................................................................24 6.2 POST-LARVAE PRODUCTION .....................................................................................................30 6.3 ALGAE PRODUCTION ..................................................................................................................49 6.4 ARTEMIA CYST HATCHING ........................................................................................................53 6.5 HATCHERY DRY OUT ..................................................................................................................54 6.6 CHEMICAL PRODUCTS MANAGEMENT ......................................................................................55

7 FORMATS – LOG SHEETS........................................................................................................56



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Summary Shrimp farmers, in Vietnam, attribute the fails of their crops to poor quality

shrimp’s post-larvae.

In order to overcome this problem, a upgrade of the existing hatcheries and a

standardization of production procedures was proposed.

The present document is divided in two sections: in the first section a set of

procedures on how to upgrade the small-medium sized shrimp hatcheries is

presented. In the second section, a detailed standard operating procedures (SOP)

are presented with, the aim to standardize high quality post-larvae production in

Vietnam.

Being supervision, one of the main task in hatcheries, a set of log sheets, for each

production unit are attached.


1 Introduction

Recurrent fails in production of shrimp farms in Vietnam are attributed to poor

quality post-larvae. Albeit, this is not the only factor affecting the result of a crop,

post-larvae quality, could be considered as one of the most important points.

There are more than 1.500 hatcheries in the country producing shrimp post-

larvae using different and variables, techniques. These techniques, most of the

time, are driven by economic factors. The increment in the cost of the inputs and

the decrement on the value of the product (post-larvae), force the producer to

adjust their production system using cheaper feed, usually of doubtful quality,

affecting at the end the quality of the post-larvae. Cross contamination and

transmission of infecto-contagious diseases is the other threat that affects the

industry.

Of the eight selected provinces, three of them account for the majority of the

hatcheries, Ca Mau having approximately 50% of the hatcheries, followed by

Khanh Hoa with 35% and Phu Yen with 6%. There are shrimp hatcheries in the

provinces of Binh Dinh, Nghe An, Soc Trang, Thanh Hoa and Ha Tinh, but all of

them together account for less than 9%.

The majority of the visited hatcheries are small to medium sized enterprises,

with variable layouts and almost all of them in very poor maintenance

conditions.

The present document is divided in two sections, one regarding the hatcheries

infrastructure and the other in reference with hatcheries operating procedures.

This document pretend to be a guideline to adapt the already existing small -

medium sized hatcheries in a way that they can be certified by the local

authorities, under the Viet-GAP standards for hatcheries or similar.

These adaptations require modifications in infrastructure and management

practices that will be described in detail along this document.

This document will not cover the legal requirements, aquatic animal movement

and product identification and requirements on human resources that should be

the same as described in the Viet-GAP.

Topics related to infrastructure, disposal of chemical products and record

keeping would be described in the present hatcheries standard.



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2 Section I – Hatchery Infrastructure

2.1 The Ideal Hatchery

One of the most critical points is the hatchery location. The quality of the

seawater supply must fulfill the requirements of the specie to be cultured. The

main water parameters (temperature, salinity, dissolved oxygen, pH, etc.) should

be as stable as possible through the year. The land topography must receive

special consideration, lower lands are difficult to drain or will require a

backfilling that will increase the construction cost.

The commercial shrimp hatcheries (usually belonging to big companies) have a

fence delimiting the land. There is an entrance with a gate guarded by a security

guard. Visitors in general, have access to the office areas, but the entrance to the

production sections is highly restricted.

Those hatcheries have separated buildings for each section; e.g.: maturation,

larviculture, nursery, algae and artemia cyst hatching.

Usually each section has its own water reservoir or at least, independent

pumping units with its own filtration systems.

The used water is collected and treated before released into environment.

The maturation’s building is divided in independent rooms, for broodstock

holding, broodstock maturation, spawning and hatching. A small lab with a

microscope and stereoscope is located near the hatching area, to check eggs

fertility and nauplii deformities, etc.

The holding tanks and maturation tanks are circular with 4 to 5 m diameter.

Many hatcheries use water recirculation systems in these tanks, to avoid sudden

changes in water parameters and to reduce the risk of pathogens introduction

into the system.

The spawning can be done individually or collectively. In both cases the tanks

are designed to easily harvest the eggs.


The hatching can be done individually or collectively, also, and the harvest of the

nauplii is done using a source of light to attract the strongest animals to the

water surface.

This section has units to manage the water temperature, usually heating systems

but in some special cases there are hatcheries that use chillers to reduce the

water temperature, also.

The larviculture building has the larviculture tanks and the laboratory for daily

observation of the animals. The tanks are rectangular with “U” shape bottom.

The water pipeline is suspended to allow the complete water drain when the

system is not in use.

The laboratory has a microscope to check the larvae. This room is used to

prepare the feed; a refrigerator and a freezer are located inside this room.

The microalgae section is divided in two areas, pure culture area and mass

production area. The pure culture area is used to keep the pure strains and to

produce algae in small volumes. The mass production area is used to produce

microalgae in big volumes (three or five tons per day) and can be done in an

open area (external) or into a room.

The artemia’s cyst hatching is an extremely dirty operation, and could be a

source of bacterial cross contamination. This room is, preferably separated from

the larviculture room and have the hatching tanks with its own air and water

pipeline system.

A properly designed hatchery must have a system for water effluent treatment.

This can be done by collecting the water in tanks or ponds, where sedimentation,

chemical and physical treatments, can be applied.

Separated warehouses for feed, chemicals and packing material storage are

commonly used. The electric generators are located in an independent room.

A schematic design of a hatchery would be as follow:



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In order to dimension the hatchery, the production objectives must be known.

The first step is to establish the quantity of post-larvae that the hatchery should

produce per cycle. Following is the procedure for a L. vannamei hatchery:

No of females Total Total

Millions of PL/ day

Survival rate

Nauplii/ day

Nauplii/ female

No female

% spawn/day

in maturation

Brood stock reserve

800,000 50% 1,600,000 150,000 11 15% 71 142 92

The numbers in red are values that the producers can adjust according with their

needs and experience.

If the hatchery want to produce 800.000 post-larvae per day, and the expected

survival rate (from nauplii to PL) is 50%, the hatchery must stock 1.6 million

nauplii per day.


If the production of nauplii per female per spawn is 150.000, the hatchery will

require the spawn of 11 females every day. Assuming that 15% of the females in

the maturation are ready to spawn every day, the maturation should have 71

females. If the ratio male:females is 1:1, the maturation must hold 142

broodstock. It is necessary to have a reserve of broodstock to replace the daily

mortality. Assuming that the reserve should have 65% of the total broodstock,

the total number of animals in reserve is 92.

Once the figure of the quantity of animals per section is established, the number

of tanks can be calculated:

In this example, the larviculture tanks (LRT) have a capacity of 12 m3 and the

stocking density is of 130 nauplii per liter

Larviculture

Volume LRT Days of Total

Stocking density Litres stocking LRT

130 12,000 10 10

If the hatchery will stock the nauplii during 10 days, will require 10 LRT.

When the PL reach the stages 5 or 6 can be transferred to a new section called

nursery. The use of nursery tanks increase the number of cycles per year in the

hatchery.

Nursey

Volume Nursery Days of Total

Stocking density Liters stocking N Tanks

40 20,000 6 6

The stocking density is reduced to 40 to 50 PL per liter. One tank of 20.000 L can

hold the 900.000 to 1 million PL produce in the LRT. As the PL will stay in these

tanks only 6 days, few tanks are required.

Doing a similar procedure for the maturation:

Maturation Reserve

Size of tank Total tanks Size of tank Total tanks

Stocking density m2 maturation Stocking density m2 maturation

12 20 1 12 12 1



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The hatchery should have one tank for maturation and one tanks for broodstock

in reserve.

Spawning can be done by individual or collective. For individual spawning a

500L plastic (or fiberglass) tank per female is enough. In this case the total

number of tanks should be around 11. For collective spawning a maximum

density of two females per m2 is recommended, this give a tank of around 6 m2

with a water column of 0.8m.

The eggs can be hatched in a plastic or fiberglass tank. The density of eggs for

hatching should be around 3 / ml. For the hatchery in consideration, a tank of

600L is enough to hatching the eggs.

After hatching the nauplii are transferred to a holding tank. The stocking density

cannot exceed 3 nauplii/ml. For this example the requirement is one tank of

approximately 600L.

The same procedure is used to estimate the volume of algae to be produce per

day. If the hatchery is producing Chaetoceros and the density of algae in the LRT

will be 80.000 cell/ml, and the LRT have a capacity of 12.000 liters, the total

daily requirements of algae is 9.6 x 1011 cells.

Chaetoceros mass culture tanks should have around 1.5 x 106 cells/ml at the

moment of transferring to the LRT. In this case the total requirement per day will

be around 700 L of algae per LRT.

Artemia cyst hatching tanks is the other component that should be considered

when dimensioning the hatchery.

The number of nauplii per feeding will depend of many factors and the

management strategy of the hatchery manager. Assuming that in the last stages

of PL the Artemia nauplii consumption will be of eight nauplii/ml per day, for

one tank of 12 m3 each, the requirements are 96 millions Artemia nauplii per

day. If the hatching rate of the cyst is 80%, the total of cyst to incubate is 120

millions. Assuming an average of 250.000 cyst per gram, the total grams to

incubate is 480. The recommendations for Artemia cyst hatching is to stock 2g of


cyst per liter of water, this mean 250 L of water will be needed every day for this

operation.

With all this information, the total volume of the seawater reservoirs can be

estimated:

Volume % water exchange Water per

Unit m3 per day day in m3

LRT 12 50% 6

Nursery 20 50% 10

Algae 1 1 1

Maturation 20 300% 60

Spawning 0.6 1 0.6

Hatching 1.2 1 1.2

Artemia 0.25 1 0.25

Ton water/day 80

2 Reservoir of 40

The effluent treatment ponds (or tanks) should have the capacity to holding the

water used in one day, in this case the volume should be 80 m3 . It is better to

have two ponds (or tanks) of 40 m3 each. The first pond will function as

sedimentation area, the second pond will receive the water coming from the

surface of the primary pond.

Then, a hatchery with a projected post-larvae production of 8 millions per cycle

should have 10 larviculture tanks, six nursery tanks, one maturation tank, one

broodstock reserve tank, one 1 m3 tank for mass algae production and 250 L (or

2 of 125L or 4 of 65L) tank for Artemia cyst hatching.

Two seawater reservoirs of approximately 40 m3 each to be used alternately

would be required.

With the previous calculations is possible to estimate the size of the buildings for

each activity.



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The land required would be: 1.000 m2 for the buildings, plus 60 m2 for the

seawater reservoirs, plus 60% for the space between the buildings, multiplied by

two for the area required for offices, accommodations, warehouse and effluent

treatment ponds, totalizing 3.400 m2.

Size, shape and materials for the tanks

LRT – The ideal shape of the LRT is rectangular with “U” shape bottom. A tank of

12 m3 (operative volume) should have around 1.4m in the deeper point, and 2m

width by 6 m long. The tanks can be constructed in concrete, with the interior

painted with epoxy paint, or can be constructed of fiberglass. The best color for

the LRT is white.

Nursery tanks – The nursery tanks can be circular or rectangular in shape, with

flat bottom. The circular shape facilitate the cleaning of the tank but use more

space than a rectangular tank of the same capacity. The size is usually bigger

than the LRT. Most hatcheries have tanks between 20 and 40 m3. These tanks

can be constructed in concrete with the interior painted with epoxy paint.

Circular lined tanks are also widely used, but the transference of temperature

with the environment is higher than the concrete tanks. In low temperature


seasons the lined tanks will lose temperature easily and the cost of energy to

heat the water will be higher. These tanks can be white, gray or sky blue color.

Maturation and reserve tanks- The maturation and broodstock holding tanks

should be circular, with flat bottom and dark color (black or dark blue). Similar

materials than the ones described for the nursery tanks can be used. As these

tanks operate with a flow through system, the loss of temperature is minimum

because the residence time of the water into the tanks is shorter than in the

nursery tanks. The maximum diameter for a circular tank should be 5 m.

Algae mass production- The most common tanks used for this activity are

cylindrical, constructed of plastic or fiberglass and white in color.

Some hatcheries use cylindrical plastic bags that are kept hanged from a frame.

The bags have a diameter of 25 to 30 cm and 1 to 1.2 m of height. The volume is

between 50 to 60 liters, then 15 to 20 bag per day are required to produce 1.000

L of algae. The glassware needed for the initial algae production are detailed in

the SOP chapter of this report.

Artemia cyst hatching tanks- The best shape of these tanks is cylindrical with

funnel shaped bottom. They are made of plastic and fiberglass and are black in

color.

Seawater reservoirs- The reservoirs are rectangular with flat bottom, with a

slope toward a collecting basin to facilitate the cleaning. Reservoirs are

constructed in concrete, but other material as liner tanks can be used. The

concrete reservoirs should be painted with epoxy paint. The white color

facilitate the cleaning operation.

Effluent treatment – Depending of the soil characteristics and space availability,

the hatcheries may use tanks or ponds to treat the effluents. Independently of

that, two units must be in place. The first unit works as a sedimentation pit. The

surface water is transferred to the second unit where the water can be treated

with chemicals for disinfection before releasing to the environment. When ponds

are used and are constructed in sandy soils, the water can be released through

percolation.



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3 Objectives

The described hatchery layout differs substantially from the local hatcheries

visited. It would be difficult to pretend to frame those hatcheries into the

standards reached for the big commercial hatcheries.

The aim of this document is to propose modifications on hatchery layout and

management techniques that can be fulfilled by the small – medium sized

hatcheries.

Probably not all the hatcheries will reach the standard, but the competent

authority can classify the hatcheries in different categories according with their

achievements.

4 Present Design of the Hatcheries

4.1 Seawater Uptake and Water Treatment

There are two modalities of seawater intake used by the hatcheries. Some of

them take the water direct from the source (no filtration) and other hatcheries

take the water through a strainer buried under the sand in the beach. The

seawater is stored in a reservoir that in many hatcheries function as

sedimentation tanks. After a variable number of hours (between 6 to 24 hours)

the water is pumped from the sedimentation tank to a secondary reservoir,

passing through sand filter. The sand filter is cleaned once the cycle is finished,

and the sand is changed for a new one. Filtered water is usually treated with

chlorine (10 to 20 ppm). From here the water go to the production units,

generally filtered with a “cloth filter” or filter bag before entering the production

tanks.

4.2 Production Building

The general layout of the hatcheries is as follow: There is one building, at the

entrance, on one side is located a small room for broodstock keeping, spawning


and hatching (used for monodon production). On the other side there is an area

used to hatch artemia cysts. Following this area are the larviculture tanks.

Usually, the tanks are square in shape, with flat bottom and with a capacity of

5.000 L. The aeration is provided by a set of air stones hanging into the tank. The

water pipeline is on the floor or, in some hatcheries, under the concrete floor.

There is not lab for microscopic observations of the larvae, and the feed is kept at

room temperature.

With few exceptions, the used water goes straight to the sea, without any

treatment.

Few hatcheries have a fence delimiting their land.

5 Proposal for Hatcheries Upgrade There are four main routes whereby a pathogen could enter the production

units; one possible entrance is with the water, another is with the animals

(shrimps or wild/domestic animals), the feed (mainly fresh feed) and cross

contamination due to men’s activities (management activities).

If the hatcheries can be upgrade in infrastructure and procedures to prevent these

four routes of contamination, they could operate under the Viet-Hatcheries

Standards.

Points 5.1 and 5.2 recommend some quarantine procedures and broodstock

transportation techniques. It is assumed that the quarantine stations belong to

the government and their infrastructure and operation are under international

standards.

5.1 Quarantine Station The broodstock are the main sources of pathogens contamination. All broodstock

must pass through a quarantine period before being introduced into the hatchery.

The small-medium sized hatcheries should use the provincial quarantine stations,

which should proceed as recommended in the next paragraph.



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5.1.1.1 Black Tiger Shrimp

The provinces should have at least one quarantine stations (as the one in Rach

Goc, Ca Mau), to keep and evaluate the broodstock quality. It is recommended

that these animals remain at the station, for at least two days. During this period,

PCR analyses can be done. A section of the last pleopod can be used as material

for DNA extraction, to run the PCR assay to detect the presence of WSV, YHV,

TSA, IHHNV and IMNV; samples of feces can be used to detect AHPND, HPV or

MBV.

If for any reason these analyses can’t be done at the quarantine station, the

offspring belonging to these animals must be checked by PCR for the detection of

the previously mentioned pathogens, before being released to the farmers.

The visual examination of the broodstock is very important, the animals should

look healthy, with bright color without black or brown patches, the gills must be

clean, and the appendages undamaged.

The size of the females should be over 200g and over 70g for the males.

5.1.1.2 White Legs Shrimp

The broodstock of L. vannamaei is imported from different countries. Only SPF

certified animals must be imported. There is a quarantine station at the Ho Chi

Minh airport that controls the entrance of these animals into the country.

5.2 Hatcheries Upgrade

5.3 Fence The hatchery must have a fence to prevent the entrance of unauthorized persons

and domestic/wild animals.

The hatchery should have only one operative entrance in order to control the

movements of visitors and workers.

Visitors must be restricted to the office area; a second fence separating the office

and accommodations from the production area must be in place.


If the hatchery has entrance for vehicles, a system for tires’ disinfection should

be implemented. This could be done by making a wheel bath or by spraying the

tires with a pressure pump. There are many brands of disinfectants in the

market (most of them used in the poultry industry), the most important is that

the product remains active with organic matter and do not affect the metallic

components of the vehicles.

5.4 Seawater Uptake and Water Treatment

Whenever possible, water uptake should be done through filtration under the

sand bed. This will reduce the introduction of many live organisms into the

system. Depending on the hatchery location, this condition can be fulfilled or not.

The surface of the walls and bottom of the sedimentation tanks and water

reservoirs should be protected with epoxy paint, plastic liner or any other

material that create a smooth surface, easy to clean and is resistant to chlorine

and other disinfectants. The sedimentation tanks and water reservoirs should be

covered. This cover could be done with shading cloth or other similar material

resistant to sun-UV emission.

The water residence time, in the sedimentation tank will depend of the quantity

of solids in suspension, this time may vary from hatchery to hatchery.

From de sedimentation tank, the water should be pumped to the reservoirs

passing through a sand filter. This filter could be a low pressure sand filter

(similar to the ones, already existing at the majority of the hatcheries) or a rapid

sand filter. If the low pressure sand filter is used, the sand must be changed after

each cycle or after 20 days of use. Rapid (pressurized) sand filters are better,

because the sand can be backwashed two or three times a day (or at any time

when needed).

The water in the reservoir must be treated with calcium hypochlorite at 10 – 20

ppm during, for 12 to 24 hours. Before using the water, the residual chlorine

must be checked. If chlorine still present, addition of sodium thiosulfate is

recommended, at a ratio of 1 ppm of thiosulfate per each ppm of residual

chlorine. The size of the reservoirs must be carefully planned, according with the



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production system to be used. It is recommended to oversize the holding

capacity of the reservoirs.

From the reservoirs, the water is pumped to the production units. Would be ideal

if each production unit have its own reservoir, this mean one set of reservoir for

maturation, other set for larviculture and another set for algae and artemia.

In the case that this is not possible, separate pumps and filter systems for each

production unit must be installed.

Each filtration systems should be integrated by an electric pump, rapid sand

filter, cartridge filters with 10 μ elements, cartridge filters with 5 μ elements and

UV filters.

The sand filters must be backwashed at least two times a day (depending on the

use) or every 6 -8 hours of continuous use. The elements of the cartridge filters

must be changed two times a day and replace for cleaned and disinfected ones.

The used elements are washed with filtered water and are immersed into a tank

with a solution of 10 ppm calcium hypochlorite. After two hours the elements are

rinsed with treated water and let to dry, to be used the following day.

The UV filter should be cleaned after every cycle. The hours of operation must be

recorded and after the period of use recommended by the manufacturer is

reached, the UV lamps must be changed.

5.5 Maturation

5.5.1.1 Maturation of Black Tiger Shrimp

The area used for maturation was designed to spawn mature females of

monodon. This room must be isolated from the larviculture area. Usually there is

a door communicating both sections, this door must be eliminated and a new

door to entre the larviculture section, must be made.

Usually, the maturation rooms are very small with tanks to hold the females and

tanks for spawning and hatching. In order to reach the standards, the

broodstock holding tanks should be separated from the spawning and hatching

area. The spawning must be conducted in tanks designed in a way that the eggs

can be harvested for counting and disinfection. The bottom of the tanks should


have a slope toward the drain and an eggs collecting area, outside the tank,

should be constructed.

If the broodstock were not analyzed by PCR, the spawn should be done in

individual tanks, in order to test the offspring before mixing it with other

spawns. Fiberglass or plastic tanks (minimum capacity 500 L) with funnel

shaped bottom can be used to do individual spawning.

After collected and disinfected, the eggs should be transferred to a hatching tank

that can hold individual or collective spawns, depending of the situation.

The maturation, spawning and hatching tanks should be painted with dark colors

(black or dark blue), preferably.

A footbath with disinfectant solution and a hand washing station should be

located at the entrance of each room.

5.5.1.2 Maturation of White Legs Shrimp

The small rooms destined for maturation, in the present hatcheries are not

suitable for the maturation process of L. vannamei (White legs shrimp).

If the hatchery is planning to hold and spawn vannamei, the construction of a

new room or total refurbishing of an existing one, must be made.

The tanks should be circular with 4 to 5 m diameter. Depending of the mating

techniques used, males and females can be in the same tank or in different tanks.

The color of the tanks should be black or dark blue.

The spawning room should be separated from the maturation room. The

spawning can be done individually or collectively. The hatching tanks are located

in a separated room. There are many modalities of hatching systems, from 2 to 3

thousand liters tanks for collective hatching to individual incubator systems. The

election of the hatching system will depend of the finality of the production,

availability of space, number of personnel, etc.

To keeping the correct water temperature during some period of the year, a

heating system must be installed. This may be a central system where the water

is heated in a boiler or may be individual electric heaters added into the tanks.

The central systems usually are used to keep the temperature in the maturation



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19


and larviculture tanks; the electric heaters are used in the spawning and

hatching tanks.



5.5.1.3 Laboratory

Independently if the hatchery is producing nauplii of monodon or vannamei, a

small laboratory to observe and evaluate the quality of the spawns and the

quality of the nauplii, is necessary.

The lab should have one microscope. The observation of the eggs will give

information of the fertility. The observation of the nauplii will reveal deformities,

differences in color, stage of development, etc.

A room of 3 meter by two meters is enough to be used as lab. The room should

have a bench top with a sink and fresh and salt water taps.


located at the entrance of the room.

5.5.1.4 Fresh Feed Preparation Room The use of fresh food is another risk of pathogens introduction into the

production system. Fresh foods are also, an important component in the

maturation process, and although the trend is to reduce the use, at the moment

cannot be replaced totally by artificial diets.

The final quality of the fresh feed depends on the initial quality of the product

and the way that this product is processed.

It is necessary to have a room where the fresh feed is processed (cleaned,

chopped, packed and frozen). This room should be separated from the

production units, should have a bench-top, a sink and fresh water tap. A balance

to weigh the feed and a set of freezers to freeze the feed, are necessary (these

freezers can be located in a different room). Door and windows must have

mosquito mesh to avoid the entrance of flies and other insects.




5.6 Larvicultura

Once separated from the maturation room, the larviculture room should have an

independent entrance.

If the room has more than ten tanks, could be convenient to divide it, into two

rooms, if possible with independent entrance.

The tanks should be painted with epoxy paint and the color should be white or

clear grey. Would be recommended to round the edges of the tanks where the

walls join the floor, this may help improving the water motion. The aeration

system inside tank can be done using a grid of perforated PVC pipe or by a set of

air stones hanging into the tank. Whatever the system used must be designed in

a way that is easy to clean and disinfect.

The water and air pipelines should be suspended on the top of the tanks; in this

way the pipelines can be kept dry after the daily use. When possible it is

preferable to have a double set of pipelines to be interchanged between

production cycles.



5.7 Algae Room

The use of live algae for the first larval stages is important from two points of

view. It is the best feed for zoea stage and also the algae will improve the water

quality of the tank by removing nitrogen and phosphorus compound, carbon

dioxide, and some species produce antibacterial products.

But, as any live feed, there is a risk of pathogens introduction, mainly bacterial

contamination.

Very few hatcheries do the complete cycle of algae production, and some of them

are using dry algae substitutes.



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To do the whole cycle of algae culture, the hatchery should have one small room

for pure strain keeping, a room for internal algal production, and an area for

mass production (can be external or internal). A separated room with an

autoclave to sterilize materials and culture media is necessary.

5.7.1.1 Pure Strain Keeping Room

This room may have a size of 2 meters by 2 meters. Must have an air condition

unit to keep the temperature around 22-23oC, and a set of shelves to hold the

tubes and plates and a bench-top to do the inoculations. Should have enough

electric tubes producing white light operating 24 hours a day. The color of the

room should white.

5.7.1.2 Internal Algal Culture The size of this room will depend of the final volume of algae required per day.

The room should have a set of shelves to hold glass flasks of different volumes

(usually Erlenmeyer of 250 ml, 1 liter, containers of 5 Liters and carboys of 20

Liters). This room should have cylindrical tanks with transparent walls with 200

L capacity.

The temperature in this room must be kept around 22 – 23oC, must have enough

electric light tubes to stimulate the photosynthesis. The floor should have very

good drain because big volumes of water are moved daily in this room.



5.7.1.3 Algae Mass Culture

This area use tanks of 2 to 5 tons, depending on the needs. Would be better if

these tanks can be into a room, to reduce the cross contamination. The room

must have translucent roof to allow the sunlight penetration.

The floor should have the proper slope to avoid water accumulation and to direct

the liquids to drainage.




5.8 Artemia Cysts Hatching Room Artemia cysts hatching is a very dirty operation and another potential source of

cross contamination.

The room must be separated from the larviculture unit. The size will be

determined by the number and size of the tanks used for hatching the cysts.

Should be big enough to allow the daily cleaning of the tanks.

Must have seawater and air pipelines, and a freshwater tap, also. Preferable, the

tanks will be made of fiberglass or plastic, with black color and cone shape

bottom. Each tank must have its own illumination system.

The floor of the room should have good slope to drain the water easily.



5.9 Laboratory for Daily Observations

This room should be very near or in a contiguous space of the larviculture room.

The room can be 3 meters by 4 meters and should have a bench-top with a sink,

a refrigerator, a freezer, an electronic balance and a microscope.



5.10 Effluent Treatment The effluents used in the production units must be treated before to be released

into the environment.

The method to be used will depend of the volume of water to be treated and the

space availability of the hatchery.

If the hatchery is moving small volume of water per day (15 to 25.000 liters per

day), the effluent can be filtered mechanically, stored in a tank and treated with



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chlorine for two or three hours. Then the chlorine must be neutralized with

Sodium Thiosulfate and then the water can be released to the environment.

Those hatcheries that move big volume of water, generally the ones that has

maturation, should have a sedimentation tank (or pond) and a second tank (or

pond) to treat the water with chlorine.

5.11 Machinery Room

Ideally, the hatchery should have a separated room for electric generators, water

boiler and other room for air blowers. The electric pumps and filters should be

under a shelter to protect the equipment from direct sunlight and rain.

Special consideration must be given to the complete electric installation of the

hatchery. The electric web must fulfill the industry standards and must be

designed and constructed in a way to ensure maximum protection to the

operators.

A system for fire fighting, including fire extinguishers in critical points must be

considered.


6 Section II - Standard Operating Procedures

6.1 Broodstock Management

6.1.1.1 Broodstock Transportation and Acclimation The feed of the broodstock must be suspended 12 hours previous to the

shipment. The animals to be packed must be at the inter-molting stage (hard

shell). The water temperature for the transportation should be reduced to 20 –

24oC depending on the distance to the hatchery. Ice should be added into the

broodstock tanks. The ice must be putted inside sealed plastic bags. The

temperature should be reduced gradually at a rate of around 1oC degree per 10 –

15 minutes.

Once acclimated, the broodstock are put into double plastic bags filled with

filtered water (5-6 liters per animal) and oxygen is injected till saturation. Ideally

each animal should be packed individually. Inserting a rubber tube in the tip of

the rostrum will prevent holes in the plastic bags. Once sealed, the plastic bags

are put into a polystyrene box and the lid sealed with tape. Ice can be added on

the top of the plastic bags if required.

Upon arrival to the hatchery, the broodstock should be acclimated to the water

parameters in the broodstock tanks. Water from the broodstock tank is slowly

added into the holding broodstock bags until the water temperature and salinity

in the bags is the same than in the tank.

Once acclimated, the animals should be immersed in a container holding a

solution of 20 ppm of Iodine – PVP, during 30 seconds. After that, the broodstock

are released in the broodstock holding tanks.

6.1.1.2 Maturation Process for P. monodon The small – medium sized hatcheries usually buy mature and mated females

(gravid). Therefore, the steps carried out in the hatchery are mainly spawning

and hatching process.

If the broodstock were not checked by PCR before entering the hatchery, it is

better to kept them in individual tanks (containers). Spawning and hatching



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should be done individually. This will allow to check the offspring by PCR and

eliminate any positive batch.

Water parameter should be keep as stable as possible, water temperature should

be 28-29o C, salinity 30-35ppt, pH 7.5-8.5, and NH3 ammonia and nitrite at <

0.1ppm.

Water exchange should be 300% per day using flow-through system.

A schedule of daily activities, including feeding, could be as follow:

Table I

Time Activity Feed Quantity

(as % of biomass)

7:00 Siphoning tank and control water exchange

8:00 Water temperature control

10:00 Feed Polychaetes 6

13:00 Pellet 1


16:00 Clams 6

19:00 Control water exchange



01:00 Pellet 1

04:00 Squid 6

For tanks disinfection the following procedures are recommended (Use this

procedures to disinfect broodstock holding, maturation, spawning and hatching

tanks)

1. Prepare a solution of Calcium Hypochlorite of 10 ppm (0.15 g of

commercial product in 10L of freshwater) in a bucket.

2. Drain the tank.

3. Rinse the tanks with treated water.

4. Soak a sponge in the chlorine solution and rub tank’s wall and bottom.

5. After 10 minutes rinse the tank with abundant freshwater.


6. Let the tank to dry.

Broodstock holding and maturation tanks should be disinfected immediately

after broodstock removal.

Spawning and hatching tanks shall be disinfected daily, after using.

This operation should be done wearing protective gloves, goggles and air filter

mask.

Only females with developmental stage IV ovary should be selected to spawn.

Stage IV female

For spawning, hatching and nauplii packing follow the described procedures:

1. Clean spawning tanks as previously described.

2. Fill the spawning tanks with treated seawater

3. Keep with air (follow the chart below)

4. Add 5 ppm of EDTA to each tank.

5. Keep record of water salinity.

6. Keep record of water temperature. (Electric heaters can be used to

keep the temperature at 30oC +/- 1).

Table II

Time Activity

5:00 Harvest, disinfect and transfer eggs to hatching tanks

8:00 Spawning tanks cleaning and disinfection

10:00 Nauplii (IV) harvest and counting

11:00 Transfer Nauplii to larviculture tanks

12:00 Hatching tanks cleaning and disinfection

15:00 Refill spawning tanks with water (put aeration and EDTA 5 ppm)

17:00 Transfer mature females to spawning tanks



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18:00 Reduce aeration in the spawning tanks

24:00 Return females to maturation tanks

04:00 Refill hatching tanks

1. Eggs will be harvest by siphoning the water into an egg collector (with

double mesh, 400 µ and 100 µ).

2. Wash the eggs with treated seawater.

3. Count the eggs per tank and estimated fertility. Spawns with fertility

under 45% should be discarded.

Fertile eggs

4. Immerse the egg in a solution of 50 ppm PVP Iodine for 1 minute.

5. Rinse the eggs with treated seawater.

6. Add 5 ppm of EDTA to the hatching tank and connect air diffuser.

7. Stock the eggs in the hatching tank (maximum density 3000/L)

8. After 14 to 15 hours harvest the nauplii using light for attraction.

(phototropic attraction)

9. Wash the nauplii with treated seawater.

10. Immerse the nauplii in a solution of 50 ppm PVP Iodine for 60 seconds.

11. Stock the Nauplii in a holding tank until transferred to larviculture

section.


12. Before transferring to larviculture section harvest the nauplii as per 8 to

11.

13. Count the nauplii and estimated hatching rate.

14. Transfer the Nauplii to Larviculture room in clean buckets or plastic bags,

maximum density 15.000 Nauplii per liter of water.

6.1.1.3 Maturation feed preparation Fresh foods are important component in the maturation process, and although

the trend is to reduce the use, at the moment cannot be replaced totally by

artificial diets.

The final quality of the fresh feed depends on the initial quality of the product

and the way that this product is processed.

In order to reduce the risk of cross contamination the fresh feed must be washed

properly; followed by immersion in PVP-iodine solution of 100 ppm by 10

minutes; rinsed, packed and frozen. The freezing process help reducing bacterial

load. Fresh feed must be checked by PCR for the main shrimp viruses, mainly

WSSV.

The use of gamma radiation has been recommended but the practical application

still to be demonstrated.

6.1.1.4 Maturation process for Litopenaeus vannamei Mating behavior of L. vannamei differ from monodon in many aspects. Tiger

shrimp females mate when they are soft shelled and retain the spermatophore

inside the thelycum (closed thelycum). Once mated, the female can spawn many

times (two or three) without the need of new copula.

Mature white legs shrimp females copulate when they are hard shelled, just few

hours before spawning. The spermatophore is attached to the female thelycum

(open thelycum) and is used to fertilize the eggs at the moment of spawning.

White legs shrimp females always must mate before spawning.



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These differences generate differences in the management of the maturation

process of both species.

The standard process start by stocking the broodstock in holding tanks. During

this period the animals are acclimated to the new environment and high quality

feed is provided to increase the nutrient reserves in the hepatopancreas. These

holding tanks keep also animals that would be used for reposition of the daily

mortality that take place in the maturation tanks. Males and females are stocked

in separate tanks. The daily water exchange should be of 300% using a flow-

through system.

This period of broodstock preparation take three to four weeks.

Following are the main activities to be carried out during this stage:

1. Stocking Density- Maximum stocking density should be 8-10 animals (500

- 600g) per m2.

2. Cleaning and feeding activities:

Table III

Time Activity Feed Quantity

(as % of biomass)

7:00 Siphoning and water exchange control




16:00 Clams 5

19:00 Water exchange



04:00 Squid 5

3. Temperature should be keep between 28 and 30ºC.

4. All utensils (hoses, buckets, etc) should be kept immersed in a tank (+/-

500 L capacity) with a solution of Calcium Hypochlorite of 10 ppm.


5. Hand net will be submerged into this tank for 5 minutes and then rinsed

with treated seawater before use.

6. All utensils will be rinse with treated water before using.

After the acclimation or preparation period, the broodstock is transferred to the

maturation section. Males and females can be stocked in the same tank or in

separated tanks.

The frequency of molt should be checked on daily basis. This is important to

choose the correct moment for females’ eyestalk ablation.

Ablation must be done in intermolt stages.

Daily water exchange should be in the rate of 300 %.

Many hatcheries use water recirculation systems. These systems have the

advantages on reducing the requirements of new water, the water parameters

are more stable and the risk of introduction of pathogens into the system is

reduced.

The remaining process of daily management, fresh feed preparation, spawning

and hatching are similar to those described for monodon, earlier in this chapter.

6.2 Post-larvae Production Procedures for cleaning and disinfection

6.2.1.1 Tanks Disinfection (Include fiberglass, concrete and plastic tanks for larviculture and artemia)

1. Prepare a solution of Calcium Hypochlorite of 10 ppm (0.15 g of

commercial product in 10L of filtered seawater).

2. Drain the tank.

3. Rinse the tank with water. If required use detergent to remove material

attached to the tank’s wall.

4. Soak a sponge in the chlorine solution and rub tank’s wall and bottom.

5. After 10 minutes, rinse the tank with abundant treated seawater.

6. Let the tank to dry.

7. Disinfected all tanks immediately after animals’ removal.

8. Disinfected artemia hatching tanks every day after using.



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9. Wear protective gloves, goggles and air filter mask to do this operation.

6.2.1.2 Reservoirs

1. Reservoirs shall be rinse every day after used and before refilling. Use

treated seawater and brush/sponge to do this operation.

2. Disinfect the walls and floor once a week with a solution of Calcium

Hypochlorite of 10 ppm (0.15 g of commercial product in 10L of filtered

seawater). After 10 minutes, reservoirs shall be rinse with treated

seawater.

6.2.1.3 Filters

1. Sand Filters: Backwash sand filters during 5 minutes (minimum) two

times a day at 7:00 am and at 5:00 pm.

2. After each cycle remove the sand; clean the filters with Calcium

Hypochlorite Solution (10 ppm) rinse with treated seawater and refill

with dry sand.

3. Filter Bag: Change filter bags every day at 8:00 am. Used filter bag shall be

rinse with treated water, immersed in Calcium Hypochlorite solution (10

ppm) for ten minutes; rinse again with abundant treated seawater,

immerse in Thiosulfate solution (5 ppm) for two minutes, rinse with

treated seawater and let air dry until next day.

6.2.1.4 Larviculture tanks preparation and management.

1. Rinse the tanks with treated seawater.

Fill the tanks with treated seawater until 60% of their volume.

2. Add 5 ppm of EDTA.

3. Inoculate micro algae around 20.000to 40.000 cells / ml.

4. Increase tank volume on daily basis reaching the total volume of

the tank in Mysis I stage.


5. All utensils (hoses, buckets, hand net, etc.) should be kept

immersed in a tank (+/- 1 T) with a solution of Calcium

Hypochlorite of 10 ppm. Utensils will be rinse with treated water

before using

6.2.1.5 Stocking Density-

Stock the nauplii at a density of 150 to 200 naups/Liter. Count the nauplii and do

a quality assessment before transferring the nauplii to the larviculture unit.

Nauplii quality assessment

Nauplii quality can be determined observing the following criteria:

Swimming behavior – The normal nauplii looks as suspended in the water

column and every few seconds do swimming movements and stop. This behavior

is repeated every few seconds. If the nauplii settle in the bottom of the beaker

could be considered as weak animals and should not be stocked.

Phototropism – When the beaker holding the nauplii is located in front of a

source of light, the animals must swim to the light.

Deformities - With the help of the microscope the nauplii must be checked for

deformities. The deformities can be seen at the level of the caudal setae.

Deformities under 5% are acceptable, if the nauplii have deformities over this

value, the possibility of discard the animals must be considered.

Color – The color of the nauplii should be dark brown. This color is indicator of

good reserves of carotenoids. Pale or clear colored nauplii should be avoided.

6.2.1.6 Larvae feeding After metamorphosis to Zoea, start feeding according with table IV.

Table IV

Algae x

1000

Feed A

ppm F

Feed B

ppm F

Artemia

n/ml F

EDTA/

ppm

Tank

Vol.

%

Water

Exc. %

N 20 5 60

Z I 50 5 70

Z II 80 0.5 * 2 0.5 * 2 5 80



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Z III 90 0.5 2 0.5 2 0.25 2 5 90

M I 50 0.5 ** 4 0.5 ** 4 0.25 4 5 100

M II 50 0.5 4 0.5 4 0.50 4 5 100

M III 50 0.5 4 0.5 4 0.75 4 5 100 10

Pl 1 0.5 4 0.5 4 1 4 5 100 10

Pl 2 1 4 1 4 1 4 5 100 10

Pl 3 1 4 1 4 1 4 5 100 20

Pl 4 1 4 1 4 1 4 5 100 20

Pl 5 1.5 *** 4 1.5*** 4 2 4 5 100 30

Pl 6 1.5 4 1.5 4 2 4 5 100 30

Pl 7 1.5 4 1.5 4 2 4 5 100 30

Pl 8 1.5 4 1.5 4 3 4 5 100 30

Pl 9 1.5 4 1.5 4 3 4 5 100 30

Pl 10 2 4 2 5 3 3 100 30

Pl 11 2 4 2 5 4 3 100 30

Pl 12 2 4 2 6 4 2 100 30

* Diet < 100 µ F= Frequency of feeding per day

** Diet 150-250 µ Artemia= Numbers are naups/ml per feeding

*** Diet 250 – 450 µ

Use these values as a reference, do daily macroscopic and microscopic

observations of the PL and the water tank conditions to adjust the quantity of

feed to supply.

6.2.1.7 Sampling and population estimation

1. Check the larvae under microscope twice a day, in the morning and

in the afternoon.

2. Estimate the tank population once a day. Increase aeration in the

tank; take three samples in different points of the tank with a 100

ml recipient. Count the larvae in each sample; do the average and

calculate the tank population according with the water volume in

the tank.


6.2.1.8 Evaluating larvae and post-larvae quality There are three levels of observation.

Level 1 consist in the visual inspection of larviculture tank and in the visual

observation of the animals in a glass jar. This level of observation give a primary

information of the condition of the larvae. Each larval stage has a characteristic

behavior. The observation of swimming behavior, response to the light

(phototropism), distribution in the water column, etc. are indicators of the

condition of the larvae.

This level of observation do not require special equipment and can be done at

the tanks side.

Level 2 consist in the observation of the animals with the microscope in the

laboratory. A sample of larvae/PL is taken and transported to the laboratory.

This level of observation allow to determine the development stage of the

animals, the degree of gut repletion, presence of epibionts (protozoa attached to

the carapace), necrosis, etc.

This level of observation require a room to be used as laboratory and one

microscope.

Level 3 consist in more complex analysis, usually done in specialized

laboratories. These test may include microbiological analyses, histology, PCR, etc.

Following are the details of this methodology, published in the FAO technical

paper 450 “Health Management and Biosecurity Maintenance in White Shrimp

(Penaeus vannamei) in Latin America” from which the consultant is co-author.

Minor changes were made in the tables.

Level 1 Observations

Level 1 observations are based on simple visual features of the larvae and water

condition that can be easily seen with the naked eye in a glass beaker of animals

taken from the tank. Special attention is paid to the behavior or activity of the



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larvae, their swimming behavior (according to the larval stage), water quality,

presence of feed and faeces and later on, size disparity and homogeneity. These

observations and the scoring system used are summarized in Table V.

Table V. Summary of Level 1 assessments of larval health.

CRITERIA SCORE STAGE OBSERVATION

Swimming activity All stages Daily (2) observations

Active (> 95%) 10

Intermediate (70-95%) 5

Weak (on bottom) (< 70%) 0

Phototropism Zoea Daily (2) observations

Positive (>95%) 10


Negative (< 70%) 0

Faecal string (cord) Zoea Daily (2) observations

Present (90-100%) 10


Absent (<70%) 0

Luminescence Mysis

Night observation of the

tank

Absent 10

Present (<10%) 5

Abundant (>10%) 0

Homogenous stage All Stages Daily (2) observation

High (80-100%) 10


Low (< 70%) 0

Intestinal contents Mysis Daily (2) observation

Full (100%) 10

Half full (50%) 5


Empty (<20%) 0

Swimming activity

The swimming activity of the larvae changes dramatically but characteristically

through the larval cycle. Zoea l stages will swim rapidly and consistently forwards,

usually in circles, filter feeding on phytoplankton. Mysis, by comparison, swim

backwards with intermittent flicks of their tails, maintaining themselves in the

water column and feeding visually on phyto- and zooplankton. PL, again turn to

swimming rapidly and consistently forward, initially planktonically, but at least

from PL4-5 onwards, benthically, searching for food, unless maintained in the

water column by strong aeration. Within these distinct modes of swimming, if

>95% of the larvae are observed to be swimming actively, they are given a score

of 10; if 70-95% are active, they are given a score of 5; and if <70% are active, they

are given a score of 0.

Phototropism

Zoea stage larvae should retain a strong positive phototaxis and move towards

light. To test this, a sample of larvae is placed in a translucent container next to a

light source and the displacement of the animals is observed. If 95% or more of

the larvae move strongly towards the light, the larvae are good and given a 10; if

70-95% respond, they are acceptable and given a 5; and if less than 70% move

towards the light, they are considered weak and given a score of 0.

Faecal string (cord)

During the zoea l stages, when the zoea are feeding almost exclusively on algae,

long faecal strings can be seen projecting from the anus and loose in the water

column. When 90-100% of the larvae have these long, continuous strings all along

the digestive tube, through their bodies and continuing outside, they are

considered well fed and given a score of 10. When 70-90% have these strings, or



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they are short or discontinuous, they are given a score of 5; and when <70% of the

larvae have these strings, the larvae are not eating and they are given a score of 0.

Luminescence

This factor is observed directly in the larval rearing tank in absolute darkness.

Larval luminescence is generally due to the presence of luminescent bacteria such

as Vibrio harveyi. If no luminescence is observed, a score of 10 is given; if the

observed luminescence appears low (up to 10% of the population), the score is 5;

and if above 10% of the population are luminescent, the score is zero.

Stage homogeneity

This indicates the uniformity of larval stages in a tank. If 80% or more of the

population is in the same stage, a score of 10 is given; if between 70 and 80% are

at the same stage, the score is 5; and if less than 70% are in the same stage, the

score is zero.

It should be noted that when larval shrimp molt, it is normal to see a decrease in

the stage homogeneity, so the time at which the stage homogeneity is determined

has to be taken into consideration. This is also true for postlarvae when they are

molting.

Intestinal contents

The intestinal contents can be observed in older larval stages. The intestine is

visible as a dark line from the hepatopancreas in the larva's head region that is

easily observed in larvae held in a clear container, such as a glass beaker. This is

useful as a guide to larval feeding and feed availability. If most of the larvae

observed are full, a score of 10 is given; if half of the larvae have food in the

intestine, a score of 5 is given; and if <20% of the larvae have food in the intestine,

the score is zero.



Level 2 observations are based on microscopic examination and squash mounts,

if necessary, of a randomly taken sample of at least 20 larvae per tank (more for

larger tanks). Special attention is paid to the state of the hepatopancreas and

intestinal contents, necrosis and deformity of limbs, fouling organisms and the

presence of baculovirus in the faeces or hepatopancreas of older larvae. These

observations and the scoring system used are summarized in Table VI.

Table VI. Summary of Level 2 assessments of larval health.

CRITERIA SCORE STAGE OBSERVATION

Hepatopancreas

(lipid vacuoles) All stages Daily (2) observations

High (>90%) 10

Moderate (70-90%) 5

Low (< 70%) 0

Intestinal content All stages Daily (2) observations

Full (>95%) 10

Moderate (70-95%) 5

Empty (< 70%) 0

Necrosis All stages Daily (2) observations

Absent (0%) 10

Moderate (<15%) 5

Severe (>15%) 0

Deformities All stages Daily (2) observations

Absent (0%) 10

Moderate (<10%) 5

Severe (>10%) 0

Epibionts All stages Daily (2) observations

Absent (0%) 10

Moderate (<15%) 5

Severe (>15%) 0

Baculovirus Mysis Daily (2) observations



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Absent (0%) 10

Moderate (<10%) 5

Severe (>10%) 0

Condition of the hepatopancreas and gut contents

The condition of the hepatopancreas gives an indication of larval feeding and

digestion. It is observed using a wet mount of a sample of larvae on a microscope

slide at a magnification of 40X. In healthy larvae showing active feeding and

digestion, the hepatopancreas and midgut will be full of small, easily observed

bubbles (digestive or "lipid" vacuoles) and strong peristalsis will be seen in the

intestine. If 90% or more of the animals sampled show abundant lipid vacuoles

and/or a full gut, a score of 10 is given; if the sample shows 70 to 90% of

individuals with lipid vacuoles and/or a moderately full gut, a score of 5 is given;

and if it is less than 70% and/or the intestine is empty, the score is zero.

Necrosis

Necrosis of the larval body and limbs, which is an indication of cannibalism or

possible bacterial infection, can be observed by light microscope under low

power. If necrosis is absent, a score of 10 is given; where <15% of the animals

show some necrosis, a score of 5 is given; and where >15% show necrosis,

indicating a severe infection is present, a score of 0 is given.

Deformities

Deformities may indicate poor quality nauplii, if in the early stages, and bacterial

infections or mishandling and stress later on. Typically, the fine setae on the limbs

of the larvae and/or their rostrums may appear bent, broken or missing; the tail

may appear bent; or the gut may terminate before the anus. Typically, no remedies

exist for these problems (unless due to rough handling), and such deformed larvae

will die. In severe cases, it may be preferable to discard the whole tank as soon as


possible to prevent infection of other tanks. Where deformities are absent, a score

of 10 is given; ff <10% have deformities, a score of 5 is given; and if >10% present

deformities, a score of 0 is given.

Epibiont fouling

The larvae may become host to a range of fouling organisms ranging from bacteria

and fungi through to protozoans of many species. These will typically attach to the

exoskeleton on the head and body, and particularly around the gills of the larvae.

Where the infections are slight, the next moult may remove the fouling without

further problems, but in severe cases, the fouling will persist or reoccur in the next

stage, indicating poor water quality and necessitating action. Where fouling is

absent, a score of 10 is given; if <15% have temporary or permanent fouling, a

score of 5 is given; and if >15% are fouled continuously, a score of 0 is given.

Baculovirus

Baculoviruses can usually be detected in whole or squashed (stained with

malachite green for Monodon baculovirus) preparations of hepatopancreas or

faecal strands from larger-sized larvae, using a high powered light microscope to

spot the characteristic viral occlusion bodies (which, in the case of MBV, are dark

coloured and spherical). The expression of bacculoviruses is often mediated by

stress, and if seen, reductions in levels of stress can often reduce prevalence and

the associated problems of growth depression. Where baculoviruses are absent, a

score of 10 is given; if <10% have baculovirus, a score of 5 is given; and if >10%

are infected, a score of 0 is given.

The value of Level 1 and 2 scoring

When all of these level 1 and 2 observations are made and recorded for each tank

of larvae at each stage and the appropriate scores given in each case, an overall

picture of larval health can be derived, with higher numbers relating to healthier

larvae and vice versa. With experience, it becomes easy to judge the overall health

of each tank of larvae and to recommend courses of action to combat the problems

encountered, depending on the scores obtained.



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Level 3 observations utilizing molecular techniques are not normally required

until the postlarvae are ready to be transferred to on-growing facilities. PCR

techniques are commonly used to test for major pathogens.

Post-larval quality assessment using Level 1 procedures

Swimming activity

The vigour of swimming activity should be assessed as a general guideline of post-

larval health using the techniques described for larvae. The post-larvae can also

be put into a bowl and the water swirled with a finger. Healthy postlarvae should

orient themselves facing the current and not fall into a pile at the bottom of the

bowl, being unable to resist the current. They should also respond to tapping the

side of the bowl by jumping.

Table VII. Summary of post-larval quality assessment using Level 1 procedures.

Criteria Observations Qualitative

Assessment

Score

Moulting Moults in the water < 5% 10

Moults not sticking to head of PL 5-10% 5

>10% 0

Swimming Activity Activity level of post-larval

swimming behavior

Active 10

Intermediate 5

Low 0

Direct Observation of

Luminescence

Night-time observation of the

tank

<5% 10

5-10% 5

>10% 0

Survival Rate and Clinical

History of Tank

Estimation of survival rate in

each tank

>70% 10

40-70% 5


<40% 0

Luminescence

The prevalence of luminescence as an indication of potentially

pathogenic Vibrio spp. infections should be determined observing the tanks in the

darkness or using Level 2 techniques described below. Presence of luminescence

requires immediate treatment (probiotic use can sometimes be successful) in

order to prevent more severe infections.

Survival rate

The survival rate of post-larvae in each tank should be estimated as an indication

of the general state of health, clinical history and lack of problems during the cycle.

Each of these Level 1 post-larval quality assessments are carried out visually on

randomly taken samples of >20 animals (where appropriate) and the scoring

system detailed in Table VII applied.

Post-larval quality assessment using Level 2 procedures.

Level 2 assessments are carried out on a randomly selected sample of >20 post-

larvae per tank which are examined using low- and high-power light microscopy.

The scoring system detailed in Table VIII is then used to score the quality of each

batch of post-larvae produced.

Muscle opaqueness

An examination should be made of the body of the PL, concentrating on the bend

of the tail around the 4th-5th abdominal segments. The normally transparent

muscles turn opaque due to various reasons, including bacterial infection.

Deformities

Post-larvae should be examined for various deformities such as bent rostrum,

enlarged head due to molting problems, or missing or damaged limbs due to

bacterial infections, to estimate general health.



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Size variation

To determine the size variation, measure individually the length of at least 50

post-larvae and calculate the mean length and the standard deviation. The

coefficient of variation (CV) is obtained by dividing the standard deviation by the

mean. If the CV is equal to or less than 15%, the size variation is considered low

(score 10); if the CV is between 15% and 25%, the size variation is moderate

(score 5); and if it is greater than 25% the size variation is high (score 0).

When post-larvae molt, it is normal that the CV will increase, so the time at which

the CV is determined has to be taken into consideration. If the CV is found to be

high, the test should be repeated after a day to give time for the whole population

to complete the molt.

Gut content

Examinations of the intestinal tract for its contents and appearance (not just the

color) should be made to assess the PL's feeding level according to the criteria

shown in Table VIII. The presence of empty guts may be the first sign of disease,

or may just be due to inadequate feeding. In either case, it should be investigated

immediately. It is important to examine post-larvae immediately following

sampling.

Color of the hepatopancreas

The hepatopancreas should not be transparent and should have a good coloration.

Typically, it should be dark yellow ferrous or ochre in color, however, the color of

the hepatopancreas can be greatly influenced by the quality and color of the diets

fed and tanks used. A darker colored hepatopancreas generally indicates better

health. Care must be taken when using some flake feeds, as these may contain dyes

that stain the hepatopancreas almost black, without necessarily contributing to

the animals' health.


Condition of the hepatopancreas

The hepatopancreas of the post-larvae should be examined for its general

condition, which is primarily indicated by the number of lipid vacuoles and its

overall size. The presence of a relatively large hepatopancreas with a large

number of lipid vacuoles is considered a sign of good health. Postlarvae with a

small hepatopancreas containing few lipid vacuoles is a sign of under feeding, and

improved feeding prior to harvest may be required in order to enhance their

quality.

Epibiont fouling

Post-larvae should be examined for any epibiont or organic matter fouling on the

exoskeleton or gills (usually consisting of protozoans such

as Zoothamnium, Vorticella, Epistylis or Acineta, filamentous bacteria or dirt and

organic matter).

Melanization

Post-larvae should be examined for melanization, which often occurs where limbs

have been cannibalized or where bacterial infections have occurred. Excessive

melanization is a cause for concern and requires treatment through water quality

and feeding regime enhancement, and sometimes reductions in stocking density,

to prevent cannibalism and reduce bacterial loads.

Gill development

The state of gill development should be examined, as it gives a good idea of when

the post-larvae are able to tolerate salinity changes, which often occur when the

shrimp are transferred to the on-growing facilities. When the gill lamellae have

become branched like Christmas trees, approximately around PL9-10, they are

generally able to tolerate fairly rapid changes in salinity (up to 1 ppt/hr down to

5 ppt, or 0.1 ppt/hr below 5 ppt) and can easily be acclimated to on-growing

conditions. Where the gill lamellae remain unbranched, the shrimp should not be



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subjected to major or rapid salinity changes and should not be considered ready

for transfer from the post-larval tanks.

Intestinal peristalsis

A high-power microscopic examination of the intestinal tract of the post-larvae

should be conducted in order to ascertain the peristaltic activity of the intestinal

muscles. Strong gut peristalsis, in combination with a full gut, is an indication of

good health and high nutritional status.

Baculovirus

Refer to page 30.

Muscle to gut ratio

A microscopic examination of the relative thickness of the ventral abdominal

muscle and the gut in the 6th abdominal segment of the tail of the post-larvae

should be conducted to determine the muscle to gut ratio. This gives a useful

indication of the nutritional status of the animal. High muscle to gut ratios are

preferable.

Table VIII. Summary of postlarval quality assessment using Level 2 procedures.

Criteria Observations Qualitative

Assessment

Score

Muscle Opaqueness Opaque muscle in tail of PL <5% 10

5-10% 5

>10% 0

Deformities Deformities in limbs and head <3% 10

3-10% 5

>10% 0

Size variation (CV) Calculation of CV of post-larval size <15% 10

15-25% 5

>25% 0


Gut content Degree of fullness of digestive tract Full 10

Moderate 5

Empty 0

Color of the

Hepatopancreas

Relative coloration of

hepatopancreas

Dark 10

Pale 5

Transparent 0

Condition of the

Hepatopancreas

Relative quantity of lipid vacuoles Abundant 10

Moderate 5

Epibiont Fouling Degree of fouling by epibionts <5% 10

5-10% 5

>10% 0

Melanization Melanization of body or limbs <5% 10

5-10% 5

>10% 0

Gill Development Degree of branching of gill lamellae Complete 10

Intermediate 5

Slight 0

Intestinal Peristalsis Movement of gut muscle High 10

Low 5

Baculovirus Daily (2) observation of Mysis Absent (0%) 10

Moderate (<10%) 5

Severe (>10%) 0

Muscle to Gut Ratio Comparison of ratio between muscle

and gut thickness

>3:1 10

1-3:1 5

<1:1 0

Stress Test If < 75%, re-testing is recommended >75% 10

Stress test

At harvest, or once the post-larvae reach PL10, a stress test can be carried out.

There are several stress tests, and the most common method is to place a

randomly selected sample of about 200 animals in a beaker with water at 0 ppt

salinity, leave them for 30 minutes and then return them to 35 ppt (or ambient)

water for another 30 minutes. Following this, the dead are counted and the



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percentage of resistant individuals calculated. Stress tests should not be carried

out when the post-larvae are molting, as they are unduly stressed at this time.

Post-larval quality assessment using Level 3 procedures.

Level 3 assessments should be carried out on a statistically determined number of

post-larvae (usually 150 for a population > 10,000) from each tank (in order to

provide a 95% confidence level at 2% prevalence in the result) using PCR

techniques for the detection of important pathogens. This testing must be done

according to standard protocols by a competent health laboratory, following all

the rules for sampling, preservation and transport of the samples.

The only acceptable result for any of these viral pathogens is a negative result

(which scores 10 points - see Table 10), where both negative and positive controls

have simultaneously given their corresponding expected results. All batches

testing positive should be destroyed.

Table IX. Summary of post-larval quality assessment using Level 3 procedures.

Analysis Observations Qualitative Determination Score

PCR WSSV Negative 10

YHV Negative 10

IMNV Negative 10

IHHNV Negative 10

TSV Negative 10

NHP Negative 10

AHPNS Negative 10

As with larval quality assessment, a summary table should be made of these three

levels of post-larval quality and the points system employed (using some or all of

the above indicators, depending on circumstances). This table then is used to

determine which tanks of post-larvae are selected for on-growing, which may

require treatment before selection, and which will be rejected. As before,

experience will guide the manager in his selection of indicators to use and of a cut-


off point for points scored, below which the post-larvae batch will be treated or

rejected.

The decision to stock or not to stock a batch of post-larvae is ultimately an

assessment of risk. No fixed guidelines or standards can be provided, as this

generally comes from experience, but the following guide can be used to reduce

the risk of experiencing mortalities or poor growth in pond culture. In this risk

analysis, the order of importance of assessment is Level 3 > Level 2 > Level 1.

The following criteria can be used:

· Post-larvae must pass Level 3 assessment.

- Post-larvae must be PCR negative for YHV, IHHNV, WSSV, TSV, IMNV, NHP and

AHPNS.

· Provided that post-larvae passed Level 3 assessment, the following guide can be

used for Level 2:

- A score greater than 100 represents a low risk of severe disease problems,

therefore recommended

- A score of 65-100 represents a moderate risk of severe disease problems.

- A score less than 65 represents a high risk of severe disease problems, therefore

not recommended.

· Provided that animals pass Level 2 assessment, the following guide can be used

for Level 1:

- A score greater than 30 represents a low risk of severe disease problems,

therefore acceptable.

- A score of 20-30 represents moderate risk of severe disease problems.

- A score less than 20 represents a high risk of severe disease problems, therefore

not recommended.



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6.2.1.9 Harvest

10. Pl’s harvesting and counting (this method is an example, other methods can

be used)

1. Reduce the water level of the selected PL tank.

2. Using a hand net catch the Pl and transfer to a cylindrical holding

tank (+/-500L capacity)

3. Concentrate the Pl reducing the water level in the holding tank until

exactly 300 L

4. Agitate vigorously the water

5. With a 100 ml beaker take 5 samples

6. Increase the water level in the tank to 500 L

7. Count the number of Pl’s in each sample and calculate the average

animals/100 ml

8. Multiply the average value by 3000 (this is the number of Pl in the

holding tank).

6.3 Algae Production

There are many species of algae that can be used in shrimp larviculture. The most

commonly used are the diatoms Chaetoceros spp. and Thalassiosira spp. and the

chlorophyta Tetraselmis spp.

1. Culture Sequence (Pure Strains Room)

Fill one test tube with 5 ml of culture medium and inoculate with 5 ml

of pure culture. (Total 4 tubes of 10 ml)

After four days transfer the content of one tube to a second tube with

90 ml of culture medium (Total 4 tubes of 100 ml)

After four days select the best three tubes and transfer the content to

250 ml Erlenmeyers (Total 3 Erlenmeyer of 250 ml)

After four days select the best two 250 ml Erlenmeyer and transfer the

content to 1000 ml Erlenmeyers (Total 2 Erlenmeyer of 1000 ml)


After four days transfer the content of 1000 ml Erlenmeyers to one 5 L

Erlenmeyer(Total 2 Erlenmeyer of 5 L)

2. Culture Sequence (Massive Production Room)

After four days transfer the content of one 5 L Erlenmeyer into one

carboy of 30 L capacity (Total 2 carboy of 30 L).

After four days transfer the content of one carboy into one 250 L

fiberglass tank (Total 2 tank of 250 L).

3. Outdoor Culture

After four days transfer the content of one 250L tank into one 1 Ton

tank (Total 2 tanks of 1 Ton).

After three days use the culture to feed the larvae.

4. The algae culture medium to be used is f/2 Guillard medium

MEDIUM GUILLARD f/2

(Guillard & Ryther 1962. Guillard 1975)

To 950 mL filtered seawater add:

Quantity Compound Stock Solution

1.0 mL NaNO3 75.0 g/L dH2O

1.0 mL NaH2PO4.H20 5.0 g/L dH2O

1.0 mL Na2SiO3.9H20 30.0 g/L dH2O

1.0 mL 172 Trace Metal Solution

(see recipe below)

0.5 mL f12 Vitamin Solution

(see recipe below)

Make final volume up to. 1.0 L with filtered seawater. Autoclave after all

additions.

Note: 172 Medium contains extensive silica precipitate and should be

used only when growing diatoms.

For other algal groups use 172-Si Medium (see below).

1/2 Trace Metal Solution



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(Guillard & Ryther 1962. Guillard 1975)

To 950 ml distilled H20 add:

Qty Compound Stock Solution

3.15 g FeCl3.6 H20 -

4.36 g Na2EDTA.2 H20 -

1.0 mL CuSO4.5H20 9.8 g/L d H20

1.0 mL Na2MoO4.2H20 6.3 g/L d H20

1.0 rnL ZnSO4.7H20 22.0 g/L d H20

1.0 mL CoC12.6H20 10.0 g/L d H20

1.0 mL MnCI2.4H20 180.0 g/L d H20

Make final volume up to 1.0 L with d H20. Autoclave.

f/2 Vitamin Solution

(Guillard& Rvther 1962. Guillard 1975)

To 950 mL dH2O add:

Quantity Compound Stock Solution

1.0 ml. Vitamin B 12 (Cyanocobalamin) 1.0 g/L d H20

l0.OmL Biotin 0.lg 1LdH2O

200.0 ing Thiamine HCI -

Make final volume up to 1.0 L with d H20. Filter sterilize into plastic vials

and store in refrigerator.

Note: Vitamin B12 and Biotin are obtained in a crystalline form. When

preparing the Vitamin B12 Stock Solution allow for approximately 11%

water of crystallization (For each 1.0 mg of Vitamin B 12 add 0.89 mL d

H20).

When preparing the Biotin Stock Solution allow for approximately 4%

water of crystallization (For each 1.0 mg of Biotin add 9.6 mL d H20)

Composition of F/2

Nitrate/Phosphate Solution

Working stock: add 75g NaNo3 + 5g NaH2PO4 to 1 liter d H20


Silicate solution

Working stock: add 30g Na2SiO3 to 1 liter d H20

Trace Metal/EDTA Solution

Primary stocks: make 5 separate stocks in 1 liter volumes

1O.Og CoCI2/1L d H20, 9.8g CuSO4/1L d H20, l8OgMnCl2/1L d H20,

6.3g Na2MoO4/1L d H20, 22.Og ZnSO4/1L d H20.

Working stock: add 1ml of each primary stock solution + 4.35g

Na2C1OH14O8N2 + 3.15g FeCI3 to 1 liter d H20

Vitamin solution

Primary stock: add 20g thiamin HCI + 0.l g biotin + 0.1 g B12 to

1 liter d H20

To obtain the final ff2 enrichment, add .1 ml each of the four working stock

solutions per liter of seawater.

For the outdoor culture the culture media is:

For one liter media

Urea 20g

Phosphate 2g

Silicate 2g

EDTA 2g

Ferric Chloride 1g

Apply 1 ml of the media per liter of culture water.

Counting algae

A hemocytometer is used to count the algae. Take a sample of the culture

flask to count and charge the hemocytometer.



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Using the microscope count the cells in the four areas indicated in the

figure. Each area has 16 squares. Multiply the total number of cells by

2.500. The obtained value is the number of algae cells per milliliter.

6.4 Artemia Cyst Hatching

The artemia cysts must be certified for freedom of WSSV by PCR analysis.

The hatching procedure is as follow:

Prepare a clean and disinfected plastic tank with brackish water at 15-20

ppt.

Weigh the required quantity of cysts and rehydrate in fresh water (with

constant aeration) during 60 minutes.

Immerse the cyst in a solution of 10 ppm calcium hypochlorite.

Stock the cysts in the hatching tank at a ratio of 1 to 2 g of cyst per liter of

water.

Keep the tank with constant aeration. A light bulb must be installed on the

top of the tank and must be keep switched on during the hatching period.

Keep the water temperature over 26o C.

After 20-24 hours harvest the nauplii (The ‘’umbrella’’ stage nauplii can be

harvested after 15 – 18 hours of incubation).


Immerse the nauplii in a solution of 10 ppm calcium hypochlorite for 2 or

3 minutes, after that rinse the nauplii with filtered seawater.

Use the nauplii to feed the larviculture tanks or put in small plastic bag to

be frozen.

Alternatively the cysts can be de-capsulated before hatching. This technique

improve the hatching rate and disinfect the cysts.

The procedure is as follow:

Put in a bucket 40g of caustic soda (NaOH). Dissolve with 2 L of water.

Add 4L of liquid chlorine (8-10% active ingredient).

Put 454 g (one can) of already hydrated cysts, in the bucket.

The mixture of NaOH and chloride produce a reaction that generate heat.

In order to keep the water temperature in 20oC add ice into the container.

Keep the mixture with constant aeration.

As soon as the cysts’ color begin to change to orange, stop the process by

adding 100g of sodium thiosulphate.

Wash the cyst with clean fresh water and transfer it to the hatching tanks.

6.5 Hatchery dry out

At the end of every larviculture cycle, the hatchery must be cleaned and

disinfected.

During this period, the rooms used for larviculture, artemia hatching and algae

must be disinfected (including walls and floor). The larviculture tanks, artemia

hatching tanks and algae culture tanks should be cleaned and disinfected

following the procedures described in 5.2.1.

6.5.1.1 Pipelines

6.5.1.1.1 Water pipelines

Disinfect water pipelines before starting a new cycle.

1. Calculate the total volume of the pipeline to disinfect.

2. Prepare in a tank a Calcium Hypochlorite solution of 50 ppm.

3. Open all the valves in the pipeline.



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4. Start pumping the disinfecting solution into the pipeline.

5. When the solution start to come out of the valves close the valves.

6. Keep the pipeline with the disinfecting solution during 24 hours.

7. Drain the line completely and let it dry.

8. Wear protective gloves, goggles and air filter mask to do this operation.

9. Before starting operation, rinse the pipeline with abundant treated

water.

6.5.1.1.2 Air pipelines

Air pipelines should be disinfected two times a year (or when required).

The disinfection must be done during the dry out of the hatchery. The pipelines

must be removed and the interior of the pipes will be disinfected with a solution

of 10 ppm calcium hypochlorite. The procedure can be done using a sponge

attached to a string and pulling the string to pass the sponge inside the tube. After

that, the pipes are allow to dry before reassembling.

6.6 Chemical Products Management All the chemical products must be located in exclusive room. One person should

be assigned to manage the chemical products warehouse. This person must keep

updated the daily movement of the different products.

When some product reach the caducity date, it must be discarded according with

the specifications of the safety sheet. In many countries there are companies

specialized in the collection and disposal of chemicals products.

Special attention must be paid to the chemicals potentially hazardous for the

human health and for the environment.

Each product must have a log sheet to control the stock, the daily movement, and

the disposal method and date.


7 Formats – Log sheets

Following are the formats to carry out the main activities in the hatchery. These

formats should be used as a reference and each hatchery should develop their

own log sheets.

The formats are ordered by production section:



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Documents

Hatchery standards