Appendix Final

APPENDICESAppendix I. Information on standard hatchery operation for trial PL and shrimp larval rearing and cultivation,

A shrimp hatchery requires the coordination of

productive units (the maturation, spawning, larval

rearing, algal production and Artemia hatching units

(and possibly quarantine), supportive systems (such

as the laboratory, staff recreation area and toilets,

cleaning area, workshop, storeroom and generator

room), management systems (feeding application, good

management practices, standard procedures, staff

training, record keeping and checklists) and

infrastructural systems (electrical supply and

backup, water delivery system, effluent waste system,

transport arrangements).

The productive systems deliver a product necessary

for shrimp hatchery production. The maturation unit

produces healthy, viable gravid and fertilised

broodstock. The spawning unit produces nauplii needed

for larval rearing. The larval rearing unit produces

the postlarvae that are to be stocked into a growout

pond. Artemia hatching units provide clean live

artemia food. Algae systems provide fresh,

exponentially growing live algae. Although the design

and layout of hatcheries varies, each has to have

these productive units established or have a supplier

of these products.

The supportive systems supply the additional

facilities needed to effectively and efficiently

operate the hatchery. Disease and contamination

prevention and control is the most important support

function. Some aspects (testing) may be delegated to

an outside professional company, or be done through

the hatchery's own laboratory, but most hygiene

routines are managed within the hatchery. A workshop

stocked with the necessary tools and spares is also

essential. Backup equipment is also needed. A

cleaning area and cleaning (sterilisation,

disinfection) routine is also essential.

The infrastructural systems consist of the

physical infrastructure needed by any business of

this type. Of primary importance is a good, reliable

water supply, and a stable electrical supply. The

management systems coordinate the units into a

functional, efficient and productive whole. Good

records help coordinate the various staff members and

keep them in communication. This presentation briefly

reviews the requirements for a functional hatchery,

looking at what is required to establish a hatchery

and then the process of postlarvae production,

starting with broodstock collection. The presentation

consists of two parts. Firstly there is a Power Point

presentation that provides a visual illustration of

hatchery components. This illustrates what is needed

to setup a hatchery and looks at the overall process

from maturation to postlarvae stocking. If you do not

have Powerpoint, you download the free Open Office

software (http://www.openoffice.org/ ) or use the

free Powerpoint viewer (

http://office.microsoft.com/downloads/2000/Ppview97.a

spx ).

This document will focus briefly on selected

aspects and management tools that focus on disease

prevention, looking at a management approach aimed at

../../../

http://office.microsoft.com/downloads/2000/Ppview97.aspx

http://office.microsoft.com/downloads/2000/Ppview97.aspx

“Total Quality Assurance” (TQA) that is required to

effectively operate a hatchery. This falls within the

category of “Good management practices” and would

compliment HACCP systems at shrimp facilities.

Whereas, the focus of HACCP (Hazard Analysis Critical

Control Point) is to identify, prevent or reduce

hazards to human health, the TQA system aims to

identify, prevent and reduce hazards to optimal

larval production.

Total Quality Management (TQA) management approach

Once the hatchery has been built, with the necessaryintegrated systems, experienced management isrequired to keep it running productively. Thisrequires training of staff in the various systems andprocedures and what to expect at any stage in theproduction cycle. To achieve this, critical controlpoints or essential activities in the productioncycle are identified. With each essential activity orroutine job function, such as “egg or naupliiwashing”, one identifies:

1. Significant hazards or important dangers to the

larvae associated with that activity.

2. Preventative measures and their critical limits or

the normal condition associated with the hazard.

3. What to check or monitor.

4. How to check or monitor the potential hazard.

5. How often to check or monitor the potential

hazard.

6. Who is delegated to handle this responsibility and

monitor the hazard.

7. Possible of suggested corrective actions in

response to a hazard exceeding it critical limits or

being out of the bounds of the normal condition.

8. The records associated with this job activity.

9. Finally, a verification activity is defined for

the hatchery manager.

This system becomes a manual for the hatchery, with

checksheets, that allow for quick training of new

staff and for retraining when errors occur. It also

allows for an efficient review of the hatchery

operation. Hatchery management reviews are important

for the progress of the hatchery. By the end of the

hatchery season, new findings or procedures may

require implementation. The TQA system is then

modified through the addition of the new procedures.

In this way a stable and controlled evolution of the

hatchery can take place. Through other hatcheries

applying similar procedures, hatcheries can be

compared and beneficial activities adopted.

Management of infrastructural systems: 1. Water systems.

Water quality is the most important factor

determining hatchery success. The setup of any new

facility should look firstly as the water source.

Unpolluted, “oceanic quality” sea water is required

(salinity of 27 to 36 ppt (parts per thousand);

temperatures of 26 -30 degrees Celsius; DO > 5 ppm;

pH 7.8 - 8.5; Ammonia N: <0.5 ppm, Nitrite-N: <0.02

ppm). This water needs to be devoid of competing or

pathogenic organisms and so needs to be well

filtered, usually down to at least 1 micron.

Depending on ambient temperatures, this water may

need heating. Following final filtration is usually

U.V. (ultraviolet) or ozone sterilisation. Some

hatcheries prefer chlorination in a reservoir at the

method of sterilisation (Browdy and Bratvold, 1998)

(calcium hypochlorite (20-30 ppm) or sodium

hypochlorite (150 ppm) for 1-2 days ensuring thorough

mixing to eliminate pathogenic microorganisms. Remove

excess chlorine from the seawater by neutralising

with sodium thiosulphate (Bioinformatics Center,

1998)).

In some cases sterilisation is followed by

probiotic addition to ensure the dominance of

harmless bacterial species in the larval system.

Probiotics are usually of two types, one that

consumes metabolites like ammonia and nitrite, so

improving water quality and one that occupies a

similar niche to potentially harmful bacteria, so out

competing these pathogens. Some highly sterile

hatcheries not using probiotics are reported to

rapidly encounter serious bacterial problems as a

specific, undesired bacterial species becomes

dominant. The textbook recommendation to this problem

is a hatchery shutdown and dryout, but recovery can

take months (Jory, 1997).

Sufficient storage capacity is needed in the case of

some type of failure, or else two backup pumping

systems should be in place should the primary pumping

system fail (motor or pump failure). Site specific

risks and water treatment procedures also need to be

evaluated in the decision of whether multiple backups

or water reservoirs are a better choice. Ideally a

combination of both water storage and system backups

should be employed.

2. Air systems.

Aeration requirements can be calculated by most

commercial suppliers, based on your specifications.

Always use a few small units rather than a single

large unit and have at least one backup blower in

storage. Always have excess aeration capacity. The

ability to aerate down to about 1.1 meters should be

sufficient. The air intakes should be filtered and it

is a good idea to have in-line filters (cotton dipped

in iodine solution is effective) on all algae

aeration up to the carboy level so as to prevent the

introduction of organisms through the air.

3. Electrical systems.

This needs to be professionally designed for safety

and easily accessible for maintenance purposes. A

backup generator is probably essential, at a minimum

to run the air-blowers. Fluorescent tube ballasts

generate significant amounts of heat, so should not

be in an air conditioned room.

4. Other infrastructural systems.

One needs to consider aspects like road access,

telephone lines, the availability of electricians and

other supportive functions. The cheapest pumps for

hatchery applications are the swimming pool pumps.

Management of production units:

The focus here is on the closed thelycum species that

molt, mate and then mature and spawn a few days

later.

Maturation Unit:

Broodstock sourcing requiring increasing levels of

technical ability are:

1. wild – artesenal fishermen, trawlers etc.

2. pond-reared and

3. Specific Pathogen Free (SPF) broodstock from

Biosecure facilities (Moss, 1998).

As soon as the breeding cycle is closed (pond-reared

and SPF broodstock), the maintenance of genetic

diversity and genetic selection become important

considerations. Few hatcheries have the facilities or

capacity to manage a genetics program.

Maturation facilities provide oceanic conditions

similar to the deep ocean floor where the wild

broodstock occur naturally.

Parameters for Penaeid Prawn Maturation and daily

mean fluctuation.

Salinity

Temperatu

re

pH Photoper

iod

D.O.

27-36ppt

+/-0.5280C +/-

20

8.0

+/- 0.2

14L, 10

D

5ppm

+

Environmental parameters need to be stable within

the specified bounds before a good diet will be

effective in stimulating egg development. Mixed

diets of formulated pellets and fresh feeds appear to

work best. A wide variety of fresh feeds, including

squid, mussels, oysters, clams, adult artemia, krill,

various shrimp species (e.g. Calianassa sp.), beef heart,

scallop gut, octopus, cuttlefish, mullet, sardines,

blood worms, cockles, shark, crab, and mysids.

Formulated pelleted diets provide specific vitamins,

minerals, antioxidants, carotenoids, cholesterol,

phospholipids and fatty acids to the diet. Linoleic,

C18:2(n-6), linolenic, 18:3(n-3), eicosapentaenoic,

C20:5(n-3), and docosahexaenoic, C22:6(n-3) are

essential fatty acids for growth and egg development.

Arachidonic acid, C20:4n-6 is often deficient in

diets for breeding.

Treece (2000) specifies maturation densities of

five to seven shrimp/m2 in large (13 ft or 4 m)

diameter, circular, maturation tanks. Jory (1996)

specifies, 3-8 animals/ m2 depending on shrimp size,

and at ratios from 1:1 to 1:4 male to female for

closed thelycum species. For P. monodon this is a good

density, while for F. indicus we put 100 shrimp (66

females and 34 males = 17/ m2 ) in a 6 m2 oval tank

(1.1m wide).

To provide improved water stability, some

maturation systems are constructed as a recirculating

system with a biofilter. pH is maintained at 8.0

through the application of sodium carbonate.

Biofilters may lead to the buildup of pathogens in

biofilms that can be detrimental to spawning success.

The broodstock system is the most likely source of

many hatchery pathogens. To counter this, the

spawning and maturation floors should be regularly

washed down with chlorine or potassium permanganate,

spawning tanks and airlines routinely sterilised and

maturation tanks routinely scrubbed down. Broodstock

and maturation tanks can be treated with 50ppm

formalin (Treece, 2000) followed by water changes to

reduce population of epibiotic microflora. This water

should not enter the biofilter. If a biofilter is not

used, flow through systems employ a daily exchange

capacity of 200% to 300% (Jory, 1996).

Unilateral eyestalk ablation is commonly

practiced with P. monodon, but is not necessary with F.

indicus. F. indicus and P. japonicus mature, mate and spawn

readily in captivity. P. monodon requires

environmental and hormonal manipulation. Wild,

gravid, female P. monodon can spawn naturally for the

first spawn. Eyestalk ablation removes the "X" organ

in their eyestalk, an organ which produces a hormone

that inhibits ovarian maturation. The easiest method

of ablation is enucleation by pinching, crushing and

squeezing the eyeball and its contents after an

incision on the eyeball has been made (Jory, 1996).

With P. monodon, this can be done rapidly by one

person.

Spawning:

Penaeus monodon, can produce 700,000 to over 1

million eggs each spawn. For example, a 290-g (10.2-

oz) female P. monodon might spawn 700,000 eggs,

whereas a 454 gram (1 lb) female might spawn 1.4 to

1.8 million eggs each spawn (Treece, 2000). Wild-

sourced Fenneropenaeus indicus tend to produce more

nauplii per spawn and more robust nauplii than pond-

reared F. indicus for the first few spawns. A wild F.

indicus can produce 120,000 nauplii, while a pond-

reared specimen will seldom deliver more than 80,000

nauplii per spawn. Treece (2000) noted that “in the

domestication process it generally takes three to

four generations to obtain pond broodstock of equal

or better quality than wild broodstock. Egg quality

and larval quality from domesticated broodstock are

generally equal to or better than wild stock.”

Spawning involves the released eggs brushing against

the spermatophore as they exit the ovipositors, while

the female is continuously swimming. If the female

stops swimming, or her swimming is interrupted by

hitting a tank wall or air lines, the eggs may fall

straight down, and are not likely to become

fertilized. Tanks with rounded walls are therefore

important. Spawning densities should be about one

third of the maturation density (F. indicus, 3-4/m2 ; P.

monodon, 1-2/m2.) We use 4.5 m2 tanks (60-70 cm deep)

and put up to 15 in each tank.

Eggs hatch within 16 hours at 28 degrees Celsius.

Nauplii can be moved the same day, once all the eggs

are hatched, or moved the following day. To wash the

nauplii, we bring 3 buckets of water from the larval

tank and of the same temperature as the spawning

system (280C). To the one, we add 20ppm providone

iodine. Nauplii are rinsed in this for 1 minute. They

are then rinsed gently in the second bucket for 1 to

2 minutes and finally placed in the third bucket and

moved to the larval rearing system. A 100ppm formalin

wash may also be employed (see ppt presentation).

Larval Rearing:

Small, medium and large hatcheries share the same

basic infrastructure. Hatcheries simulate the

natural conditions encountered by larval shrimp, but

at far higher densities than is found in the wild.

This requires water quality management and feeding

regimes that optimise commercial production and

maximize larval survival and growth to produce strong

postlarvae ready for stocking into nursery or growout

systems. The production units directly associated

with larval production are the larval rearing unit

(tanks), the microalgae laboratory and culture unit

and the Artemia production unit. Seawater treatment

is an important infrastructural component. Larval

rearing tanks can be made from any non-toxic

material and can be flat-bottomed, V-bottomed or U-

shaped. Fibreglass is robust and long-lasting. The

designs accommodate rapid water exchanges and

vigorous aeration capabilities. Tanks volumes from 5

to 50 tonnes are practical and should be based on the

number of nauplii to be stocked at any one time. A

hatchery should have up to 3 tank sizes to handle

different numbers of nauplii. Two batches (days) of

nauplii (to attain 1-1.2 million per 10 tonne tank)

can be put into the same tank, but a single batch

gives better survivals. Below (Table 1) is a typical

larval program for Fenneropenaeus indicus. The algae

added in the nauplius stage is top quality algae in

the exponential growth phase to start the initial

bloom. 90-120 liters of water from Chaetoceros cultures

at a density of 1-2 million cells per milliliter is

added. This gives us a starting algal density of

around 10,000 to 40,000 cells per milliliter. We also

add 100ml of the Guillard L1 (updated F/2) nutrient

medium. The culture density for Tetraselmis is around 100

thousand cells per milliliter at the time of feeding.

Programbased on1 million

PL's in10T

Stage

Volume(10T

tank) or%

exchange

Aeration, [EDTA],{temperature},

(salinity)

*Note-4

Algae feed inliters

Chaetoceros[Tetraselmis]

*Note-3

Artificialformulated feed in

grams/day

[Artemia]in grams*Note-2

TypicalProbiotic

(*a,b)/[typical

antibiotic(*1,2)]

*Note-1

Naupliu

s

3T Low [30g],

{28}, (32)

90 liter + 100ml F/2

0g a,b, or

[1]

Zoea 1 3T Medium [0g],

{28}, (32)

120 [0]

liter

20, [0] a,b, or

[1]

Zoea 2 6T Medium,[30g],

{28}, (32)

500 [300]

liter

30, [0] a,b, or

[-]

Zoea 3 10T High [20g],

{28}, (32)

750 [600]

liter

40, [0] a,b, or

[-]

Mysis 1 '30% High [30g],

{28}, (32)

750 [1000]

liter

50, [0]


{28}, (32)

750 [1000]

liter

70, [60]a,b, or

[2]


{28}, (32)

500 [1000]

liter

80,

[150]

a,b, or

[-]


PL's in10T

Stage

Volume(10T

tank) or%

exchange


(salinity)

*Note-4

Algae feed inliters


*Note-3


grams/day


TypicalProbiotic

(*a,b)/[typical

antibiotic(*1,2)]

*Note-1

PL 1

(*3)

'50% High [40g],

{28}, (32)

200 [1000]

liter

110,

[300]

a,b, or

[-]

PL2 '50% High [30g],

{28}, (32)

100 [1000]

liter

135,

[350]

a,b,or

[1 or 2]

PL3 '50% High [30g],

{28}, (32)

100 [0]

liter

140,

[400]

a,b

PL4 Move to nursery (15 to 20T)

High [0g],

{28}, (32)

400 [0] liter in nursery

150,

[500]

a,b

PL5 '0% High [0g],

{28}, (32)

0 [0] liter 165,

[900]

a,b

PL6 '33% High [0g],

{28}, (32)

400 [0] liter

190,

[800]

a,b

PL7 '0% High [0g],

{28}, (32)

0 [0] liter 220,

[500]

a,b

PL8 '50% High [0g],

{28}, (32)

0 [0] liter 240,

[500]

a,b

PL9 '0% High [0g], 0 [0] liter 275, [0]a,b


PL's in10T

Stage

Volume(10T

tank) or%

exchange


(salinity)

*Note-4

Algae feed inliters


*Note-3


grams/day


TypicalProbiotic

(*a,b)/[typical

antibiotic(*1,2)]

*Note-1

{28}, (32)

PL10 '50% High [0g],

{28}, (32)

0 [0] liter 330, [0]a,b

PL11 Harves

t

High [0g],

{28}, (32)

0 [0] liter 380, [0]

PL12: 0.006g, each = 6kg.; 380g feed = 6.3%

of biomass

*1 = Prefuran at 0.5g per 1000 liters*2 = F98, Furazolidone 98% at 3g per 1000 liters*3 PL = postlarvae. The larval stages are complete and the animallooks like a shrimp.*a = Clear Flo 1006 at 1 to 10g per 10 tonnes per day (5g per tonneper week in daily doses).*b = Clear Flo 1100-50x (Alken Murray, 2002) (nitrifying bacteria)at 0.5 to 1.0 ml per 10 tonnes per day.Note 1: Antibiotics and probiotics are not compatible treatments.Probiotics are preferred, with antibiotics being a last resort.Antibiotics kill bacteria, including the probiotic applications.Note 2: Artificial formulated feed times are 6h00, 9h00, 12h00,15h00, 17h30. The above is the total daily feed and is divided byfive to work out each feed amount. The type of feed for the larvalstage is specified by the commercial supplier. Note 3: Algae blooms are initiated with diatoms (Chaetoceros) innauplius from an initial density of 10-30 thousand cells permilliliter. By zoea 1, the algae density is 40 to 100 cells permilliliter. All through zoea one and sometimes zoea 2 the bloomsgrow faster than consumption by the larvae. Tetraselmis is only addedin zoea 2, as the larvae then have eyes to see this moving food.Diatoms provide essential fatty acids (20:5n3, 22:6n3) and protein.

Tetraselmis has a good amino acid profile and provides a naturalantibiotic effect. Fresh, boiled peas are a suitable feed if algaeis low for some reason. Spirulina is also suitable. Note 4: The chelating agent EDTA is routinely added to spawning andlarval rearing tanks to reduce the concentration of heavy metals.EDTA may also be involved in reducing bacterial contamination of theshrimp eggs and larvae. There are very evident benefits in its usein association with probiotics.

Algae Production Unit:

Microalgae provide food for the shrimp larval stages

and contribute to stabilizing and improving the

quality of the rearing medium by utilising

metabolites such as ammonia (Muller-Feuga, 2000). We

use the Guillard L1 nutrient medium (

http://ccmp.bigelow.org/CI/CI_01h.html or

http://www.sccap.bot.ku.dk/media/L.htm ) and a batch

algal production system (see the Powepoint

presentation). Only the diatoms need the silicate

nutrient. To simplify the daily operation, we prepare

the vitamins for daily use as follows::

600 ml distilled water, Biotin:150mg, B12: 300mg, B1:

600mg.

Usage:

http://www.sccap.bot.ku.dk/media/L.htm

http://ccmp.bigelow.org/CI/CI_01h.html

2 liter: 0.2ml; 90 liter: 9ml; 120 liter: 12ml; 350 liter: 35ml

Unused vitamins are added to 10 and 4 tonne Tetraselmis tanks each

day.

Artificial Shrimp Larval Feeds:

Formulated feeds need to be selected for various

nutritional components. Up to zoea 3 the larvae are largely

herbivorous. As filter feeders, they will also consume

bacteria and other small particles. Dried Spirulina sp. (source of

protein, mixed carotenoids (14mg/3g) and other phytonutrients,

B-Vitamins, and essential amino acids) and Haematococcus pluvialis

(Astaxanthin Content of 1.5%) are good feeds for zoea and

mysis, with high quality commercial products available. After

the zoea stages, the larvae become increasingly carnivorous.

Up to PL2, Tetraselmis can still be seen in the gut. Throughout

all larval stages the fatty acid component of the diet is

important. Highly unsaturated fatty acids (HUFAs) are needed

in the early development of the nervous system in fish and

shrimp, and also as precursors for many biologically active

compounds - such as prostaglandins - involved in regulating

growth and reproduction (Jory, 1997). Live algae provides

these in the early larval stages (Reed Mariculture), while

artificial formulated feeds also need to provide the correct

proteins (essential amino acids: lysine, methionine,

tryptophan, arginine, histidine, leucine, isoleucine), lipids

(essential fatty acids: 18:2n-6, 18:3n-3; 20:4n-6, 20:5n-3,

or 22:6n-3, depending upon species), and other nutrients like

cholesterol, carotenoids, phospholipids (Lindsey, 1998),

minerals ( P, K, Mg), trace elements (Fe, Zn, Mn, Cu, Se, I),

and vitamins (B1, B2, B6, B12, pantothenic acid, niacin,

biotin, folic acid, inositol, choline, D3, A, K3, E, and C)

(Tacon, 2002). Good commercial formulated feeds need to have

taken these requirements into account when designing their

feeds (See for example the documentation at Aquafauna

(http://www.aquafauna.com )). A commercial product called

Cyclopeeze (Argent Chemical Laboratories) serves as a partial

replacement (and possibly complete) for Artemia and

significantly reduces cannibalism in the postlarval stages of

F. indicus. The mention of commercial products in this document

does not imply that these are any better than other companies

producing similar or equivalent products, but simply that

these are the products with which we have experience.

Probiotics:

http://www.aquafauna.com/

A probiotic is defined as: “as a live microbial

adjunct which has a beneficial effect on the host by

modifying the host-associated or ambient microbial

community, by ensuring improved use of the feed or

enhancing its nutritional value, by enhancing the

host response towards disease, or by improving the

quality of its ambient environment” (Verschuere,et

al, 2000). Probiotics have a definite role in

hatcheries, especially as antibiotics such as

chloramphenicol are now banned and considered

dangerous to human health. Microbial species

composition in hatchery tanks can be changed and

controlled by adding selected bacterial species to

displace deleterious bacteria (Moriarty, 1999).

However, a scientific approach is needed, as the

potential range of probiotics is unlimited. Using

probiotics is an ecological approach, so the effect

is usually not instantaneous. It takes about two

weeks for a probiotic to dominate a hatchery system.

Daily applications are probably more effective. A

probiotic is usually specific, eliminating a single

identified set of related problems. Probiotic

applications need to be done in conjunction with

strict hygiene and implementing hygienic procedures.

Typically, bacteria originate from the broodstock,

(and spread by staff's activities) so routinely there

should be a total sterilisation of all floors,

buckets, pipes and equipment (filters, mops etc.).

Together with this, the spawning tanks should be well

sterilised and the broodstock given a 100 to 200 ppm

formalin bath (www.aquatext.com) for 1 to 2 hours or

25 ppm formalin for 24 hours. High ammonia and

nitrite levels can become a critical, even lethal,

problem in the late zoeal stage and from mysis to

postlarvae (Jory, 1997). These levels are relatively

easy to moderate through water exchanges. However we

http://www.aquatext.com/

have found the addition of nitrifying bacteria to

handle these metabolites to be especially effective

when used in combination with another that is a mix

designed to out compete Vibrio sp. Bacteria. Probiotics

may also enhance shrimp nutrition by providing

essential nutrients and enzymes, or through the

digestion of organic material and bacteria (Jory,

1997).

Appendix I I. Historical background of Probiotics

During the past 20 years, aquaculture industry

has been growing tremendously, especially that of

marine fish, shrimps and bivalves. But, as with many

other industries, this rapid growth has brought with

it the problem of environmental pollution.

Contamination of coastal waters due to aquaculture is

posing serious concerns among law makers as well as

scientists. The coastal environment has been

seriously damaged, often resulting in disease

outbreaks. Recently, shrimp culture all over the

world has been frequently affected by viral and

bacterial diseases inflicting huge loss. In China,

the production of shrimps decreased seriously. The

production of shrimps was 200,000 tons in 1992, but

was only 55,000 tons in 1994. Pathogenic

microorganisms implicated in these outbreaks were

viruses, bacteria, rickettsia, mycoplasma, algae,

fungi and protozoan parasites. For preventing and

controlling diseases, a host of antibiotics,

pesticides and other chemicals were used possibly

creating antibiotic resistant bacteria, persistence

of pesticides and other toxic chemicals in aquatic

environment and creating human health hazards. Thus,

how to improve the ecological environment of

aquaculture has become the focus of attention of

international aquaculture.

Now, researchers are trying to use probiotic bacteria

in aquaculture to improve water quality by balancing

bacterial population in water and reducing pathogenic

bacterial load. Researchers are increasingly paying

more attention to this new approach (ecological

aquaculture ), and have made considerable headway.

This review, on the basis of the new research

findings in probiotics applied to aquaculture,

analyze and summarize the mechanism of probiotic

action in aquaculture.

“Probiotics" generally includes lactobacillus

bacteria, cyanobacteria, micro algae fungi, etc. Some

Chinese researchers translate it into English as

"Normal micro biota" or "Effective micro biota"; it

includes Photosynthetic bacteria, Lactobacillus,

Actinomycetes, Nitrobacteria, Denitrifying bacteria,

Bifidobacterium, yeast, etc. Usually, it does not

include micro algae. In English literature, probiotic

bacteria are generally called the bacteria which can

improve the water quality of aquaculture, and (or)

inhibit the pathogens in water there by increasing

production. "Probiotics", "Probiont", "Probiotic

bacteria" or "Beneficial bacteria" are the terms

synonymously used for probiotic bacteria.v

The theory of ecological prevention and cure in

controlling the insect pest of terrestrial higher

grade animals and plants has been in practice for

long time, and has achieved remarkable success. The

use of beneficial digestive bacteria in human and

animal nutrition is well documented. Lactobacillus

acidophilus is used commonly to control and prevent

infections by pathogenic microorganisms in the

intestinal tract of many terrestrial animals.

Recently, the biocontrolling theory has been applied

to aquaculture. Many researchers attempt to use some

kind of probiotics in aquaculture water to regulate

the micro flora of aquaculture water, control

pathogenic microorganisms, to enhance decomposition

of the undesirable organic substances in aquaculture

water, reduces toxic gases in water and improve

ecological environment of aquaculture. In addition,

the use of probiotics can increase the population of

food organisms, improve the nutrition level of

aquacultural animals and improve immunity of cultured

animals to pathogenic microorganisms. In addition,

the use of antibiotics and chemicals can be reduced

and frequent outbreaks of diseases can be prevented.

Appendix III. Mechanism of action of the probiotic

bacteria

The mechanism of action of the probiotic bacteria

has not been studied systematically. According to

some recent publications, in the aquaculture the

mechanism of action of the probiotic bacteria may

have several aspects. 1. probiotic bacteria may

competitively exclude the pathogenic bacteria or

produce substances that inhibit the growth of the

pathogenic bacteria. 2. provide essential nutrients

to enhance the nutrition of the cultured animals. 3.

provide digestive enzymes to enhance the digestion of

the cultured animals. 4. probiotic bacteria directly

uptake or decompose the organic matter or toxic

material in the water improving the quality of the

water.

Metchnikoff - Pasteur Institute, Paris 1907

“The Prolongation of Life”

“A reader who has little knowledge of such matters may be

surprised by my recommendation to absorb large quantities of

microbes. The general belief is that microbes are harmful.

This belief is erroneous. There are many useful microbes,

amongst which the lactobacilli, have an honorable place.”

Apendix IV: Protexin Aquatech Product Details

Protexin Aquatech Product : Protexin Aquatech Program package:

Aqua-MediaAqua-1 Total Viable Count 1.0x108 CFU/ml.Aqua-2 Total Viable Count 1.0x108 CFU/ml.Aqua-3 Total Viable Count 1.0x108 CFU/ml.Aqua-4 Total Viable Count 1.0x108 CFU/ml.

The Protexin Aquatech products are complex blends of Bacillus

that promote a

healthy environment for fish, crustaceans (e.g. shrimp),

molluscs and other animals, grown in fresh, brackish and

marine waters, by lowering sludge build up, stabilising

plankton and improving water quality in fresh, brackish and

marine waters. The Protexin Aqua-Program works as a

preventative strategy and should be used from the beginning of

the crop cycle to achieve best results. To make it less likely

that disease will occur at high stocking density use should be

continued throughout the grow out cycle. The Protexin Aqua-

Program products are compatible and complementary when used

together.

The microbes contained within these products are naturally

occurring, mesophilic, gram positive spore-forming rods,

aerobic or facultative, and grow at pH 5.5 to 9.0. Their

growth is slower at salinities over 40 ppt. Activated Protexin

Aqua-1, Aqua-2, Aqua-3 and Aqua-4 will inundate aquaculture

and water systems with beneficial microbes to improve water

quality and decrease the numbers of viruses and bacteria.

These products multiply in the water to suppress the

proliferation of bacteria and viruses.

Nexge Back to Index

Activated Protexin Aqua-1, Aqua -2, Aqua -3 and Aqua -4 by

using Aqua -Media, will inundate aquaculture and water systems

with beneficial microbes to improve water quality and decrease

the numbers of viruses and bacteria.

Protexin Aquatech Product Box contains 5 different

sophisticated active and media. Protexin Aqua –Media(Separate

packet) - An innovative product that allows the activation of

large numbers of viable microbes for use in aquaculture and

water treatment systems

Protexin Aqua -1 contains concentrated viable bacteria for

sludge control and plankton stabilization to improve water

quality in aquaculture and water treatment systems

Protexin Aqua -2 contains concentrated viable bacteria for

prevention and treatment of White Spot Virus and other viruses

in aquaculture and water treatment systems

Protexin Aqua -3 contains concentrated viable bacteria

prevention and treatment of Vibrio and other bacteria in

aquaculture and water treatment systems

Protexin Aqua-4 contains concentrated viable bacteria

prevention and treatment of Vibrio and other bacteria in

aquaculture and water treatment systems

Dosage and Administration: Grow-out

The dosage rates of Aqua-Media, Aqua-1, Aqua-2, Aqua-3 and Aqua-4 will depend on the total volume of water in the pond (Litres) and the stocking density of the shrimp fry (pcs per sqm).

Author work as MODIFIED EXTENSIVE shrimp production is classedas 3-7 pcs + / m2 was there

Protexin Aqua-Media

AN INNOVATIVE PRODUCT THAT ALLOWS THE ACTIVATION OF LARGE NUMBERS OF VIABLE MICROBESFOR USE IN AQUACULTURE AND WATER TREATMENT SYSTEMS.

PRESENTATION: A cream coloured, free-flowing powder with a characteristic odour.

PROPERTIES: PROPERTIES: Protexin Aqua-Media is an easy to use proprietary formulation containing peptones, minerals and other nutrients to maximise the germination and subsequent rapid growth of Bacillus species for use in aquaculture and water treatment systems. To ensure Aquasecurity the peptones in Protexin Aqua-Media have been sterilised. Protexin Aqua-Media is a very effective and cost-effective means for growingvery large numbers of viable, beneficial bacteria in a short time. For the spores to germinate, it is necessary to provide a short ‘heat shock’ by adding the spores to freshly boiled water. Protexin Aqua-Media is essential to promote the germination and rapid growth of Protexin Aqua-1, Protexin Aqua-2, Protexin Aqua-3, and Protexin Aqua-4.

ADMINISTRATION: The following procedure will treat a pond measuring 1 Acre area by 1.5 m depth:

1. Author take 2 L of fresh clean drinking water2. And used to add 30 g, 35 gm and 40 gm of PROTEXIN AQUA-MEDIA respectively for activating 10 ml, 13 ml and 15 ml Protexin Aquatech consecutively and stir vigorously to mix thoroughly

3. He reseal PROTEXIN AQUA-MEDIA container immediately after use4. Author bring solution to the boil (for a few seconds only) then remove from heat5. Immediately add 10ml,13 ml and 15 ml (10 cc,13 cc and 15 cc) of spores (see separate datasheets for PROTEXIN AQUA-1, PROTEXIN AQUA-2, PROTEXIN AQUA-3 and PROTEXIN AQUA-4). This will ‘heat shock’ the spores6. He used to immediately add 2 L of fresh clean drinking water at room temperature7 7. . Commence activation. Activation times should be calculated from commencement of ‘heat shock’

ACTIVATION TIME IS 180 MINUTE FOR Protexin Aqua-1.ACTIVATION TIME IS 90 MINUTE FOR Protexin Aqua-2. ACTIVATION TIME IS 90 MINUTE FOR Protexin Aqua-3.ACTIVATION TIME IS 90 MINUTE FOR Protexin Aqua-4.

8. During activation time, Author stir vigorously every hour or gently aerate solution9. Author then mix the 4 L of activated spore solution with 16L of pond water10. Allow 2 hours of gentle aeration before adding to the pond11. Pond aeration is not necessary at low stocking densities (5-10 pieces/square metre),however, aeration is essential at high stocking densities (>25pieces/square metre)

STORAGE: Store below 3 °C. Shelf life is three years.PACK SIZES: 2.6 kg provides enough for 20 activations.

Protexin Aqua-1

CONCENTRATED VIABLE BACTERIA FOR SLUDGE CONTROL AND PLANKTON STABILISATION TO IMPROVE WATER QUALITY IN AQUACULTURE AND WATER TREATMENT SYSTEMS.

PRESENTATION: Reddish-brown-coloured bacterial spore liquid suspension.PROPERTIES: Protexin Aqua-1 is a special blend of Bacillus species that promote a healthy environment for fish,

crustaceans (e.g. shrimp), molluscs and other aquatic animals by lowering sludge build up,stabilising plankton and improvingwater quality in fresh, brackish and marine waters. Protexin Aqua-1microbes are naturally occurring, mesophilic, gram positive spore-forming rods, aerobic or facultative, and grow between pH 5.5 and 9.0.

Protexin Aqua-1 combines vigorous growth with high cellulase and protease enzyme activities, with control against Vibrio species, White Spot Virus and Yellow Head Virus. To achieve best results, Protexin Aqua-1 as an integral part of a preventative strategy from the beginning of the crop cycle andshuttle with Protexin Aqua-2, Protexin Aqua-3 and Protexin Aqua-4 throughout the crop cycle.

Benefits of rotating Protexin Aqua-1, Protexin Aqua-2, Protexin Aqua-3 and Protexin Aqua-4 in the shuttle program are:1. breakdown of organic flocs breakdown of organic flocs 2. accelerating breakdown of dissolved protein & fragments of

unused feed accelerating breakdown of dissolved protein & fragments of unused feed

3. zooplankton enhancement zooplankton enhancement 4. reduction in White Spot and Yellow Head Viruses, and

reduction in White Spot and Yellow Head Viruses, and Vibrio Vibrio species species

5. lessening of sludge build up lessening of sludge build up6. balancing and stabilising of existing algal blooms

balancing and stabilising of existing algal blooms7. water quality improvement water quality improvement 8. reduces the need for water exchange, provided aeration is

adequate reduces the need for water exchange, provided aeration is adequate

ADMINISTRATION: ADMINISTRATION:Activation: Author follow Protexin Aqua-Media datasheet to grow 10, 13 and 15 ml of Protexin Aqua-1 which is sufficient for Follow Protexin Aqua-Media datasheet to grow the same of Protexin Aqua-1 which is sufficient for a 1 acre x 1.5 m depth).

Prevention Program: To be continued as required.

WEEK 1 WEEK 2 WEEK 3 WEEK 4 WEEK 5AQUA-1 AQUA-2 AQUA-3 AQUA-4 Rotate back to AQUA-1

Pond Water quality control /treatment Program: To be continuedas required. Author use DOUBLE the dose rate of Aqua-1 for anyTREATMENT program.DAY 1 DAY 3 DAY 5 DAY 7 DAY 9AQUA-1 AQUA-2 AQUA-3 AQUA-4 Rotate back to AQUA-1

STORAGE: STORAGE: Store below 3 °C. Shelf life is three years.PACK SIZES: 250 ml (provides sufficient product for 5 activations of a 15 ML pond). 500 ml (provides sufficient product for 10 activations of a 15 ML pond). 1 L (provides sufficient product for 20 activations of a 15 ML pond).

Protexin Aqua-2CONCENTRATED VIABLE BACTERIA FOR PREVENTION & TREATMENT OF WHITE SPOT VIRUS & OTHER VIRUSES IN AQUACULTURE AND WATER TREATMENT SYSTEMS.

PRESENTATION: Reddish-brown-coloured bacterial spore liquid suspension.

PROPERTIES: Protexin Aqua-2 is a special blend of Bacillus species that provide a healthy environment for fish, crustaceans (e.g. shrimp), molluscs and other aquatic animals grown in fresh, brackish and marine waters. Protexin Aqua-2 microbes are naturally occurring, mesophilic, gram positive spore-forming rods, aerobic, and grow between pH 5.5 and 9.0. Protexin Aqua-2 combines high salinity tolerance with very strong anti-Vibrio species, and very strong anti-White Spot and anti-Yellow Head Virus properties. To achieve best results, use Protexin Aqua-2 as an integral part of a preventative strategy from the beginning of the crop cycle andshuttle with Protexin Aqua-1, Protexin Aqua-3 and Protexin Aqua-4 throughout the crop cycle. Benefits of rotating Protexin Aqua-1, Protexin Aqua-2, Protexin Aqua-3 and Protexin Aqua-4 in the shuttle program are:

1. breakdown of organic flocs breakdown of organic flocs 2. accelerating breakdown of dissolved protein & fragments

of unused feed accelerating breakdown of dissolved protein & fragments of unused feed

3. zooplankton enhancement zooplankton enhancement 4. ]reduction in White Spot and Yellow Head Viruses, and


5. lessening of sludge build up lessening of sludge build up

6. balancing and stabilising of existing algal blooms balancing and stabilising of existing algal blooms

7. water quality improvement water quality improvement 8. reduces the need for water exchange, provided aeration is

adequate reduces the need for water exchange, provided aeration is adequate

ADMINISTRATION: ADMINISTRATION:Activation: Athor follow Protexin Aqua-Media datasheet to grow10, 13 and 15 ml of Protexin Aqua-2 which is sufficient for Follow Protexin Aqua-Media datasheet to grow the same of Protexin Aqua-2 which is sufficient for a 1 acre x 1.5 m depth).



Pond Water quality control /treatment Program: To be continuedas required. Author use DOUBLE the dose rate of Aqua-1 for anyTREATMENT program.DAY 1 DAY 3 DAY 5 DAY 7 DAY 9AQUA-1 AQUA-2 AQUA-3 AQUA-4 Rotate back to AQUA-1


Protexin Aqua-3CONCENTRATED VIABLE BACTERIA FOR PREVENTION & TREATMENT OF VIBRIO AND OTHER BACTERIAIN AQUACULTURE AND WATER TREATMENT SYSTEMS.

PRESENTATION: PRESENTATION: Light reddish-brown-coloured bacterial spore liquid suspension.PROPERTIES: Protexin Aqua-3 is a special blend of Bacillus species that provide a healthy environment for fish, crustaceans (e.g. shrimp), molluscs and other aquatic animals grown in fresh, brackish and marine waters. Protexin Aqua-3 microbes are naturally occurring, mesophilic, gram positive spore-forming rods, aerobic, and grow between pH 5.5 and 9.0. Protexin Aqua-3 combines high salinity tolerance with very strong anti-Vibrio species properties, using adifferent blend of strains to Protexin Aqua-4. To achieve bestresults, use Protexin Aqua-3 as an integral part of a preventative strategy from the beginning of the crop cycle andshuttle with Protexin Aqua-1, Protexin Aqua-2and Protexin Aqua-4 throughout the crop cycle.

Benefits of rotating Protexin Aqua-1, Protexin Aqua-2, Protexin Aqua-3 and Protexin Aqua-4 in the shuttle program are:



3. zooplankton enhancement zooplankton enhancement 4. reduction in White Spot and Yellow Head Viruses, and




7. water quality improvement water quality improvement 8. reduces the need for water exchange, provided aeration

is adequate reduces the need for water exchange, providedaeration is adequate




Pond Water quality control /treatment Program To be continued as required. Author use DOUBLE the dose rate of Aqua-1 for anyTREATMENT program.DAY 1 DAY 3 DAY 5 DAY 7 DAY 9AQUA-1 AQUA-2 AQUA-3 AQUA-4 Rotate back to AQUA-1


Protexin Aqua-4 CONCENTRATED VIABLE BACTERIA FOR PREVENTION & TREATMENT OF VIBRIO AND OTHERBACTERIA IN AQUACULTURE AND WATER TREATMENT SYSTEMS.

PRESENTATION: Light reddish-brown-coloured bacterial spore liquid suspension.

PROPERTIES: Protexin Aqua-4 is special blend of Bacillus species that provide a healthy environment for fish, crustaceans (e.g. shrimp), molluscs and other aquatic animals grown in fresh, brackish and marine waters. Protexin Aqua-4 microbes are naturally occurring, mesophilic, gram positive spore-forming rods, aerobic, and grow between pH 5.5 and 9.0. Protexin Aqua-4 combines high salinity tolerance with very

strong anti-Vibrio species properties, strong anti-White Spot and anti-Yellow Head Virus properties, using a different blendof strains to Protexin Aqua-3. To achieve best results, use Protexin Aqua-4 as an integral part of a preventative strategyfrom the beginning of the crop cycle and shuttle with ProtexinAqua-1, Protexin Aqua-2 and Protexin Aqua-3 throughout the crop cycle.

Benefits of rotating Protexin Aqua-1, Protexin Aqua-2, Protexin Aqua-3 and Protexin Aqua-4 in the shuttle program are:



3. zooplankton enhancement zooplankton enhancement4. reduction in White Spot and Yellow Head Viruses, and




7. water quality improvement water quality improvement 8. reduces the need for water exchange, provided aeration

is adequate reduces the need for water exchange, providedaeration is adequate




Pond Water quality control /treatment Program To be continued as required. Author use DOUBLE the dose rate of Aqua-1 for anyTREATMENT program.DAY 1 DAY 3 DAY 5 DAY 7 DAY 9AQUA-1 AQUA-2 AQUA-3 AQUA-4 Rotate back to AQUA-1

STORAGE: STORAGE: Store below 3 °C. Shelf life is three years.PACK SIZES: 250 ml (provides sufficient product for 5 activations of a 15 ML pond). 500 ml (provides sufficient product for 10 activations of a 15 ML pond). 1 L (provides sufficient product for 20 activations of a 15 ML pond).Appendix V:

Appendix V : Culture system and water quality controlprogram adopt in this study

(2) MODIFIED EXTENSIVEShrimp Aquaculture Production

(3-7 pieces/m2)

Recommended to be used continuously prior to stocking of ponds in ‘closed’ systems (water exchange not exceeding 20%)

Dosage was calculated based on pond size and stocking density of pond

1) Author calculate inclusion levels for Aqua-Media and Aqua-1using Table 1 for the first weekly application to provide a highly concentrated POND PRIMING dose (first application).2) Author calculate inclusion levels for Aqua-Media and Aqua-1, Aqua-2, Aqua-3 and Aqua-4 using Table 1, then reduce the rate of administration to 30% of these levels (see Table 2). 3) Author activate Aqua-Media using Methodology or follow the pictorial diagram (see page 1 of this instruction ).

WATER QUALITY CONTROL PROGRAM

The first administration of Aqua-1 was took place after pond preparation, one day prior to stocking of shrimp.Begin the WATER QUALITY CONTROL PROGRAM where there is a cpmmon shrimp pond ecological bio-security imbalance in the local area. The frequency of administration was increased to every two days at the POND PRIMING application rate (Table 1).The frequency and inclusion rates may then be gradually reduced back to the PREVENTION PROGRAM rate when the disease risk is reduced.

Day 1 Day 3 Day 5 Day 7 Day 9 Days 11-119 etc.Aqua-1Pond

PrimingDose

Aqua-2 Aqua-3 Aqua-4 Rotateback toAqua-1

Continue rotatingAqua-1, Aqua-2,

Aqua-3 and Aqua-4

Table 1: Standard POND PRIMING and WATER QUALITY CONTROL PROGRAM administration levels depending on pond size for EXTENSIVE shrimp production.

Pondsize

Ponddepth

Pondarea

Aqua-Media

Aqua-1, Aqua-2,Aqua-3 or Aqua-4

(liters) (meters) (hectares)

(grams) (milliliters)

1,000,000

1 0.1 8.7 3.5

2,000,000

1 0.2 17.5 6.5

2,200,000

1.1 0.2 19 7.5

3,000,000

1.5 0.2 26 10

10,000,000

1 1 87 33.5

11,000,000

1.1 1 95.5 37

15,000,000

1.5 1 130 50

Table 2: Administration levels depending on stocking densityfor MODIFIED EXTENSIVE shrimp production per 1,000,000 Litres

StockingDensity(pcs/sqm)

Aqua-Media (per

1,000,000 L)

Aqua-1, Aqua-2, Aqua-3or Aqua-4

(per 1,000,000 L)5-10

pcs/sqm2.6 g 1.0 ml

11-15pcs/sqm

4.3 g 1.7 ml

16-20pcs/sqm

5.2 g 2.0 ml

21-24pcs/sqm

7.0 g 2.7 ml

25 +pcs/sqm

8.7 g 3.3 ml

Appendix VI : Activation of Protexin Aquatech in scientific way

Activation Methodologyactivation of spores

Author use Aqua-Media to activate the spores (see Instruction Leaflet or below) and added to pond once weekly according to the administration schedule (see table below).

1. Author Add calculated amount of AQUA-MEDIA to 2 L of fresh clean drinking water and Stir.

2. Bring solution to the boil (for a few seconds only) then remove from heat.

3. Immediately add calculated amount of spores (AQUA-1, AQUA-2, AQUA-3 or AQUA-4). This will ‘heat shock’ the spores.

4. Immediately add 2 L of fresh clean drinking water at room temperature.

5. Commence activation. Stir vigorously every hour or gently aerate solution.ACTIVATION TIME IS 180 MINUTES FOR AQUA-1 ACTIVATION TIME IS 90 MINUTES FOR AQUA-2, AQUA-3 and AQUA-4.

6. Mix the 4 L with 16 L of fresh clean water.7. Gently aerate for 2 hours.8. Add to the pond near the pond aerators, by boat or with a

sprayer.

Total volume of Protexin Aquatech Packs for this study will depend on: pond capacity, period of time for this study (days of culture), number of ponds.

Procedure:

Ponds in the Protexin Aquatech Treatment groups will receive Protexin at the recommended dosage rate from stocking of pondsuntil harvest.

Ponds in the Control groups was not receive any Protexin.

All data related to crop performance should be recorded and analyzed in term of:Farm Description MeasurementFarm details LocationArea sqm or hectaresDate stocked day/month/yearShrimp feed source ManufacturerDepth of water MetresSalinity %Nursery pond use Yes/No

Stocking details MeasurementTotal stocked Number per sqm.Density Number pcs/sqm.PL stage e.g. PL15Acclimatisation tank use Yes/NoFry Source HatcherySpecies of shrimp name

Water qualities DOC 0 (prior to first application with Aqua-1);DOC 1 (24 hours following first application);DOC 7 (Week 2);DOC 28 (Week 4);DOC 56 (Week 8);DOC 112 (Week 16);DOC harvest (at harvest)

Measurement

Temperature oCpH pH scaleNH3 mg/L or

ppmTransparency (estimated) cmColour (estimated description) Green/

brownDO mg/L

Harvest results MeasurementDays of culture DaysSurvival rate %Harvest Kg/haSize at harvest gFCR Ratio e.g. 1.3

The performance results from the Control ponds should then be compared to the Protexin Aquatech treated ponds.

Parameters of observation:

General ObservationsGeneral appearance of pondsGeneral appearance of shrimpMorbidityDisease status (WS or Vibrio status) / ShrimpHealth condition

The morbidity and mortality recording was substantiated by adequate post-mortem pathology and bacteriology reports.

All other treatments used during the final period was recordedas per the type and frequency of medication.

Economics

Economics MeasurementTotal Feed Consumed tonnes per cropSludge level depth, description, colour

All feed consumed was recorded through the trial period from day of stocking until day of harvest. The approximate weight of the shrimp was recorded as well. This data was subsequently used to calculate the feed conversion efficiencies.

Cost per hectare

Benefit/hectare: Extra weightLower mortality-------------------Net benefit / hectare

Ponds: Total number ponds was four with 4 replication of each treatment group (i.e. Control and Protexin Treated group), There is also a reservoir pond:

Capacity of ponds (pond area 4000 m2; pond depth m):Pond preparation: e.g. Drainage (number of days); Sludge

removal (number of days); Liming; Plankton culture (addition

of fertilizer).

Appendix VII: Pond preparation details in gher area

Pond Preparation Details :

1. Site Selection : The selection of a suitable site alwaysplays a major role in shrimp farming. After analysis ofinformation topography,ecosystem, meterological; andsopciaeconomical condition author select the four pondsfrom Shamnagar, Shatkhira. The criteria are given below:

1.1 Water quality: Physiochemical and microbiologicalcharacteristics of water are considered. In that regionPH of the water vary from 7.5 to 8.5. Dissolve oxygenusually saturated through out the water column. ButOxygen level is preferably should not be lower than 4ppm. Salinity variation is one of the important factorfor tiger shrimp production. The tiger shrimp grow faster

at 15-30 ppt. So, salinity should remain uniform atnormal weather and should not drop abruptly during rainydays.Water not to be too turbid.The water is preferableto be rich in beneficial microorganisms.

1.2 Tidal fluctuations: The tidal characteristics of theproposed site should be known knowledge, of thisparameter is of extreme importance in determining pondbottom elevation of dike, slope ration and drainagesystem.

Author choose an areas best suited from shrimp farmingwhich have moderate tidal fluctuations preferably 2-3meters. In areas where tidal range is greater than 2-3meters, the site may prove uneconomical to develop oroperate a farm, because high pond dikes will berequired. In those areas where tidal range is less thanone meter, water management will be expensive requiringthe use of pump.

1.3 Soil: Author choose the site where soil was enoughclay content. This is to ensure that the pondsconstructed will hold water. Good quality dykes areusually built from sandy clay or sandy loam materials,which harden and easily compacted.

1.4 Topography: Coastal sites where the slopes rungently towards the sea are easier for pond developmentrequiring less financial inputs since excavations isminimal.

1.5 Source of Seed : Author pay close proximity of thesite to the fry ground(Wild or hatchery) is advantageousin that the post larvae being collected for stocking arenot subject to too much transport and handling stress.

1.6 Accessibility: Author took accessibility as animportant consideration in site selection. Overhead costand delay in the transport of materials and products canbe minimized.

1.7 Other Factors: Other consideration that was takingunder custody is

a) Good electricity connections and supplyb) Good availability of laborc) Reasonable road and transportation to the sited) Reasonable communicatione) Availability of formulated and natural feed

f) Availability of equipment supply and maintenance

3. Design and construction of P. monodon Culture Pond: P.monodon is a benthic animal and it has a habit ofgathering along the wall of pond or tank. An Ideal shrimpfarm is a complex, which consisting of following parts:

a) Various size of pond for nursery and grow outb) Water control structure including embankments,

supply and drainage canals and sluice gates.

3.1 Layout of Culture pond: As rectangular ponds areappropriate for tiger shrimp culture, researcher tookrectangular pond. The longest axis of all treatment pondwere parallel to the prevailing wind direction, whichfacilitates water movement generated by wind action. Sodissolve oxygen was increased that minimized the watertemperature fluctuations in summer months.

3.2 Dykes: Dykes were prepared which not only serves asboundaries to indicate pond size and shape but alsofunction to hold water within the pond as well asprotecting other farm facilities from flood.Dykingmaterial was tested for load bearing capabilities.Design and construction of embankment must be based onsound engineering principals and economic feasibility.

To compute for the height of the dyke, following formulacould be used:

H = (HW - G) + FB/1 - % of Shrinkage

Where,H = height of designed dyke.HW = highest height water level from past record.G = ground level over mean sea levelFB = height of free board

3.3 Water Control Gate (Sluice Type): Water control gates orsluice gate was designed researcher consider tidalfluctuations and gravity in order to ensure effectivecontrol of the inflow and outflow of water within ashortest period.

The size of the gate was based on the total waterrequirement of a pond. Water intake vlume is calculatedusing the equation:

Q = CA[2g(H-h]1/2

Where,

Q = rate of flow (m/sec)C = cross section of flux(calculated by multiplying thewidth of gate opening and its depth)A = coefficient of discharge (0.61).g = gravitational constant(9.8/sec2) H = tidal level of the river or seaW = water level in the canal or pond

3.4 Supply and Drainage Canal: It is necessary to construcsupply and drainage canals for supply and drainage ofwater for shrimp pond. Author made the pond withseparate canals for drainage and supply of water.Dimension of supply and drainage canals are calculatedby using the following wquation:

Q = AV

Where, Q = volume of water discharge A = Cross section area of the canalV = velocity of water flow.V can be calculated by the following formula:

V = R2/3XS1/2 X 1/n

Where, R = depth of the water flow S = Canal bed gradientn = coefficient of roughness(0.02)

4. Culture Operations and Management:

Culture operation of tiger shrimp in Gher 9srtificial ponds)can be divided into fllowing steps:

4.1 Pond preparation: As the assigned pond is earthenculture system, bottom soil plays a major role in pondyield. Unpolluted organic matter content in neutral soiloften promotes higher primary productivity and hencehigher fish yield. So, pond preparation is a first steptowards ensuring a better pond production.

4.2 Soil sampling4.3 Prior to pond preparation, soil samples was

collected from pond bottom for PH and organic mattercontents analysis. Soil PH analysis was generallyconducted to determine lime requirement.

4.4 Leaching: The assigned pond was found to be acidic,as it was normally leached. This was done by flushing andwashing the pond bottom with water to leach awayundesirable metallic compounds like aluminum, iron andexcess sulphur ions.

4.5 Drying: After flushing out the pond, all inlet and outlet structures were sealed and the pond was allowed todry for 10 days, until the pond bottom surface can support the weight of a person without setting and the soul surface has opened in deep cracks. This drying facilitates:

a) Aerating of surface sediments, oxidizing reduces compounds such as hydrozen sulphide, nitrite,ammonia, ferrous ion, methane etc, which aretoxic to shrimp.

b) Decomposition and mineralization of organic mattersc) Disinfection of the pond bottom.d) Ploughing/tilling of the pond bottom to expose

underlying sediment layers that may be anaerobic andreduced

e) Elimination of undesirable mats of filamentous algae.

f) Elimination of fish eggs, crab larvae and potential predators.

4.5 Tilling: Researcher brought assigned pond for tilling orploughing of bottom soil quality by exposing subsoil to a depth of 5 –15 cm suing a plow, thereby speeding up the oxidation process and the realease of nutrients that are locked in the soil. Double tilling ensure through disintegration of soiul agglomerates.

4.6 Liming: At the rate of 15 kg / pond of Liming was applied as calcium and magnesium compounds to the soil for the purpose of reducing soil acidity. Just after drying it was conducted.

This liming favours as follows:a) Kills most microorganisms especially

parasites due to its caustic reations.b) It will raise pH of water when it

became acidic to neutral or sightly alkaline value.

c) It increase the alkaline reserve in waterand mud, which prevents extreme change inpH.

d) It neutralizes the harmnful action of certain substance like sulphides and acids.

e) Promotes biological productivity since itenhance the breakdown or organic substance by bacteria creating a more favorable oxygen and carbon reserve.

f) Precipitates suspended or solube organic materials, decreases biological oxygen demand(BOD), increase light penetration, enhance nitrification due to the requirement of calcium by nitrifying organism.

g) Indirectly improves fine fine texture bottom soil in the presence of organic matter.

7. Water filing: The assigned pond was filled upto30 cm with 25 ppt salinity and drain after 3days. The refilling of water upto 30 cm ispratice, recommended specially for moderateextensive culture system.The cycle of dry-plow-fill –drain treatment will improveoxidation state of the pond bottom.

8. Application of Inorganic Fertilizers: It wasadopt for the practice of incorporation urea ofsome other nitrogen source into the soil inorder to accelerate organic matter

decomposition. A dose of 5- 100 kg urea/ha maybe used before refilling the pond to 50 cm.Subsequently cowdung and TSC was mixedaccordingly.

9. Refilling the water: Addition of 10 cm waterfor everyday until the pond water is 100-120 cmdeep. Urea and NPK fertilizer was added adrequired till the color of water is brown withyellowish hue.

10. Selection of Shrimp Fry: The mostimportant criteria for selection were the stage of development of the fry.Identification of anatomical parts of the frywas require a microscope, though general farmerare not able to do well to carry a small eyepiece/magnifying lens to the hatchery forobservation of the fry before purchase, Authortook PL20 animals.

11. Transportation of Shrimp Fry: As frycollection center were

located far away from ponds and fry have to betransported to pond area, for assigned project 2,000-2,500 postlarvae in plastic bags which are oxygenated, sealed and packed incardboard cartons lined with thermocol.

12. Acclimatization and stocking of fry:Author stock the fry on early

in the morning. The collection andtransportation of seed from the hatchery,coordinated to match the stocking time. Thestocking density of fry P. monodon in differentculture treatment trial pond is 7 PL/sqm and incontrol 3 PL/sqm as per traditional system.Acclimatization of seed is as follows

a. Researcher empty the bag in a plasticcontainer on basin

b. Researcher observed the fry for level oractivity and mortality

c. Researcher lowered the basin till it floatsin the water. The water of the container wasnot mix with the pond water.

d. He hold the basin with one hand, and addwater of the pond into the container slowly.After sometime tilt the basin as the fryslowly swim out into the pond water

13. Culture technique: Researcher culture theTiger shrimp as Extensive ( stocking density 1-5/sqm) and modified Extensive (6-15 /sqm). Forcontrol treatment pond he stocked as 3 PL/sqmand for treatment trial pond he stocked 7Pl/sqm.

14. Water quality management: In any shrimpfarming , management of water quality is ofprimary consideration. Particularly in pondswith modified stocking density. As degradationof water quality is detrimental to shrimpgrowth and survival researcher concentrate hisresearch on effect of Probiotics in pondwater quality management.

a. Salinity: As it is natural issueresearcher do his label best tocontrol it. The post larvae of penaedshrimp can tolerate wide salinityfluctuation, which has little effecton their survival or growth. P.monodon can tolerate wide range ofsalinity from as low as 5 ppt to ahigh 40 ppt. Due to evaporation ratein summer season researcher faceproblem as salt concentration in pondgradually increases. Researcherchanged water frequently by pump andtidal exchange.

b. Temperature: Water temperature playsa very important role in regulatingthe activities of cultureanimal.Though the optimum temperaturefor P. monodon is 25-300C, researchertook that fluctuation must be their

for environmental nature.He wasprepared to maintain temperaturethrough water exchange. Researcherregularly took the temperature bythermometer.

c. Dissolve Oxygen: Researcher alsokeep the record of dissolve oxygen bydissolve oxygen meter. As prolongedexposure to the stress of lowconcentration of oxygen lowers theirresistance to disease and inhibitstheir growth, often resulting in massmortality. Dissolve oxygen in pondcomes from two sources by product ofphptsynthesis and from diffusion ofatmosphee. At night both plants andanimals continue to respire whileoxygen is being added to the waterfrom the atmosphere, cause totaldepletion of DO(specially at dawn).Researcher took as usual measures tocontrol normal DO by water exchangethrough renewal of pond water withfresh water by tidal flow andpumping.Besides this he installaeration system paddle wheeler.

d. pH: As the PH of the pond water isindicative of its fertility orpotential productivity, researcheruse PH meter for recording the samein whole culture period. Water withPH ranging from 7.5 to 9.0 isgenerally regarded suitable forshrimp production. The growth ofshrimp is retarded if PH falls below5.0 which can be corrected by addinglime. Water of excessive alkalinity(PH

value 9.5) may also be harmful. PH ofpond water usually exceeds 9.5 duringlate afternoon that can be correctedby water exchange.

e. Nitrogen compound: Nitrogen compondin pond exists in different formssuch as nitrate, nitrite, ammonia andvarious forms organic nitrogen. Inpond culture activities ammonianitrogen(in the form of un-ionizedammonia) is considered importantsince this compound is toxic toaquatic animals at certainconcentration.

f. H2S : Hydrogen sulphide can severelyaffect shrimp growth, which isproduced by chemical reduction oforganic matter that is produced bychemical reduction of organic matterthat accumulates and forms a thicklayer of organic deposits at thebottom. H2S meter is used to recorddata.

g. Feed and feeding management: As it ismoderate extensive shrimp cultureadequate food supply was ensured.Though traditional extensive cultureoperation researcher follow (intreatment 4/control pond) traditionalnatural feeding in which growth ofshrimp was fully depends on naturalfood. Feeding was carried out bybroadcasting over the pond.

h. Feeding method: Researcher maintainoptimal feeding rate and frequencythrough visual observation of leftover feed that was employed byfeeding tray. Periodic determinationof the stock density for appropriatefeed ration was carried out byresearcher, which is done by castnet. Feed ration was based on assumeddensity, which method of computationfeed ration, is based on theestimated survival rate.

i. Harvesting: The harvestion was doneby drainage. Around 80-90 % of thestock is removed by this method andrest is collected by hand picking.The harvested shrimp was washed andplaced immediately in chilledwater(5-150C) for about 15 minutes..They are then packed in Styrofoamboxes with alternative layers ofcrushed ice at a ration of1:1.Smaller Styrofoam box was used totransfer to lab.

Reservoir Preparation :

Documents

Appendix Final