Hatchery Management of

GROUPER

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Kingdom: AnimaliaPhylum: ChordataClass: ActinopterygiiOrder: Perciformes Family: Serranidae

Overview Most popular species in the reef food fish industry in Asia-

Pacific region Fast growing, hardy ,suitable for intensive culture Excellent characteristics for processing due to unique

culinary attributes and scarcity In 1997, the Asia-Pacific Region contributed about 90% to

the world Aquaculture prdxn w/ Grouper at 15000 ton w/ China as the biggest producer contributing 8000 tons followed closely by Indonesia. Other countries in the region commonly produces 1000-2000tons annually in 1990-1997.

Although grouper culture is widespread in Asia and Pacific, its continued dvlp. is constrained by the limited availability of fingerlings. Most economies w/recent exception of Chinese Taipei, rely almost totally on wild-caught fry and fingerlings for stocking.

The word "grouper" comes from the word for the fish, most widely believed to be from the Portuguese name, garoupa. The origin of this name in Portuguese is believed to be from an indigenous South American language.

In Australia, the name "groper" is used instead of "grouper" for several species, such as the Queensland grouper (Epinephelus lanceolatus).

In the Philippines, it is named lapu-lapu in Luzon, while in the Visayas and Mindanao it goes by the name pugapo.

In New Zealand, "groper" refers to a type of wreckfish, Polyprion oxygeneios, which goes by the Māori name hāpuku.

In the Middle East, the fish is known as hammour, and is widely eaten, especially in the Persian Gulf region.

Groupers are teleosts, typically having a stout body and a large mouth. They are not built for long-distance, fast swimming. They can be quite large, and lengths over a meter and weights up to 100 kg

They habitually eat fish, octopuses, and crustaceans. Some species prefer to ambush their prey, while other species are active predators.

Their mouths and gills form a powerful sucking system that sucks their prey in from a distance. They also use their mouths to dig into sand to form their shelters under big rocks, jetting it out through their gills. Their gill muscles are so powerful, it is nearly impossible to pull them out of a cave if they feel attacked and extend those muscles to lock themselves in.

REPRODUCTIVE BIOLOGY

Groupers are mostly Protogynous Hermaphrodites, they mature only as females and have the ability to change sex after sexual maturity

Some species of groupers grow about a kilogram per year and are generally adolescent until they reach three kilograms, when they become female.

The largest males often control harems containing three to 15 females.

Groupers often pair spawn, which enables large males to competitively exclude smaller males from reproducing.

However, some groupers are gonochoristic.

Gonochorism, or a reproductive strategy with two distinct sexes, has evolved independently in groupers at least five times. The evolution of gonochorism is linked to group spawning high amounts of habitat cover.

Both group spawning and habitat cover increase the likelihood of a smaller male to reproduce in the presence of large males.

Gonochoristic groupers have larger testes than protogynous groupers (10% of body mass compared to 1% of body mass), indicating the evolution of gonochorism increased male grouper fitness in environments where large males were unable to competitively exclude small males from reproducing

PROXIMAL CAUSES OF SEX CHANGE IN PROTOGYNOUS HERMAPHRODITE

CAUSES

Removal of males from the population leads to sex change in the dominant(largest) females.

The sex ratio would depend upon the current spawning sex ratio within the aggregation, the proportion of adults surviving to spawn in subsequent years, and the proportion of inshore juveniles surviving to mature and enter successive aggregations.

Some grouper population, males are more or less absent ‘till age 10

In case of Red grouper( Epinephalus morio), the sex ratio is not equal until age 15.

Some Grouper can live 20 years or more!

Fishing

Huntsman and Schaaf (1994) point out that both protogynous and gonochoristic (separate sexes) population tends to lose reproductive capacity as fishing pressure increases.

In protogynous stocks, however, increased fishing mortality might reduce the relative abudance of males by reducing the abundance of older-classes and diminish the probability that eggs will be successfully fertilized

Loss of behavioural interactions between male and female

Combined with continued interaction between females, (Koenig et al. 1999) suggested that the loss of male-female interaction in Gag grouper (Mycteroperca microlepis) occur at the endof aggregation period, when females purportedly return to swallower inshore waters.

To date:Only specie of grouper investigatedRed hind(Epinephalus guttatus) and coney

(Epinephalus fulva) have been histologically proven to be protogynous hermaphrodites but;

Collin(1992) groupers that aggregate are not necessarily protogynous.

Ex: Nassau grouper(Epinephalus stiatus) and Yellowfin grouper( Mycteroperca venenosa), neither of them shown to be protogynous but both known to aggregiate.

Nassau grouper (Epinephalus stiatus) spawns in very large aggregations( thousands to ten thousands of fish) for only a few weeks of the year.

In contrast, male and female Gag grouper (Mycteroperca microlepis) and Red hind (Epinephalus guttatus) co-occur only during spawning season.

GROUPER SEED IS A COLLECTIVE TERM THAT INCLUDES:

POSTLARVAE Transparent, brownish or reddish unscaled

fish, 0.1-2.5 cm(< an inch) in size nd somtimes called “Tinies”. They are collectively referred to as seed or fry.

Metamorphosed Fry scaled fish, darker than postlarvae, measure 2.5-

7.5cm (1-3 inch) w/ single continuous dorsal fin, elongated second dorsal spine and pelvic spines.

FingerlingsUsually 7.5-12.5 cm (3-5 inch) color and appearance

starting to follow the market sized stage. Size is often measured from tip of the mouth to the end of caudal fin.

ARE GROUPERS AVAILABLE ANY TIME OF THE YEAR?COLLECTION AREA

MONTH GROUPER SIZE

Peninsula Malaysia, east cost

November to December

Average 2cm

Indonesia(e.g., Banten Bay)

February to April Fry

Thailand, coastal waters near mangrove areas

January to March 7.5-10 cm fingerlings

Philippines, variable peak season in diff. Province

Peaks mostly in June to December or October to November and April to May

Tinies, fry and fingerlings

COMMONLY CULTURED SPECIES

Brown Marbled Grouper, carpet cod, flowery cod, blotchy rock cod

Epinephelus fuscoguttatus (Forsskal)

Lapu-lapu

Orange spotted grouperEpinephelus coloidesLAPU-LAPU

Dusky tail grouper Epinephelus bleekeri (Vaillant) Lapu-lapu

HATCHERY DESIGN

HATCHERY DESIGN Key

component: Regardless of scale is the implementation of biosecurity to reduce the incidence of disease, (VNN)viral nervous necrosis.

ENTRANCE OF HATCHERY FACILITY

VNN (VIRAL NERVOUS NECROSIS)

A common disease problem in marine finfish hatcheries affecting most cultured marine finfish species, including grouper .

Caused by a nodavirus also known as viral encephalopathy and retinopathy.

Symptoms are disorientation of fish ,change in color, becoming more dark.

Can be reduced by strict biosecurity. .

KEY FEATURES FOR BIOSECURITY

Separation of various functional areas; Access to hatchery limited only to

essential personnel; Disinfection and thorough cleaning of

all equipments; Routine monitoring for pathogens and

disease; Optimisation of water quality and

nutrition to improve overall health and resistance of larvae.

BROODSTOCK CONCRETE TANK

EGG INCUBATION TANKS

LARVAL REARING TANKS

HATCHERY OPERATION

BROODSTOCK ACQUISITION

1. Collecting and purchasing of wild fish in a wide range of sizes.(Epinephelinae, protogynous hermaphrodites).

In Philippines size of mature female(2.2 kg),and male (3.5 kg).

2. Grow fish produced in the hatchery (in cage, pond, or tanks) however, takes 4years to grow juvenile Tiger Grouper up to broodstock size .

CRITERIA FOR BROODSTOCK SELECTION

Normal body shape and colour Absence of skeletal deformities Overall healthy status i.e., absence of large

wounds haemorrhages, infections, and parasites

Normal behavior, i.e., good response to food distribution

Best growth and food conversion rate within this age group.

TRANSPORT REQUIREMENTS FOR BROODFISH

Transported in dark-coloured, covered tanks containing aerated or oxygenated water, to reduce stress.

Dissolved maintained at >75% saturation at all times.

Mild sedation(using approved sedatives for fish) to reduce stress and for easier and safer handling.

Fish should not be fed at least 24 hours before hand to prevent faeces and regurgitated feed from fouling transport water.

TREATMENT BEFORE STOCKING Quarantine the fish To reduce the opportunity for new fish to transmit

parasites or diseases to the established fish. Quarantine process-takes between 1and 4 weeks in small tanks(0.5-2 cubic meter)to facilitate water exchange and fish handling.

This period focuses on reducing parasite load of fish regularly by placing them in freshwater bath for 5 min. to eliminate common parasites (skin flukes, protozoans etc.)

If water quality in broodstock tanks markedly different form previous holding environment, fish acclimation by placing them in a tank filled with original water, and slowly add new tank water for up to 1 hr. before releasing them into the tank.

BROODSTOCK TANKS

Tank size. When fish is(>10kg.) size ranges bet.50-100cubic meters. Preferred tank color. Medium-range blue, brown or grey not very light or

dark-shades.

Depth. 2m preferably >2.5 to allow sufficient room for spawning behavior.

Broodstock tanks are roofed in order to reduce the growth of algae on tank walls making egg collection difficult and increasing egg parasite infestation.

Dirty tanks must be cleaned frequently w/c may stress broodstock and cause spawning failure or lower quality spawned eggs.

Continously supplied with fresh seawater at daily exchange rate of 200-300%. Seawater should be filtered and clear with stable salinity (33-35ppt) and water temperature (27-30.5 degrees celsius).

Tanks located outside are subject to the natural photoperiod, while indoor may be provided with artificial lightning to simulate different photoperiod regimes.

TANK CLEANING

Siphoning of excessive feed that accumulate on bottom to prevent water quality degradation.

After spawning remove excess dead eggs w/c decay and pollute the water .

Broodstock should be bathed in freshwater for 5-7 min. During tank cleaning to reduce incidence of parasite infestation.

BROODSTOCK MANAGEMENT

Feeding

feed to satiation (6x a week),w/fish (4x a week) ,w/ squid(2x a week)

Feeding may vary, depending on availability of fish and squid.{herrings and mackerels are commonly used as feed}

Feed is supplemented with a vitamin mix included at 1% of feed. Commercial or custom-formulated vitamin mixes can be used.

COMPONENTS OF FEED MIX

Ingredients Amount

Minced fish, squid, shrimp, etc.

793g

Rice flour or other finely ground starch product

195g

Transglutaminase B 10g

Vitamin mix 1-2g(depending on recommended inclusion rate)

Total 1kg

MAKING TRANSGLUTAMINASE-BASED WET FEED

GENDER IDENTIFICATION

Grouper broodstock held in tanks at low densities usually ,1kg/cubic meters.

Sex ratio: male to female ratios of 1:5 but can vary.

However ,groupers are protogynous, to mediate, the presence of male fish may repress sex change by females.

TO IDENTIFY GENDER:

Physical examination; abdomen of anaesthetised fish is massage in

a head-to-tail direction Male- will extrude copious milt from

urogenital pore;Female- eggs are ovulated and can be determined by egg cannulation.

PROPAGATION

NATURAL SPAWNING

o Occurs at night 9pm-3am for 3-6 nights each month during the new moon phase. However, studies shown that they spawn throughout the year.

o 0.8-6.0 M eggs each night.

o cease to spawn when temperature drops around 25 degrees celsius.

IMPORTANT ENVIRONMENTAL PARAMETERSph 7.5-8.3

temperature 25-32 degrees celsius

salinity 20-32 ppt

Dissolved oxygen 4-8 ppm

NO2-N(Nitrogen Nitrite) 0-0.05 ppm

Unionized ammonia < 0.02 ppm

Australian Center for International Agricultural Research ,2012

INDUCED SPAWNING

Prior to Induction: after acquisition of broodfish is stocked at concrete tanks at 6-12 months;

Stocking density:1/2 cubic meters; Feed: fed daily with fresh trashfish; Feeding rate: 5% body weight; Tank water:50-70% is changed daily. And condition simulating the natural

environment.

INDUCED SPAWNING CON….

o Species selected for induction: female-having mean egg diameter of 400ug. male- with running milt.o Ratio: female to male is 10:4o Weight range: female(3.6-6.5 kg),male(10-16

kg). o Hormones used: Human Chorionic

Gonadotropin(HCG), Pituitary Gland(PG),Luteinizing Releasing Hormone –a (LRH-a).

o Hormone administration type: Multiple dosages.

INDUCTION PROCEDURE

First injection, at dosages of 500 IU HCG + 3mg of PG per kg of fish, and at final injection at 1000 IU HCG + 3mg of PG per kg of fish at an interval of 24 hours, results showed that treated fish spawned naturally in spawning tank 12 hours after final injection.

o However, at lower dosages of 500 IU HCG + 3mg of PG at 24-hour intervals or using 10 mg LRH-a at 12-hour intervals, eggs can be artificially fertilized.

In cases where ovulation did not occur after the second injection,a third injection was given.

FERTILIZATION

After injection the fertilized egg that naturally spawned in tanks were collected with a fine dip net(100 micron mesh size). Planktonic organisms and detritus were removed by screening, unfertilized eggs settled down on bottom of tanks were removed by siphoning.

HATCHING

oThe eggs were placed in hatching containers and hatched out in about 15-20 hours.

TABLE 1.TYPE OF HORMONES, DOSAGES AND TIME INTERVALS USED FOR INDUCED SPAWNING

Treatment Hormones used

Dosage Time interval(hr)

A HCG + pituitary gland of Chinese carp

500 IU + 3 mg PG / kg

12

B HCG + pituitary gland of Chinese carp

500 IU + 3 mg PG / kg

24

C LRH-a 10ug/kg 12

D HCG + pituitary gland of Chinese carp

(1st) 500 IU + 3mg /kg (2nd) 1000 IU + 3mg PG/kg

24

TABLE 2. INDUCED SPAWNING OF GROUPER USING DIFFERENT HORMONES

Treatment

Fish number

Body weight(kg)

Time interval (hr)

Number of injections

Hormone used

Remarks

1 4.5 12 3 Partial ovulation, 12 hr after final injection. No fertilization.

2 3.6 12 3 Partial ovulation. No fertilization.

A 3 5.2 12 3 HCG500 IU+3 mg PG/kg of fish

Partial ovulation, 12 hr after final injection; fertilization rate 40%; hatching rate 20%. Larvae died after 6 days.

Treatment

Fish number

Body weight(kg)

Time interval (hr)

Number of injections

Hormone used

Remarks

4 4.8 12 2 Partial ovulation, 15 hr after final injection. Fertilization rate 30% but no hatching.

5 4.2 24 2 Ovulation 12 hr after final injection. Fertilization rate 60%; hatching rate 30%; larvae healthy.

Treatment

Fish number

Body weight(kg)

Time interval (hr)

Number of injections

Hormone used

Remarks

B 6 6.0 24 2 HCG 500 IU + 3 mg of fish

Partial ovulation. No fertilization.

7 5.8 24 2 Ovulation, 15 hr after final injection. Fertilization rate 30%; hatching rate 60%;larvae died after 10days .

C 8 4.1 12 3 Partial ovulation,15 hr after final injection. Fertilization rate 40%; hatching rate 50%; larvae healthy.

9 4.5 12 3 LRH-a 10u/kg of fish

Ovulation 12 hr after final injection. Fertilization rate 80%; hatching rate 40%;larvae healthy.

10 5.2 24 2 1st injection- HCG 500 IU + 3mg /kg of fish . 2nd injection – HCG 1000 IU + 3mg PG/kg of fish.

All fish spawned naturally 12 hr after final injection. Fertilization rate 30%; hatching rate 70%, larvae very healthy.

D 11 5.5 24 2

12 6.6 24 2

RESULTS FOR INDUCED SPAWNING

The fish under treatment D (Table 1),spawned naturally in tank 12 hours after the final injection(Table 2). Fish under treatment B (Table1) ovulated 12-15 hours after the final injection, but fertilization was completed only by artificial stripping.

Fish in treatments A and C required a third injection for ovulation, eggs were artificially fertilized 9-15 hours after the final injection.

Ovulation rate of the females treated with hormonal injections at 24-hour intervals was higher than those at 12-hour intervals(Table 2),this is due to handling that cause fish stress.

LARVAL AND FRY REARING

(Epinephelus salmoides) The larvae from hatching containers were collected

and stocked in nine 250-1 fiberglass larval rearing tanks at a stocking rate of 2500 larvae per tank.

3 types of feed were tested in 3 replicates on a completely randomized design.

o Type of feed fed to fry: 1st type- sea urchin eggs and Isochrysis; 2nd type-Isochrysis and Brachionus; 3rd type- Tetraselmis and Brachionus The larvae were reared indoor under intensive care

conditions. Beginning on day 3 feed was introduced. 20-30% of water was changed daily.

RESULTS OF LARVAL AND FRY REARING

Showed that sea urchin eggs are suitable feed for grouper larvae, diameter of sea urchin egg was about 50ug.

Survival rate of fry fed with:• sea urchin eggs and Isochrysis from

hatching to 20 days old was 9%;• 2% for those fed with Isochrysis and

Brachionus;• none of the fry fed with Tetraselmis and

Brachionus survived; all the larvae died after a culture period of 6 days.

MORPHOLOGICAL DEVELOPMENT OF LARVAE

Hatched larvae measure 1.4-1.7 mm TL;

Mouth opens 2-3 days after hatching; yolk is completely absorbed;

By 10-30 DAH, elongated dorsal and pelvic spines;

Metamorphoses to juvenile at about 40-45 DAH.

FERTILIZATION RATE

Should be done several hours after fertilization of taken place as well as before hatching so that embryonic development makes fertilized eggs easier to discriminate.

Fertilized preferably at 27-29 degrees Celsius.

HATCHING RATE

Dead and live eggs and hatched larvae are distributed differently in the tank, to calculate hatching rates mix eggs and larvae in the tank by swirling through disinfected beakers or small containers.

Precautionary measure should be taken not to damage the hatched larvae.

Hatching rates done on separate sample of larvae not those intended for larval rearing.

Time of hatching depends on temperature. temp. the longer to hatch

QUANTITATIVE ESTIMATION OF FERTILIZATION AND HATCHING RATES

50-80% – preferable fertilization and hatching rates hence, better quality

< 30% -poor rates hence, poor quality exhibits low survival and high incidence of deformities and health problems.

CALCULATION OF FERTILIZATION RATES

NUf + NF = (NT) total # of eggs in the sample

Where: NUf – Number of unfertilized eggs NF – Number of fertilized eggs

The fertilization rate % is NF / NT X 100.

CALCULATION OF HATCHING RATES

Nuh + NH = (NT) total # of eggs in the sample

The hatching rate % is NH / NT X 1OO

QUALITATIVE ESTIMATION OF FERTILIZATION AND HATCHING RATES

Fertilized eggs are examined under a microscope

(10× or 20× magnification is sufficient) for the following:

eggs should be regular in shape during the early stages of embryonic

development, the individual cells should be regular in size eggs and embryos should be completely

transparent, with no dark areas chorions (eggshells) should be free of any

parasites or fouling organisms.

FINGERLING PRODUCTION

At 45 DAH-larvae have metamorphosed into juveniles ranging from 2.0-2.8cm.

5-40%-survival rates at 45 DAH. 15-25%-survival rates at density of

10larvae/L. 20,000 fingerlings-expected harvest at

density of 10 larvae/L, at 10 cubic meter tank.

batch basis –in hatchery mgt. that is each batch of larvae is treated as separate production cycle, and hatchery is shut down between each production cycle.

PROPER HANDLING PRACTICES IN HATCHERY OF GROUPER

EGG HANDLING PROCEDURES

Collection Disinfection Incubation

Characteristics of Fertilized eggs

Non-adhesive Pelagic Ranges from 0.8-0.9mm diameter

COLLECTION

Fertilized eggs are collected from the overflow of egg collector tank using fine net.

Removal from collection net once the embryo has developed optic vesicles.(i.e., at the eyed stage. However, handling eggs before this stage increases mortality and deformity.

DISINFECTION

Treated with ozone to minimize the chances of vertical transmission of VNN (Viral Nervous Nacrosis) from parent to offspring.

Ozone at concentration of 1 mg/L for 1 min. Equivalent 0.8mg/L for 1.25 minutes.

Precautionary measure should be taken when treating Ozone.

INCUBATION

After treatment, eggs are rinsed with clean and disinfected seawater.

Eggs are transferred into 0.5-1.0 cubic meters with aerated seawater.

LARVAL REARING PRACTICES AND REQUIREMENTS Larval Tanks-volume of 10cubic meter, depth of 1.2m,

color of bright yellow or pale blue to allow grouper larvae to discriminate prey more easily and tank cleaning easier, roofed, and enclosed to maintain water temp., and facilitate biosecurity. Quarantined area with entry and exit of authorized person only, disinfection of all equipments.

Aeration-provided in a grid pattern to ensure even water mixture, maintained DO levels, placed in corner to prevent stagnation, should be in the light in the early stage to prevent physical damage and could be increased if the larvae become robust.

Water-should be filtered [filtered through ultraviolet or ozone disinfection],help maintain biosecurity.

LARVAL REARING PRACTICES AND REQUIREMENTS

Sea water used must be per-treated using sand filter to remove particulates, and then sterilized to reduce potential pathogen intro.

in water. 10 larvae/L-recommended initial stocking

density for grouper. Oil-added to form thin film on water

surface(around 0.2/m L-meter square)at 1-5 DAH to prevent surface aggregation mortality in early stage.

PROBLEMS IN LARVAL REARING

Surface aggregation mortality. Larvae are attracted to patches of sunlight in

the tank hence, where that patch is present they may swim to surface of tank. Results in becoming stock in water and entangled in each other spines.

Larval mortality at first feeding.

LIVE FOODS

NUTRITIONAL ENHANCEMENT Larvae of grouper require high levels of highly

unsaturated fatty acids, EPA, ARA, DHA. Provision via incorporation in live foods.

Recommended for larvae: Artemia Brachionus Isochrysis Tetraselmis

NOMU KAMSAHAMNIDA!!

DOMO ARIGATO

GOZAIMASU!!