SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.

TECH BIOTECHNOLOGY

UNIT II: Aquaculture and Fish genetics (12 hrs)

Aquaculture: Definition- Criteria of selection of aquaculture species. Culture practices of

marine fish, shrimp, crab, lobster, edible oyster, pearl oyster and seaweeds. Fish genetics:
Gynogenesis, androgensis, polyploidy, artificial insemination, eye stalk ablation-
cryopreservation.

AQUACULTURE

CRITERIA OF SELECTION OF CANDIDATE SPECIES FOR AQUACULTURE.

The choice of culture species is, in more ways than one, closely linked with the objectives of the
development and therefore the strategy/approach to be used to achieve set goals. Not all fish
species are suitable for aquaculture. By the same token, some cultivable species are more
appropriate for large-scale, commercial aquaculture rather than for small-scale operations, as
exemplified by the high-value shrimps, the production of which can hardly be undertaken
profitably on a small scale. Also, some species are best cultured using specific types of
enclosures; for example, penaeid shrimps are best cultured in fish ponds rather than in fish pens,
and certain species are more acceptable in certain countries than in others.

The choice of species for culture depends on a number of factors including the availability of
suitable sites for culture, the biological characteristics of the indigenous or introduced/exotic
species, their suitability for culture, and their acceptability in the local or international markets,
and the availability of technology and other requirements for their culture.

Principal aquaculture species in Asia

Common Name Scientific Name Culture System* Environment**

FINFISHES
Milkfish Chanos chanos E, S, I F, B, S
Freshwater eel Anguilla japonica EX, E, I F
Anguilla spp.
Grey mullet Mugil cephalus EX, E, I F, B, S
Cockup Lates calcarifer EX F
Grouper Epinephelus spp. EX S

Page | 1
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Porgy Mylio macrocephalus EX S

Mylio spp.
Red porgy Chrysophry major S, I S
Black porgy Acanthopagrus schlegeli S B, S
Tilapia Oreochromis mossambicus SI F. S
O. nilotica E, SI F, S
Tilapia zillii S F
O. aureus S F
O. mossambicus x O. niloticus S F
O. niloticus x O. aureus S F
Red tilapia Oreochromis spp. S, I F, B, S
Sweet fish, ayu Plecoglossus altivelis I F
Common carp Cyprinus carpio E, S F
Goldfish (wild) Carassius auratus E, S F
Crucian carp Carassius carassius E, S F
Puntius carp Puntius gonionotus E, S F
Puntius spp.
Rohu Labeo rohita EX, S F
Mrigal Cirrhina mrigala EX, S F
Bottom carp Cirrhina molitorella E, S F
Catla Catla catla EX, S F
Grass carp Ctenopharyngodon idellus E, S F
Black or snail carp Mylopharyngodon piceus E, S F
Silver carp Hypophthalmichthys molitrix EX, E, S F
Bighead carp Aristichthys nobilis EX, E, S F
Nilem Osteochilus hasselti EX, E F
Walking catfish Clarias batrachus E, S F
Clarias spp.
MOLLUSCS
Japanese oyster Crassostrea gigas E, I S
Hard clam Metrix lusoria I S
Small abalone Haliotis diversicolor I S
Corbiculas Corbicula fluminea E F
C. formosa E F
Purple clam Soletellina diphos E S

Page | 2
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Apple snail Ampullarius insularum S, I F

Blood clam Tegillarca granosa S S
Crassostrea malabonensis E S
C. iredalei EX, E S
C. palmipes S S
C. cuculata EX, S S
C. lugubris E S
C. belcheri E S
C. commercialis S S
Metrix metrix EX, S S
Cockle Andara granos E, S S
Green sea mussel Mytilus smaragdinus EX, E, S S
REPTILES
Soft-shell turtle Trionyx sinensis I F
Crocodile Crocodilus siamensis I F
C. porocus I F
AMPHIBIANS
Bull frog Rana catasbiana S F
Tiger frog Rana tigrina I F
SEAWEEDS
Gracilaria Gracilaria spp. E B, S
Nori Porphyra spp. E S
Wakame Undaria pinnatifida E S
Green laver Monostroma nitidum E S
*EX = experimental, E = extensive, S = semi-intensive, I = intensive
**F = freshwater, B = brackish water, S = saltwater

Source: Liao, 1988

Huet and Timmermans (1972) list the following criteria for evaluating the suitability of a species
for culture:

(i) It must withstand the climate of the region in which it will be raised. Thus, the rearing of
coldwater fish like salmonids and trout is limited to temperate regions or mountain areas of
tropical countries because they can not tolerate warm water with its low oxygen content.

(ii) Its rate of growth must be sufficiently high. Small species, even if they reproduce well in
ponds and accept formulated diets, are not the most suitable for rearing. Also, the best culture

Page | 3
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

species are those which are low in the food chain, e.g., plankton feeders, herbivores, and
detritivores. Their culture is also least expensive, even on an intensive scale, because they do not
need to be given diets which have a high content of animal protein.

(iii) It must be able to reproduce successfully under culture conditions. Species for culture should
be able to reproduce in captivity/confinement without needing special conditions that have to be
fulfilled, and which give high returns on eggs and fry. Although it is possible to rear species
whose reproduction in confinement is not possible at all (e.g., some carps) or whose reproduction
under hatchery conditions has not yet been possible on a commercial scale (e.g., milkfish in the
Philippines), the sustainability of the grow-out operations is hampered by the seasonal
unavailability of wild fry for stocking in fish pens and/or fish ponds.

(iv) It must accept and thrive on abundant and cheap artificial food. Culture species which feed
on cheap artificial feeds and give low feed conversion ratios (FCRs), also tend to give very good
production rates, thus bringing in better financial returns.

(v) It must be acceptable to the consumer. Even if all the foregoing criteria are met by a
candidate species, it is not worth culturing if there is no market for it. It is possible, though, to
promote acceptability of or encourage consumption of a particular species to ensure that it will
eventually sell in the market. (This was the situation with tilapia in the Philippines prior to the
introduction of the bigger-sized, lighter coloured S. niloticus in the early 1970s.)

(vi) It should support a high population density in ponds. Social and gregarious species which
can grow well to marketable size even under high density conditions in ponds or tanks (e.g.,
tilapia) are preferable to those which can be grown together in dense numbers only up to a
certain age beyond which they eat each other (e.g., pike).

(vii) It must be disease-resistant. Reared fish must be resistant to disease and accept handling and
transport without much difficulty. Tilapia is an ideal species for culture because of its high
resistance to disease even in highly intensive culture systems.

A wide variety of fish and aquatic resources is cultured in freshwater, brackishwater, and marine
environments world-wide using different methods (Table 4). Rabanal (1988a) estimates that
there are close to 50 species of freshwater, brackishwater, and marine finfish species; about 13
crustacean species, 13 molluscan species, 5 seaweed species, and 5 economic aquatic vertebrates
(frogs and other amphibians and turtles and other reptiles) cultivated in Southeast Asia.

Liao (1988) lists some 25 major finfish species, 18 molluscan species, 2 reptile species, 2
amphibian species, and 4 seaweed species as the principal species cultured in Asia. To the list
could be added the crustaceans consisting of the brackishwater/marine penaeid shrimps (mainly
Penaeus monodon, P. semisulcatus, P. japonicus, P. orientalis, P. merguiensis, and Metapenaeus
ensis) and the freshwater prawn of the genus Macrobrachium; the seaweeds Eucheuma,
Laminaria, and Porphyra; and marine finfishes like sea bass and groupers (Baluyut, 1989a).

Examples of aquaculture practices employed in different countries for different species

Page | 4
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Country Species Raised Mode of Culture Reference

NEPAL Common carp, Chinese carp, Integrated fish farming Pullin, 1989
Indian carp
THAILAND Penaeid shrimps, freshwater Pond and cage culture in Sirikul, et al.,
prawn (Macrobrachium) freshwater and 1988
brackishwater
Cockles and mussels Mariculture along the coast
Finfishes Cages suspended in rivers
and standing waters
Clarias batrachus
C. macrocephalus
Tricnogaster pectoralis
Pangasius sp.
Lates calcalifer (sea bass)
Epinephelus spp. (grouper)
INDIA Indian carps Integrated rice-fish culture Mukhopadhyay,
1989
YUGOSLAVIA Primary species: Pond culture Wurtz, I960
Table carp
Aischgrund sp.
Croatian sp.
Secondary species:
European catfish
Tench
Pike
POLAND Carp, tench, pike Pond culture Ackefors, 1989
EAST Carp, tench Pond culture Ackefors, 1989
GERMANY
ECUADOR Penaeid shrimps Pond culture ADCP, 1989b
EL SALVADOR T. aurea Pen culture; floating cage FAO, 1986
culture
T. mossambica
GUATEMALA Tilapia and carp Cage and pond culture FAO, 1986
COSTA RICA Tilapia Pond and cage culture FAO, 1986
Trout Semi-intensive pond
culture
Chinese carp

Page | 5
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Freshwater shrimp
Crayfish
Giant clam
(Anodontis luteola)
ECUADOR Trout Intensive pond and cage FAO, 1986
culture
Trout, marine shrimp Extensive culture
AUSTRALIA Salmonids, marine shrimps, Intensive/semi-intensive Nelson, 1988
molluscs pond culture
FRENCH
POLYNESIA
GUAM Giant clam, seaweeds, and Open water culture
pearl oysters
NEW
CALEDONIA
NEW Tilapia, milkfish, catfish, Less intensive onshore
ZEALAND freshwater prawns, and pond farming
crayfish

In Africa, the predominant species are the tilapias, carps, mullets, sea bass, and catfishes; in
addition, some salmonids, miscellaneous freshwater fish, molluscs, and crustaceans are also
cultured. Latin America grows miscellaneous exotic fish and marine shrimps, molluscs, and
salmonids. Successful experiments on the artificial reproduction and pond culture of indigenous
finfishes of the genus Colossoma and Piaractus (locally known as "tambaqui" and "pirapitinga"
in Brazil, "cachama" and "morocoto" in Venezuela, "gamitama" and "parco" in Peru, and
"cachama negra" and "cachama blanco" in Colombia) also give promise of increased yields
(Saint-Paul, 1989). The Caribbean rears tilapia, carp, marine and freshwater crustaceans, oysters,
and seaweeds; in the Mediterranean region, salmonids are the prime fish and carps are secondary
fish. In the Pacific, tilapia, milkfish, catfish, salmonids, marine and freshwater crustaceans,
molluscs (including giant clams and pearl oysters), and seaweeds are cultured but mostly on a
pilot/experimental scale (ADCP, 1989a).

FISH CULTURE (SEABASS CULTURE)

Introduction

Lates calcarifer (Bloch), commonly called the giant sea perch or seabass, is an economically
important food fish in the tropical and subtropical regions of Asia and the Pacific. It is
commercially cultivated in Thailand, Malaysia, Singapore, Indonesia, Hong Kong and Taiwan,
in both brackishwater and freshwater ponds, as well as in cages in coastal waters. Because of its

Page | 6
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

relatively high market value, it has become an attractive commodity of both large to small-scale
aquaculture enterprises. However, the major constraint to rapid expansion of seabass culture has
been the inconsistent supply of fry collected from the wild. It fluctuates widely from year to year,
making forward planning for production difficult.

In the early 1970's, Thai scientists have achieved success in the breeding of seabass under
captive conditions. Completion of its life cycle has also been accomplished. Growth performance
of the hatchery-bred fry has been shown to be comparable with that of fry collected from the
wild. Thailand is presently producing more than 100 million fry annually (Anon. 1985), with the
Satul Fisheries Station producing more than 30 million (Kungvankij 1984). Thus, the seabass
culture industry in Thailand is now assured of sufficient and consistent supply of fry.

In order to extend the technology of seabass culture, this manual is prepared to serve as a
practical guide for extension workers and farmers. Its contents are based on research findings in
addition to many years of accumulated practical experience and field observations.

1. Taxonomy

Phylum Chordata

Sub-phylum Vertebrata

Class Pisces

Sub-class Teleostomi

Order Percomorphi

Family Centropomidae

Genus Lates

Species Lates calcarifer (Bloch)

The above is an accepted taxonomic classification of seabass or giant perch. Seabass has been
placed under several families by various authors in the past (e.g. the grouper family, Serranidae
and family Latidae, etc.) However, Centropomidae is the commonly accepted familya name of
this species, and the recognized generic name is Lates. Other names such
as Perca, Pseudolates, Holocantrus, Coins, Plectropoma,Latris, and Pleotopomus were also given
by various authors who collected the fish specimens from different areas. Bloch (Schneider
1801) stated that Lates calcarifer occured in Japan Sea but named it as Holocentrus calcarifer.

Page | 7
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

The common local names of this species are listed below:

Common Giant perch, white seabass, silver seaperch, giant perch, palmer,
:
English name cock-up seabass

India : Begti, bekti, dangara, voliji, fitadar, todah

East Bengal : Kora, baor

Sri Lanka : Modha koliya, keduwa

Thailand : Pla kapong kao, pla kapong

Malaysia : Saikap, kakap

North Borneo : Ikan, salung-sung

Vietnam : Ca-chem, cavuot

Kampuchea : Tvey spong

Philippines : Kakap, apahap, bulgan, salongsong, katuyot, matang pusa

Indonesia : Kakap, pelak, petcham, telap

Australia and
Papua New
Guinea : Barramundi

2.Morphology and distinctive characters (after FAO 1974)

Body elongated, compressed, with deep caudal peduncle. Head pointed, with concave dorsal
profile becoming convex in front of dorsal fin. Mouth large, slightly oblique, upper jaw reaching
to behind eye; teeth villiform, no canine teeth present. Lower edge of preoperculum with strong
spine; operculum with a small spine and with a serrated flap above original of lateral line. Dorsal
fin with 7 to 9 spines and 10 to 11 soft rays; a very deep notch almost dividing spiny from soft
part of fin; pectoral fin short and rounded; several short, strong serrations above its base; dorsal
and anal fins both have scaly sheath. Anal fin round, with three spines and 7–8 soft rays; caudal
fin rounded. Scale large ctenoid (rough to touch).

Page | 8
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Colour: two phases, either olive brown above with silver sides and belly in marine environment
and golden brown in freshwater environment (usually juveniles). Blue-green or greyish above
and silver below (adult).

3.Distribution

3.1 Geographic distribution

Seabass is widely distributed in tropical and sub-tropical areas of the Western Pacific and Indian
Ocean, between longitude 50°E - 160°W latitude 24°N – 25°S (Fig. 1). It occurs throughout the
northern part of Asia, southward to Queensland (Australia), westward to East Africa (FAO
1974).

3.2 Ecological distribution

Seabass is a euryhaline and catadromous species. Sexually mature fish are found in the river
mouths, lakes (e.g. Songkhla lake) or lagoons where the salinity and depth range between 30–32
ppt and 10–15m, respectively. The newly-hatched larvae (15–20 days old or 0.4–0.7cm) are
distributed along the coastline of brackishwater estuaries while the 1-cm size larvae can be found
in freshwater bodies e.g. rice fields, lakes, etc. (Bhatia and Kungvankij 1971). Under natural
condition, seabass grows in freshwate and migrates to more saline water for spawning.

4. Life history

Seabass spends most of its growing period (2–3 years) in freshwater bodies such as rivers and
lakes which are connected to the sea. It has a rapid growth rate, often attaining a size of 3–5 kg
within 2–3 years. Adult fish (3–4 years) migrate towards the mouth of the river from inland
waters into the sea where the salinity ranges between 30–32 ppt for gonadal maturation and
subsequent spawning. The fish spawns according to the lunar cycle (usually at the onset of the
new moon or the full moon) during late evening (1800–2000 hours) usually in synchrony with
the incoming tide. This allows the eggs and the hatchlings to drift into estuaries. Here, larval
development takes place after which they migrate further upstream to grow. At present, it is not
known whether the spent fish migrates upstream or spends the rest of its life in the marine
environment (Fig. 2).

Smith (1965) noted that some fish spend their whole life in freshwater environment where they
grow to a length of 65 cm and 19.8 kg body weight. The gonads of such fish are usually
undeveloped. In the marine environment, seabass attaining a length of 1.7 m have been recorded
in the Indo-Australian region (Weber and Beaufort 1936).

Page | 9
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

Fig. 1. Geographic distribution of Lates calcarifer. (After FAO 1974)

Fig. 2 Migration pattern of Lates calcarifer Bloch

5. Feeding habits

Page | 10
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Although the adult seabass is regarded as a voracious carnivore, juveniles are omnivores.
Analysis of stomach content of wild specimens (1–10 cm) show that about 20% consists
plankton, primarily diatom and algae and the rest are made up to small shrimp, fish, etc.
(Kungvankij 1971). Fish of more than 20 cm, the stomach content consists of 100% animal prey:
70% crustaceans (such as shrimp and small crab) and 30% small fishes. The fish species found in
the guts at this stage are mainly slipmouths or or pony fish (Leiognatus sp.) and mullets
(Mugil sp).

6. Sex determination

Identification of the sexes is difficult except during the spawning season. There are some
dimorphic characters that are indicative of sex (Fig. 3).

 Snout of the make fish can be slightly curved while that of the female is atraight.
 The male has a more slender body than the female.
 Weight of the female is heavier than males of the same size.
 The scales near the cloaca of the males are thickers than the female during the spawning
season.
 During the spawning season, abdomen of the female is relatively more bulging than the
males.

7. Sexual maturity

In the early life stages (1.5–2.5 kg body weight) majority of the seabass appear to be male but
when they attain a body weight of 4–6 kg majority become female. After culture period of 3–4
years, however, in the same age group of seabass both sexes can be found and identified as
mentioned above. In a fully mature female, the diameter of the oocysts usually range from 0.4 ro
0.5 mm.

8. Fecundity and spawning

The fecundity of seabass is related to the size and weight of the fish. Gonad samples obtained
from 18 females of body weight ranging from 5.5 to 11 kg gave a range of 2.1 to 7.1 million
eggs (Wongsomnuk and Maneewongsa 1976) as illustrated in Table 1. Observations by the
Australian Department of Agriculture (Anon. 1975) showed that a 12 kg fish had 7.5 million
eggs; a 19 kg fish 8.5 million and a 22 kg. fish, 17 million.

Page | 11
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

Figure 3 Photograph of adult male and female seabass

Table 1. Relationship between size of fish and number of eggs ffrom

rom the gonads of
seabass (Lates
Lates calcarifer Bloch). (After Wongsomnuk and Maneewongsa 1976)

Total length Weight Fecundity (million eggs)

(cm)
Average

70 – 75 5.5 3.1

76 – 80 8.1 3.2

81 – 85 9.1 7.2

Page | 12
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

86 – 90 10.5 8.1

91 – 95 11.0 5.9

Seabass spawn all year round (Kungvankij 1984) with the peak season occuring during April-
August and large number of fry (1 cm in size) can be collected during May to August (Bhatia
and Kungvankij 1971).

Based on studies of spawning activity under tank conditions, mature male and female fish
separate from the school and cease feeding about a week prior to spawning. As the female attains
full maturity, there ia an increase in play activity with the male. The ripe male and female then
swim together more frequently near the water surface as spawning time approaches. The fish
spawns repeatedly in batches for 7 days. Spawning occurs during late evening (1800–2200
hours).

Culture of Seabass

Seabass has been commercially cultivated in brackishwater and freshwater ponds and marine
cages in many Southeast Asian countries. While the cagae culture technology is now established,
grow-out techniques in pond are still are still in the developmental stages. Although considerable
progress has been made over the past ten years, many problems remained unsolved.

The major problems that are always encountered during culture period are: (a) cannibalism
during young stage (1–20 g), (b) dependence on trash fish as a main diet which has a very
limited supply in many countries.

Despite some imperfections, the basic techniques of seabass culture are now developed and have
been considered economically viable.

1. Culture techniques

As mentioned above, cannibalism is one of the most serious problems in seabass culture. High
mortality is often encountered when uneven sizes of the fish are stocked. This has been noted to
occur mostly where the fish are very young (1–20 cm in length, the first two months of culture).
To minimize this problem, culture of seabass should be approached in two phases i.e. the nursery
phase and the grow-out phase.

1.1 Nursery

The main purpose of the nursery is to culture the fry from hatchery (1–2.5 cm in size) to juvenile
size (8–10 cm). This can solve the problem of space competition in the nursery tanks. Beyond
the nursing period, the juveniles can be graded into different size groups and stocked in separate

Page | 13
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

grow-out ponds. It has been observed that the juveniles from the nurseries perform better in
terms of growth and survival than those stocked directly into the grow-out ponds.

Nursing the fry in concrete tanks is not recommended as accumulation of excess feed on the
bottom of the tank cannot be avoided. Such accumulation can cause bacterial disease. In
addition, constant contact with the tank wall results in wounded fish and subsequent bacterial
infection

1.1.1 Nursery pond design

Nursery pond size ranges from 500 to 2000 m2 with water depth of 50–80 cm. The pond has
separate inlet and outlet gates to facilitate water exchange. Pond bottom should be flat and
sloping towards the harvesting or drainage gate. Inlet and outlet gates are provided with a fine
screen (1 mm mesh size) to prevent predators and competitors from entering and fry from
escaping the pond.

Fry ranging from 1–2.5 cm are suitable for stocking in the nursery ponds. Stocking density is
between 20–50 individuals per square meter.

1.1.2 Pond preparation

A wellprepared pond is important as predators and competitors can endanger the stocked fry.

Some farmers still practice very crude farming techniques of drying the pond bottom and
immediately filling with water and stocking fry directly for nursing. Feeding is entirely
dependent on supplementary feed such as chopped or grounded trash fish and is done twice daily
in the morning (1800 hours) and afternoon (1700 hours). In this method, the survival rate and
growth rate are low.

To enhance production, the following improved pond preparation techniques are done: The
nursery pond must be drained and dried until the bottom soil cracks to release toxic gases,
oxidize mineralized nutrients, eradicate some pests and predators. In cases where the pond
cannot be completely drained, derris root (rotinone) may be applied at the rate of 20 kg/ha
toeradicate unwanted species. Derris root is prepared by cutting them into small pieces, crushing
and soaking in water overnight. Only the solution is applied to the pond. If derris root is not
available, a mixture of 50 kg/ha of ammonium sulfate (21-0-0) with lime at a ratio of 1:50 will
be sufficient to weed out unwanted species. The mixture is applied to the portions of pond with
water. The use of any chemicals or inorganic pesticides is not recommended because the residual
effect remains for many years and can reduce the pond production. If pond soil is acidic, the
pond bottom should be neutralized with lime before letting the water in.

Production techniques of juvenile in nursery ponds have been improved recently at Satul Fishery
Station, Thailand. The improved technique is based on the live food production in the pond
supplemented with chopped or grounded trash fish. After neutralizing pond bottom by liming,
organic fertilizer (chicken manure) is applied at the rate of 500 kg/ha. Then water depth is
gradually increased for the propagation of natural food. Two to three weeks prior to stocking,

Page | 14
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

newly-hatched Artemia nauplii are inoculated into the pond (1 kg of dry cyst/ha). Artemia will
utilize the natural food as feed for growth and will reach adult stage within 10–14 days. The fry
are immediately stocked at the rate of 20–50 individual per square meter.

Another approach to the improved technique is to stock Artemia nauplii in the separate pond and
grow them into adult. Adults could be harvested daily to feed the fry.

1.1.3 Nursery pond management

Although seabass can be cultured in either freshwater or saltwater, fry must be acclimatized to
the salinity and temperature prevailing in the pond on stocking to prevent loss.

Acclimatization is done in the following manner: transfer the fry to a tank, then gradually add
nursery pond water. This can be completed within one day or more depending on the salinity
difference. If the temperature and salinity in transport bag does not differ by more than 5°C and 5
ppt with the pond water, acclimation can be done by floating the bag in the pond for sometime to
even out temperature difference. Pond water is then added gradually until both salinity become
equal and the fry can be released.

Seabass fry are stocked in the nursery pond at a density of 20–50 fry/m 2. Stocking is usually
done in the erly morning (0600–0900 hours) or early evening (2000–2200 hours) when the
temperature is cooler.

Water replenishment is needed to prevent deterioration of pond water quality due to the
decomposition of uneaten feed or excess growth of natural food. Normally, 30% of pond water is
changed daily.

Supplementary feed is given daily. The feed used for nursing seabass is chopped and grounded
(4–6 mm3) trash fish, normally at the rate of 100% of biomass given twice daily in the first week
(at 0900–1700 hours), gradually reduced to 60% for the second week and 40% in the third week.
This has been found to be most effective feeding strategy for ponds without artemia inoculation.

The application of supplementary feed is a vital operational activity that should be done
properly, if not, contamination of culture water and wastage of feeds result. Although the seabass
in nature prefer live food, the fish can be trained to feed on dead animal. Prior to feeding, the fish
should be attracted by sound (such as tapping a bamboo pole in the water) to induce them to
form a school. Feeding time and place should be fixed. After the fish have formed a school,
small amounts of feed are introduced by spreading into the water within the school of fish fry. It
must be remembered that seabass never eat the feed when it sinks to the pond bottom. Therefore,
feeding should be slow. When the fish are filled to satiation, they disappear thus feeding should
be stopped. The same procedure should be followed at every feeding time. The first few days
after stocking, feeding should be 5 to 6 times a day to teach them to accept dead feed. Once the
fish is accustomed to it which takes about 5–7 days, feeding frequency is reduced to twice daily.
In nurseries where Artemia is the main diet, once the Artemia population has thinned down,
chopped or grounded trash fish can be supplemented using above described practice.

Page | 15
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

The nursing period lasts about 30–45 days until fingerling stage (size 5–10 cm). At this stage,
they are ready for transfer to grow-out ponds.

1.1.4 Nursing in net cages

Nursing of seabass fry (1–2.5 cm to 8–10 cm) in cages is an approach to the nursery phase. The
method has been successful since conducive environmental conditions such as flow through
water, necessary for good health and growth of fish are used. It is likewise easy to maintain and
require very little capital investment.

1.1.5 Nursing cage design and investment

The most convenient cage design is a rectangular cage made of synthetic netting attached to
wooden frames. It is either (a) kept afloat by styrofoam, plastic or metal drum, or (b) stationary
by fastening to a bamboo or wooden pole at each corner. The size of cages vary from 3 cubic
meter (3 × 1 × 1m) to 10 cubic meters (5 × 2 × 1m). The mesh size of the net used for nursery
cages is 1.0 mm. The cages may be installed in the river, coastal area or in a pond. Suitable sites
for net cages should be free from biofoulers since the mesh size of a nursery cage is very small.
Cages are easily damaged in strong currents and clogging by biofoulers. (Fig. 20)

1.1.6 Nursery cage management

Seabass fry (1–2.5 cm in size) are stocked in the nursery cage at the rate of 80–100 per square
meter.

Stocking and feeding activity are the same as in nursery pond culture practice.

The net cages should be checked daily to ensure that the cages are not damaged by animals such
as crabs or clogged with fouling organisms. Cleaning of the cages should be done every other
day by brushing. This will allow water to pass through the cages naturally.

Page | 16
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Fig. 20 Floating nursery cage for seabass.

After the nursing period of 30–45 days (in pond or cages) or when the fry have reached 5–10 gm,
these are ready for transfer to grow-out ponds. Prior to stocking in grow-out ponds, grading
procedure should be applied. Fish are graded into several sizes. It will give maximum advantage
if the various sizes are stocked in separate ponds to prevent cannibalism.

1.2 Grow-out

The grow-out phase involves the rearing of the seabass from juvenile to marketable size.
Marketable size requirement of seabass vary country to country e.g. Malaysia, Thailand, Hong
Kong and Singapore. The normally accepted marketable size of seabass among these countries
and region is between 700–1200 g while in the Philippines, marketable size is between 300–400
g. The culture period in grow-out phase also vary from 3–4 months (to produce 300–400) to 8–
12 months.

Page | 17
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

2. Cage culture

Cage culture of seabass is quite well developed in Thailand, Malaysia, Indonesia, Hong Kong
and Singapore. The success of marine cage culture of seabass and its economical viability have
contributed significantly to large scale development of this aquaculture system

2.1 Suitable site for cage culture

Criteria for selecting a suitable site for cage culture of seabass include:

a. Protection from strong wind and waves. The cage culture site should preferably be
located in protected bays, lagoons, sheltered coves or inland sea.
b. Water circulation. The site should preferably be located in an area where influenc of tidal
fluctuation is not pronounced. Avoid installing cages where the current velocity is strong.
c. Salinity. Suitable site for seabass culture should have a salinity ranging from 13–30 ppt.
d. Biofouling. The site should be far from the area where biofoulers abound.
e. Water quality. The site should be far from the sources of domestic, industrial and
agricultural pollution and other environmental hazards.

2.2 Design and construction of net cages

In general, square and rectangular cages with size varying from 20 to 100 m 3 are preferable
because they are easy to construct, manage and maintain. Seabass cages usually are made of
polyethelene netting with the mesh size ranging from 2 to 8 cm. The choice of mesh size depends
on the size of the fish (Table 8).

There are two types of cages used in seabass culture:

(a) Floating cages

The net cages are attached to wooden, GI pipe or bamboo frames. The cage is kept afloat by
floating material such as metal, plastic, styrofoam drum or bamboo. The shape of the cage is
maintained with the use of concrete weights attached to the corners of the cage bottom (Fig.
22).The most manageable size for a floating cage is 50 m3 (5 × 5 × 2m). This cage dimension is
easy to change when clogged with fouling organisms.

(b) Stationary cages

The cage is fastened to the bamboo or wooden poles installed at its four corners (Fig.
21). Stationary cages are popularly used in shallow bays since they are easy to install.

2.3 Cage culture management and techniques

Prior to stocking seabass juvenile in cages, fish should be acclimatized to the ambient
temperature and salinity prevailing in the cages. The fish should be graded into several size

Page | 18
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

groups and stocked in separate cages. The stocking time should be done in the early mornings
(0600–0800 hours) or late in the evening (2000–2200 hours) when the temperature is cooler.

Stocking density in cages is usually between 40–50 fish per cubic meter. Two to three months
thereafter, when the fish have attained a weight between 150–200 g, the stocking density should
be reduced to 10–20 fish per cubic meter. Table 9 shows the growth of seabass under varying
densities in cages. There should be spare cages as these are necessary for transfer of stock and to
effect immediate change of net in the previously stocked cage once it has become clogged with
fouling organisms. Changing cages allows for grading and controlling stock density.

2.4 Feeds and feeding

Feed is the major constraint confronting the seabass culture industry. At present, trash fish is the
only known feed stuff used in seabass culture. Chopped trash fish are given twice daily in the
morning at 0800 hours and afternoon at 1700 hours at the overall rate of 10% of total biomass in
the first two months of culture. After two months, feeding is reduced to once daily and given in
the afternoon at the rate of 5% of the total biomass. Food should be given only when the fish
swim near the surface to eat.

Table shows The choice of netting mesh size of fish.

mesh size size of fish

0.5 cm 1–2 cm

1 cm 5–10 cm

2 cm 20–30 cm

4 cm bigger than 25 cm

Page | 19
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

FIG. 21 STATIONARY CAGES

FLOATING CAGES

Page | 20
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Table shows the Monthly growth of seabass at different stocking densities

in cages.

Culture Stocking density

period
(month) 16/m2 24/m2 32/m2

0 67.80 g 67.80 g 67.80 g

1 132.33 g 137.53 g 139.20 g

2 225.20 g 229.10 g 225.50 g

3 262.88 g 267.50 g 264.11 g

4 326.15 g 331.97 g 311.50 g

5 381.08 g 384.87 g 358.77 g

6 498.55 g 487.06 g 455.40 g

Since the supply of trash fish is insufficient and expensive in some countries, its use is
minimized by mixing rice bran or broken rice to the trash fish (Table 10). However, even with
these cost cutting measures, feed cost remain quite high.

A very recent development on improving the dietary intake of seabass is the introduction of
moist feed. So far, the use is still on experimental stage. The feed composition recommended is
presented at Table 11.

2.5 Fish cage management

Regular observation of cages is required. Since fish cages are immersed under water all the time,
they are vulnerable to destruction by aquatic animals such as crabs, otter, etc. If damaged, they
should be repaired immediately or replaced with a new one.

In addition to biofouling, the net walls of cages are subjected to siltation and clogging.
Biofouling is unavoidable since the net walls usually represent a convenient surface for
attachment by organisms such as amphipod, polycheate, barnacles, molluscan spats, etc. These
could lead to clogging and reduce exchange of water and may result in unnecessary stress to the
cultured fish due to low oxygen and accumulation of wastes. Feeding and growth would likewise
be affected.

Page | 21
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

To date, mechanical cleaning of fouled nets is still the most efficient and cheap method. In areas
where fouling organisms are abundant, rotational usage of net cage is highly recommended.

3. Pond culture

Although methods of pond culture of seabass have been practiced for over 20 years in Southeast
Asia and Australia, not much has been done on the commercial scale. At present, culture of
seabass in brackishwater pond has been identified in some countries as having tremendous
market potential and high profitability. These, however, can be achieved if conditions are met
such as adequate fry supply, availability of suitable site and properly designed fish farm. Supply
of fry from the wild is very limited. As with cage culture, it is one of the constraints in the
intensification of seabass culture in ponds. However, with the success in artificial propagation of
seabass, fry supply may largely come from this source in the future. A comparison of hatchery
bred and wild fry cultured in ponds did not show very significant difference in growth rate(Table
12).

There are two culture systems employed in pond culture of seabass:

(a) Monoculture

Monoculture is that type of culture where a single species of animal is produced, e.g. seabass.
This culture system has a disadvantage. It is entirely dependent on supplementary feeding. The
use of supplementary feed reduces profit to the minimal, especially where the supply of fresh
fish is limited and high priced.

Table shows the Combination of feed stuff.

Ingredient Percentage

Trash fish 70%

Rice bran or broken rice 30%

Table shows the Combination of moist diet

Ingredient Percentage

Fish meal 35%

Rice bran 20%

Soy bean meal 15%

Page | 22
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Corn meal 10%

Leaf meal 3%

Squid Oil (or fish oil) 7%

Starch 8%

Vitamin mix 2%

Table shows Comparison of growth rate of seabass (Lates calcarifer)

culture in pond between wild fry and hatchery bred fry at stocking density
of 3/m2.

Wild Hatchery bred

B.L. B.W. B.L. B.W.

Stocking 10.5 cm 40.44 g 5.2 cm 5g

1st month 13.0 88.9 7.6 12.0

2nd month 16.4 204.2 10.6 26.02

3rd month 20.9 276.3 15.2 118.1

4th month 23.4 326.5 19.5 220.9

5th month 24.1 385.2 21.8 280.6

6th month 28.2 453.5 23.2 349.6

(b) Polyculture

This type of culture approach shows great promise in reducing if not totally eliminating the
farmers' dependence on trash fish as food source. The method is achieved by simply
incorporating a species of forage fish with the main species in the pond. The choince of forage
fish will depend on its ability to reproduce continuously in quantity sufficient to sustain the
growth of seabass throughout the culture period. The forage fish must be such a species that
could make use of natural food produced in the pond and does not compete with the main species
in terms of feeding habit such as Oreochromis mossambicus, Oreochromis niloticus, etc.
Page | 23
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

4. Criteria in the selection of site for seabass culture

4.1 Water supply

The site should have enough good water quality supply all year round. Water quality includes all
physico-chemical and microbiological characteristics of water being used for culture of seabass.
The following are the parameters normally considered as suitable water supply:

Parameter Range

pH 7.5–8.5

Dissolved oxygen 4–9 ppm

Salinity 10–30 ppt

Temperature 26–32°C

NH3 less than 1 ppm

H2S less than 0.3 ppm

Turbidity less than 10 ppm

4.2 Tidal fluctuation

Area best suited for seabass should have moderate tide fluctuation range between 2–3 meters.
With this tidal characteristic even for ponds as deep as 1.5 meters, complete drainage during low
tide can be done. In addition, the pond can readily admit water during spring tide.

4.3 Topography

It is advantageous if the selected site is mapped topographically. This would reduce development
and operational costs such as for water pumping.

4.4 Soil

Ideally, the soil at the proposed site should have enough clay content to ensure that the pond can
hold water. Area with acid sulphate soil should be avoided.

Page | 24
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

4.5 Accessibility

Accessibility is an important consideration in site selection for logical reasons. Overhead cost
and delay in the transport of material and product may be minimized with good site accessibility.

Other factors in the selection of site that should be considered include availability of seed,
labour, technical assistance, market demand and suitable social condition.

5. Pond design and construction

Seabass ponds are generally rectangular in shape with size ranging from 2000 m 2 to 2 hectares
and depth of 1.2 to 1.5 meters. Each pond has separate inlet and outlet gate to facilitate water
exchange. The pond bottom is entirely flat levelling toward the drainage gate (Fig. 23).

6. Pond preparation

Preparation of grow-out ponds is similar to the procedure followed in pond system. In

monoculture, the fish are stocked immediately after neutralizing the pond soil with lime. Ponds
are filled immediately after pond preparation.

In polyculture, after the pond soil is neutralized, organic fertilizer (chicken manure) is applied at
the rate of 1 ton per hectare. Then water depth is gradually increased for propagation of natural
food. When abundance of natural food are observed, selected tilapia broodstocks are released to
the pond at the rate of 5,000–10,000 per hectare. Sex ratio of male to female is 1:3. The tilapia
are reared in pond for 1 to 2 months or until tilapia fry appear in sufficient number. Seabass
juveniles are then stocked.

Seabass juveniles (8–10 cm in size) from nursery are stocked in the grow-out pond at the rate of
10,000–20,000 per hectare in monoculture and 3,000–5,000 per hectare in polyculture system.
Prior to stocking, juveniles are acclimatized to pond culture and salinity conditions. Stocking the
fish in uniform sizes will be most ideal and should be done at cooler times of the day.

Page | 25
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

Fig. 23 Pond lay-out

out for seabass culture.

7. Pond management

Due to the need of maintaining natural food in ponds, water replenishment in polyculture system
should be minimized. Water change should be done once in three days for about 50% of
capacity.

Page | 26
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

However, in monoculture where supplemental feed is given daily, there are chances that excess
feed may pollute the water. Hence, daily water replenishment is necessary.

8. Feeds and feeding

Supplementary feed is not required in the polyculture system, but in monoculture, daily feeding
is a normal practice. The method of supplying feed in ponds follows often the practice employed
in cage culture.

Conclusion

Seabass (Lates calcarifer) culture enterprise is one of the most dynamic and potentially profitable
segments of the brackish and marine water fish farming industry in Southeast Asia. It is a
desirable fish with good flesh texture and taste, high market value and market value and demand.
It can be reared both in freshwater and seawater conditions. In the past 5 years, over 10,000
farmers engaged in cage culture of seabass and over 20,000 hectares of land have been
established in the Region for intensive pond production of the species.

PRAWN/SHRIMP CULTURE

INTRODUCTION

Coastal aquaculture has been identified by the Government of India as high potential
area for increasing the fish and shell fish production and also to achieve economic and social
benefits . India with over 8,100 Km of coastline, vast stretches of estuaries/ backwaters,
lagoons provide enormous opportunities for brackish water shrimp farming.
Commercial shrimp farming is almost three decades old in India. During the early
nineties due to proven technology in post larvae production and farming of two varieties of
shrimps viz white shrimp ( Penaeus indicus ) and tiger shrimp (Penaeus monodon ).Large scale
growth of shrimp farms and hatcheries was witnessed during a short span of ten years. It is time
for Blue Revolution to exploit the huge potential in fisheries sector.

Shrimps are called the "Pinkish Gold" of the sea because of its universal appeal, unique
taste, high unit value realisation and increasing demand in the world market. Scope for brackish
water shrimp farming. The over exploitation of shrimp from coastal waters and the ever
increasing demand for shrimp and shrimp products in the world market has resulted in the wide

Page | 27
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

gap between the demand and supply in the International market. This has necessitated the need
for exploring newer avenues for increasing shrimp production. Water quality parameters
required for maximum feed efficiency and maximum growth of Penaeus monodon are given
below :
Water Parameters Optimum level
Dissolved Oxygen 3.5-4 ppm
Salinity 10-25 ppt
Water Temperature 26-32 degree centigrade
pH 6.8-8.7
Total nitrite nitrogen 1.0 ppm
Total ammonia (less than) 1.0 ppm
Biological Oxygen Demand (BOD) 10 ppm
Chemical Oxygen Demand (COD) 70 ppm
Transparency 35 cm
Carbon dioxide (less than) 10 ppm
Sulphide (less than) 0.003 ppm

Penaeus monodon

Page | 28
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

S.NO Traditional (within CRZ) Extensive

(outside CRZ)
i. Farm Size 5 ha 5 ha
ii. Culture period 5 - 6 months 5 - 6 months
iii. Stocking density 60,000/ ha 1,00,000/ ha
(PL-20)
iv Survival 70% 70%
v Expected production 1.0-1.5 1.5-2.5
tonnes/ha/crop tonnes/ha/corp
vii Price of shrimp has been taken as Rs.600/kg

Page | 29
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Traditional aqua farming

Technical Parameters for establishing a extensive shrimp farming

Design and Construction of shrimp farm :
 An extensive shrimp farm should be of the size 0.4 - 0.5 ha. and preferably drainable
 The ponds generally should have concrete dikes, elevated concrete supply canal with
separate drain gates and adequate life supporting devices like generators and aerators.
 The design, elevation and orientation of the water canals must be related to the elevation
of the area with particular reference to the mean range of tidal fluctuation.
 The layout of the canals and dikes may be fitted as closely as technically possible to
existing land slopes and undulation for minimizing the cost of construction.
SEMI-INTENSIVE FARMING

Page | 30
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

EARTH WORK
It is normally carried out in the following order
 Site clearing
 Top soil stripping
 Staking of centre lines and templates
 Preparation of dike foundation, excavation of drainage canals
 Construction of dikes (peripheral and secondary)
 Formation and compaction of dikes, Excavation of pits for gatesa
 Levelling of pond bottom
 Construction of gates and refilling of pits,
 Construction of dike protection

Page | 31
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

The top soil may be set aside and should again be spread later to preserve pond bottom
fertility.

THE ESSENTIAL COMPONENTS OF A SHRIMP FARM

 Ponds
 Water intake structure
 Store room for feed and equipments
 An area for cleaning of the harvest
 Pump house
 Watch and ward room,
 Office and A mini laboratory.
POND PREPARATION
 Proper pond preparation will ensure higher productivity and production levels. The main
objectives of pond preparation are :
 To eradicate weed fishes and other harmful organisms
 To remove abnoxious gases
 To improve the natural productivity of the pond eco system
 To maintain high
igh water quality for proper growth and higher survival percentage.

Page | 32
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

 Eradication of unwanted organisms is usually carried out by draining out the entire water
and drying the pond bottom till it cracks
 This also helps in removal of obnoxious gases and oxygenation of the pond bottom.
 It also improves the fertility of the soil.
 Liming is done for correcting the pH and to kill pathogenic bacteria and virus.
 In undrainable ponds mahual oil should be applied @ 200 ppm to eradicate the weed
fishes.
 After around two weeks organic and inorganic fertilisers are applied to enrich the soil and
water.
 Once the thick lab-lab is formed the water level is raised and the pond is made ready for
stocking.

ACCLIMATION

SELECTIVE STOCKING
The most suitable species for culture in India are the Indian white shrimp Penaeus
indicus and
Tiger shrimp P. monodon. The stocking density varies with the type of system adopted
and the species selected for the culture. Shrimp farming with a production range of 1.5 to 2.5
tonnes/ha/crop with stocking density upto 1,00,000/ha/crop viz; 10 Nos./m 2 may be allowed. In

Page | 33
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

order to have uniform growth it is always advisable to go in for hatchery reared and PCR tested
seeds.
Food and feeding Shrimp diets may be supplementary or complete. In a extensive system the
shrimps need a complete diet. Although natural food items have good conversion values, it is
difficult to procure in large quantities and maintain a continuous supply. At present most of the
aquaculture farms depend on imported feed with a FCR of 1:1.5 - 1.8. The feeding could be done
by using automatic feed dispensers, or by broadcasting all over the pond. If feeding trays are
employed in selected pockets in the pond wastage of feed can be reduced.

HARVESTING
Complete harvesting can be carried out by draining the pond water through a bag net and
hand picking. The average culture period required is around 120 days during which time
the shrimps will grow to 25-35 gm size (depending on the species). It is possible to get
two crops in a year. Harvested shrimps can be kept between layers of crushed ice before
transporting the consignment to market.

Page | 34
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

Page | 35
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

MARKETING
Because of huge gap between supply and demand of shrimps in local as well as international
market, there may not be any problem in marketing. Shrimp can either be sold directly by the

Page | 36
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

farmers in the market or sold to exporters for processing. Shrimp can be exported in frozen form
with head on, head less, battered and breaded, or IQF products or any other form with value
addition depending on the requirement of the buyer.
WORLD FISH MARKET AT A GLANCE

Page | 37
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

====================================================================

Page | 38
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

FRESHWATER PRAWN FARMING

1. Introduction

Indian aquaculture has been evolving from the level of susbsistence activity to that of an
industry. This transformation has been made possible with the development and
standardization of many new production and associated techniques of input and output
subsystems. In recent years aquaculture has created great enthusiasm and interest among
entrepreneurs especially for shrimp farming in coastal areas. Shrimp farming is capital
intensive activity and uncontrolled mushrooming growth of it has led to outbreak of
diseases and attributed environmental issues calling for closure of shrimp farms.

Although India has vast freshwater resources they are not fully exploited except for carp
culture in limited scale. Fresh water fish culture employing composite fish culture
technology has become popular for use in large number of tanks and ponds in the country.
To meet the raw material required by the processing units for export demand there is
urgent need to expand our production base. In addition it is always stressed that there is a
need to utilise our natural resources productively to ensure the much needed food security.

2. Scope for Fresh Water Prawn Culture

Considering the high export potential, the giant fresh water prawn, Macrobrachium
rosenbergii, the scampi, enjoys immense potential for culture in India. About 4 million ha.
of impounded freshwater bodies in the various states of India, offer great potential for fresh
water prawn culture. Scampi can be cultivated for export through monoculture in existing
as well as new ponds or with compatible freshwater fishes in existing ponds. It is exported
to EEC countries and USA. Since the world market for scampi is expanding with attractive
prices, there is great scope for scampi production and export.

3. Technical Parameters

Page | 39
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

The giant freshwater prawn is suitable for cultivation in tropical and subtropical climates.
It is a hardy species by virtue of its ability to adapt to various types of fresh and brackish-
water conditions. It accepts pelleted feed and has omnivorous feeding habit. In the natural
enviroment, lower reaches of rivers, tidal inlets, where water is directly or indirectly
connected with sea are their preferred habitat specially during spawning. The breeding
takes place in low saline waters which is also needed for larval and post larval
development after incubation. Breeding of M.rosenbergii takes place in estuaries.

Though seed may be available in natural sources to a limited extent, for large scale culture
there is a need to ensure regular supply of seed. For ensuring availability of quality seed in
predictable quantity freshwater prawn hatcheries should be encouraged, technology for
which is already developed. Freshwater prawn hatcheries are coming up in many states.

The techno-economic parameters required for establishment of prawn farm and its
successful operation are briefly described in this booklet. The parameters are averaged out
and the costs are only illustrative.

3.1. Site selection

The site selection plays an important role as the entire management aspect of the farm
ultimately depends on specific conditions of the site. The aspects to be considered are
topography of the area, soil type, availability of quality water etc.. The area should be free
from pollution and flooding. Other considerations like approach roads etc. have also to be
taken into account.

3.2 Soil quality

The ideal soil for Macrobrachium culture should be clay silt mixture or sandy loam
comprising of 60% sand and 40% silt with good water retention capacity.

3.3. Water quality

There should be availability of abundant and good quality water.The water should be free
from any kind of pollution. The pH should be maintained at 7 to 8.5. The temperature
should range from 18 0 to 340C with an optimum range of 270 C to 310 C. Dissolved oxygen
content should be higher than 75% saturation.

3.4 Pond construction

Rectangular ponds are suitable mainly from the harvesting point of view. A convenient
width is 30-50 m, whereas length of the pond depends on site, topography and farm layout.
Normally a size of 0.5 to 1.5 ha is found suitable. The average depth of the ponds should
be 0.9m with a minimum of 0.75m and a maximum of 1.2m. Dike and pond slope may be
kept at 2:1. Bund must have a freeboard of at least 60 cm above the highest water level in
the pond. Designing and layout of the farms may be done keeping in view the water intake

Page | 40
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

and water outlet facilities. The drainage system should be designed carefully to prevent
mixing of outlet water with incoming water.

3.5. Water supply and drainage

Appropriate water supply and drainage systems have to be designed keeping in view the
water source and topography of the area. Tubewell and pumping system may be considered
if required for water intake/exchange. Water exchange on weekly or fortnightly basis as
required is desirable and provisions are to be made accordingly.

4. Farm Management

The type of pond preparation to be adopted before stocking is based on the type of culture
and its intensity and nature of the culture pond. Liming of the pond assumes great
importance here than in the case of freshwater fish culture. The application of fertilisers is
restricted in case pelletised feed is used. However, occasionally cow dung, single super
phosphate, urea etc. can be applied on assessing the productivity.

The stocking density normally varies from 4000 to 50000 nos. of post larvae per ha
depending on the type and intensity of the management practices. The culture system may
be monoculture or polyculture with carps. In case of polyculture with carps the more pond
depth is preferred at 4-5 feet. In case of polyculture the stocking density of prawn may
vary from 2500-20000 post larvae. The carp fingerlings may be of the order of 5000 - 2500
Nos. Nursery may be incorporated where the post larvae obtained from hatcheries could be
reared for a period of 4-5 weeks till they attain 40-50 mm or 1-3 gram.

In order to get desired production, feeding, aeration, water exchange, periodic monitoring
should be continued. The quality and type of feed is based on culture system.
Macrobrachium with its omnivorous feeding habits can make use of a variety of feeds
from common wet feed made from rice bran and oil cake to scientifically formulated
pelleted feed. The rate of feeding is determined by the stage of growth of prawn, water
quality, density of stock and other manuring practices. Generally the feeding rate my be
5% of the body weight.

The duration of culture varies from 6 to 12 months depending on the type of culture
practice. Generally in monoculture the culture period may be 6-8 months under
monoculture and 8-12 months under polyculture. The average growth of prawn may range
from 50 gms to 200 gms depending on the duration, density, water quality, feeding etc.
The survival rate may range 50% to 70% depending on the type of management practices.

5. Extension services

The borrower should have experience in prawn farming and should be conversant with
production technology, trade etc. Fish Farmers Development Agencies (FFDA) have been
established in almost all districts for providing necessary training. The offices of Marine

Page | 41
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Products Export Development Authority (MPEDA) in most of the coastal states also
provide necessary assistance.

6. Marketing

There is good demand for fresh water prawn in both local and international markets, as
such there may not be any problem in marketing the same. Fresh water prawns can be sold
directly by the farmers either in the market or to exporters for processing before export.

7. Financial outlay

Details for the financial outlay have been indicated in Annexure I. It can be seen therefrom
that the capital cost for a 1 ha. unit has been estimated as Rs. 2.075 lakh while the
operational cost for one crop works out to Rs.1.214 lakh. The items and cost indicated
under the model are indicative and not exhaustive. While preparing projects for financial
assistance the costs have to be assessed taking into account actual field conditions.

8. Margin money and bank loan

The entrepreneur is expected to bring margin money out of his own resources. The rates of
margin money stipulated are 5% for smaller farmer, 10% for medium farmer and 15% for
other farmers. For corporate borrowers the margin stipulated is 25%. NABARD could
consider providing margin money loan assistance in deserving cases.

9. Rate of Refinance

NABARD provides refinance assistance for freshwater prawn farming to commercial

banks, cooperative banks and Regional Rural Banks. The rate of refinance is fixed by
NABARD from time to time.

10. Financial viability

The following assumptions have been made for working out the financial viability of the
project.

i) Farm size 1 ha.

ii) Culture period 6-8 months
iii) Stocking density 30,000 /ha
iv) Survival 60%
v) Feed conversion ratio 2.5:1
vi) Expected production 1260 kg/ha/crop
vii) Only one crop of 6-8 months culture period has been considered
Sale price of prawn has been taken as Rs. 170 per kg.
The financial analysis has been shown in Annexure I.
The results of analysis are:

Page | 42
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

i) NPW at 15% Discounting = Rs. 2.33 lakhs

ii) BCR at 15% Discounting = 1.37:1
iii) IRR = 77%

11. Rate of interest

Interest rate to be charged would be as indicated by bank/RBI/NABARD from time to

time.

12. Repayment period

The borrower will be able to repay the bank loan in 8 years (Annex -I) with a grace period
of one year on repayment of the principal.

13. Security

Security from the ultimate beneficiaries may be obtained as per the guidelines of RBI
issued from time to time.

Annexure - I

ESTIMATED FINANCIAL OUTLAY FOR GIANT FRESH WATER

PRAWN (MACROBRAHIUM RESENBERGII) CULTURE IN 1 HA WATER

AREA

A. Capital Cost

Units Quantum Rate (Rs.) Total

1 Construction of pond including digging, bund Cum 7500 15 112500
construction and compaction and
consolidtion
2 Shallow tubewell and pumpset 5 HP Nos L/s 35,000
3 Pump house cum store room-AC roof L/s 20,000
4 Inlet/outlet sluices 10,000
5 Nets and other implements L/s 10,000
6 Aerator Nos 1 15,000 15,000
7 Miscellaneous including laying of pipe line L/s 5,000
etc.
Total A 207,500

B. Operational cost for one crop (6-8 months)

Units Quantum Rate (Rs.) Total

Page | 43
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

1 Lime Kg 300 5 1,500

2 In organic fertiliser (super phosphate) kg 75 5 375
3 Fertiliser - Organic-Cow Dung tons 2 300 600
4 Seed Nos 30,000 0.6 18,000
5 Feed-pelletted feed Kgs 3,150 20 63,000
6 Pumping and aeration charges L/s 10,000
7 Watch and Ward Mandays 240 40 9,600
8 Miscellaneous including insurance, L/s 5000
harvesting and medicine etc.
Total B 108,075
Total Cost 315,575

Annexure -I(Contd.)

C. Production

1 Survival(%) 60%
2 Average weight at harvest (gms) 70
3 Total production (Kg) 1,260
4 Farm gate price (Rs.) 170
5 Number of Crops per annum 1
6 Income during 1st year (85% of total 1,82,070
production)
Income from 2nd year onwards 214,200

D. Financial Analysis

1 2 3 4 5 6 7
Capital cost 207,500
Recurring cost 108,075 108,075 108,075 108,075 108,075 108,075 108,075
Total cost 315,575 108,075 108,075 108,075 108,075 108,075 108,075
Income 182,070 214,200 214,200 214,200 214,200 214,200 214,200
New benefit -133,505 106,125 106,125 106,125 106,125 106,125 106,125
NPW of cost 630,072
NPW of 863,222
benefit
NPW 233,150
BCR 1.37
IRR 77%

Page | 44
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

MUD CRAB FARMING

Mud crab farming is very popular in some Asian countries like Bangladesh, India, Thailand,
Philippine etc. Mud crab has huge demand and price in international market. Crab is very tasty
and many countries of the world import huge amount of crabs for consumption every year. As a
result, there are huge possibilities of earning foreign currencies by exporting crabs. The main
benefits of crab farming are, labor cost is very low, production cost is comparatively lower and
they grow very fast. Commercial crab farming business is developing the lifestyle of the people
of coastal areas. By proper care and management we can earn more from crab farming business
than shrimp farming. And small scale crab farming is gaining popularity day by day. Mud crab
farming systems in coastal areas are described below.

Types of Mud Crab

Mud crab can be found on estuaries, backwaters and coastal ares. They are member of Scylla
genus. There are two species of crabs available that are suitable for commercial production. Two
species of crabs are red claw and green mud crab.
Green Mud Crab
 Green mud crabs are larger in size.
 A green mud crab can grow to a maximum size of 22 centimeter carapace width. And it can
weights about 2 kg.
 These are free living and distinguished by the polygonal markings present on all
appendages.
Red Claw
 Generally red claws are smaller in size than green mud crab.
 A red claw can grow to a maximum size of 12.7 centimeter carapace width. And it can
weights about 1.2 kg.
 It has a burrowing habit and there are no polygonal markings on it.
Both species are suitable for commercial crab farming business. And both have good value and
huge demand in the foreign market.

Page | 45
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Mud Crab Farming Methods

You can raise mud crabs in two systems. Grow out farming and fattening systems. The systems
of farming in this two methods are shortly described below.
Grow Out System
In grow out farming system, young crabs are raised and grown for a certain period of 5 to 6
months till they reach marketing size and weight. This type of crab farming system is generally
pond based. The pond size depends on the production type. Generally ponds for crab farming
sized between 0.5 to 2 hectors. Proper bunds and tidal water exchange is a must. Small sized
ponds are very suitable for crab farming. Because they are easily maintained. Make a suitable
fence if the size of pond become small. In larger sized ponds where natural conditions are
prevailing, strengthening is necessary along the outlet area. You can stock wild collected
juvenile crabs that weights around 10 to 100 grams. Depending on the size of crabs and available
facilities the duration of production may varies between 3 to 6 months. In commercial production
with supplementary feeding you can stock 1-3 crabs per square meter. You can feed your crabs
low cost fish, shrimps, small sized crabs etc. You can visit your nearest local market and collect
rotted fish and innards of birds and animals from slaughter house. Provide the crabs 5% feed
daily of their total body weight. For example, if there are 100 kg crabs in the pond then feed 5 kg
food daily. Collect some crabs and try to determine an average weight. Regular sampling is very
necessary for monitoring the growth and general health, and to adjust the feeding rate. Keep
some pipes in the pond for shelter and the purpose of reducing mutual attacks and cannibalism.
Within 3 to 5 months they will reach marketing weight and become suitable for selling.
Fattening System
Raising soft shelled crabs for a certain period until their exoskeleton gets hardened is known as
crab fattening system. Hard shelled crabs has four to five times more value in the market than

Page | 46
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

soft shelled crabs. Farming crabs in this system take less time and the process is very profitable.
You can do crab fattening business in two systems that are described below.
 Fattening in Pond: Fattening can be done in any types of ponds between 0.025 to 0.2
hector size. Small tidal ponds with a depth of 1 to 1.5 meter is very suitable for crab
farming. Prepare the pond perfectly before stocking crabs in the pond. Pond preparation can
be done by draining the pond water, sun-drying and adding sufficient quantity of lime.
Make a fence around the pond for fattening purpose. Because the crabs have a tendency to
escape by making hole and digging the soil. Reinforce the inlet areas with bamboo matting
inside the bund. For stocking, collect soft crabs from local fisherman or crab merchants.
Collect the crabs in morning. 1-2 per squire meter stocking density is ideal for crab
fattening purpose. Divide the pond into different compartments according to the size of
crabs if it is big sized. Keeping male and female crabs separated from each other will make
good results and reduce mutual attacks and cannibalism. Depending on your location and
crabs availability 8 to 12 fattening cycles can be done in a year. Generally, crabs weight
between 300 grams to 500 grams have high demand and value in the market. Collect and
sell all the crabs when they reach the marketing weight. Always try to sell the crabs when
they are in hard shelled condition. This will ensure high profit form crab farming business.
 Fattening in Pens or Cages: Crab fattening can also be done in pens, floating net cages,
bamboo cages in shallow estuarine waterways and inside large shrimp ponds with good
tidal water influx and in tanks. You can use bamboo splits, netlon or HDPE as netting
material. 3 m * 2 m *1 m (3 m long, 2 m wide and 1 m height) is ideal cage size for crab
fattening. Arrange the cages in a row so that you can easily feed and monitor the crabs.
Stocking density of 10 crabs per squire meter in cage and 5 crabs per squire meter in pens is
ideal. Maximum stocking density can result mutual attacks and cannibalism. Fattening in
cages or pens in only used in small sale production. For commercial production fattening in
ponds is perfect and more profitable.
Between these two crab farming methods, fattening system is more profitable than grow out
system and has many advantages. Grow out crab farming system takes more time than fattening
system. But fattening system is very popular to the farmer as it take less time and highly
profitable.

Water Quality
Water quality plays an important role in the production of crabs. Change water occasionally if
possible or apply proper medicines or chemicals. See the following chart.

Page | 47
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Feeding
For commercial purpose, crabs need 5-8% food of their body weight. You can feed your crabs
low cost trash fish, chicken waste, animal innards collected form slaughter house, brackish water
clams etc. Don’t served all the feed at once. Instead give it twice a day. Give major part of the
total feeds during evening hours.

Marketing
After a certain period check the crabs for their hardening. In grow out crab farming system they
become suitable for marketing purpose within their 3 to 6 months of age. And in fattening
system the time depends on crab’s size. However, collect the crabs when they reach proper
weight and when their price remain high. Collect the crabs in the early morning hours or evening
hours. You can collect crabs from pond by using scoop net or by using alluring bait. Wash the
collected crabs with good brackish water and remove all types of dirt and mud. And then
carefully tie the crabs very carefully without breaking its legs. Then try to keep those crabs in
moist conditions. Keep them away from sunlight. Because direct sunlight has a negative effect
on their survival. After that send them to the market.

Commercial crab farming business is gaining popularity day by day in many coastal areas around
the world. Because it is a very easy, profitable and takes less time. Mud crabs have huge demand
and high value in international market. So, you can earn some extra money and make an
employment opportunity by doing commercial crab farming business.

************************************************************
Lobster culture

Page | 48
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

INTRODUCTION
• Eight species of spiny lobsters, six shallow water and two deep sea species, and
two species of slipper or sand lobsters constitute the lobster fishery of India.
• Spiny or rock lobsters have a sub-cylindrical body with long cylindrical antenna
with whip like flagellum.
• The carapace is covered with numerous spines and tubercles.
• The slipper or sand lobsters are with a dorsoventrally flattened body and short
scale like antenna without whip like flagellum.

Page | 49
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Page | 50
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

RESOURCES
• Lobster catch in India is around 2000-3000 tonnes per annum and most of it is
exported frozen, whole cooked or live.
• Export of whole lobsters since late 80's and live lobsters since 1993 and the ever
increasing demand for Indian lobsters have resulted in their regular and organised
exploitation.
• Maharashtra and Gujarat are the main lobster fishing states followed by Tamil
Nadu.

Page | 51
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

• While lobsters are landed as a bycatch in fish/shrimp trawls in the north-west

coast, they are caught by gillnets, traps and occasionally by trawls in the south-east and
south-west coasts
• Lobsters weighing 200 to 300g are best suited for whole cooked product while
those weighing over 300g (greens) and 500g (tiger) are in demand for live lobster export.
• High demand for live lobsters, which is Rs.600-1500/kg depending on size, has
recently generated considerable interest in culture/fattening of spiny lobsters.

Seed
• All commercially important species of shallow water spiny lobsters in India have
been bred in captivity, but their whole larval cycle is yet to be completed.
• Scientists at the Central Marine Fishers Research Institute (CMFRI) were
successful in rearing lobster larvae to more than half way stage and efforts are on to
complete the larval rearing process.
• In India, at present, has to start with collection of lobster juveniles from nature
and growing them to the required size.
• As there are no size regulation in our country, about a third of our commercial-
catch are undersized juveniles.
• These juveniles can be utilized for lobster culture/fattening.

Page | 52
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Nursery
• The nursery phase typically involves stocking the pueruli at 50-100/m2 into
submerged cages, consisting of mesh surrounding a steel frame.
• Each cage is placed on the sea floor at 2-5 m depth and a feeding tube from the
surface to the cage provides the means to feed the baby lobsters.
• Finely chopped trash fish, crustaceans and molluscs are used as food.
• The nursery phase lasts for 3-6 months, during which the lobsters grow to 10-30
g.

Page | 53
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Page | 54
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

Grow-out
out of tropical spiny lobsters is performed in sea cages
• cages are now deployed in deep water from floating frames that are moored to the
bottom.
• Grow-out cages are typically square in cross
cross-section
section 3 to 4 m along each side and
from 3 to 5 m deep.
• Individual farms vary in size from 10 to over 80 cages housed within one inter-
inter
linked floating framework, with narrow walkways between the cages for access.
• Each farm includes a small house for the farmer which is manned continually to
ensure security of stock and to perform routine feeding, cleaning, harvesting and re-
re
stocking.
• Lobsters are typically stocked for on
on-growing at 10-50 g each.
• These smaller lobsters may be stocked into cages with a smaller mesh size to
ensure they do not escape.
• Stocking density may be up to 30/m².
Page | 55
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

• As lobsters grow, they are periodically harvested and manually graded to

minimise the size variation within each cage.
• Larger lobsters are stocked at lower densities, typically around 5/m2 at 200 g and
2/m2 at 500 g.
• P. ornatus is usually on-grown to 1 kg which achieves the best price for export to
China.
• This typically takes 18-20 months.
• In Indonesia, where P. homarus is most commonly farmed, the desired market
size is 100-300 g, which takes ~9 months.
Fattening
• The tiger, P. ornatus is the ideal speoies due to its faster growth rate and
maximum value in live export.
• P. ornatus of 100-150g siz can be grown to 500g in about 8 months in indoor
culture systems under ideal rearing conditions.
• Since they attain maturity only at larger-size (700-800g), juveniles of this species
are more suited for farming to the target size of 500g and above.

• Fattening of larger size (300-350g to 500; 750-800g to 1000g) can be done in

shorter period of 3 to 4 months.

Page | 56
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Growth enhancement by eyestalk ablation

• Three to seven fold growth enhancement was achieved in four species of Indian
spiny lobsters by bilateral eyestalk ablation (removal of both the eyes).
• The tiger has been grown from 100g to 1500 gm 8 months by this technique.
• Research is on to find out whether the same result can be achieved by inactivating
the eyestalk hormones by laser or other modern techniques, rather than by eyestalk
ablation.

Factors influencing growth of lobsters

• Salinity, dissolved oxygen (DO), pH, temperature and nitrogenous metabolic
wastes, especially, ammonia, are the major water quality parameters regulating lobster
growth.
• Stocking density, provision of shelter, handling stress and intensity of light also
influence growth in captivity.
• Quality of feed plays a major role in obtaining optimum growth and body
colouration

Feed supply
• In nature, spiny lobsters feed predominantly mussels, barnacles, small crabs,
echinoderms and plychaete worms
• Farmed lobsters are traditionally fed a mixture of fish, crustaceans and molluscs
which come from the fish markets nearby.
• This so-called ‘trash fish’ can be highly nutritious if fresh and handled
appropriately.

Page | 57
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Page | 58
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Page | 59
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Page | 60
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Harvesting techniques
• Lobsters are easily harvested from the sea cages by pulling the net cage to the
surface and retrieving the lobsters by hand.
• Lobsters ready for market are placed in styrofoam boxes and returned to shore to
processing / export facilities.
• The farmer typically sells the lobsters at this point and the wholesaler takes on the
responsibility for further handling and transport to market.

Live export of lobsters

• Live lobster export, started in 1993, touched 24 tonnes in 1994 and is on the
upward trend reaching 99 tonnes in 1996.
• Madras is the main city for live lobster export with a share of more than 90%.
• Bombay and Thiruvananthapuram are the other cities from where lobsters are
exported live.

Page | 61
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Handling and processing

• Wholesalers and exporters of farmed spiny lobsters employ live holding systems,
consisting of tanks with clean seawater that usually involve recirculation technology to
maintain high water quality.
• Lobsters purchased from the farmers are held only briefly for 1 or 2 days to
maximise their quality and are generally not fed.
• They may be cooled to 10-15 ºC to slow their metabolism and improve
survivability during transport. Individual lobsters are normally wrapped in newspaper and
placed in styrofoam boxes before being air freighted to market.

Production costs
• Tropical spiny lobster farming is currently (2010) a profitable business with
moderate to high establishment and operating costs and high returns.
• In Vietnam the cost-benefit ratio is around 1.4 and average net revenue around
USD 15 000/yr per farm.
• The most significant operating cost is feed, which accounts for more than 60 per
cent.
• The cost of lobster seed is also significant (22 percent).

Page | 62
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

• P. ornatus harvested at 1 kg are sold at about USD 45-60/kg in Vietnam.

• In Indonesia, P. homarus harvested at 100-300 g fetch USD 30-40/kg.

Page | 63
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

EDIBLE OYSTER CULTURE

Marine animals belonging to the families Ostreidae are called oysters in common usage.
Oyster is one of the best known and most widely cultivated marine animals.The oysters are
highly esteemed sea food and considered a delicacy in USA, Europe, Japan etc.
In India there is a growing demand for oyster meat in some parts of the country. Until
recently, oyster farming has been considered as a traditional practice followed only in the
temperate countries. The awareness about the vast potentialities for development of oyster
farming in tropics is recent.Serious efforts are now being directed in its development under
tropical conditions.

Scope for oyster farming in India

• In India pioneering attempts were made by James Hornell in 1910 in developing Oyster
culture in erstwhile Madras state. Central Marine Fisheries Research Institute undertook
scientific investigations at Tuticorin from early 70's and as a result, complete package of
the technology is now available in the country. Vast stretches of backwaters, estuaries
and bays present along Indian coast harbour natural population of the oyster suggesting
suitability of the habitat for oyster culture. Being filter feeders, the oyster converts
primary production in the water into nutritious sea food.
Candidate species
Six species of oysters namely the

Page | 64
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

• Indian backwater oyster Crassostrea madrasensis,

• Chinese oyster, C.rivularis,
• West coast oyster, C.gryphoides,
• Indian rock oyster, Saccostrea cucullata,
• Bombay Oyster, Saxostrea cucullata, and
• Giant oyster Hyostissa hyotis are found in India.
The first four species mentioned above are of commercial value.
Of the six species of oysters. The Indian backwater oyster C. madrasensis is the dominant
species, more widely distributed, is euryhaline and inhabits backwaters, creeks, bays and
lagoons and occurs in the coastal areas of the States of Orissa, Andhra Pradesh, Tamil Nadu,
Kerala, Karnataka and Andamans. C.gryphoides is also euryhaline and occurs along north
Karnataka, Goa and Maharashtra coast. C.rivularis is found along Gujarat and Maharashtra
coast while Saccostrea cucullatais found all along the main land coast and Andamans and
Lakshadweep islands.
BIOLOGY, TECHNICAL PARAMETERS AND FARMING PRACTICES OF Crassostrea
madrasensis
(I) Biology of C.madrasensis
The edible oyster is a sedentary animal. The soft body of the animal is encased in two shell
valves out of which the upper valve acts as a lid to open and close by contraction and
relaxation of the adductor muscle. Oysters mainly feed on organic detritus and
phytoplanktonic organisms like diatoms and nanoplanktons. They are also capable of
absorbing dissolved organic matter in the water through the surface of gills, palps and the
mantle.

Oysters are generally dioecious but hermaphrodites are not uncommon. Young oysters
of C.madrasensis, primarily function as males (60-75 per cent) and later become females.
In zero age group upto 78 mm. in length, 75 per cent are males and in one year and above
with 80 - 115.5 mm. length, females represent 72 per cent. The peak spawning period is
reported to be during March-April and July-September. Oysters, like other bivalve molluscs,
spend the first few weeks of their lives as small, drifting larvae.When the larva is about one-
third millimetre long, it attaches to a substrate (sets) undergoes a change in its internal

Page | 65
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

organs, eventually reaches sexual maturity and spawns, thus completing its life cycle.

Page | 66
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Technology of oyster culture

The technology of oyster culture consists of two important phases namely
(A) Oyster seed production/Spat collection and
(B) Grow- out.
(A) Oyster seed production/ spat collection
The seed requirement for culture of oyster is met either from natural spat collection or
through hatchery rearing. For collection of spat from natural grounds, suitable spat collectors
or cultch materials are provided at appropriate time which may be oyster shells, coconut
shells, asbestos sheets, mussel shells or other materials. These are arranged on Nylon rope as
strings and suspended from racks in the water at suitable spots. The larval period
of C.madrasensis is 15 to 20 days and as such exposure of collectors will be ideal just after a
week or 10 days of spawning activity.
A reliable source offering sufficient quantities of spat of the desired species is critical to
successful oyster culture.Natural collection is the most important source of spat and will
continue to be so until commercial hatcheries are establishedMass production of oyster
seed is also possible in hatchery system for which technology is available, though no
commercial hatcheries are available yet. For efficient spat collection the farmer should
know the
(a) spat setting season and
(b) the sites to collect sufficient spat for stocking in the grow-out ponds.

Page | 67
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

(B) Grow out

Site Selection

For selecting suitable site for farming, several factors like water depth, bottom characteristics,
protection from wave action, tidal flow and height, turbidity, water quality including chemical
parameters, predation, fouling, pollution and accessibility are considered. Selected areas should
be sheltered from strong wave action, salinity should be from 22 to 35 ppt and temperature range
should be from 21 to 31 degree Celsius.

Page | 68
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Farming methods

• Farming methods are normally grouped as

(a) bottom culture and

(b) off bottom culture.

Raft, rack, long-line and stakes are used in various off-bottom culture practices.

The bottom culture method is yet to be experimented in India.

The off bottom culture methods are advantageous over the bottom culture due to the following
reasons :

(i) The growth and meat yield is relatively better.

(ii) It facilitates three dimensional utilization of the culture area.
(iii) Biological functions like filtration, feeding etc. become independent of tidal flow.
(iv) Silting and predatory problems are minimum.

Various off bottom culture methods are as follows.

a. Rack and string (ren) method

• It is also called ren method. This is the most common method advocated for Indian
conditions for which the oyster shell ren is used as spat collector This method is ideal for
shallow estuaries, bays and backwaters. The racks are constructed at 1 to 1.25 m depth.
Rack is a fixed structure, comprising several wooden poles vertically driven into the
substratum over which a wooden frame is made at a height of 0.5 m, above the water
level. The shell strings are suspended from these racks. A rack covering 80 m 2. area holds
90 strings and 125 racks in a ha. At the end of 7-10 months, each string may weigh 7 to
7.5 kg. and the production of oyster is estimated at 80 tons / ha. The mortality is about 45
per cent. The meat yield is about 10%.

Page | 69
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

b. Rack and tray method

The nursery reared single spat (cultch-free) measuring about 25 mm are transferred to
trays of size 40 x 40 x 10 cm at a density of 150 to 200 spat / tray. The tray is knitted with
2 mm synthetic twine of appropriate mesh size and is suspended from the rack. Once the
oyster reaches 50 mm length they are segregated and transferred to rectangular tray of
size 90 x 60 x 15 cm and these trays are placed on the rack which occupies 25 sq.m area
and holds 150-200 oysters. The average growth rate of oyster is 7 mm/ month and at the
end of 12 months, the oyster attains an average length of 85 mm.

The production estimated is 120 tons/ ha/ year which when compared to string method is
higher, however the production cost is quite high.

Page | 70
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

c. Stake culture
In this culture method, stakes with one nail on the top end and two nails on the sides are
driven into the substratum . These nails hold the shells with spat. The stakes are placed
60 cm. apart. In this method, the nursery rearing of spat is carried on the same
stake.Initially for 2 months, the spat is covered with velon screen till a size of 25-30
25 mm.
is attained and in another 10 months they reach marketable size.The production is
estimated to be 20tonnes/ ha/ year.

Page | 71
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

d. Raft culture

Raft is the most suitable farm structure in sheltered bays where the depth is 5m and
more.Wooden poles placed parallelly and tied across with coir rope to make a rigid
frame.Four empty airtight barrels of about 200 litre capacity are tied to the underside of
the raft at corners. It is moored by two anchors and a chain. The size of the raft varies
however rafts of size 6m x 5 m. are found to be quite suitable. PVC pipes instead of
wooden poles and styrofoam floats in place of barrels may be used.However, this method
has not been tried in India so far.

Page | 72
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

e. Long line culture

In this system long ropes or cables are anchored at each end and are supported at intervals
by floats. Long lines of 50-100 m length are easy to manage. Double long lines
comprising of one line on either side of the floats are also used.

Page | 73
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Farm management

Farm management practices involve periodic cleaning of the oyster, oyster rearing trays,
farm structure like racks, thinning, sorting or grading and manual removal of predators and
foulers. ‘Fouling’ includes mud, ascidians, coral, sponges and other encrusting organisms.

These agents attach themselves to trays and oysters and interfere with the feeding and
respiration of the oysters. If not attended to on time, a thick blanket of fouling organisms and
silt develops and the growth of oysters is hampered. Mortality also increases due to the
restriction of water circulation over the animals.

Harvesting

Oysters are harvested when the condition of the meat reaches high value which in case of C.
madrasensis is found to be good during March-April and August-September.Harvesting is
done manually and oysters are transported to shore in dinghies. After landing, the harvested

Page | 74
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

oysters should be brushed and any fouling organisms removed.Oysters should be depurated
to ensure they are free of bacterial contamination.

Depuration should be carried out for 36 hours. Un depurated oysters are unsafe for
consumption and may cause gastroenteritis and related diseases. Reservoir water in the
depuration unit should be replaced for each run. Oysters are marketed after depuration. Some
oysters may be sold as shucked meat.A special ‘shucking’ knife should be used to open the
oysters and remove the meat. Care must be taken not to damage the oyster meat during shucking.
The meat should be weighed and then kept on ice until sale.

Training and Extension

The Central Marine Fisheries Research Institute (CMFRI), ICAR provides technology,
training and extension services to the interested farmers to take up culture.

Page | 75
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

CHROMOSOMAL MANIPULATION

Chromosomal set manipulation can be used to produce highly inbred fish in a relatively short
period. Individual fish with F = 100% can be produced in a single generation, while inbred lines
where all fish have F = 100% can be produced in two generations. Chromosomal set
manipulation to produce inbred fish can be done in one of two basic ways, but regardless of the
technique used, the fish that are produced have only a single parent.

The first technique is to prevent the first mitotic division that occurs when the zygote nucleus
and zygote itself divides to become a two-celled embryo.To create inbred fish, this technique is
done with haploid zygotes. This technique is called either “mitotic gynogenesis” or “mitotic
androgenesis,” depending on the whether the haploid set of chromosomes of the zygote comes
from the mother or from the father.

The second technique is to prevent equational division (second meiotic division) of the
secondary oocyte (egg) after sperm penetration; this prevents the second polar body from leaving
the egg. This technique is called “meiotic gynogenesis”, because chromosomal set manipulation
is accomplished by disrupting a meiotic division; it produces fish called “meiotic gynogens”,
because all chromosomes in the offspring come from the mother.

This technology requires highly skilled labour, and the methodologies have not been perfected.
At present, these breeding programmes are important for some kinds of genetic research, but
their practical use has not been quantified. Consequently, these breeding programmes should be
done only by scientists who work at agribusinesses or research institutions that are capable of
conducting sophisticated genetics experiments.

************************************************************************

Page | 76
SBT 1304 – MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

PEARL OYSTER FARMING

Taxonomy

• Genus: Pinctada

– Includes most of the pearls

found in fashion.

• Pinctada maxima

• Pinctada fucata

– Akoya pearls (classic)

– Pinctada margartifera

– Tahitian peals (black)

Page | 77
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

– South Sea pearls

78
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Economic Importance

• Billion dollar retail industry

– Sold all over the world

• Price depends on rarity and quality

– $50 Pair of freshwater pearl earings to $100,000 strand of South Sea pearls.

Reproduction in Captivity

Thermal stimulation induces spawning.Larvae are allowed to float freely in the water under
controlled conditions until they are a few weeks old.Once the larvae develop into baby

79
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

oysters they are moved to a “nursery” area.Remain in nursery for about 1-2 years, until they
are large enough to be grafted.

80
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Saltwater Nucleation In Pearl Farming (Grafting)

Two basic methods of nucleation are used. Saltwater oysters are generally nucleated using a
"bead", prepared from mother-of-pearl. First, the bead is surrounded by a small piece of
mantle tissue taken from a donor oyster. The bead and tissue are then implanted into the
oyster's gonad.

The bead serves as a mold, or nucleus, around which the pearl develops. The resulting pearl
will contain the bead at its center and will tend to develop in the same general shape as the
original bead. The bead can be detected in the final pearl by x-rays.

81
SBT 1304/MARINE
/MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

Growing

• Raft Culturing

– Appropriate for sheltered bays

82
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

• Long-line culture method

Cages are hung from horizontal ropes or chains connected to floats.Oysters are
threaded at onto a small thread or rope that is hung from a raft.Good for open ocean
environments

• On-bottom culture

Can only be used in areas of granite or coral sand composition of the sea
bottom.

83
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

84
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

THE PEARL IS NOW ALLOWED TO GROW

After nucleating, the oysters are given a few weeks to recover from the surgery. During this
time, some of the oysters may reject and expel the implanted nuclei; others may become sick
or even die. Most, however, will fully recover. The oysters are then placed in cages or nets
and moved into the oyster bed, where they will be tended as the pearls develop.Depending on
the type of oyster, this process can require anywhere from a few additional months to several
more years!

85
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Harvest

Akoya pearls are harvested after 8 months – 2 years. All other pearls are harvested after 2 – 6
years. Harvesting is done in the winter months, when the pearl luster is highest. An x-ray can
be used to determine pearl size before harvest.

86
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

After the pearls are extracted from the oysters, they are washed, dried, and sorted into general
categories. Sometimes, the pearls are polished by tumbling in salt and water.The pearls are
then sold to jewelers, manufacturers, and pearl dealers.

87
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

MUSSEL CULTURE

INTRODUCTION
Mussels are bivalve molluscs and are found attached to rocks or any other hard substratum
by means of byssus thread secreted by the body. They belong to the family MytilidaeIn India
two species of marine mussels namely Perna viridis the Green mussel and Perna indica the
Brown mussel forms the major part of the fishery.

Kerala State can be called as the Mussel fishery zone of India since extensive beds of both
the green and brown mussel occur in this state which also account for the bulk of mussel
production in India. Of the two species commercially important the green mussel P. viridis is
widely distributed and found in the beds of Chilka lake, Visakhapatnam, Kakinada, Madras,
Pondichery, Cuddalore and Porto Nova on the East coast and extensively around Quilon,
Alleppey, Cochin, Calicut to Kasargod, Manglore, Karwar, Goa, Malwan, Ratnagiri and the
Gulf of Kutch on the West coast.

P. viridis occurs from the inter tidal zone to a depth of 15 m. On the other hand , P indica has
restricted distribution and is found along the southwest coast from Varkala near Quilon to
Kanyakumari and from there to Tiruchendur along the southeast coast. It occurs from the
inter tidal zone to 10 m depth.P.viridis is widely distributed and hence more suitable for
farming.

Perna virisis Perna indica

88
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

1. Area suitable for farming

For sea farming, coastal waters beyond surf zone at 10 - 15 mt depth is normally
selected.The area should be sheltered from strong wave action. The site should be free
from any major industrial effluent and should not interfere with transport or any other
fishing activity. Clear water with good phytoplankton production and moderate current to
bring in the food and carry away waste products is required. A salinity range of 30-35 ppt
is preferred.

2. Farming Technology of Green Mussel

1. Biology

The scientific name of the green mussel is Perna viridis. The mussel has organ
systems similar to those found in oysters with some modifications.It has a foot as in
clams though smaller in size, providing limited mobility. A mussel can discard the
byssal strands and secrete new ones for enabling it to change position.
Phytoplanktons forms the food of the mussels, and they are filter feeders.

P.viridis in the natural conditions grow to 63 mm in 6 months to 133 mm in 4

years.However , the growth in culture operations have been more than in the natural
conditions. In mussel the sexes are separate and the gonads which are located in the body

89
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

proliferate into mantle. The male gonad is creamy white in colour while in the female it is
pink or reddish. The mussel attains first maturity at 15.5 to 28 mm size.

90
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

91
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Technology of mussel culture

A) Seed collection / Availability

The spawning season of the green mussel is between July and September and the
spats are found carpeting the inter tidal and submerged rocks.At present they are
collected manually and during the peak season an individual would be able to collect
10-12 kg of seed in one hour. The seeds can also be collected using spat collectors
such as roof tiles, coir ropes and nylon ropes.

Even though the hatchery technique for commercial mussel spat production has been
perfected by Central Marine Fisheries Research Institute, Cochi, there is no
commercial hatchery at present in India.As such the culture operations have to
depend on the availability of natural seed .

92
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

B) Farming models

Three types of farming are practiced for culture of the mussels as follows:

i. Sea Farming
ii) Estuarine farming
iii) Rope culture
i. Sea Farming

Longline culture of mussel is practised in shallow waters of 10 - 15 m depth .

This method of culture can withstand the severe monsoon conditions in the west

93
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

coast. The longline unit consist of 60 mt long horizontal HPD rope of 20-24 mm
thickness anchored at both the ends with 150 Kg concrete blocks and a series of
100 liters capacity barrels as floats fixed at 3 m intervals. Vertical lines of 6 m
length seeded with mussel spats are hung at a distance of 75 cm between two
floats in the main line. A longline unit of 60x60 mt can accommodate 12
horizontal ropes and 920 - 1000 vertical ropes. The distance between two
horizontal lines is 5 mt . At every 20 mt the horizontal lines are connected using
additional horizontal lines.

ii) Estuarine farming

Pole culture and stake culture are done in estuaries at a depth of 1.5 to 3 m. The spats of 15 to 25
mm are wrapped around the poles or stakes with cotton mosquito nettings. The spats gets
attached to the poles in three or four days and by this time the cotton netting will disintegrate.
Periodical thinning is necessary.

94
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

iii) Rope culture

Rope culture of mussel is widely adopted in Northern Kerala. Ropes are suspended from rack
made of casuarina and bamboo poles. The average area of rack is 400 sq m and length of the
ropes used for seeding ranges from 1-1.25 mt depending on the depth of the water
column.Poly propylene ropes wound with coir ropes are used for seeding.

These ropes are hung down from the racks at an interval of 1 feet and nearly 500 - 550 ropes
could be suspended from one rack. The seeds collected from wild are being sold in units of
one bag and one bag of seed can be used to seed 8-10 ropes. The normal size of the seed
ranges from 35-65 mm.

Seed collected has to be seeded on the same day and it is estimated that one person can seed
around 60-70 ropes in a day. The culture period in Northern Kerala where the activity is
taken up fairly on a large scale starts from November and ends in the middle of May before
the rains. Once in a fortnight the ropes are lifted for monitoring the growth and removal of
fowling organisms.

The mussel grows to 80-100 mm size with in 6 months of culture period and it is estimated
that around 2 lakhs mussels can be harvested from 400 sq mtrs.

95
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

4. Processing

Before removing the meat from the mussel it is necessary to carry out depuration which
is a process in which the mussels are kept for 18 hours in clean sea water which will
purify the mussels of bacterial pollution. The mussels can be processed in different forms
like frozen, canned, smoked, dried and marinated. The mussel shell is used as a liming
agent in coconut plantations.

The mussel shell gives good quality lime which finds application in many industries.

96
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Financial viability

The following assumptions have been made on the basis of the farming practiced in
Kerala for working out the financial viability of the project.

1 Unit size of rack ( Area ) 400 sq m

2 Culture period 6 months
3 Size of the seed (Spats) at the time of
35-65 mm
seeding
4 Size at harvesting 80-100 mm
5 Number of mussel that could be harvested
400 sq m 2 lakh
from
6 Production
7 1st year 70%

5. Marketing

There is only limited demand for the mussel meat due to lack of awareness among the
consumers . However, there is scope for its export to Southeast Asian countries. A marketing
tie-up with the processing plants will be useful for marketing of the product.

97
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Marine algal culture (Seaweed culture)

• Seaweeds, which are macroscopic marine algae belong to the primitive non
flowering group - Thallophyta.

• They grow submerged and attached to hard substrata such as stones, rocks and
coral reefs along the shallow coasts, lagoons, estuaries and

• brackish water habitats of the Andaman - Nicobar and Lakshadweep islands and

• coastal areas of Tamil Nadu, Kerala, Karnataka, Maharashtra, Gujarat, Goa,

Orissa and Andhra Pradesh.

• Based on their pigmentation and other morphological characteristics they are

categorised into three major groups –

• Chlorophyceae which is popularly known as green seaweeds,

• Phaeophyceae or brown seaweeds and

• Rhodophyceae or red seaweeds.

Uses of seaweeds

• Seaweeds contain more than 60 trace elements in a concentration much higher

than in land plants.

• They also contain vitamins, proteins, essential amino acids, iodine, bromine and
antibiotics and several bioactive substances.

• They are used as human food, feed for livestock, poultry, fish and prawn and as
manure for many plantation crops.

• Agar is mainly produced from red seaweeds such as

Gracilaria edulis,

Gelidiella acerossa,

Gracilaria verrucosa and

• Carrageenan from

Eucheuma and

98
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Hypnea.

• Alginic acid and mannitol are manufactured from brown seaweeds such as

Sargassum and

Turbinaria

These Phycocolloids are used in

• food,

• confectionery,

• pharmaceutical,

• biomedical,

• dairy,

• textile,

• paper and

• paint industries as

• gelling,

• stabilizing and

• thickening agents.

• In Japan, Malaysia, China, Philippines and Indonesia,

Green seaweeds such as

• Ulva,

• Enteromorpha,

• Caulerpa,

• Codium and

• Monostroma;

Brown seaweeds such as

99
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

• Sargassum,

• Hydroclathrus,

• Laminaria,

• Undaria and

• Macrocystis and

• Red seaweeds such as

• Porphyra,

• Gracilaria,

• Eucheuma,

• Hypnea,

• Laurencia and

Acanthophora are consumed as vegetables, in soups, salads, porridges and pickles.

• Resource assessment surveys on seaweed conducted by CMFRI, CSMCRI and

NIO indicate that the total standing crop along the Indian coastline consists of
more than 1,00,000 tonnes

• wet weight per year belonging to 680 species of which 60 species are
economically important.

• They comprise 8,000 tonnes of agar-yielding seaweeds, 6,000 tonnes of

carrageenan-yielding seaweeds and 16,000 tonnes of algin yielding brown
seaweeds.

• Edible and other green seaweeds constitute a bulk of 70,000 tonnes.

Why cultivate seaweeds?

• In India there are about 50 seaweed industry units located in Ahmedabad,

Baroda, Cochin, Hyderabad, Madurai and Ramanathapuram.

• They depend only on natural seaweed beds for their raw material.

• A rough estimate indicated that 8,100 tonnes dry weight of agar and algin-
yielding seaweeds was utilized by the industry in the year 1995.

100
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

• As more and more new industries are coming up every year, exploitation rate
exceeds the harvestable biomass.

• The indiscriminate exploitation of these resources from the natural beds leads to
shrinking of stock.

• Hence, mariculture of seaweeds all along the Indian coast, estuaries and certain
backwaters is the only way to increase the production of phycocolloids and
thereby to make the industry commercially attractive.

Advantages of seaweed culture

a) Increases the seaweed production

b) Desirable varieties can be selected and cultivated on a large scale

c) Natural beds can be protected and conserved against over-exploitation

101
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

d) Exotic or oriental species of commercial seaweeds such as Eucheuma can be

introduced, acclimatised and cultivated in our waters

e) Can support seaweed industry by regular supply of raw materials of same quality
and maturity at low cost

How to culture seaweeds?

• The Central Marine Fisheries Research Institute has developed technologies for
mariculture of seaweeds especially the agar-yielding red seaweed Gracilaria
edulis by constant research since 1972.

• Calm and shallow coasts and bays and lagoons with sandy bottom are ideal sites
for G. edulis culture.

• For optimum growth of this weed a salinity of 28-35 ppt is desirable.

• G. edulis can easily be grown to harvestable size within 60 days from small bits of
vegetative fronds.

102
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

• This is cultivated in 5 x 2 m size net rafts made of coir ropes.

• G. edulis stock collected from natural bed are cut into small bits of approximately
5 cm size.

• These bits of about 5 g are inserted between the twists of the rope.

• These floating net rafts are tied to wooden poles that are staked from the sea
floor or tied to floats and anchors in places where wooden stakes cannot be fixed.

• Like this about 900 net rafts can be accommodated in a hectare area.

• These rafts are submerged in 30-40 cm water column to avoid desiccation during
the ebb tide.

Besides net rafts, G. edulis can also be cultivated in long-lines made of thick 10m long
coir ropes from which small seeded ropes be suspended at regular intervals.

Seed stock for mariculture can be obtained from Rameshwaram and Kilakkarai of Tamil

103
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Nadu coasts or from the lagoons of Lakshadweep islands.

Once in every fortnight cleaning the rafts is desirable to remove epiphytes and other
attached weeds.

• The harvest is made after 60 days by cutting the loosely grown fronds, leaving
the base attached to the rope.

• This forms the seed material for the second crop.

• Approximately 30 kg of seaweed per net can be harvested from 10 kg seed stock.

• In this way during the fair weather three or four crops can be cultivated in a
year.

104
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

The harvested seaweed must be cleaned and dried well before storing.
The moisture content in G. edulis ranges from 70- 75%.
Hence the dry weed weighs one fourth of the weight of fresh seaweed.
Dried seaweeds are sold to the industry at the rate of Rs.4,000 to 5,000 per tonne.
Agar is extracted from the dry seaweed.

105
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

106
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

• Production of oyster, mussels, cuttlefish, fin-fishes, shrimps, lobsters and

seaweeds through aquaculture has increased from just 10.4 million tonnes in
1980 to 22.6 million tonnes in 1990.

• This hike in production is attributed mainly due to 87.2% increase in seaweed.

107
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

• China achieved a record production of 2,75,000 tonnes dry weight of Laminaria in

1990 from just 62 tonnes in 1952.

• Today Japan stands first in the production of Nori or Porphyra, China for
Laminaria, South Korea for Undaria edulis and the Philippines for Eucheuma.

• India can take up Gracilaria culture and become the largest producer as she is
endowed with 8,041 km long coastline and 51.2 km^ of continental shelf area in
the form of sheltered bays and lagoons.

• Venturing into mariculture of G. edulis along the Kerala coast with the public
participation will be a highly profitable and spare time avocation.

108
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

109
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

FISH GENETICS:

Gynogenesis

Gynogenesis is the process of embryonic development with solely the maternal genome and
without paternal genetic input, a phenomenon similar to parthenogenesis. Gynogenesis occurs in
nature and can also be induced.

Mitotic gynogenesis

Mitotic gynogenesis can be used to create mitotic gynogens (all genes come from the mother),
fish that are 100% inbred. The technique that is used to accomplish this with species that have
the XY sex-determining system (females are XX and males are XY; virtually all aquacultured
species have this system of sex determination) is outlined in Figure.

The first step in this breeding programme is the production of first-generation mitotic gynogens.
Ultraviolet radiation is used to destroy the DNA (the genes) in sperm. The irradiated sperm are
then used to activate eggs. An irradiated sperm cannot fertilize an egg because its genes have
been destroyed. The activation causes the egg to undergo the equational division (second meiotic
division) and to extrude the second polar body. The egg now contains only a haploid egg
nucleus; this produces a haploid zygote (the zygote contains only a single chromosome
(homologue) from each chromosome pair, and each chromosome comes from the mother, which
is why they are called “gynogens”).

When the haploid zygote undergoes first cleavage, a pressure or temperature shock is used to
prevent the haploid zygote nucleus from dividing into two daughter nuclei.If the shock is timed
perfectly, the haploid zygote nucleus has replicated its chromosomes so that each daughter
nucleus will have a full and identical set of chromosomes, but the haploid zygote nucleus has not
divided.By preventing first cleavage, the zygote remains a zygote, but the chromosome number
of the zygote has doubled from the haploid state to the normal diploid state, which means that
each chromosome occurs as a pair.

110
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Since mitosis (first cleavage is a mitotic cell division) produces two identical sets of
chromosomes, each chromosome pair is composed of two identical chromosomes. Consequently,
every gene comes from the mother, and every gene is homozygous; the mitotic diploid gynogen
is 100% homozygous and 100% inbred. If first-generation mitotic gynogens are to be used in a
breeding programme to create inbred lines, a second phase of gynogenesis followed by sex
reversal is needed in order to produce the lines of 100% inbred fish, in which all fish within each
inbred line are genetically identical.

Each first-generation mitotic gynogen will be used to create a unique line of 100% homozygous
and 100% inbred fish by utilizing either of two possible types of gynogenesis. When the first-
generation mitotic gynogens mature, their eggs are stripped, and either mitotic gynogenesis is
repeated to produce second-generation gynogens or meiotic gynogenesis is used to create second
generation gynogens.

Half of the second-generation gynogens from each family are sex-reversed with anabolic
androgens (steroid hormones) to produce XX sex-reversed males. The sex-reversed males are
genetic females but phenotypic males. The fish that are not treated with hormones are raised
normally. Within each family, the sex-reversed males and their sisters are genetically identical
(genetically, they are all identical sisters); when they mate, they produce an inbred line of
genetically identical fish that is 100% female, 100% homozygous, and 100% inbred. Sex-
reversed males must be created every generation, because it is the only way males can be
produced, and it is the only way each inbred line can be perpetuated without additional
chromosomal manipulation.

111
SBT 1304/MARINE
/MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

112
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Meiotic gynogenesis

Gynogenesis can be used to create another type of inbred fish-meiotic gynogens. This type of
chromosomal manipulation is easier than mitotic gynogenesis, and meiotic gynogens have a
higher survival rate than mitotic gynogens because they have less inbreeding.Meiotic
gynogenesis is less useful in producing inbred lines because it is difficult to accurately predict
the exact amount of inbreeding produced, and the inbreeding produced each generation is quite
variable.Since regular systems of inbreeding are most useful when they produce reliable and
predictable amounts of inbreeding, meiotic gynogenesis is less useful than mitotic gynogenesis
for producing inbred lines. However, one to three generations of meiotic gynogenesis can be
used to produce highly inbred fish.

113
SBT 1304/MARINE
/MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

114
SBT 1304/MARINE
/MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

*****************************************************************************

115
SBT 1304/MARINE
/MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

Mitotic androgenesis

Mitotic androgenesis can be used to produce mitotic androgens (all genes come from the
th father),
fish that are 100% inbred. The technique that is used to produce mitotic androgens for species
with the WZ sex-determining
determining system is outlined in Figure

116
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Phase 2 of this breeding programme uses a second round of mitotic androgenesis, and half the
offspring from each family are sex-reversed with anabolic estrogens to produce sex-reversed ZZ
females. These females are genetic males but phenotypic females. Within each family, the sex-
reversed females are mated to their genetically identical brothers to produce each inbred line;
fish in each inbred line are genetically identical, 100% homozygous, and 100% inbred and all are
males. As was the case with mitotic gynogens, each line is genetically unique.

****************************************************************************

Polyploidy

An increase in the level of ploidy of an individual by the addition of one or more set(s) of
chromosomes refers to polyploidy resulting usually either in triploidy or tetraploidy.Sometimes it
may also result in penta or hexaploidy.

Natural polyploidy (triploidy/tetraploidy)

Polyploidy has been observed to occur in nature in some species of fish like the common carp
(Cyprinus carpio) and trout mainly due to chromosomal translocation and when two distantly
related fish species are crossed.The crosses between grass carp and bighead carp had produced
triploid hybrids (Marian and Krasznai, 1978).However, none of the interspecific nor intergeneric
hybrid crosses among Indian carps have been reported to produce such allotriploids.

Artificial induction of polyploidy in Indian major carps

Some preliminary attempts have been made to induce artificial induction of triploidy and
tetraploidy in Indian major carps with varied degrees of success

117
SBT 1304/MARINE
/MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

118
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Conclusion:

Chromosomal manipulation can be used to quickly produce highly inbred lines of fish.One
generation of mitotic gynogenesis or mitotic androgenesis will produce fish that are 100%
homozygous and 100% inbred. A second generation of chromosome set manipulation is needed
to produce 100% inbred fish that are capable of reproducing.

One generation of meiotic gynogenesis will produce fish that have large, but unknown, levels of
inbreeding; the amount of inbreeding produced by meiotic gynogenesis is variable and depends
on crossing over frequencies.The use of chromosomal manipulation to produce inbred lines
should be done only by scientists at large agribusinesses or at research stations.

***************************************************************************

119
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Artificial insemination
Artificial insemination (the collection of spermatozoa and ova and their mixing together in various media
that keep spermatozoa motile) is carried out in only a few species (mostly freshwater), such as
salmonids, cyprinids and acipenserids. Traditionally, fresh water (or sea water for marine species) is
used as the medium in which the male and female gametes are mixed. However, fresh water is not a
very favourable medium because hypotonic shock causes the sperm structure to deteriorate in several
minutes and the egg is activated quickly. These problems can be avoided by using as media various
saline solutions of different composition, depending on the species (125 mM NaCl pH 9 for salmonids;
50 raM Nacl pH 8 for cyprinids). These media prevent sperm deterioration, prolong slightly the duration
of motility, and prevent or defer the cortical reaction. These solutions also prevent the yolk of crushed
eggs from precipitating when it comes into contact with the water, limit motility and block the
micropyle. Fish farmers are beginning to use these media, so significantly increasing the fertilization rate
while reducing the number of spermatozoa used for insemination. The length of gamete survival is an
important factor to consider in carrying out artificial reproduction. Gamete survival in vivo (after the
release of sperm and oocytes from cysts and follicles) varies with the species. Sperm fertilizing ability
decreases during the spawning period in sea bass and trout but not in carp. Ovum survival in the general
or ovarian cavity is from one to several weeks in salmonids, several hours in carp at 20°C and only 30
min in Chinese carp. In vitro survival is from one to several weeks for sperm (under oxygen and with
antibiotics added) and several hours for ova (2–4 h in carp and 12–24 h in trout). The spermatozoa of
several species of teleosts have been stored deep frozen, but the quality of the sperm is not as good and
more spermatozoa per ovum have to be used to obtain the same percentage of fertilization as with
non‐frozen sperm.

INDUCED BREEDING AND LARVAL REARING

of
Clarias macrocephalus
by

Vijai Srisuvantach
Manob Thangtrongpiros
Regional Lead Centre in Thailand

120
SBT 1304/MARINE
/MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

National In
Inland Fisheries Institute
Bangkhen, Bangkok 10900
Thailand

1. INTRODUCTION

Walking catfish in English, or “pla duk” in Thai, is a generic name for a number of species
belonging to the family Clarridae. Five are encountered in Thailand, two of which are
popular sources of animal protein, Clarias batrachus and Clarias macrocephalus,
macrocephalus locally
known as “pla duk dan” and “pla duk oui”, respectively. C. batrachus fry is easily obtained
from the spawning pond. Unfortunately, C. macrocephalus do not readily reproduce
repro
themselves in captivity. However, it can be induced to breed if injected with extracts of fish
pituitary glands containing gonadotropin sex hormones.

Thai consumers have a preference for C. macrocephalus but, because of bottlenecks in fry
availability and slow growth, its culture is still limited in comparison to C. batrachus.
batrachus

Our biologists have worked for 20 years to develop artificial breeding methods. This attempt
was first successful for Pangasius sutchi and Chinese Carps in 1965. Further studies were
continued. Up to now, induced spawning is a rutine work for our aquaculturists.

The purpose of this review is to summarize the induced breeding and larval rearing of C.
macrocephalus practices in Thailand.

2. Characteristics and Biology

The species is closely related to C. batrachus from which it can be distinguished by the
wide occipital process (Fig. 1). Body is elongate with head broadly depressed, four pairs of
well developed barbells, and small eyes. Dorsal and anal fins are long without spine.
Pectoral
ctoral fin has a pungent spine with serrated on its inner edge. Caudal fin is not confluent
with dorsal or anal fin (Fig. 2). Body color is dark brown with purplish tint and about tent
transverse rows of small white spots on the side.

Fig. 1. Occipital process of Clarias macrocephalus (left) and Clarias batrachus (right).

121
SBT 1304/MARINE
/MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

Fig. 2. Clarias macrocephalus Günther

The species is distinguished by their ability to survive in a wide range of water conditions. It
requires a relatively small area for culture and can be stocked more densely than many
other species. They can live out of water for several hours or in waters of low oxygen
content as they have accessory organs that enable them to breathe atmospheric air. Its
range of distribution includes the areas fro
from
m Indochina penninsular. Thailand and the
Philippines.

The spawning season is between May and October. The female makes a small round
hollow nest with grassy bottom about 30 cm in diameter and 5 – 8 cm deep in shallow
water. The eggs are deposited in the nnest
est and attached to the roots of aquatic vegetation in
the nest. The male will take charge of these eggs until they are hatched out. The egg can
be hatched out within 20 hours at temperature of 25 – 30°C. A female weighing 300 – 800
gm can produce between 5,000 – 10,000 eggs. The natural diet is wide-ranging,
ranging, it includes
worms, insects, shrimps and decayed matter.

3. Selection of Spawners

The essentials in fish induced spawning are fully ripe mature brooders both female and
male. Brood fish should be carefu
carefully
lly tended for two to three months before induced
spawning operations are carried out. Males and females should be segregrated and
stocked in separate ponds. Selection of spawners is one of the most important stages in
induced spawning operations. It is ne
necessary
cessary to know how to select healthy males and
females in order to obtain maximum production of fry.

Determination of ripeness is an art and requires experience. To be good brooders the firsh
must be more than one year old or 150 gm. Sex can be distinguis
distinguished
hed by the shape of the
genital papilla (Fig. 3). The male genital papilla is pointed. The female papilla is oval shape.
The following characteristics can be used as guidelines to ascertain that the female is ready
for induced spawning operations. It has a bulging abodomen. It is clastic and soft to the
touch. The cloaca is reddish and prominent, and the contour of this ovary can be seem on
both sides of the abdomen.

4. Obtaining the Pituitary Gland

Pituitary gland contains hormone namely gonadotropin which stimulate the production of
sex steroids in the gonad which responsible for the maturation of gametes. Gonadotropin is
composed of follicle stimulating hormone (FSH) and luteinizing hormone (LH) which are
responsible for egg development and egg ovulation respectively.

122
SBT 1304/MARINE
/MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

Pituitary gland of common carp, Chinese carp, Indian carp, and Panqasius sutchi can be
used for induced spawning. The fish from which the hyopphysis is to be collected is
weighed and placed on a shopping board. The skull is cut open with a kn knife
ife (Fig. 4). After
removing a piece of the skull, fatty tissue and blood are wiped off with a cotton pad. The
pituitary gland can be seen after the mid
mid-brain
brain has been folded back by using forceps.

Fig. 3. Genital papillae of the male and female Clarias macrocephalus

5. Amount of Pituitary Gland Solution Injected

One concentration or one dose commonly used can be expressed as follow:

The gland is ground in the homoginizer: distilled water is added and the gland is again
ground. A syringe is used to take up the solution for injection (Fig. 5). The female can be
injected with aypophysis of Chinese carp in a dosage of 1.0 for the first injection and 2.0 for
the second injection with the time interval of 6 hours. Aliquots of isotomic saline solution or
distilled
lled water is added depending on the weight of recipient, about 0.5 ml for fish weigh
less than 1.0 kg of body weight and 1.0 kg body weight and 1.0 ml for fish weigh 1 – 3 kg.

The intramuscular injection is given in the area between the base of the dorsal fin and
lateral line (Fig. 6).

6. Artificial Insemination

123
SBT 1304/MARINE
/MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

Ovulation occurs about ten hours after the second injections. The water on the female's body

should be wiped off with a towel. As the abdomen is being pressed, the stripped eggs should be

collected in a dry plastic container (Fig. 7). At the same time, the milt is made to drip on the eggs

by grinding the testis with fingers and pouring the water through the fine mesh cloth. Eggs and

sperm are mixed and stirred gently with a feather. Next, a little cclean
lean water is added and gently

mixed again. After one to two minutes, water is added two or three times to cleanse the fertilized

eggs. The fertilized eggs are transferred to the hatching hapa (Fig. 8). Most of the fertilized eggs

hatch out within 24 hours.. Figure 9 shows the location of testis.

7. Fry Mursing

After yolk resorption, usually within 2 days, the larvae are transferred from hapa to the
nursery fiber glass tank. The fry develop feeding behaviour at about the same time their
yolk was absorbed. The food to be given for the first 3 weeks is live moina. Usually 3 weeks
old fry with the size of 2 – 3 cm are distributed to the fish farmers.

Fig. 4. Extraction method for removing the pituitary gland, showing transverse cut, mid-brain
mid
and pituitary gland location.

124
SBT 1304/MARINE
/MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

Fig. 5. Preparation of the pituitary gland solution

Fig. 6. Location for intramuscular injection.

125
SBT 1304/MARINE
/MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

Fig. 7. Artificial insemination.

126
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Fig. 9. Transferring fertilized eggs to incubator hapa.

127
SBT 1304/MARINE
/MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

128
SBT 1304/MARINE
/MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

Fig. 8. Location of testis with the digestiv

digestive track folded up.

8. Egg Development

The stages of egg development are given in Fig. 10.

Fig. 10. Egg development of Clarias macroccphalus, 70 X

129
SBT 1304/MARINE
/MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

Eyestalk ablation

The process of unilateral eyestalk ablation is used in almost every marine shrimp
maturation/reproduction facility in the world, both research and commercial, to
stimulate female shrimp to develop mature ovaries and spawn. This method of
inducing females to develop mature ovaries is used for two reasons:

1. most captive conditions cause in inhibitions

hibitions in females which keep them from
developing mature ovaries in captivity
2. even in conditions where a given species will develop ovaries and spawn in
captivity, use of eyestalk ablation increases total egg production and increases
the percentage of females
emales in a given population which will participate in
reproduction

Hormonal effects of eyestalk ablation

Indirect effects of eyestalk ablation

Eyestalk ablation techniques

Latency period: eyestalk ablation to ovarian development

Hormonal effects of eyestalk abl

ablation

The most commonly accepted theory is that a gonad inhibitory hormone (GIH) is produced in the
neurosecretory complexes in the eyestalk. This hormone apparently occurs in nature in the non-
non
breeding season and is absent or present only in low levels during the breeding season. By
inference, then, the reluctance of most penaeids to routinely develop mature ovaries in captivity is
a function of elevated levels of GIH, and eyestalk ablation lowers the high hemolymph titer of GIH.
The effect of eyestalk removal
emoval is not on a single hormone such as GIH, but rather effects numerous
physiological processes (Bray
Bray & Lawrence, 1992
1992).

Indirect ef
effects of eyestalk ablation

Considering that eyestalk ablation affects the hormone balance for numerous physiological
processes in addition to stimulation of gonadal hypertrophy, what are the practical effects of this
operation, and at what cost do we achieve induced ovarian developm
development
ent using eyestalk ablation?

130
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

The following observations have been made concerning use of eyestalk ablation in captive
reproduction, and may be related to either captive conditions, eyestalk ablation, or both:

 Captive spawn size (number of eggs per spawn) is smaller than in wild-matured females,
regardless of whether eyestalk ablation is used.
 Eyestalk ablation increases total egg production in captivity by producing more frequent
spawnings, but not larger spawns.
 There is not a strong trend toward diminishing spawn size over time.
 Molt cycle duration is shorter in eyestalk-ablated females than intact females.
 Higher mortality of eyestalk ablated females is often, but not always, reported.
 Eyestalk ablation has been suggested to deteriorate female condition.
 Eyestalk ablation in some instances has been observed to produce lower hatch rate of eggs
than unablated females.
 Hatch rate has been observed to decline over time under captive conditions.
 Ovarian color in captive females, especially in eyestalk ablated females, is often rather
different than wild-matured (Bray & Lawrence, 1992).

There is strong circumstantial evidence that part of the problems seen with captive
reproduction are related to a simple inability of current diets to supply required nutrients as
rapidly as required for the gonadal hypertrophy stimulated by eyestalk ablation. In nature,
an organism would not be anticipated to develop eggs, constituting some 10% of female
body weight, unless nutrients are available for first, metabolism, second, growth, and third,
reproduction. Eyestalk ablation accelerates the production of ova, regardless of whether the
proper types and balance of nutrients are available, and regardless of whether those ova are
even capable of fertilization. Dietary factors clearly have been shown to influence
percentage hatch and percentage of females spawning (Bray & Lawrence, 1992).

Eyestalk ablation techniquesShrimp should be ablated only when hard-shelled, never when in post-molt
(newly molted or seft-shelled) or premolt stages. Because molting and reproduction, especially in
females, are both energy demanding processes, they appear to be antagonistic in terms of biological
programing.

Unilateral eyestalk ablation is accomplished in the following ways:

1) Simple pinching of the eyestalk, usually performed half to two-thirds down the eyestalk. This
method may leave an open wound.

2) Slitting one eye with a razor blade, then crushing eyestalk, with thumb and index fingernail,
beginning one-half to two-thirds down the eyestalk and moving distally until the contents of eyes
have been removed. This method, sometimes called enucleation, leaves behind the transparent
exoskeleton so that clotting of hemolymph, and closure of the wound, may occur more rapidly.

3) Cauterizing through the eyestalk with either an electrocautery device or an instrument such as
a red-hot wire or forceps. If correctly performed, this method closes the wound completely and

131
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

allows scar tissue to form more readily. A variation of this technique is to use scissors or sharp
blade to sever the eyestalk, and then to cauterize the wound.

4) Ligation by tying off the eyestalk tightly with surgical or other thread. This method also has
the advantage of immediate wound closure (Bray & Lawrence, 1992).

Latency period: eyestalk ablation to ovarian development

Once females have been subjected to eyestalk ablation, complete ovarian development often
ensues within as little as 3 to 10 days, assuming the animals were removed from a breeding or
ready-to-breed population, of adequate size for reproduction, and not subjected to too much
transfer stress. If the animals have been removed from non-conducive environmental conditions
(e.g, cold, non-breeding season temperatures, or hypersaline conditions), a longer than normal
latency period between eyestalk ablation and ovarian development can be anticipated, probably
due to seasonal hormonal cycling. Duration of the latency period between eyestalk ablation and
maturation of ovaries is determined by the readiness of the population at the time of eyestalk
ablation (Bray & Lawrence, 1992).

132
SBT 1304/MARINE
/MARINE BIOTECHNOLOGY UNIT II B.TECH
.TECH BIOTECHNOLOGY
BIOTECH

Methods of eyestalk removal in Penaeids:

a) eyeball incision and squeezing;

b) ligation or tying

c) electrocautery or using a silver nitrate bar;

d) cutting;

e) pinching-crushing.

CRYOPRESERVATION

ABSTRACT

133
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Cryopreservation is a long-term storage technique to preserve the biological material without

deterioration for extended period of time at least several thousands of years. The ability to
preserve and store both maternal and paternal gametes provides a reliable source of fish genetic
material for scientific and aquaculture purposes as well as for conservation of biodiversity.
Successful cryopreservation of fish sperm have been achieved for more than 200 fish species and
many fish species have been adequated for the purpose of cryobanking. Cryopreservation of fish
embryo is not viable, mainly because of the same limitations as in fish oocytes, i.e., high chilling
sensitivity and low membrane permeability. However, cryopreservation of isolated embryonic
cells is another option for preserving both maternal and paternal genome. In this paper, an
overview of the current state of aquatic species is followed by a discussion on the sperm,
embryos, oocytes and embryonic cells - blastomeres.

Key words: cryopreservation, sperm, embryo, oocyte, blastomere

INTRODUCTION

Cryopreservation is a long-term storage technique with very low temperatures to preserve the
structurally intact living cells and tissues for extended period of time at a relatively low cost.
Cryopreservation is to preserve and store the viable biological samples in a frozen state over
extended periods of time. A very important part research in cryopreservation is to reveal the
underlying physical and biological responses of the cell and cause of cryoinjury, especially those
associated with the phase change of water in extracellular and intracellular environments (Mazur
1984). From the original slow-cooling study, another cryopreservation approach has moved to
easier and more efficient technique-vitrification, Cryoprotective agents has to gain access to all
the parts of the system. Cryopreservation considers the effects of freezing and thawing.
Therefore, the diffusion and osmosis processes have important effects during the introduction of
cryoprotective agents, the addition or removal of cryoprotectants, the cooling process, and during
thawing. These phenomena are amenable to the experimental design and analysis. Thus, reliable
methods can be developed for preserving a very wide range of cells and some tissues. These
methods have found widespread applications in biology, biomedical technology and
conservation.

Germplasm cryopreservation includes storage of the sperm, eggs and embryos and contributes
directly to animal breeding programmes. Germplasm cryopreservation also assist the ex
situ conservation for preserving the genomes of threatened and endangered species. The
establishment of germplasm banks using cryopreservation can contribute to conservation and
extant populations in the future. Since the first successful cryopreservation of bull semen (Polge
et al. 1949), cryopreserved bull semen has been used to propagate the rare and endangered
species using assisted reproduction techniques. Every year, more than 25 million cows are
artificially inseminated with frozen-thawed bull semen (Foote 1975) and many bovine calves
have been produced using the transfer of cryopreserved embryos into cow (Mapletoft and Hasler
2005). Tissues, cultured cell lines, DNA and serum samples could be frozen and store in
cryogene bank. For example, mice and sheep have been generated from frozen-thawed pieces of
ovary that have been replaced in a female and stimulated to ovulation. (Gosden et al. 1994;
Candy et al. 2000; Sapundzhiev 2008). The principle of testicular cell freezing and
transplantation has been demonstrated and is currently used for human male infertility (Clouthier

134
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

et al. 1996). Significant efforts are being made on non-mammalian species using cryobiology
techniques. In fish aquaculture, the successful cryopreservation of gametes and embryos could
offer new commercial possibilities, allowing the unlimited production of fry and potentially
healthier and better conditioned fish as required. Cryopreservation of reproductive products of
many aquatic species has been successfully achieved. Cryopreservation of aquatic sperm is
relatively common in the breeding and management of fish species, including salmonid,
cyprinids, silurids, and Acipenseridae (família) is well documented (Magyary et al. 1996;
Tsvetkova et al. 1996). However, cryopreservation of embryos and oocytes of aquatic species
have not been successful, except for eastern oyster eggs (Crassostrea virginica) (Tervit et al.
2005), larvae of eastern oyster (Paniagua-Chavez and Tiersch 2001) and larvae of the sea urchin
(Adams et al. 2006).

Cryopreservation technology applied to the preservation of fish gametes in aquaculture plays an

important role in seed production, genetic management of broodstock and conservation of
aquatic resources. Fish germplasm also plays a significant role in human genomic studies
because its relatively small size of the genome makes it easier for sequencing and ideal models
for studying the human disease. This would help in identifying the roles for human genes from
fish mutations and also in fish models for genes identified by human disease (Brownlie et al.
1998; Barbazuk et al. 2000). Aquatic species preservation would assist the development,
protection and distribution of research lines and would offer benefits for restoration of
endangered species.

Sperm

In 1949, Polge et al. (1949) successfully cryopreserved the avian spermatozoa using glycerol as a
cryoprotectant. Thereafter, cryopreservation of male gamete became possible. Blaxter (1953)
applied a similar approach for fish gametes and reported success with Atlantic herring
spermatozoa, achieving approximately 80% cellular motility after thawing. Since then,
cryopreservation of fish sperm has been studied and has been successful in more than 200
species (Kopeika et al. 2007; Tiersch et al. 2007; Tsai et al., 2010) and techniques of sperm
management have been established for freshwater and marine fish species, including carp,
salmonids, catfish, cichlids, medakas, white-fish, pike, milkfish, grouper, cod, and zebrafish
(Scott and Baynes 1980; Harvey and Ashwood-Smith 1982; Stoss and Donaldson 1983; Babiak
et al. 1995; Suquet et al. 2000; Van der Straten et al. 2006; Bokor et al. 2007; Tsai et al. 2010).
Many studies on cryopreservation of fish sperm have been carried out on economically important
freshwater species and attempts to cryopreserve sperm from the marine fish species tended to be
more successful when compared with those obtained from the freshwater fish (Tsvetkova et al.
1996). Although freshwater fish sperm are generally more difficult to cryopreserve, the
fertilization rates obtained from the cryopreserved marine fish sperm are similar to those
obtained with mammalian species (Tsvetkova et al. 1996). Controlled-rate slow cooling in
cryopreservation has been mainly used for fish sperm. Common carp has been studied using
frozen-thawed sperm with 95% fertilization and hatching rate.

Salmonid species spermatozoa have been successfully cryopreserved (Lahnsteiner 2000).

Another well studied cryopreserved group is cyprinids and some of these cyprinid fishes are
widely farmed throughout Asia and Europe. A fertilization and hatching rate of 95% using the

135
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

frozen-thawed sperm has been reported for the common carp and these results are not
significantly different from fresh sperm (Magyary et al. 1996). Tilapias are among the exotic
freshwater fishes that have been successfully established for fish farming in Taiwan; they have
been cryopreserved successfully and produced 40-80% motility with cryoprotectant DMSO
(Chao et al. 1987). The sperm of more than 30 marine fish species have been cryopreserved
successfully (Suquet et al. 2000; Gwo 2000; Van der Straten et al. 2006). Generally, high
survival and fertilization capacity has been obtained in frozen-thawed spermatozoa when
compared to freshwater species (Drokin 1993; Gwo 2000).

Successful cryopreservation of the sperm of aquatic invertebrate has been carried out for sea
urchin, oyster, starfish, abalone and coral (Adams et al. 2004a; Adams et al. 2004b; Gwo et al.
2002; Hagedorn et al. 2006; Kang et al. 2009). Dimethyl sulfoxide has also been reported as a
successful cryoprotectant for sperm cryopreservation; the concentration range used was 5 to 30%
for these species. Various levels of motility, ranging from <5% to 95%, have been reported for
the cryopreserved aquatic invertebrate sperm (Dunn and McLachlan 1973).

Embryos

Cryopreservation of embryos has become an integral part of assisted reproduction. Successful

cryopreservation of embryos is important because the biodiversity of both the paternal and
maternal genomes will be preserved. While cryopreservation techniques have been largely
established for the mammalian embryos, successful cryopreservation of intact fish embryos has
not yet been achieved. Factors limiting fish embryo cryopreservation include their
multicompartmental biological systems, high chilling sensitivity, low membrane permeability
and their large size, which gives a low surface area to volume ratio (Zhang and Rawson 1995).
The effect of such low ratio is a reduction in the rate at which water and cryoprotectants can
move into and out of the embryo during cryopreservation (Mazur 1984). Fish embryos are
osmoregulators; they are released into the external medium and activated. Then the vitelline
envelope separates from the plasma membrane and forms chorion. Studies on the chorion
permeability of zebra fish embryos clearly showed that it was permeable to electrolytes and a
range of cryoprotectant, including propane-1,2-diol, methanol, DMSO, ethylene (Zhang and
Rawson 1996). The chorion structure plays a crucial role as flexible filter for the transport of
some materials (Toshimori and Tsuzumi 1976) and protects against the microorganisms (Schoots
et al. 1982) Studies on zebra fish embryos have shown that the water permeability of the plasma
membrane at different developmental stages remained relatively stable. The permeability to
methanol (cryoprotectant) appeared to decrease during embryo development (Zhang and Rawson
1998). This also indicated that there was a gradual reduction in the permeability following the
fertilization in zebra fish embryos, as opposed to the generally held belief that the membrane
permeability of fish embryos reduced rapidly to minimum shortly after the fertilization
(Alderdice 1988).

The studies on the kinetics of subzero chilling injury in Drosophila embryos (Mazur et al. 1992)
and chilling sensitivity of zebra fish embryos have demonstrated that chilling injury plays an
important role in reduction of embryo survival during the exposure to subzero temperatures
(Zhang and Rawson 1995; Hagedorn et al. 1997). Chilling sensitivity has been shown for many
species and has been analyzed in fish embryos, including brown trout (Salmo trutta f. fario)

136
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

(Maddock 1974), rainbow trout (Oncorhynchus mykiss) (Haga 1982), carp (Cyprinus carpio)
(Dinnyes et al. 1998), fathead minnows (Pimephales promelas) (Cloud et al. 1988), goldfish
(Carassius auratus) (Liu et al. 1993) and zebrafish (Danio rerio) (Zhang and Rawson 1995;
Zhang et al. 2003). These studies demonstrated that the later stages (after 50% epiboly) were less
sensitive to chilling, but chilling sensitivity increased significantly as the temperature fell below
zero. The high chilling sensitivity of fish embryos, especially at early stages, their complex
membrane structure and large yolk are the main obstacles to achieve successful cryopreservation
of these embryos (Zhang and Rawson 1996). Chilling injury in embryos has been linked to the
inhibition of metabolic and enzymatic processes from low temperatures injuries which could be
detrimental in the embryonic development such as fish embryos (Dinnyes et al. 1998).
Cryoprotectant toxicity follows a similar pattern to chilling sensitivity with later stages being less
sensitive to cryoprotectant (Zhang et al. 2005; Zhang et al. 1993; Liu et al. 1993; Suzuki et al.
1995). Several studies have determined membrane permeability for zebra fish embryos (Zhang
and Rawson 1998; Hagedorn et al. 1997) and membrane permeability to water and most
cryoprotectants has been shown to be low (Zhang and Rawson 1996; Zhang and Rawson 1998).
Studies on the cryopreservation of zebra fish embryos demonstrated 8% embryo survival in 2M
methanol at -25 °C; however, no embryo survival was observed when frozen to -30 °C or below
(Zhang et al. 1993).

Cryopreservation studies on the embryos and larvae have been conducted on marine invertebrate
such as oysters, sea urchins, polycheate worms, coral and penaeid shrimp species (Liu et al.
2001; Gakhova et al. 1988; Lin et al. 1999; Olive and Wang 1997; Paniagua-Chavez and Tiersch
2001; Hagedorn et al. 2006; Tsai and Lin 2009). However, survivals of most of these species has
been inadequate in maintaining the structure and activity of embryos and larvae after freezing to
cryogenic temperatures. Embryonic and larval development of marine invertebrates after
cryopreservation often showed abnormalities in structure and colour (Odintsova et al. 2001). The
problems with invertebrate embryo cryopreservation associated with those identified with the
fish embryos are their low membrane permeability and high chilling sensitivity. Although
cryopreservation of the embryos has not been fully achieved, considerable progress has been
made in understanding the conditions required for fish embryo cryopreservation and this would
undoubtedly assist the successful protocol design in the future.

Oocytes

Oocyte cryopreservation is potentially the best way to preserve the female fertility.
Cryopreservation of fish oocyte has been studied (Isayeva et al. 2004; Plachinta et al. 2004;
Zhang et al. 2005; Guan et al. 2008; Tsai et al. 2009) which offers several advantages such as the
smaller sizes range, much lower water content in oocytes and absence of a fully developed
chorion that the permeability to water and solutes in oocyte is higher than embryo. Fish embryos
are too large to apply traditional cryopreservation protocol. Immature oocytes can be an
alternative for the mature eggs because of their smaller size (Hagedorn et al. 1996). However,
there is no practical technique available to induce the small oocyte to mature in vitro. A
technique to obtain the mature eggs from the late stage oocytes is available. Thus, the
combination of this technique and their cryopreservation could be a breakthrough. However, at
present, late stage oocytes cannot be successfully cryopreserved because their size is still not
small enough to result in much lower surface area to volume ratio. These reduce the rate at which

137
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

water and cryoprotectant move into and out of oocytes during the cryopreservation. Developing
the methods for cryopreservation of oocytes requires the screening of potential cryoprotectant
treatments, evaluation of tolerance to chilling, determination of the appropriate rate of freezing to
cryogenic temperatures and rate of thawing. Viability assessment methods of oocytes with trypan
blue (TB), fluorescein diacetate (FDA) + propidium iodide (PI) and adenosine triphosphate
(ATP) content assay have been developed for quick assessment of viability (Plachinta et al.
2004; Zampolla et al. 2006; Guan et al. 2008; Tsai et al. 2008; Tsai et al. 2009; Tsai et al. 2011;
Tsai and Lin 2012). A functional test based on in vitro maturation, followed by germinal vesicle
breakdown (GVBD) has also been shown effective for late stage III oocyte (Plachinta et al.
2004).

The permeability of the zebra fish oocyte membrane to water and cryoprotectants has been
studied (Zhang et al. 2005) and membrane permeability was shown to decrease with the
temperature and permeability was generally lower than those obtained from sea urchin eggs
(Adams et al. 2003) but higher than the immature medaka oocyte (Valdez et al. 2005). Studies on
zebra fish oocyte chilling sensitivity showed that those oocytes were very sensitive to chilling
and their survival decreased with decreasing temperature (Isayeva et al. 2004). Chilling
sensitivity in zebra fish oocytes was thought to be due to lipid phase transition of the oocyte
membrane (Pearl and Arav 2000). The phase transition in zebra fish oocytes showed that chilling
injury could occur when oocytes were exposed to temperatures between 12 to 22°C above the
water freezing temperatures (Drobnis et al. 1993; Pearl and Arav 2000). Cryopreservation of late
stage zebra fish oocytes has been studied using the controlled slow cooling and an optimum
cryoprotective medium and cooling rate identified. Guan et al. (2008) showed that although the
oocyte viability obtained immediately after freeze-thawing was relatively high with 88% using
TB staining; oocyte viability decreased to 29.5% after 2 h incubation at 22 °C. The study also
showed that the ATP level in the oocytes decreased significantly after thawing and all oocytes
became translucent. Cryopreservation of early stage zebra fish oocytes using the controlled slow
freezing has been reported by Tsai et al. (2009). The results suggested that 4M methanol in KCl
buffer was identified as the optimum cryoprotective medium. Although results obtained after the
cryopreservation using trypan blue and FDA+PI staining were promising with 69% and 54%,
especially with stage II ovarian follicles, the ADP/ATP ratio assay showed that the energy
system of these follicles had been compromised. Apparently the ADP/ATP ratio could be a
valuable measure of cellular injury after post-thaw incubation period as it reflected the metabolic
and energy status of population as well as indicating some measure of the potential for repair.
Furthermore, in vitro culture method is effective for assessing early stage zebra fish oocytes
growth competence in vitro. The early stage zebra fish oocytes can be cultured in vitro for 24 h,
stage I and II oocytes can grow to the sizes of early stage II and stage III oocytes after hCG
treatment. and also can be used for other teleost species (Tsai et al. 2010).

Studies on the cryopreservation of invertebrate oocytes and eggs over the past several decades
have been extraordinarily difficult to achieve (Koseoglu et al. 2001; Tsai et al. 2010; Lin et al.
2011; Lin and Tsai 2012). However, it was found that intracellular crystallization occurred in the
starfish oocytes at relatively high temperature that was very close to the temperature of
extracellular ice formation (Koseoglu et al. 2001). In order to avoid this problem, Hamaratoglu et
al. (2005) successfully cryopreserved starfish oocytes using ultra-rapid freezing technique, called
vitrification. High chilling sensitivity (Tsai et al. 2009) and low membrane permeability (Guan et

138
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

al. 2008) of zebra fish oocytes are major obstacles to the development of a successful protocol
for their cryopreservation as chilling sensitivity or cold shock can hinder slow cooling processes.
Vitrification may be another option to achieve successful cryopreservation for the oocytes.

Blastomeres

Blastomeres are the cells produced as the result of cell division and cleavage in the fertilized egg.
They are totipotent and pluripotent (depending on the stage of embryonic development) having
the ability to differentiate into any of the three germ layers or entire organism. They are different
from the muscle cells, blood cells or nerves cells. Although cultured somatic-cells from fish have
been cryopreserved successfully, their value is limited because of loss of development potential.
Cryopreservation of blastomeres can maintain the genetic diversity of both, nuclear genome and
mitochondrial DNA (Nilsson and Cloud, 1992). Blastomeres from the early embryos of fish still
retain pluripotency (Ho and Kimmel 1993) and their cryopreservation may be a promising
approach to preserve the genotypes of zygotes and reconstitution of the organism. Indeed, there
are several reports of germ-line chimeras created using the transplantation of blastomeres into
goldfish (Yamaha et al. 1997; Kusuda et al. 2004), zebra fish (Lin et al. 1992), medaka (Hong et
al. 1998; Wakamatsu et al. 2001) and rainbow trout (Takeuchi et al. 2001) embryos. Kusuda et
al. (2004) transplanted the frozen-thaw blastomeres into goldfish embryos and the blastomeres
differentiated into primordial germ cells. This report demonstrated that germ-line cells from the
cryopreserved blastomeres could develop into mature gametes of chimeric fish because the
blastomeres were not damaged by cryopreservation. Therefore, the cryopreservation techniques
are very important.

Cryopreservation of blastomeres has been successful in several fish species. In the first reported
studies, Harvey (1983) used a two-step freezing procedure, with ice-seeding at -6°C, and cooling
to -25°C, followed by immersion in liquid nitrogen. The survival rate of 84.8% was obtained
after cryopreservation of 50% epiboly zebra fish blastomeres. However, the results obtained
from a very small sample size and freezing rates were not controlled, rather tubes were allowed
to equilibrate in the cooled alcohol baths. Lin et al. (2009) demonstrated the effect of
cryopreservation on zebra fish blastomeres survival using the controlled slow cooling method. It
was shown that DMSO was the most toxic to zebra fish blastomeres. However, DMSO was the
best cryoprotectant in terms of survival of zebra fish blastomeres. Therefore, it is possible that
the cryoprotective effect of DMSO may be greater than its toxicity effect. Although the survival
rate in Lin's results progressed from 25% (Kopeika et al. 2005) to 70%, it was still lower than
that obtained by using two step methods. The comparisons between these studies must take into
consideration the different methodology. Vitrification of zebra fish blastomeres was studied
more recently and the highest blastomere survival was 93.4% (Cardona-Costa and Garcia-
Ximénez 2007). Cryopreservation of blastomeres was also carried out in rainbow trout, carp and
medaka after post-thawing. Rainbow trout blastomeres have been cryopreserved using the
controlled slow freezing procedures with a survival of 95% (Calvi and Maisse 1998). It has been
reported that the controlled slow freezing protocol adopted for rainbow trout was successfully
applied to carp blastomeres with survivals of 94% and 96% (Calvi and Maisse 1999). Lower
survival rates of cryopreserved blastomeres using controlled slow freezing have also been
reported for other fish species such as whiting (20%), medaka (34%), pejerrey (67%) and chum
salmon (59%) (Strussmann et al. 1999; Kusuda et al. 2002).

139
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

CONCLUSION

Cryopreservation of gametes and embryos are already routinely applied in the mammalian.
Cryopreserved sperm, oocytes and embryos are used for artificial insemination and embryo
transfer in the livestock industry. Cryopreservation also has enormous applications in the
artificial propagation of widely diverse aquatic organism. Sperm and embryonic cells
cryopreservaiton has been successful in a number of teleosts and invertebrate species. However,
cryopreservation of embryos and oocytes remain a major challenge. The practical application of
cryopreservation in the aquatic species needs more vigorous research efforts in this area and the
efforts may be prioritized on endangered, economical value and representative species from
various aquatic habitats. Cryopreservation of gametes, embryos and embryonic cells has become
of immense value in aquatic biotechnologies which provide an important tool for protecting the
endangered species, genetic diversity in aquatic species. The establishment of cryobanks to
utilize the cryopreservation worldwide would be a significant and promising task in the future.

***************************************************************************

140
SBT 1304/MARINE BIOTECHNOLOGY UNIT II B.TECH BIOTECHNOLOGY

Unit-II
Part-A
1. Define Aquaculture.
2. Write short notes on Semi-Intensive culture?
3. Enlist any four Lobster species.
4. Narrate Androgenesis.
5. Comment on Eye-stalk ablation.

Part-B
1. Write an illustrate account on sea bass culture?
2. Write an elaborate account on Crab culture?
3. Write an essay on tiger shrimp culture?
4. How would you produce pearls from oyster? Explain with
suitable diagrams.

5. How would you cultivate seaweeds? Explain.

6. Write an elaborate account on fish genetics?

141