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By Vivek Upadhyay

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INDEX

1. Introduction.

2. History.

3. Natural Breeding.

4. Reasons for Induced Breeding.

5. Induced Breeding of Carps

• Hormones used for induced breeding


• Preparation of P.G Extract
• Selection of brood fishes
• Making sets
• Doses of P.G Extract
• Doses of Ovaprim
• Method of injection
• Transfer of fishes to Breeding Hapa
6. Induced Breeding of fishes other than Carps.

7. Conclusion

8. References

9. Glossary
Acknowledgement

Completion of a task is a result of support and affection that we get


from our senior ones and from our family.
I would like to pay my thanks to the following personalities.
Firstly I am thankful to Dr. S.C Chandra for developing the
curiosity for fisheries in me by his valuable lectures during the
classes.
I would like to pay my special thanks to Dr. Sanjeev Shukla,
Reader Post Graduate Department of Zoology without whom M.Sc
Zoology for me is just like a dream. He has been a source of
inspiration for me.
I am also thankful to Dr.A.K Yadav, CIFE Chinhat Lucknow for
making me aware of some facts about Induced breeding which are
lacking from books.
In the end I am thankful to Almighty God for blessing me.
INTRODUCTION
INTRODUCTION

Major carps and other cultivable species of fish generally breed in running
water in rivers, during monsoon season when several environmental
conditions are favorable for spawning. These fishes become sexually mature
in ponds but do not spawn. However, the ripe fish breeders can be induced to
spawn by administration of one or more naturally occurring reproductive
hormones or their synthetic analogs. These stimulate the fish to release eggs
and sperms. This technique is based on the fact that the gonadotropic
hormones (FSH & LH) secreted by pituitary gland play a important role in
maturation and spawning of fishes. There are two main strategies used to
induce reproduction.

The first is to provide an environment similar to that in which spawning


occurs naturally. Catfish, for example, like to spawn in enclosed spaces such
as hollow logs. A farmer can simulate this by putting milk cans in a pond.
The presence of vegetation and an increase in temperature will usually work
for goldfish. Changing the photoperiod in a hatchery can accelerate or delay
maturation and ovulation in many salmon and trout species.

The second strategy is to inject the fish with one or more naturally occurring
reproductive hormones or their synthetic analogs. This is only effective in
fish that are already in breeding condition and have mature eggs in which the
germinal vesicle has migrated. Often the two strategies are used
sequentially: the first to manipulate maturation, then the second to induce
ovulation.
Numerous hormones have been used to induce reproduction. Various
methods have emerged over the past few years that seem to offer the best
chance for success at the least expense. They are injection of a GnRH
analog with dopamine antagonist, and injection of gonadotropin.

Two techniques are commonly used, sometimes in conjunction with one


another.

Methods vary from species to species and situation to situation. However, at


least two generalizations can be drawn. First, brooders are very vulnerable to
rough handling. Care should always be used to avoid damaging these
valuable animals. Second, a fish that does not have mature gametes will not
produce viable eggs or sperm no matter how many times it is injected with
hormones. Ripeness is the result of environmental factors working over a
period of time, leading to maturation of the gonads and production of viable
eggs.

Many procedures have been developed for inducing fish to undergo the last
steps of spawning. Farmers should thoroughly research the procedures that
have been developed for their species of fish through experimentation, and
select those that best suit the circumstances. In addition, once the fish have
spawned, there are many techniques involved in incubating and caring for
the eggs, and caring for the hatched fry. These too must be thoroughly
researched.
HISTORY

1. Von Ihering of Brazil (1934) developed the the technique of induced


breeding.
2. Gerbilskii (1938) of Russia followed the next.

3. In India first attempt was made by Khan(1938),which was not successful,

4. Chaudhri (1955) made the first successful attempt by injecting P.G


extract in Esmosus denricus

5. Ramaswami and Sundararaj (1956-1957) reported successful breeding of


catfishe (Heteropneustes and Clarias batrachus).

6. Chaudhri and Alikunhi (1957) made the successful attempt using P.G
extract in Labeo rohita, Cirrhinus mrigala, Cirrhinus reba).

7. Technology of induced breeding was demonstrated and transferred to


Indian fish farmers in 1970 by Cuttuck research station of CIFRI.
NATURAL BREEDING

Most fishes breed in running waters. Breeding occurs during the monsoon
months from April to August in different agro-climatic conditions. In fish,
the reproductive process involves three basic steps:

1. maturation - the development of the gametes (eggs and sperm) to a


point where fertilization can occur;
2. ovulation - the release of eggs from the ovary; and
3. spawning - the deposition of eggs and sperm so that they can unite.

In fish, as with all higher animals, hormones play a critical role in the
reproductive process. Hormones are chemical messengers released into the
blood by specific tissues, such as the pituitary gland. The hormones travel
through the bloodstream to other tissues, which respond in a variety of ways.
One response is to release another hormone, which elicits a response in yet
another tissue. The primary tissues involved in this hormonal cascade are the
hypothalamus, pituitary gland, and gonads (Fig. 1).

Fish have evolved to reproduce under environmental conditions that are


favorable to the survival of the young. Long before spawning, seasonal cues
begin the process of maturation. In many fish, this can take up to a year.
When the gametes have matured, an environmental stimulus may signal the
arrival of optimal conditions for the fry, triggering ovulation and spawning.
Examples of environmental stimuli are changes in photoperiod, temperature,
rainfall, and food availability. A variety of sensory receptors detect these
cues, including the eye, pineal gland (an organ in the dorsal part of the
forebrain that is sensitive to light), olfactory organs, taste buds, and
thermoreceptors.

The hypothalamus, located at the base of the brain, is sensitive to signals


from sensory receptors and releases hormones in response to environmental
cues. Principal among these hormones are gonadotropin releasing hormones
(GnRH), which travel from the hypothalamus to the pituitary gland. The
pituitary is responsible for a wide variety of functions, including growth and
reproduction. Certain cells of the pituitary receive GnRH and release
gonadotropic hormones into the bloodstream. The gonadotropic hormones
travel to the gonads, which synthesize steroids responsible for final
maturation of the gametes.

Maturation of the egg is a long process that involves complex physiological


and biochemical changes. One important step, vitellogenesis, is a process in
which yolk proteins are produced in the liver, transported to the ovary, and
stored in the egg, resulting in tremendous egg enlargement. The yolk is
important as a source of nutrition for the developing embryo.

Also critical are germinal vesicle migration and germinal vesicle breakdown
(GVBD). Before it migrates, the germinal vesicle, or nucleus, is located at
the center of the egg in an arrested stage of development. At this stage, the
egg is physiologically and genetically incapable of being fertilized, even
though it has the outward appearance of a fully mature egg. When conditions
are appropriate for final maturation, nuclear development resumes, and the
germinal vesicle migrates to one side. Finally, the walls of the germinal
vesicle break down, releasing the chromosomes into the cell.
The maturity of eggs can be determined using biopsy techniques. Eggs are
removed from the ovaries, cleared with a prepared solution, and viewed
under a microscope. In mature eggs, the migration of the germinal vesicle to
the side of the cell will be complete.

After the egg has matured, a class of compounds called prostaglandins are
synthesized. These stimulate ovulation, which is the rupture of the follicle
cells that hold the egg. The egg is then released into the body cavity or
ovarian lumen, where it may subsequently be released to the outside
environment. Following ovulation, the viability of the eggs can decrease
rapidly.

Fish with gametes that have not yet been released by the gonads are called
“green.” The term “ripe” is used to describe fish with gametes that have
been released from the ovary into the ovarian lumen. Ripe fish can be
stripped, green fish cannot.
REASONS FOR INDUCED BREEDING OF
FISHES

1. To overcome inadequate quantity of seed


2. To overcome mixed quality.
3. To overcome uncertainty of availability of seed.
4. To overcome cumbersome procedure of seed collection
5. To overcome high mortality of seed due to traveling and
transport.
6. To overcome high mortality due to biotic and abiotic facters.
INDUCED BREEDING OF CARPS

Induced breeding is a technique by which ripe fish breeds in confined


waters, when stimulated by administration of one or more naturally
occurring reproductive hormones or their synthetic analogs.

HORMONES USED FOR INDUCED BREEDING

1. Pituitary Gland Extract


2. Human Chorionic Gonadotropin (H.C.G)
3. Synahorin (mammalian P.G extract + H.C.G)
4. Ovaprim : This hormone was manufactured by Syndel Lab at Canada.

CONTENTS OF OVAPRIM (per


(per ml)

• 20 µg of salmon GnRH
• 10µg of Domperidon (an antagonist of dopamine)
• These are dissolved in a non-reactive oil base solvent.

5. Ovatide: It has hormonal preparations same as Ovaprim.


6. WOVA-FA: It does not contain domperidon
PREPARATION OF P.G EXTRACT
EXTRACT

Dry the preserved P.G with blotting paper


Weight the gland


Grind the tissue and homogenize in distil water


Transfer the homogenate to centrifuge tube


Centrifuge at 1200 RPM for 1 min


Transfer the supernatant discarding the tissue


Dilutye the supernatant as per requirement


Inject the sol as per doses
SELECTION OF BROOD FISHES

Female
• with glazy pectoral fin
• belly bulging
• pink vent

Male
• Pectoral fin rough
• Freely oozing milt on applying gentle pressure on vent

MAKING SETS

Brood fishes so selected for induced breeding are kept in combination with
females and males called sets.
• Set means one female with two male of equal weight of female.
• Sets could be 1:2, 2:3, 3:5 and so on.
DOSES OF P.G EXTRACT

Female
Ist dose: 2-3mg/kg body wt


4-6 hour interval


IInd dose: 5-8mg/kg body wt

Male
2-3mg/kg body wt. with sec dose to female

DOSES OF OVAPRIM (LINPE METHOD)

Female
0.3 - 0.7ml/kg body wt. depending upon species

Male
0.1 - 0.2 ml/kg body wt.
Breeding of fish through ovaprim is termed as “LINPE METHOD” named
after their inventors Dr. H.R Lin of China and Dr. R.E Peter of Canada

METHOD OF INJECTION

There are two common places to inject hormones into a fish. An


intraperitoneal (within the body cavity) injection is given through the ventral
(bottom) part of the fish behind either the pelvic or pectoral fin (Fig. 2).
Intramuscular (within the muscle) injections are commonly done on the
dorsal (upper) part of the fish above the lateral line and below the anterior
part of the dorsal fin (Fig. 3). In either case, it is important to place the
needle so that it slides under the scale rather than through it.

Two dosage levels are commonly used: a preparatory dose and a decisive, or final, dose
with a time gap generally of 12 to 24 hours between the two injections. The preparatory
dose brings the fish to the brink of spawning, and the decisive dose induces ovulation. In
general, the preparatory dose is about 10 percent of the total dose. For some fish, several
preparatory doses may be necessary.
TRANSFER
TRANSFER OF FISHES TO BREEDING HAPA
HAPA
The fishes (male and female) after injection transferred to proceeding ponds
and breeding hapa (3.5×1.5×1.0 m) within 10 – 12 hours time. Spawning
occurs midnight or a little later. Happa should not be disturbed till next
morning for observation of eggs. Fertilized eggs are crystalline, transparent
and look like pearl. They come up to surface on slight movement of water
and are transferred to hatching hapas for hatching.

INDUCED BREEDING OF FISHES OTHER

THAN CARPS

Examples
The following procedures provide examples of a variety of techniques used
to induce spawning.

Golden Shiners

Some fish can be induced to spawn simply by providing an appropriate


environment. An example is furnishing spawning substrate for golden
shiners, which naturally lay their eggs on vegetation. There are two common
ways of doing this. The first is to plant natural vegetation around the shore.
The second is to place spawning mats in the pond. The advantage of
spawning mats is that once the eggs are laid, the mats can be removed and
placed in another pond for grow-out. One common method for constructing
these mats is to sandwich Spanish moss or a similar material inside steel-
welded wire. A piece of 2” by 4” steel mesh is folded in half so that the
dimensions of the mat are about 20” by 40”. The Spanish moss is placed
inside the fold, and hognose rings are used to hold the bottom together. The
following is adapted from Third Report to the Fish Farmers, published by
the United States Fish and Wildlife Service.

1. Twenty to forty pounds of brood stock may be stocked per acre.


2. Plankton blooms can inhibit spawning. If heavy blooms occur, the
pond should be flushed with fresh water.
3. If grass is to be planted, the water level in the brood pond should be
lowered during early spring to allow growth along the shoreline. Once
the grass has been established, the ponds should be refilled.
4. If spawning mats are to be used, they should be placed in shallow
water, about one inch below the surface with one side at the edge of
the pond. Several mats may be placed end-to-end.
5. As each mat fills with eggs, it should be transferred to a rearing pond
for grow-out. Care must be taken so that the mats do not become over-
populated with eggs, because they may eventually smother each other
or be attacked by fungus.
6. If spawning activity diminishes quickly, a rapid rise in the water level
may extend activity.

Trout

In nature, trout build their nests in gravel and require cold, flowing, aerated
water to reproduce. Changes in photoperiod are critical for bringing the fish
to spawning condition. The spawning season varies with locality,
temperature, and the species or strain of fish. Brook and brown trout are fall
spawners, while rainbow trout spawn in the spring. The following steps,
summarized from Fish Culture Manual, published by the Alaska Department
of Fish and Game, illustrate one method for spawning trout by stripping
females and mixing the eggs with milt. Many other methods also exist.

1. Anesthetize the fish. Placing them in MS-222 at a concentration of


130-260 mg/l (assuming temperature between 4¡C and 15¡C) is
common. The solution should be within 5¡C of the holding or rearing
containers, and the pH should be between 6 and 8.
2. Holding the caudal peduncle with one hand and supporting the head
with the other, dip the fish in clean water to wash off the anesthetic.
3. Strip the female’s eggs into a dry, shallow container. The fish should
be held belly down in a horizontal position with the head slightly
higher than the tail. Gently press upward near the base of the pelvic
fin to start the flow of eggs. Slowly stroke the belly, moving the hand
from front to rear using constant pressure. Repeat this procedure until
no more eggs are obtained.
4. In the dry method of fertilization, sperm from the males is poured
onto the eggs. Water should not come into contact with the mixture
for it will inhibit fertilization by the sperm. The sperm, eggs, and
ovarian fluid should be gently mixed with a feather.
5. After 30 seconds, fertilization will have taken place. Add enough
water to cover the eggs and mix again.
6. Let the fertilized eggs soak for 30 seconds.
7. Rinse the fertilized eggs in clean water.
8. Water harden the eggs. This is a process whereby the eggs absorb
water and grow about 20 percent. Place the eggs into a container that
will accommodate them as they grow. Add water, then do not disturb
for an hour.
9. Put into incubators.

Catfish

Temperature is a determining factor in the control of catfish reproduction.


Spawning activity begins after water temperatures stay consistently above
70¡F. It drops off when temperatures fall below 70¡F or rise above 85¡F.
There are a variety of methods commonly used for spawning catfish,
including spawning in ponds, spawning in pens, and hand stripping. In
many, though not all cases, hormone injection may be desirable. The
following procedure from Third Report to the Fish Farmers provides an
example of spawning induction with hormones followed by hand stripping.

1. Anesthetize the fish with MS-222 at a concentration of 80 mg/l.


2. If the fish are kept in a holding tank, loosely stitch their mouths closed
to prevent them from biting one another. The thread must be loose
enough to allow respiration.
3. Using an 18-gauge needle, inject intraperitoneally with: a. 3.5-4.0 mg
of acetone-dried pituitary gland extract per kilogram of fish for the
female and about 2 mg per kilogram for the male, or b. 500-800
international units of human chorionic gonadotropin (HCG) per pound
of fish.
4. If necessary, inject again 48 hours later. Usually, the female ovulates
within 16-20 hours after an injection.
5. Strip the eggs and the milt into a 0.3 percent solution of common salt.
6. Very gently mix the eggs and milt. Fertilization should occur in 2-5
minutes.
7. Place the egg mass in a suitable incubator for hatching.
8. After 10-12 hours, stop the water flow and treat with a 0.3-0.5 percent
solution of alkaline protease enzyme for 2-3 minutes while gently
stirring. This will dissolve the sticky layer of the eggs and allow them
to float free.
9. When the eggs begin hatching, they should be taken out and placed in
a flat tray.
Conclusions
Many species of fish will not readily reproduce under certain culture
conditions. Others will, but not necessarily when the farmer desires. In these
cases, induction of spawning can be of great value.

Two techniques are commonly used, sometimes in conjunction with one


another. The first is manipulation of the culture environment to mimic some
important quality in the fish’s natural environment. The second is injection
of hormones to stimulate spawning. The hormones may be natural hormones
taken from fish or other animals, genetically engineered from bacteria, or
synthetic analogs of naturally-occurring hormones.

Methods vary from species to species and situation to situation. However, at


least two generalizations can be drawn. First, brooders are very vulnerable to
rough handling. Care should always be used to avoid damaging these
valuable animals. Second, a fish that does not have mature gametes will not
produce viable eggs or sperm no matter how many times it is injected with
hormones. Ripeness is the result of environmental factors working over a
period of time, leading to maturation of the gonads and production of viable
eggs.Many procedures have been developed for inducing fish to undergo the
last steps of spawning. Farmers should thoroughly research the procedures
that have been developed for their species of fish through experimentation,
and select those that best suit the circumstances. In addition, once the fish
have spawned, there are many techniques involved in incubating and caring
for the eggs, and caring for the hatched fry. These too must be thoroughly
researched.
Glossary

Dopamine:
A chemical that inhibits the release of hormones from the pituitary
and thereby blocks the pituitary’s response to injected LHRHa.
Dopamine antagonist:
A family of drugs that block action of dopamine.
Gonadotropin:
A pituitary hormone that controls the production by the gonads (testes
and ovary) of sperm and eggs.
Gonadotropin Releasing Hormone (GnRH):
A hormone produced by the hypothalamus that stimulates the pituitary
to release gonadotropin.
Hormones:
A chemical formed in endocrine glands that affect the functions of
specific tissues.
Human Chorionic Gonadotropin (HCG):
A commercially available, semi-purified hormone that is used to
induce ovulation and spermiation, i.e., egg and sperm production.
Hypothalamus:
A part of the brain that controls many internal body functions and the
activity of the pituitary gland; produces gonado-tropin releasing
hormones.
Intraperitoneal injection:
Injection into the abdominal cavity.
Leutinizing Hormone Releasing Hormone (LHRH):
Mammalian gonadotropin that has been used to induce the
reproductive cascade in fish.
Lumen:
The cavity of a tubular organ.
Maturation:
The growth and development of the gametes to a point where they are
ready to fuse to form a fertilized egg.
Ova:
Eggs.
Ovulation:
The release of eggs from the ovary.
Pituitary extract:
An aqueous, alcoholic, or acetone extract of the pituitary gland used
for artificial induction of spawning.
Pituitary gland:
An endocrine or hormone-producing gland found on the underside of
the brain just behind the eyes.
Prostaglandins:
A class of compounds that, among other things, stimulate ovulation.
Spawning: The deposition of eggs and sperm such that they unite to
form a fertilized egg.
Steroid:
Any of a large group of hormones, some of which are involved in
final maturation of gametes.
Testes:
The male reproductive organs.
Testosterone:
A steroid hormone produced by the testes; along with
ketotestosterone, it is responsible for the development of male
secondary sex characters.
Vitellogenesis:
A stage of egg development in which the yolk is stored, resulting in
tremendous enlargement.