Fish Genetics and Biotechnology

Chapter 6: Natural Propagation and induced breeding of fish reproductive

endocrinology, brood stock husbandary and selection

Dr. Sharmin Suraiya

B. Sc in Fisheries (Hons.) & [Link]. in Biotechnology (BAU, Bangladesh)
PhD in Biotechnology (KGSP PhD Scholar-2014, PKNU, S. Korea)
Assistant Professor
Department of Fisheries and Marine Bioscience
Jashore University of Science and Technology, Jashore.
Mobile: +8801790054236
 Management of brood fish ponds
● Brood fish is a prerequisite for all induced breeding
programmes.
● Proper brood-stock will lead to successful seed
production.
● Hence, brood fish management has assumed great
importance in hatchery management.
● Carp brood-stock ponds are generally large (0.2-2.5 ha),
1.5-2.5m deep, 20-30 m wide, rectangular, seasonal or
drainable and earthen in nature.
● Water inlet and outlet should be such that they simulate
riverine conditions, which is the natural habitat of major
carps.
Source of brood fish: Most fish seed farms raise their own carp brood-
fish for seed production.
Care of brood fish:
● Prepare brood-stock pond following standard procedure to ensure
sustained production of zooplankton.
● Stock major carps at 1,000-2,500 kg/ha, depending upon the species.
● Maintain a little lower stocking rate for catla.
● During immature stage, feed the fish with a traditional diet consisting of
rice bran and oil cake (1:1) at a feeding rate of 2-3% body weight daily.
● During the maturing phase, feed the fish with a special feed containing rice
bran, oil cake, fish meal, cereals, grams and mineral and vitamin mixture.
● Alternatively, one can use commercial floating pelleted feed (protein
content : 30%)
● In addition, feed the grass carp with a limited quantity of tender
aquatic plants.
● Segregate the common carp brood fish sex-wise and stock male
and female in separate ponds to prevent accidental spawning in
pond.
● Give special attention to catla brood fish to prevent the
deposition of abdominal fat to facilitate normal gonadal
development and check maturity periodically.
● Provide additional aeration (with paddle-wheel aerator),
particularly in catla pond.
● Segregate sexes at least one month before to increase the
affinity between male and female during spawning
● Take care to maintain water quality and plankton level by
periodic manuring.
● Prevent algal blooms and oxygen depletion by water exchange.
● Control parasites and pathogens by periodic checking of
brooders.
Identification and selection of brooders for injection
● Identification of sex of brooder is necessary to select sexual partners
● Fish is sexually dimorphic and some of the morphological/external
characteristics like size, colouration, fin characteristics, genital opening, etc.
which are prominent during season could be used
● Some of the external morphological characters which are developed
during breeding season could be used to identify sex in major carps are as
follows:

Select medium-sized (1.5 – 5 kg) carp brooders aged between 2 and 6 years, for best results.
 Fry production methods consists of:

1. Brood stock management

2. Collection of pituitary gland
3. Preservation and storage of gland
4. Preparation and preservation of extract
5. Determination of pituitary dosage
6. Ponds for induced breeding and breeding happas
7. Injections of pituitary extract to breeders
8. Spawning and fertilization
9. Stripping or artificial insemination
 Basis for induced breeding

• Reproduction in fish-regulated by environmental stimuli.

 Induced breeding in carps by hypophysation

1. Brood stock Management

• The brood pond
• Characteristic of ripe brood fish and their selection for stocking
• Stocking rate
• Caring the brooders
• Manuring and maintenance of water quality in brood-stocking
ponds
• Supplementary feeding
• Rate of feeding
The primary requirement of carp breeding is an adequate stock
of good breeders. The excellent brood fish culture is the foremost
essential material basis and key factor for successful artificial
propagation.

Brood stock: Group of mature individuals used in aquaculture for

breeding purposes

a. The brood Pond:

Ponds of moderate size (0.5 to 1 ha) & depth (1.5-2.0 m),
preferably rectangular, flat bottom.

Moderate water temp (27-32°C).

Exposure of 6-8 hrs light in a day at least for 2-3 months.

b. Characteristics of ripe brood fish and their selection for stocking
• Selection of female is very
important.
• A simple catheter can be used
to find out if the fish is having
eggs or not and shape and size
of eggs suggest that the female
is quite ripe.
• Grass carp- red colored eggs
• silver carp- blue
• Color varies with various
ecological zones and also with
the age of female.
Healthy, disease free breeders of
catla, rohu, mrigal, silver carp,
grass carp and common carp of
2-4 yrs and 1-5 kgs are normally
selected
c. Stocking rate (sex ratio)
• Sex composition of breeders is normally maintained as 1:1.
• Males and females are kept separately in brood pond so that it
increases the affinity of mating of the pair during breeding
experiment.
Stocking is done 1500-2000 kg/ha with species composition of
• 40 surface feeders (Catla 30-20 and silver carp 10-20)+
• 30 column feeders (rohu 20 and grass carp 10)+
• 30 bottom feeders (mrigal 20 and common carp 10).
d. Caring the brooders
• Fortnightly or weekly seining- for exercise, general health checkup
and gonadal maturation.
• Frequent replacing breeders from one pond to another- for food
utilization.
• In case of infection- bath in 10 ppm KMnO4

e. Manuring and maintenance of water quality in brood-stocking

ponds
•Initial manuring of brood pond- 3 weeks prior to stocking with cattle
dung or compost manure.
•7 days Before fertilization- lime treatment
•1/8th to 1/4th water change frequently.
f. Supplementary feeding
• Supplementary feed given daily.
• a complete feed is a proportionate vegetable and animal protein,
carbohydrate and fat. Mixture is prepared from: half cooked rice and
pulse (1:1, 50%)+ oil cake (20%)+ cattle dung (20%)+ Fish meal/silk
worm pupae/ cooked slaughter house refuse (10%). (Rohu, catla,
mrigal, common carp)
1-2% diet of BW before spawning and 3-4% diet after spawning. No
feed is given for one week before actual breeding.
Grass carp-fresh aquatic vegetation- Hydrilla, Vallisneria, Utricularia.
Silver carp- Spirulina, Oscillotoria, Chlorella
g. Feeding rate
• Complete stoppage of feed before spawning: for proper
defattening and physical tuning of breeder.
• Excess feeding may result in heavy accumulation of mesenteric fat
inhibiting the process of smooth ovulation.
 2. Collection of pituitary gland
Indian Major carp (IMC)-serve as Donor fishes.
However, Common carp is most preferable because of its easy
raising.
The glands from fresh and fully ripe donors are used.
Suitable period: breeding season.
 3. Preservation and storage of glands

Brazilian method of preservation in

Absolute alcohol
• Pituitary banks are maintained.
• Since pituitary hormones are water
soluble, hence Brazilian method of
preservation in absolute alcohol is
suitable.
Acetone-dried method
 SUBSTITUTES OF FISH PITUITARY GLAND:

 HCG (organon): When injected to L. Rohita @ 460-2010 IU/kg body weight did
not precipitate spawning. However it has been reported that L. Rohita could be
bred by injecting HCG @ 600 IU/kg body weight in a few cases. In the case of
silver carp, successful spawning could be achieved by injecting HCG (organon)
alone @ 630-660 IU and also with HCG 240 IU + 12 mg carp pituitary per kg
body weight.
 Synahorin: Along with carp pituitary gland was successful in breeding rohu and
silver carp, but failed to induce spawning when tried alone in rohu.
 Ovaprim: It is the new inducing hormone for fish and absolute substitute of
pituitary extract though it’s costly. Ovaprim is far superior to carp pituitary in
inducing spawning in several species of carps.

SYNTHETIC HORMONE OF FISH SPAWNING

 4. Preparation and preservation of pituitary extract

• Gland register- serially labeled phials, details of

phial no.
and
wt of gland and donor sp’s
Extract: Required quantity of gland → homogenized in tissue
homogenizer →macerated in Physiological saline (0.3% NaCl)
Intraperitoneal injection: no need of centrifugation
Intramuscular injection: suspension if centrifuged at 1000 rpm for 5
mins
Figure: Preparation of
pituitary gland to be used
for induced breeding
 5. Preservation
Glycerine preservation method
1) 1/3 of total volume of extract is made up by adding DW.
2) Solution is kept in Refrigerator for 24 hrs
3) Glycerine is added to make up 2/3 of volume (glycerine : water,
2:1)
4) Suspension again stored in refrigerator for 24 hrs and afterwards
centrifuged.
5) Supernatent is filtered and stored in air tight bottles inside
refrigerator
6) Sealed in ampoules (labeled giving details of hormones conc.,
date of prep)
 5. Dose
Calculation of doses and dilution:
I. 1-3 whole glands from ripe donor to nearly same wt as that of
recipient fish
II. For IMC 2 doses: 2-3 mg/kg BW and 5-8 mg/kg/BW
III. Exotic carps: 3-4 mg/kg/BW, 7-10 mg/kg BW
 6. Ponds for induced breeding and the breeding hapas

• Brood stock
• Brood stock ponds (No blooms, predaceous insects & fishes.
• In these ponds only breeding hapas are fixed, inside which the injected brood fishes of
both sexes are released to spawn.
BREEDING HAPA
Box shaped cloth container, like an inverted net with a cover. (fine-meshed).
Hapa should have fine thin cloth such that ovarian eggs do not pass out when released by
fish
Dimensions of hapas
a) 4 kg fish: 3.6X1.8X1.0 m
b) 1.5-4 kg: 2.4X1.2X1.0 m
c) Under 1.5 kg: 1.8X1.0X1.0 m
It has an opening on wider side
It is fixed with the help of bamboo poles
When fixed, bottom of hapa should not touch muddy pond bottom at least 15-20
cm of hapa should remain above water level.
 7. Injection of pituitary extracts to breeders

• Hypodermic syringe (2ml) with 0.1 ml graduations is used.

• Thickness and length of needle depends upon size of recipient fish.
• Generally BDH no. 22 needle- 1-3 kg wt
no. 19 needle – larger fishes
no. 24 needle – smaller fishes
Mode of injection: Intraperitoneal or intramuscular
• Intraperitoneal: injected at
the base of pelvic fin. Fish may
die due to damage of internal
organ. Practiced in US.
• Tip of needle is inserted
under a scale and is pushed
through abdominal wall into
body cavity and then directed
parallel to ventral surface
 Intramuscular: popular in Brazil and India. Administered in caudal
peduncle. While injecting the needle is first inserted under a scale
parallel to body and muscle is pierced through quickly at 45°, so that
no damage is done to scale.

• In case of single injection, this could be done in late in the evening

and spawning could be over by early hours of morning.
• Both injected males and females are kept together in breeding
hapa for spawning.
• In breeding hapa for C. carpio, hanging fibrous roots of water
hyacinth are kept because eggs of common carp are sticky.
 8. Spawning and fertilization
• After few hrs of injection, the brood fishes spawn naturally in
breeding hapa.
• Sex play is observed between male and female after final injection.
• IMC- spawn within 6-8 hrs
• In case of Chinese carps, stripping is done for better fertilization
 9. Stripping or artificial insemination
• Eggs and milt are forcible extruded out by applying pressure on
the belly of read to breed breeders kept in hapas.
Methods
Dry method and Wet method
Dry method: After 3-4 hr of second injection, abdomen is pressed
and eggs and milt are collected in tray and mixed with spoon. After
few mins fertilized eggs are transferred into breeding hapa.
Wet method: physiological saline is used to receive milt first and
eggs later
 Anesthetics in Aquaculture

Fish are easily stressed by handling and transport and stress can result in immuno-
suppression, physical injury, or even death. In aquaculture, anesthetics are used
during transportation to prevent physical injury and reduce metabolism (DO
consumption and excretion). They are also used to immobilize fish so they can be
handled more easily during harvesting, sampling and spawning procedures. An
ideal anesthetic should induce anesthesia rapidly with minimum hyperactivity or
stress. It should be easy to administer and should maintain the animal in the
chosen state. When the animal is removed from the anesthetic, recovery should
be rapid. The anesthetic should be effective at low doses and the toxic dose
should greatly exceed the effective dose so that there is a wide margin of safety.
Table 1. Stages of anesthesia in fish
 Use of anaesthetics
Protocol
Take weight and length of fish
↓
Prepare a solution of an anaesthetic (e.g. quinaldine) of
varying doses, i.e. 0, 25, 50, 75 ppm, etc.
↓
Leave fish in the anaesthetic solution and record induction
time for different stages of anaesthesia
↓
After the specified induction time, transfer the fish to a tank
containing fresh water with aeration
↓
Record recovery time
↓
Record survival of anaesthetized fish at different periods
↓
Find out optimum dose for different stages of fish
 Anesthesia of fish
Fish are usually anesthetized by immersing them in an anesthetic
bath containing a suitable concentration of drug so that the drug is
absorbed through the gills and rapidly enters the blood stream. The
simplest procedure is to prepare the required drug concentration in
an aerated container and quickly but gently transfer the fish to the
container. The anesthetic bath and recovery tank should use water (at
a similar temperature and chemistry) from which the animals
originated. Water quality needs to be carefully controlled, especially
where large numbers of animals are being handled and baths are
being reused. Main concerns involve maintaining proper
temperature, adequate dissolved oxygen, low ammonia and a
minimum amount of fecal matter. Applying an anesthetic solution to
the gills with a spray bottle can be useful with large animals or if
immersion is impractical. A 100- to 200-mg/L solution of MS-222 is
reported to be effective when applied to the gills of salmonid
broodstock. This method allows the fish to be handled without
immersion, and it has no effect on subsequent egg hatching success.
 Anesthetics used in fish

Chemical methods Non-chemical methods

MS-222 Hypothermia
Benzocaine Electro-anesthesia
Quinaldine
2-Phenoxyethanol
Aqui-S
Metomidate
Clove oil
Carbon dioxide
Table. Dose rates of major anesthetic drugs, evaluated experimentally, for a number of
commonly cultured fish species.
 Transportation and anesthesia

The stress caused by handling, grading and transporting can be considerable. It

may be preferable to anesthetize fish as they are loaded for transport and/or to
add ice to the water in transport tanks to reduce metabolic activity. Sedation can
be beneficial in the bulk transportation of fish stocks, especially over long distances
and when fish density is high. The major concerns in transporting aquatic animals
are the management of handling stress, mechanical shock, heat stress, and water
quality. Fish should not be sedated too deeply or they will sink to the bottom of
the hauling tank where very high densities can cause rapid water quality
deterioration and suffocation. As previously stated, there are no chemical
anesthetics (other than CO2) approved for use on food fish in the U. S. with zero
withdrawal time. Therefore, carbon dioxide and hypothermia are the only legal
means of sedation for transporting live food fish to market
 Schematic representation of hormone induced spawning of fish
1. Select fully gravid female fish during the spawning season
2. Transport the required number of fish to hatching facility
3. Determine the weight and number of food fish for priming and resolving dose of hormone
injection
4. Perform dosage calculations to prepare hormone solutions and maintain aseptic conditions
5. Prepare fresh hormone solutions before hormone injections
6. Label hormone vials, store hormone solutions and stock hormone solutions either in
refrigerator or freezer
7. Weigh individual fish with minimal stress
8. Load the required volume of hormone solution by intraperitoneal injection (under the
pelvic fin)
9. After 12 to 18 hours, depending on the water temperature and species, administer
resolving dose by intraperitoneal injection
10. After 12 to 26 hours (depending on species and water temperature), observe signs of
ovulated eggs
11. Prepare the sperm solution following species appropriate protocols
12. Sedate the ovulated fish in 100 ppm of MS-222 (Tricaine Methanesulfonate) solution
13. Dry the sedated fish with a dry towel, and strip the eggs in a greased container
14. Based on the estimated number of stripped eggs, add the required volume or quantity of
sperm
15. Fertilize, water harden, and hatch eggs according to species-specific protocols
Endocrine glands are ductless glands of the endocrine system that
secrete their products, hormones, directly into the blood. The major
glands of the endocrine system include the pineal gland, pituitary
gland, pancreas, ovaries, testes, thyroid gland, parathyroid gland,
hypothalamus and adrenal glands.

Exocrine glands are

glands that secrete
substances onto an
epithelial surface by way
of a duct. Examples of
exocrine glands include
sweat, salivary, mammary,
ceruminous, lacrimal,
sebaceous, and mucous.
Hormone is any member of a class of signaling molecules,
produced by glands in multicellular organisms, that are transported
by the circulatory system to target distant organs to regulate
physiology and behavior.
Hormones have diverse chemical structures, mainly of three
classes:
 Eicosanoids
 steroids
 amino acid/protein derivatives (amines, peptides, and proteins)

Pheromones are chemicals capable of acting like hormones

outside the body of the secreting individual, to impact the
behavior of the receiving individuals. There are alarm
pheromones, food trail pheromones, sex pheromones, and
many others that affect behavior or physiology.
Endocrine glands
The glands that secrete their products into the bloodstream and
body tissues along with the central nervous system to control and
regulate many kinds of body functions are known as endocrine
gland. In fishes various endocrine gland has been found
associated with different tasks and functions.
Endocrine glands of fishes: Different types of endocrine glands
are found in fishes; such as-
•The pituitary gland or Hypophysis
•Thyroid Gland
•Adrenal gland
•Corpuscles of Stannius
•Ultimobranchial Glands
•Urohypophysis
•Pancreatic islets
•Pineal gland
1. The pituitary gland or Hypophysis:
Location: Ventral surface of brain below optic chiasma.
Origin: Two parts of pituitary gland are derived from two
different components. Neurohypophysis develops from the
floor of the embryonic diencephalons. Adenohypophysis
developes from the dorsal evagination of the ectodermal
part of buccal cavity called Rathke’s pouch. This pouch
later looses its connection from the buccal cavity and
remains permanently connected to neurohypophysis during
the rest of the life. The hypophysis in adult fish remains
attached with it by a stalk is called infundibular stalk or
neurohypophyal stalk and occupies a position on the
underside of the brain; in the region of diencephalon.
Part and division of pituitary, their cell types, secretions and action of their hormones: The part of this
gland their division, cell types, secretion and functions are given below in a tabular form-
2. Thyroid Gland:
Origin: The thyroid gland in fishes arises from the floor of the pharynx as a median evagination.
Location: The location of thyroid gland varies considerably in different fish species; such as-
•In cyclostomes, follicles of thyroid are dispersed around the ventral aorta and do not form compact capsulated
structure.
•In bony fishes, it may lie under the 1st branchial arch on each side.
•In many teleosts, it is found along the afferent branchial arteries of the gills.
•In other teleosts, the follicles of thyroid migrate to distant unusual localities, such as the liver, kidney, brain, eye,
gut, spleen, gonad etc. as in platyfish.
Shape: The shape of the gland is also variable depending on various fish group; such as –
•In cyclostomes, the thyroid is in the form of follicles.
•In many teleosts, thyroid becomes a diffused structure as small masses of follicles.
•In elasmobranches and bony fishes, thyroid is compact structure.
•In dipnoi, thyroid comprises a pair of interconnected lobes.
Histology: The thyroid gland is composed of a large number of follicles forming a shape of a hollow ball and
consisting of a single layer of epithelial cells that encloses a fluid filled space. These follicles are bound together
by connective tissue. The gland is highly vascular and the epithelium surrounding the follicle may be thick or thin
and the height of the cells depends upon its secretory activity. The epithelium mainly composed of two types of
cells- the chief cells are cuboidal or columnar in shape with clear cytoplasm and colloid cells, contain droplets of
secretory material.
Secretion: Chiefly thyroxine
Function:
•Thyroid hormone’s role is oxygen consumption in fishes have been pointed out though it lacks consistency.
•Thyroid hormone influences osmoregulation in salmon and Gastrosteus.
•Thyroid along with other endocrine glands also influences migration in fishes.
•It is also known to effect growth and nitrogen metabolism in gold fish.
•Thyroxine brings about maturation in fishes.
•Scale and bone formation in fishes is also influenced by thyroxine.
3. Adrenal gland: Adrenal gland in fishes is quite different from that
of mammals. The two components of adrenal gland i.e. cortex and
medulla are separately found.
Location: One of three layers of cells lying along cardinal veins in
the region of the hemapoietic head kidney.
Origin: Mesodermal layer of embryo
Secretion: Adrenaline, Cortisol
Function:
•Promote utilization of steroid fat
•Carbohydrate metabolism
•Water metabolism
•Protein catabolism
•Sodium retention
•Electrolyte metabolism
4. The Corpuscles of Stannius:
Location: Attached to or embedded in kidneys of holosteans and
teleosts
Origin: the corpuscles of stannius orginate as outgrowths from the
pronephric or the mesonephric duct of the kidney.
Nature: Proteinous
Secretion: Hypocalcin
Colour: Pink or white
Function: Regulates calcium balance

5. The Ultimobranchial Glands: It is also known as post-branchial

bodies or suprapericardial bodies or ultimobranchial bodies.
Location: Sac-like structures between ventral wall of esophagus and
sinus venosus.
Origin: Ultimobranchial gland develops embryologically from the
epithelium of the last or ultimate gill pouch.
Secretion: Calcitonin
Function: Regulates calcium level in blood.
6. Urohypophysis: It is also known as Urophysis or caudal Neusecretory organ.
Location: This gland is in the form of a swelling at the posterior end of the caudal
spinal cord i.e. in the tail of the teleosts.
Secretion:Urotensins
Function: Metabolic regulations.

7. Pancreatic islets:
Location: Gut walls in larval Lampreys; hepatopancreas, most bony fishes
Embryonic Origin: Mesoderm
Secretion: Insulin
Function: Carbohydrate metabolism

8. Pineal gland:
Location/Origin:The Pineal organ of the fish arises as a posteromid dorsal
evagination of the epithalamus.
Secretion: Melatonin
Function: Photosensory and secretory function
Fig. 1. The hormonal chain of
events leading to the release of
mature eggs and spermatozoa
(Reproduction)
Stimuli from the environment - primarily daylength (photoperiod),
temperature, and cues associated with rainfall – are processed by
sensory receptors; the resulting neural signals, upon reaching the
hypothalamus in the base of the brain, influence the pituitary gland
through chemical messengers known as releasing hormones. These
releasing hormones, whose practical importance in induced breeding
is now firmly established, stimulate the pituitary to release into the
general circulation hormones whose target organ is the gonad. These
hormones are called gonadotropins, and they influence the
production of sex steroids in the gonad itself. Sex steroids are
responsible for gamete maturation and, if the appropriate
environmental and social signals are present, ovulation (or
spermiation) and spawning follow.
 PHEROMONES HORMONES
Pheromones are special chemicals Hormones are chemical substances
secreted by the insects like secreted by the big animals and
honeybee, etc. humans from the endocrine glands.
SECRETED BY

Pheromones are secreted during the The endocrine glands, testis and
mating season for the purpose of ovaries secrete special hormones
sexual reproduction. which help in sexual reproduction.

IMPORTANCE
The hormones secreted by the
The pheromones acts as messengers gonads (ovaries and testis) help in
to attract the male insect towards development of the secondary
the female thus aid in reproduction. sexual characters which in turn help
for sexual reproduction.
NATURE
The pheromones act outside of the The hormones act inside of the body
body of the animal or insect of the animal or human being
secreting it. secreting it.
FUNCTION
The hormones are of various types
The pheromones are single type and
like, sexual hormones, growth
they don't do many functions in the
hormones, feel-good hormones, etc,
body of animals are insects.
to help the functioning of the body.