Induced breeding

1. Basis of Induced breeding

2. Induced reproduction

3. Historical background

4. The fish Pituitary

5. Hormones of pituitary related with development of gonads and spawning

6. Induced breeding in carps by hypophysation

a. Brood stock management

b. Collection of pituitary gland

c. Preservation and storage of gland

d. Prep and preservation of extract

e. Determination of pituitary dosage

f. Ponds for induced breeding and breeding happas

g. Injections of pituitary extract to breeders

h. Spawning and fertilization

i. Stripping or artificial insemination

I) Basis for induced breeding

• Reproduction in fish-regulated by environmentalstimuli.

Environmental stimuli

Brain hypothalamus

Pituitary gland

Gonadotropichormones

Sex steroidal hormones

By sensory receptorsstimulates

Releasing hormones

Gonads

Regulation of secondary sexual chtrs, nuptial coloration, breeding

behavior

I) Provision of appropriate environmental stimuli

II) Administration of hormones for maturation and release of gonads

II) Induced reproduction

• Under culture conditions, the required environmentalfactors may not be available or persist for sufficientlength of time for spontaneous maturation to occur.

• This led to Induced reproduction or Hypophysationtechnique, where pituitary homogenates are injectedinto fish.

• This stimulates gonadotropin surge, by-passingenvironmental variables.

• Inducement of spawning in fishes that do not ordinarilybreed under conditions of confinement.

III) Historical background

• In India, first attempt to induce spawn of carps byadministering mammalian pituitary extract bymade by Hamid Khan (1938) but without success.

• Chaudhuri and Alikunhi (1957) succeeded inmaking Labeo rohita, C. mrigala, C. reba, L. bataand Puntius sarana to breed by injecting themwith fish pituitary extract.

• Chaudhuri (1959) successfully carried out crossbreeding of 12 different sps by artificialinsemination.

III) Fish pituitary• Pituitary gland or hypophysis-situated on ventral side of brain in

concavity of cranium called sella turcicia and covered by duramater.Pituitary has 2 parts:• Neurohypophysis (nervous part)• Adenophypophysis (glandular region)

Pro-adenohypophysis (ant lobe, ant glandular region)meso-adenohypophysis (middle lobe, middle glandular region)Meta-adenohypophysis (posterior glandular region)

IV) Hormones of pituitary related with development of gonads and spawning

Adenohypophysealhormones:

• GH• Somatotropic hormone• TSH• GTH• ACTH• Prolactin• Melanocyte stimulating

hormone

Neurohypophysealhormones:

• Oxytocin

• Vassopressin

• ADH

• GTH-play imp role in regulating development ofgonads and spawning.

• Gonads release: Androgen, estrogen andprogesterone.

• Further environmental factors like temp, raininfluence the gonads and other endocrine system

VI) 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

2. Collection of pituitary gland

3. Preservation and storage of gland

4.Preparation and preservation of pituitary extract

5. Determination of Pituitary dosage

6.Ponds for induced breeding and breedingHappas

7. Injections of pituitary extract to the breeders

8.Spawning and fertilization

9.Stripping or artificial insemination

The primary requirement of carp breeding is anadequate stock of good breeders. The excellent broodfish culture is the foremost essential material basis andkey factor for successful artificial propagation.

Brood stock: Group of mature individuals used inaquaculture 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.

Inductive for rapid gonadal

growth of carps

b. Characteristics of ripe brood fish and their selection for stocking

• Smooth pectoral fin.•Abdomen is soft and bulging.•Pinkish vent.•The vent of fully ripe female is slightly elevated.

•Rough pectoral fin.• Abdomen is smooth and not bulging.•Vent not pinkish.•Whitish secretion called ‘Milt’ come out on pressing the vent.

Spawner Milter

• Selection of female is very important.

• A simple catheter ca be used to find out if the fish ishaving eggs or not and shape and size of eggs suggestthat the female is quite ripe.

• Grass carp- red colored eggs

• silver carp- blue

• Color varies with various ecological zones and alsowith the age of female.

Healthy, disease free breeders of catla, rohu, mrigal,silver carp, grass carp and common carp of 2-4 yrsand 1-5 kgs are normally selected

c. Stocking rate (sex ratio)

• Sex composition of breeders is normally maintained as1:1.

• Males and females are kept separately in brood pondso that it increases the affinity of mating of the pairduring breeding experiment.

Stocking is done 1500-2000 kg/ha with speciescomposition 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 toanother- for food utilization.

• In case of infection- bath in 10 ppm KMno4

e. Manuring and maintenance of water quality inbrood-stocking ponds

•Initial manuring of brood pond- 3 weeks prior to stockingwith cattle dung or compost manure.•7 days Before fertilization- lime treatment•1/8th to 1/4th water change frequently.

Seining

f. Supplementary feeding

• Supplementary feed given daily.

• a complete feed is a proportionate vegetable and animalprotein, carbohydrate and fat. Mixture is prepared from:

half cooked rice and pulse (1:1, 50%)+ oil cake (20%)+ cattledung (20%)+ Fish meal/silk worm pupae/ cookedslaughter house refuse (10%). (Rohu, catla, mrigal,common carp)

1-2% diet of BW before spawning and 3-4% diet afterspawning. No feed is given for one week before actualbreeding.

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 ofbreeder.

• Excess feeding may result in heavyaccumulation of mesenteric fat inhibiting theprocess of smooth ovulation.

Collection of pituitary gland

IMC-serve as Donorfishes.However, Common carpis most preferablebecause of its easyraising.The glands from freshand fully ripe donors areused.Suitable period:breeding season.

Preservation and storage of glands

Brazilian MethodAcetone dried

method

Methods

Brazilian method of preservation in Abs alcohol

Glands are removed into absolute alcohol

Transferred to air-tight phials

After 24 hrs-Transferred to other air-tight phials containing

fresh alcohol

Phials are stored in dark air tight bottle containing ab Al at room temp in desssicator containing

anhydrous CaCl2 in cool place or kept inside refrigerator

• Pituitary banks are maintained.

• Since pituitary hormones are water soluble, henceBrazilian method of preservation in absolute alcohol issuitable.

Gland remain effective for 5

years

Acetone-dried methodGlands are removed

into acetone and put into refrigerator (acetone changed 2-

3 times)

After 36 hrs of storage, the phials are taken out and

kept in room temp.

Glands are removed and kept on filter paper

to dry

Glands are stored inside air-tight sterile phials and kept inside

dessicator containg anhydrous CaCl2 which is then placed inside

a refrigerator

Acetone dried glands retain potency for 6 months to 10

years

Preparation and preservation of pituitary extract

• Gland register- serially labeled phials,

details of phial no.

no. and wt of gland and

donor sp’s

Extract: Required quantity of gland → homogenized intissue homogenizer →macerated in Physiologicalsaline (0.3% NaCl)

Intraperitoneal injection: no need of centrifugationIntramuscular injection: suspension if centrifuged at 1000 rpm for 5mins

PreservationGlycerine preservation method

1) 1/3 of total volume of extract is made up by addingDW.

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 hrsand afterwards centrifuged.

5) Supernatent is filtered and stored in air tight bottlesinside refrigerator

6) Sealed in ampoules (labeled giving details ofhormones conc., date of prep)

DoseCalculation 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

Weight of spawner Provocative dose First injection

Final dose Second injection

0.5- 1 kg 0.5 ml 1 ml

1- 2 kg 0.75 ml 1.5 ml

Above 2 kg 1 ml 2 ml

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 HAPABox 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 touchmuddy pond bottom at least 15-20 cm of hapashould remain above water level.

Injection of pituitary extracts to breeders

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

• Thickness and length of needle depends uponsize 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 pelvicfin. Fish may die due to damage of internalorgan. Practiced in US.

• Tip of needle is inserted under a scale and ispushed through abdominal wall into body cavityand 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 scaleparallel to body and muscle is pierced through quicklyat 45°, so that no damage is done to scale

• In case of single injection, this could be donein late in the evening and spawning could beover by early hours of morning.

• Both injected males and females are kepttogether in breeding hapa for spawning.

• In breeding hapa for C.carpio, hanging fibrousroots of water hyacinth are kept because eggsof common carp are sticky.

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

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 ispressed and eggs and milt are collected in tray andmixed with spoon. After few mins fertilized eggs aretransferred into breeding hapa.

Wet method: physiological saline is used to receive miltfirst and eggs later

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