Induced Breeding with Pituitary Gland Extraction from Fish

The fish production from inland water which are essentially culture fisheries and are less capital intensive have growth rate at present when compared to marine fisheries which are essentially capture and are capital intensive have relatively slow growth rate. Also increased population has resulted in shortage of foodstuff from land resources and even from the capture fisheries. Fish thus can be supplied with fewer man-hours and lesser capital investment. Thus, an emphasis has been given to culture fisheries - AQUACULTURE.

          The major problem in cultivating the major carps and Chinese carps is the non-availability of seeds for ready stocking in fish ponds. Because, they do not breed in confined waters even though they mature in these water bodies. They breed in flooded rivers during monsoon when temperature is low. Though riverine seed fish resources meats nearly 90 % of the demand, but they are mixed and found not suitable for profitable fish culture. Further, the seed fish may not be available at the time of requirements and seeds are available only at some selected collection centres which are often not easily accessible and their transportation from place of collection to the culture site may also pose mortality problems. In order to overcome these problems, the induced breeding by “HYPOPHYSATION TECHNIQUE " has been development. Usually pituitary glands are used to induce the fish for spawn. The hormone secreted by pituitary gland stimulates growth, development, maturity and ovulation of eggs. These hormone secreted from pituitary are protein or peptide. 

Collection of Pituitary Gland:

• The gland should preferably be collected from fully ripe gravid fishes, as the gland is most potent at the time of breeding or just before spawning.
• Glands collected from immature or spent fishes usually do not give satisfactory results.
• Glands in induced-bred fishes collected immediately after spawning have also been found to be effective.
• Most suitable time in India for collection of pituitary glands of major carps is during May to July months, as the majority of carps attain advanced stages of their maturity during this period.
• Common carp, Cyprinus carpio is a perennial breeder, its mature individuals can be obtained almost all the year round for the collection of glands.
• The glands are usually collected from freshly killed fishes

Collection Technique

• The pituitary gland is situated on the ventral side of the brain just below the hypothalamus.
• While taking out the gland, the dorsal side of the head is first chopped with a knife.
• The brain thus exposed is carefully lifted out by detaching it from the nerves. In majority of the cyprinids, when the brain is lifted, the gland is left behind on the floor of the brain box.
• The duramatter covering the gland is then cautiously removed using a fine needle and forceps.
• The exposed gland is then picked up intact without causing any damage to it because damaged and broken glands result in loss of potency.
• Glands are also collected through foramen magnum. In this method of gland collection, the fish is required to be completely beheaded.
• While, cut open the fish and collecting the pituitary gland blood will bleeds. Here fish should be wiped to clean the blood with cotton or tissue paper. If water is used to clean the gland, the chemical composition will be weaken and readily soluble in water.
• Among the internal factors, sex stimulating hormone of the pituitary gland play an important role in the development and maturation of gonads and spawning in fishes.

Pituitary gland secretes 5 hormones. They are as follows.

1.  Somatotropic Hormone (STH)

2.  Adrenocarticotropic Hormone (ACTH)

1. Thyrotropic Hormone
2. Gonadotropic Hormone (GTH)

(Follicle-Stimulating Hormone –FSH and Leutinising Hormone – LH)

1. Prolactin (Lactogenic Hormone)

Þ    It is advisable to use the gland belonging to the same species as 

      recipient fish - Homoplastic Gland.

Þ  Use of gland belonging to the different species – Heteroplastic Gland.

Preservation of Pituitary Glands:

• The collected glands must be preserved immediately because glycol- or muco- protein contained in them are degraded by the enzymatic action.

• The pituitary glands can be preserved by three methods.

1. Absolute Alcohol

2. Acetone and

3. Freezing.

Preservation of fish pituitary gland in absolute alcohol is preferred in India.

Preservation Procedure:

1. Absolute Alcohol

• The glands after collection are immediately put in absolute alcohol for de fatting and dehydration.
• Each gland is kept in a separate phial marked serially to facilitate identification.
• After 24 hours, the glands are washed with absolute alcohol and kept again in fresh absolute alcohol in dark colour bottles and stored either at room temperature or in a refrigerator.
• Occasional changing of alcohol helps in keeping the glands in good condition for longer periods.
• In order to prevent moisture from getting inside the phials, they may be kept inside desiccators containing some anhydrous calcium chloride.
• It is preferable to keep the glands in a refrigerator. They can be stored in refrigerator up to 2-3 years and at room temperature up to one year.

2. Acetone

• It is also a good preservative. In this method, soon after collection, the glands are kept in fresh acetone or in dry ice-chilled acetone inside a refrigerator at 100⁰C for 36-48 hours.
• During this period, the acetone is changed 2-3 times at about 8-12 hours intervals for proper de-fatting and dehydration.
• The glands are then taken out of acetone, put on a filter paper and allowed to dry at room temperature for one hour.
• They are then stored in a refrigerator at 100⁰C, preferably in desiccators charged with calcium chloride or any other drying agents.

3. Freezing of gland:

• Soon after removal of gland, they should be stored inside the refrigerator. But this method is not commonly used.

Advantages of the Pituitary Gland (PG)   

1. Pituitary Gland of one species is usually active in unrelated species.
2. Out of season, spawning could be induced.
3. Fresh alcohol preserved, acetone dried or frozen glands as well as their glycerin extract and saline suspensions
4. No quantitative differences in the Pituitary Gland of male and female.

Preparation of Pituitary Gland Extract:

1) Preserved glands are weighed. This is essential for accurate determination of the dose to be given according to the weight of the breeders.

2) The weight of the gland may be taken individually or in a group. To get a more accurate weight, a gland should be weighed after two minutes of its removal from alcohol.

3) The pituitary extract should be prepared just before the time of injection.

4) The quantity of gland required for injection is at first calculated from the weight for the breeder to be injected.

5) The glands are then selected and the required quantity of glands is taken out of the phials.

6) The alcohol is allowed to evaporate, if the glands are alcohol preserved ones.

7) Acetone-dried glands are straight away taken from the phials for maceration.

8) The glands are then macerated in a tissue homogeniser by adding a measured quantity of distilled water or common salt solution or any physiological solution which is isotonic with the blood of the recipient fish.

9) The most successful results of induced breeding in the Indian major carps have so far been obtained with distilled water and 0.3% common salt solution.

10) The concentration of the extract is usually kept in the range of 1-4 mg of gland per 0.1 ml of the media i.e., at the rate of 20- 30 gm. of the gland in 1.0 ml of the media.

11) After homogenization, the suspension is transferred into a centrifuge tube. While transferring, the homogenate should be shaken well so that settled down gland particles being mixed with the solution come into the centrifuge tube.

12) The extract in the tube is centrifuged and the supernatant fluid is drawn into a hypodermic syringe for injection.

13) The pituitary extract can also be prepared in bulk and preserved in glycerine (1 part of extract with 2 parts of glycerine) before the fish breeding season so that the preparing extract every time before injection is avoided.

14)    The stock extract should always be stored in a refrigerator or in ice.

Technique of Breeding:

• The induced breeding operation of major carps is taken up when regular monsoon sets in, the fishes become fully ripe and water temperature goes down.
• Females having a round, soft and bulging abdomen with swollen reddish vent. On slight pressure on the abdomen, eggs will oozes out.
• A male breeder can be easily distinguished by roughness on the dorsal surface of its pectoral fins and males with freely oozing milt are selected for breeding.

 1. Dosage of Pituitary Extract:

a) The dose of the pituitary gland is calculated in relation to the weight of the brooders to be injected.

b) A single high dose has been found useful when the brooders are in ideal condition and the weather is favourable.

c) Rohu seed production technologies responds well to two injections while catla and mrigal to both one and two injections.

d) An initial dose as a Provocative Dose at the rate of 2-3 mg. of pituitary gland per kg body weight of fish is administered to the female breeder only.

e) In case the condition of any one of the two males is not found in the freely oozing stage, an initial injection – Provocative Dose may be administered to the male at the rate of 2-3 mg/ kg body weight.

f) After 6 hours, a second dose of 5-8 mg/kg body weight is given to the female, while both the males receive the first or second dose at the rate of 2-3 mg/kg body weight.

g) Two males against each female make a breeding set. To make a good matching set, the weight of the males together should be equal to or more than the female.

h) Slight alterations in doses may be made depending upon the condition of maturity of the brooders and the prevailing environmental factors.

i) 1-3 pituitary glands are effective for a pair of fish.

2. Method of injection:

• Intra-muscular injection is the most common practice in India and it is less risky in comparison with the other mehtods.
• Intra-peritonial injections are usually given through the soft regions of the body, generally at the base of the pelvic fin or sometimes at the base of the pectoral fin.
• Intra-peritonial injection may cause damage to the internal organs, specially the distended gonads in fully mature fishes.
• Injections are usually given at the caudal peduncle or shoulder regions near the base of the dorsal fin.
• While giving injections to the carps, the needle is inserted under a scale keeping it parallel to the body of the fish at first and then pierced into the muscle at an angle.
• Injections can be given at any time of the day and night. But since low temperature is helpful and the night time remains comparatively quieter, the injections are generally given in the late afternoon or evening hours with timings so adjusted that the fish is able to use the quietude of the night for undisturbed spawning.
• The most convenient hypodermic syringe used for the purpose is a 2 cc syringe having graduations of 0.1 cc division.
• The size of the needle for the syringe depends upon the size of the breeders to be injected. No. 22 needle is conveniently used for 1-3 kg carps, No. 19 for larger carps and No. 24 can be used for smaller carps.
• Use of anaesthetics during injection would significantly increase the survival of brood fish. Commonly used anaesthetics are MS 222 and Quinaldine.
• MS 222 may be added to water in doses of 50-100 mg/ litre. Quinaldine is used at the rate of 50-100 mg/ litre.

Intra muscular injection

3. Breeding Hapa And Spawning:

• After the injection, the brooders are released immediately inside the breeding hapa.
• A breeding hapa is generally made of fine cloth in the size of 3.5 x 1.5 x 1.0 m for larger breeders and 2.5 x 1.2 x 1.0 m for breeders weighing less than 3 kg.
• All the sides of the breeding hapa are stitched and closed except a portion at the top for introducing the brooders inside.
• Generally, one set of brooders is released inside each breeding hapa.
• After the release of the fish, the opening of the hapa is securely closed so that broosders may not jump out and escape.
• Instead of hapas, cement cisterns or plastic pools as big as hapas can also be used for breeding.
• Spawning normally occurs within 3-6 hours after the second injection – Effective Dose.
• Soon after fertilisation, the eggs swell up considerably owing to absorption of water.
• Fertilised eggs of major carps appear like shining glass beads of crystal clear transparency while the unfertilised ones look opaque and whitish.
• The size of eggs from the same species of different brooders varies considerably.
• Fully swollen eggs of the Indian major  carps measure 2.5 mm in diameter, the largest being that of catla and the smallest of rohu.
• The carp eggs are non-floating and non-adhesive type. The yolk possesses no oil globule. The Indian major carps have a profuse egg laying capacity.
• Their fecundity, on an average is 3.1 lakh in rohu, 1-3 lakh in catla and 1.5 lakh in mrigal.
• The developing eggs are retained in the breeding hapa undisturbed for a period of at least 4-5 hours after spawning to allow the eggs to get properly water-hardened.
• After this, the eggs are collected from the hapa using a mug and transferred into a bucket with a small amount of water.
• The brooders are then taken out and weighed to find out the difference before and after spawning. This gives an idea of the quantity of the eggs laid.
• The total volume and number of eggs can be easily calculated from the known volume and the number of eggs of the sample mug.
• Percentage of fertilised eggs is also assessed accordingly by conducting random sampling before and after spawning. This gives an idea of the quantity of the eggs laid.

Estimation of Eggs:

• The eggs are collected from the hapa by means of cup or tray or beaker and transferred to the buckets.
• The brooders are also removed from the hapa and their weights are noted. The difference in weights reveals approximately the number of eggs laid.
• The eggs are kept in a rectangular piece of close meshed mosquito net and allow the water to drain off.
• The eggs are measured in a beaker, mug or cup of known volume and transferred to hatcheries.
• Thus estimation of total quantity is made from total volume of the eggs measured. Percentage of fertilization can be arrived at by counting the number of fertilized eggs from egg samples of 1 ml measure.

Stripping Method for Exotic Carps:

• Chinese carps however do not spawn naturally and when they spawn, the percentage of fertilisation is generally very low.
• Stripping or artificial insemination is therefore followed. The female fish is held with its head slanting upwards and tail down and belly facing the vessel, and the eggs are collected into an enamel or plastic trough by pressing the belly portion of the female.
• The male fish is then similarly held and milt is squeezed out into the same trough.
• The gametes are then mixed as soon as possible by means of a quill feather to allow fertilisation.
• The fertilised eggs are then washed a few times with clean water to remove excess milt and allowed to stay undisturbed in freshwater for about 30 minutes. The eggs are then ready for release into the hatching tanks.

Technique of Hatching The Eggs:

• The eggs collected from breeding hapas are transferred into the hatching hapas.
• A hatching hapa consists of two separate pieces, the outer hapa and the inner hapa.
• The inner hapa is smaller in size and is fitted inside the outer hapa. The outer hapa is made up of a thin cloth in the standard size of 2 x 1 x 1 m while the inner hapa is made of round meshed mosquito net cloth in the dimension of 1.75 x 0.75 x 0.5 m.
• All the corners of the outer and inner hapas are provided with loops and ropes to facilitate installation.
• About 75,000 to 1, 00,000 eggs are uniformly spread inside each inner hapa. The eggs hatch out in 14-20 hours at a temperature range of 24-31⁰ C. The period of incubation, in fact, is inversely proportional to the temperature.
• After hatching, the hatchlings escape into the outer hapa through the meshes of the inner hapa.
• The inner hapa containing the egg shells and the dead eggs which are removed when the hatching is complete.
• The hatchlings remain in outer hapa undisturbed till the third day after hatching. During this period, they subsist on the food stored up in their yolk sac.
• By the third day the mouth is formed and the hatchlings begin directive movement and feeding. At this stage they are carefully collected from the outer hatching hapa and stocked into prepared nurseries.
• It has been found that Indian major carps could be induced to spawn twice in the same season with an interval of two months.
• The breeders after the first spawning are fed with groundnut oilcake and rice-bran in the ratio 1:1 at 2.5 % of the body weight. When favourable climatic conditions occur, they mature and are ready for spawning.

Fecundity of Major and Chinese carps

Species	Fecundity
Catla	2,50,000 eggs / kg of fish
Rohu	3,00,000 eggs / kg of fish
Mrigal	2,80,000 eggs / kg of fish
Grass carp	80,000 eggs / kg of fish
Silver carp	2,00,000 eggs / kg of fish
Common carp	1,20,000 eggs / kg of fish

Method of calculating the eggs: Both Weight and Volumetric Method can be adopted for the measuring the eggs.

In Weight Method: weigh the eggs before absorption of water, multiply the weight of the egg number per unit weight, and then total eggs can be calculated.

In Volumetric Method: Measure the volume of eggs before absorbing the water, multiply it by egg number per unit volume and then the total quantity of eggs can be calculated.

 

                                No. of fertilized eggs

1. Fertilization Rate (%) = ---------------------------- X 100

                             Total No. of eggs 

 

                               No. of individuals harvested

       2. Survival Rate (%) = ------------------------------------ X 100

                              No. of fertilized eggs

 

                            No. of hatched fry

  3. Hatching Rate (%) = ---------------------------- X 100

                             No. of fertilized eggs