COMPILED BY :
DR. ELEK WOYNAROVICH
FAO FISHERY CONSULTANT,
FOR FAO/MAG/76/002 INLAND FISHERIES
AND AQUACULTURE DEVELOPMENT PROJECT
MADAGASCAR
OCTOBER-JANUARY 1979–1980
PRACTICAL TRAINING SUBJECTS
Using the plancton net and observation with magnifying glass.
Differentiation of the main organism groups in the water : Algae, Rotifers, Copepods (Cyclops, Diaptomus), Daphniids, Larvae of Chironomids, other insect' larvae.
Estimation of the qualitative and quantitative plancton content of a pond. Plancton in developing stage ; Plancton in decreasing stage.
Investigation of the female and male common carp. The signs of readyness for induced propagation. Principles of the carp breeders' selection.
Calculation examples to the pituitary gland doses.
Preparation of the pituitary gland extract, counting, pulverization, dissolving technique practiced with carbamide grains.
Practizing the administering of hypophysis doses on rubber foam made carp like forms.
Practizing the “stitch in” of the carps on cardboard pieces.
Practice preparation of the solution, injection administering, stitching in, etc… on living carps.
Anestetizing practices with different materials (MS 222, Phenoxiethanol, Quinaldine) with fish.
Practical demonstrations of stripping eggs, collecting milt, fertilisation of the eggs, treatment against stickiness, tanninizing the eggs' mass. Preparation of the different solutions.
Observation of the eggs with magnifying glass in its early developing stages. Good and bad eggs. Practizing the incubation technique, watercurrent regulation in the jars. Observation on the embryo development.
Determination of the fertilisation rate with the help of a glass tube 2.5–3 mm inner diameter.
Preparation of malachit green basic solution. Treatment of the eggs with malachit green, against Saprolegnia.
Observation of the “ready to hatch” eggs. Transferring the eggs into the larvae rearing jars.
Practizing the larvae rearing, observation of the larvae with magnifying glass. Cleaning the jars from the debris.
Preparation of the boiled egg yolk food, feeding practice in the jars. Taking out the fry from the jars. Before that, observation of the air taking, and filling up the airbladder.
Preparation of the nursery pond. Inoculation of the nursery ponds with Rotifer plancton. In the meantime, observation of the plancton development in the nursery ponds, using plancton net and magnifying glass.
Temperature measuring practices ; Measuring the bottom water temperature Observations about transparency, using the Secchi disk.
Parallely : kakaban spawning, hatching the kakabans in hatching box. Observations on the Saprolegnia.
20a. Controlling SAPROLEGNIA with 1 : 20.000 formalin in the box.
Observation of the young fish in the nursery pond. Food quantity estimation, observing the food eaten in the living fish.
Counting the young fry.
Demonstration of the harmful activity of the CYCLOPS.
Discussion of the keeping techniques of the breeders.
Cleaning, maintenance, and storing the hatchery equipments.
Preparation Paramecium culture with dry grass in a bucket ; Demonstration of development of one cell animals.
TRAINING COURSE
Most of the fish species are living in two environments during their life. One is where they live and grow : (this is the living place, habitat) the other where they propagate. This latter is a specially chosen place, where not only the propagation occurs, but also with great probability the survival of the offsprings, during their early life stages (egg, larvae, early fry), is secured. It can be stated that the selection of the place for propagation is an instinctive activity which supposes a sort of heredited foresight of the breeders for the interest of the future generations.
Fishes can be distributed in general in four groups according to their propagation (spawning) places.
Fishes which spawn in confined waters, as lakes, ponds, reservoirs, etc… Such typical pond spawners are the Tilapia group (Cichlidae family), Black bass, Arius sp., further Crucian carp, Tench and many others.
Here, the living place of the fish is near to the spawning place although the latter is mostly limited in a characteristic place of the confined water, as shallow shore water, sandy shore, water plant covered areas, stony parts, gravel ground, etc… Usually the spawning (propagation) place of these fish species is near to the bank, in the shore area where the survival of the young generation has more chance as in deep water.
Many species of the pond spawners practise some sort of parental care, especially in the tropics and subtropics, where the confined waters used to teem with enemies of different size and kind. Such parental care is for example : preparing nest for the eggs, guarding, aerating (fanning) and protecting the eggs, larvae, early fry. Very effective parental care is for example the mouth-breeding habit.
Fishes which spawn in the rivers.
These fishes (fish species) are not all river dwellers necessarily. Their living place may be confined waters as well where they grow excellent as a sure sign of their well feeling. But for spawning, they need flowing water as rivers, streams, etc… Most of then are selecting there also a specially suitable place and time for spawning.
Especially the flooding turbid river is providing a safe place for the floating eggs of many fish species as Chinese major carps (silver, bighead, grass and black carp), Indian major carps (catla, rohu, mrigal, calbashu), Asian catfish (Pangasius) and many other species. Here the survival possibility of the eggs and larvae is better because of the lack of enemies and have good oxygen situations.
Fishes which spawn on freshly inundated lands.
Unregulated rivers during the rainy season inundate dry lands. Here the water living enemies are yet few or even nil and the food provision of the early fry is profuse. Originally the common carp selects such spawning place among natural living conditions. But the domesticated carp, most probably due to the domestication, is losing its original instinct and they used spawn in confined water (as pond) as well, if the main requirements of the spawning are met. Such as :
substrate to attaching the eggs as grass, water weeds, artificial kakaban or grass matt
clean, not too turbid water
shallow water
suitable temperature (18–22 C°)
presence of males
absence of other especially carnivorous fish species (like Black bass).
Fishes which live in fresh water but migrate to the sea.
Such fishes are the Mullet species, Chanos chanos, eels and many others.
Spawning habit means the way and mood how they do spawn and what they do before and after. According to the spawning habit the fish species also are very different.
Some Tilapia species prepare a mud nest on the bottom of the pond in which the eggs will be laid. After that the male or female takes the eggs into its mouth where they hatch and the larvae develop. Sometimes the feeding early fry find a good shelter (refuge) in danger case in the parental mouth.
The black bass prepares a nest in the sandy shallow shore area of the pond where they spawn in pair. The male afterward eagerly guards, fanns and protects the eggs and larvae.
Some other fish as american catfish not only prepares nest, guards, and protects the eggs and larvae, but also they guide the early fry in group like the brood-hen does.
The common carp, crucian carp, scatters its sticky eggs when spawns on a large area covered with suitable substrate (grass, water weeds, kakaba). The eggs adhere to the mentioned objects. After that the breeders abandon the spawning place. Usually they spawn in groups stimulating each other to spawn.
The Chinese carps (silver carp, bighead or marble carp, grass carp black carp) and Indian major carps (catla, rohu, mrigal, calbashu) are spawning in the shallow turbulent waters of flooding river. Their eggs are non adhesive, floating ones which drifted downstream and toward the shore. The eggs hatch in the river current scattered in a large area. The larvae reach in the shallow side water of the river where they may grow up to fingerling size.
The trouts usually spawn in the upper reaches of the brooks (stream) where clean, cool water is flowing on gravelly bed. They fan out a shallow plate-like place where they spawn and cover the eggs with a layer of grave for protection.
These mentioned spawning habits are only sparse examples from the huge variety of the spawning habits. We can say each fish species has a sort of different spawning habits which is developed among a given environmental conditions for the interest of the survival of the offsprings. In a totally new environment, where the dangers and enemies are different, the old inherited spawning habit usually does not work well, and the fish species in all propability cannot settle itself.
Before involving in details of the carp propagation we have to deal with :
the breeders ;
the development of the sexual products (eggs, sperm) in the gonads. Otherway some parts of the technologies and the preparation of the breeders for propagation are hardly understandable.
The first question is : which is a good breeder ?
which is healthy ;
has no injuries and wounds on its body ;
it is free from parasites ;
which has the desired body form and proportions. The distribution of its scales is typical according to the strain ;
it has not deformations in the body and fins ;
which has well developed sexual organs and so can be used for propagation ;
which has all the necessary inherited properties typical to the improved strain. (Fast growing ability, less sensitiveness especially for oxygen deficiency, and by the handling, voraciousness, omnivorous property, etc…). These all could be stated by scrutinizing the offsprings.
Not all sexually ripe common carp should be used for propagation. A sharp selection is needed in every case concerning the desired body form, scale distribution, injuries, deformations and the good development of the sexual organs.
Here we will deal with the development of female sexual product, the ova or egg, because the sperm (concentrated in the carp milt) development by the common carp does not raise any problem by the propagation. The preconditions of the sperm development are about the same as by the egg development.
The development of sexual products in the gonads (sexual organs) - in short the gonad development - is influenced by the following main factors :
Temperature.
Most probably the temperature above 17 C° is influencing essentially
the gonadal development. The sum of
Suitable protein rich food.
To build up the eggs essential amino acids (basic components of the proteins) are needed in plenty. The animal protein content of the food has a great importance for the common carp.
For building up the eggs in the ovary, the natural food is the best Artificial food has to be enriched in full values proteins (fish meal, meat meal, blood meal, etc…). By systematical manuring of the breeders' pond, natural food can be produced.
Healthy environment.
Suitable dissolved oxygen content in the water ;
Suitable water depth (above 0,6 m) ;
No high crowdedness in the pond (For the sake of natural food at least 20–40 m2 pond surface for 1 female breeder) ;
Undisturbed circumstances in the pond ;
Good lighting is also needed (not shaded ponds).
Fishes of other non carnivorous species do not disturb the carp breeders so they can be kept together in polyculture. Only by the segregation time (short before the spawning season) they are segregated from the other fish.
It is advisable to keep separated the males and females of the comcom carp throughout the year so the females can be provided with better and protein richer food.
Stages of the cocita development in the ovary :
1 - Proliferation stage. The cells are normal cell (size : 8–12 microns) they multiply in great number.
2 - Follicle starts to grow around the single cell (size : 40–200 microns).
3 - The follicle (a double cell layer) now encloses the primitive egg cell (it is important for its nurture).
4 - Vitellogenesis (the development of egg yolk) starts. First lipoid globules appear (size : 200–350 microns).
5 - Cytoplasma full with lipoid globules. Yolk composition starts (size 350–500 microns).
6 - Yolk plates push the lipoid globules toward the edge of the cell (size : 600–900 microns).
7a - The yolk synthesis is finished, the nucleoli (where the yolk synthesis happened) withdraw in the center of the nucleus (size 800–1000 microns). No more changes occur in the egg cell ; therfore from now it is named as dormant or resting stage.
Until the dormant stage, the cocita (egg cell) development is automatically influenced only by the temperature and food provision (building material provision) ; other factors (oxygen deficiency, shallow water, disturbances, crowdedness, etc…) may inhibit this development.
7b - Preovulation, the nucleus moves toward the micropyle (small opening) which is on the egg shell. Egg absorbes water (hydration), (size : 1000–1200 microns).
7c - Ovulation, nucleus membrane disappears, chromosomes become visible, first meyosis occurs. Follicle dissolves, the egg falls into the cavity of the ovary and is ready to be ejaculated and fertilised.
The development stages 7b – 7c are started and directed by gonadotropin hormones which are synthetised in the pituitary gland (hypophysis) of the breeder.
Now the female is ready to spawn. The males can sense which female is ovulating and follow it ardently. When the female is getting out a batch of eggs, males do jet in the same time sperms in millions. So the egg and sperm can meet in the water and the fertilisation of the egg may occur. (Tis is an extracorporal fertilisation).
Fertilisation.
One sperma enters into the egg cell through the micropyle (a small hole or “window” of few micron diameter on the egg shell) and meet with the nucleus of the egg. Now the second meyosis occurs, triggered by the pronucleus of the sperm.
There is two main groups of phases to be distinguished in the sexual products (eggs) development.
development phases up to the dormant (resting) stage ;
final ripening phases i.e. preovulation and ovulation.
The first group of development phases are directed by the temperature and the quality and quantity of the food. Inhibiting circumstances may be there as well : oxygen deficiency, parasite infection, disease, crowdedness, bad keeping conditions, darkness, which stop or slow down the mentioned phases of the gonadal development.
The final ripening phases of the egg development are started and directed by gonadotropin hormones. Here also can exist inhibiting or braking factors as poor oxygen supply, decreasing water level, low temperature, nervous stress (lack of calm circumstances in the keeping place). Gonadotrop hormones direct not only the spawning activity, but also the migration toward the spawning place and all preparation activity.
The gonadotrop hormones or gonadotropins.
The gonadotrop hormones are produced and stored in the pituitary gland (hypophysis) of the fish. They will be released on the order of the mid-brain ( hypothalamus) in which the impressions are collected through the sensory organs. In short the fish has to feel through its sensory organs the suitability of the spawning environment before the gonadotrop hormones of its own pituitary gland become released into the blood circulation toward the ovarium, where they cause the final ripening of the eggs as it was described.
If the breeder does not find a suitable spawning environment, its gonadotrop hormones remain in the pituitary gland and no final ripening (ovulation) will happen.
Giving injection of pituitary gland extract from other fish (donor) directly into the body of a suitable female fish (which has developed eggs in dormant stage in its ovary) “induced” ovulation or/and spawning can be achieved.
As it was told, most of the fish species have two environments ;
where they live and grow ;
where they propagate.
The common carp is also not an exception in this respect.
In the living or growing environment, if the fish meet the most important environmental requirements (temperature, food, etc…) here the gonadal products develop up to the dormant (resting) stage. The males have not such definite dormant stage of sperm development. They are ever ready to spawn when the milt is oozing from their testicles.
The propagation environment should possess all those requirements which affect the sensory organs of the fish, producing such a strong impression complex which starts inspiring the mid-brain to give order to the pituitary gland to release the gonadotrop hormones.
Factors which are necessary to trigger a natural spawning in a given environment are the follows (concerning common carp) :
suitable temperature (18–22 C°, sometimes 25 C°)
grass covered shallow water (water weeds may be good as well, or artificial egg collector facilities, like kakabans)
raising water level (or at least stable water level)
clean not too turbid water, rich in oxygen
suitable raising barometer stand (no depression)
presence of the other sex.
If all these requirements are met, natural spawning occurs. For natural spawning in ponds it is necessary to provide the suitable spawning environment for the common carp. It is to note that the common carp is very demanding in this respect, not so as crucian carp or other related species.
If the spawning conditions for the common carp are not present they do not spawn at all. Or the spawning will be partial, only, without a hope of good success.
Fish which is used for culture has to be propagated, otherwise how to obtain young fish for stocking ponds, rice fields, etc… Young fish has to be available in the demanded quality and number, year after year.
The natural propagation of the common carp is uncontrolable, its success is often very uncertain. It is hardly possible to base a planned fish-culture on that.
Therefore different techniques were worked out to improve the results and success of the natural propagation, in the interest of securing the necessary number of young carps.
The most simple techniques are the semi-artificial (or improved natural) propagation techniques.
The spawning conditions are provided according to the requirements of the common carp in a small, well controlable pond (so called spawning pond).
It is important there :
a grass covered shallow (30–40 cm deep) part in the pond where the eggs can be laid ;
clean (not too turbid) enough warm (18–22 C°) water ;
well maintained water level (the water should not decrease in the pond) ;
keeping off any predators and enemies (the inflow water has to be well filtered through a filter box) ;
males have to be stocked with the ripe females.
According to an other technique, artificial egg collectors (substratum for the eggs) as kakabans or grass matts are placed in a small pond with good water supply. The kakabans will serve as spawning place. For one female 1–1.5 m2 kakaban or grass matt surface is calculated. Here the water should be clean, of suitable temperature and free from enemies. The water level has to be maintained stable as well.
Two types of artificial or induced propagation are used in the carp propagation practice, which are based on :
induced spawning;
induced ovulation.
The ovulation (final egg ripening process) is brought about by injecting fish hypophysis (pituitary gland) extract into the dorsal muscle of the female and male. This extract contains gonatropin hormones which bring about the ovulation and are forcing the female to spawn naturally on artificial egg collectors as kakabans or grass. Because there some of the spawning conditions are met i.e. there is spawning substratum (kakaban) which stimulates the natural spawning, there are males present, and the water level is kept, the dose of the gland extract can be less. About the half dose is used for induced spawning as to induced ovulation.
The ovulation is induced by a bigger dose of hypophysis extract among totally artificial conditions, usually in a cement concrete or plastic basin, where only the suitable temperature, the oxygen supply and undisturbed circumstances are cared.
Usually two doses of gland extract are administered. The first dose is about 10% of the second dose which is given after 24–14 hours interval. For the calculation of the doses, see later.
Higher dose is necessary here because no environmental conditions help to bring about the ovulation.
The result of the induced ovulation is the well strippable so called flowing gushing “ripe for fertilisation” eggs, and profusely flowing milt. These “ripe” sexual products have to be collected “dry”, that means without coming in tuch with water. They are mixed “dry” throughgoingly and fertilised artificially with a suitable solution, which prevents adhering the eggs and promotes the fertilisation (fertilising solution).
The technology of induced ovulation and further treatment of the sexual products is given in details below :
First of all we have to answer : what is the meaning of such, rather complicated technology ? What can we gain by taking such pain of artificial propagation ? Such technology has meaning and justification only then if it provides far better protection in the early life stages for the fish and results a considerably higher survival rate for the offsprings.
Why induced ovulation ?
By this way a better fertilisation rate can be achieved ;
The sticky layer of the common carp eggs can be removed or the stickiness eliminated, and the eggs can be incubated in incubator jars. During the eggs development full controlled conditions can be provided ;
The incubation and hatching rate will be considerably higher than in nature ;
The larvae can be kept among well protected and safe conditions, securing by this way the highest possible survival rate ;
First food can be provided in the jars which has a beneficial effect on the further life of the fry ;
The first feeding fry can be stocked in a well prepared nursery pond, where most of the enemies can be eradicated, and ample food of suitable size and quantity can be provided ;
Advanced nursery management can be practiced.
But satisfactory result can be achieved from the induced ovulation only if the breeders are well prepared for propagation and their ovary development already reached the dormant stage, which means it surpassed already the vitellogenesis.
The signs of this readyness of the breeders can be stated by an external examination.
Females : have bulky soft or semi-soft belly.
Their sexual opening is reddish and protruding.
The anal ring (anal opening) is protruding and enlarged.
Males : They release a few drop of milt when pressing slightly the belly.
They have a slender appearance ; their abdomen is not bulky.
Sometimes roughness occurs on the head or on the pectoral fins.
Preparation and administering the pituitary extract.
Usually aceton dried pituitary gland of common carp is used. It should be known the average weight of the pituitary glands in milligram. It can be weighed using a fine balance.
The weight of the female individuals should be known. The weight of the females is rounded off to half and round kilogram. In the carp propagation practice exists one and two doses treatment.
The one dose treatment usually used for induced spawning.
Two doses treatment are used for induced ovulation.
The females get a very different treatment because the result of the ovulation highly depends on the quantity (hormone content) of the gland extract.
The doses are different according to the season, size of females and the temperature.
One dose for females.
(in milligram of aceton dried gland per kilogram)
| at the beginning of the season | middle of the season | out of season | in high temperature above 25–26 C° |
| 2.5 | 2 | 3–3.5 | 3–3.5 |
Two doses treatment for females.
| at the beginning of the season | middle of the season | out of season | in high temperature above 25–26 C° | ||
| 1. | first dose | 0.5 | 0.3 | 0.8 | 0.5 |
| 2. | second dose | ||||
| fish 2–5 kg | 4 | 3–3.5 | 4.5–5 | 5.2–5.5 | |
| fish 6–10 kg | 5 | 4.5 | 5.5 | 6 | |
| The males get about the same dose irrespectively to their weight. | ||||
| 2–2.5 | 0.5–1 | 2–2.5 | 2–2.5 | |
The males are injected after the females got their second dose.
When induced ovulation is the aim, to avoid scattering and unnecessary loss of eggs the common carp females have to be stitched in (sutured their genital opening).
For this operation the females are usually anaesthetized. For this purpose about 0.5 gram MS 222 is dissolved in about 50–80 cm3 water and in this solution a cotton “sausage” is put. Inserting the sausage into the mouth of the female, it will “sleep” within few minutes.
Other technique :
2 cm3 Phenoxiethanol is shaken in few cm3 water and put in 100 l. water. The fish will there sleep as well. 1 cm3 Quinaldine in 100 l. water also can be used for the above purpose.
To dissolve the gonadotrop hormones out of the dry glands, physiclogical salt solution is used. Preparation : 7–8 gram common salt (NaCl) is dissolved in 1 liter boiled and filtered drinking water.
For smaller fish, below 3 kg, the 1 kg dose is given in 1 cm3 solution (they will get maximum 3 cm3 hormone extract). For bigger fish the 1 kg dose is given in 0.5 cm3 solution. It is advisable to select about the same size females for treatment which makes easier the calculation of the doses. The first dose is administered in 1 cm3 solution.
Where to inject : The carps are injected into their dorsal muscle, in the half way between the dorsal spine and lateral line, about under the first spine of the dorsal fin.
Sequence of the activities during the administering of the second dose :
The incubation (ripening) of the females after the second injection.
The incubation time span (lapse of time) depends on the temperature of the water in which the females are kept. By changing temperature it should be measured in each hour. When added the hourly measured temperatures and their sum become 240–260 the ovulation occurs. It means 240–260 hours/grade is the ovulation time.
By stable temperature the ovulation occurs after hours shown in the table below.
| Temperature C° | mean | earlier | later |
| 17 | 15 | 14 | 15.30 |
| 18 | 14 | 13.30 | 14.30 |
| 19 | 13 | 12.30 | 13.30 |
| 20 | 12.30 | 12 | 13 |
| 21 | 12 | 11.30 | 12.30 |
| 22 | 11.30 | 11 | 12 |
| 23 | 11 | 10.30 | 11.30 |
| 24 | 10.30 | 10 | 11 |
| 25 | 10 | 9.30 | 10.30 |
| 26 | 9.30 | 9 | 10 |
Usually, but not necessarily, indicator male (males) are put into the incubation basin with the females which indicate by ardent ourting the just ovulating female.
The sexual products (egg and milt) have to be collected “dry” (without a drop of water). The fish and the collecting bowl or plate should be dried with a soft towel before stripping. Water spoils the eggs and sperms. The females usually gush out the eggs when the stitching is cut. A slight pressure on the belly helps totally emptying the ovary. When stripping, the female should be held enveloped in soft towel.
For the milt collection milt collector is used. But the males can be stripped directly over the already stripped eggs. (Attention the male may jump and destroy the eggs).
After stripping off the eggs, the egg-mass should be weighed, for possible estimation of the egg number, and mixed throughly with milt. For mixing the sexual products, feather or plastic spoon is used. After mixing the sexual products dry, the fertilisation should be made without delay.
If the stripped breeders were put in a basin paved with kakaban, they will spawn there.
The spermatozoids are motionless in the milt. They start vigorously moving when became in contact with water. Water activates the movement of the sperms. The movement (swimming) of the sperms lasts less than one minute long in normal fresh water. After that time, the again motionless sperm is no more able to fertilise egg.
One of the moving sperm enters into the egg through the small opening on the egg shell (named micropyle). The micropyle also remains open in water only about one minute long, after that it closes up and fertilisation no longer can occur. The egg becomes unfertilisable.
The sticky coating (adhesive layer) of the carp egg is activated in the water. About after 6–7 seconds the egg adheres to a substratum (grass, thread of the kakaban) or to each other.
The egg in the water starts to swell immediately. A fluid is accumulating between the egg shell and kernel. This space between the shell and kernel is named perivitellinar space. The 1000–1200 microns (1–1.2 millimeter) egg becomes among natural conditions 1600–1700 microns (1.6–1.7 millimeter) of diameter. Using salt-carbamid solution the carp egg swells up to 2.0–2.2. millimeters.
It is obvious that there is only a very short time (about 1 minute or even less) during which the egg can become fertilised. To the successful fertilisation such a solution is used which in the mean time promotes the sperm movement and the fertilisation, and impedes simultaneously the sticking together the eggs.
This solution is made of 40 grams common salt (NaCl) and 30 grams carbamid (urea) dissolved in 10 litres of clean pond water. About half the amount of this solution as the egg mass is used for fertilisation under continuous stirring. Fertilisation is made no longer than 2–4 minutes. After that stickiness dissolving (eliminating) solution is added to the egg-mass. This solution contains 40 grams common salt and 160 grams carbamid (urea) in 10 liters of clean pond water. After adding 2–3 liters of this solution the egg-mass has to be stirred only 5–8 minutes long. After that the stirring has to be made time to time only. New solution added 2–3 times by replacing the old solution which is to be poured off time to time. After 45–50 minutes the eggs are swollen and hardened. This one can feel with fingers.
When the eggs are so far, they can be treated with tannin. About 8–9 grams (a small spoonful) tannin is freshly dissolved in 10 liters water. After pouring off the stickiness dissolving solution the egg-mass is poured into 3–4 liters of tannin solution and stirred, 3–5 seconds long. After that clean water is added into about 5–6 liters. When the eggs settle down the solution is poured off and the treatment is repeated 2–3 times.
The egg-mass should be washed after the tannin treatment and put into the incubator jar.
Because the eggs in their early development stages are very sensitive for shaking and shocking, only slow waterflow has to be provided in the jars during the first day of development (0.5–1 1/min).
The eggs need during their development a continuous supply of oxygen for their life, and the waste materials produced by the living eggs should be removed. The continuous water flow in the jars does the both, supposed that the water has ample dissolved oxygen and is not loaded with waste materials of other livings.
The egg development pass through the following main stages 2-cell, 4. 8, 16. 32 cell stages, morula stage, blastula stage, gastrula stage. During this stage the cell layer grows around the yolk mass, the blastoporus is closed, and embryo development starts. It developes the head and tail. The heart starts to beat. Pigmentation occurs on the head. The embryo rotates more and more often in the egg shell. The eyes of the embryo become darker and darker ; at least eye is not only black but iridizes. This is the sign that the embryo is ready to hatch.
The eggs, if they get enough oxygen, develop well. The speed of the development depends on the temperature of the through flowing water. Some unfertilised or during the treatment injured eggs are in each batch of eggs. The living eggs are transparent, shining, the dead eggs are whitish and opaque. On the dead eggs a fungus named SAPROLEGNIA may settle and its cottonlike hairy branches spread over the living eggs and kill them. To avoid heavy losses caused by Saprolegnia attack MALACHIT GREEN (a chemical in fine cristals) is used for prevention.
A spoonful or 3 flat small spoonfuls malachit green, is added to 1 liter of water. It is a very strong solution. From this, 10–20 cm3 (3–4 small spoonfuls) are poured to each jar in the second day of incubation after stopping the waterflow. The water in the jar becomes dark blue when mixed with a stirring “oar”. New stirring the eggs and let stay 3–5 minutes long. That is the preventive treatment. After that the waterflow is restored. The treatment has to be repeated in each 6–12 hours, depending on the temperature. In each 6 hours in temperature above 25 C°. The treatment is necessary especially if the fertilisation rate is lower than 80%. Eggs short before the hatching should not be treated with malachit green. This material is very poisonous to the larvae.
The egg development lasts about 3–4 days in normal temperature (20–23 C°). The hatching, arising from the egg shell, happens so that the embryo segregates an enzyme which dissolves the shell (The mechanical breaking up the shell has only a secondary role).
For hatching the “ready-to-hatch” eggs are transfered into the larvae rearing jar or larvae rearing box.
One part of the just hatched larvae swim off and adhere to the wall of the box or jar. Others are laying on the bottom of the box or rotate in the water current in the funnel part of the jar.
The just hatched larva has no mouth, gills, gut, airbladder mentioning only the lack of the most important organs. But it has yolk sack as energy provision, special veins for the oxygen exchange and some larval organs. Its respiration proceedes through enlarged veins on the side of the yolk mass through diffusion.
The developing larvae need ample of dissolved oxygen because taking the oxygen by diffusion is not very effective.
During the larval stage, which lasts 2.5–4 days long according to the temperature, develop the mouth, the gut, airbladder, and the gills. The larva grows more 2 millimeters in length, but looses from its weight 10–20%.
At the end of the larval development the fish swims to the surface of the water and takes small air bubbles in its mouth and by pressing down in the gut, fills up its airbladder. From now the larvae can swim “fish-like”. After filling up the airbladder the larva becomes fry and it is able to take food, although its yolk sack not yet consumed totally (about 25–30% of the yolk is not yet consumed).
Hard boiled egg yolk squeezed through a fine mesh sieve cloth (plancton net material) is given to the fry in the hatchery jar before stocking them into the nursery pond.
Although the common carp fry eats boiled egg yolk it is not a full valued food for it. Getting this food only during many days, the fry will perish. At the present does not exist such artificial food which is fully suitable for carp fry. Natural food is essential for the carp fry. But it is not able to take all types of living food. The living (natural) food of the fry should be suitable according its size (not bigger than 150–300 microns) and slow moving enough to be caught by the rather awkward fry. The food animals should be enough dense around the fry, that it is able to find them easily. The fry does not take non moving food from the bottom. If it takes attached or laying food, it has to draw the attention of the fry for example with moving. The fry finds its food by seeing it. The food has to “dance” before its eyes.
The most suitable first food for the young fry are the ROTIFERS (Rotaroria) and young larvae of Copepoda (Nauplius) and very young, just born Cladocera. It eats as well many one cell animals of bigger size, which develop on rotting grass in the water. About 6–8 food animal should be in one cm3 water (6000–8000 per liter) that the fry can catch food whenever it wants. The fry needs a great quantity of food for the rapid growth and healthy life which enable its survival.
The most dangerous enemies of the young fry (and the larvae as well) are the carnivorous COPEPODS (Cyclops species) and GAMBUSIA (or other Cyprinodontidae). They are teeming in ponds which was filled up long before the nursery use, and the mentioned enemies could settle.
The nursery pond should be surely free of Gambusia, therefore when filling up the nursery pond, the water has to be filtered through a filter box, where Gambusia is present in the watersupply system (avoiding also Tilapia and other unwanted animals).
In the interest to develop an enough dense population of suitable food animals, the nursery pond has to be filled up at the same day when the eggs were obtained. In that case 5–6 days are there to develop the food animals. To promote the food animal development manure has to be put in the pond. The manure quantity is maximum 50–60 kg fresh pig or cattle manure, or 10–20 kg dry chicken dropping per are (100 m2) ; to that it is added 0.3–0.5 kg superphosphate per are (fertiliser). These have to be diluted and dispersed in water, and spread over the whole nursery surface.
The development of the food animals can be promoted, besides manuring, by fixing with poles sheaves of dry land grass along the shore of the nursery, 5–7 m distance from each other. The grass should be surely land grass otherwise the development of waterweeds will be promoted by this way.
The first treatment of the nursery pond (manuring, fertilizing) gives a good push to the development of Rotifers and other food animals. But the fry in dense population exhausts soon the natural food. Therefore 3–5 kg manure and about 20–30 gram superphosphate has to be distributed in the nursery in each 3–4 days after the stocking. The natural food animals have to be checked often with a good plancton net (or taking out a glass full of water from the nursery and observe the presence of food animals with naked eye). If they become sparse, that means the nursery is poor in natural food supply, or the fry has to be thinned out with a fine mesh net, or flour like finely grinded feed (soybeanmeal, ricebran, etc…) has to be given 1–2 times a day to the young fish, along the shore.
Usually the fry could find enough food in the nursery pond up to 7–9 days after the stocking. For the further rapid growth it needs more and more food ; now it is time to start with the systematic feeding with finely grinded flour like artificial feeds.
If the water used for filling up the nursery contains many Cyclopids (when the water is taken from an other pond for example) which are the main enemies of the early (young) fry, the nursery can be treated against those enemies to avoid greater losses.
ORGANIC PHOSPHATE COMPOUNDS (as flibol, ditrifon or other soft pesticide) in 1 ppm concentration (1 cm3 active material in 1 m3 nursery water) is used to get rid of those enemies. The pesticide treatment should be made only when it is proved by a plancton investigation that the nursery water is infested with Cyclopids. The pesticide has to be distributed evenly on the whole nursery surface. The treatment of the nursery pond can be made only about 4–5 days before the stocking. It is advisable to check the pond water after that treatment whether it is not poisonous for the fry before stocking. Unfortunately the soft pesticides do not kill the predatory insect larvae treating the pond with the mentioned concentration. These enemies have to be collected with a suitable scoopnet and killed. The tadpoles which are competing for food with the fry have to be removed or netted out, and mixed with some ricebran, can be given for the breeders, as a protein rich food.
If few fry is stocked into the nursery, its production capacity will not be used ; if too many fry is stocked, they will not find enough food for a rapid growth, and will starve and perish from hunger. It has to be experimented or find out the adequate stocking rate, when the fry is in good density and do not suffer from the lack of food.
Depending on the preparation of the nursery and its food animal density, 10000–20000 up to 50000 fry is stocked in one are. That means 100–200 up to 500 fry per m2 or 1–2 up to 5 million per hectare. It is sure that such dense population of fry can be kept in the nursery pond only about three weeks or maximum four weeks long, even when feeding. The three weeks old fry is about 2.5–3 cm in length. To keep longer the fish in the nursery pond, the stock has to be thinned out with a suitable fine mesh net.
In a well prepared and manured nursery pond 50–60% of the fry may survive, supposed the enemies are controlled and the food supply is suitable.
Besides the enemies (Cyclops predators) the food shortage causes the main losses in the nursery pond.
The three weeks old fish is named now fingerling and can be stocked with good hope of survival into other ponds, ricefields, etc…
Pituitary gland extract is used for the artificial propagation of different cultured fish species to induce the ovulation and/or spawning.
Practically the pituitary gland of all sexually ripe fish can be used for the above mentioned purpose, supposed that the donor fish is before its spawning time, and enough big (over 1 kg) so its gland can be extracted easily.
In most cases common carp pituitary gland is used because :
In the USA, the pituitary glands of pacific salmons are also extracted because they are plenty available when they migrate from the sea to the spawning places in the rivers. Silver carp, bighead carp and grass carp hypophysis also used to be extracted especially in warm climate areas where these fish become sexually ripe at 3–4 years age. The hypophysis of the Indian major carps (catla, rohu, mrigal) is also used frequently in India.
Common carp provides the best quality of hypophysis before its spawning season starts and their gonadal development reached already the dormant (resting) stage.
When the fish spawns naturally it uses the hormone content of its own pituitary gland. At that time the gland is practically empty of hormones (gonadotropins) and no use to extract. But the common carp regains quickly the hormone content in its gland, especially in the warm climate ; 2–3 months after the spawning the hormone content in its gland is again renewed.
The pituitary gland is located under the mid-brain (hypothalamus) with which it is connected through a delicate funnel named infundibulum. When the brain of the carp is removed, this funnel breaks and the gland remains on the basis of the skull embedded in fatty tissue (The whole brain is also embedded in fatty tissue).
For the extraction the skull top has to be removed ; with an electric hole drill (hole bit) a round part of the skull top, the brain with the gland is removed. When electricity is not available, the skull top is sawn off with a metal saw, or sliced off with a sharp knife. When the brain cavity is open, the brain has to be taken out or turned forward to reach the gland.
With a fine pair of pincers the gland can be freed from the fatty tissue and extracted easily without hurting it.
The gland can be used fresh for making hormone extract or put in deep-freezers and used later. Otherwise the gland can be preserved in dry form, many years long.
The preservation technique.
The fat and water content has to be removed from the fresh gland with fat free acetone. Some authors advise to use for this purpose absolute alcohol (ethyl-alcohol without any water content). The gonadotropin hormones are water solubles so a very delicate and precise work should be made by the extraction of the fat and water content from the gland.
First of all, attaching fatty tissues and non gland parts have to be removed very carefully, not to break the gland. Immediately after that, the gland is put in fat free concentrated acetone.
For good reason the fish has to be killed before removing its skull top. The best is to cut through its heart. If the fish is killed by beating its skull with a heavy object, the brain will be inundated with blood which made the gland extraction more difficult.
Practically the glands are extracted from as many fish as it is available at once.
After finishing the extraction of the glands, the acetone has to be poured off from the collected glands, and new acetone is poured over them. The change of acetone is repeated two times in 8–10 hours intervals. After 24–30 hours the actone is poured off and the glands are spread on a small plate. When the acetone totally evaporated which happens within few minutes, and the smell of the acetone is no more sensible, the glands are put in vials pressed down with a bunch of cotton and corked tightly. The vial can be sealed with wax (bee wax) or stearin (common candle material) to avoid that moisture reaches the glands.
The acetone dried glands such a way can be stored for many years without losing their hormone content.
