Some kinds of intervention by man in the course of natural propagation of cultivated or cultivable fishes may help to achieve a better survival of their offspring. The techniques of artificial propagation of fish are manifold, all of which are aimed at producing plenty of spawn, fry, and fingerlings for utilization in culture or for restocking water bodies or water courses. The demand for quality fish seeds is particularly great for modern intensive and superintensive culture systems. The polyculture system has further increased the demand for the seed of fishes of different feeding habits.
Artificial propagation, therefore, involves human intervention in the process of natural propagation and has the advantages of (a) better rates of fertilization and hatching, (b) protection against enemies and unfavourable environmental conditions, and (c) better conditions for growth and survival.
Artificial propagation as practised in different parts of the world may vary, depending on local conditions and facilities. It may start with the collection and further rearing of naturally produced egg, spawn, or fry, or with the production of the egg itself through artificial inducement (Figure 9), followed by controlled fertilization, hatching, and rearing of larvae and fry.
The collection of floating eggs, larvae, and fry of fishes spawning in rivers and inundated areas (viz., the major carps) is an age-old practice in India and China. A large number of funnel shaped nets are operated along gently sloping banks of rivers in the path of the drifting eggs or larvae. These nets are constantly watched and the eggs or larvae collecting in the tail piece emptied periodically. The seeds thus collected are placed in small earthen pits with or without continuous water flow or in cloth enclosures (hapas) fixed in the river itself for temporary holding and conditioning before transport. In the case of eggs, they are allowed to hatch in these pits or hapas and the larvae allowed to grow for a few days. The larvae are then transported to nursery ponds. Similarly, the fry collected from the rivers in special fry collection nets are also transported to nursery ponds for further rearing. The fingerlings raised are used for stocking fish ponds.
The artificial intervention in this procedure consists of (1) collection of eggs, larvae or fry; (2) their protection from natural enemies and unfavourable environmental factors; (3) raising the larvae up to the fingerling stage, and (4) distributing them for stocking in different water bodies.
This technology of river fish seed collection can be easily adopted in other parts of the world where useful river spawning fishes are available in adequate number. However, this technology has several disadvantages. The collection is generally not pure, but a mixture of several species some of which may be predators or of undesirable type. It is not easy to separate the various species, particularly in the early stages. However, some techniques have been developed both in India and in China to segregate desirable from undesirable seed. These are successful only to a limited extent, and one has to wait until the larvae develop into fry with distinguishable characteristics for proper identification. The collection technology is labour-intensive and requires special skill in handling and transport. Another disadvantage is that the brood stock is unknown and there is hardly any possibility of improving the stock.
Many authors do not consider this techniques as artificial propagation, although the helpful artificial interventions into the life cycle of the fish are obvious.
It is possible to procure fertilized eggs completely or almost free of the eggs of other fishes. Such eggs are incubated under controlled conditions and the resultant larvae can be reared to fingerling stage according to the requirements of the species concerned. The goal is to achieve the best possible survival rate, good growth, and health. The fertilized eggs can be procured in several different ways.
4.2.2.1 Procurement of fertilized eggs without hormone treatment. Fertilized eggs can be procured without resorting to hormone treatment by the following methods:
collecting the eggs of “nest spawners” by placing artificial nests in their natural spawning places, e.g., pike-perch;
imitating the natural spawning conditions in smaller artificial ponds by providing nests or grass mats to serve as egg collectors; stocked brood fish then spawn (e.g., European catfish, pike-perch, bream, common carp, giant gourami, etc.);
providing artificial holes on the sides of the ditches where the fish spawn (e.g., magur);
providing “retreat-places” or “spawning receptacles” such as drums or cans (e.g., channel catfish), and
collecting egg clump or “egg-ribbons” or eggs in foam nests from the natural spawning places during the spawning season (e.g., European perch, guabina, curito, etc.).
4.2.2.2 Procurement of fertilized eggs by induced breeding through hormone treatment. This also can be achieved by either of the following techniques:
inducing spawning in small tanks by administering human gonadotropin hormone, as shown in Figure 10 (e.g., channel catfish, Chinese carps, grey mullets, etc.);
inducing spawning in small tanks or in hapas (rectangular boxes made of close-meshed netting cloth) fixed in ponds by administering fish pituitary hormones (e.g., Indian major carps).
The above mentioned techniques are simple and inexpensive, without requiring any sophisticated installations or tools and without the difficulties and risks involved in artificial fertilization. As a result, these techniques are widely used in artificial propagation.
In artificial fertilization, the culturist handles the brood fish and is, therefore, in a position to eliminate unsuitable fish and to choose the right type of fish for eventual stock improvement. Further, this technique also enables the culturist to produce useful hybrids, combining the desirable qualities of different strains of fish of the same species and/or of different species.
The ripe sexual products required for artificial fertilization can be obtained by either of the following methods:
the fish are captured in their spawning ground during the act of natural spawning, and the sexual products (eggs and sperms) stripped off (Figure 9b) (this method is applicable to the coregonids, pike, and common carp), or
the selected brood fish are first administered human gonadotropin or fish pituitary extract, and when they are in oozing condition they are stripped to procure the ripe sexual products (Figure 9d) (this method is commonly adopted for the Chinese carp in India).
Fertilized eggs resulting from artificial fertilization are hatched and reared up to the fingerling stage under controlled conditions, thus ensuring a high rate of survival and healthy growth.
| (A) Collection of fertilized eggs and larve from a river |
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| (B) Stripping mature fish collected from nature |
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| (C) Induced spawning by hypophysation |
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| (D) Stripping after initial hypophysation |
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Figure 9 Various techniques employed to obtain fertilized eggs and larvae for controlled incubation and/or rearing
Figure 10 The course of induced ovulation
The artificial propagation of finfishes is a chain of activities, which between the various species have some points of similarity while differing in others. The various activities involved in the process of artificial propagation of finfishes are listed below:
capture of wild brood fish from spawning grounds,
selection of breeders from wild stock for natural spawning or for hormone treatment,
rearing of brood fishes,
inducement of natural spawning with or without hormone treatment,
procurement of ripe sexual products by stripping with or without hormone treatment,
artificial fertilization,
incubation and hatching of eggs, and
rearing of larvae, fry, and fingerlings.
As already indicated, not all of the listed activities must be followed in the propagation of all fishes; it depends on the stage at which artificial intervention starts and the specific requirements of the individual species.
The technology that is to be applied for the propagation of a given fish depends on:
the spawning habit of the fish,
the given or possible local conditions,
the available installations, equipment, and tools, and
the adequacy and skill of the personnel available for propagation work.
A survey of artificial propagation as practised in different parts of the world shows that even for the same species there is no hard and fast technology for artificial propagation. In most cases, the fish culturist adopts that technology which he finds easiest to apply and which provides successful results. However, the degree of success may not be the same with different methods.
The details of various activities involved in artificial propagation are given in the succeeding sections.
Sexually ripe and healthy fish are the prerequisite for any kind of artificial or semi-artificial propagation. They can be obtained from natural waters just prior to the spawning season, from the spawning ground on by fish raised on farms.
4.3.2.1 Capturing wild fish while spawning. Fish gathering in easily detectable shallow spawning grounds, or migrating en masse toward such places are vulnerable to easy capture. They can also be easily captured while actually in the act of spawning in groups or in shallow water. The object of such capture is to procure ripe sexual products for incubation and rearing under controlled conditions. Special hatcheries are generally constructed for this purpose (for coregonids, common carp, pike, etc.). This is an old technique, practised mainly in Europe, for obtaining young fish for stocking. The coregonids, pike, common carp, and bream are propagated by this method.
The gear used for such capture are traps and fyke net for pike, gillnet for coregonids, simple seine nets for common carp and bream, and castnets and covering nets for common carp and pike.
Fish caught this way can be stripped easily, since they are already ripe and in an oozing condition. Some of them may not be fully ripe, in which case they must be held in captivity for a couple of hours before they can ovulate. The spawners must be handled with care during the breeding process. This method obviates the necessity for building up and maintaining a brood stock in ponds.
4.3.2.2 Capturing brood fish in their natural habitat. Another alternative to the capturing of spawning fish from spawning grounds in the wild, is the capture of adult wild fish during their natural breeding time or when they are migrating to their spawning grounds. This is likely to yield a good number of ripe breeders, but these gravid fish are prone to easy injury during netting and transport. Moreover, wild breeders do not easily adjust to life in captivity. They become nervous, jump about wildly, and may not feed. In general, their handling is much more difficult than that of tamed stock. It is also difficult to secure adequate numbers of breeders of the required size and age.
It is necessary to use tranquillizers in the handling and transportation of such wild fish, particularly if they are large, as any injuries they sustain may endanger the success of the entire breeding operation. A further disadvantage is the high probability of introducing parasites into the hatchery. However, this method also has a definite advantage in that the farmer does not have to painstakingly build up and maintain a brood stock in his farm. Nevertheless, most commercial hatcheries generally maintain their own brood stock.
4.3.2.3 Farm rearing of brood stock. In spite of the numerous inherent difficulties, this method is widely practised all over the world, since it enables the building-up and selection of healthy breeders for eventual stock improvement. However, proper maintenance of suitable environmental conditions and adequate food supply is mandatory.
Fortunately, almost all cultivable fish either attain full sexual maturity or at least gonadal maturation up to the “resting phase” in confined waters. The river spawners and those that spawn in inundated areas are difficult cases in this regard, but it has been possible to breed most of them through artificial propagation techniques (e.g., Chinese major carps, Indian major carps, and river spawners of the Orinoco and Amazon basins).
In the brood fish ponds, various factors such as temperature, light, oxygen content, stocking density, tranquillity, size, and depth of the pond and food should generally approximate the natural requirements of the species concerned. Therefore, a through knowledge of these factors is a prerequisite for the successful rearing of breeders (Figure 11).
Temperature. Even though many fishes can withstand temperature extremes, it is advisable to rear the brood stock in a pond where extreme fluctuations of temperature do not occur. As investigations have shown, the normal gonadal development requires a sum of temperature. The sum of temperature is expressed in day-grade. In the temperate belt of the world, the gonadal development of fishes, such as common carp, Chinese carps, catfish, tench, etc. can be accelerated by warming up the environment of the brood fish. The spawning time can be advanced by a month by this treatment. During March when the atmospheric temperature is only 4°–8°C, the common carp can be made to ovulate through hypophysation after warming up its environment for two weeks. By keeping the common carp brood fish in an environment of 25°C, it can be made to breed thrice a year. On the other hand, its gonadal maturation can be significantly retarded by subjecting it to lower temperatures. In temperature, therefore, the fish culturist has a handy tool to either advance or postpone the breeding of a fish (Figure 12).
Light. The requirement for illumination varies with different species. The gonadal development in salmonids is related to photoperiods, while in warm water fishes gonadal development is adversely affected if the light conditions deviate from the normal. Many fishes, particularly catfish, are irritated in illuminated environments. Turbid waters are preferred by several species of fishes, especially river spawners. They become agitated in an illuminated, transparent environment, which exercises a negative effect on their gonadal development. Even hormone-administered breeders avoid bright light and look for hiding places.
Oxygen. Frequent fluctuations and low levels of oxygen content in pond water tend to inhibit gonadal development. To ensure healthy gonadal development, brood fish pond water should have an optimum oxygen level throughout the rearing period. Supplying fresh, oxygen-rich water, therefore, is an important requirement.
Stocking density. Although most cultivated fishes are able to tolerate crowded pond conditions, the effects of crowded conditions on gonadal development is often deleterious. Experience has shown that 20–50 ripe breeders (weighing 150–250 kg) can be reared in 0.1 ha (1 000 m2) pond area. The number of breeders can further be raised by stocking fish species of different feeding habits, as is practised in polyculture work in India and China. Examples of such stocking of combinations of Chinese carp brood fishes are shown in Table 2.
Table 2
Combinations in Multi-Species Brood Stock Rearing of Chinese Carp
| Species | No. of fish per ha | Average weight of each fish, kg |
| 1. Grass carp as major species: | ||
| Grass carp | 150–200 | 8–12 |
| Silver carp | 60–90 | 2 |
| Mud carp | 600–1 000 | 0.02–0.05 |
| 2. Bighead as major species: | ||
| Bighead | 80–100 | 7–12 |
| Grass carp | 100–120 | 5 |
| Silver carp | 30–50 | 2 |
| Common carp | 300–500 | 0.25 |
| 3. Silver carp as major species: | ||
| Silver carp | 150–250 | 3–6 |
| Grass carp | 100–130 | 2 |
| Mud carp | 500–600 | 0.02–0.05 |
| 4. Mud carp as major species: | ||
| Mud carp | 1 600–2 300 | 0.8–1.5 |
| Bighead carp | 30–50 | 5–6 |
| Silver carp | 60–100 | 2–3 |
In general it is not advisable to stock would-be breeders along with market-destined fish, especially if the latter are fed an unbalanced diet. However, young prospective breeders can be reared in restricted numbers along with ripe breeders.
Tranquillity. It is believed that frequent disturbances interfere with normal gonadal development. However, the Chinese practice of netting brood fish once or twice before hormone administration and keeping them in crowded conditions (either blocked off by nets in one corner of the pond, or suspended in seine nets) serves to acclimatize the brood fish to handling and transportation, reduces post-spawning mortality, and increases the rate of ovulation.
Size and depth of pond. The rearing pond for larger brood fish (2–10 kg or above) should be about 2 000–4 000 m2. Smaller brood fish can be raised in smaller ponds. The depth of brood fish ponds generally varies from 1 to 2 m.
Food. A suitable and adequate food supply is of foremost importance to brood fish. If the fish are left hungry or starving, the vitellogenesis phase of egg development is affected. If the food is deficient in essential nutrients, particularly the amino-acids, vitamins, and minerals, the egg development is adversely affected, ultimately leading to the failure in ovulation. Therefore, breeders reared on ample natural food or on a protein-rich artificial diet yield satisfactory results. It is better to breed a smaller number of brood fish on qualitatively and quantitatively satisfactory diet (or natural food) than to keep a huge stock under half-starved conditions. The natural diet of fishes differs from species to species. It is, therefore, important to know the food and feeding habits of the cultivated fish.
4.3.2.4 Age and weight of the breeders. The “first spawners” or “virgins” are usually used for artificial propagation. Larger fish produce more eggs, but the handling of “giants” weighting over 10–15 kg is rather difficult and tiresome. The most suitable size of spawners in the case of common carp, Indian carps, and Chinese carps is 3–5 kg. Larger specimens are convenient if breeders spawn spontaneously without having to be stripped. Very large fishes are less suitable for hormone treatment, because of the requirement of large doses of hormone and the difficulties in handling them.
The fecundity of medium size fish (2–6 kg) is generally higher than that of giant fish. Spawners of 0.5–2 kg weight are very easy to handle and strip.
Before releasing the breeders in special spawning ponds for spontaneous spawning, or before they are prepared for induced spawning, the fish culturist should make sure that they are in a “ready-for-spawning” condition. Unless their gonads have developed up to the resting or dormant stage, they will not respond to any propagation technique. Therefore, sorting out of the right breeders is very important for successful artificial propagation.
4.3.2.5 Selection of breeders for ready spawning or for hormone treatment. The symptoms indicative of the ready-to-spawn condition are somewhat similar among various fishes and are as follows (Figure 13):
Females
Well-rounded and soft abdomen, the fullness of which extends posteriorly past the pelvis to the genital opening.
The genital opening is swollen, protruding, and reddish or rose in colour; its edge is uneven or fringed.
The anus (vent) may also be swollen and reddish.
In some column-living fishes of the Orinoco river, the abdomen becomes red coloured.
Some fishes develop a nuptial colour before ovulation.
Males
The male releases a few drops of thick milt when its abdomen is pressed slightly.
In some male fishes (Chinese carps and Indian major carps) the dorsal surface of the pectoral fin becomes rough.
Some male fishes of the Orinoco and Amazon basins produce a sound when taken out of the water (coporo, curimata, curbinata).
Many fishes, such as the tench, exhibit distinctive sexual dimorphism. It is necessary to examine the females of some fishes (grass carp, channel catfish, etc.) before they are fed, to ensure that the abdominal fullness reflects the size of the gonads and not gorged food.
Some of the above-mentioned symptoms may be absent in some fishes, while there may be additional symptoms in others. In the case of cachama (Colossome oculus), the belly of the female becomes soft and rounded only a little before the actual spawning. This hard-bellied condition is a sort of adaptation for the co-existence of this fish with the aggressive piranha (caribe).
If both sexes are together in the same pond or cistern, as soon as the males indicate their readiness for spawning, the females also achieve the same condition. Since the river spawners do not breed in confined waters, there is no need to segregate their sexes. On the other hand, the segregation of sexes is mostly necessary in the case of pond spawners, since otherwise it may lead to uncontrolled spawning in the storing pond (e.g., common carp) or unnecessary fighting among males (e.g., catfishes).
It is important that the culturists carefully observe the brood fish with respect to their anatomical and behavioural changes during their readiness for spawning to ensure the success of subsequent operations.
In its natural breeding ground, a ripe ready-to-spawn brood fish can produce ripe sexual products within a short time when suitable environmental conditions occur. Elsewhere, breeding has to be induced.
Basically, there are two ways to induce ovulation (the final ripening of the eggs) and spawning (the release of eggs in the presence of the male) under artificial conditions.
Simulation of suitable environmental factors, which would trigger the fish's own hormones to govern and direct the final ripening process of the gonads.
Administration of gonadotropic hormones, which may cause the final ripening of the gonads.
A combination of these two methods is sometimes adopted, as in the case of the Chinese technique of propagation.
4.3.3.1 Inducing spawning without hormone treatment. Some pond spawners can be stimulated to spawn by providing:
Some of these methods can also be combined to obtain even better results.
Induction of spawning by providing nests. This method is commonly practised for the propagation of nest spawners, such as pike-perch, European catfish, etc. (Figures 14 and 15).
The nest used for pike-perch consists of flat bundles of dry, bushy roots of willow tree, grasses, etc. or the same materials fixed on frames. Today, an old synthetic net spread between two sticks, and “artificial grass” fixed on a frame are also used as nests. These nests are placed on the natural spawning ground shortly before the spawning season and are checked for signs of spawning every 2–3 days. The nests with eggs are collected and transferred to the hatchery.
In the farm, the framed nests (50 cm × 50 cm) are placed on a hard bottomed storing pond (500–2 000 m2 surface area), at the rate of one nest for every 8–10 m2. Depending on the number of nests, the required number of male and female pike-perch brood fish are stocked when the water temperature is about 10°C. The nests are checked every 2 or 3 days and those with clumps of eggs are removed for controlled incubation. These double-shelled eggs can be easily transported in boxes or baskets, taking care to keep them moist throughout.
In the case of European catfish, tent-like nests are made of dry bushy roots of willow tree, or branches of thuja or pine tree. Then, 3 or 4 of these nests are placed in a pond (1 000–2 000 m2 surface area) and adequate number of brood fish pair are released in it. The spawning can be easily detected because of the vigorous movements of the spawners. The nests with eggs are removed for controlled incubation.
In the case of some gouramis (e.g., the giant gourami), the mere providing of nest materials can stimulate nest building and subsequent spawning.
Induction of spawning by providing artificial spawning surface of Kakabans. The Kakabans are mat-like structures measuring a few m2 in area. They are made of dry grass, pine tree branches, or similar material fixed on a frame. The Kakabans are either fixed to the pond bottom with sticks, or are held about 20–30 cm below the water surface (Figure 16). The common carp of tropical and subtropical areas willingly spawn on Kakabans, scattering their sticky eggs on this artificial surface. They do not spawn in muddy bottomed ponds unless Kakabans are provided. After spawning has taken place, the Kakabans covered with eggs are removed from the pond and transferred to nursery ponds, where they hatch out and grow without the danger of becoming infected by parasites from their parents.
This spawning technique can be easily applied even under primitive conditions. The Kakabans with eggs can also be conveniently transported by covering the eggs with moist cloth or grass. This technique can also be adopted for other fish with spawning habits similar to that of the common carp.
Induction of spawning by providing receptacles. Some fish, such as the channel catfish, require some hiding place while spawning. They also guard and aerate their eggs. Usually 45 l milk cans or oil barrels are used as spawning receptacles in ponds or closed-off part of ponds. Soon after they are placed in the ponds, the ripe brood fish spawn in these receptacles, from which the eggs are removed and incubated under controlled conditions. Other pond-spawning catfish can also be stimulated to spawn by providing spawning receptacles (Figure 17).
Clay, plastic, or cement-concrete pipes of larger diameter (20–25 cm) can be used to stimulate spawning in other fishes, such as Plecostomus plecostomus in Venezuela and Clarias batrachus in the Far East.
All of these are based on the “nest stimulus” or “receptacle stimulus” techniques, which induce the brood fish to spawn. An essential prerequisite for this is the gravid condition of the males and females during the natural spawning season. It is always advisable to introduce one or two males fewer than females, since the males are known to fight with each other when they outnumber the females.
Induction of spawning by simulating decisive natural environmental conditions. The method of controlled propagation of common carp is based on this technique. This technique is also known as Dubisch method. Ponds used for this purpose are termed “Dubisch ponds” (Figure 18).
The decisive natural conditions to bring about spawning in common carp are as follows:
Spawning ponds with the above characteristics and measuring 100–1 000 m2 each can be easily prepared. As an alternative, a rice patch can also be converted for this purpose. A stable source of clean filtered water is an essential requirement, however.
A spawning pond of this type is constructed as follows. A ditch 2–3 m wide and 0.6–0.8 m deep is dug adjacent to the main dyke of the pond, to serve as a refuge for the breeders. A draining structure is then placed at one end of this ditch. The other parts of the pond are made to slope slightly toward the ditch and are covered with short grass. When the pond is fully inundated, about 30–50 cm water will cover the grassy area (Figure 18).
When the water temperature and weather conditions are suitable, one or two sets of spawners, each set consisting of 2 females and 3 males, are introduced into the inundated ditch. They are kept there for a few days, during which period a gentle continuous flow of water is maintained in the pond by regulating the draining structure. The draining exit is then closed and more filtered water is drawn into the pond bringing about a slow rise in water level. The rising water gradually inundates the grassy area. This operation triggers the breeders into vigorous spawning. A day after the spawning, the spent fish are carefully netted out from the pond. This removal serves to prevent cannibalism and the infection of the offspring by parasites from the parent fish.
This technique has also been successfully employed to breed some of the other fishes, such as pike, tench, crucian carp, buffalo fish, Puntius spp., etc. It should be possible to adopt this technique for the induced breeding of any pond-spawning fish that may be selected for culture.
4.3.3.2 Induction of ovulation and spawning. Induced ovulation and spawning achieved through hypophysation amounts to a “short cut” of the natural process. In nature, ovulation in a fish is regulated and brought about by its own gonadotropic hormone(s), produced and stored by the pituitary gland. The stored hormone is released into the blood when all the requisite conditions become favourable. But in the hypophysation technique, gonadotropic hormone extracted from the pituitary of some other fish (donor) is injected into the breeder and this brings about the final ovulation.
General considerations. Hypophysation is presently the most commonly used technique for the artificial propagation of fish. It is employed not only in propagation experiments, but also in the commercial production of millions of young fish.
Like all other techniques, this technique too has its own limitations. Some of the sensitive fish such as the pike-perch cannot tolerate the treatment, while others may ovulate only irregularly. Then again, the breeders whose ovaries have not yet reached the adequately ripe stage fail to respond to hypophysation. It is a fundamental rule that hypophysation will be effective only when the eggs in the ovary have reached the resting or dormant phase after the completion of vitellogenesis. The eggs are then materially ready for further development to be triggered by gonadotropin(s).
Pituitary glands of donor fishes, collected fresh or preserved, are used in hypophysation. It is necessary that these glands contain an adequate amount of stored gonadotropic hormones to bring about successful spawning.
The pituitary gland (hypophysis) acts as an intermediary between the brain and the gonads. Its cells produce and store gonadotropins, and release them only when the gland receives the necessary command. The gonadotropin content of the pituitary gland varies during different seasons and during different stages in the life of the fish. Immature fishes have only a small quantity of gonadotropin in their pituitary, while after natural spawning the spent fish are completely bereft of gonadotropins in their pituitary. On the other hand, the gonadotropin content is at its highest level in the pituitary of sexually ripe fishes when their gonads have reached or nearly reached the resting phase and throughout the duration of the resting phase. Since spawning migration is also triggered by gonadotropin, the pituitary of such migrating fish has a lower level of gonadotropin content. In view of these varying contents of gonadotropin, it is important to choose the right time for collecting the pituitary glands.
Dosage. During natural ovulation, the fish is able to precisely regulate the dosage of its own hormone. Hence, there is no wastage. In the case of hypophysation technique, wherein hormone from an external source is injected, there is usually considerable wastage. This is mainly because it is difficult to fix the exact dosage, with the result that generally more hormone than required is injected into the breeders.
Ovulation is a complicated process lasting several hours, with its exact duration depending on temperature. It is possible to differentiate ovulation into 2 phases, viz., preovulation and ovulation. In the preovulation phase, the migration of the nucleus is completed and the egg absorbs a large quantity of fluid (hydration). Its size now is nearly the same as its final size at ovulation. If hypophysation is unsuccessful, the eggs stop developing at this phase and the breeder may easily die due to the necrosis of eggs which may cause internal poisoning.
The hormone dosage required can vary significantly from fish to fish of the same species and from technique to technique. The dosage actually depends on the “readiness” of the females; their age, size, sensitivity and many other factors. In tropical and subtropical areas where the metabolism of fish is far higher (due to higher temperature) and where the probability of wastage of hormone is, therefore, greater than in temperate regions, usually two or more doses are administered. Generally, two doses are given: the introductory or preparatory dose, and the decisive or final dose.
A single, full (100 percent) or knock-out dose is given when the breeder has been in the resting phase for a long time. The preparatory dose is about 10 percent of the total dose. If a further preparatory dose is to be administered, again only 10 percent of the total dose is given. Generally, for a total dose, about 2.5–3 mg (1 gland) of hypophysis would be required per kg weight in the case of large breeders weighing over 5 kg; 1.5 mg (0.5 gland) for medium sized fishes (2–5 kg); and 0.75 mg (0.25 gland) for small fishes (0.5–2 kg). It is advisable to avoid overdose in preparatory injection, since it may lead to partial ovulation, thereby upsetting the normal schedule.
Between the preparatory and decisive doses, there must be a minimum lapse of 14 hours. The maximum lapse is 24 hours, but very rarely can it extent to 48 hours. When more than one preparatory dose is required, there should be a span of 24 hours between doses.
The males, as a rule, are only given one dose of hormone, usually at the time when the females are given the last decisive dose. It is important that the males are not administered the hormone earlier, since that may result in releasing the sperm before the females are ready to ovulate.
The dosage of gonadotropin hormone extract is expressed either in milligrammes or as a number of acetone-dried hypophysis glands. The acetone-dried pituitary gland of a 1.5–2 kg common carp, weighs 2.5–3 mg. This size of hypophysis is taken as a unit, when the dosage is expressed in terms of number of glands.
The “gland unit” is easy to use. Glands of approximately the same size are taken for preparing the dose. The other method of calculating the dosage by weight is more difficult, but is certainly more precise.
A little excess hormone in the decisive dose does not harm the fish. An overdose of 10–15 percent is generally given to be on the safer side. For the total, or 100 percent dose, usually 1 to 1.5 glands (i.e. 3.0–4.5 mg) of hypophysis are administered per kg weight of the female. If the calculated dose requires more than five glands, one more gland is usually added “for the mortar”.
If the pituitary gland is available in already pulverized form, a good balance or a spoon of known volume would be required to measure the exact dosage. The dry pituitary marketed in pulverized form can be easily adulterated with brain tissue. Therefore, it is advisable that the farmer buy his hypophysis only from reliable sources.
The recommended single decisive dose for males is 0.5 gland (1.0–1.5 mg) per kg of body weight regardless of their length. However, there is no need to administer hormone to males found to be oozing milt.
It is always advisable to be a little liberal while calculating the decisive dose. Hence, farmers are advised to increase the required dosage by 10–15 percent. A principle to follow in practice is not to administer too much of hormone in the preparatory dose and too little in the decisive dose.
When the decisive dose is administered in two or three parts, the time lapse between the injections should not be more than 6–8 hours.
Methods of hormone administration. There are several variations in hormone administration methods. Each method may have some justification, but may not be universally applicable. The technique adopted is generally dependent on the species of fish, local conditions, and working methods developed by the local scientists and technicians. However, none can be proclaimed as the only definitive technique. As a rule, the females generally require higher doses of hormone than the males, with split doses producing better results than a single large dose.
The different methods of hormone administration, as generally practised, are detailed below.
Single injection method. The calculated 100 percent dose, or the knock-out dose, is given in a single injection. It will be successful only if the female is otherwise fully ready for spawning, as those that are in their spawning migration or are captured on the spawning ground. Suitably fed fishes achieve this condition during the second half of the breeding season.
Among the majority of fish species, males are better prepared for spawning than females and, therefore, a single dose suffices. If the hormone administered to them amounts to an overdose, or is not synchronized with the gonadal maturation of the females, it may result in the wild discharge of milt before the females are ready.
Preparatory and decisive doses method. The preparatory dose, which is about 10 percent of the decisive dose, advances the gonadal development up to the preovulation stage. It is generally given about 18–24 hours prior to the decisive dose (100 percent of the calculated dose). This is a generally successful sequence of hormone administration in the temperate and subtropical regions. This method also holds good for nervous and difficult-to-handle fish.
Sometimes the interval between the preparatory and decisive doses can be shorter than that indicated above. About 14–18 hours suffice during the latter half of the spawning season, given that the water temperature is higher than the normal spawning temperature. If the females are less than 1 kg in weight and are in ripe condition for hormone treatment, an interval of only 6 hours is sufficient.
(a) One preparatory and two decisive doses
In tropical regions, where the metabolism in fish is more rapid, the decisive dose is given in two equal instalments or two instalments of 40 percent and 60 percent, with an interval of 6–8 hours between them. A preparatory dose of 5–10 percent should precede the first decisive dose by 18–24 hours.
(b) Several preparatory and two decisive doses
In certain cases where the eggs are already in the dormant stage but the ovary has yet to descend to the lower part of the body cavity, a series of several preparatory doses are required before a decisive dose can be successful. For example, the female Orinoco cachama (Colossoma oculus) requires five preparatory injections (P1–5) with an interval of 24 hours between every two injections before it is ready for the decisive dose (D1–2) which again is given in two instalments of 40 and 60 percent each with a 6-hour interval.
In this case the sequence is as follows:
Distributed doses method. In this method, the injections are given in many doses and the time span between the doses is generally short, about 6–8 hours.
The sequence and the quantity of doses may vary as follows:
These sequences have proved successful in the case of tropical fishes and those spawning confined waters.
It has been found that more hormone is needed for ovulation when the ovary is bulky. The bulkiness of the ovary can be expressed by the maximum circumference of the body, the dosage being regulated as follows:
| Maximum circumference of the body, cm | 38 | 40 | 42 | 44 | 46 | 48 | 50 | 52 | 54 | 56 | 58 | 60 | 62 |
| Dosage of dry hypophysis in mg/kg body weight | 3.0 | 3.3 | 3.5 | 3.8 | 4.0 | 4.3 | 4.5 | 4.8 | 5.0 | 5.3 | 5.5 | 5.8 | 6.0 |
This relationship is applicable to the Chinese major carps, while it is yet to be tried in the case of other fishes.
Solvent. The solvent used for gonadotropic hormone is 0.6–0.7 percent NaCl (common salt) solution. Of the solvent, 1 ml is used for the preparatory injection, regardless of the dosage ranging from 0.25 to 1 gland. The quantity of solvent for the decisive dose is calculated at the rate of 0.5 ml for each gland (2.5–3.0 mg), but the maximum quantity should not exceed 5 ml.
The quantity of solvent is not of much importance, except when too little or too much is used. In the former case the loss of one drop of solution would mean the loss of a considerable quantity of hormone, while in the latter the administration of a large volume of solution would pose a difficult problem. Therefore, a quantity varying between 1 to 5 ml is generally advised.
Preparation of pituitary gland solution. Based on the weight, number and sex of the breeders, the dose is determined, after which the requisite number or quantity of pituitary gland is counted or weighed. If the glands are not already in pulverized condition, they are thoroughly pulverized in a small porcelain mortar or a homogenizer. The mortar has to be totally dry, as otherwise the glands would become pasty while pulverizing and would not easily dissolve.
A measured quantity of solvent is then added immediately. The solvent is generally measured in a graduated syringe. It is necessary to ensure thorough mixing of the solvent and the hypophysis powder. About 10–30 minutes would be required to dissolve the hormone. The tissue residue can be removed from the solution by using a centrifuge or by simply allowing the residue to settle down and then sucking off the clear supernatant solution with a syringe (Figure 19).
The solvent is first prepared by dissolving 7 g of clean common salt, free of iodine, in 1 l of boiled and already cooled drinking water. There is no need to use distilled water. The solvent can be stored for long periods in sealed bottles.
When a number of breeders are injected at the same time, it is advisable to mark them individually by threads of different colours, bound loosely to the dorsal fin rays, immediately after they are weighed. This will facilitate the identification of the breeders and the administration of the correct doses.
Choice of body part to inject. The most commonly adopted procedure is to inject the hormone into the dorsal muscles above the lateral line and below the anterior part of the dorsal fin (Figure 20). In India, fishes are generally injected at the dorsal part of the caudal peduncle. This procedure seems to be the best for sensitive fishes.
In the case of some fishes, as in tench, the injections are administered into the body cavity. This generally yields poor results. The fish is sometimes not taken from the water while being injected. When it is taken out of the water, it is advisable to place it on a small table covered with a soft plastic foam sheet or cushion. This prevents injuries to the breeders. The fish becomes quieter if its head is covered with a piece of cloth. It is advisable to use towels while handling breeders and not to touch them with bare hands. While injecting scaly fishes, it is necessary to take the precaution of not pricking them through the scales, but to insert the needle beneath the scale and prick through the underlying muscle.
The pituitary administration does not, by itself, bring about full ovulation. A number of environmental factors, such as suitable temperature, high oxygen content, and calmness, are believed to play a decisive role (Figure 21). Among these, temperature is of vital importance. If it is too low, ovulation takes a very long time, or in most cases becomes inhibited. High temperature not only causes higher oxygen demand and rapid metabolism, but also has its own inhibitory effect. The pituitary treated fish needs about 50 percent more oxygen than before the treatment. The excitement caused by handling and treatment also results in enhanced oxygen consumption. It is, therefore, essential to keep the treated breeders in a well aerated environment or in clean, oxygen-rich flowing water. The need for calm surroundings also cannot be overlooked. Disturbed fish become agitated, swim rapidly and jump against the wall of the tank, thereby exposing themselves to injury. Their tranquillity can be secured by putting dark floating objects on the surface of the tank where the treated breeders are kept.
4.3.3.3 Inducing of ovulation and/or spawning by human chorionic gonadotropin. The preovulation phase can generally be obtained easily by administering HCG, but it is difficult to achieve full ovulation by this method in most fishes, even though some fishes do respond fully. First of all, only those fishes which are well prepared and are fully ripe for hormone treatment respond well to HCG administration as, for example, the fishes captured during their spawning migration. Carnivorous species may also respond better than the non-carnivorous. Brood fishes raised on ample natural feed respond more easily to HCG than those raised on artificial feed. Several environmental factors, such as ample flowing water, a strong current, and the availability of hiding places, may also have an important role to play in the success of HCG treatment. The only fishes that have been successfully induced to spawn on a commercial scale by HCG administration are the channel catfish (Ictalurus punctatus) the striped mullet (Mugil cephalus) and the Chinese major carps. In the case of the channel catfish, HCG is injected into the body cavity at the rate of 700–2 000 international units (IU) per kg of body weight, depending on the maturity of the fish. On the other hand, the striped mullet requires an intramuscular administration of 6 000 IU of HCG per kg body weight, given usually in two doses with an interval of 24–48 hours depending on the rate of egg development after the initial injection. The dosage in the case of Chinese major carps is only 800–1 000 IU per kg of body weight. This is administered in two doses with an interval of eight hours. Only 10–15 percent of the total quantity of HCG is administered in the first dose. The males are injected when the females are given their second injection.
4.3.3.4 Hormone induced ovulation versus hormone induced spawning
Hormone induced ovulation. Ovulation is the final phase of normal egg development. Preovulation starts when the nucleus of the egg cell starts to migrate from the centre toward the micropyle, during which time the egg absorbs fluids, a process known as hydration. The ovulation starts with the disappearance of the nuclear membrane and the appearance of the chromosomes and ends with the first meiotic division. At the same time, the follicle, which keeps the egg fixed to the wall of the ovary, splits and partly dissolves, resulting in the eggs falling into the cavity of the ovary. The mass of eggs may now flow freely through the genital opening.
This final ripening process takes time and depends largely on the temperature of the water in which the fish is kept. In practice, it is necessary to know the time interval between the last decisive injection and full ovulation. This time span is expressed in “hour-grade”, which is calculated on the pattern of “day-grade”. The temperature of water in the tank in which the breeders are held is measured every hour after the last decisive injection up to full ovulation. The readings are added to arrive at the hour-grade. The hour-grade will be higher when only one dose is administered, since egg development in that case would involve both preovulation and ovulation. In the case of common carp, the time span is about 16–18 hours when the water temperature is 21°–22°C, and hence the hour-grade works out to be 340–360. If the pituitary treatment includes one or more preparatory injections and only one final injection, the ovulation lasts 240–260 hour-grade at a water temperature of 21°–22°C; i.e., about 12–13 hours following the last injection.
A knowledge of hour-grade value would help the fish farmer to know exactly when to expect ovulation after the last injection. The hour grade value depends on the fish species treated, the type of treatment, the size of the female, and whether the fish starts spawning immediately after ovulation or not.
(a) Hour-grade in relation to fish species
At a temperature of 21°–22°C, the hour-grade of common carp is 240–260, while that of grass carp, silver carp, and bighead is 200–220.
(b) Hour-grade in relation to the type of treatment
The hour-grade is 340–360 in the case of common carp, when only one decisive dose is administered. It will be only 240–260 if a preparatory dose is given 24 hours prior to the decisive dose. The hour-grade value would go down further to 200–220 in cases where two decisive injections are given with an interval of 6–8 hours. The pattern may hold good for other species as well.
(c) Hour-grade in relation to size of the females
It is well known that the smaller females ovulate earlier than larger females. This is particularly conspicuous when the females differ greatly in size; e.g., 1–2 kg and 7–10 kg. In common carp, the hour-grade value is usually about 130–150 only when the breeders are of small size (1.0–2.0 kg).
When released with ripe active males, most of the injected females begin spawning in the “ward basin” or “ward tank”. Failure to spawn would mean that the species is not responsive, the males are inactive or already spent, the females are injured or suffering from an overdose of hormone, or some of the environmental factors are unfavourable.
Hormone induced spawning. Many of the fishes that are treated by gonadotropic hormone (fish pituitary extract or human chorionic gonadotropin) start to spawn in the presence of active males after normal ovulation. This type of spawning is termed “induced spawning” or “hormone induced spawning”. In this case the eggs are fertilized by the male breeders themselves, and the fertilized eggs can be collected easily for controlled hatching.
Fish species suitable for induced spawning are those that have non-sticky, floating, semi-floating or rolling eggs. Otherwise, the fertilized eggs will stick together in a clump. Nest spawners also can be used for induced spawning, after placing suitable nests in the breeding tank.
If the fish scatters its sticky eggs, as is the case with common carp, crucian carp, tench, etc., it is advisable to place an egg recipient like kakaban at the bottom of the tank. Even then some eggs will be scattered and become adhered to the wall of the tank.
Induced spawning has both advantages and disadvantages, and these are listed below:
Advantages:
There is no need to calculate the exact time of ovulation, or watch the females to determine whether they are ready for stripping.
It is not necessary to catch the breeders for stripping, thereby avoiding possible injuries to the breeders.
There is no need to strip the breeders and fertilize the eggs artificially, which process is not only time-consuming but would also require more working hands.
The danger of over-ripening of eggs in the ovary would not arise, since the fish would start to spawn as soon as ovulation is completed.
Disadvantages:
A special egg collector would be required to be placed on the outflow of the ward tank, in order to collect the floating fertilized eggs without causing any damage.
The collected eggs are generally mixed with particles of faeces and other alien objects carried by the water current of the ward tank. Such particles may spoil the eggs when they decompose, and the bacteria and fungi flourishing on them could also be dangerous to the developing eggs.
The estimation of the total number of fertilized eggs is more complicated and difficult than in the case of stripping.
Some of the female breeders do not spawn fully in the ward tank, with the result that the ovulated eggs remaining in the ovary become overripe and are lost. As much as 50 percent of the ovulated eggs are sometimes lost in that manner.
Some female breeders extrude their eggs in the absence of males, resulting in a high percentage of unfertilized eggs.
If the males fail to respond, spawning will be a total failure. This is often the case when only one female and one male are put in the ward tank.
To achieve successful induced spawning, it is advisable to put together one female and two-three males, two females and three males, or a maximum of three females and four males, depending on the size of the ward tank. Too many breeders in a small ward tank would disturb each other. In the case of breeders weighing 2–3 kg each, a maximum of two females and three males can be put in a ward tank of 2 m2. On the other hand, as many as three-five females and four-six males can be put in the same tank if they weigh only 0.5–1 kg each. If the breeders weigh 4–5 kg each, the putting together of only one female and two smaller males is advised. Still larger fishes would require a larger ward tank to obtain satisfactory results.
Figure 11 Important factors in broodstock rearing
Figure 12 Acceleration of gonadal development in temperate climate by raising water temperature
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Figure 13 Characteristics of breeders ready for hormone treatment
Figure 14 Nest for pond spawners for collecting fertilized eggs with or without hormone treatment. 1 and 2 for pike-perch. 3 for European catfish. 4 for common carp
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Figure 15 Facilities for induced spawning without hormone treatment
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Figure 16 Different ways of placing egg recipients (nests)
Figure 17 Spawning pens with milk can receptacles for channel catfish
Figure 18 Spawning pond for common carp: Dubisch pond
Figure 19 Preparation of pituitary gland to be used for induced breeding
Figure 20 Where and how to inject hormone
| (A) Suitable temperature | (B) Adequate oxygen |
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| (C) Tranquillity - Breeder hiding under floating dark object | |
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Figure 21 Requirement of hormone-injected breeders
Fertilized eggs are easily obtained where there is induced spawning. In cases where there is no successful induced spawning response, due to one reason or the other, the ovulated eggs and the milt are required to be stripped off the breeders and fertilization effected artificially by mixing together the stripped sexual products. It is to be noted that only the ovulated eggs can be easily stripped. Some fishes have circular muscles around their sexual opening, and if these do not relax and dilate, stripping may be difficult even though the eggs are ovulated. In some cases, it is possible to get the eggs only by cutting open the female breeder.
4.3.4.1 Artificial fertilization of fish eggs. As pointed out earlier, some fishes do not spontaneously spawn after ovulation. On the other hand, some fish, such as the common carp, are not allowed to spawn spontaneously, since their eggs need special treatment. The treatment consists of dissolving the sticky layer of the eggs, which enables the eggs to be incubated in jar-type incubators under controlled conditions. Since the common carp scatters its eggs immediately after ovulation, it is necessary to keep a watch on the females and as soon as they start to broadcast the eggs they should be captured and stripped or their genital opening sutured (see Figure 22) to prevent the scattering of the eggs. The ovulated females can be identified by introducing one or two males into the tank holding the females, since the males will follow the ovulated females and prod them in the region of the genital opening with their snout, trying to induce the females to spawn. In most species, the females do not scatter their ovulated eggs if there is no male around. In such cases, spontaneous spawning can be easily prevented by keeping the male and female breeders in different tanks.
Stripping of sexual products. Ovulation or the final ripening process of the eggs cannot be stopped or reversed. Once the process starts the eggs must either be spawned or stripped, failing which they become overripe and are no longer fertilizable. Therefore, it is essential to strip the ovulated female as soon as the main bulk of its eggs has reached the “very ripe” state. The overripening of the eggs follows shortly thereafter, the time taken being different in different species as indicated below:
The Approximate Time After Ovulation Within Which 50% of the Eggs in the Ovary Become Overripe
| min | |
| Common carp | 50–80 |
| Grass carp | 30–40 |
| Silver carp | 30–40 |
| Bighead carp | 50–80 |
| Prochilodus sp. | 20–30 |
In general, the eggs of tropical and subtropical fishes overripen earlier than those of fishes from the temperate zone.
The majority of eggs mature or become ripe and fall into the ovarian cavity at the same time. This facilitates easy and successful stripping, whence the eggs freely flow out in a thick jet. Knowledge of the hour-grade of the fish greatly helps in fixing the exact time of ovulation, within a range of only 10–20 minutes on either side. When 10–15 females are treated at the same time, those that ovulate earlier are immediately taken out for stripping, and by the time this is completed the other females also would be ready for stripping.
Most fishes eject their eggs when they are taken out of water. However, it is not necessary to take the fish out of water for testing its ripeness. The gushing out of eggs when the female is turned on its back and pressed lightly on the sides of the abdominal wall near the genital opening is an indication of ripeness of the fish.
Once the right female breeder is chosen, its body, particularly its hind part and tail, and the hand of the operator, must be dried with a soft towel before stripping. In stripping, the operator presses his thumb near the genital opening and gently squeezes out the eggs into a dry plastic or enamel bowl. The smaller fishes are held by the hands only while stripping, while the larger fishes (over 4–5 kg) are laid on a cushioned table for stripping (Figure 23).
In the case of sutured females, softness of the belly and the presence of a few eggs between the sutures would indicate their ripeness. In the alternative, “indicator” males can be employed to point out the ripe females. Here, the female fish is first wiped dry with a soft towel, after which the sutures are cut. The fully ripe females would then readily yield the eggs, which would flow out in a thick jet. Only the last few eggs may have to be stripped with a light pressure. Forced stripping (i.e., pressing out the eggs by force), should be definitely avoided as such eggs will be unfit for fertilization.
At the same time that the females are stripped, the males should either be stripped or their milt collected. In collecting, the milt is sucked with a special pipette in cases where the breeder is expected to yield only a few drops of milt, or into a milt collector where the milt yield is expected to be ample. There is no need to wipe the males in both these cases, since the milt gushing out can be easily sucked directly from the genital opening. When sufficient milt is collected, it is added on to the eggs and the sexual products are mixed “dry” immediately with a plastic spoon or a feather.
A well trained team of three or four experienced persons can complete the stripping of 10–15 females and an adequate number of males within 30–50 minutes. The various equipment required for stripping and for artificial fertilization are shown in Figure 24.
Dissolving the sticky layer of egg shell. The ovulated ripe eggs of many fishes, especially carp, have a sticky layer. This layer contains glucoprotein, a compound of sugar and proteins. It is through this sticky layer that the eggs adhere to objects in the water and develop there quite separated from one another. Unripe eggs do not have such a layer. “Dry eggs” are not sticky, since they become sticky only after coming in contact with water. The stickiness is strongest at the beginning and may disappear with time. The strength of the stickiness is different in different species. It is very weak in pike, fairly strong in cyprinids and pike-perch, and strongest in the European catfish. This may be due to different chemical compositions of the sticky material in different groups. It is known that salt water does not activate the stickiness of the carp egg, while carbamide and guanidin dissolve the sticky material from the egg surface. However, the sticky material does not lose its sticky property, but only becomes inactivated by the common salt. If the eggs are put in fresh water they will form a clump, because of the activation of the sticky material or its remnants between the eggs. Therefore, it is necessary to wash the eggs thoroughly with the salt-carbamide solution during the swelling of the eggs, in order to remove the sticky material from their surface. A better and easier technique consists of a quick washing with a weak tannin solution at the end of the swelling. Tannin denatures all protein compounds, thereby eliminating the stickiness of the eggs immediately.
Usually, both methods are applied. To start with, the eggs are washed repeatedly during their swelling period with increasing quantities of the salt-carbamide solution (fertilizing solution), after which they are washed three or four times in a tannin solution. Tannin stops the swelling of the eggs as well, because of its denaturing action on the proteins of the egg surface. Therefore, it is applied only when the swelling is complete.
Fertilization of sticky eggs. The ovulated egg that falls into the ovarian cavity has undergone only the first meiotic division. The second meiotic division will take place only when a sperm penetrates it, ending in the extrusion of the second polar body. The sperm which enters the egg through the micropyle triggers further processes, such as extrusion of the second polar body and the development of the female pronucleus, which has only half the number of chromosomes (n). The female pronucleus then fuses with the male pronucleus, which also has only half the number of chromosomes (n), and thus the first somatic cell (2n) of the new fish comes into being. This completes the process of fertilization.
The time available for the ripe egg to become successfully fertilized is rather limited. This is due to the fact that the egg when placed in water immediately starts to swell, resulting in the closure of the micropyle. In the case of common carp and Chinese carps, this closure takes place within only 45–60 seconds. As such, the time available for the sperms to penetrate the eggs is limited to only a few minutes in the case of most fishes.
Extreme care has to be taken in deciding on the amount of water or solution to be added to the mixture of sexual products. If too much water is added, many of the sperms are likely to go astray and miss the micropyle. On the other hand, if insufficient water is added, the micropyle of one egg may be covered by another egg or by the mucus of the ovary, resulting in the inability of the short-lived sperms to enter and fertilize the egg. This occurs in spite of the presence of innumerable sperms in the milt; about 10 000–20 000 million in one cubic centimetre of milt.
The procedure described below refers to the fertilization and handling of common carp eggs and is diagramatically represented in Figure 25(a-c).
The addition of water to the mixture of common carp eggs and milt will result in their sticking together in a clump within a few seconds. The swelling of eggs and their development will be hampered and they will soon die due to their inability to get oxygen. On the other hand, the use of a fertilizing solution would ensure successful fertilization and development. In this case the eggs do not stick together when they are stirred gently and continuously, and the sperms become active and move vigorously. It has been seen that the virility of sperms lasts much longer in a carbamide-salt solution (20–25 minutes) than in water (1–2 minutes).
The fertilizing solution is prepared by dissolving 30 g carbamide (urea) and 40 g common salt (NaCl) in 10 litres of clean (preferably filtered) pond water.
The quantity of the solution to be poured on to the eggs in the beginning is about 10–20 percent of the volume of the eggs to be handled. The mixture is then stirred with a plastic spoon or a feather for about three-five minutes continuously, during which time one sperm enters an egg through the micropyle and completes the process of fertilization. Subsequent stirring is carried out by hand. One person can stir two bowls continuously, or four-six by rotation.
The eggs absorb the solution without any harm and begin to swell. Larger quantities of the same solution should be added from time to time. A part of the solution with dissolved sticky material should be drained off during this operation.
The common carp egg swells about ten times when compared with its original size. Therefore, about 0.2 litres of dry eggs is placed in a 3-litre bowl to provide sufficient space for swelling.
After about one to one and a half hours, the swelling of the eggs ceases and the sticky layer of the eggs dissolve. Even so, if the eggs are transferred to water, they may tend to stick together in loose clumps, due to the presence of some sticky material between the eggs. To get rid of the sticky material completely, it is necessary to wash the eggs two or three times with the fertilizing solution. The drained solution will carry away most of the sticky material. The eggs are then transferred to a second solution, consisting of 5–8 g of tannin in 10 litres of water. Care should be taken that the solution is freshly prepared every time it is required.
About 2–4 litres of tannin solution is placed in a plastic bucket and a maximum of 2–3 litres of swollen eggs may be added to it all at once. After stirring for 3–5 seconds, clean water should be poured into the bucket. Once the eggs have settled down, the water is drained out, preferably by using a strainer. As a precautionary measure, a smaller quantity (1–2 litres) of tannin solution is then poured into the bucket, and after a brief stirring clean water is again added, followed by draining. The tannin can be harmful if it remains for long in contact with the eggs. Therefore, the eggs should be washed with clean water repeatedly, or placed immediately in the incubators and washed there. If the tannin solution is inadequate for the quantity of eggs taken, a slight sticking together may be observed. However, these water-hardened eggs can be separated from each other by hand.
The same procedure is adopted for treating the eggs of tench (Tinca vulgaris) aspius (Aspius aspius), bream (Abramis brama) and other cyprinids, to rid the eggs of the sticky material.
Fertilization of non-sticky eggs. The absence of a sticky layer renders the fertilization and handling of non-sticky eggs a much easier operation. Clean water only needs be used and there is no necessity for any “fertilizing solution”. Here also the water to be added to facilitate fertilization is about 10–20 percent of the volume of the “dry” eggs. The process lasts about five minutes, during which time stirring of the eggs should be continuous. The eggs are next transferred to incubators, taking care to put in only that many eggs which can be accommodated in the container, keeping in mind that they swell about 40–60 times their original size (Figure 26). Some fish culturists prefer to wait till the eggs are fully swollen in the bucket itself before they are transferred to the incubators. However, this operation may result in damage to a number of eggs. Therefore, it is better and more convenient to transfer the non-sticky eggs to the incubators immediately after fertilization and before they begin swelling.
The role of carbamide solution as a catalyst in fertilization. It has been observed that the sperms remain mobile longer in a carbamide solution than in natural freshwater. The carp sperms are capable of fertilizing the eggs after being in a carbamide solution for 20–25 minutes. Thus, their viability or span of life in carbamide solution is 10–20 times longer than in normal freshwater. Therefore, this solution is considered a catalyst to fertilization. In addition to prolonging the viability of sperms and thereby increasing the rate of fertilization, this solution also helps in dissolving the materials clogging the micropyle of eggs. In the case of the pike (Esox lucius), a carbamide solution (16 g carbamide in 1 litre water) increased the fertilization rate very significantly, up to as much as 80–90 percent. However, the optimal concentration of carbamide solution is different for different species of fishes. It is, therefore, advisable to determine the optimal concentration for each species concerned by testing the viability of sperms at different concentrations of carbamide solution. A concentration at which the sperms move about vigorously, for about 10–15 minutes, can be taken as the optimum. The use of carbamide fertilizing solution instead of clean water promises better results in the case of most of the fishes whose eggs are fertilized artificially. A similar catalytic effect has been seen with a common salt solution in the case of the pike.
4.3.4.2 Swelling of eggs. Ripe eggs swell when they come in contact with water or “fertilizing solution”. Even overripe eggs and those which have completed only the preovulation stage swell, but such swelling is not to the same degree as in the case of ripe eggs. The unfertilized ripe eggs also swell normally. It is, therefore, evident that fertilization is not necessary for the swelling of eggs. As the egg starts to swell, the micropyle closes, as a result of which no sperm can penetrate the egg thereafter. In the case of the common carp and the Chinese carp eggs, the micropyle closes within about one minute after coming in contact with water. This means that the eggs of these two species have only about one minute to become fertilized after they come in contact with water. Therefore, it is necessary to handle the eggs in a dry condition while stripping.
The swollen eggs consists of (i) a kernel or germ, (ii) the perivitelline space and (iii) the egg shell. The kernel contains the yolk mass, fats, etc., and the cells in cleavage. Two poles are distinguishable on the kernel, viz., the animal pole or blastodisc, and the vegetative pole or yolk mass. The animal pole includes the cell nucleus, which has by then the normal number of chromosomes as any somatic cell. Around the kernel is the so-called perivitelline space, which is filled with perivitelline liquid. This liquid contains dissolved proteins. The egg is enclosed in an egg shell, which consists of one, two, or three layers in different fish species. The thickness, hardness, and other characteristics of the egg shell may also vary with different species. The type of incubator, therefore, will have to be selected depending on the nature of the egg shell.
The egg shell is very delicate in some fishes and ruptures easily, leading to the spoilage of the egg. In other fishes, it is very tough and can hardly be ruptured even by pressing between fingers (e.g., mahaseer).
Figure 22 The method of suturing common carp
Figure 23 Techniques of stripping
Figure 24 Equipment required for stripping and artificial fertilization
Figure 25a Artificial propagation of common carp - I
Figure 25b Artificial propagation of common carp - II
Figure 25c Artificial propagation of common carp - III
| (A) Estimate the number of eggs | (B) Add fertilizing solution and stir for 5 min |
| Add milt | |
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| (C) Transfer to incubator without delay | |
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Figure 26 Procedure for handling non-sticky eggs
