Vol. 05, Issue 03, March 2020, Available Online: www.ajeee.co.in/index.php/AJEEE ARTIFICIAL REPRODUCTION IN FISH
Uma Shankar Prasad
Dept. of Zoology, Rajendra College, Chapara, Bihar, India
Abstract:- Many species of fish will not readily reproduce under certain culture conditions.
Others will, but not necessarily when the farmer desires. In these cases, induction of spawning can be of great value. Two techniques are commonly used, sometimes in conjunction with one another. The first is manipulation of the culture environment to mimic some important quality in the fish’s natural environment. The second is injection of hormones to stimulate spawning. The hormones may be natural hormones taken from fish or other animals, genetically engineered from bacteria, or synthetic analogs of naturally- occurring hormones.
Keyword:- Spawning, artificial reproduction and GnRH etc….
1. INTRODUCTION
Among the most significant advancements in the field of aquaculture during recent decades is the development of techniques to induce reproduction in fish. These techniques have allowed farmers to profitably breed and raise species that do not naturally reproduce in captivity, and to manipulate the timing of reproduction to suit production cycles.
Some species will not readily breed in captivity due to environmental or culture conditions that are different from those found in nature, such as water temperature or substrate type. These conditions may cause stress or may not provide the cues needed to complete the reproductive process. Fish in captivity may not always reproduce at the most advantageous time, and alteration of the spawning cycle may be desirable.
This allows a farmer to:-
1. Obtain fish outside of the normal spawning season either to lengthen time for grow-out or to produce hybrids with other species;
2. Improve efficiency by getting fish to spawn on a predetermined date;
and
3. Maximize survival by fertilizing and incubating eggs under hatchery conditions. Where successful, techniques for altering the spawning cycle of fish have become a valuable tool.
Semi-natural or hormone induced reproduction within ponds or concrete tanks as described above can be used on small farms to produce their own larvae and fingerlings. However, this method has not proved to be reliable for the mass production needed for larger
fish farms or fingerling distribution centres.
Therefore artificial propagation techniques under more controlled conditions, including stripping of eggs, collection of sperm, followed by egg fertilization, have been developed.
Artificial reproduction by induced breeding through hormone treatment followed by artificial fertilization and incubation of fertilized eggs and the subsequent rearing of larvae to fingerling size has several advantages (Woynarowich and Horvath, 1980) including:-
1. Better rates of fertilization and hatching.
2. Protection against enemies and unfavourable environmental conditions.
3. Better conditions for growth and survival.
The artificial reproduction of the African catfish, as for all finishes, consists of a chain of activities which is more or less similar to that which occurs during the course of natural reproduction.
2. BACKGROUND: REPRODUCTION IN NATURE
In fish, the reproductive process involves three basic steps:-
1. Maturation - the development of the gametes (eggs and sperm) to a point where fertilization can occur;
2. Ovulation - the release of eggs from the ovary; and
3. Spawning - the deposition of eggs and sperm so that they can unite.
In fish, as with all higher animals, hormones play a critical role in the reproductive process. Hormones are chemical messengers released into the blood by specific tissues, such as the
Vol. 05, Issue 03, March 2020, Available Online: www.ajeee.co.in/index.php/AJEEE pituitary gland. The hormones travel
through the bloodstream to other tissues, which respond in a variety of ways. One response is to release another hormone, which elicits a response in yet another
tissue. The primary tissues involved in this hormonal cascade are the hypothalamus, pituitary gland, and gonads (Fig. 1).
Figure 1. The Natural Hormone Cascade
Fish have evolved to reproduce under environmental conditions that are favorable to the survival of the young.
Long before spawning, seasonal cues begin the process of maturation. In many fish, this can take up to a year. When the gametes have matured, an environmental stimulus may signal the arrival of optimal conditions for the fry, triggering ovulation and spawning. Examples of environmental stimuli are changes in photoperiod, temperature, rainfall, and food availability.
A variety of sensory receptors detect these cues, including the eye, pineal gland (an organ in the dorsal part of the forebrain that is sensitive to light), olfactory organs, taste buds, and thermo receptors. The hypothalamus, located at the base of the brain, is sensitive to signals from sensory receptors and releases hormones in response to environmental cues.
Principal among these hormones are gonadotropin releasing hormones (GnRH), which travel from the hypothalamus to the pituitary gland. The pituitary is responsible for a wide variety of functions, including growth and reproduction. Certain cells of the pituitary receive GnRH and release gonadotropic hormones into the bloodstream. The gonadotropic hormones travel to the gonads, which synthesize steroids responsible for final maturation of the gametes.
Maturation of the egg is a long process that involves complex physiological and biochemical changes.
One important step, vitellogenesis, is a
process in which yolk proteins are produced in the liver, transported to the ovary, and stored in the egg, resulting in tremendous egg enlargement. The yolk is important as a source of nutrition for the developing embryo. Also critical are germinal vesicle migration and germinal vesicle breakdown (GVBD).
Before it migrates, the germinal vesicle, or nucleus, is located at the center of the egg in an arrested stage of development. At this stage, the egg is physiologically and genetically incapable of being fertilized, even though it has the outward appearance of a fully mature egg.
When conditions are appropriate for final maturation, nuclear development resumes, and the germinal vesicle migrates to one side. Finally, the walls of the germinal vesicle break down, releasing the chromosomes into the cell.
The maturity of eggs can be determined using biopsy techniques. Eggs are removed from the ovaries, cleared with a prepared solution, and viewed under a microscope. In mature eggs, the migration of the germinal vesicle to the side of the cell will be complete. After the egg has matured, a class of compounds called prostaglandins are synthesized.
These stimulate ovulation, which is the rupture of the follicle cells that hold the egg.
The egg is then released into the body cavity or ovarian lumen, where it may subsequently be released to the outside environment. Following ovulation, the viability of the eggs can decrease rapidly. Fish with gametes that have not yet been released by the gonads are called
Vol. 05, Issue 03, March 2020, Available Online: www.ajeee.co.in/index.php/AJEEE
“green.” The term “ripe” is used to describe fish with gametes that have been released from the ovary into the ovarian lumen. Ripe fish can be stripped, green fish cannot.
3. BROOD STOCK CARE AND SELECTION OF RIPE BREEDERS
In most cases brood stock selected from nature or bought at a fish farm are kept in earthen ponds at a stocking density of 0.5-1/m2 and fed regularly with agriculture waste products and sometimes with trash fish. Egg development will take place and about six weeks after a female has spawned it can be used again.
In some areas of Africa water temperatures may fall below 22oC during the dry/winter season, which in turn hampers egg development and artificial reproduction (see Figure 1). In the Republic of the Congo, this problem was overcome by careful planning i.e. catfish were reproduced artificially in the first month of the dry season and a double number of females were injected in order to guarantee a sufficient number of eggs with the result that catfish fingerlings could be produced 11 months per year.
Another method to overcome this problem is to keep the brood stock permanently indoors in a hatchery (de Graaf, 1989, Janssen, 1985a, Richter et al., 1987). A complete breakdown of the natural annual reproductive cycle can be obtained after the brood stock have been kept for one year indoors and reproduction can then be carried out continuously throughout the year.
However this method is not recommended by the authors as it depends on the availability of high quality feed and often encounters diseases such as crack head and retarded growth in the breeders, and an oedemic disease in the developing larvae.
Artificial reproduction starts with the selection of females from brood stock ponds, after which they are transferred to a holding tank within a hatchery. Ideally, brood fish weigh between 300-800 g, with larger fish being difficult to handle and often resulting in substantial egg losses prior to stripping.
In general, mature females are selected according to the following criteria:-
1. A well distended, swollen abdomen from which ripe eggs can be
obtained by slightly pressing the abdomen toward the genital papilla. Ripe eggs are generally uniform in size and a experienced hatchery operator can see the nucleus as a small dark point in the centre of the egg (see Figure 2).
2. A swollen, sometimes reddish or rose coloured genital papilla.
For male brood stock there is only one criteria: they should be larger than 200 g and not less than 7 months old.
3.1 Hormone Injection
The most common technique employed to induce final maturation and ovulation in African catfish is to inject the female with hormones or pituitary gland material (the dosages and advantages and disadvantages have been discussed in paragraph 4.3).
The required quantity of powdered acetone dried pituitary material or the required number of whole pituitaries are pulverised in a porcelain mortar, mixed with the required quantity4 of physiological salt solution (9 g of common salt/litre of water). A syringe is filled with the suspension and the injection can be given. The most common method of administering the hormone solution is by intra-muscular injection into the dorsal muscle (see Figure).
Figure 2. Injection
Note: Cover the head of the breeder with a wet towel in order to keep it quiet during injection. In general most fish keep still if their eyes are covered.
3.2 Maturation processes and stripping of eggs
The process of final maturation (migration of the nucleus to the animal pole, fusion
Vol. 05, Issue 03, March 2020, Available Online: www.ajeee.co.in/index.php/AJEEE of the yolk, breakdown of the germinal
vesicle followed by first meiotic division) and ovulation (rupture of the follicles and accumulation of the ripe eggs in the ovary cavity) cannot be stopped or reversed after administration of the correct hormone dosage.
Once these processes start the eggs must either be spawned or stripped.
The speed of the process is dependent upon water temperature, the higher the temperature the quicker the eggs ovulate.
The relationship between water temperature and the time taken for eggs to ovulate is presented in Table 1.
Table 1. The time interval between injection and stripping of female catfish in relation to water temperature (Source: Hogendoorn and Vismans, 1980)
WATER TEMPERATURE (°C)
TIME BETWEEN INJECTION AND STRIPPING (HOURS)
20 21
21 18
22 15.5
23 13.5
24 12
25 11
26 10
27 9
28 8
29 7.5
30 7
Stripping of the female spawners is carried out by gently pressing their abdomen with a thumb from the pectoral fin towards the genital papilla. Ovulated eggs will flow out easily in a thick jet from the genital vent and are usually collected into a dry plastic container (see Figure 3).
Figure 3. Stripping a female African catfish.
The ovulated eggs are more or less transparent, flattened and 1 g contains approximately 6005 eggs. Under normal conditions a “ripe” female ovulates a quantity of eggs which equals 15-20% of her own body weight (de Graaf et al., 1995). If the fish is stripped too early the eggs come out with difficulty, whereas they have a “flushy” appearance if they are stripped too late. 5 This number is not consistent as reported by different authors therefore it is recommended that you determine this number yourself.
The males of the African catfish cannot be stripped and consequently the sperm can only be obtained by sacrificing a male. The male is killed and the body surface thoroughly dried after which the testis is dissected and placed in a mortar or a teacup. The testis is rapidly cut into small pieces using a scissor and finally the milt is pressed out with a pestle or a teaspoon (see Figure).
Figure 4. Collection of milt from male African catfish
Two different methods of fertilization are used in Africa:-
1. In the Republic of the Congo (de Graaf et al., 1995) the females are first stripped, then a male killed and the milt then directly mixed with the stripped eggs. This was
Vol. 05, Issue 03, March 2020, Available Online: www.ajeee.co.in/index.php/AJEEE necessary as stripping was carried
out outdoors under all weather conditions. It should be realized that one drop of water in the bottle with sperm will destroy the sperm completely while one drop of water in the bowl of eggs will only destroy some eggs. This method is very suitable if a limited number of females are stripped.
2. In Central Africa, Kenya and Ivory Coast (Janssen, 1985a, de Graaf, 1989) milt is taken from a male spawner and diluted with a physiological salt solution (9 g of kitchen salt dissolved in 1 litre of boiled water) about half an hour before the females are stripped.
This solution is then later added to the stripped eggs. The advantage of this method is that eggs from a large number of females can be fertilized as one testis of a mature male can easily fertilize the eggs of 10-15 females.
The sperm (diluted or non-diluted) is added to the stripped eggs, and the eggs fertilized by adding an equal volume of clean water. The water and egg mass are then mixed by gently shaking the bowl.
Eggs must be stirred continuously until they are placed in the hatching tanks as the eggs become sticky and without stirring will stick together into one clump.
About 60 seconds after fertilization has taken place and the sperm has lost its activity, the fertilized eggs are then ready for incubation in tanks or happas.
3.3 Incubation of fertilized eggs
The development process from fertilized egg to hatching, like all other biological processes, is dependent upon water temperature; the higher the water temperature the faster the eggs hatch.
The relation between water temperature and the incubation time of catfish eggs is shown in Table 2.
Table 2. The time interval between fertilization and hatching of catfish eggs in relation to water temperature (Source: Hogendoorn and Vismans, 1980)
WATER TEMPERATURE (°C) TIME BETWEEN FERTILIZATION AND
HATCHING (HOURS)
20 57
21 46
22 38
23 33
24 29
25 27
26 25
27 23
28 22
29 21
30 20
A general principle of egg incubation is that water is renewed in order to provide oxygen and that after hatching the larvae are separated from the remaining egg- shells and dead eggs. The latter is of utmost importance in order to avoid fungal infections of hatchlings and consequent larval mortalities.
The following incubation techniques are usually used:-
1. The eggs are spread out on the bottom of a concrete basin. This method works well but it has the
disadvantage that dead eggs/egg- shells are not separated from the hatchlings. Daily treatment with 0.1 ppm6 malachite green is needed in order to prevent the outbreak of fungal infections.
6 ppm = parts per million or 1 mg/litre
2. The eggs are spread out on a screen (mesh size 1 mm) which is placed on the bottom of a concrete basin. This method works well as the hatchlings will pass through
Vol. 05, Issue 03, March 2020, Available Online: www.ajeee.co.in/index.php/AJEEE the screen and the dead eggs and
shells remain on the screen. By removing the screen from the basin a separation between hatchlings and dead eggs is readily achieved.
3. The eggs are allowed to “stick” to the roots of floating water hyacinth (Eichhornia crassipes) placed within a happa made from mosquito netting (mesh size 0.5 mm) located within a concrete basin with running water or in a pond. This method was developed in the Republic of the Congo (de Graaf et al., 1995). The investments are low and hatchlings easily separated from the dead eggs as long as the distance between the roots of the water hyacinth and the bottom of the happa is kept at 15-20 cm.
After hatching the larvae sink to the bottom of the happa and the egg shells remain stuck to the roots of the water-hyacinth. The dead eggs are separated from the hatchlings once the water hyacinth is removed from the happa.
4. The eggs are allowed to “stick” to the roots of floating Nile cabbage/water lettuce (Pistia stratiotes) placed within a happa located in a concrete basin. The Nile cabbage works as well as the water hyacinth; floating well and having a well developed fine root system to which the eggs stick nicely. More importantly, Nile cabbage is not a “water pest”, can be easily found and therefore more suitable than the water hyacinth.
5. The eggs are allowed to “stick” to a brush which floats inside a concrete basin or happa. This method works very well as the eggs are held completely under water, the only disadvantage being the price of the brush.
It is also possible to incubate eggs and hatchlings in stagnant water, using oil drums/barrels, or inside a happa placed in a pond. However, low egg/hatchling densities are essential and should not exceed 100-150 per litre (or 0.1 g of eggs per litre). The hatchlings (1-1.5 mg) can be kept in the incubators and do not have
to be fed as they rely on the food resource within their yolk sac.
Healthy larvae tend to stay in dark places and should not be exposed to direct sunlight. After three to four days the yolk sac will be absorbed and the hatchling is visibly developed into a small catfish.
At this stage the hatchling must be fed on external food for its further development and survival; therefore, the hatchlings should be transferred out of the incubation facilities to ponds or specialized hatchery facilities. This phase of rearing from first feeding larvae to fingerling size is usually carried out either within earthen ponds or in specialized hatcheries.
3.4 Catfish
Temperature is a determining factor in the control of catfish reproduction.
Spawning activity begins after water temperatures stay consistently above 70¡F. It drops off when temperatures fall below 70¡F or rise above 85¡F. There are a variety of methods commonly used for spawning catfish, including spawning in ponds, spawning in pens, and hand stripping.
In many, though not all cases, hormone injection may be desirable. The following procedure from Third Report to the Fish Farmers provides an example of spawning induction with hormones followed by hand stripping.
1. Anesthetize the fish with MS-222 at a concentration of 80 mg/l.
2. If the fish are kept in a holding tank, loosely stitch their mouths closed to prevent them from biting one another. The thread must be loose enough to allow respiration.
3. Using an 18-gauge needle, inject intraperitoneally with: a. 3.5-4.0 mg of acetone-dried pituitary gland extract per kilogram of fish for the female and about 2 mg per kilogram for the male, or b. 500- 800 international units of human chorionic gonadotropin (HCG) per pound of fish.
4. If necessary, inject again 48 hours later. Usually, the female ovulates within 16-20 hours after an injection.
5. Strip the eggs and the milt into a 0.3 percent solution of common salt.
Vol. 05, Issue 03, March 2020, Available Online: www.ajeee.co.in/index.php/AJEEE 6. Very gently mix the eggs and milt.
Fertilization should occur in 2-5 minutes.
7. Place the egg mass in a suitable incubator for hatching.
8. After 10-12 hours, stop the water flow and treat with a 0.3-0.5 percent solution of alkaline protease enzyme for 2-3 minutes while gently stirring. This will dissolve the sticky layer of the eggs and allow them to float free.
9. When the eggs begin hatching, they should be taken out and placed in a flat tray.
4. CONCLUSIONS
Many species of fish will not readily reproduce under certain culture conditions. Others will, but not necessarily when the farmer desires. In these cases, induction of spawning can be of great value. Two techniques are commonly used, sometimes in conjunction with one another. The first is manipulation of the culture environment to mimic some important quality in the fish’s natural environment. The second is injection of hormones to stimulate spawning.
The hormones may be natural hormones taken from fish or other animals, genetically engineered from bacteria, or synthetic analogs of naturally-occurring hormones. Methods vary from species to species and situation to situation. However, at least two generalizations can be drawn. First, brooders are very vulnerable to rough handling. Care should always be used to avoid damaging these valuable animals.
Second, a fish that does not have mature gametes will not produce viable eggs or sperm no matter how many times it is injected with hormones. Ripeness is the result of environmental factors working over a period of time, leading to maturation of the gonads and production of viable eggs. Many procedures have been developed for inducing fish to undergo the last steps of spawning.
Farmers should thoroughly research the procedures that have been developed for their species of fish through experimentation, and select those that best suit the circumstances. In addition, once the fish have spawned, there are many techniques involved in incubating and caring for the eggs, and caring for the
hatched fry. These too must be thoroughly researched.
REFERENCES
1. Fish Culture Manual. 1983. Staff, F. Alaska Department of Fish and Game, Division of Fisheries Rehabilitation, Enhancement and Development.
2. Third Report to the Fish Farmers. 1984.
Dupree, H. K. and J. V. Huner, eds. United States Department of the Interior, Fish and Wildlife Service.
3. Fish Hatchery Management. 1989. Piper, R.
G. et al. United States Department of the Interior, Fish and Wildlife Service.
4. Hatchery Manual for the White Sturgeon Acipenser
transmontanus RICHARDSON with Application to Other North American Acipenseridae. 1988. Cooperative Extension, University of California, Division of Agriculture and Natural Resources.
