4. ARTIFICIAL REPRODUCTION

4.1. Gonadal development in captivity
4.2. Induced propagation without hormone treatment
4.3. Semi-artificial reproduction through hormone treatment
4.4. Artificial reproduction

4.1. Gonadal development in captivity

The female African catfish has a fully developed ovary which contains “ripe” eggs the whole year round, if kept in ponds and once the water temperature remains above 22^o C. The eggs of a “ripe” female make up 15-20% of the body weight (i.e. a “ripe” female of 1 kg having about 150-200 g of “ripe” eggs). The oocyte development decreases once the temperature drops below 22^oC as can be seen from the results obtained in the Republic of the Congo (see Figure 12).

In the dry season (June-July-August) the water temperature drops below 22^oC and we see that the ovary usually makes up approximately 5% of the body weight of the female. Artificial reproduction is still possible but the number of eggs obtained is small and the quality of the eggs decreases as can be seen from the decreased hatching percentage.

In general the testis of a male is fully developed at an age of 8 to 12 months once they reach a weight of approximately 200 g. In the Republic of the Congo, sperm could be obtained the whole year round and no effects of temperature on the availability of sperm was found.

Figure 12. The relative fecundity (% of total body weight) hatching percentage (% of total eggs) of C. gariepinus, average monthly rainfall (cm) and air temperature (^oC) measured in the Republic of the Congo, bars indicate s.e.m. Source: de Graaf et al., 1995.

In captivity the African catfish does not spawn spontaneously since the environmental factors such as the rise in water level and inundation of shallow areas do not occur on the fish farms. Since the early seventies several techniques have been developed (with or without hormone treatment) for the artificial reproduction of the African catfish.

4.2. Induced propagation without hormone treatment

Mature breeders can be reproduced artificially by simulating the events which will occur during the rainy season which trigger off the mating and spawning processes. Ponds of about 400 m² in size are filled with 25 cm of water and stocked with six mature females (average weight 300-500 g) and four males (average weight 200-500 g). A few hours later the water level is raised up to a level of 50-60 cm. Spawning will occur at night and the following morning the breeders can be removed.

Theoretically speaking this technique can be termed artificial reproduction since some offspring are produced. However from a practical point of view it is not satisfactory as the number of fingerlings which can be harvested after 6-8 weeks is low (1-2 fingerlings/m²). This technique of semi-natural reproduction has also been applied successfully using concrete tanks (C. Nugent, pers. com.).

4.3. Semi-artificial reproduction through hormone treatment

4.3.1. Hormone induced reproduction in ponds
4.3.2. Hormone induced reproduction in a happa placed in ponds
4.3.3. Hormone induced reproduction in concrete tanks with a gravel substrate

For hormone induced reproduction (semi artificial or artificial) the following hormones are generally used;

· DOCA (Desoxycorticosteroid Acetate), 2.5-5 mg per 100 g of female. A disadvantage of using this hormone is that it is mostly suspended in oil which causes severe ulcers on the injected female.

· HCG (Human Chorionic Gonadotropin), 25 I.U. per 100 g of female. This hormone works well but is expensive.

· Common carp (Cyprinus carpio) pituitary gland material, 3-4 mg/kg of female or 1-2 whole pituitaries per female. In general the common carp pituitary gland material has to be imported from abroad which means that it is not usually accessible to small fish farms.

· Pituitaries of the African catfish (Clarius gariepinus). A female catfish will respond once it is injected with a pituitary of a catfish (male or female) of equal size.

· Pituitaries of the Nile tilapia (Oreochromis niloticus), 3-4 pituitaries of a Nile tilapia (100-150 g) per female catfish will induce ovulation.

· Pituitaries of Nile perch (Lates niloticus). 1-2 pituitaries per female catfish will induce ovulation.

4.3.1. Hormone induced reproduction in ponds

Mature females are injected with hormones in order to provoke the mating and spawning processes and then placed in completely filled ponds at a density of two females and one male per 100 m². Spawning usually takes place during the night after the hormone treatment and the breeders then removed the following morning. As for all nursing processes in ponds, the survival in the nursery ponds is a limiting factor for the mass production of fingerlings. This phenomena will be discussed in greater detail in section 5.

4.3.2. Hormone induced reproduction in a happa placed in ponds

A mature female is injected with hormones, so as to provoke ovulation, mating and the spawning processes. After injection the female and a non-injected male are then placed together in a 2-3 m³ happa (made from mosquito netting with a 0.5 mm mesh size) located within a pond (see Figure 13). Spawning usually takes place at night and the breeders then removed the following morning. One advantage of this method is that the eggs are concentrated within the happa where they eventually can be treated against fungal infections and the hatchlings easily collected after yolk sac absorption. It is essential that the happa be covered so as to prevent the breeders from jumping out.

Figure 13. Breeding happas placed within a fish pond in the Republic of the Congo.

4.3.3. Hormone induced reproduction in concrete tanks with a gravel substrate

This was one of the first techniques developed for the hormone induced reproduction of C. gariepinus in Africa (Micha, 1973; van der Waal, 1974).

Mature females are injected in the afternoon with hormonal material (DOCA, HCG or pituitaries) and placed together with a male in a concrete tank. The bottom of the concrete tank is covered with a layer of stony gravel which functions as a substrate for the released eggs. The breeders usually spawn at night after the hormonal material is injected; the released fertilized eggs sticking to the gravel or to any other substrate provided, or on the bottom of the tank if no substrate is available. The following morning the breeders are then removed from the tank. The fertilized eggs remain in the tank and hatching takes place after 24 hours (25-30^oC, see Table 2) and after 3-4 days approximately 3 000-5 000 larvae per female can be collected from the tank. However, the disadvantages of this method are that:

· brood fish often injure each other, which sometimes may result in the death of one of the breeders;

· the number of larvae obtained is relatively small as the ovulation is often only partial; the quantity of eggs released usually being only 5-10% of the total body weight, which is considerably less than the 15-20% which can be usually obtained through stripping.

4.4. Artificial reproduction

4.4.1. Introduction
4.4.2. Broodstock care and selection of ripe breeders
4.4.3. Hormone injection
4.4.4. Maturation processes and stripping of eggs
4.4.5. Incubation of fertilized eggs

4.4.1. Introduction

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:

· Better rates of fertilization and hatching.
· Protection against enemies and unfavourable environmental conditions.
· Better conditions for growth and survival.

4.4.2. Broodstock care and selection of ripe breeders

In most cases broodstock 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 22^oC during the dry/winter season, which in turn hampers egg development and artificial reproduction (see Figure 12). 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 broodstock 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 broodstock 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 crackhead and retarded growth in the breeders, and an oedemic disease in the developing larvae.

Artificial reproduction starts with the selection of females from broodstock ponds, after which they are transferred to a holding tank within a hatchery. Ideally, broodfish 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:

· 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 9).

· A swollen, sometimes reddish or rose coloured genital papilla.

4.4.3. 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.

⁴ 1 ml per fish

Figure 14. 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.

4.4.4. Maturation processes and stripping of eggs

The process of final maturation (migration of the nucleus to the animal pole, fusion 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.

Note:

Normally the females are injected in the afternoon/evening and are kept (separated from the males) in holding facilities. The holding facility can be a concrete basin inside a hatchery, a happa in a pond or even a simple plastic bucket or a half oil drum will do. Of major importance is that the breeders can be caught easily the morning after injection so as to avoid spoilage of eggs.

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

Note:

Sometimes with fluctuating water temperatures, and in particular with higher temperatures during the day, it is difficult to establish the actual mean water temperature. This can result in eggs being stripped too early with consequently very low hatching rates (5-10%). Eggs which are stripped too early tend to have a treacly consistency. It is always much safer to strip eggs later rather than earlier. If you are too early you will loose all your eggs, if you are too late you will loose some eggs. The eggs ooze out very easily if stripped at the right time.

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 15).

Figure 15. 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.

Figure 16. Collection of milt from male African catfish

Two different methods of fertilization are used in Africa:

· 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 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.

· 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.

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.

4.4.5. 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:

· 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

· 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 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.

· 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.

· 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.

· 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.

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.