{"id":4980,"date":"2022-06-24T17:41:03","date_gmt":"2022-06-24T15:41:03","guid":{"rendered":"https:\/\/veterinarska-stanica-journal.hr\/?post_type=article&p=4980"},"modified":"2022-08-19T12:19:08","modified_gmt":"2022-08-19T10:19:08","slug":"assisted-reproduction-of-clarias-gariepinus-burchell-1822","status":"publish","type":"article","link":"https:\/\/journal.h3s.org\/?article=assisted-reproduction-of-clarias-gariepinus-burchell-1822","title":{"rendered":"Assisted reproduction of Clarias gariepinus<\/em> Burchell, 1822"},"content":{"rendered":"

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Nora Mimoune<\/strong>#<\/sup>*, Abdelkader Rouabah<\/strong>#<\/sup>, Djamel Khelef<\/strong> and Rachid Kaidi<\/strong><\/p>\n

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Nora MIMOUNE<\/strong>*, (Corresponding author, e-mail: nora.mimoune@gmail.com), National High School of Veterinary Medicine, Algiers, Bab-Ezzouar, Algeria; Institute of veterinary Sciences, LBRA Laboratory, University of Blida 1, Algeria; Abdelkader ROUABAH<\/strong>, Water-Rock-Plant Laboratory; Djilali Bounaama University of Khemis-Miliana, Route de Theniet El Had 44001, Khemis-Miliana, Algeria; Djamel KHELEF<\/strong>, National High School of Veterinary Medicine, Algiers, Bab-Ezzouar, Algeria; Rachid KAIDI<\/strong>, Institute of veterinary Sciences, LBRA Laboratory, University of Blida 1, Algeria
\n#<\/sup> Equal contribution<\/div>\n
\"\"<\/a>https:\/\/doi.org\/10.46419\/vs.54.1.4<\/a><\/div>\n<\/p>\n

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Abstract<\/a>Introduction<\/a>Material and methods<\/a>Results<\/a>Discussion<\/a>Conclusion<\/a>References<\/a>Sa\u017eetak<\/a><\/span><\/div>\n<\/div>\n

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Abstract<\/h2>\n
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The objective of this work was to set up an experimental protocol concerning artificial reproduction in the catfish Clarias gariepinus<\/em> with the induction of spawning using GnRH. The experiment was carried out at an aquaculture farm in Khemis Meliana (Ain Defla), Algeria. In the study, five African catfish broodstock (3 females and 2 males) were used. Hormonal injection was made into the back muscle below the fin, and doses of GnRH were determined according to the weight of each individual. Fertilisation was performed artificially using the dry method.
\nAfter incubating the eggs, a binocular magnifying glass was used to check egg condition and embryonic development over time. The results obtained show that artificial insemination of this species, and survival and growth of larvae, are possible. After injection with GnRH, C. gariepinus<\/em> females displayed successful ovulation, fertilisation, larval hatching, and larval monitoring. For this species, a latency period of 22 hours was recorded, and approximately 35,700 larvae were obtained. The fertilization rate was 48%.
\nAt the end of this experiment, we can conclude that it is possible to improve reproduction through the proper use of hormonal stimulation techniques and by improving the diet and abiotic factors that are dominant in fish farming.<\/p>\n

Key words:<\/strong> Clarias gariepinus; GnRH; artificial insemination<\/em><\/p><\/blockquote>\n

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Introduction<\/h2>\n
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Aquaculture is a rapidly growing food production sector that currently provides nearly 50% of the fish consumed worldwide, and it is considered to have the greatest potential to meet the growing demand for aquatic food (Laffoley et al<\/em>., 2019). There are several methods of breeding farmed fish, selected on the basis of the reproductive biology of the species of interest, local environmental conditions and available facilities.
\nThese methods can be classified into three categories: natural, semi-natural, and artificial reproduction (FAO, 2012; Tkacheva et al<\/em>., 2020).<\/p>\n

The African catfish Clarias gariepinus<\/em> Burchell, 1822 is a major warmwater aquaculture species distributed in Africa and Asia (Khan and Abidi, 2011; Zidan et al<\/em>., 2020). It is an excellent intensive culture species due to its tolerance to poor water quality, its ability to maintain strong growth at high density, resilience to disease, and the ability to accept cheap food (Rouabah et al<\/em>., 2016; Shourbela et al<\/em>., 2017). Likewise, it is an excellent farmed fish, due to its economic nature compared to other production species with market value. It can constitute a piscicultural community in reservoirs and is wide used in breeding thanks to its characteristic adaptation to a wide range of temperature, dissolved oxygen concentrations, and its short trophic chain (Bakos and Gorda, 2001; G\u00e9offroy et al<\/em>., 2019).<\/p>\n

According to Legendre et al<\/em>. (1996), the seasonality of fish reproduction is one of the main features that hamper supply to the international market, and mastering artificial reproduction has thus become a mandatory step in optimising aquaculture production. To this purpose, we performed this study at an aquaculture farm in Khemis Meliana (Ain Defla, Algeria) to achieve artificial reproduction in catfish Clarias gariepinus<\/em> with spawning induction using GnRH.<\/p>\n

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Material and methods<\/h2>\n
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Study area and period<\/h3>\n

This study was carried out at the Laribi Sadek aquaculture farm, in Ain Sultane (Khemis Miliana, Ain Defla, Algeria), over a five-month period (spring season). The farm contains about ten large volume tanks (each with a diameter of 15 m), 8 medium tanks, 5 small tanks, 2 raceways and 2 small tanks for aquaponics, with a hatchery with 12 indoor basins each of 15 m\u00b3 volume. The farm is supplied with borehole water with a temperature of 21\u00b0C. This farm applies the principles of fish farming integrated with agriculture, where water is reused to irrigate orange and mandarin plantations (Figure 1).<\/p>\n

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Figure 1<\/strong>. Aquaculture farm.<\/figcaption><\/figure>\n

The breeder also uses a boiler at the entrance to the hatchery to control temperatures. There are several species raised on this farm such as: catfish, king carp, tilapia, and pike-perch.<\/p>\n

Experimental design<\/h3>\n

In this study, we used five parents of African catfish (Clarias gareipinus<\/em>), 3 females and 2 males, reared on the Laribi farm.<\/p>\n

Choice of broodstock<\/strong>
\nThe capture of adult fish consists of fishing the broodstock during their natural spawning period, or during their migration to their spawning grounds.
\nBroodstock must be kept in ponds at a maximum density of one fish per m2<\/sup> of water:
\nMature females were selected on the basis of belly roundness, indicating the proper development of ovaries at a temperature above 23\u00b0C. Artificial reproduction may be practiced all year round and the same female can lay eggs every 7 weeks (Figure 2).<\/p>\n

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Figure 2<\/strong>. Clarias female.<\/figcaption><\/figure>\n

For males, the larger ones were selected since their testes are well developed and full of semen. Males and females can easily be distinguished due to pronounced sexual dimorphism (Figure 3).<\/p>\n

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Figure 3<\/strong>. Clarias male.<\/figcaption><\/figure>\n

Anaesthesia<\/strong>
\nThe parents were anaesthetized so as not to stress them during handling to avoid any disturbance to gonad maturation. A sedative based on Eugenia Caryophyllata<\/em> (clove) was used as follows: 1 drop per 10 litres of water (Figure 4).<\/p>\n

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Figure 4<\/strong>. Sedative based on Eugenia Caryophyllata<\/em>.<\/figcaption><\/figure>\n

Hormonal induction<\/strong>
\nThe hormonal induction techniques of oocyte maturation and ovulation followed by artificial fertilisation are often favoured as they allow for better control of all phases of reproduction and larval rearing. There are mainly two hormonal induction techniques: an invasive method that consists of a hormone injection to the back muscle under the fin, and a non-invasive method that is based on inserting a probe into the genital papilla to release the hormone (Figure 5).<\/p>\n

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Figure 5<\/strong>. Intramuscular injection of GnRH under the dorsal fin.<\/figcaption><\/figure>\n

Dose calculations<\/strong>
\nThe doses of GnRH injected were determined as a function of the weight of each parent, with a total dose of 3 mg\/kg of live weight of broodstock, i.e., 3 mg hormone is applied per 1 kg bodyweight in females and per 2 kg in males.<\/p>\n

Latency<\/strong>
\nThe latency time (interval between injection and ovulation) is the maturation time of the oocytes; this is a function of the average temperature to which the females are subjected. Indeed, there is an inversely proportionate relationship between latency time and temperature, where latency time decreases as temperature rises.<\/p>\n

Fertilisation<\/strong>
\nSampling of testes and semen<\/em>
\nBoth males were sedated with clove oil before the experiment. An incision was made in the belly of the fish using a scalpel from top to bottom until reaching the anus. A finger is inserted under the incised skin to avoid damaging the internal organs. The testes are located on either side of the spine and mature testicles can be recognized by their white and opaque outer fringe. The testes were then detached from the dorsal part of the abdominal cavity (Figure 6).<\/p>\n

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Figure 6<\/strong>. Observation of testes in situ<\/em>.<\/figcaption><\/figure>\n

After the testes are removed, the males are sutured and a dose of antibiotics is given by IM.<\/p>\n

Semen collection<\/em>
\nThe testes are held over a glass container such as a beaker that has been previously dried to prevent sperm activation upon contact with water.
\nMultiple transverse incisions of the testes were made using a scalpel and the sperm was collected.<\/p>\n

Egg harvest<\/em>
\nThe eggs were collected by abdominal massage of the female; this is called \u201cstripping\u201d (Gilles et al<\/em>., 2001) (Figure 7).<\/p>\n

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Figure 7<\/strong>. Stripping.<\/figcaption><\/figure>\n

First, the genital papilla and its periphery were cleaned and dried; then, firm pressure was exerted on the abdomen with manual pressure applied to the area of the ovaries to cause the eggs to be released into a small basin or a dry basin. Ripe eggs are more or less transparent, small, with approximately 600 eggs per gram.<\/p>\n

Fertilisation<\/em>
\nArtificial insemination generally consists of mixing the eggs with dry milt, then adding a volume of water equivalent to that of the eggs in order to mobilise the spermatozoa to penetrate the eggs and thus fertilize them. Here is the approach we took for fertilisation of the eggs: a sufficient amount of milt was poured onto the eggs. It should be noted that 1 mL milt can fertilise 20 million eggs. <\/p>\n

The container containing the milt and eggs was gently shaken for two minutes to mix the sperm with the eggs before adding water. The time available for the ripe egg to be fertilised is very short since the eggs begins to swell immediately in contact with water, causing closure of the micropyle and thus preventing entry of the spermatozoa and the fertilisation of the eggs. We the added a volume of water equivalent to that of the eggs, i.e., 100 mL per 100 gram eggs, with a volume of fertilizing solution (40 g NaCl per 10 L water) to activate and mobilise the sperm. We rinsed with clean water 3 to 4 times while agitating between rinses to remove all dead and decaying cells.<\/p>\n

The fertilized eggs were mixed with whole milk at a rate of 250 mL per 100 gram eggs or with powdered milk to prevent clumping. We can also use the clay which also has a colloidal effect; it will surround the eggs to prevent their agglutination. The eggs were rinsed with clean water to remove the milk and sperm using a sieve. Eggs were then incubated under the conditions most favourable to normal development and capable of ensuring their survival.<\/p>\n

Egg incubation<\/strong>
\nImmediately upon fertilisation, the egg begins to develop, following a process of complex events. The technique consists of spreading the eggs in a single layer on frames of mosquito net or fine mesh at 1 mm. Grass-based spawning grounds can also be used as an incubation medium. The time it takes for the fertilized egg to turn into a larva depends mainly on the temperature and the dissolved oxygen content of the water.<\/p>\n

Hatching<\/strong>
\nThe movements of the fish embryo intensify until the eggshell is ruptured and larvae weighing about 1 mg are produced (Figure 8).<\/p>\n

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Figure 8<\/strong>. 1-day old larvae.<\/figcaption><\/figure>\n

All experiments were carried out according to the guidelines of the Institutional Animal Care Committee of the Algerian Higher Education and Scientific Research (Agreement Number 45\/DGLPAG\/DVA.SDA.14).<\/p>\n

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Results<\/h2>\n
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Clarias garipeinus was artificially induced. Eggs were easily collected by light abdominal massage and were already ripe with a greenish coloration (Table 1).<\/p>\n

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Table 1<\/strong>. Result of egg retrieval and latency time.<\/figcaption><\/figure>\n

Fertilisation<\/h3>\n

Fertilization was performed in vitro<\/em>.
\nThis consisted of sprinkling the milt of males on the eggs. We sprinkled the milt evenly on the surface of the eggs, and added water in the same proportion as the quantity of eggs to speed up fertilisation. The fertilisation took place in a minute. The fertilised eggs showed a marked increase in size. Egg adhesion is possible if the operation is not done with proper handling.<\/p>\n

The quantity of eggs produced was determined using two methods:<\/p>\n