African catfish, Clarias gariepinus, is a highly appreciated species for aquaculture. So, the present research was carried out to study the possibility of improving artificial reproduction of African catfish C. gariepinus brood stock by injection with some stimulating preparations. Females and males were divided into five treatments (T), each treatment had five females and two males, the females only were injected; T2, was injected with imported Pregnyl®; T3, was injected with imported Argent®; T4, was injected with native carp pituitary gland and T5, was injected with native catfish pituitary gland; but T1, set as a control, without injection. The obtained results revealed that the ovary length was significantly (p≤0.05) longer in T2, T3 and T4 than in T1 and T5. Also, T3 gave the highest Absolute Fecundity (AF), being 119917 and T2, T4 and T5 also represented higher AF, being 7.6, 7.8 and 3.9 times, respectively than T1. Yet, T3 gave the highest Relative Fecundity (RF), being 126.4 which was 8.5 times than T1. There were significant differences among treatments for serum FSH, LH and progesterone (P4) but not for estrogen. Treatment No. 2 reflected the highest (p≤0.05) level of FSH and LH among all treatments but P4 level in each of T2, T3 and T5 was significantly higher than T1 and T4. Histological examination of ovaries revealed normal structure of ovarian lamellae among all treatments and various development stages of oogenesis were observed. So, it could be concluded that T3 followed by T2 and T5 were the best treatments for improving the reproductive performance of C. gariepinus concerning the significantly heaviest egg weight and highest AF, RF, serum FSH, LH and P4 than T1 and T4.
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African catfish Clarias gariepinus is a suitable alternative to tilapia in subsistence fish farming in Africa using low grade feed composed of some local agricultural and agro-industrial by-products, the yields of catfish from ponds could be as much as 2.5 times higher than those of tilapia (Verreth et al., 1993). In addition, this species is known with its high growth rate, resistance to handling and stress, relatively low requirements for water quality, amenability to high stocking densities, excellent meat quality and preference amongst consumers in many African countries (Hecht et al., 1996). Although, the potential use of C. gariepinus in Egyptian aquaculture is very high because of several factors related to the aquaculture system, the species itself and existing market forces (Rezk, 2008). So, culturing of C. gariepinus is a growing interest, it once mass juvenile production becomes commercially and economically feasible. Furthermore, latest fish production statistics in Egypt revealed that African catfish production reached about 48750 tones (about 4.79% of the total aquaculture production in 2012, (GAFRD, 2012).
Reproductive problems are usually more serious in female brood stocks fish (Zohar and Mylonas, 2001). So, the high demand for fish fingerlings in the aquaculture industry has stimulated the need for artificial propagation of cultivable warm water fish (Nwokoye et al., 2007). Although, African catfish, C. gariepinus, is a highly appreciated species for aquaculture (De Graaf and Janssen, 1996). Yet, the seasonality of the reproductive cycle maintained catfishes in tropical fish ponds hampers the continuous production of fry and fingerlings for pond stocking (Nguenga et al., 2004), as well as catfish species are classified as single-time spawners (Tyler and Sumpter, 1996). Moreover, Dorman (2008) mentioned that, even with the genetic improvements in catfish brood stock, spawning success is often quite low, in average; only 50% of females actually spawn. Additionally, survival from the egg to fingerling stages, estimated at 60 to 70%. So, early success using injected extracts of fish pituitaries or human chorionic gonadotropin (hCG) has been exogenously administered to induce ovulation and spawning in females of captive brood stock (Nwokoye et al., 2007). Also, regulations of reproductive activity of fish by the brain-pituitary-gonad axis were reported by Yaron et al. (2003). In this manner, Carp Pituitary Extract (CPE) is the most predictable compound for the timing of ovulation (Liu et al., 1997), for induced spawn of female channel catfish (Lambert et al., 1999), as well as, catfish pituitary extract is as effective as CPE or luteinizing hormone-releasing hormone analogue (LHRHa) for inducing ovulation in females' channel catfish (Bosworth, 2005).
The need for high quality fish seeds has necessitated researchers to look for various ways of enhancing fertility to meet the growing demand (Dada et al., 2010). So, hormonal therapies have an important role in brood stock fish management (Mylonas and Zohar, 2001). Hence, various exogenous hormones have successfully been used to induce maturation and ovulation of postvitellogenic oocytes in fish (Richter and Van Der Hurk, 1982), including injection with salmon-GnRH and chicken-GnRH-II for spawning induction of C. gariepinus (Szabo et al., 2007); recently, Sharaf (2012) reported that gonadotropin-releasing hormone analog (GnRHa) is effective for induced spawning in C. gariepinus.
In Egypt, production of C. gariepinus, so far, is mainly from fisheries capture. Use of C. gariepinus for farming has encountered a number of obstacles including problems mainly related with reproduction and fry maintenance (Rezk, 2008). With regard to C. gariepinus, although artificial hatchery technology is now available to produce seeds, a few hatcheries have worked with C. gariepinus (Krouma, 2011). Therefore, the present research was designed as a comparative study to evaluate the possibility of improving artificial propagation of African catfish C. gariepinus using the intramuscular injection with some commercial imported and native preparations of reproductive hormones, by measuring different physiological, biochemical and histological parameters.
MATERIALS AND METHODS
Brood stock selection: This study was carried out at the fish farm belonging to the Egyptian Aquaculture Centre, Kafr El-Sheikh Governorate, Egypt. Brood stock African catfish in an earthen pond (8x20 m) were fed one time a day, 6 days a week for 1 month by demand feeder a tilapia commercial floating feed (manufactured by Hendrix, Egypt factory, contained 32% crude protein, 4.51% crude fat, 4.48% crude fiber and 4160 kcal kg-1 gross energy and consisted of fish meal 72%, soybean meal 44%, wheat bran, corn gluten 60%, monocalcium phosphate, lime stone, soy oil, L-lysine and vitamins and minerals mixture). This period was to make brood stock ready for spawning. Rearing water quality parameters were measured weekly at 06.0 am and 06.0 pm to determine the values of pH (using Jenway Ltd, model 350-pH meter), dissolved oxygen concentration (using an oxygen meter model d-5509) and temperature degrees centigrade (using a thermometer). The values for water temperature ranged between 28 and 30°C, pH values 7.8-8.6 and dissolved oxygen 3.9-7.1 mg L-1, these water parameters are within the acceptable ranges recommended for rearing brood stock catfish according to Boyd (1990). After this 1 month, twenty five females and ten males from brood stock in earthen pond were chosen to be equal in weight (within each sex), activity and ovary appropriate (eggs have a bright green color) approximately.
Brood stock spawning: Brood stock African catfish females (average weight 812±78.46 g fish-1 and body length 46.5±1.16 cm) and males (average weight 1065±81.36 g fish-1 and body length 53.6±1.24 cm) were taken from this earthen pond to the farm laboratory and were received in five glass tanks (50x40x50 cm) each contained 30 L fresh water mixed with 15 mL formalin as external disinfectant. After this treatment, these fishes were divided into five treatments each treatment has 5 females and 2 males. Brood stock fish were separated into 6 tanks (one tank for each treatment of females and one tank for all males). During this time, no feed was offered. The flow rate of water was 2 L-1. Extracted pituitary gland from sacrificed males was kept for female's injection. Nine hours post treatment (only females' injection), females became ready for spawning and striping. Details of the experimental treatments and the preparation of the injected materials were illustrated in Table 1.
Pituitary keeping method: After the evisceration of pituitary from under brain of males, it was transferred into test tube with 1 mL of acetone for 5 min acetone was changed after this period and repeated after 15 min, 8 h and 24 h from starting this method according to Kumar (1992). During this time, the test tube was kept at room temperature. Next these 24 h, the pituitary was dried on candidacy paper until the complete dryness and kept in a dried bottle in a refrigerator (4°C).
Egg fertilization: Eggs were collected from three females per treatment and another two females were used for blood sampling. Males were killed to collect the milt from testis to fertilize eggs of each treatment. Milt was put on eggs and mixed with about 200 mL fresh water to stimulate the fertilization. After mixing eggs with the milt in a plastic plate for 2 min to ensure that all eggs were fertilized, then these fertilized eggs were distributed on a substrate which was made of a wood frame (2x0.5x0.08 m) with American fiber net (1 cm2 contained 6 gaps, each of 20 μ). On which eggs did not accumulate over others. Each treatment was separated in a concrete pond (5 treatments/5 ponds, each of 8x3x1.25 m, whereas the water column was 60 cm), where eggs need 30 cm water depth to keep temperature between 25-28°C, tropic day. Fish were killed at the end of the experiment and soon the abdominal cavity was opened to remove gonads which were weighed individually and their length as well as size and density were measured and calculated. Gonado-Somatic Index (GSI) was calculated as:
The brood stock fish measurements were taken for the nearest 0.1 g and 0.01 cm to calculate the condition (K) factor. The males weight in average was 1065±81.36 g fish-1, body length 53.6±1.24 cm, testis weight 12.3±1.62 g fish-1, testis length 6.61±0.49 cm, k-factor 0.69±0.02 and GSI 1.14±0.11. Total egg weight and number per female as well as individual egg weight and diameter were measured too. Eggs number was counted using 1 mm insulin syringe, then related to ovary weight and body weight of fish. Absolute fecundity (AF, number of eggs/female) and relative fecundity (RF, number of eggs/g female weight) were calculated according to Bhujel (2000).
Serum hormones assay: At the end of the experiment, blood samples were collected from the remaining two females to obtain blood serum by centrifugation for 20 min at 3500 rpm. Serum Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH), as well as the sex steroids hormones, Progesterone (P4) and Estrogen (E2) were quantitatively analyzed by a calibrated DPC IMMULITE® 1000 chemiluminescent immunoassay system (DPC, Los Angeles, CA, USA), accordingly to the manufacturers instructions by test kits catalogs No. (LKFS1), (LKLH1), (LKPG1) and (LKE21), respectively.
|Table 1:||Details of the experimental treatments and the preparation of the injected materials|
|*Males without injection in all treatments, but females only injected with the tested agents|
Histological examination: At the end of the experiment, fish were sacrificed and the target organs (female gonads) were sampled. Samples were fixed in 10% neutralized formalin solution followed by washing with tab water, then dehydrated by different grades of alcohol (70, 85, 96 and 99%). Samples were cleared by xylene and embedded in paraffin wax. The wax blocks were sectioned to six micron. The sections were stained by Hematoxyline (H) and Eosin (E) and then subjected to a histological examination according to Roberts (2001).
Statistical analysis: The obtained numerical data were statistically analyzed using SAS software package (SAS, 2001) for one-way analysis of variance (ANOVA). Ratio and percent data were arcsine-transformed prior to statistical analyses and evaluated by using the following model:
Yij = μ+Ai+eij
where, Yij is an observation of body weight, length and K-factor, ovaries weight, ovaries size, ovary density, ovary length and GSI, total egg weight, egg diameter, individual egg weight, AF and RF and blood serum FSH, LH, P4 and E2; μ is least square mean; Ai is the fixed effect of treatments (T1-T5) and eij is the random error. Statistical significant (p≤0.05) differences between mean were compared by using Duncan's multiple ranges test (Duncan, 1955), which was described by Bailey (1995).
Brood stock characteristics: In respect to the female brood stock measurements (body length and K-factor) in Table 2, there were no significant (p≥0.05) differences among treatments; yet, T4 was heavier (p≤0.05) in body weight than T1 and T3.
|Table 2:||Body weight, length and K-factor data of brood stock female African catfish as affected by the experimental treatments (Mean±SE)|
|Mean having different small letters are significantly different (p≤0.05), but mean having capital letter are not significantly different (p≥0.05)|
|Table 3:||Ovarian measurements of African catfish as affected by the experimental treatments (Mean±SE)|
|Mean having different small letters are significantly different (p≤0.05), but mean having capital letter are not significantly different (p≥0.05)|
Table 3 illustrates mean±standard error (SE) of the ovarian traits including ovaries weight, size and density (specific gravity), as well as the GSI which were all not significantly (p≥0.05) different among treatments, except the ovary length which was significantly (p≤0.05) longer in T2, T3 and T4 than in T1 and T5.
Egg quality and fish fecundity: Brood stock C. gariepinus egg quality measurements are presented in Table 4, whereas no significant (p≥0.05) differences were found in egg diameter (mm) and egg weight (mg egg-1) among all treatments. However, total egg weight (g female-1) was significantly (p≤0.05) heavier in T3 (152.2±14.3 g female-1) and T2 (145.6±33.6 g female-1) than in T1 (24.2±17.9 g female-1) by 6.29 and 6.0 times, respectively. Data of AF and RF are presented also in Table 4, which revealed significant (p≤0.05) differences among treatments in both AF and RF. Fish injected with imported Argent® (T3) gave the highest egg number per female (i.e., AF, being 119917 eggs female-1) which was 9.75 times that of the control (T1). The other treatments (T2, T4 and T5) also represented higher total egg number female-1, being 7.6, 7.8 and 3.9 times, respectively than the control. The RF took the same trend, since T3 gave the highest egg number g-1 female body weight (i.e., RF, being 126.4 eggs g-1 female weight) which was 8.5 times that of the control (T1). The other treatments (T2, T4 and T5) also represented higher RF, being 8.5, 6.6 and 3.2 times, respectively than the control. However, there were no significantly (p≥0.05) differences between treatments T2 and T5 and between T1 and T5 in AF.
Blood serum hormones: Figure 1 presents the data of blood serum hormones (FSH, LH, P4 and E2) determinations. It is clear that there were significant (p≤0.05) differences among treatments for all tested hormones, except for E2. Treatment No. 2 (Pregnyl®) reflected the highest level of FSH (2.17±0.18 mIU mL-1) and LH (26.67±7.05 mIU mL-1) but P4 level was the highest in each of T2 (Pregnyl®), T3 (Argent®) and T5 (native catfish pituitary) without significant (p≥0.05) differences among these treatments which were significantly (p≤0.05) higher than the control (T1) and T4 (native carp pituitary).
Histology of the ovaries: Histological examination of ovaries of experimental African catfish brood stock revealed the presence of different stages of oocytes, which developed at various stages of oogenesis in ovaries of fish injected with experimental materials compared with fish's ovaries without injection (T1, control), as illustrated in Fig. 2a-h.
|Table 4:||Egg measurements of African catfish as affected by the experimental treatments (Mean±SE)|
|Mean having different small letters are significantly different (p≤0.05), but mean having capital letter are not significantly different (p≥0.05)|
|Fig. 1(a-d):|| |
Data of serum hormones of the experimental African catfish. Mean having different small letters are significantly different (p≤0.05) but mean having capital letter are not significantly different (p≥0.05)
In the present study, body measurements of female brood stock C. gariepinus revealed the heaviest (p≤0.05) body weight of T4 compared with T1 and T3 and the longest ovary length (p≤0.05) of T2, T3 and T4 compared with in T1 and T5 but there were no significant (p≥0.05) differences among treatments concerning body length, K-factor, ovaries weight, size and density and GSI. The non-significant differences may be attributed to the fact that the ovarian weight is usually a negligible fraction of the somatic (body) weight. De Graaf et al. (1995) found similar findings for C. gariepinus, using artificial propagation techniques. Similarly to the present findings, Sharaf (2005) noted no significant differences in GSI between C. gariepinus females as affected with single dose of synthetic GnRHs and hCG. Yet, earlier established linear relationship was found between fecundity, ovarian weight and length, GSI and somatic weight of C. gariepinus (Eyo and Mgbenka, 1992). This relationship is important in estimating fecundity from ovarian weight and length, GSI and somatic weight. In this respect, Nguenga et al. (2004) found that oocyte diameter and absolute fecundity increased with increasing the weight of Heterobranchus longifilis.
Egg quality measurement means specific parameters of egg, such as egg diameter, egg weight, total egg weight and total egg number per female (AF) or egg number per g female body weight (RF) to measure the quality of egg represented by fish treated with the tested materials, where egg quality is an important parameter for commercial fish hatcheries, as well as the quality of fry produced from a brood stock (Babin et al., 2007). The present findings concerning egg quality and fecundity showed significantly (p≤0.05) heavier total egg weight (g female-1) in T3 and T2 than in T1.
|Fig. 2(a-h):|| |
Transverse section in ovary of African catfish brood stock (stained with H and E),(a) T1 (control), showing primary yolk stage, N: nucleus, (b and c) Fish injected with Pregnyl® (T2) showing normal structure of ovarian lamellae, which contains oocytes at various stages of oogenesis, (c) Showing oocytes in mature stage, (d and e) Fish injected with carp pituitary (Argent, T3) showing oocytes in yolk globules (YG) stage, (e) Showing migratory nucleus (MN) stage, S: Spore, (f and g) Fish injected with carp pituitary (native,T4) showing normal structure of ovarian lamellae, which contains oocytes at various stages of oogenesis, (g) Showing oocytes in early yolk vesicles (EYV, arrows), stage, n: nucleolus and (h) Fish injected with catfish pituitary (native, T5) showing oocytes in late vitellogenic stage. Magnification, a, b and f (and 60); c, d, e, g and h (×120)
However, no significant (p≥0.05) differences in individual egg diameter and egg weight (mg egg-1) among all treatments were found. Data of both AF and RF revealed significant (p≤0.05) differences among treatments, whereas fish injected with experimental materials (T2, T3, T4 and T5) gave the highest AF and RF compared with the control group (T1). Similarly, in Heterobranchus bidorsalis no significant difference (p≥0.05) of egg diameter among all treatments was found (Nwokoye et al., 2007). So, Carillo et al. (1995) opined that egg diameter is not a good indicator of egg and larval quality. Increasing fecundity of C. gariepinus in this study could be attributed to the injection with native or commercial hormonal materials, which are capable of increasing the production of FSH, LH, E2 and P4, the key hormones involved in the production and maturation of eggs in the ovary. Appropriate hormonal therapies do not usually have a negative effect on egg quality (Haffray et al., 2005), whereas at times they can even improve fish fecundity compared to spontaneously maturing populations (Mikolajczyk et al., 2004).
Hormonal therapies have an important role in brood stock fish management and will continue to be a necessary tool even after fish become properly (Mylonas and Zohar, 2001). Hence, many studies confirmed the important role of gonadotropins hormone (GtHs), hCG or fish pituitary extracts injection to improve the reproductive performance of catfish sp., such as C. gariepinus due to hCG administration than CPE (Salami et al., 1994). Artificial spawning and high hatching rates (≥84%) were induced in Heterobranchus bidorsalis by single hormone injections of Carp Pituitary Suspensions (CPS), Homoplastic Pituitary Suspension (HPS) or hCG over 3 month (Adebayo and Fagbenro, 2004), as well as, they noted that using of HPS would save operational costs in fish hatchery management in African countries. Also, the highest fertilization (75-89%) and hatching (66-78%) could be obtained from the females of C. batrachus injected with at 3000-5000 IU doses of hCG with 14-17 h post- injection (Sahoo et al., 2007). Additionally, Sadek (2009) concluded that injection of Pregnyl® (hCG) have higher potent than Receptal® (GnRH) in the artificial propagation of C. gariepinus.
In contrary of the present findings, Brazilian catfish Pseudoplatystoma fasciatum treated with combined CPE/hCG was not influenced (Leonardo et al., 2004), as well as in yaqui catfish Ictalurus pricei, hCG, catfish pituitary extract and combined sGnRHa/DA treatments were ineffective (Mylonas and Zohar, 2001). Accordingly of these results, the efficacy of combinations of GtHs together with GnRHa, do not seem to be better compared to treatments using only one of the hormones for the induction of spawning in fish (Wen and Lin, 2004). Meanwhile, LH or hCG may be more appropriate in inducing oocyte maturation, ovulation and spawning (Garcia et al., 2001).
The secretion of gonadotropin in both pituitary and gonads might have important roles on sex steroids synthesis, undifferentiated gonad and in turn on sex differentiation through brain-pituitary-gonad axis (Wu et al., 2009). Data of serum hormones (FSH, LH, P4 and E2) cleared that there were significant (p≤0.05) differences among treatments for all tested hormones, except for E2. Treatment No. 2 reflected the highest level of blood FSH and LH but P4 level was the highest in each of T2, T3 and T5 without significant (p≥0.05) differences among these treatments which were significantly (p≤0.05) higher than T1 (control) and T4. In females, FSH is thought to be responsible for the regulation of vitellogenesis, while LH is responsible for oocyte maturation and ovulation (Yaron et al., 2003). Fish possess two GtHs similar to FSH and LH in other vertebrates. Furthermore, the presence of two distinct GtHRs in a single fish species was concermed by the molecular cloning of two different cDNAs in several fish species including different catfish sp. C. gariepinus (Vischer and Bogerd, 2003) and Icralurus puncrarus (Kumar et al., 2001). Based on the other scientific point of view, the exception of salmonids, there are no assays available to measure FSH, due to difficulties in producing anti-FSH antibodies from the very small FSH amounts contained in fish pituitaries (Yaron et al., 2003). Also, they added that the role of FSH during vitellogenesis was based only on measurements of FSH β-mRNA levels in the pituitary and not of the released protein in the blood (Yaron et al., 2003). However, as in the present findings, plasma FSH and LH were measured in C. gariepinus by Sayed et al. (2012).
Pituitary gonadotropins FSH (GTH-I) and LH (GTH-II) are key reproductive hormones that are involved in controlling gonadal development steroidogenesis and ovulation (Yadetie and Male, 2002). So, different pattern was illustrated for LH in C. gariepinus (Schulz et al., 1997) and black carp (Gur et al., 2000). In these species, LH levels are already detectable in juveniles, steadily increasing concomitant with gonadal growth and maturation. Swanson et al. (1991) found that plasma FSH levels remain high throughout vitellogenesis but decline later in the Coho salmon, being low during oocyte maturation and ovulation without showing the two-peak pattern as in trout. In this manner, Daghash and Hussein (1999) reported that an intramuscular injected dose of 20 μg GnRH kg-1 BW increased the serum total cholesterol (p≤0.05) and progesterone (p≤0.01) levels of treated C. gariepinus females.
Generally, from the economic point of view, a complementary study (Abdelhamid et al., 2010) related to the present study was conducted, where the produced fry from all treatments were used to alleviate the cannibalism phenomena among the African catfish, C. gariepinus fry via periodical grading to eliminate the jumpers fry. Since, the cannibalism phenomena are a major problem in the African catfish farming, which led to negative economic effects in the commercial hatcheries and in the aquaculture of this species. From the other side, concerning the human public health, usage of hCG hormone is safety, where approximately 80% of its level is metabolized, predominantly in the kidneys. Intramuscular and subcutaneous administrations of hCG were found to be bioequivalent regarding the extent of absorption and the apparent elimination half-lives of approximately 33 h (Chen et al., 1993).
Fish sexual maturity and gonadal development is associated with increased circulating levels of gonadotropins and the gonadal steroids (Huggard-Nelson et al., 2002). The histological characteristics of ovaries of experimental C. gariepinus brood stock revealed the presence of different development stages of oocytes. These observations in the present study were accordingly with those reported by West (1990). Whereas, major developmental events can be divided into six phases: Oogenesis, primary oocyte growth, cortical alveolar stage, vitellogenesis, maturation and ovulation (Tyler and Sumpter, 1996). Similar structures were reported by Hussein (1984). More histological description of different stages of fish oocytes development was reported by Selman and Wallace (1989). So, in the present study, results of C. gariepinus injected with tested hormonal materials revealed the superiority in development stages of oocytes compared with the control treatment. This superiority of injected materials in the present study may be due to mechanism to regulate fish fecundity and the physiological role of these injection hormones or fish pituitary extracts, concerning the significantly (p≤0.05) highest egg weight, absolute fecundity and relative fecundity, as well as blood hormones than the control treatment. In this trend, Miwa et al. (2001) reported that hCG stimulated 17, 20 β-P and 20 β-S production, resulting in final maturation of most mature oocytes, synchronously, in S. asotus. Meanwhile, Chowdhury et al. (2010) indicated a decrease in the number of the immature ovarian follicles, thus elucidating the rationale behind partial success of Ovaprim® induced spawning in the C. batrachus, this decrease in spawning efficiency may be attributed to a decrease in the expression of GnRH-receptor II in the catfish ovary, leading to a concomitant decrease in the development and maturation of primary follicles and subsequent ovulation.
From the aforementioned results, it could be concluded that T3 (injected with imported Argent®, carp pituitary gland) followed by T2 (injected with imported Pregnyl®) and T5 (injected with native catfish pituitary gland) were the best treatments for improving the reproductive performance of brood stock African catfish C. gariepinus compared with T1 (control, without injection) and T4 (injected with native carp pituitary gland). So, we recommend the useful usage of these materials in African catfish hatcheries, as well as further studies are needed by these materials at the commercial scale, which may be lead to the positive economic effects in the commercial hatcheries and fish farms not only for African catfish but also for fresh or marine water fish species also.
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