Male-specific DNA markers from African catfish (Clarias gariepinus)

We searched for sex-specific DNA sequences in the male and female genomes of African catfish, Clarias gariepinus (Burchell, 1822) by comparative random amplified polymorphic DNA (RAPD) assays performed on pooled DNA samples. Two sex-linked RAPD markers were identified from the male DNA pool and confirmed on individual samples, showing good agreement with phenotypic sex. Both markers were isolated, cloned and characterized. The first marker (CgaY1) was nearly 2.6 kb long, while the length of second one (CgaY2) was 458 bp. Southern blot analysis with a CgaY1 probe showed strong hybridizing fragments only in males and not in females under stringent conditions, indicating the presence of multiple copies of CgaY1 in the male genome. When tested by zoo blot on the genomes of two closely related species from the Clariidae family, CgaY1 hybridized to the DNA of Heterobranchus longifilis and generated a faint male-specific band at low stringency. CgaY2 produced similar hybridization pattern in both sexes of C. gariepinus, C. macrocephalus and H. longifilis. Specific primers were designed to the sequences and the markers were amplified in multiplex PCR reactions together with a control band common to all individuals. This allowed for rapid, molecular sexing of the species on the basis of a simple three band (male) versus one band (female) pattern. According to our knowledge these are the first sex-specific DNA markers isolated from a siluroid fish species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Free fatty acid metabolism in the air-breathing African catfish (Clarias gariepinus) during asphyxia

In several waterbreathing fish species, hypoxia induces a decrease in plasma free fatty acid (FFA) levels as opposed to an increase in air-breathing mammals. We hypothesised that this change is coupled to the mode of breathing. Therefore, we followed the metabolic response of cannulated air-breathing African catfish to an 8-h asphyxia period. The hematocrit and hemoglobin increased significantly upon asphyxia. However, no change was observed in the mean cellular hemoglobin concentration, indicating that more erythrocytes were brought into circulation. A continuous increase in plasma lactate concentration during asphyxia showed permanent activation of anaerobic glycolysis, pointing to a persistent oxygen shortage. Plasma glucose levels did not change, but FFA levels decreased significantly upon asphyxia with a concomitant increase in plasma noradrenaline levels. Thus, these results suggest that in the air-breathing African catfish noradrenaline mediated a decrease in plasma FFA levels similar to that in waterbreathing fish species.     

Evaluation of red seaweed Gracilaria arcuata as dietary ingredient in African catfish,Clarias gariepinus

The aim of this study was to evaluate the use of dried marine seaweed, Gracilaria arcuata for the first time as dietary ingredient in partial substitution of fishmeal on the growth performance, feed utilization and body composition of African catfish, Clarias gariepinus. Four experimental diets were formulated: D1 as a control group; D2; D3 and D4 which included 10%, 20% and 30% G. arcuata meal respectively. One hundred and eighty African catfish weighing 9.62 ± 0.42 g, (mean ± SE) was divided into four groups corresponding to the different feeding regimes. The final body weight of the fishes showed significant differences (P < 0.05) between the control (D1); D2 and other treated groups D3 and D4, with weights of 66.98, 59.60, 47.34 and 30.73 g recorded for D1, D2, D3 and D4, respectively. Significant differences (P < 0.05) were also evident in weight gain, specific growth rate, and feed utilization between treatment and control groups. However, no significant differences (P > 0.05) were observed between the control group and fishes fed D2 for all previous parameters. Protein productive value, protein efficiency ratio, daily dry feed intake and total feed intake were also significantly lower in fish fed with a diet containing G. arcuata than in the control group and D2 which contains 10% of G. arcuata. Overall, the results of the experiment revealed that African catfish fed a diet with G. arcuata included in 20% and 30% levels showed poorer growth and feed utilization than the control group and D2. However, the study recommended that C. gariepinus can accept this ingredient up to 10% in their diets. More defined experiments therefore seem to be necessary in order to determine the maximum level of this marine seaweed in diets with amino acid supplementation for African catfish.     

Dietary influences on product quality in African catfish (Clarias gariepinus)

In this trial a full-sib family of Clarias gariepinus was raised under standard conditions in a warmwater recirculation system. The fish were divided into two experimental groups of 1200 individuals each and were raised in separate tanks. The groups were fed two experimental diets differing in crude protein (group I 40% and group II 52% DM) and crude fat (group I 8% and group II 13% DM). Gross energy was 21.3 and 22.1 MJ/kg. Slaughtering of 100 fish per group (50 males, 50 females) took place after two experimental periods. Besides the significant effect of the diets on body weight and size, a higher degree of corpulence was observed in group II. Tissue and organ percentages were not influenced, except a higher intestinal fat deposition in intensively fed fish. This group also showed a higher carcass weight. More expressed differences between both feeding alternatives were observed in physical and chemical flesh quality criteria. In group I texture of flesh was firm and water-binding-capacity was significantly increased in group II. The appearance of the filet was affected by the diet. Intensively fed catfish showed a tendency to a lighter and more yellowish colour. The chemical composition of the flesh indicated a significant increase of dry matter caused by higher protein and fat contents in this group. A strong influence of sex and fattening age on inner and outer product quality was evident. High fat and protein content of the diet has a certain influence on product quality in African catfish. It can be concluded that there is a clear effect of diet on quantitative criteria with a tendency to higher slaughtering yields in intensively fed catfish.     

Healing of skin wounds in the African catfish Clarias gariepinus

The African catfish Clarias gariepinus was used as a model for wound healing and tissue regeneration in a scale-less fish. A temporal framework of histological and cell proliferation markers was established after wound induction in the dorsolateral cranial region, by removing the epidermal and dermal layers, including stratum adiposum (SA). Wound closure and epidermis formation was initiated within 3 h post-procedure (hpp) with migration and concomitant proliferation of epidermal cells from the wound borders. The wound was covered by this primary epidermal front 12 hpp and fusion of the opposing epidermal fronts occurred within 24 hpp. Attachment of the newly formed epidermal layer to the underlying dermis was observed 48 hpp concomitant with a second wave of cell proliferation at the wound edge. Normal epidermal thickness within the wound was achieved 72 hpp. Formation of a basement membrane occurred by 120 hpp with concomitant emergence of the SA from the wound borders. Wound healing in C. gariepinus skin involved closure of the wound and re-epithelization through cell migration with a single wave of early cell proliferation not documented in other species. Furthermore, covering of the wound by epithelium as well as the reappearance of the basement membrane and SA occurred sooner than in other fish species.     

Effects of oxytocin on semen release response in African catfish (Clarias gariepinus)

In silurid fishes, semen collection is practically impossible, even after hormonal stimulation. Instead, males are killed and testes macerated to obtain sperm. To understand the endocrine control of semen release in catfishes, we investigated the role of smooth muscle contractors in semen release and semen quality of African catfish (Clarias gariepinus). In in vitro experiments, testis slices were incubated with oxytocin (1 and 10 IU), isotocin (2 and 20 ug), vasopressin (0.2 and 2 ug), epinephrine (1 and 10 ug), PGF2alpha (1 and 10 ug), purified Clarias LH (300 ng) and partly purified Clarias pituitary extract (containing 300 ng LH). Only oxytocin increased sperm concentration of the medium (assessed by optical density measurements) compared to control incubations. Oxytocin was then tested in vivo in two groups of fish: normal males, and males that had been treated with 17alpha-methyltestosterone during larval stages to inhibit seminal vesicle development (MT males). Both groups of fish received two doses of carp pituitary suspension (8 and 10 mg/kg, respectively i.m.) with or without subsequent oxytocin treatment (5 IU/kg i.v.; cPS-OT treatment and cPS treatment, respectively). There was no effect of oxytocin on the number of strippable males. Of cPS and cPS-OT treated fish, 87% MT males and 60% normal males were strippable. The stripped semen volume was low in both groups but MT males produced higher (P < 0.001) hatching rates (63.1%) than did normal males (2.1%).     

The seminal vesicle of the African catfish,Clarias gariepinus

Summary The seminal vesicle of the African catfish, Clarias gariepinus, consists of 36–44 fingerlike lobes built up of tubules in which a fluid is secreted containing acid polysaccharides, acid-, neutral- and basic proteins, and phospholipids. In this fluid sperm cells are stored. The seminal vesicle fluid immobilizes the sperm cells. After ejaculation, it prolongs the period of sperm activity. The seminal vesicle fluid is secreted by the epithelium lining the tubules. The tubules in the proximal part of the lobes are predominantly lined by a simple cylindrical and those of the distal part by a simple squamous epithelium. These epithelial cells contain enzymes involved in energy-liberating processes, the enzyme activites being proportional to the height of the cells. Interstitial cells between the tubules have enzyme-histochemical and ultrastructural features indicative of steroid biosynthesis. Similar characteristics are found in testicular interstitial cells. The most rostral seminal vesicle lobes and the most caudal testicular efferent tubules form a network of tubules that opens at the point where the paired parts of the sperm ducts fuse with each other. The tubules of most seminal vesicle lobes, however, form a complex system that fuses with the unpaired part of the sperm duct.     

Testosterone inhibits 11-ketotestosterone-induced spermatogenesis in African catfish (Clarias gariepinus).

Male fish produce 11-ketotestosterone as a potent androgen in addition to testosterone. Previous experiments with juvenile African catfish (Clarias gariepinus) showed that 11-ketotestosterone, but not testosterone, stimulated spermatogenesis, whereas testosterone, but not 11-ketotestosterone, accelerated pituitary gonadotroph development. Here, we investigated the effects of combined treatment with these two types of androgens on pituitary gonadotroph and testis development. Immature fish were implanted for 2 wk with silastic pellets containing 11-ketotestosterone, testosterone, 5alpha-dihydrotestosterone, or estradiol-17beta; cotreatment groups received 11-ketotestosterone in combination with one of the other steroids. Testicular weight and pituitary LH content were higher (two- and fivefold, respectively) in the end control than in the start control group, reflecting the beginning of normal pubertal development. Treatment with testosterone or estradiol-17beta further increased the pituitary LH content four- to sixfold above the end control levels. This stimulatory effect on the pituitary LH content was not modulated by cotreatment with 11-ketotestosterone. However, the stimulatory effect of 11-ketotestosterone on testis growth and spermatogenesis was abolished by cotreatment with testosterone, but not by cotreatment with estradiol-17beta or 5alpha-dihydrotestosterone. Also, normal pubertal testis development was inhibited by prolonged (4 wk) treatment with testosterone. The inhibitory effect of testosterone may involve feedback effects on pituitary FSH and/or on FSH receptors in the testis. It appears that the balanced production of two types of androgens, and the control of their biological activities, are critical to the regulation of pubertal development in male African catfish.     

Development of sensory organs in larvae of African catfish Clarias gariepinus

African catfish Clarias gariepinus hatched with morphologically immature features; however, sensory organs developed rapidly with fish growth. Although the eyes of newly hatched larvae were immature without pigment, in 2 day‐old larvae, the retina of the eyes had already developed except for the rod cells. No free neuromasts were observed in newly hatched larvae. In 1 day‐old larvae, however, free neuromasts were observed on the head and trunk. Free neuromasts increased with larval growth. Newly hatched larvae had simple round‐shaped otic vesicles; however, all sensory epithelia of the inner ear were observed until the larvae were 3 days old. Two day‐old larvae swam horizontally, had sharp teeth, commenced ingesting rotifers and also artificial feed (small‐size pellets) under both light and dark conditions; by then the larvae already had many taste buds. Three day‐old larvae showed negative phototaxis and cannibalism by eating their conspecifics. Most of the free neuromasts observed in this study had the peculiar feature of many microvilli around the sensory cells on the apical surface. Detected free neuromasts as ordinary type lateral‐line organs were not observed in previous reports in teleosts. In 10 day‐old larvae, there were two lines of free neuromasts on the flank and lower edge of the trunk; presumptive canal neuromasts were oval shaped and had begun to sink under the skin. The direction of maximum sensitivity of the neuromasts was parallel with the longitudinal axis of their elliptical apical surface.     

Inhibitory and stimulatory interactions between endogenous gonadotropin-releasing hormones in the African catfish (Clarias gariepinus)

In the brain of all vertebrate classes, chicken (c) GnRH-II ([His(5), Trp(7),Tyr(8)]GnRH, cGnRH-II) is expressed in the mesencephalon. In addition, at least one other form of GnRH is expressed in the preoptical area/hypothalamus. In the human pituitary stalk and the mouse median eminence, cGnRH-II is present together with mammalian GnRH. Similarly, in the pituitary of several teleost fish (e.g., goldfish and eel, but not salmon or trout), a teleost GnRH is found together with cGnRH-II. These GnRHs are not colocalized in the same cells. Hence, these GnRH peptides may differentially regulate gonadotropin secretion and, in addition, may exert their effects simultaneously. The current study therefore investigated the effects of combinations of the two forms of GnRH present in the African catfish (Clarias gariepinus) pituitary-cGnRH-II and catfish GnRH ([His(5),Asn(8)]GnRH, cfGnRH)-on the cytosolic free calcium concentration ([Ca(2+)](i)) in single, Fura-2-loaded catfish gonadotrophs, as well as their effects on both in vitro and in vivo LH secretion. Both inhibitory and stimulatory effects of combinations of cfGnRH and cGnRH-II on [Ca(2+)](i) were observed, which were mirrored by their effects on both in vitro and in vivo LH secretion. The following pattern became apparent. The effect of intermediate or maximal effective cfGnRH doses was inhibited by the simultaneous presence of subthreshold or borderline effective cGnRH-II doses. Conversely, subthreshold or borderline effective concentrations of cfGnRH enhanced the effects of intermediate and maximal concentrations of cGnRH-II. In addition, combinations of cfGnRH and cGnRH-II concentrations that were equally active when tested separately showed an additive effect. The observed interactions between the two GnRHs may be of particular physiological relevance in the control of seasonal LH levels in the African catfish, as well as in other teleost species. Moreover, the occurrence of mutual inhibitory and stimulatory interactions between endogenous GnRHs may be a widespread aspect of GnRH action in vertebrates.     

Clock-controlled endogenous melatonin rhythms in Nile tilapia (Oreochromis niloticus niloticus) and African catfish (Clarias gariepinus)

The purpose of this work was to investigate the circadian melatonin system in two tropical teleost species characterized by different behavioral habits, Nile tilapia (diurnal) and African catfish (nocturnal). To do so, fish were subjected to either a control photoperiod (12L:12D), continuous light (LL) or darkness (DD), or a 6L:6D photoperiod. Under 12L:12D, plasma melatonin levels were typically low during the photophase and high during the scotophase in both species. Interestingly, in both species, melatonin levels significantly decreased prior to the onset of light, which in catfish reached similar basal levels to those during the day, demonstrating that melatonin production can anticipate photic changes probably through circadian clocks. Further evidence for the existence of such pacemaker activity was obtained when fish were exposed to DD, as a strong circadian melatonin rhythm was maintained. Such an endogenous rhythm was sustained for at least 18 days in Nile tilapia. A similar rhythm was shown in catfish, although DD was only tested for four days. Under LL, the results confirmed the inhibitory effect of light on melatonin synthesis already reported in other species. Finally, when acclimatized to a short photo-cycle (6L:6D), no endogenous melatonin rhythm was observed in tilapia under DD, with melatonin levels remaining high. This could suggest that the circadian clocks cannot entrain to such a short photocycle. Additional research is clearly needed to further characterize the circadian axis in teleost species, identify and localize the circadian clocks, and better understand the environmental entrainment of fish physiology.     

Effects of 4-nonylphenol on blood cells of the African catfish Clarias gariepinus (Burchell, 1822)

In the present work, the destructive effects of the 4-nonylphenol on one of the most economically important Nile fishes, namely African catfish (Clarias gariepinus) were studied. Apoptosis, erythrocytes alterations, micronucleus test and blood parameters count were used as biological indicators to detect those effects. After exposure to sublethal concentrations of 4-nonylphenol (0, 0.05, 0.08 and 0.1 mg/l), apoptotic red blood cells with many malformations and micronucleated erythrocytes were recorded. Decrease in the blood parameters such as red blood cells (RBCs), hemoglobin (Hb), package cell volume (PCV), mean corpuscular hemoglobin concentration (MCHC), platelets, white blood cells (WBCs), lymphocytes, basophils, monocytes and increase in mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), neutrophils, eosinophils indicated the negative effects of 4-nonylphenol. It was concluded that, the 4-nonylphenol caused genotoxicity in erythrocytes with many malformations in shape and number indicated with other blood parameters.     

Gonadotropin-releasing hormone-immunoreactive neuronal structures in the brain and pituitary of the African catfish,Clarias gariepinus (Burchell)

Summary The distribution of gonadotropin-releasing hormone (GnRH) immunoreactivity was studied in the African catfish, Clarias gariepinus, by means of immunofluorescence and immunoperoxidase techniques. Immunoreactive neurons were found throughout the preoptic nucleus (NPO). However, only a portion of the secretory perikarya in the NPO showed a positive reaction by use of an anti-LHRH serum. Numerous immunoreactive fibres were found to enter the pituitary and to terminate in its proximal pars distalis, the site of concentration of the gonadotropic cells. Since GnRH is present in the brain and pituitary of the African catfish, the lack of spontaneous ovulation in captivity is apparently due to an insufficient release of GnRH.     

Novel function of recombinant pituitary adenylate cyclase-activating polypeptide as stimulator of innate immunity in African catfish (Clarias gariepinus) fry

There are several studies that clearly indicate a close bidirectional communication between the neuroendocrine and immune systems. In this sense, hypothalamic releasing hormones, besides their neuroendocrine role, have been shown to influence immune functions. Despite studies developed in mammals, there is, as yet, no information available about the role of the pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP-related peptide (PRP) in the fish innate immune system. The present study has evaluated the effect of PACAP and PRP administered by bath immersion, on important parameters of innate immunity and antioxidant defenses in African catfish (Clarias gariepinus) fry. We have shown, for the first time, that administration of recombinant C. gariepinus PACAP not only promotes growth but also increases lysozyme, nitric oxide synthase-derived metabolites and antioxidant defenses in treated fry. From our results, PACAP appears to act as a regulator of the teleostean immune system, in addition to its physiological role in controlling growth of fish.     

Temperature-induced changes of early development and yolk utilization in the African catfish Clarias gariepinus

Artificially fertilized eggs and yolk-sac larvae of a freshwater tropical/subtropical fish Clarias gariepinus receiving no external food were incubated at 22, 25 and 28° C until full yolk resorption. Developmental time, size and matter composition (CHNS-O Analyzer and ashing) were assessed at egg fertilization, hatching and yolk resorption; respiration was measured every 4–5 h. The course of acceleration of C. gariepinus embryonic developmental rate with temperature ( Q_{10 dev}) was compared over the temperature range to those of Cyprinus carpio and Oncorhynchus mykiss ; they differed greatly, but were similar when compared on the basis of effective temperatures specific to each fish. Specific growth rates for energy (88, 150 and 183% per day at 22,25 and 28° C, respectively) as well as the conversion efficiencies of egg energy (64, 71 and 68%, respectively) and protein (71, 78 and 76%, respectively) in C. gariepinus larval tissues were higher than those known for the endogenous feeding period of coldwater and temperate fish species. In C. gariepinus at the end of yolk resorption, the carbon percentage and caloric values of dry weight, size (in terms of dry matter, minerals, protein and energy per larva) and transformation efficiencies were lowest at 22° C, highest at 25° C and had slightly decreased at 28° C. A tentative mechanism which leads to the positive or negative response of body size to temperature over the viable temperature range is defined.     

Growth response of African catfish, Clarias gariepinus (Burchell) to varied protein and lipid levels

To study the growth response of African catfish (Clarias gariepinus) to the diets containing two protein (30 and 35%) and two lipid levels (12 and 18%) in a closed recirculatory system for 60 days, fish were fed to satiation twice daily. The experiment was carried out in triplicate groups. Fortnightly sampling of fish and water samples was carried out. The best result in terms of growth was obtained with the diet containing 34.62% protein and 12.12% lipid. The protein to lipid ratio was 18.57 in the diet that gave best percentage weight gain. However best survival, food conversion ratio and protein efficiency ratio were recorded in the diet containing 29.86% protein and 18.08% lipid.     

Seminoma in hybrid catfish [Clarias batrachus (Linnaeus) female x Clarias gariepinus (Burchell) male

Spontaneous testicular tumors, seminoma, were noticed in four male hybrid catfish (C. batrachus female x C. gariepinus male) after the age of two years. The hybrids showed massive abdominal swelling with catchectic body and free lobulated, encapsulated tumors (> 325 g) within the serosanguinous fluid-filled peritoneal cavities. The tumor cells were large and polyhedral with prominent centrally located nuclei. Other vital organs appeared normal. It seems to be the first report of seminoma in hybrid catfish and possibly of genetic cause.