Steroidogenesis during induced oocyte maturation and ovulation in the African catfish,Clarias gariepinus

Changes in ovarian steroidogenesis accompanying oocyte maturation and ovulation were studied in the African catfish,Clarias gariepinus. Laboratory-reared females with postvitellogenic ovaries were treated with pimozide and LHRH-analogue. The plasma gonadotropin levels were determined by means of a homologous radioimmunoassay, the condition of the ovaries was studied by histological examination of the follicles, and the steroidogenetic capacity of the ovaries was analyzed byin vitro incubation of tissue fragments for 3 h with [³H]-pregnenolone and [³H]androstenedione as precursors. Data were collected at regular intervals between 0 and 16 h after pimozide-LHRH analogue administration.Until 4 h after the beginning of the experiments the plasma gonadotropin levels did not rise above 25 ng/ml, the ovaries remained in the stage of postvitellogenesis, and testosterone was the main end product of steroidogenesis. Four hours later the gonadotropin concentration in the blood had risen to more than 150 ng/ml, and the ovaries had entered the stage of germinal vesicle migration. At the same time steroidogenesis shifted towards the production of 17,20-dihydroxy-4-pregnen-3-one, 5-pregnane-3, 17-diol-20-one, 5-pregnane-3,6,17-triol-20-one, 5-pregnane-3,17,20-triol and 5-pregnane-3,6,17,20-tetrol. During the subsequent stage of germinal vesicle breakdown the plasma gonadotropin level remained high, and the synthesis of the C₂₁-steroids showed a further increase. Simultaneously, the production of some C₁₉-steroid glucuronides was enhanced. The preovulation and especially the postovulation stages were accompanied by a gradual decrease in steroidogenic capacity of the ovaries, even though the plasma gonadotropin level remained high. It is concluded that the prematuration surge of gonadotropin influences the activity of enzymes involved in steroidogenesis, leading to a reduced C_17,20-lyase and to an augmented activity of the enzymes 20-hydroxysteroid dehydrogenase (HSD), 5-reductase, 3-HSD, 6-hydroxylase and UDP-glucuronosyltransferase. During ovulation the activity of all steroidogenic enzymes, including such key enzymes as 3-HSD and 17-hydroxylase, gradually decreases.Not only 17,20-dihydroxy-4-pregnen-3-one, but also the 5-reduced pregnanes may be involved in inducing oocyte maturation and/or ovulation. The very polar triol and tetrol products may function, together with the steroid glucuronides as sex pheromones.A preliminary account of these results was presented at the XIII Conference of European Comparative Endocrinologists, Belgrade, September 7–12, 1986     

Steroid hormone secretion by testicular tissue from African catfish,Clarias gariepinus,in primary culture: identification and quantification by gas chromatography — mass spectrometry

With the final aim of identifying the testicular steroids involved in the feedback mechanism of the hypothalamus-pituitary-gonad axis, steroid secretion by the testis of African catfish, Clarias gariepinus, was studied in vitro, by means of gas chromatography followed by mass spectrometry. Testicular fragments of sexually mature catfish raised in captivity were incubated in L-15 medium with and without catfish pituitary extract (cfPE). Without adding cfPE, 22 steroids could be identified, amongst which 11β-hydroxyandrostenedione, 11β-hydroxytestosterone, 11-ketotestosterone and 11-ketoandrostenedione were dominating. After incubation in the presence of cfPE, the concentrations of the four 11-oxygenated steroids were increased about 4-fold. The amounts of pregnane derivatives in the incubation medium showed the largest increases in the presence of cfPE. 5β-Pregnane-triol levels, for example, were 60-fold higher than in the medium from control incubations. The secretion of 5α- and 5β-androstanes was also stimulated by cfPE. The stimulation was not equal for all steroids, indicating that cfPE not only stimulates total steroidogenesis by increasing the availability of cholesterol, but also by influencing specific steroid converting enzyme activities. Part of this work was presented at the IV^th International Symposium on the Reproductive Physiology of Fish, 7–12 July 1991, Norwich, U.K.     

Steroids and steroid glucuronides in the ovarian fluid of the African catfish,Clarias gariepinus,between ovulation and oviposition

As part of a series of experiments concerning a possible pheromonal function of steroids and steroid glucuronides excreted by the sex organs of the African catfish,Clarias gariepinus, qualitative and quantitative studies, using GCMS, were carried out to examine the presence of the steroids, that can be synthesized by the ovary during oocyte maturation and ovulation, and of the corresponding steroid glucuronides, in the fluid surrounding the eggs in the ovarian cavity shortly after ovulation.Full mass spectra were obtained of 5-pregnane-3,17-diol-20-one, 5-pregnane-3,17,20-triol, 5-pregnane-3,6,17-triol-20-one, 5-pregnane-3,6,17,20-tetrol, 5-androstane-3,17-diol and 5-androstane-3,17-diol-11-one. After selected ion monitoring the following steroids could be detected by the presence of at least two characteristic ions at the expected retention time: 5-pregnane-3, 17,20-triol, etiocholanolone, 5-dihydrotestosterone, 5-androstane-3,11-diol-17-one, testosterone and estradiol. After treatment with -glucuronidase the following steroids could be determined in a similar way: 5-pregnane-3,17-diol-20-one, 5-pregnane-3,17,20-triol, 5-pregnane-3,17,20-triol, 5-pregnane-3,6,17-triol-20-one, 5-pregnane,3,6,17,20-tetrol, 5-androstane-3,17-diol, etiocholanolone, 5-dihydrotestosterone, testosterone and estradiol.The free steroids 5-pregnane-3,6,17,20-tetrol and 5-pregnane-3,6,17-triol-20-one and the steroid glucuronides of testosterone, 5-dihydrotestosterone and estradiol appeared to be the most abundant of these compounds. The results indicate that very polar steroids and steroid glucuronides, synthesized in the ovary, can be excreted via the ovarian fluid shortly before and during oviposition, and possibly function as sex attractants, inducing reproductive behaviour in male conspecifics.     

Sertoli cell proliferation and FSH signalling in African catfish, Clarias gariepinus

Sertoli cell proliferation occurs mainly during the phase of rapid spermatogonial proliferation, allowing the cyst-forming Sertoli cells to form an increasingly large space for housing the growing germ cell clone. There is no information in fish on the regulation of Sertoli cell proliferation; follicle-stimulating hormone (FSH) stimulates Sertoli cell proliferation in mammals. Increasing or decreasing FSH and FSH receptor expression experimentally in male African catfish was associated with respective changes in Sertoli cell proliferation or testis growth, suggesting that also in fish, one role of FSH may be to regulate Sertoli cell numbers.     

Mismatch between patterns of circulating and testicular androgens in African catfish, Clarias gariepinus

11-Ketotestosterone (11-KT) is an important plasma androgen in male African catfish. The quantitatively predominating androgen produced by the testis, however, is 11-hydroxyandrostenedione (OHA). Our working hypothesis to explain this mismatch assumed that OHA is converted to 11-KT at extratesticular sites. First, we examined the in vivo metabolism of [³H]-OHA in mature males after sham-operation or removal of either the testes (TC), the seminal vesicles (SVC), or both (TSVC) by analysing the pattern of circulating [³H]-androgens two hours after intravenous injection of [³H]-OHA. [³H]-OHA was converted to [³H]-11-KT to the same extent in all groups, indicating that neither ablation of testes nor of seminal vesicles, or both attenuates this conversion. We then examined the in vitro metabolism of [³H]-OHA by several types of tissues. Liver and seminal vesicle tissue were found to produce significant amounts of [³H]-11-KT. The conversion capacity in vivo was assessed by injecting TSVC-castrated males with increasing doses of radioinert OHA, followed by the quantification of OHA and 11-KT plasma levels. Saturation of the conversion capacity was not reached but the 11-KT production capacity is at least 80 ng per ml of plasma per hour. Moreover, liver fragments were tested for their OHA to 11-KT conversion capacity in vitro using increasing concentrations of radioinert OHA. The 11-KT producing increased with time and OHA concentration. The production rate was 90 pg 11-KT mg^-1 liver h^-1. Considering the results of the surgical experiments and the fact that the total hepatic mass by far exceeds that of the seminal vesicles, we conclude that the hepatic conversion is of primary relevance in vivo.     

In vivo activity of native GnRHs and their analogues on ovulation in the African catfish, Clarias gariepinus (Burchell)

Four distinct forms of native gonadotropin‐releasing hormone (GnRH) and two newly designed analogues were tested for their in vivo activity to induce ovulation in African catfish. The effects of these peptides on ovulatory parameters were compared with those of carp pituitary and [d ‐Ala⁶, Pro⁹‐NEt]‐mammalian GnRH analogue (mGnRHa), two tested ovulation‐inducing agents in African catfish. Assessment of ovulation was carried out by determining the ovulation ratio and the relative quantity of egg produced. From the results of the experiments, the order of potency of the native GnRH peptides is summarized as chicken GnRH‐II (cGnRH‐II) >salmon GnRH (sGnRH) >mammalian GnRH >chicken GnRH‐I (cGnRH‐I). Chicken GnRH‐II was as potent as mGnRHa while cGnRH‐I was totally ineffective. The new d ‐Orn⁶‐cGnRH‐II and d ‐Orn⁶‐sGnRH with a substitution at position 6 with d ‐isomer residue were as potent as the most extensively used mGnRHa, indicating that the position 6 modification might be more crucial than the substitution at the C‐terminal. On the basis of our results, the potential use and incorporation of cGnRH‐II and sGnRH for the development of more generic spawning induction therapies are suggested.     

Optimum histidine requirement of fry African catfish, Clarias gariepinus (Burchell)

Dietary histidine requirement of fry African catfish, Clarias gariepinus (2.57 ± 0.02 cm; 0.22 ± 0.03 g) was quantified by feeding casein–gelatin-based isonitrogenous (40% crude protein) and isocaloric (17.90 kJ g⁻¹ gross energy) amino acid test diets with graded levels of histidine (0.25%, 0.30%, 0.35%, 0.40%, 0.45% and 0.50% dry diet) in eighteen 80 L indoor circular aqua-coloured troughs provided with the flow-through system for 12 weeks. Maximum absolute weight gain (2.66), best feed conversion ratio (1.29), highest protein efficiency ratio (1.94), protein retention efficiency (34%) and energy retention efficiency (70.4%) were achieved at 0.40% dietary histidine. Broken-line and non-linear regression models were adopted to assess dietary histidine requirement for C. gariepinus . When analysed using broken-line regression model these parameters were also best at 0.40% dietary histidine corresponding to 1.0% protein, respectively, whereas using second-degree polynomial regression analysis, histidine requirement was obtained at 0.42%, 0.41%, 0.40%, 0.41% and 0.41% of dry diet, corresponding to 1.05%, 1.02%, 1.0%, 1.02% and 1.02% protein respectively. Based on the broken-line and second-degree polynomial regression analyses of the growth and nutrient retention data, optimum histidine requirement of fry C. gariepinus was found to be in the range of 0.40–0.42% dry diet, corresponding to 1.0–1.05% of dietary protein.     

Catecholestrogens inhibit dopamine methylation in the gonadotrops of the African catfish,Clarias gariepinus

Isolated gonadotrops of the African catfish,Clarias gariepinus, were incubated with dopamine (DA) and/or catecholestrone and the activity of the enzyme catechol-O-methyltransferase (COMT) was determined by measuring the methylated products. From the apparent Km values for DA and catecholestrone of 0.4–1.3 M and 17.9–25.2 M respectively, it was concluded that catecholestrone is a better substrate for the enzyme COMT, compared to DA. Moreover, the methylation of DA is inhibited by comparatively low concentrations of catecholestrone.     

Acute toxicity of inorganic fertilizers to African catfish, Clarias gariepinus (Teugals)

Abstract. Acute toxicity of Ca(OH)₂, NPK, Na₃PO₄ 12H₂O and NaNO₃ fertilizers on Clarias gariepinus (Teugals) was investigated in glass aquaria. Results showed that Ca(OH)3 was the most toxic of the four fertilizers. Na3PO4.12H2O and NaNO3 were less toxic. The 96-h LC50 for the fertilizers ranged from 33.9 mg/l for Ca(OH)2 to 1258.9 mg/1 for NaNO3. It is concluded that secondary application of these fertilizers in stocked ponds should be avoided.     

Effect of microalga-based diet on oxidative stress enzymes of African catfish,Clarias gariepinus

Here an indigenously isolated microalgal strain Ascochloris spp. cultivated in synthetic medium was evaluated as an aquaculture feed supplement. The daily dietary supplement includes microalgal feed (AF) and commercial diet feed (CF) (as control), respectively. These diets were fed separately to the juvenile Clarias gariepinus fishes (n = 4) under controlled conditions for an experimental period of 100 days. The protein, glycogen and lipid contents in the muscle extracts were found to be marginally higher in fishes that were fed with CF than AF diet. Similarly, CF fishes showed significantly higher glutathione-s-transferase, catalase, superoxide dismutase, and lipid peroxidase activities, except glutathione content. Zero mortality of the fishes with no significant difference in the overall body mass with the two dietary supplements strongly suggests that algal biomass could supplement the requisite nutrients for their metabolic activities. This preliminary investigation helps in exploring algal biomass as a potential alternative feed additive in the aquaculture industry.     

Pubertal development of male African catfish,Clarias gariepinus. In vitro steroidogenesis by testis and interrenal tissue and plasma levels of sexual steroids

In fish, sex steroids initiate and/or accelerate the maturation of the brain-pituitary-gonad axis. In order to obtain information on the steroid milieu during the pubertal development of male African catfish, we have monitored the conversion of [³H]-pregnenolone and [³H]-androstenedione by testis and [³H]-pregnenolone by interrenal tissue fragmentsin vitro. Pubertal development occurs between two and six months of age. Testicular development proceeds through four main stages that are characterised histologically by the presence of spermatogonia (stage I), spermatogonia and spermatocytes (stage II), spermatogonia, spermatocytes and spermatids (stage III), and all germ cells including spermatozoa (stage IV). 11β-Hydroxyandrostenedione and cortisol were the main products of testes and interrenal tissue, respectively, in all stages of the pubertal development; a change in the steroidogenic pattern was not observed during this period. The interrenal tissue displayed no significant conversion of [³H]-pregnenolone to 11-oxygenated androgens. Blood plasma was analyzed for the presence of five androgens; testosterone, 11β-hydroxytestosterone, 11β-hydroxyandrostenedione, androstenetrione, and 11-ketotestosterone. 11-Ketotestosterone was the quantitatively dominating androgen in the circulation at all stages of development, which was more pronounced in stages III and IV. The obvious differences between thein vitro andin vivo results, namely 11β-hydroxyandrostenedione being the main testicular productvs. 11-ketotestosterone dominating in the blood, may indicate that 11β-hydroxyandrostenedione is converted to 11-ketotestosterone at extratesticular sites.     

GTH-cells in the pituitary of the African catfish,Clarias gariepinus,during gonadal maturation: an immuno-electron microscopical study

In an ultrastructural immunocytochemical study we investigated the development of the gonadotropic cells in the pituitary of two to six months old male African catfish in relation to testicular development. In this period, pituitary and testicular tissue samples were collected on five occasions (groups I–V). Blood samples could only be taken from the fish in groups III–V. The testicular development was divided in three stages i.e., immature (only spermatogonia, group I), early (spermatogonia and spermatocytes, groups II and III) and advanced (all germ cell stages including spermatozoa, groups IV and V) spermatogenesis. 11-Ketotestosterone blood levels were low, except for the last group. Antisera were raised against the complete catfish α,βGTH-II, as well as to the separate α- and β-subunits of catfish GTH-II. In the proximal pars distalis of immature fish, undifferentiated cells, somatotrops, putative thyrotrops (pTSH) and putative gonadotrops (pGTH) were found. In the two latter, secretory granules were labeled with anti-αGTH, but not with anti-βGTH-II. pTSH- and pGTH-cells were distinguished on the basis of the size of their secretory granules. During early spermatogenesis, two classes of putative gonadotrops could be distinguished. One type had the same immunocytochemical and ultrastructural characteristics as in immature fish; the secretory granules in the second cell type, which was more abundant, were also immunopositive for anti-βGTH-II. The mean volume of the secretory granules in these GTH-II cells was three times larger than that in the early appearing pGTH-cells. In addition, the later appearing GTH-II cells contained large inclusions, known as globules. These structures labeled with anti-αβGTH-II and with anti-βGTH-II, but not with anti-αGTH. It is assumed that the globules are involved in a differential storage and/or breakdown of the GTH-II subunits. During advanced spermatogenesis the two gonadotropic cell types could still be distinguished, but the early appearing pGTH-cell type was scarce. The present observations permit the conclusion that the early appearing cells may be GTH-I cells. However, definitive proof about their identity depends on the availability of antibodies or cDNA probes specific for GTH-I.     

Nicotinic acid supplementation of diets for the African catfish, Clarias gariepinus (Burchell)

Semi-purified diets containing 39% crude protein and 5% lipid were used to identify the qualitative requirement of African cattish, Clarias gariepinus (Burchell), for niacin and to characterize the pathologies associated with a deficiency of this vitamin. After 48 days of feeding, C. gariepinus supplied with the unsupplemented diet had developed severe deficiency symptoms and were subsequently withdrawn from the growth study. Niacin deficiency was characterized by feed refusal, listlessness, weight loss, poor feed utilization and high mortality. The skin overlaying the lateral line of the deficient fish became haemorrhagic and this clinical sign was accompanied by severe anaemia. After 126 days of feeding, fish fed diets containing 17.0 mg niacin kg^?1 had also developed a dermopathy. but without anaemia or high mortality. The feeding of diets containing less than 33.1 mg niacin kg^?1 resulted in suboptimal feed efficiency and poor protein utilisation. Allometric analysis of proximate composition indicated that carcass moisture, protein and ash were influenced by fish size, and not by dietary niacin content. However, significantly more lipid per unit of weight gain was deposited in the carcasses of fish fed the unsupplemented diet than in fish fed diets containing 17.0mgkg^?1. The indicators used in the present study could not be applied to accurately determine a value for niacin requirement. However, until a more accurate assessment is performed, it is recommended that diets for C. gariepinus contain not less than 33.1 mg nicotinic acid kg^?1 feed.     

Effect of extenders on sperm cryopreservation of African catfish, Clarias gariepinus (Burchell)

The effect of extenders was studied on the cryopreservation of sperm from African catfish, Clarias gariepinus (Burchell). The following six basic extenders were tested: fructose, glucose, sucrose, NaCl, KCl solutions and the artificial seminal plasma of the African catfish. Each of these extenders was tested both with and without buffer systems (i.e. NaHCO₃-CO₂ and Tris-HCl) by using 10% dimethyl-sulphoxide (DMSO) as a cryoprotectant. The two-step freezing was carried out in a programmable freezer by using the following freezing rates: (1) 4 °C min^–1 between 3 and –4 °C; (2) and 11 °C min^–1 between –4 and –80 °C. The best post-thaw motility (25%) was achieved with 333 mmol L^–1 fructose solution and NaHCO₃ buffer. The fertilization experiments were carried out with unbuffered fructose and glucose extenders using various amounts of sperm and two fertilization methods: (1) dry and (2) wet. The best fertilization rates were achieved with 75 μL of sperm and wet fertilization with glucose extender, or 100 μL of sperm and dry fertilization in case of fructose – both methods fertilized 96% of all eggs.     

Controlled hatchery production of African catfish, Clarias gariepinus (Burchell): an overview

Abstract In order to promote the successful supply and satisfactory survival of African catfish, Clarias gariepinus (Burchell), it is advocated that the fish be reared in hatcheries until they begin air breathing. Research conducted over a number of years to develop culture technology for intensive hatchery rearing to the stage of air breathing is brought together here as an overview. In particular, the definition of early development stages is considered as well as induced spawning, egg incubation, larval rearing and fry rearing.     

Amino acid profiles and amino acid utilization in larval African catfish (Clarias gariepinus): effects of ontogeny and temperature

The present study investigated the qualitative amino acid (AA) requirements of larval African catfish Clarias gariepinus. Yolk-sac larval AA profiles were measured at different temperatures and also in animals reared at 28 °C fed Artemia nauplii or an experimental dry diet. The AA profile of C. gariepinus larvae changed during ontogeny, especially before the start of exogenous feeding. The AA profiles of the food items (yolk, Artemia and the dry diet) differed considerably from that of the larvae. No selective absorption of yolk AA was detected. Higher temperatures led to increased absorption and depletion rates of AA, and also to a higher retention efficiency of yolk nutrients. However, changes in temperature did not induce preferential absorption or depletion of individual AA, and caused only small variations in the AA profile. Depletion rates of individual AAs varied, possibly due to differences between larval and yolk AA profiles, and also to changes in the larval AA profile during ontogeny. There was little regulation of catabolism of individual AA in yolk-sac and starved larvae, and no sparing of essential AA.     

Inherent variation in growth efficiency of African catfish Clarias gariepinus (Burchell, 1822) juveniles

Understanding the major causes of growth variation is crucial for the success of fish farming since its reduction contributes to maximize production efficiency, reduce food waste and improve water quality. The growth variation observed in aquaculture has been associated with the establishment of social hierarchies. However, some studies suggest that this variation may not be mainly a consequence of social hierarchies but mainly a result of inherent (genetic) differences. This study investigates the magnitude of individual responses, independently of group effects (fish housed individually), in growth efficiency and feeding behaviour of African catfish Clarias gariepinus (Burchell 1822). Despite the low variation in initial body weight (6.5%) and cumulative feed consumption (7.5%) over the experimental period, catfish exhibited high variation in final body weight (18.1%), specific growth rate (17.2%) and feed conversion ratio (27.9%), suggesting that individual variation in growth efficiency is important in determining growth rate. This individual variation may be related with individual differences in protein/fat deposition since faster growing fish deposited more protein and less fat than slower growing fish. Pronounced individual differences in feeding behaviour (reaction towards feed and time spent eating) were also observed and correlated with individual differences in growth efficiency. Fast eaters were the fast growers. We suggest that the growth variation observed in African catfish may be inherent and that the use of grading to increase uniformity should be further investigated.     

Characterization of the receptor for gonadotropin-releasing hormone in the pituitary of the African catfish,Clarias gariepinus

Receptors for gonadotropin-releasing hormone (GnRH) were characterized using a radioligand prepared from a superactive analog of salmon GnRH (sGnRH), D-Arg⁶-Pro⁹-sGnRH-NEt (sGnRHa). Binding of¹²⁵I-sGnRHa to catfish pituitary membrane fractions reached equilibrium after 2 h incubation at 4°C. Displacement experiments with several GnRH analogs as well as other peptides, demonstrated the specificity of¹²⁵I-sGnRHa binding. Specific binding was enhanced in the presence of the cation chelator ethylene bis (oxyethylenenitrilo) tetra-acetic acid (EGTA), indicating an inhibitory effect of cations on GnRH-receptor binding. The binding of¹²⁵I-sGnRHa to pituitary membranes was found to be saturable at radioligand concentrations of 5 nM and above. A Scatchard analysis of the saturation data suggested the presence of a single class of high-affinity binding sites (Ka=0.901±0.06×10⁹M^–1, Bmax=1678±150 fmol/mg protein). A comparative study on¹²⁵I-sGnRHa binding to pituitary membrane fractions of male and female catfish, indicated that there were no differences in binding affinity and binding capacity between both sexes. The results demonstrate the presence of specific, saturable GnRH receptors in the African catfish pituitary.