In nematodes, the structure of male copulatory organs is a significant taxonomic distinguisher and includes the morphometry of the spicules. The description of these structures mainly relies on the study of whole mounts using light microscopy. In rare instances, protruding spicules have been described with scanning electron microscopy. Even fewer studies have described the ultrastructure of isolated spicules following their isolation. In the present study, two different methods of spicule isolation were performed on two parasitic camallanid nematodes, Procamallanus (Procamallanus) pseudolaeviconchus Moravec & van As, 2015 and Paracamallanus cyathopharynx (Baylis, 1923), from African sharptooth catfish to determine the practicality and efficiency of the methodologies. The first method involved using sharpened tungsten needles and microdissection of the spicule pouch to free the spicules, followed by soft tissue digestion if necessary. Alternatively, the spicules were isolated through mechanical release instead of dissection in a method developed in the current study. This involved freeing the spicules from surrounding soft tissue by placing live specimens between a coverslip and a glass slide in a drop of water and exerting pressure with small rotational movements. Both methods yielded favourable results, but Method 2 is recommended for future studies due to the many advantages.

The objective was to evaluate the welfare implications of electrical stunning prior to gill-cutting of farmed African catfish as an alternative to live chilling in combination with gutting. Electroencephalogram (EEG) and electrocardiogram (ECG) recordings, in combination with observation of behaviour and responses to noxious stimuli, were used to assess brain and cardiac function in African catfish (body-weight 1571 ± 362 g [mean ± standard deviation], 32 males and 26 females). In the first experiment, the minimum electrical current required to induce a general epileptiform insult by head-only stunning was determined. The individual catfish were fixed in a specially designed restrainer, and applied voltages of 150 V, 200 V, 250 V, 300 V or 350 V (50 Hz AC) were delivered via scissor-model stunning tongs for approximately I s. A general epileptiform insult was observed in 31 fish, for which a successful EEG recording was obtained using 362 ± 32 V, 629 ± 180 mA for 1.2 s. The durations of the tonic, the clonic and the exhaustion phases were 8 ± 3 s, 12 ± 7 s and 7 ± 5 s as measured by EEG, respectively; a distinct exhaustion phase was not clear in II fish. The total duration of the insult was 23 ± 8 s. After the insult the fish recovered. The heart rate was 63 ± 29 beats min I prior to stunning. After stunning, the ECG revealed extrasystole and was irregular. By using an average current of 629 ± 180 mA (at approximately 360 V, 50 Hz AC), at least 91% of fish are effectively stunned with a confidence level of 95%. In the second experiment, the behaviour of 10 individual catfish, which were able to move freely in water, was observed following head-only stunning (370 V). The durations of the tonic, clonic and exhaustion phases in free-swimming fish were II ± 8 s, 20 ± 5 s and 23 ± 20 s, respectively. All fish recovered. In the third experiment, a group of seven catfish was head-only stunned followed by gill-cutting to kill them as a second procedure (ie after recovery from head-only stunning). No brain activity was seen on the EEG 12 ± 5 s after stunning. However, two fish showed responses to noxious stimuli after 2 min and 5 min. A second group of seven catfish was gill-cut only. They responded to noxious stimuli for at least 15 min. The blood loss was 1.2% and I.0% of live weight for the first and second group, respectively. It may be concluded from our results that African catfish are effectively stunned for 23 ± 8 s with a current of 629 ± 180 mA for 1.2 s, after which they recover. Since evoked responses may remain for at least 5 min after stunning and gill-cutting, we recommended that the stunning and killing procedure should be optimised.

The African catfish Clarias gariepinus Burchell 1822 is a favourite aquaculture fish in many parts of Africa and Asia because of its hardiness and fast growth rate. In this study, early, post-embryonic and larval developmental stages of C. gariepinus were examined chronologically and described. Photomicrographs of unfertilized matured oocytes from 0 min of fertilization through all cell stages to alevin, to complete yolk absorption, to free swimming larval stages are shown and documented live from lateral and top views, with the aid of a light microscope. Extruded oocytes had a mean diameter of 1 ± 0.1 mm, and possessed a thin perivitelline membrane whose space was filled with a protoplasmic layer. Heartbeat was in the range of 115–160/min prior to hatching. Hatchability rate was 85% and hatching occurred at 17 h at a controlled temperature of 28.5 ± 0.5°C, while ontogeny of the eyes and other organs were discernible. At day 4, larvae mean length was 9.3 ± 0.5 mm, exogenous feeding had commenced fully and melanophores spread cephalocaudally but were concentrated significantly on the head parts. This paper, for the first time, presents the significant chronological developmental stages of C. gariepinus embryology that will have significant implications for genetic manipulation and catfish seed production for aquaculture.

This study first reports concentrations of arsenic (As) and selenium (Se) in fish tissue of Clarias gariepinus from Sagua la Grande River in the Villa Clara Province, Cuba. We also confirm the mercury (Hg) levels in these fish obtained previously. Individuals were captured in three areas near Sagua la Grande City, where this fish is a common source of food for the city inhabitants. Concentrations range of As, Hg and Se (in wet weight) were 0.01–0.11 μg.g−1; 0.03–0.24 μg.g−1 and 0.75–3.87 μg.g−1, respectively. As and Se levels were positively correlated (n=19, ρ=0.673, P<0.05). High levels of Se were found in fish tissue and in 31.6% (n=6) of individuals captured exceeded the threshold value for Se toxicity, which means that Se is likely to produce adverse consequences on the fish themselves or on the wildlife organisms that eat them. As and Se concentrations were positively correlated with fish weight and length (P<0.05). Fish from irrigation canal have higher Se concentrations than fish captured in the other two stations. However, no significant differences were found between Hg and As concentrations in fish at the three sampling stations. Finally, as the Se concentrations in C. gariepinus were abnormally high, we suggest the need for studies about Se sources in the zone, the effect of Se in fish and the intake associated with fish consumption.

Employing practical-type diets containing graded levels of all-rac-α-tocopheryl acetate (0, 5, 15, 30, 45, 60, 75 or 100 mg/kg), the quantitative requirement for a-tocopherol by juvenile African catfish (Clarias gariepinus Burchell) was investigated. Diets, defined with respect to selenium, fatty acid and ascorbate inclusion, were offered for 12 weeks during which catfish grew from 10 g to 120 g. Dietary regime had no significant effect on growth (P > 0·05), although haematocrit values increased with increasing tocopherol dose above 15 mg/kg (assayed) (R2 = 0·71, P < 0·05). Tissue accretion of the vitamin increased concomitant with dietary dose (P < 0·001). Employing a colorimetric assay for thiobarbituric acid reactive substances (TBARS) it was evident that concentration ofTBARS in muscle and liver reflected tissue oxidation status, in that elevated dietary vitamin E resulted in decreased assayed TBARS (P < 0·05). By broken-line analysis the requirement for this species to suppress lipid peroxidation was estimated as 30 to 40 mg all-rac-a-tocopheryl acetate per kg dry diet and this agrees with published requirements of many fish species.

On termination of a 56-day feeding trial, the effect of dietary oxidized oil and all-rac-α-tocopheryl acetate on fatty acid and α-tocopherol status of juvenile African catfish (Clarias gariepinusj muscle and liver was investigated. Clarias of mean initial weight 15·8 (s.e. 0·29) g were given food at -proportionately 0·03 body weight per day on diets of two oxidative states (fresh or oxidized) at two levels of supplemental α-tocopheryl acetate (20 or 100 mg/kg dry matter).

As well as having a significant detrimental effect on final body weight (P < 0·001), oxidation of the dietary lipid source was responsible for decreasing muscle but not hepatic polyunsaturated fatty acids (PUTAs). Liver tissue was suspected of actively synthesizing longer chain PUT As in response to consumption of oxidized oils, as indicated by decreases in the proportions of linoleate (18:2 n-6) and linolenate (18:3 n-3) and the increased abundance of eicosatetraenoic acid (20:4 n-6, arachidonate) and docosahexaenoic acid (22: 6 n-3, DHA). This phenomenon has not been described in fish nutrition and therefore possible mechanisms are discussed. Additionally, oxidized oils in catfish diets caused significant depletion of a-tocopherol from both muscle and liver (P < 0·05).

Modulation of the effects of oxidized lipids was achieved at the higher dietary tocopherol concentration. Appearance of an unknown aliphatic compound (equivalent chain length (ECL) = 13·1), believed to be a product of lipid peroxidation, demonstrated that catfish given rancid/low-tocopherol diets had been subjected to heightened oxidative nutritional stress. Values obtained for this peak were significantly correlated with dietary regime (P < 0·02), Once more, dietary α-tocopheryl acetate moderated this effect. It was concluded that elevated inclusion of all-rac-α-tocopheryl acetate into diets for African catfish could abrogate the effects of oxidized dietary lipids on muscle and liver fatty acids and α-tocopherol.

The African catfish, Clarias gariepinus, is a highly appreciated species for aquaculture, because of its favourable food conversion, its resistance to discases, its relatively low requirements for water quality, the possibility for high stocking density and the excellent meat quality. For those reasons even in the Netherlands, there is a modest, but Europe's largest and still expanding, African catfish farming activity. Although this species grows and matures in captivity, there is no spontaneous reproduction. We coulddemonstrate that the failure to reproduce resides in the brain-pituiary-gonad axis. Hormones required for oogenesis and spermatogenesis are being produced in sufficient quantities. However, final oocyte maturation, ovulation, spermiation and spawning behaviour do not occur, due to the lack of a gonadotropin surge. In nature, the prespawning gonadotropin surge is induced by environmental factors such as the waterlevel in the spawning area. Under farming conditions the environmental cues are hard to identifyand/or to mimic. In combination with unavoidable stress this causes a blockade of the release ofgonadotropin releasing hormone (GnRH). Consequently, gonadotropin surge release fails to occur, which is enforced by an effective hypothalamic dopaminergic inhibition. The gonadrotropin surge induces the conversion of 17 αOH-progesterone into 17 αhydroxy-20 β-dihydroprogesterone, the final maturation inducing substance. Based on these data, several protocols for artificial propagation could be developed. They include either a treatment with a GnRH analogue in combination with a dopamine receptor antagonist, a treatment with homologous gonadotropin or HCG, or a treatment with 17 αOH-progesterone. Since a number of years we have used the African catfish as a model for fundamental research onfish reproductive endocrinology. Till now one gonadotropic hormone (GTH) could be demonstrated. Itsamino acid composition and sequence was analysed and appeared to be homologous with known forms ofthe maturational GTH (GTH-II). Specific radioimmuno assays for the complete hormone and its α- and β-subunit respectively, have been developed. cDNAS encoding the subunits have been cloned. They are applied now for Northern blotting and in situ hybridization. GnRHs were fully characterised (a specific catfish-GnRH and chicken-GnRH-II). Specific antibodiesagainst these peptides were raised and the cDNAS encoding the hormone precursor molecules werecloned and used for respectively immunocytochemical localisation and radioimmunoassays, and in situ hybridisation. The importance of the two GnRH forms for gonadotropin release was studied. Chicken-GnRH-II appears to be 10 to 100 times more potent than catfish GnRH, probably due to its higher receptor affinity. Catfish GnRH, however, is present in the brain and pituitary about 100 times more than chicken GnRH-II. Steroid hormone synthesis by ovaries, testis and seminal vescicles was analysed. The sex sieroids that play a role in the negative feedback control of gonadotopin release were identified (11-keto-testosterone and testosterone) and their interaction with hypothalamic dopamine metabolism was demonstrated as one of the possible mechanisms of action. Several steroid conjugates from the seminal vesicles were shown to have pheromonal activities, involved in reproductive behaviour. They induce under certain physiological conditions attraction between conspecifics and synchronization of ovulation.

The rates of growth and nitrogen efflux (total nitrogen and ammonia) of individual C. gariepinus ($\bar{x}~=~32.2$ g; S.D. = 4.8 g) kept under 4 feeding regimens, following a 48 h imposed fast (phase l), were measured periodically. In phase 2 (35 d), groups A, B and C were fed a 49.75%, 45.55% or 41.10% protein diet respectively at a ration of 0.5% body weight (1 d−1) Group D were not fed. In phase 3 (25 d) all groups were fed the 41.10% diet. In phase 1, the ammonia efflux rates were lower than any of the values found in either phase 2 and 3. In phase 2, group A (49.75%) had a higher mean ammonia efflux rates than the other groups and ammonia comprised 60-100% of the total nitrogen efflux in all groups. Group D showed a direct relationship between ammonia efflux rate and length of fast. In phase 3, refeeding with the 41.1% protein diet caused the ammonia efflux rates of groups B and D to converge within 7 days to values no different from those of group C, but group A maintained a significantly higher mean value until day 25. During phase 2, the growth rates in B (45.55%) were greatest, but, none of the among groups differences were significant. Group D fish (unfed) lost approximately 30% of their initial weight during phase 2. Nitrogen efflux rates, notably ammonia, showed a pattern of excretion that was directly related to the protein content of the diet, but that the source of dietary protein, dietary energy and the total available energy also influenced nitrogen metabolism. The small differences in growth found were related to between diet differences in composition. The weight loss in the unfed group of fish was probably attributable to the utilisation of lipid and/or protein reserves as metabolic fuels.

The fatty acid profile of four different genetic strains of Clarias gariepinus: gold (G), Netherlands (N), RAU (K) and Wild (W) were evaluated. Fertilised eggs and larvae of the four strains were maintained under identical environmental conditions. After nine weeks, the two faster growing strains (G and W) had less total body lipid than the others (N and R). The total saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids of the four strains were: G – 32.1; 26.8 and 37.9%; N – 33.5; 27.8 and 35.5%; R – 32.7; 29.0 and 35.4%; W – 32.9; 27.7 and 36.8%, respectively. The major fatty acids that showed statistically significant differences between strains were: C16:0, C18:1ω9, C22:5ω3 and C22:6ω3. The G strain had a significantly higher ω3/ω6 ratio (2.2) compared to that of the other strains (N – 1.9; K – 1.9 and W – 1.9). In a second trial, juveniles of strain G were fed an artificial diet for 60 days containing no lipid (A, control), or the following lipids at 10% of the diet, sunflower oil (B. a high level of C18:lω9 and C18:2ω6), cod liver oil (C, a high level of 20 and 22 Cω3 fatty acids) and tallow (D, predominantly SFA and MUFA). Muscle total lipid composition was strongly influenced by diet and contained the following SFA, MUFA and PUFA percentages, and a ω3/ω6 ratio of: A – 35.9; 45.3; 15.0% and 0.5; B – 30.6; 33.4; 33.6% and 0.1; C – 33.2; 37.2; 23.6% and 1.8; D – 38.7; 45.3; 12.1% and 0.4, respectively.

Temperature is the major abiotic factor controlling the rate of morphogenesis in fish. The present work investigated hatching time and the transition from endogenous to exogenous feeding at five constant temperatures over the range 15-35 °C. Clarias gariepinus eggs can be successfully hatched in ambient water temperatures between 20 and 35 °C, although at 30 °C the hatching rate is significantly improved. At 15 °C embryos do not survive. The period of hatching is inversely related to temperature such that synchronous hatching is encouraged by high temperature within the specific temperature range. The extent to which development rate and metabolic rate in C. gariepinus are accelerated by temperature is not the same. The duration between first feeding, yolksac absorption and the point-of-no-retum is inversely related to temperature. The same threshold temperature, below which development is theoretically arrested, can be estimated for C. gariepinus from the linear relationship between development rate and temperature up to hatching, first feeding and yolksac absorption. This closely approximates the lower lethal temperature of 15 °C estimated directly from embryo survival. The effect of temperature can be usefully modelled in C. gariepinus using the linear relationship V = a + bt to estimate t0 (threshold temperature) and Deff0 (effective day-degrees) and the hyperbolic relationship τ = Deff0/(t – t0) to determine development time. From the present work, t0 is 14.5 °C and Deff0 is 13, 26.3 and 35.7 for hatching, first feeding and yolksac absorption respectively for C. gariepinus. Unlike day-degrees, effective day-degrees remain independent of temperature over the range 20-35 °C. An approximate guide to development time at different constant temperatures within this range is given.