Induction of fertilizable eggs by conspecific vitellogenin implantation in captive female walking catfish,Clarias batrachus (Linn.)

The synthesis of vitellogenin (Vg) is induced by conspecific Vg (Vg1 and Vg2) and estradiol‐17β (E₂) as demonstrated by the pattern of ³H‐serine incorporation in the liver and plasma proteins. The incorporation studies indicated that the label was first incorporated into the liver after which it appeared in the blood in both E₂‐ and Vg‐treated male catfish. Since Vg was capable of inducing its own synthesis, experiments were conducted in females during preparatory–prespawning period (March–May) to make them gravid by implanting Vg pellets. Two implantations of 4 mg Vg1 pellets into female catfish with an interval of 15 days, followed by laboratory maintenance for 45 days of initial implantation showed a significant increment in ovarian weight with concomitant formation of yolky oocytes through synthesis and incorporation of Vg, whereas Vg2 implantation was not effective in this regard. Histological observation of yolky oocytes in Vg1‐treated group showed the peripheral migration of germinal vesicle (eccentric germinal vesicle), which indicates the onset of maturation. On 45th day, third implantation with 2 mg Vg pellets was performed and after 15 days, fish were hormonally induced with a single injection of hCG (2,000 IU/kg fish). Six groups were considered such as initial control, BSA‐implanted control, Vg1‐implanted, Vg2‐implanted, catfish collected from the field on the last day of the experiment and catfish collected during spawning period in this experiment with 3–7 fish in each group. Each of the experimental fish was sexually mature and the body weight was between 100 and 125 g. The percentage of ovulation and fertilization in the eggs of Vg1‐implanted group was 91% and 78%, respectively, which was almost similar to that of gravid female catfish collected during breeding period (July). The breeding performance in BSA‐ and Vg2‐treated females was very poor. The fertilized eggs were hatched in the laboratory conditions. Thus, in the female catfish, Vg1 not only induces vitellogenesis but also makes the oocytes viable for fertilization.     

Food consumption and digestive enzyme activity of Clarias batrachus exposed to various temperatures

The effect of temperature on the food consumption rate and the digestive enzyme activities of Clarias batrachus (80.60 ± 5.34 g) were evaluated. Fish were exposed to six different temperatures of 10, 15, 20, 25, 30 and 35 °C following an acclimation temperature of 25 °C. The rate of temperature change was 2 °C day^?1. Highest food consumption was recorded at 25 °C. It gradually reduced with decreasing water temperature. Food consumption rate was significantly (P < 0.05) lower at 10 °C compared with other treatments. Hence, 46.67, 8.20–23.58 and 1.02–6.15% reduced food consumptions were recorded in groups exposed at 10, 15 and 20 °C temperatures, respectively, compared with the 25 °C. The consumption rate was not affected in fish exposed at 30 and 35 °C. Total protease, trypsin and chymotrypsin activities were significantly (P < 0.05) higher in fish exposed at 25 °C compared with others. Lipase activity was significantly (P < 0.05) higher in fish exposed at 30 °C compared with others. Lowest enzyme activities were recorded at 10 °C. Water temperature below 25 °C affected the food consumption and digestive enzyme activities in fish that served as indicators of stress in fish.     

Biological properties of Indian walking catfish (Clarias batrachus) (L.) gonadotropins in female reproduction

The biological activities of catfish LH-like (semi-purified: s200a and purified Qa) and FSH-like (semi-purified: s200b and purified: Qb) were compared in intact and hypophysectomized female catfish, Clarias batrachus, during preparatory and the pre-spawning periods on vitellogenesis and ovarian maintenance, as well as in vitro final maturation of oocytes, germinal vesicle breakdown (GVBD). During preparatory period, in intact catfish, semi-purified FSH-like induced complete vitellogenesis through the production of estradiol-17β (E₂) and vitellogenin (Vg) accompanied by the formation of SIII yolky oocytes. On the other hand, semi-purified LH-like had induced the formation of only SII (characterized by the appearance of cortical alveoli in cytoplasm) oocytes, which indicates the initiation of vitellogenesis. In hypophysectomized female catfish, purified LH-like but not FSH-like induced the formation of SII oocytes in the ovaries. Treatment with semi-purified LH- and FSH-like at the dose level of 5 µg/fish/day for 7 days significantly maintained the yolky oocytes in gravid catfish after hypophysectomy with a significant reduction in plasma Vg, but not E₂ levels, indicating some unknown GtH-induced factor doing the job. In in vitro oocytes culture, both LH- and FSH-like induced GVBD, but the response was significantly more with LH-like than FSH-like. All these findings revealed that both LH-like and FSH-like have overlapping physiological functions, but their responses differ depending on the physiological status of the catfish.     

Dietary levamisole modulates the immune response and disease resistance of Asian catfish Clarias batrachus (Linnaeus)

In order to determine the immunomodulatory effect of dietary levamisole in Asian catfish (Clarias batrachus), fish were fed four different diets for 10 days: a formulated diet as control and the same diet supplemented with 50, 150 or 450 mg levamisole kg^?1 feed. The serum bacterial agglutination titre against Aeromonas hydrophila as a measure of specific immunity, serum haemagglutination titre, natural haemolytic complement activity (ACH₅₀), myeloperoxidase and lysozyme activities, total protein level and oxidative radical production by neutrophils as a measure of non‐specific immunity as well as disease resistance against A. hydrophila challenge to separate vaccinated and non‐vaccinated groups were evaluated at 0, 1, 2 and 3 weeks after last administration of levamisole. Levamisole supplement at the lowest level (50 mg kg^?1) significantly enhanced oxidative radical production and serum myeloperoxidase (MPO) content immediately after 10 days of feeding, which reached peak values after 3 and 2 weeks of feeding respectively. Haemolytic complement and haemagglutination titre were significantly enhanced after 3 and 1 weeks respectively. Haemolytic complement activity and MPO activities were significantly raised to 150 mg kg^?1 after 3 and 2 weeks, respectively. At the highest level of levamisole feeding (450 mg kg^?1) significant decreases in superoxide production and complement activity were measured immediately after levamisole feeding, which returned to the normal level after 1 week post‐ feeding. Fish were challenged with a virulent strain of A. hydrophila at 0, 1, 2 and 3 weeks after levamisole feeding, and the cumulative per cent survival was recorded over 10 days. Feeding levamisole at 50, 150 or 450 mg kg^?1 increased per cent survival in vaccinated fish immediately after levamisole feeding, and survival was significantly higher at 450 mg kg^?1. There was no difference in mortality patterns in non‐vaccinated fish. The results support the use of levamisole at 50 mg kg^?1 feed for 10 days as an immunostimulant in Asian catfish farming.     

Feeding gelatinized carbohydrate in the diets of magur,Clarias batrachus (Linnaeus, 1758): Effects on growth performance,enzyme activities and expression of muscle regulatory factors

This study was conducted to illustrate the effect of dietary gelatinized starch (GS) on the growth performance, enzyme activities and expression of MyoD and Myf5 in magur, Clarias batrachus fingerlings. Four iso‐nitrogenous (37%) and iso‐lipidic (6%) diets containing 15% (D‐1), 25% (D‐2), 35% (D‐3) or 45% (D‐4) GS were fed to 240 fingerlings (2.5 ± 0.5 g) in triplicates groups for 8 weeks. The maximum weight gain %, specific growth rate, protein efficiency ratio, lower FCR and higher mRNA expression of MyoD or Myf5 were found in the D‐3 group fed with 35% GS. Higher hepatosomatic index, viscerosomatic index, body lipid and lower moisture content were found in the D‐4 group. Aspartate transaminase and alanine transaminase activities were found to be higher in the D‐1 group. Amylase, glucose‐6‐phosphate dehydrogenase and blood glucose were higher in the D‐4 group. Superoxide dismutase, catalase and hexokinase activities remain unaffected by the dietary GS levels. Hence, the overall results indicate that 35% GS can improve growth performance and upregulate myogenic regulatory factors, but 45% GS level will favor lipogenesis and compromise growth. Furthermore, Myf5 gene showed more immediate response than the MyoD to the dietary carbohydrate in magur.     

Mortality and pathology of hybrid catfish,Clarias macrocephalus (Günther) × Clarias gariepinus (Burchell), associated with Edwardsiella ictaluri infection in southern Thailand

Enteric septicaemia of catfish (ESC) caused by Edwardsiella ictaluri is becoming an increasing problem in aquaculture and has been reported worldwide in a variety of fish species. This study reports ESC in hybrid catfish, Clarias macrocephalus (Günther) × Clarias gariepinus (Burchell), cultured in southern Thailand. The bacteria were identified as E. ictaluri by conventional and rapid identification systems, as well as by genetic and phylogenetic characterization. Analysis of 16S rRNA indicated 100% homology to the 16S rRNA sequence of several E. ictaluri strains in GenBank. Plasmid profiles demonstrated 4.0‐ and 5.6‐kb plasmids, compared with the 4.8‐ and 5.6‐kb plasmids in the US isolates, and representative genes of three of the four known pathogenicity islands of US isolates were present. Serologically, lipopolysaccharide (LPS) purified from the Thai isolates was not recognized by a monoclonal antibody against the LPS of US isolates. Fish experimentally infected with E. ictaluri showed 23–100% mortality within 14 days with a 168‐h LD₅₀ of 6.92 × 10⁷ CFU mL^?1 by immersion and a 96‐h LD₅₀ of 1.58 × 10⁶ CFU fish^?1 by intraperitoneal injection. Examination of tissue sections obtained from both naturally and experimentally infected fish indicated that infection of hybrid catfish with E. ictaluri produced lesions in several organs including liver, kidney, spleen, heart and brain. Histopathology findings included cellular necrosis, focal haemorrhage, infiltration of lymphocytes and multifocal granulomatous inflammation in the infected organs.     

Preliminary evaluation of the nutritive value of the variegated grasshopper (Zonocerus variegatus L.) for African catfish Clarias gariepinus (Burchell. 1822) fingerlings

A preliminary feeding trial was conducted to evaluate the nutritive value of adult variegated grasshopper (Zonocerus variegatus) meal (VGM) as a replacement for fish meal in the diets of Clarias gariepinus fingerlings. Proximate analysis result showed that VGM had 61.50% crude protein, 6.87% crude lipid, 27.33 NFE and 9.35% chitin. Five isonitrogenous (30%) and isoenergetic (18.42 MJ kg^?1) diets were formulated in which VGM progressively replaced fishmeal at five inclusion levels (0%, 25%, 50%, 75% and 100%). The best performance in terms of % weight gain was observed in the group of fish fed ED₁ (25% inclusion level) and was not significantly different (P>0.05) from the groups fed the control diet. Weight gain and specific growth rate were significantly lower (P<0.05) in the group fed ED₄ (100%VGM). Feed conversion ratio and protein efficiency ratio were numerically but not significantly different (P>0.05) among the different groups. The results indicate that apparent protein and lipid digestibility were high at all levels, although there were decreases with an increase in the inclusion levels. Carcass lipid decreased with an increase in the inclusion level of VGM. The VGM can replace fish meal up to 25% in the diets of C. gariepinus fingerlings without any adverse effect on growth and nutrient utilization.     

Dietary beta-1,3 glucan potentiates innate immunity and disease resistance of Asian catfish, Clarias batrachus (L.)

This study investigated the effects of short and prolonged administration of a yeast beta-glucan on non-specific immune parameters, growth rate and the disease resistance of Asian catfish, Clarias batrachus. Fish fed with a basal diet (control) and test diet (basal diet supplemented with 0.1% glucan) for 1, 2 and 3 weeks were assayed for superoxide production, serum myeloperoxidase (MPO) content, natural haemagglutinin level, complement and lysozyme activities. Fish were weighed at weekly intervals and specific growth rate (SGR, % increase in body weight per day) was determined. After each week, fish were challenged with Aeromonas hydrophila to measure the level of protection. Results showed that glucan administration at 0.1% in feed, significantly (P<0.05) enhanced MPO and lysozyme levels, superoxide production, haemagglutination titre and level of protection against A. hydrophila challenge, irrespective of length of exposure. The alternative complement activity and SGR were not affected by the dietary supplementation of yeast glucan. As glucan feeding at 0.1% for 1 week is able to enhance the non-specific immunity and disease resistance of catfish efficiently, short-term feeding might be used in farmed catfish diets to enhance disease resistance.     

A partial economic analysis for Nile tilapia Oreochromis niloticus L. and sharptoothed catfish Clarias gariepinus (Burchell 1822) polyculture in central Kenya

Aquaculture development in Kenya has been hampered by lack of targeted feeds. Chemical fertilizers can enhance natural food production and thus indirectly provide protein to complement energy-rich cereal brans used as feed. Consequently, an experiment was conducted at Sagana Fish Farm in central Kenya to realize least-cost combinations of rice bran and fertilizer. Twelve 800 m² (0.08 ha) ponds were stocked with juvenile (32 g) Oreochromis niloticus L. at 2 m⁻² and Clarias gariepinus (Burchell 1822) (fingerlings of 4.6 g) at 0.2 m⁻². Four treatments, calculated to approximate isonitrogenous conditions, were applied in triplicate as follows: (1) urea and diammonium phosphate (DAP) to provide 16 kg of N ha⁻¹ week⁻¹ and 4 kg of P ha⁻¹ week⁻¹; (2) urea and DAP applied to give 8 kg of N ha⁻¹ week⁻¹ and 2 kg of P ha⁻¹ week⁻¹, plus rice bran fed at 60 kg ha⁻¹ day⁻¹; (3) rice bran fed at 120 kg ha⁻¹ day⁻¹; (4) rice bran as in treatment 3 and fertilizer as in treatment 2. Ponds were sampled monthly to measure fish growth, and drained completely after 20 weeks. A partial economic analysis indicated minimal net profits of K.Sh. 18 851 (US$ 251), K.Sh. 9895 (US$ 132), K.Sh. 3299 (US$ 44) and K.Sh. 7015 (US$ 94) for treatments 1–4, respectively, because of high feed and seed costs.     

Testicular responsiveness to gonadotropic hormonein vitro and Leydig and Sertoli cell ultrastructure during pubertal development of male African catfish (Clarias gariepinus)

The gonadotropin (GTH)-stimulated testicular androgen secretionin vitro and the ultrastructure of Leydig and Sertoli cells was studied during the pubertal development in male African catfish. Testicular weight increased from less than 1 mg in the ninth week of age to nearly 600 mg in the 28th week. Immature testes (stage I: spermatogonia) were highly sensitive to GTH and secreted very high amounts of androgens per mg of tissue. The secretion per mg tissue decreased gradually in stages II (spermatogonia and spermatocytes) and III (spermatogonia, spermatocytes, and spermatids), but precipitously in stage IV (all germ cell stages, including spermatozoa). However, due to the testicular weight gain, the total androgen output per pair of testes increased slightly in stage III and strongly in stage IV. The sensitivity to GTH decreased with the appearance of haploid germ cells in stage III. Leydig cells but not Sertoli cells showed the ultrastructural characteristics of steroid producing cells. Leydig cell morphology did not change in stages I–III, while in stage IV, more smooth endoplasmic reticulum was present. The ultrastructural characteristics of Sertoli cells did not change prominently. Thus, spermatogonial multiplication and spermatocyte formation takes place when the testicular steroidogenic system is highly active and responsive to GTH; whereas the differentiation of haploid germ cells is accompanied by a reduced responsiveness to GTH and by the secretion of several-fold lower androgen amounts per mg of tissue.     

Stunning of farmed African catfish (Clarias gariepinus) using a captive needle pistol; assessment of welfare aspects

The objective of this study was to assess whether high‐pressure injection of air into the brain of African catfish (Clarias gariepinus) could render the animal unconscious and insensible immediately and permanently. In the study, 48 African catfish with a live weight of 900–1900 g were restrained and equipped with EEG and ECG electrodes and then stunned. The catfish were stunned mechanically using a captive needle pistol. The pressure to shoot the needle was 8 bar and that to inject the air was 3 bar for 1.5 s. The catfish behaviour was observed during and after stunning. τ and δ waves and spikes, which precede a stoppage in brain activity as measured on the EEG, were used as indices for the measurement of immediate induction of unconsciousness and insensibility In 23 of 42 fish, an iso‐electric line was observed after an average of 13.4 s, while in the remaining fish the τ and δ waves and spikes remained on the EEG during the recording period. In all cases, the ECG showed an irregular heart rate with fibrillation and extra systolae. Moreover, the configuration showed ischaemia. Before the captive needle stunning, free‐swimming fish (n=7) explored the tank for an average of 21±12 s before lying down at the bottom. After stunning, they showed clonic uncoordinated swimming movements. The movements stopped after an average of 38±50 s. In another group (n=7) that was stunned and subsequently placed in ice water, clonic cramps were observed in two out of seven animals. When taking into account the number of animals with a reliable EEG (n=42) and using 95% confidence intervals, it was concluded that at least 93% of the catfish were effectively stunned using a correctly positioned captive needle pistol. Furthermore, it is recommended to immobilize the stunned fish by chilling, as the post‐stun clonic cramps may hinder gutting and filleting.     

Induction,purification and partial characterization of vitellogenin in an Indian major carp Catla catla (Ham.)

Vitellogenin was purified from the serum of 17‐β estradiol (E₂)‐induced juvenile Catla catla using a simple two‐step purification procedure i.e. selective chemical precipitation followed by gel filtration chromatography. Purified protein migrated as single band in a native gradient PAGE which indicated the purity of the sample. The molecular weight of the native catla vitellogenin (~440 kDa) was determined using gel filtration chromatography. In SDS‐PAGE under reducing conditions catla vitellogenin dissociated into three major sub units at 115 kDa, 102 kDa and 73 kDa along with a few faint bands. Confirmation of purified protein as catla vitellogenin was supported by multiple physiological evidences, e.g. absence in male as well as juvenile sera and presence in matured female fish, ability to be synthesized upon estradiol injection in immature fish and certain unique biochemical properties like high molecular weight, phospholipoglycoprotein nature of the molecule. Western blot analysis showed that polyclonal antibody raised against purified protein detected vitellogenin in the sera of catla and in a few species selected from Cyprinidae family. These antisera were used to detect vitellogenin in liver tissue of hormone‐induced catla using immunohistochemistry and its applicability in other immunoassays is discussed.     

Ovoviviparously produced Artemia nauplii are a suitable live food source for the larvae of the African catfish (Clarias gariepinus: Burchell, 1822)

Brine shrimp Artemia, the most common live food organism used in larviculture, can reproduce either oviparously (production of dormant cysts) or ovoviviparously (direct production of nauplii), depending on environmental conditions. Ovoviviparous Artemia nauplii have seldom been considered as a source of live food in aquaculture, partly due to the convenience and the developed techniques associated with the production and use of the dormant cysts. In many countries in Africa, however, hatchery managers do not have access to a reliable supply of affordable good quality cysts. In this study, we therefore demonstrated the potential of a system designed for the continuous ovoviviparous production of nauplii at low salinity, using Great Salt Lake Artemia franciscana and micronized agricultural material as feed. The suitability of the produced nauplii was tested by feeding them directly to Clarias gariepinus larvae in comparison with oviparous nauplii and decapsulated cysts. Higher survival (100%), better protein efficiency ratio (2.6 ± 0.1) and food conversion ratio (1.0 ± 0.1) was observed in larvae fed with the ovoviviparous nauplii (p < 0.05). Overall, we conclude that the ovoviviparous nauplii could serve as an alternative live food for larval fish. If optimized, the system could be validated for integration in hatcheries.     

革胡子鲶出血性败血症病原菌的分离鉴定

从暴发性死亡的革胡子鲶(Clarias gariepinus)中分离出可疑病原菌株2010111403(简称1403),分别采用细菌全细胞脂肪酸鉴定系统、Biolog微生物自动鉴定系统及细菌16 S rDNA序列分析三种方法对其鉴定,结果表明菌株1403为嗜水气单胞菌(Aeromonas hydrophila)。分离菌株对健康革胡子鲶致病性测试表明其对革胡子鲶的半数致死剂量(LD50)为6.32×106菌落形成单位(CFU);实验感染革胡子鲶出现与自然发病相似症状,表明菌株1403是引起革胡子鲶发生暴发性死亡的主要致病病原。药敏试验表明,庆大霉素、四环素、阿奇霉素、氟哌酸、先锋霉素等9种抗生素对分离菌株有较强的抑制作用,但分离菌株对复方新诺明、克林霉素、罗红霉素等4种药物表现出耐药性。     

鲫卵黄脂磷蛋白的纯化鉴定及其夹心ELISA的建立

鲫(Carassius carassius)卵黄原蛋白(vitellogenin,Vtg)是检测水体环境雌激素活性常用的生物标志物。本研究利用凝胶过滤结合离子交换层析与选择性沉淀结合离子交换层析2种方法,从鲫卵巢匀浆液中纯化得到了卵黄脂磷蛋白(lipovitellin,Lv),经鉴定该蛋白含有糖、磷、脂基团,天然分子量约为521 kD,SDS变性电泳显示分子量为117 kD和103 kD的2个亚基。Western blot结果显示,金鱼(Carassius auratus)Lv抗体和斑马鱼(Danio rerio)Lv抗体都能与鲫Lv发生很好的交叉反应。利用纯化的鲫Lv与金鱼Lv抗体和斑马鱼Lv抗体建立了2种夹心ELISA,发现金鱼Lv和鲫Lv的结合曲线基本重合,并且利用鲫Lv与金鱼Lv抗体建立的夹心ELISA工作范围为15.6~1000 ng/mL,检出限约为6.8 ng/mL,显著低于利用斑马鱼Lv抗体建立的夹心ELISA,结合此前研究者建立的鲫Vtg竞争ELISA,为鲫Vtg指标的测定提供了可靠方法。     

Comparison of the lipid properties of healthy and pansteatitis‐affected African sharptooth catfish,Clarias gariepinus (Burchell), and the role of diet in pansteatitis outbreaks in the Olifants River in the Kruger National Park,South Africa

Pansteatitis has been identified in wild populations of sharptooth catfish, Clarias gariepinus (Burchell), and Nile crocodiles, Crocodylus niloticus Laurenti, inhabiting the same waters in the Olifants River Gorge in the Kruger National Park, South Africa. Mesenteric and pectoral fat tissue was investigated microscopically and by fatty acid analysis in healthy and pansteatitis‐affected catfish from both captive and wild populations. Variation in fatty acid composition between pectoral and mesenteric fat was noted. Composition of mesenteric fat differed between fish from various localities as a result of differences in diet. Pansteatitis in the captive population, resulting from ingestion of high amounts of dietary oxidized fat, reflected higher levels of unsaturated fatty acids within the mesenteric fat. Mesenteric fat of pansteatitis‐affected wild catfish was characterized by an increase in moisture content, a decrease in fat content and a decrease in stearic and linoleic acids. The n‐3 to n‐6 fatty acid ratio of mesenteric fat was higher in pansteatitis‐affected wild catfish than in healthy catfish from the same locality, reflecting higher polyunsaturated fat intake by pansteatitis‐affected fish. The possible role of alien, invasive, phytoplankton‐feeding silver carp, Hypophthalmichthys molitrix (Valenciennes), in the aetiology of pansteatitis in both catfish and crocodiles in the Olifants Gorge is discussed.     

The urogenital papillae as an indicator of sexual maturity in male African catfish,Clarias gariepinus, (Burchell, 1822)

The present study investigated a non‐invasive method based on macroscopic morphological features of male Clarias gariepinus for the assessment of sexual maturity stage. As African catfish cannot be stripped for semen as in most other fish species; they have to be killed to obtain semen from the testes. This method could be used to ensure that males used to obtain sperm are sexually mature to increase the concentration and quality of mature sperm for egg fertilization in the production of C. gariepinus. Morphological features including the length and width of the urogenital papillae (UGP), an external secondary sexual characteristic of C. gariepinus (n = 89) were measured and calculated as a percentage of total body length. The urogenital papillae length (L_UGP) and width (W_UGP) index of each fish were used and compared with the macroscopic and microscopic maturity stage of the testes. The results revealed that although positive, a significant correlation does not exist between the L_UGP or W_UGP and maturity stage of the testes. Therefore, although this procedure would be valuable as an initial non‐invasive assessment of maturity, it does not provide an accurate or reliable indication of the maturity stage of the fish.