Stocking density effects on growth and production of the threatened silurid catfish,Mystus cavasius (Hamilton) fingerlings in nursery ponds

The fingerling‐rearing experiment of the threatened catfish, Mystus cavasius was carried out at different stocking densities in earthen nursery ponds. Twelve‐day‐old fry were stocked at 200 000 ha^?1 in treatment‐1 (T₁), 250 000 ha^?1 in treatment‐2 (T₂) and 300 000 ha^?1 in treatment‐3 (T₃) respectively. The mean length and weight of fry at stocking was 1.24 ± 0.25 cm and 0.11 ± 0.04 g respectively. Fry in all the experimental ponds were supplemented with SABINCO nursery feed for the first 14 days and starter‐I feed for days 15–56. The physico‐chemical parameters and plankton population of pond water were within the suitable level for fish culture. Growth in terms of final weight, final length, weight gain, length gain and specific growth rate and survival of fingerlings were significantly higher in T₁ than those in T₂ and T₃. Feed conversion rate was significantly lower in T₁ followed by T₂ and T₃ in that order. Significantly higher number of fingerlings was produced in T₃ than that in T₂ and T₁. Even then, consistently higher net benefits were obtained from T₁ than those from T₃ and T₂. Among the treatments evaluated, 200 000 fry ha^?1 was the best stocking density considering the highest growth, production and net benefits of fingerlings of M. cavasius in nursery ponds.     

Embryonic and larval development of serpae tetra Hyphessobrycon eques (Steindachner, 1882)

In this study, the embryonic and larval development stages of one of the most important ornamental fish serpae tetra (Hyphessobrycon eques) are described. The early life stage is documented from fertilization until the beginning of the juvenile period. The fertilized eggs (the average diameter = 938.55 ± 35.20 µm) were incubated at a water temperature of 26 ± 0.5°C. The cleavage finished in 1:10 hr (=h) and the early blastula stage occurred at 1:26 hr post fertilization (hpf). The gastrulation started at 3:05 hpf, and 50% epiboly was observed at 3:25 hpf. Segmentation stage was monitored at 7:26 hpf. Embryonic developmental stage was completed and hatching occurred 20–21 hpf. The total length (TL) of newly hatched larvae was 2.64 ± 0.21 mm. The larval development of serpae tetra was divided into four different periods: Yolk‐sac larva (1–4 DAH, TL = 2.77 ± 0.09 mm ‐ 3.85 ± 0.11 mm), preflexion larva (5–12 DAH), flexion larva (13–15 DAH, TL = 5.78 ± 0.46 mm on the 15th day) and post‐flexion larva (16–30 DAH, TL = 10.7 ± 0.27 mm on the 28th–30th days). The mouth and anus are closed at 1 DAH. The mouth and anus opened at 4 DAH. Exogenous feeding started on the 4th day. The first gulping of the swim bladder was on days 3. The larva begins to swim freely, and the yolk sac was completely consumed at 4 DAH. Histological structures of the eye and brain of new hatched larva were clearly identified at 1 day after hatching (DAH). According to histological findings, the digestive system (stomach, intestine) started to develop and the liver could be seen on the ventral side of the swim bladder at 5 DAH. No histological difference was observed between the anterior intestine and the posterior intestine at 15–16 DAH. The larval metamorphosis was completed, and the larvae transformed into juveniles at 28–30 DAH.     

Conditioning,maturation and year‐round natural spawning of orange‐spotted grouper,Epinephelus coioides (Hamilton, 1822), in recirculating aquaculture system

The present experiment was aimed at studying the conditioning, maturation and natural spawning of orange‐spotted grouper, Epinephelus coioides, in a recirculatory aquaculture system (RAS). Thirty fish (n = 30; 3.35 ± 0.05 kg) were stocked in a circular tank of 125 m³ capacity fitted with an RAS for conditioning and broodstock development. After 15 days, 15 fish were implanted with 17 α methyl testosterone and letrozole at the rate of 5 mg and 0.2 mg/kg body weight, respectively, for conversion from female to male. The gonadal development started after 1 month, and by 90th day, 63.53 ± 3.78% and 2.07 ± 0.84% of the oocytes attained a size of 400–500 μm and 500–600 μm respectively. Natural spawning commenced in the RAS from 4th month onward after stocking and spawning continued round the year. The spawning pair showed courtship behaviour with a typical vertical burst of swimming just before release of gametes. The total number of eggs spawned during 1 year was 47.23 million with spawning frequency varying form 5 to 13 times per month. The association of spawning events with new moon day (lunar cycle) weakened as time progressed. The mean monthly fertilization and hatching rates varied from 77.80 ± 3.34% to 83.70 ± 1.76% and 82.80 ± 4.21% to 88.33 ± 1.39% respectively. The study proved that RAS is an efficient system that provides a stable, controllable and conducive environment for year‐round natural breeding of orange‐spotted grouper.     

Embryonic and larval development in the semelparous Nereid polychaete Hediste diversicolor (OF Müller, 1776) in Norway: Challenges and perspectives

Knowledge of broodstock manipulation, gametogenesis, artificial fertilization and larval nutrition is a prerequisite to reach a large‐scale production of the polychaete Hediste diversicolor. In this study, the characteristics of oogenesis and spermatogenesis and embryonic and larval development were observed. Moreover, the effect of temperature on embryonic development and effect of diets and sediment on earlier juvenile development were tested. Mature oocytes were characterized by a spherical shape with a diameter of about 200 μm. They showed radial symmetry with a large centric nucleus surrounded by four distinctive layers. Embryo cleavage started between 4 and 10 hr after fertilization, and developed to the swimming trochophore stage after 5–6 days after fertilization at 11°C. The rate of embryonic development increased with a temperature increase from 6.1 to 21.2°C. The trochophore larvae increased in length from day 9 and commenced differentiation into 3‐setiger stage larvae at day 12. The length growth of larvae until 7‐setiger stage was 0.43 mm/day when fed with fish feed, while those fed shellfish diet and smolt sludge both grew around 0.21–0.23 mm/day. The results suggest that fish feed is a superior diet compared to shellfish diets and smolt sludge for the early larval stage of H. diversicolor.     

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.     

Effects of dietary Leucas aspera levels on growth performance,nutrient utilization,digestive enzymes and physio-metabolic and health status of bagrid catfish,Rita rita (Hamilton, 1822)

The study was to examine the effects of dietary L. aspera on growth performance, physio-metabolic response and health status of Rita rita fish reared in tanks for 60 days. Six experimental diets were formulated containing 0 g/kg, 3 g/kg, 6 g/kg, 9 g/kg and 12 g/kg L. aspera and designated as D0, D3, D6, D9 and D12 respectively. The inclusion of L. aspera at graded levels resulted in significantly varying effects on growth performance where the weight gain (%), specific growth rate (SGR) and feed conversion efficiency (FCE) were significantly higher (p<0.05) in D6 and D9 groups than others. The protease and amylase activities were elevated in L. aspera supplemented groups than the control group (p<0.05), whereas lactate dehydrogenase, malate dehydrogenase and serum transaminase enzyme activities were significantly reduced with the increasing level of L. aspera and comparatively higher activities of the respective enzymes were found in the control group. The activities of oxidative stress enzymes were significantly higher (p<0.05) in the D9 group and lower in control. The blood glucose level significantly decreased with the increasing level of L. aspera in the experimental diets. The L. aspera supplemented fish had significantly higher liver glycogen, serum protein, albumin and globulin than control group. Red blood cell count was significantly higher (p<0.05) in D6-D12, whereas white blood cell count and haemoglobin content were significantly higher (p<0.05) in D6 and D9 groups. Regression analysis showed that L. aspera at the level of 7.3–7.9 g/kg is optimum for better growth and feed efficiency.     

Establishment and characterization of macrophage cell line from thymus of Catla catla (Hamilton, 1822)

A continuous cell line has been developed from thymus explants of Catla catla and the cells have been subcultured for 63 passages. The cells exhibited optimum growth at 30°C in L‐15 medium containing 15% foetal bovine serum. The cultured cells engulfed yeast cells and fluorescent latex beads. These cells produced reactive oxygen and nitrogen intermediates following stimulation with lipopolysaccharide and phorbol esters. The culture supernatant from the cultured cells had lysozyme activity and these cells demonstrated Fc receptors. Almost all the cells were positive for alpha‐naphthyl acetate esterase enzyme suggesting that the cells are of macrophage lineage and therefore, the cell line was designated as catla thymus macrophage (CTM) cell line. CTM cells formed aggregates around zoospores of Aphanomyces invadans, but were unable to inhibit the germination of spores. The karyotype analysis of CTM cells at 25th passage revealed a typical diploid model with 50 chromosomes per cell. Partial amplification, sequencing and alignment of fragments of two mitochondrial genes 16S rRNA and cytochrome c oxidase subunit 1 confirmed that the CTM cell line originated from C. catla. This cell line should be useful for studying the role of macrophages in differentiation and maturation of thymocytes and can be a source of macrophage‐specific enzymes and cytokines.     

First results of embryonic development,spawning and larval rearing of the Mediterranean spider crab Maja squinado (Herbst ) under laboratory conditions,a candidate species for a restocking program

The Mediterranean spider crab, Maja squinado, is depleted due to overfishing. The crab has virtually disappeared from areas where it was abundant, such as the Balearic Islands and the Catalan coast. Maja squinado, is economically and ecologically very valuable, and it is essential to obtain information on its biology and rearing conditions to attempt to repopulate the damaged stocks of the species in the Mediterranean basin. Herein, we describe the first successful rearing of M. squinado under laboratory conditions. Our results show that M. squinado is an excellent candidate for restocking using cultured juveniles. Two consecutive broods with a 1–4 day interbrood period were observed in the laboratory in wild‐caught females, the maximum observed duration of embryonic development of the egg mass being 32 days at 18.4 ± 0.9°C, and went through four different stages. The complete larval and first juvenile development was studied in laboratory cultures fed enriched Artemia metanauplius. At 19.6 ± 0.6°C, development from hatching to first crab moult took 17 days, and it comprised two zoeae stages and one megalopa stage. The survival rate at the different stages was monitored, and 7.13 ± 2.3% was achieved at the first crab instar.     

Growth and survival of olive barb,Puntius sarana (Hamilton 1822) larvae produced with cryopreserved versus fresh sperm

Despite the success in fertilization and hatching of fish eggs with cryopreserved sperm, report on growth and survival of larvae produced from frozen‐thawed sperm is inadequate. The study evaluates the applicability of cryopreserved sperm for mass seed production by comparing the growth and survival of a popular food‐fish olive barb, Puntius sarana (Hamilton 1822) larvae produced from cryopreserved and fresh sperm. The eggs were artificially fertilized with cryopreserved and freshly collected sperm, and the growth and survival of produced larvae from both group recorded up to 12 weeks. The independent sample t‐test statistic showed the difference in lengths, t(718) = 0.241; P = 0.810 and weights, t(718) = 0.412; P = 0.680 were insignificant between two groups. There was also no significant difference, t(718) = ?0.758, P = 0.448 in survival of larvae produced from cryopreserved and freshly collected sperm. The study indicates that larvae of olive barb produced from cryopreserved sperm are equally compatible in growth and survival as the larvae produced from fresh sperm. Therefore, cryopreserved sperm can be applied for artificial fertilization of P. sarana to supply quality seed for aquaculture.     

Larval rearing of the giant Azorean barnacle,Megabalanus azoricus (Pilsbry, 1916): feeding trials,larval development and settlement on artificial substrata

A series of experiments were conducted to obtain an efficient larval rearing protocol for Megabalanus azoricus. The first part of this study investigates the effect of microalgae‐based diets on survival and larval development. Mono and mixed‐diets were tested at 20 ± 1°C, in a sequence of 11‐day feeding experiments. The second part presents a preliminary study on the influence of a biofilm on recruitment and use of oyster spat collectors in a mass rearing system. A photographic record of larval development and a brief reference to the diagnostic features that enable quick larval staging are also presented, along with morphometric measurements. Of the microalgae tested (Chaetoceros sp., Chloromonas sp., Dunaliella sp., T‐Isochrysis sp. and Skeletonema sp.) the mixed‐diet Skeletonema sp. with T‐Isocrysis sp. showed the highest survival percentages: total survival ranged from 79.7 to 85.7% and 69.7–80.0% of nauplii were in stage VI after 11 days of rearing. Cypris were also present, but only represented 5.3% of the survivors at most. In the mass rearing system juveniles were found settled in the collectors after 25 days, at 20 ± 1°C. However recruitment was less than 1%. Preliminary results showed no settlement preference towards collectors with biofilm. Nevertheless, this study provides the first record of M. azoricus settlement under laboratorial conditions and represent a starting point for future larval rearing studies.     

Identification of sex‐specific markers and heterogametic XX/XY sex determination system by 2b‐RAD sequencing in redtail catfish (Mystus wyckioides)

Sex‐specific markers provide significant molecular basis for sex control breeding biotechnology to produce all‐male or all‐female fish in commercial breeding. Redtail catfish (Mystus wyckioides), one of the commercial bagrid catfishes distributed in Southeast Asian, which have a long sexual maturation period that can last 3–5 years and males have apparent growth advantage over females, but its sex determination system remains unknown. In this study, we first applied 2b‐RAD‐seq approach to identify three male‐specific 2b‐RAD‐tags and one male heterogametic SNP locus and validated by blast to the genome survey sequences and PCR amplification in both wild and breeding populations. To get longer sex‐specific region, we performed genome walking and obtained a 4,630 bp of Y‐specific sequence and 4,581 bp of X‐specific sequence from the 2b‐RAD‐tag ref189950 with 92.19% nucleotide identity between them. And 9,923 bp/3,935 bp of Y‐specific sequences and 8,491 bp/5,172 bp of X‐specific sequences were also identified with 77.49% and 57.07% nucleotide identity in ref208528 and ref210837, respectively. Subsequently, three different kinds of sex‐specific primers with different length products were designed based on the detected highly sex differentiated regions and could be used to distinguish males and females both in wild and artificially bred populations. What is more, the X‐specific fragment was discovered to produce the dosage effect association in females and in males. The data suggest that male heterogametic XX/XY sex determination system should exist in the redtail catfish. More significantly, the sex‐specific markers are of great value to protect wild resources and improve the efficiency of all‐male breeding practices for aquaculture in the redtail catfish.     

In vivo activity of salmon gonadotropin releasing hormone (GnRH), its agonists with structural modifications at positions 6 and 9, mammalian GnRH agonists and native cGnRH-II on the spawning of an Indian catfish

Effects of the native GnRHs and various agonists have been evaluated on the spawning of an Indian catfish, Heteropneustes fossilis. This study tested salmon (s) GnRH agonists and mammalian (m) GnRH agonists where a D-amino acid residue was substituted alone at position 6 or the C-terminal was modified with ethylamide. GnRH agonists with a combination of these structural modifications were also evaluated separately for their effect on the spawning of the catfish. Native sGnRH, [Pro⁹ NEt]-sGnRH agonist and chicken (c) GnRH-II exhibited similar activity and induced spawning within 14–18 h at a dose of 100 g kg^–1 body weight (BW). [D-Lys⁶]-sGnRH agonist and [D-Lys⁶ Pro⁹ NEt]-sGnRH agonist, induced spawning at a dose of 100 g kg^–1 BW and 1 g kg^–1 BW, respectively. The most notable observation in this study was the ineffectiveness of [D-Ala⁶]-mGnRH agonist and [Des Gly¹⁰ D-Ala⁶ Pro⁹ NEt]-mGnRH agonist. The results obtained suggest that substitution at position 6 alone, and in conjunction with an ethylamide-based modification at the C-terminal in the native sGnRH structure, increases the potency of the tested agonists to induce spawning in the catfish. This study also discusses the potential use and incorporation of cGnRH-II for the development of more generic spawning induction therapies.     

The effect of density in larval rearing of the pullet carpet shell Venerupis corrugata (Gmelin, 1791) in a recirculating aquaculture system

The pullet carpet shell Venerupis corrugata is an economically valuable species in several European countries, however, nowadays stocks are under high fishing pressure. Hatchery production of juveniles for release is a major contributor to strengthen the stock and consequently improve the sustainability of the natural stocks. This study aimed to determine the feasibility of rearing V. corrugata larvae with different larval densities (10, 40 and 200 larvae per mL) in a recirculating aquaculture system (RAS), compared with the traditional larval rearing methodology (Batch). The mean survival, growth and metamorphic rate of V. corrugata larvae in RAS was higher (11.1%; 71.3 μm; 21.6% respectively) than in the Batch system, in all tested densities. The larval growth was not affected by the initial density until 40 larvae per mL, however, 200 larvae per mL decreased the larval growth in length nearly 54 μm. The larval rearing time was shortened in 2 days in the RAS system. The physical, chemical and microbiologic parameters suggested that the tested densities were not excessive to disturb the biofilter stability of RAS. The V. corrugata larval rearing performed at high larval stocking densities in RAS system present a reduction in the operating costs to produce this species.     

Studies on the broodstock production and larval rearing of Coral demoiselle Neopomacentrus nemurus (Bleeker 1857)

Steps taken for broodstock development of Neopomacentrus nemurus, Coral demoiselle (Bleeker, 1857) in a hatchery, its spawning and development of egg to the juvenile stage are described. Among the three treatments tried, only the trial using Parvocalanus crassirostris nauplii as the first diet helped in the development of larvae. Larval and post‐larval growth was studied for a period of 65 days post hatch. The larva measured 2–2.4 mm in total length at the time of hatching. The mouth size of larva at the time of hatching measured 237.92 µm. By the 5th day, the width of the larval body had significantly increased. All the fins were conjoined, except for the caudal fin which had begun its appearance on the 2nd day itself. The digestive system was functional by the 5th day and the copepod remains were seen in the digestive tract. The larva metamorphosed by the 15th day with fully developed fins and fin rays. On the 20th day, the larval body had begun to display signs of fin pigmentation. The larva assumed adult pigmentation by the 35th day. By the 65th day, the larva grew to a total length of about 27 mm and weighed about 0.56 g.     

Embryonic and larval development of black skirt tetra (Gymnocorymbus ternetzi,Boulenger, 1895) under laboratory conditions

The embryonic and larval development of black skirt tetra, Gymnocorymbus ternetzi, are described under controlled laboratory conditions. In addition, major histomorphological changes and the allometric growth patterns during larval development have been described. The laboratory‐reared broodstock, that is 1 year of age, were spawned. Hatching occurred 20–21 h after spawning at 24 ± 0.5°C. The cleavage was finished in 2 h and the early blastula stage occurred at 2:04 hours after spawning. The gastrulation started at 3:20 hours and 30% epiboly was observed at 3:34 hours after spawning. Eight‐somite stage was observed at 08:33 hours. And embryonic developmental stage was completed at 21 h after spawning. The newly hatched larvae were 1442 ± 14.3 μm in mean total length (TL). The mouth opened at 3 days after hatching (DAH). The yolk sac had been totally absorbed and the larvae started to swim actively within 3–4 days. Notochord flexion began at 11 DAH. The metamorphosis was completed and the larvae transformed into juveniles at 32 DAH. In this paper, the full developmental sequence from egg to juvenile of G. ternetzi is described for the first time.     

Induced breeding and larval rearing of Surubí, Pseudoplatystoma fasciatum (Linnaeus, 1766), from the Bolivian Amazon

Brooders of Surubí (Pseudoplatystoma fasciatum) were caught in the Ichillo River (Bolivian Amazon) and adapted to captivity conditions for 1 year in the facilities of the experimental aquaculture station of ‘El Prado’ (Santa Cruz de la Sierra) under natural temperature and photoperiod conditions. Induced reproduction was obtained by means of Ovaprim^® (Syndel, Canada) injections and artificial fertilization. Sperm and ova were obtained by gentle stripping of male and female brooders. Fertilized eggs were incubated in 60 L Zug jars. A mean hatching rate of 73.7±19.0% was obtained after 24 h at 26.5 °C. For larval rearing, several protocols were tested with different settings of photoperiod, light intensity, food type and period of distribution, and stocking density. The best survival rates were obtained with Artemia nauplii feeding in total darkness. A high level of aggressiveness between larvae and precocious appearance of jumpers was observed, but these can be controlled with appropriate rearing conditions.     

Dietary Houttuynia cordata leaf extract and meal enhances the immunity and expression of immune genes in Labeo rohita (Hamilton, 1822)

A feeding trial was conducted to explore the effect of dietary Houttuynia cordata leaf extract (HCLE) and leaf meal (HCLM) on immunological responses and expression of interferon‐gamma (IFN‐γ) and tumour necrosis factor‐alpha (TNF‐α) gene in Labeo rohita fingerlings. Six isonitrogenous (350 g/kg CP) and isocaloric (17 MJ/kg DE) purified experimental diets were formulated with Houttuynia cordata leaf extract and leaf meal comprising control, C (0 g/kg HCLE and HCLM), E2.5 (2.5 g/kg HCLE), E5 (5 g/kg HCLE), E10 (10 g/kg HCLE), M10 (10 g/kg HCLM) and M20 (20 g/kg HCLM). Labeo rohita fingerlings (3.37 ± 0.23 g) were distributed in six experimental groups in triplicates following the complete random distribution. Fish were fed twice daily with respective experimental diets for a period of 60 days. A significantly (p < .05) lower lactate dehydrogenase, malate dehydrogenase, superoxide dismutase and catalase activities were registered in supplemented groups compared with control group, while respiratory burst and lysozyme activities were significantly (p < .05) higher in E10 group compared with other experimental groups. Haemoglobin, total leucocyte count, total erythrocyte count and haematocrit values were significantly (p < .05) higher in E10 group. The expression of IFN‐γ and TNF‐α in both the kidney and liver was significantly up‐regulated in leaf extract and meal supplemented groups with the highest expression in the fish of E10 group. Overall, these results suggest that the dietary supplementation of ethanolic extract of the Houttuynia cordata leaf at 10 g/kg level can enhance the immune response of L. rohita fingerlings.     

Effect of different feeds on the growth,survival and reproductive performance of zebrafish,Danio rerio (Hamilton, 1822)

Present experiment was conducted to determine the effect of different feeds with varying protein levels on the growth, survival and reproductive performance of zebrafish, Danio rerio. The control diet (T1) was wild‐collected zooplankton from local fish ponds, while test diets with 350 g kg^?1 protein (T2), 400 g kg^?1 protein (T3) and 450 g kg^?1 protein (T4) were formulated and fed to fish for a period of 210 days. The significantly (P < 0.05) highest mean weight gain and specific growth rate were observed in T1, which were similar with T3 and T4. The significantly (P < 0.05) highest number of egg production per female and relative fecundity were found in T1, followed by T4 and T3 while T2 produced lowest number of eggs. No significant (P > 0.05) differences were observed in brood survival rate, fertilization and hatching rate among the dietary treatments. The highest (P < 0.05) fry survival rate was recorded in T1, followed by T3 and T4. Thus, it is suggested that control diet i.e. mixed zooplankton exhibited better growth, reproductive performance and fry survival rate. However, diet containing 400 g kg^?1 crude protein also gave comparable results in terms of growth, survival and reproductive performance of zebrafish.