Effect of sub-lethal nitrite on selected haematological parameters in fingerling Catla catla (Hamilton)

A sub‐lethal nitrite toxicity trial was conducted using static conditions for a period of 96 h with fingerlings of Catla catla (21.4±3.6 g). Fingerlings were exposed to five concentrations of nitrite, that is, 1, 2, 4, 8 and 10.4 mg L^?1 and a nitrite‐free control to study changes in haematological parameters. Nitrite caused an increase in immature erythrocyte population (7–24%) in lower concentrations (0–4 mg L^?1) at 6 h while they were absent in higher concentrations. The total erythrocyte count was reduced at 6 h followed by an increase at 12 h with further reduction up to 96 h in all concentrations of nitrite. The 96‐h exposure resulted in 21.2–31.8% reduction in erythrocyte population in 1–10.4 mg L^?1 nitrite. The haemoglobin content decreased progressively with increasing nitrite concentrations as well as exposure periods. Total leukocyte count decreased initially at 6 h in all treatments followed by an increase after 12 h, signifying development of a protective response of the body to nitrite stress. Blood glucose decreased initially up to 24 h followed by an increase through 96 h. Serum protein level decreased continuously with increasing exposure period. The study revealed that exposure to nitrite caused changes in almost all the haematological parameters in the fingerlings depending on the concentration as well as exposure period. Nitrite being one of the important inorganic nutrients often recorded at higher levels in intensively cultured ponds, the present study highlights its adverse impact on fish and stressed the need for the management of this nutrient in culture ponds.     

Phosphorus requirement of Catla (Catla catla Hamilton) fingerlings based on growth, whole-body phosphorus concentration and non-faecal phosphorus excretion

A 120‐day feeding trial was conducted to determine the dietary requirement of phosphorus for Indian major carp, catla (Catla catla) fingerlings. Four hundred and eighty fingerlings (mean body weight: 4.23±0.87 g) were randomly distributed among eight treatment groups with three replicates each. Eight isonitrogenous and isocaloric semi‐purified diets (crude protein: 35% and crude lipid: 8.5%) were formulated with graded levels of phosphorus using KH₂PO₄ (T₁: control, 0.1%; T₂: 0.3%; T₃: 0.5%; T₄: 0.7%; T₅: 0.9%; T₆: 1.1%; T₇: 1.3%; T₈: 1.5%) and fed to the respective groups. Twenty fish were stocked in 150 L plastic tanks and fed to apparent satiation twice a day. Specific growth rate (SGR) significantly (P<0.05) increased with increasing dietary phosphorus concentration from 0.73% to 1.27%, after which there was a slight decline in growth at 1.1% available phosphorus (aP) and remained constant thereafter. The quadratic broken‐line model based on growth was Y=317.5?581(0.64?x) (0.64?x); R²=0.73. Moisture and crude protein contents of whole body were similar among all the treatments. However, the ether extract in T₁ group was significantly (P<0.05) higher than all the other treatments. The whole‐body phosphorus content increased significantly (P<0.05) with an increase in phosphorus in the diets. The one‐slope broken‐line model based on whole‐body phosphorus concentration was Y=4.07?1.63 (0.71?x); R²=0.48. The one‐slope broken‐line model for non‐faecal phosphorus excretion as inorganic phosphorus (Pi) for 24 h revealed a trend of Y=12.67+73.96 (x?0.6); R²=0.81. Minimum aP requirements based on weight gain (%), whole‐body phosphorus content and phosphorus excretion were 0.64%, 0.71% and 0.6%, respectively. Hence, the dietary aP requirement of catla fingerlings ranges from 0.6% to 0.71%.     

Culture of Catla catla in a lentic impoundment in Jammu

In May 1977, fry of Catla catla was introduced for the first time by the Regional Research Laboratory, Jammu, for culturing in the inland waters of the region. One hundred tagged specimens and 250 untagged ones were stocked along with Indian major and Chinese carps in a rain- and partially sewage-fed impoundment having an area of 2.5 ha and depth ranging from 1.50 m to 3.25 m. Tagged specimens were caught by a cast net in July 1978. The average mean weight recorded was 4,750 g and the average mean length was 58.6 cm after 14 months. The largest specimen was 7,500 g in weight and 63,00 cm in length and the smallest 4,000 g in weight and 55.00 cm in length. The pond bloomed naturally with both phyto- and zooplankton throughout the year, and no supplementary feed or fertilizer was provided.     

Dietary tryptophan requirement of fingerling Catla catla (Hamilton) based on growth,protein gain,RNA/DNA ratio,haematological parameters and carcass composition

A 12‐week feeding trial was conducted in eighteen 70 L indoor polyvinyl circular troughs provided with a water flow‐through system (1–1.5 L min^?1) at 28 ± 1 °C to evaluate the dietary tryptophan requirement of fingerling Catla catla (3.45 ± 0.24 cm; 0.60 ± 0.13 g). Six casein‐gelatin‐based amino acid test diets (330 g kg^?1 crude protein; 13.6 kJ g^?1 digestible energy) containing graded levels of L‐tryptophan (1.0, 1.4, 1.9, 2.3, 2.8, 3.4 g kg^?1 dry diet) were fed to triplicate groups of fish near to satiation at 08:00, 12:30 and 17:30 h. Absolute weight gain, feed conversion ratio, protein gain, RNA/DNA ratio, hepatosomatic index, viscerosomatic index, condition factor and haematological indices improved with the increasing levels of tryptophan from 1.0 to 2.3 g kg^?1 of dry diet. Significantly higher carcass protein was obtained at 2.3 g tryptophan per kilogram of the dry diet. Exponential analysis of absolute weight gain, feed conversion ratio, protein gain and RNA/DNA ratio against dietary tryptophan levels at 95% maximum and minimum responses displayed the tryptophan requirement at 2.5, 2.3, 2.5 and 2.1 g kg^?1 dry diet, respectively. Inclusion of dietary tryptophan in the range of 2.1–2.5 g kg^?1 dry diet, equivalent to 6.4–7.6 g kg^?1 dietary protein, is recommended in formulating tryptophan‐balanced feed for the culture of this fish species.     

Dietary threonine requirement of fingerling Indian major carp,Catla catla (Hamilton) estimated by growth,protein retention efficiency,threonine deposition,haematological parameters and carcass composition

A 12‐week feeding trial was conducted to determine the dietary threonine requirement of fingerling Indian major carp, Catla catla (3.35 ± 0.11 cm; 0.59 ± 0.06 g). Six casein‐gelatin based (33% crude protein; 3.23 kcal g^?1 digestible energy) amino acid test diets with graded levels of analysed threonine (0.74%, 0.96%, 1.21%, 1.48%, 1.72% and 1.93% dry diet) were fed to satiation to triplicate groups of fish. Absolute weight gain (g per fish), feed conversion ratio, protein retention efficiency, threonine deposition, RNA/DNA ratio and carcass protein significantly improved with the increase in dietary threonine and peaked at 1.48% of the dry diet. Haematological indices were also found to be best in fish fed at 1.48% threonine diet. Quadratic regression analysis of absolute weight gain, feed conversion ratio, protein retention efficiency, threonine deposition, RNA/DNA ratio, carcass protein, haemoglobin (g dL^?1), haematocrit (%) and RBCs (10⁶ × mm^?3) at 95% of maximum and minimum response exhibited the threonine requirement of fingerling C. catla between 1.35% and 1.48% dry diet, corresponding to 4.09–4.48% dietary protein. Present finding would be useful in formulating threonine‐balanced feeds for the intensive culture of C. catla.     

Implication of melatonin in oocyte maturation in Indian major carp Catla catla

Importance of melatonin (N-acetyl-5-methoxytryptamine) in the regulation of oocyte maturation has been studied in a carp Catla catla. Melatonin secretory cells were immunocytochemically localized only in the end vesicle. Diurnal and seasonal studies indicated that the serum levels of melatonin exhibit a minimum diurnal value in the mid-day of all seasons, but the peak value is attained either at mid-night or just before the onset of light. Moreover, highest seasonal value of melatonin was noted in the post-spawning phase. Administration of melatonin at different doses (25, 50, or 100 μg/100 g body weight) for 1, 15, or 30 days resulted in either pro- or anti-gonadal effects depending on the reproductive seasons. In vitro study revealed that incubation of oocytes with melatonin 4h prior to addition of MIH in the medium led to an accelerated rate of oocyte maturation through the formation of a complex of two proteins (MPF), cyclin B and cyclin dependant kinase, Cdc2. Moreover, melatonin pre-incubation considerably increased MIH stimulation of histone H1 phosphorylation as compared to MIH alone. Taken together, gathered information promotes the idea of a physiological role of melatonin in the maturation of oocytes in Catla catla.     

Adjuvant effect of mushroom glucan and bovine lactoferrin upon Aeromonas hydrophila vaccination in catla, Catla catla (Hamilton)

Mushroom glucan and bovine lactoferrin (Lf), known for their immunostimulatory potential, were used as adjuvant in conjunction with a formalin-killed Aeromonas hydrophila vaccine in catla, Catla catla. In vitro antigen-specific responsiveness of catla leucocytes and protective responses against experimental challenge with homologous antigen were monitored following immunization. Antigen-specific proliferation, 'macrophage activating factor' (MAF) production and antibody production were significantly higher in fish injected with glucan adjuvanted vaccine. Lf adjuvanted preparations showed a weak proliferative response and MAF production, although the antibody production was significantly higher than the controls. A good degree of protection was achieved with the glucan adjuvanted vaccine. However, in spite of producing significant anti-A. hydrophila antibody, Lf adjuvanted vaccine did not confer any protection following challenge with A. hydrophila. The potential of adjuvanticity of mushroom glucan and bovine Lf in intraperitoneal vaccination is discussed.     

In Vitro and Dietary Effects of Chitin on Cellular and Humoral Immune Parameters of Catla,Catla catla (Hamilton)

Immunostimulants increase the immunocompetency and disease resistance of fish by enhancing immune responses. This study determined the in vitro and dietary effects of chitin on immune responses of catla, Catla catla. Catla head‐kidney leukocytes were cultured in complete RPMI‐1640 medium containing 0.01, 0.1, and 1.0 mg/mL of chitin. Leucocyte viability and different cellular immune responses, namely, respiratory burst, nitric oxide production, and leucocyte peroxidase content were analyzed in vitro. In the dietary experiment, chitin‐supplemented diets (0.5, 1.0, and 2.0% of feed) were fed to catla for 2 wk, and respiratory burst, serum lysozyme, and total protein contents were measured after 1 and 2 wk of feeding. Leucocytes incubated in vitro with chitin for 72 h maintained their viability. Chitin significantly (P < 0.05) enhanced all the immune responses in vitro. The lowest dose showed the best response. Dietary chitin also significantly stimulated all the immune parameters in a dose‐dependent manner with the highest dose showing the maximum responses. All the responses were higher after 1 wk of feeding and declined thereafter. The results suggest that chitin stimulates catla cellular and humoral immune responses in vitro and in vivo.     

PCR-based segregation of one hybrid variety of Labeo rohita and Catla catla from their wild-types

High consumer preference together with its polyculture potential has undoubtedly driven Rohu (Labeo rohita) and Catla (Catla catla) to top the list of the most preferred fishes among the Indian major carps. Commonly found in these fishes are hybrids that can be natural or man-made. Increasing emphasis on biodiversity issues has necessitated proper stock management of these through molecular genetics techniques. Also with few morphological differences that can be used to differentiate wild types and hybrids properly, the problem demands a straightforward molecular approach. Here, we report a simple PCR-based technique that can differentiate the hybrid variety from wild types easily using three different microsatellite markers. Three sets of primers were used to amplify three different microsatellite markers from the genomic DNA isolated from pectoral fins. When the PCR products using all three primer sets were analyzed, ‘hybrid–Rohu’ could be distinguished from wild types. Whereas the hybrid–Rohu DNA yielded specific PCR products with all three primer pairs, only two PCR products were obtained either from wild-type Catla DNA (by primer sets 1 and 2) or from wild-type Rohu DNA (by primer sets 1 and 3). This study clearly demonstrates that a simple PCR-based technique will help the fish breeders and hatcheries to identify and differentiate suspected hybrid–Rohu carp from the wild types within a few hours.     

Development and characterization of cell lines derived from rohu, Labeo rohita (Hamilton), and catla, Catla catla (Hamilton)

Two new cell lines, designated RE and CB, were derived from the eye of rohu, Labeo rohita , and the brain of catla, Catla catla , respectively. The cell lines were maintained in Leibovitz's L-15 supplemented with 20% foetal bovine serum. The RE cell line was sub-cultured for more than 70 passages and the CB cell line for more than 35 passages. The RE cells are rounded and consist predominantly of epithelial cells. The CB cell line consists of predominantly fibroblastic-like cells. Both cell lines are able to grow at temperatures between 25 and 32 °C with an optimum of 28 °C. The growth rate of the cells increased as the foetal bovine serum concentration increased from 2% to 20% at 28 °C, with optimum growth at concentrations of 15% or 20% foetal bovine serum. The cells were successfully cryopreserved and revived at different passage levels. The cell lines were not susceptible to four marine fish viruses. Extracellular products from Aeromonas sp . were toxic to the cell lines. When the cells were transfected with plasmid eukaryotic green fluorescent protein (pEGFP [Clontech, Carlsbad, CA, USA]) vector DNA, a significant fluorescent signal was observed suggesting that these cell lines could be a useful tool for transgenic and genetic manipulation studies. Polymerase chain reaction amplification of mitochondrial 12S rRNA from rohu and catla confirmed that the cell lines originated from these fish species. The cell lines were further characterized by immunocytochemistry using confocal laser scanning microscopy.     

Molecular characterization,inductive expression and mechanism of interleukin‐10 gene induction in the Indian major carp,catla (Catla catla)

Interleukin‐10 (IL‐10) is a multifunctional cytokine and plays an important role in diseases. In this study, IL‐10 gene was cloned and characterized from catla (Catla catla), which is a highly commercially important fish species in the Indian subcontinent. The result indicated that the full‐length catla IL‐10 (cIL‐10) gene had five exons and four introns with an open reading frame of 540 nucleotides encoding a polypeptide of 179 amino acids. A phylogenetic analysis of cIL‐10 gene sequence showed that cIL‐10 clustered with freshwater carps group as expected. Quantitative real‐time polymerase chain reaction analysis showed that cIL‐10 was expressed in gill, liver, kidney, intestine, skin and heart and its expression profile was up‐regulated in bacterial infection and LPS treatment. A close relationship of high cIL‐10 expression and low pro‐inflammatory cytokine IL‐1β expression was observed in the treated group of fish, which might reveal the role of cIL‐10 as an anti‐inflammatory cytokine. Mechanism of cIL‐10 induction was investigated by blocking nuclear factor (NF)‐κB ‐signalling with BAY 11‐7082 in catla kidney cell culture. Blocking NF‐κB suppressed IL‐10 induction by LPS, and thus it revealed that cIL‐10 was induced through NF‐κB signalling. These data could be helpful to understand the function of IL‐10 in fish in response to vaccinations, probiotics and various diseases.     

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.     

Role of Stocking Density on Growth and Survival of Catla,Catla catla,and Rohu,Labeo rohita,Larvae and Water Quality in a Recirculating System

ABSTRACT

Catla, Catla catla, and rohu, Labeo rohita, fry were cultured at 6,667,8,333, and 10,000/m³ in 15-L aquaria in recirculating systems for 30 days. Larvae were fed with exogenous live plankton. Cultures at 6,667 and 8,333 larvae/m³ showed significantly (P <0.05) higher survival and growth than larvae stocked at 10,000 larvae/m³ for both species. Food was more efficiently used in low stocking density, as evident from the significantly (P <0.05) lower values of feed conversion ratio in lower density compared to those for high stocking density. Specific growth rate of both species was high in the early stage and gradually declined along with the ontogenic development. Dissolved oxygen level was higher in the low density system than in the high density one. Values of phosphate and COD increased during the experiment. Ammonia, nitrite, phosphate, and COD levels were significantly (P <0.05) higher in the 10,000 larvae/m³ density system than in the other two systems for both species. Considering the survival and growth of fish and values of water quality parameters, it appears that stocking density can be raised up to 8,333 larvae/m³ with a recirculating system for both catla and rohu.     

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.     

Acute toxicity of ammonia and its sub-lethal effects on selected haematological and enzymatic parameters of mrigal, Cirrhinus mrigala (Hamilton)

A comprehensive acute toxicity trial was conducted using a static water system to study the toxic effect of ammonia on haematology and enzyme profiles of Cirrhinus mrigala H. The LC₅₀ of total ammonia‐nitrogen (TAN) was 11.8 mg L^?1 TAN (1.029 mg L^?1 NH₃‐N). The sub‐lethal test revealed that with increasing concentration of TAN, the total erythrocyte counts were reduced in lower concentrations (1–4 mg L^?1 TAN) followed by higher levels in fish exposed to higher concentrations (8–16 mg L^?1 TAN). In contrast, the total leucocyte counts were opposite. With increasing concentration of TAN, haemoglobin and serum protein content were reduced, whereas the blood glucose level increased. As the concentration of ammonia increased, there was a reduction in acetylecholinesterase activity in the brain and liver; alkaline phosphatase activity in the serum, brain and gill; and acid phosphatase (ACP) activity in the gill. The activity of lactate dehydrogenase in the gill, liver, kidney and brain increased with increased concentration of ammonia. In addition, activities of ACP in the serum and brain, alanine aminotransferase in the serum, brain and gill, and aspartate aminotransferase in the serum, brain and gill were increased.     

Density effects of silver carp Hypophthalmichthys molitrix and catla Catla catla on the production system in all‐male freshwater prawn–finfish polyculture ponds

The effects of three different combinations of silver carp Hypophthalmichthys molitrix and catla Catla catla density on the production system in all‐male freshwater prawn–finfish polyculture ponds were evaluated in triplicate. The stocking density of silver carp and catla, respectively, were maintained at 2000 and 500 ha^?1 in treatment SC₂₀₀₀C₅₀₀, 1500 and 1000 ha^?1 in treatment SC₁₅₀₀C₁₀₀₀ and 1000 and 1500 ha^?1 in treatment SC₁₀₀₀C₁₅₀₀. Male freshwater prawn Macrobrachium rosenbergii and small fish mola Amblypharyngodon mola densities were fixed in all treatments at 12 000 and 20 000 ha^?1 respectively. Management practices were same for all treatments. Blue‐clawed male prawns were harvested twice during the 122‐day culture at 15‐day intervals before the final harvest. Plankton and macro‐benthos abundance and water quality parameters (except transparency and chlorophyll a) did not vary significantly (P>0.05) among treatments. Mean final weights of both silver carp and catla were decreased with the increasing of their own stocking density. The treatment SC₁₅₀₀C₁₀₀₀ resulted in 25–32% increased net production of silver carp plus catla (461 kg ha^?1) and 20–21% increased net production of all species combined (874 kg ha^?1) as compared with the other treatments, although the differences in production of prawn and mola among treatments were not significant.