Nutritional evaluation of fermented black gram (Phaseolus mungo) seed meal in compound diets for rohu, Labeo rohita (Hamilton), fingerlings

Six isonitrogenous (approximately 35% crude protein) and isocaloric (approximately 4.0 kcal g⁻¹) diets were formulated incorporating raw and fermented black gram, Phaseolus mungo, seed meal at 20%, 30% and 40% levels by weight into a fishmeal‐based control diet fed to rohu, Labeo rohita, fingerlings (mean weight, 1.81 ± 0.21 g) for 80 days for a study of fish performance. A particular bacterial strain (Bacillus sp.) isolated from the intestine of adult common carp (Cyprinus carpio) reared in the wild having significant amylolytic, cellulolytic, lipolytic and proteolytic activities was used for fermentation of seed meal for 15 days at 37 ± 2°C. Fermentation of P. mungo seed meal was effective in significantly reducing the crude fibre content and antinutritional factors such as tannins and phytic acid, and enhancing available free amino acids and fatty acids. In terms of growth, feed conversion ratio and protein efficiency ratio, the 30% fermented black gram seed meal incorporated diet resulted in a significantly (P < 0.05) better performance of rohu fingerlings. In general, growth and feed utilization efficiencies of diets containing fermented seed meal were superior to diets containing raw seed meal. The apparent protein digestibility (APD) values decreased with increasing levels of raw seed meal in the diets. The APD for raw seed meal was lower at all levels of inclusion in comparison to those for the fermented seed meals. The maximum deposition of protein in the carcass was recorded in fish fed the diet containing 40% fermented seed meal. The results indicate that fermented black gram seed meal can be incorporated in carp diets up to the 30% level compared to the 10% level of raw seed meal.     

Effects of amino acid supplementation on the nutritive quality of fermented linseed meal protein in the diets for rohu, Labeo rohita, fingerlings

A feeding trial was conducted for 8 weeks to examine the effects of partial substitution of fish meal (FM) protein (crude protein content: 58.5%) with linseed meal protein with and without supplemental amino acids in diets for rohu Labeo rohita (Hamilton), fingerlings (mean weight: 1.50 ± 0.3 g). Prior to incorporation into the diets, linseed meal was fermented with lactic acid bacteria ( Lactobacillus acidophilus ) to reduce/eliminate the antinutritional tannin and phytic acid factors. Twelve experimental diets (diets D1–D12) were formulated to replace the FM protein from a reference diet (RD) with linseed meal protein at different levels (four sets of diets, of which each set of three diets contained 25%, 50% and 75% replacement of FM protein by linseed meal protein, respectively). Diets D1–D3 were not supplemented with any amino acid. Lysine was supplemented in diets D4–D6. Diets D7–D9 were supplemented with methionine + cystine (together), and diets D10–D12 contained lysine and methionine + cystine (together). Lysine and methionine + cystine (together) were added to the diets at 5.7% and 3.1% of dietary protein, respectively. The groups of fish fed diets without amino acid supplementation had significantly lower percentages of weight gain, specific growth rate and high feed : gain ratio than the fish groups fed other experimental diets. The addition of lysine and methionine + cystine to the diet in which 50% of the FM protein was replaced by linseed meal protein (diet D11) significantly improved fish performance. The results of the present study suggest that rohu fingerlings can effectively utilize the supplemented amino acids and that linseed meal protein can replace up to 50% of the FM protein in rohu diets if the linseed meal is properly processed (fermented) and supplemented with the lacking amino acids.     

Digestive enzymes and metabolic profile of Labeo rohita fingerlings fed diets with different crude protein levels

Labeo rohita, commonly called rohu is one of the most important fish species for aquaculture in India. Digestive enzyme response and metabolic profile of fingerling L. rohita to different dietary crude protein (CP) levels (viz. 25, 30, 35 and 40%) were studied in an attempt to optimize a practical diet formulation for this species. After 45 days of feeding, activity of digestive enzymes and metabolite concentrations were assayed. Amylase, lipase and alkaline phosphatase (ALP) activities were not influenced by the dietary protein, but proteolytic and acid phosphatase (ACP) activities varied (P<0.05) between the treatments. Proteolytic activity showed a second order polynomial relationship with dietary crude protein (CP) as Y = 0.0734X(2) + 4.937X - 68.37, r(2)=0.97. A positive correlation was observed between dietary CP and amylase (r(2)=0.78). All the metabolites except muscle glucose showed significant change corresponding to the dietary protein levels. Glucose and glycogen levels corresponded to the dietary carbohydrate levels. Muscle and plasma pyruvic acid increased as the crude protein in the diet increased, whereas liver pyruvic acid showed the opposite trend. Muscle protein content was not affected by dietary CP. Protein fractions in plasma (total protein, albumin and globulin) showed maximum values in 30% CP fed group. It is concluded that proteolytic activity and ACP are the major digestive enzymes responsive to dietary CP in L. rohita fingerlings. Considering the cost effectiveness of the diet, and based on liver and plasma free amino acid levels and plasma protein fractions, 30% crude protein is recommended as the optimal dietary protein for L. rohita fingerlings.     

Development of monoclonal antibodies to rohu [Labeo rohita] immunoglobulins for use in immunoassays

Serum immunoglobulins [Ig] of rohu [Labeo rohita] were purified by affinity chromatography using bovine serum albumin as capture ligand. The purified rohu Ig [r-Ig] had a molecular weight [MW] of 880 kDa as determined with gel filtration chromatography. The heavy chain of r-Ig had an MW of 77.8 kDa and that of light chain was 26.4 kDa in SDS-PAGE. Purified r-Ig was used for the production of two anti-rohu Ig monoclonal antibodies [D7 and H4] that belonged to subclass IgG2b and IgG1, respectively. Both the MAbs were specific to heavy chain of r-Ig as seen in Western blotting. Anti-rohu Ig MAb was used as a diagnostic reagent in ELISA and immunocytochemical assays to demonstrate its application for sero-surveillance and for immunological studies in rohu. A competitive ELISA was used to demonstrate the antigenic relatedness of r-Ig with whole serum Ig of other fish species. Cross reactivity of anti-rohu Ig MAb was observed with serum Ig of Catla catla and Cirrihinus mrigala. No reactivity to serum Ig of Ophiocephalus striatus and Clarias gariepinus was seen. Anti-rohu Ig MAb was found to be suitable for the detection of pathogen specific [Edwardsiella tarda] antibodies in serum of immunized rohu by an indirect ELISA. In flow cytometry using D7 MAb, the mean percentage [+/-SE] of Ig positive cells in spleen and blood of rohu were found to be 64.85% [+/-2.34] and 51.84% [+/-2.55] of gated lymphocytes, respectively. Similarly, D7 MAb also stained 52.84% [+/-1.30] and 10.5% of gated lymphocytes in kidney and thymus, respectively. The anti-rohu Ig MAbs also showed specific staining of Ig bearing cells in spleen sections by the indirect immunoperoxidase test.     

Toxicity of cypermethrin in Labeo rohita fingerlings: biochemical,enzymatic and haematological consequences

The effect of exposure to sub-lethal concentrations of cypermethrin, a synthetic pyrethroid pesticide, on biochemical parameters of muscle, blood and enzyme activities in brain, liver and kidney of the Indian major carp, Labeo rohita was studied. The sub-lethal exposure studies were done for up to 45 days at 1/10 and 1/50 of 96 h LC(50) of cypermethrin. The 96 h LC(50) was found to be 0.139 ppm. RNA levels decreased while DNA levels were elevated. Acid phosphatase was unchanged while alkaline phosphatase was depleted. Brain acetylcholinesterase activity was decreased significantly (P<0.05) over a period of 45 days at both cypermethrin concentrations. Lactate dehydrogenase activity in brain and liver was elevated, but inhibited in kidney. Succinate dehydrogenase and ATPase activities were depleted in brain, kidney and liver. There was a decrease in serum protein level over control at both concentrations of the pyrethroid. Blood glucose level and total leucocytes were elevated compared with controls at either concentration from day 15 to day 45. Haemoglobin percentage and total erythrocytes decreased in both sub-lethal concentrations. Extracts of the herb Datura stramonium were effective in countering the toxicity of this pesticide. Our data suggest that sub-lethal exposure of cypermethrin alters the biochemical, haematological parameters and enzymes of organs tissue and exert stress on the fish. Plant extracts may be useful in counteracting some of these effects.     

Acute and subchronic toxicity of aflatoxin B1 to rohu, Labeo rohita (Hamilton)

Pathological alterations in various organs of rohu (L. rohita) fingerlings following acute (0, 7.50, 11.25 and 13.75 mg/kg body weight) and subchronic (0, 1.25 and 2.50 mg/kg body weight) single i.p. aflatoxin B1 exposure for 10 and 90 days, respectively, were investigated. Mortality (dose-dependent) was marked only during acute toxicosis. The changes observed in various organs were dose and time dependent. The acute dose groups revealed toxic changes viz., necrotic and vascular changes in liver and gill lamellae; meningitis, congestion in brain, degeneration and inflammatory reaction in heart along with degenerative to necrotic changes in kidney tubules and sloughing of the intestinal mucosa. During subchronic exposure to this toxin, preneoplastic lesions in liver along with changes in spleen, intestine, gill and pancreas were recorded. With low doses of aflatoxin, the fish did not reveal any mortality or external signs other than catchexia and increased pigmentation on scales. In composite culture practice of Indian major carps, this could be of economic significance.     

The immunosuppressive effect of alpha-permethrin on Indian major carp, rohu (Labeo rohita Ham.)

The immunosuppressive effects of bath exposure to a sub lethal concentration of the synthetic pyrethroid alpha-permethrin (3.05 x 10(-4) mg l(-1)) in the Indian Major carp, Labeo rohita was studied after 45 days' exposure. In some groups, the effects of alpha-permethrin on non-specific defences and serum enzymes of carp were investigated after challenge with Aeromonas hydrophila. Several nonspecific immune responses and serum enzymes were reduced after exposure of alpha-permethrin. Bactericidal activity of rohu serum was reduced significantly in pesticide and bacteria treated fish. The Glutamic Oxaloacetate Transaminase (GOT) and Glutamic Pyruvate Transaminase (GPT) activity were increased in immunosuppressed fish. Blood glucose level was elevated significantly and Hb% was reduced significantly in pesticide and bacteria treated fishes as compared to the negative control.     

Growth and body composition of rohu, Labeo rohita (Hamilton), fed compound diet: winter feeding and rearing to marketable size

A 122‐day growth trial was conducted to observe the impact of polyhouse in winter on growth, conversion efficiencies and body composition of fingerling Labeo rohita (7.9 ± 1.1 cm; 4.60 ± 0.18 g). Fish were stocked (1.5 m^?2) in polyhouse and outdoor concrete tanks (250 m²) in duplicate and fed a compound diet [35% crude protein (CP)] to apparent satiation twice daily, at 09.00 and 17.00 hours. A natural photoperiod was maintained during the trials. At the end of the trials, polyhouse‐reared fish produced significantly (P < 0.01) higher values for weight increment, specific growth rate (SGR %), protein efficiency rate (PER), protein productive value (PPV) and better feed conversion ratio (FCR). Polyhouse‐reared fish showed higher (P < 0.01) CP and fat, and lower (P < 0.01) moisture and ash contents in the muscle. In the second part of the study, which also lasted 122 days, polyhouse‐reared young L. rohita (21.7 ± 1.3 cm; 127.8 ± 0.69 g) were raised to marketable size in outdoor earthen ponds (500 m²) at a stocking density of 0.37 m^?2 to examine the effects of feeding traditional feed mixture and compound diet (30% CP) on growth and body composition of fish. Labeo rohita fed the compound diet showed higher (P < 0.01) values for weight increment, SGR (%), PER and PPV, and better (P < 0.01) FCR than those fed traditional feed. Proximate composition of fish muscle showed higher (P < 0.05) CP and fat, and lower (P < 0.05) moisture and ash contents in fish fed the compound diet. The results suggest that polyhouse may be used successfully to culture L. rohita during winter, and that thereafter the fish may be reared in earthen ponds using the compound diet to obtain better production.     

Isolation and characterization of immunoglobulin of the Indian major carp, rohu [Labeo rohita (Ham.)

Information on the structure and character of immunoglobulin of fishes is essential in health management. A study was carried out to characterize the serum immunoglobulin (IgM) of the Indian major carp, rohu Labeo rohita (Ham.). Rohu (500g) were immunised with bovine serum albumin (BSA) and the anti-BSA antibody was purified employing BSA-CL agarose affinity column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified Ig in a 3% gel under non-reduced conditions revealed a single protein having a molecular weight of 850kDa. Analysis of the purified serum in 10% SDS-PAGE under reduced conditions revealed that the immunoglobulin contained heavy and light chains with molecular weights of 85 and 23kDa, respectively. A polyclonal mouse anti-rohu IgM was prepared and used in an immunodot test which showed a specific reaction of the crude rohu anti-BSA antiserum and the purified anti-BSA IgM with BSA. Results indicate that the immunoglobulin of L. rohita is tetrameric IgM, similar to that of other fishes.     

Growth performance of two Indian major carps, catla (Catla catla) and rohu (Labeo rohita) fed diets containing different levels of Spirulina platensis

The influence of Spirulina platensis meal on the growth and carcass composition of two Indian major carps, catla, Catla catla and rohu, Labeo rohita was investigated in a 90-day culture trial. Four experimental diets were used and Spirulina replaced fish meal protein from the standard diet at 25%, 50%, 75% and 100% levels. There was no significant difference in the final weight attained by catla at all levels of Spirulina incorporation as compared to the fish-meal-based control diet. However, the replacement of fish meal by more than 25% Spirulina resulted in significantly superior growth of rohu. The specific growth rate and protein efficiency ratio recorded in rohu improved with higher levels of Spirulina inclusion, while in catla they did not differ significantly from the control treatment. In both the species, the digestibility of dry matter, protein and fat was found to improve marginally with increasing levels of Spirulina incorporation. The carcass composition showed an inverse relationship between protein and fat deposition. In general, fish fed with Spirulina diets had a significantly higher percentage of fat. The study demonstrated the usefulness of Spirulina for partial or complete replacement of fish meal in the diets of catla and rohu.     

The haematology of Rohu, Labeo rohita

Haematocrit values, haemoglobin concentration, erythrocyte counts, leucocyte counts, erythrocyte sedimentation rate and clotting time of Labeo rohita were determined. The variations in haematological parameters in relation to the size and weight of the fish were also examined. Males had higher haematological values than the females. Seasonal and variations due to maturation of gonads were also apparent.     

Dietary yeast RNA supplementation reduces mortality by Aeromonas hydrophila in rohu (Labeo rohita L.) juveniles

A feeding trial was conducted for 60 days to delineate the effect of dietary ribonucleic acid or chitin on haematological parameters, phagocyte respiratory burst and resistance to Aeromonas hydrophila of Labeo rohita juveniles. One hundred and twenty-six (avg. wt. 13.40 +/- 0.17 g) juveniles were randomly distributed in six treatment groups, each one in three replicates. Six isonitrogenous (crude protein: 34.34-35.37%) and isocaloric (414-425 kcal 100 g(-1)) purified diets were prepared with different concentrations of either ribonucleic acid or chitin except the control group, viz., control, T1 (0.1% ribonucleic acid), T2 (0.2% ribonucleic acid), T3 (0.4% ribonucleic acid), T4 (25 mg chitin kg (-1)) and T5 (50 mg chitin kg (-1)). Weight gain %, specific growth rate, feed efficiency ratio, protein efficiency ratio did not vary significantly (P > 0.05) among the experimental groups. Haemoglobin content and total erythrocyte count were observed within the normal range and were not influenced by the dietary immunostimulants. Highest total leukocyte count was found in the T(3) group. The immunomodulatory effects of dietary immunostimulants were studied by using nitroblue tetrazolium (NBT) assay and serum parameters, namely total protein, albumin, globulin and A/G ratio. The respiratory burst activity (NBT) of blood phagocytes was highest in the T3 group followed by the T2 group, which varied significantly (P < 0.05) from other groups. Significantly (P < 0.05) higher total protein, globulin and lower A/G ratio was observed in the T(3) group. The relative percent survival after challenging with Aeromonas hydrophila was highest in the T3 group, compared to the control group, followed by the T2 group. The results indicate that dietary ribonucleic acid at 0.4% enhances phagocyte respiratory burst and protection of Labeo rohita juveniles to challenge by A. hydrophila.     

Influence of ontogenetic changes in prey selection on the survival and growth of rohu, Labeo rohita and singhi, Heteropneustes fossilis larvae

When offered a mixed diet of different zooplanktonic items covering a body size range of 75–2200 μm, (a) rohu, Labeo rohita and (b) singhi, Heteropneustes fossilis larvae ingested progressively larger prey as they grew, due to age-related increase in gape. However, a nearly constant prey size/mouth size ratio was maintained for a period of 4wk after hatching. The dominance of rotifers in the diet during the first 2-wk was followed by cladocerans, particularly Moina macrocopa. Significant differences observed in the growth rates of the larvae reared on different diet regimes were related to ontogenetic changes in prey selection. An exclusive copepod diet throughout resulted in the lowest weight gain in the larvae of both species. However, copepods had no apparent adverse effects when present with the preferred rotifers and cladocerans. Although constituting a suboptimal prey size for the older larvae, rotifers alone, when present in sufficient densities, produced growth rates comparable to those obtained on a cladoceran diet. However, a mixed diet regime contributed to the maximum growth. The implications of these findings to rearing larvae of the economically important rohu and singhi are discussed.     

Stage-dependent hatching responses of rohu (Labeo rohita) embryos to different concentrations of cryoprotectants and temperatures

Hatching performances of three embryonic stages of postfertilization rohu (Labeo rohita) (9-, 12-, and 15-h) were examined after treatment with various concentrations (0.5-4.5M) of two cryoprotectants (methanol and propylene glycol) supplemented with 0.1M trehalose. Different lengths of storage (1-48 h) and temperature (-4 degrees C to ambient) were studied. Of the three stages of embryonic development, the 12-h stage proved to be the most suitable stage for low temperature storage, showing the highest percentage of hatch out (72+/-2%) with 2.0M methanol and 0.1M trehalose. Methanol was more useful for storage at higher temperatures and propylene glycol at subzero temperatures. The maximum possible duration of effective storage of 12-h embryos was 31h in 2.0M methanol at 0 degrees C. No hatch out was found beyond 31h of storage with all concentrations of methanol at 0 degrees C. The results of interactions was that the optimal concentration of methanol was 3.0M at 4 degrees C, 2.0M at 0 degrees C, and 1.5M at 4 degrees C. Among three embryonic stages 12-h stage showed better results in trehalose treatment than sucrose. Among all concentrations of trehalose tested 0.1M gave the maximal survival rate of the rohu embryos.     

Effect of multiple injections of beta-glucan on non-specific immune response and disease resistance in Labeo rohita fingerlings

The purpose of this study was to determine if multiple injections of different dosages of beta-glucan derived from barley would enhance the immune response and disease resistance against infections due to opportunistic pathogens Aeromonas hydrophila and Edwardsiella tarda in Labeo rohita fingerlings. Hence, four different dosages of beta-glucan suspension in phosphate-buffered saline at the rate of 0, 5, 10, 15 mg kg(-1) body weight of fish were injected intraperitoneally to the fingerlings of Labeo rohita at two-week intervals for four times. After every two-week interval different serum biochemical, haematological and immunological parameters of fish were evaluated. At the end of immunostimulation trial of 56 days, fish were divided into four subgroups under each major treatment group for challenge through i.p injection and bath immersion with two pathogens, A. hydrophila and E. tarda. The mortality (%) and agglutinating antibody titre was recoded on 28th day post challenge. Most of the immune parameters such as leucocyte count, phagocytic ratio, phagocytic index, lysozyme activity, complement activity, serum bactericidal activity were significantly (P < 0.05) enhanced on 42 days after three i.p injections of 10 mg of beta-glucan kg(-1) body wt. Challenge study indicated least mortality in the group of fishes injected with medium dose of 10 mg of beta-glucan kg(-1) body wt. four times. Multiple injections of beta-glucan might have maintained the activation of phagocytic cells for a long period which in turn would lead to long-term protection in fishes. Thus, injections of 10 mg of beta-glucan kg(-1) body wt. for three times can be advocated to enhance the immune response of fish species under aquaculture.     

Effect of dietary protein and carbohydrate levels on growth,nutrient utilization and body composition in fingerling rohu,Labeo rohita (Hamilton)

Twelve experimental diets (D‐1 to D‐12) in a 4 × 3 factorial design having four protein levels (25, 35, 40 and 45%) and three carbohydrate levels (15, 25 and 35%) were formulated and fed to fingerling rohu, Labeo rohita (5.48 ± 0.02 g) for 60 days in three replicates at 2% BW per day. The best performance of fish in terms of weight gain (%), specific growth rate (SGR; % per day), feed conversion ratio (FCR) and protein efficiency ratio (PER) was recorded with diet D‐9 containing 40% protein and 35% dextrin as a source of dietary carbohydrate. In general, lower protein consumption per kilogram BW was observed at all protein levels with the rise of the dextrin level. The apparent digestibilities of protein and lipid were not affected by the dietary treatments. At the end of the experiment the body composition of animals from all treatments showed lower percentages of moisture and higher percentages of protein as compared to the initial values. A consistent rise in protein retention efficiency was noted in fish fed diets with increasing dextrin levels. The highest protein sparing effect was found in fish fed the diet containing 40% protein and 35% dextrin.