Study of digestive enzyme activities and partial characterization of digestive proteases in a freshwater teleost, Labeo rohita, during early ontogeny

Digestive enzymes of freshwater fish Labeo rohita (family Cyprinidae; class Actinopterygii; infraclass Teleostei; order Cypriniformes) were studied during ontogenic development. Amylase, protease and lipase activities showed a polynomial relationship with age of rohu, whereas trypsin, chymotrypsin and lipase activities exhibited exponential trends with the age of rohu larvae. SDS‐PAGE of crude enzyme extract revealed that the proteases of higher molecular weight (MW) appeared during early ontogeny, whereas low MW proteases were observed at a later stage. Substrate SDS‐PAGE supported the quantitative study of protease activities as evidenced with the increasing number and intensity of activity bands with the age of fish. The number of protease activity bands observed in 4, 10, 12 and 24 DAH (days after hatching) larvae were 5, 7, 8 and 9, respectively. Inhibition of protease activities with soybean trypsin inhibitor (SBTI) (58.6–81.2%), phenyl methyl sulphonyl fluoride (PMSF) (55.6–70%), N‐α‐p‐tosyl‐l ‐lysine chloromethyl ketone (TLCK) (41.1–52.3%) and N‐tosyl‐l ‐phenylalanine chloromethyl ketone (TPCK) (27.9–44.5%) indicated the presence of serine proteases, trypsin‐ and chymotrypsin‐like enzymes in rohu larvae. Inhibition with SBTI and TPCK showed power, TLCK and ethylenediaminetetraacetic acid showed exponential, whereas PMSF showed polynomial relationships during the study period.     

Effects of dietary protein and lipid levels on growth, feed conversion and body composition in rohu, Labeo rohita (Hamilton), fingerlings

Twelve experimental diets (D‐1 to D‐12) in a 4 × 3 factorial design (four protein levels: 250, 350, 400 and 450 g kg^?1 and three lipid levels: 50, 100 and 150 g kg^?1) were formulated. Carbohydrate level was constant at 250 g kg^?1. Rohu fingerlings (average wt. 4.3 ± 0.02 g) were fed the experimental diets for 60 days in three replicates at 2% BW  day^?1. Weight gain (%), specific growth rate (% day^?1) and feed gain ratio (FGR) indicated that diets containing 450 g kg^?1 protein and 100 or 150 g kg^?1 lipid (diets D‐11 and D‐12) resulted in best performance, although results were not significantly different from those of diet D‐9 (400 g kg^?1 protein and 150 g kg^?1 lipid). Protein efficiency ratio was highest with diets D‐6 (350 g kg^?1 protein and 150 g kg^?1 lipid) and D‐9 (400 g kg^?1 protein and 150 g kg^?1 lipid) (P > 0.05) and declined with higher and lower protein diets at all levels of lipid tested. Elevated lipid level (50, 100 or 150 g kg^?1) did not produce better FGR in diets containing 400 and 450 g kg^?1 dietary protein (P > 0.05). The combined effects of protein and lipid were evident up to 400 g kg^?1 protein. Growth and FGR showed consistent improvement with increased lipid levels from 50 to 150 g kg^?1 at each protein level tested except with diets containing 450 g kg^?1 protein. Apparent nutrient digestibility (for protein, lipid and energy) did not show significant variation among different dietary groups (P > 0.05). Whole body protein and lipid contents increased significantly (P > 0.05) with dietary protein level. The results of this study indicate that rohu fingerlings are adapted to utilize high protein in diets with varying efficiency. The fish could utilize lipid to spare protein but there is no significant advantage from this beyond the dietary protein level of 350–400 g kg^?1 in terms of growth and body composition.     

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.     

The potential of periphyton-based culture of two Indian major carps, rohu Labeo rohita (Hamilton) and gonia Labeo gonius (Linnaeus)

The effects of periphyton, grown on bamboo substrates, on growth and production of two Indian major carps, rohu, Labeo rohita (Hamilton) and gonia, Labeo gonius (Linnaeus), were studied at the Bangladesh Agricultural University, Mymensingh. For each species, five ponds were provided with bamboo substrates and five ponds without substrate (control). Ponds were stocked at a rate of 10 000 ha^?1 in both treatments. There was no discernible difference in water quality parameters between treatments. A large number of plankton (39 genera) showed periphytic nature and colonized the bamboo substrates. Rohu grew faster, resulting in a 77% higher net production (P < 0.05) in the ponds with bamboo substrates compared with the ponds without substrate. In contrast, the growth and production of gonia did not vary significantly (P > 0.05) between the substrate and control ponds. Rohu seems to be a more suitable candidate for periphyton‐based aquaculture systems than gonia.     

The potential of deoiled sal (Shorea robusta) seed meal as a feedstuff in pelleted feed for Indian major carp, rohu, Labeo rohita (Hamilton), fingerlings

Ten isonitrogenous (350 g kg^1-crude protein) diets incorporating raw and soaked deoiled sal (Shorea robusta) seed meal at 200, 300,400, 500 and 600 g kg^?1 into a fish-meal-based diet were fed to rohu, Labeo rohita (Ham.), fingerlings for 60 days and fish performance was studied. Tannin from raw salseed meal could be reduced from 34 to 7 g kg^?1 by soaking in water for 16 h at room temperature. In terms of growth response, food conversion ratio and protein efficiency ratio, a diet containing 200 g kg^?1 soaked salseed meal resulted in significantly (P < 0.01) the best performance of the fish. Apparent protein digestibility (APD) values decreased with increasing level of oilseed meal. Carcass protein was highest in fish fed diets containing 200 g kg^?1 soaked salseed meal. The results showed that deoiled salseed meal can be incorporated in carp diets up to 200 g kg^?1 and 300 g kg^?1 in raw and treated forms respectively.     

Dietary isoleucine requirement of fingerling Indian major carp, Labeo rohita (Hamilton)

An 8‐week feeding experiment was conducted to quantify the dietary isoleucine requirement of fingerling Indian major carp, Labeo rohita (3.50 ± 0.04 cm; 0.40 ± 0.02 g) using amino acid test diets (400 g kg⁻¹ crude protein; 17.90 kJ g⁻¹ gross energy) containing casein, gelatin and l ‐crystalline amino acids. Six dietary treatments supplemented with graded levels of isoleucine (7.5, 10.0, 12.5, 15.0, 17.5 and 20.0 g kg⁻¹), in gradations of 2.5 g kg⁻¹ diet, were fed to triplicate groups of fingerlings to apparent satiation divided over two feedings at 07:00 and 17:30 h. Performance of the fish was evaluated on the basis of live weight gain, feed conversion ratio (FCR), protein efficiency ratio (PER), specific growth rate (SGR) and protein productive value (PPV). Statistical analysis of live weight gain, FCR, PER, SGR and PPV reflected significant differences among treatments. Live weight gain and conversion efficiencies were best with isoleucine at 15.0 g kg⁻¹ of diet. Live weight gain, FCR, PER, SGR and PPV data were also analysed using second‐degree polynomial regression analysis to obtain more accurate isoleucine requirement estimate which was found to be at 15.9, 15.3, 15.2, 15.8 and 15.7 g kg⁻¹ of dry diet, corresponding to 39.8, 38.3, 38.0, 39.5 and 39.3 g kg⁻¹ of dietary protein respectively. Based on the quadratic regression analysis of the live weight gain, FCR, PER, SGR and PPV, the optimum level of isoleucine for fingerling L. rohita is in the range of 15.2–15.9 g kg⁻¹ of dry diet, corresponding to 38.0–39.8 g kg⁻¹ of dietary protein. Maximum body protein, minimum moisture and fat were noted at 15.0 g kg⁻¹ of dietary isoleucine while the body ash remained constant among all the treatment levels. No mortality was recorded during the duration of the experiment.     

Stress mitigating and immunomodulatory effect of dietary pyridoxine in Labeo rohita (Hamilton) fingerlings

A 60‐day experiment was carried out to delineate stress mitigating and immunomodulatory role of dietary pyridoxine (PN) in Labeo rohita fingerlings exposed to endosulfan. Two hundred and seventy fingerlings were randomly distributed into six treatments in triplicates. Five iso‐caloric and iso‐nitrogenous purified diets were prepared with graded levels of pyridoxine. Six treatment groups were T₀ (10 mg PN+without endosulfan), T₁ (0 mg PN+endosulfan), T₂ (10 mg PN+endosulfan), T₃ (50 mg PN+endosulfan), T₄ (100 mg PN+endosulfan) and T₅ (200 mg PN+endosulfan). The role of pyridoxine on stress mitigation and immunomodulation was assessed by biochemical and haemato‐immunological parameters like aspartate aminotransaminase, alanine aminotransaminase, lactate dehydrogenase, malate dehydrogenase, superoxide dismutase and catalase were significantly (P<0.05) lower while acetylcholinesterase was significantly (P<0.05) higher in pyridoxine‐fed groups. Erythrocytes count, haemoglobin content and total serum protein, albumin, globulin, nitroblue tetrazolium and lysozyme activity were significantly (P<0.05) higher while cortisol and blood glucose were decreased significantly (P<0.05) in pyridoxine‐fed groups. Percentage survival after challenge with Aeromonas hydrophila was highest in T₀ group. The results obtained in present study indicate that dietary pyridoxine supplementation at 100 mg PN kg^?1 diet reduces the endosulfan‐induced stress and triggers immune response in L. rohita fingerlings.     

Allozyme variation of hatchery and river populations of rohu (Labeo rohita, Hamilton) in Bangladesh

The genetic variations of rohu (Labeo rohita, Hamilton) sampled from five hatchery populations (Arabpur, Brahmaputra, Comilla, Kishorganj and Natore) and three major river populations (the Halda, the Jamuna and the Padma) were analysed by allozyme electrophoresis. Ten enzymes encoded by 11 loci were screened, and six were polymorphic. Alleles at three loci (Est‐1^*, Gpi‐1^* and Gpi‐2^*) proved variable for hatchery and river populations, and the Mdh‐2^* locus exhibited heterozygous genotypes for river populations only. Polymorphic loci per population (27.3±5.3%), heterozygous loci per individual (15.5±1.2%) and relative gene diversity (0.27±0.08) in river populations were higher than those for hatchery populations (25.5±1.8%, 10.7±1.6% and 0.25±0.01 respectively). Also, the observed heterozygosity (Ho) and expected heterozygosity (He) (0.09±0.03 and 0.14±0.04 respectively) in river populations were higher than those in hatchery populations (0.08±0.01 and 0.11±0.01 respectively). The lower levels of genetic variability in hatchery populations suggested the occurrence of inbreeding and/or genetic drift. The pairwise population differentiation (F_ST) values showed a lower level of genetic differentiation between hatchery and river population pairs. The unweighted pair‐group method with arithmetic mean dendrogram of Nei's genetic distances showed a relationship between the genetic distance and geographic distance. The populations were clustered into three groups: the Padma in one group, the Halda in second group and the Jamuna, including five hatcheries, in the third group. Highly diversified rohu individuals were observed in the Padma and Halda Rivers, whereas less genetically variable individuals were found in the Jamuna River and five hatcheries. These findings can be useful for rohu hatchery propagation to enhance the sustainable aquaculture production.     

Dietary threonine requirement of fingerling Indian major carp,Labeo rohita (Hamilton)

An 8‐week feeding experiment was conducted in a water flow‐through system (26–28 °C) to determine the dietary threonine requirement of fingerling Labeo rohita (3.90±0.03 cm; 0.58±0.02 g). Growth, feed utilization and body composition of fish fed test diets (40% crude protein; 17.9 kJ g^?1 gross energy) with graded levels of l ‐threonine (0.75%, 1.0%, 1.25%, 1.50%, 1.75% and 2.0% dry diet) to apparent satiation were response variables used to assess threonine adequacy. Diets were made isonitrogenous and isoenergetic by adjusting the levels of glycine and dextrin. The amino acid profiles of the test diets were formulated to that of 40% whole chicken egg protein except for threonine. The performance of fish fed experimental diets was evaluated using calculated values for weight gain (g fish^?1), feed conversion ratio (FCR), protein efficiency ratio (PER) and protein productive value (PPV) data. Maximum weight gain (g fish^?1) (1.79), lowest FCR (1.39), highest PER (1.76) and PPV (0.33) were recorded at 1.50 g per 100 g dietary threonine. Statistical analysis of weight gain, FCR, PER and PPV data reflected significant differences (P<0.05) among treatments. Except for reduced growth performance in fish fed threonine‐deficient diets, no deficiency signs were noted. Weight gain, FCR, PER and PPV data were also analysed using second‐degree polynomial regression analysis to obtain a more accurate threonine requirement estimate, which was found, using each response variable, to be at 1.70, 1.63, 1.65 and 1.51 g per 100 g of dry diet, corresponding to 4.2, 4.07, 4.12 and 3.77 g per 100 g of dietary protein respectively. Based on the second‐degree polynomial regression analysis of the live weight gain, FCR, PER and PPV data, the optimum dietary level of threonine for fingerling L. rohita was found to be in the range of 1.51–1.70 g per 100 g of the dry diet, corresponding to 3.77–4.2 g per 100 g of dietary protein.     

Dietary leucine requirement of fingerling Indian major carp, Labeo rohita (Hamilton)

An 8-week feeding experiment was conducted to evaluate the dietary leucine requirement of fingerling Indian major carp, Labeo rohita (3.50±0.04 cm; 0.40±0.02 g) using amino acid test diets (40% crude protein; 17.90 kJ g⁻¹ gross energy) containing casein and gelatin as intact protein sources and l -crystalline amino acids. Growth performance and biochemical parameters were assessed by feeding six amino acid test diets supplemented with graded concentrations of leucine (0.75, 1.0, 1.25, 1.50, 1.75 and 2.0 g per 100 g) to triplicate groups of fingerlings to apparent satiation divided over two feedings at 07:00 and 17:30 hours. Performance of the fish was evaluated on the basis of live weight gain, feed conversion ratio (FCR), protein efficiency ratio (PER) and body protein deposition (BPD) data. Maximum live weight gain (315%), best FCR (1.35), highest PER (1.86) and BPD (33.9) were recorded at 1.50 g per 100 g dietary leucine. Statistical analysis of live weight gain, FCR, PER and BPD data reflected significant differences (P<0.05) among treatments. Live weight gain, FCR, PER and BPD data were also analysed using second-degree polynomial regression analysis to obtain more accurate leucine requirement estimate which was found to be at 1.57, 1.55, 1.52 and 1.50 g per 100 g of dry diet, corresponding to 3.92, 3.87, 3.80 and 3.75 g per 100 g of dietary protein respectively. Based on the quadratic regression analysis of the live weight gain, FCR, PER and BPD data, the optimum requirement of fingerling L. rohita for leucine is estimated to be in the range of 1.50–1.57 g per 100 g of the dry diet, corresponding to 3.75–3.92 g per 100 g of dietary protein.     

Use of different microbial probiotics in the diet of rohu,Labeo rohita fingerlings: effects on growth,nutrient digestibility and retention,digestive enzyme activities and intestinal microflora

Six iso‐nitrogenous (350 g protein kg^?1) and iso‐caloric (4100 kcal kg^?1) diets with or without probiotics supplementation namely T₁ (Basal feed (BF) without probiotics; control), T₂ (BF + Bacillus subtilis and Lactococcus lactis), T₃ (BF + L. lactis and Saccharomyces cerevisiae), T₄ (BF + B. subtilis and S. cerevisiae), T₅ (BF + B. subtilis, L. lactis and S. cerevisiae) and T₆ (BF + heat‐killed bacteria of B. subtilis, L. lactis and S. cerevisiae) were fed to Labeo rohita fingerlings (6.0 ± 0.06 g) for 60 days in triplicate tanks (30 fish per tank). In all probiotic‐supplemented diets, the probiotic concentration was maintained at 10¹¹ cfu kg^?1 feed. After 60 days of culture, the fish fed combination of three probiotics at equal proportion (T₅) had higher (P < 0.05) growth, protein efficiency ratio, nutrient retention and digestibility and lower (P > 0.05) feed conversion ratio over other treatment groups. Total heterotrophic bacterial population in intestine was drastically reduced on 15th and 30th days of sampling than the initial value (0 day of sampling) for T₃, T₄ and T₅ groups. Except T₆, the gut colonization of respective probiotics, which were supplemented through the diets, was also increased up to 30 days of culture of fish and thereafter remained constant.     

Differential inhibition of digestive proteases by tannin in two size groups of rohu (Labeo rohita,Hamilton): A biochemical and zymography study

This study was intended to evaluate tannin mediated inhibition of digestive proteases in two different size groups (fingerlings, F, 4.5 ± 0.7 cm; advanced fingerlings, AF, 18.2 ± 1.6 cm) of rohu, Labeo rohita. Graded levels (50, 100, 150 and 200 nM) of tannin (Gallotannin, 99% purity) were added to the enzyme extracts (30°C, 1 hr) prior to determination of enzyme activities. Changes in the activity of trypsin, chymotrypsin and total protease in relation to the control sets were determined through biochemical assay of enzymes and SDS‐PAGE zymography. The study revealed that tannin significantly inhibited trypsin, chymotrypsin and total protease activities in a dose dependent manner as evident from the regression equations. The degree of the inhibition appeared to be significantly higher for the F in contrast to the AF (F_5,66 = 282.311; p < 0.0001). Trypsin activities were reduced by 8.04 ± 0.19% to 52.68 ± 0.72% and 5.61 ± 0.22% to 39.46 ± 0.19% in F and AF, respectively. The reduction in Chymotrypsin activities ranged between 16.11 ± 0.03% to 38.02 ± 0.27% in F and 6.31 ± 0.07% to 22.80 ± 0.32% in AF. Total protease activities were reduced by 10.9 ± 0.07% to 49.60 ± 0.32% in L. rohitaF, whereas, it ranged between 5.19 ± 0.06% to 32.60 ± 0.13% in AF. On a comparative scale, the difference in tannin induced inhibition in F and AF were more prominent for trypsin and chymotrypsin than total protease. Further, nine protease activity bands (15.9–69 kD) with different electromobility were noticed in both the size groups. Subsequent densitometry analysis revealed that the average densities of the protease activity bands were gradually decreased with increasing level of tannin exposure (50–200 nM). The study might indicate adaptive tolerance to tannin in larger size groups and emphasizes the need for removal of tannin in the plant feedstuffs, especially for feeding the fingerlings of L. rohita.     

Effects of dietary inclusion of the beta-adrenergic agonist, salbutamol, on growth performance and whole-body composition of rohu Labeo rohita (Hamilton) fingerlings fed diets containing two protein levels

A 60-day feeding experiment was conducted in the laboratory to evaluate the effect of oral administration of the beta-adrenergic agonist (BAA), salbutamol, on growth, nutrient utilization and whole-body composition of rohu Labeo rohita (Hamilton) fingerlings (average weight 5.51 ± 0.07 g) acting as a repartitioning agent in intermediary metabolism and redistributing nutrients for muscle synthesis. Two diets (diets 30/0 and 40/0) were formulated containing 30% and 40% crude protein to serve as basal diets containing the same ingredient composition. Another four diets (diets 30/3, 30/6, 40/3 and 40/6) were prepared in the same way to contain either 3 or 6 mg kg⁻¹ BAA salbutamol at each protein level making the total of six experimental diets. Rohu fingerlings were fed with the experimental diets in three replicate treatments at a restricted feeding regime equivalent to 2% of body weight (BW) day⁻¹. A significant (P < 0.05) interaction effect was found between dietary protein level and BAA treatment. Dietary incorporation of BAA at both protein levels significantly (P < 0.05) increased growth and nutrient utilization in terms of weight gain (%), specific growth rate (SGR), feed:gain ratio (FGR) and protein efficiency ratio (PER). BAA treatment at the 6 mg kg⁻¹ concentration led to a 12% rise in growth with the 30% protein diet and a 6% rise with the 40% protein diet, above their respective controls (without BAA treatment). However, no significant differences were found by raising the BAA concentration from 3 to 6 mg kg⁻¹. The apparent digestibility values for protein, lipid and energy were only higher with the 30% protein diets containing BAA. At both protein levels tested, BAA exerted a significant (P < 0.05) positive influence on protein retention and a negative influence on lipid retention efficiency, although the differences between the 3 and 6 mg kg⁻¹ BAA diets were not significant. This indicated that BAA acted as a repartitioning agent in suppressing lipid deposition in favour of protein accretion. BAA induced significantly (P < 0.05) higher whole-body protein and lipid relative to the untreated groups. The condition factor improved significantly (P < 0.05) in the BAA-treated dietary groups. The fillet percentage displayed a direct correlation and the frame percentage a negative correlation with BAA concentration in the diets. The results indicate that the BAA, salbutamol, was effective in producing growth enhancement, improved body composition (higher protein and lower lipid accretion) and efficient nutrient utilization at the 3 mg kg⁻¹ dietary incorporation level and thus has a potential for application in formulated diets for the Indian major carp, rohu, under culture conditions.     

Effects of varying dietary protein levels on growth, reproductive performance, body and egg composition of rohu, Labeo rohita (Hamilton)

A 360‐day feeding trial was conducted to observe the influence of varying levels of dietary protein on growth, reproductive performance, body and egg composition of rohu, Labeo rohita. Twenty fish (40.4 ± 0.24 cm; 852 ± 4.9 g), stocked in outdoor concrete tanks (200 m²), in duplicate, were fed diets with varying levels (200, 250, 300, 350 and 400 g kg^?1) of crude protein exchanged with carbohydrate to apparent satiation, twice daily, at 09:00 and 17:00 h. Higher (P < 0.05) weight increment was discernible in fish fed dietary protein ≥300 g kg^?1. Gonadosomatic index was comparable (P > 0.05) among fish of different dietary groups except those fed 200 g kg^?1 protein diet which produced least values. Egg diameter remained unaffected (P > 0.05) by variations in levels of dietary protein. Relative fecundity was maximum (P < 0.05) in fish fed 250 and 300 g kg^?1 protein diets. With the exception of fish fed 200 g kg^?1 protein diet, fertilizability (%) remained unaffected (P > 0.05) by variations in dietary protein level. Hatchability (%) followed the trend of variations almost similar to that of fertilizability. Proximate composition of muscle and eggs varied significantly (P < 0.05) with dietary protein levels. For broodstock L. rohita, a dietary protein level of 250 g kg^?1 was found optimum with regard to its reproductive performance, egg quality and composition.     

Effects of paddy straw and sugarcane bagasse on water quality, bacterial biofilm production and growth and survival of rohu, Labeo rohita (Hamilton)

Sugarcane bagasse (T₁), paddy straw (T₂) and their mixture (T₃) were added to mud‐bottomed, manured cement tanks in triplicate to study the effect on bacterial biofilm production and growth and survival of rohu, Labeo rohita. All tanks were stocked with fingerling rohu at 10 000 ha^?1. The total plate count (TPC) of bacteria in water and on substrate did not differ significantly between treatments. The mean phytoplankton and zooplankton density in water was the highest in T₂, followed by T₃ and T₁. The growth of fish was significantly (P<0.05) higher in T₂ and T₃ compared with T₁. Significantly higher RNA values were recorded in T₂ and T₃ compared with T₁. Similarly, T₂ and T₃ registered higher DNA values than T₁. RNA:DNA ratio was higher in T₂ compared with T₁. However, T₃ did not differ significantly from T₁ and T₂. Both intestinal and hepatopancreatic protease and lipase activity was higher in T₁ and T₂ compared with T₃ and both differ significantly from T₁. Intestinal amylase activity was higher in T₂ and T₃ compared with T₁, whereas hepatopancreatic amylase activity was higher in T₂ compared with T₁ and T₃. The results demonstrated that production of L. rohita can be significantly increased by the introduction of biodegradable substrates into culture systems where fertilization is employed.     

Interacting effects of dietary lipid level and temperature on growth, body composition and fatty acid profile of rohu, Labeo rohita (Hamilton)

Three isonitrogenous (320 g kg^?1 crude protein, casein and gelatine) semi‐purified diets with 80 (L8), 130 (L13) and 180 (L18) g kg^?1 lipid (sunflower oil at increasing levels and cod liver oil fixed at 50 g kg^?1) at three digestible energy levels (12 096, 13 986 and 15 876 kJ kg^?1 dry weight) and were tested, in triplicate, on rohu fingerlings (3.2 ± 0.08 g) at two different temperatures (21 and 32 °C). Fish were fed to apparent satiation, twice daily, at 09.00 and 15.00 h, 7 days a week for 56 days. Maximum growth was obtained at a lipid level of 80 g kg^?1 (L8) at 21 °C (439.37%) and 130 g kg^?1 (L13) at 32 °C (481.8%). In general growth rate was higher at 32 °C than at 21 °C at all lipid levels. Tissue monounsaturated fatty acid (MUFA) contents decreased with increasing lipid level at 32 °C, but the reverse occurred at 21 °C. At 21 °C, Polyunsaturated fatty acid (PUFA) level increased significantly (P > 0.05) over initial values, but was affected insignificantly by dietary lipid level. At 32 °C, fish fed diet L13 had more n‐3 fatty acid (FA) in liver and muscle than the other two dietary groups while at 21 °C, both liver and muscle FA profiles exhibited significant change (P > 0.05) in n‐3 and n‐6 FA content which corresponded to variation in percent addition of dietary lipid. However, n‐3/n‐6 ratio was higher for fish fed diet L13 at 32 °C and diet L8 at 21 °C and may be correlated with fish growth.     

Dietary Phosphorus Requirement of Fingerling Indian Major Carp,Labeo rohita (Hamilton)

Dietary phosphorus requirement of fingerling Labeo rohita (6.1 ± 0.13 cm; 1.88 ± 0.05 g) was quantified by feeding seven isonitrogenous (350 g/kg crude protein) and isocaloric (16.72 kJ/g gross energy) purified diets with different levels of phosphorus as 3.5 (basal diet), 4.6, 5.7, 6.5, 7.8, 8.9, and 10.1 g/kg. Triplicate groups of fish were fed at 0800, 1200, and 1600 h to apparent satiation for 8 wk. Live weight gain (LWG; 494.68%), specific growth rate (3.18%/d), feed conversion ratio (1.54), feed efficiency (0.65), protein gain (PG; 1.26 g/fish), protein efficiency ratio (1.86), and phosphorus utilization efficiency (98.78%) improved significantly (P < 0.05), with increasing dietary phosphorus level up to 6.5 g/kg. However, phosphorus contents of vertebrae and scale increased significantly up to 7.8 g/kg. Dietary phosphorus levels significantly affected serum phosphorus concentration and alkaline phosphatase activity. Broken‐line analysis based on LWG; PG; and whole‐body, vertebrae, and scale phosphorus against dietary phosphorus indicated the optimal phosphorus requirement of fingerling L. rohita at 6.56, 6.58, 6.56, 8.02, and 8.44 g/kg diet, respectively. In order to restrict superfluous phosphorus in the diet, inclusion of 6.56 g/kg phosphorus is recommended for optimal growth of fingerling L. rohita.     

Evaluation of the nutritive value of Leucaena leucocephala leaf meal, inoculated with fish intestinal bacteria Bacillus subtilis and Bacillus circulans in formulated diets for rohu, Labeo rohita (Hamilton) fingerlings

Nine isonitrogenous (35% crude protein approximately) and isocaloric (18.37 kJ g^?1) experimental diets (RLL20–BCFL40) were formulated with either raw or treated (inoculated with fish intestinal bacteria) Leucaena leucocephala leaf meal at 20%, 30% and 40% levels replacing other ingredients partially from a fish meal based reference diet (RD). Two specific strains of fish intestinal bacteria, Bacillus subtilis (isolated from Cyprinus carpio) and B. circulans (isolated from Oreochromis mossambicus) having extracellular cellulolytic and amylolytic activities, were used to inoculate Leucaena leaf meal for 15 days at 37°C. The crude fibre, cellulose and hemicellulose contents and the antinutritional factors, tannin, phytic acid and mimosine in the leaf meal decreased due to inoculation. However, free amino acids and fatty acids increased in the treated leaf meal. The response of rohu, Labeo rohita, fingerlings fed the experimental diets for 80 days was compared with fish fed a RD. Both the inclusion level and type of Leucaena leaf meal in diets significantly affected the growth performance of rohu. Fish fed diets containing inoculated Leucaena leaf meal performed better in comparison with those with the RD. On the basis of growth response, feed conversion ratio, protein efficiency ratio and apparent net protein utilization, diet formulated with 30%Leucaena leaf meal inoculated with B. circulans resulted in the best performance of rohu fingerlings followed by diet with 40%B. subtilis inoculated Leucaena leaf meal. The apparent protein digestibility (APD) was better in fish fed diets containing B. circulans inoculated leaf meal. An increasing level of raw Leucaena leaf meal was associated with a decrease in the carcass protein content of rohu fingerlings. The activity of α‐amylase increased with the increasing level of treated leaf meal in diets. Cellulase activity increased with increasing level of inclusion of raw leaf meal, and was comparatively lower in fish fed diets with treated leaf meal. Activities of protease and lipase were higher in fish fed the RD. The results showed that it is possible to incorporate Leucaena leaf meal inoculated with enzyme‐producing fish intestinal bacteria in carp diets up to 40% level of inclusion.     

Effect of fermentation on the nutritive value of sesame seed meal in the diets for rohu, Labeo rohita (Hamilton), fingerlings

Six isonitrogenous (350 g kg⁻¹ crude protein) and isoenergetic (17573 kJ kg⁻¹) experimental diets incorporating raw and fermented sesame ( Seasamum indicum ) seed meal at 200, 300, and 400 g kg⁻¹ into a fishmeal based diet were fed to rohu Labeo rohita fingerlings for 60 days and the growth performance and feed utilization efficiency of the fish was studied. The antinutritional factor phytic acid, from raw sesame seed meal, could be reduced below detection limit by fermentation with lactic acid bacteria ( Lactobacillus acidophilus ). Fermentation of the oilseed meal resulted in reduction of the tannin content from 20 to 10 g kg⁻¹. In terms of growth response, feed conversion ratio and protein efficiency ratio, a diet containing 400 g kg⁻¹ fermented sesame seed meal resulted in a significantly ( P  < 0.01) best fish performance. In general, growth and feed utilization efficiencies of fish fed fermented sesame seed meal diets were superior to those fed raw oilseed meal diets. Apparent protein digestibility (APD) values decreased with increasing levels of raw oilseed meal. APD was, however, significantly ( P  < 0.01) higher at all levels of incorporation of fermented sesame seed meal, while diets containing raw oilseed meal resulted in poor protein and lipid digestibility. Carcass protein and lipid contents of fish fed fermented sesame seed meal diets increased with increasing level of incorporation, being highest with 400 g kg⁻¹ fermented oilseed meal-containing diet. The results showed that sesame seed meal may be incorporated in carp diets up to 200 g kg⁻¹ and 400 g kg⁻¹ in raw and treated (fermented) forms respectively.