Microbial Phytase Supplementation in Rohu, Labeo rohita, Diets Enhances Growth Performance and Nutrient Digestibility

Abstract.— A feeding trial was conducted for 60 d to study the effect of dietary microbial phytase on growth performance and nutrient digestibility of rohu, Labeo rohita , fingerlings. One hundred and twenty fingerlings (average initial weight: 9.17 g/fish) were equally distributed into five experimental tanks, each with four replicates. Five isonitrogenous (35%) and isocaloric (16.79 kJ/g) diets were prepared from plant-based ingredients, supplemented with microbial phytase at the level of 0, 250, 500, 750, and 1000 U/kg diets and fed to T₀, T₁, T₂, T₃, and T₄ groups, respectively. Weight gain %, food conversion ratio, protein efficiency ratio, and apparent net protein utilization were significantly ( P  < 0.05) improved in groups fed phytase-supplemented diets compared to control, the highest being observed in T₃ group. Maximum apparent digestibility of phosphorus and crude protein was recorded in T₃ group. Bone ash, phosphorus (P), and calcium (Ca) contents were also significantly ( P  < 0.05) increased in phytase-fed groups. However, maximum was recorded in T₃ group. Results from the present study indicated that addition of 750 U microbial phytase/kg diets effectively improved nutrient utilization, bone mineralization, and hence growth of L. rohita fingerlings.     

Dietary Tryptophan Requirement for the Growth of Rohu,Labeo rohita

ABSTRACT

The dietary tryptophan requirement for rohu, Labeo rohita (an Indian major carp) was determined by conducting a growth study. Diets (40% protein) contained casein and gelatin supplemented with crystalline amino acids to provide an amino acid profile similar to rohu muscle, except for tryptophan. The experimental diets contained graded levels of tryptophan (0.15, 0.38, 0.63, 0.88, 1.13, 1.38% dietary protein). Each test diet was fed to triplicate groups of rohu fingerlings twice daily to satiation for 8 weeks. The dietary tryptophan requirement estimated by employing broken-line regression analysis was 1.13% of dietary protein. Maximum survival, food efficiency, and specific growth rate were obtained by fish fed the diet containing above level of tryptophan.     

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.     

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.     

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.     

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.     

Effects of probiotic doses on the survival and growth of hatchlings,fry, and advanced fry of Rohu (Labeo rohita Hamilton)

ABSTRACT

Rohu (Labeo rohita) is the most popular fish in South Asia. Its farming is often constrained by shortages of seed due to low survival (20%–30%) during nursing. Hypothesizing that probiotics might help, varying doses of two multistrain commercial probiotics were tested at hatchling (days 8–38), fry (days 38–68), and advanced fry (days 68–98) stages. The first probiotics (P1) consisted of Bacillus subtilis, Pediococcus acidilactici, and yeast (Saccharomyces cerevisiae) and the second probiotics (P2) of Bifidobacterium and Lactobacilli plus yeast (Saccharomyces cerevisiae), microalgae (Spirulina sp.), and phytase. Low, medium, and high doses of P1 (1.0, 2.0, and 3.0 g/kg) and P2 (0.5, 1.0, and 2.0 g/kg) were tested, supplementing the control diet, which consisted of mustard oil cake (40%), rice bran (30%), wheat flour (20%), and fish powder (10%). At the hatchling stage results showed that medium and high doses of P1 increased survival by 14.4% and 16.2% respectively over the control. Similarly, medium and high doses of P2 increased survival by 22.1% and 22.3% respectively compared to the control. Survival of hatchlings and fry increased linearly (P < 0.01) with the increase in the doses of P1. The dose of P2 had a quadratic relationship with the survival based on which dose of 1.5 g and 1.7 g/kg feed was found to maximize the survival of hatchlings (72%) and fry (99%) respectively. Regression analysis also showed that maximum specific growth rate (SGR%/day) of fry could be obtained with the estimated dose of 3.3 g/kg diet for P1 and for 1.5 g/kg diet for P2. Therefore, tested probiotics are beneficial during the nursing of Rohu hatchlings and fry or before the age of 68 days but not for nursing afterwards.     

Water Quality and Growth of Rohu,Labeo rohita,in a Biofloc System

We evaluated the technical feasibility of reducing water dependency of rohu, Labeo rohita, culture with biofloc under light-limited indoor culture. Biofloc and control treatments were conducted in 700-L indoor tanks at three different stocking densities (STD): 1.3, 2.6, and 3.9 Nos. fish m^?2 of surface area of tank for a period of 90 days. In biofloc treatment, fish were fed 20% crude protein feed and extra organic carbon in the form of wheat flour, whereas in case of control treatment they were fed 30% crude protein feed only. Fish survival was 100% in both the treatments. Lower stocking density produced larger fish, but growth was similar within stocking densities among control and biofloc treatments. The nutritional quality of biofloc was found to be quite suitable for rohu. Frequency of water exchange was significantly less in biofloc treatments as compared to the control ones.     

Growth Response and Biochemical Composition of Rohu,Labeo rohita,Fed Salt-Incorporated Diets

Abstract

Rohu, Labeo rohita, fingerlings (av. wt. 1.6 g) were fed pelleted diets supplemented with 0,0.5,1.0,1.5 and 2.0% sodium chloride (NaCl) for 120 days in 25-m³ cement tanks. The final weight gain of fish fed 0.5 and 1.0% NaCl-incorporated diets was significantly (P < 0.05) higher than that of the control (0% NaCl). Net protein retention and carcass protein and fat contents were higher under different treatments as compared to the control. Growth indices and carcass composition indicate inclusion of 1.0% NaCl in the diet to be beneficial for growth promotion in rohu.     

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.     

Optimum formulation of feed for rohu, Labeo rohita (Hamilton), with biofloc as a component

The present study was conducted to design an optimum feed mix for the growth of rohu (Labeo rohita) in light-limited indoor culture with biofloc as a component along with fish feed. Eighteen 700-L tanks were utilized for producing biofloc using aquaculture effluent from a nearby carp culture pond. Fifteen different feed mixes were prepared using fish feed and biofloc in dry (4 % moisture content) and wet (90 % moisture content) forms at different proportions and used in feeding trials (three replications) conducted in forty-five 50-L glass aquarium stocked with three rohu fingerlings with average individual weight of 20 ± 1.5 g for a period of 90 days. Fish survival was 100 % in all the treatments. Mixture design was used to obtain a solution of best combination of feed source to obtain the optimum growth parameters of rohu. Optimum growth parameters (net yield, specific growth rate, protein efficiency ratio and feed conversion ratio) of rohu were obtained at feed mix containing 50 % fish feed and 50 % wet floc. The nutritional quality of biofloc was found to be quite suitable for rohu. The images of 3-week-old biofloc captured in scanning electron microscope (SEM) indicated the presence of different types of bacteria, algae, protozoa, rotifers, etc. in different sizes ranging from 10 to 100 μm.     

Dietary protein level, microbial phytase, citric acid and their interactions on bone mineralization of Labeo rohita (Hamilton) juveniles

A feeding trial was conducted for 60 days to study the effect of dietary protein, microbial phytase and citric acid on intestinal digesta pH, bone ash and bone mineral contents in Labeo rohita juveniles. Eight experimental diets were prepared in 2 × 2 × 2 factorial arrangement with crude protein levels (25% and 35%), microbial phytase (0 and 500 U kg^?1), and citric acid (0 and 3%). The 25% crude protein level feed was supplemented with phytase (U kg^?1) and citric acid (%) at the level of 0,0 (C₂₅); 500,0 (T₁); 0,3 (T₂); 500,3 (T₃), and 35% crude protein level feed at 0.0 (C₃₅); 500,0 (T₄); 0,3 (T₅) and 500,3(T₆) respectively. One hundred and twenty juveniles of L. rohita (av. wt. 12.61–13.72 g) were distributed randomly in eight treatments, each of with three replicates. Addition of citric acid in the 25% crude protein feed significantly decreased (P<0.001) feed pH with concurrent decrease in intestinal digesta pH (P<0.001) and increased the bone ash content (P<0.05) by 4.6%. An interaction between citric acid and phytase (P<0.05) was also observed for bone ash content. Increasing the dietary protein content from 25% to 35% significantly decreased (P<0.01) bone Zn content by 14.9%, which was more prominent with the addition of citric acid, resulting in significant interaction between protein and citric acid (P<0.05), but the bone Cu content was significantly increased (P<0.01) with increasing dietary protein content. Dietary supplementation of microbial phytase (500 U kg^?1) significantly increased (P<0.05) bone Na, Ca, K, P and Fe contents by 15%, 12.1%, 17.4%, 9.2% and 40.7%, respectively, whereas bone P and Mn content was significantly increased (P<0.05) by addition of citric acid (3%). Addition of phytase to plant‐based diets increased the bioavailability of minerals, thereby increasing bone mineralization. The effect of phytase was increased because of addition of citric acid (3%).     

Arginine and histidine requirements of the Indian major carp, Labeo rohita (Hamilton)

Growth studies were conducted to quantify requirements for the essential amino acids (EAA), arginine and histidine in Labeo rohita (Hamilton). Diets incorporating casein and gelatine, as sources of intact protein, supplemented with crystalline amino acids were formulated to a crude protein content of 400 g kg^?1. Diets with six graded levels of arginine (14.6, 17.0, 19.0, 21.0, 23.0 and 25 g kg^?1) and histidine (3.2, 5.0, 7.0, 9.0, 11.0 and 13.0 g kg^?1) were formulated and fed to triplicate groups of juvenile rohu twice a day up to satiation for 60 days. Dietary requirements for arginine and histidine for rohu, estimated using break point analysis, were 23 and 9 of the diet respectively (57.5 and 22.5 g kg^?1 of dietary protein). Food conversion rate, specific growth rate and survival were better in treatments with diets containing optimum levels of EAA.     

Utilization of electron beam irradiated Jatropha kernel meal in the diet of Labeo rohita (Hamilton, 1822) fingerlings

Defatted Jatropha kernel meal (DJKM) was irradiated through electron beam radiation at 25 kGy (IJKM). After irradiation, PEs and phytate were decreased by 36.67% and 55.27%, respectively, with slight reduction in total hydrolysed amino acids in IJKM. A 45‐day feeding trial was conducted to evaluate the utilization of irradiated Jatropha kernel meal (IJKM) in the diet of rohu (Labeo rohita) fingerlings. Five isonitrogenous (300 g/kg CP) and isoenergetic (15 MJ/kg GE) diets such as T0 (control, without IJKM), T5 (50 g/kg IJKM), T10 (100 g/kg IJKM), T15 (150 g/kg IJKM) and T20 (200 g/kg IJKM) were prepared and fed to fish of respective treatments. Fish fed diets containing T15 and T20 groups exhibited significantly lower (p < .05) weight gain, FCE, PER, ANPU, HSI, ISI, survival rate, nutrient and energy digestibility, than the other groups. Fish of higher IJKM fed groups (T15 and T20) also showed lower muscle moisture, protein, ash and higher muscle lipid content. The liver catalase and SOD activities significantly decreased in the higher IJKM fed groups. It is concluded that IJKM (irradiated by 25 kGy electron beam) can be incorporated up to 100 g/kg in carp feed with the replacement of 33% soybean meal and 28% ground nut oil cake without compromising growth performances of Labeo rohita.     

Production performance of Labeo calbasu (Hamilton) in polyculture with three Indian major carps Catla catla (Hamilton), Labeo rohita (Hamilton) and Cirrhinus mrigala (Hamilton) with provision of fertilizers,feed and periphytic substrate as varied inputs

Inclusion of kalbasu, Labeo calbasu as a candidate species in the Indian major carps based polyculture system was evaluated through a six-month grow-out trial in earthen ponds of 0.08 ha each. Species performance was assessed through provision of varied inputs viz., fertilizers (T-1), fertilizers + supplementary feed (T-2) and fertilizers + supplementary feed + periphytic substrate (T-3) as the three treatments, which were evaluated in replicates. Catla (35%), rohu (35%), mrigal (15%) and kalbasu (15%) were stocked at combined density of 7500 fingerlings/ha. While ponds were fertilized with cowdung, urea and single super phosphate, mixture of groundnut oilcake and rice bran at 1:1 (w/w) was provided as supplementary feed. The periphytic substrate, comprised stripe bamboo mat, was provided at 10% of the pond surface area. Provision of each additional input caused significantly higher increase in overall mean survival, growth, SGR and net biomass yield of carps. Among the carp species, while only rohu and kalbasu showed significantly higher weight gain (234.4 g and 170.3 g, respectively) in T-3, no such increase was noticed either in catla or mrigal. The net production in T-3 (1516.1 ± 24.3 kg ha^− 1 6 months^− 1) was 13.0 and 73.2% higher than those of T-2 (1341.7 ± 15.5 kg ha^− 1 6 months^− 1) and T-1 (875.2 ± 15.6 kg ha^− 1 6 months^− 1), respectively. The study revealed the relative advantage of using periphytic substrates in carp polyculture systems with kalbasu as a component species.     

Kinetics of Total Volatile Basic Nitrogen and Trimethylamine Formation in Stored Rohu (Labeo rohita) Fish

Fish freshness estimation has been mostly studied in terms of total volatile basic nitrogen (TVB-N) and trimethylamine nitrogen (TMA-N) determination and partially addresses the kinetics, which limits its practical utility. In this study, TVB-N and TMA-N content of Rohu stored under limited refrigerated (5 and 0°C) and frozen (?5°C) conditions for 42 days showed an increasing trend, respectively. TVB-N and TMA-N formation in stored Rohu was higher at higher temperature and their initial and final values were found 4.57 ± 0.321, 0.14 ± 0.047 mg/100g and 46.56 ± 0.994, 3.24 ± 0.112 mg/100g, respectively. The volatile formations in Rohu at ?5°C were slow, as the frozen storage arrests the enzymatic and microbial activities in comparison to 0°C and above. The kinetics of TVB-N and TMA-N were investigated using zero, first, and nth-order reaction model. First-order kinetics exhibited the best fit model for TVB-N (R² = 0.98; χ² = 0.045), whereas TMA-N formation showed nth-order kinetics (n = ?0.4608, R² = 0.96, χ² = 0.028) as best fit model. The first-order kinetic reaction contributed activation energy of 58.16 kJ/mol for TVB-N and 10.86 kJ/mol for TMA-N. TVB-N and TMA-N formations were influenced by storage temperature and duration as expected. Kinetic parameters were also significantly (P ≤ 0.05) affected by storage temperatures.     

Secondary stress responses in Indian major carps Labeo rohita (Hamilton), Catla catla (Hamilton) and Cirrhinus mrigala (Hamilton) fry to increasing packing densities

Glycogen content and metabolic enzyme activities viz. lactate dehydrogenase (LDH), malate dehydrogenase (MDH), aspartate amino transferase (AST) and alanine amino transferase (ALT) in Indian major carps, Labeo rohita, Catla catla and Cirrhinus mrigala, were investigated after a 6 h transportation trial to compare the species‐specific variation and the effect of increased packing density on the metabolism. Fish (45±5 mm, 0.5±0.1 g) were packed in three densities (100, 150 and 200 L^?1) for the experiment, and 12 specimens of each species were randomly sampled from all the treatments at the end of transportation. The glycogen content of L. rohita ingerlings decreased significantly (P<0.05) with increasing packing density. The activities of enzymes LDH, MDH, AST and ALT showed a rising trend with increasing packing density in all the three species. Species‐specific differences were observed in various tested parameters at the lowest packing density (100 fry L^?1). Alanine amino transferase and LDH activities were significantly (P<0.05) lower in C. mrigala as compared with the other two species. However, glycogen reserves and MDH activity were not significantly different (P>0.05) among the species. The present study reveals that the optimum packing density for Indian major carp fry (100 fry L^?1) for transportation up to 6 h and metabolic regimes are species specific during transportation.