Digestive proteases of three carps Catla catla, Labeo rohita and Hypophthalmichthys molitrix: partial characterization and protein hydrolysis efficiency

Characteristics and functional efficacy of digestive proteases of Catla catla, catla, Labeo rohita, rohu and Hypophthalmichthys molitrix, silver carp were studied. Total protease activity was significantly (P < 0.05) higher in rohu (1.219 ± 0.059 U mg protein⁻¹ min⁻¹) followed by silver carp (1.084 ± 0.061 U mg  protein⁻¹ min⁻¹), and catla (0.193 ± 0.006 U mg  protein⁻¹ min⁻¹). Trypsin activity of silver carp and rohu was 89–91% higher than catla. Chymotrypsin activity was significantly (P < 0.05) higher in silver carp compared with rohu and catla. The protease activity of rohu and silver carp displayed bell‐shaped curves with maximum activity at pH 9; whereas in catla, maximum activity was found between pH 8 and 11. Inhibition of protease activity with soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF) revealed the presence of serine proteases and inhibition of activity with N‐α‐p‐tosyl‐L‐lysine‐chloromethyl ketone (TLCK) and N‐tosyl‐L‐phenylalanychloromethane (TPCK) indicated the presence of trypsin‐like and chymotrypsin‐like enzymes in all these three carps. SDS‐PAGE showed the presence of several protein bands ranging from 15.3 to 121.9 kDa in enzyme extracts of catla, rohu and silver carp. The substrate SDS‐PAGE evidenced the presence of various protease activity bands ranging from 21.6–93.7, 21.6–63.8 and 26.7–98.5 kDa for catla, rohu and silver carp respectively. In pH‐stat hydrolysis of Chilean fishmeal showed significantly (P < 0.05) higher degree of hydrolysis compared with soybean meal, silver cup (a commercial fish feed of Mexico) and wheat flour, with enzyme preparations of three fishes. The rate of hydrolysis was significantly (P < 0.05) higher in silver carp compared with others.     

Improved survival of rohu, Labeo rohita (Hamilton-Buchanan) and silver carp, Hypophthalmichthys molitrix (Valenciennes) fingerlings using low-dose quinaldine and benzocaine during transport

The effects of fingerlings immersion in low‐dose benzocaine (15 and 30 mg L⁻¹, silver carp and rohu) and quinaldine (100 μL L⁻¹ silver crap and 250 μL L⁻¹ rohu) for 1, 3 and 6 h on stress responses and survival of rohu, Labeo rohita and silver carp, Hypophthalmichthys molitrix fingerlings were evaluated in a transport simulation experiment. Both quinaldine and benzocaine showed low mortalities (0–2%). The total mortality in control (with no anaesthesia) was 30% for rohu and 14% for silver carp. Quinaldine and benzocaine‐treated fingerlings had significantly higher plasma chloride levels than the control in both species. Benzocaine, quinaldine, as well as the control, had an initial elevation of plasma cortisol levels. Benzocaine lost its effectiveness after 3 h exposure while quinaldine persisted throughout the 6 h experimental period. Both sedatives reduced bacterial build‐up compared with the control. No post‐exposure mortality was observed for any of the transport methods assessed 48 h after the treatment. This study suggests that the use of low‐dose benzocaine or quinaldine during transport has positive effect on the survival and health of rohu and silver carp fingerlings.     

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.     

Effect of plant protease inhibitors on digestive proteases in two fish species, Lutjanus argentiventris and L. novemfasciatus

This work provides a comparative study of the inhibitory effect of several plant protein sources on digestive proteases of two snappers: yellow snapper (Lutjanus argentiventris) and dog snapper (Lutjanus novemfasciatus). Seed extracts did not affect gastric proteases whereas they significantly inhibit intestinal proteases. Inhibition of alkaline proteases showed that pancreatic proteases of L. argentiventris were more sensitive to seed protease inhibitors than those of L. novemfasciatus. Legume seeds showed the highest inhibitory capacity on alkaline proteases causing inhibition higher than 50% in total proteolytic activity. Protease inhibition on digestive extracts was assessed using different relative concentration of seed extracts and represented by constructing dose response curves. In order to reduce the inhibitory effect, seed extracts were acid-treated before the inhibition assay. Results showed that acid treatment did not affect the inhibitory capacity of seeds on alkaline proteases in both species. However, when the action of gastric enzymes was simulated on seed extracts, the inhibitory capacity was reduced significantly, mainly in the case of L. novemfasciatus. The responses of fish enzymes to heat-treated seed extracts were also tested. Only higher temperatures were capable of reducing the inhibitory capacity of seed, with the specific response to the snapper species. The use of biochemical assays allows us to quantify the action of inhibitors on total proteolytic activity. In addition, zymograms obtained by substrate-SDS-PAGE provided qualitative information about the number and type of proteases affected by each inhibitor. Each seed extract produces a characteristic profile of inhibition on alkaline protease. The results obtained are important for future formulation of feeds for these snapper species.     

Use of probiotics Bacillus coagulans,Rhodopseudomonas palustris and Lactobacillus acidophilus as growth promoters in grass carp (Ctenopharyngodon idella) fingerlings

The present research evaluated the effects of three probiotics on the growth performance and intestinal digestive enzyme activity in fingerlings of grass carp, Ctenopharyngodon idella. Three treatments (T‐1, T‐2 and T‐3) were fed with diets containing different viable bacteria with a final concentration 10⁶ CFU g^?1 feed (Bacillus coagulans, Rhodopseudomonas palustris and Lactobacillus acidophilus, respectively) and the control was fed with basal diet without probiotics. All the diets supplemented with probiotics resulted in better (P < 0.05) final weight, daily weight gain and relative gain rate than control but no significant differences were observed among the treatment groups. The highest protease activity was observed in T‐1. However, T‐2 and T‐3 showed no difference (P > 0.05) in protease and cellulase activities compared with the control. The protease activity was higher in the foreintestine than in the hindintestine (P < 0.05), and the reverse was observed in cellulase activity. As for amylase activity, there was no difference between foreintestine and hindintestine. In conclusion, the three selected probiotics increased the growth performance of grass carp fingerlings. Furthermore, different digestive enzyme activity was observed in different intestine segment.     

Effects of dietary vitamin C on immunity, growth and survival of Indian major carp Labeo rohita, fingerlings

An 8‐week feeding trial was conducted to evaluate the efficacy of dietary l ‐ascorbic acid (AA) dosages on immunity, growth and survival of the fingerlings of Labeo rohita. AA was supplemented at 0, 100, 200 and 500 mg kg⁻¹ basal diets. Each diet was fed for 8 weeks to duplicate groups of fingerlings of rohu with initial body weight of 35 ± 5 g. Selected biochemical and haematological analyses were evaluated every second week. At the end of the feeding trial of 56 days, fish were divided into two subgroups under each major treatment group for challenge with two pathogens, Aeromonas hydrophila and Edwardsiella tarda. The mortality (%) and agglutinating antibody titre were recorded on 28th day postchallenge. Non‐specific immune parameters like superoxide anion production, lysozyme activity and complement activity increased independently of the levels of AA in the diet. Whereas phagocytic ratio, phagocytic indices, lymphokine production indices and serum bactericidal activity were better in the group of fish fed with 200 mg AA kg⁻¹ feed for 42 days. However, growth parameters such as specific growth rate, feed conversion ratio and survival were better in the group of fish fed with the highest level of AA through feed for 56 days. These data indicated that elevated levels of dietary AA could be recommended for optimum immunity, growth and survival of fingerlings of L. rohita.     

Phytic Acid‐Induced Inhibition of Digestive Protease and α‐Amylase in Three Indian Major Carps: An in vitro Study

Antinutritional effects of phytic acid (myoinositol hexaphosphate, IP6) on growth and digestibility in fish have been reported. However, specific effect of IP6 on the digestive enzymes in fish has not been addressed. In this study, inhibitory effect of synthetic IP6 (Phytic acid sodium salt, 90% purity) on the activity of the digestive protease and α‐amylase in rohu, Labeo rohita; catla, Catla catla; and mrigal, Cirrhinus mrigala has been investigated in vitro. Graded levels (12.5, 25, 50, 100, 150, and 200 µg/mL) of IP6 were added to the reaction mixtures containing enzyme extracts and substrate solution in triplicate to detect any change in enzyme activity. Results of the experiment revealed that IP6 significantly inhibit/lower activities of the digestive enzymes in a dose‐dependent manner, as evident from the regression equations (F values significant at P < 0.001 level). Apparently, irrespective of the fish species studied IP6‐induced inhibition of α‐amylase activity was greater than protease activity. Among the three fish species studied, C. mrigala appeared to be more sensitive to IP6 for both α‐amylase and protease activity. Enzyme activity was least affected in C. catla. Results of the study might raise concern while incorporating IP6 rich plant‐derived feed ingredients in aqua feed preparation.     

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.     

Partial characterization of digestive proteases of the three‐spot cichlid Cichlasoma trimaculatum (Günter 1867)

Partial characterization of digestive proteases in the three‐spot cichlid Cichlasoma trimaculatum juveniles was conducted. It was determined that there is higher alkaline proteases activity (3.95 ± 0.32 IU mg^?1 protein) compared to acidic proteases (2.01 ± 0.57 IU mg^?1 protein). Optimal temperature for alkaline proteases is 60 °C which resulted in more thermostability to temperature changes. On the other hand, optimal temperature for acidic proteases is 50 °C. Optimal pH for acidic proteases was pH 2, while for alkaline proteases, it was pH 10, which resulted in more stability in relation to pH changes than acidic protease. The use of specific inhibitors and the SDS‐PAGE electrophoresis analysis revealed seven types of bands for alkaline proteases, which make evident the main presence of serine proteases. In acidic proteases, more than 98 g kg^?1 of the activity was inhibited with pepstatin A inhibitor. Therefore, it is evident that C. trimaculatum digestion is composed by acidic and alkaline proteases; thus, it should be considered an omnivorous fish.     

Study of digestive proteinases and proteinase inhibitors of Daphnia carinata

Quantification of proteases activities and their class structure have been studied in a cladoceran, Daphnia carinata. Protease activity ranged from 0.28 to 0.55 Unit mg⁻¹ protein min⁻¹ with an average value of 0.42±0.06 Unit mg⁻¹ protein min⁻¹. Chymotrypsin activity was more than twofold higher (0.49±0.09 Unit mg⁻¹ protein min⁻¹) than the trypsin activity (0.21±0.02 Unit mg⁻¹ protein min⁻¹). Protease activity and reduction of activity in bands of samples treated with specific inhibitors were documented in photometric assay and substrate SDS–PAGE. Proteinase activity against azocasein was inhibited (91.4±1.5%) with SBTI. PMSF reduced the enzyme activity by 53.1±6.5%, and the azocasein hydrolysis was reduced up to 64.6±3.8% by the specific inhibitor of trypsin, TLCK. In the present investigation, the molecular weight of various activity bands ranged from 16.3 to 51.1 kDa. The molecular weights of several protein bands are similar to protease activity zones. The knowledge of digestive enzyme profiles of fish food organisms generated in the present study may assist in the formulation of age-specific feed.     

Effects of replacing soybean meal with rubber seed meal on digestive enzyme activity,nutrient digestibility and retention in tilapia (Oreochromis niloticus × Oreochromis aureus)

A feeding trial was conducted to evaluate the effects of replacing soybean meal (SBM) with rubber seed meal (RSM) on digestive enzyme activity, nutrient digestibility and retention in juvenile tilapia (Oreochromis niloticus × Oreochromis aureus). Five isonitrogenous and isoenergetic diets were formulated with 0 (control), 100, 200, 300 and 400 g kg⁻¹ RSM replacing graded levels of SBM respectively. Each diet was randomly assigned to triplicate groups of 30 fish (initial average weight 5.2 g) per aquarium in a rearing system maintained at 29 ± 1°C for 8 weeks. The hepatic protease and lipase activities gradually decreased with increasing dietary RSM level, but no significant differences were observed among the low inclusion level (0–200 g kg⁻¹) groups. The apparent digestibility coefficients of dry matter, crude protein, crude lipid and ash showed a similar trend as the hepatic protease and lipase activities. The retentions of protein and individual essential amino acid (except lysine, threonine and leucine) in fish fed diet with 200 g kg⁻¹ RSM were similar to those in fish fed the control diet. These results indicate that dietary RSM inclusion level up to 200 g kg⁻¹ did not markedly affect the digestive enzyme activity, nutrient digestibility and retention in tilapia, whereas these were depressed by the inclusion of 400 g kg⁻¹ RSM.     

Purification and characterization of stomach protease from the turbot (Scophthalmus maximus L.)

Proteolytic activity in the different parts of the digestive tract of the turbot (Scophthalmus maximus L.) were studied in this work. One pure protease was isolated from turbot stomach and its behavior was studied. Results showed the optimum pH for proteases in the different parts of the digestive tract of the turbot were pH 2.0 for the stomach, pH 8.0 for the pylorus cecum, pH 8.0 for the foregut, pH 8.5 for the midgut, and pH 8.0 for the hindgut. The activity of proteases in the different parts of the digestive tract were in the sequence pylorus cecum protease > stomach protease > foregut protease > midgut protease > hindgut protease. The stomach protease was purified by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose F.F. and Sephadex G-100. The purified enzyme gave a single band in SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Its molecular weight was found to be approximately 42,000 Da. The enzyme is stable at pH 1.0–9.0 and at temperatures below 40°C. Its activity was maximum at pH 2.0 and 40°C. When reaction time was prolonged the optimum temperature of the enzyme tended to decline. The enzyme was activated by Mn²⁺ and Cu²⁺ and inactivated by Fe³⁺. It was fully inhibited by pepstatin and partially inhibited by PMSF, TPCK, PCMB, and NBS. These results imply the enzyme is a pepsin.     

Viscera of Labeo rohita: A Potential Source of Trypsin for Industrial Application

ABSTRACT

The serine protease trypsin was isolated and purified from the digestive system of carp Labeo rohita rohu by ammonium sulphate precipitation, ion exchange, and affinity chromatography. The purified enzyme showed high activity between pH 7.0 and 9.0. The activity was maximum at 40°C. Incubation of the purified enzyme with CaCl₂ (2 mM) stabilized the enzyme activity for 8 h. The enzyme showed stability at 30 and 40°C for 1 h, but above 40°C, enzyme activity was reduced. The kinetic constants were recorded as K_m (0.104 mM), k_cat (44.25 s^?1), and catalytic efficiency (427.54 s^?1 mM^?1). Monovalent, bivalent, and trivalent ions (Li⁺, K⁺, Hg²⁺, Al³⁺, Mg^2+, Cd^2+, Co^2+, Zn²⁺, and Al³⁺) influenced the enzyme activity. Phenylmethylsulfonylflouride, soybean trypsin inhibitor, and N-α-p-tosyl-_L-lysine chloromethyl ketone completely inhibited the enzyme activity, while ethylenediaminetetraacetate caused partial inhibition. Molecular mass of the purified enzyme was 22.46 kDa. The pH and temperature stability of enzyme may be useful for its industrial applications.     

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.     

Nursing duration and pond fertilization level affect polycultures of Indian major carp (rohu Labeo rohita and mrigal Cirrhina mrigala) with monosex Nile tilapia Oreochromis niloticus

This study investigated the effects of nursing duration on the subsequent performance of rohu (R) Labeo rohita and mrigal (M) Cirrhina mrigala in polyculture with monosex male Nile tilapia (T) Oreochromis niloticus at four levels of pond fertilization. Nile tilapia, rohu and mrigal were stocked at a ratio of 4:1:1 in a 90‐day trial based on 40 20‐m² pens fixed in four 400‐m² earthen ponds. Growth of carp fingerlings during prolonged nursing (5 or 12 months) was stunted compared with fish nursed over a conventional duration of 3 months (3) but showed superior growth subsequently. Mean daily weight gain of stunted rohu (12) ranged from 2.2 to 2.8 g per fish day^?1 compared with 1.1–1.6 g per fish day^?1 for younger fish (3). The comparable ranges for mrigal were 1.9–2.8 and 1.4–2.1 g per fish day^?1. Growth of Nile tilapia was inversely related to duration of carp nursing at the four levels of fertilization. Nile tilapia showed more response to increasing levels of fertilizer input (Y=?1.421+1.716X, where Y is the daily weight gain of Nile tilapia and X is the fertilizer level, r²=0.98, P<0.01, n=12). At a high level of fertilization (3.0 kg N:1.5 kg P ha^?1 day^?1), performance of stunted fingerlings (5 and 12) of both rohu and mrigal was similar (range 2.3–2.8 g per fish day^?1, P>0.05), but younger mrigal (M3) grew faster than rohu (2.1 g per fish day^?1 and 1.6 g per fish day^?1 respectively). Older rohu (12) appeared to perform particularly well, and Nile tilapia poorly at the lowest level of fertilization (1.5 N:0.75 kg P ha^?1 day^?1), suggesting the impact of age of seed on competition within polycultures. The net fish yield (NFY) of tilapia was not affected significantly (P>0.05) by differential stocking age of carps; therefore, combined NFY of the three experimental fish species was not affected by the age of carp, as tilapia was the dominant species in polyculture. The highest combined NFY of all species in the most intensively fertilized pond (3.0 N:1.5 P kg ha^?1 day^?1) was calculated at 4.06±0.08 g·m^?2 day^?1, which was significantly higher (P<0.001) than the yield (1.82±0.12 g·m^?2 day^?1) from the pond with the lowest fertilization. At the highest fertilizer level, tilapia, rohu and mrigal contributed 72%, 14% and 14%, respectively, to the NFY, whereas the ratio was 60%, 20% and 20% at the lowest fertilization level. The study indicated that yields from tilapia in polyculture with the two carp species in more eutrophic water can be optimized if advanced nursing of carps is practised. Moreover, higher inputs of inorganic fertilizer and advanced nursing of carp are economically attractive under Bangladeshi conditions. Advanced nursing of rohu also improves its performance in more extensive systems when tilapia densities are high.     

Ontogenic changes in the digestive enzyme patterns and characterization of proteases in Indian major carp Cirrhinus mrigala

Digestive enzymes of Cirrhinus mrigala (Ham.) were studied during ontogenic development. Specific amylase activity was detected in first feeding fish. The enzyme activity decreased up to day‐18 and then it increased with the age of fish to reach the highest level on day‐34. Protease activity was 28.61 ± 8.90 mU mg protein^?1 min^?1 on day‐4 and increased with the age throughout the study period. Trypsin activity was 31.86 ± 1.12 mU mg protein^?1 min^?1 on day‐4. The activity decreased up to day‐10 and from day‐12 onwards increased up to day‐26. Chymotrypsin activity was 14.56 ± 2.74 mU mg protein^?1 min^?1on day‐4 and constantly increased up to day‐26. A significant increase in lipase activity was observed between days‐24 and 34. SDS‐PAGE and substrate SDS‐PAGE showed the diversity of protein (17.4–127.8 kDa) and protease activity bands (16.6–88.8 kDa) during ontogenesis. Soybean trypsin inhibitor, phenyl methyl sulphonyl fluoride, N‐α‐p‐tosyl‐l ‐lysine chloromethylketone and N‐tosyl‐l ‐phenylalanine chloromethylketone inhibited the protease activity up to 79.72–97.21, 65.55–94.83, 45.41–75.31 and 40.78–64.72%, respectively. Inhibition study in substrate SDS‐PAGE revealed the abundance of serine proteases and the presence of isoforms of trypsin and chymotrypsin. Ethylenediamine‐tetraacetate showed 5.56–22.78% inhibition of metal ion‐specific enzyme activity.     

Evaluation of raw and hydrothermically processed leguminous seeds as supplementary feed for the growth of two Indian major carp species

To study the effect of four different raw and hydrothermically processed leguminous seeds, namely Glycine max (soybean), Vigna radiata (moong), Vigna ungiculata (cowpea) and Cyamopsis tetragonaloba (guar) on growth in the fingerlings of Cirrhinus mrigala (mrigal) and Labeo rohita (rohu), two experiments were conducted. The first experiment was conducted under laboratory conditions (LD 12:12 at 25 °C) and the mrigal fingerlings were fed @2% BW d^?1 for 30 days. Before using the seeds as feeds, they were hydrothermically processed (15 lbs at 121 °C for 15 min) for the elimination of antinutrient factors (ANF). The results indicate that 15 minute autoclaving significantly reduces the trypsin inhibitor activity. Phytase‐phosphorous and tannins were also analysed from raw and treated beans. Studies on mrigal have revealed that weight gain was significantly low (P < 0.05) in the fish fed on raw beans compared with the fingerlings fed on processed bean proteins. Among the various raw diets used, significantly highest growth (P < 0.05) was observed in fish fingerlings fed on raw moong, followed by raw soybean, cowpea and guar. On the other hand, when beans were processed, the highest growth was observed in fingerlings fed on processed soybean. Studies have further revealed that hydrothermal processing also affected the values of the feed conversion ratio, gross conversion efficiency, protein efficiency ratio, gross protein retention, gross energy retention and ammonia excretion. Proximate composition of the carcass revealed high accumulation of protein, fat and energy in fingerlings fed on processed soybean in comparison with other diets. Keeping in view the higher weight gain in fish fed on hydrothermically processed bean proteins, only processed bean diets were fed to the fingerlings of mrigal and rohu stocked in fish ponds (experiment 2). A significant (P < 0.05) increase in mean fish weight, and specific growth rate was observed in fingerlings fed on hydrothermically processed soybean, followed by moong and cowpea. Weight gain for mrigal fingerlings remained significantly low in comparison to the rohu fingerlings fed on similar diets. An investigation on the effects of feeds on water quality parameters indicate that the trophic status of the ponds where soybean was used as fish feed remained high in comparison to the other treatments. A significant positive correlation of nutrients, NPP and plankton population with fish growth was also observed.     

Effects of a nonsteroidal aromatase inhibitor on gonadal differentiation of bluegill sunfish Lepomis macrochirus

In the present study, the efficacy of Letrozole, a potent nonsteroidal aromatase inhibitor (AI), on gonadal sex differentiation and sex reversal was examined in bluegill sunfish (Lepomis macrochirus). In Experiment 1, using AI diet treatments (50, 150, 250 and 500 mg kg⁻¹) from 30 to 90 days posthatch (dph), AI interrupted ovarian cavity formation at a dose of 500 mg kg¹ diet and one intersex fish was identified in this group. The proportions of males in all the treated groups were significantly higher than those in the control group. In Experiment 2, using AI immersion treatments (250, 500 and 1000 μg L⁻¹) during 30–50 dph, the treated groups of 500 and 1000 μg L⁻¹ produced significantly more males than the control and 250 μg L⁻¹ groups. Histological examination revealed no differences in ovary or testis tissue between control and AI‐treated fish. There were no significant differences detected in body weight and length among the AI treated and control groups (P>0.05) for both experiments. The results from these two experiments suggest that inhibition of aromatase activity by AI could influence sex differentiation in bluegill sunfish.     

Effect of different oil cake sources on growth, nutrient retention and digestibility, muscle nucleic acid content, gut enzyme activities and whole-body composition in silver barb, Puntius gonionotus fingerlings

Six iso‐nitrogenous (30% crude protein) and iso‐energetic (15 kJ g⁻¹) diets were prepared using different oil cake sources, viz. groundnut, soybean, sunflower, sesame, mustard and mixed oil cakes as major ingredients, and protein sources along with a minimum of 5% fish meal in each diet and were fed to silver barb Puntius gonionotus fingerlings (16.20±0.11 g) ad libitum four times a day close to an apparent satiation level for a period of 60 days to determine the effect of diets on growth, nutrient utilization, apparent digestibility coefficient (ADC) of the nutrients in the diets, gut enzyme activity, muscle nucleic acid content and whole‐body chemical composition of fish. Significantly higher (P<0.05) weight gain, specific growth rate, protein efficiency ratio, nutrient retention, ADC of nutrients in the diets, DNA:RNA ratio, protease and amylase activity with lower (P<0.05) feed:gain values were recorded in fish‐fed groundnut and soybean oil cake‐based diets than other diets tested. Among the dietary treatment groups, significantly higher (P<0.05) whole‐body protein, lipid and energy were also found in groundnut oil cake‐ and soybean oil cake‐based diets. The study suggests that the groundnut and soybean oil cake‐based diets, which led to significantly higher (P<0.05) growth and nutrient utilization than the other oil cake‐based diets in P. gonionotus fingerlings, may be used for pond culture of this species.     

Protein requirement of silver barb, Puntius gonionotus fingerlings

Five iso‐energetic (15.05 MJ kg^?1) semi‐purified diets with graded levels of crude protein, i.e. 200 (D‐1), 250 (D‐2), 300 (D‐3), 350 (D‐4) and 400 (D‐5) g kg^?1 diet were fed to Puntius gonionotus fingerlings (average weight 0.88 ± 0.03 g) in triplicate groups (15 healthy fish per replicate) for a period of 90 days to determine the optimum protein requirement of the fish. Fifteen flow‐through cement tanks of 100‐L capacity with a flow rate of 0.5 L min^?1 were used for rearing the fish. Specific growth rate (SGR), food conversion (food gain) ratio (FCR), nutrient digestibility and retention, digestive enzyme activity, RNA : DNA ratio and tissue composition were used as response parameters with respect to dietary protein levels and feed intake. The mean weight gains of fish after 90 days were 10.84 ± 0.27, 11.07 ± 0.12, 14.09 ± 0.20, 11.27 ± 0.12 and 10.91 ± 0.25 g for D‐1, D‐2, D‐3, D‐4 and D‐5, respectively. Maximum SGR (3.13 ± 0.02% per day), RNA : DNA ratio (10.09 ± 0.09), tissue protein content (160 ± 0.1 g kg^?1 wet weight), protease activity (25.27 ± 0.47 μg of leucine liberated mg tissue per protein h^?1 at 37 °C) and minimum FCR (1.60 ± 0.02) was found in D‐3 group fed with 300 g kg^?1 protein level. All these parameters were negatively affected with the further increase in protein level in the diet. Digestibility of protein, lipid and energy was not affected because of variation in dietary protein levels and nitrogen intake of fish. Maximum energy retention (27.68 ± 0.12%) was recorded at 300 g kg^?1 dietary crude protein fed group. However, using broken line regression analysis, the maximum growth was found to be at 317.7 g kg^?1 dietary protein. Hence, it may be concluded that the protein requirement of P. gonionotus fingerling is 317.7 g kg^?1 diet with a resultant P/E ratio of 21.1 g protein MJ^?1.