Comparative Study on Precipitation Techniques for Protease Isolation and Purification from Labeo rohita Viscera

Precipitation techniques play a vital role in the industrial extraction of enzymes. The present study aimed to extract the proteases from the viscera of Labeo rohita (commonly called Rohu) and to compare the precipitation techniques for the isolation and purification of the enzyme. The enzyme is usually discarded as tons of waste during processing. Hence, a trial has been carried out to isolate the protease enzymes from viscera of the freshwater fish Labeo rohita. The proteases were precipitated with ammonium sulfate, ethanol, and acetone. Acetone precipitation was found to be the best option for the recovery of enzymes (54%) from the viscera of Rohu, and two caseinolytic protease bands were shown in the zymogram. The precipitates with highest proteolytic activity were further subjected to dialysis, and their molecular weight was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).     

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.     

In vitro assay of plant protease inhibitors from four different sources on digestive proteases of rohu, Labeo rohita (Hamilton), fingerlings

The in vitro inhibitory effect of protease inhibitors from four seed extracts (soybean, grasspea, black gram and horse gram) on digestive proteases of rohu was assessed by enzyme inhibition assay and substrate sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. High proteolytic activity was detected in the intestinal extract of rohu (Labeo rohita) fingerlings at two different pH ranges (8–8.5 and 10–11). That protein digestion occurs mainly in the alkaline condition in this fish without a stomach is evident from very high trypsin activity (0.95±0.04 benzoyl‐dl ‐arginine‐p‐nitroanilide U mg protein⁻¹) in the intestine. In case of grass pea seed, more than 50% inhibition of alkaline protease activity was recorded when the ratio of inhibitor to enzyme was 9.41 μg U⁻¹. More than 40% inhibition of protease activity was recorded in case of horse gram seed when the ratio of inhibitor to enzyme was 5.51 μg U⁻¹. Black gram at 11.0 μg U⁻¹ and soybean seed proteins at 62.75 μg U⁻¹ resulted in 50% and more than 30% inhibition of digestive protease activity in rohu fingerlings respectively. A plot of the inhibition values obtained by changing the relative concentrations of enzyme/inhibitor resulted in different dose–response curves for different protein sources. The use of substrate gel electrophoresis allowed the visualization of the aforementioned differences in inhibition. Each seed extract produced a characteristic profile of protease inhibition. It is concluded that protease inhibitors present in plant protein sources adversely affect the digestive proteases in fish and hence there is a need to eliminate/reduce the amount of such inhibitors through proper processing before incorporation into aquafeeds.     

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.     

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.     

Ontogenic development of enzymatic activity and digestive system in Jullien's golden carp (Probarbus jullieni Sauvage, 1880)

Ontogenic development of the main enzymes and histological structure of digestive organs were studied in Jullien's golden carp (Probarbus jullieni) from hatching until 50 days after hatching (DAH). The larval fish were produced by artificial insemination and fed only Moina sp. till end of experiment. Body weight (mg) and total length (cm) of Jullien's golden carp increased exponentially and linearly. The results indicate the fish weight grew fast with increasing rate, while length increased at a constant rate over the studied period. Up‐regulation of acid protease was observed in newly hatched larvae and the specific activity gradually decreased with time. Trypsin specific activity was relatively stable within the first 35 DAH, while fluctuations in chymotrypsin were observed. Among these three proteolytic enzymes, acid proteases exihibited relatively high specific activity in newly hatched larvae, suggesting a role in yolk protein degradation. Alkaline proteases became more prominent with age and correlated with an abrupt decrease in acid proteases. Increased lipase‐specific activity appeared within 3 DAH and then gradually decreased with time, indicating the capacity to digest yolk lipid reserve. Amylase and cellulase‐specific activities changed in a similar manner, and the sensitivity to time was higher in amylase than in cellulase. The digestive organs and accessory organs developed around 3–5 DAH. However, intestinal histology was almost fully developed around 18 DAH. These findings should be useful for deciding the preferred timing for weaning, as well as on developing artificial diets referenced to the physiological changes of digestive enzymes.     

Partial characterisation of digestive proteases of the Mayan cichlid Cichlasoma urophthalmus

The characterisation of digestive proteases in native freshwater fish such as the Mayan cichlid Cichlasoma urophthalmus provides scientific elements that may be used to design balanced feed that matches with the digestive capacity of the fish. The purpose of this study was to characterise the digestive proteases, including the effect of the pH and the temperature on enzyme activity and stability, as well as the effect of inhibitors using multienzymatic extracts of the stomach and intestine of C. urophthalmus juveniles. Results showed that the optimum activities of the acid and alkaline proteases occurred at pH values of 3 and 9, respectively, whereas their optimum temperatures were 55 and 65 °C, respectively. The acid proteases were most stable at pH values of 2–3 and at temperatures of 35–45 °C, whereas the alkaline proteases were most stable at pH values of 6–9 and at 25–55 °C. The inhibition assays recorded a residual activity of 4 % with pepstatin A for the acid proteases. The inhibition of the alkaline proteases was greater than 80 % with TPCK, TLCK, EDTA and ovalbumin, and of 60 and 43.8 % with PMSF and SBT1, respectively. The results obtained in this study make it possible to state that C. urophthalmus has a sufficiently complete digestive enzyme machinery to degrade food items characteristic of an omnivorous fish species, although specimens showed a tendency to carnivory.     

Effects of day and night on swimming,grazing and social behaviours of rohu Labeo rohita (Hamilton) and common carp Cyprinus carpio (L.) in simulated ponds

Diel rhythmicity of grazing, swimming, resting and social interactions of rohu (Labeo rohita) (weight 66.5–68.3 g) and common carp (Cyprinus carpio) (79.9–82.0 g) were observed in 1 m² simulated ponds using video images. Fish behaviour was monitored during a full 24‐h period, starting at 08:00 hours with a 15‐min recording, which was repeated every 3 h. Rohu spent more time grazing during the day than at night. Grazing activity peaked at the beginning and at the end of the day. Grazing and resting of rohu were negatively correlated. Common carp grazed day and night, showing no distinct grazing peak. Considering the grazing time of rohu and common carp, a feeding schedule with two feeding times at 07:30 and 16:30 hours might be appropriate for better food intake and conversion in rohu–common carp polyculture system.     

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.     

Fermentative Properties of Proteolytic Pediococcus Strains Isolated from Salt Fermented Fish Hydrolysate Prepared Using Freshwater Fish Rohu (Labeo rohita)

Salt fermented fish hydrolysate (SFFH) was prepared from freshwater fish Rohu (Labeo rohita), an Indian major carp, using salt at room temperature for 50 days. Total lactic acid bacteria (LAB) counts of SFFH prepared using 20% and 30% NaCl (w/w) were found to be in the range of 5.83–7.25 log cfu/ml and 3.93–5.46 log cfu/ml, respectively, whereas the log cfu/ml values for the halotolerant LAB counts varied from 2.09 to 7.17 and 2.02 to 4.77, respectively. Out of 96 isolates from SFFH, 21 isolates were screened based on their pH reducing ability, total titratable acidity, and proteolytic properties. Morphological, biochemical, and molecular characterization of four selected LAB strains were carried out. The isolates FSAP3-3 (Pediococcus pentosaceus), FSBP4-40 (Pediococcus pentosaceus), FSBP16-40 (Pediococcus pentosaceus), and FSBP28-50 (Pediococcus acidilactici) showed both halotolerant and proteolytic properties. The identified LAB isolates showed antagonistic properties against several human pathogens and exhibited sensitivity toward a broad spectrum of antibiotics. All the identified LAB isolates showed excellent proteolytic activity and phosphohydrolase activity. These halotolerant LAB isolates that had both proteolytic properties and acidifying ability have the potential for application in an acceleration of fermentation to produce salted fish products (i.e., fish sauce, fish paste, fish mince, etc.).     

草鱼消化道碱性蛋白酶的分析鉴定

试验结果表明,草鱼肠道粗提取物在pH 10.3有最高酶活力。SDS-底物-PAGE电泳表明,草鱼肠道粗酶中有4种碱性蛋白酶,其中3种是丝氨酸蛋白酶(胰蛋白酶),1种是非丝氨酸蛋白酶,它们的相对分子质量分别为:26 400、30 750、43 000、约105 000。     

Characterization of protease activity in developing discus Symphysodon aequifasciata larva

The activity of different protease classes was monitored in developing discus (Symphysodon spp.) larvae using a combination of biochemical assays and substrate SDS–PAGE techniques. Results showed the presence of alkaline proteases of serine proteases such as trypsin with a significant increase in activity levels detected beginning 3 days after hatching. Other alkaline proteases such as metallo‐proteases and chymotrypsin, a type of serine protease, were only detected in older larval stages, at around 20–30 days after hatching. Acidic protease activity was very low during the first 20–25 days of development before showing a significant (P < 0.01) rise. This is despite the formation of a stomach observed 10 days after hatching. Based on the development of the protein digestive system observed, the use of microdiets to replace Artemia should be considered 25 days after hatching.     

Effects of dietary protein levels on activities of protease and expression of ingestion and protein digestion‐related genes in Nile tilapia juveniles

While some studies have investigated the effects of dietary protein on the activities of protease in Nile tilapia, little information exists about these enzymes and hormones regulating appetite at the molecular level. Therefore, we investigated the performance, protease activities and expression of genes related to protein intake and digestion in juvenile tilapia fed with diets containing different levels of crude protein (CP): 25.14, 31.12, 36.60 and 42.05%. The fish were fed to apparent satiation three times a day for 65 days. The animal performance and the retention rates of protein and energy linearly decreased with an increase in dietary CP content. A positive quadratic effect of CP on pepsinogen was observed, although the acid protease activity was not affected. Trypsinogen and trypsin activity in the intestine presented similar patterns, showing a positive quadratic response to dietary protein levels. A linear increase in intestinal chymotrypsinogen expression was observed, but the activity of chymotrypsin showed a positive quadratic response. In addition, the expression of cholecystokinin (cck) and peptide yy (pyy) increased linearly with increasing CP. Dietary protein modulates the activity of alkaline proteases as well as the expression of these protease genes and hormones involved in appetite regulation. These changes can help expand our understanding of feed intake and digestion control, thereby improving feed formulations for this species.     

Food consumption and digestive enzyme activity of Clarias batrachus exposed to various temperatures

The effect of temperature on the food consumption rate and the digestive enzyme activities of Clarias batrachus (80.60 ± 5.34 g) were evaluated. Fish were exposed to six different temperatures of 10, 15, 20, 25, 30 and 35 °C following an acclimation temperature of 25 °C. The rate of temperature change was 2 °C day^?1. Highest food consumption was recorded at 25 °C. It gradually reduced with decreasing water temperature. Food consumption rate was significantly (P < 0.05) lower at 10 °C compared with other treatments. Hence, 46.67, 8.20–23.58 and 1.02–6.15% reduced food consumptions were recorded in groups exposed at 10, 15 and 20 °C temperatures, respectively, compared with the 25 °C. The consumption rate was not affected in fish exposed at 30 and 35 °C. Total protease, trypsin and chymotrypsin activities were significantly (P < 0.05) higher in fish exposed at 25 °C compared with others. Lipase activity was significantly (P < 0.05) higher in fish exposed at 30 °C compared with others. Lowest enzyme activities were recorded at 10 °C. Water temperature below 25 °C affected the food consumption and digestive enzyme activities in fish that served as indicators of stress in fish.     

Effects of Neutral Phytase Supplementation on Biochemical Parameters in Grass Carp,Ctenopharyngodon idellus,and Gibel Carp,Carassius auratus gibelio,Fed Different Levels of Monocalcium Phosphate

Two 8‐wk studies were conducted to evaluate the effects of neutral phytase supplementation on hemato‐biochemical status, liver biochemical parameter, and intestinal digestive enzyme activity of grass carp, Ctenopharyngodon idellus, and gibel carp, Carassius auratus gibelio, fed with different levels of monocalcium phosphate (MCP). The control diet was prepared with 2% MCP but without phytase (P2.0). The other three experimental diets were prepared with the addition of 1.5, 1.0, and 0.5% MCP, respectively, when supplemented with 500 U/kg neutral phytase in each diet and designated as PP1.5, PP1.0, and PP0.5, respectively. The results indicated that the serum alkaline phosphatase (ALP), alanine transaminase (ALT), and aspartate transaminase (AST) activities, as well as the albumin (ALB) content were increased in grass carp (P < 0.05) and gibel carp (P > 0.05) fed with phytase‐supplemented diets. Meanwhile, the serum cholesterol, high‐density lipoprotein, and total protein contents of the two species of fish were increased in comparison to the control. In addition, dietary phytase inclusion did not significantly affect hepatic ALP, ALT, and AST activities in the two species of carp fed with different levels of MCP. Amylase activity increased in foregut and hindgut of both species when fed with the phytase‐supplemented diets while lipase activity was reduced in the foregut and hindgut in both fish. This study suggests that neutral phytase supplementation increases serum ALP, ALT, and AST activities but does not notably affect these enzyme activities in the liver of the two species of carp when fed different levels of MCP. On the other hand, amylase activity increased while lipase activity was reduced in the intestine of the species of carp fed with phytase‐supplemented diets.     

Effect of dietary iron levels on growth,body composition and intestinal enzyme activities of juvenile Jian carp (Cyprinus carpio var. Jian)

A 60‐day feeding trial was carried out to investigate the effect of iron on growth, body composition and digestive enzyme activities. Diets with seven levels of iron (53.9, 90.0, 115.6, 146.1, 176.0, 215.8 and 266.0 mg iron kg^?1 diet) were fed to Jian carp (initial weight 11.4 ± 0.0 g). Per cent weight gain (PWG), feed efficiency (FE) and protein efficiency ratio were the lowest in fish fed the basal diet (P < 0.05). Body protein content was increased with the increasing iron levels (P < 0.05), but moisture, lipid and ash of fish were not significantly affected by dietary iron levels (P > 0.05). Activities of trypsin, lipase, α‐amylase, Na⁺, K⁺‐ATPase, alkaline phosphatase and gamma‐glutamyl transpeptidase were improved with increasing dietary iron levels. Serum iron were significantly enhanced with dietary iron levels up to 146.1 mg iron kg^?1 diet, and plateaued. In conclusion, iron improved digestive enzyme activities of juvenile Jian carp and the dietary iron requirement for serum iron of juvenile Jian carp (11.4–64.0 g) was 147.4 mg iron kg^?1 diet with ferrous fumarate as the iron source.     

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.     

Ontogeny of protease, amylase and lipase in the alimentary tract of hybrid Juvenile tilapia (Oreochromis niloticus × Oreochromis aureus)

Juvenile tilapias (Oreochromis niloticus × O. aureus), with average body weights of groups I, II and III of 55.14, 122.82, and 225.68 g respectively were used to study changes in the activity of protease, amylase, and lipase in different organs and sections of the alimentary tract. Additionally, tract containing digesta, which is closer to physiological conditions, may help reveal the mechanisms of whole chemical digestion process. Another group of tilapias (mean body weight of 111.81 ± 0.14 g) was used to analyze changes in activity of protease, lipase, and amylase in response to varying pH and temperatures. The activity of enzymes in all digestive organs increased when body weight increased from 55 to 122 g, and the activity further increased when the weights increased from 122 to 225 g. The rates of increase in digestive enzyme activity were significantly greater than the rates of growth of the fish. When the activity was compared in different sections of the tract, a similar regularity was found for the three enzymes in all fish. The highest amounts of activity were seen in the foregut and the lowest in the rectum, with the middle amount of activity in the hindgut. The pH ranges for the maximum activity of tilapia protease in the stomach and intestine were 1.6–3.3 and 9.5–10.5 respectively and the optimal temperature in these organs was 55°C. The pH ranges for the maximum amylase activity in the intestine and hepatopancreas were 6–7 and 7.5 respectively, and high amylase activity in these organs was found at 25–35°C. The activity of lipase in the intestine remained unchanged at pH 6.0–9.0 and with temperatures between 25 and 35°C. These results suggest that the ability of tilapia to utilize feed varies at different growth stages and remains relatively stable with changes in environmental pH and temperature, which should be taken into account when designing feed formulas for tilapia.     

Effects of dietary thiamin supplement on growth,body composition and intestinal enzyme activities of juvenile Jian carp (Cyprinus carpio var. Jian)

A total of 1050 juvenile Jian carp (Cyprinus carpio var. Jian) (8.20 ± 0.02 g) were fed diets containing seven graded levels of thiamin (0.25, 0.48, 0.79, 1.06, 1.37, 1.63 and 2.65 mg kg^?1) for 60 days to investigate the effects of thiamin on growth, body composition and digestive enzyme activities. Percent weight gain (PWG), feed intake and feed efficiency (FE) were the lowest in fish fed the basal diet (P < 0.05). Protein productive value and lipid productive value increased with increasing dietary thiamin levels up to 1.06 and 0.79 mg kg^?1 diet, respectively (P < 0.05). Body protein and lipid increased with increasing dietary thiamin levels (P < 0.05), while moisture and ash of fish carcasses decreased with the increase in dietary thiamin supplementation (P < 0.05). Intestinal folds height had a similar trend to PWG (P < 0.05). Activities of α‐amylase, lipase, trypsin, Na⁺, K⁺‐ATPase, alkaline phosphatase and gamma‐glutamyl transpeptidase in intestine were all affected by the dietary thiamin (P < 0.05). In conclusion, thiamin could improve growth and intestinal enzyme activities of juvenile Jian carp. The dietary thiamin requirement of juvenile Jian carp (8.0–60.2 g) based on PWG was 1.02 mg kg^?1 diet.     

Growth,body composition,intestinal enzyme activities and microflora of juvenile Jian carp (Cyprinus carpio var. Jian) fed graded levels of dietary phosphorus

A 9‐week feeding trial was carried out with juvenile Jian carp (Cyprinus carpio var. Jian) to study the effects of dietary phosphorus on growth, body composition, intestinal enzyme activities and microflora. Quadruple groups of juvenile Jian carp (7.17 ± 0.01 g) were fed practical diets containing available phosphorus 1.7 (unsupplemented control), 3.6, 5.5, 7.3, 9.2 and 11.0 g kg^?1 diet to satiation. Feed intake, specific growth ratio and feed efficiency were the lowest in fish fed the basal diet (P < 0.05). Body moisture, protein, lipid content and ash were all significantly affected by dietary available phosphorus levels (P < 0.05). Activities of trypsin, amylase, Na⁺, K⁺‐ATPase, alkaline phosphatase and gamma‐glutamyl transpeptidase were improved with increasing dietary phosphorus levels. Intestinal Aeromonas and Escherichia coli decreased with increasing dietary phosphorus up to 3.6 and 5.5 g kg^?1 diet respectively (P < 0.05), while Lactobacillus increased with the increasing dietary phosphorus up to 9.2 g kg^?1 diet (P < 0.05). These results suggested that phosphorus could enhance intestinal enzyme activities of juvenile Jian carp and the minimum dietary available phosphorus requirement for SGR of juvenile Jian carp (7.2–63.8 g) was 5.2 g kg^?1 diet.