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.     

Modulation of appetite,lipid and glucose metabolism of juvenile grass carp (<Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis>) by different dietary protein levels

This study was undertaken to explore the systemic metabolic strategies of juvenile grass carp (Ctenopharyngodon idellus) to maintain growth when fed with different dietary protein levels. The optimal growth group and two growing discomfort groups were selected through the basic data, to explain the growth difference from appetite regulation and lipid and glucose metabolism perspective. Three experimental diets were formulated with three dietary protein levels at 200.3, 296.1 and 442.9 g kg^?1, named P1, P2 and P3, respectively. Juvenile grass carp (initial body weight 12.28 ± 0.14 g) were fed with three diets with 3 replications per dietary treatment in an indoor recirculation system for an 8-week feeding trial. Fish fed with diet P2 dietary group showed significantly higher WG, SGR, FI and PER than other groups. Compared with other groups, mRNA expressions of NPY, Y8a and Y8b in fish fed with P2 significantly down-regulated, while the expressions of CCK and CART in fish fed with P3 significantly down-regulated (P < 0.05). With increasing dietary protein levels, G6Pase, GK, PK and PEPCK were all significantly inhibited (P < 0.05). For lipid metabolism, the mRNA expression of ACC in P1 dietary group was significantly higher than P3 dietary group; besides, LPL expression in P3 group was significantly higher than other two groups (P < 0.05). PPARα expression in P2 was significantly lower than other groups (P < 0.05). These results suggested that grass carp fed with P2 (296.1 g kg^?1 protein level) showed highest weight gain, contributed to more balanced nutrient metabolism and appetite regulation. Too high dietary protein (442.9 g kg^?1) should be avoided because it induced lowest PER, body lipid and liver lipid, and inhibited glucose and lipid metabolism in juvenile grass carp.     

Dietary taurine can improve the hypoxia-tolerance but not the growth performance in juvenile grass carp Ctenopharyngodon idellus

This study was conducted to evaluate the effects of dietary taurine, as a feed additive, on the hypoxia-tolerance and growth performance of the juvenile grass carp Ctenopharyngodon idellus, one of the most important and intensively cultured freshwater fish, with the largest production in China. Graded levels of taurine (0, 0.5, 1, 1.5, 2 and 2.5 g kg^–1 dry diet) were fed to grass carp juveniles (mean weight: 5.26 ± 0.03 g) for 8 weeks. The survival time during acute hypoxia increased as dietary levels of taurine increased, with the highest dose of taurine resulting in the best acute hypoxia-tolerance. The erythrocyte osmotic fragility in grass carp was significantly improved when dietary taurine level was at least 1.5 g kg^?1 diet and can be improved much more when dietary taurine level was up to 2.5 g kg^?1 diet. A significant correlation between hemolysis rate of the erythrocyte osmotic fragility test and the survival time of acute hypoxia (r = ?0.873, P = 0.023 < 0.05) strongly suggested that the biomembrane stabilization function of taurine may contribute to its role of enhancing acute hypoxia-tolerance in grass carp. Dietary taurine cannot improve growth performance of grass carp, but it can increase the value of mesenteric fat index, indicating that dietary taurine influences the lipid metabolism. This study provides valuable information to improve hypoxia-tolerance of grass carp.     

Inhibitory properties of some heavy metals on carbonic anhydrase I and II isozymes activities purified from Van Lake fish (<Emphasis Type="Italic">Chalcalburnus Tarichi</Emphasis>) gill

In this study, CA I and II isoenzymes were purified from Van Lake fish gills by using Sepharose-4B-L-tyrosine-sulfanilamide affinity chromatography and to determine the effects of some metals on the enzyme activities. For purified CA I isoenzyme, yield, specific activity, and purification fold were obtained as 42.07%, 4948.12 EU/mg protein, and 116.61 and for CA II isoenzyme, 7%, 1798.56 EU/mg protein, and 42.38 respectively. Activity of CA was determined by measuring “CO₂-hydratase activity”. Purity control was checked by SDS-PAGE. In vitro inhibitory effect of Cu²⁺, Ag⁺, Cd²⁺, Ni²⁺ metal ions, and arsenic (V) oxide were also examined for both isozymes activities. Whereas Cu²⁺, Ag⁺, Cd²⁺, and Ni²⁺ ions showed inhibitory effects on both isozymes, arsenic (V) oxide showed activation effect. IC₅₀ values were calculated by drawing activity %-[I] graphs for metal ions exhibiting inhibitory effects. IC₅₀ values were determined as 3.39, 6.38, 13.52, and 206 μM for CA I isozyme and 6.16, 20.29, 46, and 223 μM for CA II isozyme respectively.     

Cloning and molecular characterization of cationic amino acid transporter y+LAT1 in grass carp (Ctenopharyngodon idellus)

The solute carrier family 7A, member 7 gene encodes the light chain- y⁺L amino acid transporter-1 (y⁺LAT1) of the heterodimeric carrier responsible for cationic amino acid (CAA) transport across the basolateral membranes of epithelial cells in intestine and kidney. Rising attention has been given to y⁺LAT1 involved in CAA metabolic pathways and growth control. The molecular characterization and function analysis of y⁺LAT1 in grass carp (Ctenopharyngodon idellus) is currently unknown. In the present study, full-length cDNA (2,688 bp), which encodes y⁺LAT1 and contains a 5′-untranslated region (319 bp), an open reading frame (1,506 bp) and a 3′-untranslated region (863 bp), has been cloned from grass carp. Amino acid sequence of grass carp y⁺LAT1 contains 11 transmembrane domains and shows 95 %, 80 % and 75 % sequence similarity to zebra fish, amphibian and mammalian y⁺LAT1, respectively. The tissue distribution and expression regulation by fasting of y⁺LAT1 mRNA were analyzed using real-time PCR. Our results showed that y⁺LAT1 mRNA was highly expressed in midgut, foregut and spleen while weakly expressed in hindgut, kidney, gill, brain, heart, liver and muscle. Nutritional status significantly influenced y⁺LAT1 mRNA expression in fish tissues, such as down-regulation of y⁺LAT1 mRNA expression after fasting (14 days).     

Respiratory response of grass carp <Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis> to dissolved oxygen changes at three acclimation temperatures

Respiratory parameters of grass carp were studied during dissolved oxygen (DO) changes from normal DO to hypoxia, then return to normal DO at 15, 25, and 30 °C acclimation, respectively. The results showed that with increases of acclimation temperature at normoxia the respiratory frequency (f_R), oxygen consumption rate (VO₂), respiratory stroke volume (V_S.R), gill ventilation (V_G), and V_G/VO₂ of grass carp increased significantly, but the oxygen extraction efficiency (EO₂) of fish decreased significantly (P < 0.05). With declines of DO levels, the f_R, V_S.R, V_G, and V_G/VO₂ of fish increased significantly at different acclimation temperatures (P < 0.05). A slight increase was found in VO₂, and the EO₂ of fish remained almost constant above DO levels of 3.09, 2.91, and 2.54 mg l^?1 at 15, 25, and 30 °C, while the VO₂ and EO₂ began to decrease significantly with further reductions in DO levels (P < 0.05). After 0.5 h of recovery to normoxia from hypoxia at three acclimation, the f_R, V_S.R, V_G, and V_G/VO₂ of the fish decreased sharply; meanwhile, the VO₂ and EO₂ increased sharply (P < 0.05). The respiratory parameters of fish gradually approached initial values with prolonged recovery time to normoxia, and reached their initial values in 2.5 h at 25 and 30 °C acclimation. The critical oxygen concentrations (C_c) of fish for VO₂ were 2.42 mg l^?1 at 15 °C, 2.02 mg l^?1 at 25 °C, and 1.84 mg l^?1 at 30 °C, respectively. The results suggest that grass carp are highly adapted to varied DO and short-term hypoxia environments.     

Effect of dietary lysine on growth,intestinal enzymes activities and antioxidant status of sub-adult grass carp (Ctenopharyngodon idella)

The dietary lysine requirement of sub-adult grass carp (460 ± 1.5 g) was assessed by feeding diets supplemented with grade levels of lysine (6.6, 8.5, 10.8, 12.9, 15.0 and 16.7 g kg^?1 diet) for 56 days. The test diets (28 % CP) contained fish meal, casein and gelatin as sources of intact protein, supplemented with crystalline amino acids. Weight gain (WG), feed intake and feed efficiency were significantly improved with increasing levels of lysine up to 12.9 g kg^?1 diet and thereafter declined (P < 0.05). Quadratic regression analysis of WG at 95 % maximum response indicated lysine requirement was 10.9 g kg^?1 diet. Activities of trypsin, chymotrypsin, lipase, Na⁺, K⁺-ATPase and alkaline phosphatase in intestine, creatine kinase activity in proximal and mid-intestine responded similar to WG (P < 0.05). In addition, lipid and protein oxidation decreased with increasing levels of lysine up to certain values and increased thereafter (P < 0.05); the anti-hydroxyl radical capacity, dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase (GST) activities and glutathione content were increased with increasing dietary lysine levels up to certain values in the detected tissues, except for hepatopancreatic GST. Requirement estimated on the basis of malondialdehyde content in intestine and hepatopancreas was 10.6 and 9.53 g lysine kg^?1 diet, respectively.     

Water quality and growth of Pacific white shrimp Litopenaeus vannamei (Boone) in co-culture with green seaweed Ulva lactuca (Linaeus) in intensive system

An indoor trial was conducted for 28 days to evaluate the effects and interactions of biofloc and seaweed Ulva lactuca in water quality and growth of Pacific white shrimp Litopenaeus vannamei in intensive system. L. vannamei (4.54 ± 0.09 g) were stocked in experimental tanks at a density 132 shrimp m^?2 (566 shrimp m^?3) and the U. lactuca was stocked at a density 0.46 kg m^?2 (2.0 kg m^?3). Biofloc with seaweed (BF-S) significantly reduced (P < 0.05) total ammonia nitrogen (TAN) by 25.9 %, nitrite–nitrogen (NO₂–N) by 72.8 %, phosphate (PO ₄ ³ -P) by 24.6 %, and total suspended solids by 12.9 % in the water and significantly increased (P < 0.05) settleable solids by 34.2 % and final weight of shrimp by 6.9 % as compared to biofloc without seaweed. The BF-S can contribute by reducing nitrogen compounds (TAN and NO₂–N), phosphate (PO ₄ ³ -P), and total suspended solids in water and increased final weight of shrimp.     

Characterization and virus susceptibility of a skin cell line from red-spotted grouper (Epinephelus akaara)

A red-spotted grouper Epinephelus akaara skin (RGS) cell line was established and characterized. RGS cells had a normal diploid chromosome number of 2n = 48, the morphology of which was fibroblastic-like in 3 days and epithelial-like over 5 after 16 passages. The cells multiplied well in Dulbecco’s modified Eagle’s medium supplemented with 10% of fetal bovine serum at 25°C. Susceptibilities of RGS and grass carp ovary (GCO) cells to two viruses were tested, and the results showed that the titer of an iridovirus Rana grylio virus (RGV) in RGS cells was 10^3.5 TCID₅₀ ml^?1, which was much higher than a rhabdovirus spring viremia of carp virus (SVCV) in the cells (10^0.5 TCID₅₀ ml^?1). The titers of RGV and SVCV in GCO were 10^6.0 TCID₅₀ ml^?1 and 10^8.0 TCID₅₀ ml^?1, respectively, which were higher than those in RGS cells. The data may imply that RGS cells could be selectively resistible to some viruses during infection. RT-PCR analysis of RGV-infected RGS cells showed that RGV could replicate in RGS cells. Further study of virus replications in RGS cells was conducted by electron microscopy and immunofluorescence microscopy has shown that virus particles scattered in the cytoplasm and virus protein appeared in both the cytoplasm and nucleus. The results suggested that RGS cells could be used as a potential in vitro model to study the cutaneous barrier function against virus infection.     

Preliminary study on zeolite materials used to control of heavy metal pollution during the culture of mud clam Tegillarca granosa L.

To control heavy metal pollution during the culture of the mud clam, Tegillarca granosa L., the isothermal static adsorption capacities of three adsorption materials [natural zeolites, zeolites synthesized from kaolin and zeolites synthesized from coal fly ash (ZFA)] towards Cu²⁺, Cd²⁺, Cr⁶⁺, Zn²⁺, Pb²⁺ and Hg²⁺ in seawater were investigated. The adsorption capacity of the best material was investigated during the culture of mud clam in an artificially polluted environment. Results showed that the ZFA adsorption capacity was the highest and conformed to the Freundlich model. The theoretical maximum adsorption capacities of the adsorbent towards Cu²⁺, Cd²⁺, Cr⁶⁺, Zn²⁺, Pb²⁺ and Hg²⁺ in seawater were 3.057, 1.123, 0.325, 13.101, 6.116 mg g^?1 and 6.527 μg g^?1 respectively. For seawater artificially polluted by six heavy metals, heavy metal adsorption by ZFA followed the order Zn²⁺ > Cd²⁺ > Hg²⁺ > Cu²⁺ > Pb²⁺ > Cr⁶⁺. For the mud clam culturing experiment that simulated a continuously polluted water source, the Cu²⁺, Cd²⁺, Cr⁶⁺, Zn²⁺, Pb²⁺ and Hg²⁺ contents of mud clam tissues decreased by 40.9%, 25.8%, 9.6%, 28.2%, ?27.8% and 25.7%, respectively, after 21 days of culturing compared with the contents before the experiment.     

Supplementation of microbial levan in the diet of Cyprinus carpio fry (Linnaeus, 1758) exposed to sublethal toxicity of fipronil: effect on growth and metabolic responses

A 60-day feeding trial was conducted to study the effect of dietary microbial levan on growth performance and metabolic responses of Cyprinus carpio fry exposed to sublethal dose (1/10th LC₅₀) of fipronil [(±)-5-amino-1-(2,6-dichloro-α,α,α-trifluoro-p-tolyl)-4-trifluoromethylsulfinylpyrazole-3-carbonitrile]. Two hundred and twenty five fry were randomly distributed in five treatments in triplicates. Four purified diets were prepared with graded levels of microbial levan. Five different treatment groups were levan control L₀P₀ (basal feed + 0 % levan without exposure to pesticide); pesticide control L₀P₁ (basal feed + 0 % levan with exposure to pesticide); L_0.25P₁ (basal feed + 0.25 % levan with exposure to pesticide); L_0.50P₁ (basal feed + 0.50 % levan with exposure to pesticide); and L_0.75P₁ (basal feed + 0.75 % levan with exposure to pesticide). Weight gain% and specific growth rate were significantly higher (p < 0.05) in levan fed groups compared to their non-levan fed counterpart. Highest (p < 0.05) content of ascorbic acid in muscle, liver and brain tissues was observed with higher level of dietary levan. Glucose-6-phosphate dehydrogenase activity decreased with the increasing level of dietary levan in the liver and muscle. Aspartate aminotransferase activity exhibited a second order polynomial relationship with the dietary levan, both in liver (Y = ?1.001x ² + 5.366x + 5.812, r ² = 0.887) and muscle (Y = ?0.566x ² + 2.833x + 6.506, r ² = 0.858) while alanine aminotransferase activity showed third order polynomial relationship both in liver (Y = 1.195x ³ ? 12.30x ² + 35.23x + 9.874, r ² = 0.879) and muscle (Y = 0.527x ³ ? 8.429x ² + 31.80x + 8.718, r ² = 0.990). Highest (p < 0.05) superoxide dismutase activity in gill was observed in the group fed with 0.75 % levan supplemented diet. Overall results indicated that dietary microbial levan at 0.75 % in C. carpio fry ameliorated the negative effects of fipronil and augmented the growth.     

The effect of irradiance and temperature on the photosynthesis of two agarophytes Gelidium elegans and Pterocladiella tenuis (Gelidiales) from Kagoshima,Japan

The effect of irradiance and temperature on the photosynthesis of two Japanese agarophytes, Gelidium elegans and Pterocladiella tenuis (Gelidiales), was determined using dissolved oxygen sensors and pulse amplitude modulated (PAM) fluorometry. Gross photosynthesis and dark respiration rates were determined over a range of temperatures (8–36 °C). The highest gross photosynthetic rates were 40.3 and 37.0 mg O₂ g _ww ^?1  min^?1 and occurred at 24.3 and 25.5 °C [95 % Bayesian credible interval (BCI) 20.7–28.0 and 23.4–28.3 °C], respectively. The dark respiration rate in G. elegans and P. tenuis increased with increasing temperature at a rate of 0.10 and 0.31 mg O₂ g _ww ^?1  min^?1 °C^?1 , respectively. Modeling the net photosynthesis–irradiance (P–E) responses of G. elegans and P. tenuis at 20 °C revealed that the net photosynthetic rates quickly increased at irradiance levels below the estimated saturation irradiance of 88 and 83 µmol photons m^?2 s^?1, with a compensation irradiance of 14 and 19 µmol photons m^?2 s^?1, respectively. The highest value of the maximum effective quantum yield (Φ _PSII) occurred at 20.1 °C (BCI 18.9–21.5 °C) and 21.3 °C (BCI 20.2–22.5 °C) for G. elegans and P. tenuis and was 0.49 and 0.45, respectively. These optimal temperatures of Φ _PSII were relatively lower than those determined by the photosynthesis–temperature model of oxygen evolution. The temperature response of these species indicates that they are probably well adapted to the current range of seawater temperatures in the study site but that they are near the boundary of their tolerable limits.     

Fat deposition pattern and mechanism in response to dietary lipid levels in grass carp, <Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis>

This study aimed to evaluate the fat deposition pattern and lipid metabolic strategies of grass carp in response to dietary lipid levels. Five isonitrogenous diets (260 g kg^?1 crude protein) containing five dietary lipid levels (0, 20, 40, 60, 80 g kg^?1) were fed to quadruplicate groups of 15 fish with initial weight 200 g, for 8 weeks. The best growth performance and feed utilization was observed in fish fed with lipid level at 40 g kg^?1. MFI and adipose tissue lipid content increased with increasing dietary lipid level up to 40 g kg^?1, and higher lipid level in diet made no sense. Fish adapted to high lipid intake through integrated regulating mechanisms in several related tissues to maintain lipid homeostasis. In the present study, grass carp firstly increased PPARγ and CPT1 expressions in adipose tissue to elevate adipocyte differentiation and lipolysis to adapt to high lipid intake above 40 g kg^?1. In liver, fish elevated hepatic lipid uptake but depressed biosynthesis of hepatic FAs, resulted in no difference in HSI and liver lipid content among the groups. Only in muscle, fish showed a significant fat deposition when the lipid intake above 40 g kg^?1. The excess lipid, derived from enhanced serum TC and TG contents, was more likely to induce deposition in muscle rather than lipid uptake by adipose tissue in grass carp fed with high dietary lipid, indicating the muscle of grass carp might be the main responding organ to high lipid intake.     

Grass carp (Ctenopharyngodon idella) grazing on duckweed (Spirodela polyrhiza)

The aims of this experiment were (1) toquantify the ability of grass carp to processduckweed and (2) to assess indirect changes inwater chemistry and phytoplankton community,caused by grass carp feeding. Yearling grass carp sized 126 ± 7.7 mm (TL) and19.6 g in weight were kept in 9 laminate tanksof 1 m³ for 14 days. Two stockingdensities (2 and 6 fish per m³) anda control without fish were used. Standard growthrate (SGR) of grass carp fed exclusively onduckweed was 0.70% body weight (BW) d^–1and food conversion ratio (FCR) reached 2.0(average water temperature =21.1 ± 3.8 °C). Daily food intakewas 0.2 g of duckweed dry weight (DW), i.e.,1% of average BW of grass carp. SGR ofduckweed growing in 20 × 20 cm floatingenclosures, differed significantly[F(6,2) = 417.9; p = 0.002] between the twostocking densities of grass carp and thecontrol tanks (without fish). Mean SGR ofduckweed was 0.02 g g^–1 day^–1 and thehighest SGR was recorded in the control tanks.Both decrease in NH₄-N and increase inNO₂-N concentrations differedsignificantly between the treatments[F(2,2) = 45.3; p = 0.02 and F(2,2) = 19.2; p = 0.04 respectively]. Changes in other nitrogenand phosphorus components (NO₃-N, TN, TPand PO₄-P) caused by stocking of grasscarp were not significant. Biomass ofphytoplankton, dominated by filamentous algaeand blue-greens, increased proportionately tostocking density of grass carp. Althoughduckweed has a large potential for nutrientremoval, the most common pathway for thenutrients released through grass carp grazingif duckweed cover is loose is theirincorporation into phytoplankton biomass.     

Silymarin inhibits adipogenesis in the adipocytes in grass carp <Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis> in vitro and in vivo

In this study, two experiments were performed to explore the function of silymarin in adipogenesis in grass carp (Ctenopharyngodon idellus) using in vitro and in vivo models. In experiment 1, differentiated grass carp pre-adipocytes were treated with silymarin for 6 days. Treatment with 100 μg mL^?1silymarin (SM100 group) significantly reduced triglyceride accumulation at day 6. The adipogenic gene expression levels of PPARγ, C/EBPα, SREBP1c, FAS, SCD1, and LPL, and the protein expression level of PPARγ were significantly down-regulated in the SM100 group. Additionally, the SM100 group had significantly lower reactive oxygen species production and reduced glutathione contents compared with the control in vitro. In experiment 2, the juvenile grass carp (mean body weight= 27.4 ± 0.17 g) were fed six isonitrogenous and isocaloric diets in a factorial design containing 0, 100, or 200 mg kg^?1 silymarin (SM0, SM100, SM200) associated with either 4 or 8% lipid levels (low lipid, LL, and high lipid, HL, respectively) for 82 days. The results demonstrated that dietary silymarin supplementation significantly reduced the elevated intraperitoneal fat index in grass carp fed with high-lipid diets, and the gene expression of adipogenesis (PPARγ, FAS) when supplemented with dietary silymarin was notably lower than when no silymarin was supplemented under the high-lipid diets. Thus, our data suggest that silymarin suppressed lipid accumulation in grass carp both in vitro and in vivo, and the effect might be due to an influence on the expression of adipogenesis factors and ROS production partly associated with effects on antioxidant capability.     

The effects of starvation on fast-start escape and constant acceleration swimming performance in rose bitterling (<Emphasis Type="Italic">Rhodeus ocellatus</Emphasis>) at two acclimation temperatures

To investigate the effects of starvation and acclimation temperature on the escape ability of juvenile rose bitterling (Rhodeus ocellatus), we measured the fast-start escape and constant acceleration swimming performance of fish fasted for 0 (control), 1 and 2 weeks and half-lethal periods (6 or 4 weeks) at two temperatures (15 and 25 °C). Fish acclimated at a high temperature exhibited shorter response latency (R), higher maximum linear velocity (V _max) and longer escape distance during escape movement (D _120ms) than those at the low temperature. Starvation resulted in a significant decrease in V _max and D _120ms at either low or high temperature and a significant increase in R at only the high temperature in the half-lethal period groups (P < 0.05). The relationship between V _max (Y, m s^?1) and starvation time (X, week) was Y ₁₅ = ?0.062X + 1.568 (r = ?0.665, n = 36, P < 0.001) at low temperature and Y ₂₅ = ?0.091X + 1.755 (r = ?0.391, n = 40, P = 0.013) at high temperature. The relationship between U _cat (Y, cm s^?1) and starvation time (X, week) was Y ₁₅ = ?1.649X + 55.418 (r = ?0.398, n = 34, P = 0.020) at low temperature and Y ₂₅ = ?4.917X + 62.916 (r = ?0.793, n = 33, P < 0.001) at high temperature. The slopes of equations showed a significant difference between low and high temperature (F _1,63 = 9.688, P = 0.003), which may be due to the different energy substrate utilization when faced with food deprivation at different temperatures.     

Immunomodulatory effect of cimetidine in common carp (Cyprinus carpio L.)

The main indication of cimetidine is being H₂-receptor antagonist, but studies suggest that cimetidine may also act as a non-specific stimulant of cell-mediated immunity and immunomodulator. In order to determine the immunomodulatory effect of dietary intake of cimetidine in the common carp (100 ± 10 g), subjects were fed diets containing 0 (control), 50, 100 and 200 mg cimetidine kg^?1 of dry diet for a period of 6 weeks. TLC and NBT assays were significantly (P < 0.05) stimulated in cimetidine-supplemented groups displaying the highest value in 200 mg kg^?1 group. A decrease (P < 0.05) in cortisol and ACH50 value was recorded in fish treated with cimetidine. Serum protein, albumin and serum globulin levels were not significantly changed. The findings of the present investigation suggest that the incorporation of cimetidine in the diet of common carp enhances the non-specific immunity.     

Low water exchange Gracilariopsis bailiniae Zhang & B.M. Xia culture in intensive milkfish culture effluents: effects of seaweed density on seaweed production and effluent treatment

This study tested the feasibility of a low-cost seaweed biofiltration system for pond-based aquaculture through an indoor-integrated fish-seaweed culture experiment using weekly nutrient supply regime and different seaweed stocking densities. The culture experiment was conducted in glass aquaria that were stocked with Gracilariopsis bailiniae at 3 densities (low = 0.5 kg m^?2, middle = 2 kg m^?2 and high = 3.5 kg m^?2) and provided either with effluents from intensive milkfish (Chanos chanos) culture or with effluent-free seawater (control) as nutrient source. Stocking density was used as a factor in optimizing nutrient availability for growth and nutrient removal under such low water exchange conditions. Our results showed that G. bailiniae cultivated in milkfish effluents had higher growth, biomass and nitrogen yields than those cultivated in effluent-free seawater. Among the different stocking densities tested, highest growth rate (1.03 % day^?1) was obtained in the middle density. Increasing biomass and nitrogen yields were also obtained at this density until the end of the culture period. Poorer growth rates at low and high stocking densities were attributed to light limitation from phytoplankton and self-shading, respectively. Due to seaweed treatment, average outflow concentration of NH₄ ⁺ was reduced from half of its pretreated level. This study showed that a weekly effluent supply at 2 kg m^?2 seaweed stocking density can sustainably support the growth of G. bailiniae as long as the dissolved nutrients are present at high levels.     

A study of the damage of the intestinal mucosa barrier structure and function of <Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis> with <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

The aim of this study is to explore the effect of Aeromonas hydrophila on the intestinal mucosal barrier structure and intestinal permeability in grass carp (Ctenopharyngodon idella). Histopathological examinations showed that A. hydrophila induced severe intestinal lesions, including inflammatory cell infiltration and intestinal villus fusion and swelling. Messenger RNA (mRNA) expression of the inflammatory cytokines TNF-α, IL-1β, IL-8, IL-10 and MyD88 was significantly increased after infection with A. hydrophila. The permeability of intestinal mucosa was determined using Evans blue (EB) and d-lactic acid. The results indicated that the levels of EB and serum d-lactic acid were significantly increased after infection with A. hydrophila (p < 0.05). Our results also indicated that the intestinal mucosal barrier injury induced by A. hydrophila infection was closely associated with the expression of the tight junction (TJ) protein zonula occludens-1 (ZO-1), occludin, claudin b and claudin c as well as the activity of Na⁺, K⁺-ATPase and Ca²⁺, Mg²⁺-ATPase. Lower mRNA levels of occludin and lower Na⁺, K⁺-ATPase and Ca²⁺, Mg²⁺-ATPase activity in the intestines were observed after challenge. ZO-1 and claudin c were significantly increased 24 h after infection with A. hydrophila. The most interesting finding was that claudin b also significantly increased 24 h after challenge and then decreased to lower levels at 72, 120 and 168 h post-infection compared to the PBS-treated control group. The results demonstrated that grass carp infection with A. hydrophila induced intestinal inflammation and impaired the structure and function of the intestinal mucosal barrier.