Growth and metabolic responses of tambaqui (Colossoma macropomum) fed different levels of protein and lipid

Growth and metabolism were investigated in juvenile tambaqui (Colossoma macropomum) fed with isoenergetic diets in four treatments with protein (P) lipid (L) in g kg^?1: P350L49; P301L81; P253L113 and P205L145, respectively. The experiments were carried out in the laboratory facilities. Fish growth, the intermediary metabolites (total sugar, lactate, pyruvate, ammonia, protein, fatty acids, triacylglycerols, amino acids and glycogen), and the enzyme activities of lactate dehydrogenase, malate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase were assayed. Fish from treatments P350L49 and P301L81 depicted the best growth. Increase in glycolytic, glycogenolytic and lipolytic activities in liver was observed following dietary lipid increase. In fish fed with high lipid levels, the energetic demand was supplied by enhancement of muscular glycogenesis and lipolysis. The plasma metabolic profile reflected the blood function on metabolites delivery to tissues underlining biochemical adaptations because of changes in the feeding. In conclusion, the increase in dietary lipid plus protein decrease resulted in (i) reduction in fish growth, (ii) augment of liver glycolysis and glycogenolysis (iii) increase in muscular glycogenesis and (iv) predominance of lipolytic metabolism.     

Estrogen treatment and its influence on protein synthesis and amino acid metabolism in Zoarces viviparus (L) males

Overall protein synthesis was studied in estradiol-treated and control Zoarces viviparus males. Significant increases in total RNA, the RNA:DNA and the RNA:protein ratios in the liver were correlated with a 6-fold increase in total calcium levels in the plasma, indicating increased protein synthetic activity and the presence of circulating vitellogenin respectively in the induced males. The time-course incorporation of ¹⁴C-phenylalanine into protein in a cell free liver system showed a linear increase during the first 30 min after poly (U) was added to the incubation medium in control as well as in the estradiol-treated males. The incorporation rate in the induced liver, however, was increased significantly above the level of the control livers. Estradiol-treatment altered the concentration of most amino acids in the plasma and a few in the liver with particular marked effects on alanine and tyrosine in the plasma and glutamate in the liver.     

Metabolic responses of matrinxã, Brycon amazonicus (Spix & Agassiz, 1829), exposed to environmental nitrite

Nitrite is usually found in aquatic environments where nitrification process occurs. This ion can cause several injuries to aquatic species, particularly fish. Nitrite reacts with haemoglobin yielding the non‐functional methaemoglobin, which leads to many physiological consequences such as functional anaemia and supposed hypoxia. Metabolism of the freshwater teleost matrinxã, Brycon amazonicus, exposed to environmental 0.6 mg L^?1 of nitrite N‐NO₂^? for 96 h was studied, and the fermentative/oxidative preference was gauged. Concentrations of glycogen, glucose, lactate, pyruvate and ammonia, plus the activities of lactate, glutamate and malate dehydrogenase were assayed. The exposure resulted in a metabolic profile that allowed inferring the continuity of oxidative metabolism. Catabolism of amino acids prevailed or was apparently exacerbated by inferred branchial injury and consequent impairment of nitrogen excretion. Moreover, the studied enzymes glutamate dehydrogenase, lactate dehydrogenase and malate dehydrogenase from brain and heart were little affected by nitrite. The expected fermentative metabolism due to the high methaemoglobin formation was not observed.     

Biochemical responses of matrinxãBrycon cephalus (Günther, 1869) after sustained swimming

Juvenile matrinxã, Brycon cephalus, were submitted to sustained swimming for 72 days at 1.0 body length s^?1. Exercised fish (EF) grew more than non‐EF and their feed conversion ratio (FCR) improved; haematological responses demonstrated a decrease in haemoglobin and mean cell haemoglobin contents and increase in the mean cell volume. In the plasma, sodium, ammonia and amino acid concentrations increased; plasma triglycerides decreased while free fatty acids increased. Liver glucose, free amino acids, ammonia, the rate protein per fish weight and total lipid content increased, while the glycogen per fish ratio declined. Glutamate dehydrogenase (GDH) activity increased while pyruvate kinase (PK) and lactate dehydrogenase (LDH) decreased. White muscle glucose, lactate, the glycogen per fish‐weight ratio and total lipid content exhibited a decrease in their values; ammonia, free amino acids and the protein per fish‐weight ratio increased. GDH and PK decreased their activities. In the red muscle glycogen store, the glycogen per fish‐weight ratio and glucose were reduced. Juvenile matrinxãs, under sustained swimming, were physiologically and biochemically adapted to exercise as indicated by improved blood flow, transport and oxygen uptake, FCR, amino acid and protein incorporation and growth. Continuous exercise is a good practice for B. cephalus cultivation.     

Purification and characterization of glucose 6-phosphate dehydrogenase (G6PD) from grass carp (Ctenopharyngodon idella) and inhibition effects of several metal ions on G6PD activity in vitro

Glucose 6-phosphate dehydrogenase (G6PD) is a key enzyme catalyzing the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in various organisms, including fish. In the present study, G6PD was purified from grass carp (Ctenopharyngodon idella) hepatopancreas using the methods of 2′,5′-ADP-Sepharose 4B affinity chromatography followed by DEAE Sepharose Fast Flow ion exchange chromatography. The characterization of G6PD and inhibition effects of several metal ions on G6PD activity in vitro were also determined. Grass carp hepatopancreas G6PD, with a specific activity of 18 U/mg protein, was purified 1,066-fold with a yield of 19.5 % and Mr of 71.85 kDa. The enzyme had a temperature optimum of 42 °C, pH optimum of 7.5 and 9.0. The K _m values for G6-P and NADP⁺ were determined to be 0.026, 0.0068 mM, respectively. The V _max values for G6-P and NADP⁺ were 2.20 and 2.27 μM min^?1 mg protein^?1, respectively. The catalytic efficiency for G6-P and NADP as the substrates was 0.085 and 0.334 × 10^?6 min^?1 mg protein^?1, respectively. Inhibition effects of metal ions on the purified G6PD activity indicated that IC₅₀ values of Zn⁺², Mn⁺², Al⁺³, Cu⁺², and Cd⁺² were 0.42, 0.54, 0.94, 1.20, and 4.17 mM, respectively. The Ki constants of Zn⁺², Al⁺³, Cu⁺², and Cd⁺² were 0.52, 1.12, 0.26, and 4.8 mM, respectively. Zn⁺², Al⁺³, and Cd⁺² showed competitive inhibition, while Cu⁺² inhibited the G6PD in a noncompetitive inhibition manner. Our study provided important information about the control of the grass carp liver PPP, the biosynthesis of several important related biomolecules, and the status of detoxification systems in grass carp liver in relation to metabolism.     

Enzyme activity levels associated with the production of glycerol as an antifreeze in liver of rainbow smelt (Osmerus mordax)

Rainbow smelt (Osmerus mordax) tolerate temperatures close to the freezing point of sea water, in part, through the use of glycerol as an antifreeze. Potential mechanisms for glycerol production by liver were assessed by comparing activities of key enzym es of carbohydrate and amino acid metabolism in rainbow smelt to those in Atlantic tomcod (Microgadus tomcod) and smooth flounder (Liopsetta putmani). The latter two species inhabit the same environment but do not maintain high levels of blood glycerol. The enzyme profile of liver from rainbow smelt is substantially different from those of the other species and is poised for glycerol production. With respect to carbohydrate metabolism, glycerol-3-phosphate dehydrogenase activity in rainbow smelt liver was 156 µmoles min^-1 g^-1, a level which was 28 and 12-fold higher than activities in tomcod and flounder liver, respectively. Glycerol-3-phosphatase activity in smelt liver was 1.95 µmol min^-1 g^-1. This activity was 2.7 and 5.4-fold higher than those in tomcod and flounder liver, respectively. As such, the production of glycerol appears to be dependent upon the concerted action of glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. The enzyme profile also suggests that amino acids are a potential source of carbon for glycerol. Aspartate aminotransferase activity in rainbow smelt was 7 to 14-fold higher in comparison to tomcod and flounder liver, respectively. Activities of alanine aminotransferase and glutamate dehydrogenase in liver were between 2 and 3-fold higher in rainbow smelt than in the other two species. Finally, it is shown that in vitro preparations of smelt liver sections produce glycerol at 0°C lending support to the concept that liver is a site of glycerol synthesis in vivo.     

Carbohydrate and amino acids metabolic response to heat stress in the intestine of the sea cucumber Apostichopus japonicus

The sea cucumber Apostichopus japonicus is an economic species mainly distributed along the coast of northern China, south‐eastern Russia, Japan, Republic of Korea and Democratic People's Republic of Korea. Aquaculture industry of A. japonicus has been facing severe challenge of high temperature. In this study, we studied the mRNA expression profiles of eight key metabolic enzymes involved in carbohydrate and amino acids metabolism in A. japonicus under heat stress. The expression of hexokinase, pyruvate kinase and malate dehydrogenase showed downregulated response to heat stress, while the expression of glutamate dehydrogenase, alanine transaminase and branched‐chain aminotransferase showed upregulated response. In addition, the expression of lactate dehydrogenase and glutamate synthase showed no significant difference. We also applied ¹H NMR‐based metabolomics to investigate metabolic changes in the intestine tissue of A. japonicus under heat stress, the results of which revealed nine increased and 10 decreased metabolites in the heat stress group. These response genes and metabolites have potential to become markers for identifying severity of heat stress. More importantly, our findings suggest significant links between gene expression and metabolites changing, highlighting regulation networks of carbohydrate and amino acid metabolism in heat‐stressed A. japonicus.     

Effects of salinity on growth,nutrient composition,fatty acid composition and energy metabolism of Scylla paramamosain during indoor overwintering

Three salinities (4‰, 12‰ and 25‰) were selected to determine the effects of different salinities on the growth, survival, nutrition and energy metabolism of Scylla paramamosain during indoor overwintering. Growth performance (survival rate, condition factor, hepatopancreatic index, weight gain rate and specific growth rate), nutrient composition (ash, moisture, crude fat, crude protein and total nitrogen), fatty acids, energy metabolites (glycogen, glucose, triglycerides, cholesterol and lactic acid) and energy metabolic enzymes (hexokinase, pyruvate kinase, lactate dehydrogenase and succinate dehydrogenase) were measured and calculated. The results showed that S. paramamosain exhibited the best growth and survival at 25‰ and worst at 4‰ after overwintering. In addition to consuming crude fat, excess protein was also broken down to provide energy in the 4‰ and 12‰ groups. The content of unsaturated fatty acids in the 25‰ group was higher after overwintering. Glycogen and glucose consumption and triglycerides, cholesterol and lactic acid production were lowest in the 25‰ group. The activity of hexokinase, pyruvate kinase, lactate dehydrogenase and succinate dehydrogenase was highest in the 4‰ group and lowest in the 25‰ group. The activity of hexokinase, pyruvate kinase, lactate dehydrogenase and succinate dehydrogenase in the hepatopancreas was higher than in the muscle. In conclusion, S. paramamosain uses less energy, has more unsaturated fatty acids and has a higher survival rate at a salinity of 25‰ after overwintering. The results of this study provide helpful information the indoor overwintering S. paramamosain in aquaculture production systems.     

Motility of spermatozoa ofAlburnus alburnus (Cyprinidae) and its relationship to seminal plasma composition and sperm metabolism

The composition of seminal plasma and metabolism of sperm of the cyprinid fishAlburnus alburnus were investigated. Statistically significant correlations were found between motility parameters and seminal fluid osmolality, pH, Na⁺, K⁺ and protein levels (negative correlations: % immotile spermatozoa-Na⁺, K⁺; positive correlations: % motile spermatozoa-osmolality, pH, Na⁺, K⁺, protein; % linear motile spermatozoa-pH protein; swimming velocity of spermatozoa-pH, Na⁺, protein). Spermatozoan motility and ATP metabolism and glycolysis were correlated as indicated by measurement of ATPase, pyruvate kinase, adenylate kinase and lactate dehydrogenase activity. The physiological meanings of these correlations and their possible significance for quality control of semen are discussed.Abbreviations used ACP acid phosphatase - ADP adenosine diphosphate - AK adenylate kinase - ALP alkaline phosphatase - ASPAT aspartate aminotransferase - ATP adenosine triphosphate - ATPase magnesium dependent adenosine triphosphatase - CRPO creatine phosphate - -GLU \-D-glucuronidase - ICDH isocitrate dehydrogenase - LDH lactate dehydrogenase - PK pyruvate kinase     

Protein and amino acid composition from the mantle of juvenile O ctopus vulgaris exposed to prolonged starvation

Protein and amino acid composition of the mantle of juvenile O ctopus vulgaris (Cuvier, 1797) during fasting for 27 days were determined. Average protein content of octopus mantle was of 711.19 ± 46.80 g kg^?1 DW, and it decreased with increasing fasting days. The non‐essential amino acids content was higher (486.18 ± 11.08 g kg^?1 protein) than essential amino acids (425.82 ± 9.15 g kg^?1 protein) at the start of the experiment (unstarved animals). The results suggest that the amino acid profile of the mantle where the most abundant amino acids are Arg, His, Lys, Gly, Leu and Pro could indicate a prolonged fasting condition (>20 days) or poor nutrition of O . vulgaris. This study supports the idea of using mantle for metabolic needs of starved O . vulgaris suggesting that the degradation pathway of amino acids to pyruvate and tricarboxylic acid cycle intermediates was favoured contrary to the degradation pathway of ketogenic amino acids. Special considerations should be taken concerning Thr, Ile, Ser, Ala, Asx (Asp, Asn), Glx (Glu, Gln) (because of their fast intake) and Lys and His (due to their stable contents) during a prolonged period of fasting.     

The essential amino acid requirements of the Dungeness crab, Cancer magister

The essential amino acid requirements for juveniles of the Dungeness crab Cancer magister were determined by the radiometric method. Both the protein and free amino acids were quantified and their specific activities compared.Injection of five crabs with [U-¹⁴C] glucose resulted in the labelling of the amino acids cysteine, aspartate, serine, glutamate, proline, alanine, and glycine. One crab injected with [U-¹⁴C] glutamate showed results similar to the glucose-injected crabs, with the notable exception that proline did not become labelled. An animal injected with [U-¹⁴C] phenylalanine produced radioactive tyrosine, alanine, and glutamate. Since fumarate is one of the breakdown products of phenylalanine in those species that have been studied, the lack of labelled aspartate is of interest.These results suggest that cysteine, aspartate, serine, glutamate, alanine, proline, and glycine are dispensable and that tyrosine is dispensable if phenylalanine is supplied. The non-labelled amino acids threonine, valine, methionine, isoleucine, leucine, phenylalanine, tryptophan, lysine, histidine, and arginine are inferred to be essential.     

Influence of oral administration of estradiol-17β and testosterone on growth,digestion, food conversion and metabolism in the underyearling red sea bream,Chrysophrys major

Estradiol-17 (E2) administered in the diet to the red sea bream Chrysophrys major did not affect appetite, food conversion efficiency, protein efficiency ratio and specific growth rate. Serum concentrations of total protein, albumin, globulin, vitellogenin, -amino acids, total lipid, free fatty acids, cholesterol and calcium were elevated. The hepatosomatic index was also increased. Activities of hepatic enzymes including lactate dehydrogenase, isocitrate dehydrogenase and glutamate-pyruvate transaminase were higher than found in untreated control fish. Intestinal activity of leucine aminopeptidase was augmented. However, there were no changes in muscle water, protein, lipid and glycogen content. In contrast, testosterone (T) given by the same route increased appetite, food conversion efficiency, protein efficiency ratio and specific growth rate. There were no alterations in serum protein and calcium concentrations but serum glucose, ammonia and triglyceride levels were elevated. Hepatic glycogen content was increased. The activities of hepatic fructose- 1,6-diphosphatase, glucose-6-phosphatase and glycogen synthetase and intestinal activities of alkaline phosphatase and -glutamyltransferase were higher than noted on control fish. The results reveal that estradiol-17 and testosterone exerted different metabolic effects in the red sea bream and they suggest that testosterone exerts its anabolic actions by increasing appetite, food conversion efficiency and activities of digestive enzymes.     

Utilization of dietary starch by juvenile white sea bream Diplodus sargus at different feeding frequencies

To assess the effect of feeding frequency on the utilization of dietary starch by white sea bream juveniles, triplicate groups of fish were fed an experimental diet (400 g kg^?1 protein, 140 g kg^?1 lipids and 350 g kg^?1 pregelatinized maize starch) to apparent visual satiation 2, 3 or 4 times a day for 63 days. Growth performance, feed utilization efficiency, glycaemia, cholesterolaemia, plasma triacylglycerides, liver lipids and glycogen content were unaffected by feeding frequency. α‐Amylase activity increased from the pyloric caeca to the posterior intestine and was higher in fish fed twice a day than in fish fed 3 or 4 times a day. Hepatic glucokinase (GK) activity decreased with the increase in feeding frequencies, whereas fructose‐1,6‐bisphosphatase (FBPase) activity increased. Hepatic glucose‐6‐phosphate dehydrogenase and glutamate dehydrogenase activities were unaffected by feeding frequency. Overall, feeding frequency did not improve white sea bream dietary starch utilization. α‐Amylase, GK and FBPase activities responded to dietary starch consumed at each meal, denoting a good metabolic adaptation of the fish to the feeding conditions.     

Effects of salinity on the ions important and sodium‐potassium ATPase in osmoregulation,cortisol, amino acids,digestive and immune enzymes in Scylla paramamosain during indoor overwintering

Overwintering is an important part of the conservation of Scylla paramamosain, and salinity has an important effect on the conservation of S. paramamosain during overwintering. Three salinities (4‰, 12‰ and 25‰) were selected as the overwintering salinities to reveal the effects of different salinities on the relevant important ions in osmotic pressure regulation, plasma cortisol, digestive enzymes, immune enzymes and amino acids of S. paramamosain during indoor overwintering. Results indicated that after overwintering, Cl^‐, Na⁺ and the osmotic pressure of serum have the highest salinity at 25‰, and the lowest salinity at 4‰. Na⁺/K⁺‐ATPase activity and cortisol were found to increase with decreasing salinity. The activity of digestive and immune enzymes was highest at 25‰, and was the lowest at 4‰. The amount of total amino acids (TAA), umami amino acids (UAA) and essential amino acids (EAA) in 25‰ were significantly higher than in 4‰ and 12‰. After overwintering, the essential amino acid index (EAAI) in the salinity range of 12–25‰ was 54.04–59.00, compared to 48.56–54.04 in the salinity range of 4–12‰. As a result, S. paramamosain at 25‰ had higher digestion and immunity than at 4‰ and 12‰, due to requiring more energy for osmotic pressure adjustment. In addition, S. paramamosain at 25‰ had the best meat quality. The results of this study are helpful for aquaculture production for indoor overwintering of S. paramamosain.     

Alteration in certain enzymological parameters of an Indian major carp, Cirrhinus mrigala exposed to short- and long-term exposure of clofibric acid and diclofenac

The extensive use of pharmaceuticals in human and veterinary medicine may enter the aquatic environment and pose a serious threat to non-target aquatic organisms like fish. In this study, Indian major carp Cirrhinus mrigala was exposed to different concentrations (1, 10 and 100 μg L^?l) of most commonly used pharmaceutical drugs clofibric acid (CA) and diclofenac (DCF) to evaluate its impacts on certain enzymological parameters during short- and long-term exposures. During short-term (96 h) exposure period, plasma glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and gill Na⁺/K⁺-ATPase activity were significantly altered at all concentrations of both the CA- and DCF-treated fish. In long-term exposure (35 days), gill Na⁺/K⁺-ATPase activity was found to be significantly increased at all concentration of CA and DCF exposures throughout the study period (except at the end of 7th day in 10 and 100 µg L-1) . However, a biphasic trend was observed in plasma GOT and GPT activity when compared to the control groups. In both short- and long-term exposure, a significant (P < 0.01 and P < 0.05) changes were observed in all enzymological parameters of fish C. mrigala exposed to different concentrations of CA and DCF. The alterations of these enzymological parameters can be effectively used as potential biomarkers in monitoring of pharmaceutical toxicity in aquatic environment and organisms.     

Interaction of dietary energy levels and culture density on growth performance and metabolic and oxidative status of rainbow trout (Oncorhynchus mykiss)

Rainbow trout (100 g initial weight) were subjected to the combined effect of two culture densities (15 and 40 kg m⁻³, D15 and D40, respectively) and two dietary energy levels (22 and 27 MJ kg⁻¹ E22 and E27, respectively) during a 75-days experimental period. At the end of the experiment, the growth rate as well as the metabolic and oxidative status of liver and muscle of fish were studied.The results showed that combination of culture density and dietaryenergy level negatively affected growth, cholesterol and LDL plasma levels and oxidative stress in muscle. Higher culture density negatively affected the values of total protein, triglycerides, and HDL in plasma, values of hepatic and muscular metabolic activities pyruvate kinase (PK), citrate synthase (CS), and hydroxiacil-CoA dehydrogenase (HOAD); glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT) activities in plasma, liver, and muscle; glucose 6P dehydrogenase (G6PDH) activity in muscle; and oxidative stress in liver.High energy intake, adversely affected the hepatic activity of G6PDH, HOAD, GPT and oxidative stress in muscle.Consequently our results indicate that a combination of high culture density and a high level of dietary energy (27 MJ kg⁻¹ in diet) exert a negative impact on the physiology and consequently on the welfare of the farmed fish.     

Metabolic enzyme activities in larvae of the African catfish,Clarias gariepinus: changes in relation to age and nutrition

The influence of ontogeny and nutrition on metabolic enzyme activities in larvae of the African catfish, Clarias gariepinus, was studied. After start of exogenous feeding, the larvae were reared for 10 days under three different nutritional conditions: Artemia nauplii, a dry starter diet, and starvation. The live feed gave the best growth (96 mg within 10 days) whereas the dry diet resulted in low growth (33 mg). This growth difference was reflected in larval RNA and DNA concentrations, but not in the levels of soluble protein. Enzymes representing the following aspects of metabolism have been analysed: NADPH generation (G6PDH, ME), glycolysis (PFK, PK), gluconeogenesis (FDPase), amino acid catabolism (GOT, GPT) and oxidative catabolism (CS). All enzymes were present from the start of exogenous feeding onwards, but their maximum specific activities displayed different developmental patterns. In catfish larvae fed on Artemia, G6PDH and ME activities steadily increased with age and weight of the larvae. CS levels remained, after an immediate enhancement upon onset of exogenous feeding, on a rather stable plateau. The amino acid-degrading enzymes GOT and GPT showed maximum levels at days 3–5 of feeding or at a body weight of 10–20 mg, but decreased thereafter. Activities of PFK, PK and FDPase showed low initial levels, and increased significantly with age and size. Based on the ontogenetic patterns of metabolic enzymes, in C. gariepinus larvae an early and a late developmental phase can be distinguished. During the early phase, the glycolytic and gluconeogenetic capacities are low, whereas they are enforced during the later phase. The oxidative capacity is high both during the early and the late phase. The metabolic changes in catfish development coincide with other major ontogenetic events, e.g., alterations of muscle organization, gill morphology, respiration and stomach structure and function. Rearing catfish larvae on a dry diet instead of Artemia partly altered the developmental pattern described: The ontogenetic elevation of CS, PFK and FDPase was delayed and the early peak in GOT and GPT activities was not realized. Particularly during the early developmental phase, the enzyme behaviour of the larvae fed on dry food was similar to that of starved larvae.Abbreviations CS citrate synthase - FDPase fructose-1,6-diphosphatase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase - G6PDH glucose-6-phosphate dehydrogenase - ME malic enzyme - PFK phosphofructokinase - PK pyruvate kinase     

The role of maslinic acid in the pentose phosphate pathway during growth of gilthead sea bream (Sparus Aurata)

NADPH produced in the pentose phosphate pathway (PPP) plays a central role in the reductive biosynthesis of membrane lipids, as well as in the maintenance of cell integrity; also, it has a key part in the synthesis of protein, the other member element. For that NADPH is involved in the growth processes. Our main objective is to study the effects of maslinic acid (MA) on kinetics and its molecular nature of NADPH‐generating systems in the gilthead sea bream (S. aurata) as well as the possible changes of these enzymes related with several feeding conditions; for this, we have studied the kinetic and expression levels of glucose 6‐phosphate dehydrogenase (G6PDH), 6‐phosphogluconate dehydrogenase (6PGDH), both PPP enzymes, in liver and white muscle. MA, a triterpene, stimulates growth, protein turnover rates and hyperplasia in fish. G6PDH and 6PGDH showed hyperbolic kinetics under all experimental conditions. With MA feeding, the specific activity, maximum velocity and catalytic efficiency of both enzymes increased in both tissue. The Michaelis constant changed with MA and fixed diet, and these changes being in relation to the substrate affinity. Moreover, we found that MA increased the protein levels studied, and this behaviour being consistent with the regulation of the number of enzyme molecules. These results show that G6PDH and 6PGDH are two inducible enzymes regulated by MA. Our findings corroborate that MA affect to the activity and expression of G6PDH and 6PGDH, confirming its role as markers of growth.     

Characterization and expression of NADPH oxidase genes in stimulated splenic neutrophils of tilapia,Oreochromis niloticus by a mix of traditional Chinese medicine and Bacillus species (TCMBS)

Traditional Chinese medicine and Bacillus species (TCMBS) mixture is an immunostimulant with considerable promise as an alternative in improving fish health. However, nothing is known on its effects on the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase genes and production of reactive oxygen species (ROS) in the neutrophils of fish. The full lengths of tilapia phagocytic NADPH oxidase genes gp91^phox, p22^phox, p40^phox, p47^phox, and p67^phox were cloned and their expression profiles after TCMBS stimulus investigated. The cDNAs of tilapia gp91^phox, p22^phox, p40^phox, p47^phox, and p67^phox contained open reading frames of 1698 bp, 561 bp, 1053 bp, 1584 bp, and 1470 bp respectively, encoding 561, 186, 350, 527, and 489 amino acids respectively. Comparison of the deduced amino acid sequences showed that tilapia NADPH oxidase genes shared 58%–91% and 21%–67% identity with those of other teleost and mammals respectively. Besides, tilapia NADPH oxidase genes contain conserved domains and motifs required for ROS generation. Phylogenetic analysis suggested tilapia NADPH oxidase genes were close to those of Fundulus heteroclitus. After 2 weeks of TCMBS application showed significant upregulation in expression of NADPH oxidase genes, antioxidant genes (i.e., superoxide dismutase, catalase, and glutathione‐disulphide reductase), and an increase in the production of ROS compared to the control in splenic neutrophils of tilapia. Collectively, our study provides evidence of the structure of tilapia NADPH oxidase genes and demonstrate that TCMBS application could modulate their activity in neutrophils to improve immunity in tilapia.