Trout Enzyme Research Articles

Effects of swimming activity and feed restriction on antioxidant and digestive enzymes in juvenile rainbow trout: Implications for nutritional and exercise strategies in aquaculture.

In this study, we investigated the effects of swimming activity and feed restriction on digestion and antioxidant enzyme activities in juvenile rainbow trout (average body weight of 26.54±0.36g). The stomach, liver and kidney tissues were obtained from four distinct groups: the static water group (fish were kept in static water and fed to satiation), the feeding restricted group (fish were kept in static water with a 25% feed restriction), the swimming exercised group (fish were forced to swimming at a flow rate of 1 Body Length per second (BL/s)) and the swimming exercised-feed restricted group (subjected to swimming exercise at a 1BL/s flow rate along with a 25% feed restriction). We determined the levels of glutathione, lipid peroxidation and the activities of catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and lactate dehydrogenase, as well as the presence of reactive oxygen species in the tissues obtained from the fish. Additionally, the activities of pepsin, protease, lipase and arginase in these tissues were measured. Swimming activity and feed restriction showed different effects on the enzyme activities of the fish in the experimental groups. It can be concluded that proper nutrition and exercise positively influence the antioxidant system and enzyme activities in fish, reducing the formation of free radicals. This situation is likely to contribute to the fish's development.

Time-dependent changes in oxidative stress biomarkers and activities of lysosomal and antioxidant enzymes in hepatic tissue of rainbow trout (Oncorhynchus mykiss Walbaum) following vaccination against Yersinia ruckeri

Abstract This study analyzed time-dependent effects of vaccination against Y. ruckeri on the oxidative mechanism underlying those effects by detecting relevant lipid peroxidation (2-thiobarbituric acid reactive substances, TBARS) and protein oxidation biomarkers [aldehydic and ketonic derivatives of oxidatively modified proteins (OMP)], antioxidant defenses [activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), total antioxidant capacity (TAC)], as well as activities of lysosomal functioning [alanyl aminopeptidase (AAP), leucyl aminopeptidase (LAP), acid phosphatase (AcP), and β-N-acetylglucosaminidase (NAG)] in hepatic tissue of rainbow trout, Oncorhynchus mykiss (Walbaum) following anti-Y. ruckeri vaccination in the first, second, and sixth months. A concentrated vaccine with Y. ruckeri strains was enclosed in fish feed and was administered three times every other day. Rainbow trout from each group were euthanized 31, 61, and 181 days following vaccination, and hepatic tissue was sampled for analysis. In the current study, vaccination against Y. ruckeri resulted in a no statistically significant change in TBARS levels, while aldehydic and ketonic derivatives of OMP in hepatic tissue decreased, especially after the first and second months following immunization. Moreover, the activities of glutathione-dependent enzymes increased, especially after the first and sixth months. The highest TAC levels were observed two and six months after vaccination. It has been shown that vaccination-related oxidative stress in hepatic tissue is involved in adaptive responses through the temporary mobilization of antioxidant and lysosomal enzymes in rainbow trout. The present study showed the effect of vaccination on lysosome membrane permeability for carbohydrate cleavage after the development of immunity against Yersinia, whereas antioxidant defence was reduced. Our results confirmed that the concept of preserving antioxidant enzyme function after vaccination was also evident when CAT, GR, and GPx activities either increased or were unchanged following vaccination.

Efficacy of vitamin E with or without probiotic, astaxanthin or rosemary extract on growth performance, survival, haematological parameters, antioxidant activity and liver enzymes in rainbow trout ( Oncorhynchus mykiss )

Growth and health status of rainbow trout (Oncorhynchus mykiss) (170.46 ± 1.16 g) fed diets containing 50 mg kg−1 vitamin E (control), 250 mg kg−1 vitamin E, 500 mg kg−1 probiotic (Bacillus lichiniformis + Bacillus subtilis each at 108 cfu g−1), 50 mg kg−1 astaxanthin and 500 mg kg−1 rosemary (Rosmarinus officinalis) extract were assessed for 4 months. No significant differences were seen in growth performance among treatments and between treatments and control (p > 0.05). The highest values of protein and lipid efficiency ratios were observed in 250 mg vitamin E, astaxanthin and rosemary trails. Probiotic trail demonstrated lower activity of liver superoxide dismutase (p < 0.05), while rosemary trail was superior (p < 0.05). Vitamin E trail exhibited an improvement in liver glutathione peroxidase compared to other treatments (p < 0.05), while catalase activity was insignificant among the treatments. Total leucocytes, neutrophil, red blood cells and haemoglobulin were insignificantly higher in fish fed probiotic and rosemary than other treatments. The lowest and the highest levels of activity of alanine aminotransferase in fish liver were measured in fish fed diets astaxanthin and control fish, respectively (p < 0.05), while no difference was seen in aspartate aminotransferase (p > 0.05). Fish fed with bacilli probiotic and rosemary extract resulted in higher survival rates compared to other treatments. Overall, these data exhibited that application of diets containing 50 mg vitamin E plus each rosemary extract (50 mg kg−1) or bacilli probiotic (500 mg kg−1) could be effective in maintaining health status of rainbow trout.

Effects of different LED light spectra on rainbow trout (Oncorhynchus mykiss): in vivo evaluation of the antioxidant status.

Rainbow trout (Oncorhynchus mykiss) farming is one of the major aquacultures in Turkey. Some conditions in fish farming can induce oxidative stress leading to the deterioration in properties such as appearance/color, texture, and flavor in fish meat. This situation may cause the consumer not to prefer edible fish. Although there are some studies on the impacts of light intensity on fish welfare, the changes in the antioxidant enzyme activities have not been elucidated. In the current study, it was intended to examine in rainbow trout how cultivating under different wavelengths affects the antioxidant enzymes and acetylcholine esterase (AChE) activity, because its activity is associated with oxidative stress, and also the determination of which light is suitable for fish welfare was aimed. Rainbow trout larvae were grown under four lights with different wavelengths: natural sunlight and incandescent long-wave (red light), medium-wave (green light), and short-wave (blue light) LED light. The experiment lasted for 64days. Biochemical assays were carried on in the brain, gill, and liver of rainbow trout. Antioxidant enzymes and AChE activity, which play an important role in the central nervous system, were assayed. In gill tissues, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glucose 6 phosphate dehydrogenase (G6PD), glutathione reductase (GR), glutathione S-transferase (GST), and AChE activities increased under all three light wavelengths. In the liver, while activities of antioxidant enzymes and AChE decreased in red light, all of them increased in blue and green light. In the brain, GPx, GST, G6PD, and SOD activities were reduced but AChE activity did not alter under all three light sources. In conclusion, light sources with different spectral structures caused important changes in the activities of antioxidant enzymes in rainbow trout. On this basis, it may be thought that this may be a response to the changing redox status of a cell. Based on our results, blue light sources may be suggested for fish welfare in rainbow trout culture, and providing fish welfare by changing light sources can be easy and cheap in fish farming.

Fishmeal alternative from renewable CO2for rainbow trout feed

The rapid global growth in fish farming and limited supply of fish meal (FM) has consequently reduced FM inclusion levels in compound feeds leading to a higher reliance on alternative protein sources. Sasya is a single cell protein (SCP) product that has a similar amino acid profile as FM. The aim of this study was to evaluate the effects of replacing FM with SCP on in vivo digestibility, growth, feed efficiency, whole-body proximate/amino acid composition and gene expression levels of various hepatic enzymes in rainbow trout (Oncorhynchus mykiss). Three isonitrogenous (470 g/kg crude protein) and isolipidic (18 g/kg crude lipid) diets were formulated as follows: Diet 1: control (30% FM); Diet 2:24% FM + 6% SCP and Diet 3:18% FM + 12% SCP. Each diet was hand-fed to triplicate tanks containing 30 rainbow trout fingerlings (4.99 ± 0.20 g) for 9 weeks. Apparent digestibility coefficients of SCP for dry matter, crude protein, lipid and energy were 60, 80, 93 and 74% respectively. Growth performance (final weight: 69–71 g), feed conversion ratios (0.91–0.94) as well as whole-body protein and amino acid composition were unaffected by diets. However, Diet 3 significantly increased whole-body crude fat and energy. Fish fed the SCP-based diets had significantly higher expression for carnitine palmitoyltransferase-1b (CPT1b), fatty acid delta 6 desaturase (FADS6) and fatty acid elongase 5 compared to the control. Overall, the quality of the SCP was similar as FM. Therefore, this product could enlarge the portfolio of alternative protein sources that can be used in fish diets and thus open a new market opportunity for use of a new feed resource in the feed industry.

Dietary supplementation of lemon verbena (Aloysia citrodora) improved immunity, immune-related genes expression and antioxidant enzymes in rainbow trout (Oncorrhyncus mykiss)

The use of veterinary drugs for the treatments of fish diseases has often health and environmental side effects and thus alternative more sustainable strategies are needed. In this study we have explored the effect of dietary administration of lemon verbena (Aloysia citrodora) leaves powder on growth, immune parameters and antioxidant enzyme activity of rainbow trout (Oncorrhyncus myskiss). The results show that adding lemon verbena (LV) leaves powder (0.5, 1 and 2%), as feed supplement does not affect significantly rainbow trout growth, but increases the levels of some immune parameters. All enriched-diets induced an increase in the levels of lysozyme activity and total immunoglobulin in rainbow trout serum (P < 0.05), whilst 1 and 2% enriched-diets increased lysozyme and immunoglobulin in skin mucus. Expression of interleukin-1β (IL-1β) was enhanced in all fish supplemented with LV leaves powder (0.5, 1 and 2%) whilst only the diet with the highest dose (2%) induced a significantly higher expression of IL-8 and tumour necrosis factor (TNF-α). Fish fed with LV leaves powder at 1 and 2% had significantly lower expression of transforming growth factor (TGF-β, P < 0.05). Furthermore, all enriched-diets significantly increased the activity of super oxide dismutase (SOD, P < 0.05) and diets containing 1 and 2% of LV leaves powder also increased activity of glutathione-S-transferase (GST) and glutathione peroxidase (GPx). In conclusion, LV leaves powder administered as feed supplement at 2% enhances the expression of some immune-related genes (IL-1β, IL-8 and TNF- α), rises the levels of lysozyme and total immunoglobulin in serum and skin mucus and increases the activity of antioxidant enzymes (SOD, GST and GPx) in rainbow trout.

Thermal sensitivity of white muscle lactate dehydrogenase isolated from a lake trout, (Salmo trutta), inhabiting lake Plav, Montenegro

To shed light on thermoadaptive properties of Salmo trutta from lake Plav (Montenegro), we undertook kinetic studies of pyruvate reduction rates and thermal stability analyses of white muscle LDH. We compared these with the data obtained for trout of the same, confirmed by us, Danubian lineage living in rivers and streams of Serbia and Montenegro. We also tested the effect of acclimation in captivity at 4 and 14 °C. The lake trout was of a typical smoltified phenotype (the size, the elongated silver colored body). At physiological substrate concentration, the breaks in the Arrhenius plots (critical temperature - Tc) correlated with acclimation temperatures or habitat water temperatures. Q10 values for temperatures above Tc were close to one, in all cases except 4 °C acclimated trout. At temperatures below Tc Q10 was close to two, except in the case of 14 °C acclimated trout. Lake trout had a highest Q10 values at temperatures below Tc. It was conspicuous that within the entire range of tested temperatures the differences in Q10 resulted from the effect of environmental temperature. Higher Q10 values were obtained with LDH isolated from trout acclimated to 4 °C compared with LDH acclimated to 14 °C. Ea values were much lower at a temperature below Tc compared with temperatures above Tc. Thermal stability of muscle LDH was lower after acclimation to 14 compared to 4 °C, while extremely high thermostability was obtained with the lake trout enzyme. Our data support the concept that Tc values have distinct physiological significance.

The effect of mycotoxin deoxynivalenol (DON) on the oxidative stress markers in rainbow trout (Oncorhynchus mykiss,Walbaum 1792)

Summary Deoxynivalenol (DON) is one of the most frequently detected mycotoxins in agricultural commodities used as animal feedstuff in Central Europe. This study focuses on determining effects of diets containing DON on oxidative stress markers and detoxifying enzymes in rainbow trout (Oncorhynchus mykiss). The fish were fed with commercial pellets containing DON at a concentration of 2 mg kg−1. Selected enzymes were measured in liver, gill and caudal kidney of the fish after 23 and 32 days of the experiment. Significant differences between the control and experimental groups were observed concerning activities of glutathione peroxidase (GPx) in kidney, glutathione reductase (GR) in gill and kidney, catalase (CAT) in kidney and liver and glutathione S-transferase (GST) in gill and liver. No significant differences were found for superoxide dismutase (SOD) gene expression, lipid peroxidation (TBARS) and the ferric reducing ability of plasma (FRAP). The data show that DON in the diet at the concentration below EC recommendation (2006/576/EC) induces oxidative stress in the rainbow trout.

Nutritional Regulation of the Rainbow Trout Long Chain‐Polyunsaturated Fatty Acid Biosynthetic Enzymes

Rainbow trout (Oncorhynchus mykiss) is a commercially important aquaculture species. The use of the vegetable oil‐derived omega‐3 polyunsaturated fatty acid (PUFA) α‐linolenic acid (18:3n‐3; ALA) instead of fish oil as a sustainable lipid source in feeds is being investigated. The metabolism of ALA to eicosapentaenoic acid (20:5n‐3; EPA) and docosahexaenoic acid (22:6n‐3; DHA) found in fish oil requires three desaturation and three elongation steps performed by the enzymes Δ6desaturase, Δ5desaturase, Elovl5 and Elovl2. However, the biosynthetic capabilities of rainbow trout are largely unknown. The objectives of this study were to (a) functionally characterise the elongases, Elovl5 and Elovl2, using a yeast expression system and (b) investigate the nutritional regulation of the desaturase and elongase enzymes in juvenile rainbow trout fed diets rich in ALA, EPA or DHA and any combination thereof. The Elovl5 activity was specific to C18–20 PUFA substrates and not C22 PUFA. In contrast, the Elovl2 activity was specific to C20‐22 PUFA only, with no activity for C18 PUFA. Furthermore, we determined the change in Δ6desaturase, Δ5desaturase, elovl5 and elovl2 gene expression in response to dietary omega‐3 PUFA. Our study was the first to identify an Elovl2 in rainbow trout and it is only the third Elovl2 to be functionally characterised from fish. This study has elucidated that rainbow trout is dependent on Elovl2 for docosapentaenoic acid (22:5n‐3; DPA) to 24:5n‐3 synthesis and ultimately DHA synthesis.

The Effects of Fish Meal Replacement by Yeast Based Nucleotides on Growth, Body Composition and Digestive Enzyme Activity in Rainbow Trout Juveniles (Onchorchyncus mykiss)

This 60 day study investigated the effects of organically certified nucleotide yeast-derived protein source (Nu-Pro®) on growth, feed efficiency, fillet proximate composition and digestive enzymes in rainbow trout (Onchorhyncus mykiss). Diets were isonitrogenous and isocaloric. Three experimental diets wherein 20%, 40% and 60% of the fish meal content was replaced by Nu-Pro®, were compared to the control diet in which the crude protein content was anchovy fish meal and corn gluten meal. The rainbow trout (initial weight 27 g/fish) were fed twice daily in 200-l Aqaria. Live weight gain increase ranged from 125-195% in fish fed the experimental diets. Results indicated that up to 40% fish meal protein can be replaced by Nu-Pro® without compromising growth rates, feed efficiency or the fillet biochemical composition in the rainbow trout. The effect of the dietary Nu-Pro® supplementation on digestion was partly observed by assaying the activity of pepsin, intestinal amylase, trypsin and lipase. Apart from amylase activity, results indicated that dietary supplementation of nucleotides is beneficial and may also have differential effects upon digestive enzyme activities.

Determantion of Growth Parameters, Digestibility and Nitrogen-Phosphorus Enviromental Impacts of Enzyme Supplementation to Soybean Based Diet in Rainbow Trout (Oncorhynchus mykiss)

This study was carried out to determine the effects on growth performance, feed utilization, apparent digestibility and reduce environment pollution waste output of nitrogen and phosphorus of diets based on Soybean Meal (40%) supplemented with exogenous enzymes in rainbow trout (Oncorhynchus mykiss) culture. Trout groups was began (initial mean weight 87.00±1,5g) method of random plots with 3 replications of 50 fish/pond with the ratio of 1050 fish with 6,0 metrik tonne in concrete ponds. 43% protein - 12% fat diet was used as the with no added enzyme in control group (K0) and diet supplemented with %02 protease enzyme (P), %02 mix enzyme (cellulose, xylanase, endo-s-1,3:1,4-glucanase (M), %02 pyhtase enzyme (F), %01 protease + %02 mix enzyme (PM), %01 protease + %01 phytase (PF) and %01 mix enzyme + %01 phytase (MF) were tested in total 7 groups. About of growth performance were found while condition factor (1,21-1,23) were statistically similar (p>0.05) however growth rate (173,68-177,27), specific growth rate (1,118-1,340) and feed conversion ratio (1,26-1,30) significantly different among groups (p<0.05) at the end of the experiment. P and F groups significantly improved specific growth rate, feed conversion ratio better than control group. In this study, showed the highest nitrogen apparent digestibility coefficient (ADC-N) (85,49±1,98) in group of P while the poorest value (72,82±0.01) group of K0 obtained, respectively (p<0.05). Also the best (58.57±0.49) and lowest (42.85±1.98a) ADC-P for phosphorus was obtained from F and K0, respectively (p<0.05). The highest (53,71%) and lowest (17,69%) retained nitrogen was held in the P and K0 groups (p<0,05). Also the dropped to water (discarded), nitrogen effluent was lowest (23,31 g/kg) in the P group and highest (46,91 g/kg) in the K0 group, respectively (p<0,05). In addition, highest (68,74%) and lowest (46,12%) retained phosphorus levels were obtained in the F and K0 groups, respectively. Also the dropped to water (discarded) while phosphorus levels reached the maximum (6,.94 g/kg) in K0 group and minimum value was obtained in the F group (4,85 g/kg)(p<0,05).

Isolation and characteristics ofβ-N-acetylglucosaminidase present in rainbow trout (Oncorhynchus mykiss) and Siberian sturgeon (Acipenser baerii) milt

Summary The aim of this study was to isolate and characterise the enzyme s-N-acetylglucosaminidase (β-NAGase) in rainbow trout and Siberian sturgeon milt, in order to find similarities or differences in its involvement in the fertilization process of these two species. While sturgeon sperm possess an acrosome, teleosts do not. Applying ion exchanging chromatography to β-NAGase of rainbow trout sperm, two peaks of β-NAGase activity were obtained from the plasma material and from the sperm. The molecular weight was 74 kDa for both milt plasma forms and 127 kDa for rainbow trout spermatozoa peak. Optimum pH of purified rainbow trout enzyme peaks ranged from 4.6 to 5.0. Michaelis–Menten constants were 11.59 × 10−4 and 11.48 × 10−4 m for I and II milt plasma peaks. Incubation of purified peaks I and II at 56°C resulted in a 45 to 50% decrease in activity of both forms. Km value for rainbow trout spermatozoa β-NAGase was 5.4 × 10−4 m. Enzyme incubation tests at high temperature proved this form of the enzyme to be most resistant to high temperatures, since its activity after 20 min of incubation at 56°C decreased only by 25%. The molecular weight of the enzyme originating from Siberian sturgeon milt plasma was about 265 and 113 kDa for I and II milt plasma peaks, respectively, and 271 kDa for the sperm extract. Optimum pH for all sturgeon purified peaks ranged from 3.8 to 5.0. The Km constant was 5.12 × 10−4, 5.28 × 10−4 and 5.0 × 10−4 m for I and II milt plasma and spermatozoa peaks, respectively. The loss of s-NAGase activity at 56°C was 58% for both milt plasma peaks and 35% for the sturgeon spermatozoa peak. The kinetic parameters, especially thermostability of s-NAGase was similar to the homologous enzymes observed in mammalian semen.

Effects of pulp and paper mill effluent extracts on liver anaerobic and aerobic metabolic enzymes in rainbow trout

This study investigates whether pulse exposure to Chilean pulp and paper mill effluent solid phase extracted (SPE) extracts via intraperitoneal injection (IP), would result in changes in the activities of the respiratory metabolic enzymes citrate synthase (CS) and lactate dehydrogenase (LDH) in rainbow trout livers. It also investigated if an alteration in liver metabolic capacity influenced the liver detoxification processes and estrogenic effects previously reported. Besides, a comparison of those enzymatic activities with fish IP injected with SPE extracts of two model effluents coming from industries that process 100% different type of feedstock (softwood, SW and hardwood, HW) was also evaluated. An initial induction of the anaerobic metabolism (increase in LDH enzymatic activity) was detected in all Chilean pulp mill effluent extracts evaluated, contrary to the initial unaltered aerobic metabolism (CS enzymatic activity) observed. A compensatory relationship in energy metabolism (Pasteur effect) was observed when comparing both enzymatic activities of fish exposed to those effluent extracts. LDH and CS activities observed in fish injected with Chilean extracts seem to be related to the effects observed in fish injected with SW extracts. This study showed that intraperitoneal injection of pulp and paper mill effluent extracts affected the anaerobic and aerobic metabolic capacities in rainbow trout livers, but this metabolic alteration did not affect detoxification capability or estrogenic effect previously reported.

Modifications of digestive enzymes in trout ( Oncorhynchus mykiss) and sea bream ( Sparus aurata) in response to dietary fish meal replacement by plant protein sources

Three experimental diets in which fish meal was partially (50 and 75%, named PP50 and PP75 diets, respectively) or totally replaced (PP100 diet) by plant protein sources were fed to rainbow trout and gilthead sea bream. We studied the effects of these diets on digestive enzymes in comparison to fish fed fish meal diets (FM). A mixture of vegetal ingredients (corn gluten meal, wheat gluten, extruded peas and rapeseed meal) was used to meet the amino acid requirements of the fish. Over a 12-week trial, four groups of the two species were fed one experimental diet twice a day until they showed satiety. After the growth period, post-prandial protease and α-amylase activities in proximal intestine were measured. Trout fed fish meal as the sole protein source (FM diet) showed a peak in total protease activity 3 h post-feeding; however, trout fed plant protein based diets (PP diets) did not register a peak in activity of these enzymes. In FM fed sea bream, proteolytic activity peaked in the most proximal intestinal segment 6 h post-feeding. This peak was also observed in PP fed sea bream, although the magnitude tended to decrease as the percentage of plant protein increased. This reduction was due to the specific inhibition of chymotrypsin-like bands, although a slight increase in trypsin secretion was detected in the zymograms of this species. The replacement of fish meal by plant protein did not affect α-amylase activity in either trout or sea bream. In sea bream proximal intestine histology was also studied. The PP100 fed group showed shorter folds and smaller goblet cell population when compared to FM fed group. In response to feeding the PP75 and PP100 diets, sea bream and trout showed a significant increase in relative intestinal length. Despite this compensatory mechanism, final weights of the two species diminished; however, this decrease exceeded 20% only in the case of groups fed the PP100 diets.

In Vitro Treatment with 17,20 -Dihydroxy-4-pregnen-3-one Regulates mRNA Levels of Transforming Growth Factor Beta Superfamily Members in Rainbow Trout (Oncorhynchus mykiss) Ovarian Tissue.

Transforming growth factor beta (TGFB) superfamily members are important paracrine/autocrine regulators of ovarian development and steroidogenesis in mammals, but their reproductive role in fishes is not well understood. Our objectives were 3-fold: to determine if key TGFB superfamily transcripts are regulated during the final stages of 17,20β-dihydroxy-4-pregnen-3-one (17,20βP)-induced oocyte maturation, to determine if this regulation is affected by ovarian development stage and to determine the steroid specificity of this regulation. In the first experiment we used real-time PCR to measure mRNA levels of key TGFB superfamily members in rainbow trout follicle-enclosed oocytes progressing through oocyte maturation and the resumption of meiosis. Briefly, ovarian tissue was removed from 6 fish and verified to be at similar developmental stages. After careful dissection, groups of isolated follicle-enclosed oocytes were placed in culture plates and incubated with either a control medium or 17,20βP, which is the maturation inducing hormone (MIH) in rainbow trout. The MIH-stimulated oocytes were monitored for progression through maturation and sampled at 3 different time points, overnight, yolk vesicle fusion, and germinal vesicle breakdown (GVBD), over a 96h period. Control samples were also collected at these times, although they were not progressing through maturation. While inhibin alpha and inhibin beta-B subunits did not significantly change with treatment or time period, inhibin beta-A (Inhba) mRNA levels increased throughout the 96h incubation in control tissues, but were drastically attenuated in 17,20βP-stimulated tissues. The inhibin beta subunits form either hetero- or homodimers to make activins. Furthermore, mRNA levels of bambi (bmp and activin membrane-bound inhibitor), a type 1 psuedoreceptor for bone morphogenetic proteins and activins, increased in control tissues throughout the 96h incubation, but were further increased in MIH-stimulated tissues. Ovarian development stage specificity of these responses was investigated in samples from female rainbow trout that were classified as late vitellogenic, non competent, or competent based on histology and responsiveness to MIH. The MIH-induced decrease of inhba and increase of bambi was corroborated at all stages. However, the response of bambi was greatly enhanced in the competent tissues, suggesting limited stage specificity. Lastly, the specificity of these responses to MIH was investigated by incubating competent ovarian fragments in the presence of serial diluted aliquots (29nM 2.9ìM) of estradiol (E2), testosterone (T), cholesterol, 17,20βP, 17 hydroxyprogesterone (17HP), and progesterone (P4). Stimulation with E2, T and cholesterol did not alter transcript levels in comparison with controls, whereas all of the progestins were effective at attenuating inhba and augmenting bambi in a dose-responsive manner. However, the effectiveness of MIH to alter inhba and bambi transcript levels was far superior to 17HP or P4, which were only effective at the highest dose (2.9ìM). When taken together with previously reported data describing MIH-induced regulation of steroidogenic enzymes in rainbow trout and the functions of TGFB members in mammals, these results support a model for an MIH positive feedback system at the level of the ovary and therefore suggest the involvement of key TGFB members and inhibitors in the final stages of rainbow trout ovarian development.

The role of hepatic, renal and intestinal gluconeogenic enzymes in glucose homeostasis of juvenile rainbow trout

Rainbow trout is unable to utilize high levels of dietary carbohydrates and experiences hyperglycemia after consumption of carbohydrate-rich meals. Carbohydrates stimulate hepatic glycolytic activity, but gene expression of the rate-limiting gluconeogenic enzymes glucose-6-phosphatase (G6Pase), fructose-1,6-bisphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK) remains high. Although there is significant mRNA expression and activity of gluconeogenic enzymes in trout intestine and kidney, the regulation of these enzymes by diet is not known. We tested the hypothesis that dietary carbohydrate modulates intestinal and renal G6Pase, FBPase and PEPCK. Fish were either fasted or fed isocaloric carbohydrate-free (CF) or high carbohydrate (HC) diets for 14 days. As expected, fish fed HC exhibited postprandial hyperglycemia and enhanced levels of hepatic glucokinase mRNA and activity. Dietary carbohydrates had no significant effect on the expression and activity of PEPCK, FBPase and G6Pase in all three organs. In contrast, fasting enhanced the activity, but not the mRNA expression of both hepatic and intestinal PEPCK, as well as intestinal FBPase. Therefore, the activity of rate-limiting gluconeogenic enzymes in trout can be modified by fasting, but not by the carbohydrate content of the diet, potentially causing hyperglycemia when fed high levels of dietary carbohydrates. In this species consuming low carbohydrate diets at infrequent intervals in the wild, fasting-induced increases in hepatic and intestinal gluconeogenic enzyme activities may be a key adaptation to prevent perturbations in blood glucose during food deprivation.

Divergent cyclooxygenase responses to fatty acid structure and peroxide level in fish and mammalian prostaglandin H synthases

Prostanoid synthesis in mammalian tissues is regulated at the level of prostaglandin H synthase (PGHS) cyclooxygenase catalysis by the availability and structure of substrate fatty acid and the availability of peroxide activator. Two major PGHS isoforms, with distinct pathophysiological functions and catalytic regulation, have been characterized in mammals; a functionally homologous PGHS isoform pair has been cloned from an evolutionarily distant vertebrate, brook trout. The cyclooxygenase activities of recombinant brook trout PGHS-1 and -2 were characterized to test the generality of mammalian regulatory paradigms for substrate specificity, peroxide activation, and product shifting by aspirin. Both trout cyclooxygenases had much more restrictive substrate specificities than their mammalian counterparts, with pronounced discrimination toward arachidonate (20:4n-6) and against eicosapentaenoate (20:5n-3) and docosahexaenoate (22:6n-3), the latter two prominent in trout tissue lipids. Aspirin treatment did not increase lipoxygenase-type catalysis by either trout enzyme. Both trout enzymes had higher requirements for peroxide activator than their mammalian counterparts, though the preferential peroxide activation of PGHS-2 over PGHS-1 seen in mammals was conserved in the fish enzymes. The divergence in cyclooxygenase characteristics between the trout and mammalian PGHS proteins may reflect accomodations to differences among vertebrates in tissue lipid composition and general redox state.

Evaluation of an Exposure Assay to Measure Uptake of Sediment Pah by Fish

The ecological risks of polynuclear aromatic hydrocarbons (PAH) in aquatic sediments will vary with both toxicity and bioavailability to aquatic biota. While there are standardized protocols to test the acute toxicity of sediment-borne compounds to aquatic invertebrates, there are none for assessing bioavailability to fish. We found that sediment-borne PAH caused an exposure-dependent induction of cytochrome P450 (CYP1A) enzymes in rainbow trout (Oncorhynchus mykiss) fingerlings exposed in semi-static 96 h bioassays, as shown by increased activity of ethoxyresorufin-o-deethylase (EROD). Assuming that PAH are taken up by trout due to partitioning from organic and inorganic constituents of sediments, we tested the effect of different test variables on bioaccumulation using induction as an index of exposure. EROD activity increased with exposure of fish to increasing volumes of sediments containing PAH, i.e., with increasing ratios of sediment to water. Uptake of single compounds from sandy sediments did not differ from uptake from clay or low organic (7% LOI - loss on ignition) sediments, but decreased when organic content was very high (58% LOI). Maximum induction was observed within 24 h of exposure and at 7.5 degrees C relative to 15, 22, or 28 degrees C. Storage and handling techniques had minor effects on sediment EROD induction potency. Absolute EROD activity was greater in white sucker (Catostomus commersoni) a benthic species, than in trout, a pelagic species. However, when basal (negative control) activity was accounted for, there was no difference in response between the species. Together, these experiments provide a basis for developing a standard protocol to test the bioavailability to fish of sediment-borne PAH.

Cross effects of the strain of dietary Saccharomyces cerevisiae and rearing conditions on the onset of intestinal microbiota and digestive enzymes in rainbow trout, Onchorhynchus mykiss, fry

Two strains of Saccharomyces cerevisiae were tested as probiotics for rainbow trout fry, during the first month of feeding. Each strain was introduced into separate diets, at the rate of 10 6 CFU g − 1 and their effects were compared with those of a control diet. Two rearing conditions were simultaneously compared, to test the adaptability of the probiotic treatment. From start feeding onwards, the water supply came from either spring or river, resulting in two different temperature ranges, 11–11.5 and 7–8 °C respectively. Growth and development were optimal in spring water, while some delay was observed with colder river water. A slight but significant increase in mortality was also observed in the river group. In all groups, the counts of bacteria associated with trout intestine were maximum 10 days post start feeding (dpsf; 10 7 CFU g − 1 ). The counts of probiotic yeast were also maximum at 10 dpsf (10 4–10 5 CFU g − 1 ), but the decrease was slower in river than in spring water. An autochthonous yeast, Debaryomyces hansenii, was also retrieved associated to the intestine of the control group in high numbers after 240 degree days of experiment (10 4–10 5 CFU g − 1 ), while the colonization level was significantly less in trout fed the probiotic diets. The effect of the dietary yeast was observed by assaying the activity of three enzymes in the brush border membrane of the enterocytes: alkaline phosphatase (AP), γ-glutamyl-transpeptidase (GGT), and leucine-amino-peptidase N (LAP). At 10 and 20 dpsf, the trout reared in spring water had higher activities of the three enzymes when they were fed the strain S. cerevisiae var. boulardii, suggesting an earlier maturation of the digestive system in this group, compared with trout fed either the other strain of S. cerevisiae or the control diet. The effect was not observed in trout reared in river water with slower growth. Both S. boulardii and D. hansenii seemed to stimulate digestive maturation in fish, but the natural colonization by D. hansenii was likely too late for trout reared at optimal temperature. The supplementation of trout starter diet with S. boulardii may be particularly useful in fast growing conditions.

Development of a simple and efficient method for assaying cytidine monophosphate sialic acid synthetase activity using an enzymatic reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide converting system

A new reliable method to assay the activity of cytidine monophosphate sialic acid (CMP-Sia) synthetase (CSS) has been developed. The activation of sialic acids (Sia) to CMP-Sia is a prerequisite for the de novo synthesis of sialoglycoconjugates. In vertebrates, CSS has been cloned from human, mouse, and rainbow trout, and the crystal structure has been resolved for the mouse enzyme. The mouse and rainbow trout enzyme have been compared with respect to substrate specificity, demonstrating that the mouse enzyme exhibits a pronounced specificity for N-acetylneuraminic acid (Neu5Ac), while the rainbow trout CSS is equally active with either of three Sia species, Neu5Ac, N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (KDN). However, molecular details that explain the pronounced substrate specificities are unknown. Understanding the catalytic mechanisms of these enzymes is of major importance, since CSSs play crucial roles in cellular sialylation patterns and thus are potential drug targets in a number of pathophysiological situations. The availability of the cDNAs and the obtained structural data enable rational approaches; however, these efforts are limited by the lack of a reliable high-throughput assay system. Here we describe a new assay system that allows product quantification in a reduced nicotinamide adenine dinucleotide (NADH)-dependent color reaction. The activation reaction catalyzed by CSS, CTP + Sia → CMP-Sia + pyrophosphate, was evaluated by a consumption of Sia, which corresponds to that of NADH on the following two successive reactions: (i) Sia → pyruvate + ManNAc (or Man), catalyzed by a sialic acid lyase (SAL), and (ii) pyruvate + NADH → lactate + oxidized nicotinamide adenine dinucleotide (NAD +), catalyzed by a lactate dehydrogenase (LDH). Consumption of NADH can be photometrically monitored on a microtiter plate reader for a number of test samples at the same time. Furthermore, based on the quantification of CSS used in the SAL/LDH assay, relative activities toward Sia derivatives have been obtained. The preference of mouse CSS toward Neu5Ac and the ability of the rainbow trout enzyme to activate both KDN and Neu5Ac were confirmed. Thus, this simple and time-saving method is suitable for a systematic comparison of enzyme activity of structurally mutated enzymes based on the relative specific activity.