Changes of carotenoids contents and analysis of astaxanthin geometrical isomerization in Haematococcus pluvialis under outdoor high light conditions

In this study, carotenoid contents of Haematococcus pluvialis during outdoor high light cultivation were measured, moreover, changes of astaxanthin geometrical isomers and biotic factors which may affect isomerization were investigated. During the incubation, contents of both astaxanthin and its precursors (zeaxanthin and canthaxanthin) increased over time, whereas the relative content of lutein decreased. Contents of all astaxanthin isomers increased, while the proportion of different astaxanthin geometrical isomers fluctuated during the incubation. All‐trans‐astaxanthin of total astaxanthin (T/A) was higher during the astaxanthin accumulation phase than that in the cell transformation phase, which was in contrast to results of 13‐cis‐astaxanthin of total astaxanthin (13C/A) and 9‐cis‐astaxanthin of total astaxanthin (9C/A). Farnesyl diphosphate synthase (trans (2E,6E)‐FPPS, EC2.5.1.10) and geranylgeranyl diphosphate synthase (GGPS, EC2.5.1.29), the key proteins involved in geometrical isomerization, decreased during the astaxanthin accumulation phase. Moreover, the presence of cis (2Z,6E)‐FPPS was firstly confirmed in H. pluvialis by HPLC‐MS/MS shotgun method. The results indicated the biotic factor (trans‐FPPS, cis‐FPPS and GGPS) may play an important role in astaxanthin geometrical isomerization of H. pluvialis, but not a crucial role. This study would help in optimizing the regulation of astaxanthin geometrical isomers in H. pluvialis, with great significance in theory and production.     

Effects of dietary astaxanthin on the growth,innate immunity and antioxidant defence system of Paramisgurnus dabryanus

The effects of astaxanthin supplementationon the growth, innate immunity and antioxidant defence system of loach (Paramisgurnus dabryanus) were investigated in this study. A total of 450 fish (initial average weights of 3.00 ± 0.10 g) were fed five diets with graded levels of astaxanthin (0.00, 50.00, 100.00, 150.00 and 200.00 mg/kg) for 56 days. The results showed that astaxanthin supplementation significantly stimulated the growth (FBW, WG, FER, HSI), innate immunity (AKP activity in the hepatopancreas, intestinal tract, muscle and skin and LZM activity in the hepatopancreas, intestinal tract and skin), antioxidant ability (T‐SOD, GSH‐PX and CAT activities and T‐AOC, GSH and MDA contents in the hepatopancreas, intestinal tract, muscle and skin) of loach. Moreover, dietary astaxanthin supplementation significantly up‐regulated the relative mRNA levels of Nrf2 and Maf, and down‐regulated the relative mRNA level of Keap1 in the hepatopancreas when the supplementation levels of astaxanthin were 50–200 mg/kg. In conclusion, the diets with 100–151.06 mg/kg astaxanthin supplementation were optimal for loach, which based on the growth, immunity and antioxidant‐related indicators, and the astaxanthin supplementation regulated the antioxidant ability partially referring to Keap1‐Nrf2 signallings.     

Effects of dietary astaxanthin on growth,blood biochemistry,antioxidant, immune and inflammatory response in lipopolysaccharide‐challenged Channa argus

The present study investigated the effects of dietary astaxanthin on the growth, blood biochemical, antioxidant, immune and inflammatory response in lipopolysaccharide‐challenged Channa argus. A total of 0, 50, 100 and 200 mg/kg of astaxanthin were added to the basal die for 56 days. After the feeding experiment, each group was subjected to a lipopolysaccharide challenge (except for the Control group). The results showed that adding astaxanthin to the diet can significantly increase the weight gain and specific growth rate and decrease the feed conversion ratio of C. argus; the highest weight gain, specific growth rate and minimum feed conversion ratio occurred in the 100 mg/kg group. Furthermore, dietary astaxanthin supplementation can alleviate the negative effects of lipopolysaccharides by increasing the levels of alkaline phosphatase, lysozyme, complement 3, complement 4, total serum protein, albumin, globulin, superoxide dismutase, catalase and glutathione peroxidase and decrease the serum cortisol, aspartate aminotransferase, alanine aminotransferase, glutathione peroxidase, and malondialdehyde. Dietary astaxanthin supplementation also can decrease the relative expression of inflammatory genes (nuclear factor κB, interleukin‐1, interleukin‐8 and tumour necrosis factor‐α) in the liver, spleen, kidney and intestine. To summarise, dietary astaxanthin addition can improve the growth performance and attenuate the negative effects of lipopolysaccharide challenge in C. argus. The optimal amount of astaxanthin is 100 mg/kg.     

Comparative study on nutrient composition and quality evaluation in a new variety and wild‐typed ridgetail white prawn (Exopalaemon carinicauda)

Kesuhong No. 1 with high concentration of astaxanthin was a newly approved aquaculture variety of ridgetail white prawn (Exopalaemon carinicauda) in China, and the information on the nutrient composition and quality evaluation of this new variety is insufficient. Thus, the proximate composition, amino acid and fatty acid profiles were analysed and compared between the new variety and the wild‐typed prawn in the present study, for its better application of the new variety. It showed that there were no significant differences (p > 0.05) of the proximate composition, essential amino acid and saturated fatty acid (SFA) of muscle tissue between the wild‐typed prawn and the new variety. However, for the content of some delicious amino acids (Ala and Tyr), it was significantly higher (p < 0.05) than that in the wild‐typed prawn. Similarly, for some ω3 polyunsaturated fatty acids (ω3 PUFA), such as docosahexaenoic acid (DHA) and eicosapntemacnioc acid (EPA), it was also significantly higher (p < 0.05) than that in the wild‐typed prawn. In addition, higher PUFA/SFA ratio and lower ω6/ω3 ratio also improved muscle quality in the new variety. Information obtained in the present study indicates a promising natural source of ω3 PUFA with high nutritional value, and has strong potential application value in this new variety of E. carinicauda.     

Digestibility,growth and pigmentation of astaxanthin,canthaxanthin or lutein diets in Lake Kurumoi rainbowfish,Melanotaenia parva (Allen) cultured species

New cultured ornamental fish namely Lake Kurumoi rainbowfish Melanotaenia parva (Allen) run into reduced of colour performances when reared in the aquaria, consequently, fish feed must be added with carotenoids as a pigment source. The aim of this study was to evaluate the digestibility, growth and pigmentation of astaxanthin, canthaxanthin and lutein in diet. Apparent digestibility coefficients (ADC) of dry matter, lipid, protein, carotenoids, growth and pigmentation were studied in twenty fish after 14 and 56 days of observation. The single‐dose supplementation of 100 mg/kg of astaxanthin, canthaxanthin, or lutein diets on fish was fed by apparent satiation. The basal diet without carotenoids was used as control. The result showed that the ADC of carotenoids of test diets was higher compared to control. Fish fed astaxanthin diet had higher survival rate (96.67 ± 2.89%), colour measurements of lightness (57.60 ± 7.46%), a*‐values (4.66 ± 1.20), total carotenoids content in skin (33.75 ± 5.02 mg/kg) and muscle (2.16 ± 0.74 mg/kg). Astaxanthin also increased the growth after 14 days (2.00% ± 0.19%/days) but there was no significantly different at the end of experiment. The yellowish‐orange colour performance was more rapidly achieved by fish fed astaxanthin diet after 28 days experimentation. These values suggested that dietary carotenoids were required and astaxanthin diet was superior to other diets for skin pigmentation of Lake Kurumoi rainbowfish.     

PCR‐DGGE analysis of the autochthonous gut microbiota of grouper Epinephelus coioides following probiotic Bacillus clausii administration

The autochthonous microbiota in the foregut, midgut and hindgut of juvenile grouper Epinephelus coioides following the dietary administration of probiotic Bacillus clausii for 60 days were assessed using polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE). A complex and generally similar bacterial composition along the digestive tract of E. coioides was detected in the DGGE profiles, while several bacteria showed regional specialization. Similarity dendrogram revealed that the bacterial composition of the foregut was more similar to the midgut than the hindgut. Samples collected from the probiotic group and the control group showed generally similar DGGE patterns, while no significant difference in the total number of bands and Shannon index were observed between the probiotic group and the control group, suggested that probiotic B. clausii exerted no significant effect on the gut microbiota of E. coioides. However, various potentially beneficial bacteria, such as Enterococcus sp.‐like and Bacillus pumilus‐like bacterium were selectively stimulated by probiotic B. clausii, while some potential harmful species, like Staphylococcus sp.‐like and Vibrio ponticus‐like bacterium were depressed. These indicated that the gut microbiota was modified to some degree by probiotic B. clausii. Sequences analysis showed that the autochthonous gut bacteria of E. coioides could be classified into four groups, i.e. Proteobacteria, Firmicutes, Actinobacteria and unclassified bacteria.     

Effects of dietary fish oil replacement by microalgae raw materials on growth performance,body composition and fatty acid profile of juvenile olive flounder,Paralichthys olivaceus

Replacing dietary fish oil with DHA‐rich microalgae Schizochytrium sp. and EPA‐rich microalgae Nannochloropsis sp. for olive flounder (Paralichthys olivaceus) was examined. Three experimental isonitrogenous and isolipidic diets with lipid source provided by 50% fish oil (F50S50), 50% (M50F25S25) and 100% microalgae raw material (M100) respectively were compared with a soybean oil (S100) diet as control. Triplicate groups of olive flounder juveniles (16.5 ± 0.91 g) were fed the experimental diets, and a group was fed the control diets for 8 weeks in a recirculation system. Results showed feed efficiency and growth performance were not significantly changed when fish oil (FO) was totally substituted by soybean oil (SO) or microalgae raw material (MRM). The whole‐body composition, lipid content of liver and muscle, and lipid composition of plasma were not significantly influenced by the total substitution of FO by MRM. The polyunsaturated fatty acids (PUFA) content of muscle and liver declined in fish fed S100 diet, whereas it was not significantly reduced in fish fed M50F25S25 and M100 diets. The total substitution of FO by MRM not only maintained the levels of arachidonic acid, EPA or DHA but also increased n‐3/n‐6 ratio. In conclusion, MRM as the sole lipid source is sufficient to obtain good feed efficiency, growth performance and human health value in olive flounder juveniles.     

Response of the intestinal mucosal barrier of carp (Cyprinus carpio) to a bacterial challenge by Aeromonas hydrophila intubation after feeding with β‐1,3/1,6‐glucan

The effect of dietary β‐glucan on the bacterial community in the gut of common carp (Cyprinus carpio) was examined after oral application of Aeromonas hydrophila. Carp received either feed supplemented with 1% MacroGard^®, a β‐1,3/1,6‐glucan, or a β‐glucan‐free diet. Fourteen days after feeding, half of the carp from each group were intubated with 10⁹ colony‐forming units (CFU) of a pathogenic strain of A. hydrophila. Gut samples were taken 12 hr to 7 days after application and analysed using microbiological and molecular biological techniques (NGS, RT‐PCR‐DGGE). The reaction of the mucosa and the microbiota to an A. hydrophila intubation differed in carp fed with β‐glucan compared to carp from the control group. In β‐glucan fed carp, the total bacterial amount was lower but the number of bacterial species was higher. Bacterial composition was different for carp from both treatment groups. The number of mucin filled goblet cells was reduced in carp fed the β‐glucan diet. Mucus was obviously released from the goblet cells and was probably washed out of the gut together with high numbers of bacteria. This might be protective against pathogenic bacteria and, therefore, feeding with β‐glucan may provide protection against infections of the gut in carp.     

Chemical composition of Lippia spp. essential oil and antimicrobial activity against Aeromonas hydrophila

Phytotherapy can replace antibiotic administration as an alternative to control Aeromonas hydrophila, one of the main bacteria involved in the aetiology of farmed fish diseases. Given that plants of the Lippia spp. genus show biological potential for antimicrobial activity, this study evaluated the chemical composition of essential oils extracted from Lippia alba, Lippia origanoides and Lippia sidoides and their activity against A. hydrophila. The oils were obtained by steam distillation in a Clevenger‐type apparatus and their composition determined by gas chromatography and mass spectrometry (CG/MS). Antibacterial activity was assessed by calculating the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using broth microdilution method. The main compounds identified were geranial (25.4%) and neral (16.6%) in L. alba oil, carvacrol (40.4%) and p‐cymene (11.4%) in L. origanoides oil and thymol (76.6%) and ortho‐cymene (6.3%) in L. sidoides oil. The three Lippia species showed bacteriostatic and bactericidal action against A. hydrophila, with MICs and MBCs ranging from 1250 to 5000 μg mL^?1. Of the species tested, the best performance was obtained with essential oil of L. sidoides.     

Maltoporin (LamB protein) contributes to the virulence and adhesion of Aeromonas veronii TH0426

Aeromonas veronii is one of the main pathogens causing freshwater fish sepsis and ulcer syndrome. More and more cases have shown that it has become an important zoonotic and aquatic agent. In this study, a A. veronii TH0426 mutant strain (ΔlamB) with an in‐frame deletion removed nucleotides 10–1,296 of the lamB gene was firstly constructed to investigate its functions. The results showed that the LD₅₀ value of the mutant ΔlamB to zebrafish and mice was 13.7‐fold and 5.6‐fold higher than those of the wild‐type strain, respectively. The toxicity of wild‐type strain to EPC cells was 2.1‐fold and threefold higher than those of ?lamB when infected for 1 and 2 hr. Furthermore, the ability of biofilm formation and the adhesion and invasion to EPC cells of ?lamB significantly decreased for 5.6‐fold and 1.8‐fold separately. In addition, motility detection result indicated that ?lamB lost the swimming ability. The results of flagellar staining and TEM demonstrated that the flagella of ?lamB were shed. In general, the deletion of lamB gene caused a significant decrease in the virulence and adhesion of A. veronii TH0426, and it can be known that the lamB gene of A. veronii plays a crucial role in the pathogenesis.     

The effect of insect meal as a feed ingredient on survival,growth, and metabolic and antioxidant response of juvenile prawn Palaemon adspersus (Rathke, 1837)

For successful prawn aquaculture, feeds should be based on readily consumed ingredients that promote survival and optimal growth performance. This pilot study investigates the rearing of juvenile Baltic prawn Palaemon adspersus. Two feeding trials were carried out for 60 days; both incorporated insect meals, the first one in fishmeal‐based diets, whereas the second one in plant meal ones. Insect meals derived from larvae of Tenebrio molitor (TM), Hermetia illucens (HI) and Musca domestica (MD) were tested as feed ingredients. This study indicated that the inclusion of HI in fishmeal diets resulted in significantly higher growth performance and survival of the prawns, whereas the MD diet led to similarly high growth performance reducing significantly their survival. Growth performance was not affected by the insect inclusion in the plant‐based diets, but survival was higher in the TM and HI inclusion diets. The inclusion of TM and HI resulted in higher protein and energy content of the prawns’ muscle when incorporated in fishmeal and plant meal diets respectively. No significant differences were observed in the activities of hepatopancreas’ amino acid‐catabolizing enzymes. Concluding, the combinations fishmeal–HI and plant meal–TM can be used for the successful rearing of Baltic prawn.     

Replacement of fish oil with a DHA‐rich Schizochytrium meal on growth performance,activities of digestive enzyme and fatty acid profile of Pacific white shrimp (Litopenaeus vannamei) larvae

This trial was conducted to evaluate the effects of replacing dietary fish oil with Schizochytrium meal for Pacific white shrimp (Litopenaeus vannamei) larvae (initial body weight 4.21 ± 0.10 mg). Six test microdiets were formulated using Schizochytrium meal to replace 0 g/kg, 250 g/kg, 500 g/kg, 750 g/kg, 1000 g/kg or 1500 g/kg fish oil DHA. No significant differences were observed in survival, growth, final body length and activities of digestive enzyme among shrimp fed different diets (p > .05). No significant differences were observed in C20:5n‐3 (EPA) in muscle samples (p > .05). C18:3n‐3 and C20:4n‐6 in muscle increased as Schizochytrium meal replacement level increased (p < .05). No significant differences were observed in C22:6n‐3 (DHA) and n‐3 fatty acids among shrimp fed diets that algae meal replaced 0 g/kg ‐ 1000 g/kg of fish oil. Shrimp fed diet R150 had higher DHA content than other groups and had higher n‐3 fatty acids than that of shrimp fed diets R50, R75 and R100 (p < .05). C18:2n‐6, PUFA and n‐6 fatty acids in muscle increased, while n‐3/n‐6 ratio decreased with increasing algae meal replacement level from 0 g/kg to 1000 g/kg (p < .05). In conclusion, Schizochytrium meal could replace 1500 g/kg fish oil DHA in the microdiets without negatively affecting shrimp larvae survival, growth and activities of digestive enzyme.     

Effects of substituting dietary fish oil with crude palm oil and palm fatty acid distillate on growth,muscle fatty acid composition and the activities of hepatic lipogenic enzymes in snakehead (Channa striatus,Bloch 1793) fingerling

Three diets were formulated to be iso‐nitrogenous (450 g kg^?1), iso‐lipidic (65 g kg^?1) and iso‐energetic (18.5 KJ g^?1), varying only in their lipid sources and designated as 100% fish oil (FO), 100% crude palm oil (CPO) and 100% palm fatty acid distillate (PFAD). Feed were hand fed to homogenous groups of 12 Channa striatus fingerlings (mean weight 3.5 ± 0.3 g) per tank in triplicate for 12 weeks, in a recirculation system. The growth performance and feed intake in the CPO and PFAD treatments were significantly (P<0.05) higher than those in the fish fed the control diet (FO), respectively, whereas the feed conversion ratio was better in PFAD than that in the other treatments respectively. The biological indices monitored (hepatosomatic index and viscerosomatic index) as well as carcass yield did not vary significantly among all the treatments respectively. The muscle fatty acid (FA) profile of fish was influenced by the composition of the diets fed, whereas no differences were recorded in the activities of the hepatic lipogenic enzymes monitored (fatty acid synthetase, citrate cleavage enzyme and malic enzyme). Whole‐body proximate composition analysis revealed that PFAD treatment, compared with others, contained significantly higher protein and ash, but lower lipid contents, although the muscle content of these nutrients was similar among all the treatments. Based on the results of this trial, CPO and PFAD could be used to partially substitute FO in the diet for C. striatus fingerling, to achieve good growth performance without any negative effects or compromising the muscle n‐3 FA composition (especially in the docosa hexaenoic acid and eicosa pentaenoic acid content).     

The efficacy of MS‐222 as anaesthetic agent in four freshwater aquarium fish species

The efficacy of anaesthetic tricaine methanesulfonate (MS‐222) was evaluated in four freshwater aquarium fish species, Zebrafish (Danio rerio), Guppy (Poecilia reticulata), Discu (Symphysodon discus) and Green swordtail (Xiphophorus helleri). The correct dose of anaesthetic should induce the plane 4 of anaesthesia in less than 180 s, recovery in less than 300 s and must survive when exposed during 30 min to anaesthetic. Fishes were exposed to six concentrations of anaesthetic (75, 100, 125, 150, 200 and 250 mg L^?1) and the time of fish reaching plane 4 of anaesthesia, post exposure recovery, and the percentage of survival when fish were subject to 30 min in the anaesthetic were recorded. The optimal doses varied according to the species: D. rerio – 75, 100 and 125 mg L^?1, P. reticulata – 125, 150 and 200 mg L^?1, S. discus – 75 and 100 mg L^?1 and X. helleri – 125 and 150 mg L^?1. The induction time generally decreased significantly with increasing concentration of MS‐222 for all of the species evaluated. The recovery time had a tendency to increase with the increase of the MS‐222 concentration for D. rerio, P. reticulata and S. discus. On the other hand, X. helleri recovery time decreased with the increase of MS‐222 concentration. MS‐222 proved to be effective in anaesthesia for all the freshwater ornamental species studied. The main results clearly show that the optimal dose to anesthetize is fish species dependent and it is completely wrong to extrapolate optimal anaesthetic concentrations between different species.     

The effects of synthetic pigments on pigmentation of Pethia conchonius (Hamilton, 1822)

This study evaluated the effects of diets containing 20, 40, 60, 80 and 100 mg kg^?1 diet astaxanthin or canthaxanthin on Pethia conchonius (Hamilton, 1822) pigmentation. A completely randomized experimental design was developed with ten treatments and three replicates. Three hundred rosy barb with a mean weight of 0.92 ± 0.06 g were assigned to thirty aquaria for period of eight weeks. Carotenoid contents of fish fed canthaxanthin were always lower than those fed astaxanthin. Yellowness (b*) was not affected by pigments. While Luminosity (L*) decreased in fish fed astaxanthin diets, this parameter increased by feeding on canthaxanthin. The most pronounced effect was higher a* values in fish fed astaxanthin. Astaxanthin retention rate was higher than that of canthaxanthin. The present results demonstrate that canthaxanthin cannot be considered as a proper replacement with astaxanthin. Inclusion of 80 and 100 mg astaxanthin kg^?1 diet can be suitable dietary levels to ensure pigmentation and this condition may improve market value of rosy barb.     

Immune effects of bathing European eels in live pathogenic bacteria,Aeromonas hydrophila

The specific and non‐specific immune parameters and protection of European eels (Anguilla anguilla) were evaluated after bathing eels with Aeromonas hydrophila. Two hundred eels were distributed into two equal groups and bathed with Phosphate‐buffered saline (Control group) or 1.0 × 10⁷ cfu mL^?1 A. hydrophila (Test group) for 1 h respectively. Then, eels were bled aseptically from the caudal sinus on 1, 4, 7, 14 and 28 days post treatment. The blood cells were used to evaluate the cellular immunity and the serum was used to determine the titres of specific antibody as well as the activities of superoxide dismutase (SOD) and lysozyme. Eels from both groups were challenged by intraperitoneal injection of 1.0 × 10⁷ cfu of A. hydrophila on 28 and 42 days post bathing. The results show that eels bathed in A. hydrophila significantly (P < 0.05) enhanced the proliferation of different types of blood cells and the serum titres of anti‐A. hydrophila antibody. The Relative Percent Survival (RPS) after challenge on 28 and 42 days post bathing in Test group vs. Control group was 40% and 50% respectively. These results suggest that bathing European eels in A. hydrophila would positively affect specific as well as non‐specific immune parameters and protect against A. hydrophila infection in freshwater farming.     

Effects of dietary supplementation of Haematococcus pluvialis powder on gonadal development,coloration and antioxidant capacity of adult male Chinese mitten crab (Eriocheir sinensis)

The green algae Haematococcus pluvialis is an important source of natural astaxanthin as feed additive. This study was conducted to investigate the effects of dietary supplementation of H. pluvialis powder on gonadal development, coloration and antioxidant capacity of adult male Chinese mitten crab Eriocheir sinensis, and four experimental diets were formulated to contain 0, 0.2%, 0.4% and 0.6% of H. pluvialis powder. There were four treatments (defined as D1~D4) in this study and each treatment had three replicates. Dietary H. pluvialis contents had no significant effects on survival, body weight gain rate and gonadal development of male E. sinensis. For colour parameters, the total carotenoids content in carapace and hepatopancreas as well as hepatopancreatic lightness (L*) and carapace redness (a*) increased significantly with increasing dietary H. pluvialis (p < 0.05). For the antioxidant indices in the serum, D4 had the lowest activities of superoxide dismutase (SOD) and peroxidase (POD), but the highest glutathione peroxidase (GSH‐Px) and lactic dehydrogenase (LDH), while the malondialdehyde (MDA) in serum and hepatopancreas decreased significantly with the rising content of dietary H. pluvialis (p < 0.05); D1 had the highest levels of SOD, POD and GSH‐Px in hepatopancreas. For the non‐specific immune indices, the highest activities of acid phosphatase (ACP) and γ‐glutamyl transpeptidase (γ‐GT) were found on the serum of D3 and D4 (p < 0.05). D1 had the highest levels of ACP and alkaline phosphatase (ALP) in hepatopancreas, while D2 and D3 had the lowest levels of ALP and ACP respectively. These results suggested the optimal dietary natural astaxanthin level was around 40 mg/kg diets.     

Effects of crude extracellular protein and crude intracellular polysaccharides of Vibrio alginolyticus on the growth,energy metabolism regulation and WSSV resistance of Litopenaeus vannamei

This study aimed to investigate the effects of adding crude extracts of the extracellular protein (Ex‐Pro) and intracellular polysaccharides (In‐Poly) of Vibrio alginolyticus to diets, at a dosage of 10 g/kg, on the growth, energy metabolism and WSSV (White Spot Syndrome Virus) resistance of Litopenaeus vannamei (initial weight, 0.88 ± 0.04 g/shrimp). Growth and survival rate were not significantly affected by the crude extracts (p > 0.05). Shrimp fed Ex‐Pro crude extracts showed higher succinate dehydrogenase (SDH) activity in the hepatopancreas and muscle but lower lactate dehydrogenase (LDH) activity in the muscle (p < 0.05). The activities of hexokinase (HK), pyruvate kinase (PK), LDH and SDH in the hepatopancreas and the activity of SDH in the muscle were significantly increased by feeding In‐Poly crude extracts (p < 0.05). In contrast, the content of fatty acid synthase (FAS) in the muscle was significantly reduced by the crude extracts (p < 0.05). The contents of glucose and triglyceride and the activity of the electron transport system in the hepatopancreas were significantly increased by the crude extracts (p < 0.05), and the WSSV resistance of the shrimp was increased. These results indicated that the Ex‐Pro and In‐Poly crude extracts of V. alginolyticus could affect energy metabolism, and there was a correlation between WSSV resistance and energy metabolism in L. vannamei.     

Development of a method to assess binding of astaxanthin to Atlantic salmon Salmo salar L. muscle proteins

Several methods were examined to characterize the binding between astaxanthin and salmon muscle protein(s) in order to provide tools for evaluation of the role of muscle proteins on astaxanthin retention in Atlantic salmon Salmo salar L. flesh. The methods included gel filtration chromatography, displacement of a hydrophobic probe and ultrafiltration. With gel filtration chromatography, aggregation of astaxanthin under the experimental conditions was a major problem for the separation of bound astaxanthin from free astaxanthin because the apparent molecular weight of aggregated astaxanthin or astaxanthin micelles was in the range of protein–astaxanthin complexes. Displacement of the fluorescent probe 8‐anilino‐1‐naphthalenesulphonate (ANS) was not effective as astaxanthin quenched the fluorophore so that displacement could not be observed. An ultrafiltration method was developed using 200‐mM sodium cholate for dispersion of astaxanthin aggregates. This allowed unbound astaxanthin to be separated from bound astaxanthin using a 30‐kDa filter. After salmon muscle proteins were solubilized in different fractions by sequential extraction using low ionic strength solutions, the astaxanthin binding of different fractions was assessed using the ultrafiltration method. The significant difference (P<0.05) observed in the astaxanthin binding of the various fractions suggests an application of this assay to detect differences in affinity of proteins for astaxanthin. The results also suggest that proteins other than actomyosin or actin can bind astaxanthin in Atlantic salmon flesh. This method can be used for the identification of astaxanthin‐binding proteins in salmon flesh and other tissues.