Characterization of the properties of human- and dairy-derived probiotics for prevention of infectious diseases in fish

The present study aimed to investigate the potential probiotic properties of six lactic acid bacteria (LAB) intended for human use, Lactobacillus rhamnosus ATCC 53103, Lactobacillus casei Shirota, Lactobacillus bulgaricus, L. rhamnosus LC 705, Bifidobacterium lactis Bb12, and Lactobacillus johnsonii La1, and one for animal use, Enterococcus faecium Tehobak, for use as a fish probiotic. The strains for human use were specifically chosen since they are known to be safe for human use, which is of major importance because the fish are meant for human consumption. The selection was carried out by five different methods: mucosal adhesion, mucosal penetration, inhibition of pathogen growth and adhesion, and resistance to fish bile. The adhesion abilities of the seven LAB and three fish pathogens, Vibrio anguillarum, Aeromonas salmonicida, and Flavobacterium psychrophilum, were determined to mucus from five different sites on the surface or in the gut of rainbow trout. Five of the tested LAB strains showed considerable adhesion to different fish mucus types (14 to 26% of the added bacteria). Despite their adhesive character, the LAB strains were not able to inhibit the mucus binding of A. salmonicida. Coculture experiments showed significant inhibition of growth of A. salmonicida, which was mediated by competition for nutrients rather than secretion of inhibitory substances by the probiotic bacteria as measured in spent culture liquid. All LAB except L. casei Shirota showed tolerance against fish bile. L. rhamnosus ATCC 53103 and L. bulgaricus were found to penetrate fish mucus better than other probiotic bacteria. Based on bile resistance, mucus adhesion, mucus penetration, and suppression of fish pathogen growth, L. rhamnosus ATCC 53103 and L. bulgaricus can be considered for future in vivo challenge studies in fish as a novel and safe treatment in aquaculture.     

Expression of immune-related genes in rainbow trout (Oncorhynchus mykiss) induced by probiotic bacteria during Lactococcus garvieae infection

The aim of the present study was to investigate the effect of lactic acid bacteria (LAB) on the control of lactococcosis as well as to assess the impact of probiotics on the expression of immune-related genes in the head kidney and intestine of rainbow trout (Oncorhynchus mykiss). Lactobacillus plantarum, Lactococcus lactis and Leuconostoc mesenteroides, were administered orally at 10? CFU g?1 feed to fish for 36 days. Twenty-one days after the start of the feeding period, fish were challenged with Lactococcus garvieae. Only the fish fed the diet containing Lb. plantarum showed significantly (P < 0.05) improved protection against L. garvieae compared to the control. Subsequently, real-time PCR was employed to determine the mRNA levels of IL-1β, IL-8, IL-10 and TNF-α in the head kidney, and IL-8, Tlr5 and IgT in the intestine of the control and Lb. plantarum groups. IL-1β, IL-10 and TNF-α gene expression were significantly up-regulated by Lb. plantarum. Moreover, the mRNA levels of IL-10, IL-8 and IgT were significantly higher in the Lb. plantarum group after L. garvieae infection, suggesting that Lb. plantarum can stimulate the immune response of rainbow trout. PCR-DGGE revealed no detectable levels of the probiotics or the pathogen present on the distal intestinal mucosa. These findings demonstrate that direct probiotic-host interactions with the intestine are not always necessary to induce host stimulatory responses which ultimately enhance disease resistance. Furthermore, as L. garvieae did not colonise the intestinal tract, and therefore likely did not infect via this route, the antagonistic properties of the probiotic candidate towards L. garvieae were likely of little influence in mediating the improved disease resistance which could be attributed to the elevated immunological response.     

Innate immune responses in rainbow trout (Oncorhynchus mykiss, Walbaum) induced by probiotics

Carnobacterium maltaromaticum B26 and Carnobacterium divergens B33, which were isolated from the intestine of healthy rainbow trout (Oncorhynchus mykiss, Walbaum), were selected as being potentially useful as probiotics with effectiveness against Aeromonas salmonicida and Yersinia ruckeri. Thus, rainbow trout administered with feed supplemented with B26 or B33 dosed at >10(7) cells g(-1) feed conferred protection against challenge with virulent cultures of the pathogens. Moreover, both cultures persisted in the gut for up to 3 weeks after administration. The cultures enhanced the cellular and humoral immune responses. Specifically, fish fed with B26 demonstrated significantly increased phagocytic activity of the head kidney macrophages, whereas the use of B33 led to significant increases in respiratory burst and serum lysozyme activity. Also, the gut mucosal lysozyme activity for fish fed with both cultures was statistically higher than the controls.     

Characterization of probiotic properties of lactic acid bacteria isolated from an estuarine environment for application in rainbow trout (Oncorhynchus mykiss, Walbaum) farming

Bacterial attachment to fish surfaces and the capacity to compete with pathogens for adhesion sites are essential characteristics in order to select a candidate probiotic for aquaculture. Twelve lactic acid bacteria (LAB) isolated from fish and sediments from Bahía Blanca Estuary, Argentina, were examined for in vitro adhesion to rainbow trout mucus, cell surface properties and competitive exclusion against two salmonid pathogens, Yersinia ruckeri and Aeromonas salmonicida. In order to assess their survival through the digestive tract, pH and rainbow trout bile tolerance were evaluated. All LAB strains survived for 1.5 h incubation in 10% rainbow trout bile. Most of the strains survived 1.5 h at pH 3.0 and three of them showed a reduction of viable counts lower than 2 logarithms, with respect to control (pH 6.5). Only a few strains showed tolerate pH 2.0. All the strains were able to attach to rainbow trout skin mucus (10⁴–10⁶ cells/cm²), to glass (10⁴–10⁵ cells/cm²) and to stainless steel (10³–10⁴ cells/cm²). Sixty percent of LAB strains were capable of competing with and successfully excluding Y. ruckeri and all strains were able to displace it. Against A. salmonicida, 75% of LAB strains competed successfully, 50% were capable of displacing and 60% excluded this pathogen. Our data suggest the potential of these strains as anti-infective agents for use in rainbow trout culture. This study is the first report on the probiotic potential of LAB strains isolated from an estuarine environment from Argentina.     

Yeast colonizing the intestine of rainbow trout (Salmo gairdneri) and turbot (Scophtalmus maximus)

Yeast were isolated from the intestine of farmed rainbow trout (Salmo gairdneri), turbot (Scophtalmus maximus), and free-living flat-fish (Pleuronectes platessa and P. flesus). The average number of viable yeasts recovered from farmed rainbow trout was 3.0 × 10³ and 0.5 × 10² cells per gram homogenized intestine for white and red-pigmented yeasts, respectively. The dominant species were Debaryomyces hansenii, Saccharomyces cerevisiae, Rhodotorula rubra, and R. glutinis. In 5 of 10 free-lving marine fish, > 100 viable yeast cells per gram intestinal mucus were recovered. Red-pigmented yeasts dominated and composed >90% of the isolates. Colonization experiments were performed by inoculating rainbow trout and turbot with fish-specific, isolated yeast strains and by examining the microbial intestinal colonization at intervals. Inoculation of experimental fish with pure cultures of R. glutinis and D. hansenii HF1 yielded colonization at a level several orders of magnitude higher than before the inoculation. Up to 3.8 × 10⁴, 3.1 × 10⁶, and 2.3 × 10⁹ viable yeast cells per gram intestine or feces were recovered in three separate colonization experiments. The high level of colonizing yeasts persisted for several weeks. The concentrations of yeasts in the tank water never exceeded 10³ viable cells per milliliter. No traces of fish sickness as a result of high yeast colonization were recorded during any of the colonization experiments. For periods of the experiments, the concentration of aerobic bacteria in the fish intestine was lower than the intestinal yeast concentration. Scanning electron microscopy studies demonstrated a close association of the yeasts with the intestinal mucosa. The mucosal colonization was further demonstrated by separating intestinal content, mucus, and tissue. All compartments were colonized by >10³ viable yeast cells per gram. No bacteria were detected on the micrographs, indicating that their affinity for the intestinal mucosa was less than that of the yeasts. Correspondence to: Thomas Andtid     

Direct Transmission of a Hemoflagellate,Cryptobia salmositica (Kinetoplastida: Bodonina) Between Rainbow Trout Under Laboratory Conditions1

Transmission of Cryptobia salmositica occurred when infected and uninfected rainbow trout were held in the same tank. In tanks where infected and uninfected fish were allowed to mix freely, 67–80% of the uninfected fish acquired detectable infections by the 27th week. None of the control fish in another tank was infected. In another tank where the infected and uninfected fish were separated by a wire screen, 9 of 20 uninfected fish acquired detectable infections by the 22nd week. This is the first demonstration of direct transmission of a hemoflagellate via the water medium in aquatic vertebrates. Cryptobia salmositica was found in the mucus on the body surface of 9 of 10 fish examined 6 weeks after infection. These parasites were infective and some of them were morphologically similar to those in the blood or peritoneal fluid. It is suggested that the vascular species of Cryptobia were originally ectoparasitic on fishes and that these ectoparasitic species were descended from the free-living Procryptobia.     

Comparative susceptibility of rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta to Myxobolus cerebralis, the cause of salmonid whirling disease.

The susceptibility of rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta to Myxobolus cerebralis, the cause of salmonid whirling disease, was assessed following dosed exposures to the infectious stages (triactinomyxons). Parallel groups of age-matched brown trout and rainbow trout were exposed to 10, 100, 1000 or 10,000 triactinomyxons per fish for 2 h and then placed in aquaria receiving single pass 15 degrees C well water. Severity of infection was evaluated by presence of clinical signs (whirling and/or black tail), prevalence of infection, severity of microscopic lesions, and spore counts 5 mo after exposure. Clinical signs of whirling disease, including a darkened caudal region (black tail) and radical tail chasing swimming (whirling), occurred first among rainbow trout at the highest dose at 6 to 7 wk post exposure. Black tail and whirling occurred among rainbow trout receiving 1000 and 100 triactinomyxons per fish at 8 to 9 wk post exposure. Only 1 of 20 fish had a black tail among rainbow trout receiving 10 triactinomyxons per fish, although 30% of the fish were infected at 5 mo post exposure. Black tails were observed in brown trout at 1000 and 10,000 triactinomyxons per fish beginning at 11 and 7 wk post exposure, respectively. There was no evidence of the tail chasing swimming (whirling) in any group of brown trout. The prevalence of infection, spore numbers, and severity of microscopic lesions due to M. cerebralis among brown trout were less at each exposure dose when compared to rainbow trout. Infections were found among rainbow trout at all doses of exposure but only among brown trout exposed to doses of 100 triactinomyxons per fish or greater. Risk of infection analyses showed that rainbow trout were more apt to be infected at each exposure dose than brown trout. Spore counts reached 1.7 x 10(6) per head among rainbow trout at the highest dose of exposure compared to 1.7 x 10(4) at the same exposure dose among brown trout. Spore numbers increased with dose of exposure in rainbow trout but not in brown trout. As microscopic lesion scores increased from mild to moderate, spore numbers increased in rainbow trout but not brown trout. The mechanisms by which brown trout resist infections with M. cerebralis were not determined. Cellular immune functions, including those of eosinophilic granular leukocytes that were more prominent in brown trout than rainbow trout, may be involved.     

Phylogenetic analysis and in situ identification of the intestinal microbial community of rainbow trout (Oncorhynchus mykiss, Walbaum)

AIMS: To identify the dominant culturable and nonculturable microbiota of rainbow trout intestine. METHODS AND RESULTS: Microbial density of rainbow trout intestine was estimated by direct microscopic counts (4',6-diamidino-2-phenylindole, DAPI) and by culturing on tryptone soya agar (TSA). Differential gradient gel electrophoresis analysis of bacterial DNA from intestinal samples, re-amplification of bands and sequence analysis was used to identify the bacteria that dominated samples where aerobic counts were < or =2% of the DAPI counts. 16S rDNA gene sequences of 146 bacterial isolates and three sequences of uncultured bacteria were identified. A set of oligonucleotide probes was constructed and used to detect and enumerate the bacterial community structure of the gastrointestinal tract of rainbow trout by fluorescence in situ hybridization (FISH). Members of the gamma subclass of Proteobacteria (mainly Aeromonas and Enterobacteriaceae) dominated the bacterial population structure. Acinetobacter, Pseudomonas, Shewanella, Plesiomonas and Proteus were also identified together with isolates belonging to the beta subclass of Proteobacteria and Gram-positive bacteria with high and low DNA G + C content. In most samples, the aerobic count (on TSA) was 50-90% of the direct (DAPI) count. A bacterium representing a previously unknown phylogenetic lineage with only 89% 16S rRNA gene sequence similarity to Anaerofilum pentosovorans was detected in intestinal samples where aerobic counts were < or =2% of direct (DAPI) counts. Ten to 75% of the microbial population in samples with low aerobic counts hybridized (FISH) with a probe constructed against this not-yet cultured bacterium. CONCLUSIONS: Proteobacteria belonging to the gamma subclass dominated the intestinal microbiota of rainbow trout. However, in some samples the microflora was dominated by uncultivated, presumed anaerobic, micro-organisms. The bacterial population structure of rainbow trout intestine, as well as total bacterial counts, varied from fish to fish. SIGNIFICANCE AND IMPACT OF THE STUDY: Good correlation was seen between cultivation results and in situ analysis, however, a molecular approach was crucial for the identification of organisms uncultivated on TSA.     

Bacillus subtilis AB1 controls Aeromonas infection in rainbow trout (Oncorhynchus mykiss, Walbaum)

AIM: To develop a probiotic with effectiveness against Aeromonas sp., which was pathogenic to rainbow trout. METHODS AND RESULTS: When Bacillus subtilis AB1, which was obtained from fish intestine, was administered for 14 days to rainbow trout in feed at a concentration of 10(7) cells per gram either as viable, formalized or sonicated cells or as cell-free supernatant, the fish survived challenge with the pathogen. AB1 stimulated immune parameters, specifically stimulating respiratory burst, serum and gut lysozyme, peroxidase, phagocytic killing, total and alpha1-antiprotease and lymphocyte populations. CONCLUSIONS: Bacillus subtilis AB1 was effective as a probiotic at controlling infections by a fish-pathogenic Aeromonas sp. in rainbow trout. SIGNIFICANCE AND IMPACT OF THE STUDY: Disease control in fish is possible by means of the oral application of live and inactivated cells and their subcellular components with the mode of action reflecting stimulation of the innate immune response.     

Occurrence of Flavobacterium psychrophilum in fish-farming environments

Occurrence of Flavobacterium psychrophilum in fish farms and fish-farming environments was studied using agar plate cultivation, the immunoflourescence antibody technique (IFAT) and nested PCR. Characteristics of 64 F. psychrophilum isolates from rainbow trout Oncorhynchus mykiss, fish farm rearing water, ovarian fluid and wild fish were serotyped, ribotyped and compared biochemically. Virulence of F. psychrophilum isolates from different sources was compared by injection into rainbow trout. Additionally, the number of F. psychrophilum cells shed by naturally infected rainbow trout was determined. F. psychrophilum was detected and isolated from skin mucus, skin lesions and internal organs of diseased rainbow trout and from fish without clinical disease. The pathogen was also present in wild perch Perca fluviatilis, roach Rutilus rutilus, and ovarian fluids of farmed rainbow trout brood fish. Isolates were biochemically homogenous, excluding the capability to degrade elastin. Five different agglutination patterns with different antisera against F. psychrophilum were found among the isolates studied. Although several different ribopatterns were found (ClaI: 12 ribopatterns and HaeIII: 9 ribopatterns), ribotype A was the most dominant. Farmed rainbow trout brood fish carried a broad-spectrum of serologically and genetically different F. psychrophilum in ovarian fluids. Virulence of the tested isolates in rainbow trout varied and naturally infected rainbow trout shed 10(4) to 10(8) cells fish(-1) h(-1) of F. psychrophilum into the surrounding water.     

Social dominance and interrenal cell activity in rainbow trout,Salmo gairdneri (Pisces,Salmonidae)

Synopsis The relationship between standing in a dominance hierarchy and physiological stress was studied in rainbow trout. Individual fish were assigned relative dominance ranks, based on behavioral observations in a large, simulated stream tank. These ranks were compared to histometric measures of interrenal cell activity. Fish, isolated individually in the stream tank had significantly lower levels of interrenal activity than fish from the crowded holding tank. Groups of fish in the stream tank formed stable, linear dominance hierarchies. Interrenal activity correlated inversely with dominance rank, with the exception that top ranking fish had higher activity than expected. Possible cause and effect relationships are discussed.     

Proteinase inhibitor(s) in seminal plasma of teleost fish

Anti-proteinase activity has been found in seminal plasma of three teleost fish: rainbow trout, whitefish and yellow perch. The activity was effective against trypsin (bovine and cod) and acrosin (boar), but not bovine chymotrypsin. Inhibitor activity against fish trypsin was nine- to ten-fold higher than against bovine trypsin. All anti-proteinase activity remained in the retentate after filtration through molecular filter with 30 kDa cut-off membrane and eluted from a column of Sephacryl S-200 HR at the volume characteristic for molecular weight of approximately 90 kDa (data for rainbow trout). Inhibitor(s) had low thermal stability (50–60% activity remained after 15 min at 60° C). The discovery of proteinase inhibitor(s) in the seminal plasma of teleosts raised the question of the regulatory function of this protein in the systematic group of fishes having anacrosomal spermatozoa.     

Immunomodulatory effects of decaffeinated green tea (Camellia sinensis) on the immune system of rainbow trout (Oncorhynchus mykiss)

In order to study the immunomodulatory effects of decaffeinated green tea extract on rainbow trout, a study with a 30-day feeding trial was conducted. Commercial diets with graded levels of decaffeinated green tea extract, 20 mg (T1), 100 mg (T2), 500 mg (T3) per kg feed were prepared. 120 rainbow trout (35 ± 3 g) were randomly assigned to 4 groups in triplicates and fed one of the 3 experimental diets formulated or control diet. After feeding trial, 12 fish from each group were sampled for analysis of some immunological parameters. Remaining fish were injected with 0.5 ml of chicken red blood cell (C-RBC) suspension (2%) intraperitoneally on days 5 and 15 after feeding trial. Results of the current study showed that the inclusion of 20 mg kg-1 green tea (T1) in fish diet enhanced the serum bactericidal activity against Yersinia ruckeri, while significant elevation of lysozyme activity was shown in T2 group. Anti-trypsin activity due to α1-antiprotease was significantly higher in T1 and T2 groups while peroxidase content showed significant increase in all treatment groups compared to control group. Hemagglutination antibody titer against C-RBC was significantly higher in fish administered with 100 mg kg(-1) green tea (T2). Our findings showed that decaffeinated green tea in lower doses of administration could be optimum to enhance the immunity of rainbow trout.     

Effects of dietary Ergosan on cutaneous mucosal immune response in rainbow trout (Oncorhynchus mykiss)

The effects of dietary Ergosan on the growth performance and mucosal immunity in rainbow trout skin were investigated. 60 rainbow trout (100-110 g) were randomly assigned to 2 groups in triplicates and fed one of the experimental diet formulated with 5 g kg⁻¹ Ergosan or control diet for 50 days. Results showed that on the 45th day of feeding trial, Ergosan supplementation significantly enhanced the growth performance compared to control group. Various enzyme activities, namely lysozyme, protease, alkaline phosphatase and esterase in treatment group were also enhanced on the 45th and 50th day. Skin mucus in Ergosan-fed fish showed the agglutination of erythrocytes while in control group, no visible agglutination was shown. In addition, skin mucus in treatment group showed strong antibacterial activity against Yersinia ruckeri. In conclusion, the major immune components of rainbow trout mucus that are involved in the non-specific immunity were enhanced by administration of Ergosan in 5 g kg⁻¹.     

Quantitative measurement of endotoxin in rainbow trout (Salmo gairdneri) serum by the chromogenic substrate method

The amount of endotoxin in serum collected from normal rainbow trout (Salmo gairdneri) and trout inoculated with viable Vibrio anguillarum or lipopolysaccharide (LPS) extracted from bacteria was determined by the chromogenic substrate method. The mean values of endotoxin in four different groups of normal rainbow trout sera ranged from 31.9 to 65.3 pg/ml. When fish were inoculated with viable bacteria (1 x 10(8], they became septicaemic and a large amount of endotoxin ng/ml) was detected in the sera. In fish inoculated with a smaller number of bacteria the amount of endotoxin was several times higher than that of normal fish in spite of failure of bacterial isolation. Although the endotoxin level in serum increased rapidly (greater than 100 ng/ml) after intraperitoneal inoculation with purified V. anguillarum LPS (540 micrograms), no fish died during the experiment. The high level of endotoxin in normal rainbow trout and the resistance of trout to endotoxin are in striking contrast to those of mammalian and avian species.     

Observational and experimental studies on the acquisition of Anisakis sp. larvae (nematoda: Ascaridida) by trout in fresh water

In the enclosed fresh-water environmsnt of Hanningfield Reservoir, Essex, England, Anisakis sp. larvae (parasites of marine fish) were found in 55 per cent of 40 brown trout and in 26·2 per cent of 61 rainbow trout. Experimental infection by intubating larvae into the stomach was more successful in brown trout (50·6 per cent recovery rate) than in rainbow trout (27 per cent recovery rate). Some larvae reached the body-cavity as early as 2 h after infection. They penetrated the region between the oesophagus and intestine immediately posterior to the caecal openings. Fewer larvae successfully penetrated the gut wall of brown trout within 24 h at 8°C than at 15 ± 1°C. It appears that the reservoir trout acquired Anisakis by being fed as juveniles on untreated marine fish offal containing live larvae.     

Degranulation of eosinophilic granule cells induced by capsaicin and substance P in the intestine of the rainbow trout (Oncorhynchus mykiss Walbaum)

Summary Adult rainbow trout (Oncorhynchus mykiss) were injected intraperitoneally with capsaicin, substance P, serotonin, or a control of saline vehicle or bovine serum albumin (0.5 g/g body weight). Fish were sacrificed 30 min and 1,2 and 4 h post-injection, the gut was dissected out, and a small section of the upper intestine was processed for electron microscopy. A significant proportion of eosinophilic granule cells (EGCs) of the intestine were in close association with non-myelinated neuronal bundles in all fish (4 fish per treatment and time period), but there was no significant difference between treatment or time, suggesting that the association was unaffected by these factors. Close examination of EGC ultrastructure showed that fish treated with capsaicin and substance P exhibited limited degranulation of the EGCs in the stratum compactum and extensive crinophagic-like degranulation in the lamina propria. Cells of the lamina propria contained characteristic multivesicular-like bodies. The degranulation was reminiscent of both mast cell degranulation and endocrine cell crinophagy. EGCs of fish treated with serotonin or a control were unaffected, suggesting that the serotoninergic neurons, believed to be involved in gut motility, were not responsible for degranulation. It is apparent that EGCs of the trout intestine may be under nervous control, as has been demonstrated previously for mammalian mast cells.     

Cytokine expression in the intestine of rainbow trout (Oncorhynchus mykiss) during infection with Aeromonas salmonicida

Gene expression of a number of cytokines in the intestine of rainbow trout (Oncorhynchus mykiss) was investigated after challenge with a pathogenic strain of Aeromonas salmonicida. Fish were exposed to A. salmonicida by immersion in a bacterial suspension (bath challenge) and tissue samples of the distal and proximal intestine were collected at days 0, 2, 4, 6 and 8 post-exposure. Head kidney tissue was also collected to assess the effect in a systemic immune tissue. A classic profile of pro-inflammatory cytokine upregulation was observed in the proximal intestine of fish infected by bath challenge, as determined by semi-quantitative RT-PCR. Expression of IL-1beta, IL-8, TNF-alpha and IFN-gamma was increased in the proximal intestine. TGF-beta was significantly decreased in the distal intestine. In the head kidney, infection with A. salmonicida by bath challenge caused decreased expression levels of IL-1beta, IL-8, TNF-alpha and TGF-beta. The results are discussed in the context of potential immune mechanisms in the gut to prevent infection.     

饲料中添加枯草芽孢杆菌对青鱼生长、消化酶活性和鱼体组成的影响

用不加枯草芽孢杆菌的对照饲料和分别添加2×10⁶、2×10⁷、2×10⁸和5×10⁸ cfu/g枯草芽孢杆菌的4种实验饲料, 分别喂养初始均重为(4.16±0.03) g的5组三重复的青鱼鱼种8周。饲养试验在15只容积为80 L循环式水簇箱内进行, 水温控制为(25±0.5) ℃。结果表明: 饲料中添加枯草芽孢杆菌的实验组青鱼鱼体终重和鱼体增重比不添加的饲料组显著提高, 而饲料系数则显著降低(P<0.05), 不同枯草芽孢杆菌添加量对青鱼鱼体增重和饲料系数均无显著影响(P>0.05)。饲料中添加枯草芽孢杆菌对青鱼的成活率无显著影响(P>0.05)。青鱼摄食添加枯草芽孢杆菌的实验饲料后其肠蛋白酶活性比不添加组显著升高(P<0.05), 并随着枯草芽孢杆菌添加量的增加而显著上升(P<0.05), 但添加量到2×10⁷ cfu/g后继续增加则不再显著变化(P>0.05)。青鱼摄食添加枯草芽孢杆菌的实验饲料后, 其肠淀粉酶活性均比不添加组显著升高(P<0.05), 且随枯草芽孢杆菌添加量增大呈不断升高的趋势。青鱼摄食添加2×10⁷、2×10⁸和5×10⁸ cfu/g枯草芽孢杆菌的饲料后, 其肝胰脏蛋白酶活性显著高于摄食不添加或添加2×10⁶ cfu/g枯草芽孢杆菌的实验组(P<0.05)。青鱼摄食添加枯草芽孢杆菌的实验饲料后, 其肝胰脏淀粉酶、肝胰脏脂肪酶和肠脂肪酶的活性均与不添加的实验组无显著差异(P>0.05)。青鱼鱼种摄食添加枯草芽孢杆菌的饲料与摄食不添加枯草芽孢杆菌的饲料相比, 其全鱼营养组成均无显著差异(P>0.05)。因此, 在实验条件下, 饲料中添加2×10⁶—5×10⁸ cfu/g的枯草芽孢杆菌能促进青鱼鱼种的生长和降低饲料系数, 而对鱼体组成没有影响。     

Effects of stress on growth, cortisol and glucose levels in non-domesticated Eurasian perch (Perca fluviatilis) and domesticated rainbow trout (Oncorhynchus mykiss)

Eurasian perch (Perca fluviatilis) is a promising aquaculture candidate, but the growth performance of this non-domesticated species may be negatively affected by its stress responsiveness to intensive culture conditions. To evaluate this potential problem, juvenile Eurasian perch were exposed to a standardized handling stressor twice a week for an 8-week period. A similar study was conducted on domesticated rainbow trout (Oncorhynchus mykiss) for comparison of intra- and inter-specific differences. The stressed fish of both species showed lower body growth than the non-stressed control fish, however, the final mean body mass was 35.4% lower in the stressed Eurasian perch than in the non-stressed controls, compared to 22.8% difference between the two groups in rainbow trout. The stress responsiveness was examined by comparing the post-stress cortisol and glucose levels in repeatedly stressed fish and fish exposed to the stressor only once. The cortisol stress response in both species strongly indicated a habituation to the repeated stressor. Thus, repeatedly stressed Eurasian perch reached maximum cortisol levels of 130 ng/mL after 0.5 h compared to 200 ng/mL in the fish stressed once, while considerably smaller differences in cortisol levels were shown between the repeatedly and single stressed rainbow trout. Rainbow trout also showed lower post-stress glucose levels in the repeatedly stressed fish compared to the single stressed fish. In contrast, the glucose levels in both groups of Eurasian perch increased abruptly after stress treatment and remained elevated at approximately 19 mM for 6 h; levels were three times as high as the peak levels 3 h post-stress in rainbow trout. Together, the habituation of the stress response shown in both species did not eliminate the growth difference found in the repeatedly stressed fish versus the control fish. Further, the lower growth performance of Eurasian perch compared to rainbow trout could partly be due to the increased energy consumption in the more stress responsive Eurasian perch.