Chinese Journal of Oceanology and Limnology   2016, Vol. 34 Issue(1): 13-18     PDF       
http://dx.doi.org/10.1007/s00343-015-4333-1
Institute of Oceanology, Chinese Academy of Sciences
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Article Information

Najmeh SHEIKHZADEH, Fatemeh CHEHRARA, Marzieh HEIDARIEH, Katayoon NOFOUZI, Behzad BARADARAN
Eff ects of irradiated Ergosan on the growth performance and mucus biological components of rainbow trout Oncorhynchus mykiss
Chinese Journal of Oceanology and Limnology, 2016, 34(1): 13-18
http://dx.doi.org/10.1007/s00343-015-4333-1

Article History

Received Nov. 24, 2014
accepted in principle Feb. 26, 2015;
accepted for publication Apr. 1, 2015
Eff ects of irradiated Ergosan on the growth performance and mucus biological components of rainbow trout Oncorhynchus mykiss
Najmeh SHEIKHZADEH1 , Fatemeh CHEHRARA1,2, Marzieh HEIDARIEH2 , Katayoon NOFOUZI3, Behzad BARADARAN4       
1 Department of Food Hygiene and Aquatic Animals, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran;
2 Nuclear Science and Technology Research Institute, Karaj, Iran;
3 Departments of Pathobiology, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran;
4 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
ABSTRACT:Eff ects of irradiated and non-irradiated Ergosan extract (alginic acid) on rainbow trout growth performance and skin mucosal immunity were compared. Ergosan was irradiated at 30 kGy in a cobalt-60 irradiator. A total of 252 fi sh (128.03±9.4 g) were randomly divided into four equal groups, given the basal diet either unsupplemented with Ergosan (control group) or supplemented with crude Ergosan (5 g/kg), ethanol-extracted Ergosan (0.33 g/kg) or irradiated Ergosan (0.33 g/kg) according to this protocol: basal diet for 15 days, treatment diet for 15 days, basal diet for 10 days and treatment diet for 15 days. Highest growth performance was observed in fi sh fed irradiated Ergosan ( P < 0.05). Dietary administration of diff erent Ergosan types did not cause any changes in mucus protein level, but improved alkaline phosphatase level and hemagglutination titer compared with the control (basal diet without Ergosan) on day 55 of feeding trial ( P < 0.05). Furthermore, the highest value of lysozyme activity was observed in gamma-irradiated Ergosan on day 55. In conclusion, gamma-irradiated Ergosan at 0.33 g/kg was found to improve growth performance and mucus biological components signifi cantly in comparison with the control group (basal diet without Ergosan).
Keywordsgamma-irradiation     Ergosan     growth performance     mucus     immune system     rainbow trout    
1 INTRODUCTION

Fish mucus contains many active components such as lysozymes, protease, immunoglobulins, complement proteins, C-reactive protein, lectins, proteolytic enzymes and various other antibacterial proteins and peptides(Subramanian et al., 2007). Since fish are in intimate contact with a wide range of pathogenic and non-pathogenic microorganisms, enhancement of biological systems in fish epidermal mucus can result in better resistance(Sheikhzadeh et al., 2012).

Gamma-ray irradiation of natural polysaccharides, such as chitosan, carrageenan and sodium alginate, off ers as a clean method for the formation of low molecular weight oligomers. These irradiated natural polysaccharides can function as antibiotic, antioxidant, and plant-growth promoting substances(Duy et al., 2010). Many studies have been made on the application of these degraded polysaccharides in fields such as agriculture(Haji-Saeid et al., 2010; Naeem et al., 2012). For example, sodium alginate depolymerised by gamma irradiation showed various biological eff ects on plants, including enhanced seed germination, shoot elongation, and root growth(Sarfaraz et al., 2011; Idrees et al., 2012). However, there is no information on the impact of these gammairradiated polysaccharides on aquatic animal health.

Ergosan contains 1% alginic acid extracted from two brown seaweeds, Laminaria digitata and Ascophyllum nodosum. In in vitro model, the remaining powder after solubilization of Ergosan was irradiated with diff erent doses of gamma rays(10, 20, 30, 40, and 50 kilogray(kGy)). Structural changes produced by UV absorption markedly increased UV absorbance at 300 nm when Ergosan was irradiated at 30 kGy, perhaps due to the formation of carboxyl groups at this dose. Meanwhile, DPPH(2, 2-diphenyl- 1-picrylhydrazyl)assay showed a higher antioxidant activity in comparison with crude Ergosan and other doses of gamma-irradiation(Heidarieh et al., 2012a). Since the biological eff ects of irradiated polysaccharides on aquatic animals could also be important, the growth performance and immunomodulatory role of Ergosan before and after the gamma irradiation were studied in rainbow trout. Therefore, the aim of this work has been to investigate the eff ects of Ergosan before and after gammairradiation at 30 kGy on rainbow trout growth and biological components.

2 MATERIAL AND METHOD 2.1 Preparation of gamma-irradiated Ergosan

Five grams of commercial Ergosan(Schering- Plough Aquaculture, UK)was suspended in sterile 0.15 mol/L phosphate buff ered saline(pH 7.2). Samples were sonicated for 30 min in a water bath sonicator(Jencons, Engl and ) and centrifuged at 5 000 r/min for 15 min(Peddie et al., 2002). After precipitation in 2.5 volumes of 96% ethanol, Ergosan samples were dried at 40°C and then milled. Remaining powder(0.33 g)was irradiated at 30 kGy from a Cobalt-60 gamma irradiator(PX-30 IssIedovapel, Russia)at a dose rate of 0.22 Gy/sec(Heidarieh et al., 2012a, 2014a, b). Dosimetry was performed with a Fricke reference st and ard dosimetry system and after irradiation; the irradiated-Ergosan was stored at 4°C for further experiments.

2.2 Animals and treatments

Rainbow trout(mean body weight 128.03±9.4 g, mean±st and ard error)were obtained from a commercial fish farm in Karaj, Iran. Fish were subjected to a 2-week conditioning period at the Agricultural Research School, Karaj prior to the feeding trial. Fish were r and omly distributed into 12 fiberglass tanks at 21 fish per tank and maintained in continuously aerated free-flowing dechlorinated freshwater with a flow rate of 1.5 L/s and water temperature 15±1°C. Three experimental diets including 5 g/kg crude Ergosan, 0.33 g/kg ethanolextracted Ergosan(equivalent to 5 g/kg crude Ergosan) and 0.33 g/kg Ergosan-irradiated particles(equivalent to 5 g/kg crude Ergosan)were used. The basal diets were supplemented via Ergosan of these diff erent types, they were added to palletized feed and then the batches of feed were separately dried in an oven at 30°C. Palletized feed batches were sent to an oiling process in order to be covered with fish oil. Pellets were packed and stored in tightly sealed plastic bags at 8-10°C until they were used in the feeding experiments. The proximate composition of the basal diet(Behparvar, Iran)was 38% crude protein, 18% crude fat, 10% crude moisture, and 3% crude fiber. Each experimental diet was fed to three replicate tanks of fish according to the manufacturer’s recommendations for using Ergosan: 15 days of basal diet; 15 days of treatment diet; 10 days of basal diet; 15 days of treatment diet. The controls were fed on a basal diet solely. All groups were fed three times per day at a rate of 2.5% body weight. At the end of feeding trial, the final weight and the feed conversion ratio(FCR)were determined by selecting five fish from each tank.

2.3 Skin mucus preparation

On days 40 and 55 of feeding trial, eight representative fish from each tank were obtained r and omly and anaesthetized(clove oil 50 μL/L). Mucus was sampled by gently scraping the surface of each fish with the blunt side of a sterile scalpel blade and homogenizing with 4 volumes of Tris-buff ered saline(TBS, 50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl)on ice(Palaksha et al., 2008). Mucus samples from four fish of the same tank were pooled to reach the requested volume for the subsequent assays. After sample collection, mucus was centrifuged at 4 000 r/min for 30 min at 4°C and the supernatant was lyophilized(Alpha-1-2LD, Germany)following freezing at -20°C and -80°C respectively. Three hundred(300)milligrams of the lyophilized skin mucus was added to 1 mL TBS buff er, and centrifuged at 4 000 r/min for 30 min at 4°C to obtain the supernatant. Aliquots of the mucus extract was introduced into 2 mL tubes and stored at -20°C for subsequent analysis. Prior to the assays, protein content in mucus samples was analyzed using the Bradford assay(Bradford, 1976)using bovine gamma globulin as the st and ard to calculate the specific enzyme activities.

2.4 Lysozyme activity of mucus extract

The lysozyme activity was determined using a turbidometric assay(Sheikhzadeh et al., 2012). Twenty-five μL mucus extract was mixed with 175 μL of 0.75 mg/mL suspension of Micrococcus lysodeikticus(Sigma)in 0.1 mol/L phosphate citrate buff er, pH 5.8 in 96-well microtiter plates. The O.D. was measured at 450 nm continuously for 30 min. A unit of lysozyme activity was defined as the amount of mucus that caused a decrease in the absorbance of 0.001/min and expressed as U/mg of mucus sample.

2.5 Alkaline phosphatase activity of skin mucus

The alkaline phosphatase activity assay was followed by the method described in Palaksha et al.(2008). Equal volumes of mucus extracts were incubated with 4 mmol/L p-nitrophenol phosphate in 100 mmol/L NH4HCO3, pH 7.8, 1 mmol/L MgCl2. The change in optical density at 405 nm was measured over a 2 h period at 30°C. One unit(U)of activity was defined as the amount of enzyme required to release 1 mmol of p-nitrophenol product in 1 min. For p- nitrophenol, extinction coefficient=1.78×104 /(mol/L)/ CM.

2.6 Hemagglutination assay

A 2.5% chicken red blood cell(C-RBC)suspension in TBS was prepared following Sheikhzadeh et al.(2012). In round-bottomed micro plates, 45 μL of mucus extract(equivalent to 31.5 μg protein)was added to first well follow by a dilution series in TBS buff er from the second well to the eighth. An equal volume of 2.5% C-RBCs was added to all the wells. After incubation at room temperature for 1 h, the reciprocal of the highest dilution with visible agglutination was considered as the hemagglutinating titer.

2.7 Antibacterial activity of skin mucus

The minimum inhibitory concentration(MIC)of the mucus samples was estimated according to the method of Sheikhzadeh et al.(2012). Fish bacterial pathogen, Yersinia ruckeri(BCCM5/LMG3279), was inoculated into tryptic soy broth(Merck, Germany) and incubated at 25°C for 16 h. Then, bacterial culture was adjusted to 1 McFarl and turbidity st and ard. Forty-five μL mucus extract(equivalent to 31.5 μg protein)was added to the first well followed by a dilution series with sterile tryptic soy broth from the second well to the eighth. Finally, 45 μL of bacterial suspension was added to all wells and incubated at room temperature(25°C)for 24 h. Minimal inhibitory concentration in each sample was determined by considering the last well that gave bacterial inhibition.

2.8 Statistical analysis

One-way analysis of variance followed by LSD multiple comparison test was used to determine diff erences among groups. The level for accepted statistical significance was P <0.05.

3 RESULT

During this study, no mortality was observed in the diff erent groups. Significant increase in weight gain after 55 days was observed in fish fed with irradiated Ergosan compared to other groups. The lowest significant FCR was also observed in fish fed the diet supplemented with irradiated Ergosan, compared with the control group(basal diet without Ergosan), but no significant diff erences were observed among the other treatment groups(Table 1).

Table 1 Growth performance of diff erent experimental groups of rainbow trout at the end of 55 dayfeeding trial

Tables 2 and 3 show the mucus components of rainbow trout fed with experimental diets containing diff erent Ergosan sources after 40 and 55 days. In this study, there were no significant changes in mucus total protein level among the diff erent groups on days 40 or 55 of feeding trial. On day 40, no significant diff erences in lysozyme activity was observed among the various groups but on day 55, the highest lysozyme activity was observed in fish fed irradiated Ergosan, compared with the other groups. On day 40, alkaline phosphatase in fish mucus was aff ected by the irradiated dietary Ergosan, but it was not significant in comparison with ethanol-extracted Ergosan. On day 55, alkaline phosphatase in fish fed treatment diets was significantly higher than fish in the control group.

Table 2 Mucus components of diff erent experimental groups administrated with Ergosan, Ergosan-sonicated and irradiated nanoparticles on day 40 of feeding trial

Table 3 Mucus components of diff erent experimental groups administrated with Ergosan, Ergosan-sonicated and irradiated nanoparticles on day 55 of feeding trial

Antibacterial activity did not show significant changes on day 40, but on day 55, visible bacterial inhibition was shown in the first wells in all of treatment groups, compared with the control group(basal diet without Ergosan). No significant diff erence in hemagglutination titer was observed when fish were sampled on day 40, whereas on day 55, fish fed diff erent Ergosan sources had significantly higher hemagglutination titers compared with control group(basal diet without Ergosan)(Tables 2, 3).

4 DISCUSSION

Gamma-irradiation is a physically eff ective, simple, reproductive, and environmentally friendly technique(Byun et al., 2008; Pasanphan et al., 2010). Gamma irradiation has been extensively used to generate nanoscale metals and nanocomposites at room temperature and normal pressure(Karim et al., 2007). Heidarieh et al.(2014b)also synthesized alginic acid nanoparticles via using irradiation method(at 30 kGy)to detect target organ in rainbow trout. In this study, higher significant growth performance was noted in fish fed irradiated Ergosan and significant changes in FCR were shown, compared with control group(basal diet without Ergosan). On day 40, fish administrated irradiated Ergosan showed significant increase in alkaline phosphatase activity whereas, over all the groups, no significant changes in other mucus parameters were noted. On day 55, all mucus components showed significant increases in treatment groups compared with control group.

For the optimal use of immunostimulant agents, the dose and the duration are always important(Sakai, 1999). It seems that short-duration administration of Ergosan resulted in non-significant diff erences in growth performance compared with control group, but confirms Heidarieh et al.’s(2012b)previous study, which showed that after 45 days’ continuous feeding, Ergosan had positive eff ects on rainbow trout growth parameters. Considering the stronger eff ect of irradiated Ergosan on fish growth parameters in the same time period, it can be concluded that gammairradiation made alginic acid a more potent stimulant of growth performance. In previous studies, polysaccharides such as alginate, carrageenan, and chitosan in their depolymerised form, enhanced some biological functions(Haji-Saeid et al., 2010; Sarafraz et al., 2011; Naeem et al., 2012). Nanoparticles are able to exhibit a high rate of uptake in the gastrointestinal tract even though the extent of absorption depends on the nature of the particles employed, their surface charge, their colloidal stability, the dose given and the species of animal(Florence, 2005). Therefore, it was proposed that improved intestinal mucosal morphology following the administration of nanoparticles may be another reason for the increase in growth performance(Han et al., 2012). This was supported by Heidarieh et al.’s(2014a)study, changing alginic acid to nanoparticles that could result in positive eff ects on intestine villi and pyloric caeca folds in rainbow trout Similarly, fish fed nanoparticles of iron, selenium or chitosan showed faster rate of growth(Wang and Li, 2011; Zhou et al., 2011).

Biologically active compounds in skin mucus of fish play a role in the prevention of colonization by parasites, bacteria and fungi(Hellio et al., 2002). In this study, following the administration of all Ergosan types, stronger antibacterial activity was shown. Administration of these substances in fish diet may activate some antimicrobial compounds, resulting in stronger antibacterial activity. In the current study, activity of some antibacterial agents, namely lysozyme and alkaline phosphatase, were also enhanced. Higher hemagglutination titer was also shown in treatment groups, which indicates that the skin mucus in treatment groups had stronger activity of agglutinin-type substances. In this study, it seems that extraction of Ergosan by ethanol and then irradiation activated the biological components. Even though the exact mechanism of ethanol extraction and gamma-irradiation is not clear, the final Ergosan nanoparticles may be enhancing the biological components in fish mucus. Immunogenic properties of alginates isolated from several species of brown algae, have been shown by Draget and Taylor(2011). The signaling cascade leading to the immunological response and proinflammatory cytokine induction of mannuronate-rich alginates seems to involve pattern recognition receptors(PPR’s), especially toll-like receptors TLR4 or TLR2 together with CD 14(Draget and Taylor, 2011). Changing the crude Ergosan, a source of alginic acid, to nanoparticles can change the character of the substance. In fact, nanoparticles can be readily loaded or coupled with immunomodulatory agents such as cytokines or immune cell-specific lig and s and can be administered through a variety of routes to improve immunity against infectious disease(Look et al., 2010). Nanoparticle interactions with the immune system depend on the physicochemical properties of these substances such as size, surface charge, solubility, and surface functionality, which may influence their biological properties(Dobrovolskaia et al., 2008). For example, due to the special characters of chitosan nanoparticles, more efficient uptake by phagocytotic cells induces stronger systemic and mucosal immune responses than occurs with chitosan(Wen et al., 2011). Even though the direct exposure of bacterial pathogens to nanoparticles might lead to disruption of cell membranes and the leakage of cytoplasm(Qi et al., 2004), the strong antibacterial eff ects in fish mucus that we observed act probably via the activation of immunological components, which occurred after the administration of Ergosan nanoparticles.

In summary, the current study demonstrates that irradiated Ergosan is more eff ective in enhancing fish growth performance. Extraction of Ergosan followed by gamma-irradiation might be used to augment some biological components in rainbow trout mucus.

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