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Discussion

An important point that should be considered in all studies with nanotechnology researchers is determining the character of nano materials. Especially in the commercial ones that the information have been provided by the manufacturers for researchers are often incomplete and may be false.

Therefore, re-evaluation of its properties by researchers is essential. In addition, the storage period and storage conditions of nanomaterials (temperature and light) can be a significant effect on their properties (Kittler et al, 2010; Pinto et al, 2010).

In this study also nanosilver colloid properties have been measured including particle diameter and the visible and ultraviolet absorption spectra. The absorption spectra of silver colloid particles were determined in the range of 410 nm, which was similar to the other studies results (Petit et al, 1993; Kong and Jang, 2006;Shahverdi et al, 2007;

Bhui et al, 2009).

Scanning electron microscopy images confirmed the presence of spherical silver nanoparticles formed on the surface of food. X-ray diffraction pattern of the spectra was shown that there are clear differences between the sample and the food covered with silver nanoparticles, which confirmed crystal structure of silver in the sample the food coated by nanoparticles. On the other hand, sometimes it is possible that the small size of the crystal causes the nanosilver particles

are not detectable by X-ray diffraction spectra (Liu et al, 2001).

However, the ability of antimicrobial nanosilver colloid and its usage on other aquatic devices and aquaculture already have been approved (Prochorov et al, 2002; Fu-ping et al, 2009; Li and Wang , 2011, Alishahi et al,2011; Zhou et al, 2011). This study is the first usage of antimicrobial silver nanoparticles in colloid mode for food antibacterial in aquatic breeding and reproduction system. Also Prochorov(2002) and Fu-ping (2009) reported that food covered by nanoparticles were useful to increase growth, reduce mortality, combating microorganisms and control them in aquatic breeding and reproduction system.

Based on some reports, there are different opinions about negative effects of silver nanoparticles on the environment, some researchers believe that AgNps are eco-friendly because its short half-life (Reynolds et al, 2001; Kim et al, 2005; Gajjar et al, 2009). The results showed that Zebra was very sensitive to the food covered by nano particle for instance LC50-96h was 195.208 mg/kg. Soltani et al in 2009 reported that LC50-96h was 5 mg/l for Rainbow trout. (Soltani et al, 2009).Bar-Ilan in (2009) had similarly results for Zebra embryos.

In the present study, the acute mortality test results were showed that the sensitivity of Zebra fish at high concentrations is much more than the concentration of low. Grosell et al in (2002) article was mention that that smaller fish have higher sensitivity to silver Zystmndan, More because of the greater surface area to body mass, especially the gills which both affect the process of ion exchange, osmoregulation and absorption of chemicals from the environment influence.

Histological studies can be applied a useful tools for assessment the damage because of nano-materials. In this study the greatest effects nanosilver colloid was observed in liver and gill tissue. The high impact of AgNps in gills especially in high concentrations of was shown by hyperplasia and hypertrophy. Nano materials that made gills histology are considered of respiratory toxins (Shaw and Handy, 2011).Copper nanoparticles were caused Epithelial cells proliferation and edema primary and secondary lamalea gills in zebra fish (Griffitt et al, 2007).Also exposure of adult zebra fish with copper nanoparticles at 48­

hour, gill lamalea diameter increasing were observed, while exposure of zebra fish with nanosilver and titanium dioxide those damages were not

observed (Griffitt et al, 2009).

Smith et al in (2007), trout was exposured with a concentration of 0.1 mg/L with single-wall carbon nanotubes, union organizer Perez, the inflation and Shorten the mucous membrane was observed due to the ingestion of nanomaterials. Swallowing water containing nano-particles, not only have direction effects on tissue structure, but also could lead to absorption this material into body and tract through with endocytosis(Handy et al, 2008). In the present study, due to the ingestion of food containing nanosilver, have been affects direct influence on gastrointestinal structure. Liver is the filtration of toxins in body, so obviously are used impression of pollutants and harmful substances (Federici et al,2007). The silver present in the nanoparticles have harmful effects such as hepatic degeneration and vacuolization.

There are not many studies on oral administration of nanoparticles on renal tissue damage in Zebra fish.

In this study, renal tubular degeneration and some necrosis are evidence of destructive effects of silver nanoparticles on kidney.

The results of Silver nanoparticles accumulation in tables and graphs, was shown that the bio accumulation causes silver accumulation in the liver, gills and muscle. The greatest bioaccumulation of silver occurred in the liver, gills and muscle of fish respectively. Web and Wood (1998) reported that after 21 days of exposure of rainbow trout with 14.5 |ig/L silver, the highest concentration of silver in liver, gills, intestine and muscle tissues were seen. Silver accumulation in liver tissue reflects of increased uptake and transport of silver compounds (silver nano particles or ions released) through the intestine or via flow blood to the tissue. According to this, the concentration of silver granulation was result that liver effects would be delayed; due to this fact fishes able to store metals in granular deposits in the liver(Lanno et al, 1987; Shaw and Handy, 2007).

In gill tissue, the part of the silver that was measured in gills due to gills Mucus secretion, while was absorbed into gill tissue. But silver was accumulated on the gill surface (Shaw and Handy, 2011). In study Scown

et al (2010) have been observed increasing concentration of silver in the liver and gills of rainbow trout after exposure to nanosilver. the rate of accumulation was twice in the liver than gills. Also bioaccumulation of silver in gills tissue after were exposured in silver nanoparticles, has been reported in other studies (Griffitt et al, 2009; Farmen et al, 2012).

Conclusion

Silver Nanoparticles in monitoring pollution in aquatic ecosystems, entrance concentration to AgNps, will affect physiological living things. We conclude that the Ag-nps have the potential to cause health and ecotoxicity in a concentration-dependent manner. The Ag-np treated zebra fish exhibited phenotypic defects, altered physiological functions, namely bioaccumulation silver in tissues, axial curvatures and degeneration of body parts. The TEM images showed the presence of nanoparticles in the liver of the zebra fish. The pathological events following long-term deposition of nanoparticles in fish tissues. Furthermore, the exposure of Ag- Nps resulted in accumulation of blood in different parts of the body, thereby causing edema and necrosis. Further studies will be directed towards the genotoxicity and the gene expression profile of Ag-np treated fishes. This study pointed out the adverse effects of Ag-np in aquatic species and all applications involving silver nanoparticles should be given special attention and promoted only after detailed studies. The release of untreated nanoparticle waste to the environment should be restricted for the well being of human and aquatic species.

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Short Papers

Experimental contamination of Artemia Urmiana cyst with Aspergillus niger and the effect of contamination on hatching rate and the study of effect formalin as a

treatment

Mahdluy Afshin1, Ownagh Abdolghaffar2, Agh Naser3, Adibhesami Masood4*

1 Graduated veterinary Medicine, Urmia university, Iran

2 Department of Microbiology, Faculty of Veterinary Medicine, Urmia University, Iran

3 Artemia and Aquatic Animals Research Center, Urmia University, Iran

4 PhD student of microbiology, Faculty of Veterinary Medicine, Urmia University, Iran * Email: masood.adibhesami@gmail.com

Introduction

Urmia Lake is one of the biggest natural Artemia habitats in the world. It is a landlocked thalassohaline, Sodium Chloride Lake located in the northwestern region of Iran. Gunther (1899) reported for the first time the presence of Artemia in Urmia Lake. The species was designated as A. urmiana. Artemia are able to avoid cohabitating with most types of predators, such as fish, by their ability to live in waters of very high salinity up to 250%. Fish farm owners search for a cost-effective, easy to use, and available food that is preferred by the fish. From cysts, brine shrimp nauplii can readily be used to feed fish and crustacean larvae just after one-day incubation. Artemia urmiana is one of the important species of it. Its high nutritional values and various forms with many uses have caused this creature to be considered as the most valuable live feed for the cultured aquatic animals. Hatching of cyst depend too many factors like genetic and environmental for example microbial agents. Bacterial and fungal agents have important role in contamination of Artemia Urmiana cyst and delay hatching rate of cysts. Among of fungal agents, the role of Aspergillus niger in more prominent than others.

Materials and Method

Cysts collected from surface of Urmia Lake with 120 M plankton net.

Then samples process that with diphase flotation method and dehydration. For preparation of seawater with a salinity of 35 ppt, the salinity of the sea water (150-180 ppt), distilled water was added. With using sodium bicarbonate PH of water was maintenance between 8/5 to 9 and regulated temperature (25-280C), light and aeration operation (up 2 mg/Lit).

Saburo dextrose agar was used for growing Apergillus niger. Different dilutions of the spores of the fungus were prepared. Artemia Urmiana cysts were contaminated for a period of 14 days with 102, 104, 106 and 108 spores from Apergillus niger per ml containing a cyst gram. Hatching percentage of contaminated cysts was calculated after 24 and 48 hour incubation under standard condition experiment was performed adding 80 ppm formalin to the hatching cones as antifungal agent. To determine the percentage of hatching cysts, 250 |il of hatched cysts isolated and were poured separate micro plate.

Samples with lugol stabilized, nauplii were counted (n1 - n6) and the average was calculated (N). Then umbrella modes were counted (u1 -u2) and the average was calculated (U). By Formula [ H% = (Nx100) : (N+U+E) ] percent hatching was calculated in each container.

Result

Fungi in contact with cysts hatching percentage were greatly reduced. With the increasing number of fungal spores, percent hatching was reduced at 24 h incubation. Formaldehyde associated with increased concentrations of Fungi, percent hatching was reduced in all groups. With increasing spore concentration with formaldehyde, formalin disinfecting power declined. Formalin associated with increased concentrations of fungi after 48 h incubation, hatching percentage dropped. Between 106 and 108 spore-containing with formalin groups and controls there was a significant difference (p<00005).

Conclusion

Results of this study were to investigate how to increase the concentration of Aspergillus niger spores in contact with the cysts;

percentage hatching Artemia cysts are reduced. Formaldehyde (80ppm) with various concentrations of Aspergillus niger spores can cause disinfection, and percent hatching cysts has increased. The power of formalin disinfection with the increasing number of fungal spores is reduced.

References

Sanaz Khosravi, Naser Agh, Saber Khodabandeh. Comparative study on mycosporine-like amino acids accumulation in the total body of Artemia urmiana and parthenogenetic population of Artemia. Journal of Biotechnology 2010,

(150)128.

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5(4): 395-297.

Alireza ASEM, Nasrullah RASTEGAR-POUYANI1, Naser AGH. Biomedical Study of Artemia urmiana (Anostraca: Artemiidae( Cysts Harvested from Lake Urmia

(West Azerbaijan, Iran). Turk J Zool. 31 (2007) 171-180.

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LXIX). Aquaculture 254 (2006) 442-454.

Effects of Flower horn fish larvae fed with Artemia and artificial food treated with different on growth and

coloration

Mohammad Hassan Elahi, Che Roos Saad, Sharr Azni bin Harmin, Mustafa Kamal Abdul Sattar

One of the most popular species in ornamental fish trade is the Flower horn. The flower horns fish are a hybrid of several kinds of cichlid. They were created for the first time in Malaysia by a cross breeding. The flower horns are fertile but we could not find any wild population in the world (Malaysia cichlosoma Hauluohan 2002).

The feed used usually nauplii of ArtemiaIn another study, Sargeloos et al. (2003) currently there is no artificial feed formulation available which is suitable to completely substitute for Artemia feeding to young fish larvae in commercial hatchery operations.

Carvalho et al (2003) suggested that the artificial diets for fish larvae should have a nitrogen solubility and molecular weight profile similar to that found in live food.

It is very difficult to culture larvae of ornamental fish, and using an appropriate food source to produce the best quality of flower horns fish larvae will lead to the improvement of survival rate, increase growth and quality of color.

To compare the growth performance of flower horn fed with Artemia (nauplii and decapsulate) and artificial diets.

To compare the growth performance of flower horn fed with Artemia (nauplii and decapsulate) and artificial diets.

Materials and Methods

Experiment 1. Four treatment diets was used, namely Diet 1(Pellet) for control, Diet 2 (formulated artificial diet), such as fish and shrimp meal 50% skimmed milk ,soy been, heart cow, wheat powder, vitamin premix 50% Diet 3(nauplii) and Diet 4 (decapsulate of Artemia) Each treatment is replicated three times. The fish larvae fed is divided

randomly in twelve aquarium tanks (30x20x30) stocked with 60 Flower horn larvae, each randomly arranged. Larvae were fed four times daily (8.00, 11.00, 14.00 and 17.00h). Excess and uneaten food was siphoned away and the weight was recorded.

Mean initial weight (mg) and total length (mm) of the larvae were measured every 2 weeks for 12 weeks. At the end of the experiment, the final weight, length and survival of fish were calculated. FCR and SGR were also calculated.

Experiment 2 for color determination a colorimeter Minolta was used and a calibration of CR-300 for chromo meter and measurement of color for fish that used four diets was carried out.

Data were analyzed by one way analysis using ANOVA all data while Turkey's test was used to compare mean differences .The analysis was conducted using the SPSS version 16.

Result

The weight, length and survival between artificial foods, decapsulate and nauplii were significantly different (P<0.05). However, there was not any significant difference between micro pellet and artificial food as well as between decapsulate and nauplii of Artemia.

The value L* corresponds to a dark and bright scale a*for red and green b*for yellow and blue

The colors of fish use with Decapsulate and pellet food were significantly (p<0.05)higher than two other foods

The pellet foods are contained carotinoid pigment may be the best color for fish by using Decapsulate and pellet for food pigmentation. The Nauplii pigment reduced in hatching stage.

Measurement of total Aflatoxin in the diet of farmed rainbow trout with using ELISA

Dostkam Farahmand1, Ownagh Abdolghaffar2, Tokmehchi Amir3, Adibhesami Masood4*, Rahman Bentolhoda4

1 Graduated veterinary Medicine, Urmia university, Iran

2 Department of Microbiology, Faculty of Veterinary Medicine, Urmia University, Iran

3 Artemia and Aquatic Animals Research Center, Urmia University, Iran

4 PhD student of microbiology, Faculty of Veterinary Medicine, Urmia University, Iran * Email: masood.adibhesami@gmail.com

Introduction

Tried for human consumption in that year instead of planting crops they grow and eventually mass produced product to the warehouse for a long time. Storage of seeds, grains, cereals and fruits in addition to the maintenance costs of the food provides biological problems. Molds are parasitic organisms that can grow in the food and produce toxins such as aflatoxins. Aflatoxins are naturally occurring mycotoxins that are produced by many species of Aspergillus, a fungus, the most notable ones being Aspergillus flavus and Aspergillus parasiticus. Continuous feeding of low levels of aflatoxins in food may not cause visible damage physiologic but small amounts can cause low performance and the resulting economic significant losses. When grains and legumes are consumed by animals and humans, fungal toxins absorbed by biological systems, they will have devastating effects on different organs of the body, including the reproductive organs (ovaries and testes) will be created. For years, several studies on the effects of anti-carcinogens and reproductive toxins, fungal done. This study aimed to search for total aflatoxins in the diet of rainbow trout are farmed ELISA.

Material and method

In the ten salmon farm of West Azerbaijan, according to age, rearing of fish food samples of random is done sampling. 100 grams of each food type and quantity of the food samples were taken. A total of 44 samples

were collected in sterile as soon as were shipped Artemia and Aquatic Animals Research Center microbiology laboratory. Solution with 70% methanol in water (v / v) total anatoxins (including B1, B2, G1 and G2) were extracted from samples of food. The 20 grams of food were weighed and ground in a clean container with 100 ml of methanol 70% mixture for 3 minutes at room temperature was shaking. Using a clean pipette supernatant collected using filter paper in a clean container with a score filtered and until the ELISA procedure was stored at refrigerator temperature. To measure the total aflatoxin in food samples using ELISA kits (Agraqunt, Romer Laboratory Co, USA) was used. To obtain results for pouring solutions of multi-channel pipette was used. Turbidity of samples by ELISA Reader for converting data obtained by the absorption of light in terms of ppb concentrations of aflaoxin.

Result

In this study, 42 samples of different types of rainbow trout diets were analyzed. Separating the sample included 9 food samples SFT, 11 food samples FFT, 14 food samples from food BFT, and 8 GFT samples, respectively. Maximum and minimum numbers of meals sampled GFT BFT food meals were sampled. The results of this study show that the highest levels of aflatoxins in food samples with FFT ppb 2/13 from the field is. The average concentration of total aflatoxins in food samples was higher than the rest of BFT.

Conclusion

Mycotoxins cause significant financial losses are in the range of animal feed and on the other hand due to health complications in the form of consumer. In hot and humid countries each year a large percentage of grains and oilseeds are contaminated to various mycotoxins such as aflatoxins. Unfortunately, not fed aflatoxin-contaminated grain is not always practical because it is not balanced feed rations with other components of the cost of these components is higher. The results of this study indicate that aflatoxin levels measured in a variety of foods is less

than the limit. Feeding America national law and advise Philippine Institute of Animal Industry standard limit of aflatoxin in a variety of animal foods (especially fish) is to determine the amount 20 ppb. In the present study, the highest value measured 2/13 ppb which is less than the allowable amount. It is concluded that the quality of raw materials used for the preparation of foods sampled at the appropriate level.

References

Wayne L. Bryden . (2012). Animal Feed Science and Technology. 173(1): 134-158.

Motalebi, A.A., Ardalani, K., Jamili, S. (2008). Effect of Temperatureon the produced of Anatoxins in Rainbow trout feed in west Azarbaijan province, Journal of Fishery and Aquatic Science, 3(6): 392-397.

Lovel, R.T. (2001). Nutrition and feeding of Fish. 2nd edition, Haworth Press, New York, USA, pp. 24-28.

Gulshen, A., Gulsun. B., Gelish, T. 2003. Level of Aflatoxin in Some Fish Feeds from Fish Farming Processes, Feed Factories and Imported Feeds. Turkish Journal of Veterinary Animal Sciences, 27: 1247-1252.

Identification of juvenile Litopenaeus vannamii bacterial flora - Introducing the probiotic, in vivo and

in vitro

Mahmoud Hafezieh , IFRO; Shahram Shakiba Introduction

Aquaculture, which is the fastest growing primary production sector in the world at present, having annual growth rate of 11% year-1 over the last decade (Silva et al., 2006). This attractive industrial development has been accompanied by some practices which are potentially harmful to human and animal health ( Naylor and Burke, 2005) that consist of releasing huge amounts of veterinary drugs and chemical, using for prevention and remediation of infected disease, into the environment (Boxal et al., 2004). A substitute disease avoidance method is applying of non-pathogenic bacteria as biocontrol agents or probiotic (Moriarty, 1997). Probiotics have been considered as microorganism feed additive which provide the host organism with the beneficial effects (Fuller, 1989). The benefits of such complement consist of improving feed value, enzymatic involvement to digestion, inhibition of pathogenic microorganisms, antimutagenic and anticarcinogenic activity, growth-promoting factors, and increased immune response (Verschuere et al., 2000).

The aims of this study are:

i. Isolation and identification of bacteria flora of hatchery reared juvenile L. vannamii.

ii. Screening potential bacteria of this shrimp species as probiotics using antibacterial activity test and estimate their antibacterial potential in different growth condition.

iii. Evaluating growth potential of candidate probiotic/probiotics in different growth condition.

iv. assessing the pathogenicity of candidate probiotic/probiotics against L. vannamii, and evolution of its effect on survival of

shrimp at stressful condition.

v. Determining the best level of candidate probiotic to enhance survival and growth of L. vannamii and evaluation of it through pathogenicity challenge test.

vi. Appraising possible human risk of using candidate probiotic / probiotics application in shrimp farming.

Material and Methods

Post larvae of western white leg shrimp Litopenaeus vannamii, was reared in Madani hatchery - Chabahar up to juvenile stage. Bacterial flora of different body parts, sediment and rearing water was determined using conventional biochemical methods. Antagonistic ability of bacterial flora isolates was examined against the most detrimental shrimp pathogens (Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio alginolyticus) using cross streak technique. Further observation on growth potential of the candidate probiotic, B.glumae, in different pH, salinity, temperature and time was performed using RSM. The safety of candidate probiotic for white leg shrimp was subsequently examined. Consequently, invivo study of candidate probiotic was assessed through feeding shrimp treatment groups by supplementing commercial shrimp feed with 4 levels of candidate probiotic (T1; 104, T2; 106, T3; 109 and T4; 1012cfu/g).

Results

Seven different genera were identified from different body parts, sediment and rearing water comprised of 72% Gram negative bacteria. Burkholderia glumae was the predominant bacteria in digestive system , which is indirect evidence for its colonization in digestive system. Highest amount of total bacterial count, total Vibrio count and total Pseudomonas count were observed in sediment followed by digestive system (hepatopancrease and intestine). The largest inhibitory zones

against mentioned shrimp pathogen were produced by B.glumae followed by Bravibacterium linens with producing of anti-Vibrio substances. The significant highest feed conversion ratio, specific growth rate, protein efficiency ratio and survival was observed in 3rd treatment, this treatment consisted of significantly better bacterial balance and exhibited highest survival rate after 2 weeks challenging with V. harveyi. The possible human risk associated with using of B. glumae was derived from lethal dose 50%, LD50, on mammalian model, mice. LD50 of candidate probiotic was about 1030 via oral rout of administration which exhibited its safety for mammalian. Generally, B. glumae exhibited quite good probiotic characteristics and can be introduced for further commercial and industrial studies.

References

Boxal, A.B., Fogg, L.A., Blackwell, P.A., Kay, P., Pemberton, E.J. and Croxford, A. (2004). Veterinary medicines in the environment. Reviews of Environmental Contamination and Toxicology. 180: 1-91.

De Silva, S.S., Nguyen, T.T.T., Abery, N.W. and Amarasinghe, U.S. (2006). An

evaluation of the role and impacts of alien finfish in Asian inland aquaculture. Aquaculture Research. 37: 1-17.

Fuller, R. (1989). A review, probiotics in man and animals. Journal of Applied Bacteriology. 66: 365-378.

Moriarty, D. (1997). The role of microorganisms in aquaculture ponds. Aquaculture.

151: 333-349.

Naylor, R., and Burke, M. (2005). Aquaculture and ocean resources: Raising tigers of the sea. Annual Review of Environment and Resources. 30: 158-218.

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