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1、 Mcdwdi Research Journal of Biological Sciences 2 (3): 221-224, 2007 Medwell Journals, 2007 _ The Use of Oxolinic Acid to Enrich Artemia urmiana from Urmia Lake, Iran Wiry sef Yahyazadeh， 2AminEimanifar， 3William N. Camargo， 4Mehdi Soltani and 5Feridon Mohebbi Iranian Artemia Research Center, P.O. B

2、ox: 368 Urmia, Iran Iranian Artemia Research Center, P.O. Box: 57135-1367 Urmia, Iran fisheries and Illinois Aquaculture Center, Southern Illinois University at Carbondale, Carbondale, IL 62901-6511, USA department of Aquatic Health and Disease, Faculty of Veterinary Medicine, P.O. Box: 14155-6453 T

3、ehran， Iran Abstract: The live food enrichment technique using emulsions for preventing fish diseases was investigated as a tool for transferring therapeutics through the food chain. Nauplii and adult of the brine shrimp, Artemia urmiana were enriched with Oxolinic Acid (OXA) as an antibacterial dru

4、g to determine its content after different enrichment intervals. Various concentrations of OXA ranging from 25? 50? 75 and 100 mg L_1 were mixed with emulsion oil. Enriched nauplii and adult were sampled at 2? 4? 6? 8 h intervals. An analytical method for the quantification of OXA in a biological ma

5、trix, A. urmiana nauplii and adult, was developed using High Performance Liquid Chromatography (HPLC). The highest and lowest accumulation rate of OXA in enriched nauplii body tissue occurred at 100 and 75 mg L_1 at intervals 6 and 8 h? respectively and for adult at was 50 and 25 mg L_1 at 4 and 8 h

6、 enrichment period, respectively. The average amounts of this were estimated to be 39 and 2.5 jag g_1 WW? respectively for nauplii and 192 and 3 jag g_1 WW? respectively for adult of A. urmiana. The obtained results showed that there is a significant outcome between enrichment dosage and period with

7、 content of OXA inboth4. urmiana nauplii and adult (p0.001). However, a direct correlation could not be observed between enrichment dosage and period with OXA level in all enriched groups. Key words: Artemia urmiana, oxolinic acid, enrichment dosage and period INTRODUCTION Various bacterial diseases

8、 produce many problems in fish culture such as massive mortalities and economic loses. Therefore， antimicrobial agents are widely used to prevent and treat bacterial diseases in fish cultures (Trust， 1986; Alderman， 1988). Treating microbial infections in fish and shrimp larvae is most often done by

9、 dissolving relatively high doses of broad spectrum antibiotics in the culture water or or mixing them with food (Brown， 1989; Yanong， 2006). A disadvantage of this method is the large amounts of expensive drugs which are used and discharged in the environment, placing animal and human health at ris

10、k (Cabello, 2006). Traditional methods to control microbial fish diseases are inefficient and impose high costs of the drug waste and treatment repetitions on farmer (Leger et !， 1986; Alderman，1988; Samuels en a/. ? 1999). Nowadays, a direct treatment is available through the food chain, which uses

11、 much smaller quantities and is far more effective to control fish disease and safer for the environment than other traditional methods. Among live food organisms, the brine shrimp Artemia as filter feeder has successfully been used as a biological carrier for transferring essential nutrients to pre

12、dator larvae (Leger et ah, 1986; Stottrup and McEvoy, 2004). This is achieved through enrichment or bioencapsulation techniques (Gapasin et ah, 1996; Aguilar-Aguila et ah, 1994). This technique is a suitable and controlled method to deliver pharmaceutical agents into cultured organisms and to preven

13、t the manifestation of many problems which occur when using traditional methods (Nelis et al, 1991; Duis et ah, 1995; Touraki etal, 1999). Artemia nauplii are used as an essential food source in fish and crustacean larviculture and Artemia biomass is used in adult fish and shrimp cultures (Sorgeloos

14、, 1973). Noshirvani et al. (2006) reported enriching freshly hatched Artemia urmiana nauplii with Ascorbic Acid (AA) and the AA concentration was determined during various enrichment dosage and periods. Further, Artemia Corresponding Author: Amin Eimanifar, Iranian Artemia Research Center, P.O. Box:

15、 57135-1367 Urmia, Iran 221 Res. J. Biol. Set, 2 (3): 221-224, 2007 enrichment with pharmaceutical agents and its application in bacterial diseases inhibition was reported by Verpraet etal. (1992), Dhcrt etal. (1993) and Chair etal. (1996). Oxolinic acid belongs to the quinolons group, which is more

16、 effective on gram negative bacteria and is widely used to control infectious disease in the aquaculture industry (Varvarigos, 2003). The aim of the present study was to enrich, urmiana nauplii and adult with Oxolinic Acid (OXA) as antibacterial pharmaceutics and to determine the variation of OX A c

17、ontent in A. urmiana body during various enrichment dosages and periods. MATERIALS AND METHODS Artemia enrichment: A. urmiana cysts were hydrated and decapsulated according to the method proposed by Van Stappen (1996). Decapsulated cysts were incubated in a 2-L conical incubator containing natural f

18、iltered sea-water (30 g L_1) at 281C under strong aeration and illumination (2000 lux) according to the method suggested by Sorgeloos (1997). After 24 h, Instar I nauplii were harvested and separated from hatching debris and thoroughly rinsed. A part of the freshly nauplii were transferred into coni

19、cal beakers and remainder were reared till adult stage (density 400 individuals mL_1? salinity 30 g L_1) and kept for 2, 4, 6 and 8 h enrichment The enrichment medium consisted of a commercial live food (INVE) into which various levels (25， 50， 75 and 100 mg L_1) of oxolinic acid (Sigma) was incorpo

20、rated. The 25 individual mL_1 of nauplii and adults were enriched with aeration at 281C for various intervals (2? 4， 6 and 8 h). Sampling was done at the end of each enrichment period Nauplii and adults were taken after each interval, rinsed in ambient freshwater to remove excess surface oxolinic ac

21、id from their bodies. Oxolinic acid analysis (OXA analysis): A total mass of 500 mg of each nauplii and adult from each enrichment and interval was weighed; all specimens were homogenized in methanol. After centrifugation the residue was separated from the methanol fraction. This step was repeated t

22、wice on the residue. The three methanol fractions were combined. The interfering lipids and caretenoids were removed from the methanol fraction by a double hexane extraction at pH 7.0. Samples were kept frozen (-20C) until examination for OXA determination. Using the autoinjector, 20 juL of aqueous

23、extract was injected into the column. A Knauer High Performance Liquid Chromatography (HPLC) system was used to determine the OXA levels in A. urmiana nauplii and adults. The system consisting of a K-1001 pump, an injection valve model D-l 4163 fitted with a 20 juL injection loop. A variable wavelen

24、gth UV-Vis detector 2501 and Model V 7566 version 0696 interface box. The system was driven by a personal computer using EuroChrom 2000 HPLC software for Windows. The analytical cartridge (Knauer 125x4 mm I .D.) was packed with 5 jurn Spherimage 80 and ODS as end capping sorbent (Merk). The mobile p

25、hase (PH 4.01) used comprised of 30% methanol and 70% water and detection of OXA was made at a wavelength 254 nm. Oxolinic acid contents were expressed per gram Wet Weight (WW). Three replicates from each enrichment and intervals were examined and OXA levels in all specimens determined as an average

26、 of obtained values for each treatment. The statistical differences between means and homogeneity of variance were evaluated by one-way ANOVA and Laven s test. All analysis was performed using the Statistical Package for the Social Sciences (SPSS V.l 1.5). RESULTS AND DISCUSSION The role of feeding

27、aquacultural organisms with antibacterial pharmaceutics, vaccines and hormones incorporated into the live foods especially via Artemia nauplii and adults facilitates the remedy of bacterial diseases in fish species (Joosten et aL， 1995; Campbell etal, 1993; Skjeimo etal, 1995). Enrichment of A. fran

28、ciscana nauplii with Sarafloxacin at various dosages (1? 5? 15, 20 and 40%) at 2-24 h intervals to control four Vibrio strain through Antibiotic Susceptibility Test was carried out by Dixon et al. (1995). They concluded that 15% dosage at 6 h enrichment period was effective against four Vibrio strai

29、ns. Dhert et al. (1993) demonstrated the efficiency of sulphadrugs agents against Vibrio angillarum through feeding sea-bass and trout larvae with enriched Artemia nauplii. The oral feeding of larvae fish after 4 h was effective against V. angillarum. As, no evidence has been published on A. urmiana

30、 to explain the variation of OXA content during enrichment, then, the present study was concentrated on to introduce optimum dosage of OXA as antibacterial agent to control bacterial diseases in fish species through enrichment of it at various dosage and intervals with A. urmiana nauplii and adults.

31、 In this research, A. urmiana has shown a different process of variation in OXA concentration during enrichment periods. The highest accumulation rate of OXA in nauplii occurred in the 100 mg mL_1 dosage with 6 h enrichment time. In contrast, the lowest accumulation rate of OXA in nauplii was in the

32、 75 mg mL_1 dosage with 8 h enrichment time. The accumulated amounts of OXA in 6 and 8 h enrichment time were estimated to be 39 and 2.5 jugg-1 222 Res. J. Biol. Set, 2 (3): 221-224, 2007 WW? respectively. Mohney et al (1990) studied the franciscana nauplii enriched with Romet-30 and they concluded

33、that the higher absorbance rate of this drug in nauplii occurred at the 3 mg L_1 dosage with 4 h interval to be 0.1 jug nauplii-1. In comparison, the highest and lowest OXA accumulation rate in adults body tissue was in dosage 50 and 25 mg mL_1 at intervals 4 and 8 h, respectively. The accumulated a

34、mounts of OXA in 4 and 8 h enrichment time were estimated to be 192 and 3 jug g_1 WW? respectively. The results of the present study revealed that there is a significant outcome between enrichment dosages and intervals with OXA concentration in A. urmiana nauplii and adult of (p0.001). Although, the

35、 obtained results was not in accordance to the findings of Aguilar-Aguila et al. (1994) in which there was a dependence among accumulation rate of Romet-30 with enrichment dosage and periods. Their results suggest that there is a possible saturation threshold for OXA in. urmiana nauplii under certai

36、n enrichment dosages and periods. As many quantitative analysis methods have been developed to assay different pharmaceutical agents concentrations in Artemia nauplii (Nelis et al., 1991) then, we can indicate that pharmaceutical agents accumulation rates not only depend on enrichment time and perio

37、d, but also involves other parameters such as nauplii density ， species and enrichment condition. Therefore, further investigations regarding using of different enrichment methods to detect OXA inhibitoiy concentration via Artemia as a biologic carrier against fish and shrimp bacterial diseases are

38、recommended ACKNOWLEDGMENT The authors are thankful to S. Shiri for laboratoiy assistance of this research. We are grateful to members of Urmia University due to analytical performance during this research. Financial support was obtained from the Iranian Fisheries Research Organization (IFRO). REFER

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