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Values followed by the same letters within the same column are not significantly different (P<0.05).

Discussion

The goal of the present study was to find the effect of different dose rates of MT treatment on survival rate in C. nigrofasciatum. It was observed that the MT was effective at various dose levels in increasing mortality in the population of C. nigrofasciatum larvae. However, the present result is consistent with reported results from studies with such fish species as Sciaenochromis ahli, Cyprinus carpio and Cichlasoma nigrofasciatum, all of which were treated with synthetic hormone. According to Marjani et al. (2009), in present study different doses of MT, significantly effected on survival of C. nigrofasciatum, and all the treatments which received MT, showed more average of mortality rate than the controls. MT treatment for 21 days showed an increasing in mortality rate in O. mossambicus (Marjani et al. 2009). These results are

in line with the findings regarding anabolic effect of MT in fish and all male culture of Cichlids by different authors observed more mortality rate of hormone treated fish. Hanson et al. (1984) reported that MT-treatment showed less survival than control, these are also in line with Dan and Little (2000) that compared the culture performance of different strains of O. niloticus and found that considering all strains, MT treatment resulted more mortality in treated fish. As a finally result in present study and about the C. nigrofasciatum larvae, best selected dose rate which resulted in maximum male population with the least loss of fish was the 100 mg MT /kg of feed.

References

Axelrod, H.R., C.V. Emmens, D. Sculthorpe, W.V. Wingler and N. Pronek, 1971. Exotic tropical fishes. TFH Publ, Jersey City.

Barras, E. and C. Melard, 1997. Individual growth patterns of juvenile Nile tilapia, Oreochromis niloticus (L): emergence and dynamics of sexual growth dimorphism. In: Fitzsimons, k. (Ed), Tilapia Aquaculture. North-east reg. agric.

Eng. Serv., 106:169-177.

Dan, N.C. & D.C. little, 2000. The culture performance of monosex and mixed-sex new season and overwintered fry in three strains of Nile tilapia in North Vietnam. Aquaculture, 184:221-231.

George, T. and T.J. Pandian, 1996. Hormonal induction of sex reversal and progeny testing in the zebra cichlid Cichlasoma nigrofasciatum. J Exp Zool., 275: 374­382.

Hanson, R., R.O. Sinith Evman, H.W. Shelton and R.A. Dunham, 1984. Growth Comparison of monosex tilapia produced by separation of sex's hybridization and sex reversal, P.570. M.Sc thesis, Auburn University, Alabama US.

Marjani, M., Sh. Jamili, P.G. Mostafavi, M. Ramin and A. Mashinchian, 2009. Influence of 17-alpha methyl testosterone on masculinization and growth in tilapia (Oreochromis mossambicus). Journal of Fisheries and Aquatic Science,

4(1): 71-74.

Mousavi Sabet H., Kamali A., SoltaniM., Bani A., Esmaeili H.R., Rostami H., Vatandoust S.,Moradkhani Z. 2011. Age, reproduction, and fecundity of a population of Cobitis sp. (Actinopterygii: Cypriniformes: Cobitidae) from the Babolrud River in the southern Caspian Sea basin. Acta Ichthyol. Piscat. 41 (2):

117-122.

Mousavi Sabet, H., A. Kamali, M. Soltani, A. Bani, H.R. Esmaeili, H. Khoshbavar Rostami, S. Vatandoust and Z. Moradkhani, 2011. Reproductive biology of the Spined Loach, Cobitis sp. (PISCES, COBITIDAE) from the Talar River in South of the Caspian Sea Basin. Iranian journal of fisheries sciences. Accepted.

Myers, J.M., J. Penman, K.J. Rana, N.R. Bromage, S.F. Powell and B.J. McAndrew, 1995. Applications of induced androgenesis with tilapia. Aquaculture, 137(1-4):150.

Nelson, J., 1994. Fishes of the world, 3rd edn. Wiley, New York.

Piferrer, F. and L.C. Lim, 1997. Application of sex reversal technology in ornamental fish culture. Aquarium Sciences and Conservation, 1: 113-118.

Shelton, W.L., F.H. Meriwether, K.J. Semmens and E. Calhoun, 1983. Progeny sex ratio from intraspecific pair spawning of Tilapia aureus and Tilapia nilotica. In: Fishers L, Yaron Z (eds) International Symposium on Tilapia in Aquaculture.

T.A. University, T.A., 270-280

Effect of enriched Artemia nauply with unsaturated fatty acid and vitamin C on survival rate in Oscar Astronotus ocellatus larvae

Nasrollah Latif1, Hamed Mousavi-Sabet1*, Akbar Nasrollahzade1

1 Deptartment of Fisheries, Faculty of Natural Resources, University of Guilan, Someh

Sara, Guilan, Iran. * Mousavi-sabet@guilan.ac.ir

Abstract

Enriched Artemia nauply with unsaturated fatty acid and vitamin C was added to diets of Oscar Astronotus ocellatus larvae and survival rate was determined. Oscar larvae were kept separately in a 50 L aquarium. Artemia + fatty acid emulsion +1.0 g vitamin C resulted in the highest larvae survival (92.0%) which was also significantly higher than the control group (P < 0.05). Adding Artemia decreased larval mortality rate and would be a useful ingredient to include in diets for larvae and is likely to be economical for hatchery operations because of increased efficiency even though the feed cost would be higher.

Introduction

Ornamental fishes are popular pets around the world and their cultivation can be quite lucrative. Fresh water ornamental fishes are found in different areas in the world and the aquarium industry has developed methods for cultivation and propagation of many common species. Oscar Astronotus ocellatus is native of Central America and can live and adapt to a variety of climate or environmental conditions. This fish is commonly sexually mature at less than three year of age. Its life spawn depends upon how well proper culture conditions are maintained, but it seems that this fish would survive for 5-8 years under ideal conditions. Successful fish production depends upon access to suitable

food for feeding that is healthy and supports growth for the newborn stages for the larvae (Lim 2002a). Among numerous sources and variety in live food, Artemia is particularly important. Artemia can be used as carrier of some nutritive materials such as long chain unsaturated fatty acids and vitamins especially vitamin C (Coutteau et al. 1997). Researchers indicate that fatty acids play a critical role in the reproductive physiology of teleost fishes (Kim et al. 1996) such as maintaining immune function, osmoregulatory systems and endocrine system function. Vitamin C plays an important role in maintaining immune response and is required for numerous biological functions in fish and other vertebrates, for example, maintaining skeletal integrity, growth and survival and physiologic coefficient such as resistance against stresses, poisoning and immune activities improve in different species of aquatic larvae with usage of vitamin C complements (Dhert and Sorgeloos 1995). In this research the affect of feeding Oscar larvae with Artemia which had been specifically fed an emulsion of unsaturated fatty acids and vitamin C was tested to determine if feeding these enriched artemia would improve the survival rate of Oscar larvae.

Materials and methods

Oscar larvae were divided into 4 treatment groups and each treatment replicated 3 times. Artemia nauply was hatched in the ornamental fish farm and enriched in 1.5 L dishes with 33 g/L salt, 28 °C and under mild aeration. The density of the Artemia was 200 animals per liter when fed to the fish. To produce the artemia high in unsaturated fatty acid the following feeding regime was followed: Artemia was suspended in a

small tank containing an emulsion INVE Co (ICES 30/4/C, INVE Co.,

Belgium) which consist of 30% unsaturated fatty acid methyl esters at 30% dry weight of the emulsion). This emulsion was 4 to 1 of dicosahexenoic (DHA) and eicosapentaenoic (EPA). Preparation of standard emulsion is done on the basis of Leger's formulation 1989 (Lim 2003). Five ml of the fatty acid emulsion was blended with 50ml dechlorinated fresh water and blended in an electric blender for 3 minutes

at room temperature. For Artemia enriching with fatty acids and vitamin C, ascorbyl palmitate was added at a ratio of either 10% or 20% of the fatty acid emulsion as proposed by (Merchie et al. 1995, 1997) and Agh and Sorgeloos (2005). The ascorbyl palmitate at 0.05 g or 1 g per 5 ml of emulsion was blended with 50 ml of water for a total of 55 ml. Five ml of this blend was then added to the 1.5 L tank for enrichment. These prepared emulsions blends were stored in tightly sealed containers which excluded light under a nitrogen head space to limit oxidation and the suspension kept in the refrigerator for up to 10 days until it was used for enriching the Artemia. For artemia enrichment, 2ml of one of the emulsions described above was added to per 1liter of water (Leger et al. 1987; Coutteau and Sorgeloos 1997). After 12 hr, enrichment solution effective saturation of the artemia was achieved. A treatment of 4 ml per liter water was added with the Artemia and incubated for 24 hr. After 24 hr, the enriched Artemia were recovered. At this point, we wrapped the artemia in a clean and wet cloth and held them in a refrigerator (10°C) until use. The enriched artemia were fed within 24 hours to Oscar larvae. Larva were feed 4 meals a day at 7:00, 12:00, 17:00, 23:00 hours. 5 to 7 days after hatching, the number of actively swimming larvae was counted. This provided data for computing larvae survival rate. To determine uptake of long chain fatty acids in the enriched Artemia, fatty acid profiles were measured (n=3) by the gas chromatography system (GC). Data were analyzed by analysis of one-way ANOVA, and Duncan's test in P<0.05 for comparison of averages using SPSS 16 software.

Results

The concentration of EPA and DHA in the artemia increased after enrichment with the emulsion from 3.48-3.82g and 6.89-6.77 g percent of the total fatty acids and vitamin C for EPA and DHA respectively. The level of EPA was 1/25(%) and DHA was 1/59(%) in concentrate food. The survival rate were 61.8, 73.0, 92.0 and 81.7 in different treatments including Artemia + unsaturated fatty acid, Artemia + unsaturated fatty

acid + 0.5g Vitamin C, Artemia + unsaturated fatty acid + 1g Vitamin C, and nun-enriched Artemia, respectively.

Discussion

The effect of enriching live artemia with long chain unsaturated fatty acid and Vitamin C is has been evaluated in different species of fishes and shrimp in salt and fresh water, but only in a few ornamental fishes. Similar research with Acipenser sp., Indian white shrimp post larvae (Tenaeus indicus), rainbow trout larvae (Oncorhynchus mykiss), giant freshwater prawn (Macrobranchium rosenbergii) and milk fish larvae (Chanos chanos) reported and that enriched artemia with unsaturated fatty acid and Vitamin C increased larvae survival rate (Dhert et al. 2004; Gapasin 1998; Girri et al. 2002; Lim 2001; Lim 2002a). Here we saw an increase in survival between the control and the treatments using fatty acid enrichment, also adding vitamin C in addition to the unsaturated fatty acids increased survival. Moradkhani (2008) studied Cichlasoma severum and noticed effects of using live food include enriched foods on increased fecundity. Also, Tamaru et al. (2003) noted that the use of live food for Angel brood stock and Carassius auratus increases fertilization percent.

Lim et al. (2003), Tamaru et al. (2003), Dhert (2004) and Moradkhani

(2008) researched feeding for Poecilia reticulata, Xiphophorus helleri, Xiphophorus maculates, Poecilia sphenops, Hyphessobrycon herbertaxelrodi, Cichlasoma severum, Carassius auratus and Symphysodon aequifasciata and found that enrichment of Artemia with unsaturated fatty acid and ascorbic acid and its use in feeding of brood stocks improved larvae survival rate. Adding Artemia with unsaturated fatty acids and Vitamin C to broodstocks diets improved larval survival rate (Moradkhani 2008; Lim 2001a; Lim 2001b; Merchie 1997; Sorgeloos 1980; Tamaru et al. 2003). About larvae survival rate, acquired results showed that all treatments have significant difference with testimonial group (P < 0.05). Since Vitamin C decreases stress, as result; decreasing effect of environmental stresses on larvae cause to increase its survival in treatment which had been enriched with ascorbic acid. Increasing

unsaturated fatty acid and vitamin C content in the larvae food could increasing the strength of the resulting larvae. Improving efficiency of hatchery operations would make them more economically viable and also increase the number of offspring from breeding pairs of Oscar.

References

Coutteau P, Sorgeloos P, Leger P. 1997. Manipulation of dietary lipids fatty acid and vitamin in zooplankton cultures. Freshwater Biol 38: 501-512.

Dhert PH, Sorgeloos P. 1995. Live feeds in aquaculture, in: Nambiar KPP, Singh T. (Ed.) (1995). Aquaculture towards the 21th Century: Proceedings of INFOFISH-AQUATECH '94. International Conference on Aquaculture. pp. 209-219.

Dhert P, Lim L, Yen Chew W, Dermaux V, Nelis H, Sorgeloos P. 2004. Enhancement of Stress Resistance of the Guppy (Poecilia reticulata) through Feeding with Vitamin C Supplement. J World Aquacult Soc 33(1): 32-40.

Gapasin RSJ. 1998. Enrichment of live food with essential fatty acids and vitamin C: effects on milkfish (Chanos chanos) larval performance. Aquaculture 162: 269­286.

Girri SS, Sahoo SKBB, Saha AK, Mohanty SN, Mohanty PK, and Ayyappan S, 2002.

Larval survival and growth in Wallago attu (Bloch and Schnider): effect of light, photoperiod and feeding regime. Aquaculture 213: 157-161.

Javahery M. 2006. Study on use of enriched Artemia with unsaturated fatty acid & vitamin C in survival rate of Salmo trutta caspius larvae. Ph.D thesis, Islamic Azad University, Science & research campus, Tehran, Iran.

Kim J, Massee KC, Hardy RW. 1996. Adult Artemia as food for first feeding coho salmon (Oncorhynchus kisutch). Aquaculture 144: 277-226.

Leger P. 1986. The use and nutritional value of Artemia as food source.Occagar. Oceanogr. Mar Biol Ann Rev 24: 521-623.

Leger P. 1989. Advances in the enrichment of rotifers and Artemia as food sources in marine larviculture. In: Aquaculture Europe '89. European Aquaculture Society Special Publication N 10. Bredene, Belgium, 344 p.

Lim LC. 2001b. Feeding of discus juveniles with on-grown Artemia, in: Hendry, C.I. (Ed.) (2001), 3rd fish and shellfish larviculture symposium Gent, Belgium, September 3-6, 2001. Special Publication European Aquacul Soc 30: 315-317.

Lim LC. 2002a. Enhancement of stress resistance of the guppy (Poecilia reticulate) through feeding with vitamin C supplement. J World Aquacul Soc 33(1): 32-40.

Lim LC. 2002b. Use of decapsulated Artemia cysts in ornamental fish culture. Aquacult

Res 33(8): 575-589.

Lim LC. 2003. Recent developments in the application of live feeds in the freshwater ornamental fish culture. Aquaculture 21: 319-331.

Merchie G. 1995. Live food mediated vitamin C transfer to Dicentrarchus labraxand and Clarias gariepinus. J Appl Ichthyol 11(3-4): 336-341.

Moradkhani K. 2008. Effect of using enriched Artemia with highly unsaturated fatty acid and vitamin C on the propagation of Astronotus ocellatus. J Fisheries 4: 15­22.

Sorgeloos P. 1980. The use of the brine shrimp Artemia in aquaculture, in: Persoone, G. (Ed.) (1980). The brine shrimp Artemia: Proceedings of the International Symposium on the brine shrimp, Corpus Christi, Texas, USA, August 20-23, 1979: 3. Ecology, culturing, use in aquacul. pp. 25-46.

Tamaru clyde S. Ako H. 2003. Enrichment of Artemia for use in fresh water ornamental fish production. Center for Tropical and Subtropical Aquaculture, Number 48.

Growth performance and stress resistance of Persian

sturgeon (Acipenser persicus) larvae fed live food (Artemia franciscana) enriched with fish and Soybean oils supplemented with vitamin E

Mahdi Naderi Koshk1, Abdolmohammad Abedian Kenari1

1 Aquaculture Department, Tarbiat Modares University, Noor, P.O. Box 64414-356, Mazandaran, Iran

Introduction

Persian sturgeon (Acipenser persicus) inhabits in the southern part of the Caspian Sea, being one of the most important fishes in this area. Because of the critically endangered status of this species (It is currently included in the IUCN Red Data List) most research in recent years has been focused on the culture of Persian sturgeon for restocking and commercial aquaculture programs. Live foods such as Artemia is widely uses in larvae culture of sturgeon fishes. Survival of Persion sturgeon larvae is too low when using inert diet, so live food such as Artemia is main food for larval early stage of this fish. Thus using of Artemia nauplii as a starter food seems to be one of the methodological choices to enhance Persion sturgeon larviculture. Artemia are naturally low in essential fatty acids, therefore, enrichment of live foods with lipids contain EFA before feeding is necessary (Copeman et al., 2002).

Some previous studies showed positive effects of enriched Artemia with oils in sturgeon larvae (Agradi et al., 1993; Hafezieh et al., 2009). But no studies exist to test enriched Artemia with two sources of EFA (animal and vegetal) with Vitamin E on the performance of Persion sturgeon larvae. The study also aims at evaluating the role of HUFA and PUFA supplemented with vitamin E, which acts as a lipid soluble antioxidant to find their interaction effects on the growth performance and stress resistance of Persian sturgeon larvae.

One thousand and five hundred of seven-days old post-hatch larvae of A. persicus with yolk-sac obtained from Shahid Rajaee Sturgeon Hatchery Center (Sari, Mazandaran, Iran; Lat 36°37 ' N, Long 53°05 ' E) and in a oxygenated plastic bags transported to the hatchery of marine sciences faculty of Tarbiat Modares University (Noor, Mazandaran, Iran). They were then stocked in a round fiberglass tank with final volume of 100 liter. One hundred randomly collected larvae (in three replicates) were distributed into 15 tanks containing 25 L water. Each Feeding rate was adjusted to the actual fish biomass in each treatment. All treatments fed non-enriched Artemia for the initial 5 days after first feeding (tenth day) and then fed experimental foods for 17 days. Larvae were fed at 25% body weight per day, with a feeding frequency of four times per day. The diurnal light/dark cycle was at 12:12 h. The pH (7­7.5), temperature (19-20 ± 1 C) and dissolved oxygen level (8-8.5 mg L-1) of each tank were monitored daily.

Fish (Kilka species from Clupeonella) and Soybean oils and all-rac-a-tocopheryl acetate (Merck Company, Germany) were used as lipid and vitamin E sources. Five feeding treatments included : Artemia franciscana nauplii enriched with Soybean oil+15% and 30% vitamin E (S15 and S30), fish oil+15 % and 30% vitamin E (F15 and F30) and non-enriched Artemia (control) were tested. Vitamin E was added as percent of oil to the solution. Cysts of A. franciscana were hatched in 1L cylindro-conical glass vessels under standard conditions(Sorgeloos et al., 1986). Artemia nauplii were washed with salt water and put into the enrichment tanks. The enrichment solution at doses of 0.5 mL L-1 was given to the Artemia nauplii. After 12 h incubation with enrichment, the nauplii were washed with salt water (28 g L-1) to discard non-absorbed lipids and kept aerated until served to fish (Jalali et al., 2008). Each day, a new batch of enriched Artemia nauplii was used. The enrichment solution was prepared by adding 20 g oil to 20 g water containing 2 cc sodium polysurbate as an emulsifier and stirring with a magnetic stirrer at room temperature. Tree or six grams (for treatments contain 15% and

30% vitamin E, respectively) of all-rac-a-tocopheryl acetate were then added to the solution and stirred for 5 min (Noshirvani et al., 2006).

At day 31, all of the fishes from each tank were removed for wet weight measurements. The dead larvae were counted daily and removed from each culture tank. Also 15 fishes from each tank were removed and submersion in salt water (12 ppt for 3 days) and survival was recorded for each tank. This experiment was performed in order to evaluate larvae resistance to salinity stress. The fish did not feed during the stress tests. The confinement stress consisted of netting 15 fish from each tank, holding them out of water for 1 min and survival was recorded for 4 days. Sufficient aeration was supplied to prevent additional stress from oxygen depletion (Carey and McCormick, 1998).

Data analyzed by one-way analysis of variance (ANOVA) using the statistical software SPSS, version 16.0. When ANOVA identified differences among groups, multiple comparisons among means were made with Duncan's new multiple-range tests. The values of P < 0.05 were considered significantly different.

Results

A significant growth difference between treatments was observed (P < 0.05), larvae fed with enriched Artemia (F30 and S30) and control group (C) had higher growth than the other groups and lowest final weight was related to F15 (Fig. 1). Survival of fishes not significantly (P > 0.05) difference at the end of the feeding trial.

Results related to the salinity stress showed that after 24 and 48 h, survival was not significantly different (P > 0.05) among all treatments. But after 72 h stress resistance in the C, S30 and F30 groups was significantly higher (P < 0.05) than others (Fig. 2).

Results of confinement stress test showed that after 24, 48, 72 and 96 h survival was not significantly affected by the food treatments. Survival in the treatments was more than 80%.

Figure 1 Final weight of Acipenser persicus larvae fed Arternia enriched with soybean oil+15% and 30% vitamin E(S15 and S30), fish oil+15 % and 30% vitamin E(F15 and F30) and non-enriched Artemia (control). Bars with different letters above are significantly different from each other.

On the first day       On the second day       On the third day

120 la a a aaaa a

- 100 t t t a a b t t t t a t ta ab §    80                      T TT                              b T

•-    go ^1 ^1 ^1        ^1 ^1

4o ^1 ^fl ^1        ^1 ^fl

20

a a a       a a

aa

S15

S30

Treatment

F15

F30

Figure 2 Survival of Acipenser persicus larvae on the first day until third day after salinity stress (12 ppt) fed Artemia enriched with Soybean oil+15% and 30% vitamin E(S15 and S30), fish oil+15 % and 30% vitamin E(F15 and F30) and non-enriched Artemia (control); groups having different letters are significantly different (P < 0.05).

C

In the present work we found that larvae fed with enriched Artemia (F30 and S30) and control group (C) had higher growth than the other groups. This means that non- enriched Artemia nauplii is suitable for this fish and provide nutritional requirements for them. More size of the Artemia nauplii after 12 h enrichment could be another reason for not significantly effect of enriched Artemia. Results showed that fishes were fed S30 and F30 had higher performance compare to the other enriched groups. Firstly this means Persion sturgeon larvae are able to use both n6 and n3 fatty acids which previously proven for white sturgeon (Deng 1996; Deng et al., 1998) and secondly expression that vitamin E is effective to use both vegetal and animal oils. The nutritional value of high levels of (n-3) HUFA was confirmed by enhanced growth in many marine fish larvae, while the different results were reported did not find a relationship between (n-3) HUFA levels in live food fed to larval turbot and larval growth (Rainuzzo et al., 1994). Enrichment of Artemia with (n-3) HUFA supplemented with vitamin E did not have a significant effect on walleye (Stizostedion vitreum) larvae growth (Kolkovski et al., 2000) but Similar to our study, enrichment of Artemia with fish oil and vitamin E has positive effect on growth of Beluga (Huso huso) larvae (Jalali et al., 2008). Different results maybe is due to the species, age, water condition, rearing condition and etc. Resistance of Persion sturgeon larvae after third day to 12 ppt salinity stress test was better in C, S30 and F30 groups and had significantly differences with other enriched groups. Also there were no significant differences between groups for confinement stress test. This is similar to the results of growth and survival obtained from the normal condition. This means enrichment of Artemia with both animal and vegetal oils is not so necessary for Persion sturgeon larvae probably because of their ability to elongation and desaturation (Xu et al., 1996). Similar result to no efficiency of fish oil enrichment for Persion sturgeon larvae against salinity stress test is mentioned (ABEDIAN et al., 2007) but enhancing resistance against salinity stress has been reported in Beluga (Jalali et al., 2008, 2010).

Results obtained from this study showed that enrichment of Artemia with both animal and vegetal oils is not so necessary for Persion sturgeon larvae might be due to their ability to elongation and desaturation. But they can use both of the oils with almost similar trend. It is also proved that using vitamin E is necessary to enhance both vegetal and animal oils efficiency might be due to its anti oxidant role.

References

ABEDIAN, K.A.A.M., OVEYSIPOUR, M., Nazari, R., 2007. Effects of n3-HUFA enriched Daphnia magna on growth, survival, stress resistance, and fatty acid composition of larvae of Persian sturgeon (Acipenser persicus). Iranian Journal of Fisheries Sciences 7, 1-14.

Agradi, E., Abrami, G., Serrini, G., McKenzie, D., Bolis, C., Bronzi, P., 1993. The role of dietary n-3 fatty acid and vitamin e supplements in growth of sturgeon (< i> Acipenser naccarii</i>). Comparative Biochemistry and Physiology Part A: Physiology 105, 187-195.

Carey, J.B., McCormick, S.D., 1998. Atlantic salmon smolts are more responsive to an acute handling and confinement stress than parr. Aquaculture 168, 237-253.

Copeman, L., Parrish, C., Brown, J., Harel, M., 2002. Effects of docosahexaenoic, eicosapentaenoic, and arachidonic acids on the early growth, survival, lipid composition and pigmentation of yellowtail flounder (< i> Limanda ferruginea</i>): a live food enrichment experiment. Aquaculture 210, 285-304.

Deng, D.F. (1996) qualitative requirement of essential fatty acids for white sturgeon (Acipenser transmontanus). MS dissertation, University of California, Davis, California.

Deng, D.F.. Hung. S.S.O. and Conklin. D.E. (1998) White sturgeon (Acipenser transmontanus) requir both n-3 and n-6 fatty acids. Aquaculture 161,333.

Hafezieh, M., Kamarudin, M.S., Saad, C.R.B., Sattar, M., Agh, N., Hosseinpour, H., 2009. Effect of Enriched Artemia urmiana on Growth, Survival and Composition of Larval Persian Sturgeon. Turkish Journal of Fisheries and Aquatic Sciences 9,

201-207.

Jalali, M.A., Hosseini, S.A., Imanpour, M.R., 2008. Effect of vitamin E and highly unsaturated fatty acid-enriched Artemia urmiana on growth performance, survival and stress resistance of Beluga (Huso huso) larvae. Aquaculture

Research 39, 1286-1291.

Jalali, M.A., Hosseini, S.A., Imanpour, M.R., 2010. Physiological characteristics and stress resistance of great sturgeon (Huso huso) juveniles fed with vitamins C, E, and HUFA-enriched Artemia urmiana nauplii. Fish physiology and biochemistry

36, 555-564.

Kolkovski, S., Czesny, S., Yackey, C., Moreau, R., Cihla, F., Mahan, D., Dabrowski, K., 2000. The effect of vitamins C and E in (n-3) highly unsaturated fatty acids-enriched Artemia nauplii on growth, survival, and stress resistance of fresh water walleye Stizostedion vitreum larvae. Aquaculture Nutrition 6, 199.

Noshirvani, M., Takami, A.G., Rassouli, A., Bokaee, S., 2006. The stability of ascorbic acid in Artemia urmiana following enrichment and subsequent starvation. Journal of Applied Ichthyology 22, 85-88.

Rainuzzo, J.R., Reitan, K.I., J0rgensen, L., Olsen, Y., 1994. Lipid composition in turbot larvae fed live feed cultured by emulsions of different lipid classes. Comparative Biochemistry and Physiology Part A: Physiology 107, 699-710.

Sorgeloos, P., Lavens, P., Leger, P., Tackaert, W., Versichele, D., 1986. Manual for the culture and use of brine shrimp Artemia in aquaculture.

Xu, R; Hung, S.S.O. and German, J.B. (1996) Effects of dietary lipids on the fatty acid composition of triglycerides and phospholipids in tissues of white sturgeon. Aquaculture Nutrition 2, 101-109.

The microbial bioremediation of effluent of cultivation system for reuse in rearing tanks of Common Carp (Cyprinus carpio) larvae

*Naderi, M.1, Jafaryan, H. 1, Gholipour, H.1, Harsig, M. 1, Farhangi, M.1

1 Departement of fishery, University of Gonbade kavos, Iran * Corresponding author: hojat.jafaryan@gmail.com

Abstract

Bacillus can act positively on cultured organisms by enhancing survival and growth, by stimulating the digestive and immune systems and by improving water quality in terms of bioremediation. The bioaugmentor bacterial strain Bacillus licheniformisi, B. subtilis, B. polymyxa, B. laterosporus and B. circulans (Protexin Aquatech, UK) were used in this study. The effluent or waste water of Common Carp cultivation system was used in this experiment. In trial of probiotic (T1), the blend of bacillus were added directly to the waste water in bacterial treatment tanks at a concentration of 1x106 CFU/ liter at the period of 72 h. In treatment of T2 and treatment of T3 the raw waste water (untreated waste water) and the tap water were used in trial tanks respectively. This experiment was conducted in 3 treatments, each with three replicates. The replacement of water in all of rearing tanks of Common Carp (Cyprinus carpio), in each treatment was done in rate of 50% daily. At the end of the experiment (24 days) all the fish were sampled and some growth and feeding parameters of fish larvae were calculated. The blends of bacillus had a good effect for adjustment of total ammonium nitrogen, nitrate nitrogen, nitrite nitrogen and turbidity in treatment tanks of effluent waste water. The incorporation of bacteria in the rearing tanks increased significantly (P<0.05) the mean weight, F.B.W (g), F.B.L

(mm), CF and ADG (%) of Carp larvae in T1 (rearing in treated waste water) in comparison with T2 (rearing in raw waste water).

Key words: Bacillus, water quality, bioaugmentor, effluent, total ammonium nitrogen

Introduction

Organic enrichment and nitrogenous waste, including ammonium and ammonia are a serious concern in aquaculture. Nitrification is a process of transformation of ammonia to nitrate by two groups of bacteria, i.e. ammonia-oxidizing bacteria and nitrite-oxidizing bacteria. This process can help to prevent to build up of toxic ammonia.

Bacteriologicalnitrification is the most practical method for the removal of ammonia from closed aquaculture systems. The dissolved and suspended organic matter contains mainly carbon chains and is highlyavailable to microbes and algae. A good bioremediator must contain microbes that are capable of effectively clearing carbonaceous wastes from water. Additionally, it helps if these microbes multiplyrapidly and have good enzymatic capability. Members of the genus Bacillus, like Bacillus subtilis, Bacillus licheniformis and Bacillus cereus, are good examples of bacteria suitable for bioremediation of organic detritus (Singh, et al. 2001). Among probionts, Bacillus is gram positive, spore forming bacteria, used commercially as probiotics. Bacillus can act positively on cultured organisms by enhancing survival and growth, by stimulating the digestive and immune systems and by improving water quality in terms of bioremediation. The use of probiotic bacillus has been suggested as an important strategy to accomplish reproducible outputs through biocontrol in cultivation systems for marine fish larvae and crustaceans (Nogami and Maeda, 1992). The bacterial flora in the larval gut originates from the bacteria associated with the eggs, the water in the rearing tanks, and the live food (Olafsen and Hansen, 1992). The probiotic bacteria can increase the digestive enzymes

activity, digestibility of the ingested nutrients and enhancement of growth and feeding performance in fish larvae. The objective of the present study was to investigate the ability of using of bioremediated wast water in cultivation of Common Carp larvae (Cyprinus carpio) in rearing tanks.

Materials and Methods

Bacterial suspension. The bioaugmentor bacterial strain Bacillus licheniformisi, B. subtilis, B. polymyxa, B. laterosporus and B. circulans (Protexin Aquatech, UK) were used in this study. The spores of B. circulans and B. licheniformis were rehydrated to vegetative bacteria according to manufacturer's instructions and the suspension of bacteria in level of 1x108 CFU/L was determined by optical density at 610 nm in a spectrophotometer.

Cultureing system of fish larvae. Twenty-day old Common Carp (Cyprinus carpio) larvae with average weight of 500±10 mg were obtained from Woshmgir Fish Hatchery, Golestan, Iran. Fish larvae were acclimatized to laboratory condition for 5 days and fed with starting food of Biomar. Each experimental tank was supplied with non-chlorinated water from a deep tube well with continuous aeration. The fish were transferred and randomly allocated at 30 fish per tank to 9 circular fiberglass tanks (capacity of 10 liters).

Samples of tank water from each treatment were collected. Serial dilutions of the samples were done in autoclaved distilled water and 0.9% (wt/vol) NaCl and number of cells per ml of suspension was determined by spread plating in triplicate on Tryptic Soy agar (TSA) and incubated at 30 °C for 24 h. After 24 h the colony forming unit (CFU) was counted (Gomez-Gil, et al., 1998). At the end of the experiment all the fish were sampled and their growth parameters were measured.

Bioremediation of waste water. The effluent or waste water of Common Carp cultivation system was used in this experiment. In trial of probiotic (T1), the blend of bacillus were added directly to the waste water in bacterial treatment tanks at a concentration of 1x108 CFU/ liter

at the period of 72 h. and the colony forming units (CFU) was counted. All of trial tanks were aerated water temperature was 20-22 °C.

In treatment of T2 and treatment of T3 the raw waste water (untreated waste water) and the tap water were used in trial tanks respectively. This experiment was conducted in 3 treatments, each with three replicates.

The replacement of water in all of rearing tanks in each treatment was done in rate of 50% daily.

The makeup water in treatment of T1, T2 and T3 were treated waste water, untreated waste water and tap water respectively.

The feeding rate of larvae was 5% of wet body weighs per day. The period of experiment was 25 days. At the end of the experiment all the fish were sampled and some growth and feeding parameters of fish larvae were calculated by mathematical formula.

Statistical analysis. Statistical analysis of data was performed by analysis of variance (ANOVA) using SPSS-17 followed by Duncan's multiple range tests.

Results

As indicated in table 1, the blends of bacillus had a good effect for adjustment of total ammonium nitrogen, nitrate nitrogen, nitrite nitrogen and turbidity in treatment tanks of effluent waste water. The lowest level of total ammonium nitrogen, turbidity and highest nitrate nitrogen were obtained in tank of treated waste water during 72 h.

Tablel. Changes in the water quality parameters of raw and treated waste water by adding of bacillus in the treatment tanks after 72 h.

Treatments

Total ammonium nitrogen (mg/l)

Nitrate nitrogen

(mg/l)

Nitrite nitrogen

(mg/l)

Turbidity

(NTU)

1RWW (Starting time)

2.78 ±0.21a

23.5 ±1.20b

0.9 ±0.05c

6.40±0.14a

RWW (After 72 h.)

1.37±0.11b

19.80 ±1.70c

3.8 ±0.10a

5.20 ±0.14b

TWW (After 72 h.)

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