Int. J. Aquat. Biol. (2013) (1): 28-32 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2013 Iranian Society of Ichthyology Sulfuric acid treatment for Artemia cyst decapsulation Seyyed Morteza Hoseini *1, Mohammad Hadi Abolhassani1, Rasool Ghorbani1 1 Department of Fisheries, Faculty of Fisheries and Environment, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran. Article history: Received 2 March 2013 Accepted 12 April 2013 Available online 14 April 2013 Keywords: Acipenseridae Decapsulation Artemia Sulfuric acid Hypochlorite solution Abstract: In the present study, sulfuric acid was used for Artemia cysts decapsulation. Cysts of Artemia franciscana were hatched out in regular manner or following hypochlorite or acid decapsulation. Two acid concentrations (1 and 5%), three acid immersion times (10, 30 and 50 min) were used and hatching rates were recorded after 15, 18 and 24 h incubation. Hatching rates increased but hatching time decreased in line with acid concentration and acid immersion time increment. Hypochlorite-treated cysts had significantly higher hatching rate (97%) compared to other groups. However, among the acid- treated cysts, the best hatching rate (92.4%) was achieved in cysts treated with 1% acid over 50 min. Acid treatment could be used as a decapsulation method which saves cost and labor because of increasing the hatching rate and speed. Introduction Live food is necessary for fish larvae, especially in marine species. Undeveloped digestive system of larvae, makes them completely dependent on live food. Live foods have other advantages over the formulated feed including appropriate size, palatability due to high water content, immersion in water column and stimulation of larval predatory behavior (Bengston, 2003). Nauplius of Artemia sp. is one of the important live foods with the widespread use in aquaculture. It contains 41-62% protein and 12-23% lipid which is a good source of linolenic acid and eicosapentaenoic acid (Dhot and Van Stappen, 2003). Small size, acceptable nutritional values, easy to produce and suitability for use in bio-encapsulation process are the advantages of Artemia nauplii. The common technique to produce Artemia nauplii is to collect and hatch the cysts under artificial condition. The cysts have a chorion shell constituted of lipoproteins, chitin and haematin (García-Ortega et al., 1998). This layer is completely indigestible by all known cultured species and may cause gut obstruction * Corresponding author: Seyyed Morteza Hoseini E-mail address: seyyedmorteza.hoseini@gmail.com (Dhot and Van Stappen, 2003). During nauplii production, some cysts might not hatch. Consumption of these cysts may cause gut obstruction in fish larvae, which should be considered. Decapsulation is a process in which the chorion layer is dissolved and removed. Decapsulated cysts could be used as a food source for fish larvae, although its application is limited compared to the nauplii. Decapsulated cysts have been used to rear larvae of freshwater catfish (Clarias gariepinus), common carp (Cyprinus carpio), and marine shrimp (Penaeus indicus and Penaeus monodon) and milkfish (Chanos chanos) (Verreth et al., 1987; Vanhaecke et al., 1990; Stael et al., 1995; Ribeiro and Jones, 1998; Sui, 2000). The decapsulated cysts offer a number of advantages over nauplii and non-decapsulated cysts, in larval production (Dhot and Van Stappen, 2003): 1. Use of decapsulated cysts instead of nauplii eliminates the need for labor and additional hatching-related facilities. 2. Cyst shells are not introduced into the culture tanks. Non-decapsulated cysts introduce shell to the 29 Hoseini et al./ Int. J. Aquat. Biol. (2013) (1) 28-32 rearing tanks. It is not easy to separate all shells. In addition, hatching rate is not always 100% and the remaining unhatched cysts may cause gut obstruction in larvae. 3. Decapsulated nauplii have higher energy content and individual weight (30–55% depending on strain) compared to nauplii hatched out of non-decapsulated cysts. Where cysts have relatively a low energy content, the hatchability could be improved by decapsulation as the larvae need a lower energy to break out of a decapsulated cyst. 4. Cysts would be completely disinfected by decapsulation. 5. Cysts with poor hatching quality or even non- hatching cysts can still be used as a food source. Decapsulation is normally performed by hypochlorite solution. The detailed protocol was described by Dhot and Van Stappen (2003). It would be of interest to investigate other solvent for decapsulation. Although cysts are washed following hypochlorite exposure, the risk of hypochlorite residual is not eliminated. Hypochlorite releases chlorine, which is highly toxic to fish (Brungs, 1973). The present study aimed to investigate the efficiency of sulfuric acid on decapsulation of Artemia cysts. The residual of sulfuric acid is sulfate which is a natural water anion. Materials and methods Hatching technique was based on Dhont and Van Stappen (2003). Briefly, cysts of A. franciscana were artificially hatched by adding 1 g cyst to 1 liter water (33 ppt). Cysts were incubated for 24 h. Temperature and light intensity was maintained at 28 ºC and 2000 lux, respectively. Continuous aeration was provided to ensure dissolved oxygen up to 5 ppm and suitable turbulence for cysts. Hatching rate was determined by counting and averaging the nauplii in three samples (0.1 ml) under stereoscopic loupe (Olympus, SZX7, Japan). Control cysts were hatched following the aforementioned method. The cysts were decapsulated using the method by Dhont and Van Stappen, 2003), cysts were allowed to hydrate for 1 h in clean aerating water. Then, cysts were added to hypochlorite (liquid bleach, NaOCl, 12%) or acid solution (1 and 5% sulfuric acid, Mihanjaz Co., Iran). Cysts were removed from hypochlorite solution after 5 min. The cysts were washed with clean water 5 times prior to incubation. In acid treatments, cysts remained 10, 30 and 50 min in both 1 and 5% acid solution. Thereafter, cysts were washed 5 times prior to incubation. Hatching rates were recorded at 15, 18 and 24 h after incubation. To compare hatching rates of acid treated cysts, data were analyzed using a split-plot design, which incubation time considered as the whole plot and acid immersion time and concentration as split plots Table 1. Analyses of variance of effect of acid concentration (%), acid immersion time (min) and incubation time (h) on hatching rate of Artemia cysts. Source Type III Sum of Squares df Mean Square F P value IT1 24298.7 2 12149.3 830 <0.0001 Error 87.7 6 14.6 354.8 AT2 4250.5 2 2125.2 35.2 <0.0001 IT × AT 844.35 4 211.1 10.3 <0.0001 CON3 61.65 1 61.65 0.25 0.003 IT × CON 2.96 2 1.48 59.5 0.486 AT × CON 712.65 2 356.32 2.24 <0.0001 IT × CON × AT 53.61 4 13.4 0.09 Error 179.69 30 5.99 Corrected total 30491.84 53 1. Incubation time, 2. Acid immersion time, 3. Acid concentration. 30 Hoseini et al./ Int. J. Aquat. Biol. (2013) (1) 28-32 on the incubation time. To compare hatching rate of acid-treated groups, control group and hypochlorite- treated group, data of 24 h incubation were analyzed using a one-way ANOVA. Duncan test was used as Post Hoc to determine significant difference among the treatments. P<0.05 was considered as significant difference. Data are presented as mean ± SE. Results Results showed that acid concentration, acid immersion time, incubation time, interaction between acid concentration × acid immersion time as well as acid immersion time × incubation time had significant effect on hatching rate (Table 1). Hatching rates of acid-treated cysts are shown in table 2. There was significant difference in hatching rate among 15, 18 and 24 h incubated cysts as hatching rate increased with increase of the incubation time (Fig. 1). Hatching rates increased with increase of the acid immersion time from 10 to 50 min (Fig. 2). Hatching rate of the cysts exposed to 5% acid was significantly higher than those exposed to 1% acid (Fig. 3). Comparison of hatching rates among acid- treated, hypochlorite-treated and control cysts are shown in Fig. 4. The hatching rates were in following order: 1% acid over 10 min < 5% acid over 10 min < control < 1% acid over 30 min and 5% acid over 30 min < 5% acid over 50 min < 1% acid over 50 min < hypochlorite. Table 2. Hatching rate (mean and SE) in different acid concentration × acid immersion time × incubation time combinations. 95% Confidence Interval CON (%) AT (min) IT (h) Mean SE Lower Bound Upper Bound 1 10 15 12.16 a 1.499218 9.126112 15.20722 18 25.90 bc 1.499218 22.85945 28.94055 24 51.23 f 1.499218 48.19278 54.27389 30 15 29.46 c 1.499218 26.42611 32.50722 18 43.43 e 1.499218 40.39278 46.47389 24 80.10 h 1.499218 77.05945 83.14055 50 15 34.16 d 1.499218 31.12611 37.20722 18 50.93 f 1.499218 47.89278 53.97389 24 92.40 j 1.499218 89.35945 95.44055 5 10 15 23.36 b 1.499218 20.32611 26.40722 18 29.90 cd 1.499218 26.85945 32.94055 24 59.36 g 1.499218 56.32611 62.40722 30 15 23.60 b 1.499218 20.55945 26.64055 18 40.16 e 1.499218 37.12611 43.20722 24 77.53 h 1.499218 74.49278 80.57389 50 15 22.56 b 1.499218 19.52611 25.60722 18 41.96 e 1.499218 38.92611 45.00722 24 84.76 i 1.499218 81.72611 87.80722 a b c 0 10 20 30 40 50 60 70 80 H a tc h in g r a te ( % ) Incubation time (h) Figure 1. Effect of incubation time on hatching rate of acid-treated cysts. Different letters above the bars show significant difference. P<0.05. SE=3.63.combinations. 31 Hoseini et al./ Int. J. Aquat. Biol. (2013) (1) 28-32 Discussion Hypochlorite oxidation is the common method for Artemia cysts decapsulation. However, if hypochlorite is not neutralized appropriately, it may damage the fish larvae. The present study showed that acid digestion could be used instead of hypochlorite oxidation for Artemia cyst decapsulation. This method would be safe, since residual of acid, if any, may only change the pH slightly, depending on the water alkalinity. However, 3-5 times washing would completely eliminate the risk of pH change. Previous studies showed that hatching rate of A. franciscana cysts were about 70 % (Bruggeman et al., 1980; Triantaphyllidis et al., 1994). In the present study, similar hatching rate was also obtained in non- decapsulated cysts. Data of this study showed that decapsulation has a significant impact on hatching rate of Artemia cysts. On the other hand, the present results showed that increase in acid immersion could speed up hatching process. This feature is important because this could be cost and labor saving in practice. Increase in acid concentration and acid immersion time cause increase in hatching rate. This could be due to increased shell digestion allowing nauplii to hatch out spending lees energy and over a shorter time. Similar results were reported in Artemia parthenogenetica decapsulated with hypochlorite solution over 2-5 min (Hosseini Najd Gerami and Agh, 2008). The best hatching rate (97%) was observed in hypochlorite-treated cysts. Although, hatching rate of the cysts treated with 1% acid over 50 min was significantly lower than hypochlorite-treated ones, it was still high (92.4%). However, all acid-treated cysts showed significantly higher hatching rate compared with control, except those exposed to acid over 10 min. This suggests that 10 min acid exposure is not suitable for decapsulation. More studies are needed to illustrate effects of acid-treatment on cysts in term of pH, shell thickness and embryo condition. It is concluded that acid-treatment could be a useful method for cysts decapsulation. There is no risk of hypochlorite residuals in this method. Decapsulation with acid cause higher hatching rate and shorter hatching time compared to control. Acid concentration and acid immersion time are important Figure 2. Effect of acid immersion time on hatching rate of acid- treated cysts. Different letters above the bars show significant difference. P<0.05. SE = 7.48. a b c 0 10 20 30 40 50 60 H a tc h in g r a te ( % ) Acid immersion time (min) a b 40 41 42 43 44 45 46 47 48 49 50 H a tc h in g r a te ( % ) Acid concentration (%) Figure 3. Effects of acid concentration on hatching rate of Artemia cysts. Different letters above the bars show significant difference. P<0.05. SE = 0.50 c g a d f b d e 0 20 40 60 80 100 120 C o n tro l H P C A -1 -1 0 A -1 -3 0 A -1 -5 0 A -5 -1 0 A -5 -3 0 A -5 -5 0 H a tc h in g r a te ( % ) Treatments Figure 4. Twenty four hours hatching rate of control, hypochlorite- treated (HPC) and acid-treated (A-1-10 = 1% acid over 10 min, A- 1-30 = 1% acid over 30 min, A-1-50 = 1% acid over 50 min, A-5- 10 = 5% acid over 10 min, A-5-30 = 5% acid over 30 min, A-5-50 = 5% acid over 50 min) cysts. Different letters above the bars show significant difference. P < 0.05. 32 Hoseini et al./ Int. J. Aquat. Biol. (2013) (1) 28-32 factors affecting hatching rate and time. The best hatching rate (92.4%) in acid-treated cysts was achieved when cysts were exposed to 1% acid over 50 min. References Bengston D. A. (2003). Status of marine aquaculture in relation to live prey: Past, present and future. In: J. G. Støttrup, and L. A. McEvoy (Eds.) Live Feeds in Marine Aquaculture, Wiley-Blackwell. pp: 1-16. Bruggeman E., Sorgeloos P., Vanhaecke P. (1980). Improvements in the decapsulation technique of Artemia cysts. In: The brine shrimp Artemia, Vol. 3. (Eds: Persoone, G., Sorgeloos, P., Roels, O., Jaspers, E.). University press, Western Belgium. 456 p. Brungs W.A. (1973). Effects of residual chlorine on aquatic life. Journal (Water Pollution Control Federation), 2180-2193. Dhot J., Van Stappen G. (2003). Biology, Tank Production and Nutritional Value of Artemia. In: J.G. Støttrup and L.A. McEvoy (Eds.) Live Feeds in Marine Aquaculture, Wiley-Blackwell. pp: 65- 112. García-Ortega A., Verreth J. A. J., Coutteau P., Segner H., Huisman E. A., Sorgeloos P. (1998). Biochemical and enzymatic characterization of decapsulated cysts and nauplii of the brine shrimp Artemia at different developmental stages. Aquaculture, 161: 501–514. Hosseini Najd Gerami E., Agh N. (2008). Optimization of decapsulation method for parthenogenetic Artemia, Artemia parthenogenetica originated from Urmia Lake. Pajouhesh and Sazandegi, 77: 42-47. Ribeiro F.A., Jones D.A. (1998). The potential of dried, low-hatch, decapsulated Artemia cysts for feeding prawn post-larvae. Aquaculture International, 6: 421-440. Stael M., Sanggontanagit T., Van Ballaer E., Puwapanich N., Tunsutapanich A., Lavens P. (1995). Decapsulated cysts and Artemia flakes as alternative food sources for the culture of Penaeus monodon postlarvae. In: P. Lavens, E. Jaspers, I. Roelants (Eds.) Larvi’ 95, Book of Abstracts. European Aquaculture Society, Special Publication No. 24, Ghent. pp: 342–345 Sui L. (2000). Use of Artemia biomass in practical diets and decapsulated cysts as food source for common carp (Cyprinus carpio L.). MSc Thesis, Ghent University, Ghent. Triantaphyllidis G.V., Pilla E.J., Thomas K.M., Abatzopoulos T.J., Beardmore J.A., Sorgeloos P. (1994). International Study on Artemia L II. Incubation of Artemia cyst samples at high temperature reveals mixed nature with Artemia franciscana cysts. Journal of Experimental Marine Biology and Ecology, 183: 273-282. Vanhaecke P., De Vrieze L., Tackaert W., Sorgeloos P. (1990). The use of decapsulated cysts of the brine shrimp Artemia as direct food for carp Cyprinus carpio L. larvae. Journal of World Aquaculture Society, 21: 257–262. Verreth J., Segner H., Storch V. (1987). A comparative study on the nutritional quality of decap-sulated Artemia cysts, micro-encapsulated egg diets and enriched dry feeds for Clarias gariepinus (Burchell) larvae. Aquaculture, 63: 269–282.