Artemia salina

Journal of Applied Aquaculture, 25:1–7, 2013 Copyright © Taylor & Francis Group, LLC ISSN: 1045-4438 print/1545-0805 online DOI: 10.1080/10454438.2013.817182

Encapsulated Spirulina Powder Feed for the Nutritional Enrichment of Adult Brine Shrimp (Artemia salina) P. ARUMUGAM1 , D. INBAKANDAN2 , M. S. RAMASAMY3 , and M. MURUGAN4 1

Centre for Advance Studies in Botany, School of Life Science, University of Madras, Guindy Campus, Chennai, India 2 Centre for Ocean Research, Sathyabama University, Chennai, Tamil Nadu, India 3 The AU-KBC Research Centre, Madras Institute of Technology, Anna University, Chennai, Tamil Nadu, India 4 Department of Microbial Technology, School of Biological Science, Madurai Kamaraj University, Madurai, Tamil Nadu, India

Liposomes were prepared in the laboratory with lipids obtained from chicken egg yolk to encapsulate Spirulina powder. Artemia napulii were hatched and fed with lipid-encapsulated Spirulina or Spirulina powder. After 20 days of rearing, the experimental A. salina were harvested and measured for total body length and total content of protein and carbohydrates. Total body length was 45% higher, protein content was 49% higher, and carbohydrate was 35% higher in Artemia fed with liposomes as compared with those fed only Spirulina powder.

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KEYWORDS Aquaculture, Artemia salina, enrichment, liposome, Spirulina

INTRODUCTION Artemia are widely utilized as partial or sole feed by over 85% of cultured marine animals (Wurtsbaugh and Gliwicz 2001; Teresita et al. 2005; Lakshmana Senthil et al. 2011). However, the nutritional quality of commercially available Artemia is relatively poor in terms of eicosapentaenoic and Address correspondence to Dr. P. Arumugam, Centre for Advance Studies in Botany, University of Madras, Guindy Campus, Chennai-25, Tamil Nadu, India. E-mail: [email protected] 1

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docosahexaenoic essential fatty acids (Hanaee et al. 2005). Bioencapsulation is used to enrich Artemia to make it suitable for marine species (Sorgeloos et al. 1998; Teresita et al. 2005; Mohebbi 2010; Khairy et al. 2012). Microalgae, e.g., Chlorella minutisima, Euglena gracilis, and Isochrysis galbana have been used for this purpose (Watanabe et al. 1978; Olsen et al. 1997). Liposomes are a type of microencapsulation that can hold enriching nutrients within their multilamellar phospholipid bilayers. Phospholipids such as lecithin from egg yolk are commonly used to prepare liposomes by reverse-phase evaporation and dehydration–rehydration (Szoka and Papahadjopoulos 1978; Matsuzaki et al. 1989, Kirby and Gregoriadis 1984; Nordgreen et al. 2007). The present study was designed to test liposome encapsulated Spirulina to enrich the Artemia.

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MATERIALS AND METHODS Certified organic Spirulina capsules (500 mg containing 20%–25% carbohydrates, 56%–69% protein, 5%–7% fat, 15%–19% phycocyanin, 0.15%–0.25% betacarotene, 1.20%–1.60% chlorophyll, and 0.25%–0.47% xanthophyll) were obtained from Parry Nutraceuticals, India. Artemia nauplii were obtained by hatching of EG grade Inve NV (Ghent, Belgium) cysts. Lipids for liposome production were extracted from chicken egg yolk. Lipid was extracted as describe by Folch et al. (1957). Egg yolk was separated manually from the white and homogenized with chloroform and methanol (2:1) to a final volume of 20 times the volume of yolk (i.e., 1 g in 20 ml solvent mixture). After dispersion, the whole mixture was agitated for 15–20 min in an orbital shaker at 28◦ C. The homogenate was centrifuged to recover the liquid phase. The solvent was washed with 1/5th volume of 0.9% sodium chloride solution and vortexed for 10 seconds. After vortex, the contents were centrifuged at 2,000 rpm and organic phase containing lipids separated and concentrated with a rotary vacuum evaporator. Liposomes were prepared using the dehydration–rehydration method described by Shimizu et al. (1993) as modified by Chang et al. (2002). Extracted lipid (100 mg) was mixed well with 20 ml of chloroform containing 40 mg of cholesterol. The mixture was concentrated using a vacuum evaporator. Concentrated lipids and Spirulina powder (10 mg) were added into 10 ml of phosphate buffered saline and sonicated for 10 min at 30◦ C until a uniform emulsion formed. A drop of solution was examined under the microscope (100x and 20x) to confirm liposome formation (Figure 1). Artemia napulii were hatched in the laboratory and fed with one of two randomly assigned test diets: 1) lipid encapsulated Spirulina and 2) Spirulina powder alone. Enrichment was carried out by placing nauplii at an approximate density of 100 nauplii per liter in triplicated glass bowls (3 bowls per treatment) containing filtered seawater (33 ppt) at a temperature

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Nutritional Enrichment of Adult Brine Shrimp

Liposomes at 20X

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Liposomes at 100X

FIGURE 1 Microscopic images of liposomes. (Color figure available online.)

of 28◦ C with strong aeration and light. Experimental animals were fed twice a day with either 5–10 mg of liposome or 5–10 mg of Spirulina powder. Nauplii were allowed to grow for 20 days and then harvested. Nutritional enrichment was assessed by estimating total Artemia body length (average of 10 individuals each from the three replicates), total protein, and carbohydrate content (each the average of 10 individuals pooled from each replicate). Protein was estimated according to the methods of Peterson (1977). Lipid-encapsulated Spirulina and Spirulina fed Artemia salina were harvested and 500 mg of each were homogenized with 10 ml phosphate buffer saline solution. One ml of the stock solution was made up to 5 ml with distilled water, of which 200 µl was taken into a test tube to which was added 2 ml of alkaline copper sulphate. The resulting solution was incubated at 28◦ C for 10 min. Then, 0.2 ml of Folin Ciocalteau reagent (2 ml commercial reagent + 2 ml water) was added and incubated for another 30 minutes. Optical density was read at 595 nm in a Beckman (DU-20) spectrophotometer. Carbohydrate estimation was carried out using the dinitrosalicylic acid (DNSA) method (Lindsay 1973). One hundred µl of the stock solution described above was added to 1 ml of DNSA and made up to 5 ml using distilled water and heated at 90◦ C for 10 min to develop a red brown color. The color was stabilized by adding 1 ml of 40% potassium sodium tartarate. After cooling to room temperature, absorbance was read at 540 nm. Results were presented as mean ± standard deviation for three triplicates of experimental and control. Statistical analyses were performed by oneway ANOVA using SPSS Software Version 12.0. Values of P ≤ 0.05 were considered to be significant.

RESULTS AND DISCUSSION Average growth of Artemia salina was 1.6 ± 0.07 cm in Artemia fed with liposome and 1.1 ± 06 cm in Artemia fed with Spirulina powder, a

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statistically significant 45% improvement in experimental animals fed with liposome compared to those animals fed with only Spirulina (Figures 2 100 and 3). Enhanced growth rate was probably the result of a combination of improved enhancement and increased lipid in the diet as a result of the liposome itself. Average protein content of Artemia salina was 360 ± 5.55 µg/g in animals fed with liposomes and 242 ± 7.00 µg/g in animals fed with Spirulina 105 powder, a 49% increase of protein in experimental animals fed with liposome over the animals fed with Spirulina (Figure 4). Average carbohydrate content of Artemia salina was 350 ± 12.31 µg/g when fed with liposomes and

FIGURE 2 Graphical representation of the growth rate by measuring the total body length of Artemia salina fed with liposome and Spirulina powder.

FIGURE 3 Graphical representation of the protein values of Artemia salina fed with liposome and Spirulina powder.


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FIGURE 4 Graphical representation of the Carbohydrate values of Artemia salina fed with liposome and Spirulina powder.

258 ± 7.16 µg/g when fed with straight Spirulina powder. Carbohydrate levels were 35% higher in experimental animals fed with liposomes compared 110 to the animals fed with Spirulina. Enhanced protein and carbohydrate in treated animals may have been the result of improved nutrient uptake from liposomes. This relatively simple method for liposome production using chicken egg yolk lipids represents a practical means for marine animal culturists to 115 improve the quality of their larval diets.

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