Highly Unsaturated Fatty Acid (HUFA) Retention Quality in Freshwater Cladoceran, Moina macrocopa, Enriched With Lipid Emulsions

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1 The Israeli Journal of Aquaculture - Bamidgeh, IJA_ The IJA appears exclusively as a peer-reviewed on-line Open Access journal at Sale of IJA papers is strictly forbidden. Highly Unsaturated Fatty Acid (HUFA) Retention Quality in Freshwater Cladoceran, Moina macrocopa, Enriched With Lipid Emulsions Jiun Yan Loh 1,2, Han Kiat Alan Ong 3, Yii Siang Hii 4, Thomas J. Smith 5, Malcolm W. Lock 5, Gideon Khoo 2 * 1 Faculty of Science & Biotechnology, Universiti Industri Selangor, Jalan Timur Tambahan, Bestari Jaya, Selangor, Malaysia 2 Faculty of Science, Universiti Tunku Abdul Rahman, Jalan Universiti, Bandar Barat, Kampar, Perak, Malaysia 3 Faculty of Medicine & Health Sciences, Universiti Tunku Abdul Rahman, Bandar Sungai Long, Cheras, Kajang, Selangor, Malaysia 4 Faculty of Agro-technology & Food Science, Universiti Malaysia Terengganu, Kuala Terengganu, Terengganu, Malaysia 5 Biomedical Research Centre, Sheffield Hallam University, Sheffield, United Kingdom (Received, Accepted) Key words: arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, Moina macrocopa, enrichment Abstract Enrichment time, treatment concentration and type of lipid emulsion were investigated in this study to elucidate enrichment capability of the freshwater live food, Moina macrocopa. Fatty acid profile was reviewed by comparing the fatty acids content of M. macrocopa enriched with animal and plant source lipids coupled with a commercial enrichment diet. In this study, arachidonic acid (AA) level of M. macrocopa was significantly increased by A1 DHA Selco at 1 g/l to 0.28 mg/g BW (body weight) from that less than 0.01 mg/g BW of *Corresponding author. gideonkhoo@utar.edu.my

2 2 Khoo et al. the control organism. Similarly, the use of canola oil also increased AA level to 0.38 mg/g BW at 2 g/l with both enrichments for 12 h. Results showed that A1 DHA Selco was comparatively more effective in docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) enhancement. Enriching M. macrocopa with 1 g/l A1 DHA Selco for 12 h markedly improved the levels of EPA and DHA to 0.38 mg/g BW and 0.09 mg/g BW, respectively. Our data showed that AA/EPA ratio of enriched M. macrocopa was highest when treated with 2 g/l canola oil at 12 h and squid oil at 24 h. AA/DHA and DHA/EPA ratios of M. macrocopa were highest when enriched with 1 g/l canola oil at 24 h. Statistical analysis indicated that treatment concentration had a more predominant effect on the fatty acid content of M. macrocopa compared with enrichment time. AA, EPA and DHA levels of M. macrocopa increased substantially with increasing treatment concentration. The notable exceptions were AA level for A1 DHA Selco and DHA level for canola oil. In short, DHA level was improved by 8% with 2 g/l A1 DHA Selco at 24 h enrichment. The EPA level was raised 253.3% using 1 g/l A1 DHA Selco at 12 h, and the AA level to 38% with 2 g/l canola oil at 12 h. The AA/EPA and AA/DHA ratios could be significantly improved with 2 g/l canola oil, whilst the DHA/EPA ratio was enhanced by 2 g/l squid oil when M. macrocopa was enriched with canola or squid oil for 12 h. This study suggests that cheaper lipid sources (squid and canola oils) may competitively enhance different groups of fatty acids and improve the n-3/n-6 lipid ratios when compared to commercial enrichment diets. Introduction Artemia nauplius is the most widely used live food species for fish larval feeding, and in nutrient or drug bio-encapsulated purposes (Sargent et al., 1997; Sargent et al., 1999b). However, this live food involves some operational constraints such as the high costs of importing the product in many countries. A major drawback of feeding these saltwater live foods to freshwater cultured species is that the Artemia nauplii usually die in freshwater within or after an hour, and therefore intermittent feeding to the fish larvae is necessary (Merchie, 1996). To overcome these problems, freshwater zooplankton should be developed, e.g. Moina sp., specifically for larval feeding of freshwater fish and crustaceans (Alam et al., 1993). Moina macrocopa is one of the most abundant zooplankton that inhabits aquatic ecosystems, e.g., paddy fields, ponds, swamps, rivers and mangrove regions in South East Asia. These Cladocerans are rich in protein and other nutrients, making them superior live food for fish and prawn larvae compared to other live foods such as Artemia (Alam et al., 1993). However, the fatty acid contents of Moina are variable when they are cultured with different media (Watanabe and Kiron, 1994). Like Artemia, Moina do not meet the HUFAs (highly unsaturated fatty acids) requirements for fish or crustacean larvae in terms of fatty acid deficiencies (He et al., 2001). It is thus important to improve their nutritional quality by enriching them with HUFAs prior to feeding to fish larvae. Fatty acids play a major role as an energy source and may influence cellular membrane functions which are, in turn, vital for cell growth, differentiation, and metabolism (Sargent et al., 1999a; Cahu et al., 2003; Tocher, 2003). Increasing the levels of dietary HUFAs in fish larvae helps to increase stress resistance against starvation or osmotic shock (Koven et al., 2003), assists in skeletal formation (Cahu et al., 2003), aids the development of neural and visual systems (Sargent et al., 1997, 1999b), improves survival rate (Watanabe, 1993), and enhances the pigmentation of fish larvae (Bransden et al., 2005). In many hatcheries, the common practice of enriching fatty acid composition in Artemia and other live foods such as rotifers comprised microalgae, microalgae paste and marine fish oils (Papandroulakis et al., 2002). Improvement of fatty acid contents, particularly essential HUFAs in Artemia nauplii, has been achieved in many finfish and crustacean hatcheries and laboratories (Alam et al., 1993; Sargent et al., 1997, 1999b).

3 Unsaturated Fatty Acid (HUFA) Retention Quality in Freshwater Cladoceran, Moina macrocopa 3 Some studies have found that the nutritional value of Moina could be similarly enhanced by enrichment with various culture media as demonstrated in Artemia nauplii and rotifers (Watanabe et al., 1993; Das et al., 2007). There is, however, limited information and no significant study has been conducted on M. macrocopa fatty acid enrichment, especially on selection of culture media, enrichment concentration and appropriate treatment time. Many HUFAs, especially AA, DHA and EPA, are necessary for the physiological development of fish and crustaceans (Watanabe, 1993). Fatty acid ratios such as n-6/n- 3, n-3/n-3 and n-3/n-6 are of considerable importance for determining the effects of HUFAs fed to fish (Sargent et al., 1999a; Corraze, 2001), because fatty acid ratios would influence the production of hormone-like substances that are critical in a very wide range of physiological processes in aquatic biota (Logue et al., 2000; Lund et al., 2007). To improve the specific lipid contents in live food, fatty acid retention time and biosynthesis of fatty acids in the gut are required to formulate the enrichment protocol. The present study was therefore conducted to determine the HUFA retention quality and HUFAs ratio improvement of M. macrocopa fed with lipid emulsions of squid oil and canola oil, and to compare these results with a commercial enrichment diet, A1 DHA Selco. The effects of dietary types, treatment concentration and enrichment time on the fatty acid profile of M. macrocopa enriched with different lipid emulsions were investigated in this study. Materials and Methods Preparation of diets. Canola oil/co (Sime Darby Edible Product Ltd., Singapore), squid oil/so (Asia Star Laboratory Ltd., Thailand) and commercial formulated emulsion, A1 DHA Selco /ADS (INVE Ltd., Belgium) were selected for this experiment. ADS served as a reference diet. All treatment diets were prepared at the concentrations of 1.0 and 2.0 g/l. The concentration of 1.0 g/l used in this study was based on the standard enrichment protocol for ADS commercial enrichment diet, whilst 2.0 g/l was selected to determine if doubling the enrichment concentration would significantly increase the HUFA levels in M. macrocopa as compared to 1.0 g/l. Oil emulsions of CO and SO were prepared based on methods modified from Estévez et al (1998), and Agh and Sorgeloos (2005). Soybean-extracted emulsifier, L- phosphatidylcholine (Sigma-Aldrich TM, USA) was added to CO and SO at a ratio of 1:4 (w:w). The mixtures were blended vigorously with de-chlorinated water at o C using an electric blender (Waring Commercial, USA) at maximum speed for 3 mins. ADS emulsion was prepared according to the manufacturer s instructions, where the ADS emulsion was weighed and subsequently mixed with de-chlorinated water (at ambient temperature) to the required concentrations for the enrichment experiment. Enrichment experiments. An 80 L plastic barrel was filled with tap water, which was de-chlorinated by strong aeration for several days prior to use. Wild-type M. macrocopa were obtained from oxidation ponds located in residential areas within Kuala Lumpur, Malaysia. M. macrocopa were acclimatized in the barrel overnight before the experiments commenced. Mild aeration was provided during the acclimatization period to maintain the dissolved oxygen level at 4 ppm. Only actively moving Moina individuals were selected for the experiments. During the selection, aeration was turned off to reduce the water current in the tank. Moina was collected with a fine sieve near the tank s edge. All Moina adults were concentrated into a glass beaker for quantification. For sample counting, 40 ml of test organisms was transferred using a syringe onto a Petri dish, and counting was performed under a dissecting microscope. Approximately 300 individuals per ml of Moina were added into different treatment tanks for enrichment (1 L of treatment diet per 1.5 L aquarium tank). Mild aeration was provided during the experiment, where dissolved oxygen was measured at 5.0 ± 0.1 ppm. The average water temperature was 26 to 30 o C, and the ph ranged from 7.2 to 8.1. The experiments were conducted at natural photoperiod (12 h light : 12 h dark). Since M. macrocopa obtained from natural habitats were found to vary greatly in their HUFA levels depending on the food sources in their habitats (unpublished data), fasting was conducted for 12 h before enrichment experiments so that the HUFA

4 4 Khoo et al. levels were uniform and could be standardized across all un-enriched Moina samples. Enriched Moina were collected after 12, 24 and 36 hours of incubation in the respective experimental diets. Samples were rinsed thoroughly with distilled water to remove excess oil emulsions prior to storage in 1.5 ml tubes at -20 o C for further analysis. Lipid extraction and fatty acid determination. Fatty acid methyl ester (FAME) of M. macrocopa was performed according to a method modified from AOAC (1995). Qualitative and quantitative analyses by gas chromatography were conducted by a certified commercial analytical food chemistry laboratory (Chemical Laboratory Sdn. Bhd., Malaysia). Statistical analysis. All the fatty acid data shown are the means of four data sets, while the experimental diets were in two data sets. The data was tested for normality and homogeneity-of-variance, and analyzed by 2-way ANOVA (with Tukey s test) to determine the effects of diet levels (1.0 and 2.0 g/l), enrichment period (t 12, t 24 and t 36 ) and the interaction of both these factors. A 95% significance level (P < 0.05) was used throughout the data analysis. All statistical analysis was performed using Minitab ver. 13. Results The fatty acid contents varied among the groups of un-enriched and enriched Moina macrocopa. Table 1 shows the fatty acid profile of un-enriched M. macrocopa (12 h) and the various lipid emulsions that were used as negative controls in the study. Tables 2 & 3 show the fatty acid compositions of M. macrocopa after enrichment with various types of oil emulsion, at different incubation times and concentrations. The initial levels of linoleic acid (18:2n-6), and linolenic acid (18:3n-3) in M. macrocopa after 12 h starvation were 0.23 and 0.14 mg/g BW (body weight), respectively. Linoleic acid increased to a maximum level of 0.50 mg/g BW when M. macrocopa had been fed with 1 g/l SO diet for 12 h, and 0.40 mg/g BW with 2 g/l CO diet at 36 h (Tables 2 & 3). The detected linolenic acid levels increased as the SO or CO concentration was raised to 2 g/l, yielding a 0.70 mg/g BW for SO and 0.86 mg/g BW for CO after 12 h enrichment (Table 3). The concentrations of essential fatty acids such as arachidonic acid (AA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) were found to be low in un-enriched M. macrocopa (Table 1). With the addition of 1 g/l ADS (Table 2) after 12 h, the level of AA was elevated to 0.28 mg/g BW. AA levels were raised to 0.38 mg/g BW by the addition of 2 g/l CO after a 12 h enrichment period (Table 3). The level of EPA was significantly (P < 0.05) increased to 0.38 mg/g BW through enrichment with 1 g/l ADS for 12 h. Enrichment with ADS showed promising results with regards to the fatty acid DHA. The levels of DHA (22:6n-3) increased to 0.09 mg/g BW at 1 g/l and 0.08 mg/g BW at 2 g/l for ADS enrichment after 12 and 24 h, respectively (Tables 2 & 3), while trace levels were undetectable in the un-enriched cultures (Table 1). Figures 1 and 2 show the fatty acid enrichment kinetics of M. macrocopa (in ratio per body weight in grams) following enrichment with the three types of oil emulsions under varying conditions. The AA/EPA ratios of M. macrocopa enriched with CO (46.5) and SO (14.0), each at 1 g/l, were maximum after 12 h of enrichment (Fig. 1a) but they subsequently decreased as enrichment time was extended. When the concentration of the oil component of the diet was increased to 2 g/l, the AA/EPA ratios of M. macrocopa enriched with CO were noticeably elevated to the highest level (2476.3) at 12 h enrichment. When SO was added at the same concentration, the AA/EPA ratio of M. macrocopa was maximum (167.2) at 24 h (Fig. 2a). With the treatment diet containing CO at 1 g/l, the AA/DHA and DHA/EPA ratios showed very similar trends, i.e., reaching the maximum after 24 h enrichment (Figs. 1b & 1c). This trend changed at the treatment concentration of 2 g/l, where AA/DHA ratio of M. macrocopa fed with CO was highest after 12 h enrichment (856.3) and decreased significantly (P < 0.001) after 24 h (Fig. 2b). The DHA/EPA ratio of M. macrocopa enriched with 1 g/l of CO was the highest (16.0) at 24 h (Fig. 1c), but it did not show a consistent trend where DHA/EPA ratio of M. macrocopa fed with SO was the highest with 2 g/l enrichment at 12 h (210.0) (Fig. 2c).

5 Unsaturated Fatty Acid (HUFA) Retention Quality in Freshwater Cladoceran, Moina macrocopa 5 Table 4 shows the F-values and P-values of the two-way ANOVA that was used to analyze the effects of concentration, time and the interaction of these two factors on the M. macrocopa fatty acid composition. Diet concentration was shown to be the predominant factor in comparison with enrichment time. AA, EPA and DHA levels increased significantly (P-value ranged from < to 0.027) when treatment concentrations were raised. One notable exception was the AA level for ADS enrichment (P = 0.292), in which low measured levels of this fatty acid was noted in M. macrocopa during enrichment (Table 4). The ratios of n-6 to n-3 HUFAs were significantly affected by the enrichment time in ADS. Positive trends were also observed for the AA/DHA ratios with both SO and CO enrichments. Continued enrichment using SO and CO for more than 24 h with either treatment concentration did not alter the EPA and DHA levels or the AA/EPA and DHA/EPA ratios. Enrichment with ADS gave the highest overall EPA and DHA contents, showing significant interactions (P < 0.05) between treatment concentration and enrichment time (Table 4). Discussion Live food enrichment with oil emulsions is a common practice in fish and crustacean hatcheries. Various commercial enrichment products have been formulated for this purpose. Although enrichment methodologies have been standardized and commercially adopted for Artemia nauplii and rotifers (Agh and Sorgeloos, 2005), enrichment protocols have not been clearly identified for use with the freshwater cladoceran, Moina macrocopa. Lipids play crucial roles in physiological and biological functions of living organisms (Sargent et al., 1997, 1999b). Marine and freshwater fish species require n-3 and n-6 fatty acids in their daily diet, especially during the early stages of development as lipids are the main source of energy at the gastrula stage of fish embryo development (Vetter et al., 1983). Inadequate lipid content in cultured fish diets can adversely affect the performance of larvae during the grow-out stage, since larvae often have low energy reserves and require substantial energy sources for their high somatic growth rates and development of their bodies (Fraser et al., 1988). Lipids contain 2.5 times the energy compared to carbohydrates or proteins per consumed weight (Parker, 2002). In addition, lipids which are the energy source for cellular housekeeping functions, are required for hormone synthesis, and serve as carriers for fat soluble vitamins (Parker, 2002). In the present study, the data clearly indicate that lipid levels in M. macrocopa can be improved through changes to the enrichment process, in a manner similar to that of Artemia nauplii (Das et al., 2007). Enrichment diets and conditions (initial stage of neonate/nauplius, and duration and dose of the enrichment) are the most obvious factors known to influence the eventual enrichment result (Léger et al., 1987). In this study, enrichment time, concentration and different types of emulsified lipids were investigated to elucidate the lipid profile of M. macrocopa. The treatment concentration had a more predominant effect on the fatty acid content of M. macrocopa when compared with enrichment time. Essential fatty acids such as arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are desirable components of live foods, are naturally low in un-enriched Moina. A study by Piedecausa et al. (2007) demonstrated that a fish species, Diplodus puntazzo, which was fed with soybean and linseed oil emulsions, produced only low levels of AA in the muscles. The canola oil used in this study increased the AA levels in the enriched Moina for 12 h to 0.38 mg/g at 2 g/l (Table 3). AA is an essential dietary fatty acid which has been measured in relatively high amounts in the neural and visual tissues of fish (Tocher and Harvie, 1988). This fatty acid is the main precursor to hormone-like compounds, e.g., eicosanoids, which are important in the development of neural tissues and the immune system, specifically for signal transduction (Sargent et al., 1999a) and inflammatory responses (Tocher, 2003). The DHA levels in un-enriched M. macrocopa (< 0.01 mg/g) indicated that this zooplankton is naturally deficient in this fatty acid. EPA levels were, however, higher (0.15 mg/g) than DHA in un-enriched M. macrocopa (Table 1), as was reported in Moina micrura by Das et al (2007). It has been hypothesized that Moina sp. cannot synthesize

6 6 Khoo et al. DHA de novo and metabolize PUFA as rapidly as Artemia (Estévez et al., 1998). The elevation of DHA will generally increase the level of EPA in the organisms, as shown by the marked retroconversion of DHA to EPA that takes place during enrichment (Navarro et al., 1999). This retroconversion occurred in Moina enriched with 1 and 2 g/l of ADS, although this zooplankton took a longer time to synthesize and metabolize the DHA from ADS (Tables 2 & 3). The DHA content in Artemia nauplii increased approximately two fold from 12 h to 24 h of the enrichment period (Han et al., 2000). Although DHA levels did not increase greatly over the enrichment time, the DHA levels from the three experimental diets were significantly higher than in the un-enriched Moina (range of mg/g) (Tables 1 3). Since DHA is important for vision and membrane fluidity functions (Koven, 2003), insufficient levels of this essential fatty acid in fish larval diets is likely to impair the neural and visual development (Sargent et al., 1999a). The EPA values of Moina dropped markedly after the enrichment period except with 1 g/l ADS. During the duration of the supplementation, it was anticipated that EPA would be metabolized to eicosanoids particularly 3-series prostanoids and 5-series leukotrienes (Sargent et al., 1999a). The levels of EPA were not increased by SO and CO in this study. Highly unsaturated fatty acids (HUFAs) are important for the survival, growth and pigmentation of almost all fish and crustacean larvae (Watanabe, 1993). Nevertheless, the effects of HUFAs cannot be determined solely on the absolute amount of a particular HUFA that is fed to the larvae. The absolute requirement for any given HUFA in fish is proportional to the absolute amounts of other HUFAs in the diet due to the competitive inhibition by fatty acids, which could affect the quality of the provided HUFAs (Sargent et al., 1999a; Corraze, 2001). AA and EPA compete for binding to the cycloxygenase and lipoxygenase enzymes (Hamre et al., 2005). Such competitions can result in biological imbalances, which ultimately affect the cell membrane receptors (Sargent et al., 1999a). Within the fish tissues, EPA and DHA bind to these cycloxygenase and lipoxygenase, and subsequently determine the ratio of n-2 and n-3 eicosanoids in the muscles of fish larvae. These two series of eicosanoids cause different degrees of physiological changes (Logue et al., 2000), such as the malpigmentation of fish due to excessive production of eicosanoids which induces biochemical stress for the fish (Sargent et al., 1999b). It has been suggested that increasing the ratio of n-3/n-6 could reduce or inhibit the synthesis of AA-derived prostaglandins, and consequently influence the rate and type of eicosanoids produced (Lund et al., 2007). The results of this study indicate that lipid ratio is strongly influenced by the lipid ratio of the live foods given to the targeted species. The commercial enrichment diet, ADS, was found to be superior in enhancing DHA levels. Nonetheless, it does not efficiently improve n-3/n-6 or n-6/n-3 ratios as shown by canola oil and squid oil in the present study (Figs. 1 & 2). For a better enrichment process, AA/EPA and AA/DHA ratios were comparatively more effective when M. macrocopa was enriched with 2 g/l CO for 12 h and 24 h, respectively. The malpigmentation of some species, e.g., turbot could be mitigated if the n-6/n-3 ratio is increased in the diet during larval feeding (Reitan et al., 1994). In our study, the DHA/EPA ratio was found to increase when M. macrocopa was enriched with 2 g/l SO at 12 h. A study on the megalopa stage of the Chinese mitten crab, Eriocheir sinensis, showed that it could tolerate a greater range of salinity fluctuations when the diet was supplemented with DHA/EPA (Sui et al., 2007). Increasing the ratios of HUFAs could also reduce solidification of the total bio-membrane lipid pool, which acts as an anti-freeze mechanism for many aquatic poikilotherms in order to protect them from cold water temperatures (Brett and Mϋller-Navarra, 1997). The bio-encapsulated lipid ratios in M. macrocopa tissues during enrichment indicated a positive trend with the levels measured in the experimental diets. Other fatty acids such as linoleic acid/la (18:2n-6) and linolenic acid/lna (18:3n-3) are also relatively important for larval performance. The LA levels showed better improvement (0.50 mg/g) when M. macrocopa was enriched with 1 g/l SO at 12 h (Table 2) and LnA level could be improved further (0.86 mg/g) by using 2 g/l CO at 12 h (Table 3). LA is a metabolic precursor of -linolenic acid (GLA) to AA. It functions to prevent abnormal coloration in freshwater fish, while LnA could be elongated to DHA via EPA catabolism (Tocher, 2003).

7 Unsaturated Fatty Acid (HUFA) Retention Quality in Freshwater Cladoceran, Moina macrocopa 7 As demonstrated in this study, LnA-enriched M. macrocopa could be an alternative source of DHA and EPA for freshwater fish larvae. In conclusion, this study suggests that the fatty acid content in M. macrocopa could be improved with lipid emulsions. Animal- and plant-based oil emulsions, such as squid and canola oils, possess a competitive advantage for enhancing the distribution of different fatty acids as well as increasing the n-3/n-6 lipid ratios of this zooplankton. The oil emulsions are also comparatively cheaper than the commercial enrichment diet A1 DHA Selco, thus making oil emulsions a viable alternative for the enrichment of M. macrocopa. Acknowledgements This research was funded by the UTAR postgraduate research fund (6202/L10). The authors wish to thank Assoc. Prof. Dr Chuah Tse Seng of Universiti Malaysia Terengganu for his invaluable advice on statistical analysis, and Mr. Rodgers for the manuscript preparation. This work was also partially supported via a PMI-2 grant from the British Council (to TJS and HKAO). References Agh, N. and P. Sorgeloos, Handbook of Protocols and Guidelines for Culture and Enrichment of Live Food for Use in Larviculture. Artemia and Aquatic Animals Research Center, Urmia Univ., Iran. 60 pp. Alam, M.J., Ang, K.J. and S.H. Cheah, Weaning of Macrobrachium rosenbergii larvae from Artemia to Moina micrura. Aquaculture, 112: AOAC, Official Methods of Analysis of AOAC International, Vol. 1, 16th ed. Association of Official Analytical Chemists, Arlington, Virginia, USA pp. Bransden, M.P., Butterfield, G.M., Walden, J., McEvoy, L.A. and J.G. Bell, Tank colour and dietary arachidonic acid affects pigmentation, eicosanoid production and tissue fatty acid profile of larval Atlantic cod (Gadus morhua). Aquaculture, 250: Brett, M.T. and D.C. Müller-Navarra, The role of highly unsaturated fatty acids in aquatic foodweb processes. Freshw. Biol., 38: Cahu, C., Zambonino Infante, J. and T. Takeuchi, Nutritional components affecting skeletal development in fish larvae. Aquaculture, 227: Corraze, G Lipid nutrition. pp In: Guillaume, J., Kaushik, S., Bergot, P. and Metailler, R. (Eds.). Nutrition and Feeding of Fish and Crustaceans. Springer-Praxis, UK. 408 pp. Das, S.K., Tiwari, V.K., Venkateshwarlu, G., Reddy, A.K., Parhi, J., Sarma, P. and J.K. Chettri, Growth, survival and fatty acid composition of Macrobrachium rosenbergii (de Man, 1879) post larvae fed HUFA-enriched Moina micrura. Aquaculture, 269: Estévez, A., McEvoy, L.A., Bell, J.G. and J.R. Sargent, Effects of temperature and starvation time on the pattern and rate of loss of essential fatty acids in Artemia nauplii previously enriched using arachidonic acid and eicosapentaenoic acid-rich emulsions. Aquaculture, 165: Fraser, A.J., Gamble, J.C. and J.R. Sargent, Changes in lipid content, lipid class composition and fatty acid composition of developing eggs and unfed larvae of cod (Gadus morhua). Mar. Biol., 99: Hamre, K., Moren, M., Solbakken, J., Opstad, I. and K. Pittman, The impact of nutrition on metamorphosis in Atlantic halibut (Hippoglossus hippoglossus L.). Aquaculture, 250: Han, K.M., Geurden, I. and P. Sorgeloos, Enrichment strategies for Artemia using emulsions providing different levels of n-3 highly unsaturated fatty acids. Aquaculture, 183: He, Z.H., Qin, J.G., Wang, Y., Jiang, H. and Z. Wen, Biology of Moina mongolia (Moinidae, Cladocera) and perspective as live food for marine fish larvae: review. Hydrobiologia, 457:25-37.

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9 Unsaturated Fatty Acid (HUFA) Retention Quality in Freshwater Cladoceran, Moina macrocopa 9 Table 1. Fatty acid composition of un-enriched Moina macrocopa at 12 h and the respective oil emulsified diets. Un-enriched Oil emulsions Fatty acid Moina ADS SO CO 12:0 0.01±< :0 0.02±< ±< ±< :1 0.01±< ±< ±< :0 0.10±< ±< ±< ±< :0 0.04±< ±< ±< :0 0.40± ±< ±< :0 0.31± ±< ±< ±< :1n ± ±< ±< ±< :2n ± ±< ±< :0 0.03±< ±< ±< :3cis ± ±< ±< :3n ± ±< ±< :0 0.01±< ±< ±< ±< :3n ±< ±< :3n ±< ±< ±< :4n ±< :5n ± ±< ±< :6n ±< ±< ±<0.01 SAFA ± ± ± ±0.14 MUFA ± ± ± ±0.15 PUFA ± ± ± ±0.28 n-3 PUFA 90.14± ±< ± ±0.25 n-6 PUFA 1.51± ± ± ±0.11 AA/EPA 0.41± ± ± ±0.01 AA/DHA 3.13± ± ± ±0.02 DHA/EPA 0.66± ±< ± ±0.01 Values are given as mean SEM with M. macrocopa (control) in 4 data sets and experimental data in 2 data sets. Dash ( ) indicates that the fatty acid content was non-detectable or < Oil emulsions, sources and abbreviations are as shown below: ADS = A1 DHA Selco (INVE, Belgium) SO = squid oil (Asia Star Lab. Ltd., Thailand) CO = canola oil (Sime Darby Edible Product Ltd., Singapore) SAFA = total saturated fatty acids MUFA = total monounsaturated fatty acids PUFA = total polyunsaturated fatty acids

10 10 Khoo et al. Table 2. Fatty acid composition (mg/g body weight) of Moina macrocopa enriched with 1 g/l ADS, SO and CO over three enrichment periods. ADS SO Fatty acid 12 h 24 h 36 h 12 h 24 h 36 h 12:0 0.03±< ±< ±< :0 0.02±< ±< ±< ±< :1 0.01±<0.01 b a ab 15:0 1.36±0.12 a 0.11±<0.01 b 0.44±0.05 ab 0.01±< :0 0.36±0.03 b 0.02±<0.01 a 0.05±<0.01 a 0.01±< :0 8.50±0.70 b 1.36±0.14 a 1.78±0.16 a 0.05± ±< :0 1.37±0.12 b 0.05±<0.01 a 0.23±0.03 a 0.07± ±< :1n ± ± ± ±0.03 e 0.45±0.01 e 0.02±<0.01 d 18:2n ± ± ± ± ±< :0 0.20± ± ±< ±< ± :3cis ±0.01 b 0.10±<0.01 a 0.06±<0.01 a 0.05±< ±< :3n ±< ±< ±< ±< :0 0.08±0.01 b a 0.02±<0.01 a 0.01±< ±< :3n ± ± ± ± ±< :3n ±< ±< :4n ±0.01 b 0.03±<0.01 a 0.04±<0.01 a 0.01±< ±< :5n ±0.02 b 0.03±<0.01 a 0.05±<0.01 a 0.02±< ±< :6n ±0.01 b a a SAFA ±2.99 ±30.59 ±22.91 ±10.09 d ±19.78 e ±3.68 e MUFA ±8.80 a ±38.69 ab ±26.06 b ±19.01 e ±19.77 d ±2.87 d PUFA ±11.67 b ±8.15 ab ±3.16 a ±9.13 ±0.22 ±0.81 n-3 PUFA ±1.86 b ±0.20 a ±0.24 a ±0.59 ±0.09 ±0.16 n-6 PUFA ±0.39 ±2.48 ±0.95 ±1.42 ±0.07 ±0.33 AA/EPA 7.35± ± ± ± ± ±<0.01 AA/DHA 4.27± ±1.41 DHA/EPA 4.23± ±0.25 Values are given as mean SEM (n = 4). Superscripts indicate significant variation (p < 0.05) within the enrichment periods for the individual diet. Dash ( ) indicates that the fatty acid content was < 0.01.

11 Unsaturated Fatty Acid (HUFA) Retention Quality in Freshwater Cladoceran, Moina macrocopa 11 Table 2 (continuation). Fatty acid composition (mg/g body weight) of Moina macrocopa enriched with 1 g/l ADS, SO and CO over three enrichment periods. CO Fatty acid 12 h 24 h 36 h 12:0 g 0.01±<0.01 h g 14:0 0.44±0.01 h 0.02±<0.01 g g 14:1 0.04±<0.01 h g 0.01±<0.01 g 15:0 0.29±0.01 h g 0.03±<0.01 g 16:0 3.22±0.16 h g 0.03±<0.01 g 17:0 0.10± ± :0 0.04±< ±< :1n ±0.03 h 0.13±0.01 g 0.01±<0.01 g 18:2n ±< ±< ±< :0 0.04±< ± :3cis ± :3n ±< ±< :0 g 0.10±0.01 h 0.01±<0.01 g 20:3n ±< :3n-3 20:4n ±< ±< ±< :5n ±< ±< :6n ±<0.01 SAFA ±22.49 h ±4.57 g ±2.76 g MUFA ±21.56 g ±2.35 h ±15.96 g PUFA ±0.95 g ±6.58 g ±13.97 h n-3 PUFA ±0.36 ±0.10 ±0.72 n-6 PUFA ±0.16 ±0.18 ±0.14 AA/EPA 46.50± ± ±0.41 AA/DHA 25.25± ±2.30 DHA/EPA 13.63± ±1.12 Values are given as mean SEM (n = 4). Superscripts indicate significant variation (p < 0.05) within the enrichment periods for the individual diet. Dash ( ) indicates that the fatty acid content was < 0.01.

12 12 Khoo et al. Table 3. Fatty acid composition (mg/g body weight) of Moina macrocopa enriched with 2 g/l ADS, SO and CO over three enrichment periods. ADS SO Fatty acid 12 h 24 h 36 h 12 h 24 h 36 h 12:0 0.01±< ±< ±< :0 0.01±< ±< ±<0.01 d 0.03±<0.01 e 0.04±<0.01 e 14:1 0.01±<0.01 a a 0.01±<0.01 b d 0.01±<0.01 e 0.01±<0.01 e 15:0 0.22±< ±< ±<0.01 d 0.21±<0.01 e 0.16±<0.01 e 16:0 0.09±< ±< ±< ±<0.01 d 0.11±<0.01 e 0.09±<0.01 e 17:0 1.70± ± ±<0.01 d 4.33±0.02 e 3.83±0.04 e 18:0 0.16±< ±< ±< ±0.08 e 0.14±<0.01 d 0.11±<0.01 d 18:1n ±< ±< ±< ±< ±< ±< :2n ±< ±< ±< ± ±< ±< :0 0.01±< ±< ±< ±0.13 e 0.02±<0.01 d 0.02±<0.01 d 18:3cis ±<0.01 b 0.65±<0.01 b 0.10±0.01 a 0.78±0.08 e 0.05±<0.01 d 0.08±<0.01 d 18:3n ±< ±< ± ±< ±< :0 0.01±< ±< ±< ± ±< ±< :3n ±< ±< ± ±<0.01 d 0.20±<0.01 e 0.35±0.01 f 20:3n ±< ±< :4n ±< ±< ±< ±<0.01 d 0.14±<0.01 e 0.14±<0.01 e 20:5n ±< ±< ±< ±< ±< :6n ±< ±< ±< ±< ±<0.01 SAFA ±1.01 ±1.17 ±0.92 ±3.99 ±0.27 ±1.08 MUFA ±0.25 ±0.37 ±0.48 ±24.52 ±2.44 ±1.76 PUFA ±0.87 ±1.37 ±0.51 ±27.54 ±2.58 ±2.51 n-3 PUFA ±0.20 b ±0.29 b ±0.04 a ±0.98 ±0.10 ±0.29 n-6 PUFA ±0.53 ±0.07 ±1.21 ±0.31 d ±0.37 e ±1.10 f AA/EPA ±0.34 a ±0.05 a ±0.40 b ±19.80 ±8.54 ±7.64 AA/DHA ±0.86 a ±0.06 a ±23.06 b ±2.20 d ±9.84 e ±2.37 e DHA/EPA 4.25±0.02 bc 5.86±0.07 b 0.93±0.07 ac ± ± ±0.44 Values are given as mean SEM (n = 4). Superscripts indicate significant variation (p < 0.05) within the enrichment periods for the individual diet. Dash ( ) indicates that the fatty acid content was < 0.01.

13 Unsaturated Fatty Acid (HUFA) Retention Quality in Freshwater Cladoceran, Moina macrocopa 13 Table 3 (continuation). Fatty acid composition (mg/g body weight) of Moina macrocopa enriched with 2 g/l ADS, SO and CO over three enrichment periods. CO Fatty acid 12 h 24 h 36 h 12:0 0.01±<0.01 g 0.01±<0.01 h 0.01±<0.01 h 14:0 0.02±< ±< :1 0.01±< ±< :0 g 0.13±0.01 h 0.12±<0.01 g 16:0 0.06±< ±< ±< :0 g 2.21±0.13 h 2.68±0.06 h 18:0 2.31±0.12 h 0.11±0.01 g 0.09±<0.01 g 18:1n ±< ±< ± :2n ±< ±< ± :0 2.24±0.06 h 0.02±<0.01 g 0.03±<0.01 g 18:3cis ± ± ± :3n ±0.10 h 0.04±<0.01 g 0.01±<0.01 g 22:0 0.06±< ±< ±< :3n ±<0.01 g 0.01±<0.01 g 0.79±0.01 h 20:3n ±< :4n ±0.03 h 0.03±<0.01 g 0.03±<0.01 g 20:5n ±< ±< :6n ±<0.01 SAFA ±14.78 ±13.89 ±3.34 MUFA ±2.17 ±5.99 ±1.45 PUFA ±12.66 g ±19.87 g ±4.67 h n-3 PUFA ±7.92 ±0.08 ±0.10 n-6 PUFA ±3.11 h ±0.23 g ±0.95 h AA/EPA ± ±0.27 ±0.04 AA/DHA ±48.14 h ±2.47 g ±6.33 g DHA/EPA 25.00± ± ±0.03 Values are given as mean SEM (n = 4). Superscripts indicate significant variation (p < 0.05) within the enrichment periods for the individual diet. Dash ( ) indicates that the fatty acid content was < 0.01.

14 14 Khoo et al. Table 4. F- and P-values derived from 2-way ANOVA analysis, with further comparisons using Tukey s test to describe the effects of diet concentration, time and interaction of these two variables. Diet Fatty acid Concentration Time Concentration x Time F-value P-value F-value P-value F-value P-value ADS 20:4n *** ** *** 20:5n ** ** 22:6n * * ** AA/EPA *** * *** AA/DHA ** * ** DHA/EPA ** ** * SO 20:4n *** *** *** 20:5n *** :6n ** AA/EPA *** AA/DHA *** ** *** DHA/EPA CO 20:4n ** * ** 20:5n ** :6n * * AA/EPA AA/DHA *** *** *** DHA/EPA Significance level: *p < 0.05, **p < 0.01, ***p <

15 DHA/EPA ratio AA/EPA ratio AA/DHA ratio Unsaturated Fatty Acid (HUFA) Retention Quality in Freshwater Cladoceran, Moina macrocopa a b control control c 0 control 12 Time (h) Fig. 1. Variation of AA, EPA and DHA ratios (ratio of fatty acids/g body weight) of un-enriched Moina macrocopa (control) and ADS-, SO- and CO-enriched Moina after 12, 24 and 36 h at the concentration of 1 g/l. Error bars indicate mean standard deviation. Symbols: ADS, SO, CO.

16 DHA/EPA ratio AA/EPA ratio AA/DHA ratio 16 Khoo et al a 1000 b control control c Fig. 2. Variation of AA, EPA and DHA ratios (ratio of fatty acids/g body weight) of un-enriched Moina macrocopa (control) and ADS-, SO- and CO-enriched Moina after 12, 24 and 36 h at the concentration of 2 g/l. Error bars indicate mean standard deviation. Symbols: ADS, SO, CO. 0 control Time (h) Title Authors Addresses (Received x.x.x, Accepted x.x.x)

17 Unsaturated Fatty Acid (HUFA) Retention Quality in Freshwater Cladoceran, Moina macrocopa 17 Key words: Abstract

18 18 Khoo et al. Introduction Materials and Methods Results Acknowledgements References