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Effects of dietary fish oil to soybean oil ratio on survival, development, and growth of early juveniles of the blue swimmer crab Portunus pelagicus.

ABSTRACT Identifying suitable alternatives to fish oil for inclusion in formulated diets for aquaculture species is becoming increasingly important; however, relatively few studies have assessed the potential of terrestrial oils as possible replacements for fish oil in diets for marine crustaceans. This study examined the potential of soybean oil as a partial or complete replacement for fish oil in diets for blue swimmer crab Portunus pelagicus juveniles. Seven iso-nitrogenous and iso-lipidic diets were formulated to contain soybean oil to fish oil ratios of 1:0. 3:1, 2:1, 1:1, 1:2, 1:3, and 0:1. They were fed to first-stage crabs over four molts and survival, development, and growth of the crabs were measured. Crabs fed the diet containing soybean oil as the sole lipid source (i.e., soybean oil:fish oil ratio 1:0 treatment) recorded the lowest survival among treatments, significantly longer intermolt duration, and significantly lower specific growth rates compared with other treatments. Improved performance was shown by crabs fed diets containing fish oil at the same, or higher, dietary level than soybean oil but the best survival, shortest intermolt duration, and fastest growth rates were recorded for crabs fed the diet containing the soybean oil:fish oil ratio of 1:1. The results indicated that P. pelagicus juveniles require a balance of dietary n-3 and n-6 fatty acids and thus dietary lipid requirements can be met partially by soybean oil. Diets containing soybean oil are cheaper to produce than traditional fish oil-based diets and soybean oil is a more sustainably sourced ingredient than fish oil. Our results should support further development of the P. pelagicus aquaculture industry.

KEY WORDS: fish oil replacement, soybean oil, survival, development, growth, blue swimmer crab, Portunus pelagicus, juveniles

INTRODUCTION

Within the animal husbandry subsectors, aquaculture is now the largest user of fish meal and fish oil (FAO 2011). It is estimated, for example, that around 69% (>3.8 million tonnes) of global fish meal production and more than 81% (0.82 million tonnes) of global fish oil production were used by aquaculture in 2007 (Tacon et al. 2010); however, the use of wild-captured fish to produce fish meal and fish oil as the key ingredients in formulated feeds for aquaculture has become a controversial topic and there are questions relating to the sustainability of this practice as well as perceived inefficiency and wastefulness associated with feeding "fish to fish" (Naylor et al. 2000, Shepherd 2005, FAO 2011). In addition, fish oil has become an increasingly expensive resource and further expansion of the aquaculture industry will rely on identification and use of suitable alternatives, such as more sustainably sourced vegetable oils (Tacon & Metian 2008). Among the various vegetable oils, soybean oil is the most commonly investigated for partial or complete replacement of fish oil in aquaculture feeds; it is also one of the most widely produced plant oils, and is relatively cheap and well digested by aquatic animals (Turchini et al. 2009, 2010). Soybean oil contains high levels of n-6 polyunsaturated fatty acids (PUFA), particularly linoleic acid (LA, 18:2//-6), but low levels of n-3 PUFA, such as [alpha]-linolenic acid (ALA, 18:3/7-3), which are nutritionally essential for the majority of marine animals. Both n-3 and n-6 PUFA are considered essential dietary components for crustaceans because they cannot be synthesized de novo.

Whereas both LA and ALA are precursors to long-chain PUFA (LC-PUFA), it is generally accepted that marine crustaceans have limited ability to synthesize essential fatty acids, such as docosahexaenoic acid (DHA; 22:6n-3) and eicosapentaenoic acid (EPA; 20:5n-3), which are known to promote survival, growth, and physiological functions of crustaceans (Baum et al. 1990, Merican & Shim 1997, Glencross & Smith 1999, Glencross et al. 2002, Gonzalez-Felix et al. 2003). On this basis, deficiencies in the LC-PUFA content of vegetable oils, including soybean oil, is a major limiting factor in their use in aquaculture feeds. Investigations into the partial replacement of dietary marine oils with soybean oil for crustaceans are relatively few, although most have reported encouraging results (Deering et al. 1997, Gonzalez-Felix et al. 2002, Gonzalez-Felix et al. 2010, Unnikrishnan et al. 2010). For example, various combinations of dietary soybean oil, sunflower oil, and cod-liver oil promoted comparable growth of mud crab Scylla serrata juveniles to that of a cod-liver oil-based diet (Unnikrishnan et al. 2010). For the Pacific white shrimp Litopenaeus vannamei, complete replacement of dietary menhaden oil with soybean oil resulted in significantly reduced growth (Gonzalez-Felix et al. 2002); however, when soybean oil was used in varying proportions with fish oil, no significant growth differences were reported (Gonzalez-Felix et al. 2010). It was suggested that the reduced growth rates of shrimp receiving only dietary soybean oil probably resulted from a lack of dietary LC-PUFA (Gonzalez-Felix et al. 2002).

Pond-raised crabs from the genera Eriocheir, Scylla, and Portunus have a large market based on strong demand (He et al. 2014, Wu et al. 2010, Trino & Rodriguez 2002); however, the blue swimmer crab Portunus pelagicus is a relative newcomer to this market with current annual production of around 20 tonnes (FAO 2013). It is a commercially important species throughout the Indo-Pacific region (Romano & Zeng 2006) and fisheries landings of P. pelagicus have increased substantially from 75,968 tonnes in 1990 to around 180,000 tonnes in 2012 (FAO 2013). With increasing market demand, P. pelagicus is considered to be a good candidate for increasing aquaculture production because of its relatively short larval duration (~14 days) and ability to be propagated using relatively simple culture methods (Noordin 2011). Key factors supporting increasing growth of the P. pelagicus aquaculture industry include greater knowledge of the culture requirements of this species and improved nutrition. The aim of this study was, therefore, to assess the feasibility of partial and total replacement of fish oil with soybean oil in formulated diets for P. pelagicus juveniles as a basis for developing a practical, cost-effective diet supporting further aquaculture development of this species.

MATERIALS AND METHODS

Source of Crabs

Sexually mature male and female Portunus pelagicus were collected in estuarine areas around Townsville, North Queensland, Australia, using baited crab pots. Broodstock crabs were maintained in 1,000-l outdoor-holding tanks at the Marine and Aquaculture Research Facility Unit at James Cook University within a recirculating seawater system. They were fed daily on an alternating diet of squid, prawn, and mussel at a rate of ~5%-8% of crab body weight. Any berried females identified during daily feeding were closely monitored until the color of their egg mass changed from orange to grey (indicating the development of eye spots). These berried females were then transferred to indoor 300-1 hatching tanks supplied with 1-[micro]m filtered and ultraviolet-treated recirculating seawater at a rate of ~1.5 l/min. Water temperature and salinity in the hatching tanks were maintained between 26-29[degrees]C and 30-35, respectively. At this stage, feeding was ceased to maintain water quality and the tanks were siphoned every morning to remove feces and any discarded eggs.

Hatching occurred in the early morning, and newly hatched larvae were collected and transferred to larval culture tanks where they were cultured using the methods described by Romano and Zeng (2006). Actively swimming newly hatched larvae were stocked into several flat bottomed, 300-l circular tanks for rearing. The tanks were initially filled halfway with 1-[micro]m filtered and ultraviolet-treated seawater at an initial salinity of 25 [+ or -] 2 (the salinity was gradually increased to 28 as the larvae developed) and water temperature was maintained at 28 [+ or -] 2[degrees]C. The water in larval culture tanks was treated with antibiotic (10 g/l streptomycin sulfate; Sigma-Aldrich Australia Pty Ltd, S6501) only once upon initial stocking.

Newly hatched Zoea I larvae were fed rotifers. Brachionus sp., at a density of 20-40 individuals/ml until they molted to stage II. When the majority of larvae had reached the Zoea II stage, newly hatched Artemia nauplii were added at a density of ~0.5 individual/ml and from the Zoea III stage onwards, Artemia metanauplii enriched with Selco DC DHA (INVE) were added daily at a density of 1-4 individuals/ml until most larvae had metamorphosed to the first-stage crabs (C1).

Diet Preparation

Prior research in this laboratory has shown that Portunus pelagicus juveniles fed a diet supplemented with 4% neutral lipids along with 9% phospholipids showed a significant increase in survival and best growth performance (Noordin 2011). On this basis, the practical diets used in this study were formulated to contain the same level of 4% neutral lipids but with different ratios of soybean oil:fish oil (1:0, 2:1, 3:1, 1:1, 1:2, 1:3, and 0:1). The fish oil used in this study was menhaden oil (Sigma-Aldrich, Australia) and soybean oil was produced and supplied by CSD Grains Pty Ltd, Australia. Prior to diet preparation, the fish meal used in all diets was defatted by vigorous mixing with a chloroform:methanol (2:1, v:v) solvent (Folch et al. 1957) using a highspeed magnetic stirrer for 1 h. To ensure that the defatting procedure was effective, the process was repeated three times before the fish meal was dried in a fume hood for 24 h before being sieved to remove particles larger than 100 [micro]m.

Both dry and wet ingredients (Table 1) used in the experimental diets were individually weighed using a Sartorius TE2145 electronic balance (resolution: 0.1 mg) and then combined using an electric mixer to form a homogenous blend. Agar was used as a binder for the experimental diets (Sheen 2000) and prepared by dissolving in distilled water at a temperature of >80[degrees]C. As the solution became clear, the heat was turned off but the solution was continually stirred until the temperature decreased to 40[degrees]C. Individual diet mixtures were added slowly to the agar solution using vigorous stirring until all ingredients were thoroughly mixed. The agar quickly gelatinized as the temperature dropped below 40[degrees]C and, once set, the diets were cut into small 2 [mm.sup.3] pieces and stored at -20[degrees]C for later use.

The diets formulated in this study were made iso-caloric by incorporating the same levels of energy yielding nutrients; protein, lipid, and carbohydrate in all diets (Table 1). Energy contents of the diets were calculated using standard energy equivalent value of 23.01, 38.07, and 17.15 MJ/kg for protein, lipid, and carbohydrates, respectively (Anderson & De Silva 2003).

Experimental Design and Setup

Toward the end of larval culture, when mass metamorphosis of postlarvae (megalopae) to the C1 crabs was expected to occur the next day (approximately day 14 of the culture), the tank was meticulously searched to locate and remove any C1 crabs that had already appeared. This was done to ensure that all crabs used in this study were newly settled C1 crabs within 12 h of metamorphosis. In the early morning of the following day, healthy C1 crabs with intact appendages were collected using a 1 [mm.sup.2] sieve and briefly rinsed with fresh seawater before being placed individually into 750-ml circular culture vessels (8 cm in diameter) to begin the feeding experiment. Because of the cannibalistic nature of Portunus pelagicus juveniles, each crab was held in an individual culture unit throughout the experiment. Each of the culture units was labeled for identification so that molting intervals of successive molts could be accurately traced for each crab. All culture units remained static and were kept in a water bath to maintain water temperature at 28 [+ or -] 2[degrees]C throughout the experiment.

There were 40 crabs per treatment, held individually in a total of 280 culture units. The mean ([+ or -]SE) weight of the newly settled C1 crabs at the start of the experiment was 1.11 [+ or -] 0.27 mg, whereas mean carapace width and length were 3.25 [+ or -] 0.22 mm and 2.38 [+ or -] 0.11 mm. respectively. During the experiment, a daily 100% water exchange was carried out in the morning by transferring crabs individually into new culture units containing clean seawater, whereas the old units were sanitized using chlorine and dried overnight for reuse in the next day. Any mortality or molting observed during the daily water exchange was recorded, and after the water exchange, crabs were fed to satiation with their designated diet. Throughout the experiment, salinity and pH were maintained at 30.0 [+ or -] 2.0 and 7.5-8.1, respectively, and photoperiod was set at light:dark, 14:10.

Because the C4 crab stage was the end point of this study (requiring ~15-21 days depending on treatment), toward the end of the experiment during the daily morning water exchange and inspection, any crabs that were found to have molted to the C4 stage were recorded and removed from culture containers in the afternoon for determination of final carapace length and width as well as dry weight. This was done to allow sufficient time for newly molted crabs to harden their new exoskeleton before measurement. Carapace length was determined as the distance from the longest rostral spike to the abdomen line, whereas carapace width was defined as the distance between the tips of two lateral spines (Romano & Zeng 2006). Following carapace measurement, crabs were rinsed several times with distilled water to remove residual salt and then individually dried in an oven at 60[degrees]C for 24 h before being weighed on a digital balance (Sartorius TE2145; resolution = 0.1 mg) to determine dry weight. The experiment was terminated when all crabs had either molted to the C4 stage or died.

Dietary Fatty Acid Analysis

The fish oil and soybean oil used in the experimental diets as well as all experimental diets were analyzed for their fatty acid compositions. For fatty acid analysis, lipid was first extracted from the samples according to the method of Folch et al. (1957). Fatty acid methyl esters were then derived using 14% boron trifluoride-methanol (Wijngaarden 1967) and analyzed using an Agilent Technologies 6890 gas chromatograph with split injection using helium as the carrier gas and flame ionization detection. A DB23 fused silica capillary column (30 mm X 0.25 mm) with a 0.25 [micro]m coating (Agilent Technologies) was held at a temperature of 140[degrees]C for 5 min. It was then elevated at a rate of 3[degrees]C/min to 210[degrees]C where it was held until all fatty acid methyl esters of interest had been eluted. Fatty acid methyl esters were identified by comparing their retention times with standards (Sigma-Aldrich Co.) and they were quantified by comparison with an internal standard (heneicosanoic acid; 21:0).

Data Analysis and Statistics

Molt death syndrome (MDS) observed in this study was calculated as:

(MDS/TM) X 100

where MDS represents the number of crabs that died during molting or within 48 h of molting and TM represents total number of crabs that died. Because of the limited number of crabs, these data were not subjected to statistical analysis.

The specific growth rate (SGR) based on dry weight or carapace size increases of crabs used in the experiment from C1 to C4 were calculated using the formula:

SGR = [(ln F - ln I)/T] X 100

where F is the mean value of dry weight or carapace width/ length of newly molted C4 crabs, I is the mean initial value of dry weight or carapace width/length of the experimental crabs (C1), and T is the average duration (days) for the crabs to develop from C1 to C4.

Data for survival, intermolt period, and SGR of dry weight and carapace size of crabs from different treatments are presented as mean [+ or -] SE and were subjected to one-way analysis of variance to determine any significant differences (P < 0.05). Significant differences between treatment means were identified using Tukey's posthoc test. Before analysis, all data were tested to confirm normality and homogeneity of variance. Log transformations were performed before further analysis, when homogeneity of variance was violated. Survival data were arcsine transformed before analysis. All statistical analyses were conducted using SPSS, version 17.

RESULTS

Fatty Acid Composition of Oils and Experimental Diets

The fatty acid profiles of soybean oil and fish oil used in the experimental diets are shown in Table 2. Soybean oil contained predominately C18 fatty acids with LA(18:2n-6) being the most dominant and making up more than half of total fatty acids (54.45%). Among the different fatty acid classes, soybean oil contained 14.25% saturated fatty acids (SFA), 23.00% monounsaturated fatty acids (MUFA), and 62.74% PUFA, but was virtually void of any LC-PUFA ([greater than or equal to] 20:3n) (Table 2). Soybean oil also contained only one n-3 group fatty acid, that is, ALA (18:3n-3), which made up 8.29% of the total fatty acids. In contrast, the fish oil used in this study had a more diverse fatty acid profile: containing 28.98% SFA, 26.54% MUFA, 10.55% PUFA, and 28.49% LC-PUFA (Table 2). Compared with soybean oil, fish oil contained substantially less LA (5.15%) and ALA (1.26%), but significantly higher levels of EPA (20:n-3) (11.53%) and DHA(22:6n-3) (11.84%). Fish oil also contained n-6 group LC-PUFA (i.e., arachidonic acid; 20:4n-6) at 0.90%, which was absent in soybean oil.

As the fatty acid compositions of all seven formulated practical diets used in the experiment reflected the different soybean oil:fish oil ratios adopted in diet formulation (Table 3), the diet containing only soybean oil (i.e., soybean oil:fish oil ratio 1:0) had the highest level of PUFA among all diets, mainly due to the very high level of LA. Dietary LA and ALA decreased, whereas LC-PUFA, particularly EPA and DHA, increased concomitantly with increasing dietary fish oil inclusion (Table 3).

Survival, Growth, and Development of Juvenile Crabs

Highest survival was recorded for crabs fed the diet containing soybean oil:fish oil at a ratio of 1:1 where 75.0% of crabs survived to the C4 stage, whereas the lowest survival of 37.5% was recorded for crabs fed the diet with a soybean oil:fish oil ratio of 1:0 (i.e., soybean oil only) (Table 4). Substantially, higher incidences of death during molting or within 48 h of molting were also observed for crabs fed the diet with soybean oil as the sole lipid source (Table 4).

Dietary soybean oil:fish oil ratio also had a significant effect on the SGR of the crabs from C1 to C4. The SGR (for dry weight and carapace width) were highest for crabs fed the diet with a soybean oil:fish oil ratio of 1:1, whereas the lowest SGR were shown by crabs fed the diet without fish oil (Table 4). In general, diets that contained no or low levels of fish oil (i.e., soybean oil:fish oil ratios of 1:0, 3:1, and 2:1) showed lower SGR than those fed diets with higher fish oil inclusion levels (i.e., soybean oil:fish oil ratios of 1:1, 1:2, 1:3, and 0:1).

Following the same trend, development of Portunus pelagicus juveniles was better supported when they were fed diets containing fish oil to a level equal to, or higher than, that of the soybean oil (Table 5). No significant differences for the intermolt period of the C1 crabs were detected (P > 0.05) among all diet treatments and significant differences in development only became apparent from the second molt onward. For example, crabs fed the diet without fish oil had a significantly longer intermolt duration for the C2 crabs than the crabs in all other treatments except those fed the diet with a soybean oil:fish oil ratio of 2:1 (P< 0.05). The shortest accumulated development time from C1 to C4 was recorded for crabs fed the diet with a soybean oil to fish oil ratio of 1:1 (14.9 days), and this was more than 4 days shorter than the longest development time (19.1 days) shown by crabs fed the diet without fish oil (P <0.01) (Table 5).

DISCUSSION

Dietary essential fatty acid deficiencies are suggested to be unlikely when a diet is supplied with sufficient fish meal to satisfy these requirements (Turchini et al. 2009), but because fish meal shows great variation in quality due to factors that include lipid oxidation (i.e., that occur during processing and storage) (Laohabanjong et al. 2009, Ricque-Marie et al. 1998), dietary oils become the principal ingredients that supply essential fatty acids. Our results show that incorporating soybean oil with fish oil at a ratio of 1:1 into formulated diets for Portunus pelagicus juveniles supported higher survival, more rapid development and the best growth performance among all diets tested, including that with fish oil as the sole lipid source. This result is consistent with previous studies on other crustacean species that have reported better performance when marine and vegetable oil blends are used in formulated diets compared with the use of marine oils only (Deshimaru et al. 1979, Kamarudin & Roustaian 2002, Holme et al. 2006). For example, when megalopal larvae of the portunid crab Scylla serrata were fed microbound diets with different fish oil to corn oil ratios, a diet with a fish oil:corn oil ratio of 1:1 supported the best survival to the C1 crabs with the resulting C1 crabs having a similar dry weight and carapace width to those fed a diet containing fish oil only (Holme et al. 2006). Similarly, with Kuruma prawn Marsupenaues japonicas juveniles, a dietary soybean oil to Pollack liver oil ratio between 3:1 and 1:1 resulted in markedly higher growth rates and feeding efficiency compared with diets containing only Pollack liver oil or soybean oil (Deshimaru et al. 1979).

These results may be explained by the fact that blends of fish oil and vegetable oil may provide appropriate ratios of dietary n-3 and n-6 fatty acids that better meet the nutritional requirements of the target species. These requirements of appropriate dietary n-3 and n-6 fatty acids ratios are thought to reflect natural feeding habits (Ackman 1967). Carnivorous marine fish, for example, have natural diets that are rich in both n-3 and n-6 LC-PUFA. This has been suggested as a cause for an evolutionary downregulation of their fatty acid desaturation and/or elongation abilities. In contrast, freshwater animals have natural diets that are rich in PUFA and this may have led to their enhanced capacity to synthesize LCPUFA from precursor PUFA and/or elongate lower n-3 LC-PUFA (Sargent et al. 2002). On this basis, there is likely to be greater success in the utilization of fish oil alternatives by freshwater animals than by marine animals (Tacon & Metian 2008). For example, a total replacement of dietary fish oil with corn oil for the freshwater prawn Macrobrachium rosenbergii was recently shown to support superior growth (Kim et al. 2012).

The optimal dietary soybean oil:fish oil ratio for early Portunus pelagicus juveniles was found to be 1:1 in this study, indicating that this species requires a more balanced dietary n-3/n-6 ratio. This probably reflects the natural diet of P. pelagicus that includes benthic invertebrates and (to a lesser extent) teleost fish, as well as plant materials (Williams 1982, Wassenberg & Hill 1987, Lestang et al. 2003). In addition, complete replacement of dietary fish oil with soybean oil or use of soybean oil at a higher ratio, negatively impacted juvenile crab performance and this is likely to result from their limited ability to elongate and desaturate LA and ALA to LC-PUFA. There is broad consensus that dietary LC-PUFA is essential for survival, development, and growth of decapod crustaceans (Xu et al. 1993, D'Abramo 1997, Boonyaratpalin 1998, Coutteau et al. 2000). For example, when larvae of the mud crab Scylla serrata and the swimming crab Poriunus trituberculatus were fed LC-PUFA-deficient Artemia and rotifers, respectively, both showed lower survival and prolonged intermolt periods (Hamasaki et al. 1998, Takeuchi 2000, Suprayudi et al. 2004).

Whereas molt death syndrome in the past has been particularly associated with an inability of the larvae to successfully extract themselves from the old exoskeleton during molting, subsequent deformity and death shortly after molting should also be considered to be clinical signs of MDS (Bowser & Rosemark 1981) and, on this basis, mortalities up to 48 h postmolt were interpreted as MDS in this study. More than four times higher incidence of MDS was recorded for crabs fed the diet with soybean oil as the sole lipid source (i.e., soybean oil: fish oil ratio 1:0) compared with other treatments (Table 4). This may be related to the function of LC-PUFA as precursors of eicosanoids, the molting hormones of crustaceans, generated in situ through cyclooxygenases lipoxygenases pathways (Sargent et al. 1999, Gonzalez-Felix et al. 2003).

The dietary fish oil requirement of Portunus pelagicus juveniles could also be related to the role played by LC-PUFA in improving cholesterol absorption and utilization, because cholesterol is essential for ecdysis in crustaceans (D'Abramo 1997, Boonyaratpalin 1998). Unlike fish, crustaceans cannot synthesize cholesterol de novo (Sheen 2000) and often require a source of dietary cholesterol to ensure normal function and development (Grieneisen et al. 1993). It has been reported that diets containing only soybean oil resulted in lower cholesterol levels in both muscle and the hepatopancreas of shrimp Litopenaeus vannamei juveniles compared with those fed a diet with fish oil as the sole lipid source (Cheng & Hardy 2004). Similarly, juvenile black sea bream Acanthoparus schlegeli had significantly lower plasma cholesterol levels when fed soybean oil-based diets, which was suggested to result from elevated levels of dietary oleic acid (18:1), LA and ALA, because all these fatty acids from soybean oil are capable of reducing cholesterol absorption (Peng et al. 2008). Furthermore, plant oils, including soybean oil, are known to contain phytosterols (Phillips et al. 2002) that may decrease levels of cholesterol and low-density lipoprotein-cholesterol in teleost fish by reducing intestinal absorption efficiency (Vanstone et al. 2002, Gilman et al. 2003). Whether such mechanisms also exist in crustaceans is unknown and this warrants further research, particularly because crustaceans are known to use cholesterol more extensively (Baum et al. 1990, Boonyaratpalin 1998, Cheng & Hardy 2004).

Our results are encouraging because the use of soybean oil in formulated diets for aquaculture is considerably cheaper than using fish oil only, and it is also more readily available and sustainably produced (Naylor et al. 2009). The results of this study indicate that Portunus pelagicus juveniles can be cultured with high productivity using diets that are cheaper and contain more sustainably sourced ingredients than traditional fish oil based diets. This study also generates nutritional information that will provide a basis for further investigation of the nutritional requirements of P. pelagicus juveniles that will support development of more appropriate feeds and facilitate aquaculture development.

ACKNOWLEDGMENTS

We would like to thank Mr. Ian Brock from the Department of Employment, Economic Development and Innovation of Queensland (DEEDI), Australia for carrying out fatty acid analyses and Mr. Luke Berrei from CDS Grains Pty Ltd, Toowomba, Queensland, Australia, for supplying the soybean oil used in this study.

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NOORDIYANA MAT NOORDIN, (1,2) CHAOSHU ZENG, (1) * PAUL C. SOUTHGATE (1,4) AND NICHOLAS ROMANO (1,3)

(1) Centre for Sustainable Tropical Fisheries and Aquaculture, College of Marine & Environmental Sciences, James Cook University, Townsville, Queensland 4811, Australia; (2) School of Fisheries and Aquaculture Sciences, University Malaysia Terengganu, 21030, Kuala Terengganu, Terengganu Darul Iman, Malaysia; (3) Faculty of Agriculture, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysia; (4) Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Maroochydore, Queeensland 4558, Australia

* Corresponding author. E-mail: chaoshu.zeng@jcu.edu.au

DOI: 10.2983/035.034.0333
TABLE 1.
Percentage composition of the experimental diets used in this
study with varying soybean oil:fish oil ratios. Diet formulation
was modified from that of Sheen (2000).

Ingredients                                        Level (g/100 g)
Basal diet composition
  Defatted fish meal *                                 50.0
  Phospholipid ([dagger]) (a)                           9.00
  Cholesterol ([dagger]) (b)                            1.0
  Vitamin mix ([double dagger]) (a)                     4.0
  Mineral mix ([double dagger]) (b)                     4.0
  Choline chloride ([dagger]) (c)                       1.0
  Dibasic calcium phosphate ([dagger]) (d)              0.6
  Starch ([dagger]) (e)                                10.0
  Cellulose ([dagger]) (f)                              4.4
  Agar (binder) ([dagger]) (g)                         12.0
Soybean oil ([section]):fish oil ([dagger]) (h)
  1:0                                                   4.0:0
  3:1                                                   3.0:1.0
  2:1                                                   2.7:1.3
  1:1                                                   2.0:2.0
  1:2                                                   1.3:2.7
  1:3                                                   1.0:3.0
  0:1                                                   0:4.0

 * Skretting, Tasmania.

([dagger]) Sigma-Aldrich Australia Pty Ltd (a) P3644, (b) C8667,
(c) 98% powder C7527, (d) dibasic calcium phosphate C4131, (e) S4126
(corn), (f) C8002 alpha, (g) A7002, (h) F8020 from menhaden.

([double dagger]) Rabar Pty Ltd, Australia (a) ZZ600 DPI, each 1 kg
contains vitamin A 2 mlU, vitamin [D.sub.3] 0.8 mlU, vitamin E 40 g,
vitamin K 2.02 g, inositol 50 g, vitamin [B.sub.3] 30.40 g, vitamin
B5 9.18 g, vitamin B9 2.56 g, vitamin B2 4.48 g, vitamin [B.sub.12]
0.004 g, biotin 0.1 g, vitamin B6 4 g, vitamin B| 3.4 g, vitamin C
44.4 g, para amino benzoic acid 20 g, tixosil 5 g, antioxidant 30 g;
(b) ZZ603 DO 067 DPI, each 1 kg contains copper 1 g, cobalt 100 mg,
magnesium 59.4 mg, manganese 5 g, iodine 800 mg, selenium 20 mg,
iron 8 mg. zinc 20 g, aluminum 100 mg, chromium 100 mg.

([section]) CSD Grains Pty Ltd, Australia.

TABLE 2.
Fatty acid compositions (% of total fatty acids) of the soybean
oil and fish oil used in the experimental diets formulated for
this study.

                                                   Soybean   Fish
Major fatty acids                                    oil      oil

14:0                                                  --      7.75
15:0                                                  --      0.70
16:0                                                10.04    16.56
18:0                                                 3.84     2.86
22:0                                                 0.37      --
16:1n-7                                               --     11.01
18:1n-9                                             21.45     8.40
18:1n-7                                              1.38     2.90
18:2n-6                                             54.45     5.15
18:3n-3                                              8.29     1.26
20:4n-6                                               --      0.90
20:5n-3                                               --     11.53
22:6n-3                                               --     11.84
[SIGMA] SFA                                         14.25    28.98
[SIGMA] MUFA                                        23.00    26.54
[SIGMA] PUFA ([greater than or equal to] 18:2K)     62.74    10.55
[SIGMA] HUFA ([greater than or equal to] 20:3n)       --     28.49
[SIGMA] n-3                                          8.30    31.85

[SIGMA] n-6                                         54.50     6.70
n-3/n-6                                              0.15     4.77
DHA/EPA                                               --      1.03

[SIGMA] includes minor fatty acids percentage that is not shown in
the table.

"--" indicates less than 0.005% (% of total fatty acids).

TABLE 3.
Fatty acid compositions (g/kg diet dry weight) of the seven
experimental diets used in this study.

                                            Soybean oil:fish oil

Fatty acids                             1:0     3:1     2:1     1:1

14:0                                    0.16    0.87    0.99    1.35
15:0                                     --     0.12    0.14    0.17
16:0                                   14.71   14.48   14.69   14.58
16:1n-7                                 0.16    1.23    1.44    2.05
17:0                                    0.14    0.18    0.20    0.22
18:0                                    4.29    3.82    3.84    3.65
18:1n-9                                15.68   13.00   12.75   11.43
18:1n-7                                 1.48    1.58    1.65    1.72
18:2n-6 (LA)                           56.93   47.42   47.00   42.13
18:3n-3 (ALA)                           7.31    6.09    6.08    5.42
18:4n-3                                  --     0.36    0.45    0.63
20:0                                    0.24    0.20    0.20    0.18
20:1n-9                                 0.14    0.29    0.32    0.40
20:2n-6                                  --      --      --      --
20:4n-6                                  --     0.14    0.17    0.20
  (arachidonic acid)
20:5n-3 (EPA)                            --     1.45    1.80    2.52
22:0                                    0.33    0.27    0.28    0.24
22:5n-3                                  --     0.44    0.52    0.63
22:6n-3 (DHA)                           0.21    1.70    2.05    2.89
[SIGMA]SFA                             19.89   20.0    20.33   20.40
[SIGMA]MUFA                            17.47   16.1    16.16   15.60
[SIGMA]PUFA ([greater than or equal    64.24   53.9    53.53   48.18
  to] 18:2n)
[SIGMA]HUFA ([greater than or equal     0.21    3.7     4.53    6.24
  to] 20:3n)
[SIGMA]n-6                             56.93   47.56   47.17   42.33
[SIGMA]n-3                              7.51   10.05   10.88   12.09
n-6/n-3                                 7.58    4.73    4.33    3.50
DHA/EPA                                  --     1.13    1.11    1.16

                                        Soybean oil:fish oil

Fatty acids                             1:2     1:3     0:1

14:0                                    1.78    1.86    2.63
15:0                                    0.22    0.23    0.30
16:0                                   15.11   14.91   15.87
16:1n-7                                 2.62    2.79    3.82
17:0                                    0.26    0.27    0.33
18:0                                    3.57    3.48    3.42
18:1n-9                                10.39    9.76    8.44
18:1n-7                                 1.81    1.81    2.03
18:2n-6 (LA)                           38.53   36.45   32.17
18:3n-3 (ALA)                           4.95    4.68    4.14
18:4n-3                                 0.81    0.87    1.20
20:0                                    0.17    0.16    0.15
20:1n-9                                 0.49    0.51    0.67
20:2n-6                                 0.11    0.12    0.14
20:4n-6                                 0.26    0.29    0.38
  (arachidonic acid)
20:5n-3 (EPA)                           3.21    3.45    4.75
22:0                                    0.23    0.21    0.19
22:5n-3                                 0.77    0.81    1.10
22:6n-3 (DHA)                           3.60    3.87    5.31
[SIGMA]SFA                             21.34   21.12   22.90
[SIGMA]MUFA                            15.42   15.01   15.37
[SIGMA]PUFA ([greater than or equal    44.39   42.12   37.65
  to] 18:2n)
[SIGMA]HUFA ([greater than or equal     7.96    8.42   11.71
  to] 20:3n)
[SIGMA]n-6                             39.01   36.86   32.85
[SIGMA]n-3                             13.34   13.69   16.50
n-6/n-3                                 2.93    2.69    1.99
DHA/EPA                                 1.13    1.11    1.10

includes minor fatty acid that is not shown in the table.
"--" indicates less than 0.05 g/kg.

TABLE 4.
Mean survival (%), mortality due to molt death syndrome (%), and mean
([+ or -] SE) SGR for dry weight, carapace width, and carapace
length, of newly molted C4 crabs of Portunus pelagicus cultured from
newly settled CI crabs and fed diets with different soybean oil
to fish oil ratios.

Dietary
soybean oil:
fish oil ratio   Survival (%)   Molt death syndrome (%)

1:0                  37.5                20.0
3:1                  60.0                 2.5
2:1                  60.0                 0
1:1                  75.0                 2.5
1:2                  55.0                 2.5
1:3                  60.0                 2.5
0:1                  60.0                 5.0

Dietary                                               SGR
soybean oil:            Dry weight               Carapace width
fish oil ratio       (% [day.sup.-1])           (% [day.sup.-1])

1:0               8.87 [+ or -] 0.21 (a)    4.06 [+ or -] 0.42 (a)
3:1               9.92 [+ or -] 0.85 (ab)   4.93 [+ or -] 0.42 (bc)
2:1               9.14 [+ or -] 0.32 (a)    4.72 [+ or -] 0.13 (ab)
1:1              10.89 [+ or -] 0.31 (b)    5.52 [+ or -] 0.11 (c)
1:2              10.04 [+ or -] 0.33 (ab)   4.92 [+ or -] 0.13 (bc)
1:3              10.77 [+ or -] 0.30 (b)    5.27 [+ or -] 0.11 (bc)
0:1              10.55 [+ or -] 0.32 (b)    5.24 [+ or -] 0.12 (bc)

Dietary
soybean oil:      Carapace length
fish oil ratio    (% [day.sup.-1])

1:0              3.50 [+ or -] 0.42
3:1              3.28 [+ or -] 0.10
2:1              3.20 [+ or -] 0.15
1:1              3.68 [+ or -] 0.08
1:2              3.26 [+ or -] 0.09
1:3              3.54 [+ or -] 0.11
0:1              3.52 [+ or -] 0.10

Different superscripts within a column indicate significant
differences (P < 0.05).

TABLE 5.
Mean ([+ or -] SE) intermolt duration (days) of the first three crab
stages and cumulative development time from CI to C4 of Portunus
pelagicus early juveniles fed semipurified diets with different
soybean oil to fish oil ratios.

Dietary soybean oil:   C1-C2 intermolt       C2-C3 intermolt
fish oil ratio             duration             duration

1:0                    4.6 [+ or -] 0.2   7.0 [+ or -] 0.4 (a)
3:1                    4.2 [+ or -] 0.1   5.9 [+ or -] 0.2 (b)
2:1                    4.4 [+ or -] 0.2   6.1 [+ or -] 0.2 (ab)
1:1                    4.2 [+ or -] 0.2   5.4 [+ or -] 0.2 (b)
1:2                    4.5 [+ or -] 0.2   5.7 [+ or -] 0.2 (b)
1:3                    4.0 [+ or -] 0.2   5.2 [+ or -] 0.1 (b)
0:1                    4.6 [+ or -] 0.2   5.8 [+ or -] 0.3 (b)

Dietary soybean oil:      C3-C4 intermolt      Cumulative development
fish oil ratio               duration            time from CI to C4

1:0                    6.9 [+ or -] 0.4 (a)    19.1 [+ or -] 1.74 (a)
3:1                    6.0 [+ or -] 0.3 (ab)   15.6 [+ or -] 0.4 (b)
2:1                    6.3 [+ or -] 0.2 (ab)   16.3 [+ or -] 0.3 (b)
1:1                    5.7 [+ or -] 0.1 (b)    14.9 [+ or -] 0.2 (b)
1:2                    6.2 [+ or -] 0.2 (ab)   15.8 [+ or -] 0.3 (b)
1:3                    6.8 [+ or -] 0.3 (ab)   15.3 [+ or -] 0.4 (b)
0:1                    5.8 [+ or -] 0.3 (ab)   15.8 [+ or -] 0.5 (b)

Different superscripts within a column indicate significant
differences (P < 0.05).
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Author:Noordin, Noordiyana Mat; Zeng, Chaoshu; Southgate, Paul C.; Romano, Nicholas
Publication:Journal of Shellfish Research
Article Type:Report
Date:Dec 1, 2015
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