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GROWTH PERFORMANCE AND FEED UTILIZATION OF LOW-COST ARTIFICIAL FEEDS FOR JUVENILE ASIAN HORSESHOE CRAB CULTURE.

INTRODUCTION

Destruction of spawning habitats with coastal developments led to the significant decline of Asian horseshoe crab populations in mainland China (Weng et al. 2009), Hong Kong (Shin et al. 2009, Kwan et al. 2016, Lee & Morton 2016), Japan (Itow 1993), Taiwan (Hsieh & Chen 2009), Malaysia (Tan et al. 2012), Philippines (Schoppe 2002), and Singapore (Cartwright-Taylor et al. 2011). In addition, Asian horseshoe crabs, especially Tachypleus spp., are heavily harvested for local food consumption, for Tachypleus amebocyte lysate production, and for baits in fish traps (Chen et al. 2004, Christianu & Saad 2009, Cartwright-Taylor et al. 2011, Kwan et al. 2017a). In recent years, the culture of juvenile horseshoe crabs for field release has become popular, thereby enhancing horseshoe crab populations in nursery shores (Chatterji et al. 2004, Lee & Morton 2005, Carmichael et al. 2009, Chen et al. 2010, Kwan et al. 2017b). Artificial breeding is one of the effective ways of enhancing the population of horseshoe crabs. The success of this culture method depends on the survival and growth of the juveniles, which in turn relies on the provision of an optimal diet (Hu et al. 2014, John et al. 2017). The food and diet supplements used for juvenile horseshoe crab culture range from brine shrimp, bivalves, squid/fish, worms, crabs/shrimp, other invertebrates, and fish food to algae (Carmichael & Brush 2012). Tzafrir-Prag et al. (2009, 2010) estimated nutrient and protein requirements for the Atlantic horseshoe crab (Limulus polyphemus). Hu et al. (2013) compared the effect of different frozen natural food on survival and growth of juvenile Chinese horseshoe crab (Tachypleus tridentatus). Despite these abovementioned investigations, relevant research on Asian horseshoe crabs is still limited. Carmichael and Brush (2012) suggested that research on diet quality for laboratory-reared stocks should be performed if these stocks are to be used for propagation and restoration. The high cost of natural diet is a limitation; therefore, researchers need to focus on supply and quality of artificial food that will yield nutritionally balanced yet cost-effective feeds for successful mass culture.

The quality of dietary protein ingredients plays an important role in regulating growth and feed utilization of aquatic animals under culture (Watanabe 2002). Fish meal (FM) is the primary protein source included in most aquafeeds; thus, manufacturing of these feeds relied heavily on FM (Taco & Akiyama 1997). Declining production and increasing price of FM have made it one of the most expensive macro-ingredients of an aquaculture diet (Yang et al. 2004). As a result, considerable nutritional research has focused on the use of animal or plant protein to replace FM in formulated feeds (Yang et al. 2004, Zhou et al. 2005, Pham et al. 2007); however, the application of plant protein ingredients as FM substitutes is limited by the presence of compounds that may interfere with nutrient absorption (Francis et al. 2001), their high-fiber trait, indigestible carbohydrate content (Refstie et al. 2005), and poor palatability (Escaffre et al. 1997, Opstvedt et al. 2003). Rendered animal by-product protein ingredients are the most promising replacement for FM because they contain a high amount of total protein than plant ingredients (Polan et al. 1997, Schreibman & Zarnoch 2009, Tzafrir-Prag et al. 2010) and are suitable FM substitutes in the artificial feeds for many aquatic animals (Yang et al. 2004, Hu et al. 2008). Therefore, it is feasible to replace FM with suitable animal by-product protein in horseshoe crab feeds.

Different kinds of rendered animal by-product protein ingredients, such as blood meal (BM), meat and bone meal (MBM), and poultry by-product meal (PBM), are widely used in aquatic feeds as substitutes of FM because of their high protein content (Yang et al. 2004), high digestibility (El-Haroun et al. 2009), reasonable price, and steady supply (Bureau et al. 2000, Watanabe 2002). Blood meal is produced from clean, fresh animal blood, exclusive of all extraneous material such as hair and stomach contents. Blood meal may be dried by several processes but most often spray-dried. Spray-dried BM is preferred for its exceptionally high protein (85%-90%) and lysine (7%-8%) contents (Chiba 2001); however, BM becomes unpalatable after drying (Meeker & Hamilton 2006). Meat and bone meal is the dry rendered product derived from mammalian tissues, exclusive of hair, hoof, horn, manure, and paunch contents. The relatively high fat content of MBM apparently helps protect lysine from degradation during rendering (Meeker & Hamilton 2006). Meat and bone meal is primarily used in the formulation of animal feed to improve its amino acid profile as lysine and methionine are highly available for metabolism (Wang & Parsons 1998). The energy digestibility of MBM is, however, usually lower than that of PBM because of the high content of saturated fatty acids and ash, which contain no energy (Bureau et al. 2000, Yang et al. 2004, Hu et al. 2008). Poultry by-product meal is made by grinding clean, rendered parts of poultry carcasses and can contain bones, offal, and undeveloped eggs; these have protein content in the range 49%-69% depending on the source (Dozier et al. 2003). Digestibility of PBM also varies, but it is generally high and between 80% and 90%; however, PBM is limited in lysine and methionine (Taco & Jackson 1985, Nengas et al. 1999). Replacement of FM protein by the combination of BM, MBM, and PBM at different levels can eliminate the imbalance in essential amino acids (EAA) in these ingredients (Wang et al. 2006, Guo et al. 2007); thus, a combination of these protein sources may be the best option for an artificial diet.

Several studies reported that different proportions of PBM and MBM in combinations have different effects on the growth and feed utilization of various aquatic animals (Guo et al. 2007, Hu et al. 2008). Yang et al. (2004) found that a PBM to MBM ratio of around 1:1 could nutritionally and economically replace FM; however, Guo et al. (2007) and Hu et al. (2008) indicated that the proportion of PBM should be twice that of MBM in the combination to replace FM in the practical feed for aquatic animals. The application of a combination of rendered animal protein ingredients to replace FM protein in juvenile horseshoe crab diets may have economic and nutritional benefits, but more study is needed to define the optimal level for replacement of FM (Schreibman & Zarnoch 2009, Tzafrir-Prag et al. 2010).

The present study identified the optimal level for FM replacement by a combination of rendered animal protein ingredients (PBM, MBM, and BM) at different proportions of PBM and MBM (2:1 or 1:1) to formulate nutritionally balanced and cost-effective feeds for juvenile Tachypleus tridentatus and Carcinoscorpius rotundicauda.

MATERIALS AND METHODS

Ingredients, Diet Formulation, and Preparation

Fish meal was provided by the Asian Regional Office of the National Renderers Association (USA) based in Hong Kong. The other feed ingredients were obtained from the Guangdong Haid Group in China. The proximate composition of PBM, MBM, and BM is depicted in Table 1 and their EAA profiles are shown in Table 2. Other ingredients, such as soybean meal, rapeseed meal, and wheat flour, were similar to the batch used by Hu et al. (2014). The feed ingredients were ground with a hammer mill and passed through a 0.5-mm sieve to make the ingredients homogeneous. Contents of digestible protein (DP) and digestible energy (DE) in the experimental feeds were estimated using published digestible coefficients (Catacutan et al. 2003, Tuan et al. 2006). Nine isonitrogenous (ca. DP 40%) and isoenergetic (ca. DE 14 MJ k[g.sup.-1]) diets were formulated based on the results of the ideal combination of DP and energy levels, which was obtained from a previous nutritional requirement study of juvenile horseshoe crabs (Hu et al. 2014). Diet 1 contained 100% FM and acted as the control. In diets 2-9, FM was replaced with a combination of PBM and MBM at ratios of 2:1 (diets 2-5) or 1:1 (diets 6-9). The quantity of FM replacement was 25% (diets 2 and 6), 50% (diets 3 and 7), 75% (diets 4 and 8), and 100% (diets 5 and 9). Blood meal constituent contents in the experimental diets increased from 1.5% in diet treatments of 25% FM replacement to 6% in 100% FM replacement. The proximate composition and EAA profile of these nine diets are presented in Tables 3 and 4.

The experimental feeds were prepared by the same procedures described by Hu et al. (2014). Nine diet treatments were formulated by mixing the dry ingredients in an electric mixer before adding the lipid source. Wheat flour was mixed last and the resulting dough was passed thrice through a laboratory-scale screw extruder fitted with a 0.5-mm-diameter die. The diet was oven-dried at 45[degrees]C for 72 h and cut into 2- to 3-mm-sized pellets.

Experimental Conditions, Animals, and Feeding Regime

The feeding experiment was conducted in an indoor system that contained 27 plastic tanks (25 cm X 15 cm X 10 cm) for each species. The tanks were filled with filtered seawater up to 10 cm depth, which were cleaned daily. The entire volume of water was replaced to maintain good water quality. To achieve better growth of the two horseshoe crab species, some water parameters were suitably adjusted to replicate natural environmental conditions (Hu et al. 2014). Water temperature was maintained at 28[degrees]C-30[degrees]C using electric heaters, salinity at 33[per thousand]-34[per thousand], pH at 8.1, dissolved oxygen between 6.0 and 8.0 mg [L.sup.-1], and ammonia concentration below 0.05 mg [L.sup.-1]. The experimental setup was kept in the dark with a black plastic sheet cover.

Juveniles of Tachypleus tridentatus were hatched from fertilized eggs spawned by mating pairs of adult horseshoe crabs collected from Hong Kong waters, whereas juvenile Carcinoscorpius rotundicauda were obtained from their natural habitat at Luk Keng, Hong Kong. Before experimentation, juveniles of similar size with prosomal width of 14-17 mm were maintained in a sheltered indoor pool and fed with an equally proportioned mixture of all the experimental diets once daily (17:00 h) ad libitum (about 3%-4% body weight) until molting. Horseshoe crabs of such size can ingest diets very well and are suitable for culture with artificial feeds under laboratory conditions, according to Hu et al. (2014). Newly molted juvenile T. tridentatus at fifth instar stage (body weight 0.935 [+ or -] 0.001 g [ind.sup.-1], mean [+ or -] SE, n = 54) and C. rotundicauda at sixth instar stage (body weight 1.856 [+ or -] 0.012 g [ind.sup.-1], mean [+ or -] SE, n = 54) were selected and weighed. For each species, two horseshoe crabs were randomly allocated to one tank, and three tanks were randomly assigned to each diet.

The feeding experiment lasted 84 days, during which the juveniles were fed with an excess amount of feed once daily (17:00 h), 7 days per week. Uneaten feed was collected at 24 h after feeding, dried at 60[degrees]C, and reweighed. Leaching of uneaten feed was estimated by placing weighed samples of each diet into the experimental tanks without horseshoe crabs for 24 h, then recovered, dried, and reweighed. The average leaching value was used to correct the amount of uneaten feed.

Sampling and Chemical Analysis

Methods of recording mortality, measuring wet body weight, testing and calculating feeds, and analyzing horseshoe crab chemical composition followed the same procedures described by Hu et al. (2014). Data on mortality of juvenile horseshoe crabs over the study period were recorded, and dead animals were removed from the experimental tanks. Wet body weight was measured at the start and at the end of the experiment using an electronic balance to 0.1 mg. Before experimentation, 10 juveniles of each horseshoe crab species were sampled, freeze-dried, and analyzed for their amino acid profiles.

For the experimental feeds, dry matter content was determined by drying the feeds to a constant weight at 105[degrees]C (AOAC 2000), whereas dry matter digestibility of each feed ingredient was estimated from published literature as stated earlier for DP and DE. For the nutrient content, nitrogen was tested using an elemental analyzer (PerkinElmer 2400), crude protein content was calculated from nitrogen content by multiplying the value with a factor of 6.25 (AOAC 2000), crude lipid was analyzed by ether extraction using a Soxtec system (Soxtec System HT6, Sweden), and ash was estimated by combustion at 550[degrees]C (AOAC 2000). All measurements were made in duplicates. Carbohydrate content was estimated by taking the percentage sum of crude protein, crude lipid, and ash from 100%. Gross energy was determined by calculating the sum of the energy contents of the protein, lipid, and carbohydrate using the nutritive value of 21.3, 17.2, and 39.5 kJ [g.sup.-1] of protein, carbohydrate, and lipid, respectively (Cuzon & Guilaume 1997).

Analysis of amino acids in the feed ingredients, different diet treatments, and initial horseshoe crab carcasses was performed in a commercial laboratory (Analysis Laboratory of Beijing Institute Nutritional Resources, China) using high-performance liquid chromatography and according to AOAC (2000) Official Method 982.30, Part E guidelines. Samples were hydrolyzed in 6 M HC1 at 108[degrees]C for 24 h in nitrogen-flushed glass vials. A reversed-phase HPLC in a Pico-Tag amino acid analysis system (Waters Corporation) was used with norleucine as the internal standard. Amino acids, except tryptophan and sulfur amino acids, were measured after acid hydrolysis (Moore 1963). Sulfur amino acids were analyzed separately after performing acid oxidation and acid hydrolysis. Tryptophan was destroyed by the acid hydrolysis; thus, it was excluded from analysis.

Data Treatment and Analysis

Apart from final individual body weight (FBW, g [ind.sup.-1]), data on survival rate, thermal-unit growth coefficient (TGC), feeding rate (FR), and feeding efficiency ratio (FER) were calculated using the following formulae:

[mathematical expression not reproducible]

where I (g) = total amount of consumed feed on a dry weight basis, [W.sub.0] (g) = total initial body weight (bw), [W.sub.t] (g) = total final body weight (bw), [DELTA]W(g) = total body weight gain, and t (day) = duration of the experiment.

Results of all data were expressed as mean [+ or -] SE. Before statistical analysis, data normality was evaluated using Shapiro-Wilk test, and the homogeneity of variances was checked by Levene's test. Values of FER in Carcinoscorpius rotundicauda were cosine-transformed because these values were not normally distributed. Differences in the abovementioned variables among treatments and control were statistically tested using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test if a significant difference (P < 0.05) was found. Significant difference of survival rates among different treatments were checked by Chi-square test. All statistical analyses were undertaken using SPSS, version 16.0 (SPSS, Inc., Chicago, IL).

RESULTS

The survival rate of Tachypleus tridentatus ranged from 67% to 100%, with the lowest record in both 100% FM replacement groups (PBM:MBM of 2:1 and 1:1). For Carcinoscorpius rotundicauda, the survival rate varied from 50% to 100%, and the lowest record was observed in 75% FM replacement with PBM:MBM of 1:1 (Table 5). No significant differences (Chi-square test, P > 0.05) were found in the survival rates of both horseshoe crabs in the different treatments, although low survival rates were observed in high FM replacement treatments.

For Tachypleus tridentatus, the means of FBW ranged from 0.98 to 1.37 g [ind.sup.-1] (Fig. 1 A). The TGC ranged from 0.007 to 0.056 [g.sup.1/3] [([degrees]C day).sup.-1] (Fig. 2A). The FR ranged from 0.43% to 0.90% bw [day.sup.-1] (Fig. 3A). The FER ranged from 13.72% to 64.60% (Fig. 4A). For Carcinoscorpius rotundicauda, means of FBW varied from 1.25 to 2.63 g [ind.sup.-1] (Fig. 1B). Thermal-unit growth coefficient varied from 0.021 to 0.148 [g.sup.1/3] [([degrees]C day).sup.-1] (Fig. 2B). Feeding rate varied from 0.62% to 3.34% bw [day.sup.-1] (Fig. 3B). Feeding efficiency ratio varied from 18.05% to 39.91% (Fig. 4B).

For Tachypleus tridentatus, ANOVA results of FBW (F= 8.806, P < 0.001), TGC (F = 12.439, P < 0.001), FR (F = 3.086, P = 0.022), and FER (F = 7.063, P < 0.001) were significantly different among various diets. For Carcinoscorpius rotundicauda, significant differences in ANOVA results were only found in FBW (F = 17.227, P < 0.001), TGC(F= 15.861, P<0.001), and FR(F = 16.183, P < 0.001), whereas FER was statistically the same (F = 1.642, P = 0.182) among different diets. For growth performance, FBW and TGC values of T. tridentatus at 25% and 50% FM replacement levels were comparable with that of the control diet. At higher FM replacement levels, both FBW and TGC showed lower values than the control (Figs. 1A and 2A). For C. rotundicauda, FBW and TGC values at 75% FM replacement were similar to those of the control among all diet treatments. Both FBW and TGC values of C. rotundicauda in all FM-replaced diets were also lower than that of the control diet, except for the diet treatment with 75% FM replacement and PBM:MBM of 2:1 (Figs. 1B and 2B). Comparisons of feed utilization at different FM replacement levels were less notable among the diet treatments for both species. Feeding rate and FER values of T. tridentatus at 100% FM replacement were lower than those of the control diet (Figs. 3A and 4A), whereas a similar result was noted in FR of C. rotundicauda only (Figs. 3B and 4B). For both species, at the same FM replacement level, FR and FER of the diets with PBM:MBM in 2:1 generally showed higher values than that of the diets with proportions of PBM and MBM in 1:1, except for FER at 25% FM replacement level in T. tridentatus and 100% FM replacement level in C. rotundicauda (Figs. 3 and 4).

DISCUSSION

In the present study, diets with various FM replacement levels showed different effects on the two juvenile horseshoe crab species in terms of survival rate, growth performance (FBW and TGC), and feed utilization (FR and FER) indices. Survival and growth performance are the most important criteria for selecting the optimal feed formulation for certain animal species (Lovell 1989). The survival rates noted in the present study were generally in line with the reported survival data of greater than 50% on captive juvenile horseshoe crabs (Carmichael & Brush 2012). Based on the growth performance and feed utilization of the two species, the optimal FM replacement level for Tachypleus tridentatus was estimated at 50%, whereas that of Carcinoscorpius rotundicauda was at 75%. The present results may be used to develop cost-effective feeds for the culture of juvenile horseshoe crabs. The raw material cost for PBM and MBM is about 20%-30% of that for FM (Bureau 2007). According to Yu (2006), 50% FM replacement in the feeds by rendered animal protein ingredients, such as PBM and MBM, would result in 15%-25% cost reduction. A higher FM replacement level of greater than 60% by meat meal and plant proteins could even reduce feed cost by up to 40% in fish aquaculture (Allan et al. 2000).

High FM replacement levels were normally used in feed formulation because of the good protein quality of the substitutes that are beneficial for animal growth (Polan et al. 1997, Nengas et al. 1999, Tan & Zheng 2003). Amino acids are precursors of protein, which is a basic building block of animals and serves biological, structural, and mechanical functions. From the perspective of animal nutrition, the single most important factor affecting the efficiency of protein utilization in an animal's body is the profile of digestible EAA entering the small intestine through diet consumption (Boisen et al. 2000). Essential amino acid requirements of an organism can be approximated from its body tissue amino acid profile (De Silva & Anderson 1995) because dietary amino acids are used for protein synthesis, and the concentration of amino acids in the lean tissues of an animal should indicate the balance of amino acids required in its diet (Bautista-Teruel & Millamena 1999). Thus, replacing FM with substitutes that contain similar EAA profiles to the target animal is considered an important aspect of feed formulation.

Rendered animal protein ingredients, such as MBM, PBM, and BM, are generally considered inferior and are rarely included in commercial diets as main protein sources because they lack some EAA, such as lysine and methionine (Yang et al. 2004, Hu et al. 2008). Previous studies have shown that deficiencies or excess of one or more EAA limit protein synthesis and growth of test animals (Gunasekera et al. 1999, Litaay et al. 2001). The nutritional value of proteins could be evaluated based on the amino acid composition of the study animals by the essential amino acid index (EAAI). Meister (1965) suggested that optimum growth is best achieved with dietary proteins containing an EAAI value that is similar to those in the tissues of the study animals. Oser (1959) graded diets with EAAI ranging from 0.90 to 1.00 as good-quality proteins; an EAAI value of around 0.80 was considered useful and values below 0.70 were inadequate. In the present study, all EAAI values of the experimental diets based on the carcasses of juvenile Tachypleus tridentatus and Carcinoscorpius rotundicauda were in a narrow range between 0.87 and 1.07. Therefore, dietary proteins of the experimental feeds for the two horseshoe crab species were of good quality, thereby indicating that the proportion adjustment was able to eliminate the EAA imbalance for the mixture of PBM, MBM, and BM.

In the present study, the use of BM constituents in the experimental diets increased from 1.5% to 6% with increasing FM replacement level (see Table 3). For both species, the highest FR was found in the control Diet 1 without BM content. Feeding rate values of Tachypleus tridentatus in Diet 9 (100% FM replacement, PBM:MBM = 1:1) and that of Carcinoscorpius rotundicauda in diets 7-9 (50%, 75%, and 100% FM replacement, respectively, PBM:MBM = 1:1) were also significantly lower than their corresponding control groups. Blood meal was unpalatable after the drying process (Meeker & Hamilton 2006), which might explain the reduced FR values among horseshoe crabs fed with higher replacement diets (75%-100%). Similar results were also observed in some fish, for example, the Murray cod (Maccullochella peelii) and rainbow trout (Oncorhynchus mykiss) expressed negative growth responses when fed diets with high BM levels (Abery et al. 2002, Bahrevar & Hamid 2015). Thus, reduced FR and growth in horseshoe crab or fish fed high BM diets were possibly related to low palatability and digestibility of BM diets (Burr et al. 2012, Bahrevar & Hamid 2015).

Feeding efficiency ratio is the ratio measuring the efficiency by which the body converts consumed feed into growth (Prato et al. 2010). Feeding efficiency ratios of Tachypleus tridentatus fed with diets 2 and 6 and diet 3 (25% and 50% FM replacement levels, respectively) were significantly higher than those fed with other diets, but no significant difference of FER was found in Carcinoscorpius rotundicauda fed with the different diets tested in this study. For juvenile T. tridentatus, FM replaced by blends of rendered animal protein ingredients at suitable levels (25% or 50%) improved the FER of the feeds but showed no significant effect on FER of C. rotundicauda. Hence, the present data reflected the potential differences in nutritional requirements between the two horseshoe crab species. Stable isotope-based trophic assessments of native horseshoe crabs in the field also found that major food sources for these two species of horseshoe crabs are different. For example, stable isotope [delta]13C reflects slightly higher seagrass-derived carbon in T. tridentatus diet, whereas influences of lighter sediment particulate organic matter were predominant in C. rotundicauda diet (Fan et al. 2017). The natural diet composition of C. rotundicauda is more complex than that of T. tridentatus. Thus, C. rotundicauda may show similar FER for various diet treatments.

From an economic standpoint, replacement of FM with cheaper rendered animal protein ingredients in a diet for juvenile horseshoe crabs can alleviate the problem of low FM availability and high cost. The combinations of PBM, MBM, and BM had good nutritive values and could be more suitable alternative animal protein sources as feeds for juvenile Tachypleus tridentatus and Carcinoscorpius rotundicauda than FM. Relatively high FM replacement levels (50% for T. tridentatus, 75% for C. rotundicauda) by the combination of these rendered animal protein ingredients can be adopted as artificial feeds for juvenile horseshoe crabs, if the EAA profile of the FM-replaced feeds is formulated similar to that of the juveniles. In conclusion, Diet 3 (50% FM replaced by the blend of 8% PBM, 4% MBM, and 3% BM) and Diet 4 (75% FM replaced by the blend of 12% PBM, 6% MBM, and 4.5% BM) were the most nutritionally balanced and cost-effective feed for laboratory culture of juvenile T. tridentatus and C. rotundicauda, respectively.

ACKNOWLEDGMENTS

We express our thanks to Raymond W. M. Chan and Amy M. Y. Chong of the Department of Biology and Chemistry, City University of Hong Kong, for their technical assistance. Funding support from the Ocean Park Conservation Foundation Hong Kong is greatly appreciated.

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MENGHONG HU, (1,2,3) SHUN YAN O, (4) PAUL K. S. SHIN, (4,5) SIU GIN CHEUNG, (4,5) MINGY AN YAN (1,2,3) AND YOUJI WANG (1,2,6*)

(1) National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, No 999 Huchenghuan Road, Shanghai, China; (2) International Research Center for Marine Biosciences at Shanghai Ocean University, Ministry of Science and Technology, No 999 Huchenghuan Road, Shanghai, China; (3) Shanghai Engineering Research Center of Aquaculture, No 999 Huchenghuan Road, Shanghai, China;(4) Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China; (5) State Key Laboratory in Marine Pollution, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China; (6) Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, No 999 Huchenghuan Road, Shanghai, China

(*) Corresponding author. E-mail: youjiwang2@gmail.com

DOI: 10.2983/035.037.0311
TABLE 1. Gross nutrient composition and energy content of the feed
ingredients in PBM, MBM, and BM.

Ingredients  Dry matter (%)  Crude protein (%)  Crude lipid (%)  Ash (%)

PBM          92.95           71.08              14.32            14.11
MBM          93.42           59.23              13.49            23.18
BM           94.41           96.12               0.00             3.87

Ingredients  Carbohydrate (%)  Gross energy content (MJ [kg.sup.1])

PBM          0.49              17.33
MBM          4.10              15.65
BM           0.01              16.05

Crude protein, crude lipid, ash, and carbohydrate are expressed on a
dry matter basis and given as means (n = 2). Blood meal is spray-dried.

TABLE 2. Essential amino acid profile (%) of the feed ingredients in
PBM, MBM, and BM.

Ingredient  Thr   Val   He    Leu   Tyr   Phe   Lys   His   Arg   Met +
                                                                  Cys

PBM         2.99  3.42  2.80  4.77  1.79  3.25  4.84  1.36  5.47  2.13
MBM         2.11  2.84  1.72  3.38  1.32  2.24  3.90  1.01  4.85  1.34
BM          0.37  0.81  0.05  1.29  0.22  0.75  0.91  0.77  0.45  0.14

Essential amino acids threonine (Thr), valine (Val), isoleucine (He),
leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), lysine (Lys),
histidine (His), arginine (Arg), methionine (Met), and cysteine (Cys)
are expressed on a dry matter basis and given as means (n = 2).

TABLE 3. Formulation and chemical composition of the diet treatments.

                                                  Diet
Ingredients                       1      2      3      4      5

Formulation (%)
 FM                              28     21     14      7      0
 PBM                              0      4      8     12     16
 MBM                              0      2      4      6      8
 BM                               0      1.5    3      4.5    6
 Soybean meal                    25     25     25     25     25
 Rapeseed meal                   15     15     15     15     15
 Wheat flour                     15.18  14.68  14.18  13.68  13.18
 Celufil (cellulose filler)       8      8      8      8      8
 Fish oil                         1      1      1      1      1
 Soybean oil                      0.5    0.5    0.5    0.5    0.5
 Soybean lecithin (*)             2      2      2      2      2
 Cholesterol                      0.5    0.5    0.5    0.5    0.5
 Dicalcium phosphate              1.5    1.5    1.5    1.5    1.5
 Sodium chloride                  0.2    0.2    0.2    0.2    0.2
 Vitamin mix ([dagger])           1.5    1.5    1.5    1.5    1.5
 Mineral mix ([double dagger])    0.5    0.5    0.5    0.5    0.5
 Antioxidant ([section])          0.02   0.02   0.02   0.02   0.02
 Mildew inhibitor ([paragraph])   0.1    0.1    0.1    0.1    0.1
 Binder (sodium alginate)         1      1      1      1      1
Gross nutrient composition (%)
 Dry matter                      95.1   95.1   95.1   95.1   95.1
 Crude protein                   43.32  43.69  44.07  44.44  44.82
 Crude lipid                      7.63   7.79   7.96   8.12   8.28
 Ash                             15.43  15.4   15.36  15.33  15.29
 Carbohydrate                    33.62  33.12  32.62  32.11  31.61
 Gross energy                    15.72  15.76  15.8   15.84  15.88
 Digestible dry matter           84.59  83.72  82.84  81.97  81.1
 DP                              39.83  39.76  39.68  39.61  39.54
 DE                              14.15  14.18  14.22  14.25  14.29
 DP/DE                           28.16  28.03  27.91  27.79  27.67

                                          Diet
Ingredients                       6      7      8      9

Formulation (%)
 FM                              21     14      7      0
 PBM                              3      6      9     12
 MBM                              3      6      9     12
 BM                               1.5    3      4.5    6
 Soybean meal                    25     25     25     25
 Rapeseed meal                   15     15     15     15
 Wheat flour                     14.68  14.18  13.68  13.18
 Celufil (cellulose filler)       8      8      8      8
 Fish oil                         1      1      1      1
 Soybean oil                      0.5    0.5    0.5    0.5
 Soybean lecithin (*)             2      2      2      2
 Cholesterol                      0.5    0.5    0.5    0.5
 Dicalcium phosphate              1.5    1.5    1.5    1.5
 Sodium chloride                  0.2    0.2    0.2    0.2
 Vitamin mix ([dagger])           1.5    1.5    1.5    1.5
 Mineral mix ([double dagger])    0.5    0.5    0.5    0.5
 Antioxidant ([section])          0.02   0.02   0.02   0.02
 Mildew inhibitor ([paragraph])   0.1    0.1    0.1    0.1
 Binder (sodium alginate)         1      1      1      1
Gross nutrient composition (%)
 Dry matter                      95.1   95.1   95.1   95.1
 Crude protein                   43.57  43.82  44.07  44.32
 Crude lipid                      7.78   7.94   8.09   8.25
 Ash                             15.49  15.55  15.6   15.66
 Carbohydrate                    33.16  32.7   32.24  31.77
 Gross energy                    15.74  15.76  15.79  15.81
 Digestible dry matter           83.76  82.92  82.08  81.25
 DP                              39.61  39.4   39.19  38.98
 DE                              14.17  14.19  14.21  14.23
 DP/DE                           27.97  27.77  27.58  27.39

Crude protein, crude lipid, ash, and carbohydrate are expressed on a
dry matter basis and given as means (n = 2).
(*) From Sigma-Aldrich Pty., Ltd.
([dagger]) Vitamin mix with the following vitamins (mg or MIU
[kg.sup.-1] dry feed): thiamin, 54; riboflavin, 108; pyridoxine, 84;
cyanocobalamine, 0.3; niacin, 216; pantothenic acid, 6; biotin, 0.015;
folic acid, 18; inositol, 450; stable C, 1500; menadione, 36;
tocopherol, 247; carotene, 3.0 MIU; and calciferol, 0.6 MIU.
([double dagger]) Mineral mix containing the following minerals (g
[kg.sup.-1] dry feed): P, 9.6; Ca, 9.6; Mg, 1.2; Fe, 0.12; Zn. 0.336;
Cu, 0.168; K, 6; Co, 0.0088; Mn, 0.128; Se, 0.0008; Mo, 0.0004; Al,
0.002; I, 0.32; B, 0.0008; and Ni, 0.00008.
[(section)] Antioxidant: ethoxyquin 66%.
([paragraph]) Mildew inhibitor: check mold 50%.

TABLE 4. Essential amino acid profile (%) of different diet formulation.

Diet                          Thr   Val   Ile   Leu   Tyr   Phe   Lys

1                             1.54  1.94  1.63  2.98  1.29  1.95  2.86
2                             1.53  1.91  1.58  2.89  1.23  1.90  2.75
3                             1.51  1.88  1.53  2.80  1.17  1.85  2.64
4                             1.50  1.85  1.49  2.72  1.12  1.80  2.54
5                             1.48  1.82  1.44  2.63  1.06  1.76  2.43
6                             1.52  1.91  1.57  2.88  1.23  1.89  2.74
7                             1.50  1.87  1.51  2.78  1.17  1.84  2.63
8                             1.47  1.83  1.45  2.67  1.10  1.77  2.51
9                             1.45  1.80  1.39  2.57  1.04  1.71  2.39
Tachypleus tridentatus        1.39  2.47  1.04  3.66  3.60  1.45  1.68
Carcinoscorpius rotundicauda  1.54  2.60  1.02  3.68  3.66  1.53  1.60

Diet                          His   Arg   Met + Cys  EAAI (T) (*)

1                             1.10  3.35  1.20       1.07
2                             1.05  3.19  1.14       1.04
3                             1.01  3.03  1.08       1.00
4                             0.97  2.86  1.03       0.97
5                             0.92  2.70  0.97       0.93
6                             1.05  3.18  1.13       1.03
7                             1.00  3.02  1.07       0.99
8                             0.96  2.85  1.00       0.95
9                             0.91  2.68  0.94       0.91
Tachypleus tridentatus        1.29  1.88  0.82       1.00
Carcinoscorpius rotundicauda  1.41  2.33  0.84       -

Diet                          EAAI (C) ([dagger])

1                             1.02
2                             0.99
3                             0.95
4                             0.92
5                             0.89
6                             0.98
7                             0.95
8                             0.91
9                             0.87
Tachypleus tridentatus        -
Carcinoscorpius rotundicauda  1.00

Essential amino acids: threonine (Thr), valine (Val), isoleucine (Ile),
leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), lysine (Lys),
histidine (His), arginine (Arg). methionine (Met), and cysteine (Cys)
are expressed on a dry matter basis and given as means (n = 2).
(*) Essential amino acid index, EAAI = [([aa.sub.1]/[AA.sub.1] X
[aa.sub.2]/[AA.sub.2] x ... X [aa.sub.n]/[AA.sub.n]).sup.I/n], where
[aa.sub.1] is the A/E ratio (i.e., ratio of a given EAA to the total
EAA) in the diet, [AA.sub.1] is the A/E ratio in the experimental
animal, and n is the number of EAA (Penaflorida 1989). EAAI (T) = EAAI
of experimental diet to juvenile Tachypleus tridentatus.
([dagger]) EAAI (C) = EAAI of experimental diet to juvenile
Carcinoscorpius rotundicauda.

TABLE 5. Survival rate (%) of juvenile horseshoe crabs fed with
experimental diets.

      FM replacement  Tachypleus   Carcinoscorpius
Diet  (%)  PB:MBM     tridentatus  rotundicauda

1       0  -          100           83
2      25  2:1        100          100
3      50  2:1        100           83
4      75  2:1         83          100
5     100  2:1         67           67
6      25  1:1        100          100
7      50  1:1         83          100
8      75  1:1         83           50
9     100  1:1         67           67
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Publication:Journal of Shellfish Research
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Date:Aug 1, 2018
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