Replacement of fishmeal with soybean meal and mineral supplements in diets of Litopenaeus vannamei reared in low-salinity water

Accepted Manuscript Replacement of fishmeal with soybean meal and mineral supplements in diets of Litopenaeus vannamei reared in lowsalinity water

Fei Huang, Ling Wang, Chun-xiao Zhang, Kai Song PII: DOI: Reference:

S0044-8486(17)30248-X doi: 10.1016/j.aquaculture.2017.02.011 AQUA 632523

To appear in:

aquaculture

Received date: Revised date: Accepted date:

17 July 2016 3 January 2017 6 February 2017

Please cite this article as: Fei Huang, Ling Wang, Chun-xiao Zhang, Kai Song , Replacement of fishmeal with soybean meal and mineral supplements in diets of Litopenaeus vannamei reared in low-salinity water. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Aqua(2017), doi: 10.1016/j.aquaculture.2017.02.011

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ACCEPTED MANUSCRIPT Replacement of fishmeal with soybean meal and mineral supplements in diets of Litopenaeus vannamei reared in low-salinity water Fei Huang, Ling Wang, Chun-xiao Zhang *, Kai Song

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The Key Laboratory of Healthy Mariculture for the East China Sea, Xiamen Key Laboratory for Feed Quality Testing and Safety Evaluation, Fisheries College, Jimei University, Xiamen 361021, China.

* Corresponding author: Chun-Xiao Zhang, Tel.: +86 592 6181054; Fax: +86 592 6181054;

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E-mail: [email protected] (C.X. Zhang). Address: Fisheries College, Jimei University, Yindou Road 43, Xiamen, 361021 China

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Abstract To date, few studies have examined the impacts of the mineral profiles of diets high in plant protein on shrimp in aquaculture. Therefore, a feeding trial was conducted to determine how the

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mineral levels of diets formulated with soybean meal replacement of fishmeal affected the growth performance, osmoregulation and tissue mineralization of Litopenaeus vannamei at low

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salinity. Six diets were designed: two high fishmeal diets (FMN and FMR) contained 300 g/kg fishmeal, and four high soybean meal diets (SBN, SBF, SBR and SBRR) were formulated by

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soybean meal replacement of 60% fishmeal of FMN with various mineral supplements; FMN and SBN had no mineral supplements; SBF was supplemented with minerals according to the

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mineral composition of FMN; FMR and SBR with mineral supplements were based on dietary requirements of L. vannamei; SBRR had the same macro-element level as SBR but double

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micro-element content. After an 8-week feeding trial, the growth performance, serum osmotic pressure, gill ATPase activity and tissue mineralization of Ca and P of shrimp were significantly negatively affected by the replacement of fishmeal with soybean meal. With proper mineral

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supplementation, however, the growth performance, muscle protein and lipid deposition, and osmoregulation of shrimp fed diets high in soybean meal were markedly improved. Additionally,

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shrimp fed high soybean protein diets with supplementation of relevant minerals had similar tissue mineralization to those fed high fishmeal diets. Anyhow, there were no significant

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differences in experiment indexes between the shrimp fed the FMN and FMR diets, nor between the shrimp fed the SBR and SBRR diets. In conclusion, high soybean meal diets need to be supplemented with appropriate minerals to compensate for the negative effects of their mineral

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composition on L. vannamei reared in low-salinity water.

Statement of relevance No competing interests exist in commercial aquaculture.

Keywords: Litopenaeus vannamei; Soybean meal; Fishmeal; Mineral elements; Low salinity

1. Introduction

ACCEPTED MANUSCRIPT Fishmeal (FM) is a high-quality protein containing 60-72% crude protein and is a rich source of minerals, vitamins and other nutrients, and is highly digestible and palatable to aquatic animals (Riche, 2015). As a result of these qualities, FM has been used as a preferred protein source in aquaculture feeds. The typical FM concentration in commercial feeds for the shrimp Litopenaeus vannamei is between 250 and 500 g/kg (Samocha et al., 2004; Amaya et al., 2007; Ye et al., 2012). However, recently the cost of L. vannamei feed has intensively risen as a result

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of the limited availability and increasing price of fishmeal (Tacon and Metian, 2008). Therefore, the aquaculture industry has searched for alternative protein sources to reduce dependence upon

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FM and to facilitate the development of a sustainable aquaculture industry (Naylor et al., 2009; Hardy, 2010; Khaoian et al., 2014).

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One of the most promising and common fishmeal replacements in aquafeeds is soybean meal (SBM), which is high in crude protein and has one of the best amino acid profiles among

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plant protein sources, but also contains anti-nutritional compounds such as phytic acid (NRC, 2011). Previous studies have demonstrated that 40%–75% of FM in shrimp diets (containing

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more than 300 g/kg FM) can be replaced with SBM without affecting the growth performance of shrimp in seawater (Paripatananont et al., 2002; Rahman et al., 2010; Shiu et al., 2015). Although SBM is readily available at a lower price than FM and is a sustainable source of

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dietary proteins, the concentrations of lysine (Lys), methionine (Met), histidine (His), glycine (Gly), alanine (Ala), calcium (Ca), phosphorus (P), sodium (Na) and iron (Fe) in SMB are much

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lower than those in FM (Table 1). A significant replacement of fishmeal with plant proteins has been shown to alter the amino acid composition of aquafeeds (Amaya et al., 2007; Suárez et al.,

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2009; Ye et al., 2012; Hien et al., 2015), but little is known about how replacing FM with SBM impacts the mineral element profile of feeds and the growth performance of cultured marine

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organisms.

Marine organisms are able to take up minerals and trace elements via ingested seawater. However, shrimp cannot meet their physiological needs for certain essential ions from ingestion of seawater alone and therefore require a dietary supplement for healthy growth and metabolism (Davis and Gatlin, 1996; Saoud et al., 2003; Shiau and Bai, 2009; Bharadwaj et al., 2014). Previous studies have estimated the dietary mineral requirements of the shrimp L. vannamei to be 5 g/kg calcium (Ca) and 5–10 g/kg phosphorus (P) (Cheng et al., 2006), 10–15 g/kg potassium (K) (Roy et al., 2007; Liu et al., 2014), 2.6–3.5 g/kg magnesium (Mg) (Cheng et al., 2005), 32 mg/kg copper (Cu) (Davis et al., 1993a), 70 mg/kg manganese (Mn) (Liu and Lawrence, 1997) and 33 mg/kg zinc (Zn) (Davis et al., 1993b). Given the high concentration of

ACCEPTED MANUSCRIPT sodium (Na) in seawater and of iron (Fe) in feed ingredients, the growth performance of shrimp was not affected by diets without supplementation of Na or Fe (Davis and Gatlin, 1996). These mineral elements are essential in skeletal tissue metabolism, neuromuscular transmission, and immune capacity of aquatic animals, and may be especially limiting for marine animals reared in freshwater or low-salinity water (Lall, 2002). FM is a major source of minerals and trace elements for cultured fish and crustaceans

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(Kaushik, 2001; Lall, 2002; Prabhu et al., 2014). Compared with FM-based protein diets, the unbalanced mineral compositions of high SBM-based protein diets may affect the normal growth

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and metabolism of shrimp. Furthermore, the presence of certain antagonists in plant feeds may reduce the availability of minerals, including trace elements. For example, phytic acid binds with

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divalent cationic minerals (such as Ca and Mg), rendering them unavailable to animals (Davis and Gatlin, 1996; Lall, 2002). The reduced availability and potential deficiency of minerals are

with switching to diets high in SBM protein.

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vital for the growth, metabolism and health of shrimp, therefore a concern has been associated

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As a result of exploitation of natural resources and increased consumer demands, L. vannamei farming in low-salinity water and freshwater has increased recently in many regions including the United States, Ecuador, Thailand and China (Saoud et al., 2003; Cheng et al., 2006;

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Roy et al., 2010; Li et al., 2015). Mineral elements are the major osmolytes in the hemolymph of crustaceans and are required to maintain the osmotic pressure of shrimp reared in low-salinity

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media (Pequeux, 1995; Charmantier, 1998; Roy et al., 2010; Huong et al., 2010). The mineral content of low-salinity water is substantially lower than that of seawater (Table 2), which may

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lead to an increased dependence upon dietary minerals. Thus, adding essential mineral elements to high plant protein diets to either make them equivalent to those of fishmeal diets or to meet

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the estimated requirements of the species may be beneficial for shrimp reared in low-salinity water. However, limited research has examined the impacts of dietary mineral deficiencies on shrimp in aquaculture, especially the high plant protein diets. The specific objectives of this study were therefore to determine the effects of replacing FM with SBM in the presence and absence of mineral supplementation on the growth, body and mineral composition, serum and tissue mineral content and osmotic regulation of L. vannamei reared in low-salinity water. 2. Materials and methods 2.1. Experimental diets Six isonitrogenous and isolipidic diets were formulated to contain 400 g/kg crude protein, 90 g/kg crude lipid and 100 g/kg ash (Table 3). Proximate compositions, amino acid and mineral

ACCEPTED MANUSCRIPT profiles of fishmeal and soybean meal were shown in Table 1. The two diets high in fishmeal (FMN and FMR) contained 300 g/kg fishmeal and 200 g/kg soybean meal, whereas the other four diets were high in soybean meal (SBN, SBF, SBR and SBRR) and contained 120 g/kg fishmeal and 466 g/kg soybean meal. The FMN and SBN diets were not supplemented with minerals. The SBF diet was supplemented with minerals to reach a profile equivalent to that of FMN. The FMR and SBR diets were supplemented with different mineral premixes to meet the

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dietary requirements of L. vannamei (Table 5; 5 g/kg Ca, 10 g/kg P, 10 g/kg K, 2.6 g/kg Mg, 32 mg/kg Cu, 70 mg/kg Mn and 33 mg/kg Zn). Finally, the SBRR diet had the same macro-element

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contents as the SBR diet, but had the double micro-element content to address the potentially reduced bioavailability of trace minerals in plant-based diets (Bharadwaj et al., 2014; Read et al.,

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2014). Mineral premix of each experimental diet was presented in Table 4. In addition, fish oil, soybean oil, amino acid premix and silicon dioxide (SiO2) were added (according to Table 3) to

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minimize the differences of fatty acid, amino acid and ash among the diets. [Insert Table 1, Table 2, Table 3 and Table 4 here]

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The dry ingredients were ground using a hammer mill and then passed through a 180 μm mesh. The filtered ingredients were thoroughly mixed with compound lipids (fish oil, soybean oil and lecithin) before water was added to produce a mash. The dough was pelleted through a 1-

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and 2-mm die, using multifunctional spiral extrusion machinery (CD4XITS, South China University of Technology, Guangzhou, China). The pellets were dried at 22 °C in an

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air-conditioned room overnight to a moisture content of 8%–10% and then sealed in plastic bags stored in a freezer at −20 °C until used. The analyses on mineral element concentration of

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experimental diets were presented in Table 5. [Insert Table 5 here]

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As shown in Table 5, the SBN diet, where 60% of fishmeal in the FMN diet was replaced with soybean meal without mineral supplementation, contained lower levels of Ca, P and Na than the FMN diet. Based on the mineral differences between fishmeal and soybean meal, CaCl 2, Ca(H2PO4)2, NaCl and Fe-Met were added to SBF to make Ca, P, Na and Fe levels equal to those in the FMN diet. Because the K, Mg, and Mn contents of the FMN diet and the P, Mg, and Mn contents of the SBN diet were lower than the dietary requirements of L. vannamei, the FMR and SBR diets were supplemented with Mg-Gly, Mn-Met, KCl or NaH2PO4·2H2O. Through supplementation of NaH2PO4·2H2O, Mg-Gly, Cu-Met, Mn-Met and Zn-Met, the SBRR diet had the same concentrations of macro-elements as the SBR diet and the double concentrations of micro-elements in contrast with the FMR and SBR diets, except for Fe, which was high in all

ACCEPTED MANUSCRIPT diets. P was supplemented in the form of Na salts (NaH2PO4·2H2O) in the SBR and SBRR diets rather than via Ca or K salts because of the higher K content and lower Na content in the high-soybean-meal diet (SBN). As a result, the concentrations of dietary K and Na in the FMR, SBF, SBR and SBRR diets were balanced. There were no substantive differences in the rest of the analyzed minerals and all of the analyzed dietary mineral concentrations were proportionate to the targeted supplementation concentrations (Table 5).

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2.2. Feeding trial The study was conducted at the experimental station of Jimei University (Xiamen, China).

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L. vannamei postlarvae were purchased from commercial hatcheries (Zhangpu, Fujian, China) and reared in four circular fiberglass tanks (1,000 L) with newly hatched Artemia nauplii and

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commercial shrimp feed for juveniles. The postlarvae were gradually acclimated from 30‰ to low salinity (2‰) water by adding exposed tap water to reduce the salinity of the rearing water

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by 1‰–2‰ daily. After the shrimp were acclimated to the low salinity water for two weeks, healthy juvenile L. vannamei of a uniform size (average initial weight 0.18 ± 0.02 g, mean ± SD)

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were selected and randomly distributed among 18 aquaria (100 L) at a density of 35 shrimp per aquarium. The aquaria were part of a recirculating system consisting of a reservoir with a biological filter, a circulation pump and an automatic temperature control device supplied with

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aerated water (salinity 2‰). Three aquaria were randomly assigned to each of the six experimental diets and shrimp were hand-fed to apparent satiation four times each day (at 7:00,

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12:00, 17:00 and 22:00). At the beginning of each feeding, water recirculating systems were turned off; feeds were consumed within one hour by feeding every 15 min to reduce the leaching

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loss of feeds in the water; and then uneaten feeds were collected, dried and weighted to determine the daily feed intake. After that, faeces and molts were removed from the aquaria by

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siphoning one hour after each feeding. Each aquarium had a water flow velocity of 5 L/min, and the system had a water renewal rate of 150% per day. During the trial, water was maintained at 29.0 ± 0.5 °C, pH 7.6–8.2, total ammonia–nitrogen 0.05–0.10 mg/L, nitrite nitrogen 0.12–0.17 mg/L and dissolved oxygen > 6.5 mg/L. The natural light/dark regime was maintained throughout the growth trial. Rearing water stored in the circular tanks (5,000 L) was adjusted to a salinity of 2‰ by adding appropriate amounts of seawater into exposed tap water. The mineral compositions of seawater and rearing water used in this study are presented in the Table 2. 2.3. Sample collection and analysis At the end of the feeding trial, shrimp fasted for 24 h before the remaining individuals and

ACCEPTED MANUSCRIPT total wet body weights of shrimp in each aquarium were determined. Six shrimp from each aquarium were randomly sampled and stored at −20 °C for whole-body mineral composition analysis. Hemolymph of another 15 shrimp from each aquarium was withdrawn from the pericardial cavity using a 1 ml syringe and then kept at 4 °C overnight. Serum was obtained after centrifugation at 4,000 × g at 4 °C for 10 min and was pooled across individuals within each aquarium. The exoskeleton, hepatopancreas, gills and abdominal muscle of the fifteen shrimp

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from each aquarium were dissected and pooled together, then stored at −80 °C for further analysis.

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Crude protein, lipid, ash and moisture content of feed ingredients, diets and muscle samples were measured according to the standard methods of the Association of Official Chemists

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(AOAC, 1995). Amino acid profiles of fishmeal and soybean meal were determined by a large-scale comprehensive testing and inspection organization named PONY TEST (Shanghai,

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China). Samples of feed ingredients, diets, whole bodies, muscle tissues and exoskeletons were homogenized and dried at 105 °C to a constant weight prior to mineral analysis. Mineral

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concentrations of the diets, whole-body, muscle, exoskeleton, hepatopancreas, serum, rearing water and seawater were analyzed using an inductively coupled plasma-atomic emission spectrophotometer (ICP-OES, Prodigy7, LEEMAE LABS, USA) after nitric acid digestion as

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described by Read et al. (2014). Osmolality of the serum and water samples were measured by using a freezing-point osmometer (OM806, Loser, Germany). Gill Na+/K+-ATPase and

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Ca2+/Mg2+-ATPase activities were measured according to Roy and Chainy (1996) and Pan et al. (2007). The gill samples were suspended in a standard ice-cold homogenization media (0.25 M

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sucrose and 50 mM Tris–HCl buffer, pH 7.4) to provide a 20% weight by volume preparation and then homogenized at 10,000 RPM for 5 min. The crude extract was obtained by centrifuging

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the homogenate at 11,000 RPM for 20 min at 4 °C. Enzyme activity was expressed as the amount of enzyme that hydrolyses ATP to produce 1.0 μmol inorganic phosphorus (Pi) per hour (μmol Pi/mg protein/h). Protein concentration was determined by the method of Bradford (1976). 2.4. Data calculation Lastly, shrimp were weighed and counted to calculate weight gain (WG), feed conversion ratio (FCR), protein efficiency ratio (PER), survival rate (SR) and daily feeding intake (DFI), according to the formulae: WG = (final weight (g) − initial weight (g)) / initial weight (g) × 100 FCR = feed consumed (g) / weight gain of shrimp (g) PER = weight gain of shrimp (g) / weight of protein consumed (g)

ACCEPTED MANUSCRIPT SR = final number of shrimp/ initial number of shrimp × 100 DFI = (weight of feed consumed/number of shrimp)/days 2.5. Statistical analysis All data were presented as means of three replicates and analyzed using one-way analysis of variance (ANOVA) and Duncan’s multiple range tests. The level of significance was set at P < 0.05. Analyses were performed using SPSS 17.0 (SPSS Inc., Michigan Avenue, Chicago, Illinois,

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USA). 3. Results

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3.1. Rearing water

In this study, the mineral composition and osmolality of rearing water were approximately

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one-tenth of those of seawater, and Na content was highest in comparison to other ions in the water (Table 2).

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3.2. Growth performance and muscle proximate composition

There was no significant difference in survival rate among shrimp fed different diets. Final

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average weight and weight gain of shrimp fed the FMR, SBF, SBR and SBRR diets were also not significantly different (P > 0.05, Table 6). In contrast, the growth performance of shrimp fed the FMN diet was significantly lower than those fed the SBR and SBRR diets (P < 0.05, Table 6).

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PER in shrimp fed the FMR diet was significantly higher than that of shrimp fed the four high-soybean-meal-based diets (P < 0.05, Table 6), but the PER of shrimp fed the FMN diet was

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not significantly different from that of shrimp fed the SBF or SBRR diets (P > 0.05, Table 6). Similarly, FCR and DFI in shrimp fed the high fishmeal diets (FMN, FMR) were significantly

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lower than those of shrimp fed the four high-soybean-meal diets (P < 0.05, Table 6), while the FCR and DFI of shrimp fed the SBF, SBR and SBRR diets were not significantly different from

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each other (P > 0.05, Table 5). The growth performance, PER, FCR and DFI of shrimp fed the FMN diet did not differ significantly from those of shrimp fed the FMR diets (P > 0.05, Table 6). However, shrimp fed the SBN diet had significantly lower WG and PER and significantly higher FCR compared with shrimp fed the other diets (P < 0.05, Table 6). Moisture and crude ash in the muscle tissue of shrimp did not significantly differ among individuals fed different diets (P > 0.05, Table 7). The crude protein content was the lowest for shrimp fed the SBN diet, but the value was not significantly different from those in shrimp fed the other diets, except for the group fed the SBRR diet. The crude lipid levels of shrimp fed the SBF, SBR and SBRR diets were significantly higher than those of shrimp fed the other diets (P < 0.05, Table 7).

ACCEPTED MANUSCRIPT [Insert Table 6 and Table 7 here] 3.3. Gills ATPase activity and serum osmolality In contrast to gills of shrimp fed the other diets, gills of shrimp fed the SBN diet showed significantly greater activities of Na+/K+-ATPase and Ca+/Mg+-ATPase (P < 0.05, Table 8). There were no significant differences among shrimp fed the FMN, SBF, SBR and SBRR diets in their gill Na+/K+-ATPase activity (P > 0.05, Table 8), but shrimp fed the FMR diet had significantly

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lower gill Na+/K+-ATPase activity than those fed the SBF, SBR and SBRR diets (P < 0.05, Table 8). Gill Ca+/Mg+-ATPase activity and serum osmolality of shrimp were not significantly different

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among shrimp fed the FMN, FMR, SBF, SBR and SBRR diets (P > 0.05, Table 8). Although the serum osmolality of shrimp fed the SBN diet was not significantly different from that of shrimp

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fed either the FMN or FMR diets (P > 0.05, Table 8), it was significantly lower than that of shrimp fed the SBF, SBR and SBRR diets which had been supplemented with mineral premixes

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(P < 0.05, Table 8). [Insert Table 8 here]

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3.4. Mineral compositions of whole bodies, muscles and exoskeletons Neither protein sources nor dietary mineral levels had significant effects on Na, K and Mg contents of whole bodies, muscles or exoskeletons of shrimp, nor did they affect muscle P

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content or exoskeleton Fe, Cu, Mn, Zn contents (P > 0.05, Tables 9-11). Whole-body, muscle, and exoskeleton Ca concentrations of shrimp fed the SBN diet were significantly lower than

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those of shrimp fed the other diets (P < 0.05, Tables 9-11). Shrimp fed the SBR and SBRR diets showed significantly higher whole-body Ca contents compared with shrimp fed the FMN diet (P

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< 0.05, Table 9), but no significant differences were found between shrimp fed the FMR and SBF diets (P > 0.05, Table 9). Muscle Ca contents of shrimp fed high fishmeal diets were significantly

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higher than those of shrimp fed high-soybean-meal diets (P < 0.05, Table 10), but muscle Ca content data showed no differences between the shrimp fed the FMN and FMR diets (P > 0.05, Table 10), nor among the shrimp fed the SBF, SBR and SBRR diets. Exoskeleton Ca content did not differ significantly among shrimp fed the FMN, FMR, SBF, SBR and SBRR diets (P > 0.05, Table 11). Whole-body P content was only significantly different between shrimp fed the SBN and SBRR diets (P < 0.05, Table 9), whereas exoskeleton P content only was significantly different between shrimp fed the SBF diet and those fed either the SBN or FMR diets (P < 0.05, Table 11). Although the differences were not always statistically significant, the Fe, Mn and Zn contents of whole bodies and muscle tissue were lowest for shrimp fed the SBF diet. Shrimp fed the SBRR diet (which contained double micro-element levels in comparison with other diets)

ACCEPTED MANUSCRIPT had significantly higher whole-body Cu content and muscle Cu and Mn contents than shrimp fed the other diets (P < 0.05, Tables 9 and 10), but did not show significant differences in the Zn content of whole bodies or muscle tissues (Tables 9 and 10). The muscle Mn content of shrimp fed the FMR diet was significantly higher than that of shrimp fed the FMN, SBN and SBF diets (P < 0.05, Table 10), but not significantly different from shrimp fed the SBR diet (P > 0.05, Table 10).

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[Insert Table 9, Table 10 and Table 11 here] 3.5. Mineral compositions of hepatopancreas and serum

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There were no significant differences in the Ca, P, Na, K, Mg, Zn and Fe contents of either the hepatopancreas or serum of shrimp, regardless of their diets (P > 0.05, Tables 12 and 13). Shrimp

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fed the SBRR diet displayed significantly higher Cu levels in the hepatopancreas and serum than those fed the other diets (P < 0.05, Tables 12 and 13). Hepatopancreas Mn contents of shrimp fed

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the FMR, SBR and SBRR diets were significantly higher than those of shrimp fed the FMN, SBN and SBF diets (P < 0.05, Table 12). Serum Mn and Zn contents were lowest in shrimp fed

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the SBF diet, and were highest in shrimp fed the SBR and SBRR diets (Table 13). [Insert Table 12 and Table 13 here] 4. Discussion

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The growth performance of shrimp during an 8-week feeding trial was not significantly influenced by soybean meal substituting for 60% fishmeal in diets as long as the feed was

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supplemented with minerals. These results indicate that the fishmeal content of a practical L. vannamei diet can be as low as 120 g/kg, and adding minerals to feeds high in soybean meal (and

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thus low in fishmeal) is essential for L. vannamei reared in low-salinity water. Although this species can tolerate a wide range of salinities from 0.5 to 40 g/L (Saoud et al., 2003), the

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elements available from seawater are not able to meet requirements for growth and metabolism of shrimp (Davis and Gatlin, 1996; Saoud et al., 2003; Shiau and Bai, 2009; Bharadwaj et al., 2014). Water used in this study was at a salinity of 2 ‰, which had substantially lower ionic concentrations than seawater (Table 2). Such a low-salinity medium could increase the dependence on dietary minerals for shrimp (Li et al., 2015). In contrast to fishmeal, soybean meal used in this study had lower levels of Ca, P and Na, resulting in significantly lower contents of these mineral elements in the diet with 60% substitution of fishmeal by soybean meal but no mineral supplementation (SBN). Ca, P and Na, three of the essential macronutrients, are of great physiological importance in aquatic animals, affecting tissue mineralization, blood clotting, muscle function, nerve impulse transmission, enzymatic processes, osmoregulation and

ACCEPTED MANUSCRIPT energy-yielding reactions (NRC, 2011). Therefore, deficiencies of these minerals were likely to cause the significantly lower WG and PER and higher FCR in shrimp fed the SBN diet compared with those fed the other diets. In addition, increasing the soybean meal content to replace fishmeal in feeds can result in a further reduction in the availability of Ca, P and some trace minerals due to the presence of phytic acid, which is the storage form of P and can readily bind divalent cations (Davis and Gatlin, 1996; Lall, 2002). Dietary supplementation of key minerals

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potentially counteracted the reduced availability and potential deficiency of minerals in diets containing high level of soybean meal. Consequently, the present study showed that supplying

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relevant minerals to high soybean meal diets had positive effects on growth performance, PER and FCR for shrimp reared in low-salinity water.

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Interestingly, without Ca supplementation, high soybean meal diets containing 9.8 g/kg total Ca and 11.8 g/kg total P appeared to meet the requirements of dietary Ca and P for L. vannamei,

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considering that the best growth performance was observed in shrimp fed the SBR and SBRR diets. Similarly, Cheng et al. (2006) reported that diets containing 5 g/kg total Ca and 10 g/kg

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total P were adequate for the optimal growth of L. vannamei reared in low-salinity water (63 mg/L Ca). The Ca requirement of shrimp could be met either from the basal diet alone or in conjunction with direct uptake from the water (Cheng et al., 2006). Furthermore, high dietary Ca

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levels demanded higher levels of dietary P, otherwise excess Ca could have negative effects on growth or tissue mineralization, as shown in Penaeus monodon and L. vannamei (Davis et al.,

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1993c; Peñaflorida, 1999; Cheng et al., 2006). For this reason, Ca content in practical shrimp feeds should be reduced. Conversely, adequate dietary P was necessary for better growth

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performance for L. vannamei reared in either low-salinity or sea water relative to its low concentration compared with other macroelements in natural water (Davis et al., 1993c; Gong et

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al., 2004; Cheng et al., 2006; Roy et al., 2007 ). Especially when fishmeal is replaced by soybean meal, the first limiting mineral in shrimp feed formulation is P as only 30–40% of total P content in soybean meal is available for L. vannamei, and hence supplemental P is essential for optimal shrimp growth as fishmeal was removed (Sookying et al., 2013). In this study, P supplements might be the main reason for the better growth performances of shrimp fed the SBF, SBR and SBRR diets than those fed the SBN diet. Although no optimal dietary Ca:P ratio had been determined for L. vannamei, Cheng et al. (2006) found that dietary Ca:P ratios for good growth varied from 0.21 to 0.73. Therefore, the higher ratios of Ca to P in the FMR, FMN and SBF diets (Ca:P ≈ 1.3, Table 5) might not be suitable for the optimal growth of shrimp. Supporting this, the FMR and SBR diets had similar mineral compositions except for the FMR diet having an

ACCEPTED MANUSCRIPT elevated Ca concentration, but the growth performance of shrimp fed the SBR diet seemed to be better than that of shrimp fed the FMR diet. These results suggest that, unlike P, dietary Ca supplementation is not necessary for L. vannamei fed high soybean meal diets and reared in low-salinity conditions. It is known that aquatic animals can obtain maximum growth in isosmotic media, for it would be expending the minimal amount of energy in osmotic regulation (Le Moullac and

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Haffner, 2000). In the present study, shrimp had to face a diffusive loss of salts from the hemolymph to the low salinity medium (Pequeux, 1995), given the large difference in osmolality

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and ion concentrations between the rearing water and the shrimp serum (Tables 2, 8, 13). Shrimp in low salinity water must regulate constantly to maintain homeostasis depending largely on

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osmoregulation via the change of various enzymes and transporters, for instance, an increased Na+/K+-ATPase activity in gills, but the physiological adaptations to these functional changes are

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highly energy demanding (Pequeux, 1995; Furriel et al., 2000; Tseng and Hwang, 2008; Huong et al., 2010). Then lipids, proteins and carbohydrates from tissues or diets were more utilized as

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energy substrates to meet the increased energy consumption for osmotic and ionic regulation, which could negatively affect feed efficiency, growth and energy storages of shrimp at low salinity (Mommsen et al., 1999; Palacios et al., 2004; Li et al., 2015). Generally, fishmeal had a

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higher apparent digestibility of energy than soybean meal for L. vannamei (NRC, 2011), which potentially reduced the energy availability of diets high in soybean meal. Hence, in order to meet

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the increased energy requirement for growth and osmoregulation at low salinity, shrimp were likely to actively ingest more soybean meal diets compared to fishmeal diets. In a way, this

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might explain the increase of feed consumption and feed conversion ratio of shrimp fed high soybean meal diets.

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Soluble minerals including calcium, sodium, potassium and chloride, are closely related to the osmoregulatory function (Pequeux, 1995). Accordingly, dietary supplements of selected minerals could partly increase the osmoregulatory ability and facilitate better survival, growth and feed efficiency of shrimp reared in low-salinity conditions (Saoud et al., 2003; Roy et al., 2010; Li et al., 2015). For example, supplementation of KCl, MgO, NaCl, cholesterol and lecithin into diets improved the osmoregulatory capacity of L. vannamei reared in low salinity waters in Arizona (Gong et al., 2004). Likewise, in this study, shrimp fed the SBF, SBR and SBRR diets with mineral supplements had an increased osmoregulatory ability in terms of their significantly higher serum osmolality than that of shrimp fed the SBN diet. Dietary supplements of selected minerals could possibly mitigate a lack of ions at the gill-water interface by

ACCEPTED MANUSCRIPT increasing their availability and absorption in the digestive tract (Roy et al., 2010). Therefore, it was more likely that shrimp fed the SBN diet without mineral supplements had to cost more energy substrates form tissues or feeds on maintaining the internal osmotic and ionic homeostasis through fortifying the participation of various ion exchange enzymes and transporters, which could be reflected by the significantly high ATPase activities in gills and the significantly low growth and feed efficiency in the SBN group. Correspondingly, higher levels of

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protein and lipid in muscle and lower ATPase activities in gills of shrimp potentially indicated that energy of the SBF, SBR and SBRR diets could be more devoted to growth, not just

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osmoregulation of shrimp at low salinity, which explained the increased growth and PER and the decreased FCR in these groups compared with the SBN group. However, these results suggest

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that dietary supplementation of key minerals to high soybean meal diets is necessary for normal growth, osmoregulation and energy deposition of L. vannamei reared in low-salinity media.

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Mineral compositions of edible and non-edible portions of farmed fish and shrimp is linked to the mineral levels of their feeds (Cheng et al., 2006; Roy et al., 2007; Fallah et al., 2011;

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Bharadwaj et al., 2014; Prabhu et al., 2014). In the present study, unlike other mineral elements, Ca and P concentrations in whole bodies, muscle tissue and exoskeletons were affected by the replacement of fishmeal with soybean meal, although the differences were not always

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statistically significant. Kousoulaki et al. (2010) demonstrated that cod could actively preserve P but not Ca when there was a dietary inadequacy of P. This agreed with our observations that Ca

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accumulation was significantly reduced, other than P accumulation, in shrimp fed the SBN diets which contained lower P than the other diets. However, Cheng et al. (2006) reported that in the

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presence of adequate dietary P (over 10 g/kg total P), L. vannamei reared in low-Ca water and fed diets with 0–10 g/kg supplemental Ca had similar mineral profiles of their muscle and

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exoskeleton. Similarly, P, Ca, Mg, Mn and Zn in whole body of cod significantly increased with increasing dietary P supplementation, whereas dietary Ca supplementation had no such effect (Kousoulaki et al., 2010). These results indicated that dietary P was important for tissue mineralization, which was consistent with the results of this study. Dietary supplementation of P but not Ca in the SBR and SBRR diets (approximate 9.8 g/kg total Ca and 11.9 g/kg total P) significantly increased the Ca contents of whole bodies, muscle tissues and exoskeletons in relation to shrimp fed the SBN diet which contained 9.7 g/kg total Ca but only 8.5 g/kg total P. This result suggests that the reduced tissue calcification in shrimp fed the SBN diet is the result of a lack of dietary P. Therefore, the potential deficiency of P in high soybean diets should be of concern, as P was a vital macro element required for tissue mineralization and growth of shrimp.

ACCEPTED MANUSCRIPT Interestingly, although the soybean meal used in this study had a much lower Na content than the fishmeal (Table 1), the Na content of shrimp was not significantly affected by replacement of fishmeal with soybean meal. Regardless of salinity, L. vannamei are typically strong hypo-hyperosmoregulators that maintain a relatively stable haemolymph osmolality and internal ionic homeostasis, especially the Na+ and Cl– content as major osmolytes in haemolymph, which are regulated mainly by the posterior pairs of gills (Pequeux, 1995;

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Charmantier, 1998; Lucu and Towle, 2003). The higher level of Na+/K+-ATPase activity in the gills indicates that shrimp fed the SBN diet without mineral supplements potentially had actively

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transferred more Na+ from cell to hemolymph for maintaining a steady Na concentration (Roy et al., 2010), in spite of the relatively Na content in the SBN diet. Moreover, compared to other

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mineral elements, farming waters were adequate in Na which could be readily obtained by shrimp (Saoud et al., 2003), dietary Na supplements always seemed to be unnecessary for shrimp

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reared in seawater and low salinity waters (Davis and Gatlin, 1996; Roy et al., 2007, 2010; Li et al., 2015). Therefore, although supplementation of Na with other minerals into the high soybean

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meal diets improved the osmoregulatory capacity of L. vannamei, it was needed to further examine the role of dietary Na for this specie acclimated to low salinity. In contrast to the patterns seen for Ca, P and Na, the K and Mg concentrations of soybean

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meal were not lower than those of fishmeal, with the result that the SBN diet had higher dietary K and Mg concentrations than the FMN diet. Both K and Mg are essential elements for normal

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growth, survival, and osmoregulatory function of crustaceans (Pequeux, 1995; Gong et al., 2004; Cheng et al., 2005; Roy et al., 2007). In low salinity waters (2–4 g/L), dietary Mg and K

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requirements for optimal growth of L. vannamei were 2.6–3.4 and 10–15 g/kg diet, respectively (Cheng et al., 2005; Roy et al., 2007; Liu et al., 2014), which were close to the Mg and K levels

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in the diets used for this study. Additionally, replacement of fishmeal by soybean meal had no significant influences on the tissue mineralization of K and Mg in shrimp. These findings indicate that for shrimp, it seems that K and Mg are unnecessary to be supplemented into diets containing high levels of soybean protein. Likewise, results from this study showed that tissue mineralization levels of Fe, Cu, Zn and Mn in shrimp were not affected by substitution of fishmeal with soybean meal, despite the large differences in mineral concentrations of the two protein sources (Table 1). Although these microelements were essential for normal growth, metabolism and health of shrimp (Davis and Gatlin, 1996), double levels of Cu, Zn and Mn in the SBRR diet (relative to the SBR diet) failed to further improve growth performance, indicating that shrimp were more likely to have met

ACCEPTED MANUSCRIPT their needs for these trace elements from farming water and testing diets which already contained dietary required levels of Cu 32 mg/kg, Mn 70 mg/kg and Zn 33 mg/kg for L. vannamei (Davis et al., 1993a; Davis et al., 1993b; Liu and Lawrence, 1997). In regards to Fe, Davis et al. (1992) observed that practical diets of shrimp should not require Fe supplementation, by reason of the high concentration of Fe in base ingredients. This was in agreement with our observations that fishmeal and soybean meal were both rich in Fe and subsequently that the Fe levels of the six

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test diets were very high and quite similar to each other. Interestingly, shrimp fed the soybean-heavy diet supplemented with Ca salts (SBF) had lower Fe, Mn and Zn deposition in

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whole body, which aligned with the results from other studies that Ca supplementation induced a significant decrease in other bivalent minerals in fish (Storebakken et al., 1998; Hossain and

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Furuichi, 2000; Kousoulaki et al., 2010; Prabhu et al., 2014; Song et al., 2016). This pattern was probably due to competitive inhibition of these cations during intestinal absorption (Roy and Lall,

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2003). Conversely, the proportions of bivalent minerals in the SBR diet appeared to be more suitable for the growth and tissue mineralization of shrimp. However, to date, few studies have

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examined either the antagonistic interactions of these bivalent minerals (including their effects on absorption and utilization) or the dietary requirements of these trace elements for shrimp reared in low-salinity water.

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5. Conclusions

These results suggest that the mineral composition of soybean meal is one of the key factors

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underlying the growth and physiological effects seen with the replacement of dietary fishmeal for L. vannamei reared in 2‰ salinity water. Therefore, it is necessary to develop practical low

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fishmeal diets based on a balanced mineral composition, especially for diets containing a high proportion of conventional plant protein sources.

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Acknowledgements

The authors wish to acknowledge the Special Fund for Agro-scientific Research in the Public Interest (Grant No. 201303053) and National Natural Science Foundation of China (Grant No. 31572625) for supporting this research work. References Amaya, E.A., Davis, D.A., Rouse, D.B., 2007. Replacement of fish meal in practical diets for the pacific white shrimp (Litopenaeus vannamei) reared under pond conditions. Aquaculture 262, 393-401. AOAC (Association of Official Analytical Chemists), 1995. Official Methods of Analysis of the Association of Official Analytical Chemists International, 16th ed. Association of Official Analytical Chemists, Arlington, V.A.

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Bharadwaj, A.S., Patnaik, S., Browdy, C.L., Lawrence, A.L., 2014. Comparative evaluation of an inorganic and a commercial chelated copper source in pacific white shrimp Litopenaeus vannamei, (Boone) fed diets containing phytic acid. Aquaculture 422, 63-68. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248-254. Charmantier, G., 1998. Ontogeny of osmoregulation in Crustaceans: a review. Inv. Repr. Dev. 33, 177-190. Cheng, K.M., Hu, C.Q., Liu, Y.N., Zheng, S.X., Qi, X.J., 2005. Dietary magnesium requirement and physiological responses of marine shrimp Litopenaeus vannamei, reared in low salinity water. Aquac. Nutr. 11, 385-393. Cheng, K.M., Hu, C.Q., Liu, Y.N., Zheng, S.X., Qi, X.J., 2006. Effects of dietary calcium, phosphorus and calcium/phosphorus ratio on the growth and tissue mineralization of Litopenaeus vannamei reared in low-salinity water. Aquaculture 251, 472-483. Davis, D.A., Lawrence, A.L., Gatlin III, D.M., 1992. Evaluation of the dietary iron requirement of Penaeus vannamei. J. World Aquacult. Soc. 23, 15-22. Davis, D.A., Lawrence, A.L., Gatlin III, D.M., 1993a. Dietary copper requirement of Penaeus vannamei. Nippon Suisan Gakk. 59, 117-122. Davis, D.A., Lawrence, A.L., Gatlin III, D.M., 1993b. Evaluation of the dietary zinc requirement of Penaeus vannamei and effects of phytic acid on zinc and phosphorus bioavailability. J. World Aquacult. Soc. 24, 40-47. Davis, D.A., Lawrence, A.L., Gatlin III, D.M., 1993c. Response of Penaeus vannamei to dietary calcium, phosphorus and calcium:phosphorus ratio. J. World Aquacult. Soc. 24, 504-515. Davis, D.A., Gatlin III, D.M., 1996. Dietary mineral requirements of fish and marine crustaceans. Rev. Fish. Sci. 4, 75-99. Fallah, A.A., Saei-Dehkordi, S.S., Nematollahi, A., Jafari, T., 2011. Comparative study of heavy metal and trace element accumulation in edible tissues of farmed and wild rainbow trout (Oncorhynchus mykiss) using ICP-OES technique. Microchem. J. 98, 275-279. Furriel, R.P.M., McNamara, J.C., Leone, F.A., 2000. Characterization of (Na+, K+)-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii. Comp. Biochem. Physiol. 126B, 303-315. Gong, H., Jiang, D.H., Lightner, D.V., Collins, C., Brock, D., 2004. A dietary modification approach to improve the osmoregulatory capacity of Litopenaeus vannamei cultured in the Arizona desert. Aquac. Nutr. 10, 227-236. Hardy, R.W., 2010. Utilization of plant proteins in fish diets: effects of global demand and supplies of fishmeal. Aquac. Res. 41, 770-776. Hien, T.T.T., Be, T.T., Lee, C.M., Bengtson, D.A., 2015. Development of formulated diets for snakehead (Channa striata and Channa micropeltes): Can phytase and taurine supplementation increase use of soybean meal to replace fish meal? Aquaculture 448, 334-340.

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Pan, L.Q., Zhang, L.J., Liu, H.Y., 2007. Effects of salinity and pH on ion-transport enzyme activities, survival and growth of Litopenaeus vannamei postlarvae. Aquaculture 273, 711-720. Paripatananont, T., Boonyaratpalin, M., Pengseng, P., Chotipuntu, P., 2002. Substitution of soy protein concentrate for fishmeal in diets of tiger shrimp Penaeus monodon. Aquac. Res. 32, 369-374. Peñaflorida, V.D., 1999. Interaction between dietary levels of calcium and phosphorus on growth of juvenile shrimp, Penaeus monodon. Aquaculture 172, 281-289. Pequeux, A., 1995. Osmotic regulation in crustaceans. J. Crustacean Biol. 15, 1-60. Prabhu, P.A.J., Schrama, J.W., Mariojouls, C., Godin, S., Fontagné-Dicharry, S., Geurden, I., 2014. Post-prandial changes in plasma mineral levels in rainbow trout fed a complete plant ingredient based diet and the effect of supplemental di-calcium phosphate. Aquaculture 430, 34-43. Rahman, S.H.A., Razek, F.A.A., Goda, A.S., Ghobashy, A.F.A., Taha, S.M., Khafagy, A.R., 2010. Partial substitution of dietary fish meal with soybean meal for speckled shrimp, Metapenaeus monoceros (Fabricius, 1798) (Decapoda: Penaeidae) juvenile. Aquac. Res. 41, 299-306. Read, E.S., Barrows, F.T., Gaylord, T.G., Paterson, J., Petersen, M.K., Sealey, W.M., 2014. Investigation of the effects of dietary protein source on copper and zinc bioavailability in fishmeal and plant-based diets for rainbow trout. Aquaculture, 432, 97-105. Riche, M., 2015. Nitrogen utilization from diets with refined and blended poultry by-products as partial fishmeal replacements in diets for low-salinity cultured Florida pompano, Trachinotus carolinus. Aquaculture, 435, 458-466. Roy, A., Chainy, G.B.N., 1996. Age-related change in rat testicular ATPase activities in response to HCH treatment. Bull. Environ. Contam. Toxicol. 56, 165-170. Roy, L.A., Davis, D.A., Saoud, I.P., Henry, R.P., 2007. Supplementation of potassium, magnesium and sodium chloride in practical diets for the pacific white shrimp, Litopenaeus vannamei, reared in low salinity waters. Aquac. Nutr. 13, 104-113. Roy, L.A., Davis, D.A., Saoud, I.P., 2010. Shrimp culture in inland low salinity waters. Rev. Aquacult. 2, 191-208. Roy, P.K., Lall, S.P., 2003. Dietary phosphorus requirement of juvenile haddock (Melanogrammus aeglefinus L.). Aquaculture 221, 451-468. Samocha, T.M., Davis, D.A., Saoud, I.P., Debault, K., 2004. Substitution of fish meal by co-extruded soybean poultry by-product meal in practical diets for the pacific white shrimp, Litopenaeus vannamei. Aquaculture 231, 197-203. Saoud, I.P., Davis, D.A., Rouse, D.B., 2003. Suitability studies of inland well waters for Litopenaeus vannamei culture. Aquaculture 217, 373-383. Shiau, S.Y., Bai, S.C., 2009. Micronutrients in shrimp diets. In: Browdy, C.L., Jory, D.E. (Eds.), The Rising Tide, Proceedings of the Special Session on Sustainable Shrimp Farming, World Aquaculture 2009. The World Aquaculture Society, Baton Rouge, Louisiana, USA.

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ACCEPTED MANUSCRIPT Table 1 Composition and proximate analyses of fish meal1and soybean meal2 (dry-matter basis). Fish meal

Soybean meal

Dry matter (%) Crude protein (%) Crude lipid (%) Crude ash (%) Amino acids

91.92 67.68 7.86 16.42

89.14 47.82 1.56 5.94

Alanine (Ala %) Arginine (Arg %) Aspartic (Asp %) Cysteine (Cys %) Glutamate (Glu %) Glycine (Gly %) Histidine (His %) Isoleucine (Ile %)

3.52 3.24 5.17 0.54 7.14 3.25 1.83 2.39 4.27 4.66 2.07 2.46 2.23 2.50 0.52 1.88 2.77

3.40 3.10 0.44 2.25 2.20 1.79 0.46 1.42 2.08

Mineral elements Calcium (Ca %) Phosphorus (P %) Sodium (Na %) Potassium (K %) Magnesium (Mg %) Iron (Fe mg/kg) Copper (Cu mg/kg)

3.03 2.16 1.11 0.85 0.30 912.03 30.76

0.28 0.55 0.03 1.60 0.29 136.03 29.52

Manganese (Mn mg/kg) Zinc (Zn mg/kg)

12.66 48.14

38.72 55.74

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Leucine (Leu %) Lysine (Lys %) Methionine (Met %) Phenylalanine (Phe %) Serine (Ser %) Threonine (Thr %) Tryptophan (Trp %) Tyrosine (Tyr %) Valine (Val %)

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1.92 3.04 4.93 0.53 8.05 1.89 1.18 1.93

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Ingredients

1

Menhaden fish meal, obtained from Tecnológica de Alimentos S.A., Peru.

2

Soybean meal, obtained from Quanzhou Fuhai cereals and oils industry Co., Ltd.

ACCEPTED MANUSCRIPT Table 2 The analysis on mineral element concentration and osmolality of seawater and rearing water in this study. Sea water

Farming water

Ca (mg/kg)

593.28

54.12

P (mg/kg)

1.66

0.60

Na (mg/kg)

8432.23

623.40

K (mg/kg)

685.91

Mg (mg/kg)

1331.55

Fe1 (mg/kg)

0.001

Cu1 (mg/kg)

<0.001

<0.001

Mn1 (mg/kg)

0.004

<0.001

Zn1 (mg/kg)

0.003

<0.001

30

2

1288

106

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Salinity (‰)

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Osmolality (mOsm/kg) 1

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Mineral elements

63.77 83.65 <0.001

The microelement concentration of seawater or rearing water in this study was below

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the detection limit of the instrument, being regarded as <0.001 mg/kg.

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Table 3 Formulation and proximate composition of experimental diets (dry-matter basis). FMN

FMR

SBN

SBF

SBR

SBRR

Fish meal Soybean meal Shrimp meal

300 200 100

300 200 100

120 466 100

120 466 100

120 466 100

120 466 100

Microcrystalline cellulose

43.0

43.0

－

－

－

－

Alpha-starch Fish oil Soybean oil Lecithin Choline chloride Cholesterol Vitamin premixa

260 20 20 10 5 5 5

260 20 20 10 5 5 5

Amino acid premixb

－

－

188 35 16 10 5 5 5 5.3

188 35 16 10 5 5 5 5.3

188 35 16 10 5 5 5 5.3

188 35 16 10 5 5 5 5.3

8.0

－

27.2

13.7

14.6

14.6

6.6

27.2

－

13.5

12.6

0.5 5 10 1.5 0.5

0.5 5 10 1.5 0.5

0.5 5 10 1.5 0.5

0.5 5 10 1.5 0.5

0.5 5 10 1.5 0.5

0.5 5 10 1.5 0.5

72.5 390.1 94.9 103.4 19.2

69.8 394.3 94.1 102.8 19.3

73.3 408.2 95.7 107.6 19.0

79.3 401.1 92.3 108.2 18.7

74.3 402.4 96.0 108.5 18.9

74.4 401.1 94.1 107.9 18.8

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SiO2d Vitamin C Yeast nucleic acid Sodium alginate Antiseptic Antioxidant Proximate composition Moisture (g/kg) Crude protein (g/kg) Crude lipid (g/kg) Ash (g/kg) Gross energy (kJ/g) e

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－

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Mineral premix

c

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Ingredients (g/kg)

a

Vitamin premix was according to Ye et al. (2012).

b

Amino acid premix (g/kg diet): Met 2.8 g, His 0.15 g, Lys 0.12 g, Gly 0.86 g, Ala 1.32 g.

c

Mineral premix of each experimental diet was presented in Table 4.

d

SiO2 was used to keep the same ash level of each diet treatment.

e

Calculated values based on 23.6, 39.5 and 17.2 kJ/g for protein, lipid and carbohydrate,

respectively.

ACCEPTED MANUSCRIPT Table 4 Mineral premix of each experimental diet (dry-matter basis). FMR

SBF

SBR

SBRR

－

9.8

－

－

Ca(H2PO4)2

－

10.8

－

－

NaH2PO4·2H2O

－

－

12.4

12.4

KCl

4.3

－

－

－

NaCl

－

5.5

－

－

Mg-Gly 1

3.3

－

1.0

1.0

Fe-Met 1

－

1.1

－

－

Cu-Met 1

－

－

－

0.24

Mn-Met 1

0.4

－

0.3

0.78

Zn-Met 1

－

－

－

0.18

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1

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Minerals (g/kg) CaCl2

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The metal amino acid chelates Mg-Gly, Fe-Met, Cu-Met, Mn-Met and Zn-Met respectively contain 11% Mg, 13% Fe, 17% Cu, 15% Mn and 19% Zn.

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Table 5 The analysis on mineral element concentration of experimental diets (dry-matter basis) 1.

T P

SBRR

DR1

9.74±0.09

5.00

11.90±0.44

11.74±0.82

10.00

13.56±0.64

13.56±0.35

13.44±0.84

10.00

6.26±0.40

8.43±0.56

8.42±0.76

－

2.55±0.16

2.49±0.20

2.47±0.16

2.60±0.19

2.59±0.36

2.60

719.68±39.31

690.86±40.15

707.10±10.63

N A

8.44±0.16

710.03±71.02

701.80±18.01

709.84±16.84

－

Cu (mg/kg)

42.08±1.99

39.78±2.93

41.05±1.02

41.04±2.50

44.16±1.12

78.50±2.59

32.00

Zn (mg/kg)

46.41±1.10

49.74±1.15

45.94±1.91

45.43±1.69

47.80±2.57

74.06±3.23

33.00

Mn (mg/kg)

23.56±2.07

88.33±4.25

36.00±2.25

33.33±2.20

88.44±2.99

167.00±12.63

70.00

Ca/P

1.37±0.10

1.13±0.09

1.27±0.02

0.83±0.03

0.83±0.07

0.50

Treatments

FMN

FMR

SBN

SBF

SBR

Ca (g/kg)

16.12±0.24

16.08±0.12

9.70±0.05

14.28±0.13

9.89±0.09

P (g/kg)

11.78±0.81

11.66±0.75

8.53±0.54

11.29±0.20

K (g/kg)

9.78±0.60

11.57±0.61

13.12±0.83

Na (g/kg)

8.56±0.63

8.73±0.98

Mg (g/kg)

2.24±0.24

Fe (mg/kg)

D E

T P E

C C

1.38±0.07

1

I R

C S U

M

Values were presented as means ± standard deviation of three replications. 2 DR, dietary mineral requirements of L. vannamei; Ca, P and Ca/P from Cheng et al. (2006); K from Roy et al. (2007); Mg from Cheng et al. (2005); Cu from Davis et al. (1993a); Mn from Liu and Lawrence (1997); Zn from Davis et al. (1993b); a supplementation of dietary Na or Fe was needless (Davis and Gatlin, 1996).

A

ACCEPTED MANUSCRIPT

Table 6 Growth performance of Litopenaeus vannamei fed experimental diets for 8 weeks1.

1206.05±25.39 1.80±0.05a 1.39±0.0

81.11±1.1

49.37±1.7

b

1

2b

WG (%)

2.35±0.05 b

PER (%)

b

ab

1.37±0.0

83.33±1.9

50.85±1.7

3c

2

3b

1.93±0.0

77.78±1.1

60.39±0.7

d

3a

1

6a

2.49±0.10a 1285.68±53.50 1.62±0.10

1.58±0.0

77.78±1.1

61.85±2.4

b

4b

1

0a

1.60±0.0

80.00±1.9

64.17±2.2

3b

2

6a

1347.04±19.05 1.77±0.09

1.56±0.0

77.78±2.9

63.87±1.7

a

1b

4

6a

ab

SBR

2.59±0.09a

SBRR

2.60±0.03a

bc

1338.15±48.14 a

1.56±0.03c

MA

SBF

1.85±0.05a 1.25±0.05

1.93±0.06c 971.23±32.84c

SBN

3c

2

RI

b

FCR

2

PT

FAW (g)

2

2.41±0.04a 1239.26±21.20

FMR

b

Values were presented as means ± standard error of three replications. Means in the

D

1

SR (%)

DFI 2 (mg/d)

2

SC

FMN

2

NU

Treatmen ts

PT E

same column with different superscripts are significantly different (P <0.05). 2 FAW, final average weight; WG, weight gain; PER, protein efficiency ratio; FCR, feed conversion ratio; SR, survival rate; DFI, daily feed intake.

Moisture

Crude protein

Crude lipid

Crude ash

78.67±0.13

17.89±0.12ab

0.71±0.02b

1.19±0.03

78.72±0.11

17.79±0.14ab

0.72±0.00b

1.15±0.03

SBN

78.79±0.14

17.71±0.23b

0.70±0.01b

1.17±0.09

SBF

78.62±0.14

18.14±0.33ab

0.80±0.01a

1.18±0.03

SBR

78.32±0.29

18.24±0.10ab

0.79±0.02a

1.18±0.04

SBRR

78.68±0.06

18.35±0.17a

0.79±0.02a

1.14±0.05

FMN FMR

1

AC

Treatments

CE

Table 7 Muscle proximate composition of Litopenaeus vannamei fed experimental diets for 8 weeks (%) 1.

Values were presented as means ± standard error of three replications. Means in the same column with different superscripts were significantly different (P <0.05).

ACCEPTED MANUSCRIPT Table 8 Gills ATPase activity and serum osmolality of Litopenaeus vannamei fed experimental diets for 8 weeks1. Serum osmolality (mOsm/kg)

FMN

4.17±0.13bc

2.56±0.12b

568.33±1.76ab

FMR

3.96±0.16c

2.26±0.16b

571.33±4.67ab

SBN

7.13±0.25a

4.25±0.15a

SBF

4.80±0.20b

2.23±0.19b

SBR

5.00±0.34b

2.35±0.20b

SBRR

4.78±0.32b

2.45±0.09b

PT

Treatments

Na+/K+-ATPase Ca+/Mg+-ATPase (μmol Pi/mg (μmol Pi/mg protein/h) protein/h)

SC

RI

557.67±2.90b

1

578.67±5.24a 577.67±4.48a 577.00±5.03a

AC

CE

PT E

D

MA

NU

Values were presented as means ± standard error of three replications. Means in the same column with different superscripts were significantly different (P <0.05).

ACCEPTED MANUSCRIPT Table 9 Whole body mineral composition of Litopenaeus vannamei fed experimental diets for 8 weeks (dry-matter basis) 1. Treatment s

Ca (g/kg)

P (g/kg)

Na (g/kg)

K (g/kg)

Mg (g/kg)

Fe (mg/kg)

FMN

34.48±0.49b

13.02±0.44 ab

11.49±0.29

14.96±0.20

3.50±0.11

38.04±3.13a

FMR

35.37±0.58ab

13.01±0.35ab

11.74±0.62

13.94±0.25

3.55±0.05

SBN

32.93±0.24c

12.63±0.10b

11.86±0.15

13.98±0.81

3.40±0.21

SBF

35.82±0.19ab

13.12±0.08ab

11.70±0.97

14.52±0.59

3.49±0.01

SBR

36.14±0.09

a

12.98±0.10ba

36.33±0.15

a

SBRR 1

b

D E

11.44±0.30

T P

E C

13.63±0.04

C A

a

12.27±0.97

Mn (mg/kg)

58.20±1.56b

7.46±0.03a

38.56±1.88a

57.14±2.50b

7.80±0.11a

32.07±2.67a

57.42±2.32b

6.84±0.04a

b

59.02±1.46b

5.27±0.09b

T P

I R

C S

U N

A M

Cu (mg/kg)

25.80±1.39

14.47±0.58

3.43±0.06

33.40±2.96a

56.91±2.17b

7.47±0.07a

14.46±0.63

3.52±0.05

33.15±2.82a

67.09±3.03a

7.49±0.03a

Zn (mg/kg) 72.06±1.6 2ab 73.87±2.2 9a 73.78±3.3 7a 65.88±1.6 3b 71.52±1.6 7ab 71.22±2.8 4ab

Values were presented as means ± standard error of three replications. Means in the same column with different superscripts were significantly different (P <0.05).

ACCEPTED MANUSCRIPT Table 10 Muscle mineral composition of Litopenaeus vannamei fed experimental diets for 8 weeks (dry-matter basis) 1. Treatment s

Ca (g/kg)

P (g/kg)

Na (g/kg)）

K (g/kg)

Mg (g/kg)

Fe (mg/kg)

FMN

6.52±0.08a

9.60±0.36

7.56±0.18

13.52±0.17

1.87±0.05

30.57±1.21ab

FMR

6.47±0.10a

9.57±0.37

7.61±0.16

13.57±0. 22

1.92±0.05

36.28±3.16a

SBN

5.60±0.03c

9.13±0.12

7.35±0.29

13.13±0.29

1.90±0.06

SBF

6.07±0.07b

9.58±0.35

7.38±0.19

13.53±0.23

SBR

6.14±0.04b

9.73±0.21

7.41±0.19

D E

b

9.82±0.20

SBRR 1

6.03±0.13

C A

E C

T P

7.50±0.23

Cu (mg/kg)

Mn (mg/kg)

17.64±1.35b

1.68±0.09c

19.61±0.53ab

1.89±0.05b

36.71±4.75a

18.39±1.75b

1.68±0.03c

1.87±0.02

27.65±1.35b

19.37±0.09ab

1.62±0.01c

13.73±0.19

1.92±0.05

32.43±1.28a

19.82±1.12ab 1.72±0.06bc

13.74±0.21

1.92±0.06

32.92±1.34a

23.05±2.21a

I R

C S

U N

A M

T P

2.16±0.09a

Zn (mg/kg) 72.49±3.38 ab

74.67±0.30 a

75.93±2.22 a

66.38±0.77 b

71.71±1.92 ab

74.73±1.59 a

Values were presented as means ± standard error of three replications. Means in the same column with different superscripts were significantly different (P <0.05).

ACCEPTED MANUSCRIPT Table 11 Exoskeleton mineral composition of Litopenaeus vannamei fed experimental diets for 8 weeks (dry-matter basis) 1. Treatment s

Ca (g/kg)

P (g/kg) a

Na (g/kg)

K (g/kg)

Mg (g/kg)

Fe (mg/kg)

b

10.28±0.30

11.29±0.22

4.98±0.07

60.06±2.03

10.56±0.23

11.23±0.29

5.03±0.13

62.24±4.02

9.60±0.47

11.20±0.30

4.89±0.01

9.89±0.34

11.11±0.24

14.59±0.09a

FMN

87.17±1.12

FMR

89.81±0.47a

14.35±0.14b

SBN

80.64±0.53

b

b

SBF

89.19±1.24a

15.56±0.09a

SBR

87.59±0.66

a

15.27±0.11a

88.93±1.47

a

SBRR

14.32±0.08

b

15.32±0.02a b

T P

10.01±0.15

E C

1

Mn (mg/kg)

Zn (mg/kg)

60.01±4.17

10.25 ±0.63

68.30±0.23

C S

61.54±4.40

10.32 ±0.49

66.60±2.72

57.66±3.44

62.77±3.82

11.20±0.76

5.03±0.08

59.57±0.30

61.92±2.61

10.47±0.22

10.97±0.14

5.07±0.13

59.70±1.65

64.82±2.27

10.78±0.27

10.99±0.07

5.11±0.10

58.95±5.34

64.08±2.51

11.61 ±0.47

D E

10.27±0.53

Cu (mg/kg)

I R

U N

A M

T P

65.01± 1.24 63.03± 2.97 67.01± 0.71 66.86± 0.40

Values were presented as means ± standard error of three replications. Means in the same column with different superscripts were significantly different (P <0.05).

C A

ACCEPTED MANUSCRIPT Table 12 Hepatopancreas mineral composition of Litopenaeus vannamei fed experimental diets for 8 weeks (wet-matter basis) 1. Treatments

Ca (g/kg)

P (g/kg)

Na (g/kg)

K (g/kg)

Mg (g/kg)

Fe (mg/kg)

Cu (mg/kg)

T P

Mn (mg/kg)

Zn (mg/kg)

71.88±2.41b

3.52±0.18b

32.35±1.55

70.77±4.15b

4.99±0.33a

32.18±1.06

72.12±4.83b

3.39±0.13b

31.86±0.61

FMN

1.07±0.06

3.22±0.10

3.89±0.14

3.48±0.11

0.33±0.01

54.20±4.09

FMR

1.02±0.12

3.27±0.07

3.95±0.03

3.58±0.05

0.35±0.01

55.43±4.26

SBN

1.09±0.05

3.13±0.10

3.87±0.09

3.34±0.17

0.34±0.01

51.82±4.49

SBF

1.14±0.08

3.28±0.12

4.09±0.21

3.46±0.17

0.33±0.01

52.88±5.44

71.25±2.83b

2.89±0.05b

31.54±0.92

SBR

1.04±0.04

3.13±0.11

3.91±0.16

3.38±0.06

0.32±0.02

53.37±4.04

75.93±6.33b

5.33±0.15a

34.50±1.44

SBRR

1.15±0.09

3.23±0.08

3.90±0.05

3.38±0.05

0.32±0.02

53.06±5.18

121.60±6.95a

4.84±0.46a

35.97±1.64

1

C S

U N

A M

I R

Values were presented as means ± standard error of three replications. Means in the same column with different superscripts were significantly different (P <0.05).

D E

T P

C A

E C

ACCEPTED MANUSCRIPT

Table 13 Serum mineral composition of Litopenaeus vannamei fed experimental diets for 8 weeks (mg/L) 1. Treatments FMN FMR SBN SBF

Ca

P

777.30±16.3

116.57±4.5

7

3

775.78±15.2

114.45±3.1

7 762.92±3.97 782.05±7.51

SBR

778.01±9.85

SBRR

779.37±4.11

1

Na

5720.96±130.5 558.89±10.8 7

1 114.28±6.3 5 122.23±3.7 2 120.37±3.6 4

C A

4

5775.67±73.76

557.90±11.3 1

Mg

Fe

84.41±0.15

3.60±0.34

88.50±1.62

SC

U N

A M

3.65±0.46

88.61±2.12

3.60±0.27

5718.85±81.87 570.36±8.96

86.98±2.11

3.90±0.30

88.14±2.17

3.77±0.19

87.41±1.04

3.79±0.22

D E

PT

5781.44±89.56 5734.23±89.59

562.10±11.7 5

573.17±11.0 1

T P

I R

5619.73±86.54 571.73±7.57

E C

126.94±6.2 4

K

Cu

Mn

120.64±1.5

0.087±0.003

7b

cd

122.45±3.8

0.095±0.004

b

8

c

121.39±2.3

0.106±0.004

6b

b

126.08±5.4

0.076±0.001

4b

d

131.45±2.4

0.135±0.005

b

7

146.71±3.9 9

a

a

0.136±0.005 a

Zn 15.55±0.48 16.40±0.28 16.21±0.94 14.99±0.91 17.56±0.82 17.16±0.34

Values were presented as means ± standard error of three replications. Means in the same column with different superscripts were significantly different (P <0.05).

ACCEPTED MANUSCRIPT Highlights of the manuscript

PT RI SC NU MA D PT E



CE



Replacing fishmeal with soybean meal affects the osmoregulation and tissue mineralization of shrimp in low-salinity water. Mineral composition of soybean meal determines the growth of shrimp in low-salinity water. Formulating practical low-fishmeal diets should balance mineral composition.

AC

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