Defatted Haematococcus pluvialis meal can enhance the coloration of adult Chinese mitten crab Eriocheir sinensis

Aquaculture 510 (2019) 371–379

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Defatted Haematococcus pluvialis meal can enhance the coloration of adult Chinese mitten crab Eriocheir sinensis

T

⁎⁎

Nan Maa,1, Xiaowen Longa,1, Jianguo Liub, Guoliang Changc, Deng Dengd, , Yongxu Chenga,e,f, ⁎ Xugan Wua,e,f, a

Centre for Research on Environmental Ecology and Fish Nutrition of the Ministry of Agriculture and Rural Affairs, Shanghai Ocean University, Shanghai 201306, China National and Local Joint Engineering Laboratory of Ecological Mariculture, Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China c Jiangsu Engineering Laboratory for Characteristic Aquatic Species Breeding, Huaiyin Normal University, Huai'an 223300, China d Shenzhen Alpha Feed Co. Ltd., Shenzhen, Guangdong 518054, China e Shanghai Engineering Research Center of Aquaculture, Shanghai Ocean University, Shanghai 201306, China f National Demonstration Centre for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Carotenoids Antioxidant capacity Feed additive Functional feed Chinese mitten crab

Defatted algal meal, a by-product of carotenoid extraction from Haematococcus pluvialis, still contains a certain level of astaxanthin and other nutrients, which is considered as a potential ingredient or additive for aquafeeds. This study was conducted to investigate the effects of dietary supplementation of defatted H. pluvialis on gonadal development, coloration, and antioxidant and immune capacity of adult Chinese mitten crab, Eriocheir sinensis. Three isonitrogenous (crude protein: 42% of dry diet) and isolipidic (crude lipid: 16% of dry diet) experimental diets were formulated to contain 1% defatted H. pluvialis meal (Diet 1), 0.2% whole H. pluvialis powder (Diet 2), and without supplementation of astaxanthin (Diet 3, control diet). Therefore, there were three treatments with four replicate tanks for each treatment, and each tank had 12 crabs (6 males and 6 females). For the color parameters, no significant differences were found between Diet 1 and 2 treatments. The values of hepatopancreas lightness (L* = 62.65) and carapace redness (a* = 20.29) of male crabs from Diet 1 treatment were significantly higher compared to those (L* = 53.94, a* = 17.87) of Diet 3 treatment; while the a* values of ovaries (a* = 26.28) and carapace (a* = 19.93) of female crabs from Diet 1 treatment were significantly higher than those (ovarian a* = 18.28, carapace a* = 16.30) of Diet 3 treatment. The total carotenoids contents in the hepatopancreas (243.73 mg kg −1 dry matter) and carapace (73.44 mg kg −1 dry matter) of male crabs fed Diet 1 were higher than those (133.50, 43.14 mg kg −1 dry matter) of Diet 3 treatment. The contents of total carotenoids in the ovaries (356.50 mg kg −1 dry matter), hepatopancreas (120.88 mg kg −1 dry matter) and carapace (79.61 mg kg −1 dry matter) of females fed Diet 1 were significantly higher than those (290.92, 76.04 and 61.77 mg kg −1 dry matter, respectively) of Diet 3 treatment; while the astaxanthin contents in the ovaries (174.69 mg kg −1 dry matter) and carapace (19.97 mg kg −1 dry matter) of females from Diet 1 treatment were significantly higher than those (25.65, 13.87 mg kg −1 dry matter) in the Diet 3 treatment. For the antioxidant capacity indices, the male crabs from Diet 1 treatment had the lower levels of superoxide dismutase, peroxidase, glutathione reductase and malondialdehyde in the serum as well as the activities of glutathione peroxidase and peroxidase in the hepatopancreas than those of Diet 3 treatment (P < .05), but no significant differences were found between Diet 1 and 2 treatments. For the immune indices, dietary supplementation with defatted H. pluvialis significantly increased the activities of acid phosphatase, alkaline phosphatase in the serum of male crabs as well as the levels of serum alkaline phosphatase, hemocyanin and nitric oxide of female crabs compared to Diet 3 treatment (P < .05). In conclusion, dietary supplementation of defatted H. pluvialis meal can enhance the coloration, antioxidant and immune capacity, and reduce the cost of a fattening diet for E. sinensis.

⁎ Corresponding author at: Centre for Research on Environmental Ecology and Fish Nutrition of the Ministry of Agriculture and Rural Affairs, Shanghai Ocean University, Shanghai 201306, China. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (D. Deng), [email protected] (X. Wu). 1 These authors contributed equally to this work.

https://doi.org/10.1016/j.aquaculture.2019.05.063 Received 18 October 2018; Received in revised form 26 May 2019; Accepted 26 May 2019 Available online 27 May 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.

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1. Introduction

Table 1 Formulations, proximate composition, and carotenoid contents of experimental diets.

When dried, the green alga Haematococcus pluvialis contains 1.5–6.0% astaxanthin (Orosa et al., 2005; Sandesh et al., 2008), making it an important source of natural astaxanthin. Astaxanthin is widely used as a nutraceutical, and in cosmetics, pharmaceutical products and for other applications (Arad and Yaron, 1992; Capelli et al., 2013; Cenariu et al., 2015; Fiedor and Burda, 2014; Guerin et al., 2003; Kidd, 2011; Palozza et al., 2009; Tominaga et al., 2012). The astaxanthin in H. pluvialis is stable, mostly esterified, and generally present as the 3S, 3′S - enantiomer that can be well-utilized by humans and aquatic animals (Chien and Jeng, 1992; Chien and Shiau, 2005). A large amount of defatted algal meal is produced during astaxanthin extraction from H. pluvialis, and the meal contains many valuable nutrients, including proteins, lipids, carbohydrates and some astaxanthin. This may make the meal suitable for inclusion in aquafeeds (Ju et al., 2012; Shi et al., 2017). The Chinese mitten crab Eriocheir sinensis, is a commercially important aquaculture species in China, with production being 750, 945 tons in 2017 (Bureau of Fisheries and Management, 2018). The crab is popular because of its high nutritional value and flavor (Shao et al., 2013; Wang et al., 2016). Although aquaculture production of E. sinensis is high, pond-reared crabs generally have a light colored, yellowish carapace after cooking, whereas wild or lake-stocked crabs are red or deep-orange (Kong et al., 2012). The light-yellow carapace of pond-reared crustaceans may be related to low levels of dietary carotenoids (Long et al., 2017; Wade et al., 2017). Color is one of the most important factors that affect a consumer's decision to purchase commercial crustacean species (Chien and Jeng, 1992; Tume et al., 2009). Cultured crustaceans with a light-yellow carapace generally have a lower market price than those with a red or deep orange carapace (Erickson et al., 2007; Parisenti et al., 2011; Wade et al., 2017); therefore, it is important to improve the coloration of pond-reared crabs. Supplementation of feeds with astaxanthin can enhance the coloration, antioxidant capacity, and tissue astaxanthin concentrations of E. sinensis, and may improve the nutritional quality of pond-reared crabs (Gong et al., 2014; Long et al., 2017; Wu et al., 2017). Micro-algal meals have been used as pigment sources in crustacean feeds (Boonyaratpalin et al., 2001; Daly et al., 2013; Ju et al., 2011; Wade et al., 2017), but the astaxanthin originating from H. pluvialis cannot be widely applied as an additive in aquafeeds because of its high cost and limited supply (Long et al., 2017; Parisenti et al., 2011). Defatted H. pluvialis meal contains some astaxanthin along with a range of nutrients (Damiani et al., 2010; Liu et al., 2015), and its price per unit of astaxanthin is lower than that of H. pluvialis powder. As such, defatted H. pluvialis meal may have potential for inclusion in formulated feeds to enhance the color and immune response of E. sinensis. The aims of this study were to examine the effects of dietary supplementation with defatted H. pluvialis on gonad development, color, and antioxidant and immune capacity of adult Chinese mitten crab, E. sinensis; and to compare the efficacy of defatted H. pluvialis with that of whole H. pluvialis at the same dietary astaxanthin concentration.

Items

Diet 1

Diet 2

Diet 3

Ingredients (%) Soybean meal Rapeseed meal Wheat gluten Fish meal Chicken meal Wheat flour Brewer's yeast Squid meal Vitamin premix a Mineral premix b Ca(H2PO4)2 Choline chloride (50%) Inositol Vitamin C (35%) Soy lecithin Pork lard Salt Fish oil Soybean oil Rapeseed oil Defatted Haematococcus pluvialis meal Haematococcus pluvialis powder

20.00 11.15 2.00 18.00 6.00 13.00 4.00 8.00 0.60 1.20 2.00 0.40 0.30 0.15 2.00 2.00 0.20 4.00 2.00 2.00 1.00 0

20.00 11.15 2.00 18.00 6.00 13.80 4.00 8.00 0.60 1.20 2.00 0.40 0.30 0.15 2.00 2.00 0.20 4.00 2.00 2.00 0 0.20

20.00 11.15 2.00 18.00 6.00 14.00 4.00 8.00 0.60 1.20 2.00 0.40 0.30 0.15 2.00 2.00 0.20 4.00 2.00 2.00 0 0

Analyzed composition Moisture (% dry diet) Crude protein (% dry diet) Crude lipid (% dry diet) Ash (% dry diet) Total carotenoids (mg kg −1 dry diet) Astaxanthin (mg kg −1 dry diet) Lutein (mg kg −1 dry diet) Canthaxanthin (mg kg −1 dry diet) β-carotene (mg kg −1 dry diet)

9.67 42.01 15.96 10.05 82.36 64.80 1.09 3.24 0.75

10.02 42.16 16.00 9.93 90.28 65.79 0.87 2.46 0.61

9.34 42.49 15.97 9.85 16.94 1.00 0 0 0.02

a Vitamin premix (per kg diet): vitamin A, 62500 IU; vitamin D3, 15,000 IU; vitamin E, 1.75 g; vitamin K3, 35.4 mg; vitamin B1, 100 mg; vitamin B2, 150 mg; vitamin B6, 150 mg; vitamin B12, 0.2 mg; biotin, 4 mg; D‑calcium pantothenate, 250 mg; folic acid, 25 mg; nicotinamide, 300 mg; vitamin C, 700 mg. b Mineral premix (per kg diet): FeSO4.H2O, 200 mg; CuSO4.5H2O, 96 mg; ZnSO4.H2O, 360 mg; MnSO4.H2O, 120 mg; MgSO4.H2O, 240 mg; KH2PO4, 4.2 g; NaH2PO4, 0.5 g; KI, 5.4 mg; CoCl2.6H2O, 2.1 mg; Na2SeO3, 3 mg.

Co., Ltd., China. Three isonitrogenous and isolipidic experimental diets were formulated to contain 1.0% defatted H. pluvialis meal, 0.20% whole H. pluvialis powder, and were defined as Diet 1 and Diet 2, respectively, while the control diet (Diet 3) was not supplemented with astaxanthin. All the dry ingredients for the experimental diets were ground to pass through a 250 μm sieve before mixing, then combined and thoroughly mixed to homogeneity by manual mixing. Lipid ingredients, defatted H. pluvialis meal and H. pluvialis powder were added to the dry components and thoroughly mixed; then water (30% dry ingredients mixture) was added and mixed. The mixtures were extruded through a 5 mm orifice and then air-dried at room temperature. All experimental diets were stored in black plastic bags at −20 °C until use. The proximate composition, fatty acid composition, amino acid, and carotenoid contents of the defatted H. pluvialis meal (DHM) and whole H. pluvialis powder (WHP) were shown in Table 2 (the description of the methods used for the biochemical analysis were provided later in in Section 2.5).

2. Materials and methods 2.1. Experimental diets The formulation, proximate composition and actual carotenoid contents of the three experimental diets are shown in Table 1. Soybean meal, rapeseed meal and fish meal were used as the main protein sources, while fish oil, pork lard, soybean oil and rapeseed oil were used as lipid sources. The defatted H. pluvialis meal (astaxanthin: 0.66% of dry matter) and whole H. pluvialis powder (astaxanthin: 3.01% of dry matter) were used as astaxanthin sources, and whole H. pluvialis powder was dry disruption of algae cells. The defatted H. pluvialis meal and whole H. pluvialis powder were donated by the Yunnan Alphy Biotech

2.2. Experimental setup and culture management This experiment was conducted at the Chongming research base of Shanghai Ocean University. The adult E. sinensis were obtained from a local mitten crab farm. Only healthy, active and intact crabs with immature gonads were selected for the experiment. In order to confirm the gonadal development status, at the beginning of the experiment, 15 372

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Table 2 Proximate composition, fatty acid composition, amino acid, and carotenoid contents of the defatted H. pluvialis meal (DHM) and whole H. pluvialis powder (WHP). Items

Content DHM

Items

Content

WHP

Proximate composition (% dry weight) Moisture Crude protein Crude lipid Ash Total carbohydrate

9.23 26.95 6.48 3.65 28.08

7.10 18.00 39.69 2.79 20.11

Fatty acids (% total fatty acids) C14:0 C15:0 C16:0 C17:0 C18:0 C20:0 ∑ SFA C14:1n5 C16:1 C17:1n7 C18:1n9 C18:1n7 C20:1n9 ∑ MUFA C18:2n6 C18:3n6 C18:3n3 C20:2n6 C20:4n6 C20:3n3 C20:5n3 ∑ PUFA (≥18:2n) ∑ n-3 PUFA ∑ n-6 PUFA ∑ LC-PUFA(≥20:3n)

2.56 2.50 23.82 1.03 2.79 0.55 32.91 0.42 1.31 1.59 7.76 3.99 0.39 15.33 14.92 1.05 10.54 1.19 0.44 0.18 0.21 28.53 10.93 17.60 0.83

0.89 0.19 24.40 0.33 1.83 0.42 28.06 0.04 1.81 1.83 14.92 3.73 1.38 23.71 21.61 1.19 12.13 1.22 0.72 0.23 0.66 37.76 13.02 24.74 1.61

Amino acids (mg g Threonine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Tryptophan Methionine Cysteine Aspartic acid Serine Glutamic acid Glycine Alanine Histidine Arginine ∑ TAA

DHM

WHP

13.79 18.02 10.21 22.99 7.55 12.11 11.26 4.05 7.03 2.32 21.48 11.43 24.08 14.70 23.26 4.82 12.09 233.94

8.63 10.14 6.72 15.22 4.49 7.42 9.11 2.59 2.54 1.48 14.27 8.19 18.11 9.05 13.49 3.08 8.53 152.19

7261.25 6649.35 97.09 289.63 65.27

34,105.46 30,142.43 414.47 1138.62 279.02

−1

)

Carotenoid composition (mg kg Total carotenoids Astaxanthin Canthaxanthin Lutein β-carotene

−1

)

∑EAA: total essential amino acids; ∑NEAA: total no-essential amino acids; ∑ TAA: total amino acids; ∑ SFA: total saturated fatty acids; ∑ MUFA: total monounsaturated fatty acids; ∑PUFA: total polyunsaturated fatty acids; ∑ LC-PUFA: total long chain polyunsaturated fatty acids.

each day at 18:00 and feed waste was removed the next morning. The amount of feeds was adjusted in relation to water temperature and feed waste (Long et al., 2017); the daily ration was 1.5–3% of the total biomass when temperature was > 20 °C, and was reduced to 1–1.5% of total biomass when the temperature was 15–20 °C. The amount of feeds and feed waste were recorded each day for all tanks. The ammonia-N, nitrite, dissolved oxygen (DO) and pH of the water were measured every three days and the water was exchanged based on the water quality. During the experiment, the water quality parameters were maintained as follows: ammonia-N < 0.5 mg L−1; nitrite: < 0.15 mg L−1; DO: > 4 mg L−1 and pH: 7.0–9.0.

females and 15 males were randomly selected and dissected to obtain gonads. The gonad from each crab was subsequently weighed to calculate gonadosomatic index [GSI (%) = 100 × gonad wet weight/body wet weight]. The mean GSI of males and females was 0.80% and 0.35%, respectively. The mean initial body weight ranges of males and females were 100–120 g and 80–90 g, respectively. In this study, all crabs had completed their puberty molt, and were used for fattening culture. The adult crabs that completed puberty molt were determined based on the external characteristics: 1) > 70% area of the chelipeds is covered with hairs and the hair length is > 5 mm for adult males; and 2) the male petasma of adult crabs become hard and raised compared to immature males (Xu et al., 2016). For the females, the mature crabs were characterized by a yellowish green color on the carapace, and an abdominal flap that was semicircular in shape that was covered by short hair; in contrast, immature females had a khaki colored carapace, and the abdomen was more triangular (Wang, 2013). The feeding trial was conducted in a water recirculating system, which was equipped with circular polyethylene tanks (diameter × height = 108 cm × 120 cm). Six polyvinyl chloride (PVC) tubes (diameter × height = 15 cm × 20 cm) were provided as shelters for the crabs in each tank. There were three dietary treatments (defined as Diet 1 - Diet 3), and each treatment had four replicate tanks with 12 crabs (6 males and 6 females) per tank. During the experiment, the water level in each tank was maintained at 50 cm. Prior to the feeding experiment, the crabs were acclimated to the indoor culture for 3–5 days and fed Diet 3. This experiment started 28 August 2015 and lasted 60 days. During the feeding trial, all tanks were provided with continuous aeration. Photoperiod was set at 12 h light: 12 h dark with fluorescent lamps (40 W) as the light source. The crabs were fed their allotted diets

2.3. Sample collection At day 30 of the experiment, the crabs were fasted for 24 h prior to sampling. Two males and two females were randomly sampled from each tank and their body weights were measured using a digital balance (precision = 0.01 g). Subsequently, hemolymph was withdrawn using 1.0 mL syringe to collect at the base of the third walking limb of the crab twice; therefore, a total of 2 mL hemolymph was collected from each crab. The hemolymph sample of each crab was stored in 2 mL tubes at −40 °C until use. The gonad (ovaries or testis) and hepatopancreas of each crab was dissected and weighed for calculation of GSI and hepatosomatic index [HSI (%) = 100 × hepatopancreas wet weight/body wet weight]. The hemolymph and hepatopancreas samples at day 30 were separately stored at −40 °C, which was only used for the later analysis of antioxidant and immune indices. Therefore, each diet treatment had 8 crabs for GSI, HSI, antioxidant and immune indices of males and females. 373

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supernatant was then retrieved and vacuum dried at 50 °C to remove the initial HCl, followed by the addition of 2–5 mL of 0.02 mol L−1 HCl. Finally, 1 mL of final sample was analyzed by a Hitachi L-8900 amino acid analyzer (Hitachi, Ltd., Tokyo, Japan). The concentration of tryptophan analysis was determined by hydrolyzation using a solution containing 5 M NaOH and 5% SnCl2 (w/v) for 20 h at 110 °C. After hydrolysis, the hydrolysate was neutralized with 6 M HCl, centrifuged at 2659 ×g, and filtered using filter paper. The subsequent analysis was done similar to the methods described above. The concentrations of methionine and cysteine in the samples were determined according to the method of formic acid oxidation and hydrolyzation (Spindler et al., 1984). Prior to extraction of total carotenoids in crab tissues, the gonads, hepatopancreas and carapace of each crab from day 60 sampling were freeze-dried and ground separately. The same tissues of crabs with same gender at each experimental tank were pooled and mixed prior to carotenoids analysis, and there were four replicates for the carotenoid analysis of each diet treatment. The total carotenoids in all samples were extracted with acetone and their levels were determined using an ultraviolet-visible spectrophotometer (T6 New Century, Beijing Purkinje General Instrument Co., Ltd., Beijing, China) at 470 nm (Johnston et al., 2000). The esterified carotenoids were first hydrolyzed by enzymatic hydrolysis (Wade et al., 2005), and then the astaxanthin, lutein, zeaxanthin, canthaxanthin and β-carotene were analyzed with an Agilent high performance liquid chromatograph (HPLC) using an Agilent 1260 HPLC system (Agilent Technologies Inc., CA, USA) following the methods by Peng et al. (2008). The Agilent 1260 HPLC system was equipped with a YMC™ Carotenoid C30 column (4.6 × 150 mm, diameter of packing = 3 μm, YMC Co., Ltd., Kyoto, Japan), while the gradient mobile phases were consisting of methanol (A) and methyl tert-butyl ether (B). The detailed procedure of carotenoid analysis for crab tissues was described by Long et al. (2017).

Similarly, at day 60 of the experiment, two males and two females were randomly sampled from each tank and their weights measured after which they were dissected to obtain the gonads and hepatopancreas. The carapace and hepatopancreas from each crab, and ovary from each female crab were stored separately at −40 °C for later color measurement and carotenoid analysis. 2.4. Color measurement Male gonads (testes) were milky white without redness or yellowness, so were not used for the determination of color parameters and carotenoid composition. Prior to the measurement of color parameters, the female gonads (ovaries), along with the hepatopancreas and carapace of both males and females sampled on Day 60 were pretreated as follows. The ovaries were freeze-dried, ground and then heated at 100 °C for 2 h. the carapaces were cooked in a steamer for 10 min and cooled at room temperature; the hepatopancreases were not freezedried or cooked because the cooking process would lead to a loss of lipid from the hepatopancreas, which would affect its color. The color measurement was followed the previous publication (Long et al., 2017). The L*a*b system were used for the assessment of color values, which were referred from International Commission on Illumination (CIE, 2004). The L* value represents lightness with a scale of 0 (pure black) to 100 (pure white). The a* value (redness) represents color along a spectrum from green to red, and the b* value (yellowness) represents color spectrum from blue to yellow. The color values (L*, a* and b*) were assessed using a colorimeter (CR-400, Konica Minolta, Marunouchi, Tokyo, Japan) over a 1 cm diameter circle standardized under D65 illumination. Six relatively smooth points on each carapace surface were selected for L*, a* and b* measures, and the results were presented as the average value for each crab. 2.5. Biochemical analysis

2.6. Measurements of antioxidant and immune indices The analysis of moisture, crude protein and ash in experimental diets, H. pluvialis meal and H. pluvialis powder were conducted according to AOAC procedures (AOAC, 1995). Total lipids were extracted with chloroform-methanol (2:1, v/v) according to the method described by Folch et al. (1957). Total carbohydrate content in the defatted H. pluvialis meal and H. pluvialis powder was measured using the phenolsulfuric acid method as detailedly described by Zhang (1987). For the fatty acid analysis, total lipids of the defatted H. pluvialis meal and H. pluvialis powder were esterified with boiling 14% boron trifluoride/ methanol (w/w), and the fatty acid methyl esters (FAMEs) were extracted by hexane (Wu et al., 2007). FAMEs were analyzed using an Agilent 7890B GC/5977A gas chromatograph-chromatograph-mass spectrometer (GC–MS) with an Omegawax 320 fused silica capillary column (30 m × 0.32 mm × 0.25 um; Supelco, Billefonte, PA, USA). The injector and detector temperature were maintained at 260 °C. The column temperature was initially set at 40 °C, it was then increased at a rate of 10 °C min−1 to 170 °C and held for 1 min, followed by an increase at a rate of 2 °C min−1 to 220 °C and held for 1 min. It was then further increased at a rate of 2 °C min−1 to the final temperature of 230 °C and held for 30 min until all FAMEs had been eluted. The peaks were identified by comparing retention times with a 37-component FAMEs standard mixture (18919-1AMP, Sigma-Aldrich Co., St. Louis, MO, USA). Fatty acid composition was expressed as the percentage of each fatty acid to the total fatty acids (% total fatty acids). The amino acid composition of the defatted H. pluvialis meal and H. pluvialis powder were analyzed following the methods described by Blackburn (1968). Approximately 0.1 g from each sample was placed separately in 40 mL ampules with 8 mL of 6 mol L−1 HCl. The ampules were vacuumed-sealed, and samples were hydrolyzed at 110 °C for 24 h. Hydrolysates were dissolved to a volume of 50 mL using distilled water and centrifuged at 2659 ×g, and the supernatants were filtered through a 0.22 μm filter (Millipore, Bedford, MA). 1 mL of the filtered

The thawed hemolymph was homogenized for 30 s with an IKA homogenizer (T10B, IKA Co., Germany), and then centrifuged at 12, 000 ×g for 20 min at 4 °C. The supernatant (serum) was collected and stored at −40 °C, until subsequent analysis. The hepatopancreas tissue plus the 5-fold volume (v/w) of physiological saline solution (at 4 °C) were combined into a 5 mL tube and homogenized using an IKA homogenizer. The homogenate was centrifuged at 12, 000 ×g for 10 min at 4 °C, and the supernatant was then collected for later analysis. The superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione peroxidase (GSH-Px), glutathione reductase (GR), total antioxidant capacity (T-AOC), lactic dehydrogenase (LDH), lactic acid (LD), nitric oxide (NO), malonaldehyde (MDA) and acid phosphatase (ACP) in the serum and hepatopancreas were analyzed for utilizing commercial kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). The phenoloxidase (PO) and hemocyanin (Hc) in the serum were determined using the methods of Hernández-López et al. (1996) and Nickerson and Holde (1971), respectively. The activities of alkaline phosphatase (ALP) and γ-glutamyl transpeptidase (γ-GT) in serum and hepatopancreas were determined using an automatic biochemical analyzer (BS-200, Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China) and the respective commercial bio-kits were purchased from Shanghai Jinxi Biotech Co., Ltd. (Shanghai, China). 2.7. Statistical analysis Data are expressed as mean ± standard error (SE), and the homogeneity of variance of data was tested with Levene's test. When necessary, arcsine-square root or logarithmic transformation was performed prior to analysis. For all statistical analyses, each tank was treated as one replicate. The data of GSI, HSI, color parameters, antioxidant and immune indices for each tank (replicate) was the mean value. The 374

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Table 3 Effects of dietary supplementation of defatted H. pluvialis meal and sampling time on hepatosomatic index (HSI) and gonadosomatic index (GSI) of adult E. sinensis. Items

Diet 1

Diet 2

Diet 3

Two-way ANOVA (P-value) Treatment

Time

Treatment × Time

Male GSI 30 60

1.82 ± 0.08 2.85 ± 0.10⁎

1.84 ± 0.06 2.78 ± 0.11⁎

1.87 ± 0.05 2.81 ± 0.09⁎

0.887

< 0.001

0.925

HSI Day 30 Day 60

9.06 ± 0.18 7.58 ± 0.30⁎

8.50 ± 0.23 7.04 ± 0.25⁎

8.82 ± 0.20 7.24 ± 0.40⁎

0.295

< 0.001

0.983

Female GSI Day 30 Day 60

4.09 ± 0.20 8.85 ± 0.20⁎

4.00 ± 0.15 8.55 ± 0.25⁎

4.15 ± 0.20 8.36 ± 0.25⁎

0.415

< 0.001

0.473

HSI Day 30 Day 60

11.35 ± 0.27 8.39 ± 0.20⁎

11.38 ± 0.30 8.35 ± 0.21⁎

10.52 ± 0.25 8.00 ± 0.22⁎

0.078

< 0.001

0.239

Data are presented as mean ± SE (n = 4). The data with Asterisk within the same column indicates significant differences (P < .05) between two sampling time for the same dietary treatment. Two-way ANOVA was used to determine the interactions between dietary treatments and sampling time.

statistical test used was one - way analysis of variance (ANOVA), followed by Duncan's multiple range tests. When a normal distribution and/or homogeneity of the variances were not achieved, data was subjected to the Kruskal-Wallis H non-parametric test, followed by the Games-Howell nonparametric multiple comparison test. For GSI and HSI, two-way ANOVA was used to determine the interactions between treatments and sampling time, while student t-test was used to detected the differences between Day 30 and Day 60 samples for the same treatment. P < .05 was considered as the statistically significant level. All statistical analyses were implemented by using the SPSS package (version 16.0). 3. Results 3.1. Gonadosomatic index (GSI) and hepatosomatic index (HSI) Fig. 1. Color of dried cooked ovaries of adult female E. sinensis fed three experimental diets for 60 days.

Table 3 showed that there were no significant differences (P > .05) in the GSI and HSI among all diet treatments (Table 3). From day 30 to day 60, GSI increased significantly while HSI decreased significantly for all treatments (Table 3, P < .001). No significant interactions were found between diet treatments and sampling time (P > .05).

carotene content was detected in the Diet 3 treatment (P < .05). For either males or females, the levels of total carotenoids in the hepatopancreas, as well as the total carotenoids and astaxanthin in the carapace were significantly higher in the Diet 1 and Diet 2 treatments than the Diet 3 treatment (P < .05). The highest total carotenoid content in the hepatopancreas of males, as well as the lutein in the carapace of females were found in the Diet 1 and Diet 2 treatments (P < .05) respectively.

3.2. Color parameters and carotenoid composition After 60 days, the dried, cooked ovaries of the crabs fed diets 1 and 2 had a deeper red color than those of the crabs fed diet 3, and the ovaries of the crabs fed diet 2 were slightly darker red than the ovaries of crabs fed diet 1 (Fig. 1). Table 4 shows the color values of ovaries, hepatopancreas and carapace. For the males, the values of hepatopancreas L* and carapace a* in Diet 1 and Diet 2 treatments were significantly higher than those in Diet 3 (P < .05), while no significant differences were found for the value of hepatopancreas a* among all diet treatments as well as the L* and b* of carapace (P > .05). For the females, the significantly higher a* value of ovaries and carapace were found in the Diet 1 and Diet 2 treatments compared to Diet 3, while the highest L* and b* of ovaries were observed in the Diet 3 treatment (P < .05). As expected, the visible redness of the cooked carapace of adult males and females were enhanced by supplementing the diet with defatted H. pluvialis meal or H. pluvialis powder (Fig. 2A and B). The carotenoid composition in the gonad, hepatopancreas and carapace of adult E. sinensis are shown in Table 5. The Diet 1 and Diet 2 treatments had significantly higher levels of total carotenoids, astaxanthin, lutein and zeaxanthin in the ovaries, while the highest β-

3.3. Antioxidant indices The antioxidant indices recorded in the serum and hepatopancreas are reported in Table 6. The serum of males from the Diet 3 treatment had the highest levels of SOD, POD, GR and MDA, while the highest content of LDH was detected in the animals from the Diet 1 and Diet 2 treatments (P < .05). For the females, the activities of GSH-Px and CAT in serum of subjects under Diet 1 and Diet 2 treatments were significantly higher than those under the Diet 3 treatment. The highest content of MDA was detected in the Diet 3 treatment (P < .05). For the antioxidant indices in the hepatopancreas of males fed Diet 3 had the highest levels of GSH-Px, POD and MDA (P < .05), whereas no significant differences were found for the other antioxidant indices among all treatments (P > .05). For the females, the highest levels of GR and MDA were detected in the Diet 3 treatment (P < .05). No significant differences were found for the other antioxidant indices among all 375

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Table 4 Effects of dietary supplementation of defatted H. pluvialis meal on color values of gonad, hepatopancreas and carapace of adult E. sinensis. Items

Male

Female

Diet 1

Diet 2

Diet 3

Diet 1

Diet 2

Diet 3

Gonad L* a* b*

– – –

– – –

– – –

56.87 ± 0.69b 26.28 ± 0.60a 25.19 ± 0.90b

55.61 ± 0.33b 27.07 ± 0.28a 23.42 ± 0.63b

68.91 ± 1.15a 18.25 ± 0.64b 39. 86 ± 0.68a

Hepatopancreas L* a* b*

62.65 ± 1.38a 16.34 ± 1.14 38.56 ± 1.06ab

66.27 ± 1.07a 15.81 ± 0.30 40.16 ± 0.82a

53.94 ± 1.14b 15.05 ± 0.74 35.97 ± 0.96b

54.72 ± 1.84 8.46 ± 0.49 19.47 ± 0.82ab

54.75 ± 0.56 8.64 ± 0.20 20.87 ± 0.45a

53.29 ± 1.66 7.48 ± 0.44 16.81 ± 1.15b

Carapace L* a* b*

58.68 ± 1.41 20.29 ± 0.51a 29.06 ± 1.04

59.98 ± 0.63 21.60 ± 0.43a 30.32 ± 0.40

58.07 ± 1.82 17.87 ± 0.21b 28.18 ± 0.90

57.04 ± 1.69ab 19.01 ± 0.35a 30.27 ± 1.06

58.38 ± 0.42a 19.93 ± 0.25a 32.09 ± 0.39

52.46 ± 1.43b 16.30 ± 0.58b 29.23 ± 1.14

Data are presented as mean ± SE (n = 4). Values within the same row with different letters mean significant difference (P < .05). ‘—’ indicates values are undetectable or not detected. L*: lightness; a *: redness; b*: yellowness.

the serum of E. sinensis under the Diet 1 and Diet 2 treatments had significantly higher values of ALP, Hc and NO compared to those of Diet 3 treatment. The hepatopancreas of animals under Diet 3 treatment had the highest levels of ACP, ALP, γ-GT and NO among three treatments (P < .05). 4. Discussion The hepatopancreas and ovary are two important edible parts of adult E. sinensis, and the hepatosomatic index (HSI) and gonadosomatic index (GSI) generally can be used to evaluate the edible and economic value of crabs (Wu et al., 2007). In this study, the results showed that dietary supplementation of defatted H. pluvialis meal has no significant effects on the HSI and GSI of E. sinensis. The possible reason for this result was that dietary astaxanthin, as a natural antioxidant and immunopotentiator (Wang et al., 2018a), mainly affects the antioxidant capacity of E. sinensis but does not significantly affect their gonadal development (Long et al., 2017; Wu et al., 2017). In addition, the GSI increased significantly for all treatments during the fattening period between day 30 and day 60, while the HSI showed a decreasing trend. Such results could be explained by the fact that all crabs used in this study had completed their puberty molt (last molt), therefore they developed their gonads rapidly, and the hepatopancreatic nutrients being transferred to the developing gonads during gonadal development (Wu et al., 2017; Xu et al., 2016). Coloration of crustaceans generally affects consumer acceptance and their market price (Chien and Jeng, 1992; Kong et al., 2012), which is affected by the dietary composition and contents of different carotenoids (especially astaxanthin) (Parisenti et al., 2011; Tume et al., 2009; Wade et al., 2008, 2015). In juvenile kuruma shrimp Marsupenaeus japonicus, the redness (a*) and yellowness (b*) of head and body increased with increasing dietary astaxanthin (Wang et al., 2018b). Similarly, the Pacific white shrimp Litopenaeus vannamei fed the diets containing natural astaxanthin exhibited a deep red color, while the shrimp fed the diets without supplementation of astaxanthin showed the light pink color (Ju et al., 2011). Moreover, the juvenile red king crab Paralithodes camtschaticus had low hue and brightness values, but high saturation when the crab fed diets containing astaxanthin (Daly et al., 2013). In this study, dietary supplementation of the defatted H. pluvialis meal or H. pluvialis powder significantly increased the carapace a* of males and females and ovary a* of females. These results indicate that dietary supplementation with the defatted H. pluvialis meal could effectively improve the coloration of E. sinensis. As crustacean species cannot synthesize carotenoids de novo, which must be obtained from their diets (Tanaka et al., 1976a, 1976b). In this study, crabs fed Diet 1 and Diet 2 had higher contents of total

Fig. 2. Color of cooked carapace of adult E. sinensis fed three experimental diets for 60 days. (A): Male crabs; (B): Female crabs.

treatments (P > .05).

3.4. Non-specific immune indices Table 7 shows the non-specific immune indices of adult E. sinensis. For the males, the activities of ACP, ALP and γ-GT in the serums of animals under Diet 1 and Diet 2 treatments were significantly higher than those of Diet 3 treatment (P < .05). Significant higher activities of ACP, ALP and γ-GT, and NO content in the hepatopancreas were detected in animals under Diet 3 treatment (P < .05). For the females, 376

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Table 5 Effects of dietary supplementation of defatted H. pluvialis meal on carotenoids contents (mg kg−1 dry matter) in gonad, hepatopancreas and carapace of adult E. sinensis. Items

Male

Female

Diet 1

Diet 2

Diet 3

Diet 1

Diet 2

Diet 3

Gonad Total carotenoids Astaxanthin Lutein Zeaxanthin β-Carotene

– – – – –

– – – – –

– – – – –

356.50 ± 13.33a 174.69 ± 20.59a 1.99 ± 0.13a 12.88 ± 0.56a 67.37 ± 16.76b

377.12 ± 13.35a 210.07 ± 15.49a 2.36 ± 0.07a 14.49 ± 1.23a 64.54 ± 8.27b

290.92 ± 14.36b 25.65 ± 0.38b 0.30 ± 0.08b 5.56 ± 0.07b 115.79 ± 3.79a

Hepatopancreas Total carotenoids Astaxanthin β-Carotene

243.73 ± 8.33a 2.39 ± 0.27 78.78 ± 0.15a

249.68 ± 6.65a 2.68 ± 0.28 87.14 ± 2.36a

133.50 ± 8.90b – 31.57 ± 1.78b

120.88 ± 3.05a 6.90 ± 0.27 40.63 ± 3.99b

134.67 ± 5.20a 7.68 ± 0.56 57.62 ± 4.68a

76.04 ± 4.11b – 27.86 ± 1.26c

Carapace Total carotenoids Astaxanthin Lutein Zeaxanthin

73.44 ± 2.36a 12.95 ± 0.51a 1.98 ± 0.20a 2.29 ± 0.08

77.32 ± 2.36a 13.91 ± 0.31a 2.02 ± 0.13a 2.30 ± 0.04

43.14 ± 2.40b 9.32 ± 0.25b 1.38 ± 0.14b 1.99 ± 0.17

79.61 ± 2.51a 19.97 ± 1.38a 1.71 ± 0.22 2.87 ± 0.31

86.25 ± 2.17a 20.62 ± 0.85a 1.98 ± 0.31 3.01 ± 0.14

61.77 ± 1.50b 13.87 ± 0.52b 1.24 ± 0.18 2.23 ± 0.20

Data are presented as mean ± SE (n = 4). Values within the same row with different letters mean significant difference (P < .05). ‘–’ indicates values are undetectable or not detected.

whole H. pluvialis powder decreased the levels of serum SOD, POD and GR, and the hepatopancreas GSH-Px and POD of male crabs as well as the hepatopancreas GR of females, which indicated lower free radicals in the tissues of crabs fed astaxanthin supplemented diets. This is because astaxanthin may serve as a good antioxidant and has stronger O2 quenching activity than antioxidant enzymes. This resulted in decreasing the substrates of antioxidant enzymes in tissues, and consequently led to the decreasing activities of some antioxidant enzymes (Long et al., 2017; Zhang et al., 2013). MDA is an important indicator of lipid peroxidation (Janero, 1990). In this study, dietary supplementation of defatted H. pluvialis meal or H. pluvialis powder resulted in a lower MDA level in the hepatopancreas and serum of E. sinensis. These results indicated that the addition of defatted H. pluvialis meal or whole H. pluvialis powder in the diet could inhibit lipid peroxidation and improve the antioxidant capacity of E. sinensis. Moreover, it has been reported that dietary carotenoids also could affect the immune performance of crustaceans (Wang et al., 2018a; Wu

carotenoids and astaxanthin in the ovaries, hepatopancreas and carapace compared to Diet 3 treatment, which suggested that E. sinensis could utilize the carotenoids from defatted H. pluvialis meal and deposit them in the tissues. It is worth noting that the content of β-carotene in Diet 1 was greater than the control diet (Diet 3), but the highest βcarotene content of ovaries was found on the Diet 3 treatment. Such results may be due to the lack of astaxanthin in Diet 3, with the βcarotene preferentially accumulated in the ovaries of females, which may reduce the negative impacts of astaxanthin deficiency (Long et al., 2017). However, the actual underlying physiological mechanism remains to be further studied. The antioxidant capacity indices generally can reflect the antioxidant and health status of aquaculture crustaceans (Xie et al., 2018). The antioxidant enzymes (e.g. SOD, GSH-Px and POD) are responsible for scavenging free radicals and preventing tissue damage by radical process and phagocytosis (Chien and Shiau, 2005). The present study showed that dietary supplementation of defatted H. pluvialis meal or

Table 6 Effects of dietary supplementation of defatted H. pluvialis meal on antioxidant indices in the serum and hepatopancreas of adult E. sinensis. Items

Male

Female

Diet 1

Diet 2

Diet 3

Diet 1

Diet 2

Diet 3

Serum SOD (U g −1 protein) GSH-Px (U g −1 protein) POD (U g −1 protein) CAT (U g −1 protein) GR (U g −1 protein) LDH (U g −1 protein) T-AOC (U g −1 protein) LD (mmol L −1) MDA (nmol mL −1)

0.40 ± 0.01b 292.51 ± 9.17 431.27 ± 5.72b 17.26 ± 0.65 0.45 ± 0.03b 0.63 ± 0.03a 68.08 ± 1.62 8.28 ± 0.19 3.92 ± 0.15b

0.40 ± 0.01b 293.98 ± 2.16 410.31 ± 3.45b 17.14 ± 0.54 0.42 ± 0.01b 0.63 ± 0.00a 66.19 ± 2.37 8.23 ± 0.15 4.10 ± 0.12b

0.44 ± 0.00a 293.86 ± 7.53 491.28 ± 6.57a 18.51 ± 1.08 0.54 ± 0.02a 0.27 ± 0.03b 66.74 ± 1.51 7.51 ± 0.23 7.62 ± 0.40a

0.33 ± 0.00 216.48 ± 2.94a 494.20 ± 15.37 18.15 ± 0.49a 0.45 ± 0.02 0.53 ± 0.03 60.82 ± 2.13 11.72 ± 0.35 4.90 ± 0.15b

0.33 ± 0.00 219.57 ± 4.84a 468.50 ± 14.35 19.03 ± 1.64a 0.43 ± 0.03 0.51 ± 0.03 60.37 ± 3.69 11.99 ± 0.22 4.45 ± 0.12b

0.34 ± 0.01 199.21 ± 4.32b 552.97 ± 17.27 10.02 ± 0.26b 0.51 ± 0.03 0.63 ± 0.01 68.47 ± 2.59 10.42 ± 0.28 6.18 ± 0.15a

Hepatopancreas SOD (U mg −1 protein) GSH-Px (U g −1 protein) POD (U mg −1 protein) GR (U g −1 protein) T-AOC (U mg −1 protein) LD (mmol g −1 tissue) MDA (nmol mg −1 protein)

7.14 ± 0.24 50.84 ± 1.87b 12.81 ± 0.40b 6.48 ± 0.46 2.27 ± 0.10 0.25 ± 0.01 1.64 ± 0.11b

7.24 ± 0.38 50.28 ± 1.75b 12.50 ± 0.42b 6.36 ± 0.28 2.25 ± 0.02 0.25 ± 0.02 1.63 ± 0.03b

7.41 ± 0.55 66.63 ± 2.02a 16.55 ± 0.51a 5.84 ± 0.96 2.43 ± 0.10 0.21 ± 0.02 2.40 ± 0.25a

5.37 ± 0.28 55.62 ± 1.55 5.68 ± 0.42 5.93 ± 0.54b 2.20 ± 0.07 0.32 ± 0.02 1.58 ± 0.11b

5.24 ± 0.19 55.86 ± 2.65 5.83 ± 0.30 5.75 ± 0.46b 2.18 ± 0.11 0.34 ± 0.02 1.56 ± 0.08b

6.02 ± 0.25 52.99 ± 2.02 6.46 ± 0.48 7.45 ± 0.38a 2.47 ± 0.12 0.26 ± 0.03 1.92 ± 0.15a

Data are presented as mean ± SE (n = 4). Values within the same row with different letters mean significant difference (P < .05). SOD: superoxide dismutase; GSHPx: glutathione peroxidase; POD: peroxidase; CAT: catalase; GR: glutathione reductase; LDH: lactate dehydrogenase; T-AOC: total antioxidant capacity; LD: lactic acid; MDA: malonaldehyde. 377

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Table 7 Effects of dietary supplementation of defatted H. pluvialis meal on non-specific immune indices of adult E. sinensis. Items

Male

Female

Diet 1

Diet 2

Diet 3

Diet 1

Diet 2

Diet 3

Serum ACP (U g −1 protein) ALP (U g −1 protein) γ-GT (U g −1 protein) PO (U mg −1 protein) Hc (mg mL −1) NO (μmol L −1)

0.25 ± 0.02a 2.00 ± 0.02a 0.23 ± 0.01a 28.59 ± 0.96 52.73 ± 1.01 13.19 ± 0.35

0.25 ± 0.00a 1.95 ± 0.02a 0.24 ± 0.01a 27.96 ± 0.69 52.33 ± 0.71 12.27 ± 0.30

0.19 ± 0.01b 1.57 ± 0.03b 0.16 ± 0.01b 32.24 ± 1.09 51.22 ± 1.29 12.41 ± 0.32

0.22 ± 0.01 1.89 ± 0.04a 0.14 ± 0.01 17.76 ± 0.52 64.46 ± 1.28a 23.81 ± 0.65a

0.21 ± 0.00 1.86 ± 0.03a 0.14 ± 0.01 18.11 ± 0.92 65.79 ± 1.89a 25.00 ± 1.50a

0.27 ± 0.02 1.70 ± 0.04b 0.12 ± 0.01 19.67 ± 1.35 58.11 ± 0.92b 10.37 ± 0.32b

Hepatopancreas ACP (U 100 g −1 protein) ALP (U g −1 protein) γ-GT (U g −1 protein) NO (μmol g −1 protein)

4.61 ± 0.35b 29.22 ± 1.56b 109.45 ± 4.50b 1.48 ± 0.08b

4.33 ± 0.26b 29.12 ± 1.20b 134.49 ± 4.28b 1.44 ± 0.10b

7.02 ± 0.53a 55.04 ± 3.38a 189.10 ± 10.62a 1.91 ± 0.12a

6.37 ± 0.38b 41.11 ± 1.26b 225.63 ± 7.23b 1.44 ± 0.08b

6.17 ± 0.26b 40.67 ± 2.02b 226.96 ± 8.86b 1.43 ± 0.06b

11.94 ± 1.24a 51.47 ± 3.56a 264.42 ± 5.26a 2.10 ± 0.12a

Data are presented as mean ± SE (n = 4). Values within the same row with different letters mean significant difference (P < .05). ACP: acid phosphatase; ALP: alkaline phosphatase; γ-GT: γ-glutamyl transpeptidase; PO: phenoloxidase; Hc: hemocyanin; NO: nitric oxide.

formulation, improve the coloration, enhance antioxidant and immune capacity of adult E. sinensis. This study provides valuable information on a high-value application of defatted H. pluvialis meal.

et al., 2017), and ALP and ACP play important roles in the immune process of crustaceans (Liu et al., 1999; Wu et al., 2017). In this study, higher activities of ALP and ACP in the male serum as well as higher activities of ALP in female serum were found for Diet 1 and Diet 2, which indicated that dietary supplementation of defatted H. pluvialis meal or whole H. pluvialis powder could improve the immune ability of E. sinensis. Furthermore, Hemocyanin (Hc) is the major protein in hemolymph of crustaceans, where it plays an important role in oxygen transportation and immune function (Long et al., 2017; Velayutham and Munusamy, 2016). Dietary astaxanthin only significantly improved the Hc content in the female serum, which indicated that gender differences are existed on the immune response of dietary astaxanthin on adult E. sinensis. A different trend was observed in the hepatopancreas, with the control treatment (without astaxanthin supplementation) recording the highest activities of ACP, ALP and γ-GT than the dietary treatment with the supplementation of astaxanthin. This result was consistent with previous studies (Long et al., 2017; Wu et al., 2017). This could be because astaxanthin, acts as an antioxidant, which can eliminate free radicals and reduce the oxidative stress (Ambati et al., 2014; Chien and Shiau, 2005), so the immune enzymes are less stimulated by undesirable factors (such as free radicals), and their activities are then activated to a lesser extent. Currently, the natural astaxanthin from H. pluvialis is widely accepted as a pigment or a feed additive in animal feeds or nutraceuticals (Long et al., 2017). However, its market price is several times than that of synthetic astaxanthin (Capelli et al., 2013; Wade et al., 2015). Currently, the commercial crustacean feeds are recommended to contain 50–100 mg kg −1 of synthetic astaxanthin (Ju et al., 2012; Niu et al., 2014). The price of per unit astaxanthin originated from defatted H. pluvialis meal (astaxanthin: 0.66% of dry matter), whole H. pluvialis powder (astaxanthin: 3.0% of dry matter) and synthetic astaxanthin (astaxanthin: 10.0% of dry matter) are quite different. If the fattening diet for E. sinensis was formulated to contain around 60 mg kg −1 of astaxanthin using 1% defatted H. pluvialis meal, or 0.2% whole H. pluvialis powder, or 0.06% synthetic astaxanthin (10% of astaxanthin), as astaxanthin sources, it would result in additional cost of USD 0.09 kg −1 diet, USD 0.40 kg −1 diet, and USD 0.15–0.18 kg −1, respectively, compared to the control diet (without astaxanthin supplementation). In this study, no significant differences were detected on gonadal development, color parameters, astaxanthin contents, antioxidant and immune capacity of E. sinensis between Diet 1 (with the supplementation of defatted H. pluvialis meal) and Diet 2 (with the supplementation of whole H. pluvialis powder) treatments. In conclusion, using the defatted H. pluvialis meal as an astaxanthin source in the fattening feed could significantly reduce the cost of feed

Acknowledgements This work was supported by two projects (No.31873041 and No. U1706209) from the Natural Science Foundation of China and a “Shu Guang” Project (No.17SG50) from Shanghai Education Commission and Shanghai Education Development Foundation. The analysis cost was partially funded by the general project (No.HSXT217) of the Jiangsu Collaborative Innovation Center of Regional Modern Agriculture & Environmental Protection, the Policy Guidance Cooperation Project (BY2016062-04) from the Science and Technology Department of Jiangsu Province, the Special Fund (CARS-48) of Chinese Agriculture Research System from Ministry of Agriculture of China, and the R&D project (D-8006-15-0054) from the Shenzhen Alpha Feed Co. Ltd. Infrastructure costs were supported by the construction and improvement project (No. A1-2801-18-1003) for high level university in Shanghai from Shanghai Education Commission and the innovation and extension project (Y2017-4) of Science and technology from Jiangsu Ocean and Fisheries Bureau. The authors would like to thank Renfu Wu and Zehua Liu from the Crustacean Nutrition and Reproduction research group of Shanghai Ocean University for their help with sampling and biochemical analysis. We thank Dr. John van der Meer (PanAmerican Marine Biotechnology Association, USA) and Prof. Giovanni M Turchini (Deakin University, Australia) for their comments and assistance with language and proofreading. Sincere thanks are also due to the anonymous reviewers for their constructive comments to improve the quality of this manuscript. References Ambati, R.R., Phang, S., Ravi, S., Aswathanarayana, G.R., 2014. Astaxanthin: sources, extraction, stability, biological activities and its commercial applications-a review. Mar. Drugs 12, 128–152. AOAC, 1995. Official Methods of Analysis of Association of Official Analytical Chemists, 16th ed. Method, Arlington, VA, pp. 13. Arad, S.M., Yaron, A., 1992. Natural pigments from red microalgae for use in foods and cosmetics. Trends. Food. Sci. Tech. 3, 92–97. Blackburn, S., 1968. Amino Acid Determination, Methods and Techniques. Marcel Dekker Inc, New York, pp. 271. Boonyaratpalin, M., Thongrod, S., Supamattaya, K., Britton, G., Schlipalius, L.E., 2001. Effects of β-carotene source, Dunaliella salina, and astaxanthin on pigmentation, growth, survival and health of Penaeus monodon. Aquac. Res. 32, 182–190. Bureau of Fisheries and Management, 2018. China Fishery Statistical Yearbook of 2017. China Agriculture Press, Beinjing, China (in Chinese). Capelli, B., Bagchi, D., Cysewski, G.R., 2013. Synthetic astaxanthin is significantly inferior to algal-based axtaxanthin as an antioxidant and may not be suitable as a human nutraceutical supplement. Nutrafoods 12, 145–152.

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