Dietary supplementation of astaxanthin enhances hemato-biochemistry and innate immunity of Asian seabass, Lates calcarifer (Bloch, 1790)

Aquaculture 512 (2019) 734339

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Dietary supplementation of astaxanthin enhances hemato-biochemistry and innate immunity of Asian seabass, Lates calcarifer (Bloch, 1790)

T

Keng Chin Lima, Fatimah Md. Yusoffa,b,c, , Mohamed Shariffb,d, Mohd. Salleh Kamarudina,c, Norio Nagaob,e ⁎

a

Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia c Institute of Aquaculture and Aquatic Sciences, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia d Aquatic Animal Health Unit, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia e Bluescientific Shinkamigoto Co. Ltd., 102 Naname-go, Shinkamigoto-cho, Minami Matsuura-gun, Nagasaki 857-4214, Japan b

ARTICLE INFO

ABSTRACT

Keywords: Asian seabass Dietary astaxanthin Feeding phase Hemato-biochemistry Innate immunity Supplementation

This study aimed to evaluate the impacts of dietary astaxanthin supplementation on hematology, blood biochemistry and innate immunity of Asian seabass Lates calcarifer, with special reference to dose-response associations and variations over different phases of feeding (short-term, medium-term and long-term). Triplicate groups of fish (n = 30) with an average weight of 28 g were fed astaxanthin-incorporated diets (AX50, 50 mg kg−1 diet; AX100, 100 mg kg−1 diet; AX150, 150 mg kg−1 diet) for 90 days. A diet without astaxanthin supplement (CD) served as the control. Our findings demonstrated that fish displayed significant enhancements (P < .05) in hematological indices (white blood cell count, red blood cell count, hemoglobin and hematocrit) when fed various diets with elevated doses of astaxanthin throughout the specified phases of feeding. Serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), glucose, and cortisol in fish fed the supplemented diets decreased significantly (P < .05) with increasing dietary inclusion levels. Moreover, the provision of dietary astaxanthin at escalating doses markedly reduced (P < .05) the circulating levels of serum cholesterol (proportionately) and triglyceride (dose-dependently) in fish, following three consecutive feeding phases. Correspondingly, the supplemented fish exhibited much higher (P < .05) serum total protein content associated with astaxanthin administration. Immunological parameters (respiratory burst activity, lysozyme activity, phagocytic activity, and serum total immunoglobulin) of fish were significantly stimulated (P < .05) in response to dietary intervention with astaxanthin. The present investigation highlights the ameliorating effect of dietary astaxanthin on hemato-biochemical and immunological variables of Asian seabass and could be administered in culture protocols to improve fish immunocompetence and health.

1. Introduction

widespread and unrestricted application of prophylactic antimicrobials, particularly in developing countries, to treat and forestall infections. The indiscriminate use of antimicrobials has been associated with the emergence of antimicrobial-resistant pathogens (Watts et al., 2017). An interesting alternative approach to immunoprophylactic control is the administration of natural immunostimulants (e.g., microalgae and carotenoid pigments) whereby these substances play crucial roles in modulating and enhancing the immune response of aquatic animals against many disease causative agents. Asian seabass (Lates calcarifer Bloch) is among the most important food fishes for commerce and subsistence in southeastern Asia and Australia. Aquaculture production of the Asian seabass is a rapidly developing enterprise with market expansion as a popular seafood item

The unceasing pressure on aquaculture to bridge the gap between supply and demand for food fish has caused a spurt in the growth of intensive fish farming. Nevertheless, intensive fish culture operations frequently subject animals to various stressful conditions (e.g., grading, handling, transporting, vaccinating and overcrowding), which contributes to physiological dysfunction, poor fish performance, immune suppression, and increased susceptibility to pathogens (Liu et al., 2016; Lim et al., 2018). Outbreaks of infectious diseases, especially during the early production stages, constitute the largest single cause of severe economic losses worldwide and remain the greatest challenge for the aquaculture industry. This situation has inevitably led to the ⁎

Corresponding author at: Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia E-mail address: [email protected] (F.Md. Yusoff).

https://doi.org/10.1016/j.aquaculture.2019.734339 Received 8 March 2019; Received in revised form 23 July 2019; Accepted 23 July 2019 Available online 24 July 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.

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Table 1 Proximate chemical compositions of the four experimental diets. Diet designation

Control diet (CD)

AX50

AX100

AX150

Proximate composition Moisture (%) Fiber (%) Ash (%) Lipid (%) Protein (%) Nitrogen-free extract (%)⁎ Astaxanthin (mg kg−1 diet)

10.91 ± 0.05a 4.11 ± 0.04a 11.42 ± 0.07a 8.73 ± 0.07a 43.45 ± 0.12a 21.38 ± 0.13a 0

10.95 ± 0.07a 4.22 ± 0.05a 11.27 ± 0.09a 8.78 ± 0.08a 43.59 ± 0.13a 21.19 ± 0.12a 48.95 ± 0.35

11.11 ± 0.05a 4.17 ± 0.06a 11.23 ± 0.05a 8.64 ± 0.06a 43.21 ± 0.12a 21.64 ± 0.20a 97.98 ± 0.42

11.04 ± 0.04a 4.19 ± 0.04a 11.38 ± 0.06a 8.56 ± 0.08a 43.34 ± 0.09a 21.49 ± 0.13a 148.83 ± 0.48

Values are expressed as the mean ± SEM, n = 4. Means that do not share a common superscript within each row are significantly different (P < .05). ⁎ Nitrogen-free extract = 100 − (% moisture + % fiber + % ash + % lipid + % protein).

in many European and North American countries (Harrison et al., 2014). The global production of this species has seen a surge over the past decades, and this trend is anticipated to resume (FAO, 2018). The Asian seabass is a euryhaline carnivorous fish, and farming activities have been predominantly initiated based on hatchery-reared stocks in either fresh, brackish and inshore coastal waters using net cages and ponds. However, the intensifying production of Asian seabass under stressful environmental conditions and lack of adequate health management measures has presented its own unique disease challenges that may adversely impact future sustainability. Therefore, considerable effort in fish nutrition research must be directed towards mitigating stress and enhancing the immunity of farmed Asian seabass. Astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) is a naturally occurring deep red-orange carotenoid pigment found primarily in the carapace of many crustaceans (e.g., crab, crayfish, lobster, krill and shrimp), the flesh of salmonids and in other marine organisms including microalgae (Begum et al., 2016; Lim et al., 2018) and microbes (Barredo et al., 2017). Fish, as do other animals, lack the capacity to biosynthesize astaxanthin de novo, and it must be acquired from the diet. Wild fish derive astaxanthin from their prey organisms, whereas the pigment is incorporated directly into the diet of farmed fish. Hitherto, Haematococcus pluvialis is generally regarded as one of the most promising sources of natural astaxanthin (2–5% on a dry weight basis) and the exclusive producer of this secondary carotenoid (Ho et al., 2018). Astaxanthin is dubbed as one of nature's most potent antioxidants with multiple biological functions and important applications in animal health and nutrition (Lim et al., 2018). While substantial research effort has focused on improving the immunocompetence of aquatic animals by incorporating synthetically derived astaxanthin (Amar et al., 2012; Alishahi et al., 2015; Cheng et al., 2018), prospective studies on the utilization of H. pluvialis-derived astaxanthin remain relatively scarce. Moreover, several earlier studies found that astaxanthin from synthetic sources only enhanced certain aspects of the immune defense mechanism in fish (Smith et al., 2013; Alishahi et al., 2015; Cheng et al., 2018). The present study was designed to evaluate the modulatory effects (dose-response manner) of various dietary astaxanthin inclusion levels on hemato-biochemical parameters and innate immune capacity in Asian seabass.

2.2. Preparation of diets and proximate composition Lyophilized H. pluvialis microalgal cells (containing approximately 37.94 ± 0.41 mg astaxanthin g−1 dry weight) (ScienceGates Biotech, Malaysia) were administered to commercial pellets (Star feed, CP Group, Malaysia) to produce experimental diets with varying dietary levels of astaxanthin designated as AX50, 50 mg (1.32 g dry weight kg−1 feed); AX100, 100 mg (2.64 g dry weight kg−1 feed); and AX150, 150 mg (3.95 g dry weight kg−1 feed). The preparation of these diets followed Lim et al. (2019). The proximate chemical compositions (including moisture, fiber, ash, lipid, protein, and nitrogen-free extract) of the experimental diets were determined based on methods described in AOAC (2016). Final astaxanthin concentration of each respective diet was quantified according to the procedures described by Inbaraj et al. (2006) with high-performance liquid chromatography (HPLC) (Agilent 1200, Agilent Technologies, Santa Clara, California, USA). Dietary compositions and final astaxanthin concentrations of the experimental diets are listed in Table 1. 2.3. Fish husbandry and experimental conditions Healthy Asian seabass (L. calcarifer Bloch) juveniles were obtained from a local commercial aquaculture farm (Oasis Long Diann Bio-tech, Banting, Malaysia). The fish were transported to the study site in polythene bags filled with oxygen. Before the feeding trials, fingerlings were subjected to a preventive bath of diluted formaldehyde (150 ppm; 30 min) and acclimatized in 1,000-L cylindrical polycarbonate tanks under natural photoperiod and continuous aeration for two weeks. Fish were fed the control diet to apparent satiation daily. After the acclimatization period, fish of approximately the same size (28.24 ± 0.14 g, mean ± SEM) were then randomly distributed into one control group and three test groups, containing 30 fish each, in triplicate. The experiment was carried out using 200-L glass aquaria. Airlift pumps equipped with under-gravel biofilters were used to supply aeration to the experimental units. Water quality variables including temperature (26–28 °C), salinity (18–20‰), pH (7–8), dissolved oxygen (5–6 mg L−1) and ammonia (0–0.25 mg L−1) were carefully monitored and regulated closely within optimal levels. Bottom debris and fecal matter were siphoned out and approximately 20–30% of the water was exchanged daily with dechlorinated seawater to ensure excellent water quality.

2. Materials and methods 2.1. Ethical statement

2.4. Feeding and sampling

All fish handling procedures were conducted in accordance with relevant guidelines on the care and use of animals for scientific purposes developed by the Institutional Animal Care and Use Committee (IACUC) of Universiti Putra Malaysia (UPM), Malaysia (approval number: UPM/IACUC/AUP-R072/2017).

Specimens in each experimental aquarium (three replications) were hand-fed with one of the experimental diets at a rate of 4% body weight day−1 over a 90-day trial period. The quantity of food was adjusted accordingly every 15 days based on fish body weight increment. Samplings were conducted repeatedly throughout the experimental period (at 30, 60, and 90 days of feeding). Fish were starved for 24 h prior to sampling. The fish were netted quickly and placed in 2

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120 mg L−1 MS-222 (Tricaine methanesulfonate) for rapid, deep anesthesia. Peripheral blood samples were collected from five anesthetized fish per tank using heparinized (30 IU sodium heparin mL−1) tuberculin syringes following venipuncture and each divided into two portions. One portion was transferred into blood collection tubes and used for hematological measurements and immunological assays. The second portion was transferred into sterile Eppendorf microcentrifuge tubes and left to clot at 4 °C. Serum samples were isolated via centrifugation (2000 g, 10 min, 4 °C) and frozen at −80 °C prior to further biochemical analyses.

solution (NBT) (Sigma-Aldrich Corp., St. Louis, United States) in a microtitre plate well, and the final solution was homogenized prior to incubation for 30 min at room temperature. The NBT solution was prepared in phosphate-buffered saline (PBS) at pH 7.4. After incubation and a second homogenization, 50 μL of the NBT-blood cell suspension was loaded in a glass vial containing 1 mL of N, N-dimethylformamide (DMF) (Sigma-Aldrich Corp., St. Louis, United States). The mixture was homogenized and centrifuged at 3000 g for 5 min. The optical density (OD) of the supernatant was immediately measured at 620 nm using a UV–Vis spectrophotometer (Shimadzu UV-2600, Kyoto, Japan). The blank solution comprised the same components except that distilled water was substituted for blood.

2.5. Hemato-biochemistry Total red blood cell (RBC) and white blood cell (WBC) counts, hemoglobin (Hb) and hematocrit (Ht) were analyzed using the Abbott CELL-DYN 3700 system multiparameter automated hematology analyzer (Abbott Laboratories, Illinois, USA) (Fazio et al., 2017). Biochemical indices including serum total protein, cholesterol, triglyceride, glucose, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were quantitatively determined via electrochemiluminescence using a Biolis 24i Premium automated clinical analyzer (Tokyo Boeiki Machinery Ltd., Tokyo, Japan), with assay kits supplied by BioREX Mannheim Malaysia Sdn Bhd, Selangor, Malaysia. Serum cortisol levels were quantified using a fish cortisol ELISA kit (CUSABIO, Wuhan, China), according to the manufacturer's guidelines. The optical density of each well was detected within 10 min, with the Sunrise absorbance microplate reader (Tecan Group Ltd., Mannedorf, Switzerland) set to 450 nm. Cortisol concentration of each sample was calculated according to the established standard curve.

2.6.4. Phagocytic activity Leucocyte phagocytic function was assayed following the method of Anderson and Siwicki (1995) and Ragap et al. (2012), with modifications. In short, 50 μL of each heparinized fish blood sample was added to 100 μL of Vibrio alginolyticus culture (5 × 108 CFU mL−1 in PBS suspension) in a microtitre plate well and mixed homogeneously with a sterile pipette to ensure contact of bacteria to leucocytes. Each mixture was then incubated for 20 min at room temperature with occasional shaking. After incubation, a drop of each suspension was placed on a methanol-cleansed microscope slide to produce a smear (similar to a blood smear) and subsequently air-dried at room temperature. This was followed by fixation in 95% ethyl alcohol for 5 min. The cells were then stained with 7% Giemsa stain for 10 min and washed gently with distilled water. Finally, each prepared slide was observed under the compound light microscope, and approximately 100 cells were examined in 10 random fields of view to assess numbers of phagocytized bacterial cells. Data were expressed as percent phagocytosis based on the following equation: Percent phagocytosis (%) = 100 × (total number of phagocytized bacterial cells/total number of counted leucocytes).

2.6. Immunological assay 2.6.1. Lysozyme activity Serum lysozyme activity was measured using the turbidimetric method as described by Ellis (1990) and Milla et al. (2010), with some modifications. The assay relied on the lysis of the lysozyme-sensitive Gram-positive bacterium Micrococcus lysodeikticus (Sigma-Aldrich Corp., St. Louis, United States) via the lysozyme present in the serum. In brief, 50 μL of fish serum was mixed with 950 μL of M. lysodeikticus suspension (0.15 mg mL−1 of 0.05 M potassium phosphate buffer at pH 6.24). The decrease in absorbance min−1 was immediately determined within 5 min with a spectrophotometer (Shimadzu UV-2600, Kyoto, Japan). The blank solution consisted of the same components, except that distilled water was substituted for serum. One unit of lysozyme activity (U) corresponded to the amount of enzyme that produced a decrease in absorbance of 0.001 min−1 at 450 nm.

2.7. Statistical analysis Significant differences among means of independent groups were analyzed using one-way analysis of variance (ANOVA) with evenly spaced distances between treatments (i.e., graded levels of dietary astaxanthin). The polynomial contrast procedure was employed to identify the trends of relationships that may exist among pairs of means when a significant effect has been found. This made it possible to test statistically if the studied effects were linear, i.e., directly proportional to the supplementation level of astaxanthin, or quadratic (curvilinear), i.e., dose-dependent influence (Davis, 2010; Yossa and Verdegem, 2015). The optimal supplementation dose was estimated by evaluating the resulting polynomial regression function between the independent quantitative (dietary astaxanthin levels) and dependent (response) variables according to the procedures described by Yossa and Verdegem (2015). Differences between treatment means were explored using orthogonal contrasts within individual sampling time. Quantitative data were presented as the mean ± standard error of the mean (SEM). Differences were considered statistically significant when P < .05. All statistical analyses were conducted using IBM SPSS software version 23 for Windows (IBM Corporation, Armonk, New York, USA).

2.6.2. Total immunoglobulin (Ig) The analysis of total immunoglobulin (Ig) in serum was performed essentially as previously established (Siwicki and Anderson, 1993). Immunoglobulins were precipitated from the serum with 10,000 kD polyethylene glycol (PEG) (Sigma-Aldrich Corp., St. Louis, United States). Briefly, the serum sample was mixed homogeneously with an equal volume of 12% PEG solution (suspended in deionized water) and incubated at room temperature for 2 h under constant shaking. The supernatant was collected after centrifugation (5000 g, 10 min) and assayed for total protein content. Serum total immunoglobulin concentration was quantified by subtracting the obtained value from the total protein concentration in the serum prior to precipitation with PEG.

3. Results . 3.1. Hematological parameters

2.6.3. Respiratory burst activity The respiratory burst activity procedure was conducted in accordance with the protocol outlined by Anderson and Siwicki (1995), with slight modifications. In brief, 100 μL of each heparinized blood sample was added to an equal volume of 0.2% nitroblue tetrazolium

White blood cell (WBC) counts of fish were affected by experimental diets and elevated significantly (P < .05) in a linear fashion as the supplementation level of astaxanthin increased from 0 to 150 mg kg−1 diet through all phases of feeding (Table 2). Significantly higher 3

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Table 2 Hematological parameters of Asian seabass (Lates calcarifer) fed diets supplemented with various levels of astaxanthin during short-term, ST (30 days), medium-term, MT (60 days) and long-term, LT (90 days) feeding phases, respectively, throughout the trial period. Parameter

WBC (×108 L−1) RBC (×1012 L−1) Hb (g L−1) Ht (%)

Feeding phase

ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days)

Diet CD

AX50

AX100

AX150

5.35 ± 0.05 5.39 ± 0.07 5.54 ± 0.09 3.03 ± 0.07 3.14 ± 0.08 3.30 ± 0.07 104.97 ± 2.69 105.67 ± 1.76 111.67 ± 1.45 32.67 ± 0.88 33.33 ± 0.67 34.33 ± 0.33

5.51 ± 0.08 5.95 ± 0.09a 6.16 ± 0.13a 3.25 ± 0.09 3.38 ± 0.06a 3.54 ± 0.08a 106.67 ± 2.33 115.00 ± 2.08a 120.67 ± 1.20a 34.00 ± 0.58 37.67 ± 0.88a 38.00 ± 0.58a

5.74 ± 0.06a 6.11 ± 0.08a 6.61 ± 0.12a 3.31 ± 0.10 3.51 ± 0.07a 3.62 ± 0.06a 112.40 ± 2.66 124.67 ± 2.19a 126.07 ± 1.09a 34.67 ± 0.67 40.00 ± 0.58a 42.33 ± 0.33a

5.67 ± 0.09a 6.19 ± 0.11a 6.58 ± 0.08a 3.27 ± 0.08 3.57 ± 0.05a 3.65 ± 0.08a 110.33 ± 2.03 128.27 ± 2.02a 128.33 ± 1.45a 34.33 ± 0.67 41.33 ± 0.88a 42.67 ± 0.33a

Polynomial relationship

Significance†

Linear Linear Linear – Linear Linear – Linear Linear – Linear Linear

P< P< P< ns P< P< ns P< P< ns P< P<

.05 .05 .05 .05 .05 .05 .05 .05 .05

Values are expressed as the mean ± SEM, n = 3. WBC, white blood cell count; RBC, red blood cell count; Hb, hemoglobin; Ht, hematocrit. ns = not statistically significant (P > .05). † Polynomial contrasts of dependent variables across diets. a Significantly different from the control (P < .05) within each row.

(P < .05) WBC counts were observed in fish fed AX100 and AX150 diets compared to the control group over short-term feeding (30 days). For both medium-term and long-term feedings (60 and 90 days, respectively), the WBC count of fish receiving the control diet was significantly lower (P < .05) than the supplemented groups. Regardless of diet, WBC counts of fish increased progressively with feeding duration in all treatments. Red blood cell (RBC) count, hemoglobin and hematocrit values of fish did not vary significantly (P > .05) among different treatments during the first 30 days of feeding (short-term); albeit discernible improvements were seen in the astaxanthin-fed groups (Table 2). Nevertheless, these variables increased significantly (P < .05) in a linear trend with escalating levels of dietary astaxanthin when the fish were fed different diets over both medium-term and long-term feedings; as evidenced by the significant polynomial contrasts. During the previously mentioned periods, dietary astaxanthin-supplemented fish (AX50, AX100 and AX150 groups) exhibited significantly higher (P < .05) RBC counts, hemoglobin and hematocrit concentrations compared to no supplemented fish (CD group). It is worth noting that these components increased consistently across all fish groups as the duration of feeding lengthened.

cholesterol level of fish throughout all feeding phases (short-term, medium-term and long-term) (Table 3). In contrast, increasing supplementary astaxanthin levels (0–150 mg kg−1 diet) in fish diets exhibited a significant quadratic effect (P < .05) on serum triglyceride level of fish through different feeding phases (short-term, medium-term and long-term) (Fig. 2a, b and c). The lowest estimates in each of the earlier-mentioned feeding phases were obtained with supplementation levels of 146.25, 150 and 142.14 mg astaxanthin kg−1 diet, respectively. Serum cholesterol and triglyceride concentrations in fish fed AX100 and AX150 diets were significantly lower (P < .05) than in the control group over the short-term feeding phase. Nonetheless, fish fed supplemented diets at all dietary levels (AX50, AX100, and AX150) displayed significantly reduced (P < .05) contents of serum cholesterol and triglyceride compared with those in the control group during the medium-term and long-term feeding phases. Higher levels of serum cholesterol and triglyceride were noted in all fish groups as feeding duration lengthened. Serum total protein concentration was, on average, significantly elevated (P < .05) in all fish fed with various dietary doses of astaxanthin (AX50, AX100, and AX150) in relation to the control group (CD) at each phase of the feeding trial (short-term, medium-term and longterm) (Table 3). The increase in supplementation level (0–150 mg astaxanthin kg−1 diet) corresponded to a significant positive linear effect (P < .05) on serum total protein content of Asian seabass over the short-term and medium-term phases of feeding. With regard to longterm feeding, the presence of a significant quadratic effect (P < .05) was associated with a higher calculated estimate at the optimal supplementation dose of 107.61 mg astaxanthin kg−1 diet and a negligible decline when the dietary dose was further increased to 150 mg astaxanthin kg−1 diet (Fig. 1d). According to the patterns of gradual changes, the levels of serum total protein rose proportionately across all fish groups as feeding proceeded. Dietary administration of astaxanthin markedly influenced serum cortisol content of fish, which significantly declined (P < .05) linearly with the increase in supplementation levels (0–150 mg astaxanthin kg−1 diet) over the first two phases of feeding (Table 4). However, there was a significant quadratic relationship (P < .05) between serum cortisol concentration of fish and dietary levels of astaxanthin during the long-term feeding phase. Accordingly, the optimal supplementary dose that led to the lowest serum cortisol concentration in fish was estimated to be 118.75 mg astaxanthin kg−1 diet (Fig. 1e). Regardless of dietary level or enriched diet, serum cortisol concentrations of supplemented fish were significantly lower (P < .05) than those of control fish throughout the specified phases of feeding. Seemingly, serum

3.2. Serum biochemical indices Serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and glucose were not significantly different (P > .05) among fish groups during the short-term phase of feeding, despite being lower in the astaxanthin-treated groups (Table 3). The first order polynomial (linear) for serum ALT, AST and glucose responses over the medium-term phase of feeding were significant (P < .05), denoting substantial linear decrements in these variables as fish fed on diets with increasing levels of dietary astaxanthin. Significant quadratic effects (P < .05) of supplementary level of astaxanthin on the above indices were observed over the long-term phase of feeding. The lowest estimates for serum levels of ALT, AST and glucose were determined at the optimal doses of 122.5, 113.57 and 142 mg astaxanthin kg−1 diet, respectively (Fig. 1a, b, and c). Diets containing astaxanthin at all inclusion levels (AX50, AX100, and AX150) led to significantly lower (P < .05) serum contents of ALT, AST, and glucose in fish when compared to the control (CD) through the medium-term and long-term phases of feeding. The values of these indices were comparatively higher in different fish groups as feeding progressed. In general, the provision of dietary astaxanthin at increasing levels exerted a significant negative linear effect (P < .05) on serum 4

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Table 3 Serum biochemistry profile of Asian seabass (Lates calcarifer) fed diets supplemented with various levels of astaxanthin during short-term, ST (30 days), mediumterm, MT (60 days) and long-term, LT (90 days) feeding phases, respectively, throughout the trial period. Parameter

Feeding phase

ALT (U L−1) AST (U L

−1

)

Glucose (mmol L−1) Cholesterol (mmol L

−1

)

Triglyceride (mmol L−1) Total protein (g L−1)

ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days)

Diet CD

AX50

AX100

AX150

26.33 ± 0.33 33.33 ± 0.67 41.67 ± 1.20 34.67 ± 0.88 43.33 ± 0.67 48.00 ± 1.00 2.53 ± 0.15 4.07 ± 0.09 5.53 ± 0.18 3.80 ± 0.21 4.63 ± 0.12 5.37 ± 0.15 2.91 ± 0.06 3.50 ± 0.10 4.38 ± 0.07 36.00 ± 1.06 42.20 ± 1.17 44.63 ± 1.22

25.33 ± 0.88 30.00 ± 0.58a 32.67 ± 1.45a 32.00 ± 1.16 38.33 ± 0.67a 41.00 ± 0.58a 2.40 ± 0.17 3.67 ± 0.15a 4.43 ± 0.18a 3.27 ± 0.15 4.07 ± 0.18a 4.83 ± 0.09a 2.71 ± 0.08 2.95 ± 0.05a 3.65 ± 0.11a 42.03 ± 1.53a 47.30 ± 1.49a 50.80 ± 1.55a

25.00 ± 0.58 27.33 ± 0.88a 30.67 ± 1.33a 31.67 ± 0.33 35.67 ± 0.88a 37.67 ± 0.33a 2.37 ± 0.13 3.27 ± 0.12a 4.03 ± 0.20a 2.17 ± 0.19a 3.73 ± 0.12a 4.53 ± 0.09a 1.90 ± 0.07a 2.63 ± 0.04a 3.03 ± 0.07a 44.63 ± 1.66a 48.07 ± 1.16a 54.33 ± 1.33a

25.67 ± 0.67 26.00 ± 0.58a 31.33 ± 0.33a 33.33 ± 0.67 34.33 ± 0.33a 38.67 ± 1.20a 2.20 ± 0.10 3.10 ± 0.06a 4.27 ± 0.23a 2.27 ± 0.18a 3.40 ± 0.06a 4.23 ± 0.12a 2.02 ± 0.04a 2.42 ± 0.09a 2.98 ± 0.06a 48.97 ± 0.73a 52.70 ± 1.04a 54.23 ± 1.19a

Polynomial relationship

Significance†

– Linear Quadratic – Linear Quadratic – Linear Quadratic Linear Linear Linear Quadratic Quadratic Quadratic Linear Linear Quadratic

ns P< P< ns P< P< ns P< P< P< P< P< P< P< P< P< P< P<

.05 .05 .05 .05 .05 .05 .05 .05 .05 .05 .05 .05 .05 .05 .05

Values are expressed as the mean ± SEM, n = 3. ALT, alanine aminotransferase; AST, aspartate aminotransferase. ns = not statistically significant (P > .05). † Polynomial contrasts of dependent variables across diets. a Significantly different from the control (P < .05) within each row.

cortisol levels in all fish groups increased gradually as feeding continued.

(Table 5). The in vitro phagocytic activity of fish fed diets supplemented with astaxanthin (AX50, AX100, and AX150) was significantly augmented (P < .05) compared to the unsupplemented counterpart (CD). This activity tended to increase steadily across all fish groups as the feeding trial advanced to later stages.

3.3. Immunological indices 3.3.1. Respiratory burst activity (RBA) Dietary incorporation of astaxanthin significantly enhanced (P < .05) RBA in fish, which also increased significantly (P < .05) in a linear manner with escalating amounts of supplemented astaxanthin (0–150 mg astaxanthin kg−1 diet) throughout different phases of the trial period (Table 5). RBA of fish fed the control diet was significantly lower (P < .05) than that of fish fed the supplemented diets (AX50, AX100, and AX150) during all phases of feeding (short-term, mediumterm and long-term). Additionally, the RBA intensified with the extension of feeding duration as fish grew, irrespective of diet. The described effect was much more pronounced in the supplemented groups.

3.3.4. Total immunoglobulin (Ig) Dietary supplementation with astaxanthin significantly increased (P < .05) the serum total Ig concentrations in fish, regardless of the administered dose, when compared to the control group at various phases of the whole feeding period (Table 5). In turn, the serum total Ig content of fish increased significantly in a linear fashion (P < .05) as the supplementary dose of astaxanthin rose with different diets (0–150 mg astaxanthin kg−1 diet) during each feeding phase, as confirmed by the significant polynomial responses. Serum total Ig levels of fish fed the supplemented diets (AX50, AX100, and AX150) rose concomitantly with feeding duration, whereas the increment was marginal in the control group (CD).

3.3.2. Lysozyme activity The serum lysozyme activity of fish fed with different diets exhibited a significant linear increase (P < .05) accompanying the increment of astaxanthin inclusion level (0–150 mg astaxanthin kg−1 diet) during the short-term and medium-term phases (Table 5). Additionally, there was a significant quadratic effect (P < .05) of astaxanthin content on lysozyme activity of serum with long-term feeding, with the highest estimate observed being for supplementation with 125.2 mg astaxanthin kg−1 diet (Fig. 3). Levels of serum lysozyme activity of fish fed AX100 and AX150 were significantly greater (P < .05) than those of fish fed the control diet through the short-term phase. In medium-term and long-term feedings, the serum lysozyme activity of fish receiving the control diet was significantly lower (P < .05) than in all supplemented groups. Gradual increments of serum lysozyme activity were immediately obvious in the astaxanthin-fed groups (AX50, AX100, and AX150) with the progress of feeding duration, whereas a decrement occurred for the control group.

4. Discussion 4.1. Hemato-biochemistry 4.1.1. Hematological parameters Through the years, not many attempts have been made to observe the effects of dietary astaxanthin on hematological parameters in fish. Earlier studies revealed that dietary supplementation of astaxanthin was not effective in enhancing hematological indices of fish, including Atlantic salmon Salmo salar (Christiansen et al., 1995) and large yellow croaker Pseudosciaena crocea (Li et al., 2014). Notwithstanding, our results concur with previous findings in common carp Cyprinus carpio supplemented with astaxanthin (Jagruthi et al., 2014). The contrasting effects might be attributed to variations in size or physiological stage of fish, type or biological source of pigment, supplementation dose, feeding duration, environmental rearing conditions, and species-specific responses. A variety of natural antioxidants, including carotenoids, function to neutralize endogenously produced harmful free radicals, thereby preventing peroxidation damage in cytomembranes and maintaining the structural integrity of important immune cells and

3.3.3. Phagocytic activity On average, increasing dietary astaxanthin diet (0–150 mg astaxanthin kg−1 diet) caused a significant positive linear effect (P < .05) on total phagocytic activity in fish through separate phases of feeding 5

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Fig. 1. Significant quadratic relationships (P < .05) between serum ALT (alanine aminotransferase) (a), AST (aspartate aminotransferase) (b), glucose (c), total protein (d), and cortisol (e) concentrations of Asian seabass (Lates calcarifer) and dietary levels of astaxanthin during the long-term, LT (90 days) feeding phase.

tissues (Kattappagari et al., 2015; Foo et al., 2017; Lim et al., 2018). The potent antioxidant property of astaxanthin may well indicate its superior effects on immune system stimulation and functions of organs related to hematopoiesis, such as bone marrow, head kidney, thymus, and spleen.

the levels of ALT, AST, and glucose in fish (Martinez-Porchas et al., 2009; Tang et al., 2019). The current study revealed that a longer duration of dietary astaxanthin supplementation is necessary for modulations of serum ALT, AST, and glucose contents in Asian seabass. Similar observations have been made in several fish species (Pan et al., 2011; Liu et al., 2016; Cheng et al., 2018), as a direct result of dietary astaxanthin provision. Thus, we acknowledge the instrumental role of astaxanthin in stress alleviation and liver enhancement, in turn improving the health of fish. Our results clearly demonstrated the antihyperglycemic potential of dietary astaxanthin, as previously documented in corpulent rat and mice models (Hussein et al., 2007; Bhuvaneswari et al., 2014). These findings infer that dietary

4.1.2. Serum biochemical indices The activity levels of serum ALT and AST represent critical indicators in the diagnosis of digestive function and liver integrity (Sookoian and Pirola, 2015). Glucose is the primary source of energy that fish use to cope with physiological stress. It is well accepted that any stress exposure or disease infection would substantially heighten 6

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Fig. 2. Significant quadratic relationship (P < .05) between serum triglyceride concentration of Asian seabass (Lates calcarifer) and dietary levels of astaxanthin during short-term, ST (30 days) (a), medium-term, MT (60 days) (b) and long-term, LT (90 days) (c) feeding phases.

astaxanthin may be potentially beneficial in stimulating the insulin sensitivity of fish, as demonstrated by Uchiyama et al. (2002) in a diabetic mouse model. Changes in the levels of serum cholesterol and triglyceride may occasionally signify liver dysfunction or disturbance of lipid metabolism, in response to physiological stressors (Canli et al., 2018). Our findings corroborate the results of Sheikhzadeh et al. (2012) and Li et al. (2014), in which dietary astaxanthin and H. pluvialis stimulated considerably lower serum total cholesterol and triglyceride of fish when fed at increasing levels. Therefore, our study suggests that the antioxidative property of astaxanthin may be of value in alleviating hyperlipidemia through the augmentation of endogenous cholesterol and triglyceride clearance mechanisms, which consequently relieve stress in fish. The anti-hyperlipidemic potential of astaxanthin has been

investigated in rat and mice models with promising outcomes (Ryu et al., 2012; Kumar et al., 2017). Astaxanthin was also found to ameliorate lipid metabolism in human subjects with hyperlipidemia (Yoshida et al., 2010; Choi et al., 2011). The functional mechanism for the supposed anti-hyperlipidemic effect of dietary astaxanthin has been speculated to be modulated by peroxisome proliferator-activated receptors (PPARs) (Tsubakio-Yamamoto et al., 2008). An elevation in serum total protein content is believed to correspond with the stronger innate immune response in fish (Shiu et al., 2017; Rebl and Goldammer, 2018). This is broadly reflected by the stronger innate immunity of astaxanthin-supplemented fish. Limited studies are available concerning the effect of astaxanthin on plasma or serum total protein in teleostean fish. Li et al. (2014) did not observe any notable improvements in the serum total protein level of large

Table 4 Serum cortisol concentrations of Asian seabass (Lates calcarifer) fed diets supplemented with various levels of astaxanthin during short-term, ST (30 days), mediumterm, MT (60 days) and long-term, LT (90 days) feeding phases, respectively, throughout the trial period. Feeding phase

Diet CD

−1

Cortisol concentration (ng mL

)

ST (30 days) MT (60 days) LT (90 days)

9.46 ± 0.11 10.95 ± 0.10 12.09 ± 0.13

AX50

AX100 a

7.86 ± 0.06 9.48 ± 0.12a 9.53 ± 0.11a

Values are expressed as the mean ± SEM, n = 3. ns = not statistically significant (P > .05). † Results from polynomial contrasts of dependent variables across diets. a Significantly different from the control (P < .05) within each row. 7

Polynomial relationship

Significance†

Linear Linear Quadratic

P < .05 P < .05 P < .05

AX150 a

7.08 ± 0.08 8.72 ± 0.06a 9.55 ± 0.09a

6.79 ± 0.09a 7.39 ± 0.07a 8.87 ± 0.04a

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Table 5 Innate immune parameters of Asian seabass (Lates calcarifer) fed diets supplemented with various levels of astaxanthin during short-term, ST (30 days), medium-term, MT (60 days) and long-term, LT (90 days) feeding phases, respectively, throughout the trial period. Parameter

Respiratory burst activity (OD620nm) Lysozyme activity (U mL−1) Phagocytic activity (%) Total immunoglobulin (g L−1)

Feeding phase

ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days)

Diet CD

AX50

AX100

AX150

0.52 ± 0.01 0.58 ± 0.02 0.61 ± 0.01 235.87 ± 10.78 203.53 ± 11.16 201.13 ± 8.80 76.33 ± 1.20 80.67 ± 1.45 81.67 ± 1.20 19.33 ± 0.67 20.30 ± 0.97 20.53 ± 0.94

0.58 ± 0.01a 0.67 ± 0.01a 0.72 ± 0.02a 238.13 ± 11.39 242.20 ± 6.06a 261.77 ± 7.77a 70.67 ± 0.88a 85.33 ± 1.20a 86.33 ± 0.88a 23.03 ± 0.88a 25.47 ± 0.78a 26.97 ± 0.62a

0.63 ± 0.01a 0.73 ± 0.01a 0.79 ± 0.02a 285.00 ± 10.94a 287.87 ± 8.84a 298.87 ± 3.39a 83.67 ± 1.45a 86.33 ± 0.88a 88.67 ± 1.33a 25.63 ± 0.83a 29.67 ± 0.95a 31.40 ± 0.61a

0.65 ± 0.02a 0.76 ± 0.02a 0.83 ± 0.01a 291.87 ± 10.99a 294.40 ± 9.98a 295.27 ± 9.04a 85.33 ± 1.33a 89.67 ± 1.20a 93.33 ± 1.45a 26.30 ± 0.57a 33.23 ± 0.66a 35.37 ± 0.73a

Polynomial relationship

Significance†

Linear Linear Linear Linear Linear Quadratic Linear Linear Linear Linear Linear Linear

P P P P P P P P P P P P

< < < < < < < < < < < <

.05 .05 .05 .05 .05 .05 .05 .05 .05 .05 .05 .05

Values are expressed as the mean ± SEM, n = 3. ns = not statistically significant (P > .05). † Polynomial contrasts of dependent variables across diets. a Significantly different from the control (P < .05) within each row.

cortisol levels in fish. This effect may be ascribed to its role in diminishing the production and secretion of cortisol by the inter-renal cells through the inhibition of ACTH, as elucidated by Haddad et al. (2013). 4.2. Immunological indices 4.2.1. Respiratory burst activity (RBA) RBA involves the swift release of reactive oxygen species (ROS) from an array of phagocytes generated by the membrane-bound NADPH oxidase for antimicrobial host defense (Nguyen et al., 2017). Little is known about the effect of astaxanthin on the RBA of aquatic animals. Our findings were consistent with observations of common carp C. carpio fed with β-carotene (Anbazahan et al., 2014). In vitro evidence suggests that phytochemical antioxidants can either scavenge or generate more ROS under certain conditions, which may be cell-type specific or dose-dependent (Halliwell, 2008; Ivanova et al., 2013; Leite et al., 2014). Dietary carotenoids, including β-carotene and lycopene, have been observed to exert in vivo antioxidant or prooxidant activity (Young and Lowe, 2001). According to our findings, the possibility exists that dietary astaxanthin may act as a prooxidant by stimulating NADPH oxidase-generated ROS for a more robust RBA in Asian seabass.

Fig. 3. Significant quadratic relationship (P < .05) between serum lysozyme activity of Asian seabass (Lates calcarifer) and dietary levels of astaxanthin during the long-term, LT (90 days) feeding phase.

yellow croaker P. crocea that received dietary astaxanthin. This contradictory finding may likely be due to variation in fish species and size, metabolic aspects, experimental rearing conditions, feeding management, and source and dietary inclusion levels of astaxanthin. As the majority of plasma or serum proteins are synthesized and secreted by the liver (Magnadottir et al., 2005), we assumed that the antioxidative role of astaxanthin may have amplified the mechanism of protein synthesis in the livers of fish, but this warrants further investigation. There is a paucity of information regarding the effects of dietary carotenoids, especially astaxanthin, on the serum cortisol concentration of fish. Cortisol is mainly secreted by the inter-renal cells of the head kidney into the bloodstream via the neuroendocrine system under the stimulatory action of ACTH (adrenocorticotropic hormone) following stressful circumstances (Montero et al., 2015; Reyes-Lopez et al., 2018). As an adaptive hormone, cortisol mediates the redistribution of energy (through liver glycogenolysis and gluconeogenesis) to restore homeostatic regulation (Gorissen and Flik, 2016). Numerous scientific articles consider as a rule of thumb that fish undergoing stressful conditions display elevated blood cortisol and glucose (Conde-Sieira et al., 2013; Birceanu and Wilkie, 2018). In this regard, we noticed higher serum glucose levels in fish with greater serum cortisol content, particularly the control group. Meilisza et al. (2018) showed that dietary carotenoids lowered cortisol levels and increased the resistance of Lake Kurumoi rainbow fish Melanotaenia parva in response to transportation stress. Our findings put forward some confirmatory evidence about the dietary effect of astaxanthin on stress mitigation through reduction in

4.2.2. Lysozyme and phagocytic activities Many in-depth studies have investigated the effective use of astaxanthin for stimulating phagocytic and serum lysozyme activities in European seabass D. labrax (Saleh et al., 2018), Japanese flounder P. olivaceus (Galindo-Villegas et al., 2006), large yellow croaker P. crocea (Li et al., 2014), and oscar Astronotus ocellatus (Alishahi et al., 2015). Lysozyme is among the few antimicrobial proteins of the innate immune system of living teleosts that exerts bacteriolytic activity by acting in concert with the complement system and phagocytes (Ragland and Criss, 2017). Phagocytes (lymphocytes, monocytes, and neutrophils) annihilate infectious agents principally through the production of ROS and lysozyme-catalyzed hydrolysis (Panase et al., 2017; Grayfer et al., 2018), which reflect greater respiratory burst and lysozyme activities in the astaxanthin-fed fish. In agreement with these findings, Jagruthi et al. (2014) and Ali et al. (2018) associated increased leucocyte count and phagocytic activity to greater lysozyme activity of fish. Furthermore, the higher proportion of WBCs reinforces the notion of much intensive phagocytic activity in supplemented fish. 4.2.3. Total immunoglobulin (Ig) Immunoglobulins (Igs) are a class of heterodimeric antibodies that neutralize pathogens of broad specificity (Mashoof and Criscitiello, 8

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Table 6 Estimated minimum effective and optimal supplementary doses of dietary astaxanthin that exerted clinically significant responses on the measured parameters of hemato-biochemistry in Asian seabass (Lates calcarifer) during each distinctive feeding phase. Parameter Hematology White blood cell count Red blood cell count Hemoglobin count Hematocrit level

Serum biochemistry ALT concentration AST concentration Glucose level Cholesterol level Triglyceride level Total protein content Cortisol concentration

Feeding phase

Minimum effective dose (mg astaxanthin kg−1 diet)

Optimal supplementary dose (mg astaxanthin kg−1 diet)

ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days)

50 50 50 – 50 50 – 50 50 – 50 50

150 150 150 – 150 150 – 150 150 – 150 150

ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days)

– 50 50 – 50 50 – 50 50 100 50 50 100 50 50 50 50 50 50 50 50

– 150 122.5 – 150 113.57 – 150 142 150 150 150 146.25 150 142.14 150 150 107.61 150 150 118.75

ALT, alanine aminotransferase; AST, aspartate aminotransferase. Table 7 Estimated minimum effective and optimal supplementary doses of dietary astaxanthin that exerted clinically significant responses on the measured parameters of innate immunity in Asian seabass (Lates calcarifer) during each distinctive feeding phase. Parameter

Feeding phase

Minimum effective dose (mg astaxanthin kg−1 diet)

Optimal supplementary dose (mg astaxanthin kg−1 diet)

Respiratory burst activity

ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days) ST (30 days) MT (60 days) LT (90 days)

50 50 50 100 50 50 50 50 50 50 50 50

150 150 150 150 150 125.2 150 150 150 150 150 150

Serum lysozyme activity Phagocytic activity Total Immunoglobulin (Ig)

2016). A relatively high concentration of Igs is present in the serum of fish, specifically synthesized by B lymphocytes (Adedeji et al., 2013; Zhu et al., 2013). Very little information is available related to the effects of astaxanthin administration on fish Ig production. Supporting these findings, Amar et al. (2000) reported that serum total Ig content was markedly enhanced in rainbow trout O. mykiss fed with β-carotene. In studies with mice, dietary incorporation of carotenoids was demonstrated to improve in vivo antibody production in response to Tdependent antigens (TD-Ags) (Jyonouchi et al., 1993, 1994). There was also evidence that dietary astaxanthin can enhance in vitro immunoglobulin production by human peripheral blood nuclear cells (Jyonouchi et al., 1995). The beneficial effect of astaxanthin on Ig

synthesis in Asian seabass suggests its possible immunomodulatory mechanism on the activation of fish T lymphocytes, which is arguably the most prominent factor regulating the differentiation and proliferation of peripheral B lymphocytes that produce Igs (Nakanishi et al., 2015; Ashfaq et al., 2019). 4.3. Administrative dose Based on our findings, we consider that supplementing fish with an ideal dose of astaxanthin over a correct feeding duration is necessary for any notable positive impacts and the best achievable performance (Tables 6 and 7). Collectively, our results suggest that diet 9

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supplemented with 50–100 mg astaxanthin kg−1 diet is beneficial for short-term feeding, whereas dietary intake of 50 mg astaxanthin kg−1 diet in fish is adequate for medium- to long-term feeding. It is worth mentioning that a minimum effective dose of astaxanthin should be used to produce desirable outcomes, as a requisite to minimize feed production costs. Supplemented astaxanthin represents approximately 10–15% of total feed cost in the global aquaculture industry (Lim et al., 2018, 2019). The aforementioned could serve as a useful feeding guideline to commercial fish farms, ultimately generating economic benefits.

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