Introduction

Astaxanthin (3,3-dihydroxy-4,4-diketo-β,β-carotene) is an orange-reddish ketocarotenoid pigment used as feed supplement to improve the coloration and health condition of aquatic animals in the aquaculture and ornamental fish industries, thereby influencing its high market value (Lim et al. 2018; Elbahnaswy and Elshopakey 2023). It has been shown to have higher antioxidant activity and oxygen radical absorbance capacity compared with other carotenoids (Nakagawa et al. 2011; Merhan 2017). This biological pigment can generate the red/pink hue that attracts hobbyists in the ornamental trade (Lim et al. 2018). Astaxanthin can be obtained from natural or artificial sources where the former can be synthesized mainly from either plants, algae, phytoplankton, bacteria, or a few fungi (Grung and Metzger 1994; Qin et al. 2008; Katsumata et al. 2014; Novoveská et al. 2019; Yadavalli et al. 2021). Also, natural sources of astaxanthin can be acquired indirectly in our diet by eating crustaceans (such as krill, copepods, and shrimp) and Salmonidae (such as rainbow trout and salmon) species (Liu et al. 2016; Wang et al. 2018; Lu et al. 2021). On the other hand, artificial sources can be produced through chemical synthesis to meet up the global demand for this pigment in the feed market (Li et al. 2011). Depending on the fish species, time of culture, source, and concentration of astaxanthin are the major contributing components that can promote the skin pigmentation of fish (Ha et al. 1993). Aquatic animals especially fish cannot biosynthesize these pigments de novo. The pigmentation requires dietary supplementation (Kalinowski et al. 2007; Li et al. 2018a).

Previous studies have shown that dietary astaxanthin promoted growth performance and enhanced skin pigmentation in Astronotus ocellatus (oscar fish), Cichlasoma synspilum × Cichlasoma citrinellum (blood parrotfish), Symphysodon spp. (discus fish), and Pagrus pagrus (red porgy) by Alishahi et al. (2015), Li et al. (2018a), Song et al. (2017), and Nogueira et al. (2021), respectively. However, the information on the effects of astaxanthin on the integrity of chromatic assessment, some biochemical indices, and tissue morphology of these species is scarce. Also, astaxanthin digestion coefficients and carotenoid composition of the plasma, liver, muscle, and whole kidney in Atlantic salmon and Atlantic halibut show the ability to metabolically change ingested carotenoids among fish species and flesh coloration (Bjerkeng and Berge 2000). Hence, the large differences between salmonid fish species to absorb and deposit carotenoids in the muscle are a result of low uptake from the intestinal tract and low retention of the absorbed astaxanthin in the muscle (Storebakken and No 1992; March and MacMillan 1996). Total carotenoid concentration in the muscle of rainbow trout (Oncorhynchus mykiss) fed an astaxanthin (50 mg/kg) diet increased its redness deposition (Rahman et al. 2016). The study conducted by Song et al. (2017) demonstrated that astaxanthin accumulation reduced and later rapidly increased as time progresses which is like the results obtained on rainbow trout (Zhang et al. 2012). Australian snapper (Pagrus auratus) fed 39 mg/kg dietary astaxanthin improved skin astaxanthin concentration compared with fish fed 13 to 26 mg/kg dietary astaxanthin after a 63-day feeding trial (Ben et al. 2009). Inayah and Qin (2010) reported that false clownfish (Amphiprion ocellaris) fed enriched dietary astaxanthin enhanced the skin zeaxanthin content after 5 weeks of feeding trial.

Johnson and Bergmann (1984) suggested the use of histomorphology in the analysis of the effects of dietary supplements. The liver is considered the primary organ responsible for storing and metabolizing carotenoids in fish (Segner et al. 1989). Carotenoids are transported via lipoproteins to their target organs or tissues including muscle, skin and scale, testes, ovaries, and intestine (Bjerkeng 2008; Parker 1996). Segner et al. (1989) reported that dietary astaxanthin of red tilapia (Oreochromis niloticus) and sunset thick-lipped gourami (Coliosa labiosa) fed astaxanthin at 71–132 mg/kg and 32 mg/kg, respectively, enhanced liver histomorphology, thereby improving hepatic parenchyma arrangement consisting of hepatocytes. Furthermore, Page et al. (2005) established that the supplementation of dietary carotenoid pigment improves positively the structure of liver cells. However, astaxanthin has been reported to improve the gastrointestinal tract of golden pompano (Trachinotus ovatus) by increasing the length and width of the gut villus (Niu et al. 2020). Their findings revealed that the supplementation of dietary astaxanthin impacted positively the integrity of gut morphology. Nevertheless, higher supplementation impacted negatively on the gut morphology. Thus, the required quantity of dietary astaxanthin supplements necessary for gut morphology integrity needs to be managed.

Blood parrotfish, a hybrid between Vieja melanurus♀× Amphilophus citrinellus ♂, is a well-known ornamental freshwater fish globally. Because of their colorful red appearance and plump body, they are widely accepted in China and Japan (Sui et al. 2016). The red hue plays a significant role in the quality and market price of this ornamental fish. Some studies have used varying sources of dietary astaxanthin in blood parrotfish to promote body coloration. For instance, Li et al. (2016a, b) and Song et al. (2016) demonstrated that synthetic astaxanthin and alfalfa saponins with the addition of astaxanthin promoted body coloration. Also, blood parrotfish fed with natural astaxanthin from green microalgae Haematococcus pluvialis enhanced skin coloration (Li et al. 2018a, b). However, information on the effects of astaxanthin on the integrity of tissues morphology and chromatic and biochemical assessment on blood parrotfish has not been fully documented. Consequently, this study was aimed at investigating the integrity of astaxanthin on the skin and muscle (chromatic assessment); liver, intestine, muscle, and blood (biochemical indices); and liver, spleen, anterior intestine, mid-intestine, and posterior intestine (histomorphology characteristics) of juvenile blood parrotfish.

Materials and methods

Animal care

All experimental protocols involving animals were approved by the animal care and welfare committee of the College of Fisheries and Life Science of Shanghai Ocean University, Shanghai, China (SHOU-DW-2021-027).

Experimental feed preparation and proximate composition

Two test diets with the same basal composition (Table 1) were prepared. The treatment diet was supplemented with 0.45 g/kg of synthetic astaxanthin (Carophyll® pink 10% CWS, DSM Nutritional Products Ltd). Feed ingredients were finely ground, sieved, and thoroughly mixed. Distilled water was added to form a dough and the dough was pelleted into 1-mm pellet size, air-dried, sieved, and sorted into uniform sizes. The formulated diets were later stored at −20 °C to prevent astaxanthin degradation.

Table 1 Dietary ingredients and proximate analysis of the basal and treatment diets
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The proximate composition analysis of basal and treatment diets was measured via standard methods of the Association of Official Analytical Chemists (AOAC 1995). Moisture was determined by oven-drying at 105 °C to a constant weight and dry matter content by subtracting moisture value from 100. Crude protein (N × 6.25) contents of diets were determined by the Kjeldahl method after acid digestion using an Auto Kjeldahl System (2300-Autoanalyzer Foss Tecator, Sweden). Crude lipid was determined by a chloroform-methanol extraction method according to Cejas et al. (2004). Astaxanthin concentration in the basal and treatment diets was analyzed using a fish astaxanthin ELISA kit purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. (mlbio) Shanghai, China, following the manufacturer’s procedures.

Experimental fish

A total of 360 juvenile blood parrotfish with an average weight of 10.16 ± 0.38 g were acquired from a commercial fish farm in Hainan, China. All fish were acclimated to laboratory conditions and fed the control diet for 14 days before the start of the trial. The fish were assigned to 18 glass aquaria measuring 48 cm × 45 cm × 30 cm with 20 fish per aquarium, and divided into three groups with sextuplicate each. The dissolved oxygen, water temperature, pH, and ammonia level were maintained at 7.2 ± 0.1 mgL−1, 26 ± 1 °C, 7.1 ± 0.2, and 0.02 ± 0.01 mgL−1, respectively. This was done during acclimatization and experimentation condition. Hanna equipment (model H198194) was used to measure the dissolved oxygen, water temperature, and pH by inserting the probe inside the water while ammonia was measured using HI-700. A total of 30% of the water was changed everyday from the bottom of the tanks as the fish were cultured in a recirculating aquaculture system. The fish were hand-fed twice until apparent satiation at 10:00 am and 4:00 pm daily. The fish were subjected to a natural photoperiod during the 84-day experimental period. The control group were fed a diet containing no supplemental astaxanthin for 84 days. The coloration (ASX) group were fed a diet containing 0.45 g/kg synthetic astaxanthin for 84 days. The decoloration (ASX-) group were initially fed the same diet as the ASX group for 42 days then fed the same diet as the control group for another 42 days (Fig. S1).

Sample collection

At the end of the trial, approximately 24 h after the last feeding, three fish from each aquarium were randomly taken then euthanized with tricaine methanesulfonate (MS-222, 50 mg/L) (Shanghai Reagent Corporation, China). After blood collection, the same fish were dissected. Skin, liver, spleen, and intestine were removed and stored at −80 °C for further analysis.

Color analysis

Five individual fish from each aquarium were randomly taken and euthanized with MS-222 for skin and muscle colorimeter analysis after the feed trial using a Minolta CM-600 Chroma Meter (Minolta Camera Co. Ltd, Asaka, Japan). The Chroma Meter was set to take absolute measurements in the L*, a*, and b* measuring mode CIE (1976) using D65 illuminant. Color measurements were taken in the center (Fig. S2) of the lateral body. L* is the lightness variable where L* = 100 is white and L* = 0 is black; a* is for the red chromaticity coordinates where + a* stands for red and −a* stands for green, and b* is for the yellow chromaticity coordinates where + b* stands for yellow and −b* stands for blue. Hue (H°ab) and chroma (Cab*) values were calculated from the a* and b* values. Hue (e.g., red and yellow, which are the observable colors) is an angular measurement where 0° indicates a red hue and 90° denotes a yellow hue, and is calculated by the equation, H°ab = tan−1 (b*/ a*) (Stokes and Brill 1992). Chroma is an expression of saturation or intensity of the color attained and is calculated by the equation \(C\text{ab} \ *= \sqrt{(a^{*2}+b^{*2})}\) (Wyszecki and Stiles 1967).

Histomorphology and chromatophore analysis

At the end of the experiment, tissue samples of the liver, spleen, mid-intestine, anterior intestine, and posterior intestine (Fig. S3) were removed and immersed immediately in Bouin’s solution after dissection. This was done on three fish randomly taken from each aquarium (the same as in the sample collection). Samples were then dehydrated in graded ethanol solution after 24 h, embedded in paraffin, and transversely sectioned into 4-µm cuts. The sectioned tissue was mounted on glass slides and stained with hematoxylin and eosin. Slides were examined under a NIKON Eclipse Ci microscope (Nikon Corporation, Japan) and photomicrographs of sectioned liver, spleen, mid-intestine, anterior intestine, and posterior intestine tissues were taken (×200 magnification). The muscular thickness, villus width, and villus height were determined by electronic images using the technique described by Poolsawat et al. (2021).

At the end of the experiment, skin samples of two randomly taken fish (control, ASX, and ASX-) were removed with forceps from the side area of each fish. The skin and scale of each were then carefully placed upon a 76 × 26 mm glass micro slide (Kimble micro slides, Owens-Illinois Co., USA) and covered with a translucent coverslip to immobilize it and then transferred to a compound fluorescent microscope (Nikon Eclipse 80i with digital sight DS-Ri1, Nikon Corporation, Japan). Both ×20 and ×10 amplitude objectives (Plan Achromatic series, Nikon Corporation, Japan) were used.

Biochemical analysis

High-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were determined using assay kits obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), and were used according to the manufacturer’s instruction. The above parameters were measured spectrophotometrically (UV-2365, Unico Instruments Co. Ltd., Shanghai, China) at 546-nm wavelength and measurements were performed on the blood plasma, liver, intestine, and skin samples at day 84. A heparinized tube was used to collect the blood then centrifuged for 2500 rpm for 10 min and plasma was retrieved. The liver, intestine, and skin samples were homogenized with 0.86% saline in the ratio of 1:9 using a tissue homogenizer and then centrifuged in a low temperature (4°C) at 2500 rpm for 10 min to retrieve the supernatant. The optical densities (OD) of the blank and standard were used to generate the exact concentrations of the biochemical parameters. This assay was done on three randomly taken fish (control, ASX, and ASX-) per replicate. The activities were expressed as units of millimole per gram of protein.

Determination of astaxanthin concentration in intestine, liver, scale, and skin

Astaxanthin concentrations in the intestine, liver, scale, and skin on three randomly taken fish (control, ASX, and ASX-) per replicate were analyzed using a fish astaxanthin ELISA kit purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. (mlbio) Shanghai, China, which was used following the manufacturer’s instruction. The intestine, liver, scale, and skin samples were homogenized with 0.86% saline in the ratio of 1:9 using a tissue homogenizer and then centrifuged in a low temperature (4°C)  at 2500 rpm for 10 min to retrieve the supernatant. Optical density at 450-nm wavelength was read using a microplate reader with 96 wells and actual activities of enzymes were calculated with reference to OD of different concentrations of a standard solution.

Data analysis

The data obtained from this experiment were subjected to a test of homogeneity to test if they were normally distributed. The differences in these parameters between treatments were compared  using one-way analysis of variance in SPSS V24.0 (SPSS Inc., Chicago, IL, USA) for Windows. The differences between means were tested by Tukey’s multiple range test. All data are presented as means ± SD. Two-way ANOVA was used to establish the effects of the interaction between treatment and section on intestinal morphometrics analysis. P values < 0.05 were considered to indicate statistical significance.

Results

Chromatic and chromatophore analysis

Color parameters such as redness (a*), yellowness (b*), chroma (Cab*), and hue (H°ab) for the ASX group showed a significant effect (*P < 0.05, **P < 0.01) for skin and muscle color assessment of blood parrotfish compared to the ASX- and control groups (Fig. 1a, b). However, lightness (L*) shows no significant difference (P > 0.05) in muscle among treatments.

Fig. 1
figure 1

L*, a*, b*, Cab*, and H°ab of blood parrotfish showing the a skin and b muscle. Data are expressed as mean ± SD (n = 90). The asterisk indicates significant difference (*P < 0.05, **P < 0.01)

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The fish fed the astaxanthin-enriched diet (ASX group) showed significantly concentrated red-colored erythrophore cells (pigment cells) compared with the ASX- with dispersed red-colored erythrophore cells, whereas erythrophore cells were absent in the control diet (Fig. 2a–c) after 84 days of feed trial.

Fig. 2
figure 2

The skin chromatophores of blood parrotfish fed control, ASX, and ASX- diets, respectively. a Absence of erythrophores in control, b concentrated red-colored erythrophores in ASX, and c dispersed structure of red-colored erythrophores in the ASX- group (magnification ×200)

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Astaxanthin concentration in the intestine, liver, scale, and skin of blood parrotfish

Astaxanthin impacts positively on the intestine, scale, and skin of the ASX group (**P < 0.01) compared to the ASX- and control groups (Fig. 3a–c), whereas astaxanthin was significantly higher in the liver of the ASX- group (*P < 0.05) in contrast to the ASX and control groups (Fig. 3d).

Fig. 3
figure 3

Concentration of astaxanthin in a scale, b skin, c intestine, and d liver of blood parrotfish fed control, ASX, and ASX- diets, respectively. Data are expressed as mean ± SD (n = 54). The asterisk indicates significant difference (*P < 0.05, **P < 0.01) between treatments

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Blood plasma lipoproteins assay

In Fig. 4a and b, blood plasma HDL-C and LDL-C differed significantly; fish fed the astaxanthin-enriched diet (ASX group) showed increased (**P < 0.01) HDL-C and LDL-C compared with the ASX- and control groups. The HDL-C and LDL-C of blood plasma in the ASX and ASX- groups were significantly higher than in the control group.

Fig. 4
figure 4

The lipoprotein-cholesterol of blood parrotfish fed control, ASX, and ASX- diets, respectively. a HDL-C in plasma and b LDL-C in plasma. Data are expressed as mean ± SD (n = 18). The asterisk represents a highly significant difference (**P < 0.01) between treatments

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Skin, liver, and intestine HDL-C assay

The results for skin, liver, and intestine HDL-C are shown in Fig. 5a–c. Dietary ASX showed significantly higher (*P < 0.05, **P < 0.01) HDL-C in contrast to the ASX- group. The lowest HDL-C was observed in the control group.

Fig. 5
figure 5

The high-density lipoprotein-cholesterol of blood parrotfish fed control, ASX, and ASX- diets, respectively. a Skin, b liver, and c intestine. Data are expressed as mean ± SD (n = 54). The asterisk indicates significant difference (*P < 0.05, **P < 0.01) between treatments

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Skin, liver, and intestine LDL-C assay

Dietary astaxanthin (ASX) positively enhanced LDL-C (*P < 0.05, **P < 0.01) in the skin and intestine tissue than the ASX- and control groups, though the skin LDL-C of ASX- fish was higher compared to that of control fish and no significant difference was observed between the ASX- and control groups of intestine LDL-C. However, the LDL-C concentration in the liver of the group fed the control diet was observed to be higher (**P < 0.01) than in the ASX and ASX- groups (Fig. 6a–c).

Fig. 6
figure 6

The low-density lipoprotein-cholesterol of blood parrotfish fed control, ASX, and ASX- diets, respectively. a Skin, b liver, and c intestine. Data are expressed as mean ± SD (n = 54). The asterisk indicates significant difference (*P < 0.05, **P < 0.01) between treatments

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Intestinal histomorphology and liver and spleen morphology of blood parrotfish

This is the first study that provides experimental evidence on the effects of astaxanthin on the integrity of the intestine, liver, and spleen of blood parrotfish. As shown in Table 2, the ASX group improved (*P < 0.05) muscular thickness (MT) of the posterior intestine (PI), mid-intestine (MI), and anterior intestine (AI) and villus height (VH) (**P < 0.01) of AI, followed by PI and MI of blood parrotfish than the control and ASX- groups. Nevertheless, villus width (VW) shows no significant (P > 0.05) effect on the PI, AI, and MI among the ASX, ASX-, and control groups (Table 2). Moreover, it caused a significant increment in the PI, followed by AI and MI of the ASX group compared to the ASX- and control groups. The histomorphology of AI, MI, and PI, stained with hematoxylin and eosin (H&E) displaying the MT, VW, and VH morphometrics, is shown in Fig. 7.

Table 2 Intestinal morphometrics of anterior intestine, mid-intestine, and posterior intestine of blood parrotfish fed control, ASX (0.45 g/kg), and ASX- (0.45 g/kg + 0 g/kg) diets
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Fig. 7
figure 7

The anterior intestine, mid-intestine, and posterior intestine (H&E-stained sections) morphology of blood parrotfish showing the VH (villus height); VW (villus width); and MT (muscular thickness) of a control, b ASX, and c ASX- groups (magnification ×200) for 84 days of feeding trial

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The hepatocytes (HP), sinusoids (SN), and Kupffer cells (KCs) of hepatocytes show a better parenchyma arrangement in the ASX group than in the ASX- and control groups (Fig. 8a–c). Nevertheless, the control group showed a better hepatocyte, sinusoids, and Kupffer cells parenchyma arrangement than the ASX- group. The red pulp (RP) was highly promoted in the ASX group compared to the ASX- and control groups (Fig. 9a–c). Furthermore, the white pulp (WP) was significantly higher in the ASX group in contrast to the ASX- and control groups. The central arteriole (CA) is responsible for the supply of oxygenated blood to the spleen of all the groups.

Fig. 8
figure 8

The liver (H&E-stained sections) morphology of blood parrotfish showing the hepatocytes (HP), sinusoids (SN), and Kupffer cells (KCs) of a control, b ASX, and c ASX- groups (magnification ×200) for 84 days of feeding trial

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Fig. 9
figure 9

Spleen (H&E-stained sections) morphology of blood parrotfish showing the white pulp (WP), the red pulp (RP), and central arteriole (CA) of a control, b ASX, and c ASX- groups (magnification ×200) for 84 days of feeding trial

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Discussion

Astaxanthin is a carotenoid of the xanthophyll family of hydroxy-carotenoids usually supplemented in cultured fish and crustaceans’ diets to boost color pigmentation in the ornamental and aquaculture industry (Schuchardt and Izquierdo 2005; Kop 2008; Flores and Chien 2011; Li et al. 2016a, b; Abd El-Gawad et al. 2019). Previous studies have shown that astaxanthin impacts positively on fish blood serum, flesh, liver, and intestine lipoprotein-cholesterol (Torrissen et al. 1989; Torrissen 1995). Also, astaxanthin enhanced the liver histology of rainbow trout, Nile tilapia, and thick-lipped gourami (Segner et al. 1989; Page et al. 2005) and the gastrointestinal tract of golden pompano (Niu et al. 2020).

In the present study, the dietary astaxanthin in the astaxanthin-fortified diet influenced redness, yellowness, chroma, and hue of the skin compared to the muscle in blood parrotfish. Body color is attributed to the health status, quality, and market price of fish and crustaceans in the aquaculture and ornamental industry (Sathyaruban et al. 2021). The saturation and deposition of astaxanthin take place in the skin faster than in the muscle which may be due to the transforming ability of the alimentary tract to ingest and transform carotenoids, thereby storing it in the skin of blood parrotfish though the exact mechanism of this action is not known. This is similar to the results obtained for koi carp (Cyprinus carpio) that were fed beetroot, carrot peel, tomato peel powder, and powder mixture for 45 days (Maiti et al. 2017) and 56 days in blood parrotfish (Li et al. 2016a, b).

This study showed that synthetic astaxanthin is effective in concentrating the pigment cells (erythrophores). Erythrophores are red pigment cells that contain carotenoid vesicles filled with carotenoids (Ligon and Mccartney 2016). Skin color or concentration of pigment cells may be dependent on the duration of the feeding trial. Nevertheless, color comes from carotenoid precipitation in the fish tissue (skin) which contains chromatophores (Chatzifotis et al. 2011). This result was also buttressed by the redness (a*) value (Fig. 1a). Astaxanthin concentrations of the scale, skin, and intestine of blood parrotfish were significantly higher in the ASX group than in the control and ASX- groups, which indicates adequate absorption and uptake of the carotenoid. Our skin and scale carotenoid concentration results are in accordance with the studies of Song et al. (2017) on discus fish (Symphysodon spp.), Gouveia and Rema (2005) on goldfish (Carassius auratus), and Kalinowski et al. (2007) on red porgy (Pagrus pagrus). However, astaxanthin concentration of the liver was higher in the ASX- group followed by the control and ASX groups, suggesting a higher rate of carotenoid conservation in the ASX- group. Also, the fish may probably have the ability to store astaxanthin after withdrawal. The uptake and transport efficiency can affect carotenoid accumulation (Ho et al. 2013).

Dietary astaxanthin actuates increase in the HDL-C concentration thereby regulating LDL-C concentration in blood plasma and liver which indicates that astaxanthin could help maintain tissue integrity observed in the ASX group. This can be attributed to continuous dietary astaxanthin administration over a long period of time. Furthermore, astaxanthin can swiftly influence the production of HDL-C which is a transporter of long-chain polyunsaturated fatty acid that plays a vital role in carotenoid metabolism before their deposition in the integument of the fish. This present study concurred with a previous study of Aas et al. (1999) that observed a significant effect of 14C-astaxanthin in Atlantic salmon (Salmo salar L.) for lipoproteins (HDL and LDL) transport. However, they suggested that the majority of carotenoid transportation may be associated with serum albumin. Moreover, the observation of the effect of carotenoids on blood serum lipoproteins was also documented in rainbow trout (Oncorhynchus mykiss) (Salvador et al. 2009). In addition, dietary astaxanthin positively enhanced HDL-C (skin, liver, and intestine) and LDL-C (skin and intestine) of blood parrotfish but decreased the activity of liver LDL-C compared to the ASX- and control groups. The enhancement may probably be because astaxanthin has been absorbed in the free state and biotransformed by oxidation and reduction. This is in agreement with the results of Choubert and Heinrich (1993) and Aas et al. (1999). Furthermore, when the activity for liver LDL-C increases, it can cause lipid peroxidation which can cause cell death of the fish. Astaxanthin is an antioxidant with a protective effect on lipid peroxidation (Wang et al. 2006; Xie et al. 2017; Li et al. 2018b).

Previous studies have been reported on the mechanisms responsible for carotenoid absorption in man and mammal gut (Erdman et al. 1993; Parker 1996; Furr and Clark 1997; Van den Berg 1999) but limited or no information related to activities in blood parrotfish. The increased VH, VW, and MT in the ASX group of this present study indicate that astaxanthin improves intestinal health and surface area for nutrient uptake of the fish. Torrissen et al. (1989) and White et al. (2002) demonstrated that absorption of carotenoids occurs primarily at the proximal and mid-intestine of salmonids though there is a need for further research to ascertain this hypothesis. In addition, several literatures have reported that absorption of dietary nutrients takes place rapidly in the anterior or posterior intestine due to the higher number and length of the intestinal villi (Collie 1985; Buddington and Diamond 1987; Dabrowski 1990; Bakke-McKellap et al. 2000; Jutfelt et al. 2007; Firdaus-Nawi et al. 2013).

To maintain the normal physiological health status of fish, the liver integrity and morphological structure must be in a good state because of their metabolic role in nutrient metabolism (Yusuf et al. 2021). Johnson and Bergman (1984) reported that a histological approach can be used to evaluate the impact of food additives. The supplementation of dietary carotenoid pigment improved positively the structure of liver cells in our present study. Kupffer cells are a spindle-like structure part of the reticular endothelial system called macrophages whereas hepatocytes are round-like structures separated by sinusoids that help detoxify harmful materials. Also, Kupffer cells are attached to the walls of the sinusoids together with the spleen which participate in removing worn-out erythrocytes (Ferri and Sesso 1981). Excessive Kupffer cells along the portal vein lining sinusoid were highly produced in the ASX group because abundant Kupffer cells play a significant role in improving hepatic homeostasis and boosting the fish immune system against disease-causing organisms. Even so, the presence of hepatocyte cells in all the groups is an indication of protein synthesis. These findings were in line with those of Page et al. (2005) for Oncorhynchus mykiss fed carotenoid pigment for 3 weeks by improving the structure of the liver, and Segner et al. (1989) for Oreochromis niloticus and Colisa labiosa fed astaxanthin for 23 weeks by also observing improvement in liver structure histologically. Our histological results show that astaxanthin can positively improve the metabolic effect of blood parrotfish.

More red pulp was present in the spleen of the ASX group which indicates the removal of worn-out erythrocyte cells, whereas the increase in white pulp is an indication of the proliferation of macrophages, thereby promoting the well-being of the fish against microscopic organisms. This corroborates our findings on liver morphology. Milani et al. (2017) stated that carotenoids can stimulate the proliferation of macrophages, and B- and T- lymphocytes in humans. However, liver and spleen histochemistry analysis to identify reticular fibers, glycogen, neutral glycoprotein, ceroid, hemosiderin, and lipofuscins would be necessary to understand the metabolic activities of macrophages (Sales et al. 2017).

Conclusion

In conclusion, dietary astaxanthin improves the concentration of pigment cells, chromatic parameters, villus height, and muscular thickness in blood parrotfish. Astaxanthin increases blood HDL-C and decreases liver LDL-C in blood parrotfish. Furthermore, the fish probably have the mechanism to store astaxanthin in the liver after withdrawal of the astaxanthin-enriched diet. Consequently, this study unfolded more data about the effects of astaxanthin on the coloration and physiology of blood parrotfish.