2.1. Experimental fish
P. leopardus sub-adults with an average initial weight of 450.3 ± 20.5 g were procured from a private grouper farm in Pingtung, Taiwan. The fish were stocked and acclimatized in the breeding tanks for four weeks, and fed a commercial diet of 1% of their average body weight once a day. Before the experiment, fish were starved for 24 h. The protocol used for the animal experiments was approved by the Institutional Animal Care and Use Committee of the National Kaohsiung University of Science and Technology (approval number: 0102-AAAP-001).
2.2. Experimental set-up and treatments
Four experiments were conducted in a high-efficiency grouper breeding circulation system that was developed, designed, and assembled in-house. The system was set up in outdoor earth ponds (300 metric ton capacity) at the National Kaohsiung University of Science and Technology. The effects of tank color, shade, and feed additives on fish pigmentation were evaluated 28 d after the introduction of these factors. Experiment I was designed to evaluate the effects of tank color (white, black, bright red, and green). Experiment II was designed to assess the effects of shade in the cages (0%, 50%, and 80% shade rates). Each culture net cage measured 350 cm × 58.3 cm × 150 cm, and a black net was used to cover the top to provide shade. Experiments III and IV were conducted to determine the effects of trace elements in the diet: astaxanthin (1000 mg, 2000 mg, and 3000 mg kg-1), vitamin E (1000 mg, 2000 mg, and 3000 mg kg-1), and plant extracts (250 mg, 500, and 750 mg kg-1), as well as feed additives in the form of 0%, 10%, 20%, and 30% krill meal kg-1.
Each treatment was replicated with ten subadult groupers, as different groups of fish were used in each experiment. During the feeding trials, the fish were kept in net cages. The fish were fed once a day in the morning at 1% of their body weight. A recirculating aeration system was used to prevent build-up of fish waste and the presence of other types of organic nutrients, to effectively maintain water quality and substrate stability, and to inhibit algal growth. During the experiments, the following water parameters were maintained: salinity (25–27 ppt), temperature (26–28°C), dissolved oxygen concentration (5–7 mg l-1), ammonia concentration (0.1–0.2 mg l-1), nitrogen concentration (0.1–0.2 mg l-1), and oxidation-reduction potential (130–180 mv). The color and luster of the fish body surfaces were compared among different tank colors, shade rates, and feed additives. Body surface chromaticity was determined for each group weekly. All experiments were conducted in triplicate.
2.3. Preparation of plant extracts
Plant pre-extracts were prepared according to protocols described by Yi et al. (2018). Carotenoids are pigments that occur naturally in plant leaves. Fresh leaves of Angelica sinensis (Danggui), Eleutherococcus senticosus (Siberian ginseng), Schisandra chinensis (Omija), and Ligusticum chuanxiong (Chuan Xiong), a traditional Chinese herbal medicine, were sourced from local markets in Nanzih District, Kaohsiung City, Taiwan. Leaves were washed thoroughly with sterilized water. They were then were air-dried at room temperature, pulverized using a mechanical grinder, and oven-dried at 70°C until a constant weight was obtained. The four dried and powdered plant species were thoroughly mixed in a dough mixer in equal proportions and placed in an airtight, resealable plastic bag. The powdered dry samples were then extracted using 80% ethanol for 48 h; this procedure was repeated thrice to ensure complete extraction. The ethanolic extract was filtered through No. 1 Whatman paper and concentrated under reduced pressure in a vacuum rotary evaporator until all solvents evaporated (Zhang et al. 2014). Then the extract was poured into a bottle and stored at -20°C for further use in the preparation of test diets.
2.4. Preparation of feeds
The basal diet formulated in this study was used as the control (Table 1). The raw feed materials were mixed uniformly with 30% distilled water during the production process, pelletized, and oven-dried. The feed formulations for color enhancement using trace elements and raw materials as feed additives are listed in Tables 2 and 3. The test diets with different inclusion levels of astaxanthin, vitamin E, plant extracts, and krill meal were added, mixed well with 30% distilled water to prepare extrusion feeds, and then pelletized. The pellets were prepared to the desired size and oven-dried at 40°C for 24 h, then placed in a sealed, double-layered, airtight, and resealable plastic bag, labeled, and stored at -20°C for further use and to avoid rancidity. Prior to feeding trials, the control and test diets were maintained at room temperature for 24 h.
Table 1
Composition of the basal diet.
Ingredient
|
Amount (%)
|
Brown fishmeal
|
40.5
|
Krill meal
|
30.0
|
Wheat flour
|
24.94
|
Carophyll yellow
|
0.06
|
Sodium glutamate
|
0.05
|
Vitamin premixa
|
2.0
|
Mineral premixb
|
2.0
|
Proximate composition (%)
|
|
Crude protein
|
49.4
|
Crude lipid
|
9.5
|
a, b Sourced from Yisheng (Quanxing) International Aquatic Products Co., Ltd.
Table 2
Composition of experimental diets using different inclusion of trace elements (1000 mg, 2000 mg, and 3000 mg of astaxanthin kg-1; 1000 mg, 2000 mg, and 3000 mg of vitamin E kg-1, and 250 mg, 500 mg, and 750 mg of plant extracts kg-1diet) as feed additives.
Ingredients
|
Control
|
Inclusion amount (g kg− 1)
|
Astaxanthin
|
Vitamin E
|
Plant extracts
|
1
|
2
|
3
|
1
|
2
|
3
|
0.25
|
0.5
|
0.75
|
Brown
fishmeal
|
405
|
404
|
403
|
402
|
404
|
403
|
402
|
404.75
|
404.5
|
404.25
|
Astaxanthin
|
0
|
1
|
2
|
3
|
0
|
0
|
0
|
0
|
0
|
0
|
Vitamin E
|
0
|
0
|
0
|
0
|
1
|
2
|
3
|
0
|
0
|
0
|
Plant Extracts
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0.25
|
0.5
|
0.75
|
Krill meal
|
300
|
300
|
300
|
300
|
300
|
300
|
300
|
300
|
300
|
300
|
Carophyll
yellow
|
249.4
|
249.4
|
249.4
|
249.4
|
249.4
|
249.4
|
249.4
|
249.4
|
249.4
|
249.4
|
Sodium glutamate
|
0.6
|
0.6
|
0.6
|
0.6
|
0.6
|
0.6
|
0.6
|
0.6
|
0.6
|
0.6
|
Vitamin premixa
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
Mineral premixb
|
20
|
20
|
20
|
20
|
20
|
20
|
20
|
20
|
20
|
20
|
Table 3
Composition of experimental diets using krill meal as feed additive.
Ingredient
|
Inclusion amount (% kg− 1)
|
0%
|
10%
|
20%
|
30%
|
Brown fishmeal
|
70.50
|
60.50
|
50.50
|
40.50
|
Krill meal
|
0
|
10.00
|
20.00
|
30.00
|
Wheat flour
|
24.70
|
24.70
|
24.70
|
24.70
|
Carophyll yellow
|
0.30
|
0.30
|
0.30
|
0.30
|
Sodium glutamate
|
0.50
|
0.50
|
0.50
|
0.50
|
Vitamin premixa
|
2.00
|
2.00
|
2.00
|
2.00
|
Mineral premixb
|
2.00
|
2.00
|
2.00
|
2.00
|
a, b Sourced from Yisheng (Quanxing) International Aquatic Products Co., Ltd.
2.5. Determination of body surface chromaticity
The three-dimensional color space of the CIE 1976 L*a*b* system developed by the International Commission on Illumination and Van der Salm et al. (2004) is shown in Fig. S1 and S2 (supplementary data). The dorsal surface of the leopard coral grouper was quantified using a colorimeter (NR 3000; Nippon Denshoku) with the color parameters L*, a*, b*, C*, and H*, where L* represents lightness (brightness and tendency to white; a* represents redness; b* represents yellowness; C* represents chroma; and H* represents hue (pure red at 0° and pure yellow at 90°).
The formulas for C* and H* are as follows:
$${C}^{*}={\left({a}^{*2}+{b}^{*2}\right)}^{0.5}$$
$${H}^{*}={tan}^{-1}\frac{{b}^{*}}{{a}^{*}}$$
2.6. Statistical analysis
The obtained data were statistically analyzed by one-way ANOVA using the Statistical Analysis System (SAS-PC, SAS Institute) and further tested using Tukey’s HSD test to test for significant differences between the means of treatments at p < 0.05.