Aquaculture Reports 32 (2023) 101705

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Aquaculture Reports
journal homepage: www.elsevier.com/locate/aqrep

Effects of dietary supplementation of turmeric (Curcuma longa) extract on

growth, feed and nutrient utilization, coloration, hematology, and
expression of genes related immune response in goldfish (Carassius auratus)
Anurak Khieokhajonkhet a, b, *, Tanaphum Roatboonsongsri b, Piluntasoot Suwannalers c,
Niran Aeksiri a, b, Gen Kaneko d, Kumrop Ratanasut a, b, Wilasinee Inyawilert a, b,
Wutiporn Phromkunthong e
a
Center for Agriculture Biotechnology, Faculty of Agriculture, Natural Resources, and Environment, Naresuan University, Phitsanulok 65000, Thailand
b
Department of Agricultural Science, Faculty of Agriculture, Natural Resources, and Environment, Naresuan University, Phitsanulok 65000, Thailand
c
Faculty of Agriculture Technology, Chiang Mai Rajabhat University, Chiang Mai 50300, Thailand
d
College of Natural and Applied Science, University of Houston-Victoria, 3007 N. Ben Wilson, Victoria, TX 77901, USA
e
Kidchakan Supamattaya Aquatic Animal Health Research Center, Aquatic Science and Innovative Management Division, Faculty of Natural Resources, Prince of
Songkla University, Songkhla 90112, Thailand

A R T I C L E I N F O A B S T R A C T

Keywords: To examine the effect of turmeric extract (TE) on the growth, feed and nutrient utilization, coloration, hema­
Ornamental fish tology, and expression of immunity genes in goldfish (Carassius auratus), a 10-week feeding trial was conducted.
Perennial herb Goldfish (7.56 ± 0.01 g/fish) were subjected to four feeding treatments using diets containing 0 (control group,
Color
TE0), 1, 2, and 3 g/kg (TE1 – TE3, respectively) of TE. Dietary TE supplementation did not significantly affect
Carotenoid
growth performance measured every two weeks (~ 1 g/week weight gain), feed intake, or nutrient utilization
Blood
but significantly increased the value of a* in the head, abdominal, and tail regions. The TE3 group had signif­
icantly higher total carotenoids in serum compared to other groups. Also, TE supplementation increased the total
carotenoid content in the fin, skin, and liver with significant differences between TE0 vs. TE3 and TE2 groups.
Moreover, dietary supplementation of TE increased white and red blood cells, total protein, albumin, and
globulin with linear and/or quadratic effects. LDL-cholesterol and triglycerides were significantly decreased by
increasing TE supplementation with the opposite tendencies in HDL-cholesterol levels. Lysozyme and IL-10
transcripts were significantly increased in the TE supplemented groups. Conversely, IL-1β transcript levels
were significantly decreased in the liver by TE supplementation, and no significant differences were observed for
HSP70 gene expression among all tested groups. Plasma glucose and cortisol levels were linearly decreased in all
TE groups, and a quadratic effect was observed for plasma glucose levels. Taken together, the results of this study
indicate that supplementing 2–3 g/kg of TE in diets could improve coloration, strengthen immunity, and alle­
viate stress in goldfish.

1. Introduction resistance is in place due to concerns about the development of bacterial

resistance and the potential negative impacts on the environment and
The development of aquaculture on a global scale has accelerated fish health (Alagawany et al., 2021). Thus, in the past few years, tradi­
recently, making it one of the fastest-expanding sectors in the produc­ tional medicinal herb powders and their extracts have been wildly tested
tion system (FAO, 2020; Tadese et al., 2022). Despite the high level of in aquaculture due to their anti-stress functions, pharmacological
intensification, disease outbreaks still pose a serious barrier to sustain­ characteristics (Jiang et al., 2016), and immunomodulatory effects
able production (Tadese et al., 2022). Currently, the global restriction on (Mahmoud et al., 2017) for controlling and preventing diseases (Ala­
the use of drugs and chemicals to promote growth and bacterial gawany et al., 2021). Several recent reports have demonstrated that

* Correspondence to: Department of Agricultural Science, Faculty of Agriculture, Natural Resources, and Environment, Naresuan University, 99 M. 1, T. Thapo, A.
Muang, Phitsanulok Province 65000, Thailand.
E-mail address: [email protected] (A. Khieokhajonkhet).

https://doi.org/10.1016/j.aqrep.2023.101705
Received 26 June 2023; Received in revised form 13 August 2023; Accepted 23 August 2023
Available online 28 August 2023
2352-5134/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
A. Khieokhajonkhet et al. Aquaculture Reports 32 (2023) 101705

medical herbs improve the immunological state and growth perfor­ genes related immunity in ornamental goldfish (Carassius auratus).
mance as safe and eco-friendly food additives (Khieokhajonkhet et al.,
2022a; Tadese et al., 2022). 2. Materials and methods
Curcuma longa Linn., commonly known as turmeric, is a perennial
herb characterized by its rhizomatous roots that belongs to the Zingi­ 2.1. Preparation of turmeric extract
beraceae family. Turmeric is wildly cultivated in tropical countries,
especially in South East Asian countries. This rhizomatous plant is the Fresh turmeric rhizome was bought from a wholesale market and
most popular herb worldwide due to its culinary, aesthetic, and thera­ delivered to Naresuan University in Phitsanulok, Thailand. Turmeric
peutic features (Pal et al., 2020; Ibáñez and Blázquez, 2021). This tu­ was briefly washed two times with running tap water, peeled, and cut
berous species is a great supply of bioactive ingredients including into ~ 0.2–0.3 cm thickness. Sliced turmeric rhizome was air-dried
phenolic compounds, flavonoids, β-carotene, vitamins C, and vitamin E. overnight at 50◦ C using a hot air oven. An obtained dry turmeric
Curcumin, the most extensively studied active component, constitutes rhizome was meticulously ground to a fine powder using a household
approximately 0.3–5.4% of raw turmeric (Akram et al., 2010). It has kitchen blender. Afterward, the obtained powder was carefully stored in
been demonstrated that curcumin has hypoglycemic, antimicrobial, aluminum-plastic sip bags and maintained at a temperature of − 20◦ C
antifungal, antiviral, antioxidant, anti-inflammatory, anticancer, and until used.
hepato- and neuroprotective effects (Akram et al., 2010; Ibáñez and Approximately 200 g of turmeric powder was macerated with 800
Blázquez, 2021; Meng et al., 2018; Pal et al., 2020). Curcumin is also mL of 90% ethanolic alcohol, and thereafter the mixture solution was
known to have a variety of biological functions in teleost fish species partitioned with hexane of an equal volume (1: 1, v/v). The upper phase
including growth improvement (Hu et al., 2003; Sruthi et al., 2018; layer was evaporated using a Bunchi Labortechnik rotary evaporation,
Wang and Wu, 2007), antioxidant (Ming et al., 2020), antipathogenic Ratavapor R-210 (Flawil, Switzerland), and subsequently lyophilized
bacteria and immunomodulatory (Ming et al., 2020; Mahmoud et al., using a freeze-dryer (Martin Christ, Beta 2–8 LD plus, Germany). To
2017; Yonar et al., 2019), and hepatic and renal protection (Cao et al., ensure protection against light sensitivity, the extracted TE powder was
2015; Mohamed et al., 2020). Turmeric extracts (TE) are also reported as stored in aluminum-plastic zip bags and maintained at − 20 ◦ C until
a good herbal feed additive to modulate oxidative status and stress re­ used.
sponses in fish (Xavier et al., 2021). To our knowledge, however, no
report has been conducted on the effects of TE supplementation on stress 2.2. Diets preparation
response and immunity in ornamental goldfish in spite of their com­
mercial importance. Four experimental diets (iso-lipidic, 9% and iso-nitrogenous, 42%)
Goldfish (Carassius auratus, Cyprinidae family), is a well-known were prepared by formulating with 350 g/kg of fish meal (FM), 375 g/kg
ornamental fish species that have been used in a variety of biological of soybean meal (SBM), and 28 g/kg of fish oil (FO) (Table 1) as the basal
investigations (Talaat et al., 1998; Omori and Kon, 2019; Khieokha­ feed ingredients. A basal diet was formulated without TE supplemen­
jonkhet et al., 2022c; Yousefi et al., 2022). The ornamental value of this tation. The other three diets consisted of the basal diet containing 1, 2,
fish is attributed to its beautiful colors, remarkable ability to adapt to and 3 g/kg of TE and were referred to as TE1, TE2, and TE3, respec­
artificial diets, and high level of environmental tolerance in captivity tively. The experimental diets were made according to the previously
(Gumus et al., 2016), all of which can be improved by taking advantage published protocols in our laboratory (Khieokhajonkhet et al., 2021).
of natural feed additives. Indeed, in commercial feeds for ornamental Briefly, all ingredients were weighed by following each formulation as
fish commonly contain synthetic astaxanthin. This carotenoid is widely shown in Table 1, and homogenized using a kitchen homogenizer for 10
recognized as the most effective and commonly used substance in min. The micro-ingredients including TE powder, methionine, lysine,
aquaculture to enhance pigmentation (Paripatananont et al., 1999). vitamin and mineral mixtures, and vitamin C were mixed for another 5
However, it should be noted that synthetic astaxanthin is relatively min. Finally, FO, lecithin, and 35% (v/w) distilled water was added and
costly, causing a feed cost increase of approximately 10–20% (Mora homogenized for 15 min. The feeding pellets were produced using a
et al., 2006). The utilization of synthetic carotenoids results in a faster meat mincer (diameter of 2 mm), air-dried overnight at 50◦ C, and
accumulation (~ 28–42 days) compared to natural carotenoids (Par­ maintained at a temperature of − 20◦ C until used for feeding experi­
ipatananont et al., 1999; Booth et al., 2004; Song et al., 2017), reaching ments and chemical analyses.
a plateau more quickly (Jiang et al., 2019). However, the color attained
at the plateau is consistently less appealing compared to the natural 2.3. Experimental conditions and ethical statement
equivalent, even when using the same concentration and duration of
exposure (Jiang et al., 2019). Aquaculture and the ornamental industries Goldfish (Carassius auratus) of initial body weight 5.24 ± 0.5 g/fish
are thus particularly interested in natural, sustainable, and affordable were bought at Chatuchak market, Bangkok, Thailand, and adapted for
sources of astaxanthin as a replacement for synthetic chemicals to one month to the indoor experimental facilities of The Aquatic Animal
improve color. Several studies have identified natural carotenoids for Feed Laboratory, Department of Agriculture Science, University of
goldfish such as algae meal (Gouveia and Rema, 2005; Kargun and Naresuan (Phitsanulok, Thailand). Goldfish were acclimatized in a 500 L
Dikbas, 2020), shrimp or lobster waste meal (Bell et al., 2019; Weer­ fiber tank under a natural photoperiod (approximately 12 L: 12D) and
atunge and Perera, 2016), yeast (Xu et al., 2006), red pepper (Khieo­ temperature conditions (26.50 ± 2.20◦ C). Goldfish were manually
khajonkhet al, 2023), and other natural carotenoid sources (Dananjaya hand-fed until satiation trice daily (8:00, 11:30, and 17:30) with a
et al., 2015; Yarnar et al., 2008). Curcuminoids are polyphenolic pig­ commercial diet (7% fat, 40% protein, and 4% fiber).
ments responsible for the yellow/brown color of the peel of turmeric and Before the commencement, the goldfish were subjected to fasting for
the deep orange color of its inner part (Akram et al., 2010; Meng et al., 24 h. Following this, they were weighed after being desensitized with
2018). These active compounds are widely used as a spice in food clove oil stock solution (ethanol: clove oil, 9: 1) at a concentration of 30
manufacturing to enhance both flavor and color (Amalraj et al., 2017). ppm. A total of 240 healthy goldfish (initial body weight, IBW, 7.56 ±
Given the cardio- and hepatoprotection, hypoglycemia, antifungal, 0.01 g/fish) were chosen randomly and allocated among 12 glass tanks
antiparasite, anti-amyloidogenic, anti-oxidant, and chemo- and with 3 replicate tanks per treatment. Each tank had a capacity of 150 L
radio-resistance activities (Amalraj et al., 2017; Li et al., 2011), TE can with dimension of 0.45 × 0.45 × 0.90 m3, and was equipped with 24 h
be a promising food additive for goldfish. Therefore, the primary aeration using 2 submerged air stones per tank. The goldfish were
objective of this study was to examine the effects of TE on growth, feed stocked at a density of 20 fish per tank, with each treatment assigned to
and nutrient utilization, coloration, hematology, and the expression of three tanks. The goldfish were manually fed trice daily (8:00–8:30,

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A. Khieokhajonkhet et al. Aquaculture Reports 32 (2023) 101705

Table 1 = 9 per group) to determine the colorimetric measurement. Another

Experimental diets and proximate composition used in the present study (dry three goldfish were also randomly collected from each replicate and
matter). pooled (9 fish per treatment, n = 3) to determine total carotenoid con­
Items TE0 TE1 TE2 TE3 tents in serum. Goldfish in each replicate were bulk-weighed for every 2
Feed formula (g/kg)
weeks to determine growth performance. Prior to the weight measure­
FM 350 350 350 350 ments, goldfish were fasted for 24 h and desensitized with 30 ppm of
SBM 375 375 375 375 clove oil stock solution. At the termination, all remaining fish of each
Squid meala 87 87 87 87 tank were weighted to determine growth, feed efficiency, and survival
Rice bran 80 80 80 80
parameters. Three goldfish of each replicate (9 fish per group, n = 3)
Corn meal 11 11 11 11
Wheat flour 30 29 28 27 were randomly collected, pooled, and kept at − 20◦ C for the whole-body
Turmeric extract 0 1 2 3 chemical composition analyses. Another set of fish samples (three fish
Vitamin pre-mixb 10 10 10 10 from each replicate tank) was used to measure the total length, and their
Mineral pre-mixc 10 10 10 10 liver and visceral organs were excised out and weighed to calculate
FOd 28 28 28 28
Lecithin 3 3 3 3
organosomatic indexes: hepatosomatic index (HSI), condition factor (K-
Vitamin C 4 4 4 4 value), and viscerosomatic index (VSI). Thereafter, their fin, muscle,
Methionine 5 5 5 5 skin, and liver were used for the measurement of total carotenoid con­
Lysine 7 7 7 7 tent. Blood samples for hematology and serum biochemistry analyses
Total 1000 1000 1000 1000
were obtained from six fish selected from each tank. Blood samples were
Crude protein (%) 42.40 42.83 42.03 42.97
Crude fat (%) 9.79 9.29 9.54 9.37 collected from the caudal vein of the fish for subsequent blood analyses.
Ash (%) 6.96 6.92 6.54 6.93 After collection, the blood samples were left to stand on ice at 4◦ C for 2
Dry matter (%) 89.48 90.12 90.22 90.26 h. Afterward, the samples were centrifuged at 4◦ C for 15 min at 3000g.
Total carotenoids (g/kg) 0.07 0.26 0.39 0.41 The resulting serum was carefully separated and immediately stored at
Gross energy (MJ/kg)e 19.09 19.12 19.14 19.17
− 80◦ C until utilized for biochemical analysis. To determine immune-
FM, SBM, rice bran, wheat flour, and corn meal used in the present study were related gene expression, two fish (n = 6 per treatment) were collected
supplied by Praepan Animal feed Corporation, Phitsanulok, Thailand. from each tank, and the liver was collected with approximately 0.3–0.5
a
Pro-squid, high protein concentrate powder, Bangkok, Thailand. g/fish. Liver tissue samples were immersed in RNA preservative solution
b
Vitamin pre-mix (mg or IU/kg diet): A, 5000 IU; Thiamine, 2500 mg; riboflavin, (RNAlater, Amnion, Cambridgeshire, UK) and immediately kept at −
1000 mg; Pyridoxine, 1000 mg; cobalamin, 10 mg; D3, 1000 IU; E, 5000 mg; K,
80◦ C.
2000; biotin, 10 mg; folic acid, 300 mg; inositol, 1000 mg; niacin acid, 3000 mg;
pantothenic acid, 3000 mg; C, 10,000 mg.
c
Mineral pre-mix (mg/kg diet): calcium lactate, 100; calcium phosphate, 80; 2.5. Growth, feed and nutrient utilization, organosomatic indexes, and
ferrous sulphate, 1.24; Magnesium chloride, 2.16; zinc oxide, 1.6; copper sul­ survival
phate, 1.2; manganese oxide, 1.2; potassium chloride, 0.23; potassium iodine,
0.23; cobalt carbonate, 0.2; sodium selenite, 0.10. Growth, feed and nutrient utilization, and survival parameters
d
Origin Nature, fish oil, Norway. including weight gain (WG), feed conversion ratio (FCR), specific
e
The gross energy value was determined by calculating the combustion values of growth rate (SGR), protein efficiency ratio (PER), protein productive
various macronutrients. According to the NRC (2011), the combustion values value (PPV) were calculated by following equations: weight gain (WG, g
used for calculation were 23.6 kJ/g protein, 39.5 kJ/g fat, and 17.2 kJ/g / fish) = (FBW - IBW); specific growth rate (SGR, % / day) = [(Ln FBW -
carbohydrates.
Ln IBW) / days] × 100; feed conversion ratio (FCR) = individual feed
intake (g) / individual weight gain (g); protein efficiency ratio (PER) =
11:30–12:00, and 17:30–18:00). Every feeding lasted for 30 min until wet weight gain (g) / protein intake (g); protein productive value (PPV,
the fish in the tank appeared to be apparent satiation throughout the %) = protein gain (g) / protein intake (g) × 100; survival (%) = 100 ×
experimental period of 10 weeks. Feed consumption was measured and [(final number of fish) / (initial number of fish)].
recorded every 2 weeks to determine growth performance and feed To determine the data related to fish carcasses, fish samples were
utilization (Section 2.5). eviscerated and measured for carcass weight, liver weight, and visceral
To ensure the constant operation of the raising system and to organ weight. Organosomatic indexes including condition factor (K-
maintain water quality, feces and debris were removed immediately value), viscerosomatic index (VSI), and hepatosomatic index (HSI) were
after each feeding using a siphon. Approximately 70% of the water was also calculated by following equations: condition factor (K, g / cm3) =
siphoned out and refreshed with dechlorinated water. The fish received 100 × [(final body weight, g) / (final body length, cm)3]; hepatosomatic
a photoperiod of 12 h of light and 12 h of darkness, with the light period index (HSI, %) = 100 × [(liver weight, g) / (whole body weight, g)]; and
lasting ~ 7:00–18:30. Water quality parameters, including water tem­ viscerosomatic index (VSI, %) = 100 × [(viscera weight, g) / (whole
perature, pH, dissolved oxygen (DO), and total ammonia, were moni­ body weight, g)].
tored twice per week during the feeding trial. Temperature, pH, and DO
was determined using a portable multi-probe meter, Mettler Toledo 2.6. Chemical scrutiny
(Ulm, Germany). Total ammonia was determined using a portable YSI
9300 m (OH, USA). These parameter ranged 24.9–27.9 ◦ C, 6.8–7.6, The proximate composition was analyzed according to the standard
5.6–7.4 mg/L, and 0.04–0.07 mg/L, respectively. All experimental protocol of The Association of Official Analytical Chemists (AOAC,
procedures conducted in this study adhered to the ethical guidelines 1990). The methods including moisture, crude protein, crude lipid, and
established by the National Research Council of Thailand (NRCT) for ash contents were previously described in our report (Khieokhajonkhet
Animal Experimentation Ethics, under license No.: U1/00704/2558. et al., 2023).
This study was carried out under the approved protocol No.: NUAA­
CUC.0016/2564 by Naresuan University committee, ensuring the wel­ 2.7. Colorimetric determination
fare and ethical treatment of the animal involved.
During week 5 and week 10 of the feeding trial, fish samples were
2.4. Sample collection collected from three distinct regions on the left side of the head, the
trunk part above the lateral line, and the caudal fin. Coloration analysis
At week 5, three goldfish from each tank were randomly sampled (n was conducted using a Hunter Lab MiniScan EZ 4500 L instrument

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A. Khieokhajonkhet et al. Aquaculture Reports 32 (2023) 101705

(Hunter Associates, USA). The references (dark and white plates) were of the first strand cDNA, the revertAid First-Strand Synthesis System kit
calibrated before analysis. The L* value represents the lightness mea­ (Thermo Scientific, Fermentas, USA) was employed, adhering to the
surement, where 100 corresponds to white and 0 corresponds to black. guidelines provided by the manufacturer. The cDNA templates were
The a* value measures the dimension of redness/greenness, while the stored at − 20◦ C until used for RT-qPCR.
b* value represents the dimension of yellowness/blueness. These di­
mensions are defined according to guidelines provided by The Interna­ 2.11. Quantitative real-time poly chain reaction
tional Commission on Illumination (CIE, 1976).
After the cDNA synthesis, it was diluted to a 1:100 ratio using sterile
2.8. Determination of total carotenoid distilled water. The diluted cDNA was employed as a template for the
reverse transcription-quantitative polymerase chain reaction (RT-
At week 10, the total carotenoid content in the fin, muscle, skin, qPCR). The RT-qPCR assay was conducted using the Maxima SYBR
liver, and experimental diets was determined using a modified protocol Green/ROX qPCR Master Mix (2X) from Thermo Fisher Scientific. The
based on the Torrissen and Naevdal (1988) method. In brief, organ PCR amplification and detection were carried out using the PCRmax
samples of ~ 1 g were weighed and homogenized with 5 mL of cold ECO48 Real-time qPCR system from Staffordshire, UK. The primer se­
acetone and 1 g of sodium anhydrous (Na2SO4). An extracted solution quences specific to the target gene and reference (β-actin) genes are
was collected into a fresh tube. Then, the extraction was continued with shown in Table 2. The thermal cycling steps for the RT-PCR assay, begin
the same amount of solution mixture until there was no longer any color with an initial denaturation at 95◦ C for 10 min. This was followed by 40
in the tissue samples. The solution mixture was centrifuged at 3500g at cycles consisting of denaturation at 95◦ C for 15 s, and annealing/
4◦ C for 10 min. After centrifugation, the supernatant was carefully extension at 59◦ C for 42 s. All cDNA reactions were analyzed in triplicate
collected and analyzed using a spectrophotometer at a wavelength of (n = 6). The relative mRNA levels were calculated using a comparative
450 nm (UV-1800, Shimadzu, Kyoto, Japan). Total carotenoid level in cycle threshold (Ct) method, with β-actin serving as the internal control
serum was carried out by following Barbosa et al. (1999)’s protocol. Two gene. In this method, the Ct values of the target gene are compared with
hundred microliter of blood serum were homogenized with the Ct values of the β-actin gene. The ratio between the target gene and
ethanol-hexane (4: 1, v/v) solution. The mixture solution was vortexed the Ct value and the β-actin is calculated to determine the relative mRNA
briefly and subsequently subjected to centrifugation at 2000g at 4◦ C for levels.
15 min. The obtained upper layer was then collected and used for
analysis at a wavelength of 450 nm, using an ethanol-hexane (4:1)
2.12. Statistical analysis
mixture solution as the reference.

All raw data were tested for the normality of distribution and ho­
2.9. Hematological and biochemical determination
mogeneity of variance. The Levene’s test was employed to evaluate the
homogeneity of variance, while the Kolmogorov-Smirnov test was used
The Neubauer hemocytometer was used to evaluate hematological
to examine the normality of the data distribution using the SPSS for
parameters, including the red blood cell count (RBC) and white blood
Windows version 17.0 software (IL, USA). One-way analysis of variance
cell count (WBC), employing the established technique outlined by
(ANOVA) was employed to analyze the data. To identify differences
Hesser (1960). The hematocrit (Hct) was analyzed using the micro­
between each experimental group and the TE0 group, Dunnett’s
hematocrit method with hematocrit centrifugation (Beijing, China).
multiple-comparison test was applied. Furthermore, the orthogonal
Hemoglobin (Hb) was analyzed according to the colorimetric method
polynomial contrast was applied to evaluate the linear and/or quadratic
using Drabkin’s assay kit (Sigma Chemical, MO, USA) using absorbance
trend of dietary TE supplementation.
at 540 nm. Blood glucose concentrations were analyzed using Gluc­
ometer (Accu-Check Active, Roche, Mannheim, Germany). Total protein
3. Results
and albumin content in serum were carried out by following the
Bromocressol-green (Scoffone and Fontana, 1975) and Biuret (Drupt
3.1. Growth, feed and nutrient utilization, and organosomatic parameters
et al., 1974) methods. Globulin content in serum was calculated by the
following equation: total globulin (mg/L) = total protein - albumin
Dietary TE supplementation exerted no linear or quadratic effects on
concentration. The levels of serum biochemical parameters including
growth at weeks 2, 4, 6, 8, and 10 (Supplementary Table 1). In addition,
aspartate aminotransferase (AST), alanine aminotransferase (ALT), and
the feed intake, FCR, nutrient utilization, and organosomatic parameters
alkaline phosphatase (ALP) activities, and levels of triglycerides, total
also showed no linear or quadratic tendencies (P > 0.05) at week 10
cholesterol, low-density lipoprotein cholesterol (LDL-c), and
(Table 3). Estimation of TE ingested in each treatment was the highest in
high-density lipoprotein cholesterol (HDL-c) were conducted by
TE3 (0.093 g/fish), followed by TE2 (0.061 g/fish) and TE1 (0.031 g/
following Coz-Rakovac et al. (2008)’s protocols. This evaluation was
fish). The survival of goldfish in each group varied between 97.78% and
performed through a colorimetric method using an automated serum
100%. However, no significant impact of dietary TE supplementation on
biochemistry analyzer (Roche Diagnostics, Switzerland). Serum cortisol
survival was observed according to the results of orthogonal polynomial
was analyzed using a Cusabio Biotech Co., Ltd. enzyme-linked immu­
nosorbent assay kit (ELISA, Wuhan, China).
Table 2
2.10. RNA extraction and cDNA synthesis Primer sequences used in this study.
Genes Primer sequences (5–3′) References
Approximately 1 g of liver tissue was used to extract total RNAs with IL-1β F-GATGCGCTGCTCAGCTTCT Tu et al. (2019)
1 mL of Qiazol lysis buffer (Qiagen, Hilden, Germany). Subsequently, R-AGTGGGTGCTACATTAACCATACG
the mixture solution was processed using RNeasy Mini Kit (Qiagen, IL-10 F-CAAGGAGCTCCGTTCTGCAT Tu et al. (2019)
R-TCGAGTAATGGTGCCAAGTCATCA
Hilden, Germany). To eliminate any genomic DNA contamination, the
Lysozyme F-GTATCTTCAAGCGAGAGGGACT Rashmeei et al. (2020)
extracted total RNAs were then treated with DNase I (Thermo Fisher R-CCCTGTGGGTCTTATACTTACTC
Scientific, Waltham, USA) in accordance with the manufacturer’s in­ HSP70 F-GGCAGAAGGTGACAAATGCA Khosravi-Katuli et al. (2018)
structions. The obtained total RNAs were subjected to measurement of R-TGGGCTCGTTGATGTTCTCA
the purity and concentration at a ratio of 260: 280 nm using a microplate β-actin F-GATGCGGAAACTGGAAAGGG Tu et al. (2019)
R-ATGAGGGCAGAGTGGTAGACG
reader BioTek Instrument Inc., (VT, USA). For the reverse transcription

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Table 3
Growth, feed efficiency, survival, and organosomatic parameters of goldfish fed graded levels of turmeric extract for 10 weeks.
Parameters TE0 TE1 TE2 TE3 SEM Linear Quadratic

Growth and survival

IBW (g/fish) 7.56 ± 0.01 7.57 ± 0.01 7.57 ± 0.01 7.55 ± 0.01 0.002 0.677 0.965
FBW (g/fish) 17.10 ± 0.27 17.17 ± 0.77 17.23 ± 0.54 17.46 ± 1.01 0.494 0.550 0.853
WG (g/fish) 9.56 ± 0.27 9.60 ± 0.76 9.66 ± 0.54 9.91 ± 1.01 0.491 0.563 0.806
SGR (%/day) 1.30 ± 0.03 1.30 ± 0.07 1.31 ± 0.05 1.33 ± 0.09 0.004 0.595 0.752
Survival (%) 97.78 ± 3.85 100.00 ± 0.00 100.00 ± 0.00 97.78 ± 3.85 7.407 1.000 1.000
Feed utilization
Feed intake (g/fish) 30.75 ± 0.78 30.50 ± 0.76 30.57 ± 0.54 31.11 ± 0.50 0.436 0.526 0.330
FCR 3.22 ± 0.04 3.19 ± 0.17 3.17 ± 0.12 3.16 ± 0.28 0.031 0.690 0.916
PER 0.63 ± 0.05 0.64 ± 0.06 0.66 ± 0.06 0.69 ± 0.03 0.003 0.150 0.818
PPV (%) 16.32 ± 1.65 17.10 ± 2.92 17.11 ± 1.49 17.67 ± 1.39 4.604 0.051 0.645
Estimated TE ingestion (g/fish) 0.00 ± 0.00 0.031 ± 0.001 0.061 ± 0.001 0.093 ± 0.002 N.D. N.D. N.D.
Organosomatic indexes
K-value (g/cm3) 10.19 ± 0.40 9.37 ± 1.06 9.48 ± 1.65 10.23 ± 1.29 1.423 0.903 0.095
HSI (%) 1.69 ± 0.67 1.25 ± 0.26 1.37 ± 0.36 1.56 ± 0.36 0.196 0.729 0.074
VSI (%) 11.09 ± 3.52 10.82 ± 2.92 10.60 ± 1.60 10.18 ± 2.74 7.769 0.539 0.947

The data presented in the study are reported as mean values (n = 3 for growth, survival, and feed efficiency, n = 9 for organosomatic indexes). N.D. = not determined.

contrast or analysis of variance (ANOVA) (Table 3). The high survival the abdominal region (Table 5). In fact, according to Dunnett’s test, all
rates were well associated with the high water quality achieved by dietary supplementation of TE levels of all three regions caused a sig­
frequent water change. Although the nitrite and nitrate concentrations nificant increase in the a* value compared to those obtained with a TE0
were not determined in this study, total ammonia concentration diet. A linear effect on the L* value was found at the head region (P =
remained very low (0.04–0.07 mg/L) throughout the experiment. 0.044, Table 5).

3.2. Chemical scrutiny of the whole-body goldfish 3.4. Total carotenoid content

The chemical compositions of whole-body fish fed different levels of At weeks 5 and 10, the content of total carotenoid in the serum was
TE supplementation are depicted in Table 4. Dietary supplementation of found to be significantly influenced by dietary TE supplementation in
TE did not significantly affect (P > 0.05) dry matter and fat contents. both linear and quadratic manners (Supplementary Table 3 and Table 6,
However, positive linear and quadratic responses to the dietary TE respectively). At week 5 and week 10, the TE3 supplementation showed
supplementation were observed for whole-body crude protein and only the highest total serum carotenoid content (P < 0.05, Supplementary
a linear response for ash content (P < 0.001). Dietary supplementation Table 3 and Table 6). By week 10, total carotenoid contents of the fish in
of TE2 and TE3 had significant effects on whole-body protein content the TE3 group were significantly higher when compared with the con­
when compared to a TE0 group (P < 0.05, Dunnett’s test). A similar trol (TE0) group (Dunnett’s test, P < 0.05).
response was also found in whole-body ash content (Dunnett’s test, P < Increasing levels of dietary TE supplementation resulted in a linear
0.05) with a linear effect (Table 4). and quadratic increase in total carotenoid contents in the fin, liver, and
skin. However, in muscle, only a linear effect (P < 0.05) was observed
3.3. Color parameters at week 5th and week 10th (Table 6). The highest content of total carotenoid in muscle, skin, and
liver was observed in goldfish in the TE3 group, while the TE2 group
Color parameters of goldfish fed dietary TE supplementation at week showed the highest total carotenoid content in the fin region. According
5 and week 10 are demonstrated in Supplementary Table 2 and Table 5, to Dunnett’s test, dietary supplementation of TE2 and TE3 significantly
respectively. Our results showed no noteworthy linear or quadratic increased total carotenoids in the fin and muscle compared with the TE0
patterns (P > 0.05) in the b* value at the head position, L* value at the group. The supplementation of TE at all levels resulted in a noteworthy
abdominal position, and L* and a* values at the tail part at week 5 elevation (P < 0.05) in the total carotenoid content present in both skin
(Supplementary Table 2). Conversely, there were notable positive linear and liver (Table 6).
and quadratic tendencies (P < 0.05) in the a* value at both the
abdominal and head regions. In addition, all TE supplementation levels 3.5. Hematology and biological characteristics
exhibited a significant increase in the a* value at both the abdominal
body and head parts, in comparison to the group that was not supple­ Dietary supplementation of TE exhibited a linear effect on RBC and
mented with TE in their diet (Dunnett’s test, P < 0.05). The L* value was WBC levels. However, no quadratic or linear tendencies (P > 0.05) were
quadratically increased at the head region, and the b* value was linearly observed in Hct and Hb levels (Table 7). Regarding blood biochemical
increased at the tail part (P < 0.05) at week 5 (Supplementary Table 2). parameters, total protein, and globulin in serum were found to increase
At week 10, positive linear and quadratic effects were found in a* linearly (P < 0.05) with increasing dietary TE supplementations.
value at the abdominal, tail, and head regions (P < 0.05), and b* value at Moreover, notable linear and quadratic effects were detected in albumin

Table 4
Whole-body composition of goldfish fed graded levels of turmeric extract for 10 weeks (%).
Composition TE0 TE1 TE2 TE3 SEM Linear Quadratic

Dry matter 28.84 ± 0.18 27.97 ± 0.67 28.53 ± 0.43 28.60 ± 0.33 0.501 0.633 0.431
Crude protein 15.54 ± 0.23 15.28 ± 0.13 15.96 ± 0.18 * 16.17 ± 0.05 * 0.339 < 0.001 0.016
Crude fat 14.68 ± 0.04 13.83 ± 0.12 14.36 ± 0.17 14.38 ± 0.16 0.258 0.110 0.130
Ash 3.29 ± 0.15 3.45 ± 0.13 3.55 ± 0.09 * 3.69 ± 0.09 * 0.186 < 0.001 0.925

The data presented in the study are reported as mean values (n = 3, three fish per replicate was pooled). Asterisks (*) above individual dietary TE supplementation
levels indicate significant differences compared to a control group (TE0, Dunnett’s test, P < 0.05).

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A. Khieokhajonkhet et al. Aquaculture Reports 32 (2023) 101705

Table 5
Color parameters fed different levels of turmeric extract in goldfish for 10 weeks.
TE0 TE1 TE2 TE3 SEM Linear Quadratic

Head
Luminosity (L*) 61.93 ± 3.54 65.56 ± 6.41 * 67.83 ± 4.72 66.21 ± 3.81 * 2.2617 0.044 0.144
Redness (a*) 13.90 ± 2.68 23.37 ± 4.27 * 23.73 ± 2.21 * 26.59 ± 2.98 * 9.799 < 0.001 0.003
Yellowness (b*) 26.03 ± 3.86 23.75 ± 4.96 27.55 ± 4.62 28.60 ± 3.44 8.159 0.080 0.250
Abdominal region
Luminosity (L*) 62.15 ± 5.71 63.90 ± 5.64 61.47 ± 4.71 63.83 ± 5.51 9.288 0.747 0.867
Redness (a*) 10.97 ± 3.30 20.48 ± 1.59 * 24.24 ± 4.05 * 25.67 ± 2.66 * 9.222 < 0.001 0.001
Yellowness (b*) 37.13 ± 4.05 48.22 ± 4.63 * 41.84 ± 4.88 43.75 ± 3.46 * 8.423 0.043 0.003
Tail
Luminosity (L*) 47.29 ± 4.19 47.23 ± 6.12 44.20 ± 2.31 44.47 ± 3.14 17.543 0.096 0.829
Redness (a*) 12.66 ± 2.89 21.42 ± 3.26 * 24.29 ± 5.08 * 25.00 ± 2.22 * 2.424 < 0.001 0.002
Yellowness (b*) 41.24 ± 3.31 42.70 ± 5.44 43.74 ± 2.95 44.51 ± 5.13 8.888 0.103 0.811

The data presented in the study are reported as mean values (n = 9, three fish per tank). Asterisks (*) above individual dietary TE supplementation levels indicate
significant differences compared to a control group (TE0, Dunnnett’s test, P < 0.05).

Table 6
Total carotenoids in goldfish fed varying amount of turmeric extract for 10 weeks.
Items TE0 TE1 TE2 TE3 SEM Linear Quadratic

Fin (µg/g) 67.41 ± 9.85 78.60 ± 11.65 116.37 ± 17.29 * 92.04 ± 12.91 * 17.463 0.001 0.008
Muscle (µg/g) 30.96 ± 7.21 39.29 ± 7.88 74.04 ± 15.58 * 86.27 ± 17.02 * 1.474 < 0.001 0.736
Skin (µg/g) 93.76 ± 11.50 177.65 ± 21.27 * 159.06 ± 13.61 * 183.03 ± 19.79 * 29.206 < 0.001 0.001
Liver (µg/g) 14.02 ± 3.43 33.93 ± 4.46 * 47.89 ± 6.50 * 54.05 ± 4.80 * 4.302 < 0.001 0.007
Serum (µg/mL) 1.13 ± 0.44 1.77 ± 0.17 1.72 ± 0.16 2.01 ± 0.18 * 0.071 < 0.001 0.044

The data presented in the study are reported as mean values. Asterisks (*) above individual dietary TE supplementation levels indicate significant differences compared
to a control group (TE0, Dunnnett’s test, P < 0.05).

Table 7
Hematological and biochemical parameters of goldfish fed different levels of turmeric extract for 10 weeks.
Blood parameters TE0 TE1 TE2 TE3 SEM Linear Quadratic

Hematological parameters
RBC (×106/µl) 0.93 ± 0.04 1.17 ± 0.09 * 1.27 ± 0.11 * 1.37 ± 0.13 * 0.010 0.011 0.386
WBC (×104/µl) 5.70 ± 0.71 6.47 ± 2.12 6.66 ± 1.59 6.90 ± 1.06 * 2.164 0.001 0.061
Hct (%) 30.71 ± 3.87 32.52 ± 2.54 32.87 ± 1.38 34.45 ± 0.75 5.988 0.920 0.920
Hb (g/dL) 7.78 ± 0.86 7.28 ± 0.98 7.53 ± 1.00 7.72 ± 0.74 0.819 0.966 0.462
Biochemical parameters
Total protein (g/dL) 1.96 ± 0.21 2.00 ± 0.14 2.37 ± 0.18 2.68 ± 0.21 * 0.024 0.006 0.286
Albumin (g/dL) 0.45 ± 0.04 0.46 ± 0.03 0.52 ± 0.02 0.68 ± 0.02 * 0.001 0.002 0.032
Globulin (g/dL) 1.51 ± 0.16 1.53 ± 0.10 1.85 ± 0.21 2.00 ± 0.00 0.021 0.017 0.587
AST (U/L) 13.42 ± 0.77 11.75 ± 1.05 12.75 ± 1.06 13.96 ± 1.39 1.192 0.785 0.090
ALT (U/L) 46.73 ± 1.21 42.85 ± 4.03 43.48 ± 0.70 42.14 ± 1.97 0.532 0.152 0.488
ALP (U/L) 35.31 ± 0.80 35.61 ± 1.93 35.92 ± 1.30 37.41 ± 0.61 1.769 0.173 0.545
Total cholesterol (mg/dL) 122.99 ± 3.46 106.47 ± 3.72 * 114.42 ± 3.61 113.98 ± 4.24 14.225 0.185 0.039
Triglycerides (mg/dL) 262.43 ± 3.46 264.71 ± 6.07 247.44 ± 3.44 260.98 ± 1.41 15.677 0.015 0.019
HDL-c (mg/dL) 23.02 ± 0.74 26.41 ± 0.65 * 27.48 ± 0.70 * 25.08 ± 0.14 0.374 0.020 0.003
LDL-c (mg/dL) 119.48 ± 10.61 84.26 ± 8.09 * 76.25 ± 4.70 * 82.57 ± 3.40 * 2.904 0.007 0.038

The data presented in the study are reported as mean values (n = 3, 3 fish per replicate were pooled). Asterisks (*) above individual dietary TE supplementation levels
indicate significant differences compared to a control group (TE0, Dunnett’s test, P < 0.05).

levels (P = 0.002, P = 0.032). The levels of AST, ALT, and ALP activities 0.05) LDL-c in all TE supplementation levels (Table 7).
did not have significant linear or quadratic effects on the TE supple­
mentation levels. Total cholesterol levels quadratically decreased with 3.6. Blood stress markers
increasing TE supplementation levels (Table 7). Likewise, there were
notable linear and quadratic trends observed in the levels of tri­ The effect of goldfish-fed dietary TE supplementation on blood
glycerides and LDL-c (P < 0.05). While dietary TE treatment dramati­ glucose and serum cortisol levels is shown in Table 8. Goldfish fed with
cally elevated HDL-c levels with both linear and quadratic effects (P = TE2 supplementation exhibited the lowest glucose concentration among
0.020, P = 0.003). Dunnett’s test showed a significantly decreased (P < the treatment groups. Dietary TE supplementation exerted linear and

Table 8
Plasma cortisol and glucose levels of goldfish fed different levels of curcumin extract for 10 weeks.
Items TE0 TE1 TE2 TE3 SEM Linear Quadratic

Glucose (mmol/dL) 43.50 ± 0.70 36.15 ± 9.19 17.53 ± 0.70 * 29.49 ± 2.13 2.515 0.015 0.047
Cortisol (mg/mL) 15.50 ± 0.71 13.00 ± 0.70 * 11.13 ± 0.09 * 11.04 ± 0.08 * 0.254 0.001 0.087

The data presented in the study are reported as mean values (n = 3, 3 fish per replicate were pooled). Asterisks (*) above individual dietary TE supplementation levels
indicate significant differences compared to a control group (TE0, Dunnett’s test, P < 0.05).

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A. Khieokhajonkhet et al. Aquaculture Reports 32 (2023) 101705

quadratic effects on the blood glucose level (P = 0.015, P = 0.047). applications of this important herb.
Additionally, the TE0 group exhibited the highest blood cortisol level, The current study showed that TE supplementation exhibited no
and a significant impact of dietary TE was observed (P = 0.001) with a significant linear or quadratic effects on growth, feed efficiency, and
linear trend (Table 8). In comparison to the TE0 group, all levels of di­ organosomatic parameters in goldfish. There have been some discrep­
etary TE supplementation resulted in a significant decrease in cortisol ancies regarding the effect of curcumin on these parameters. An intra­
levels (Dunnett’s test, P < 0.05). peritoneal injection of 100 nmol of curcumin significantly decreased
feed intake in goldfish (Kang et al., 2011). According to Akdemir et al.
3.7. Hepatic mRNA expression related to immunity (2017), the study reported that the administration of curcumin did not
show any significant effect on the growth or FCR of rainbow trout, even
The mRNA expression levels of goldfish fed TE supplemented diets after being received an experimental diet for 60 days. However, con­
are shown in Fig. 1. Goldfish fed dietary supplementation of TEs down- trasting findings have been observed in other studies where the use of
regulated the expression of IL-1β transcripts in comparison to the TE0 turmeric powder has shown positive effects on growth and feed utili­
group. Fish fed TE0 showed the highest IL-1β expression level, while TE2 zation parameters (FCR and feed intake) in silver catfish (Rhamdia
and TE3 showed significantly lower levels than the TE0 group (Fig. 1A). quelen) and Nile tilapia, (Cui et al., 2013; Mahmoud et al., 2017; Bal­
The lysozyme and IL-10 transcripts were generally up-regulated in fish dissera et al., 2018; Sruthi et al., 2018). In a study conducted by Wojno
fed TEs, and the highest expression levels (P < 0.05) of these genes were et al. (2021), curcumin extract suppressed weight gain and significantly
observed in the TE2 group (Fig. 1B). Furthermore, the Dunnett’s test increased FCR in common carp. Conversely, in some other studies, a
showed significant enhancements in the expression of IL-10 and lyso­ significant increase in weight gain and decrease in FCR were found in
zyme mRNAs in all groups that fed dietary TE supplementation, when rainbow trout and grass carp when exposed to curcumin extract (Yonar
compared to the control group (Figs. 1B, 1C). On the other hand, fish fed et al., 2019; Akdemir et al., 2017; Ming et al., 2020). A multitude of
TE supplementation showed no significant difference in HSP70 mRNA factors, including genetics, species, nutritional composition, rearing
expression levels compared to that fed diet without TE supplementation condition, and environment, can account for these variations. However,
(P > 0.05, Fig. 1D). it would be possible to conclude that TE supplementation up to 3 g/kg is
unlikely has adversely severe affect growth and feed efficiency of
4. Discussion goldfish. Also, under the favorable experimental condition, TE supple­
mentation did not significantly increase the survival rate. Given the
Currently, aquaculture fish is generally reared under intensive and potential immune system enhancement observed in TE (see below), it is
stressful conditions due to the rapid expansion of the aquaculture sector. possible to ascertain the immunomodulatory effects of TE.
Such practices have caused the emergence of infectious diseases (Van TE supplementation significantly increased the crude protein and ash
Doan et al., 2018). Herbal remedies, especially turmeric or curcumin, contents in this study. Consistent with our findings, dietary TE supple­
are a promising substitute for antibiotics in aquaculture (Grenni et al., mentation increased whole-body composition of crude protein and ash
2018; Liu et al., 2019) as shown in various fish species (Mahmoud et al., in common carp (Pirani et al., 2021), crucian carp (Carassius auratus)
2017; Yonar et al., 2019). The primary objective of this study was to (Jiang et al., 2016), and Nile tilapia (Mahmoud et al., 2017; Mohamed
investigate the impact of dietary TE supplementation on an ornamental et al., 2020). This may be related to effective nutrient utilization by gut
fish species, with the aim of exploring the potential benefits and microbiota affected by curcumin (Mahmoud et al., 2017). According to

Fig. 1. The expression of IL-1β (A), IL-10 (B), lysozyme (C), and HSP70 (D) in the liver of goldfish (Carassius auratus) fed a diet containing 0 (TE0), 1 (TE1), 2 (TE2),
and 3 (TE3) g/kg diet of turmeric extract for 10 weeks. The data presented in the study are reported as mean values for six individuals (n = 6) per treatment.
Significant differences in Dunnette’s test are shown by an asterisk (P 0.05).

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A. Khieokhajonkhet et al. Aquaculture Reports 32 (2023) 101705

Jiang et al. (2016), the administration of TE in crucian carp (Carassius leakage through damaged plasma membranes (Yang and Chen, 2003). In
auratus) has been found to yield positive effects on digestive enzymes the present study, supplementing with dietary TE had no effect on the
such as amylase, lipase, and trypsin. The coloration of fish can be activity of these hepatic enzymes, suggesting no adverse effects on
enhanced through dietary supplementation of natural carotenoids, as protein metabolism and liver function in experimental fish (Yang and
fish lack the ability to synthesize carotenoids de novo (Gupta et al., Chen, 2003; Kumar et al., 2011; Khieokhajonkhet et al., 2021).
2007), which is especially important in ornamental fish species (Belay Consistent with our results, dietary supplementation of curcumin did not
et al., 1996; Khieokhajonkhet et al., 2023; Tulli et al., 2012). Turmeric affect AST and ALT activities in Nile tilapia, which was also confirmed
contains total carotenoids of approximately 2.8 – 6.0 mg/g (Sontsa-­ by liver histology (Amer et al., 2022).
Donhoung et al., 2022). The primary pigment of turmeric is lutein Serum cholesterol and triglyceride significantly decreased in the fish
(Krishnamurthy et al., 1976; Safitri et al., 2020), which is responsible for fed an increasing TE level with linearly and quadratically effects. These
the yellowish-orange color and accounts for 90% of total carotenoid in results are in line with the hypolipidemic effect reported in fingerlings of
the integument of goldfish (Hata and Hata, 1970). A recent study also Nile tilapia (Abdel-Tawwab et al., 2022). Similarly, dietary adminis­
showed that the administration of 50 mg/kg of lutein demonstrated the tration of turmeric, curcumin, and nano-encapsulated curcumin signif­
ability to enhance survival and stimulate carotenoid pigmentation in the icantly decreased total cholesterol and triglyceride in Pacific white
skin of goldfish after fed for 84 days (Besen et al., 2019). In addition, shrimp (Penaeus vannamei) (Moghadam et al., 2022). Furthermore, the
turmeric contains 2–5% of polyphenolic curcuminoids that have supplementation of TE demonstrated linear and quadratic increase in
distinctive bright yellowish to orange-yellowish colors (Sandur et al., HDL-c, as well as a decrease in LDL-c levels in goldfish. A reduction of
2007; Pal et al., 2020). These compounds are likely responsible for the cholesterol, LDL-c, triglyceride, and increased HDL-c was also found in
improved coloration (Table 5) and increased total carotenoid contents large yellow croaker (Larimichthys crocea) fed dietary curcumin sup­
(Table 6) observed in this study. In agreement with our study, the sup­ plementation (Ji et al., 2020). These results could be attributed to the
plementation of turmeric powder in the diet also improved the colora­ active compounds in turmeric, which limit cholesterol biosynthesis and
tion in several tissues of red tilapia after fed for 56 days (Boonyaratpalin absorption, leading to the reduction of plasma and hepatic cholesterol
and Unprasert, 1989), guppy (Poecilia reticulata) after fed for 95 days concentrations (Ji et al., 2020; Abdel-Tawwab et al., 2022).
(Mukherjee et al., 2009), and yellow tail Cichlid (Pseudotropheus acei) In response to microbial invasion and tissue damage, IL-1β, also
after fed for 90 days (Öngun et al., 2021). Although carotenoid-rich known as a pro-inflammatory cytokine, plays crucial role in regulating
source powders are beneficial for pigmentation, some studies revealed the inflammation triggered by pathogenic bacteria (Yousefi et al., 2022).
that dietary supplementation with natural extracts led to a more sig­ While, the IL-10 gene plays a pivotal role in regulating both
nificant rise in total carotenoid content and skin pigmentation compared anti-inflammatory and immune responses during bacterial pathogen
to the use of natural carotenoid powder (Pham et al., 2014; Baron et al., infection (Cao et al., 2018; Khieokhajonkhet al, 2023). In goldfish, genes
2008). This finding implies that extracting natural carotenoids from rich encoding IL-1 and IL-10 were found to be significantly upregulated upon
sources shows greater promise and effectiveness than utilizing the dietary supplementation with medicinal plants, herbs, or probiotics.
powdered form. However, Nascimento et al. (2019) revealed that di­ This finding suggests that these dietary components may play a role in
etary administration of 0–25 g/kg turmeric powder could not improve modulating the immune response in goldfish by upregulating the
skin coloration in the thick-lipped gourami (Trichogaster labiora). expression of these specific genes (Cao et al., 2018; Khieokhajonkhet
Hematological parameters are impacted by dietary nutrition and et al., 2023; Yousefi et al., 2022). In this study, supplementation of TE in
environmental stressors and are often used to monitor fish health and common carp significantly decreased IL-1β and upregulated the IL-10 of
physiological states. In this study, RBC and WBC linearly increased with anti-inflammatory cytokine mRNA expression levels in goldfish. Giri
increasing dietary TE supplementation (P < 0.05). WBC count is one of et al. (2019) reported that curcumin had a similar effect in decreasing
the crucial fish health markers. Several factors including age, sex, hor­ mRNA transcripts of IL-1β and tumor necrosis factor-alpha (TNF-α),
monal changes, stress, temperature, inflammation, and diseases change which was also observed in terrestrial animals (Literat et al., 2001; Mito
WBC (Clauss et al., 2008; Khieokhajonkhet et al., 2022b; Kumar et al., et al., 2011). Turmeric is known to reduce several inflammatory cyto­
2011). An increase in WBC counts within the normal range is interpreted kines (e.g. IL-1β, TNF-α, IL-2, IL-6, IL-8, IL-12, and chemokines) by
that fish have higher cellular defenses against infections by the suppressing TNF-α binding and NF-kB pathways (Anthwal et al., 2014;
nonspecific immunity activation (Clauss et al., 2008). Since curcumin is Giri et al., 2019). TNF-α and IL-1β expression may be negatively regu­
one of the active compounds of turmeric that have antioxidant, anti­ lated by IL-10 (Lee et al., 2001; Wei et al., 2015). Lysozyme, a crucial
pathogenic bacterial, and immunomodulatory properties, this active immune defense component, participates in the innate immunity de­
compound may account for the increase in WBC. Similar effects of fense since it lyses the cell walls peptidoglycans of bacteria and acts as
turmeric powder have been reported for several fish species including an opsonin to stimulate the complement system and phagocytes (Mag­
ornamental dotty back (Pseudochromis dilectus), climbing perch (Anabas nadóttir, 2006). In the case of goldfish, the incorporation of medicinal
testudineus), and rohu (Labeo rohita) (Devi et al., 2016; Manju et al., herbs containing various bioactive compounds into their diet is believed
2013; Sahu et al., 2008). to elevate the level of lysozyme mRNA expression (Rashmeei et al.,
The total serum protein levels serve as a commonly utilized indicator 2020; Yousefi et al., 2022). This, in turn, is thought to contribute to the
to assess the nutritional status and overall health conditions of fish enhancement of their innate immune system (Magnadóttir, 2006). In
(Yang and Chen, 2003). An elevation of total serum protein levels this study, lysozyme transcripts were significantly increased in the TE2
generally suggests an improvement in fish health and immunity. The group as also reported in common carp (Giri et al., 2019), rainbow trout
present study found that dietary TE supplementation resulted in a sig­ (Yonar et al., 2019), and Nile tilapia (Mahmoud et al., 2017). HSP70 is
nificant increase in total serum protein, albumin, and globulin levels. associated with environmental stress factors and plays a pivotal role in
These results are in line with observations in Nile tilapia fed 0.1% and the cellular machinery responsible for protein folding. This function aids
0.2% curcumin (Diab et al., 2014) and those fed 1% turmeric powder in the repair and protection of cellular proteins against damage caused
(Ayoub et al., 2019). Likewise, Abdel-Tawwab et al. (2022) and Sahu by various stressors in several fish species including goldfish (Ming et al.,
et al. (2008) revealed that turmeric powder and curcumin nanoparticles 2010; Magouz et al., 2021; Kim et al., 2014). In the present study, di­
could potentially improve total serum proteins and serum biochemistry etary TE supplementation did not alter HSP70 mRNA expression levels.
in fish. The measurement of AST, ALT, and ALP provides information on Similar effects of turmeric supplementation were also studied in other
the overall nutritional state, as well as the liver function and circulatory fish species including gilthead seabream (Sparus aurata) (Teixeira et al.,
system (Kumar et al., 2011). An increase in these enzyme activities may 2022) and common carp (Giri et al., 2019; Giri et al., 2021).
indicate increased hepatic enzyme production or potential enzyme In aquaculture studies, various herbs have been investigated for their

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A. Khieokhajonkhet et al. Aquaculture Reports 32 (2023) 101705

stress-relieving effects on fish, such as oak leaf extract (Paray et al., References
2020), essential oil of eucalyptus (Taheri Mirghaed et al., 2018), white
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