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Journal of Applied Aquaculture
ISSN: 1045-4438 (Print) 1545-0805 (Online) Journal homepage: https://www.tandfonline.com/loi/wjaa20
Growth performance and color enhancement of
goldfish, Carassius auratus, fed diets containing
natural dyes extracted from annatto (Bixa orellana)
seeds
S.H.S Dananjaya, Prabuddha Manjula, A. S. Dissanayake, M. Edussuriya, K.
Radampola, Bae Keun Park & Mahanama De Zoysa
To cite this article: S.H.S Dananjaya, Prabuddha Manjula, A. S. Dissanayake, M. Edussuriya,
K. Radampola, Bae Keun Park & Mahanama De Zoysa (2019): Growth performance
and color enhancement of goldfish, Carassius�auratus, fed diets containing natural dyes
extracted from annatto (Bixa�orellana) seeds, Journal of Applied Aquaculture, DOI:
10.1080/10454438.2019.1629371
To link to this article: https://doi.org/10.1080/10454438.2019.1629371
Published online: 02 Jul 2019.
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Growth performance and color enhancement of goldfish,
Carassius auratus, fed diets containing natural dyes
extracted from annatto (Bixa orellana) seeds
S.H.S Dananjaya
a
, Prabuddha Manjula
b
, A. S. Dissanayake
c
, M. Edussuriya
c
,
K. Radampola
d
, Bae Keun Park
a
, and Mahanama De Zoysa
a
a
College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungnam National
University, Yuseong-gu, Daejeon, Republic of Korea;
b
Division of Animal and Dairy Science, Chungnam
National University, Yuseong-gu, Daejeon, Republic of Korea;
c
Department of Chemistry, Faculty of
Science, University of Ruhuna, Matara, Sri Lanka;
d
Department of Fisheries & Aquaculture, Faculty of
Fisheries and Marine Sciences and Technology, University of Ruhuna, Matara, Sri Lanka
ABSTRACT
The present study was conducted to evaluate growth perfor-
mance and color enhancement of goldfish, Carassius auratus,
fed diets containing 0, 50, 100, 200, and 250 mg kg
−1
diet of
annatto dye (AD) for 60 days. The survival rate was significantly
higher in fish fed 100, 200, and 250 mg AD kg
−1
diet over than
these fed control and 50 mg AD kg
−1
diet (p< 0.05). AD sig-
nificantly (p<0 .05) increased the pigmentation in the skin and
caudal fin of goldfish in a concentration dependent manner
(R
2
= 0.995, 0.997). The highest amount of total carotenoid
deposition in fish skin and fins were given by diets containing
200–250 mg AD kg
−1
diet. The highest redness (a*) of 43.21
and yellowness (b*) of 12.53 were obtained by 250 and 50 mg
AD kg
−1
, respectively. The present results show that AD can be
successfully used as an alternative natural carotenoid source in
goldfish diets at levels of 200–250 mg AD kg
−1
diet.
KEYWORDS
Goldfish; Carassius auratus;
carotenoid; annatto dye;
pigmentation
Introduction
Ornamental fish farming is one of the commercially important sectors in the
aquaculture industry all over the world. Beside the size of the fish, body
shape and coloration (skin pigmentation) are vital characters, which directly
affect the marketability of any ornamental fish. Carotenoids, which are lipids
soluble pigments, are responsible for the skin color of fish. Level of carote-
noid directly determines the commercial value of fish (Gouveia and Rema
2005; Liang et al. 2012; Paripatananont et al. 1999). It also influences fish
growth, metabolism, and reproduction (Miki 1991). Fish are unable to per-
form de novo synthesis of carotenoids (Goodwin 1984) and therefore rely on
the dietary supply of pigments to achieve their natural pigmentation.
CONTACT Mahanama De Zoysa mahanama@cnu.ac.kr College of Veterinary Medicine and Research
Institute of Veterinary Medicine, Chungnam National University, Yuseong-gu, Daejeon, 34134, Republic of Korea
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/wjaa.
JOURNAL OF APPLIED AQUACULTURE
https://doi.org/10.1080/10454438.2019.1629371
© 2019 Taylor & Francis
Goldfish, Carassius auratus, is one of the most popular freshwater ornamental
fish, which has a high market value (Yanar et al. 2008). This fish must be
pigmented to have an orange-red color to achieve good consumer acceptance
(Gouveia and Rema 2005). Goldfish are generally reared in eutrophic ponds,
with a high level of Chlorophyta and Cyanobacteria that are rich sources of
carotenoids (Moreira et al. 2011). Goldfish cultured in such environments
exhibit an excellent intensive coloring, nevertheless, if carotenoid sources are
not included in the fish diets, and this coloration will not be achieved under
intensive rearing conditions (Yanar et al. 2008). Therefore, skin pigmentation of
goldfish in aquaria has been accomplished by supplementing their diets with
synthetic or extracted carotenoids, such as zeaxanthin, lutein or astaxanthin
(Matsuno, Matsutaka, and Nagata 1981; Ohkubo et al. 1999; Paripatananont
et al. 1999). However, recent efforts have focused on natural compounds as
alternative sources to synthetic carotenoids because of concerns on the use of
synthetic additives as well as the high cost of pigmented feeds (Gupta et al. 2007).
It has been reported that natural carotenoid sources, such as red yeast,
Xanthophyllomyces dendrorhous (Xu et al. 2006), Spirulina (Kiriratnikom,
Zaau, and Suwanpugdee 2005), Chlorella vulgaris, Haematococcus pluvialis and
Arthrospira maxima (Gouveia et al. 2003), enhance the pigmentation of goldfish.
Annatto (Bixa orellana) is a tropical shrub originated from the American
continent that bears an inedible, red fruit containing about 50 red seeds. The
extract of annatto seeds are characterized by a high content of red pigments
with a high absorption coefficient in the visible part of the solar spectrum.
Among all natural colorants, annatto ranks second in economic importance
worldwide and is widely used in industries such as textiles, varnishes, cos-
metics, tattoos, and medicinal purposes (Scotter 2009; Yolmeh, Habibi Najafi,
and Farhoosh 2014). The amont of total carotenoids in annatto seed was
reported about 4.5–5.5% of seed weight (Giridhar, Venugopalan, and
Parimalan 2014). The pericarps of the annatto seed contain a high concen-
tration of carotenoids, and bixin makes up between 70 and 80% of the total
mass of these carotenoids (Gomezortiz et al. 2010). In addition to bixin,
a smaller quantity of many other carotenoids and related compounds are also
present (Mccullagh and Ramos 2008). These include methyl bixin, norbixin,
all-E- geranylgeraniol, geranylgeraniol esters phytofluene, neurosporene and
phytoene (Figure 1). The annatto seed extract also has documented antiox-
idant and antimicrobial properties (Yolmeh, Habibi Najafi, and Farhoosh
2014). Previously we have purified bixin from AD and showed bixin based
diet can enhance the skin color and pigmentation in goldfish (Dananjaya
et al., 2017). Based on our results we continue the present study to investigate
whether AD could have similar effects (enhance the skin color and pigmen-
tation) on goldfish which may be useful to develop an inexpensive carotenoid
source for fish diet formulations.
2S. H. S. DANANJAYA ET AL.
In the present study, we applied the ultrasound assisted extraction proce-
dure to extract AD (from annatto seed) with a higher recovery rate. Four
diets were formulated by varying levels of AD and tested with goldfish to
evaluate its effect on the growth and enhancement of skin and fin coloration.
Finally, the optimum AD concentration was determined for formulating feed
to obtain effective color enhancement in goldfish.
Materials and methods
Experimental design
Three months old red variety of goldfish, Carassius auratus,wereobtainedfrom
a local commercial breeder and kept under quarantine conditions for three
weeks and then acclimatized to the experimental conditions in aquarium system
O
OH
CH3CH3
CH3CH3
CH3
O
O
CH3
O
OH
CH3CH3
CH3CH3
CH3
OH
O
O
O
CH3CH3
CH3CH3
CH3
O
O
CH3
H3C
H3C
CH3CH3CH3CH3
OH
H3C
CH3CH3CH3CH3
O
O
CH3
(a)
(b)
(c)
(d)
(e)
Figure 1. Chemical structures of the main carotenoid compounds in annatto dye. (a) bixin; (b)
ethyl bixin; (c) nor-bixin (d); geranylgeraniol and (e); geranylgeraniol esters.
JOURNAL OF APPLIED AQUACULTURE 3
for two weeks before the commencement of the experiment. During this period,
fish were fed by a control diet (CD).
A total of 315 fish (7 treatment X 3 replicates x 15 fish/tank) with an average
body weight 7–11 g and similar in color were randomly distributed in 21 tanks (30
x30×60cm
3
) at a stocking density of 15 fish/tank. Six experimental diets with
different levels of AD (0, 50, 100, 150, 200, and 250 mg/kg) were prepared and
labeled as CD, 50 AD, 100 AD, 150 AD, 200 AD, and 250 AD, respectively.
A commercial color feed (CCF) containing astaxanthin was used as the seventh
treatment to compare the coloration of fish in aquarium conditions. Fish were
fed ad libitum with one of the tested diets in triplicates twice a day for 60 days. Feed
consumption was recorded daily. Body weight (g) of fish were measured initially
and then 10 days at the intervals. Total carotenoid content of the skin and the fin of
fish (n = 3) from each tank were analyzed at 0, 20, 40, and 60 days. Uneaten feed
and fish faces were siphoned out daily and water in the aquarium was changed at
a rate of 60% volume per day. Water temperature and pH were determined daily
with a glass electrode (Thermo Orion, Beverly, Massachusetts, USA). Total ammo-
nia (un-ionized ammonia) (ng L
−1
) was measured by an indophenol method and
nitrite was measured (ug L
−1
) weekly by an azo method (Boyd and Tucker, 1992).
Tanks were aerated continually and natural photoperiod (12 h: 12 h light –dark
cycle) was maintained in the aquarium during the experimental period.
Annatto dye extraction
The annatto dye was extracted according to the ultrasound-assisted extrac-
tion method according to Yolmeh, Habibi Najafi, and Farhoosh (2014) with
some modifications. First, annatto seeds (50 g) were soaked in n-hexane
(Sigma, USA) for 6 hours in order to remove oils and defatted seeds were
used for dye extraction. Samples were placed in capped glass tubes and mixed
with chloroform (400 mL, Sigma, USA) and then immersed in a water bath at
50
°
C. The ultrasound-assisted extraction process was performed using ultra-
sonic device (20 kHz, 550 W, Misonix, Germany). The working frequency,
power and time were fixed at 20 kHz, 200 W, and 12 min, respectively. After
ultrasonic extraction, the extracts were filtered through Whatman filter paper
(No.1) and then vacuum-dried to make dye powder. The obtained powder
was weighed and the mass ratio (powder to seeds) was taken into account as
the extraction yield. Consequently, the extraction yield was calculated
according to the following formula; Extraction yield % = (W1 -W2)/W1
X100, where W1 = weight of powder and W2 = weight of annatto seeds.
Diet preparation
The control diet was formulated using fish meal, soybean meal, wheat flour,
wheat gluten, meat meal, coconut oil, fish oil and vitamin-mineral mixture as
4S. H. S. DANANJAYA ET AL.
main ingredients, and AD was added in different amounts (0, 50, 100, 150,
200 and 250 mg/kg) to each diet to prepare other five experimental diets.
Dietary feed ingredients were ground using a laboratory grinder and then
blended into homogenous dough matter by adding water and pellets that
were made by pressing through a die of 3 mm diameter in a grinding
machine. Then, prepared pellets were dried and stored in plastic containers
in a freezer at −20 °C until taken for the feeding trial. The formulation and
proximate composition are given in Table 1. Proximate compositions of
formulated diets were determined according to analytical procedures of
Association of Official Analytical Chemists (AOAC) (2005). Dry matter
was calculated after drying in an oven at 105 °C to a constant weight; ash
content was determined by incineration in a muffle furnace at 550 °C for
12 h; crude protein (N- 6.25) by the Kjeldahl method after acid digestion;
amount of crude fiber was analyzed after digestion with H
2
SO
4
and NaOH.
Crude lipid content was measured by petroleum ether extraction in a Soxlet
apparatus for 40 min in a Tecator HT6 equipment. Triplicate samples of each
ingredient or diet were used for each analysis.
Analysis of carotenoid content
The carotenoid content was extracted according to the method of Torrissen and
Naevdal (1984), with some modifications. Sample (skin or fin) of 200–1000 mg
were taken from both sides of the abdominal and dorsal regions of the fish, being
Table 1. Feed ingredients and nutrient composition (% dry weight) of the control diet.
Ingredients % dry weight
Fish meal 45
Wheat flour 30
Soy bean meal 7.5
Wheat gluten 5.5
Meat meal 2.5
Coconut oil 3.5
Fish oil 3.5
Vitamin-Mineral premix 2.5
Proximate composition
Crude protein 38 ± 1.6
Crude lipid 12 ± 0.8
Dry matter 85 ± 2.4
Crude fiber 1.4 ± 0.5
Ash 10 ± 0.6
Each value is mean of three samples.
a
Vitamin-Mineral premix consisted of (mg/kg) Vitamin A 500,000 IU, Vitamin D3 100,000 IU, Vitamin E 3,000
IU, Vitamin B1 3,000 mg, Vitamin B2 100 mg, Vitamin B6 200 mg Vitamin B12 1 mg, Vitamin C 2,000 mg,
Vitamin K3 200 mg, Pantothenic acid 500 mg, Nicotinic acid 1,000 mg, Inositol 1,000 mg, Choline chloride
10,000 mg, Folic acid 100 mg, Biotin 2 mg, Dl-methionine 1,000 mg, Zinc sulfate 5,000 mg, Ferrous sulfate
4,000 mg, Copper sulfate 1,000 mg, Cobalt sulfate 250 mg, Magnesium oxide 1,000 mg, L-Lysine 1,000 mg,
DC Methionine 1,000 mg. Fish meal with 66% crude protein level (TripleNine Group A/S, Denmark), and
Soy bean meal with 46% crude protein content (The Delong Co.Inc, USA) was used.
JOURNAL OF APPLIED AQUACULTURE 5
careful to remove adhering adipose tissue. The grounded sample was transferred to
10 mL pre-weighed glass tubes containing 10 mL acetone (98%, Merck, Germany)
and 1.5 g of anhydrous sodium sulfate. It was homogenized using a Bio homo-
genizer (Biospec and Samro, USA). The samples were stored for one day at 4°C,
and then extracted with acetone, two or three times until no more color could be
obtained. The solutions were centrifuged at 3500 rpm for 10 min, and then
absorptions were measured at 476 nm by a spectrophotometer (Hach DR/4000U
Spectrophotometer, USA). A similar method was adapted for the analysis of total
carotenoid in the feed samples, but anhydrous sodium sulphate was not used and
absorption was measured at 450 nm (Ramamoorthy et al. 2010).
Analysis of skin color
Skin color analysis was performed by reflectance spectroscopy. The color para-
meters of L* (lightness), a* (greenness & redness), and b* (blueness & yellowness)
were obtained according to the CEB lab criteria using a portable ‘Minolta colori-
meter’(CR-300, AE.PT.01) calibrated towards a white standard. The color of the
dorsal region of the fish skin from each tank was measured at the beginning of the
experiment and also the end of the experimental period of 60 days.
Analysis of biological indices
Biological indices (specific growth rate, weight gain and feed conversion
ratio) were obtained according to Gouveia et al. (2003) and Kalinowski
et al. (2005).
Specific growth rate (% weight gain/day) = [(Final weight-initial weight)/
number of days] *100.
% SGR
Weight gain (WG; g) = Final weight-initial weight
Feed intake = feed intake (g) per 100 g fish for the period
Feed conversion ratio (FCR) = Feed intake/average daily weight gain
Statistical analysis
All the data were expressed as the mean ± SD. The results were subjected
to one-way analysis of variance (ANOVA) using Minitab 16 software
(Minitab, Inc., USA). Turkey’s studentized range test for mean separation
procedure was applied to compare differences between the means at 5%
significance level. The functional relationship between diets including
annatto dye and carotenoid content of the skin and caudal fin of the
fish was analyzed by using second order polynomial regression fit in
6S. H. S. DANANJAYA ET AL.
R (3.2.1 for windows, 2007). The function Ŷ=b
0±
b
1
x±b
2
x
2
±…±b
k
x
k
wasusedtofitourdata,whereŶis the predicted carotenoid content (mg/
kg), x is the annatto dye level (mg/kg) in diet and b
0
,b
1
,andb
2
are the
constants determined by the regression. Subjective goodness of fit was
assessed by plotting the data and the fitted curve.
Results
Annatto dye extraction and diet preparation
The extraction yield of annatto dye was 4.18%. The feed ingredients and
proximate compositions of the experimental diet are given in Table 1.The
carotenoid content of the control diet and the experimental diet with
different amounts of annatto dye are shown in Table 2.
Biological indices of experimented gold fish
The water temperature was maintained at 27.5 ± 2
°
C using an aquarium
heater. Other physico-chemical parameters throughout the experimental
period were pH 7.8 ± 0.9, total ammonia, 90 ± 15 ng/L and nitrite,
34.56 ± 5.7 µg/L. All diets were readily accepted by fish, which proved
that there was a relationship between palatability and feed intake of AD
supplementeddiets.Allfishshowed a good health condition and grew
normally. Thereby, zero mortality was observed in all groups except the
control group during the experimental period. However, the survival rate
in each treatment at the end of the experimental time was significant
(p< 0.05) compared to control and 50 mg/kg AD group (Table 2).
The biological indices revealed that the initial body weight of the fish
was ranged from 6.77 to 7.77 g and fish body weight increased by 2.2–2.6
folds at the end of the experimental period of 60 days (Table 2). The
inclusion of AD in fish diets did not affect the final weight, weight gain
and specific growth rate of fish (p> .05). Furthermore, the highest and
the lowest mean final body weights of 18.85 g and 16.26 g were observed
in fish fed with 250 mg/kg AD in diet and the control diet, respectively.
The highest TWG and SGR values were 11.65 g and 1.60% g/day, respec-
tively and the lowest TWG and SGR values were 8.91 g and 1.11% g/day
in that order. The FCR of the fish was adversely affected by the higher
level of dietary AD in diets. The lowest significant FCR value of 1.42 was
observed in fish fed with 250 AD, whereas, the highest FCR value of 1.72
was exhibited by the control group (p<0.05).
JOURNAL OF APPLIED AQUACULTURE 7
Table 2. Growth parameter and total carotenoid level in the skin and caudal fin of goldfish fed formulated diets for 60 days of the experimental period.
(Annatto dye (AD) supplementation level mg/kg)
Rearing parameter Control 50AD 100AD 150AD 200AD 250AD CCF
Initial body weight (g) 7.34 ± 0.86 7.77 ± 0.70 7.01 ± 0.41 6.77 ± 0.49 7.01 ± 0.40 7.21 ± 0.45 7.18 ± 0.58
Final body weight (g) 16.26
a
± 1.22 17.44
a
± 0.51 16.75
a
± 0.45 16.89
a
± 0.66 18.17
a
± 1.03 18.85
a
± 1.37 17.77
a
± 1.19
Total weight Gain (g) 8.91
a
± 1.56 9.66
a
± 1.21 9.75
a
± 0.07 10.11
a
± 1.05 11.16
a
± 0.73 11.65
a
± 1.74 10.58
a
± 1.77
Specific growth rate (SGR) (%g/day) 1.33
a
± 0.24 1.114
a
± 0.19 1.45
a
± 0.20 1.45
a
± 0.06 1.53
a
± 0.17 1.601
a
± 0.22 1.51
a
± 0.07
Feed conversion ratio (FCR) 1.72
a
± 0.06 1.70
a
± 0.07 1.64
a
± 0.11 1.56
a
± 0.07 1.43
b
± 0.06 1.42
b
± 0.10 1.58
a
± 0.07
Initial total carotenoids in skin (mg/kg) 16.02 ± 1.20 16.02 ± 1.20 16.02 ± 1.20 16.02 ± 1.20 16.02 ± 1.20 16.02 ± 1.20 16.02 ± 1.20
Initial carotenoids in fin (mg/kg) 26.68 ± 1.60 26.68 ± 1.60 26.68 ± 1.60 26.68 ± 1.60 26.68 ± 1.60 26.68 ± 1.60 26.68 ± 1.60
Final total carotenoids in skin (mg/kg) 17.23
a
± 1.38 36.77
b
± 5.06 46.44
cb
± 4.41 59.14
d
± 3.69 66.28
eg
± 1.97 76.95
fg
± 4.29 73.34
g
± 2.15
Final total carotenoids in fin (mg/kg) 27.23
a
± 2.98 50.84
b
± 3.44 65.68
c
± 3.62 77.64
d
± 2.76 88.51
eg
± 1.04 95.11
fg
± 4.23 87.12
g
± 3.17
Fish survival (%) 87.25
a
± 00 87.25
a
± 00 100
b
± 00 100
b
± 00 100
b
± 00 100
b
± 00 100
b
±00
Values are a means ± S.D. Means within the same row with different superscript letters differ significantly (P< 0.05)
8S. H. S. DANANJAYA ET AL.
Analysis of the effect of annatto dye on pigmentation of goldfish skin and
caudal fin
The results of pigmentation in goldfish skin and caudal fin were significant
(p< .05). Initial carotenoid value of 16.02 mg/kg in fish skin for all the treatments
was increased in an ascending rate of accumulation in the first 20 days. However,
the rate of carotenoid accumulation was observed to be at a descending rate at day
60. The total carotenoid level was increased with time in all treatments
(Figure 2A). Thereby, highest carotenoid amount of 76.95 mg/kg was given by
fish fed with the diet of 250 mg AD/kg diet at the 60 days of sampling (Table 2)
(p< .05). The range of total carotenoid levels at each sampling time (i.e. 20d, 40d,
60d) were 16.45–53.10; 17.08–17.65; 17.24 −76.95 mg/kg, respectively.
Furthermore, the highest significant total carotenoid amount in the skin at each
sampling period was observed in fish fed with 250 mg AD/kg, followed by
a commercial diet and 200 mg AD/kg diet.
A similar pattern of total carotenoid level in the caudal fin was observed
during the experimental period. The initial carotenoid level was 1.6 times
higher than that of the skin (26.68 mg/kg). The highest rate of carotenoid
accumulation during the first 20 days was observed and became constant
at day 60 (Figure 2A). Mean total carotenoid amount range of fish fin during
20d, 40d, and 60d were 26.66 −75.55; 28.47–87.65 and 27.23–95.11 mg/kg,
respectively. The diets of 250, 0.0 (commercial diet) and 200 mg AD/kg diets
gave the highest significant mean total carotenoid amounts in the caudal fin
(p< .05) in descending order at each sampling period. The lowest carotenoid
amount in both skin and fin at each sampling period was observed in fish fed
with the control diet.
The relationship between the levels of AD and pigmentation in skin and fin
was significantly (P<.05)correlated(Figure 3 A &B). A regression analysis
results on skin and fin carotenoid amount with the level of dietary AD at 60 days
Figure 2. Effect of AD supplemented diets on total carotenoid amount in goldfish during the
experimental period. a) skin and b) caudal fin. Value is a means ± S.D (n = 3).
JOURNAL OF APPLIED AQUACULTURE 9
have nonlinear relation. The coefficient of correlation (R
2
) values for skin and fin
were 0.995, and 0.997, respectively.
Analysis of external skin color of goldfish
An effect of dietary AD on the body color was investigated using CIElab
coordinating criteria. Average values for L* (lightness), a* (redness), b*(intensity
of yellowness) and H° (hue) were shown in Table 3. According to the results only
H°, a* and L* values of the fish skin were significant (p< .05) while b* values
were not significant among treatments (p> .05). Feeding of the control diet
(a)
(b)
Carotenoid amount (mg/ kg)
Level of annatto dye (mg/kg) in diets
y = -0.0007x2+ 0.4455x + 28.22
R2= 99.69%
Level of annatto dye (mg/kg) in diets
y = -0.0004x2+ 0.3256x + 18.675
R2 = 98.85%
Carotenoid amount (mg/ kg)
Figure 3. The relationship between total carotenoid content of goldfish and AD supplemented
diet at the end of the experiment period (60 days). a) skin and b) caudal fin.
10 S. H. S. DANANJAYA ET AL.
increased L*, H° compared to the other diets. However, the highest a* and b*
values of 12.53, 43.21, were observed in fish fed with diets included 50 and
250 mg AD/kg diet respectively.
The L* and a* and H° of fish skin were significantly affected by (p<.05)
higher levels of dietary AD while b* values were not significant (p>.05)with
both treatment and carotenoid amount in the skin. Furthermore, as shown in
Figure 4 (A) negative linear correlation was indicated by L*, b* and H°,
whereas, a positive correlation for a* with a carotenoid amount in fish skin
Table 3. Summary of color values of fish skin.
Color Parameter
Treatment L* a* b* H°
Control 67.59
a
±1.73 10.33
d
±1.18 11.70
a
±0.71 48.73
a
±3.44
50AD 59.37
b
±2.07 15.17
a
±1.76 12.53
a
±1.48 39.76
b
±6.32
100AD 50.69
c
±1.43 21.33
b
±0.74 12.01
a
±0.67 29.33
c
±0.54
150AD 45.04
d
±1.13 27.82
c
±1.68 12.23
a
±1.20 23.94
c
± 3.40
200AD 37.50
e
±1.59 35.57
e
±1.38 11.89
a
±0.85 18.46
d
±0.79
250AD 29.19
f
±1.86 43.21
h
±1.37 11.64
a
±0.66 15.05
d
±0.45
CCF 65.43
ag
±1.65 35.97
e
±2.02 11.69
a
±0.58 18.05
d
±1.09
L*: lightness, a*: redness, b*: yellowness, H°: hue, CCF: commercial color feed, AD: annatto dye, Value are
a means ± S.D. Means with different superscript letters within the same Colom differ significantly
(P< 0.05)
ab
cd
r = -0.608
p = 0.003
r = 0.964
p = 0.000
r = -0.126
p = 0.586 r = -0.947
p = 0.000
Figure 4. Comparisons between the carotenoid concentration in skin color parameters. A)
lightness (L*); B) redness (a*); C) yellowness (b*) and D) hue (H°) in goldfish fed on a different AD
supplemented diet for 60 days.
JOURNAL OF APPLIED AQUACULTURE 11
was obtained. The correlation coefficient (R
2
) for L*, b*, H° and a* were
−0.608, −0.126, −0.947 and 0.964, respectively.
Discussion
In the present study, the effect of AD formulated diets on growth, skin and fin
pigmentation in goldfish were investigated. The primary source of coloration of
fish skin is reported as carotenoid (Villar-Martinez et al. 2013). According to the
results of the present study, it was found that carotenoid levels in goldfish skin
and the caudal fin were increased by increasing the incorporating level of AD in
the fish diet (50–250 mg/kg diet). Dietary carotenoid plays a significant role in
the regulation of skin and muscle color in fish (Ezhil, Jeyanthi, and Narayanan
2008). Carotenoids have a positive role in the intermediary metabolism that
could enhance nutrient utilization and ultimately results in improved growth of
fish (Villar-Martinez et al. 2013). However, the inclusion of AD (50–250 mg/kg
diet) in fish diets was not significantly affected final body weight, weight gain and
specific growth rate of goldfish in the present study. In similar study, Zhengyu
et al. (2006) reported that astaxanthin in goldfish diets had no effect on the weight
gain and survival rate. In contrast, a similar level of annatto seed meal in the
formulated diets has shown significant growth rate (SGR) of rainbow trout
(Safari and Atash 2015). Furthermore, their findings indicated a positive correla-
tion between the levels of annatto seed meal and the final body weight and SGR,
while a negative correlation between FCR and blood carotenoid. Similarly, the
negative correlation with FCR was observed when the AD level was increased in
the diet of goldfish. The lowest FCR was observed in fish fed with diets containing
250 mg/kg diet. The lower FCR may be due to the pre-biotic effect of annatto seed
extraction and ability to improve the fish gut bacterial colonization that ulti-
mately increases the feed utilization (Safari and Atash 2015).
Most importantly, the effectiveness of carotenoids as a source of pigmentation
is species dependent (Ha et al. 1993). Goldfish have the ability to use different
feed materials effectively as sources of enhancing pigmentation. Especially they
can utilize lutein and zeaxanthin from alfalfa (Yanar et al. 2008), spirulina
(Kiriratnikom, Zaau, and Suwanpugdee 2005), A. maxima (Gouveia et al.
2003), red yeast, X.dendrorhous (Xu et al. 2006), and Marigold (Villar-
Martinez et al. 2013). In the present study, dietary AD played the most impor-
tant role in increasing pigmentation in skin and fin of goldfish with its high
concentration. According to the findings of Safari and Atash (2015), bixin and
nor-bixin in annatto seed meal have been identified as the main carotenoid
sources responsible for the pigmentation. Furthermore, bixin is categorized as
oil soluble, whereas nor-bixin as water soluble carotenoid. According to
Giridhar, Venugopalan, and Parimalan (2014), total carotenoids availability in
annatto seed meal is about 4.5–5.5% of seed weight. Moreover, his finding
revealed that annatto seed as an effective natural carotenoid source for
12 S. H. S. DANANJAYA ET AL.
pigmentation of rainbow trout. Carotenoids metabolism and pigmentation of
ornamental fish depends on the source of feed, availability and other parameters
such as carotenoids extraction methods, body size and weight, life cycle, envir-
onment, proximate composition of the feed ingredients and especially the lipid
content that should be carefully considered when presenting the results of fish
coloration and growth (Safari and Atash 2015).
The pigmentation rate of goldfish varied during different time periods
depending upon whether they ingest the diets with astaxanthin or not. In this
regard, goldfish can absorb un-esterified axtaxanthin and transform into an
esterified type for deposition (Xu et al. 2006). Similarly, the inclusion of 10% of
S. platensis as a natural source that contains zeaxanthin resulted in a high
carotenoid deposition in tissues (Mahdi, Amirkolaie, and Yeganeh 2013).
These results support for evidence that physical and chemical properties of the
fish diet play a crucial role in fish body metabolism and growth. Similarly, the
outcomes of some previous experiments showed that artificial diets result in
different skin coloration in goldfish (Yeşilayer et al. 2012). Different concentra-
tions of the AD used in the feed resulted in considerably higher differences in
color of skin and caudal fin. Interestingly Xu et al. (2006) showed that incor-
poration of different dosage of astaxanthin (60–80 mg/kg) by supplementing
with red yeast on goldfish diets significantly increased the carotenoid concen-
tration. Villar-Martinez et al. (2013) stated that tiger barbs (Barbus tetrazona)
fed diets containing carotenoid from shrimp meal, marigold petal meal, and
annatto seed extract had successfully increased the skin color. The correlation
between body coloration and dose of dietary carotenoids is supported by similar
findings by Sun et al. (2012) using dietary carotenoids of xanthophyll in Japanese
ornamental carp.
The concentration of carotenoids in fish skin and the caudal fin has shown an
increasing pattern with a decreasing rate of deposition. The regression analysis
showed 76.95 and 95.11 mg/kg as the highest levels of carotenoids in goldfish
skin and caudal fin, respectively at 250 mg AD/kg diet. A similar pattern of
increasing carotenoid levels in the skin was observed by Yanar et al. (2008), in
which goldfish diets contained dietary alfalfa for 60 days. Even though saturation
level for goldfish has not previously been published for AD supplemented diets,
Yanar et al. (2008) reported the saturation level of carotenoid in gold fish skin
was 102 mg/kg when it was fed with alfalfa 26–31% in the diet. Moreover,
Rezende et al. (2012) have shown four levels (125, 250, 375 and 500 mg of bixin/
kg of diet) of red carotenoid extracted from annatto plant (B. orellana) included
in diets granted the greatest intensity of red –yellowish color in pearl Gouramy
(Trichogaster leeri) skin. These findings support the assumption that the addi-
tional carotenoid accumulationmay be possible with feeding the fish beyond the
current AD level of 250 mg/kg diet.
When evaluating a goldfish, body conformation and fin placement are the
most important criteria. In addition, color variation and brightness are the
JOURNAL OF APPLIED AQUACULTURE 13
essences of goldfish. The scale type strongly influences the intensity and stability
of color. Each scale consists of different integumentary cells called cromato-
phores. The orange/red color is produced by the combination effect of red
pigmented cells, (erythrophores), yellow pigmented cells (xanthophores), and
white cells (leucophores) in the scales and skin (Kottler et al. 2014). Diet and
environment may influence the color saturation, but seldom changes the hue
(Gouveia and Rema 2005). Linear correlations are shown in redness (a*) light-
ness (L*) and yellowness (b*) and hue (Ho) in the current study is supported by
the findings of Mahdi, Amirkolaie, and Yeganeh (2013) and Wathne et al. (1998).
The higher survival rate of goldfish was observed in AD containing treat-
ments during the experiment, compared to that of the control one. Reasons for
this observation may be due to the identified influence of carotenoids on fish
immunity (Miki 1991) and also the antibacterial and antifungal effect of annatto
seed extraction (Akshatha and Parvatam 2012). Furthermore, carotenoid intake
affects both humoral and cell mediated immunity in Atlantic salmon, ultimately
producing higher survival rates (Paripatananont et al. 1999). Nevertheless,
a previous finding (Yeşilayer et al. 2012) showed that the higher survival rates
of goldfish fed diets containing gammarus and canthaxanthin compared to
diets supplemented with astaxanthin and oleoresin.
According to the obtained results, it can be concluded that the goldfish can
utilize annatto dye effectively. The inclusion of 200–250 mgAD/kg diet in
goldfish diets showed the most effective level in pigmentation and higher
survival rate compared to the control diet. Therefore, annatto dye can be
suggested as an effective alternative to natural carotenoid source for goldfish
color enhancement.
Acknowledgments
This work was supported by research fund of Chungnam National University (2017).
Disclosure Statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by research fund of Chungnam National University (2017).
ORCID
Mahanama De Zoysa http://orcid.org/0000-0003-2814-659X
14 S. H. S. DANANJAYA ET AL.
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