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The effect of dietary pigments on the coloration of Japanese ornamental carp (koi, Cyprinus carpio L.)

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This study evaluated the effects of dietary supplementation with four pigment sources on the coloration of Japanese ornamental carp (Showa koi) (Cyprinus carpio L.). Showa koi (which are colored black with scattered red patches and white spots) initially weighing 18.04 ± 0.92 g were fed five dietary treatments in triplicate: a control diet with no added pigments, a diet with 1.5 g kg− 1 Carophyll® red (synthetic, CR diet), a diet with 200 g kg− 1 wet weight of a photosynthetic bacterium (Rhodopseudanonas palustris, PB diet), a diet with 200 g kg− 1 wet weight of effective microorganisms (EM diet) and a diet with 75 g kg− 1 dry weight feed-grade Spirulina platensis (SP diet). After a 99 day feeding trial, the fish's color was evaluated with a colorimeter to measure the chroma, lightness, redness and yellowness of different color zones. The carotenoid and xanthophyll concentration in the skin and the scales of the fish's red, black and white zones were tested. S. platensis significantly increased the growth and feeding efficiency of koi (P < 0.05). S. platensis and Carophyll® red significantly improved the chroma of the black zone, the redness and the chroma of the red zone, and the lightness of the white zone (P < 0.05). S. platensis and Carophyll® red increased the carotenoid content of the black and red scales and the xanthophyll content of the black and red skin and scales (P < 0.05). The results indicate that Showa koi pigmentation can be modified by supplementing the diet with 1.5 g kg− 1 Carophyll® red or 75.0 g kg− 1S. platensis. Dietary R. palustris, at levels up to 1.0 g dry matter kg− 1 of diet, does not appear to affect the coloration of Showa koi. Furthermore, body coloration was generally correlated with the dose of dietary carotenoids and xanthophylls, and carotenoids had a deeper and greater influence than xanthophylls.
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The effect of dietary pigments on the coloration of Japanese ornamental carp
(koi, Cyprinus carpio L.)
Xiangjun Sun
a,1
, Yu Chang
c,1
, Yuantu Ye
b
, Zhihong Ma
a
, Yongjun Liang
a
, Tieliang Li
a
, Na Jiang
a
,
Wei Xing
a
, Lin Luo
a,
a
Beijing Fisheries Research Institute, Beijing 100068, China
b
School of Basic Medical and Biological Sciences, Soochow University, Suzhou 215123, China
c
Beijing University of Technology, Beijing 100124, China
abstractarticle info
Article history:
Received 7 December 2011
Received in revised form 10 February 2012
Accepted 15 February 2012
Available online 25 February 2012
Keywords:
Koi (Cyprinus carpio L.)
Spirulina platensis
Rhodopseudanonas palustris
Carophyll® red
Coloring
Pigmentation
This study evaluated the effects of dietary supplementation with four pigment sources on the coloration of
Japanese ornamental carp (Showa koi) (Cyprinus carpio L.). Showa koi (which are colored black with scat-
tered red patches and white spots) initially weighing 18.04±0.92 g were fed ve dietary treatments in
triplicate: a control diet with no added pigments, a diet with 1.5 g kg
1
Carophyll® red (synthetic, CR
diet), a diet with 200 g kg
1
wet weight of a photosynthetic bacterium (Rhodopseudanonas palustris,PB
diet), a diet with 200 g kg
1
wet weight of effective microorganisms (EM diet) and a diet with
75 g kg
1
dry weight feed-grade Spirulina platensis (SP diet). After a 99 day feeding trial, the sh's color
was evaluated with a colorimeter to measure the chroma, lightness, redness and yellowness of different
color zones. The carotenoid and xanthophyll concentration in the skin and the scales of the sh's red,
black and white zones were tested. S. platensis signicantly increased the growth and feeding efciency
of koi (P b0.05). S. platensis and Carophyll® red signicantly improved the chroma of the black zone,
the redness and the chroma of the red zone, and the lightness of the white zone (Pb0.05). S. platensis
and Carophyll® red increased the carotenoid content of the black and red scales and the xanthophyll con-
tent of the black and red skin and scales (P b0.05). The results indicate that Showa koi pigmentation can
be modied by supplementing the diet with 1.5 g kg
1
Carophyll® red or 75.0 g kg
1
S. platensis. Dietary
R. palustris, at levels up to 1.0 g dry matter kg
1
of diet, does not appear to affect the coloration of Showa
koi. Furthermore, body coloration was generally correlated with the dose of dietary carotenoids and xan-
thophylls, and carotenoids had a deeper and greater inuence than xanthophylls.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Ornamental carp (koi) are characterized by a wide diversity of
colors and color patterns (Gomelsky et al., 1996). More than 100 dif-
ferent types of coloration have been developed (Kuroki, 1981;
Tamadachi, 1990) for these sh, which are valued as pets. Color is
one of the most important quality criteria dictating the market
value of koi. Because sh cannot synthesize carotenoids de novo,
they rely on a dietary supply of these pigments to achieve their natu-
ral skin pigmentation (Gouveia et al., 2003; Paripatananont et al.,
1999). Fish use carotenoids, one of the most important groups of nat-
ural pigments, for pigmentation of their skin and esh. Carotenoids
commonly occurring in freshwater food sources include β-carotene,
lutein, taraxanthin, astaxanthin, tunaxanthin, α-, β-doradexanthins,
and zeaxanthin (NRC (National Research Council), 1983, 1993).
Various synthetic pigments (β-carotene, canthaxanthin, zeaxan-
thin, and astaxanthin) and natural sources (yeast, bacteria, algae,
higher plants, and crustacean meal) have been used as dietary sup-
plements to enhance the pigmentation of sh and crustaceans
(Kalinowski et al., 2005; Shahidi et al., 1998). Natural sources of ca-
rotenoids are usually composed of several carotenoids in various
forms, and these sources vary in their digestibility, making their pig-
mentation efciency complicated to interpret. In contrast, studies can
clearly determine the pigmentation efciency of synthetic caroten-
oids (which are always a single carotenoid). Given the high costs of
synthetic pigments, however, efforts have been made to evaluate
the potential of natural compounds. Some studies have shown that
Chlorella vulgaris is as efcient as synthetic pigments in the pigmenta-
tion of rainbow trout Oncorhynchus mykiss (Gouveia et al., 1996b),
gilthead seabream Sparus aurata (Gouveia et al., 2002), koi Cyprinus
carpio and goldsh Carassius auratus (Gouveia et al., 2003). Pigments
obtained from red yeast Phafa rhodozyma (Bon et al., 1997), the ma-
rine bacteria Agrobacterium aurantiacum (Yokoyama and Miki, 1995),
Chlorococcum sp. (Zhang et al., 1997), the green algae Haematococcus
Aquaculture 342-343 (2012) 6268
Corresponding author. Tel./fax: +86 10 67588781.
E-mail address: luo_lin666@sina.com (L. Luo).
1
The rst two authors contributed equally to this work.
0044-8486/$ see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2012.02.019
Contents lists available at SciVerse ScienceDirect
Aquaculture
journal homepage: www.elsevier.com/locate/aqua-online
pluvialis (Harker et al., 1996; Yuan and Chen, 2000), Chlorella zon-
giensis (Bar et al., 1995) and C. vulgaris (Gouveia et al., 1996a) were
used as sources of dietary carotenoids.
In practice, we nd out that some ornamental sh culturists used
green algae Spirulina platensis to improve body color of sh such as
goldsh (Carassius auratus auratus), ornamental Cichlid (Cichlidae
sp.) and so on. On the other hand, we found that red color of kohaku
koi had been signicantly improved by photosynthetic bacteria (Rho-
dopseudanonas palustris) when we splashed this bacteria liquid into
koi cultural ponds to improved the water quality of (Sun et al.,
2010). Based on the practice and above researches, the present
study evaluated the potential of these natural microorganisms, in-
cluding S. platensis, single R. palustris, and effective microorganisms
including R. palustris, as sources of dietary pigments for coloring the
skin of Showa koi (C. carpio L.). These sources were compared to a
positive control of synthetic astaxanthin (Carophyll® red) and a neg-
ative control, a diet with no pigment added.
2. Materials and methods
2.1. Experimental diets
A basal diet containing 300 g kg
1
crude protein and 50 g kg
1
crude fat without added pigments was used as a control diet. Using
this basal mixture, four experimental diets were formulated by add-
ing pigments in accordance with recommended doses: diet CR con-
taining 1.5 g kg
1
Carophyll® red (DSM Nutritional Products Ltd.),
diet PB containing 200 g kg
1
(wet weight) of the photosynthetic
bacteria R. palustris (University of Science and Technology of Beijing,
China), diet EM containing 200 g kg
1
(wet weight) of effective mi-
croorganisms, primarily photosynthetic bacteria (Shanghai Sanzhi
Biotech. Co., Ltd., China), and diet SP containing 75 g kg
1
of the
algae S. platensis (Beijing Sunpu Biochemical and Technology Co.,
Ltd., China). The ingredients and biochemical composition of the
diets are shown in Table 1.
The pigments in the CR and SP diets were mixed with the basal
diet completely and dry-pelleted using a steamless pelleting machine
tted with a 2.5-mm diameter screen. During the pelleting process,
the temperature varied from 40 to 50 °C. The pigments in the PB
and EM were evenly spray-coated onto the surface of the basal pellets
and air-dried. To preserve the pigments, all diets were stored at 4 °C
and were protected from light throughout the experiment.
2.2. Fish and rearing conditions
The homogeneous Showa koi carp were obtained from a commer-
cial sh farm in Beijing, China. Before the beginning of the experi-
ment, sh were fed with the control diet for 2 weeks to acclimatize
them to the laboratory culturing system. At the beginning of the
test, the sh, with an initial mean body weight of 18.04±0.92 g,
were randomly divided into fteen groups of 20 sh each (5 treat-
ments in triplicate). Each group of sh was stocked in a 0.21 m
3
in-
door tank with a freshwater input of 0.5 l min
1
. The oxygen level
was more than 7 mg l
1
. The sh were subjected to a natural photo-
period, and the average temperature of the water was 23 °C during
the 99 day experimental period.
The sh were fed the appropriate experimental pellet correspond-
ing to 35% of their body weight three times per day (8:00, 13:00, and
18:00). The amount fed was corrected every month after the sh
were individually weighed. Mortality was recorded daily. During the
experiment, sh in each tank were batch-weighed every 2 weeks to
adjust the amount of feed and clean the tank. At initiation and at
the end of feeding, after 1 day starvation, three sh were randomly
selected from each tank, killed by means of a sharp blow to the
head and batch weighed. At the end of the trial, three sh from
each tank were sampled randomly for color analysis. Three other
sh in each treatment were sacriced by a blow to the head, and sam-
ples of the black, red and white skin and scales of each sh were ex-
cised, immediately frozen in liquid nitrogen, and kept at 50 °C until
measurement of their carotenoid and xanthophyll contents.
2.3. Biochemical analysis
2.3.1. Feed analysis
Biochemical analysis of feed was conducted in triplicate according
to the AOAC (1995). Briey, the crude protein (N × 6.25) content was
determined by the Kjeldahl method after acid digestion using an Auto
Kjeldahl System (2100-Auto-analyzer, Foss, Hillerød, Denmark). The
crude fat content was determined by the ether extraction method
using a Soxtec System HT (Soxtec System HT6, Foss, Hillerød, Den-
mark). The moisture content was determined by oven-drying at
105 °C for 24 h. The ash content was determined by combustion at
550 °C for 12 h.
2.3.2. Total carotenoid and xanthophyll analysis
The total carotenoid and xanthophyll content from feed, skin and
scale samples were measured according to the methods of the AOAC
(1995) with some modications. At rst, the black, red or white
scales at sample area were pulled out by forceps separately, and
skin under the scale was picked from the meat. Individual feed(1 g,
dry weight), skin or scale samples (1 g, wet weight) were placed
into a brown 25 ml Erlenmeyer ask and mixed with 7.5 ml of n-
hexane/acetone/ethanol/toluene (10:7:6:7 v/v), and the ask was
rmly closed and shaken vigorously by hand for 1 min. A total of
1 ml 40% KOH methanol solution was then added. The ask was
Table 1
Ingredients and proximate composition of the experimental diets (g kg
1
).
Ingredients Control diet CR diet PB diet EM diet SP diet
Fishmeal 150.0 150.0 150.0 150.0 150.0
Soybean meal 170.0 170.0 170.0 170.0 170.0
Soybean, full fat 80.0 80.0 80.0 80.0 20.0
Solvent-extracted
cottonseed meal
110.0 110.0 110.0 110.0 80
Wheat shorts 250.0 250.0 250.0 250.0 240.0
Wheat our 150.0 148.5 150.0 150.0 180.0
Attapulgite meal 40.0 40.0 40.0 40.0 40.0
Vitamin/minerals premix
a
10.0 10.0 10.0 10.0 10.0
Soybean oil 20.0 20.0 20.0 20.0 15.0
Ca(H
2
PO
4
)
2
·H
2
O 20.0 20.0 20.0 20.0 20.0
Carophyll® red
b
1.5
Photosynthetic bacterium
(wet weight)
c
200.0
Effective microorganism
(wet weight)
d
200.0
Spirulina platensis
e
75.0
Proximate composition (%) Control
diet
CR diet PB diet EM diet SP diet
Dry matter 88.3 88.3 88.3 88.3 87.6
Crude protein 30.7 30.7 30.7 30.7 30.2
Crude fat 5.3 5.3 5.3 5.3 5.3
Ash 10.4 10.4 10.4 10.4 9.55
Total carotenoid (mg kg
1
) 4.72 23.27 7.97 6.83 12.07
Xanthophyll (mg kg
1
) 2.47 15.84 2.71 3.01 5.19
a
Vitamin premix (mg kg
1
): thiamine-HCl, 8.0; riboavin, 8.0; niacin mix, 100.0;
pyridoxine-HCl, 20.0; cyanocobalamine, 0.1; pantothenate, 20.0; biotin, 1.0; inositol,
100.0; folic acid, 5.0; ascorbic acid, 250.0; Vitamin A, 20.0; Vitamin D, 8.0; Vitamin E,
150.0; Vitamin K, 10.0; BHT,10.0; α-cellulose, 1289.9. Mineral premix (mg kg
1
):
MgSO
4
·7H
2
O, 300.0; FeSO
4
·7H
2
O, 180.0; ZnSO
4
·7H
2
O, 120.0; MnSO
4
·7H
2
O, 35.0; KI,
0.65; Na
2
SeO
3,
0.5; CoCl·6H
2
O (1%), 7.0; CuSO
4
·5H
2
O, 5.0; zeolite, 7351.85.
b
Pigments: 10% canthaxanthin.
c
Pigments: 1.5 g dry PB in per kg PB liquid.
d
Pigments: 0.7 g dry PB in per kg EM liquid.
e
Pigments: 1 g β-carotene and 2.6 g zeaxanthin per kg algae.
63X. Sun et al. / Aquaculture 342-343 (2012) 6268
heated in a 55.5 °C water bath for 20 min after being shaken for
1 min to mix the solution and sample. After cooling, 7.5 ml of n-
hexane was added and the ask contents were stirred for 1 min,
then a 10% sodium sulfate solution was added to bring the sample
up to 25 ml and shaken vigorously by hand for 1 min. After 1 h in
the dark, the saponied sample solution was separated chromato-
graphically. The separation was performed using a tandem-
installed ChromSpher 5 μmC
18
(100×3 mm I.D.) column with a
guard column of C
18
material (Chromsep guard column SS) preced-
ing the main column. A total of 10 ml saponifying sample solution
was injected into the column, and then an elution solution of n-
hexane/acetone (96/4, v/v) was slowly added into the column until
all of the carotenoids were eluted. The total carotenoid content
from the supernatant was measured using a spectrophotometer
(Shimadzu, UV-120-02) at 448 nm against a hexane (+BHT) blank,
using an E
1%,1 cm
of 2500.
Total carotenoid content mg kg1

¼AKVðÞ=EGðÞ
Here, A is the absorbency, K is dilution, V is the amount of super-
natant (ml), E is the absorbency index (2500) and G is the sample
weight (g).
After the carotenoids were eluted, the xanthophyll remained in
the column. A xanthophyll elution solution of n-hexane/acetone/
methanol (80:10:10, v/v) was slowly added into the column until
all of the xanthophylls were eluted. The total xanthophyll content
from the supernatant was measured by spectrophotometer (Shi-
madzu, Kyoto, Japan, UV-120-02) at 474 nm. Each sample was ana-
lyzed in triplicate.
Xanthophyll content mg kg1

¼A474 1000 fðÞ=263 bdðÞ
Here A
474
is absorbency, f (instrument error)= 0.561/observed
A
474
, b is the comparison tube length (cm), d (diluents index)=(the
sample weight(g)×saponication (ml))/(top phase value (ml)×the
last diluents (ml)).
2.3.3. Color analysis
At the end of the trial, three sh from each treatment were ran-
domly selected to evaluate their skin color. Because koi are ornamen-
tal sh, investigators need a simple, rapid and accurate way to
analyze color on living animals. In this study, skin color was assessed
with reectance spectroscopy with transformation into color param-
eters based on the tristimulus values, L*, a*, b* and dE, representing
lightness, redness, yellowness and chromatic aberration, respectively
(Skrede, 1987), using a portable Hanpu Chroma Meter HP-200
(Hanpu, Shanghai, China) calibrated with a white standard (the orig-
inal adjusted value of the white standard was L* = 97.40 ±0.01; a* =
0.10±0.01; b* = 1.92 ± 0.01). The measurements were performed
on the largest zone of black, red and white from each sh. L* and dE
were measured in the black color zones; L* and b* were measured
in white color zones; L*, a* and dE were measured in red color zones.
2.4. Statistics
All data were subjected to a one-way analysis of variance
(ANOVA) using the Statistica 8.0 software environment to test the ef-
fects of the experimental diets. Duncan's multiple range test and crit-
ical ranges were used to test differences among the individual means.
The differences were regarded as signicant when Pb0.05. All of the
results are expressed as the means±S.E.M. The slopes of the color pa-
rameters and scale and skin pigment responses to the diets were
compared after tting a linear regression model using Statistica 8.0.
Correlations were regarded as signicant when the correlation coef-
cient R> 0.5.
3. Results
3.1. Effects of experimental diet on the growth performance
The changes in growth during the experiment are shown in
Table 2. The weight gain and feed conversion ratio were signicantly
affected by dietary treatment. The sh in the SP diet group had a sig-
nicantly higher rate of weight gain (WGR) and specic growth rate
(SGR) than the other sh groups (Pb0.05). The food conversion
Table 2
Growth performance and feed utilization of koi fed experimental diets for 99 days
1
.
Diets Control diet CR diet PB diet EM diet SP diet
WGR
2
(%) 2.11±0.10
a
2.04±0.08
a
1.93±0.09
a
1.89±0.04
a
2.71±0.12
b
SGR
3
(%) 1.14±0.03
a
1.12±0.02
a
1.10±0.06
a
1.10±0.02
a
1.32±0.03
b
FCR
4
1.86±0.08
b
1.93±0.07
bc
2.02±0.10
cd
2.08±0.04
d
1.45±0.07
a
K factor
5
2.85±0.08 2.84 ± 0.06 2.83±0.13 2.87 ± 0.10 2.81 ± 0.41
VSI
6
(%) 5.63±0.56 6.39 ± 0.52 5.73± 0.43 6.16 ± 0.76 5.98 ± 0.26
HSI
7
(%) 1.56± 0.23 1.79 ± 0.16 1.42± 0.36 1.68± 0.30 1.66 ± 0.25
1
Values are expressed as the means ±S.E.M. Values in same row with different superscripts are signicantly different (P b0.05).
2
WGR (weight gain rate) = 100% × (Wf + Wd Wi)/Wi, where Wf is the total nal body weight (g), Wd is the total dead body weight (g), and Wi is the total initial body
weight (g).
3
SGR (Specic growth rate) = 100% × (ln Wfln Wi) / days.
4
FCR (feed conversion ratio)= total dry feed offered (g) / total wet weight gain (g).
5
K factor=100 × (L/W
3
), where L is sh length (cm) and W is sh weight (g).
6
VSI (Viscerosomatic index) = 100% × visceral weight (g)/sh weight (g).
7
HSI (Hepatosomatic index) = 100%× hepatopancreas weight (g)/sh weight (g).
Table 3
The concentration of total carotenoids and xanthophylls in scale of koi (mg kg
1
).
Items Control diet CR diet PB diet EM diet SP diet
Total
carotenoids
White scale 4.49±0.07 6.40 ±1.07 5.57± 0.30 6.77± 0.8 6.13±1.11
Black scale 3.66± 0.37
a
9.72±1.99
b
4.47±0.02
ab
3.24±1.62
a
4.45±0.14
ab
Red scale 6.75±0.66
ab
10.69±1.28
b
4.14±1.90
a
4.87±1.14
a
7.85±1.40
ab
Xanthophylls White scale 0.56±0.15 0.62 ±0.12 0.55± 0.10 0.77± 0.20 0.41 ±0.18
Black scale 0.27± 0.07
a
1.16±0.44
b
0.87±0.16
ab
0.61±0.08
ab
0.89±0.25
ab
Red scale 1.24±0.21
a
1.72±0.31
a
1.11±0.35
a
1.56±0.53
a
3.45±0.70
b
Values are expressed as the means ± S.E.M. Values in same row with different superscripts are signicantly different (P b0.05).
64 X. Sun et al. / Aquaculture 342-343 (2012) 6268
ratio (FCR) of the sh fed the SP diet was signicantly lower than that
of the other sh groups (P b0.05). The FCR of the control sh was sig-
nicantly lower than that of sh fed the PB diet or the EM diet
(Pb0.05). The FCR of the sh fed the CR diet was signicantly lower
than that of the sh fed the EM diet (P b0.05).
The K factor, VSI and HSI of the sh were unaffected by the exper-
imental diets (P> 0.05). No mortality was associated with experi-
mental treatments.
3.2. Effects of the experimental diets on total carotenoid and xanthophyll
concentrations of koi skin and scales
The amounts of total carotenoid and xanthophyll found in koi skin
are shown in Table 3. No signicant differences were found in the
total carotenoid content of white scales among all of the experiment
diet groups (P > 0.05). The sh on the CR diet had a signicantly
higher total carotenoid content in their black scales compared with
the control and EM diets and a higher content in their red scales com-
pared with the PB and EM diets (P b0.05).
The xanthophyll concentration of the white scales did not differ
among any of the diets (P> 0.05). All pigments increased the xantho-
phyll content in black scales, and there were signicant differences
between the CR and the control diets (Pb0.05). The sh fed the SP
diet had markedly higher xanthophyll content in their red scales
than the sh fed other diets (P b0.05).
The relationship between dietary pigments and scale pigments
is shown in Table 4. The level of black scale pigments correlated
well with dietary pigment doses, with correlation coefcients (R
values) of 0.96 and 0.73 for total carotenoids and xanthophylls, re-
spectively. The level of total carotenoids in red scales was correlat-
ed with the total dietary carotenoid dose (R = 0.85). A regression
analysis of the scale pigment level revealed that black and red
scale deposition was linearly related to the total carotenoid levels
in the feed, with R
2
values of 0.93 and 0.72 for black and red
scales, respectively. An increase in the total feed carotenoids
resulted in signicantly elevated levels of carotenoids in the
black scales (P b0.01).
Table 5 shows the total carotenoid and xanthophyll content in koi
skin. The CR and SP diet groups had signicantly lower total caroten-
oid content in their white skin than the sh in the PB, EM and control
diet groups (P b0.05). Fish fed the EM diet had markedly higher total
carotenoid content in their black skin than the other diet groups did
(Pb0.05). Fish fed the PB diet had signicantly higher total carotenoid
content in their red skin than that of sh fed the CR or control diets
(Pb0.05). There were no signicant differences in the xanthophyll
content of the white skin among all diets (P > 0.05). Fish fed the SP
diet had signicantly higher xanthophylls in their black skin than
the sh fed the PB or EM diets (P b0.05). Fish fed the CR and SP
diets had markedly higher xanthophyll content derived from red
skin than the sh fed the PB or EM diets (P b0.05).
The relationship between feed pigments and skin pigments is
shown in Table 6. The total carotenoid level in white skin was signif-
icantly correlated with dietary carotenoid dose (R = 0.92). A regres-
sion analysis showed that carotenoid levels in the white skin of sh
were linearly related to the feed carotenoid level (R
2
=0.85). An in-
crease in the total amount of carotenoids in the feed resulted in a sig-
nicant decline of carotenoids in the white skin (P b0.05). Red skin-
derived xanthophyll levels were correlated with the dietary dose of
xanthophylls (R=0.87). A regression analysis showed that the xan-
thophyll levels in red skin were linearly related to the xanthophyll
level in the feed (R
2
=0.75).
3.3. Effects of experimental diet on the body color
The intensity of color, red and yellow tonalities, and chromatic
aberration for the chromatic varieties of Showa koi are shown in
Table 7. All of the pigments increased the lightness (L*) of the
black zones, and there was a signicant difference between the SP
and the control diet groups (P b0.05). The black and white chro-
matic aberrations (dE of the black zones) of the CR and PB diet
groups were signicantly higher than those of the EM, SP and con-
trol diet groups (Pb0.05).
The CR diet groups showed a signicantly lighter red zone than
that of groups fed the control, PB or SP diets (P b0.05). The group
fed the control diet showed weak red tonality (a*) and dE of the red
Table 4
The correlation of sh scale pigments with dietary pigments.
Items Correlation Regression models
White scale carotenoid
by feed carotenoid
R= 0.48; Pb0.5
Black scale carotenoid
by feed carotenoid
R= 0.96; Pb0.01 y =1.34 +0.34x; R
2
=0.93
Red scale carotenoid by
feed carotenoid
R= 0.85; Pb0.1 y= 3.57+ 0.30x; R
2
=0.72
White scale xanthophyll
by feed xanthophyll
R= 0.04; P> 0.5
Black scale xanthophyll
by feed xanthophyll
R= 0.73; Pb0.5 y= 0.51+ 0.04x; R
2
=0.54
Red scale xanthophyll
by feed xanthophyll
R= 0.13; P> 0.5
Table 5
The concentration of carotenoids and xanthophylls in skin of koi (mg kg
1
).
Items Control diet CR diet PB diet EM diet SP diet
Total
carotenoids
White skin 7.54± 1.07
b
2.78±0.23
a
6.35±0.25
b
7.44±0.41
b
3.89±0.28
a
Black skin 2.15 ± 0.20
a
2.20±0.33
a
1.93±0.44
a
9.81±2.59
b
2.38±0.87
a
Red skin 2.44 ± 0.39
a
2.61±0.30
a
4.25±0.54
b
3.58±0.44
ab
3.42±0.35
ab
Xanthophylls White skin 0.48± 0.06 0.67 ± 0.15 0.46 ± 0.15 0.68 ± 0.12 0.85 ± 0.21
Black skin 0.54 ± 0.15
ab
0.65±0.02
ab
0.27±0.05
a
0.44±0.10
a
0.89±0.03
b
Red skin 1.05 ± 0.27
ab
2.42±0.77
b
0.72±0.07
a
0.76±0.14
a
1.92±0.56
b
Values are expressed as the means ± S.E.M. Values in same row with different superscripts are signicantly different (P b0.05).
Table 6
The relationship between sh skin pigments and feed pigments.
Items Correlation Regression models
White skin carotenoid
by feed carotenoid
R=0.92; P b0.05 y= 8.55 0.27x; R
2
=0.85
Black skin carotenoid
by feed carotenoid
R=0.30; P >0.5
Red skin carotenoid
by feed carotenoid
R=0.32; P >0.5
White skin xanthophyll
by feed xanthophyll
R= 0.31; P> 0.5
Black skin xanthophyll
by feed xanthophyll
R= 0.37; P> 0.5
Red skin xanthophyll
by feed xanthophyll
R= 0.87; Pb0.1 y= 0.70+ 0.12x; R
2
=0.75
65X. Sun et al. / Aquaculture 342-343 (2012) 6268
zone (red and white chromatic aberration), which differed signi-
cantly from values found for groups fed the CR or SP diets (P b0.05).
In the white zones, the CR, EM and SP diet groups had signicantly
stronger L* than that of the PB or the control diet groups (Pb0.05). All
of the pigments increased the yellow hue (b*) of the white zones, and
there were signicant differences between the SP and the control diet
groups (Pb0.05).
The relationship between dietary pigments and body color pa-
rameters is shown in Table 8. A regression analysis revealed that
the dE of the black zones and the, Land dE of the red zones
were linearly related to the feed carotenoid levels, with R
2
values
of 0.662, 0.671 and 0.802, respectively. The Land dE of the red
zones were linearly related to the level of xanthophylls in the
feed, with R
2
values of 0.792 and 0.726, respectively. An increase
in the total carotenoid levels in the feed resulted in a signicant in-
crease in the dE of the red zones (P b0.05); increasing the xantho-
phyll levels in the feed level resulted in a signicant increase in the
Lof the red zones (P b0.05).
4. Discussion
In this study, the growth and feed utilization parameters were not
markedly improved by any pigments except S. platensis (P b0.05). S.
platensis has been identied as a potential protein source for animal
feed owing to its high protein content and the presence of essential
amino acids, vitamins and minerals. In addition, this type of microal-
gae has been reported to have no cell wall, which results in improved
digestion and absorption (Becker and Venkataraman, 1984). Im-
provement in the growth of sh by the dietary inclusion of Spirulina
has been reported earlier in a number of studies (Mustafa et al.,
1994; Nakazoe et al., 1986). Some studies have revealed that S. pla-
tensis could be used as the sole source of protein in common carp
and catla (Catla catla) diets (Nandeesha et al., 1998, 2001). In this
trial, S. platensis replaced 75% full-fat soybean and 27% solvent-
extracted cottonseed meal to keep the diet isonitrogenous. If S. pla-
tensis replaced other plant proteins, it could improve the growth of
the sh in this trial and replace shmeal. These results are also in
agreement with detailed investigations on the utilization of microal-
gae as a feed for sh in Israel, which found that sh grew better on
algae-enriched diets than on any conventional sh feed (Sandbank
and Hepher, 1978).
In colorimetry and color theory, hue is one of the main proper-
ties of a color, dened technically (in the CIECAM02 model), as
the degree to which a stimulus can be described as similar to or
different from stimuli that are described as red, green, blue, and
yellow. Chroma is the colorfulness relative to the brightness of
another color that appears white under similar viewing conditions.
Lightness is a property of a color, or a dimension of a color space,
that reects the subjective brightness perception of a color to
humans along a lightnessdarkness axis. With three attributes
colorfulness (or chroma or saturation), lightness (or brightness),
and hueany color can be described (http://en.wikipedia.org/
wiki/Colorfulness). The black, red and white zones of koi were
tested separately in this trial. Lightness (L*) and chroma (dE)
were the main indexes for the black zones. Higher dE of the
black zones correlated with better black zones. Meanwhile, a
higher L* of the black zone indicates that the black color is
brighter. The sh fed the SP diet had the highest L* and dE of
the black zones, followed by the sh fed the CR diet, which had
signicantly higher dE. A higher level of red hue (a*) indicates
that the red zones are more similar to red. Higher a*, L* and dE in-
dicated signicant improvement in the coloration of the sh fed
the CR and SP diets. Less yellowness (b*) means that white
zones is more pure. A higher yellow hue in the white zones of
the sh fed the experimental diets indicates that these zones
could become more variegated with increased amounts of dietary
pigments. The reason might be that Showa koi is colored black
with scattered red and white patches, and the grounding color of
the white patches is black. The white zones would not be snow-
white when the black grounding became darker and brighter.
Also for this reason, those sh fed SP and CR diets had a high yel-
lowness morphologically when the diets reduced carotenoids con-
tent in the white skin. Overall, the CR and SP diets yielded better
coloring for the Showa koi in this trial.
Carotenoids can be broadly classied into two classes, carotenes
(which are purely hydrocarbons and contain no oxygen) and xan-
thophylls (which contain hydroxyl groups or pairs of hydrogen
atoms that are substituted by oxygen atoms,http://en.wikipedia.
org/wiki/Xanthophylls). The total carotenoid and xanthophyll con-
centrations in the diets were measured to analyze the relationship
between dietary pigment content and sh body color. The feed pig-
ment doses targeted in this study were selected to be in range with
doses commonly employed in the market. Thus, the carotenoid and
xanthophyll content in the experimental diets were not equal
Table 8
The relationship of color parameters and feed pigments.
Items Correlation Regression models
L* of black zone by feed carotenoid R= 0.13; P > 0.5
dE of black zone by feed carotenoid R =0.81; Pb0.1 y = 11.78 + 1.83x;
R
2
=0.662
L* of red zone by feed carotenoid R=0.82; Pb0.1 y = 50.06 +0.82x;
R
2
=0.671
a* of red zone by feed carotenoid R= 0.68; Pb0.5 y = 15.51 +0.97x;
R
2
=0.471
dE of red zone by feed carotenoid R= 0.90; P b0.05 y =14.32+ 1.44x;
R
2
=0.802
L* of white zone by feed carotenoid R = 0.46; Pb0.5
b* of white zone by feed carotenoid R= 0.14; P> 0.5
L* of black zone by feed xanthophyll R =0.04; P > 0.5
dE of black zone by feed xanthophyll R=0.72; P b0.5 y= 19.65 + 2.09x;
R
2
=0.514
L* of red zone by feed xanthophyll R= 0.89; Pb0.05 y = 52.31 +1.16x;
R
2
=0.792
a* of red zone by feed xanthophyll R= 0.59; Pb0.5 y =19.83 + 1.07x;
R
2
=0.347
dE of red zone by feed xanthophyll R= 0.85; P b0.1 y = 19.73+ 1.77x;
R
2
=0.726
L* of white zone by feed xanthophyll R =0.42; Pb0.5
b* of white zone by feed xanthophyll R =0.02; P >0.5
Table 7
Color parameters (L*, a*, b* and dE) for koi fed experimental diets.
Items Control diet CR diet PB diet EM diet SP diet
L* of black zone 35.47± 6.50
a
44.77 ±5.82
ab
48.35±4.85
ab
52.13±2.17
ab
55.80 ±5.72
b
dE of black zone 19.40± 1.90
a
49.15 ±2.85
b
24.13±2.88
a
16.25±0.45
a
50.45 ±4.75
b
L* of red zone 54.75 ± 3.75
a
71.25 ±10.55
b
54.20±2.80
a
60.90±2.61
ab
54.17 ±2.46
a
a* of red zone 14.80±2.30
a
34.15 ±0.05
b
16.80±0.76
a
26.33±1.18
ab
38.40 ±8.46
b
dE of red zone 20.75 ±1.35
a
45.77 ±5.72
b
18.90±4.20
a
25.77±3.03
a
39.23 ±2.69
b
L* of white zone 74.55± 2.05
a
83.80 ±2.00
b
71.70±3.10
a
85.45±1.25
b
85.63 ±2.82
b
b* of white zone 4.05± 1.05
a
7.30±0.84
ab
11.40±1.80
ab
7.10±4.29
ab
13.37 ±0.70
b
Values are expressed as the means ± S.E.M. Values in same row with different superscripts are signicantly different (P b0.05).
66 X. Sun et al. / Aquaculture 342-343 (2012) 6268
because of the different pigment contents of the sources and the dif-
ferent amounts of the sources added to the diets. The control diet
contained some carotenoids and xanthophylls because it included
plant proteins and solvent-extracted cottonseed meal.
As showed in Table 4, the level of dietary pigments is correlat-
ed with the level of black scale pigments for carotenoids (P b0.01)
and xanthophyll (Pb0.5). Feeding sh with carotenoids signicant-
ly (R
2
=0.93) increases the level of carotenoid pigments in their
black scales. Therefore, dietary supplementation with carotenoids
primarily affects the level of carotenoids in black scales. In addi-
tion, the level of dietary carotenoid pigments correlates with the
level of white-skin carotenoid pigments because the P value is
less than 0.05. As more carotenoid was fed, the level of white-
skin carotenoid pigment decreased (R
2
=0.85). Dietary supplemen-
tation with xanthophylls correlates with the level of red-skin xan-
thophyll pigments (Pb0.1), but their linear relationship is not
signicant because the R
2
value is only 0.75. All of these results
suggested that the applied dietary carotenoids were correlated
with the carotenoids levels of the colored scales of koi, directly af-
fecting body coloration. The results based on Tables 4 and 6 also
suggested that scales might play more important role than skin
on showing the body color. Dietary supplementation with caroten-
oids is positively and linearly dependent on the dE of the red zone
(Table 8,R
2
=0.802, P b0.05) whereas dietary supplementation
with xanthophlls can increase the L* of red zones (R
2
=0.792,
Pb0.05). The color-improving functions of xanthophylls are
Fig. 1. The differences in coloration from different treatments.
67X. Sun et al. / Aquaculture 342-343 (2012) 6268
thought to mainly improve red coloration. The results here suggest
that the carotenoid and xanthophyll content in sh feeds were
highly correlated with sh body coloration, and the carotenoids
had a deeper and greater inuence compared to the xanthophylls.
Gouveia et al. (2003) showed that Showa koi fed 80 mg total co-
lorings kg
1
from Arthrospira maxima (Spriulina) had similar body
color and total carotenoid content with sh fed the same content col-
orings from synthetic astaxanthin. This trial got similar even better
results compared with Gouveia's. Showa koi fed SP diet containing
less total pigment content (diet total carotenoids + xanthophylls,
17.26 mg kg
1
) had similar body color with sh fed CR diet contain-
ing more total pigment (39.11 mg kg
1
). Koi are a type of red carp
that can convert zeaxanthin, canthaxanthin and lutein into astax-
anthin and store it in the body (Simpson, 1981). Spriulina contains
mainly zeaxanthin and β-carotene (Liao et al., 1993; Soejima et al.,
1980). One reason that these compounds can produce an improved
body coloration might be that zeaxanthin in S. platensis is able to be
used by koi effectively. These results suggested that S. platensis
could be a useful, even competitive pigment for inclusion in the
diets of koi carp to improve skin pigmentation.
Li and Wang (1997) indicated that the main pigments in the cells
of photosynthetic bacteria are bacteriochlomphyll a, bacteriophaeo-
phytin and 3 carotenoids with absorption maxima at 485 nm and
516 nm, 481 nm and 510 nm, 486 nm and 521 nm. In the present
study, the total pigment content in the PB and EM diets was 3.49
and 2.65 mg kg
1
, respectively. Both of these values are higher
than the control diet values. Because body coloration generally cor-
related with the dietary pigment dose and koi can use astaxanthin,
zeaxanthin, canthaxanthin and lutein effectively, the poorer colora-
tion of the sh consuming the PB and EM diets may be due to the
low total pigment content and low utilization rate of koi for
chlorophyll.
The present study suggested that Showa koi pigmentation could
be modied by supplementing the diet with 1.5 g kg
1
Carophyll®
red or 75.0 g kg
1
S. platensis. Dietary R. palustris at levels up to
1.0 g dry matter kg
1
of diet does not appear to affect the coloration
of Showa koi (Fig. 1). Furthermore, body coloration generally correlat-
ed with the dose of dietary carotenoids and xanthophylls. Future
work will attempt to optimize the concentration of S. platensis in
the diet and the period of pigment supplementation, and determine
the optimal size of the sh to begin supplementation.
Acknowledgments
The authors would like to acknowledge Chaolin Xiang, Lei Zhu,
and Xianqiong Hu for their assistance with analytical experiments.
This work was supported by the Beijing Municipal Science & Technol-
ogy Commission (China) through the project D09060500430000.
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68 X. Sun et al. / Aquaculture 342-343 (2012) 6268
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... The Haematococcus produces Astaxanthin, which gives salmon its pink hue [134]. Additionally, Spirulina contains additional carotenoids that ornamental koi and other fish can convert to astaxanthin and other brightly colored pigments [135]. Phaeodactylum tricornutum produces large quantities of fucoxanthin, which has been shown to contribute to the golden yellow coloration of gilthead seabreams [136]. ...
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