31

Research Journal of Fisheries and Hydrobiology, 8(2): 31-37, 2013

ISSN 1816-9112
This is a refereed journal and all articles are professionally screened and reviewed

ORIGINAL ARTICLES
 
The effect of marigold (Tagetes erecta) as natural carotenoid source for the
pigmentation of goldfish (Carassius auratus L.).
1
Alma A. del Villar-Martínez, 1Juan C. Orbe-Rogel, 1Pablo E. Vanegas-Espinoza, 1Adrián G. Quintero-
Gutiérrez, 2Maurilio Lara-Flores
1
Centro de Desarrollo de Productos Bióticos del Instituto Politécnico Nacional. Carretera Yautepec-Jojutla
Km. 6 calle CEPROBI No. 8. Col. San Isidro, Yautepec, Morelos, México. C.P. 62731, Apartado Postal 24.
2
Instituto de Ecología, Pesquerías y Oceanografía del Golfo de México de la Universidad Autónoma de
Campeche. Av. Agustín Melgar y Juan de la Barrera S/N Col. Buenavista, San Francisco de Campeche,
Campeche, México.

ABSTRACT

This study was conducted to evaluate the effects of diets containing 0, 100, 200 and 300 mg of
carotenoid/kg diet from marigold meal on skin pigmentation, growth, feed utilization and survival of goldfish
(Carassius auratus), with average initial weight of 1.8 g for a rearing period of 63 days. The pigmentation
degree in skin of goldfish increased significantly with increasing inclusion of marigold meal at 200 mg of
carotenoid/kg diet. However, the marigold inclusion over that level did not lead to more total carotenoid
accumulation in the skin of fishes. No significant difference was observed in the survival, growth or feed
utilization of the fishes. The present results demonstrate that marigold meal can be successfully used as an
alternative natural carotenoid source in goldfish diets. Our data indicates that 200 mg of carotenoid/kg diet from
marigold meal is a suitable dietary level to ensure good pigmentation, acceptable growth and feed utilization in
goldfish.

Key words: Carassius auratus, marigold, carotenoid, pigmentation.

Introduction

The growing interest in aquarium fish has resulted in steady increase in aquarium fish trade globally.
Today, with a turnover of US$ 9 billion a year and an annual growth of 8%, production of ornamental fish is an
important business activity as well as one of the most popular hobbies in the world. It is thought that this sector
can contribute to the economic development in underdeveloped countries, especially in the tropics (Yanar et al.,
2008). Ornamental fish are characterized by a wide diversity of colors and color patterns and success in the
ornamental fish trade is very much dependent on the vibrant color of the fish. The commercial value of these
fish reflects this requirement; hence, ornamental fish growers are constantly exploring methods of enhancing
skin coloration. This color is derived from the deposition of carotenoids in its tissue (Simpson et al., 1981). The
carotenoids are also vital nutrient for healthy growth, metabolism, and reproduction as well as color (Miki,
1991). Since fish, like other animals, are not able to perform denovo synthesis of carotenoids (Goodwin, 1984),
they have to obtain them from dietary sources.
Goldfish (Carassius auratus) is the most popular variety of ornamental fish (NRE, 2002). Besides body
shape, fin shape, and size, an important characteristic affecting the market price of goldfish is body color. To
achieve consumer acceptance and optimal price, the goldfish must be pigmented to have and orange-red color
(Parippatananont et al., 1999). Because fish cannot synthesize these pigments, pigmentation of fish is due to
ingested carotenoids. Different sources of carotenoid pigments like pure carotenoid pigments, animal source and
plant source are included in fish diet. The use of plant source in fish feed presents a double advantage: besides
being rich in carotenoid pigments, they are a direct source of nutrient like protein, lipids and vitamins. Many
studies have emphasized the interest in utilization of plant sources of carotenoid pigments such as paprika
(Capsicum annum) (Tsushima et al., 1998), yeast (Rhodotorulasanneii) (Savolainen and Gyllenberg, 1970),
chesnut flowers (Neamtu et al., 1976), dried flowers (Torrissen et al., 1989) and hippophae oil
(Hippophaerhamnoides) Kamata et al., 1977). Chapman (2000) has concluded that to enhance coloration in
ornamental fish, a combination of synthetic and natural carotenoid pigments should be added at a level of 0.04-
2% of the diet. Marigold (Tagetes erecta), contains various carotenoids of which lutein is the principal
(Navarrete-Bolaños et al., 2005). The flower is used for obtaining pigments, which are then used in the
production of pasta, oils, dairy products and poultry. Keeping in view the importance of marigold as a color
Corresponding Author: Maurilio Lara-Flores. Instituto de Ecología, Pesquerías y Oceanografía del Golfo de México,
Universidad Autónoma de Campeche, Av. Agustín Melgar y Juan de la Barrera S/N, San
Francisco de Campeche, Campeche, México, C.P. 24039.
E-mail: [email protected]; [email protected]
 32
Res. J. Fish & Hydrobiol., 8(2): 31-37, 2013

enhancer, this study was conducted to evaluate the effects of dietary supplementation of goldfish feed with three
concentrations of marigold carotenoid sources. The growth rate, survival, and carotenoid content in body fish
were measured.

Materials and Methods

Experimental design:

This study was carried out in an indoor system. A red variety of goldfish, Carrasius auratus, was obtained
from a local commercial breeder, kept under quarantine conditions for three weeks, and then acclimatized to the
experimental conditions for two weeks before the experiment. During this period, the fish were fed a basal diet
(or control diet). The fish with the same color hue, initial weights of 1.7-1.9 g and about 6 weeks ages were
selected from the general population. Eleven goldfish were stocked per glass aquarium (25 L) with three
replicates for four experimental diets.
The fish were fed by hand three times a day at a rate of 6% of body weight during the experimental period
of 63 days. Body weight were measured every week and skin carotenoid content every 15 days.
Compressed air with air stone was used for maintaining soluble oxygen throughout the experiment. Water
in each aquarium changed at a rate of 100% volume per week. Uneaten feed and faces were siphoned out daily.
Aquaria were checked daily, and mortalities (if present) were recorded. Temperature, pH and dissolved oxygen
level in water of aquaria were measured daily. Throughout the experimental period, the water temperature was
between 25 and 27 °C, dissolved oxygen level was above 5.5 g/L, pH was around 7.4. All aquaria were
maintained under a constant photoperiod (12 h light and 12 h dark) created by fluorescent lamps.
Growth performance parameters evaluated along bioassay included: initial and final body weight, growth
rate [(final weight (g)/initial weight (g))x100], specific growth rate [Ln final weight-Ln initial weight/no
days)x100], feed intake [feed intake (g) per fish for the period], weight gain, feed conversion rate (feed intake
(g)/weight gain (g)] and survival rate [(final live fish/initial live fish)x100] (Gouveia et al., 2003; Kalinowski et
al., 2005).

Diet preparation:

Four experimental diets were prepared after re-milling the NUTRIPECC4510H diet (45% crude protein,
10% fat; Purina®, Mexico). The diet was ground through a mill and mixed with 5% pre-gelatinized potato starch
to assist in re-binding. The mash was thoroughly re-mixed with desired inclusion level of marigold (100, 150
and 200 mg of carotenoid/kg of feed) to develop the test diets and distilled water was added to form pellets
through a meat mincer fitted with 1.4 mm die. The same procedure was applied to the basal diet. Diets were
dried at 45 °C for 12 h and stored at 4° C until required.

Proximate composition analysis:

Proximate compositions of finished diets were determined according to analytical procedures described in
AOAC (1990). Triplicate samples were used for each analysis (Table 1).

Table 1: Proximate composition of experimental diets (g/kg).

Experimental diets1
T0 T1 T2 T3 MM
Dry matter 25.9 36.7 33.4 31.4 88.6
Crude protein 381.8 378.8 377.0 373.0 81.1
Crude fiber 412.3 412.2 411.5 416.0 730.0
Crude fat 102.5 95.4 100.9 103.0 58.6
Ash 77.5 76.9 77.2 76.1 44.9
Total carotenoid (mg kg-1)2 209.68 303.01 347.79 405.44 16937.33
1
T0=Control diet, T1=Diet with marigold meal (100 mg of carotenoid supplementation kg-1 of fed), T2=Diet with marigold meal (150 mg of
carotenoid supplementation kg-1 of feed), T3=Diet with marigold meal (200 mg of carotenoid supplementation kg -1of feed, MM=Marigold
meal.
2
Total carotenoid=carotenoid on basal diet + carotenoid of marigold meal.

Analysis of pigments on body of goldfish:

The carotenoid content of fish skin was extracted according to the method of Torrissen and Naevdal (1984).
Three fish were randomly sampled from each diet treatment per sampling period and used for carotenoid
analyses, which were carried out in triplicate. Sample of 200-300 mg skin were collected from head, dorsal
regions, and caudal fin of the fish and then transferred to 10 mL pre-weighed glass tubes. After the samples

 
 
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Res. J. Fish & Hydrobiol., 8(2): 31-37, 2013

were ground in acetone containing anhydrous sodium sulphate, the extractions were made up to 10 mL with
acetone. The samples were stored for 3 days at 4 °C in a refrigerator, and then extracted three or four times until
no more color could be obtained. The solutions were centrifuged at 5000 rpm for 5 min, and then absorptions
were measured in a spectrophotometer (Shimadzu, UV 16UA).
The samples of carotenoids described previously was used to determinate the total carotenoids content by
High-Performance Liquid Chromatography (HPLC) according to method described by Meléndez-Martínez et al.
(2005).

Statistical analysis:

All data on growth, feed conversion ratio, survival and carotenoid content in skin of the fish are expressed
as means±s.e. Data from each treatment diet for each sampling period were analyzed by one-way ANOVA, and
significant differences were ranked with Tukey´s multiple comparison test at the 5% level of significance by
using Statgraphics Centurion XV.

Results:

Growth performance, feed conversion and survival:

All fish grew normally, and no specific signs of disease were observed. All diets were accepted equally well
by the fish. Fish weight increased 2-2.5 fold at the end of the feeding trial. The carotenoid-supplemented diets
did not appear to have any effect on goldfish growth rate (Table 2).

Table 2: Growth performance and feed utilization of goldfish fed experimental diets1
Diets
T0 T1 T2 T3
Survival (%) 85.71±14.28 80.95±21.82 100.00±0.00 95.24±8.24
Initial weight (mg) 176±9 179±4 180±3 187±7
Final weigh (mg) 398±4.7 403±4.2 366±1.9 411±1.9
Feed conversion rate 3.02±0.20 2.93±0.53 3.44±0.12 2.96±0.47
Specific growth rate 1.30±0.06 1.29±0.07 1.13±0.03 1.25±0.07
Weight gain (mg) 221.4±38.9 223.7±46.3 185.9±19.5 224.1±25.9
Feed intake (mg) 664±79 641±27 640±42 656±31
Values are means ± s.e. No significant differences were observed (p>0.05)
1
T0=Control diet, T1=Diet with marigold meal (100 mg of carotenoid supplementation kg-1 of fed), T2=Diet with marigold meal (150 mg of
carotenoid supplementation kg-1 of feed), T3=Diet with marigold meal (200 mg of carotenoid supplementation kg -1of feed), MM=Marigold
meal.

Effects of carotenoids of marigold on the distribution of pigments of goldfish:

The distribution of the pigments in the body of goldfish was not found homogeneous (Table 3). The
observed differences were significant (p<0.05) and apparently not affected by the diet composition. The highest
concentration of pigments in the goldfish tested was observed in the caudal fin followed by that in the dorsal
region. Head was found to have the lowest concentration of pigments.
Supplementing 200 mg/kg carotenoids from marigold meal in the diet changed the color significantly over
the time of the study (Figure 1).

Table 3: Pigment distribution (%) of different body parts of goldfish fed experimental diets1
Diets Body part 3º week 6º week 9º week
Head 18.67±6.4 20.04±6.04 5.92±3.8
T0 Dorsal region 16.21±9.5 14.60±3.4 4.77±1.8
Caudal fin 65.11±15.6 65.34±2.8 89.30±3.0
Head 12.13±2.1 21.22±7.1 13.25±8.0
T1 Dorsal region 10.57±5.6 24.09±5.2 11.83±7.3
Caudal fin 77.28±5.6 54.68±28.31 74.90±5.14
Head 16.51±7.6 15.36±8.8 24.20±8.0
T2 Dorsal region 3.64±1.2 11.35±7.5 15.79±13.5
Caudal fin 79.84±8.0 73.28±15.8 60.00±20.8
Head 14.21±7.7 34.67±24.2 7.37±6.2
T3 Dorsal region 16.97±8.6 9.98±4.3 6.02±5.3
Caudal fin 68.81±14.4 55.33±25.6 86.59±10.1
1
T0=Control diet, T1=Diet with marigold meal (100 mg of carotenoid supplementation kg-1 of fed), T2=Diet with marigold meal (150 mg of
carotenoid supplementation kg-1 of feed), T3=Diet with marigold meal (200 mg of carotenoid supplementation kg -1of feed)

 
 
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Res. J. Fish & Hydrobiol., 8(2): 31-37, 2013

Fig. 1: Color differences of goldfish fed experimental diets. 1T0=Control diet, T1=Diet with marigold meal (100
mg of carotenoid supplementation kg-1 of fed), T2=Diet with marigold meal (150 mg of carotenoid
supplementation kg-1 of feed), T3=Diet with marigold meal (200 mg of carotenoid supplementation kg -
1of feed).

Effects of the experimental diets on total carotenoid concentration of goldfish skin:

After three weeks of feeding, the carotenoid content in body of fish fed diets supplemented with increasing
pigment levels started to differ from that of fish fed the control diet (Table 4).
At the beginning of the experiment, while carotenoid content in body of the fish was 6.2 µg/g, this value
increases significantly at the end of the experiment to 10.64-49.56 µg/g in fish diet containing 100 mg and 200
mg of carotenoid per kg. On the other hand, the carotenoid content of the control group decreased to only 4.22
µg/g in the same period. Total carotenoid content in body of goldfish increased significantly with 200mg of
carotenoid per kg in the diet (P<0.05). However, inclusion of 300 mg/kg of pigment did not lead to more total
carotenoid accumulation in the fish (Table 4).

Table 4: Total carotenoid concentration in the body of goldfish fed experimental diets (µg of carotenoids/ g of tissue).
Diets Initial 3° week 6° week
T0 6.2 4.0 4.22
T1 6.2 4.3 10.64
T2 6.2 15.63 49.56
T3 6.2 3.2 5.10
1
T0=Control diet, T1=Diet with marigold meal (100 mg of carotenoid supplementation kg-1 of fed), T2=Diet with marigold meal (150 mg of
carotenoid supplementation kg-1 of feed), T3=Diet with marigold meal (200 mg of carotenoid supplementation kg -1of feed)

Discussion:

In nature, carotenoids have been implicated in diverse functions such as pigmentation, antioxidant activity,
immunostimulation and reproduction, and they play a positive role in intermediary metabolism (McGraw and
Ardia, 2003; Watanabe and Vasallo-Agius, 2003; Chatzifotis et al., 2005). Fish use carotenoids as one of the
most important group of natural pigment for pigmentation of the skin and flesh. As fish cannot synthesize these
pigments, they rely on a dietary supply of carotenoids to achieve their natural skin pigmentation, one of the
quality criteria most in demand for the market value of ornamental high-value species such as goldfish (Lovell

 
 
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Res. J. Fish & Hydrobiol., 8(2): 31-37, 2013

2000; Gouveia et al., 2003; Sinha and Asimi, 2007). Further, there also reports that link carotenoids to growth
enhancement in Atlantic salmon fry (Salmo salar) (Christiansen et al., 1995), rainbow trout (Oncorhyncus
mykiss) (De la Mora et al., 2006) and goldfish (Carassius auratus) (Sinha and Asimi, 2007); or to improvement
of survival rate in kuruma prawn (Penaeus japonicas) (Chien and Jeng, 1992) and characins (Hyphessobrycon
callistus) (Pan et al., 2010). Yet there is no equivocal agreement on this issue, as in species like red porgy
(Pagrus auratus and Pagrus pagrus) (Chatzifotis et al., 2005; Kalinowsky et al., 2005), characins
(Hyphessobrycon callistus) (Wang et al., 2006), Cichlasoma severum (Kop et al., 2010); carotenoids do not
cause any notable increase in growth. In agreement with the latter studies, our results showed that marigold meal
do not promote the growth of goldfish.
The effectiveness of a carotenoid source for pigment deposition is species specific (Ha et al., 1993). Fish
species may exhibit different pathways for carotenoid metabolism (Matsuno, 2001). In rainbow trout
(Oncorhynchus mikiss) the dietary astaxanthin is partially metabolized to zeaxanthin (Schiedt et al., 1985).
Nakazoe et al. (1984) reported that the content of skin carotenoid in fish fed the β-carotene-supplemented diets
was low and the dietary β-carotene failed to increase the reddish color red porgy. It also likely that the
accumulation of β-carotene in skin is low and its conversion to astaxanthin in the skin is minimal. On the
contrary, other species such shrimps can convert β-carotene to astaxanthin (Boonyaratpalin et al., 2001).
Differing from rainbow trout and red porgy (Nakazoe et al., 1984; Schiedt et al., 1985), the current results
indicate that goldfish were able to utilize lutein and zeaxanthin from marigold efficiently. Similar results were
obtained for goldfish by feeding different natural carotenoid sources, such as Spirulina (Kiriratnikom et al.,
2005), microalgal biomass (Gouveia and Rema, 2005), red yeast (Xanthophyllomyces dendrorthous) (Xu et al.,
2006) and alfalfa (Medicago sativa) (Yanar et al., 2008).
Although the pigmentation in skin of goldfish increased with a level of 200 mg of carotenoids from
marigold meal, over that level did not result in additional carotenoid accumulation. Yanar et al. (2008)
mentioned that this may indicate that the carotenoid uptake or transportation to the tissue was saturated due to
carotenoid inclusion level in goldfish, only this work reported a saturation level for this specie.
Our results showed that marigold meal as a natural carotenoid source was effective on skin pigmentation of
goldfish. Also, this study demonstrated that since marigold meal led to nearly maximum carotenoid
accumulation in the skin of goldfish, it should be considered as a valuable source of carotenoids, such as
zeaxanthin, lutein or astaxanthin (Hata and Hata, 1971; Matsuno et al., 1981; Ohkubo et al., 1999; Paripatanont
et al., 1999).

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