Int J Biol Med Res.

2011; 2(3): 621-626

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Volume 2, Issue 3, July 2011

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Original Article
Influence of Four ornamental flowers on the growth and colouration of orange
sword tail Chicilidae fish (Xiphophorus hellerei, Heckel, 1940)
Baby Joseph, S. Sujatha*, J. Jemima Shalin and A. Palavesam
*International Centre for Bioresources Management, Malankara Catholic College Mariagiri, Kaliyakkavilai-629153.

ARTICLE INFO ABSTRACT

Keywords: The present research was designed to study the effect four botanical additives
Carotenoid (H. rosasinensis, Rosa indica, Ixora coccinea and Crossandra infundibuliformiss) on the growth
Medicinal plants
X. hellerei and body colouration of an ornamental fish of red sword tail Xiphophorus hellerei (Heckel).
Pigmentation This experiment was conducted in adult female fish for a period of 75 days. The carotenoid
pigment sources were added to the supplementary diet at 1.5, 2.5 and 3.5 percent levels of
concentrations respectively. The fishes were treated with at the rate of 5 to ten percent level of
body weight. Three fold increases in growth was observed in H. rosasinensis fed fishes
followed by R. indica. Furthermore, the similar weight gain has been observed rest of the two
flower petals (I. coccinea and C. infundibuliformiss). The percentage of colour pigments
obtained in adult fish were maximum in I. coccinea one percent level then remaining flowers
peal showed another highest pigment production was R. indica, H. rosasinensis and C.
infundibuliformiss. Consequently a significant difference was found between individuals fed
by natural pigment material and those by unpigmented feeds (p≤0.05). It was demonstrated
that natural pigment substances have an impact on coloration of cichlid and the groups did not
exhibit any distinctions in feed conversion and growth rates. Therefore, it was determined that
these pigment sources have an effect on the colour of cichlid fish.
c Copyright 2010 BioMedSciDirect Publications IJBMR -ISSN: 0976:6685. All rights reserved.

1. Introduction
Ornamental fishes are nowadays rapidly gaining importance into various tissues. The molecular properties of rainbow trout
because their aesthetic value and also due to their immense (Oncorhynchus mykiss) retinol-binding protein (rtRBP), the
commercial value in the export trade world over. Attractive specific retinol carrier in vertebrate plasma, were studied to
colouration determines the commercial value of ornamental fish elucidate its role in transporting retinols to developing fish
pigmentation in the skin is responsible for colouration in the fish oocy[6,7]. The red colouration of salmonids, crustaceans and some
[1].Carotenoids are the primary source of the pigmentation on the aquarium fish have become of interest in the cultivation. Dietary
skin of fishes [2]. In natural environment, the fishes meet their carotenoids play significant part in the regulation of skin and
carotenoid requirements by ingesting aquatic plants or through muscle color in fish [8]. Astaxanthin is the main carotenoid
their food chain. But fishes can not synthesis the carotenoid pigment of red-pink coloured aquatic animals, being widely used
denovo [3,4,5]. Carotenoids are absorbed in animal diets, in aqua-cultural processes because it is a standardized and
sometimes transformed into other carotenoids, and incorporated chemically stable product with a high carotenoid concentration
[9]. The colour enhancing diets should contain additional natural
pigments to enhance the colour of ornamental fish[10]. In earlier
* Corresponding Author : Dr.S. Sujatha days Ali and Salim [11] established the fish do not possessed that
International Centre for Bioresources Management fish do not possess the ability to synthesize carotenoids. Hence the
Malankara Catholic College carotenoid pigmentation of fish results depends upon the
Mariagiri, Kaliyakkavilai-629153.
E.mail: sujatharbs@rediffmail.com supplementary feed contains the carotenoid amount. Several

c
Copyright 2010 BioMedSciDirect Publications. All rights reserved.
 S. Sujatha et.al / Int J Biol Med Res. 2011; 2(3): 621-626
622

authors have been proved that the fish can be pigmented by 2.2.Qualitative estimation of total Carotenoids:
including processing wastes and botanical plant source[12,13,1].
The Qualitative analysis of carotenoid was carried out by the
Since, there is no more works has been conducted this kind of
Thin Layer Chromatography (TLC) method [15].The
orange word tail fish with these four kinds of flower petals
identification of compounds carried by comparing the Rf values of
carotenoid pigments. Hence the present study were undertaken
samples.
to find out the influence of four botanical additives on growth and
colouration of orange sword tail. 2.3.TLC Plate Preparation
The TLC plates are made by spreading of silica gel G for TLC over
2. Materials and Methods
the glass plates of size 20cm x 20 and the thickness of 250µ.The
The experimental fish of Orange swordtails (Xiphophorus plates were allowed to dry at 100◦c in the oven. After drying, the
helleri) (H.) of uniform size group (1g) were purchased from a plates were ready to use. Before using the plates for separation it
commercial aquarium fish farm from Nagercoil from should be activated at 120°C for2-3hrs for obtaining better results
Kanyakumari District, Tamilnadu and were acclimatized to and clear separation.
laboratory conditions for one week before the start of the
experiment. The experiments were conducted for a period of 21 2.4. Developing of TLC
days and were carried out in 5 plastic troughs of 20 litre capacity. Saturation of the developing chamber has a strong influence on
The fish were weekly weighed and recorded. For the present separation and reproducibility hence recommended in
investigation, the feed was prepared in the form of dry pellets carotenoid work. The saturation chamber was rapidly charged
[4].The experimental diet composed of the basic ingredients like with the Mobile phase (Acetone/Methanol 9:1) described by
flower petals of (H. rosasinensis, R. indica, I. coccinea and C. Hector et al. [16].
infundibuliformiss) (Table-1). Using the four ingredients, diet
2.5.Detection of Spots In TLC
with 40% protein was prepared using the square method. They
were mixed with different quantities of four types of flower petal A Smaller amount of samples and reference compounds were
meal content in the diet. These flowers were purchased from the spotted at the level of minimum quantities (2-5 µl).Then the TLC
local area of in and around the college campus. Four flower petal plate was placed on the chamber with mobile phase
meals were added to the diets just before pelletization with (Acetone/Methanol) to develop the color spots. After mobile
respective concentration of 0,2,3,4 mg /100 g of basal diet. The solvent searching the upper limit, plates were taken out and dried
prepared foods were provided two times daily to the experimental for few minutes. The developed and dried chromatogram was
fishes (X. helleri) to satiation. The control food for control tank stained by 5gms iodine vapours the distance traveled by each spot
feed 1, feed 2, feed 3 for experimental demand. The unfed was in the experimental samples was measured from the base line and
collected from the tank; the feeding experiment was continued for relative Rf values were calculated .By comparing the standard Rf
a period of 21 days. For the present study, four different tanks of values for the chosen mobile phase, the number of carotenoids
orange sword tail were cultured in glass tanks. Among the four present in samples were identified.
tanks, one was used as control tank remaining three tanks were
experimental tank F1, F2 and F3 respectively. The experimental Distance traveled by the substance
Rf=
was carried out at seven days interval for 21 days. During the
Distance traveled by the solvent
experimental period the fishes were fed on control and
experimental diets added with carotenoid at the rate of two times 2.6.Method of TLC
a day. Water quality parameters were maintained by aeration. The
physicochemical parameters such as temperature, dissolved Thin-layer chromatography consists of a stationary phase
oxygen, ammonia pH in all the experimental tanks were immobilized on a glass or plastic plate, and an organic solvent. The
estimated. Standard methods were employed for the analysis of sample, either liquid or dissolved in a volatile solvent, is deposited
the water quality parameters as a spot on the stationary phase. The constituents of a sample can
be identified by simultaneously running standards with the
2.1.Pigment Extraction in Fish Tissue unknown. The bottom edge of the plate is placed in a solvent
The method used for pigment extraction from the red sword- reservoir, and the solvent moves up the plate by capillary action.
tail tissue was as described in Five gram of entire red sword-tail When the solvent front reaches the other edge of the stationary
body tissue (without head and alimentary canal) was taken in a 10 phase, the plate is removed from the solvent reservoir. The
ml screw capped clear glass vials and 2.5 g of anhydrous sodium separated spots are visualized with ultraviolet light or by placing
sulphate was added. The sample was gently meshed with a glass the plate in iodine vapor. The different components in the mixture
rod against the side of the vial and then 5ml of Chloroform was move up the plate at different rates due to differences in their
added and left overnight at 0°C. When the chloroform formed a partitioning behavior between the mobile liquid phase and the
clear 1-2 cm layer above the caked residue, the optical density was stationary phase.
read at 380, 450, 470 and 500 nm in a spectrophotometer. A blank
2.7. Statistical Analysis
prepared in a similar manner was used for comparison. The
wavelength at which maximum absorption, was used for the Sigmastat 3.5 was used for statistical analysis. A one way
calculation. ANOVA was applied to find out the significant differences among
average values of total carotenoid content and the difference
between the mean treatments were tested with Tukey test. A two
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623

way ANOVA was applied to establish significant differences between the values of nutritional parameters and the difference between the
mean treatments were tested with Tukey test.

3. Result
The present work was carried out to determine whether carotenoids of Hibiscus rosa-sinensis, Rosa indica, Ixora Coccinea, Crossandra
infundibuliformiss petals could induce pigmentation to make orange swordtail more color. The amount of total caroteniod was higher in C.
infundibuliformiss-1.802 mg/gm followed by Hibiscus rosasinensis, Ixora Coccinea, Rosa indica contains 1.52, 1.014 and 0.47 respectively.
An ornamental fish does not have the capacity of synthesizing the carotenoid itself hence it must be supplied through diet.

Table: 1. List of Ingredients used for carotenoid based food preparation

Ingredients Percentage of contents (included food stuff)

Control Experimental Experimental Experimental

diet -1 (1%) Diet-2 (3%) diet -3 (5%)

Fish meal (gm) 10 20 25 30

Casein (gm) 10 20 25 30

Soya meal (gm) 10 20 25 30

Tapioca powder (gm) 10 20 25 30

Wheat flour (gm) 10 20 25 30

Rice bran (gm) 15.5 20 25 30

Vitamin and Mineral Mixture 0.05 0.05 0.05 0.05

Table-2. The growth of adult orange sword tail with four different botanical additives at three different concentrations

Treatment Mean Weight

Concentration 7th 14th 21st 28th Mean wt Wt gain / Specific P-value

(%) (day in g) growth rate

H. rosasinensis 1 3.80 3.91 4.67 5.10 1.55 0.522 0.46

3 4.22 4.97 4.98 5.18 1.84 0.781 0.51 4.21x10-5

5 4.11 5.22 5.36 5.40 2.34 0.235 0.61

1 4.35 4.41 4.58 4.72 3.54 0.542 0.54

I. coccinea 3 4.65 4.69 4.84 4.98 3.84 0.165 0.35 0.0035

5 5.12 5.21 5.46 5.68 3.97 0.321 0.35

1 4.03 4.14 4.25 4.57 3.45 0.310 0.52

3 4.21 4.34 4.67 4.61 3.56 0.256 0.54 0.0018

C. infundibuliformiss 5 4.58 4.48 4.73 4.89 2.97 0.455 0.67

1 3.10 3.45 3.89 4.10 2.11 2.013 0.59 0.0057

R. indica 3 3.54 3.58 3.93 4.10 1.38 0.254 0.73

5 3.85 3.91 4.11 4.35 2.58 0.041 0.56

Control optimum 5.12 5.49 5.49 5.60 4.48 0.054 0.45

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624

The data regarding the growth of adult orange sword tail with Table 4. Preliminary phytochemical screening of fraction on
four different botanical additives at three different concentrations carotenoid supplementary feed 15-18 with Recolumn
are presented in Table 2. The mean weight of the adult orange sword chromatography
tail fish on the first day of stocking in the control was 4. 48g. The
initial mean weight of the H. rosasinensis fed fish group at three Compounds Solvents
different concentrations viz. 1, 3 and 5 percent were 1.55, 1.84 and
Steroids Distilled water Acetone Methanol
2.34g and the initial mean weight in the I. coccinea were recorded to
be 2.54, 2.84 and 2.97g respectively. Furthermore, the mean weight Triterpenoids + + +
gains of the fish fed with C. infundibuliformiss were found to be 3.45, Reducing sugar - - -
3.56 and 2.07mg. Moreover, R. indica showed optimum level of
Carbohydrate - - -
mean weight gain observed in this flower fed fish such as 2.11, 1.38
and 2.58 on 1, 3 and 5% respectively. Among the four flowers Alkaloids + + +
treated diet, I. coccinea fed fishes having maximum mean weight Phenolic compounds + + +
4.72, 4.98 and 5.68mg in 1, 2 and 5% respectively. Though, the four
Saponin - + +
different kinds of diet revealed better specific growth rate was
observed on two flowers such as C. infundibuliformiss and R. indica. Xantho protein + + +
Tannins + + +
3.1.Column Chromatography
Flavonoids + + +
The extracts obtained from chloroform and benzene mixture
(525: 225ml) was subjected to column chromatographic study
+ means for presence of the compound
which eluted successively with chloroform, glacial acetic acid,
diethyl ether and methanol. Totally twenty five (each of 10ml) - means Absence of the compound
fractions were eluded from the column. Various fractions were Fig.1. External morphology of orange sword tail
grouped individually by monitoring TLC behaviour and their Rf (Xiphophorus helleri) after treated with three concentration
value is presented in (Table. 3) the prepared supplementary feed. A of flower based carotenoid based supplementary feed
light greenish yellow elution obtained from the botanical based
carotenoid feed. Of these various colour fractions only 15-18 CONTROL
fractions was selected for further purification. However, fraction
was obtained while Glacial acetic acid and Chloroform (70:30) at
30-50 C. From this fraction yields a 0.78mg of dark green colored
product. It was again subjected to column chromatography eluted
successively Benzene and Methanol with different proportion. In
order to that 19 fractions were obtained from these compounds. For
instance, the various groups of other compounds are also subjected
in TLC assay were determined based upon the RF values. Apart from
these RF values of peculiar compounds assessed with the help of
TLC chromatography and qualitative phytochemical analysis
presented in table (Table 5). The RF value recorded for the fraction
of 9 to 13 (Table 4).

Table 3. TLC Pattern of Recolumn (15-18) fractions of

botanical based carotenoid supplementary feed for Red
sword tail Xiphophorus hellerii
F1 (a)
Fraction from RF values
carotenoid food
I II
0.540 -
1 0.540 -
2 0.510 -
3 0.320 -
4 0.380 0.67
5 0.401 0.64
6 0.405 0.66
7 0.425 0.67
8 0.35 0.57
9 0.34 0.55
10 0.39 0.34
11 0.39 -
12 0.28 -
13 0.26 -
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625

F2 (b) demonstrated positive results for speed of skin color change may
be suitable for use in a pond-water culture system based on our
overall results for goldfish reared in pond water and given a feed
that contained pigments (Zeigler Tropical) (carotenoid based
referred only commercial feeds are continually modified, as they
are subject to least-cost formulation, new research, and updates
on nutrient requirements of fish [22].In this study, we
demonstrated how feed formulations dramatically affect color
development in the skin of fancy goldfish, and how the type and
amount of pigment in a feed formulation may impart yellow,
orange and red skin coloration.
The protective function of carotenoids associated with the
reaction centers and antenna complex are so critical those an
inability to form cyclic carotenoid due to a block in carotenoid
biosynthesis [23].We also observed that inhibition of carotenoid
denaturation by NF does not lead to phytoene accumulation in the
F3 (c) dark. This can be explained by a reduced flux into the
carotenogenic pathway and would be in line with the above
mentioned hypothesis that a strongly reduced carotenoid
synthesis occurs in darkness [24,25].

According to our knowledge, the hypolipidemic and

hypocholesterdemic effects of carotenoids have not previously
been investigated. Dietary carotenoids source and concentration
had no effect on the growth and survival of red devil agree with the
following studies '[26] and ] also found that there were no
differences in growth and feeding rates among rainbow trout fed
AX and BC. Neither growth nor feed efficiency of gilthead sea
bream was affected by diets supplemented with synthetic AX or H.
pluvialis or no carotenoids [28]. Furthermore, , , suggested when
rainbow trout fed with Dunaliella salina, which contains BC, and P.
rhodozyma, which contains free AX, and found no difference in
growth rate between the treatment groups and control group.
4. Discussion
This study presents data on the effect of carotenoid sources on
Carotenoids are also vital nutrients for healthy growth, skin coloration of Orange sword tail (X. hellerei). The physical and
metabolism, and reproduction [17].Further work is needed to chemical characteristic of the diet plays an important role on
determine the optimum types, concentrations and combinations species. Similarly the result from the experiment shows that the
of carotenoids (specifically astaxanthin, zeaxanthin, and lutein) artificial diets definitely imparted different skin coloration,
that are required to produce acceptable levels of the natural growth and biochemical changes in orange sword tail. The
orange-red skin coloration in juvenile Red Oranda gold fish by carotenoid based artificial feeds reduced the best orange red skin
Use of practical or semi purified diets in this type of study is colouration in orange sword tail. The quantity of biochemical,
imperative for limiting the effects of dietary deficiencies or energy growth and carotenoid analysis objectively demonstrated the
difference on pigment assimilation that may occur with different concentrated diet source imparted large differences in
commercial diet formulations. These types of studies would allow skin color types and amounts. More studies were reported only
investigators and feed manufacturers to further refine which diet carotenoid source that had a significant effect on skin hue,
formulation will produce the optimal level of orange-red skin promoting a reddish coloration to the dorsal skin area and a
coloration. Investigation of color-enhancing diets as finishing ventral hue similar to wild red porgy (Fig 1). No apparent effect of
feeds for goldfish in well-water culture systems is also warranted carotenoid source on skin melanin content was observed. In
as is done in the trout and salmon culture industries [19]. contrast, dietary protein/carbohydrate ratio affected melanin
Overall results for natural orange-red skin coloration content in the skin. Finally these results indicate that the
development, speed of skin color change, feed formulation efficiency of carotenoid assimilation by orange sword tail is not
carotenoid profile, survival and growth demonstrated that the age dependent; some individuals will begin carotenoid
VibraGro feed should be a suitable diet for juvenile Red Oranda assimilation sooner than others depending on their concentration
goldfish reared in a well-water culture system [20,21].The of diet.
carotenoid astaxanthin appears to impart the orange-red
coloration in this variety of goldfish. Other feeds that
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626

Acknowledgement [20] Choubert GJR. Tentative utilization of spirulina algae as a source of

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Rev.Fr. Premkumar, (M.S.W) Malankara Catholic College, Mariagiri [21] Chatzifotis S, Pavlidis M, Jimeno C, Vardanis G, Sterioti A, Divanach P. The
effect of different carotenoid sources on skin coloration of cultured red
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porgy (<I>Pagrus pagrus</I>). Aquac Res.2005; 36 (16): 1517-1525.
preparation of this manuscript.
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