Animal Nutrition and Feed Technology (2015) 15: 417-425

doi: 10.5958/0974-181X.2015.00042.6

Marigold Petal Meal: A Natural Carotenoid Source for

Pigmentation in Swordtail (Xiphophorus helleri)
G.H. Pailan*, Parimal Sardar and B.K. Mahapatra
Central Institute of Fisheries Education, Kolkata Centre
Salt Lake, Kolkata-700 091, India
(Received January 23, 2014)

ABSTRACT
Pailan, G.H., Sardar, P. and Mahapatra, B.K. 2015. Marigold petal meal: A natural carotenoid source
for pigmentation in Swordtail (Xiphophorus helleri). Animal Nutrition and Feed Technology, 15: 417-425.
An experiment was conducted to see the effect of feeding graded levels of marigold petal meal
on pigmentation of skin, growth performance, nutrient utilization and muscle biochemical composition
in ornamental fish, swordtail (Xiphophorus helleri). Different iso-caloric and iso-nitrogenous experimental
diets were formulated with supplementing five levels (0, 2, 4, 6 and 8%) of marigold flower meal. The
experimental fish (n=225) were divided into five equal groups having three replicates under each group
and fed respective experimental diets for a period of 60 days. The biochemical composition of fish muscle
was similar in all the groups. Total carotenoids concentration (g/g) in the skin of sword tail at the beginning
and end of feeding was similar in control diet (2.94 vs 3.17). The supplementation of marigold petal meal
in the diet significantly increased (P<0.05) the total carotenoid concentration in the muscle and skin of
sword tail from 2.87 to 6.17 g/g. Growth performance and nutrient utilization in fish were similar in
all the experiment groups. The study revealed that marigold petal meal at the 4% level can be supplemented
in the diet of swordtail for improvement of skin coloration through increase in the carotenoids concentration
in skin without any adverse effect on body composition, growth and feed conversion efficiency.
Key words: Carotenoids, Marigold petal meal, Pigmentation, Swordtail.

INTRODUCTION
The ornamental fish trade is very much dependent on achieving vibrant colour.
Colour is one of the major factors which determine the price of the ornamental fish
in the world market (Saxena, 1994). The freshwater ornamental fish industry has
experienced the problem of faded coloration in fish, especially when the fishes are
kept under captivity for long duration and also in intensive culture condition. Generally
diet for aquarium fish should not only be nutritionally balanced, palatable, and
resistant to crumbling, water stable, and buoyant but it should also enhance pigmentation

*Corresponding author: ghpailan@cife.edu.in

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Pailan et al.

in the fish. Fish like other animals cannot synthesize carotenoids and in natural
conditions, they acquire carotenoid pigments from various sources, including micro
algae, crustaceans and insects but in artificial conditions, supplementing diets with
carotenoid pigments obtains this pigmentation. Hence, a direct relationship between
dietary carotenoids and pigmentation exists in them (Halten et al., 1995).
Carotenoids are a group of naturally occurring lipid soluble organic pigment
that are responsible for the red, orange and yellow colour in the skin, shell and
exoskeleton of aquatic animals. To date, over 600 carotenoids have been identified
in nature which are primarily produced in phytoplankton, algae and plants. Carotenoids
are absorbed in animals, sometimes transformed into other carotenoids and incorporated
into various tissues. Carotenoids commonly occurring in freshwater fish include betacarotene, lutein, taraxanthin, astaxanthin, tunaxanthin, alpha, beta-doradexanthins,
and zeaxanthin. Research also indicates additional benefits from dietary carotenoids
beyond the resulting coloration. Astaxanthin, for example, has biological functions
related to growth, reproduction and tissue health in salmonids and shrimp, possibly
due to the compounds strong antioxidant properties (Bell et al., 2000).
Coloration in ornamental fish is a very important phenomenon and if enhancement
of colouration can be done by administering pigment enriched feed, it will definitely
improve the quality and cost of the fish. However, detail studies on colouration
enrichment in ornamental fish are lacking. Plant sources have been utilized for
enhancing pigmentation in fish. For example, Spirulina have been used as a source
of carotenoid pigments for rainbow trout and fancy carp (Choubert, 1979; Boonyaratpalin
and Phromkunthony, 1986; Alagappan et al., 2004) and marigold petal meal was used
for tiger barb (Boonyaratpalin and Lovell, 1977) and for Swordtail (Ezhil et al.,
2008).
In view of the above the present work was carried out to know whether
marigold petal meal could be used as a low cost natural pigmenting source in order
to enhance the colouration of experimental fish, swordtail (Xiphophorus helleri), one
of the colourful ornamental fish having great market demand.

MATERIALS AND METHODS

Experimental design
Swordtail (Xiphophorus helleri) of uniform length and size was procured from
a commercial fish trader of Kolkata, West Bengal for the experiment. Fish were
subjected to bath treatment with 5 ppm KMnO4 for disinfection. The fish were
acclimatised to fibre tanks of 500 litre capacity with continuous aeration prior to
start of the experiment. The feeding study was conducted for a period of 60 days in
15 plastic troughs of 50 litre capacity. These 15 troughs were grouped into five sets
with three in each set. The fishes were stocked at the rate of 15 per trough. The
length and weight of fishes were recorded at fortnightly intervals.

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Marigold petal meal as a natural carotenoid source for fish

Experimental diets and feeding trial

Fish were fed on dry pelleted feed. The basal diet (T0) was formulated (Table
1) and prepared using the basic ingredients like fish meal, soyabean meal, and
groundnut oil cake as protein sources, rice bran, wheat flour as carbohydrate sources
starch powder as binder and soya oil as lipid sources. The ingredients were ground,
thoroughly mixed and dough was prepared by adding required amount of water. This
dough was autoclaved at 121C for 10 min., cooled for a while and other ingredients
like vitamins and minerals premixes at 1% were then mixed thoroughly. Basal diet
was considered as control diet (T0). Marigold petal meal was added to the basal diet
at five different levels of 2, 4, 6 and 8 g/100g by replacing same quantity of rice
bran for the preparation of treatment diets T1, T2, T3 and T4, respectively (Table
1). The dough was taken into hand pelletizer to make 2.0 mm pellets. The pelleted
feed was air dried and kept in airtight zipper bags until further use.
The fish were fed twice daily in the morning and evening at 3% of body weight
per day. The feed was adjusted at 15 days interval according to the body weight of
the fish. Plastic troughs were cleaned properly by siphoning off feed particles and
metabolic wastes twice in a day from the bottom of the trough. Water exchange was
carried out once in a day for the sufficient supply of oxygen to the fish. The bottom
of the troughs was cleaned twice weekly by scrubber.
Sample collection and analysis
Water quality parameters viz. water temperature, pH, dissolved oxygen, total
hardness and alkalinity from all the troughs were analysed at fortnightly intervals as
per standard methods (APHA, 1998). The fish tissue and feed were analysed for
moisture, crude protein, ether extract and total ash (AOAC, 1990). The Cyanotech
(2002) method was adopted to estimate the total carotenoids in marigold petal meal
and experimental diets. All procedures were performed in low light and temperature
Table 1. Ingredient composition of experimental diets (% DM basis) fed to swordtail
T0

T1

Dietary treatments
T2

T3

T4

Fish meal

15

15

15

15

15

Soybean meal

22

22

22

22

22

Groundnut cake

25

25

25

25

25

Ingredients

Rice bran

20

18

16

14

12

Wheat flour

12

12

12

12

12

Starch powder

Soybean oil

Vit. mixture

Marigold flower meal

20.45

22.83

25.21

27.59

29.97

Cost (Rs./kg)

Basal diet supplemented with marigold petal meal at 0 (T0), 2 (T1), 4 (T2), 6 (T3), and 8 (T4) percent levels.

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Pailan et al.

as carotenoids are very sensitive to light, temperature and oxygen. Analysis of total
carotenoids in the fish tissue was carried out prior to the start of the experiment and
after the termination of the experiments following Olson (1979).
Calculation
Initial body weight, final body weight, total feed intake, protein intake were
used to calculate absolute growth rate (AGR), relative growth rate (RGR), feed
conversion ratio (FCR), protein efficiency ratio (PER) using standard procedures.
The survival (%) was also calculated.
Statistical analysis
The data obtained were statistically analysed according to Snedecor and Cochran
(1994). The data were subjected to analysis for one-way analysis of variance (ANOVA)
and significant (P<0.05) difference among mean values of different treatment were
compared by DMR test (Duncan, 1995).

RESULTS AND DISCUSSION

Proximate composition of the experimental diets
The Proximate composition of the diet was similar in all the experimental
diets (Table 2). The similarities in the nutrient compositions in the experimental
diets can be correlated to the level feed ingredients inclusions which were only
differentiated by the level of carotenoids. The development of manufactured feed
could be considered as one of the contributing factors to the tremendous growth of
this hobbys widespread popularity over the past 50 years (Earle, 1995). The increased
acceptability of reliance upon manufactured feed for ornamental fish has focused the
attention on the nutritional requirements of these beautiful aquatic vertebrates. In the
present experiment the proximate composition of the diets was well in agreement
with the earlier reports. In general high protein feed is not required for rearing and
maintenance of ornamental fishes. For the growth of guppy (Poecilia reticulate) 30%
protein in diet is optimum (Fah and Leng, 1986). Shirley et al. (2006) reported 30%
protein and 12% lipids level are optimum for growth of sword tail (Xiphophorus
Table 2. Proximate composition (%) of experimental diets fed to swordtail
T0

T1

Dietary treatments
T2

T3

T4

DM

91.37

91.41

91.46

91.72

91.87

CP

31.56

31.62

31.68

31.49

31.60

EE

9.13

9.17

9.21

9.11

9.04

CF

8.69

8.54

8.52

8.48

8.46

Ingredients

TA

6.38

6.34

6.24

6.33

6.19

NFE

43.93

44.33

44.28

44.46

44.34

Basal diet supplemented with marigold petal meal at 0 (T0), 2 (T1), 4 (T2), 6 (T3), and 8 (T4) percent levels.

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Marigold petal meal as a natural carotenoid source for fish

Table 3. Physico-chemical parameters of water recorded in experimental troughs
T0

T1

Dietary treatments
T2

T3

T4

Temperature (C)

24.5

24.3

24.3

24.5

24.5

Dissolved oxygen (ppm)

6.4

6.5

6.8

6.3

6.6

pH

7.4

7.7

7.7

7.9

7.8

Hardness (ppm)

194

198

187

207

211

Alkalinity (ppm)

291

282

284

276

279

Ingredients

Basal diet supplemented with marigold petal meal at 0 (T0), 2 (T1), 4 (T2), 6 (T3), and 8 (T4) percent levels.

helleri). Loachmann and Phillips (1994), found 29% protein sufficient for optimum
weight gain, feed conversion, and protein efficiency ratio of goldfish (C. auratus). So
the diets prepared for this experiment was nutritionally balanced for the growth and
maintenance of the fish.
Water quality parameters
Physico-chemical parameters of water in all experimental tanks was estimated
fortnightly and found within the acceptable range (Alzieu, 1990) throughout the
experimental period (Table 3). Ornamental fishes are highly adaptable in culture
condition and capable of living in wide range of environment (Chapman, 2000). The
hardness of water from which many ornamental fishes originate can vary from 5 to
20 mg/l to over 300 mg/l CaCO3 (Chapman, 2000). Several species of aquarium fishes
(Mollienesia latipinna, M. senops, Xiphophorus maculatus) are well adapted in hard
water environment and grew faster than in low water hardness.
Growth performance
The absolute growth rate, specific growth rate as well as relative growth rate
in sword tail was similar in all the groups (Table 4). Similarly, increase in length
was also not varied among the groups (Table 5). The increase in length was observed
to be positively correlated with weight gain. Carotenoid known to have a positive role
in metabolism in fish (Tacon, 1981) might have better nutrient utilization and improved
growth (Amar et al., 2001). Kim et al. (1999) observed that the Korean rose bitterlings
(Rhodeus uyeki) fed with lutein, -carotene and astaxanthin supplemented diet showed
higher growth rate than the control. A positive effect of dietary carotenoid on growth
has also found in red tilapia Oriochromis niloticus L (Boonyaratpalin and Unprasert,
1989), however, in the present study no influence of dietary supplementation of
carotenoids on growth was observed.
Feed conversion ratio (FCR) in different experimental groups ranged from 1.54
to 1.76 highest being in T0 and lowest being in T4 (Table 4) and the variation among
the groups was not significant (P>0.05). Similarly, protein efficiency ratio (PER)
value was also highest in T0 and lowest in T4, however variation among the groups
was not significant. Feed conversion ratio and PER are considered as indices for feed
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Pailan et al.
Table 4. Growth performance of swordtail maintained under different experimental diets for 60 days
period
Initial
Treatments weight (g)

Final
weight (g)

AGR
(g/d)

RGR
(%)

FCR

PER

Survival
%

T0

0.350.04

0.690.06

0.340.05 97.146.92 1.760.09

0.560.04

90.0

T1

0.330.07

0.670.09

0.340.06 103.037.92 1.720.09

0.540.03

90.0

T2

0.320.07

0.680.09

0.360.06 112.507.38 1.670.07

0.530.03

93.3

T3

0.340.06

0.710.08

0.370.07 105.889.24 1.620.11

0.510.06

90.0

T4

0.300.06

0.690.08

0.390.08 130.0011.16 1.540.14

0.490.06

90.0

Basal diet supplemented with marigold petal meal at 0 (T0), 2 (T1), 4 (T2), 6 (T3), and 8 (T4) percent levels.

Table 5. Mean length of swordtail maintained under different experimental diets for 60 days period
Treatments

Initial length (cm)

Final length (cm)

Increase in length (cm)

T0

2.760.19

3.740.25

0.980.13

T1

2.810.11

3.760.24

0.950.12

T2

2.850.14

3.820.24

0.970.11

T3

2.860.09

3.670.15

0.810.13

T4

2.720.17

3.790.23

1.070.16

Basal diet supplemented with marigold petal meal at 0 (T0), 2 (T1), 4 (T2), 6 (T3), and 8 (T4) percent levels.

utilization. In the present study results clearly showed that dietary supplementation
of carotenoids has no influence on FCR and PER. However, Christiansen and Torrissen,
(1996) observed better FCR in Atlantic salmon juveniles fed with astaxanthin
supplemented diet. Similarly, in Penaeus monodon, better FCR was observed with
prawn fed on the diet containing spirulina.
The survival (%) of fish in control group was 90 and the corresponding values
ranged from 90 to 93 in different treatment groups (Table 4). The results showed that
dietary supplementation of marigold petal meal has no effect on survival of fish. The
survival of Atlantic salmon fry (Christiansen et al., 1995) and juveniles (Christiansen
and Torrissen, 1996) fed the diet without asthaxanthin supplementation was lower
than that in the groups that were fed the diets containing asthaxanthin. The different
results obtained in the present investigation may be attributed to lower densities and/
or the hardiness of species, since swordtail are very hardy species.
Biochemical composition and carotenoid level
The biochemical composition of muscle of fish in terms of moisture, crude
protein, ether extract and total ash at the end of 60 days of experimental feeding
period in all the groups was similar (Table 6). In the present study results showed
that incorporation of carotenoids in the diet through marigold petal meal have no
effect on biochemical composition of muscle of fish. Christiansen et al. (1995)
observed higher moisture and protein content and lower lipid content in the Salmo
salar fry that were fed the diets low in astaxanthin. Similar results were observed
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Marigold petal meal as a natural carotenoid source for fish

Table 6. Proximate composition of muscle and total carotenoid level of skin of swordtail fed with different
experimental diets
Treatments

Moisture
(%)

Crude
protein (%)

Ether
extract (%)

Ash
(%)

Total carotenoids (g/g)

Initial
Final

T0

76.381.39

17.501.31

4.160.26

2.540.14

2.940.38

3.17a0.31

T1

77.111.32

16.451.42

4.130.28

2.670.14

2.920.26

3.73a0.29

T2

77.171.18

16.041.72

4.320.34

2.710.16

2.870.26

5.82b0.24

T3

76.931.46

17.351.55

3.960.23

2.620.11

2.960.31

5.60b0.38

T4

76.541.62

16.251.63

4.090.31

2.590.18

3.040.19

6.17b0.27

Basal diet supplemented with marigold petal meal at 0 (T0), 2 (T1), 4 (T2), 6 (T3), and 8 (T4) percent levels.
Values bearing different superscript in a column differ significantly (P<0.05)

ab

in the Salmo salar juveniles (Christiansen and Torrissen, 1996). The different observation
in the present investigation may be due to the species difference as well as due to
the stage of maturity.
Result clearly showed that the total carotenoids concentration in the skin of
swordtail linearly increased with the increasing supplementation of marigold petals
meal in the diet (Table 6) with significantly (P<0.05) higher value in T2, T3 and
T4 groups in comparison to T0 and T1 groups. Pigmentation in fish is affected by
the concentration of dietary pigments and duration of supplementation into the basal
formulation (Torrissen, 1985). As fish cannot synthesize these pigments, they rely on
dietary supply of carotenoids to achieve their natural skin pigmentation, one of the
most important quality criteria informing the market value of ornamental high value
species (Gouveia et al., 2003). In corroboration with present observation,
Boonyaratpalin and Lovell (1977) reported that feeding of marigold petal meal to
tiger barb (Puntius tetrazona) increased the bright colouration of the fish. Total
carotenoid levels in the skin of rainbow trout, Oncorhynchus mykiss increased
significantly with increase in concentration of green alga Haematococcus pluvialis in
the feed (Sommer et al., 1992). In rainbow trout, the quantity of total pigments in
skin was greater in the fish receiving the diet supplemented with increasing amount
of spirulina (Choubert, 1979). Continuous feeding of marigold might increase
pigmentation and growth in red Xiphophorus helleri (Ezhil et al., 2008).
It can be concluded that marigold petal meal, the natural carotenoid source
successfully promoted pigmentation in swordtail (Xiphophorus helleri). This is a
highly significant development for commercial ornamental fish culture because this
carotenoid source is very cheap and abundantly available. It is therefore, recommended
that marigold petal meal as natural carotenoid source can be incorporated at 4%
level in the diet of sword tail to obtain desired pigmentation without any adverse
effect on growth, feed conversion efficiency and body composition of fish.

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Pailan et al.

ACKNOWLEDGEMENT
The authors gratefully acknowledged Dr. W.S. Lakra, Director & Vice
Chancellor, Central Institute of Fisheries Education (CIFE), Mumbai for providing
all necessary support and facilities for conducting the research work at Kolkata
Centre of CIFE, Mumbai.

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