Use of Tomato, Carrot, and Beetroot Powder as Fish

Feed Additive and Their Effects on Growth,

Carotenoid Deposition, Muscle Composition, and
Palatability of Barbonymus Gonionotus (Silver Barb)
Md. Rony Hosen
University of Rajshahi
Md. Shahidul Islam
University of Rajshahi
Atik Uj Jaman
University of Rajshahi
Shihab Hasan
University of Rajshahi
Md. Mahabubur Rahman (  mratan1980@gamil.com )
University of Rajshahi

Research Article

Keywords: Fish Pigment, Carotenoid, Growth, FCR, Proximate Composition, Taste

Posted Date: November 29th, 2022

DOI: https://doi.org/10.21203/rs.3.rs-2285544/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License.
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Abstract
The present study was conducted to investigate the effects of three plant-based dietary pigment sources
on the growth, carotene deposition, muscle quality, and palatability of Barbonymus gonionotus. A 90-day
feeding trial was conducted in twelve cages under four treatments and three replicates. A commercial
feed was used as control (CF), where three other feeds were formulated by adding 2% tomato (TM), carrot
(CR), and beetroot (BR) powder to the commercial feed. Fifteen B. gonionotus juveniles with average
weight ranging from 75.31 ± 6.43g to 76.44 ± 6.51g were stocked in each cage and the experimental
feeds were supplied at 5% of their body mass. Fish were sampled fortnightly and growth and feed
utilization indices were estimated at the end of the trial. The highest mean weight gain (193.89 ± 12.31g)
and specific growth rate (1.41 ± 0.08%) were found in CR while the lowest in CF (124.35 ± 9.20g and 1.07
± 0.07%). No significant difference was found in the survival rate among the treatments. CR also showed
the best feed conversion (1.59 ± 0.16), and protein efficiency ratio (3.07 ± 0.31) followed by TM, BR, and
finally CF. Significantly higher carotenoid content was recorded in CR (4.31 ± 0.30mg/g) while the lowest
was found in CF(0.29 ± 0.19mg/g). Results of proximate composition analysis of fish muscle showed no
significant difference across the treatments. For the palatability test, a significantly higher organoleptic
score was recorded in CR (25.54 ± 0.57) while the lowest was found in CF (19.52 ± 0.77). The study
concludes that 2% carrot powder supplementation in feed is more suitable for enhancing the growth,
flesh color, and palatability of B. gonionotus.

Trial Registration No.: RU-MSR1611067151

Introduction
Fish is one of Bangladesh's major and cheap sources of animal protein. As Bangladesh has achieved
self-sufficiency in fish production in recent times (DoF 2020), the future of aquaculture research lies in
sustainable production methods, value addition, and quality enhancement of aquaculture products. The
demand and consumer preference for fish and fish products are not only dependent on geographic
location and sociocultural norms (Pieniak et al. 2011) but also driven by the smell, taste, and coloration
of that product (Uddin et al. 2019). Coloration and smell may have both psychological and physiological
impacts on the mind and can dictate the buying behavior of consumers (Kaushik 2011) which may
ultimately reflect in the market demand for fish. Chromatophores are primarily responsible for the
coloration in fish and they are broadly classified into four types, melanin, purin, preidum, and carotenoid
(Hoar and Randall 1992). Carotenoids produce red to yellow color in the tissue and skin of fishes and
their presence can facilitate various physiological function as growth, survival rate, and disease
resistance (Ezhil et al. 2008). Like many other higher vertebrates, fish can’t synthesize carotenoids in their
body (Goodwin 1984) and therefore it needs to be supplemented through carotene enriched diet.

Many vibrantly colored vegetables such as tomato (Solanum lycopersicum), carrot (Daucus carota), and
beetroot (Beta vulgaris) are rich sources of carotenoids and prior studies have proved their significance in
fish coloration and growth when supplemented with diet (Maiti et al. 2017; Neverian et al. 2014; Jain et al.

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2019). Being an agriculturally enriched country, Bangladesh produces a significant amount of fruits and
vegetables year-round where the post-harvest loss of crops prevails as a major challenge to overcome
(Noman 2021). Additionally, a large amount of production during the peak production season leads to a
significant devaluation of the crops as well. Under such circumstances, the integration of agricultural
crops as a potential additive and carotenoid sources in fish feed manufacturing can ensure the
multipurpose use of agricultural crops, reduce loss and prove beneficial for both industries.

Barbonymus gonionotus (Silver barb) is one of the most important and fast-growing freshwater species.
It is a short-cycle species that can be farmed with easy technology and relatively less effort, thus being
popular as a farmed fish in Bangladesh. The flesh color of this species is whitish gray and if natural
carotenoid sources can be effectively used for the enhancement of the color, it will boost its market
demand in an eco-friendly way. Although, there were some research works on the effects of dietary
natural pigment on the growth and coloration of ornamental fish all over the world, no research work has
been carried out to investigate the effects of natural pigment sources on culturable minor carp species
like the Silver barb. Therefore, the present study was conducted to evaluate the change in growth, muscle
composition, carotene deposition, and palatability of B. gonionotus under a tomato, carrot, and beetroot
powder enriched feeding regimen.

Methodology
Collection and Preparation of Pigment Source

The experiment evaluated the effects of three natural pigment sources, tomato, carrot, and beetroot which
were purchased from a local vegetable market called Shaheb Bazar, Rajshahi. After collection, they were
washed and cut into thin slices, and dried under sunlight until gaining constant weight. After drying, these
slices were ground and converted into powder, and stored in a polythene bag for subsequent use.

Experimental Design

A 90-day feeding trial from July to September 2021 was conducted in 12 cages set in an earthen pond.
Each of the cages was 96ft3 in volume and covered with synthetic knotless nets with a mesh size of 5
mm. The cages were also equipped with a feeding tray and an opening at the top for feeding and
sampling purpose. A commercial carp grower feed from ACI Agrovet was used as control (CF) and three
additional types of feed were formulated by incorporating tomato (TM), carrot (CR), and beetroot (BR)
powder separately at a rate of 2% per kg selected commercial feed. These four types of feed were
considered as four treatments and were arranged in a complete randomized design (CRD) across the
settled cages with 3 replications for each. The proximate composition analysis of the experimental feeds
exhibited no statistically significant difference among them and the feeds were isoproteinic and isolipidic
in composition (Table 1).

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 Table 1
Chemical composition of experimental feeds (% wet basis)
Component (%) CF TM CR BR

Moisture 13.18 ± 0.50a 13.51 ± 0.41a 13.44 ± 0.78a 13.36 ± 0.35a

Protein 20.45 ± 0.21a 20.59 ± 0.31a 20.47 ± 0.18a 20.51 ± 0.25a

Lipid 5.22 ± 0.47a 5.35 ± 0.30a 5.18 ± 0.42a 5.57 ± 0.15a

Ash 9.7 ± 0.35a 9.01 ± 0.23a 9.22 ± 0.18a 9.26 ± 0.14a

Fiber 8.34 ± 0.17a 8.26 ± 0.09a 8.45 ± 0.12a 8.14 ± 0.15a

Carbohydrate 43.11 ± 0.60a 43.28 ± 0.52a 43.24 ± 0.41a 43.16 ± 0.25a

Values in the same row with different superscripts are significantly different (P<0.05).

Collection, Stocking, and Rearing of Experimental Fish

180 juveniles of B. gonionotus of similar size and weight were purchased from a local fish farmer and
were transported in the presence of an aeration system and acclimatized in a circular cemented tank for a
week and fed with the selected commercial feed. After the acclimation period, fifteen fish were randomly
selected, weighed, and distributed in each cage under the four treatments (CF, TM, CR, and BR). The initial
weight of the experimental fish ranged from 75.31 ± 6.43g to 76.44 ± 6.51g across the treatments with no
significant difference among them (Table 4). During the feeding trial, fish were fed with a meal size of 5%
of body weight divided into two rations, one in the morning and the other in the afternoon. Each fish from
the cages were fortnightly sampled for weight measurement to adjust the ration size throughout the trial.
Additionally, leftover feed in each feeding tray was collected and dried after one hour of providing the
ration and the total amount of leftover feed was culled while estimating the feed utilization parameters at
the end of the trial.

Monitoring Water Quality Parameters

During the experimental period, water temperature, pH, DO, total alkalinity, and NH3-N were measured
fortnightly with standard methods (Table 2). There was no significant difference in water quality
parameters among the treatments and were within the suitable range.

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 Table 2
Water quality parameters across the treatments
Parameters CF TM CR BR

Temperature (°C) 28.53 ± 0.91a 29.46 ± 0.69a 27.45 ± 0.71a 28.92 ± 0.67a

DO (mg/l) 6.55 ± 0.46a 6.48 ± 0.72a 6.64 ± 0.43a 6.18 ± 0.45a

pH 7.53 ± 0.19a 7.56 ± 0.29a 7.35 ± 0.23a 7.41 ± 0.27a

Alkalinity (mg/l) 145.28 ± 4.14a 149.31 ± 4.07a 143.5 ± 10.25a 147.43 ± 5.54a

NH3-N (mg/l) 0.024 ± 0.006a 0.039 ± 0.012a 0.031 ± 0.009a 0.047 ± 0.005a

Values in the same row with different superscripts are significantly different (P<0.05).

Analysis of Growth and Feed Utilization

After the feeding trial, the final weights of the fish were measured individually. Growth performance was
estimated as mean weight gain (MWG), percent weight gain (PWG), specific growth rate (SGR), and
survival rate (SR) whereas, feed utilization was evaluated by feed conversion ratio (FCR), and protein
efficiency ratio (PER) according to the standard formulae.

M W G = M eanF inalW eight (M F W ) − M eanInitialW eight(M IW )………………………..(1)

M F W − M IW
PWG = × 100 … … … … … … … … … … … … … … (2)
M IW

ln (M F W ) − ln (M IW )
SGR = × 100 … … … … … … … … … . (3)
C ultureDuration (Days)

N o. ofF ishH arvested

SR = × 100 … … … … … … … … … … … . (4)
N o. ofF ishStocked

F eedF ed
F CR = … … … … … … … … … … … … … … … … (5)
LiveW eightGain

LiveW eightGain
P ER = … … … … … … … … … … … … … … … … (6)
P roteinF ed

Carotenoid Deposition Analysis

The fresh fish muscle samples from each replicate were weighted and ground by mortar and pestle using
90% methanol. After grinding, the sample mixtures were subsequently centrifuged at 12000 rpm for 12
minutes and the supernatant was transferred into another centrifuge tube. Finally, the optical density
(OD) of the supernatant was recorded at 662 nm, 653 nm, and 470 nm by using a spectrophotometer
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(Analytic Gena, Germany). The total concentration of carotenoids was measured by using the following
equation (Lichtenthaler and Wellburn 1983).

Where, A666 = OD at 666 nm, A653 = OD at 653 nm, and A470 = OD at 470 nm.

Chemical Analysis of Feed and Fish Flesh

Crude protein, lipid, fiber, carbohydrate, ash, and moisture content in the formulated feeds and muscle of
the experimental fish from each replicate were determined by using the standard method (AOAC 2005).

Palatability Test

250g of fish flesh was collected from each of the treatments and was boiled-cooked following the
traditional procedure. After cooking, the cooked fish was served to a panel of twelve members for sensory
evaluation according to a specific structured scaling system (Table 3) described by Huss (1995). The
panelists blindly gave their scores on the odor, taste, and texture of the cooked fish.

Table 3
Organoleptic scoring sheet for palatability evaluation
Sensory Attributes Score

Odor Taste Texture

Species-specific Meaty flavor Firm/elastic 10

Fresh fish Sweet Firm/springy 8

Slightly fishy or slightly sour Slightly fishy Less firm 6

Sour and stale Slightly sour/some off flavor Softer 4

Strong ammonia Slightly rotten Very soft 2

Rotten smell Spoiled Slippery 0

Statistical Analysis

After determining the normality of the data set through Levine’s test, a one-way analysis of variance
(ANOVA) was conducted to test for the statistical difference by using SPSS 21 (statistical package for

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social science). The significant difference among the mean values was determined by Duncan’s multiple
range test at level 5% (P < 0.05).

Results
Growth Performance

All the feeds with added natural pigment sources (TM, CR, and BR) exhibited significantly better growth
performance compared to the control group CF (Table 4). The highest MWG, PWG, and SGR were
recorded in CR followed by TM, BR, and finally CF. However, the growth performance of TM and BR didn’t
differ significantly from each other. Additionally, natural pigment supplementation didn’t affect the SR of
the experimental fish as it was found to be constant across the treatments.

Table 4
Growth performance of the experimental groups
Parameters CF TM CR BR

Initial weight (g) 76.44 ± 6.51a 75.31 ± 6.43a 76.13 ± 4.80a 76.06 ± 5.01a

Final weight (g) 200.81 ± 250.19 ± 269.63 ± 244.06 ±

10.20c 11.99b 12.45a 13.03b

Weight gain (g) 124.35 ± 9.20c 174.86 ± 193.89 ± 168.56 ±

11.50b 12.31a 12.87b

Percent Weight gain (%) 162.83 ± 1.28c 232.18 ± 1.04b 255.90 ± 1.16a 222.53 ± 1.19b

Specific Growth Rate (% 1.07 ± 0.07c 1.33 ± 0.01b 1.41 ± 0.08a 1.30 ± 0.08b
bwd− 1)

Survival rate (%) 100 ± 00a 100 ± 00a 100 ± 00a 100 ± 00a

Values in the same row with different superscripts are significantly different (P<0.05).

Feed Utilization

The FCR and PER values of TM, CR, and BR were found to be significantly better compared to the control
group CF while CR demonstrated the best values in these parameters (Table 5). Similar to the growth
performance, no significant difference was observed between the FCR values of TM and BR. The PER
values of TM, CR, and BR didn’t show any significant difference among them as well.

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 Table 5
Feed utilization parameters of the experimental groups
Parameters CF TM CR BR

FCR 2.5 ± 0.28c 1.74 ± 0.04b 1.59 ± 0.16a 1.84 ± 0.19b

PER 1.97 ± 0.22b 2.78 ± 0.06a 3.07 ± 0.31a 2.66 ± 0.28a

Values in the same row with different superscripts are significantly different (P<0.05).

Carotenoid Deposition

The concentrations of carotenoid in the fish muscles of TM, CR, and BR were significantly higher than in
the control CF. Figure 1 shows that the carotenoid content in the fish muscle of CR (4.31 ± 0.30 mg/g)
was the highest followed by TM (1.57 ± 0.09 mg/g) and BR (1.44 ± 0.40 mg/g) whereas the lowest
content was recorded in CF (0.29 ± 0.19 mg/g). However, no significant difference in carotenoid
deposition was found between TM and BR. The apparent change in flesh coloration due to the possible
effect of carotenoid deposition is shown in Fig. 2 which suggests a relatively higher amount of deposition
around the spinal region.

Muscle composition

Changes in muscle compositions of the fish fed with pigments included feeds are shown in Table 6. No
significant differences in moisture, protein, lipid, ash, carbohydrate and fiber content were found among
the treatments. However, relatively higher muscle protein and ash content were recorded in BR whereas
relatively higher moisture, lipid, and fiber content were found in CR compared to the control group CF.

Table 6
Muscle compositions of the experimental fish
Component (%) CF TM CR BR

Protein 14.50 ± 1.16a 14.82 ± 0.23a 14.44 ± 0.04a 15.15 ± 0.07a

Lipid 2.18 ± 0.03a 2.06 ± 0.11a 2.23 ± 0.33a 2.22 ± 0.007a

Ash 1.70 ± 0.02a 1.82 ± 0.07a 1.70 ± 0.31a 1.86 ± 0.27a

Carbohydrate 9.22 ± 0.83a 10.68 ± 1.09a 9.32 ± 1.86a 10.37 ± 0.39a

Fiber 1.05 ± 0.19a 1.03 ± 0.11a 1.25 ± 0.007a 1.11 ± 0.18a

Moisture 70.64 ± 0.62a 70.47 ± 0.0a 71.06 ± 1.87a 69.60 ± 0.72a

Values in the same row with different superscripts are significantly different (P<0.05).

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Palatability Test

After the consumption of cooked fish, a number of consumers made their comments on odor. taste and
texture of fish through the specific score on a score sheet and the mean scores are shown in Table 7. In
the present study, it was observed that cooked fish muscle in CR was ranked 1st according to the odor
and taste scores given by the consumers followed by TM, BR, and CF.

Table 7
Mean sensory test scores for the palatability test of cooked fish
Organoleptic Criteria CF TM CR BR

Odor 6.51 ± 0.45c 7.5 ± 0.45b 8.52 ± 0.52a 7.7 ± 0.63b

Taste 6.5 ± 0.65c 7.66 ± 0.47b 8.61 ± 0.78a 7.8 ± 0.63b

Texture 6.59 ± 1.23c 7.4 ± 1.65b 8.41 ± 0.43a 6.1 ± 1.63c

Total Score 19.52 ± 0.77c 22.56 ± 0.85b 25.54 ± 0.57a 21.6 ± 0.96b

Rank 4th 2nd 1st 3rd

Values in the same row with different superscripts are significantly different (P<0.05).

Discussion
In the present investigation, different natural pigments (tomato, carrot, and beetroot powder) were added
to a commercial feed at the same doses (2%) in order to investigate the possible effects on the growth
and feed utilization of Silver barb. The inclusion of natural pigments in the feed appeared to have
positive effects on the growth of fish. Fish supplemented with a carotenoid enriched diet generally
exhibits better growth performance possibly due to improved metabolism and nutrient utilization (Amar et
al. 2001). According to the findings of this study, fish treated with commercial feed (CF) without
additional natural pigments showed the poorest growth performance and feed utilization. Contrastingly,
the highest growth performance and feed utilization were obtained in the fish fed with carrot powder
treated feed (CR) followed by tomato powder (TR) and beetroot powder (BR) treated feed (Tables 4 and
5). This result is in agreement with Maiti et al. (2017) where significantly better weight gain was reported
in Cyprinus carpio by supplementing carrot powder treated feed followed by tomato and beetroot. This
outcome also coincides with the findings of Tiewsoh et al. (2019) where the highest specific growth rate
of goldfish (Carassius auratus) was found in 6% carrot powder treated fish compared to the control group
(0% carrot). Lakshmi et al. (2015) also reported an improved growth rate of albino Tiger barb by using
carrot powder enriched feed compared to the dietary provision of beetroot. Supporting evidence of the
current study has also been found in the reports of Swian et al. (2014) who studied the positive effects of
natural pigments on the growth of Koi carp. The result also associates with the findings of Jain et al.

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(2019) where the highest weight gain and specific growth rate of Cyprinus carpio were found in carrot
(5%) enriched feed than the fish treated without it.

Besides the effectiveness of carotenoids in growth and carotenoid deposition, it has also a positive effect
on the nutrition and survival rate of fish. Torrissen and Christiansen (1995) reported that carotenoid
sources like astaxanthin are essential for the growth and survival of fish and crawfish. However, in this
experiment, the result showed that there was no significant difference in survival rates among the
treatment. This could be the possible outcome of stocking relatively larger-sized fish whereas dietary
carotenoids are more likely to improve larval or fingerling survival rates according to prior studies.

The findings of this experiment suggest that natural pigment inclusion in feed leads to a positive effect
on carotenoid deposition in fish muscle. The fish's digestive tract progressively converts the β-carotene
they consume into astaxanthin (Tietze 1998). When an adequate amount is supplemented through diet,
they slowly get deposited in the chromatophore cells and modify the coloration of the muscle (Gupta et
al. 2006). The current experiment result showed that fish groups treated with pigments-enriched feed had
higher carotenoid content than the control groups (Fig. 1). A similar result was obtained by Maiti et al.
(2017) where the inclusion of natural pigments (carrot, tomato beetroot, and their mixture) in feed
increased the carotenoid deposition in Cyprinus carpio. Similarly, Tiewsoh et al. (2019) found that the
gradual higher inclusion of carrots in feed increases the carotenoid content in the goldfish. The present
study result also associates with the findings of Swian et al. (2014) and Ezhil et al. (2008) where higher
carotenoid deposition was found in the Xiphophorus helleri and Cyprinus carpio treated with natural
pigments enriched feed. Neverian et al. (2014) have also reported improved fillet coloration of rainbow
trout when supplemented with beetroot juice enriched feed.

No research on pigment inclusion in fish diet and its effect on muscle composition in B. gonionotus was
found in the existing literature. Nonetheless, numerous researchers have conducted experiments to find
out the effect of various types of diet on the body composition of B. gonionotus. The result of the present
experiment showed no significant difference in moisture, crude protein, fiber, and ash content of B.
gonionotus. The result was in conformity with the findings of Kamaruddin et al. (2018) who reported that
there was no difference in moisture, crude protein, and ash content of B. gonionotus treated under
different dietary fiber levels. In the present study, the findings of carcass protein composition exhibited no
significant difference and similar crude protein content in the carcass of B. gonionotus was reported by
Mohanta et al. (2009) when trialed under diets with varying carbohydrate levels. In the present experiment
carcass lipid content coincides with the results of Das et al. (2018) where similar lipid content was found
in B. gonionotus when supplemented with an Azolla-enriched diet. Overall, the present findings indicate
that the inclusion of natural pigment sources in the diet does not impart any adverse effect on the
carcass composition of B. gonionotus.

The palatability test of the experimental groups suggests that dietary pigment source inclusion can
positively impact the odor, taste, and texture of B. gonionotus. Arguably, this could the possible outcome
of increased carotenoid deposition in fish tissue that may have contributed to the development of

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characteristic odor, taste, and texture compared to the control group. However, supporting evidence of
such an outcome can’t be found in the existing literature and therefore requires further investigation for a
more accurate assumption.

Conclusion
The present experiment indicates that the natural sources of carotenoids can be effectively utilized by
silver barb along with carotenoids deposition in skin and muscle and growth of fish. The fish fed with 2%
carrot-enriched feed was much more effective in enhancing the growth and carotenoid deposition in B.
gonionotus. The findings can be helpful to develop a more economically viable production of B.
gonionotus for the promising aquaculture industry in our country and can also establish an alternate
usage and reduce the economic effect of post-harvest loss of agricultural products.

Declarations
Funding

The experiment was funded by Rajshahi University Research Grant Commission (Grant No: RUSG-
202107).

Competing Interests

The authors state that they have no known conflicting financial or non-financial interests or personal ties
that may be seen as having influenced the work described in this manuscript.

Author Contributions

All authors contributed to the conception and design of the experiment. Material preparation, data
collection, and analysis were performed by Md. Rony Hosen, Md. Shahidul Islam, Atik Uj Jaman, and
Shihab Hasan. The first draft of the manuscript was written by Md. Rony Hosen and Md. Shahidul Islam.
All authors read and approved the final manuscript for submission. The collection of the fund and overall
supervision of this investigation was done by Md. Mahabubur Rahman.

Data Availability

The datasets generated during and/or analyzed during the current study are available from the
corresponding author upon reasonable request.

Ethics Approval

The Rajshahi University Research Ethics Committee has exempted the need for ethics approval for this
investigation in accordance with the National Wildlife Protection and Conservation Act, 2012 as the fish
species used in this study are commercially produced and not wild or endangered species. However, all

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procedures performed in this investigation were in accordance with the ethical guidelines provided by The
International Council for Laboratory Animal Science (ICLAS) for researchers.

Consent to Participate

Evaluators of the organoleptic test gave their verbal informed consent to use their data in this
submission.

Consent to Publish

All authors confirm that this original article is not published before and is currently not under
consideration for publication anywhere else and approval for the submission of this manuscript is
provided.

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https://doi.org/10.1080/10498850.2019.1597004

Figures

Figure 1

Flesh carotene content of the experimental groups

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Figure 2

Flesh coloration of the experimental groups

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