Effect of Citrus Peels Mingled Diets on Carassius auratus Coloration

Sumaira Abbas1, Muhammad Sultan Haider2,*, Fatima Kafayet1, Sana Ashraf2, Atifa Masood3 and Moazma Batool4

1Department of Fisheries and Aquaculture, University of Veterinary and Animal Sciences, Lahore

2Department of Zoology, The University of Lahore, Lahore

3Department of Botany, The University of Lahore, Lahore

4Department of Zoology, GC Women University, Faisalabad

ABSTRACT

Present project was an endeavor to use natural carotenoid sources to enhance skin color of gold fish Carassius auratus. Citrus peels were collected from local market dried and grinded. Organic solvent extraction was carried out by hexane and acetone mixture (1:1 v/v). Carotenoid concentration was determined by thin layer chromatography (TLC) and found satisfactory. By mixing fish meal, sunflower meal and rice polish four treatment diets, T1, T2, T3, were prepared and citrus peel powder was added @ 200g, 400g and 600g/ 1000g, respectively. While fourth one, control diet (To) was without citrus peels. These is nitrogenous diets (30% protein) were offered to Carassius auratus juvenile having body weight 20±7.54g in powder form for 92 days. Weight gain, length gain and FCR were calculated fortnightly. At completion of feeding trial, color intensity and pigment concentration was measured in Carassius auratus skin. Statistical analysis of results showed non-significant differences (P<0.05) among all treatments in weight gain and FCR. Maximum color intensity 1.52±0.08 and carotene concentration in lateral and tail region, 0.4±0.01 and 0.05 ±0.02 were recorded in T3 without any harmful effect. It is concluded that citrus peels are good, natural, low price Carotene source for color enhancement.

Article Information

Received 07 November 2016

Revised 22 July 2018

Accepted 23 October 2018

Available online 17 January 2020

Authors’ Contribution

MSH and S Abbas planned the research and wrote the manuscript. FK conducted research work. AM, MB and S Ashraf helped in data analysis and paper-writing.

Key words

Carotene, Goldfish, TLC, Growth, Color.

DOI: https://dx.doi.org/10.17582/journal.pjz/20161107041109

* Corresponding author: [email protected]

0030-9923/2020/0002-0519 $ 9.00/0

Copyright 2020 Zoological Society of Pakistan

Introduction

Now a days, ornamental fishes are rapidly gaining importance because of their aesthetic and commercial value in the export trade all over the world. Attractive colouration determines the commercial value of any ornamental fish. Pigmentation in the skin is responsible for colouration in the fish (James and Sampaths, 2003). Ornamental fish keeping is also a popular hobby, among young and old alike. It has been estimated that 1.5 to 2 million people worldwide keep marine ornamental fish aquaria (Mandal et al., 2010; Lim and Wong, 1997). Estimated value of marine ornamental trade is 200-330 million US$ per year (Degnai and Gur, 1992).

Carotenoids is the primary source of the pigmentation in fish skin. Fishes cannot synthesis carotenoid in natural environment, so they meet their carotenoid requirements by ingesting specific aquatic plants (Zhao et al., 2003). Dietary carotenoids play significant role in regulation of skin and muscle color in fish after absorption carotenoids are transformed into other carotenoids if necessary, and incorporated into tissues. Carotenoids are vital for healthy growth, metabolism, and reproduction, as well as color, in fish (Li et al., 2005). Astaxanthin is the main carotenoid pigment of red-pink coloured aquatic animals, being widely used in aqua-cultural processes due to its chemical stability (Madhupratap et al., 2001). The colour enhancing diets should contain additional natural pigments to enhance the colour of ornamental fish (Rickman et al., 2007).

Carotenoids in fish feed give yellow, red and pink color to fish skin, flesh and eggs. Some 600 species of plant and water organism contain these colorant substances (Irie and Seki, 2002; Furuita et al., 2003). Astaxanthin, which is the most effective coloring pigment exsists densely in water organisms such as gammarus, copepods etc and starfish (Ando et al., 1992).

Astaxanthin and canthaxanthin are widely prepared artificially and used as supplements in diets for goldfish and other ornamental fishes to induce the desired coloration (Kithara, 1983).

Goldfish (Carassius auratus) is a widely cultured and commonly traded ornamental fish in the global aquarium business (Shahidi et al., 1998). Color is a major factor that determines the quality and market value of any ornamental fish (Kitahara, 1983). Skin pigmentation of goldfish has been accomplished by supplementing their diets with synthetic or extracted carotenoids, such as zeaxanthin, lutein or astaxanthin (Royes et al., 2006; Seyedi et al., 2013). From natural sources containing Carotenoids, the Red Yeast, Xanthophyllomyces dendrorhous (Wang et al., 2006), Spirulina (Wallat et al., 2002), Chlorella vulgaris, Haematococcus pluvialis and Arthrospira maxima (Gouveia et al., 2003) have already been used for pigmentation of Goldfish. Citrus (Citrus reticulate) peels have been tested as a protein source in some ornamental fish species (Reitain et al., 1997; Passos et al., 2007). However, nothing has been reported on its use as a pigment source for goldfish or other aquatic animals, except in freshwater crayfish, Cheraxquadri carinatus (Paulo and Antonio, 2005). So, recent effort has focused on use of this natural carotenoid source citrus (Citrus reticulate) peels as alternative to synthetic carotenoids in goldfish diet for color enhancement.

 

Materials and methods

Site and sample collection

Present experiment was conducted in Fish Hatchery and Laboratory, University of Veterinary and animal Sciences, Lahore, Pakistan. Goldfish of uniform size 20±7.54g were collected from ornamental fish market, Akbar Chowk, Lahore and acclimatized to laboratory conditions for one week before initiation of experiment. Complete randomized design (CRD) was used in this study. Citrus (Citrus reticulate) peels were collected from local market, dryed and grinded in powder form by electric grinder machine.

Pigment extraction from citrus peel

Veronica et al. (2010) was followed for pigment extraction from citrus peels. Citrus peel powder (200g) was weighed and immersed in 400 ml acetone solution and shaked well. The mixture was kept at room temperature, in darkness, for 12 h and continuously shaken by magnetic stirrer. Afterward mixture was filtered and evaporated on hot plate. Residues were extracted with petroleum ether and extract was left to saponify for 12 h at room temperature. Later, it was washed with 100 ml ether and diluted with 200 ml distilled water. Two phases were successively partitioned at this stage. The upper lipophilic phase contains the carotenoids. These carotenoids were concentrated on hot plate almost to dryness and stored till further use. The stored extract was dissolved in acetone and submitted to TLC on silica using petroleum ether and diethyl ether in same ratio.

The total carotenoids contents were calculated as μg per g weight of sample as follows:

Where, 10 is the dilution factor and 0.25 is the extinction coefficient.

Feeding trial

Fish feed was prepared by mixing traditional feed ingredients i.e. fish meal, sunflower meal and rice polish following Gupta et al. (2007) and were termed as T1, T2, T3 and T0. All diets were iso-nitrogenous with 30% protein level and were offered to fish in powdered form. Already ground citrus peels were mixed @200, 400, 600g/1000 g of basal diet in T1, T2 and T3, respectively, while fourth treatment diet T0 was control and prepared without citrus peels.

After preparation, proximate analysis of diets was performed for determination of total moisture, proteins, lipids and ash contents following AOAC (2000). When nutritional value of prepared diets was found satisfactory, prepared diets were offered to experimental fish in troughs @ 4% body weight for 90 days. The experiment was conducted in triplicate in 4 troughs having 70 liter volume capacity. In each experimental group for 90 days. Experimental groups were termed as G1, G2 and G3. In each group, fifteen fish approximately of equal size (20±7.54 g) were randomly selected and kept. Water quality parameters (dissolve oxygen level, temperature and pH) were recorded on daily basis while growth parameters like weight gain (WG), length gain (LG), feed conversion ratio (FCR), specific growth rate (SGR) and survival rate (SR) were calculated fortnightly. At the end of feeding trial, three fish were randomly selected from each treatment group and were subjected to pigment extraction procedure.

Fish growth parameters were calculated by following formulas:

 

Pigment extraction from fish tissue

At the end of feeding trial, color pigment was extracted from fish tissue following Sant’Anna et al. (2005). One gram of fish body tissue was taken and mixed with 2.5 g of anhydrous sodium sulphate and gently meshed. Then 5ml of chloroform was added in it and left overnight at 0°C. Chloroform formed a clear 1-2 cm layer of carotenoids on upper surface of this mixture. The layer was removed and its optical density was recorded at ƛ470nm using spectrophotometer, taking 0.3 ml of chloroform diluted aliquots mixed with 3ml absolute ethanol. Blank tube was used as standard for comparison. Wavelength at which maximum absorption was recorded was used for calculation of carotenoid concentration. Calculations were made by formula given below:

Where, C is concentration (µg/g for tissue), 2100 is E (1%, 1 cm) or extinction coefficient of carotenoids in hexane at 472 nm and 10000 is the scale factor.

Skin colour analysis

For color analysis skin samples were taken from from lateral and tail regions. Reflectance spectroscopy and Portable Minolta Chroma Meter CR-300 (Konica USA) was used for color analysis following (Storebakken, 1987).

Statistical analysis

For statistical analysis SAS Version 9.4 was used. One way analysis of variance (ANOVA) was applied on data to find out the significant differences among means. Results were considered significant at (p< 0.05).

 

Results and discussions

Proximate analysis

Four treatment diets were prepared by mixing fish meal, sun flower meal and rice bran. Citrus peel (Citrus reticulate) powder was incorporated in diets at different ratios. According to Govind (2013) nutrients required for commercial and ornamental fish are same as per other animals.

 

Table I.- Nutritive evaluation of different treatment diets based on different ratio of citrus peel powder.

Tr.	Moisture	Protein	Fat	Ash	Orange peel g/kg
T1	4.89±0.57b	31.88±0.52a	7.53±1.32b	6.58±0.60b	200
T2	6.69±0.43a	32.03±0.15a	9.40±0.63a	8.41±0.76a	400
T3	5.08±0.83b	32.10±0.29a	6.69±0.52b	8.79±0.58a	600
To	6.05±0.66ab	31.81±0.21a	4.75±0.84c	5.70±0.50b	-

Means with similar letters in a column are statistically non-significant (p<0.05).

 

According to Wang et al. (2006) various nutrients vary in proportion according to fish type. Protein concentrations vary from 30-32% in diets. Proximate analysis of four treatment diets was performed following AOAC (2000). All treatment diets were isonitrogenous and protein level was maintained at 30%. While moisture, fat and ash ratio vary in all diets, the values are presented in Table I.

According to Ho et al. (2014) protein is most crucial nutrient in fish feed and its requirements vary according to ornamental fish species, size, feeding rate and water quality parameters. According to Sim et al. (2005) protein requirement of guppy (C. reticulate) is 30-35%%, goldfish (C. auratus) is 30%. By observing these arguments it can be said that our prepared feed is suitable for ornamental fish. According to Wilson (1994) and Halver and Hardy (2002) fish growth rate mostly solely depends upon protein utilization rate. Remaining parameters like moisture, protein, fat and ash contents were also within range as described by different authors (Sargent et al., 1995; Li and Galtin, 2008). However, the concentrations of orange peel vary in all treatment diets.

Rf value is ratio between distance travelled by a pigment, solute and the solvent. In other words Rf = (distance moved by solute) / (distance moved by solvent) (Hartely and Kennedy, 2006).

Calculated Rf values of extracted pigments are presented in Table II. According to our results T1 showed lycopene and xanthophyl with RF value 0.88 and 0.86, respectively. In T2 leutine and pheophytin were found with Rf value 0.68 and 0.71. While in T3 xanthophyl, α and ß-carotene were found having Rf value of 0.89 and 0.90. These values are in line with values as described by Gouveia et al. (2003) and Donato et al. (2003). They described Rf values for xanthophyl, chlorophyl b and a and carotene, as 0.16, 0.32, 0.44, and 0.91, respectively. So our selected orange peel powder has enough concentration of carotenoids and can be used as color enhancer in ornamental fish diet.

 

Table II.- Rf value and colors pattern of different plates showed under UV lamp.

Tr. No	Replicate No	Rf Value	Colors	Pigment name
1	1	0.88	Red-Orange	ß-carotene Zeaxanthin Astaxanthin
	2	0.86	Yellow
	3	0.87	Yellow
2	1	0.88	Yellow – brown	ß-carotene α−carotene Zeaxanthin
	2	0.89	Yellow – brown
	3	0.81	Olive Green
3	1	0.89	Yellow – Orange	−carotene Xanthophyll ß-carotene
	2	0.90	Yellow – orange
	3	0.88	Yellow – orange

 

Growth parameters

Fish growth parameters like weight gain, length gain, FCR, SGR and survival rate were recorded fortnightly (Table III). All treatments showed non-significant variation for weight gain among all treatment diets, however in case of FCR T1 and T3 showed significant difference (p<0.05) from other two treatments T2 and T4. Total weight gain was recorded as 39.76±1.12, 40.63±1.43, 34.85±1.61and 42.34±1.21 among T1, T2, T3 and T0. While FCR was recorded as 3.31±1.74, 2.93±0.85, 3.87±1.07 and 2.43±1.37 among T1, T2, T3 and T0. This variation in weight gain can be justified by Macartney (1996) and Sales and Janssens (2003), who described that in captivity ornamental fish may show variation in protein digestibility. The difference in FCR value among four treatments can be justified by Pannevis (1993) and Lim et al. (2001). They stated that along with protein other components like lipids, carbohydrates and minerals also effect growth rate of fish. As there is variation in feed proximate analysis results so there is also variation in FCR values in our treatment diet results.

 

Table III.- Weight gain, length gain, FCR, SGR, and survival rate of Carassius auratus fed on different treatment diets.

Tr.	Total weight gain	Total length gain	FCR	SGR	Survival %
T1	39.76±1.12a	12.51±2.56c	3.31±1.74a	0.95a	100%
T2	40.63±1.43a	13.37±3.18b	2.93±0.85b	1.27b	100%
T3	34.85±1.61ab	13.81±1.12b	3.87±1.07a	0.91a	100%
T0	42.34±1.21a	15.50±1.25a	2.43±1.37b	1.29b	100%

Means with similar letters in a column are statistically non-significant (p<0.05).

 

Table IV.- Carotenoid concentration in lateral, tail region (μg/g) and color intensity in muscle region of Carassius auratus in different treatment groups of fish tissue.

Sample	T0	T1	T2	T3
Lateral region	0.03±0.03a	0.3±0.04b	0.4±0.01b	0.8±0.01c
Tail region	0.02±0.01a	0.03±0.02a	0.05±0.02b	0.06±0.01b
Muscle color	1.23±0.15a	1.32±0.17ab	1.52±0.08b	1.72±0.11c

Muscle color was observed by comparing muscle sample to Salmonids Roche TM Color Card. Means with similar letters in a column are statistically non-significant (p<0.05).

 

Color assessment

Recoded color assessment and total number of carotenoid calculated are presented in Table IV. During experiment all groups were refractive to color enhancer. Skin color was estimated by comparing muscle sample with Salmonids Roche TM Color Card (Lim et al. 2001). In lateral region, carotenoid contents were measured as 0.3±0.04, 0.4±0.01, 0.8±0.01 and 0.03±0.03 in T1, T2, T3 and T0, while in tail region the carotenoid concentration was recoded as 0.03±0.02, 0.05±0.02, 0.06±0.01 and 0.02±0.01 for T1, T2, T3 and T0, respectively. Carotenoids are categorized as micronutrient and attractant for fish. For ornamental fish addition of carotenoids in fish diet enhance color pigment concentration in fish muscle (Hardy and Barrows, 2002). As the concentration of carotenoids increase in fish diet its color also enhanced.

Kruger et al. (2001) reported that when swordtail fish was fed with 40-80 mg kg-1 astaxanthin equivalent of orange peel carotenoids for eight weeks, it scored the highest for customer preferences and its muscle color had no difference with those treated with astaxanthin (Carophyll Pink TM) at same concentration. All groups showed enhancement in muscle color. Muscle color concentration was recorded as 1.32±0.17, 1.52±0.08, 1.72±0.11 and 1.23±0.15 for T1, T2, T3 and T0, respectively. These results are in agreement with results of Woods (2003), who reported similar findings when rainbow trout fed with from H. roretzis extract.

 

Conclusions

It can be concluded from above experiment that citrus (Citrus reticulate) peel is a good source of carotenoids and when given in feed positively effect color pattern in goldfish (Carrassius auratus).

 

Acknowledgements

The authors would like to express their gratitude to Higher Education Commission of Pakistan (HEC) and University of Veterinary and Animal Sciences, Lahore for facilitating the current project.

 

Statement of conflict of interest

The authors declare no conflict of interest.

 

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