COLOR ENHANCEMENT IN THE ORNAMENTAL RED ......also be absorbed by colorants such as dye or pigment...
Transcript of COLOR ENHANCEMENT IN THE ORNAMENTAL RED ......also be absorbed by colorants such as dye or pigment...
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COLOR ENHANCEMENT IN THE ORNAMENTAL RED ZEBRA CICHLID, PSEUDOTROPHEUS ESTHERAE BY ADDITION OF CAROTENOIDS TO THE DIET
By
SERDAR YEDIER
A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
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© 2013 Serdar Yedier
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To my parents, who supported me in every stage of my Master of Science
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ACKNOWLEDGMENTS
I thank Allah, my source of strength every day and the reason I constantly strive
to be a better person. I thank my parents and friends from Turkey, who gave me a
strong moral code, knowing that eventually I’d straighten up my act.
I would like to express my sincere appreciation to my supervisor Dr. Frank
Chapman for his encouragement. He is really friendly and helpful; I have always
admired his relationship with his students. His philosophy of science will lead me in my
career as a scientist.
I thank my committee members Dr. Daryl C. Parkyn and Dr. Mark Brenner for
useful directions and suggestions. They helped me to place the results of my
experiments in a wider perspective. Special thanks to my good friend and colleague
Elisa Livengood for the help she gave at every step of my study. I would also like to
thank Emir Yasun, PhD candidate in the chemistry department at UF, who helped in the
total carotenoid analysis of fish diets. I really appreciate Enrique Schmalbach of
Schmalbach Aquaculture, who donated all the red zebra cichlids.
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TABLE OF CONTENTS
page
ACKNOWLEDGMENTS .................................................................................................. 4
TABLE OF CONTENTS .................................................................................................. 5
LIST OF TABLES ............................................................................................................ 6
LIST OF FIGURES .......................................................................................................... 8
ABSTRACT ..................................................................................................................... 9
CHAPTER
1 INTRODUCTION .................................................................................................... 11
2 MATERIALS AND METHODS ................................................................................ 24
Experimental Diets .................................................................................................. 24 Fish and Experimental Design ................................................................................ 25 Fish Images and Fish Skin Colors Analysis ............................................................ 27
Total Carotenoid Analysis ....................................................................................... 29 Growth and Survival Rate ....................................................................................... 30
Statistical Analysis .................................................................................................. 30
3 RESULTS ............................................................................................................... 32
4 DISCUSSION ......................................................................................................... 58
LIST OF REFERENCES ............................................................................................... 63
BIOGRAPHICAL SKETCH ............................................................................................ 73
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LIST OF TABLES
Table page 2-1 Proximate analysis of rations given to the experimental fish .............................. 25
3-1 Mean weight gain (WG) (per fish), specific growth rate (SGR), and survival data of red zebra cichlid ..................................................................................... 32
3-2 Red zebra cichlid average weight and total length of initial and final data, from day 0 to day 35 (5 weeks) .......................................................................... 32
3-1 Skin coloration in red zebra cichlid, five weeks after being fed diets containing astaxanthin (0.3%), lutein (corn protein concentrate at 12%), and Spirulina (12%). The control diet contained no predominant pigment type ......... 33
3-3 Predominant skin color in red zebra cichlid and averages of total carotenoid concentrations in the diet .................................................................................... 34
3-4 Color results for red zebra cichlids on initial color, and the most colorful and lighter sampled after 5 weeks ............................................................................. 40
3-5 Statistical analysis of darker red zebra cichlids weight ....................................... 41
3-6 Multiple comparisons of darker red zebra cichlids weight ................................... 41
3-7 Statistical analysis of lighter red zebra cichlids weight ....................................... 42
3-8 Multiple comparisons of lighter red zebra cichlids weight ................................... 42
3-9 Statistical analysis of lighter red zebra cichlids total length ................................ 43
3-10 Multiple comparisons of lighter red zebra cichlids total length ............................ 43
3-11 Statistical analysis of darker red zebra cichlids total length ................................ 44
3-12 Multiple comparisons of darker red zebra cichlids total length ........................... 44
3-13 Red zebra cichlids CIE L* mean, Min, and Max values after 5 weeks ................ 45
3-14 Red zebra cichlids CIE a* mean, Min, and Max values after 5 weeks ................ 45
3-15 Red zebra cichlids CIE b* mean, Min, and Max values after 5 weeks ................ 45
3-16 Statistical color analysis of lighter red zebra cichlids for CIE L* values .............. 46
3-17 Multiple comparisons CIE L* values for lighter red zebra cichlids ....................... 46
3-18 Statistical color analysis of lighter red zebra cichlids for CIE a* values .............. 47
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3-19 Multiple comparisons CIE a* values for lighter red zebra cichlids ....................... 47
3-20 Statistical color analysis of lighter red zebra cichlids for CIE b* values .............. 48
3-21 Multiple comparisons CIE b* values for lighter red zebra cichlids ....................... 48
3-22 Statistical color analysis of darker red zebra cichlids for CIE L* values .............. 49
3-23 Multiple comparisons CIE L* values for darker red zebra cichlids ...................... 49
3-24 Statistical color analysis of darker red zebra cichlids for CIE a* values .............. 50
3-25 Multiple comparisons CIE a* values for darker red zebra cichlids ...................... 50
3-26 Statistical color analysis of darker red zebra cichlids for CIE b* values .............. 51
3-27 Multiple comparisons CIE b* values for darker red zebra cichlids ...................... 51
3-28 Darker red zebra cichlids replicate L* values for Diet-1, Diet-2,Diet-3, and Diet-4 .................................................................................................................. 52
3-29 Darker red zebra cichlids replicate a* values for Diet-1, Diet-2, Diet-3, and Diet-4 .................................................................................................................. 53
3-30 Darker red zebra cichlids replicate b* values for Diet-1, Diet-2, Diet-3, and Diet-4 .................................................................................................................. 54
3-31 Lighter red zebra cichlids replicate L* values for Diet-1, Diet-2, Diet-3, and Diet-4 .................................................................................................................. 55
3-32 Lighter red zebra cichlids replicate a* values for Diet-1, Diet-2, Diet-3, and Diet-4 .................................................................................................................. 56
3-33 Lighter red zebra cichlids replicate b* values for Diet-1, Diet-2, Diet-3, and Diet-4 .................................................................................................................. 57
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LIST OF FIGURES
Figure page 1-1 Chemical structure of astaxanthin ...................................................................... 15
1-2 Chemical structure of zeaxanthin ....................................................................... 15
1-3 Chemical structure of lutein ................................................................................ 15
1-4 Chemical structure of β-carotene ....................................................................... 15
1-5 Chemical structure of lycopene .......................................................................... 16
1-6 The red zebra cichlid Pseudotropheus estherae ................................................ 22
2-1 Experimental Diets, Diet-1 (Control), Diet-2 (Astaxanthin), Diet-3 (Lutein) and Diet-4 (Spirulina) ................................................................................................. 24
2-2 Experimental tank was covered on three sides with black plastic....................... 26
2-3 Experimental tanks ............................................................................................. 26
2-4 Color Machine Vision System and sampling materials ....................................... 27
2-5 Sony camera with Color Machine Vision System ............................................... 27
2-6 Color standard cards .......................................................................................... 28
2-7 Photographing the side of the fish ...................................................................... 28
3-2 Two distinct color tones were visible within the same treatment group .............. 34
3-3 Absorbance of carotenoids which are in the diets. Diet 1 (Control), Diet-2 (Astaxanthin), Diet-3 (Lutein), and Diet 4 (Spirulina) .......................................... 40
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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science
COLOR ENHANCEMENT IN THE ORNAMENTAL RED ZEBRA CICHLID
PSEUDOTROPHEUS ESTHERAE BY ADDITION OF CAROTENOIDS TO THE DIET
By
Serdar Yedier
May 2013
Chair: Frank Chapman Major: Fisheries and Aquatic Sciences
The color of ornamental fish is one of the most important factors that attract
buyers. Thus, the ability to influence the color of fishes can increase the probability of
attracting a buyer. Color in these fishes can be enhanced or changed by feeding them
diets supplemented with pigments. In this work, a popular ornamental fish, the red zebra
cichlid (Pseudotropheus estherae) was fed fish diets supplemented with different
carotenoids (organic pigments) and with Spirulina, a blue-green algae (cyanobacterium)
that contains carotenoids. Four experimental diets were provided. Diet 1 did not contain
a carotenoid pigment and was used as a control. Diets 2 and 3 were supplemented with
the carotenoids astaxanthin and lutein, respectively. Diet 4, was supplemented with
Spirulina, and contained mainly zeaxanthin and β- carotene carotenoids. Pigmentation
in the red zebra cichlid fed the special diets was compared to the control group using a
Color Machine Vision System (CMVS). Color values for the red zebra cichlid fed with
the diet containing carotenoid supplements were significantly different from those of the
red zebra cichlid fed the control diet. I conclude that diets supplemented with
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carotenoids induce a color change in red zebra cichlid. Moreover, fish readily accepted
the carotenoid-supplemented diets, remained healthy, and gained significant weight and
length during the trial period.
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CHAPTER 1 INTRODUCTION
Color depends basically on three things: a light source, an object, and an
observer (Bull, 2009). When light hits an object, some of it is absorbed and the rest is
reflected. The reflected portion of the light is responsible for the color of the object. A
light source is the first important aspect in the process of seeing color. It is impossible to
see the color of an object without a light source. Sometimes a light source is confused
with an illuminant. Whereas a light source is a natural source of light, an illuminant is a
man-made energy source of wavelengths that represent the spectral characteristics of
specific type of light source. Average Daylight (C) and Noon Daylight (D65) are
examples of a light source, and Incandescent (A) and Cool White Fluorescent (F2) are
examples of illuminants. The light source normally emits light that appears to be white.
When light passes through a prism, it can be seen that it is composed of all visible
wavelengths, which can be measured in nanometers (nm). The wavelength range of the
visible spectrum is from approximately from 400 to 700 nm (Powles, 1984).
The object is the second important part of the process of seeing color. Light can
be absorbed, reflected or transmitted by objects. Black objects, for instance, absorb all
the light hitting them. As a result, we see them as black because no visible wavelength
of light is reflected and reaches our retina. White objects, on the other hand, reflect all
the wavelengths, whereas transparent objects such as glass transmit them. Light may
also be absorbed by colorants such as dye or pigment in the objects, making them
appear different colors.
The observer is a third important factor in the process of seeing or in detecting
color. Whereas a light source such as sunlight contains all possible wavelengths,
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receptors in our eyes enable us to differentiate only certain wavelengths, i.e., the visible
spectrum. The human retina contains billions of specialized photoreceptor cells, which
fall into two types, rods and cones. Whereas rod photoreceptors are very sensitive to
light, cone receptors are capable of color vision and are responsible for high spatial
acuity. The population of these receptors affects a person’s vision, and thus different
individuals may see the same color differently. Generally, cone and rod photoreceptors
are visually similar, and contain retinal proteins called opsins that are extremely
important in determining color. Rhodopsin, the most important retinal protein for night
vision, is found in the rod cells. There are three types of cone receptors that contain
slightly different opsins, which account for the differences in peak wavelength
absorption for each pigment. Human cone receptors are most sensitive to orange (558
nm), green (531 nm), and violet (419 nm).
Color is often described as having three properties: 1) hue being the pure or
actual name of the color; 2) value, the lightness or darkness of the color and 3)
saturation (same as intensity or chroma) is the purity or vividness of the color. Two
colors can have the same value and or saturation. Each color has a specific lightness
level, and the amount of chroma or saturation can change the original color. In terms of
the object color and the lightness level, values are not as easily distinguished by
people. To facilitate measurements, colors were characterized or defined within
coordinates of an X (red), Y (luminance) and Z (blue) space. In 1931, the XYZ color
space model, the first to employ mathematically defined color space values, was
created by the International Commission on Illumination (Smith at al., 1931-1932, CIE,
1931). Therefor to measure color, three things are required: a light source, a specimen
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and an instrument like a spectrophotometer or colorimeter that can measure reflectance
or transmittance spectra over a wide range of wavelengths. Some colorimeters or
spectrophotometers have three broadband filters and a photodetector, that obtain the
relative spectral energy distribution of the object or sample, that are transformed into
three numbers that can be converted directly to color space values. Today, many
different color models are in use to determine color levels, the most widespread being
RGB, HSB, Hunter L, a, b and CIE L*, a*, b*. The CIE L*, a*, b*, (1976) and Hunter L, a,
b (1948) models are mostly used in academic research. CIE L*a*b* has an almost
uniform color scale that is determined between points of color space. CIE organizes the
color space in cube form. In this model, L* is lightness, the maximum value of which is
100, which means white, and minimum is 0, which means black. The a* and b* have no
specific numerical limits; however a positive value of a* is red, a negative green, a
positive value of b* is yellow, a negative blue. Hunter (1948) organized the color space
in rectangular form, and this model has a 3-dimensional rectangular color space
coordinate system. Its axes, L, a and b, are similar to the L*, a* and b* axes in the CIE
L*, a*, b* model. L is lightness, the maximum value of which is 100, meaning white, and
the minimum 0, meaning black. The a and b have no specific numerical limits; however,
a positive value of a is red, a negative is green and 0 is neutral, and a positive value of
b is yellow, a negative value blue and 0 neutral. Although Hunter L, a, b and CIE L*, a*,
b* are similar models, exact numerical color values can be different. These differences
result from a different root transformation of the color coordinates on the coordinate
system. While CIE L*, a*, b* model coordinates are based on a cube root transformation
of the color data, those of the other models are based on a square root transformation.
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Fish, amphibians and reptiles have multilayer color patches known as dermal
chromatophores (Grether et al., 2004). The presence of dermal chromatophores can
affect color values. Their structure is basically composed of three cell layers:
melanophore, which contains melanin pigments that appear dark brown or black
(Bagnara, 1966); iridophore, which reflects light like a mirror (Taylor, 1969); and
xanthophore, which contains pteridine pigments and carotenoids (Bagnara, 1976).
Pigments are compounds that can absorb wavelengths of light. Deposited in the
integument of fish, they assign to color to the biological pieces of the dermal
chromatophore. Carotenoids and melanin pigments are commonly studied in the field of
aquatics, especially in relation to coloration. Fish use colors for some important
biological functions such as camouflage, and competition (Grether, 2000). In cultured
fish, which have no access to carotenoids in their food, pigments must be added to the
diet to maintain their bright coloration.
Carotenoids are the most widespread and important pigment classes in living
organisms, and are widely distributed in terrestrial as well as aquatic animals such as
prawns and fish (Yamada et al., 1990). More than 600 forms of carotenoids are known.
They can be split into two large groups: xanthophylls, which contain oxygen molecules
in their chemical structure, and include astaxanthin (Fig. 1.1), zeaxanthin (Fig. 1.2), and
lutein (Fig. 1.3); and carotenes, which contain carbon and hydrogen in their chemical
structure, such as α-carotene, β-carotene (Fig. 1.4), and lycopene (Fig. 1.5). Oxygen
molecules are not present in the structure of carotenes.
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Figure 1-1. Chemical structure of astaxanthin
Figure 1-2. Chemical structure of zeaxanthin
Figure 1-3. Chemical structure of lutein
Figure 1-4. Chemical structure of β-carotene
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Figure 1-5. Chemical structure of lycopene
Ong and Tee reported (1992) that most carotenoids are biosynthesized by
plants, algae, and certain yeasts and bacteria. Only phytoplankton, algae and plants
produce natural carotenoids (Davis, 1985) that are lipid soluble. Spirulina, for instance,
is a type of blue-green algae (cyanobacteria) rich in carotenoids (Annapurna et al.,
1991). This alga contains several types of carotenoids such as zeaxanthin and β-
carotene (Careri et al., 2001), and is a rich dietary source for humans (Yu et al., 2012).
In aquaculture and the aquarium industry, it is used as a diet supplement (Vonshak,
1997). Carotenoids play an important role in photosynthesis (Armstrong, 1997), and are
found widely in natural systems, especially in aquatic animals, leaves and fruits. Many
colors in nature come from carotenoids such as astaxanthin, β-carotene, lutein,
zeaxanthin, canthaxanthin, and lycopene. Aquatic animals cannot biosynthesize
carotenoids from mevalonic acid, but they can alter dietary carotenoids by oxidation and
accumulate them in their tissues.
Bjerkeng et al. (1990) noted that many animals take advantage of carotenoids in
their diet, and can modify these chemicals. Davis (1985) reported that some aquatic
animals such as koi and various species of crustaceans need an enzymatic mechanism
to modify carotenoids. In addition, carotenoids allow chromatic adaptation to
environments through coloration of body, tissue and biological fluids (Ghidalia, 1985).
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Generally, carotenoids are responsible for the red, orange, and yellow colors of
fish and crustaceans (Packer 1992). Because fish cannot synthesize carotenoids like
other animals, yet carotenoids are vital to their reproduction, growth, and metabolic
activities, they must absorb them from their diet (Hata & Hata 1972a; Torrissen et al.,
1989; Storebakken & No, 1992). A number of researchers (Stevens, 1947; Goodwin,
1954; Fox, 1957) have reported that the pink coloration of wild trout results from their
consumption of crustaceans. Peterson et al. (1966) used extracts of crawfish, paprika
and marigold petals in the first studies of the dietary supplementation of pigments to
cultured rainbow trout. Torrissen and Naevdal (1988) reported that individual size,
weight, age, sexual maturity, and genetic factors influenced deposition of carotenoid
pigment in Atlantic salmon Salmo salar. Carp, Cyprinus carpio, were fed diets including
alfalfa meal, mysis-stage shrimp, lutein, and astaxanthin. Results showed red color in
the skin with high concentrations of lutein, and that astaxanthin generated a brighter
color (Iwahashi & Haruo, 1976).
Astaxanthin was identified by Simpson et al. (1981) as the primary red pigment in
the skin of goldfish. Nevertheless, Simpson et al. (1981) found contradictory reports in
the literature about the ability of goldfish to synthesize astaxanthin. In a study in which
goldfish were fed with lutein and carotene, these were converted to astaxanthin, and the
total amount of carotenoids in the fish increased (Hirao et al., 1963). Hata and Hata
(1972a, 1972b), however, reported poor conversion of lutein and β-carotene to
astaxanthin in goldfish.
There are two main types of carotenoids, synthetic and natural. In the natural
aquatic environment, primary producers microalgae or phytoplankton synthesize
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astaxanthin. This is absorbed by herbivorous insects, zooplankton and crustaceans,
which are eaten by fish. Synthetic astaxanthin is the main pigment used in the
aquaculture industry worldwide (Higuera-Ciapara et al., 2006). Goodwin (1984) reported
that fish are unable to perform de novo synthesis of carotenoids, and therefore rely on
dietary supply to achieve their natural pigmentation. Under intensive farming conditions
and aquarium rearing, ornamental fish are given exclusively compound feeds, which
must be supplemented with carotenoids. Various synthetic carotenoids, such as
canthaxanthin, astaxanthin, and lutein (Choubert & Storebakken, 1989), as well as
natural sources (Chien & Shiau, 2005), have been used as dietary supplements to
enhance the pigmentation of ornamental fish (Gouveia et al., 2003). Because
astaxanthin is the naturally occurring carotenoid in salmonid flesh (Storebakken &
Choubert, 1991), synthetic astaxanthin is administered in preference to synthetic
canthaxanthin, giving the flesh a more yellow-orange coloration (Johnson, 1992).
It is therefore necessary to supply astaxanthin to cultured fish through their feed.
The added astaxanthin is absorbed, and accumulates in the tissues to reproduce the
animals’ natural appearance. However, synthetic astaxanthin is expensive, and
significantly increases the cost of feed and production (Johnson, 1991).
In modern aquaculture, carotenoids are represented by astaxanthin,
canthaxanthin, or lutein pigmentation of the flesh of salmonids. Astaxanthin is the
natural carotenoid found in salmonids (Storebakken & Choubert, 1991), but synthetic
astaxanthin applied along with synthetic canthaxanthin gives the flesh a more yellow-
orange coloration (Johnson, 1992).
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In 1992, Koteng found that the pigmentation of Atlantic salmon, Salmo salar, and
rainbow trout, Oncorhynchus mykiss, is regarded as the most important criterion after
product freshness for consumers. It is therefore of vitally importance for the salmon
farmer to achieve satisfactory pigmentation of the salmon flesh. Torrissen (1989)
reported on factors influencing the absorption and deposition of carotenoids, and
showed that salmonids absorb and deposit astaxanthin and canthaxanthin in the muscle
during the growth period. At the time of sexual maturation, they mobilize the carotenoid
store and transport the accumulated astaxanthin or canthaxanthin to the skin, and the
eggs of females. In addition, the effectiveness of carotenoid sources in terms of
deposition and pigmentation is species-specific (Ha et al., 1993). For example, goldfish
convert the yellow pigment zeaxanthin to the red pigment astaxanthin (Hata & Hata,
1972a).
Winterhalter and Rouseff (2002) reported that astaxanthin is a carotenoid
classified as a xanthophyll, first found in yellow leaves. Supplementation of astaxanthin
in the fish diet improved the skin redness of farm-reared Australian snapper, Pagrus
pagrus, whereas skin redness decreased over time in fish without astaxanthin
supplementation (Booth et al., 2004). Similarly, the addition of astaxanthin to the diet of
goldfish, Carassius auratus, increased the red pigmentation density of the skin (Xu et
al., 2006). The study of the role of carotenoids in changing fish pigmentation has
therefore become an important aspect of ornamental fish culture. The deposition of
carotenoids into fish skin depends on the species (White et al., 2002, 2003; Kalinowski
et al., 2005) and dosage-dependent (Paripatananont et al., 1999; Matsuno, 2001; Wallat
et al., 2005). Pigmentation can be accomplished by supplementing the diet with
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astaxanthin, canthaxanthin and lutein. Choubert and Storebakken (1989) reported that
all fish can utilize both astaxanthin and canthaxanthin very well.
There are several methods of measuring and analyzing fish skin or flesh color.
One is visual inspection and hand-grading by experienced individuals and this
traditional method is used by the ornamental or tropical fish industry to select
marketable live fish (Chapman, 2000). Duncan and Lovell (1994) employed panels of
trained persons to evaluate the color of tropical fish fed with various amounts of
pigments. Hata and Hata (1971) described in detail the different phases of skin color
development in goldfish by visually inspecting the dorsal side of the fish. Skrede et al.
(1990) reported that the processing industry used color cards, chips, tiles or fans for
quality standardization of color in salmonid meat. Nevertheless, Ling et al. (1996)
reported that human evaluation of color was subjective and affected by differences in
color perception and lighting conditions. Besides, the scoring accuracy of color in
salmonid meat by color cards decreased as pigment levels exceeded 4mg/L (Torrissen
et al., 1989; March & MacMillan, 1996). Also, various research groups have used a
colorimeter to appraise pigment content in the meat of salmonids (Skrede &
Storebakken, 1986; Gentles & Haard, 1991; King, 1996).
A variety of food items such as fish fillets (Hatano et al., 1989), shrimp (Balaban
et al., 1994; Luzuriaga et al., 1997), beef and carrots (Ling et al., 1996) as well as plant
tissue (Alchanatis et al., 1993) have been analyzed by Color Machine Vision Systems
(CMVS). A computerized vision system that offers objective measurements under
uniform conditions can analyze a much larger surface area than the traditional
colorimeter. This system is useful to measure the skin color of fish in the water; it can
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also sample the fish safely and quickly because it does not require a standard color
analysis process or anesthesia. Wallet et al. (1997) reported on an application of CMVS
technology for measurement and analysis of skin color development in live goldfish, an
ornamental fish of high commercial value.
Trade in aquarium fish depends on their sparkling colors and patterns, and most
often dictates the market value of the fish (Saxena, 1994; Ramamoorthy et al., 2010;
Dharmaraj & Dhevendaran, 2011). Under intensive farming conditions and aquarium
rearing, ornamental fish are fed exclusively on compound feeds, which must therefore
be supplemented with carotenoids to enhance their color. The source and concentration
of carotenoids play an important role in the pigmentation of fish (Gouveia & Rema,
2005). Various synthetic carotenoids, such as β-carotene, astaxanthin (Choubert &
Storebakken, 1989; Storebakken & No, 1992), as well as natural sources (Coral, et al.,
1998; Chien & Shiau, 2005; Kalinowski, et al., 2005) have been used as dietary
supplements to enhance the pigmentation of ornamental fish (Gouveia, et al, 2003).
Chapman (2000) 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. The efficiency of different carotenoids can vary within fish species
such as red porgy (Kalinowski et al., 2005) and the guppies (Mirzaee et al., 2012). Fish
feeds are usually enhanced with relatively expensive astaxanthin or canthaxanthin
carotenoids. Therefore, there is a growing need to find cheaper carotenoid substitutes.
Natural carotenoid sources are usually composed of several carotenoids in various
forms, and vary in terms of digestibility, making their pigmentation efficiency
complicated to interpret and predict. The green unicellular freshwater alga
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Haematococcus pluvials, a potent producer of esterified astaxanthin (Czygan, 1968;
Borowitzka, et al., 1991; Grung, et al., 1992) also contains other carotenoids, such as
canthaxanthin, β-carotene, lutein and echinenone (Czygan, 1968; Choubert & Heinrich,
1993). This alga has been demonstrated to enhance the pigmentation of rainbow trout
(Sommer et al., 1991, 1992; Choubert & Heinrich, 1993), gilthead seabream (Gomes et
al., 2002), koi carp and goldfish (Gouveia et al., 2003).
Efficiency in pigmentation from the above sources can be attributed to target
animal species, type, composition, and concentration of the pigments, digestibility of the
material itself, and possibly the presence of cofactors in the material involved in
absorption and deposition (Torrissen, et al., 1989; Storebakken & No, 1992; Wang, et
al., 2006).
There have been few studies examining the relationship between fish coloration
and pigment enriched diets, and color is an important factor in the commercial value of
ornamental fish. The red zebra cichlid, Pseudotropheus estherae, a species of cichlid
(Figure 1.1), is popular among ornamental fish hobbyists.
Figure 1-6. The red zebra cichlid Pseudotropheus estherae
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The color variations of this species, which include orange and red, make it an
attractive addition to freshwater aquaria, and specimens command a high market value
in the trade. These cichlids are freshwater perciform fish, endemic to the northern
coastal region of Lake Malawi in East Africa (McKaye, 1983). Despite the fact that they
are called red zebra cichlid, they are in fact orange in color. Male and female red zebra
cichlids are sexually dimorphic, and differences of morphology, size, ornamentation and
behavior are found in the same species. For instance, males are more orange than
females, and most males have reddish stripes (Kuwamura, 1986). Most African cichlids
are mouth brooders: females hatch the young in their mouths and keep them there for a
little more than a month before releasing them (Fryer & Iles, 1972; McKaye, 1983;
Kuwamura, 1986). In their natural habitat, this cichlid grows to about 9 cm in length,
although they have been known to reach 15 cm in captivity (Fryer & Iles, 1972).
Pigmentation efficiency of various carotenoids in the red zebra cichlid and the
preferred pigment sources are largely unknown. This was the incentive for this study.
The objective of the present study was therefore: to observe the pigmentation effect on
the red zebra cichlid fed with different types of carotenoid supplemented diets.
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CHAPTER 2 MATERIALS AND METHODS
Experimental Diets
Fish were divided into four experimental groups and each group was given a diet
containing a different pigment. One group was offered the control diet that only
contained basic feed ingredients that do not a pronounced effect on body hue. The
other three diets were prepared using the same ingredients as the control, diet but with
the addition of pigments from different natural sources. Pigments obtained were from
the red-carotenoid pigment astaxanthin, produced by algae (Cyanotech Corporation,
Kailua-Kona, HI), yellow lutein contained in the protein concentrate of corn seeds
(Cargill Corn Milling, Wayzata MN), and a combination of several natural pigments
(primarily yellow/orange) found in the blue-green alga Spirulina (Carbon Capture
Corporation, La Jolla CA). Ingredients containing the pigments astaxanthin, lutein, and
those from Spirulina were incorporated into the diets at 0.3%, 12% and 12%,
respectively (Figure 2-1).
Figure 2-1. Experimental Diets, Diet-1 (Control), Diet-2 (Astaxanthin), Diet-3 (Lutein)
and Diet-4 (Spirulina)
25
The diets were prepared to be isocaloric and isonitrogenous based primarily on
inclusion of wheat flour and sardine meal to a basal cichlid feed formulation at 34.3 and
19.8 percent, respectively (Table 2-1; Royes et al., 2006). Raw ingredients were
weighed, reduced in size with a grinder, blended, extruded, and dried in pellet form. The
size of the final pellets was 0.8 mm as they were passed through a sieve.
Table 2-1. Proximate analysis of rations given to the experimental fish
Diet No Calories Protein Fat Carbohydrate Ash Moisture
1.Control 391 41.4 9.6 35 7.1 7.3
2.Astaxanthin 390 41.6 9.3 35 7.3 7.1
3.Lutein 389 41.4 9.6 34 7.0 7.1
4.Spirulina 394 41.5 10.1 34 6.6 7.6
Fish and Experimental Design
About 400 juvenile red zebra cichlid, Pseudotropheus estherae, were donated by
an ornamental fish farm in Miami, FL and transported to the laboratory of Fisheries and
Aquatic Sciences, University of Florida, Gainesville, USA. All the fish were
prophylactically treated with a sodium chloride bath and acclimatized in two tanks 90 x
40 x 45 cm (W*H*L) (200 fish per tank) for three weeks, and fed with a control diet until
the experiments began. After acclimation, fish were randomly assigned to 16 tanks 30 x
30 x 35 cm (W*H*L) (20 fish per tank) with four replicates for each treatment, and were
maintained at 24-26 ºC. Each aquarium contained a biochemical air pump with double
sponge filter that provided constant aeration and mechanical filtration. All aquaria were
supplied with filtered freshwater from a reservoir.
Ten juvenile cichlids were sampled randomly from each tank, weighed and
photographed, at the start of the study. Feeding trials were carried out for five weeks,
and the juvenile cichlids were hand-fed the experimental diets to apparent satiation
twice a day (10 AM and 4 PM), except on Sundays. Feces and uneaten feed were
26
removed from each tank daily, and about one-third of the water was changed.
Experimental water conditions were maintained as temperature of 24.0 ± 1.0 °C,
dissolved oxygen at 5-7 mg L-1, pH of 7.0-7.3 and NH3 of 0.05-0.1 ppm. One week later,
I covered three sides of each tank with black plastic to reduce stress on the fish (Figs.
2-2 and 2-3).
Figure 2-2. Experimental tank was covered on three sides with black plastic
Figure 2-3. Experimental tanks
The natural photoperiod was enhanced by a florescent light at an intensity of
1200 lux during daylight hours. After five weeks, nine more colorful and five lighter
young cichlids were sampled from each tank for carotenoid analyses.
27
Fish Images and Fish Skin Colors Analysis
I used a Color Machine Vision System (CMVS) (Fig. 2-4) to measure changes in
the skin color of the red zebra cichlid in this study.
Figure 2-4. Color Machine Vision System and sampling materials
The major components of the CMVS include an illumination chamber covered
with a Rosco Polarizing Filter Sheet, 43 cm x 51 cm, a medium-resolution color video
camera (Sony MVC-CD500 Digital Camera 5MP), Carl Zeiss 6X lens, disks with a
52mm Sony lens tube adaptor, and a 52 mm Sony polarizing lens (Fig. 2-5).
Figure 2-5. Sony camera with Color Machine Vision System
The camera saved all pictures on small 156MB RW Mavica multi-speed disks.
We set the camera adjustments at focal length = 13.9 mm, f-number = F/5, and
28
exposure time=1/15 sec. We used a standard yellow color card (Fig. 2.6) to compare
fish color in the pictures.
Figure 2-6. Color standard cards
Each fish was briefly removed from its culture aquarium and placed with a yellow
standard card in a small, water-filled glass chamber that was inserted into the
illumination chamber, where the fish’s color image was captured by the Sony MVC-
CD500 camera. The entire procedure lasted approximately five minutes and did not
require the fish to be anaesthetized.
I took three to four images from the right or left side of each fish (Fig. 2-7).
Figure 2-7. Photographing the side of the fish
29
The image format was jpg, so I used a procedure similar to that in Luzuriaga et
al. (1997) and Wallat et al. (2003). The images were transferred to a Sony Viao laptop,
and each image was resized from 100 to 50% and converted to bitmap using Adobe
Photoshop CS6. The resized and reformatted images were then transferred to the
computer in the Fisheries and Aquatic Science laboratory for color analysis.
For color analysis of the fish images, I used the LensEye Color Expert software
program (version 10.0.0). This program analyzed each image, and provided its color
values (RGB, HSB, and CIE L*, a*, b*) in an Excel spreadsheet. The color spectrum of
this program was adjusted to 4096 representative colors based on the CIE system of
the color analysis program. This program can count all the pixels in an image, but for
this analysis, I selected only the images of the fish themselves. Each pixel was
compared with 4096 representative color blocks, the defining pixel of each color was
calculated, and the closest color found by the CMVS program.
Total Carotenoid Analysis
Total carotenoid was analyzed for each diet in the chemistry department
laboratory at the University of Florida. The carotenoid content of the fish diet was
extracted by a method similar to that used by Torrissen and Naevdal (1984). 10-mg
samples of each diet were ground and transferred to 10-mL pre-weighed glass tubes
with 0.2 ml chloroform. Samples were shaken for 15 minutes at 1400 rpm at 25 °C in
chloroform. Solutions were centrifuged at 5000 rpm for five minutes, the supernatant
was removed, and absorbance was measured at a wavelength between 250 and 900
nm using a spectrophotometer. Total carotenoid concentration in the diet was
determined spectrophotometrically in chloroform using extinction coefficients (E1%, 1
cm) of astaxanthin 1692.2 (Aquasearch Inc. 1999) at 485 nm, zeaxanthin 2540 at 487
30
nm, lutein 2369 at 454 nm, and β-carotene at 462 nm 2330 (Neal and Joseph, 1992).
We converted the values from E1% to ε.
The amount of carotenoid in each sample was calculated on a sample dry
weight. Total pigment content was calculated as μg carotenoid per g diet, using the
formula:
Total carotenoid content SWl
FWA
A = Absorption at maximum wavelength
FW = Molecular weight
= Extinction coefficient
l = Length of cuvette
SW = Sample weight Growth and Survival Rate
Growth performance of red zebra cichlid fed with four different diets including
different forms of carotenoids was evaluated by calculating weight gain (WG), specific
growth rate (SGR), and survival rate (SR). These growth parameters were calculated
using the formulas:
WG (g) = mean final weight (g) - mean initial weight (g)
SGR (SGR % day-1) = {[ln mean final weight (g)-ln mean initial weight (g)]/feeding period (day)} x100
SR (%) = (final number of fish/initial number of fish) x 100
Statistical Analysis
All the experimental data were statistically analyzed using SPSS (version 16) to
evaluate the effect of the experimental diets (astaxanthin, lutein, and Spirulina) on the
coloration of red zebra cichlid. A one way ANOVA and Tukey HSD were used to test the
effect of carotenoid type on fish color and growth parameters SPSS (version 16.0).
Differences were considered to be significant at P < 0.05. A Chi squared analysis was
31
used to calculated the ratio of lighter to darker fish in diets that had distinguishable color
differences (astaxanthin and Spirulina).
32
CHAPTER 3 RESULTS
All fish readily accepted and consumed the 0. 8 mm food pellet that was offered
to them, regardless of the pigment type incorporated into the pellet. With the
expectation of two individuals that died during the first week, all fish survived and
remained healthy from the beginning to the end of the trial. Weight gain, specific growth
rate, and survival were not significantly different among fish fed different diets (Table
3-1).
Table 3-1. Mean weight gain (WG) (per fish), specific growth rate (SGR), and survival data of red zebra cichlid
Diet WG, g SGR, % day-1 Survival, %
1 0.76 ± 0.4 2.3 ± 1.2 100
2 0.75 ± 0.3 2.3 ± 1.2 97.5
3 0.75 ± 0.3 2.3 ± 1.3 100
4 0.81 ± 0.4 2.5 ± 1.3 97.5
Weight Gain (WG) (per fish), Specific Growth Rate (SGR), and Survival Rate (SR) data for (1) Control, (2) Astaxanthin, (3) Lutein, and (D-4) Spirulina.
Fish in all treatment groups, gained significant weight and length during the
experimental period of 35 days (Table 3-2). Average weight gain alone was greater than
one standard-deviation from the initial average mean of approximately 0.61 ± 0.2 g to
an average mean of 1.38 ± 0.5 g at 5 weeks.
Table 3-2 Red zebra cichlid average weight and total length of initial and final data, from day 0 to day 35 (5 weeks)
Diet Average Weight (g) Average Total Length (mm)
Initial Final Initial Final
1 0.60 ± 0.1 1.36 ± 0.4 32 ± 3 41 ± 4
2 0.60 ± 0.2 1.35 ± 0.4 31 ± 4 42 ± 4
3 0.63 ± 0.2 1.38 ± 1.2 32 ± 3 43 ± 4
4 0.62 ± 0.2 1.43 ± 0.5 32 ± 3 43 ± 5
(1) Control, (2) Astaxanthin, (3) Lutein, and (4) Spirulina.
33
The type of pigment in the diet significantly affected skin color in the red zebra
cichlid (Table 3-3). At the initiation of the trial the fish were primarily light yellowish-
brown in color. About two weeks thereafter, clearly distinguishable differences in skin
color were observed. After five weeks, most of these fish had completely changed with
distinct colors that covered 6% or more of their body surface (Figure 3-1). Fish fed the
diet containing the carotenoid astaxanthin at 0.3% developed the most orange-red
coloration. Fish fed the diet containing 12% corn protein concentrate as a lutein
(xanthophyll) source became dark yellow. Fish fed 12% Spirulina (primarily beta-
carotene and zeaxanthin) in their diets became dark orange-yellow in color, had the
most vivid colors, and had the most visible ‘egg spots’ on the anal fin.
No predominant pigment type Astaxanthin
Lutein Spirulina Figure 3-1. Skin coloration in red zebra cichlid, five weeks after being fed diets
containing astaxanthin (0.3%), lutein (corn protein concentrate at 12%), and Spirulina (12%). The control diet contained no predominant pigment type
The diet that had the highest amounts of carotenoid pigments was that which
contained 12% Spirulina and 0.3% astaxanthin (Table 3-3). Pigment content in the diet
34
prepared with corn protein concentrate was only 8-10% a great. The fish fed the control
diet (that not having discrete types of pigments) had no significant changes in their skin
coloration and lacked vivid colors. Absorbance of carotenoid in the diets is presented in
Figure 3-3.
Table 3-3. Predominant skin color in red zebra cichlid and averages of total carotenoid concentrations in the diet
(1) Control, (2) Astaxanthin, (3) Lutein, and (4) Spirulina.
In addition to skin color differences between fish fed different pigment types, light
and dark shades of skin color were distinguishable among individuals within the same
treatment group after 5 weeks later (Figure 3-2). These were more apparent in the fish
fed the astaxanthin and Spirulina rich diets. The two shades were not significantly
apparent between the control group at the initiation and end of the trial with those fish
fed the low pigment and lutein diets.
Figure 3-2. Two distinct color tones were visible within the same treatment group
A chi-squared analysis indicated that the number of fish with in treatment group
having light and dark skin coloration did not differ significantly from a 50:50 ratio.
Source of Pigment
Initial color Final color Amount of Pigment
1.Control Light yellowish brown Light yellowish brown 0
2.Astaxanthin Light yellowish brown Moderate orange 348.7 mg/kg
3.Lutein Light yellowish brown Dark yellow 42.2 mg/kg
4.Spirulina Light yellowish brown Dark orange-yellow 409.5-448.5 mg/kg
35
Examination of the gonads and histology revealed light color fish were females and
those with the vivid colors were principally males.
Total carotenoids were calculated and are shown in Table 3-1. According to the
total carotenoid results, Diet 1 did not include any carotenoid; Diet 4 contained two type
of carotenoids, β-carotene (448 mg/kg) and zeaxanthin (409 mg/kg); Diet 2 contained
one type of pigment, astaxanthin (448 mg/kg); and Diet 3 contained lutein (42 mg/kg).
Carotenoid amount differed greatly, with Diet 3 containing only about 10% of that in the
other experimental diets. Absorbance of carotenoid in the diets is presented in Figure
3-3.
For zebra cichlid, weight gain (WG), specific growth rate (SGR), and survival rate
(SR) data for each diet were calculated, and are displayed in Table 3-1. I did not find a
significant difference in weight or length within diets for the lighter and more colorful red
zebra cichlid. For Diets 2 and 4 survival was 97.5%, because one fish died in Diet 2 and
one in Diet 4 during the first week of the experiment. Specific growth rates were very
similar for the colorful red zebra cichlids on each diet. However, the lighter red zebra
cichlid fed on Diet 1 showed lower specific growth rates than those on the other diets.
After two weeks, the red zebra cichlid fed with Diet 1, 2, 3 and 4 had visually
distinguishable differences in color. We determined the exact color of the fish for initial
sampling, selecting the more colorful and lighter fish sampling after five weeks for the
groups (C) Control, (A) Astaxanthin, (L) Lutein, and (S) Spirulina (β-carotene and
zeaxanthin). The exact color results are displayed in Table 3-4. Fish colors were
represented by more than 6% of the total image in all treatments (Table 3-4).
36
The initial results showed that the shade of the more strongly colored zebra
cichlid was the same, a light yellowish brown, for all diets. After five weeks, however,
most of these fish had changed color, to moderate orange on Diet 2, dark yellow on Diet
3, and dark orange-yellow on Diet 4, while Diet 1 produced no change of color.
According to the data, after five weeks, the lighter red zebra cichlid changed color only
on Diet 2. Diet 1, Diet 2, and Diet 3 did not result in any change of color in the lighter
zebra cichlid. In addition, we determined the best color values of red zebra cichlid fed
with Diets 1, 2, 3 and 4 after five weeks (Fig.3-1).
Average values of total length and weight for the darker and lighter red zebra
cichlid are given in Table 3-2. We did not find any significant differences in the total
length and weight of red zebra cichlid fed on Diets 1, 2, 3 and 4. Statistical results
showing the weight of both the darker and lighter red zebra cichlids are given in Tables
3-5 and 3-6 and Tables 3-7 and 3-8 respectively.
Moreover, the total length of the lighter zebra cichlid is presented in Tables 3-9
and 3-10, and that of the darker zebra cichlid in Tables 3-11 and 3-12. Average,
minimum and maximum values of L* for the red zebra cichlids are shown in Table 3-13.
Minimum, maximum, and average values of a* for both the lighter and darker zebra
cichlids are displayed in Table 3-14. Moreover, maximum, minimum and average values
of b* are presented in Table 3-15: L* is lightness, a* is scale green (-) to yellow (+), and
b* is scale blue (-) to red (+).
For the L* color values of the lighter zebra cichlid, there were in general no
significant (P>0.05) differences between fish fed on Diets 1, 2, 3 and 4. L* color values
37
for the lighter zebra cichlid are detailed in Table 3-13. Significant information about
lighter red zebra cichlid L* color values are shown in Tables 3-16 and 3-17.
The a* color values of the lighter zebra cichlid (P<0.05) differed significantly,
however. This means that the red color level is different among the lighter fish fed on
different diets. The a* color values of the lighter red zebra cichlid are detailed in Table 3-
14. Significant information about lighter zebra cichlid a* color values is shown in Tables
3-18 and 3-19. Diets 1, 3 and 4 did not produce a noticeable difference, but the lighter
red zebra cichlid fed with Diet 2 (astaxanthin) contained significantly more red pigment
than the others. This diet can therefore be used to enrich the red color of fish. The b*
color values of lighter red zebra cichlid showed no significant (P>0.05) differences
between those fed on Diets 1, 2, 3 and 4. Lighter red zebra cichlid b* color values are
detailed in Table 3-15. Significant information about lighter red zebra cichlid b* values is
displayed in Tables 3-20 and 3-21. We did not find any significant difference in the
yellow-blue color level of lighter red zebra cichlid fed on different diets.
We know that L* is lightness, a* is scale green (-) to yellow (+), and b* is scale
blue (-) to red (+). These values affected the color of the fish. There were significant
differences between the L* color values (P<0.05) of the brighter red zebra cichlid. L*
color values for the darker zebra cichlid are detailed in Table 3-13. Statistical
information about the L* color values of these fish is shown in Tables 3-22 and 3-23.
The darker zebra cichlid fed on Diet 1 showed significantly higher light levels than those
on Diet 2, while the Diet 4 group was significantly different to the fish fed on Diets 2 and
1 but not significantly different from those on Diet 3. Moreover, there were no significant
differences between fish on Diets 2, 3 and 4.
38
There were significant differences (P<0.05) between the a* color values of the
darker zebra cichlids, as shown in Table 3-14. Additional statistical analysis information
about the a* values of these fish is given in Tables 3-24 and 3-25. The red levels of the
more highly colored zebra cichlid fed on Diet 2 were significantly different to those of the
others. In addition, there were significant differences between those on Diets 3 and 4.
The fish fed with Diet 4 contained more reddish pigment than those fed on Diet 3. Diet 4
can therefore be an effective means of increasing the reddish color of zebra cichlid.
However, we did not find significant differences between Diet 1 and Diet 3, or
between Diet 1 and Diet 4. There were significant differences (P<0.05) between the b*
color values of the darker zebra cichlid; these values are displayed in Table 3-15.
Significant information about the b* values of these fish is shown in Tables 3-26 and
3-27.
The yellow levels of the more highly colored zebra cichlid fed on Diet 3 and Diet
4 were significantly different to those of the others. Moreover, fish fed on Diet 4
contained more yellow pigment than those fed on Diets 1, 2 and 3. Diets 3 and 4 can
therefore be used to enhance the yellow color of red zebra cichlid. There were
significant differences between Diets 2 and 3. The red zebra cichlid fed on Diet 2
contained less yellow pigment than those fed on Diet 3. In addition, the red zebra cichlid
fed on Diet 3 contained more yellow pigment than those fed on Diet 1. However, we did
not find any significant differences between Diets 1 and 2 for b* color value.
We found significant differences between L*, a* and b* in replicate samples.
These differences are shown in Tables 3-28, 3-29 and 3-30 for the more highly colored
zebra cichlid, while statistical values for the replicate samples of lighter zebra cichlid are
39
displayed Tables 3-31, 3-32 and 3-33. According to the results, the Diet 1 and Diet 2
replicate samples showed significant differences, especially in their L* and a* values.
These differences could arise from genetic variations between individual fish and how
they affected pigmentation, size, and competition for food in each tank.
40
Figure 3-3. Absorbance of carotenoids which are in the diets. Diet 1 (Control), Diet-2
(Astaxanthin), Diet-3 (Lutein), and Diet 4 (Spirulina)
Table 3-4. Color results for red zebra cichlids on initial color, and the most colorful and
lighter sampled after 5 weeks
Diet Initial Color 5 week Colorful 5 week Lighter
1 L*67.94 a*6.75 b*20.20 light yellowish brown
L*63.85 a*6.46 b*29.70 light yellowish brown
L*66.98 a*5.68 b*21.02 light yellowish brown
2 L*65.72 a*7.01 b*23.45 light yellowish brown
L*62.15 a*15.48 b*32.93 moderate orange^
L*67.37 a*7.44 b*18.63 moderate yellowish pink^^
3 L*64.28 a*6.35 b*21.36 light yellowish brown
L*62.66 a*5.66 b*39.23 dark yellow^
L*67.25 a*6.09 b*20.27 light yellowish brown
4 L*64.50 a*7.45 b*23.75 light yellowish brown
L*61.13 a*8.13 b*45.50 dark orange yellow^
L*67.45 a*5.40 b*21.49 light yellowish brown
(1) Control, (2) Astaxanthin, (3) Lutein, and (4) Spirulina. (^)Different color for Colorful red zebra cichlids. (^^)Different color for Lighter red zebra cichlids.
41
Table 3-5. Statistical analysis of darker red zebra cichlids weight
Colorful Weight Sum of Squares df Mean Square F P
Between Groups 4.652 3 1.551 2.262 0.084
Within Groups 95.969 140 0.685
Total 100.622 143
Table 3-6. Multiple comparisons of darker red zebra cichlids weight
Colorful Weight Tukey HSD Test
95% Confidence Interval
(I) Diet (J) Diet Mean Difference (I-J) Std. Error P Lover Bound
Upper Bound
1 2 -0.01944 0.19515 1.000 -0.5269 0.4880
3 -0.41389 0.19515 0.152 -0.9213 0.9035
4 0.01944 0.19515 1.000 -0.4880 0.5269
2 1 0.01944 0.19515 1.000 -0.4880 0.5269
3 -0.39444 0.19515 0.185 -0.9019 0.1130
4 0.03889 0.19515 0.997 -0.4685 0.5463
3 1 0.41389 0.19515 0.152 -0.0935 0.9513
2 0.39444 0.19515 0.185 -0.1130 0.9019
4 0.43333 0.19515 0.123 -0.0741 0.9408
4 1 -0.01944 0.19515 1.000 -0.5269 0.4880
2 -0.03889 0.19515 0.997 -0.5463 0.4685
3 -0.43333 0.19515 0.123 -0.9408 0.0741
(D-1) Control, (D-2) Astaxanthin, (D-3) Lutein, and (D-4) Spirulina.
42
Table 3-7. Statistical analysis of lighter red zebra cichlids weight
Lighter Weight Sum of Squares df Mean Square F P
Between Groups 1.561 3 0.520 2.650 0.55
Within Groups 14.921 76 0.196
Total 16.482 79
Table 3-8. Multiple comparisons of lighter red zebra cichlids weight
Lighter Weight Tukey HSD Test
95% Confidence Interval
(I) Diet (J) Diet Mean Difference (I-J) Std. Error P
Lover Bound
Upper Bound
1 2 -0.20500 0.14012 0.465 -0.5731 0.1631
3 -0.20000 0.14012 0.486 -0.5681 0.1681
4 -0.39500* 0.14012 0.031* -0.7631 0.0269
2 1 0.20500 0.14012 0.465 -0.1631 0.5731
3 0.00500 0.14012 1.000 -0.3631 0.3731
4 -0.19000 0.14012 0.531 -0.5581 0.1781
3 1 0.20000 0.14012 0.486 -0.1681 0.5681
2 -0.00500 0.14012 1.000 -0.3731 0.3631
4 -0.19500 0.14012 0.508 -0.5631 0.1731
4 1 0.39500* 0.14012 0.031* -0.0269 0.7631
2 0.19000 0.14012 0.531 -0.1781 0.5581
3 0.19500 0.14012 0.508 -0.1731 0.5631
(D-1) Control, (D-2) Astaxanthin, (D-3) Lutein, and (D-4) Spirulina. *. The mean difference is significant at the P < 0.05.
43
Table 3-9. Statistical analysis of lighter red zebra cichlids total length
Lighter Length Sum of Squares df Mean Square F P
Between Groups 93.138 3 31.046 1.410 0.246
Within Groups 1673.750 76 22.023
Total 1766.888 79
Table 3-10. Multiple comparisons of lighter red zebra cichlids total length
Lighter Length Tukey HSD Test
95% Confidence Interval
(I) Diet (J) Diet Mean Difference (I-J) Std. Error P
Lover Bound
Upper Bound
1 2 -1.75000 1.48402 0.642 -5.6482 2.1482
3 -2.15000 1.48402 0.473 -6.0482 1.7482
4 -2.95000 1.48402 0.202 -6.8482 0.9482
2 1 1.75000 1.48402 0.642 -2.1482 5.6482
3 -0.40000 1.48402 0.993 -4.2982 3.4982
4 -1.20000 1.48402 0.850 -5.0989 2.6982
3 1 2.15000 1.48402 0.473 -1.7482 6.0482
2 0.40000 1.48402 0.993 -3.4982 4.2982
4 -0.80000 1.48402 0.949 -4.6982 3.0982
4 1 2.95000 1.48402 0.202 -.9482 6.8482
2 1.20000 1.48402 0.850 -2.6982 5.0982
3 0.80000 1.48402 0.949 -3.0982 4.6982
(D-1) Control, (D-2) Astaxanthin, (D-3) Lutein, and (D-4) Spirulina.
44
Table 3-11. Statistical analysis of darker red zebra cichlids total length
Colorful Length Sum of Squares df Mean Square F P
Between Groups 52.389 3 17.463 0.814 0.488
Within Groups 3003.167 140 21.451
Total 3055.556 143
Table 3-12. Multiple comparisons of darker red zebra cichlids total length
Colorful Total Length Tukey HSD Test
95% Confidence Interval
(I) Diet (J) Diet Mean Difference (I-J) Std. Error P
Lover Bound
Upper Bound
1 2 -0.80556 1.09167 0.882 -3.6441 2.0329
3 -1.58333 1.09167 0.470 -4.4218 1.2552
4 -0.27778 1.09167 0.994 -3.1163 2.5607
2 1 0.80565 1.09167 0.882 -2.0329 3.6441
3 -0.77778 1.09167 0.892 -3.6163 2.0607
4 0.52778 1.09167 0.963 -2.3107 3.3663
3 1 1.58333 1.09167 0.470 -1.2552 4.4218
2 0.77778 1.09167 0.892 -2.0607 3.6163
4 1.30556 1.09167 0.630 -1.5329 4.1441
4 1 0.27778 1.09167 0.994 -2.5607 3.1163
2 -0.52778 1.09167 0.963 -3.3663 2.3107
3 -1.30556 1.09167 0.630 -4.1441 1.5329
(D-1) Control, (D-2) Astaxanthin, (D-3) Lutein, and (D-4) Spirulina.
45
Table 3-13. Red zebra cichlids CIE L* mean, Min, and Max values after 5 weeks
Diet No N Mean Min. Max.
1 56 64.97357 ± 3.0 56 63.63304 ± 4.3 56 64.30357 ± 3.4 56 63.39000 ± 3.7
57.80 70.94
2 61.62 71.80
3 62.65 73.03
4 62.87 70.46
(1) Control, (2) Astaxanthin, (3) Lutein, and (4) Spirulina after 5 weeks later. Table 3-14. Red zebra cichlids CIE a* mean, Min, and Max values after 5 weeks
Diet No N Mean Min. Max.
1 56 6.185893 ± 1.7 56 11.36464 ± 5.3 56 5.819643 ± 1.7 56 7.157679 ± 2.0
2.19 9.71
2 4.94 37.69
3 1.59 9.24
4 2.83 11.94
(1) Control, (2) Astaxanthin, (3) Lutein, and (4) Spirulina after 5 weeks later Table 3-15. Red zebra cichlids CIE b* mean, Min, and Max values after 5 weeks
Diet No N Mean Min. Max.
1 56 26.60125 ± 8.3 56 27.55054 ± 11.2 56 32.46625 ± 12.7 56 36.92946 ± 13.7
7.34 44.81
2 11.94 55.20
3 11.77 57.15
4 10.96 62.83
(1) Control, (2) Astaxanthin, (3) Lutein, and (4) Spirulina after 5 weeks later.
46
Table 3-16. Statistical color analysis of lighter red zebra cichlids for CIE L* values
CIE L* value Sum of Squares df Mean Square F P
Between Groups 2.567 3 0.856 0.113 0.952
Within Groups 575.897 76 7.578
Total 578.464 79
Table 3-17. Multiple comparisons CIE L* values for lighter red zebra cichlids
CIE L* value Tukey HSD Test
95% Confidence Interval
(I) Diet (J) Diet Mean Difference (I-J) Std. Error P
Lover Bound
Upper Bound
1 2 -0.38950 0.87049 0.970 -2.6761 1.8971
3 -0.27550 0.87049 0.989 -2.5621 2.0111
4 -0.47500 0.87049 0.947 -2.7616 1.8111
2 1 0.38950 0.87049 0.970 -1.8971 2.6761
3 0.11400 0.87049 0.999 -2.1726 2.4006
4 -0.08550 0.87049 1.000 -2.3721 2.2011
3 1 0.27550 0.87049 0.989 -2.0111 2.5621
2 -0.11400 0.87049 0.999 -2.4006 2.1726
4 -0.19950 0.87049 0.996 -2.4861 2.0871
4 1 0.47500 0.87049 0.947 -1.8116 2.7616
2 0.08550 0.87049 1.000 -2.2011 2.3721
3 0.19950 0.87049 0.996 -2.0871 2.4861
(D-1) Control, (D-2) Astaxanthin, (D-3) Lutein, and (D-4) Spirulina.
47
Table 3-18. Statistical color analysis of lighter red zebra cichlids for CIE a* values
CIE a* value Sum of Squares df Mean Square F P
Between Groups 48.949 3 16.316 6.274 0.001*
Within Groups 197.662 76 2.662
Total 246.611 79
*. The mean difference is significant at the P < 0.05 P < 0.05 (one way ANOVA). Table 3-19. Multiple comparisons CIE a* values for lighter red zebra cichlids
CIE a* value Tukey HSD Test
95% Confidence Interval
(I) Diet (J) Diet Mean Difference (I-J) Std. Error P
Lover Bound
Upper Bound
1 2 -1.75900* 0.50998 0.005* -3.0986 -0.4194
3 -0.41250 0.50998 0.850 -1.7521 0.9271
4 0.27950 0.50998 0.947 -1.0601 1.6191
2 1 -1.75900* 0.50998 0.005* 0.4194 3.0986
3 1.34650* 0.50998 0.048* 0.0069 2.6861
4 2.03850* 0.50998 0.001* 0.6989 3.3781
3 1 -0.41250 0.50998 0.850 -0.9271 1.7521
2 1.34650* 0.50998 0.048* -2.6861 -0.0069
4 0.69200 0.50998 0.530 -0.6476 2.0316
4 1 -0.27950 0.50998 0.947 -1.6191 1.0601
2 2.03850* 0.50998 0.001* -3.3781 -0.6989
3 -0.69200 0.50998 0.530 -2.0316 0.6476
(D-1) Control, (D-2) Astaxanthin, (D-3) Lutein, and (D-4) Spirulina. *. The mean difference is significant at the P < 0.05.
48
Table 3-20. Statistical color analysis of lighter red zebra cichlids for CIE b* values
CIE b* value Sum of Squares df Mean Square F P
Between Groups 94.087 3 31.362 0.909 0.441
Within Groups 2623.122 76 34.515
Total 2717.208 79
Table 3-21. Multiple comparisons CIE b* values for lighter red zebra cichlids
CIE b* value Tukey HSD Test
95% Confidence Interval
(I) Diet (J) Diet Mean Difference (I-J) Std. Error P
Lover Bound
Upper Bound
1 2 2.38700 1.85781 0.575 -2.4931 7.2671
3 0.74450 1.85781 0.978 -4.1356 5.6246
4 -0.47100 1.85781 0.994 -5.3511 4.4091
2 1 -2.38700 1.85781 0.575 -7.2671 2.4931
3 -1.64250 1.85781 0.813 -6.5226 3.2376
4 -2.85800 1.85781 0.420 -7.7381 2.0221
3 1 -0.74450 1.85781 0.978 -5.6246 4.1356
2 1.64250 1.85781 0.813 -3.2376 6.5226
4 -1.21550 1.85781 0.914 -6.0956 3.6646
4 1 0.47100 1.85781 0.994 -4.4091 5.3511
2 2.85800 1.85781 0.420 -2.0221 7.7381
3 1.21550 1.85781 0.914 -3.6646 6.0956
(D-1) Control, (D-2) Astaxanthin, (D-3) Lutein, and (D-4) Spirulina.
49
Table 3-22. Statistical color analysis of darker red zebra cichlids for CIE L* values
CIE L* value Sum of Squares df Mean Square F P
Between Groups 161.245 3 53.748 7.409 > 0.001*
Within Groups 1015.672 140 7.255
Total 1176.917 143
*. The mean difference is significant at the P < 0.05. Table 3-23. Multiple comparisons CIE L* values for darker red zebra cichlids
CIE L* value Tukey HSD Test
95% Confidence Interval
(I) Diet (J) Diet Mean Difference (I-J) Std. Error P
Lover Bound
Upper Bound
1 2 2.30167* 0.63486 0.002* 0.6509 3.9524
3 1.19528 0.63486 0.240 -0.4555 2.8460
4 2.72722* 0.63486 0.000* 1.0765 4.3780
2 1 -2.30167 0.63486 0.002* -3.9524 -0.6509
3 -1.10639 0.63486 0.306 -2.7571 0.5443
4 0.42556 0.63486 0.908 -1.2252 2.0763
3 1 -1.19528 0.63486 0.240 -2.8460 0.4555
2 1.10639 0.63486 0.306 -0.5443 2.7571
4 1.53194 0.63486 0.079 -0.1188 3.1827
4 1 -2.72722* 0.63486 0.000* -4.3780 -1.0765
2 -0.42556 0.63486 0.908 -2.0763 1.2252
3 -1.53194 0.63486 0.079 -3.1827 0.1188
(D-1) Control, (D-2) Astaxanthin, (D-3) Lutein, and (D-4) Spirulina. *. The mean difference is significant at the P < 0.05.
50
Table 3-24. Statistical color analysis of darker red zebra cichlids for CIE a* values
CIE a* value Sum of Squares df Mean Square F P
Between Groups 1358.506 3 452.835 47.663 > 0.001*
Within Groups 1330.120 140 9.501
Total 2688.626 143
*. The mean difference is significant at the P < 0.05. Table 3-25. Multiple comparisons CIE a* values for darker red zebra cichlids
CIE a* value Tukey HSD Test
95% Confidence Interval
(I) Diet (J) Diet Mean Difference (I-J) Std. Error P
Lover Bound
Upper Bound
1 2 -7.07861* 0.72652 0.000* -8.9677 -5.1896
3 0.79889 0.72652 0.690 -1.0902 2.6879
4 -1.66694 0.72652 0.104 -3.5560 0.2221
2 1 -7.07861* 0.72652 0.000* 5.1896 8.9677
3 7.87750* 0.72652 0.000* 5.9884 9.7666
4 5.41167* 0.72652 0.000* 3.5226 7.3007
3 1 -0.79889 0.72652 0.690 -2.6879 1.0902
2 -7.87750* 0.72652 0.000* -9.7666 -5.9884
4 -2.46583 0.72652 0.005* -4.3549 -0.5768
4 1 1.66694 0.72652 0.104 -0.2221 3.5560
2 -5.41167* 0.72652 0.000* -7.3007 -3.5226
3 2.46583* 0.72652 0.005* 0.5768 4.3549
(D-1) Control, (D-2) Astaxanthin, (D-3) Lutein, and (D-4) Spirulina. *. The mean difference is significant at the P < 0.05.
51
Table 3-26. Statistical color analysis of darker red zebra cichlids for CIE b* values
CIE b* value Sum of Squares df Mean Square F P
Between Groups 5420.369 3 1806.790 21.189 > 0.001*
Within Groups 11937.599 140 85.269
Total 17357.968 143
*. The mean difference is significant at the P < 0.05. Table 3-27. Multiple comparisons CIE b* values for darker red zebra cichlids
CIE b* value Tukey HSD Test
95% Confidence Interval
(I) Diet (J) Diet Mean Difference (I-J) Std. Error P
Lover Bound
Upper Bound
1 2 -2.80278 2.17650 0.572 -8.4620 2.8565
3 -9.53694* 2.17650 0.000* -15.1962 -3.8777
4 -15.80444* 2.17650 0.000* -21.4637 -10.1452
2 1 2.80278 2.17650 0.572 -2.8565 8.4620
3 -6.73417* 2.17650 0.013* -12.3934 -1.0749
4 -13.00167* 2.17650 0.000* -18.6609 -7.3424
3 1 9.53694* 2.17650 0.000* 3.8777 15.1962
2 6.73417* 2.17650 0.013* 1.0749 12.3934
4 -6.26750* 2.17650 0.024* -11.9267 -0.6083
4 1 15.80444* 2.17650 0.000* 10.1452 21.4637
2 13.00167* 2.17650 0.000* 7.3424 18.6609
3 6.26750* 2.17650 0.024* 0.6083 11.9267
(D-1) Control, (D-2) Astaxanthin, (D-3) Lutein, and (D-4) Spirulina. *. The mean difference is significant at the P < 0.05.
52
Table 3-28. Darker red zebra cichlids replicate L* values for Diet-1, Diet-2,Diet-3, and Diet-4
Diet-1 L* Replicate values
Sum of Squares df Mean Square F P
Between Groups 7.806 3 2.602 0.425 0.739
Within Groups 195.949 32 6.123
Total 203.755 35
Diet-2 L* Replicate values
Sum of Squares df Mean Square F P
Between Groups 104.594 3 34.865 3.329 0.32
Within Groups 335.128 32 10.473
Total 439.722 35
Diet-3 L* Replicate values
Sum of Squares df Mean Square F P
Between Groups 3.867 3 1.289 0.186 0.905
Within Groups 221.770 32 6.930
Total 225.636 35
Diet-4 L* Replicate values
Sum of Squares df Mean Square F P
Between Groups 16.795 3 5.598 1.381 0.26
Within Groups 129.763 32 4.055
Total 146.558 35
53
Table 3-29. Darker red zebra cichlids replicate a* values for Diet-1, Diet-2, Diet-3, and Diet-4
Diet-1 a* Replicate values
Sum of Squares df Mean Square F P
Between Groups 26.447 3 8.816 2.998 0.045
Within Groups 94.103 32 2.941
Total 120.550 35
Diet-2 a* Replicate values
Sum of Squares df Mean Square F P
Between Groups 95.905 3 31.968 1.151 0.343
Within Groups 888.48 32 27.765
Total 984.389 35
Diet-3 a* Replicate values
Sum of Squares df Mean Square F P
Between Groups 4.374 3 1.458 0.413 0.745
Within Groups 113.064 32 3.533
Total 117.438 35
Diet-4 a* Replicate values
Sum of Squares df Mean Square F P
Between Groups 3.810 3 1.270 0.391 0.760
Within Groups 103.932 32 3.248
Total 107.742 35
54
Table 3-30. Darker red zebra cichlids replicate b* values for Diet-1, Diet-2, Diet-3, and Diet-4
Diet-1 b* Replicate values
Sum of Squares df Mean Square F P
Between Groups 476.164 3 158.721 4.332 0.11
Within Groups 1172.458 32 36.639
Total 1648.622 35
Diet-2 b* Replicate values
Sum of Squares df Mean Square F P
Between Groups 955.261 3 318.420 3.258 0.034
Within Groups 3127.191 32 97.725
Total 4082.452 35
Diet-3 b* Replicate values
Sum of Squares df Mean Square F P
Between Groups 151.345 3 50.449 0.454 0.717
Within Groups 3559.471 32 111.233
Total 3710.817 35
Diet-4 b* Replicate values
Sum of Squares df Mean Square F P
Between Groups 33.833 3 11.278 0.147 0.931
Within Groups 2461.875 32 76.934
Total 2495.708 35
55
Table 3-31. Lighter red zebra cichlids replicate L* values for Diet-1, Diet-2, Diet-3, and Diet-4
Diet-1 L* Replicate values
Sum of Squares df Mean Square F P
Between Groups 24.765 3 8.255 0.887 0.469
Within Groups 148.942 16 9.309
Total 173.707 19
Diet-2 L* Replicate values
Sum of Squares df Mean Square F P
Between Groups 96.900 3 32.300 11.165 0.000
Within Groups 46.287 16 2.893
Total 143.187 19
Diet-3 L* Replicate values
Sum of Squares df Mean Square F P
Between Groups 61.534 3 20.511 3.196 0.052
Within Groups 102.675 16 6.417
Total 164.209 19
Diet-4 L* Replicate values
Sum of Squares df Mean Square F P
Between Groups 17.906 3 5.969 1.242 0.327
Within Groups 76.887 16 4.805
Total 94.794 19
56
Table 3-32. Lighter red zebra cichlids replicate a* values for Diet-1, Diet-2, Diet-3, and Diet-4
Diet-1 a* Replicate values
Sum of Squares df Mean Square F P
Between Groups 14.600 3 4.867 3.237 0.050
Within Groups 24.053 16 1.503
Total 38.653 19
Diet-2 a* Replicate values
Sum of Squares df Mean Square F P
Between Groups 35.298 3 11.766 3.392 0.440
Within Groups 55.496 16 3.468
Total 90.794 19
Diet-3 a* Replicate values
Sum of Squares df Mean Square F P
Between Groups 1.142 3 .381 0.151 0.928
Within Groups 40.412 16 2.526
Total 41.554 19
Diet-4 a* Replicate values
Sum of Squares df Mean Square F P
Between Groups 4.122 3 1.374 0.975 0.429
Within Groups 22.539 16 1.409
Total 26.661 19
57
Table 3-33. Lighter red zebra cichlids replicate b* values for Diet-1, Diet-2, Diet-3, and Diet-4
Diet-1 b* Replicate values
Sum of Squares df Mean Square F P
Between Groups 320.675 3 106.892 1.987 0.157
Within Groups 860.583 16 53.786
Total 1181.257 19
Diet-2 b* Replicate values
Sum of Squares df Mean Square F P
Between Groups 165.738 3 55.246 3.205 0.051
Within Groups 275.804 16 17.238
Total 441.542 19
Diet-3 b* Replicate values
Sum of Squares df Mean Square F P
Between Groups 89.422 3 29.807 1.032 0.405
Within Groups 461.915 16 28.870
Total 551.337 19
Diet-4 b* Replicate values
Sum of Squares df Mean Square F P
Between Groups 120.527 3 40.17 1.957 0.161
Within Groups 328.459 16 20.529
Total 448.985 19
58
CHAPTER 4 DISCUSSION
In this study, red zebra cichlids were fed carotenoid-supplemented commercial
diets, which induced skin color changes. Differences in skin color of the fish over time,
after consumption of the experimental diets, were analyzed by the Color Machine Vision
System method. The CMVS analysis can provide the L*, a*, and b* color values or
identify the exact skin color of fish. With this method, skin color of individual or multiple
fish can be analyzed depending on the image captured. Because this method is rapid
and non-lethal, CMVS is preferred for live fish analysis.
The diets used in this study contained astaxanthin, lutein and Spirulina as
coloration pigments. After 35 days of feeding the fishes those diets, results obtained
from the CMVS method showed that different dietary carotenoids can cause changes in
skin coloration in the red zebra cichlid. These results are in agreement with the previous
reports, in which it was indicated that dietary carotenoids can induce skin coloration of
fishes (Pan and Chien, 2009; Yasir and Qin, 2010).
The impact of dietary carotenoids on skin color change was more easily
detectable by direct analysis of the skin pigments in red zebra cichlid compared to
image analysis of the whole fish. Coloration is clearly defined in Figure 3.1. Results
indicate that carotenoid-supplemented diets trigger the skin pigmentation of red zebra
cichlid, in agreement with the findings of Xu (2006) and Yanar et al. (2008) for gold fish.
After 14 days of feeding fishes with the carotenoid-supplemented diets, the
change in skin color of the red zebra cichlids could be easily distinguished with the
naked eye. Color differences of the fishes after the 35th day of feeding were confirmed
by CMVS. Previous reports showed that the time required for color change may differ
59
among species and may depend on the level of carotenoid in the fish diet. For instance,
Paripatananont et al. (1999) reported that after seven days, the skin color of goldfish fed
with an astaxanthin-supplemented diet started to differ from fish that were fed a control
diet. Similarly, Wallat et al. (2005) found that the oranda goldfish developed its color
with supplemented diets that contain astaxanthin, lutein and zeaxanthin after 20 weeks
of feeding. Storebakken et al. (1987) reported that more astaxanthin was deposited in
the skin of Salmo salar after 21 days of feeding on a supplemented diet compared to
fish on a control diet. Similarly, after a 21-day period of feeding with an astaxanthin-
enhanced diet, 40 mg kg-1 astaxanthin was found in the skin of the gilthead sea bream
Sparus auratus (Gomes et al., 2002).
These carotenoid supplements in fish diets mainly contain β-carotene and
zeaxanthin and they are commonly used coloration pigments for ornamental fishes.
Previous studies showed that dietary carotenoids such as canthaxanthin, astaxanthin,
and β-carotene led to the deposition of astaxanthin in Parribacus japonicus (Yamada, et
al., 1990; Chien & Jeng 1992).
The type of pigment in the diet affected skin color in the red zebra cichlid (Table
3.3). Astaxanthin, lutein, and Spirulina affected the skin coloration of red zebra cichlid.
Effects of the different carotenoids on skin coloration in the fish are seen in Figure 3.1.
Differing effectiveness of the carotenoids was also reported previously, such as
Yamada et al. (1990) indicated that astaxanthin is a more effective carotenoid than
canthaxanthin or β -carotene in skin coloration of fish.
When the effects of carotenoid-supplemented diets on fish skin coloration were
analyzed by the CMVS, diet 2, which was supplemented with astaxanthin, increased the
60
red-orange color of the fish skin, this was confirmed by the higher a* values obtained by
CMVS compared to the ones obtained for the other diets. Diet 4 supplements with
Spirulina increased orange and yellow color because the b* value was higher than in
others. A study has shown that dietary astaxanthin increased pinkish orange color,
whereas zeaxanthin led to light orange color (Tanaka et al., 1992). Although astaxanthin
led to a lower hue value or reddish color in fish studied by Kalinowski et al., (2005),
similar to my results, β-carotene did not develop the red color in red porgy (Chatzifotis
et al., 2005). Moreover, Doolan et al. (2008) reported that astaxanthin was an effective
carotenoid for develop pigmentation in Australian snapper, using L*, a*, and b* color
values and skin coloration. My results are in agreement with this finding because diet 2
supplements with astaxanthin influenced the L*, a*, and b* color values of red zebra
cichlid (Table.3.23, 3.25, and 3.27).
My result showed that although the diet 3, supplemented with lutein, is almost 10
times lower than other diets supplemented with astaxanthin or Spirulina, it still affected
the skin color of red zebra cichlid. These results indicate that different levels of
carotenoid can affect skin color in red zebra cichlids. Nevertheless, Nakazoe et al.
(1984) found that diet supplements with low levels of β–carotene failed to develop the
reddish color in red porgy. Kim et al. (1999) reported that lutein is the best diet
supplement to enhance the color.
Some studies have suggested that a mixed diet is more effective than diets with
single pigments. For instance, Wang et al. (2006) reported that a mixed diet with
astaxanthin and β–carotene is more effective than a diet with only one type of
carotenoid. We used Spirulina as diet 3, which is a mixed diet containing β –carotene
61
and zeaxanthin. Diet 3 had a pronounced impact on fish color, because the Spirulina
contributed to development of all the color values, such as L*, a*, and b*.
The role of carotenoids sexual selection has been studied in only a few
ornamental fish species. In addition, carotenoids have many functions in sexual
ornamentation (Kolluru et al., 2006). Coloration with carotenoids, however, is now
recognized as being important for sexual selection. Endler (1980) reported that after a
few generations without predation, the color pattern of male guppies in a population
increased. Carotenoid concentration alone cannot be used as a criterion for fish color
(Little et al., 1979). Torrissen and Naevdal (1988) reported that individual size, weight,
age, sexual maturity, and other factors influenced deposition of carotenoid pigment in
Atlantic salmon (Salmo salar). I found that carotenoids affect the coloration of fish
differently based on sex. Male red Carotenoids influenced skin color in male zebra
cichlid more so than in female fish. In contrast, Goodwin (1952) reported that a lack of
carotenoids had a negative effect on the performance of fish.
This study showed that different dietary carotenoids did not affect the growth and
survival of the red zebra cichlid. Similar findings were also reported with rainbow trout
and other salmonid fishes supplemented with β-carotene and astaxanthin (Bell et al.,
2000; Amar et al., 2001; Ramamoorthy et al., 2010). Similar results were also reported
by Boonyaratpalin et al. (2001), who used astaxanthin as a dietary carotenoid and found
it did not affect growth, survival or health of Penaeus monodon significantly. Some
studies, however, reported dietary carotenoids influenced the survival or growth of
certain fish. For instance, whereas the survival rate for goldfish fed astaxanthin was
higher than goldfish fed the control diet, astaxanthin did not affect their growth
62
(Paripatananont et al. 1999). Growth was highest in Korean rose bitterling Rhodeus
uyekii fed a diet supplemented with astaxanthin, however no effect was observed when
the diet was supplemented with β-carotene or lutein (Kim et al. 1999).
The influence of different types and concentrations of dietary carotenoids is a
good topic for future study and it will be important to determine the optimum dose of
carotenoids for coloration of fish. If the optimum dose of carotenoid for ornamental
fishes can be determined, it will decrease the price of the diet, reducing costs for
aquaculturists. This study of carotenoid pigments in ornamental fishes provides a model
for researchers who study color in larger, more difficult taxa destined for human
consumption.
This work examined the effects of concentration of dietary colorants astaxanthin,
lutein, and Spirulina on pigmentation in the red zebra cichlid. If used judiciously in
farming red zebra cichlid, such diets can bring large economic benefits to farmers who
raise fish with different coloration. Color is a really important criterion for the fish
consumer (Dharmaraj & Dhevendaran, 2011). Some studies with the Australian
snapper, Pagrus auratus, showed that color in wild fish is more red than in farmed fish
(Booth et al., 2004; Cejas et al., 2003). Thus, supplementing the diet of farm fish with
carotenoids can develop the color and increase their market value.
63
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BIOGRAPHICAL SKETCH
Serdar Yedier was born in Nigde, Turkey. He started his undergraduate degree
at 19 Mayis Samsun University in 2005, majoring in biology. Upon graduation in 2009,
he started his master of science at Ordu University, but did not complete the program.
He won a full scholarship from the Turkish government to take his Master of Science
and Doctor of Philosophy in the United States of America. He came to the University of
Florida in 2011 and started his Master of Science there under the supervision of Frank
Chapman.