COLOR ENHANCEMENT IN THE ORNAMENTAL RED ......also be absorbed by colorants such as dye or pigment in the objects, making them appear different colors. The observer is a third important

1

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

2

© 2013 Serdar Yedier

3

To my parents, who supported me in every stage of my Master of Science

4

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.

.

5

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

6

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

9

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

10

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.

11

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,

12

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

13

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.

14

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.

15

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

16

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).

17

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

18

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).

19

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

20

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

21

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

22

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

23

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.

24

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*62.15 a*15.48 b*32.93 moderate orange^

L*67.37 a*7.44 b*18.63 moderate yellowish pink^^


L*62.66 a*5.66 b*39.23 dark yellow^



L*61.13 a*8.13 b*45.50 dark orange yellow^


(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


Total 16.482 79

Table 3-8. Multiple comparisons of lighter red zebra cichlids weight

Lighter Weight Tukey HSD Test


(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



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


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



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


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


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


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


Total 578.464 79

Table 3-17. Multiple comparisons CIE L* values for lighter red zebra cichlids

CIE L* value Tukey HSD Test



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


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*


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



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


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



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


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



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


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



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


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



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


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


Total 203.755 35



Between Groups 104.594 3 34.865 3.329 0.32

Within Groups 335.128 32 10.473

Total 439.722 35



Between Groups 3.867 3 1.289 0.186 0.905


Total 225.636 35



Between Groups 16.795 3 5.598 1.381 0.26


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


Between Groups 26.447 3 8.816 2.998 0.045


Total 120.550 35



Between Groups 95.905 3 31.968 1.151 0.343


Total 984.389 35



Between Groups 4.374 3 1.458 0.413 0.745


Total 117.438 35



Between Groups 3.810 3 1.270 0.391 0.760


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


Between Groups 476.164 3 158.721 4.332 0.11

Within Groups 1172.458 32 36.639

Total 1648.622 35



Between Groups 955.261 3 318.420 3.258 0.034

Within Groups 3127.191 32 97.725

Total 4082.452 35



Between Groups 151.345 3 50.449 0.454 0.717

Within Groups 3559.471 32 111.233

Total 3710.817 35



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



Between Groups 24.765 3 8.255 0.887 0.469


Total 173.707 19



Between Groups 96.900 3 32.300 11.165 0.000


Total 143.187 19



Between Groups 61.534 3 20.511 3.196 0.052


Total 164.209 19



Between Groups 17.906 3 5.969 1.242 0.327


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



Between Groups 14.600 3 4.867 3.237 0.050


Total 38.653 19



Between Groups 35.298 3 11.766 3.392 0.440


Total 90.794 19



Between Groups 1.142 3 .381 0.151 0.928


Total 41.554 19



Between Groups 4.122 3 1.374 0.975 0.429


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



Between Groups 320.675 3 106.892 1.987 0.157

Within Groups 860.583 16 53.786

Total 1181.257 19



Between Groups 165.738 3 55.246 3.205 0.051

Within Groups 275.804 16 17.238

Total 441.542 19



Between Groups 89.422 3 29.807 1.032 0.405

Within Groups 461.915 16 28.870

Total 551.337 19



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

http://www.acronymgeek.com/CMVS/Color_Machine_Vision_System

http://www.acronymgeek.com/CMVS/Color_Machine_Vision_System

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.

Documents

COLOR ENHANCEMENT IN THE ORNAMENTAL RED ......also be absorbed by colorants such as dye or pigment in the objects, making them appear different colors. The observer is a third important