Group Title: Factors affecting the measurement and utilization of xanthophylls in the egg yolk and broiler skin /
Title: Factors affecting the measurement and utilization of xanthophylls in the egg yolk and broiler skin
CITATION PDF VIEWER THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00098095/00001
 Material Information
Title: Factors affecting the measurement and utilization of xanthophylls in the egg yolk and broiler skin
Physical Description: x, 7l leaves : graphs ; 28 cm.
Language: English
Creator: Fletcher, Daniel Lake, 1949-
Publication Date: 1977
Copyright Date: 1977
 Subjects
Subject: Poultry -- Feed utilization efficiency   ( lcsh )
Poultry -- Housing   ( lcsh )
Light -- Physiological effect   ( lcsh )
Poultry Science thesis Ph. D
Dissertations, Academic -- Poultry Science -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 61-70.
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Daniel Lake Fletcher.
 Record Information
Bibliographic ID: UF00098095
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000011486
oclc - 03403541
notis - AAB3987

Downloads

This item has the following downloads:

PDF ( 1 MBs ) ( PDF )


Full Text






























































I


FACTORS AFFECTING THE MEASUREMENT
AND UTILIZATION OF XANTHOPHYLLS
IN THE EGG YOLK AND BROILER SKIN

















By

DANIEL LAKE FLETCHER














A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY



UNIVERSITY OF FLORIDA





ACKNOWLEDGMENTS


The author wishes to express his most sincere appreciation to

Dr. D. M. Janky, Co-chairman of the Supervisory Committee, for his day

to day guidance and support, both professionally and as a friend. He is

also indebted to Dr. R. H. Harms, Chairman of the Supervisory Committee,

for his valuable research guidance.

The author also extends his sincerest thanks to Dr. H. R. Wilson,

Dr. J. L. Oblinger and Dr. R. L. West, members of the Supervisory

Committee, for their valued support and encouragement. Special thanks

are extended to his fellow graduate students, Winston Nesbeth, Jin Ho

Choi and Bob Bloomer, for making the Manor liveable. He also wishes to

thank the staff, faculty and graduate students of the Poultry Science

Department for their help.

He is especially indebted to his parents, family and close friends

who consistently lent their support and encouragement in this endeavor.

And finally to Sharon, to whom I dedicate this final work.





TABLE OF CONTENTS





ACKNOWLEDGMENT .. .. .. .. .. .. .. .. .. ii

LIST OF TABLES ... .. . .. ... v

LIST OF FIGURES .. .. .. .. .. .. .. ... . .. .. vii

ABSTRACT ... .. .. .. ... .. .. .. .. . ... viii

INTRODUCTION .. .. . .... .. ... .. .. .. .. 1

LITERATURE REVIEW .. .. ... .. . .. ... .. .. 3

Importance of Pigmentation in Poultry .. ..... 3
History and Nomenclature of the Carotenoids .. .. .. .. 5
The Xanthophylls Commonly Found in Egg Yolks and
Broilers and the Feed Sources that Supply these
Pigments in the Diet .. ... .. . ... . .. .. 7
Factors Affecting Pigmentation and Xanthophyll
Utilization .. .. .. ... .. .. . ... . .. 10
Methods of Evaluating Broiler and Egg Yolk
Pigmentation ... .. .. .. .. ... .. .. 17

CHAPTER I THE INFLUENCE OF LIGHT ON BROILER
PIGMENTATION I. .. ... ... .. . .. ... 20

Experimental Procedure .. .. .. .. .. .. 2
Results and Discussion ... .. .. .. .. .. 24

CHAPTER II STRAIN DIFFERENCES IN EGG YOLK PIGMENTATIONJ .. 30

Experimental Procedure ... .. .. .. .. .. 30
Results and Discussion .. ... .. . .... 32

Tr~ial 1 .. .. .. ... .. ... .. 32
Trial 2 .. ... .. . ... .. ... 34

CHAPTER III YOLK COLOR CHARACTERISTICS, XANTHOPHYLL
AVAILABILITY, AND A MODEL SYSTEM FOR PREDICTING
EGG YOLK COLOR USING BETA-APO-8'-CAROTENAL AND
CANTHAXANTHIN . .... . .. ... 38










TABLE OF CONTENTS (Continued)


40
43

59

61

71


Experimental Procedure;.
Results and Discussion .

SUMMARY AND CONCLUSIONS. .. .

LIST OF REFERENCES. .

BIOGRAPHICAL SKETCH.. .


. . . . . .
. . . . . .

...........

...........

...........














LIST OF TABLES


Table Page

1 Xanthophyll concentrations and ranges of variation
for different feedstuffs .. .. ... .. .. ... 11

2 Continuous lighting program used for windowless
houses .. .. .. .. .. .. . . .. . 21

3 Starter, finisher and withdrawal feed formulas
used in both the windowless and the open-type
houses .. .. . .. . .. . ... 22

4 Body weight, feed consumption, feed efficiency,
blood xanthophyll, skin color score and shank
dominant wavelength (DWL), excitation purity (EP)
and luminosity (Lum) for open venusLc windowless
houses .. .... .. .. .. .. .. .. .. 25

5 Dominant wavelength (DWL), excitation purity (EP),
luminosity (Lum) and light intensity for broilers
raised in either outer or inner pens . ... .. .. 27

6 Dominant wavelength (DWL), excitation purity (EP),
and luminosity (Lum) of shanks from birds raised
in either outside oriented pens or inside pens in
four previous experiments .. .. .. .. . 29

7 Composition of diets (Trials 1 and 2) ... .. .. 31

8 Dominant wavelength (DWL), excitation purity (EP)
and luminosity (Lum) of yolks from eggs produced by
12 strains of laying hens in cage and floor houses
(Trial 1) . . . . . . . . . . . . 33

9 Dominant wavelength (DWL), excitation purity (EP)
and luminosity (Lum) of yolks from eggs produced by
12 strains of laying hens in cage and floor houses
(Trial 2) . . . . . . . . . . . . 35

10 Dominant wavelength (DWL), excitation purity (EP)
and luminosity (Lum) of yolks from eggs produced by
laying hens in cage and floor houses (Trials 1 and
2) . . . . . . . 37









LIST OF TABLES (Continued)


Table Page

11 Composition of white corn basal diet . .. .. .. .. 41

12 Total xanthophyll concentrations (mg./kg.) of the
25 diets at the corresponding levels of beta-apo-
8'-carotenal and canthaxanthin .... .. .. .. 42

13 Dominant wavelength (DWL) and luminosity (Lum) of
the 25 dietary levels of beta-apo-8'-carotenal and
canthaxanthin on undiluted yolk samples .. .. .. .. 44

14 Visual color scores using the Heiman-Carver Color
Rotor (HCCR) and the Roche Color Fan (RCF) for the
25 fed dietary levels of beta-apo-8'-carotenal and
canthaxanthin on undiluted yolk samples with cor-
relation coefficients (r) with dominant wavelength
(DWL), excitation purity (EP) and luminosity (Lum)
of the same sample . .. .. . .... . . . 45

15 Excitation purity values (%) of the 25 dietary
levels of beta-apo-8'-carotenal and canthaxanthin
on undiluted and diluted yolk samples .. ... .. 46

16 Dominant wavelength (DWL), excitation purity (EP)
and luminosity (Lum) of blended yolk samples and
yolks which were obtained from hens receiving a
similar dietary level of beta-apo-8'-carotenal (A)
and canthaxanthin (C) .. .. .. .. .. .. .. 52





LIST OF FIGURES


Figure Pg

1 Excitation purity values for undiluted and
diluted yolks from hens fed various levels of
beta-apo-8'-carotenal and canthaxanthin . .. .... 48

2 Linear regression model of diluted excitation
purity values for beta-apo-8'-carotenal and
canthaxanthin between 2.2 and 17.6 mg./kg.
total xanthophyll when fed separately .. .. .. .. 51

3 Diluted excitation purity values for blended
vetsnud fed xanthophyll concentrations . ... .. .. 54

4 C.I.E. x-y chromaticity diagram with the 5 x 5
beta-apo-8'-carotenal and canthaxanthin fed
yolk sample coordinates and the Roche Color Fan
values .. .. .... . ... .. .. . .. .. 57





Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy



FACTORS AFFECTING THE MEASUREMENT
AND UTILIZATION OF XANTHOPHYLLS
IN THE EGG YOLK AND BROILER SKIN

By

Daniel Lake Fletcher

August, 1977
Chairman: R. H. Harms
Co-chairman: D. M. Janky
Major Department: Animal Science

Three experiments were conducted to determine xanthophyll utiliza-

tion in both broilers and layers. The first experiment was conducted to

determine the effects of rearing broilers in windowless houses vennuo

open-type houses on skin and shank pigmentation. In addition, data from

five previous experiments conducted on broiler pigmentation were ana-

lyzed according to the positions of the birds in the house to compare

the pigmentation of broilers exposed to different light intensities.

Skin and shank of broilers raised with constant low intensity light-

ing in the windowless houses, exhibited a significantly lower dominant

wavelength (less orange) than birds reared in open-type houses. With a

natural diurnal light cycle present, broilers reared in pens along open

outer walls had a higher dominant wavelength (more orange) than broilers

reared in interior pens where light intensity was lower. These results

indicate that light has an effect on broiler pigmentation.

The second experiment was divided into two parts and was conducted

to evaluate egg yolk pigmentation from eggs produced by 12 commercial





laying strains of hens housed in both floor and cage conditions. Egg

yolk pigmentation was evaluated using reflectance colorimetry.

Significant differences in dominant wavelength, excitation purity
and luminosity were found between the 12 strains. Egg yolks from

several strains were more highly pigmented regardless of housing condi-

tions, however, yolks from several other strains appeared to vary with

the housing condition.

The third experiment was conducted to evaluate the egg yolk pig-

menting ability of beta-apo-8'-carotenal and canthaxanthin using

reflectance colorimetry to describe color and determine relative bio-

logical availability. Laying hens, previously depleted of xanthophylls,

were fed diets containing 0, 2.2, 4.4, 8.8 and 17.6 mg./kg., of each of

the two xanthophylls, in a 5 x 5 factorial treatment design. The yolk

color was evaluated, undiluted and diluted with a white diluent for each

treatment, for the YCIE values of dominant wavelength (DWL), excitation

purity (EP) and luminosity (Lum) using the IDL COLOR-EYE~ Selected

treatment combinations of egg yolks were blended and also evaluated

undiluted and diluted.

DWL increased from 572.8 nm. for the 0 level to 577.9 and 590.6 nm.

for the 17.6 mg./kg. levels of beta-apo-8'-carotenal and canthaxanthin,

respectively. Analysis of the diluted EP values showed that beta-apo-8'-
carotenal had a biological availability of 82% and a pigmenting effi-

ciency of 92% when compared to canthaxanthin. Blended samples yielded

almost identical color scores when compared at the same level of

xanthophyll concentration as fed. From these data a model was formulated

by which yolk color could be predicted from the feed concentrations of

these two xanthophylls. It is suggested that egg yolks of a desired








color could be obtained by using the model to predict the feed concen-

tration of the two test xanthophylls required in the diet. It would

also be possible to produce a yolk product of desired color by blending

yolks produced from feeds with known concentrations of xanthophyll.














INTRODUCTION


Pigmentation is an important factor in the consumer acceptance and

quality judgement of broilers, egg yolks and certain products utilizing

egg yolk as a color source. The desired degree of broiler pigmentation

usually depends upon consumer preferences in a particular geographical

area based upon tradition, availability of product and marketing

promotion. Though yolk color for consumer eggs is less critical than

the color of broilers in the selection of the product by consumers, it

is frequently cited as a major factor in judging egg acceptability.

Egg yolk color, however, is an important criterion to egg breakers who

supply egg yolks to processors who utilize eggs in such products as

mayonnaise, egg noodles and certain bakery products. Quality of these

products is often judged on the "richness" of color as reflected by

apparent egg yolk content.

Depending on regional preferences and market demand, the broiler

and egg producer can often demand and receive a premium for a well

pigmented product. Therefore, considerable effort has been focused on

supplying necessary xanthophylls to the diet of the bird to get maximum

pigmentation at the least cost. The two major xanthophyll sources in

most common poultry diets are yellow corn and alfalfa meal, however,

these do not supply the necessary amount and type of xanthophylls to

produce adequate pigmentation in many geographical areas. Therefore,

high xanthophyll feed sources which are often quite expensive in








relation to their nutritive value must be added to the diet. This has

resulted in a strong economic stimulus to identify and control the

factors that influence pigmentation and thus increase the efficiency of

the expensive xanthophyll sources.

The general purpose of these studies was to examine some of the

factors that can influence both broiler and egg yolk pigmentation. More

specifically, the objectives of these studies were as follows:

1. to determine the influence of light intensity on broiler

pigmentation;

2. to determine the differences in egg yolk pigmentation as

related to layer strain and housing condition; and

3. to determine xanthophyll availability and the resulting yolk

color characteristics of two synthetic xanthophylls, beta-apo-8'-

carotenal and canthaxanthin.














LITERATURE REVIEW


Importance of Pigmentation in Poultry

It has been observed that color is an important factor in the con-

sumer acceptance of many food products. Pomeranz and Meloan (1971, p. 72)

state, ". . Although they [food colors] do not necessarily reflect

nutritional, flavor, or functional values, they relate to consumer pref-

erences based on the appearance of the product." The role played by

color in foods is well stated by Bunnell and Bauernfeind (1962, p. 36):

Nature has so liberally endowed the world with color that the
association of color with food is inseparable. The rich
yellow of an egg yolk, the bright red of a ripe tomato, the
appetizing appeal of the red color of a freshly cooked lobster
are so strongly associated that a green egg yolk, a blue
tomato, or a white lobster would be completely unappetizing
and even repulsive. Color, therefore, increases our enjoyment
and influences our selection of food.

The influence of both broiler and egg yolk pigmentation on the con-

sumer acceptance of these products, as well as products utilizing egg

yolk as a source of color, has been shown by several researchers. The

actual color or degree of pigmentation preferred by consumers for either

table egg yolks or broiler skin seems to depend upon regional preferences

based on tradition and availability.

Palmer (1915) stated that in some sections of the country the

poultry trade demanded a "highly colored" flesh, but the "fancy" trade

demanded a flesh with the "least color" possible. Fritz and Wharton

(1957) reported that consumers preferred a moderately pigmented bird.





In a consumer preference study in Tennessee conducted by Raskopf et at.

(1961), it was reported that consumers preferred more darkly pigmented

birds when faced with a choice of light and dark pigmented birds;

however, 23% of the consumers did prefer light skinned birds. When

offered only light skinned birds, the consumers did not show any dis-

crimination against these birds. Heffner at at. (1964) reported that,

according to sales records, consumers in New Orleans preferred dark to

moderately pigmented birds over non-pigmented birds. About 44% preferred

heavily pigmented, 33% moderately pigmented and 23%/ non-pigmented birds.

It was also reported that the degree of pigmentation was related to the

geographical area in which the birds were produced, but not the area

marketed (Raskopf et al., 1961).

Ashton and Fletcher (1962) stated that the desired egg yolk color

for home use varies with geographical location and customs of the people,

but, in general, a fairly light color was preferred. In a consumer

preference study, conducted in Columbus, Ohio, it was reported that more

consumers preferred medium yolk color than either light or dark colored

yolks, however, more housewives were concerned with uniformity than with

shade of color (Jasper and Cray, 1953). De Groote (1970) reported that

consumers in Belgium preferred dark pigmented egg yolks. Of 23,304

participants, 51%/ preferred egg yolks corresponding to a Roche color fan

value of 15, while 21% preferred egg yolks corresponding to a value of

12. Slocum and Swanson (1954) conducted a consumer preference study in

Seattle, Washington, in which 44% of the consumers preferred medium yolk

color, 26% preferred light yolk colors and only 15% preferred dark


orange yolks.








It is widely accepted that consumers will use yolk color as an

indicator of quality (Sullivan and Holleman, 1962). Goffinet and Ledent

(1967) allowed consumers to choose from the following 10 categories of

egg qual ity factors. volume of ai r cell, taste of egg, color of white,

color of yolk, position of yolk in egg, color of shell, odor of egg,

firmness of white, firmness of yolk or other. They reported that 42.5%

of the consumers stated that yolk color was the Main factor used in

judging egg quality and 57% stated that it was one of several factors.

The color of egg yolks is also important to food processors who

utilize eggs in their products and depend upon the yolk pigments to

color their products. The consumer tends to judge richness of the prod-

uct by the apparent amount of egg color it contains (Dalby, 1948).

Therefore, egg breakers generally prefer much darker yolks than con-

sumers prefer for table eggs (Ashton and Fletcher, 1962, Sullivan and

Holleman, 1962).


History and Nomenclature of the Carotenoids


The pigments responsible for the yellow-red color in poultry belong

to a group of compounds called carotenoids. A particular class of

carotenoids, called xanthophylls, is usually the most prominent in

poultry pigmentation. These two terms are often used interchangeably in

the literature to mean all the fat soluble pigments found in poultry.

This confusion dates back to the earliest isolations and namings of

these compounds. Wachenroder in 1826 prepared a crystalline product of

the yellow pigments from carrot roots, which he named carotinn" (Weis

and Bisbey, 1947). The yellow alcohol-soluble pigments of autumn leaves

were termed "xanthophylls" by Berzelius in 1837 (Goodwin, 1954).





Tswett in 1911 (Goodwin, 1954) used chromiatographic separation of leaf

xanthophylls which resulted in a complex mixture of "polichromes."

These were divided into two classes: caroteness," which were very solu-

ble in hydrocarbon solvents, and "xanthophylls," which were less soluble

in hydrocarbon solvents but very soluble in ethanol. These two classes

were grouped together under the general term "carotenoids" (Goodwin,

1954). M~any of these early ambiguities have been resolved by the "Union

International de Chemie" (C.I.E.) based upon Karrer's definition of

carotenoids (Goodwin, 1954, p. 1):

Carotenoids are yellow to red pigments of aliphatic or
alicyclic structure, composed of isoprene units (usually eight)
linked so that the two methyl groups nearest the centre of the
molecule are in positions 1:6 whilst all other lateral methyl
groups are in positions 1:5, the series of conjugated double
bonds constitutes the chromophoric system of the carotenoids.

The carotenes are defined as the carotenoid hydrocarbons; however, there

are still some confusing problems in the nomenclature of other

carotenoids. The C.I.E. recognizes the oxygen containing carotenoids as

being derivities of carotenes. Xanthophyll was the name given to one

particular oxycarotenoid which was first isolated in pure form by Karrer.

Later, Kuhn isolated the same pigment and called it lutein and sug-

gested xanthophyll as a group name for the hydroxy carotenoids (Goodwin,

1954). Goodwin (1954) suggested the use of the name lutein to designate

Karrer's individual pigment and the use of the name xanthophyll as a

group term for the hydroxy carotenoids. This nomenclature is the con-

vention in many countries.

The above definition of carotenoids includes such naturally occur-

ring compounds as vitamin A and azafrin in so far as they are considered

breakdown products (apocarotenoids) of carotenoids containing 40 carbon

atoms. There are at least 100 carotenoids and they can all be related





to the parent substance lycopene, the red pigment of tomatoes. Lycopene

is derived from some color-polyene, such as phytoene. By cyclization,

the ends of the chain are closed to form beta-carotene and by further

chemical changes, a wide range of pigmlents may be derived from the

parent carotenoid (Bunnell and Bauernfeind, 1962).

Bauernfeind (1972, p. 456) gave the following classification of
the carotenoids:

Chemically, they may be divided into (a) the carotenes, made
up of carbon and hydrogen only, and (b) the oxycarotenoids
containing oxygen in addition to carbon and hydrogen, sub-
divisions of which would be (c) the epoxiy, (d) the furanoxy1,
(e) the hydroxy or xanthophylls (monols, diols, polyols),
(f) the methoxy, (g) the keto, (h) esters, etc. A different
classification system subdivides the carotenoids into (i)
acyclic, (j) monocyclic, and (k) bicyclic derivatives.
Functionally, they can be divided into (1) provitamines A or
vitamin A precursors, (m) vitamin A precursors which also
function as animal tissue pigmenters, (n) animal tissue pig-
menters without vitamin A activity, and (0) compounds which
do neither.


The Xanthophylls Commaonly Found in Egg Yolks
and Broilers and the Feed Sources that
Supply these Pigments in the Diet

In 1912, Willstatter and Escher (Goodwin, 1954) were the first to

investigate the pigments found in the domestic hen's egg yolk and they

considered it to be a homogeneous pigment which was an isomer of "leaf

xanthophyll." They termed this pigment lutein. Eighteen years later

Karrer and Helfenstein (Goodwin, 1954) showed that the yolk pigment,

although having the same melting point as leaf xanthophyll luteinn),

differed from it in optical rotation. Other workers (Goodwin, 1954)

then showed that the yolk pigment of hens on common rations was a mix-

ture consisting of 70% lutein and the remainder principally zeaxanthin.

The xanthophylls are stored in the egg in the free form, with only 8%









esterified xanthophylls present. Carotene constitutes only 2-10%/ of the

total carotenoids present (Goodwin, 1954). In general, it can be said

that hens deposit in the yolk at least "part" of any carotenoid fed

(Goodwin, 1954).

The carotenoids commonly isolated from egg yolks are lutein,

zeaxanthin, cryptoxanthin, carotene, capsanthin, lycopene, neoxanthin,

flavoxanthin, isolutein, astaxanthin, echinenone and canthaxanthin

(Goodwin, 1954; Smith and Perdue, 1966, Rodriguez e~t at., 1976). It was

also noted that lutein and zeaxanthin account for the largest percent of

the xanthophylls present. Other compounds have also been isolated from

egg yolks but these are often from exotic sources or isomers of the al-

ready mentioned compounds.

Palmer's work in the early 1920's (Goodwin, 1954) showed that in

hens, dietary xanthophylls, but not carotenes, occur in the blood

plasma, fat and skin, especially the shanks and toes. The main pigment

is lutein which is esterified in the skin. Xanthophylls are stored in

the liver of hens and turkeys and in the skin, fat, face and bills of

15 species of wild birds (Goodwin, 1954). Xanthophylls that have been

isolated from poultry skin are lutein, zeaxanthin, astaxanthin,

galioxanthin, carotenes and cryptoxanthin (Goodwin, 1954; Smith and

Perdue, 1966).

A great many sources of dietary xanthophylls have been examined and

evaluated over the years. Steenbock (1919) related fat-soluble vitamin

activity to the yellow pigment of yellow corn. Palmer and Kempster

(1919) also compared yellow corn venus white corn in pigmenting ability.

They reported that green feeds were effective as yellow pigmenters.

Some pigmenting ability was noted for hemp seed, barley, gluten feed and








red corn but virtually none for wheat, wheat bran, oats, cottonseed meal,

rape seed, meat scrap and blood meal. Yellow corn, corn gluten meal,

alfalfa meal and grass meals, which are the major sources of xanthophyll

in practical poultry feeds, have been extensively evaluated and compared

for pigmenting ability (Holleman and Sullivan, 1959; Sunde, 1962; Hall

et at., 1966; Kuzmicky eat aL., 1968; Tortuero, 1968; Wilkinson and

Barbee, 1968; Livingston et: al., 1969, Halloran et at., 1971-, Herrick

eat at., 1972; Guenthner et at., 1973; Hurst et at., 1973; Sullivan et

dt., 1974; Fry and Harms, 1975).

Many local sources and exotic feedstuffs with a high xanthophyll

content have also been evaluated. The influence of pigments from waste

pimento peppers fed in layer diets on yolk color was examined by Norgan

and Woodroof (1927) and Brown (1938). Sewage grown algae (Grau and

Klein, 1957), algae meal (Morehouse, 1961), dried lake weed (Madiedo and

Sunde, 1962) and seaweed carotenoids (Jensen, 1963) have been evaluated

for skin and yolk pigmentation value. Carotenoids from different levels

of paprika extract (Mackay eat al., 1963), cow manure (Littlefield eAt al.,

1973) and anaerobic mud, tomato paste, lobster shells and bacteria

(Nelson and Baptist, 1968) have been evaluated as to their pigmenting

effects in egg yolks. Dehydrated broccoli leaf (Runnels et aE., 1951),

dried Kenaf tops (Fry eat at., 1967), shrimp waste (Chawan and Gerry,

1974) and soybean oil by-products and soapstock (Lipstein e~t al., 1967;

Menge and Beal, 1973) have been evaluated as proiler pigmenters. Waste

citrus sludge (Angalet eat at., 1976) and sweet potato vine meal (Garlich

et ae., 1974) have also been, evaluated as possible poultry pigmenting

agents. Work has been focused on the pigmenting ability of marigold

meal (Taguete ehecta) in both broilers and egg yolks (Brambila eat at.,

































































~


1963; Coon and Couch, 1971; Waldroup and Hazen, 1974; Coon and Couch,

1976).

Research has also been focused on the use of extracted naturally

occurring xanthophylls and synthetically produced xanthophylls for use

as poultry pigments. These compounds have been evaluated alone and in

combination with each other and with natural feed ingredients to deter-

mine their pigmenting ability in both egg yolks and broilers (Marusich

et al., 1958; Mlarusich at at., 1960; Tarver, 1961; Bunnell eat aR., 1962;

Farr et at~., 1962; Camp et at., 1963; Couch and Farr, 1970; Farr and

Couch, 1970; Marusich and Bauernfeind, 1970a; Marusich and Bauernfeind,

1970b; Couch and Farr, 1971; Hinton eat at., 1973; Weber and Philip, 1975;

Mlarusich eat al., 1976).


Factors Affectin Pgetio
and Xanthophyll Utilization


There are a variety of factors that can influence pigmentation and

xanthophyll utilization in poultry. Variations between different sources

of feeds as well as within a single source; type of bird; dietary factors

affecting absorption, both natural and additive; general bird health;

and processing and post processing conditions all can influence pigmen-

tation and xanthophyll utilization.

Palmer (1915) showed that the degree of yolk pigmentation was

directly proportional to the amount of xanthophyll in the feed. This

has been observed by numerous authors, for a wide variety of xanthophyll

sources (Hammond and Harshaw, 1941; Heiman and Tighe, 1943; Fritz and

Wharton, 1957; Bunnell et at., 1962). The differences in xanthophyll

concentration between different feedstuffs and the variations within a

particular feedstuff are shown in Table 1.


































Alfalfa (20% protein)

Alfalfa (17% protein)

Yellow corn

Grass meal

Corn gluten meal
(60% protein)


Table 1. Xanthophyll concentrations
different feedstuffs


and ranges of variation for


Xanthophyll concentration Ranges of variation
(mg./kg.) (mg./kg.)

Anonymous Scott eA at. Anonymous
(1971) (1969) (1971)

260 400-500 60-450

100 185-350 60-450

17 20- 25 8- 40

300 185-350 130-480

150 330 30-300


Feedstuff





The differences within a particular source can be attributed to

biological variation, local conditions and time of year grown. The

carotenoids as a group are very susceptable to oxidation due to the

conjugated system of double bonds that are also responsible for their

rich, intense colors (Bunnell and Bauernfeind, 1962). Thus, length and

conditions of storage would also have a pronounced effect on source

variations. Moster and Quackenbush (1952) found that temperature and

light would cause variations in the relative percent concentration of

several of the carotenoids present in corn.

The types of xanthophyll present also would have an impact on pig-

mentation since different xanthophylls will impart different colors in

poultry. Moster et~ at. (1952) isolated 15 carotenoids from corn

seedlings. Bickoff eat at. (1954) isolated over 40 xanthophyll fractions

from dehyrdated alfalfa meal. Smith and Perdue (1966) summarized data

from several researchers which outlined the principal hydroxy carotenoids

in alfalfa meal, yellow corn and dried algae meal. Of the total hydroxy

carotenoids of alfalfa, yellow corn and algae meal, lutein made up 46,

54 and 86% and zeaxanthin made up 4, 23 and 2%, respectively. Another

factor which could account for these variations in total xanthophyll

between sources and within sources is the procedure used for xanthophyll

analysis.

Collins at a. (1955) found that the shank color of New Hampshires

was significantly darker than that of White Plymouth Rocks; that pigmen-

tation within strains was significantly different and that males had

significantly darker shanks than females. Scott at al. (1968) found

that the genetic capability to absorb and deposit xanthophylls in the

egg yolk varied among individual hens within a single strain. Marusich








et at. (1960) reported that the degree of pigmentation depended upon the

breed of the hens. Differences in the ability of birds to deposit pig-

ment has been found among strains of broilers (Harms et dt., 1977).

Several feedstuffs and feed supplements have also been found to

affect pigmentation. Culton and Bird (1941) presented evidence indicat-

ing that certain common poultry feedstuffs such as meat scraps, fish

meal and soybean oil meal contain a factor, or factors, which inhibit the

deposition of yellow pigment in the shanks of growing chicks. They also

found some variation in pigment-inhibiting properties among different

lots of menhaden meal and soybean oil meal. Large quantities of cod

liver oil or small quantities of sulphur in the diet of a laying hen

interferred with the transfer of pigment from the hen's diet through her

eggs to the shanks of her chicks (Hammond et cat., 1942). Carver (1959)

reported that yellow grease, No. 1 tallow, hydrolyzed animal and vege-

table fat and methyl esters of vegetable fat reduced pigmentation in

broilers to varying degrees. Heath and Shaffner (1972) reported that as

the percentage of dietary oil was increased, a significant increase in

tissue lipid and xanthophyll deposition in back skin occurred. Further,

as the percentage of oil in the diet and tissue lipid increased, a

greater proportion of xanthophyll was deposited per gram of sample.

Waibel ~At at. (1972) observed that hens receiving diets containing 3%

activated charcoal produced significantly lighter yolks than those re-

ceiving diets with no charcoal. The reduced pigmentation from feeding

charcoal was attributed to the fact that charcoal is not absorbed by the

hen and that some of the xanthophylls were adsorbed by the charcoal thus

rendering them unavailable to the hen. Lecithin was shown to have no

effect on broiler pigmentation (Smidt et al., 1965) when fed with 2% fat





or with 4% fat (Ratcliff eat at., 1959). Beef tallow at 5% of the

broiler diet was shown to increase pigmentation (Day and Williams, 1958),

but Carver (1959) reported that No. 1 tallow reduced pigmentation.
Bird (1943) reported that dried skim milk was found to be a more satis-

factory source of animal protein than meat and bone meal which depressed

pigmentation. Substitution of neutralized, dried soapstock for oil in

laying hen diets resulted in deeper pigmented egg yolks without reducing

performance (Menge and Beal, 1973; Littlefield czt ae., 1975). Ratcliff
et al. (1959) reported that changing the protein-calorie ratio, between

35 and 60, had no influence on broiler pigmentation.

Several researchers have examined the effects of antioxidants on

pigmentation, often with a disparity of results. The ability of the
antioxidant ethoxyquin to improve broiler and egg yolk pigmentation has

been shown by Griminger and Fisher (1960), Waldroup eat al. (1960) and

Ratcliff et al. (1961). Bartov and Bornstein (1966) and Bushong et cLe.

(1972) also showed that ethoxyquin was an in v~itho enhancer of pigmenta-

tion, i.e., it stabilized dietary xanthophylls. Potter at al. (1956)

reported a small increase in blood plasma and liver xanthophyll feeding

BHT (Butylated hydroxytoluene) in broilers. Ratcliff eat al. (1961)

reported that BHT increased pigmentation in one trial while decreasing

pigmentation in a second trial while Day and Williams (1958) had pre-
viously reported that BHT reduced pigmentation in broilers. The use of

D.P.P.D. (diphenyl-p-phenylenediamine) was shown to significantly im-

prove pigmentation by Potter a-t al. (1956); however, Harms et st. (1958)

reported that D.P.P.D. significantly reduced pigmentation. Other feed
additives shown to increase pigmentation are menadione (Griminger and

Fisher, 1960; Smidt eA al~., 1965), lipamone (Herrick et al., 1970),





vitamin B12 (Potter et CLt., 1956) and sodium bicarbonate (Bushong et ak.,

1972).

Since the carotenoids are absorbed in the small intestine, any

factor that impares the absorption of lipid material will affect

pigmentation. Thus, several investigators have examined the effect of

diseases and general bird health on pigmentation. Squibb eat nt. (1955)

reported that coryza, cholera and Newcastle Disease all significantly

reduced total serum carotenoids. A great deal of research has been

conducted on the effects of coccidia on both broiler and egg yolk

pigmentation. Barnett and Stephens (1963) reported that hens producing

pale yolks tended to have more coccidial oocysts than birds producing
dark yolks. The intestinal microflora (total aerobes, total anaerobes,

lactobacilli, coliforms, enterococci, staphylococci, yeasts and

filamentous fungi) of both groups of birds was similar. The reduction

of serum xanthophyll levels and visual pigmentation scores as a result

of coccidial infections by EA~menii maxim~a, E. nceaLvLtinaa E. mivat~i, E.

neestrrix, E. baiunattt and E. Laenettea has been shown by a number of

researchers (Bletner at at., 1966, Marusich eat ae., 1971b; Ruff eat at.,

1974; and others). Marusich e~t ite. (1972) reported that E. pahecox and

E. teneffar did not depress pigmentation, while Ruff and Britton (1976)

reported that upper intestinal species decreased yolk color but that in-

fection with cecal species did not affect yolk color. Littlefield et at.

(1972) located the site of xanthophyll absorption in chickens using

surgical techniques and concluded that absorption takes place in the
area of the jejunum-ileum (same section in which E. maxima infections

occur), that a small amount of absorption, if any, takes place in the

duodenum and large intestine and that none takes place in the ceca.








The effect of aflatoxicosis on poultry pigmentation has also been

studied. Wyatt et at. (1972) reported that serum carotenoids were

significantly lowered at an aflatoxin level of 1.25 ppm in the diet of

broilers. Huff eat a-e. (1975) reported that serum carotenoid and egg

yolk pigmentation were increased in layers as a result of,aflatoxicosis.

This was attributed to a decreased egg production (69%) an' the result-

ing increased deposition of xanthophylls from the diet rather than de

novo synthesis in the liver as occurs with other yolk lipids.

The addition of several arsenicals, coccidostats and antibiotics

have been evaluated for pigmentation enhancing effect. Roxarsone has

been shown to be an effective pigment enhancing factor by 01son at at.

(1972), Ott eat at~. (1974), Fry and Harms (1974) and Kowalski and Reid

(1975), but it was found to have no effect on pigmentation with continu-

ous administration (Marusich et at., 1971a). Robenidine was shown to

enhance pigmentation by Fry and Harms (1974) as was 3-nitro-4-

hydroxyphenylarsonic acid by Ratcliff eat nt. (1959) and Smidt eat at.

(1965). The positive pigmentation effect of flavomycin and 3-nitro-10

with a series of coccidiostats has been well documented by Fry eat al.

(1976a), Fry et at. (1976b) and Harms at al. (1976).

Several other factors have been implicated in influencing

pigmentation. Stone eat al. (1971) reported that age, hatch, housing and

sex influenced variation in carotenoid concentration in chicken tissues.

Collins eat al. (1955) reported that the environment markedly influenced

shank color. Cox (1973) reported in an experiment of bin ~votsua return

feed from a drag chain feeding system on the performance of Single Comb

White Leghorn pullets. Egg yolks from the birds fed the return feed

were considerably darker in color.








Methods of Evaluating Broiler
and Egg Yolk Pigmentation


There are numerous procedures available for the evaluation of

poultry pigmentation, both directly and indirectly, and subjectively or

objectively. Indirect methods are those that measure some aspect of

xanthophyll absorption or deposition and which have a high correlation

to actual final color as perceived by the consumer. Examples are blood

serum xanthophyll and shank color. Direct methods are those that mea-

sure the final actual color such as visual analysis and colorimetry.

Subjective methods normally consist of visual analysis against set

standards or simply consumer acceptance surveys. Objective methods in-

clude primarily chemical, spectrophotometric and colorimetric analyses.

Each type of procedure has its advantages and disadvantages and should

be selected according to the type of information needed.

The simplest, fastest and least expensive method is subjective

visual analysis of final product color with or without visual standards.

The earliest evaluations of poultry pigmentation were simply descriptive

in nature, based upon visual observations such as "bright yellow,"

"highly colored," etc. (Palmer, 1915; Parker eft at., 1925). One of the

earliest successful color scales developed to standardize egg yolk color

description was developed by Kupsch in 1934 and consisted of 10 rec-

tangular sheets chosen from the Oswald system and numbered 1 to 10,

corresponding to a range of white-yellow to yellow-orange yolks (Francis

and Clydesdale, 1972). Heiman and Carver (1935) developed a "yolk color

index," which was widely used and issued by the U.S. Department of Agri-

culture, made up of 24 convex colored glasses mounted on a black circu-

lar board. The next major visual color standard was the Hoffman-LaRoche





color fan developed first in 1956. The history, development and use of

this standard was reveiwed by Vuilleumier (1969). Although developed

primarily as an egg yolk pigmentation standard, it has also been used

extensively for measuring broiler shank color (Marusich, 1969). Another

visual scale was developed by Ashton and Fletcher (1962) utilizing 15

colored metal caps with circular holes.

Several researchers have shown some of the inadequacies of simple

visual methods to accurately describe poultry pigmentation or xantho-

phyll utilization. Waldroup and Johnson (1974) reported the lack of

repeatability of scores among persons using the Roche color fan to

assess the shank color of broilers. Bornstein and Bartov (1966), in a

comparison of visual scoring of yolk color and a colorimetric assay of

yolk carotenoids, discussed the relative limitations of each method.

Several methods of objective pigmentation evaluation have been developed

that can be classified as being either chemical-spectrophotometric or

colorimetric.

The chemical-spectrophotometric techniques usually involve a pig-

ment extraction followed by spectrophotometric analysis of the pigments

at a standard wavelength. Heiman and Tighe (1943) described the first

such technique for evaluating chicken shank skin. Wilson (1956) used an

acetone extraction of blood and read absorbance at 445 nm. to determine

blood carotenoid levels. Davis and Kratzer (1958) found a high correla-

tion between the serum xanthophyll level and shank pigmentation. The

nost widely used procedures of this type for analyzing pigmentation are

the N.E.P.A. (National Egg and Poultry Association) number method,

A.0.A.C. (Association of Official Analytical Chemists) method and the

A.N.R.C. (Animal Nutrition Research Council) method. The N.E.P.A.








number method used dichromate standard solutions read at 450 nm. against

an acetone extraction of liquid yolk (Dalby, 1948). The A.0.A.C. method

(A.0.A.C., 1970) compares acetone extracted yolk noaterial against

standard beta-carotene solutions at 450 nm. The A.N.R.C. method as

described by Marusich (1967) compared acetone-chloroform yolk extracts

to standard solutions of beta-apo-8'-carotenal at 440 nm.

The use of tristimulus colorimeters to .measure and describe color

has been used extensively by several researchers. Tortuero (1968) used

a tristimulus spectrophotometer to describe the color of egg yolks in

the trichromatic coefficients. Hunter color difference meters have also

been used to evaluate both yolk and broiler color (Davies a-t at., 1969,

Francis and Clydesdale, 1972). The IDL COLOR-EY~L has been used to

describe and evaluate pigmentation in egg yolks, egg yolk products and

broilers (Fry at at., 1969; McCready cat al., 1973; Fry eat nPt., 1974).

Tristimulus values obtained with the color eye can be converted to the

C.I.E. system values of dominant wavelength (the actual color), excita-

tion purity (the color intensity) and luminosity (degree of brightness).














CHAPTER I
THE INFLUENCE OF LIGHT ON BROILER PIGMENTATION


Stone et at. (1971) found that housing was one of several factors

that influenced the carotenoid concentration in chicken tissue, while

Collins at al. (1955) found that environment markedly influenced shank

color. It has also been noted in the field (privileged communications)

that broilers reared in windowless houses did not pigment as well as

those reared in conventional houses. This study was conducted to deter-

mine the influence of light on broiler pigmentation.


Experimental Procedure

Equal numbers of day-old male and female Cobb color-sexed broiler

chicks were randomly distributed into each of six houses (92 to 100

chicks/house) so that densities in the houses were equal (0.11 mP/bird).

Three of the houses were of the open-type, and three were fan-ventilated

and windowless, so that all outside light was excluded. Birds in the

open houses received natural daylight only, while birds in the window-

less houses received the continuous commercial-type lighting regime as

shown in Table 2. All birds were brooded on peanut hull litter floors

with infrared lights used for heat. The same commercial-type diet and

feeding schedule were used for both treatments as follows: starter feed

from 0-28 days, finisher feed from 29-49 days and a withdrawal feed from

50-56 days of age. A11 diets contained 55 mg. of xanthophyll per kg. of

feed, and are shown in Table 3. At 56 days of age the experiment was


20


























Table 2. Continuous lighting program used for windowless
houses


Age (days) Light source Foot candles


0- 7 40 watt bulb and heat lamp 2.00

8-14 25 watt bulb 0.90

15-21 10 watt bulb 0.35

22-56 7.5 watt bulb 0.15






















Ingredient Starter (%) Finisher (%) Withdrawal (%)

Corn meal 51.70 46.97 54.32
Soybean meal (49% protein) 32.13 29.00 22.88
Gluten (60% protein) 4.49 12.02 12.08
Stabilized fat 6.21 6.96 5.67
Meat meal 2.58 2.00 2.00
Dikal 185 1.22 1.41 1.46
Limestone 0.76 0.71 0.66
Salt 0.46 0.52 0.52
Coban --- 0.12 0.12
Choline chloride 0.10 0.10 0.10
Trace minerals 0.50 0.05 0.05
Vitamin premix 0.50 0.05 0.05
Lysine HCI --- 0.035--
Lincomix --- 0.025 0.025
CuSO4 0.013 0.0125 0.0125
3-Nitro 0.010 0.010 -
DL-Methionine (98%) 0.142 0.009--
Vitamin E 0.0005 0.0005 -
Nicarb (25%) 0.05-- -
Baciferm 0.031----
Marigold 0.0005 --- 0.0085
Ethoxyquin 0.008----
SE-200 --- --- 0.05


Table 3. Starter, finisher and withdrawal feed formulas
the windowless and the open-type houses


used in both


L





terminated, birds were weighed and feed consumption was calculated.

Five birds from each sex were selected at random from each replicate and

blood samples obtained by anterior heart puncture. These birds were

killed, skin color scored and then the shanks removed for later analysis.

Subjective skin color scores were determined on a 1 to 5 scale with

1 being the most "acceptable" and 5 being the least "acceptable."

Acceptability was determined by the degree or deepness of pigmentation

with deeply pigmented birds being designated as the most acceptable.

Blood xanthophyll was determined as follows: a 10-15 ml. blood

sample was centrifuged to separate the plasma and red blood cells.

Subsequently, 0.5 ml. of plasma was removed and placed in a 15 ml. tube

with 5 ml. acetone and mixed vigorously by shaking. The tube was then

centrifuged at 2000 g. for 3 min., decanted and absorbance determined

at 423 nm. Xanthophyll in mg.% was determined by multiplying the

absorbance at 423 nm. by the constant 62.147 (Perdue Inc., 1976).

Evaluation of shank pigmentation, which is highly correlated with

skin pigmentation, was made with the IDL COLOR-EYE according to the

procedure of Fry et al. (1969). The raw data from the COLOR-EYE~ were

converted to values for domiinant wavelength (DWL), excitation purity

(EP) and luminosity (Lum) using the computer program developed by Fry

and Damron (1971). These values quantify the color or hue (DWL) of the

sample, the purity or intensity of that color (EP) and the brightness

of the color (Lum).

Data from five previous experiments on broiler shank pigmentation

were re-evaluated. In these experiments, the replications consisted of

pens positioned in such a way as to receive varying levels of light
intensity. The house in which these experiments were conducted was








divided into 72 pens in four rows of 18 pens each. The outer rows were

along the open sides of the house with two adjacent rows along the

center. Pigmentation experiments were conducted by dividing treatments

equally among the four rows. Four of these experiments had been con-

ducted when the side walls were semi-closed without large window areas.

The house was remodeled by installing large openings at the level of the

birds, after which, one additional experiment was conducted. Data were

calculated from those previous experiments according to pen position

with regard to outside vehllua inside pens, in order to determine differ-

ences in pigmentation due to light differences. Light meter (Spectra

COMBI-500, Model S-501) readings were taken after remodeling at noon on

a partly cloudy day.

All data were analyzed using analysis of variance procedures of the

Statistical Analysis System (SAS) (Service, 1972).


Results and Discussion


The shanks of birds reared in open-type houses had a significantly

higher dominant wavelength (hue) and a significantly lower (more

"acceptable") skin color score (Table 4). The excitation purity values

(color intensity) were not significantly different, indicating that

total shank pigment was equal in birds from both types of houses.

Shanks from birds reared in open-type houses had significantly lower

luminosity values than shanks from birds reared in windowless houses

indicating that the shanks of open-type housed birds were darker.

Birds reared in the open houses had a significantly higher feed

consumption and slightly higher body weights than birds reared in window-

less houses, however, the feed efficiencies and thus, xanthophyll intake



















Table 4. Body weight, feed consumption, feed efficiency,
blood xanthophyll, skin color score and shank
dominant wavelength (DWL), excitation purity (EP)
and luminosity (Lum) for open vennun windowless
houses"


House type

Open Windowless

b
1.3 2.1a

581.2 a 580.2 b

68.31a 68.62a

48.45b 52.16a

2167 a' 2098 a

72.7 a 70.2 b

1.94a 1.93a

17.24b 20.13a


Measurement


Skin color score *

Dominant wavelength (nm.)

Excitation purity (%)

Luminosity (%)

Body weight (g.)

Feed consumption (g./bird/day)

Feed efficiency (g. feed/g. gain)

Blood xanthophyll (mg.%)


Means within each row with different superscripts are
significantly different (P < 0.05).
**
Skin color scores ranged from I to 5 with 1 being the
most "acceptable" and 5 being the least "acceptable."








per gram body weight, were equal for the two treatments. The xantho-

phyll concentration in the blood of birds reared in the open houses was

significantly lower than blood xanthophyll concentration of windowless-

housed birds (Table 4).

Excitation purity for shanks from birds reared in both houses was

not significantly different (Table 4). Dominant wavelength was higher

for shanks from birds reared in open-type houses, however, and this was

associated with a significant improvement in skin color score. These

data indicated that the hue or color (DWL) o~f the pigment and/or the

brightness (Lum) was responsible for the difference in visual pigmenta-

tion scoring, since the actual concentration of the pigments was the

same for birds from the two treatments.

In the experiment in the remodeled house with outside wall pens

vehcud inside pens, dominant wavelengths were significantly higher for

the outside pens while excitation purity and luminosity were signifi-

cantly lower (Table 5). Therefore, the birds in the outside pens were

more orange and darker (higher DWL and lower Lum) than those in the

inside pens. The significantly lower excitation purity indicated a

lower pigment content in the birds from the outside rows (lower inten-

sity of color). Although light intensities of outside and inside pens

were not measured at the time the experiment was conducted, later obser-

vation at noon on a partly cloudy summer day in this house resulted in

a reading of 200 ft. candles for outside pens and 15 ft. candles for

inside pens at bird level.

Four experiments conducted in the same house before remodeling

resulted in the same or greater dominant wavelength values for shank

samples from birds housed in the outside pens than for birds housed in





Table 5. Dominant wavelength (DWL), excitation purity (EP),
luminosity (Lum) and light intensity for broilers
raised in either outer or inner pens*


Pen position
Measurement
Outside Inside

Dominant wavelength (nm.) 579.1 a 578.7 b

Excitation purity (%) 55.62b 56.71a

Lumninosity (%) 55.78b 56.49a

Foot candles 200 15


Means within each row with different superscripts are
significantly different (P < 0.05).








the inside pens, while luminosity and excitation purity varied in effect

(Table 6). Since these experiments were conducted when the outside

walls were partially closed, less difference in light intensity would

have been expected.

There are two possible explanations that can account for this

effect of light intensity on broiler pigmentation: light intensity can

cause an isomerization or alteration of xanthophyll structure in the

feed, or light intensity can trigger a metabolic pathway within the bird

that will alter some xanthophylls to a redder pigment. Evidence for the

first possibility can be found in the work by Moster and Quackenbush

(1952) on the effects of temperature and light on the carotenoids of

seedlings grown from three corn hybrids. They found that high light

intensity and low temperature tended to favor the accumulation of

zeaxanthin and the reduction of beta-carotene. The changes in

zeaxanthin were often approximately quantitatively equal and opposite

to simultaneous changes in beta-carotene. They went on to state that

this suggests that these two pigments may be either interconvertible or

formed from a common precursor. They also found similar interrelation-

ships between lutein and violaxanthin. Thommen (1971) described a meta-

bolic pathway in the game pheasant by which zeaxanthin is transformed

by the bird into astaxanthin, a red pigment, which is found in the

papillae surrounding the eye. It is possible that these changes are

stimulated by light and thus would affect broiler pigmentation.













Table 6. Dominant wavelength (DWL), excitation purity (EP)
and luminosity (Lum) of shanks from birds raised in
either outside oriented pens or inside pens in four
previous experiments*


Pen position
Experiment --- ----
Outside Inside


579.3 a 579.1 b

67.42a 67.25a

55.70b 56.62a


1 Dominant wavelength (nm.)

Excitation purity (%)

Luminosity (%)


2 Dominant wavelength (nm.)

Excitation purity (%)

Luminosity (%)


3 Dominant wavelength (nm.)

Excitation purity (%)

Luminosity (%)



4 Dominant wavelength (nm.)

Excitation purity (%)

Luminosity (%)

*Means within each row with different
significantly different (P < 0.05).


578.7 a

61.96a

53.56a


578.5 a

61.93a

57.69a


578.8 a

67.10a

55.88a


578.7 a

60.79b

53.94a


578.4 a

61.95a

57.62a



578.8 a

65.69b

55.98a


superscripts are





CHAPTER II
STRAIN DIFFERENCES IN EGG YOLK PIGMENTATION


The genetic capability .to absorb and deposit xanthophylls in the

egg yolk has been found to vary among individual hens within a single

strain (Scott eat al., 1968) and also between breeds of hens (Marusich

et al., 1960). Strain differences in pigmentation have also been

observed with broilers by Harms at a-e. (1977). The purpose of this

study was to evaluate the yolk pigmenting ability of 12 laying strains

housed on the floor (peanut hull litter) and in cages.





All eggs were obtained from the Florida -Poultry Evaluation Center,

Chipley, Florida, during the 23rd Performance Evaluation Trial Year.

Eggs were collected from 12 strains in both cage and floor houses

and transported to the Poultry Science Department, University of

Florida, Gainesville, for yolk color evaluation. Eggs were collected

in two trial groups, trial 1, January 17 through February 13, and trial

2, July 3 through July 30. All pertinent production records were

supplied for the dates as listed above. All birds were fed the same

diet (Table 7) and received the same commercial lighting program.

Yolk color evaluation and sample preparation were conducted using

the IDL COLOR-EYE according to the procedure of Fry eat at. (1974)

utilizing sample holders identical in design to those described by

Little (1969). Two replicates of three eggs each were selected at




















Ingredient %


Table 7, Composition of diets (Trials 1 and 2)


Yellow corn meal

Soybean meal (44%)

Fish meal (60%)

Alfalfa meal (17%)

Dicalcium phosphate

Ground limestone

Iodized salt


65.99

19.80

2.50

3.00

1.92

6.04

0.25

0.50


16.90

2800

3.00

0.75

19.7


(18% P + 23% Ca)


Florida micro-mix*


Protein (%)

Metabolizable energy (kcal./kg.)

Calcium (%)

Phosphorus (%)

Xanthophyll (mg./kg.)


*Micro-ingredient mix supplied per kg. of diet: 6,600
I.U. vitamin A, 2,200 I.C.U. vitamin D3, 500 mg. choline
chloride, 40 mg. niacin, 4.4 mg. riboflavin, 13 mg. panto-
thenic acid, 22 mcg. vitamin Bl,125m.ehxqi,2 g
iron, 2 mg. copper, 198 mcg. cogalt 1.1 mg. itodqine, 100 mg.
zinc, 71 mg. manganese, and 2.2 mg. menadione sodium
bisulfite.








random from each strain-house combination. Each replicate was evaluated

in duplicate. The raw data were converted to values for dominant wave-

lengthi (DWL), excitation purity (EP) and luminosity (Lum) by the use of

the computer program of Fry and Damron (1971).

The data were subjected to the analysis of variance and Duncan's

multiple range test using the Statistical Analysis System (SAS) de-

scribed by Service (1972).


Results and Discussion

Trial 1


Significant differences in yolk pigmentation were found among the

12 strains for dominant wavelength, excitation purity and luminosity in

both the floor and cage houses (Table 8).

Dominant wavelength ranged from highs of 578.58 (strain 7) for egg

yolks from birds in the floor house and 578.90 (strain 5) for egg yolks

from birds in the cage house to lows of 577.75 (strain 2) and 578.05

(strain 2), respectively. Egg yolks from strains 7 and 5 had a signifi-

cantly different color (golden as opposed to yellow) than egg yolks from

strain 2 in the floor and cage house, respectively, as indicated by the

higher dominant wavelengths.

Excitation purity ranged from highs of 92.73 (strain 7) for egg

yolks from birds in the floor house and 92.76 (strain 7) for egg yolks

from birds in the cage house to lows of 89.90 (strain 11) and 90.71

(strain 1), respectively. These results indicated that egg yolks pro-

duced by strain 7 had higher intensity of color in both the floor and

cage house than egg yolks produced by strain 11 or 1, respectively, as

indicated by the higher excitation purities.






















UU u U U 0
E~- .0 h U 0 d d .0) CU

3m h m a d m m m m N








3o a



CC
o a o ouo o .0





o N O T D CJ d a 0n o ,m I
E u o o io o oo l U3 o o 0

ummmmmmemmomm
C O 5- O L 0 L O L D D L O L
r -
II E" I a


a O CO O IDC C l

OI Ih- CO d t aI t n

II I u ., N \ \



--r Ilu 0 o sa -









Luminosity ranged from highs of 35.20 (strain 2) for egg yolks

produced by birds in the floor house and 34.51 (strain 3) for egg yolks

from birds in the cage house to lows of 32.28 (strain 7) and 31.30

(strain 7), respectively. The lower luminosities exhibited by egg yolks

from this strain had the "darkest" yolks (furthest from white).

Trial 2


Significant differences in yolk pigmentation were again found among

the 12 strains examined for domiinant wavelength, excitation purity and

luminosity in both the floor and cage houses (Table 9).

Dominant wavelength ranged from highs of 578.28 (strain 8) for egg

yolks produced by birds in the floor house and 577.93 (strain 10) for

egg yolks from birds in the cage house to lows of 576.93 (strain 6) and

576.90 (strain 1), respectively. Excitation purity ranged from highs of

93.91 (strain 5) for egg yolks produced in the floor house and 92.49

(strain 5) for egg yolks from birds in the cage house to lows of 88.63

(strain 6) and 88.45 (strain 1), respectively. Luminosity ranged from

highs of 33.28 (strain 6) in the floor house and 33.83 (strain 4) in the

cage house to lows of 30.94 (strain 10) and 30.48 (strain 10),

respectively.

Although the 12 strains behaved somewhat differently in the two

housing conditions and the two trials, several of the strains showed a

consistent trend. Strains 5, 7 and 8 were generally good pigmenting

strains, i.e., high dominant wavelengths and excitation purities but

with low luminosities. Strains i, 3 and 6 were generally poor pigment-

ing strains, L~e., low dominant wavelengths and excitation purities but

with high luminosities.

























U U U U
U ~ V1I)13 ~01)

E or cu cg m F, o a h, c\I a m ~t
rrJ m to a, o m o
-Ir V d F; m(UC~~--NNO(UPI~
VI[] F7
mmmmmmmm~mmm h
=~U rl E
rn
C,
II -1
E
V)U
rCI
O II I I U U U V U V U U
U~~~O r
L~~~~~~y~~V~~ lC
E O N d U3 ~t U3 00 00 h Ln ~f C) O r
VI O m Nm~~~~~~~~ r

a II I III~ cu - C) o o cu N -r
~CJ~ m m m~m~~m~mmm vl
e

r a
L
ah II I
w~ II I I u u U U
V~ C1
a
rJ, to m o cn a, N m to ~O m r
CIO ~I ~f m h o NcocOloh~a, L
r u
vl II I I a, o o, o cu d u
lc cocnaocno~r~~nor ~nororor L
TZr
a
II a
a
w
oo
vl
'r vl II I I u u u u
c, ouo oouo u cr
C
O rU h to m r, to cn Ln a,
'' O O Io N ~D O, ~D (1 h E? to L
uh
a
xo II I LL I '- N hi Cr) CO r\l CU N rC
a cno~o~rorolo3oro~o~~coo~r r
n
o
-IsJ
~oc rJI
o E
0~ ~ U

II Ia O a in m m m o m m c? Ln o
r cn cn m in h d h o,
cr~nr~
cncn, II lu ID h h h h h
hhh~hhh~~~h~ E
V)I~
arE 3~
o~n o o
u
4LQ)
L~c vl II I I o o o o o o o u rcr^
U~Um~~~~U~V~ Ln
C~ VI O L in m o m to m o a, o o o o Eo
~yy O h O rt CU Ln O N in m r
o ro
~OL ~~~~~~~~h~hh C,
'r hO LL h h h -r V

ooie a
mu
r
r~o
cn arr
ra,
r
a, II , c L
a
cr h a o N
o L
LC
4 11 CI
C II LO ''
~CI








Significant differences were also found between the two housing

conditions across strains in both trials (Table 10). Birds from the

cage house produced egg yolks which had significantly higher dominant

wavelength and excitation purity but lower luminosity than egg yolks

from birds in the floor house in trial 1. This indicated that egg yolks

from the cage birds were darker and more highly pigmented than egg yolks

from the floor birds. In trial 2, egg yolks from the floor housed birds

had significantly higher dominant wavelengths and excitation purities

but did not differ significantly in lumiinosities. The differences in

yolk pigmentation between floor and cage houses might have been due to

differences in light intensity, which has been shown to affect broiler

pigmentation (Chapter I). Trial 1 was conducted during the winter

months of January and February when the floor houses (with closeable

shutters) would be subjected to less natural light than the cage houses.

In trial 2, conducted during the summer month of July, the floor house

would receive approximately the same amount of natural light as the

birds in the cage houses.

Data fromr this experiment indicated that yolk pigmentation did vary

among layer strains and housing conditions.




















Table 10. Dominant wavelength (DWL), excitation parity (EP)
and luminosity (Lum) of yolks from eggs produced by
laying hens in cage and floor houses (Trials 1 and
2)*

Housing Type
Trials Parameter
Floor Cage

1 Dominant wavelength (nm.) 578.12b 578.43a

Excitation purity (%) 91.04b 92.00a
Luminosity (%) 34.20a 33.06b


2 Dominant wavelength (nm.) 577.74a 577.57b
Excitation purity (%) 91.71a 90.88b
Luminosity (%) 31.91a 32.21a


Means within a row having different
significantly different (P < 0.05).


superscripts are





CHAPTER III
YOLK COLOR CHARACTERISTICS, XANTHOPHYLL AVAILABILITY,
AND A MODEL SYSTEM FOR PREDICTING EGG YOLK COLOR
USING BETA-APO-8'-CAROTENAL AND CANTHAXANTHIN


The possibility of utilizing synthetic xanthophylls in feedstuffs

for promoting both egg yolk and broiler pigmentation has been investi-

gated by several researchers. Although not approved as a feed additive

by the Food and Drug Administration for use in the United States, syn-

thetic xanthophylls have found commercial applications in several

European and South American countries. In addition, synthetic xantho-

phylls are useful experimental tools for studying the influence of dif-

fering xanthophyll compounds in both egg yolk and broiler pigmentation.

For many years investigators have evaluated the pigmenting effects

in both broilers and egg yolks of naturally occurring carotenoids in

many differing feedstuffs. In 1957, Isler and Zeller reported the

chemical synthesis of pure carotenoid substances. With increased

availability of these compounds, several researchers have evaluated them

for suitability as feed additives for pigmenting both broilers and egg

yolks. Marusich at at. (1960) fed several pure carotenoids as compounds

for pigmenting egg yolk with evaluation by visual scoring and spectro-

photometric analysis of acetone extracts at 440 nm. They reported that

beta-apo-8'-carotenal produced a "pleasing yellow color" and would

appear to be the "preferred carotenoid" for egg yolk pigmentation.

Canthaxanthin was found to be one of several carotenoids that "impart an

orange tinge to the yolks which is not acceptable." Bunnell et at.





(1962) studied the use of beta-apo-8'-carotenal as an egg yolk pigmenter

and reported that a stabilized beadlet of this compound fed at a level

of 4.36 g./ton (4.81 mg./kg.) with either a white corn or milo ration

and only 1 or 2% alfalfa will provide adequate color for table eggs.

Couch and Farr (1971) used the Hoffman-LaRoche Color Fan and acetone

extraction to evaluate the pigmenting capabilities of canthaxanthin and

beta-apo-8'-carotenal when added to diets containing yellow corn and

alfalfa. They found that the addition of 4.4 mg./kg. canthaxanthin re-

sulted in a large increase in NEPA score. They found a further increase

in egg yolk pigmentation as the canthaxanthin was increased in the diet

to 8.8 and 13.2 mg./kg., respectively; however, there was no increase in

NEPA number of egg yolks resulting from the addition of beta-apo-8'-

carotenal in combination with canthaxanthin. They observed that the

addition of beta-apo-8'-carotenal to the diet containing canthaxanthin

did not improve visual scores.

It is apparent from published data that it has not been possible to

evaluate yolk pigmenting characteristics of canthaxanthin and beta-apo-

8'-carotenal which were fed in diets containing yellow corn. This prob-

lem is apparently due to the different dominant wavelengths (actual

color) of the pigments. Since color can be accurately described using

the three variables of dominant wavelength, excitation purity and

luminosity, it is unlikely that accurate evaluation of pigmentation in

egg yolks can be accomplished by either visual or spectrophotometric

analysis of acetone extracts using a constant wavelength. The purpose
of this experiment was to analyze and define color characteristics of

egg yolks when beta-apo-8'-carotenal and canthaxanthins when fed

separately or in combination. These two xanthophylls were selected








because of the great difference in color imparted to the yolk by the

two different compounds.


Experimental -Procedure

Babcock B-300 laying hens, individually caged, were fed a white

corn basal, xanthophyll-free diet (Table 11) for 21 days to deplete body

and yolk xanthophyll stores. After depletion, 250 hens were selected

and randomized into two replicates of 25 pens with five individually

caged birds/pen. Each replication group received one of 25 diets with

0, 2.2, 4.4, 8.8 and 17.6 mg./kg. canthaxanthin and beta-apo-8'-

carotenal (Table 12). After 21 days on the experimental diets, eggs

were collected over a five-day period and held (120 C. and 85% Relative

Humidity) for yolk color analysis.

Yolk color evaluation and sample preparation were conducted as

detailed in the experimental procedure of Chapter II. Yolk samples were

also evaluated after diluting 50:50 (w./w.) with a white diluent (Haley's

M.0. ). The white diluent was used to reduce the tendency of excita-

tion purity values to plateau at high xanthophyll concentrations (Fry

et aLe., 1974). The color of undiluted yolks was also visually scored

using the Roche Color Fan and the Heiman-Carver Color Rotor. Color eye

data were used to calculate relative biological availability of the two

compounds and to postulate a system by which yolk color could be pre-

dicted from known feed concentrations of these two synthetic compounds.

Various yolk samples were then blended to produce selected

xanthophyll combinations. The blended samples were then analyzed both

undiluted and diluted with the white diluent as previously described

using the COLOR-EYE ),



























Ingredient %o


White corn meal 71.02

Soybean meal (50%) 19.00

Limestone 6.88

DynaFos (18.5% P, 24% Ca) 2.25

Iodized salt 0.35

Florida micro-mix* 0.50


*Micro-ingredient mix supplied per kg. of diet: 6,600
I.U. vitamin A, 2,200 I.C.U. vitamin D3, 500 mg. choline
chloride, 40 mg. niacin, 4.4 mg. riboflavin, 13 mg. panto-
thenic acid, 22 mcg. vitamin B12, 125 mg. ethoxyquin, 20 mg.
iron, 2 mg. copper, 198 mcg. cobalt, 1.1 mg. iodine, 100 mg.
zinc, 71 mg. manganese and 2.2 mg. menadione sodium
bisulfite.


Table 11. Composition of white corn basal diet

























Table 12. Total xanthophyll concentrations (mg./kg.) of the
25 diets at the corresponding levels of beta-apo-
8'-carotenal and canthaxanthin


B-apo-8'- Canthaxanthin (mg./kg.)
carotenal
(mg./kg.) 0 2.2 4.4 8.8 17.6

0 0 2.2 4.4 8.8 17.6

2.2 2.2 4.4 6.6 11.0 19.8

4.4 4.4 6.6 8.8 13.2 22.0

8.8 8.8 11.0 13.2 17.6 26.4

17.6 17.6 19.8 22.0 26.4 35.2








Results and Discussion


Dominant wavelength of the yolk samples increased from 572.8 nm.

for yolks from hens fed the xanthophyll free diet to 577.9 nm. and 590.6

nm. for yolks from hens fed 17.6 mg./kg. beta-apo-8'-carotenal or

canthaxanthin, respectively (Table 13). Canthaxanthin had the greater

effect on dominant wavelength (DWL) of yolks from hens fed mixed concen-

trations of the two compounds than did beta-apo-8'-carotenal (Table 13).

Luminosity decreased from 42.42 for yolks from hens fed the xanthophyll

free diet to 34.91 and 19.73 for yolks from hens fed 17.6 mg./kg. beta-

apo-8'-carotenal or canthaxanthin, respectively (Table 13). The lower

luminosity scores for yolks from hens fed canthaxanthin were due to the

greater absorbance (less reflectance) of Tight across the wider spectrum

of color.

Correlation coefficients for visual scores and the color eye values

indicated that dominant wavelength had the greatest effect on visual

scoring followed by luminosity and excitation purity (Table 14).

Because of the narrow range of colors that can be accurately compared to

these visual standards, it was difficult to assign a visual score to

many of the samples.

Excitation purity increased from 34.09% for yolks from hens fed the

xanthophyll free diet to 85.03% and 89.39% for yolks from hens fed 17.6

mg./kg. beta-apo-8'-carotenal or canthaxanthin, respectively (Table 15).

The diluted excitation purity values increased from 17.33% up to 54.01%

and 59.68% for the same yolk samples and indicated the reduced tendency

of excitation purity to plateau at relatively high xanthophyll concen-

trations (Figure 1). The excitation purity values of the yolk samples


















Table 13. Dominant wavelength (DWL) and luminosity (Lum) of
the 25 dietary levels of beta-apo-8'-carotenal and
canthaxanthin on undiluted yolk samples


B-apo-8'- Color Canthaxanthin .(mg./kg.)
carotenal values
(mg./kg.) 0 2.2 4.4 8.8 17.6

0 DWL (nm.) 572.8 583.0 585.2 587.8 590.6

Lum (%) 42.42 30.96 27.82 24.48 19.73

2.2 DWL (nm.) 574.0 582.3 585.2 588.4 592.0

Lum (%) 39.36 31.02 27.39 23.69 17.90

4.4 DWL (nm.) 574.9 582.1 585.1 587.7 590.8

Lum (%) 39.64 30.74 26.02 22.88 19.62

8.8 DWL (nm.) 576.6 581.6 584.4 587.6 591.3

Lum (%) 36.51 30.40 27.86 23.37 18.44

17.6 DWL (nm.) 577.9 581.9 584.7 588.0 591.0

Lum (%M) 34.91 29.31 25.99 22.99 19.11












Table 14. Visual color scores using the Heiman-Carver Color
Rotor (HCCR) and theRoche Color Fan (RCF) for the
25 fed dietary levels of beta-apo-8'-carotenal and
canthaxanthin on undiluted yolk samples with cor-
relation coefficients (r) with dominant wavelength
(DWL), excitation purity (EP) and luminosity (Lum)
of the same sample


B-apo-8' Score Canthaxanthin (mg./kg.)
carotenal
(mg./kg.) Sytm 0 2.2 4.4 8.8 17.6

0 HCCR 3.4 17.7 17.0 18.7 21.3

RCF 1.0 12.0 12.7 13.0 15.0

2.2 HCCR 6.0 16.7 17.7 19.0 22.3

RCF 2.3 11.0 13.0 14.7 15.0

4.4 HCCR 9.0 16.0 17.7 20.0 22.0

RCF 3.7 11.0 13.0 14.7 14.7

8.8 HCCR 11.0 16.7 18.0 19.7 21.7

RCF 5.0 10.7 12.7 14.3 15.0

17.6 HCCR 13.0 17.0 18.0 19.7 22.0

RCF 7.3 10.7 12.7 15.0 15.0

DWL EP Lum RCF

HCCR .959** .839** -.956** .981**

RCF .959** .782** -.948** --


**Significant (P < 0.01).



















Table 15. Excitation purity values (%) of the 25 dietary
levels of beta-apo-8'-carotenal and canthaxanthin
on undiluted and diluted yolk samples


B-apo-8'- Canthaxanthin (mg./kg.)
carotenal
(mg./kg.) 0 2.2 4.4 8.8 17.6

0 Undiluted 34.09 60.58 68.88 79.23 89.39

Diluted 17.33 29.53 38.53 45.91 59.68


2.2 Undiluted 52.98 67.16 77.15 83.58 91.33

Diluted 26.92 35.45 41.85 52.32 62.26


4.4 Undiluted 62.57 75.16 80.38 84.05 90.38

Diluted 33.43 40.14 46.03 54.07 64.46


8.8 Undiluted 77.79 80.70 82.50 87.94 92.42

Diluted 45.81 48.29 50.46 58.01 66.96


17.6 Undiluted 85.03 87.50 89.90 90.65 93.09

Diluted 54.01 58.36 60.74 65.56 69.94




























c


EC


cC,






rC

Or

CCu

"OO

r m.


ZU
o











MO


Cr- >


C,
OO(
-m *


*m-
U "
XO










~LL





mO % S ,O

r(ll~nd uo!lell3x3 ~





were slightly less from hens fed beta-apo-8'-carotenal than values ob-

tained when canthaxanthin was fed at the same feed concentrations. It

was observed, however, that the relative amount of pigment supplied to

the yolk from the feed is reflected by the excitation purity values,

regardless of source. Excitation purity values of diluted egg yolk

samples were used to calculate the relative biological availability and

the pigmenting efficiency of the two compounds when fed separately.
Biological availability was determined as the relative amount of beta-

apo-8'-carotenal that would be required in the feed to give the equiva-
lent egg yolk excitation purity when hens were fed canthaxanthin. It

was determined that beta-apo-8'-carotenal was 82.2% as available as

canthaxanthin. The relative pigmenting efficiency was determined using

a linear regression model to compare the excitation purity values of

yolks from hens fed the two compounds separately between 2.2 mg./kg. and

17.6 mg./kg. (Figure 2). It was determined that beta-apo-8'-carotenal

was 92.1% as efficient as canthaxanthin in producing yolk excitation

purity at the same feed concentrations. There was a high degree of
similarity in the color (DWL, EP and Lum) of blended yolk samples and

yolks which were obtained from hens receiving a similar dietary level of

the two xanthophylls as that calculated for a particular blended sample

(Table 16). Since the excitation purity of blended yolks was predicted

by yolks from hens fed similar levels of xanthophyll (Figure 3), regard-
less of actual color, it appeared that excitation purity is an accurate

measure of pigment concentration.

The data obtained in these experiments were used to develop a model

by which yolk colors could be predicted from known feed concentrations

of the two synthetic xanthophylls, beta-apo-8'-carotenal and canthaxanthin.





Figure 2. Linear regression model of diluted excitation purity
values for beta-apo-8'-carotenal and canthaxanthin
between 2.2 and 17.6 mg./kg. total xanthophyll when
fed separately















60 -- Canthaxanthin
(y = 28.22 + 1.84x; r2 = .94)


55 -




50 -1









S40 -C






/ Beta-apo-8'-carotenal
(y = 25.90 + 1.71x; r2 = .90)

30 /C





2 4 8 10 12 14 16 18
Total Xanthophyll (mg./kg.)




















31.39
31.02


28.25
27.39


23.49
23.69


30.98
30.74


27.29
26.02


22.73
22.88


31.12
30.40


27.87
27.86


23.76
23.37


~Xanthophyll concentration of blended samples
a 1:1 blending of yolks from hens fed the selected


was calculated using
levels of xanthophyll.


DWL EP Lum
(nm.) () (%)


Treatment



Blended
Fed


Blended
Fed


Blended
Fed


Blended
Fed


Blended
Fed


Blended
Fed


Blended
Fed


Blended
Fed


Blended
Fed


Table 16. Dominant wavelength (DWL), excitation purity (EP) and
luminosity (Lumn) of blended yolk samples and yolks which were
obtained from hens receiving a similar dietary level of beta-
apo-8'-carotenal (A) and canthaxanthin (C)


Calculated yolk

concentrations
(mg./kg.)


C2.2A2.2
C2.2A2.2


C4.4A2.2
C4.4A2.2


C8.8A2.2
C8.8A2.2


C2.2A4.4
C2.2A4.4


C4.4A4.4
C4.4A4.4


C8.8A4.4


C2.2A8.8
C2.2A8.8


C4.4A8.8
C4.4A8.8


C8.8A8.8
C8.8A8.8


Feed xanthophyll
concentrations
(mg./kg. )


C4.4Ao + CoA4.4
C2.2A2.2


C8.8Ao + CoA4.4
C4.4A2.2


C17.6Ao + CoA4.4
C8.8A2.2


C4.4Ao + CoA8.8
C2.2A4.4


C8.8Ao + CoA8.8
C4.4A4.4


C17.6Ao + CoA8.8
C .A4.4


C4.4Ao + CoA17.6
C2.2A8.8


C8.8Ao + CoA17.6
C4.4AS8.8


C17.6Ao + CoA17.6
C8.8A8.8


581.6
582.3


584.1
585.2


587.4
588.4


581.7
582.1


585.0
585.1


588.2
587.7


582.0
581.6


584.2
584.4


587.2
587.6


67.29
67.16


73.97
77.15


83.23
83.58


73.58
75.16


79.10
80.38


87.05
84.05


79.47
80.70


82.45
82.50


87.88
87.94
















































th









-r-



0 r-









-3


aC





CO
V-


*m





Lt.

















































D
ar

r
~D
CI
Y-


;CC




O


~71~nd uo~7e~i3x3 ~


































































L


This model is based upon utilizing the x-y chromaticity diagram of the

C.I.E. system for plotting the spectral colors. The procedure for

calculating dominant wavelength and excitation purity as well as an

excellent discussion of the C.I.E. system has been described by

Clydesdale (1969) and Clydesdale and Francis (1969). The x-y values of

the 25 dietary treatments were plotted on the portion of C.I.E. chro-

maticity diagram that corresponds to the yellow-orange range (Figure 4).

The 25 coordinates were connected by lines corresponding to the increas-

ing levels of the two pigments, from 0 to 17.6 mg./kg., when fed alone

or in combination, forming a triangular grid within the chromaticity

diagram. The curved line is the area of the chromaticity diagram repre-

sented by the Roche color fan, with respect to the 25 dietary treatments.

By locating the feed concentration of the two test xanthophylls in the

triangular area and determining the x-y values that correspond, it is

possible to predict the yolk color that will be produced from that feed

level. Also, it is possible to produce yolk colors of a predetermined

color designation in the C.I.E. system by feeding the concentration of

beta-apo-8'-carotenal and canthaxanthin that corresponds to the x-y

coordinates of the color selected.

It is proposed that this model could be expanded and with better

characterization of the naturally occurring xanthophylls in feedstuffs as

to type and quantity, yolk color produced by these natural sources could

also be predicted. Better methods of xanthophyll assays, both for total

concentration and for relative pigmenting efficiencies, would be needed.

It is apparent from these data, that the colorimetric analysis of

yolk color, as affected by different xanthophylls in the diet, is more

applicable in determining the effects of different pigments on resulting


























































































































~


C
a $.




c Ju
o
10











LD



xar

ER


Sr-


oa

MCL
Or

.CC




OO






"QC


*rC1
C.)4 L










tco





oV /i
\F/ / l


\ Iv o







Ot





yolk color than visual analysis. This is due to the lack of precise

color description using visual standards and the inability of the eye to

discern the difference in pigment concentration accurately, as seen by

comparison of the visual scores to the objective description of the same

samples. By analyzing the color as determined by the COLOR-EYE it is

possible to describe the color in terms of the dominant wavelength,

excitation purity and luminosity and relate it to what the human eye

actually perceives. There are several advantages to using colorimetric

analysis of yolks and the effects of dietary xanthophylls on color over

more conventional visual or chemical-spectrophotometric analysis

techniques. The COLOR-EYE~ can be used to describe color accurately

over a wide range of variations, by determining the dominant wavelength

(the actual color), excitation purity (intensity of color at that wave-

length) and the luminosity (lightness or total reflected light). Visual

comparisons based on standards can only give a relative degree of simi-

larity between one color and the set standards. Chemical spectrophoto-

metric techniques usually rely on determining the absorption maxima of

an extract of xanthophylls based on the absorption maxima of some

standard such as beta-carotene or dichromate. This method is only ap-

plicable for xanthophylls with an absorption maxima very close to that

of the standard and will give very poor results for xanthophylls that

have differing absorption maxima.





SUMMARY AND CONCLUSIONS


Three experiments were conducted to determine some of the factors

that can influence the measurement and utilization of xanthophylls in

both broiler skin and egg yolks. The results of the first experiment

indicated the effects of light intensity on broiler pigmentation. Birds

reared with constant low intensity lighting in windowless houses ex-

hibited significantly lower dominant wavelengths (less orange color) and

a significantly higher luminosity (less dark) than birds reared in open-

type houses. Excitation purity values were not significantly different

between the two housing types indicating that total pigment in the birds

was equal. It was also shown that birds reared along the outside of

houses had shanks with higher dominant wavelengths and thus were also

more orange than those of birds reared in the interior of houses with a

greatly reduced light intensity. It was concluded that light intensity

does indeed affect broiler pigmentation.

The second experiment was conducted to evaluate egg yolk pigmenta-

tion from eggs produced by 12 commercial laying strains of hens housed

in both floor and cage conditions. Significant differences in dominant

wavelength, excitation purity and luminosity were found between the 12

strains. Egg yolks from several strains were more highly pigmented

regardless of housing conditions; however, yolks from several other

strains appeared to vary with housing conditions.





The third experiment was conducted to evaluate the egg yolk pig-

menting ability of beta-apo-8'-carotenal and canthaxanthin and to deter-

mine the relative biological availability and pigmenting efficiency of

these two synthetic pigments. Dominant wavelength increased from 572.8

nm. for the 0 level to 577.9 and 590.6 nm. for the 17.6 mg./kg. levels

of beta-apo-8'-carotenal and canthaxanthin, respectively. Analysis of

the diluted excitation purity values showed that beta-apo-8'-carotenal

had a biological availability of 82% and a pigmenting efficiency of 92%

when compared to canthaxanthin. Blended yolk samples yielded almost

identical color scores when compared to yolks produced by hens fed the

same concentrations of xanthophyll. From these data a model was formu-

lated by which yolk color could be predicted from the feed concentra-

tions of these two xanthophylls. Egg yolks of a desired color could be

obtained by using the model to predict either the feed concentration of

the two test xanthophylls required in the diet or by blending yolks pro-

duced from feeds with known xanthophyll concentrations.




































































1


LIST OF REFERENCES


Angalet, S. A., J. L. Fry, B. L. Damron and R. H. Harms, 1976.
Evaluation of waste activated sludge (citrus) as a poultry feed
ingredient. Poultry Sci. 55:1219-1225.

Anonymous, 1971. Egg yolk pigmentation with carophyll. F. Hoffmann-
LaRoche and Co. Ltd., Basle, Switzerland.

Ashton, H. E., and D. A. Fletcher, 1962. Development and use of color
standards for egg yolks. Poultry Sci. 41:1903-1909.

Association of Official Analytical Chemists, 1970. Official Methods of
Analysis. 11 ed. Washington, D. C., p. 283.

Barnett, B. D., and J. F. Stephens, 1963. Variation in egg yolk
pigmentation. Poultry Sci. 42:1254.

Bartov, I., and S. Bornstein, 1966. Studies .on egg yolk pigmentation.
2. Effect of ethoxyquin on xanthophyll utilization. Poultry Sci.
45:297-305.

Bauernfeind, J. C., 1972. Carotenoid vitamin A precursors and analogs
in foods and feeds. J. Agr. Food Chem. 20:456-473.

Bickoff, E. M., A. L. Livingston, G. F. Bailey and C. R. Thompson, 1954.
Alfalfa carotenoids: Xanthophylls in fresh and dehydrated alfalfa.
J. Agr. Food Chem. 2:563-567.

Bird, H. R., 1943. Increasing yellow pigmentation in shanks of
chickens. Poultry Sci. 22:205-208.

Bletner, J. K., R. P. Mitchell, Jr. and R. L. Tugwell, 1966. The effect
of E~menla maximcz on broiler pigmentation. Poultry Sci. 45:689-
694.

Bornstein, S., and I. Bartov, 1966. Studies on egg yolk pigmentation.
1. A comparison between visual scoring of yolk color and colori-
metric assay of yolk carotenoids. Poultry Sci. 45:287-296.

Brambila, S., J. A. Pino and C. Mendoza, 1963. Studies with a natural
source of xanthophylls for the pigmentation of egg yolks and skin
of poultry. Poultry Sci. 42:294-300.

Brown, W. L., 1938. The influence of pimento pigments on the color of
the egg yolk of fowls. J. Bio1. Chem. 122:655-659.





Bunnell, R. H., and J. C. Bauernfeind, 1962. Chemistry, uses, and
properties of carotenoids in foods. Food Technol. 16(7):36, 38,
40, 42-43.

Bunnell, R. H., W. L. Marusich and J. C. Bauernfeind, 1962. Beta-apo-
8'-carotenal as an egg yolk pigmrenter. Poultry Sci. 41:1109-1115.

Bushong, R. D., Jr., B. C. Dilworth and E. J. Day, 1972. The influence
of ethoxyquin and sodium bicarbonate on egg yolk pigmentation.
Poultry Sci. 51:1768-1770.

Camp, A. A., C. R. Creger and J. R. Couch, 1963. Canthaxanthin as a
broiler pigmienter. Poultry Sci. 42:1258-1259.

Carver, D. S., 1959. Variation in the effects of fat supplements on
broiler pigmentation, growth and feed conversion. Poultry Sci.
38:71-76.

Chawan, C. B., and R. W. Gerry, 1974. Shrimp waste as a pigment source
in broiler diets. Poultry Sci. 53:671-676.

Clydesdale, F. M., 1969. The measurement of color. Food Technol. 23:
16-22.

Clydesdale, F. M., and F. J. Francis, 1969. Color measurement of foods:
VIII Developmient of the CIE system. Food Product Development
2(6) :50-56.

Collins, W. M., S. C. Thayer and W. C. Skoglund, 1955. Breed and strain
differences in shank pigmentation in growing chickens. Poultry
Sci. 34:223-228.

Coon, C. N., and J. R. Couch, 1971. Pigmentation studies of TagQete
steels on laying hens. Poultry Sci. 50:1566.

Coon, C. N., and J. R. Couch, 1976. Effect of storage and fatty acid
esters on the utilization of xanthophyll from marigold meal by
laying hens. Poultry Sci. 55:841-847.

Couch, J. R., and F. M. Farr, 1970. Canthaxanthin and beta-apo-8'-
carotenal as feed additives for increasing egg yolk pigmentation.
Poultry Sci. 49:1377.

Couch, J. R., and F. M. Farr, 1971. The effect of adding canthaxanthin
and beta-apo-8'-carotenal to laying diets containing yellow corn
and alfalfa on egg yolk pigmentation. Brit. Poultry Sci. 12:49-55.

Cox, A. C., 1973. Effects of bin ve~nun return feed from a drag chain
feeding system on the performance of S.C.W. Leghorn pullets.
Poultry Sci. 52:2015.

Culton, T. G., and H. R. Bird, 1941. Effect of certain protein supple-
ments in inhibiting pigment deposition in growing chicks. Poultry
Sci. 20:432-436.





Dalby, G., 1948. The determination and definition of color in eggs.
Cereal Chem. 25:413-417.

Davies, R. E., M. L. Jones and H. Yacowitz, 1969. Direct instrumental
measurement of skin color in broilers. Poultry Sci. 48:1800.

Davis, P. N., and F. H. Kratzer, 1958. The relation of serum xantho-
phyll in chickens to the pigmentation of their shanks. Poultry
Sci. 37:851-854.

Day, E. J., and W. P. Williams, Jr., 1958. A study of certain factors
that influence pigmentation in broilers. Poultry Sci. 37:1373-
1381.

De Groote, G., 1970. Research on egg yolk pigmentation and its
practical application. World's Poultry Sci. J. 26:435-441.

Farr, F. M., and J. R. Couch, 1970. Pigmentation of broilers with
canthaxanthin. Poultry Sci. 49:1384.

Farr, F. M., C. R. Creger and J. R. Couch, 1962. Egg yolk pigmentation.
Poultry Sci. 41:1643.

Francis, F. J., and F. M. Clydesdale, 1972. Color measurements of
foods: XXXV. Miscellaneous: part V. egg yolks. Food Product
Development 6(5):47-55.

Fritz, J. C., and F. D. Wharton, Jr., 1957. The influence of feed on
broiler pigmentation. Poultry Sci. 36:118.

Fry, J. L., E. M. Ahmed, G. Ml. Herrick and R. H. Harms, 1969. A
reflectance method of determining skin and shank pigmentation.
Poultry Sci. 48:1127-1129.

Fry, J. L., and B. L. Damron, 1971. Computer calculation of poultry and
egg pigmentation data. Food Technol. 25:44-45.

Fry, J. L., and R. H. Harms, 1974. Effect of Robenidine and/or Roxarsone
on broiler pigmentation. Poultry Sci. 53:1404-1407.

Fry, J. L., and R. H. Harms, 1975. Yolk color, candled egg grade and
xanthophyll availability from dietary natural pigmenting
ingredients. Poultry Sci. 54:1094-1101.

Fr~y, J. L., R. H. Harms and M. W. Moeller, 1976a. Effect of Flavomycin
and 3-nitro-10 on broiler pigmentation when used with different
coccidiostats. 1. Amprol Hi-E. Poultry Sci. 55:744-747.

Fry, J. L., R. H. Harms, M. W. Moeller and H. F. Kling, 1976b. Effect
of Flavomycin and 3-nitro-10 on broiler pigmentation when used with
different coccidiostats. 2. C10pidol. Poultry Sci. 55:1112-1116.





Fry, J. L., .G. M. Herrick and R. H. Harms, 1967. Yolk pigmnenting value
of dried kenaf tops. Quart. J. Florida Acad. Sci. 30:295-300.

Fry, J. L., C. F. Hinton and R. H. Harms, 1974. Reflectance colori-
metric evaluation of egg yolk pigmentation. J. Food Sci. 39:508-
510.

Garlich, J. D., D. M. Bryant, H. M. Covington, D. S. Chamblee and A. E.
Purcell, 1974. Egg yolk and broiler skin pigmentation with sweet
potato vine meal. Poultry Sci. 53:692-699.

Goffinet, R., and A. Ledent, 1967. Eisen van de Belgische konsument
ten aanzien van eieren. L.E.I. Schriften-nr. 73/R-39. From
De Grotte, 1970.

Goodwin, T. W., 1954. Caratenoids: Their comparative biochemistry.
Chemical Publishing Co., Inc., New York, N.Y.

Grau, C. R., and N. W. Klein, 1957. Sewage-grown algae as a feedstuff
for chicks. Poultry Sci. 36:1046-1051.

Grimiinger, P., and H. Fisher, 1960. A pigmentation enhancing effect of
2-methyl-1, 4-naphtoquinone in growing chickens. Poultry Sci. 39:
706-707.

Guenthner, E., C. W. Carlson, 0. E. 01son, G. 0. Kohler and A. L.
Livingston, 1973. Pigmentation of egg yolks by xanthophylls from
corn, marigold, alfalfa and synthetic sources. Poultry Sci. 52:
1787-1798.

Hall, G. M., P. W. Waldroup, J. L. Fry, C. B. Amrmerman and R. H. Harms,
1966. A comparison of the pigmenting value of alfalfa meals dif-
fering in protein and xanthophyll content. Poultry Sci. 45:639-
641.

Halloran, H. R., J. B. Lyle and W. T. Trana, 1971. Nutritional and
pigmenting evaluations of high xanthophyll alfalfa meal. Poultry
Sci. 50:1583.

Hammond, J. C., and H. M. Harshaw, 1941. Some factors influencing shank
and skin color in the growing chicken. Poultry Sci. 20:437-444.

Hammond, J. C., D. Miller and D. Whitson, 1942. The effect of the diet
of the hen on the shank color of her day-old chicks. Poultry Sci.
21:525-527.

Harms, R. H., J. L. Fry and B. N. McPherson, 1977. Evidence of differ-
ences in pigmentation among strains and crosses of broilers.
Poultry Sci. 56:86-90.

Harms, R. H., J. L. Fry, M. W. Moeller and H. F. Kling, 1976. Effect
of Flavomycin and 3-nitro-10 on broiler pigmentation when used with
different coccidiostats. 3. Monensin and Zoalene. Poultry Sci.
55:2214-2217.








Harms, R. H., J. H. Quisenberry and J. R. Couch, 1958. The effects on
broiler pigmentation of incorporating milo, dehydrated alfalfa
meal and diphenyl-p-phenylene-diamine (D.P.P.D.) in the diet.
Poultry Sci. 37:143-147.

Heath, J. L., and C. S. Shaffner, 1972. The effect of dietary soybean
oil on the deposition of xanthophyll in broiler skin. Poultry Sci.
51:502-506.

Heffner, J. E., P. Roy, B. H. Davis and W. B. Hilton, 1964. Consumer
preference for broiler pigmentation in New Orleans, Louisiana.
Agr. Exp. Stat. Bull. 586, Louisiana State University.

Heiman, V., and J. S. Carver, 1935. The yolk color index. U.S. Egg
Poultry Mag. 41:40-41.

Heiman, V., and L. W. Tighe, 1943. Observations on the shank pigmenta-
tion of chicks. Poultry Sci. 22:102-107.

Herrick, G. M., J. L. Fry, B. L. Damron and R. H. Harms, 1970. Evalua-
tion of dienestrol diacetate (Lipamone) supplementation of broiler
finisher feeds on pigmentation, growth characteristics and market
quality. Poultry Sci. 49:222-225.

Herrick, G. M., J. L. Fry and R. H. Harms, 1972. The use of beta-apo-
8'-carotenoic acid ethyl ester as a standard for determination of
xanthophyll utilization from natural sources by broilers. Poultry
Sci. 51:428-435.

Hinton, C. F., J. L. Fry and R. H. Harms, 1973. Subjective and colori-
metric evaluation of the xanthophyll utilization of natural and
synthetic pigments in broiler diets. Poultry Sci. 52:2169-2180.

Holleman, K. A., and T. W. Sullivan, 1959. Egg yolk color as influenced
by xanthophyll and feedstuffs containing related pigments.
Poultry Sci. 38:1214.

Huff, W. E., R. D. Wyatt and P. B. Hamilton, 1975. Effects of dietary
aflatoxin on certain egg yolk parameters. Poultry Sci. 54:2014-
2018.

Hurst, R. E., E. J. Day and B. C. Dilworth, 1973. An evaluation of
xanthophyll sources for egg yolk pigmentation. Poultry Sci. 52:
2043.

Isler, 0., and P. Zeller, 1957. Total synthesis of carotenoids.
Vitamins and Hormones, 15:31-71.

Jasper, A. W., and R. E. Cray, 1953. Consumer preferences, practices
and demands in purchasing eggs and poultry in Columbus, Ohio.
Ohio Agr. Exp. Stat. Bull. 736.

Jensen, A., 1963. The effect of seaweed carotenoids on egg yolk
coloration. Poultry Sci. 42:912-916.





Kowalski, L. M., and W. M. Reid, 1975. Effects of Roxarsone on pigmen-
tation and coccidiosis in broilers. Poultry Sci. 54:1544-1549.

Kuzmicky, D. D., G. O. Kohler, A. L. Livingston, R. E. Knowles and J. W.
Nelson, 1968. Pigmentation potency of xanthophyll sources.
Poultry Sci. 47:389-397.

Lipstein, B., S. Bornstein and P. Budowski, 1967. By-products of the
refining of soybean oil as pigment sources for poultry. 1.
Pigmentation studies with broilers. Poultry Sci. 46:626-638.

Little, A. C., 1969. Reflectance characteristics of canned tuna.
1. Development of an objective method for evaluating color on an
industry-wide basis. Food Technol. 23:1301-1318.

Littlefield, L. H., J. K. Bletner and 0. E. Goff, 1973. The effect of
feeding laying hens various levels of cow manure on the pigmenta-
tion of egg yolks. Poultry Sci. 52:179-181.

Littlefield, L. H., J. K. Bletner, H. V. Shirley and 0. E. Goff, 1972.
Locating the site of absorption of xanthophylls in the chicken by a
surgical technique. Poultry Sci. 51:1721-1725.

Littlefield, L. H., C. W. Coursey and R. E. Beal, 1975. Neutralized,
dried soapstock (NDSS) as a fat substitute in the laying hen diet.
Poultry Sci. 54:1341.

Livingston, A. L., D. D. Kuzmicky, R. E. Knowles and G. 0. Kohler, 1969.
The nature and deposition of the carotenoids from alfalfa and corn
gluten meal in chicken skin. Poultry Sci. 48:1678-1683.

Mackay, E., G. J. Mountney and E. C. Naber, 1963. Yolk color resulting
from different levels of paprika extract in the ration. Poultry
Sci. 42:32-37.

Madiedo, G., and M. L. Sunde, 1962. The effect of algae, dried lake
weed, alfalfa meal and ethoxyquin on yolk color. Poultry Sci. 41:
1660.

Marusich, W. L., 1967. Collaborative ANRC yolk pigmentation standard
study--Final report. Feedstuffs 39(4):48-50.

Marusich, W. L., 1969. Feasibility of broiler pigmentation standard.
Feedstuffs 41(3):26, 28.

Marusich, W. L., and J. C. Bauernfeind, 1970a. Oxycarotenoids in
poultry pigmentation. 1. Yolk studies. Poultry Sci. 49:1411.

Marusich, W. L., and J. C. Bauernfeind, 1970b. Oxycarotenoids in
poultry pigmentation. 2. Broiler studies. Poultry Sci. 49:1411.





Marusich, W. L., E. De Ritter and J. C. Bauernfeind, 1960. Evaluation
of carotenoid pigments for coloring egg yolks. Poultry Sci. 39:
1338-1345.

Marusich, W. L., H. Kadin and J. C. Bauernfeind, 1958. Evaluation of
carotenoid pigments for coloring yolk and skin of poultry. Poultry
Sci. 37:1224.

Marusich, W. L., E. F. Ogrinz, P. R. Brown and M. Mitrovic, 1971a.
Effect of xanthophylls, canthaxanthin and Roxarsone on broiler pig-
mentation. Poultry Sci. 50:1602.

Marusich, W. L., E. F. Ogrinz, N. Camerlengo, J. McCanbley and M.
Mitrovic, 1976. Zeaxanthin as a broiler pigmenter. .Poultry Sci.
55:1486-1494.

Marusich, W. L., E. Schildknecht, E. F. Ogrinz, P. R. Brown and M.
Mitrovic, 1971b. Effect of coccidiosis on pigmentation in
broilers. Poultry Sci. 50:1603.

Marusich, W. L. E. G. Schildknecht, E. F. Ogrinz, P. R. Brown and M.
Mitrovic, 1972. The effect of Eimetia P/raecox and Eimenia Teneffa
on pigmentation in broilers. Poultry Sci. 51:1832.

McCready, S. T., J. L. Fry,.C. F. Hinton and R. H. Harms, 1973.
Colorimetric characterization of egg yolk and egg yolk products.
J. Food Sci. 38:175-176.

Menge, H., and R. E. Beal, 1973. The use of neutralized soybean oil
soapstock for broilers. Poultry Sci. 52:219-222.

Morehouse, A. L., 1961. Dried algae meal as a source of xanthophyll for
egg yolk pigmentation. Poultry Sci. 40:1432.

Morgan, W. A., and J. G. Woodroof, 1927. Waste pimento pepper for
coloring egg yolks. Georgia Exp. Stat. Bull. 147.

Moster, J. B., and F. W. Quackenbush, 1952. The effect of temperature
and light on the carotenoids of seedlings grown from three corn
hybrids. Arch. Biochem. Biophys. 38:297-303.

Moster, J. B., F. W. Quackenbush and J. W. Porter, 1952. The
carotenoids of corn seedlings. Arch. Biochem. Biophys. 38:287-296.

Nelson, T. S., and J. N. Baptist, 1968. Feed pigments. 2. The in-
fluence of feeding single and combined sources of red and yellow
pigments on egg yolk color. Poultry Sci. 47:924-931.

01son, G., W. R. Cobb, A. D. McManus, A. Van Iderstine and W. H. Ott,
1972. Bacitracin and Roxarsone for growth, feed efficiency and
pigmentation in broiler chickens. Poultry Sci. 51:1846-1847.





Ott, W. H., W. R. Cobb, A. D. McManus, G. 01son and A. Van Iderstine,
1974. Penicillin and Roxarsone for growth, feed efficiency and
pigmentation in broiler chickens. Poultry Sci. 53:1964.

Palmer, L. S., 1915. Xanthophyll, the principal natural yellow pigment
of the egg yolk, body fat, and blood serum of the hen. The physio-
logical relation of the pigment to the xanthophyll of plants. J.
Bio1. Chem. 23:261-279.

Palmer, L. S., and H. L. Kempster, 1919. The influence of specific
feeds and certain pigments on the color of the egg yolk and body
fat of fowls. J. Bio1. Chem. 39:331-337.

Parker, S. L., S. S. Gossman and W. A. Lippincott, 1925. Studies on egg
quality: 1: Introductory note on variations in yolk color.
Poultry Sci. 5:131-145.

Perdue, Inc., 1976. A method of xanthophyll analysis. Personal
communications.

Pomeranz, Y., and C. E. Meloan, 1971. Food Analysis: Theory and
practice. AVI Publishing Co., Inc., W~estport, Conn., p. 72.

Potter, L. M., R. H. Bunnell, L. D. Matterson and E. P. Singsen, 1956.
The effect of antioxidants and a vitamin B12 concentration on the
utilization of carotenoid pigments by the chicks. Poultry Sci.
35:452-456.

Raskopf, B. D., I. H. Kidd and 0. E. Goff, 1961.. Effects of diets con-
taining milo on broilers and consumer acceptance of broilers dif-
fering in degree of skin pigmentation. Tennessee Agr. Exp. Stat.
Bull. 324. University of Tennessee.

Ratcliff, R. G., E. J. Day and J. E. Hill, 1959. Broiler pigmentation
as influenced by dietary modifications. Poultry Sci. 38:1039-1048.

Ratcliff, R. G., E. J. Day and J. E. Hill, 1961. Comparison of two
antioxidants and two sources xanthophyll in a pigmentation study
with broilers. Poultry Sci. 40:716-720.

Rodriguez, D. B., P. T. Arroyo, A. S. Bucoy and C. O. Chichester, 1976.
Identification of pigments of the egg yolk from Philippine-bred
ducks. J. Food Sci. 41:1418-1420.

Ruff, M. D., and W. M. Britton, 1976. Reduced yolk color in layers with
coccidiosis. Poultry Sci. 55:1712-1716.

Ruff, M. D., W. M. Reid and J. K. Johnson, 1974. Lowered blood
carotenoid levels in chickens infected with coccidia. Poultry Sci.
53:1801-1809.





Runnels, T. D., E. F. Waller, E. G. Kelley and S. Krulick, 1951.
Growth promotion and pigmentation properties of dehydrated broccoli
leaf meal in a practical broiler diet. Poultry Sci. 30:930.

Scott, M. L., I. Ascarelli and G. 01son, 1968. Studies of egg yolk
pigmentation. Poultry Sci. 47:863-872.

Scott, M. L., M. C. Nesheim and R. J. Young, 1969. Nutrition of the
Chicken. M. L. Scott and Associates, Ithaca, New York.

Service, J., 1972. A Users Guide to the Statistical Analysis System.
Student Supply Stores, North Carolina State University, Raleigh,
N.C.

Slocum, W. L., and H. S. Swanson, 1954. Egg consumption habits, purchas-
ing patterns and preferences of Seattle consumers. Washington Agr.
Exp. Stat. Bull. 556, Pullman, Wash.

Smidt, M. J., L. J. Classen, F. Pagoria and J. C. Fritz, 1965. Factors
affecting pigmentation of broilers. Fed. Proc. 24:687.

Smith, I. D., and H. S. Perdue, 1966. Isolation and tentative identifi-
cation of the carotenoids present in chicken skin and egg yolks.
Poultry Sci. 45:577-581.

Squibb, R. L., J. E. Braham, M. Guzman and N. S. Scrimshaw, 1955. Blood
serum total proteins, riboflavin, ascorbic acid, carotenoids and
vitamin A of New Hampshire chickens infected with coryza, cholera
or Newcastle disease. Poultry Sci. 34:1054-1058.

Steenbock, H., 1919. White corn us~. yellow corn and a probable relation
between the fat-soluble vitamin and yellow plant pigments. Science
50:352-353.

Stone, H. A., W. M. Collins and W. E. Urban, Jr., 1971. Evaluation of
carotenoid concentration in chicken tissues. Poultry Sci. 50:675-
681.

Sullivan, T. W., G. W. Froning, D. H. Kahler, A. L. Livingston and G. 0.
Kohler, 1974. Evaluation of freeze dried and dehydrated alfalfa
for egg yolk pigmentation in layer diets. Poultry Sci. 53:1983.

Sullivan, T. W., and K. A. Holleman, 1962. Effect of alfalfa meal, corn
gluten meal and other dietary components on egg yolk color.
Poultry Sci. 41 :1474-1478.

Sunde, M. L., 1962. The effect of different levels of vitamin A, beta
apo 8' carotenal and alfalfa on yolk color. Poultry Sci. 41:532-
541.

Tarver, F. R., Jr., 1961. The influence of yolk color intensity upon
yolk shadow values, albumien quality and yolk color index--
Deposition and color intensity of abdominal fat of pullet carcasses.
Poultry Sci. 40:987-991.





































































I


Thommen, H., 1971. Carotenoids. VIII. Metabolism, edited by 0. Isler,
H. Gutmann and U. Solms, F. Hoffman-LaRoche & Co. Ltd., Basle,
Switzerland.

Tortuero, F., 1968. Effects of different levels of dehydrated alfalfa
meal in practical rations of laying hens on the pigmentation of egg
yolk, their influence in the utilization of red xanthophylls
(capsantin and capsorrubin). Poultry Sci. 47:376-383.

Vuilleumier, J. P., 1969. The 'Roche Yolk Color Fan'--An instrument for
measuring yolk color. Poultry Sci. 48:767-779.

Waibel, G. P., G. M. Speers and P. E. Waibel, 1972. Effects of DDT and
charcoal on performance of White Leghorn hens. Poultry Sci. 51:
1963-1967.

Waldroup, P. W., C. R. Douglas, J. T. McCall and R. H. Harms, 1960. The
effects of Santoquin on the performance of broilers'. Poultry Sci.
39:1313-1317.

Waldroup, P. W., and K. R. Hazen, 1974. Performance of hens fed combina-
tions of alfalfa meal and dehydrated marigold meal in high pigment
diets. Poultry Sci. 53:1989.

Waldroup, P. W., and Z. B. Johnson, 1974. Lack of repeatability among
persons using the Roche color fan to assess the shank color of
broilers. Poultry Sci. 53:437-439.

Weber, C. W., and T. W. Philip, 1975. Evaluation of various types of
luteins in egg yolk pigmentation. Poultry Sci. 54:1827-1828.

Weis, A. E., and B. Bisbey, 1947. The relation of the carotenoid pig-
ments of the diet to the growth of young chicks and to the storage
in their tissues. Missouri Agr. Exp. Stat. Bull. 405. Colombia,
Mo.

Wilkinson, W. S., and C. Barbee, 1968. The relative value of xantho-
phyll from corn gluten meal, alfalfa, coastal bermudagrass and
pearl millet for broiler pigmentation. Poultry Sci. 47:1579-1587.

Wilson, W. 0., 1956. Identifying non-laying chicken hens. Poultry Sci.
35:226-227.

Wyatt, R. D., H. T. Tung, J. P. Thaxton and P. B. Hamilton, 1972. New
aspects of aflatoxicosis in chicks. Poultry Sci. 51:1873.















BIOGRAPHICAL SKETCH


Daniel Lake Fletcher was born January 1, 1949, in Detroit, Michigan.

He moved to Pompano Beach, Florida, in 1953 and graduated from Pompano

Beach Senior High School in 1967. In June, 1969, he received his Asso-

ciate of Arts degree in biology from Palm Beach Junior College. He then

enrolled at the University of Florida in September, 1969, and in August,

1971, received his Bachelor of Science degree in zoology.

In September, 1973, he re-entered the University of Florida as a

graduate assistant in Food Science. While in Food Science he was an

active member of the Food Science Club, became a member in Alpha Zeta

Honorary Fraternity and was awarded the Alpha Zeta Outstanding Graduate

Student Award for the College of Agriculture, 1975. After receiving his

Master of Science in Agriculture degree in August, 1975, he transferred

to the Poultry Science Department, University of Florida, to pursue a

Doctor of Philosophy degree. He served as Secretary-Treasurer and

President of the Poultry Science Club, was admitted to Gamma Sigma Delta

and Sigma Xi Honorary Societies and was awarded the Ruby V. Voitle

Outstanding Graduate Student Award, 1977. In September, 1977, he will

begin his professional career as an Assistant Professor in Poultry

Science at the University of Georgia following his expected graduation

with a Doctor of Philosophy degree in August, 1977.





I certify that I have read this study and that in my opinion it
conforms, to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.





R. H. Harms, Chairman
Professor of Poultry Science



I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.





D. M. Jank~y, 9 chaim
Assistant Professor o oultry Science



I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.





H. R. Wilson
Professor of Poultry Science



I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.





J. IOb nger
A cia Professor o Food Science








I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.





R. L. West
Assistant Professor of Animal Science



This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate Council, and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.

August, 1977



Dean, 11lege of Agricultur


Dean, Graduate School




University of Florida Home Page
© 2004 - 2010 University of Florida George A. Smathers Libraries.
All rights reserved.

Acceptable Use, Copyright, and Disclaimer Statement
Last updated October 10, 2010 - - mvs