SOME PHYSiCAL Am} Ci-{EMECAL ASPECTS OF
MOWLEQ £653 YOLKS

Thesis for fha :30ng 0? Ph. D.

MECHIGAN STATE UNNERSH‘Y

Benny Angala Siivasfi'rini
19.5335

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This is to certify that the
thesis entitled

SOLE PH DEM; Al’D CHEAICAL ASPECTS
OFI JOTTLED EEG YCLKS

presented by

Denny Angelo Silvestrini

has been accepted towards fulfillment
of the requirements for

__P_h-__D:._degree in__F00d Science

Major professor

 

Date November 20, 1961

 

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AETRACT

SOME PHISICAL AND CHEMICAL ASPECTS OF
MOT'I‘IED EEG YOLKS

by Denny Angelo Silvestrini

Mottled egg yolks, chemically induced, were obtained from two
groups of 12 White Leghorn hens which were fed diets supplemented with
0.0125 and 0.001 percent Nicarbazin, respectively, and control eggs
from a third group of 12 White Leghorns which was fed a basal ration.
ZDuring the first three weeks of the experiment, all eggs laid were
collected, broken-out and scored for degree and incidence of mottled
yolks and albumen quality. All other eggs laid were stored for various
periods at 35°F. and broken-out and scored for incidence and severity
of mottled yolks.

Paper electrophoresis analyses were employed to study and
separate the proteins of the mottled and normal yolks. After separation
and isolation of the lipoproteins, lipovitellin and lipovitellenin, from
both normal and mottled yolks, the lipids were extracted from the
lipoproteins and analyzed on eilicic acid chromatography columns. Gas
chromatography was used to separate the fatty acids of the phospholipids
and triglycerides of the lipoproteins obtained from silicic acid column
chromatographic analyses. Amino nitrogen, total nitrogen, phosphorus,
choline and cholesterol determinations were carried out on the phospho-
lipids and triglycerides obtained from the lipovitellin and lipovitellenin
of mottled and normal yolks.

A direct relationship was found between the number of mottled
yolks and length of time hens were fed the supplemented diets. Storage

of eggs from hens fed the drug caused an increase in degree and number

of mottled egg yolks. Twenty-five percent fewer eggs were laid by
hens fed 0.0125 percent Nicarbazin, but drug-supplemented diets fed
to hens had no effect on albumen quality.

Electrophoresis and lipoprotein isolation analyses showed
that mottled yolks contained less lipovitellin and lipovitellenin but
more water soluble protein. A direct relationship was observed between
the mottled yolk score and the proportion of ovalbumin in the yolk.

Iipovitellin of mottled and normal yolks contained the same
percentage of ether and alcohol extracted lipids but lipovitellenin of
mottled yolks contained 6.3 percent more other extracted lipids than
I normal yolks.

' Triglycerides, cholesterol, cephalins and lecithins in ether
extracted lipids from lipovitellin of mottled and normal yolks showed
no differences. More lecithins and less cephalins were found in alcohol
extracted lipids from lipovitellin of mottled yolks than normal yolks,
but no differences in triglycerides and cholesterol were observed
between the alcohol extracted lipids of normal and mottled yolks.

Ether extracted lipids from lipovitellenin from mottled and
normal yolks contained the same percentages of triglycerides, cephalins
and lecithins but mottled yolks contained more cholesterol than normal
yolks. Less triglycerides and cholesterol but more cephalins and
lecithins were found in alcohol extracted lipids from lipovitellenin
of mottled yolks than normal yolks.

Mottled yolks contained more oleic acid in triglycerides of
the ether extracted lipids of lipovitellin and in triglycerides of the

ether and alcohol extracted lipids from lipovitellenin than normal. yolks.

the appearance of mottled areas in yolks from hens fed Nicarbazin

was probably due to the presence of varying amounts of water and ovalbumin

under the vitelline membrane of the mottled yolks.

SOME PHYSICAL AND CHEMICAL ASPECTS OF

WPI'IEDEGGYOIKB

By

Denny Angelo Silvestrini

ATHEBIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MOTOR OF PHIIOSOPHY

Department of Food Science

1961

ACKNOWLEDGMENTS

The author wishes to express his thanks to Dr. L. E. Dawson
for his constant personal interest, motivation and continual guidance
throughout this work. He is also indebted to.Dr. R. J. Evans for the
use of the laboratory facilities and for his assistance in contributing
much useful information concerning chemical techniques which were
employed throughout the work.

The writer is indebted to Drs. J.'W. Goff, F. R. Peabody,
B. S. Schweigert and H. C. Zindel for their helpful suggestions and
constructive review of this thesis.

Finally, the author is indebted, above all, to his wife,
Betty, for her patience and encouragement during this strenuous

period of study and research.

ii

TABLE OF CONTENTS

INTRODUCTION

LITERATURE REVIEW

History of Mottled Egg Yolks

Chemical Composition of Mottled Yolks
Lipoprotein Analyses

Lipoprotein Composition

Phospholipids In Egg Yelks'

EXPERIMENTALIPROCEDURE

Source and Quality of Mottled Egg Yolks
Paper Electrophoresis of Yelk Proteins
Preparation of egg yolk samples
Method of paper electrophoresis
Paper strip analysis
Preparation of Egg Yolk Proteins
Lipovitellin
Lipovitellenin
Livetin
Lipid Extraction of Lipoproteins
Silicic Acid Chromatography
Silicic acid preparation
Column and apparatus preparation

Addition of sample to column and solvent
systems used

Fraction separation and evaporation

iii

*o
m
(D

seemsaeselm

l8
l9

Analytical Methods
Amino nitrogen
Total nitrogen
Phosphorus
Iodine number
Choline
Total cholesterol
Fatty acid methyl esters
RESULES AND DISCUSSION
Photographs of Mottled Yblks
Incidence of Mottled.!blk$
Newly-laid eggs
Sterage eggs
Egg production and egg quality

Protein Distribution in Yblks of Normal and
Mottled Eggs

Lipoprotein Isolation
Lipoprotein Lipid Extraction
Lipid Composition of Lipoproteins
SUMMARY AND CONCLUSIONS
REFERENCES

iv

#3
43
5o
50
61

Figure

10

LIST OF FIGURES

Photograph of an egg with a slightly mottled
yolk

Photograph of an egg with a moderately mottled
yolk

Photograph of an egg with a severely mottled
yolk

Photograph of an egg with a very severely
mottled yolk

Photograph of 4 egg yolks

Filter paper strip and Analytrol scanner-
integrator graph of strip for normal egg yolk
proteins

Filter paper strip and Analytrol scanner-
integrator graph of strip for slightly mottled
egg yolk proteins

Filter paper strip and Analytrol scanner-
integrator graph of strip for moderately
mottled egg yolk proteins

Filter paper strip and Analytrol scanner-
integrator graph of strip for severely
mottled egg yolk proteins

Filter paper strip and Analytrol scanner-
integrator graph of strip for very severely
mottled egg yolk proteins

33

3#

35
36

“5

47

Table

10

ll

13

1#

LIST OF TABLES

Percentage of eggs in each mottled yolk
classification from hens fed different levels
of Nicarbazin up to 132 days

Percentage of fresh eggs from.hens fed
Nicarbazin to 132 days with moderate to very
severe mottled yolk scores

Percentage of eggs in each mottling score, from
hens fed diff rent levels of Nicarbazin, after
storage at 35 F., up to 107 days

Percentage of eggs from hens fed Nicarbazin for
6 or more days with moderate to severely mottled
yolks

Percentage of eggs with yolk mottling scores from
mderate to very severe from hens fed Nicarbazin
7 weeks or more and stored up to 13 months

Percentage protein composition of egg yolks with
different degrees of’mottling, as determined by
paper electrophoresis

Percentage yield of lipoproteins of normal and
mottled egg yolks

Yield of lipoproteins in normal and mottled
egg yolks

Percent lipid in normal and mottled yolk
lipoproteins

Lipid distribution from lipovitellin and
lipovitellenin of normal and mottled yolks

Composition of lipids from lipovitellin
Composition of lipids from lipovitellenin

Iodine numbers, distribution and unsaturation of
fatty acids in lipids of lipovitellin

Iodine numbers, distribution and unsaturation of
fatty acids in lipids of lipovitellenin

38

#1

42

51

52

53

54
56
57

59

6O

INTRODUCTION

The appearance of an abnormality in eggs, referred to as
mottled yolks, has 'bee‘n'recognized for at least 28 years, but little
is known of it's incidence in eggs from farm flocks. Although mottled
yolks have been found most frequently in stored eggs, they have also
been observed in fresh eggs.

The mottled condition observed on egg yolks has been described
as transparent, orange-yellow, oily appearing spots which appear on the
surface of the yolks.

Some investigators have shown that slight mechanical pressure
on the yolk membrane will. produce a condition similar to a mottled area
and others found that mottled yolks may be induced by drugs, antioxidants
and other compounds. It is still uncertain, however, which factors are
responsible for mottled yolks which occur in eggs obtained from hens not
fed diets containing specific drugs. Long storage periods and high
temperatures have been found to increase the intensity and incidence of
mottled yolks. 1

Nicubazini, a coccidiostat, usually recommended for young
chickens, has been fed experimentally to laying hens to produce eggs
with mottled yolks. Due to carelessness in mixing feed, the drug has
been fed to laying hens on farms and mottled yolks have resulted.

Limited data are available concerning the physical and

chemical differences between normal and mottled egg yolks.

 

1 1
Nicarbazin is a trade name for an equinolatmlar complex of 1+, it -dinitro-

carbanilide (DNC) and 2 - hydroxy - it, 6-dimethylpyrimidine.
1

1“

2
The purposes of this study were to determine differences in

egg yolk proteins and lipids between normal and mottled egg yolks; to
evaluate the influence of egg storage on the incidence of mottled yolks;
and to evaluate the influence of the length of Nicarbazin feeding period

on yolk mottling and other egg characteristics.

LITERATURE REVIEW
History of Mottled Egg Yolks

Almquist (1933) first observed mottled yolks in eggs from
hens fed cottonseed meal in their diet. Yolks from newly-laid eggs
appeared normal but the incidence and degree of mottling increased
during storage. Mottled yolks contained more water, more egg white
protein and a higher protein-fat ratio than normal yolks. Almquist
concluded that mottled yolks were caused by diffusion of egg white
protein into the yolk because the vitelline membrane was more permeable
than that in normal eggs. Schaible e_t_ 59;. (1936) observed a mottled
yolk condition apparently similar to that described by Alnquist (1933),
but the mottled areas of the egg yolks were not caused by feeding cotton
seed meal in the diet of the hens. They found that a mottled area could
be produced by slight mechanical pressure on the yolk membrane, as done
by pushing with the tip of the finger.

Jeffrey (1945) frequently observed mottled yolks in the eggs
laid by Barred Plymouth Rock and Rhode Island Red hens but not in the
eggs from Single Comb White Leghorns or Legbars. Another defect similar
to mottled yolks was caused by the mixing of fragmented yolk with albumen.
Romanoff and Romanoff (19159) reported that mottled yolks were seen in
stored eggs more frequently than in fresh eggs.

Nicarbazin, a new coccidiostat introduced in 1954, adversely
influenced hatchabi'lity, thus was not recommended for adult chickens.
Within two years after the inclusion of Nicarbazin in broiler rations
the presence of mottled yolks in eggs from hens fed Nicarbazin was
discovered and reported by Polin at g. (1956 b).

Polin _e_t_ 2;. (1957) concluded that the accidental feeding

g h
of Nicarbazin to layers might result from (1) erroneous use of starter,

broiler or grower feeds which contained Nicarbazin and fed to hens, or
(2) through contamination of layer rations with such feeds. Mottled
yolks could occur in eggs even when the hens did not receive Nicarbazin
in the ration. No significant increase in the occurrence of mottled
yolks was shown until the level of Nicarbazin in the ration was increased
to a minimum of 0.005 percent. Polin and Porter (1956) found similar
results and reported further that when levels as high as 0.03 percent
Nicarbazin were fed a direct relationship was found between degree of
nottling and the percentage of Nicarbasin in the ration.

Polin gt ~331;. (1956 a) developed a method of analysis for
Midnitrocarbanflide (DNC) in the yolks of eggs. The rate at which
DNC was deposited in the eggs of hens fed Nicarbazin depended upon the
concentration of the compound in the blood and tissues and upon the
rate of yolk deposition.

Weiss (1957) showed that Nicarbazin fed at a level of 0.0125
percent for 10 to 15 days to small groups of white Rock and White Leghorn
pallets and to hybrid hens caused a “whitening of the tinted eggs" after
3 days treatment. " Egg production was reduced an average of 8 percent
and egg weight an average of 5 percent from control levels. Shell
thickness and albumen scores were not affected. One out of nine White
Leghorn hens fed Nicarbazin laid fresh eggs with severely mottled yolks.

Polin e_t_ 21; (1958) reported that 0.00? percent Nicarbasin
in the ration of laying hens decreased egg size, by reducing the yolk
size. When eggs from hens fed a minim of 0.005 percent Nicarbasin
were stored for 7 to 10 days, significantly more mottled yolks were found
than in normal eggs. Extremely mottled yolks were associated with lower

albumen quality, but no effect on albumen quality was evident in eggs

which were only slightly mottled.

Severely mottled yolks were observed in eggs stored for 5 or
more days at room temperature (Miller 33 _al_. 1957). These investigators
reported that an increase in level of dietary fat did not increase the
number of severely mottled eggs.

Baker at g. (1957) reported that laying hens fed Nicarbazin
produced eggs with ”blemished" or mottled yolks. When a level of 0.0125
percent Nicarbazin was fed, nearly all of the eggs were severely mottled.
At lower levels of Nicarbazin (0.006 to 0.009 percent) only a few
severely mottled yolks were observed and only a minimum of 0.0015 per-
cent Nicarbasin was required to increase the incidence of mottled yolks.
Storage for l to 2 weeks increased the incidence of severely mottled
yolks from hens fed 0.006 percent Nicarbazin in the ration. Egg production
was reduced markedly by feeding 0.0125 percent Nicarbazin to Leghorns
and by feeding 0.007 percent Nicarbazin to the heavy breed hens. Egg
sisewas also reduced by feeding a minimum of 0.006 percent Nicarbazin
in the diet. The heavy breeds produced white-shelled eggs when fed
0.009 percent Nicarbazin while a level of 0.003 percent had no effect
on shell color, but produced some mottled yolks. I

The results of McLaughlin gt :1. (195?) agree with those of
Weiss (1957) and Baker 33 2. (1957) in that heavy breed hens laid
white-shelled eggs after the hens had been fed 0.0125 percent Nicarbazin
for 3 days. During the second week of treatment, hens fed Nicarbezin
laid only one-half as may eggs as the controls. This difference in
egg production was maintained more than a week after Nicarbasin feeding
was discontinued. No significant differences were reported in egg
weight, shell thickness or interior quality of eggs between those produced

by hens fed a control ration and hens fed Nicarbasin.

6

Miller it; -a_l_. (1957) showed that holding eggs at room
temperature for 3 days resulted in 35 percent slightly mottled yolks and
25 percent moderately mottled yolks. Polin it; 31. (1956 b) found an
incidence of 12 percent mottled yolks in eggs stored at 50°F. Similar
results were observed by Baker _e_t 31. (1957), McNally and Brant (1958)
and Mitchell and Stadelman (1958).

The antioxidants, gallic acid and n-propylgallate, when fed
at a level above that which occurred during normal feed consumption,
increased the incidence of mottled yolks of eggs stored for 2 to 3 days
at 55°F. (McNally and Brant, 1958). Tannic acid fed to hens caused an
increase in number of mottled yolks. Dawson (1960) stated that certain

worm medicants‘and ammonia gas have been known to cause mottled yolks.
Chemical Composition of Mottled Yolks

Mottled yolks were reported to contain more water and less
solids, lipids and nitrogen than non-mottled yolks according to Polin
(1957). He found yolk material intermingled with the inner thin layer
of albumen in most eggs with extremely mottled yolks. Polin (1957)
agreed with the theory of Baker 21;. 9.1.: (1956) that the water passing
from the albumen into the yolk appeared to be involved in yolk mottling.
Baker gt a_l_. (1956) reported that extremely mottled yolks from hens fed
Nicarbazin had a more alkaline pH than normal yolks.

Van Tienhoven 21 :1. (1958) observed yolk weight differences
between mottled and non-mottled egg yolks which were similar to those
reported by Polin (1957). Mottled yolks contained a significantly
higher water content than normal yolks. No significant difference was
observed between mottled and normal eggs in behavior of the yolk contents

to a standard membrane. The lack of difference in ash content between

7
the mottled and normal yolks would substantiate the theory that yolk

composition.was not responsible for the added.water uptake.

Polin (1960), in a review of Qyolk-mottling', reported that
the predominant change in mottled yolks was a movement of water from the
albumen to the yolk. He stated that Qmottling' occurred when.yolk
:material passed into solution because the water, carrying with it
buffering salts from the albumen, shifted the pH of the yolk to the
alkaline side. Polin (1960) thus described the mottled yolks as “watered-
down! yolks.

Three general observations concerning mottled.yolks which Baker
gt 3.3;. (1956), Polin (1957) and van Tienhoven (1958) have reported are
as follows:

1. Incidence and.severity of mottled yolks in eggs laid
by hens fed Nicarbazin increased during storage.

2. The mottled areas on yolks produced by hens fed Nicarbazin
were associated with a movement of water from the albumen to the yolk
and this change accompanied a decrease in the fat content or an increase
in the protein-fat ratio of the egg yolks.

3. The change in pH of mottled.yolks from acid to alkaline

was accompanied by'a water movement from.albumen to yolk.

Lipqproteinrinalzggg

 

Romanoff and Romanoff (19h9) reviewed the literature prior to
l9h9 on yolk composition and reported that yolk was composed of 16.6
percent proteins, 32.6 percent lipids, 1.0 percent carbohydrates, 1.1
percent minerals and other constituents and “8.7 percent water. The
yolk lipids contained 62.3 percent neutral fats, 32.8 percent phospholipids,

#.9 percent sterols and a trace of cerebrosides. Yolk fatty acid

8
fraction was composed of approximately 50 percent oleic, 27 percent
palmitic, 11 percent linoleic, 6 percent stearic, and 6 percent
palmitol eic, linolenic, clupanodonic and myristic acids.

Yolk protein consisted of three main fractions, lipovitellin,
lipovitellenin and livetin. Dumas and Cahours (1842) first studied the
egg yolk protein, ovovitellin. Osborne and Campbell (1900) isolated
lecithovitellin from egg yolks and found that it contained lecithin,
which could be removed with ethyl alcohol, leaving the insoluble vitellin.
Chargaff (1942) and Alderton and Fevold (1945) reported different methods
for isolating lipovitellin, and later Fevold and Lausten (1946) found
that like lecithovitellin was also composed of two lipoproteins, lipo-
vitellin and lipovitellenin. Evans and Bandemer (1957) reported a
method for the separation and study of egg yolk proteins by paper

electrophoresis.
lipoprotein Composition

Wittcoff (1951), after a review of literature, stated that
lipovitellenin contained approximately twice as much lipid as lipo-
vitellin. lipovitellenin contained 36 to 41 percent lipids and comprised
40 percent of the total lipoprotein of the egg yolk. When lipoprotein
was emulsified in water, the protein was slowly liberated and became
increasingly less soluble in salt solutions. The globular nature of the
lipoproteins appeared to depend on the phosphatide present and the
emulsifying power of the egg yolk appeared to be due to the phosphatide
present in complex formation with the protein.

Chargaff (1942) found that lipovitellin prepared by his method
contained 18 percent phosphatides which could be removed by extraction

with alcohol. lipovitellin contained 1.5 percent phosphorus, 13.0 per-

fl

9

cent nitrogen, 0.9 Percent sulfur and 3.9 percent ash. Composition of
lipovitellin isolated by'Alderton and Fevold (1945) agreed well with
that of Chargaff (19u2). Lipovitellin isolated by'Sugano (1957)
contained 13 to 14 percent lipid and 2.2 to 2.4 percent phosphorus compared
to 16 to 18 percent lipid and 2.3 to 2.4 percent phosphorus in that obtained
by Fevold and Lausten (1946).

Lipovitellenin, isolated by Fevold and Lausten (1946) contained
9.9 to 10.2 percent nitrogen and 1.56 to 1.74 percent phosphorus and 36
to 41 percent lipid. Composition of egg lipovitellenin isolated by

811ng (1957) agreed with that of Fevold and Lausten (19%).
Phospholipids in Egg Yelks

As early as 1793 the existence of complex fatty compounds was
‘well established. Thudicum.(1884) differentiated lecithin from cephalin
by separating the two phospholipids in alcohol. He found that cephalin
was insoluble in alcohol but lecithin dissolved readily in alcohol. Folch
(1942) reported “cephalin? was actually a mixture of several components,
two of which were identified as phosphatidylethanolamine and phosphatidyl-
serine. Another cephalin component identified was inositol.

Levene and Rolf (1921, 1927) developed a method for isolating
lecithin.by'precipitation with cadmium chloride and then washing the
precipitate with ether. Pangborn (1941) presented data on an improved
quantitative lecithin isolation. Chargaff (1942) isolated lecithin
and cephalin from egg yolk, and later Levine and Chargaff (1951)
reported further analyses of egg yolk phospholipids. Sinclair (1948)
isolated an egg yolk lecithin of good purity that was not contaminated
by amino nitrogen.

Aluminum.oxide and silicic acid columns have recently'been

10
used to separate phospholipids. Hanahan, Turner and J ayko (1951)
reported good yield of highly purified lecithin from aluminum ofide
columns. Lea and Rhodes (1953, 1954) used cellulose chromatographic
columns to separate egg yolk phospholipids. More recently, Lea, Rhodes
and Stoll (1955) prepared egg lecithin with a high degree of purity by
chromatographic separations on alumina columns. Rhodes and Lea (1957)
reported separating egg phospholipids into two fractions, one containing
phosphatidylethanolamine and the remaining fraction containing components

which phosphatidylcholine predominated.

EXPERIMENTAL PROCEDURE

Source and Quality of Mottled Egg Yolks

Thirty-six (36) white Leghorn hens (18 Dekalb strain and 18
Michigan State University strain) were placed in single bird laying
cages. The Michigan State strain was put in the cages on one side of
the battery while the other strain was placed on the opposite side of
the battery. The hens on both sides of the battery were randomized into
three groups; Group 1 was fed the Michigan State University 2-4 Battery
all-mash laying ration while Groups 2 and 3 received the same ration
swplemented with Nicarbazin (DNC) at levels of 0.001 and 0.0125 percent,
respectively. The composition of the Z-4 basal ration was as follows:
ground corn, 34.5 percent; ground oats, 20.0 percent; wheat bran, 15.0
percent; flour middlings, 10.0 percent; dehydrated alfalfa meal, 3.0
percent; meat scraps, 3.0 percent; dried skim milk, 2.0 percent;
Henhaden fish meal, 2.5 percent“; ground oyster shell flour, 5.0 per-
cent; steamed bone meal, 1.5 percent; salt, 0.6 percent; and cod liver
oil (2,000 A and 400 D), 0.4 percent. The Nicarbazin added to the basal
ration was an equimolecular complex of 4, 4' -dinitrocarbanilide (DNC)
and 2-hydroxy-4, 6-dinethylpyrimidine and has the following structural

formula; (’0

C\

\/Nl1 NH\ \ 0H3\c/c\c/CH3
' I |
N’ / /‘~02 "\c/N
I

4, 4' -dinitrocarbanilide hydr .1; 6 0591.111 thy].
2 9 03W ’ ’ e .-
NICARBAZIN pyrimidine

ll

12

The feed supplements were added after all hens had been fed
the basal ration for 2 weeks and feeding started on November 13, 1959
and was continued until. March 31, 1960.

During the first 3 weeks after the Nicarbazin feeding had been
started, eggs were collected twice daily and broken for fresh egg quality
evaluation. All of these eggs were broken-out at intervals of from 1
to 6 days after being laid. All other eggs collected to the termination
of the experiment were stored for various periods. Every egg was weighed,
broken-out and the height cf the albumen measured with an Ames tripod
micrometer. The eggs were broken out on a glass plate supported between
mirrors so that all Surfaces of the egg yolk could be observed. Haugh
score values for all the eggs were calculated according to the method
of Brant and Shrader (1952).

The yolks were scored for degree 01‘ mottling according to a
method similar to that of Polin 93 :1_. (1957), except that they were
scored from 1 to 5, in units of one. The scoring system was established
after samples of yolks with different degrees of mottling had been
observed. A yolk with a score of l was very slightly mottled; however,
only on very close yolk examination was the mottled area detected. Talks
with a score of 2 were classified as slightly mottled, easily detectable
but not too objectionable in appearance. Yolks with moderate mottling
were assessed a score of 3, while severely mottled yolks received a
score of 4. Very severely mottled yolks were given a score of 5 and
were characterized, in new instances, by being devoid of the bright
yellow pigment and appeared orange, oily and transparent. To reduce
personal error, all eggs were weighed by one individual and the breaking
and quality measurements made by a second individual.

Eggs which were collected after the three-week daily-breaking

f‘

-k.‘

13
schedule were stored at 35°F. until further examination. At intervals
during and after the feeding trial, eggs were removed from storage and
quality evaluations carried out as previously outlined.

Paper Electrophoresis of Yolk Proteins

 

The paper electrophoresis procedures of Eyans and Bandemer
(1956, 1957) were used.

a) Preparation of the egg yolk samples

The yolks were separated from the whites, and amr adhering

egg albumen was removed from the yolks by rolling them over cheese cloth.
The chalaziferous layer and chalazae were removed with the aid of a pair
of tweezers. Care had to be taken with severely mottled yolks when
removing the albumen and chalazae because the vitelline membranes were
weakened due to internal pressure, and they ruptured easily. In general,
the samples of yolk were obtained as whole egg yolks, diluted with an
equal weight of 10 percent sodium chloride solution. In some cases, a
1 milliliter syringe was used to remove fluid from the mottled areas of
the yolk.

b) Method of paper electrophoresis

The Spinco Model R paper electrophoresis apparatus, a

ridge-pole type cell, manufactured by the Beckman Spinco Division, was
used. A ten microliter sample of egg yolk was applied to each paper
strip by means of a special stripper (furnished with Model R) and each
sample was run in duplicate on each of two consecutive days. A constant
direct current of 5 milliamperes (2.2 to 2.7 volts per centimeter) was
applied over the paper strips for 16 hours at room temperature (approx «-
imately 25°c). Prior to applying the sample to the Whatman Number 3 MM
paper strips, the filter papers were allowed to equilibrate in the buffer

4‘

14
solution for one hour. At the end of the 16 hour run, the papers were
dried at 125 to 130°C. for 30 minutes. The strips were allowed to cool
and were placed in a tray to be dyed over-night (16 hours) at room
temperature. After immersion in the dye solution.;{ the strips were
rinsed in two consecutive rinses with 5 percent acetic acid, each rinse
for 6 minutes. Then the paper strips were rinsed in a fixing solution?
for another 6 minute period. Excess fixative was allowed to drain from
the strips. The strips were dried carefully between two sheets of clean
paper towelling after'which.they'were oven dried for 15 minutes at 120
to 130°C.

The buffer solution used for the electrophoretic yolk protein
analysis was a diethylbarbiturate (barbital) buffer of pH 6.8. The
buffer was made by dissolving 10. 309 grams or sodium dietkwlbarbiturate
and 1.8419 grams of dietbylbarbituric acid and diluting to 1 liter with
distilled water. This buffer gave a solution which was 0.05 M in sodium
diethylbarbiturate and 0.01 Miin diethylbarbituric acid.

c) Paper strip analysis

Some of the stained filter strips were scanned on a
Spinco Mbdel RA.Analytro1 recording scanner and integrator. The distance
travelled by eaCh.protein was determined by'measuring the distance of the
recorded peaks from.tho origin. ‘Distribution of yolk proteins was

determined quantitatively after cutting each strip into several parts.

 

l/ The dye solution contained 0.10 gram of bromophenol b1ue, 50.0
grams zinc sulfate (7320) and 5 percent acetic acid to make 1 liter.
In order to speed up solution of the dye, a few milliliters of
alcohol were added before dissolving the zinc sulfate in acetic acid.

y 3.0 grams of sodium acetate (3 1120) were diluted to 1 liter with
5 percent acetic acid.

15
Each part of the paper strip was placed in a 100 milliliter beaker and
then 25 milliliters of 0.01 N sodium twdroxide solution were added in
each beaker. The color was extracted for one hour with fmquent swirling.
After color extraction, the solution was mixed well and the absorbance
measured at a wave length of 590 m )1 in the Beckman Model B spectro-

photometer.
Preparation of EgLYolk Proteins

a) lipovitellin
The lipoprotein, lipovitellin, was prepared by a modification
of the method of Alderton and Fevold (1916) . After removing all adhering
albumen and chalazae, 12 yolks were pooled for analysis. Removal of the
albumen was aided by rolling each yolk on cheesec10th, and the chalazae
were removed with tweezers. The yolks were well mixed with tirice their
weight in distilled water, and the diluted yolk emulsion was centrifuged
in a Servall centrifuge, at full speed, for 30 minutes. The precipitate,
lipovitellin, had a waxy, light-yellow appearance. After dissolving
the precipitate in a 5 percent sodium chloride solution, the lipovitellin
mixture was filtered through cheesecloth to remove foreign materials such
as vitelline membranes or chalasae not previously removed. The filtrate
was placed in cellophane tubes and dialyzed with distilled water in a
walk-in refrigerator (temperature at 2 to 3°C). When dialysis was
complete, (no chloride ion detected in dialysate,) the lipovitellin
solution was shell frozen, lyophilised, weighed and stored at .30 to
40°C.
b) lipovitellenin
This yolk protein was prepared by the method described by

Evans and Bandemer (1957). The supernatant from the lipovitellin preparation

16

was dialyzed in a refrigerator at 2 to 3°C. until the dialysate was free
of chloride ions. The pH of the mixture was adjusted to 6.2 with
sodium tudroxide and the mixture was centrifuged at room temperature for
30 minutes and allowed to cool in a walk-in refrigerator to 2 to 3 0C.
Cooling the mixture solidified the oily lipovitellenin precipitate which
had risen to the top of the centrifuge tubes during centrifugation.
Cooling the precipitate provided a clear-cut separation of the solid and
liquid fractions. The lipovitellenin was a dark yellow, fatty-looking
solid. The precipitate was dissolved in 5 percent sodium chloride and
dialyzed to remove the chloride ions. After complete dialysis, the
lipovitellenin mixture was shell frozen, lyophilized, weighed and stored
at -30 to 490°C.

c) Livetin

The filtrate remaining after centrifugation of the lipo-
vitellenin mixture contained the livetin fraction. This fraction appeared
as a mlky-yellow, somewhat opalescent-like solution. After complete
dialysis, the solution was shell frozen, lyophilized, weighed and stored
at -30 to 440°C. Since the experimental analyses in this study did
not involve this protein, no attempt was made to purify it.

The above separation for lipoproteins was used for both
normal and mottled yolks. The two lots of yolks were analyzed separately,
so as to complete the whole separation procedure in as short a time as
possible and thereby reduce loss of lipoproteins due to oxidation or

other changes.
Lipid Extraction of Lipoproteins

The lipovitellin and lipovitellenin fractions of both the

normal and mottled yolks were used for the extraction of total lipids.

 

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1?

Duplicate 10.0 gram samples of each freeze-dried lipoprotein
were extracted for 30 minutes in 125 milliliter glass-stoppered Erlenmeyer
flasks by shaking with three 50 milliliter portions of peroxide-free
diethyl . ether. The extraction was carried out at room temperature and
as much as possible under a nitrogen gas atmosphere. Immediately after
extraction, the lipids were poured into tared 50 milliliter beakers and
the ether evaporated off under nitrogen gas on a controlled 60°C. hot
plate. At no time were the lipids in the beakers allowed to become
even slightly warm since occasional swirling of the beaker prevented
the bottom layer from becoming too warm. when the odor of ether was no
longer detected in the lipids, the beaker containing the lipids was
placed in a desiccator. With the aid of a vacuum pump the desiccator
was evacuated and then the desiccator was placed in a walk-in refrigerator
at 2 to 3°C. This desiccator evacuating procedure was continued until
the lipid weights remained constant.

The residue remaining from the ether extraction was extracted
again, this time using absolute ettwl alcohol. The residue was shaken
for 30 minutes at room tenperature using three 50 milliliter portions
of alcohol and kept as much as possible under a stream of nitrogen
gas. The same solvent evaporating system as used above with the ether
samples was carried out for these samples. Following evaporation, the
beakers were placed in a desiccator and evacuated until the weight of
the beakers containing the lipids was constant. The lipid samples were

stored at all times in a walk-in refrigerator held at 2 to 3°C.
Silicic Acid Column Chromium

a) Silicic acid preparation
Hallinckrodt's analytical reagent (100 mesh powder)

~ 7 v
.
f r ‘
V A
' N
V d'
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,‘ ‘
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,
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1‘

18

silicic acid suitable for chromatographic analysis was used throughout
this study. The general column chromatographic procedure used was
essentially the same as that outlined by Lea gt El. (1955), except
that no 'celite” was added to the silicic acid slurry. The silicic
acid was prepared by drying overnight at 110 to 120°C. and stored in a
moisture-proof brown glass bottle. Ten grams of the dried silicic acid
were mixed with 25 to 30 milliliters of chloroform-methanol 98:2 (v/v)
to make a thin slurry. Settling and draining of the column was aided
by applying slight nitrogen gas pressure.

b) Column and apparatus preparation

The columns used in this study measured 19 x 200 millimeters

and had a standard taper glass disk (fritted) at the bottom of the column.
The top of the column.was attached by a cork stopper to a separatory
funnel which in turn was connected to a tube from a nitrogen gas tank.
‘When the silicic acid slurry was added to the column, the reservoir
funnel was connected and a slight nitrogen pressure used to regulate the
flow rate at 1 to 2 milliliters per minute. It was washed with 50
milliliters of the solvent.

c) Addition of sample to column and solvents systems used

After the column.was washed with the initial 50 milli-

liters of chloroformamethanol 98:2 (v/v) and when the level of this
solvent reached the surface of the adsorbent, a sample of mixed lipid
containing 0.5 to 0.6 gram in 15 to 20 milliliters of chlorofonm-
methanol 98:2 (v/v) was added. The sample was allowed to flow slowly
on to the column without disturbing the surface of the adsorbant. The
sample adsorbed on the column.was washed with 100 milliliter portions
of the following solvents: chloroformsmethanol (98:2, v/v) to elute

triglycerides, sterols and sterol esters; chloroformpmethanol (70:30, v/v)

 

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....I.

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- - . . f \
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19
to elute cephalins; methanol-chlorofom-water (75 :20 :5 by volume) to
elute lecithins.

The solvent system mployed in the chromatography of a
mixed phospholipid sample in this study followed the procedure of Rhodes
and Lea (1957).

(1) Fraction separation and evaporation

After each fraction was collected, the solvents were
evaporated to a small volume on a 60°C. hot plate under 8 stream of
nitrogen. The fractions were transferred to. tared 30 milliliter beakers
and then evaporated to dryness. The beakers, containing the fractions,
were placed in a desiccator and the desiccator was evacuated. After
the samples had reached constant weight, they were weighed and diluted
with chloroform and transferred to glass-stoppered volumetric flasks

and stored at 2 to 3°C.

Meal Methods

a) Amino nitrogen
The quantitative ninlvdrin method described by Lea and

Rhodes (1954) was used for determining the amino nitrogen content of
the phospholipids and triglyceride fractions. Duplicate samples
containing 0.3 milligrams of lipid, dissolved in chloroform, were pipetted
into 5 x 5/8- pyrex tubes and the ehlorofon evaporated off under a
stream of nitrogen. The residue was taken up in 1 milliliter of methyl
cellosolve in which it dissolved quite readily. Two milliliters of
ninhydrin solution were added and each tube shaken and placed in a

Reagent contained stannous chloride, 0.016 gram per milliliter

in citrate buffer (pH 5). and ninhydrin, 0.4 gram per milliliter in

methyl cellosolve. The solutions were mixed, and allowed to cool.

On mixing these two solutions, a brown color appeared which slowly
faded to yellow when the reagent was ready for use. This reagent

was freshly prepared for each analysis.

r‘

20
water bath at 33°C. for 20 minutes. . When the tubes cooled, 7 milliliters
of methyl cellosolve were added and the contents of the tube mixed well.
The samples were allowed to stand for 10 to 15 minutes and the absorbance
of the samples was read on a Beclonan Model B Spectrophotometer at 570 m )1.
The samples were read against a blank containing the evaporated residue
from 0.5 milliliters chloroform.

Each time an amino-nitrogen determination was conducted a
standard curve was also mm. For purposes of this study, a standard
ranging from 0 to 6 micrograms was adequate. The following volumes of
the final standard solution]., (10 micrograms per milliliter) were added
to pyrex tubes: 0.1, 0.2, 0.3, 0.11», 0.5, 0.6 milliliter and made up
to a one milliliter volume with methyl cellosolve.

b) Total nitrogen 7

A modified micro-K3 eldahl method, developed in the
Agricultural Chemistry Department, Michigan State University, was used.

The sample in chloroform was pipetted into the digestion
flask and the chloroform evaporated off with the aid of low heat and
nitrogen. The size of sample used to provide accurate and valid results
was in the range of 30 to 50 milligrams of lipid. Following the chloroform
evaporation, the following substances were added to the digestion flask:

 

y Ten milliliters of Z-amino-ethanol (pH about 13) were weighed into
a 100 milliliter beaker. The beaker was placed in a cold water
bath and the pH of the 2-amino-ethanol adjusted to about 8.5
using approximately 15 to 20 milliliters of concentrated Indra-
chloric acid. Hydrochloric acid diluted 1 to 5 with water was
added dropwise to the Z-amino-ethanol to adjust the pH to exactly
5. This solution was added to a 100 milliliter volumetric flask
and diluted with distilled water. The correct concentration for
the standard curve was obtained by further diluting 5 milliliters
of the stock solution to 100 milliliters with water and then
diluting 0. 5 milliliter of this dilution to 50 milliliters with
distilled water (10 micrograms amino-nitrogen per milliliter).

21
l milliliter 10 percent copper sulfate, 2 milliliters concentrated
sulfuric acid, 0.5 gram potassium sulfate and several glass beads to
prevent bumping. ‘When all the black (carbon) materials had disappeared
and digestion was thought to be almost complete, 3 drops of "superoxal"
(hydrogen peroxide) was also added at three different intervals. The
blank sample contained the residue from.evaporation of chloroform.
When frothing appeared to be a problem a drop of capryl alcohol was
added to the digestion flask prior to heating. The distillation was
carried out in a closed steam distillation system. The receiver flask
contained 20 milliliters of 2 percent boric acid and 5 drops of bromo
creosol green (0.1 percent bromcreosol green in alcohol). The boric
acid solution containing the distillate (ammonia) changed the color
of the solution from olive green to a blue shade. Titration of the
final distilled solution from blue to green was done with 0.0142 normal
sulfuric acid. A sample calculation shows the procedure for determining
the nitrogen content.

Milliliters of sulfuric acid X equivalent weight of nitrogen (1’4)= grams of nitrog
1000

 

Grams of nitrogen X 100 = percent nitrogen
Grams of sample

 

One (1) milliliter of 0.0142 normal sulfuric acid was equivalent
to 0.198 milligrams of nitrogen. From this relationship the weight or
percent of nitrogen in the original sample was calculated.

c) Phosphorus

The method of Fisk and Subbarow as described by Hawk,
Oser and Summerson (19h?) was employed to determine the phosphorus
content of the triglycerides and phospholipids.

Samples containing 0.7 to 0.9 milligram of lipid were

transferred to pyrex test tubes graduated at 10 milliliter, and the

22

solvent was evaporated under a stream.of nitrogen. To each tube was

added 0.6 milliliter of 1:1 sulfuric acid and several boiling chips,

and the tube was heated on a micro burner until the material appeared

charred. The tubes were removed from the microburner and 1 drop of

30 percent hydrogen peroxide was allowed to drop on the reaction

mixtures followed by heating again. The hydrogen peroxide additions

were repeated until the contents became colorless (usually 5 to 7

drops of hydrogen peroxide were required). After clearing, digestion

of the mixture was carried on for 3 minutes. When the tubes had

cooled, 3 milliliters of distilled water were added to each tube and

then the mixture was again brought to a boil to decompose pyrophosphate.
After the samples had cooled, 1.2 milliliters of 5 normal

sodium.hydroxide (saturated sodium.hydroxide diluted 1:1.with distilled

water) were added and the samples were mixed thoroughly. One milliliter

of molyhdate reagent was added and the mixture shaken thzroughly.

Then 0.“ milliliter of aminonaphthosulphonic acid reagent was added

 

5] Twenty-five grams of reagent grade ammonium.mb1ybdate were dissolved
in about 200 milliliters of distilled water. Five hundred milliliters
of 10 normal sulfuric acid were placed in a 1 liter volumetric flask,
the molybdate solution added and diluted with washings to the liter
mark with distilled water. This solution was stable indefinitely.

é] One hundred and ninety-five milliliters of 15 percent sodium
bisulfite solution were added to a glass-stoppered cylinder and
0.5 gram of l,2,4-aminonaphtholsulfonic acid was added. Then 5
milliliters of 20 percent sodium sulfite were added and the cylinder
shaken until the powder dissolved. (15 percent sodium bisulfite:
to 30 grams of sodium.bisulfite in a beaker, 200 milliliters of
distilled water were added from a graduated cylinder and stirred
until the powder dissolved). (20 percent sodium sulfite: 20 grams
of anhydrous sodium sulfite were dissolved in distilled water
and diluted to 100 milliliters).

23
and the volume in each tube made to 10 milliliters with distilled water.
After the samples were allowed to stand for 10 to 30 minutes, their
absorbance was read in the Beclouan Model B Spectrophotometer at 660 m )1.
A blank containing 1 milliliter of chloroform was treated in the same
manner as the samples.

The standard solutions were treated in the same manner as
the samples except that to each tube 0.6 milliliter of sulfuric acid
(1:1) was added and the solution evaporated on a microburner, until
white fumes appeared in the tubes. Then one drop of hydrogen peroxide
was added to each tube and the procedure continued as for the samples.

d) Iodine number

The method of Trappe (1938) was employed since it was
applicable to micro amounts of lipids. Samples containing 0.5 to 0.8
milligram of lipid were transferred to 50 milliliter glass-stoppered
Erlenmeyer flasks. Two milliliters of 0.01 normal bromine solution

é/ .
in sodium bromide saturated methyl alcohol were added to each sample.

 

Z] Exactly 0.351 gram of pure dry monopotassium phosphate was dissolved
in distilled water and transferred quantitatively to a 1 liter
volumetric flask. Ten milliliters of 10 normal sulfuric acid
were added and the solution was made up to 1 liter with distilled
water. This solution contained 0.4 milligrams of phosphorus in 5
milliliters. It was found to be stable indefinitely. Five milliliters
of this stock phosphate standard, containing 0.4 milligrams of
of phosphorus were added to a 50 milliliter volumetric flask, made
up to volume with distilled water and mixed thoroughly. One
milliliter of the last solution contained 0.008 milligrams of
phosphorus. To establish a standard curve 0, 1, 2, 3, 4, and 5
milliliter portions of this last solution were added to 10
milliliter graduated pyrex test tubes.

8/ Bromine in sodium bromide saturated methyl alodnl was made in the
following way: one milliliter of bromine was added to 100
milliliters of methanol saturated with sodium bromide. Then 2.5
milliliters of this were made to 100 milliliters with sodium
bromide saturated methanol.

24
The flasks were stoppered and allowed to stand in the dark for 4
hours with’occasional swirling. To each flask was added 1.0 milliliter
of 2 percent potassium iodide solution and two drops of freshly pre-
pared, 1 percent starch solution. A solution of 0.002 normal sodium
thiosulfate solutiongjwas used to titrate this sample. The blank
sample co ntained 1 milliliter of chloroform. A sample calculation
for determining the iodine number is shown below.

Iodine number = Titration difference I 0.2538 X 100 X Normality of thiosulfate
Weight of lipid .002

 

e) Choline
The choline content of the lecithin fraction was determined

by the method of Enth gt a]: (1944). The determination of choline
by this method required at least 20 to 30 milligrams of lipid material
per sample. Each sample was transferred to a 50 milliliter Maker
and the solution made basic to phenolphthalein with saturated barium
Ivaroxide, and then 25 milliliters more saturated barium hydroxide were
added. The beakers were covered with watchglasses and Ivdrolyzed for 2
hours at about 100°C. on a controlled taupe rature hot plate. Some
reduction in the volume of the samples did occur; however, distilled
water was added occasionally to prevent the samples from becoming dry.
When hydrolysis was complete, the samples were neutralized with glacial
acetic acid and filtered directly into 50 milliliter centrifuge tubes

using Whatman No. 42, 7.0 centimeter filter papers. The beakers were

 

2/ The sodium thiosulfate solution was made by dissolving 24.83
grams of sodium thiosulfate in distilled water and making to 1
liter, and further diluting 10 milliliters to 500 milliliters with
distilled water. The thiosulfate solution was standardized using
5 milliliters of standard potassium iodate (.3568 grams per liter),
1 gram potassium iodide, 10 milliliters of distilled water,
0.5 milliliters of glacial acetic acid and 1 milliliter of 1

percent starch.

25

rinsed with distilled water and the washings added to the filter paper;
however, the combined washings did not exceed 25 milliliters. Five
milliliters of a 2 percent solution of reineckate salt in methanol were
added to the filtrate and the samples were allowed to stand overnight
in a refrigerator at 2 to 3° . to insure complete precipitation of

the choline reineckate. The samples were centrifuged at 2800 revolutions
per minute for 15 minutes to pack the precipitate. After decanting the
supernatant, the precipitate was washed with cold etlwl alcohol and
then centrifuged for the same time as previously outlined. me
supernatant was again discarded and the precipitate washed with cold
ethanol and centrifuged as before. After centrifagstion, the supernatant
was decanted and the tube inverted on clean towels to drain the emcess
ethanol. me washed choline reineckate precipitate was dissolved in

a small amomt of acetone and then passed through What-Ian No. 1.2 filter
paper and collected in 25 milliliter voluntric flasks. After rinsing
eat the centrifuge tubes with acetone, the washings were also passed
through the filter paper. he filter paper was washed with acetone
until no choline reinechte precipitate reamined on the filter paper.
The filtrate was nde up to the 25 milliliter mark of the voluntric
flasks with acetone and read in the spectrophotometer at 525 mm. The
blank contained acetone only. A standard curve was obtained from
results of the following standard preparation. me-tenth gram of a
reagent grade choline chloride was nde up to 10 milliliters with
distilled water giving a solution containing 10 milligrams per milli-
liter. men 5 milliliters of this latter solution were diluted to 50
milliliters (l milligram per milliliter). Aliquots of l, 2. 3, h. 5.
6, 7, 8 and 9 milliliters of the 1 milligram per milliliter solution

were transferred with a pipette to 50 milliliter beakers. The solutions

26
were made basic to phenolphthalein with saturated barium hydroxide
and the remainder of the procedure was continued as with the lipid
samples.
f) Total cholesterol
The method of Schoenheimer and Sperry as cited in Hawk,
Oser and Summerson (1947) was employed to determine the total cholesterol
content of the triglyceride fraction. Samples containing 3.0 to 5.0
milligrams of lipid.were transferred to 15 milliliter centrifuge
tubes. After the chloroform.was evaporated under nitrogen, three
milliliters of acetone-ethanol (121) were added to dissolve the
residue. Three drops of potassium.hydroxide solution (10 grams potassium
hydroxide in 20 milliliters of distilled water) were added from a
dropping bottle. A stirring rod was added to each graduated
centrifuge tube and the contents stirred vigorously; The centrifuge
tubes, with the stirring rods, were placed in a preserving jar con-
taining a layer of sand about 3 centimeters deep, previously'heated

to 45°C. The cover was placed on the jar and then placed in an
incubator at 37 to hoOC. for 35 minutes(included.was 5 minutes for

temperature equilibration). After incubation, the tubes were removed
to a wire tube rack and allowed to cool to room temperature. The
stirring rods in the tubes were raised momentarily and 3 milliliters
of acetone-ethanol were used to wash the rods and sides of the tubes.
After mixing, one drop of phenolphthalein solution was added to each
sample and then 10 percent acetic acid was added drop by drop until
the red color just disappeared and then 1 drop in excess. Three
milliliters of digitonin solution (0.4 gram of digitonin was dissolved
in 100 milliliters distilled water and the solution filtered) were

added to each tube. After a thorough stirring, the tubes, with

27
stirring rods, were placed in a quart size preserving jar, covered
tightly and allowed to stand at room temperature overnight.

The tubes were transferred to a test tube rack and the
samples stirred gently to free the particles of precipitate from the
tube walls. The rods were removed and placed on a numbered.wire rack
being very careful not to lose any precipitate particles. The samples
were centrifuged for 15 minutes at 2800 revolutions per minute. After
decanting the supernatant, the tubes were inverted and allowed to drain
for a few minutes on a clean.paper towel. The stirring rods were
replaced in the tubes and the walls of the tubes washed with 1.5 to
2.0 milliliters of acetone-ether (1:2) from a dropping bottle. The
samples were thoroughly'mixed, centrifuged for 5 minutes, and
after decanting off the supernatant, the precipitate was washed with
diethyl ether. The stirring rods were added to the samples and set
aside until ready for color development.

A layer of sand about 3 centimeters deep was placed in a
shallow pan and heated to 110 to 115°C. in an oven. The tubes
containing the washed cholesterol digitonide precipitate were placed
in the pro-heated sand and the pan.was returned to the oven for 30
minutes. Two milliliters of glacial acetic acid were used to wash
the stirring rod and walls of the tube while the tube was still in
the hot sand. The samples were stirred well and allowed to stay
in the sand for 2 minutes more (no longer). The tubes, including
the blank and standard, were placed in a water bath at 25°C. in
the dark and allowed to come to temperature equilibrium. Four

milliliters of cold acetic anhydride-sulfuric acid

28

10 / ‘
reagent_- (freshly prepared) were added to each tube, mixed well

and returned to the water bath. The tubes were allowed to stand 27
minutes in the dark and read after 30 minutes in the spectrophotometer
set at 625 m.n.

No standard curve was required, however, the absorbance of
the samples was read against a blank and a standard cholesterol solutiongl/
containing 0.2 milligrams per milliliter. The blank contained 2
milliliters of glacial acetic acid. Calculation for the cholesterol
content of the samples was done by the following procedure:

‘Qensity'of unknown X .2 X 100 _= milligrams total cholesterol
Density of standard weight of sample per 100 milliliters

 

__/ A glass-stoppered, graduated cylinder was placed in a cold room
at 2-3° C. along with the acetic anhydride bottle and concentrated
sulfuric acid. Twenty milliliters of chilled acetic anhydride
'and 1 milliliter of concentrated sulfuric acid (a little at a
time) were mixed together by placing the stopper on the cylinder
and shaking vigorously for a few moments. The reagent was not
prepared until ready to be used and at all times kept chiIbd.

ll] One hundred milligrams of pure cholesterol were dissolved in 50
milliliters of glacial acetic acid by'warming on an electric hot
plate and stirring, then transferring this mixture with the
rinsings to a 100 milliliter volumetric flask. The solution was
diluted to 100 milliliters with acetic acid. For making up the
working solution, the latter stock solution.was warmed to
room temperature and 5 milliliters of this were diluted to 50
milliliters with glacial acetic acid (contained 0.2 milligrams
per 2 milliliter).

29
g) Fatty acid.methy1 esters
Samples from the silicic acid columns were transferred

to 100 milliliter beakers and saponified at room temperature by standing
overnight in 10 milliliters of ethanol containing 1.5 grams of potassium
hydroxide (12 grams potassium hydroxide and 80 milliliters ethanol).
Twenty-five milliliters of distilled water were added to dissolve the
mixture and then 0.75 milliliters concentrated sulfuric acid in 5
milliliters of distilled water (6.0 milliliters sulfuric acid + 40
milliliters water) were used to acidify the samples. The solutions
were allowed to cool and extracted three times in a separatory funnel
with peroxide free diethyl ether (using 20 milliliters ether for each
extraction). The combined extracts were evaporated in a beaker on a
hot plate at 60°C. under a stream of nitrogen. After a large part
of the ether had evaporated, the samples were transferred to glass
stoppered volumetric flasks and made to volume with ether.

DiazomethaneEEKwas added dropwise to 15 to 20 milligrams of
the mixedfatty acids in cold ether to convert the fatty acids to the
methyl esters, and ether was removed under a stream of nitrogen. The
residues were taken up in 100 microliters of pure benzene and transferred

to a vial and tightly stoppered until ready for use.

 

l_2_/ Preparation of Diazomethane: To a distilling flask was added
0.5 gram.potassium.hydroxide, 0.8 milliliter water and 2.5

milliliters of ethanol. After mixing, the flask was heated to
65°C. Two receivers in ice water were connected through a cold

water condenser. One receiver contained 3.0 milliliters of ether.
To the distilling flask was added a solution of 2.15 grams of
'Diazold” in 13 milliliters of ether. Two more milliliters of
ether were added and the rate of distillation.was regulated to
equal the rate of addition of the 'Diazold” solution.

30

Aliquots of 5 to 10 microliters of fatty acid methyl esters
dissolved in pure benzene were injected into the gas-liquid chromato-
graphic apparatus for separation of the fatty acid methyl esters. The
determinations were conducted on a Wilkens Aerograph Model A-90-C
gas-liquid chromatographic instrument with a weston, 1 millivolt single
point recorder. The fatty acid.methyl esters were separated on a
diethylene glycol succinate column operated at 200°C. with helium
flow rate regulated at 100 milliliters per minute.

The percentages of fatty acids found in each sample were
proportional to the areas under the peaks on the graph and the areas
‘were calculated in square millimeters. The area under each peak was
measured by'multiplying the height of the peak by the width at a point
middway between the base and top of peak. The percentage of each
fatty acid was obtained by dividing the area under each peak by the ’
sum of the areas under all the peaks. Percentage of fatty acids in
a sample were calculated as follows:

Height of ‘Width of curve Area under Percent of

 

Fatty acid curve at midgpoint curve fatty acid
mm. nmn sq. mun
Myristic 5 2 10 0.b
Palmitic 193 3 675 29.2
Palmitoleic 21 5 105 4-5
Stearic 35 5 201 8.7
Oleic lhB 6 926 “0.0
Linoleic 53 7 398 17.2

 

Total 23l3 100.0

RESULTS AND DISCUSSION
Photographs of Nettled Yelks

Eggs containing yolks varying in incidence and intensity
of mottled areas were obtained from.hens fed Nicarbazin and from hens
fed a basal ration. Each egg observed was classified and given a
visual score of O to 5, depending on the presence and appearance of
the mottled areas. The typical appearances of a number of mottled
egg yolks are shown in Figures 1 to 5. Yolks with mottled scores
of 0 to 1 appeared similar, thus no photograph of an egg yolk with a
score of 1 is included. Figure 1 is a photograph of a slightly
mottled yolk classified.with a visual score of 2; Figure 2, a moderately
mottled yolk with a scorecf 3; Figure 3, a severely mottled yolk with
a score of #g and Figure 4, a very severely mottled yolk with a score
of 5. A series of four yolks - one normal and three very severely
mottled - are shown in Figure 5. One of these mottled yolks was
obtained from a commercial farm flock and the others were laid by

hens in this experiment.
Incidence of Mottled Yolks

a) Newly laid eggs
A direct relationship was found between the number of
mottled yolks and the length of time the birds were fed Nicarbazin-
supplemented rations. One-to 2-day 01d eggs from hens in Group 3 fed
for 10 to 11 days were moderately to severely mottled while hens in
Group 2 had a smaller percentage of moderately mottled yOlks (Tables

1 and 2). At different intervals, all eggs produced during a 2-day

31

32

 

 

44“.; m‘

Figurel. Photograph ofaneggwitha slightlyuttledyelk
given a visual mettling score of 2.

33

 

 

 

Figurez. Photographer“ euwithamederatelynottledyelk
given a visual notth score of 3.

 

 

 

Iigure3. Photon-aphefameggwithauvmly-ottledyolk
given a visual nottling score of ‘l'.

35

 

 

 

Figure ‘1'. Photograph of an egg with a severely mottled yolk
given a visual Iottlimg score of 5.

36

.0
so 1

Figure 5. Photograph of 1; egg yolks.

 

 

Yolk upper left - very severely mottled obtained in present study.
Iolk upper right - very severely mottled obtained in present study.
Yolk lower left - normal obtained in present study.

Iolk lower right - very severely mottled obtained from cos-ercial flock.

37

 

 

 

 

 

 

 

 

 

 

 

 

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mm o: n ma m N: on NH 0 me on NH :m-mm
m: ‘ on 5 3H 0 om om ma 0 om om ma mauma
0: mm mm ma 0 aw as ea 0 0 so ma manna
mm m mm ma 0 am on ma 0 3H om 3H HH-OH
0 ma Hm HH 0 o ooa HH 0 o ooa :a my:
0 m mm NH 0 o ooa Hm o ma no ma m-~

geochom ammo.oz pcoohom mwwo.oz acooaom ammo.oz when

mum, mum fine muzl. mum Hap; Imwa, m-~ Hro eosoom

oooom mafiaoooz ogoom mneappozv oncomvmcaappo: mcaoomm

pcoohom mNHo.

pzoohom Hoo.

Houpnoo

 

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naoaomnnom :Humnhooaz

 

mm mma on u :fismnnmofiz .
Ho xaow ooappoz scam ca mwmm.mo ommpcoonom

 

.H wands

38

1
Table 2. Percentage of Fresh Eggs‘lfrom Hens Fed Nicarbazin to 132
Dale'with Mederate to Vegz;Severe‘Mottled Yelk Scores

Nicarbazin sgpplement

 

 

 

 

Control 0.001 percent 0.0125 percent
Mottled yolk MOttled yolk Mottled yolk
Time on Feed Total score of 3, Total score of Total score of
(days) Eggs 4. 5 Eggs 3.4.5. Eggs 3.“.5

NUmber No. Percent fiifiber No. Percent Number No. Percent
2-3 15 0 21 O 12 l 8
#~5 14 0 ll 0 11 l 9
10-11 1h 0 l7 3 18 13 10 77
15-16 13 0 17 l 6 15 8 53
18-19 15 1 7 15 1 7 14 9 64
23-24 12 ' o 12 4 33 15 13 87
131-132 13 O 11 l 9 7 a 58

 

1] Eggs broken 1 or 2 days after they were laid.

39
period were broken and the yolks observed and given a mottled yolk
score. The incidence and severity of mottled yolks increased in
eggs from hens in Group 3 fed the supplemented ration for 10 days
or more. Only minor differences were apparent in yolk scores
between eggs from.hens in Group 1 and Group 2. These results agree
with data reported by Polin e_t_ 51. (1957), Baker gr; 31. (1957),
'Weiss (1957), and Polin.gt a}. (1958).
b) Storage eggs

In Tables 3 and h are reported results which show the
influence of storage and drug level in the feed on incidence of
mottled yolks. Storage of eggs from hens in Groups 2 and 3 increased
the number of mottled yolks compared to the control group, and this
increase in number was most noticeable in yolks from hens in Group 3.

‘When eggs obtained from.hens fed Nicarbazin were stored
up to l3 months, more mottled yolks were observed (Table 5). Even
some eggs from hens fed a basal ration showed a substantial increase
in percentage of mottled yolks.after storage for long periods. The
mottled yolks which were observed in stored eggs from hens of Groups
2 and 3 appeared to be similar to the condition described by Aimquist
(1933) who reported that ”blemished" yolks occurred in storage eggs
of hens fed diets containing cottonseed meal or oil. Polin gt_g}.
(1957). Weiss (1957). Baker .e_t 31; (1957). Meloughlin 2:2 5;. (1957)
and van Tienhoven gt 2;;‘1958) have shown that the incidence of mottled
yolks may be drastically increased by addition of 0.0125 percent
Nicarbazin to the ration of laying hens.

c) Egg production and egg quality

Hens in Group 3 laid 25 percent fewer egg; while‘those

40

Table 3. Percentage of Eggs in Each Mottling Score, from Hens Fed
ngfsgeggygevels of Nicarbazin, After Storage at 35°F., Up

 

r

 

r

Nicarbazin supplement

 

 

 

 

 

 

 

 

Control .001 percent .0125 percent

Storage Mottling score Mottling score Mottling score
Time 0-1 2-3 1-5 0-1 2.3 ‘15 0-1 2-3 '47::
Days . Percent of eggsf _ Percent of eggs. Percent of eggs
0 85 15 0 55 M5 0 M3 #3 ~1h
6-7 85 15 0 50 36 14 33 17 50
18-19 100 0 0 82 18 0 17 50 33
49-50 100 0 0 100 0 0 0 25 75
82-83 100 0 0 79 21 0 9 9 82

106-107 82 18 0 7h 26 0 0 O 100

 

#1

Table 4. Percentage of Eggs from Hens Fed Nicarbazin for 6 or
‘More Days with Moderate to Severely'Mottled Yblks

 

 

 

Time in storage Nicarbazin supplement
at 35 F. Control 0.001%7DNC 0.0125%’DNC
Days Percent Percent Percent
o o 9 55
6-7 5 29 66
18-19 0 10 66
49-50 0 V 0 88
82-83 0 17 91

106-107 0 O 100

 

42

Table 5. Percentage of Eggs with Yolk Mottling Scores from Moderate
to Very Severe from Hens Fed Nicarbazin 7 Weeks or More

and Stored Up to 13 Months

Nicarba zin supplement

 

 

Time in storage Time on

at 35°F. feed Control 0.001% DNC 0.01253’3 DNC
Months Weeks Percent Percent Percent

13 7 4 146 92

12 11 3 63 97

ll 15 15 73 97

10 19 5 77 98

 

1P3

in Group 2 laid about the same number of eggs as the control group.
No difference in albumen quality was found between the eggs of the
hens fed a basal ration and those fed the supplemented rations.

The decrease in total number of eggs laid by hens in
Group 3 is in agreement with the data reported by Weiss (1957), Baker
33; 31; (1957), and McLoughlin gt _a_1_. (1957). Others (Weiss, 1957,
Baker 92 a1. 1957, and McLoughlin 93: 31. 1957) have also noted no
adverse effect on albumen quality of eggs from hens fed Nicarbazin

up to levels of 0.0125 percent.

Protein Distribution in.Yolks of Normal and Mottled Eggs

Protein distribution data for yolks of normal and mottled
eggs as determined by filter paper electrophoresis are presented
in Table 6 and Figures 6 to 10.

Lipovitellin content of mottled yolks decreased as the
mottled yolk score increased, whereas, the lipovitellenin-lipovitellin
ratio increased showing that lipovitellenin increased relative to
lipovitellin. The ovalbumin content of the mottled yolks increased
until more than 20 percmt of the yolk protein was ovalbumin in the
severely mottled yolks. Electrophoretic mobility of the lipovitellenin
increased as the degree of yolk mottling increased as shown by
increased distances between the lipovitellin and lipovitellenin peaks.
The distance between the lipovitellin and lipovitellenin in normal
yolk was 9 millimeters while the distance between peaks of the mottled
yolk was 1“ millimeters. The composition of normal yolk protein

agreed with data presented by Evans gt_al. (1958).

Dipoprotein Isolation

Mbttled yolks contained less lipovitellin and lipovitellenin

Table 6e

   

- ”hfi —. -..._—-a- ---.. *hwv »~-.

—— _—-—.._. H-.. “h...

 

Mottling score

Percentage Protein Composition of Egg Yelks with Different
Degrees of Mottling, as Determined by Paper Electrophoresis

hm

 

 

 

Protein Fraction 0 2 3 4 ~5
Percent

Unidentified protein (a) 111.1 13.3 13.7 16.7 12.6

Lipovitellin (b) 39.1 38.4 33.9 2n.9 25.2

Lipovitellenin (c) 38.5 38.5 39.9 37.9 33.2

Livetin (d) 8.3 7.0 8.5 7.5 6.5

Ovalbumin (e) - - 2.8 4.0 12.9 22.5

 

 

 

 

 

Figure 6. Filter paper strip and Analytrol scanner-integrator graph
efthestrip fernomleuyolkproteins.

a) water soluble protein
tellim

b) lipovi
o) lipovitellenin
1!) 117““

e) «albumin

 

obc d ”e

 

Figure 7. Filter paper strip and Analytrol sooner-integrator graph
of the strip for slightly mottled egg yolk proteins.

a) water soluble protein
b) lipovitellin

c) lipovitellenin

11) until!

e) evalbunin

4?

on ‘d -e

 

 

obc d e

flgms.mterpeperstripesdlnflytre1soamr-istegretergreph
of the strip fer moderately mottled egg yolk proteins.

 

 

abc d s

Figure 9. Filter paper strip and Analytrol soenner-intepster graph
eftbe strip fore severely-ottledeggyolkproteins.

a) water soluble
b) 11mm
o) lipovitellenin
d) livetin

e) smelt-in

 

 

abc d s

Figure 10. Filter paper strip and AnsJytrol scanner-integrator graph
of the strip for very mottled egg yolk proteins.

a) water soluble protein

50
but more water soluble protein than normal yolks (Table 7). Table 8
shows that by calculation of the data, mottled yolks contained
0.92 grams more water per yolk than normal egg yolks. This value was
somewhat lower than that reported by Polin (1957). The difference
in the percentages of the lipoproteins found in Tables 6 and 7 are
explained by the fact that the electrophoresis data in Table 6
measured onky'the protein part of.the lipoprotein while the isolation

data in Table 7 included both protein and lipid of the lipoprotein.

Lipoprotein Lipid Extraction

As shown in Table 9, the total lipid extracted from the
lipovitellin of mottled and normal yolks was about the same. More
of the lipids were extracted by ether than by alcohol from the
lipovitellin and lipovitellenin. Lipovitellenin isolated from
mottled yolks contained 6.3 percent more lipid than lipovitellenin
from normal yolks. Lipovitellin contained about 31.7 percent lipid
compared to 21.3 percent in that isolated by Evans and Bandemer (1961).
Lipovitellenin isolated from normal yolks contained about 77.“ percent lipid
compared to 82.0 percent in that of Evans and Bandemer (1961).
Differences in source of eggs and methods of protein isolation and
lipid extraction might account for these variations and those reported

by Evans and Bandener (1961).

Lipid Composition of Lipoproteins

 

No differences were observed between normal and mottled
yolks in triglycerides, cephalins and lecithins in the ether ex-
tracted lipids of lipovitellin and lipovitellenin (Table 10). The
alcohol extracted lipids of lipovitellin of mottled yolks contained

less cephalins and more lecithins than that of normal egg yolks.

51

Table 7. Percentage Yield of Lipoproteins of Normal and Mottled
Egg Yelks

 

 

 

 

 

 

 

TREATMENT
Control Mottled
Whole yolk Dried yolk ‘Whole yolk Dried yolk
(percent) (percent) (percent) (percent)
LipOVithlin ll. 5 23 e 6 9 e 9 21 e 8
Lipovitellenin 35.5 72.6 31.7 69.“
water soluble
protein 1.9 3.8 3.9 8.?
Tom]. ’48 e 9 1+5 0 5

Water 51.1 54.5

 

     

 

   

52

 

 

 

 

11.1.51. 0.9 - Wi “M1 ed} - ' Yolk:
Treatment
Control Mottled
gm. per dozen eggs
Whole yolk 216.3 223.2
Lipovitellin 29.9 22.3
Lipovitellenin 76.7 70.5
‘Water soluble protein 4.1 8.9
Water 110 . 6 121. 6

 

53

Ieble ?.#_Percent Ligid 1n Normal and Mottled Yo1k‘L1-o-roteine

mt

   

 

 

 

Lipovitellin Lipovitellenin
Normal 'Mbttled Normal Mottled
Percent iPercent Percent Percent
Ether extract 18.9 17.1 59.7 67.8
Alcohol extract 12.8 14.5 17-7 15-9
Total lipid 31.7 31.6 77.“ 83.7
Protein 68.3 68.4 22.6 16.3

 

 

Total Lipoprotein 100.0 100.0 100.0 100.0

 

54

Table 10. Lipid Distribution from Lipovitellin and Lipovitellenin of
Normal and Mettled 10153

 

 

 

 

 

Triglycerides Cholesterol Ce halin Lecithin .
Normal Mottled Normal Mottled Norma? Mottled NormaI Mottled
Percent

Ether Extracted Lipids

Lipovitellin 81.0 81.0 5.7 5.0 5.1 4.7 8.2 8.9

Lipovitellenin 84.3 82.8 3.6 0.5 0.9 “.2 7.8 8.6

Alcohol Extracted Lipids

Lipovitellin 18.3 18.2 3.1 3.9 #2.6 31.9 36.0 #7.0

Lipovitellenin 12.3 5.0 2.6 1.7 49.5 52.h 35.6 00.9

 

55
No differences were observed between normal and mottled yolks in
cholesterol in the ether and alcohol extracted lipids of lipovitellin.
More cholesterol was found in the ether extracted lipids of lipovitellenin
of mottled yolks than that of normal yolks. .Less cholesterol was
obtained in the alcohol extracted lipids of this lipoprotein from
mottled yolks than that of normal yolks. The cholesterol content of
lipovitellenin from.normal yolks was similar to that found by MoIndoe
(1959). The alcohol extracted lipids of lipovitellenin from mottled
yolks contained more cephalins and lecithins but less triglycerides
than that found in normal yolks. The decrease in cephalins in
lipovitellin of mottled yolks was not due to any contamination by
triglycerides or lecithins during separation on the columns. Cal-
culation of the phosphorus content showed that the triglyceride
fraction did not contain enough phosphorus to account for the
difference found in the cephalins.

The amount of nitrogen and phosphorus in phospho1ipids has
been used as a measure of their purity. Theoretically, both cephalin
and lecithin should contain approximately 1.7 percent nitrogen and
h.0 percent phosphorus. Inouye and Noda (1958) reported that their
sample of egg phosphatidylcholine contained 1.76 percent nitrogen and

3.87 percent phosphorus, whereas, Pangborn (1941) found 1.81 percent
nitrogen and 3.82 percent phosphorus for egg lecithin. Egg lecithin
prepared.bw'1evine and Chargaff (1951) contained 1.78 percent nitrogen
and 3.88 percent phosphorus. Differences in amino nitrogen, nitrogen

and phosphorus indicated that separation of the fractions on silicic

acid columns was fairly satisfactory (Tables 11 and 12). The choline

content of the lecithins of normal and.mottled yolks was similar,

however, the choline content was about 5 percent higher than that

56

 

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mm.m em.e mm.m H~.m
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23233 endgame

noodhoohHwane

 

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57

 

mo.HN :0.NN I I I I I I I I :m.am Ho.om I I I I I I I I ocaaono

 

 

 

Hm.& Hm.m mm.m Nw.m 0.0 0m. nN.m 00.N NB.N mn.N 0.0 0.0 mononmmonm
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PCOOHOA . RGOOHOA
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pomnpxm Hoaooaa somnpxm tease

3’1

cacoaaopa>omqq gone neaaaa mo gossamoaaoo .NH «anus

 

58
reported by Hanahan (1954). This difference may have been due to
differences in techniques used for the choline determination.

In Tables 13 and 14 are reported the iodine numbers,
distribution and degree of unsaturation of fatty acids of lipids
extracted from the lipovitellin and lipovitellenin of normal and
mottled yolks. The iodine number is defined as the number of grams
of iodine absorbed by 100 grams of fat or oil. Since the iodine is
absorbed by the double bonds in the molecule, the iodine number is a
measure of the degree of unsaturation of the lipid.

It is difficult to explain these data due to the incon-
sistencies of the observed iodine numbers and calculated iodine numbers
obtained from the fatty acid analyses. Furthermore, degree of un-
saturation of the fatty acids did not always agree with the iodine
number obtained. In general, that fatty acid analyses would be most
reliable for evaluating the data on differences between the normal and
mottled yolks.

Fatty acid gas chromatographic analyses showed more oleic
acid was found in triglycerides of ether extracted lipids of lipovitellin
from mottled yolks than normal yolks, but less stearic acid.was found
in cephalins from alcohol extracted lipids of mottled yolks than normal
yolks. Mottled yolks contained more oleic acid in the triglycerides
of ether and alcohol extracted lipids from lipovitellenin than normal
yolks.

The chemical characterization of the triglycerides and
phospholipids may have some merit; however, due to variability in
the data, it was difficult to state whether or not the chemical
differences which existed between the mottled and normal yolks were

significant.

59

 

 

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easeaoog casemmoo oooaeoowwwaee heeeaoog oaaeheoo oooaeoowwmehe

eooeexm Hosooae someoxm heaps

 

 

 

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eaaaoeaemmmq do needed as moaoe evade mo cosponsored: one eoapooaepoaa .oeooeez odaooH .mH canoe

 

60

 

 

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:aooaaoeaeoqu mo nomads ea ooaoe.hppoa no nonpoeeeoeee one ooaeehaeemaa .oeooeez oeaooH .ea canoe

SUMMARY AND CONCLUSIONS

Thirtyesix (36)'White Leghorn hens were placed at random
in a single hen laying cage battery in three groups of 12 hens each.
Group 1 was fed a control ration while Group 2 and Group 3 were fed
diets supplemented with 0.001 and 0.0125 percent Nicarbazin, respectively.
Nicarbazin was used to chemically induce mottled yolks in eggs from
hens fed the drug.

A direct relationship was observed between the time and
level of feeding the drug and the incidence of mottled yolks. 'When
eggs from hens in Group 3 were stored 10 to 11 days, a marked increase
in the number and.degree of mottled yolks occurred and.eggs from hens
in Group 2 showed a similar trend but fewer mottled yolks were observed.
Storage of eggs for 13 months from hens in Groups 2 and 3 caused a
further increase in number and degree of mottled yolks and a low
percentage of yolks from hens fed the basal ration were slightky
mottled. Hens in Group 3 laid 25 percent fewer eggs while hens in
Groups 1 and 2 laid about the same number of eggs; however, the
albumen duality of the three groups was similar.

Chemical and physical determinations of an exploratory
nature were used to ascertain what differences existed between mottled
and normal egg yolks obtained in this study; Electrophoresis analyses
showed a direct relationship between.mottled yolk score and the
proportion of ovalbumin in the yolks. Furthermore, the lipovitellenin
fraction of mottled yolks showed a change in.mobility'whbh did not
occur in nonmal yolks. This protein mobility difference was attributed

to a chemical or physical change in the composition of the lipoprotein.

61

62

Electrophoretic differences in the lipoproteins observed between
normal and mottled yolks were substantiated by similar differences
obtained in the percentages of isolated lipoproteins. Mettled
yolks contained less lipovitellin and lipovitellenin but more
water soluble protein than normal yolks.

Lipovitellin of mottled yolks and normal yolks contained
the same percentage of ether and alcohol extracted lipids but
lipovitellenin of mottled yolks contained 6.3 percent more ether
extracted lipids than normal yolks.

No differences were observed in the triglycerides, cholesterol,
cephalins and lecithins in the ether extracted lipids from lipovitellin
of mottled and normal yolks. Lipovitellin of mottled yolks con-
tained.more lecithin and less cephalin than normal yolks, but no
differences were observed in triglycerides and cholesterol between
the alcohol extracted lipids of normal and.mottled yolks.

Ether extracted lipids from.1ipovitellenin from mottled
and normal yolks contained the same percentage of triglycerides,
cephalins and lecithins but mottled.yolks contained.more cholesterol
than normal yolks. Alcohol extracted lipids from lipovitellenin of
mottled yolks contained less triglycerides and cholesterol but more
cephalins and lecithins than normal yolks.

Amino nitrogen,total nitrogen and phosphorus determinations
showed that the separation of the fractions on silicic acid columns
was fairly good. Choline determinations indicated that no differences
existed between the lecithins of normal and mottled yolks.

Fatty acid gas chromatographic analyses showed more oleic

acid was found in triglycerides of ether extracted lipids of lipovitellin

63
from.mottled than normal yolks, but less stearic acid was found in
cephalin from alcohol extracted lipids of mottled yolks than normal
yolks. Mettled yolks contained.more oleic acid in the triglycerides
of ether and alcohol extracted lipids from lipovitellenin than normal
yolks.

Due to variabilities in some of the chemical determinations,
a complete evaluation of the differences between mottled and normAl
yolks was not possible, however, other results in this study would
indicate that definite differences exist between the composition
of the lipoproteins and lipids of mottled and normal yolks.

Under conditions of this experiment, the appearance of a‘
mottled area on egg yolks from hens fed Nicarbazin was probably due
to the presence of ovalbumin and water. The variation in appearance
or intensity of the mottled areas on the yolk was probably caused by
the different proportions of water and ovalbumin present under the

vitelline membrane of the yolks.

REFERENCES

Alderton, G., and H. L. Fevold, 1945. Preparation of the egg yolk
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