THE INFLUENCE OF SPECIFIC FEEDS AND CERTAIN

PIGMENTS ON THE COLOR OF THE EGG

YOLK AND BODY FAT OF FOWLS.*
BY LEROY S. PALMER AND HARRY L. KEMPSTER.
(From the Dairy Chemistry Laboratory and department of Poultry
Husbandry, University of Missouri, Columbia.)

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(Received for publication, July 9, 1919.)

A previous paper1 has reported the success which was attained

in raising White Leghorn chickens from hatching to maturity on
rations devoid of carotinoids. This result, together with the
fact that the hens in our carotinoid-free flock exhibited normal
fecundity, made it possible to make a more critical study of the
effect of various coloring matters upon the pigmentation of the
adipose tissue, egg yolk, and visible skin parts of fowls of the White
Leghorn breed than is possible with birds normally pigmented
with carotinoids.

Injluence of Carotin and Xanthophyll.

We were particularly interested, among other things, in con-

firming the observation made previously by one of us2 that fowls
use little if any carotin for the pigmentation of adipose tissue,
blood serum, and egg yolk, xanthophyll being the chief plant
carotinoid responsible for the yellow pigmentation normal to
animals of this species.
Five of the cockerels raised on carotinoid-free rations were
fattened for 2 weeks by feeding heavily with white corn and skim
milk. The birds gained in weight from an average of 2.57 to an
average of 3.08 pounds. Birds 400 and 345 were.fed the carotin
ration and Birds 480,482, and 485, the xanthophyll ration.

* Published by permission of the Director of the Missouri Agricultural

Experiment Station.
1 Palmer, L. S., and Kempster, H. L., J. Biol. Chem., 1919, xxxix, 299.
* Palmer, L. S., J. Viol. Chem., 1915, xxiii, 261.
331
332 Effect of Pigments on Egg Yolks

Influence of Curotin.-Carotin was fed in the form of naturally

very highly colored butter secured from the colostrum milk of a
Jersey cow. The butter was thoroughly mixed with the ration
of white corn and skim milk.
At the beginning of the experiment neither bird showed any
visible traces of pigmentation in the ear lobes, beak, shanks, etc.
The carotin ration was continued with Bird 400 for 20 days and
with Bird 345 for 26 days, after which the birds were killed and
their carcasses examined for the presence of pigment. The proto-
cols of the two examinations follow.

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Bird @O.-Base of beak and shanks very faintly tinted yellow in com-
parison with a check bird from the carotinoid-free flock. Tissue fat ap-
parently colorless; rendered, melted fat in 1 inch layer had color of 1.3
units yellow, 0.2 unit red with Lovibond tintometer. Blood serum con-
tained traces of carotin. Ether extract of oil sac on base of tail had faint
yelldw color.
Bird .%&.-Ear lobes colorless. Eye rings colorless. Base of beak
faintly yellow. Shanks faintly tinted in comparison with check bird.
Oil sac contained small amount of colorless oil. Blood serum contained
traces of carotin. Adipose tissue faintly yellow; rendered, melted fat in 1
inch layer had color of 4.5 units yellow, 0.5 unit red with Lovibond
tintometer.

Influence of XanthophyZL-Xanthophyll was fed in the form of

yellow corn, which replaced the white corn in the previous ration.
None of the birds showed a trace of yellow pigmentation at the
beginning of the xanthophyll feeding. The xanthophyll ration
was continued with Bird 485 for 3 days,. with Bird 480 for 8
days, and with Bird 482 for 42 days. Birds 485 and 480 were
killed at the end of the xanthophyll feeding but Bird 482 was put
back on the carotinoid-free ration. The protocols of the three
birds at the close of the xanthophyll feeding are as follows.
Bird 485.-Pigment began to appear in beak and eye rings after 72
hours. On killing, the beak, eye rings, ear lobes, and shanks were dis-
tinctly yellow in comparison with check birds. Blood plasma rich in
xanthophyll; tintometer reading of extracts of 5 cc. in 14 cc. volume and
1 inch layer was 10.0 units yellow, 0.2 unit red. Oil sac full of bright
yellow oil. Tissue fat not examined for xanthophyll as bird had pre-
viously been used for feeding Sudan III. (See experiment with Sudan
111.)
Bird 480.-Xanthophyll quite noticeable in shanks and beak on 5th
day of yellow corn feeding. On 6th day yellow was distinctly visible in
 L. S. Palmer and H. L. Kempster
eye rings, comb skin, and ear lobes. When slaughtered on 8th day all
visible skin parts were quite yellow, that in the beak extending about
two-thirds out from the base. Skin on body showed no pigment. Blood
serum rich in xanthophyll-ether extracts of 5 cc. showing tintometer read-
ing of 14.0 units yellow, 0.5 unit red in 1 inch layer and 13 cc. volume.
Tissue fat not highly pigmented; tintometer reading of 1 inch layer of
rendered, melted fat was 4.2 units yellow, 0.8 unit red.
Bird @Z.-Bird was fed yellow corn until all visible skin parts were
deeply yellow and the feathers had a smooth, creamy appearance. Pig-
ment began to appear in body on 5th day of xsnthophyll feeding. On
6th day the shanks were distinctly yellow. At the end of the 42 days
feeding the color of the beak, shanks, ear lobes, and skin at base of tail

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feathers was measured with the Bradley color top with the results shown
in Table I.

TABLE I.
Color of Visible Skin Parts after Feeding Yellow Corn.

Pslt of fowl. YdlOW. Orsnge. White.

per cent per mt pet cent

Shanks.. . . _. . _. _. . . . . . . . . . _. . . . . . 55 25 20
Beak. . . . __. . . . . . . . . . . . . . . . . . . . . . 55 25 20
Ear lobes.. . . . . . . . . . . . . . . . . . . . . . . . . 55 0 45
Base of tail. . . . ._.. . . . . . . . . . . . . . . . 60 0 40

Injhxce of Sudan III and Annatto.

Experiment with Sudan III.-Cockerel 485, raised on carotinoid-free
ration, and fattened for 17 days on white corn and skim milk, was fed
Sudan III for 8 days. An ether solution.of the dye was poured over the
white corn-meal and the ether driven off leaving the corn with a bright
pink color. The amount of Sudan III fed each day by this method was
not estimated. However, on the 8th day of pigment feeding 0.075 gm.
of Sudan III powder was mixed with the corn-meal.
There was no visible effect of the pigment feeding in any of the visible
skin parts of the bird; i.e., the ear lobes, beak, shanks, etc.
The bird was put back on carotinoid-free ration for 4 days after which
the white corn was changed to yellow corn. 3 days later the bird was
killed. Autopsy showed that the adipose tissue in the mesentery, abdom-
inal, and crop regions was deeply tinted pink with Sudan III, as were
also the bone marrow and subcutaneous adipose tissue. No trace of the
dye was evident in t.he epidermis in any part of the body. The blood
plasma and the bile were also examined for the presence of Sudan III but
no pigment was found. The effect of the xanthophyll feeding on the blood
plasma has been noted in connection with the experiment already reported.
 Effect of Pigments on Egg Yolks
Experiment with Ann&to.-Cockerel 332, raised on a carotinoid-free
ration from hatching, was fed annatto for 17 days. The pigment was fed
in the form of an ether extract poured over white corn-meal until the
corn, on evaporation of the ether, had a deep yellow color. The artificial
yellow corn-meal thus prepared was fed in the form of a wet mash with
skim milk.
After 17 days of annatto feeding there was no visible pigmentation of
the ear lobes, beak, eye rings, vent, or shanks. The bird was then bled
to death and the oxalated blood plasma and carcass carefully examined for
annatto. No test for annatto could be obtained from extracts of the
blood plasma or the liver. The adipose tissue was apparently colorless
but the rendered, melted fat had the faint yellow color of 1.1 units yellow,

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0.1 unit red in a 1 inch layer, when examined with the Lovibond tintom-
eter. This color was not regarded to be due to the annatto.
The gizzard, small intestine, ceca, and large intestine were examined
separately for annatto and a positive test was obtained in each case using
the stannous chloride test on stains left on filter paper by ether extracts of
the material. Especially strong tests were obtained from the contents of
the gizzard and large intestine.

The significance of the failure of Sudan III to stain any but

adipose tissue has been discussed in a previous paper.s
We regard the failure of the pigment of annatto to be resorbed
as a confirmation of the observation that none of the yellow plant
pigments other than xanthophyll is of significance in the pig-
mentation of fowls. Schunck4 has shown that annatto is related
to the carotinoids in many of its properties and states that it has
been given the empirical formula, G~H~~OE..

Influence of Specijic Feeds on the Color of Egg Yolk.

The normal fecundity of the hens of our flock raised on caroti-

noid-free rations and the resulting absence of xanthophyll from
the yolks of the eggs suggested the more or less practical experi-
ment of ascertaining the relative effect of specific feeds on the
coloration of the egg yolks. At the same time we expected to
ascertain whether the adipose tissue and visible skin parts of the
hens would be pigmented on xanthophyll rations while the birds
were exhibiting fecundity.
Fourteen laying hens were taken from the carotinoid-free flock
and placed in separate small cages with sand-covered f)oors and
J Palmer, L. S., and Kempster, H. L., J. Biol. Chem., 1919, xxxix,313.
4 Schunck, C. A., Proc. Roy. Sot. London, 1964, lxxii, 165.
 L. S. Pahner and H. L. Kempster 335

were fed the following rations, respectively, for a period of 4 weeks.

Tap water and oyster shell were kept before the birds at all times.
Ration I.-Check ration: Baked mixture of corn-starch and skim milk
powder, dried and ground.
Ration 9.-Wheat ration: Whole wheat fed as scratch feed with mash of
white corn-meal. Pork liver fed at intervals and skim milk ad libitum.
Ration 8.-Yellow corn ration: Yellow corn as scratch feed and yellow
corn-meal as mash, supplementing with pork liver and skim milk.
Ration &-Red corn ration: Same as Ration 3, the red corn replacing
the yellow corn.

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Ration 5.-Gluten feed ration: White corn as scratch feed, mash of
gluten feed and white corn-meal, 1: 3, supplementing with pork liver and
skim milk.
Ration 6.-Oat ration: White corn as scratch feed, oatmeal as mash,
supplementing with pork liver and skim milk.
Ration 7.-Barley ration: Scratch feed of barley and white corn, 1: 2,
mash of white corn-meal and barley meal, 1: 3, supplementing with pork
liver and skim milk.
Ration 8.-Cottonseed meal ration: White corn as scratch feed, mash
of cottonseed meal and white corn-meal, 1: 3, supplementing with pork
liver and skim milk.
Ration 9.-Hemp seed ration: 25 per cent hemp seed in mash and scratch
feed, the remainder being white corn-meal and white corn. Perk liver
and skim milk as in other rations.
Ration iO.--Rape seed ration: Similar in every way to hemp seed ration.
Ration Il.-Green feed ration: White corn, liver, and milk, with green
feed in abundance.
Ration I,!?.-Meat scrap ration: White corn for scratch feed, mash of
white corn-meal and meat scrap, 3: 1. No milk or liver.
Ration Id.--Blood meal ration: White corn for scratch feed, mash of
white corn-meal and blood meal, 3: 1. No milk or liver.
Ration I&-Wheat bran ration: White corn for scratch feed, mash of
white corn-meal and bran, 3: 1, supplementing with pork liver and skim
milk.

The egg production of the birds during the experiment and the
color of the egg yolks are shown in Table II. The egg yolk colors
are given for both the raw and hard boiled yolks using the Bradley
color top. In the case of the yellow corn and green feed rations
the color of the raw yolks was too deep to be matched with the
Bradley color top. The color readings were made for these eggs
with the Lovibond tintometer.
Especially interesting was the fact that the rations rich in xan-
thophyll had no effect on the visible skin coloring of ear lobes,
336 Effect of Pigments on Egg Yolks

beak, shanks, etc., of the laying hens. This was also true of the
adipose tissue which failed to show the effects of the heavy xan-
thophyll feeding, although the egg yolks were very highly colored.
These results show that all but traces of the xanthophyll of the feed
goes into the egg yolk during fecundity and that it is therefore
impossible to color the body fat or skin of a laying hen with the
plant carotinoids natural to this species of animals.
TABLE II.
Effect of Feeds on Color of Egg Yolk.
-

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Raw yolk. Hard boiled yolk.
Bird - -T
Ration. NO.
5 0 B
9 %
__ ..F h g
Pm Per 1De7 Per per per Per Per
cent :enl : c ent cent cent cent cent unt

Carotinoid-free. ......... 2942 17 55 0 5 40 7.5 0 2.590.0

Wheat ........... ........ 2938 13 65 10 5 20 30.0 0 2.567.5
Yellow corn. ..... ........ 3152 10 * 70.0 12.5 0 17.5
Red. ...... ........ 2906 14 60 10 5 20 37.5 .o 0 62.5
Gluten feed ...... ......... 2909 6 80 10 0 10 27.5 0 0 72.5
Oats. ............ ........ 2939 9 65 10 5 20 27.5 0 2.5 70.0
Barley. .......... ........ 2910 7 70 15 5 10 35.0 0 0 65.0
Cottonseed meal. ........ 3477 t 4 55 0 5 40 20.0 0 5.075.0
Hemp seed ....... ........ 3163 7 62 23 5 10 40.0 0 0 60.0
Rape ....... ........ 3188 8 65 5 0 30 20.0 0 5.075.0
Green food ....... ........ 2950 15 f 72.5 17.5 0 10.0
Meat scrap ....... ...... ..293 4 12 65 0 0 35 20.0 0 2.577.5
Bloodtieal...... ........ 2943 10 60 0 0 40 12.5 0 2.585.0
Wheat bran ...... ........ 2944 10 60 0 5 35 20.0 0 2.577.5

* 33.0 yellow, 4.2 red with Lovibond tintometer.

t Bird died on 21st day of experiment.
$47.0 yellow, 4.5 red with Lovibond tintometer.

Some interesting results were secured in egg yolk coloration

with the specific feeds. The data in Table II show that yellow
corn and green feed are the only common poultry feeds rich in
xanthophyll. Hemp seed, barley, gluten feed, and red corn con-
tain small amounts of xanthophyll but the remainder of the feeds
contain quantities which are practically negligible as far as their
effects on the color of the egg yolk is concerned.
As stated in a previous paper by one of US,~yellow corn is the
best winter chicken food for keeping up the color of the egg yolks
or for coloring the adipose tissue during fattening.
 L. S. Palmer and H. L. Kempster 337
SUMMARY.

1. Carotin and the orange-yellow pigment of the annatto seed

are without influence on the color of the adipose tissue of poultry.
2. Sudan III colors the adipose tissue only of non-laying fowls,
and is without effect on the color of the visible skin parts.
3. Xanthophyll, fed in the form of yellow corn, has an imme-
diate effect on the color of both adipose tissue and visible skin
parts of fowls of the type of White Leghorns.
4. The relative xanthophyll content of various chicken feeds

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was tested separately by feeding them to laying hens raised on
carotinoid-free rations and laying eggswith xanthophyll-free yolks.
Yellow corn and green feed only were found to be rich in xantho-
phyll. A little pigment was found in hemp seed, barley, gluten
feed, and red corn. Wheat, wheat bran, oats, cottonseed meal,
rape seed, meat scrap, and blood meal were found to contain
negligible quantities of xanthophyll.
THE INFLUENCE OF SPECIFIC FEEDS
AND CERTAIN PIGMENTS ON THE
COLOR OF THE EGG YOLK AND BODY
FAT OF FOWLS
Leroy S. Palmer and Harry L. Kempster
J. Biol. Chem. 1919, 39:331-337.

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