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THE INFLUENCE OF CARUTENE FEEDTNE ON THE
VITAMIN A CBNTENT OF THE YULKS 0F EBBS
AND OF THE UVERS 0T CHTCKENS

TflESIS FOR DEGREE 0T M. S.

HARVEY B. OHM ER
1936

THE INFIUENCE OF CAROTENE FEEDING OB TfiE VITAMIN A
CONTENT OF THE YOLKS OF EGGS AND OF THE LIVERS 0F
CHICKENS

THE INFLUENCE OF GARGTTNE FEEDING ON THE VITAMIN A
CONTENT OF THE YOLKS 0F EGGS AND GETHE LIVERS' 0E” "
CHICKENS

by THE”
y“
Hervey B. Ohmer

Submitted in partial fulfillment of the requirements

for the Degree or Maeter of Science

Department of Chemistry
Division of Applied Science
Michigan State College

1936

The author wishes to express his
indebtedness and gratitude to Dr. C. A.
Hoppert, Associate Professor of Chemistry
for his assistance and advice in planning
and conducting the experiments and in the
preparation of this manuscript and to Mr.
J. A. Davidson of the Poultry Department
for making possible this investigation.

16233"? T. f)

Introduction

During the past decade, a great deal of emphasis
has been placed on the importance of vitamin A in the
nutrition of man and.animals. In recent years this
trend has been especially strong in poultry nutrition.

Fraps, Treichler and associates (1, 2) working
at the Texas experiment station, have contributed a
great deal in the study of the vitamin A.content of
foods and feeds in conjunction with a rather comp-
rehensive study of the vitamin A requirements of
poultry for various physiological functions. Their
work and that of other investigators (3,4,5) in this
field has shown in a general way what the vitamin A
requirements in poultry are and that these requirements
may be met by the feeding of vitamin A containing con-
centrates such as cod liver oil or by the use of feeds
containing certain pigments which are convertible to
vitamin A in the animal organism.

Carotene has been known for several years to take
the place of vitamin A in the diet. At the present
time three different forms of carotene are recognized,
each of which is convertible to vitamin A. As can be
seen from the structural formulae given below, the alpha
form.gives rise to one molecule of vitamin A, the beta

form, two, and the gamma form.one.

-2-

 

33 /C33 CH; 0% CH3 Th3\ 330\ C33

H.,/°\2-:-¢.l.§-§-w it” - - 52-5-0233- -21 5-3”

ch /c-ca, ' H3C' c\\CH cl/ CE.
H H:

1. Alpha Carotene (040356)

a. c\ fag TH. CH3 CH: CH3 HsC\ /CHs

H.c;\o-Idi=g-c=g-§=E-c 3333.03.33.03. -c’c‘caa

Hake/EL- CHa HaC-C\c/CHg
H. H.

2. Beta Carotene (040356)

HOC\ Cfis T33 ('31s ([333 1233 33 c\ /CHa
3.5% \f-E-E-c §-§=§-c=%-E.§-§=c-Riga-E E-fi KGB
B‘cm/J- CEO HQC'cY/ifl.

H; H“

3. Gamma Carotene (040356)

3.0 \/ CH; (‘33 CH;
HsC/ 0:0‘E=E'C =g-gzg-C |=§’§-OH
H. \ l-CH3

E.

4. Vitamin A (020330 0)

-3-

Recently (6) another pigment has been isolated from
yellow corn, which from.its structural configuration

is capable of forming vitamin A, molecule for molecule.

H c CH CH on a c on.
H:\c/EH:1HHTHE HHTEiH 01:;\/

’c\c- -c=¢-c=<‘:-c=c-c=c =¢-¢= -c=é-g= -'=¢-fi’v\(I:H.

HOBCKCIC-Cfia Hsc—C\c/CH2
He He

5. Kryptoxanthin (0403560)

This pigment called kryptoxanthin, the formula for
which is given above, apparently lies intermediate between
beta carotene and zenitpth‘ih, the latter having no vitamin
A value at all. Whether there are still other pigments
capable of forming vitamin A is not definitely known. It
is, however, quite likely that others may be found.

While it has been shown.that certain pigments from
various plant materials are an important source of
vitamin A, relatively little has been done in the study
of the value and effectiveness of feeding carotene
concentrates. This then becomes the object of the
present study, the criteria being the vitamin A content

of the eggs and livers of chickens.

-4-

Experimental
In this work the problem involved was to make a

study of the vitamin A content of the yolks of the
eggs and of the livers of hens that had been fed a
carotene supplement in conjunction with an adequate
ration. In determining the amount of vitamin A in
the egg yolk, the Sherman-Todhunter single feeding
method was used, whereas in the case of the livers,
the Carr-Price antimony trichloride reaction.was

employed.

management of the chickens
The birds used were from.the Michigan State
College ”White Leghorn" flock. The flock was
range raised on pasture and the birds used were
pullets, six months old at the beginning of this
experiment. The two general rations used in feeding
the chicken had the following composition:
Corn Ration 6
Ground yellow corn(Mich,).................18%
Bran (Mich.)..............................20%
Ground oats...............................13%
Middlings ................................20%
Meat scrap(50% protein)...................lo%
Dried skim milk .......................... 5%
White fish meal .......................... 5f
Alfalfa meal (Mich.)...................... 8%
Sodium.chloride........................... 1%

Sardine oil (Source of vitamin D)......... 2%

-5—

Grain

Whole corn ad lib (in happers)

Whole oats ad lib (in hoppers)

40 lbs. of wheat per month scattered in the litter

Ration 10 was the same as ration 6, except
that barley was used in the place of corn in the dry
mash and in the whole grain supplement. Rations 4
and 8 were supplemented with carotene ef'en amount
calculated to supply each bird with the equivalent
of 11,200 U.S.P.X.(1934) vitamin A.units per month.
The carotene was obtained from the S.M.A. corporation
in the form of a solution in oil and was incorporated
in the dry mash. The monthly allowance of carotene
was fed in such a way that it would be consumed in the
course of a week. This practice seemed desirable
because of the uncertainty of the stability of the
carotene in the feed mixture. To check on the keeping
quality of the mixture a determination.was made of
the vitamin A equivalence of the ration after a weeks
storage. The results of these tests indicated that
there was no appreciable loss.
Analysis of Egg Yolks

The technique used in determining the amount of
vitamin A.in the egg yolks was the Sherman-Todhunter
single feeding method (7). This nethod was selected
because of several appreciable advantages which it
possesses over the official biological method. First

of all there is a considerable saving of time, because

-6-

a single feeding replaces the daily feeding over a
period of from six to eight weeks. Then there is the
elimination of the danger of the loss of vitamin
potency incidental to the storage of the material to
be tested. Finally the number of failures is likely
to be reduced because of the fact that a relatively
large amount of the material is fed at one time. This
brings about a more uniform response to the vitamin A
supplement than would be the case with very small daily
doses.

The animals selected were white and striped rats
of the chemistry department colony. They were at least
21 days old and weighed between 55 and 45 grams. The
usual care was observed in having a uniform distribution
of the rats with regard to size and sex in the various
series. The animals were placed on a basal ration
consisting of the following:

Dextrin.....-.................... 60%
Casein(Vitamin free)............. 18%
Yeast .........-................. 7%
Yeast(irradiated)................ 1%
Salt mixture..................... 4%
Crisco .......................... 10%

The animals were weighed weekly for three weeks
and after that every other day until the weights
became constant. This constancy in weight marked

the end of the depletion period. At this stage

-7-

the rats were put into separate cages and fed the
various supplements.

The arrangement of the groups was as follows:

Group 1 Controls

Group 3 60 U.S.P.X.(1934)A units
Group 4 1 gram of yolk 4

Group 6 1 gram of yolk 6

Group 8 1 gram of yolk 8.

Group 10 1 gram of yolk 10

The supplement was placed in a china cup and was
consumed usually in less than a day. After the feed-
ing of the supplements the rats were weighed at weekly
intervals. The average gains and losses in weight
were plotted against time. Using the average loss in
weight of the control animals for the first week follow-
ing the depletion period as a base line, the areas under
the growth curve were measured by the use of a planimeter
and the amount of vitamin A calculated by comparing the
areas with that given by the animals receiving the 60
U.S.P.X. (1934) vitamin A units of standard reference oil.
The curves for the first series only are given to
illustrate the manner of obtaining the areas mentioned
above. In the subsequent series were obtained in a
similar manner, but the results are presented in tabular

form,

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-9-

Series 3 (May)

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: ’3 : : :
: Group : Supplement : Area in : Calculated Vitamin A :
z : : sq. in. : units per gram. :
z : : e :
: 1' : z z :
z 3 3 60 U.S.P.X.: 3.44 z :
: : 1934 Vit A z z :
‘_i fir : units +I: pf ,:_ “v v :
: : : : :
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z : 15g§;4: : : :
: “' ""f z ’ t ’ W :
z 0 3 1 gm. of z 1.90 : 33.12 :
: _» w}lyolk.6 gin :, :
: : z : :
x 8 : 1 gm. of : 2.81 : 49.01 :
:fi' : yolk 8’ _} x :
x : : a :
s 10 : 1 gm. of : .79 x 15.78 s
z : yolk_lo : z :

 

An examination of the above tables indicates that
the feeding of carotene definitely and appreciably
increases the vitamin A potency of the eggs. This was
true in the case of the corn ration as well as the
barley ration. In general the eggs from the corn groups
were somewhat richer in vitamin A than those frem the
barley groups. This would be expected because yellow
corn is a better source of the vitamin A producing
pigments than barley. The inconsistencies where they
occur are due in part to the limitations of the biological
methods of assay when a relatively small number of rats
is used and in part to the fact that a small number of
eggs was used in the study. Undoubtedly the data would
have been more satisfactory if more complete assays with

a greater number of eggs had been made.

-10-

Analysis of Livers

In determining the vitamin A potency of the
livers it was decided to use the chemical method
because of its rather general adOption during the past
few years. The method used was that accredited to
Carr and Price and involves the measurement of the
blue color produced when vitamin Axis treated with
antimony tri-chloride.

The livers were obtained frem the same group
of hens from which the eggs were derived. In those
cases in which the livers could not be analyzed soon
after the hens were killed, the livers were stored
in an electric refrigerator. The period of storage
in no case exceeded two or three days so that there
was little opportunity for loss in vitamin A potency.

In determining the vitamin A content of the livers,
the Guilbert and Hart (8) procedure was used. This
involved the following steps: The livers were weighed
to the nearest tenth of a gram and 10 grams of the
liver used for each determination. 20 to 40 c.c.
of 10 percent potassium.hydroxide was added to the
liver in a 300 c.c. crlenmeyer flask and the mixture
heated on a boiling water bath until the liver went
into solution. The contents of the flask was then
cooled and removed to a sepratory funnel. After

the addition of 20 to 30 c.c. of ethyl alcohol,

-11..

two extractions were made with ethyl ether. The
alkaline layer was drawn off and the ether solution
washed free from.alkali with distilled water.. The
ether solution was then transferred to a flask con-
taining 5 to 10 grams of anhydrous sodium.sulphate
and the flask shaken vigorously to remove any water
that might be present. The ether solution was decanted
off and the sodium.sulphate residue washed twice with
anhydrous ether. The ether was distilled off under
reduced pressure at 50 “ C. and the residue dissolved
in 10 c.c. of anhydrous chloroform.

For the determination of vitamin A, l 0.0. of the
chloroform.solution obtained by the above method was
treated with 2 c.c. of a saturated solution of antimony
tri-chloride in anhydrous chloroform. Theblue color
which developed was measured by the use of the Lovibond
tintometer. The number of Lovibond blue units were
calculated by the use of the following formula:

Blue units per gram - B V‘Vl

Where:
B - Blue units on the tintometer
V - Number of c.c. in the cell
Yi- Total volume of chloroform solution
W . Grams of liver sample

N.- Number of c.c. of chlorofonn
solution used

-12..

The values are given in terms of Lovibond blue
units as well as U.S.P.X. 1934 vitamin A units, it
being assumed that l Lovibond blue unit is equivalent
to 2 1/2 U.S.P.X. 1934 vitamin A units. This conversion
factor was determined by Mr. Moore of the dairy department
in an extensive comparison between the biological and

chemical methods.

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-14-

The data obtained on the livers indicates that
a considerable storage of vitamin A is effected by
supplementing poultry rations with a carotene concent-
rate. In the case of the corn ration the increase
in vitamin A.potency appeared to be considerably
greater than was observed in the case of the barley
group. In view of the limited scepe of the experiment
no explanation for this difference can be offered.

A determination of the yellow units was made, but
the results were rather inconsistent and therefore
are not included. The results however, do suggest that

very little carotene is stored in the liver.

Conclusions

1. Feeding of carotene concentrates, definitely
increases the vitamin A potency of yolks of eggs.

2. A considerable storage of vitamin A in the
livers results from feeding a carotene concentrate.
Apparently very little carotene is stored as such

in the liver.

1.

2.

3.

4.

5.

8.

-15..

Bibliography

Texas Agric. Expt. Sta. Bull. 477

Texas Agric. Expt. Sta. Bull. 493

Plimmer, R.H.A., Rosedale, J.L. and Raymond, W.H.,
Biochem. J. 21, 940 ,

Capper,N.s., McKibbin, 1.11.11. and Prentice, 3.11.,
Biochem. J. 1931, 25, 265

Russell, W.C. and Weber, A.L., Proc. of the Sec.
for‘lxper. Biol. and med. 1931, XXIX, 297

Kuhn and Grundmann, Berichte 1934, 67, 593

Sherman, 8.8. and Todhunter, E.N., J. of Nutrition
1934, a, 347

Guilbert, H.R. and Hart, G.H., J. of Nutrition
1954, s, 25

 

 

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”The’lnfluence of carotene
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