A10-1037-8M-L180

TEXAS AGRICULTURAL EXPERIMENT STATION

A. B. CONNER, DIRECTOR
COLLEGE STATION, BRAZOS COUNTY, TEXAS

BULLETIN NO. 557 OCTOBER, 1937

DIVISION OF CHEMISTRY

Losses of Vitamin A and Carotene
From Feeds During Storage

L I B R A R Y
Agricultural & Mechanical Ballege of Texas

 

AGRICULTURAL AND MECHANICAL COLLEGE OF TEXAS
T. O. WALTON, President

Some manufacturers of mixed feeds, having recognized that some
of the commercial mixed poultry feeds did not carry enough vita-
min A potency, have begun to correct these deficiencies by adding
fish liver oils, fish liver oil concentrates or solutions of carotene in
oil, or yellow corn or alfalfa leaf meal of high potency. Since
vitamin A and carotene are both unstable, it is important to know
whether these substances would remain in commercial mixed feeds
long enough to have the desired effect.

Cod liver oil, other fish liver oils, concentrates of cod liver oil,
and carotene dissolved in oil were added to various feed mixtures,
the mixtures stored in several different ways and examined for
vitamin A or carotene after definite intervals of time had elapsed.
It was found that practically all the vitamin A, added in the form
of cod liver oil, other fish oils, or cod liver oil concentrates, was lost
after four weeks of storage. When hydroquinone equal to 0.1% of
of the feed was used as a stabilizer, the vitamin A did not dis-
appear so quickly, but even then most of it was lost after 3 weeks.
The use of fish liver oils in a commercial mixed feed for the purpose
of supplying vitamin A appears to be of little or no value, since
most of the vitamin A may disappear before the feed is used. If
such oils are mixed in a feed to supply vitamin A, the mixture
should all be used in 10 days after it is made up, to avoid serious
losses of vitamin A.

The carotene in solution in vegetable oil after being added to
feeds was more stable than vitamin A in cod liver oil, especially
when the mixture was stored at low temperatures. At a tem-
perature of 42-49°F. only 3 to 6% of the carotene was lost in 8
weeks. However, at room temperatures of 77-82°F., from 7 to
27% of the carotene was lost in 4 weeks and from 12 to 53%
in 8 weeks.

Carotene in alfalfa products and cryptoxanthin in yellow corn
were also found to be unstable, though they were not lost as rapidly
as carotene dissolved in oil. At high temperatures there was con-
siderably more loss than at low temperatures. The method of
storage had considerable effect on the loss. Large compact sam-
ples of the feeds lost carotene at a less rapid rate than small
samples loosely packed. Alfalfa leaf meal stored in tightly packed
vials at refrigerator temperatures lost only from 0 to‘ 3% carotene
per month. Mixtures of feeds with carotene are likely to lose part
of the carotene when stored under ordinary conditions, so that when
the mixture is fed, the animals will not receive the quantity of
carotene originally placed in the feed. Being more stable, carotene
is a better source of vitamin A potency than cod liver oil for
mixing with feeds. However, most of the carotene may be lost
unless the mixture is fed within two or three months after it is
made. '

CONTENTS

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 5
Previous Work . . . . . . . . . . . . . . . . . . . . .' . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Method of Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Method for carotene in feeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Losses of vitamin A from cod liver oil and other fish liver oils or
concentrates when added to feed mixtures . . . . . . . . . . . . . . . . . ., . . . 12
Losses of carotene from yellow corn, alfalfa products, and other
feeds during storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 15
The effect of storage upon losses of carotene added in oil to feed
mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 23
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 25

References . . . w . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 27

BULLETIN NO. 557 OCTOBER, 1937

LOSSES OF VITAMIN A AND CAROTENE FROM FEEDS
DURING STORAGE

G. S. FRAPS, Chief, and A. R. KEMMERER, Assistant Chemist,
Division of Chemistry

Vitamin A has been known for some time to be necessary for the life
and good health of animals. If the quantity in the diet of young animals
is too small, they will stop growing in a short time and then begin t0 lose
weight. With an insufiicient quantity of vitamin A, young animals may
grow slowly but not make a normal growth. Night blindness is a symptom
of a deficiency of vitamin A (15). Animals sufiering from this trouble
cannot see well at night and may run into all kinds of obstacles. Sore eyes
are another symptom of deficiency of vitamin A. An animal having a
deficiency of vitamin A becomes weak and susceptible to respiratory troubles
and other diseases. A symptom of extreme deficiency in steers and dairy
cows is convulsions (16).

An adequate supply of vitamin A is necessary for the growth of young
animals (27), for the production of eggs (26), and for a good production
of milk (5) or healthy offspring (3). High quantities of vitamin A potency
must be fed if cows are to produce milk (5) or if hens are to produce
eggs (26) high in vitamin A. Sherwood and Fraps (26) have pointed out
that the ration of laying hens should contain about 3 to 7.5 Sherman-
Munsell units of vitamin A units per gram. Fraps, Copeland, and Treichler
(5) have shown that the requirements of dairy cows for vitamin A are
large. The possibility of a deficiency of vitamin A in the diet of range
animals has been pointed out by Guilbert and Hart (12), while Converse
and Meigs (3) have shown that such deficiencies may occur with dairy
cows fed upon low-grade roughages. Vitamin A has a greater importance
in animal feeding than it was formerly supposed to have.

Vitamin A is a colorless substance which occurs in fish liver oils, butter,
eggs, and the livers of various animals. Carotene is a yellow substance
which has vitamin A potency and occurs in alfalfa, carrots, sweet potatoes,
and other yellow or green plants or plant products. Cryptoxanthin is a
yellow substance having vitamin A potency which occurs with carotene in
yellow corn. Other yellow-colored “substances occur in plants but have no
vitamin A potency. Carotene eaten by animals may be converted into
vitamin A which can be stored in the animal body, chiefly in the liver.
Animals which receive abundant supplies of carotene (or vitamin A) may

store enough vitamin A in the liver to last for several months even though '

the feed used later is deficient in this vitamin (14).

Since the knowledge of vitamin A has become more and more widespread,
and its importance in feeds for livestock more generally recognized, some
feed manufacturers have attempted to increase the vitamin A potency of
some of their commercial mixed feeds (especially chicken feeds) by addi-
tions of carotene dissolved in oil, cod liver oils, fish oil concentrates, or

6 BULLETIN NO. 557 TEXAS AGRICULTURAL EXPERIMENT STATION

alfalfa meals high in carotene. Feeds often have to be stored over winter
or for even longer periods of time. While it is known that both carotene
and vitamin A are unstable, it is not known definitely how long either oi
them will remain in a feed. Therefore, it is a matter of considerable prac-
tical importance to determine how stable carotene and vitamin A are in
feeds alone, and when they are added to commercial feeds, and in what
way they can best be added to a feed to give the greatest vitamin A potency
for the greatest length of time. The work presented here attempts to give
some of this needed information. '

PREVIOUS WORK

Fraps and Treichler (7) have reported appreciable losses of vitamin A
potency from alfalfa, dried milk, yellow corn and other materials during
storage. Guilbert (10) has shown that carotene in alfalfa decreased 30 te
50% during storage for 8 Weeks at room temperature, While at from -5 t:
0° C. there was practically no destruction in the same length of time. Ii
has been shown by others (1, 21, 30) that carotene dissolved in oil is
unstable. Holmes, Corbet, and Hartzler (18) have reported that the
vitamin A in cod liver oil was completely destroyed in 6 Weeks if the oil
was stored at room temperature in a bottle one-fourth filled with the oil.
When the oil contained 0.1% hydroquinone and 0.1% lecithin, 75% of the
vitamin remained after 42 weeks of storage. Marcus (20) found that
when a cod liver oil concentrate very rich in vitamin A was added to the
U. S. P. basal ration used for vitamin A determinations and stored in the
dark, 85% of the vitamin A had been destroyed after 10 days. A summary
of the results obtained by the above and other workers is given in Tables

1 and 2.

i
a

 

Table 1. Previous work on the stability of vitamin A to storage

LOSSES OF VITAMIN A AND CAROTENE FROM FEEDS DURING STORAGE 7

 

  

    
  

  

   
   
   

 

 Source of vitamin A and Temperature Loss Period of Literature
.__ treatment of storage per cent storage reference
i 'but liver oil. 10 cc. in 40 cc. brown
 bottle in diffused light . . . . . . . . . . . . . . . . . Room . . . . . . 100 21 weeks 18
 ibut liver oil + 1% hydroquinone +
_ A 5% lecithin. 10 cc. in 40 cc. brown
{bottle in diffused light . . . . . . . . . . . . . . . . Room... . . . 32 62 weeks 18
 - t liver oil. 10 cc. in 40 cc. brown bottle
v _' in diffused light . . . . . . . . . . . . . . . . . . . . . . Room . . . . . . 100 6 weeks 18
’ d liver oil with . 1% hydroquinone. 1O
 cc. in 40 cc. brown bottle in diffused
Y‘ light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Room. .  . . 54 42 weeks 18
Il- liver oil_with .1% hydroquinone and
 ._l% lecithin. l0 cc. in 4O cc. bottle in
diffused light . . . . . . . . . . . . . . . . . . . . . . . . . Room . . . . . . 25 42 weeks 18
 tamin A, concentrate added to U.S.P.
g hasal ration for vitamin A assay. Stored
.f 1n dark . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Room . . . . . . 85 10 days 20
itamin A concentrate added to hydro-
.; quinone. Stored in dark . . . . . . . . . . . . . . Room... . . . 15 15 days 20
itamin A concentrate added to granu-
i lated lactose. Stored in dark . . . . . . . . . . . Room... . . . 90 8 days 2O
_ita.min A concentrate added to granu-
lated starch with .1% hydroquinone. . . . . Room . . . . . . 5 15 days 2O
nsaponifiable residue of cod liver oil— Refrigerator
: in peanut oil . . . . . . . . . . . . . . . .- . . . . . . . 100 6 weeks_ 22
in olive oil . . . . . . . . . . . . . . . . . . . . . . . . . . 30 6 weeks 22
c in coconut oil . . . . . . . . . . . . . . . . . . . . . . . 0 6 weeks 22
__ nsaponifiable residue of cod liver oil with
" -.05% hydroquinone— Refrigerator
‘ in peanut oil . . . . . . . . . . . . . . . . . . . . . .. 0 6 Weeks 22
in olive oil . . . . . . . . . . . . . . . . . . . . . . . .. 18 6 weeks 22
in coconut oil . . . . . . . . . . . . . . . . . . . . .. 5 6 Weeks 22
Jmples of cod liver oil placed on market
 for two to four years, not opened . . . . . . . . Room . . . . . . 0 . . . . . . . . 4
0d liver oil irradiated with ultra-violet
ligh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 100 8 hours 2
_d liver oil mixed into a ration contain-
. ing 2O to 25 per cent very rancid lard. . . . . . . . . . . . . . . . 100 Few days 23
alibut liver oil. 3O cc. in 5O cc. flask
i‘ exposed to air in diffused light . . . . . . . . . . Room . . . . . . 96 31 days 19
‘ alibut liver oil with 5% coarsely ground
-. 1; whole oats. 3O cc. in a 5O cc. flask ex-
 posed to air in diffused light . . . . . . . . . . . . Room . . . . . . 94 31 days 19
almon oil. 5O cc. in a 125 cc. flask. Ex-
._ posed to air in diffused light . . . . . . . . . . . . Room . . . . . . 63 31 day ~» 19
-'= on oil with 5% coarsely ground whole
-_oats. 50 cc. 1n a 125 cc. flask. Exposed
Room... . . . 0 85 days 19

f to air in diffused light . . . . . . . . . . . . . . . . . .

 

  

 

 

8 BULLETIN NO. 557 TEXAS AGRICULTURAL EXPERIMENT STATION

Table 2. Previous work on the stability of carotene to storage
Source of carotene and Temperature Loss Period of Literatun
treatment of storage per cent storage reference
Alfalfa leaves . . . . . . . . . . . . . . . . . . . . . . . . . . . — 5 to 0° C 0 8 weeks 10

Dehydrated alfalfa leaf meal . . . . . . . . . . . . . . — 5 to 0° C 0 8 weeks 10

Alfalfa leaves in dark . . . . . . . . . . . . . . . . . . . . 20 to 30° C 30.5 8 weeks 10

Alfalfa leaves in dark . . . . . . . . . . . . . . . . . . . . 60° C 61.9 9 clays 10

Alfalfa leaves in dark . . . . . . . . . . . . . . . . . . . . 80° C 86.8 9 days 10
Alfalfa leaves exposed to sunlight in the
field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69.5 8 weeks 10
Alfalfa cured in the field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70—9O From_
_ curing 24
Alfalfa hay artificially dried . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 Drying 25

Alfalfa dried in air . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67-88 Air drying 25

Alfalfa hay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Barn . . . . . . . 50—67 7 months 29

Alfalfa leaf meal . . . . . . . . . . . . . . . . . . . . . . . . . Room . . . . . . 50 11 months 7

Baled alfalfa at Mesa, Arizona . . . . . . . . . . . . Summer 5O 3 months 28

Baled alfalfa at Mesa, Arizona . . . . . . . . . . . . Winter.. . . . 0.0 3 months 28

Baled alfalfa at Mesa, Arizona . . . . . . . . . . . . All year 75 12 months 28

Yellow corn, ground . . . . . . . . . . . . . . . . . . . . . ‘Room . . . . . . 85 30 months 7

Yellow corn, unground . . . . . . . . . . . . . . . . . . . oom. . 60 5 months 7

Carotene dissolved in cottonseed oil . . . . . . . . 4° C 10 2 months 1
Carotene dissolved in cottonseed oil and

hydroquinone . . . . . . . . . . . . . . . . . . . . . . . . . 4° C 12 2 months 1
Carotene dissolved in sesame oil . . . . . . . . . . 4° C 15 3 months 1
Carotene dissolved in olive oil. . . . . . . . . . . . . 4° C 28 2 months 1
Carotene dissolved in olive oil and hydro-

quinone . . . . . . . . . . . . . . . . . . . . .< . . . . . . . . . 4° C 2O 2 months 1
Carotene dissolved in corn oil . . . . . . . . . . . . . 4° C 20 2 months 1
Carotene dissolved in corn oil and hydro-

quinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4° C 23 2 months 1
Carotene dissolved in coconut oil . . . . . . . . . . 4° C 24 2 months 1
Carotene dissolved in coconut oil and

hydroquinone . . . . . . . . . . . . . . . . . . . . . . . . . 4° C 10 2 months 1
Carotene dissolved in ethyl laurate . . . . . . . . 4° C 72 2 months 1
Carotene dissolved in ethyl laurate and

hydroquinone . . . . . . . . . . . . . . . . . . . . . . . . . 4° C 11 2 months 1
Carotene dissolved in ethyl sebacate . . . . . . . 4° C 63 2 months 1
Carotene dissolved in ethyl sebacate and

hydroquinone . . . . . . . . . . . . . . . . . . . . . . . . . 4° C 11 2 months 1
Carotene dissolved in cottonseed oil 0.46

mg. carotene per cc . . . . . . . . . . . . . . . . . .. Room 31 2 months 1
Carotene dissolved in cottonseed oil 0.01

mg. carotene per cc . . . . . . . . . . . . . . . . . .. Room.. 0.0 2 months 1
Carotene dissolved in olive oil . . . . . . . . . . . . . 100° 100 12 days 30
Carotene dissolved in olive oil and hydro-

quinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100° 25 6 months 30

Carotene dissolved in olive oil . . . . . . . . . . . . . Room . . . . . . 100 30 days 30

Carotene dissolved in peanut oil . . . . . . . . . . . Room 100 4 weeks 21

Carotene dissolved in corn oil . . . . . . . . . . . . . Room . . . . . . 27 8 weeks 21

Carotene dissolved in Wesson oil . . . . . . . . . . . Room . . . . . . 20 8 weeks 21
Carotene dissolved in peanut oil in vacuum. . 37° 44 8 weeks 21
Carotene dissolved in Wesson oil in vacuum. . 37° 15 8 weeks 21
Carotene dissolved in corn oil in vacuum. . . . 37° 15 8 weeks 21

These tables show that vitamin A and carotene will be destroyed if the
materials containing them are not kept under proper conditions. Light,
exposure to air, and ordinary room or high temperatures destroy both of
them. A low temperature, protection from light and air and, in case of
vitamin A, the use of an antioxidant such as hydroquinone, appear under
some conditions to be favorable to the preservation of vitamin A and
carotene.

Method of Procedure

To study the stability of vitamin A in feeds, fish liver oils, which are
carriers of this vitamin, were mixed with various feeds and stored under
different conditions. The amount of vitamin A was determined by means

LOSSES OF VITAMIN A AND CAROTENE FROM FEEDS DURING STORAGE 9

of the spectrograph at the beginning of the test and at the end of various

p intervals. Details are given on subsequent pages.

A similar procedure was followed for feed mixtures that contained
carotene. A number of samples containing carotene were stored under

- various conditions. Details of this procedure are given in connection with

E

the discussion of the work. The method used for carotene is described
below. Since the method is colorimetric, the results obtained by it may
be too high, because products that have the same color and solubility as

fcarotene may be formed from the carotene during storage. In such case,

. the flasks.

the loss of carotene would be greater than shown by the analysis.

Method for Carotene in Feeds

The procedure used for the determination of carotene is based on the

cmethod of Guilbert (11) and is as follows:

1 to 6 grams feed are refluxed 30 minutes with 20 to 120 cc. of saturated
alcoholic potassium hydroxide, free from aldehydes and ketones. Care is
exercised to keep portions of the sample from collecting on the sides of
If any material does collect on the side of the flask, it is
washed down with alcohol. After refluxing, the contents of the flask are
cooled with water, 50 cc. ethyl ether added, and after shaking for a minute
and allowing the sediment to settle, the ether-alcohol mixture is decanted
into a liter separatory funnel. This extraction is repeated two more times
with 15 cc. portions of ether. Then the residue is broken up by shaking
first with 5 cc. of 95% ethyl alcohol and then with 15 cc. of ethyl ether.
Usually after 2 or 3 additional extractions with ether, no more color is
extracted and the residue is discarded.

To the combined ether-alcohol mixtures i.n the separatory funnel is
added 100 cc. of cold distilled water. The alkaline alcohol water solution
containing most of the chlorophyllines and flavines separates is drawn
off from the bottom of the funnel and is re-extracted by shaking gently
twice with ether in another funnel. If an emulsion is formed, it is cleared
by adding 1 cc. of ethyl alcohol. The ether extracts are combined and
washed with cold distilled water until free from chlorophyllines and alkali.
Washing three or four times by pouring the water through the solution
and down the sides of the funnel removes most of the alkali. The remainder
is removed by gently shaking the ether solution with 25 cc. portions of
water until the wash water no longer gives a color with phenolphthalein.

The ether solution containing the combined carotene and xanthophyll is
transferred to a flask and the ether distilled off with diminished pressure.
The residue containing both carotene and xanthophyll is dissolved in 30 cc.
light petroleum ether, added in 3 portions of 10 cc. each, and transferred
to a small separatory funnel. The petroleum ether solution is shaken for
2 minutes with 85% methanol to remove the xanthophyll. This extraction
is repeated until the methanol layer is colorless. About 5 or 6 extractions
with 85% methanol are usually suflicient. If during the first extraction
the lower layer is cloudy, it is drawn off and extracted with 20 cc. petro-
leum ether. This petroleum ether extract is added to the other petroleum

10 BULLETIN NO. 557 TEXAS AGRICULTURAL EXPERIMENT STATION

   
    
 
  
   
     
  
    
   
     
   
  
   

ether fraction and the shaking with 85% methanol continued. After 5
petroleum ether solution is shaken with 85% methanol, it is shaken 
90% methanol to remove any traces of xanthophyll which still rem1
Usually no color is obtained in the lower layer after 2 or 3 extracti
with 90% methanol. The petroleum ether layer is finally washed Vi
Water, dried over anhydrous sodium sulphate, concentrated in vacuo 
made up to 10, 25, or 5O cc., depending on the amount of color in i‘
solution. . i’

The amount of carotene in the petroleum ether solution is estim 
by comparing it with 0.1% potassium dichromate. The carotene solu"
is placed in the left cup of a micro-colorimeter set at a depth-of 0.5, I
2.0, 3.0, or 4.0 cm. according to the density of the color. The depthQ:
the dichromate solution, contained in the right cup of the colorimeter, y‘
varied until the density of color is equal. An averageof 8 independe
readings in millimeters is taken. This average should be between 4 a
12 mm. of the dichromate solution. By use of Table 3, the millime

Table 3. Carotene dissolved in petroleum ether, equivalent in color to a .l% solution.“
potassium dichromate “

_ Potassium Carotene, parts Potassium Carotene, p ~
dichromate, mm. per million dichromate, mm. ‘ per million}
4.0. . . . . . . . . . . . . . . . . . . . .. 2.5 8.0 . . . . . . . . . . . . H‘. 4.9

4.2 . . . . . . . . . . . . . . . . . . . . .. 2.6 8.2 . . . . . . . . . . . . . . . . . . .. 5.0

4.4 . . . . . . . . . . . . . . . . . . . . .. 2.7 8.4. . . . . . . . . . . . . . . . . . .. 5.2

4.6 . . . . . . . . . . . . . . . . . . . . .. 2.8 8.6 . . . . . . . . . . . . . . . . . . .. 5.3

4.8 . . . . . . . . . . . . . . . . . . . . .. 2.9 8.8 . . . . . . . . . . . . . . . . . . .. 5.4

5.0 . . . . . . . . . . . . . . . . . . . . .. 3.1 9.0 . . . . . . . . . . . . . . . . . . .. 5.6

5.2 . . . . . . . . . . . . . . . . . . . . .. 3.2 9.2 . . . . . . . . . . . . . . . . . . .. 5.8

5.4 . . . . . . . . . . . . . . . . . . . . .. 3.4 9.4 . . . . . . . . . . . . . . . . . . .. 5.9

5.6 . . . . . . . . . . . . . . . . . . . . .. 3.5 9.6 . . . . . . . . . . . . . . . . . . .. 6.0

5.8 . . . . . . . . . . . . . . . . . . . . .. 3.6 9.8 . . . . . . . . . . . . . . . . . . .. 6.1

6.0 . . . . . . . . . . . . . . . . . . . . .. 3.8 10.0 . . . . . . . . . . . . . . . . . . .. 6.3

6.2 . . . . . . . . . . . . . . . . . . . . .. 3.9 10.2 . . . . . . . . . . . . . . . . . . .. 6.5

6.4 . . . . . . . . . . . . . . . . . . . . .. 4.0 10.4 . . . . . . . . . . . . . . . . . . .. 6.7

6.6 . . . . . . . . . . . . . . . . . . . . .. 4.1 10.6 . . . . . . . . . . . . . . . . . . .. 6.8

6.8 . . . . . . . . . . . . . . . . . . . . .. 4.2 10.8 . . . . . . . . . . . . . . . . . . .. 6.9

7.0 . . . . . . . . . . . . . . . . . . . . .. 4.3 11.2 . . . . . . . . . . . . . . . . . . .. 7.3

7.2 . . . . . . . . . . . . . . . . . . . . .. 4.5 11.4 . . . . . . . . . . . . . . . . . . .. 7.4

7.4 . . . . . . . . . . . . . . . . . . . . .. 4.6 11.6 . . . . . . . . . . . . . . . . . . .. 7.5

7.6 . . . . . . . . . . . . . . . . . . . . .. 4.7 11.8.. . . . . . . . . . . . . . . . . .. 7.6

7.8 . . . . . . . . . . . . . . . . . . . . .. 4.8 12.0 . . . . . . . . . . . . . . . . . . .. 7.8

depth of the 0.1% dichromate is transformed into parts per million of ca 
tene. Then the parts per million of carotene (c) in the sample are w 1k"
culated by use of the following formula No. 1: 

CZPS/ GD.

P is parts per million carotene (from Table 3). S is volume of soluti
in cc. G is grams sample and D is depth of carotene solution in cm.

The parts per million of carotene equivalent to the millimeters of dichr
mate given in Table 3 were ascertained by comparing 0.1% potassi -1

dichromate in a micro-colorimeter against a petroleum ether solution c0
taining 10 parts per million of purified carotene. To purify the carote 
0.1 gm. of carotene obtained from the S. M. A. Corporation of Clevela%
Ohio, was dissolved in about 2 cc. of chloroform, precipitated by additio;

is
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3i
1+.

 

 

if
E LOSSES OF VITAMIN A AND CAROTENE FROM FEEDS DURING STORAGE 11

50f 15 cc. absolute methanol, the precipitated carotene filtered ofi’ and

  
  
   
  
  
   
  
  
  
  
  

sible. Ten mg. of this purified carotene was dissolved in 1 cc. of chloroform
‘and diluted to1000 cc. with light petroleum ether. This solution was placed
in the left cup of a micro-colorimeter and the depth of solution set at

, 7, 10, 12, 15, 17, 20, and 25 millimeters. The 0.1% potassium dichromate
was placed in the right cup and the settings at which its color matched
the color for the carotene settings determined.

Four carotene solutions and four potassium dichromate solutions pre-
pared at different times were thus examined. The data are tabulated in
.Table 4. From the average of these data a curve was plotted with mm.
10f carotene solution as the abscissa and mm. of .1% potassium dichro-
fmate as the ordinate, and the values in Table 3 were finally obtained by
reading them directly from the curve. These values are for a petroleum
tether solution containing the carotene from 1 gram of sample in 1 cc.
with the left cup of the colorimeter set at a depth of 1 cm. Formula
jNo. 1 must be used along with Table 3 for any other concentrations and
colorimeter setting. a

‘Table 4. Colorimetric comparison of a 0.1% solution of potassium dichromate with petnfleum
' ether containing l0 parts per million of carotene

Colorimetric readings of bichromate solutions
Colorimeter setting of First Second Third Fourth
carotene solution carotene carotene carotene carotene Average

solution solution solution solution

mm. mm. mm. mm. mm. mm

2 . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 2.8 2.6 2.8 2.9 2.8

~ 5 . . . . . . . . . . . . . . . . . . . . . . . . . . .. 8.0 8.0 8.5 8.7 8.3

K. 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 11.2 11.0 11.1 11.3 11.2

T10 . . . . . . . . . . . . . . . . . . . . . . . . . . .. 15.2 14.1 14.3 15.4 14.8

:12 . . . . . . . . . . . . . . . . . . . . . . . . . . .. 17.0 16.3 16.0 17.2 16.6

§l5 . . . . . . . . . . . . . . . . . . . . . . . . . . .. 18.0 19.1 18.6 19.2 18.7

17 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 21.6 20.3 21.4 21.1

20 . . . . . . . . . . . . . . . . . . . . . . . . . . _ . . . . . . . . . . . .. 23 8 23.3 23 5 23.5

25 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 25 0 25 3 25 7 25 3

    
 

é-the several vitamin A active pigments in feeds. Alpha carotene, beta
F carotene, gamma carotene, and cryptoxanthin are all estimated as “caro-
tene.”

In order to test the method, known amounts of pure carotene were put
through the entire procedure with the result that from 91 to 98% of the
l; added carotene was recovered. The method has also been checked with
éanimals in work already published (9). Weighed amounts of carotene were
‘dissolved in cottonseed oil and fed to rats according to the modified
iilSherman-Munsell method (8) used in this laboratory. One microgram of
Ythis carotene was found to be equal to 1.4 Sherman-Munsell units. The
 amount of carotene in a number of alfalfa meal samples was determined
.9 by this method for carotene. These same alfalfa samples were fed to rats

I,’ and it was found that one microgram of carotene in the alfalfa was on
l an average equal to 1.4 Sherman-Munsell units.

 The method for carotene described above does not differentiate between

 

dried in vacuo over concentrated sulphuric acid in as short a time as pos-y

12 BULLETIN NO. 557 TEXAS AGRICULTURAL EXPERIMENT STATION

Losses of Vitamin A from Cod Liver Oil and Other Fish Liver Oils or
Concentrates When Added to Feed Mixtures

Cod liver oil or other fish oils are frequently added to commercial chicken
feeds for the purpose of supplying vitamin D, and they are also used at
times for supplying vitamin A. Since Marcus (20) has reported that 85%
of the vitamin A added t0 a feed mixture was destroyed in 1O days, a
further study of the matter was desirable.

Several different kinds of tests were made upon mixtures of feed with
oils containing vitamin A. Some experiments were made to ascertain the
effect of temperature, of the antioxidant hydroquinone, and of materials
(yeast and soy bean oil meal) which have been claimed to contain sub-
stances that delay the destruction of the vitamin A. These experiments
were made upon small quantities of material.

The desired quantity of cod liver oil Was first mixed with 8 grams of
the feed and then diluted to 100 grams with white corn meal. The feeds
with which the cod liver oil was mixed were white corn meal alone, white
corn meal and hydroquinone, yeast, and soy bean oil meal. Some of these
mixtures were stored at refrigerator temperature of 6° C., at room tem-
perature (26° G.) and at 35° C. Mixtures exactly the same, except that
no cod liver oil was added, were made up and kept under the same con-
ditions. The estimation of vitamin A was made by the spectrographic
method used in this laboratory for vitamin A in butter (6). This method,
in brief, consists of measuring the density of absorption of light at 328
millimicrons by the unsaponified residue of the feeds by means of a spectro-
graph, and calculating the total spectro vitamin A by use of an appro-
priate factor. Since feeds and other substances contain materials that
absorb light at the same Wave length as vitamin A but are not vitamin A,
the total spectro vitamin A had to be corrected for this pseudo vitamin A.
This was done by making the estimation of the pseudo vitamin A in the
mixtures that did not contain the cod liver oil, at the same time and under
the same conditions as the estimation of the total spectro vitamin A in the
mixtures containing the cod liver oil. The amount of pseudo vitamin A was
then subtracted from the total amount of spectro vitamin A. If there
had been a decrease in the pseudo vitamin A in the sample containing no
cod liver oil during the storage, the quantity of vitamin A in the sample
with which the vitamin A was mixed would have appeared to be greater
than it really was; however, the analyses showed that the pseudo spectro
vitamin A was practically unchanged during the period of the experiment,
and that no appreciable error could be introduced from this source.

The results of this Work are given in Table 5. All the feed mixtures
lost vitamin A rapidly. Stored at 6° C., the feeds lost from 34 to 74%
in 2 weeks; at room temperature, the loss was 73 to 89% in two Weeks
and 93 to 100% in 4 weeks; at 35° C., the loss was 73 to 100% in 2 weeks.
When hydroquinone had been added, there was no loss the first Week, but
71 to 84% of the vitamin A was lost at the end of the third week, and from
S3 to 87% had been lost at the end of the fifth Week. Storing at 6° C.
gave a slightly better stability than storing at room temperature or at

LOSSES OF VITAMIN A AND CAROTENE FROM FEEDS DURING STORAGE 13

35° C. for the first 2 weeks, but no better stability at the end of 4 weeks.
At the end of 4 weeks practically all the vitamin A had disappeared from
the samples which did not contain hydroquinone at either the high or low
temperature of storage.

The use of hydroquinone, which is a powerful antioxidant, even at the
high rate of 0.1% of the feed, prevented the destruction of vitamin A only
during the first week. After that the destruction was very rapid and the
hydroquinone had lost its effect, for nearly all" the vitamin A (83 to
87.0%) had been destroyed at the end of the fifth week. The quantity of
hydroquinone used was greatly in excess of that usually added to fish liver
oils, which is about .01% of the oil. The yeast and the soy bean oil meal
like hydroquinone had no practical effect in delaying the oxidation of the
vitamin A (Table 5).

Table 5. Stability of vitamin A in mixed feeds

Vitamin A lost during storage—per cent
_ Temperature 4
Constituents of feed of storage One Two Three Four Five
week weeks weeks weeks weeks
Corn meal + cod liver oil. . . .. 6° C 17.2 34.4 . . . . . . .. 90.0 . . . . . . ..
26°C 30.0 73.3 . . . . . . .. 93.3 . . . . . . ..
35°C 43.3 73.3 . . . . . . .. 93.3 . . . . . . ..
~Corn meal + 8% yeast + cod
liver oil . . . . . . . . . . . . . . . . . .. 6°C 74.2 74.2 . . . . . . .. 84.0 . . . . . . ..
26°C 66.7 88.9 . . . . . . .. 100.0 . . . . . . ..
35°C 59.2 100.0 . . . . . . .. 100.0 . . . . . . ..
‘Corn meal + 8% soybean oil
meal + cod liver oil . . . . . . .. 6° C 38.0 70.0 . . . . . . .. 100.0 . . . . . . ..
26°C ‘ 40.0 78.5 . . . . . . .. 100.0 . . . . . . ..
35°C 29.0 100.0 . . . . . . .. 100.0 . . . . . . ..
»Corn_ meal + 0.1% hydro-
quinone . . . . . . . . . . . . . . . . . .. 6° C O 0 . . . . . . .. 84.0 . . . . . . . .
26°C O0 . . . . . . .. 87.0 . . . . . . .. 87 0
35° C 0 0 . . . . . . . . 71.4 . . . . . . . .

There are different kinds of cod liver oils and cod liver oil concen-
trates, and also other liver oils, so that there may be differences in the
stability of the vitamin A from different sources when added to mixed feeds.
Further tests were made, therefore, upon such different kinds of liver oils

-or concentrates as were obtainable. The procedure was the same as that
described above, and the feed used was corn meal. The results of tests

on 12 samples are tabulated in Table 6. The vitamin A lost in 2 weeks
ranged from 29 to 100%, while in 4 weeks the loss was from 79 to 100%.

The rate of loss of vitamin A was somewhat different with different oils,

but not sufficient to be of practical importance or to justify the use of any

-of themin mixed feeds which are to be stored over one t0 two weeks. We

do not consider it necessary to give the names of these products, but the
samples represented the various kinds of such materials on the market.

It was considered possible that vitamin A might be less stable in small
samples of 100 grams than in larger packages of 10 kilos. Experiments
were made to test this point. The procedure was similar to that with

;the 100 gram samples, except that larger quantities were used. 200 cc. of

14 BULLETIN NO. 557 TEXAS AGRICULTURAL EXPERIMENT STATION

cod liver oil high in vitamin A were mixed well with about 1000 grams of
white corn meal. This mixture was then diluted to 10 kilos with corn meal,

“mixed thoroughly in a mechanical mixer, placed in a small sack and stored

at room temperature. A smaller sample of 100 grams of the same mixture
was placed in an 8 oz. bottle and stored under the same conditions. A sack
of 10 kilos of the corn meal without the cod liver oil was stored under the
same condition.

Table 6. Loss of vitamin A at room temperature (26° C.) when 12 dilferent oils were added
to corn meal
_ _ Specto vitamin A in feeds Added vitamin
Liver oil or parts per million destroyed, per cent
Number concentrate added
of oil per cent _ _ Stored Stored
Beginning 14 days 28 days In 14 days In 28 days
61005 2.0 . . . . . . . . . . . . . . . .. 1.5 0.2 . . . . . . . . .. 86.7 . . . . . . . . ..

62435 2.0 . . . . . . . . . . . . . . . .. 6.8 0.2 . . . . . . . . .. 97.1 . . . . . . . . ..

61946 1.0 . . . . . . . . . . . . . . . .. 7.5 0.0 . . . . . . . . .. 100.0 . . . . . . . . ..

59875 2.0 . . . . . . . . . . . . . . . .. 2.9 0.9 . . . . . . . . .. 70.0‘ . . . . . . . . ..

43363 0.5 . . . . . . . . . . . . . . . .. 6.2 2.6 . . . . . . . . .. 58.1 . . . . . . . . ..

45551 0.5 . . . . . . . . . . . . . . . .. 3.8 2.7 0.8 28.9 78.9

67017 2.0 . . . . . . . . . . . . . . . .. 5.4 2.3 0.0 57.4 100.0

67092 2.0 . . . . . . . . . . . . . . . .. 0.9 0.0 0.0 100.0 100.0

67057 2.0 . . . . . . . . . . . . . . . .. 5.1 2.8 0.3 45.1 94.1

67261 0.5 . . . . . . . . . . . . . . . .. 5.2 2.6 0.1, 50.0 98.1

67333 0.5 . . . . . . . . . . . . . . . .. 4.7 0.9 0.0 80.9 100.0

67132 2.0 . . . . . . . . . . . . . . . .. 12.0 6.8 2.4 43.3 80.0

After definite periods of time had elapsed, samples were taken from the
large sample with a fertilizer sampling tube. Parts of all the samples
were analyzed at the same time. The results from the two mixtures are
tabulated in Table 7. There is only a slight difference in the rate of destruc-
tion in the large and small sample after 7, 14, and 28 days. These differ-
ences are not sufiicient to be of significance. The vitamin A in the large
samples was no more stable than in the small ones.

Table 7. The elfect of the size of the samples upon the loss of vitamin A from corn meal and
cod liver oil stored at room temperatures '
Vitamin A destroyed in storage
——per cent
Size of sample

7 days 14 days 28 days
Mixture A. 10 kilo sacks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54.1 70.6 91.9
100 gram bottle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 60.9 81.5 89.2
Mixture B. 10 kilo sacks . . . . . . . . . . . . . . . . . . . . . . . . . . .. 12.3 76.7 98.6
100 gram bottle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 70.8 100.0

The results presented here show that vitamin A, when mixed with feed.
mixtures, is rapidly destroyed and is practically all gone at the end of 4
weeks. Other workers have shown that vitamin A in cod liver oil may be
destroyed, sometimes in a short time, when left in bottles partly filled
with air (Table 1). When these oils are mixed with feeds, they coat the-
feed particles so that very large surfaces are exposed to air. It is, there-

LOSSES OF VITAMIN A AND CAROTENE FROM FEEDS DURING STORAGE 15

fore, reasonable that the vitamin A is rapidly destroyed, and that under
such conditions, antioxidants would be of no practical value in retarding
the oxidation. It would appear that if cod liver oil is to be fed in a mixed
feed, it should be mixed with the feed just before feeding, or not over one
week to ten daysbefore feeding. The liver oil itself should be stored in
completely filled containers kept at a low temperature.

Losses of Carotene from Yellow Corn, Alfalfa Products, and Other Feeds
During Storage

Work done in this and other laboratories already referred to (Table 2)
shows that carotene is lost from feeds during storage, especially at elevated
temperatures. In order to study the matter further, carotene was esti-
mated from time to time in samples which were stored at different temper-
atures, in small containers and in large quantities.

The results of the analysis of various feeds stored in pint and quart
fruit jars are presented in Tables 8 and 9. At room temperature there
were losses of 3 to 4% per month in 2 periods, 10 to 13% in 6 periods,
and 15 to 35% per month in 6 periods. Thus the losses may be high at
room temperatures.

Table 8. Loss of carotene in feeds during storage in fruit jars at room temperature

Approximate
Month Carotene Carotene rate of loss
Laboratory Description sample parts per lost, per month,
number analyzed million per cent per cent of
total at
beginning

41967 Dehydrated alfalfa leaf meal. . . July 53.8
Oct. 35.6 34 11

42622 Dehydrated alfalfa leaf meal. . . Sept. 66.7
Oct. 60.0 10 10

41730 Alfalfa meal . . . . . . . . . . . . . . . . . Mar. 30.6
' May 16.3 47 24
July 10.5 66 1O
Dec. 5.4 82 3

41736 Alfalfa meal . . . . . . . . . . . . . . . .. May 17.2
June 13.9 19 19
July 7.8 55 36

41745 Alfalfa leaf meal . . . . . . . . . . . . . May 53.2
Oct. 43.3 19 4

41727 Alfalfa meal . . . . . . . . . . . . . . . . . May 50.0
Dec. 5.4 89 13

60960 Alfalfa meal . . . . . . . . . . . . . . . . . May 9.7
July 7 . 5 28 12

60750 Alfalfa meal . . . . . . . . . . . . . . . . . April 17.5
May 14.6 17 17

-41729 Peanut hay . . . . . . . . . . . . . .  . . May 7.5
July 5.3 29 15

41724 Peanut hay . . . . . . . . . . . . . . . . .. May 11.8
Oct. 5.4 54 11

41105 Sorghum hay . . . . . . . . . . . . . . .. April 5.0
May 3.8 24 24

16 BULLETIN NO. 557 TEXAS AGRICULTURAL EXPERIMENT STATION

When stored at refrigerator temperature (Table 9) the losses per
month were at the rate of 0 to 4% in 12 periods, 5 to 9% in 2 periods and
10 to 14% in 7 periods. The losses are appreciably lower at refrigerator
temperature than at room temperature. Later work shows that the higher
losses of 10 to 14% may have been due to exposure t0 Warm air and stir-
ing, when the samples were taken out of the refrigerator from time to
time in order to secure portions for analysis.

Table 9. Losses of carotene in feeds during storage in fruit jars at refrigerator temperature

Approximate
Description Carotene Carotene rate of loss
Laboratory _ _ _ parts per st, per month,
number Stored in fruit Jars at refrigerator million per cent per cent of
temperature total at
beginning
43280 Dehydrated alfalfa leaf meal . . . . . . . . . . . . . 63.5 . . . . . . . . . .

Stored 1 month . . . . . . . . . . . . . . . . . . . . . . 55.3 13 13

Stored 2 months . . . . . . . . . . . . . . . . . . . . . . 53.2 16 3

43638 Dehydrated alfalfa leaf meal . . . . . . . . . . . . . 70.0 . . . . . . . . . .

Stored 1 month . . . . . . . . . . . . . . . . . . . . . . 61.3 12 12

Stored 2 months . . . . . . . . . . . . . . . . . . . . . . 55.6 21 9

Stored 3 months . . . . . . . . . . . . . . . . . . . . . . 52.3 25 4

Stored 4 months . . . . . . . . . . . . . . . . . . . . . . 53.8 23 0

43336 Dehydrated alfalfa leaf meal . . . . . . . . . . . . . 65.6 . . . . . . . . . .

Stored 3 months . . . . . . . . . . . . . . . . . . . . . . 56.3 14 5

44085 Dehydrated alfalfa leaf meal . . . . . . . . . . . . . 60. 9 . . . . . . . . . .

Stored 1 month . . . . . . . . . . . . . . . . . . . . . . 62.3 O

Stored 2 months . . . . . . . . . . . . . . . . . . . . . . 52.5 14 14

44048 Alfalfa meal . . . . . . . . . . . . . . . . . . . . . . . . . . . 36.8 . . . . . . . . . .

Stored 1 month . . . . . . . . . . . . . . . . . . . . . . 36.8 0 0

Stored 2 months . . . . . . . . . . . . . . . . . . . . . . 36.9 0 0

Stored 3 months . . . . . . . . . . . . . . . . . . . . . . 36.3 1 1

Stored 4 months . . . . . . . . . . . . . . . . . . . . . . 31.3 15 14

Stored 5 months . . . . . . . . . . . . . . . . . . . . . . 32.5 12 0

43576 Alfalfa meal . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.9 . . . . . . . . . .

Stored 1 month . . . . . . . . . . . . . . . . . . . . . . 16.8 11 11

Stored 3 months . . . . . . . . . . . . . . . . . . . . . . 12.9 32 l0

43204 Alfalfa meal . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.7 . . . . . . . . . .

Stored 1 month . . . . . . . . . . . . . . . . . . . . .. 18.3 2 I

Stored 2 months . . . . . . . . . . . . . . . . . . . . . 17.9 4 2

43789 Alfalfa meal . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.9 . . . . . . . . . .

Stored 4 months . . . . . . . . . . . . . . . . . . . . . . 10.7 17 4

44194 Buffalo grass . . . . . . . . . . . . . . . . . . . . . . . . . . 49.3 . . . . . . . . . .

Stored 2 months . . . . . . . . . . . . . . . . . . . . . . 36.3 26 13

44058 Mesquite grass . . . . . . . . . . . . . . . . . . . . . . . . . 42.2 . . . . . . . . . .

Stored 1 month . . . . . . . . . . . . . . . . . . . . . . 42.5 0 O

In order to study the stability of the carotene when the air was not
changed by opening the container, and stirring was avoided, samples of feeds.
were tightly packed in vials, of about 10 cc. capacity which were tightly
stoppered, and stored at refrigerator temperature. The contents of the
vials were discarded after a portion was taken from it for analysis. The
results of this work are given in Table 10. The losses of carotene per
month were 0 to 3% in 19 cases, 5 to 8% in 5 cases, and 12% in one
case. These losses were very low.

;-,,-t-a,y-v‘wyy.g,,.~.,,.. W, .,  . .

 

LOSSES OF VITAMIN A AND CAROTENE FROM FEEDS DURING STORAGE 17

Table 10. Losses of carotene during storage in tightly packed vials at refrigerator temperature
Carotene, Carotene
Laboratory Description parts, per st,
number million per cent
44397 Alfalfa hay meal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.3 . . . . . . . . . .

Stored 2 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.6 0

44276 Alfalfa hay meal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29.8 . . . . . . . . . .

Stored 1 month . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27.3 8

Stored 3 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28.3 5

44194 Buffalo grass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.0 . . . . . . . . . .

Stored 1 month . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.8 0

Stored 3 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.3 O

44184 Buffalo grass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 . . . . . . . . . .

Stored 1 month . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 4.5 O

Stored 3 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 2

44188 Buffalo grass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.2 . . . . . . . . . .

Stored 1 month . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2 12

44065 Feather sage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.2 . . . . . . . . . .

Stored 1 month . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.8 2

Stored 2 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.3 0

44482 Oak leaves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118.8 . . . . . . . . . .

Stored 2 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121.3 0

Stored 3 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124.0 0

Stored 4 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117.5 1

44386 Yellow corn meal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 . . . . . . . . . .

Stored 1 month . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 3

Stored 2 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 0

Stored 3_months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 6

44387 Yellow corn meal . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . 5.2 . . . . . . . . . .

Stored 1 month . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 0

44388 Yellow corn meal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 . . . . . . . . . .

Stored 1 month . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 0

Stored 2 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 7

44395 Dehydrated alfalfa leaf meal . . . . . . . . . . . . . . . . . . . . . . . . . 38.8 . . . . . . . . . .

Stored 1 month . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 42.5 0

Stored 2 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45.0 0

Stored 3 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.8 0

45223 Alfalfa leaf meal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115.0 . . . . . . . . . .

Stored 1 month . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117.5 0

Stored 2 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107.5 7

Stored 3 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.5 2

The storage at refrigerator temperature of feed firmly packed in
homeopathic vials seems to be a very good method of preserving the caro-
tene and is suitable for small quantities of feeds to be used for biological
assays for vitamin A.

In order to ascertain further the effect of exclusion of air on the
stability of carotene, portions of a sample of dehydrated alfalfa leaf meal
high in carotene were placed in homeopathic vials, the air displaced with
nitrogen and the vials tightly stoppered and stored at refrigerator tempera-
ture (6° C.) and 35° C. Other portions of this same sample were placed
in 8 oz. bottles, stirred twice a week with a spatula to allow the feed
to come into contact with air, and stored at the same temperatures.
All these samples were analyzed for carotene after definite periods of
time had elapsed. For each analysis of the samples in the vials, an un-

18 BULLETIN NO. 557 TEXAS AGRICULTURAL EXPERIMENT STATION

opened vial was used while the large samples Were Well mixed and a
small portion taken for analysis. The data obtained from this experiment
are tabulated in Table 11 and show that samples that are stored over
nitrogen and not allowed to come into contact with air lose carotene less
rapidly than samples that are stirred and aerated. However, storing the
samples in nitrogen is only slightly better than storing them firmly

packed in vials (Table 10). The effect of a high temperature is shown'

in Tables 11 and 12. In Table 12 the loss in the first period of one week
at 35° C. was from 22 to 38%, in the second week 2 to 9% and in the
third period of 2 weeks, 2 to 6%. The rate of loss during a period of 8
months was then about 4% a month with the alfalfa leaf meal and 1%
with the corn. In Table 11, the loss at 35° C. was 50 to 57% the first
month, 3 to 5% in the second month, and 13 to 16% in the third month,
after which it was 3% or less, except in one period. Thus the carotene
is lost rapidly when first exposed to a high temperature, and then much
more slowly. This is in accordance with most oxidation-reduction proc-
esses. There is usually a short inductive period, then a period of rapid
oxidation and a final period of slow oxidation. Also the carotene first
destroyed may be on the exterior portion of the material, and the caro-
tene destroyed later may be embedded in such a way as to be partly pro-
tected by plant material. It is also possible that fairly stable yellow
degradation products of carotene may be formed during storage, and since
the loss of carotene was estimated by loss of yellow color, there may
appear to be a small loss of carotene where there is really a relatively
large loss.

The carotene equivalent (as we term it) in yellow corn was destroyed
less rapidly than in alfalfa (Table 12). This may be due to the fact that
the active pigment in yellow corn consists for the greater part of‘ cryp-
toxanthin (40 to 70% according to our analysis), while in alfalfa it is
practically all beta carotene (17). However, it is to be noted that the
carotene in alfalfa meal No. 43070 was more slowly destroyed than that
in Sample 43005. The same factors that caused this difference may have
operated to a greater extent in the yellow corn, and the_ cryptoxanthin
content may have had nothing to do with the more rapid rate of destruction.

Because of the high vitamin A potency of some samples of alfalfa leaf
meal, they are diluted with corn starch in running biological tests, so as
to enable the quantity fed to be weighed more exactly. Since it is possible
that a greater loss of carotene might take place with these mixtures than
with the unmixed feed, the following experiments were made. One part
alfalfa meal was mixed with 9 parts of corn starch that had previously
been heated at 105° for 24 hours; portions of the mixture were placed
in 8 oz. bottles and stored at refrigerator and at room temperatures. For
a control equal amounts of the undiluted alfalfa meal were stored in
the same manner. The results of the experiment are given in Table 13.
The loss of carotene was considerably more rapid in the first 3 months
in the diluted samples than in the undiluted samples. During the fourth
month, however, more carotene was lost from the undiluted samples
than from the diluted samples. It is possible that the carotene may be

l9

LOSSES OF VITAMIN A AND CAROTENE FROM FEEDS DURING STORAGE

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20 BULLETIN NO. 557 TEXAS AGRICULTURAL EXPERIMENT STATION

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4.4 030E

LOSSES OF VITAMIN A AND CAROTENE FROM FEEDS DURING STORAGE 21

lost more rapidly from a mixed feed than from the same carrier of
carotene when not mixed with another feed.

It appears that samples of feed stored in a compact condition 10st less
carotene than those stored in a less compact condition and that those
stored at a high temperature lost more than those stored at a low tempera-
ture. These experiments were all made on small quantities of material.

It seemed possible that the carotene might be more stable when the
feed was stored in larger amounts than those used in the work reported
above. That such differences might occur was found in an experiment
with a sack of alfalfa leaf meal kept for experimental purposes. This
sack of meal weighing about 15 pounds was placed in a cold storage
(4 to 6° C.), and a small portion of it was placed in a fruit jar and stored
in a refrigerator (6°). Samples from both the sack and the jar were taken
for analysis from time to time. A large difference in the carotene content
of the two samples developed. The results are shown in Table 14, sample
43005. The sample in the fruit jar contained 14-0 parts carotene per million
at the end of 4 months and 108 at the end of 5 months, which were losses
of 32 and 47% respectively, while the large sample in the sack had not
lost any carotene at the end of six months, and had lost only 27% at the
end of 12 months. The difference may be due partly to the stirring of
the small sample and partly to exposure to warm air when it was
taken from the refrigerator for sampling.

Analyses of portions of large samples of alfalfa meal were made at
regular intervals on samples drawn from lots of several hundred pounds
used in work on the vitamin A requirements of animals. These samples
were stored in unheated rooms; sample 44086 was stored in a large dark
bin at the Spur Substation by the Department of Range Animal Hus-
bandry; 43051, 44675, and 45419 were stored in large sacks at the Feeding
and Breeding Station by the Division of Poultry Husbandry; 44504 was
stored in large sacks at College Station by the Division of Veterinary
Science; and 45318 was stored in sacks by the Division of Swine Hus-
bandry. The loss of carotene in these samples is shown in Table 14. The
loss of carotene took place slowly in the winter months and proceeded at a
relatively rapid rate during the summer months except in sample 44504, in
which the rate of destruction was as high in the Winter months as it
was in the summer months for other samples. The more rapid rate
of destruction in this sample must have been caused by something other
than the conditions of storage, for the temperature and method of storage
were apparently the same for all the samples stored at College Station.

If the data in Table 14 are compared with the data in Table 8, it is
seen that the carotene in the large samples was considerably more stable
than it was in the smaller samples stored in pint and quart Mason jars.
The reason for this is probably that in the large samples the alfalfa on
the exterior protects that in the interior from the air. The total amount
is, therefore, less exposed to the air, and consequently the oxidation of
carotene takes place less rapidly.

22 BULLETIN NO. 557 TEXAS AGRICULTURAL EXPERIMENT STATION
Table 14. The effect of storage upon the amount of carotene in large samples of alfalfa
Carotene
Mean Carotene Carotene lost,
Lab. Month monthly partsper , rate per
N0_ Description analyzed tempera- million total month
ture per cent per cent
43051 Dehydrated 7alfalfa leaf meal Dec., 1935 50.6F 75.0 0 0
stored in large sacks at room Jan., 1936 49.0 70.2 6 6
temperature at College Sta- Feb., 1936 45.8 65.8 12 6
tion‘ by ' the Division of Mar., 1936 65.2 65.6 13 1
Poultry Husbandry April, 1936 65.2 64.3 14 1
May, 1936 73.7 67.1 11 0
June, 1936 83.0 66.7 11 0
July, 1936 81.2 60.9 19 8
Aug.. 1936 83.6 44.7 40 21
Oct., 1936 65.2 40.7 46 6
44675 Dehydrated alfalfa leaf meal Oct., 1936 65.2 103.8 0 0
stored in large sacks at room Nov., 1936 54.8 92.5 11 11
temperature at College Sta- Dec., 1936 55.0 90.0 13 2
tion by the Division of Jan., 1937 50.2 91.7 12 0
Poultry Husbandry Feb., 1937 54.8 91.3 12 0
44086 Alfalfa hay stored in large dark July, 1936 82 .2 40.1 0 0
bin at the Spur Substation Aug, 1936 82.4 32.6 19 19
by the Division of Range Sept., 1936 73.2 32.7 19 0
i Animal Husbandry Oct., 1936 60.0 22.7 43 24
Nov., 1936 '49.6 23.2 42 0
Dec., 1936 45.7 22.9 43 1
Feb. 1937 43.5 21.9 45 1
Mar., 1937 47.6 23.8 41 0
44504 Alfalfa hay stored at room Sept., 1936 79.4 F 25.0 0 O
temperature in large sacks at Oct., 1936 65.2 20.9 16 16
College Station by the Divi- Nov., 1936 54.8 22.1 12 0
sion of Veterinary Science Dec., 1936 55.0 18.5 26 14
Jan., 1937 50.2 16.6 34 8
Feb., 1937 54.8 13.3 47 13
Mar., 1937 56.6 14.1 44 0
April, 1937 68.2 10.3 59 15
43005 Dehydrated alfalfa leaf meal At start . . . . . . . .. 205.0 0 0

stored in a 15 lb. sack in a 5th . . . . . . . . . 210.0 0 0

refrigerator at 4°—9° C 6th . . . . . . . . . 211.7 0 0

7th . . . . . . . . . 195.0 5 5

9th . . . . . . . . . 160.0 22 9

10th . . . . . . . . . 150.0 27 5

11th . . . . . . . . . 145.0 29 2

12th . . . . . . . . . 150.0 27 0

43005 Dehydrated alfalfa leaf meal At start . . . . . . . .. 205.0 . . . . . . . . . . . . . . . . ..

stored in quart fruit Jar in a 1st . . . . . . . . . 208.0 0 0

refrigerator at 4°—9° C 4th . . . . . . . . . 140.4 32 11

5th . . . . . . . . . 108.3 47 15

45419 Dehydrated alfalfa leaf meal Feb., 1937 54.8 F 180.0 0 0
stored in large sacks at room Mar., 1937 56.6 173.8 3 3
temperature at College Sta- April, 1937 68.2 155.0 14 11

tion by the Division of May, 1937 77.4 137.5 24 10

Poultry Husbandry June, 1937 83.7 133.3 ‘ 26 2

45318 Alfalfa leaf meal stored atroom Jan., 1937 50.2 F 65.0 0 0
temperature at College Sta- Feb., 1937 54.8 61.7 5 5

tion by the Division of Mar_ , 1937 56.6 60.7 7 2

Swine Husbandry April, 1937 68.2 56.3 13 6

May, 1937 77.4 52.2 2O 7

June, 1937 83.7 56.6 13 0

July, 1937 85.3 46.6 28 15

The results of these tests show that the losses of carotene are lower
if the dried material containing carotene is stored in a compact mass
than in a loose, mass exposed to the air. They also show that the loss

LOSSES OF VITAMIN A AND CAROTENE FROM FEEDS DURING STORAGE 23

is greater when the temperature is high than when it _is low. When ex-
posed to a high temperature, a considerable portion of the carotene may
be 10st rapidly, after which the remainder is lost more slowly. During
the winter months there may be comparatively slight losses of carotene
from yellow corn or alfalfa meal properly stored. In the summer, the
losses may be considerable, especially if the storage is in Warehouses
that are highly heated by exposure to the sun.

The Effect of Storage Upon Losses of Carotene
Added in Oil to Feed Mixtures

Since technical advances have made possible the preparation of strong
solutions of carotene dissolved in oil, at a price permitting their use in
mixed feeds, ‘some manufacturers of mixed feeds have begun to use
these preparations in their chicken feeds for the purpose of supplying
vitamin A potency. The question of the stability of such preparation
when added to mixed feeds has thus become a matter of practical importance.

In order to study the stability of carotene added in oil to mixed feeds,
a number of mixtures were tested. The desired quantity of carotene
solution Was mixed with 8 gm. of feed and then diluted to 100 gm.
with white corn meal. The feeds with which the carotene was mixed were
white corn meal alone, white corn meal with yeast, dried skim milk and
wheat gray shorts. These mixtures were stored both in the refrigerator
and at room temperatures and the amount of carotene determined at the
end of different periods of time.

Two samples of carotene were used. Sample No. 1 was a commercial
preparation of carotene in oil used by a manufacturer of commercial feeds.
Sample N0. 2 was a commercial crystallized carotene purified and dis-
solved in a purified cottonseed oil. A very small amount of chloroform was
used in sample No. 2 to dissolve the carotene before it was diluted with the
oil. -

The results are tabulated in Table 15. The experiment continued for
16 weeks with one series and 28 weeks with the other. From the data it
is apparent that the carotene in all the mixtures was more stable at
7° C. (45° F.) than at 28° C. (82° F.). The feeds stored at 7° C. (45° F.)
lost from 2 to 3% of their carotene in four weeks, 3 to 6% in 8 weeks
and 13% in 20 weeks. The feeds stored at 28° C. lost from 7 to 27% of
their carotene in four weeks, from 12 to 53% in 8 weeks, 17 to 67% in
12 weeks, and from 24 to 70% in 16 weeks. The losses of carotene from
the different samples were different, but the quantities lost in 8 weeks
at 28° C. from all the samples was appreciable. The presence of skim
milk powder, wheat gray shorts, or yeast which might be assumed to
contain stabilizing agents, did not increase the stability of carotene to

any practical extent. However, it is of interest to note that yeast de-i

creased the rate of destruction of carotene to a small extent.

Table 15 also shows that the commercial preparation of carotene (No. 1)
was more stable in the feed mixtures than our solution of carotene (No. 2).
The lower stability of our preparation may have been .due to the chloro-

24 BULLETIN NO. 557 TEXAS AGRICULTURAL EXPERIMENT STATION

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.2 2.3-.

LOSSES OF VITAMIN A AND CAROTENE FROM FEEDS DURING STORAGE 25

form used to help dissolve it in the oil. Tests were made in order to de-
termine whether the lower stability of carotene No. 2 was due to the ef-
fect of the chloroform. A saturated solution of carotene in Wesson oil
was prepared. The amount of carotene in this solution was then esti-
mated colorimetrically and enough added to 100 grams of corn meal to
make the mixture contain approximately 4 parts carotene per million.
To a portion of the above carotene solution was added the same amount of
chloroform as was added to our carotene preparation No. 2, namely, 0.05 cc.
of U. S. P. chloroform for each microgram of carotene. This solution was
then added to corn meal in the same proportion as the other. The two
feed mixtures were stored at room and at refrigerator temperatures and
the carotene determined at monthly intervals. The results are listed in
Table 16. In both tests the small amount of chloroform increased the
rate of the destruction of the carotene, especially during the first part
of the storage at room temperature. Consequently, there is very little
doubt that the chloroform in carotene No. 2 was the cause of its being
less stable than carotene No. 1. This is in accordance with some previ-
ous work in which it was found that a vitamin A preparation, ordinarily
very stable in methanol, was rapidly destroyed when dissolved in chloro-
form (6).

While the losses of the individual mixtures varied, the average rate of
loss of carotene at 28° C. for period of 2 weeks as shown in Table 15
was remarkably uniform, being from 4.0 to 4.6% of the carotene at the
beginning over a period of 24 weeks, for mixtures with carotene No. 1,
and from 8.1 to 9.3% per period for 12 weeks with carotene No. 2. In
both cases, the loss is less during the last period. Alfalfa leaf meal stored
at 35° C. (Table 12) lost carotene rapidly the first week, and then losses
decreased and later were at the rate of 4% a month. The carotene in oil
at a lower temperature of 28° was lost at a uniform rate of about 8%
a month. This would indicate that some of the carotene in the alfalfa is
protected by the plant tissue.

Appreciable amounts of carotene (from 17 to 67%) may be lost from
carotene dissolved in oil added to feeds stored at ordinary temperature
during a period of 3 months, and larger quantities are lost when stored
for longer periods of time. If carotene dissolved in oil is added to com-
mercial mixed feeds to supply vitamin A potency, there should be a
liberal allowance for losses during storage.

SUMMARY

1. When fish oils or their concentrates were added to feed mixtures to
increase their vitamin A potency, from 79 to 100% of the vitamin A dis-
appeared after 4 weeks at either 7° C. or 28° C. The use of hydroquinone
delayed the loss in the first week or two but the loss at the end of 5
weeks was practically the same as if it had not been used.

2. Feeds stored in large amounts lost their vitamin A from fish oils
as rapidly as feeds stored in small amounts.

26 BULLETIN NO. 557 TEXAS AGRICULTURAL EXPERIMENT STATION

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A: 0E5.

LOSSES OF VITAMIN A AND CAROTENE FROM FEEDS DURING STORAGE 27

3. The method used for the determination of carotene in feeds is
given.

4. Carotene in alfalfa‘ meal was found to be more stable when the
meal was stored at 6° C. than it was when stored at room temperatures.
The destruction at room temperature for samples stored in pint or quart
jars varied from 6 to 70% in 8 weeks and at refrigerator temperatures
from 0 to 26%.

5. The destruction of carotene in samples packed in homeopathic vials at
refrigerator temperature was very low, usually from 0 to 3% per month.

6. Alfalfa kept at a temperature of 35° C. lost carotene rapidly at
first, and then quite slowly, indicating that some of the carotene may
be easily destroyed, while a portion may be so protected that it is much
less easily destroyed.

7. A sample of alfalfa leaf meal diluted 1 to 9 with corn starch lost
carotene at a more rapid rate than it did when it was not diluted.

8. Large samples of alfalfa meal stored at ordinary temperatures lost
carotene slowly A during the winter months and comparatively rapidly
during some of the summer months. The losses during the. summer
months were less than for samples stored in pint and quart jars at
laboratory temperatures. Losses of as much as 40% were found after
storage for three months in the summer.

9. When feed mixtures containing carotene in oil were stored at 7° C.,
there was a loss of 2 to 3% carotene in 4 weeks of storage, 3 to 6% in
8 weeks, and 5 to 14% in 12 Weeks. At 28° C. there was a loss of 7 to
27% carotene in 4 weeks of storage, 12 to 53% in 8 weeks, 17 to 67% in
12 weeks, and 24 to 70% in 16 weeks. If carotene in oil is added to mixed
feeds to supply vitamin A potency, there should be liberal allowance for
losses in storage.

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