Studies on Encephalomalacia in the Chick

1. THE INFLUENCE OF A VITAMIN E DEFICIENCY ON THE

PERFORMANCE OF BREEDING HENS AND THEIR CHICKS 1
E. P. SINGSEN, L. D. MATTERSON, ANNA KOZEFE AND R. H. BUNNELL
Department of Poultry Husbandry

AND

E . L . TUNGHERR

Department of Animal Diseases, Storrs Agricultural Experiment Station, University of Connecticut, Storrs
(Received for publication June 17, 1953)

T HE present status of the encephalo-

malacia problem in chicks has been
reviewed by Tungherr, Singsen and Mat-
terson (1952). Of particular interest is the
observation by Jungherr (1949) of the oc-
currence of encephalomalacia in day-old
hatchery-cull chicks and his suggestion of
an egg transmitted deficiency. Numerous
studies have been conducted in which vit-
amin E low diets were fed to normal
chicks, but very little work has been done
with adult birds. Dju el al. (1950) re-
ported on the utilization of pure a-, y-,
and S-tocopherols by laying hens fed a
purified vitamin E free diet, but did not
attempt to produce any chicks from these
hens. Adamstone, Krider and James
(1949) observed severe muscular incoordi-
nation and high mortality in the offspring
of sows fed a vitamin E low diet indicat-
ing that maternal nutrition is a worth-
while avenue of approach to the vitamin
E deficiency problem.
The most outstanding single difficulty
in the study of chick encephalomalacia has
been the failure of research workers to
1
This study was supported in part by grants from
the encephalomalacia committee of the American
Feed Manufacturers Association; the Yantic Grain
and Products Company, Norwich, Connecticut;
Eastern States Farmers Exchange, Inc., West
Springfield, Massachusetts; Merck and Co., Rail-
way, New Jersey; and the National Institutes of
Health.
192
produce the syndrome consistently on any
diet resembling a normal poultry feed.
Purified laboratory diets high in oil or fat
will produce a brain condition which his-
tologically is identical with field en-
cephalomalacia, and can be prevented
with vitamin E. In general growth on
these diets has been very poor, and there
has been a general reluctance on the part
of poultry men and feed manufacturers to
accept the laboratory-produced condition
as identical with field encephalomalacia.
This reluctance has been based largely on
two facts: (1) The condition was seldom
produced in the laboratory on a practical
poultry ration; and (2) the rations on
which the disease did occur, on the farm,
were believed to contain adequate amounts
of vitamin E.
The experiments reported here were un-
dertaken in an attempt to produce through
maternal nutrition, encephalomalacia sus-
ceptible chicks which could then be used
in a study of the factors effecting the field
condition.
EXPERIMENTAL MATERIALS AND
METHODS
A. Breeding Hens
The composition of the vitamin E low
basal ration fed to the hens is shown in
Table 1. On analysis it was found that this
diet contained 1.69 percent total fat and
2.54 mg. of total tocopherols per 100 gm.
 ENCEPHALOMALACIA IN THE CHICK 193

of ration. Because of the observations of TABLE 1.- -Composition of the vitamin E

low basal rations
Morgulis and Spencer (1936 a and b) on
muscular dystrophy, Jukes and Babcock Breeding
Chicks
birds
(1938) on encephalomalacia, and Scott
% %
(1951) on enlarged hocks in turkeys, that White corn meal 1 79.25 68.40
Soybean oil meal (50%) 5.00 10.00
a water-soluble factor might work in con- Crude casein 7.00
2.00
15.00
Brewers dried yeast
junction with vitamin E in the prevention Fish meal (60%) 2.50
Dicalcium phosphate 3.00 2.00
of deficiency symptoms, the basal diet was Ground limestone 3.00 1.50
Salt 0.50 0.50
also designed to be deficient in certain B- MnSO, 0.0125 0.0125
Vitamin A a n d D feeding oil 0.20
complex vitamins. Six lots of 25 Barred
Plymouth Rock pullets were housed in Vitamin supplement per 100 pounds of basal ration

floor pens on litter and fed the basal diet Vitamin A* (5,000A/gm.) 70.0 gm. 113.5 gm.
Vitamin D 3 (l,S00D/gm.) 25.0 gm. 45.4 gm.
with the variations shown in Table 2. Two Vitamin B12 Cone. (6 mg./lb.) 4 5 . 4 gm.
—
Rhode Island Red cockerels were placed Vitamin B12 (crystalline)
Niacin —
900 mg.
600 gamma
1,500 mg.
Riboflavin 200 mg. 227 mg.
in each pen, and were rotated every two Ca pantothenate
— 635 mg.
weeks so as to minimize the influence of Pyridoxine
— 227 mg.
P-aminobenzoic acid
— 227 mg.
the males on the performance of the fe- Inositol
— 227 mg.
Bio tin
— 4.54 mg.
males. The amount of a-tocopherol added Thiamine
— 90 mg.
Folic acid (crude 3%)
— 605 mg.
to the diets of lots 2, 4 and 6 was thought Vitamin K
— 45 mg.
Choline chloride
— 40 gm.
to be high, but was chosen on the basis of 1
A de-germed, de-branned product manufactured b y the
the work of Dju el al. (1950) so as to be Quaker Oats Co., Chicago, 111.
2
Synthetic vitamin A beadlets in gelatin manufactured by
more than adequate to meet the require- the Chas. Pfizer Co., Brooklyn, N . Y., and dispersed in white
corn meal.
ments of laying birds. After a preliminary
period of three weeks during which all
and liver (hepatonephrosis) and will be re-
birds were fed the basal ration, the lots
ported in detail later. Facilities for to-
were placed on their respective diets for a
copherol determinations were not available
period of 9 months (275 days), and com-
until the last two months of the experi-
plete records of egg production, mortality,
ment, at which time total blood tocoph-
fertility and hatchability were kept. All
erols of both the breeders and their day-
birds that died were autopsied at the di-
old chicks were determined.
agnostic laboratory of the Department of
All eggs incubated were candled on the
Animal Diseases. Mortality was due
7th and 18th day of incubation. Embryos
primarily to regressive changes in kidney
dying during the first seven days were
TABLE 2.—Dietary variables fed to the breeding hens in the encephalomalacia experiments

Lot No.
Diet variables

Basal ration (%) 100.0 100.0 98.0 98.0 100.0 100.0

Vitamin A and D oil (%) 2.0 2.0 — —
Alpha tocopheryl acetate (mg./lb.) 13.62 —
— 13.62 — 13.62
Ca pantothenate (mg./lb.) — — 5.00 5.00
Pyridoxine (mg./lb.) — — 2.27 2.27
P-aminobenzoic acid (mg./lb.) — — 2.27 2.27
Inositol (mg./lb.) — — 2.27 2.27
Thiamine (mg./lb.) — — .90 .90
Folic acid (crude 3%) (mg./lb.) — — 6.05 6.05
Vitamin K (mg./lb.) — — .045 .045
Ascorbic acid (gm./Ib.) — — .075 .075
Choline chloride (gm./lb.) — — .30 .30
194 SINGSEN, MATTERSON, KOZEFF, BUNNELL AND JUNGHERR

classified as early dead germs, and all oth- 0.50 mg. percent was present as gamma
ers, including those dead-in-shell at hatch- and delta tocopherols with the balance be-
ing time, as late dead germs. Samples of ing alpha tocopherol. The composition of
brain for histological examination, were the practical chick starting ration used in
taken at regular intervals during the mid- some of the tests appears in Table 3. The
dle of the experimental period, from 21 chicks were reared in electrically heated,
day-old pipped, but unhatched embryos, metal batteries, and given feed and water
and also from normal appearing day-old ad libitum. Any chick showing definite
chicks. signs of incoordination was sacrificed and
the brain removed for histological exam-
B. Chick Experiments ination. Some chicks died very suddenly
The chicks hatched from the six lots of without previously exhibiting symptoms
breeding hens were used in a series of ex- of incoordination. Brain samples were not
periments designed (1) to develop a simpli- saved from chicks judged to have been
fied chick diet which would support good dead more than an hour. I t must be kept
growth with low mortality, but which un- in mind, therefore, that the data on inci-
der conditions of physiological stress would dence of encephalomalacia presented in this
consistently produce a high incidence of report refer specifically to the specimens
encephalomalacia; and (2) to study the examined histologically and not to all
influence of maternal diet on the incidence chicks that died in the lot. The data are
of encephalomalacia in the chicks. The presented in the form of a fraction in
composition of the vitamin E low chick which the numerator is the number of
basal ration is shown in Table 1. This ra- positive cases of encephalomalacia, and
tion contained 1.1 percent total fat, ap- the denominator the number of brains
proximately 23 percent protein and 1.94 examined. Borderline or questionable cases
mg. percent of total tocopherols, of which were not included.
Approximately equal numbers of male
TABLE 3.—Composition of the practical chick
starting ration and female chicks were used whenever
possible in these experiments, and body
Percent weights are based on a 1:1 weighted aver-
Ground yellow corn 55.0 age.
Std. wheat middlings 10.0 Two oils were used as stress factors in
Soybean oil meal (44%) 15.0
Fish meal (60%) 7.5 the chick experiments reported here. The
Meat and bone scrap (50%) 2.5 first, an oil of fish origin having a very
Alfalfa meal (17%) 2.5
Corn gluten meal 2.5 high level of free fatty acids, had an offen-
Butyl fermentation solubles sive odor and an iodine number of 145.6.
(BY-500) 2.0
Liver meal 1.0 The second, a good quality, fresh, com-
Ground limestone 1.0 mercial brand of fortified vitamin A and D
Salt 0.5
Manganese sulphate 0.025 oil had an iodine number of 161.8. The
Vitamin A and D oil (300D- work of Dam (1943) has indicated that
l,500A/gm.) 0.10 the exudative diathesis and encephalo-
Vitamin D 3 (dry, 1,500 D/gm.) 0.025
Choline chloride (dry, 25%) 0.15 malacia-producing ability of an oil is re-
Niacin 900 mg. lated to the presence of highly unsatu-
Aureomycin cone. (Aurofac)1 0.20
Sulfaquinoxaline 0.0125 rated fatty acids which can be measured by
1
the iodine combining ability of the oil. A
Containing 1.8 gms. aureomycin and 1.8 mgs. completely rancid oil, having a low iodine
vitamin Bi /lb.
2
 ENCEPHALOMALACIA IN THE CHICK 195

TABLE 4.—The influence of a-tocopherol, vitamin A and D oil and a complex vitamin
mixture on breeding hens and their chicks

Lot No.
Breeding hens 1 2 3 4 5 6
Results (274 days)
% Egg production 58.9 61.5 58.1 58.2 64.4 57.4
% Mortality 45.8 36.0 20.0 36.0 20.0 12.0
Total no. eggs set 2,148 2,196 2,389 1,823 2,785 2,287
% Fertile 71.5 82.1 76.1 91.6 84.7 85.6
(The Following Data Are Based on
Fertile Eggs)
% Early dead germs 12.4 10.5 18.5 11.9 10.6 10.3
% Late dead germs 16.9 18.5 32.0 24.5 10.3 12.7
% Crippled chicks 1.7 3.8 1.7 2.6 1.4 1.7
% Hatchability 49.1 67.2 47.8 61.0 77.7 75.3
Blood Tocopherol Levels (mg. %)
Breeders 0.08 0.32 •— — 0.05 0.31
Day old chicks 0.16 0.74 0.16 0.70 0.39 1.18
Encephalomalacia at Hatching Time
21 day pipped embryos
No. examined 28 20 34 37 27 36
Pos. encephalomalacia 0 1 5 0 0 0
Day old chicks
No. examined 48 49 48 46 45 44
Pos. encephalomalacia 0 2 2 2 0 0

number was ineffective in producing vi- received both a-tocopherol and the com-
tamin E deficiency symptions in Dam's ex- plex vitamin mixture. The presence of a-
periments. tocopherol alone, did not appear to exert
any consistent influence on adult mortal-
RESULTS AND DISCUSSION
ity.
A. Breeding Hens
A summary of 29 hatches (Table 4)
A complete summary of the perform-
TABLE 5.—Rate of decrease of total blood tocopherols
ance of the six lots of breeding birds ap- of breeding hens changed from a normal
pears in Table 4. Egg production,expressed ration to the vitamin E low ration
on a hen-day basis, was fairly good in
all lots throughout the experiment. The Time in weeks
Hen
very low levels of total blood tocopherols No. 0 1 2 3 .. 7
apparently did not affect egg production, mg. percent
as lots 1 and 3 laid just as well as lots 2, 4 46447 0.59 0.28 0.25 0.19 0
and 6. Data obtained later in the year, 46653 0.36 0.24 0.07 0.07 0
46654 0.30 0.23 0 0.27 0.08
and presented in Table 5, indicate that the 46655 0.20 0.16 0.12 0.11 0.14
blood tocopherol levels of birds changed 46684 0.31 0.27 0.17 0.17 0.08
46694 0.38 0.23 0.22 0.17 0
from a regular breeding ration to the sim- 46701 0.22 0.15 0.55 0.15 *
plified vitamin E deficient ration dropped 46703 0.22 0.16 0.12 0.23 0
46734 0.63 0 0 0.10 0
to near zero in approximately seven weeks. 40736 0.09 0 0 0 0
It is apparent, therefore, that these birds 46740 0.36 * 0.13 0.15 0
46754 0.32 0.27 0.21 0.16 0
were deficient in tocopherols throughout
most of the experiment. Mortality was Av. 0.33 0.18 0.15 0.15 0.03
greatest in lot 1 and least in lot 6, which !
Missing value.
196 SINGSEN, MATTEESON, KOZEFE, BUNNELL AND JUNGHERR
spaced at weekly or bi-weekly intervals
throughout the experimental period re-
veals the interesting fact that fertility was
consistently higher in lots 2 and 4 which
received the tocopherol supplemented
diet, than in lots 1 and 3. Lots 5 and 6,
which received the complex vitamin mix-
ture did not show this response to tocoph-
erol. The percent fertility data for each
hatch were converted to "Angles Corre-
sponding to Percentage" (Snedecor, 1940)
and examined by analysis of variance for
significance. The " F " value obtained,
4.938, where 4.10 represents the 0.1 per-
cent level of significance, indicates that the
differences observed were very highly sig-
nificant. Although some differences exist
between lots 2, 4, 5 and 6, it is interesting
that the percent fertility in all these lots
was significantly higher than the fertility
of lots 1 and 3. This suggests that one or
more of the vitamins in the mixture added
to the diets of lots 5 and 6 was assisting in
the utilization of the tocopherol. In the
absence of the second vitamin(s), larger
amounts of tocopherol were required to
maintain fertility. There is a possibility
that the fertility differences observed were
actually due to very early embryo mortal-
ity, which could not be detected by can-
dling, but this question cannot be answered
with the data available.
Histological examination of the brains
of 21 day old, pipped, embryos and day-
old chicks revealed several positive cases
of encephalomalacia. This supports Jung-
herr's (1949) observation of the occur-
rence of encephalomalacia in day-old
hatchery-cull chicks and provides definite
evidence that some cases of field encepha-
lomalacia occurring at an early age may
be traceable to a maternal vitamin defi-
ciency rather than to a fault in the chick
ration. Breeder lot 3 produced the most
cases, although the number was not large
for any of the lots. The absence of positive
cases on the basal diet, lot 1, was probably
chance, since in later experiments three
chicks hatched from this breeder lot had
typical lesions at 1 day of age. The addi-
tion of a-tocoperhol to the diet of breeder
lots 2 and 4 did not prevent the appear-
ance of encephalomalacia in the embryos
and day-old chicks, but no cases were ob-
served from lots 5 and 6.
The data on total tocopherol levels in
the blood presented in Table 4, are based
on pooled samples of blood. The values for
the breeder hens represent at least ten
hens per lot, bled from the wing vein.
Heart punctures were tried, but resulted
in nearly 100 percent mortality probably
due to an abnormal cardiac condition
which may develop in the presence of vi-
tamin E deficiency. These cardiac abnor-
malities are being studied in cooperation
with Dr. Paul Sturkie of Rutgers Univer-
sity, New Brunswick, New Jersey, and
will be reported in detail at a later date.
The values for the day-old chicks repre-
sent over 100 specimens per lot that were
bled by heart puncture. The hens fed the
unsupplemented ration, lots 1 and 5,
showed very low blood tocopherol levels,
while those receiving a-tocopherol in the
diet, lots 2 and 6, had average values close
to the normal indicated in Table 5. Un-
published data reported to the authors by
Glista (1952) also indicated a total blood
tocopherol level for breeding hens fed a
normal ration of near 0.35 mg. percent.
The blood tocopherol levels of the chicks
at hatching time were two to three times
as high as those of the breeding hens, but
followed a definite pattern depending up-
on the diet fed the parents. Those from
the a-tocopherol deficient breeders lots 1
and 3, showed extremely low blood tocoph-
erol levels, while those from the supple-
mented lots, 2 and 4, approached the val-
ues expected for our normal chicks (0.78
mg. percent). Of particular interest is the
 ENCEPHALOMALACIA IN THE CHICK 197

marked increase in blood tocopherols of Further experiments on this phase of the

the chicks from breeder lots 5 and 6. These
breeder lots received the same amount of
dietary tocopherol as lots 1 and 3, and 2
and 4, respectively, but in addition they
received the vitamin mixture indicated in
Table 2. The marked rise in blood tocoph-
erols, based on pooled samples from a
large number of chicks, provides definite
evidence that one or more of the vitamins
added to the breeding ration is assisting in
the utilization of tocopherols. This obser-
problem are being planned.
B. Chicks Experiments
The simplified, vitamin E low chick
basal ration shown in Table 1 was chosen
following three pilot experiments involv-
ing variations in its protein and vitamin
content. Both normal and vitamin E defi-
cient chicks fed this ration showed good
growth, low mortality, and there were no
cases of encephalomalacia. The growth

TABLE 6.—The effect of different oils on the growth, mortality and incidence
of encephalomalacia of vitamin E deficient chicks

Lot No.
Diet variables
1 2 3 4 5 6

High free fatty acid oil — 1 2 2

Normal vitamin A and D feeding oil 2 2
Alpha tocopheryl acetate (mg./lb.) 13.62 13.62

Results (40 days)

Av. body wt. (gm.) 565 498 372 505 476 491
% mortality 8 25 50 67 8 8
Positive encephalomalacia1 0/0 0/1 3/4 3/4 0/0 0/0

1
The data are presented in the form of a fraction in which the numerator is the number of positive cases
of encephalomalacia, and the denominator is the number of brains examined.

vation substantiates the two factor con-
cept of vitamin E utilization suggested by
Morgulis and Spencer (1936 a, b), and
Jukes and Babcock (1938) and is also in
accord with our reported differences in
fertility among these breeding pens. Just
which vitamin(s) was improving the utili-
zation of tocopherol, either in the hen or
the embryo, cannot be stated from the ev-
idence now available, but it is worth not-
ing that Dam el al. (1951) and Zacharias
et al. (1950) obtained partial protection
against experimental encephalomalacia
with ascorbic acid, and that Milhorat and
Bartels (1945) reported that the substance
active in reducing creatinuria in patients
with progressive muscular dystrophy is a
combination of inositol with a-tocopherol.
was comparable to that obtained on the
practical chick starting ration shown in
Table 3. The effect of adding either the oil
having a high free fatty acid content or
the normal vitamin A and D feeding oil to
this ration is shown in Table 6. The chicks
used in this experiment were hatched from
breeder lot 5. The body weight was mark-
edly depressed by the high free fatty acid
oil, and to a lesser extent, by the normal
A and D oil. This growth depression was
partially overcome by adding a-tocopherol
to the diet. In evaluating the effect of the
diet on encephalomalacia in this and oth-
er chick experiments, it is necessary to
consider both the percent mortality and
the positive encephalomalacia, because as
indicated earlier, some chicks died very
198 SINGSEN, MATTERSON, KOZEIT, BUNNELL AND JUNGHERR
suddenly and were not discovered until it
was too late to use the brains for histologi-
cal examination. It is reasonable to as-
sume, however, that the bulk of the chicks
died from encephalomalacia since approxi-
mately two-thirds of the chick brains ex-
amined showed positive symptoms of the
disease, and since the unsupplemented
basal ration showed very low mortality in
the absence of oil throughout these experi-
ments. It is obvious that mortality in-
creased sharply with increasing amounts
of oil, and that this effect could be com-
pletely controlled by adding a-tocopherol
to the ration. The normal vitamin A and
D feeding oil actually produced a higher
mortality than did the high free fatty acid
oil. This finding is in accord with the high-
er iodine number of the former oil, and
also agrees with the findings of Dam
(1943). Since it was more effective as a
stress factor and also more typical of prod-
ucts available to the poultry industry,
the normal vitamin A and D feeding oil
was used in all experiments except the
first three.
There was some question as to the
length of the experimental period required
in order to get a true picture of the effect
of the diet. The relationship between age
and incidence of encephalomalacia (121
cases) in seven experiments varying in
length from 28 to 49 days is presented in
Figure 1. The period of highest incidence
was between the tenth and twentieth
days, with 88.5 percent of all cases occur-
ring by the 21st day and 98.35 percent by
the 28th day. It was decided, therefore,
that all future experiments would be ter-
minated when the chicks were 28 days old.
The period of highest incidence was ap-
proximately 10 days earlier than that re-
ported by Jungherr el al. (1952) for field
encephalomalacia and undoubtedly re-
flects the use of both vitamin E deficient
chicks and a vitamin E low chick diet. Of
interest also is the fact that of 104 positive
cases for which sex was recorded, 67 were
males and 37 were females, or a sex inci-
dence ratio of approximately 2:1.
40-
35-
30-
-1 2 25-
1<— 1o 2 0 -
oH <
X 15"
Q.
U. u 10-
o o 5-
.. UJz
S« 0-
AGE IN DAYS
FIG. 1. Average age and incidence of encephal-
omalacia experimental chicks (121 cases).
Six experiments were conducted in
which chicks from the six lots of breeding
birds were fed a uniform ration containing
2 percent of added oil as a stress factor, in
an attempt to measure the influence of the
maternal diet on growth and the incidence
of encephalomalacia. In three of these ex-
periments, Table 7, the simplified vi-
tamin E low chick basal ration was used,
and in the other 3, Table 8, the practical
chick starting ration shown in Table 3 was
fed. The body weight data did not at first
show any clear cut chick growth response,
but close study revealed the fact that in
13 of 18 comparisons in these six experi-
ments, the chicks hatched from hens
whose diet was supplemented with a-to-
copherol were heavier than their controls,
at the close of the experiment. Normal
chicks of a similar genetic background
(lot 7) attained approximately the same
body weight on these diets as the chicks
hatched from the vitamin E deficient
breeding lots.
The pattern of mortality in experiments
82D, G and J, Table 7, is fairly consistent.
In the absence of high levels of oil in the
breeder ration, the addition of a-tocoph-
erol to the breeder ration reduced the
mortality of the chicks. Comparing first
 ENCEPHALOMALACIA IN THE CHICK 199

TABLE 7.—The influence of maternal nutrition on the growth, mortality and incidence
of encephalomalacia in chicks fed a vitamin E low diet

Age Chicks from breeder lot N o . Normal

Expt. Chicks chicks
in Stress factor Results
No. days per lot
1 2 3 4 5 6 7
82-D 28 15 2 % High free Av. W t . 288 285 301 308
80
293-
60
334 —
fatty acid oil % Mortality 67 40 47 7
Positive E M ' 5/7 3/5 2/6 5/8 2/3 1/1
82-G 49 18 2 % Vitamin A & Av. Wt. 768 859 788 914 799 872 846
D feeding oil % Mortality 61 17 61 33 39 39 17
Positive E M . 1 9/U 1/2 4/8 1/4 5/6 6/6 2/2
82-J 42 12 2 % Vitamin A & Av. Wt. 695 748 602 738 745 634 695
(6 in lot 3) D feeding oil % Mortality 1 33 0 33 25 25 17 17
Positive E M . 1/4 0/0 1/2 3/3 1/3 0/2 2/2
% Mortality—3 expts. 56 20 51 47 42 22 17
Total Encephalomalacia 1 —3 expts. 15/22 4/7 7/16 9/15 8/12 7/9 4/4
1
Numerator of fraction is the number of positive cases of encephalomalacia and the denominator is the number of brains ex-
amined.

the chicks hatched from breeder lots 1 and
2, it is apparent that the addition of a-to-
copherol to the diet of breeder lot 2 defi-
nitely reduced the mortality of their
chicks. The incidence of encephalomalacia
among the chicks that did die was approxi-
mately the same as for lot 1, but the total
mortality was reduced to a level similar to
that of the normal chicks (lot 7) reared on
the same ration. This protective action of
a-tocopherol was not present between
chicks from breeder lots 3 and 4 due un-
doubtedly to the addition of 2 percent of
vitamin A and D oil to the breeder ration.
Comparing the performance of chicks
hatched from breeder lots 5 and 6, it is
again apparent that the a-tocopherol in
the diet of the breeding hens reduced the
mortality of the chicks.
The chicks in experiments 82F, H and
K, Table 8, which were fed the normal
chick starting ration, showed approxi-
mately one-half as much total mortality
as the chicks fed the vitamin E low ration,
and a much lower incidence of encepha-
lomalacia. The appearance of occasional
cases of encephalomalacia in chicks from
breeder lots 1, 3 and 5 indicated clearly,
however, that under the conditions of
stress used in these experiments, chicks
hatched from vitamin E deficient breeding
hens did come down with encephaloma-
lacia whereas those hatched from hens in
lots 2 and 6, fed the vitamin E supplement

TABLE 8.—The influence of maternal nutrition on the growth, mortality and incidence
of encephalomalacia in chicks fed a normal ration

Chicks from breeder lot N o . Normal

Age Chicks chicks
Expt. in per
No. Stress factor Results
days lot 4 6 7
1 2 3 5
82-F 31 14 2 % High free Av. Wt. 406 393 357 368 368 420
fatty acid oil % Mortality 36 7 14 36 21 0
Positive E M . 1 1/3 0/1 1/2 1/5 3/3 0/0
82-H 42 18 2 % Vitamin A & Av. Wt. 709 720 725 741 699 770 718
D feeding oil % Mortality 6 41 17 11 6 0 6
Positive E M . 1 0/1 0/7 1/3 0/2 1/1 0/0 0/1
82-K 42 16 2 % Vitamin A & Av. W t . 574 710 695 682 782 706 705
D feeding oil % Mortality 31 37 0 25 13 7 0

3
(12 in lot Positive E M . 1 1/5 0/6 0/0 0/4 1/2 0/1 0/0
(15 in lot
% Mortality—3 expts. 23 29 11 23 12 2 3
1
Total Encephalomalacia —3 expts. 2/9 0/14 2/5 1/H 5/6 0/1 0/1
1
Numerator of fraction is the number of positive cases of encephalomalacia and the denominator is the number of brains ex-
amined.
200 SINGSEN, MATTERSON, KOZEIT, BUNNELL AND JUNGHERR
did not. The only exception was lot 4,
where again it may be reasoned that the
high level of vitamin A and D oil in the
breeder diet interfered with the utilization
of the added a-tocopherol.
Of particular interest is the very low
total mortality of the chicks hatched from
breeder lot 6 and fed the normal ration
with added oil. It is obvious that chicks
hatched from hens whose diet contained
both the complex vitamin mixture and the
a-tocopherol supplement, were able to live
and show no encephalomalacia despite the
presence of the dietary stress factor. The
chicks hatched from lot 2 also showed no
encephalomalacia, but had a much higher
level of total mortality due to other vita-
min deficiencies in the breeding diet.
SUMMARY
The experiments reported here were
completed during the first year of a three
year study of the encephalomalacia prob-
lem in chicks. The data indicate that
very low levels of vitamin E intake, as re-
flected by low blood tocopherol levels, do
not affect egg production or adult mortal-
ity over a period of nine months. The
addition of a-tocopherol to breeding ra-
tions that were deficient in several other
vitamins, resulted in a highly significant
increase in fertility. This response to a-to-
copherol was not obtained when a mix-
ture containing ascorbic acid, vitamin K
and B-complex vitamins was added to the
breeding ration. Data on the total blood
tocopherol levels of day-old chicks defi-
nitely indicate that one or more of the vi-
tamins in the above mixture was assisting
in the utilization of tocopherols by either
the breeding hen or the developing em-
byro.
Several cases of encephalomalacia were
observed in unhatched embryos and day-
old chicks.
Chicks hatched from vitamin E defi-
cient breeding hens, and fed a vitamin E
low diet containing 2 percent of oil of fish
origin, consistently showed from 33 to 67
percent mortality. This mortality was
largely due to encephalomalacia, and
could be prevented by the additon of high
levels of a-tocopherol to the chick diet.
The addition of a-tocopherol to breeder
diets having low levels of vitamin A and
D oil markedly reduced the chick mortal-
ity from encephalomalacia, but did not
prevent it entirely when the chicks were
fed the vitamin E low ration containing
added oil. The addition of a-tocopherol to
breeders diets having low levels of vi-
tamin A and D oil did prevent the appear-
ance of encephalomalacia in the chicks
when the chicks were fed a normal ration
containing 2 percent of added vitamin A
and D oil. It was necessary to add both
a-tocopherol and the complex vitamins
mixture to the breeding ration in order to
reduce total mortality to its lowest level.
In 13 out of 18 comparisons, chicks
hatched from breeders whose diet was
supplemented with a-tocophreol were
heavier at the end of the experimental pe-
riod than chicks hatched from breeders
not receiving the supplement.
The incidence of encephalomalacia was
approximately twice as high in the male
as in the female chicks.
ACKNOWLEDGMENTS
The authors wish to thank Dr. Philip
L. Harris, Distillation Products Indus-
tries, Rochester, New York, for his assist-
ance in carrying out some of the vitamin
E determinations included in this report
and for supplying the vitamin E. We are
also indebted to the Chas. Pfizer Co.,
Brooklyn, New York for the synthetic vi-
tamin A, New England By-Products, Inc.,
Boston, Massachusetts, for the high free
fatty acid oil, and to Merck and Com-
pany, Inc., Rahway, New Jersey, for crys-
 METHIONINE METABOLISM IN THE H E N
talline vitamin B12 used in these experi-
ments.
REFERENCES
Adamstone, F., J. L. Krider and M. F. James,
1949. Response of swine to vitamin E deficient
rations. Ann. New York Acad. Sci. 52: 260.
Dam, H., 1943. Effect of cod liver oil and rancidity
on certain vitamin E deficiency symptoms. Proc.
Expt. Biol. Med. 52: 285-287.
Dam, H., I. Kruse, I. Prange and E. Sondergaard
1951. Substances affording partial protection
against certain vitamin E deficiency symptoms.
Acta Physiol. Scand. 22: 299-310.
Dju, M. Y., M. L. Quaife and P. L. Harris, 1950.
Utilization of pure a-, y-, and o'-tocopherols by
laying hens. Am. J. Physiol. 160: 259-263.
Glista, W. A., 1952. Unpublished data.
Jukes, J. H., and S. H. Babcock, 1938. Experiments
with a factor promoting growth and preventing
paralysis in chicks on a simplified diet. J. Biol.
Chem. 125: 169-181.
Jungherr, E. L., 1949. Ten-year incidence of field
encephalomalacia in chicks and observations on
its pathology. Ann. New York Acad. Sci. 52:
104-112.
Methionine Metabolism in the Laying Hen
1. EFFECT OF CHANGE IN THE DIETARY PROTEIN OR
TRYPTOPHAN LEVEL ON DEPOSITION OF S35 IN THE EGG 1
LAWRENCE J. MACHLIN
Bureau of Animal Industry, U. S. Department of Agriculture, Beltsville, Maryland
(Received for publication Tune 18, 1953)
A LTHOUGH the studies on the chem-
•£*• ical composition and nutritive value
of egg proteins have been voluminous,
comparatively few reports have been con-
cerned with the formation of egg proteins.
This subject has been reviewed by Jukes
and Kay (1932) and Conrad and Scott
(1938). Data on the dietary requirements
for amino acids for egg production are also
1
This project was supported in part by the U. S.
Atomic Energy Commission. Part of a thesis sub-
mitted by L. J. Machlin to Georgetown University,
Washington, D. C. in partial fulfillment of the
requirements for the Ph.D. degree, 1953.
201
Jungherr, E. L., E. P . Singsen and L. D. Matterson,
1952. The present status of the encephalomalacia
problem in chicks. Proceed. 89th Ann. Meeting
Am. Vet. Med. Assn.: 301-310.
Milhorat, A. T., and W. E. Bartels, 1945. The defect
in the utilization of tocopherols in progressive
mucular dystrophy. Science, 101:93.
Morgulis, S., and H. C. Spencer, 1936a. A study of
the dietary factors concerned in nutritional
muscular dystrophy. J. Nutrition, 11: 573-591.
Morgulis, S., and H. C. Spencer, 1936b. Metabolism
studies in nutritional muscular dystrophy. J.
Nutrition, 12: 191-204.
Scott, M. L., 1951. Studies on the enlarged hock
disorder in turkeys. III. Evidence of the detri-
mental effect of fish liver oil and the beneficial
effect of dried brewers yeast and other materials.
Poultry Sci. 30: 846-855.
Snedecor, G. W., 1940. Statistical Methods Applied
to Experiments in Agriculture and Biology,
3rd. ed., The Iowa State College Press, Ames,
pp. 382-383.
Zacharias, L. P. Goldhaber and V. E. Kinsey, 1950.
Vitamin E deficiency in chicks. I. The effects of
dietary supplements on plasma tocopherol levels
and vitamin E deficiency symptoms. J. Nutri-
tion, 42: 359-373.
meager. It was thought that a study of the
fate of radioactive labeled amino acids in
the laying hen would aid in the elucidation
of the mechanism of egg protein formation
and possibly provide a basis for a new ap-
proach to the determination of amino acid
requirements.
In the experiments reported here, the
uptake of S36 in the egg after administra-
tion of S35-labeled methionine was deter-
mined. In addition, the effect of reducing
the level of protein or tryptophan in the
diet on subsequent S36-deposition in the
egg albumen was studied.