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CHOLINE IN CHICK RATIONS

by
Albert Wade Brant

M

A THESIS
Submitted to the Graduate School of Michigan
State College of Agriculture and Applied
Science in partial fulfilment of the
requirements for the degree of

MASTER OF SCIENCE

Department of Poultry Husbandry

1942

THESIS

ACKNOWLEDGEMENT
The author is indebted to Dr. P. J. Schaible, Research
Associate in Chemistry; J. A. Davidson, Associate Professor
of Poultry Husbandry; and C. G. Card, Professor of Poultry
Husbandry, Michigan State College. Their helpful guidance

and c00perative efforts are greatly appreciated.

i

142884

TABLE OF CONTENTS

Page
INTRODUCTION‘ -------------- 1
LITERATURE REVIEW --------- 2
CHEMICAL DETERMINATION
OF CHOLINE ----------- ll
CHICK EXPERIMENTS -------- 14
DISCUSSION --------------- 22
SUMMARY ------------------ 25

BIBLIOGRAPHY ------------- 26

INTRODUCTION

The existence of choline in the living organism has
been recognized for almost a century. Its importance as a
dietary essential, however, was demonstrated only within the
last ten years. At the present time there is evidence that
choline functions in at least three says; to stimulate the
formation of phospholipids, to make possible the production
of acetylcholine, or to supply labile methyl groups.

The experimental work reported herein has been along
two distinct lines: (1) an attempt to produce choline defi-
ciency symptoms in the chick, (2) the adaption of a chemical

procedure to the analysis of choline in poultry feedstuffs.

LITERATURE REVIEW*

It had been known for some years that depancreatized
dogs develOped fatty livers. However, it was not possible to
study this phenomonen prOperly until after the discovery of
insulin. Allen and co-workers (1924) and Fisher (1924) re-
ported that depancreatized dogs, even though maintained with
insulin, develOped fatty livers after some months. A nephro-
sis was also produced, secondary to the liver damage. The
addition of fresh beef pancreas or crude egg-yolk "lecithin"
(Hershey, 1930; and Hershey and Soskin, 1931) to the diet
prevented the deveIOpment of these pathological conditions.

Best §t_al (1952) produced dietary fatty livers in nor-
mal rats and found lecithin to be lipotrOpically active.
Further investigation by Best, Hershey, and Huntsman (1932)
and by Best and Hershey (1952) revealed that the component
of the lecithin molecule responsible was the nitrogenous base
choline. Later Best, Ferguson and Hershey (1953) found that
choline would also prevent fatty livers in depancreatized
dogs. Fatty livers, preventable by choline, have also been
deve10ped in mice (Best et a1, 1952) and chickens (Abbott
and DeMasters, 1940).

The condition referred to as "fatty" liver is character-
ized by an accumulation of large amounts of neutral fat in

the liver. As a result the organ becomes light in color,

*The literature on this subject has also been reviewed by Best
and Ridout (1959), Griffith (1941), McHenry (1941), Best (1941).

5.
soft, and much enlarged. Diets high in fat or sucrose, but
containing no added choline, result in "fat" type fatty liver;
those containing considerable cholesterol produce the "cho-
lesterol" type (Best g§_al, 1954; Channon and Wilkinson,
1954). A large quantity of choline is necessary to prevent
or cure the "cholesterol" fatty liver. The effect of choline
is on the glyceride fraction of the fat in the liver (Best
Ridout, 1959). Choline is considered lipotropic because it
promotes the formation of phospholipids (McHenry, 1941; Welch,
1956; Perlman and Chaikoff, 1959).

Choline, however, is only one of many lipotropic com-
pounds (Moyer and du Vigneaud, 1942). It is necessary that
cystine (Beeston and Channon, 1956) and thiamin (McHenry,
1957; Best and Ridout, 1958) be present in a low-choline diet
to produce the greatest concentration of fat in the liver.

Another important biological function of choline is its
ability to afford a labile methyl supply to the organism (du-
Vigneaud, 1941). Homocystine will support growth of rats fed
a methionine-free diet only in the presence of choline (du -
Vigneaud etwgl, 1959, 1959a; Welch, 1941; Klose and Almquist,
1941). The transfer of methyl groups from choline to homo-
cystine was theorized since triethylcholine does not support
growth in the presence of homocystine (du Vigneaud et_al,
1959, 1959a). Labelling the methyl groups of choline and
methionine with deuterium, these methyl groups were traced

throughout the body of the rat.

 

' ' 4 o
_The results of these methylation studies may be repre—

sented schematically as follows (du Vigneaud, 1941).

 

 

 

 

 

 

035 NH2
HO\| l
/NFCH2-CH2-OH C=NH
CH5 l I
CH3 NH-CHQCOOH
Choline Guanido acetic acid
4
P THs NHg
l
1“ ‘1‘” an
CH2 (CH5) CH2 if , 1 N-CHg-COOH
I I TCHs) 7 l
CHZM/ fH2 CH3
HC-NH2.______+,HC-NH2 -Creatine
I <————— L 1
COOH OOH NH
/ \
Homocysteine Methionine HN=C C=O
T CH5-N———-CH2
Creatinine

By using N15 in aminoethanol it has been shown that this
compound may be a precursor of choline in vivo if labile meth—
yl groups are available (Stetten, 1941, 1941a).

Hats, 21 to 26 days of age, maintained on a diet adequate
in all respects except choline develOped severe hemorrhagic
renal degeneration within 10 days (Griffith and Wade, 1959,
1940; Griffith, 1940). The renal hemorrhage disappeared lat-
er but renal pathology persisted. Hemorrhagic degeneration
was prevented by choline, methionine and betaine (Griffith
and Mulford, 1941). By restricting food intake Griffith (1941)
prevented signs of choline deficiency on a diet that produced

severe deficiency symptoms when fed ad libitum.

 

 

5.

Low-choline diets also produced deficient vagus function
in rats and a deficient formation of acetylcholine at the
nerve endings (Solandt and Best, 1959). The administration
of choline depresses hematOpoiesis induced in dogs by cobalt
(Davis, 1959). It prevents papillomatous lesions in the fona-
stomach of rats on a diet containing 89% white flour (Sharp-
less, 1940). It is indiSpensable for lactation in adult rats
and for growth and prevention of paralysis in suckling rats
(Sure, 1940). Small amounts of lecithin prevents the occurr-
ence of experimental arteriosclerosis caused by high-choles-
terol feeding of rabbits (Downs, 1955).

Choline and lecithin are concerned in maintaining acid-
base equilibria and excretion of phosphate by the kidney
(Bloor, 1940). Sharpe (1925) suggested that choline is used
in the elaboration of guanidine in the hens' egg during incu-
bation since choline decreased preportionately as guanidine
increased. Choline chloride, injected subcutaneously in rab-
bits and dogs, caused hyperglycemia but no glycosuria in rab-
bits with no effect on dogs (Underhill and Petrelli, 1929).

The earliest work indicating that choline was an access-
ory food factor in the diet of the domestic fowl was presented
by Abbott and DeMasters (1940) who employed a basal diet con-
sisting of skimmilk and alcohol-extracted rice plus vitamin
supplements. Rhode Island Red pullets, when placed on this
basal diet at 5 to 6 months of age, laid an average number
of 16 eggs; mortality was high. When birds were given one
gram of lecithin per day in addition, egg production was im-

proved and mortality decreased. Chicks reared from 2 weeks

6.
of age on the basal diet matured very slowly and many did not

come into production. AutOpsy showed they had large fatty
_livers and many aborted yolks. Additions of 25 mg., 50 mg.,
and 75 mg. of choline were given daily to three other groups
of birds on the basal diet. The last group produced the most
eggs, aborted the fewest yolks, and showed apparantly normal
livers. The 25 mg. and 50 mg. improved the basal diet, dut
not to the same extent.) Males reared on the lowest amount of
choline showed normal testes and produced live and numerous
sperm as contrasted to the underdeveloped testes and fewer
sperm produced in cockerels on the basal diet alone.

Additions of choline caused a marked reduction in the
fatty acid content of the livers. The smallest quantity of
fatty acids was found in the livers of birds fed the 75 mg.
of choline per day. It was also noted that the concentration
of fatty acids in the liver of males was lower than that in
females, on the same diet, with or without choline.

Jukes (1940) noted that choline stimulated the growth of
chicks on a simplified diet. In earlier experiments, he was
unable to prevent perosis in poults receiving as high as 1440
parts per million of manganese(Jukes, 1959). The diet was
composed of yellow corn meal, dried skimmilk, washed casein,
and alfalfa meal, plus vitamin and mineral supplements.

Since Hogan gtflgl (1940) had reported that liver contained
an alcohol-soluble organic factor which prevented a perosis,
an examination of the known B-vitamins was prompted. When

a mixture of vitamins including thiamin, riboflavin, nicotinic

7.
acid, pyridoxine, and pantothenic acid plus choline chloride
was added to the basal diet (Jukes 1959, 1940a, 1940b), no
perosis occurred. Testing the various ingredients separately
he found that choline was the active agent. Using a more
simplified diet consisting of glucose ("cerelose"), washed
casein, dried yeast, and soy bean oil plus vitamin and mineral
supplements, Jukes (1940a, 1940b) confirmed his earlier find-
ings. Betaine accelerated the occurrence of perosis and ar-
senocholine exhibited limited anti-perotic activity; neither
biotin nor inositol gave any preventive action. Table I sum-
marizes the compounds known at present to have antiperotic
and growth-promoting activity in poultry.

Table I
Compounds similar to choline which have been experiment-

ally tried in the diet of the fowl. Taken from a compilation
by Moyer and du Vigneaud (1942).

 

Preven—

tion of Fowl
Compound Perosis Growth
Aminoethanol - -
Arsenocholine + +
Betaine — -
Betaine aldehyde - i
Choline + +
Creatine - :
Diethylmethylhydroxy-
ethylammonium chloride + —
a,a-dimethy1choline - -
Dimethylethylhydroxy-
ethylammonium chloride + +
Lecithin + +

 

8.
Table I (Cont.)

 

Preven-

tion of Fowl
Compound Perosis Growth
Methionine - -
B-methylcholine q: -
Triethylcholine - -

Jukes (1940a, 1940b) reported that choline stimulated
growth in poults and that .l% of choline added to the basal
ration was sufficient to promote growth but .5% was required
to prevent perosis. Jukes (1941) tested various feedstuffs
to determine their anti-perotic activity, concerned with cho-
line content, with the results as shown in Table II.

Table II

Anti-perotic Activity (Choline Content) of Various Feedstuffs
Tested by Jukes

Level Anti-perotic

 

 

Material Fed activit of Compara-
_.fi_,_,,__ jl leveI,£gd._ __ Live rating

Yellow corn 65 as None 'Very poor

50 b** None
Wheat 65 a Very slight Poor

50 b Sligh
Barley 65 a Fair' Fair

40 b Fair
Oats 50 b None Very poor
Rice bran 25 a Fair Poor

20 b None
Wheat bran 25 a Very slight Poor
Soybean meal 25 a Good Good

40 b Very good
Sardine meal 17 a Fair Good

40 b Good
Alfalfa meal 15 a Very slight Poor
Cottonseed meal 40 b Fair Fair
Dried pork liver 5 a Fair Very good
Dried skimmilk 15 a None Very poor
Fish oil blend 2 a None Very poor
ane molasses 10 a Slight Poor

 

 

*a. Tested (Jukes, 1940b) with a basal diet consisting
principally of yellow corn, skimmilk, and casein and contain-
ing 0.1 percent of added MnSO4.

**b. *Tested, (Jukes, 1941) on a basal diet consisting
principally of glucose, casein, and yeast, and containing
0.05 percent of added MnSO4.

 

9.

It was observed (Jukes, 1941) that Wheat and especially
corn increased the severity of perosis. Lecithin prepared
from.egg yolk had an effect similar to choline, but methionine,
inositol, and creatine were ineffective. The anti-perotic
activity was removed from soy bean meal and meat scrap by
extraction with boiling alcohol (Jukes, 1941b).

Using a diet similar to diet b, Table II, Jukes (1940b)
(produced perosis also in White Leghorn chicks. Choline pre—
vented perosis and stimulated growth on this ration. The I
"rice factor"(Almquist pt 31, 1940) was supplied by adding
gelatin and sodium alginate to the basal diet since if it were
not included, no perosis resulted. Jukes (1941a) used gum
arabic to supply the carbohydrate component of the "rice fact-
or" and found creatine just as effective as gelatin. Since
Almquist and Mecchi (1940) had shown that a dietary deficiency
of creatine or its precursors resulted in muscular dystrOphy,
it was speculated that in the absence of creatine, muscular
tension Was so reduced that the hook joint remained intact.

McElroy and Jukes (1940) have observed perosis accompany—
ing the egg-white syndrome in chicks. Choline was ineffective
in preventing this condition but biotin, when injected, com-
pletely protected chicks (Jukes and Bird, 1942).

Hogan 2212; (1940) reported a perosis occurring on a sim-
plified diet in the presence of an adequate supply of manganese.
The factor, provisionally termed Bp, was present in the hot
alcohol extract of dried liver but not in the residue nor the
hot water extract. Choline prevented the perotic conditions

develOped (Hogan et a1, 1941). The ration employed consisted

 

F ||V|||

10.
of casein, corn starch, cane molasses, lard and cellulose plus
vitamin and mineral supplements. Hogan gt_gl (1941) describe
the following conditions as indicative of this type of pero-
sis: (l) the tendon of Achilles is out of position, and the
tarsometatarsal bone is rotated in such a way as to point the
toes to one side; (2) the tibial-tarsometatarsal joint is
greatly enlarged; (5) the tarsometatarsal bone is markedly
short and thick; (4) because of curvature of the tarsometar
tarsal bone the chicks are bow legged. Curvature of the tibia
is less pronounced; (5) the long bones are all shorter and
thicker than is normal, but the deformity would pass unnot-
iced on casual examination. The fifth set of symptoms was
found to be the most common. In addition, choline deficiency
causes a "frayed" condition of the feathers (Warren, 1958;,
Jukes, 1941b).

In studying growth factors, Hegsted 221%} (1941) encount-
ered a perosis in chicks fed a ration of dextrin, casein, soy
bean oil, cartilage, and yeast plus vitamin and mineral sup?
plements. The addition of .05% choline to the ration was
sufficient to prevent perosis but .2% choline gave the maxi-
mum growth. These chicks did not show fatty livers nor have
a low bone phosphatase (as reported for manganese-deficient
chicks by Wiese gt_gl, 1959).

Norris (1941) reported that chicks fed a simplified diet
deficient in choline would deve10p perosis. However, the
work at Cornell indicates that there is another organic anti-

perotic factor found in yeast. This conclusion was based on

 

11.
the finding that all the anti-perotic activity of brewers'
yeast could not be due to its very low choline content alone.

Record and Bethke (1942) also reported that choline pre-
vents perosis in chicks fed a simplified diet of corn meal,
peanut meal, casein, cane molasses, and soy bean oil plus
vitamin and mineral supplements. Growth was stimulated by
choline; soybean lecithin was found effective to the extent
of its choline content. The feathering condition of the
chicks was improved by added choline. It was found that .15%
choline in the ration gave maximum effects.

CHEMICAL DETERMINATION OF CHOLINE
Choline is an organic homologue of ammonium hydroxide

having the following structural formula:

H Choline has been determined by its oxi-
_ 0
CH5 I H H dation to trimethylamine, the formaticn
v>N-C-C-0H
CH5 CHH H of an insoluble complex with iodine,
5

the isolation of the chlorOplatinate

or chloroaurate, and its precipitation as the reineckate. In
the latter method choline is separated from the other reine-
ckates by virtue of its insolubility in water and ethyl a1-
cohol. When dissolved in acetone it forms a red solution
which can be measured colorimetriCally. The procedure final-
ly adOpted was that reported by Jacobi e§_gl (1941), modified
as follows:

About 2 grams of finely ground sample is weighed and
transferred to a dried Gooch crucible with an asbestos mat.
The crucible with the sample is placed in a Bailey-Walker

extraction flask containing 50 m1. of 99% methyl alcohol;

12.
a condensor is placed in the neck of the flask and the mater-
ial is extracted overnight on an electric hot plate. The
alcohol containing the extract is transferred to a 100 ml.
evaporating dish, covered with a watch glass, and the extract
evaporated to 2232 dryness. Care must be exercised to pre-
vent the material from becoming completely dry. Fifteen ml.
of saturated Ba(OH)2 are added to the evaporating dish, the
watch glass replaced and the mixture hydrolyzed for 2 hours
at 800 C. on a steam bath. The excess Ba(0H)2 is then neut-
ralized with glacial acetic acid using phenolphthalein as
indicator. The residue is filtered by suction on hardened
filter paper in a small Bachner funnel and washed with a few
ml. of distilled HOH. The filtrate is directed into a 50 m1.
centrifuge tube and 5 ml. of a freshly prepared 2% solution
of ammonium.reineckate in methanol is added.

The centrifuge tubes are covered with small watch glass-
es and placed overnight in the refrigerator to insure complete
precipitation. The precipitate is filtered on to hardened
filter paper in a small Bfiohner funnel and washed with 15 m1.
of 95% ethyl alcohol at 100 C. or less. A 25 m1. volumetric
flask is then placed in the suction flask to receive the col-
ored solution. A few ml. of acetone is then poured into the
funnel. After most of the choline reineckate is in solution,
suction is applied and the filter paper washed free of the
red precipitate by directing a stream of acetone from a wash
bottle upon it. The volume of the acetone solution in the
volumetric flask is brought to exactly 25 ml. and the color

intensity measured in a photelometer (Sheard-Sanford

 

l5.
photelometer, filters #552 and #401). In a similar manner
the reference curve on the next page was prepared by using
known quantities of choline. Table III lists a number of
poultry feedstuffs analyzed for choline.
Table III
The Choline Content of Certain Poultry Foodstuffs

 

Material Mg. Choline

Analyzed ,per Gm.
Ground yellow corn 0.40
Ground Whole oats 0.45
Ground wheat 0.70
Wheat bran 0.85
Flour middlings 0.95
Dehydrated alfalfa meal 1.00
Dehydrated leafy alfalfa meal 1.10
Soy bean oil meal (Expeller) 2.10
Dried skimmilk 0.85
Dried buttermilk 0.75
Sardine meal 2.00
Jhite fish meal 2.55
Meat Scraps 1.50
Yeast* 2.00
Blood meal 1.55
Tankage 2.00
Corn gluten meal 0.00

*Fleischmann's, type 2019

 

 

 

14.

CHICK EXPERIMENTS
Preliminary trials —- These were concerned with the pro-
duction of perosis upon manganese- and choline-deficient ra-
tions. Two groups of 25 S. C. White Leghorn chicks were fed
the rations given in Table IV.
Table IV
Percentage Composition of Rations Used in Preliminary Trials

Ingredients Rations

 

Ground yellow corn
Dried skimmilk
Meat scraps (55%)
Alfalfa meal (17%)
Salt

Fish oil (85 D)
Yeast

Corn starch
Casein

Bonemeal

00>

000101000
0 e w

HHm
uanomooq
O . O O O
mwmuwowuoq
O O O O O 0
com omo 00

Lot 1 received Ration A which is the manganese-deficient
diet used previously at this station (Schaible g§_gl, 1958).
Lot 2 received Ration B which was compounded to simulate Jukes'
(1940a) choline-deficient ration used to produce perosis in
poults. All chicks were brooded in a battery. Table V sums
marizes the incidence of perosis and periodic weights of these
two lots.
Table V

Growth, Mortality and Perosis in S. C. White Leghorn
Chicks Fed the Rations in Table IV

 

 

 

 

Lot 1 Lot 2
Age in Ave. No. of Chicks Ave. No. of Chicks
days Wt.-Gms* Total Perotic Wt.-Gms% Total Perotic
10 71 25 0 59 25 0
24 156 25 0 108 25 0
58 264 25 12 177 21 9
45 550 25 15 190 21 7
52 258 20 16

*Pullet cockerel average

 

15.

Ration A produced a considerably faster growth rate than
did Ration B. The incidence of perosis suddenly increased as
the chicks reached an average weight of 200 grams. This in-
dicates that the weight supported by the "hook" joint has a
direct bearing on its ability to remain in a normal state.

Three lots of 18 Barred Plymouth Rock chicks were treated
in a similar manner. Lot 5 received Ration A, lot 4 received
Ration B, and lot 5 received Ration B plus 50 p.p.m. of man-
ganese. Table VI gives the periodic weights of these birds
and records the incidence of perosis.

Table VI

Growth, Mortality, and Perosis in Barred Plymouth
Rgcks Fed the Rations in Table IV

A 15? 2‘

Lot 1” Lot 2“ _H Lot 5*
Age Ave. No. chicks Ave. No. chicks Ave. No. chicks
in Wt. Tot- Per- Wt. Tot- Per- Wt. Tot- Per-
Days Gmg. al otic Gms. a1 otic Gms. a1 otic

 

 

 

 

 

 

 

1 ‘58 18 o 58 18 59 18 o
8 os 18 o 59 16 63 18 o
15 94 18 o 79 16 96 18 o

22 145 18 8 105 15

29 205 18 14 145 14

56 270 17 15 187 15

45 542 17 16 250 12 1
*Very mild cases

145 18 1*
209 18 1%
292 17 1*
540 16 O

OQOIOOOO

As in the trial with Leghorns, Ration B did not produce
nearly as much growth as Ration A. However, the addition of
50 p.p.m. of manganese to Ration B resulted in growth as good
as that produced by Ration A. Ration B, therefore, was lack-
ing in some unknown growth requirement since Ration A was
known to be deficient in manganese. Ration B did not produce
perosis from choline deficiency since the added manganese gave
practically complete protection. Inasmuch as perosis was not
obtained by choline deficiency, it was decided to follow ex-

actly Jukes' rations and technic.

 

16.

Experiment I

Six lots of day-old Barred Plymouth Rock chicks were

placed in battery brooders.

Fifteen chicks were included in

each lot. The composition of the basal diet and of the salt

mixture are given in Table VII.

Table VII

The Basal Diet and Salt Mixture of Jukes Used in
Experiment I

 

 

Basal Diet Salt Mixture
Ingredient Parts Ingredient Part5
Cerelose 55.0 Bone ash 58.0
Vit.-free casein 18.0 CaCOg 20.0
Yeasts 6 O Iodized NaCl 22.0
Gum arabic 5 0 MgSO4 5.9
Soy bean oil 5 O KH2P04 7.5
Salt mixture 5 0 Ferric citrate 5.5
Fish oil (400 D) 0 5 MnSO4 2.0

CuCOg 0.1
aAnheuser-Busch, Strain G ZnO 0.1

Lots 1 and 2 received no gelatin but lot 2 received add-

ed choline chloride.

The remaining four lots received gela-

tin plus different amounts of added choline chloride. Table

VIII records the kinds and amounts of supplements added to

the basal ration for each lot as well as the weights at week-

ly intervals.

No perosis occurred.

Table VIII

Growth of Barred Plymouth Rock Chicks Fed the
Basal Ration in TableXKEIPlus Certain Supplements

1Average Weight in Grams

Age'in

days

8
15
22
29
56
45

Lot 1
" 2
I! 5
n 4 __
fl 5
ll 6
1

“WWW”

52

44 44

55 54

66 70

88 82

107 122
151%? 15099

Basal Ration
Basal Ration
Basal Ration
Basal Ration
Basal Ration
Basal Ration

+-+4-+'+

55 55 55 55
59 50 47 48
55 71 51 51
68 95 89 87

90 123 111 102
106 151 126 116
152 ‘3' 182 9(- 162 e:- 169 .x.

0.1% Choline Chloride

8% Gelatin

8% Gelatin + 0.1% Choline Chloriie
8% Gelatin + 0.2% Choline Chloride
8% Gelatin + 0.3% Choline Chloride

cockerel average

 

l7.

Barred Plymouth Rock chicks did not deve10p perosis upon
any of these Rations. The chicks, however, did not reach an
average weight of 200 grams in any case. It was noted in the
preliminary trials that, upon reaching this weight, the inci-
dence of perosis was markedly increased. Added choline, with
or without gelatin, produced some increase in weight. There
was some improvement in feathering and feather quality result-
ing from added choline.

Experiments II and III

In Experiment II, lots of 15 S. C. White Leghorn chicks
were fed the same diets fed lots 5 and 4 of Experiment I.
Mortality was so high no quantitative data are given. No ex-
planation for this mortality was apparent. No perosis was
observed.

Upon the recommendation of Jukes (1942a) 50 parts of
corn meal were substituted for an equivalent amount of cere-
lose in the basal ration given in Table VII. This modified
basal ration was fed to two lots of 15 S. C. White Leghorn
chicks. One lot received the basal ration and the other 0.1%
of added choline chloride. Mortality of unknown origin was
again so high that any interpretation of the results would
be questionable.and therefore data is not given. However,
the addition of corn meal tended to increase the growth rate
slightly. A few cases of perosis were noted in the group
receiving no choline.

Experiment IV
Since mortality terminated Experiments II and III before

the chicks were old enough to show a definite reaction to the

 

18.

ration, it was decided to repeat the Experiment again. Two
lots of 20 S. C. White Leghorn chicks were placed in batter-
ies and fed the modified basal ration of Jukes'. However,
neither lot received any added choline. Lot 1 received the
basal ration and lot 2 received the basal ration plus certain
synthetic vitamins. The added vitamins in mg./kg. were thia-
min, 2; riboflavin, 2; pyridoxine, 2; alpha tocopherol, 80;
2 methyl-1, 4-napthoquinone, 10. Table IX records the pero-
sis and weekly weights of the birds.

Table IX

Growth, Mortality,_and Perosis in S. C.
White Leghorn Chicks Fed Jukes' Modified Ration

“1111813111--.. .._..__ Lot 2 ,.
Age in Average Total Number Average Total Number
days Wt.in gm. No. ‘Perotic Wt.in gm. No. Perotic
l 55 O

 

20 O 56 20
9 57 19 O 60 19 O
16 75 15 1 78 14 2
25 110 14 4 115 14 4
50 155* 14 6 159* 14 11

*Pullet cockerel average

The added vitamins did not produce any increase in growth;
there was, however, an increase in the incidence of perosis.
The incidence of perosis and the growth rate were much lower
than that reported by Jukes using the basal ration containing
no corn meal.

Experiment V

The slow chick growth and infrequent occurrence of pero—
sis on the rations employed by Jukes prompted Experiment V.
The ration used was similar to that reported by Hegsted gt_gl
(1941). The Composition of this ration and of the salt mix-

ture are given in Table X.

 

19.

The cartilage was prepared by removing the cartilagenous
tips of the scapulae from beef cattle and grinding them as
fine as possible in an ordinary meat grinder. 'This material
was dried overnight at about 45° C. over steam pipes. The
dried material was then ground and incorporated in the ration.

Table X .

Hegsted's Basal Ration and Salt Mixture

 

_ Basal Ration Salt Mixture
Ingredient Parts Ingredient Farts
Cerelose 4607 03.005 - 5000
Vit.-free casein 18.0 KH2P04 52.0
Salt mixture 5.0 CaHP04°2H20 7.6
805'" bean Oil 500 MgSO4'7H20 1000
Cartilage 15.0 NaCl 17.0
Yeastw 10.0 Fe(CgH507)°6H20 2.7
Fish oil (400 D) 0.5 KI 0.1
Thiamine mg/k 2.0 MnSO4°4H20 0.5
Riboflavin mg kg 2.0 Zn012 0.04
Pyridoxine mg/kg 2.0 Cuso4-5H20 0.06

*Anheuser-Busch, Strain G

Two lots of 20 S. C. White Leghorn chicks were placed in
a battery and fed Hegsted's ration for 50 days. Lot 1 re-
ceived the basal ration unsupplemented; lot 2 received the
basal ration plus 0.1% of added choline chloride. The weekly
weights and incidence of perosis in these two lots are record-
ed in Table XI.

Table XI

'Growth, Mortality, and Perosis in S. C. White
Leghorn Chicks Fed Hegsted's Ration

Vgpt 1 _fi 1. _Lot 2
Age in Average Total Number Average Total Number
days Wt.in gm. No. Perotic Wt.in gm. No. Perotic

 

 

l 56 2O 0 56 2O 0

9 61 18 O 61 2O 0
16 91 14 6 121 15 O
25 145 14 6 185 15 - O
50 191* 14 11 261* 15 1**

*Pullet cockerel average %%Very mild case

 

20.

This ration produced a very marked perosis preventable
by choline. The percentage of perotic birds was greater and
the growth rate of the chicks fed Hegsted's ration far ex-
ceeded that of any chicks on Jukes' rations. In addition to

completely preventing perosis, 0.1% of added choline gave a

very marked increase in growth.

 

21.
In Figure I are the photographs of two typically pero-
tic chicks and two normal ones. The perotic chicks received
Hegsted's basal ration, whereas the normal chicks received
0.1% of choline in addition.
Figure I

Perotic Chicks Developed on Hegsted's Choline-deficient Ration
and Normal Chicks Produced When Choline Was Added

 

are typical cases of severe perosis.
are normal chicks with sturdy legs.

 

 

22.

DISCUSSION

The preliminary Trials confirmed Jukes' findings that
chicks would not deve10p perosis when fed the choline-defi-
cient ration which Jukes found produced perosis in poults.
The growth of Barred Plymouth Rock chicks in Experiment I
was much lower than that reported by Jukes (1940b) for S. C.

V White Leghorn chicks fed the same rations. On normal rations,
Barred Plymouth Rock chicks weigh more than S. C. White Leg—
horn chicks of the same age. No reason for_the difference
noted was apparent. However, the slow growth of the chicks
in Experiment I may account for the lack of perosis, since
perosis does not seem to occur as frequently in underdevelop-
ed chicks.

The high mortality in Experiments II and III did not
appear to result from the rations fed, since in subsequent
identical trials livability was very good. Although conclu-
sive results were not obtained, the substitution of corn meal
for part of the cerelose stimulated growth and increased the
incidence of perosis.

The addition of the synthetic vitamins to the modified
diet of Jukes did not materially accelerate growth, but the
incidence of perosis was increased. Two possible explana-
tions may apply here. One or more of the added vitamins may
aggravate choline deficiency or the added vitamins might re-
sult in a slight increase in metabolic rate resulting in
more acute choline-deficiency symptoms. The growth of the
S. C. White Leghorns in Experiment IV compares very closely

with that Obtained by Jukes (1940b, 1941a, 1941b) upon his

 

25.
ration containing no corn meal or added vitamins. The data
does not explain why the chicks in these Trials grew at a
slower rate than that reported by Jukes for chicks fed the
same or even more simplified rations.

It was difficult to record accurately the conditions of
feathers and feathering encountered during these Trials.
However, an improvement in feather quality and condition of
feathering appeared to result from choline added to the defi-
cient ration of Jukes'. The feather quality and feathering
condition of choline-supplemented birds was still much below
that of birds on ordinary rations. However, no difference
was noted between the two groups in Experiment V. Cartilage
may also contain a factor involved in feathering of chicks.
Such a conclusion is purely speculative, however, since Re-
cord and Bethke (1942) reported an improvement in feathering
obtained by adding choline to a diet much different from any
used in these Trials.

The growth of chicks in Experiment V slightly exceeded
that reported by Hegsted §t_gl (1941) for S. C. White Leghorns
and was far greater than any reported by Jukes for a like per-
iod of time. The very marked increase in growth of chicks
fed Hegsted's ration indicates very strongly that cartilage
contains a growth factor or factors other than choline not
present in Jukes' ration. Hegsted's ration was further im-
proved by added choline and perosis was completely prevented
by this addition. Cartilage must contain the factor or fact—
ors because Hegsted's ration differed but little from Jukes'

ration in all other respects.

 

24.

On the basis of the choline content of the various
poultry feedstuffs analyzed, it appears that poultry rations
composed of naturally occurring materials will contain a
large quantity of this factor. The chick starting mash de-
ve10ped at the Michigan Agricultural Experiment Station
(Spartan Chick Starter) contains approximately 0.1% of cho-
line. This quantity was more than sufficient in all of the
experimental rations. The most potent sources of choline in

poultry rations are soy bean oil meal, fish meals, and meat

scraps.

 

25.

SUMMARY

The chemical method for determining choline reported by
Jacobi gt_gl (1941) has been modified in order to determine
choline in poultry feedstuffs. The quantity of choline in
certain poultry feedstuffs is reported. On the basis of the
analyses presented, the Spartan Chick Starter was found to
contain an ample supply of naturally occurring choline.

A choline-deficient ration similar to that used by Jukes
to produce perosis in poults was found to be deficient in
undetermined growth requirements of chicks. Cartilage was
found to improve this ration, possibly because it contains
growth factors not present in gelatin or gum arabic.

Choline improved the growth of chicks fed the simplified
rations reported by Jukes and by Hegsted. Neither Barred
Plymouth Rock nor S. C. White Leghorn chicks developed pero-
sis on the diets reported by Jukes. S. C. White Leghorn
chicks did, however, deve10p perosis when this diet was modi-
fied to contain 50 parts of corn meal. Added choline im-
proved the feathering condition of chicks fed Jukes' rations,
but no improvement was noted in the chicks fed Hegsted's

ration.

‘

 

26.

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28.

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