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r ‘ :-
_ , \ ‘ : -
- w; _ _.;_ ._ ‘ - F .__ - _ _ E
. - This is to certify that the -’
: thesis entitled I"
f? I
, .« EFFECT OF DIETARY PE‘LTICILLIN
: ON SOME B VITAMIN REQUIRE-EMS
x" IN THE CHICK
I" presented bl]
\ Jr 1 f
l . David. A. Libby ;
‘ _ has been accepted towards fulfillment .
.~ \. of the requirements for
J Master of §gieng§ degree ians‘bandry ,
.‘w I
' 4 g M I
#1— ! t -
6*}. Major professor ‘I
i' " Date J’ //’/€a !
:’ '1‘

 

 

 

 

333% OF DIETARY .'.-;IICILLIH c2: SOLE
B vmezm REQUIREEITS

IN “2'23 CHICK

by

David A. Libby

A TEESIS
Submitted to the School of Graduate Studies of I'inchigan
State College of Agriculttme and Applied Science
in partial fulfillment of the requirements
for the degree of
I-LASTER OF SCIEJCE
Department of Poultry Htsbandry

1952

TH 55$

“S

\ ".

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\

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‘4‘ (:1

x .
\J

Acknowledgement

The author wishes to express his appreciation to Doctor A, C.
Groschke and Professor C. G. Card of the Department of Poultry
Husbandry, and Doctor Re J. Evans, Agricultural Chemist of Michigan
State College, for their guidance, assistance and cooperation in
making this work possible.

The crystalline vitamins and procaine penicillin were supplied

by Merck and Company, Rahway, New Jersey. The author wishes to thank

this concern for generously supplying these materials.

”Wt-1.538 1

my!

HEIRS 03' 0031113?

I Z:TRODU T I QII O O O D O O O O O O O O O O O O O O O O .

ELECTED RaiLREKCES CH RE EXT AKIIBIOTIC STUDIES

1- “!th '1- . .1?
IL .L‘\ kl llLArI 4L Cd 0 n o o o o o o o o o o o o o o 0 o

m ~r~*.;*\"*3'7

to y 1‘
‘as U Lbs-‘1 . o o o o o o o o o o o o o o o o a I o o 9

WOW v‘ Tg—afl-m

nnrnnlulhi . The Effects of Dietary Penicillin on
the Response of Chicks to Graded Levels
of Calcium Pantothenate . . . . . . .

Selected References on Pantothenic Acid . . . . .

Results and Discussion . . . . . . . . . . . . .

“A!

EKinniMZEl 2. The niiects of Dietary Penicillin on
the Response of Chicks to Graded Levels
of 1‘: 13C in 0 0 o o o o o o o o o o o . .

Selected References on Niacin . . . . . . . . . .
Results and Discussion . . . . . . . . . . . . .
Oh: Tram ? W‘ cm

the Response of Chicks to Graded Levels
Of Clloline O I O O O O O O O O O O O 0
Selected References on Choline . . . . . . . . .
Results and Discussion . . . . . . . . . . . . .
ZIPlilhifiT b. The Effects of Dietary Penicillin on
the Response of Chicks to Graded Levels
Of Rib Ofla‘rin O O O O I O O O C O O 0
Selected References on Riboflavin . . . . . . . .
Results and Discussion . . . . . . . . . . . . .

SL1 3 my 0 o a o o o o n o o o o a g o o o o o o a o o

BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . .

Page

10

10

37
37
39

1&6

LIST CF TABLES AID FIGUPSS

Table l. Basal Diet . . . . . . . . . . . . . . . . . . .

Table 2. Effects of graded levels of calcium pantothenate
with and xvithout penicillin on body weight, feed
efficiency and the incidence of deficiency
symptoms . . . . . . . . . . . . . . . . . . . .

Table 3. Effect of graded levels of niacin with and withr
out penicillin on body weight. feed efficiency
and the in ncidence of deficiency s5 mptoms . . . .

F3
m

ble h. Effect of graded levels of choline with and
without penicillin on body weight, feed efficien-
cy and the incidence of deficiency symptoms . .

Table 5. Effect of fraded levels of riboflavin with and
without penicillin on body weight, feed efficien-
cy and the incidence of deficiency symptoms . .

Figure 1. Response of chicl :s to graded levels of ca lciim
pe.ntothenate with and without penic1llin . . .

Figure 2. Comparison of control chick with calcium pan-
tothenate deficient chicks . . . . . . . . . .

Figpre 3. Chicks showing calcium pe.ntothenate deficiency.
(Dermatosis of beak and eyes) . . . . . . . .

Figure 4. Ch iels showing calcium pentothenate deficiency.
(Dermatosis of feet and hooks) . . . . . . . .

Figure 5. Typical perosis produced by pantothenic acid
deficiency . . . . . . . . . . . . . . . . . .

Figure 6. Re ponse of chicks to gr ded levels of n1 iacin
with and without penicillin . . . . . . . . .

Figure 7. Response of chicks to graded levels of choline
with and without penicillin . . . . . . . . .

LIST cr TABIES AZTD Helms (cont.)

Page
Figure 8. Comparison of control chicks with choline
deficient chicks . . . . . . . . . . . . . . 35
Figure 9. Typical perosis induced by a choline
deficiency . . . . . . . . . . . . . . . . . 36

Figure 10. Response of chicks to graded levels of ribo-
flavin with and without penicillin . . . . . 42

EITECT or DIETARY mmcILLm on s it?
B VIM-nu murmmvrs

Ill TILE CHICK

IETRODUCTICN

The science of nutrition is a relatively new one when compared
with the evolution of some of the other sciences such as mathematics,
astronomy, physics and chemistry. In its earliest phase, it was
generally'believed that the nutritional requirements of animals were
satisfied by three classes of compounds, namely, carbohydrates, fats
and proteins, tOgether with water and necessary minerals. Eijkman
(1897) however, was one of the earlier investigators to report the
existence of an additional dietary principle. His work showed that
rice polishings contained a nutrient which prevented a peculiar kind
of paralysis in chickens which was caused by feeding a diet of pol-
ished rice.

Hopkins (1906) extended this concept when he stated, "No animal
can live on a mixture of pure protein, fat and carbohydrate, and even
when the necessary inorganic minerals are carefully supplied, the
animal cannot flourish. The animal body is adjusted to live either
on plant tissues or other animals, and these contain countless sub-
stances other than protein, carbohydrate and fat".

It remained for Funk in 1911 to clarify further these substances
as essential factors in the diet of the animal. Funk chemically

analyzed the principle reported by Eijkman and located an "amine"

group, and chose the name "vital-amine", later shortened to "vitamine"
and now referred to as "vitamin“.

Later workers discovered essential substances incorporated in the
lipid portions of plant and animal tissues indicating that two classes
of vitaminic factors were involved; one a fat-soluble principle, and
the other a water—soluble principle.

Within the last two decades, progress was rapid in the isolation
and identification of most of the water-soluble vitamins. The latest
B vitamin to be elucidated was vitamin 312 in 1948. Extensive work
with the Animal Protein Factor, a complex of which vitamin 312 has been
shown to be the most important member, disclosed complications indicat-
ing a multiple nature of this factor. In addition to vitamin 312, some,
but not all animal protein factor supplements were shown to possess
extra growth stimulating principles. Independent work by several inves-
tigators however, soon showed that the presence of small amounts of
antibiotics which were present in certain animal protein factor supple—
ments were responsible for the hitherto unexplained extra growth.

Antibiotics are products of living organisms which inhibit the
growth or destroy other living organisms. Examples are penicillin,
streptomycin, aureomycin and terramycin. Antibiotics have been shown
to increase the rate of growth of poultry and swine, and to increase
the efficiency of feed utilization in these animals. Antibiotics will
decrease, but not eliminate the vitamin 312 and other water-soluble
vitamin requirements, exert a "protein-sparing" effect, and increase
livability. The exact mode of action of antibiotics is unknown, but

it is generally assumed at the present time that the growth stimulation

is an indirect effect, mediated in some manner through changes exerted
on the intestinal microflora. This may result in a suppression of
"unfavorable" types of bacteria which would compete with the host for
ingested nutrients, or ma also aid in the establishment of "favorable"
types which.would synthesize vitamins and other factors required for
the well being of the animal.

It has also been speculated that antibiotics might stimulate
growth as a result of their cleansing action on the intestinal lumen,
thereby creating a greater unobstructed surface area, resulting in
more efficient absorption of nutrients by the villi.

Regardless of the fact that the exact growth stimulating mech-
anism of antibiotics remains unknown, antibiotics are playing an
important role in the feeding of livestock. It can safely be stated
that practically all manufactured feeds prepared for young growing
chickens and turkeys today contain one or more antibiotics.

Inasmuch as the exact mechanism of antibiotic growth stimulation
is unknown, it seemed desirable to obtain some information which in—
directly, at least might lend support to one theory, namely, that
antibiotics stimulate growth as a result of limiting the growth of
intestinal organisms which compete with the host for ingested nutri~
ents. In such a situation the host animal should be favored because
a greater quantity of nutrients would be available for growth. Fur-
thermore, it would appear that if this concept of host-microorganism
competition were rue then the feeding of an antibiotic should show
a marked sparing action on selected vitamins which may be marginal

in the diet.

This study was initiated to determine the effect of penicillin
on the growth response of chicks to graded levels of calcium panto-
thenate, niacin, choline and riboflavin, and to determine the extent
to which penicillin might reduce the chicks' requirements for these

vitamins.

SELECTED “WmREK ES ON RECENT AETIBIOTIC STUDIES

N hUTdITICN

Briggs, Luckey Elvehjem and Hart (194M), in work with chicks on
purified diets deficient in certain vitamins contained in liver pre~
parations, showed additional growth stimulation due to the inclusion
of sulfasuxadine in the diet. Subsequently Moore and co-workers
(19b6), showed similar growth stimulation in chicks fed purified diets
where sulfasuxadine and in addition, streptomycin were fed.

Stokstad and co-workers (l9h9), reported the animal protein
factor to be multiple in nature, containing an unidentified chick
growth factor in addition to vitamin 312. The unidentified chick
growth factor was contained in a fraction from Streptomyces aureo—
faciens cultures. In further work on this unidentified chick growth
factor, Stokstad and Jukes (1950), reported it to be aureomycin,
which was found to increase the rate of growth of chicks.

Groschke and Evans (1950), in an independent study confirmed
the work of Stokstad and Jukes (1950;, by showing that the growth
effects resulting from feeding an animal protein factor supplement
could be duplicated by feeding supplements of aureomycin or
streptomycin.

Oleson and co-workers (1950), showed aureomycin and vitamin 312
to have a sparing action on each other, while Stoke ad and Jukes
(1951), showed aureomycin to have a sparing effect on vitamin 312
in some instances.

The effects of antibiotics on sparing vitamins was further shown

by Linkswiler et a1 (1951). These workers showed aureomycin had a

sparing action on the requirement of the rat for pyridoxine.

Lih and Bemmann (1951), using rats, demonstrated that aureomycin,

’13
'3

e.ici11in and streptomycin, but not terramycin or chloromycetin would
spare the vitamins thiamin, riboflavin and pantothenic acid. The anti-
biotics were most effective in the diets that contained onLy enough
vitamin for half maximum growth, and the growth responses due to the
antibiotics were approximately equal to those observed when the vitamin
content of the diet was considered adequate.

Eiely and March (1951), using a practical type diet, indicated
that sub-Optimal levels of folic acid, riboflavin and nicotinic acid

may be adequate for chicks when aureomycin is included in the diet.

Procedure

Four experiments were conducted in this study. Day old, straight—
run Rhode Island Red chicks were used in each experiment. The maternal
diet consisted of a 20 percent protein, practical-type breeder mash
complete in all known essential nutrients. A grain mixture of corn and
wheat was fed with the mash in an amount to equal one—half of the total
daily feed intake.

Twelve groups, of eight chicks, comprised each experiment. The
chicks were segregated at random, wingbanded and placed in electrically
heated.battery brooders with raised wire floors, and fed the experi-
mental diets for a four week period. Feed and water were kept before
the chicks at all times. The basal diet used in these experiments is
given in Table 1.

The batteries used in this study were equipped on each side with
a.bank of 40 watt bulbs, vertically suspended at mid-section. This
arrangement allowed equal illumination of each deck level. Continuous
lighting was provided throughout each experiment. The room temperature
was main ained between 80° and 85° Fahrerflieit. All chicks were weighed
at weekly intervals, kept under daily observation and examined closely
for the classical deficiency symptoms.

At the end of the four week period, all chicks were sacrificed
by dislocating the neck. The hearts and livers were removed, the
livers weighed and both organs were then frozen on dry ice. The frozen
tissues were stored in a deep freeze compartment to be assayed for B
vitamins at a later date. These assays are part of another study and

are not reported in this dissertation. At the end of each experiment

Table 1

Composition of Basal Diet

 

Eercent

Cerelose 61.0
Gelatin 10.0
Casein (Vitamin Test) 18.0
*Mineral Mixture 6.0
Soy Bean Oil (Crude Degummed) 3.0
Fish 011 (@001). 2000A) 1.0
**Vitamin Mixture O.#
Methionine 0.3
Choline Chloride 0.2
Inositol 0.1

 

* Mineral Mixture: (in grams) 021003 1.2100, csajPoLp 1.7000, KZHPO4
1.0000, 2:2.ch .9000, 172:2qu .6000, MgSOb’°7320 .5000, resoLP-mzo
.0570, Mason-£20 .0250, K1 .0050, 011504-5320 .0020, zn012 .0009,

** Vitamin l-iixturel: (mg./kilo) Riboflavin 8.0, Thiamin E01 4.0, Cal-
cium Pantothenate 20.0, Ryridoxine HCl 6.0, Niacin 100.0, Biotin
.2, Folic Acid 1.0, Menadione 1.0, ArtOCOpheryl Acetate 5.0, 312
(NaCl triturate) 30.0, Para.Amino Benzoic Acid 2.0.

1 The above vitamins were mixed thoroughly with enough cerelose
so as to make up 0.4 percent of the diet.

feed consumption over the four week period was determined, from which

feed efficiency Values were calculated.

10

Experiment 1
THE EFFECT OF DIETARY PEEICILLIN OE THE RESPONSE OF CHICKS

TO sewers LEVELS or CALCIUM PAHTCTEEEATE

Selected References of Pantothenic Acid

The importance of pantothenic acid in chick nutrition has been
well established. Norris and Ringrose (1930), were the first to report
on a pellagra~like syndrome in chicks. Ringrose, Norris and Heuser
(1931), further described this pellagra-like syndrome. Their work in-
dicated a requirement by the chick for a relatively heat-stable growth
promoting factor. Present day knowledge of vitamins however, indicates
that this early work was complicated with a multiple vitamin deficiency
involving pantothenic acid, biotin and riboflavin.

Kline, Keenan, Elehjem and Hart (1932), produced a very severe
type of dermatosis by subjecting a purified diet, similar to that of
Ringrose and co-workers (1931), to heat in a dry atmosphere.

Ringrose and Norris (1936), working with a pork liver extract found
it to be effective in preventing the pellagra—like syndrome in chicks.
After various manipulations of the extract in an attempt to identify
the active substance, the activity was found to remain in the filtrate.
Hence the active principle was desingated the "filtrate factor".

The "filtrate factor" was identified by Jukes (1939) and wooiey
and co—workers (1939), as pantothenic acid, and the synthesis of panto—
thenic acid was reported by Williams et al. (l9fi0).

The manifestations of a pantothenic acid deficiency as described

by many workers are very similar in all cases. Ewing (1951) sums up

11

the pantothenic acid deficiency symptoms as poor growth, loss of vita1~
ity, poor feathering, inflamation of the skin, sores and incrustations
at the corners of the mouth, on the eyelids and sometimes on the feet,
cracks on the feet, and scales on the shanks become thickened. Jukes
(1943) observed. paralytic symptoms in addition to the above mentioned
syndrome.

Bauernfiend, Norris and Heuser (1942), reported that Single Comb
White Leghorn chicks require 500-550 micrograms percent of pantothenic
acid for the prevention of dermatosis, and 600 micrograms percent for
maximum growth. In this work, Rhode Island Red chicks were found to
require 75 micrograms percent less than the Leghorn chicks.

Using a purified diet, Jukes, Stokstad and Franklin (1943), re-
ported that the requirement of pantothenic acid for New Hampshire
chicks appeared to be in excess of 1.0 milligram percent after a de-
pletion period of six days. These results were confirmed in a later
study by Jukes and McEloroy (1949), while Hegsted and Riggs (1949),

reported 900 micrOgrams percent of pantothenic acid for maximum growth.

Five levels of calcium pantothenate were fed with and without
penicillin. These levels ranged from 0.2 milligrams percent to 1.0
milligram percent. On the basis of values reported in the literature,
it was believed that the highest value used, nameLy 1.0 milligram per-
cent of calcium pantothenate would satisfy the Chick's requirement
for this vi a.in.

The results pertaining to growth are graphically presented in
Figure 1. The data presented clearly shows that penicillin exerts a
sparing action on calcium pantothenate. This action is especially
evident in the groups receiving 0.4 milligrams percent pantothate,
the group receiving penicillin in addition having a final average
body weight 48 percent greater than the non-penicillin group.

As expected, the growth response of the non-penicillin groups
increased as the level of pentothenate was increased, with maximum
growth appearing to be at the highest level used. The maximum growth
response of the groups receiving penicillin was at the 0.8 milligrams
percent level, and additional supplements of pantothenate gave essen-
tially the same growth. The greatest percentage increase in growth
from penicillin appeared in the groups receiving sub-marginal levels
of pantothenate. This confirms the works of Lih and Baumann (1951),
and.Beily and March (1951), whose work indicated that antibiotics
appeared to lower the requirement of rats and chicks for certain
vitamins.

Figure 2 compares a normal chick with two chicks from group 9

which exhibit poor growth and symptoms of dermatosis. A close—up of

13

the same two deficient chicks may be seen in Figure 3 which clearly
shows the encrustations at the corners of the mouth and the granular
condition of the eyelids.

The data on feed utilization, mortality and occurrence of defi-
ciency symptoms tOgether with average body weights are summarized in
Table 2.

It will be noted that feed efficiency values were improved by
penicillin in those groups receiving the lower levels of pantothenate
(compare groups 1, 2, 3, and 4 with groups 7, 8, 9, and 10). Feed
efficiency values were not materially affected by penicillin in those
groups receiving the higher levels of pantothenate (compare groups 5,
and 6 with groups 11, and 12).

In the absence of supplemental pantothenate, penicillin reduced
mortality slightly. However, in connection with the occurrence of
deficiency symptoms, penicillin tended to accentuate the expression
of typical pathological lesions about the beak, eyes, hooks, and feet,
especially in those groups fed sub-marginal levels of pantothenate.
It is suggested that this accentuation of the deficiency syndrome is
probably due to the stress produced.by the increased growth stipulat—
ing preperties of penicillin. This increase is produced on a limited
supply of pantothenate, and therefore, the pantothenic acid deficiency
symptoms were enhanced.

It should be stated here that the observation of dermatosis in
the hook region is a new finding and has not been reported previousky
in the literature in descriptions of pantothenio acid deficiency in

the chick. Moreover, the production of perosis is a hitherto unreported

l4

observation which this study has associated with pantothenio acid
deficiency. The above mentioned, newly observed syndromes are shown
in Figures 4 and 5.

This study confirms the earlier work of Jukes (1943), that some
chicks exhibit paralytic symptoms as a result of pantothenio acid
deficiency. A.greater number of chicks with parakysis were observed,
however, in the groups receiving sub—marginal levels of pantothenate
and penicillin than in the comparable groups not receiving penicillin
(see groups 2 and 8, 9 and 10). Again this shows the tendency of
penicillin to enhance the expression of deficieno" when a sub—marginal
level of the vitamin is fed.

The first pantothenio acid deficiency symptom to appear was
diarrhea on the 6th day in groups 1, 2, and 7. On the 10th day,
perosis appeared, and by the 13th day the corners of the mouths were
inflamed, and on the 14th day, the typical dermatosis appeared. The
scales and skin of the shanks and feet became dry and thickened and
by the 16th day fissures and cra ks appeared on the feet. The eyelids
becam granular and tended to stick together, and on several occasions
it became necessary to open the eyes so that the chicks could see to
eat and drink. By the 24th day nervous symptoms were evident, ex-
pressed by tremors and ataxia.

The foregoing results tend to support one theory of the growth
stimulating mechanism of antibiotics, specifically the host-microor-

anism competition concept. The sparing action of penicillin might

h

\

be explained by the limited growth of those intestinal organisms which

require pantothenio acid and other vitamins for growth, thereby

15

reducing competition for the vitamin in favor of the host animal.
Moreover, the possible establishment of a type of intestinal micro-
organism which would synthesize vitamins or essential factors can
not be overlooked.

The complete absence of the dermatosis syndrome in the group
receiving 0.6 milligram percent pantothenate and penicillin, and the
occurrence of dermatosis in its domparable non-penicillin group (see
groups 4 and 10), further demonstrates the pantothenate sparing action

of penicillin.

Penicillin

.300 No Penicillin

 

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* Figures indicate percent
advantage of penicillin
groups over comparative

.250 non-penicillin groups

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Supplement None .2 mgm.% .4 mgm,% .6 mgm.% .8 mgm.% 1.0 mgm.%
to Basal Diet: Calpan Calpan Calpan Calpan Calpan

Fig. 1. Response of Chicks To Graded Levels of Calcium
Pantothate With and Without Penicillin

 

17

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Fig. 5 A typical case of perosis produced by pantothenio
acid deficiency.

21

22

Experiment 2
THE EFFECT OF DIETARY PEEICILLIH ON THE RlSPOHSE

OP CHICKS TO GRADED LEVELS OF NIACIl

Selected References on Niacin

 

The vitamin, now referred to as nicotinic acid or niacin was first
shown to have nutritional significance by Elvehjem (1937). This worker
reported the prevention or cure of canine "black~tongue" through the
administration of niacin. Bethke (1942), reported that poultry either
do not require niacin or that its needs are so low as to be of no signi-
ficant importance. Jukes and Almquist (1942/ were unable to produce a
dietary deficiency in poultny. The failure of these early workers to
produce a niacin deficiency in poultry is now clear and can.be explained
by the fact that the diets employed were of a good amino acid pattern
high in tryptOphane.

Briggs, Mills, Elvehjem and Hart (1922), reported that chicks
require a minimum level of 1.8 milligrams percent for maximum growth,
but 0.5 milligrams percent was sufficient to prevent chick "black—
tongue". Occasional perosis was noted. Further work by Briggs (1946).
indicated that 3 to 5 milligrams percent niacin would prevent perosis
in turkey poults.

{rehl and co-workers (1946) reported that tryptophane or niacin
counteracts a growth retardation in rats caused by the addition of
corn grits to a low protein diet.

Briggs (1945), produced typica niacin deficiency symptoms by

feeding 10 percent of gelatin as a source of arginine and glycine, and

the addition of 5.0 milligrams percent niacin or 200 milligrams percent
dl~trypt0phane counteracted the symptoms and growth depression. This
work indicated tryptophane to be a precursor of niacin.

Scott, Singsen and Ma terson (19Q6) reported that niacin is re—
quired to prevent perosis in chicks on a diet high in corn and contain~
ing 5.0 percent gelatin. Sarma and Elvehjem (1946) confirmed this
report by adding 40 percent corn grits to a purified ration which pro—
duced a growth depression which niacin counteracted.

Briggs and co-workers (1943) reported their obserVations on a
niacin def‘ciency to include chick "black—tongue", decreased feed con-

sumption, poor feather development, occasional perosis and a lowering

of the niacin and coenzyme 1 content of the breast muscle.

RESULTS AND DISCUSSION

Five levels of niacin were fed with and without penicillin. The
levels ranged from 1.25 milligrams percent to 10.00 milligrams percent
based on the values reported in the literature. The highest value
used (10.00 milligrams percent) was believed to be more than sufficient
to satisfy the chicks‘ requirements for niacin.

The results pertaining to growth are presented graphically in
Figure 6. It may be seen that penicillin produced additional growth
at all levels of niacin supplementation. When growth advantages are
considered in terms of percent increase, penicillin afforded the
greatest increases in growth in the groups receiving 1.25 milligrams
percent and 10.00 milligrams percent of niacin. It is doubtful
whether the growth advantages shown in groups 11 and 12 represent real
differences which can be attributed to penicillin. Rather, it is
believed that these differences are fortuitous, and represent the
high degree of variability which is known to exist in the strain of
Rhode Island Reds used in these studies.

The results indicate that the growth response of the non~penicillin
groups reaches its maximum at the 3.75 milligrams percent level of
niacin, with additional supplements of the vitamin showing no material
advantage.

The data on feed utilization, mortality and the occurrence of de-
ficiency symptoms together with average body weights are summarized in
TmfleB.

The feed efficiency value for group 1 was not calculated due to

25

the severe mortality (75 percent). Except in groups 3 and 9, penicillin
improved the feed efficiency Values.

Penicillin exerted a protective action in the absence of supple-
mental niacin, reducing the mortality significantly (compare group 1 -
75 percent mortality, with group 7 - 12.5 percent mortality).

The niacin deficiency symptoms appeared very early; the chick
"black-tongue", and cankers or lingual ulcers being noticed on the
fifth day. Chicks of group 1 which died at about 2 weeks were ex~
tremely dehydrated and the oral cavities were severely inflamed. By
the 18th day, the chicks of groups 1 and 7 (no supplemental niacin)
were raggy and wet in appearance caused by excessive salivation.

Unlike the effects on pantothenate deficiency symptoms, penicillin

had no material effects on the incidence or severity of the chick
"black—tongue" or ca.kers. .fter the third week, the deficiency syn-
drome tended to lessen in severity, the chick "black-tonne" and cankers
completely disappearing in many cases, and the chicks appeared to make
a noticable recovery.

The perosis resulting from this diet was not severe and only ten
mild cases in all were noted. This confirms the observations of Briggs
and co-workers (l9fi3) who reported "occasional perosis" in their de-
scription of the niacin deficiency syndrome. Penicillin exerted no
apparent effect on the perosis encountered in this experiment. Two
and one-half milligrams percent niacin seemed marginal for the complete
prevention of the deiiciency syndrome with and without penicillin,

while the 3.75 milligrams percent niacin supplement completely prevent-

ed the appearance of any symptoms.

26

In this study, penicillin exhibited its beneficial effects on
young growing chicks by increasing the growth rate and by reducing
mortality. These effects were especially noted in chicks fed diets
totally deficient or sub-marginal in their niacin content. These

results definitely show a sparing action of penicillin on niacin in

the chick.

27

 

‘\

1‘9

@ Penicillin
G lie Penicillin

 

goo

* Figures indicate

percent advantage
of penicillin ~

L\\\\ a

1

groups over com-
parable non-
L§5O penicillin groups

we

 

k\\\\:k~§:

 

goo
11%
,_4,
__so

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Supplement None 1.25 mgm.% 2.50 mgm,% 3.75 mgm.% 5.00 mgm.% 10.00 mgm.%
to Basal Niacin Niacin Niacin Niacin Niacin

Fig. 6 Response of Chicks to Graded Levels of Nicotinic
Acid With and Without Penicillin

 

 

 

 

 

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Experiment 3
TEES IE'E'L'CTS 03‘ DIE TARY P21? ICILL I1? CEI 'I'E-IE RESPOZTSE

or CHICKS T0 GPJ-JJED LP. 135 or cnoLm

:11

Selected eferences on Choline

The elucidation of choline was unique when compared to th oe dis—
covery and isolation of other vitamins. The biochemical importance
of choline was known for a n imber of years before its nutritional
significance was disclosed.

Best and Huntsman (1932) reported the occurrence of fatty liver
in rats fed a high fat low choline ration, and the prevention of the
deposition of excess liver lipid by dietai' supplements of choline.

Elvebiem (1937) sug 5estcd that choline be considered a member of
the vitamin B complex since it definitely had been shown to play a
significant role in the nutrition of several animals.

J‘Ees (1939), reported a high incidence of peros is on a simpli-
fied r ation for turkey poults in spite of the addition of as high as
0.@ percent of manganese sulphate. Continuing the work on perosis,
Jukes (lQQO) presented evidence showing choline to prevent a type of
perosis often encountered in poults. He also observed choline to be
growth-promoting in this work. In further work on perosis Juhes (1940a)
found that choline prevented perosis and promoted growth in chicks fed
a purified ration. Choline at a level of 0.1 percent was found to be
effective for promoting growth and the prevention of perosis in chicks.

He qsted and co—workers (19+l) also reported 0.1 percent choline

to be effective in the prevention of perosis and for growth promotion.

Berry and co-workers (l9h3) reported that the choline requirement

for the prevention of perosis is less than the requirement for growtl

’..—

ent

fl.

[do

in chickens. One hundred and fifty milligrams percent was suff c
for maximum growth in chicks, while considerably less prevented the
occurrence of perosis.

A great deal of work has been carried on to determine the inter-

relationship of choline, methionine, betaine and vitamin B12, but the

work is so voluminous, and since this relationship was not to be

studied in this experiment, it was decided not to report on this other—

wise interesting phenomen of choline.

31

Results and Discussion

Five levels of choline were used with and without penicillin.
These levels ranged from 40 milligrams percent to 200 milligrams per—
cent. On the basis of values reported in the literature, it was
believed that the highest level used in this experiment, namely 200

illigrams percent, supplied enough choline to satisfy the requirements
of the chick for this vitamin.

The results representing the growth responses are presented

graphically in Figure 7. It will be noted that penicillin exerted a

(+-

ow h advantage in each of the six groups w.ere it was fed. The

$5

paring action of penicillin on choline is most evident in those groups

(I)

receiving 80 and 120 milligrams percent choline.

Maximum growth among the non~penicillin groups was reached at the
160 milligrams percent level of choline. daximum growth among the
penicillin supplemented groups was attained at 80 milligrams percent
choline and the additional supplements of choline did not materially
increase growth in the presence of penicillin.

Figure 8 compares two normal chicks with two chicks from group 1
which exhibit the poor growth and feathering resulting from the choline
deficiency. The ungainly position of the two deficient chicks is the
result of the perosis brought about by the choline deficiency. (See
close-up, Figure 9).

The data on feed utilization, mortality and occurrence of deficiency
symptoms together with average body weights are summarized in Table #.

The efficiency of feed utilization values were improved as the

supplementation of choline was increased, and the addition of penicillin

32

further improved these values at all supplemented levels of choline.
This improvement was not noticed in the groups not receiving a supple-
ment of choline (compare group 1 with group 7).

The mortality which occurred in this experiment was slight and
scattered. Although the basal diet employed is deficient in choline,
it would appear that S‘dficient amounts of precursors are available to
allow the chick to synthesize enough of this vitamin compatable with
maintenance needs.

The incidence of perosis in this trial was one hundred percent in
the deficient and sub-marginal groups (1, 2 and 7 and 8). Penicillin
did not appear to have any effect on the incidence or severity of
perosis while on the other hand, it did show a sparing action on the
requirement of choline for growth.

Inasmuch as it has been established in the literature that the
requirement of choline for the prevention of perosis is much less than
the requirement for rowth, the failure of penicillin to sharply affect
the incidence of perosis in this study might be explained by reasoning

that the levels of choline employed were too wide to allow the sparing

action of penicillin to be observed in regard to the perosis pattern.

3’3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Vl/A Penicillin , 1
7
114% / §
- , . 2 ,
ho PeniCillin /
f 2 a
* Figures indicate percent V/’ 5::
advantage of penicillin f//
groups over comparable ///
non-penicillin groups S::
_250 z.
P
“53 a
Q)
3
;?
+5200
:1)
5,
u
(5
m
our-1
4.3
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e 12%
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r: A
9
Si
100
-50
SLroolement None no mgm % 80 mgm 4 120 m. % 160 % 2
i- - n" @- mam. 00 mgmfl
‘to basal Choline Choline Choline Choline Choline

Fig. 7 Response of Chicks to Graded Levels of Choline Chloride

With and.Without Penicillin

 

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35

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36

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37

E.periment 4
THE aerCTS OF DIElARY FEJICILL H OH THE RESPCESE

OF CHICKS TO GRADED LEVELS OF RIBOFLAVIN

“0

Selected References on aibofla i

Range and Garrick (l 26) reported results Which indicated that
the chick required a growthepromoting factor for the maintenance of
life and growth. This factor, although unnamed at the time, was
probably riboflavin.

These results were confirmed by a number of workers in the follow-
ing years, and Norris and co—workers (1936) concluded that this growth—
promoting factor was flavin or vitamin G.

Heuser and co—workers (1938) further pointed out that the Chick's
requirement for riboflavin is not constant, but varies for different
ages. Evidence was also presented to show that the amount of riboflavin
required by the chick is correlated with the rate of growth.

Norris and co~workers (1936) reported that chicks need 290 micro-
grams percent of riboflavin to attain normal weight at eight weeks of
age; 300 micrograms percent at six weeks and 325 micrograms percent at
four weeks.

Stokstad and Manning (1938) prevented curled-toe paralysis (a
specific riooflavin deficiency syndrome) at six weeks of age in chicks

hat had been depleted for two weeks by additions of about 275—300
micrOgrams percent riboflavin to their diet.

Culton and Bird (19%0) reported that 300 micrograms percent of

crystalline riboflavin added to a ration containing approximately 175

38

micrograms percent riboflavin was not sufficient to prevent curled-toe
paralysis. Likewise, approximately #15 microg ams percent riboflavin
in dried skimmilk or dried whey did not fully prevent curled-toe
paralysis.

Bethke and Record (19h2) found that synthetic and naturally
occurring riboflavin were equally effective in preventing curled—toe
paralysis and in promoting growth in chicks. They further stated that
chicks require greater amounts of riboflavin per unit of feed for the
prevention of curled-toe paralysis than for promoting maximum growth,
while Bird and associates (1946) stated that less riboflavin wa re-
quired for the prevention of curled-toe paralysis than was required for
optimum growth. They reported that between 275-325 micrograms percent

riboflavin was required for optimum growth to four weeks.

y
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(1
1.
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c...
(0
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Five levels of riboflavin were fed with and without penicillin.
These levels ranged from 100 micrOgrams percent to 800 micrOgrams per-
cent. Besed on the values reported in the literature the highest levels
used in this experiment, namely 800 micrograms percent riboflavin, was
believed to be more than sufficient to satisfy the Chick's requirement
for growth ano the prever .tion of curleo.-toe paralysi .

The results pertaining to growth are graph ice ly presented in
Figure 10. hese results definitely show that penicillin exerts a spar—
ing action on riboflavin. This action is most evident in the lowest
riboflavin supplemented groups, (compare groups 1, 2, 3 and 4 with groups
7,8,9 and 10) w: th.penicillin showing percent advantages between 29
and 39 percent.

As expected the growth responses of the non~penici11in groups were
correlated with t} e increased supplementation of ribof 1 vin the data
indicates however, that the strain of Rhode Island Reds used has a high
requirement for riboflavin, needing more than 400 micrograms percent for
maximum growth.

With the penicillin supplement, maximum growth was achieved with
300 micrograms percent of riboflavin. In terms of percent advantage,
penicillin showed its greatest growth stimulating effect when fed to
chicl :s receiving 100 micrOgr& ms percent of riboflavin. The maximum
growth response due to the penicillin supplement occurred at the 300 mi—
crograms percent level of riboflavin, and considering that additional

supplements of riboflavin resulted in lesser growth responses this

#0

growth advan age is believed to be due to the previously mentioned
variability in this strain of Rhode Island Reds rather than attributed
to the growth stimulation produced by penicillin.

The data on feed utilization, mortality, and the occurrence of
deficiency symptoms, together with the average bo‘v weights are sum—
marized in Table 5. It will be noted that penicillin improved the feed
efficiency values over all non—penicillin groups. The improved values
were very marked at the 100 micrOgrams percent level of riboflavin, in
which case penicillin reduced by almost one~third the amount of feed
required per unit-gain.

Severe mortality was experienced among the chicks of group 1 receiv-
ing no supplemental riboflavin, 75 percent of the chicks having died
before the 4 week period was completed. The protective action of pen-
icillin, noted previously (see experiment 2) increased the livability
of the chicks on the riboflavin deficient diet and allowed 100 percent
survival.

The incidence and severity of curled-toe paralysis was slight,
making its appearance about the end of the fourth week. The curled—toe
paralysis did not a_pear in the deficient or sub~marginal levels of
riboflavin. This confirms the work of Stokstad and Manning (1933) that
in general, if no riboflavin was added to the basal diet, curled-toe
parakysis did not appear, but small supplements of the vitamin increased
the incidence and large amounts of ribof avin completely prevented it.

Curled—toe paralysis appeared at the 200 micrograms percent level
of riboflavin both with and without penicillin and these results agree

with the observations of Bird and associates (1946) that less riboflavin

41

is required for the prevention of curled-toe paralysis than is required
for the optimum growth.

Penicillin had but slight effect on the incidence (comnar groups
3 and 9) and no effect on the severity of curled~toe paralysis in this

experiment.

 

7," Penicillin
"200 -No Penicillin

l\\‘1€a

* Figures indicate
Percent advantage
_g50 0f penicillin groups
over comparable non-

penicillin groups

B\\\\\\\\Vlg
R\V_¥a

b\\\\\<§v~;

__200

"

Average Weight in Grams at b weeks

l\\\\\1:§

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

g...
0 ,4
P5
Supplement None 100 ug.73 200 ug.% 300 ugfi #00 ug.% 800 um:
to Basal Riboflavin Riboflavin Riboflavin Riboflavin Riboflavin

Fig. 10. Response of Chicks to Graded Levels of Riboflavin With
and Without Penicillin

43

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do nHHHHoHdom pdoanz can ng3 sHbmHmopHn Mo mHobmH dodwnm mo pommmm .m magma

SULJARY

A series of four experiments were carried out to determine the
effects of dietary penicillin on the biological response of chicks fed
graded levels of calcium pantothenate, niacin, choline, and riboflavin.
These experiments were undertaken to obtain information of a positive
nature which indirectly might lend support to the theory that anti-
biotics stimulate growth, in part at least, by altering the intestinal
microflora in a manner that would be advantageous to the host.

This speculative approach embraces he concept that the normal
animal harbors a countless number of intestinal microorganisms, of
various types and species, which compete with the host for ingested
nutrients. Should this be true, then it is conceivable that anti-
biotics might stimulate growth indirectly by limiting the growth of
these bacteria with the consequence that competition for food, from
the animal's point of view, is reduced and a greater quantity of nutri—
ents is made available to the host. ioreover, it would seem that the
amount of any given nutrient that might be spared by an antibiotic
would represent a more critical part of the total quantity of that
nutrient in diets that were subumarginal for it as compared with diets
that contained an optimum level. Therefore, the feeding of an anti-
biotic should lead to greater percentage growth increases in chicks
fed sub-marginal diets as compared with an adequate diet.

The procedure of feeding graded levels of micro-nutrients (calCium
pantothenate, niacin, choline, and riboflavin) with and without penicil-

lin yielded data which may be interpreted as supporting the host-bacteria

competition theory. These data, in general, showed that penicillin
exerted a greater growth stimulating effect (percent increase in growth)
on chicks receiving very low or s1b—marginal levels of vitamins than it
did on chicks fed marginal or optimum levels of vitamins. It was also
observed that feed efficiency values were improved to a greater extent
by penicillin in the groups of chicks fed sub—marginal levels of
vitamins as compared with those fed optimum levels. Under conditions

of extreme vitamin deficiency penicillin improved the livability of

chicks to four weeks of age.

46

BIBLIOGRAPHY

Bauernfiend, J. C., L. C. Horris, 83L G. F. Heuser, 1942. The panto-
thenio acid requirement of chicks. Poultry Sci. 21:1h2-146.

tr}

eily, J. and B. March, 1951. The effect of aureomycin and vitamins
on the growth rate of chicks. Science 114:330-331.

Berry, B. P., C. W. Garrick, R. B. Roberts, and S. M. Bauge, 1943.
A deficiency of choline in soybean oil and soybean oil meal.
Poultry Sci. 2:"2-h&5.

Best, C. H. and Huntsman, 1932. The effects of the components of
lecithine upon depostion of fat in the liver. J. Physiology 75:
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Bag;Red

Fla

Bethke, R. M., 19h2. Vitamin reviews in poultry. The Bee
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Bethke, R. M. and P. R. Record, 1942. The relation of riboflavin to
growth and curled-toe paralysis in chicks. Poultry Sci. 21:147-154.

Bird, F. H., V. S. Asmundson, F. H. Xratzer and S. Lepkovsky, 1946. The
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Briggs, G. h., R. C. Mills, C. A. Elvehjem and E. B. Hart, 1942.
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51:59-61.

, 19Q2a. flew vitamin important for poultry. Wise. Agr. Expt.

Briggs, G. M., T. D. Luckey, C. A, Blvehjem and B. B. Hart, 1944. Effect
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Briggs, G. M., 19b5. Influence of gelatin and tryptophane on nicotinic
acid requirement of chicks. Jour. Biol. Chem. 161:7h9-750.

, 19h6. Iieotinic acid deficiency in turkey poults and the
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Culton, T. G. and H. R. Bird, 1940. The effect of some riboflavin
supplements on chick growth and curled—toe paralysis. Abstracts
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Association, Ithaca, New York.

Daniel, L. J., F. A. Farmer and L. C. Norris, l9h6. Polio acid and
perosis. Jour. Biol. Chem. 163:3Q9—350.

I

K

47

Bijkman, C., 1897. Line beri beri-a1zuliche dranlflleit der hukner.
Virchows Arch. Path. Anat. 143:523— ~532.

, 1897a. Ein ver such zur beltnmpfung der beri—beri. Virchows
Arch. Path. Anat. 149: 187- 194.

Blvehjem, C. A., 1937. Vitamin B fractions. Jour. Amer. Chem. Soc.

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Ewing, W. R., 1951. Pantothenic acid. Poultry Nutrition. 4th Ed.
1127-1145.

Punk, 0., 1911. On the chemical nature of t? e subs t.a nce which cures
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of antibiotics, syn-
. supplement on chick

Groschke, A. C. and R. J. Evans, 1950. Effec
thetic vitamins, vitamin B12 and an.A.P.
growth. Poultry Sci. 29: 616~613.

4.
v

w-q

.2

Range, S. M. and C. W. Garrick, 1920. A differentiation between the
water-soluble grow h—promotin g and antineuritic substances. Biol.

Chem. 69:403.

Regsted, D. K., R. C. Mills, C. R. Bivehjem and E. B. Hart, 194
Choline in the nutrition of chicks. Jour. Biol. Chem. 138:466.

Regsted, D. M. and T. R. Riggs, 194 . The pantothenic acid require—
ments of chicks receiving a purified diet. J. Nutrition 37:
361—367.

Reuser, G. P., H. S. Wilgus and L. C. horr is, 1933. The quantitative
vitrmin G requirements of chicks. Poultry Sci. 17:227-234.

Repkins, G. P., 1906. The a.oLrst and the medic: 1 man. Ana lyst 31: 3? 5.

Jukes, T. 3., 1940. Effect of choline and other Supplements on perosis.
J. Nutrition 20:445—458.

, 1940a. Prevention of perosis by choline. Jou . Biol. Chem.
1343739-790-

Jukes, T. R. and H. J. Alnquist, 1942. Avie n biochemistry. Ann. Rev.
Biochemistry 11:511-530.

Jukcs, T. H. and L. W. KcElroy, 1943. Observations on the pantothenio
acid requirements of c Hic:s. Poultry Sci. 22:439—441.

Jukes, T. H., E. L. R. Stokstad and A. L. Franklin, 1949. Observation
on the vitamin requirement of chicks on a purified diet. Poultry
Sci. 28:770.

48

Kline, O. L., J. A. Keenan, C. A. Elvehjem and E. B. Hart, 1932. The
use of the chick in vitamin BI and B2 studies. Jour. Biol. Chem.

99:295-307.

Krehl, W. A., P. S. Sarma and C. A. Blvehjem, 1946. The effect of
protein on the nicotinic acid and tryptophane requirement of the
growing rat. Jour. Biol. Chem. 162:403—411.

Lih, H. and C. A» Baumann, 1951. Effects of certain antibiotics on
the growth of rats fed diets limiting in thiamin, riboflavin or
pantothenio acid. J. Nutrition 45:143.

Lillie, R. J. and G. M. Briggs, 1947. Folic acid requirements of New
Hampshire chicks receiving synthetic diets. Poultry Sci. 26:

295-298.

Linkswiler, H., C. A. Baumann and B. E. Snell, 1951. Effect of aureo-
mycin on the response of rats to various forms of vitamin B6.
J. Nutrition 43:565.

Luckey, T. D., P. R, Moore, C. Am Elvehjem and E. B. Hart, 1946. Effect
of diet on the response of chicks to folic acid. Proc. Soc. Exp.

Moore, P. P., H. Evenson, T. D. Luckey, E. McCoy, C. A. Elvehjem and
E. B. Hart, 1946. Use of sulasuxadine, streptothricin and strep-
tomycin in nutritional studies with the chick. J. Biol. Chem.

165:437-441.

Horris, L. C. and A. T. Ringrose, 1930. The occurrence of a pellagra~
like syndrome in chicks. Science 71:643-6A4u

Norris, L. C., H. S. Wilgus,.A. T. Ringrose, V. Heiman and G. S. Heuser,
1936. The vitamin G requirement of poultry. Cornell Agr. Exp.
Sta. Bul. 660.

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