STUDiES ON AN UNKNOWN
DIETARY FACTOR
(AMMAL PROTEIN FACTOR)
REQUIRED BY CHICKS

Thesis for fho Degree of M. S.
MICHIGAN STATE COLLEGE

Donald C. Miller
1949

This is to certify that the

thesis entitled

STUDIES on m UNKNOWN DIETARY FACTOR
(ANIMAL PROTEIN FACTOR)
REQUIRED BY CHICKS

presented by

Donald C . Miller

has been accepted towards fulfillment
of the requirements for

Master omdegwe in Powlt Husbandry

Major professor

Date March 10 .1242 _

 

 

STUDIES ON AN UNKNOWN DIETARY FACTOR
(ANIMAL PROTEIN FACTOR)
REQUIRED BY CHICKS.

by
Donald C. Killer

N

A THESIS

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

(MASTER OF SCIENCE)
Department of Poultry Husbandry

1949

ACKNOWLEDGEMENT

The author wishes to eXpress his appreciation to
Dr. A. C. Groschke, and Professor C. G. Card of the Depart-
ment of Poultry Husbandry, Dr. C. F. Huffman and R. J.
Flipse of the Department of Dairy Husbandry, Dr. E. P.
Reineke of the Department of Physiology and Dr. R. J.
Evans, Agricultural Chemist of Michigan State College, for
their guidance, assistance and cooperation in making this
work possible.

The crystalline vitamins and 812 concentrate were
supplied by Merk and Company,_Rahway, New Jersey; Butyl
fermentation solubles (B-Y feed) was supplied by Commercial
Solvents Corporation, Terre Haute, Indiana; and the Liqua-
fish and condensed fish solubles were supplied by
Dehydrating Process Company, Boston, Massachusetts. The
author wishes to thank these concerns for generously

supplying these materials.

Introduction,
Literature Survey , , , ,
Materials and Experimental Procedure,

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

Table

Table

Table

Table

Table

Table

Table

Table

Table

l.
2.
3.

4.

5.

9.

Table 10.

Table 11.

TABLE OF CONTENTS

0 O O O O O O O O O O O O O O O O O 0 O
O 0 O O O O O O O O O O O O

Maternal diet (Breeder mash formula C-l).

Basal "animal protein free" diet, , , , ,
Assay of liver concentrate and beef
extract for "animal protein factor“
aetivj'ty o o o o o o o o o o o o o o o o o
Assay of dried fresh horse manure and
dried incubated horse manure for "animal
protein factor" activity. , . , , , , , ,
Re-assay of dried fresh horse manure and
dried incubated horse manure for "animal
protein factor" activity. . . , , , , , ,

Effect of vitamin mixture on assay of
liver concentrate, incubated horse

manure and B-Y feed for "animal protein
factor“ activity. . . . . . . . . . . . .

Assay of several products of animal
origin for "animal protein factor"
aetivj-tyo O O O 0 o o o o o o o o o o o o

Assay of dried fresh rumen content and
dried fresh cow manure for “animal
protein factor" activity. . . . . . . . .

Effect Of vitamin mixture on assay of
dried fresh rumen content and dried fresh
cow manure for "animal protein factor"
activity. . . . . . . . . . . . . . . . .

Assay of vitamin 312 for "animal protein
factor" activity. . . . . . . . . . . . .

protein factor" activ

O O 0 O O O O O O

Re-assay of vitamin Blg for "animal
y

16

4O
41

42

43

44

46

A7

48

5O

Table of contents (cont'd)

Summary. . . . . . . . . .
Conclusions. . . . . . . .

Bibliography . . . . . . .

STUDIES ON AN UNKNOWN DIETARX FACTOR (ANINAL PROTEIN FACTOR)
REQUIRED BY CHICKS.

INTRODUCTION

During the past fifteen years soybean oil meal has
increased in importance as a protein supplement in poultry
feeds from a relatively minor ingredient to one of major
usage. Prior to World War II most of the commercially
prepared mashes relied, principally, on protein concentrates
of animal origin as sources of protein, namely, dried skim-
milk or buttermilk, fish meal, and meat scrap, while soy-
bean oil meal rarely exceeded 5 percent of the mash compo-
sition. With the outbreak of the war, enormous demands
were placed on the poultry industry for increased production
of poultry and poultry products. The strain of this
increased production was immediately felt in the feed indus—
try which was called upon to increase its output of manu-
factured feeds. It was immediately recognized that the
country's available and potential supply Of protein concen-
trates, of animal origin, was seriously lacking. To meet
this crisis, it was imperative that poultry mash formulas
had to be revised in order to stretch the supply of animal
protein concentrates to the greatest extent possible.
Experiment Stations throughout the country quickly took
action and the results of research showed that soybean Oil

meal could be used in poultry mashes at much higher levels

than it had ever been used before. Practical application
of this knowledge in the feed industry resulted in an out-
put of high quality, manufactured feeds in volume great
enough to meet the demands Of the poultry industry, thus
enabling the poultry industry to achieve the greatest pro-
duction goal in its history. I

Subsequent war-time research, however, uncovered
.valuable fundamental, as well as practical, information
concerning the utilization of soybean oil meal in poultry
diets. It was found that soybean oil meal could not totally
replace animal protein products in starting and breeding
mashes. Numerous investigations showed that small amounts
of proteins of animal origin had to be in the diet for
maximum growth of chicks and hatchability of eggs. The
subsequent effect of these protein materials was found not
to be associated with any of the known vitamins or amino
acids carried by these proteins. Thus, it was established
that an unidentified factor or factors, vitamin-like in
nature, existed. Because the factor was contained in ani-
mal protein concentrates, nutritionalists called it the
“animal protein factor“.

That sources of the ”animal protein factor“ existed
other than in animal proteins was eventually shown. Cow
manure was found to be a potent source of the factor.
Concentrated extracts of this material proved to be highly
active and demonstrated further the existence of an unident-

ified vitamin-like factor required by poultry. Moreover,

3.

it was shown that soybean oil meal could be used as the
major source of protein for poultry when the diet was supple-
mented with small amounts of the concentrate containing the
“cow manure factor” which possessed "animal protein factor"
activity.

This study was undertaken with the following objectives:
(1) To develop a reliable chick bioassay method for assay-
ing various materials for "animal protein factor” activity,
and (2) to study the occurrence and distribution of the
"animal protein factor“ in certain selected feedstuffs and

experimental preparations.

LITERATURE SURVEY

A number of conflicting reports have appeared in the
literature concerning the ability of soybean oil meal to
adequately supplement poultry diets. Phillips, Carr, and
Kennard (1920) reported that chicks could utilize soybean
meal to an advantage. Kennard, Holder, and White (1922)
reported that the addition of calcium and phOSphorus to
the diet increased the value of soybean meal as a supple-
ment to the diet of chicks. Pratt (1942) obtained as good
growth with a ration containing 20 per cent soybean oil
meal, as the only protein supplement, as with rations con-
taining some animal protein concentrate.

Byerly, Titus and Ellis (1932, 1933); Deobald, Halpin
and Holmes (1937); and Wilgus and Gassner (1941) observed
a marked decrease, especially during the winter months, in
the hatchability of eggs produced by hens whose diets con-
tained soybean oil meal as the chief source of protein.
Nestler, Byerly, Ellis and Titus (1936) were not able to
correct the deficiency by the addition of 10 per cent
dried whey to increase the intake of riboflavin. However,
Christiansen, Halpin, and Hart (1939, 1940) demonstrated
that this low hatchability was overcome by the addition
of synthetic riboflavin or manganese sulfate to the diet.

They concluded that the diet was deficient in riboflavin

5.

and in some ways the manganese acted to alleviate this
deficiency.

Bird and Groschke (1942) reported that in five out of
seven CXperiments, growth was inferior when soybean oil
meal replaced 4 per cent menhaden meal making soybean oil
meal the sole protein supplement in the chick ration.
Domestic meat and bone meal, domestic meat meal, and blood
meal failed to satisfactorily replace the menhaden meal.
EXperimental meat and bone meal, which contained consider-
able amounts of visceral material, showed an appreciable
advantage over the domestic meat and bone meal. The
domestic meat and bone meal consisted, largely, of bone,
muscle and connective tissue. Menhaden meal, being made
of the Whole fish, contains considerable amounts of visceral
material. Moderately good results were obtained when 4 per
cent dried skimmilk replaced the 4 per cent menhaden meal.
Better results were obtained when 7 per cent dried skimmilk
was used in lieu of the 4 per cent menhaden meal.

It was reported by Nelson, Volz, Parkhurst, and Park-
inson (1944) that soybean oil meal and corn distillers‘
by-products replaced all of the dried skimmilk,fish meal
and meat scraps in the starting ration of chicks.

Wilgus and Zander (1944, 1945) reported that soybean
oil meal was inadequate, as the sole protein supplement,
for growing chicks, fed on such a diet, suffered high mor-
tality, non-Specific enteritis, and were severely stunted.

Five per cent meat and bone scraps was adequate as a

supplement to such a diet. The gains, obtained by the
supplementation of meat and bone scraps, were not improved
by the addition of dried whey.
It was reported by Winter (1943), and Scott, Avery,
and Matterson (1943) that, when soybean oil meal was used
as the sole protein supplement, reasonably good growth
resulted but the efficiency Of feed utilization was inferior
to diets containing some protein from animal source.
Almquist (1943), in summarizing the reports of a number
of Experiment Stations, indicated that the relative growth
on soybean oil meal rations represented 55 to 99 per cent
of the growth obtained on the good rations. The addition
of 3 to 5 per cent of animal protein concentrate was
lrequired to equal the best growth. It was concluded by
Heuser and Norris (1944) that to obtain Optimum growth and
efficiency of feed utilization, a diet, in which soybean
Oil meal is the chief source of protein, should contain a
minimum of 2 to 3 per cent of animal protein concentrate.
These conflicting reports indicate a marked degree of
variability in the quality of the materials used. Incon-
sistencies in the procedures used in condUcting the experi-_
ments may be reaponsible for part of the variability. The
variability occurring among commercial soybean oil meals
has been investigated by Bird and Burkhardt (1943).
Hammond and Titus (1944) reported that they obtained
better growth when feeding diets consisting of wheat and

soybean oil meal than with corn and soybean oil meal.

7.

‘Whitson, Hammond, Titus, and Bird (1945) reported a sig-
nificant improvement in growth when a corn-soybean Oil meal
diet was supplemented with a combination of choline chloride,
nicotinic acid, pyridoxine, inositol, and para-aminobenzoic
acid in one eXperiment and with nicotinic acid, choline
chloride and bone meal in another experiment. When the
same supplements were used with a wheat-soybean oil meal
diet, the growth response was significantly greater than
that for chicks on the corn-soybean oil meal diet. Mishler,
Garrick, Roberts, and Range (1946) reported that the
deficiencies in a corn-soybean oil meal diet could be over-
come by the addition of choline chloride, calcium panto-
thenate, riboflavin, and nicotinic acid. Bird and Rubin
(1946) concluded that it would appear that either calcium
pantothenate or pyridoxine hydrochloride, when fed at
exceptionally high levels, is capable of exciting an
important effect on the growth of chicks fed a corn-soybean
oil meal diet which was high in choline.

In a study of the effect of dietary level of soybean
oil meal in an all plant protein diet, Whitson, Titus, and
Bird (1946) reported that as the level of soybean Oil meal
was increased from O to 40 per cent (in increments of 10
per cent) the hatchability decreased. In another study
these workers found that wheat supplementation of soybean
oil meal, at 20 per cent level, was more effective than
corn supplementation. They concluded from this that soy-

bean oil meal had a direct depressing effect on hatchability

or that wheat or high levels of corn supply an unknown
dietary essential.

It was reported by Bird, Rubin, and Groschke (1947)
that some hens were able to maintain high hatchability,
over extended periods of time, when fed a diet deficient
in animal protein factor. It was thought that this was
an inborn characteristic.

Seasonal trends in hatchability were also observed
by Groschke, Rubin and Bird (1948) when the birds fed a
corn-soybean diet were confined to Open front houses and
permitted access to their own feces. Similar birds kept
in hen batteries showed no increase in hatchability of eggs
in the spring and summer. The seasonal variation was
eliminated in those groups characterized by low hatchability
by the addition of an unidentified factor in cow manure to
their diet. The improved hatchability of the low hatch-
ability group was thought to have been due to COprOphagy.
Conditions, for the synthesis of the essential dietary
factor in the voided feces, were more favorable during the
warmer months than the cooler months. Bethke, Pensack,
and Kennard (1947); Rubin and Bird (1946, 194%» and
Mc Ginnis and Carver (1947) found that the growth factor
associated with protein of animal origin is transmitted
through the egg to the chick. These findings emphasize
the importance of breeder diets and/or, the use of a
depletion period for the chicks which are to be used in

the assaying of animal proteins.

Mishler, Garrick, and Hauge (1948) found that by add-
ing condensed fish solubles to their corn-soybean oil meal
diet containing supplements Of the known vitamins an addi-
tional growth response resulted. From this they concluded
that the fish solubles contain an unidentified factor or
factors not present in the vitamin supplemented plant
protein diet.

Fish and fish by—products have been reported as good
animal protein supplements to poultry diets by Bird and
Groschke (1942); Whitson, Hammond, Titus, and Bird (1945);
Bird, Rubin, Whitson, and Haynes (1946); Mo Ginnis and
Carver (1947) and Bethke et. a1. (1947).

Robblee, Nichol, Cravens, Elvehjem, and Halpin (1947)
reported the following prOperties Of the unidentified
chick growth factor found in condensed fish solubles. It
was found to be soluble in water, 70 per cent methanol,

70 per cent ethanol. It was insoluble in ether and acetone.
It was dialyzable through a cellophane membrane and heat
stable over a range of pH 3 to 9; no loss in activity was
caused by enzymatic digestion.

It was reported by Usuelli and Fiorini (1939) that
growth was increased by the supplementation, with fractions
of rumen contents, of an all plant diet. Hammond (1942)

'reported increased gains in weight and increased growth of
comb and wattles when a low grade starter mash was supple-
mented with dried cow manure and increased gains in weight
when rumen content was used as the supplement. The stimu-

"lation of comb and wattle growth was later attributed, by

10.

Riley and Hammond (1942), to an androgenic (male) hormone
present in cow manure.

Whitson, Titus, and Bird (1946Q conducted some experié
ments to study the effects of cow manure SUpplementation on
hatchability and egg production. They were able to show
that when the androgenic factor was destroyed by heat,cow
manure supplementation did not impare egg production.
Hatchability was significantly improved and seasonal vari-
ations were largely eliminated by the addition of a cow
manure supplement (8%) to a diet high in soybean Oil meal
and devoid of both animal protein and alfalfa. The work
of Groschke et. a1. (1948) confirmed their report that the
addition of the growth factor in cow manure increased
hatchability and largely eliminated seasonal variations
regardless of exposure to sun light. Whitson et. a1. (1946)
concluded therefore, that cow manure contained an unident-
ified factor or factors which permitted good hatchability
and growth on a vegetable-protein diet. Whitson et. a1.
(1945) concluded further that the factor was neither a
protein nor any of the chemically characterized vitamins.

Rubin, Bird and Rothchild (1946) reported that the
growth factor present in cow manure was also present in
hen feces. Since the factor was not in the hen's diet,
they suggested that the factor was synthesized in the
digestive tract. Mo Ginnis, Stevens, and Groves (1947)
demonstrated, by incubating hen feces, that the synthesis

of the growth factor took place after defecation rather

11.

than in the digestive tract.

Bird, Rubin, Whitson, and Haynes (1946) reported that
the deficiency due to feeding a diet of corn-soybean oil
meal could be corrected by the supplementation of 10 per-
cent sardine meal, or 10 percent dried Skimmilk or 5 per-
cent cow or steer manure to the basal diet. Me Ginnis and
Carver (1947) reported that 3 percent fisn solubles or 0.5
percent alcohol soluble liver fraction in the diet of the
hen permitted adequate storage of the factor to meet the
first four weeks requirement for the chicks. They further
concluded that the factor or factors were distinct from
vitamin A, vitamin D, thiamine, riboflavin, pyridoxine,
pantothenic acid, niacin, choline, biotin, folic acid,
para-aminobenzoic acid, and inositol,. Rubin and Bird
(1946) reported that the factor was not one of the non-
chemically identified known chick growth factors (L. casei
factor, factor U, factors R and S, vitamins Bio or Elli
These facts further established the chick growth factor as
a new unidentified factor in poultry nutrition.

An attempt to identify the factor by Rubin and Bird
(1946a) was successful in extracting the factor from cow
manure. Potent concentrates were effectively used at 3.75
to 7.5 milligrams per 100 grams of diet (as compared with
8000 milligrams of cow manure per 100 grams of diet). In
studying its prOperties they found the factor to be stable
to heat, moderately water soluble, insoluble in ether, and
non-dialyzable through cellOphane. In further studies,

Bird, Rubin, and Groschke (1948) reported that this factor

12.

was soluble in water at pH 3.0 if the protein were prev-
iously removed by digestion with papain or by precipitation
from half saturated (NH4)2804 solution or 2 percent CaCl2
solution. The factor was soluble in 80 percent acetone.

It was destroyed by autoclaving 1 hour with 2N acid. It
was demonstrated by several workers, Rubin and Bird (1946),
Bethke et. a1. (1947), Rubin and Bird (1947L and Mc Ginnis
and Carver (1947), that the factor was influenced by tne
diet of tne hen and that it was transmitted to the egg.

The distribution or tne factor within the egg was shown

by Rubin and Bird (1947) to be chiefly in the yolk.

The diets used contained high amounts of soybean oil
mea1.and, since increasing the amount of soybean oil meal
in the diet aggravated the deficiency, it was thought that
this material might contain an inhibiting factor for growth
and hatchability. At least one substance is known to
interfere with growth, an inhibitor for trypsin has been
shown by Ham and Sandstedt (1944) to be present. The fact
that the trypsin inhibitor is liable to heat ruled out
this possibility.

It has been snown by many investigators that soybean
oil meal is low in methionine. Bird et. a1. (1947) snowed
that the addition of methionine, to tne diet of chicks,
produced good growth wnen the cow manure factor was present
in the diet of the hens, but was less effective when the
maternal diet did not contain this factor. As suggested

by Rubin and Bird (1947) it was conceivable that the factor

13.

might facilitate the liberation of methionine from soybean
oil meal in the digestive tract; it might function in the
metabolism of methionine after absorption; or it might have
an entirely different function which could be performed in
part by methionine. The work of Rubin and Bird (1947)
demonstrated that wnen excessively high levels (70%) of
soybean oil meal was fed, methionine was wholly ineffect-
ive in counteracting the detrimental effects wnich resulted.
It was postulated that soybean oil meal (and possibly other
plant proteins) contain a heat-stable growth inhibitor and
that the growth factor, for chicks, found to be present in
cow manure, apparently overcomes this heat-stable growth
inhibitor.

Another explanation has been offered by Gary and
Hartman (1947), the requirement for the animal protein
factor or chick growth factor might be increased as the
quantity of protein is increased in the diet, thus demon-
strating that the factor has a role in protein metabolism.

An announcement by Rickes, Brink, Koniuszy, Wood, and
Folkers (1948) was made of the isolation of a new crystal-
line vitamin from liver. It was tentatively named vitamin
B1:'

The purification of two amorphous forms of the anti-
pernicious anemia factor from liver was announced by Smith
(1948). The material, though not as pure as the vitamin
812 of Rickes et. al. (1948), exhibited highly similar

physical and chemical prOperties.

l4.

Ott, Rickes, and Wood (1948) announced that when
vitamin B12 was added in amounts as low as 6’7 per kilogram
to purified diets in which 40-70% soybean oil meal furnished
the only source of protein, it exerted animal protein activ-
ity on the growth of chicks. It is possible that vitamin
B12 is identical with or closely related to the “animal
protein factor". Shorb (1948) announced that the new
cyrstalline vitamin B12 had activity for the growth of
Lactobacillus lactis Dorner, having an assigned potency of
1000 LLD units per milligram, vitamin B12 had a potency of
approximately 11,000,000 LLD units per milligram. This
makes B12 one of the most potent of microbiological active
compounds. A

It was announced by Smith (1948a) and confirmed by
Rickes, Brink, Koniuszy, Wood and Folkers (1948a) that

cobalt was present in the structure of B Rickes et. a1.

12'
(1948a) reported also the presence of some nitrogen but no
sulfur in vitamin B12. It has a probable molecular weight
between 1550 and 1750. Cobalt had heretofore been unknown
as a component of a natural organic compound.

It has long been recognized that cobalt, as a trace
element, was required by ruminants, Martin (1944). Tosic
and Mitchell (1948) reported that the cobalt content of the
micro-organisms, taken from the rumen content in adequately
fed sheep, was three times the content of similar microbial

fractions from the rumen of sheep fed cobalt-deficient hay.

But in sheep fed cobalt-deficient hay and dosed with l

15.

milligram of cobalt daily, the cooalt content of microbial
fractions was five times that of similar sheep receiving no
cobalt supplement.

Thymidine (the desoxyriboside of thymine), a crystal-
line factor functionally related to folic acid was isolated
from liver by Shive, Eakin, Harding, Ravel, and Sutherland
(1948). Thymidine was found by Shive, Ravel, and Eakin
(1948), and Wright, Skeggs, and Huff (1948), to be capable
of replacing vitamin B12 in the nutrition of Lactobacillus
lactis. Thymine, however, does not replace 312. Because
of the large amounts of thymidine required to replace B12,
the two are not considered identical. wright et. al. (1948)
have interpreted the data to indicate that 812 acts as a
ccenzyme in the processes involved in the conversion of
thymine to thymidine. Shive, Ravel, and Harding (1948)
confirmed the postulated role of B12 in the biosynthesis of
thymine and thymidine and indicate also its functional

identity with the "animal protein factor'-in this activity.

16.

MATERIALS AND EXPERIMENTAL PROCEDURE

Nine experiments were conducted. Day-old Rnode
Island Bed chicks of mixed sexes and of known pedigree,
secured from six breeding pens located at the college
farm, were used throughout this study. The composition
of the maternal diet is given in table 1.

Equal parts of mash (0-1) and the following grain
mixture were fed: Yellow corn 5, wheat 4, and oats 1.

Eggs were collected daily and held at a temperature
of 50°F until set. Settings of eggs were made at two week
intervals.

The first objective of this investigation was the
develOpment of a reliable method for assaying various
materials for animal protein factor activity (hereafter
referred to as APF) utilizing the chick as a test animal.
The work of other investigators, previously mentioned,
showed that APF was transmitted from the hen to the chick
through the egg, and that high levels of soybean oil meal
in the diet of the chick increased the chick's require-
ment for APF. 0n the basis of these facts most workers
resorted to the use of chicks, in their studies, which
had been produced from dams which had received an APF
deficient diet and in turn the chicks were placed on a
high soybean oil diet which further facilitated the pro-

duction of an APF deficiency.

17.

The procedure of securing chicks from hens receiving
an APF deficient diet was not satisfactory for the facili-
ties available in this study because APF has been shown
to be not only a chick growth factor but also a hatchabil-
ity factor. Since a limited amount of Space was to be had
for hens it seemed desirable to feed a high quality breeder
mash with the purpose of hatching as great a number of
chicks as possible and then subject the chicks to a
depletion period before using them in assay work.

In attempts at standardization of a procedure to be
used in assay work, it was found during preliminary trials
that chicks which had been fed a diet containing 40 per-
cent of soybean oil meal for a two week depletion period
did not adequately deplete their body stores of APF and.
consequently were not satisfactory as test animals. 0n
the other ham; when the soybean oil meal in the diet was
raised to 70 percent it was found that a two week depletion
period was too severe and the chicks again were poor test
animals because of exceedingly high mortality rates.

A basal APF deficient diet containing 50 percent
soybean oil meal was found to be satisfactory for the pur-
poses of this investigation. Its composition is given in
EQQ;§,§. This diet was employed in all of the experiments
reported in this thesis.

All of the soybean oil meal used in preparing the
basal diet was from the same lot of solvent extracted, 44

percent protein, meal.

18.

All of the chicks used in this study were confined in
electrically heated steel batteries with wire floors. The
batteries were located in a well lighted, and well ventil-
ated, cement-floored room. Room temperature was thermo-
statically controlled and was maintained at a range between
75-800F. Feed and water were available to the chicks at
all times.

The procedure used in all of the chick bio-assay
experiments presented in this thesis was as follows: Day-
old chicks were fed the basal APF deficient diet for two
weeks. This constituted the depletion period. At the end
of two weeks individual weights were recorded and only
those chicks which averaged 75-90 grams (grouped in 5 gram
weight intervals) were kept for assay use. The eXperimen-
tal groups were then formed on an equivalent weight basis
so that each group contained an equal number of chicks
from each weight class. The assay period was of 14 days
duration. Individual weighings were recorded on the 7th
and 14th days. At the time of the last weighing feed
consumption was determined for each group (except eXperi-
ment 1) and efficiency of feed utilization was calculated

using the following formula:

 

total grams gained x 100 _ efficiency of feed
total grams feed consumed utilization

In order to measure APF activity of various supple-
ments it was necessary to include a negative control group
(fed basal diet only) and a positive control group (fed

basal diet + liver concentrate). The liver concentrate

19.

(Wilsons liver 1:20; one part derived from 20 parts of
fresh liver) is known to be a potent source of APF and
therefore was used as a reference standard in the major-
ity of this work. In a few experiments where liver concen-
trate was not used in the positive control group (eXperi-
ments 6 and 7) a material was substituted (incubated horse
manure) which had been standardized against the liver
concentrate.

Prior to the initiation of the present study available
evidence indicated that bacteria probably were responsible
for the occurrence of an unidentified chick growth factor
in cow manure. The role played by rumen bacteria in the
synthesis of the factor was not known but it was Specu-
lated that these bacteria were involved. The concept of
bacterial synthesis was further strengthened when it was
shown that fresh chicken feces, Obtained from hens fed a
diet deficient in the chick growth factor, possessed little,
if any, growth promoting activity when fed to chicks. How-
ever, the same feces caused marked growth stimulation after
several hours of incubation.

It seemed of interest to study the problem of bacter-
ial synthesis of unidentified chick growth factors in
fecal matter further. Accordingly, the horse was chosen
as the manure source animal because, (1) it's dietary
regime, like the cow's, consists of all-vegetable matter
and is therefore devoid of APF, (2) it's well developed

caecum offers a favorable site for bacterial growth, and

20.

(5) no reports exist in the literature concerning studies
on unidentified growth factors for chicks in horse manure.
The horse manure used in the eXperiments reported
herein was obtained freshly defecated from nine stallions.
The animals were fed the following “all-vegetable" diet:l

Mixed timothy and alfalfa hay plus a daily supplement of
cats 8 pounds, wheat bran % pound, and linseed oil meal
1/8 pound. Manure collections were made on three separate
mornings, within a period or one week in December, when
the temperature averaged approximately 20°F. The samples
were promptly placed in a walk-in refrigerator Operated

at 00F, and stored until collections were completed. Upon
removal, the manure samples were mixed and one-half the
total amount was dried immediately at 80°C for 24 hours.
This was designated "dried fresh horse manure". The
remaining portion was incubated at 57°C for 4 days. Small
additions of water were added at the end of the first and
third days to keep the sample moist. After incubation

the manure was dried at 800C for 24 hours. This was
designated "dried incubated horse manure".

After drying both manures were ground through a 1/16
inch screen of a hammer mill. Supplementation to the
basal diet was made in this form, the appearance and
texture approximating that of a low quality alfalfa meal.

A mixture of synthetic vitamins listed in tgblg'gg
was used to supplement the basal diet in experiments 4, 7

and 8.

21.

Table 2a

Vitamin mixture (numerals indicate milligrams of each
vitamin added per each 100 grams of basal diet).

WWW

 

Thiamine Hydrochloride 0.2 Menadione 0.1
Riboflavin 0.5 p-aminobenzoic acid 0.2
Ca pantothenate 1.5 Alpha toc0phenol 0.5
Pyridoxine Hydrochloride 0.4 Folic acid 0.1
Biotin 0.02 Nicotinic acid 5.0

 

Several products of animal origin were assayed in
experiment 5 for APF at the 4.0 percent level of supple-
mentation. They are described as follows:

Fishmeal - An unidentified sample. It was guaranteed to
contain not less than 60% crude protein. Many fine bones,
scales and pieces of fish heads were Observed in this meal
indicating that it was prepared largely from fish trim-
mings. Such a preparation is regarded as inferior to a
meal prepared from whole fish.

Meatscrap - Purchased as a meat and bone meal and guaran-
teed to contain not less than 50 percent protein. This
sample was regarded as an inferior grade of meatscrap as
evidenced by its rather high content of hair.

Blood meal - Dried whole beef blood and contained approxi-’
mately 80 percent of crude protein.

Li!§£.£2§l - Prepared from animal livers and glandular
tissues (largely spleens) and was guaranteed to contain
not less than 65 percent protein.

Dried whey - Prepared from cheese whey and was guaranteed

to contain not less than 12 percent crude protein.

22.

Condensed fish solubles - Prepared from an aqueous extract
of fish (run-off water in the making of fisnmeal) free of

oils. It was condensed to 50 percent solids and contained
approximately 30 percent crude protein.

Liqua fish - Prepared from homogenized whole fisn and fish

 

trimmings plus fisn solubles. It contained 55.8 percent
solids and approximately 35 percent crude protein.

The rumen content and cow manure used in eXperiments
S and 7 was Obtained from a heifer at the college eXperi-
mental dairy barn which was equipped with a surgical
fistula. Her only feed was dried brome grass hay and she
had received this feed for a period of several weeks.
Rumen and manure samples were collected on the same day.
The entire rumen sample was obtained at once and immedi-
ately immersed in 95 percent ethyl alcohol to stob bact-
erial action. The manure sample was collected from
several defecations which occurred during the morning.
After each defecation the manure was likewise immersed,
in a separate vessel, containing 95 percent alcohol. Upon
completion of collections the alconol was poured off from
each sample, and the samples dried at 80°C for 24 hours.
The dried manure and rumen content were then ground through
a 1/16 inch screen of a hammer mill and supplemented to the
basal diet in this form.

The vitamin 812 preparation used in experiments 8 and
9 was a concentrated adsorbate wnich was standardized by

microbiological assay to contain 5 milligrams of vitamin

312 per pound. This concentrate contained charcoal (40%)
as the carrier or adsorbing agent and soybean flour (60%)
as the diluent.

In all of the groups where supplementation was made
to the basal diet, such supplementation was made at the
expense of corn.

All of the data presented were analyzed for signifi-
cance of differences by Fisher's method of analysis of

variance (Fisher, 1950).

24.

RESULTS AND DISCUSSION

Egperiment l

The results of this experiment as shown in tgble E
definitely show that liver concentrate possesses APF activ-
ity. The basal diet, when fed unsupplemented, produced
deficiency symptoms in that the growth of the chicks in the
control group was depressed, thus making it a suitable diet
for assaying APF activity. When used at the 0.2 percent
level, liver concentrate adequately supplemented the basal
diet. The difference between the average gain of the chicks
in the control group and the average gain of the group
receiving supplementation of 0.2 percent liver concentrate
was highly significant. There was an increase, although
not significantly so, in the average gains of chicks when
the basal diet was supplemented with 0.4 and 0.8 percent
liver concentrate over the group which received 0.2 per-
cent supplementation. The level of 0.8 percent liver
concentrate showed no appreciable increase in APF activity
over the supplementation with liver concentrate at the 0.4
percent level. Activity, for APF, was shown for liver
concentrate at the 0.05 percent level. The difference
between the average gain for that group and the control
group was significant. From these results it was con-
cluded that 0.4 percent liver concentrate could be used

as a reliable supplement to the basal diet for a positive

25.

control in assaying materials for APF activity.
Supplementation of the basal diet with 0.2 and 0.4
percent beef extract did not make significant differences
between the gains of chicks in those groups and the control
group, thus indicating that beef extract did not possess

APF activity at the levels used in this eXperiment.

Experimentlg

In this experiment liver concentrate was re-assayed
at the 0.1, 0.2, and 0.4 percent levels. Dried fresh
horse manure and dried incubated horse manure were both
‘assayed for APF at levels of 1.25 and 2.5 percent. The
results are given in tgblg 4.

The data clearly show that the liver concentrate, as
was found in experiment 1, was a potent source of APF. A
.highly significant growth response was obtained with each
of the three levels of supplementation used as compared
with the negative control group. The greatest increment
of gain was obtained with 0.4 percent liver concentrate,
which is in line with the results obtained in experiment 1.

The initial assays of the two samples of horse manure
gave striking results. Supplementation of the basal diet
with dried fresh horse manure at the 1.25 and 2.5 percent
levels produced a slight growth response, but the apparent
advantages were not significant, (compare groups 5 and 6
‘with group 1). In contrast, however, supplementation of
the basal diet, at the same levels, with dried incubated

liorse manure produced growth responses which were highly

26.

significant, (compare groups 7 and 8 with group 1). The
gains were essentially in line with the gains obtained
with liver concentrate supplements, (compare groups 7 and 8
with groups 2, 5, and 4). These results indicate that
appreciable synthesis of APF does not occur in the intest—
inal tract of the horse. 0n the other hand, the data show
quite conclusively that large amounts of APF occur in

horse manure which has been subjected to incubation.
Presumably APF originated in this instance through bacter-
ial synthesis, although proof of this assumption is not
given in this work. Supporting evidence for the bacterial
synthesis theory is found, however, in the work of McGinnis
et. al. (1947) who showed that fresh chicken feces obtained
from chickens fed an APF free diet possessed no APF activ-
ity when fed to chicks. However, when the same feces were
incubated and then fed to chicks a significant growth res-
ponse was obtained. These workers concluded that the
growth reSponse was due to the presence of APF which origin-
ated as a result of bacterial synthesis, and that aerobic
bacteria were involved in the synthesis. In a similar
study Stokstad et. al. (1948) found that a nonmotile, rod-
shaped organism from hen feces, when grown aerobically on
simplified media containing no appreciable quantities of
APF, could produce this factor as indicated by assay with
chicks on diets containing all the known B-complex factors
together with high levels of soybean oil meal.

It is interesting to note in this eXperiment as well

27.

as others which follow, that the efficiency of feed utiliz-
ation is increased considerably when adequate amounts of APF
are present in the diet. In this connection, this is the
first instance, as far as the writer is aware, where data
banibeen obtained on the effect of APF in the diet on the

efficiency of feed utilization.

Experiment,fi

Dried fresh horse manure and dried incubated horse
manure were re-assayed at higher levels in this eXperiment.
The results are given in tablg,5.

Dried fresh horse manure did not exhibit activity for
APF when supplemented to the basal diet at the 2.5, 5.0 and
10 percent levels (compare groups 5, 4, and 5 with group 1).
However, significant increases in growth were obtained when
the same levels of dried incubated horse manure were used
in a similar manner (compare groups 6, 7, and 8 with group
1). These results are essentially the same as those
obtainedin experiment 2 and substantiate further, the con-
tention that little APF is synthesized by intestinal micro-
organisms in the horse. The data also suggest that aerobic
conditions are conducive to the synthesis of APF by micro-
organisms in horse manure.

In comparing feed efficiency figures it may be seen
again that the presence of adequate amounts of APF improved
the efficiency of feed utilization (compare groups 2 and 6
‘with group 1). Although a maximum growth reSponse was

attained with the addition of 2.5 percent dried incubated

28.

horse manure, additional increments of 5.0 and 10 percent
appeared to depress growth and lower the efficiency of
feed utilization. This effect may have been caused by the
high amount of fiber in the manure, or by some toxic sub-
stances known to occur in fecal matter such as indole and
skatole, or to the action of both fiber and toxic materials.
Although the data of this eXperiment indicate that
fresh horse manure contains no appreciable amounts of APF,
it would not be safe to conclude that bacterial synthesis
of the factor in the intestines does not occur. It is
conceivable that minute amounts of APF are produced by
intestinal bacteria but because of interfering substances in
feces, pointed out previously (fiber and toxins) potency
couldnnm'be demonstrated in the whole manure even though a
relatively high level was used. It is possible that
concentrates prepared from fresh horse manure would exhibit

some activity for APF.

Experiment,&

This eXperiment was designed to test the influence of
additional vitamins in the basal diet on the assays of liver -
concentrate, dried fresh horse manure, and B-Y feed for APF.
Tgplg Q shows a summary of the results obtained.

Other investigators have shown (Rubin and Bird (1946a),
and Whitson et. al. (1945))that the type of diet such as
was used in this work is adequate in all the known nutrient
essentials required by the chick. The data of this experi-

ment show that the addition of vitamins (see table fig for

29.

composition of vitamin mixture) to the basal diet produced

a growth reaponse which was highly significant. The supple-
ments of 0.4 percent liver concentrate and 2.5 percent
incubated horse manure likewise stimulated growth to a
degree which was highly significant. B-Y feed at a level

of 2.5 percent did not significantly improve growth.

The favorable reSponse obtained with the vitamin mix-
ture might be interpreted as being caused by counteraction
of vitamin deficiencies existing in the basal diet. Argu-
ment against such an interpretation is presented in the
report of Bird and Rubin (1946) who showed that high levels
of pantothenic acid and pyridoxine supplements to a Corn-
soybean diet significantly improved the growth rate of
chicks. Apparently these vitamins in combination at high
levels have a aparing action on APF in the chick which
results in stimulated growth. The vitamin mixture used in
this eXperiment contained relatively high amounts of these
two vitamins, and they conceivably could have been respons-
ible for the favorable results obtained although some of
the other vitamins of the mixture could possibly have
played contributing roles. Massive doses of riboflavin
apparently have no supplemental effect as evidenced by the
failure of B-Y feed, an excellent source of riboflavin,
to stimulate growth when fed at the 2.5 percent level,
(compare groups 4 and 1).

It is interesting to note that the group receiving
the liver concentrate and vitamins exhibited a greater

gain than the groups receiving either of these supplements

alone

30.

alone showing that the effect of each was additive (the
differences between groups 5 and 6 are highly significant).
The group receiving dried incubated horse manure and
vitamins also showed a greater gain than the groups receiv-
ing either of these supplements alone but the differences
were not significant. The combination of liver concentrate
and B-Y feed did not improve growth over that which was
obtained from liver concentrate alone (compare groups 8 and

2).

Exocriment,5

Several proteins of animal origin were assayed for
APF in this experiment. The results are given in 33212,].

It can be seen that meatscrap, blood meal, and dried
whey had little activity for APF when assayed at the 4
percent level of supplementation (compare groups 4, 5, and
9 with group 1). It has been indicated elsewhere in this
thesis (materials and procedure) that the sample of meat-
scrap used was considered to be of inferior quality
because of its appearance and high content of extraneous
matter. Numerous investigators have found considerable
variability among different lots of meatscraps in regard
to APF potency. 0n the basis of the findings of other
workers, together with the results obtained in this experi-
ment, it would appear unsafe to rely upon meatscrap as the
sole carrier of APF in chick starting and breeding mashes
unless unusually high levels of this product were employed

in an attempt to offset a possible deficiency of the

31.

factor in the sample of meatscrap used.,

When the basal diet was supplemented with 4 percent
of blood meal,growth was significantly depressed and
efficiency of feed utilization was lowered. This is in
agreement with the work of Bird and Groschke (1942) who
obtained similar results with this product. Increasing
the protein level of an APF deficient diet with additions
of soybean oil meal, which is devoid of APF, has been
shown to aggravate an APF deficiency in the chick as
characterized by a depressed growth rate. If bloodmeal
were low in APF, which apparently is true, the depression
in growth obtained in this eXperiment could conceivably
be caused by the high protein effect, since the supplement
of 4 percent of bloodmeal would add approximately 3 per-
cent more protein to the basal diet.

The growth of chicks fed the basal diet supplemented
with 4 percent of dried whey was essentially the same as
that of the group fed the basal diet alone (compare groups
1 and 9). Since the addition of dried whey was made at
the expense of corn the percent of protein in the diet was
not appreciably changed. The results indicate that dried
whey is not a good source of APF:

Fishmeal, although it appeared to be an inferior
sample, was shown to contain APF as evidenced by its
ability to promote growth in chicks at the 4 percent level

of supplementation. The increased growth was significant

but not highly significant (compare groups 1 and 3).

32.

Liver meal, condensed fish solubles, beef extract,
and liqua-fish were shown to be excellent sources of APF.
When these products were added to the basal diet, at a
level of 4 percent, highly significant gains were made
(compare groups 6, 7, 8, and 10 with group 1). In eXperi-
ment 1 (see tablg 3) it was found that beef extract did
not possess APF activity at levels of 0.2 and 0.4 percent.
The results of this experiment show that beef extract does
carry APF, but higher levels than those previously employed
must be used to demonstrate the occurrence of the growth
factor in this product.

There were no significant differences between the
gains made by the chicks receiving liver meal, condensed
fish solubles, beef extract and liqua-fish as compared
with the group fed 0.4 percent liver concentrate (positive
control).

In comparing percent feed efficiencies, again it was
shown that when adequate amounts of APF were present in the

diet feed utilization was improved.

Experiment g

 

The Beltsville workers have demonstrated that cow
manure contains an unidentified chick growth factor.
According to presumptive evidence, the “cow manure factor"
is either identical with or closely related to APF. The
site of origin of the “cow manure factor" is still obscure.

It is known, however, that bacteria present in the rumen

synthesize appreciable amounts of all of the known water-

33.

soluble vitamins and in view of this biological phenomenon
it has been postulated that the “cow manure factor",
because it is water-soluble (Rubin and Bird (1946b)), may
also be synthesized by rumen bacteria. It seemed of
interest to study this problem further since a rumen-
fistula heifer was available which afforded an excellent
Opportunity to assay rumen content and feces from the

same animal, and thus establish more precisely the site

of origin of the "cow manure factor".

The procedure followed for collecting and drying the
rumen content and cow manure has been described (see
materials and procedure). In this experiment both dried
rumen content and dried fresh cow manure were assayed for
APF at levels of 2.5 and 5 percent. The growth promoting
activity of these supplements was compared with the
growth promoting activity of dried incubated horse manure
fed at the same levels (2.5 and 5 percent). The horse
manure preparation was used as a positive control in this
instance because it was established in previous experiments
that either 0.4 percent of liver concentrate or 2.5 per-
cent of dried incubated horse manure produced maximum
growth stimulation in the chick. The results of the
assays are given in tablg §. -

The data clearly show that dried incubated horse
manure, as was found previously, is a very potent source
of APF. Highly significant gains were produced with the

2.5 and 5 percent levels of supplementation.

34.

The results obtained with the dried rumen content are
extremely interesting. In this instance both the 2.5 and
5 percent levels depressed growth although the growth
depression was not significant. Efficiency of feed
utilization was also impared. These data are difficult to
interpret. The results suggest however that APF is not
synthesized in appreciable quantities by rumen bacteria.
The recent work of Nichol et. al. (1948) involving studies
on the distribution of an unidentified chick growth factor
(APF) showed that among two samples of rumen content
extract assayed, one sample stimulated growth at a level
of 1.5 percent while the second sample depressed growth
when fed at the same level. It is obvious that more work
must be done to clarify the picture of microbial synthesis
of unidentified growth factors in the rumen.

The assay of dried fresh cow manure showed activity
for APF at the 2.5 percent level of supplementation and
the growth reSponse in this instance was significant.
Efficiency of feed utilization was also slightly improved
(compare groups 1 and 6). However, when the level of
supplementation was increased to 5 percent the growth
reSponse was of the same magnitude as that of the negative
control and the efficiency of feed utilization was impaired
(compare groups 1 and 7). Increased fiber and/or fecal
toxins could possibly have caused inferior results obtained
with the higher level of supplementation.

The data of this experiment would seem to indicate

that bacterial synthesis of APF does occur in the intest-
inal tract of the cow and that this is a more important

site of origin of the factor than the rumen.

Experiment 7

The effect of vitamins on the assay of dried fresh cow
manure and dried rumen content for APF was studied in this
experiment. The vitamin mixture (Lgélg gg) was added to
all eXperimental groups. The results obtained are given in
James.

It was shown in the previous experiment (experiment 6)
that supplementation of the basal diet with dried rumen
content depressed growth and efficiency of feed utilization.
In the present experiment these deleterious effects were
not observed and it would appear that certain vitamins when.
fed at high levels are effective in counteracting the growth
depression caused by the dried rumen content. Tne mechan-
isms involved in this favorable effect cannot be eXplained
on the basis of the limited data at hand. Inasmuch as the
dried rumen content again failed to produce a growth
reSponse when fed at either the 2.5 or 5 percent levels
(compare groups 6 and 7 with groups 1, 2, and 3) these
results suggest that very little, if any, APF is synthesized
by rumen bacteria.

The results obtained with the feeding of dried fresh
cow manure in this eXperiment conflict the the results
obtained in the previous eXperiment (eXperiment 6) where a

significant growth reaponse was obtained at the 2.5 percent

36.

level of supplementation (see tgblg é, group 6). It should
be pointed out, however, that the dietary conditions were
not the same in both instances; vitamins were added to the
diets of all eXperimental groups in this experiment, while
none of the eXperimental groups received supplemental
vitamins in eXperiment 6. The differences obtained can
possibly be eXplained on the basis of the results of a
previous eXperiment (see tghlg Q) where it was shown that
the addition of vitamins to the basal diet significantly
improved growth and that a combination of vitamins and APF
produced an additional significant growth response showing
that the growth effect of each was additive. With this in
mind it might be argued that the growth reSponse obtained
vith the 2.5 percent level of the cow manure preparation in
experiment 6 was caused, largely, by vitamins of the known
category synthesized by intestinal bacteria rather than to
the presence of appreciable amounts of APF. The failure
of cow manure to improve growth in this experiment over
that of the negative control, which received vitamins,
suggests that APF was lacking and therefore it seems
questionable whether a significant amount of the factor is

produced by intestinal bacteria in the cow.

Exgeriment §

During the progress of this work simultaneous announce-
ment was made from two laboratories working independently
(Rickes et. al. (1948) and Smith (1948)), that a red

crystalline substance had been isolated from liver fractions

having high activity for the human anti-pernicious anemia
factor. Rickes and co-wcrkers named the factor vitamin B12.
Subsequent work by Ott et. a1. (1948) showed that vitamin
812 had APF activity for the chick and that the Chick's
requirement for the new vitamin appeared to be less than
3.0 micrograms per 100 grams of diet.

It was of interest to determine whether vitamin B12
had APF activity as measured by the chick bio-assay proced-
ure developed in this study. Accordingly, a standardized
vitamin B12 concentrate was obtained and several levels of
vitamin B12 were fed. The results are summarized in 2&212
lQo .
It should be pointed out that all of the eXperimental
groups received a supplement of the vitamin mixture (see
tablg gg). The vitamin mixture was employed as a precaut-
ionary measure in order to obtain maximum growth stimu-
lation from the vitamin B12 supplements since previous
eXperiments showed that the vitamin mixture as well as APF
produced a growth reSponse but that the growth effect of
each was additive.

The data of this eXperiment definitely demonstrate

that vitamin B has APF activity for the chick. Each

12
level of vitamin B12 used produced essentially the same
gain as was achieved with either the supplement of 0.4L
percent liver concentrate or 5 percent dried incubated

horse manure. It would appear that the Chick's requirement

for vitamin 812 is at least 1.0 micrograms per 100 grams of

38.

diet, since significant increases in growth were not
obtained with levels above this figure.
Lillie et. al. (1948) showed that as little as 1.0

micrograms percent of pure crystalline vitamin B com-

12
pletely replaced the need for the "cow manure factor"
(presumably the same as APF) in chicks. In this experi-
ment as little as 1.0 micrograms of vitamin B12 per 100
grams of diet produced a maximum growth response and com-
pletely replaced the need for APF which was derived from
either liver concentrate or dried incubated horse manure.

All of the eXperimental groups receiving the various
levels of vitamin B12 showed marked improvement in effic-
iency of feed utilization and were comparable to the
efficiency figures obtained with the feeding of liver
concentrate or dried incubated horse manure.

The results of this experiment establish a close
relationship between APF and vitamin B12 and indicate that

they may be identical factors.

Experiment‘g

In this experiment the vitamin B12 concentrate was
re—assayed for APF activity at lower levels and the vitamin
mixture, in contrast to experiment 8, was omitted from all
the eXperimental groups. A summary of the results obtained
is given in t§3;2_11.

Again, as in experiment 8, the vitamin B12 concentrate
exhibited APF activity. Highly significant weight gains

were obtained with supplements of 0.5, 1.0, 3.0, and 5.0

39.

micrograms of vitamin B12 per 100 grams of diet (compare
groups 4, 5, 6, and 7 with group 1). The greatest growth
response was obtained with the highest level of vitamin
B12 (5.0 micrograms percent) but this value was not signi-
ficantly greater than that of the positive control (compare
groups 2 and 7). The difference in gains between the 0.5
microgram percent level (group 4) and the 5.0 microgram
percent level (group 7) was significant. However, the
difference in.gains between the 1.0 microgram percent level
(group 5) and the 5.0 microgram percent level (group 7)
were not significant. Differences between groups 6 and 7
were also not significant. It would appear, therefore,
that under the conditions of this experiment, the chick's
(Rhode Island Reds) requirement for vitamin B12 for maxi—
mum growth is in the region of 1.0 micrograms per 100
grams of diet. This value is in agreement with the work
'of Lillie et. al. (1948) whose data indicated that the
requirement of vitamin 812 for chicks was between 1.0 and
2.0 micrograms percent. Agreement is also found with the
work of Ott et. al. (1948) who reported that the require—
ment appeared to be below 3.0 micrograms percent. The data
of this eXperiment indicate that vitamin 312 is identical
with or closely related to APF.

Efficiency of feed utilization was increased with all
levels of vitamin B12 supplementation as compared with the

negative control.

40.

Table l.-Materna1 diet (Breeder mash formula C-l).

 

 

 

Ingredient Percentage

Ground yellow corn I 17.5
Wheat bran 15.0
Wheat middlings ‘ . a 15.0
Ground oats 12.5
Soybean oil meal (44%) 12.51
Dehydrated alfalfa meal (17%) 7.5
Meal scraps (55%) 5.0
Fish meal 5.0
Dried skimmilk 3.0
Steamed bone meal 3.0

round oyster shell flour 2.0
Salt (iodized) 1.0
Fish oil (400 D - 3000 a) 0.8
Manganese sulfate 8 oz./ton

 

Calculated analysis:

Protein 20.8%, Calcium 2.42%, PhOSphorus 1.27%

41.

Table 2.-Basal "animal protein-free“ diet.

 

 

u

 

Ingredient Percentage
Soybean oil meal (44%) 50.0
Ground yellow corn 39.5
Dehydrated alfalfa meal (17%) 5.0
Steamed bone meal 3.0
Ground limestone 1.5
B-Y feed (500 riboflavin per gram) 0.3
Cod liver oil (400 D - 2000 A) 0.2
Salt (iodized) 0.5
Choline chloride 0.1
Nicotinic acid 0.005 '.
Manganese sulfate 0.023

 

Calculated analysis:

Protein 26.6%, Calcium 1.5%, Phosphorus 0.8%

According to the recommended nutrient allowances, as

established by the National Research Council, this diet

meets the requirements of all the known nutrient essentials

'for the chick.

42.

Table 3.-Assay of liver concentrate and beef extract for

”animal protein factor".

L
f

(14 chicks per group)

f

 

 

av. wt. av. wt. av. %
Group Supplement 2 wk. 4 wk. gain mor-
ng. ems. gms. tality
1 None 88.9 161.4 72.5 0
2 0.05% Liver concen- 87.0 187.3 100.3* 7.14
trate
3 0.1 % Liver concen- 86.9 187.9 101.0* 0
trate
4 0.2 % Liver concen- 87.9 208.5 120.6** 0
trate
5 0.4 % Liver concen- 86.1 224.5 138.4** 0
trate
6 0.8 % Liver concen- 85.1 223.6 l38.5** 0
trate
7 0.2 % Beef extract 86.6 .160.1 73.5 7.14
8 0.4 % Beef extract 87.1 180.3 93.2 0

 

* Significant at the 5% level.

** Significant at the 1% level.

L‘
K)!
0

Table 4.~Assay of dried fresh horse manure and dried
incubated horse manure for "animal protein factor".

(14 Chicks per group)

av. wt. av. wt. av.
Group Supplement 2.wk.. 4 wk. gain

gms. gms. gms.

 

% feed %
effic- mor-
iency tality

 

1 None 85.0 136.4 51.8

2 0.1% Liver concen- 85.5 201.5 116.0**
. trate -

3 0.2% Liver concen- 85.4 189.5 104.1%“

trate

4 0.4% Liver concen- 85.7 217.9 132.2**
trate

5 1.25% Dried fresh 86.5 159.2 72.6

horse manure

6 2.5% Dried fresh 86.5 175.8 89.3
horse manure

7 1.25% Dried Incubated 86.6 196.3 109.7**
horse manure

8 2.5% Dried Incubated 85.9 210.4 124.5*§
horse manure

25.20 7.14
43.50 0
39.57 0
44.44 0
31.90 0
31.85 0
44.82 0
40.87 0

 

** Significant at tne 1% level.

Table 5.-Re-assay of dried fresh horse manure and
dried incubated horse manure for "animal

protein factor". (14 chicKs per group)

_:-
_--

44.

 

av. wt. av. wt. av. % feed %
Group Supplement 2 wk. 4 wk. gain effic- mor-
gms. gms. gms. iency tality
1 None 87.2 149.5 62.3 29.40 0
2 0.4% Liver concen- 85.7 217.1 131.4** 44.56 0
trate
3 2.5% Dried fresh 86.6 161.8 75.6 32.28 0
horse manure -
4 5 % Dried fresh 87.4 171.3 83.9 34.31 0
horse manure
5 10 % Dried fresh 85.7 153.4 67.7 28.16 7.14
horse manure
6 2.5% Dried incubated 86.4 218.6 132.2** 42.22 0
horse manure
7 5 % Dried incubated 86.2 208.1 122.6** 34.71 0
horse manure
8 10 % Dried incubated 85.2 202.2. 116.8** 36.54 0
horse manure

 

** Significant at

the 1% level.

45.

Table 6.-Effect of vitamin mixture on assay of liver
concentrate, incubated horse manure and B-Y feed
for "animal protein factor". (14 cnicks per group)

 

 

av. wt. av. wt. av. % feed %
Group Supplement 2 wk. 4 wk. gain effic- mor-
gms. gms. gms. iency tality
1 None 84.0 149.5 65.5 28.46 0
2 0.4% Liver concen- 84.5 197.6 115.1** 43.65 7.14'
trate
3 2.5% Dried incubated 82.1 186.2 104.1** 38.22 0
norse manure
4 2.5% B-Y feed 80.9 162.8 81.9 32.66 0
5 Vitamin mixturel 83.0 185.4 102.4** 45.06 7.14
6 0.4% Liver concen- 82.7 217.1 134.4** 45.17 0
trate and vitamin
mixture
7 2.5% Dried incubated 82.0 202.6 120.6** 41.22 0
horse manure and
vitamin mixture1
8 2.5% B-Y feed and 81.5 198.0 116.5** 59.56 0

0.4% liver
concentrate

 

** Significant at tne 1% level.

1 See table 22 for composition or vitamin mixture.

46.

Table 7.-Assay of several products of animal origin for
"animal protein factor". (12 chicks per group)

u:
I.

 

T

 

 

1:“

av. wt. av. wt. av. % feed %
Group Supplement 2 wk. 4 wk. gain effic- mor-
gms. gms. gms. iency tality

 

1 .None 87.5 165.3 77.8 32.05 0
2 0.4% Liver concen- 87.0 208.0 122.0** 42.09 0
trate
3 4 % Fish meal 87.7 185.4 97.7“ 39.21 0
4 4 % Meat scrap 87.4 168.5 81.1 31.33 0
5 4 % Blood meal 86.6 129.6 43.6** 21.32 0
6 4 % Liver meal 85.8 197.0 111.2** 40.71 0
7 4 % Condensed 86.6 191.2 104.6** 36.79 0
- fish solubles

8 4 % Beef extract 86.4 192.6 106.2** 34.05 0
9 4 % Dried whey 86.0 157.1 71.1 26.42 8.33
10 4 % Liqua-fish 86.0 208.1 122.1** 40.52 0

2 Significant at the 5% level.
** Significant at the 1% level.

47.

Table 8.-Assay of dried fresh rumen content and dried
fresh cow manure for ”animal protein factor“.
(12 chicks per group)

 

 

+

 

 

av. wt. av. wt. av. % feed %
Group Supplement 2 wk. 4 wk. gain effic- mor-
gms. gms. gms. iency tality
None 84.7 148.3 63.6 30.52 0
2.5% Dried incubated 84.8 198.8 114.0** 40.70 0
horse manure '
5 % Dried incubated 84.2 193.7 109.5** 38.20 0
horse manure
2.5% Dried rumen 85.7 129.5 45.6 15.24 8.55
content
5 % Dried rumen 83.9 132.0 48.1 18.49 8.33
content
2.5% Dried fresn 89.2 172.6 89.4* 34.90 0
cow manure
5 % Dried fresh 85.1 151.7 68.6 24.80 0
cow manure
* Significant at the 5% level.
** Significant at the 1% level.

48.

Table 9.-Effect of vitamin mixture on assay of fresh
dried rumen content and fresh dried cow manure for
”animal protein factor“. (12 ch10ks per group)

 

 

1 av. wt. av. wt. av. % feed %
Group Supplement 2 wk. 4 wk. gain effic- mor-

gms. gms. gms. iency tality

 

1 None 79.6 166.7 87.1 31.02 0
2 2.5% Dried incubated 80.1 201.2 121.2* 42.11 8.33
horse manure

3 5 % Dried incubated 79.3 203.7 124.4** 39.03 8.33
horse manure

4 2.5% Dried fresh 79.5 173.6 94.1 34.96 0
- cow manure

5 5 % Dried fresh 78.0 156.4 78.4 31.16 0
cow manure

6 2.5% Dried rumen 79.3 167.8 88.5 34.80 8.3:
content

7 5 % Dried rumen 87.5 156.3 68.8 31.93 8. 3
content

 

* Significant at the 5% level.
** Significant at the 1% level.

1 Vitamin mixture (table 2a) added to all groups.

49.

Table 10.-Assay of vitamin B12 for "animal protein
factor“ activity. (12 chicks per group)

 

“I

 

 

 

 

 

1 av. wt. av. wt. av. % feed %
Group Supplement 2 wk. 4 wk. gain effic- mor-
gms. gms. gms. iency tality

 

ww—v

1 None 86.1 155.4 69.3 29.46 0
2 0.4% Liver concen- 86.6 213.8 127.2** 42.69 0
trate

5 5 % Dried incubated 85.8 210.5 124.5** 58.42 0
horse manure

4 1” 7. B12 86.1 214.8 128.7M 45.56 0
5 2'7% B12 85.1 206.4 121.5** 41.95 0
6 57% B12 84.9 218.7 155.8** 43.67 0
7 5rr% 512 85.2 211.6 126.4** 42.49 0
8 77% 1312 85.2 216.8 151.6" 57.87 0

** Significant at the 1% level.

Vitamin mixture (table 2a) added to all groups.

Table 11.-Re-assay of vitamin B12 for"anima1 protein
factor" activity. (12 chicks per group)

 

 

av. wt. av. wt. av. % feed %
Group Supplement 2 wk. 4 wk. gain effic- mor-
gms. gms. gms. iency tality

 

1 None 76.7 139.2 62.5 21.61 0

2 0.4% Liver concen- 77.2 192.6 115.4** 43.93 0

trate
5 0.17% 1312 77.4 152.2 74.8 27.51 8.55
4 0.5735812 77.1 177.6 100.5" 57.09 0
5 1.07% 812 77.9 195.2 115.5M 40.84 0
6 5.07% 312 76.5 179.6 105.1M 57.91 8.33
7 5.07% 1312 76.3 205.0 128.7** 45.91 0

 

* Significant at the 5% level.
** Significant at the 1% level.

A reliable procedure was developed for assaying
various protein concentrates and certain preparations for
"animal protein factor“ (APF) activity. It was demon-
strated that chicks from hens fed a high quality breeder
mash (containing APF) could be used for bio-assay purposes
provided the chicks were fed an APF deficient basal diet
for a two week period after hatch. This preliminary treat-
ment was necessary in order to deplete the chicks of their
bodily stores of APF and thus make them responsive to
supplements carrying the growth factor. Following the
depletion period the chicks were weighed and only those.
within a certain weight range (75-90 grams) were utilized
as assay animals. EXperimental groups were then formed,
carefully equalized with respect to weight, and continued
on the basal diet for two weeks (the assay period) except
that each test group received as a supplement one of the
materials to be tested. In each assay a negative control
group was fed the basal diet alone and a positive control
group the basal diet plus 0.4 percent liver concentrate,
or a preparation which had the potency equivalent to 0.4
percent liver concentrate.

Assays of dried fresh horse manure and dried incubated 4
horse manure for APF activity showed very little, if any,
activity in the fresh manure, whereas the incubated manure
exhibited high activity. These results indicate that, at

most, bacterial synthesis of APF in the intestines of the

 

52.

horse is very small and that aerobic conditions faVor the
synthesis rather than anaerobic.

Although the basal diet employed in this work was
formulated to meet the requirements of the chicks for all
the known nutrient essentials it was found that the addi-
tion of a vitamin mixture stimulated growth which was
highly significant. However, a combination of vitamins
and APF produced a further highly significant growth
response, thus demonstrating that the growth effect of each
was different and additive.

When several proteins of animal origin were assayed
for APF activity at a level of 4 percent it was found that
the meatscrap, bloodmeal, and dried whey samples were
inadequate sources.of APF. Growth was significantly depress-
ed by the bloodmeal. The fishmeal sample, although
considered to be of inferior qualftv, significantly improved
growth. Condensed fish-solubles, liqua-fish, and beef
extract were shown to be excellent s urces of APF.

Dried fresh cow manure and dried rumen content were
assayed for APF. Neither of these materials appeared to
possess APF activity. Thus it would seem that if bacterial
synthesis of the factor does occur in the rumen or in the
intestines of the cow, at most, the amount produced is very
small.

A standardized concentrate of vitamin B12 was assayed
and was found to possess APF activity. As little as 1.0

microgram per 100 grams of diet was shown to produce a

53.

maximum growth stimulation. The growth reSponse from
vitamin B12 supplementation was of the same magnitude as
that obtained from supplementation with liver concentrate
or dried incubated horse manure.

Efficiency of feed utilization was shown to be markedly

improved by adequate levels of APF or vitamin 812 supple-

mentation.

54:

CONCLUSIONS

From the data obtained in this investigation, the

following conclusions may be made:

1.

Chicks which are suitable for assaying APF activity
can be obtained from hens fed‘a high quality breeder
diet (containing APF) provided the chicks are
depleted of their bodily stores of APF prior to the
assay period. ‘

Incubated horse manure was found to be a potent
source of APF, whereas fresh horse manure contained
no measureable amounts of the factor. Hence it
would appear that if intestinal synthesis of APF

in the horse does occur, at most, the amount pro-
duced is very small.

Among various samples of protein concentrates of
animal origin assayed for APF at a level of 4 per-
cent supplementation, condensed fish solubles,
liqua-fish, liver meal and beef extract were found
to be excellent sources of the factor. Fishmeal
appeared to be a good source, and meatscrap, blood-
meal, and dried whey exhibited no potency.

Dried rumen content and dried fresh cow manure
appeared to have no APF activity at levels of
either 2.5 percent or 5.0 percent supplementation.
This finding suggests that if bacterial synthesis

of A2F does occur in the rumen or in the hfiestines

5.

55.

of the cow, at most,. the amount produced is very
small.

Vitamin B12 is either identical with or closely
related to APF. The vitamin 812 requirement of the
chicks for maximum growth was found to be in the

region of 1.0 microgram per 100 grams of diet.

Vitamin-B and APF greatly improve the efficiency

12
of feed utilization of chicks fed a corn-soybean

diet.

56.

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58.

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'Jan 21 '55

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