Effect of Moldy Diet and Moldy Soybean Meal on the Growth of

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					Effect of Moldy Diet and Moldy Soybean                                   Meal
on the Growth of Chicks and Poults
                            L. R. RICHARDSON, STELLA WILKES, JACKIE GODWIN
                            ANDK. R. PIERCE
                            Departments of Biochemistry and Nutrition and Veterinary Pathology
                            Texas Agricultural Experiment Station, Texas A and M Colleqe
                            College Station, Texas

   Grain that has undergone          spontaneous   used in those with poults. The procedure
heating due to the growth of various fungi         used to mold the soybean meal was briefly
is considered unsuitable       for human foods,    as follows:    Two hundred pounds of soy
but large quantities       are used widely in      bean meal were mixed and then divided in

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livestock and poultry feeds. Some animals          to two lots. One lot containing      10% mois
consuming     certain    lots of moldy grain       ture was stored in covered containers           at
have developed       characteristic     symptoms   ambient temperature      as a control for the
and died (Ewing, '51; Semeniuk, '54; Mor
rison, '56). These conditions were attrib          moldy meal. There was no fungal growth
                                                   in this lot and it is designated     as the con
uted to a toxicosis.        Other animals con      trol. The second lot, after adjusting         the
suming moldy grains          were affected only    moisture content to 19% by the direct addi
slightly or not at all.     The effect of moldy    tion of water, was placed in a series of 4-
grain on the growth         of different species   liter Dewar flasks. The flasks containing
of animals has been         variable.   An excel   the meal were then stored in a heating cabi
lent review (Forgacs,     '62) on the mycotoxi-    net at 31 °C and 78% relative humidity for
coses that have developed in different spe         6 weeks. Growth of the naturally occuring
cies of animals       has   been    published      fungi produced heating in 5 to 6 days and
recently.                                          the temperature    of the meal in the flask
    Renewed interest has developed in the          was an average of 43°C during the entire
possible toxicity of moldy feeds because           storage period. This continuous       high tem
of the widespread mortality in poults that         perature   shows that fungi were growing
received certain ground nut meals. This            during the entire storage period.         To in
disease has been called "Turkey X" disease
                                                   sure that all the soybean meal within a
(Blount, '61) and the toxic substance has
                                                   flask was subjected to fungal activity, the
been associated with the growth of Asper-          contents    of each      flask    were     mixed
gillus flavus (Lancaster  et al., '61).
                                                   thoroughly at the end of three weeks. At
   In view of the high mortality in poults         the end of 6 weeks, the meal was dried in
receiving   certain ground nut meals and           an air circulating    drier at 40°C for 24
the retarded growth obtained in this lab           hours. The entire lot which had a mois
oratory in chicks receiving a moldy diet, it       ture content of approximately         10% was
was decided to investigate      the nutritive      reground, mixed thoroughly and stored at
properties   of moldy soybean meal using           room temperature     until used. This same
poults as the test animal.    A summary of         procedure was used to mold the diet for
the tests with chicks and the results with         chicks with the exception        that the feed
poults receiving    diets containing   moldy       was allowed to mold for periods of 2, 4, 6
soybean meal are described in this report.         and 10 weeks.
                                                       Using Czapek solution agar and potato
                EXPERIMENTAL                       dextrose agar, the predominant         fungi on
   Preparation of moldy feed and soybean           moldy soybean meal were Pénicillium sp.
meal.    A moldy diet was used in the tests         and Aspergillus glaucus group.
with chicks and moldy soybean meal was               Received for publication June 8, 1962.
J. NUTRITION, 8: '62

   Extracted soybean meal. If the re                                      TABLE 1
tarded growth was due to a toxic sub                        Composition        of   typical   diet
stance, it appeared that it might be pos
sible to remove it by extraction with some
solvent. With this possibility in view,        (milo)Soybean
                                               Sorghum grain
moldy and control soybean meals were                     bone
each extracted with 70% ethanol. In this
extraction, 5 kg of soybean meal were
stirred one hour with 10 liters of 70%         oil3DL-MethionineCholine
ethanol. After the mixture had settled for
an hour, the extract was filtered off on a
large Buchner funnel. This procedure was
repeated 4 times and the combined ex           AVitamin
tracts were concentrated to 33% moisture.      D3Menadionea-TocopherolThiamine-HClRiboflavinPyridoxine-HClCa
The concentrate from the control soybean
meal is designated as control extract and
that from the moldy meal is designated
as moldy extract. The residues left from

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the extraction were dried to approximately
10% moisture at a maximum temperature
of 45°C. The residue from the control         pantothenateNiacinBiotinFolie
meal is designated control residue and
that from the moldy meal is designated         acidVitamin
moldy residue.                                 Bi2Inositolgm58.
    Care of poults. Day-old white broad
breasted mixed sex (Nicholas) poults ob          1The author is indebted to Merck and Company,
tained from a commercial hatchery were         Rahway, N. J., for generous supplies of vitamins; to
                                               Lederle Laboratories, Division of American Cyanamid
used to test the effect of the moldy soy       Pearl River, N. Y. for folie acid.
                                                 2Control and moldy soybean meal analyzed 48%
bean meals and moldy residues. Ten poults      and 57% protein, respectively.
                                                 3Mazola, Corn Products Company, New York.
per group with essentially the same aver
age weight were housed in electrically
heated battery-type brooders. Feed and         permitted the growth of the naturally oc
water were supplied ad libitum and the         curring fungi. Storage periods of 2, 4, 6
poults were weighed weekly during the ex       and 10 weeks were used. As a control for
perimental period indicated in the tables.     the moldy diet, a second portion of the
In these studies, deaths occuring during       same diet was stored under the same tem
the first week were due usually to a weak      perature conditions, but the moisture con
poult or an accident and these poults were     tent was below that which permitted the
not counted in the observation. The com        growth of fungi.
position of a typical diet is given in table      The diet, which had been allowed to
 1. Control and moldy soybean meal and         mold two weeks, supported a rate of
the residues were substituted for the soy      growth essentially equal to that of the
bean meal in this diet at a level of 35% .     control or nonmoldy diet. When the diet
                                               was allowed to mold 4, 6, or 10 weeks, the
        RESULTS AND DISCUSSION                 chicks grew at a progressively slower rate
   Moldy diet for chicks. A few tests          as the molding period increased. Supple
have been carried out in this laboratory       menting the moldy diet with known vita
on the effect of a moldy diet on the growth    mins, protein or fat did not improve it to
of chicks. The details of these tests were     a significant degree. There were no defi
described in another paper (Richardson         ciency symptoms in the chicks or evidence
and Webb, 62) and only a brief summary         of a disease to account for the retarded
of the results will be given in this report.   growth and it was tentatively concluded
In these tests, a diet that was adequate       that the fungi produced some substance
for the growth of chicks was stored under      which was sufficiently toxic to retard the
moisture and temperature conditions which      growth of chicks.
                                 MOLDY        SOYBEAN      MEAL      FOR    POULTS                           303

   Moldy soybean meal for poults.           The              necrosis with the formation of small round
average gain in weight at three weeks of                     homogeneous        pale-pink     globules of pro-
poults receiving     various   soybean    meals              teinaceous-type      residues.     In the kidney
and residues is summarized          in table 2.              there was extensive         degeneration        or ne
The data for groups 1 and 2 show that                        crosis of tubule epithelium          and some de
the nutritive   value of the meal was de                     generation     in the glomerular         tufts.    The
creased markedly by the molding process.                     viable epithelium         of tubules       was very
Poults receiving either control (group 3) or                 swollen. In the liver lipidosis was observed
moldy residue (group 4) grew at a slower                     that was more severe in some areas than
rate than those receiving control soybean                    in other areas.        Nucleoli of the hepatic
meal (group 1). Five of 10 poults receiv                     cells were very distinct.          The final diag
ing the moldy residue died during the sec                    nosis was a toxic hepatitis and nephritis.
ond and third week. None receiving the                       The symptoms         in the other poult were
control residue died.                                        similar but less severe.           Histological     ex
   Addition of the moldy extract         to the              amination     of the tissue of the poults in
moldy residue (group 5) did not improve                       this group (no. 4, table 2) which survived
the rate of growth, but addition of the                      to three weeks showed no characteristic
moldy extract (group 7) to control soy                       changes from poults receiving control soy

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bean meal increased        the rate of growth                bean meal.        Histopathological         examina
slightly over that obtained by the control                    tions were carried out on the livers of 28
soybean meal ( group 1 ). Addition of con                     additional poults receiving control soybean
trol extract to control soybean meal (group                  meal and 28 receiving moldy meal. The
6) supported essentially the same rate of                    hepatic epithelium        in most of the poults
growth as the control meal (group              1).           from both groups exhibited morphological
These data show that extraction          of the               alterations.   The cytoplasm of the affected
moldy soybean meal with 70% ethanol did                      epithelial cells was distended by an exces
not remove any substance which retarded                      sive amount of water soluble substance
the growth of poults when the extract was                     (presumably     glycogen).      The chromatin of
fed with control soybean meal.          On the               the nuclei was marginated              and the nu
other hand, the high mortality in groups 4                   cleoli were enlarged.        These lesions were
and 5 which received the moldy residue                       most severe in two of the last 5 poults killed
suggests that the residue may have been                      at 19 days. Livers of 10 poults receiving
more detrimental      to the poults than the                 moldy soybean meal and 0.8% of lysine
original moldy soybean meal (group 2).                       were less severely affected although                in
   Pathology.    Two of the poults receiv                    creased fat or glycogen content was dem
ing the moldy residue (table 2, group 4)                     onstrable in some hepatic epithelial cells.
appeared    moribund     and were killed for                     Amino acid supplements.            Total amino
gross and histopathological       examination.               acids were determined          in acid hydrolyzed
The following changes were observed in                       control and moldy soybean meals by the
one poult.     There was focal myocardial                    ion exchange        procedure      (Moore et al.,

                                                        TABLE 2
           Various   fractions   of control    and moldy   soybean    meals   (SBM) in diets for poults

        Group                                   of                         poultsInitial'            gain,
         no.1234567Fractions                                                                      3 weeksam2407313111898238292

                         extract2Control + moldy
                         extract2Control + control
                                    SBM + moldy extract2No.of
      1 Initial no. = number surviving at one week.
      2 Fed at equivalent of three times 35% soybean meal.
304               L. R. RICHARDSON,        S. WILKES,      J.   GODWIN    AND   K. R. PIERCE

'58). The meals were hydrolyzed by re-                     These data suggested that lysine was pres
fluxing 1.5 gm of soybean meal with 150                    ent in borderline amount and a prelimi
ml of constant boiling hydrochloric acid                   nary test was run supplementing the diet
for 24 hours. Hydrochloric acid was re                     containing the moldy meal with 0.8% of
moved by evaporating under reduced pres                    lysine. With this amount of lysine added,
sure. Distilled water was added and the                    the moldy meal supported a rate of growth
evaporation was repeated 6 times. Free                     equal to that obtained with the control
amino acids were obtained from the meals                   meal.
with 70% alcohol by the method described                       One group of 6 broad breasted bronze
by De Vay ('52). The amino acids were                      poults was given the moldy soybean meal
determined in the extract by the ion ex                    diet for 4 weeks. A photograph of a poult
change procedure. In general, most of the                  from this group is given in figure 1 and
free amino acids were slightly higher in                   the development of white bands in wing
moldy meal than in the control meal, but                   feathers shows that the diet supplied in
the relative amounts of free amino acids                   adequate lysine.
in both meals was small in comparison                          After the preliminary test had shown
with the total amino acids. The concentra                  that lysine essentially corrected the in
tion of total amino acids in both meals is                 adequacy of the moldy soybean meal, an

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given in table 3.                                          other series was set up to test the effect
   Of the essential amino acids, lysine and                of lysine and arginine on both control and
probably arginine were the only ones that                  moldy meals. There were 10 poults per
appeared to be significantly less in the                   group and the experimental period was
moldy meal. Using the data for lysine in                   4 weeks. These results are summarized in
table 3, the amount of lysine supplied by                  table 4. As in previous tests, growth of
the control and moldy soybean meals was                    poults (series 1) receiving moldy soybean
0.830% and 0.637%, respectively. The                       meal was severely retarded. Supplement
estimated amount of lysine supplied by                     ing with 0.6% of lysine (series 2) im
the sorghum grain was approximately                        proved the moldy meal but did not cor
0.174% in each case. The lysine require                    rect it completely.          When lysine was
ment for poults has been shown to range                    increased to 0.8% (series 3), the poults re
from approximately 1.1 to 1.46% (Alm-                      ceiving the moldy meal grew slightly faster
quist, '52; Griminger and Scott, '59).
                                                           than those receiving the control meal.
                                                           When the lysine was further increased to
                         TABLE 3
                                                            1.2%, growth of poults receiving the
      Amino acid content of control and moldy              moldy meal was depressed.                Since this
                     soybean meals                         amount did not depress growth of the
           Amino                                           poults receiving control soybean meal, it
       acidAspartic                                        seemed probable that some amino acid
                                                           other than lysine may have been affected
                                                           by growth of the fungi. Total amino acid
                                                           analysis showed that moldy meal was
                                                           lower in arginine than the control meal
                                                           and tests were run to determine whether
                                                            this amino acid might be limiting under
                                                           some conditions. Both moldy and con
                                                            trol soybean meals were supplemented with
                                                            1.0% (series 5) and 1.5% (series 6) of
                                                            arginine. Growth of poults receiving the
                                                            moldy meal was definitely increased with
                                                            1.5% of arginine, but the most rapid rate
                                                           of growth (series 7) was obtained when
                                                            the moldy meal was supplemented with
                                                            0.8% of lysine and 1.0% of arginine.
                                                            These data show that retarded growth of
                               MOLDY      SOYBEAN     MEAL      FOR   POULTS                                  305

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  Fig. 1   Broad breasted bronze poult that had received a moldy soybean meal diet for 4 weeks.

                                                    TABLE 4
    Lysine and arginine as supplements       to control and moldy soybean meals in diets for poults

                   0.8 + Arginine, 1.0Seriesno.1234567No.
         1 L-Lysine and L-arginine monochlorides were fed, but the amounts shown represented the calculated
    free base.
     ice    UH9BI
         2 There were no deaths in any groups after the first week.
         3 Observed average gain at 4 weeks.

poults receiving moldy soybean meal is                   are needed to determine how the growth
due primarily to some important change                   of fungi affect the amino acids so that
in the availability of lysine. Arginine may              they become limiting factors for the growth
be involved also, but this amino acid does               of animals. The amino acid analysis indi
not necessarily become a limiting factor                 cated that the effect is more than merely
for the growth of poults until a fairly large            destruction of the amino acids. The pos
excess of lysine is added. Further studies               sibility that some fungi may produce a
306          L.   R.   RICHARDSON,   S. WILKES,   J.   GODWIN   AND   K. R.   PIERCE

metabolite that is antagonistic to lysine         soybean meals; to Dr. C. E. Miller, Biology
has been considered, but the observations         Department and Dr. H. D. Schroeder,
with 70% ethanol-extracted meal appears           Plant Science Department, Texas A and M
to discredit this probability.                    College, for identifying the predominant
   The fact that maximal growth was not           fungi on the moldy soybean meal.
obtained with less than 0.8% of lysine
indicates that the moldy meal contained                          LITERATURE CITED
very little available lysine. Whether the         Almquist, H J. 1952 Amino acid requirements
problem with moldy soybean meal is re               of chickens and turkeys.        A Review. Poult.
lated to "Turkey X" disease produced by             Sci., 31: 966.
                                                  Blount, W. F      1961 Turkey "X" Disease. Tur
certain peanut meals has not been estab             keys, 9: 52.
lished.                                           De Vay, J. E. 1952 A note on the effect of
                  SUMMARY                           mold growth and increased moisture content
                                                    on the free amino acids in hard Red Spring
    Retarded growth occurred in poults that         wheat. Cereal Chem., 29: 309.
received a diet containing moldy soybean          Ewing, W. R. 1947 Poultry Nutrition, ed. 3 W.
meal. This moldy meal was produced by               Ray Ewing Publication, South Pasadena, Cali
subjecting commençai soybean meal to               fornia.
                                                  Forgacs, J. 1962 "Mycotoxicoses" the neglected
fungal growth for 6 weeks. In general,              disease. Feedstuffs, 34: 124.

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mortality was low, but gross and histo-           Griminger, P., and H. M. Scott 1959 Growth
pathological examination of two poults that         rate and lysine requirement of the chick. J.
were moribund showed a toxic hepatitis              Nutrition, 68: 429.
                                                  Lancaster, M. C., F. P. Jenkins, J. McL. Philp also
and nephritis. Poults receiving the moldy            K. Sargeant, A. Sheridan and J. O'Kelly 1961
soybean meal and 0.8% of lysine grew at              Toxicity associated with certain samples of
a normal rate and the livers were less               ground nuts. Nature, 192: 1095.
severely affected.     Supplementing     the      Moore, S., D. H. Spackman and W. H. Stein
moldy meal with 1.5% of arginine indi                1958 Chromatography        of amino acids on
                                                     sulfonated   polystyrene   resin.  Anal. Chem.,
cated that the availability of this amino            30: 1185.
acid also may have been affected by the           Morrison, F. B. 1957 Feeds and Feeding, ed. 22
fungal growth. When 1.2% of lysine was               Morrison Publishing      Company, Ithaca, New
 added to the moldy meal, arginine defi              York.
                                                  Richardson, L. R., and B. D. Webb 1962 Ef
 nitely became a limiting amino acid for             fect of a moldy diet on the growth and mortal
 growth.                                             ity of chicks and poults. Texas Feed Service
                                                     Report no. 23, p. 3.
           ACKNOWLEDGMENTS                        Semeniuk, G. 1954 In Storage of Cereal Grains
                                                     and Their Products, vol. 2, eds., J. A. Anderson
  Acknowledgment is made to Martha                   and A. W. Alcock. Am. Assoc. Cereal Chem.
Cannon for the amino acid analyses of                Monograph Series, p. 132.

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