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 Downloaded from jn.nutrition.org by guest on March 12, 2012 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 Â°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 43Â°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 40Â°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 PÃ©nicillium 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. 7 J. NUTRITION, 8: '62 301 302 L. R. RICHARDSON, S. WILKES, J. GODWIN AND K. R. PIERCE 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 meal2Steamed moldy and control soybean meals were bone mealNaClMnSCX,H2OCorn 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 chlorideVitaminsVlOO an hour, the extract was filtered off on a large Buchner funnel. This procedure was gmVitamin 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 Downloaded from jn.nutrition.org by guest on March 12, 2012 the extraction were dried to approximately 10% moisture at a maximum temperature of 45Â°C. The residue from the control pantothenateNiacinBiotinFolie meal is designated control residue and that from the moldy meal is designated acidVitamin moldy residue. Bi2Inositolgm58.035.02.00.22.214.171.124.33000IU400ICUm0.752.01.01.01.04.02.00.020.20.0110.0 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 Downloaded from jn.nutrition.org by guest on March 12, 2012 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, soybean mealControl no.1234567Fractions 3 weeksam2407313111898238292 Final20201010891920191056919Avg SBMMoldy SBMControl residueMoldy residueMoldy residue extract2Control + moldy SBM 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 Downloaded from jn.nutrition.org by guest on March 12, 2012 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 acidThreonineSerineProlineGlutamic 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 acidGlycineAlanineValineCystineMethionineIsoleucineLeucineTyrosinePhenylalanineLysineHistidineArginineControlmg/gm53.617.931.115.474.42 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 Downloaded from jn.nutrition.org by guest on March 12, 2012 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 Controlacids%NoneLysine, ofpoults2981010789Gain**gm223411518461238399543 ofpoults210899885amgm510535501501519515501MoldyNo. 0.6Lysine, 0.8Lysine, 1.2Arginine, 1.0Arginine, 1.5Lysine, 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 commenÃ§ai soybean meal to fornia. Forgacs, J. 1962 "Mycotoxicoses" the neglected fungal growth for 6 weeks. In general, disease. Feedstuffs, 34: 124. Downloaded from jn.nutrition.org by guest on March 12, 2012 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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