Detection of estrogenically active substances in diets for sows by an in vitro bioassay supported by HPLC analysis

There is considerable evidence that exogenous estrogenic compounds can have adverse effects on fertility. The main reason cited in literature for hyperestrogenism in pigs is contamination of feedstuffs by the mycotoxin zearalenone (Boehm, 2000), but further estrogenically active substances might also be involved in cases of impaired fertility with symptoms like enlarged, red-coloured vulvae in piglets, irregular estrus cycles and anestrus of sows (Bennetts et al., 1946; Drane et al., 1981). It is well known that soy used in diets for pigs as a main protein source contains phytoestrogens. Amongst them, isoflavones like genistein and daidzein are of particular interest. Aim of this study was to optimize and use an established bioassay (Kluczka, 2003) to determine estrogenic activity in feedstuffs for pigs related to isoflavones and further substances with estrogenic potential. This bioassay is a reporter gene assay based on stably transfected human embryonal kidney cells (HEK 293) that contains either alpha or beta estrogen receptor (alpha- or beta-HEK). The estrogenic activity measured in the luciferase assay was expressed in estradiol-equivalents (EEQ) and the results were compared with the isoflavone content (genistein, daidzein) obtained by chemical analysis using high performance liquid chromatography-Ultraviolet (HPLC-UV). Mean estrogenic activity in diets fed to sows in herds with altered fertility was 275.8 microg EEQ/kg feed in alpha-HEK cells and 295.0 microg EEQ/kg feed in beta-HEK cells. Feedstuffs from herds without any altered fertility showed an average estrogenic activity of 204.9 microg EEQ/kg feed in alpha-HEK and 213.3 microg EEQ/kg feed in beta-HEK. The estrogenic activity was strongly related to the concentration of the isoflavones (alpha-HEK, r(2)=0.9488; beta-HEK, r(2)=0.9427). Clinically relevant zearalenone concentrations (>50-150 microg/kg feed) displayed estrogenic effects in the bioassay that did not differ significantly from those caused by high isoflavone concentration because of the use of soy as protein source.     

Determination of the estrogenic activity of wild phytoestrogen-rich Pueraria mirifica by MCF-7 proliferation assay

The aim of this study was to evaluate the estrogenic activity of tuberous samples of wild, phytoestrogen-rich Pueraria mirifica collected from 28 out of 76 provinces of Thailand by MCF-7 proliferation assay. The plant extracts were administered to MCF-7, ER alpha positive human mammary adenocarcinoma cell cultures, for 3 days at dosages of 0.1, 1, 10, 100 and 1,000 microg/ml and were compared with 17 beta-estradiol at concentrations of 10(-12)-10(-6) M. The mean P. mirifica population at 1 mug/ml exhibited significant proliferation. Two plant samples exhibited levels of proliferation in MCF-7 that were similar to 17beta-estradiol. The mean P. mirifica populations at 100 and 1,000 microg/ml exhibited significant cytotoxicity in MCF-7. Analysis of the estrogenic activity of puerarin, representative of major isoflavonoids in P. mirifica tubers, revealed proliferation in MCF-7 only at the highest dose (10(-6) M) that was 10(2)-10(5) times less active than 17 beta-estradiol. Puerarin and 17 beta-estradiol at concentration of 10(-12)-10(-6) M exhibited no cytotoxicity in MCF-7.     

Influence of zearalenone on reproductive system cell proliferation in gilts

Zearalenone (ZEA) is a macrocyclic lactone, estrogenic, diet-depending and fusaric micotoxin, which is produced on many kinds of cereals and feeds in the favourable conditions of humidity and temperature. The structure of ZEA is similar to the structure of estrogens and it enables binding to the estrogenic receptors. The stimulation of protein synthesis in the cells of the reproductive system, which causes intensification of cell proliferation, is one of the effects of ZEA actions. Oedema and vulva reddening are the clinical, external signs of ZEA intoxication in pigs. The aim of this study was to designate the degree of reproductive cell proliferation after low doses of ZEA were applied per os in sexually immature gilts with simultaneous monitoring of zearalenone and alpha-zearalenol levels in peripheral blood. The following were observed in the gilts examined fluctuations of zearalenone and alpha-zearalenol levels in blood, which were connected with entero-hepatic circulation and also numerous histopathological changes in ovarian follicle structure. These changes were present in the reproductive system of sexually immature gilts with a big contribution of PCNA-positive cells. The studies show that zearalenone application in sexually immature gilts caused ovarian follicle atresia and apoptoso-like changes in granule cells. Intensified cell proliferation, which was expressed with the growth of PCNA index, was observed in uterus and oviduct.     

The impact of zearalenone on the level of the selected estrogens in blood serum of sexually immature gilts

The aim of the study was to evaluate the influence of low dose (LOEL - lowest observed effect level) of zearalenone (200 microg/kg b.w.), applied per os for 7 days (short-term intoxication), on sexual behavior, concentration of the examined xenobiotic and its metabolite and selected estrogens in sexually immature gilts: ovariohysterectomised (group D1) and intact (group D2) animals. Clinical signs of oestrus (reddening, oedema and hyperaemia of the vulva and serorhoea from the reproductive tract--lack of standing reflex) were obserwed in group D1 on day 6 and in group D2 on day 4 of the experiment. Laboratory analyses of blood plasma were carried out determine the presence of zearalenone and alpha-zearalenole. They revealed an increase in the level of alpha-zearalenol before the oestrus, decrease in total amount of both examined substances on day when the oestrus appeared and increase in the level of both examined xenobiotics in the post oestrus period together with the higher share of zearalenone. Medium concentrations of estrone and estradiol within the borders of method determination in the majority of periods examined. Higher levels of estrone (32.0 pg/ml) were found on day 4, in the group D2 and estradiol (6.5 pg/ml) on day 6 in the D2 group. The presents study revealed that zearalenone applied per os at LOEL dose causes the incidence of apparent sexual readiness (without standing reflex) in sexually immature gilts with the somatically immature reproductive system.     

Basis of melengestrol acetate action as a progestin

To provide insight into potential mechanisms contributing to the various biological responses of cattle to treatment with progesterone, norgestomet, and melengestrol acetate (MGA), MCF-7 cells were utilized to determine the relative binding affinity of the progesterone receptor for MGA, norgestomet, progesterone, and a progesterone agonist (R5020), and to determine if progesterone, MGA, or norgestomet have estrogenic and/or anti-estrogenic activities. The progesterone receptor had greater affinity (P<0.05) for MGA, R5020, and norgestomet than for progesterone; and the affinity for norgestomet exceeded (P<0.05) that of MGA and R5020. Estrogen stimulates proliferation of MCF-7 cells; therefore these cells have been utilized as a bioassay to detect estrogenic and anti-estrogenic activity. Progesterone (10(-11) to 10(-5)M) did not promote cellular proliferation. However, MGA (10(-8), 10(-7), and 10(-6)M) increased (P<0.05) cell proliferation compared to the control group (10(-11) to 10(-9) and 10(-5)M MGA did not stimulate cell proliferation), and MGA-induced cell proliferation (10(-8)M) was reduced (P=0.095) by an estradiol antagonist (ICI 182,780; ICI). Cellular proliferation increased (P<0.05) with norgestomet (10(-5)M) compared to the control group (10(-11) to 10(-6)M norgestomet did not stimulate cell proliferation) and the increased proliferation was decreased (P<0.05) by ICI. Neither progesterone nor MGA demonstrated anti-estrogenic activity. Norgestomet (10(-10) to 10(-6)M) did reduce (P<0.05) maximal estrogen-stimulated cell proliferation, but not to basal levels. In summary, the affinities of the progesterone receptor for norgestomet, MGA, and progesterone are consistent with their effective dose to inhibit ovulation in vivo, but their progestin and their estrogenic/anti-estrogenic activities cannot fully explain why progesterone and norgestomet are more capable of reprogramming the reproductive axis in anestrous postpartum cows compared to MGA.     

Conflict of estrogenic activity by various phthalates between in vitro and in vivo models related to the expression of Calbindin-D9k

Phthalates are suspected to disrupt the endocrine system, especially through estrogenic effects. In the present study, we investigated the effects of various phthalates and compared them with those of estrogenic compounds that disrupt the female reproductive system. To assess the effects of these phthalates, alteration of the Calbindin-D9k (CaBP-9k) gene was measured as a biomarker because rat CaBP-9k gene carries an estrogen response element (ERE) which is involved in estrogen responsiveness of the gene during the estrous cycle. In this study, phthalates were tested for estrogenic properties in in vitro and in vivo models. First, the E-Screen assay was used to measure the proliferation of MCF-7 cells, a human breast cancer cell line. Treatments with 17beta-estradiol (E2; 9-fold) and 17alpha-estradiol (EE; 9-fold) induced MCF-7 cell proliferation at concentrations of 10(-9) M. Phthalates induced an increase in MCF-7 proliferation at concentration of 10(-6) M up to 10(-4) M. Nbutyl benzyl phthalate (BBP; 6-fold vs. vehicle), dicyclohexyl phthalate (DCHP; 8-fold), 2-ethylhexyl phthalate (DEHP; 6-fold) and di-n-butyl phthalate (DBP; 7-fold) at the concentration of 10(-4) M induced in an increase in MCF-7 proliferation after 6 d of treatment compared to vehicle. However, significant increase in MCF-7 proliferation was induced by diethyl phthalate (DEP). Second, we investigated the expression of CaBP-9k in the uterus of immature rats after oral treatment with BBP, DCHP, DEHP, DBP or DBP (600 mg/kg per day) in this in vivo model, because the immature rat model is highly sensitive to exposure to estrogenic chemicals. None of the phthalates induced the expression of CaBP-9k mRNA and its protein in the neonatal uterus as analysed by Northern and Western blot analyses, respectively. Although phthalates induced an increase in MCF-7 cell proliferation by an estrogenic effect, they could not induce CaBP-9k expression in the in vivo system, suggesting that the assays of estrogenic effects of various phthalates conducted in vitro and in vivo expression of CaBP-9k may produce conflicting results.     

Influence of zearalenone on some metabolic, physiological and pathological aspects of female rabbits at two different ages.

Thirty six female rabbits half of which at four months of age while the other half aged eight months. Each group of age was divided into three subgroups of an equal number and received 0.0, 0.5, and 1.0 ppm zearalenone (F-2) in the feed of young animals and 0.0, 1.0 and 4.0 ppm in the feed of the old one for 18 days. The data showed that zearalenone administration to young rabbit diets elevated body weight gain, feed intake, water consumption, digestibility and digesta contents of dry matter and ash. Haemoglobin percent, packed cell volume, and serum calcium, phosphorus and vitamin C were also increased in response to dietary F-2. Liver dry matter, ether extract and ash contents as well as bone density, ash and silica contents were considerably elevated in the young treated animals. The opposite trend however, was seen for all studied parameters of old rabbits fed on diets supplemented with F-2. On the other hand, zearalenone application caused a noticeable histopathological changes in liver, kidneys, lungs, heart, adrenal glands, spleen and uterus. Thus, and in spite of its improving the performance of young rabbits, it would not be recommended to use F-2 as anabolic agent in rabbits diet.     

Low concentrations of zearalenone in diets of boars for a prolonged period of time

Thirty-two boars were fed diets containing 0, 3, 6 or 9 ppm zearalenone from 32 d of age up to 145 or 312 d of age. Rate of gain, feed intake and gain:feed were not significantly different among treatments. Feeding of diets containing 9 ppm zearalenone did not affect the libido in seven boars. There was a tendency for boars fed 9 ppm dietary zearalenone to produce lower total and gel free volumes of semen, with lower total motile sperm; however, only the percent motility of the sperm was reduced (P less than .05).     

Mycotoxins and reproduction in domestic livestock.

Molds are parasitic plants that are ubiquitous in livestock feedstuffs. Even though molds themselves reduce the quality of grains, their synthesis of chemical substances termed mycotoxins causes the greatest monetary loss to the animal industry. Five major mycotoxins that impair growth and reproductive efficiency in North America are aflatoxins, zearalenone, deoxynivalenol, ochratoxin, and ergot. Aflatoxins are produced by Aspergillus flavus and Aspergillus parasiticus. Consumption of grains containing aflatoxins by swine affects reproduction indirectly by reducing feed intake and growth. In swine, aflatoxins impair liver and kidney function, delay blood clotting, increase susceptibility to bruising, and interfere with cellular humoral immune systems. Ruminants are comparatively resistant to aflatoxicosis, but presence of aflatoxins in milk of dairy cows is closely monitored for human safety. Depending on environmental conditions, Fusarium roseum can produce either zearalenone or deoxynivalenol. Days 7 to 10 postmating seem to be a critical period of gestation for zearalenone to exert its detrimental actions on early embryonic development. Presence of deoxynivalenol in swine feedstuffs decreases feed intake, causes feed refusal, and induces occasional vomiting. Several species of Penicillium and Aspergillus produce ochratoxin, a mycotoxin that causes necrosis of kidney tissue. Ergot alkaloids produced by Claviceps purpurea on wheat can cause reproductive problems and are associated with lactational failure in swine. Various methods have been developed to remove mycotoxins from infected feedstuffs. Chemical analyses in laboratories as well as diagnostic kits suitable for use at the elevator or farm can be used successfully to identify which mycotoxins are present in suspect feedstuffs.     

Effects of Dietary Contamination by Zearalenone and Its Metabolites on Serum Anti‐Müllerian Hormone: Impact on the Reproductive Performance of Breeding Cows

We investigated the effects of in vivo exposure to low zearalenone levels on the anti‐Müllerian hormone endocrine levels and the reproductive performance of cattle. Urine and blood samples and reproductive records were collected from two Japanese Black breeding female cattle herds with dietary zearalenone contamination below the threshold levels (<1 ppm) at 30 days after calving. Urinary zearalenone, α‐zearalenol and β‐zearalenol concentrations were measured by chromatography‐tandem mass spectrometry, and serum anti‐Müllerian hormone concentrations were determined along with serum biochemical parameters. Urinary concentrations of α‐zearalenol were significantly higher (p < 0.05) in cattle in Herd 1 than in cattle in Herd 2, reflecting the different amounts of zearalenone in the diet of the two herds. Although the number of 5‐mm and 10‐mm follicles of the herds and their fertility after artificial insemination were similar, the serum anti‐Müllerian hormone concentrations in herds 1 and 2 were 438.9 ± 48.6 pg/ml and 618.9 ± 80.0 pg/ml, respectively, with a trend towards a significant difference (p = 0.053), which may indicate differences in the antral follicle populations between herds. Thus, zearalenone intake from dietary feed, even when below the threshold zearalenone contamination level permitted in Japan, may affect the ovarian antral follicle populations, but not the fertility, of post‐partum cows.     

Bone ossification and carcass characteristics of wethers given silastic implants containing estradiol

Administration of growth promotants with estrogenic activity via hastening closure of the growth plate could have an economic impact on lambs because closure results in ovine carcasses being classified as yearlings. Twelve wether lambs approximately 12 mo old were given silastic implants filled with estradiol-17 beta and allotted randomly to be slaughtered 30, 60, 90, or 220 d after implantation to determine time of growth plate closure in relation to date of implanting. Seven comparable wethers not implanted served as controls. Four wethers implanted at 2 mo of age and slaughtered 220 d later also were included. Implanted wethers had serum concentrations of estradiol averaging 13.9 pg/ml over the 220-d implant period; controls averaged 2.7 pg/ml. Estradiol implants increased carcass maturity scores but fat deposition was not altered. Growth plate widths decreased (P less than .05) as wethers grew older and implants were in place for longer periods of time. Metacarpal growth plates in 12-mo-old lambs were completely ossified 220 d after implanting, but control wethers and wethers implanted at 2 mo maintained growth plate widths. Neither metacarpal nor metatarsal bone lengths differed (P less than .05) between control and implanted wethers. Growth plate ossification was not complete until 570 d of age in implanted lambs, even though bone length had stopped increasing by 408 d. Therefore, even though estradiol and other growth stimulants with estrogenic activity increase rate of ossification of the metacarpal growth plate, bone length or mature size is not limited.(ABSTRACT TRUNCATED AT 250 WORDS)     

Combined action of plant estrogens, F-2 toxin and natural estrogens

The investigation concerns the interaction of plant estrogens, F-2 toxin, and natural estrogen (estradiol-benzoate) as causative factors of eostrogenic changes. Plant estrogens and F-2 toxin were applicated at three and estradiol-benzoate at two dosage levels. The estrogenic effect was studied by bioassay on immature rats taking uterine weight, uterine liquid and vaginal opening as criteria. When uterine weight is taken as a criterion, plant estrogens and F-2 toxin, no matter if they are administered alone or together, regularly increase the effect of estradiol-benzoate, but when uterine fluid is taken as a criterion they always decrease the effect of estradiol-benzoate. According to the present results it seems obvious, that the mode of action of estrogenic substances occurring in plants considerably differ from that of natural estrogens. Thus the mechanisms which cause disturbances may be different from those of natural estrogens.     

Effect of feeding sheep on naturally spoiled rice straw, clover hay and concentrate feed mixtures

The effects of feeding naturally contaminated feedstuffs to sheep were investigated through feeding of contaminated and uncontaminated clover hay, rice straw, and concentrate feed mixture were compared. The highest level of contamination with fungi was observed in the concentrate feed mixture followed by clover hay and rice straw. The most dominant mycotoxin in the contaminated feedstuffs was zearalenone. Contamination resulted in increases of crude protein, ash and silica in feedstuffs while both ether extract and crude fibre decreased. In vivo digestibility of different diet constituents in both contaminated and uncontaminated feedstuffs were determined using four mature Rahmani rams. Contamination resulted in significant increase in the digestibility of dry matter, crude fibre and energy. Contamination affected feed intake of the different feedstuffs particularly the concentrate feed mixture. A metabolism trial was also conducted to study the nitrogen and mineral balance. Retention of nitrogen and calcium was reduced when mouldy feeds were given. On the other hand, magnesium was relatively increased. Blood analysis for sheep at the end of the trials resulted in elevated values of inorganic phosphorus and phospholipid phosphorus and declined total nitrogen, calcium, cholesterol, calcium/phosphorus ratio and cholesterol/calcium ratio due to feeding on mouldy ration. Results of the urine analysis during the metabolism trials proved that mouldy ration increased the estimated values of the concentration of calcium, magnesium and vitamin C; whereas pH values, total nitrogen, ammonia nitrogen and inorganic phosphorus were declined during the feeding on the mouldy ration. Mouldy feeds tested, seemed to have toxic, nephritic and hepatic effect.     

Evaluation of brine shrimp (Artemia salina) larvae as a bioassay for mycotoxins in animal feedstuffs.

Brine shrimp larvae was tested as a possible simple biological screening system to identify specimens of animal feedstuffs that should be examined further by chemical analytical procedures for mycotoxins. All extracts of the control, nonmouldy feedstuffs increased larval mortality, this being most marked in the case of silage. Chemical and biological testing of diagnostic specimens indicated that the bioassay identified two of four chemically positive specimens and 59 of 135 chemically negative specimens and 59 identified larvicidal compounds present in normal feedstuffs gave a high percentage (56%) of false-positive bioassay results when compared to the results of chemical analyses for three mycotoxins. The use of brine shrimp larvae did not materially reduce the necessity of conducting chemical analyses for mycotoxins.     

Estrogen receptor in bovine skeletal muscle

In connection with investigations of the anabolic action of estrogens, we examined skeletal muscle of veal calves for estradiol receptors. The high speed supernatant of muscle homogenate was incubated with .5 nM 3H-estradiol and for the determination of nonspecific binding with .5 nM 3H-estradiol plus 13 nM estradiol at 0 C overnight. After treatment with charcoal two times, the supernatant was analyzed by agar gel electrophoresis. Specific binding was found in the typical position of cytosolic estradiol receptor. Ninety percent of 3H-estradiol binding was suppressed by estradiol-17 beta, zeranol, estrone or diethylstilbestrol, but was not affected by testosterone, dihydrotestosterone, trenbolone or progesterone. The specific binding activity varied between .3 and 2.0 fmol/mg protein and the dissociation constant of the receptor was Kd = 60 pM. After an enrichment up to 42 fmol/mg cytosolic protein using heparin sepharose, the receptor remained unchanged as determined by agar gel electrophoresis. Although uterine tissue generally contains 1,000 times more estradiol receptors, these results clearly demonstrate that skeletal muscle also contains estradiol receptors with identical properties. This indicates that one possible component of the anabolic action of estrogens may be the direct stimulation of the muscle via the estradiol receptor.     

Assessing estrogenic activity of pyrethroid insecticides using in vitro combination assays

Pyrethroid insecticides are among the most commonly used classes of insecticides worldwide, but their endocrine disrupting activities remain unclear. Therefore, in the present study, we examined the estrogenic activities of pyrethroid insecticides in E-screen and competition binding assays. In addition, we measured estrogen receptor (ER) protein and pS2 mRNA levels in human breast cancer cells (MCF-7 BUS) to clarify the mechanism of their estrogenicity. Seven pyrethroid insecticides (bioallethrine, cypermethrin, deltamethrin, fenvalerate, permethrin, sumithrin, and tetramethrin) were tested because of their worldwide usage. In addition, 17beta-estradiol was tested as a positive control. As expected, 17beta-estradiol significantly increased MCF-7 BUS cell proliferation at concentrations of 10(-11) M and above. Of the pyrethroid insecticides tested, only sumithrin increased MCF-7 BUS cell proliferation in a dose-dependent manner; the maximum induction of cell proliferation was observed at a dose of 10(-5) M. In the anti-estrogenic activity test, bioallethrin, fenvalerate, and permethrin significantly inhibited 17beta-estradiol-induced MCF-7 BUS cell proliferation at 10(-6) M, a concentration comparable to the effective dose (10(-9) M) of ICI 182,780, a pure ER antagonist. However, none of the pyrethroid insecticides competitively inhibited the binding of [(3)H]estradiol to rat uterus ERs in competition binding assays. Both 17beta-estradiol (10(-10) M) and sumithrin (10(-5) M) decreased the levels of cytosolic ERalpha and ERbeta protein expression significantly as compared with the vehicle control. In addition, 17beta-estradiol (10(-10) M) increased pS2 mRNA expression markedly, and sumithrin significantly increased pS2 mRNA levels in a dose-dependent manner. The other six compounds tested in the present study did not affect ER protein levels or pS2 mRNA levels. These results suggest that certain pyrethroid insecticides may be considered to be estrogen-like chemicals that act through pathways other than direct ER binding, and may function as endocrine modulators in both wildlife and humans.     

抽样调查北京地区猪场饲料及饲料原料玉米赤霉烯酮污染状况

本研究采用免疫亲和柱高效液相色谱法测定了北京地区猪场饲料原料及全价饲料中玉米赤霉烯酮（ＺＥＮ）的含量,以了解北京地区饲料中 ＺＥＮ的污染情况。试验抽样采集北京市昌平区、大兴区、延庆区、平谷区、顺义区 ５个区县 １５个猪场 １３１份饲料样［５５份玉米、豆粕、麸皮、干酒糟及其可溶物（ＤＤＧＳ）原料,７６份猪全价饲料］进行 ＺＥＮ含量的测定。结果表明：玉米、豆粕、麸皮、ＤＤＧＳ和全价饲料中 ＺＥＮ的检出率分别为 １００．００％、５４．４５％、１００．００％、１００．００％和 ５８．８８％,超标率分别为 ０．００％、０．００％、０．００％、４１．１８％和 ０．００％,平均含量分别为 １０９．０８、９．１９、１４．９２、８８２．６８和 ５８．８８μｇ／ｋｇ。结果提示,不同饲料原料中 ＺＥＮ含量存在差异,ＤＤＧＳ中 ＺＥＮ平均含量超标,猪全价饲料及玉米、豆粕等饲料原料中 ＺＥＮ平均含量均未超标。     

玉米赤霉烯酮的代谢、毒性及其预防措施

玉米赤霉烯酮(ZEA)是由镰孢属真菌产生的类雌激素毒素.由于气候条件不同,食品和饲料中ZEA浓度差异很大(从几个mg/kg至几千个mg/kg).本文综述了ZEA的吸收、代谢和生物转化机制,ZEA的生殖毒性、细胞毒性、免疫毒性和遗传毒性,并且从预防土壤中镰孢菌及其相关霉菌的污染、收获和收获后真菌毒素的控制、污染颗粒的物理...     

Modification of zearalenone structure in model and natural conditions

Zearalenone is a mycotoxin widely occurring in cereals and animal feed, and it is associated with hyperestrogenism and other reprodutive disorders in animals. A new method of detoxication of feedstuffs involves alkaline hydrolysis of toxic macrolactone (1) (as well as model compounds (2a, 2b)). The method caused modification of zearalenone structure under mild conditions and the toxin underwent irreversible hydrolysis with high efficiency.     

Survey of vomitoxin-contaminated feed grains in midwestern United States, and associated health problems in swine

During the 1981 corn harvest season in Illinois and surrounding states, cold wet weather enhanced the growth of Fusarium graminearum, with resulting contamination by vomitoxin and, to a lesser extent, zearalenone. Of 342 feed samples analyzed, 274 contained vomitoxin at a concentration ranging from 0.1 to 41.6 ppm (mean, 3.1 ppm) and 40 samples contained zearalenone at a concentration ranging from 0.1 to 8 ppm (mean, 0.66 ppm). Animal health problems and reduced growth performance were observed mainly in swine fed vomitoxin-contaminated rations. The predominant clinical complaints, in decreasing frequency were: reproductive problems (50%), feed refusal (43%), reduced weight gain (25%), diarrhea (17%), death (14%), and emesis (11%).