Author Correction: Regulation of volatile and non-volatile pheromone attractants depends upon male social status.

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Regulation of volatile and non-volatile pheromone attractants depends upon male social status.

We investigated the regulation of chemical signals of house mice living in seminatural social conditions. We found that male mice more than doubled the excretion of major urinary proteins (MUPs) after they acquired a territory and become socially dominant. MUPs bind and stabilize the release of volatile pheromone ligands, and some MUPs exhibit pheromonal properties themselves. We conducted olfactory assays and found that female mice were more attracted to the scent of dominant than subordinate males when they were in estrus. Yet, when male status was controlled, females were not attracted to urine with high MUP concentration, despite being comparable to levels of dominant males. To determine which compounds influence female attraction, we conducted additional analyses and found that dominant males differentially upregulated the excretion of particular MUPs, including the pheromone MUP20 (darcin), and a volatile pheromone that influences female reproductive physiology and behavior. Our findings show that once male house mice become territorial and socially dominant, they upregulate the amount and types of excreted MUPs, which increases the intensities of volatiles and the attractiveness of their urinary scent to sexually receptive females.

Pru p 3, a marker allergen for lipid transfer protein sensitization also in Central Europe.

In the Mediterranean area, lipid transfer proteins (LTPs) are important causes of plant-food allergies often associated with severe allergic reactions. There, peach LTP (Pru p 3) seems to be the primary sensitizer, whereas in Central Europe, little is known about the importance of LTP sensitization. In this region, allergen extract-based diagnosis is often complicated by co-sensitization to Bet v 1, the major birch pollen allergen, its cross-reactive food allergens, and profilins. We investigated the role of LTP sensitization in Central European patients displaying strong allergic reactions to plant-derived food. Analysis of IgE reactivity revealed that ten of thirteen patients were sensitized to Pru p 3, nine to Bet v 1, and two to profilin. Our results showed that LTP sensitization represents a risk factor for severe allergic symptoms in Central Europe. Furthermore, the strong IgE reactivity detected in immunoblots of plant-food extracts indicated that Pru p 3 can be used as a marker allergen for LTP sensitization also in Central European patients.

Detection and first characterization of an uncommon haptoglobin in porcine saliva of pigs with rectal prolapse by using boronic acid sample enrichment.

Salivary glycoprotein profiles, obtained after boronic acid enrichment, were studied for the first time in pigs in order to search for specific overall alterations related to acute inflammatory condition. Five healthy pigs and five pigs suffering from rectal prolapse were used, and the levels of acute phase proteins were measured to determine the degree of inflammation of the animals. The enriched glycoprotein profiles, achieved by two-dimensional gel electrophoresis (2DE) were statistically evaluated and spots that appeared differentially regulated between states were subjected to MS analysis for protein identification. Spots from three unique proteins were identified: carbonic anhydrase VI (CA VI), α-1-antichymotrypsin and haptoglobin (Hp). CA VI appeared as two adjacent horizontal spot trains in the glycoprotein profile of healthy animals in its regular isoelectric points (pI). One spot of α-1-antichymotrypsin was found in saliva from pigs with rectal prolapse in an unusual basic pI, and was considered as a breakdown product. Hp was identified as several spot trains in saliva from pigs with rectal prolapse in an unusual alkaline pI and was consequently further investigated. SDS-PAGE and 2DE of paired serum and saliva samples combined with Western blot analysis showed that the unusual Hp position observed in saliva samples was absent in serum. Furthermore, N-glycans from serum and saliva Hp glycopatterns were evaluated from SDS-PAGE Hp bands and showed that the serum N-glycan distribution in Hp ß-chain was comparable in quantity and quality in both groups of animals. In saliva, no Hp ß-chain derived N-glycans could unambiguously be identified from this sample set, thus needing further detailed investigations in the future.

Detection of potential markers for systemic disease in saliva of pigs by proteomics: a pilot study.

Animals with different health status have been studied in order to extend the knowledge about protein composition of porcine saliva samples and to discover potential salivary markers for systemic disease in porcine production. Clinical examination of animals was performed at farm level where 10 healthy pigs and 10 animals with evident clinical signs of disease were randomly selected. Saliva and blood samples were obtained and afterwards animals were humanely sacrificed to perform a complete necropsy. Levels of two acute phase proteins, haptoglobin and C-reactive protein, were used to identify possible active infections of the animals. Moreover, serological analysis, to the main porcine infectious diseases in the area, was performed. Salivary proteins were separated by two-dimensional gel electrophoresis followed by mass spectrometry for the identification of specific proteins. A total of 58 spots out of 75 were successfully identified by MS, which correspond to 20 unique proteins. Two different approaches were used to perform a statistical comparison of saliva protein patterns from healthy and diseased animals using the relative spot volume (% spot volume/total volume of all spot in the gel, approach "A") or taking also into account the total protein content of each saliva sample (µg of spot/mL of saliva, approach "B"). Both analyses showed three proteins in common that are differentially regulated between states. However, approach B was selected for biomarker searching since it gave an estimation of protein concentration and showed differential expression of proteins between both health states in a total of 10 proteins, which were up-regulated in disease. Mass spectrometric analysis identified those proteins as salivary lipocalin, lipocalin 1, double headed protease inhibitor protein, adenosine deaminase, haptoglobin, albumin fragments, S100-A8, S100-A9, S100-A12 and pancreatic alpha amylase. These proteins could be considered as potential salivary markers of disease.

The levels of zearalenone and its metabolites in plasma, urine and faeces of horses fed with naturally, Fusarium toxin-contaminated oats.

Concentration profile of zearalenone (ZON) and its metabolites in plasma, urine and faeces samples of horses fed with Fusarium toxin-contaminated oats is described. In plasma, ß-zearalenol (ß-ZOL) was detected at high levels on day 10 of the study (3.21-6.24 µg/l). ß-Zearalenol and α-zearalenol were the major metabolites in urine. Zearalenone, α-ZOL and ß-ZOL were predominantly found in faeces. Zearalanone could also be detected in urine (1.34-5.79 µg/l) and faeces (1 µg/kg). The degree of glucuronidation was established in all sample types, approximately 100% in urine and plasma. Low per cent of glucuronidation (4-15%) was found in faeces samples. The results indicate the main conversion of ZON into ß-ZOL in horse. This finding could explain why horse is not susceptible to ZON in comparison with swine which produce α-ZOL as a predominant metabolite.

A proteomic analysis of serum from dogs before and after a controlled weight-loss program.

The objective of this study was to investigate how weight-loss program would alter the proteome of the serum of Beagle dogs. For this purpose, serum samples from 5 Beagle dogs, before and after weight loss, were analyzed using 2-dimensional electrophoresis. Protein profiles of all samples were obtained, divided into 2 classes (obese and lean), and compared using specific 2-dimensional software, giving a total of 144 spot matches. Statistical analysis revealed 3 spot matches whose expressions were modulated in response to weight loss: 2 protein spots were upregulated and 1 protein spot was downregulated in the obese state in comparison with the lean state of the dogs. Mass spectrometric identification of differentially regulated spots revealed that these protein spots corresponded to retinol-binding protein 4, clusterin precursor, and α-1 antitrypsin, respectively, which could be considered potential markers of obesity and obesity-related disease processes in dogs.

Aflatoxin B(1) in affecting broiler's performance, immunity, and gastrointestinal tract: a review of history and contemporary issues.

Aflatoxin B(1) is a common contaminant of poultry feeds in tropical and subtropical climates. Research during the last five decades has well established the negative effects of the mycotoxin on health of poultry. However, the last ten years of relevant data have accentuated the potential of low levels of aflatoxin B(1) to deteriorate broiler performance. In this regard, any attempt to establish a dose-effect relationship between aflatoxin B(1) level and broiler performance is also complicated due to differences in types of broilers and length of exposure to the mycotoxin in different studies. Contrary to the prevalent notion regarding literature saturation with respect to aflatoxicosis of chicken, many areas of aflatoxicosis still need to be explored. Literature regarding effects of the mycotoxin on the gastrointestinal tract in this regard is particular scanty and non-conclusive. In addition to these issues, the metabolism of aflatoxin B(1) and recently proposed hypotheses regarding biphasic effects of the mycotoxin in broilers are briefly discussed.

Comparison of manual and automatic sampling for monitoring ochratoxin A in barley grain.

Automatic and manual sampling for ochratoxin A (OTA) in barley grain was compared under industrial conditions considering sampling uncertainty as well as practical and technical aspects. Ten tonnes of barley inoculated with Penicillium verrucosum were incubated until the OTA concentration reached approximately 15 µg kg(-1) and sampled with manual and automatic sampling. A nested experimental design and ANOVA was used to estimate variance components from sampling, sample reduction, sample preparation and analysis. Manual sampling resulted in a high sampling uncertainty and OTA concentrations in aggregate samples ranged from 2 to 80 µg kg(-1). When aggregate samples were formed by automatic sampling the uncertainty arising from nugget effects and spatial distribution was practically eliminated. Results from this study show that an automatic sampler mounted after a mixer or conveyer can provide representative samples of OTA from a moving stream of barley. Automatic sampling might present a practical and economical alternative to manual sampling for feed mill operators when monitoring low levels of mycotoxins in grain or other commodities. Despite careful precautions, sample preparation and analysis resulted in a relative uncertainty of ±40% (p = 0.95), which was attributed to the sub-sampling following the two grinding steps. Size fractionation of the coarsely ground barley showed that 40% of the total amount of OTA was present in a small fraction of fine particles with a strong tendency to aggregate or stick to equipment and containers. Thus, in order to take advantage of the automatic sampling, it is crucial to apply an appropriate sub-sampling to prevent segregation of particles which may affect the OTA measurements.

Proteomic analysis of porcine saliva.

Saliva contains a number of proteins that may be useful as biomarkers of health and disease and can be easily obtained from large numbers of animals in a non-invasive, stress-free way. The objective of this study was to explore the protein composition of porcine saliva from 10 specific pathogen free pigs using first one-dimensional SDS-PAGE and then two-dimensional electrophoresis and mass spectrometry. A reference proteome pattern for porcine saliva was established with the identification of 13 different, mainly saliva-specific, proteins. These reference data will facilitate the investigation of salivary proteins potentially altered in disease and could serve as novel diagnostic biomarkers.

Effects of B-trichothecenes on luminal glucose transport across the isolated jejunal epithelium of broiler chickens.

Trichothecenes are closely-related sesquiterpenoids (ring structure) with a 12, 13 epoxy ring and a variable number of hydroxyl, acetyl or other substituents. In chickens, D-glucose and amino acid absorption occurs via carrier-mediated transport. Recently, it has been observed that deoxynivalenol (DON) alters the gut function and impairs glucose and amino acid transport in chickens. The purpose of this work was to determine the effects of different B-trichothecenes [DON, Nivalenol (NIV), 15-Ac-DON and Fusarenon X (FUS X)] on intestinal carrier-mediated sodium co-transport of D-glucose in the small intestine of broiler chickens. Intestinal transport was determined by changes in the short-circuit current (Isc), proportional to ion transmembrane flux, in the middle segment of the jejunum of broilers with the Ussing chamber technique. D-glucose produced an increase of the Isc, and this effect was reverted by different B-trichothecene mycotoxins, indicating that the glucose induced Isc was altered by B-trichothecenes. The addition of glucose after pre-incubation of the tissues with B-trichothecenes had no effect (p > 0.05) on the Isc, suggesting that B-trichothecenes afflicted the Na(+)-D-glucose co-transport. However, FUX had no obvious effect on the measured parameters. It could be concluded from the present study that the glucose co-transporter activity appears to be more sensitive to DON, NIV and 15-Ac-DON suppression than by FUS X in the jejunum of broilers.

Detection of 3-nitropropionic acid and cytotoxicity inMucor circinelloides.

Mucorales are regarded as the aetiological agents of Mucormycosis. Their capabilities to produce mycotoxins are not profoundly investigated, in contrast to those of the fungi from the generaPenicillium, Aspergillus, orFusarium. The aim of this study was to isolate and identify fungi of the order Mucorales and investigate mycotoxins production. Twelve samples of visibly moulded grass silage and eight samples of damaged whole crop maize silage were analysed. Malt extract agar plates were used for sub cultivation. Three fungal species of the order Mucorales were isolated from grass silage, which were identified by their macro-and micro-morphology asAbsidia corymbifera, Mucor circinelloides andRhizopus stolonifer. The cytotoxicity ofMucor circinelloides extract was analysed using the cytotoxicity test (MTT assay) and the result, showed a low cytotoxicity. Additionally extracts fromAbsidia corymbifera, Mucor circinelloides andRhizopus stolonifer were tested for mycotoxin-production using an LC/MS/MS-based multimycotoxin method. 3-nitropropionic acid was detected in the culture extract ofMucor circinelloides.

Influence of deoxynivalenol on the D-glucose transport across the isolated epithelium of different intestinal segments of laying hens.

Deoxynivalenol (DON) decreases glucose absorption in the proximal jejunum of laying hens in vitro and this effect is apparently mediated by the inhibition of the sodium D-glucose co-transporter. DON could modulate the sugar transport of other intestinal regions of chickens. For this purpose, we have measured the effects of DON on the Na(+) D-glucose co-transporter, by addition of DON after and before a glucose addition in the isolated epithelium from chicken duodenum, jejunum, ileum, caecum and colon by using the Ussing chamber technique in the voltage clamp technique. The data showed in all segments of the gut that the addition of D-glucose on the mucosal side produced an increase in the current (Isc) compared with the basal values, the Isc after glucose addition to the small intestine was greater than the Isc of the large intestine compared with the basal values, specially of the jejunum (p < 0.002), indicating that the jejunum is the segment that is the best prepared for Na(+)-D-glucose co-transport. Further addition of 10 microg DON/ml to the mucosal solution decreased the Isc in all segments and the Isc returned to the basal value, especially in the duodenum and mid jejunum (p < 0.05). In contrast, the addition of 5 mmol D-glucose/l on the mucosal side after incubation of the tissues with DON in all segments had no effect on the Isc (p > 0.05), suggesting that DON previously inhibited the Na(+)D-glucose co-transport. The blocking effects of DON in duodenum and jejunum were greater than the other regions of the gut. It can be concluded that the small intestine of laying hens has the most relevant role in the carrier mediated glucose transport and the large intestine, having non-significant capacity to transport sugars, appears to offer a minor contribution to glucose transport because the surface area is small. The effect of D-glucose on the Isc was reversed by DON in all segments, especially in the duodenum and jejunum, suggesting that DON entirely inhibited Na(+)-D-glucose co-transport. This finding indicates that the inhibition of Na(+) co-transport system in all segments could be an important mode of action for DON toxicity of hens.

In vitro effects of deoxynivalenol on small intestinal D-glucose uptake and absorption of deoxynivalenol across the isolated jejunal epithelium of laying hens.

Deoxynivalenol (DON) is a common mycotoxin contaminant in feedstuffs. It has been shown to cause diverse toxic effects in animals. The aim of the present study was to evaluate the effects of DON on the glucose transport capacity in chickens' jejunum and to investigate the permeation of DON itself by the Ussing chamber technique. Glucose uptake into chicken jejunal epithelia was measured after the addition of 200 mumol/L of (14)C-labeled glucose to the mucosal solution. Glucose uptake under control condition was 3.28 +/- 0.53 nmol/cm(2) x min. The contribution of sodium glucose-linked transporter 1 (SGLT-1) to total glucose uptake was estimated by inhibiting SGLT-1 with phlorizin (100 micromol/L). In the presence of phlorizin, glucose uptake was reduced (P < 0.05) to 1.21 +/- 0.19 nmol/cm(2) x min. Deoxynivalenol decreased (P < 0.05) the glucose uptake in the absence of phlorizin to 1.81 +/- 0.24 nmol/cm(2) x min but had no additional effect on the glucose uptake in the presence of phlorizin (0.97 +/- 0.17 nmol/cm(2) x min). Mucosal-to-serosal permeation of DON was proportional to the initial DON concentration over a concentration range from 1 to 10 mug/mL on the mucosal side. Apparent permeability at 10 microg/mL of DON measured 60 to 90 min after DON application was 1.7 x 10(-05) cm/s. It can be concluded that DON (10 mg/L) decreases glucose uptake almost as efficiently as phlorizin. The similarity between the effects of phlorizin and DON on glucose uptake evidences their common ability to inhibit Na(+)-D-glucose cotransport. In addition to local effects, DON can be absorbed from the jejunum. A predominant part of DON passes across the chicken intestinal epithelium by passive diffusion, which is likely on the paracellular pathway. The results imply that the exposure to DON-contaminated feeds may negatively affect animal health and performance by local (i.e., inhibition of intestinal SGLT-1) and systemic effects.

Occurrence of deoxynivalenol (DON) and ochratoxin A (OTA) in dog foods.

The commercially available dog food samples (29 dry foods and 11 wet foods) were analysed for deoxynivalenol (DON) and ochratoxin A (OTA) using ELISA. All (100%) dry foods were contaminated with DON with various amount of the toxin (22-1837 µg/kg). In wet food 3 samples were found to be positive for DON in the range of 95-170 µg/kg. There were a few samples contaminated with OTA: 3 samples in dry foods (7-40 µg/kg) and 2 samples in wet foods (45 and 115 µg/kg).

Effects of Fusarium toxin-contaminated wheat and feed intake level on the biotransformation and carry-over of deoxynivalenol in dairy cows.

An experiment was carried out to examine the effects of feeding Fusarium toxin-contaminated wheat (8.21 mg deoxynivalenol (DON) and 0.09 mg zearalenone (ZON) per kg dry matter) at different feed intake levels on the biotransformation and carry-over of DON in dairy cows. For this purpose, 14 ruminal and duodenal fistulated dairy cows were fed a diet containing 60% concentrate with a wheat portion of 55% (Fusarium toxin-contaminated wheat (mycotoxin period) or control wheat (control period)) and the ration was completed with maize- and grass silage (50 : 50) on a dry matter basis. Daily DON intakes ranged from 16.6 to 75.6 mg in the mycotoxin period at dry matter intakes of 5.6-20.5 kg. DON was almost completely biotransformed to de-epoxy DON (94-99%) independent of the DON/feed intake, and the flow of DON and de-epoxy DON at the duodenum related to DON intake ranged from 12 to 77% when the Fusarium toxin-contaminated wheat was fed. In the serum samples, de-epoxy DON was detected in the range of 4-28 ng ml-1 in the mycotoxin period, while concentrations of DON were all below the detection limit. The daily excretion of DON and de-epoxy DON in the milk of cows fed the contaminated wheat varied between 1 and 10 microg and between 14 and 104 microg, respectively. The total carry-over rates as the ratio between the daily excretion of DON and de-epoxy DON into milk and DON intake were in the ranges of 0.0001-0.0002 and 0.0004-0.0024, respectively. Total carry-over rates of DON as DON and de-epoxy DON into the milk increased significantly with increasing milk yield. In the urine samples, de-epoxy DON was the predominant substance as compared with DON with a portion of the total DON plus de-epoxy DON concentration to 96% when the Fusarium toxin-contaminated wheat was fed, whereas the total residues of DON plus de-epoxy DON in faeces ranged between 2 and 18% of DON intake in the mycotoxin period. The degree of glucuronidation of de-epoxy DON was found to be approximately 100% in serum. From 33 to 80% of DON and from 73 to 92% of de-epoxy DON, and from 21 to 92% of DON and from 86 to 100% of de-epoxy DON were glucuronidated in the milk and urine, respectively. It is concluded that DON is very rapidly biotransformed to de-epoxy DON in the rumen and only negligible amounts of DON and de-epoxy DON were transmitted into the milk within the range of 5.6-20.5 kg day-1 dry matter intake and milk yields (fat corrected milk) between 10 and 42 kg day-1.

Determination of zearalenone and its metabolites in urine, plasma and faeces of horses by HPLC-APCI-MS.

The paper describes a method for the sensitive and selective determination of zearalenone and its metabolites in urine, plasma and faeces of horses by high performance liquid chromatography and atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS). While only one step sample clean-up by an immunoaffinity column (IAC) was sufficient for plasma samples, urine and faeces samples had to be prepared by a combination of a solid-phase extraction (SPE) and an immunoaffinity column. The method allows the simultaneous determination of zearalenone and all of its metabolites; alpha-zearalenol, beta-zearalenol, alpha-zearalanol, beta-zearalanol and zearalanone. Dideuterated zearalanone was used as internal standard for quantification and the study of the matrix effect. Recovery rates between 56 and slightly above 100% were achieved in urine samples, and more than 80% in plasma and faeces samples. The limits of detection ranged from 0.1-0.5 microg/l or microg/kg, the limits of quantification from 0.5-1.0 microg/l or microg/kg. The practical use of the method is demonstrated by the analysis of spiked and naturally contaminated urine, plasma and faeces of horses.

Effect of addition of a probiotic microorganism to broiler diets contaminated with deoxynivalenol on performance and histological alterations of intestinal villi of broiler chickens.

An experiment was conducted to study the effects of deoxynivalenol (DON) on the performance of broilers, organ weights, and intestinal histology and to evaluate the efficacy of a probiotic feed additive (PB, Eubacterium sp.) with the ability to deepoxidize DON. Two hundred seventy-seven 1-d-old broiler chicks were randomly assigned to 1 of the 3 dietary treatments for 6 wk. The dietary treatments were 1) control; 2) artificially contaminated diets with 10 mg of DON/kg of diet; 3) DON-contaminated diets plus probiotic feed additive (DON-PB). The BW and the efficiency of feed utilization were not adversely affected (P > 0.05) by the inclusion of DON in the diets. A slight improvement in feed intake and BW gain over the course of the experiment was observed in broilers fed DON-PB with no change in feed efficiency. The absolute or relative organ weights were not altered (P > 0.05) in broilers fed the diet containing DON compared with controls and the DON-PB group. The absolute liver weights were numerically increased (P < 0.1) for broilers receiving the diet containing DON-PB. There were no significant differences in the absolute and relative weights of the gizzard, duodenum, pancreas, heart, and spleen. However, the absolute and relative weights of the jejunum and cecum were increased for DON-PB-fed broilers compared with the controls and DON group. No pathological lesions were found in the gut of birds fed DON-contaminated diets during the feeding trial, but mild intestinal changes were observed. The DON altered small intestinal morphology, especially in the duodenum and jejunum, where villi were shorter and thinner (P < 0.05). The addition of the eubacteria to the DON-contaminated feed of the broilers effectively alleviated the histological alterations caused by DON and led to comparable villus length as in the control group. In conclusion, diets with DON contamination below levels that induce a negative impact on health and performance could affect small intestinal morphology in broilers. The histological alterations caused by DON were reduced by supplementing the DON-containing diets with PB. This indicates that in case of DON contamination of feedstuffs, the addition of PB would be a proper way to counteract the possible effects caused by this mycotoxin.

Effects of feeding deoxynivalenol contaminated wheat on growth performance, organ weights and histological parameters of the intestine of broiler chickens.

A feeding trial was conducted to evaluate the effects of moderate dietary concentrations of deoxynivalenol (DON) during a 21-day feeding experiment on the performance of broilers. Fifteen 1-day-old broiler chicks were randomly divided into two groups. The control group was fed non-contaminated diet. Another group of broilers was fed a diet naturally contaminated with 5 mg DON/kg diet. Deoxynivalenol had no effect (p > 0.05) on feed consumption, feed conversion, body-weight gain, live body weight or mortality. The absolute and relative weight of the organs (gizzard, pancreas, heart, spleen, colon and caecum) were not altered by the dietary inclusion of DON contaminated grain. However, both the absolute and relative weight of small intestine was decreased (p < 0.01) in DON fed broilers compared to the controls. No gross lesions were detected in any of the organs of birds fed contaminated wheat during the feeding trial. The microscopic examination revealed that, the height and the width of villi in duodenum decreased (p < 0.05) in birds fed DON contaminated wheat compared to controls. On the other hand the height and the width of jejunum villi were not affected (p > 0.05). This study indicates that feeding DON for 21 days to broiler chickens at a concentration of up to 5 mg/kg of diet influenced the weight of the small intestine as well as intestinal histology, especially the duodenum, as evidenced by shorter and thinner villi. In conclusion, diets with DON contamination below levels that induce negative impact on health and performance could affect small intestinal morphology in broilers.

Effects of Fusarium toxin-contaminated wheat grain on nutrient turnover, microbial protein synthesis and metabolism of deoxynivalenol and zearalenone in the rumen of dairy cows.

The aim of the present study was to examine the effects of feeding Fusarium toxin-contaminated wheat to dairy cows on nutrient utilization in the rumen and on duodenal flow of deoxynivalenol (DON), zearalenone (ZON) and their metabolites. Six dairy cows fitted with a large rumen cannula and a simple T-shaped cannula at the proximal duodenum was used in two experiments. The experiments included a control period in which the uncontaminated control wheat was fed and a period in which the control wheat was replaced by the Fusarium toxin-contaminated wheat (8.05 and 7.15 mg DON/kg and 0.26 and 0.1 mg ZON/kg in Expts 1 and 2 respectively). The wheat portion of the daily ration amounted to 50% on a dry matter (DM) basis and rations were completed with hay or grass silage. Five of the six cows were non-lactating and the total daily DM-intake ranged between 4 and 12 kg. The pH-values and the concentration of volatile fatty acids in ruminal fluid were not significantly influenced by feeding the contaminated wheat. In contrast, the postprandial ammonia concentration was consistently higher when the mycotoxin-contaminated wheat was fed. Moreover, the flow of microbial protein and utilizable protein at the duodenum were reduced at the same time. The concentrations of DON and ZON and of their metabolites in freeze-dried duodenal digesta were either not detectable or negligible during the control periods whereas distinct concentrations were measured during the periods where the contaminated wheat was fed. DON was nearly completely metabolized to de-epoxy-DON and the flow at the duodenum ranged between 4% and 28% of DON-intake. The ZON metabolites alpha-zearalenol (ZOL) and beta-ZOL were recovered at the duodenum beside the parent toxin ZON. Their recovery as a percentage of ZON-intake ranged between 43% and 132%. In conclusion, feeding of Fusarium toxin-contaminated wheat altered the ruminal protein utilization. The question of whether this effect was a result of the mycotoxin being present in the rumen or of Fusarium growth-related structural (cell wall) changes of the wheat grain needs to be clarified. The low recovery of DON at the duodenum would indicate either a nearly complete degradation of the molecule in the rumen or an absorption by the mucosa of the rumen, whereas the higher ZON recovery would suggest a lower degradation of the parent toxin in the rumen and/or recovery of some bile-originating entero-hepatic cycling ZON/metabolites.