Janine Winkler

Residues of zearalenone (ZEN), deoxynivalenol (DON) and their metabolites in plasma of dairy cows fed Fusarium contaminated maize and their relationships to performance parameters.

A feeding trial with dairy cows fed graded proportions of a Fusarium toxin contaminated maize containing mainly deoxynivalenol (DON) was carried out to relate the plasma levels of DON, zearalenone (ZEN) and their metabolites to the performance. German Holstein cows (n=30) were divided into three groups (n=10 in each): CON (0.02mgZEN and 0.07mgDON, per kg dry matter, DM), FUS-50 (0.33mg ZEN and 2.62mgDON, per kg DM), FUS-100 (0.66mgZEN and 5.24mgDON, per kg DM). The average performance level was characterised by daily DM intake, energy balance and milk yield which were not affected by the DON and ZEN levels in feed. DON, de-epoxy-DON (de-DON) and ZEN were detected simultaneously in all plasma samples. A linear relationship between toxin intake and plasma levels could be established. Moreover, a linear relationship between DON and de-DON concentration could be derived. It was concluded that DON and ZEN intake of 0.5mgZEN/kg and 5mgDON/kg (current guidance values) had no considerable effects on the performance parameter of dairy cows. Furthermore, increased plasma concentrations of ZEN, DON and de-DON may hint on toxin exposure through the diets.

Invited review: Diagnosis of zearalenone (ZEN) exposure of farm animals and transfer of its residues into edible tissues (Carry over)

The aim of the review was to evaluate the opportunities for diagnosing the zearalenone (ZEN) exposure and intoxication of farm animals by analyzing biological specimens for ZEN residue levels. Metabolism is discussed to be important when evaluating species-specific consequences for the overall toxicity of ZEN. Besides these toxicological facts, analytics of ZEN residues in various animal-derived matrices requires sensitive, matrix-adapted multi-methods with low limits of quantification, which is more challenging than the ZEN analysis in feed. Based on dose-response experiments with farm animals, the principle usability of various specimens as bio-indicators for ZEN exposure is discussed with regard to individual variation and practicability for the veterinary practitioner. ZEN residue analysis in biological samples does not only enable evaluation of ZEN exposure but also allows the risk for the consumer arising from contaminated foodstuffs of animal origin to be assessed. It was compiled from literature that the tolerable daily intake of 0.25 μg ZEN/kg body weight and day is exploited to approximately 8%, when a daily basket of animal foodstuffs and associated carry over factors are assumed at reported ZEN contamination levels of complete feed.

Effect of increasing body condition on key regulators of fat metabolism in subcutaneous adipose tissue depot and circulation of nonlactating dairy cows

In response to negative energy balance, overconditioned cows mobilize more body fat than thin cows and subsequently are prone to develop metabolic disorders. Changes in adipose tissue (AT) metabolism are barely investigated in overconditioned cows. Therefore, the objective was to investigate the effect of increasing body condition on key regulator proteins of fat metabolism in subcutaneous AT and circulation of dairy cows. Nonlactating, nonpregnant dairy cows (n=8) investigated in the current study served as a model to elucidate the changes in the course of overcondition independent from physiological changes related to gestation, parturition, and lactation. Cows were fed diets with increasing portions of concentrate during the first 6wk of the experiment until 60% were reached, which was maintained for 9wk. Biopsy samples from AT of the subcutaneous tailhead region were collected every 8wk, whereas blood was sampled monthly. Within the experimental period cows had an average BW gain of 243±33.3 kg. Leptin and insulin concentrations were increased until wk 12. Based on serum concentrations of glucose, insulin, and nonesterified fatty acids, the surrogate indices for insulin sensitivity were calculated. High-concentrate feeding led to decreased quantitative insulin sensitivity check index and homeostasis model assessment due to high insulin and glucose concentrations indicating decreased insulin sensitivity. Adiponectin, an adipokine-promoting insulin sensitivity, decreased in subcutaneous AT, but remained unchanged in the circulation. The high-concentrate diet affected key enzymes reflecting AT metabolism such as AMP-activated protein kinase and hormone-sensitive lipase, both represented as the proportion of the phosphorylated protein to total protein, as well as fatty acid synthase. The extent of phosphorylation of AMP-activated protein kinase and the protein expression of fatty acid synthase were inversely regulated throughout the experimental period, whereas the extent of phosphorylation of hormone-sensitive lipase was consistently decreasing by the high-concentrate diet. Overcondition in nonpregnant, nonlactating dairy cows changed the expression of key regulator proteins of AT metabolism and circulation accompanied by impaired insulin sensitivity, which might increase the risk for metabolic disorders.

Simultaneous determination of zearalenone, deoxynivalenol and their metabolites in bovine urine as biomarkers of exposure

A feeding trial with 30 dairy cows which were fed rations with three different concentrations of zearalenone (ZEA) and deoxynivalenol (DON) contaminated maize was carried out to examine the ZEA and DON concentration in urine. German Holstein cows (n=30) were divided into three groups (n=10 in each) which received diets with following toxin concentrations: CON (0.02 mg ZEA and 0.07 mg DON, per kg dry matter (DM)), FUS-50 (0.33 mg ZEA and 2.62 mg DON, per kg DM), FUS-100 (0.66 mg ZEA and 5.24 mg DON, per kg DM). For urine analysis, a reliable, cost-efficient and sensitive method for simultaneous determination of ZEA, DON and their metabolites was developed. The method comprises a solid phase extraction clean-up on Oasis HLB cartridges followed by LC-MS/MS measurement. ZEA, α-zearalenol, β-zearalenol, DON and de-epoxydeoxynivalenol (DOM) could be detected in the urine samples of the feeding trial. Thereby, DON was almost completely metabolised to DOM (83-98%) independent of the DON exposure. Moreover, conjug...

Description of a bovine model for studying digestive and metabolic effects of a positive energy balance not biased by lactation or gravidity

Physiological consequences of adaptation to and continued feeding of a high-energetic diet were studied in eight non-pregnant, non-lactating dairy Holstein cows over a period of 16 weeks. The first six weeks served as an adaptation period from the low energetic straw-based diet (3.8 MJ NEL/kg DM) to the high-energetic ration (7.5 MJ NEL/kg DM). Intake of dry matter (DM) increased with dietary energy concentration from 9 to 20 kg/d up to week 9 to 12 and decreased thereafter. The initial live weight (LW) of 550 ± 60 kg was increased linearly and corresponded to an average daily LW gain of 2.3 ± 0.3 kg. Energy balance increased approximately nine-fold to a maximum of 114 MJ NEL/d in week 10. Ruminal fermentation pattern was completely changed from an acetate dominating profile to a propionate based one, which was paralleled by a marked increase in the rumen fluid endotoxin concentration. Unlike blood glucose concentration, which increased continuously, that of cholesterol and triglycerides started to increase after an initial stagnation. In conclusion, both ruminal adaptation to a high-energetic diet and the continued feeding of such a diet induced digestive and metabolic adaptations in non-pregnant, non-lactating cows characterised by a progressing positive energy balance.

Diagnostic opportunities for evaluation of the exposure of dairy cows to the mycotoxins deoxynivalenol (DON) and zearalenone (ZEN): reliability of blood plasma, bile and follicular fluid as indicators

To investigate the usefulness of follicular fluid (FF) in relation to blood plasma and bile as indicators of exposure of dairy cows to ZEN, DON and their metabolites, a dose-response study was performed with 30 dairy cows. The cows, 10 in each group (named CON; FUS-50, FUS-100), received a diet with three different concentrations of Fusarium toxin-contaminated maize. Thereby, the following dietary concentration were reached: CON (0.02 mg ZEN and 0.07 mg DON, per kg dry matter, DM), FUS-50 (0.33 mg ZEN and 2.62 mg DON, per kg DM) and FUS-100 (0.66 mg ZEN and 5.24 mg DON, per kg DM). ZEN, DON and de-epoxy-DON (de-DON) were detected in FF. Based on the linear regression between toxin concentration in plasma and FF, it seems that about 50% (m = 0.5) of ZEN present in plasma is present in FF while an increase of 1 ng/ml DON or de-DON in plasma is paralleled by an increase of 1.5 ng/ml DON or 1.1 ng/ml de-DON in FF. ZEN, DON and their metabolites, except zearalenone (ZAN), were also detected in bile. Contrary to DON and de-DON, ZEN and its metabolites were accumulated in bile so that the concentration of ZEN and metabolites was much higher than for DON and de-DON. The main compound was β-zearalenol (β-ZEL). The biliary ZEN, α-zearalenol (α-ZEL) and β-ZEL concentration correlated linearly with each other with an uncertainty of <15% (r(2) ≥ 0.86), whereas the ratio between ZEN: α-ZEL: β-ZEL was about 1.5:1:11. With the help of established linear relationship between toxin intake and toxin concentration, bile could be used as diagnostic indicator to assess the exposure of cows.

Development of a multi-toxin method for investigating the carryover of zearalenone, deoxynivalenol and their metabolites into milk of dairy cows

A dose–response study was carried out to examine the carryover of zearalenone (ZEN), deoxynivalenol (DON) and their metabolites into bovine milk. Therefore, a feeding trial with 30 dairy cows fed with three different levels of Fusarium (FUS) toxin-contaminated maize was performed. A control group (0.02 mg ZEN kg–1 dry matter (DM) and 0.07 DON kg–1 DM) was compared with two groups fed contaminated diets. The first diet contained 0.33 mg ZEN kg–1 DM and 2.62 mg DON kg–1 DM (group FUS-50) and the second diet contained 0.66 mg ZEN kg–1 DM and 5.24 mg DON kg–1 DM (group FUS-100). For milk sample analysis, a new cost-efficient sample preparation method was developed for the simultaneous determination of ZEN, DON and their metabolites. The method comprised the separation of the milk fat followed by an SPE clean-up on Oasis HLB and a LC-MS/MS measurement. The less toxic metabolite de-epoxy-DON had the highest detected concentration (5.6 ng ml–1 milk) in the milk samples obtained from the feeding trial. Additionally, ZEN (up to 0.29 ng ml–1), α-zearalenol (up to 0.17 ng ml–1), β-zearalenol (up to 0.95 ng ml–1) and DON (up to 2.5 ng ml–1) were detected in these samples. The milk toxin concentrations of cows fed the control diet were significantly lower compared with cows fed the contaminated diet. The calculated carryover rates ranged between 0 and 0.0075 for ZEN and metabolites and between 0 and 0.0017 for DON independent of exposure. It can be concluded that dietary toxin concentrations in the feed below or close to the current guidance values do not pose a risk for consumers due to negligible carryover rates.

Haematological and immunological adaptations of non-pregnant, non-lactating dairy cows to a high-energetic diet containing mycotoxins.

abstract Diet change and fatness are supposed to challenge the immune system of the cow. Therefore, immunological and haematological consequences of adaptation to and continued feeding of a high-energy diet were studied in eight non-pregnant, non-lactating Holstein cows over 16 weeks. Blood haptoglobin concentration remained unaltered, suggesting that an acute phase reaction was not induced. Stimulation ability of peripheral blood mononuclear cells and stimulated oxidative burst capacity of granulocytes increased significantly in the course of the experiment after an initial drop. While total leucocyte counts increased, the proportion of granulocytes increased and that of lymphocytes decreased at the same time as the ratio of CD4+/CD8+ lymphocytes did. Capability of rumen microbes to detoxify the immune-modulating mycotoxin deoxynivalenol (DON) was not compromised as indicated by the exclusive presence of de-DON as the detoxified DON metabolite in blood. In conclusion, both diet change and prolonged positive energy balance influenced the bovine immune system.

Studies on the Bioavailability of Deoxynivalenol (DON) and DON Sulfonate (DONS) 1, 2, and 3 in Pigs Fed with Sodium Sulfite-Treated DON-Contaminated Maize

Deoxynivalenol (DON) exposure of pigs might cause serious problems when critical dietary toxin concentrations are exceeded. As DON contamination of agricultural crops cannot be completely prevented, detoxification measures are needed. Wet preservation with sodium sulfite resulted in a significant DON reduction of naturally-contaminated maize in previous experiments. The preserved material had a characteristic DON sulfonates (DONS) pattern. DONS is known to be less toxic than DON but its stability was shown to depend on pH, which gives rise to the question if a back-conversion to DON occurs in vivo. Therefore, the toxicokinetics and bioavailability of DON and DONS were studied in pigs. After the administration of a single oral or intravenous bolus of DON or DONS, serial blood samples were collected and subsequently analyzed. DONS was not detectable after oral administration of DONS mixtures. The results showed further that the bioavailability of DONS as DON in pigs fed maize preserved wet with sodium sulfite was significantly decreased compared to untreated control maize (DON), indicating that DONS obviously did not convert back to DON to a large extent in vivo. Moreover, the fact that DONS was not detectable in systemic blood requires further investigations regarding their ingestive and/or metabolic fate.

Effects of oral exposure to sodium sulphite-treated deoxynivalenol (DON)-contaminated maize on performance and plasma concentrations of toxins and metabolites in piglets

ABSTRACT The objective of the present study was to demonstrate the efficiency of the decontamination process applied to deoxynivalenol (DON)-contaminated maize by sodium sulphite (Na2SO3) treatment in vivo. Additionally, in vitro characterisation of the toxicity of the DON sulphonates (DONS 1, 2 and 3 denote structurally different forms), the resulting DON metabolites, on peripheral blood mononuclear cells (PBMC) should substantiate the inactivation of DON. In a piglet experiment, both DON-contaminated maize and -uncontaminated control maize either untreated (DON−, CON−) or Na2SO3-treated (DON+, CON+) were mixed into feed and fed for 42 d starting from weaning. The results showed that feed intake and daily weight gain of animals fed DON− were significantly lower compared to animals fed CON− and CON+, whereas group DON+ reached the control level or even exceeded it. The feed-to-gain ratio was unaffected (p = 0.45). Furthermore, DON concentrations in plasma markedly reflected the diets’ DON concentrations. These were < 0.1, < 0.1, 5.4 and 0.8 mg/kg feed for CON−, CON+, DON− and DON+, and amounted to 0.3, 0.4, 33.0 and 9.3 ng/ml in plasma, respectively. Whereas DONS 2 and 3 were detected in the DON+ diet, only DONS 2 was recovered in plasma. Regarding the toxicity of DONS, no or much lower toxicity was found compared to DON. DONS 1 and Na2SO3 did not affect the viability of PBMC. At 32.71μM DONS2 the viability was reduced by 50% and thus this compound was less toxic than DON by a factor of 73. Consequently, wet preservation of maize with Na2SO3 was an effective tool to avoid the adverse effects of DON on performance of piglets.