Anti-mycotoxin feed additives: effects on metabolism, mycotoxin excretion, performance, and total tract digestibility of dairy cows fed artificially multi-mycotoxin-contaminated diets.

The aim of this study was to evaluate the effects of different anti-mycotoxin feed additives on the concentration of mycotoxins in milk, urine, and blood plasma of dairy cows fed artificially multi-mycotoxin-contaminated diets. Secondarily, performance, total-tract apparent digestibility of nutrients, and blood parameters were evaluated. Twelve multiparous cows (165 ± 45 d in milk, 557 ± 49 kg body weight, and 32.1 ± 4.57 kg/d milk yield at the start of the experiment) were blocked according to parity, milk yield, and days in milk and used in a 4 × 4 Latin square design experiment with 21-d periods, where the last 7 d were used for sampling and data analysis. Treatments were: 1) Mycotoxin group (MTX), basal diet (BD) without anti-mycotoxin feed additives; 2) Hydrated sodium calcium aluminosilicate (HSCA), HSCA added to the BD at 25g/cow/d; 3) Mycotoxin deactivator 15 (MD15), MD (Mycofix® Plus, dsm-firmenich) added to the BD at 15 g/cow/d; and 4) Mycotoxin deactivator 30 (MD30), MD added to the BD at 30 g/cow/d. Cows from all treatments were challenged with a blend of mycotoxins containing 404 µg aflatoxins B1 (AFB1), 5,025 µg deoxynivalenol (DON), 8,046 µg fumonisins (FUM), 195 µg T2 toxin (T2), and 2,034 µg of zearalenone (ZEN) added daily to the BD during the last 7 d of each period. Neither performance (milk yield and composition) nor nutrient digestibility was affected by treatments. All additives reduced aflatoxin M1 (AFM1) concentration in milk, whereas MD15 and MD30 group had lower excretion of AFM1 in milk than HSCA. DON, FUM, T2, or ZEN were not detected in milk of MD15 and MD30. Concentrations in milk of DON, FUM, T2, and ZEN were similar between MTX and HSCA. Except for AFM1, none of the analyzed mycotoxins were detected in urine of MD30 group. Comparing HSCA to MD treatments, the concentration of AFM1 was greater for HSCA, whereas MD30 was more efficient at reducing AFM1 in urine than MD15. AFM1, DON, FUM, and ZEN were not detected in the plasma of cows fed MD30, and DON was also not detected in MD15 group. Plasma concentration of FUM was lower for MD15, similar plasma FUM concentration was reported for HSCA and MTX. Plasma concentration of ZEN was lower for MD15 than MTX and HSCA. Serum concentrations of haptoglobin and hepatic enzymes were not affected by treatments. Blood concentration of sodium was lower in HSCA compared with MD15 and MD30 groups. In conclusion, the mycotoxin deactivator proved to be effective in reducing the secretion of mycotoxins in milk, urine, and blood plasma, regardless of the dosage. This reduction was achieved without adverse effects on milk production or total-tract digestibility in cows fed multi-mycotoxin-contaminated diets over a short-term period. Greater reductions in mycotoxin secretion were observed with full dose of MD.

Duration of Zearalenone Exposure Has Implications on Health Parameters of Lactating Cows.

There is a limited research focus on evaluating the detrimental effects of prolonged zearalenone (ZEN) intake on dairy cows' health under controlled conditions. This experiment was conducted to evaluate whether the length of exposure to a ZEN-contaminated total mixed ration (TMR) at a level of 9.45 mg per day can negatively influence animal health parameters, such as milk composition, rumen and fecal fermentation, and the chewing activity of lactating dairy cows. For this experiment, we used 18 lactating Simmental cows that were fed a diet of 60% forage and 40% concentrate (on dry matter basis) for 26 consecutive days. The first 4 days were for adaptation prior to the first sampling day (day 0). The sampling events took place on day 0 (baseline) without ZEN, followed by day 1, day 7, day 14, and day 21 (with toxin). Dry matter intake (DMI) and ruminating chews per minute increased on the third week of ZEN inclusion; meanwhile, ruminating, eating, and drinking times were not affected. Most milk composition variables were also unaffected. Rumen fluid osmolality increased on day 21 and total short-chain fatty acids (SCFA) of ruminal fluid decreased on day 7. Fecal SCFA increased on day 21 and the acetate-to-propionate ratio increased from day 1 onwards, showing the influence of toxin intake. Animal health parameters, like heart rate, respiratory rate, and body temperature, were negatively influenced by ZEN intake, all increasing consistently on days 4 and 6, 9 and 12, and 16 and 18, respectively. The liver enzyme glutamate dehydrogenase decreased in response to ZEN intake on day 7. A total daily ZEN intake at the level of 9.45 mg did not show detrimental effects on DMI. Nevertheless, certain health parameters were negatively affected, including body temperature, respiratory rate, and heart rate, starting from the 7th day of ZEN intake, with additional signs of possible loss of water balance on the last sampling day.

Correction: Penagos-Tabares et al. Mixtures of Mycotoxins, Phytoestrogens, and Other Secondary Metabolites in Whole-Plant Corn Silages and Total Mixed Rations of Dairy Farms in Central and Northern Mexico. Toxins 2023, 15, 153.

In the original publication [...].

Effects of Aflatoxins and Fumonisins, Alone or in Combination, on Performance, Health, and Safety of Food Products of Broiler Chickens, and Mitigation Efficacy of Bentonite and Fumonisin Esterase.

The current study evaluated the effects of feeding diets contaminated with aflatoxin B1 (AFB1), fumonisins (FBs), or both on the performance and health of broiler chickens and the safety of their food products as well as the efficacy of bentonite and fumonisin esterase to mitigate the effects of these mycotoxins under conditions representative for sub-Saharan Africa (SSA). Four hundred one-day-old Cobb 500 broiler chickens were randomly assigned to 20 treatments with either a control diet, a diet with moderate AFB1 (60 µg/kg feed) or high AFB1 (220 µg/kg feed), or FBs (17,430 µg FB1+FB2/kg feed), alone or in combination, a diet containing AFB1 (either 60 or 220 µg/kg) and/or FBs (17,430 µg FB1+FB2/kg) and bentonite or fumonisin esterase or both, or a diet with bentonite or fumonisin esterase only. The experimental diets were given to the birds from day 1 to day 35 of age, and the effects of the different treatments on production performance were assessed by feed intake (FI), body weight gain (BWG), and feed conversion ratio (FCR). Possible health effects were evaluated through blood biochemistry, organ weights, mortality, liver gross pathological changes, and vaccine response. Residues of aflatoxins (AFB1, B2, G1, G2, M1 and M2) were determined in plasma, muscle, and liver tissues using validated UHPLC-MS/MS methods. The results obtained indicated that broiler chickens fed high AFB1 alone had poor FCR when compared to a diet with both high AFB1 and FBs (p = 0.0063). Serum total protein and albumin from birds fed FBs only or in combination with moderate or high AFB1 or detoxifiers increased when compared to the control (p < 0.05). Liver gross pathological changes were more pronounced in birds fed contaminated diets when compared to birds fed the control or diets supplemented with mycotoxin detoxifiers. The relative weight of the heart was significantly higher in birds fed high AFB1 and FBs when compared to the control or high AFB1 only diets (p < 0.05), indicating interactions between the mycotoxins. Inclusion of bentonite in AFB1-contaminated diets offered a protective effect on the change in weights of the liver, heart and spleen (p < 0.05). Residues of AFB1 were detected above the limit of quantification (max: 0.12 ± 0.03 µg/kg) in liver samples only, from birds fed a diet with high AFB1 only or with FBs or the detoxifiers. Supplementing bentonite into these AFB1-contaminated diets reduced the levels of the liver AFB1 residues by up to 50%. Bentonite or fumonisin esterase, alone, did not affect the performance and health of broiler chickens. Thus, at the doses tested, both detoxifiers were safe and efficient for use as valid means of counteracting the negative effects of AFB1 and FBs as well as transfer of AFB1 to food products (liver) of broiler chickens.

Co-occurrence of mycotoxins and other fungal metabolites in total mixed rations of cows from dairy farms in Punjab, Pakistan.

After India and the USA, Pakistan is the third country leading in global dairy production, a sector of very high socioeconomic relevance in Asia. Mycotoxins can affect animal health, reproduction and productivity. This study analysed a broad range of co-occurring mycotoxins and fungal secondary metabolites derived from Alternaria, Aspergillus, Fusarium, Penicillium and other fungal species. To complete this, a validated multi-metabolite liquid chromatography/electrospray ionization-tandem mass spectrometric (LC/ESI-MS/MS) method was employed, detecting 96 of > 500 tested secondary fungal metabolites. This first preliminary study demonstrated that total mixed rations (TMRs) (n = 30) from big commercial dairy cattle farms (> 200 lactating cows) in Punjab, Pakistan, presented ubiquitous contamination with mixtures of mycotoxins. The mean of mycotoxins per sample was 14, ranging from 11 to 20 mycotoxins among all TMR samples. Metabolites derived from other fungi and Fusarium spp. showed the highest levels, frequency and diversity among the detected fungal compounds. Among the most prevalent mycotoxins were Fusarium toxins like fumonisins B1 (FB1) (93%), B2 (FB2) (100%) and B3 (FB3) (77%) and others. Aflatoxin B1 (AFB1) was evidenced in 40% of the samples, and 7% exceeded the EU maximum limit for feeding dairy cattle (5 µg/kg at 88% dry matter). No other mycotoxin exceeds the EU guidance values (GVs). Additionally, we found that dietary ingredients like corn grain, soybean meal and canola meal were related to increased contamination of some mycotoxins (like FB1, FB2 and FB3) in TMR from the province of Punjab, Pakistan. Among typical forage sources, the content of maize silage was ubiquitous. Individually, the detected mycotoxins represented relatively low levels. However, under a realistic scenario, long-term exposure to multiple mycotoxins and other fungal secondary metabolites can exert unpredictable effects on animal health, reproduction and productivity. Except for ergot alkaloids (73%), all the groups of metabolites (i.e. derived from Alternaria spp., Aspergillus spp., Fusarium spp., Penicillium spp. and other fungi) occurred in 100% of the TMR samples. At individual levels, no other mycotoxins than AFB1 represented a considerable risk; however, the high levels of co-occurrence with several mycotoxins/metabolites suggest that long-term exposure should be considered because of their potential toxicological interactions (additive or synergistic effects).

Zearalenone and Its Emerging Metabolites Promptly Affect the Rumen Microbiota in Holstein Cows Fed a Forage-Rich Diet.

The study investigated the short-term effects of a single oral bolus of zearalenone (ZEN) on the rumen microbiota and fermentation patterns in four rumen-cannulated Holstein cows fed a forage diet with daily 2 kg/cow concentrate. During the baseline day, cows received uncontaminated concentrate, followed by ZEN-contaminated concentrate on the second day, and again the uncontaminated concentrate on day three. Free rumen liquid (FRL) and particle-associated rumen liquid (PARL) were collected at different hours post-feeding on all days to analyze the prokaryotic community composition, absolute abundances of bacteria, archaea, protozoa, and anaerobic fungi, as well as short-chain fatty acid (SCFA) profiles. The ZEN reduced the microbial diversity in FRL but not in the PARL fraction. The abundance of protozoa was higher after ZEN exposure in PARL, which may be related to their strong biodegradation capacity that, therefore, promoted protozoal growth. In contrast, α-zearalenol might compromise anaerobic fungi as indicated by reduced abundances in FRL and fairly negative correlations in both fractions. Total SCFA significantly increased in both fractions after ZEN exposure, while the SCFA profile only changed marginally. Concluding, a single ZEN challenge caused changes in the rumen ecosystem soon after intake, including ruminal eukaryotes, that should be the subject of future studies.

Mixtures of Mycotoxins, Phytoestrogens, and Other Secondary Metabolites in Whole-Plant Corn Silages and Total Mixed Rations of Dairy Farms in Central and Northern Mexico.

Mycotoxins and endocrine disruptors such as phytoestrogens can affect cattle health, reproduction, and productivity. Most studies of mycotoxins in dairy feeds in Mexico and worldwide have been focused on a few (regulated) mycotoxins. In contrast, less known fungal toxins, phytoestrogens, and other metabolites have been neglected and underestimated. This study analyzed a broad spectrum (>800) of mycotoxins, phytoestrogens, and fungal, plant, and unspecific secondary metabolites in whole-plant corn silages (WPCSs) and total mixed rations (TMRs) collected from 19 Mexican dairy farms. A validated multi-metabolite liquid chromatography/electrospray ionization-tandem mass spectrometric (LC/ESI-MS/MS) method was used. Our results revealed 125 of >800 tested (potentially toxic) secondary metabolites. WPCSs/TMRs in Mexico presented ubiquitous contamination with mycotoxins, phytoestrogens, and other metabolites. The average number of mycotoxins per TMR was 24, ranging from 9 to 31. Fusarium-derived secondary metabolites showed the highest frequencies, concentrations, and diversity among the detected fungal compounds. The most frequently detected mycotoxins in TMRs were zearalenone (ZEN) (100%), fumonisin B1 (FB1) (84%), and deoxynivalenol (84%). Aflatoxin B1 (AFB1) and ochratoxin A (OTA), previously reported in Mexico, were not detected. All TMR samples tested positive for phytoestrogens. Among the investigated dietary ingredients, corn stover, sorghum silage, and concentrate proportions were the most correlated with levels of total mycotoxins, fumonisins (Fs), and ergot alkaloids, respectively.

Efficacy of Bentonite and Fumonisin Esterase in Mitigating the Effects of Aflatoxins and Fumonisins in Two Kenyan Cattle Breeds.

The objective of the study was to investigate the efficacy of bentonite and fumonisin esterase, separately or combined, in mitigating the effects of aflatoxins (AF) and fumonisins (FUM) in Boran and Friesian-Boran crossbreed cattle. These effects were studied by measuring mycotoxins, their metabolites, and biomarkers that relate to animal health, productivity, and food safety. The study was divided into three experiments each lasting for 2 weeks. Cows in experiment 1 received in random order aflatoxin B1 (AFB1) [788 µg/cow/day (69.7 µg/kg dry matter intake (DMI)) for Borans and 2,310 µg/cow/day (154 µg/kg DMI) for crossbreeds], bentonite (60 g/cow/day), or both AFB1 and bentonite. Boran cows in experiment 2 received in random order FUM (12.4 mg/cow/day (1.1 mg/kg DMI)), fumonisin esterase (120 U/cow/day), or both FUM and fumonisin esterase. Boran cows in experiment 3 received in random order AFB1 (952 µg/cow/day (84.2 µg/kg DMI)) + FUM (30.4 mg/cow/day (2.7 mg/kg DMI)), bentonite (60 g/cow/day) + fumonisin esterase (120 U/cow/day), or both AFB1 + FUM and bentonite + fumonisin esterase. Feeding AFB1 and/or FUM contaminated feed with or without the addition of the detoxifiers for 14 days did not affect DMI, milk composition, hematology, and blood biochemical parameters. The addition of bentonite in a diet contaminated with AFB1 led to a decrease in milk aflatoxin M1 (AFM1) concentration of 30% and 43%, with the carry-over subsequently decreasing from 0.35% to 0.20% and 0.08% to 0.06% for crosses and Borans, respectively. No significant change was observed in the sphinganine/sphingosine (Sa/So) ratio following feeding with FUM alone or in combination with fumonisin esterase; however, the ability of fumonisin esterase to hydrolyze FUM into less toxic fully hydrolyzed FUM and partially hydrolyzed FUM was evident in the rumen fluid and feces. These results indicate bentonite was effective in decreasing AFM1 concentration in milk, and AFB1 and AFM1 in plasma, while fumonisin esterase can convert FUM into less toxic metabolites and can be a suitable addition to feed cocontaminated with AFB1 and FUM.

Residues of pesticides and veterinary drugs in diets of dairy cattle from conventional and organic farms in Austria.

Modern agriculture depends highly on pesticides and pharmaceutical preparations, so controlling exposure to these substances in the feed and food chain is essential. This article presents the first study on residues of a broad spectrum of pesticides and veterinary drugs in the diets of dairy cattle. One hundred and two representative samples of the complete diets, including basal feed rations and additional fed concentrate, were collected in three Austrian provinces (Styria, Lower and Upper Austria) in 2019 and 2020. The samples were tested for >700 pesticides, veterinary drugs and related metabolites using a validated method based on liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS). In total, 16 residues (13 pesticides and three veterinary drug residues) were detected. > 90% of the diets contained pesticide residues and <10% veterinary drug residues, whereas banned pesticides were not found. The most frequent pesticide residues were fluopyram (62%), piperonyl butoxide (39%) and diethyltoluamide (35%). The following pesticides exceed the default EU maximum residue level (MRL) (10 µg kg-1) for products exclusively used for animal feed production: Benzovindiflupyr (proportion of samples > MRLs: 1%), bixafen (2%), fluopyram (6%), ipconazole (1%) and tebuconazole (3%). Three residues (dinitrocarbanilide, monensin and nicarbazin) of veterinary drugs were identified, all below the MRLs. Over 60% of the evaluated samples contained mixtures of two to six residues/sample. Only one pesticide (diethyltoluamide) presented a significant difference among regions, with higher concentrations in Upper Austria. Brewery's spent grains were the dietary ingredient that showed the strongest correlation to pesticide residues. These findings evidence the realistic scenario of highly occurrent low doses of pesticides cocktails in the feed/food chain, which may affect the animal, human and environmental health. Since the risk assessments are based on single pesticides, the potential synergistic effect of co-occurring chemicals ("cocktail effect") requires further investigations.

Mixtures of mycotoxins, phytoestrogens and pesticides co-occurring in wet spent brewery grains (BSG) intended for dairy cattle feeding in Austria.

Spent brewery grains (BSG) are the main by-product of beer production and are incorporated in rations of food-delivering animals, mainly dairy cows. Like other agricultural commodities, BSG can be contaminated by a broad spectrum of natural and synthetic undesirable substances, which can be hazardous to animal and human health as well as to the environment. The co-occurrence of mycotoxins, phytoestrogens, other fungal and plant secondary metabolites, along with pesticides, was investigated in 21 BSG samples collected in dairy farms in Austria. For this purpose, a validated multi-metabolite liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) was employed. Metabolites derived from Fusarium, Aspergillus, Alternaria and pesticide residues, were ubiquitous in the samples. Zearalenone (ZEN), T-2 and HT-2 toxins were the only regulated mycotoxin detected, albeit at concentrations below the European guidance values for animal feeds. Ergot alkaloids, Penicillium-derived metabolites, and phytoestrogens had occurrence rates of 90, 48 and 29%, respectively. Penicillium metabolites presented the highest levels among the fungal compounds, indicating contamination during storage. Aflatoxins (AFs), ochratoxins and deoxynivalenol (DON) were not detected. Out of the 16 detected pesticides, two fungicides, ametoctradin (9.5%) and mandipropamid (14.3%) revealed concentrations exceeding their respective maximum residue level (MRL) (0.01 mg kg-1) for barley in two samples. Although based on European guidance and MRL values the levels of the detected compounds probably do not pose acute risks for cattle, the impact of the long-time exposure to such mixtures of natural and synthetic toxicants on animal health and food safety are unknown and must be elucidated.

Cocktails of Mycotoxins, Phytoestrogens, and Other Secondary Metabolites in Diets of Dairy Cows in Austria: Inferences from Diet Composition and Geo-Climatic Factors.

Dairy production is a pivotal economic sector of Austrian and European agriculture. Dietary toxins and endocrine disruptors of natural origin such as mycotoxins and phytoestrogens can affect animal health, reproduction, and productivity. This study characterized the profile of a wide spectrum of fungal, plant, and unspecific secondary metabolites, including regulated, emerging, and modified mycotoxins, phytoestrogens, and cyanogenic glucosides, in complete diets of lactating cows from 100 Austrian dairy farms. To achieve this, a validated multi-metabolite liquid chromatography/electrospray ionization−tandem mass spectrometric (LC/ESI−MS/MS) method was employed, detecting 155 of >800 tested metabolites. Additionally, the most influential dietary and geo-climatic factors related to the dietary mycotoxin contamination of Austrian dairy cattle were recognized. We evidenced that the diets of Austrian dairy cows presented ubiquitous contamination with mixtures of mycotoxins and phytoestrogens. Metabolites derived from Fusarium spp. presented the highest concentrations, were the most recurrent, and had the highest diversity among the detected fungal compounds. Zearalenone, deoxynivalenol, and fumonisin B1 were the most frequently occurring mycotoxins considered in the EU legislation, with detection frequencies >70%. Among the investigated dietary factors, inclusion of maize silage (MS) and straw in the diets was the most influential factor in contamination with Fusarium-derived and other fungal toxins and metabolites, and temperature was the most influential among the geo-climatic factors.

Short-term exposure to the mycotoxins zearalenone or fumonisins affects rumen fermentation and microbiota, and health variables in cattle.

Zearalenone (ZEN) and fumonisins (FUM) jeopardize fertility and health in cattle; yet, their toxigenic effects on rumen health and microbiota, both being crucial for animal health, are not clarified. This study determined the effects of a short-term exposure to ZEN or FUM on the rumen ecosystem, and further evaluated acute implications on health parameters. Six cows were fed a basal diet with 40% grain (dry matter basis) and exposed to either 5 mg of ZEN or 20 mg of FUM daily for two consecutive days each, separated by a 7-days washout period. The exposure to ZEN or FUM led to a reduction of Lachnospiraceae and Prevotellaceae in the rumen. Similarly, ZEN lowered the ruminal pH and total short-chain fatty acid concentration, despite increased rumination activity of the cows. Fumonisins increased the number of observed features and significantly impacted ß-diversity structure and metagenome predicted function. At the systemic level, FUM exposure suggested an immediate hepatotoxic effect, as evidenced by increased liver enzyme concentrations, which were accompanied by altered heart and respiratory rates. Similarly, ZEN increased the body temperature up to a mild fever. Concluding, short-term exposure to ZEN and FUM can harm the rumen ecosystem and acutely impair systemic health.

Fungal species and mycotoxins in mouldy spots of grass and maize silages in Austria.

Fungi and mycotoxins in silage can have detrimental consequences for both cattle and human health. This pilot study identified, via the routinary direct plating method, the dominant cultivable fungi in mouldy grass silages (GS) (n = 19) and maize silages (MS) (n = 28) from Austria. The profiles of regulated, modified, and emerging mycotoxins together with other fungal metabolites were analysed via LC-(ESI)MS/MS. Penicillium roqueforti, Saccharomyces spp., Geotrichum candidum, Aspergillus fumigatus and Monascus ruber were the most frequent fungal organisms identified. Other species including Mucor circinelloides, Fusarium spp. and Paecilomyces niveus were detected at lower frequencies. The presence of complex mixtures of toxic and potentially toxic compounds was evidenced by high levels and occurrences (≥ 50%) of Penicillium-produced compounds such as mycophenolic acid (MPA), roquefortines (ROCs), andrastins (ANDs) and marcfortine A. Mouldy silages contained toxins commonly produced by genus Fusarium (e.g. zearalenone (ZEN) and trichothecenes), Alternaria (like tenuazonic acid (TeA) and alternariol (AHO)) and Aspergillus (such as sterigmatocystin (STC)). Compared to those in GS, mouldy spots in MS presented significantly higher fungal counts and more diverse toxin profiles, in addition to superior levels of Fusarium spp., Penicillium spp. and total fungal metabolites. Generally, no correlation between mould counts and corresponding metabolites was detected, except for the counts of P. roqueforti, which were positively correlated with Penicillium spp. metabolites in mouldy MS. This study represents a first assessment of the fungal diversity in mouldy silage in Austria and highlights its potential role as a substantial contributor to contamination with complex mycotoxin mixtures in cattle diets.

Supplementing a Clay Mineral-Based Feed Additive Modulated Fecal Microbiota Composition, Liver Health, and Lipid Serum Metabolome in Dairy Cows Fed Starch-Rich Diets.

Starch-rich diets are a commonly adopted strategy in order to sustain high milk yields in dairy cows. However, these diets are known to increase the risk of gut dysbiosis and related systemic health disorders. This study aimed to evaluate the effects of supplementing a clay mineral-based feed additive (CM; Mycofix® Plus, BIOMIN) on fecal microbiota structure, fecal short-chain fatty acid (SCFA) fermentation, serum metabolome, and liver health in primiparous (PP, n = 8) and multiparous (MP, n = 16) early-lactation Simmental cows (737 ± 90 kg of live body weight). Cows were randomly assigned to either a control or CM group (55 g per cow and day) and transitioned from a diet moderate in starch (26.3 ± 1.0%) to a high starch diet (32.0 ± 0.8%). Supplementation of CM reversed the decrease in bacterial diversity, richness, and evenness (p < 0.05) during high-starch diet, demonstrating that CM supplementation efficiently eased hindgut dysbiosis. The CM treatment reduced levels of Lactobacillus in PP cows during starch-rich feeding and elevated fecal pH, indicating a healthier hindgut milieu compared with that in control. Butyrate and propionate levels were modulated by CM supplementation, with butyrate being lower in CM-treated MP cows, whereas propionate was lower in MP but higher in PP cows. Supplementing CM during high-starch feeding increased the concentrations of the main primary bile salts and secondary bile acids in the serum and improved liver function in cows as indicated by reduced levels of glutamate dehydrogenase and γ-glutamyl-transferase, as well as higher serum albumin and triglyceride concentrations. These changes and those related to lipid serum metabolome were more pronounced in PP cows as also corroborated by relevance network analysis.

Effects of Deoxynivalenol and Fumonisins Fed in Combination to Beef Cattle: Immunotoxicity and Gene Expression.

We evaluated the effects of a treatment diet contaminated with 1.7 mg deoxynivalenol and 3.5 mg fumonisins (B1, B2 and B3) per kg ration on immune status and peripheral blood gene expression profiles in finishing-stage Angus steers. The mycotoxin treatment diet was fed for a period of 21 days followed by a two-week washout period during which time all animals consumed the control diet. Whole-blood leukocyte differentials were performed weekly throughout the experimental and washout period. Comparative profiles of CD4+ and CD8+ T cells, along with bactericidal capacity of circulating neutrophils and monocytes were evaluated at 0, 7, 14, 21 and 35 days. Peripheral blood gene expression was measured at 0, 7, 21 and 35 days via RNA sequencing. Significant increases in the percentage of CD4-CD8+ T cells were observed in treatment-fed steers after two weeks of treatment and were associated with decreased CD4:CD8 T-cell ratios at this same timepoint (p ≤ 0.10). No significant differences were observed as an effect of treatment in terms of bactericidal capacity at any timepoint. Dietary treatments induced major changes in transcripts associated with endocrine, metabolic and infectious diseases; protein digestion and absorption; and environmental information processing (inhibition of signaling and processing), as evaluated by dynamic impact analysis. DAVID analysis also suggested treatment effects on oxygen transport, extra-cellular signaling, cell membrane structure and immune system function. These results indicate that finishing-stage beef cattle are susceptible to the immunotoxic and transcript-inhibitory effects of deoxynivalenol and fumonisins at levels which may be realistically encountered in feedlot situations.

Mycotoxins, Phytoestrogens and Other Secondary Metabolites in Austrian Pastures: Occurrences, Contamination Levels and Implications of Geo-Climatic Factors.

Pastures are key feed sources for dairy production and can be contaminated with several secondary metabolites from fungi and plants with toxic or endocrine-disrupting activities, which possess a risk for the health, reproduction and performance of cattle. This exploratory study aimed to determine the co-occurrences and concentrations of a wide range of mycotoxins, phytoestrogens and other secondary metabolites in grazing pastures. Representative samples of pastures were collected from 18 Austrian dairy farms (one sample per farm) between April to October 2019. After sample preparation (drying and milling) the pastures were subjected to multi-metabolite analysis using LC-MS/MS. In total, 68 metabolites were detected, including regulated zearalenone and deoxynivalenol (range: 2.16-138 and 107-505 µg/kg on a dry matter (DM) basis, respectively), modified (3-deoxynivalenol-glucoside, HT-2-glucoside) and emerging Fusarium mycotoxins (e.g., enniatins), ergot alkaloids and Alternaria metabolites along with phytoestrogens and other metabolites. Aflatoxins, fumonisins, T-2 toxin, HT-2 toxin and ochratoxins were not detected. Of the geo-climatic factors and botanical diversity investigated, the environment temperature (average of 2 pre-sampling months and the sampling month) was the most influential factor. The number of fungal metabolites linearly increased with increasing temperatures and temperatures exceeding 15 °C triggered an exponential increment in the concentrations of Fusarium and Alternaria metabolites and ergot alkaloids. In conclusion, even though the levels of regulated mycotoxins detected were below the EU guidance levels, the long-term exposure along with co-occurrence with modified and emerging mycotoxins might be an underestimated risk for grazing and forage-fed livestock. The one-year preliminary data points out a dominant effect of environmental temperature in the diversity and contamination level of fungal metabolites in pastures.

Co-Occurrence of Regulated and Emerging Mycotoxins in Corn Silage: Relationships with Fermentation Quality and Bacterial Communities.

Sixty-four corn silages were characterized for chemicals, bacterial community, and concentrations of several fungal metabolites. Silages were grouped in five clusters, based on detected mycotoxins, and they were characterized for being contaminated by (1) low levels of Aspergillus- and Penicillium-mycotoxins; (2) low levels of fumonisins and other Fusarium-mycotoxins; (3) high levels of Aspergillus-mycotoxins; (4) high levels of non-regulated Fusarium-mycotoxins; (5) high levels of fumonisins and their metabolites. Altersetin was detected in clusters 1, 3, and 5. Rugulusovin or brevianamide F were detected in several samples, with the highest concentration in cluster 3. Emodin was detected in more than 50.0% of samples of clusters 1, 3 and 5, respectively. Kojic acid occurred mainly in clusters 1 and 2 at very low concentrations. Regarding Fusarium mycotoxins, high occurrences were observed for FB3, FB4, FA1, whereas the average concentrations of FB6 and FA2 were lower than 12.4 µg/kg dry matter. Emerging Fusarium-produced mycotoxins, such as siccanol, moniliformin, equisetin, epiequisetin and bikaverin were detected in the majority of analyzed corn silages. Pestalotin, oxaline, phenopirrozin and questiomycin A were detected at high incidences. Concluding, this work highlighted that corn silages could be contaminated by a high number of regulated and emerging mycotoxins.

Metabolism of Zearalenone in the Rumen of Dairy Cows with and without Application of a Zearalenone-Degrading Enzyme.

The mycotoxin zearalenone (ZEN) is a frequent contaminant of animal feed and is well known for its estrogenic effects in animals. Cattle are considered less sensitive to ZEN than pigs. However, ZEN has previously been shown to be converted to the highly estrogenic metabolite α-zearalenol (α-ZEL) in rumen fluid in vitro. Here, we investigate the metabolism of ZEN in the reticulorumen of dairy cows. To this end, rumen-fistulated non-lactating Holstein Friesian cows (n = 4) received a one-time oral dose of ZEN (5 mg ZEN in 500 g concentrate feed) and the concentrations of ZEN and ZEN metabolites were measured in free rumen liquid from three reticulorumen locations (reticulum, ventral sac and dorsal mat layer) during a 34-h period. In all three locations, α-ZEL was the predominant ZEN metabolite and ß-zearalenol (ß-ZEL) was detected in lower concentrations. ZEN, α-ZEL and ß-ZEL were eliminated from the ventral sac and reticulum within 34 h, yet low concentrations of ZEN and α-ZEL were still detected in the dorsal mat 34 h after ZEN administration. In a second step, we investigated the efficacy of the enzyme zearalenone hydrolase ZenA (EC 3.1.1.-, commercial name ZENzyme®, BIOMIN Holding GmbH, Getzersdorf, Austria) to degrade ZEN to the non-estrogenic metabolite hydrolyzed zearalenone (HZEN) in the reticulorumen in vitro and in vivo. ZenA showed a high ZEN-degrading activity in rumen fluid in vitro. When ZenA was added to ZEN-contaminated concentrate fed to rumen-fistulated cows (n = 4), concentrations of ZEN, α-ZEL and ß-ZEL were significantly reduced in all three reticulorumen compartments compared to administration of ZEN-contaminated concentrate without ZenA. Upon ZenA administration, degradation products HZEN and decarboxylated HZEN were detected in the reticulorumen. In conclusion, endogenous metabolization of ZEN in the reticulorumen increases its estrogenic potency due to the formation of α-ZEL. Our results suggest that application of zearalenone hydrolase ZenA as a feed additive may be a promising strategy to counteract estrogenic effects of ZEN in cattle.

Multi-Mycotoxin Occurrence in Dairy Cattle and Poultry Feeds and Feed Ingredients from Machakos Town, Kenya.

Mycotoxins are common in grains in sub-Saharan Africa and negatively impact human and animal health and production. This study assessed occurrences of mycotoxins, some plant, and bacterial metabolites in 16 dairy and 27 poultry feeds, and 24 feed ingredients from Machakos town, Kenya, in February and August 2019. We analyzed the samples using a validated multi-toxin liquid chromatography-tandem mass spectrometry method. A total of 153 mycotoxins, plant, and bacterial toxins, were detected in the samples. All the samples were co-contaminated with 21 to 116 different mycotoxins and/or metabolites. The commonly occurring and EU regulated mycotoxins reported were; aflatoxins (AFs) (70%; range 0.2-318.5 µg/kg), deoxynivalenol (82%; range 22.2-1037 µg/kg), ergot alkaloids (70%; range 0.4-285.7 µg/kg), fumonisins (90%; range 32.4-14,346 µg/kg), HT-2 toxin (3%; range 11.9-13.8 µg/kg), ochratoxin A (24%; range 1.1-24.3 µg/kg), T-2 toxin (4%; range 2.7-5.2 µg/kg) and zearalenone (94%; range 0.3-910.4 µg/kg). Other unregulated emerging mycotoxins and metabolites including Alternaria toxins, Aspergillus toxins, bacterial metabolites, cytochalasins, depsipeptides, Fusarium metabolites, metabolites from other fungi, Penicillium toxins, phytoestrogens, plant metabolites, and unspecific metabolites were also detected at varying levels. Except for total AFs, where the average contamination level was above the EU regulatory limit, all the other mycotoxins detected had average contamination levels below the limits. Ninety-six percent of all the samples were contaminated with more than one of the EU regulated mycotoxins. These co-occurrences may cause synergistic and additive health effects thereby hindering the growth of the Kenyan livestock sector.

A Review of the Impact of Mycotoxins on Dairy Cattle Health: Challenges for Food Safety and Dairy Production in Sub-Saharan Africa.

Mycotoxins are secondary metabolites of fungi that contaminate food and feed and have a significant negative impact on human and animal health and productivity. The tropical condition in Sub-Saharan Africa (SSA) together with poor storage of feed promotes fungal growth and subsequent mycotoxin production. Aflatoxins (AF) produced by Aspergillus species, fumonisins (FUM), zearalenone (ZEN), T-2 toxin (T-2), and deoxynivalenol (DON) produced by Fusarium species, and ochratoxin A (OTA) produced by Penicillium and Aspergillus species are well-known mycotoxins of agricultural importance. Consumption of feed contaminated with these toxins may cause mycotoxicoses in animals, characterized by a range of clinical signs depending on the toxin, and losses in the animal industry. In SSA, contamination of dairy feed with mycotoxins has been frequently reported, which poses a serious constraint to animal health and productivity, and is also a hazard to human health since some mycotoxins and their metabolites are excreted in milk, especially aflatoxin M1. This review describes the major mycotoxins, their occurrence, and impact in dairy cattle diets in SSA highlighting the problems related to animal health, productivity, and food safety and the up-to-date post-harvest mitigation strategies for the prevention and reduction of contamination of dairy feed.