Changes in the nutrient profile and the load of mycotoxins, phytoestrogens, and pesticides in horse pastures during spring and summer in Austria.

Pastures are used for grazing and the production of conserved roughage in horses. Yet, the nutritional profile of the forage varies from spring to late summer, affecting equine nutrient supply and health. In addition, environmental factors may also favor plant contaminants such as mycotoxins. This study aimed to determine the nutritional profile and contaminant load of selected horse pastures from early spring till late summer. The nutrient composition (main macronutrients, macro elements and trace elements), as well as mycotoxins, metabolites, pesticides, and plant-derived compounds of seven horse pastures were analyzed. Each pasture was sampled three times and the samples were categorized according to the status of the pasture plants: ear emergence, early- till full bloom, and drought-damaged vegetation. Drought-damaged pastures demonstrated a rise in the acid to neutral detergent fiber ratio, calcium, iron, and magnesium but lower potassium contents. Mycotoxins and other contaminants were found in the pastures including 64 fungal compounds (ergot alkaloids (13) and metabolites from Fusarium (21), Aspergillus (2), Penicillium (8), Alternaria (8) and other fungal species (12), one bacterial metabolite (cereulide), twelve plant metabolites (including eight phytoestrogens and three cyanogenic glycosides (linamarin, lotaustralin and prunasin)), 11 nonspecific metabolites and six pesticides. Fusarium metabolites showed the highest concentrations among the fungal metabolites and drought-induced stress increased the contamination levels (range: 123-3873 µg/kg DM). In conclusion, there was a dominant effect of the developmental stages of the plants, botanical composition of the pastures and weather conditions on the nutritional composition and presence of contaminants on pastures.

Magnesium in dairy cattle nutrition: A meta-analysis on magnesium absorption in dairy cattle and assessment of simple solubility tests to predict magnesium availability from supplemental sources.

Supplemental Mg sources differ in bioavailability, and solubility is one of the determining factors. We explored whether and which in vitro solubility tests could reliably differentiate the quality of supplemental Mg sources. In experiment 1, we compared 3 chemical methods using an acetic acid solution (50 mL/L, termed vinegar test), a 1 M ammonium nitrate solution, and an artificial rumen buffer fluid without rumen microbiota. The Mg solubility results suggested the vinegar test was the best method due to its robustness, simplicity, and reproducibility. In experiment 2, we validated the reliability of the vinegar test using 4 MgO sources from experiment 1 and 12 new MgO sources plus a laboratory-grade MgO as a standard. Accordingly, we repeated the vinegar test with short (0.5 h) and long (3.0 h) incubation times on these sources and then conducted ruminal incubations in 24-h batch culture experiments. The repeated vinegar test resulted in similar results as in experiment 1. Linear regression across both experiments showed the soluble Mg content (g/kg) = 44.46 (±2.55) × pH - 142.9 (±14.9), root mean square error (RMSE) = 10.2, P slope <0.001, and concordance correlation coefficient (CCC) = 0.953. The predictable pH range was from 4 to 6. The equation cannot be applied to low-alkaline sources such as Mg sulfate, Mg acetate, or a group of MgO with exceptionally high alkaline properties showing a cluster of pH above 8.5. Solubility of the MgO sources in the vinegar test ranged from 5 to 35%, whereas the 24-h ruminal incubations led to more solubility (15-70%). Nevertheless, the differences among most MgO sources were parallel to the data from the in vitro rumen solubility. Next, we performed a meta-analysis of published studies (21 studies, 94 treatments) to assess the true Mg absorption in vivo and potential factors affecting Mg absorption in dairy cows. It appeared that on average dairy cows absorbed about 20% of the Mg intake (range 10-40%), regardless of their lactation status. We revealed a new strategy to predict Mg absorption relative to dietary K as follows: true Mg absorption (g/d) = 0.3395 (±0.025, P < 0.001) × Mg intake (g/d) - 1.9273 (±1.16, P = 0.11) when dietary K ≤20 g/kg DM, and 0.154 (±1.06, P = 0.05) + 0.209 (±0.026, P < 0.001) × Mg intake (g/d) when dietary K >20 g/kg DM (RMSE = 2.19). This strategy improved the accuracy of prediction as compared with the existing prediction (CCC = 0.922 vs. 0.845). Still, over- or underestimations inherent to individual studies were evident and might be related to unaccountable factors, especially the quality of supplemental Mg sources. In conclusion, the vinegar test is a useful tool to rank inorganic Mg sources with alkaline properties. Including in vitro solubility data in Mg nutrition research could help to refine the prediction of bioavailable Mg contents and increase precision in feed formulation.

Effect of inclusion of bakery by-products in the dairy cow's diet on milk fatty acid composition.

Bakery by-products (BP), rich in fats and sugars, are unconventional feed sources for cows whose effects on milk fat composition have not yet been evaluated. This research paper aimed to assess the effects of dietary BP inclusion rate and feeding period on the milk fatty acid composition. Twenty-four Simmental cows were fed a diet without BP (CON) for 1 week. Then they either continued with the CON diet or switched to one of the BP diets (with 15% or 30% BP in diet dry matter) for 3 weeks. Milk samples were taken before diet change and three times during BP feeding and analysed for fatty acid composition. Data showed that increasing BP content in the diet increased total fatty acid intake, especially of 18 : 1 n9. In the milk fat, the percentages of total monounsaturated fatty acids especially of the 18 : 1 origin linearly increased with increasing dietary BP level. The percentage of fatty acids de novo synthesized in the mammary gland (the sum of 4 : 0-14 : 0) remained similar among diets (32-34% of total fatty acids). The 16 : 0 percentage dropped from 32.5 to 29.6% and from 33.6 to 28.3% for 15% and 30% BP, respectively. Only 30% BP elevated the percentage of conjugated linoleic acids (CLA: by 59%) compared with CON throughout the 3 weeks. Proportions of 18 : 2 n6 and 18 : 3 n3 and the n6:n3 ratio were unaffected by BP and feeding time. BP feeding improved all those estimated health indices of the milk fat that are suggested to be related to coronary health. In summary, the inclusion of BP in dairy rations beneficially shifted the milk fatty acid profile to more 18 : 1 fatty acids at the expense of 16 : 0. At a 30% inclusion rate, BP feeding showed an additional benefit of increased CLA content in milk fat.

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.

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.

Physicochemical stressors and mixed alkaloid supplementation modulate ruminal microbiota and fermentation in vitro.

The drop of ruminal pH and heat are common physicochemical stressors challenging ruminal microbiota, nutrient digestion and cattle performance. We characterized the ruminal microbiota and digestive activity in response to different pH (6.0 and 6.6) and temperature (39.5 and 42 °C), as well as established the effective dose of alkaloid supplementation (0, 0.088 and 0.175% of feedstock DM) to modulate ruminal fermentation under these conditions. The acidotic condition decreased microbial diversity and abundances of minor bacterial families whereas most of the highly abundant families like Lactobacillaceae, Prevotellaceae, and Bifidobacteriaceae thrived under the stress. Abundances of all three methanogenic archaea taxa detected increased with heat, as did methane production. However, while Methanomassiliicoccaceae benefited from the low pH, Methanomicrobiaceae diminished and methane production decreased. The low dose of alkaloid addition shifted the fermentation to more propionate and less acetate and the high dose decreased methane and ammonia concentration under the low pH. In conclusion, physicochemical stressors shape the microbial community and function. Mixed alkaloid supplementation facilitates the activity of rumen microbial community under acidotic stress.

Methods of emulsifying linoleic acid in biohydrogenation studies in vitro may bias the resulting fatty acid profiles.

The effects of three emulsifying methods on ruminal fatty acid biohydrogenation (BH) in vitro were compared. Using a static in-vitro gas test system, four replicates of each treatment were incubated in buffered ruminal fluid. Hemicellulose (300 mg dry matter) was supplemented either with or without linoleic acid (9c12c-18:2, 5% in diet dry matter) and incubated for 4 and 24 h. Three methods of emulsifying 9c12c-18:2 were tested: (1) ethanol, (2) Tween 80, and (3) sonication. The products were then compared to non-emulsified 9c12c-18:2. Out of the three emulsifying methods tested, ethanol and sonication resulted in stable 9c12c-18:2 emulsions, indicating good 9c12c-18:2 distribution, while the Tween 80 emulsion was less stable. BH was strongly inhibited by treating 9c12c-18:2 with ethanol and sonication at different steps of the BH-pathway, resulting in changed concentrations of certain BH intermediates. The fatty acid profile generated from the major BH-pathways of 9c12c-18:2 with Tween 80 was comparable to that without emulsification after 24 h of incubation. We conclude that it is not recommended to emulsify lipids before incubating them in vitro when investigating fatty acid BH. If emulsification of 9c12c-18:2 is necessary, Tween 80 seems to be the method that interferes least with BH.