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.

Winery by-products as a feed source with functional properties: dose-response effect of grape pomace, grape seed meal, and grape seed extract on rumen microbial community and their fermentation activity in RUSITEC.

BACKGROUND: Grape and winery by-products have nutritional values for cattle and also contain functional compounds like phenols, which not only bind to protein but can also directly affect microbiota and their function in the rumen. We characterized the nutritional and functional effects of grape seed meal and grape pomace as well as an effective dosage of grape phenols on ruminal microbiota and fermentation characteristics using a rumen simulation technique. RESULTS: Six diets (each n = 8) were compared including a control diet (CON, no by-product), a positive control diet (EXT, CON + 3.7% grape seed extract on a dry matter (DM) basis), two diets with grape seed meal at 5% (GS-low) and 10% (GS-high), and two diets with grape pomace: at 10% (GP-low) and 20% (GP-high), on a DM basis. The inclusion of the by-product supplied total phenols at 3.4%, 0.7%, 1.4%, 1.3%, and 2.7% of diet DM for EXT, GS-low, GS-high, GP-low, and GP-high, respectively. Diets were tested in four experimental runs. All treatments decreased ammonia concentrations and the disappearances of DM and OM (P < 0.05) compared to CON. EXT and GP-high lowered butyrate and odd- and branch-chain short-chain fatty acids while increased acetate compared to CON (P < 0.05). Treatments did not affect methane formation. EXT decreased the abundance of many bacterial genera including those belonging to the core microbiota. GP-high and EXT consistently decreased Olsenella and Anaerotipes while increased Ruminobacter abundances. CONCLUSION: The data suggest that the inclusion of winery by-products or grape seed extract could be an option for reducing excessive ammonia production. Exposure to grape phenols at a high dosage in an extract form can alter the rumen microbial community. This, however, does not necessarily alter the effect of grape phenols on the microbial community function compared to feeding high levels of winery by-products. This suggests the dominant role of dosage over the form or source of the grape phenols in affecting ruminal microbial activity. In conclusion, supplementing grape phenols at about 3% of diet DM is an effective dosage tolerable to ruminal microbiota.

Sigla storax (Liquidambar orientalis) mitigates in vitro methane production without disturbances in rumen microbiota and nutrient fermentation in comparison to monensin.

AIM: The aim of this study was to investigate the in vitro dose-dependent effects of sigla storax (Styrax liquidus) on rumen microbiota and rumen microbial fermentation in comparison to monensin as a positive control. METHODS AND RESULTS: This study was carried out using a rumen simulation model (Rusitec). Treatments consisted of no additive (control), 10 mg l-1 of monensin sodium salt, 100 mg l-1 (Low-Sigla), and 500 mg l-1 (High-Sigla) of sigla storax (n = 6/treatment). In addition to rumen fermentation characteristics, rumen microbial composition was investigated using 16S rRNA sequencing. The methane variables and the acetate to propionate ratio decreased in the both High-Sigla and monensin groups (P < 0.05). High-Sigla had no effect on ammonia, total SCFA and nutrition degradation, while monensin decreased these parameters (P < 0.05). Unlike monensin, the sigla storax treatments did not affect the alpha or beta diversity indexes of the microbiota. The relative abundance of Methanomethylophilaceae and Ruminococcaceae decreased with High-Sigla and monensin (P < 0.05), and Atopobiaceae and Eggerthellaceae decreased with the both doses of sigla storax as well as monensin treatments (P < 0.05). Syntrophococcus, DNF00809, and Kandleria were among the genera that most decreased with High-Sigla and monensin (Q < 0.07) and were strongly positively correlated with methane production (r = 0.52-0.56). CONCLUSIONS: The high dose of sigla storax (500 mg l-1) decreased methane in the rumen ecosystem without adverse effects on nutrient degradation and SCFA production, and without dramatically impacting the microbial composition. Sigla storax might be a novel feed additive to mitigate methane in cattle.

Changes in eating time, chewing activity and dust concentration in horses fed either alfalfa cubes or long-stem hay.

BACKGROUND: Chewing is an essential physiological process in horses; yet, the physical form of feeds may affect their chewing and feeding behaviour with consequences for equine digestion and health. OBJECTIVES AND METHODS: The present study evaluated the potential of a commercial forage cubes made from alfalfa and mixed meadow grasses to maintain chewing activity when compared with a traditional long and fibre-rich hay. An additional aim was to measure the dust formation during feeding. The experiment was a crossover design with six horses (11 ± 4 years old, mean ± SD), fed with 5 kg (as-fed basis) of their feed (long hay or alfalfa cubes) overnight. Eating and chewing activity was measured using the EquiWatch system with a sensor-based halter. RESULTS: Data showed that by feeding the same amount of feed overnight, cube feeding led to 24% shorter eating time (on average 67 min less) and 26% fewer total chews compared to the long hay. On an hourly basis, horses also spent more time eating and chewing the long hay compared to the cubes. The cube feeding led to a higher dust concentration of the inhalable fraction (<100 µm), but not of the thoracic (<10 µm) fraction. Nevertheless, average dust concentrations were generally low for both cubes and hay, whereby both were of sound hygienic status. CONCLUSION AND RECOMMENDATION: Our data suggest that feeding alfalfa-based cubes overnight generated shorter eating time and less chews than the long hay without major differences in the thoracic dust. Therefore, because of the decreased eating time and chews, alfalfa-based cubes should not be fed a sole forage source, especially when it is to be fed without restriction.

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.

Moringa oleifera and Propolis in Cattle Nutrition: Characterization of Metabolic Activities in the Rumen In Vitro.

Moringa oleifera by-products such as seed cake and leaves are protein-rich ingredients, while raw propolis has the potential to influence ruminal protein metabolism. These substances are also known to be sources of functional compounds. With these properties, they could modulate ruminal fermentation activities. Using the rumen simulation technique, we investigated ruminal fermentation and the antioxidant properties of four dietary treatments. These included a control diet (CON) without supplementation; the CON diet top-dressed on a dry matter (DM) basis, either with moringa seed cake (MSC, containing 49% crude protein (CP)), moringa leaf powder (ML, containing 28% CP), or raw propolis (PRO, 3% CP). MSC, ML, and PRO accounted for 3.8, 7.4, and 0.1% of the total diet DM, respectively. Both ML and MSC resulted in 14 and 27% more ammonia concentration, respectively than CON and PRO (p < 0.05). MSC increased the propionate percentage at the expense of acetate (p < 0.05). Both ML and MSC decreased methane percentages by 7 and 10%, respectively, compared to CON (p < 0.05). The antioxidant capacity of the moringa seed cake, moringa leaf powder, and raw propolis were 1.14, 0.56, and 8.56 mg Trolox/g DM, respectively. However, such differences were not evident in the fermentation fluid. In conclusion, the supplementation of moringa seed cake desirably modulates rumen microbial activities related to protein and carbohydrate metabolism.

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.

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.

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.

Models to predict the risk of subacute ruminal acidosis in dairy cows based on dietary and cow factors: A meta-analysis.

The present research aimed at developing practical and feasible models to optimize feeding adequacy to maintain desired rumen pH conditions and prevent subacute ruminal acidosis (SARA) in dairy cows. We conducted 2 meta-analyses, one using data from recent published literatures (study 1) to investigate the prediction of SARA based on nutrient components and dietary physical and chemical characteristics, and another using internal data of our 5 different published experiments (study 2) to obtain adjustments based on cow status. The results of study 1 revealed that physically effective neutral detergent fiber inclusive of particles >8 mm (peNDF >8) and dietary starch [% of dry matter (DM)] were sufficient for predicting daily mean ruminal pH {y = 5.960 - (0.00781 × starch) + (0.03743 × peNDF >8) - [0.00061 × (peNDF >8 × peNDF >8)]}. The model for time of pH suppression (<5.8 for ruminal pH or <6.0 for reticular pH, min/d) can be predicted with additionally including DMI (kg/d): 124.7 + (1.7007 × DMI) + (20.9270 × starch) + (0.2959 × peNDF >8) - [0.0437 × (DMI × starch × peNDF >8)]. As a rule of thumb, when taken separately, we propose 15 to 18% peNDF >8 as a safe range for diet formulation to prevent SARA, when starch or NFC levels are within 20 to 25% and 35 to 40% ranges, respectively. At dietary starch content below 20% of DM, grain type was insignificant in affecting ruminal pH. However, increasing dietary starch contents by using corn as the sole grain source could lead to more severe drops of pH compared with using grain mix based on barley and wheat, as underlined by an interaction between starch content and grain type. Data from study 2 emphasized an increased risk of SARA for cows in the first and second lactation with lower mean pH (0.2 units) and double amounts of time at pH <5.8 compared with the cows with ≥3 parities. Given that a lower ruminal pH is expected in these high-risk cows, it is advisable to keep the lower end of recommended starch (20%) and higher peNDF >8 (18%) contents in the diet of these cows. Overall, the present study underlines the possibility of predicting SARA based on dietary factors including peNDF >8 and starch contents, as well as DMI of the cows, which can be practically implemented for optimal diet formulation for dairy cows. With more data available, future studies should attempt to improve the predictions by including additional key dietary and cow factors in the models.

Betaine Modulates Rumen Archaeal Community and Functioning during Heat and Osmotic Stress Conditions In Vitro.

Rumen archaea play an important role in scavenging ruminal hydrogen (H2) and thus facilitate rumen fermentation. They require optimum temperature and osmolality for their growth and metabolism; however, a number of external factors may put archaea under heat and osmotic stress. Betaine is an osmolyte, molecular chaperone, and antioxidant; therefore, it bears potential to combat against these stressors. In this in vitro study, three betaine levels, namely, 0 (control), 51 (low), and 286 (high) ppm, were used. Each of these was subjected to two temperatures (39.5 and 42°C) and two osmolality conditions (295 and 420 mOsmol kg-1) with n = 6 per treatment. Sequencing analyses of the solid phase (which use solid materials containing primarily fibrous materials of low-density feed particles) and the liquid phase (rumen fermenter liquid) using 16S rRNA revealed that more than 99.8% of the ruminal archaea in fermenters belong to the phylum Euryarchaeota. At the genus level, Methanobrevibacter was the most prevalent in both phases, and Methanosaeta was only detected in the liquid phase. The genera Methanobrevibacter and Methanobacterium both showed a positive correlation with methane (CH4) formation in the liquid and solid phases, respectively (P < 0.05). Heat stress increased the relative abundance of genus Methanimicrococcus at the expense of candidate archaeal genus Vadin CA11 (P < 0.05). In the solid phase, osmotic stress significantly reduced the Shannon and Simpson indices of diversity, and relative abundance was higher for Methanobrevibacter at the expense of Methanimicrococcus. In the liquid phase, osmotic stress increased not only the abundance-based coverage estimator (ACE) and singles parameters of diversity but also the relative abundances of Methanosphaera and Methanobacterium. The overall decrease in all gas parameters and estimated metabolic hydrogen ([2H]) utilization was observed during osmotic stress conditions (P < 0.05). Betaine enhanced the diversity of solid phase archaea as indicated by the increase in ACE and singles during heat stress, and only a high dose improved all diversity parameters in the liquid phase during osmotic stress (P < 0.05). Thus, betaine alleviates the effects of heat stress and osmotic stress on the archaea community.

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.

Betaine addition as a potent ruminal fermentation modulator under hyperthermal and hyperosmotic conditions in vitro.

BACKGROUND: Climatic and dietary shifts predispose ruminal microbes to hyperthermal and hyperosmotic stress, leading to poor fermentation and subsequently adverse effects on ruminant productivity. Betaine may function as substrate, osmolyte, antioxidant, and methyl donor for microbes. However, its effect depends on the extent of microbial catabolism. This study revealed the ruminal disappearance kinetics of betaine and its dose effect on ruminal fermentation during thermal and osmotic stress using a rumen simulation technique. RESULTS: Three different betaine doses were used: 0, 50, and 286 mg L-1 ; each was assigned to two incubation temperatures (39.5 and 42 °C) and two osmotic conditions (295 and 420 mOsmol kg-1 ). Betaine disappeared rapidly within the first 6 h of incubation; however, the rate was lower during hyperosmotic stress (P < 0.05), the stress condition that also suppressed the overall fermentation and degradation of organic nutrients and decreased the bacterial diversity (P < 0.001). During hyperosmotic stress, betaine shifted the fermentation pathway to more propionate (P < 0.05). Betaine counteracted the negative effect of hyperthermal stress on total short-chain fatty acid concentration (P < 0.05) without affecting the composition. Both stress conditions shifted the bacterial composition, but the effect of betaine was minimal. CONCLUSION: Despite its rapid ruminal disappearance, betaine modulated microbial fermentation in different ways depending on stress conditions, indicating the plasticity of the betaine effect in response to various kinds of physicochemical stress. Although betaine did not affect the abundance of ruminal microbiota, the enhanced fermentation suggests an improved microbial metabolic activity under stress conditions. © 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Correction: Temporal dynamics of in-situ fiber-adherent bacterial community under ruminal acidotic conditions determined by 16S rRNA gene profiling.

[This corrects the article DOI: 10.1371/journal.pone.0182271.].

Diet-induced inflammation: From gut to metabolic organs and the consequences for the health and longevity of ruminants.

Dietary shifts play an important role in decreased longevity in ruminant livestock. Ruminants evolved as cellulose fermenters adapt to fiber-rich diets. Instead, high-producing ruminants nowadays are commonly fed with grain-based diets to increase intake and productivity. Such diets, however, trade off the health of the animal. One negative aspect of such feeding is related to elevated levels of bacterial endotoxin (lipopolysaccharide, LPS) in the gut lumen and the likelihood of LPS translocation across the gut causing systemic and local (tissue) inflammation with consequences for production and longevity. However, the view for toxicity of gut LPS is oversimplified, overlooking the physicochemistry of LPS and the translocation route that determine the fate and immune reactive activity of LPS within the host. The barrier and defensive mechanisms of rumen morphology and intestinal mucus are understated. LPS cross the epithelial barrier paracellularly through impaired tight-junction and transcellularly through receptor-mediated transcytosis and the lipoprotein pathway transporting lipids. The lipoprotein pathway delivers LPS to the circulation before reaching the liver for detoxification and is believed to be the major natural route of gut LPS translocation at least in non-ruminants. Ruminant research has focused on endotoxemia and systemic inflammation but with little success and conflicting results, not to mention that low-grade inflammation is not easy to detect. In fact, LPS in the circulation must be effectively removed to avoid an adverse effect of rising level of LPS in the circulation. Circulating LPS could be transported towards target tissues in various organs, leading to local inflammation and altered metabolic activity in the tissues. Therefore, it might be feasible to capture tissue inflammation, especially in the metabolic organs including the liver, adipose tissues, and mammary gland. The present review gathers research updates and presents a comprehensive view of the physicochemical properties and bioactivity of LPS and the possibilities of translocation as well as other possible fate of LPS at each gut site in ruminants. Furthermore, we describe the involvement of three key metabolic organs including the liver, adipose tissue, and mammary gland in response to gut-derived LPS that lead to inflammation in the tissue posing consequences for the health and longevity of dairy cows.

Changes in fibre-adherent and fluid-associated microbial communities and fermentation profiles in the rumen of cattle fed diets differing in hay quality and concentrate amount.

The rumen microbiota enable important metabolic functions to the host cattle. Feeding of starch-rich concentrate feedstuffs to cattle has been demonstrated to increase the risk of metabolic disorders and to significantly alter the rumen microbiome. Thus, alternative feeding strategies like the use of high-quality hay, rich in sugars, as an alternative energy source need to be explored. The aim of this study was to investigate changes in rumen microbial abundances in the liquid and solid-associated fraction of cattle fed two hay qualities differing in sugar content with graded amounts of starchy concentrate feeds using Illumina MiSeq sequencing and quantitative polymerase chain reaction. Operational taxonomic units clustered separately between the liquid and the solid-associated fraction. Phyla in the liquid fraction were identified as mainly Firmicutes, Proteobacteria and Bacteroidetes, whereas main phyla of the fibre-associated fraction were Bacteroidetes, Fibrobacteres and Firmicutes. Significant alterations in the rumen bacterial communities at all taxonomic levels as a result of changing the hay quality and concentrate proportions were observed. Several intermicrobial correlations were found. Genera Ruminobacter and Fibrobacter were significantly suppressed by feeding sugar-rich hay, whereas others such as Selenomonas and Prevotella proliferated. This study extends the knowledge about diet-induced changes in ruminal microbiome of cattle.

Temporal dynamics of in-situ fiber-adherent bacterial community under ruminal acidotic conditions determined by 16S rRNA gene profiling.

Subacute rumen acidotic (SARA) conditions are a consequence of high grain feeding. Recent work has shown that the pattern of grain feeding can significantly impact the rumen epimural microbiota. In a continuation of these works, the objective of this study was to determine the role of grain feeding patterns on the colonization and associated changes in predicted functional properties of the fiber-adherent microbial community over a 48 h period. Eight rumen-cannulated Holstein cows were randomly assigned to interrupted or continuous 60%-grain challenge model (n = 4 per model) to induce SARA conditions. Cows in the continuous model were challenged for 4 weeks, whereas cows of interrupted model had a 1-wk break in between challenges. To determine dynamics of rumen fiber-adherent microbial community we incubated the same hay from the diet samples for 24 and 48 h in situ during the baseline (no grain fed), week 1 and 4 of the continuous grain feeding model as well as during the week 1 following the break in the interrupted model. Microbial DNA was extracted and 16SrRNA amplicon (V3-V5 region) sequencing was done with the Illumina MiSeq platform. A significant decrease (P < 0.001) in fiber-adherent rumen bacterial species richness and diversity was observed at the end of a 4 week continuous SARA challenge in comparison to the baseline. A total of 159 operational taxonominc units (OTUs) were identified from the microbial population representing > 0.1% relative abundance in the rumen, 18 of which were significantly impacted by the feeding challenge model. Correlation analysis of the significant OTUs to rumen pH as an indicator of SARA showed genus Succiniclasticum had a positive correlation to SARA conditions regardless of treatment. Predictive analysis of functional microbial properties suggested that the glyoxylate/dicarboxylate pathway was increased in response to SARA conditions, decreased between 24h to 48h of incubation, negatively correlated with propanoate metabolism and positively correlated to members of the Veillonellaceae family including Succiniclasticum spp. This may indicate an adaptive response in bacterial metabolism under SARA conditions. This research clearly indicates that changes to the colonizing fiber-adherent rumen microbial population and their predicted functional genes occur in both the short (48 h) and long term (4 wk) under both continuous and interrupted SARA challenge models.