Review: Strategies and technologies in preventing regulated and emerging mycotoxin co-contamination in forage for safeguarding ruminant health.

Ruminants are often considered less susceptible to mycotoxins than monogastrics, owing to rumen microflora converting mycotoxins to less toxic compounds or several compounds present in the rumen-reticulum compartment, being able to bind the mycotoxin "mother" molecule that make them unavailable for absorption process in the gastro-intestinal tract of host animals. However, if ruminants consume feed contaminated by mycotoxins for long periods, their growth, development, and fertility can be compromised. Among regulated mycotoxins, the most studied and known for their effects are aflatoxins (AFs) AFB1, AFB2, AFG1 and AFG2, as well as the AFM1 for its high importance in dairy sector, deoxynivalenol (DON) and its metabolites 3/15 acetyl-DON and 3-glucoside DON, T-2 and HT-2 toxins, zearalenone, fumonisins, in particular that belong to the B class, and ochratoxin A. Furthermore, because of the emergence of multiple emerging mycotoxins that are detectable in feed utilised in ruminant diets, such as ensiled forage, there is now a growing focus on investigating these compounds by the scientific community to deepen their toxicity for animal health. Despite the enhancement of research, it is remarkable that there is a paucity of in vivo trials, as well as limited studies on nutrient digestibility and the impact of these molecules on rumen and intestinal functions or milk yield and quality. In this review, recent findings regarding the occurrence of regulated and emerging mycotoxins in forage and their possible adverse effects on dairy cattle are described, with special emphasis on animal performance and on rumen functionality.

Near-infrared calibration models for estimating volatile fatty acids and methane production from in vitro rumen fermentation of different total mixed rations.

Volatile fatty acids (VFA) and methane (CH4) are the major products of rumen fermentation. The VFA are considered an energy source for the animal and rumen microbiota, and CH4 (which is released by eructation) is considered an energy loss. Quantification of these fermentation products is fundamental for the evaluation of feeds and diets, and provides important information regarding the use of nutrients by ruminants. Near-infrared (NIR) spectroscopy is increasingly used for the evaluation of animal feeds because it is rapid, nondestructive, noninvasive, and inexpensive; does not require reagents; and the results are reproducible. The aim of this study was to develop NIR calibration models for estimating the production of VFA (acetic, propionic, butyric, valeric, isovaleric, and isobutyric acids), total gas, and CH4 using in vitro gas production tests with buffered rumen inoculum throughout fermentation. Fifty-four total mixed rations (TMRs) were examined, and rumen fluid was manually collected from 2 dry Holstein dairy cows that had ruminal fistulas and were fed at maintenance energy levels. Then, 30 mL of buffered rumen fluid was incubated in bottles with ~220 mg of TMR. The total gas, VFA, and CH4 were measured after 2, 5, 9, 24, 30, 48, and 72 h of rumen incubation for each TMR. The VFA were measured on 32 randomly selected TMR. In particular, 7 bottles were used for each TMR, one for each incubation time. Methane was measured in the headspace and VFA were measured in the buffered rumen fluid. The bottles were considered experimental units for calibration purposes. The production of CH4 was quantified from the bottle headspaces by gas chromatography, and total gas production was measured using a pressure transducer at each incubation time. Two aliquots of the fermented liquids were sampled by opening the bottles at each incubation time, and (1) the concentrations of VFA were determined by gas chromatography or (2) spectra were obtained from Fourier-transform NIR spectroscopy. The data were randomly divided into calibration and validation data sets. The average concentrations of acetic acid (45.30 ± 11.92 and 43.86 ± 11.93 mmol/L), propionic acid (14.97 ± 6.08 and 14.38 ± 6.56 mmol/L), butyric acid (8.47 ± 3.47 and 8.65 ± 3.79 mmol/L), total gas (111.34 ± 81.90 and 116.46 ± 82.44 mL/g of organic matter), and CH4 (9.65 ± 9.45 and 10.35 ± 9.33 mmol/L) were similar in the 2 data sets. The best calibration models were retained based on the coefficient of determination (R2) and the ratio of prediction to deviation (RPD). The R2 values for prediction of VFA ranged from 0.69 (RPD = 3.28) for valeric acid to 0.94 (RPD = 4.20) for acetic acid. The models also provided good predictions of CH4 (R2 = 0.89, RPD = 3.05) and cumulative gas production (R2 = 0.91, RPD = 3.30). The models described here precisely and accurately estimated the production of CH4 and VFA during in vitro rumen fermentation tests. Validations at additional laboratories may provide more robust calibrations.

Milk metabolome reveals pyrimidine and its degradation products as the discriminant markers of different corn silage-based nutritional strategies.

The purpose of this study was to evaluate the effect of 6 different feeding systems (based on corn silage as the main ingredient) on the chemical composition of milk and to highlight the potential of untargeted metabolomics to find discriminant marker compounds of different nutritional strategies. Interestingly, the multivariate statistical analysis discriminated milk samples mainly according to the high-moisture ear corn (HMC) included in the diet formulation. Overall, the most discriminant compounds, identified as a function of the HMC, belonged to AA (10 compounds), peptides (71 compounds), pyrimidines (38 compounds), purines (15 compounds), and pyridines (14 compounds). The discriminant milk metabolites were found to significantly explain the metabolic pathways of pyrimidines and vitamin B6. Interestingly, pathway analyses revealed that the inclusion of HMC in the diet formulation strongly affected the pyrimidine metabolism in milk, determining a significant up-accumulation of pyrimidine degradation products, such as 3-ureidopropionic acid, 3-ureidoisobutyric acid, and 3-aminoisobutyric acid. Also, some pyrimidine intermediates (such as l-aspartic acid, N-carbamoyl-l-aspartic acid, and orotic acid) were found to possess a high discrimination degree. Additionally, our findings suggested that the inclusion of alfalfa silage in the diet formulation was potentially correlated with the vitamin B6 metabolism in milk, being 4-pyridoxic acid (a pyridoxal phosphate degradation product) the most significant and up-accumulated compound. Taken together, the accumulation trends of different marker compounds revealed that both pyrimidine intermediates and degradation products are potential marker compounds of HMC-based diets, likely involving a complex metabolism of microbial nitrogen based on total splanchnic fluxes from the rumen to mammary gland in dairy cows. Also, our findings highlight the potential of untargeted metabolomics in both foodomics and foodomics-based studies involving dairy products.

Kinetics of gas production in the presence of Fusarium mycotoxins in rumen fluid of lactating dairy cows.

Little is known about the effects of Fusarium mycotoxins on the fermentation potential of rumen fluid sampled from lactating dairy cows ingesting diets contaminated at regular levels of these mycotoxins (i.e., contamination levels that can normally be found on dairy farms). In the current experiment, rumen donor animals received diets contaminated with both deoxynivalenol (DON) and fumonisins (FB) with or without a mycotoxin-deactivating product. The rumen fluid donor animals were 12 lactating Holstein dairy cows that received one of 3 experimental diets in agreement with a 3 × 3 Latin square design (3 periods and 3 treatments). The 3 diets were as follows: (1) a TMR contaminated with a regular level of Fusarium mycotoxins [340.5 ± 161.0 µg of DON/kg of dry matter (DM) and 127.9 ± 43.9 µg of FB/kg of DM; control diet, CTR], (2) a TMR contaminated with Fusarium mycotoxins at levels higher than CTR but below US and European Union guidelines (733.0 ± 213.6 µg of DON/kg of DM and 994.4 ± 323.2 µg of FB/kg of DM; MTX), and (3) the MTX diet (897.3 ± 230.4 µg of DON/kg of DM and 1,247.1 ± 370.2 µg of FB/kg of DM) supplemented with a mycotoxin-deactivator product (Mycofix, Biomin Holding GmbH; 35 g/animal per day; MDP). Each experimental period lasted 21 d, and rumen fluid was individually sampled from all cows on the last day of each intoxication period. Then, the 4 rumen fluids sampled from cows receiving the same experimental diets were pooled into a single rumen inoculum, which was used in the in vitro gas production test. For the gas production test, 3 different rumen inocula (i.e., CTR, MTX, and MDP) were buffered (buffer:rumen ratio of 2:1, vol/vol) and then used in 3 fermentation runs to evaluate gas production dynamics in the presence of 8 feeds (i.e., corn meal, barley meal, corn silage, sorghum silage, alfalfa hay, ryegrass hay, dry brewers barley grains, and dried distillers grains with solubles). The kinetic parameters of gas production and volatile fatty acid concentrations were evaluated at the end of fermentation. The block run (i.e., fermentation day) effect influenced all of the fermentative and kinetic parameters. Greater final volumes or rates of gas production over time were observed for MDP compared with MTX rumen inocula (i.e., 172.6 vs. 147.8 mL/g of organic matter or 0.078 vs. 0.063 h-1, respectively). However, the increase in rate of gas production was not consistent among tested feeds, meaning that a treatment by feed interaction was observed. Volatile fatty acid concentrations were not different among treatments, except for a slight increase of acetic acid in CTR compared with MTX (i.e., 71.0 vs. 67.9 mmol/L). This study showed that Fusarium-produced mycotoxins negatively affected the kinetics of gas production in feeds, whereas the presence of the mycotoxin-deactivator product in the diets of donor animals resulted in an increase in rumen fermentation potential, thus safeguarding the rumen environment.