Mixed Feed Samples Research Articles

Co-occurrence and toxicological relevance of secondary metabolites in dairy cow feed from Thailand

ABSTRACT The occurrence of secondary metabolites and co-contaminants in dairy cow feed samples (n = 115), concentrate, roughage, and mixed feed, collected from Ratchaburi and Kanjanaburi provinces, Thailand, between August 2018 and March 2019 were investigated using LC-MS/MS based multi-toxin method. A total of 113 metabolites were found in the samples. Fungal metabolites were the predominant compounds, followed by plant metabolites. Among major mycotoxins, zearalenone and fumonisins were most frequently detected in concentrate and mixed feed samples, while deoxynivalenol and aflatoxin B1 were found at the frequency lower than 50%. Other metabolites, produced by Aspergillus, Fusarium, Penicillium, and Alternaria species, occurred in the samples. Flavoglaucin, 3-nitropropionic acid, averufin, and sterigmatocystin were the most prevalent Aspergillus metabolites. Common Fusarium metabolites occurring in the samples included moniliformin, beauvericin, and enniatins. For Penicillium metabolites, mycophenolic acid, questiomycin A, quinolactacin A, oxaline, citrinin, and dihydrocitrinone were frequently detected. The toxic Alternaria metabolites, alternariol, and alternariol monomethyl ether showed the high incidence in the samples. Plant metabolites were commonly found, mainly cyanogenic compounds and isoflavones, from cassava and soybean meal used as feed ingredients. Overall, 96.6% of feed samples contained at least two metabolites, in a range from 2 to 69. According to co-contamination of mycotoxins found in feed samples, zearalenone were mostly found in combination with fumonisin B1, deoxynivalenol, and aflatoxin B1. Fumonisin B1 co-occurred with aflatoxin B1 and deoxynivalenol. The mixtures of deoxynivalenol and aflatoxin B1, and of zearalenone, fumonisin B1 and deoxynivalenol were also found. Due to known individual toxicity of fungal and plant metabolites and possible additive or synergistic toxic effects of multi-mycotoxins, the occurrence of these metabolites and co-contaminants should be monitored continuously to ensure food safety through the dairy supply chain.

Determination of fungal genera composition and total toxicity of feed produced in the Republic of Crimea

Animal mycotoxicoses caused by ingestion of toxicogenic micromycete-contaminated feed are of major concern for agricultural industry and of great importance for production of agricultural products. In 2017–2019, feed and raw feed materials produced in the Republic of Crimea were tested for mold fungi and genera composition thereof; the feedstuffs were tested for total toxicity by bioassay in rabbits. A total of 252 samples including 124 grain feed samples, 70 forage samples, 58 mixed feed samples were selected for testing. Tests showed that the major detected contaminants were members of the following genera: Mucor (67.9%), Penicillium (26.6%), Aspergillus (13.1%), Fusarium (9.1%), Alternaria (8.7%), Stachybotrys (3.6%) and Rhizopus (2.0%). It was revealed that feed were exposed to mold fungi contamination during vegetation and harvesting as well as during transportation and storage. Thus, in spring the feed were more often contaminated with micromycetes of Penicillium genus (37.8%) and Stachybotrys genus (6.7%); feed collected and tested in autumn were more often contaminated with toxicogenic mold fungi of Fusarium genus (14.9%), Alternaria genus (13.9%) and Rhizopus genus (3.0%); in winter members of Mucor genus (78.0%) and Aspergillus genus (22.0%) were most often detected in feed. Tests for determination of total toxicity showed that 9 (7.3%) and 10 (8.1%) samples out of 124 tested grain feed samples were low toxic and evidently toxic, respectively. Tests of mixed feed samples for toxicity showed that 5 samples (8.6%) and 2 (3.4%) samples out of 58 mixed feed samples were low toxic and evidently toxic, respectively. It was shown that the proportion of contaminated feed was the highest in spring (25.0%) as compared to proportion of the contaminated feed in winter (18.2%), in autumn (13.7%) and in summer (4.5%).

Estimation of dry matter, crude protein and starch values in mixed feeds by near-infrared reflectance (NIR) spectroscopy

This study aims to determine the levels of dry matter, crude protein, and starch widely used in ruminant feeding, in dairy and beef cattle mixed feeds based on the measurements taken from two different Near-Infrared Reflectance (NIR) devices. The chemical analyses of the mixed feed samples used in the study were performed and the reference values (REF) were determined. In the research, two different NIR devices, a Benchtop-NIR and a Portable-NIR, were used and the nutrient values of mixed feeds were analyzed with these devices. It was determined that there were statistically significant differences between the reference method and NIR results for protein and starch content. Although the differences between the mean values were significant, the reference analyses results and NIR measurements were similar in some ways. According to the correlation coefficients calculated between the reference analyses with Benchtop-NIR and Portable-NIR devices, there was a weak correlation in the dry matter content, while a strong positive correlation existed in the protein (r = 0.72 for Portable, r = 0.93 for Benchtop NIR) and starch content. In the study, 30 different mixed feed (dairy cattle feed, beef cattle feed) values that are commonly used in ruminant feeding were measured.

Determination of crude protein and metabolized energy with near infrared reflectance spectroscopy (NIRS) in ruminant mixed feeds

This study aims to determine the crude protein and metabolizable energy values in ruminant mixed feeds based on the measurements obtained from Near Infrared Reflectance Spectroscopy (NIRS) device. The mixed feeds used in the study were produced in Balıkesir. Reference analyses were determined by making chemical analyses of mixed feed samples used in the study. Metabolic energy value of mixed feed samples was calculated by using the determined nutrient values in equations. Portable NIRS device was used in the study. The relationship between estimates obtained from NIRS device and reference values was statistically evaluated. In regression analysis, R2 value was found as 0.0064 for crude protein while it was 0.9397 for metabolizable energy. It has been demonstrated that the NIRS method is a fast, reliable and good estimation method for quantitatively determining the metabolic energy value in ruminant mixed feeds.

CONDITIONALITY OF AMINO ACID COMPOSITION OF EXPERIMENTAL SAMPLES OF FEED FOR YOUNG CATTLE

The amino acid composition of protein in mixed feed samples of experimental formulations for young cattle is Analyzed. The variation of the content of individual amino acids depending on the component composition of the feed was revealed. The possibilities of combining the high content of individual essential amino acids in one version of feed are considered.

Detection of Spray-Dried Porcine Plasma (SDPP) based on Electronic Nose and Near-Infrared Spectroscopy Data

Since the first proposal to use spray-dried porcine plasma (SDPP) as an animal-based protein source feed additive for piglets in the late 1980s, a large number of studies have been published on the promotion effect of SDPP on piglets. SDPP contains biologically active components that support pig health during weaning stress and may be more economical to use compared to similar bovine-milk-derived protein sources. Unfortunately, animal blood proteins have been suspected as a source for African Swine Fever Virus (ASFV) spread in China. Furthermore, there are no offcially recognized methods for quantifying SDPP in complex feed mixtures. Therefore, it is essential to develop rapid, high-effciency analytical methods to detect SDPP. The feasibility of detecting SDPP using an electronic nose and near-infrared spectroscopy (NIRS) was explored and validated by a principal component analysis (PCA). Both discrimination experiments and prediction experiments were implemented to compare the detect feature of the two techniques. On this basis, partial least squares discriminant analysis (PLS–DA) under various preprocessing methods was used to develop a qualitative discriminant model for estimating the prediction performance. Before selecting a specific regression model for the quantitative analysis of SDPP, a continuum regression (CR) model was employed to explore and choose the potential most appropriate regression model for these two different types of datasets. The results showed that the optimal regression model adopted partial least squares regression (PLSR) with the Savitzky–Golay first derivative and mean-center preprocessing for the NIRS dataset (Rp2 = 0.999, RMSEP = 0.1905). Overall, combining the NIRS technique with multivariate data analysis methods shows more possibilities than an electronic nose for rapidly detecting the usage of SDPP in mixed feed samples, which could provide an effective way to identify the use of SDPP in feed mixtures.

The Assessment of Certain Minerals Content in Mixed Feeds in Relation to the Laying Hens’ Exploitation System (Organic vs. Conventional)

Feedstuffs grown in certain geographic areas may be marginal or deficient in specific elements. Thus, poultry diets usually require supplementation to ensure an adequate intake of trace minerals. The aim of this paper was the assessment of calcium (Ca), phosphorus (P), magnesium (Mg) and sodium (Na) content in the mixed feeds from two different production systems (organic and conventional). Were analyzed ten samples of mixed feed used for the feeding laying hens from Brown hybrid at the age of 27 weeks in two units in Romania. Sampling and the preparation of samples were performed in compliance with the rules of the standards in force in Romania and harmonized with EU legislation (ISO 6869:2002); the method for the determination used was spectrophotocolorimetry method (UVmini 1240 SHIMADZU) for P, and for Ca, Mg and Na was was assessed by atomic absorption spectrometry method. Analysis of mixed feed samples revealed statistically significant differences (p≤0.05) between the two systems (organic vs. conventional) for the P content (0.25 vs. 0.26 %) and Mg content (0.26 vs. 0.28 %). Statistically insignificant differences (p≥0.05) between the two systems (organic vs. conventional) were found for the Ca content (2.93 vs. 2.97 %) and Na content (1.706 vs. 1.712 % ).

Simultaneous determination of six resorcylic acid lactones in feed using liquid chromatography–tandem mass spectrometry and multi-walled carbon nanotubes as a dispersive solid phase extraction sorbent

A simple and cost-effective pre-treatment procedure was developed for six resorcylic acid lactones (RALs) in feed using dispersive solid phase extraction (dSPE) with multi-walled carbon nanotubes (MWCNTs). The sample was analysed after purification by ultra-high performance liquid chromatography–negative electrospray ionisation tandem mass spectrometry (UHPLC–ESI-MS/MS). After extraction with acetonitrile/water (80:20, v/v) and dilution with water, a dSPE procedure was carried out with MWCNTs. The pH value of the extract, the extraction time for MWCNTs, the type and amount of MWCNTs and the type of eluent were optimised to increase the sample throughput and the sensitivity. The samples were quantified using the internal standard zearalenone-D6. The absolute recoveries of the target compounds from feed samples were most efficient when using 100mg of MWCNTs with an outer diameter of less than 8nm and a length of 10–30μm, and ethyl acetate was shown to be the most suitable solvent for desorbing the target compounds from the MWCNTs. The mean recoveries from fortified swine mixed feed samples ranged from 95.3% to 107.2% and had relative standard deviations lower than 10%; the limits of detection and quantification for RALs were in the ranges of 0.20–0.29μg/kg and 0.54–0.78μg/kg, respectively.

Occurrence of trichothecenes, zearalenone and ochratoxin A in cereals and mixed feed from central Lithuania.

A total of 92 samples - 23 winter wheat, 12 summer barley, 5 oats and 52 mixed feed - were collected from a state factory in Kaunas, Lithuania and were analysed for the presence of trichothecenes, zearalenone (ZEN) and ochratoxin A (OA) using gas chromatography with electron capture detection and immunoaffinity column/high performance liquid chromatography with fluorescence and UV detections. Deoxynivalenol (DON), nivalenol (NIV), T-2 toxin and HT-2 toxin were detected at concentrations above 10 μg/kg in 68%, 48%, 38% and 8% of cereal samples, respectively, and in 98%, 88%, 12% and 8% of samples of mixed feed for swine and poultry. More than 10 μg/kg of zearalenone and ochratoxin A were found in 58% and 92% of the mixed feed samples, respectively. The highest concentrations of all analysed trichothecenes in Lithuanian mixed feed and cereal grains, with an exception of T-2 toxin in one oat lot and one sample of mixed feed and OA in two mixed feed samples, were lower than those reported as Lithuanian advisory or tolerance limits.

APPLICATION OF GENE AMPLIFICATION IN CONJUNCTION WITH A HYBRIDIZATION SENSOR FOR RAPID DETECTION OF SALMONELLA SPP. AND FECAL CONTAMINATION INDICATORS IN ANIMAL FEEDS

AbstractThe rapid detection of pathogenic microbial species in feed is of paramount importance considering its implications for animal production and food safety. To demonstrate the feasibility of rapidly detecting Salmonella spp. and fecal pollution microbial indicators in feed using gene amplification protocols, commercial and mixed feed samples were inoculated with two levels of a marker strain of S. typhimurium. Liquid extracts of the feed samples were used as templates in gene amplification reactions to amplify sequences associated with fecal contamination indicators. The sequence specificity of the amplification products (amplicons) were confirmed using biotin and fluorescein labeled probes in a navel dual probe based hybridization sensor. Using the combination of gene amplification and the hybridization sensor, the presence of sequences associated with fecal contamination were detected in 15 different feed matrices without employing preenrichment steps. Using this detection methodology, fecal pollution can be confirmed in feed at naturally occurring concentrations. The study demonstrates that it is possible to rapidly detect and confirm the presence of pathogenic bacterial genera in feed matrices by combining robust gene amplification reactions with appropriate post amplification detection systems.

Amino Acid Analysis of Feeds in The Netherlands: Four-Year Proficiency Study

To improve accuracy and precision of amino acid analysis in 12 Dutch feed laboratories, a proficiency study was organized twice annually over a 4-year period. The method used included reflux acid hydrolysis for 22 h followed by evaporation, separation on a cation-exchange resin in an amino acid analyzer, and photometric detection after post-column derivatization with ninhydrin. For the determination of sulfur-containing amino acids, samples were oxidized prior to hydrolysis. For the determination of tryptophan, samples underwent an alkaline hydrolysis excluding oxygen, were separated by liquid chromatography on a Hypersil ODS analytical column, and were assayed by UV or fluorescence detection. The average relative standard deviations within (CVr) and between (CVR) laboratories were 3 and 7%, respectively. For some mixed feed samples, the results from the proficiency study were compared with those obtained by the International Analytical Group. For those samples, the relative standard deviation of the difference between IAG and Dutch groups was only 1.9%. For samples that were analyzed twice during this 4-year period, relative standard deviation of between-series differences was only 2.1%.

Radioimmunoassay of Deoxynivalenol in Wheat and Corn

With the availability of antibody against deoxynivalenol triacetate (DON-triacetate), a radioimmunoassay (RIA) for DON in wheat was developed. DON is extracted from the sample with acetonitrile-water (84 + 16), defatted with hexane, and then reacted with acetic anhydride to form DON-triacetate. The reaction mixture is loaded onto a C-18 cartridge to remove excess reagents and impurities. Acetylated DON is eluted from the cartridge with 50% methanol in water, and then analyzed by radioimmunoassay utilizing antiserum against DON-triacetate and tritiated DON-triacetate. Overall recovery for DON added to wheat between 50 and 5000 ppb was 86% with a standard deviation of 7% and coefficient of variation of 8%. The limit of detection for DON was about 20 ppb. Analysis of 12 naturally contaminated wheat, corn, and mixed feed samples for DON revealed that RIA results agreed well with thin layer chromatographic analyses performed by other laboratories.

Improved Gas Chromatographic Method for Quantitation of Deoxynivalenol in Wheat, Corn, and Feed

A gas chromatographic (GC) method is described to determine deoxynivalenol in wheat and corn at levels as low as 20 ppb. Ground samples are extracted with water, adsorbed onto a Clin Elut column, extracted with ethyl acetate, and passed through a silica gel Sep-Pak cartridge. The final extract is then derivatized with N-heptafluorobutyrylimidazole and quantitated by GC using an electron capture detector. Recoveries are greater than 85% for spiked samples at levels of 50-1000 ppb. Results for wheat, corn, and mixed feed samples are given as well as the results of an interlaboratory study on a naturally contaminated wheat sample.

Natural occurrence of Fusarium toxins in feedstuff.

The mycotoxins diacetoxyscirpenol, deoxynivalenol, and zearalenone, produced by Fusarium roseum, were found naturally occuring in mixed feed samples. In all cases analyzed, deoxynivalenol occurred together with zearalenone. The natural occurrence of zearalenone in sesame seed is reported for the first time. Strains of F. roseum isolated in various parts of the world form feed implicated in animal mycotoxicosis produced monoacetoxyscirpenol, diacetoxyscirpenol, deoxynivalenol, and zearalenone.