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Macro- and microscopic evaluation of feed includes detection of animal proteins, botanical ingredients and impurities, and prohibited ingredients such as packaging material (PM), according to Regulation (EC) 767/2009. In addition, detection of micro-plastics (possible degradation products of some of the PM) is getting attention. PM can harm animals or disturb their feed intake, pollute the environment, and are considered as undesired impurities in feeds. These materials do not consist of a definite molecule, group of molecules, living species or definite bodies. They can be plastic foil, hard plastic, metal pieces, paper, wood or some combination of materials. Their features (sharp, pointed) can be as important as the material itself. This is a typical topic for microscopy detection and evaluation. This short review presents the work done on detection of PM in 15 monitoring entities (institute, laboratories). Since 2011, some institutes have analysed more than 20 samples each year and the incidence of non-compliant samples will be presented here. Thirteen out of 15 entities have an active monitoring, whereas others have passive surveillance (done while performing other microscopy analyses). The protocols used by the different entities depend on sample types and analysts, highlighting a need for harmonisation. Highlights: Former food products as ingredients for animal feed reduce food losses but contain residues of packaging material (PM). The microscopic examination and evaluation of feeds contribute to the safety of ingredients issued from food re-cycling and by-products valorisation. The lack of a prescribed method and limit of tolerance for PM cause variability in survey results. [ABSTRACT FROM AUTHOR]

The potential of utilizing inorganic constituents in processed animal proteins (PAPs) for species identification in animal feeds was investigated, with the aim of using these constituents to ensure the quality and authenticity of the products. This study aimed to quantify the inorganic content across various PAP species and assess whether inorganic analysis could effectively differentiate between PAP species, ultimately aiding in the identification of PAP fractions in animal feeds. Four types of PAPs, namely bovine, swine, poultry, and fish-based, were analyzed and compared to others made up of feathers of vegetal-based feed. Also, three insect-based PAPs (Cricket, Silkworm, Flour Moth) were considered in this study to evaluate the differences in terms of the nutrients present in this type of feed. Ionic chromatography (IC) was used to reveal the concentrations of NO3−, NO2, Cl−, and SO42−, and inductively coupled plasma optical emission spectroscopy (ICP-OES) to detect Al, Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, Si, Sr, Ti, and Zn. The application of multivariate chemometric techniques to the experimental results allowed us to determine the identification capability of the inorganic composition to identify correlations among the variables and to reveal similarities and differences among the different species. The results show the possibility of using this component for discriminating between different PAPS; in particular, fish PAPs are high in Cd, Sr, Na, and Mg content; swine PAPs have lower metal content due to high fat; feathers and vegetal feed have similar Al, Si, and Ni, but feathers are higher in Fe and Zn; and insect PATs have nutrient levels comparable to PAPs of other origins but are very high in Zn, Cu, and K. [ABSTRACT FROM AUTHOR]

Tetracyclines are a group of antibiotic substances largely administered through medicated feed to control diseases in food-producing animals. Fine dosing of antibiotics contained in medicated feed is crucial for the success of the treatment as well as minimising potential threats such as the spread of antimicrobial resistance and the transfer of antibiotic residues in food. A rapid analytical method based on HPLC with diode array detection (HPLC–DAD) was developed to quantify oxytetracycline, chlortetracycline and doxycycline in medicated feed. The reported method underwent in-house validation and was found to be suitable for the quantification of three target tetracyclines within the concentration range of 40–1000 mg kg−1 in official routine analysis. The method was applied to 103 official samples in the framework of the Italian National Plan on animal feed during the years 2021–2023 and nine non-compliant concentrations were identified in swine and fish feed samples. [ABSTRACT FROM AUTHOR]

Early in this century, the crisis connected to the spread of bovine spongiform encephalopathy caused a great concern related to the use of animal by-products (ABPs). According to the Commission Regulation (EU) No 1069/2009, these materials are classified in three categories according to their related risk. In 2011 Commission Regulation (EU) No 142/2011 established that meat and bone meal (MBM) and fat deriving from ABPs not intended for human consumption (category 1 and 2) are required to be permanently marked with glyceroltriheptanoate (GTH), at a minimum concentration of 250 mg kg–1 of fat, while category 3 processed animal proteins (PAPs) must not contain this compound. PAPs are bio resources, which could be used in a renewable and regenerative way in a circular economy model for a conscious usage of raw materials. The aim of this study was to provide information on GTH occurrence in MBM and, if any, in PAPs. Samples were collected from 2017 to 2021 and analysed by GC-MS. Detected non-compliant samples were exclusively of MBM category 1 and 2, probably due to the addition of an inadequate amount of GTH during the manufacturing processes. These results highlighted the importance of National Monitoring Programs as a useful tool to minimise safety related risk due to the misuse of GTH. Thus, investigating the critical points in feed supply-chain and sharing the information on its occurrence may help to improve animal and human wellness and safety. [ABSTRACT FROM AUTHOR]

Background: Harmful botanical impurities may contaminate feed and feed materials and be a potential danger to animal or human health, or to the environment. The aim of this study was to establish rapid and sensitive methods that can be used in routine official controls to determine botanical impurities such as Datura stramonium, Ricinus communis, Crotaliaria spp., and Ambrosia spp. in animal feed and raw materials. Claviceps sclerotia were also detected in cereals, due to the similarities of the targets and the analytical procedure. Regulation (EU) 625/2017, which replaces Reg. 2004/882/EC, states that EU member states should conduct official controls in assessed and accredited laboratories and that the analytical methods must be validated before use by considering parameters such as specificity, precision, recovery, and measurement uncertainly. Results and Conclusion: The results demonstrate that all of the methods tested are suitable for the official quantitative analyses required by EU official legislation. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Valorisation of former foodstuff products (FFP) in feed is part of a long-term strategy for sustainability. An approach to valorise FFP outside the waste value chain is their use as an alternative source of feed materials, with a subsequent optimisation of the environmental impact of products. In the current practice of food production, food packaging is provided to ensure the maintenance of food quality and safety during transport and storage. One of the problems of reusing FFP is how to deal with packaging materials or remains that can become residues in the feed. The aim of this study is to propose a fast and sensitive gravimetric method, fit for routine official controls, for the determination of packaging residues in feed. The developed method can briefly be summarised as: (1) visual selection of the undesired ingredients which can be identified as remnants of packaging materials; (2) weighing of the selected materials; (3) defatting; (4) dehydration; (5) final weighing; and (6) reporting of weight and percentage. Moreover, the method has been validated through the determination of some of the parameters listed in Council Regulation 2004/882/EC (i.e., specificity, limit of quantification (LOQ), recovery, repeatability, within-laboratory reproducibility and measurement uncertainty). [ABSTRACT FROM PUBLISHER]

Bone fragments are essential structures for the detection of processed animal proteins (PAPs) in feed by light microscopy for official controls according to Annex VI of European Union Regulation EC/152/2009. The preparation of samples submitted for analysis requires a grinding step to make them suitable for microscopic slide preparation and observation. However, there are no technical guidelines set down for this step despite the fact that it can lead to an increase in bone numbers due to fragmentation. This was demonstrated by an in-house study carried out by the Irish National Reference Laboratory (NRL) for animal protein detection. The present collaborative study investigated the possible effects of three different grinding conditions on the final result for a feed adulterated with 0.05 and 0.01% (w/w) of PAP. The microscopic analysis either combined or not with an Alizarin Red staining was carried out by 10 different laboratories. The results demonstrated that although a large variation in the numbers of bone fragments was noted, five of the six different grinding/staining combinations applied at two levels of PAP adulteration did not significantly (atp= 0.05) differ from one another. The only exception occurred when grinding the feed containing 0.05% of PAP with a rotor mill equipped with a 0.5-mm sieve and combined with a staining which resulted in a greater number of bone fragments by forced fragmentation. Overall, the impact of the grinding/staining combinations on the final results was shown to be negligible when considering the regulatory limit of detection (LOD) requirement for the method and the current rules of implementation of the light microscopic method. From a total of 180 analyses carried out on the feed matrix containing 0.05% of PAP no false-negative result was observed, and at a level of 0.01% PAP only 10 false-negative results occurred. [ABSTRACT FROM PUBLISHER]

The aim of this study was to evaluate the use of light microscopy with differential staining techniques for the discrimination of insect material from marine arthropods – classified as fishmeal. Specifically, three samples of single-species insect material,Hermetia illucens(HI),Bombyx mori(BM) andTenebrio molitor(TM), and two samples of marine arthropods, shrimp material and krill, were analysed and compared after staining by two reagents to enhance fragment identification. Alizarin Red (AR) and Chlorazol Black (CB), which react respectively with calcium salts and chitin, were tested for their potential efficacy in distinguishing between insect and marine materials. Results indicated that AR failed to stain HI, BM and TM materials. By contrast, the three insect species materials tested were stained by CB. When shrimp fragments and krill were considered, AR and CB stained marine materials reddish-pink and light blue to black, respectively. By combining these results, it can be suggested that CB staining may efficiently be used to mark insect materials; AR does stain shrimp fragments but does not stain the tested insect material, indicating a possible approach for discriminating between insects and marine arthropods. However, since the present study was performed on pure materials and a small set of samples, possible implementation of this technique still needs to be confirmed in complex matrices such as compound feed. [ABSTRACT FROM PUBLISHER]

The aim of this study was to evaluate the potential of image analysis measurements in combination with the official analytical method for the detection of constituents of animal origin in feedstuffs, in distinguishing between bovine and swine (bone containing) material. Authentic samples of controlled origin containing bovine or swine meat and bone meals were analysed by the microscopic method, in accordance with the official analytical method. Sediment fractions of each sample were observed with a compound microscope at X40. A total of 362 bone fragment lacunae images were recorded and processed through image analysis software, deriving 30 geometric variables for each lacuna. Results indicated that not only were most variables significantly (P<0.001) different between bovine and swine samples, but also that two thirds of the same variables were bigger in bovine than in swine. This information, however, does not seem to be so effective in practice since bovine and swine features and measurements overlapped. It can be concluded that the microscopic method even when combined with image analysis does not fit all the requirements for accurately identifying prohibited ingredients of animal origin. A combined approach with other methods is therefore recommended. [ABSTRACT FROM AUTHOR]

Abstract: The general term “pesticide” includes a large number of substances that belong to many different chemical classes. Pesticides are applied to crops at various stages of cultivation to provide protection against weeds and pests, and during post-harvest storage to preserve quality. The list of which pesticides are authorized for use in Europe is available on EU pesticides Database. The QuEChERS approach is a method designed for the analysis of pesticides in fruits and vegetables. This method is based on an extraction and clean-up step; it has been designed to be Quick Easy Cheap Effective Rugged and Safe. The aim of this study was to modify the QuEChERS method to be applied in the analysis of 9 organophosphate and 1 pyrethroid pesticides in raw materials for animal feeding introducing an additional liquid–liquid partition step. This additional step allowed us to concentrate the samples, avoiding any solvent evaporation, prior to the instrumental analysis. Once the method was optimized, it was carried out a pesticides quantization study using a GC–MS SIM multi-residue analysis. 45 samples of maize and soy coming from Northern Italy (Piedmont Region) were analysed during ten months. In 30 samples organophosphate pesticides were found up to 12.4 mg kg−1 of Chlorpyrifos, while no Deltamethrin was detected. [Copyright &y& Elsevier]

Directive 2003/126/EC defines the method for the determination of constituents of animal origin for official control of feedingstuffs. One of the hardest problems for microscopist is the differentiation between mammalian and poultry bones on the basis of some characteristics as colour and borders of the fragments, shape and density of osteocyte lacunae. The shape of osteocyte lacuna in poultry and mammals is often described in different way, elliptic or roundish according with the Author(s). The aim of this study was to analyze the characteristics of lacunae in chicken bones of different type. For this purpose, smashed fragments and histological sections of the same bone were compared in order to evaluate the microscopic aspect of lacunae in different breaking and trimming planes. According to the observations carried out, it was possible to infer that chicken osteocyte has a biconvex lens shape; however the different arrangement and some size variation of the osteocytes in the several bone segments influence the microscopic features of corresponding lacunae. Chicken bone is made of a parallel-fibered tissue, without osteons. This structure probably determines the plane fracture of the bone and consequently the different aspect of lacunae (from spindle-shaped to elliptic-roundish) we can see in chicken derived PAP (processed animal protein). For example, in the fragments obtained from smashed diaphysis, the prevalence of spindle-shaped lacunae is depending on the preferential breaking of the bone along longitudinal plane. Likewise, for the epiphysis, being made mostly by bone trabeculae with strange directions, the breaking happens along different planes, creating lacunae of various shape. Performing the official check of animal feedingstuffs, the presence of bone fragments with roundish or elliptic osteocyte lacunae induces the analyst to thinking that the meat and bone meal comes respectively from mammals and poultry or vice versa depending to the reference Author(s); apart from the final evaluation (also based on some other features of the fragments), it is important to consider that the chicken bones could show lacunae of different shapes (spindle, elliptic and roundish), in accordance with the type and the breaking of the involved skeleton segments. [ABSTRACT FROM AUTHOR]

The bovine spongiform encephalopathy epidemic is thought to have occurred as a consequence of feeding prion-infected material to cattle. To avoid the risk of bovine spongiform encephalopathy diffusion, the European Commission (Directive 2003/126/EC) established an official method to detect the presence of animal-derived constituents in feedstuffs, using microscopic examination. This method allows easy identification of bone fragments among other animal constituents. The analysis is based on morphological conformation of the fragments and their characterization (mainly of the shape of lacunae) to discriminate among mammalian, poultry, and fish tissues. The aim of this study was to assess the performances of nine European laboratories through a ring trial of the official microscopic method, and to calculate accuracy and reproducibility of the method. In general the reproducibility of the microscopic method was very good (K overall = 0.83), with a high sensitivity for all laboratories. Concerning the analysis on the different animal-derived constituents, the results show values of sensitivity with large variability between fish and poultry or mammal. It was generally more difficult to discriminate between mammalian and poultry tissues than fish tissue. [ABSTRACT FROM AUTHOR]

In 2013, the European Union (EU) lifted the feed ban restriction, authorizing the use of non-ruminant (NR) processed animal proteins (PAPs) as ingredient in aquafeed. A further relaxation is soon expected, and NR PAPs will be allowed in next future in poultry and pig feed, avoiding cannibalism. Other potential hazards linked to PAPs as raw material should be evaluated. Antibiotics administered along the lifecycle of animals may leave residue in tissues and bones and still be present in PAPs. This monitoring study aimed to determine tetracyclines (TCLs), known to cumulate in bones, in PAPs and their possible residual antibiotic activity (RAC). A sensitive Liquid Chromatography coupled to Tandem Mass Spectrometry (LC-MS/MS) method for the quantification of TCLs in PAPs was developed and applied to 55 PAPs from EU manufactures. Most PAP samples (n = 40) contained TCLs (concentrations 25.59 ÷ 456.84 µg kg−1). Among samples containing more than 25 µg kg−1 for at least three TCLs, three PAPs were chosen for RAC test before and after TCLs extraction procedure applying an in vitro acidic digestion: in two out of those three samples, RAC was observed after in vitro digestion. TCLs were determined in the digested PAPs (concentrations 26.07 ÷ 64.55 µg kg−1). The detection of TCLs in PAPs should promptly target the risk assessments of this unconsidered way of exposure to antibiotic residues. [ABSTRACT FROM AUTHOR]

The existing European Regulation (EC n° 51/2013) prohibits the use of animals meals in feedstuffs in order to prevent Bovine Spongiform Encephalopathy infection and diffusion, however the legislation is rapidly moving towards a partial lifting of the “feed ban” and the competent control organisms are urged to develop suitable analytical methods able to avoid food safety incidents related to animal origin products. The limitations of the official methods (i.e. light microscopy and Polymerase Chain Reaction) suggest exploring new analytic ways to get reliable results in a short time. The combination of spectroscopic techniques with optical microscopy allows the development of an individual particle method able to meet both selectivity and sensitivity requirements (0.1% w/w). A spectroscopic method based on Fourier Transform micro-Raman spectroscopy coupled with Discriminant Analysis is here presented. This approach could be very useful for in-situ applications, such as customs inspections, since it drastically reduces time and costs of analysis. [ABSTRACT FROM AUTHOR]

Insects have recently emerged as a new protein source for both food and feed. Some studies have already demonstrated that insects’ meal can be successfully added to animal feed without threaten animals’ growth indices. However, effective and validated tests to individuate insects’ meal in feed are strongly needed to meet traceability and safety concerns and to support the European legislation under development. Spectroscopic techniques represent valuable rapid and non-destructive methods that can be applied for in-situ analysis in feed production plants or in farms. In this work a Fourier Transform Near Infrared spectroscopy imaging (FT NIR) as a potential screening method for the detection and quantification of insects’ meal in feed is presented. Discriminant analysis was used for the automatic recognition of insects’ meal fragments into the feed matrix. Moreover, the possibility to quantify insect’s meal in feed sample was successfully tested. The proposed method is a rapid and green strategy for feed contamination screening analysis. [ABSTRACT FROM AUTHOR]

Swine farms are considered a hotspot of antimicrobial resistance and may contribute to the spread of antibiotic-resistant and/or pathogenic bacteria into the environment as well as to farm workers. In this study, swine fecal samples have been collected over the primary production, selecting three categories, i.e. , "Suckling piglets", "Weaning pigs" and "Fatteners", in six intensive swine farms, for two years. Feces were analysed for the detection and abundance of class 1 integrons (used as proxy of antibiotic resistance and of anthropogenic pollution), and of enterococci [fecal indicator bacteria (FIB) and potentially pathogenic for humans] by quantitative Real Time PCR. Furthermore, Enterococcus faecalis and Enterococcus faecium were isolated, analysed for the presence of the intI 1 gene by Real Time PCR and genetically typed by Pulsed-Field Gel Electrophoresis. Both enterococci and class 1 integrons were significantly more abundant in the Suckling piglets (p = 0.0316 and 0.0242, respectively). About 8% of the isolated enterococci were positive for the intI 1 gene by Real Time PCR. E. faecalis and E. faecium were found genetically heterogeneous and no specific pattern could be identified as the driver for their presence along the pig primary production. These findings suggest that the "Suckling piglets" category of production represents the key point where to mitigate the risk of transmission of enterococci and class 1 integrons with associated antibiotic resistance genes to humans and spread into the environment. [Display omitted] • Class 1 integrons and enterococci are more abundant in Suckling piglets category. • Enterococci might be carriers of class 1 integrons. • E.faecalis and E.faecium are genetically heterogeneous in pig farms. [ABSTRACT FROM AUTHOR]

Highlights: [•] We present a rapid and sensitive method to detect melamine in milk based on SERS. [•] Interaction of melamine with AuNPs enhances melamine Raman signal in the analysis. [•] Different size AuNPs were tested to achieve the highest SERS enhancement. [•] Melamine detection in milk is in accordance with the European law limits. [•] SERS analysis is validated for sensitivity, linearity, recovery and repeatability. [Copyright &y& Elsevier]

The article presents findings of the study which aims to evaluate the performances of a dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) for rapid screening of 17 β-estradiol in bovine serum. The researchers performed the validation based on the European Union (EU) Decision 2002/657/EC. Accordingly, they found that the method is appropriate for qualitative screening analysis of 17 β-estradiol.