The increasing spread of invasive alien species (IAS) represents one of the major causes of biodiversity loss, making containment practices necessary. In this regard, the circular economy framework proposes to reuse the biomass from IAS in growing sectors such as aquaculture, in which more sustainable practices are required. This study evaluated the possibility of using biomass derived from two widespread freshwater IAS, Procambarus clarkii and Silurus glanis, as dietary ingredients for Oncorhynchus mykiss. Experimental diets were formulated by incorporating 20% of IAS-derived muscle powder into a commercial feed, and their effects were assessed through a 35-day feeding trial. Chemical analyses confirmed the nutritional suitability of the formulated diets and the absence of antibiotic residues. No mortality or significant differences in growth performance were observed among treatments. Blood biochemical parameters showed limited variations, remaining within physiological ranges, while oxidative stress biomarkers indicated only minor, diet-specific responses without evidence of oxidative damage. An increase in Hsp70 expression suggested adaptive physiological responses rather than pathological stress. Histological analyses of liver and gut tissues revealed no structural alterations across experimental groups. Overall, the results demonstrate that the inclusion of IAS-derived biomass at 20% is well tolerated by O. mykiss and does not impair fish health. These findings support the potential of invasive species valorization as a sustainable strategy for aquaculture feed production, contributing to both resource efficiency and ecosystem management.

The prohibition of ethoxyquin as a feed additive in the European Union has highlighted the need for reliable analytical methods to monitor its occurrence in feed materials, including trace-level concentrations. This study describes the development and validation of a high-performance liquid chromatography–tandem mass spectrometry method for ethoxyquin determination in feed and fish meal. Sample preparation involved a simple solid–liquid extraction with an organic solvent, while chromatographic separation was completed within seven minutes. The method was validated and evaluated in terms of selectivity, linearity, sensitivity, precision, accuracy, and recovery. Excellent linearity was achieved across both high- and trace-level concentration ranges. Limits of detection (LODs) were 3 µg/mL for both matrices at high concentrations and 0.8 and 3 ng/mL for feed and fish meal, respectively, at trace levels. Corresponding limits of quantification (LOQs) were 10 µg/mL and 2.5 and 10 ng/mL. Precision, expressed as relative standard deviation, remained below 10%, accuracy was within ±20%, and recoveries ranged from 93.8% to 108%. Application to real feed and fish meal samples confirmed the method suitability for quantitative analysis. Its rapid analysis, simple sample preparation, and robust performance make it well suited for routine monitoring and regulatory control.

Enterococcus spp. are opportunistic and nosocomial pathogens. Intensive pig farms have been recently described as important hotspots for antibiotic resistance and reservoirs of potentially pathogenic enterococci, including other species than the most known E. faecalis and E. faecium. Here, we identified Linezolid-resistant non-E. faecalis and E. faecium (NFF) Enterococcus strains isolated from different production stages (suckling piglets, weaning pigs, and fatteners) across six intensive pig farms. The transferability of the linezolid-resistance determinants was assessed by bacterial conjugation and strains were also characterized for biofilm production, hemolytic and gelatinase activity. Among 64 identified NFF Enterococcus strains, 27 were resistant to at least three different antibiotic classes and 8/27 specifically to Linezolid. E. gallinarum and E. casseliflavus both transferred their Linezolid resistance determinants to the main pathogenic species E. faecium. Remarkably, this is the first report of the optrA gene transfer from E. casseliflavus to E. faecium by conjugation, which can greatly contribute to the spread of antibiotic resistance genes among pathogenic enterococcal species. The “weaning pigs” stage exhibited a significantly higher number of antibiotic-resistant enterococci than the “fatteners”. These findings highlight the importance of monitoring pig farms as hotspots for the spread of antibiotic-resistant enterococci, especially in the early stages of production. Furthermore, they underscore the significant role of NFF Enterococcus species as carriers of antibiotic resistance genes, even to last-resort antibiotics, which may be transferable to the major enterococcal species.

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.

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.

Antimicrobial resistance is a global threat, and pet-associated strains may pose a risk to human health. Equine veterinarians are at high risk of carrying methicillin-resistant staphylococci (MRS), but specific risk factors remain elusive, and few data are available for other personnel involved in the horse industry. The prevalence, characteristics, and risk factors for nasal carriage of MRS in horses and their caregivers were studied in northwestern Italy. Nasal swabs from 110 asymptomatic horses housed at 21 barns and 34 human caregivers were collected. Data on barns, horses, and personnel were acquired through questionnaires. The samples were incubated in selective media, and the bacterial isolates were identified by mass spectrometry. Risk factors were investigated by Poisson regression. MRS were isolated from 33 horses (30%), 11 humans (32.4%) and 3 environmental samples (14.2%). Most isolates were multidrug resistant (MDRS). The prevalence of MRS and MDRS was greater in racehorses and their personnel than in pleasurable and jumping/dressing horses. MRS carriage in caregivers was associated with an increased prevalence of MRS carriage in horses. The frequency of antimicrobial treatments administered in the barn during the last 12 months was a risk factor for MRS carriage in horses [prevalence ratio (PR) 3.97, 95% CI 1.11, 14.13] and caregivers (PR 2.00, 95% CI 1.05, 3.82), whereas a good ventilation index of the horse tabling environment was a protective factor (PR 0.43, 95% CI 0.20, 0.92). Our data reveal relevant interactions occurring between bacterial communities of horses and humans that share the same environment, suggesting that One Health surveillance programs should be implemented.

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.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder determined by a combination of both genetic and environmental factors. Despite wide investigations, the role of chronic exposure to environmental pollutants is still rather unknown. Among natural toxins, the mycotoxins have received major attention only in the last few years, due to both technical and scientific achievements that allowed to disentangle many important features of the complex fungal biology. Whereas the effects of acute and high-dose mycotoxin exposure are well known, the potential effects of chronic and low-dose exposure on neurodegeneration have not been broadly elucidated. In this review, we have summarized all the studies concerning environmental exposure to unknown substances that caused ALS outbreaks all over the world, reinterpreting in light of the new scientific acquisitions and highlighting the potential and neglected role of mycotoxins. Then, we focused on recent papers about food exposure to mycotoxin, mycobiome and fungal infections in ALS and other neurodegenerative diseases. We analyzed the gaps of current literature that lead to an undervaluation of mycotoxins as detrimental molecules. By listing all the most important mycotoxins and analyzing all the biological pathways that they can affect, we explained the reasons why they need to be considered in the next epidemiological studies on ALS and other neurodegenerative and neuroinflammatory diseases. In conclusion, after suggesting some possible solutions to mitigate mycotoxin exposure risk, we affirm that future collaborations between scientists and policymakers are important to develop sustainable interventions and promote health through dietary diversity.

The Farm to Fork strategy promotes the development of organic farming and discourages the use of artificial fertilizers. The sustainable agricultural system increases the reduction of waste, allows the reuse of by-products and prefers renewable resources for the production of fertilizers. The European Union (EU) Regulation 2019/1009, called the Regulation on Fertilizer Products (FPR), enhances biomass conversion in organic fertilizers through the exploitation of certain animal by-products. High- and medium-risk animal by-products (Category 1 and 2 materials) must not be recycled in the food chain and therefore must be traceable and permanently marked with glyceroltriheptanoate (GTH) to discriminate them from low-risk materials [processed animal proteins (PAPs) and category 3 fat]. The EU regulates the use of category 2 meat and bone meal (MBM) and PAPs for fertilizers’ production which can ensure a proper supply of macro, micronutrients and organic matter. This study aimed to improve fat extraction and to quantify with gas chromatography mass spectrometry (GC–MS) the detectable amount of GTH in organic fertilizer containing MBM, PAPs, and dry manure in different percentages. The following performance parameters were evaluated: fat recovery (

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 intI1 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 intI1 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.

Background: Processed animal products (PAPs) could be a great alternative to common protein supplements and represent a good example of recycling and valorization of by-products. Due to the reintroduction of certain types of PAPs in feed, a deeper knowledge of these heterogeneous matrices is needed. Thus, the aim of this study is to evaluate the levels of essential elements and inorganic contaminants in 55 PAPs considered as potential alternatives to common protein supplements. Methods: PAPs samples were analysed for essential (cobalt, nickel, chromium, copper, zinc, iron and manganese) and non-essential elements (arsenic, cadmium, lead and mercury) by Inductively Coupled Plasma Mass Spectrometer (ICP-MS), Graphite Furnace Atomization Atomic Absorption Spectrometer (GF-AAS) and dual cell Direct Mercury Analyzer spectrometer (DMA-80). Results: Essential elements were found with the following decreasing order iron > zinc > copper > manganese > chromium > nickel > cobalt (mg kg(-1)). Only one sample was found non-compliant to lead concentration according to the European Union Regulation while negligible values of others non-essential elements were found. Conclusions: This study suggests that PAPs could be a useful supplement for animal diet due to their natural content of essential elements. A careful monitoring of chemical elements should be required and eventually guidelines have to be drafted for a correct use of PAPs to ensure a safe and sustainable feed production.

Insects represent a valuable and environmentally friendly protein alternative in food and feed. The Farm to Fork strategy encouraged the reintroduction of animal by-products in feed production to optimise recycling and to valorise under-used resources. In order to grant safe and valuable feed products, this study investigated the black soldier fly (BSF) (Hermetia illucens) chemical risk. Samples collected in different steps of production (8 samples of substrate for culturing, 7 samples of larvae, 15 samples of protein meal, 18 samples of spent substrate) were analysed for microessential elements (chromium, copper, iron, nickel, selenium and zinc) and inorganic contaminants (aluminium, arsenic, cadmium, lead, tin and vanadium) by Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Microessential elements were found in the following order: Fe > Zn > Cu > Ni > Se > Cr (mg kg−1). Non-essential element concentrations were found lower than the set limits according to the European Union Regulations. The growing demand for alternative protein sources for feed production could be partially compensated by black soldier fly (BSF) (Hermetia illucens) meal, as it appears a good source for high-quality proteins and microessential elements which play a pivotal role in animal growth. In the foreseeable future the current legislation and the official monitoring plans may be implemented and broaden, to focus and assess limits for upcoming matrices, and to ensure feed and food safety.

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.

Background Following the bovine spongiform encephalopathy (BSE) in 2001, processed animal proteins (PAPs) reintroduction is envisaged in non-ruminant feed thanks to their high protein content, easy availability and cost-effective characteristics. PAPs must be submitted to rendering practices, providing sterilization of products, under standardized conditions of temperature and pressure, according to Regulation (EC) No 142/2011. However, the chemical risk associated to these raw materials has been never evaluated. The aim of this study was to develop and validate a reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination and quantification of tetracycline residues in PAPs at µg kg − 1 level. Methods The LC-MS/MS method performances were evaluated in terms of specificity, linearity (25–500 µg kg − 1 ), limit of quantitation (LOQ) (25 µg kg − 1 ), accuracy and precision (CV% <25%), uncertainty, recovery (80–120%) and ruggedness. Results All the evaluated parameters fulfilled the analytical performance criteria and the validated LC-MS/MS method fits for purpose as confirmatory method on the occurrence of residues (µg kg − 1 ) of TCLs in PAPs. Conclusions PAPs are a powerful product which could be used both as raw material in feed and in organic fertilizer production in a circular economy context. Therefore the lack of regulation and control over antibiotics occurrence should be implemented to avoid a misuse and an increment of antibiotic resistance pressure over the environment and to ensure safety of the feed and food chain.

Since the beginning of the feed ban regarding the use of processed animal protein (PAP) in feedstuffs, one of the main challenges has been to develop methods to detect and identify the type and origin of PAP or animal products. By now, in the context of a controlled relaxation of the ban, complementary methods are sought to characterize the product when positive responses are delivered by current official methods, with special attention on feed materials (whether or not authorized) originating from ruminants. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics was identified as the most promising method to meet this challenge. This study presents the first inter-laboratory test constructed to evaluate and compare the LC-MS/MS methods currently developed in six different laboratories from four countries. Nine samples consisting of blank feed matrix or feed adulterated with bovine processed animal proteins and/or milk products were analyzed using in-house protocols and the results obtained were discussed. This study confirms the potential of LC-MS/MS-based proteomics to resolve the current analytical gaps in the detection and differentiation of PAP and highlights the technical challenges that need to be addressed in future.

With the diminishing availability of farmland, climate change and the threat of declining water resources, livestock needs to meet the growing demand for food and feed by using fewer resources. The re-use of food losses as sustainable ingredients for feed formulations could represent a promising alternative to cereal grains for both monogastrics and ruminants, increasing livestock sustainability and reducing the competition between animal and human nutrition. The acceptance of food leftover for feeding animals it is still far to be completely welcomed in several countries, where the outdated stereotypical image of the garbage used as feed is still existing. To implement this practice, a renewed image of food leftover as feed is needed, mainly disseminating the most recent findings about their properties, the new technologies applied for their production and their impact on the environment. This paper aims to disseminate a wide understanding of food losses and explores the potential benefits of using two main categories of food leftovers, namely former food products (FFPs) and bakery by-products (BBPs), as alternative feed ingredients in pig and ruminant nutrition. Several characteristics of those two categories of food losses are examined and compared to a standard diet, such as nutritional-related properties, safety, efficiency and environmental implications. The literature shows that both categories of food leftovers hold a significant nutritional value and are a sustainable alternative to traditional feed ingredients. They resulted as a low risk category for animal health. In addition, when used in complete feed to replace traditional feed ingredients, neither FFPs nor BBPs do not decrease animal’s growth performances. These findings valorize food losses into animal feed as a well-suited strategy to contribute to a reduced environmental and climate footprint of animal products and food waste prevention. However, a greater participation by feed/food processors and stakeholders is crucial to allow the sector to increase its contribution in the entire EU food and feed chain.

Medicated feeds are widely used to treat bacterial infection in poultry, pig and aquaculture.During feed production process, carry-over can occur as an unintentional but unavoidable presence of traces of active substances in a feed batch to the next ones.Sharing information overall among different countries is a crucial step in order to contain development of microbial drug resistance, which represent a key concern and a research priority.So far, European Union (EU) rules do not provide any carry-over action limit for antibiotics in feed, whereas since 2015 the Italian Competent Authorities established action limits based on the 'as low as reasonably achievable' (ALARA) principles.This study aimed to investigate antibiotics at carry-over level as part of the official feeding-stuffs controls carried out between 2017 and 2020 in North-West of Italy (Piedmont, Liguria and Aosta Valley).Analyses were carried out applying accredited in-house methods, performed with high-performance liquid chromatography with diode-array detector (HPLC-DAD), with fluorescence detector (HPLC-FLD) and with electrospray tandem mass spectrometry (HPLC-MS/MS), to detect different classes of antibiotics.Collected data highlight a decreasing trend of non-compliant samples along the examined period.The most identified compound were tetracyclines which are widely used because of their cost-effective activity.Indeed, swine are confirmed to be the most commonly treated species.Systematic measurement of antimicrobial, at carry-over level in feed is an effective tool to contain the development of antimicrobial resistance.Focused and effective steps on the optimisation antimicrobials use may help to improve feed safety and enhance animal and human welfare.

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.