Brain-Derived Neurotrophic Factor, Kynurenine Pathway, and Lipid-Profiling Alterations as Potential Animal Welfare Indicators in Dairy Cattle.

Complete animal welfare evaluation in intensive farming is challenging. With this study, we investigate new biomarkers for animal physical and mental health by comparing plasma expression of biochemical indicators in dairy cows reared in three different systems: (A) semi-intensive free-stall, (B) non-intensive tie-stall, and (C) intensive free-stall. Additionally, protein levels of mature brain-derived neurotrophic factor (mBDNF) and its precursor form (proBDNF) and indoleamine 2,3-dioxygenase (IDO1) specific activity were evaluated in brain samples collected from 12 cattle culled between 73 and 138 months of age. Alterations in plasma lipid composition and in the kynurenine pathway of tryptophan metabolism were observed in the tie-stall-reared animals. The total plasma BDNF concentration was higher in tie-stall group compared to the two free-housing groups. Brain analysis of the tie-stall animals revealed a different mBDNF/proBDNF ratio, with a higher level of proBDNF (p < 0.001). Our data are similar to previous studies on animal models of depression, which reported that inhibition of the conversion of proBDNF in its mature form and/or elevated peripheral kynurenine pathway activation may underlie cerebral biochemical changes and induce depressive-like state behavior in animals.

Optimization and validation of a quick and responsive LC-ESI-MS/MS method to evaluate tetracycline residues in processed animal proteins (PAPs).

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. 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. 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 tetracyclines in PAPs. PAPs are a powerful product which could be used both as raw materials in feed and in organic fertilizer production in a circular economy context. Therefore, the lack of regulation and control over antibiotic 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.

Preliminary Data on Essential and Non-essential Element Occurrence in Processed Animal Proteins from Insects.

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.

Multi-Level System to Assess Toxicity in Water Distribution Plants.

The toxicity of water samples from water distribution plants needs to be investigated further. Indeed, studies on the pro-oxidant effects driven by tap water are very limited. In this study, the water quality, pro-oxidant effects, and potential health risks driven by exposure to groundwater samples from two water plants (sites A and B) located in Northwestern Italy were investigated in a multi-level system. Physicochemical parameters and the absence of pathogens, cyanotoxins, and endocrine active substances indicated a good water quality for both sites. The 25 metals analyzed were found under the limit of quantification or compliant with the maximum limits set by national legislation. Water samples were concentrated by the solid-phase extraction system in order to assess the aquatic toxicity on Epithelioma papulosum cyprini (EPC) cell line. Levels of superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, and glutathione reductase were evaluated through the Integrated Biomarkers Response (IBRv2) index. EPC cell line was found a sensible model for assessing the antioxidant responses driven by both water concentrates. A similar antioxidant response was shown by plots and IBRv2 suggesting a muted risk for the two sampling sites.

Trace and rare earth element bioaccumulation in the spotted dogfish (Scyliorhinus stellaris).

Metals (trace elements and rare earth elements, REEs) were analysed by inductively coupled plasma-mass spectrometry in blood, the liver, the kidney and muscle of ex situ spotted dogfish (Scyliorhinus stellaris). The controlled environment in which these elasmobranchs were hosted allowed to assess a baseline level of metals in the different organs since exposure via water and food can be easily monitored. The highest arsenic, chromium, copper, and iron values were found in the liver, cobalt in the kidney, and cadmium and rubidium in muscle. The highest total trace elements content was found in the trend liver (75 mg kg-1) > blood (33 mg kg-1) > muscle (31 mg kg-1) > kidney (10 mg kg-1), while the ΣREEs was the liver (30 µg kg-1) > muscle (15 µg kg-1) > kidney (13 µg kg-1) > blood (4.1 µg kg-1). Between REEs, the most represented element was scandium. Significant differences in the concentration of metals among organs were observed for almost all elements. Nonessential elements were generally lower and essential elements higher in the examined specimens compared to wild elasmobranchs, suggesting a close relationship between a balanced diet and animal welfare.

Glyceroltriheptanoate (GTH) occurrence in animal by-products: a monitoring study to minimise safety-related risk of misuse.

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.

Processed animal proteins (PAPs) in animal nutrition: Assessment of the chemical risk of essential and non-essential elements.

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.

Occurrence of trace elements in Mediterranean mussels (Mytilus galloprovincialis Lamarck, 1819) from an experimental pilot farm in the Calich Lagoon (Sardinia, Italy).

The present study aimed to determine trace elements in Mediterranean mussels (Mytilus galloprovincialis) from an experimental pilot farm of the Calich Lagoon, a typical Sardinian brackish area (Italy). Two sampling sessions were scheduled in February and May 2019 and the occurrence of 24 metals (Hg, Ag, Al, As, Be, Bi, Cd, Co, Cr, Cu, Fe, Ga, In, Mg, Mn, Mo, Ni, Pb, Rb, Se, Sn, Ti, V, Zn) in bivalves was considered. Environmental conditions of water (temperature, salinity, pH, dissolved oxygen, and chlorophyll a) were also measured in situ. A high significant (P<0.001) difference was reported for temperature, pH, and dissolved oxygen. Our results showed a significant sessional variation of Mo (P<0.001); Cd, V (P<0.01); Ni, Pb and Co (P<0.05) in examined M. galloprovincialis samples; as all values were higher in February than those for May session samples, meanwhile the highest levels were reported for Mg (mean±s.d. 1151±263 mg kg-1 wet weight), Al (mean±s.d. 341±192 mg kg-1 w.w.), and Fe (mean±s.d. 212 ±75 mg kg-1 w.w.) in February samples. The European Union uppermost values (EC Reg. 1881/2006) for Cd, Hg, and Pb were never overpassed. The results confirmed the role of M. galloprovincialis as one of the most appropriate biological indexes to track the presence of trace elements in brackish environments. It could be concluded that the current ecology of the Calich Lagoon suggests that compatibly with the transitional ecosystem, the classification as a bivalves' production area and the implementation of extensive shellfish farming can improve its production capacities. The knowledge of the lagoon ecology is an essential tool for its sustainable exploitation, preserving biodiversity, and mitigating the effects of anthropogenic activities on public health.

Microplastics as vectors of metals contamination in Mediterranean Sea.

Microplastics are contaminants of great concern all over the world. Microplastics constitute pollutants themselves; moreover, other contaminants such as metals are easily absorbed on their plastic surface, becoming bioavailable to marine biota such as zooplankton.We collected marine zooplankton from Mediterranean Sea to investigate trace elements associated with microplastics. Samples were subjected to visual sorting by a stereomicroscope, collected with sterile tweezers, pooled and subjected to sonication, filtration, and drying before being subjected to acid extraction. An ICP-MS was utilized for multi-elemental determination.Aluminum, iron, chromium, zinc, nickel, molybdenum, manganese, lead cobalt, and copper were found at concentrations of mg/kg while arsenic, vanadium, rubidium, and cadmium at level of µg kg-1. Other elements such as silver, beryllium, bismuth, selenium, tin, and thallium were under the limit of quantitation. Lower levels of iron and manganese in samples from Italy were found in comparison to England and Brazil, while aluminum, copper, and zinc registered comparable values. The presence of metals in marine waters is strictly related to sediment lithology and anthropogenic inputs, but plastic plays a key role as vectors for metal ions in the marine system, being able to concentrate metals several order of magnitude higher than in surrounding waters and exerting potential toxicity for living beings after chronic exposure.

Tetracyclines in Processed Animal Proteins: A Monitoring Study on Their Occurrence and Antimicrobial Activity.

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.

Seasonal accumulation of trace elements in native Mediterranean mussels (Mytilus galloprovincialis Lamarck, 1819) collected in the Calich Lagoon (Sardinia, Italy).

The occurrence of 21 trace elements in native Mediterranean mussel (Mytilus galloprovincialis) from the Calich Lagoon, a typical brackish area of the northwest of Sardinia (Italy), was investigated. The seasonal variation of metals in bivalves was considered, and the highest values were found in spring and summer; in particular, a high significant (P < 0.001) temporal variation was reported for silver (Ag) and mercury (Hg). The highest and similar concentrations were registered for aluminium (Al, mean 32 mg kg-1wet weight), iron (Fe, mean 32 mg kg-1 w. w.), and zinc (Zn, mean 25 mg kg-1 w. w.). The maximum limits set by European Regulations for cadmium (Cd), mercury (Hg), and lead (Pb) were never exceeded. Speciation analysis revealed negligible risk related to inorganic arsenic (iAs). Therefore, M. galloprovincialis confirmed its role as suitable bioindicator to monitor the contamination of coastal environments. Although the recommended tolerable weekly intake (TWI) was not exceeded, the levels of aluminium should be carefully evaluated in monitoring plans in the studied lagoon. Graphical abstract.

Occurrence and persistence of enteric viruses, arsenic and biotoxins in Pacific oysters farmed in an Italian production site.

The presence of Norovirus (NoV) and Hepatitis E virus (HEV) in non-depurated and depurated oysters raised in the north-west Italian coast was investigated by quantitative real-time RT-PCR. Total and inorganic arsenic (As) and the presence of marine biotoxins (DSP, ASP, PSP) by LC-MS were also investigated. NoV was detected through all the sampling period in non depurated and depurated oysters with highest levels during wintertime (>104 genome copies per gram, gc/g) and minimum values in summer below the LOQ (<130/140 gc/g). HEV has never been found as well as biotoxins. Total As concentration was found in oysters in the range 0.45-3.0 mg/kg, while inorganic As was found in all samples in concentrations below the LOQ (<0.020 mg/kg). The study highlights how the 24 h depuration process didn't reduce significantly NoV levels and therefore the high concentration of NoV in oysters could represent a risk for consumers especially during winter and spring months.

Distribution and bioaccumulation of trace elements and lanthanides in apples from Northwestern Italy.

BACKGROUND: Transfer of metals from soil to plant is a possible route of contamination for the food chain. This investigation focused on the occurrence of 40 elements in the "Red Apple of Cuneo", an Italian excellence and a Protected Geographical Indication (P.G.I.). Four cultivars were considered: Red Delicious (Jeromine) and Gala (Bukeye, Brookfield, Schniga). METHODS: Trace elements and rare earth elements (REEs) detection was performed by an Inductively Coupled Plasma-Mass Spectrometer (ICP-MS) previous homogenization of samples and microwave acid digestion. One-way analysis of variance (ANOVA) with Bonferroni correction was employed to for statistical analysis. RESULTS: Concentrations of metals in the four apples cultivars were quite comparable, Al and Rb were the most represented nonessential elements while Fe, Cu and Zn between the essential; REEs were found at negligible concentrations. Bioaccumulation factors demonstrated an extremely low capacity of bioaccumulation from soil to fruit. CONCLUSIONS: The analysis performed on the "Red Apple of Cuneo" has shown that this product is safe for human consumption since metals were recovered at concentrations of no concern and lower than those registered in apples from other countries. Since each production area is characterized by a typical elemental pattern the multielemental profile based on the analysis of 40 elements could be useful to relate products to their region of origin. Differences between the four apple cultivars were instead not significant to discriminate between them.

Trace and rare earth elements in monofloral and multifloral honeys from Northwestern Italy; A first attempt of characterization by a multi-elemental profile.

BACKGROUND: Metals are a minor constituent in honey, but they have been suggested to be a potential tool to characterize honeys according to their botanical or geographical origin. METHODS: A total of 40 metals were analyzed by Inductively Coupled Plasma-Mass Spectrometry in monofloral and multifloral honeys from Northwestern Italy. PCA and ANOVA were used to discriminate honeys according to the various floral types. RESULTS: The highest levels of trace elements and rare earth elements (REEs) were found in chestnut honey, while the lowest concentrations were recorded in acacia and rhododendron honeys. Rubidium and aluminum were the most represented nonessential elements, while manganese, iron, zinc and copper had the highest values of the essential elements. Potentially toxic elements arsenic, cadmium and lead were close to or below the limit of quantification. CONCLUSIONS: The present study showed significant differences in metal concentrations according to honey floral type, reflecting the strong influence of botanical origin on the chemical composition of this particular food.

Trace elements and rare earth elements in honeys from the Balkans, Kazakhstan, Italy, South America, and Tanzania.

Honey is a very unusual food - a natural substance produced by bees from the nectar or secretion of plants and elaborated by the bees themselves. Differences in trace elements and rare earth elements (REEs) are related to botanical and geographical origin, and, as such, honey could be a suitable indicator for metal occurrence in the environment within the bee forage area. A total of 40 metals were analyzed by inductively coupled plasma-mass spectrometry in multi-floral honey samples from different geographical areas. The highest levels of trace elements and REEs were found in honey from Tanzania, while lower concentrations were recorded in Italian, Kazakhstan, the Balkans, and South American honeys. Tanzanian honey is of interest due to its different content of metals, related to the peculiar lithology and the mineral resources of this country. Rubidium and aluminum were the most represented nonessential elements, while manganese, iron, zinc, and copper registered the highest values of the essential elements. Very low concentrations were found for the toxic elements lead, cadmium, and arsenic. The present study showed differences in metal concentrations in multi-floral honey from different countries, confirming the strong influence of the area of origin on the chemical composition of honeys.