New insights into aflatoxin B1 mechanistic toxicology in cattle liver: an integrated approach using molecular docking and biological evaluation in CYP1A1 and CYP3A74 knockout BFH12 cell lines.

Aflatoxin B1 (AFB1) is a pro-carcinogenic compound bioactivated in the liver by cytochromes P450 (CYPs). In mammals, CYP1A and CYP3A are responsible for AFB1 metabolism, with the formation of the genotoxic carcinogens AFB1-8,9-epoxide and AFM1, and the detoxified metabolite AFQ1. Due to climate change, AFB1 cereals contamination arose in Europe. Thus, cattle, as other farm animals fed with grains (pig, sheep and broiler), are more likely exposed to AFB1 via feed with consequent release of AFM1 in milk, posing a great concern to human health. However, knowledge about bovine CYPs involved in AFB1 metabolism is still scanty. Therefore, CYP1A1- and CYP3A74-mediated molecular mechanisms of AFB1 hepatotoxicity were here dissected. Molecular docking of AFB1 into CYP1A1 model suggested AFB1 8,9-endo- and 8,9-exo-epoxide, and AFM1 formation, while docking of AFB1 into CYP3A74 pointed to AFB1 8,9-exo-epoxide and AFQ1 synthesis. To biologically confirm these predictions, CYP1A1 and CYP3A74 knockout (KO) BFH12 cell lines were exposed to AFB1. LC-MS/MS investigations showed the abolished production of AFM1 in CYP1A1 KO cells and the strong increase of parent AFB1 in CYP3A74 KO cells; the latter result, coupled to a decreased cytotoxicity, suggested the major role of CYP3A74 in AFB1 8,9-exo-epoxide formation. Finally, RNA-sequencing analysis indirectly proved lower AFB1-induced cytotoxic effects in engineered cells versus naïve ones. Overall, this study broadens the knowledge on AFB1 metabolism and hepatotoxicity in cattle, and it provides the weight of evidence that CYP1A1 and CYP3A74 inhibition might be exploited to reduce AFM1 and AFBO synthesis, AFB1 toxicity, and AFM1 milk excretion.

Plant Essential Oils as a Tool in the Control of Bovine Mastitis: An Update.

Bovine mastitis is a major concern for the dairy cattle community worldwide. Mastitis, subclinical or clinical, can be caused by contagious or environmental pathogens. Costs related to mastitis include direct and indirect losses, leading to global annual losses of USD 35 billion. The primary treatment of mastitis is represented by antibiotics, even if that results in the presence of residues in milk. The overuse and misuse of antibiotics in livestock is contributing to the development of antimicrobial resistance (AMR), resulting in a limited resolution of mastitis treatments, as well as a serious threat for public health. Novel alternatives, like the use of plant essential oils (EOs), are needed to replace antibiotic therapy when facing multidrug-resistant bacteria. This review aims to provide an updated overview of the in vitro and in vivo studies available on EOs and their main components as an antibacterial treatment against a variety of mastitis causing pathogens. There are many in vitro studies, but only several in vivo. Given the promising results of treatments with EOs, further clinical trials are needed.

Enantiospecific pharmacokinetics of intravenous dexmedetomidine in beagles.

The goal of this study was to investigate the pharmacokinetic (PK) behaviour of dexmedetomidine in dogs administered as a pure enantiomer versus as part of a racemic mixture. Eight unmedicated intact purpose-bread beagles were included. Two intravenous treatments of either medetomidine or dexmedetomidine were administered at 10- to 14-day intervals. Atipamezole or saline solution was administered intramuscularly 45 min later. Venous blood samples were collected into EDTA collection tubes, and the quantification of dexmedetomidine and levomedetomidine was performed by chiral LC-MS/MS. All dogs appeared sedated after each treatment without complication. Plasma concentrations of levomedetomidine were measured only in the racemic group and were 51.4% (51.4%-56.1%) lower than dexmedetomidine. Non-compartmental analysis (NCA) was performed for both drugs, while dexmedetomidine data were further described using a population pharmacokinetic approach. A standard two-compartment mammillary model with linear elimination with combined additive and multiplicative error model for residual unexplained variability was established for dexmedetomidine. An exponential model was finally retained to describe inter-individual variability on parameters of clearance (Cl1 ) and central and peripheral volumes of distribution (V1 , V2 ). No effect of occurrence, levomedetomidine or atipamezole could be observed on dexmedetomidine PK parameters. Dexmedetomidine did not undergo significantly different PK when administered alone or as part of the racemic mixture in otherwise unmedicated dogs.

Exposure to perfluoroalkyl substances through human milk in preterm infants.

Perfluoroalkyl substances (PFASs) are environmental contaminants that have been shown to exert toxic effects, which are dependent upon concentration, in animals and humans. No specific data on the exposure of preterm infants to PFASs are available. We aimed to quantify the potential exposure of preterm infants to PFASs through human milk (HM), to be compared to the exposure data recently reported for infants by EFSA. The amount of PFASs in ten preterm (PHM) and ten donor HM (DHM) samples was evaluated, and the expected daily intake (EDI) at full enteral feeding was calculated. This EDI was compared to the mean and the 95th centile dietary exposure ranges at the lower bound for infants issued by EFSA. The calculated median EDI for total PFASs was 20.72 ng/kg/day (range 10.72-107.84) for PHM and 17.92 ng/kg/day (range 6.4-28.96) for DHM, which were both higher than mean exposure ranges reported for infants (2.4-12.2 ng/kg/day). The calculated EDI for DHM was far more similar to the 95th centile (4.5-27.9 ng/kg/day) dietary exposure ranges. For PHM samples, higher EDI values were obtained, with 4 out of 10 samples exceeding the upper limit of the 95th centile range.Conclusion: The exposure of preterm infants to PFASs through HM feeding might exceed reference values reported for older and healthier infants. Given the immunological and developmental vulnerability of preterm infants, the risks related to their exposure to PFASs should be further investigated, also focusing on how maternal exposure and subsequent transfer through HM feeding can be reduced. What is Known: • Perfluoroalkyl substances (PFASs) are environmental contaminants that have been shown to exert toxic effects, which are dependent upon concentration, in animals and humans. The EFSA has recently issued reference values for PFASs exposure for different age groups. • Infants might be exposed to PFASs prenatally, as these substances can cross the placenta, and postnatally, through breastfeeding. No specific data about exposure of preterm infants through human milk (HM) feeding are currently available. What is New: • The exposure of preterm infants to PFASs through HM feeding might exceed reference values reported for older and healthier infants. • Given the immunological and developmental vulnerability of preterm infants, the risks related to their exposure to PFASs deserve further investigation. As HM represents the optimal feeding for preterm infants, it will be fundamental to focus on how maternal exposure and subsequent transfer through HM feeding can be reduced.

Pharmacokinetics of S-ketamine and R-ketamine and their active metabolites after racemic ketamine or S-ketamine intravenous administration in dogs sedated with medetomidine.

OBJECTIVE: To assess the differences in the pharmacokinetic profiles of S-ketamine, R-ketamine and their metabolites, S-norketamine and R-norketamine, and to measure relevant physiologic variables after intravenous administration of racemic (RS) ketamine or S-ketamine alone in Beagle dogs sedated with medetomidine. STUDY DESIGN: Experimental, blinded and randomized crossover study. ANIMALS: A total of six (three female and three male) adult Beagle dogs. METHODS: Medetomidine (450 µg m-2) was administered intramuscularly, followed by either S-ketamine (2 mg kg-1) or RS-ketamine (4 mg kg-1) 20 minutes later, both administered intravenously. Blood samples were collected before medetomidine administration and at multiple time points 1-900 minutes following the ketamine administration. Plasma samples were analysed using liquid chromatography-tandem mass spectrometry. Heart rate, respiratory rate, noninvasive blood pressure, haemoglobin saturation with oxygen and body temperature were measured at baseline, before ketamine administration, and 1, 2, 5, 10, 15, 20 and 30 minutes after ketamine administration. All cardiovascular variables, blood glucose, haemoglobin and lactate concentrations were analysed using different linear mixed effects models; the significance was set at p < 0.05. RESULTS: S-ketamine showed a two-compartment kinetic profile; no statistically significant differences were observed between its concentrations or in the calculated pharmacokinetic parameters following S- or RS-ketamine. When the racemic mixture was administered, no differences were detected between R- and S-ketamine concentrations, but the area under the curve (AUC) for R-norketamine was significantly lower than that for S-norketamine. Clinically relevant physiologic variables did not show statistically significant differences following the administration of the racemic mixture or of S-ketamine alone. CONCLUSIONS AND CLINICAL RELEVANCE: This study performed in dogs showed that RS-ketamine and S-ketamine combined with medetomidine showed enantioselective pharmacokinetics as S- and R-norketamine AUCs were different, but S-ketamine levels were identical.

Evaluation of methadone concentrations in bitches and in umbilical cords after epidural or systemic administration for caesarean section: A randomized trial.

OBJECTIVE: To measure plasma methadone concentrations in bitches and the umbilical cords of their puppies after systemic or epidural administration. STUDY DESIGN: Prospective, randomized, clinical study. ANIMALS: A total of 27 healthy pregnant female dogs undergoing caesarean section, 4.3 ± 2.3 years of age and weighing 19.9 ± 13.2 kg. METHODS: The dogs were randomly divided into three groups: 1) intramuscular methadone (0.3 mg kg-1) (group MET; n = 9); 2) epidural methadone (0.1 mg kg-1) (group METEPI; n = 9); and 3) epidural lidocaine (4.4 mg kg-1) [group CON (control group); n = 9]. Ten minutes before induction, methadone was administered intramuscularly to the group MET dogs. Anaesthesia was induced with propofol and maintained with isoflurane. Cardiovascular and respiratory parameters were monitored throughout the anaesthesia. After induction, epidural anaesthesia was administered to dogs in groups METEPI and CON. Before any treatment (T0) and, as soon as the last foetus was removed from the uterus (T1), venous blood samples were collected from each dog into heparinized tubes; the umbilical cords were collected and stored at -80 °C until pharmacological analysis was carried out. The samples were analysed using ultra performance liquid chromatography. RESULTS: The cardiorespiratory parameters of the bitches and of the puppies at birth, and the Apgar scores did not differ significantly between groups. At T1 both the median maternal methadone plasma concentration and the median methadone umbilical cord concentration were higher in group MET compared to group METEPI (p = 0.0018 and p = 0.004, respectively). The maternal plasma concentration was higher than the concentration in the umbilical cords (p = 0.05) in group METEPI but not in group MET (p = 0.25). CONCLUSIONS AND CLINICAL RELEVANCE: Epidural methadone (0.1 mg kg-1) administered to bitches undergoing caesarean section is associated with lower umbilical cord methadone concentrations as compared with intramuscularly administered methadone at higher dosages (0.3 mg kg-1).

Pharmacokinetics of buprenorphine following constant rate infusion for postoperative analgesia in dogs undergoing ovariectomy.

OBJECTIVE: To investigate the pharmacokinetics of buprenorphine and its main active metabolite, norbuprenorphine, after administration of an intravenous loading dose followed by constant rate infusion (CRI) in dogs. STUDY DESIGN: Prospective, clinical study. ANIMALS: A total of seven healthy dogs undergoing elective ovariectomy. METHODS: Buprenorphine was administered as a loading dose (intravenous bolus of 15 µg kg-1) followed by CRI (2.5 µg kg-1 hour-1 for 6 hours). Moreover, intraoperative analgesia was supplemented by an intramuscular carprofen (4 mg kg-1) injection, administered prior to surgery, and by lidocaine, administrated through subcutaneous infiltration and through a splash on the ovarian vascular pedicle during surgery. Pain and sedation were scored for all animals throughout the 24-hour study period and rescue analgesia was administered when a visual analogue scale score was > 40 mm. Blood samples were collected from a jugular catheter at regular intervals, and plasma concentrations of buprenorphine and norbuprenorphine were determined by a validated liquid chromatography-tandem mass spectrometry method. RESULTS: Buprenorphine showed a two-compartment kinetic profile. Maximum concentration was 23.92 ± 8.64 ng mL-1 at 1 minute (maximum time); elimination half-life was 41.87 ± 17.35 minutes; area under the curve was 486.68 ± 125.66 minutes ng-1 mL-1; clearance was 33.61 ± 13.01 mL minute-1 kg-1, and volume of distribution at steady state was 1.77 ± 0.50 L kg-1. In no case was rescue analgesia required. Norbuprenorphine resulted below the lower limit of quantification in almost all samples. CONCLUSIONS AND CLINICAL RELEVANCE: The results suggest that a buprenorphine CRI can be a useful tool for providing analgesia in postoperative patients, considering its minor side effects and the advantages of a CRI compared to frequent boluses. The negligible contribution of norbuprenorphine to the therapeutic effect was confirmed.

Short communication: Monitoring the presence of perfluoroalkyl substances in Italian cow milk.

Perfluoroalkyl substances (PFAS) are fully fluorinated compounds widely used during the last 60 yr in the production of multiple industrial and consumer applications, such as food packaging, nonstick cookware, cleaning agents, and many more. These emerging contaminants have recently become of concern for human health because of their potential negative effects. The risk of exposure to PFAS for humans is mainly related to diet, and the increasing interest in food safety has led the European Commission to call Member States to monitor these contaminants in food matrices. The purpose of the present work was to perform the first monitoring on the presence of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), the 2 main and most widely investigated molecules of this family, in cow milk commercially available in Italy. We used an analytical protocol consisting of liquid-liquid extraction followed by 2 purification steps through solid-phase extraction cartridges and injection on an ultra-performance liquid chromatography-tandem mass spectroscopy system. The analysis of 67 samples of different types of cow milk from Italy demonstrated that contamination by PFOS was often present, although at relatively low concentrations (up to 97 ng/L), whereas PFOA was rarely found. On the basis of these results and data reported in the literature on this matrix, milk does not seem to be a major source of PFAS compared with other food categories such as fish and seafood. However, variability among different types of milk must be taken into account, and surveys of milk-derived products would be helpful to better define the risk for consumers.

Pharmacokinetics of florfenicol in serum and seminal plasma in beef bulls.

The objectives of this study were to compare the serum and seminal plasma pharmacokinetic profiles of florfenicol (FLO) and florfenicol amine (FLA) after the administration of FLO either by IM or SC routes in beef bulls. Four clinically healthy Hereford bulls underwent a comprehensive physical exam, including breeding soundness examination, CBC, and chemistry profile panel. Bulls were healthy and classified satisfactory potential breeders. In one group (n = 2), a single dose of FLO was administered SC in the middle of the neck at a dose of 40 mg/kg of body weight. In the second group (n = 2), a single dose was administered IM in the muscles of the neck at a dose of 20 mg/kg. Concentrations of FLO and FLA in serum and seminal plasma were determined by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Blood and semen samples were collected before the administration of FLO and at 12, 24, 36, 48, 72, 96, 120, 144, and 168 h after injection. The blood was collected from the coccygeal vessels, and semen was collected by electroejaculation. All samples were immediately refrigerated, processed within the first hour after collection, and finally stored at -80 °C. The mean level of total FLO in serum was higher when administered by the SC route (1,415.5 ng/mL) than by the IM route (752.4 ng/mL; P = 0.001). Differences were observed between the percentage of FLA in serum (1.8%; ranging from 1.3 to 2.9) and in seminal plasma (27.5%; ranging from 15.9 to 34.2; P = 0.0001). The mean level (±SD) of FLA was higher in seminal plasma compared to serum (467 ± 466 ng/mL and 18 ± 16 ng/mL, respectively; P = 0.001). The mean level of total FLO in seminal plasma was 1,454.8 ng/mL for the SC route and 1,872.9 ng/mL for the IM route without differences between the two routes (P = 0.51). Differences in the mean level of total FLO between serum and seminal plasma were detected (1,187 ± 2,069 ng/mL and 1,748 ± 1,906 ng/mL, respectively; P = 0.04). From the present investigation, it was concluded that FLO is a suitable antibiotic based on its pharmacokinetic attributes and may be employed for the treatment of bull genital infections when its use is indicated. To study the pharmacokinetics of FLO in seminal plasma, the analysis of FLA should be incorporated.

The Use of Antibiotics and Antimicrobial Resistance in Veterinary Medicine, a Complex Phenomenon: A Narrative Review.

As warned by Sir Alexander Fleming in his Nobel Prize address: "the use of antimicrobials can, and will, lead to resistance". Antimicrobial resistance (AMR) has recently increased due to the overuse and misuse of antibiotics, and their use in animals (food-producing and companion) has also resulted in the selection and transmission of resistant bacteria. The epidemiology of resistance is complex, and factors other than the overall quantity of antibiotics consumed may influence it. Nowadays, AMR has a serious impact on society, both economically and in terms of healthcare. This narrative review aimed to provide a scenario of the state of the AMR phenomenon in veterinary medicine related to the use of antibiotics in different animal species; the impact that it can have on animals, as well as humans and the environment, was considered. Providing some particular instances, the authors tried to explain the vastness of the phenomenon of AMR in veterinary medicine due to many and diverse aspects that cannot always be controlled. The veterinarian is the main reference point here and has a high responsibility towards the human-animal-environment triad. Sharing such a burden with human medicine and cooperating together for the same purpose (fighting and containing AMR) represents an effective example of the application of the One Health approach.

Florfenicol and Florfenicol Amine Quantification in Bull Serum and Seminal Plasma by a Single Validated UHPLC-MS/MS Method.

Florfenicol is a broad-spectrum antibiotic belonging to the amphenicols class that inhibits protein synthesis by binding to bacteria's ribosomal subunits. This drug is commonly used in veterinary medicine to treat bacterial infectious diseases in cattle, swine, poultry, and fish. The proposed method uses a quick protein precipitation with acetonitrile for the extraction of florfenicol and florfenicol amine in serum and seminal plasma, followed by analysis in UHPLC-MS/MS for their simultaneous quantification. A BEH C18 reversed-phase column was chosen for analyte separation, allowing to obtaining sharp and symmetrical peak shapes in a chromatographic run of just 3.5 min under programmed conditions. Two specific transitions were observed for each analyte, and florfenicol-d3 was used as the internal standard. The approach was fully validated in each matrix over ranges suitable for field concentrations of florfenicol and florfenicol amine, showing good linearity during each day of testing (R2 always >0.99). Excellent accuracy and precision were demonstrated, for both analytes, by calculated bias always within ±15% and CV% always below 15% at all QC levels tested. The satisfactory outcomes obtained during recovery, matrix effect, and process efficiency investigations in serum and seminal plasma confirmed the strength of the method for the quantification of target compounds. To our knowledge, this is the first LC-MS/MS-validated approach for the quantification of florfenicol and florfenicol amine in serum and seminal plasma and was successfully applied for the determination of their concentration-time profiles in bulls. This paves the way to understanding the pharmacokinetics of this antibiotic and its active metabolite in bull's seminal plasma, which will enable the design of more appropriate treatment protocols.

Discovering the Protective Effects of Quercetin on Aflatoxin B1-Induced Toxicity in Bovine Foetal Hepatocyte-Derived Cells (BFH12).

Aflatoxin B1 (AFB1) induces lipid peroxidation and mortality in bovine foetal hepatocyte-derived cells (BFH12), with underlying transcriptional perturbations associated mainly with cancer, cellular damage, inflammation, bioactivation, and detoxification pathways. In this cell line, curcumin and resveratrol have proven to be effective in mitigating AFB1-induced toxicity. In this paper, we preliminarily assessed the potential anti-AFB1 activity of a natural polyphenol, quercetin (QUE), in BFH12 cells. To this end, we primarily measured QUE cytotoxicity using a WST-1 reagent. Then, we pre-treated the cells with QUE and exposed them to AFB1. The protective role of QUE was evaluated by measuring cytotoxicity, transcriptional changes (RNA-sequencing), lipid peroxidation (malondialdehyde production), and targeted post-transcriptional modifications (NQO1 and CYP3A enzymatic activity). The results demonstrated that QUE, like curcumin and resveratrol, reduced AFB1-induced cytotoxicity and lipid peroxidation and caused larger transcriptional variations than AFB1 alone. Most of the differentially expressed genes were involved in lipid homeostasis, inflammatory and immune processes, and carcinogenesis. As for enzymatic activities, QUE significantly reverted CYP3A variations induced by AFB1, but not those of NQO1. This study provides new knowledge about key molecular mechanisms involved in QUE-mediated protection against AFB1 toxicity and encourages in vivo studies to assess QUE's bioavailability and beneficial effects on aflatoxicosis.

A Novel UHPLC-MS/MS Method for the Measurement of 25-Hydroxyvitamin D3 in Canine Serum and Its Application to Healthy Dogs.

Several studies have shown the importance of vitamin D3 supplementation in small animals. In dogs, a low vitamin D3 status is associated not only with bone metabolism but also with different kinds of disorders, such as congestive heart failure, gastrointestinal diseases, chronic kidney diseases, and some types of cancer. However, it is crucial to maintain balance and monitor the introduction of this essential nutrient through the diet because over-supplementation can result in toxicity. Due to the clinical importance of assessing the vitamin D3 status in small animal patients, a quick, simple, and highly performing analytical method for its measurement is needed. In this study, we describe the development of a novel liquid chromatography-tandem mass spectrometry method for 25-hydroxyvitamin D3 quantification in canine serum. The approach was successfully validated following current European guidelines, proving excellent linearity (R2 always ≥0.996), accuracy (always within ±13%) and precision (always <10%). The application of the validated approach to samples collected from 40 healthy dogs made possible the definition of a reliable reference interval for 25-hydroxyvitamin D3, the main biomarker of vitamin D3. In addition, variations below 5% in the results obtained quantifying the same samples using a water-based calibration curve demonstrated that a surrogate matrix may be used without affecting data accuracy. Thanks to its simplicity, the proposed technique represents a useful tool for supporting clinical routine and investigating correlations between serum concentrations of this metabolite and multiple diseases. Additionally, it could enable the monitoring of supplementation in small animal patients in veterinary clinical practice.

Validation of a single liquid chromatography-tandem mass spectrometry approach for oxytetracycline determination in bull plasma, seminal plasma and urine.

Oxytetracycline is a broad-spectrum antibiotic, which inhibits protein synthesis and is generally used for the treatment of pneumonia, shipping fever, leptospirosis and wound infections in cattle and swine. The present work proposes a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for oxytetracycline quantification in bull plasma, seminal plasma and urine, requiring limited sample treatment before analysis. Extraction with trichloroacetic acid followed by dilution of the supernatant in mobile phase proved to be effective in all three matrices, allowing to rapidly process large batches of samples. Sharp and symmetrical peak shape was obtained using a BEH C18 reversed-phase column in a chromatographic run of just 3.5 min. The mass spectrometer operated in positive electrospray ionization mode and monitored specific transitions for oxytetracycline (461.1 → 425.8) and the internal standard demeclocycline (465.0 → 447.6). The method was validated over concentration ranges suitable for field concentrations of oxytetracycline found in each matrix, showing good linearity during each day of testing (R2 always >0.99), as also confirmed by analysis of variance (ANOVA) and lack-of-fit tests. Excellent accuracy and precision were demonstrated by calculated bias always within ±15% and CV% below 10% at all quality control (QC) levels in the three matrices. Matrix effect and recovery were investigated for both analytes, which showed consistent and comparable behaviour in each matrix. To our knowledge, this is the first validated approach for mass spectrometric determination of oxytetracycline in seminal plasma and urine. The method was successfully applied to samples collected during a pharmacokinetic study in bulls, allowing to assess the oxytetracycline concentration-time profile in plasma, seminal plasma and urine.

A single LC-MS/MS validated method for tulathromycin quantification in plasma, seminal plasma, and urine to be applied in a pharmacokinetic study in bull.

Tulathromycin is a macrolide antibiotic generally used for the treatment of respiratory diseases in cattle and swine. This work proposes an improvement of a previously published LC-MS/MS method for tulathromycin determination in pig serum, here validated in three different bull matrices: plasma, seminal plasma, and urine. The approach is based on a quick protein precipitation with acetonitrile, filtration, and sample dilution before injection, allowing to rapidly process large batches of samples. Analytes separation was obtained using a BEH C18 (50 × 2.1 mm, 1.7 µm) column, maintained at 40°C with a chromatographic run of 5 min. The method was fully validated over concentration ranges suitable for field levels of tulathromycin found in each matrix (0.01-1 µg/ml for plasma, 0.05-5 µg/ml for seminal plasma, and 0.1-10 µg/ml for urine), showing good linearity during each day of testing (R2 always >0.99). Accuracy and precision were within ±15% at all QC concentrations in all the three matrices. Furthermore, the use of tulathromycine-d7 as internal standard mitigated the potential impacts of matrix effect. The validated technique was successfully applied to samples collected during a pharmacokinetic study in bulls, allowing to monitor tulathromycin concentrations over time in the three matrices. To our knowledge, this is the first validated approach for LC-MS/MS quantification of tulathromycin in seminal plasma and urine.

Effects of Turmeric Powder on Aflatoxin M1 and Aflatoxicol Excretion in Milk from Dairy Cows Exposed to Aflatoxin B1 at the EU Maximum Tolerable Levels.

Due to the climatic change, an increase in aflatoxin B1 (AFB1) maize contamination has been reported in Europe. As an alternative to mineral binders, natural phytogenic compounds are increasingly used to counteract the negative effects of AFB1 in farm animals. In cows, even low dietary AFB1 concentrations may result in the milk excretion of the genotoxic carcinogen metabolite aflatoxin M1 (AFM1). In this study, we tested the ability of dietary turmeric powder (TP), an extract from Curcuma longa (CL) rich in curcumin and curcuminoids, in reducing AFM1 mammary excretion in Holstein-Friesian cows. Both active principles are reported to inhibit AFM1 hepatic synthesis and interact with drug transporters involved in AFB1 absorption and excretion. A crossover design was applied to two groups of cows (n = 4 each) with a 4-day washout. Animals received a diet contaminated with low AFB1 levels (5 ± 1 µg/kg) for 10 days ± TP supplementation (20 g/head/day). TP treatment had no impact on milk yield, milk composition or somatic cell count. Despite a tendency toward a lower average AFM1 milk content in the last four days of the treatment (below EU limits), no statistically significant differences with the AFB1 group occurred. Since the bioavailability of TP active principles may be a major issue, further investigations with different CL preparations are warranted.

Does Bentonite Cause Cytotoxic and Whole-Transcriptomic Adverse Effects in Enterocytes When Used to Reduce Aflatoxin B1 Exposure?

Aflatoxin B1 (AFB1) is a major food safety concern, threatening the health of humans and animals. Bentonite (BEN) is an aluminosilicate clay used as a feed additive to reduce AFB1 presence in contaminated feedstuff. So far, few studies have characterized BEN toxicity and efficacy in vitro. In this study, cytotoxicity (WST-1 test), the effects on cell permeability (trans-epithelial electrical resistance and lucifer yellow dye incorporation), and transcriptional changes (RNA-seq) caused by BEN, AFB1 and their combination (AFB1 + BEN) were investigated in Caco-2 cells. Up to 0.1 mg/mL, BEN did not affect cell viability and permeability, but it reduced AFB1 cytotoxicity; however, at higher concentrations, BEN was cytotoxic. As to RNA-seq, 0.1 mg/mL BEN did not show effects on cell transcriptome, confirming that the interaction between BEN and AFB1 occurs in the medium. Data from AFB1 and AFB1 + BEN suggested AFB1 provoked most of the transcriptional changes, whereas BEN was preventive. The most interesting AFB1-targeted pathways for which BEN was effective were cell integrity, xenobiotic metabolism and transporters, basal metabolism, inflammation and immune response, p53 biological network, apoptosis and carcinogenesis. To our knowledge, this is the first study assessing the in vitro toxicity and whole-transcriptomic effects of BEN, alone or in the presence of AFB1.

Deepening the Whole Transcriptomics of Bovine Liver Cells Exposed to AFB1: A Spotlight on Toll-like Receptor 2.

Aflatoxin B1 (AFB1) is a food contaminant metabolized mostly in the liver and leading to hepatic damage. Livestock species are differently susceptible to AFB1, but the underlying mechanisms of toxicity have not yet been fully investigated, especially in ruminants. Thus, the aim of the present study was to better characterize the molecular mechanism by which AFB1 exerts hepatotoxicity in cattle. The bovine fetal hepatocyte cell line (BFH12) was exposed for 48 h to three different AFB1 concentrations (0.9 µM, 1.8 µM and 3.6 µM). Whole-transcriptomic changes were measured by RNA-seq analysis, showing significant differences in the expression of genes mainly involved in inflammatory response, oxidative stress, drug metabolism, apoptosis and cancer. As a confirmatory step, post-translational investigations on genes of interest were implemented. Cell death associated with necrosis rather than apoptosis events was noted. As far as the toxicity mechanism is concerned, a molecular pathway linking inflammatory response and oxidative stress was postulated. Toll-Like Receptor 2 (TLR2) activation, consequent to AFB1 exposure, triggers an intracellular signaling cascade involving a kinase (p38ß MAPK), which in turn allows the nuclear translocation of the activator protein-1 (AP-1) and NF-κB, finally leading to the release of pro-inflammatory cytokines. Furthermore, a p38ß MAPK negative role in cytoprotective genes regulation was postulated. Overall, our investigations improved the actual knowledge on the molecular effects of this worldwide relevant natural toxin in cattle.

Pharmacokinetics of oxytetracycline long-acting on plasma and semen of beef bulls.

The objectives of this investigation were to evaluate the pharmacokinetic parameters of oxytetracycline long-acting in plasma and seminal plasma after a single administration through either subcutaneous or intramuscular route at 10 mg/kg or 20 mg/kg dose. Four Simmental bulls, healthy and satisfactory potential breeders, were used. The route of administration either subcutaneous or intramuscular did not affect the mean values for 10 mg/kg dose in plasma (1,470 ng/mL vs. 1,330 ng/mL; P = 0.82) or seminal plasma (5,710 ng/mL vs. 5,390 ng/mL; P = 0.88), or for 20 mg/kg dose in plasma (2,540 ng/mL vs. 2,590 ng/mL; P = 0.96) or seminal plasma (25,600 ng/mL vs. 19,400 ng/mL; P = 0.58), respectively. Comparison between the 10 mg/kg and 20 mg/kg doses showed a difference in terms of mean plasma levels (1400 ng/mL vs. 2570 ng/mL; P = 0.07) and mean seminal plasma levels (6,480 ng/mL vs. 26,200 ng/mL; P = 0.004), respectively. After the dose of 10 mg/kg, plasma Cmax was 2,841 ng/mL at 12 h (Tmax) with a half-life of 20.1 h; seminal plasma Cmax was 11,515 ng/mL at 24 h (Tmax) with a half-life of 23.7 h. After the dose of 20 mg/kg, plasma Cmax was 5,269 ng/mL at 12 h (Tmax) with a half-life of 18.1 h; seminal plasma Cmax was 55,040 ng/mL at 24 h (Tmax) with a half-life of 15.7 h. Oxytetracycline long-acting may be an appropriate antibiotic, owing to its pharmacokinetic properties, that could be used for treating bulls' genital infections when its usage is indicated.

Pharmacokinetics of tulathromycin on plasma and semen of beef bulls.

The objective of this investigation was to evaluate the pharmacokinetic parameters of tulathromycin in plasma and semen of beef bulls after administering a single sc dose at two different sites in the neck. Four Simmental bulls with excellent temperament received a comprehensive physical exam that included breeding soundness examination. In addition, blood was collected and analyzed for CBC and chemical panel in order to rule out any subclinical liver or kidney disease. All bulls were diagnosed as healthy and satisfactory potential breeders. The mean plasma levels of tulathromycin for the two neck sites of sc administration were not different between posterior aspect of the ear where it attaches to the head (RP; regio parotidea; 77.9 ± 43.3 ng/mL; X ± SD) and to the middle of the neck (RC; regio collis lateralis; 73.7 ± 39.7 ng/mL; P = 0.84). The mean seminal plasma levels of tulathromycin after administration in the RP was 608 ± 374 ng/mL and for RC was 867 ± 599 ng/mL without differences between both sites (P = 0.29). The mean level of tulathromycin in plasma was 75.8 ± 40.2 ng/mL, which was lower than mean seminal plasma levels of 781 ± 482 ng/mL (P = 0.001). The plasma peak tulathromycin concentration (Cmax) was 160 ± 27 ng/mL at 21 ± 6 h (Tmax) post-administration. The seminal plasma Cmax was 1539 ± 44.4 ng/mL at 33.00 ± 18.00 h (Tmax) post-administration. The Cmax between plasma and seminal plasma were different (P = 0.008) without any differences in Tmax between plasma and seminal plasma (P = 0.35). The terminal half-life for plasma tulathromycin (81.4 ± 27.6 h) showed a tendency to be shorter than in seminal plasma (114.7 ± 21.7; P = 0.10). The plasma area under the curve concentration time from the first to the last sample (AUC0-last) was 15,440 ± 1717 ng/mL/h, which was significatively smaller compared with 171,071 ± 58,556 ng/mL/h for seminal plasma AUC0-last (P = 0.01). The plasma means residence time from the first to the last sample (MRT0-last) was 89.3 ± 5.1 h and it was shorter than for seminal plasma of 96.6 ± 5.0 h (P = 0.05). From the present investigation, it was concluded that tulathromycin is a suitable antibiotic based in its pharmacokinetic properties that could be used for treatment of bull genital infections when its application is indicated.