Sub-acute exposure of male guppies (Poecilia reticulata) to environmentally relevant concentrations of PFOA and GenX induces significant changes in the testis transcriptome and reproductive traits.

Poly- and perfluoroalkyl substances (PFAS) are frequently detected in the environment and are linked to adverse reproductive health outcomes in humans. Although legacy PFAS have been phased out due to their toxicity, alternative PFAS are increasingly used despite the fact that information on their toxic effects on reproductive traits is particularly scarce. Here, we exposed male guppies (Poecilia reticulata) for a short period (21 days) to an environmentally realistic concentration (1 ppb) of PFOA, a legacy PFAS, and its replacement compound, GenX, to assess their impact on reproductive traits and gene expression. Exposure to PFAS did not impair survival but instead caused sublethal effects. Overall, PFAS exposure caused changes in male sexual behaviour and had detrimental effects on sperm motility. Sublethal variations were also seen at the transcriptional level, with the modulation of genes involved in immune regulation, spermatogenesis, and oxidative stress. We also observed bioaccumulation of PFAS, which was higher for PFOA than for GenX. Our results offer a comprehensive comparison of these two PFAS and shed light on the toxicity of a newly emerging alternative to legacy PFAS. It is therefore evident that even at low concentrations and with short exposure, PFAS can have subtle yet significant effects on behaviour, fertility, and immunity. These findings underscore the potential ramifications of pollution under natural conditions and their impact on fish populations.

Survey on antimicrobial prescribing patterns in small animal veterinary practice in Emilia Romagna, Italy.

This investigation provides for the first time a general view of the prescribing patterns of antimicrobials in small animal practice in Emilia Romagna, Italy. In the context of a project on antimicrobial resistance managed by the Regional Veterinary Service, veterinary clinicians were invited to voluntarily complete an online questionnaire. This was designed to gather information on antimicrobial prescribing practices and biosecurity measures and to understand the perception of the issue specific to this region of Italy. In total, 266 questionnaires correctly completed were collected. Although clinicians seemed to follow different approaches when using antimicrobials, the data analysis revealed a general awareness on resistance. Penicillins were the most commonly prescribed class, followed by (fluoro)quinolones and cephalosporins. Among those who use laboratory testing more or less frequently (microbiological analysis and susceptibility testing) to support their prescribing habits, only 7 per cent make a habit of always waiting for the results before starting the treatment. Seventy-eight per cent of the respondents declared the use of antimicrobials licensed for human beings. Biosecurity measures were carefully taken into account by the majority of the veterinarians. The results identified the antimicrobial classes that are commonly prescribed and highlighted that perioperative hygiene measures and the use of laboratory diagnosis are critical aspects that need to be emphasised in drawing up guidelines on the prudent use of these drugs in pets.

A quick LC-MS-MS method for the determination of flunixin in bovine muscle.

A simple, fast and cost-effective liquid chromatographic/tandem mass spectrometry (LC-MS-MS) method for the quantitative determination of flunixin (FLU) in bovine muscle was developed and validated. The sample preparation procedure involved an extraction with acetonitrile, followed by evaporation and reconstitution. Chromatographic separation was achieved on a reverse-phase column under programmed conditions. FLU detection was performed with positive electrospray ionization in selected reaction monitoringmode, monitoring one precursor and two products ions. For quantification purposes, FLU-d3 was used as an internal standard. The matrix effect on the analysis of FLU in bovine muscle was evaluated by comparison between calibration curves prepared with standard solution and in blank matrix extracts. The equivalent responses obtained confirmed the absence of signal suppression or/and enhancement. The method was extensively validated according to the parameters requested by European Commission Decision 2002/657/EC in terms of specificity, limit of detection, linearity, trueness, precision, decision limit (CCα) and detection capability (CCß). FLU stability was also investigated in matrix and in sample extracts at different times and storage conditions.

Technical note: development and validation of a method using ultra performance liquid chromatography coupled with tandem mass spectrometry for determination of vitamin B12 concentrations in milk and dairy products.

A method using ultra performance liquid chromatography coupled with tandem mass spectrometry was developed to measure cobalamins in naturally enriched raw milk and to evaluate their fate during thermal treatments and along the process of cheese making. After addition of methotrexate as internal standard, samples were submitted to heat treatment in the presence of cyanide, which converts all the less-stable cobalamins into cyanocobalamin; then, purification was performed by a solid-phase extraction step. Reverse-phase ultra performance liquid chromatography separation coupled with tandem mass spectrometry provided a fast and reliable determination. Mass spectrometric analysis was carried out in multiple reaction monitoring mode. The monitored transitions were m/z 678.36 → 147.10 and 678.36 → 359.30 for vitamin B12 and m/z 455.22 → 175.13 and 455.22 → 308.22 for methotrexate (internal standard). The limit of quantification was 2 ng/g. The method showed good linearity from 2 to 20 ng/g (R(2) ≥ 0.98) and intra- and interday precisions were always less than 19%.

Development and validation of a liquid chromatography/tandem mass spectrometry method for quantitative determination of amoxicillin in bovine muscle.

A simple, quick and economical liquid chromatographic/tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of amoxicillin in bovine muscle was developed and validated. The sample preparation procedure involved a liquid extraction with water, followed by a protein precipitation step with acetonitrile. The extract was purified by a liquid-liquid partition with dichloromethane and the upper aqueous layer was directly injected into the LC-MS/MS system. Chromatographic separation was achieved on a reversed phase column, using a mixture of acetonitrile, water and 0.005% formic acid in water as mobile phase. Gradient elution was performed at a flow rate of 0.2 mL min⁻¹. Amoxicillin was detected using positive electrospray ionization in selected reaction monitoring (SRM) mode and was quantified using terbutaline as internal standard. The responses for standards prepared in solvent and in matrix were equivalent and additionally the absence of signal suppression was confirmed by the post column infusion technique. Amoxicillin stability in standard solution and in matrix was investigated at different times and storage conditions. Amoxicillin standards prepared in water were stable on storage up to 20 days at -20°C. Amoxicillin stability in matrix (spiked bovine muscle samples) was assessed up to 15 days at -20°C. The method was validated according to the parameters requested by European Commission Decision 2002/657/EC in terms of specificity, linearity, trueness, precision, decision limit (CCα) and detection capability (CCß). All the trueness values fell within a range between 14.5% and 6.3%. Precision values for all levels of concentration tested were lower than the relative limit calculated by the Horwitz equation. The amoxicillin MRL is set at 50 µg kg⁻¹ and the CCα and CCß of the method were 61.2 µg kg⁻¹ and 72.4 µg kg⁻¹, respectively.

[Pulmonary physiopathology in scleroderma: study of respiratory function in 86 patients]. / La fisiopatologia polmonare nella sclerodermia: esame della funzionalità respiratoria in 86 pazienti.

OBJECTIVE: To contribute to an early diagnosis of pulmonary involvement in scleroderma by evaluating the correlation between respiratory symptoms and functional respiratory data observed. PATIENTS AND METHODS: 86 patients affected by scleroderma, 76 women and 10 men, age 14-75, underwent lung function tests, blood gas sample, CO diffusing capacity in setting and supine position, respiratory drive measurement through P0.1 and evaluation of the respiratory muscles efficiency with Maximum Inspiratory Pressure (MIP). RESULTS: Data obtained suggested us to divide our patients in four different groups: first group where both spirometric data and pulmonary diffusion were normal; a second group with a clear reduction of pulmonary diffusion likely due to the reduction of vascular bed; a third group where we observed a restrictive ventilatory impairment due to the reduction of the compliance and a reduction of the pulmonary diffusion likely related to interstitial damage; finally, a fourth group where beside a restricted spirometric outline we have detected a more accentuated reduction of pulmonary diffusion likely due to pulmonary hypertension. Moreover, our study has highlighted a progressive decrease of MIP and Maximum Voluntary Ventilation (MVV) shifting from the first to the fourth group, suggesting reduction of the muscular efficiency with an increase of P0.1 index of activity in the respiratory drive. CONCLUSIONS: The results could explain the dyspnea often reported by the patients affected by scleroderma even without spirometric alteration.

[Possible effects of non invasive mechanical ventilation on respiratory drive and muscles]. / Possibili effetti della ventilazione meccanica non invasiva su drive e muscolatura respiratoria.

OBJECTIVE: To evaluate whether long-term Non-Invasive Mechanical Ventilation (NIMV) might have an effect on respiratory drive and respiratory muscles strength, measuring mouth occlusion pressure (P0,) and maximal inspiratory pressure (MIP). PATIENTS AND METHODS: 20 consecutive patients with hypercapnic respiratory failure underwent measurements of dyspnea, respiratory drive and respiratory muscles strength before hospital treatment with NIMV; those patients who showed significant improvement of gas-exchange continued home ventilation for a period of four weeks, and were readmitted to hospital for re-evaluation of Borg's scale for dyspnea, P0,1 and MIP. RESULTS: Data obtained show a mild reduction of P0,1 and a significant improvement of respiratory muscles strength, with satisfactory dyspnea relief. CONCLUSIONS: We conclude that unloading respiratory muscles through mechanical ventilation results in better muscle performance in the long-term that could act, together with normalization of gas-exchange, on neuromuscular respiratory drive and contribute to dyspnea relief.