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Pigs are becoming an important pre-clinical animal species for translational ophthalmology, due to similarities with humans in anatomical and physiological patterns. Different models of eye disorders have been proposed, and they are good candidates to assess biocompatibility/functionality of retinal prostheses. Electroretinography is a common tool allowing to gain information on retinal function, with several types of electroretinogram (ERG) been implemented including full field (ff-ERG), multifocal (mf-ERG) and pattern (p-ERG). p-ERG represents a valuable tool to monitor Retinal Ganglion Cells (RGCs) activity and can be used to calculate p-ERG spatial acuity. Unfortunately, scarce methodological data are available regarding recording/interpretation of p-ERG and retinal acuity in biomedical pigs yet enhancing knowledge regarding pig vision physiology will allow for more refined and responsible use of such species. Aim of this study was to record p-ERG in juvenile pigs to functionally assess visual acuity. Six female hybrid pigs underwent two p-ERG recording sessions at 16 and 19 weeks of age. Photopic ff-ERG were also recorded; optical coherence tomography (OCT) and histology were used to confirm retinal integrity. ff-ERG signals were repeatable within/across sessions. All p-ERG traces consistently displayed characterizing peaks, and the progressive decrease of amplitude in response to the increment of spatial frequency revealed the reliability of the method. Mean p-ERG spatial acuities were 5.7 ± 0.14 (16 weeks) and 6.2 ± 0.15 cpd (19 weeks). Overall, the p-ERG recordings described in the present work seem reliable and repeatable, and may represent an important tool when it comes to vision assessment in pigs.

Background The use of adrenocorticotropic hormone stimulation test as method to monitor efficacy of trilostane treatment of hypercortisolism (HC) in dogs has been questioned. Objectives To evaluate and compare 12 methods with which to monitor efficacy of trilostane treatment in dogs with HC. Animals Forty‐five client‐owned dogs with HC treated with trilostane q12h. Methods Prospective cross‐sectional observational study. The dogs were categorized as well‐controlled, undercontrolled, and unwell through a clinical score obtained from an owner questionnaire. The ability to correctly identify trilostane‐treatment control of dogs with HC with the following variables was evaluated: before trilostane serum cortisol (prepill), before‐ACTH serum cortisol, post‐ACTH serum cortisol, plasma endogenous ACTH concentrations, prepill/eACTH ratio, serum haptoglobin (Hp) concentration, serum alanine aminotransferase (ALT), gamma‐glutamyl transferase (γGT) and alkaline phosphatase activity, urine specific gravity, and urinary cortisol : creatinine ratio. Results Ninety‐four re‐evaluations of 44 dogs were included; 5 re‐evaluations of 5 unwell dogs were excluded. Haptoglobin was significantly associated with the clinical score (P < .001) and in the receiver operating characteristic analysis, Hp cutoff of 151 mg/dL correctly identified 90.0% of well‐controlled dogs (specificity) and 65.6% of undercontrolled dogs (sensitivity). Alanine aminotransferase (P = .01) and γGT (P = .009) were significantly higher in undercontrolled dogs. Cutoff of ALT and γGT greater than or equal to 86 U/L and 5.8 U/L, respectively, were significantly associated with poor control of HC by trilostane. Conclusions and Clinical Importance Of all the 12 variables, Hp, and to a lesser degree ALT and γGT, could be considered additional tools to the clinical picture to identify well‐controlled and undercontrolled trilostane‐treated dogs.

The present review aims to summarize the main features of mammary gland anatomy, and the physiology of lactation and colostrum/milk in the most commonly used animal species for regulatory toxicity. The final goal is the selection of a preferred animal species to be enrolled in studies investigating the potential transfer of drugs and exogenous molecules through milk, within the Innovative Medicines Initiative (IMI) funded project ConcePTION. Reference data regarding humans were also collected and analyzed in order to highlight critical similarities and differences with the studied species. Additional practical considerations were also taken into account, such as ethical consideration regarding the chosen species which affects the group size, financial implications and technical feasibility of lactation trials (e.g., ease of sampling, volume of sampling, husbandry requirements and scientific recognition). In conclusion, the present analysis of the literature confirms the complexity of the decisional process behind the choice of an animal model for in vivo trials. For some of the evaluated species, data were either poor or missing, highlighting the necessity to generate more physiological background studies for species that are routinely used in laboratory settings. Overall, when taking into consideration ethical factors, feasible group size, milk volume and ease of milk collection, and physiological similarities with humans, minipigs seem to represent the most appropriate choice.

The red deer (Cervus elaphus L., 1758) is one of the largest deer species in the world. Females are seasonal polyestrous, with negative photoperiod: the increase of the night peak of melatonin determines the secretion of GnRH and, therefore, LH and FSH. To date there is little information regarding the hormonal control during pregnancy for this species; this could be due to the difficulty of sampling wild subjects, while farmed animals’ hormonal concentrations may not reflect the physiology of the animal in a natural state. In this study we evaluated the concentration of cortisol and progesterone, extracted from blood and hair, on 10 wild and pregnant red deer females. Belonging to the population of the Bolognese Apennines (Italy), the hinds were sampled in the January–March 2018 period, according to the regional selective hunting plan. Plasma progesterone (P4) ranged from a minimum of 1.9 to a maximum of 7.48 ng/mL; while hair P4 concentrations varied from 41.68 to 153.57 pg/mg. The plasma and hair cortisol ranges are respectively 0.4–2.97 ng/mL and 0.03–0.55 pg/mg; the only significant correlation was found between hair concentration of P4 and the date of death. The results of this preliminary study represent a small step towards a better knowledge of this species’ physiology during pregnancy.

Steroids, providing information regarding several biological patterns including stress and sexual behavior, have been investigated in different matrices in laboratory mice. Data regarding hair quantification, indicative of longer timespans when compared to blood and saliva, are lacking. The aim of the work was to analyze the hormonal hair profile of laboratory male mice and to investigate potential relationships with age and housing, as a potential tool for welfare assessment. Fifty-six adult male C57BL/6J and C57BL/6OlaHsd substrain mice were included in the study, housed in pairs or groups. Testosterone (T) and dehydroepiandrosterone (DHEA) were quantified by radioimmunoassay, corticosterone (CORT) by ELISA. Mean hormone levels were 6.42 pg/mg for T, 23.16 pg/mg for DHEA and 502.1 pg/mg for CORT. Age influenced all hormones by significantly increasing T and DHEA levels and decreasing CORT; only DHEA, significantly higher in grouped mice, was influenced by housing conditions. The influence of age indicates the need for accurate age-related reference intervals, while the higher levels of DHEA in grouped animals suggests that such housing practice may be beneficial for social interactions. In conclusion, it seems that hair hormones quantification may be a good tool for welfare assessment in laboratory mice and may help in refining husbandry.

The first weeks of life represent a crucial stage for microbial colonization of the piglets’ gastrointestinal tract. Newborns’ microbiota is unstable and easily subject to changes under stimuli or insults. Nonetheless, the administration of antibiotics to the sow is still considered as common practice in intensive farming for pathological conditions in the postpartum. Therefore, transfer of antibiotic residues through milk may occurs, affecting the piglets’ colon microbiota. In this study, we aimed to extend the knowledge on antibiotic transfer through milk, employing an in vitro dedicated piglet colon model (MICODE—Multi Unit In vitro Colon Model). The authors’ focus was set on the shifts of the piglets’ microbiota composition microbiomics (16S r-DNA MiSeq and qPCR—quantitative polymerase chain reaction) and on the production of microbial metabolites (SPME GC/MS—solid phase micro-extraction gas chromatography/mass spectrometry) in response to milk with different concentrations of amoxicillin. The results showed an effective influence of amoxicillin in piglets’ microbiota and metabolites production; however, without altering the overall biodiversity. The scenario is that of a limitation of pathogens and opportunistic taxa, e.g., Staphylococcaceae and Enterobacteriaceae , but also a limitation of commensal dominant Lactobacillaceae , a reduction in commensal Ruminococcaceae and a depletion in beneficial Bifidobactericeae . Lastly, an incremental growth of resistant species, such as Enterococcaceae or Clostridiaceae , was observed. To the authors’ knowledge, this study is the first evaluating the impact of antibiotic residues towards the piglets’ colon microbiota in an in vitro model, opening the way to include such approach in a pipeline of experiments where a reduced number of animals for testing is employed. Key points • Piglet colon model to study antibiotic transfer through milk. • MICODE resulted a robust and versatile in vitro gut model. • Towards the “3Rs” Principles to replace, reduce and refine the use of animals used for scientific purposes (Directive 2010/63/UE). Graphical abstract

To fully harness mesenchymal-stromal-cells (MSCs)’ benefits during Normothermic Machine Perfusion (NMP), we developed an advanced NMP platform coupled with a MSC-bioreactor and investigated its bio-molecular effects and clinical feasibility using rat and porcine models. The study involved three work packages: 1) Development ( n  = 5): MSC-bioreactors were subjected to 4 h-liverless perfusion; 2) Rat model ( n  = 10): livers were perfused for 4 h on the MSC-bioreactor-circuit or with the standard platform; 3) Porcine model ( n  = 6): livers were perfused using a clinical device integrated with a MSC-bioreactor or in its standard setup. MSCs showed intact stem-core properties after liverless-NMP. Liver NMP induced specific, liver-tailored, changes in MSCs’ secretome. Rat livers exposed to bioreactor-based perfusion produced more bile, released less damage and pro-inflammatory biomarkers, and showed improved mithocondrial function than those subjected to standard NMP. MSC-bioreactor integration into a clinical device resulted in no machine failure and perfusion-related injury. This proof-of-concept study presents a novel MSC-based liver NMP platform that could reduce the deleterious effects of ischemia/reperfusion before transplantation. MSCs-based therapies have broad application potential in reducing ischemia/reperfusion injury, but their use for treating isolated organs before transplantation remains limited. Here, the authors present a new technology, which enables to better exploit the benefits of cell-based therapies during NMP.

Antimicrobial resistance, an ever-growing global crisis, is strongly linked to the swine production industry. In previous studies, Melaleuca alternifolia and Rosmarinus officinalis essential oils have been evaluated for toxicity on porcine spermatozoa and for antimicrobial capabilities in artificial insemination doses, with the future perspective of their use as antibiotic alternatives. The aim of the present research was to develop and validate in vitro and ex vivo models of porcine uterine mucosa for the evaluation of mucosal toxicity of essential oils. The in vitro model assessed the toxicity of a wider range of concentrations of both essential oils (from 0.2 to 500 mg/mL) on sections of uterine tissue, while the ex vivo model was achieved by filling the uterine horns. The damage induced by the oils was assessed by Evans Blue (EB) permeability assay and histologically. The expression of ZO-1, a protein involved in the composition of tight junctions, was assessed through immunohistochemical and immunofluorescence analysis. The results showed that low concentrations (0.2–0.4 mg/mL) of both essential oils, already identified as non-spermicidal but still antimicrobial, did not alter the structure and permeability of the swine uterine mucosa. Overall, these findings strengthen the hypothesis of a safe use of essential oils in inseminating doses of boar to replace antibiotics.