IntroductionCardiac implantable electronic devices manage arrhythmias but are limited by mechanical failures, infection risks, and poor long-term biocompatibility. Developing a biological alternative that restores intrinsic pacemaking remains a key clinical challenge.MethodsWe developed cardiac scaffolds from porcine atrioventricular nodes using an optimized Tergitol-based decellularization protocol. Morphological, ultrastructural, proteomic, and mechanical analyses were conducted to assess ECM integrity and preservation of native architecture.ResultsThe decellularization process effectively removed cellular and nuclear components while preserving three-dimensional structure, collagen content, and overall ECM organization. Analyses confirmed that key features essential for pacemaker tissue support were maintained.DiscussionOur findings demonstrate that the scaffold retains native characteristics suitable for biologically inspired pacemaker applications. This work provides a foundation for ECM-derived hydrogel development, cytocompatibility testing, and integration with cardiomyocytes in next-generation tissue-engineered cardiac scaffolds.

Tracheal replacement is an unmet clinical need, as patients with long or complex airway defects are managed with tracheostomy or permanent stents. Experimental and clinical research is ongoing in order to find safe airway substitutes; however, the strategies under investigation suffer from major limitations, such as unsatisfactory re-epithelialization, insufficient long-term mechanical support, and complex ex vivo procedures. A ready-to-use and stable patch, able to support airway functionality and tissue regeneration, remains a significant challenge. Here we present the development of an off-the-shelf composite patch consisting of a resorbable polymer to aid epithelial restoration and a 3D-printed multimaterial structure to guarantee effective mechanical stability. To evaluate the prosthesis performance, we designed a pilot study on a large animal setting, monitoring postoperative survival and airway healing for up to 60 days. An anterior cervical tracheal defect was created on four domestic pigs and patched with the prosthesis. The results were satisfactory in terms of postoperative survival, as only one animal died before the end of the study. However, endoscopic findings revealed a worsening stenosis due to wound contraction, granulation tissue formation, and partial displacement of the prosthesis. These findings were confirmed at histology, where a prominent inflammatory infiltrate was evident. Blood tests performed during follow-up did not reveal any systemic inflammatory reaction. Overall, we believe that further optimization of the prosthesis design and materials is necessary in order to create an ideal "off-the-shelf" tracheal substitute. Nevertheless, this pilot study provides promising results and novel insights into a clinically relevant research area.

Background and Aim:Propranolol is a widely used non-selective beta-adrenergic blocker in human medicine, with well-characterized pharmacokinetics (PK) in humans but virtually no data available for pigs, a species of growing biomedical relevance. Furthermore, no validated bioanalytical methods exist for propranolol or its primary metabolite, 4-hydroxy-propranolol, in porcine matrices. This study aimed to develop and validate a rapid, sensitive, and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of propranolol and 4-hydroxypropranolol in pig plasma and dried blood spots (DBS), and to apply it in a preliminary PK investigation in pigs. Materials and Methods:Sample preparation involved simple protein precipitation (plasma) or solvent extraction (DBS) using acetonitrile-water mixtures, followed by chromatographic separation on a Bridged ethyl hybrid C18 column (50 × 2.1 mm, 1.7 μm; 4-min run). Detection was performed in Multiple reaction monitoring mode with propranolol-d7 as the internal standard. Validation followed EMA ICH M10 guidelines, assessing linearity, accuracy, precision, matrix effects, recovery, and stability. The method was then applied to plasma samples from five juvenile female pigs receiving oral propranolol (3 mg/kg, q8 h). Results:The method demonstrated excellent linearity (r2 > 0.99) and acceptable accuracy and precision (±15%) across 2-500 ng/mL (propranolol) and 1-400 ng/mL (4-hydroxypropranolol). Recoveries ranged from 83% to 116% (plasma) and 81%-113% (DBS), with no matrix interference or carry-over. In vivo PK data revealed rapid absorption (Tmax 1.14 ± 0.63 h), moderate elimination (t½ 2.19 ± 0.86 h), and a mean Cmax of 112.02 ± 81.87 ng/mL. Notably, 4-hydroxypropranolol was undetectable in all plasma samples, suggesting species-specific metabolic differences. Conclusion:This study reports the first validated LC-MS/MS assay for propranolol and 4-hydroxypropranolol in pigs and demonstrates its successful application in a PK study. The method's simplicity, short runtime, and compatibility with DBS microsampling make it ideal for preclinical and veterinary research, minimizing animal stress and sampling volume. Absence of 4-hydroxypropranolol highlights interspecies metabolic variability and warrants further investigation into propranolol biotransformation pathways in swine and other translational models.

Animals with seasonal reproductive cycles, as the Roe deer (Capreolus capreolus), have developed mechanisms to synchronize reproduction with the environmental cycle in order to optimize reproductive success through melatonin. Angiogenesis and oxidative stress are key processes in spermatogenesis, contributing to testicular remodeling and recovery after reproductive effort. This study carried out a gene expression analysis on 18 samples of mature male Roe deer testicles, collected during the local hunting season in pre-rut (N = 9) and post-rut (N = 9) periods. A quantitative real-time PCR (qPCR) array targeting 84 genes involved in oxidative stress and 84 in angiogenesis were used, followed by validation through individual qPCR of selected genes and related protein quantification by ELISA assays. Post-rut animals showed upregulation of several antioxidant genes: Peroxiredoxin-4 (PRDX4), Scavenger receptors class A member 3 (SCARA3), Superoxide Dismutase 3 (SOD3). Instead, Leptin (LEP) and Thrombospondin Ⅱ (THBSⅡ), a known angiogenesis inhibitor, are downregulated. A novel insight is represented by the upregulation of Neuropilin (NRP2) in post-rut period that, given to its posttranscriptional silencing too, needs better investigations. The pleiotropic nature of NRP2, including roles in neurodevelopment, immune modulation, and vascular remodeling, makes this gene an interesting candidate for further study, cause its function in reproductive tissues remains poorly understood.

Teleost embryos utilise conserved zinc-binding astacin proteinases for the chemical modification of the glycoproteins composing the zona radiata. European eels, an evolutionary basal catadromous species, have multiple isozymes with similar catalytic functions for this task. Researchers are focusing on developing effective rearing practices for this critically endangered species as assisted reproduction efforts are currently obtaining variable embryonal hatching and survival. Thus, the objective was to investigate whether the variant hatching rate could be due to lacking high choriolytic enzyme 1-like, eel hatching enzyme expression and/or it's collagenase-like activity, while examining also the regulatory intercellular Zn2+ concentrations in developing embryos after fertilisation and pre-hatching. Six assisted reproduction was conducted for the collection of buoyant (vital) and sunk (non-vital) fertilised embryo pools two hours post-spawning (t0) and pre-hatching (t55). Real-time PCR was used together with Collagen degradation fluorometric assay, and Inductively Coupled Plasma-Optical Emission Spectrometry to investigate the intracellular Zn2+ concentrations. Transcripts of the eel hatching enzyme were detected in all sample pools with significant difference between vital and non-vital embryos even 2 h post-fertilisation, suggesting maternal derivation, or the onset of hatching enzyme expression before the known maternal-to-zygotic transition. Relative mRNA quantity depended on all factors (time, state, interaction) demonstrating an overall increase of expression. The collagenase-like activity was only time-dependent, and only non-vital samples contained higher intracellular Zn2+ concentrations than 0.6 mg kg(-1), suggesting a similar threshold as for other species. Correlation analysis revealed reverse correlations between Zn2+ concentration and the weight of the reproductive female at ovulation.

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.

The inside-out submitochondrial particles (IO-SMPs) showed a strong protective effect against mitochondrial permeability transition pore (mPTP) opening in mitochondria isolated from swine hearts 3 h after explantation. The latter condition was used to emulate situation of mitochondrial damage. We identified that the protective effect of IO-SMPs cannot be attributed to a functional modulation of the enzymatic complexes involved in mPTP formation. Indeed, oxidative phosphorylation and F1FO-ATPase activity were not affected. Conversely, mPTP desensitization might be caused by structural modification. IO-SMP incorporation into the mitochondria can modulate the membrane-bound enzyme complexes' functionality, inducing F1FO-ATPase to be unable to carry out the conformational changes useful for mPTP opening. Thus, the data are a valid starting point for IO-SMP application in the treatment of impaired cardiovascular conditions supported by mPTP opening.

Evaluating milt samples before their preservation and/or reproductive use is critical to correctly develop assisted reproductive strategies for teleost species. This is especially true in case of “critically endangered” species, like the European eel (Anguilla anguilla), whose life cycle is not closed yet, leading to the necessity of capturing younglings from natural populations for both research and productive purposes. Despite androgens being involved in spermatogenesis and playing pivotal roles in the pre-migratory morphological and physiological changes, their quantification in different biological tissues is still to be refined in European eels. The aim of this study was to evaluate the feasibility of extraction and quantification of testosterone (T) and 11-ketotestosterone (11-KT) from male European eel milts. The secondary aim was to investigate potential differences in androgens’ profiles and/or functional characteristics of spermatozoa between wild-caught and farmed specimens. Twenty-four male European eels obtained from the North Adriatic region (14 wild-caught and 10 farmed) underwent a maturation-inducing hormonal treatment for milt production; blood and milt samples were then collected for androgens quantification upon ELISA and RIA methodologies. Spermatozoa were also evaluated for viability and objective motility. Plasma showed average T and 11-KT levels of 2.32 and 6.88 ng/mL, respectively, while milt 0.24 (T) and 1.09 ng/mL (11-KT). Significantly higher spermatozoa concentrations and motility were observed in farmed eels’ milts. This work contributes to deepening the knowledge on teleost physiology, being the first report quantifying T and 11-KT from European eels’ seminal plasma and comparing the quality of milts of farmed vs wild specimens.

After decades of dramatic reductions in their populations, Italian wolves have begun recolonizing parts of their historic range. This growth in populations can lead to potential conflicts with human activities, which remain the main cause of wolf mortality. With the aim of examining animal welfare and their health status, a hormonal dosage on hair was conducted on 20 wolf carcasses originating from the Tuscany and Emilia-Romagna regions. The concentrations of cortisol, testosterone, dehydroepiandrosterone (DHEA), progesterone, and oestradiol (E2) were measured using radioimmunoassay (RIA). Our results, listed as mean +/- SD, were cortisol 1.81 +/- 1.17, testosterone 2.74 +/- 1.6, DHEA 62.92 +/- 24.99, P4 53.22 +/- 32.8 and E2 0.46 +/- 0.23 pg/mg. The study employed a novel approach to analyse hormone levels in this species, but its opportunistic nature resulted in uneven data distribution across sex and age groups. Consequently, comparisons of hormone levels by these categories did not yield relevant differences. Further studies with larger sample sizes are necessary to analyse hormone trends throughout the year and explore differences based on factors such as sex, age, and causes of death.

Soy contributes to the environmental impact of Italian pork production. This study aimed to evaluate the effect of a reduced soybean meal (SBM) and crude protein (CP) finishing diet on growth performance, health, behaviour and gut health of Italian heavy pigs. 1920 pigs (35.6 kg body weight; (BW)) balanced by sex and BW were assigned to the control diet (CO), or the treated diet (TRT) formulated by reducing SBM by 31%, 67% and 69% (replaced by pea and sunflower meal) in 3 feeding phases, respectively, and 1.2% CP in the third phase. 251 pigs were individually weighed at d11, d94 and d181. Feed intake (FI), behavioural indices and air gases at pen-level were monitored monthly. Faecal samples (20/pigs/group) for microbiota, ammonia and volatile fatty acids (VFAs) and hair for stress biomarkers were collected. Diet did not affect final BW, faecal ammonia, cortisol and dehydroepiandrosterone. From d11-d94, the CO group had higher gain to feed (G:F) (p = .007), favourable faecal VFAs profile and a lower environmental ammonia (p < .0001). From d10-d181, the TRT diet increased the ADG (p = .04) and G:F (p = .01), reduced FI (p < .0001) and the lesions score index at d102 (p = .03) and promoted Methanobrevibacter (d94; padj. = 0.013) and Clostridium sensu stricto (d181; padj. = 0.001). Overall, the TRT diet combined with the stress of the transport and acclimatisation to the farm may limit the growth of pigs in the initial period, but it can increase their growth in the long term. Concluding, replacing 56% of SBM with sustainable alternatives seems promising for heavy pig.

Healthcare-associated infections (HAIs) pose significant risks, leading to increased morbidity, mortality, and costs, exacerbated by multi-drug-resistant microorganisms. This study aimed to evaluate pharmacological prophylaxis targeting sympathetic reflex control of immunity to mitigate systemic infections, offering a novel approach to combating HAIs. The study included animal experiments and a retrospective analysis of orthopedic surgery patients in Romagna, Italy. Young female pigs were intravenously inoculated with Escherichia coli (E. coli) and divided into two groups: propranolol-treated (non-selective β-blocker; 3mg/kg; 3x/day orally) and vehicle-treated, starting two days before infection. Parameters such as bacteraemia, serum cytokines, biochemical profile, blood count, lactate, glycemia, and flow cytometry were assessed. Additionally, a retrospective analysis of 92,649 orthopedic surgery hospitalizations (2017–2022) examined the effects of non-selective and selective β1-blockers on HAIs development using conditional logistic regression. Propranolol-treated pigs exhibited a disinhibited immune response to systemic infection, clearing circulating bacteria much earlier than vehicle-treated animals. The retrospective analysis showed that patients on non-selective beta-blockers had a 71.7% reduced risk of developing HAIs, while those on selective β1-blockers had an 18% higher risk. These findings suggest that targeting sympathetic reflex control of immunity via pharmacological prophylaxis may reduce HAIs in surgical patients.

The application of essential oils as potential alternatives to antibiotics in swine semen storage is promising, due to their antioxidant and antibacterial properties. However, detrimental effects on spermatozoa should be clarified first. The aim of this study was to evaluate 9 essential oils (EOs; Satureja montana, Pelargonium graveolens, Cymbopogon nardus, Melaleuca leucadendron, Eucaliptus globulus, Citrus limon, Lavandula angustifolia, Lavandula hybrida, Mentha piperita) and a blend (GL mix) on key morpho-functional parameters of swine spermatozoa. Test compounds were firstly chemo-characterized and experimental doses were prepared by suspending a fixed number of spermatozoa with 3 different concentrations (0.1, 0.5, 1 mg/mL) of EOs. Experimental doses were stored at 16 °C and sampled after 3 and 120 h for analysis. Overall, S. montana, P. graveolens and L. angustifolia EOs induced the strongest alterations, with C. nardus and E. globulus EOs being the best tolerated. Swine spermatozoa represent a good preliminary testing platform to screen toxicity and its different patterns. The comprehensive overview on the potential mechanisms of action of some of the most common EOs, despite of the direct aim of the study being swine reproduction, may be exploited in other fields of research within both veterinary and human medicine.

Introduction Insufficient supply of cardiac grafts represents a severe obstacle in heart transplantation. Donation after Circulatory Death (DCD), in addition to conventional donation after brain death, is one promising option to overcome the organ shortage. However, DCD organs undergo an inevitable more extended period of warm unprotected ischemia between circulatory arrest and graft procurement. Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have shown remarkable protective effects against ischemia-reperfusion injury. Thus, we aimed to enhance grafts preservation from DCD donors, through treatment with MSC-EVs. Methods Female pigs were euthanized by barbiturate overdose and after 20 min of a flat EKG, the chest was opened, the heart harvested and subsequently connected to an extracorporeal perfusion machine. MSC-EVs, isolated by ion exchange chromatography, were added to the perfusion solution (1×1011 particles) and the heart was perfused for 2 h. Then, heart tissue biopsies were taken to assess histological changes, mitochondrial morphology, antioxidant enzyme activity and inflammation mediators’ expression. Biochemical parameters of myocardial viability were assessed in the perfusate. Results The treatment with MSC-EVs significantly prevented mitochondria swelling, mitochondrial cristae loss and oxidative stress in cardiac tissue. The protective effect of MSC-EVs was confirmed by the delayed increase of the cardiac-specific enzymes CK and TnC in the perfusate and the reduction of caspase-3+ cells in tissue sections. Conclusion MSC-EVs improve graft quality by preserving the mitochondrial ultrastructure protecting the myocardium against oxidative stress, reducing apoptosis of cardiac cells and preventing the increase of pro-inflammatory cytokines.

The use of phytoextracts has been proposed as a method to improve animal welfare, also in pigs, by reducing stress and anxiety and improving performances. Lavandula angustifolia (Miller) essential oil (LaEO) is an interesting calming phytoextract that could be administered by inhalation for prolonged periods of time to help pigs coping with on-farm conditions. The aim of this study was to assess the effects of daily inhalation of vaporized LaEO on pigs' welfare and health indicators, and nasal microbiota, trying to understand whether this phytoextract represents a feasible tool to improve animal welfare under intensive farming conditions. Eighty-four crossbred barrows were randomly divided into 3 experimental groups: control (C); lavender (L): 3 vaporization sessions of 10 min each of a custom made 1% solution of LaEO; sham (S): same vaporization sessions of L group but only using the solution vehicle. Experimental readouts included growth parameters, behavioural traits, tail and skin lesions, hair steroids and nasal microbiota. L group animals did not show altered growth performance and seemed calmer (increased recumbency time), with decreased amount of skin lesions also associated with lower severity class for tail lesions. They also showed decreased CORT/DHEA ratio, potentially suggesting a beneficial effect of LaEO. Inhalation of LaEO significantly affected the nasal pig microbiome by reducing its diversity. Overall, the study suggests how inhalation of Lavender essential oil may be capable of improving welfare in growing pigs, yet it is pivotal to consider the microbial modulatory capabilities of essential oils before exploiting them on larger scale.

Background: Early gut microbiota (GM) dysbiosis can affect a child’s health and has been linked to the onset of pathologies later in life. Breast milk is recognized as a major driver of the structure and dynamics of an infant’s GM. In addition to nutritious and prebiotic compounds, milk contains a microbiota that is shaped by several maternal factors, including gut microorganisms and medications. However, the impact of the latter on the milk microbiota is still largely unknown. Here, we investigated the effects of amoxicillin on the milk microbiota and GM of lactating Göttingen Minipigs sows, a promising model for studying medication transfer during lactation. Methods: Three sows were given amoxicillin (7 mg/kg/day) for three weeks starting from the second week after farrowing. Fecal and milk samples were collected before and after treatment and profiled by 16S rRNA amplicon sequencing. Results: Göttingen Minipigs’ milk microbiota showed similarities to that of humans and conventional sows, with minor compositional shifts after treatment. At the genus level, we observed a decrease in Staphylococcus and o_Bacteroidales;Other;Other, and an increasing trend in the abundance of Streptococcus, Stenotrophomonas, f_Rhodobacteraceae;Other, Proteiniclasticum, f_Propionibacteriaceae;Other and Gemella. In contrast, as expected, the GM was strongly affected by amoxicillin, even at the phylum level. Conclusions: In addition to demonstrating the relevance of Göttingen Minipigs as a valid model for studying the impact of medications on maternal milk and GM, our findings suggest that the milk microbiota may be more stable during antibiotic treatment than the GM.

Gene expression emerges from DNA sequences through the interaction of transcription factors (TFs) with DNA cis-regulatory sequences. In eukaryotes, TFs bind to transcription factor binding sites (TFBSs) with differential affinities, enabling cell-specific gene expression. In this view, DNA enables TF binding along a continuum ranging from low to high affinity depending on its sequence composition; however, it is not known whether evolution has entailed a further level of entanglement between DNA-protein interaction. Here we found that the composition and length (22 bp) of the DNA sequence interposed between the CRX and NRL retinal TFs in the proximal promoter of RHODOPSIN (RHO) largely controls the expression levels of RHO. Mutagenesis of CRX-NRL DNA linking sequences (here termed “DNA-linker”) results in uncorrelated gene expression variation. In contrast, mutual exchange of naturally occurring divergent human and mouse Rho cis-regulatory elements conferred similar yet species-specific Rho expression levels. Two orthogonal DNA-binding proteins targeted to the DNA-linker either activate or repress the expression of Rho depending on the DNA-linker orientation relative to the CRX and NRL binding sites. These results argue that, in this instance, DNA itself contributes to CRX and NRL activities through a code based on specific base sequences of a defined length, ultimately determining optimal RHO expression levels.

Background and Aim:Environmental enrichment (EE) is a pivotal tool for creating ideal housing conditions that allow animals to fully display their ethograms. At the micro-environmental level, they should elicit cognitive and social responses and increase physical activities. Hair steroids may be among the best biomarkers to evaluate the effects of prolonged exposure to different enrichments because they are non-invasive and provide information regarding a longer period. This study aimed to compare the hair steroid profiles, specifically corticosterone, cortisol, and dehydroepiandrosterone (DHEA), of Wistar rats exposed to two different EE settings. Materials and Methods:Twenty (n = 20) outbred Wistar rats were enrolled in this study. First hair collection (T0) was performed 3 days before weaning (at 28 days of life), and then Wistar rats were randomly divided into two equal groups with a sex ratio of 1:1: standard EE group, provided with one rat tunnel, and extra EE group, provided with an additional tunnel hanging from the top of the cage. Environmental conditions were 20°C-24°C, 45%-65% relative humidity, and a 12:12 dark/light cycle, with water and pelleted diet ad libitum. The rats were housed in ventilated cages with poplar bedding and nesting material. Hair was sampled again after 3 months (T1). Steroids were quantified using radioimmunoassay or enzyme-linked immunosorbent assay after methanol extraction. Results:Cortisol was not quantifiable, while corticosterone and DHEA were. After 3 months of exposure (T1), no differences were noted between the experimental groups. On data categorization per sex, females showed higher levels of all steroids than males. In males, the extra EE group had higher corticosterone levels. Conclusion:This study showed that corticosterone and DHEA are quantifiable in rats' hair, yet bigger datasets are needed to better understand the physiological levels of these hormones in such a matrix. Different enrichment settings induced differences between and within sex.

Electrocorticography is an established neural interfacing technique wherein an array of electrodes enables large-area recording from the cortical surface. Electrocorticography is commonly used for seizure mapping however the implantation of large-area electrocorticography arrays is a highly invasive procedure, requiring a craniotomy larger than the implant area to place the device. In this work, flexible thin-film electrode arrays are combined with concepts from soft robotics, to realize a large-area electrocorticography device that can change shape via integrated fluidic actuators. We show that the 32-electrode device can be packaged using origami-inspired folding into a compressed state and implanted through a small burr-hole craniotomy, then expanded on the surface of the brain for large-area cortical coverage. The implantation, expansion, and recording functionality of the device is confirmed in-vitro and in porcine in-vivo models. The integration of shape actuation into neural implants provides a clinically viable pathway to realize large-area neural interfaces via minimally invasive surgical techniques.