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Roberta Salaroli,1 Domenico Ventrella,1 Chiara Bernardini,1 Alberto Elmi,1 Augusta Zannoni,1,2 Maria Laura Bacci,1,2 Monica Forni,1,2 Fiorella Calanni,3 Antonella Ferrieri,4 Fabio Baldi5 1Department of Veterinary Medical Science, University of Bologna, Ozzano Emilia, Bologna, Italy; 2Health Sciences and Technologies Interdepartmental Center for Industrial Research, University of Bologna, Bologna, Italy; 3Pre-Clinical Research Department, Alfasigma, Bologna, Italy; 4Division of Clinical Research, Department of Research and Development, Alfasigma, Bologna, Italy; 5Center for the Study of Diseases of the Esophagus, University of Bologna and Gruppo Villa Maria Care & Research, Ravenna, ItalyCorrespondence: Monica ForniDepartment of Veterinary Medical Science, University of Bologna, Via Tolara, 50, Ozzano Emilia 40064, Bologna, ItalyTel +39 051 20 9 7913Email monica.forni@unibo.itPurpose: AL2106 is a new medical device based on a mixture of chondroitin sulphate in a xyloglucan and glycerol solution made to maximize its bioadhesive capability to the esophageal mucosa. The aim of the present study was twofold to evaluate the AL2106 protective effect on the esophageal mucosa when exposed to an acidic solution mimicking gastric reflux and to assess the resilience of this effect to saline washing.Materials and Methods: A porcine ex vivo model was used and the effects of the new medical device were compared to a sodium alginate suspension (SAS) already present on the market which was assumed as reference. Mucosal damage was induced in 19 porcine esophagi by perfusion with an acidic solution added with pepsin, and Evans blue dye (EBD) tissue uptake was used as an indicator of mucosal permeability. The EBD penetration, expressed as EBD μg/g of dry tissue, was assessed in specimens of untreated damaged mucosa and in specimens treated with AL2106 or SAS. The same evaluation was carried out after washing with normal saline.Results: Both topical agents tested significantly reduced the EBD uptake by more than 60% (AL2106 8.4± 4.5, SAS 3.6± 2.7 vs control 23.2± 13.1, p< 0.01). The saline washing did not cause any significant reduction in the protective effect of AL2106 (8.6± 5.9), while it significantly reduced that of SAS (5.9± 4.3, p< 0.05).Conclusion: The new AL2106 medical device showed a good barrier effect against a reflux-like damaging solution and preserved this effect after the mucosal washing test, thus suggesting its possible relevance for the treatment of gastroesophageal reflux disease.Keywords: bioadhesion, Evans blue dye, EBD, animal model, esophagus, gastroesophageal reflux disease, GERD

Organic agriculture is an ecological production management system aimed at sustainable production of safe food, environmental protection and maintenance of soil health. The objective of this study was to evaluate, in a period of organic transition, the effects of different organic fertilizers on wheat yield, grain quality and soil characteristics. The research was performed in an experimental farm of Southern Italy (Foggia), over a three year period. An organic commercial fertilizer and compost, obtained from municipal wastes, with and without a commercial bioactivator, were compared in a randomized block design. Grain yield and quality and soil total organic carbon content (TOC) were measured. The Henin-Dupuis model was also tested to simulate soil organic C dynamics after organic fertilizer application. Among the years, significant differences were observed in grain yield and quality, indicating that wheat crop response was influenced by the weather conditions. The grain and straw yield was not significantly affected by the organic fertilizer used, though the compost resulted in significantly lower grain quality compared with the organic commercial fertilizer. In any case, at the end of the experimentation, the compost resulted in significantly higher TOC (+4%) compared to the organic commercial fertilizer. The results showed that the compost may be used as an alternative to sustain wheat productivity, feasible also from an economic point of view and to conserve soil fertility. Finally, under the local Mediterranean conditions, the soil organic C dynamics could be defined by the Henin-Dupuis model, also considering the C supply of annual root residues. [ABSTRACT FROM AUTHOR]

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&#95;Bacteroidales;Other;Other , and an increasing trend in the abundance of Streptococcus , Stenotrophomonas , f&#95;Rhodobacteraceae;Other , Proteiniclasticum , f&#95;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.

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 (1×10 11 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

Background and Aim: Seasonal changes, especially temperature and photoperiod, are well-known determining factors of swine reproductive capacity, but the underlying mechanisms remain unknown. This study aimed to investigate the effect of age and seasonal variations on boar seminal plasma steroids (dehydroepiandrosterone [DHEA], cortisol [CORT], and testosterone [TEST]) over 1 year., Materials and Methods: Four commercial hybrid adult boars (Large White × Duroc), aged between 12 and 44 months, were repeatedly evaluated at the Department of Veterinary Medical Sciences of the University of Bologna. Daily temperature and light hours relating to the collection date were considered for each observation within the four astronomical seasons: Winter, spring, summer, and autumn. Hormones were quantified using radioimmunoassay. The association between seasonal factors and hormone concentrations was evaluated using linear regression models. Univariate models were estimated for each hormone to assess the influence of the independent variables; two multivariate models were assessed to evaluate the effect of temperature and daylight hours, including boar and season factors., Results: Age significantly affected all analyzed hormones (CORT p < 0.0001; DHEA p < 0.0001; and TEST p < 0.0001). The highest average levels were found for each hormone during summertime, suggesting a positive correlation between steroid concentrations with temperature and light hours., Conclusion: The results of this study support the hypothesis that the increase in external temperature and light hours is somehow associated with higher levels of steroid concentrations in the seminal plasma of in-housed boars. These findings may help further investigate seasonal fluctuations in reproductive outcomes, which are well-known for porcine species., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Aniballi, et al.)

Two main classes of perivascular multipotent populations have been described: the microvascular pericytes and the vascular wall mesenchymal stem cells (VW-MSCs). VW-MSCs are isolated from large vessels in many species and they participate in vascular remodeling together with other cellular components such as endothelial cells. Considering that the Göttingen Minipigs are widely used in Europe as a translational model in the field of cardiovascular diseases, the aim of the present research was to isolate VW-MSCs from the adult aorta of Göttingen Minipigs while preserving and also collecting endothelial cells. The results obtained in the present research demonstrated that this new protocol allows us to obtain a pure population of VW-MSCs and endothelial cells. VW-MSCs from Göttingen Minipigs responded fully to the MSC minima international criteria, being positive to CD105, CD90, and CD44 and negative to CD45 and CD34. Moreover, VW-MSCs presented a differentiative potential towards osteogenic, chondrogenic, and adipogenic lineages. Overall, the present protocol, preserving the viability and phenotypic features of the two isolated populations, opens future possibilities of using minipig VW-MSCs and endothelial cells in in vitro vascular remodeling studies.

Introduction: We have recently developed a new miniaturized device for extracorporeal ultrafiltration (UF) to be used in patients with fluid overload: Artificial Diuresis-1 (AD1) (Medica S.p.A., Medolla, Italy). The device has a reduced priming volume, operates at very low pressures and flow regimes, and is designed to perform extracorporeal UF at bedside. After accurate experiments were carried out in vitro, we report in this paper the results of in vivo UF sessions carried out in selected animals according to veterinary best practice., Materials and Methods: The AD1 kit is pre-filled with sterile isotonic solution and operates with a polysulfone mini-filter, MediSulfone (polysulfone at 50,000 Dalton). A collection bag with a volumetric scale is connected to the UF line, and the ultrafiltrate is obtained by gravity based on the height at which the ultrafiltrate collection bag is placed. Animals were prepared and anesthetized. The jugular vein was cannulated with a double-lumen catheter. Three 6-h sessions of UF were scheduled with a target fluid removal of 1,500 mL. Heparin was used as anticoagulant., Results: In all treatments, the target value of UF was obtained in the absence of major clinical or technical problems with a maximum deviation from the scheduled UF rate lower than 10%. The device resulted to be safe, reliable, accurate, and easily usable thanks to a user-friendly interface and its very small dimensions., Conclusions: This study opens the way for clinical trials in different settings including departments with low intensity of care and even in ambulatory centers or patient's home., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

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)., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

The increasing incidence of calcific aortic valve disease necessitates the elaboration of new strategies to retard the progression of the pathology with an innovative solution. While the increasing diffusion of the transcatheter aortic valve replacements (TAVRs) allows a mini-invasive approach to aortic valve substitution as an alternative to conventional surgical replacement (SAVR) in an always larger patient population, TAVR implantation still has contraindications for young patients. In addition, it is liable to undergo calcification with the consequent necessity of re-intervention with conventional valve surgery or repeated implantation in the so-called TAVR-in-TAVR procedure. Inspired by applications for non-cardiac pathologies or for vascular decalcification before stenting (i.e., coronary lithotripsy), in the present study, we show the feasibility of human valve treatment with a mini-invasive device tailored to deliver shockwaves to the calcific leaflets. We provide evidence of efficient calcium deposit ruptures in human calcified leaflets treated ex vivo and the safety of the treatment in pigs. The use of this device could be helpful to perform shockwaves valvuloplasty as an option to retard TAVR/SAVR, or as a pretreatment to facilitate prosthesis implantation and minimize the occurrence of paravalvular leak., Competing Interests: EF, DB, MCP, and EP are collaborators of the equity-financed company AorticLab S.r.l. AorticLab S.r.l. is an equity-financed Company for development of the TDD as a novel minimally invasive calcium debridement procedure in human pathologic valves. GG and CR were employed by ASST Fatebenefratelli Sacco. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bernava, Fermi, Gelpi, Rizzi, Benettin, Barbuto, Romagnoni, Ventrella, Palmieri, Agrifoglio, Polvani, Bacci, Pasquino and Pesce.)

The ConcePTION project aims at generating further knowledge about the risks related to the use of medication during breastfeeding, as this information is lacking for most commonly used drugs. Taking into consideration multiple aspects, the pig model has been considered by the consortium as the most appropriate choice. The present research was planned to develop an efficient method for the isolation and culture of porcine Mammary Epithelial Cells (pMECs) to study the mammary epithelial barrier in vitro. Mammary gland tissues were collected at a local slaughterhouse, dissociated and the selected cellular population was cultured, expanded and characterized by morphology, cell cycle analysis and immunophenotyping. Their ability to create a barrier was tested by TEER measurement and sodium fluorescein transport activity. Expression of 84 genes related to drug transporters was evaluated by a PCR array. Our results show that primary cells express epithelial cell markers: CKs, CK18, E-Cad and tight junctions molecules ZO-1 and OCL. All the three pMEC cellular lines were able to create a tight barrier, although with different strengths and kinetics, and express the main ABC and SLC drug transporters. In conclusion, in the present paper we have reported an efficient method to obtain primary pMEC lines to study epithelial barrier function in the pig model.

Roe deer are seasonal breeders with a complete yearly testicular cycle. The peak in reproductive activity is recorded during summer, the rutting period, with the highest levels of androgens and testicular weight. Melatonin plays a pivotal role in seasonal breeders by stimulating the hypothalamus-pituitary-gonads axis and acting locally; in different species, its synthesis within testes has been reported. The aim of this study was to evaluate the physiological melatonin pattern within roe deer testes by comparing data obtained from animals sampled during pre- and post-rut periods. Melatonin was quantified in testicular parenchyma, along with the genetic expression of enzymes involved in its local synthesis (AANAT and ASMT) and function (UCP1). Melatonin receptors, MT1-2, were quantified both at protein and gene expression levels. Finally, to assess changes in reproductive hormonal profiles, testicular dehydroepiandrosterone (DHEA) was quantified and used for a correlation analysis. Melatonin and AANAT were detected in all samples, without significant differences between pre- and post-rut periods. Despite DHEA levels confirming testicular involution during the post-rut period, no correlations appeared between such involution and melatonin pathways. This study represents the first report regarding melatonin synthesis in roe deer testes, opening the way for future prospective studies in the physiology of this species.

Breastfeeding plays a major role in the health and wellbeing of mother and infant. However, information on the safety of maternal medication during breastfeeding is lacking for most medications. This leads to discontinuation of either breastfeeding or maternal therapy, although many medications are likely to be safe. Since human lactation studies are costly and challenging, validated non-clinical methods would offer an attractive alternative. This review gives an extensive overview of the non-clinical methods (in vitro, in vivo and in silico) to study the transfer of maternal medication into the human breast milk, and subsequent neonatal systemic exposure. Several in vitro models are available, but model characterization, including quantitative medication transport data across the in vitro blood-milk barrier, remains rather limited. Furthermore, animal in vivo models have been used successfully in the past. However, these models don't always mimic human physiology due to species-specific differences. Several efforts have been made to predict medication transfer into the milk based on physicochemical characteristics. However, the role of transporter proteins and several physiological factors (e.g., variable milk lipid content) are not accounted for by these methods. Physiologically-based pharmacokinetic (PBPK) modelling offers a mechanism-oriented strategy with bio-relevance. Recently, lactation PBPK models have been reported for some medications, showing at least the feasibility and value of PBPK modelling to predict transfer of medication into the human milk. However, reliable data as input for PBPK models is often missing. The iterative development of in vitro, animal in vivo and PBPK modelling methods seems to be a promising approach. Human in vitro models will deliver essential data on the transepithelial transport of medication, whereas the combination of animal in vitro and in vivo methods will deliver information to establish accurate in vitro/in vivo extrapolation (IVIVE) algorithms and mechanistic insights. Such a non-clinical platform will be developed and thoroughly evaluated by the Innovative Medicines Initiative ConcePTION., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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.

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.

Aim: The aim of the study was to set up a porcine ex vivo model of acid-induced damage and to evaluate its performance by means of multichannel intraluminal impedance and pH (MII-pH) live recording, histology, and Evans blue (EB) permeability assay., Materials and Methods: Thirteen esophagi, collected at a slaughterhouse, were ablated of their sphincters, pinned upright on a support, and placed in a thermostatic hood at 37°C with two infusion tubes and an MII-pH probe inserted in the top end. Three esophagi (histology controls) were only left in the hood for 3.5 h before sampling, while the remaining organs underwent the experimental protocol including saline infusion and recovery recording, and acid solution infusion and recovery recording., Results: MII-pH analysis highlighted a significantly stronger decrease during acid infusion when compared to saline, but a better post-infusion recovery for saline solution. At the end of the protocol, MII was still statistically lower than baseline. The acid-damaged esophagi significantly absorbed more EB dye, and histology revealed strong mucosal exfoliation., Conclusion: The proposed model of esophageal acid damage seems to be repeatable, reliable, and achievable using organs collected at the slaughterhouse. MII recording proved to have good sensitivity in detecting mucosal alterations also in ex vivo trials., (Copyright: © Ventrella, et al.)

The progressive degeneration of retinal photoreceptors is one of the most significant causes of blindness in humans. Conjugated polymers represent an attractive solution to the field of retinal prostheses, and a multi-layer fully organic prosthesis implanted subretinally in dystrophic Royal College of Surgeons (RCS) rats was able to rescue visual functions. As a step toward human translation, we report here the fabrication and in vivo testing of a similar device engineered to adapt to the human-like size of the eye of the domestic pig, an excellent animal paradigm to test therapeutic strategies for photoreceptors degeneration. The active conjugated polymers were layered onto two distinct passive substrates, namely electro-spun silk fibroin (ESF) and polyethylene terephthalate (PET). Naive pigs were implanted subretinally with the active device in one eye, while the contralateral eye was sham implanted with substrate only. Retinal morphology and functionality were assessed before and after surgery by means of in vivo optical coherence tomography and full-field electroretinogram (ff-ERG) analysis. After the sacrifice, the retina morphology and inflammatory markers were analyzed by immunohistochemistry of the excised retinas. Surprisingly, ESF-based prostheses caused a proliferative vitreoretinopathy with disappearance of the ff-ERG b-wave in the implanted eyes. In contrast, PET-based active devices did not evoke significant inflammatory responses. As expected, the subretinal implantation of both PET only and the PET-based prosthesis locally decreased the thickness of the outer nuclear layer due to local photoreceptor loss. However, while the implantation of the PET only substrate decreased the ff-ERG b-wave amplitude with respect to the pre-implant ERG, the eyes implanted with the active device fully preserved the ERG responses, indicating an active compensation of the surgery-induced photoreceptor loss. Our findings highlight the possibility of developing a new generation of conjugated polymer/PET-based prosthetic devices that are highly biocompatible and potentially suitable for subretinal implantation in patients suffering from degenerative blindness., (Copyright © 2020 Maya-Vetencourt, Di Marco, Mete, Di Paolo, Ventrella, Barone, Elmi, Manfredi, Desii, Sannita, Bisti, Lanzani, Pertile, Bacci and Benfenati.)

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.

The roe deer ( Capreolus capreolus ) represents a spontaneous model of testicular inactivation: During winter, bucks show a suspension of spermatogenesis that starts again in spring and peaks during the breeding season (July-August). The underlying mechanisms to the regulation of the cyclic testicular changes are still not fully clear but seem to be imputable to the spermatogenic cell line since other testicular cell populations remain stable without apoptotic phenomena. The aim of the study was to investigate apoptosis, gelatinases (MMP2 and 9), their inhibiting factors (TIMP 1-2), and two isoforms of vascular endothelial growth factor (VEGF121 and 165) with its receptors (VEGFR1-2) in testes collected during pre- and post-rut periods, and to correlate them with testicular weight (TW) and testosterone (TEST). Testes from 18 adult sexually mature bucks were collected in Bologna Apennines (Italy). Samples were weighed and parenchyma collected. Radioimmunoassay, real-time PCR, and zymography were performed. The results showed a post-rut decrease in TW and TEST and an increase in proMMP2, also highlighting a correlation between the gelatinases and the testicular functionality. The VEGF pattern did not show modifications nor correlation with TW and TEST. Overall, gelatinases and their inhibitors, described herein for the first time in roe deer testes, seem to play an important role in the testicular cycle., Competing Interests: The authors declare no conflict of interest.

Vascularization is a crucial factor when approaching any engineered tissue. Vascular wall-mesenchymal stem cells are an excellent in vitro model to study vascular remodeling due to their strong angiogenic attitude. This study aimed to demonstrate the angiogenic potential of experimental highly porous scaffolds based on polylactic acid (PLA) or poly-e-caprolactone (PCL) doped with calcium silicates (CaSi) and dicalcium phosphate dihydrate (DCPD), namely PLA-10CaSi-10DCPD and PCL-10CaSi-10DCPD, designed for the regeneration of bone defects. Vascular wall-mesenchymal stem cells (VW-MSCs) derived from pig thoracic aorta were seeded on the scaffolds and the expression of angiogenic markers, i.e. CD90 (mesenchymal stem/stromal cell surface marker), pericyte genes α-SMA (alpha smooth muscle actin), PDGFR-β (platelet-derived growth factor receptor-β), and NG2 (neuron-glial antigen 2) was evaluated. Pure PLA and pure PCL scaffolds and cell culture plastic were used as controls (3D in vitro model vs. 2D in vitro model). The results clearly demonstrated that the vascular wall mesenchymal cells colonized the scaffolds and were metabolically active. Cells, grown in these 3D systems, showed the typical gene expression profile they have in control 2D culture, although with some main quantitative differences. DNA staining and immunofluorescence assay for alpha-tubulin confirmed a cellular presence on both scaffolds. However, VW-MSCs cultured on PLA-10CaSi-10DCPD showed an individual cells growth, whilst on PCL-10CaSi-10DCPD scaffolds VW-MSCs grew in spherical clusters. In conclusion, vascular wall mesenchymal stem cells demonstrated the ability to colonize PLA and PCL scaffolds doped with CaSi-DCPD for new vessels formation and a potential for tissue regeneration., Competing Interests: The authors declare no conflict of interest.

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.