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Macrophages are the foremost controllers of innate and acquired immunity, playing important roles in tissue homeostasis, vasculogenesis, and congenital metabolism. In vitro macrophages are crucial models for understanding the regulatory mechanism of immune responses and the diagnosis or treatment of a variety of diseases. Pigs are the most important agricultural animals and valuable animal models for preclinical studies, but there is no unified method for porcine macrophage isolation and differentiation at present; no systematic study has compared porcine macrophages obtained by different methods. In the current study, we obtained two M1 macrophages (M1_IFNγ + LPS, and M1_GM-CSF) and two M2 macrophages (M2_IL4 + IL10, and M2_M-CSF), and compared the transcriptomic profiles between and within macrophage phenotypes. We observed the transcriptional differences either between or within phenotypes. Porcine M1 and M2 macrophages have consistent gene signatures with human and mouse macrophage phenotypes, respectively. Moreover, we performed GSEA analysis to attribute the prognostic value of our macrophage signatures in discriminating various pathogen infections. Our study provided a framework to guide the interrogation of macrophage phenotypes in the context of health and disease. The approach described here could be used to propose new biomarkers for diagnosis in diverse clinical settings including porcine reproductive and respiratory syndrome virus (PRRSV), African swine fever virus (ASFV), Toxoplasma gondii (T. gondii), porcine circovirus type 2 (PCV2), Haemophilus parasuis serovar 4 (HPS4), Mycoplasma hyopneumoniae (Mhp), Streptococcus suis serotype 2 (SS2), and LPS from Salmonella enterica serotype minnesota Re 595.

Impairment of development, migration, or function of inhibitory interneurons are key features of numerous circuit-based neurological disorders, such as epilepsy. From a therapeutic perspective, symptomatic treatment of these disorders often relies upon drugs or deep brain stimulation approaches to provide a general enhancement of GABA-mediated inhibition. A more effective strategy to target these pathological circuits and potentially provide true disease-modifying therapy, would be to selectively add new inhibitory interneurons into these circuits. One such strategy, using embryonic medial ganglionic (MGE) progenitor cells as a source of a unique sub-population of interneurons, has already proven effective as a cell transplantation therapy in a variety of preclinical models of neurological disorders, especially in mouse models of acquired epilepsy. Here we will discuss the evolution of this interneuron-based transplantation therapy in acquired epilepsy models, with an emphasis on the recent adaptation of MGE progenitor cells for xenotransplantation into larger mammals.

The aims of this study were to investigate the effects of different equilibration times with cryoprotectants on viability and metaphase plate morphology of vitrified-warmed porcine mature oocytes (Experiment 1) and to evaluate the effects of supplementation with 10(-9) M melatonin during in vitro maturation on these parameters (Experiment 2). In Experiment 1, 2,392 mature oocytes were vitrified using different equilibration times of oocytes with cryoprotectants (3, 10, 15, 20, 30, 40, 60 and 80 min). Fresh oocytes matured in vitro for 44 hr (n = 509) were used as controls. In Experiment 2, a total of 573 COCs were used. COCs were matured with 10(-9) M melatonin supplementation or without melatonin (control). Some oocytes from each group were vitrified with a 60-min equilibration time with cryoprotectants according to the results of Experiment 1. The remaining oocytes from each maturation group were used as fresh control groups. In both experiments, oocytes were stained with 2 ,7 -dichlorodihydrofuorescein diacetate and Hoechst 33342 to assess viability and metaphase plate morphology, respectively. Vitrification and warming affected (p < .01) oocyte viability compared with controls, which were all viable after 44 hr of IVM. In Experiment 1, the longer the equilibration time with cryoprotectants, the higher the viability. Oocytes equilibrated for 60 and 80 min had the highest (p < .05) viability and similar metaphase plate characteristics to the fresh control oocytes. In Experiment 2, supplementation with melatonin during in vitro maturation had no effect on oocyte viability or metaphase plate morphology of vitrified-warmed oocytes. In conclusion, under our experimental conditions, vitrified porcine mature oocytes equilibrated with cryoprotectants for 60 or 80 min exhibited the highest viability and similar metaphase plate characteristics to fresh controls. Furthermore, supplementation with 10(-9) M melatonin during in vitro maturation had no effect on these parameters. Funding Agencies|Madrid, Spain [MCIN/AEI/10.13039/501100011033]; ERDF A way of making Europe, Madrid, Spain; Fundacion Seneca, Murcia, Spain [19892/GERM/15]

The present study was stimulated by the continuous growth of commercially available high intensity focused ultrasound (HIFU) systems for fat reduction. Herein, HIFU was utilised for fat ablation using a single-element ultrasonic transducer operating in thermal mode.The custom-made concave transducer that operates at 1.1 MHz was assessed on excised porcine adipose tissue for its ability to reduce fat. Ultrasonic sonications were executed on the adipose tissue utilising acoustical power between 14 and 75 W and sonication time in the range of 1-10 min. The mass of the adipose tissue sample was weighed afore and after ultrasonic sonications and the effect of the sonication on the mass change was recorded.Mass change was linearly dependent with either increasing acoustical power or sonication time and was in the range of 0.46-1.9 g. High acoustical power of 62.5 W for a sonication time of 1 min and a power of 75 W for a sonication time of 5 min, respectively resulted in the formation of a lesion or possible cavitation on the piece of excised adipose tissue.The study demonstrated the efficacy of the proposed transducer in achieving a reduction of excised fat tissue. The present findings indicate the potential use of the transducer in a HIFU system indicated for the reduction of subcutaneous adipose tissue where increased values of acoustical power can result in increased amounts of fat reduction.

PCR sex determination assays must be reliable and cost effective due to the frequent and integral use of these assays in biological research and the animal production industry. Thus, the design of proof of a primer pair with a built-in control is warranted to not only bypass the extra cost of a multiplex reaction, but also to prevent anomalous results that have been documented with other primer pairs. The objective of this study was to design primer pairs with built in PCR amplification control to identify sex in Equus caballus (domestic horse), Homo sapiens (humans), Macaca mulatta (rhesus macaque), and Sus scrofa (domestic pig) DNA samples. The procedures utilized in this study were to first align the DDX3X gene with the Y chromosome homolog and create primer pairs to flank Y chromosome specific indels with each species. PCR with gel electrophoresis results showed confirmation of the hypothesis that the primer pairs can be used to accurately identify separate X and Y chromosome specific sequences via species-specific single primer pairs in Equus caballus, Homo sapiens, Macaca mulatta, and Sus scrofa genomic samples. Additionally, in silico and qualitative gel analyses were completed to assess the efficacy of the Equus caballus specific primers in alternative species, which yielded no results. Summarily, results concluded that there are suitable indel regions for PCR amplification in Equus caballus, Homo sapiens, Macaca mulatta, and Sus scrofa. The results presented herein represent a meaningful contribution to the field from this methodology, these indel regions can be identified via the designed primers and utilized in an efficient, quick, and cost-effective way to identify sex without the need for multiplex or the risk of false identification.

In this study, to analyze whether artificial regulation of apoptosis in the development of somatic cells can affect the stable growth and development of cells, 20 alpha-hydroxysteroid dehydrogenase (20α-HSD) and rapamycin were treated to induce apoptosis and autophagy in the both skin and muscle cells. Respectively, and 3-methyladenine was supplemented to inhibit cell death. Our results show that stimulation with rapamycin activated autophagy in both types of cells, but increased apoptosis more than autophagy in the case of skin cells. These results indicate that there was a difference in the expression of survival factors and cell development depending on the type of cell. In particular, in the expression of autophagy-related gene (MAP1LC3A) was higher than that of Casp-3, an apoptosis factor. Furthermore, cell development was the highest in all cell groups cultured by artificially inducing autophagy, however the lowest in the apoptosis-inhibited group. Especially, the noteworthy result of this study was that when apoptosis was induced using 20α-HSD, it was possible to induce apoptosis in both skin and muscle cells. Therefore, the main point of this study is that apoptosis induced during cell culture plays a pivotal role in cell remodeling.

DNA-methylation profiles have been used successfully to develop highly accurate biomarkers of age, epigenetic clocks, for many species. Using a custom methylation array, we generated DNA methylation data from n = 238 porcine tissues including blood, bladder, frontal cortex, kidney, liver, and lung, from domestic pigs (Sus scrofa domesticus) and minipigs (Wisconsin Miniature Swine™). Samples used in this study originated from Large White X Landrace crossbred pigs, Large White X Minnesota minipig crossbred pigs, and Wisconsin Miniature Swine™. We present 4 epigenetic clocks for pigs that are distinguished by their compatibility with tissue type (pan-tissue and blood clock) and species (pig and human). Two dual-species human-pig pan-tissue clocks accurately measure chronological age and relative age, respectively. We also characterized CpGs that differ between minipigs and domestic pigs. Strikingly, several genes implicated by our epigenetic studies of minipig status overlap with genes (ADCY3, TFAP2B, SKOR1, and GPR61) implicated by genetic studies of body mass index in humans. In addition, CpGs with different levels of methylation between the two pig breeds were identified proximal to genes involved in blood LDL levels and cholesterol synthesis, of particular interest given the minipig’s increased susceptibility to cardiovascular disease compared to domestic pigs. Thus, breed-specific differences of domestic and minipigs may potentially help to identify biological mechanisms underlying weight gain and aging-associated diseases. Our porcine clocks are expected to be useful for elucidating the role of epigenetics in aging and obesity, and the testing of anti-aging interventions. Supplementary Information The online version contains supplementary material available at 10.1007/s11357-021-00439-6.

The efficacy of two locally produced oxfendazole (OFZ) formulations against cysticercosis at 22,5% and 10%, versus a commercial formulation (Synanthic 9,06%) was evaluated in twenty-two naturally infected pigs that received a single oral dose of 30 mg/kg. Pigs were sacrificed at eight weeks post-treatment to evaluate the cysts found in their carcasses, and to determine the cysticidal efficacy, which was defined as the proportion of degenerated cysts over total cysts. Only degenerated cysts were found in muscle, heart, and tongue of pigs treated with OFZ in all groups, which shows an efficacy of 100%. Viable and degenerated cysts were found in brains, being the efficacy lower in all groups (65% [commercial OFZ], 47% [local OFZ 22.5%] and 31% [local OFZ 10%], p = 0.355). Locally produced OFZ formulations were similarly effective to the commercial formulation and may provide a practical alternative for the treatment of porcine cysticercosis. Se evaluó la eficacia de dos formulaciones de oxfendazol (OFZ) contra cisticercosis producidas localmente, al 22,5% y 10% en comparación con una formulación comercial (Synanthic 9,06%) en 22 cerdos naturalmente infectados, que recibieron una dosis oral de 30 mg/kg. Los cerdos fueron sacrificados a las ocho semanas postratamiento para evaluar quistes en en sus carcasas, y se determinó la eficacia cisticida a través de la proporción de quistes degenerados sobre el total. Solo se encontraron quistes degenerados en la musculatura, corazón y lengua de los cerdos tratados con OFZ en todos los grupos, lo cual muestra una eficacia del 100%. En los cerebros se encontraron quistes viables y degenerados, con una eficacia menor en todos los grupos (65% [OFZ comercial], 47% [OFZ local 22,5%] y 31% [OFZ local 10%], p = 0,355. Las formulaciones de OFZ producidas localmente fueron igual de efectivas que la formulación comercial y pueden proporcionar una alternativa para el tratamiento de la cisticercosis porcina

Morbilliviruses are highly contagious pathogens. The Morbillivirus genus includes measles virus, canine distemper virus (CDV), phocine distemper virus (PDV), peste des petits ruminants virus, rinderpest virus, and feline morbillivirus. We detected a novel porcine morbillivirus (PoMV) as a putative cause of fetal death, encephalitis, and placentitis among swine by using histopathology, metagenomic sequencing, and in situ hybridization. Phylogenetic analyses showed PoMV is most closely related to CDV (62.9% nt identities) and PDV (62.8% nt identities). We observed intranuclear inclusions in neurons and glial cells of swine fetuses with encephalitis. Cellular tropism is similar to other morbilliviruses, and PoMV viral RNA was detected in neurons, respiratory epithelium, and lymphocytes. This study provides fundamental knowledge concerning the pathology, genome composition, transmission, and cellular tropism of a novel pathogen within the genus Morbillivirus and opens the door to a new, applicable disease model to drive research forward.

Objective: Traditional negative pressure wound therapy (tNPWT) systems can be large and cumbersome, limiting patient mobility and adversely affecting quality of life. PICO™, a no canister single-use system, offers a lightweight, portable alternative to tNPWT, with improved clinical performance. The aim of this study was to determine the potential mechanism(s) of action of single-use NPWT (sNPWT) versus tNPWT. Approach: sNPWT and tNPWT were applied to an in vivo porcine excisional wound model, following product use guidelines. Macroscopic, histological, and biochemical analyses were performed at defined healing time points to assess multiple aspects of the healing response. Results: Wounds treated with single-use negative pressure displayed greater wound closure and increased reepithelialization versus those treated with traditional negative pressure. The resulting granulation tissue was more advanced with fewer neutrophils, reduced inflammatory markers, more mature collagen, and no wound filler-associated foreign body reactions. Of note, single-use negative pressure therapy failed to induce wound edge epithelial hyperproliferation, while traditional negative pressure therapy compromised periwound skin, which remained inflamed with high transepidermal water loss; features not observed following single-use treatment. Innovation: Single-use negative pressure was identified to improve multiple aspects of healing versus traditional negative pressure treatment. Conclusion: This study provides important new insight into the differing mode of action of single-use versus traditional negative pressure and may go some way to explaining the improved clinical outcomes observed with single-use negative pressure therapy.

Mitochondria are complex organelles that play a critical role within the cell; mitochondrial dysfunction can result in significant cell damage or death. Previous studies have demonstrated the promising therapeutic effects of autologous mitochondria transplantation into ischemic cardiac tissue; however, few studies have examined the in vivo effects of mitochondria infusion into the brain. The aim of this study is to report a procedure for carotid infusion of autologous mitochondria into porcine brains. By using this infusion technique, we propose that a selective and minimally invasive administration is feasible and may provide benefits in the treatment of various central nervous system disorders Las mitocondrias son organelas complejas que desempeñan un papel fundamental en la célula, la disfunción mitocondrial puede ocasionar daños celulares significativos o la muerte. Estudios previos han demostrado los prometedores efectos terapéuticos del trasplante de mitocondrias autólogas a un tejido cardiaco isquémico, sin embargo, pocos estudios han evaluado los efectos in vivo de la infusión de mitocondrias en el cerebro. El presente trabajo tiene como objetivo dar a conocer el procedimiento para la infusión vía carótida de mitocondrias autólogas en cerebros porcinos. Mediante esta técnica de infusión, proponemos que una administración selectiva y mínimamente invasiva es factible y puede proporcionar beneficios en el tratamiento de diversas patologías del sistema nervioso central

Background: Invasive ventilation during cardiopulmonary resuscitation (CPR) is very complex due to unique thoracic pressure conditions. Current guidelines do not provide specific recommendations for ventilation during ongoing chest compressions regarding positive end-expiratory pressure (PEEP). This trial examines the cardiopulmonary effects of PEEP application during CPR. Methods: Forty-two German landrace pigs were anaesthetised, instrumented, and randomised into six intervention groups. Three PEEP levels (0, 8, and 16 mbar) were compared in high standard and ultralow tidal volume ventilation. After the induction of ventricular fibrillation, mechanical chest compressions and ventilation were initiated and maintained for thirty minutes. Blood gases, ventilation/perfusion ratio, and electrical impedance tomography loops were taken repeatedly. Ventilation pressures and haemodynamic parameters were measured continuously. Postmortem lung tissue damage was assessed using the diffuse alveolar damage (DAD) score. Statistical analyses were performed using SPSS, and p values

Access to the central vasculature is critical for hemodynamic monitoring and for delivery of life-saving therapeutics during emergency medicine and battlefield trauma situations but requires skill often unavailable in austere environments. Automated central vascular access devices (ACVADs) using ultrasound and robotics are being developed. Here, we present an ex vivo lower-body porcine model as a testing platform for evaluation of vascular devices and compare its features to commercially available platforms. While the commercially available trainers were simpler to set-up and use, the scope of their utility was limited as they were unable to provide realistic anatomic, physiologic, and sonographic properties that were provided by the ex vivo model. However, the ex vivo model was more cumbersome to set-up and use. Overall, both have a place in the development and evaluation pipeline for ACVADs before testing on live animals, thus accelerating product development and translation.

Made available in DSpace on 2021-06-25T11:03:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Culture conditions regulate the process of pluripotency acquisition and self-renewal. This study aimed to analyse the influence of the in vitro environment on the induction of porcine induced pluripotent stem cell (piPSCs) differentiation into primordial germ cell-like cells (pPGCLCs). piPSC culture with different supplementation strategies (LIF, bFGF, or LIF plus bFGF) promoted heterogeneous phenotypic profiles. Continuous bFGF supplementation during piPSCs culture was beneficial to support a pluripotent state and the differentiation of piPSCs into pPGCLCs. The pPGCLCs were positive for the gene and protein expression of pluripotent and germinative markers. This study can provide a suitable in vitro model for use in translational studies and to help answer numerous remaining questions about germ cells. Graphical abstract: [Figure not available: see fulltext.] Department of Animal Reproduction School of Veterinary Medicine and Animal Science University of São Paulo Department of Veterinary Medicine School of Animal Sciences and Food Engineering Department of Pharmacology Institute of Biosciences São Paulo State University (UNESP) Department of Surgery Faculty of Veterinary Medicine and Animal Sciences University of Sao Paulo Department of Biochemistry Paulista School of Medicine Federal University of São Paulo (UNIFESP) Department of Preventive Veterinary Medicine and Animal Reproduction Faculty of Agricultural and Veterinary Sciences São Paulo State University (UNESP) Department of Animal Science Faculty of Animal Science and Food Engineering University of São Paulo Department of Pharmacology Institute of Biosciences São Paulo State University (UNESP) Department of Preventive Veterinary Medicine and Animal Reproduction Faculty of Agricultural and Veterinary Sciences São Paulo State University (UNESP)

Glutamine supplementation to porcine embryo culture medium improves development, increases leucine consumption, and enhances mitochondrial activity. In cancer cells, glutamine has been implicated in the activation of mechanistic target of rapamycin complex 1 (mTORC1) to support rapid proliferation. The objective of this study was to determine if glutamine metabolism, known as glutaminolysis, was involved in mTORC1 activation in porcine embryos. Culture with 3.75 mM GlutaMAX improved development to the blastocyst stage compared to culture with 1 mM GlutaMAX, and culture with 0 mM GlutaMAX decreased development compared to all groups with GlutaMAX. Ratios of phosphorylated to total MTOR were increased when embryos were cultured with 3.75 or 10 mM GlutaMAX, which was enhanced by the absence of leucine, but ratios for RPS6K were unchanged. As another indicator of mTORC1 activation, colocalization of MTOR and a lysosomal marker was increased in embryos cultured with 3.75 or 10 mM GlutaMAX in the absence of leucine. Culturing embryos with glutaminase inhibitors decreased development and the ratio of phosphorylated to total MTOR, indicating reduced activation of the complex. Therefore, glutaminolysis is involved in the activation of mTORC1 in porcine embryos, but further studies are needed to characterize downstream effects on development.

The combination of estrus synchronization and superovulation (SS) treatments causes alterations in ovarian and endometrial gene expression patterns, resulting in abnormal follicle and oocyte growth, fertilization, and embryo development. However, the impact of combined SS treatments on the transcriptome of the surviving embryos remains unidentified. In this study, we examined gene expression changes in day 6 blastocysts that survived a brief regimen of synchronization treatment combined with superovulation. The sows were included in one of three groups: SS7 group (n = 6), sows were administered Altrenogest (ALT) 7 days from the day of weaning and superovulated with eCG 24 h after the end of ALT treatment and hCG at the onset of estrus; SO group (n = 6), ALT nontreated sows were superovulated with eCG 24 h postweaning and hCG at the onset of estrus; control group (n = 6), weaned sows displaying natural estrus. Six days after insemination, the sows underwent a surgical intervention for embryo collection. Transcriptome analysis was performed on blastocyst-stage embryos with good morphology. Differentially expressed genes (DEGs) between groups were detected using one-way ANOVA with an un-adjusted p-value < 0.05 and a fold change 1.5. The effect of SO treatment on the number of altered pathways and DEGs within each pathway was minimal. Only four pathways were disrupted comprising only a total of four altered transcripts, which were not related to reproductive functions or embryonic development. On the other hand, the surviving blastocysts subjected to SS7 treatments exhibited moderate gene expression changes in terms of DEGs and fold changes, with seven pathways disrupted containing a total of 10 transcripts affected. In this case, the up-regulation of certain pathways, such as the metabolic pathway, with two up-regulated genes associated with reproductive functions, namely RDH10 and SPTLC2, may suggest suboptimal embryo quality, while the down-regulation of others, such as the glutathione metabolism pathway, with down-regulated genes related to cellular detoxification of reactive oxygen species, namely GSTK1 and GSTO1, could depress the embryos’ response to oxidative stress, thereby impairing subsequent embryo development. The gene expression changes observed in the present study in SS7 embryos, along with previous reports indicating SS7 can negatively affect fertilization, embryo production, and reproductive tract gene expression, make its use in embryo transfer programs unrecommendable.

Gelatin (bovine/porcine)-based edible films are considered as an excellent carrier for essential oils (EOs) to preserve food quality and extend their shelf life. Spearmint essential oil (SEO) is known for its potential antioxidant and antimicrobial effects; nevertheless, its food applications are limited due to the volatile nature of its active components. Thus, edible films loaded with essential oil can be an alternative to synthetic preservatives to improve their food applications. In the present study, the effect of SEO addition was investigated on the physicochemical properties of bovine and porcine gelatin films, and antioxidant activity was assessed. GCMS (Gas chromatography mass spectrometry) analysis revealed the presence of carvone (55%) and limonene (25.3%) as major components. The incorporation of SEO into the films decreased the opacity, moisture content, water solubility, and elongation at break of bovine and porcine gelatin films. However, with the addition of EO, the thickness and water vapor permeability of bovine and porcine-based gelatin films increased. Moreover, the addition of SEO increased the tensile strength (TS) of the porcine-based film, whereas bovine samples demonstrated a decrease in tensile strength. XRD (X-ray diffraction) findings revealed a decrease in the percentage crystallinity of both types of gelatin films. SEM (scanning electron microscope) results showed the changes in the morphology of films after the addition of SEO. Antioxidant properties significantly (p < 0.05) increased with the incorporation of EO when compared with control films. Therefore, the addition of SEO to gelatin-based edible films could be an effective approach to prepare an active food packaging material to prevent food oxidation.

Aflatoxin B1 (AFB1) is a type of mycotoxin produced by the fungi Aspergillus flavus and Aspergillus parasiticus and is commonly found in cereals, oils and foodstuffs. In order to understand the toxic effects of AFB1 exposure on Porcine alveolar macrophages (3D4/2 cell), the 3D4/2 cells were exposed to 40 μg/mL AFB1 for 24 h in vitro, and several methods were used for analysis. Edu and TUNEL analysis showed that the proliferation of 3D4/2 cells was significantly inhibited and the apoptosis of 3D4/2 cells was significantly induced after AFB1 exposure compared with that of the control group. Whole-transcriptome analysis was performed to reveal the non-coding RNA alteration in 3D4/2 cells after AFB1 exposure. It was found that the expression of cell-cycle-related and apoptosis-related genes was altered after AFB1 exposure, and lncRNAs and miRNAs were also significantly different among the experimental groups. In particular, AFB1 exposure affected the expression of lncRNAs associated with cellular senescence signaling pathways, such as MSTRG.24315 and MSTRG.80767, as well as related genes, Cxcl8 and Gadd45g. In addition, AFB1 exposure affected the expression of miRNAs associated with immune-related genes, such as miR-181a, miR-331-3p and miR-342, as well as immune-related genes Nfkb1 and Rras2. Moreover, the regulation networks between mRNA-miRNAs and mRNA-lncRNAs were confirmed by the results of RT-qPCR and immunofluorescence. In conclusion, our results here demonstrate that AFB1 exposure impaired proliferation of 3D4/2 cells via the non-coding RNA-mediated pathway.

This study aimed to determine uterine blood flow indices by transabdominal Doppler ultrasound in sows (n = 18) under different conditions: (i) sows after estrus detection (day 0, D0); (ii) sows 2 h after artificial insemination (AI), performed 24 h after detection of estrus (day 1, D1); (iii) sows in early diestrus (day 5, D5). Moreover, three different types of seminal doses were used for AI depending on the ejaculate fraction included (F1: doses containing only the rich fraction of the ejaculate; F2: F1 + the transition fraction between rich and poor fractions; F3: F2 and poor fraction). The statistical analysis revealed significant differences in some indices regarding the period of analysis (D0, D1, and D5). Diastolic velocity and mean velocity showed lower values at D5 in comparison with D0 and D1 (p < 0.01). On the other hand, the pulsatility index and the relationship systolic velocity/diastolic velocity indicated higher values at D5 in comparison with D0 and D1 (p < 0.01). No differences were observed regarding the type of seminal dose used in any of the time points analyzed (p > 0.05). Neither insemination per se nor the type of ejaculate fraction used immediately modified the uterine vascularity, but some indices are affected by the stage of the estrus cycle (estrus vs. early diestrus). This research was supported by the Ministry of Science and Innovation (PID2019-106380RB-I00/AEI/10.13039/501100011033).

Early in life and particularly around weaning, piglets are susceptible to infections because of abrupt social, environmental, and dietary changes. Dietary interventions with probiotic bacteria have gained popularity because of the increased awareness of the direct link between diet and health. In this study, piglets received the probiotic strain Escherichia coli Nissle 1917 (EcN) or a control treatment perorally from day 2 after birth until 2 weeks post-weaning. To investigate spatio-temporal effects of EcN on the gut microbiota composition, intestinal epithelial gene expression and immune system, feces, digesta, blood, scraping material and mesenteric lymph node tissue were collected at different time points. In addition, oral vaccinations against Salmonella enterica serovar Typhimurium were administered on days 21 and 45 of the study to assess the immunocompetence. EcN-treated pigs showed a reduced diversity of taxa within the phylum Proteobacteria and a lower relative abundance of taxa within the genus Treponema during the pre-weaning period. Moreover, EcN induced T cell proliferation and Natural Killer cell activation in blood and enhanced IL-10 production in ex vivo stimulated mesenteric lymph node cells, the latter pointing toward a more regulatory or anti-inflammatory state of the local gut-associated immune system. These outcomes were primarily observed pre-weaning. No significant differences were observed between the treatment groups with regards to body weight, epithelial gene expression, and immune response upon vaccination. Differences observed during the post-weaning period between the treatment groups were modest. Overall, this study demonstrates that the pre-weaning period offers a ‘window of opportunity’ to modulate the porcine gut microbiota and immune system through dietary interventions such as EcN supplementation.

Tissues, such as skin, bones, and tendons, exhibit a piezoelectric effect, which may be an important phenomenon in terms of tissue renewal and regeneration as well as the possibility of modifying their mechanical behavior. In this article, we present the design and development of an in-house system for the precise measurement of electrical potentials and mechanical properties of tendons. The system was validated using tendon fascicle bundles derived from positional as well as energy-storing tendons from various adult mammals (porcine, bovine, and deer samples). The presented system is able to capture changes in elastic and viscoelastic properties of tissue as well as its time–voltage response and, thus, may be used in a broad spectrum of future studies to uncover factors influencing piezoelectric phenomena in tendons. This, in turn, will help to optimize current methods used in physiotherapy and postoperative treatment for effective tendon recovery.

In mammals, imprinted genes often exist in the form of clusters in specific chromosome regions. However, in pigs, genomic imprinting of a relatively few genes and clusters has been identified, and genes within or adjacent to putative imprinted clusters need to be investigated including those at the SGCE/PEG10 locus. The objective of this study was to, using porcine parthenogenetic embryos, investigate imprinting status of genes within the genomic region spans between the COL1A2 and ASB4 genes in chromosome 9. Whole-genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) were conducted with normal and parthenogenetic embryos, and methylome and transcriptome were analyzed. As a result, differentially methylated regions (DMRs) between the embryos were identified, and parental allele-specific expressions of the SGCE and PEG10 genes were verified. The pig imprinted interval was limited between SGCE and PEG10, since both the COL1A2 and CASD1 genes at the centromere-proximal region and the genes between PPP1R9A and ASB4 toward the telomere were non-imprinted and biallelically expressed. Consequently, our combining analyses of methylome, transcriptome, and informative polymorphisms revealed the boundary of imprinting cluster at the SGCE/PEG10 locus in pig chromosome 9 and consolidated the landscape of genomic imprinting in pigs.

Mesenchymal stem cells (MSCs) have been widely used as donor cells for somatic cell nuclear transfer (SCNT) to increase the efficiency of embryo cloning. Since replicative senescence reduces the efficiency of embryo cloning in MSCs during in vitro expansion, transfection of telomerase reverse transcriptase (TERT) into MSCs has been used to suppress the replicative senescence. Here, TERT-transfected MSCs in comparison with early passage MSCs (eMSCs) and sham-transfected MSCs (sMSCs) were used to evaluate the effects of embryo cloning with SCNT in a porcine model. Cloned embryos from tMSC, eMSC, and sMSC groups were indistinguishable in their fusion rate, cleavage rate, total cell number, and gene expression levels of OCT4, SOX2 and NANOG during the blastocyst stage. The blastocyst formation rates of tMSC and sMSC groups were comparable but significantly lower than that of the eMSC group (p < 0.05). In contrast, tMSC and eMSC groups demonstrated significantly reduced apoptotic incidence (p < 0.05), and decreased BAX but increased BCL2 expression in the blastocyst stage compared to the sMSC group (p < 0.05). Therefore, MSCs transfected with telomerase reverse transcriptase do not affect the overall development of the cloned embryos in porcine SCNT, but enables to maintain embryo quality, similar to apoptotic events in SCNT embryos typically achieved by an early passage MSC. This finding offers a bioengineering strategy in improving the porcine cloned embryo quality.

Objectives: The use of thermal ablative therapies in the pancreatic tumors is limited because of the risk of the vessel injury and potential pancreatitis or fistula formation. Electrochemotherapy (ECT) is an ablative therapy with established role in the treatment of cutaneous and liver tumors. This study was designed to evaluate the safety and feasibility of ECT of the pancreas in a porcine survival model. Methods: In the first group, 4 animals underwent computed tomography (CT)%guided percutaneous ECT with bleomycin of the pancreatic tail. In the second group (4 animals), the intraoperative ECTwith bleomycin of pancreatic tail and head was performed. Animals were followed for 7 days and then killed. Clinical parameters, CT imaging, laboratory, and histologic analysis were performed. Results: All pigs survived the ECT procedure and none of them developed clinical signs of acute pancreatitis or related complications. There were no signs of acute pancreatitis or damage to the large vessels present in the follow-up CT scans. No significant change in laboratory parameters was obtained after procedure. Conclusions: This study shows that ECT with bleomycin is feasible and safe in the pancreatic parenchyma. Clinical studies are needed to evaluate the efficacy of ECT in pancreatic cancer.

The cytokine platelet factor 4 (PF4) enhances differentiation and cell viability of different stem cells lines in vitro. This study investigated whether PF4 addition to customary pig embryo semi-defined culture media can improve their developmental outcome (Experiment 1) and ultimately replace the need for bovine serum albumin (BSA, Experiment 2). Experiment 1 added PF4 (100-1000 ng/mL, 0 = control) to NCSU-23 with 0.4 mg/mL BSA culturing 3430 presumptive zygotes. Experiment 2 added PF4 (100 -1000 ng/mL, 0 = Control-PVA) to a BSA-free medium (NCSU-23 with 0.3 mg/mL PVA) culturing 3820 presumptive zygotes. Zygote culture in NCSU-23 with 0.4 mg/mL BSA was used as overall control. All groups of Experiment 1 displayed similar rates of day 2-cleavage (range: 65.0 +/- 10.9 to 70.0 +/- 5.8%); of day 7-blastocyst rates (range: 46.6 +/- 10.0 to 56.4 +/- 8.2%) and of total day 7-blastocyst efficiency (range: 32.3 +/- 8.3 to 37.2 +/- 7.3%). Addition of PF4 did not affect total cell numbers of day 7 blastocysts (range: 44.1 +/- 23.2 to 50.5 +/- 26.4). In Experiment 2, PF4 accelerated embryo development, increasing (P < 0.01) blastocyst yield compared to 0-PF4, and blastocyst formation by day 5 adding PF4 100-500 ng/mL (range: 29.9 +/- 7.8 to 31.8 +/- 5.5%; P < 0.05) compared with BSA-control (17.2 +/- 8.2%) and PF4 1000 ng/mL (15.5 +/- 7.9%); showing similar blastocyst rates (range: 42.0 +/- 11.5 to 49.3 +/- 10.0%), total efficiency (28.0 +/- 8.2 to 32.3 +/- 7.1%) total cell numbers (range: 42.6 +/- 19.3 to 45.7 +/- 23.9) as BSA-controls. In conclusion, although PF4 did not show additive improvement under usual semi-defined, BSA-supplemented embryo media, it successfully replaced BSA sustaining porcine blastocyst production in chemically defined conditions. (C) 2019 Elsevier Inc. All rights reserved. Funding Agencies|Ministry of Science, Innovation and Universities-ERDF, Madrid, Spain [RTI2018-093525-B-I00]; Seneca Foundation, Murcia, SpainFundacion Seneca [19892/GERM/15]; Research Council FORMAS, Stockholm, Sweden [2017e00946]

In the early development of parthenogenetic embryo, cytoplasm and nucleic acid fragmentation may be a cause of lower embryo development. The purpose of this study was to evaluate whether embryonic development and apoptosis factors can be reduced by controlling the in-vitro culture environment by the addition of hormones, pregnancy serum and uterine milk. Our study showed that the activity of Casp-3 increased within the cytoplasm when artificially used hormones to induce the incubation environment, and PCNA's manifestation was low. However, the addition of pregnant serum appeared to lower the Casp-3 activity compared to the other groups. In addition, MMP-9 activity was increased and early embryo development and cytoplasmic fidelity were also increased. Therefore, the results of the present study showed that the use of gestational serum in the development of parthenogenetic embryo inhibit apoptosis and increases cytoplasmic reorganization by natural environmental control in in vitro culture.

The etiology of Porcine respiratory disease complex is complicated by infections with multiple pathogens, and multiple infections increase the difficulty in identifying the causal pathogen. In this present study, we developed a detection system of microbes from porcine respiratory by using TaqMan real-time PCR (referred to as Dempo-PCR) to screen a broad range of pathogens associated with porcine respiratory diseases in a single run. We selected 17 porcine respiratory pathogens (Actinobacillus pleuropneumoniae, Boldetella bronchiseptica, Haemophilus parasuis, Pasteurella multocida, Pasteurella multocida toxin, Streptococcus suis, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynovie, porcine circovirus 2, pseudorabies virus, porcine cytomegalovirus, swine influenza A virus, porcine reproductive and respiratory virus US strain, EU strain, porcine respiratory coronavirus and porcine hemagglutinating encephalomyelitis virus) as detection targets and designed novel specific primer-probe sets for seven of them. In sensitivity test by using standard curves from synthesized DNA, all primer-probe sets showed high sensitivity. However, porcine reproductive and respiratory virus is known to have a high frequency of genetic mutations, and the primer and probe sequences will need to be checked at a considerable frequency when performing Dempo-PCR from field samples. A total of 30 lung samples from swine showing respiratory symptoms on six farms were tested by the Dempo-PCR to validate the assay’s clinical performance. As the results, 12 pathogens (5 virus and 7 bacteria) were detected and porcine reproductive and respiratory virus US strain, Mycoplasma hyorhinis, Haemophilus parasuis, and porcine cytomegalovirus were detected at high frequency. These results suggest that Dempo-PCR assay can be applied as a screening system with wide detection targets.

The Duroc sire line has a smaller litter size compared to the Landrace dam line and we have previously observed fewer surface follicles on Duroc ovaries one day after weaning. In that same study, a broader cumulus expansion and faster nuclear maturation were observed for Duroc oocytes at 20 h of in vitro maturation (IVM), while Landrace oocytes showed more advanced stages of cortical granule distributions. However, no differences between breeds were observed after the final IVM period. The aim of this study was to assess subsequent in vitro embryo production (IVP) in Duroc and Landrace. Furthermore, follicle diameter and steroid hormone levels in follicular fluid (FF) were measured to study possible relation to oocyte developmental competence. Follicular phase sow ovaries were collected one day after weaning and follicle size of the 10 largest follicles were measured per ovary before aspiration. Cumulus-oocyte complexes (COCs) were matured in vitro, and cumulus expansion was analysed by assessing individual COC areas at 0 and 20 h. Fertilization of Duroc and Landrace oocytes was performed with sperm from both a Duroc and a Landrace boar. A larger follicle diameter was observed for Landrace animals (5.7 vs. 4.8 mm, P < 0.0001) and individual COC area was additionally larger at 0 h after aspiration (P < 0.0001) compared to Duroc. Contrary, cumulus expansion from 0 to 20 h of maturation was broader for Duroc oocytes than for Landrace (407 ± 67% vs. 319 ± 31%, P < 0.0001). After fertilization, cleavage rate was higher for Duroc oocytes, and the highest blastocyst yield was obtained for Duroc oocytes fertilized with the Landrace sperm. Steroid hormone analysis of the follicular fluid showed differences in the pathways between breeds with a higher total level of estrogens (P = 0.01) and aromatase products/substrates ratio (P < 0.01) in Landrace compared to Duroc. In conclusion, results suggest that Duroc oocytes have a better in vitro oocyte developmental competence when cultured under the same in vitro conditions and breed differences in steroidogenesis were found in the early follicular phase.

In this study, bovine sodium caseinate (NaCas) was hydrolyzed with four proteases, alcalase, savinase, subtilisin A, and flavourzyme. In addition to the structural changes occurred through the enzymatic hydrolysis, the solubility, oil binding capacity, zeta potential, emulsification properties, and in vitro antioxidant capacity, anti-carcinogenic and antidiabetic properties of hydrolysates were determined. FTIR combined with hierarchical cluster analysis (HCA) made in Amide I region enable to classification of the samples based on the changes of the secondary structure depending on the enzyme type and degree of fragmentation. Technological properties of NaCas were enhanced through the enzymatic hydrolysis, and those were more prominent in serine-type enzymes, regardless of the enzyme type, all hydrolysates showed high antioxidant capacities. All hydrolysates, specifically those produced by savinase and alcalase, reduced the viability of the carcinogenic Caco-2 cells in a dose-dependent manner and showed a very low level of cytotoxicity against healthy HEK-293 cells. The hydrolysis treatment made a significant contribution to the antidiabetic activity of NaCas. Particularly alcalase and savinase hydrolysates suppressed the activity of alpha- amylase and alpha- glucosidase. Therefore, the generated milk protein hydrolysates could be used in functional food developments for specific dietary purposes.

Candida albicans is a common cause of opportunistic mycoses worldwide and a major contributor in wound infections. The purpose of this study was to establish a fungal wound model and analyze the effects of a common antifungal agent against the proliferation of three C. albicans strains. Second degree burns were created, and then inoculated with one of three different C. albicans ATCC strains: 10261 reference strain, 64550 fluconazole resistant and 26310 fluconazole sensitive. After fungal inoculation, every wound was covered with dressings for 4 h to allow fungal colonization on every wound bed. After 4 h, the dressings were removed, and each wound was treated either once or twice daily with a topical terbinafine hydrochloride or left untreated. On days 2, 4 and 7 post inoculation, three wounds from each treatment group were scrub cultured and quantified. On day 2, wounds infected with the sensitive strains 26310 and 10261 and treated twice showed a significant reduction when compared against those infected wounds receiving once daily treatment. On day 4, wounds which were infected with C. albicans fluconazole sensitive (ATCC 26310) showed a significant reduction in fungal cell counts with treatment applied twice daily. A significant reduction in the colony counts was exhibited in all three strains at the seventh day with active as compared to the non-treated wounds. Twice daily treatment resulted in a lower fungal count than once daily treatment. Neither treatment was able to entirely eradicate C. albicans during the duration of this study. Establishing a reliable fungal wound model will help in the translational goal of identifying new antifungal that could be used clinically by wound care providers.

BackgroundPrevious studies observed that the intervertebral disc experiences the greatest forces during spinal manipulative therapy (SMT) and that the distribution of forces among spinal tissues changes as a function of the SMT parameters. However, contextualized SMT forces, relative to the ones applied to and experienced by the whole functional spinal unit, is needed to understand SMT’s underlying mechanisms.AimTo describe the percentage force distribution between spinal tissues relative to the applied SMT forces and total force experienced by the functional unit.MethodsThis secondary analysis combined data from 35 fresh porcine cadavers exposed to a simulated 300N SMT to the skin overlying the L3/L4 facet joint via servo-controlled linear motor actuator. Vertebral kinematics were tracked optically using indwelling bone pins. The functional spinal unit was then removed and mounted on a parallel robotic platform equipped with a 6-axis load cell. The kinematics of the spine during SMT were replayed by the robotic platform. By using serial dissection, peak and mean forces induced by the simulated SMT experienced by spinal structures in all three axes of motion were recorded. Forces experienced by spinal structures were analyzed descriptively and the resultant force magnitude was calculated.ResultsDuring SMT, the functional spinal unit experienced a median peak resultant force of 36.4N (IQR: 14.1N) and a mean resultant force of 25.4N (IQR: 11.9N). Peak resultant force experienced by the spinal segment corresponded to 12.1% of the total applied SMT force (300N). When the resultant force experienced by the functional spinal unit was considered to be 100%, the supra and interspinous ligaments experienced 0.3% of the peak forces and 0.5% of the mean forces. Facet joints and ligamentum flavum experienced 0.7% of the peak forces and 3% of the mean forces. Intervertebral disc and longitudinal ligaments experienced 99% of the peak and 96.5% of the mean forces.ConclusionIn this animal model, a small percentage of the forces applied during a posterior-to-anterior SMT reached spinal structures in the lumbar spine. Most SMT forces (over 96%) are experienced by the intervertebral disc. This study provides a novel perspective on SMT force distribution within spinal tissues.

The reprogramming of cells into induced neural stem cells (iNSCs), which are faster and safer to generate than induced pluripotent stem cells, holds tremendous promise for fundamental and frontier research, as well as personalized cell-based therapies for neurological diseases. However, reprogramming cells with viral vectors increases the risk of tumor development due to vector and transgene integration in the host cell genome. To circumvent this issue, the Sendai virus (SeV) provides an alternative integration-free reprogramming method that removes the danger of genetic alterations and enhances the prospects of iNSCs from bench to bedside. Since pigs are among the most successful large animal models in biomedical research, porcine iNSCs (piNSCs) may serve as a disease model for both veterinary and human medicine. Here, we report the successful generation of piNSC lines from pig fibroblasts by employing the SeV. These piNSCs can be expanded for up to 40 passages in a monolayer culture and produce neurospheres in a suspension culture. These piNSCs express high levels of NSC markers (PAX6, SOX2, NESTIN, and VIMENTIN) and proliferation markers (KI67) using quantitative immunostaining and western blot analysis. Furthermore, piNSCs are multipotent, as they are capable of producing neurons and glia, as demonstrated by their expressions of TUJ1, MAP2, TH, MBP, and GFAP proteins. During the reprogramming of piNSCs with the SeV, no induced pluripotent stem cells developed, and the established piNSCs did not express OCT4, NANOG, and SSEA1. Hence, the use of the SeV can reprogram porcine somatic cells without first going through an intermediate pluripotent state. Our research produced piNSCs using SeV methods in novel, easily accessible large animal cell culture models for evaluating the efficacy of iNSC-based clinical translation in human medicine. Additionally, our piNSCs are potentially applicable in disease modeling in pigs and regenerative therapies in veterinary medicine.

This dataset contains data collected during an in vivo experiment with pigs at the Wageningen University as part of the PhD Thesis Projects of Mirelle Geervliet and Hugo de Vries (first authors of the manuscript). This research project was made possible by The Netherlands Organisation for Scientific Research and Vereniging Diervoeders Nederland (VDN).

Adipose tissue-derived mesenchymal stromal/stem cells (ASCs) are considered important tools in regenerative medicine and are being tested in several clinical studies. Porcine models are frequently used to obtain adipose tissue, due to the abundance of material and because they have immunological and physiological similarities with humans. However, it is essential to understand the effects and safe application of ASCs from pigs (pASCs) as an alternative therapy for diseases. Although minipigs are easy-to-handle animals that require less food and space, acquiring and maintaining them in a bioterium can be costly. Thus, we present a protocol for the isolation and proliferation of ASCs isolated from adipose tissue of farm pigs. Adipose tissue samples were extracted from the abdominal region of the animals. Because the pigs were not raised in a controlled environment, such as a bioterium, it was necessary to carry out rigorous procedures for disinfection. After this procedure, cells were isolated by mechanical dissociation and enzymatic digestion. A proliferation curve was performed and used to calculate the doubling time of the population. The characterization of pASCs was performed by immunophenotyping and cell differentiation in osteogenic and adipogenic lineages. The described method was efficient for the isolation and cultivation of pASCs, maintaining cellular attributes, such as surface antigens and multipotential differentiation during in vitro proliferation. This protocol presents the isolation and cultivation of ASCs from farm pig as an alternative for the isolation and cultivation of ASCs from minipigs, which require strictly controlled maintenance conditions and a more expensive process.

As a member of the neurotrophic family, brain-derived neurotrophic factor (BDNF) provides a key link in the physiological process of mammalian ovarian follicle development, in addition to its functions in the nervous system. The emphasis of this study lay in the impact of BDNF on the proliferation of porcine follicular granulosa cells (GCs) in vitro. BDNF and tyrosine kinase B (TrkB, receptor of BDNF) were detected in porcine follicular GCs. Additionally, cell viability significantly increased during the culture of porcine GCs with BDNF (100 ng/mL) in vitro. However, BDNF knockdown in GCs decreased cell viability and S-phase cells proportion—and BDNF simultaneously regulated the expression of genes linked with cell proliferation (CCND1, p21 and Bcl2) and apoptosis (Bax). Then, the results of the receptor blocking experiment showed that BDNF promoted GC proliferation via TrkB. The high-throughput sequencing showed that BDNF also regulated the expression profiles of miRNAs in GCs. The differential expression profiles were obtained by miRNA sequencing after BDNF (100 ng/mL) treatment with GCs. The sequencing results showed that, after BDNF treatment, 72 significant differentially-expressed miRNAs were detected—5 of which were related to cell process and proliferation signaling pathways confirmed by RT-PCR. Furthermore, studies showed that BDNF promoted GCs’ proliferation by increasing the expression of CCND1, downregulating miR-127 and activating the ERK1/2 signal pathway. Moreover, BDNF indirectly activated the ERK1/2 signal pathway by downregulating miR-127. In conclusion, BDNF promoted porcine GC proliferation by increasing CCND1 expression, downregulating miR-127 and stimulating the MAPK-ERK1/2 signaling cascade.

Industrial farming of livestock is increasingly focused on high productivity and performance. As a result, concerns are growing regarding the safety of food and feed, and the sustainability involved in their production. Therefore, research in areas such as animal health, welfare, and the effects of feed additives on animals is of significant importance. In this study, an in vitro co-culture model of the piglet gut was used to investigate the effects of two phytogenic feed additives (PFA) with similar compositions. Intestinal porcine epithelial cells (IPEC-J2) were co-cultivated with peripheral blood mononuclear cells (PBMC) to model the complex porcine gut environment in vitro. The effects of treatments on epithelial barrier integrity were assessed by means of transepithelial electrical resistance (TEER) in the presence of an inflammatory challenge. Protective effects of PFA administration were observed, depending on treatment duration and the model compartment. After 48 h, TEER values were significantly increased by 12–13% when extracts of the PFA were applied to the basolateral compartment (p < 0.05; n = 4), while no significant effects on cell viability were observed. No significant differences in the activity of a PFA based mainly on pure chemical compounds versus a PFA based mainly on complex, natural essential oils, and extracts were found. Overall, the co-culture model was used successfully to investigate and demonstrate beneficial effects of PFAs on intestinal epithelial barrier function during an inflammatory challenge in vitro. In addition, it demonstrates that the two PFAs are equivalent in effect. This study provides useful insights for further research on porcine gut health status even without invasive in vivo trials.

Luteal angiogenesis is regulated by pro-angiogenic hormones including fibroblast growth factor 2 (FGF2) and angiopoietin 1 (Ang1), which are regulated by the adipokine leptin during development. Another adipokine, adiponectin, exhibits an inverse relationship with leptin and has been identified in the CL. Therefore, it is hypothesized that adiponectin will influence pro-angiogenic hormones in the developing porcine CL. Crossbred sows were randomly allocated to one of two days of the estrous cycle, day 5 (D5; n = 4) or day 7 (D7; n = 5) for CL collection. Tissue was processed for immunohistochemical localization of adiponectin receptor 2 (AdipoR2), gene expression of FGF2, Ang1, leptin, AdipoR2, and cell culture for adiponectin treatment. The expression of AdipoR2 tended (p = 0.09) to be higher in D7 lutea and was more prevalently localized to the cell surface of large and small luteal cells than in D5 tissue. Adiponectin influenced (p ≤ 0.05) FGF2, leptin, and AdipoR2 gene expression relative to the dose and day (D5 or D7). Collectively, the evidence supports the supposition that adiponectin influences angiogenic factors in the developing CL.

Advances in medical care, improvements in sanitation, and rising living standards contribute to increased life expectancy. Although this reflects positive human development, it also poses new challenges. Among these, reproductive aging is gradually becoming a key health issue because the age of menopause has remained constant at ~50 years, leading women to live longer in suboptimal endocrine conditions. An adequate understanding of ovarian senescence mechanisms is essential to prevent age-related diseases and to promote wellbeing, health, and longevity in women. We here analyze the impact of aging on the ovarian extracellular matrix (ECM), and we demonstrate significant changes in its composition and organization with collagen, glycosaminoglycans, and laminins significantly incremented, and elastin, as well as fibronectin, decreased. This is accompanied by a dynamic response in gene expression levels of the main ECM- and protease-related genes, indicating a direct impact of aging on the transcription machinery. Furthermore, in order to study the mechanisms driving aging and identify possible strategies to counteract ovarian tissue degeneration, we here described the successful production of a 3D ECM-based biological scaffold that preserves the structural modifications taking place in vivo and that represents a powerful high predictive in vitro model for reproductive aging and its prevention.

Background Reactive oxygen species (ROS) modulator 1 (ROMO1) is a mitochondrial membrane protein that is essential for the regulation of mitochondrial ROS production and redox sensing. ROMO1 regulates ROS generation within cells and is involved in cellular processes, such as cell proliferation, senescence, and death. Our purpose is to investigates the impact of ROMO1 on the mitochondria during porcine embryogenesis. Results We found that high expression of ROMO1 was associated with porcine preimplantation embryo development, indicating that ROMO1 may contribute to the progression of embryogenesis. Knockdown of ROMO1 disrupted porcine embryo development and blastocyst quality, thereby inducing ROS production and decreasing mitochondrial membrane potential. Knockdown of ROMO1 induced mitochondrial dysfunction by disrupting the balance of OPA1 isoforms to release cytochrome c, reduce ATP, and induce apoptosis. Meanwhile, ROMO1 overexpression showed similar effects as ROMO1 KD on the embryos. Overexpression of ROMO1 rescued the ROMO1 KD-induced defects in embryo development, mitochondrial fragmentation, and apoptosis. Conclusions ROMO1 plays a critical role in embryo development by regulating mitochondrial morphology, function, and apoptosis in pigs.

The use of frozen peripheral blood mononuclear cells (PBMC) is common in immunological studies. The impact of freezing PBMC has been assessed using human and mice cells, but little information is available regarding domestic animals. In the present study, the phenotype and functionality of frozen porcine PBMC were examined. In a preliminary experiment, three freezing media: fetal bovine serum plus 10% dimethyl sulfoxide, PSC cryopreservation kit, and Cryostor CS10, were compared regarding the preservation of cell viability and the response of PBMC to mitogens after thawing. After being stored one month in liquid nitrogen, cell viability was above 89% for all freezing media. The ELISPOT IFN-gamma (IFN-γ) results in response to PHA and of IgG ELISPOT in response to R848+IL-2 were similar to those obtained using fresh PBMC. In the second set of experiments, PBMC were obtained from five pigs vaccinated against Porcine reproductive and respiratory syndrome virus (PRRSV) and then frozen using Cryostor CS10. Recovered cells were phenotyped by flow cytometry using anti-CD3, CD4, CD8, and CD21 antibodies and were used to assess the PRRSV-specific responses in a proliferation experiment, an IFN-γ ELISPOT, and an IgG ELISPOT, and compared to the results obtained with fresh cells. The antigen-specific responses of frozen cells were significantly (p

Background The evaluation of the DNA damage generated in cumulus cells after mature cumulus-oocyte complexes vitrification can be considered as an indicator of oocyte quality since these cells play important roles in oocyte developmental competence. Therefore, the aim of this study was to determine if matured cumulus-oocyte complexes exposure to cryoprotectants (CPAs) or vitrification affects oocytes and cumulus cells viability, but also if DNA damage is generated in cumulus cells, affecting fertilization and embryo development. Results The DNA damage in cumulus cells was measured using the alkaline comet assay and expressed as Comet Tail Length (CTL) and Olive Tail Moment (OTM). Results demonstrate that oocyte exposure to CPAs or vitrification reduced oocyte (75.5 ± 3.69%, Toxicity; 66.7 ± 4.57%, Vitrification) and cumulus cells viability (32.7 ± 5.85%, Toxicity; 7.7 ± 2.21%, Vitrification) compared to control (95.5 ± 4.04%, oocytes; 89 ± 4.24%, cumulus cells). Also, significantly higher DNA damage expressed as OTM was generated in the cumulus cells after exposure to CPAs and vitrification (39 ± 17.41, 33.6 ± 16.69, respectively) compared to control (7.4 ± 4.22). In addition, fertilization and embryo development rates also decreased after exposure to CPAs (35.3 ± 16.65%, 22.6 ± 3.05%, respectively) and vitrification (32.3 ± 9.29%, 20 ± 1%, respectively). It was also found that fertilization and embryo development rates in granulose-intact oocytes were significantly higher compared to denuded oocytes in the control groups. However, a decline in embryo development to the blastocyst stage was observed after CPAs exposure (1.66 ± 0.57%) or vitrification (2 ± 1%) compared to control (22.3 ± 2.51%). This could be attributed to the reduction in both cell types viability, and the generation of DNA damage in the cumulus cells. Conclusion This study demonstrates that oocyte exposure to CPAs or vitrification reduced viability in oocytes and cumulus cells, and generated DNA damage in the cumulus cells, affecting fertilization and embryo development rates. These findings will allow to understand some of the mechanisms of oocyte damage after vitrification that compromise their developmental capacity, as well as the search for new vitrification strategies to increase fertilization and embryo development rates by preserving the integrity of the cumulus cells.

A significant amount of knowledge has been gained with the use of cell-based assays to elucidate the mechanisms that mediate heart valve calcification. However, cells used in these studies lack their association with the extra-cellular matrix or the influence of other cellular components of valve leaflets. We have developed a model of calcification using intact porcine valve leaflets, that relies upon a biological stimulus to drive the formation of calcified nodules within the valve leaflets. Alizarin Red positive regions were formed in response to lipopolysaccharide and inorganic phosphate, which could be quantified when viewed under polarized light. Point analysis and elemental mapping analysis of electron microscope images confirmed the presence of nodules containing calcium and phosphorus. Immunohistochemical staining showed that the development of these calcified regions corresponded with the expression of RUNX2, osteocalcin, NF-kB and the apoptosis marker caspase 3. The formation of calcified nodules and the expression of bone markers were both inhibited by adenosine in a concentration-dependent manner, illustrating that the model is amenable to pharmacological manipulation. This organ culture model offers an increased level of tissue complexity in which to study the mechanisms that are involved in heart valve calcification.

Bisphenol A (BPA) is an endocrine disruptor widely distributed in the environment due to its common use in the plastics industry. It is known that it has a strong negative effect on human and animal organisms, but a lot of aspects of this impact are still unexplored. This includes the impact of BPA on the enteric nervous system (ENS) in the large intestine. Therefore, the aim of the study was to investigate the influence of various doses of BPA on the neurons located in the descending colon of the domestic pig, which due to similarities in the organization of intestinal innervation to the human gastrointestinal tract is a good animal model to study processes occurring in human ENS. During this study, the double immunofluorescence technique was used. The obtained results have shown that BPA clearly affects the neurochemical characterization of the enteric neurons located in the descending colon. The administration of BPA caused an increase in the number of enteric neurons containing substance P (SP) and vasoactive intestinal polypeptide (VIP) with a simultaneously decrease in the number of neurons positive for galanin (GAL) and vesicular acetylcholine transporter (VAChT used as a marker of cholinergic neurons). Changes were noted in all types of the enteric plexuses, i.e., the myenteric plexus, outer submucous plexus and inner submucous plexus. The intensity of changes depended on the dose of BPA and the type of enteric plexus studied. The results have shown that BPA may affect the descending colon through the changes in neurochemical characterization of the enteric neurons located in this segment of the gastrointestinal tract.

Combining somatic cell nuclear transfer (SCNT) with genome editing technologies has emerged as a powerful platform for the creation of unique swine lineages for agricultural and biomedical applications. However, successful application of this research platform is still hampered by the low efficiency of these technologies, particularly in attaining complete cell reprogramming for the production of cloned pigs. Treating SCNT embryos with histone deacetylase inhibitors (HDACis), such as Scriptaid, has been routinely used to facilitate chromatin reprogramming after nuclear transfer. While increasing histone acetylation leads to a more relaxed chromatin configuration that facilitates the access of reprogramming factors and DNA repair machinery, it may also promote the expression of genes that are unnecessary or detrimental for normal embryo development. In this study, we evaluated the impact of inhibiting both histone deacetylases and RNA synthesis on pre- and post-implantation development of pig SCNT embryos. Our findings revealed that transcription can be inhibited for up to 40 h of development in porcine embryos, produced either by activation, fertilization or SCNT, without detrimentally affecting their capacity to form a blastocyst and their average number of cells at this developmental stage. Importantly, inhibiting RNA synthesis during HDACi treatment resulted in SCNT blastocysts with a greater number of cells and more abundant transcripts for genes related to embryo genome activation on days 2, 3 and 4 of development, compared to SCNT embryos that were treated with HDACi only. In addition, concomitant inhibition of histone deacetylases and RNA synthesis promoted the full reprograming of somatic cells, as evidenced by the normal fetal and full-term development of SCNT embryos. This combined treatment may improve the efficiency of the genome-editing + SCNT platform in swine, which should be further tested by transferring more SCNT embryos and evaluating the health and growth performance of the cloned pigs.

In vitro embryo research is an important stage for the advancement of many reproductive technologies in research and agriculture. For this reason, the improvement of in vitro embryo development is a strategic field worthy of investigation. Relatively little is known about miR-143 and its effects on autophagy associated with embryo development and in vitro embryo culture. In this study, we examined the effect of miR-143 (via mimics and inhibitors) on embryonic development threatened by microinjection after parthenogenetic activation. We evaluated rates of cleavage, blastocyst, and total cell number of blastocyst; additionally, we performed LC3 immunofluorescence analysis and mRNA expression analyses of genes associated with autophagy, endoplasmic reticulum (ER)-phagy, ER stress, embryo quality, and apoptosis. The inhibition of miR-143 positively influenced embryo development by increasing the activity of autophagy and ER-phagy and the expression of embryo quality-related genes, while reducing apoptosis. In contrast, treatment with miR-143 mimics increased ER stress-related gene expression and apoptosis, and reduced embryo development. Together, our findings indicate that miR-143 plays a role in the interplay between autophagy, ER-phagy, and embryo quality during early porcine embryo development.

Simple Summary CRISPR/Cas9 driven multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to DNA double-strand breaks at multiple loci simultaneously. To overcome this problem in porcine cells we utilized Target-AID, a base editing system, to edit multiple loci in the porcine genome. We showed that the Target-AID system works well in porcine fibroblasts with up to 63.15% efficiency. This is the first report demonstrating that the Target-AID system works well in porcine cells and can be used to generate genome-edited pigs. Abstract Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3–5 base editing windows 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

Candida auris readily colonizes skin and efficiently spreads among patients in healthcare settings worldwide. Given the capacity of this drug-resistant fungal pathogen to cause invasive disease with high mortality, hospitals frequently employ chlorhexidine bathing to reduce skin colonization. Using an ex vivo skin model, we show only a mild reduction in C. auris following chlorhexidine application. This finding helps explain why chlorhexidine bathing may have failures clinically, despite potent in vitro activity. We further show that isopropanol augments the activity of chlorhexidine against C. auris on skin. Additionally, we find both tea tree (Melaleuca alternifolia) oil and lemongrass (Cymbopogon flexuosus) oil to further enhance the activity of chlorhexidine/isopropanol for decolonization. We link this antifungal activity to individual oil components and show how some of these components act synergistically with chlorhexidine/isopropanol. Together, the studies provide strategies to improve C. auris skin decolonization through the incorporation of commonly used topical compounds.

Promyelocytic leukemia (PML) protein is a crucial component of PML-nuclear bodies (PML-NBs). PML and PML-NBs are involved in the regulation of various cellular functions, including the antiviral immune response. The human PML gene can generate several different isoforms through alternative splicing. However, little is known about the porcine PML alternative splicing isoforms and their expression profiles during Japanese encephalitis virus (JEV) infection. In the present study, we cloned seven mature transcripts of porcine PML, all of which contained the same N-terminal sequence but differed in the C-terminal sequences due to alternative splicing. These seven transcripts encoded five proteins all of which had the RBCC motif and sumoylation sites. Amino acid sequence homology analysis showed that porcine PML-1 had relatively high levels of identity with human, cattle, and goat homologs (76.21, 77.17, and 77.05%, respectively), and low identity with the mouse homolog (61.78%). Immunofluorescence analysis showed that the typical PML-NBs could be observed after overexpression of the five PML isoforms in PK15 cells. Quantitative reverse transcription PCR (RT-qPCR) analysis showed significant upregulation of PML isoforms and PML-NB-associated genes (Daxx and SP100) at 36 and 48 h post-infection (hpi). Western blotting analysis indicated that the PML isoforms were upregulated during the late stage of infection. Moreover, the number of PML-NBs was increased after JEV infection. These results suggest that porcine PML isoforms may play essential roles in JEV infection.

Background: Ischemia-reperfusion and cardiac remodeling is associated with cardiomyocyte death, excessive fibrosis formation, and functional decline, eventually resulting in heart failure (HF). Glucagon-like peptide (GLP)-1 agonists are reported to reduce apoptosis and myocardial infarct size after ischemia-reperfusion. Moreover, mineralocorticoid receptor antagonists (MRAs) have been described to reduce reactive fibrosis and improve cardiac function. Here, we investigated whether combined treatment with GLP-1R agonist exenatide and MRA potassium canrenoate could minimize cardiac injury and limit HF progression in animal models of chronic HF.Methods and Results: Forty female Topigs Norsvin pigs were subjected to 150 min balloon occlusion of the left anterior descending artery (LAD). Prior to reperfusion, pigs were randomly assigned to placebo or combination therapy (either low dose or high dose). Treatment was applied for two consecutive days or for 8 weeks with a continued high dose via a tunneled intravenous catheter. Using 2,3,5-Triphenyltetrazolium chloride (TTC) staining we observed that combination therapy did not affect the scar size after 8 weeks. In line, left ventricular volume and function assessed by three-dimensional (3D) echocardiography (baseline, 7 days and 8 weeks), and cardiac magnetic resonance imaging (CMR, 8 weeks) did not differ between experimental groups. In addition, 36 C57Bl/6JRj mice underwent permanent LAD-occlusion and were treated with either placebo or combination therapy prior to reperfusion, for two consecutive days via intravenous injection, followed by continued treatment via placement of osmotic mini-pumps for 28 days. Global cardiac function, assessed by 3D echocardiography performed at baseline, 7, 14, and 28 days, did not differ between treatment groups. Also, no differences were observed in cardiac hypertrophy, assessed by heart weight/bodyweight and heart weight/tibia length ratio.Conclusion: In the current study, combined treatment with GLP-1R agonist exenatide and MR antagonist potassium canrenoate did not show beneficial effects on cardiac remodeling nor resulted in functional improvement in a small and large animal chronic HF model.

Simple Summary Poor in vitro embryo development is a major obstacle in porcine assisted reproduction. In the current study, we utilized modified Spirulina maxima pectin nanoparticles as a supplement to improve porcine in vitro maturation medium. Results showed that modified Spirulina maxima pectin nanoparticles at 2.5 µg/mL improved oocyte maturation in form of first polar body extrusion, reduced oxidative stress, and increased the developmental competence of the oocytes after parthenogenetic activation and somatic cell nuclear transfer. Moreover, the relative transcripts quantification showed significant increase in the pluripotency-associated transcripts in the resultant cloned embryos after modified Spirulina maxima pectin nanoparticles supplementation. Therefore, we provide an optimum in vitro maturation condition to improve the in vitro embryo production in porcine. Abstract Molecular approaches have been used to determine metabolic substrates involved in the early embryonic processes to provide adequate culture conditions. To investigate the effect of modified Spirulina maxima pectin nanoparticles (MSmPNPs) on oocyte developmental competence, cumulus–oocyte complexes (COCs) retrieved from pig slaughterhouse ovaries were subjected to various concentrations of MSmPNPs (0, 2.5, 5.0, and 10 µg/mL) during in vitro maturation (IVM). In comparison to the control, MSmPNPs-5.0, and MSmPNPs-10 groups, oocytes treated with 2.5 µg/mL MSmPNPs had significantly increased glutathione (GSH) levels and lower levels of reactive oxygen species (ROS). Following parthenogenetic activation, the MSmPNPs-2.5 group had a considerably higher maturation and cleavage rates, blastocyst development, total cell number, and ratio of inner cell mass/trophectoderm (ICM:TE) cells, when compared with those in the control and all other treated groups. Furthermore, similar findings were reported for the developmental competence of somatic cell nuclear transfer (SCNT)-derived embryos. Additionally, the relative quantification of POU5F1, DPPA2, and NDP52 mRNA transcript levels were significantly higher in the MSmPNPs-2.5 group than in the control and other treated groups. Taken together, the current findings suggest that MSmPNP treatment alleviates oxidative stress and enhances the developmental competence of porcine in vitro matured oocytes after parthenogenetic activation and SCNT.