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Background Methods Results Conclusions Both global ischemia caused by circulatory arrest and extracorporeal circulation circuits have been shown to trigger cytokine release. We hypothesized that inserting a hemoadsorption device during thoraco‐abdominal normothermic regional perfusion (TA‐NRP) in the donation after circulatory death setting would mitigate the inflammatory response, potentially resulting in improved cardiac allograft function.In 15 pigs, circulatory arrest was induced by hypoxia. After a 15‐min no‐touch‐period, TA‐NRP was performed for 60 min. Eight pigs had a hemoadsorption device incorporated in the ECC, while seven did not. Plasma concentrations of IFN‐α, IFN‐γ, TNF‐α, IL‐1β, IL‐4, IL‐6, IL‐8, IL‐10, and IL‐12p40 were assessed by ELISA at baseline, immediately at start of TA‐NRP, 60 min after start of TA‐NRP (just before weaning from ECC), and at 30 and 60 min after weaning from ECC. Cardiac function was assessed with pressure–volume loop analysis.Hemoadsorption had no relevant effects on systemic cytokine levels post TA‐NRP. IL‐6 plasma levels gradually rose throughout the procedure for both groups. Hemoadsorption did not affect systolic or diastolic left ventricular function, nor were global hemodynamics improved by hemoadsorption.The insertion of a hemoadsorption device did not significantly affect plasma cytokine levels or cardiac function. Further research is necessary to assess the role of the inflammatory response in DCD heart transplantation and its modulation by TA‐NRP. [ABSTRACT FROM AUTHOR]

This study aimed to compare the effectiveness, in terms of viability and quality post‐warming, when vitrifying in vitro‐produced (IVP) pig blastocysts with either a modified Cryotop (n = 161; 20 blastocysts/device) or the conventional Superfine Open Pulled Straw (SOPS; n=160; 5‐6 blastocysts/device systems. Blastocyst viability, morphology, and apoptosis parameters were evaluated after 24 h of in vitro culture. The Cryotop system yields better results in terms of higher embryo viability and total cell numbers (p <.05) and lower apoptosis (p <.05) parameters than the SOPS procedure, defining a high effectiveness to simultaneously vitrify 20 pig IVP blastocysts at one time, thus increasing the yield of IVP blastocysts readily available for embryo transfer. [ABSTRACT FROM AUTHOR]

Parabens (PBs) are widely used in the cosmetic, pharmaceutical, and food industries as preservatives of products. Because of its great use, humans and other organisms are highly exposed daily. However, little is known about the effect of PBs on male infertility. Therefore, the aim of the present study was to evaluate the effect of methylparaben (MePB) and propylparaben (PrPB), alone or in combination, on the physiological characteristics of pig in vitro exposed sperm to different concentrations (0, 200, 500, and 700 μM) for viability, motility, and acrosome integrity evaluation and (0, 200, 500, 700, 1000, and 2000 μM) for DNA fragmentation index evaluation, after 4 h of exposure. The results showed that sperm viability decreased after exposure to MePB from the concentration of 500 μM. In the PrPB and mixture groups, viability decreased at all concentrations except for the control. The decrease in viability of sperm exposed to PrPB was greater than that of the mixture and MePB groups. Sperm motility decreased in all the experimental groups exposed to PBs, at all concentrations, except for the control group. Acrosome integrity was not decreased in the MePB group; however, in the PrPB group, it decreased at a concentration of 200 μM and in the mixture at 500 μM. All groups exhibited DNA damage at different concentrations, except for the control group. Additionally, the effect of PBs on sperm quality was concentration‐dependent. The results demonstrated that MePB and PrPB alone or in combination can have adverse effects on sperm quality parameters. MePB had lower toxicity than did both PrPB and the mixture. The mixture did not have an additive effect on any of the parameters evaluated. This could partially explain the link between PB exposure and infertility. Sperm viability decreased after exposure to MePB from the concentration of 500 μM. In the PrPB and mixture groups, viability decreased at all concentrations. Sperm motility decreased in all the experimental groups exposed to PBs at all concentrations. Acrosome integrity was not decreased in the MePB group; however, in the PrPB group, it decreased at a concentration of 200 μM and in the mixture at 500 μM. All groups exhibited DNA damage at different concentrations. [ABSTRACT FROM AUTHOR]

Clonazepam causes sedation and psychomotor impairment in people. Due to similarities between people and swine in response to benzodiazepines, clonazepam may represent a viable option to produce mild‐to‐moderate tranquillization in pigs. The objective of this study was to determine the pharmacokinetic profile of a single oral dose (0.5 mg/kg) of clonazepam in eight healthy, growing commercial cross pigs. Serial plasma samples were collected at baseline and up to 96 h after administration. Plasma concentrations were quantified using reverse‐phase high‐performance liquid chromatography, and compartment models were fit to time–concentration data. A one‐compartment first‐order model best fits the data. Maximum plasma concentration was 99.5 ng/mL, and time to maximum concentration was 3.4 h. Elimination half‐life was 7.3 h, mean residence time 7.4 h, and apparent volume of distribution 5.7 L/kg. Achieved plasma concentrations exceeded those associated with psychomotor impairment in people although pharmacodynamic effects have not been investigated in pigs. A simulated oral regimen consisting of 0.35 mg/kg administered every 8 h to pigs would achieve plasma concentrations above 32 ng/mL which are shown to produce psychomotor impairment in people. Further studies to test the clinical efficacy of these dosages in commercial and miniature pigs are warranted. [ABSTRACT FROM AUTHOR]

Background: Currently, high polyspermy remains a significant obstacle to achieving optimal efficiency in in vitro fertilization (IVF) and in vitro embryo production (IVP) systems in pigs. Developing strategies that would prevent polyspermy is essential in overcoming this challenge and maximizing the potential of this reproductive biotechnology. Previous results have demonstrated that using boar spermatozoa subjected to a high‐extension and reconcentration procedure and then cryopreserved resulted in significant improvements in IVF/IVP systems with high rates of monospermy and penetration. Objective: The aim of the present study was to unveil the molecular mechanisms that may underlie the changes in fertilization patterns exhibited by highly extended and cryopreserved boar spermatozoa. Materials and methods: To achieve this goal, we used quantitative proteomic analysis (LC‒ESI‒MS/MS SWATH) to identify differentially abundant proteins (DAPs) between highly extended (HE) and conventionally (control; CT) cryopreserved boar spermatozoa. Prior to the analysis, we evaluated the in vitro post‐thawing fertilizing ability of the sperm samples. The results demonstrated a remarkable improvement in monospermy and IVF efficiency when using HE spermatozoa in IVF compared with CT spermatozoa. Results: At the proteomic level, the combination of high‐extension and cryopreservation had a significant impact on the frozen–thawed sperm proteome. A total of 45 proteins (24 downregulated and 21 upregulated) were identified as DAPs (FC > 1 or ≤1; p < 0.05) when compared with CT spermatozoa. Some of these proteins were primarily linked to metabolic processes and the structural composition of sperm cells. The dysregulation of these proteins may have a direct or indirect effect on essential sperm functions and significantly affect spermatozoa–oocyte interaction and, therefore, the sperm fertilization profile under in vitro conditions. While these findings are promising, further research is necessary to comprehend how the disturbance of specific proteins affects sperm fertilization ability. [ABSTRACT FROM AUTHOR]

Background: Ex vivo normothermic machine perfusion (NMP) is a promising tool for assessing an isolated kidney prior to transplantation. However, there is no consensus on the perfusate's optimal oxygen‐carrying capacity to support renal function. To investigate the association of hemoglobin levels with renal function parameters, a retrospective analysis of isolated, normothermically, perfused porcine kidneys was performed. Methods: Between 2015 and 2021, a total of 228 kidneys underwent 4 h of NMP with perfusates that varied in hemoglobin levels. A generalized linear model was used to determine the association of hemoglobin levels with time‐weighted means of renal function markers, such as fractional sodium excretion (FENa) and creatinine clearance (CrCl). Stratified by baseline hemoglobin level (<4.5, 4.5–6, or >6 mmol/L), these markers were modeled over time using a generalized linear mixed‐effects model. All models were adjusted for potential confounders. Results: Until a hemoglobin level of around 5 mmol/L was reached, increasing hemoglobin levels were associated with superior FENa and CrCl. Thereafter, this association plateaued. When hemoglobin levels were categorized, hemoglobin <4.5 mmol/L was associated with worse renal function. Hemoglobin levels were neither significantly associated with proteinuria during NMP nor with ATP levels at the end of NMP. Hemoglobin levels >6 mmol/L showed no additional benefits in renal function. Conclusion: In conclusion, we found an association between baseline hemoglobin levels and superior renal function parameters, but not injury, during NMP of porcine kidneys. Furthermore, we show that performing a retrospective cohort study of preclinical data is feasible and able to answer additional questions, reducing the potential use of laboratory animals. [ABSTRACT FROM AUTHOR]

Chlorogenic acid (CGA) is an effective phenolic antioxidant that can scavenge hydroxyl radicals and superoxide anions. Herein, the protective effects and mechanisms leading to CGA‐induced porcine parthenogenetic activation (PA) in early‐stage embryos were investigated. Our results showed that 50 μM CGA treatment during the in vitro culture (IVC) period significantly increased the cleavage and blastocyst formation rates and improved the blastocyst quality of porcine early‐stage embryos derived from PAs. Then, genes related to zygotic genome activation (ZGA) were identified and investigated, revealing that CGA can promote ZGA in porcine PA early‐stage embryos. Further analysis revealed that CGA treatment during the IVC period decreased the abundance of reactive oxygen species (ROS), increased the abundance of glutathione and enhanced the activity of catalase and superoxide dismutase in porcine PA early‐stage embryos. Mitochondrial function analysis revealed that CGA increased mitochondrial membrane potential and ATP levels and upregulated the mitochondrial homeostasis‐related gene NRF‐1 in porcine PA early‐stage embryos. In summary, our results suggest that CGA treatment during the IVC period helps porcine PA early‐stage embryos by regulating oxidative stress and improving mitochondrial function. [ABSTRACT FROM AUTHOR]

A potbelly pig was evaluated for anorexia and icterus. Clinicopathologic abnormalities suggested an active inflammatory hepatobiliary process. Ultrasound and CT of the abdomen revealed an extrahepatic biliary obstruction of the common bile duct (CBD). Surgical exploration and choledochotomy revealed a markedly dilated CBD containing a large volume of intraluminal inspissated biliary material. This case report describes the imaging findings of an extrahepatic biliary obstruction secondary to abscessation within the CBD in a pig. [ABSTRACT FROM AUTHOR]

Background: Validated quantitative biomarkers for assessment of renal graft function during normothermic machine perfusion (NMP) conditions are lacking. The aim of this project was to quantify cortex microperfusion during ex vivo kidney perfusion using laser speckle contrast imaging (LSCI), and to evaluate the sensitivity of LSCI when measuring different levels of renal perfusion. Furthermore, we aimed to introduce LSCI measurements during NMP in differentially damaged kidneys. Methods: Eleven porcine kidneys were nephrectomized and perfused ex vivo. Cortex microperfusion was simultaneously monitored using LSCI. First, a flow experiment examined the relationship between changes in delivered renal flow and corresponding changes in LSCI‐derived cortex microperfusion. Second, renal cortical perfusion was reduced stepwise by introducing a microembolization model. Finally, LSCI was applied for measuring renal cortex microperfusion in kidneys exposed to minimal damage or 2 h warm ischemia (WI). Results: Cortex microperfusion was calculated from the LSCI‐obtained data. The flow experiment resulted in relatively minor changes in cortex microperfusion compared to the pump‐induced changes in total renal flow. Based on stepwise injections of microspheres, we observed different levels of cortex microperfusion that correlated with administrated microsphere dosages (r2 = 0.95–0.99). We found no difference in LSCI measured cortex microperfusion between the kidneys exposed to minimal damage (renal cortex blood flow index, rcBFI = 2090–2600) and 2 h WI (rcBFI = 2189–2540). Conclusions: Based on this preliminary study, we demonstrated the feasibility of LSCI in quantifying cortex microperfusion during ex vivo perfusion. Furthermore, based on LSCI‐measurements, cortical microperfusion was similar in kidneys exposed to minimal and 2 h WI. [ABSTRACT FROM AUTHOR]

Background: Pigs for biomedical research are administered commercial fluids made for human consumption when they receive intravenous (IV) fluid therapy. Although pigs and humans have slightly different bodily fluid compositions, the composition shift happens at the same rate as their rapid growth. Objective: The study aimed to analyse the composition of porcine blood according to breeding cycle using a portable blood analyser and provides data for developing customized IV fluids for pigs. Methods: Pigs were sorted 25, 50, 100 and 120 days after birth, and sows were classified into candidate, pregnant and farrowing groups. A blood sample was collected from the external jugular vein and analysed using the EPOC blood analysis system using haematological, biochemical and gas parameters. Results: There was no difference among pig groups by age, but hematocrit and haemoglobin amounts decreased in sows after farrowing, but their concentrations were higher as compared to pigs. Glucose gradually reduced as age increased in pigs and during pregnancy in sows. Conclusion: This study provided a comprehensive analysis of porcine blood composition by breeding cycle and highlighted the importance of glucose supplementation for IV fluid therapy in pigs. [ABSTRACT FROM AUTHOR]

Background: Malignant liver tumors seriously endanger human health. Among different therapeutic approaches, high‐frequency irreversible electroporation (H‐FIRE) is a recently emerging tumor ablation technique. The objective of this study was to evaluate the feasibility and safety of ultrasound‐guided percutaneous H‐FIRE using four electrode needles in porcine livers. Methods: Twelve experimental pigs underwent percutaneous H‐FIRE ablation using a compound steep‐pulse therapeutic device. Liver tissues adjacent to the gallbladder, blood vessels, and bile ducts were selected as the ablation targets. Pigs were randomly divided into three groups: (1) immediately after ablation (N = 4), (2) 2 days after ablation (N = 4), and (3) 7 days after ablation (N = 4). Blood routine, liver and kidney function, and myocardial enzyme levels were measured before and after ablation. Ultrasound, contrast‐enhanced ultrasound (CEUS), contrast‐enhanced magnetic resonance imaging (MRI), and hematoxylin–eosin staining were performed to evaluate the ablation performance. Results: Ultrasound‐guided percutaneous H‐FIRE ablations using four electrode needles were successfully performed in all 12 experimental pigs. The general conditions of the pigs, including postoperative activities and feeding behaviors, were normal, with no significant changes compared with the preoperative conditions. The imaging features of ultrasound, CEUS, and MRI demonstrated no significant changes in the gallbladder walls, bile ducts, or blood vessels close to the ablation areas. Laboratory tests showed that liver function indices and myocardial enzymes increased temporarily after H‐FIRE ablation, but decreased to normal levels at 7 days after ablation. Histopathological examinations of porcine liver specimens showed that this technique could effectively ablate the target areas without damaging the surrounding or internal vascular systems and gallbladder. Conclusions: This study demonstrated the feasibility and safety of ultrasound‐guided percutaneous H‐FIRE ablation in porcine livers in vivo, and proposed a four‐needle method to optimize its clinical application. [ABSTRACT FROM AUTHOR]

Background: Pig islet xenotransplantation is a potential treatment for type 1 diabetes. We have shown that maintenance immunosuppression is required to protect genetically modified (GM) porcine islet xenografts from T cell‐mediated rejection in baboons. Local expression of a depleting anti‐CD2 monoclonal antibody (mAb) by the xenograft may provide an alternative solution. We have previously reported the generation of GGTA1 knock‐in transgenic pigs expressing the chimeric anti‐CD2 mAb diliximab under an MHC class I promoter (MHCIP). In this study, we generated GGTA1 knock‐in pigs in which MHCIP was replaced by the β‐cell‐specific porcine insulin promoter (PIP), and compared the pattern of diliximab expression in the two lines. Methods: A PIP‐diliximab knock‐in construct was prepared and validated by transfection of NIT‐1 mouse insulinoma cells. The construct was knocked into GGTA1 in wild type (WT) porcine fetal fibroblasts using CRISPR, and knock‐in cells were used to generate pigs by somatic cell nuclear transfer (SCNT). Expression of the transgene in MHCIP‐diliximab and PIP‐diliximab knock‐in pigs was characterised at the mRNA and protein levels using RT‐qPCR, flow cytometry, ELISA and immunohistochemistry. Islets from MHCIP‐diliximab and control GGTA1 KO neonatal pigs were transplanted under the kidney capsule of streptozotocin‐diabetic SCID mice. Results: NIT‐1 cells stably transfected with the PIP‐diliximab knock‐in construct secreted diliximab into the culture supernatant, confirming correct expression and processing of the mAb in β cells. PIP‐diliximab knock‐in pigs showed a precise integration of the transgene within GGTA1. Diliximab mRNA was detected in all tissues tested (spleen, kidney, heart, liver, lung, pancreas) in MHCIP‐diliximab pigs, but was not detectable in PIP‐diliximab pigs. Likewise, diliximab was present in the serum of MHCIP‐diliximab pigs, at a mean concentration of 1.8 μg/mL, but was not detected in PIP‐diliximab pig serum. An immunohistochemical survey revealed staining for diliximab in all organs of MHCIP‐diliximab pigs but not of PIP‐diliximab pigs. Whole genome sequencing (WGS) of a PIP‐diliximab pig identified a missense mutation in the coding region for the dixilimab light chain. This mutation was also found to be present in the fibroblast knock‐in clone used to generate the PIP‐diliximab pigs. Islet xenografts from neonatal MHCIP‐diliximab pigs restored normoglycemia in diabetic immunodeficient mice, indicating no overt effect of the transgene on islet function, and demonstrated expression of diliximab in situ. Conclusion: Diliximab was widely expressed in MHCIP‐diliximab pigs, including in islets, consistent with the endogenous expression pattern of MHC class I. Further investigation is required to determine whether the level of expression in islets from the MHCIP‐diliximab pigs is sufficient to prevent T cell‐mediated islet xenograft rejection. The unexpected absence of diliximab expression in the islets of PIP‐diliximab pigs was probably due to a mutation in the transgene arising during the generation of the knock‐in cells used for SCNT. [ABSTRACT FROM AUTHOR]

Low‐density lipoproteins (LDL), quercetin (Q) and resveratrol (R) have been used for sperm preservation to improve sperm quality in different species. To evaluate the effects of LDL, Q and R during the cooling of boar semen. Fifteen boar semen samples were diluted in a BTS extender supplemented with the treatments: LDL at 6%, Q at 10 μM (Q10), 30 μM (Q30) and 50 μM (Q50), or R at 10 μM (R10), 30 μM (R30) and 50 μM. A control without supplementation was included. The semen was stored by cooling at 16°C for 96 h. Every 24 h, sperm motility and kinetics were evaluated using a computer‐assisted sperm analyzer (IVOS). At 24 and 96 h of cooling, functional membrane integrity and mitochondrial membrane potential (ΔΨM) of sperm were evaluated by the hypoosmotic swelling test (HOST) an flow cytometry with JC‐1 probe, respectively, LDL improved progressive motility of sperm during cooling. Likewise, LDL increased average path velocity (VAP) and straight‐line velocity (VSL) and/or curvilinear velocity (VCL) during the first 48 h of cooling. The use of Q between 10 and 30 μM caused a reduction in total motility, progressive motility and amplitude of the lateral head displacement during the entire cooling period, as well as a decrease in VAP, VSL and VCL at 96 h of cooling. LDL, Q10, Q30 and Q50 modulated mitochondrial activity by reducing high‐ΔΨM sperm at 0 and 96 h of cooling. During the cooling of the boar semen prior to artificial insemination, the parameters of sperm quality that could influence fertility decrease; however, the inclusion of antioxidants and additives that protect the plasma membrane, such as LDL, could mitigate the damaging effects on spermatozoa. It is concluded that LDL can improve the motility and kinetics of boar semen during cooling while it could modulating the sperm's mitochondrial activity. On the contrary, Q could alter the motility and kinetics of boar sperm during the cooling period. [ABSTRACT FROM AUTHOR]

Objective: Posterior tracheomalacia (TM) is characterized by excessive intraluminal displacement of the tracheal membranous wall. Recently, novel surgical strategies for repair of posterior TM have been introduced. To our knowledge, these strategies have not been evaluated in a model of posterior TM. Thus, we sought to design an ex vivo mechanical model of posterior TM to evaluate potential repair interventions. Methods: A model for posterior TM was created with partial thickness longitudinal incisions to the posterior aspect of ex vivo porcine trachea. Three groups of tracheas were tested: (1) control (unmanipulated), (2) posterior TM (injury), and (3) intervention (repair). Interventions included external splinting with 0.3 and 0.5 mm bioresorbable plates, posterior tracheopexy, and injection tracheoplasty with calcium hydroxylapatite. An airtight tracheal system was created to measure tracheal wall collapse with changes in negative pressure. A bronchoscope and pressure transducer were connected to either end. Cross‐sectional area of the tracheal lumen was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD). Results: Average percent reduction in cross‐sectional area of the tracheal lumen was compared using a two‐tailed paired t‐test. Significant differences were found between control and TM groups (p < 0.019). There was no significant difference between control and external splinting and posterior tracheopexy groups (p > 0.14). Conclusion: We describe an ex vivo model for posterior TM that replicates airway collapse. External splinting and tracheopexy interventions showed recovery of the injured tracheal segment. Injection tracheoplasty did not improve the TM. Level of Evidence: N/A Laryngoscope, 133:2000–2006, 2023 [ABSTRACT FROM AUTHOR]

The application of flap surgery is becoming more and more widespread with the development of microsurgical techniques. Currently, postoperative blood flow monitoring of flaps is still mainly assessed by medical staff for traditional clinical parameters, which has the disadvantage of being subjective and unable to monitor in real‐time. This study describes a self‐contained infrared wireless infrared thermometry device for flap blood supply monitoring and evaluates its effectiveness on eight porcine flap models. A scapular muscle flap model was established using eight small pigs, and the vessels were ligated at irregular intervals using a lumir ligature to simulate arterial crisis and venous crisis. Laser Doppler flowmetry (LDF), the wireless infrared thermometry device, and traditional clinical observation methods were applied to monitor the blood supply of the flap and evaluate the effect. The time to the determination of blood supply disturbance by wireless infrared thermography (IRT) was 28.75 ± 3.30 min and 96.5 ± 27.09 min for the arterial and venous groups, respectively; by LDF was 6.00 ± 1.41 min and 52.75 ± 15.76 min; by clinical observation was 42.00 ± 8.60 min and 156.50 ± 40.91 min, respectively. Paired t‐tests were performed between the wireless IRT device and clinical observations, and the statistical results were significantly different in the arterial group and not significantly different in the venous group. Paired t‐testing of the wireless infrared thermometry device with the LDF also showed significant differences in the arterial group and non‐significant differences in the venous group. This wireless infrared thermometry device outperforms traditional clinical observation methods in monitoring blood supply in a porcine skin flap model. Because of its low cost, real‐time monitoring, simple operation, and non‐invasive features, it has the potential to be used in clinical practice as a routine means of postoperative blood supply monitoring in flap surgery. [ABSTRACT FROM AUTHOR]

Y‐box binding protein 1 (YBX1) is a member of the family of DNA‐ and RNA‐binding proteins that play crucial roles in multiple aspects, including RNA stabilization, translational repression, and transcriptional regulation; however, its roles in embryo development remain less known. In this study, to investigate the function of YBX1 and its mechanism of action in porcine embryo development, YBX1 was knocked down by microinjecting YBX1 siRNA at the one‐cell stage. YBX1 is located in the cytoplasm during embryonic development. The mRNA level of YBX1 was increased from the four‐cell stage to the blastocyst stage but was significantly decreased in YBX1 knockdown embryos compared with the control. Moreover, the percentage of blastocysts was decreased following YBX1 knockdown compared with the control. Defecting YBX1 expression increased maternal gene mRNA expression and decreased zygotic genome activation (ZGA) gene mRNA expression and histone modification owing to decreased levels of N6‐methyladenosine (m6A) writer N6‐adenosine‐methyltransferase 70 kDa subunit (METTL3) and reader insulin‐like growth factor 2 mRNA‐binding protein (IGF2BP1). In addition, IGF2BP1 knockdown showed that YBX1 regulated the ZGA process through m6A modification. In conclusion, YBX1 is essential for early embryo development because it regulates the ZGA process. [ABSTRACT FROM AUTHOR]

Background: Hemolysis testing of new devices to treat heart failure is a regulatory requirement. The ASTM F1841–97 standard for hemolysis testing was developed for continuous flow pumps and does not specify test rig design. When research groups use different methodologies, results are difficult to compare. Pulsatile flow pump rigs require compliance chambers, and thus, the Aachen rig (Gräf et al) was developed for the pulsatile Reinheart TAH. The study objective was to use this rig to test the early Realheart TAH prototype V11C hemolysis performance compared to literature. Methods: The experimental control was the continuous flow pump BPX‐80 (Medtronic) and pooled heparinized porcine blood was used. Results: The mgNIH of BPX‐80 and V11C was 5.42 ± 1.47 and 25.20 ± 5.46 mg/100 L, respectively. The NIH ratio of V11C over BPX‐80 was 5.5. Conclusion: The absolute and the relative hemolysis of the V11C are lower compared to both the large and small Reinheart TAH devices published values. Pulsatile pumps create more hemolysis in the Aachen rig, and it is not known if this is because how the rig handles pulsatile flow or due to the devices. Future studies will, therefore, use a pulsatile pump such as the SynCardia as clinical comparator and human blood to test the performance of future Realheart TAH prototypes. [ABSTRACT FROM AUTHOR]

Background: Peripheral blood vessels in pigs are not easily accessible, making placement of intravenous catheters difficult. Alternative methods to intravenous administration of fluids, such as administering fluids via the rectum (proctoclysis), are warranted in pigs. Hypothesis: Administration of polyionic crystalloid fluids via proctoclysis results in hemodilution changes similar to intravenous administration. The objectives of this study were to evaluate the tolerance for proctoclysis in pigs and compare analytes before and after intravenous or proctoclysis therapy. Animals: Six healthy, growing, academic institution‐owned pigs. Methods: Randomized, cross‐over design clinical trial, with 3 treatments (control, intravenous, and proctoclysis) with a 3‐day washout period. The pigs were anesthetized and jugular catheters were placed. A polyionic fluid (Plasma‐Lyte A 148) was administered at 4.4 mL/kg/h during the intravenous and proctoclysis treatments. Laboratory analytes, including PCV, plasma, and serum total solids, albumin, and electrolytes were measured over 12 h at T0, T3, T6, T9, and T12. Effects of treatment and time on analytes were determined by analysis of variance. Results: Proctoclysis was tolerated by pigs. Albumin concentrations decreased during the IV treatment between T0 and T6 (least square mean of 4.2 vs 3.9 g/dL; 95% CI of mean difference = −0.42, −0.06; P =.03). Proctoclysis did not significantly affect any laboratory analytes at any time points (P >.05). Conclusions and Clinical Importance: Proctoclysis did not demonstrate hemodilution similar to intravenous administration of polyionic fluids. Proctoclysis might not be an effective alternative to the intravenous administration of polyionic fluids in healthy euvolemic pigs. [ABSTRACT FROM AUTHOR]

Background: Pacemaker implantation in infants and small children is limited to epicardial lead placement via open chest surgery. We propose a minimally invasive solution using a novel percutaneous access kit. Objective: To evaluate the acute safety and feasibility of a novel percutaneous pericardial access tool kit to implant pacemaker leads on the epicardium under direct visualization. Methods: A custom sheath with optical fiber lining the inside wall was built to provide intrathoracic illumination. A Veress needle inside the illumination sheath was inserted through a skin nick just to the left of the xiphoid process and angled toward the thorax. A needle containing a fiberscope within the lumen was inserted through the sheath and used to access the pericardium under direct visualization. A custom dilator and peel‐away sheath with pre‐tunneled fiberscope was passed over a guidewire into the pericardial space via modified Seldinger technique. A side‐biting multipolar pacemaker lead was inserted through the sheath and affixed against the epicardium. Results: Six piglets (weight 3.7–4.0 kg) had successful lead implantation. The pericardial space could be visualized and entered in all animals. Median time from skin nick to sheath access of the pericardium was 9.5 (interquartile range [IQR] 8–11) min. Median total procedure time was 16 (IQR 14–19) min. Median R wave sensing was 5.4 (IQR 4.0–7.3) mV. Median capture threshold was 2.1 (IQR 1.7–2.4) V at 0.4 ms and 1.3 (IQR 1.2–2.0) V at 1.0 ms. There were no complications. Conclusion: Percutaneous epicardial lead implantation under direct visualization was successful in six piglets of neonatal size and weight with clinically acceptable acute pacing parameters. [ABSTRACT FROM AUTHOR]

Intestinal epithelial stem cells (ISCs) are responsible for intestinal epithelial barrier renewal; thereby, ISCs play a critical role in intestinal pathophysiology research. While transgenic ISC reporter mice are available, advanced translational studies lack a large animal model. This study validates ISC isolation in a new porcine Leucine Rich Repeat Containing G Protein‐Coupled Receptor 5 (LGR5) reporter line and demonstrates the use of these pigs as a novel colorectal cancer (CRC) model. We applied histology, immunofluorescence, fluorescence‐activated cell sorting, flow cytometry, gene expression quantification, and 3D organoid cultures to whole tissue and single cells from the duodenum, jejunum, ileum, and colon of LGR5‐H2B‐GFP and wild‐type pigs. Ileum and colon LGR5‐H2B‐GFP, healthy human, and murine biopsies were compared by mRNA fluorescent in situ hybridization (FISH). To model CRC, adenomatous polyposis coli (APC) mutation was induced by CRISPR/Cas9 editing in porcine LGR5‐H2B‐GFP colonoids. Crypt‐base, green fluorescent protein (GFP) expressing cells co‐localized with ISC biomarkers. LGR5‐H2B‐GFPhi cells had significantly higher LGR5 expression (p <.01) and enteroid forming efficiency (p <.0001) compared with LGR5‐H2B‐GFPmed/lo/neg cells. Using FISH, similar LGR5, OLFM4, HOPX, LYZ, and SOX9 expression was identified between human and LGR5‐H2B‐GFP pig crypt‐base cells. LGR5‐H2B‐GFP/APCnull colonoids had cystic growth in WNT/R‐spondin‐depleted media and significantly upregulated WNT/β‐catenin target gene expression (p <.05). LGR5+ ISCs are reproducibly isolated in LGR5‐H2B‐GFP pigs and used to model CRC in an organoid platform. The known anatomical and physiologic similarities between pig and human, and those shown by crypt‐base FISH, underscore the significance of this novel LGR5‐H2B‐GFP pig to translational ISC research. [ABSTRACT FROM AUTHOR]

Objective: Porcine models of ocular disease are becoming increasingly utilized. A recently commercialized ocular tonometer, the Reichert Tono‐Vera® Vet, has not been evaluated for use in pigs. The purpose of this study was to calibrate this device for use in porcine eyes and to determine which settings are most appropriate for use in pigs. Procedure: The anterior chambers of five freshly enucleated normal porcine eyes were cannulated then connected to a reservoir of balanced salt solution and a physiologic monitor. Triplicate measurements were obtained with the four available settings: dog, cat, horse, and rabbit at intraocular pressures ranging from 5– to 80 mmHg. Bland–Altman analysis was utilized to determine bias and 95% limits of agreement for each setting. Results: There was a strong positive linear regression trend for all settings (dog r2 = 0.986, horse r2 = 0.947, cat r2 = 0.977, and rabbit r2 = 0.982). The linear regression equations for the dog, horse, cat, and rabbit setting were y = 1.0168x – 2.6128, y = 0.8743x – 3.4959, y = 0.9394x – 7.3188, and y = 1.1082x – 3.4077. The average bias and 95% limits of agreement for dog, horse, cat, and rabbit settings were − 2.00, −8.32, −9.58, and 0.57 mmHg, and (−7.52, 3.53), (−19.00, 2.37), (−16.66, −2.50), and (−7.79, 8.93), in mmHg. Conclusion: The Tono‐Vera® Vet dog setting was most accurate and precise setting compared to true intraocular pressures. This setting is likely to be appropriate for in vivo use in pigs, with the appropriate correction formula applied. [ABSTRACT FROM AUTHOR]

Segmental bone defects are often performed with cryopreserved allografts. They provide immediate stability, but risk nonunion, infection and late stress fracture. Improving the rate and extent of bone revitalization may improve results. Angiogenesis from surgically placed arteriovenous (AV) bundles improves bone blood flow and vitality in cryopreserved rat femora, augmented by vasculogenic growth factors. This study tests the same principal in Yucatan mini‐pigs with a tibial diaphyseal defect, combining surgical angiogenesis with angiogenic gene therapy within cryopreserved orthotopically‐placed allografts. Tibial diaphyseal defects were reconstructed with cryopreserved allografts and rigid internal fixation in 16 mini pigs. Half of the cranial tibial AV bundles placed within the allograft medullary canal were transfected with an adeno‐associated virus containing vascular endothelial growth factor (VEGF) and platelet‐derived growth factor (PDGF) genes (AAV9.VEGF.PDGF). Bone remodeling, angiogenesis, and allograft healing were assessed. During the postoperative survival period 5 of 8 transfected animals developed cutaneous benign vascular lesions at sites remote from the operated hindlimb, causing excessive bleeding. Within the allograft, both medullary (p = 0.013) and cortical (p = 0.009) vascular volumes were higher and vessels more mature than nontransfected allografts. Bone turnover (p = 0.013), bone mineralization (p = 0.018), bone healing (p = 0.008) and graft incorporation (p = 0.006) were all significantly higher in the gene therapy group. In a large animal tibial defect model, gene therapy of implanted AV bundles improved revascularization, remodeling and healing of cryopreserved allografts used for limb reconstruction. However, benign vascular lesions causing excessive bleeding developed in 5 out of 8 pigs transfected with AAV containing genes for VEGF and PDGF. This unforeseen complication makes vasculogenic gene therapy unacceptable for clinical use. [ABSTRACT FROM AUTHOR]

New Findings: What is the central question of this study?Invasive cardiovascular instrumentation can occur through closed‐ or open‐chest approaches. To what extent will sternotomy and pericardiotomy affect cardiopulmonary variables?What is the main finding and its importance?Opening of the thorax decreased mean systemic and pulmonary pressures. Left ventricular function improved, but no changes were observed in right ventricular systolic measures. No consensus or recommendation exists regarding instrumentation. Methodological differences risk compromising rigour and reproducibility in preclinical research. Animal models of cardiovascular disease are often evaluated by invasive instrumentation for phenotyping. As no consensus exists, both open‐ and closed‐chest approaches are used, which might compromise rigour and reproducibility in preclinical research. We aimed to quantify the cardiopulmonary changes induced by sternotomy and pericardiotomy in a large animal model. Seven pigs were anaesthetized, mechanically ventilated and evaluated by right heart catheterization and bi‐ventricular pressure–volume loop recordings at baseline and after sternotomy and pericardiotomy. Data were compared by ANOVA or the Friedmann test where appropriate, with post‐hoc analyses to control for multiple comparisons. Sternotomy and pericardiotomy caused reductions in mean systemic (−12 ± 11 mmHg, P = 0.027) and pulmonary pressures (−4 ± 3 mmHg, P = 0.006) and airway pressures. Cardiac output decreased non‐significantly (−1329 ± 1762 ml/min, P = 0.052). Left ventricular afterload decreased, with an increase in ejection fraction (+9 ± 7%, P = 0.027) and coupling. No changes were observed in right ventricular systolic function or arterial blood gases. In conclusion, open‐ versus closed‐chest approaches to invasive cardiovascular phenotyping cause a systematic difference in key haemodynamic variables. Researchers should adopt the most appropriate approach to ensure rigour and reproducibility in preclinical cardiovascular research. [ABSTRACT FROM AUTHOR]

Pigs are at risk of vomiting from medical conditions as well as the emetic side effects of drugs administered for peri‐operative manipulations, but there is a lack of pharmacokinetic data for potential anti‐emetic therapies, such as maropitant, in this species. The main objective of this study was to estimate plasma pharmacokinetic parameters for maropitant in pigs after a single intramuscular (IM) administration dosed at 1.0 mg/kg. A secondary objective was to estimate pilot pharmacokinetic parameters in pigs after oral (PO) administration at 2.0 mg/kg. Maropitant was administered to six commercial pigs at a dose of 1.0 mg/kg IM. Plasma samples were collected over 72 h. After a 7‐day washout period, two pigs were administered maropitant at a dose of 2.0 mg/kg PO. Maropitant concentrations were measured via liquid chromatography/mass spectrometry (LC–MS/MS). A non‐compartmental analysis was used to derive pharmacokinetics parameters. No adverse events were noted in any of the study pigs after administration. Following single IM administration, maximum plasma concentration was estimated at 412.7 ± 132.0 ng/mL and time to maximum concentration ranged from 0.083 to 1.0 h. Elimination half‐life was estimated at 6.7 ± 1.28 h, and mean residence time was 6.1 ± 1.2 h. Volume of distribution after IM administration was 15.9 L/kg. Area under the curve was 1336 ± 132.0 h*ng/mL. The relative bioavailability of PO administration was noted to be 15.5% and 27.2% in the two pilot pigs. The maximum systemic concentration observed in the study pigs after IM administration was higher than what was observed after subcutaneous administration in dogs, cats, or rabbits. The achieved maximum concentration exceeded the concentrations for anti‐emetic purposes in dogs and cats; however, a specific anti‐emetic concentration is currently not known for pigs. Further research is needed into the pharmacodynamics of maropitant in pigs to determine specific therapeutic strategies for this drug. [ABSTRACT FROM AUTHOR]

The fractionated picosecond laser produces microscopic lesions in the epidermis and dermis due to laser‐induced optical breakdown (LIOB). There have been multiple histological reports, but the present literature lacks detailed in vivo studies after treatment with high‐power laser systems. Our study aimed to characterize the healing patterns of microlesions induced with 150 ps duration 1064 nm MLA‐type picosecond laser. The induced picosecond laser‐tissue reactions with pulse energy of 50–250 mJ and different treatment modes were observed in in vivo porcine skin model over 10 days after the laser procedure. A macroscopic evaluation was combined with microscopic histological analysis to observe the healing dynamics of laser‐induced microlesions. Superficial, intraepidermal cavitation bubbles were induced using microbeam fluence of 4–20 J/cm2. Skin irritation scores positively correlated with pulse energy and dose. Our findings demonstrate that dose and pulse energy had a direct impact on epidermal thickness and lesions healing dynamics. [ABSTRACT FROM AUTHOR]

An approximately 12‐year‐old female Vietnamese Pot‐Bellied Pig was presented to the Mississippi State College of Veterinary Medicine Food Animal Service for anorexia of 2 days duration. On physical examination, the patient appeared depressed and lethargic with significantly pale mucus membranes, open mouth breathing, and nostril flaring. On abdominal palpation, the abdomen was tense and uncomfortable. A complete blood count (CBC) and chemistry profile were performed. The CBC revealed significant anemia and mild leukocytosis characterized by mild neutrophilia with a left shift. Mast cells were rarely observed. Hematocrit = 8.1% (RI 22‐50), RBC = 1.25 × 106/μL (RI 3.6‐7.8), WBC = 19.85 × 103/μL (RI 5.2‐17.9), Neutrophils = 15.08 × 103/μL (RI 0‐11.4), and Bands = 0.993 × 103/μL (RI 0‐0.019). The chemistry profile was unremarkable with a mildly elevated BUN and slightly decreased total protein and albumin (BUN = 39 mg/dL [RI 4.2‐15.1], total protein = 6.2 g/dL [RI 6.6‐8.9], and albumin = 2.5 g/dL [RI 3.6‐5.0]). An abdominal ultrasound revealed numerous hypoechoic nodules diffusely scattered throughout the hepatic parenchyma. An FNA of one of the hepatic nodules was performed. A mild suppurative component and numerous variably granulated mast cells were observed. A presumptive cytologic diagnosis of mast cell tumor was made. Histopathology was performed, confirming the cytologic interpretation. [ABSTRACT FROM AUTHOR]

Oxidative stress influences the embryo production efficiency in vitro. We investigated the effects of alpha lipoic acid (ALA) treatment during the in vitro maturation (IVM) period on the porcine somatic cell nuclear transfer (SCNT) embryo production. After IVM, maturation rates of the 12.5‐ and 25‐μM ALA‐treated groups were not significantly different from those of the 0‐μM ALA‐treated group. Compared to those in the 0‐μM ALA‐treated group, the reactive oxygen species and glutathione levels were significantly decreased and increased, respectively, in the cytoplasm of matured oocytes in the 12.5–50‐μM ALA‐treated groups. Apoptosis rate in cumulus cells after IVM was significantly lower in the 12.5–50‐μM ALA‐treated groups than in the 0‐μM ALA‐treated group. Blastocyst formation rate was significantly higher in parthenogenetic oocytes treated with 12.5‐μM ALA than in the 0‐, 25‐, and 50‐μM ALA‐treated groups. Similarly, in SCNT embryos, the 12.5‐μM ALA‐treated group showed a significantly higher blastocyst formation rate than the 0‐μM ALA‐treated group. Apoptosis rate in SCNT blastocysts was significantly decreased by 12.5‐μM ALA treatment. The results showed that treatment with 12.5‐μM ALA during IVM improves porcine SCNT embryo development and partial quality. [ABSTRACT FROM AUTHOR]

The process of wound healing is critical to maintaining homeostasis after injury. Although a considerable amount has been learned about this complex process, much remains unknown. Whereas, studies with human volunteers are ideal given the unique nature of the human skin anatomy and immune system, the lack of such clinical access has made animal models prime candidates for use in preclinical studies. This review aims to discuss the strengths and limitations of the commonly used mammalian species in wound healing studies: murine, rabbit and porcine. Thereafter, a survey of models of various acute wounds such as cutaneous, ear, and implant are presented and representative studies that use them are described. This review is intended to acquaint readers with the vast spectrum of models available, each of which has a distinct utility. At the same time, it highlights the importance of utilising clinical samples to complement investigations conducted in animal models. Through this strategy, it is hoped that forthcoming research may be more reflective of the acute wound healing process as it occurs in humans. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Seminal plasma (SP) affects reproduction, inducing cell and molecular changes in the female genital tract. A main active component in SP is the modulatory transforming growth factor‐β (TGF‐β), particularly its TGF‐β1 isoform, which affects the synthesis of other cytokines as granulocyte‐macrophage colony‐stimulating factor, relevant for embryo development and pregnancy. This study evaluated the effect of pooled frozen–thawed SP and commercial TGF‐β1 infused during oestrus in sows post‐cervically inseminated with liquid extended semen, containing ~4 ml of residual SP, on their fertility and prolificacy. For this, 250 sows in their post‐weaning oestrus were used. Sows were randomly assigned to one of the following groups to be post‐cervically treated 30 min before insemination: (i) SP group: infused with 40 ml of SP (N = 57); ii) Group TFGß1: infused with 40 ml of BTS extender containing 3 ng/ml of porcine TGF‐β1 (N = 64); iii) BTS group: infused with 40 ml of BTS extender (N = 60); and iv) Control Group: sows catheterized but not infused prior to AI (N = 69). Farrowing rates (range: 86.7% to 91.3%) and numbers of live‐born piglets (range: range: 12.8 ± 2.9 to 13.4 ± 3.1) were not affected by any treatment compared with Controls, indicating that neither pre‐infusions of SP nor TGF‐ß1 30 min before AI influenced subsequent fertility and prolificacy. [ABSTRACT FROM AUTHOR]

The quality of pre‐implantation embryos could affect developmental efficiency after embryo transfer. However, the assessment of pre‐implantation embryos was unsatisfactory, especially in pig embryos to date. Therefore, this study was designed to investigate available and applicable parameters that indicate developmental potential and quality of porcine pre‐implantation embryos produced by handmade cloning (HMC), and parthenogenetic activation without zona pellucida (PAZF) and with zona pellucida (PAZI). Firstly, a common division behaviour was detected, that is the formation of uneven division with two unequal size blastomeres (UD 2‐cell), especially in HMC embryos; then, the proportion of UD 2‐cell was found to be significantly higher than that of even division with equal size blastomeres (ED 2‐cell) (72.56 ± 4.56 vs. 24.57 ± 1.92). The formation of UD 2‐cell might be due to the spindle migration along the long axis in 1‐cell stage, and the cleavage furrow was not formed in the centre of cytoplasm. In the two sister blastomeres of UD 2‐cell, uneven distribution of organelles (mitochondria and lipid droplet) was observed with lower proportion in the smaller one (p <.05). Although no difference in blastocyst rate was observed between UD and ED 2‐cell embryos, the cell number per blastocyst from UD 2‐cell embryos was lower than that from ED 2‐cell embryos (44.15 ± 2.05 vs. 51.55 ± 1.83). Besides, because of non‐synchronized division of each blastomere, the following three cleavage routes were observed in all HMC/PAZF/PAZI embryos: T1 (2‐cell → 3‐cell → 4‐cell → ≥5‐cell → morula → blastocyst), T2 (2‐cell → 3‐cell → 4‐cell → morula → blastocyst) and T3 (2‐cell → 3‐cell/4‐cell → morula → blastocyst). Therefore, in pig in vitro‐produced embryos, division behaviours of uneven volume of cytoplasm and non‐synchronized cell cycles were observed at the early embryonic developmental stage, which might be another potential factor to evaluate embryonic development. [ABSTRACT FROM AUTHOR]

Knowledge on cortical development is based mainly on rodents besides primates and carnivores, all being altricial. Here, we analyzed a precocial animal, the pig, looking at dorsoparietal cortex from E45 to P90. At E45, most ionized calcium‐binding adapter molecule 1‐positive (Iba1+) cells had a macrophage‐like morphology and resided in meninges and choroid plexus. Only a few cells were scattered in the ventricular and subventricular zone (VZ and SVZ). At E60/E70, all laminar compartments displayed microglia cells at a low‐to‐moderate density, being highest in VZ and SVZ followed by intermediate zone/white matter (IZ/WM). The cortical plate and marginal zone displayed only a few Iba1+ cells. Cells were intensely labeled, but still had poorly arborized somata and many resembled ameboid, macrophage‐like microglia. Concurrent with a massive increase in cortical volume, microglia cell density increased until E85, and further until E100/E110 (birth at E114) to densities that resemble those seen postnatally. A fraction of microglia colabeled with Ki67 suggesting proliferation in all laminar compartments. Cell‐to‐cell distance decreased substantially during this time, and the fraction of microglia to all nuclei and to neurons increases in the laminar compartments. Eventually, of all cortical DAPI+ nuclei 7–12% were Iba1+ microglia. From E70 onwards, more and more cells with ramified processes were present in MZ down to IZ/WM, showing, for instance, a close association with NeuN+, NPY+, and GAD65/67+ somata and axon initial segments. These results suggested that the development of microglia cell density and morphology proceeds rapidly from mid‐gestation onwards reaching near‐adult status already before birth. [ABSTRACT FROM AUTHOR]

Background: An increase in the total nucleated cell count (TNCC) of amniotic fluid can detect inflammation in the amniotic cavity. Traditionally, a manual count is employed, but the technique suffers from inaccuracy and is labor intensive. Automated cell counting could enhance precision and timeliness. Objectives: We aimed to analyze the cell counts in the amniotic fluid of preterm pigs using an automated method and compared the results with manual cell counts. We also tested if increased TNCC is associated with an inflammatory group in a porcine model of chorioamnionitis. Methods: Amniotic fluids from 34 preterm pigs were analyzed blindly using two automatic settings of the ADVIA 2120i hematology analyzer (whole blood [WB] and cell poor [CP] settings) and manually by two observers using Neubauer chambers. Inter‐ and intra‐variability were calculated. The correlation analysis of TNCC/μL in amniotic fluid was determined between the methods, including Deming and Bland‐Altman analyses. Fischer's exact tests compared the known health status of the pigs to the outcomes of the automatic and manual TNCC. Results: Inter‐ and intra‐observer variability of the manual TNCC were high, although the correlation of TNCC between (r =.95, P <.0001) and within observers (r =.98, P <.0001; r =.89, P <.0001) was good. Correlation between the manual and CP TNCC was moderate and significant (r =.50, P =.014). Manual and WB TNCC were not correlated. The CP (P =.003) and manual (P =.0001) analyses accurately classified the disease state of the pigs. Conclusions: An acceptable correlation between automatic CP TNCC and manual counting was demonstrated. Both methods could accurately classify the disease state of the pigs. [ABSTRACT FROM AUTHOR]

This study developed an efficient method for liquid storage of in vitro‐derived porcine blastocysts at ambient temperature for 24 hr. We evaluated the effects of new chemically defined media (cell wash and preservation solution, Cellstor®‐W [Cell‐W] and cell suspension and preservation solution, Cellstor®‐S [Cell‐S]) for short‐term storage. In the first experiment, in vitro‐derived blastocyst were stored at 25ºC for 24 hr in Cell‐W solution, Cell‐S solution and pig embryo culture (PBM) medium. There were no differences in the rates of survival and development of stored blastocysts between the Cell‐S and Cell‐W solutions, but the total cell number of embryos that survived after storage in Cell‐S solution was significantly higher (p <.05) than that in the Cell‐W solution. In the second experiment, Cell‐S solution was used to store the in vitro‐derived blastocysts at 20°C, 25°C and 30°C. Storage at 20°C resulted in a significant decrease in the rates of survival and development of stored blastocysts compared to storage at 25°C or 30°C. No differences in survival and development rates were observed between storage at 25°C and 30°C, but the damage to the embryo quality after storage and culture was significantly lower at 25°C than at 30°C. In the third experiment, Cell‐S solution was supplemented with β‐mercaptoethanol and curcumin, either alone or in combination, as antioxidant agents. Although the supplementation with curcumin did not improve survival, it significantly increased the development rate of stored blastocysts compared with the control blastocysts stored without antioxidants. In conclusion, when porcine blastocysts were stored at 25°C for 24 hr, a Cell‐S solution may be effective for maintaining the survival and development of in vitro embryos. [ABSTRACT FROM AUTHOR]

The reproductive function of animals is often affected by climatic conditions. High‐temperature conditions can cause damage to oocyte maturation and embryonic development in a variety of ways. The purpose of this study was to prove that supplementation idebenone (IDB) to the maturation medium can improve the maturation and development of porcine oocytes after heat stress (HS). Porcine cumulus‐oocyte complexes (COCs) were cultured in the maturation medium with different concentrations of IDB (0, 0.1, 1 and 10 μM) for 44 hr at either 38.5°C or under the HS conditions. The cumulus oophorus expansion, nuclear maturation and blastocyst rate after parthenogenetic activation (PA) were measured. We found that HS (in vitro maturation 20–24 hr, 42°C) exposure significantly reduced cumulus expansion index and maturation rate of oocytes and the blastocyst rate of PA embryos, while IDB supplementation significantly improved oocyte maturation and development to the blastocysts stage after PA. Moreover, the addition of IDB decreased the intracellular level of ROS and increased GSH content, hence enhancing the antioxidant capacity of oocytes under HS. Meanwhile, IDB treatment also obviously improved the mitochondrial membrane potential and ATP synthesis of oocytes under HS conditions. Furthermore, IDB treatment increased the expression of GDF9 and BMP15 in IVM oocytes which attribute to improve the quality and outcome of IVM oocytes and the development competence of PA embryos in pigs. In summary, we demonstrated that IDB supplementation into the maturation medium exerted protective effects and improved the ability of maturation and developmental competence of porcine oocytes exposed to HS. [ABSTRACT FROM AUTHOR]

The key to successful in vitro embryo production (IVEP) is to mimic the natural in vivo oviductal microenvironment. Although the chemically defined media in extensive use for the in vitro culture of mammalian embryos is based on the composition of oviductal fluid, the IVEP systems in current use must still bypass the oviduct to produce embryos in vitro. Extracellular vesicles (EVs) in the oviduct are versatile intercellular delivery vehicles for maternal–embryo communication, and a lack of them can be associated with failed early embryonic development under in vitro culture conditions. Herein, we isolated EVs from porcine oviduct fluid and confirmed that oviductal EV supplementation improves the embryonic development of parthenogenetically activated (PA) embryos in terms of blastocyst formation rates and total cell numbers. Our experiments also revealed that a beneficial effect of oviductal EVs on PA embryos was achievable, at least in part, by relieving endoplasmic reticulum stress. These results suggest that the maternal–embryo communication mediated by oviductal EVs benefits early embryonic development. Given the contribution of oviductal EVs to early embryonic development, these findings offer novel insights for the optimization of current IVEP systems. [ABSTRACT FROM AUTHOR]

We aimed to develop a simple method for the short-term preservation of in vitro-produced porcine zygotes at 25°C for up to 2 days. Firstly, we evaluated the efficiency of three storage solutions to preserve porcine zygotes at 25°C for 24 h. Two of these were commercially available defined media for cell storage (Cell-W and Cell-S), and the third was fetal bovine serum (FBS). Thereafter, we examined the effects of storing the zygotes in the Cell-W solution for 24-72 h at 25°C. The Cell-W solution was the most efficient for 24 h storage of porcine zygotes at 25°C, with no apparent effects on blastocyst quality. However, short-term storage of porcine zygotes for 24 h reduced the blastocyst formation rate compared with the fresh control group. As storage duration increased from 24 to 48 or 72 h, blastocyst formation rates were significantly decreased from 11.3% to 4.4% and 0%, respectively. In conclusion, during zygote storage, the developmental competence to the blastocyst stage decreased with time. Storage of zygotes in chemically defined media did not affect blastocyst quality, but the storage in 100% serum had an adverse effect on developing embryos causing apoptosis. [ABSTRACT FROM AUTHOR]

We aimed to develop a simple method for the short‐term preservation of in vitro‐produced porcine zygotes at 25°C for up to 2 days. Firstly, we evaluated the efficiency of three storage solutions to preserve porcine zygotes at 25°C for 24 h. Two of these were commercially available defined media for cell storage (Cell‐W and Cell‐S), and the third was fetal bovine serum (FBS). Thereafter, we examined the effects of storing the zygotes in the Cell‐W solution for 24–72 h at 25°C. The Cell‐W solution was the most efficient for 24 h storage of porcine zygotes at 25°C, with no apparent effects on blastocyst quality. However, short‐term storage of porcine zygotes for 24 h reduced the blastocyst formation rate compared with the fresh control group. As storage duration increased from 24 to 48 or 72 h, blastocyst formation rates were significantly decreased from 11.3% to 4.4% and 0%, respectively. In conclusion, during zygote storage, the developmental competence to the blastocyst stage decreased with time. Storage of zygotes in chemically defined media did not affect blastocyst quality, but the storage in 100% serum had an adverse effect on developing embryos causing apoptosis. [ABSTRACT FROM AUTHOR]

The widely used porcine artificial insemination procedure involves the use of liquidstored semen because it is difficult to control the quality of frozen–thawed porcine sperm. Therefore, there is a high demand for porcine semen. The control and enhancement of sperm function are required for the efficient reproduction of pigs. We previously reported that gamma-aminobutyric acid (GABA) enhanced sperm capacitation and acrosome reaction in mice. In this study, we demonstrated the presence of GABAA receptors in porcine sperm acrosome. Furthermore, we investigated the GABA effects on porcine sperm function. We did not detect any marked effect of GABA on sperm motility and tyrosine phosphorylation of sperm proteins. However, GABA promoted acrosome reaction, which was suppressed by a selective GABAA receptor antagonist. GABA binds to GABAA receptors, resulting in chloride ion influx. We found that treatment with 1 μM GABA increased the intracellular concentration of chloride ion in the sperm. In addition, the GABA concentration effective in the acrosome reaction was correlated with the porcine sperm concentration. These results indicate that GABA and its receptors can act as modulators of acrosome reaction. This study is the first to report the effects of GABA on porcine sperm function. [ABSTRACT FROM AUTHOR]

Clinical application of skin substitute is typically a two‐stage procedure with application of skin substitute matrix to the wound followed by engraftment of a split‐thickness skin graft (STSG). This two‐stage procedure requires multiple interventions, increasing the time until the wound is epithelialised. In this study, the feasibility of a one‐stage procedure by combining bioengineered collagen‐chondroitin‐6‐sulfate (DS1) or decellularised fetal bovine skin substitute (DS2) with autologous skin cell suspension (ASCS) in a porcine full‐thickness wound healing model was evaluated. Twelve full‐thickness excisional wounds on the backs of pigs received one of six different treatments: empty; ASCS; DS1 with or without ASCS; DS2 with or without ASCS. The ASCS was prepared using a point‐of‐care device and was seeded onto the bottom side of DS1, DS2, and empty wounds at 80 000 cells/cm2. Wound measurements and photographs were taken on days 0, 9, 14, 21, 28, 35, and 42 post‐wounding. Histological analysis was performed on samples obtained on days 9, 14, 28, and 42. Wounds in the empty group or with ASCS alone showed increased wound contraction, fibrosis, and myofibroblast density compared with other treatment groups. The addition of ASCS to DS1 or DS2 resulted in a marked increase in re‐epithelialisation of wounds at 14 days, from 15 ± 11% to 71 ± 20% (DS1 vs DS1 + ASCS) or 28 ± 14% to 77 ± 26 (DS2 vs DS2 + ASCS) despite different mechanisms of tissue regeneration employed by the DS used. These results suggest that this approach may be a viable one‐stage treatment in clinical practice. [ABSTRACT FROM AUTHOR]

ACSS1/2 converts acetate into acetyl‐coenzyme A, which contributes to histone acetylation in the mitochondria and cytoplasm. Zygotic genome activation (ZGA) is critical for embryo development involving drastic histone modification. An efficient crRNAs‐Cas13a targeting strategy was employed to investigate the ACSS1/2 function during ZGA. The results showed that nuclear accumulation of ACSS1 and ACSS2 occurs during ZGA. Knockdown of ACSS1/2 did not affect blastocyst formation when using a normal medium. On culturing embryos in a medium with acetate and no pyruvate (−P + Ace), knockdown of ACSS1 did not affect histone acetylation levels but significantly reduced ATP levels, whereas knockdown of ACSS2 significantly reduced histone acetylation levels in porcine embryos. Inhibition of fatty acid beta‐oxidation by etomoxir significantly reduced ATP levels, which could be restored by acetate. The histone acetylation levels in the ACSS1 and ACSS2 knockdown groups both decreased considerably after etomoxir treatment. Moreover, acetate showed dose‐dependent effects on SIRT1 and SIRT3 levels when under metabolic stress. The C‐terminus of ACSS1 regulated the nuclear translocation. In conclusion, ACSS1/2 helps to maintain ATP and histone acetylation levels in porcine early embryos under metabolic stress during ZGA. [ABSTRACT FROM AUTHOR]

Differences in total number of piglets born per litter are observed between the Norwegian Duroc (ND) sire and Norwegian Landrace (NL) dam line. The aim of this study was to evaluate ovarian characteristics, and in vitro nuclear and cytoplasmic oocyte maturation in both breeds. One day after weaning, follicular phase ovaries were collected. Ovary length and weight were measured and the number of follicles (< 3 mm and 3–8 mm) was counted. Cumulus‐oocyte complexes (COCs) were collected and matured for 48 hr. To assess cumulus expansion, COC area was analysed at 0 and 20 hr. Nuclear maturation and cortical granule (CG) distribution were analysed at 20 and 48 hr, and total glutathione (GSH) was measured at 48 hr to further elucidate cytoplasmic maturation. In first parity sows, a smaller ovary length and fewer 3 to 8 mm follicles were observed in ND compared to NL. For all sows, ND COCs covered a significantly smaller area at 0 hr, but a higher cumulus expansion ratio was observed at 20 hr compared to NL (364 ± 46% versus. 278 ± 27%, p < 0.001). At 20 hr, more ND oocytes exhibited advanced stages of nuclear maturation, while more NL oocytes showed advanced stages of CG distribution. Nuclear maturation to MII stage at 48 hr did not differ between ND and NL oocytes (90.1% and 87.7%, respectively). Moreover, no significant differences were observed for GSH content or CG distribution after maturation. In conclusion, differences with regard to ovarian characteristics as well as to cumulus expansion, and nuclear and cytoplasmic oocyte maturation at 20 hr were observed between the breeds. Further studies are required to determine if this subsequently affects in vitro fertilization and embryo development. [ABSTRACT FROM AUTHOR]

Whole‐organ engineering is emerging as an alternative source for xenotransplantation in end‐stage diseases. Utilization of decellularized whole lung scaffolds created by detergent perfusion is an effective strategy for organ replacement. In the current study, we attempted to decellularize porcine whole lungs to generate an optimal and reproducible decellularized matrix for future clinical use. Porcine whole lungs were decellularized via perfusion of various detergents (sodium dodecyl sulfate (SDS)/Triton X‐100, sodium lauryl ether sulfate (SLES)/Triton X‐100, dextrose/SDS/Triton X‐100 and dextrose/SLES/Triton X‐100) through the pulmonary artery and bronchus of the lung. The decellularized scaffolds were evaluated for decellularization efficiency, extracellular matrix (ECM) component preservation, xenoantigen removal and compatibility. The resulting lung scaffolds obtained from treatment with the dextrose/SLES/Triton X‐100 cocktail showed minimal residual cellular components and xenoantigens, including DNA and protein, and good preservation of ECM composition. Evaluation of the porcine lung ECM by specific staining and immunofluorescence confirmed that the three‐dimensional ultrastructure of the ECM was noticeably preserved in the SLES‐treated groups. In addition, the decellularized lung scaffolds originating from the dextrose/SLES/Triton X‐100 cocktail supported cell adhesion and growth. In summary, the novel detergent SLES alleviated the damage to retain a better‐preserved ECM than SDS. Sequential Triton X‐100 perfusion eliminated SLES. Moreover, performing dextrose perfusion in advance further protected scaffold components, especially collagen. We developed an optimal dextrose/SLES/Triton X‐100 cocktail method that can be used for the decellularization of porcine whole lung to obtain a clinical‐scale bioengineered scaffold. [ABSTRACT FROM AUTHOR]

Overall efficiency of cell reprogramming for porcine fibroblasts into induced pluripotent stem cells (iPSCs) is currently poor, and few cell lines have been established. This study examined gene expression during early phase of cellular reprogramming in the relationship to the iPSC colony morphology and in vitro pluripotent characteristics. Fibroblasts were reprogrammed with OCT4, SOX2, KLF4 and c‐MYC. Two different colony morphologies referred to either compact (n = 10) or loose (n = 10) colonies were further examined for proliferative activity, gene expression and in vitro pluripotency. A total of 1,697 iPSC‐like colonies (2.34%) were observed after gene transduction. The compact colonies contained with tightly packed cells with a distinct‐clear border between the colony and feeder cells, while loose colonies demonstrated irregular colony boundary. For quantitative expression of genes responsible for early phase cell reprogramming, the Dppa2 and EpCAM were significantly upregulated while NR0B1 was downregulated in compact colonies compared with loose phenotype (p <.05). Higher proportion of compact iPSC phenotype (5 of 10, 50%) could be maintained in undifferentiated state for more than 50 passages compared unfavourably with loose morphology (3 of 10, 30%). All iPS cell lines obtained from these two types of colony morphologies expressed pluripotent genes and proteins (OCT4, NANOG and E‐cadherin). In addition, they could aggregate and form three‐dimensional structure of embryoid bodies. However, only compact iPSC colonies differentiated into three germ layers. Molecular signature of early phase of cell reprogramming coupled with primary colony morphology reflected the in vitro pluripotency of porcine iPSCs. These findings can be simply applied for pre‐screening selection of the porcine iPSC cell line. [ABSTRACT FROM AUTHOR]

Selenium (Se) is an essential trace element in organism. Se deficiency can cause many diseases, including vascular disease. Studies have shown that inflammation is the main inducement of vascular disease, microRNA (miRNA) can influence inflammation in various ways, and Se deficiency can affect miRNAs expression. To study the mechanism of aorta damage caused by Se deficiency, we constructed a Se deficiency porcine aorta model and found that Se deficiency can significantly inhibit miR‐223, which downregulates the expression of nucleotide‐binding oligomerization domain‐like receptor family 3 (NLRP3). Subsequently, we found that in Se deficiency group, NLRP3, and its downstream (caspase‐1, apoptosis‐related spot‐like protein [ASC], IL‐18, IL‐1β) expression was significantly increased. In vitro, we cultured pig iliac endothelium cell lines, and constructed miR‐223 knockdown and overexpression models. NLRP3 messenger RNA and protein levels were significant increased in the knockdown group, and decreased in the overexpression group. The results of this study show that Se deficiency in porcine arteries can induce inflammation through miR‐223/NLRP3. [ABSTRACT FROM AUTHOR]

Background: Neonatal porcine islets (NPIs) can restore glucose control in mice, pigs, and non‐human primates, representing a potential abundant alternative islet supply for clinical beta cell replacement therapy. However, NPIs are vulnerable to inflammatory insults that could be overcome with genetic modifications. Here, we demonstrate in a series of proof‐of‐concept experiments the potential of the cytoplasmic ubiquitin‐editing protein A20, encoded by the TNFAIP3 gene, as an NPI cytoprotective gene. Methods: We forced A20 expression in NPI grafts using a recombinant adenovirus 5 (Ad5) vector and looked for impact on TNF‐stimulated NF‐κB activation and NPI graft function. As adeno‐associated vectors (AAV) are clinically preferred vectors but exhibit poor transduction efficacy in NPIs, we next screened a series of AAV serotypes under different transduction protocols for their ability achieve high transduction efficiency and suppress NPI inflammation without impacting NPI maturation. Results: Forcing the expression of A20 in NPI with Ad5 vector blocked NF‐κB activation by inhibiting IκBα phosphorylation and degradation, and reduced the induction of pro‐inflammatory genes Cxcl10 and Icam1. A20‐expressing NPIs also exhibited superior functional capacity when transplanted into diabetic immunodeficient recipient mice, evidenced by a more rapid return to euglycemia and improved GTT compared to unmodified NPI grafts. We found AAV2 combined with a 14‐day culture period maximized NPI transduction efficiency (>70% transduction rate), and suppressed NF‐κB‐dependent gene expression without adverse impact upon NPI maturation. Conclusion: We report a new protocol that allows for high‐efficiency genetic modification of NPIs, which can be utilized to introduce candidate genes without the need for germline engineering. This approach would be suitable for preclinical and clinical testing of beneficial molecules. We also report for the first time that A20 is cytoprotective for NPI, such that A20 gene therapy could aid the clinical development of NPIs for beta cell replacement. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress is a major contributor to embryonic development failure. Mammalian oocytes have a high risk of exposure to cellular stress during in vitro embryo production. We investigated the effects of zinc supplementation during in vitro maturation under ER stress. We evaluated cumulus expansion, embryonic development derived by parthenogenetic activation, reactive oxygen species, protein expression of X‐box binding protein 1 (XBP1), and expression of genes related to ER stress. Supplementation with 1 μg/ml zinc significantly increased the nuclear maturation of oocytes, cleavage and blastocyst formation rates, and total blastocyst cell number (p <.05). Under ER stress, zinc significantly reduced protein expression of XBP1, and increased cleavage and blastocyst rates (p <.05). Concomitantly, zinc supplementation upregulated the expression of zinc transporters (SLC39A14 and SLC39A10), PTGS2, and downregulated ER stress‐related genes (sXBP1, uXBP1, ATF4, and PTPN1/PTP1B), and caspase 3. These results suggest that zinc supplementation alleviates ER stress by providing essential metal‐ion transporters for oocyte maturation and subsequent embryonic development. [ABSTRACT FROM AUTHOR]

Objective: To compare surface topography of porcine and human root dentin and to develop a new in vitro model for class II furcation defects. The hypothesis for this study was that porcine mandible blocks can function as a model for class II furcation defects. Background: Treatment of mandibular class II furcation defects is unpredictable. There is a need for in vitro models to investigate new treatment methods. Methods: A model to investigate the surface topography of porcine and human root dentin was developed and the two tissues compared by SEM imaging and profilometer. A novel method for studying class II furcation defects was then tested. Blocks of porcine mandibles with molar 3 were prepared. Buccal class II furcation defects were created. The furcation area was isolated and bioluminescent Staphylococcus epidermidis Xen43 was used to form a biofilm in the furcation area to test the functionality of the novel furcation model. Results: Micromechanical damage caused by debridement on porcine and human root dentin showed similar pattern. No significant difference in the surface morphological parameters was observed between the corresponding porcine and human samples. The model allowed for assessment of the root surface inside the furcation area. While the number of viable bacteria in the furcation following debridement could be quantified, no significant difference between the treatment groups was detected, likely due to bacterial colonization within the periodontal ligament space. Conclusion: Porcine and human root dentin show similar surface topography following surface debridement. Porcine mandible blocks can function as a model for class II furcation defects. However, further development and refinement of the novel in vitro model is warranted. [ABSTRACT FROM AUTHOR]

Objectives: S‐nitrosoglutathione reductase (GSNOR), a protein denitrosylase, protects the mitochondria from mitochondrial nitrosative stress. Mammalian preimplantation embryos are mitochondria‐rich, but the effects of GSNOR on mitochondrial function in preimplantation embryos are not well‐studied. In the present study, we investigate whether GSNOR plays a role in mitochondrial regulation during porcine preimplantation embryo development. Materials and Methods: GSNOR dsRNA was employed to knock down the expression of GSNOR, and Nω‐Nitro‐L‐arginine methyl ester hydrochloride (L‐NAME), a pan‐NOS inhibitor, was used to prevent protein S‐nitrosylation. Mitochondrial amount and function in embryo development were assessed by performing immunofluorescence staining, Western blot, fluorescent probe and real‐time reverse transcription PCR. Results: GSNOR knock‐down significantly impaired blastocyst formation and quality and markedly induced the increase in protein S‐nitrosylation. Notably, GSNOR knock‐down‐induced overproduction of S‐nitrosylation caused mitochondrial dysfunction, including mitochondrial membrane potential depolarization, mitochondria‐derived reactive oxygen species (ROS) increase and ATP deficiency. Interestingly, GSNOR knock‐down‐induced total mitochondrial amount increase, but the ratio of active mitochondria reduction, suggesting that the damaged mitochondria were accumulated and mitochondrial clearance was inhibited. In addition, damaged mitochondria produced more ROS, and caused DNA damage and apoptosis. Importantly, supplementation with L‐NAME reverses the increase in S‐nitrosylation, accumulation of damaged mitochondria, and oxidative stress‐induced cell death. Interestingly, autophagy was downregulated after GSNOR knock‐down, but reversed by L‐NAME treatment. Thus, GSNOR maintains mitochondrial homeostasis by promoting autophagy and the clearing of damaged mitochondria in porcine preimplantation embryos. [ABSTRACT FROM AUTHOR]