Mesenchymal stem cells in animal reproduction: sources, uses and scenario.

Studies regarding mesenchymal stem cells turned up in the 1960's and this cell type created a great number of questions about its functions and applicability in science and medicine. When used with therapeutic intent, these cells present an inclination to migrate to sites of injury, inflammation or disease, where they secrete bioactive factors that stimulates the synthesis of new tissue. In this context, studies using rodents reported that MSCs promoted positive effects in the ovarian function in mice with premature aging of follicular reserve. In female bovines, experimental stem cell-based therapies have been used to either generate new oocytes with in vitro quality or stimulate such action in vivo. It is also reported, that the intraovarian application of mesenchymal stem cells generates a greater production of embryos in vitro and the production of early and expanded blastocysts. Additionally, analysis of ovarian tissue in animal subjected to treatment showed an increase in the number of developing follicles. Nevertheless, the treatments involving stem cells with different modes of application, different sources and different species were able to act on the hormonal, tissue, cellular and metabolic levels, generating positive results in the recovery and improvement of ovarian functions.

Estudos sobre células-tronco mesenquimais surgiram na década de 1960 e esse tipo de célula gerou muitas dúvidas sobre suas funções e aplicabilidade na ciência e na medicina. Quando utilizadas com intuito terapêutico, essas células apresentam tendência a migrar para locais de lesão, inflamação ou doença, onde secretam fatores bioativos que estimulam a síntese de novos tecidos. Nesse contexto, estudos utilizando roedores relataram que as CTM promoveram efeitos positivos na função ovariana em camundongos com envelhecimento precoce da reserva folicular. Em fêmeas bovinas, terapias experimentais baseadas em células-tronco têm sido utilizadas para gerar novos oócitos com qualidade in vitro ou estimular tal ação in vivo. É relatado também que a aplicação intraovariana de células-tronco mesenquimais gera maior produção de embriões in vitro e produção de blastocistos precoces e expandidos. Além disso, a análise do tecido ovariano em animais submetidos ao tratamento mostrou aumento no número de folículos em desenvolvimento. Apesar disso, os tratamentos envolvendo células-tronco com diferentes modos de aplicação, diferentes fontes e diferentes espécies foram capazes de atuar nos níveis hormonal, tecidual, celular e metabólico, gerando resultados positivos na recuperação e melhora das funções ovarianas.

Comparative Analysis of Fluorescent Labeling Techniques for Tracking Canine Amniotic Stem Cells.

The utmost aim of regenerative medicine is to promote the regeneration of injured tissues using stem cells. Amniotic mesenchymal stem cells (AmMSCs) have been used in several studies mainly because of their easy isolation from amniotic tissue postpartum and immunomodulatory and angiogenic properties and the low level of rejection. These cells share characteristics of both embryonic/fetal and adult stem cells and are particularly advantageous because they do not trigger tumorigenic activity when injected into immunocompromised animals. The large-scale use of AmMSCs for cellular therapies would greatly benefit from fluorescence labeling studies to validate their tracking in future therapies. This study evaluated the fluorophore positivity, fluorescence intensity, and longevity of canine AmMSCs. For this purpose, canine AmMSCs from the GDTI/USP biobank were submitted to three labeling conditions, two commercial fluorophores [CellTrace CFSE Cell Proliferation kit - CTrace, and CellTracker Green CMFDA - CTracker (CellTracker Green CMFDA, CT, #C2925, Molecular Probes®; Life Technologies)] and green fluorescent protein (GFP) expression after lentiviral transduction, to select the most suitable tracer in terms of adequate persistence and easy handling and analysis that could be used in studies of domestic animals. Fluorescence was detected in all groups; however, the patterns were different. Specifically, CTrace and CTracker fluorescence was detected 6 h after labeling, while GFP was visualized no earlier than 48 h after transduction. Flow cytometry analysis revealed more than 70% of positive cells on day 7 in the CTrace and CTracker groups, while fluorescence decreased significantly to 10% or less on day 20. Variations between repetitions were observed in the GFP group under the present conditions. Our results showed earlier fluorescence detection and more uniform results across repetitions for the commercial fluorophores. In contrast, fluorescence persisted for more extended periods in the GFP group. These results indicate a promising direction for assessing the roles of canine AmMSCs in regenerative medicine without genomic integration.

The Influence of Recombinant Bovine Somatotropin (rbST) on the Metabolic Profile and Milk Composition of Lactating Murrah Buffalo.

The use of recombinant bovine somatotropin (rbST) leads to an increase in variable amounts of milk production in buffalo, but there is a lack of information on the influence of rbST on their metabolism. This study looked at the effects of a single 500 mg dose of rbST on the lipid profile, liver and kidney function, and physical, chemical, and cellular constitution of milk in 14 buffalo over 14 days, from the 100th day of lactation, compared with 14 animals in a control group. From the first day after rbST, there was a rise in beta-hydroxybutyrate (ß-HBO), possibly due to higher dry matter intake or the biotransformation of NEFA into ß-HBO. The treatment did not influence blood glucose, non-esterified fatty acids (NEFAs), triglycerides, cholesterol, total protein, albumin, AST, GGT, bilirubin, urea, or creatinine levels. In 71.3% of the buffalo, there was a gradual increase in milk production, with the maximal response occurring in the first week followed by a gradual decrease, whilst in 21.4%, the increase in production occurred between 7 and 10 days. Only 7.1% of the animals did not respond. On the 3rd, 5th, 7th, and 10th days after treatment, an increase was found in daily milk production between the two groups equal to 1.04, 1.52, 1.42, and 1.06 L, respectively. In relative terms, this means an increase in milk production, respectively, of 15.1%, 21.0%, 19.8%, and 15.1%. The constitution of the milk showed no difference in the amounts of fat, lactose, total solids, or somatic cell count; however, on the third day after rbST administration, there was a decrease in protein. Notably, from the fifth day, the protein values showed no statistical difference. It can be concluded that the use of rbST in buffalo from the 100th day of lactation is metabolically safe since the treatment neither caused imbalances in fat metabolism nor overloaded the liver or renal function, and the changes in milk composition were transient and limited to a decrease in milk protein.

Exogenous OCT4 and SOX2 Contribution to In Vitro Reprogramming in Cattle.

Mechanisms of cell reprogramming by pluripotency-related transcription factors or nuclear transfer seem to be mediated by similar pathways, and the study of the contribution of OCT4 and SOX2 in both processes may help elucidate the mechanisms responsible for pluripotency. Bovine fibroblasts expressing exogenous OCT4 or SOX2, or both, were analyzed regarding the expression of pluripotency factors and imprinted genes H19 and IGF2R, and used for in vitro reprogramming. The expression of the H19 gene was increased in the control sorted group, and putative iPSC-like cells were obtained when cells were not submitted to cell sorting. When sorted cells expressing OCT4, SOX2, or none (control) were used as donor cells for somatic cell nuclear transfer, fusion rates were 60.0% vs. 64.95% and 70.53% vs. 67.24% for SOX2 vs. control and OCT4 vs. control groups, respectively; cleavage rates were 66.66% vs. 81.68% and 86.47% vs. 85.18%, respectively; blastocyst rates were 33.05% vs. 44.15% and 52.06% vs. 44.78%, respectively. These results show that the production of embryos by NT resulted in similar rates of in vitro developmental competence compared to control cells regardless of different profiles of pluripotency-related gene expression presented by donor cells; however, induced reprogramming was compromised after cell sorting.

Porcine Germ Cells Phenotype during Embryonic and Adult Development.

Primordial germ cells (PGCs) are the precursors of gametes. Due to their importance for the formation and reproduction of an organism, understanding the mechanisms and pathways of PGCs and the differences between males and females is essential. However, there is little research in domestic animals, e.g., swine, regarding the epigenetic and pluripotency profiles of PGCs during development. This study analyzed the expression of epigenetic and various pluripotent and germline markers associated with the development and differentiation of PGCs in porcine (pPGCs), aiming to understand the different gene expression profiles between the genders. The analysis of gonads at different gestational periods (from 24 to 35 days post fertilization (dpf) and in adults) was evaluated by immunofluorescence and RT-qPCR and showed phenotypic differences between the gonads of male and female embryos. In addition, the pPGCs were positive for OCT4 and VASA; some cells were H3k27me3 positive in male embryos and adult testes. In adults, the cells of the testes were positive for germline markers, as confirmed by gene expression analysis. The results may contribute to understanding the pPGC pathways during reproductive development, while also contributing to the knowledge needed to generate mature gametes in vitro.

3D culture of mesenchymal stem cells from the yolk sac to generate intestinal organoid.

Organoids are in vitro models that originated from the three-dimensional culture of stem cells with the ability to reproduce part of the in vivo structural and functional specificities of body organs. Intestinal organoids have great relevance in cell therapy, as they provide more accurate information about tissue composition and architecture in relation to two-dimensional culture, in addition to serving as a study model for host interaction and drug testing. The yolk sac (YS) is a promising source of mesenchymal stem cells (MSCs), which are multipotent stem cells with self-renewal ability and potential to differentiated into mesenchymal lineages. Besides this, the YS is responsible for the formation of intestinal epithelium during embryonic development. Thus, the aim of this study was to verify if the three-dimensional in vitro culture of stem cells derived from the canine YS is capable of developing intestinal organoids. MSCs from the canine YS and gut cells were isolated and characterized, then three-dimensionally cultured in Matrigel. In both cells lineages, spherical organoids were observed and after 10 days the gut cells formed crypt-like buds and villus-like structures. Despite having the same induction of differentiation process and having the expression of intestinal markers, the MSC from the YS morphology was not in the form of crypt budding. The hypothesis is that these cells could generate structures equivalent to the intestinal organoids of the colon that other studies showed formed only spherical structures. The culture of MSC from the YS, as well as the establishment of protocols for 3D cultivation of this tissue, is relevant, as it will serve as a tool in various applications in basic and scientific biology.

Different prenatal supplementation strategies and its impacts on reproductive and nutrigenetics assessments of bulls in finishing phase.

This study investigated the effect of different prenatal nutrition approaches in 126 pregnant Nellore cows on reproductive and nutrigenetic traits of the male offspring during the finishing phase. For that purpose, three nutritional treatments were used in these cows during pregnancy: PP - protein-energy supplementation in the final third, FP - protein-energy supplementation during the entire pregnancy, and NP - (control) only mineral supplementation. The male progeny (63 bulls; 665 ± 28 days of age) were evaluated for scrotal circumference, seminal traits, number of Sertoli cells and testicular area. We performed a genomic association (700 K SNPs) for scrotal circumference at this age. In addition, a functional enrichment was performed in search of significant metabolic pathways (P < 0.05) with inclusion of genes that are expressed in these genomic windows by the MetaCore software. With the exception of major sperm defects (P < 0.1), the other phenotypes showed no difference between prenatal treatments. We found genes and metabolic pathways (P < 0.05) that are associated with genomic windows (genetic variance explained >1%) in different treatments. These molecular findings indicate that there is genotype-environment interaction among the different prenatal treatments and that the FP treatment showed greater major sperm defects compared to the NP treatment.

Delivery anatomy kits help to keep practical veterinary classes during the COVID-19 pandemic.

To address the restrictions caused by the COVID-19 pandemic and to search for assistive learning tools for the subject of Animal Anatomy II and Applied Anatomy, 123 anatomical kits were prepared at the Animal Anatomy Laboratory for students of the Veterinary Medicine course at the University of São Paulo, Faculty of Animal Science and Food Engineering (FZEA/USP) in Pirassununga city, São Paulo, Brazil. The kits contained anatomical pieces for teaching splanchnology and topographic anatomy (two different classes), and they were elaborated based on effective preservation techniques for the preparation of animal anatomical pieces. At the end of each course, we sent an online questionnaire to the students for evaluation of the methodology used. Alternative methods were used to minimize the odour and non-generation of chemical or microbiological contaminants. The acceptance of the kits was unanimous with adherence by all the students, who had the opportunity to experience the Anatomy class in its entirety, without leaving their homes.

Complications following transcutaneous cecal trocarization in horses with a cattle trocar and a cecal needle.

Percutaneous decompression of the cecum is a procedure that could be considered for horses with cecal gas distension. The aim of this study was to identify complications such as peritonitis and clinically relevant peritonitis (CRP) after transabdominal cecal trocarization in healthy horses using a cattle trocar and a cecal needle. Mixed breed horses were assigned to three groups (n = 6): horses that underwent trocarization with a cecal needle (G1) or a cattle trocar (G2), and a control group (CG) without cecal trocarization. The same horses were used in each group, respecting a three-month washout period between studies. A physical examination, serial blood, and peritoneal fluid sampling were performed, prior to cecal trocarization and 2, 6 and 12 hours after the first collection and 1, 2, 3, 7, and 14 days after the procedure. Acute-phase proteins in blood and peritoneal fluid were analyzed by polyacrylamide gel electrophoresis. Horses with a high cell count in the peritoneal fluid (i.e., 10,000 cells/µl) were considered to have peritonitis and CRP if they met at least two of the following clinical criteria: anorexia, lethargy, tachycardia, tachypnea, fever, ileus, abnormal oral mucous membrane color, abnormal white blood cells count, or high blood fibrinogen concentration (> 5 g/L). All horses recovered from cecal trocarization and abdominocentesis with no major complications. Cecal trocarization caused cytologic evidence of peritonitis in G1 and G2 during the 14 days of evaluation. CRP was not observed, although a decrease in cecal motility was observed in G1 and G2 during the experimental period and three horses, one from G1 and two from G2, showed a single moment of fever. None of the groups showed leukopenia or leukocytosis, although blood neutrophil count decreased at D7 and D14 in G1 and at D14 in G2 (p ≤ 0.05). After cecal trocarization, an increase in the total nucleated cells count, total proteins, globulins, alkaline phosphatase and acute phase proteins were observed in the peritoneal fluid of G1 and G2 during the 14 days of evaluation (p ≤ 0.05), without causing clinically relevant peritonitis. Transcutaneous cecal trocarization promotes peritonitis, which is more intense with a cattle trocar than with a cecal needle. The cecal needle should be considered for cecal trocarization of horses with cecal tympany.

In vitro induced pluripotency from urine-derived cells in porcine.

BACKGROUND: The generation of induced pluripotent stem cells (iPSC) has been a game-changer in translational and regenerative medicine; however, their large-scale applicability is still hampered by the scarcity of accessible, safe, and reproducible protocols. The porcine model is a large biomedical model that enables translational applications, including gene editing, long term in vivo and offspring analysis; therefore, suitable for both medicine and animal production. AIM: To reprogramme in vitro into pluripotency, and herein urine-derived cells (UDCs) were isolated from porcine urine. METHODS: The UDCs were reprogrammed in vitro using human or murine octamer-binding transcription factor 4 (OCT4), SRY-box2 (SOX2), Kruppel-like factor 4 (KLF4), and C-MYC, and cultured with basic fibroblast growth factor (bFGF) supplementation. To characterize the putative porcine iPSCs three clonal lineages were submitted to immunocytochemistry for alkaline phosphatase (AP), OCT4, SOX2, NANOG, TRA1 81 and SSEA 1 detection. Endogenous transcripts related to the pluripotency (OCT4, SOX2 and NANOG) were analyzed via reverse transcription quantitative real-time polymerase chain reaction in different time points during the culture, and all three lineages formed embryoid bodies (EBs) when cultured in suspension without bFGF supplementation. RESULTS: The UDCs were isolated from swine urine samples and when at passage 2 submitted to in vitro reprogramming. Colonies of putative iPSCs were obtained only from UDCs transduced with the murine factors (mOSKM), but not from human factors (hOSKM). Three clonal lineages were isolated and further cultured for at least 28 passages, all the lineages were positive for AP detection, the OCT4, SOX2, NANOG markers, albeit the immunocytochemical analysis also revealed heterogeneous phenotypic profiles among lineages and passages for NANOG and SSEA1, similar results were observed in the abundance of the endogenous transcripts related to pluripotent state. All the clonal lineages when cultured in suspension without bFGF were able to form EBs expressing ectoderm and mesoderm layers transcripts. CONCLUSION: For the first time UDCs were isolated in the swine model and reprogrammed into a pluripotent-like state, enabling new numerous applications in both human or veterinary regenerative medicine.

Porcine Primordial Germ Cell-Like Cells Generated from Induced Pluripotent Stem Cells Under Different Culture Conditions.

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.

Feeding strategies impact animal growth and beef color and tenderness.

The impact of growth rate (GR) and finishing regime (FR) on growth and meat quality traits of Angus x Nellore crossbred steers, harvested at a constant body weight (530 ± 20 kg) or time on feed (140 days), was evaluated. Treatments were: 1) feedlot, high GR; 2) feedlot, low GR; 3) pasture, high GR and 4) pasture, low GR. Live body composition, carcass and meat quality traits were evaluated. High GR had greater impact on muscle and fat deposition in feedlot-finished, but not in pasture-finished animals. Feedlot animals had higher Longissimus muscle area, backfat thickness, meat luminosity and tenderness when compared to pasture groups. Moreover, pasture- and feedlot-finished animals with similar GR did not differ in the chromatic attributes of non-aged meat, regardless of endpoint. Thus, GR appeared to be the main factor driving beef chromatic parameters, while FR had a major impact on achromatic attributes and tenderness of meat.

Transient Pulmonary Artery Hypertension in Holstein Neonate Calves.

The neonatal period is a challenging phase for calves, and during this phase constant adaptations are required. The aim of the present study was to evaluate the invasive hemodynamics with the Swan-Ganz catheter in neonate calves to understand adaptive changes during the first 30 days of life. A prospective and observational study was conducted with 10 Holstein calves. Assessments of the right atrial pressure (RAP), right ventricular pressure (RVP), pulmonary artery pressure (PAP), pulmonary capillary pressure (PW), cardiac output (CO), heart rate (HR), pulmonary vascular resistance (PVR), and blood gas levels were performed. The analyses of PAP, PVR, PW, HR, sO2, and arterial blood gases differed (p < 0.05) between the evaluated periods. Our results indicated transient pulmonary artery hypertension during the process of extrauterine adaptation during the first 30 days of life. This hypertension must be considered as physiological and consequent to the neonatal adaptation process.

Generation of neural progenitor cells from porcine-induced pluripotent stem cells.

In this study, porcine embryonic fibroblasts (pEFs) were reprogrammed into porcine-induced pluripotent stem cells (piPSCs) using either human or mouse specific sequences for the OCT4, SOX2, c-Myc, and KLF4 transcription factors. In total, three pEFs lines were reprogrammed, cultured for at least 15 passages, and characterized regarding their pluripotency status (alkaline phosphatase expression, embryoid body formation, expression of exogenous and endogenous genes, and immunofluorescence). Two piPSC lines were further differentiated, using chemical inhibitors, into putative neural progenitor-like (NPC-like) cells with subsequent analyses of their morphology and expression of neural markers such as NESTIN and GFAP as well as immunofluorescent labeling of NESTIN, ß-TUBULIN III, and VIMENTIN. NPC-like cells were positive for all the neural markers tested. These results evidence of the generation of porcine NPC-like cells after in vitro induction with chemical inhibitors, representing an adequate model for future regenerative and translational medicine research.

Characterization of post-edited cells modified in the TFAM gene by CRISPR/Cas9 technology in the bovine model.

Gene editing in large animal models for future applications in translational medicine and food production must be deeply investigated for an increase of knowledge. The mitochondrial transcription factor A (TFAM) is a member of the HMGB subfamily that binds to mtDNA promoters. This gene maintains mtDNA, and it is essential for the initiation of mtDNA transcription. Lately, we generated a new cell line through the disruption of the TFAM gene in bovine fibroblast cells by CRISPR/Cas 9 technology. We showed that the CRISPR/Cas9 design was efficient through the generation of heterozygous mutant clones. In this context, once this gene regulates the mtDNA replication specificity, the study aimed to determine if the post-edited cells are capable of in vitro maintenance and assess if they present changes in mtDNA copies and mitochondrial membrane potential after successive passages in culture. The post-edited cells were expanded in culture, and we performed a growth curve, doubling time, cell viability, mitochondrial DNA copy number, and mitochondrial membrane potential assays. The editing process did not make cell culture unfeasible, even though cell growth rate and viability were decreased compared to control since we observed the cells grow well when cultured in a medium supplemented with uridine and pyruvate. They also exhibited a classical fibroblastoid appearance. The RT-qPCR to determine the mtDNA copy number showed a decrease in the edited clones compared to the non-edited ones (control) in different cell passages. Cell staining with Mitotracker Green and red suggests a reduction in red fluorescence in the edited cells compared to the non-edited cells. Thus, through characterization, we demonstrated that the TFAM gene is critical to mitochondrial maintenance due to its interference in the stability of the mitochondrial DNA copy number in different cell passages and membrane potential confirming the decrease in mitochondrial activity in cells edited in heterozygosis.

Generation of induced pluripotent stem cells from large domestic animals.

BACKGROUND: Induced pluripotent stem cells (iPSCs) have enormous potential in developmental biology studies and in cellular therapies. Although extensively studied and characterized in human and murine models, iPSCs from animals other than mice lack reproducible results. METHODS: Herein, we describe the generation of robust iPSCs from equine and bovine cells through lentiviral transduction of murine or human transcription factors Oct4, Sox2, Klf4, and c-Myc and from human and murine cells using similar protocols, even when different supplementations were used. The iPSCs were analyzed regarding morphology, gene and protein expression of pluripotency factors, alkaline phosphatase detection, and spontaneous and induced differentiation. RESULTS: Although embryonic-derived stem cells are yet not well characterized in domestic animals, generation of iPS cells from these species is possible through similar protocols used for mouse or human cells, enabling the use of pluripotent cells from large animals for basic or applied purposes. Herein, we also infer that bovine iPS (biPSCs) exhibit similarity to mouse iPSCs (miPSCs), whereas equine iPSs (eiPSCs) to human (hiPSCs). CONCLUSIONS: The generation of reproducible protocols in different animal species will provide an informative tool for producing in vitro autologous pluripotent cells from domestic animals. These cells will create new opportunities in animal breeding through transgenic technology and will support a new era of translational medicine with large animal models.

Characterization and Immunomodulation of Canine Amniotic Membrane Stem Cells.

PURPOSE: Amniotic membrane stem cells have a high capacity of proliferation, cell expansion, and plasticity, as well as immunomodulatory properties that contribute to maternal-fetal tolerance. Owing to the lack of research on human amniotic membrane at different gestational stages, the canine model is considered ideal because of its genetic and physiological similarities. We aimed to characterize the canine amniotic membrane (CAM) cell lineage in different gestational stages and evaluate the expression of immunomodulatory genes. MATERIALS AND METHODS: Twenty CAMs from early (20-30 days) (n=7), mid- (31-45 days) (n=7), and late gestation (46-63 days) (n=6) stages were studied. The cell features were assessed by cell viability tests, growth curve, colony-forming units, in vitro differentiation, cell labeling for different immunophenotypes, and pluripotent potential markers. The cells were subjected to RT-PCR and qPCR analysis to determine the expression of IDO, HGF, EGF, PGE2, and IL-10 genes. RESULTS: CAM cells exhibited a fibroblastoid morphology and adherence to plastic with an average cell viability of 78.5%. The growth curve indicated a growth peak in the second passage and we obtained an average of 138.2 colonies. Osteogenic, chondrogenic, and adipogenic lineages were confirmed by in vitro differentiation assays. Cellular immunophenotyping experiments confirmed the presence of positive mesenchymal markers (CD90 and CD105) and the low or negative expression of hematopoietic markers (CD45 and CD34). Qualitative analysis of the immunomodulatory functions indicated the expression of the IDO, HGF, EGF5, and PGE2 genes. When stimulated by interferon-gamma, CAM cells exhibited higher IDO levels throughout gestation. CONCLUSION: The CAMs from different gestational stages presented features consistent with mesenchymal stem cell lineage; better results were observed during the late gestation stage. Therefore, the gestational stage is a key factor that may influence the functionality of therapies when using fetal membrane tissues from different periods of pregnancy.

In Vitro Induction of Pluripotency from Equine Fibroblasts in 20% or 5% Oxygen.

The cellular reprogramming into pluripotency is influenced by external and internal cellular factors, such as in vitro culture conditions (e.g., environmental oxygen concentration), and the aging process. Herein, we aimed to generate and maintain equine iPSCs (eiPSCs) derived from fibroblasts of a horse older than 20 years and to evaluate the effect of different levels of oxygen tension (atmospheric 20% O2, 5% O2, or 20% to 5% O2) on these cells. Fibroblasts were reprogrammed, and putative eiPSCs were positive for positive alkaline phosphatase detection; they were positive for pluripotency-related genes OCT4, REX1, and NANOG; immunofluorescence-positive staining was presented for OCT4 and NANOG (all groups), SOX2 (groups 5% O2 and 20% to 5% O2), and TRA-1-60, TRA-1-81, and SSEA-1 (only in 20% O2); they formed embryoid bodies; and there is spontaneous differentiation in mesoderm, endoderm, and ectoderm embryonic germ layers. In addition to the differences in immunofluorescence analysis results, the eiPSC colonies generated at 20% O2 presented a more compact morphology with a well-defined border than cells cultured in 5% O2 and 20% to 5% O2. Significant differences were also observed in the expression of genes related to glucose metabolism, mitochondrial fission, and hypoxia (GAPDH, GLUT3, MFN1, HIF1α, and HIF2α), after reprogramming. Our results show that the derivation of eiPSCs was not impaired by aging. Additionally, this study is the first to compare high and low oxygen cultures of eiPSCs, showing the generation of pluripotent cells with different profiles. Under the tested conditions, the lower oxygen tension did not favor the pluripotency of eiPSCs. This study shows that the impact of oxygen atmosphere has to be considered when culturing eiPSCs, as this condition influences the pluripotency characteristics.

Thoracolumbar epidural anaesthesia with 0.5% bupivacaine with or without methadone in goats.

BACKGROUND: Epidural anaesthesia is one of the most commonly used locoregional techniques in ruminants. The lumbosacral epidural technique is reasonably easy to perform and requires low volumes of local anaesthetic drug to allow procedures caudal to the umbilicus. However, surgical procedures in the flank of the animal would require an increased volume of drugs. The anaesthetized area provided by thoracic epidural technique is larger than the lumbosacral technique; however the former is rather challenging to perform. Therefore, access through lumbosacral area to introduce a catheter into the thoracolumbar space is a potential alternative to thoracic access. Epidural anaesthesia is achieved with local anaesthetics; opioids can be added to improve analgesia. This study aimed to evaluate the effects of 0.5% bupivacaine with or without methadone, administered through an epidural catheter inserted through the lumbosacral access and advanced to the thoracolumbar space, on thoracolumbar epidural anaesthesia in goats. METHODS: Six animals received two treatments each in a randomized crossover study: BUP treatment consisted of 0.5% bupivacaine (1 mL per each 10 cm of spine column; 1 ± 0.2 mg/kg BW) and BMT treatment was the same; however 1 mL of bupivacaine was replaced by 1 mL (0.22 ± 0.03 mg/kg BW) of methadone (10 mg/mL). The treatments were administered near to T11-T12 through an epidural catheter. Motor blockade and analgesia were evaluated by electrical stimulation. RESULTS: Heart rate, respiratory rate, ruminal motility and rectal temperature were evaluated before and after the treatment. Motor blockade was observed on both treatments, up to 6 h post-treatment. Analgesia was observed on BUP up to 4 h and on BMT up to 6 h post-treatment. Physiological values did not change at any moment. CONCLUSIONS: Bupivacaine-methadone combination promoted longer-lasting analgesia in goats compared to bupivacaine alone when administered through an epidural catheter into the thoracolumbar space.

Estudo comparativo da resistência mecânica da força de compressão entre biomateriais naturais, sintéticos e mistos / Comparative study of mechanical strength of compression between natural, synthetic and mixed biomaterials

As afecções ortopédicas com perda de tecido ósseo são um desafio tanto na medicina veterinária quanto na medicina humana. Portanto, não é raro ortopedistas se depararem com fraturas cominutivas irredutíveis de ossos longos, neoplasias ósseas ou não-uniões, que necessitam de procedimentos cirúrgicos reparadores, por meio da substituição de segmento ou preenchimento de falha óssea com o uso de biomateriais. Pretende-se com esta pesquisa avaliar e comparar a resistência mecânica entre biomateriais naturais, sintéticos e mistos. Foram utilizados sete grupos experimentais compostos por seis corpos de provas cada: Grupo 1 , tecido ósseo cortical de coelho (OSSO - controle); Grupo 2, cimento ósseo (CO); Grupo 3, cimento ósseo autoclavado (COA); Grupo 4, cimento ósseo e macrofragmento ósseo cortical de cão (COMaFO); Grupo 5, cimento ósseo e macrofragmento ósseo autoclavado de cortical de cão (COMaFOA); Grupo 6, cimento ósseo e microfragmento ósseo cortical de cão (COMiFO) e Grupo 7, cimento ósseo e microfragmento ósseo cortical de cão (COMiFOA). Os corpos de prova foram submetidos a ensaios mecânicos de compressão axial controlada em máquina universal de ensaio Emic®. O teste era interrompido quando ocorria queda brusca na curva do gráfico indicando falência da amostra. Em relação à Força máxima, os grupos COA, COMaFOA e COMiFOA não diferiram estatisticamente do grupo controle (OSSO; p>0,01). Já os grupos CO, COMaFO e COMiFO diferiram estatisticamente do grupo controle (OSSO; p<0,01). Quanto a rigidez relativa, os grupos COMaFOA e COMiFOA não diferiram estatisticamente do grupo controle (OSSO; p>0,01). Já os grupos CO, COA, COMaFO e COMiFO diferiram estatisticamente do grupo controle (OSSO; p<0,01). Comparando a deformação, os grupos COMaFo, COMaFOA e COMiFO não diferiram estatisticamente do grupo controle (OSSO; p>0,01). Já os grupos CO, COA e COMiFOA diferiram estatisticamente do grupo controle (OSSO; p<0,01). Conclui-se que apenas os grupos COMaFOA e COMiFOA apresentaram propriedades mecânicas muito semelhantes às do grupo controle (OSSO). Por isso, devido a essas características, esses dois biomateriais (COMaFOA e COMiFOA) seriam os mais indicados como substitutos na reparação de falhas ósseas.(AU)

The orthopedic diseases with bone loss are the challenge in both veterinary and human medicine. Therefore, the orthopedist commonly deal with irreducible comminuted fractures of long bones, bone tumors or non-unions, which require repairers surgical procedures, through the segment replacement or bone defect filling with biomaterials. The aim of this research is to evaluate and compare the mechanical strength of natural, synthetic and mixed biomaterials. Seven experimental groups of six test samples each were used: Group 1 rabbit cortical bone (BONE - control); Group 2, bone cement (CO); Group 3, bone cement autoclaved (COA); Group 4, bone cement and dog cortical bone macrofragment (COMaFO); Group 5, bone cement and bone autoclaved macrofragment dog cortical (COMaFOA); Group 6, bone cement and dog cortical bone microfragment (COMIFO) and Group 7, bone cement and dog cortical bone microfragment (COMiFOA). The specimens were subjected to axial compression mechanical tests controlled universal testing machine EMIC®. The test was stopped when there was sharp decline in the graph curve indicating failure of the sample. In relation to the maximum force, the COA groups COMaFOA and COMiFOA not statistically different from the control group (BONE; p> 0.01). Already the CO groups, COMaFO and COMIFO difeririram statistically the control group (BONE; p <0.01). The relative rigidity, the COMaFOA and COMiFOA groups did not differ statistically from the control group (BONE; p>0.01). Already the CO groups, COA, COMaFO and COMIFO differed significantly from the control group (BONE; p<0.01). Comparing the deformation, the COMaFo groups COMaFOA and COMIFO not statistically different from the control group (BONE; p>0.01). Already the CO groups, COA and COMiFOA differed significantly from the control group (BONE; p<0.01). It is concluded that only COMaFOA and COMiFOA groups showed very similar mechanical properties to the control group (BONE). Therefore, due to these characteristics, these two biomaterials (COMaFOA e COMiFOA) would be the most suitable as a substitute in the repair of bone defects.(AU)