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Somatic cell nuclear transfer (SCNT) is a powerful tool to understand the mechanisms driving the cellular reprogramming, as well as addressing fundamental questions on developmental biology. Hundreds of animals have already been generated through this technology, however its large-scale use is still hampered by the low efficiency and disrupted phenotypes reported in SCNT derived newborns. Therefore, this review aims to expose some of the consequences often seen in cloned animals, as well as to discuss and propose some possibilities and perspectives to avoid epigenetic failures during reprogramming.

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.

Encephalic vascular accident, or stroke, is the most common pathology of the central nervous system in humans, the second leading cause of death and physical and cognitive disabilities, in developing countries. It presents as an ischemic (more common) or hemorrhagic form. Ozone therapy has been shown to be effective in neuromodulation, neuroprotection, and nerve regeneration. The present study aimed to evaluate the effect of targeted mild ozone after inducing cerebral ischemia in vitro. Neuroblastoma lineage cells (SH-SY5Y) and canine amniotic membrane stem cells were subjected to 24 hours of hypoxia in an incubator culture chamber. The cells were evaluated by MTT assay, colorimetric assay spectrophotometry, fluorescence microscopy, and flow cytometry. Treatment with low concentrations of ozone (2–10 µg/mL), indicated a possible neuroregenerative effect at low concentrations, correlated with lower levels of apoptosis and oxidative stress compared to cells not subjected to hypoxia. High concentrations of ozone (18–30 µg/mL) promoted an increase in rate of apoptosis and cell death. We developed a novel protocol that mimics ozone therapy for ischemic stroke, using ozonized culture medium after hypoxia induction. Although more studies are needed, we conclude that ozone has a dose-dependent hormetic effect and can reverse the effect of ischemia in vitro at low concentrations.

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 (β-HBO), possibly due to higher dry matter intake or the biotransformation of NEFA into β-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.

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.

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.

Abstract 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.

Alessandra de Oliveira Pinheiro,1 Valéria M Lara,1 Aline F Souza,1 Juliana B Casals,2 Fabiana F Bressan,1 Paulo Fantinato Neto,1 Vanessa C Oliveira,1 Daniele S Martins,1 Carlos E Ambrosio1 1Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Pirassununga, São Paulo, Brazil; 2Private Veterinary Practice, Pirassununga, São Paulo, BrazilCorrespondence: Carlos E AmbrosioDepartment of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, FZEA- Av. Duque de Caxias Norte, 225, ZMV, Pirassununga 13635-900, São Paulo, BrazilTel +55 19 3565-4113 Email ceambrosio@usp.brPurpose: 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.Keywords: canine stem cells, immunomodulation, fetal annexes

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.

Testicular degeneration by heat is the leading cause of infertility in bulls. Beef cattle are generally farmed under hot and humid conditions, and consequently, the thermotolerance of each breed must be considered in their natural environment. This study aimed to evaluate the reproductive characteristics of Brahman bulls maintained in the grazing system, with or without shadow availability. Ten Brahman bulls aging between 24 and 30 months were allocated in two different paddocks, with or without shadow availability. The heat tolerance test was performed on three non-consecutive typical summer days. The semen samples were collected at four times points in a 14 days interval. The climate conditions were monitored throughout the experiment; and clinical evaluation, testicular consistence and scrotal circumference were measured before every semen collection. In addition, semen was evaluated regarding volume, aspect, turbulence, motility, straight movement, sperm concentration, and morphological exam. The studied Brahman bulls showed a high thermolysis capacity, high heat tolerance, and no differences in semen quality were observed between groups.

The aim of the present study is to evaluate the erythrogram and iron serum profiles of neonatal calves born spontaneously or born by elective cesarean section with or without dexamethasone induction. The research was performed on 38 newborn Nellore calves. Three groups of calves were assigned according to the type of birth: calves born by spontaneous vaginal calving (n = 10), calves born by elective cesarean section without inducing labor (n = 14), and calves born by elective cesarean section with labor induction with dexamethasone (n = 14). Blood samples to assess red blood cell count (RBC), hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), concentration of mean corpuscular hemoglobin (MCHC), serum iron (SFe), total capacity to bind iron to transferrin (TIBIC), and transferrin saturation index (TSI) were performed at calving (0, 3, 6, and 12 h of life) and on 1, 2, 3, 5, 7, 10, 15, and 30 days of life. Regardless of the experimental group (calves born spontaneously, or born by elective cesarean section with or without dexamethasone induction), in the first day of life there was a decrease in the number of red blood cells, hemoglobin rates, and values of the globular volume. In the period of the first 10 days of life, animals from spontaneous vaginal delivery quickly recovered values of erythrocytes, hemoglobin, and packed cell volume, whereas animals born by elective C-section (induced and uninduced) did not recover as quickly in their rates of hemoglobin and packed cell volume values. In calves born by elective C-section (induced and uninduced), it was observed in their period between 10 and 30 days of life that the MCV and MCH were reduced by passing the presenting microcytic hypochromic when compared with calves obtained by spontaneous vaginal delivery. In the period between 10 and 30 days of life, the levels of SFe and TSI in animals born by elective C-section (induced and uninduced) are significantly lower. The differences in the erythrogram values between Nellore calves born spontaneously and those by elective C-section with or without induction must be considered consequent to the process of neonatal adaptation to extrauterine life. Iron supplementation in the first month of life in calves from cesarean could be recommended to prevent anemia of this iron deficiency.

The veterinarian has to frequently assess blood tests in equine medicine. The reference values are adapted from international literature, based on examinations of healthy horses, under a standardized climate and physical activity. This study aimed to compare the blood values obtained from sports horses and traction horses, taking into account the type of activity performed. Anamnesis and hematology data from 76 animals were used: 32 members belonged to the Traction Group (TG), corresponding to equines from traction activity (cart horses) and 44 members belonged to the Sport Group (SG), corresponding to equines used in obstacle jumping, both groups were from the Pirassununga region, State of São Paulo. Complete blood counts and plasmatic fibrinogen dosage were evaluated. The animals from the TG had lower erythrocyte count and hematocrit values. The animals from the TG had greater leukocyte values in the leukogram compared to the animals from the SG. Cart horses were found to have lower means of hematological values compared to the jumping horses. Moreover, these values were below the normal physiological range for the species. Keywords: correlation; hematological evaluation; physical activity; horses.

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.

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.

Abstract 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.

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.

This review aim to present some clinical problems found in IVP-derived animals focusing on NT procedures and to discuss the possible role of epigenetics in such process. Also, as cell-secreted vesicles have been reported as possible regulators of important physiological reproductive processes such as folliculogenesis and fertilization, it is also presented herein a new perspective of manipulating the pre-implantation period trough effector molecules contained in such vesicles.

This study aimed to evaluate the possible renal and hepatic toxicity of tepoxalin administered before or after isoflurane-induced hypotension, as well as for five consecutive days. Twelve healthy mixed-breed cats, adult males, weighing 4.0±0.8kg were allocated into two groups. They received 25mgkg-1 of tepoxalin orally, two hours before the anesthetic procedure (PRE) or after the procedure (POST) and daily for five days. Cats were anesthetized with isoflurane and the concentration was increased until mean arterial pressure reached 40-60mmHg and kept at this level for 60 minutes. During hypotension, the physiological variables were measured at time 0 and every 10 minutes until 60 minutes, and bleeding time was measured at time 0, 30 and 60 minutes. Blood samples were drawn for a hemogram and determination of concentrations of alanine aminotransferase, alkaline phosphatase, urea, creatinine and Na+ at baseline, 24 hours, 48 hours and 7 days post-hypotension. Urine was collected at baseline, 24 hours, 48 hours and 7 days post-hypotension for determination of concentrations of creatinine, gamma-glutamyltransferase, urine specific gravity, protein, albumin and Na+. During the anesthetic procedure there were no important variations in physiological variables and bleeding time. There were differences only in fractional excretion of Na+, which was elevated at 7 days of evaluation in PRE and in the urine protein/creatinine ratio in PRE, which was higher than in POST at 24 and 48 hours post-hypotension. We conclude that tepoxalin does not cause alterations in hepatic enzymes but can cause discrete renal injury, resulting in proteinuria, in cats subjected to 60min of hypotension.

Com o intuito de caracterizar a anemia decorrente da verminose gastrintestinal e avaliar as alterações no leucograma, foram realizadas hemogramas de 96 caprinos da raça Saanen adultos, sendo que 57 destes estavam anêmicos. O diagnóstico da enfermidade foi realizado pelos sintomas característicos da doença como mucosas conjuntival esbranquiçadas, edema submandibular, emagrecimento e pelame arrepiado, confirmados por exame de fezes no qual se detectou a presença de ovos da superfamília Trichostrongyloidea. As amostras de sangue foram colhidas por punção da veia jugular, utilizando-se o EDTA como anticoagulante. Realizaram-se as seguintes provas: contagem do numero de hemácias, determinação do volume globular, dosagem de hemoglobina, cálculo dos índices hematimétricos (VCM, HCM e CHCM), contagem do número total de leucócitos e a contagem diferencial de leucócitos, efetuada em esfregaços sanguíneos, corados pelo método de Rosenfeld. Os animais foram divididos em grupos, baseado na magnitude da diminuição do volume globular, (1) animais sem anemia, (2) anemia de grau leve, (3) anemia moderada e (4) anemia intensa. Nos processos anêmicos leves ou moderados a anemia era do tipo normocítico e normocrômico, enquanto nos animais com anemia intensa (diminuição maior que metade dos valores normais de He, Ht, Hb) observou-se anemia do tipo normocítico e hipocrômico. O estado anêmico decorrente da verminose gastrintestinal determinou normoleucocitemia, neutrofilia sem desvio a esquerda, monocitose e linfopenia absoluta. Observou-se inversão do padrão leucocitário, que passou de linfocitário para neutrofílico. Essa inversão acentuou-se à medida que a gravidade da anemia verminótica tornava-se mais intensa.

Este estudo teve como objetivo avaliar a influência de recursos de climatização, ventilação e nebulização, sobre a fisiologia e o comportamento de vacas Holandesas alojadas em free-stall, durante o verão do sudeste brasileiro. Foram utilizadas 20 vacas Holandesas submetidas a dois tratamentos com e sem climatização. Os parâmetros ambientais registrados foram temperatura de bulbo seco, umidade relativa do ar e temperatura de globo negro. As variáveis fisiológicas avaliadas foram temperatura retal e frequência respiratória. As variáveis comportamentais registradas foram postura e suas atividades dentro da instalação. Para análise estatística utilizou-se a metodologia de quadrados mínimos por meio do procedimento PROC MIXED e PROC GLM. Apesar das diferenças estatísticas obtidas para as variáveis fisiológicas, as mesmas não foram biologicamente efetivas e indicaram que os animais se encontravam em conforto térmico. Os animais que dispunham de ventilação e nebulização alimentaram-se mesmo nas horas mais quentes do dia. A climatização é uma estratégia que permite maior conforto térmico aos animais e por consequência pode aperfeiçoar a produção leiteira através do aumento no consumo alimentar.

BACKGROUND: To investigate mechanisms of fetal-maternal cell interactions in the bovine placenta, we developed a model of transgenic enhanced Green Fluorescent Protein (t-eGFP) expressing bovine embryos produced by nuclear transfer (NT) to assess the distribution of fetal-derived products in the bovine placenta. In addition, we searched for male specific DNA in the blood of females carrying in vitro produced male embryos. Our hypothesis is that the bovine placenta is more permeable to fetal-derived products than described elsewhere. METHODOLOGY/PRINCIPAL FINDINGS: Samples of placentomes, chorion, endometrium, maternal peripheral blood leukocytes and blood plasma were collected during early gestation and processed for nested-PCR for eGFP and testis-specific Y-encoded protein (TSPY), western blotting and immunohistochemistry for eGFP detection, as well as transmission electron microscopy to verify the level of interaction between maternal and fetal cells. TSPY and eGFP DNA were present in the blood of cows carrying male pregnancies at day 60 of pregnancy. Protein and mRNA of eGFP were observed in the trophoblast and uterine tissues. In the placentomes, the protein expression was weak in the syncytial regions, but intense in neighboring cells on both sides of the fetal-maternal interface. Ultrastructurally, our samples from t-eGFP expressing NT pregnancies showed to be normal, such as the presence of interdigitating structures between fetal and maternal cells. In addition, channels-like structures were present in the trophoblast cells. CONCLUSIONS/SIGNIFICANCE: Data suggested that there is a delivery of fetal contents to the maternal system on both systemic and local levels that involved nuclear acids and proteins. It not clear the mechanisms involved in the transfer of fetal-derived molecules to the maternal system. This delivery may occur through nonclassical protein secretion; throughout transtrophoblastic-like channels and/or by apoptotic processes previously described. In conclusion, the bovine synepitheliochorial placenta displays an intimate fetal-maternal interaction, similar to other placental types for instance human and mouse.

Resumo: Na clínica médica de equinos, explora-se o perfil hematológico do animal, geralmente, com a finalidade de encontrar alterações que não foram constatadas ao exame clínico. A pesquisa de hematozoários em equinos, muitas vezes, apresenta resultados conflitantes entre o quadro clínico apresentado pelo animal e o resultado laboratorial, levantando a hipótese de que a técnica de pesquisa de hematozoários seja a responsável por falhas diagnósticas. Este estudo visa comparar os valores obtidos em exames hematológicos de 15 equinos de esporte e 15 equinos de tração (carroceiros), levando-se em consideração diferenças como características nutricionais, estado de higidez e tipo de atividade realizada, e comparar as diferentes técnicas de pesquisa de hematozoários, como esfregaço sanguíneo e PCR. Verificou-se que apenas os equinos de tração apresentaram valores médios de hemácias, hematócrito e hemoglobina abaixo do considerado fisiológico para a espécie, embora 100% dos animais, de ambos os grupos experimentais, tenham sido considerados positivos para hemoparasitoses por PCR. Verifica-se a superioridade do método de pesquisa de hemoparasitas por PCR, em comparação com esfregaço sanguíneo, realizado por diferentes técnicas, visto que apenas 33,3% dos animais foram considerados positivos para Theileria equi por esta técnica, enquanto que o PCR revelou 100% de positividade, para Theileria equi, Babesia caballi e infecção mista. Nenhum dos animais estudados foi diagnosticado com Anaplasma phagocytophilum (Ehrlichia equi) e Ehrlichia risticcii (Neoricketsia risticii). Verifica-se, então, que muitos dos diagnósticos de ausência de hemoparasitose por exame hematológico e ou esfregaço sanguíneo são errôneos, devido à baixa sensibilidade da técnica e podem repercutir em falha no tratamento ou disseminação dos hemoparasitos e das hemoparasitoses. Ressalta-se, então, a importância de exames como o PCR na elaboração de diagnóstico definitivo.

RESUMO: 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; p0,01). Já os grupos CO, COA, COMaFO e COMiFO diferiram estatisticamente do grupo controle (OSSO; p0,01). Já os grupos CO, COA e COMiFOA diferiram estatisticamente do grupo controle (OSSO; p

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Several opportunities for embryo development, stem cell maintenance, cell fate, and differentiation have emerged using induced pluripotent stem cells (iPSCs). However, the difficulty in comparing bovine iPSCs (biPSCs) with embryonic stem cells (ESCs) was a challenge for many years. Here, we reprogrammed fetal fibroblasts by transient expression of the four transcription factors (Oct4, Sox2, Klf4, and c-Myc, collectively termed “OSKM” factors) and cultured in iPSC medium, supplemented with bFGF, bFGF2i, leukemia inhibitory factor (LIF), or LIF2i, and then compared these biPSC lines with bESC to evaluate the pluripotent state. biPSC lines were generated in all experimental groups. Particularly, reprogrammed cells treated with bFGF were more efficient in promoting the acquisition of pluripotency. However, LIF2i treatment did not promote continuous self-renewal. biPSCs (line 2) labeled with GFP were injected into early embryos (day 4.5) to assess the potential to contribute to chimeric blastocysts. The biPSC lines show a pluripotency state and are differentiated into three embryonic layers. Moreover, biPSCs and bESCs labeled with GFP were able to contribute to chimeric blastocysts. Additionally, biPSCs have shown promising potential for contributing to chimeric blastocysts and for future studies.

Solar radiation is responsible for bull body temperature elevation. An alternative to minimize heat stress is to use artificial shade. Thus, this study aimed to evaluate the effect of thermal stress reduction, through shade availability, on reproductive characteristics of Nellore bulls (Bos indicus). For this, ten bulls were divided in: Available artificial shade (AS, n = 5) and Unavailable shade (US, n = 5). Each group was kept in two hectare paddocks, in which shade availability for group AS was artificially created. Animals were submitted to a clinical-reproductive evaluation and seminal analyses. No interaction was observed between treatments (AS and US) and time (8 collections) for all analyzed variables (P>0.05). No significant effect (P > 0.05) of treatment was observed for all parameters analyzed. So, it can be concluded that the absence of shaded areas during summer does not negatively affect reproductive characteristics such as: scrotal circumference, testicular consistency, progressive motility, percentage of rapidly moving cells (Computer Assisted Semen Analysis - CASA), morphology or sperm viability in Nellore bulls raised in southeastern Brazil, considering that results could be different in other regions of the country where average temperature is higher.

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., Competing Interests: Conflict of interests: ATO, AFRB, JRFM, PFN, CEA – No conflict of interest.

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 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 (β-HBO), possibly due to higher dry matter intake or the biotransformation of NEFA into β-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.