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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

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.

Transposon-mediated transgenesis is a well-established tool for genome manipulation in small animal models. However, translation of this active transgenesis method to the large animal setting requires further investigation. We have previously demonstrated that a helper-independent piggyBac (PB) transposon system can efficiently transpose transgenes into the bovine genome [Alessio et al. 2014 Reprod. Domest. Anim. 49 (Suppl. 1), 8]. The aims of the current study were a) to investigate the effectiveness of a hyperactive version of the PB transposase, and b) to determine the ability of the genetically modified cells to support early embryo and fetal development upon somatic cell nuclear transfer (SCNT). Bovine fetal fibroblasts (BFF) were chemically transfected with either pmGENIE-3 (a helper-independent PB transposon conferring genes for hygromycin resistance and enhanced green fluorescent protein (EGFP); Urschitz et al. 2010 PNAS USA 107, 8117-8122), pmhyGENIE-3 (carrying an hyperactive version of the PB transposase; Marh et al. 2012 PNAS USA 109, 19184-19189), or pmGENIE-3/Δ PB (a control plasmid lacking a functional PB transposase). Upon transfection, cell cultures were subjected to 14 days of hygromycin selection. Antibiotic-resistant and EGFP+ colonies were counted and data analysed by ANOVA and Tukey´s test. For SCNT, pmhyGENIE-3 and pmGENIE-3 polyclonal cell lines were selected by FACS and individual cells used as nuclear donors. Day 7 blastocysts were nonsurgically transferred to synchronized recipients. Conceptuses were recovered by Day 35 of gestation, observed under fluorescence excitation, and genotyped. The mean number of colonies in pmhyGENIE-3 group was significantly higher than those in pmGENIE-3 and the control group (324.0±17.8 v. 100.0±16.1 and 2.8±0.8 respectively, n=4-7; P

This study aimed to verify the influence of the transport in open or closed compartments, followed by two resting periods (1 and 3 hours) for the slaughter process on the levels of cortisol as a indicative of stress level in lambs. The slaughterhouse was located 85 km away from the place of confinement and the transportation of the lambs was carried out in a cage truck type, each cage had a divider in the middle making the front without external visual access to the environment, and the back portion of the cages with visual access to the road. At the slaughterhouse, blood samples were taken from 86 lambs after the transport and before slaughter (1 or 3 hours of resting) for plasma cortisol analysis. Variables were evaluated through the PROC GLM procedure from the Statistical Analysis System ©, version 9.1.3 software.The method of transport influenced in the cortisol concentration (P

Este estudo teve como objetivo avaliar a influência do recurso de ventilação sobre a fisiologia de vacas Holandesas alojadas em free-stall, durante o verão do sudeste brasileiro. Foram utilizadas 20 vacas Holandesas submetidas a dois tratamentos: controle e ventilação. Os parâmetros ambientais registrados foram temperatura e umidade relativa do ar e temperatura de globo negro. As variáveis fisiológicas avaliadas foram temperatura retal e frequência respiratória. Para obtenção das estatísticas descritivas utilizou-se a metodologia de quadrados mínimos. 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.

Under heat stress conditions, testicular degeneration can occur, even on zebu animals. Therefore, the aim of this work was to verify the occurrence of any possible effect of shadow availability on pasture on spermatic morphology of Brahman Bulls on Brazil Southeast. Five animals were allocated on pasture with shadow availability, and five bulls were allocated on pasture without any kind of shadow. No difference was observed (P>0.05) in any of the characteristics analyzed when Brahman bulls were maintained on pastures with or without shadow availability.