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.

Adipose and amnion-derived mesenchymal stem cells: Extracellular vesicles characterization and implication for reproductive biotechnology.

The stem cell-based research for reproductive biotechnology has been widely studied and shows promise for repairing defective tissue or degenerated cells to treat different diseases. The adipose tissue and amniotic membrane have awakened great interest in regenerative medicine and arises as a promising source of mesenchymal stem cells. Both types, adipose and amniotic derived mesenchymal stem cells (AMSCs) are multipotent cells with an enhanced ability to differentiate into multiple lineages.. We aimed to evaluate the effect of basal supplementation of exosomes in cell cultures with canine amniotic mesenchymal stem cells (MSCs). Mesenchymal stem cells derived from canine amniotic and adipose tissue were isolated and cultured performing cell passages until 80-90% confluence was reached. The growth curve was determined and peak cell growth was observed in the second passage. The cells were then characterized and differentiated into adipogenic, chondrogenic and osteogenic lineages. Extracellular vesicles from amnion were isolated using an ultracentrifugation protocol and characterized by nanosight analysis. To evaluate their ability to improve cellular viability in naturally inefficient passages, exosomes were co-cultures to the MSC cells. The results showed a 15-20% increase in the expansion rate of cultures supplemented with vesicles extracted in the first and second passages when compared to the control group. Statistical analysis using the Dunnett test (p ≤ 0.05) corroborated this result, showing a positive correlation between supplementation and expansion rate. These results indicate not only the importance of exosomes in the cell communication process but also the feasibility of the culture supplementation protocol for therapeutic purposes. The potential of the AMSCs for reproductive biotechnology is undoubted, however, their application to repair reproductive disorders and the involved mechanisms remain elusive. The strategies to enable the Adipose Stem Cells and AMSCs application in reproductive biotechnology and optimize their use for tissue regeneration open new venues using exosomes interactions.

Preliminary analysis of reproductive, behavioral and physiological characteristics of military working dogs.

The use of dogs in military work environments has always aroused great interest in the general population and determining the stress levels they go through is extremely important to maintain their welfare. The aim of this research was to evaluate if the work shifts in military working dogs leads to stress conditions and if this working influences on the reproductive performance and life quality. The study was conducted at the Military Police Kennel located at Campinas, Sao Paulo, Brazil. Eight male dogs of four different breeds (German Shepherd, Belgian Malinois Shepherd, Doberman, and Rottweiler) were evaluated during two different shifts: Working Shifts: animals working 12 hours a day with 2 hour-interval; and Control Shifts: animals that were on their day off (36 hours). Saliva samples were collected for cortisol analysis at the control and working shifts. The study was carried out over 60 days and analyzed behavior, physiology, and reproduction quality. Saliva samples, behavior observation of stereotyping, resting and moving activities and semen analysis were collected by digital stimulation (for combined second and third fractions). The salivary cortisol levels during the control and working shifts were between 0.361-0.438 and 0.312-0.592 µg/dL, respectively; the highest values were found at the end of working shifts. The animals were resting during most of the observation period, but few showed stereotypic behaviors. The testicular consistency was firm and semen parameters were within the normal values in German Shepherd, Belgian Malinois Shepherd, and Doberman dogs. However, Rottweiler dogs had a higher rate of sperm abnormalities, higher salivary cortisol levels, and more stereotypic behaviors. Nevertheless this work highlights the importance of further research relating reproduction and cortisol levels in military dogs.

Gonadal differentiation during embryonic and fetal development of male New Zealand rabbits (Oryctolagus cuniculus) / Diferenciación gonadal durante el desarrollo embrionario y fetal de conejos machos neozelandeses (Oryctolagus cuniculus)

SUMMARY: The rabbit is considered an ideal animal model for studies that describe abnormalities in the testicles due to the similar morphogenetic mechanisms of sexual development and diseases commonly found in humans. The aim of this study was to determine the male sexual differentiation of the New Zealand rabbit (Oryctolagus cuniculus) through development. The gestational age was estimated and classified as 9, 12, 14, 16, 18, 20, 23 and 28 gestational days. The morphological and sexual determination were performed by histological analysis of the reproductive tract in the embryos and fetuses (9-28 days) as well as by immunohistochemistry- Desert hedgehog-Dhh- (testis-specific protein on Y chromosome- 16, 20, 23 days and adult rabbits). Gonads were observed from the 14th day in an undifferentiated stage and with homogeneous aspect. Sexual differentiation was observed from the 16th day with presence of cells forming gonadal cords and Dhh+ cells in the gonadal parenchyma. From the 18th gestational day testicular cords were observed, which evolved into organized seminiferous tubules. The formation of the efferent ducts and ductus deferens and epididymis was observed on the 20th and 23rd days, respectively. The differentiation of the external genitalia occurred from the 23rd days from the anogenital distance and was identified to identify the penile structures. In summary, the features of the sexual differentiation were determined by observation of the Dhh+ protein in embryos from the 16th day to adulthood, and the morphological particularities observed from the 18th gestational day, determined by differentiation of the external genitalia from the 23rd day.

RESUMEN: El conejo se considera un modelo animal ideal para estudios que describen anomalías a nivel testícular debido a que presenta mecanismos morfogenéticos similares al desa- rrollo sexual y enfermedades que se encuentran comúnmente en los seres humanos. El objetivo de este estudio fue determinar la diferenciación sexual masculina del conejo de Nueva Zelanda (Oryctolagus cuniculus) a través del desarrollo. La edad gestacional se estimó y clasificó en 9, 12, 14, 16, 18, 20, 23 y 28 días gestacionales. La determinación morfológica y sexual se realizó mediante análisis histológico del tracto reproductivo en los embriones y fetos (9 - 28 días) así como mediante inmunohistoquímica -Desert hedgehog-Dhh- (proteína testicular específica en el cromosoma Y- 16, 20, 23 días y conejos adultos). Las gónadas se observaron a partir del día 14 en un estadio indiferenciado y con aspecto homogéneo. Se observó diferenciación sexual a partir del día 16 con presencia de células formadoras de cordones gonadales y células Dhh+ en el parénquima gonadal. A partir del día 18 de gestación se observaron cordones testiculares, que evolucionaron a túbulos seminíferos organizados. La formación de los conductos eferentes, deferentes y del epidídimo se observó a los 20 y 23 días, respectivamente. La diferenciación de los genitales externos ocurrió a partir del día 23 desde la distancia anogenital y se utilizó para identificar las estructuras del pene. En conclusión, las características de la diferenciación sexual se determinaron mediante la observación de la proteína Dhh en embriones desde el día 16 hasta la edad adulta, y las particularidades morfológicas observadas a partir del día 18 de gestación, determinadas por diferenciación de los genitales externos a partir del día 23.

Actions and Roles of FSH in Germinative Cells.

Follicle stimulating hormone (FSH) is produced by the pituitary gland in a coordinated hypothalamic-pituitary-gonadal (HPG) axis event, plays important roles in reproduction and germ cell development during different phases of reproductive development (fetal, neonatal, puberty, and adult life), and is consequently essential for fertility. FSH is a heterodimeric glycoprotein hormone of two dissociable subunits, α and ß. The FSH ß-subunit (FSHß) function starts upon coupling to its specific receptor: follicle-stimulating hormone receptor (FSHR). FSHRs are localized mainly on the surface of target cells on the testis and ovary (granulosa and Sertoli cells) and have recently been found in testicular stem cells and extra-gonadal tissue. Several reproduction disorders are associated with absent or low FSH secretion, with mutation of the FSH ß-subunit or the FSH receptor, and/or its signaling pathways. However, the influence of FSH on germ cells is still poorly understood; some studies have suggested that this hormone also plays a determinant role in the self-renewal of germinative cells and acts to increase undifferentiated spermatogonia proliferation. In addition, in vitro, together with other factors, it assists the process of differentiation of primordial germ cells (PGCLCs) into gametes (oocyte-like and SSCLCs). In this review, we describe relevant research on the influence of FSH on spermatogenesis and folliculogenesis, mainly in the germ cell of humans and other species. The possible roles of FSH in germ cell generation in vitro are also presented.

Step by Step about Germ Cells Development in Canine.

Primordial germ cells (PGCs) have been described as precursors of gametes and provide a connection within generations, passing on the genome to the next generation. Failures in the formation of gametes/germ cells can compromise the maintenance and conservation of species. Most of the studies with PGCs have been carried out in mice, but this species is not always the best study model when transposing this knowledge to humans. Domestic animals, such as canines (canine), have become a valuable translational research model for stem cells and therapy. Furthermore, the study of canine germ cells opens new avenues for veterinary reproduction. In this review, the objective is to provide a comprehensive overview of the current knowledge on canine germ cells. The aspects of canine development and germ cells have been discussed since the origin, specifications, and development of spermatogonial canine were first discussed. Additionally, we discussed and explored some in vitro aspects of canine reproduction with germ cells, such as embryonic germ cells and spermatogonial stem cells.

Pluripotent stem cells proliferation is associated with placentation in dogs.

Pluripotent stem cells have been studied as source of cells for regenerative medicine and acquire or genetic diseases, as an innovative therapy. Most tissues have stem cells populations, however in few quantities or impossible to be used during adult life, which lead to scientists look for new sources. Thus, this study aimed to analyze the presence of pluripotent cells in the uterus and placenta, following up non-pregnant, pregnant (begin, middle, and final), and postpartum periods in dogs. The uteri were obtained from social castration programs for population control in Pirassununga, Sao Paulo, Brazil. It was collected 20 uteri at different stages. The samples were fixed and processed for immunohistochemical analysis of NANOG, OCT4 and SOX2 expression, knowing as pluripotent stem cells makers. Our results showed positive expression for NANOG, OCT4 and SOX2 in all stages of gestation and nonpregnant uterus; however, we highlight some quantitative different between stages. OCT4 showed more expression in non-pregnant uterus than NANOG and SOX2, and its expression increased in pregnant uterus. In pregnant uterus there was more expression of NANOG than OCT4 and SOX2. Interesting, no difference was found between these markers in the other periods. In conclusion, it was possible to identify pluripotent stem cells in all periods in dog placenta and uterus, however during the early stage of pregnancy we observed more pluripotent stem cells than in all the others periods confirming the high plasticity and regeneration capacity of the uterine tissue.

Somatic feather follicle cell culture of the gallus domesticus species for creating a wild bird genetic resource bank.

The creation of a genetic resource bank of avian species aims to prevent the decline and fragmentation of wild bird populations, which in turn lead to the loss of genetic diversity and, in more serious cases, the extinction of the most threatened species. In order for the collected genetic material to be stored in a bank and useful when necessary, it is essential to improve the technique ensuring its effectiveness. Thus, our study used feather follicle cells from the domestic gallus species to standardize the technique of cell culture and subsequent cryopreservation. This study aimed to establish a protocol, in vitro, of isolation and primary culture of somatic cells derived from the feather follicle, with the purpose of establishing a cell lineage, and evaluate its viability for the biobank formation. Developing feathers of gallus domesticus were collected at 12, 21 and 34 days of age. The feathers were morphologically analyzed and then we selected the region of the calamus due to the presence of pulp for cell culture and cryopreservation. The results showed that it is possible to find cells with distinct morphology; cells in elliptical shape with central nucleus also in elliptical shape, cells with shape and round nucleus, cells compatible with the fibers of the barbules, cell agglomerates and cells adhered to the bottom of the plate with fibroblastatoid shape. After 24 hours of culture there was the presence of primary culture with 80% of confluence and after cryopreservation the average viability after freezing was 68.8%, with cellular morphologies being maintained. Therefore, we proved the isolation of somatic cells from the follicle of bird's feathers, suggesting that this is a source of great value, viable and effective for obtaining biological material for the elaboration of a biobank.

Morphology of male and female reproductive tract of the ocelot (Leopardus pardalis).

The Ocelot (Leopardus pardalis) is the largest species of this genus, despite having broad distribution in the Americas; it is included in the main list of endangered species. Their conservation is widely studied, but there is a lack of studies about their morphology. In order to contribute to the knowledge of its reproductive system, five male and female ocelots were examined macro- and microscopically by histological techniques. Macroscopic analysis of the male reproductive system revealed presence of prostate and bulbourethral gland located caudally to the urinary bladder and a penis with small spicules. Microscopically, the testes were encased by the tunica albuginea and divided it into lobules with 5-10 tubules per lobe. In females, macroscopic analysis demonstrated two ovaries position dorsally in the sublumbar region and caudal to the kidneys. The bicornuate uterus is composed by uterine horns (12 to 14 cm in length), which travels from the ovaries in a caudal direction to form a small uterine body (4 cm in length). The ovary analysis revealed, in longitudinal section, medullary region composed of loose connective tissue, a stroma rich in blood vessels, and an external parenchymal region surrounded by a tunica albuginea. The results of the study confirmed the similarity between ocelot's reproductive system as domestic cat's ones and showing for the first time the complete morphological tool to highlight these organs and tissue in this male and female endangered wild felid specie. The present study open venue for other researchers to consider morphological and preservationist features and aimed to help at long-term conservation of wild felines.

Neurons-derived extracellular vesicles promote neural differentiation of ADSCs: a model to prevent peripheral nerve degeneration.

Potential mechanisms involved in neural differentiation of adipocyte derived stem cells (ADSCs) are still unclear. In the present study, extracellular vesicles (EVs) were tested as a potential mechanism involved in the neuronal differentiation of stem cells. In order to address this, ADSCs and neurons (BRC) were established in primary culture and co-culture at three timepoints. Furthermore, we evaluated protein and transcript levels of differentiated ADSCs from the same timepoints, to confirm phenotype change to neuronal linage. Importantly, neuron-derived EVs cargo and EVs originated from co-culture were analyzed and tested in terms of function, such as gene expression and microRNA levels related to the adult neurogenesis process. Ideal neuron-like cells were identified and, therefore, we speculated the in vivo function of these cells in acute sciatic nerve injury. Overall, our data demonstrated that ADSCs in indirect contact with neurons differentiated into neuron-like cells. Neuron-derived EVs appear to play an important role in this process carrying SNAP25, miR-132 and miR-9. Additionally, in vivo neuron-like cells helped in microenvironment modulation probably preventing peripheral nerve injury degeneration. Consequently, our findings provide new insight of future methods of ADSC induction into neuronal linage to be applied in peripheral nerve (PN) injury.

Canine fetus immune system at late development.

The immune system is mainly responsible for protecting the organism against agents that may interfere in its homeostasis. Thus, understand how this system develops and operates is very important, for create new therapies to assist this system in its operation, such as its failure. In domestic dogs, few studies show how actually occurs the development, maturation and functioning of the immune system. Therefore, this study demonstrates the development and possible activation of it on dog fetus from late gestational period by in situ and microscopic analyzes.

Stem cells derived from bone marrow and preclinical trials in Veterinary Medicine / Células-tronco derivadas da medula óssea e suas aplicações na Medicina Veterinária

The bone marrow is the largest reserve of stem cells into the body, consisting of stromal cells/mesenchymal niches and hematopoietic system. These cells can differentiate as different lineages as osteogenic, chondrogenic and adipogenic. The bone marrow stem cells population has widely used and an attractive target for potential therapeutic treatment for preclinical trials, due its high plasticity of differentiation. Thus, with this review we aimed to show an overview of stem cells and mainly bone marrow cells with basic concepts, as well as their potential new venues for treatment in regenerative veterinary medicine.

A medula óssea é a maior reserva de células-tronco do corpo, cujo consistem em células mesenquimais e hematopoiéticas. Estas células têm a capacidade de diferenciar-se em varias linhagens, como osteogênicas, condrogênicas e adipogênicas. A população de células-tronco derivadas da medula óssea tem sido um alvo atraente para tratamentos terapêuticos e ensaios pré-clínicos, devido sua elevada plasticidade e capacidade de diferenciação. Assim, esta revisão objetiva mostrar uma visão geral das célulastronco e principalmente conceitos básicos sobre as células da medula óssea, bem como os seus potenciais e novos espaços para o tratamento regenerativo na medicina veterinária.

Anatomia da cavidade bucofaringeana de Sorubim trigonocephalus (Siluriformes, Osteichthyes) / Anatomy of the buccopharyngea cavity of Sorubim trigonocephalus (Siluriformes, Osteichthyes)

Sorubim trigonocephalus Miranda et Ribeiro, 1920 conhecido popularmente como chinelo, é uma espécie migratória, de importância econômica. Bastante apreciado na gastronomia por apresentar sabor agradável e sem ossos intermusculares (espinhos). Os exemplares foram coletados em um trecho no Rio Teles Pires, localizado no município de Alta Floresta/MT. Anestesiados e sacrificados em solução aquosa de benzocaína, fixados com solução de formoldeído a 10%, transferidos para o Laboratório de Anatomia Animal/Unemat para descrição das características da cavidade bucofaringeana. Esta espécie apresentou uma pré-maxila bem desenvolvida e cabeça achatada dorsoventralmente, boca localizada na porção ventral média da cabeça, com fenda bucal ampla. Os lábios superiores apresentaram pigmentação cinza escuro com pequenas áreas claras, sendo mais largos que os inferiores e não apresentaram pigmentação. Foram identificadas quatro regiões dentígeras na porção anterior da cavidade bucal e duas na região da faringe, todas com dentes viliformes. A porção posterior da cavidade bucal era limitada lateralmente por quatro pares de arcos branquiais que decresciam em tamanho do primeiro ao último par, crânio-caudal, formados por dois ramos: o superior, mais curto, e o inferior, mais longo. A cavidade bucofaringeana de S. trigonocephalus se mostrou semelhante à de outros teleósteos descritos na literatura, estando adaptado ao hábito alimentar com dieta carnívoro-ictiofágica e diferindo apenas pela ausência de língua estrutural com pré maxila bem desenvolvida apresentando dentes viliformes.

Sorubim trigonocephalus Miranda et Ribeiro, 1920 is popularly known as slipper, migratory specie of economic importance. It is much appreciated in gastronomy because it has good flavor and no intermuscular bones (spines). Specimens were collected on a stretch of the Teles Pires river, located in Alta Floresta/MT, anesthetized and euthanized in aqueous solution of benzocaine, fixed with 10% formaldehyde and transferred to the Laboratory of Animal Anatomy of Unemat to be analyzed and described the characteristics of the buccopharyngeal cavity. With a premaxilla well developed and head dorsoventrally flattened, S. trigonocephalus show out located on the ventral portion of the head mouth wide slit. The upper lip pigmentation had dark gray with small light areas, wider than the and without pigmentation. Was identified four dentigerous regions in in the anterior oral cavity and two in the pharynx region, both with viniliform tooth. The posterior portion of the oral cavity was bounded laterally by four pairs of gill arches that decreased in size from first to last pair, formed by two branches: the higher, shorter, and the lower, longer. The buccopharyngea cavity of S. trigonocephalus showed similar to other teleosts described in the literature, and is adapted to eating habits with diet ictiophagic carnivore and differing only by the absence of a structural tongue and pre maxilla well developed showing viniliform tooth.

Modelo de suprimento sanguíneo do intestino delgado e grosso da preguiça de coleira (Bradypus torquatus) / Blood supply model of the small and large intestine of the maned three-toed sloth (Bradypus torquatus)

O Bradypus torquatus, conhecido como preguiça de coleira, está classificado na lista vermelha da IUCN como vulnerável. Este animal é um folívoro estrito que se alimenta de um pequeno número de plantas. O suprimento sanguíneo do intestino delgado e grosso de oito Bradypus torquatus, pertencentes ao acervo da Anatomia dos Animais Domésticos e Sivestres da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, foi estudado. O método incluiu preparação de relatório macroscópico, perfusão do sistema arterial com água (40°C), injeção de látex corado (Neoprene® 650, Sulvinil® 2350-0003), fixação com formaldeído (10%), conservação em solução modificada de Laskowiski e dissecação por mesoscopia de luz (Lupa LTS® 3700). A irrigação dos intestinos delgado e grosso dependeu da aorta abdominal, cujo ramo visceral ventral identificado como artéria mesentérica comum distribuiu-se no mesentério e mesocólon. Uma sequência de 9 a 25 ramos colaterais primários craniais destinaram-se ao duodeno, jejuno, íleo e parte da bolsa cecal. Outra sequência de 4 a 11 ramos caudais destinaram-se à bolsa cecal e cólons. No animal adulto, o modelo de vascularização do intestino diferiu dos outros vertebrados recentes em razão de não ocorrer coalescência peritoneal ao longo do intestino delgado e grosso.

The Bradypus torquatus also known as maned three-toed sloth is listed as vulnerable in the IUCN red list. This species is strict folivorous that feeds on a relatively small number of food plants. The blood supply in the small and large intestines of eight Bradypus torquatus was studied. These animals are from the Anatomia dos Animais Domésticos e Silvestres collection of the Universidade de São Paulo . The method included preparation of the macroscopic collection report, perfusion of the arterial network with water (40°C), injection of colored latex (Neoprene 650®, 2350-0003 Suvinil® dye), fixation in formaldehyde (10%), preservation in modified Laskowski solution and dissection under mesoscopic light (Lupa LTS® 3700). The blood supply of small and large intestine depends on the abdominal aorta, whose ventral visceral branch identified as the common mesenteric artery was distributed in the mesentery and mesocolon. A sequence of 9 to 25 primary collateral branches cranial is allocated to the duodenum, jejunum, ileum and part of the cecum. Another sequence of 4 to 11 caudal branches are destined to the cecum and colon pouch. The vascular pattern of adult maned three-toed sloth intestine differs from those of other previously described vertebrates, because there is no occurrence of coalescence peritoneal through small and large intestine.

Protocols for obtainment and isolation of two mesenchymal stem cell sources in sheep.

PURPOSE: To evaluate different protocols to isolate stem cells from ovine umbilical cord blood and adipose tissue. METHODS: There were used 5 samples of umbilical blood and 5 samples of perirenal adipose tissue from 10 female sheep. All the samples were obtained through surgery, to harvest aseptic samples. There were used 3 protocols for obtainment and culture of umbilical cord blood stem cells and 4 protocols for ovine adipose tissue stem cells. RESULTS: It was possible to observe only one successful protocol for the obtainment of umbilical cord blood stem cells. When analyzing the techniques used to obtain adipose tissue stem cells, only one of the methods was effective as well. Through colony forming unit assay, there were obtained 58 colonies of cells after seven days in culture. Flow citometry tests revealed the cells were positive to CD44 and exhibited negative reaction to CD38, CD45, CD41/61. These cells showed a growth curve with very well defined phases LOG, LAG and PLATEAU. This phases are typically seem in mesenchymal stem cells growth curves. CONCLUSIONS: The isolation and culture of mesenchymal stem cells from ovine umbilical cord blood are complex and request more detailed assays. Stem cells from fat tissue sheep showed mesenchymal characteristics, according to their cell growth curve, ability to origin colonies of fibroblastoid cells and positive reactivity with the antibody CD44 by flow citometry.

Protocols for obtainment and isolation of two mesenchymal stem cell sources in sheep / Protocolos para obtenção e isolamento de células tronco mesenquimais de duas fontes em ovinos

PURPOSE: To evaluate different protocols to isolate stem cells from ovine umbilical cord blood and adipose tissue. METHODS: There were used 5 samples of umbilical blood and 5 samples of perirenal adipose tissue from 10 female sheep. All the samples were obtained through surgery, to harvest aseptic samples. There were used 3 protocols for obtainment and culture of umbilical cord blood stem cells and 4 protocols for ovine adipose tissue stem cells. RESULTS: It was possible to observe only one successful protocol for the obtainment of umbilical cord blood stem cells. When analyzing the techniques used to obtain adipose tissue stem cells, only one of the methods was effective as well. Through colony forming unit assay, there were obtained 58 colonies of cells after seven days in culture. Flow citometry tests revealed the cells were positive to CD44 and exhibited negative reaction to CD38, CD45, CD41/61. These cells showed a growth curve with very well defined phases LOG, LAG and PLATEAU. This phases are typically seem in mesenchymal stem cells growth curves. CONCLUSIONS: The isolation and culture of mesenchymal stem cells from ovine umbilical cord blood are complex and request more detailed assays. Stem cells from fat tissue sheep showed mesenchymal characteristics, according to their cell growth curve, ability to origin colonies of fibroblastoid cells and positive reactivity with the antibody CD44 by flow citometry.

OBJETIVO: Testar diferentes protocolos para o isolamento de células tronco a partir de sangue de cordão umbilical e tecido adiposo de ovinos. MÉTODOS: Foram utilizadas cinco amostras de sangue de cordão umbilical e cinco amostras de tecido adiposo perirrenal de 10 fêmeas de ovelha. A coleta das amostras foi realizada através de procedimento cirúrgico para coleta do material de forma mais asséptica possível. Foram realizados três protocolos de isolamento e cultivo das células-tronco do cordão umbilical e quatro protocolos para o isolamento e cultivo das células-tronco de gordura de ovinos RESULTADOS: Somente um dos protocolos utilizados para o isolamento das células-tronco de cordão umbilical foi efetivo. Dos quatro protocolos utilizados para isolamento das células-tronco de gordura, da mesma forma, apenas um obteve sucesso. Foi realizado o ensaio de unidades formadoras de colônias destas células, sendo contadas 58 colônias ao final de sete dias. Na citometria de fluxo essas células mostraram-se positivas para CD44 e negativas para CD38, CD45, CD41/61. Estas células apresentaram curva de crescimento com fases de LOG, LAG e PLATEAU bem definidas, características das curvas de crescimento das células-tronco de origem mesenquimal. CONCLUSÕES: O isolamento e cultivo das células-tronco mesenquimais do cordão umbilical de ovinos é de difícil realização, exigindo maiores ensaios e estudos profundos. Células tronco do tecido adiposo de ovelhas demonstraram características mesenquimais, de acordo com a curva de crescimento, habilidade de formação de colônias, células com morfologia fibroblastóide e reação positiva ao anticorpo CD44.

Propolis and amnion reepithelialise second-degree burns in rats.

Burns are serious consequences of trauma in terms of both imminent mortality and prolonged periods of morbidity. They are often accompanied by unsatisfactory cosmetic as well as functional and psychological outcomes. These complications emphasise the need for stronger efforts in achieving greater diversity and effectiveness in the treatment of skin burns. This study aimed to verify the effectiveness of gross and microscopic epidermal and dermal responses in the process of regenerative repair or healing of burns in rats that were treated either daily with 5% propolis ointment or by autologous amnion graft. Second-degree burns were inflicted in the neck region of female rats by contact with a hot metal (at 130 °C) for 5 s. Propolis treatment accelerated the process of tissue repair and led to decreased local inflammation, which indicates that treatment with propolis was successful in the initial period (7 days) and stimulated the production of collagen fibre (assessed by morphometry) in all the periods evaluated (14 and 21 days). Amnion treatment inhibited local inflammation (assessed macroscopically), stimulated local epithelial regeneration (assessed microscopically) and stimulated the production of collagen fibre (assessed by morphometry) in the days following burn. These treatments offer new therapeutic strategies for treating severe skin burns; these strategies may allow the minimisation of scar formation, a more rapid return of function and, ultimately, a better quality of life for burn patients.

Successful transplant of mesenchymal stem cells in induced osteonecrosis of the ovine femoral head: preliminary results / Sucesso no transplante de células tronco mesenquimais em ovinos com osteonecrose induzida da cabeça do fêmur: resultados preliminares

PURPOSE: Evaluate the bone tissue recovery following transplantation of ovine mesenchymal stem cells (MSC) from bone marrow and human immature dental-pulp stem cells (hIDPSC) in ovine model of induced osteonecrosis of femoral head (ONFH). METHODS: Eight sheep were divided in three experimental groups. First group was composed by four animals with ONFH induced by ethanol through central decompression (CD), for control group without any treatment. The second and third group were compose by two animals, six weeks after ONFH induction received transplantation of heterologous ovine MSC (CD + oMSC), and hIDPSC (CD + hIDPSC), respectively. In both experiments the cells were transplanted without application of any type of immunosupression protocol. RESULTS: Our data indicate that both cell types used in experiments were able to proliferate within injured site providing bone tissue recovery. The histological results obtained from CD+hIDPSC suggested that the bone regeneration in such animals was better than that observed in CD animals. CONCLUSION: Mesenchymal stem cell transplant in induced ovine osteonecrosis of femoral head by central decompression technique is safe, and apparently favors bone regeneration of damaged tissues.

OBJETIVO: Verificar os efeitos das células-tronco mesenquimais da medula óssea de ovinos e da polpa dentária imatura humana em ovinos com osteonecrose induzida, da cabeça do fêmur. MÉTODOS: Oito ovelhas foram distribuídas em três grupos experimentais. O primeiro grupo foi composto por quatro animais com osteonecrose da cabeça do fêmur induzida por etanol através da descompressão central, que não receberam nenhum tratamento. O segundo e o terceiro grupo, cada um composto por dois animais, receberam transplante heterólogo de células tronco mesenquimais de ovinos e polpa dentária imatura humana seis semanas após a indução da osteonecrose da cabeça do fêmur, respectivamente. Em ambos os grupos experimentais as células foram transplantadas sem o uso de drogas imunossupressoras. RESULTADOS: Os achados demonstram que as células-tronco mesenquimais injetadas na cabeça do fêmur se encontravam viáveis após o transplante no novo sítio e proliferaram em pouco tempo. Os dados histológicos sugerem que a regeneração óssea nos animais transplantados com polpa dentária imatura humana foi mais rápida do que nos animais submetidos somente a descompressão central. CONCLUSÃO: O transplante de células tronco mesenquimais na osteonecrose da cabeça do fêmur induzida em ovinos através da técnica de descompressão central é um procedimento seguro, e aparentemente favorece a regeneração óssea de tecidos lesados.

Successful transplant of mesenchymal stem cells in induced osteonecrosis of the ovine femoral head: preliminary results.

PURPOSE: Evaluate the bone tissue recovery following transplantation of ovine mesenchymal stem cells (MSC) from bone marrow and human immature dental-pulp stem cells (hIDPSC) in ovine model of induced osteonecrosis of femoral head (ONFH). METHODS: Eight sheep were divided in three experimental groups. First group was composed by four animals with ONFH induced by ethanol through central decompression (CD), for control group without any treatment. The second and third group were compose by two animals, six weeks after ONFH induction received transplantation of heterologous ovine MSC (CD + oMSC), and hIDPSC (CD + hIDPSC), respectively. In both experiments the cells were transplanted without application of any type of immunosupression protocol. RESULTS: Our data indicate that both cell types used in experiments were able to proliferate within injured site providing bone tissue recovery. The histological results obtained from CD+hIDPSC suggested that the bone regeneration in such animals was better than that observed in CD animals. CONCLUSION: Mesenchymal stem cell transplant in induced ovine osteonecrosis of femoral head by central decompression technique is safe, and apparently favors bone regeneration of damaged tissues.

Ultra-sonografia abdominal e pélvica em cães da raça golden retriever sadios, portadores e afetados pela distrofia muscular progressiva / Abdominal and pelvic ultrasonography in healthy golden retriever dogs, carriers and affected by gradual muscular dystrophy

A distrofia muscular de Duchenne (DMD) é um tipo de distrofia muscular em humanos caracterizada por uma doença genética ligada ao cromossomo X. O cão golden retriever portador da distrofia muscular (GRMD) tem sido intensamente estudado e considerado o modelo mais representativo para a doença observada em humanos. Assim, como forma de verificar anormalidades em órgãos internos nesses animais, foi realizado o exame ultra-sonográfico de 24 cães golden retriever saudáveis, portadores e afetados pela distrofia muscular. O exame ultra-sonográfico do GRMD diagnosticou aumento hepático de moderado a severo, incluindo os vasos hepáticos e seus ramos e aumento de ecogenicidade da vesícula biliar e vesícula urinária. Entretanto, não foram observadas imagens claras de alterações no baço e nos vasos ramos da aorta. A partir disso, acreditamos que o exame ultra-sonográfico constitui-se em um procedimento útil no acesso de órgãos abdominais em cães afetados pela distrofia muscular.

Duchenne muscular dystrophy (DMD) is one type of human’s muscular dystrophy characterized by a genetic disorder linked to the X chromosome. The Golden Retriever muscular dystrophic (GRMD) has been extensively studied and considered the best resembling model to the human disease. Therefore, for identifying internal organs abnormality in GRMD, abdominal and pelvic ultrasonography was performed in 24 golden retriever dogs, either healthy or muscular dystrophic in different levels of disease. The GRMD ultrasonographic exams diagnosed moderate to severe liver enlargement, including hepatic vessels and their branches and increase of echogenicity in gallbladder and urinary bladder. However was not-clearly recognized pathologic images from spleen and aortic vessels were accessed. Therefore, we believe, the ultrasonographic exam was an useful procedure to the assessment of abdominal organs in dogs affected by muscular dystrophy.