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

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

Nitric oxide in frozen-thawed equine sperm: Effects on motility, membrane integrity and sperm capacitation.

Nitric oxide (NO) is a reactive nitrogen species (RSN) that, over the years, has been shown to be integrated with biological and physiological events, including reproductive processes. NO can affect the functionality of spermatozoa through free radical scavenging, deactivating and inhibiting the production of superoxide anions (O2.-). However, the role of NO in mammalian spermatozoa physiology seems paradoxical. The aim of this study was to investigate the effects of NO on motility, hyperactivation, membrane integrity, peroxidation, and capacitation in cryopreserved equine sperm. Ejaculates were collected and cryopreserved. After thawing, samples were centrifuged, suspended in an in vitro fertilization (IVF) medium and incubated with the following treatments: 1) C = control (IVF); 2) A = l-arginine (10 mM - In); 3) L = L-NAME (1 mM - Ih); 4) M = methylene blue (100 mM - Re); 5) AL = L-arginine + L-NAME (In + Ih); 6) AM = L-arginine + methylene blue (In + Re). The samples were evaluated for spermatic kinetics by CASA and other analyses [plasma and acrosomal membranes used the propidium iodide (PI) and Pisum sativum agglutinin (PSA), detection of tyrosine residues phosphorylation in the membrane (F0426), nitric oxide (DAF-2/DA), lipid peroxidation (C11-BODIPY581/591)] by flow cytometry. The l-arginine treatments reduced MOT, PROG, RAP and LIN only at time 0 min compared to the control and L-NAME. These treatments (MT and MP, VAP, VSL, LIN, RAP) also reduced the sperm movement characteristics but only at the beginning of the incubation period. After this period of incubation, motility recovered. NO removal by methylene blue almost completely inhibited sperm motility, but these treatments had the highest percentages of intact membranes. l-arginine treatments improved acrosome reactions and differed from M and AM. NO production, tyrosine phosphorylation and lipid peroxidation did not differ among treatments, except for M and AM, where a reduction in these variables was detected. Therefore, equine sperm capacitation and the acrosome reaction are part of an oxidative process that involves the participation of ROS, and NO plays an important role in the maintenance and regulation of motility, hyperactivation, induction of acrosome reaction and possibly in capacitation, which are indispensable processes for the fertility of equine sperm.

In vitro identification of a stem cell population from canine hair follicle bulge region.

Skin is an extensive and easily accessible organ possessing various cell types that are constantly renewed. Previous studies have suggested the presence of a stem cell niche at the bulge region of the hair follicle, which contains cells positive for CD200 and CD34. Thus, this study sought to identify these cell populations in canine skin cells using the following methods 1- collecting samples of adult and fetal skin and isolating and culturing these cells using a method of simple enzymatic digestion and 2- testing the cell cultures for CD200 and CD34 in vitro and comparing them with skin tissue samples (in situ). Immunofluorescence results were negative for both CD200 and CD34 in frozen and paraffin embedded tissue, whereas the analysis showed that cultured cells positive for CD34, CD200 and double positive cells could be visualized in different percentages. Additionally, the pluripotency marker OCT4 was positive in the isolated cells. Analysis of CD34, CD200 and OCT4 by RT-qPCR showed that there is expression in fetal and adult cells, although no difference was observed between groups. Our results suggest that bulge stem cells from both fetuses and adult dogs were reported with the use of CD34 and CD200 markers in this study, and further techniques for cell isolation and in vitro cultivation are needed in order to obtain enriched populations of skin stem cells in dogs.

Regulation of neural stem cells by choroid plexus cells population.

The choroid plexus is a tissue on the central nervous system responsible for producing cerebrospinal fluid, maintaining homeostasis and neural stem cells support; though, all of its functions still unclear. This study aimed to demonstrate the niches of choroid plexus cells for a better understanding of the cell types and functions, using the porcine as the animal model. The collected material was analyzed by histology, immunohistochemistry, and cell culture. The cell culture was characterizated by immunocytochemistry and flow cytometry. Our results showed OCT-4, TUBIII, Nestin, CD45, CD73, CD90 positive expression and GFAP, CD105 negative expression, also methylene blue histological staining confirmed the presence of telocytes cells. We realized that the choroid plexus is a unique and incomparable tissue with different niches of cells as pluripotent, hematopoietic, neuronal progenitors and telocyte cells, which provide its complexity, differentiated functionality and responsibility on brain balance and neural stem cells regulation.

Caracterização comparativa do intestino das espécies da Ordem Xenarthra / Comparative characterization of the intestine of species from the Xenarthra Order

O sucesso na manutenção de uma espécie depende de vários fatores entre eles a eficiência digestiva, sendo assim parâmetros morfométricos do tubo digestório são necessários para o conhecimento dos processos digestivos dos alimentos no organismo animal além de indicar a preferência alimentar de uma espécie. Este trabalho visou descrever morfologicamente os intestinos delgado e grosso, órgãos do sistema digestório de representantes da ordem Xenarthra a fim de fornecer subsídios para a avaliação da dieta e realização de procedimentos clínicos nestes animais, sejam eles de vida livre ou de cativeiro. Foram utilizados 7 espécimes entre preguiças-de-coleira (Bradypus torquatus), tatu-verdadeiro (Dasypus novemcinctus) e tamanduá-bandeira (Myrmecophaga tridactyla). Todos as amostras foram processadas seguindo procedimentos de rotina efetuados nos laboratórios de Anatomia Animal e Histologia da FZEA/USP. Os intestinos de B. torquatus se apresentaram curtos e simples, enquanto que nos exemplares de D. novemcintus e M. tridactyla o intestino era longo e com algumas peculiaridades. No duodeno de todos os espécimes notamos a presença das glândulas de Brünner e estruturas para aumentar a superfície de absorção. Apenas em preguiças, o mesentério mantém o jejuno preso à parede dorsal da cavidade abdominal. O íleo representou a menor porção nas preguiças e tatus, exceto em tamanduáque apresentava o íleo como a maior parte depois do jejuno. O ceco em tatus e tamanduás apresentavam tamanho considerável e a presença de glândulas na mucosa, nestas espécies destacamos a funcionalidade do ceco, uma vez que este se apresentou repleto de restos alimentares. Na mucosa do cólon de todos os espécimes, haviam criptas de Lieberkühn, sendo mais numerosas em D. novemcinctus e M. tridactyla. Apenas em B. torquatus, o reto apresentou maior diâmetro e rigidez em relação ao cólon. No reto de todas as espécies estudadas, a superfície glandular era numerosa e com grande quantidade de células caliciformes, que produzem muco, para facilitar a defecação. Nossos resultados demonstram que a conformação e estruturas do sistema digestório reflete mais o tipo de alimentação e necessidades digestórias do animal do que à família que ele pertence.(AU)

Morphometric parameters of the digestive tract are required for an understanding of the digestive processes of the food in the animal organism, besides indicating the feeding preference of specie. This study aimed to describe morphologically the small and large intestines, organs of the digestive system of representatives of Xenarthra order to provide data for the evaluation of diet and conduct clinical procedures in these animals, whether free-living or captive. At this research, were used in total 7 specimens from three-toed sloths (Bradypus torquatus), nine-banded armadillo (Dasypus novemcinctus) and giant anteater (Myrmecophaga tridactyla). The intestines of B. torquatus were short and simple, but at the specimens of D. novemcintus and M. tridactyla the intestines were long and had some peculiarities. We notice the presence of Brunner's glands and structures to increase the surface absorption at the duodenum of all specimens. Only in B. torquatus, we notice that the mesentery remains the jejune attached to the dorsal wall of the abdominal cavity. The ileum represented the lower portion of the intestines in all studied specimens except in M.tridactyla. The cecum in D. novemcinctus and M. tridactyla showed considerable size, glands at the mucosa and was full of food debris, indicating that it was functional. In the mucosa of the colon of all specimens had crypts of Lieberkühn, being more numerous in D. novemcinctus and M. tridactyla. Only in B. torquatus, the rectum showed greater diameter and stiffness compared to the colon. In all species studied, we notice a large glandular surface and lots of goblet cells that produce mucus to facilitate defecation. Our results demonstrate that the conformation and structure of the digestive system reflects more the kind of diet and digestive needs of the animal, than to the family he belongs.(AU)

Morfologia da glândula mamária do quati (Nasua nasua) / Morphology of mammary gland of the coati (Nasua nasua)

Nasua nasua é um animal onívoro, encontrado em todo Brasil. A reprodução da espécie ocorre somente uma vez ao ano, na primavera. As fêmeas são matriarcas e amamentam suas crias até os 5 meses de idade, vivem com seus filhotes em bandos de até 30 indivíduos. Para descrição morfológica da glândula mamaria do Nasua nasua foram utilizados seis animais provenientes do Criatório Cientifico (Cecrimpas), Unifeob. Autorizado pelo Ibama (Proc.02027.002322/98-99). Para análise macroscópica um animal foi injetado com látex neoprene, sendo a artéria femoral injetada com látex de cor vermelha e a veia jugular de cor azul. Os demais animais foram fixados em solução aquosa a 10% de formaldeído. Para análise microscópica, fragmentos glandulares foram coletados e submetidos ao processo rotineiro, embebido em parafina e corados com Hematoxilina e Eosina, Picrossírius e Azul de Toluidina. Macroscopicamente foram evidenciados três pares de glândulas mamárias, sendo dois pares posicionados na região abdominal e um par na região inguinal. Microscopicamente, notou-se epitélio de revestimento externo das papilas mamárias, epitélio pavimentoso estratificado queratinizado, o qual seguia por toda glândula de forma irregular. Na entrada do óstio, o epitélio da epiderme era modificado ocorrendo uma transição de epitélio pavimentoso estratificado para um epitélio cúbico no ducto papilar. O parênquima glandular era caracteristicamente túbulo alveolar com células secretoras, evidenciado principalmente no animal lactente. Os resultados macroscópicos e microscópicos assemelham-se aos já descritos nas cadelas (Canis familiaris) e os do Procyon cancrivorus pertencente à mesma família do quati, Família Procyonidae.

Coati is an omnivorous animal, found throughout of Brazil. The reproduction of this species occurs only once a year during spring. Females are matriarchs and nurse their young until 5 months old, living with their young in groups of up to 30. For morphological description of the mammary gland Nasua nasua six animals were used coming from the hatchery Scientific (Cecrimpas), Unifeob. Authorized by IBAMA (Proc. 02027.002322/98-99). For the macroscopic analysis one animal was injected with neoprene latex, was injected into the femoral artery with red latex and the jugular vein with blue colored one. The others animals were fixed in a 10% aqueous solution of formaldehyde. For microscopic analysis, glandular fragments were collected and submitted to routine process, embedded in paraffin and stained with hematoxylin and eosin, toluidine blue and picrossirius. Macroscopically were evidenced three pairs of mammary glands, two pairs positioned in the abdominal region and one pair in the inguinal region. Microscopically, was found a simple epithelium lining external mammary papillae, keratinized stratified epithelium, which continued throughout the gland. At the entrance of the papillae ostium the epithelium of the epidermis was modified occurring a transition from stratified epithelium to cubical epithelium into the papillary duct. The glandular parenchyma was characteristically alveolar with secretory cells observed predominantly in the lactating female. The macroscopic and microscopic results are similar to those already described in bitches (Cannis familiaris) and Procyon cancrivorus belonging to the same family of coati, the family Procyonidae.

Forma e função dos músculos perineais da viscacha (Lagostomus maximus) / Shape and function of the perineal muscles of viscacha (Lagostomus maximus)

Entre as espécies de roedores já estudadas podemos destacar a grande variação na morfologia do aparelho reprodutor masculino. Assim, considerando a importância ecológica dos roedores, e a grande representatividade numérica quanto geográfica destes animais, bem como a escassez que aborda a anatomia reprodutiva, desenvolvemos este trabalho com um roedor histricomorfo da América do Sul, a viscacha. Este animal apresenta algumas características reprodutivas bem peculiares, desta forma descreveremos a anatomia macroscópica da musculatura perineal, e o papel dos músculos no comportamento copulatório desta espécie. A região perineal da viscacha é composta por cinco músculos, sendo que três músculos encontram-se dispostos no diafragama urogenital superficial, Musculus ischiocavernosus, M. bulbocavernosus e M. bulbospongiosus, e pelos músculos que se encontram no diafragma pélvico, M. levator ani e M. retractor penis. Sendo assim, destacamos que o estudo do assoalho pélvico em animais silvestres é de grande valia, uma vez que contribuem com o melhor entendimento dos mecanismos relacionados â ereção e ejaculação; ou seja colaboram com os estudos sobre a reprodução dos animais.

Among the rodent species studied we can highlight the wide variation in the morphology of the male reproductive system. Thus, considering the ecological importance of rodents, and the large number and geographical representation of this animal, as well as shortages regarding the reproductive anatomy, we developed this study with viscacha, a South American histricomorph rodent. As this species has some very peculiar reproductive features, we described the gross anatomy of the perineal muscles and the role of copulatory behavior. The perineal region of viscacha is composed of five muscles, three of which are arranged in the superficial genitourinary diaphragm, as Musculus ischiocavernosus, M. bulbocavernosus and M. bulbospongiosus, and the muscles that lie at the pelvic diaphragm, M. levator ani and M. retractor penis. Therefore, we emphasize that the study of the pelvic floor in wild animals is of great value, then contribute to a better understanding of the mechanisms related to erection and ejaculation or collaborate with studies on the reproduction of animals.

Classificação morfofuncional dos dentes de quati, Nasua nasua / Teeth morphofunctional classification of coati, Nasua nasua

O quati (Nasua nasua), é um mamífero onívoro, amplamente distribuído pela América do sul e encontrado em todo Brasil. A evolução provocou nos dentes dos mamíferos múltiplas variações, adequando às mais diversas dietas. Por essas variações nos dentes é possível classificar os vertebrados em ordem, gênero e família. Portanto este trabalho teve como objetivo descrever e classificar morfologicamente os dentes de machos e fêmeas de N. nausa, a fim de classificá-los e compará-los entre si e com a morfologia dos dentes de Canis familiaris descrita na literatura. Neste estudo foram utilizados cinco animais Nasua nasua, adultos, de ambos os sexos, cedidas pelo Criatório Cientifico Cecrimpas (Unifeob). Dois deles macerados tiveram a cavidade oral exposta para a realização da documentação fotográfica da posição dos dentes, outros três, os dentes foram submetidos à descalcificação e processamento e embebição em parafina para observação histológica. Na cavidade oral de N. nasua, devido ao dimorfismo sexual, foi possível comparar a dentição do macho e da fêmea macroscopicamente. A fêmea apresentou dentes menores, caninos arredondados e o macho, caninos maiores. Os animais apresentaram a fórmula dentária 2x(I3/3, C1/1, P4/4, M2/2) (I=incisivos, C=caninos, P=pré-molares e M=molares) = 40. Microscopicamente todos os dentes foram classificados como braquidontes, pois o esmalte é depositado sobre a dentina. Este tipo de dente não tem erupção constante como o hipsodonte, pois seu esmalte permanece intacto até a época de erupção. Seus dentes também foram classificados como anelodontes, por ter um período limitado de características de crescimento.

Nasua nasua, also known as coati, is an omnivorous mammal with wide distribution in South America and found all over Brazil. The evolution adapted the teeth of the multiple mammals for most different diets. Through these variations it is possible to classify the vertebratesï teeth of vertebrates in classes, genera and families. The objective of this paper is to describe and classify morphologically the teeth of Nasua nasua, comparing them with the teeth of Canis familiaris described in the literature. Five adult male and female Nasua nasua were studied. Photographs of the oral cavity of two macerated skulls were taken for documentation of teeth position. From other three animals, teeth were processed for decalcification and embedded in paraffin for histology. It was possible to reveal macroscopically male and female differences in N. nasua, due to a sexual dimorphism. Female have smaller round canine teeth, and male canine teeth are larger than the female ones. The animals presented the dental formula 2x (I3/3, C1/1, P4/4, M2/2) (I=incisive, C=canine, P=premolar, M=molar), with a total of 40 teeth. Microscopically, the teeth were classified as brachyodont with enamel in the most external region, covering the dentine. This type of teeth does not erupt constantly as the hypsodonts, because the enamel stays intact until the time of eruption. Teeth were also classified as anelodont, with a limited period of growth characteristics.

The use of animal models for stroke research: a review.

Stroke has been identified as the second leading cause of death worldwide. Stroke is a focal neurologic deficit caused by a change in cerebral circulation. The use of animal models in recent years has improved our understanding of the physiopathology of this disease. Rats and mice are the most commonly used stroke models, but the demand for larger models, such as rabbits and even nonhuman primates, is increasing so as to better understand the disease and its treatment. Although the basic mechanisms of stroke are nearly identical among mammals, we here discuss the differences between the human encephalon and various animals. In addition, we compare common surgical techniques used to induce animal models of stroke. A more complete anatomic knowledge of the cerebral vessels of various model species is needed to develop more reliable models for objective results that improve knowledge of the pathology of stroke in both human and veterinary medicine.