Structural plasticity and isolation of umbilical cord progenitor cells of agouti (Dasyprocta prymnolopha) raised in captivity / Plasticidade estrutural e isolamento de células progenitoras do cordão umbilical de cutias (Dasyprocta prymnolopha) criadas em cativeiro

The agouti has been used as an experimental model in several studies focused on reproductive biology. The umbilical cord, an embryonic attachment that connects the foetus to the placenta, has been reported as an important anatomical site for obtaining stem cells. The objective of this study was to describe macro- and microscopically the umbilical cord of agoutis at different stages of gestation, to expand and cultivate in vitro the progenitor cells and to report their morphological characteristics. Seven cutias were submitted to caesarean section to collect the umbilical cords: five were destined for studies of cord structure in different stages of gestation (30, 35, 50, 75 and 100 days postcoital), and two were collected in the third stage of gestation for isolation and cell culture. The umbilical cord of cutias assumes a spiral arrangement, with veins and arteries on it starting 50 days after coitus. The arteries present an outer layer of smooth muscle fibres in a longitudinal and circular arrangement and a medium layer of smooth muscle fibres with only longitudinal and intimate orientation and coated by the endothelium. The veins consist of longitudinal smooth muscle fibres with an extract of smooth muscle cells, and the endothelium, in all analysed gestational phases, is a structure bounded by simple pavement epithelial tissue originating from the amnion, adhered to Whartons Jelly and forming the umbilical vessels and allantoid duct. The proposed protocol allowed the collection of a high cellular concentration of umbilical cord progenitor cells from viable cutias.(AU)

A cutia vem sendo utilizada como modelo experimental em diversos estudos voltados à biologia reprodutiva. O cordão umbilical, anexo embrionário que une o feto à placenta, tem sido relatado como um importante sítio anatômico para obtenção de células-tronco. O objetivo deste estudo foi descrever macro e microscopicamente o cordão umbilical de cutias, em fases diferentes da gestação, expandir e cultivar in vitro as células progenitoras e relatar suas características morfológicas. Foram utilizadas sete cutias submetidas à cesariana para a coleta dos cordões umbilicais, cinco foram destinadas aos estudos da estrutura do cordão, em diferentes estágios de gestação (30, 35, 50, 75 e 100 dias pós-coito), e duas, no terço final da gestação, para isolamento e cultivo celular. O cordão umbilical de cutia assume disposição espiralada, com veias e artérias sobre ele a partir dos 50 dias após o coito. As artérias apresentam camada externa de fibras musculares lisas, disposição longitudinal e circular, camada média de fibras musculares lisas, apenas com disposição longitudinal e íntima revestida pelo endotélio. As veias constituídas por fibras musculares lisas longitudinais com um extrato de células musculares lisas e pelo endotélio. Em todas as fases gestacionais analisadas é uma estrutura delimitada por tecido epitelial simples pavimentoso, proveniente do âmnio, aderido a Geleia de Wharton e com formação de vasos umbilicais e ducto alantóide. O protocolo proposto permitiu a coleta de células progenitoras do cordão umbilical de cutias, viáveis com elevada concentração celular.(AU)

Structural plasticity and isolation of umbilical cord progenitor cells of agouti (Dasyprocta prymnolopha) raised in captivity / Plasticidade estrutural e isolamento de células progenitoras do cordão umbilical de cutias (Dasyprocta prymnolopha) criadas em cativeiro

The agouti has been used as an experimental model in several studies focused on reproductive biology. The umbilical cord, an embryonic attachment that connects the foetus to the placenta, has been reported as an important anatomical site for obtaining stem cells. The objective of this study was to describe macro- and microscopically the umbilical cord of agoutis at different stages of gestation, to expand and cultivate in vitro the progenitor cells and to report their morphological characteristics. Seven cutias were submitted to caesarean section to collect the umbilical cords: five were destined for studies of cord structure in different stages of gestation (30, 35, 50, 75 and 100 days postcoital), and two were collected in the third stage of gestation for isolation and cell culture. The umbilical cord of cutias assumes a spiral arrangement, with veins and arteries on it starting 50 days after coitus. The arteries present an outer layer of smooth muscle fibres in a longitudinal and circular arrangement and a medium layer of smooth muscle fibres with only longitudinal and intimate orientation and coated by the endothelium. The veins consist of longitudinal smooth muscle fibres with an extract of smooth muscle cells, and the endothelium, in all analysed gestational phases, is a structure bounded by simple pavement epithelial tissue originating from the amnion, adhered to Wharton’s Jelly and forming the umbilical vessels and allantoid duct. The proposed protocol allowed the collection of a high cellular concentration of umbilical cord progenitor cells from viable cutias.

A cutia vem sendo utilizada como modelo experimental em diversos estudos voltados à biologia reprodutiva. O cordão umbilical, anexo embrionário que une o feto à placenta, tem sido relatado como um importante sítio anatômico para obtenção de células-tronco. O objetivo deste estudo foi descrever macro e microscopicamente o cordão umbilical de cutias, em fases diferentes da gestação, expandir e cultivar in vitro as células progenitoras e relatar suas características morfológicas. Foram utilizadas sete cutias submetidas à cesariana para a coleta dos cordões umbilicais, cinco foram destinadas aos estudos da estrutura do cordão, em diferentes estágios de gestação (30, 35, 50, 75 e 100 dias pós-coito), e duas, no terço final da gestação, para isolamento e cultivo celular. O cordão umbilical de cutia assume disposição espiralada, com veias e artérias sobre ele a partir dos 50 dias após o coito. As artérias apresentam camada externa de fibras musculares lisas, disposição longitudinal e circular, camada média de fibras musculares lisas, apenas com disposição longitudinal e íntima revestida pelo endotélio. As veias constituídas por fibras musculares lisas longitudinais com um extrato de células musculares lisas e pelo endotélio. Em todas as fases gestacionais analisadas é uma estrutura delimitada por tecido epitelial simples pavimentoso, proveniente do âmnio, aderido a Geleia de Wharton e com formação de vasos umbilicais e ducto alantóide. O protocolo proposto permitiu a coleta de células progenitoras do cordão umbilical de cutias, viáveis com elevada concentração celular.

Centrifugación en gradiente de densidad: alternativa para sexar espermatozoides caninos? / Density gradient centrifugation: alternative for sexing sperm in dogs? / Centrifugação em gradiente de densidade: alternativa para sexagem espermática em cães?

La evolución de técnicas para la separación del sexo de esperma en los últimos años ha permitido avances significativos, pero todavía no hay método que ha sido utilizado con éxito en perros. Apear de la citometría de flujo ser un método eficaz para el sexado de esperma, en perros puede ser considerado difícil de implementar en vista del alto costo de la aplicación de técnica y la recuperación baja de espermatozoides después de la separación. En contraste, la centrifugación en gradiente de densidad también puede ser utilizado para la separación de espermatozoides en los perros. Esta técnica también se basa en la diferencia de contenido en DNA entre los espermatozoides portadores de los cromosomas X e Y. Separación de esperma puede ser confirmada por diversos métodos tales como citometría de flujo, hibridación in situ fluorescente (FISH), coloración con quinacrina mostaza y PCR. El objetivo de esta revisión es abordar el centrifugacion en gradiente de densidad como alternativa a la separación del sexo de espermatozoides en los perros.

The technical progress concerning sperm sexing in recent years has enabled significant advances, but still none method was used successfully in dogs. Despite the flow cytometry to be an effective method for sperm sexing, in dogs it can be considered difficult to implement in view of high cost of application of the technique and low sperm recovery after separation. In contrast, density gradient centrifugation can also be used for separating sperm in dogs. This technique is also based on the DNA content difference between sperm with chromosomes X and Y. Sperm separation can be confirmed by various methods such as flow cytometry, fluorescence in situ hybridization (FISH) staining with quinacrine mustard and PCR. The objective of this review to address the density gradient centrifugation as an alternative to sperm sexing in dogs.

A evolução das técnicas de sexagem espermática nos últimos anos permitiu avanços significativos, mas ainda nenhum método foi empregado com sucesso na espécie canina. Apesar da citometria de fluxo ser um método eficiente para sexagem espermática, nos cães pode ser considerada de difícil aplicação, em vista do alto custo da aplicação da técnica e da baixa recuperação espermática após a separação. Em contrapartida, a centrifugação em gradiente de densidade também pode ser utilizada para separação espermática em cães. Essa técnica também se baseia na diferença do conteúdo de DNA entre os espermatozoides portadores dos cromossomos X e Y. A separação espermática pode ser confirmada por diferentes métodos, como citometria de fluxo, hibridização in situ fluorescente (FISH), coloração com quinacrina mostarda e PCR. Objetivou-se, com esta revisão abordar a centrifugação em gradiente de densidade como uma alternativa a sexagem espermática em cães.

Centrifugación en gradiente de densidad: alternativa para sexar espermatozoides caninos? / Density gradient centrifugation: alternative for sexing sperm in dogs? / Centrifugação em gradiente de densidade: alternativa para sexagem espermática em cães?

La evolución de técnicas para la separación del sexo de esperma en los últimos años ha permitido avances significativos, pero todavía no hay método que ha sido utilizado con éxito en perros. Apear de la citometría de flujo ser un método eficaz para el sexado de esperma, en perros puede ser considerado difícil de implementar en vista del alto costo de la aplicación de técnica y la recuperación baja de espermatozoides después de la separación. En contraste, la centrifugación en gradiente de densidad también puede ser utilizado para la separación de espermatozoides en los perros. Esta técnica también se basa en la diferencia de contenido en DNA entre los espermatozoides portadores de los cromosomas X e Y. Separación de esperma puede ser confirmada por diversos métodos tales como citometría de flujo, hibridación in situ fluorescente (FISH), coloración con quinacrina mostaza y PCR. El objetivo de esta revisión es abordar el centrifugacion en gradiente de densidad como alternativa a la separación del sexo de espermatozoides en los perros.(AU)

The technical progress concerning sperm sexing in recent years has enabled significant advances, but still none method was used successfully in dogs. Despite the flow cytometry to be an effective method for sperm sexing, in dogs it can be considered difficult to implement in view of high cost of application of the technique and low sperm recovery after separation. In contrast, density gradient centrifugation can also be used for separating sperm in dogs. This technique is also based on the DNA content difference between sperm with chromosomes X and Y. Sperm separation can be confirmed by various methods such as flow cytometry, fluorescence in situ hybridization (FISH) staining with quinacrine mustard and PCR. The objective of this review to address the density gradient centrifugation as an alternative to sperm sexing in dogs.(AU)

A evolução das técnicas de sexagem espermática nos últimos anos permitiu avanços significativos, mas ainda nenhum método foi empregado com sucesso na espécie canina. Apesar da citometria de fluxo ser um método eficiente para sexagem espermática, nos cães pode ser considerada de difícil aplicação, em vista do alto custo da aplicação da técnica e da baixa recuperação espermática após a separação. Em contrapartida, a centrifugação em gradiente de densidade também pode ser utilizada para separação espermática em cães. Essa técnica também se baseia na diferença do conteúdo de DNA entre os espermatozoides portadores dos cromossomos X e Y. A separação espermática pode ser confirmada por diferentes métodos, como citometria de fluxo, hibridização in situ fluorescente (FISH), coloração com quinacrina mostarda e PCR. Objetivou-se, com esta revisão abordar a centrifugação em gradiente de densidade como uma alternativa a sexagem espermática em cães.(AU)

Omental adipose tissue is a more suitable source of canine Mesenchymal stem cells.

BACKGROUND: Mesenchymal Stem Cells (MSCs) are a promising therapeutic tool in veterinary medicine. Currently the subcutaneous adipose tissue is the leading source of MSCs in dogs. MSCs derived from distinct fat depots have shown dissimilarities in their accessibility and therapeutic potential. The aims of our work were to determine the suitability of omental adipose tissue as a source of MSCs, according to sampling success, cell yield and paracrine properties of isolated cells, and compared to subcutaneous adipose tissue. RESULTS: While sampling success of omental adipose tissue was 100% (14 collections from14 donors) for subcutaneous adipose tissue it was 71% (10 collections from 14 donors). MSCs could be isolated from both sources. Cell yield was significantly higher for omental than for subcutaneous adipose tissue (38 ± 1 vs. 30 ± 1 CFU-F/g tissue, p < 0.0001). No differences were observed between sources regarding cell proliferation potential (73 ± 1 vs. 74 ± 1 CDPL) and cell senescence (at passage 10, both cultures presented enlarged cells with cytoplasmic vacuoles and cellular debris). Omental- and subcutaneous-derived MSCs expressed at the same level bFGF, PDGF, HGF, VEGF, ANG1 and IL-10. Irrespective of the source, isolated MSCs induced proliferation, migration and vascularization of target cells, and inhibited the activation of T lymphocytes. CONCLUSION: Compared to subcutaneous adipose tissue, omental adipose tissue is a more suitable source of MSCs in dogs. Since it can be procured from donors with any body condition, its collection procedure is always feasible, its cell yield is high and the MSCs isolated from it have desirable differentiation and paracrine potentials.

Conception rate of lactating cows and heifers (Bos taurus X Bos indicus) for sexed and conventional semen in artificial insemination / Taxa de concepção de vacas lactantes e novilhas (Bos taurus X Bos indicus) com o uso de sêmen sexado e convencional na inseminação artificial

The objective of this study was to evaluate the conception rate of crossbred heifers (n=50) and cows (n=50) inseminated with sexed and conventional semen between 18 and 24 hours after estrous detection. The synchronization protocol of the estrous cycle started on day zero (D0) by inserting the intravaginal device with 1g progesterone (Sincrogest® Ourofino, Brazil) and injecting 2 mg of estradiol benzoate, intramuscularly (Sincrodiol® Ourofino, Brazil). On the fifth day (D5), 200 IU of equine chorionic gonadotrophin was injected intramuscularly (Folligon®, Intervet, Brazil). On the eighth day (D8), after removing the progesterone device, 500 g of sodium cloprostenol was injected intramuscularly (Sincrocio®, Ourofino, Brazil). After that, the animals were checked for estrus 3 times daily, and inseminated 18 to 24 hours after estrus detection. Pregnancy diagnosis was performed 30 to 40 days after insemination. Conception rate did not differ (P> 0.05) according to animal category, but was higher for conventional semen compared to sexed semen when evaluating the total of animals and lactating cows (P < 0.05). Artificial insemination of heifers with sexed semen 18 to 24 hours after estrus detection was effective, however, conventional semen was more efficient in lactating cows.(AU)

Considerando os benefícios do uso de sêmen sexado e também os danos causados pelo processo de separação dos espermatozoides, o objetivo do presente estudo foi avaliar a taxa de concepção de novilhas (n=50) e vacas (n=50) mestiças inseminadas com sêmen sexado e convencional após 18 a 24 horas a observação do cio. O protocolo de sincronização do ciclo estral consistiu em inserção de dispositivo intravaginal com 1g de progesterona (Sincrogest® Ourofino, Brasil) e aplicação intramuscular de 2mg de benzoato de estradiol (Sincrodiol® Ourofino, Brasil) no dia zero (D0). No quinto dia (D5), foi realizada uma aplicação intramuscular de 200UI de gonadotrofina coriônica equina (Folligon®, Intervet, Brasil). No oitavo dia (D8), o dispositivo de progesterona foi retirado, e aplicado por via intramuscular 500g de cloprostenol sódico (Sincrocio®, Ourofino, Brasil). A partir deste momento, o estro foi observado 3 vezes ao dia e os animais foram inseminados 18 a 24 após a detecção do cio. O diagnóstico de gestação foi realizado 30 a 40 dias após a inseminação. Não foi observada diferença na taxa de concepção de acordo com a categoria animal (P > 0,05), entretanto, animais inseminados com sêmen convencional apresentaram melhor taxa de concepção do que com sêmen sexado quando se avaliou o total de animais e vacas lactantes (P < 0,05). A inseminação artificial de novilhas com sêmen sexado 18 a 24 horas após detecção de estro mostrou-se eficaz, entretanto, para vacas lactantes não foi observada a mesma eficiência ao se comparar com o sêmen convencional.(AU)

Método imunomagnético associado ao meio MesenCult® na obtenção de células mononucleares da medula óssea de coelhos negativas para o anticorpo monoclonal CD45 / Immunomagnetic method associated with MesenCult® medium to obtain bone marrow mononuclear cells from rabbits negative for CD45 monoclonal antibody

O objetivo detse artigo é de descrever um protocolo de isolamento das células mononucleares da medula óssea de coelhos, seguido de purificação celular por depleção negativa com o anticorpo monoclonal CD45 e posterior expansão em meio de cultura MesenCult®. Dez coelhos machos adultos, da raça Nova Zelândia, com idade média de 1,0±0,2 anos e peso médio 3,5±0,24kg, foram utilizados para padronização da metodologia. O isolamento das células mononuclares da medula óssea foi realizado pelo gradiente de densidade Ficoll-paque® e a purificação e obtenção das células- pela depleção negativa com o anticorpo monoclonal CD45 em base imunomagnética. A população celular obtida foi expandida posteriormente em meio de cultura MesenCult®. No isolamento pelo gradiente de icoll-Paque® foi obtido um rendimento médio de 7,31x106 células/mL. Após purificação e obtenção das possíveis células-tronco mesenquimais pela base imunomagnética, houve um decréscimo do rendimento para 2,28x106 células/mL, mas o processo de expansão foi incrementado pelo cultivo celular. Os resultados indicaram que as células obtidas da fração mononuclear da medula óssea, cultivadas in vitro foram capazes de gerar células aderentes 24 horas após o cultivo, com predominância de células fibroblastóides sugestivas de células-tronco mesenquimais. Concluiu-se que a obtenção de células-tronco mesenquimais pode ser alcançada após purificação das células mononucleares da medula óssea de coelhos pelo método imunomagético, o meio de cultura MesenCult® proporciona um ambiente adequado para a rápida expansão in vitro e o número de passagens exerce influência negativa sobre as características morfológicas das células.

The objective of this study was to describe guidelines for the isolation of bone marrow mononuclear cells from rabbits, followed by cell purification by negative depletion with CD45 monoclonal antibody, and further expansion in MesenCult® medium. Ten adult male New Zealand White rabbits, age average of 1.0±0.2 years and weighting 3.5±0.24kg, were used to obtain a standardized method. The mononuclear cells of the bone marrow were isolated with Ficoll-paque® density gradient centrifugation, and the cell purification and acquisition was completed by negative depletion with CD45 monoclonal antibody in immunomagnetic base. The cell population obtained was expanded in MesenCult® medium. Through isolation with Ficoll-paque® density gradient was possible to obtain an average yield of 7.31x106 cells/mL. After purification and acquisiton of potential mesenchymal stem cells by the immunomagnetic base, there was a yield decrease to 2.28x106 cells/mL; however the expansion process was increased in cell culture. The results indicated that cells obtained from the mononuclear fraction of bone marrow and cultivated in vitro were capable to generate adherent cells 24 hours after culture, with predominance of fibroblastoid cells suggestive of mesenchymal stem cells. It can be concluded that mesenchymal stem cells can be achieved with purified rabbit bone marrow mononuclear cells through the immunomagnetic method, as the MesenCult® medium provides a suitable environment for a quick in vitro expansion, and the number of passages exerts negative influence on the morphological characteristics.

Método imunomagnético associado ao meio MesenCult® na obtenção de células mononucleares da medula óssea de coelhos negativas para o anticorpo monoclonal CD45 / Immunomagnetic method associated with MesenCult® medium to obtain bone marrow mononuclear cells from rabbits negative for CD45 monoclonal antibody

O objetivo detse artigo é de descrever um protocolo de isolamento das células mononucleares da medula óssea de coelhos, seguido de purificação celular por depleção negativa com o anticorpo monoclonal CD45 e posterior expansão em meio de cultura MesenCult®. Dez coelhos machos adultos, da raça Nova Zelândia, com idade média de 1,0±0,2 anos e peso médio 3,5±0,24kg, foram utilizados para padronização da metodologia. O isolamento das células mononuclares da medula óssea foi realizado pelo gradiente de densidade Ficoll-paque® e a purificação e obtenção das células- pela depleção negativa com o anticorpo monoclonal CD45 em base imunomagnética. A população celular obtida foi expandida posteriormente em meio de cultura MesenCult®. No isolamento pelo gradiente de icoll-Paque® foi obtido um rendimento médio de 7,31x106 células/mL. Após purificação e obtenção das possíveis células-tronco mesenquimais pela base imunomagnética, houve um decréscimo do rendimento para 2,28x106 células/mL, mas o processo de expansão foi incrementado pelo cultivo celular. Os resultados indicaram que as células obtidas da fração mononuclear da medula óssea, cultivadas in vitro foram capazes de gerar células aderentes 24 horas após o cultivo, com predominância de células fibroblastóides sugestivas de células-tronco mesenquimais. Concluiu-se que a obtenção de células-tronco mesenquimais pode ser alcançada após purificação das células mononucleares da medula óssea de coelhos pelo método imunomagético, o meio de cultura MesenCult® proporciona um ambiente adequado para a rápida expansão in vitro e o número de passagens exerce influência negativa sobre as características morfológicas das células.(AU)

The objective of this study was to describe guidelines for the isolation of bone marrow mononuclear cells from rabbits, followed by cell purification by negative depletion with CD45 monoclonal antibody, and further expansion in MesenCult® medium. Ten adult male New Zealand White rabbits, age average of 1.0±0.2 years and weighting 3.5±0.24kg, were used to obtain a standardized method. The mononuclear cells of the bone marrow were isolated with Ficoll-paque® density gradient centrifugation, and the cell purification and acquisition was completed by negative depletion with CD45 monoclonal antibody in immunomagnetic base. The cell population obtained was expanded in MesenCult® medium. Through isolation with Ficoll-paque® density gradient was possible to obtain an average yield of 7.31x106 cells/mL. After purification and acquisiton of potential mesenchymal stem cells by the immunomagnetic base, there was a yield decrease to 2.28x106 cells/mL; however the expansion process was increased in cell culture. The results indicated that cells obtained from the mononuclear fraction of bone marrow and cultivated in vitro were capable to generate adherent cells 24 hours after culture, with predominance of fibroblastoid cells suggestive of mesenchymal stem cells. It can be concluded that mesenchymal stem cells can be achieved with purified rabbit bone marrow mononuclear cells through the immunomagnetic method, as the MesenCult® medium provides a suitable environment for a quick in vitro expansion, and the number of passages exerts negative influence on the morphological characteristics.(AU)

Pregnancy rate and birth rate of calves from a large-scale IVF program using reverse-sorted semen in Bos indicus, Bos indicus-taurus, and Bos taurus cattle.

Obtaining sexed sperm from previously frozen doses (reverse-sorted semen [RSS]) provides an important advantage because of the possibility of using the semen of bulls with desired genetic attributes that have died or have become infertile but from whom frozen semen is available. We report the efficiency of RSS on the pregnancy rate and birth rate of calves in a large-scale program using ovum pick-up and in vitro embryo production (IVEP) from Bos indicus, Bos indicus-taurus, and Bos taurus cattle. From 645 ovum pick-up procedures (Holstein, Gir, and Nelore), 9438 viable oocytes were recovered. A dose of frozen semen (Holstein, Nelore, Brahman, Gir, and Braford) was thawed, and the sperm were sex-sorted and cooled for use in IVF. Additionally, IVF with sperm from three Holstein bulls with freeze-thawed, sex-sorted (RSS) or sex-sorted, freeze-thawed (control) was tested. A total of 2729 embryos were produced, exhibiting a mean blastocyst rate of 29%. Heifers and cows selected for adequate body condition, estrus, and health received 2404 embryos, and 60 days later, a 41% average pregnancy rate was observed. A total of 966 calves were born, and 910 were of a predetermined sex, with an average of 94% accuracy in determining the sex. Despite the lower blastocyst rate with freeze-thawed, sex-sorted semen compared with sex-sorted semen, (P < 0.05), the pregnancy rate (bull I, 45% vs. 40%; II, 35% vs. 50%; and III, 47% vs. 48% for RSS and control, respectively; P > 0.05) and sex-sorted efficiency (bull I, 93% vs. 98%; II, 96% vs. 94%; and III, 96% vs. 97% for RSS and control, respectively; P > 0.05) were similar for each of the three bulls regardless of the sperm type used in the IVF. The sexing of previously frozen semen, associated with IVEP, produces viable embryos with a pregnancy rate of up to 40%, and calves of the desired sex are born even if the paternal bull has acquired some infertility, died, or is located a long distance from the sexing laboratory. Furthermore, these data show the feasibility of the process even when used in a large-scale IVEP program.

Function of milk polymorphonuclear neutrophil leukocytes in bovine mammary glands infected with Corynebacterium bovis.

Corynebacterium bovis is one of the most commonly isolated bacteria from aseptically collected bovine milk samples. The objective of the current study was to characterize the bovine innate immune response by evaluating milk polymorphonuclear neutrophilic leukocytes (PMNL) in mammary glands infected with C. bovis. Twenty quarters infected with C. bovis and 28 culture-negative quarters (with milk somatic cell count <1×10(5) cells/mL) were used. The percentages of milk PMNL and the PMNL expression of L-selectin (CD62L), ß2-integrin (CD11b), and one of the endothelial-selectin ligands (CD44), as well as the levels of intracellular reactive oxygen species (ROS) and the phagocytosis of Staphylococcus aureus, were evaluated by flow cytometry. The apoptosis and necrosis rates of the PMNL were quantified using dual-color flow cytometry with fluorescein-labeled annexin and propidium iodide. The present study revealed a higher percentage of PMNL in the milk from C. bovis-infected quarters, although no significant differences were found in levels of CD44, CD62L, or CD11b expression among the PMNL. A lower percentage of apoptotic PMNL was observed in C. bovis-infected quarters, as well as higher percentages of viable PMNL and of PMNL that produced intracellular ROS. However, no alterations were observed in phagocytosis of Staph. aureus by the PMNL or in intensity of intracellular ROS production by PMNL. Thus, results from this investigation of the PMNL function support, at least in part, the fact that intramammary infections by C. bovis may offer protection against intramammary infections by other bacteria.

Isolamento, caracterização e diferenciação de células-tronco mesenquimais do líquido amniótico equino obtido em diferentes idades gestacionais / Isolation, characterization and differentiation of mesenchymal stem cells derived from equine amniotic fluid obtained from different gestacional ages

O interesse nas pesquisas com células-tronco derivadas de anexos fetais de diversas espécies cresceu exponencialmente nas últimas décadas em virtude de serem fontes de células-tronco adultas com potencial de diferenciação em diversas linhagens celulares que apresentam pouca ou nenhuma imunogenicidade, apresentando-se assim como alternativa de grande importância para a formação de bancos celulares. Apesar do crescente interesse, os estudos para espécie equina ainda são escassos. O objetivo deste trabalho foi isolar, caracterizar e diferenciar células-tronco mesenquimais (CTMs) derivadas do líquido amniótico equino obtidas do terço inicial, médio e final da gestação (LA-CTMs), comparando suas características. Foram colhidas 23 amostras de líquido amniótico as quais foram submetidas às análises morfológica, imunocitoquímica, imunofenotípica por citometria de fluxo e às diferenciações osteogênica, adipogênica e condrogênica in vitro. Todas as amostras demonstraram adesão ao plástico e morfologia fibroblastóide. No ensaio imunocitoquímico as células de todos os grupos foram imunomarcadas para CD44, PCNA e vimentina com ausência de marcação para citoqueratina e Oct-4. Na citometria de fluxo observou-se a expressão de CD44 e CD90 e ausência de expressão de CD34, sendo que os marcadores CD44 e CD90 mostraram padrão de expressão decrescente em relação ao desenvolvimento gestacional. As amostras obtidas de todas as fases da gestação foram capazes de diferenciação nas linhagens osteogênica, condrogênica e adipogênica. Portanto, as células obtidas do líquido amniótico apresentaram características morfológicas, imunofenotípicas e potencial de diferenciação típicos das CTMs, demonstrando que a colheita pode ser realizada em qualquer fase gestacional. No entanto, mais pesquisas devem ser realizadas principalmente quanto à expressão de marcadores de pluripotencialidade (como o Oct-4) e ao seu potencial de diferenciação em linhagens extra mesodermais já relatados na literatura.

The interest in stem cells derived from fetal annexes of many species has exponentially increased during the last decades, because they are adult stem cell sources with potential of differentiation in several cell lineages; which present little or no immunogenicity and are an alternative with great importance for storage cell banks. Despite the rising interest, studies for the equine species are still rare. The aim of this study was to isolate, characterize and differentiate mesenchymal stem cells derived from equine amniotic fluid obtained from initial, middle and late third of gestation (AF-MSCs), and compare their results. Twenty three samples from equine amniotic fluid were evaluated by morphological, immunocytochemical and immunophenotypical (Flow cytometer) assays and osteogenic, adipogenic and chondrogenic in vitro differentiation. All samples demonstrated plastic adhesion and fibroblastoid morphology. The immunocytochemical assay demonstrated cells from all the studied groups were positive for CD44, PCNA and vimentin and negative for cytokeratin and Oct-4. Flow cytometry demonstrated expression of CD44 and CD90 and no expression of CD34, where CD44 and CD90 markers presented decreasing pattern of expression in relation to the gestational development. All samples collected from all gestational phases were capable to differentiate in osteogenic, chondrogenic and adipogenic lineages. Thus, cells obtained from equine amniotic fluid presented morphological and immunophenotypical characteristics and potential of differentiation typical of MSCs showing that the collection can be performed at any stage of pregnancy. However, more studies should be performed about the expression of pluripotent markers as Oct-4 and the differentiation potential for extra mesodermal lineages prior demonstrated in the literature.

Isolamento, caracterização e diferenciação de células-tronco mesenquimais do líquido amniótico equino obtido em diferentes idades gestacionais / Isolation, characterization and differentiation of mesenchymal stem cells derived from equine amniotic fluid obtained from different gestacional ages

O interesse nas pesquisas com células-tronco derivadas de anexos fetais de diversas espécies cresceu exponencialmente nas últimas décadas em virtude de serem fontes de células-tronco adultas com potencial de diferenciação em diversas linhagens celulares que apresentam pouca ou nenhuma imunogenicidade, apresentando-se assim como alternativa de grande importância para a formação de bancos celulares. Apesar do crescente interesse, os estudos para espécie equina ainda são escassos. O objetivo deste trabalho foi isolar, caracterizar e diferenciar células-tronco mesenquimais (CTMs) derivadas do líquido amniótico equino obtidas do terço inicial, médio e final da gestação (LA-CTMs), comparando suas características. Foram colhidas 23 amostras de líquido amniótico as quais foram submetidas às análises morfológica, imunocitoquímica, imunofenotípica por citometria de fluxo e às diferenciações osteogênica, adipogênica e condrogênica in vitro. Todas as amostras demonstraram adesão ao plástico e morfologia fibroblastóide. No ensaio imunocitoquímico as células de todos os grupos foram imunomarcadas para CD44, PCNA e vimentina com ausência de marcação para citoqueratina e Oct-4. Na citometria de fluxo observou-se a expressão de CD44 e CD90 e ausência de expressão de CD34, sendo que os marcadores CD44 e CD90 mostraram padrão de expressão decrescente em relação ao desenvolvimento gestacional. As amostras obtidas de todas as fases da gestação foram capazes de diferenciação nas linhagens osteogênica, condrogênica e adipogênica. Portanto, as células obtidas do líquido amniótico apresentaram características morfológicas, imunofenotípicas e potencial de diferenciação típicos das CTMs, demonstrando que a colheita pode ser realizada em qualquer fase gestacional. No entanto, mais pesquisas devem ser realizadas principalmente quanto à expressão de marcadores de pluripotencialidade (como o Oct-4) e ao seu potencial de diferenciação em linhagens extra mesodermais já relatados na literatura.(AU)

The interest in stem cells derived from fetal annexes of many species has exponentially increased during the last decades, because they are adult stem cell sources with potential of differentiation in several cell lineages; which present little or no immunogenicity and are an alternative with great importance for storage cell banks. Despite the rising interest, studies for the equine species are still rare. The aim of this study was to isolate, characterize and differentiate mesenchymal stem cells derived from equine amniotic fluid obtained from initial, middle and late third of gestation (AF-MSCs), and compare their results. Twenty three samples from equine amniotic fluid were evaluated by morphological, immunocytochemical and immunophenotypical (Flow cytometer) assays and osteogenic, adipogenic and chondrogenic in vitro differentiation. All samples demonstrated plastic adhesion and fibroblastoid morphology. The immunocytochemical assay demonstrated cells from all the studied groups were positive for CD44, PCNA and vimentin and negative for cytokeratin and Oct-4. Flow cytometry demonstrated expression of CD44 and CD90 and no expression of CD34, where CD44 and CD90 markers presented decreasing pattern of expression in relation to the gestational development. All samples collected from all gestational phases were capable to differentiate in osteogenic, chondrogenic and adipogenic lineages. Thus, cells obtained from equine amniotic fluid presented morphological and immunophenotypical characteristics and potential of differentiation typical of MSCs showing that the collection can be performed at any stage of pregnancy. However, more studies should be performed about the expression of pluripotent markers as Oct-4 and the differentiation potential for extra mesodermal lineages prior demonstrated in the literature.(AU)

Nanoscale differences in the shape and size of X and Y chromosome-bearing bovine sperm heads assessed by atomic force microscopy.

Sperm dimensions and the question of whether X and Y chromosome-bearing sperm differ in size or shape has been of great interest, especially for the development of alternative methods to sort or classify sperm cells. The aim of the present study was to evaluate possible differences in the shape and size of the sperm head between X and Y chromosome-bearing sperm by atomic force microscopy (AFM). One ejaculate per bull (n = 4) was used. Each ejaculate was separated into four fractions: non-sexed (NS), sexed for X-sperm (SX), sexed for Y-sperm (SY) and a pooling of SX and SY samples (SXY). Using AFM, 400 sperm heads per group were measured. Twenty three structural features were assessed including one-, two- and three-dimensional parameters and shape descriptors. These measurements determine the micro- to nanoscale features of X- and Y-bearing chromosomes in sperm cells. No differences were observed for any individual variables between SX and SY groups. Next, a simultaneous evaluation of all features using statistical discriminant analysis was performed to determine if it was possible to distinguish to which group belong each individual cells. This analysis clearly showed, a distinct separation of NS, SXY, SX and SY groups. The recognition of this structural possibility to distinguish between X and Y sperm cell might improve the understanding of sperm cells biology. These results indicated that the associations of several structural measurements of the sperm cell head are promising candidates for development of a new method of sperm sexing.

Cryopreservation with α-tocopherol and Sephadex filtration improved the quality of boar sperm.

The objectives were to evaluate postthaw sperm quality and the response to an inducer of in vitro sperm capacitation in boar sperm, cryopreserved with (T) or without (C) α-tocopherol. Boar sperm frozen in 0.2-mL pellets were thawed and washed (W) or selected by three methods: Percoll discontinuous gradient (PS) or Sephadex (Sigma-Aldrich, St. Louis, MO, USA) (neutral [S] or with ion exchange [S+IO] columns). All separation methods enhanced sperm motility, plasma membrane integrity, and functionality and acrosome integrity for both C and T samples (P < 0.05). The best results were obtained with S and ionic Sephadex column. There was a decrease (P < 0.05) in capacitation-like changes in C samples separated with Sephadex (W: 19 ± 0.9%, PS: 22 ± 2.5%, S: 17 ± 1.2%, and S+IO: 17 ± 2.0%). Cryopreservation with α-tocopherol decreased (P < 0.05) the percentage of cryocapacitated sperm (W: 14 ± 0.7%, PS: 14 ± 1.0%, S: 13 ± 1.0%, and S+IO: 14 ± 0.9%) compared with C samples, without differences among selection techniques. Freezing with α-tocopherol and subsequent selection decreased lipid peroxidation (W: 20.79 ± 2.64 nmol thiobarbituric acid reactive substances (TBARS)/10(8) sperm; PS: 13.15 ± 2.39 nmol TBARS/10(8) sperm; S: 13.20 ± 2.18 nmol TBARS/10(8) sperm, and S+IO: 13.62 ± 2.76 nmol TBARS/10(8) sperm), with respect to washed and selected C samples (W: 37.69 ± 5.34 nmol TBARS/10(8) sperm, PS: 25.61 ± 5.85 nmol TBARS/10(8) sperm, S: 19.16 ± 3.28 nmol TBARS/10(8) sperm, and S+IO: 22.16 ± 6.09 nmol TBARS/10(8) sperm). In vitro capacitation levels were significantly higher for neutral Sephadex-selected T samples in comparison with C and unselected samples. These results were confirmed with a follicular fluid-induced acrosome reaction. In conclusion, cryopreserved sperm with α-tocopherol and subsequent Sephadex selection, improved postthaw quality and functionality of boar sperm, which could be useful for assisted reproductive techniques.

Isolamento e caracterização das células mesenquimais derivadas da membrana amniótica dos gatos domésticos / Isolation and characterization of Mesenchymal cells from cat amniotic membrane

As células tronco mesenquimais derivadas do âmnio (AMSCs) são células multipotentes com alto potencial para se diferenciar em múltiplas linhagens. Podem ser isoladas sem recurso a procedimentos invasivos e usadas sem levantar quaisquer implicações éticas. O presente estudo visa isolar e caracterizar as células mesenquimais progenitoras da membrana amniótica de gatos domésticos para futura aplicação em terapia celular. As células foram isoladas de quatro membranas fetais, coletadas durante as campanhas rotineiras de castração em gatas no último terço de gestação, após anestesia geral. A porção dorsal do âmnio foi separada mecanicamente, lavada com PBS e submetida à digestão com colagenase. As células coletadas foram propagadas em cultivo (DMEN-F12/-MEM) e criopreservadas em várias passagens enquanto se efetuava a avaliação da cinética de crescimento e das características morfológicas. Em cultivo, as AMSCs demonstraram aderência à placa e uma morfologia similar a dos fibroblastos.

The amnion derived mesenchymal stem cells (AMSCs) are multipotent cells with a high ability to differentiate into multiple lineages. They can be obtained by non-invasive methods and therefore are exempt from the normal ethical problems involving stem cell use. The aim of this study was to isolate and characterize the progenitor mesenchymal cells from the cat amniotic membrane for future application in cell therapy. The cells were isolated from four fetal membranes collected after a routine ovarian hysterectomy process from cats in their third gestational trimester, under general anesthesia. The dorsal portion of amnion was mechanically separated, washed with PBS and subjected to collagenase digestion. The isolated cells were propagated in culture media (DMEMF12 or -MEM) and frozen in various passages while the growing kinetics and cell morphology were analyzed. In culture medium, AMSCs were adherent to the plastic culture dish and had a morphology similar to fibroblasts.

α-6 integrin expression in bovine spermatogonial cells purified by discontinuous Percoll density gradient.

The study of spermatogonial stem cells (SSCs) provides a model to better understand adult stem cell biology. Besides the biomedical potential to perform studies of infertility in many species, SSCs hold a promising application at animal transgenesis. Because stem cells are thought to be associated with basement membranes, expression of α-6 integrin has been investigated as a marker of type A spermatogonial cells, which are considered SSCs because of their undifferentiated status and self-renewal ability. In this manner, the aim of this study was to isolate type A SSCs from adult bulls by a two-step enzymatic procedure followed by a discontinuous Percoll density gradient purification and verify the expression of α-6 integrin by flow cytometry and real-time RT-PCR before and after Percoll purification. Spermatogonial cells were successfully obtained using the two-step enzymatic digestion. An average of 1 × 10(5) viable cells per gram of testis was isolated. However, the discontinuous Percoll did not purify isolated cells regarding α-6 integrin expression. Flow cytometry analysis demonstrated no differences in the α-6 integrin expression between cell samples before and after Percoll purification (p = 0.5636). The same was observed in the real-time PCR analysis (p > 0.05). In addition to α-6 integrin, the expression of GFRa-1 and PGP9.5, known bovine SSCs markers, was detected in all samples studied. Considering that Percoll can reduce cell viability, it is possible to conclude that Percoll density gradient is not suitable to purify bovine SSC, according to α-6 integrin expression.

Effects of discontinuous Percoll gradient centrifugation on the quality of bovine spermatozoa evaluated with computer-assisted semen analysis and fluorescent probes association.

The objective of this study was to evaluate the quality of bovine frozen-thawed sperm cells after Percoll gradient centrifugation. Frozen semen doses were obtained from six bulls of different breeds, including three taurine and three Zebu animals. Four ejaculates per bull were evaluated before and after discontinuous Percoll gradient centrifugation. Sperm motility was assessed by computer-assisted semen analysis and the integrity of the plasma and acrosomal membranes, as well as mitochondrial function, were evaluated using a combination of fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide. The procedure of Percoll gradient centrifugation increased the percentage of total and progressive sperm motility, beat frequency, rectilinear motility, linearity and rapidly moving cells. In addition, the percentage of cells with intact plasma membrane and mitochondrial membrane potential was increased in post-centrifugation samples. However, the percentage of sperm cells with intact acrosomal membrane was markedly reduced. The method used selected the motile cells with intact plasma membrane and higher mitochondrial functionality in frozen-thawed bull semen, but processing, centrifugation and/or the Percoll medium caused damage to the acrosomal membrane.

In vitro embryo production in llamas (Lama glama) from in vivo matured oocytes with raw semen processed with Androcoll-E using defined embryo culture media.

The aim of this study was to carry out in vitro fertilization using spermatozoa selected with Androcoll-E™ and to evaluate the efficiency of the culture medium DMEM-F12 for in vitro embryo development in the llama. Twelve adult females from 18 superstimulated (67%) were used as oocyte donors. They were superstimulated with 1500 IU of eCG and after 5 days, received a single dose of buserelin. Twenty hours post-injection, follicular aspiration was conducted by flank laparotomy. Semen collections were performed under general anesthesia by electroejaculation of the male. The ejaculates were processed with a solution of collagenase (0.1%) and an Androcoll-E™ column was used to improve the sample. Sixty nine COCs were recovered from 79 aspirated follicles (87% recovery). Only expanded COCs were used (n = 67); they were randomly placed in groups of 1-5 in Fertil-TALP and the sperm suspension (20 × 10(6) live spermatozoa/ml) was added to each fertilization microdroplet. After 24 h, they were randomly placed in one of two culture media: SOF (n = 34) or DMEM-F12 (n = 33) and incubated for 6 days in humidified atmosphere of 5% CO(2) , 5% O(2) and 90% N(2) at 38°C. The blastocyst rate was 20% (7/34) in SOF medium (3 hatched, 2 expanded and 2 early blastocysts) and 15% (5/33) in DMEM medium (all expanded blastocysts). In conclusion, using Androcoll-E™ it is possible to select good quality spermatozoa from llama ejaculates for in vitro fertilization and to produce blastocysts in DMEM-F12 medium. This is also the first time that hatched llama blastocysts have been produced after culture in a defined medium such as SOFaa.

Transmission electron microscopy for characterization of acrosomal damage after Percoll gradient centrifugation of cryopreserved bovine spermatozoa.

The objective of this study was to characterize acrosomal ultrastructure following discontinuous Percoll gradient centrifugation of cryopreserved bovine sperm. Semen was collected from six bulls of different breeds and three ejaculates per bull were evaluated. Frozen semen samples were thawed and the acrosomal region of sperm cells was evaluated by transmission electron microscopy (TEM) before (n = 18) and after (n = 18) Percoll centrifugation. The evaluation of 20 sperm heads from each of the 36 samples analyzed ensured that a large number of cells were investigated. The data were subjected to analysis of variance at a level of significance of 5%. Percoll centrifugation reduced the percentage of sperm exhibiting normal acrosomes (from 61.77 to 30.24%), reduced the percentage of sperm presenting atypical acrosome reactions (from 28.38 to 4.84%) and increased the percentage of sperm exhibiting damage in the acrosome (from 6.14 to 64.26%). The percentage of sperm with typical acrosome reactions was not significantly different before (3.70%) and after (0.67%) centrifugation. TEM distinguished four different types of acrosomal status and enabled ultrastructural characterization of acrosomal injuries. The percentage of sperm exhibiting normal acrosomes decreased and damage in the acrosome was the most frequent acrosomal injury with the Percoll gradient centrifugation protocol utilized.

Identification of CD3+ T lymphocytes in the green turtle Chelonia mydas.

To understand the role of the immune system with respect to disease in reptiles, there is the need to develop tools to assess the host's immune response. An important tool is the development of molecular markers to identify immune cells, and these are limited for reptiles. We developed a technique for the cryopreservation of peripheral blood mononuclear cells and showed that a commercially available anti-CD3 epsilon chain antibody detects a subpopulation of CD3 positive peripheral blood lymphocytes in the marine turtle Chelonia mydas. In the thymus and in skin inoculated with phytohemagglutinin, the same antibody showed the classical staining pattern observed in mammals and birds. For Western blot, the anti-CD3 antibodies identified a 17.6k Da band in membrane proteins of peripheral blood mononuclear cell compatible in weight to previously described CD3 molecules. This is the first demonstration of CD3+ cells in reptiles using specific antibodies.