Sphingosine 1-phosphate promotes activation of aprine preantral follicle in vitro / Esfingosina 1-fosfato promove ativação de folículos pré-antrais de caprinos in vitro

This study describes the effect of sphingosine 1-phosphate (S1P) for development of preantral follicle, therefore the activation and follicular viability of caprine follicles cultured in vitro. Ovarian fragments were cultured for 1 or 7 days in Minimum Essential Medium with different S1P concentrations (0, 1, 10, 50, 100 or 200ng/mL). All ovarian fragments were processed for histological analysis in optical microscopy, transmission electron microscopy and fluorescence analysis. The treatment using 1ng/mL of S1P was able to maintain the percentage of normal follicles with the progression of the culture from day 1 to 7. At end of the 7-day culture period there was a significant reduction (P<0.05) in the percentage of primordial follicles in all groups treated with S1P, compared with fresh control (FC) and Control Culture (CC), which was followed by an increase of activated follicles (intermediary, primary and secondary). In addition, the culture for 7 days with media supplemented with S1P with 1ng/mL preserved the ultrastructure of organelles and kept the preantral follicular viability when evaluated by fluorescence microscopy. In conclusion, after 7 days of culture, the 1ng/mL of S1P activates the development of preantral caprine follicles, cultured in situ and maintains the oocitary and follicular viability...

Este estudo descreve o efeito da esfingosina 1-fosfato (S1P) no desenvolvimento de folículos pré-antrais, portanto da ativação e viabilidade de folículos caprinos cultivados in vitro. Fragmentos de ovários foram cultivados por um ou sete dias em meio essencial mínimo com diferentes concentrações de S1P (0, 1, 10, 50, 100 ou 200ng/mL). Os fragmentos de ovário foram processados para análise histológica em microscopia óptica, microscopia eletrônica e microscopia de fluorescência. O tratamento usando 1ng/mL de S1P foi capaz de manter a porcentagem de folículos normais durante o período de cultivo de sete dias. Ao final do período de cultivo, houve uma redução significativa (p<0,05) na porcentagem de folículos primordiais em todos os grupos tratados com S1P, comparados com os grupos controle (FC e CC), seguida por um aumento do número de folículos ativados (intermediários, primários e secundários). Adicionalmente, na cultura por sete dias com meio suplementado com S1P (1ng/mL), houve preservação da ultraestrutura das organelas e manteve-se a viabilidade dos folículos pré-antrais avaliados por microscopia de fluorescência. Em conclusão, após sete dias de cultura, o meio suplementado com 1ng/mL de S1P ativa o desenvolvimento de folículos pré-antrais de caprino, cultivados in situ e mantém as viabilidades oocitária e folicular...

NaOCl effect on biofilm produced by Staphylococcus aureus isolated from the milking environment and mastitis infected cows / Efeito do hipoclorito de sódio em biofilmes produzidos por Staphylococcus aureus isolados do ambiente de ordenha e de vacas com mastite

Biofilms constitute a physical barrier, protecting the encased bacteria from detergents and sanitizers. The objective of this work was to analyze the effectiveness of sodium hypochlorite (NaOCl) against strains of Staphylococcus aureus isolated from raw milk of cows with subclinical mastitis and Staphylococcus aureus isolated from the milking environment (blowers and milk conducting tubes). The results revealed that, in the presence of NaOCl (150ppm), the number of adhered cells of the twelve S. aureus strains was significantly reduced. When the same strains were evaluated in biofilm condition, different results were obtained. It was found that, after a contact period of five minutes with NaOCl (150ppm), four strains (two strains from milk , one from the blowers and one from a conductive rubber) were still able to grow. Although with the increasing contact time between the bacteria and the NaOCl (150ppm), no growth was detected for any of the strains. Concerning the efficiency of NaOCl on total biofilm biomass formation by each S. aureus strain, a decrease was observed when these strains were in contact with 150 ppm NaOCl for a total period of 10 minutes. This study highlights the importance of a correct sanitation protocol of all the milk processing units which can indeed significantly reduce the presence of microorganisms, leading to a decrease of cow´s mastitis and milk contamination.

Biofilmes são constituídos de bactérias aderidas a uma superfície e aderidas entre si envolvidas por um polissacarídeo de constituição proteica, lipídica e glicídica que conferem uma barreira física às bactérias dentro deste microambiente. O objetivo deste trabalho foi analisar a eficácia do hipoclorito de sódio (NaOCl) contra estirpes de Staphylococcus aureus isoladas de leite cru de vacas com mastite subclínica e Staphylococcus aureus isolados do ambiente de ordenha (borrachas de ordenhadeiras e mangueiras condutoras de leite). Os resultados revelaram que, na presença de hipoclorito de sódio (150ppm), o número de células aderidas das 12 estirpes de S. aureus analisadas foi significativamente reduzido. Quando as mesmas estirpes foram avaliadas em condições de biofilme, diferentes resultados foram obtidos. Verificou-se que, após um período de contato de cinco minutos com NaOCl (150ppm), quatro estirpes (duas estirpes de leite, uma estirpe das borrachas das ordenhadeiras e uma estirpe de uma mangueira condutora de leite) ainda eram capazes de crescer. Com o aumento do tempo de contato do hipoclorito e as bactérias, cada vez maior, na concentração de 150ppm, não foi detectado o crescimento das estirpes. Em relação à eficácia do NaOCl na formação total da biomassa do biofilme por cada uma das estirpes de S. aureus, observou-se decréscimo da biomassa dos biofilmes quando estas estirpes estavam em contato com o NaOCl na concentração de 150ppm durante um tempo total de 10 minutos. O estudo demonstra a importância de um protocolo de saneamento correto de todas as unidades de processamento de leite, que pode, efetivamente, reduzir a presença de microrganismos de forma significativa, conduzindo a uma diminuição da mastite e da contaminação do leite.

Mitochondrial and DNA damage in bovine somatic cell nuclear transfer embryos

The generation of reactive oxygen species (ROS) and subsequent mitochondrial and DNA damage in bovine somatic cell nuclear transfer (SCNT) embryos were examined. Bovine enucleated oocytes were electrofused with donor cells and then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. The H2O2 and .OH radical levels, mitochondrial morphology and membrane potential (DeltaPsi), and DNA fragmentation of SCNT and in vitro fertilized (IVF) embryos at the zygote stage were analyzed. The H2O2 (35.6 +/- 1.1 pixels/embryo) and .OH radical levels (44.6 +/- 1.2 pixels/embryo) of SCNT embryos were significantly higher than those of IVF embryos (19.2 +/- 1.5 and 23.8 +/- 1.8 pixels/embryo, respectively, p < 0.05). The mitochondria morphology of SCNT embryos was diffused within the cytoplasm. The DeltaPsi of SCNT embryos was significantly lower (p < 0.05) than that of IVF embryos (0.95 +/- 0.04 vs. 1.21 +/- 0.06, red/green). Moreover, the comet tail length of SCNT embryos was longer than that of IVF embryos (515.5 +/- 26.4 microm vs. 425.6 +/- 25.0 microm, p < 0.05). These results indicate that mitochondrial and DNA damage increased in bovine SCNT embryos, which may have been induced by increased ROS levels.

Development and characterization of a potential diagnostic monoclonal antibody against capsid protein VP1 of the chicken anemia virus

Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. In this study, a potential diagnostic monoclonal antibody against the CAV VP1 protein was developed which can precisely recognize the CAV antigen for diagnostic and virus recovery purposes. The VP1 gene of CAV encoding the N-terminus-deleted VP1 protein, VP1Nd129, was cloned into an Escherichia (E.) coli expression vector. After isopropyl-beta-D-thiogalactopyronoside induction, VP1Nd129 protein was shown to be successfully expressed in the E. coli. By performing an enzyme-linked immunoabsorbent assay using two coating antigens, purified VP1Nd129 and CAV-infected liver tissue lysate, E3 monoclonal antibody (mAb) was found to have higher reactivity against VP1 protein than the other positive clones according to the result of limiting dilution method from 64 clones. Using immunohistochemistry, the presence of the VP1-specific mAb, E3, was confirmed using CAV-infected liver and thymus tissues as positive-infected samples. Additionally, CAV particle purification was also performed using an immunoaffinity column containing E3 mAb. The monoclonal E3 mAb developed in this study will not only be very useful for detecting CAV infection and performing histopathology studies of infected chickens, but may also be used to purify CAV particles in the future.

Erythropoiesis and erythrophagocytosis in bovine haemal nodes / Eritropoyesis y eritrofagocitosis en nodos linfáticos hemales de bovino

Whether bovine haemal nodes are involved in turnover of red blood cells has been a subject of some controversy In this study, fluorescent and conventional optical microscopy of conventionally or immunohistochemically stained node sections, together with transmission electron microscopy, showed the presence of erythrocyte precursors and megakaryocytes, and evidence of active involvement in the destruction and replacement of old or degenerate red cells and the platelets.

Si nodos linfáticos hemales bovinos están involucrados en la cantidad de volumen de glóbulos rojos ha sido objeto de cierta controversia. En este estudio, secciones de nodos linfáticos teñidas convencionalmente o inmunohistoquimicamente fueron analizadas con microscopía óptica fluorescente y convencional, junto con microscopio electrónico de transmisión, los que revelaron la presencia de precursores eritrocíticos y megacariocitos, y la evidencia de participación activa en la destrucción y sustitución de glóbulos rojos viejos o degenerados y plaquetas.

Characterization and localization of the unique Marek's disease virus type 2 ORF873 gene product

Studies on Marek's disease virus (MDV)-unique genes are important for understanding the biological nature of the virus. Based on complete DNA sequence analyses of the MDV genomes, the MDV genomes contain presumably at least five MDV-unique genes, which are commonly conserved among the three MDV serotypes. A recombinant baculovirus that contains the MDV serotype 2 (MDV2)-unique gene, ORF873, under the polyhedrin promoter was constructed and designated rAcORF873. Polyclonal and monoclonal antibodies, which recognize the recombinant MDV2 ORF873 protein in Spodoptera frugiperda clone 9 (Sf9) cells infected with rAcORF873, were prepared by immunizing mice with a recombinant fusion protein expressed in Escherichia coli. Immunoblot analyses with the antibodies revealed a major protein band with a molecular mass of 108-kDa in both MDV2-infected chick embryo fibroblasts (CEF) and rAcORF873-infected Sf9 cells. By indirect immunofluorescence analyses using monoclonal antibody, the authentic ORF873 protein was localized in the cytoplasm of MDV2-infected CEF cells. The monoclonal and polyclonal sera, which were generated in the present study and reacted effectively to MDV2 ORF873 protein, are considered to be useful reagents for further studying the role(s) of the ORF873 protein in MDV2 infection.

Thickness of cumulus cell layer is a significant factor in meiotic competence of buffalo oocytes

This study evaluated the meiotic competence of buffalo oocytes with different layers of cumulus cells. A total of 588 oocytes were collected from 775 ovaries averaging 0.78 oocytes per ovary. Oocytes with homogenous cytoplasm (n = 441) were selected for in vitro maturation (IVM) and divided into four groups based on their cumulus morphology: a) oocytes with > or == 3 layers of cumulus cells, b) 1-2 layers of cumulus cells and oocytes with partial remnants or no cumulus cells to be cocultured c) with or d) without cumulus cells. Oocytes in all four groups were matured in 100 microL drop of TCM-199 supplemented with 10microgram/mL follicle stimulating hormone (FSH), 10microgram/mL luteinizing hormone (LH), 1.5microgram/mL estradiol, 75microgram/mL streptomycin, 100 IU/mL penicillin, 10 mM Hepes and 10% FBS at 39degrees C and 5% CO2 for 24 hours. After IVM, cumulus cells were removed from oocytes using 3 mg/mL hyaluronidase, fixed in 3% glutaraldehyde, stained with DAPI and evaluated for meiotic competence. The oocytes with > or ==3 layers of cumulus cells showed higher maturation rates (p <0.05: 64.5%) than oocytes with partial or no cumulus cells (8.6%) and oocytes co-cultured with cumulus cells (34.5%) but did not differ from oocytes having 1-2 layers of cumulus cells (51.4%). The degeneration rates were higher (p < 0.05) for oocytes with partial or no cumulus cells (51%) than rest of the groups (range: 13.8% to 17.4%). These results suggest that buffalo oocytes with intact layers of cumulus cells show better IVM rates than oocytes without cumulus cells and the co-culture of poor quality oocytes with cumulus cells improves their meiotic competence.