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OBJECTIVE: To quantify the platelet concentration in the blood of SHR rats, by means of different centrifugation protocols, and to evaluate what the most effective method for obtaining platelets is. METHODS: We used 40 male rats of the isogenic SHR lineage. The animals were divided into three groups: control, using whole blood without centrifugation; single centrifugation, using whole blood subjected to a single centrifugation at 200 × gand 400 × g; and double centrifugation, using whole blood subjected one centrifugation at different rotations, followed by collection of whole plasma subjected to another centrifugation at different rotations: 200 × g+ 200 ×g; 200 × g+ 400 × g; 200 × g+ 800 × g; 400 ×g+ 400 × g; 400 × g+ 800 × g. Samples of 3 ml of blood were drawn from each animal by means of cardiac puncture. The blood was stored in Vacutainer collection tubes containing 3.2% sodium citrate. The blood from the control group animals was analyzed without being subjected to centrifugation. After the blood from the other groups of animals had been subjected to centrifugation, the whole plasma was collected and subjected to platelet counting in the lower third of the sample. RESULTS: We obtained greatest platelet enrichment in the subgroup with two centrifugations comprising 400 × gfor 10 min + 400 ×gfor 10 min, in which the mean platelet concentration was 11.30 times higher than that of the control group. CONCLUSION: It was possible to obtain a high platelet concentration using viable simple techniques, by means of centrifugation of whole blood and use of commonly used materials. The most effective method for obtaining platelet concentrate was found in samples subjected to two centrifugations. RESUMO OBJETIVO: Quantificar a concentração de plaquetas do sangue de ratos SHR, por meio de diferentes protocolos de centrifugação, e avaliar qual o método mais eficaz de obtenção de plaquetas. MÉTODOS: Usamos 40 ratos machos da linhagem isogênica SHR. Os animais foram divididos em três grupos: Controle (GCT) - sangue total sem centrifugação; Única Centrifugação (GUC) - sangue total submetido a uma única centrifugação: 200 g e 400 g; Dupla Centrifugação (GDC) - sangue total submetido a uma centrifugação, seguido de coleta do plasma total, e realizado uma centrifugação, em diferentes rotações: 200 g + 200 g; 200 g + 400 g; 200 g + 800 g; 400 g + 400 g; 400 g + 800 g. Foram retirados 3 ml de sangue de cada animal por meio de punção cardíaca. O sangue foi acondicionado em tubo de coleta vacutainer com citrato de sódio 3,2%. O sangue dos animais do grupo controle não foi submetido à centrifugação e foi analisado. Após a centrifugação do sangue dos animais, submetido à centrifugação, o plasma total foi coletado e submetido à contagem de plaquetas no terço inferior da amostra. RESULTADOS: Obtivemos maior enriquecimento de plaquetas no subgrupo de duas centrifugações (400 g por 10 minutos + 400 g por 10 minutos), no qual ocorreu uma concentração média de plaquetas 11,30 vezes superior em relação ao grupo controle. CONCLUSÃO: Foi possível obter uma alta concentração plaquetária, com técnica simples e viável, por meio de centrifugação do sangue total e uso de materiais de uso corriqueiro; e método mais eficaz de obtenção de concentrado de plaquetas ocorreu nas amostras submetidas a duas centrifugações.

Foram avaliados os efeitos da centrifugação associada ao uso de dois diluentes na manutenção da viabilidade espermática em tatus-peba (Euphractus sexcinctus) ao longo do teste de termorresistência (TTR). Amostras de sêmen (n=12), oriundas de 04 machos adultos coletados por eletroejaculação, foram divididas em quatro alíquotas, sendo duas imediatamente diluídas em Tris ou água de coco em pó (ACP-119(r)), e as outras duas centrifugadas (800g10min-1) previamente à diluição. As amostras foram incubadas a 34°C por 3h, e os parâmetros seminais avaliados em intervalos de 1h. Em termos gerais, verificou-se uma redução da viscosidade espermática imediata à diluição em ambos os diluentes, independente do uso da centrifugação. Aos 60 minutos, verificou-se uma redução dos parâmetros avaliados (P0,05). Ainda, verificou-se um efeito deletério da centrifugação sobre a qualidade do sêmen de tatus-peba durante todo o teste de termorresistência. Nas condições do presente estudo, conclui-se que o diluente Tris mostrou-se superior ao ACP-119(r) para a manutenção da viabilidade do sêmen de tatus-peba, sendo desnecessária a realização de centrifugação prévia à diluição. The effects of the centrifugation associated to the use of two extenders on the viability of six-banded armadillo's (Euphractus sexcinctus) sperm were evaluated during a thermo resistance test (TRT). Semen samples (n=12) derived from 04 stud males collected by electroejaculation were divided in four aliquots; two of that were immediately diluted in Tris or powdered coconut water (ACP-119(r)); the two others were centrifuged (800g10min-1) prior to the dilution. Samples were incubated at 34ºC per 3h, and the semen parameters were evaluated at each hour. In general, dilution promoted a reduction in semen viscosity in the use of both diluents using centrifugation or not. At 60min, a decrease was verified for all semen parameters (P

Three commercial kits of One-Step RT-qPCR were evaluated for the molecular diagnosis of Canine Distemper Virus. Using the kit that showed better performance, two systems of Real-time RT-PCR (RT-qPCR) assays were tested and compared for analytical sensitivity to Canine Distemper Virus RNA detection: a One-Step RT-qPCR (system A) and a One-Step RT-qPCR combined with NESTED-qPCR (system B). Limits of detection for both systems were determined using a serial dilution of Canine Distemper Virus synthetic RNA or a positive urine sample. In addition, the same urine sample was tested using samples with prior centrifugation or ultracentrifugation. Commercial kits of One-Step RT-qPCR assays detected canine distemper virus RNA in 10 (100%) urine samples from symptomatic animals tested. The One-Step RT-qPCR kit that showed better results was used to evaluate the analytical sensitivity of the A and B systems. Limit of detection using synthetic RNA for the system A was 11 RNA copies µL-1 and 110 RNA copies µl-1 for first round System B. The second round of the NESTED-qPCR for System B had a limit of detection of 11 copies µl-1. Relationship between Ct values and RNA concentration was linear. The RNA extracted from the urine dilutions was detected in dilutions of 10-3 and10-2 by System A and B respectively. Urine centrifugation increased the analytical sensitivity of the test and proved to be useful for routine diagnostics. The One-Step RT-qPCR is a fast, sensitive and specific method for canine distemper routine diagnosis and research projects that require sensitive and quantitative methodology. RESUMO: Três kits comerciais de One-Step RT-qPCR foram avaliados para o diagnóstico molecular do Vírus da Cinomose Canina.Utilizando o kit que apresentou melhor desempenho, dois sistemas de RT-PCR em tempo real (RT-qPCR) foram comparados quanto à sensibilidade analítica na detecção do RNA do Vírus da Cinomose Canina:One-Step RT-qPCR (Sistema A) e One-Step RT-qPCR seguido da NESTED-qPCR (Sistema B).Os limites de detecção dos dois sistemas foram determinados utilizando diluição seriada de RNA sintético do Vírus da Cinomose Canina ou de uma amostra de urina positiva. Adicionalmente, uma amostra de urina foi avaliada com centrifugação ou ultracentrifugação prévia. Os kits comerciais de One-Step RT-qPCR amplificaram o RNA do vírus da cinomose canina em 10 (100%) amostras de urinas de animais sintomáticos. O kit de One-Step RT-qPCR que apresentou melhor resultado foi utilizado para avaliar a sensibilidade analítica dos sistemas A e B. Na reação da curva padrão com RNA sintético, o limite de detecção do sistema A foi de 11 cópias de RNA µL-1. No sistema B foi de 110 cópias de RNA µL-1 na One-Step RT-qPCR e 11 cópias de RNA µL-1 na NESTED-qPCR. A relação entre os valores de Ct e concentração de RNA foi linear. O RNA extraído das diluições da urina foi detectado nas diluições de 10-3 e10-2 pelos sistemas A e B, respectivamente. A centrifugação prévia da urina aumentou a sensibilidade analítica da análise e mostrou ser importante para a rotina diagnóstica. A reação de One-Step RT-PCR é um método rápido, sensível, específico e aplicável na rotina de diagnóstico molecular da cinomose e em projeto de pesquisa que requer metodologia quantitativa e sensível.

Artificial insemination is routinely used in the swine industry to reduce the costs of production through to increase the efficiency of the refrigerated boar semen process. The objective of this study was to evaluate the effect of different levels of cysteine (CYS) added to the Beltsville Thawing Solution (BTS) extender semen during cooling for up to 72 hours. Ejaculated from three boars were collected with the gloved-hand technique and semen aliquots were diluted in BTS as follow: BTS only (BTS), BTS + 0.1mM cysteine (CYS0.1), BTS + 0.5mM cysteine (CYS0.5), BTS + 1.0mM cysteine (CYS1.0), BTS + 2.5mM cysteine (CYS2.5), BTS + 5.0mM cysteine (CYS5.0), BTS + 10.0mM cysteine (CYS10.0), and BTS + 20.0mM cysteine (CYS20.0). Evaluation of sperm integrity were analyzed using 0.5mg/ml propidium iodide (plasma membrane), 100µg/ml isothiocynate-conjugated Pisum sativun agglutinin (acrosomal membrane) and 153µM 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (mitochondria potential) after semen dilution at specific times (0, 24, 48 and 72 hours). Additionally, we also evaluated the effects of 5.0 mM CYS addition in the BTS extender on the maintenance of sperm quality and their influence on fertility in the swine production. After artificial insemination, animals were evaluated based on the estrous return and the number of piglet's born. Cysteine at concentrations of 10.0 and 20.0mM resulted in more pronounced reductions even at the time zero. Semen viability decreased to levels below 10% at these high levels of CYS in the first 24 hour of storage at 17ºC. At the end of the storage time, less than 65% of sperm cells had intact plasma membrane in all groups. The sperm viability decreased significantly when the semen was added at high concentrations of CYS (time "0"; CYS10.0 and CYS20.0; p

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:56:30Z No. of bitstreams: 1 S0102-09352011000300002.pdf: 233493 bytes, checksum: 5094bd900d00f38cb747d4ff63b4af05 (MD5) Made available in DSpace on 2013-08-22T18:56:30Z (GMT). No. of bitstreams: 1 S0102-09352011000300002.pdf: 233493 bytes, checksum: 5094bd900d00f38cb747d4ff63b4af05 (MD5) Previous issue date: 2011-06-01 Made available in DSpace on 2013-09-30T18:02:04Z (GMT). No. of bitstreams: 2 S0102-09352011000300002.pdf: 233493 bytes, checksum: 5094bd900d00f38cb747d4ff63b4af05 (MD5) S0102-09352011000300002.pdf.txt: 30539 bytes, checksum: ae43cbd6b0eb5b6c0e7561636e91399e (MD5) Previous issue date: 2011-06-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:15:20Z No. of bitstreams: 2 S0102-09352011000300002.pdf: 233493 bytes, checksum: 5094bd900d00f38cb747d4ff63b4af05 (MD5) S0102-09352011000300002.pdf.txt: 30539 bytes, checksum: ae43cbd6b0eb5b6c0e7561636e91399e (MD5) Made available in DSpace on 2014-05-20T13:15:20Z (GMT). No. of bitstreams: 2 S0102-09352011000300002.pdf: 233493 bytes, checksum: 5094bd900d00f38cb747d4ff63b4af05 (MD5) S0102-09352011000300002.pdf.txt: 30539 bytes, checksum: ae43cbd6b0eb5b6c0e7561636e91399e (MD5) Previous issue date: 2011-06-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) O objetivo do presente estudo foi determinar o enriquecimento de espermatozoides portadores do cromossomo X após a centrifugação em gradiente de densidade contínuo de Percoll ou OptiPrep, utilizando reação em cadeia da polimerase quantitativa em tempo real (qPCR) do DNA do espermatozoide e dos embriões bovinos produzidos in vitro resultantes pela PCR convencional. Espermatozoides descongelado foram depositados em gradientes de densidade e os tubos foram centrifugados. Os sobrenadantes foram gentilmente aspirados e os espermatozoides recuperados do fundo dos tubos. As taxas de clivagem e de blastocisto foram determinadas pela produção in vitro de embriões e a PCR foi realizada para a identificação genética do sexo dos embriões. Verificou-se diferença na taxa de blastocistos entre os grupos Percoll e OptiPrep (P

Tullgren funnels are commonly used when extracting mites from substrates for ecological and taxonomic studies. During this process, extracted mites are often contaminated with soil particles and other debris, making the processing of those organisms difficult, especially for the smaller mites, such as Tarsonemidae. The Water Sugar-Water (WSW) method, here described here, is intended to facilitate the separation of the extracted mites from those fouling material. It consists of a modification of the Jenkins method, used for the extraction of nematodes. Through a combination of centrifugation with two steps of floatation (first in water and then in 1.5 molar sucrose solution), this method separates mites from contaminants. In this study, the WSW method was tested in different conditions including three soil textures, two storage times (3 days or 6 months) and leaf litter. We also evaluated the possibility to use this method for separation of the broad mite, Polyphagotarsonemus latus (Banks) (Tarsonemidae), given its common occurrence in fields where the present work was conducted. Eight of nine treatments performed consistently well with extraction efficiencies >95% (mites collected in relation to total mites present in the funnel extraction), with no significant differences among them. One treatment, using the addition of kaolin clay to the sample, retained some Phytoseiidae in the sediment pellets, reducing extraction efficiency to 88.3%. Mites of the taxa Acaridae, Cunaxidae, Eupodidae, Laelapidae, Phytoseiidae, Scutararidae, Tarsonemidae, Tydeidae, Rhodacaroidea and Orbatida were successfully removed from contaminated samples with this method. The frequency of these taxa was equally distributed between the water and sugar-water steps of the two-step floatation method. This method is fast, uses inexpensive and non-toxic compounds and has great potential for use to determine the presence of mite groups most commonly found in extractions of Tullgren funnels.

O presente estudo teve por objetivo avaliar a capacidade de concentração plaquetária e sua correlação com os níveis do fator de crescimento TGF-B1, a presença de leucócitos e de hemácias nos diferentes protocolos utilizados na obtenção do plasma rico em plaquetas (PRP) de equinos, através do método manual. Dez equinos, sadios, com idade média de 7 anos (±2,39), pesando em média 500kg (±67,1) foram utilizados neste estudo. Os protocolos testados variaram na velocidade e no tempo nas duas centrifugações. As variáveis analisadas nas amostras de PRP foram: concentração de plaquetas, presença de leucócitos e hemácias, e níveis de TGF-β1 quantificados pelo teste ELISA. Os protocolos testados não diferiram na capacidade de concentração de plaquetas e nos níveis de TGF-β1. Entretanto, houve diferença significava entre o protocolo I e os demais por este apresentar maior número de hemácias e leucócitos nas amostras de PRP, sendo por esse motivo considerado um protocolo inadequado para processamento do volume de sangue utilizado. Os demais protocolos podem ser utilizados para obtenção de PRP terapêutico em equinos. PRP is plasma that contains a high numbers of platelets and growth factors in a small volume. The aim of this study was to evaluate seven different protocols to obtain PRP by the manual method according to their capacity to concentrate platelets, leukocyte and erythrocyte contamination and correlation between platelet count and TGF-β1 growth factor levels in PRP samples. Ten healthy horses with a mean age of 7 years (±2.39), weighing on average 500 kg (±67.1) were used in this study. The protocols tested varied according to the speed and time used at the two centrifugations. PRP samples were analyzed regarding platelet concentration, leukocyte and erythrocyte contamination and TGF-β1 levels quantified by ELISA. No significant differences among protocols were observed regarding the ability to concentrate platelets and TGF-β1. However, protocol I showed significantly higher erythrocyte and leukocyte counts in PRP samples than the other protocols, reason why it was considered inadequate for the volume of blood processed in this experiment. The remaining protocols are suitable for extracting PRP.