Validation of a reference control for an SYBR-Green fluorescence assay-based real-time PCR for detection of bovine herpesvirus 5 in experimentally exposed bovine embryos

The objective of this study was to optimize an internal control to improve SYBR-Green-based qPCR to amplify/detect the BoHV-5 US9 gene in bovine embryos produced invitro and experimentally exposed to the virus. We designed an SYBR-Green-based binding assay that is quick to perform, reliable, easily optimized and compares well with the published assay. Herein we demonstrated its general applicability to detect BoHV-5 US9 gene in bovine embryos produced invitro experimentally exposed to BoHV-5. In order to validate the assay, three different reference genes were tested; and the histone 2a gene was shown to be the most adequate for normalizing the qPCR reaction, by considering melting and standard curves ( p<0.05). On the other hand, no differences were found in the development of bovine embryos invitro whether they were exposed to BoHV-5 reference and field strains comparing to unexposed embryos. The developed qPCR assay may have important field applications as it provides an accurate BoHV-5 US9 gene detection using a proven reference gene and is considerably less expensive than the TaqMan qPCR currently employed in sanitary programs. © 2013 Elsevier Ltd.

Modelo experimental com caprinos e cobaias para avaliação da eficácia de vacinas contra o herpesvírus bovino tipo 1 e o vírus da diarreia viral bovina tipos 1 e 2

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Programmed cell death-associated gene transcripts in bovine embryos exposed to bovine Herpesvirus type 5

In vitro-produced bovine embryos become infected after exposure to bovine Herpesvirus type 5 (BoHV-5), yet no changes in developmental rates, mitochondrial activity and inhibition of apoptosis are detected in comparison to unexposed embryos. Thus, the aim of the present study was to assess the transcription of mitochondria-mediated apoptosis genes using TaqMan real-time polymerase chain reaction. Transcripts of mcl-1, caspase-2, -3, Apaf-1 and Bax genes were measured after exposure to BoHV-5 in vitro. Mitochondrial dehydrogenase activity was evaluated by MIT test and compared between groups of exposed and unexposed embryos, at day 7 of development. The rate of oocyte maturation was assessed by the extrusion of the first polar body. In summary, BoHV-5 exposed embryos retained their viability, mitochondrial dehydrogenase activity and displayed up-regulation of transcription of survival mcl-1 gene and down-regulation of Bax transcription in relation to mitochondria-mediated pathway which might improve embryo viability. These findings demonstrate that BoHV-5 exposed embryos maintain their viability and mitochondrial dehydrogenase activity with no compromise of embryos produced in vitro. (c) 2013 Elsevier Ltd. All rights reserved.

Effect of hydrogen peroxide on thawed ovine sperm motility

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mediadores da via intrínseca da morte celular programada relacionados à infecção in vitro pelo herpesvirus bovino tipo 5

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fate of non O157 Shigatoxigenic Escherichia coli in ovine manurecomposting

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)