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Capabilities and Challenges of Gene Expression Examination for Quality Assessment of Domestic Cat Embryos
R.H. Waurich Hribal, B.C. Braun, J...
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OBJECTIVES AND METHODS: Next to morphology, cleavage proportions and kinetics, the expression of specific genes important for embryo development is used as a tool to evaluate embryo quality. Those methods, mainly applied in bovine embryos, were proven to be far more sensitive and revealed differences even between embryos that did not differ regarding conventional quality markers (1). For cats, the examination of transcript abundances could also be promising to examine the impact of different invasive methods and culture systems used to produce embryos in vitro within the context of assisted reproduction. Indeed, few studies on cat embryos of different origins were carried out so far. Expression of genes involved in the maintenance of pluripotency, histone acetylation and DNA methylation was different in cloned embryos compared to in vitro derived embryos (2). Although the effect was not significant, morulae produced by IVF with fresh sperm tended to have higher mRNA expression levels compared to those produced with cryopreserved sperm (3).
In a semiquantitative endpoint-RT-PCR assay the temporal pattern of embryonic gene activation was examined on the basis of DNMT1, DNMT3A, GJA1, OCT4, IGF1R, IGF2R and ACTB expression in embryos produced by IVF or ICSI, either with fresh or frozen-thawed semen. For that, different developmental stages (2 cell, 4 cell, 8-16 cell embryo, morula, blastocyst) were analyzed.
RESULTS: In contrast to the morula stage, where higher relative mRNA levels were found in embryos generated with fresh sperm, in blastocysts mRNA abundances tended to be higher in those, that were obtained by fertilization with cryopreserved semen compared to those produced by IVF with fresh semen. This effect was observed independently of the fertilization method used (IVF or ICSI).
CONCLUSION: The use of frozen-thawed spermatozoa seems to influence the temporal pattern of relative mRNA abundances in early cat embryos (higher levels in morulae produced with fresh semen, in contrast higher levels in blastocysts generated with frozen-thawed semen). Although the differences between the two examined groups were not significant (due to high variation) it might be suggested that the onset of gene expression is delayed in embryos fertilized with cryopreserved sperm cells.
Relative gene expression in early embryos can be used as a tool to define influences of different methods of assisted reproduction in cats. High variation between single or small pools of embryos could be due to differences in oocyte and semen quality and/or by differing individual embryo quality. To overcome those challenging issues embryos of different origin that are to be compared should be produced in parallel from equal oocyte and semen pools. In addition, large sample sizes are required.
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