The exposure of embryos to serum during in vitro culture can affect morphology, metabolism, tolerance to cryopreservation, and expression of specific transcripts. On the other hand, serum-free medium seems to avoid some of those serum effects. KnockoutTMSR (GIBCO Laboratories, Grand Island, NY, USA) is a serum replacer optimized to support embryonic stem cells in culture and can also be used to replace serum during culture of in vitro-fertilized bovine embryos. The expression of genes associated with stress response, such as heat shock proteins (HSP), can be affected by in vitro culture conditions, being easily induced by a variety of stress agents, including culture medium components. This study aimed to determine whether KnockoutSR or serum in culture medium alters the relative abundance of HSP70.1 transcripts in in vitro-fertilized bovine embryos. Cumulus–oocyte complexes obtained from slaughterhouse ovaries were matured and feritlized in vitro. Presumptive zygotes were randomly cultured with their own cumulus cells in CR2aa medium supplemented with 10% fetal calf serum (GIBCO-BRL, Paisley, UK; FCS group), 10% KnockoutSR (GIBCO-BRL; KSR group), or 3 mg mL-1 of polyvinyl alcohol (PVA group). All steps were performed at 38.5�C, under 5% CO2 in air and 95% humidity. Blastocysts on Day 8 post-fertilization were rapidly frozen in liquid nitrogen and subsequently thawed for RNA extraction (3 replicates for each group). Total RNA extraction was performed using an Rneasy� Micro kit (Qiagen, Valencia, CA, USA), and the first strand was synthesized using SuperscriptTM III First Strand Synthesis kit (Invitrogen, Chicago, IL, USA). Relative quantification was performed in duplicate using real-time PCR (ABI Prism� 7000 Applied Biosystems, Foster City, CA, USA); reactions consisted of a mixture of iTaqTM SYBR� Green Supermix with ROX (Bio-Rad, Waltham, MA, USA) with cDNA equivalent to 0.8 embryos and gene-specific primers. Expression of the glyceraldehyde 3-phosphate dehydrogenase gene was used as endogenous reference. Calculations of relative quantification were performed by comparative Ct method, using the value found in the PVA group as calibrator. Expression levels for the FCS and KSR groups were 1.2 � 0.06- and 1.4 � 0.08-fold differences relative to the PVA group without differences (P > 0.05). These data show that bovine embryos cultured in medium supplemented with KSR have the same HSP70-1 expression pattern as those in medium with added FCS, suggesting that embryos in both groups are under the same stress conditions.
This work was supported by FAPEMIG, MG, Brazil, and CNPq, DF, Brazil. Thanks to Agrogenetica, Vi�osa, Brazil, for the real-time PCR machine.
Evaluation of the inhibitory effects of drugs on the growth of Babesia Parasites using real-time PCR method