Effects of guaiazulene on in vitro bovine embryo production and on mRNA transcripts related to embryo quality.

Reactive oxygen species (ROS) are between the major contributors for the reduced rate of in vitro bovine embryo production. It is believed that they can cause abnormal meiosis of oocytes, developmental arrest or cell death of embryos. Reports on the effectiveness of various antioxidants on embryo yield are rather conflicting mainly due to the nature and the concentration of the substances used. Here we report the effects of guaiazulene--an exogenous antioxidant, without known properties that could interfere with the biological process of IVF--on embryo development and on the quality of the produced blastocysts. Bovine cumulus oocyte complexes (COCs) were aspirated from abattoir ovaries and COCs were matured in TCM199 with FCS and EGF at 39 °C under an atmosphere of 5% CO(2) in air, with maximum humidity. After 24 h oocytes were inseminated with frozen/thawed semen and co-incubated for further 24 h. Zygotes were cultured in groups of 25 in 25 μl of SOF with 5% FCS at 39 °C under an atmosphere of 5% CO(2) , 5% O(2) in air with maximum humidity. In the first experiment the maturation medium was modified with addition of 0.1 mM of G (n = 497), or 0.01 mM of guaiazulene (n = 468), 0.05% DMSO--the guaiazulene diluent (Control(+), n = 467), and 459 oocytes were used as Control(-). In the second experiment, the culture medium was modified with the addition of 0.1 mM of guaiazulene (n = 344), 0.01 mM of guaiazulene (n = 345), 0.05% DMSO (Control(+), n = 347) and 355 were the Control(-). Blastocyst yield was recorded on days 6, 7, 8 and 9. Day 7 blastocysts from each experiment and group were snap frozen and stored for mRNA extraction. Quantification of transcripts for mRNA of genes related to metabolism (AKR1B1, PTGS2, GADPH, SLC2A5, G6PD); oxidation (GPX1); and implantation (PLAC8) was carried out by real time quantitative RT-PCR. Data for embryo development and on transcript abundance were analysed by χ(2) and anova respectively. In the first experiment no differences were found between groups in terms of cleavage rate (Control(-): 74.20%; Control(+): 74.58%; 0.01 mM: 71.61%; 0.1 mM: 71.63%) or day 9 blastocyst yield (Control(-): 28.26%; Control(+): 25.80%; 0.01 mM: 25.86%; 0.1 mM: 25.25%). In the second experiment, cleavage rate tended to be higher in 0.01 mM group than in Control(-) (77.87% vs 71.41% respectively, p = 0.07). No other differences were detected in cleavage rate (Control(+): 71.32%; 0.1mM: 72.75%) or in the overall blastocyst yield on day 9 (Control(-): 25.50%; Control(+): 26.71%; 0.01 mm: 29.58%; 0.1 mM: 25.75%). In both experiments the relative abundance of genes studied varied between groups but these differences were not statistically significant. Our results imply that oxidation has minimal effect on the in vitro embryo production. Guaiazulene, a compound possessing no biological properties other than those of a strong antioxidant, while it increased cleavage rate, it failed to improve either the blastocyst formation rate, or the quality of the produced embryos under 5% O(2) .