Superovulation is a routine procedure to stimulate growth and ovulation of multiple follicles. However, the hormonal changes in the reproductive tract after superovulation treatment affect the environment and subsequently the early embryo development. The aim of the study was to examine the effect of superovulation pretreatment on embryo development and gene expression of IVM/IVF derived embryos subsequently cultured in vivo. The cumulus‐oocyte complexes derived from slaughterhouse ovaries were in vitro matured and fertilized. The denuded presumptive zygotes were cultured in CR1 medium with 5% oestrous cow serum. A total of 788 cleaved embryos at Day 2 were transferred by transvaginal endoscopy into the oviduct of synchronized and superovulated heifers (superstimulated group, SS) and 784 cleaved embryos were transferred into the ipsilateral oviduct of single ovulated synchronized heifers (single ovulation group, SO). In total, 10 Simmental heifers were used for in vivo culture in a crossover design. The in vivo culture was repeated once at an interval of at least 6 weeks in the same animal. At Day 7, embryos were recovered by combined flushing of the oviducts by endoscopy and the adjacent part of the uterine horns by conventional procedure. The numbers of recovered blastocysts were recorded and the embryos were cultured for the following 48 h to determine the blastocyst rate at Days 8 and 9. Simultaneously, 410 cleaved embryos were cultured in vitro for 9 days (control group, C). Triplicate pools of 10 blastocysts recovered at Day 7 from each treatment group were used for RNA isolation. Real-time PCR using sequence specific primers was performed in StepOnePlus™ real time PCR system (Applied Biosystem, Foster City, CA, USA). A comparative threshold cycle method was used to quantify expression levels of the candidate genes compared to the internal control GAPDH gene. The number of recovered embryos after in vivo culture was significantly lower in the SS group compared with the SO group (66.9 v. 79.5%, respectively; P < 0.05). The blastocyst rates at Days 7, 8, and 9 in the SS, SO, and C groups were not significantly different (31.9, 43.3, and 47.1% v. 35.2, 48.5, and 53.5% v. 37.8, 50, and 56.1%, respectively). Molecular analysis of selected genes playing important roles during pre-implantation development revealed significantly lower expression levels of IL6, IL18, and ABCC2 between both experimental in vivo culture groups and the C-group. The IL18 was also significantly down-regulated in the SS-group compared to the SO-group. The transcription factor NFκB was found to be down-regulated in the SS-group compared to the SO and C groups (P < 0.05). In conclusion, we showed that the superovulation pretreatment did not affect blastocyst yield during the culture period but seemed to influence the expression of developmentally important genes in the resulting embryos.
Evaluation of housekeeping genes in Listeria monocytogenes as potential internal control references for normalizing mRNA expression levels in stress adaptation models using real-time PCR
