Master of Science (MS)
Animal Science (Animal, Dairy, and Poultry Sciences)
Numerous studies have reported aberrant gene expression levels attributed to suboptimal in vitro culture conditions presented to embryos. Since the culture environment is a common aspect of both in vitro production (IVP) and nuclear transfer (NT), research focusing on the in vitro culture system will have the potential to improve both techniques. This study investigated the effects of different culture systems and protein sources on the developmental competence of IVP embryos measured by cleavage and blastocyst rates, cell number, and relative abundance of oct-4, nanog, connexin 43, and GLUT-1 transcripts when compared to in vivo embryos. Experiment 1 compared IVP embryos cultured in either synthetic oviductal fluid (SOFaa) or potassium simplex optimized medium (KSOMaa) supplemented with amino acids. Experiment 2 compared the same two culture systems with and without the addition of calf serum (CS). Results from both experiments indicated that despite similar developmental rates, significant differences were observed at the mRNA level. In Experiment 1, oct-4 was the only transcript to have a mean abundance level significantly higher in KSOMaa blastocysts when compared with both SOFaa and in vivo embryos. The same pattern of upregulation of oct-4 in KSOMaa or KSOMaa with CS blastocysts was noted in Experiment 2. There were no significant alterations of the ICM specific transcript nanog in either experiment. In contrast to reports by others, connexin 43 was not expressed at detectable levels in in vivo embryos analyzed in our studies. Connexin 43 was not detected in IVP blastocysts used in Experiment 1. Connexin 43 was detected in KSOMaa, SOFaa, and SOFaa with CS blastocysts in Experiment 2. Blastocysts cultured in SOFaa with CS or KSOMaa had a significant upregulation of GLUT-1 when compared with other treatments and in vivo embryos. Overall, the transcript levels of the majority of the genes analyzed were significantly altered by an in vitro culture condition. Differences continue to be observed between in vitro cultured and in vivo embryos, and until these differences are minimized, aberrations in in vitro development will continue to arise.
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Purpera, Megan Nicole, "Effect of culture conditions on gene expression in manipulated bovine embryos" (2007). LSU Master's Theses. 2162.
Kenneth R. Bondioli