Influence of Estradiol on In vitro Maturation of Porcine Oocytes

In vitro production of embryos allows efficient management of herd genetics, reduction of disease impact, and if used in combination with other reproductive technologies it could aid in preserving the threatened genetic diversity of swine. In vitro maturation (IVM) is identified as a deficient step in porcine in vitro production (IVP) of embryo systems, which decreases the overall success of IVP. There are problems encountered in each step of IVP; chromosomal abnormalities and decreased cell numbers in blastocysts during in vitro culturing (IVC), and low monospermic fertilization rates during in vitro fertilization (IVF) may be a result of insufficient IVM. As an addition to maturation media, porcine follicular fluid (pFF) can affect IVM. Estrogen can be found in high concentrations in pFF; possibly contributing to the effects seen when pFF is added to IVM. The objective of this thesis was to investigate the effects of estrogen supplementation during IVM on IVP of porcine embryos. The first objective was to evaluate the in vitro maturation rates of porcine oocytes in two maturation media: protein-free and 10% pFF supplemented. Nuclear maturation of oocytes was evaluated using Lamin/Dapi staining of oocytes matured in protein-free and 10% pFF maturation media to ensure the efficiency of the protein-free media. Protein-free and 10% pFF media mature oocytes at similar rates (91% and 89% respectively). The transcripts within the oocyte can be altered based on the in vitro maturation environment, so the second objective was to observe the expression of four chosen maternal effect genes: Basonuclin-1 (BNC1), Nucleoplasmin 2 (NPM2), Zygote arrest 1 (ZAR1), and Tripartite-motif protein-24 (TRIM24), using oocytes matured in 50 ng/ml, 100 ng/ml, or 1000 ng/ml of estradiol 17-β (E₂), 10% pFF, or protein-free maturation media. Expression of maternal effect genes, was shown by the ΔCt (cycle threshold) values, obtained from the difference between the Ct values of the normalizing gene (GAPDH) and the genes of interest evaluated through QRT-PCR. Values of ΔCt were analyzed in place of fold change to avoid data manipulation. The ΔCt expression of TRIM24 in 0 ng/ml E₂ maturation medium and the 10% pFF maturation medium were significantly different (p