Master of Science (MS)
Animal Science (Animal, Dairy, and Poultry Sciences)
In vitro maturation (IVM) is a reproductive technique critical to in vitro embryo production (IVP) in commercial livestock industries, research, and human infertility treatment. Currently, IVP has low efficiency due to an inadequate IVM system in which premature meiotic resumption results in low oocyte viability. Meiotic arrest is regulated primarily by 3’,5’-cyclic adenosine monophosphate (cAMP), and the most successful methods of improving IVM utilize cAMP modulators to maintain high intra-oocyte cAMP, delaying the onset of maturation. This thesis includes experiments comparing standard bovine IVM to a novel extended IVM method similar to the procedure described by Albuz and colleagues (Albuz et al., 2010). Bovine oocytes were obtained from mixed breed cattle by transvaginal ultrasound-guided aspiration. Oocytes from each cow were divided into two groups: standard IVM and extended IVM. Standard IVM consists of a 23-hour maturation composed of TCM-199 based media supplemented with 10% fetal bovine serum, sodium pyruvate, pen/strep, glutamine, and FSH, and cultured in 5% CO2 at 39⁰C. Extended IVM is composed of two steps: a pre-IVM of HEPES-TALP supplemented with 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methylxanthine (IBMX) for 2 hours at 39⁰C, and then an extended IVM consisting of standard maturation medium supplemented with 20 µM cilostamide for 31 hours (5% CO2, 39⁰C). Oocytes were sampled at various times throughout maturation depending on the experiment. Data was collected either by staining with aceto-orcein to determine nuclear status or by a cAMP ELISA after freezing in groups of ten. Data from the initial experiments showed that cAMP modulators significantly delayed maturation, but overall maturation rates were significantly less than standard IVM (44.5% vs. 81%). Results of the cAMP assay indicated a significant increase in cAMP within the first three hours of oocyte collection after using FSK and IBMX in collection media, but cAMP was not maintained in the cilostamide-only extended IVM medium. Additionally, cilostamide may have had a negative effect on the oocytes since there was a higher percentage arrested at MI in extended IVM.
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Farmer, Sarah, "Regulation of oocyte meiotic resumption using cAMP modulators in bovine in vitro maturation" (2014). LSU Master's Theses. 3977.