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Browsing by Author "Krisher, Rebecca, committee member"

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    Investigation of assisted reproductive technologies (ART) for conservation of Bovidae
    (Colorado State University. Libraries, 2022) Benham, Hayley Marie, author; Barfield, Jennifer, advisor; Graham, James, advisor; Krisher, Rebecca, committee member; Duncan, Colleen, committee member
    There is an ongoing loss in global biodiversity in both wildlife and domestic species, creating a need to protect and preserve valuable genetics for the maintenance and sustainability of these populations. One of the proposed strategies to combat this loss is through the use of assisted reproductive technologies (ART), not as a replacement to natural breeding but as a strategy to augment and increase the tools we have to preserve genetic diversity. Currently, the application of ARTs is not broadly used as there are gaps in knowledge of species-specific reproductive biology and gamete biology, hindering the ability to make fast progress in implementing these techniques for conservation purposes in species and populations that are rapidly declining. ARTs used in domestic species in the Bovidae family, including cattle, have been developing over the past century, and recently are being adapted for use in non-domestic Bovidae. Arguably, one of the most critical ARTs developed for genetic rescue and conservation goals has been the successful utilization of cryopreservation techniques, which has allowed for the creation of genetic resource banks (GRBs), which are biorepositories for gametes, embryos, and tissues. Through GRBs breeding programs can achieve similar or greater levels of heterozygosity and increase the effective population size as represented by embryos and germplasm, compared to captive populations. In this work, we describe studies developing in vitro oocyte maturation (IVM) and in vitro embryo production (IVP) techniques used for the preservation of valuable bison genetics using gametes collected post-mortem, and cryopreservation techniques of bovine oocytes and ovarian tissue for fertility preservation. Using oocytes collected post-mortem, from bison within the YNP herd (a population with endemic brucellosis) we created disease-free embryos for subsequent transfer into healthy surrogate bison. The transfer of brucellosis-free embryos resulted in the live birth of a healthy brucellosis-negative bison calf. Next, by assessing the follicle size and duration of in vitro maturation (IVM) of bison oocytes collected from abattoir ovaries, out of season, we determined that seasonality does impact oocyte competence and blastocyst production, although viable bison embryos can be created independent of seasonal effects. While cryopreservation of female gametes in bovid species remains experimental, vitrification of oocytes may provide an opportunity for infusing greater genetic diversity into future generations. By assessing mitochondrial function of bovine oocytes during vitrification, we found that germinal vesicle (GV) stage oocytes may require additional support through the vitrification process, as they demonstrated a reduced ability to handle cryo-induced oxidative stress post-vitrification. Additionally, vitrification of bovine ovarian cortical tissue coupled with techniques for vitro activation (IVA) of primordial follicles may be an alternative way to preserve female germplasm. Follicle viability in bovine ovarian cortical tissue was partially preserved after vitrification, making it feasible to biobank vitrified tissue from valuable domestic or wild Bovidae, while in vitro tissue culture and/or IVA treatments significantly reduced tissue and follicle viability was unsuccessful. Further investigation of germline preservation in Bovidae is needed before these techniques can be broadly implemented. These ARTs are a toolbox of approaches to conserve biodiversity and valuable genetics through production, use, and preservation of tissue, gametes and embryos.
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    Metabolic support of preimplantation embryo growth and viability
    (Colorado State University. Libraries, 2024) Fresa, Kyle Joseph, author; Carnevale, Elaine, advisor; Chicco, Adam, advisor; Tesfaye, Dawit, committee member; Krisher, Rebecca, committee member; Gentile, Christopher, committee member
    Early embryo metabolism involves essential and dynamic biological reactions that support viability, growth, and pregnancy establishment. Embryo metabolism not only serves to provide energy through ATP synthesis, but also facilitates the production of macromolecules such as proteins, nucleotides, and lipids. The ways in which embryos balance catabolic and anabolic activity during the preimplantation stage are not well understood; however, understanding these processes may lead to improved fertility treatments, embryo culture, and pregnancy outcomes. The studies described in this dissertation utilize innovative methods, such as stable isotope tracer analysis to track carbon and nitrogen flux through various pathways, oxygen microsensors to determine individual embryo respiration under various conditions, and proteomic analysis to determine the impacts of metabolic disturbances on embryo viability. The overarching hypothesis of this dissertation is that embryo viability is dependent on efficient and tightly regulated metabolic activity, and disturbances to metabolic function ultimately lead to reduced developmental potential. To test this hypothesis, a series of projects were conducted to 1) evaluate the importance of phosphoenolpyruvate carboxykinase (PEPCK) during early development, 2) uncover the function of PEPCK to support catabolic and anabolic activity in early embryos, and 3) determine the impacts of delayed embryo development on embryo metabolism and pathway regulation. These projects revealed important insights into the impact of embryo metabolism on development, including the discovery of a novel, PEPCK-mediated pathway that embryos utilize to balance energy production and biosynthesis. Furthermore, the impact of delayed embryo development was demonstrated to alter embryo metabolic activity and pathway regulation, including increased aerobic activity and altered protein expression. These findings improve our understanding of metabolic activity and regulation during preimplantation development, highlighting the impact of metabolic activity to promote ATP production, biosynthesis, developmental kinetics, and ultimately survival. The experimental outcomes presented in this dissertation provide a foundation for targeted approaches to improve embryo development and reproductive success.