LaVelle, Gerica Anne Helton, authorBarfield, Jennifer, advisorTesfaye, Dawit, advisorCairo, Betsy, committee memberPinedo, Pablo, committee member2023-06-012023-06-012023https://hdl.handle.net/10217/236667The assisted reproductive technologies (ART) area of research within bovine encompasses a diverse group of technologies including artificial insemination, cryopreservation of gametes and embryos, in vitro fertilization, induction of multiple ovulations, sex determination of sperm and embryos, and more. These techniques have been studied thoroughly within the last forty years, yet there is a plethora of knowledge to still be discovered on a molecular basis extending to longitudinal effects of ART interference generationally. One large sector of ART research in the bovine industry is to gain information that will aide in the advancement of current protocols and practices to increase productivity and cost effectiveness. This includes improving cryopreservation of gametes, creating higher in vitro embryo production yield, reducing costs, and developing easier practices for technicians with hopes of providing a more efficient system, from both a labor and fiscal standpoint. The following dissertation contributes specifically to this research sector by providing novel information regarding cryopreservation and capacitation timing of bovine spermatozoa treated with cholesterol prior to cryopreservation, embryo production rates when such spermatozoa are used within an in vitro fertilization (IVF) system, and the use and metabolomic components of spent media from bovine embryo culture. The first chapter of this dissertation will provide background information on the complex sperm capacitation and acrosome reaction process, and methods on how to assess and modify the capacitation process via molecular processes, both pre- and post-cryopreservation. This lays the foundation for the research presented here, which focuses on the loading of bovine spermatozoa with cholesterol prior to cryopreservation, and what effect this treatment has on the cells post-thaw in relation to capacitation timing analyzed using flow cytometry within the second chapter. The first chapter also provides background to the fertilization process and in vitro production of embryos which is examined by using cholesterol-loaded spermatozoa for IVF presented in the third chapter. Additionally, contextual information is explained regarding the type of culture systems within IVF, where embryos are individually cultured or in groups. This information is required for the fourth chapter which includes experimentation with spent culture media and the study of the metabolomic components of media exposed to group embryo culture. Pre-loading bovine sperm with cholesterol prior to freezing is known to increase cryosurvival, though the timing of capacitation in these sperm has not yet been evaluated. The objective of the study performed in chapter two was to determine if there is a potential delay in capacitation timing in these sperm due to the increased cholesterol content. Flow cytometric evaluation was utilized to assess viability, and stain technology to assess acrosome intactness (Propidium Iodide/FITC-PNA), intracellular calcium levels (Propidium Iodide/FLUO 3-AM) and membrane fluidity (Merocyanine 540/YO-PRO-1). Through this investigation it was determined that treatment of sperm with cholesterol-loaded cyclodextrin (CLC) (2 mg/mL) increased cryosurvival and viability over time, though longer time to capacitate was required compared with non-treated sperm. Treatment with CLC also significantly decreased membrane fluidity in sperm (P<.05). by 1 hour, resulting in increased intracellular calcium and increased acrosome reaction, and consequently viability loss by 3 hours. Taken together, these results determined sperm pre-loaded with cholesterol display significantly improved post-thaw viability but require longer to capacitate which may hinder fertilization capacity and/or require adjustments to timing of in vitro fertilization. Given the delayed capacitation timing of sperm treated with CLC, it was then investigated if CLC-treated sperm could be successful in IVF to determine if the additional retained cholesterol hindered fertilization ability and consequently, embryo production. The research summarized in chapter three examines the fertilization ability of CLC-treated sperm and subsequent in vitro embryo development by offsetting sperm preparation timing in order to adjust for delayed capacitation due to the increased cholesterol content of CLC-treated sperm. Additionally, treatment with methyl-β-cyclodextrin (MβCD) was utilized as a capacitation inducing agent by causing an efflux of cholesterol from the sperm plasma membranes during the sperm preparation incubation period. Bovine sperm pre-loaded with cholesterol via CLC prior to cryopreservation did not result in significant difference in cleavage or blastocyst formation (P>0.05) when sperm preparations were completed 90 minutes prior to co-incubation to adjust for delayed capacitation timing. There was significant decline (P<0.05) in embryo cleavage percentage in both control and CLC-treated sperm groups when MβCD was used as a capacitation inducer at 2 mg/ml. The findings presented in chapter three demonstrate that bovine sperm treated with CLC resulted in a nonsignificant difference in embryo production when used in IVF, even when delayed capacitation timing is adjusted for in sperm preparation. This concludes that the fertilization ability of the spermatozoa treated with CLC was not impaired, and that the most important application of CLC-treated sperm is not within in vitro fertilization examination, but rather, within in vivo models utilizing AI synchronization timing adjusted for delayed capacitation. Here the benefit of improved cryosurvival rates can be best utilized, potentially resulting in the ultimate goal of increased fertility. One of the most widely pursued research avenues to improve embryo development is advancements in culture media and conditions. Many attempts have been made to optimize in vitro production of embryos by component supplementation and recipe adjustment. An additional culture condition with a dramatic impact on bovine preimplantation embryogenesis is whether embryos are cultured individually or in groups, where increased embryo production is observed in group culture conditions. These items together, have created an active area of research which analyzes culture media after exposure to embryo culture to examine the artifacts secreted by embryos including various amino acids, proteins, and other metabolites that are considered to be embryotrophic, or positive signals from companion embryos. Chapter four experiments include culturing embryos both individually and in groups with spent culture media from a group culture donor cycle with embryo production analyzed via cleavage and blastocyst formation with time-lapse imaging. Similar cleavage percentages between control and spent media were observed, however, an increase in blastocyst formation was observed in grouped embryos compared with single embryos. No difference (P>.05) was observed in the timing of developmental events in embryos cultured in control or spent media within individual culture. Analysis of the metabolomic profile of spent culture media with gas chromatography-mass spectrometry (GC-MS) was also completed, suggesting potential embryotrophic factors in the spent media. These findings conclude that bovine embryos progress in a more timely and developmentally differentiated degree when cultured within groups, and artifacts left within the spent culture media were not sufficient to result in significant increase in embryo production within spent media culture.born digitaldoctoral dissertationsengCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.bovinein vitro fertilizationandrologyspermembryologyText