Microfluidic Selection, Zeta Potential Characterization, and Sperm Transcriptomics for the improvement of Equine Embryo Production
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Microfluidic Selection, Zeta Potential Characterization, and Sperm Transcriptomics for the improvement of Equine Embryo Production


In horses, in vitro embryo production is limited to a technique called Intracytoplasmic Sperm Injection (ICSI), in which a sperm is manually selected and injected into the oocyte. Therefore, it is essential to select a sperm that is functionally intact and capable of both fertilizing an embryo and maximizing developmental potential by relying on measurable parameters indicative of sperm quality. However, sperm quality is often oversimplified to prioritize parameters such as motility and morphology, which do not necessarily account for the true ability of the cell to fertilize an oocyte and produce a viable offspring. Thus, understanding more complex properties of sperm is highly beneficial to maximizing the success of ICSI. In non-equid species, researchers have investigated a novel property called Zeta Potential (ZP). ZP is an estimation of surface charge, thought to originate from charged proteins within the sperm glycocalyx, that has been correlated with basic sperm quality parameters, fertilization, and embryo development in other species including humans. This parameter has not been investigated in stallions and is characterized, for the first time, in this thesis. In addition to understanding sperm quality, it is common to select for a population of more fertile sperm based on sperm quality parameters using established sperm selection techniques. Equine breeding facilities utilize sperm selection techniques to enrich the population of sperm portraying superior fertility parameters, including motility and morphology, but also accounting for characteristics such as cell viability, mitochondrial potential, and DNA integrity. Conventional selection techniques include swim up (SU), density gradient centrifugation (DGC), and DGC-SU combination (DG-SU). A novel technique used in humans that has not yet been studied for stallion sperm selection is the microfluidic chip (MF), which selects sperm based upon their rheotactic motion similar to that seen within the female tract. In this work, we will present a thorough comparison of conventional sperm selection methods to MF. Lastly, the sperm transcriptome has become a focus of fertility research, as sperm RNAs are thought to contribute to regulation of the female environment as well as to early embryo development. However, recovery of sperm RNAs is often low, which makes downstream analysis more difficult. As there is success in sperm RNA extraction in non-equine species, we aim to compare sperm RNA extraction methods for both sperm RNA yield and quality. In this thesis, we will be discussing normal sperm morphology and physiology, as well as conventional and novel selection techniques, we will characterize ZP as a novel parameter of equine sperm fertility, and we will compare RNA extraction methods for optimal yield and quality of sperm specific transcripts.

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