Mammalian embryo quality is often assessed by morphology, the timing of mitotic events, and aneuploidy. These parameters are more commonly investigated in non-equid mammalian species, therefore, the parameters which indicate embryo quality in the horse remains to be fully elucidated. The overall objective of this dissertation was to optimize the in vitro production (IVP) of equine embryos by understanding the consequences of errors that can arise during pre-mitotic and post-mitotic events. This was accomplished in 3 chapters. Chapter 1 is an introduction to the dissertation. Chapter 2: Review of relevant literature provides a review of the literature pertaining to early mammalian embryo development, knowledge gaps, and implications for equine embryo quality assessment. The specific aims of the experiments conducted in Chapter 3: Pronuclear formation and cytoplasmic extrusion and Chapter 4: Aneuploidy in the early cleavage stage equine embryo were to determine the pre- and post-mitotic events in the zygote using fluorescence microscopy, and the exact percentage of aneuploid embryos in the cleavage stage equine embryo. Currently, the general timing of pronuclear (PN) apposition and fusion is unknown, and this information is critical to bridge the gap in understanding the transition from the PN phase to first cleavage. We found that in vitro PN formation is delayed by more than 6 hours compared to previous observations of PN in the in vivo equine model. The sperm head is still intact at 12 hours post intracytoplasmic sperm injection (ICSI). Additionally, cytoplasmic extrusion (CE) is a pre-cleavage event unique to the horse, and previous studies have shown that CE is a critical event in equine embryo development and occurs as it is associated with developmental success. While CE has also been described as a fragmentation event, there was little previous evidence that the extruded material contains DNA. Using fluorescence microscopy, we confirmed that no DNA was present in the extruded CE material and at this moment, the zygote was at the metaphase of mitosis. In Chapter 4, euploid and aneuploid blastomeres were determined by single-cell sequencing for the first time in the horse. To accomplish this, embryos were removed from culture between days 2 and 3 of embryo culture following ICSI. Because embryonic endpoints were unknown, a prediction model using machine learning using time-lapse data from Chapter 3 was used to determine if the embryos would have failed or survived to blastocyst stage and whether the early developmental stages were predictive of aneuploidy. According to the results from the sequencing data and prediction model, the blastocyst prediction model could not distinguish embryos with a higher percentage of euploid blastomeres in the horse. The percentage of aneuploid embryos was alarmingly high (81.25%), which could be explained by the frequent multipolar divisions observed during the early cleavage divisions. The findings from this work inform future research efforts to improve IVP practices in the horse with potential applications for improving embryo outcome predictions, selection methods as well as pregnancy outcomes.

Mammalian sperm quality is often clinically investigated by assessing parameters such as sperm motility and morphology as indicators of sperm health and male fertility. Though commonly assessed, the physiology and pathophysiology underlying sperm quality parameters remains to be fully elucidated. This knowledge gap limits the clinical and scientific knowledge of male fertility across mammalian species. Study of mammalian sperm physiology is complicated by interspecies variation, which has left differing knowledge gaps between species. The interspecies variation also challenges efforts to advance the field of human male fertility, for which more appropriate models than the commonly used rodent models are needed. The overall objective of this dissertation research was to investigate the physiology and pathophysiology of mammalian sperm quality parameters frequently assessed in the clinical setting, specifically morphology and motility. This dissertation includes assessment of bull, stallion and canine sperm to address specific knowledge gaps in sperm physiology and pathophysiology of said species. I aimed to further explore the physiologic significance of sperm quality parameters by investigating their relationship with mitochondrial functionality and oxidative metabolism in multiple mammalian species. This was accomplished in four chapters. Chapter 1, Mammalian sperm quality and oxidative metabolism- a review, provides a review of the literature pertaining to mammalian sperm quality and oxidative metabolism, emphasizing knowledge gaps, disagreements in existing literature, and implications for male fertility. In Chapter 2, Effects from disruption of mitochondrial electron transport chain function on bull sperm motility, I investigate the relationship between mitochondrial function and motility in bull sperm by simultaneously measuring motility parameters and mitochondrial oxygen consumption in the presence of several mitochondrial effector drug treatments. The bull was chosen as the model for this chapter due to conflicting results in the literature regarding the role of mitochondria in fueling bull sperm motility. Motility parameters were only observed to differ significantly from the vehicle with antimycin (inhibitor of complex III of the electron transport chain) treatment, for which significant decreases in numerous parameters, including total motility (p=0.007), progressive motility (p=0.01), and velocity parameters, such as VAP and VSL (p<0.05), were identified. These results indicate that bovine sperm motility is impacted by mitochondrial functionality. In Chapter 3, the novel use of imaging flow cytometry was employed to investigate the relationship between sperm morphology and reactive oxygen species generation in fresh and cool-stored stallion sperm. The stallion was used as the model in this chapter in part due to literature associating the percentage of morphologically abnormal sperm with elevated ROS and decreased fertility, but limited information regarding ROS generation of specific morphologic abnormalities. Compared to morphologically normal sperm, ROS production was significantly higher in cells with abnormal heads, proximal droplets and abnormal midpieces in both fresh and cooled semen. These results indicate that excessive oxidative stress contributes to the pathophysiology of morphologic abnormalities, which may contribute to findings previously reported in the literature linking certain morphologic abnormalities to decreased fertility in stallions. In Chapter 4, we evaluated the repeatability and accuracy of the tablet-based Canine iSperm® instrument compared to computer-assisted sperm analysis (CASA) for assessment of motility (total and progressive) in fresh and frozen-thawed canine sperm. This tablet-based system is available for both stallion and canine sperm, but prior to our study, published validation was only available for stallion sperm. Correlational analysis revealed significant positive correlations between CASA and iSperm® assessment for both fresh and frozen-thawed samples. These results indicate the iSperm® system offers an accurate alternative to CASA for measurement of canine sperm motility. This tablet-based system for motility assessment is substantially more affordable to veterinary practitioners than conventional CASA systems and could allow for more clinical data collection of canine sperm motility, which would be invaluable to future research efforts to expand knowledge of canine sperm physiology. The multispecies work in this dissertation reports findings in sperm quality, physiology and bioenergetics. The results of this work directly contribute to current clinical knowledge by improving the diagnostic power of commonly employed sperm quality measures and by elucidating some of the underlying pathophysiology of sperm morphologic abnormalities and sperm motility. Further, the findings from this dissertation research informs future research efforts, with potential applications in advancement and improvement of mammalian male fertility, sperm preservation techniques, species conservation efforts and mitochondrial physiology.

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.

Not all sires have sperm suitable for chilled or frozen storage, and success in artificial insemination (AI) varies highly among individual dogs and breeds. Fertilizing potential is further complicated as sperm quality declines with the aging process. The overall objective of this work was to study the effects of age and mitochondrial function on sperm quality and bioenergetics using traditional methods assessed in the clinical setting, as well as emerging methods of investigating metabolic flexibility and the heterogenous nature of sperm. This dissertation explores the physiology and pathophysiology of canine sperm through the study of a breeding colony of highly fertile Labrador retrievers, and a rapidly aging purebred population of North American Great Danes. This was accomplished in three chapters. Chapter 1, Comparative Oxidative Metabolism in Mammalian Sperm, provides a review of the literature and current understanding of mitochondrial physiology and metabolic control of essential sperm functions in mammalian species of veterinary importance. We discuss the differences in fundamental oxygen and ATP substrate balance in stallions, bulls, and dogs, with an emphasis on the maintenance of sperm motility, electron transport chain (ETC) function, reactive oxygen species (ROS) production, and the balance of glycolytic and oxidative phosphorylation production of ATP in sperm. In Chapter 2, Sperm Parameters in the Great Dane: Influence of Age on Semen Quality, I characterized the distribution of sperm quality parameters within a purebred population of actively showing North American Great Danes, a rapidly aging breed currently suffering declining fertility. I identified progressive declines in sperm quality that accompany aging, detailed the relationship between ROS production and sperm motility and morphology, and distinguished sources of variation in sperm quality within the breed. Age was negatively associated with several sperm motility parameters associated with sperm fertility such as total and progressive motility, and amplitude of lateral head displacement (p<.05), with a predicted -9.9%, -9.0%, and +8.3% change per year of age. Sperm of younger GD dogs aged 12 ≤ x <24 months had significantly higher total and progressive motility (TM, PM), amplitude of lateral head displacement (ALH), and nonlinear motility (p<.05) than older dogs (x ≥ 48 months). Results support anecdotal reports of decline of the fertility with the advance of age in this breed, and indicate that age and ROS have significant influences on sperm parameters in the GD. The influence of selection for breed specific phenotypes could help explain the functional significance of the diversity among GD males. In Chapter 3, Effects of Substrate Availability and Mitochondrial Disruption on Oxidative Metabolism and Sperm Motility in Fertile Dogs, I investigated the adaptability of canine sperm bioenergetics under differing nutrient conditions and the effects of mitochondrial dysregulation on sperm motility. With simultaneous measurement of mitochondrial oxygen consumption (MITOX) and sperm kinematics, I observed a high adaptability and metabolic flexibility of high-quality canine sperm. While all energetic substrates tested successfully maintained sperm motility, significant differences were found in MITOX and mitochondrial spare respiratory capacity (SRC), the strength with which mitochondrial energy production may rise to meet energetic challenge. In addition, the ability to maintain sperm kinematics when supplemented with various energetic substrates differed significantly with mitochondrial disruption by mitochondrial effectors. A population of Labrador retrievers from a highly fertile breeding colony managed by Guide Dogs for the Blind was chosen for this study due to their high semen quality, good health, and known fertility.

Assisted reproductive technologies (ART) like in vitro production (IVP) of embryos in the horse is an important tool in the equine industry. In particular, the aspiration of antral follicles and the collection of immature oocytes is the most common and widely practiced procedure to obtain female gametes for IVP. However, in vitro maturation (IVM) of these immature oocytes remains a significant limitation for efficient IVP. This is mainly due to suboptimal maturation media formulations and overall suboptimal maturation culture conditions. Maturation of the oocyte is a critical and delicate phase where the oocyte acquires its capacity to be fertilized by one spermatozoon and then undergoes embryogenesis. In Chapter 1, a review of the pertinent literature regarding oocyte maturation and associated processes is presented.In the studies presented in Chapter 2 of this dissertation, it is described for the first time, that the gene expression profile of oocytes and surrounding cumulus cells (CC) and the associated changes that occur during maturation. Oocytes expressed 13,918 genes, with 68.46% being protein-coding genes. 749 differentially expressed genes (DEG) were observed between the immature (GV) and Mature (MII) groups. Within these DEG, 330 presented a higher expression in the OC-MII group and were associated with pathways of the cell cycle and chromosome segregation. 419 genes showed a lower expression in the OC-MII group and were associated with ribonucleoprotein complex biogenesis and nucleolus. In CC samples, it was established 927 DEG with 609 upregulated and 318 downregulated genes. It was determined that many pathways related to GTPase activity were part of the upregulated genes. Downregulated genes were mainly related to the extracellular matrix organization, collagen trimer, and collagen fibril. A weighted gene co-expression network analysis (WGCNA) of maturation revealed that important pathways are associated with nuclear maturation and the meiotic cycle. Moreover, the expression of the follicle-stimulating hormone receptor (FSHR) increased while the luteinizing hormone receptor (LHCGR) decreased during maturation. The studies presented in Chapter 3 of this dissertation were designed to compare IVM to in vivo maturation (IVV) by evaluating the gene expression profile of the CC. There were over six thousand DEG between the two groups with 2180 genes upregulated and 4481 downregulated in the IVM group. Genes associated with extracellular matrix and cumulus expansion were downregulated in the IVM group. Also, the expression of FSHR was higher, while LHCGR expression was lower in the IVM compared with the IVV group. Additionally, cumulus expansion during IVM exhibited an increasing linear rate of expansion. The results from Chapters 2 and 3 combined suggested that gonadotropins (FSH and LH) might have an important role during oocyte maturation in the horse, thus a re-evaluation of the maturation media formulations, like the concentration of FSH used in the formulation, could result in the improvement of maturation outcomes. Additionally, the reduced activation of genes and pathways associated with cumulus expansion is another important factor that should be addressed and investigated in future studies.