Investigating sire fertility : the relationship between spermatozoa characteristics and early embryonic development

[EMBARGOED UNTIL 12/1/2023] Early embryonic mortality in cattle occurs between days 1-27 of gestation and is one of the primary contributors to economic failure in the dairy industry. Specifically, by day 7 of gestation, up to 50 percent of all embryo mortality will have already occurred. Many critical events that determine an embryo's viability occur within the first week of development. Interestingly, it is during this time that the sire exhibits its greatest effects on pregnancy success. Commercial dairy sires differ in their ability to produce viable blastocysts, yet our understanding of the malfunction of cellular mechanisms regulated by sire during early embryo development is still lacking. This study aimed to elucidate novel biomarkers of fertility status in spermatozoa and how those differences impact mechanisms in a subsequent embryo, with a focus on cellular stress. The elucidation of which spermatozoa characteristics impact early embryonic development has the potential to reduce the use of subfertile bulls and the incidence of early embryo mortality in the dairy cattle industry. Using modern techniques such as flow cytometry, thousands of sperm cells per sample can be accurately analyzed for a feature of interest in just minutes, which significantly increases the sample size used and decreases variability that is seen in manual, subjective evaluation under a microscope. In this study, up to four high performing sires and four low performing sires with varying SCR values were selected for experiments based on previous classification as having high or low capacity to produce embryos in vitro. Fertilization, cleavage, and blastocyst rates were all recorded for embryos produced by either high or low performing sires. Next, cellular stress mechanisms in early embryos were investigated--autophagic activity and reactive oxygen species (ROS) production were measured at multiple stages of embryo development (2-6 cell, 8-16 cell, morula, and blastocyst) in embryos produced by high or low performing sires under normal culture conditions, via fluorescent microscopy. Autophagy and ROS production were also measured in 2-6 cell embryos under induced stress conditions in the form of a heat shock. Additionally, DNA damage and lipid oxidation were assessed in 2-6 cell embryos via fluorescent microscopy. Embryos produced by high performing sires had significantly higher cleavage and blastocyst rates compared to embryos produced by low performing sires, yet there were no differences in fertilization rates. Embryos at the 2-6 cell stage of development produced by low performing sires exhibited an increase in both ROS production and autophagic activity compared to embryos produced by high performing sires--indicating that these embryos begin development under increased cellular stress. The 2-6 cell embryos produced by low performing sires, that were exposed to a heat shock, continued to exhibit increased ROS production. There was no difference observed in the DNA damage or lipid oxidation of 2-6 cell embryos. Next, biomarkers of sire fertility status were investigated in the spermatozoa. DNA damage was evaluated in spermatozoa both prior to gradient purification and following. Additionally, morphological assessments were performed both prior to, and following gradient purification, utilizing immunocytochemistry and epifluorescent microscopy to visualize the sperm head acrosome and nucleus, and the tail midpiece and principal piece. Finally, image-based flow cytometry was performed to quantify the presence of defective aggregated proteins that were initially identified in the spermatozoa with altered morphology, capacitation status, and membrane integrity. In samples prior to gradient purification, or pre-gradient samples, there was an increased incidence of DNA damage in the low performing sires. Alternatively, in samples following gradient purification, or post-gradient samples, the high performing sires had increased DNA damage. Pre-gradient samples from low performing sires had increased aggregated protein content, termed aggresomes, located in the head of spermatozoa compared to high performing sires. Yet, high performing sires had increased tail defects. Using image-based flow cytometry, there were no differences found in aggresome defects of pre-gradient samples from high and low performing sires. Although, in post-gradient samples, spermatozoa from low performing sires had an increased incidence of aggresomes located in the head compared to high performing sires. Finally, post-gradient samples from low performing sires had a greater percentage of spermatozoa that were undergoing premature capacitation and had accrued membrane damage. Currently, we have demonstrated that sire variability can impact cellular stress mechanisms and developmental competence to the blastocyst stage in early embryos, independently of SCR. Embryos at the 2-6 cell stage of development, produced by low performing sires, exhibit increased cellular stress in the form of ROS production and autophagic activity, and go on to have decreased developmental rates compared to embryos produced by high performing sires. Spermatozoa from low performing sires that have undergone gradient purification have increased aggresome defects in their heads. This indicates that the aggresomes may be incorporated into the fertilized oocyte--overwhelming protein degradation machinery in the zygote, such as autophagic machinery, leading to higher levels of cellular stress.