Broiler breeder rooster fertility has been gradually declining as a result of intense selection for bodyweight gain in broiler chickens. The nature of this issue is multifaceted, stemming from genetic, physiologic and behavioral influencers resultant from selection focused on growth rate. The goal of this work was to investigate strategies for determining and improving broiler breeder rooster fertility.First, sperm-associated antigen 6 (SPAG6) was characterized for its utility as a biomarker of rooster sperm mobility, due to its importance in sperm flagellar structure and previously being documented as associated with decreased rooster fertility. SPAG6 was found to be expressed throughout the male reproductive tract and to increase in abundance as sperm mature, localizing mainly in the flagellar axoneme but also in the midpiece and surrounding the acrosomal region of the sperm head. SPAG6 abundance was not found to be a biomarker suitable as a stand-alone predictor of sperm mobility.Next, sperm protamination was investigated as an indicator of rooster sperm quality. In order to assess rooster sperm protamination, a combined method of sperm protamine extraction and heparin-stabilized gold nanoparticle-based quantification of sperm protamination was optimized. Sperm protamination was not found to be a strong indicator of sperm quality orrooster fertility, despite its recent implication in determining fertility of various mammalian species.Last, glyphosate-based herbicide (GBH) residues were studied for their effects as a reproductive toxicant in broiler breeder roosters. Residues GBH ingredients like those found in animal feeds produced from conventionally grown sources were shown to negatively impact rooster testis morphology and sperm mobility. Additionally, humic acids incorporated into rooster feeds remedied the effect of the glyphosate present in the feed. Through adsorption of glyphosate found on feeds, humic acids improved rooster testis morphology and sperm mobility, thereby improving the overall reproductive health of roosters fed conventionally grown feed crops.The findings of factors influencing rooster fertility explored in this work may be applied in future broiler breeding and broiler breeder management programs in order to improve the productive efficiency of the broiler industry.