Fertility preservation in prepubertal boys undergoing gonadotoxic cancer treatments remains a major clinical challenge, as they cannot produce mature sperm for standard cryopreservation. Testicular organoids offer a promising in vitro platform to support germ cell maintenance and differentiation from immature testicular tissue, with the long-term goal of restoring fertility. My DPhil research investigates the generation, characterization, and potential applications of long-term bovine testicular organoids derived from prepubertal tissue as a physiologically relevant model. Organoids were cultured for up to 56 days and analyzed at multiple time points (days 0, 7, 14, 28, and 56) using viability assays, morphometric analysis, and RT-qPCR to track dynamic changes in gene expression across key testicular cell types. To understand how external factors influence organoid development, various culture conditions were tested, including the addition of growth factors and exposure to clinically relevant gonadotoxic agents. Single-cell RNA sequencing was employed to dissect the transcriptional landscape and cellular heterogeneity of the organoids at different time points, providing high-resolution insights into somatic and germ cell populations and their developmental trajectories in vitro. Together, this research provides foundational data on the stability, plasticity, and functional potential of bovine testicular organoids over long-term culture.