DEVELOPMENT OF NOVEL BIOTECHNOLOGICAL APPROACHES FOR ACCELERATING GENETIC GAIN IN THE BOVINE

Modern livestock production is increasingly challenged to improve reproductive efficiency while maintaining sustainability and animal welfare standards. Accelerating genetic gain in cattle (Bos taurus) is essential for breeding animals that are more resilient to climate change, more resistant to disease, and capable of higher meat and milk productivity—all while minimizing their environmental footprint. One possible strategy to achieve this goal is to significantly shorten the generation interval by producing gametes directly from embryos. Current evidence indicates that a fully developed offspring can be obtained by injecting a spermatid into a fully matured oocyte. In contrast, fertilization cannot be achieved by injecting a mature sperm into an immature oocyte. Based on this, the focus of this work was placed on the generation of male haploid cells in bovine species. However, in vitro spermatogenesis remains a major challenge in large mammals and has, to date, been successfully accomplished only in rodents. To address this limitation, a stepwise approach was adopted to develop targeted techniques for some of the phases of the process required to recapitulate the differentiation of embryonic cells into the spermatogonial lineage and to subsequently support their progression through complete meiosis. The initial experiments aimed to establish a protocol for isolating immature bovine spermatids and evaluating their fertilization potential using micromanipulation techniques. This was followed by the optimization of a decellularization protocol to create a testicular biological scaffold that mimics the native tissue microenvironment, preserving its structural, biomechanical, and biochemical properties. This scaffold was designed to support in vitro meiosis. The final set of experiments focused on isolating and characterizing key functional testicular cell types— Sertoli cells and spermatogonia—which are essential for meiotic progression and intended to repopulate the scaffold. The results demonstrated successful isolation of bovine spermatids, with significant enrichment of haploid subpopulations. These cells exhibited high haploidy rates, expressed spermatid-specific transcripts (PRM1, PRM2, SPACA9, SPERT), and showed cytoplasmic localization of SPERT protein. DNA integrity was maintained after 24 hours at both 4°C and 37°C, although mitochondrial activity and reactive oxygen species (ROS) levels increased over time. Despite these promising features, spermatids showed limited fertilization capacity following intracytoplasmic injection into in vitro matured oocytes. The second phase of the study established an efficient decellularization protocol for bovine testicular tissue, achieving thorough removal of cellular components while preserving the extracellular matrix architecture. A 12-hour exposure to SDS provided the optimal balance between decellularization and matrix integrity. These scaffolds supported fibroblast repopulation, confirming their biocompatibility. In the final part of the thesis, fibroblasts, Sertoli cells, and spermatogonia were successfully isolated and characterised using specific markers—vimentin, SOX9, and PGP9.5, respectively. These cell populations represent the essential building blocks for developing a functional 3D in vitro model of the testicular microenvironment, which may support spermatogonial differentiation and, potentially, completion of the meiotic process. Overall, this work established protocols for a few key steps toward the ultimate goal of generating male haploid cells suitable for injection into mature oocytes. Although the task confirmed to be highly complex, the gradual and systematic approach adopted in this study contributes to laying the groundwork for future advancements in the field.