ADVANCING IN VITRO EMBRYO PRODUCTION IN PREPUBERTAL SMALL RUMINANTS: OPTIMIZING CULTURE SYSTEMS TO ACCELERATE GENETIC GAIN AND ENHANCE ENVIRONMENTAL SUSTAINABILITY

In vitro embryo production (IVEP) from prepubertal donors represents a promising strategy to accelerate genetic gain in livestock; however, the reduced developmental competence of prepubertal oocytes remains a major limitation. This is mainly due to intrinsic oocyte immaturity and suboptimal in vitro maturation (IVM) conditions. Therefore, improving culture systems and identifying reliable biomarkers of oocyte quality are crucial to enhance the efficiency of juvenile IVEP programs. Three-dimensional (3D) culture systems, such as the Liquid Marble (LM) microbioreactor, have emerged as innovative approaches to better mimic the physiological microenvironment compared to conventional two-dimensional (2D) systems. In this context, the LM system was evaluated during IVM of prepubertal goat oocytes, showing comparable nuclear maturation and blastocyst rates to standard culture conditions, while improving mitochondrial activity and distribution without detrimental effects on oocyte competence. These findings support the use of LM as a cost-effective and biologically relevant 3D culture system. In parallel, the molecular characterization of cumulus cells (CCs) provides valuable insights into oocyte quality. Transcriptomic analysis of CCs derived from prepubertal and adult sheep oocytes revealed significant differences in gene expression profiles, with over 1000 differentially expressed genes. These genes are involved in key biological processes, including mitochondrial function, cell–cell communication, extracellular matrix organization, and growth factor signalling, highlighting the impact of donor age on CC functionality and identifying potential non-invasive biomarkers of oocyte competence. To further improve oocyte developmental potential, a pre-IVM (PRE-IVM) strategy based on the maintenance of meiotic arrest using C-type natriuretic peptide (CNP) combined with oestradiol (E2) was investigated under both 2D and 3D culture conditions. The combination of CNP/E2-mediated PRE-IVM with the LM system significantly enhanced oocyte quality by improving cumulus–oocyte communication, cytoplasmic maturation, and mitochondrial organization, while reducing apoptotic incidence in resulting blastocysts. These improvements ultimately translated into enhanced embryo quality. In conclusion, the integration of advanced 3D culture systems with physiological PRE-IVM strategies and molecular characterization of cumulus cells represents a powerful approach to improve the developmental competence of prepubertal oocytes. This combined strategy may contribute to increasing the efficiency and sustainability of juvenile IVEP programs in livestock species.