Abstract: This investigation aimed to optimize the in vitro culture system for small cohorts of ovine COCs(5 per group), and enhance in vitro maturation quality and subsequent developmental potential. Experimental variables included culture volume (50 COCs/500 μL,5 COCs/500 μL, 5 COCs/50 μL,) supplemented with exogenous BMP15 and GDF9 proteins (200 ng·mL^-1), and maturation durations tested (18, 21, 24 h). Comprehensive evaluations encompassed cumulus expansion index, HAS2 gene expression profiles, apoptosis-related gene expression (BAX, BCL2, C-Myc) in cumulus-oocyte complexes, oocyte redox homeostasis parameters (GSH, ROS, GPX-1, GSR), and embryonic developmental indexes (cleavage rate, blastocyst rate, mean cell numbers/blastocyst). Addition of the combination of BMP15 and GDF9 into maturation medium significantly enhanced cumulus expansion indexes (P<0.01), upregulating HAS2 expression and improving redox balance through elevated GSH levels and reduced ROS accumulation. The optimized protocol employing 500 μL culture medium with 21 h maturation achieved superior outcomes, yielding cleavage rate of 70.23%±2.78% and blastocyst rate of 35.33%±1.53% at 48 h after in vitro fertilization of oocytes cultured in small group. Notably, the 21-hour maturation group exhibited significantly lower BAX expression and BAX/BCL2 ratios compared to the 24-hour group. Culturing 5 COCs in 500 μL microdrops yielded better results than culturing 5 COCs in 50 μL microdrops. Co-supplementation with BMP15 and GDF9 proteins reduced apoptosis and enhanced intracellular redox capacity. Shortening the in vitro maturation (IVM) duration from 24 h to 21 h reduced apoptosis, promoted OCT4 and CCNB1 gene expression in blastocysts, and increased the average blastocyst cell count.

Key words: ovine, oocyte, small group cultivation