Effects of Opti-MEM®-based maturation and stage-specific glucose supplementation on ovine embryo development in vitro

In vitro embryo production (IVEP) in sheep remains constrained by suboptimal in vitro maturation (IVM) and culture conditions. This study evaluated Opti-MEM® as an alternative IVM medium for ovine oocytes and investigated the effects of stage-specific glucose supplementation on embryonic development and blastocyst quality. Cumulus–oocyte complexes (COCs) recovered from slaughterhouse-derived ovaries were matured for 24 h in either TCM-199 or Opti-MEM®, both supplemented with hormones and serum. Following in vitro fertilization, embryos were cultured in a two-step synthetic oviduct fluid medium supplemented with amino acids (SOFaa) under two glucose strategies: glucose-free medium transitioning to 1.5 mM (G0→G1.5), or 1.5 mM transitioning to 3.0 mM glucose (G1.5→G3.0). Development was monitored through Day 8, and blastocyst quality was assessed by differential staining of inner cell mass (ICM) and trophectoderm (TE) cells. Nuclear maturation rates were comparable between groups, with metaphase II rates of 74.62% and 66.66% in TCM-199 and Opti-MEM®, respectively (p>0.05). Embryo development was significantly influenced by both IVM medium and glucose supplementation strategy. The G1.5→G3.0 protocol yielded superior blastocyst formation and quality, with the highest blastocyst rate and ICM cell count recorded in the TCM-199 group (p