Received: Nov 18, 2025 / Accepted: Apr 22, 2026 / Published: Jun 30, 2026
Hypothermic storage at 4°C offers a practical and low-cost approach for short-term handling of bovine in vitro-produced (IVP) embryos; however, optimal buffering conditions and storage limits for preserving embryo viability remain insufficiently defined. This study evaluated how varying HEPES concentrations and storage durations influence post-chilling survival and developmental competence of Day 7 IVP blastocysts. Across treatments, TCM199 supplemented with 50% FBS supported the highest embryo recovery, and the response to HEPES showed a clear concentration-dependent pattern. Moderate buffering markedly improved embryo stability, with embryos stored in 25-mM HEPES exhibiting the highest post-storage survival and hatching rates (approximately 63% and 37%, respectively). In contrast, insufficient (0 mM) or excessive supplementation (≥ 50mM) resulted in substantially reduced developmental performance, confirming that an intermediate buffering range provides the greatest protection during chilling. Storage duration further shaped embryo outcomes. Embryos maintained relatively high viability between 24-48h of hypothermic exposure (survival ~54-62%), whereas prolonged storage led to a sharp decline in developmental potential, with hatching becoming rare or absent beyond 72h. The total cell number also decreased markedly after extended storage, indicating progressive structural compromise. These findings identify an optimal HEPES range and a practical storage window that together enhance the success of hypothermic preservation of bovine IVP embryos. The study establishes biologically validated parameters that can support the development of reliable, low-cost protocols for embryo storage and transport in research and field applications.
