Porcine blastocyst viability and developmental potential is maintained for 48 h of liquid storage at 25 °C without CO₂ gassing

Short- and medium-term storage of pig embryos has become relevant for commercial application of non-surgical deep uterine embryo transfer (NsDU-ET) in the light of the strict legal and administrative requirements posed by the International Association for Air Transport (IATA) to allow shipment of liquid nitrogen (LN₂) containers and the technical drawbacks when using vitrified embryos. Therefore, this study developed an efficient method for the liquid storage of in vivo-derived porcine blastocysts for a moderate duration (48 h) without controlled CO₂ gassing. We evaluated two storage temperatures (25 °C and 37 °C) and three HEPES-supplemented media: the chemically defined media TL-PVA and NCSU-PVA and the semi-defined medium NCSU-BSA. We observed no differences in survival, hatching rate or final developmental stage between the two temperatures, but storage at 25 °C was more efficient to preserve zona pellucida (ZP) integrity. Blastocysts were successfully stored for 24 h in a chemically defined medium. Yet, only 48 h storage in NCSU-BSA medium supported blastocyst development. Although all storage conditions resulted in an embryonic developmental delay, blastocysts stored in NCSU-BSA at either tested temperature could hatch and attain the same final developmental stage as control blastocysts when cultured under standard conditions after storage. Moreover, blastocysts stored at 25 °C for 48 h in NCSU-BSA medium could produce pregnancies after surgical transfer. In conclusion, porcine blastocysts maintain their viability and developmental potential after storage in the semi-defined medium NCSU-BSA for at least 48 h at 25 °C.