Eventual re-vitrification or storage in liquid nitrogen vapor does not jeopardize the practical handling and transport of vitrified pig embryos

Alicia Nohalez, Cristina A Martinez, Inmaculada Parrilla, Carolina Maside, Jordi Roca, María A Gil, Heriberto Rodriguez-Martinez, Emilio A Martinez, Cristina Cuello

This study aimed (1) to evaluate the in vitro post-warming survival of porcine embryos after re-vitrification and (2) to assess the efficacy of transport of embryos in dry shipper (DS) in maintaining the viability and quality of vitrified embryos for a 3-day period. Embryos at the compacted or cavitating morula (CCM) and unhatched blastocyst (UBL) stages were surgically obtained from weaned, crossbred sows. In the first experiment, more than 85% of the embryos survived an initial vitrification and warming and achieved comparable survival rates to those of their fresh counterparts. In contrast, those embryos subjected to a second vitrification and warming had clearly lower survival rates (60% and 64% for re-vitrified embryos from the CCM and UBL groups, respectively) compared to the survival rates of the initial vitrification and fresh control groups (P < 0.01). Hatching rates were similar in re-vitrified blastocysts derived from vitrified CCMs and fresh control groups (50.8% and 55.3%, respectively). However, differences (P < 0.01) in hatching rates were recorded in re-vitrified blastocysts derived from vitrified UBLs and fresh control blastocysts (14.7% and 90.0%, respectively). In the second experiment, vitrified embryos were stored in a liquid nitrogen tank for one month. Then, the straws containing the embryos were transferred to a DS (DS group) or to another liquid nitrogen tank (control group) for an additional three days. Embryos from the DS and control groups had similar survival and hatching rates, regardless of the embryonic stage considered. The DS storage of CCMs and UBLs did not affect their development after culturing, including total cell numbers, compared to the control, although their apoptotic index was slightly higher (P < 0.05), regardless of the developmental stage. In conclusion, although re-vitrification negatively affects embryo survival, this study demonstrated that >60% of vitrified embryos could be successfully re-vitrified and re-warmed. The present study also showed the effectiveness of the DS for the storage of vitrified porcine CCMs and UBLs for at least three 3 days.