Survival of Porcine Delipated Oocytes and Embryos after Cryopreservation by Freezing or Vitrification

Abstract

The present study examined whether delipated porcine oocytes and embryos at various stages of development can be cryopreserved by conventional slow cooling or vitrification. Most (93%) of the 27 delipated morulae developed to blastocysts after freezing with 1.5 M propanediol + 0.1 M sucrose. Late morulae and early blastocysts delipated at 2-4 cell stage and cultured in vitro survived freezing either with 1.5 M glycerol + 0.25 M sucrose (10/18, 56%) or 1.8 M ethylene glycol + 0.25 M sucrose (14/19, 74%). Delipated 2-4 cell stage embryos and oocytes could be cryopreserved by vitrification with 40% ethylene glycol, 1 M sucrose and 20% fetal calf serum. Half (7/14) of the vitrified, delipated embryos developed to blastocysts after thawing. Of 48 delipated oocytes, 27 (56%) maintained an intact outline of the ooplasm after vitrification and underwent subzonal sperm injection. Fertilization was confirmed in 12 (25%) of these oocytes and 3 (6%) developed to morula stage. This study also aimed at developing a non-invasive method for cryopreserving porcine embryos after reducing their cytoplasmic lipid content without micromanipulation. Morulae and early blastocysts were centrifuged in the presence of cytochalasin B and cryoprotectants and then frozen immediately. More than half (14/24, 58%) of the centrifuged morulae developed to blastocycts after freezing with 1.5 M propanediol + 0.1 M sucrose. Greater than 70% of centrifuged early blastocysts survived freezing either with 1.5 M propanediol (30/31, 97%), 1.5 M glycerol (22/29, 76%) or 1.8 M ethylene glycol (21/29, 72%). These results demonstrated that delipation (lipid removal) from porcine oocytes and embryos at various stages enables their cryopreservation. A new insight into the development of a non-invasive method for cryopreserving porcine embryos was also provided.