121VITRIFICATION OF MOUSE MORULAE BY A NEW METHOD: PULLULAN FILM-STRAW VITRIFICATION

Abstract

Recent technical improvements have resulted in higher cryosurvival of oocytes and embryos of various species. However, almost all methods require thawing and washing the embryos under microscopic observation and, therefore, cannot be conveniently used for large animal ET in the field. The purpose of the present work was to develop a new embryo cryopreservation method using a water-soluble film made of pullulan (Hayashibara, Okayama, Japan) that might, in the future, be readily adaptable to field conditions. Morula-stage mouse embryos were collected from superovulated ICR donors 72h after hCG injection. Embryos were first exposed to 10% DMSO+10% ethylene glycol (EG) in DPBS+20% FCS (mPBS) for 2min, and then equilibrated for 30s in a vitrification solution composed of 20% DMSO+20% EG+0.6M sucrose in mPBS. In the pullulan film-straw vitrification method, the embryos were loaded onto the pullulan film (20μm thick, 5mm long and 1mm wide) and were directly plunged into LN2. The pullulan film was inserted into a pre-frozen 0.25mL plastic straw (<−150°C) containing 0.15mL mPBS and sealed with a plastic screw cap. For thawing, the medium in the straw was rapidly warmed in 37°C water while the pullulan film remained frozen by placing the top of the straw in contact with a cold iron block (<−150°C). As soon as the medium thawed, the pullulan film was immersed in the medium by a rapid downward swinging of the straw. Five min later, embryos were recovered from the straw and washed for 2min in mPBS, for 2min in 0.1% BSA-PBS and for 2min in KSOM sequentially, and then cultured at 37°C in 5% CO2 for 38h. Noncryopreserved embryos and embryos cryopreserved by the cryoloop method (Lane et al., 1999 Nat. Biotech. 17, 1234) served as controls. Data were analyzed by χ2 test and Student’s t-test. Results are shown in Table 1. There are no significant differences (P>0.05) in either developmental abilities or cell numbers between vitrified and non-vitrified embryos. This study demonstrates that mouse morulae can be successfully vitrified and thawed by the PFSV method. This method may eventually be applied to bovine ET under field conditions. Table 1 Development of mouse morulae in culture following vitrification by the PFSV method