121 VITRIFICATION OF MOUSE MORULAE BY A NEW METHOD: PULLULAN FILM-STRAW VITRIFICATION
Y. Takagi A , M. Shimizu A , T. Kato A , A. Danguri A and M. Sakamoto AFaculty of Agriculture, Shinshu University, Kamiina, Japan. email: ytakagi@gipmc.shinshu-u.ac.jp
Reproduction, Fertility and Development 16(2) 183-183 https://doi.org/10.1071/RDv16n1Ab121
Submitted: 1 August 2003 Accepted: 1 October 2003 Published: 2 January 2004
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 72 h after hCG injection. Embryos were first exposed to 10% DMSO + 10% ethylene glycol (EG) in DPBS + 20% FCS (mPBS) for 2 min, and then equilibrated for 30 s in a vitrification solution composed of 20% DMSO + 20% EG + 0.6 M sucrose in mPBS. In the pullulan film-straw vitrification method, the embryos were loaded onto the pullulan film (20 μm thick, 5 mm long and 1 mm wide) and were directly plunged into LN2. The pullulan film was inserted into a pre-frozen 0.25 mL plastic straw (<−150°C) containing 0.15 mL 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 2 min in mPBS, for 2 min in 0.1% BSA-PBS and for 2 min in KSOM sequentially, and then cultured at 37°C in 5% CO2 for 38 h. 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.
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