BRIEF COMMUNICATION QUICK FREEZING OF MOUSE EMBRYOS BY DIRECT PLUNGE INTO LIQUID NITROGEN VAPOR: EFFECTS OF SUGARS

Conventionally, cryopreservation of mammalian embryo has been accomplished by suitable slow cooling in the presence of a cryoprotectant to very low temperatures before storage in liquid nitrogen (LN2). However, it has been demonstrated in mouse,7,13) cattle 1 ,4) and rabbit 10 ) that embryos can be frozen by using a more rapid technique with the two-step freezing method: i. e., rapid cooling to a subzero holding temperature (- 20° to -79°C), followed by rapid cooling into LN2 • Moreover, it was reported recently that mouse embryos can endure quick freezing by being plunged directly into LN z . 5,6,9, 11,12) In these studies, embryos were frozen with glycerol and nonpermeable sugars such as sucrose 5 ,6, 11,12) and trehalose,6) or with the vitrification solution. 9) Up to now, there have been no reports mentioning the effect of other sugars and the viability of embryos upon transfer to the recipient animals after the quick freezing. In the present study, the effects of several types of sugars under various glycerol concentrations on the viability of frozen-thawed embryos were ex­ amined, and normal young were obtained after the transfer of embryos frozen by the quick freezing procedure. Female ddY mice, aged 4 to 5 weeks, were induced to superovulate by intraperi­ toneal injections of 5 IU of pregnant mare's gonadotrophin and 5 IU of human chorionic gonadotrophin (hCG) given 48 hours apart. The mice were mated with males of the same strain after the injection of hCG. Embryos at the morula stage were flushed from the uteri with Dulbecco's phosphate-buffered saline supplemented with 5% heat­ treated calf serum (PBS + CS) at 76 to 78 hours after the injection of hCG, The embryos were washed with fresh PBS + CS before transfer to the freezing medium, and then pipetted into different freezing media: PBS + CS containing 1, 2, 3 or 4 M-glycerol and 0.25 M-sugar (raffinose, lactose, sucrose, glucose or xylose), In the