Effect of dimethyl sulfoxide and protein concentration on the viability of two-cell mouse embryos frozen with a rapid freezing technique.
Two-cell mouse embryos were frozen by direct plunging into liquid nitrogen after a 3-min exposure to solutions containing 0.25 M sucrose with 1.5, 3, or 4.5 M dimethyl sulfoxide (Me2SO), and 0, 4, 8, 16, or 32 mg/ml bovine serum albumin (BSA). In the absence of BSA, significantly more embryos were lost or damaged during freezing and thawing. Increasing the BSA concentration from 4 to 32 mg/ml had no significant effect on subsequent embryo viability in vivo or in vitro. Blastocyst formation in vitro was greater than 90% in embryos exposed to the cryoprotective solutions only. Although development to blastocysts was not significantly different to nonfrozen controls in most groups frozen in 3 and 4.5 M Me2SO (up to 92% blastocysts), it was significantly reduced when embryos were frozen in 1.5 M Me2SO (up to 65% blastocysts). The development to fetuses of embryos frozen in 3 M Me2SO (64 to 74% fetuses) was not significantly different from nonfrozen controls (68 to 79% fetuses) or embryos frozen by a conventional slow cooling method (70%). Frozen thawed two-cell embryos developed into normal adults which were able to reproduce normally. We conclude that this freezing method can efficiently cryopreserve early cleavage stage mouse embryos.
Shaw
J M
JM
Centre for Early Human Development, Monash Medical Centre, Monash University, Clayton, Victoria, Australia.
Trounson
A O
AO
D000818:Animals; D001755:Blastocyst / Q000187:drug effects / Q000502:physiology*; D000013:Congenital Abnormalities; D015925:Cryopreservation; D004121:Dimethyl Sulfoxide / Q000008:administration & dosage / Q000494:pharmacology*; D004624:Embryo Transfer; D005314:Embryonic and Fetal Development; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D012710:Serum Albumin, Bovine / Q000008:administration & dosage / Q000494:pharmacology*; D014021:Tissue Preservation
D012710:Serum Albumin, Bovine; D004121:Dimethyl Sulfoxide