Embryo cryopreservation

Key words Genetically modified mice, inbred mice, sperm cryopreservation, embryo cryopreservation, IVF, in vitro fertilization, genetic drift, genetic contamination. 

Wada, 1., Macnamee, M.C., Wick, K., Bradfield, J.M. and Brinsden, P.R. (1994). Birth characteristics and perinatal outcome of babies conceived from cryopreserved embryos. Hum. Reprod. 9 543-546. 

Reduced survival of cryopreserved embryos, due to cellular damage caused by diffusion of ionic components Tg ... 

Shibahara H, Mitsuo M, Fujimoto K et al (1999) Prenatal sonographic diagnosis of a fetal renal mesoblastic nephroma occurring after transfer of a cryopreserved embryo. Hum Reprod 14 1324-1327 Skari H, Bjornland K, Bjornstad Ostensen A et al (1998) Consequences of prenatal ultrasound diagnosis a preliminary report on neonates with congenital malformations. Acta Obstet Gynecol Scand 77 635-642 Spitzer A (1996) The current approach to the assessment of fetal renal function fact or fiction. Pediatr Nephrol 10 230-235... 

Rail, W.F. (1987). Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiol. 24, 387-402. 

Steponkus, P.L., Caldwell, S. (1993). An optimized procedure for the cryopreservation of Drosophila melanogaster embryos. Cryo-Letters 14, 375-380. 

Trounson, A. Mohr, L. (1983). Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 305, 707-710. 

Pienta RJ, Poiley JA, Lebherz WB III. 1977. Morphological transformation of early-passage golden Syrian hamster embryo cells derived from cryopreserved primary cultures as a reliable in vitro bioassay for identifying diverse carcinogens. Int J Cancer 19 642-655. 

Pienta, R.J., Lebherz, W.B., III Schuman, R.F. (1981) The use of cryopreserved Syrian hamster embryo cells in a transformation test for detecting chemical carcinogens. In Stich, H.F. San. R.H.C., eds, Short-Term Tests for Chemical Carcinogens, Berlin, Springer-Verlag, pp. 323-337... 

Thus upon comparing the economics of the two methods, embryos and sperm, it is very apparent that the freezing of embryos is considerably more expensive due to the need for more resources for example, with a C57BL/6 background, to produce 250 two-cell embryos for cryopreservation by IVF requires >15 females. If strains are never recovered or only recovered once or twice, then the bulk of this expense remains forever frozen. In contrast, cryopreserving sperm has a low initial cost as only few animals (1-3 males) and relatively little labor and materials are required (12). It is upon recovery from sperm by in vitro fertilization (IVF) that animals and labor are used, but then only the required number animals per recovery are used as the IVF process can be scaled to produce the desired number of offspring. 

Two cell embryo cryopreservation (heterozygotes embryos generated via IVF) Moderately simple Needs only 1-3 carrier males Only half the genome is preserved Needs IVF to make embryos Needs female (wild type) oocyte donors. Strain dependent 5-50 oocytes/female Only half the genome is persevered... 

Two to eight cell embryo cryopreservation (flushed homozygous embryos) Simple Provides homozygous storage Needs a large colony of strain to be cryopre-served (expense) to provide embryos Simple to implant Can be used to achieve rederivation of strain Expensive resource to restock Not scalable ... 

Embryos can now be cultured, transferred to a pseudopregnant animal, or cryopreserved (see Notes 14 and 15). 

T6. Toner, J. P., Veeck, L. L., and Muasher, S. J., Basal follicle-stimulating hormone level and age affect the chance for and outcome of pre-embryo cryopreservation. Fertil. Steril. 59, 664-667 (1993). 

This procedure is used for cryopreservation of 8-32-cell morula stage embryos (14). Mouse cleavage-stage embryos can be harvested from the oviducts of 2-3-d pc pregnant mothers or generated by culturing fertilized one-celled embryos in Ml6 medium for 2-3 d. 

Rat cryopreservation is less well researched. This protocol describes a method for freezing one-cell rat embryos (15). 

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