Fundamental cryobiology of early mouse embryos and rat zygotes

Embryo cryopreservation has become a widely applied and efficient technology. Various protocols and methods for the cryopreservation of embryos of several species have been published during the last two and a half decades. Successful cryopreservation is still limited to a few species, and the survival rates often differ among developmental embryonic stages. Virtually all the currently existing protocols have been developed from numerous empirical experiments. The objectives of the studies in this thesis were to analyze the cell membrane permeability of mouse oocytes, zygotes, and 2-cell, 4-cell 8-cell cleavage stage embryos to water (Lp), cryoprotectant ( PCPA), and the reflection coefficient sigma (σ), and to analyze the membrane permeability of rat zygotes for three strains and CPAs in regard to Lp, PCPA, σ, and activation energies (Ea) for L p, PCPA, and σ, followed by a cryopreservation and transfer experiment of rat zygotes using the evaluated CPAs and two different plunging temperatures. The mouse study demonstrated that data can be obtained from cleavage stage embryos, but there are significant differences (p \u3c 0.05) between measurement methods depending on whether they are taken on the entire embryo level or individual cells. Measurements the entire cleavage stage embryos lead to elevated Lp and lowered PDMSO values. Oocytes up to the 4-cell stage had similar permeability estimates, but 8-cell stage embryos had significantly lower PDMSO and significantly higher Lp mean values. No differences were detected between rat strains used in this study. The Lp, PCPA, σ, and Ea mean values were similar between rat strains and CPAs used in this study. Fetal development at days 16–18 did not support the hypothesis that the different CPAs would afford similar protection during cryopreservation. In vivo development of cryopreserved zygotes after transfer into pseudo-pregnant recipient rats ranged from 8–61%. The highest fetal developmental rates, were obtained with EG and plunging at −30°C, and statistically not different (p \u3e 0.05) from the control (non-frozen) results (∼61% vs. ∼55%). The fetal development was significantly higher (p \u3c 0.05) for zygotes plunged at −30°C than at −80°C, and different (p \u3c 0.05) between the three CPAs despite similar permeability estimates