Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Biological Sciences


Wheeler, Alfred Hap" P"

Committee Member

Boone , William R

Committee Member

Wourms , John P

Committee Member

Gibbons , John R


Embryo cryopreservation is an integral part of assisted reproduction because it allows for future use of these embryos. Cryopreservation occurs when there are supernumerary embryos or when an embryo transfer cannot be performed.
There are two main methods to cryopreserve embryos. The most recent is vitrification, which uses high concentrations of cryoprotectants, a short time to cool and avoids ice crystals. The 'gold standard' is the slow-cool method, which uses low concentrations of cryoprotectants, a long time to cool embryos and produces extracellular ice crystals.
Prior to introducing vitrification as part of the human cryopreservation regime, it is important that vitrification be evaluated through a research protocol using a mammalian embryo model. In this research, we used the two-cell, mouse embryo model to develop a closed system for vitrification, observe the toxicity of vitrification solutions, and compare blastocyst rates, pup rates and DNA damage between the vitrification and slow-cool method.
Two commercially available devices were used to vitrify embryos. Both devices were studied as an open-system and a closed-system using two-cell and eight-cell mouse embryos. These devices and systems produced similar blastocyst rates. Two-cell mouse embryos were used for subsequent studies due to the increased potential to observe a detrimental effect with the earlier cell stage.

A toxicity study demonstrated that two-cell mouse embryos, after remaining in vitrification solution for 32 minutes, produced fewer blastocysts than did two-cell mouse embryos that were removed from the vitrification solution at earlier time intervals (1, 2, 4, 8 and 16 minutes). However, when surviving blastocysts were transferred to recipients, they produced pups.
Comparison studies between the vitrification and slow-cool method demonstrated that there were no significant differences in blastocyst rate, pup rate or percentage of normal embryos (not demonstrating DNA damage).
In conclusion, a closed system was developed to vitrify two-cell mouse embryos. The toxicity of vitrification solutions were observed and the comparison of vitrification and slow-cool methods using blastocyst rate, pup rate and DNA damage as determinants showed that both methods were similar.

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