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Use of sucrose during removal of cryoprotectants after thawing eight-cell mouse embryos
T Takeda1, R P Elsden, G E Seidel
1Animal Reproduction Laboratory, Colorado State University, Fort Collins, CO 80523, USA.
Abstract:
Eight-cell mouse embryos were frozen in 0.5-ml plastic straws in modified Dulbecco's phosphate buffered saline (PBS) plus 5% steer serum plus either 1.32 M dimethyl sulfoxide (DMSO) or 1.32 M glycerol. Upon thawing, embryos were diluted 1:4 with 0.0, 0.2, 0.6, or 1.0 M sucrose solutions within the straws. Thawing was either in air at ambient temperature or in 8 degrees C or 38 degrees C water. After 48 h of culture, more embryos frozen in DMSO and thawed in 8 degrees C and 37 degrees C water developed to blastocysts (87 and 93%, respectively) than embryos thawed in air (75%; P<0.05). No significant differences in development were noted among the three thawing regimens when embryos were frozen with glycerol. There was no significant effect of concentration of sucrose during dilution on development of embryos postthaw. With glycerol as the cryoprotectant, damage to zonae pellucidae increased as thawing rates increased, whereas the opposite was observed with DMSO as the cryoprotectant (P<0.05).
Insights
Cryoprotectant choice impacts mouse embryo survival post-thaw. Dimethyl sulfoxide (DMSO) with rapid water-bath thawing improved blastocyst development compared to air thawing, unlike glycerol.
Area of Science:
- Reproductive Biology
- Cryobiology
- Developmental Biology
Background:
- Efficient cryopreservation of mammalian embryos is crucial for assisted reproductive technologies and research.
- Cryoprotective agents (CPAs) like dimethyl sulfoxide (DMSO) and glycerol are essential for preventing ice crystal damage during freezing.
- Thawing rates and methods can significantly influence post-thaw embryo viability and developmental potential.
Purpose of the Study:
- To investigate the effects of different thawing methods and cryoprotectants on the survival and development of eight-cell mouse embryos.
- To evaluate the impact of varying sucrose concentrations during post-thaw dilution on embryo development.
- To compare the effects of thawing rates on zona pellucida integrity when using DMSO versus glycerol.
Main Methods:
- Eight-cell mouse embryos were frozen using dimethyl sulfoxide (DMSO) or glycerol as cryoprotectants in modified PBS with steer serum.
- Post-thaw dilution involved sucrose solutions (0.0-1.0 M) within straws, followed by thawing in air, 8°C water, or 38°C water.
- Embryo development to blastocyst stage was assessed after 48 hours of in vitro culture; zona pellucida integrity was also examined.
Main Results:
- Embryos frozen with DMSO showed significantly higher blastocyst development rates when thawed in 8°C (87%) and 38°C (93%) water compared to air thawing (75%).
- Thawing regimens did not significantly affect embryo development when glycerol was used as the cryoprotectant.
- Sucrose concentration during dilution had no significant impact on post-thaw embryo development; however, zona damage patterns differed between DMSO and glycerol with varying thawing rates.
Conclusions:
- Rapid thawing in warm water significantly enhances the developmental competence of DMSO-frozen mouse embryos compared to slower air thawing.
- Glycerol-based cryopreservation appears less sensitive to thawing rates regarding blastocyst development, but zona pellucida integrity is compromised at faster rates.
- Optimizing thawing protocols, particularly the choice of cryoprotectant and thawing rate, is critical for successful mouse embryo cryopreservation.
