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In-vitro development of refrozen mouse embryos

N J Vitale1, M W Myers, R S Denniston

  • 1Department of Animal Science, School of Veterinary Medicine, Louisiana State University, Baton Rouge 70803, USA.

Human Reproduction (Oxford, England)
|February 1, 1997
PubMed
Summary

Mouse embryos can survive multiple freeze-thaw cycles, though repeated freezing damages their zona pellucida and reduces cell count. Survival rates decrease with successive freezing, but in-vitro development is still possible after three cycles.

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Area of Science:

  • Reproductive Biology
  • Cryobiology
  • Developmental Biology

Background:

  • Cryopreservation is crucial for preserving embryos.
  • Understanding the impact of repeated freeze-thaw cycles on embryo viability is essential for improving assisted reproductive technologies.

Purpose of the Study:

  • To investigate the effects of sequential, repetitive freezing and thawing on the in-vitro development of mouse embryos.
  • To assess embryo survival, developmental capacity, and morphological integrity after multiple cryopreservation cycles.

Main Methods:

  • Mouse embryos (8-16 cell stage) underwent one of five treatments: control, single freeze-thaw, two freeze-thaw cycles, freeze-thaw-culture-refreeze, or three successive freeze-thaw cycles.
  • Embryos were cultured in vitro to the hatched blastocyst stage.

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  • Cell number was determined using Hoechst 33342 staining for hatched blastocysts.

    • Main Results:

    • Embryos frozen once showed higher development rates to hatched blastocysts than those subjected to refreezing.
    • No significant difference in post-thaw development was observed between culture-refreeze and direct-refreeze procedures.
    • Repeated freezing caused significant zona pellucida damage and reduced the mean cell number per embryo at hatching compared to controls.

    Conclusions:

    • Mouse embryos can survive up to three successive freeze-thaw cycles and retain in-vitro developmental potential.
    • Repeated cryopreservation negatively impacts embryo quality, evidenced by zona damage and reduced cell counts.
    • Further research may optimize protocols to mitigate damage from sequential freezing.