Yonsei Med J.  2006 Jun;47(3):399-404. 10.3349/ymj.2006.47.3.399.

Simplified EM Grid Vitrification Is a Convenient and Efficient Method for Mouse Mature Oocyte Cryopreservation

• Kim SH
• Ku SY
• Sung KC
• Kang MJ
• Kim SA
• Kim HS
• Oh SK
• Jee BC
• Suh CS
• Choi YM
• Kim JG
• Moon SY

• KMID: 1715860
• DOI: http://doi.org/10.3349/ymj.2006.47.3.399

Abstract

This study was performed to evaluate the efficiency of simplified EM grid vitrification, skipping the step of removing the cryoprotectant (5.5M EG + 1.0M sucrose) droplet on the grid after loading oocytes, compared to conventional cryopreservation protocols for mouse mature oocytes. Firstly, the recovery, survival, fertilization and hatching rates of simplified EM grid vitrification were compared with those of the slow freezing method using 1.5M DMSO. Then, conventional EM grid vitrification was compared with simplified EM grid vitrification. Simplified EM grid vitrification showed higher survival, fertilization and hatching rates than those of the slow freezing method (85.6% vs. 63.2%; 51.0% vs. 22.3%; 38.7% vs. 12.5%, p < 0.01, respectively). Moreover, simplified EM grid vitrification showed higher recovery, survival and fertilization rates than those of conventional EM grid vitrification (100% vs. 95.0%, p=0.024; 90.0% vs. 78.9%, p=0.033; 56.7% vs. 38.7%, p=0.021, respectively). Hatching rate tended to be higher for simplified EM grid vitrification compared to conventional EM grid vitrification (41.1% vs. 24.1%). In conclusion, simplified EM grid vitrification is a convenient and efficient method for cryopreservation of mouse mature oocytes, compared to conventional EM grid vitrification and slow freezing methods.

Keyword

EM grid vitrification; cryopreservation; mouse oocyte

MeSH Terms

Pregnancy
Oocytes/*cytology
Mice, Inbred DBA
Mice, Inbred C57BL
Mice
Male
*Fertilization in Vitro
Female
Cryopreservation/*instrumentation/*methods
Cell Survival
Animals

Figure

• 

  Fig. 1 Conventional and simplified vitrification methods. Top panel: In the simplified vitrification method, the cryoprotectant solution on the grid is not removed with the underlying sterilized filter paper (Wipes) as in conventional EM grid vitrification. Bottom panel: Most cryopreserved-thawed oocytes were retrieved at step I (TS I) following the simplified EM grid vitrification method, enabling the oocytes to avoid exposure to room air. In contrast, following conventional EM grid vitrification, it was necessary to move the grid to step II (TS II). Note the exemplary numbers of oocytes retrieved from both methods after 5 oocytes were loaded on each grid. TS: thawing solution, TS I: 1.0 M sucrose for 2.5 min, TS II: 0.5 M sucrose for 2.5 min, TS III: 0.25 M sucrose for 2.5 min, TS IV: 0.125 M sucrose for 2.5 min.

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