Mitochondrial function in vitrified versus slow-frozen murine embryos


  • Razif Dasiman Universiti Teknologi MARA
  • Mimi-Sophia Sarbandi Universiti Teknologi MARA
  • Nor-Shahida Abdul Rahman Universiti Teknologi MARA
  • Salina Othman Universiti Teknologi MARA
  • Mastura Malek Universiti Teknologi MARA
  • Fathiah Abdullah Universiti Teknologi MARA
  • Fatin Nadzirah Zakaria Universiti Teknologi MARA
  • Nina Keterina Hashim Universiti Teknologi MARA
  • Nor-Ashikin Mohamed Noor Khan Universiti Teknologi MARA



Mitochondria, slow freezing, vitrification, murine embryos, Confocal Laser Scanning Microscope (CLSM)


The effects of vitrification and slow-freezing on mitochondrial functions of in vitro produced murine embryos at various developmental stages were investigated using the Confocal Laser Scanning Microscope (CLSM). Oocytes were obtained from superovulated females, fertilized with sperm and cultured. Resulting 2-, 4- and 8-cell embryos were collected and cryopreserved by vitrification and slow-freezing. Mitochondria were stained with MitoTracker Red (CMXRos). Images were viewed by CLSM and analyzed using QWin SoftwareV.3. Fluorescent intensities were used to indicate viability. Results showed that mitochondrial fluorescence intensities of cryopreserved embryos were significantly lower as compared to non-cryopreserved embryos (p<0.01). Vitrification was found to be superior to slow-freezing at all developmental stages, based on mitochondrial function.

Author Biographies

Razif Dasiman, Universiti Teknologi MARA

Maternofetal and Embryo Research Group (MatE), Faculty of Medicine

Mimi-Sophia Sarbandi, Universiti Teknologi MARA

Maternofetal and Embryo Research Group (MatE), Faculty of Medicine

Nor-Shahida Abdul Rahman, Universiti Teknologi MARA

Maternofetal and Embryo Research Group (MatE), Faculty of Medicine

Salina Othman, Universiti Teknologi MARA

Maternofetal and Embryo Research Group (MatE), Faculty of Medicine

Mastura Malek, Universiti Teknologi MARA

Maternofetal and Embryo Research Group (MatE), Faculty of Medicine

Fathiah Abdullah, Universiti Teknologi MARA

Maternofetal and Embryo Research Group (MatE), Faculty of Medicine

Nor-Ashikin Mohamed Noor Khan, Universiti Teknologi MARA

Maternofetal and Embryo Research Group (MatE), Faculty of Medicine


Al-Mehdi, A-. B., Pastukh, V. M., Swiger, B. M., Reed, D. J., Patel, M.R., Bardwell, G. C. Pastukh, V. V., Alexeyev, M. F., & Gillespie, M. N. (2012). Perinuclear mitochondrial clustering creates an oxidant-rich nuclear domain required for hypoxia-induced transcription. Science Signaling, 5(231), 1-20.

Dalcin, L., Silva, R. C., Paulini, F., Silva, B. D. M., Neves, J. P., & Lucci, C.M. (2013). Cytoskeleton structure, the pattern of mitochondrial activity and ultrastructure of frozen or vitrified sheep embryos. Cryobiology, 67(2), 137-145.

Dasiman, R., Nor-Shahida, A. R., Salina, O., Mohd-Fazirul, M., Norhazlin, J. M. Y., Wan-Hafizah, W. J., Mohd-Hamim, R., Gabriele, R. A. F., & Nor-Ashikin, M. N. K. (2013). Cytoskeletal alterations in different developmental stages of in vivo cryopreserved preimplantation murine embryos. Medical Science Monitor Basic Research, 19, 258-266.

Dewitt, D. A., Hurd, J. A., Fox, N., Townsend, B. E., Griffioen, K. J. S., Ghribi,O., & Savory, J. (2006). Peri-nuclear clustering of mitochondria is triggered during aluminum maltolate induced apoptosis. Faculty Publications and Presentations, Paper 11.

Faten, F. A., Nina, D., Ahmed, M. A.S, Falcone, T., & James, G. (2010). Slow freezing, vitrification and ultra-rapid freezing of human embryos: A systematic review and meta-analysis. Reproductive Biomedicine Online,20, 209-222.

Harvey, A., Gibson, T., Quebedeaux, T., & Brenner, C. (2007). Impact of assisted reproductive technologies: A mitochondrial perspective of cytoplasmic transplantation. Current Top Development Biology, 77, 229-249.

Kasai, M. (2002). Advances in the cryopreservation of mammalian oocytes and embryos: Development of ultrarapid vitrification. Reproductive Medicine and Biology, 1, 1-9.

Lieberman, J., Isachenko, V., Rahimi, G., & Tucker, M.J. (2002). The potential importance of vitrification in reproductive medicine. Biology Reproduction,67, 1671-1680.

Ludwig, T.E., Squirell, J.M., Palmenberg, A.C., & Bavister, B.D. (2001). The relationship between development, metabolism and mitochondrial organization in 2-cell hamster embryos in the presence of low level of phosphates. Biology Reproduction, 65, 1648-1654.

Nagai, S., Mabuchi, H., Hirata, S., Kasai, T., & Hoshi, K. (2006). Correlation of abnormal mitochondrial distribution in mouse oocytes with reduced developmental competence. Tohoku Japan Experimental Medicine, 210, 137-144.

Nagy A., Gertsensten M., Vintersten K. and Behringer R. (2003). Manipulating the mouse embryo: A laboratory manual.(3rd edition). Cold Spring Harbor Laboratory Press, New York.

Nor Ashikin, M. N.K. (2006). In vitro and in vivo development of preimplantation murine embryos following gender identification using the polymerase chain reaction (PCR) technique. Ph.D. Thesis, University of Malaya.

Nor-Shahida, A. R., Dasiman, R., Salina, O., Gabrielle, R. A. F., & Nor-Ashikin,M. N. K. (2009). Immunofluorescence staining of the murine preimplantation embryos: DAPI vs. Hoechst 33342. Proceedings of the 18th Scientific Conference of Electron Microscopy Society of Malaysia (EMSM), Kuala Lumpur. 2009.

Rall, W. F. & Fahy, G. M. (1985) Ice-free cryopreservation of mouse embryos at −196 degrees C by vitrification. Nature, 313, 573-575.

Saragusty, J., & Arav, A. (2011). Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification. Reproduction, 141(1), 1-19.

Suzuki, H., Satoh, M., & Kabashima, K. (2006). Distributions of mitochondria and the cytoskeleton in hamster embryos developed in vivo and in vitro. Journal Mammalian Ova Research, 23, 128-134.

Takeuchi, T., Neri, Q. T., Katagiri, Y., Rosenwak, Z., & Palermo, G. D. (2005).Effect of treating induced mitochondrial damaged on embryonic development and epigenesis. Biology Reproduction, 72, 584-592.

Valojerdi R. M., Yazdi E. P., Karimian L., Hassani F., & Movaghar B. (2009).Vitrification versus slow freezing gives excellent survival, post warming embryo morphology and pregnancy outcomes for human cleaved embryos. Journal of Assisted Reproduction and Genetics, 26, 347-254.

Van Blerkom, J., Davis, P., & Alexander, S. (2000). Differential mitochondrial distribution in human pronuclear embryos leads to disproportionate inheritance between blastomeres: Relationship to the microtubular organization, ATP content, and competence. Human Reproduction, 15, 2621–2633.

Wennerholm, U. B. (2000). Cryopreservation of embryos and oocytes: Obstetric outcome and health in children. Human Reproduction, 15, 18-25.

Whittingham, D. G., Leibo, S. P., & Mazur, P. (1972). Survival of mouse embryos frozen to −196 degrees and −269 degrees C. Science, 178, 411-414.

Zeng, H.T., Ren, Z., Yeung, W.S., Shu, Y.M., & Liang, X.Y. (2007). Low mitochondrial DNA and ATP contents contribute to the absence of birefringent spindle imaged with PolScope in in vitro matured human oocytes. Human Reproduction, 22, 1681-1686.