Open Access
Research (Published online: 02-11-2018)
1. The profiling of pre- and post-warming DNA in mouse embryos with microsatellite method
Widjiati Widjiati, Soeharsono Soeharsono and Yeni Dhamayanti
Veterinary World, 11(11): 1526-1531

Widjiati Widjiati: Department of Veterinary Anatomy, Faculty of Veterinary Medicine University of Airlangga, Surabaya, Indonesia.
Soeharsono Soeharsono: Department of Veterinary Anatomy, Faculty of Veterinary Medicine University of Airlangga, Surabaya, Indonesia.
Yeni Dhamayanti: Department of Veterinary Anatomy, Faculty of Veterinary Medicine University of Airlangga, Surabaya, Indonesia.

doi: 10.14202/vetworld.2018.1526-1531

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Article history: Received: 26-06-2018, Accepted: 24-09-2018, Published online: 02-11-2018

Corresponding author: Widjiati Widjiati


Citation: Widjiati W, Soeharsono S, Dhamayanti Y (2018) The profiling of pre- and post-warming deoxyribonucleic acid in mouse embryos with microsatellite method, Veterinary World, 11(11): 1526-1531.

Aim: This research aimed to identify the deoxyribonucleic acid (DNA) profile and changes of post-warming embryo after being frozen with vitrification method using microsatellite method.

Materials and Methods: This research examined the mouse embryo blastocysts that were divided into four groups: Post-warming living blastocyst, post-warming living blastocyst with half fragmented cell, post-warming dead blastocyst, and pre-freezing living blastocyst. The isolation sample applied phenol-chloroform method. After obtaining polymerase chain reaction results, all the samples of pre-freezing fresh embryo, post-warming living embryo, dead embryo, and degenerated embryo were then examined by single-strand conformation polymorphism (SSCP).

Results: The amplification with D18mit14 primer was 100 bp and 150bp with D18mit87 primer, 150bp with D7mit22, and 300bp with D7mit25. The result of SSCP with D18mit14 primer showed that the blastocysts were fragmented and dead after warming process and formed into two DNA strand fragments, while the fresh embryos which passed freezing process did not form any fragment. D18mit87 primer SSCP indicated different fragments for each treatment. The result of SSCP using D7mit22 formed two different fragments for each treatment. While using D7mit25, the SSCP result formed some different fragments for each sample. Post-warming living embryo had similar ribbon to pre-freezing fresh embryo.

Conclusion: D7mit222, D7mit25, and D18mit87 primers could be used as the aneuploidy marker on mouse embryos that were induced by post-warming process. The profile of living blastocyst, dead blastocyst, and post-warming fragmented blastocyst had different DNA tapes.

Keywords: blastocyst, DNA mutation, single-strand conformation polymorphism, vitrification.


1. Pooyanfar, P., Foroutan, T. and Dashtizad M. (2018) Effects of blastocyst artificial collapse prior to vitrification on hatching and survival rates and the expression of kl4 gene in mouse embryos. Vet. Res. Forum, 9(1): 87-92. [PubMed] [PMC]

2. Choi, Y.H., Ross, P. and Velez, I.C. (2015) Cell lineage allocation in equine blastocysts produced in vitro under varying glucose concentrations. Reproduction, 150(1): 31-41. [Crossref] [PubMed]

3. Kim, Y.M., Uhm, S.J., Gupta, M.K., Yang, J.S., Lim, J.G., Das, Z.C., Heo, Y.T., Chung, H.J., Kong, K., Kim, N.H., Lee, H.T. and Ko, D.H. (2012) Successful vitrification of bovine blastocysts on paper container. Theriogenology, 78(5): 1085-1093. [Crossref] [PubMed]

4. Inaba, Y., Aikawa, Y., Hirai, T., Hashiyada, Y., Yamanouchi, T., Misumi, K., Ohtake, M., Somfai, T., Kobayashi, S., Saito, N., Matoba, S., Konishi, K. and Imai K. (2011) In-straw cryoprotectant dilution for bovine embryos vitrified using cryotop. J. Reprod. Dev., 57(4): 437-443. [Crossref]

5. Kuwayama, M. (2007) Highly efficient vitrification for cryopreservation of human oocytes and embryos: The cryotop method. Theriogenology, 67(1): 73-80. [Crossref] [PubMed]

6. Larman, M.G., Katz-Jaffe, M.G., Mc Callie, B., Filipovits, J.A. and Gardner, D.K. (2011) Analysis of global gene expression following mouse blastocyst cryopreservation. Hum. Reprod., 26(10): 2672-2680. [Crossref] [PubMed]

7. Zenon, O., Katerina, A., Sharma, C., Antonia, S., Louisa, O., Basil, C.T. and Efstratios, K. (2017) Effects of vitrification on blastomere viability and cytoskeletal integrity in mouse embryo. Zygote, 25(1): 75-84. [Crossref] [PubMed]

8. Wenhong, M., Xingfang, H., Xing, Y. and Xiaoyan, L. (2016) Maternal obesity in mice not only affects fresh embryo quality but also aggravates injury due to vitrification. J. Assist. Reprod. Genet., 33(1): 1515-1523.

9. Leme, L.O., Dufort, I., Spricigo, J.F.W., Braga, T.F., Sirard, M., Franco, M.M. and Dode, M.A.N. (2016) Effects of vitrification using the cryotop method on the gene expression profile of in vitro-produced bovine embryos. Theriogenology, 85(4): 724-733. [Crossref] [PubMed]

10. Sudano, M.J., Caixeta, E.S., Paschoal, D.M., Martins, A., Machado, R., Buratini, J. and Landim-Alvarenga, F.D. (2014) Cryotolerance and global gene-expression patterns of Bos taurus indicus and Bos taurus taurus in vitro- and in vivo produced blastocysts. Reprod. Fertil. Dev., 26(8): 1129-1141. [Crossref] [PubMed]

11. Saenz-de-Juano, M.D., Marco-Jimenez, F., Viudes-de-Castro, M.P., Lavara, R. and Vicente, J.S. (2014) Direct comparison of the effects of slow freezing and vitrification on late blastocyst gene expression, development, implantation, and offspring of rabbit morulae. Reprod. Domest. Anim., 49(3): 505-511. [Crossref] [PubMed]

12. Inaba, Y., Miyashita, S., Somfai, T., Geshi, M., Matoba, S., Dochi, O. and Nagai T. (2016) Cryopreservation method affects DNA fragmentation in trophectoderm and the speed of re-expansion in bovine blastocysts. Cryobiology, 72(2): 86-92. [Crossref] [PubMed]

13. Carroll, J. and Marangos, P. (2013) The DNA damage response in mammalian oocytes. Rev. Art. Front. Genet., 4(3): 117. [Crossref]

14. Ashwood-Smrth, M.J. and Edwards, R.G. (1996) Deoxyribonucleic acid repair by oocytes. J. Mol. Hum. Reprod., 2(1): 46-51.

15. Coutinho, A.R., Mendes, C.M., Caetano, H.V., Nascimento, A.B., Oliveira, V.P., Hernadez-Blazquez, F.J., Sinhorini, I.L., Visintin, J.A. and Assumpcao, M.E. (2007) Morphological changes in mouse embryos cryopreserved by different techniques. Microsc. Res. Tech., 70(4): 296-301. [Crossref] [PubMed]

16. Kader, A., Falcone, T., Sharma, R.K., Mangrola, D. and Agarwal, A. (2010) Slow and ultrarapid cryopreservation of biopsied mouse blastocysts and its effect on DNA integrity index. J. Assist. Reprod. Genet., 27(8): 509-515. [Crossref] [PubMed] [PMC]

17. Niimura, S. (2003) Time-lapse video micrographic analyses of contractions in mouse blastocysts. J. Reprod. Dev., 49(6): 413-423. [Crossref]

18. Rachel, W., Teri, O., Marisa, S.B., Christos, C. and Monica, M. (2017) The superovulated environment, independent of embryo vitrification, results in low birthweight in a mouse model. Biol. Reprod., 97(1): 133-142. [Crossref] [PubMed]

19. Albert, B., Bray, D., Hopkin, K., Johnson, A., Lewis, J. and Raff, M. (2013) Essential Cell Biology. 4th ed. Garland Science, New York. p170-181.

20. Zheng, X., Yang, S., Zhang, D., Zhong, Z., Tang, X. and Deng, K. (2016) Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism. Plant Cell Rep., 35(7): 1545-1554. [Crossref] [PubMed]

21. Widjiati, W., Epy, M.L. and Benjamin, C.T. (2017) Effectivity of insulin transferrin selenium and bovine serum albumin addition on in vitro culture medium on fertilization and blastocyst rate of mice (Mus musculus). J. Int. Dent. Med. Res., 10(3): 1080-1083.

22. Epy, M. Luqman, W.W. and Suryo, K. (2017) Effect of combined cryoprotectant of ethylene glycol and propanediol on embryo cryopreservation to blastomere cell apoptosis and blastocyst quality. Experimental article. J. Int. Dent. Med. Res., 10(3): 1074-1079.

23. Widjiati, E.M.L., Viski, F.H. and Portia, S. (2017) Comparison of Morula and Blastula Embryo Vitrification by Using Cryoprotectant Ethylene Glycol, Propanediol, DMSO and Insulin Transferrin Selenium. KnE Life Sciences. The Veterinary Medicine International Conference (VMIC). p1-5. [Crossref]

24. John, T.C. (2014) Basic Molecular Protocols in Neuroscience: Tips, Tricks, and Pitfalls. Agarose gel Electrophoresis. Elsevier Inc. p21-25.

25. Summer, H., Gramer, R. and Droge P. (2009) Denaturing urea polyacrylamide gel electrophoresis (urea page). J. Vis. Exp., 29(32): 1485-1488. [Crossref]

26. Benbouza, H., Jean-Maria, J., Jean-Pierre, B. and Guy, M. (2006) Optimization of a reliable, fast, cheap, and sensitive silver staining method to detect SSR markers in polyacrylamide gels. Biotechnol. Agr. Soc. Environ., 10(2): 1-9.

27. Fang, C., Yue, C.M.R., Huang, L.W. and Jia L. (2016) Pregnancy outcomes of blastocysts cultured overnight after thawing. Arch. Gynecol. Obstet., 293(6): 1347-1356. [Crossref] [PubMed]

28. Hendriks, W.K., Roelen, B.A.J., Colenbrander, B. and Stout, T.A.E. (2015) Cellular damage suffered by equine embryos after exposure to cryoprotectants or cryopreservation by slow-freezing or vitrification. Equine Vet. J., 47(6): 701-707. [Crossref] [PubMed]

29. Kakavas, V.K., Plageras, P., Vlachos, T.A., Papaioannou, A. and Noulas, V.A. (2008) PCR SSCP: A method for the molecular analysis of genetic diseases. Mol. Biotechnol., 38(2): 155-163. [Crossref]