INFLUENCES OF INCUBATION TIME AND SUCROSE CONCENTRATION ON MICE (Mus musculus L.) OOCYTE VIABILITY FOR ENUCLEATING PROCEDURE

Ekayanti Mulyawati Kaiin, Muhammad Gunawan, Madihah Madihah, Ghina Nafisah

Abstract


This study aimed to find out the optimum incubation time to complete mouse oocyte maturation at Metaphase II (MII) stage and determine the optimum sucrose concentration enabling to induce nuclear swelling for visualization that is important for enucleating process at the initial procedure of somatic cell nuclear transfer (SCNT). In this current study, mice were used as animal model. Completely randomized design was arranged, consists of 2 trials with 4 treatments and 7 replications. In the first trial, the oocytes were cultured at 0-2, 4-6, 8-10, and 12-14 h in 5% CO2 incubator at 37 C. Second, the MII oocytes obtained from previous trial were cultured in M199 medium containing different concentrations of sucrose (0, 1.5, 3, and 6%). The parameters measured were the oocyte viability at various stages, i.e germinal vesicle (GV), metaphase I (MI), anaphase/telophase I (A/T I), and metaphase II (MII), and the viability of swollen nuclear oocytes using Hoechst/PI staining. The results showed that the optimum incubation time required by oocytes to reach MII stage was 12-14 h with a percentage of 57.14±12.67%, while the optimum sucrose concentration for nuclear swelling was found at 3% with a percentage of 100±0.00%. Our findings provided preliminary results related to the maturation process of the mouse oocyte nucleus, which is meaningful for the initial procedure of SCNT.

Keywords


metaphase II; mice; nuclear swelling; oocyte; SCNT

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References


Campbell, K.H., J. MacWhir, W.A. Ritchie, and I. Wilmut. 1996. Sheep cloned by nuclear transfer from a cultured cell line. Nature. 54: 380 (6959).

Campbell, N.A., J.B. Reece and L.G. Mitchel. 2000. Biology, 5th ed.Terjemahan Wasmen Manali. Erlangga. Jakarta.

Chang, M.C. 1995. The maturation of rabbit oocytes in culture and their maturation, activation, fertilization and subsequent development in the fallopian tubes. J. of Exp. Zoology.128: 379-405.

Fulka, J. Jr, R.M. Moor and J. Fulka. 1995. Mouse oocyte maturation: meiotic checkpoints. Exp. Cell Research. 219: 414-419.

Gunawan, M., M. Fahrudin, dan A. Boediono. 2014. Perkembangan embrio sapi setelah fertilisasi menggunakan metode Intracytoplasmic Sperm Injection (ICSI) dan aktivasi dengan strontium. J. Kedokteran Hewan 8(2): 154-157.

Hafez, B. and E.S.E Hafez. 2000. Reproduction in Farm Animals. 7th ed. Lea and Febiger, Philadelphia. pp. 96-107.

Heidari, M., A.K. Malekshah, N. Azami and S.M. Mirhoseini. 2012. In vitro maturation of mouse preantral follicles in two culture media: A comparative study. Annals of Biol. Res. 3(10): 4937-4941.

Hogan, B., F. Constantini and E. Lacy. 1986. Mouse Husbandry. In Monk, M. (ed). Mammalian Development a Practical Approach. IRL Press. Washington DC.

Kono, T., Y. Sotomaru, Y. Sato and T. Nakahara. 1993. Development of androgenetic mouse embryos produced by in vitro fertilization of enucleated oocytes. Mol. Reprod. and Dev. 34 : 43-46.

Liu, J., L. Sung, M. Barber, and X. Yang. 2002. Hypertonic medium treatment for localization of nuclear material in bovine metaphase II oocytes. Biol. of Reprod. 66: 1342-1349.

Mahmoudi, R., A. Subhani, P. Pasbakh, F. Abolhasani, I. Amiri, M. Salehnia, and F. Etesam. 2005. The effects of cummulus cells on in vitro maturation of mouse germinal vesicle stage oocytes. Iranian J. of Reprod. Med.3 (2) : 74-78.

Masui,Y. and H.J. Clarke. 1979. Oocyte maturation. International Rev. of Cyto. 57: 185-282.

Mitalipov, S.M., Q. Zhou, J.A. Byrne, W.Z. Ji, R.B. Norgren, and D.P. Wolf. 2007. Reprogramming following somatic cell nuclear transfer in primates is dependent upon nuclear remodeling. Human Reprod. 22 : 2232-2242.

Moor, R.M. and A.O. Trounson. 1977. Hormonal and follicular factor affecting maturation of sheep oocytes in vitro and their subsequent developmental capacity. J. Reprod. Fert. 74 : 657-660.

Murti, H. 2013. Produksi Embrio Kloning Mencit dengan Pengembangan Teknik Transfer Inti Sel Somatis. Disertasi. Sekolah Pascasarjana Institut Pertanian Bogor. Bogor.

Polanski, Z. 1986. In vivo and in vitro maturation rate of oocytes from two strains of mice. J. Reprod. Fert. 78 : 103-109.

Song , H.B. and A. Iritani. 1987. Studies on in vitro maturation of follicular oocytes in the immature goats. Korean J. Anim. Sci. 29, pp.303-309.

Sudjana. 2012. Metode Statistik. Tarsito. Bandung.

Sumer, J.L. and J.V. Paul. 2011. Cellular programming of somatic cells. Indian J. of Exp. Biol. 49: 409-415.

Voronina, E. and G.M. Wessel. 2003. The regulation of oocyte maturation. Current Topics in Dev. Biol. 58: 53-110.

Wang, M., D. Chen, J. Liu, G. Li and Q. Sun. 2001. In vitro fertilization of mouse oocytes reconstructed by transfer of metaphase II chromosomes result in live births. Zygote 9 (1) : 9-14.

Wang, M., J. Liu, G. Li, L. Lian and D. Chien. 2001. Sucrose pretreatment for enucleation : an efficient and non-damage method for removing the spindle of the mouse MII oocyte. Mol. Reprod. and Dev. 58 : 432-436.

Wun, I. and E. Ralph. 2008. Human somatic cell nuclear transfer. Chinese J. of Physiol. 41 (4) : 208-213.




DOI: https://doi.org/10.21157/j.ked.hewan.v12i3.10896

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