THE TECHNIQUE FOR CRYOPRESERVATION OF CATTLE EGGS

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Lucia Olexiková
Alexander V. Makarevich
Linda Bédeová
Elena Kubovičová

Abstract

The aim of this investigation was to establish a methodology of cryopreservation of cattle eggs (oocytes) under our laboratory conditions. For cryopreservation of in vitro matured oocytes, the freezing in minimum volume by ultra-rapid cooling technique was used. Oocytes with at least three layers of cumulus cell were placed into the equilibration solution (ES: 3 % ethylene glycol in M199-HEPES, supplemented with 10 % foetal calf serum) for 12 min. Following equilibration, the oocytes were transferred to vitrification solution (30 % EG + 1M sucrose in M199-HEPES with 10 % foetal bovine serum) at room temperature for 25 sec. Then the oocytes were placed onto nickel grid (electron microscopy grade) and plunged into liquid nitrogen. After thawing the oocytes were fertilized in vitro. Development of produced embryos (cleavage on day 2, and blastocyst yield on day 7) and total cell number of blastocysts after DAPI staining were determined. We obtained a relatively high cleavage rate (55.81 %) of fertilized oocytes after thawing; of them 11.24 % developed to the blastocyst stage. The quality of blastocysts obtained from vitrified oocytes was similar to the control blastocysts, as evidenced by a comparable total cell number (84.45 vs 97.29, resp.). In conclusion, the designed freezing technique proved to be suitable for cryopreservation of cattle oocytes, nevertheless further optimization is required.

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References

Bulgarelli, D.L., Vireque, A.A., Pitangui-Molina, C.P., Silva-De-Sá, M.F., De Sá Rosa, E. & Silva, A.C.J. (2017). Reduced competence of immature and mature oocytes vitrified by Cryotop method: assessment by in vitro fertilization and parthenogenetic activation in a bovine model. Zygote 25(2), 222-230. doi: 10.1017/S0967199416000381.
Diez, C., Duque, P., Gómez, E., Hidalgo, C.O., Tamargo, C., Rodríguez, A., Fernández, L., De La Varga, S., Fernández, A., Facal, N. & Carbajo, M. (2005). Bovine oocyte vitrification before or after meiotic arrest: effects on ultrastructure and developmental ability. Theriogenology, 64(2), 317-333.
Fuku, E., Kojima, T., Shioya, Y., Marcus, G.J. & Downey, B.R. (1992). In vitro fertilization and development of frozen thawed bovine oocytes. Cryobiology, 29(4), 485-492.
Chian, R.C., Kuwayama, M., Tan, L., Tan, J., Kato, O. & Nagai, T. (2004). High survival rate of bovine oocytes matured in vitro following vitrification. Journal of Reproduction and Development, 50(6), 685-696.
Ishii, T., Tomita, K., Sakakibara, H. & Ohkura, S. (2018). Embryogenesis of vitrified mature bovine oocytes is improved in the presence of multi-layered cumulus cells. Journal of Reproduction and Development, 64(1), 95–99. doi: 10.1262/jrd.2017-095.
Kuwayama, M., Vajta, G., Kato, O. & Leibo, S.P. (2005). Highly efficient vitrification method for cryopreservation of human oocytes. Reproductive Biomedicine Online, 11(3), 300-308.
Lane, M. & Gardner, D.K. (2001). Vitrification of mouse oocytes using a nylon loop. Molecular Reproduction and Development, 58(3), 342-347.
Magnusson, V., Feitosa, W.B., Goissis, M.D., Yamada, C., Tavares, L.M., D'avila Assumpção M.E. & Visintin, J.A. (2008). Bovine oocyte vitrification: effect of ethylene glycol concentrations and meiotic stages. Animal Reproduction Science, 106(3-4), 265-273.
Martino, A., Songsasen, N. & Leibo, S.P. (1996). Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biology of Reproduction, 54(5), 1059-1069.
Nowshari, M.A. & Brem, G. (1998). Effect of cryoprotectants and their concentration on post-thaw survival and development of expanded mouse blastocysts frozen by a simple rapid-freezing procedure. Theriogenology, 50(7), 1001-1013.
Ortiz-Escribano, N., Smits, K., Piepers, S., Van Den Abbeel, E., Woelders, H. & Van Soom, A. (2016). Role of cumulus cells during vitrification and fertilization of mature bovine oocytes: Effects on survival, fertilization, and blastocyst development. Theriogenology, 86(2), 635-641. doi: 10.1016/j.theriogenology.2016.02.015.
Papis, K., Shimizu, M. & Izaike, Y. (2000). Factors affecting the survivability of bovine oocytes vitrified in droplets. Theriogenology, 54(5), 651-658.
Prentice, J.R., Singh, J., Dochi, O. & Anzar, M. (2011). Factors affecting nuclear maturation, cleavage and embryo development of vitrified bovine cumulus-oocyte complexes. Theriogenology, 75(4), 602-609. doi: 10.1016/j.theriogenology.2010.09.027.
Sprícigo, J.F., Morais, K., Ferreira, A.R., Machado, G.M., Gomes, A.C., Rumpf, R., Franco, M.M. & Dode, M.A. (2014). Vitrification of bovine oocytes at different meiotic stages using the Cryotop method: assessment of morphological, molecular and functional patterns. Cryobiology, 69(2), 256-265. doi: 10.1016/j.cryobiol.2014.07.015.
Vajta, G., Holm, P., Kuwayama, M., Booth, P.J., Jacobsen, H., Greve, T. & Callesen, H. (1998). Open Pulled Straw (OPS) vitrification:a new way to reduce cryoinjuries of bovine ova and embryos. Molecular Reproduction and Development, 51(1), 1998, p.53-58.
Vanderzwalmen, P., Bertin, G., Debauche, C.H., Standaert, V., Bollen, N., Van Roosendaal, E., Vandervorst, M., Schoysman, R. & Zech, N. (2003). Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing. Human Reproduction, 18(7), 1504-1511.
Wiesak, T., Wasielak, M., Złotkowska, A. & Milewski, R. (2017). Effect of vitrification on the zona pellucida hardening and follistatin and cathepsin B genes expression and developmental competence of in vitro matured bovine oocytes. Cryobiology, 76, 18-23. doi: 10.1016/j.cryobiol.2017.05.001.
Zhou, X.L., Al Naib, A., Sun, D.W. & Lonergan, P. (2010). Bovine oocyte vitrification using the Cryotop method: effect of cumulucells and vitrification protocol on survival and subsequent development. Cryobiology, 61(1), 66-72. doi: 10.1016/j.cryobiol.2010.05.002.

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