Effect of duration of сycloheximide and 6‐dimethylaminopurine (6‐DMAP) treatments on development competence of cloned embryos in cattle

Cycloheximide and 6‐dimethylaminopurine (6‐DMAP) are widely used in protocols of somatic cell nuclear transfer (SCNT) for inhibition of maturation promoting factor (MPF) activity in SCNT-oocytes in the post-activation period of their culture. Nevertheless, you should remember that these agents have a wide range of activity and can conflict with other cell processes. Therefore, a definition of the optimal period of culture of SCNT-oocytes with the previously mentioned inhibitors may help to prevent the undesirable negative consequences. In this research the effects of cycloheximide (10 µg/ml) and 6‐DMAP (2mM) treatments duration (3.0, 4.0 or 5.0 h) on the reprogramming of somatic nuclear was estimated by the cleavage and blastocyst rates, and by the total cell number and a level of apoptotic cell in the obtained cloned blastocysts. The cleavage rate did not differ between the experimental groups, varying from 63.7 to 77.0 %. Also, there was not found an effect of treatment duration of the investigated factors on the development of activated SCNT-oocytes before blastocyst stage. For 3-hr treatment, the blastocyst rate was 19.6 ± 1.8%. The prolonged up to 4 and 5 hours duration did not change this rate. Meanwhile, we found out the effect of culture duration with сycloheximide and 6‐DMAP on quality of cloned embryos. In case of 3-hr treatment, the total cell number in cloned blastocyst was 58.8 ± 2.4. With prolongation of duration up to 4 hours the result was growing up to 76.6 ± 1.4 (p<0.05), but prolongation up to 5 hours reduced the total cell number in blastocyst as compared to that in 4-hr group (p<0.05). The apoptosis rate had no difference between the treated groups and had its variety between 5.4 to 7.0 %. Our date indicate that efficiency of bovine cloned embryo production depends on duration of 6-DMAP and cycloheximide treatment of the SCNT-oocytes in the post-activated period of their culture. The optimal duration according to the described protocol of SCNT for the best quality of embryos is 4 hours.

1. Brophy B. Cloned transgenic cattle produce milk with higher levels of beta-casein and kappa-casein / B. Brophy, G. Smolenski, T. Wheeler, D. Wells, P. L’Huillier, G. Laible // Nature Biotechnology. – 2003. – Vol. 21. – P. 157–162. doi: 10,1038/nbt783.

2. Richt J. A. Production of cattle lacking prion protein / J. A. Richt, P. Kasinathan, A. N. Hamir, J. Castilla et. al. // Nature Biotechnology. – 2007. – Vol. 25. – P.132–138. doi: 10.1038/nbt1271.

3. Wu H. TALE nickase-mediated SP110 knock in endows cattle with increased resistance to tuberculosis / H. Wu, Y. Wang, Y. Zhang, M. Yang, J. Lv, J. Liu, Y. Zhang // Proceedings of the National Academy of Sciences. – 2015. – Vol. 112(13). – E1530–E1539. doi: 10.1073/pnas.1421587112.

4. Proudfoot C. Genome edited sheep and cattle / C. Proudfoot, D. F. Carlson, R. Huddart, C. R. Long, J. H. Pryor, T. J. King, S. G. Lillico, A. J. Mileham, D. J. McLaren, C. B. Whitelaw, S. C. Fahrenkrug // Transgenic Research. – 2015. – Vol. 24(1). – P. 147–153. doi: 10,1007/s11248-014-9832-х.

5. Gao Y. Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects / Y. Gao, H. Wu, Y. Wang, X. Liu, L. Chen, Q. Li, C. Cui, X. Liu, J. Zhang, Y. Zhang // Genome biology. – 2017. – Vol. 18(1). – P. 13 doi:10.1186/s13059-016-1144-4.

6. Farin P.W. Errors in development of fetuses and placentas from in vitro-produced bovine embryos / P. W. Farin, J. A. Piedrahita, C. E. Farin // Theriogenology. – 2006. – Vol. 65. – P. 178-191. doi: 10.1016/j.theriogenology.2005.09.022.

7. Bertolini M. Developmental problems during pregnancy after in vitro embryo manipulations / M. Bertolini, L. R. Bertolini, R. P. C. Gerger, C. A. Batchelder, G. B. Anderson // Rev. Bras. Reprod. Anim. – 2007. – Vol. 31. – P. 391-405.

8. Su J. Aberrant mRNA expression and DNA methylation levels of imprinted genes in cloned transgenic calves that died of large offspring syndrome / J. Su, Y. Wang, Q. Liu, B. Yang, Y. Wu, Y. Luo, G. Hu, Y. Zhang // Livestock Science. – 2011. – Vol. 141. – P. 24–35. doi: 10.1016/j.livsci.2011.04.012.

9. Latham K. E. Early and delayed aspects of nuclear reprogramming during cloning / K. E. Latham // Biol. Cell. – 2005. – Vol. 97. – P. 119–132. doi: 10.1042/BC20040068.

10. Milazzotto M. P. Effect of chemical or electrical activation of bovine oocytes on blastocyst development and quality / M. P. Milazzotto, W. B. Feitosa, A. R. S. Coutinho, M. D. Goissis et. al. // Reprod. Dom. Anim. – 2008. – Vol. 43. – P. 319–322. doi: 10.1111/j.1439-0531.2007.00900.x.

11. Wakai T. Artificial activation of mammalian oocytes for cloning / T. Wakai, I. Ito, R.A. Fissore // Pinciples of cloning. – 2014. – P. 3-10. doi: 10.1016/B978-0-12-386541-0.00001-1.

12. Susko-Parrish J.L. Inhibition of protein kinases after an induced calcium transient causes transition of bovine oocytes to embryonic cycles without meiotic completion / J. L. Susko-Parrish, M. L. Leibfried-Rutledge, D. L. Northey, V. Schutzkus, N. L. First // Dev Biol. – 1994. – Vol. 166. – P. 729–739. doi: 10.1006/dbio.1994.1351.

13. Akagi S. Timing of the first cleavage and in vitro developmental potential of bovine somatic cell nuclear transfer embryos activated by different protocols / S. Akagi, S. Tamura, K. Matsukawa // Cellular Reprogramming. – 2020. – Vol. 22(1) P. 36-42. doi: 10.1089/cell.2019.0074.

14. Vichera G. Chemical activation with a combination of ionomycin and dehydroleucodine for production of parthenogenetic, ICSI and cloned bovine embryos / G. Vichera, J. Alfonso, C.C. Duque, M.A. Silvestre, F. Pereyra-Bonnet, R. Fernández-Martín and D. Salamone // Reprod. Domest. Anim. – 2010. – Vol. 45. – P. 306-312.

15. Akagi S. Recent progress in bovine somatic cell nuclear transfer / S. Akagi, M. Geshi, T. Nagai // Animal Science Journal. – 2013. Vol. 84(3). – P. 191-199. doi: 10.1111/asj.12035). 16.

16. Ross P. J. Bovine somatic cell nuclear transfer / P. J. Ross, J. B. Cibelli // Cellular Programming and Reprogramming. – 2010. – Vol. 636. – P. 155-177. doi: 10.1007/978-1-60761-691-7_10.

17. Bavister B.D. Development of preimplantation embryos of the golden hamster in a defined culture medium / B. D. Bavister, M. L. Liebfried, G. Lieberman // Biology of Reproduction. – 1993. – Vol. 28. – P. 235–247. doi: 10.1095/biolreprod28.1.235.

18. Rosenkrans C. F. Jr. Effect of free amino acids and vitamins on cleavage and develop-mental rate of bovine zygotes in vitro / C. F. Jr. Rosenkrans, N. L. First // Journal of Animal Science. – 1994. – Vol. 72(2). – P. 434-437. doi: 10.2527/1994.722434x.

19. German S. D. Livestock somatic cell nuclear transfer. In: Sustainable food production / S. D. German, K. H. S. Campbell P. Christou, R. Savin, B. A. Costa-Pierce, I. Misztal, C. B. A. Whitelaw (eds.) Springer, New York. – 2013. – P. 1067-1095. doi: 10.1007/978-1-4614-5797-8_2.

20. Lan G. C. Effects of duration, concentration, and timing of ionomycin and 6‐dimethylaminopurine (6‐DMAP) treatment on activation of goat oocytes / G. C. Lan, D. Han, Y. G. Wu, Z. B. Han, S. F. Ma, X. Y. Liu, C.L. Chang, J. H. Tan // Molecular Reproduction Development. – 2005. – Vol. 71(3). – P. 380-8. doi: 10.1002/mrd.20267.