Authors: A. S. Barkova, doctor of veterinary sciences, senior scientific researcher, ORCID 0000-0002-2602-6810, AuthorID 610629;
V. A. Makutina, candidate of biological sciences, senior scientific researcher, ORCID 0000-0003-1127-2792, AuthorID 612436.
M. V. Modorov, candidate of biological science, senior scientific researcher, ORCID 0000-0003-1877-2378, AuthorID 178678.
A. G. Isaeva, doctor of veterinary sciences, associate professor, leading scientific researcher, ORCID 0000-0001-8395-1247, AuthorID 665717:
A. S. Krivonogova, doctor of veterinary sciences, associate professor, leading scientific researcher, ORCID 0000-0003-1918-3030, AuthorID 6832391.
Ural Federal Agrarian Scientific Research Centre – Ural Branch of the Russian Academy of Sciences, Ekaterinburg, Russia.
Abstract. To carry out genome editing in cattle, an effective and well-functioning system for obtaining gametes, fertilizingeggs and their cryopreservation is necessary. Aim of the work: review and research of present day existing methods of obtaining, insemination and cryopreservation of donor material, in order to provide genome editing in cows. Methods andmaterials. The work is completed according to the theme No. 0532-2019-0001 “Development of complex technology ofmarker based genome selection of agricultural animals” within State Order of Ministry of Education and Science of the Russian Federation. The analysis of open scientific literature on the issues of in vitro fertilization in animals, cryopreservation of oocytes and embryons, sperm preparation and methods of insemination of cows’ oocytes, and cryopreservation of oocytesand embryons of animals is done. Features of the preparation of biological material of cattle for genome editing by microinjectionin to ooplasm are described. Results of research and duscussion. At present time there are two ways to obtain donormaterial from cattle: from live animals and taking ovaries after slaughtering cows. Material transportation is carried out ata temperature of 30–37 °C depending on the distance to the laboratory and expected time period of transportation. Oocytecumulus complexes can be removed by ovarian dissection and aspiration of visible follicles. In both cases, immature eggsare predominantly obtained. Subsequent ripening is carried out in vitro using special media in a CO2 incubator. The culture medium for oocyte maturation should contain hormones that mimic the peak of LH (luteinizing hormone), which occursin vivo during the maturation of oocytes before ovulation. To accumulate a certain number of eggs at the stage of MII, it is recommended to carry out their cryopreservation by the method of vitrification, having previously released the oocyte from the cumulus cells. After thawing, oocytes need to be incubated for 2–3 hours 38.5 °C in 5–6.5% CO2 to restore the spindle. Inorder to make editing more effective, the introduction of genetic material is recommended to be carried out in parallel with the fertilization method “icsi”. In humans, mice and rabbits, an injection of sperm into the cytoplasm is sufficient to activatethe oocyte, however, in cattle, just microinjection of the sperm is not enough and often the male pronucleus does not form.To solve the problem, various methods are used, including freezing thawing of sperm, resulting in damage of a membrane, oraddition of heparin glutathione into the medium that in creases decondensation of the sperm DNA.
Keywords: genetic modeling, in vitro maturation of oocytes, artificial insemination in cows, oocyte vitrification.
For citation: Barkova A. S., Makutina V. A., Modorov M. V., Isaeva A. G., Krivonogova A. S. Features of the preparation of biological material for genome editing in cattle // Agrarian Bulletin of the Urals. 2019. No. 12 (191). Pp. 40‒44.DOI: 10.32417/1997-4868-2019-191-12-40-44.
