• P. A. Trotskiy Institute of Animal Breeding and Genetics nd. a. M.V.Zubets of NAAN, (Chubynske, Ukraine)
Keywords: cryopreservation, oocyte-cumulus complexes, cryoprotector, vitrification solution, in vitro maturation, embryos


Implementation of biotechnological process in livestock should be considered not only in terms of selection process intensification (obtain of embryos in vitro, their transplantation) and to a greater extent as development of effective methods of freezing and long storage of mammalian cells, including ova and embryos. Application of biotechnology in livestock breeding increases rate of genetic progress, preservation of gene pool of breeds via banks of sperm, embryos and cryobank of oocytes, obtaining and regulation of progeny of the desired sex, providing genetic evaluation of gametes and embryos, and it will enable to use genetic potential of animals after culling by age, replicate and create new genotypes with desired properties repeatedly.

Solution to this problem is to improve medium and conditions of gametes and embryos freezing. Although the overall development of cryopreservation method is through simplification of the equilibration and vitrification solutions which would be able to ensure the full development frozen-thawed gametes. Addition of biologically active substances to the solution for cryopreservation contributes to protection of gametes during freezing and thawing, and determination of the consistent patterns of these substances will improve procedures of frozen-thawed oocyte cultivation outside the body. So it is necessary to deepen the fundamental research on the mechanisms of formation of a mature ovum of cows obtained from frozen-thawed oocytes to obtain embryos.

The aim of the research is to conduct comparative analysis of different biologically active substances in equilibration and vitrification solutions at cryopreservation of oocyte-cumulus complexes of cows.

Material and methods of the research. The objects of experimental studies were oocyte-cumulus complexes of black-and-white cows. The oocytes with homogeneous fine-grained ooplasm, undamaged pellucid zone, thick or partially loosened cumulus were used for freezing.

The gametes of cows were treated by equilibration solution before freezing and then were transferred into vitrification solution. All the equilibration (10% glycerol + 20% propanediol) and vitrification (25% glycerol + 25% propanediol) solutions for cryopreservation of cows’ oocyte-cumulus complexes were prepared in Dulbecco phosphatebuffered saline with addition of 20% fetal serum of cows, 1х10-4 M unithiol, 1х10-6 M acetylcholine and without addition of bioactive substances.

The research on adding some biologically active substances (fetal serum of cows – version A, unithiol – version B, acetylcholine – version C, without addition of biologically active substances – version D and not frozen cells of the control group (K)) in equilibration and vitrification solutions at freezing the oocyte-cumulus complexes of cows was carried out.

It was found by the results of experimental studies that introduction of the above-mentioned components into the equilibration solution for freezing cows’ oocyte-cumulus complexes increased their cryoresistance, as evidenced by increasing indicator of maturation of frozen-thawed gametes outside the body to metaphase-2 of meiosis after 27-hour cultivation by 5,4-23,0% and decreasing indicator of number of oocytes with chromosomal abnormalities by 2,9-15,3%.

The introduction of biologically active substances into the vitrification solution and subsequent cultivation during 27 hours after freezing and thawing cows’ oocyte-cumulus complexes showed that the indicator of maturation of frozen-thawed gametes outside the body to metaphase-2 of meiosis increased by 3,9-16,4% and the indicator of number of oocytes with chromosomal abnormalities decreased by 2,9-8,4%.

Comparative analysis of the results of in vitro fertilization of cows’ frozen-thawed ova which were frozen using fetal serum of cows (version A) and without it (version - B) has shown a positive effect of adding it to equilibration and vitrification medium at freezing gametes of cows; it led to increase of obtaining bovine embryos in vitro by 11,5%.

Thus, the analysis of experimental results showed different efficiency of using fetal serum of cows, unithiol, acetylcholine in the equilibration solution for freezing cows’ oocyte-cumulus complexes. The advantage of using these biologically active substances in the vitrification solution for cryopreservation of cow’s gametes by the indicator of maturation of frozen-thawed oocyte-cumulus complexes outside the body to metaphase-2 of meiosis wasn’t established.


  1. Introduction of fetal serum into cryopreservation solution increases cryoresistance of cow’s oocytes to cooling leading to increasing the indicator of the matured frozen-thawed gametes outside the body to metaphase-2 of meiosis by 23,0% and the obtained bovine embryos in vitro by 11,5%.


1. Kovtun, S. I. 2007. Novi biotekhnolohichni metody zberezhennya henetychnykh resursiv tvaryn – New biotechnological methods for preserving animal genetic resources, Problemy zberezhennja genofondu tvaryn : materialy tvorchoi' dyskusii' – Preservation of the gene pool of animals: Materials creative discussion. Kyiv, Ahrarna nauka, 44–45 (in Ukrainian).
2. Guzjeva, I. V., and Ju. F. Mel'nyka. 2009. Prohrama zberezhennya henofondu osnovnykh vydiv sil's'kohospodars'kykh tvaryn v Ukrayini na period do 2015 roku – Program preserve the gene pool of the main types of farm animals in Ukraine for the period up to 2015, zah. nauk. red. I.V. Huzyeva, konsul'tatsiya ta spetsyfikatsiya Yu.F. Mel'nyka – common. Science. ed. I.V Huzyeva, consultation and specification Yu.F. Melnyk Kyiv, Aristey, 132 (in Ukrainian).
3. Woods, E. J., J. D. Benson, Y. Agca, and J. K. Crister. 2004. Fundamental cryobiology of reproductive cells and tissue. Cryobiology. 48:146–156.
4. Halyts'ka, T. V., and P. A. Trots'kyy. 2015. Osoblyvosti otrymannya embrioniv svyney in vitro v systemi zberezhennya bioriznomanittya tvaryn – Peculiarities of pig embryos in vitro system biodiversity of animals. Rozvedennya i henetyka tvaryn – Animal Breeding and Genetics. Kyiv. (49): 243–247 (in Ukrainian).
5. Caamano J.N., E. Gomez, B. Trigal, et al. M. Munoz, S. Carrocera, D. Martin, and C. Diez. 2015. Survival of vitrified in vitro–produced bovine embryos after a one-step warming in-straw cryoprotectant dilution procedure. Theriogenology. 83(5): 881–890.
6. Arav, A. 2014. Cryopreservation of oocytes and embryos. Theriogenology. 81(1):96–102.
7. Christopher G. Grupen. 2014. The evolution of porcine embryo in vitro production. Theriogenology. 81(1):24–37.
8. Trots'kyy, P.A. 2011. Otsinka zhyttyezdatnosti dekonservovanykh ootsyt-kumulyusnykh kompleksiv koriv zamorozhenykh nadshvydkym metodom – Assessment of the viability of canned oocyte-cumulus complexes cows on frozen fast method. Visnyk Ukrayins'koho tovarystva henetykiv i selektsioneriv – Bulletin of the Ukrainian Society of geneticists and breeders. Kyiv. 9(2): 283–287 (in Ukrainian).
9. Parrish, J.J., J.L. Susko-Parrish, R.R. Handrow, M.M. Sims, and N.L. First. 1989. Capacitation of bovine spermatozoa by oviduct fluid. Biol. Reprod. 40:1020–1025.
How to Cite