doi: 10.15389/agrobiology.2019.6.1206eng

UDC: 636.2:591.39

Supported by grant from Russian Foundation for Basic Research (project No. 17-29-08035) and the Ministry of Science and Higher Education of the Russian Federation



G.N. Singina, E.N. Shedova

Ernst Federal Science Center for Animal Husbandry, 60, pos. Dubrovitsy, Podolsk District, Moscow Province, 142132 Russia, e-mail (✉ corresponding author),

Singina G.N.
Shedova E.N.

Received September 17, 2019


In vitro maturation (IVM) of the oocytes is an important step for in vitro embryo production (IVP). In vitro culture during maturation decreases oocyte quality and therefore, IVM conditions need to be improved. Routinely, IVM of bovine oocytes is performed using one-step medium, the TC-199 complemented with fetal bovine serum (FBS) and gonadotropins. However, the asynchrony of oocyte nuclear and cytoplasm maturation processes may require the differential hormonal environment during IVM. In the present work, for the first time we have compared the efficiency of the two-step IVM protocols, which include the first step in standard conditions and final maturation in hormone-free mediums TC-199 or Fert-TALP; the last one is a routine medium for in vitro fertilization (IVF). The objective was to study the effects of two-step IVM protocols to chromosome modifications and apoptotic events in mature oocytes and a quality of in vitro produced embryos. Oocyte-cumulus cells complexes (ОСС) were in vitro matured during 16 h in medium TC-199 complemented with 10 % FBS, 10 mg/ml follicle-stimulating hormone and 10 mg/ml luteinizing hormone, and then transferred to either medium TC-199 without gonadotropins (System 1) or Fert-TALP medium (System 2) for additional 8 h of IVM. After final IVM, nuclear state and apoptosis were checked in a part of the mature oocytes. IVF was performed on remaining OCC to analyze in vitro embryo development. No difference was observed in nuclear maturation rates between the systems 1 and 2 after 24 h IVM: 83.3 % and 84.7 % of the oocytes reached metaphase-II. Apoptosis rate were significantly lower (p < 0.05) in the oocytes matured in System 2 (11.7±0.7 %) compared to System 1 (19.4±1.1 %). In addition, blastocyst rate after 7 days of in vitro embryo development was significantly higher (p < 0.05) in System 2 (30.0±2.9 %), than in System 1(17.4±0.4 %). Oocyte cleavage rate, cell number and apoptosis rate in the blastocysts were similar in both IVM systems. In conclusion, two-step IVM system using final 8-hour maturation in Fert-TALP medium is more advantageous compared to a system including hormone-free TC-199 medium at the end of IVM. Fert-TALP IVM system decreased apoptosis rate in the oocytes and increased their competence to in vitro embryo development after IVF.

Keywords: bovine oocytes, in vitro maturation, maturation media, apoptosis, embryo development.



  1. Lonergan P., Fair T. In vitro-produced bovine embryos: dealing with the warts. Theriogenology, 2008, 69: 17-22 CrossRef
  2. Galli C., Duchi R., Crotti G., Turini P., Ponderato N., Colleoni S., Lagutina I., Lazzari G. Bovine embryo technologies. Theriogenology, 2003, 59(2): 599-616 CrossRef
  3. Ponsart S., Le Bourhis D., Knijn H., Fritz S., Guyader-Joly C., Ottern T., Lacaze S., Charreaux F., Schibler L., Dupassieux D., Mullaart E. Reproductive technologies and genomic selection in dairy cattle. Reproduction, Fertility and Development, 2013, 26(1): 12-21 CrossRef
  4. Blanco M.R., Demyda Peyrás S., Moreno M.M., Genero E. Developmental competence of in vivo and in vitro matured oocytes: a review. Biotechnology and Molecular Biology Review, 2011, 6: 155-165.
  5. Nagano M. Acquisition of developmental competence and in vitro growth culture of bovine oocytes. Journal of Reproduction and Development, 2019, 65(3): 195-201 CrossRef
  6. Hyttel P., Xu K.P., Smith S., Greve T. Ultrastructure of in-vitro oocyte maturation in cattle. J. Reprod. Fertil., 1986, 78: 615-625 CrossRef
  7. Abd El-Aziz A.H., Mahrous U.E., Kamel S.Z., Sabek A.A. Factors influencing in vitro production of bovine embryos: a review. Asian Journal of Animal and Veterinary Advances, 2016, 11: 737-756 CrossRef
  8. Niemann H., Wrenzycki C. Alterations of expression of developmentally important genes in preimplantation bovine embryos by in vitro culture conditions: implications for subsequent development. Theriogenology, 2000, 53: 21-34 CrossRef
  9. Dominko T., First N.L. Timing of meiotic progression in bovine oocytes and its effect on early embryo development. Molecular Reproduction and Development, 1997, 47: 456-467 CrossRef
  10. Ward F., Enright B., Rizos D., Boland M., Lonergan P. Optimization of in vitro bovine embryo production: effect of duration of maturation, length of gamete co-incubation, sperm concentration and sire. Theriogenology, 2002, 57(8): 2105-2117 CrossRef
  11. Russell D.F., Baqir S., Bordignon J., Betts D.H. The impact of oocyte maturation media on early bovine embryonic development. Molecular Reproduction and Development, 2006, 73(10): 1255-1270 CrossRef
  12. McKiernan S.H., Bavister B.D. Different lots of bovine serum albumin inhibit or stimulate in vitro development of hamster embryos. In Vitro Cell Dev. Biol. — Animal, 1992, 28A: 154-156 CrossRef
  13. Sagirkaya H., Misirlioglu M., Kaya A., First N.L., Parrish J.J., Memili E. Developmental potential of bovine oocytes cultured in different maturation and culture conditions. Animal Reproduction Science, 2007, 101: 225-240 CrossRef
  14. Singina G.N., Lebedeva I.Yu., Shedova E.N., Taradajnic T.E., Mityashova O.S., Tsyndrina E.V., Danch S.S. Bovine oocyte ability to embryonic development when maturing in different two-phase culture systems. Agricultural Biology [Sel’skokhozzyaistvannaya Biologiya], 2017, 52(4): 776-784 CrossRef
  15. Parrish J.J. Bovine in vitro fertilization: in vitro oocyte maturation and sperm capacitation with heparin. Theriogenology, 2014, 81(1): 67-73 CrossRef
  16. Lebedeva I.Yu., Singina G.N., Shedova E.N., Lopukhov A.V., Mityashova O.S. Resistence of bovine oocytes to age-realated changes after exposure to luteotropic factors during the second phase of culture till the metaphase II stage. Agricultural Biology [Sel’skokhozzyaistvannaya Biologiya], 2018, 53(6): 1202-1211 CrossRef
  17. Singina G.N., Bagirov V.A., Danch  S.S., Taradainik T.E., Dotsev A.V., Zinovieva N.A. In vitro production of interspecies hybrid embryos of cattle (Bos taurus) and wisent (Bison bonasus). Agricultural Biology [Sel’skokhozzyaistvannaya Biologiya], 2016, 51(6): 824-829 CrossRef
  18. Lebedeva I.Yu., Singina G.N., Lopukhov A.V., Zinovieva N.A. Dynamics of morphofunctional changes in aging bovine ova during prolonged culture in vitro. Cell and Tissue Biology, 2014, 8(3): 258-266 CrossRef
  19. Guemra S., da Silva Santo E., Zanin R., Monzani P.S., Sovernigo T.C., Ohashi O.M., Verde Leal C.L., Adona P.R. Effect of temporary meiosis block during prematuration of bovine cumulus-oocyte complexes on pregnancy rates in a commercial setting for in vitro embryo production. Theriogenology, 2014, 81(7): 982-987 CrossRef
  20. Salhab M., Tosca L., Cabau C., Papillier P., Perreau C., Dupont J, Mermillod P., Uzbekova S. Kinetics of gene expression and signaling in bovine cumulus cells throughout IVM in different mediums in relation to oocyte developmental competence, cumulus apoptosis and progesterone secretion. Theriogenology, 2011, 75(1): 90-104 CrossRef
  21. Mastromonaco G.F., Semple E., Robert C., Rho G.J., Betts D.H., King W.A. Different culture media requirements of IVF and nuclear transfer bovine embryos. Reproduction in Domestic Animals, 2004, 39: 462-467 CrossRef
  22. Lonergan P., Kearney-Flynn M.O., Boland M.P. Effect of protein supplementation and presence of an antioxidant on the development of bovine zygotes in synthetic oviduct fluid medium under high or low oxygen tension. Theriogenology, 1999, 51: 1565-1576 CrossRef
  23. Abe H., Yamashita S., Itoh T., Satoh T., Hoshi H. Ultrastructure of bovine embryos developed from in vitro-matured and fertilized oocytes: comparative morphological evaluation of embryos cultured either in serum-free medium or in serum-supplemented medium. Molecular Reproduction and Development, 1999, 53: 325-335 CrossRef
  24. Wrenzycki C., Herrmann D., Carnwath J.W., Niemann H. Alterations in the relative abundance of gene transcripts in preimplantation bovine embryos cultured in medium supplemented with either serum or PVA. Molecular Reproduction and Development, 1999, 53: 8-18 CrossRef






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