Specifics of vitrification of in vitro-produced cattle embyos at various development stages

  • V. V. Kovpak National University of Life and Environmental Sciences of Ukraine
  • O. S. Kovpak Limited Liability Company “BioTechCom”
  • O. A. Valchuk National University of Life and Environmental Sciences of Ukraine
  • Y. V. Zhuk National University of Life and Environmental Sciences of Ukraine
  • S. S. Derkach National University of Life and Environmental Sciences of Ukraine
Keywords: equilibration; protocol of cryoconservation; biotechnology of reproduction of ruminants; morula; blastocyst


Producing embryos in vitro is an important technology used to improve the genetic potential of cattle and perfect the programs of their breeding. Regardless of the way they are produced, all embryos that had not been used for transplantation to recipients must be conserved. Because of significantly increased interest in the problem of cryoconservation of embryos, both coming from scientists and businesses, there are emerging new commercial environments that allow the facilitation of cryoconservation and the increase in the embryo survival. Oocyte-cumulus complexes obtained from the ovaries of slaughtered clinically healthy cows matured in 22–24 h in in vitro conditions. The oocytes were co-cultured with spermatozoids in Fertilization medium, and the obtained zygotes were cultured in Culture medium with Sodium-Pyruvate for 4 or 7 days up to the stage of morula or blastocyste, respectively. For the vitrification of cow embryos, we used a commercial kit for the vitrification of human embryos, having compared the duration of equilibration. According to the results of the studies, we observed high efficiency of cryoconservation of cow embryos using the commercial kit for vitrification of human embryos. The results revealed the significant effect of equilibration on survival and further development of embryos. In addition, we described the dependence of development stage of cattle embryo on the duration of the contact of embryo with equilibration solution. Therefore, optimal time of contact of cattle embryos at the morula stage with equilibration solution was 12 minutes. On the 24th h after thawing, 46.7 ± 3.3% of the embryos were observed to undergo blastulation, and on 48th h, this parameter increased to 96.7 ± 3.3%, which corresponded to the parameters in the group of embryos that had not been subjected to cryoconservation. In the conditions of further cultivation, the percentage of blastocystes that hatched in the experimental group was no different from that of the control. At the same time, the highest efficiency of vitrification of blastocystes of cows was seen after the contact with the equilibration solution for 15 min, since the percentage of hatched blastocystes was the same as in the control group. Therefore, using the commercial kit for vitrification of human embryos is beneficial, for it promotes the parameters of cow embryos after vitrification/thawing that are similar to such of intact embryos (without freezing). The data we analyzed and presented in the paper could help to increase the efficiency of cryoconservation of cattle embryos for both scientific and commercial purposes.


Aardema, H., Bertijn, I., van Tol, H., Rijneveld, A., Vernooij, J., Gadella, B. M., & Vos, P. (2022). Fatty acid supplementation during in vitro embryo production determines cryosurvival characteristics of bovine blastocysts. Frontiers in Cell and Developmental Biology, 10, 837405.
Abdalla, H., Shimoda, M., Hara, H., Morita, H., Kuwayama, M., Hirabayashi, M., & Hochi, S. (2010). Vitrification of ICSI- and IVF-derived bovine blastocysts by minimum volume cooling procedure: Effect of developmental stage and age. Theriogenology, 74(6), 1028–1035.
Aono, A., Nagatomo, H., Takuma, T., Nonaka, R., Ono, Y., Wada, Y., Abe, Y., Takahashi, M., Watanabe, T., & Kawahara, M. (2013). Dynamics of intracellular phospholipid membrane organization during oocyte maturation and successful vitrification of immature oocytes retrieved by ovum pick-up in cattle. Theriogenology, 79(8), 1146–1152.
Arshad, U., Sagheer, M., González-Silvestry, F. B., Hassan, M., & Sosa, F. (2021). Vitrification improves in-vitro embryonic survival in Bos taurus embryos without increasing pregnancy rate post embryo transfer when compared to slow-freezing: A systematic meta-analysis. Cryobiology, 101, 1–11.
Asgari, V., Hosseini, S. M., Forouzanfar, M., Hajian, M., & Nasr-Esfahani, M. H. (2012). Vitrification of in vitro produced bovine embryos: Effect of embryonic block and developmental kinetics. Cryobiology, 65(3), 278–283.
Bó, G., & Mapletoft, R. (2013) Evaluation and classification of bovine embryos. Animal Reproduction, 54, 344–348.
Boland, M. P., Goulding, D., & Roche, J. F. (1991). Alternative gonadotrophins for superovulation in cattle. Theriogenology, 35, 5–17.
Campos-Chillòn, L. F., Walker, D. J., de la Torre-Sanchez, J. F., & Seidel Jr., G. E. (2006). In vitro assessment of a direct transfer vitrification procedure for bovine embryos. Theriogenology, 65(6), 1200–1214.
Capper, J. L., Cady, R. A., & Bauman, D. E. (2009). The environmental impact of dairy production: 1944 compared with 2007. Journal of Animal Science, 87(6), 2160–2167.
Do, V. H., & Taylor-Robinson, A. W. (2020). Cryopreservation of in vitro-produced bovine embryos by vitrification: In pursuit of a simplified, standardized procedure that improves pregnancy rates to promote cattle industry use. Biotechnology in Animal Husbandry, 36(3), 251–270.
Dobrinsky, J. R. (2002). Advancements in cryopreservation of domestic animal embryos. Theriogenology, 57(1), 285–302.
Dochi, O. (2019). Direct transfer of frozen-thawed bovine embryos and its application in cattle reproduction management. The Journal of Reproduction and Development, 65(5), 389–396.
Egger-Danner, C., Cole, J. B., Pryce, J. E., Gengler, N., Heringstad, B., Bradley, A., & Stock, K. F. (2015). Invited review: Overview of new traits and phenotyping strategies in dairy cattle with a focus on functional traits. Animal, 9(2), 191–207.
Estudillo, E., Jiménez, A., Bustamante-Nieves, P. E., Palacios-Reyes, C., Velasco, I., & López-Ornelas, A. (2021). Cryopreservation of gametes and embryos and their molecular changes. International Journal of Molecular Sciences, 22(19), 10864.
Ferré, L. B., Kjelland, M. E., Taiyeb, A. M., Campos-Chillon, F., & Ross, P. J. (2020). Recent progress in bovine in vitro-derived embryo cryotolerance: Impact of in vitro culture systems, advances in cryopreservation and future considerations. Reproduction in Domestic Animals, 55(6), 659–676.
Gerber, P. J., Steinfeld, H., Henderson, B., Mottet, A., Opio, C., Dijkman, J., Falucci, A., & Tempio, G. (2013). Tackling climate change through livestock – a global assessment of emissions and mitigation opportunities. Food and Agriculture Organization of the United Nations, Rome.
Gómez, E., Carrocera, S., Martín, D., Pérez-Jánez, J. J., Prendes, J., Prendes, J. M., Vázquez, A., Murillo, A., Gimeno, I., & Muñoz, M. (2020). Efficient one-step direct transfer to recipients of thawed bovine embryos cultured in vitro and frozen in chemically defined medium. Theriogenology, 146, 39–47.
Gupta, A., Singh, J., Dufort, I., Robert, C., Dias, F. C. F., & Anzar, M. (2017). Transcriptomic difference in bovine blastocysts following vitrification and slow freezing at morula stage. PLoS One, 12(11), e0187268.
Gutnisky, C., Alvarez, G. M., Cetica, P. D., & Dalvit, G. C. (2013). Evaluation of the Cryotech Vitrification Kit for bovine embryos. Cryobiology, 67(3), 391–393.
Hasler, J. F. (2014). Forty years of embryo transfer in cattle: A review focusing on the journal Theriogenology, the growth of the industry in North America, and personal reminisces. Theriogenology, 81(1), 152–169.
Hasler, J. F., Bilby, C. R., Collier, R. J., Denham, S. C., & Lucy, M. C. (2003). Effect of recombinant bovine somatotropin on superovulatory response and recipient pregnancy rates in a commercial embryo transfer program. Theriogenology, 59, 1919–1928.
Inaba, Y., Aikawa, Y., Hirai, T., Hashiyada, Y., Yamanouchi, T., Misumi, K., Ohtake, M., Somfai, T., Kobayashi, S., Saito, N., Matoba, S., Konishi, K., & Imai, K. (2011). In-straw cryoprotectant dilution for bovine embryos vitrified using Cryotop. Journal of Reproduction and Development, 57(4), 437–443.
Inaba,Y., Miyashita, S., Somfai, T., Geshi, M., Matoba, S., Dochi, O., & Nagai, T. (2016). Cryopreservation method affects DNA fragmentation in trophectoderm and the speed of re-expansion in bovine blastocysts. Cryobiology, 72(2), 86–92.
Janati Idrissi, S., Le Bourhis, D., Lefevre, A., Emond, P., Le Berre, L., Desnoës, O., Joly, T., Buff, S., Maillard, V., Schibler, L., Salvetti, P., & Elis, S. (2021). Lipid profile of bovine grade-1 blastocysts produced either in vivo or in vitro before and after slow freezing process. Scientific Reports, 11(1), 11618.
Jennifer, A., Hernandez, G., & Craig, A. G. (2013). Role of reproductive biotechnologies in enhancing food security and sustainability. Animal Frontiers, 3(3), 14–19.
Juanpanich, T., Suttirojpattana, T., Takayama, M., Liang, Y., Dochi, O., Parnpai, R., & Imai, K. (2018). Survival and developmental competence of bovine embryos at different developmental stages and separated blastomeres after vitrification in different solutions. Animal Science Journal, 89(1), 42–51.
Kocyigit, A., & Cevik, M. (2016). Correlation between the cryosurvival, cell number and diameter in bovine in vitro produced embryos. Cryobiology, 73(2), 203–208.
Liebermann, J., & Tucker, M. J. (2006). Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application. Fertility and Sterility, 86(1), 20–26.
Løvendahl, P., Difford, G. F., Li, B., Chagunda, M. G. G., Huhtanen, P., Lidauer, M. H., Lassen, J., & Lund, P. (2018). Review: Selecting for improved feed efficiency and reduced methane emissions in dairy cattle. Animal, 12(s2), s336–s349.
Mahmoudzadeh, A. R., Van Soom, A., Ysebaert, M. T., & de Kruif, A. (1994). Comparison of two-step vitrification versus controlled freezing on survival of in vitro produced cattle embryos. Theriogenology, 42(8), 1389–1397.
Martínez-Rodero, I., García-Martínez, T., Ordóñez-León, E. A., Vendrell-Flotats, M., Olegario Hidalgo, C., Esmoris, J., Mendibil, X., Azcarate, S., López-Béjar, M., Yeste, M., & Mogas, T. A. (2021). Shorter equilibration period improves post-warming outcomes after vitrification and in straw dilution of in vitro-produced bovine embryos. Biology, 10(2), 142.
Moore, S. G., & Hasler, J. F. (2017). A 100-year review: Reproductive technologies in dairy science. Journal of Dairy Science, 100, 10314–10331.
Morató, R., Izquierdo, D., Paramio, M. T., & Mogas, T. (2010). Survival and apoptosis rates after vitrification in cryotop devices of in vitro-produced calf and cow blastocysts at different developmental stages. Reproduction, Fertility and Development, 22, 1141–1147.
Mucci, N., Aller, J., Kaiser, G. G., Hozbor, F., Cabodevila, J., & Alberio, R. H. (2006). Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification. Theriogenology, 65(8), 1551–1562.
Najafzadeh, V., Bojsen-Møller Secher, J., Pihl, M., Ærenlund, A., Jørgensen, N., Jensen, K. K., Jensen, M. T., Fenner, M. F., Strøbech, L., & Hyttel, P. (2021). Vitrification yields higher cryo-survival rate than slow freezing in biopsied bovine in vitro produced blastocysts. Theriogenology, 171, 44–54.
Nedambale, T. L., Dinnyés, A., Groen, W., Dobrinsky, J. R., Tian, X. C., & Yang, X. (2004). Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification. Theriogenology, 62, 437–449.
Oliveira, C. S., da Silva Feuchard, V. L., de Freitas, C., da Silva Rosa, P. M., dos Camargo, A. J. R., & Saraiva, N. Z. (2020). In-straw warming protocol improves survival of vitrified embryos and allows direct transfer in cattle. Cryobiology, 97, 222–225.
Parrish, J. J., Susko-Parrish, J. L., Leibfried-Rutledge, M. L., Critser, E. S., Eyestone, W. H., & First, N. L. (1986). Bovine in vitro fertilization with frozen-thawed semen. Theriogenology, 25(4), 591–600.
Petrescu-Mag, R. M., Burny, P., Banatean-Dunea, I., & Petrescu, D. C. (2022). How climate change science is reflected in people’s minds: A cross-country study on people’s perceptions of climate change. International Journal of Environmental Research and Public Health, 19(7), 4280.
Pugh, P. A., Tervit, H. R., & Niemann, H. (2000). Effects of vitrification medium composition on the survival of bovine in vitro produced embryos, following in straw-dilution, in vitro and in vivo following transfer. Animal Reproduction Science, 58, 9–22.
Rall, W. F., & Fahy, G. M. (1985). Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification. Nature, 313(6003), 573–575.
Raney, T., Skoet, J., & Steinfeld, H. (2009). The state of food and agriculture: Livestock in the balance. Food and Agriculture Organization of the United Nations, Rome.
Rienzi, L., Gracia, C., Maggiulli, R., LaBarbera, A. R., Kaser, D. J., Ubaldi, F. M., Vanderpoel, S., & Racowsky, C. (2017). Oocyte, embryo and blastocyst cryopreservation in ART: Systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance. Human Reproduction Update, 23(2), 139–155.
Rocha, J. C., Passalia, F., Matos, F. D., Maserati Jr., M. P., Alves, M. F., Almeida, T. G., Cardoso, B. L., Basso, A. C., & Nogueira, M. F. (2016). Methods for assessing the quality of mammalian embryos: How far we are from the gold standard? JBRA Assisted Reproduction, 20(3), 150–158.
Sanches, B. V., Lunardelli, P. A., Tannura, J. H., Cardoso, B. L., Colombo Pereira, M. H., Gaitkoski, D., Basso, A. C., Arnold, D. R., & Seneda, M. M. (2016). A new direct transfer protocol for cryopreserved IVF embryos. Theriogenology, 85, 1147–1151.
Shirazi, A., Nazari, H., Ahmadi, E., Heidari, B., & Shams-Esfandabadi, N. (2009). Effect of culture system on survival rate of vitrified bovine embryos produced in vitro. Cryobiology, 59(3), 285–290.
Siberski-Cooper, C. J., & Koltes, J. E. (2021). Opportunities to harness high-throughput and novel sensing phenotypes to improve feed efficiency in dairy cattle. Animals, 12(1), 15.
Sommerfeld, V., & Niemann, H. (1999). Cryopreservation of bovine in vitro produced embryos using ethylene glycol in controlled freezing or vitrification. Cryobiology, 38(2), 95–105.
Souza, J. F., Oliveira, C. M., Lienou, L. L., Cavalcante, T. V., Alexandrino, E., Santos, R. R., Rodrigues, A. P. R., Campello, C. C., Figueiredo, J. R., & Dias, F. E. F. (2018). Vitrification of bovine embryos followed by in vitro hatching and expansion. Zygote, 26(1), 99–103.
Stinshoff, H., Wilkening, S., Hanstedt, A., Brüning, K., & Wrenzycki, C. (2011). Cryopreservation affects the quality of in vitro produced bovine embryos at the molecular level. Theriogenology, 76(8), 1433–1441.
Szurek, E. A., & Eroglu, A. (2011). Comparison and avoidance of toxicity of penetrating cryoprotectants. PloS One, 6(11), e27604.
Uchikura, A., Matsunari, H., Nakano, K., Hatae, S., & Nagashima, H. (2016). Application of hollow fiber vitrification for cryopreservation of bovine early cleavage stage embryos and porcine morula-blastomeres. Journal of Reproduction and Development, 62(2), 219–223.
Vajta, G., Holm, P., Greve, T., & Callesen, H. (1996). Overall efficiency of in vitro embryo production and vitrification in cattle. Theriogenology, 45(3), 683–689.
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), 53–58.
Valente, R. S., Marsico, T. V., & Sudano, M. J. (2022). Basic and applied features in the cryopreservation progress of bovine embryos. Animal Reproduction Science, 239, 106970.
Vanderzwalmen, P., Bertin, G., Debauche, C., Standaert, V., van Roosendaal, E., Vandervorst, M., Bollen, N., Zech, H., Mukaida, T., Takahashi, K., & Schoysman, R. (2002). Births after vitrification at morula and blastocyst stages: Effect of artificial reduction of the blastocoelic cavity before vitrification. Human Reproduction, 17(3), 744–751.
Viana, J. (2020). 2019 statistics of embryo production and transfer in domestic farm animals: Divergent trends for IVD and IVP embryos. Embryo Technology Newsletter, 38(4), 1–15.
Vieira, A. D., Forell, F., Feltrin, C., & Rodrigues, J. L. (2007). In-straw cryoprotectant dilution of IVP bovine blastocysts vitrified in hand-pulled glass micropipettes. Animal Reproduction Science, 99, 377–383.
Wang, X., Hua, T. C., Sun, D. W., Liu, B., Yang, G., & Cao, Y. (2007). Cryopreservation of tissue-engineered dermal replacement in Me2SO: Toxicity study and effects of concentration and cooling rates on cell viability. Cryobiology, 55(1), 60–65.
Wilmut, I., & Rowson, L. E. A. (1973). Experiments on the low-temperature preservation of cow embryos. Veterinary Rehabilitation, 92, 686–690.
How to Cite
Kovpak, V. V., Kovpak, O. S., Valchuk, O. A., Zhuk, Y. V., & Derkach, S. S. (2022). Specifics of vitrification of in vitro-produced cattle embyos at various development stages . Regulatory Mechanisms in Biosystems, 13(3), 265-271. Retrieved from https://medicine.dp.ua/index.php/med/article/view/822

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