Skip to main content Accessibility help

Several aspects of animal embryo cryopreservation: anti-freeze protein (AFP) as a potential cryoprotectant

  • A. V. Makarevich (a1) (a2), E. Kubovičová (a2), M. Popelková (a3), D. Fabian (a4), Š. Čikoš (a4), J. Pivko (a2) and P. Chrenek (a2) (a5)...


With the development of embryo technologies, such as in vitro fertilization, cloning and transgenesis, cryopreservation of mammalian gametes and embryos has acquired a particular interest. Despite a certain success, various cryopreservation techniques often cause significant morphological and biochemical alterations, which lead to the disruption of cell organelles, cytoskeleton damages, cell death and loss of embryo viability. Ultrastructural studies confirm high sensitivity of the cell membrane and organelle membrane to freezing and thawing. It was found that many substances with low molecular weights have a protective action against cold-induced damage. In this concern, an anti-freeze protein (AFP) and anti-freeze glycoproteins (AFGPs), which occur at extremely high concentrations in fish that live in Arctic waters and protect them against freezing, may be of potential interest for cryostorage of animal embryos at ultra-low temperatures. This mini-review briefly describes several models of AFP/AFGP action to preserve cells against chilling-induced damages and indicates several ways to improve post-thaw developmental potential of the embryo.


Corresponding author

All correspondence to: A.V. Makarevich. Animal Production Research Centre (APRC) Nitra, 95141 Luzianky near Nitra, Slovak Republic. Tel: +421 37 6546 335. Fax: +421 37 6546 480. e-mail:


Hide All
Ahn, H.J., Sohn, I.P., Kwon, H.C., Jo Do, H., Park, Y.D. & Min, C.K. (2002). Characteristics of the cell membrane fluidity, actin fibers, and mitochondrial dysfunctions of frozen–thawed two-cell mouse embryos. Mol. Reprod. Dev. 61, 466–77.
Ali, J. & Shelton, N. (1993). Vitrification of preimplantation stages of mouse embryos. J. Reprod. Fertil. 98, 459–65.
Arav, A., Rubinsky, B., Seren, E., Roche, J.F. & Boland, M.P. (1994). The role of thermal hysteresis proteins during cryopreservation of oocytes and embryos. Theriogenology 41, 107–12.
Asada, M., Ishibashi, S., Ikumi, S. & Fukui, Y. (2002). Effect of polyvinyl alcohol (PVA) concentration during vitrification of in vitro matured bovine oocytes. Theriogenology 58, 1199–208.
Baguisi, A., Arav, A., Crosby, T.F., Roche, J.F. & Boland, M.P. (1997). Hypothermic storage of sheep embryos with antifreeze proteins: development in vitro and in vivo. Theriogenology 48, 1017–24.
Baguisi, A., Lonergan, P., Overstrom, E. & Boland, M. (1999). Vitrification of bovine embryos: incidence of necrosis and apoptosis. Theriogenology 55, 162.
Bautista, J.A. & Kanagawa, H. (1998). Current status of vitrification of embryos and oocytes in domestic animals: ethylene glycol as an emerging cryoprotectant of choice. Jpn. J. Vet. Res. 45, 183–91.
Beebe, L.F., Cameron, R.D., Blackshaw, A.W., Higgins, A. & Nottle, M.B. (2002). Piglets born from centrifuged and vitrified early and peri-hatching blastocysts. Theriogenology 57, 2155–65.
Berthelot, F., Martinat-Botte, F., Locatelli, A., Perreau, C. & Terqui, M. (2000). Piglets born after vitrification of embryos using the open pulled straw method. Cryobiology 41, 116–24.
Cervera, R.P. & Garcia-Ximenez, F. (2003). Vitrification of zona-free rabbit expanded or hatching blastocysts: a possible model for human blastocysts. Hum. Reprod. 18, 2151–6.
Chen, S.U., Lee, T.H., Lien, Y.R., Tsai, Y.Y., Chang, L.J. & Yang, Y.S. (2005). Microsuction of blastocoelic fluid before vitrification increased survival and pregnancy of mouse expanded blastocysts, but pretreatment with the cytoskeletal stabilizer did not increase blastocyst survival. Fertil. Steril. 84, 1156–62.
Cocero, M.J., De La Espina, S.M.D. & Aguilar, B. (2002). Ultrastructural characteristics of fresh and frozen–thawed ovine embryos using two cryoprotectants. Biol. Reprod. 66, 1244–58.
Devries, A.L. & Wohlschlag, D.E. (1969). Freezing resistance in some antarctic fishes. Science 163, 1073–5.
Dobrinsky, J.R. (1996). Cellular approach to cryopreservation of embryos. Theriogenology 45, 1726.
Dobrinsky, J.R., Pursel, V.G., Long, C.R. & Johnson, L.A. (2000). Birth of piglets after transfer of embryos cryopreserved by cytoskeletal stabilization and vitrification. Biol. Reprod. 62, 564–70.
Emiliani, S., Van Den Bergh, M., Vannin, A.S., Biramane, J. & Englert, Y. (2002). Comparison of ethylene glycol, 1,2-propanediol and glycerol for cryopreservation of slow-cooled mouse zygotes, 4-cell embryos and blastocysts. Hum. Reprod. 15, 905–10.
Eroglu, A., Toner, M. & Toth, T.L. (2002). Beneficial effect of microinjected trehalose on the cryosurvival of human oocytes. Fertil. Steril. 77, 152–8.
Eroglu, A., Elliott, G., Wright, D.L., Toner, M. & Toth, T.L. (2005). Progressive elimination of microinjected trehalose during mouse embryonic development. Reprod. BioMed. Online 10, 503–10.
Fabian, D., Gjorett, J.O., Berthelot, F., Martinat-Botte, F. & Maddox-Hyttel, P. (2005). Ultrastructure and cell death of in vivo derived and vitrified porcine blastocysts. Mol. Reprod. Dev. 70, 155–65.
Fair, T., Lonergan, P., Dinnyes, A., Cottell, D.C., Hyttel, P., Ward, F.A. & Boland, M.P. (2001). Ultrastructure of bovine blastocysts following cryopreservation: effect of method of blastocyst production. Mol. Reprod. Dev. 58, 186–95.
Fletcher, G.L., Goddard, S.V. & Wu, Y.L. (1999). Antifreeze proteins and their genes: from basic research to business opportunity. Chemtech 29, 1728.
Gaida, B. (1996). Vitrification of rabbit embryos at 1-cell to morula stage in an ethylene glycol-based solution. CryoLetters, 7, 368–70.
Hasler, J.F., Hurtgen, P.J., Jin, Z.Q. & Stokes, J.E. (1997). Survival of IGF-derived bovine embryos frozen in glycerol or ethylene glycol. Theriogenology 48, 563–79.
Hincha, D.K., De Vries, A.L. & Schmitt, J.M. (1993). Cryotoxicity of antifreeze proteins and glycoproteins to spinach thylakoid membranes – comparison with cryotoxic sugar acids. Biochem. Biophys. Acta 1146, 258–64.
Kagabu, S. & Umezu, M. (2000). Transplantation of cryopreserved mouse, Chinese hamster, rabbit, Japanese monkey and rat ovaries into rat recipients. Exp. Anim. 49, 1721.
Kasai, M., Komi, J.H., Takakama, A., Tsudera, H., Sakurai, T. & Machida, T. (1990). A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J. Reprod. Fert. 89, 91–7.
Kasai, M., Hamaguchi, Y., Zhu, S.E., Miyake, T., Sakurai, T. & Machida, T. (1992). High survival of rabbit morulae after vitrification in an ethylene glycol based solution by a simple method. Biol. Reprod. 46, 1042–8.
Kohli, V., Robles, V., Cancela, M.L., Acker, J.P., Waskiewicz, A.J. & Elezzabi, A.Y. (2007). An alternative method for delivering exogenous material into developing zebrafish embryos. Biotechnol. Bioeng. 98, 1230–41.
Kuleshova, L., Gianaroli, L., Magli, C., Ferraretti, A. & Trounson, A. (1999). Birth following vitrification of a small number of human oocytes: case report. Hum. Reprod. 14, 3077–9.
Lagneaux, D., Huhtinen, M., Koskinen, E. & Palmer, E. (1997). Effect of anti-freeze protein (AFP) on the cooling and freezing of equine embryos as measured by DAPI-staining. Equine Vet. J. Suppl. 25, 85–7.
Landel, C.P. (2005). Archiving mouse strains by cryopreservation. Lab. Anim. NY 34, 50–7.
Lane, M., Maybach, J.M., Hooper, K., Hasler, J.F. & Gardner, D.K. (2003). Cryosurvival and development of bovine blastocysts are enhanced by culture with recombinant albumin and hyaluronan. Mol. Reprod. Dev. 64, 70–8.
Lee, S.H., Shin, C.S., Ko, J.J., Lee, H.C., Park, C. & Lee, K.A. (2000). In vitro culture of the human adult ovarian tissues after vitrification: comparison among detection methods of the culture effects. Fertil. Steril. 74 (Suppl.), 1161 (Abstr. P-208).
Liebermann, J. (2003). Recent developments in human oocyte, embryo and blastocyst vitrification: where are we now? Reprod. Biomed. Online 7, 124134.
Lopez-Bejar, M. & Lopez-Gatius, F. (2002). Non-equilibrium cryopreservation of rabbit embryos using modified (sealed) open pulled straw procedure. Theriogenology 58, 1541–52.
Madura, J.D., Baran, K. & Wierzbicki, A. (2000). Molecular recognition and binding of thermal hysteresis proteins to ice. J. Molec. Recog. 13, 101–13.
Martinez-Paramo, S., Perez-Cerezales, S., Robles, V., Anel, L. & Herraez, M.P. (2008). Incorporation of antifreeze proteins into zebrafish embryos by a non-invasive method. Cryobiology 56, 216–22.
Makarevich, A.V., Chrenek, P., Olexikova, L., Popelkova, M., Turanova, Z., Ostro, A. & Pivko, J. (2008). Post-thaw survival, cell death and actin cytoskeleton in gene-microinjected rabbit embryos after vitrification. Theriogenology 70, 675–81.
Marquez-Alvarado, Y.C., Galina, C.S., Castilla, B., Leon, H. & Morena-Mendoza, N. (2004). Evidence of damage in cryopreserved and fresh bovine embryos using the Tunel technique. Reprod. Dom. Anim. 39, 141–5.
Martino, A., Songsasen, N. & Leibo, S.P. (1996). Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol. Reprod. 54, 1059–69.
Mavrides, A. & Morroll, D. (2005). Bypassing the effect of zona pellucida changes on embryo formation following cryopreservation of bovine oocytes. Eur. J. Obstet. Gynecol. Reprod. Biol. 118, 6670.
Moore, K. & Bonilla, A.Q. (2006). Cryopreservation of mammalian embryos: the state of the art. ARBS Annu. Rev. Biomed. Sci. 8, 1932.
Moreira Da Silva, F. & Metelo, R. (2005). Relation between physical properties of the zona pellucida and viability of bovine embryos after slow-freezing and vitrification. Reprod. Dom. Anim. 40, 205–9.
Naik, B.R., Rao, B.S., Vagdevi, R., Gnanprakash, M., Amarnath, D. & Rao, V.H. (2005). Conventional slow freezing, vitrification and open pulled straw (OPS) vitrification of rabbit embryos. Anim. Reprod. Sci. 86, 329–38.
Nguyen, B.X., Sotomaru, Y., Tani, T, Kato, Y. & Tsunoda, Y. (2000). Efficient cryopreservation of bovine blastocysts derived from nuclear transfer with somatic cells using partial dehydration and vitrification. Theriogenology 53, 1439–48.
Palasz, A., Alkemade, S. & Mapletoft, R.J. (1993). The use of sodium hyaluronate in freezing media for bovine and murine embryos. Cryobiology 30, 172–8.
Palasz, A.T., Gustafsson, H., Rodrigues-Martinez, H., Gusta, L., Larsson, B. & Mapletoft, R.J. (1997). Vitrification of bovine IVF blastocysts in an ethylene glycol/sucrose solution and heat-stable plant-extracted proteins. Theriogenology 47, 865–97.
Papis, K., Sypecka, J., Korwin-Kossakowski, M., Wenta-Muchalska, E. & Bilska, B. (2005). Banking of embryos of mutated, paralytic tremor rabbit by means of vitrification. Lab. Anim. 39, 284–9.
Park, S.Y., Kim, E.Y., Cui, X.S., Tae, J.C., Lee, W.D., Kim, N.H., Park, S.P. & Lim, J.H. (2006). Increase in DNA fragmentation and apoptosis-related gene expression in frozen–thawed bovine blastocysts. Zygote 14, 125–31.
Pivko, J., Kubovicova, E., Grafenau, P. & Oberfranc, M. (1998). Ultrastructural Analysis of Early Cow Embryos after Freezing. pp. 22–6. Acta Fytotech. Zootech. Univ. Agric. Nitra.
Pivko, J., Kubovicova, E., Grafenau, P., Riha, Ľ. & Zibrin, M. (2003). Ultraštrukturálne zmeny in vitro produkovaných embryí kráv po vitrifikácii metódou OPS a následnej kultivácii. J. Farm Anim. Sci. 36, 1116.
Popelkova, M., Chrenek, P., Pivko, J., Makarevič, A.V., Kubovičová, E. & Kačmarik, J. (2005). Survival and ultrastructure of gene-microinjected rabbit embryos after vitrification. Zygote 13, 283–93.
Popelkova, M., Turanova, Z., Koprdova, L., Ostro, A., Toporcerova, S., Makarevich, A.V. & Chrenek, P. (2008). Effect of vitrification technique and assisted hatching on rabbit embryo developmental rate. Zygote 17, 5761.
Rall, W.F. & Fahy, G.M. (1985). Ice–free cryopreservation of mouse embryos at –196 °C by vitrification. Nature 313, 573–5.
Robles, V., Barbosa, V., Herraez, M.P., Martinez-Paramo, S. & Cancela, M.L. (2007). The antifreeze protein type I (AFP I) increases seabream (Sparus aurata) embryos tolerance to low temperatures. Theriogenology 68, 284–9.
Rubinsky, B., Arav, A., Mattioli, M. & De Vries, A.L. (1990). The effect of antifreeze glycopeptides on membrane potential changes at hypothermic temperatures. Biochem. Biophys. Res. Commun. 173, 1369–74.
Scholander, P.F., Van Dam, L., Kanwisher, J.W., Hammel, H.T. & Gordon, M.S. (1957). Supercooling and osmoregulation in Arctic fish. J. Cell. Comp. Physiol. 49, 524.
Shaw, J., Oranratnachai, A. & Trounson, A. (2000). Fundamental cryobiology of mammalian oocytes and ovarian tissue. Theriogenology 53, 5972.
Silvestre, M.A., Saeed, A.M., Escriba, M.J. & Garcia-Ximenez, F.Vitrification of in vitro cultured rabbit morulae. Anim. Reprod. Sci. 76, 113–24.
Sommerfeld, V. & Niemann, H. (1999). Cryopreservation of bovine in vitro produced embryos using ethylene glycol in controlled freezing or vitrification. Cryobiology 38, 95105.
Son, W.V., Yoon, S.H., Yoon, H.J, Lee, S.M. & Lim, J.H. (2003). Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Hum. Reprod. 18, 137–9.
Sugimoto, M., Miyamoto, H., Kabasawa, T. & Manabe, N. (1996). Follicle survival in neonatal rat ovaries cryopreserved by vitrification. CryoLetters 17, 93–8.
Tharasanit, T., Colenbrander, B. & Stout, T.A.E. (2005). Effect of cryopreservation on the cellular integrity of equine embryos. Reproduction 129, 789–98.
Vajta, G., Booth, P.J., Holm, P., Greve, T. & Callesen, H. (1997). Successful vitrification of early stage bovine in vitro produced embryos with the open pulled straw (OPS) method. CryoLetters 18, 191–5.
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. Mol. Reprod. Dev. 51, 53–8.
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. Hum. Reprod. 17, 744–51.
Vincent, C., Turner, K., Pickering, S.J. & Johnson, M.H. (1991). Zona pellucida modifications in the mouse in the absence of oocyte activation. Mol. Reprod. Dev. 28, 394404.
Visintin, J.A., Martin, J.F.P., Bevilacqua, E.M., Mello, M.R.B., Nicacio, A.C. & Assumpcao, M.E.O.A. (2002). Cryopreservation of Bos taurus vs Bos indicus embryos: are they really different? Theriogenology 57, 345–59.
Wang, T., Zhu, Q., Yang, X., Layne, J.R. & De Vries, A.L. (1994). Antifreeze glycoproteins from Antarctic notothenioid fishes fail to protect the rat cardiac explant during hypothermic and freezing preservation. Cryobiology 31, 185–92.
Yeh, Y. & Feeney, R.E. (1996). Antifreeze proteins: structures and mechanisms of function. Chem. Rev. 96, 601–17.
Yoon, T.K., Chung, H.M., Lim, J.M., Han, S.Y., Ko, J.J. & Cha, K.Y. (2000). Pregnancy and delivery of healthy infants developed from vitrified oocytes in a stimulated in vitro fertilization-embryo transfer program (letter). Fertil. Steril. 74, 180–1.


Several aspects of animal embryo cryopreservation: anti-freeze protein (AFP) as a potential cryoprotectant

  • A. V. Makarevich (a1) (a2), E. Kubovičová (a2), M. Popelková (a3), D. Fabian (a4), Š. Čikoš (a4), J. Pivko (a2) and P. Chrenek (a2) (a5)...


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed