Skip to main content Accessibility help

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

  • Alessandra Corallo Nicacio (a1) (a2), Renata Simões (a1), Fabiola Freitas de Paula-Lopes (a1), Flavia Regina Oliveira de Barros (a1), Maria Angelica Peres (a1), Mayra Elena Ortiz D'Avila Assumpção (a1) and Jose Antonio Visintin (a3)...


The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7–9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen–thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows®. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.


Corresponding author

All correspondence to: José Antonio Visintin. Department of Animal Reproduction, College of Veterinary Medicine and Animal Science, University of São Paulo, Avenida Prof. Dr. Orlando Marques de Paiva, 87–Cidade Universitária–05508–270–São Paulo, SP, Brazil. Tel: +55 11 3091 7915. Fax: +55 11 3091 7412. e-mail:


Hide All
Assumpção, M.E.A.O., Milazzotto, M.P., Simões, R., Nicacio, A.C., Mello, M.R.B. & Visintin, J.A. (2008). In vitro survival of in vitro-produced bovine embryos cryopreserved by slow freezing, fast freezing and vitrification. Anim. Reprod. 5, 116–20.
Barceló-Fimbres, M. & Seidel, G.E. Jr (2007), Effects of fetal calf serum, phenazine ethosulfate and either glucose or fructose during in vitro culture of bovine embryos on embryonic development after cryopreservation. Mol. Reprod. Dev. 74, 1395–405.
Campos-Chillon, L.F., Wlaker, D.J., De La Torre-Sanchez, J. & Seidel, G.E. Jr (2006). In vitro assessment of a direct transfer vitrification procedure for bovine embryos. Theriogenology 65, 1200–14.
Corcoran, D., Rizos, D., Fair, T., Evans, A.C.O. & Lonergan, P. (2007). Temporal expression of transcripts related to embryo quality in bovine embryos cultured from the two-cell to blastocysts stage in vitro or in vivo. Mol. Reprod. Dev. 74, 972–7.
Fahning, M.L. & Garcia, M.A. (1992). Status of cryopreservation of embryos from domestic animals. Cryobiology 29, 118.
George, F., Daniaux, C., Genicot, G., Verhaeghe, B., Lambert, P. & Donnay, I. (2008). Set up of a serum-free culture system for bovine embryos: embryo development and quality before and after transient transfer. Theriogenology 69, 612–23.
Hochi, S., Seemple, E. & Leibo, S.P. (1996). Effect of cooling and warming rates during cryopreservation on survival of in vitro-produced bovine embryos. Theriogenology 46, 837–47.
Jin, B., Kusanagi, K., Ueda, M., Seki, S., Valdez, D.M. Jr, Edashige, K. & Kasai, M. (2008). Formations of extracellular and intracellular ice during warming of vitrified mouse and its effect on embryo survival. Cryobiology 56, 233–40.
Kaidi, S., Bernard, S., Lambert, P., Massip, A., Dessy, F. & Donnay, I. (2001). Effect of conventional controlled-rate freezing and vitrification on morphology and metabolism of bovine blastocysts produced in vitro. Biol. Reprod. 65, 1127–34.
Kaidi, S., Van Langendockt, A., Massip, A., Dessy, F. & Donnay, I. (1999). Cellular alteration after dilution of cryoprotective solutions used for the vitrification of in vitro-produced bovine embryos. Theriogenology 52, 515–25.
Leibo, S.P. (2008). Cryopreservation of oocytes and embryos: optimization by theoretical versus empirical analysis. Theriogenology 69, 3747.
Leoni, G., Bogliolo, L., Berlinguer, F., Rosati, I., Pintus, P.P., Ledda, S. & Naitana, S. (2002). Defined media for vitrification, warming, and rehydration: effects on post-thaw protein synthesis and viability of in vitro derived ovine embryos. Cryobiology 45, 204–12.
Lonergan, P., Fair, T., Corcoran, D. & Evans, A.C.O. (2006). Effect of culture environment on gene expression and developmental characteristics in IVF-derived embryos. Theriogenology 26, 137–52.
Marquez-Alvarado, Y.C., Gallina, C.S., Castilla, B., Leon, H. & Moreno-Mendoza, N. (2004). Evidence of damage in cryopreserved and fresh bovine embryos using the Tunnel technique. Reprod. Dom. Anim. 39, 141–5.
Martinez, A.G., Valcárcel, A., De Las Heras, M.A., De Matos, D.G., Furnus, C. & Brogliatti, G. (2002). Vitrification of in vitro produced bovine embryos: in vitro and in vivo evaluations. Anim. Reprod. Sci. 73, 1121.
Mello, M.R.B., Queiroz, V.S., Lima, A.S., Tavares, L.M.T., Assumpção, M.E.O.A., Wheeler, M.B. & Visintin, J.A. (2001). Cryopreservation of mouse morulae through different methods: slow-freezing, vitrification and quick-freezing. Braz. J. Vet. Res. Anim. Sci. 38, 160–4.
Mucci, N., Aller, J., Kaiser, G.G., Hozbor, F., Cabodevilla, J. & Alberio, R.H. (2005). Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification. Theriogenology 65, 1551–62.
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, 437–49.
Pereira, D.C., Dode, M.A.N. & Rumpf, R. (2005). Evaluation of different culture systems on the in vitro production of bovine embryos. Theriogenology 63, 1131–41.
Robert, C., McGraw, S., Massicotte, L., Pravetoni, M., Gandolfi, F. & Sirard, M.A. (2002). Quantification of housekeeping transcript levels during the development of bovine preimplantation embryos. Biol. Reprod. 67, 1465–72.
Russell, D.F., Baqir, S., Bordignon, J. & Betts, D.H. (2006). The impact of oocyte maturation media on early bovine embryonic development. Mol. Reprod. Dev. 73, 1255–70.
Silva, F.M. & 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.
Tavares, L.M.T., Feitosa, W.B., Mello, M.R.B., Nicacio, A.C., Lima, A.S., Assumpção, M.E.A.O. & Visintin, J.A. (2008). Is the early reduction of fetal calf serum concentration in bovine in vitro embryo culture beneficial? Anim. Reprod. 5, 34–8.
Thompson, J.G. (1997). Comparison between in vitro-derived and in vitro-produced pre-elongation embryos from domestic ruminants. Reprod. Fertil. Dev. 9, 341–54.
Tominaga, K., Iwaki, F. & Hochi, S. (2007). Conventional freezing of in vitro-produced and biopsied bovine blastocysts in the presence of a low concentration of glycerol and sucrose. J. Reprod. Dev. 53, 443–7.
Vajta, G., Holm, P., Greve, T. & Callesen, H. (1996). Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration. Anim. Reprod. Sci. 45, 191200.
Van Wagtendonk-De Leeuw, A.M., Den Daas, J.H.G., Kruip, T.H.A.M. & Rall, W.F. (1995). Comparison of the efficacy of conventional slow freezing and rapid cryopreservation methods for bovine embryos. Cryobiology 32, 157–67.
Wrenzycki, C., Herrmann, D. & Niemann, H. (2007). Messenger RNA in oocytes and embryos in relation to embryo viability. Theriogenology 68, 7783.



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