Skip to main content Accessibility help

Sperm-mediated gene transfer: effect on bovine in vitro embryo production

  • Renata Simões (a1), Alessandra Corallo Nicacio (a2), Mario Binelli (a3), Fabiola Freitas de Paula-Lopes (a4), Marcella Pecora Milazzotto (a1), José Antonio Visintin (a3) and Mayra Elena Ortiz D'Ávila Assumpção (a5)...


The technique of sperm-mediated gene transfer (SMGT) can be used to delivery exogenous DNA into the oocyte. However, it has low repeatability and produces inconsistent results. In order to optimize this technique, it is necessary to study the mechanism by which DNA enters the sperm cell and integrates in the sperm genome. Furthermore, studies must focus in the maintenance of sperm cell viability and function. The aim of this study was to evaluate different SMGT protocols of sperm electroporation or capacitation (CaI) aiming to maintain sperm viability in the production of bovine embryos in vitro. Frozen–thawed semen was divided in two experimental groups (electroporation or CaI) and one control group (non-treated cells). For the electroporation method, five different voltages (100, 500, 750, 1000 or 1500 V) with 25 μF capacitance were used. For CaI treatment, combinations of two CaI concentrations (250 nM or 500 nM), two incubation periods of sperm cells with CaI (1 or 5 min) and two incubation periods that mimicked time of sperm cell interaction with exogenous DNA molecules (1 or 2 h) were evaluated. According to our data, electroporation and CaI treatments do not prevent sperm penetration and oocyte fertilization and can be an alternative method to achieve satisfactory DNA delivery in SMGT protocols.


Corresponding author

All corresponding to: Mayra Elena Ortiz D'Avila Assumpção. Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Av. 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
Camaioni, A., Russo, M.A., Odorisio, T., Gandolfi, F., Fazio, V.M. & Siracusa, G. (1992). Uptake of exogenous DNA by mammalian spermatozoa: specific localization of DNA on sperm heads. J. Reprod. Fertil. 96, 203–12.
Canovas, S., Adan, A.G. & Gadea, J. (2010). Effect of exogenous DNA on bovine sperm functionality using the sperm mediated gene transfer (SMGT) technique. Mol. Reprod. Dev. 77, 687–98.
Carvalho, J.O., Sartori, R., Machado, G.M., Mourão, G.B. & Dode, M.A.N. (2010). Quality assessment of bovine cryopreserved sperm after sexing by flow cytometry and their use in in vitro embryo production. Theriogenology 74, 1521–30.
Chen, C., Smye, S.W., Robinson, M.P. & Evans, J.A. (2006). Membrane electroporation theories: a review. Med. Biol. Eng. Comput. 44, 514.
Feitosa, W.B., Milazzotto, M.P., Simões, R., Rovegno, M., Nicacio, A.C., Nascimento, A.B., Gonçalves, J.S.A., Visintin, J.A. & Assumpçào, M.E.O.A. (2009). Bovine sperm cells viability during incubation with or without exogenous DNA. Zygote 17, 315–20.
Gagné, M.B., Pothierm, F. & Sirard, M.A. (1991). Electroporation of bovine spermatozoa to carry foreign DNA in oocytes. Mol. Reprod. Dev. 29, 615.
Galli, C., Crotti, G., Notari, P., Duchi, T.R. & Lazzari, G. (2003). Embryo production by ovum pick up from live donors. Theriogenology 55, 1341–57.
Humblot, P., Bourhis, D., Fritz, S., Colleau, J.J., Gonzalez, C., Joly, C.G., Malafosse, A., Heyman, Y., Amigues, Y., Tissier, M. & Ponsart, C. (2010). Reproductive technologies and genomic selection in cattle. Vet. Med. Int. 2010, 192787.
Iranpour, F.G., Nasr-Esfahani, M.H., Valokerdi, M.R. & Al-Taraihi, T.M. (2000). Chromomycin A3 staining as a useful tool for evaluation of male fertility. J. Assist. Reprod. Genet. 17, 60–6.
Jonák, J. (2000). Sperm-mediated preparation of transgenic Xenopus laevis and transmission of transgenic DNA to the next generation. Mol. Reprod. Dev. 56, 298300.
Kuznetsov, A.V., Kuznetsova, I.V. & Schit, I.Y. (2000). DNA interaction with rabbit sperm cells and its transfer into ova in vitro and in vivo. Mol. Reprod. Dev. 56, 292–7.
Landim-Alvarenga, F.C., Graham, J.K., Alvarenga, M.A. & Squires, E.L. (2004). Calcium influx into equine and bovine spermatozoa during in vitro capacitation. Anim. Reprod. 1, 96105.
Lavitrano, M., Busnelli, M., Cerrito, M.G., Giovannoni, R., Manzini, S. & Vargiolu, A. (2006). Sperm-mediated gene transfer. Reprod. Fertil. Dev. 18, 1923.
Müller, F., Ivics, Z., Erdélvi, F., Papp, T., Váradi, L., Horváth, L. & Maclean, N. (1992). Introducting foreign genes into fish eggs with electroporation sperm as carrier. Mol. Marine Biol. Biotechnol. 1, 276–81.
Nishikage, S., Koyama, H., Miyata, T., Ishii, S., Hamada, H. & Shigematsu, H. (2004). In vivo electroporation enhances plasmid-based gene transfer of basic fibroblast growth factor for the treatment of ischemic limb. J. Surg. Res. 120, 3746.
Parrish, J.J., Susko-Parrish, J.L., Winer, M.A. & First, N.L. (1988). Capacitation of bovine sperm by heparin. Biol. Reprod. 38, 1171–80.
Pereira, R.J.T.A., Tuli, R.K., Wallenhorst, S. & Holtz, W. (2000). The effect of heparin, caffeine and calcium ionophore A 23187 on in vitro induction of the acrosome reaction in frozen–thawed bovine and caprine spermatozoa. Theriogenology 54, 185–92.
Perry, A.C.F., Wakayama, T., Kishikawa, H., Kasai, T., Okabe, M., Toyoda, Y. & Yanagimachi, R. (1999). Mammalian transgenesis by intracytoplasmic sperm injection. Science 284, 1180–3.
Rieth, A., Pothier, F. & Dirard, M.A. (2000). Electroporation of bovine spermatozoa to carry DNA containing highly repetitive sequences into oocytes and detection of homologous recombination events. Mol. Reprod. Dev. 57, 338–45.
Sin, F.Y.T., Mukherjee, U.K., Mckenzie, J.C. & Sin, I.L. (1995). Electroporation of abalone sperm enhances sperm-DNA association. J. Fish Biol. 47, 20–8.
Sin, F.Y., Walker, S.P., Symonds, J.E., Mukherjee, U.K., Khoo, J.G. & Sin, I.L. (2000). Electroporation of salmon sperm for gene transfer: efficiency, reliability, and fate of transgene. Mol. Reprod. Dev. 56, 285–8.
Tervit, H.R., Whittingham, D.G. & Rowson, L.E. (1972). Successful culture in vitro of sheep and cattle ova. J. Reprod. Fertil. 30, 493–7.
Tsai, H.J. (2000). Electroporated sperm mediation of a gene transfer system for finfish and shellfish. Mol. Reprod. Dev. 56, 281–4.
Yamada, C., Caetano, H.V., Simões, R., Nicacio, A.C., Feitosa, W.B., Assumpção, M.E.O.A. & Visintin, J.A. (2007). Immature bovine oocyte cryopreservation: comparison of different associations with ethylene glycol, glycerol and dimethylsulfoxide. Anim. Reprod. Sci. 99, 384–8.


Sperm-mediated gene transfer: effect on bovine in vitro embryo production

  • Renata Simões (a1), Alessandra Corallo Nicacio (a2), Mario Binelli (a3), Fabiola Freitas de Paula-Lopes (a4), Marcella Pecora Milazzotto (a1), José Antonio Visintin (a3) and Mayra Elena Ortiz D'Ávila Assumpção (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