Electroactivation of zebrafish (Danio rerio) eggs

  • J. Cardona-Costa Laboratory of Animal Reproduction and Biotechnology (LARB-UPV). Universidad Politécnica de Valencia (UPV).
  • M. Perez-Camps Laboratory of Animal Reproduction and Biotechnology (LARB-UPV). Universidad Politécnica de Valencia (UPV).
  • F. Garcia-Ximenez Laboratory of Animal Reproduction and Biotechnology (LARB-UPV). Universidad Politécnica de Valencia (UPV).
Keywords: egg activation, fish, nuclear transplant, oocyte


In zebrafish, initial egg activation by water after being discharged from the ovarian stroma is followed by normal cleavages only in inseminated eggs. When sperm (genetically active or not) is not used as activating agent, reproductive techniques (as either nuclear transplant or intracytoplasmic sperm injection are ineff icient. In this work, three experiments of egg activation by electric stimuli were performed: In the first, six activation treatments were compared (Voltage × Pulses: 2.76 × 1; 2.76 × 2; 2.76 × 3 and 5.40 × 1; 5.40 × 2; 5.40 × 3). The group 5.40 × 3 showed the best results (32% activated). In the second experiment, an electrical treatment of 20 min was carried out. It consisted in a sequence of three equal electrical stimuli every 10 min (of 1 or 3 consecutive direct current square pulses for 20 μs each and applied at two voltage levels, 2.76 V or 5.4 V). It was observed that the number of pulses negatively affected the rates of damaged and lysed eggs. Moreover, only the 20 min treatment with the combination of 3 consecutive pulses at 2.76 V showed significant differences with their respective control group (43% vs 18% activated eggs, p < 0.05). In the third experiment, negative effects of egg ageing were observed. In conclusion, the best activation treatment for intact (non manipulated) zebrafish eggs concerns sequence B3. The electro-activation stimulus proposed here would be the only one available once the egg has already been activated by water.


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Bubenshchikova E., Kaftanovskaya E., Motosugi N., Fujimoto T., Arai K., Kinoshita M., Hashimoto H., Ozato K., Wakamatsu Y., 2007. Diploidized eggs reprogram adult somatic cell nuclei to pluripotency in nuclear transfer in Medaka fish (Oryzias latipes). Develop Growth Differ 49, 699–709. http://dx.doi.org/10.1111/j.1440-169X.2007.00963.x

Cardona-Costa J., Pérez-Camps M., García-Ximénez F., Espinós F.J., 2009. Effect of gametes aging on their activation and fertilizability in zebrafish (Danio rerio). Zebrafish 6(1), 93-95. http://dx.doi.org/10.1089/zeb.2008.0578

Chang D.C., Chassy B.M., Saunders J.A., Sowers A.E., 1992. Guide to electroporation and electrofusion. Academic Press, 581 pp.

Collas P., Robl J.M., 1990. Factors affecting the efficiency of nuclear transplantation in the rabbit embryo. Biol Reprod 43, 877-884. http://dx.doi.org/10.1095/biolreprod43.5.877

Elsheikh A.S., Takahashi Y., Tanaka H., Hishinuma M., Kanagawa H., 1995. Electrofusion of zona-free mouse embryonic cells in electrolytes and their development in vitro. Jpn J Vet Res 43(3-4), 125-134.

Escribá M.J., García-Ximénez F., 1999. Electroactivation of rabbit oocytes in an hypotonic pulsing medium and parthenogenetic in vitro development without cytochalasin B diploidizing pretreatment. Theriogenology 51, 963-973. http://dx.doi.org/10.1016/S0093-691X(99)00042-4

Escribá M.J., García-Ximénez F., 2000. Influence of sequence duration and number of electrical pulses upon rabbit oocyte activation and parthenogenetic in vitro development. Anim Reprod Sci 59(1-2), 99-107. http://dx.doi.org/10.1016/S0378-4320(00)00069-5

Grush J., Noakes D.L.G., Moccia R.D., 2004. The efficacy of clove oil as an anesthetic for the zebrafish, Danio rerio (Hamilton). Zebrafish 1(1), 46-53. http://dx.doi.org/10.1089/154585404774101671

Howley C., Ho R.K., 2000. mRNA localization patterns in zebrafish oocytes. Mech Dev 92, 305-309. http://dx.doi.org/10.1016/S0925-4773(00)00247-1

Huang H., Ju B., Lee K., Lin S., 2003. Protocol for nuclear transplant in zebrafish. Cloning Stem Cells 5, 333-337. http://dx.doi.org/10.1089/153623003772032835

Kaufman M.H., 1981. Parthenogenesis: a system facilitating understanding of factors that influence early mammalian development. In: Progress in anatomy (Harrison R.J. and James R.L., eds), Vol. 1, Cambridge Univ Press. 1-34 pp.

Lee K.W., Webb S.E., Miller A.L., 1999. A wave of free cytosolic calcium traversas zebrafish eggs on activation. Dev Biol 214, 168-180. http://dx.doi.org/10.1006/dbio.1999.9396

Macháty Z., 2006. Activation of oocytes after nuclear transfer. In: Nuclear transfer protocols: Cell reprogramming and transgenesis (Verma P.J. and Trounson A., eds.), Methods Mol Biol 348, 43-58.

Mansour R., Fahmy I., Tawab N.A., Kamal A., El-Demery Y., Aboulgar M., Serour G., 2009. Electrical activation of oocytes after intracytoplasmic sperm injection: a controlled randomized study. Fertil Steril 91(1), 133-139. http://dx.doi.org/10.1016/j.fertnstert.2007.08.017

Mylonas C.C., Cardinaletty G., Sigelaki I., Polzonetty-Magni A., 2005. Comparative efficacy of clove oil and 2-phenoxyethanol as anesthetics in the aquaculture of european sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) at different temperatures. Aquaculture 246, 467-481. http://dx.doi.org/10.1016/j.aquaculture.2005.02.046

Nüsslein-Volhard C., Dahm R., 2002. Zebrafish: a practical approach. Oxford Univ Press, Oxford, England. 332 pp.

Okahara-Narita J., Tsuchiya H., Takada T., Torii R., 2007. Cloned blastocysts produced by nuclear transfer from somatic cells in cynomolus monkeys (Macaca fascicularis). Primates 48(3), 232-240. http://dx.doi.org/10.1007/s10329-006-0032-z

Onishi A., Iwamoto M., Akita T., Mikawa S., Takeda K., Awata T., Hanada H., Perry A.C., 2000. Pig cloning by microinjection of fetal fibroblast nuclei. Science 289(5482), 1188-1890. http://dx.doi.org/10.1126/science.289.5482.1188

Pérez-Camps M., Cardona-Costa J., Francisco Simao M., García-Ximénez F., 2009. Definition of three somatic cell nuclear transplant methods in zebrafish (Danio rerio): before, during and after egg activation by sperm fertilization. Zygote 12, 1-7.

Poleo G.A., Dennisnton R.S., Reggio B.C., Godke R.A., Tiersch T.R., 2001. Fertilization of eggs of zebrafish, Danio rerio, by intracytoplasmic sperm injection. Biol Reprod 65, 961-966. http://dx.doi.org/10.1095/biolreprod65.3.961

Rickords L.F., White K.L., 1992. Effect of electrofusion pulse in either electrolyte or nonelectrolyte fusion medium on subsequent murine embryonic development. Mol Reprod Dev 32(3), 259-264. http://dx.doi.org/10.1002/mrd.1080320311

Sakai N., Burgess S., Hopkins N., 1997. Delayed in vitro fertilization of zebrafish eggs in Hank's saline containing bovine serum albumin. Mol Mar Biol Biotech 6, 84-87.

Simao M.F., Pérez-Camps M., Carcía-Ximénez F., 2007. Short communication. Zebrafish embryo development can be reversibly arrested at the MBT stage by exposure to a water temperature of 16°C. Span J Agric Res 5(2), 181-185. http://dx.doi.org/10.5424/sjar/2007052-244

Siripattarapravat K., Busta A., Steibel J.P., Cibelli J., 2009a. Characterization and in vitro control of MPF activity in zebrafish eggs. Zebrafish 6(1), 97-105. http://dx.doi.org/10.1089/zeb.2008.0527

Siripattarapravat K., Pinmee B., Venta P.J., Chang C.C., Cibelli J.B., 2009b. Somatic cell nuclear transfer in zebrafish. Nat Methods 6, 733-5. http://dx.doi.org/10.1038/nmeth.1369

Stice S.L., Robl J.M., 1988. Nuclear reprogramming in nuclear transplant rabbit embryos. Biol Reprod 39, 657-664. http://dx.doi.org/10.1095/biolreprod39.3.657

Streisinger G., Singer F., Walker C., Knauber D., Dower N., 1986. Segregation analyses and gene-centromere distances in zebrafish. Genetics 112(2), 331-339.

Tanaka H., Kanagawa H., 1997. Influence of combined activation treatments on the success of bovine nuclear transfer using young or aged oocytes. Anim Reprod Sci 49, 113-123. http://dx.doi.org/10.1016/S0378-4320(97)00070-5

Ungar A.R., Helde K.A., Moon R.T., 1998. Production of androgenetic haploids in zebrafish with ultraviolet light. Mol Mar Biol Biotechnol 7(4), 320-326.

Wakamatsu Y., 2008. Novel method for the nuclear transfer of adult somatic cells in medaka fish (Oryzias latipes): Use of diploidized eggs as recipients. Develop Growth Differ 50, 427–436. http://dx.doi.org/10.1111/j.1440-169X.2008.01050.x

Westerfield M., 2007. The zebrafish book: A guide for the laboratory use of zebrafish (Danio rerio). 5th Ed. Univ Oregon Press, Eugene, OR, USA. 385 pp.

Zhang J., Blaszczyk A., Grifo J., Ozil J.P., Adler A., Berkeley A., Licciardi F., Noyes N., Krey L., 1997. Electrical activation and in vitro development of human oocytes which failed fertilization following intracytoplasmic sperm injection (ICSI). Fertil Steril 1, 141. http://dx.doi.org/10.1016/S0015-0282(97)90920-1

How to Cite
Cardona-Costa, J., Perez-Camps, M., & Garcia-Ximenez, F. (1). Electroactivation of zebrafish (Danio rerio) eggs. Spanish Journal of Agricultural Research, 9(1), 59-65. https://doi.org/10.5424/sjar/20110901-110-10
Animal breeding, genetics and reproduction