Electroactivation of zebrafish (Danio rerio) eggs

Abstract

The zebrafish, has recently acquired a complex role in many emerging areas of knowledge, mainly in biomedical research (Nüsslein-Volhard and Dahm, 2002), becoming a new scientific tool in multitude of labs around the world. In this sense, several and varied are the topical areas where the zebrafish has established as a both efficient and capable vertebrate model for developing knowledge. As Streisinger (1986) pointed out, the reasons were their many useful biological characteristics.In zebrafish, egg activation takes place after they are discharged from the ovarian stroma and come into AbstractIn 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.Additional key words: egg activation; fish; nuclear transplant; oocyte. Resumen Electroactivación de oocitos de pez cebra (Danio rerio)En pez cebra, una vez que los oocitos son descargados del estroma ovárico, su activación inicial por contacto en agua da lugar a divisiones normales únicamente cuando son fertilizados. Cuando no se utiliza semen (inactivado genéticamente o no), las técnicas reproductivas (como transplante nuclear o inyección espermática intracitoplasmática) son ineficientes. En el presente trabajo se desarrollaron tres experimentos de activación oocitaria por estímulos eléctricos: en el primero de ellos, se compararon seis tratamientos de activación (Voltaje × Pulsos: 2,76 × × 1; 2,76 × 2; 2,76 × 3 y 5,40 × 1; 5,40 × 2; 5,40 × 3). El grupo 5,4 × 3 mostró los mejores resultados (32% de activación). En el segundo experimento, se evaluó un tratamiento eléctrico de 20 min de duración. El tratamiento consistió en la aplicación de una secuencia de tre...