electroporation. Other available methodologies for introducing key
nutrients into fish eggs may include microinjection and ultrasound
cavitation (sonophoresis). The later, similarly to electroporation, is a
high throughput approach although lower survival (around 60%) was
reported implementing this approach on sea bream fertilized eggs
(Engrola et al., 2014).
The selected protocol using the present technique was established
by taking into account both hatchability and the extent of fluorescein
dye incorporation into the egg. The use of fluorescein as an indicator
for electroporation success was also described by Jung et al. (2013),
who showed that the egg inner membrane and not the chorion of the
eggs of medaka fish (Oryzias latipes) presents the main barrier against
the diffusion of small molecules. Our findings also suggest a similar phenomenon
is at play in the gilthead sea bream eggs. This is because the
incorporation of fluorescein occurred only when the inner membrane
phospholipid integrity was successfully disrupted by the electroporation
pulses. Both Heller et al. (1996) and Mir et al. (1999) emphasized
that the electrical pulse intensity and duration have a direct effect on
the level of permeability of the phospholipid membrane and also on
cell mortality during the process. The pulse intensity and duration in
the chosen electroporation protocol were sufficient to induce the
permeability of the fertilized eggs inner membrane to a small molecule
such as fluorescein (332 Da), while retaining high hatching rates.