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Scientific Programme38
PAPER PRESENTATIONS
Chilled storage of Zebrafish embryos, development and evolution of a promising
tool
Zebrafish embryo is a valid model already widely used thanks to its particular features
(ease of maintenance, small size, high fecundity, rapid development, optical transparency
of the embryo). Furthermore, according to the 3Rs principle (Replacement, Reduction
and Refinement), it is considered as a replacement method to animal experimentation.
Its widespread use in scientific research requires the development of techniques aimed at
preserving embryos. In fact, the availability of stored embryos would have an important
impact not only on the storage of genetically modified lines, but also on trade. Currently,
there is not a single and functional protocol aimed at preserving the structure, functionality
and morphology of the Zebrafish embryo. Therefore, the identification of a practical
procedure that can be used in different laboratories could broaden new horizons in a
collaborative context between scientific teams as well as in reaching an essential step for
understanding embryo preservation. The aim of the present investigation is to use Zebrafish
embryos as an alternative model to explore the development of a storage protocol and to
identify the limiting factors associated with fish embryo cryopreservation. Several variables
can play a key role in the identification of a cooling protocol for Zebrafish embryos: the
stage of embryo’s development is extremely important for its correct conservation. In
addition, further to identifying the exact time window, the choice of the more suitable
cryoprotective agent (CPA) should not be underestimated. To this, is added both the search
for the appropriate exposure time and the analysis of what could be the proper exposure
temperature at which the embryos would be incubated without undergoing alterations.
Furthermore, it is possible to set the same temperature for the whole cooling period,
otherwise it is possible to set a (more or less) rapid decrease of the thermal condition.
In this study embryos were obtained from a breeding stock of wild-type Zebrafish with
well-documented fertilisation rate (≥70 %). As preliminary experiments, the viability of
Zebrafish embryos was investigated in Dimethyl sulfoxide (DMSO) and Methanol (MeOH),
both at 1M concentration, while Embryo Medium (EM) was chosen as negative control.
Viable fertilised eggs correctly developed were divided into two experimental groups: 1)
embryos with chorion, 2) embryos treated with Pronase® (50 mg/mL) in order to remove
this protective barrier. Then, the two groups were exposed to CPAs as MeOH and DMSO
and to the negative control (EM). After 24 hours at 4°C, embryos were brought back to
room temperature for 2 hours and subsequently transferred in Embryo Medium at the
target development temperature of 28°C. A very high mortality was observed at 120
hours post fertilisation (hpf), especially for the dechorionated ones, thus it was decided to
continue testing only embryos with chorion and also to reduce the concentration of both
CPAs at 0.1M. The following experiment was carried out with the final concentration of
cryoprotectants halved to 0.05M and 0.1M of sucrose was added to all three solutions, in
order to verify its conservative function on the whole organism. This preliminary step was
followed by additional experiments: embryos at 24hpf and 48hpf were exposed for each
protocol devised, but further development stages will be investigated in order to find the
most applicable storage protocol. In conclusion, the results obtained so far are promising
and open up different possibilities in this under investigated area: additional targeted
experiments are needed for the optimization of a complete and feasible cooling protocol
of Zebrafish embryos.
Wednesday 24th March
14.30 – 15.00 hrs
