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Early steps of cryopreservation of day one honeybee (Apis mellifera) embryos treated with low-frequency sonophoresis

Abstract : Honeybees, major providers of pollination, are endangered in many areas. Embryo cryopreservation may be avery useful tool to maintain their genetic diversity. However, it is complex in insects, because embryos are chillsensitive and are surrounded by two protectant membranes, the chorion and vitelline. These membranes preventpenetration of cryoprotectant in the embryos. This study aimed to test different conditions of embryo pre-paration before cryopreservation, including low-frequency sonophoresis, a physical method of permeabilization,and passages through cryoprotectant solutions.Apis mellifera ligusticaembryos were collected in artificial cellplugs 7.5 h after queens had been caged, in two different seasons (winter, spring) and were then incubatedinvitroovernight (16.5 h). Embryos were individually sonicated and then incubated in three cryoprotectant baths(B1 = 10%, B2 = 20% and B3 = 40% of cryoprotectant) and quenched in liquid nitrogen. Artificial cell plugsandin vitroincubation device were efficient in producing future embryos hatching. Embryos stained ruby redwith rhodamine B after sonophoresis treatment indicated that low-frequency ultrasound had permeabilizedembryos. According to the treatment, different significant hatching rates were obtained after sonophoresis (up to25%). After three cryoprotectant incubations, best hatching rates were obtained after 10 min in B1 and B2, and40 s in B3. These results show that sonophoresis is an efficient tool to permeabilize the chorion and vitellinemembrane of the day one honeybee embryo allowing a hatching rate of more than 20%. They also show that theseason is an important variability factor
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Florence Guignot, Dominique Fortini, Stéphane Grateau, Cécile Burette, Virginie Dreyfus, et al.. Early steps of cryopreservation of day one honeybee (Apis mellifera) embryos treated with low-frequency sonophoresis. Cryobiology, Elsevier, 2018, 83, pp.27-33. ⟨10.1016/j.cryobiol.2018.06.009⟩. ⟨hal-02622796⟩



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