Preliminary studies on the cryopreservation of silkworm (Bombyx mori)

Developing a technique for the cryopreservation of Bombyx mori L. germplasm may provide significant benefits for supplying good strains to farmers and facilitate the conservation of living material in genetic banks. Producing species-specific protocols for the selection of the most apt embryonic stage, dechorionation/permeabilization of eggshells, and the acclimation at low temperature before the treatment of eggs at cryogenic temperature is a prerequisite for cryopreservation of B. mori embryos. In this research, non-diapausing eggs of B. mori Nistari strain were used to study cold hardiness; the final aim was to select the most apt embryonic development stages, and in addition, reliable permeabilization, and vitrification treatments. Polyhybrid B. mori eggs of a diapausing strain were also used for comparison in experiments of embryogenesis, permeability, and acclimation under low temperature before cryopreservation. The Nistari eggs in the stage of 24 hours post-oviposition (h PO) showed a scarce survival rate (0.1%) after their submersion in liquid nitrogen (LN), while the highest survival in loading and dehydration with cryoprotective agents was assessed in 40h PO eggs. Overwintering diapausing eggs preserved for three months at 5 degrees C and then treated with various vitrification solutions gave rise to good hatching rates. The achieved results show that it is possible to improve the method of cryopreservation of B. mori eggs by obtaining a good permeability with cryoprotectants at loading/dehydration steps and by decreasing the toxicity, reducing the treatment time.