3. Cryopreservation straws (0.25 mL or 0.5 mL) and either sealing powder or a heat sealer.
4. Cryopreservation security gear (gloves, forceps/tweezers, protective eyewear).
5. Timer.
6. Liquid nitrogen (~1.5 L).
7. Prepare a solution of 15% (v/v) dimethyl sulfoxide in sterile
natural seawater (FSW) or artificial filtered seawater (ASW)
(i.e., passed through a 0.22 μm filter and/or UVA-treated).
2.2 Cryopreservation
of Sea Cucumber
(Holothuria
forskali) Sperm
1. General equipment for liquid nitrogen vapor cryopreservation
(see Fig. 1).
2. Polystyrene rack, height 6 cm above the liquid nitrogen surface
(for cryopreservation at an approximate cooling rate of
~16
C/min).
3. Prepare a solution of 15% (v/v) dimethyl sulfoxide in sterile
natural seawater or artificial seawater (i.e., passed through a
0.22 μm filter and/or UVA-treated).
2.3 Cryopreservation
of Mussel
(Mytilus galloprovincialis) Sperm
1. General equipment for liquid nitrogen vapor cryopreservation
(see Fig. 1).
2. Polystyrene rack, height 5 cm above the liquid nitrogen
surface.
3. Prepare a solution of 15% (v/v) dimethyl sulfoxide in filtered
(0.22 μm + UVA) natural seawater or artificial seawater.
Fig. 1 Equipment used in cryopreservation in liquid nitrogen vapor: (1) Styrofoam box, (2) straws, (3) comb to
place the straws prior to applying sealing powder, (4) hand-made floating racks, and (5) close-up of two
floating racks with different distance to the liquid nitrogen surface (i.e., different cooling rates)
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