4. The cryoprotecting agents (CPAs) are added stepwise. Add
1 mL of CPA solution to 1 mL of embryo suspension in
seawater, in 15 equimolar steps 1 min apart (1:1 final dilution)
at 19 Æ 1
C.
5. Thawing is performed by immersion in a 17 Æ 1
C water bath
until the ice is melted upon visual inspection.
6. CPAs should then be diluted with clean ASW in 12 equimolar
steps 1 min apart at room temperature 19 Æ 1
C, and embryos
can finally be rinsed with clean ASW.
7. Post-thaw quality assessment: embryos can be incubated for
96 h (Fig. 4c) until reaching the four-armed pluteus stage.
Cryopreserved sea urchin embryos take up to 96 h to develop
to four-armed pluteus, while fresh embryos take 48 h
(18–20
C). After 96 h cryopreserved embryos will lead to
>90% healthy four-armed pluteus larvae that are typically half
the size of control larvae (Fig. 4) (see Note 6).
3.6 Cryopreservation
of Mussel (Mytilus
galloprovincialis)
D-Larvae
1. Collect separately the spawned mussels. In the case of males,
put separately in backers with the umbo facing upward. Try to
transfer the minimum quantity of seawater to conserve the
sperm “dry” and inactivated.
2. Fertilized M. galloprovincialis are incubated for 72 h (18
C),
with gentle aeration. Incubation density should be between
20 and 40 fertilized eggs/mL.
3. Collect and concentrate the larvae, and add the CPA in a single
step (1:1 dilution so the final concentration is ethylene glycol
10% + trehalose 0.4 M).
4. Allow for an equilibration time of 60 min at 18
C while
loading into straws and sealing them. After 60 min place the
straws into the controlled rate freezer, making sure they are
completely dry on the outside.
5. The cryopreservation protocol starts with holding at 4
C,
cooling at 1
C/min to À12
C, and then holding for 2 min
for seeding. After this, the protocol continues cooling at 1
C/
min to À35
C. Finally, quickly transfer the straws to liquid
nitrogen for storage.
6. Prepare the water bath with water at 35
C for thawing. Hold
the straws in the warm bath for 6 s. Then, transfer the content
of the straw to a vial, and add seawater in a 1:1 proportion.
7. Post-thaw quality assessment: D-larvae survival can be assessed
short-term in several ways, for example, by monitoring feeding
activity or swimming behavior. Long-term assessment will
require the assessment of growth and development during
larval rearing and settlement capacity (see Note 7).
422
Estefania Paredes et al.
1 mL of CPA solution to 1 mL of embryo suspension in
seawater, in 15 equimolar steps 1 min apart (1:1 final dilution)
at 19 Æ 1
C.
5. Thawing is performed by immersion in a 17 Æ 1
C water bath
until the ice is melted upon visual inspection.
6. CPAs should then be diluted with clean ASW in 12 equimolar
steps 1 min apart at room temperature 19 Æ 1
C, and embryos
can finally be rinsed with clean ASW.
7. Post-thaw quality assessment: embryos can be incubated for
96 h (Fig. 4c) until reaching the four-armed pluteus stage.
Cryopreserved sea urchin embryos take up to 96 h to develop
to four-armed pluteus, while fresh embryos take 48 h
(18–20
C). After 96 h cryopreserved embryos will lead to
>90% healthy four-armed pluteus larvae that are typically half
the size of control larvae (Fig. 4) (see Note 6).
3.6 Cryopreservation
of Mussel (Mytilus
galloprovincialis)
D-Larvae
1. Collect separately the spawned mussels. In the case of males,
put separately in backers with the umbo facing upward. Try to
transfer the minimum quantity of seawater to conserve the
sperm “dry” and inactivated.
2. Fertilized M. galloprovincialis are incubated for 72 h (18
C),
with gentle aeration. Incubation density should be between
20 and 40 fertilized eggs/mL.
3. Collect and concentrate the larvae, and add the CPA in a single
step (1:1 dilution so the final concentration is ethylene glycol
10% + trehalose 0.4 M).
4. Allow for an equilibration time of 60 min at 18
C while
loading into straws and sealing them. After 60 min place the
straws into the controlled rate freezer, making sure they are
completely dry on the outside.
5. The cryopreservation protocol starts with holding at 4
C,
cooling at 1
C/min to À12
C, and then holding for 2 min
for seeding. After this, the protocol continues cooling at 1
C/
min to À35
C. Finally, quickly transfer the straws to liquid
nitrogen for storage.
6. Prepare the water bath with water at 35
C for thawing. Hold
the straws in the warm bath for 6 s. Then, transfer the content
of the straw to a vial, and add seawater in a 1:1 proportion.
7. Post-thaw quality assessment: D-larvae survival can be assessed
short-term in several ways, for example, by monitoring feeding
activity or swimming behavior. Long-term assessment will
require the assessment of growth and development during
larval rearing and settlement capacity (see Note 7).
422
Estefania Paredes et al.