cryobanking purposes, they should be sealed according to the
manufacturer’s instructions. For laboratory experiments, they
can be used without sealing.
5. A portable programmable biofreezer programmed with a freezing rate of 6
C/min from 0 to À70
C is used for freezing
straws. After freezing, straws are directly stored in a liquid
nitrogen container.
6. Thaw the frozen straws in a water bath at 37
C for 10 s.
7. Post-thaw quality assessment: Sperm quality is assessed using
motility and cell viability. Motility is evaluated using computerassisted sperm analysis (CASA system). Several commercial
CASA softwares are available. Analyses are carried out in a
Makler chamber under a 10Â negative-phase-contrast objective coupled with a digital camera set for 25 fps. Samples need
to be diluted 1:10 in artificial seawater prior to analysis. Motility analyses are performed by placing 10 μL of diluted sperm
into a Makler chamber. After activation, sperm moves for several days. To assess sperm viability, two methods can be used:
fluorescence microscope or flow cytometer. For fluorescence
microscope analysis, mix 15 μL of diluted sperm, 0.5 μL SYBR
Green (final concentration 100 nM), and 1.5 μL propidium
iodide (PI), and observe under a fluorescence microscope.
Count at least 100 cells, distinguishing live (SYBR Green positive, green cells) and dead cells (PI stained, red cells). For flow
cytometer analysis, dilute 5 μL of sperm in 500 μL of 1% NaCl
buffer. Add 2 μL PI at a concentration of 2.4 mM to the
suspension. Analyze in a flow cytometer after 5 min of incubation in the dark. After freezing and thawing, motile sperm with
no agglutination decreases drastically (approximately 2% motility), and the percentage of viable cells is approximately 60%.
For fertilization, use the sperm immediately after thawing as
the viability of spermatozoa decreases with time (see Note 5).
3.5 Cryopreservation
of Sea Urchin
(Paracentrotus lividus)
Embryos
1. P. lividus early blastulas are obtained with 7–8 h incubation at
20
C. For this purpose a male and female sea urchin should be
dissected and gametes collected directly from the gonads (see
Fig. 4). Upon quick examination of gamete quality (spherical
oocytes with homogeneous color and motile sperm), fertilization should be conducted and a % of fertilization success calculated. Do not use batches with a fertilization rate under 80%.
2. P. lividus cells are incubated for 8 h (20
C), with gentle
aeration. Incubation density should be between 40 and 80 fertilized eggs/mL.
3. Filter the embryos carefully with a 40–60 μm mesh filter and
concentrate them. Calculate the cell density prior to
cryopreservation.
Cryopreservation of Marine Invertebrates
421
manufacturer’s instructions. For laboratory experiments, they
can be used without sealing.
5. A portable programmable biofreezer programmed with a freezing rate of 6
C/min from 0 to À70
C is used for freezing
straws. After freezing, straws are directly stored in a liquid
nitrogen container.
6. Thaw the frozen straws in a water bath at 37
C for 10 s.
7. Post-thaw quality assessment: Sperm quality is assessed using
motility and cell viability. Motility is evaluated using computerassisted sperm analysis (CASA system). Several commercial
CASA softwares are available. Analyses are carried out in a
Makler chamber under a 10Â negative-phase-contrast objective coupled with a digital camera set for 25 fps. Samples need
to be diluted 1:10 in artificial seawater prior to analysis. Motility analyses are performed by placing 10 μL of diluted sperm
into a Makler chamber. After activation, sperm moves for several days. To assess sperm viability, two methods can be used:
fluorescence microscope or flow cytometer. For fluorescence
microscope analysis, mix 15 μL of diluted sperm, 0.5 μL SYBR
Green (final concentration 100 nM), and 1.5 μL propidium
iodide (PI), and observe under a fluorescence microscope.
Count at least 100 cells, distinguishing live (SYBR Green positive, green cells) and dead cells (PI stained, red cells). For flow
cytometer analysis, dilute 5 μL of sperm in 500 μL of 1% NaCl
buffer. Add 2 μL PI at a concentration of 2.4 mM to the
suspension. Analyze in a flow cytometer after 5 min of incubation in the dark. After freezing and thawing, motile sperm with
no agglutination decreases drastically (approximately 2% motility), and the percentage of viable cells is approximately 60%.
For fertilization, use the sperm immediately after thawing as
the viability of spermatozoa decreases with time (see Note 5).
3.5 Cryopreservation
of Sea Urchin
(Paracentrotus lividus)
Embryos
1. P. lividus early blastulas are obtained with 7–8 h incubation at
20
C. For this purpose a male and female sea urchin should be
dissected and gametes collected directly from the gonads (see
Fig. 4). Upon quick examination of gamete quality (spherical
oocytes with homogeneous color and motile sperm), fertilization should be conducted and a % of fertilization success calculated. Do not use batches with a fertilization rate under 80%.
2. P. lividus cells are incubated for 8 h (20
C), with gentle
aeration. Incubation density should be between 40 and 80 fertilized eggs/mL.
3. Filter the embryos carefully with a 40–60 μm mesh filter and
concentrate them. Calculate the cell density prior to
cryopreservation.
Cryopreservation of Marine Invertebrates
421