3. Add the cryoprotecting agent (CPA) to the sperm suspension
in a 1:1 proportion in one step, and allow 5 min for reaching
osmotic equilibrium while loading the straws with the solution.
Place the straws in the comb to make room for the seal, and seal
them with PVC powder. Allow the powder to polymerize by
immersion of the tip of the straw into the water bath for a few
seconds, and dry the straws carefully.
4. After equilibration, put the straws immediately on the polystyrene rack and cover the box. Allow 15 min for cooling and then
plunge the straws into liquid nitrogen. The samples can subsequently be stored in liquid nitrogen for as long as necessary.
5. For thawing, prepare a water bath at 28
C, and plunge the
straws into the water for 10 s. Then, transfer the content of
each straw to TCD plates, and dilute with seawater the cryoprotective agent 1:1 to minimize toxic effects.
6. Post-thaw quality assessment: This protocol produces motile
sperm, cells are free moving without excessive aggregation, and
movement is slower than controls. Fertilization with a 100:1
sperm oocyte ratio leads to 50% production of normal 4-armed
pluteus larvae after 48 h. Fertilization occurs in a matter of
seconds; therefore for that shorter period of time, the sperm/
oocyte ration can be increased until reaching 100% fertilization, and then the excess sperm should be removed prior to
incubation (see Note 1).
3.2 Cryopreservation
of Sea Cucumber
(Holothuria
forskali) Sperm
1. Sperm can be obtained by the direct collection of sperm from
the gonads after dissection of a male. Pure concentrated sperm
is stored in a 50 mL vial at room temperature until use.
2. Cryoprotecting agents (CPA) are added in a single step:
500 mL of CPA solution is mixed with 50 μL of pure sperm
and allowed a 15-min equilibration period at 18
C while
loading and sealing the 0.25 mL straws.
3. Cryopreservation is carried out by placing the straws over a
floating platform (6 cm above the liquid nitrogen level) in a
closed Styrofoam box for 30 min before plunging the samples
into liquid nitrogen for storage.
4. Thawing can be performed by immersion of the straws in a
controlled temperature water bath (28
C) until the samples
melted (8 s).
5. Sperm can later be diluted with FSW (1:1) to dilute the CPA.
6. Post-thaw quality assessment can be undertaken by observation of sperm activity under the microscope: sperm cells should
be homogeneously moving in the samples, without aggregation, and movement should be vigorous and continuous.
Assessing fertilization can be challenging due to the need for
natural-spawned oocytes (see Note 2).
Cryopreservation of Marine Invertebrates
419
in a 1:1 proportion in one step, and allow 5 min for reaching
osmotic equilibrium while loading the straws with the solution.
Place the straws in the comb to make room for the seal, and seal
them with PVC powder. Allow the powder to polymerize by
immersion of the tip of the straw into the water bath for a few
seconds, and dry the straws carefully.
4. After equilibration, put the straws immediately on the polystyrene rack and cover the box. Allow 15 min for cooling and then
plunge the straws into liquid nitrogen. The samples can subsequently be stored in liquid nitrogen for as long as necessary.
5. For thawing, prepare a water bath at 28
C, and plunge the
straws into the water for 10 s. Then, transfer the content of
each straw to TCD plates, and dilute with seawater the cryoprotective agent 1:1 to minimize toxic effects.
6. Post-thaw quality assessment: This protocol produces motile
sperm, cells are free moving without excessive aggregation, and
movement is slower than controls. Fertilization with a 100:1
sperm oocyte ratio leads to 50% production of normal 4-armed
pluteus larvae after 48 h. Fertilization occurs in a matter of
seconds; therefore for that shorter period of time, the sperm/
oocyte ration can be increased until reaching 100% fertilization, and then the excess sperm should be removed prior to
incubation (see Note 1).
3.2 Cryopreservation
of Sea Cucumber
(Holothuria
forskali) Sperm
1. Sperm can be obtained by the direct collection of sperm from
the gonads after dissection of a male. Pure concentrated sperm
is stored in a 50 mL vial at room temperature until use.
2. Cryoprotecting agents (CPA) are added in a single step:
500 mL of CPA solution is mixed with 50 μL of pure sperm
and allowed a 15-min equilibration period at 18
C while
loading and sealing the 0.25 mL straws.
3. Cryopreservation is carried out by placing the straws over a
floating platform (6 cm above the liquid nitrogen level) in a
closed Styrofoam box for 30 min before plunging the samples
into liquid nitrogen for storage.
4. Thawing can be performed by immersion of the straws in a
controlled temperature water bath (28
C) until the samples
melted (8 s).
5. Sperm can later be diluted with FSW (1:1) to dilute the CPA.
6. Post-thaw quality assessment can be undertaken by observation of sperm activity under the microscope: sperm cells should
be homogeneously moving in the samples, without aggregation, and movement should be vigorous and continuous.
Assessing fertilization can be challenging due to the need for
natural-spawned oocytes (see Note 2).
Cryopreservation of Marine Invertebrates
419