Cryopreservation of juvenile stages of barnacles
A method for the cryopreservation of eggs, nauplii and juvenile of barnacles. Cryopreserved feed and a use of the preserved organisms as live feed organism in marine aquaculture typically replacing rotifers and Artemia and other life feed organisms.
1. A method for cryopreservation of eggs, nauplii and/or juveniles of barnacles, the method comprising the following chronological steps:
(a) providing a source of organisms with water,
(b) draining water from the organisms to provide drained organisms,
(c) adding a cryoprotectant solution to the drained organisms to form a mixture, the cryoprotectant solution comprising a cryoprotectant selected from the group consisting of ethylene glycol, propylene glycol, glycerol, and dimethyl sulfoxide or a mixture thereof with a concentration within the range of between 5 M and 8 M,
(d) freezing the mixture in a container using the following procedure:
(i) freezing at a first slow freezing rate being not faster than −1° C. min −1 until a temperature wherein the organisms in the mixture begin to crystallize;
(ii) freezing at a second slow freezing rate being not faster than −0.1° C. min −1 until the mixture of the organisms and the cryoprotectant are entirely crystallized; and
(iii) freezing the entirely crystallized organisms and cryoprotectant until a cryogenic storage temperature is achieved.
2. The method according to claim 1 , wherein the volume ratio between the cryoprotectant solution and the drained organisms in the mixture is at least 1:4.
3. The method according to claim 1 , wherein the first slow freezing rate is −0.5° C. min −1 or less.
4. The method according to claim 1 , comprising an equilibration step lasting 5-60 minutes performed before the first freezing step.
5. The method according to claim 4 , wherein the equilibrium step is combined with application of a first slow freezing rate of −0.5 to −1° C. min −1 .
6. The method according to claim 1 , wherein the second slow freezing rate is between −0.015 and −0.1° C. min −1 .
7. The method according to claim 1 , wherein the second slow freezing rate lasts until a temperature of −30° C. or lower.
8. The method according to claim 1 , wherein the drained organisms have a dry weight content of 6 to 14% after drainage.
9. The method according to claim 1 , wherein the cryoprotectant solution comprises 2.0-4.5% NaCl.
10. The method according to claim 1 , wherein the concentration of the cryoprotectant added to the drained organisms is within the range of 6-8 M.
11. The method according to claim 1 , wherein the concentration of the cryoprotectant added to the drained organisms is at least 7 M.
12. The method according to claim 1 , wherein the cryoprotectant is ethylene glycol.
13. The method according to claim 4 , wherein the equilibration step is performed at a temperature within the range of between 0° C. and 10° C.
14. The method according to claim 1 , wherein the amount of material to be cryopreserved in one unit is at least within the range of 5 g-2000 g.
15. The method according to claim 1 , comprising an equilibration step performed before the first freezing step, wherein the mixture of the cryoprotectant and organisms are placed in a container suitable for cryopreservation before the equilibrium step or directly thereafter, and wherein said mixture in the container has a maximal thickness during the freezing process of no more than 100 mm.
16. The method according to claim 1 , wherein the first slow freezing rate is finished when the material has a homogenous temperature within the range of −10 to −13° C.
17. The method according claim 1 , wherein step (d) of freezing lasts at least 5 hours.
18. The method according to claim 1 , wherein the organisms are cryopreserved in individual volumes of 5-50 ml in molds.
19. A method for revitalization cryopreserved organisms that have been cryopreserved via the method of claim 1 , comprising one or more from the group consisting of:
thawing in a liquid bath having a temperature within the range of 30-40° C.;
crushing the frozen material and washing the crushed frozen material in sea water having a temperature of preferably 10° C. or less to form washed crushed material, and lowering the temperature of the washed crushed material at temperature of less than 5° C. for up to 36 hours; and
crushing the frozen material and incubating the crushed frozen material in sea water with aeration at a temperature of preferably 10° C. or less to form incubated crushed frozen material, and lowering the temperature of the incubated crushed frozen material to a temperature of less than 5° C. for up to 36 hours.
20. The method according to claim 19 , wherein the thawed organisms are adapted after thawing to the water temperature in the fish culture to be fed to.