DE4205386C1 - Cryo:preservation and revitalisation of fish eggs, crab larvae etc. - involves treating with e.g. DMSO, freezing and revitalising by warming - Google Patents

Abstract

Cryopreservation and revitalisation of complete fish eggs, isolated embronic development stages, small water organism and/or the larvae of crabs comprises introducing the organisms into a water bath with a temp. of 10-30 deg.C and treating with 0-12% glycerol soln. or DMSO contg. added bovine serum albumin (BSA); (b) sucking the organisms up into thin plastic straws, and closing (c) freezing in a cryostat by cooling to -5 to -10 deg.C at a rate of 0.1 - 5 deg.C/min., and maintaining the temp. in the cryostat or a freezer) so that the organisms remain continuously in the liq. phase; and revitalising by (d) warming by placing in a water bath and heating to 10-30 deg.C at a rate of 40 deg.C/minute. Pref. the organisms are eggs of Zebra barbling, carp, golden orfe, small fresh-water fish or marine flat fish or cod family; Daphnia magno, Artemia saklina; or decapoda larval stages. ADVANTAGE - Process is simple. Revitalisation is good.

Description

The present invention relates to a method for Cryopreservation and subsequent revitalization from complete fish eggs and / or isolated embryonic Stages of development and / or small crabs and / or Larvae of higher crayfish.

Early life stage tests with early life stages at Fish are well suited for capturing sublethal effects due to potentially water-polluting substances. (Early Life Stage Test, ELST, Journal for Applied Zool. 76 (1989) 195-220).

The implementation of such tests is, however, by Seasonal spawning season for native species limited.

FARRANT & ASHWOOD-SMITH, 1980 treat the general Aspects of cryopreservation of cells, tissues and Organisms.

(FARRANT, J., and ASHWOOD-SMITH, M. J .: Practical aspects. In ASHWOOD-SMITH, M. J. and J. FARRANT (eds.): Low Temperature Preservation in Medicine and Biology, Pitman Medical Ltd. Turnbridge Wells, Kent, (1980) 285-310).

The biochemical foundations of cryobiology are at FRANKS (1985) summarized.

(FRANKS, F., Biophysics and biochemistry at low temperatures. Cambridge University Press. (1985) 209 pp).

STOSS (1983) gives an overview of the state of knowledge the cryopreservation of fish gametes.  (STOSS, J.,: Fish Gamete Preservation and Spermatozoan Physiology. In: HOAR, W. S., RANDALL, D. J. and E. M. DONALDSON (eds.): Fish Physiology, Vol. IX, Reproduction, 477 pp. (1983) 305-350).

Freezing sperm and later use for artificial insemination is in cattle breeding well examined and has been part of the breeding for a long time Practice. Freezing is similar to warm-blooded animals known from sperm in fish. In numerous Different methods will work in different ways Variations on cryopreservation of fish sperm presented.

The processes are based on techniques in which dry ice or liquefied nitrogen is used. The cooling preservation of fish eggs or insulated There is a difference between embryonic stages of development this is still at the beginning and there are only a few orientation Investigations.

WITTINGHAM & ROSENTHAL (1978) described a partial successful process for freezing isolated Herring embryos.

(WITTINGHAM, D.G. and ROSENTHAL, H .: Attemps to preserve herring embryos at subzero temperatures. Arch. Fisheries Science 29: 75-79 (1978).

HAGA (1982) and STOSS & DONALDSON (1983) put similar ones Investigations with the rainbow trout and a Pacific salmon species.

(HAGA, Y.,: On the Subzero Temperature Preservation of Fertilized Eggs of Rainbow Trout. Bull Japan. Soc. Sci. Fish. 11 (1982) 1569-1572;  STOSS, J. and DONALDSON, E. M .: Studies on Cryopreservation of Eggs from Rainbow Trout (Salmo gaidneri) and Coho Salmon (Oncorhynchus kisutch). Aquaculture. 31 (1983) 51-65).

Supercooling experiments on various salmonids have been carried out by HARVEY & ASHWOOD-SMITH (1982) and by HARVEY et al. (1983).

(HARVEY, B. and ASHWOOD-SMITH, M. J .: Cryoprotectant Penetration and Supercooling in the Eggs of Salmonid Fishes. Cryobiol. 19 (1982) 29-40; HARVEY, B., STOSS, J. and BUTCHART, W .: Supercooled Storage of Salmonid Ova. Canadian Technical Report of Fisheries and Aquatic Sciences No. 1222 (1983) 13 pp).

The optimistic report of surviving salmonid Eggs after freezing with liquid nitrogen could not be determined by follow - up examinations of the Author confirmed by other experimenters gene.

(ZELL, S. R.,: Cryopreservation of Gametes and Embryos of Salmonid Fishes. Ann. Biol. Anim. Bioch. Biophys. 4th (1978) 1089-1099; ERDAHL, D.A. and GRAHAM, E. F .: Preservation of gametes of freshwater fish. Int. Congr. Anim. Reprod. Artif. Insem. (proc), 9th, (1980) 317-326.

In the currently mandatory fish test (DIN 38 412-L 31), gold throws are used. The DIN standard dictates to use animals 5-7 cm in length. Since the spawning period is limited by Goldwerfen, the Test must be feasible at any time, the ge  demanded length range only through manipulated nutrition the fish are kept.

This inevitably leads to the test fish being different are old and differently sensitive. The fish size specified in the standard and the corresponding one Obesity increases the basic deficiency of different seasons only apparently on. The reproducibility of the test results is not improved.

With regard to the "reproduction" parameter, there are suitable ones Bio-tests with local fish species for registration sublethal effects such as B. ELST, reproduction test or the life cycle test. Are for enforcement these tests are currently not practical, because their applicability to the limited time Availability of standardized egg material tert.

Around now egg material from fish is constantly available to try to freeze fish eggs, around the above Conduct standardized tests. Unlike small cell organisms like bacteria, unicellular blue or green algae or vertebrae Animal cell cultures are the main difficulty when freezing fish eggs in their high water content and in size.

So it turned out that with simple means in liquid nitrogen frozen eggs after the Thawing deformations and enveloping strong mechanical destruction of the embryonic systems grasslands.

It was therefore an object of the invention to provide a method for To provide with the standardized, ge  Freeze-preserved material from fish eggs is available can be put. Subject of the invention is a process for cryopreservation and subsequent Revitalization of complete fish eggs and / or isolated embryonic developmental stages and / or Small crabs and / or larvae of higher crabs, which is characterized in that

• a) the fish eggs and / or isolated embryonic ent stages of development and / or small crabs and / or Larvae of higher crayfish in a water bath with a Temperature transferred between 10 ° C to 30 ° C and with a 0-12% glycerol solution or dimethyl sulfoxide treated with bovine serum albumin additive will,
• b) then the fish eggs and / or isolated embryonic Stages of development and / or small crabs and / or larvae of higher crabs on commercially available thin plastic tubes (straws; mini sequins) opened and the straws sealed will,
• c) the straws are then immediately transferred to a programmable cryostat for freezing according to the following temperature profile.
  • Cooling from + 10 ° C to + 30 ° C to -5 ° C to -10 ° C with a freezing rate between 0.1 ° C / min and 5 ° C / min,
  • - keeping the temperature in the cryostat or after transferring it in the freezer,
• d) the organisms are constantly in the liquid phase remain,
• e) then the straws directly from the cryostat or the Freezer in a water bath with temperatures introduced between 10 ° C to 30 ° C and with a Rate of 40 ° C / min reheated and thus revitalized will.

Particularly preferred embodiments of the invention Processes are characterized in that

• - zebrafish and / or carp eggs and / or the gold orfe and / or small fish of the Freshwater and / or as sea fish the groups of Uses flatfish and / or the codfish,
• - Daphnia magna and / or Artemia as small crustaceans salina,
• - you use the larval stages of the Decapoda.

Still other preferred embodiments of the invention Processes are characterized in that

• - the temperature profile
  • - Cooling from + 28 ° C to -10 ° C with a freezing rate of 0.5 ° C / min
• amounts to  
• - the temperature profile
  • - Cooling from + 18 ° C to -5 ° C with a freezing rate of 0.5 ° C / min
• amounts to
• - the temperature profile
  • - Cooling from + 22 ° C to -5 ° C with a freezing rate of 0.5 ° C / min
• amounts to
• - the temperature profile
  • - Cooling from + 10 ° C to + 30 ° C to -5 ° C with a freezing rate of 0.5 ° C / min
• is.

After the revitalization, the condition of the organisms controlled in a binocular and the organisms are immediately incubated or used for tests.

The method according to the invention in particular achieved the following advantages:

• - Provision of uniform test material (Sex products from only one pair of parents or a strain, genetically homogeneous starting material); this is particularly useful for toxicological studies important to the naturally high varia  restrict the biological material and thus the reproducibility and comparability of the Increase results that are indispensable to justice of a test system,
• - Long-term preservation of fish eggs is the Basic requirement for the introduction of test procedures with early stages of life of indigenous fish ten;
• - the availability of biological material for Such a method makes test purposes independent from the seasonally limited spawning season to indigenous Fish species;
• - a maintenance of spawning fish stocks related to elaborate maintenance procedure is connected can be significantly simplified or by shipping of eggs through a central hatchery for many users are completely eliminated;
• - for aquaculture and fisheries interesting aspects because the use of cryopreserved material new breeding opportunities opened like they already do in cattle breeding are widely used and there are production processes and restocking can be carried out not from the seasonal maturity of the spawning animals are dependent
• - The test with daphnia to be carried out according to DIN 38 412 as well as testing with other small crabs can be essential be simplified because of their rearing and keeping could be omitted in root culture.

The invention is illustrated by the following examples explained in more detail. Here mean  

Fig. 1 spawning aquarium for zebrafish,

Fig. 2 Working scheme for freezing and revitalizing

Example 1 material and methods

The experimental animals of the species Brachydanio rerio BUCH had an age between four and twelve months. Adult animals can easily be differentiated according to male and female. The size of the animals spawned was 50-70 mm. The keeping took place under constant Conditions at a water temperature of 27 ° C, a pH around the neutral point, an oxygen content of almost 100% and an artificially controlled one Day-night rhythm of 12 hours each. The Feeding was done with commercially available ornamental fish food the Tetramin brand.

The oviposition of the parent animals was stimulated and controlled as follows: For spawning, eight males and four females were placed in prepared spawning aquariums. Spawning is done by placing a rectangular glass bowl with a number of green glass plant imitations on the eve of the spawning aquarium ( Fig. 1).

For sexually mature zebrafish that are among natural Conditions only over submerged aquatic plants spawning is this artificial spawning substrate the trigger around at dawn the following Eggs and sperm of the day over the "glass tree"  to deliver. The eggs sink down and can be used for further Treatment together with the glass bowl from the Aquarium can be removed.

The freezing experiments were with fertilized eggs carried out at different stages of development. Here, a cryostat from SyLab, type Cryocell 1200, used with the defined temperature profiles freeze and reheat could. This device was used as a field device for cryopreservation developed by bull semen, but is suitable very good around small diameter fish eggs as well Freeze fish sperm samples. The eggs will here on thin plastic tubes, so-called "straws" or "mini sequins" and placed in a sample chamber transferred from solid aluminum. The cooling takes place by a cold conduction rod attached to the chamber, also made of solid aluminum, which in liquid Nitrogen is immersed.

The desired temperature profile when freezing is programmable and can consist of up to twenty individual steps with different freezing rates. Before the The eggs were frozen with the antifreeze Glycerin or dimethyl sulfoxide with bovine serum albumin addition treated to undesirable egg crystal formation to prevent.

Results

The freezing experiments with zebrafish eggs were carried out at various stages of embryonic development. A cryostat was used to check the suitability of numerous different temperature profiles during the freezing process and reheating. The procedure is shown schematically in Fig. 2.

First, the zebrafish (Fig. 2.1) were prepared as described brought to spawn. The eggs were then transferred to a water bath and treated there with a glycerol solution so that an 8% final concentration was reached ( Fig. 2.2).

The eggs were immediately pulled out of the water bath on mini sequins ( Fig. 2.3) and the straws were then sealed with stoppers made of polyvinyl alcohol powder. The straws were immediately transferred to the cryostat and frozen there ( Fig. 2.5). Very flat temperature gradients have proven to be favorable here. The numerous experiments with different temperature profiles had shown that reheating as quickly as possible, ie a very steep temperature gradient during the thawing process, had a more favorable effect on the eggs. For this reason, the straws were thawed directly from the cryostat into a water bath at 28 ° C and swiveled slightly so that the thawing process only took a few seconds ( Fig. 2.6). The eggs were rinsed simultaneously and the glycerol solution was removed.

In the last step, the condition of the egg shell and the embryonic system in the binocular was checked or transferred to a incubation cuvette ( Fig. 2.7 and 2.8).

The first orientation tests showed that

• - still on the use of antifreeze could not be waived and that
• - a more careful freeze with small ones Freeze rates the eggs less damaged.

With the "CryoCell" device from SyLab then experiments with varying profiles performed, with freezing rates between 0.2 ° / min and 10 ° / min checked and with different on tauraten were combined.

Common to all trials was the start of the trial and that End of experiment at a water temperature of 28 ° C, the incubation temperature of zebrafish.

Fig. 3 shows an example of one of the very similar profiles, which in this case consists of 10 individual steps. The temperature profiles were developed and changed in small steps, based on how well the eggs had been damaged during the procedure.

Having a large percentage in the first freeze attempts of the thawed eggs strong deformations and mechanical destruction of the cortex as well of the embryonic system were increasingly successful better to design the procedure so that the damage decreases. The results were confirmed of the preliminary examinations that indicated that a shallow temperature gradient when freezing one less harmful influence on the eggs or embryonic systems has as a quick freeze or even a "Flash Freeze".

Conversely, when reheating is as steep as possible Gradient cheaper. The best result where  for the first time, a small proportion of the frozen ones succeeded Revitalizing eggs was accomplished with the following Temperature profile achieved:

• - Cooling from + 28 ° C to -10 ° C at a rate from 0.5 ° C / min,
• - keeping the temperature for more than 30 min,
• - Reheat in a water bath at 28 ° C with a Rate of approx. 40 ° C / min.

In this experiment, 45 zebrafish eggs were used. Of these were after reheating eight embryos alive. In the following hours after the experiment two of the embryos died, at Hatch two more. They came to a successful hatch four larvae that have no externally visible damage or malformations. The experiment was repeated under the same boundary conditions. The results could be reproduced, whereby however, the low N is a statistical evaluation excludes.

In addition to determining a favorable temperature profile two other parameters were of particular importance to: the type, concentration and exposure time of the Antifreezing agent and the development stage of Embryos.

At a water temperature of approx. 28 ° C, zebrafish have a development time until hatching of approx. 3.5 days. Freezing attempts were initially relative early stages of development. Here were as Orientation the experience from freezing treatment  used by bovine embryos: freezing them Embryos work best in the early morula stage. Transferred to the development of zebrafish this means an age of about 3-5 hours after the Oviposition. The embryonic system is at this early stage not yet differentiated, it lies as a flat one Disk of the yolk sphere.

Surprisingly, the results described above were not with these very early stages of development achieved, but with larvae that largely were differentiated, the yolk regrow to 360 ° C had and already showed the first pigmentation. The larvae were 55 hours old, from which Egg laying counted.

During the investigations it became clear that the exact Control of the temperature profile in the freezing process a crucial prerequisite for successful Ge freezing experiments.

In the freezing experiments with fertilized eggs of the Zebrafish could gradually improve the quality of the reheated eggs or their embryonic systems a gradual change in the temperature profile ver be improved. Eventually, a number of eggs actually succeeded to revitalize and larvae without deformities to hatch. The knowledge according to the invention, who had this result can be as follows sum up:

• 1. Temperature profiles proved to be favorable  very shallow when freezing and reheating were set very steep.
• 2. It is cheaper to keep the eggs refrigerated to keep in the liquid phase and the egg to avoid crystal formation. Possibly is however, by refining the "seeding" - Procedure by which the crystallization is triggered will compensate.
• 3. The first resuscitated embryos were in place at the time of freezing in a very advanced Stage of development. The larvae found itself shortly before hatching. According to the experience the cryopreservation of bovine oocytes this was surprising: here are possible early stages frozen.

Table 1 below shows the results of Experiments summarized. Resuscitation rates of To achieve 100%, d. H. all tried frozen Eggs could be revitalized and hatched. It will shown that this even with the earliest stages of life of the zebra bear succeeds and that this is due to the addition of antifreeze can be completely dispensed with can or only use low concentrations are.  

Table 1

Resuscitation rates (%) at different concentrations of glycerin and at different stages of development

Example 2

It was examined whether the method of cooling preservation from fish eggs to other groups of organisms can be transferred. For this purpose experiments with the little ones were carried out cancer types Daphnia magna and Artemia salina leads.

D. magna is native and wide in local waters spread. It is used in aquatic toxicology as a test organism (daphnia test according to DEV, DIN 38 412 L 11 and L 30) and must be in the laboratories that are regular perform this daphnia test, in permanent culture being held.

The benefits cryopreserved animals would offer are obvious: the quality of the daphnia could be standardized by a central shipping office, the Maintaining a regular culture could be dispensed with and thus would be institutions are also able to perform this test easy to perform, which has no regular culture feature.

There were two attempts with D. magna and one attempt performed with A. salina. Here the daphnia pretreated as required in DIN 38 412 L 11, d. H. the animals were "DIN-compliant" Individuals from the 0.3 mm sieve with a size of 0.3 to 0.6 mm in size and less than an age 24 hours.  

The daphnia were made up of about 10 animals each on "mini sequins" reared, with a surrounding glycerol concentration of 2%. The initial temperature was the housing temperature of 18 ° C, final temperature -5 ° C. The Freezing gradient, as in the experiments with fish eggs, 0.5 ° C / min. The reheating was carried out as in experiments with fish eggs, in a water bath, however not at 28 ° C, but at 18 ° C.

After reheating were in both trials D. magna all animals alive, active and showed one compared to the non-frozen animals completely "normal" vitality.

The same result was obtained in an experiment with 56 individuals the tropical cancer Artemia salina. The boundary conditions were slightly modified: No glycerin was added, since the surrounding water contains artificial sea salt ("tropic marin ") in a concentration of 3%, and such ice formation was impossible. Home and The final temperature was 22 ° C.

Here, too, all animals were alive after thawing, active and vital.

List of reference symbols

Fig. 1:
A full glass aquarium
E eggs
G glass tree
L spawning bowl
M stitches
Fig. 2:
Ek incubation cuvette
G glass cuvette
Kr cryostat
K refrigeration guide
L spawning pool
M mini sequins
S syringe
V closure
W water bath

Claims (12)

1. A method for cryopreservation and subsequent revitalization of complete fish eggs and / or isolated embryonic stages of development and / or small crabs and / or larvae of higher crayfish, characterized in that

• a) the fish eggs and / or isolated embryonic developmental stages and / or small crayfish and / or larvae of higher crayfish are transferred to a water bath at a temperature between 10 ° C to 30 ° C and treated with a 0-12% glycerol solution or dimethyl sulfoxide with bovine serum albumin additive ,
• b) the fish eggs and / or isolated embryonic developmental stages and / or small crayfish and / or larvae of higher crayfish are then grown and sealed on thin plastic tubes (straws),
• c) the straws are then immediately transferred to a programmable cryostat for freezing according to the following temperature profile
  • Cooling from + 10 ° C to + 30 ° C to -5 ° C to -10 ° C with a freezing rate between 0.1 ° C / min and 5 ° C / min,
  • - keeping the temperature in the cryostat or after transferring it in the freezer,
• d) the organisms remain constantly in the liquid phase,
• e) The straws are then placed directly from the cryostat or the freezer into a water bath at temperatures between 10 ° C to 30 ° C and reheated at a rate of approx. 40 ° C / min and thus revitalized.

2. The method according to claim 1, characterized in that that you have eggs of the zebrafish and / or carp and / or the gold orfe and / or small fish of fresh water and / or as sea fish Groups of flatfish and / or cod-like starts. 3. The method according to claim 1, characterized in that Daphnia magna and Artemia salina. 4. The method according to claim 1, characterized in that the larval stages of the Decapoda are used. 5. The method according to claim 1 and / or 2, characterized in that the temperature profile

• - Cooling from + 28 ° C to -10 ° C with a freezing rate of 0.5 ° C / min

is.   6. The method according to claim 1 and / or 3, characterized in that the temperature profile

• - Cooling from + 18 ° C to -5 ° C with a freezing rate of 0.5 ° C / min

is. 7. The method according to any one of claims 1 and / or 3, characterized in that the temperature profile

• - Cooling from + 22 ° C to -5 ° C with a freezing rate of 0.5 ° C / min

is. 8. The method according to claim 1 and / or 4, characterized in that the temperature profile

• - Cooling from + 10 ° C to + 30 ° C to -5 ° C with a freezing rate of 0.5 ° C / min

is.