JPS5819255B2 - Low-temperature preservation method for fertilized fish eggs - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a low-temperature preservation method for cooling fertilized fish eggs to a predetermined temperature and preserving them for a desired period of time.

In aquaculture, if seedlings can be obtained regardless of the spawning season of fish, there are many advantages such as effective use of aquaculture ponds, shipping throughout the year, and saving on feed.

One possible way to do this is to cryopreserve the spermatozoa and eggs of fish, and while there are many examples of research on the cryopreservation of sperm, there are only a few examples of research on the cryopreservation of eggs.

Here, regarding the cryopreservation of eggs, cryopreservation of fertilized eggs is considered to be effective from the viewpoint of ease of hatching operation after cryopreservation and obtaining a high hatching rate.

Regarding cryopreservation of these fertilized eggs, the fertilized eggs of aquatic fish are cooled to about -10℃ and stored in the presence of a cryoprotectant that prevents the eggs from freezing, with a hatching rate of about 80℃. Although there are studies showing that it is advantageous, hatching rates close to 100% have not been obtained, and there are no examples of freshwater fish.

The present invention was made in response to the above-mentioned circumstances, and an object of the present invention is to provide a method for preserving fertilized fish eggs at a low temperature so as to obtain a high hatching rate close to 100q6.

The details of the low temperature preservation method for fish fertilized eggs according to the present invention are as follows.
cExplain.

The method for preserving fertilized fish eggs at a low temperature according to the present invention includes, for example, preserving fertilized eggs of rainbow trout, etc. using an inorganic salt aqueous solution, a cryoprotectant that prevents the fertilized eggs from freezing inside the egg, and an auxiliary agent that enhances the antifreeze effect of the cryoprotectant. immersed in a container containing a medium consisting of
This is a method of low-temperature preservation of fertilized eggs by cooling the fertilized eggs together with the container to a predetermined temperature of 0° C. or lower and preserving the cooled state for a desired period of time.

The inorganic salt aqueous solution contains potassium chloride (KCA'),
It is an aqueous solution containing calcium chloride (CaC122020) and sodium chloride (NaCl).

The above-mentioned cryoprotectant includes dimethyl sulfoxide ((CH3)2S), which is used for cryopreservation of eggs and blood of mammals.
O) (hereinafter referred to as "DMSO") is used.

This DMSO increases the osmotic pressure of the medium and further increases the permeability of the egg membrane of the fertilized egg, thereby dehydrating free water within the egg.

Therefore, in the presence of DMSO, free water within the fertilized egg is removed, and freezing within the egg is prevented even if the surrounding medium freezes.

Here, regarding the determination of the concentration of DMSO in the medium, the results shown in FIG. 1 were obtained.

That is, DMSO was added to an inorganic salt aqueous solution at O~4.2M (
When fertilized eggs were immersed in a medium with DMSO added and cooled at -7°C, no eggs were observed to hatch without DMSO added, but when 1.4 to 2.1 M of DMSO was added, Showing a hatching rate of 40-47 hatches, DMSO of 2.8-
When 4.2M was added, almost no hatching was observed.

In addition, in the figure, the broken line shown as a solid line indicates that the concentration of sodium chloride in the inorganic salt aqueous solution is 154 mM (mmol), and the broken line shown as a chain line indicates that the concentration of sodium chloride is <188 mM.

In addition, no hatching was observed in the cooling treatment at −10° C. or lower.

From this result, the optimal concentration of DMSO is 1.4-2.1M.
It can be seen that it is.

Next, if only DMSO is added to the medium, the deterioration rate will be only about 50, as shown in FIG. is added to the medium.

Examples of this adjuvant include glucose fraction serum or carbokinine methylcellulose (CMC).

Although it is not fully clear why such adjuvants enhance the antifreeze effect of DMSO, DMSO has the ability to aid in membrane permeation of substances that would not normally cross biological membranes.
By using the above adjuvant together with DMSO, 1,
It is presumed that the adjuvant penetrates into the egg and acts on the egg from within.

The hatching rate when glucose is added as an adjuvant is
As shown in the figure.

That is, a fertilized egg is immersed in a medium prepared by adding glucose to an inorganic salt aqueous solution containing 2.1M DMSO, and then incubated at 17°C.
When 5.6 mM glucose was added, the hatching rate was 40 to 57, and the hatching rate was 11.1.
The hatching rate of about 95 was obtained with the addition of mM.

In addition, in the figure, the broken line shown as a solid line indicates that the concentration of sodium chloride in the inorganic salt aqueous solution is 154 mM, and the broken line shown as a chain line indicates that the concentration of sodium chloride in the aqueous solution of inorganic salts is 171 mM.

Still, no eggs were observed to hatch after cooling to −10° C. or lower.

Next, the hatching rate when conidial serum is added as an adjunct to the cryoprotectant is as shown in Figure 3.
Fertilized eggs were immersed in an aqueous inorganic salt solution containing 2.1M MSO and conidial serum was added, and the eggs were cooled at -7°C. The hatching rate was approximately 95.

In addition, in the figure, the broken line shown as a solid line indicates that the concentration of k jJium chloride in the inorganic salt aqueous solution is 154 mM, and the broken line shown as a chain line indicates that the concentration is also 171 mM.

In addition, when conidial serum is added in this way, as shown in Figure 4, when the conidial serum concentration is IO%, the hatching rate of about 17 chicks is increased by cooling treatment at -10°C, but when cooling at -12°C. In the treatment as well, a hatching rate of about 14 inches was observed.

From this, it can be seen that conidic serum is superior to the above-mentioned glucose as an adjunct to the cryoprotectant for cryopreservation of fertilized eggs.

The cooling treatment and treatment of the fertilized eggs is carried out by placing the fertilized eggs in a test tube or the like containing a medium and placing the tubes in a cooling tank at about -20°C.

- In this way, once cooled to a predetermined temperature of -7°C to -20°C, the cooled state is maintained and stored for a desired period.

After that, when you want to hatch the eggs, take out the test tube from the cooling tank, return it to room temperature, and place it in the hatching tank to hatch.As explained above, in the present invention, the test tube is added to the medium in which the fertilized fish eggs are immersed. Dimethyl sulfoxide is added as an antifreeze agent, and as an auxiliary agent that enhances its antifreeze effect,
Alternatively, by adding conidic serum, the hatching rate of fertilized eggs after low-temperature storage can be increased to nearly 100 times.

Therefore, the eggs collected from the parent fish can be hatched without wasting them, and can be hatched at the desired time, making it possible to obtain seedlings regardless of the spawning season of the fish, allowing for effective use of the aquaculture pond, and year-round operation. Out of the blue.

It can provide many advantages in aquaculture, such as saving on cargo and feed.

Next, the present invention will be specifically explained with reference to Examples.

Examples Eggs used in experiments were squeezed out from brood stock of rainbow trout, artificially fertilized by an egg-washing method, and housed in an Adkins-type hatching species kept at a water temperature of 12° C. until used for experiments.

The medium in which the fertilized eggs were immersed was 3.2 mM potassium chloride, 2.3mM calcium chloride, and 154ml VI.
An inorganic salt aqueous solution containing ~188mM sodium chloride, 1.4~2.1M dimethyl sulfoxide as a cryoprotectant, and 5.6M glucose as an adjuvant.
mM ~ 11.1mM or 10% by volume of calf serum
1 Using the above-mentioned hatching tank, 30 eyed eggs 24 to 28 days after fertilization were prepared using 4.0%
20ml of medium cooled to ~7.2°C! The test tube was placed in a cooling bath at -20°C.

The cooling rate at this time is 0.4 to 0.9° up to -5°C.
C/ml n, below which it is 0.7-8
．． The temperature was 9°C/h.

After cooling the developed eggs to a predetermined temperature of -7°C to -20°C at the above cooling rate, the constant temperature state was maintained for 3 hours.

After this cooling treatment, the test tube was taken out from the cooling tank, immersed in water at about 12°C to bring it back to room temperature, and the developed eggs were transferred to the hatching tray along with the medium, and thoroughly rinsed with water to wash away the DMSO. They were housed in an incubation tank.

The survival of eggs after cooling treatment was investigated by observing the hatching status in the hatching tank.

The results showed that when glucose or calf serum was added as a supplement to DMSO, -7
When treated at ℃, a maximum hatching rate of about 95% was shown.

[Brief explanation of the drawing]

Figure 1 shows DMSO as a cryoprotectant in -7°C treatment.
Figure 2 is a graph showing the hatching rate when glucose is added as an adjunct to the cryoprotectant in treatment at -7°C. Figure 3 is a graph showing the hatching rate when glucose is added as an adjunct to the cryoprotectant in treatment at -7°C. Figure 4 is a graph showing the hatching rate when calf serum is added and the cooling temperature is lowered to 17.
It is a graph showing the hatching rate when the temperature is further lowered than °C.

Claims (1)

[Scope of Claims] 1 Fertilized fish eggs are prepared using a medium containing an inorganic salt aqueous solution, a cryoprotectant that prevents the fertilized eggs from freezing inside the egg, and an adjuvant that enhances the antifreeze effect of the cryoprotectant. Immerse the fertilized eggs in a container and cool the fertilized eggs together with the container to a predetermined temperature of 0°C or less,
A method for preserving fertilized fish eggs at a low temperature, the method comprising preserving the fertilized fish eggs for a desired period of time by keeping the eggs in a cooled state. 2 The cryoprotectant in the above medium is dimethino 1. 2. The method for low-temperature preservation of fertilized fish eggs according to claim 1, characterized in that sulfoxide is used. 3. The low-temperature preservation method for fertilized fish eggs according to claim 2, wherein the auxiliary agent in the medium is glucose or serum.