JPS5834135B2 - Freezing methods for fertilized eggs, sperm, etc. - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for freezing fertilized eggs, sperm, etc.

BACKGROUND ART In recent years, artificial insemination has become popular for the purpose of breeding and breeding livestock, and fertilized eggs and sperm are now being frozen and preserved.

Conventionally, as a method for freezing fertilized eggs, sperm, etc., it is known to place the fertilized eggs and sperm in a buffer solution already stored in a storage tube, and then freeze this buffer solution.

By the way, the temperature change over time when a pure substance is cooled below a certain pressure is generally known as a cooling curve, and according to this, solidification does not necessarily begin immediately when the substance reaches its freezing point; Generally, solidification begins after being supercooled to a temperature lower than the freezing point.
At the same time, the temperature rises and reaches the true freezing point, and then once all the substances have solidified, the temperature begins to drop again.

Therefore, when using the above-mentioned conventional freezing method, which involves simply cooling, the buffer solution becomes supercooled during the freezing process, which then causes a rapid temperature rise. It has a serious defect that causes the eggs and sperm to die.

There is also a known freezing method in which a buffer solution that has been cooled to the freezing point temperature is taken out and the storage tube is clamped with tweezers suspended in liquid nitrogen, thereby allowing the solidification to grow from the clamped area to avoid thermal shock. However, taking out the storage tube affects the temperature of the buffer solution, preventing solidification, and the complicated operation makes automatic control difficult, making it impractical.

Therefore, as a result of studies in view of the above-mentioned conventional circumstances, the present invention has been developed to make it possible to coagulate the buffer solution without supercooling, thereby increasing the survival rate of fertilized eggs and sperm. The aim is to provide a freezing method that is suitable for

The present invention will be described in detail based on the drawings shown as embodiments. As shown in FIG.
For example, dimethyl sulfoxide DMSO and glucose,
A buffer solution 2 prepared by dissolving glycerin and sodium citrate in distilled water is contained, and a to-be-frozen object 3 such as a fertilized egg or sperm is placed in the buffer solution 2.

At this time, the lower end of the storage tube 1 is closed with a cotton plug 4 or the like, while the object to be frozen 3 is stored in an unevenly distributed position approximately in the lower half of the buffer solution 2.

In addition, if you want to divide the buffer solution and store fertilized eggs, sperm, etc. in one of the divided buffer solutions, please refer to Figures 1 and 2.
As shown in B in the figure, a segmented gap 5 may be formed by air bubbles at a suitable location in the buffer solution 2, or a cotton plug 6 may be fitted to form a segmented gap as shown in A in FIG. 2. In this case, The gap 5 is made within approximately 2 rrun.

In the present invention, the storage tube 1 as described above is cooled.
Rather than simply cooling the entire interpipe 1, the buffer solution 2a in the lower storage area in which the objects 3 to be frozen are unevenly distributed in the case shown in the figure;
By setting a phase difference in the cooling temperature with the upper non-storage buffer solution 2b, the non-storage buffer solution 2b is first solidified to form a crystal nucleus, and this nucleus grows up to the storage buffer solution 2a. The object 3 to be frozen is then frozen.

Here, FIGS. 3 and 5 show examples of apparatuses that can be used to carry out the present invention, and in the case of FIG. 3, a double-formed low-temperature constant temperature bath 8 containing a refrigerant 7 is used, The storage buffer solution 2a is immersed in the refrigerant 7, and the non-storage buffer solution 2b is immersed in the refrigerant 7.
The storage pipe 1 is arranged so as to protrude from the liquid level to the gas phase part 9 in the tank, and in this case, as shown in Figures 1 and 2, the height h from the liquid level to the division gap 5 is about 30 mm or more. It's good to keep it.

In the same device, LN is placed between the double walls of the thermostatic chamber 8.
2 and He gas, etc., and impentane or the like is used as the refrigerant 7. In the refrigerant 7, a heater 10, a stirrer 11, and a temperature sensing element 12 are provided, and a control (not shown) is provided. The temperature of the refrigerant 7 is controlled by the refrigerant so that the buffer solution 2 is maintained at the freezing point (for example, -4.2°C).

In this way, the temperature of the buffer solution 2 is kept constant at the freezing point, but at this time, the vaporized refrigerant is sufficiently distributed in the vapor phase part 9 in the tank, and as a result, the temperature becomes lower than that of the refrigerant 7, and the temperature reaches the freezing point. On the other hand, the temperature drops to approximately -30°C.

For this reason, the non-accommodating buffer solution 2b is placed above the liquid level of the refrigerant 7.
becomes a supercooled state as shown in the cooling curve X in FIG. 4, and solidifies with a rapid temperature rise from the supercooling point X1.

On the other hand, the storage buffer solution 2a in the refrigerant 7 is not affected by the above-mentioned temperature rise of the non-storage buffer solution 2b, and is maintained at a constant temperature, which is the freezing point, as shown by the cooling curve Y in FIG. It will be.

Then, by solidifying the non-storage buffer solution 2b as described above, crystal nuclei are generated, and these nuclei grow into the storage buffer solution 2a.

At this time, it is preferable to slowly lower the temperature of the coolant 7 in order to encourage the growth of the nuclei, so that the crystal nuclei rapidly grow into the storage buffer solution 2a, resulting in a supercooled state. Instead, the buffer solution 2a in the refrigerant 7 solidifies.

Here, X2 in FIG. 4 is the solidification start point of the non-storage buffer solution 2b and also the solidification start point of the storage buffer solution 2a, and the solid liquid 2a has completely solidified at the freezing point. Thereafter, as shown by the cooling curve Y, the temperature is lowered to about -100° C. using the refrigerant 7, and the storage tube 1 is stored in liquid nitrogen or the like.

Next, in the apparatus shown in FIG. 5, the storage tube 1 housed in the low-temperature constant temperature bath 8 is vertically defined by a partition plate 13, and the storage buffer solution 2a receives the refrigerant from the liquefied gas source 14. The upper non-storage buffer solution 2b is separately solidified using the refrigerant from the liquefied gas source 14, thereby generating crystal nuclei, The storage buffer solution 2b is cooled as it is at a constant temperature, or the temperature is gradually lowered to grow crystals, and the buffer solution 2a is solidified to freeze the frozen object 3 such as a fertilized egg or sperm. Also, as mentioned above, the temperature is lowered to the desired temperature.

Here, a heat exchanger 17.18 is disposed in each of the illustrated refrigerant supply lines 15.16, and the heat exchanger 17.18 is
The temperature of each buffer solution 2a, 2b can be adjusted to a desired temperature by a temperature controller 21.22 connected to a temperature detection element 19.20 that detects the temperature of each buffer solution 2a, 2b.
The temperature of the refrigerant injected to a and 2b can be controlled separately.

As explained above, according to the method for freezing fertilized eggs, sperm, etc. according to the present invention, the storage buffer solution 2a containing fertilized eggs, sperm, etc. is placed under cooling but is not solidified and is first stored in the non-storage area. Since the solid liquid 2a is solidified by coagulating the buffer solution 2b with a refrigerant and growing the resulting crystal nuclei toward the buffer solution 2a, it is possible to solidify the solid liquid 2a, so that fertilized eggs, sperm, etc. In the freezing process, the buffer solution 2a freezes without becoming supercooled during freezing, and death of fertilized eggs, sperm, etc. due to rapid temperature rise can be avoided.
As a result, freezing with a high survival rate can be carried out, the method can be carried out using various appropriate devices, and automatic control can be introduced, making it suitable for mass production.

[Brief explanation of drawings]

FIG. 1 is a longitudinal sectional front explanatory view of a storage tube used in the method for freezing fertilized eggs, sperm, etc. according to the present invention, and A and B in FIG. 2 are longitudinal sectional front explanatory views showing different examples of the same storage tube. Figures 3 and 5
The figures are longitudinal sectional front views of devices that can be used to implement the freezing method,
FIG. 4 is a graph showing the cooling curve of the buffer solution in the same method. 1... Storage tube, 2... Buffer solution, 2a.
...Buffer solution in non-accommodating area, 2b...Buffer solution in storage area, 3...Object to be frozen such as fertilized eggs and sperm.

Claims (1)

[Scope of Claims] 1. Objects to be frozen, such as fertilized eggs and sperm, are unevenly stored in a buffer solution in a storage tube, and the buffer solution in the non-storage area of the object to be frozen is lower than the buffer solution in the storage section. By cooling with a desired refrigerant, the non-storage buffer solution is solidified and crystal nuclei are generated, and then the buffer solution is cooled to grow the crystal nuclei to the storage buffer solution. A method for freezing fertilized eggs, sperm, etc., characterized in that the object to be frozen is frozen by coagulation. 2. The method for freezing fertilized eggs, sperm, etc. according to claim 1, wherein the cooling temperature of the storage buffer solution is maintained at the solid-liquid freezing point. 3. Fertilized eggs, sperm, etc. according to claim 1, wherein the temperature of the refrigerant for cooling the storage buffer solution is gradually lowered after crystal nuclei are generated in the non-storage buffer solution. Freezing method.