JPH084601B2 - Sperm freezing method and sperm freezing device - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for storing sperm by using a liquefied gas as a refrigerant, and a sperm freezing apparatus suitable for this method.

[0002]

2. Description of the Related Art At present, as a freezing method of bovine sperm, a quick freezing method using liquid nitrogen gas (LNG) as a refrigerant is generally used. At the time of freezing, conventionally, a compact device was used, and a large number of sperm-enclosed containers were placed vertically in a refrigerant container and frozen for the purpose of freezing at once. In this work, when the temperature was set to about 4 ° C., a straw-shaped sperm enclosure container was manually erected in a single mesh frame. For example, it took about 30 minutes to build 200 bottles.

[0003]

The conventional sperm freezing method as described above requires a great deal of labor, and it is necessary to examine the sperm survival rate in the cooling process. Therefore, the present invention aims to save labor in freezing sperm and to improve the survival rate of sperm in the cooling process.

[0004]

As a result of studying the above-mentioned problems, it was found that the difference in the retention of straws, particularly when frozen, affects the viability and fertility of sperm after thawing. is there. The present invention is characterized in that when the spermatozoa in the straw-shaped sperm-enclosed container are frozen with the refrigerant liquefied gas, a plurality of straw-shaped sperm-enclosed containers are aligned and frozen in a substantially horizontal direction.

Further, when freezing the sperm in the straw-shaped sperm enclosing container with the refrigerant liquefied gas, before immersing the sperm in the refrigerant liquefied gas, the horizontally oriented sperm enclosing container is almost frozen and then vaporized. It is a sperm freezing method characterized by lowering to a predetermined temperature in a refrigerant gas and then lowering to a target temperature in a refrigerant liquefied gas.

The apparatus used in this method for freezing sperm comprises an aligning device 5 for a sperm-enclosed vessel 1 in the form of a straw, and an alignment freezing stand 2 for aligning and holding the sperm-enclosed vessel 1 in a substantially horizontal direction by being immersed in a refrigerant. The alignment / freezing base 2 is supported by a suspension device 3 at an appropriate position in the refrigerant.

An aligning device 5 for aligning and holding the sperm enclosing container 1 on the aligning / freezing base 2 has a sperm enclosing container feeding slope 5b on one side of the plate-shaped base 5a, and the aligning / freezing mount 2 is located substantially at the center. An aligning plate 7 to be fitted is provided, and an aligning groove 8 having the same pitch as the groove of the gantry 2 is provided in the aligning plate 7 in a direction perpendicular to the slope.

The aligning / freezing base 2 for aligning and holding the sperm-enclosing containers 1 preferably has a large number of container aligning grooves 4. Further, it is preferable that the suspension device 3 of the aligned freezing stand 2 for aligning and holding the sperm-enclosed containers 1 in the refrigerant tank has a variable support position.

[0009]

According to the present invention as described above, by using the aligning device 5, it is possible to align a plurality of sperm-enclosed containers 1 in the aligning grooves 4 of the frame only by grasping them by hand and scattering them onto the aligning device. The sperm enclosure 1 is cooled almost uniformly in all parts, the sperm survival index is increased, and the conception rate is also increased. In addition, it is not necessary to manually address the straw-shaped sperm enclosure container, so that the work is simplified.

[0010]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The sperm freezing method and sperm freezing apparatus of the present invention will be described in detail below with reference to the accompanying drawings. FIG. 1 is a side view of a sperm freezing device implementing the sperm freezing method of the present invention. FIG. 2 is a plan view of only the mount portion. FIG. 3 is a plan view of an alignment table on which the sperm-enclosed container 1 is placed on the gantry, and FIG. 4 is a side view of the same. FIG. 5 is a plan view of an essential part showing an example of the alignment plate, and FIG.
FIG. 7 is a side view showing the same, and FIG. 7 is a side view showing a state in which the alignment racks are stacked in multiple stages. FIG. 8 is a side view of the refrigerant gas generator.

The sperm freezing apparatus of the present invention shown in FIG. 1 has an aligned freezing stand 2 capable of holding a large number of straw-shaped sperm-enclosing containers 1 aligned in a substantially horizontal direction and immersing them in the refrigerant in a cooling tank 10. The suspension unit 3 is capable of supporting the aligned freezing base 2 at an appropriate position in the refrigerant. A large number of container aligning grooves 4 are provided on the aligning / freezing base 2 and are shown in FIGS.
By using the aligning device 5 shown in FIG. 6, the sperm-enclosed container 1 can be efficiently placed on a large number of container aligning grooves 4.
As described above, the alignment freezing base 2 has a frame shape having irregularities on the upper surface so that a large number of sperm enclosure containers 1 can be aligned and held. Also,
When the alignment freezing bases 2 are stacked in a plurality of stages, as shown in FIG. 7, the container alignment grooves 4 are arranged so that the top and bottom are arranged in a staggered manner.
It is better to arrange.

An aligning device 5 has been developed so that the sperm enclosing container 1 can be placed on the aligning groove 4 well, and its appearance is shown in FIGS. The aligning device 5 is a plate-shaped base 5a larger than the aligning / freezing base 2 of the sperm enclosure container 1.
On one side (left side of the drawing), there is a feeding slope 5b of the sperm enclosure container 1. This feed slope 5b is rotatably supported by a shaft 5c, and its tip portion serves as a holding portion 5d. The alignment freezing base 2 comprises an outer frame 2a and an alignment groove frame 2b which is an inner frame. The alignment groove frame 2b has the container alignment groove 4 described above. Outer frame 2a is an alignment groove frame
Since it is formed slightly higher than 2b, sperm enclosure 1
Are aligned within the outer frame 2a, and are prevented from lateral displacement during movement. Inside the alignment groove frame 2b, the plate-shaped base 5a
An alignment plate 7 having alignment grooves 8 having the same pitch as the grooves of the pedestal 2 in the direction perpendicular to the slopes as shown in FIGS.

In order to align and hold one sperm enclosure container 1 in the container alignment groove 4 of the alignment freezing stand 2 using the alignment device 5,
First, the aligning and freezing stand 2 is held by the holding portion 5d by holding the aligning groove frame on the outer periphery of the aligning plate on the plate-shaped base 5a, and a large number of the sperm enclosure containers 1 are sent to the sending slope 5b. Good. By doing this, the alignment freezing stand 2
Since the alignment plate 7 is provided therein, the sperm-enclosed container 1 is fitted into each of the alignment grooves 8 provided in parallel with the alignment plate 7 and is in an aligned state.

An aligned freezing stand 2 holding the sperm enclosure 1 in an aligned manner can be supported in a cooling tank 10 by a suspension device 3. In the example shown in FIG. 1, the suspension device 3 has a stainless steel net cage 3a capable of accommodating the aligned freezing stand 2, a pole 3b for supporting the stainless steel net cage in the cooling tank 10, and a supporting position to be selected. And a plurality of hooks 3c that can be locked to the opening edge of the cooling tank 10. In the stainless steel net basket 3a, the aligned freezing racks 2 of the sperm enclosure containers 1 are housed in a plurality of rows and a plurality of stages. Stainless steel basket
At the bottom of 3a, there is a hanging strip 3d that allows you to know when it has landed on the refrigerant liquefied gas by smoking. In the example of FIG. 1, a tray 6 containing a refrigerant liquefied gas is placed on the aligned freezing stand 2 accommodating the sperm enclosing container 1 to further cool it from above.

The above-mentioned drooping strip is for generating a low-temperature gas by boiling the liquefied refrigerant gas inside the cooling tank 10. The refrigerant gas generator shown in FIG. 8 is for actively generating the low-temperature gas. It is 9. In this device, a non-heat generating portion 9a from the opening of the cooling tank 10 down to the refrigerant liquefied gas and a heat generating portion 9b supported below the heat generating portion 9b are formed in a substantially L shape so that electricity can be conducted inside. With the heat generated by the heater, the refrigerant liquefied gas can be boiled to generate a low-temperature gas. Although the heat generating portion 9b is formed in a circular shape, a plurality of heat generating portions having different capacities may be provided in parallel to increase or decrease the heat generation amount.

Using the apparatus of the present invention as described above, first, bovine sperm having the properties shown in Table 1 are prepared according to a conventional method, and yolk,
Treated with citrate buffer, dispensed into 0.5 ml sperm enclosure 1 and set on the alignment freezing stand 2 and put in one frame.
As shown in Figs. 1 and 2, 6 frames for 50 bottles were housed in the suspension device 3, and 5 to 10 cm from the upper edge of the cooling tank with the straw-shaped sperm enclosing container 1 aligned and held in the horizontal direction. The inside of the sperm enclosure 1 was frozen by keeping it aligned. After that, the sperm enclosure 1 together with the cradle is lowered to a position of 2 to 5 cm above the liquid nitrogen liquid level, which is the refrigerant liquefied gas in the cooling tank, and the inside of the sperm enclosure 1 is filled with the low temperature nitrogen gas by the refrigerant gas generator 9. The temperature was lowered until it fell below -150 ° C. After confirming this, the alignment freezing stand 2 was further lowered, and the whole sperm enclosure 1 was immersed in liquid nitrogen and lowered to -196 ° C. When it reaches -196 ℃,
The sperm enclosure 1 was quickly collected and transferred to another storage container.

As a comparative example, a sperm enclosure container was held vertically on a commercially available mount, and the mount was similarly immersed in liquid nitrogen,
Freezing was performed according to the same cooling method as in the above example.

[0018]

[Table 1]

Cooling curves for the examples and comparative examples,
The viability and the vitality of the frozen sperm after thawing were examined.
FIG. 9 shows cooling curves of the case (a) of the present invention in which the sperm enclosure is frozen in the horizontal direction and the conventional example (b) in which it is frozen in the vertical direction. The measurement was carried out by providing sensors at two places, a cotton plug part and a tip part of the sperm enclosure container. In the case of the present invention, the inside of the sperm sealing container is cooled uniformly, whereas in the conventional example, the cooling process differs depending on the site of the sperm sealing container.

FIG. 10 is a cooling curve in the case of the present invention when the aligned freezing pedestals 2 accommodating the sperm enclosure 1 are stacked in four stages and frozen. As it goes up, there is a tendency that the cooling curve becomes dull.

FIG. 11 is a comparison of the viability of the molten sperm in each stage in the case of the four-tier stack of the present invention as a sperm survival index, compared with the conventional vertical type. The sperm survival index of the comparative example was 40 to 55, whereas the sperm survival index of the present invention was 40 to 50 in the third and fourth stages from the bottom, which is slightly inferior to the comparative example, but the first stage was
From 45 to 60, the second stage has an excellent result of 45 to 60.

FIG. 12 shows the results of examining the time-dependent viability of sperm thawed after freezing. In the case of the horizontal type of the present invention, the viability after melting is good as in the case of the conventional vertical type. In particular, in the first and second steps of the method of the present invention, far superior results were obtained as compared with the conventional comparative example. Therefore, the vertical type of the comparative example has a sperm count of 45 million per sperm enclosure. Of frozen comparative semen and the semen prepared by the horizontal method of the present invention
A field insemination test was performed using frozen semen with a sperm count of 2 (22.5 million). The results are shown in Table 2.

[0023]

[Table 2]

As seen in Table 2, according to the present invention,
Even when the sperm count of the comparative example is 1/2, the conception rate is unchanged and it can be used sufficiently in the field. From this, many inseminations can be performed with a small number of sperms. Therefore, conventionally, only one container for sperm containing 200 to 400 sperm could be produced with one sperm, but the present invention doubled the number to 400 to 800, and highly efficient results were obtained.

[0025]

Industrial Applicability According to the present invention, the labor of freezing sperm can be reduced, a large LNG tank can be used for freezing, and a large amount of homogeneous storage can be achieved. Allowed insemination.

[Brief description of drawings]

FIG. 1 is a side view of a sperm freezing device implementing the sperm freezing method of the present invention.

FIG. 2 is a plan view of only a pedestal portion.

FIG. 3 is a plan view of an alignment device that mounts a sperm enclosure container on a mount.

FIG. 4 is a side view of the alignment device.

FIG. 5 is a main part plan view showing an example of an alignment plate.

FIG. 6 is a side view of a main part of an alignment plate.

FIG. 7 is a side view showing a state in which aligned freezing mounts are stacked in multiple stages.

FIG. 8 is a side view of the refrigerant gas generator.

FIG. 9 is a cooling curve for each holding method when frozen.

FIG. 10 is a cooling curve for each stage according to the present invention.

FIG. 11 is a graph showing the sperm survival index at each stage.

FIG. 12 is a graph showing changes over time in the sperm survival index.

[Explanation of symbols]

 1 Sperm Encapsulation Container 2 Alignment Freezing Stand 3 Suspension Device 4 Container Alignment Groove 5 Alignment Device 6 Tray 7 Alignment Plate 8 Alignment Groove 9 Refrigerant Gas Generator 10 Cooling Tank

Claims (3)

[Claims] 1. When freezing spermatozoa in a straw-like sperm enclosure with a liquefied refrigerant gas , the straw-like sperm enclosure is first arranged in a horizontal plane before being immersed in the liquefied refrigerant gas.
Bring the multiple sperm enclosures that have been
In the cooled refrigerant gas to a predetermined temperature
A method for freezing sperm, which comprises lowering the temperature to a target temperature in the activated gas . 2. A plurality of straw-shaped spermatozoa in a substantially horizontal plane.
Alignment freezing base 2 for aligning and holding the enclosure 1 and the alignment freezing
A suspension device 3 for supporting the gantry 2 at an appropriate position in the refrigerant
The alignment / freezing stand 2 includes a plurality of sperm enclosures 1.
Alignment device 5 for aligning and holding in a substantially horizontal plane is provided.
Well, the aligning device 5 has sperm on one side of the plate-shaped base 5a.
There is a feed slope 5b for the enclosure and it is aligned in the center.
An alignment plate 7 for fitting the row freezing mount 2 is provided, and the alignment plate 7 has
Same pitch as the groove of the gantry 2 in the direction perpendicular to the slope
A sperm freezing device provided with the alignment groove 8 of FIG . 3. A substantially suspension device alignment freeze frame 2 and said alignment freeze frame 2 for aligning holding a straw-shaped sperm enclosure 1 a plurality of in one horizontal plane for supporting the appropriate location in the refrigerant tank 10 3
Because Do Ri, the suspension device 3 is located variable support height,
Refrigerant tank 10 generates refrigerant gas for cooling before immersion in liquefied gas
A sperm freezing device comprising the device 9 .