JPS5834136B2 - Freezing methods for fertilized eggs, sperm, etc. - Google Patents
Freezing methods for fertilized eggs, sperm, etc.Info
- Publication number
- JPS5834136B2 JPS5834136B2 JP12499781A JP12499781A JPS5834136B2 JP S5834136 B2 JPS5834136 B2 JP S5834136B2 JP 12499781 A JP12499781 A JP 12499781A JP 12499781 A JP12499781 A JP 12499781A JP S5834136 B2 JPS5834136 B2 JP S5834136B2
- Authority
- JP
- Japan
- Prior art keywords
- buffer solution
- temperature
- storage
- sperm
- fertilized eggs
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】 本発明は、受精卵、***等の凍結方法に関する。[Detailed description of the invention] The present invention relates to a method for freezing fertilized eggs, sperm, etc.
近年、家蓄の品種改良や増殖を計るべく人工授精が盛ん
に実施されていることから、受精卵、***を凍結させて
保存することが行なわれている。In recent years, artificial insemination has become popular in order to improve breeds and propagate family stock, and fertilized eggs and sperm are now being frozen and preserved.
従来、上述受精卵、***等の凍結方法としては、既に収
納管に受精卵、***等を緩衝液と共に投入し、この緩衝
液を冷却することで、受精卵等を凍結保存することが知
られている。Conventionally, as a method for freezing fertilized eggs, sperm, etc., as described above, it is known that the fertilized eggs, sperm, etc. are put into a storage tube together with a buffer solution, and the fertilized eggs, etc. are cryopreserved by cooling this buffer solution. ing.
ところで一般に純粋物質が一定圧力下で冷却されたとき
の時間に対する温度変化は、冷却曲線として知られてお
り、これによると当該物質が凝固点に達した時点で直ち
に凝固が始まるとはかぎらず、一般的には、当該凝固点
よりも低い温度まで過冷却された後、凝固が始まり、こ
れと同時に温度が上昇し、真の凝固点に達し、さらに全
物質の凝固が終わってから再び温度が低下していくこと
になる。By the way, the temperature change over time when a pure substance is cooled under a constant pressure is generally known as a cooling curve.According to this, solidification does not necessarily begin immediately when the substance reaches its freezing point; Specifically, after being supercooled to a temperature lower than the freezing point, solidification begins, the temperature rises at the same time, reaching the true freezing point, and then the temperature decreases again after all the substances have solidified. I'm going to go.
従って従来の単に冷却するだけの上記凍結方法によると
きＧＬ凍結過程において緩衝液が凍結時に過冷却状態と
なり、その後に急激な温度上昇をもたらすことになるか
ら、この温度急変による当該熱衝撃により受精卵、***
等が斃死してしまうという重大な欠陥を有するのである
。Therefore, when using the above-mentioned conventional freezing method that simply involves cooling, the buffer solution becomes supercooled during freezing during the GL freezing process, which then causes a rapid temperature rise. It has a serious defect in that spermatozoa, etc., die.
そこで凝固点温度まで冷却した緩衝液を取り出して、当
該収納管を液体窒素につげたピンセットで挟持すること
により当該挟持箇所から凝固を成長させ、熱衝撃を回避
しようとする凍結方法も知られているが、収納管を取出
すことにより緩衝液の温度に影響を与えて凝固が行なわ
れなかったり、また操作が繁雑であるため自動制御が困
難となり、実用性に乏しいものとなっている。There is also a known freezing method in which the buffer solution cooled to the freezing point temperature is taken out and the storage tube is clamped with tweezers attached to liquid nitrogen, thereby causing solidification to grow from the clamped area to avoid thermal shock. However, when the storage tube is taken out, the temperature of the buffer solution is affected and solidification may not take place, and the operation is complicated, making automatic control difficult, making it impractical.
そこで上述事情に鑑みて検討の結果、収納管全体を単に
冷却するのではなく、受精卵、***等が偏在している部
分である収納部緩衝液を、その凝固点付近の温度に制御
しておき、一方弁収納部緩衝液は充分に冷却して、先ず
固液を凝固させ、これにより得られた結晶の核を収納部
緩衝液まで成長させることにより、当該受精卵等を凍結
すれば、上述過冷却による熱衝撃を受けることなく、同
受精卵等の生存率を高め得ることが判明したが、本発明
ではさらに上記の如く収納部緩衝液を凝固点に保持させ
るに際し、使用する緩衝液が違うたびに、温度制御用の
冷媒温度を決定するため、当該緩衝液の凝固点を測定し
、その測定値に基づき制御温度をセットしなげればなら
ないことの低作業性に鑑み、これを改善しようとするの
がその目的である。Therefore, after considering the above circumstances, we decided that instead of simply cooling the entire storage tube, we should control the temperature of the storage buffer solution, which is the part where fertilized eggs, sperm, etc. are unevenly distributed, to around its freezing point. On the other hand, if the valve storage buffer is sufficiently cooled to first solidify the solid liquid, and the resulting crystal nucleus is allowed to grow to the storage buffer, the fertilized eggs, etc. can be frozen. It has been found that the survival rate of fertilized eggs, etc. can be increased without being subjected to thermal shock due to supercooling. However, in the present invention, as described above, when maintaining the storage buffer solution at the freezing point, the buffer solution used is different. In order to determine the refrigerant temperature for temperature control each time, the freezing point of the buffer solution must be measured and the control temperature must be set based on the measured value. Its purpose is to.
以下、本発明の実施に好適な装置例を示した図面によっ
て詳述すれば、第１図に示したように、ストロ−管等の
収納管１に緩衝液２と共に受精卵、***等の凍結物３を
収納し、この際収納管１の下端は綿栓４等にて閉塞して
おき、当該被凍結物３は収納管１の下部等に偏在させて
収納する。Hereinafter, the present invention will be described in detail with reference to the drawings showing an example of a suitable apparatus for implementing the present invention.As shown in FIG. The objects 3 are stored, and at this time the lower end of the storage tube 1 is closed with a cotton plug 4 or the like, and the objects 3 to be frozen are stored unevenly in the lower part of the storage tube 1 or the like.
そして上記収納管１は例えば第２図に示したように、低
温恒温槽５内に収容するが、この際被凍結物３が収納さ
れた収納部緩衝液２ａは槽内設置の区画板６の下位にあ
って噴出する冷媒７により冷却され、一方被凍結物３の
ない非収納部緩衝液２ｂの方は区画板６の上位にあって
噴出する冷媒７′により冷却される構成となっている。The storage tube 1 is stored in a low-temperature constant temperature bath 5 as shown in FIG. It is configured to be cooled by the refrigerant 7 that is located at the lower level and jets out, while the non-storage buffer solution 2b where there is no frozen object 3 is cooled by the refrigerant 7' that is located at the upper part of the partition plate 6 and jets out. .
ここで、上記緩衝液２としては、ジメチルスルオキトや
ブドウ糖、グリセリン、クエン酸ナトリウムを蒸留水に
溶解したもの等が用いられる。Here, as the buffer solution 2, a solution obtained by dissolving dimethyl sulfuric acid, glucose, glycerin, sodium citrate in distilled water, etc. is used.
さらに上記恒温槽５には、前記冷媒７，７′を液化ガス
源８から供給するため、冷媒７については、熱交換器９
を介設した下位の冷媒供給路１０が設けられ、その先端
ノズル１１から収納部緩衝液入に冷媒７を噴出させると
共に、当該緩衝液２ａの温度を感知する温度センサ１２
が温度制御器１３に接続され、同制御器１３により上記
熱交換器９の熱源たる図示しない電気ヒータへの電流が
制御される構成になっている。Furthermore, since the refrigerants 7 and 7' are supplied to the thermostatic chamber 5 from the liquefied gas source 8, the refrigerant 7 is supplied to the heat exchanger 9.
A lower refrigerant supply path 10 is provided with a lower refrigerant supply path 10 interposed therein, and a temperature sensor 12 is provided for spouting the refrigerant 7 from the tip nozzle 11 into the buffer solution container of the storage section and sensing the temperature of the buffer solution 2a.
is connected to a temperature controller 13, and the controller 13 controls the current to an electric heater (not shown) which is the heat source of the heat exchanger 9.
一方冷媒７′についても上記の場合と同じく、熱交換器
９′をもち、先端ノズル１１′から非収納部緩衝液２ｂ
に冷媒７′を噴当する冷媒供給路１０′が設けられると
共に、当該緩衝液２ｂの温度センサ１２′が温度制御器
１３′に接続され、同制御器１３′が熱交換器９′の熱
源温度を設定値に制御することになるが、さらに上記の
両温度制御器１３．１３’＃″−接続されており、これ
により温度制御器１３′に入来した検知信号が後述のよ
うに最高値となったとき、当該最高値としての測定値が
、温度制御器１３の制御すべき温度設定値として印加さ
れることになる。On the other hand, the refrigerant 7' also has a heat exchanger 9' as in the above case, and a non-storage buffer liquid 2b is supplied from the tip nozzle 11'.
A refrigerant supply path 10' for injecting refrigerant 7' is provided, and a temperature sensor 12' of the buffer solution 2b is connected to a temperature controller 13', which controls the heat source of the heat exchanger 9'. The temperature will be controlled to the set value, but the above-mentioned temperature controllers 13 and 13'#'' are also connected, so that the detection signal that enters the temperature controller 13' will reach the highest level as described later. When the maximum value is reached, the measured value as the maximum value is applied as the temperature setting value to be controlled by the temperature controller 13.
而して上記構成において、これを用いて受精卵、***等
の被凍結物３を凍結するには、収納管１を上述のように
低温恒温槽５に収容した後、液化ガス源８から冷媒供給
路１０ 、１０’を介して低温恒温槽５に液化ガスであ
る冷媒７，７′を供給し、収納部緩衝液２ａと非収納部
緩衝液２ｂを夫夫各別に降温させていくが、この際冷媒
７′の温度を充分低くすることにより、第３図の冷却曲
線Ｘのように非収納部緩衝液２ｂの温度が低下し、凝固
点以下の過冷却状態となり、同図の過冷却点Ｘ１から急
激な温度上昇を伴なって固液２ｂが凝固し、結晶の核が
生成されるに至る。In the above configuration, in order to freeze objects 3 such as fertilized eggs and sperm, the storage tube 1 is placed in the low-temperature thermostat 5 as described above, and then a refrigerant is supplied from the liquefied gas source 8. Refrigerants 7 and 7', which are liquefied gases, are supplied to the low-temperature constant temperature chamber 5 through supply channels 10 and 10', and the temperature of the storage buffer solution 2a and the non-storage buffer solution 2b is lowered separately. At this time, by lowering the temperature of the refrigerant 7' sufficiently, the temperature of the non-storage buffer liquid 2b decreases as shown in the cooling curve The solid-liquid 2b solidifies with a rapid temperature rise from X1, leading to the generation of crystal nuclei.
そしてこのように凝固が開始されると固液２ｂが完全に
凝固するまで最高温度に保持され、当該温度が緩衝液２
の凝固点を示すこととなり、これを検知した温度センサ
１２界当該検知信号を温度制御器１３′から温度制御器
１３に印加し、さらに同制御器１３は上記測知の凝固点
よりも実際上例えば０．５℃程度低い温度を設定値とし
て熱交換器９を制御し、これにより冷媒７による冷却に
よって、収納部緩衝液２ａの温度を、第３図の冷却曲線
Ｙの如く凝固点近傍に制御することとなる。Once coagulation has started, the solid liquid 2b is held at the highest temperature until it is completely solidified, and the buffer solution 2b is kept at this temperature.
The temperature sensor 12 detects this and applies the detection signal to the temperature controller 13 from the temperature controller 13', and the controller 13 actually indicates the freezing point, for example, 0. The heat exchanger 9 is controlled by setting a temperature approximately 5° C. lower as a set value, and thereby the temperature of the storage buffer solution 2a is controlled to be near the freezing point as shown by the cooling curve Y in FIG. 3 by cooling with the refrigerant 7. becomes.
かくて上記の非収納部緩衝液２ｂに生成された結晶の核
は、凝固点近傍にある収納部緩衝液２ａまで成長して行
き、従って同緩衝液２ａは過冷却状態となることなく凝
固して被凍結物３が凍結されることになる。In this way, the crystal nuclei generated in the non-storage buffer solution 2b grow to the storage buffer solution 2a, which is near the freezing point, and therefore the buffer solution 2a solidifies without becoming supercooled. The object to be frozen 3 will be frozen.
本発明は上記実施例によって具現される通り収納管内の
緩衝液中に受精卵、***等の被凍結物を偏在して収納し
、当該被凍結物の非収納部緩衝液を収納部緩衝液よりも
低温となるよう所望冷媒にて冷却することにより、非収
納部緩衝液を凝固させて結晶の核を生成させた後、当該
結晶の核を前記収納部緩衝液まで成長させることにより
被凍結物を凍結するようにした方法において、非収納部
緩衝液に結晶の核が生じた時点での当該最高液温を測知
し収納部緩衝液の冷却温度を、上記測知温度近傍に自動
制御するようにしたものであるから、受精卵、***等の
凍結過程において、当該緩衝液２ａが凍結時に過冷却状
態となることなく凍結に至り、急激な温度上昇による精
子等の斃死を回避でき、生存率の高い凍結が可能となっ
て、しかも同方法の実施に自動制御を導入することがで
きるだけでなく、非収納部緩衝液の凝固時点を利用して
、緩衝液の凝固点を測知し、これにより収納部緩衝液の
温度が自動制御されるから、別途使用すべき各種の緩衝
液につき、その凝固点を測定する操作が不必要となり、
効率的な凍結を実施できると共に、結晶の核を虫取させ
る最適条件の設定が自動化され、常に望ましい凍結の実
現を保証することができる。As embodied by the above-mentioned embodiment, the present invention stores objects to be frozen, such as fertilized eggs and sperm, unevenly distributed in a buffer solution in a storage tube, and transfers the buffer solution in the non-storage portion of the object to be frozen from the buffer solution in the storage tube. By cooling with a desired refrigerant so that the non-storage buffer solution is cooled to a low temperature, the non-storage buffer solution is solidified and crystal nuclei are generated, and then the crystal nuclei are grown to the storage buffer solution to freeze the object. In the method of freezing the non-storage buffer solution, the maximum liquid temperature at the time when crystal nuclei are generated in the non-storage buffer solution is measured, and the cooling temperature of the storage buffer solution is automatically controlled to be close to the measured temperature. Therefore, during the freezing process of fertilized eggs, sperm, etc., the buffer solution 2a achieves freezing without becoming supercooled during freezing, avoiding death of sperm, etc. due to sudden temperature rise, and ensuring survival. Not only is it possible to achieve high freezing rates and to introduce automatic control into the implementation of the method, but also to use the solidification point of the non-storage buffer to determine the freezing point of the buffer. Since the temperature of the storage buffer solution is automatically controlled, there is no need to separately measure the freezing point of each buffer solution.
Not only can efficient freezing be performed, but the optimal conditions for removing crystal nuclei can be automatically set, ensuring that desired freezing is always achieved.
第１図は本発明の実施に用いる収納管の縦断側面説明図
、第２図は本発明の実施に用い得る装置例の全体説明図
、第３図は収納管内の非収納部緩衝液と収納部緩衝液の
時間に対する温度変化を示した冷却曲線のグラフである
。
１・・・・・・収納管、２・・・・・・緩衝液、２ａ・
・・・・・収納部緩衝液、２ｂ・・・・・・非収納部緩
衝液、３・・・・・・非凍結物。Fig. 1 is a longitudinal side explanatory view of a storage tube used in carrying out the present invention, Fig. 2 is an overall explanatory view of an example of an apparatus that can be used in carrying out the present invention, and Fig. 3 is a diagram showing the buffer solution and storage in the non-storage area in the storage tube. 2 is a graph of a cooling curve showing the temperature change of a buffer solution over time. 1... Storage tube, 2... Buffer solution, 2a.
...Storage buffer solution, 2b...Non-storage buffer solution, 3...Non-frozen material.
Claims (1)
偏在して収納し、当該被凍結物の非収納部緩衝液を収納
部緩衝液よりも低温となるよう所望冷媒にて冷却するこ
とにより、非収納部緩衝液を凝固させて結晶の核を生成
させた後、当該結晶の核を前記収納部緩衝液まで成長さ
せるよう冷却して、当該緩衝液を凝固させることにより
被凍結物を凍結するようにした方法において、非収納部
緩衝液に結晶の核が生じた時点での当該最高液温を測知
し、収納部緩衝液の冷却温度を、上記測知温度近傍に自
動制御するようにしたことを特徴とする受精卵、***等
の凍結方法。1 Store objects to be frozen, such as fertilized eggs and sperm, unevenly distributed in a buffer solution in a storage tube, and cool the buffer solution in the non-storage part of the object to be frozen with a desired refrigerant so that the temperature is lower than that of the buffer solution in the storage part. By doing so, the non-storage buffer solution is solidified to generate a crystal nucleus, and then the crystal nucleus is cooled to grow to the storage buffer solution, and the buffer solution is solidified to generate a crystal nucleus. In a method that freezes objects, the highest temperature of the liquid at the time when crystal nuclei are formed in the non-storage buffer solution is measured, and the cooling temperature of the storage buffer solution is automatically adjusted to near the measured temperature. A method for freezing fertilized eggs, sperm, etc., characterized in that it is controlled.
Priority Applications (9)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12499781A JPS5834136B2 (en) | 1981-08-10 | 1981-08-10 | Freezing methods for fertilized eggs, sperm, etc. |
| US06/404,400 US4429542A (en) | 1981-08-10 | 1982-08-02 | Method of freezing fertilized ova, spermatozoa or the like and apparatus therefor |
| AU86705/82A AU592747B2 (en) | 1981-08-10 | 1982-08-03 | Method of freezing fertilized ova, spermatozoa or the like and apparatus therefor |
| NZ201489A NZ201489A (en) | 1981-08-10 | 1982-08-03 | Method of and apparatus for freezing fertilized ova and spermatazoa |
| EP82304167A EP0072225B1 (en) | 1981-08-10 | 1982-08-06 | Method of freezing fertilized ova, spermatozoa or the like and apparatus therefor |
| DE8282304167T DE3267799D1 (en) | 1981-08-10 | 1982-08-06 | Method of freezing fertilized ova, spermatozoa or the like and apparatus therefor |
| CA000409052A CA1197467A (en) | 1981-08-10 | 1982-08-09 | Method of freezing fertilized ova, spermatozoa or the like and apparatus therefor |
| DK356782A DK158822C (en) | 1981-08-10 | 1982-08-09 | PROCEDURE FOR FREEZING FERTILIZED EGGS AND SPERMATOZOES AND APPARATUS FOR EXERCISING THE PROCEDURE |
| US06/533,932 US4487033A (en) | 1981-08-10 | 1983-10-26 | Method of freezing fertilized ova, spermatozoa or the like and apparatus therefor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12499781A JPS5834136B2 (en) | 1981-08-10 | 1981-08-10 | Freezing methods for fertilized eggs, sperm, etc. |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5827555A JPS5827555A (en) | 1983-02-18 |
| JPS5834136B2 true JPS5834136B2 (en) | 1983-07-25 |
Family
ID=14899329
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP12499781A Expired JPS5834136B2 (en) | 1981-08-10 | 1981-08-10 | Freezing methods for fertilized eggs, sperm, etc. |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5834136B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2633862B2 (en) * | 1987-09-06 | 1997-07-23 | 日本信号株式会社 | Automatically responsive traffic signal controller |
| JP2633864B2 (en) * | 1987-09-29 | 1997-07-23 | 日本信号株式会社 | Traffic signal linked controller |
-
1981
- 1981-08-10 JP JP12499781A patent/JPS5834136B2/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5827555A (en) | 1983-02-18 |
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