JPH02117601A - Frozen fertilized ovum of pig and freezing preservation thereof - Google Patents
Frozen fertilized ovum of pig and freezing preservation thereofInfo
- Publication number
- JPH02117601A JPH02117601A JP63269422A JP26942288A JPH02117601A JP H02117601 A JPH02117601 A JP H02117601A JP 63269422 A JP63269422 A JP 63269422A JP 26942288 A JP26942288 A JP 26942288A JP H02117601 A JPH02117601 A JP H02117601A
- Authority
- JP
- Japan
- Prior art keywords
- fertilized ovum
- fertilized
- preserved
- freezing
- ovum
- 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.)
- Granted
Links
- 238000007710 freezing Methods 0.000 title abstract description 6
- 230000008014 freezing Effects 0.000 title abstract description 6
- 238000004321 preservation Methods 0.000 title abstract description 4
- 102000002322 Egg Proteins Human genes 0.000 title abstract 9
- 108010000912 Egg Proteins Proteins 0.000 title abstract 9
- 210000004681 ovum Anatomy 0.000 title abstract 9
- 235000013601 eggs Nutrition 0.000 claims description 38
- 238000000034 method Methods 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 abstract description 19
- 241000282887 Suidae Species 0.000 abstract description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 4
- 239000007788 liquid Substances 0.000 abstract description 4
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 abstract description 2
- 238000012258 culturing Methods 0.000 abstract description 2
- 230000000740 bleeding effect Effects 0.000 abstract 1
- 239000003223 protective agent Substances 0.000 abstract 1
- 210000002459 blastocyst Anatomy 0.000 description 12
- 239000000243 solution Substances 0.000 description 9
- 235000011187 glycerol Nutrition 0.000 description 8
- 238000009395 breeding Methods 0.000 description 7
- 230000001488 breeding effect Effects 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 5
- 238000005138 cryopreservation Methods 0.000 description 5
- 239000002577 cryoprotective agent Substances 0.000 description 5
- 230000003205 diastolic effect Effects 0.000 description 5
- 210000001161 mammalian embryo Anatomy 0.000 description 5
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 230000004720 fertilization Effects 0.000 description 3
- 210000004340 zona pellucida Anatomy 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000011067 equilibration Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 230000013011 mating Effects 0.000 description 2
- 210000000472 morula Anatomy 0.000 description 2
- 210000000582 semen Anatomy 0.000 description 2
- 239000010902 straw Substances 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 241000283707 Capra Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 210000004952 blastocoel Anatomy 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000010583 slow cooling Methods 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は豚の凍結受精卵及び受精卵の凍結保存方法に関
するものである。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to frozen fertilized pig eggs and a cryopreservation method for fertilized eggs.
一般に、養豚業においては生産効率を高めるために遺伝
的に優秀な豚を多く生産することが望まれている。Generally, in the pig farming industry, it is desired to produce a large number of genetically superior pigs in order to increase production efficiency.
従来の豚の繁殖は、繁殖豚に誰豚を交配せしめることに
より妊娠、分娩させることで行なわれてきた。この方法
によると、交配から分娩まで約１１４日の日数を要する
ことから、−繁殖雌豚が産業上可能な範囲での分娩回数
は、高々ＩＯ数回までである。Conventional pig breeding has been carried out by mating breeding pigs with other pigs, causing them to become pregnant and give birth. According to this method, it takes about 114 days from mating to farrowing, and therefore, the number of farrowings a breeding sow can produce within an industrially possible range is at most several IO times.
このように−繁殖雌豚が次の世代に継代できる子豚の数
には限度があり、これを克服する方法として受精卵移植
が利用されてきた。しかしその方法は、受精卵を提供す
る豚から回収された受精卯を少なくとも数日以内に借り
腹となる豚に移植しなければならなかった。Thus, there is a limit to the number of piglets that a breeding sow can pass on to the next generation, and fertilized egg transfer has been used as a way to overcome this. However, this method required that the fertilized rabbits recovered from the donor pigs be transplanted into the borrowed pigs within at least a few days.
ところで、受精卵の凍結保存技術はマウス、兎、山羊、
羊、牛などについて開発されており、凍結保存用には一
般に桑実期胚あるいは初期胚盤胞が使用されている。一
方、豚の受精卵の保存技術はまだ開発されていない。By the way, cryopreservation technology for fertilized eggs is available in mice, rabbits, goats,
It has been developed for sheep, cattle, etc., and morula stage embryos or early blastocysts are generally used for cryopreservation. On the other hand, techniques for preserving pig fertilized eggs have not yet been developed.
受精卵移植は家畜の育種繁殖上重要な技術であるが受精
卯は長期にわたって保存することができないためその効
果を十分に発揮することが出来なかった、受精卵を凍結
保存できればこの問題は解決することができるが、豚に
ついての成功例はまだ知られていない。Fertilized egg transplantation is an important technique for breeding and breeding livestock, but fertilized rabbits cannot be stored for long periods of time, so it has not been able to demonstrate its full effect. This problem could be solved if fertilized eggs could be cryopreserved. However, there are no known success stories for pigs.
本発明者は、豚受精卵を長期に保存する方法を開発すべ
く種々検討を重ねた結果、種々の時期の豚受精卵を摂氏
マイナス３５度以下に凍結保存し、その後これを凍結融
解したところ囲卵腔の無くなるまで・発育したものが生
存していることを確認して本発明を完成するに至った。As a result of various studies aimed at developing a method for long-term preservation of pig fertilized eggs, the present inventor has found that pig fertilized eggs at various times were frozen and preserved at temperatures below -35 degrees Celsius, and then frozen and thawed. The present invention was completed after confirming that the perivitelline cavity remained until the perivitelline cavity disappeared.
すなわち本発明は、囲卵腔の無くなるまで発育し又はそ
れ以降の発育期にあって摂氏マイナス３５度以下に保存
された豚の受精卵とその保存方法に関するものである。That is, the present invention relates to a fertilized pig egg that has developed until the perivitelline cavity disappears or is in the subsequent developmental stage and is stored at -35 degrees Celsius or lower, and a method for preserving the same.
受精卵は１細胞期胚が分裂して２細胞期胚になり、さら
に分裂を繰返して４細胞期胚、８細胞期胚、桑実期胚、
初期胚盤胞、拡張期胚盤胞、透明帯脱出胚盤胞と成長し
ていって子宮に着床する。The fertilized egg divides from a one-cell stage embryo to become a two-cell stage embryo, which then undergoes further division to become a four-cell stage embryo, an eight-cell stage embryo, a morula stage embryo, and a fertilized egg.
The blastocyst develops into an early blastocyst, a diastolic blastocyst, and a blastocyst that has escaped the zona pellucida and implants in the uterus.
一方、胚盤胞が拡張期にはいると受精卵と透明帯の間の
空隙部である囲卵腔が無くなる。本発明ではこの拡張期
胚盤胞又は透明帯脱出胚盤胞を使用する。受精後の日数
では５〜８日目に相当し、５〜７日目のものが好ましい
、受精後拡張期胚盤胞又は透明帯脱出胚盤胞になるまで
の成長は受精卵を提供する豚の体内で行なわせればよい
が、人工培養によって行なってもよい。培養液には哺乳
動物細胞を培養する公知のものを用いればよく、例えば
ＴＣＭ−１９９、Ｈａｍ’ｓ Ｆ−１０、Ｂｒ１ｎｓｔ
ｅｒ液（ＢＭＯＣ−３）、Ｍｅｎｅｚｏ’ｓ Ｂ−２、
ＳＯＦ修正ＰＢＳなどを必要によりウシ胎児血清等を加
えて利用できる。培養条件も牛の受精卵等の培養条件と
同様でよい。On the other hand, when the blastocyst enters the diastolic phase, the perivitelline space, which is the space between the fertilized egg and the zona pellucida, disappears. In the present invention, this diastolic blastocyst or blastocyst that has escaped the zona pellucida is used. The number of days after fertilization corresponds to the 5th to 8th day, preferably the 5th to 7th day, and the pig that provides the fertilized egg grows until it becomes a diastolic blastocyst or a zona prolapsed blastocyst after fertilization. It may be carried out in the body of a person, but it may also be carried out by artificial culture. The culture solution may be one known for culturing mammalian cells, such as TCM-199, Ham's F-10, Br1nst.
er fluid (BMOC-3), Menezo's B-2,
SOF-modified PBS etc. can be used with addition of fetal bovine serum etc. if necessary. The culture conditions may be the same as those for fertilized bovine eggs, etc.
このような受精卵を細胞凍結に用いられる適当な凍結保
護剤を含む培養液に入れ２０〜４０°Ｃで一定時間静置
して平衡させる。培養液には例えば上記のものを使用す
ることができる。凍結保護剤にはグリセリン、ジメチル
スルホキシド、エチレングリコール、ポリビニルピロリ
ドン、シｇ糖等を利用すればよい、凍結保護剤は数段階
の濃度に分け、受精卵を順次濃度の高い液に浸漬してい
くのがよい。グリセリンの濃度は最終濃度で１〜２モル
濃度程度がよく、例えば０．２．０．３５．０．６５．
０．８５．１．０の５段階の液を用いることができる。The fertilized eggs are placed in a culture solution containing a suitable cryoprotectant used for cell freezing and allowed to stand at 20 to 40°C for a certain period of time to equilibrate. As the culture solution, for example, those mentioned above can be used. Glycerin, dimethyl sulfoxide, ethylene glycol, polyvinylpyrrolidone, sig sugar, etc. may be used as the cryoprotectant.The cryoprotectant is divided into several concentrations, and the fertilized eggs are immersed in solutions with successively higher concentrations. It is better. The final concentration of glycerin is preferably about 1 to 2 molar, for example 0.2.0.35.0.65.
A five-stage solution of 0.85.1.0 can be used.
各々の液の浸漬時間は５〜１０分間程度でよく、最終濃
度の液には１５分ないし１時間程度浸漬する。ＤＭＳＯ
も最終濃度で１〜２モル濃度程度がよく、やはり段階濃
度に分けた液を用いることが好ましい。The immersion time in each solution may be about 5 to 10 minutes, and the immersion time in the final concentration solution is about 15 minutes to 1 hour. DMSO
The final concentration is preferably about 1 to 2 molar, and it is also preferable to use a solution divided into graded concentrations.
凍結保護剤で平衡した受精卵は凍結保存用容器に入れて
−５〜−１０″Ｃ程度まで徐冷し植木を行なう。容器に
は受精卵の凍結保存用に使用されている一般的なものを
使用すればよく、例えば小試験管、小アンプル、凍結精
液用ストロ−管などを利用できる。冷却はプログラムフ
リーザーなどを用いて一定速度で行なうことが好ましい
、植木は小氷片の投入、容器外側に液体窒素で冷却した
金属片を接触させる、冷気を吹き付ける、軽い振動を与
えるなどの手段によって行なえばよい。The fertilized eggs equilibrated with the cryoprotectant are placed in a cryopreservation container and slowly cooled to around -5 to -10"C before planting. The container is a typical one used for cryopreservation of fertilized eggs. For example, small test tubes, small ampoules, straw tubes for frozen semen, etc. can be used. Cooling is preferably performed at a constant rate using a program freezer, etc. This can be done by bringing a metal piece cooled with liquid nitrogen into contact with the outside, blowing cold air, or applying light vibrations.
植木後−３０〜−４０°Ｃ程度まで更に除冷して受精卵
を凍結させる。この除冷もプログラムフリーザー等を用
いて一定速度で行なうのがよい。After planting, the fertilized eggs are frozen by further cooling to about -30 to -40°C. This slow cooling is also preferably carried out at a constant speed using a program freezer or the like.
こうして得られた凍結受精卵はフリーザーまたは液体窒
素中等に保存する。The frozen fertilized eggs thus obtained are stored in a freezer or liquid nitrogen.
受精卵を使用する際の融解は急速融解でもよく、緩慢融
解であってもよい。When fertilized eggs are used, the fertilized eggs may be melted rapidly or slowly.
本発明を適用しうる豚の種類は問うところではない。There is no question as to the type of pig to which the present invention can be applied.
ランドレース種と大ヨーク種との交配種またはハンプシ
ャ一種に雌にデュロック種誰の精液を人工授精し、受精
後５ないし６日後に外科的に受精卵を回収した。Females of the Landrace-Great York breed or the Hampshire breed were artificially inseminated with the semen of a Duroc breed, and fertilized eggs were surgically collected 5 to 6 days after fertilization.
回収された受精卵は２０％ウシ胎児血清を添加した燐酸
緩衝液に凍結保護剤としてグリセリンを加えた液に浸漬
した。グリセリン濃度は５段階とし、濃度の薄いものか
ら順次濃い濃度の液に浸漬した（グリセリン平衡）。グ
リセリン平衡した後受精卵を０．２５ｄ容量のプラスチ
ックスドロー内に吸引封入した。封入されたストロ−は
プログラムフリーザーに入れ摂氏３５度から摂氏マイナ
ス６．８度まで１分間摂氏１度の速度で冷却し摂氏マイ
ナス６．８度で植木をおこない１０分間保持した。その
後摂氏マイナス３５度まで１分間に摂氏０．３度の速度
で、さらに摂氏マイナス３６度まで１分間に摂氏０．１
度の速度で冷却し、液体窒素（摂氏マイナス１９６度）
中に投入して保存した。このようにして凍結保存された
受精卵を摂氏３７度の温湯中に直接投入して溶解後グリ
セリン平衡と逆の手順によりグリセリンを除去した。こ
のようにした受精卵を摂氏３７度、炭酸ガス濃度５％の
培養器内で培養した。The recovered fertilized eggs were immersed in a phosphate buffer solution containing 20% fetal bovine serum and glycerin as a cryoprotectant. The glycerin concentration was set to 5 levels, and the samples were immersed in solutions from the lowest concentration to the highest concentration (glycerin equilibrium). After glycerin equilibration, the fertilized eggs were suction-sealed into a 0.25 d capacity plastic suction drawer. The encapsulated straw was placed in a program freezer and cooled from 35 degrees Celsius to -6.8 degrees Celsius at a rate of 1 degree Celsius for 1 minute, and the plants were planted at -6.8 degrees Celsius and held for 10 minutes. Thereafter, the temperature is reduced to -35 degrees Celsius at a rate of 0.3 degrees Celsius per minute, and further to -36 degrees Celsius at a rate of 0.1 degrees Celsius per minute.
liquid nitrogen (minus 196 degrees Celsius)
I put it inside and saved it. The thus cryopreserved fertilized eggs were directly poured into hot water at 37 degrees Celsius, and after dissolution, the glycerin was removed by the reverse procedure of glycerin equilibration. The fertilized eggs thus obtained were cultured in an incubator at 37 degrees Celsius and a carbon dioxide gas concentration of 5%.
その結果、凍結時に受精卵の発育段階が初期胚盤胞（胚
盤胞腔は形成されてまもなく囲卵腔の認められる受精卵
）より若いものでは２８個凍結保存するも凍結後に生存
していたものは１個のみであったのに対し、拡張期胚盤
胞以降の受精卵では１３４個凍結し４９個が生存してい
た。As a result, 28 fertilized eggs whose developmental stage was younger than early blastocysts (fertilized eggs with a perivitelline cavity recognized shortly after the blastocyst cavity was formed) survived after freezing. In contrast, 134 fertilized eggs after diastolic blastocyst were frozen, and 49 survived.
豚受精卵を凍結することにより長期にわたる保存が可能
となった。従来の豚の受精卵移植では、受精卵の保存が
出来なかったため遺伝子の保存には役立たなかったが、
本発明により保存が可能となった。例えば受精卵を産出
した雌豚が死亡した後においても産子を得ることができ
豚の育種改良、繁殖上多大な効果をもたらすことができ
る。Freezing fertilized pig eggs has made it possible to preserve them for a long period of time. Conventional pig fertilized egg transplantation was not useful for preserving genes because the fertilized eggs could not be preserved.
The present invention has made preservation possible. For example, even after the sow that produced the fertilized egg dies, it is possible to obtain offspring, which can have a great effect on the breeding improvement and reproduction of pigs.
Claims (2)
期にあって摂氏マイナス３５度以下に保存された豚の受
精卵(1) Fertilized pig eggs that have developed until the perivitelline cavity disappears or are in the subsequent developmental stage and are stored at -35 degrees Celsius or below.
期にある豚の受精卵を摂氏マイナス３５度以下に保存す
ることを特徴とする豚の受精卵の保存方法(2) A method for preserving fertilized pig eggs, which comprises storing fertilized pig eggs that have developed until the perivitelline cavity is exhausted or are in the subsequent developmental period at -35 degrees Celsius or below.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP26942288A JPH0651601B2 (en) | 1988-10-27 | 1988-10-27 | Frozen fertilized egg of pig and method for cryopreservation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP26942288A JPH0651601B2 (en) | 1988-10-27 | 1988-10-27 | Frozen fertilized egg of pig and method for cryopreservation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02117601A true JPH02117601A (en) | 1990-05-02 |
| JPH0651601B2 JPH0651601B2 (en) | 1994-07-06 |
Family
ID=17472202
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP26942288A Expired - Fee Related JPH0651601B2 (en) | 1988-10-27 | 1988-10-27 | Frozen fertilized egg of pig and method for cryopreservation |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0651601B2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02238837A (en) * | 1989-03-14 | 1990-09-21 | Kachiku Jiyuseiran Ishiyoku Gijutsu Kenkyu Kumiai | Method for preserving swine fertilized egg at low temperature |
| WO1995005075A1 (en) * | 1993-08-13 | 1995-02-23 | Bresatec Limited | Cryopreservation of porcine embryos |
| CN103190391A (en) * | 2013-03-28 | 2013-07-10 | 金�一 | Method for improving integrity of cytoskeletons after freeze thawing of preserved boar sperm |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5891617A (en) * | 1993-09-15 | 1999-04-06 | Organogenesis Inc. | Cryopreservation of harvested skin and cultured skin or cornea equivalents by slow freezing |
-
1988
- 1988-10-27 JP JP26942288A patent/JPH0651601B2/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02238837A (en) * | 1989-03-14 | 1990-09-21 | Kachiku Jiyuseiran Ishiyoku Gijutsu Kenkyu Kumiai | Method for preserving swine fertilized egg at low temperature |
| WO1995005075A1 (en) * | 1993-08-13 | 1995-02-23 | Bresatec Limited | Cryopreservation of porcine embryos |
| CN103190391A (en) * | 2013-03-28 | 2013-07-10 | 金�一 | Method for improving integrity of cytoskeletons after freeze thawing of preserved boar sperm |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0651601B2 (en) | 1994-07-06 |
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