JPH02238837A - Method for preserving swine fertilized egg at low temperature - Google Patents
Method for preserving swine fertilized egg at low temperatureInfo
- Publication number
- JPH02238837A JPH02238837A JP5957489A JP5957489A JPH02238837A JP H02238837 A JPH02238837 A JP H02238837A JP 5957489 A JP5957489 A JP 5957489A JP 5957489 A JP5957489 A JP 5957489A JP H02238837 A JPH02238837 A JP H02238837A
- Authority
- JP
- Japan
- Prior art keywords
- fertilized
- blastocyst
- eggs
- stage
- fertilized egg
- 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
- 238000000034 method Methods 0.000 title claims description 12
- 241000282898 Sus scrofa Species 0.000 title abstract 5
- 210000002459 blastocyst Anatomy 0.000 claims abstract description 32
- 238000007710 freezing Methods 0.000 claims abstract description 7
- 230000008014 freezing Effects 0.000 claims abstract description 7
- 238000012258 culturing Methods 0.000 claims abstract description 5
- 235000013601 eggs Nutrition 0.000 claims description 38
- 241000282887 Suidae Species 0.000 claims description 6
- 238000009395 breeding Methods 0.000 claims description 5
- 230000001488 breeding effect Effects 0.000 claims description 3
- 210000004291 uterus Anatomy 0.000 claims description 2
- 238000004321 preservation Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 12
- 238000005138 cryopreservation Methods 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 210000002257 embryonic structure Anatomy 0.000 description 6
- 241000124008 Mammalia Species 0.000 description 5
- 210000001161 mammalian embryo Anatomy 0.000 description 5
- 210000004340 zona pellucida Anatomy 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 210000004952 blastocoel Anatomy 0.000 description 4
- 230000035899 viability Effects 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000000472 morula Anatomy 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000010902 straw Substances 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 206010011732 Cyst Diseases 0.000 description 1
- 101100010303 Drosophila melanogaster PolG1 gene Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 101150078890 POLG gene Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 230000001055 chewing effect Effects 0.000 description 1
- 208000031513 cyst Diseases 0.000 description 1
- 230000012173 estrus Effects 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000002350 laparotomy Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000003761 preservation solution Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Meat, Egg Or Seafood Products (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は豚の受精卵の保存方法に関するものである。ま
た本発明はこのように保存された豚の受精卵を用いるこ
とによる受精卵の移植を効率的に実施する方法に関する
ものである。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for preserving fertilized pig eggs. The present invention also relates to a method for efficiently carrying out fertilized egg transplantation using the thus preserved fertilized pig eggs.
更に具体的には本発明は豚の受精卵をその生存性を損な
うことな＜１５℃以下の低温に保存する方法およびこの
ように保存された豚の受精卵を用いる受精卵の移植を効
率的に実施する方法に関する。More specifically, the present invention provides a method for storing fertilized pig eggs at a low temperature of <15°C or less without impairing their viability, and an efficient method for transplanting the fertilized eggs using the fertilized pig eggs stored in this manner. Concerning how to carry out this.
ヒトを含む大型の噛乳動物、例えば牛、馬、豚、羊、山
羊などおよび小型の啼乳動物、例えば兎、マウス、ラッ
トなどの繁殖方法として、体内または体外で受精した受
精卵を体内に戻して受胎させることは既に実験的に行な
なわれており、多くの成功例が報告されている。この方
法をより容易化し、また遠隔地で採取された受精卵を利
用可能とするために、受精卵の低温又は凍結保存の多く
の試みがこれまでになされている。そして豚以外に上記
した啼乳動物の受精卵ではこのような試みが成切してお
り、かかる受精卵の低温又は凍結保存技術の確立によっ
て受精卵の遠隔地への輸送が可能となり、夫々の動物種
の品種改良、繁殖手段の多様化に役立っている。As a breeding method for large chewing mammals, including humans, such as cows, horses, pigs, sheep, and goats, and for small mammals, such as rabbits, mice, and rats, fertilized eggs that have been fertilized internally or externally are placed inside the body. Returning animals to impregnate them has already been carried out experimentally, and many successful cases have been reported. In order to make this method easier and to make it possible to utilize fertilized eggs collected in remote locations, many attempts have been made to preserve fertilized eggs at low temperatures or cryopreservation. Such attempts have been successful with fertilized eggs of the above-mentioned mammals other than pigs, and the establishment of low-temperature or cryopreservation technology for such fertilized eggs has made it possible to transport fertilized eggs to remote areas, and It is useful for improving the breeding of animal species and diversifying breeding methods.
豚の受精卵に対しても同様の低温又は凍結保存の試みが
これまでになされたが、豚の受精卵は１５℃以下の低温
に曝されただけで死滅すること（例えばＣ． Ｐｏｌｇ
ｅらによるＣｒｙｏｂｉｏｌｏｇＹ第１１巻第５６０頁
（１９７４）参照）から、かかる試みは成功せず、従っ
て豚受精卵の低温保存は全く不可能と考えられていた。Similar low-temperature or cryopreservation attempts have been made for fertilized pig eggs, but fertilized pig eggs die when exposed to temperatures below 15°C (for example, C. Polg
(see Cryobilog Y, Vol. 11, p. 560 (1974)), such attempts were not successful, and it was therefore thought that low-temperature preservation of pig fertilized eggs was completely impossible.
このような状況に鑑み、本発明者らは豚の受精卵をその
生存性を損なうことなく−２０℃以下の低温において保
存しうる条件を研究し本発明を完成したのである。In view of this situation, the present inventors completed the present invention by researching conditions under which fertilized pig eggs can be stored at a low temperature of -20° C. or lower without impairing their viability.
上記した啼乳動物の受精卵に対する低温又は凍結保存の
試みは従来から２細胞期、４細胞期、８細胞期、桑実胚
期および胚盤胞期までの受精卵に対して行なわれている
。そしてこれらの発生段階の受精卵は透明帯と呼ばれる
卵膜に包まれかかる膜の保護作用下にあることから一般
に受精卵が低温による障害を受けないＩ；めには上記発
生段階の受精卵を取り扱うことが好結果をもたらすもの
と予想されたのである。事実この予想は豚以外の啼乳動
物の受精卵では正しいものであったが豚の受精卵に対し
ては予想通りではなかったのは上記した通りである。The above-mentioned attempts at low-temperature or cryopreservation of fertilized eggs of mammals have been carried out on fertilized eggs up to the 2-cell stage, 4-cell stage, 8-cell stage, morula stage, and blastocyst stage. . Since the fertilized eggs at these developmental stages are protected by a membrane called the zona pellucida, the fertilized eggs are generally not affected by low temperatures. It was expected that handling it would bring about good results. In fact, as mentioned above, this prediction was correct for fertilized eggs of mammals other than pigs, but it was not as expected for fertilized pig eggs.
本発明者らは研究の結果、以外にも豚の受精卵では上記
した透明帯脱出直前の拡大期胚盤胞の段階の受精卵が低
温による障害を受けないことを見出して本発明を完成さ
せたのである。As a result of our research, the present inventors have also completed the present invention by discovering that, in the case of fertilized pig eggs, fertilized eggs at the expanding blastocyst stage, just before the zona pellucida, are not affected by low temperatures. It was.
すなわち本発明は、拡大期胚盤胞の段階の豚の受精卵を
凍結することを特徴とする豚の受精卵の保存方法に関す
る。That is, the present invention relates to a method for preserving a fertilized pig egg, which comprises freezing a fertilized pig egg at the expanded blastocyst stage.
更に本発明は、拡大期胚盤胞の段階の豚の受精卵を凍結
して保存し、このようにして保存された受精卵を移植す
ることを特徴とする豚の繁殖方法にも関する。Furthermore, the present invention also relates to a method for breeding pigs, which comprises freezing and preserving fertilized pig eggs at the expanding blastocyst stage and transplanting the thus preserved fertilized eggs.
本発明において凍結保存しうる拡大期胚盤胞（ｅｘｐａ
ｎｄｉｎｇ ｂｌａｓｔｏｃｙｓｔ）の段階の豚の受精
卵とは、受精卵における桑実期胚の次の発達段階である
胚盤胞がその胞胚腔を拡大させた段階の受精卵であって
、次の発達段階の脱穀すなわち透明帯からの脱出（ハツ
チング）を終了する直前の受精卵を意味し、一般的には
胚盤胞を適当な培養液中に培養し、その胞胚腔が拡大し
たことを目視または他の手段、例えば光電的監視手段で
確認して得られたものでありうるが、かかる培養の手段
を経ることなく、他の手段、例えば交配した雌豚の子宮
からかかる拡大期胚盤胞が入手しえたならばそのものの
使用が可能であることは勿論である。In the present invention, expanded stage blastocysts (expa
A fertilized pig egg at the nding blastocyst stage is a fertilized egg at the stage where the blastocyst, which is the next developmental stage after the morula stage embryo in the fertilized egg, has expanded its blastocoel, and is at the next developmental stage. This refers to the fertilized egg just before it completes its threshing, or hatching, from the zona pellucida.Generally, the blastocyst is cultured in an appropriate culture medium, and the expansion of its blastocoel is checked visually or by other means. However, such expanded blastocysts may be obtained by other means, such as from the uterus of a mated sow, without going through such culturing means. Of course, if you can do it, you can use it.
しかしながら、確実な拡大期胚盤胞の入手の手段として
は胚盤胞の人工的培養の手段で発達させて胞胚腔の拡大
を待つことが挙げられる。However, one way to reliably obtain expansion-stage blastocysts is to develop them by artificial culturing of blastocysts and wait for the blastocoel to expand.
この場合の培養は既知の種々の手段で行ないうる。また
用いる培養液にも種々のものを選択することができ、例
えばＷｈｉｔｔｉｎｇｈａｍの液（Ｍ−１６； １９７
１） 、Ｗｈｉｔｔｉｎｇｈａｍの修正ＰＢＳ（ＦＢＩ
． １９７］）、Ｗｈｉｔｔｅｎ液（１９５７）、Ｂｒ
ｉｎｓｔｅｒ液（ＢＭＯＣ−１． １９６３）、Ｂｒ
ｉｎｓｔｅｒ液（ＢＭＯＣ−２． １９６５）、Ｂｒ
ｉｎｓｔｅｒ液（ＢＭＯＣ−３． １９７１）、Ｗｈ
ｉＬｔｉｎ液（１９６８）、Ｂｉｇｇｅｒｓ液（１９７
１）、ＴＹＨ液（１９７１）、ＢａｖｉｓＬｅｒ液（１
９６９）（以上タレプスーリンゲル重炭酸液を改良した
もの）などおよびこれらを適宜修正したものが培養に使
用可能である。Cultivation in this case can be carried out by various known means. Furthermore, various culture solutions can be selected for use, such as Whittingham's solution (M-16; 197
1), Whittingham's Modified PBS (FBI
．． 197]), Whitten solution (1957), Br
inster liquid (BMOC-1. 1963), Br
inster liquid (BMOC-2. 1965), Br
inster liquid (BMOC-3. 1971), Wh
iLtin solution (1968), Biggers solution (197
1), TYH solution (1971), BavisLer solution (1
969) (improved version of the above Talep-Ringer bicarbonate solution) and the like, as well as appropriate modifications thereof, can be used for culture.
胚盤胞はこれらの培養液中で３７゜Ｃ前後の温度で培養
すると数時間〜３０時間で拡大期胚盤胞となる。When the blastocyst is cultured in these culture solutions at a temperature of around 37°C, it becomes an expansion-stage blastocyst in several to 30 hours.
このようにして得られた拡大期胚盤胞は適当な保存液、
例えば１０〜２０％の仔牛血清、牛胎仔血清、豚血清、
ｌ ＝ １６ｍｙ／ ｒｘ（ｌの牛血清アルブミ拡大期
胚盤胞（ｅｘｐａｎｄｉｎｇ ｂｌａｓｔｏｃｙｓｔ）
を採取しン等を含むダルペッコのリン酸緩衝液に１．５
Ｍ前後のグリセロールあるいはＤＭＳＯを加えたものを
使用して凍結保存される。The expanded blastocysts obtained in this way are stored in an appropriate preservation solution,
For example, 10-20% calf serum, fetal bovine serum, pig serum,
l = 16my/rx (l bovine serum albumin expanding blastocyst)
1.5 in Dulphecco's phosphate buffer containing
It is cryopreserved using glycerol around M or DMSO.
一定時間保存後、試料を２５℃温水浴に移して約３０秒
間加温することによって融解し、融解後胚を回収し、培
養あるいは移植によって生存性を判定した。この結果、
−２０°Ｃに凍結しても胚が生存することが確認された
。After storage for a certain period of time, the sample was transferred to a 25° C. hot water bath and heated for about 30 seconds to thaw, and after thawing, the embryo was collected and viability was determined by culturing or transplantation. As a result,
It was confirmed that the embryos survived even when frozen at -20°C.
本願方法で用いる拡大期胚盤胞は胚盤胞の次の発達段階
である胞胚腔が拡大する時期の受精卵であれば、次の発
達段階の脱殼の直前のものであっても良い。The expanded blastocyst used in the method of the present application may be a fertilized egg at the stage where the blastocoel expands, which is the next developmental stage of the blastocyst, and may be one that is just before exhullation at the next developmental stage.
以下に本発明を実施例によって詳細に説明する。The present invention will be explained in detail below using examples.
（！ｉｔ！施例〕
Ｌ ＷＩｉ成熟雌豚にデュロック種の雄を交配させ発情
終了後５〜６日目に開腹手術を行ない、た。こうして採
取した胚をＷｈｉｔｔｉｎｇｈａｍ（１９７１）のＭ−
１６培養液とＤｕｌｂｅｃｃｏのリン酸緩衝液（ＰＢＳ
）に牛胎仔血清（１６％）を加えた液との混合培養液に
浮遊させた。この培養液中の胚の一部については２４時
間培養した。この培養処置によって、胚の発達段階は以
下の様に進行した。(!it!Example) L WIi mature sows were mated with Duroc male pigs and laparotomy was performed 5 to 6 days after the end of estrus.The embryos thus collected were transferred to the M-
16 culture medium and Dulbecco's phosphate buffer (PBS)
) and a solution containing fetal bovine serum (16%). Some of the embryos in this culture solution were cultured for 24 hours. By this culture treatment, the developmental stages of the embryo progressed as follows.
拡大期胚盤胞（ｅｘｐａｎｄｉｎｇ ｂｌａｓＬｏｃｙ
ｓｔ） →透明帯脱出期胚盤胞（ｈａｔｃｈｅｄ ｂｌ
ａｓｔｏｃｙｓｔ）この胚の名称は、ｉｎ ｖｉｔｒｏ
透明帯脱出胚盤胞とした。expanding blastocyst
st) → zona pellucida exit stage blastocyst (hatched bl
astrocyst) The name of this embryo is in vitro
The zona pellucida prolapsed blastocyst.
このようにして得られた雌豚より採取直後の拡大期胚盤
胞、透明帯脱出胚盤胞および「培養により発達段階を進
行させた胚盤胞」を以下の凍結保存に供した。Expanding-stage blastocysts, zona pellucida-exited blastocysts, and "blastocysts whose developmental stages have been advanced through culture" immediately after collection from the sows thus obtained were subjected to cryopreservation as described below.
低温保存法
胚を１．５Ｍ ＤＭＳＯまたは１．５Ｍグリセロールを
含む１６％牛胎仔血清加ＰＢＳとともに、０．２５＄１
βプラスティックストローに吸引し、ゼラチンパウダー
で封入した。このサンプルを約１℃／分の冷却速度で−
５〜７℃に冷却後植氷処理しさらに０．３℃／分の速度
で−２０℃に冷却し凍結した。Cryopreservation embryos were incubated with 1.5M DMSO or 1.5M glycerol in PBS supplemented with 16% fetal bovine serum for 0.25$1.
It was sucked into a β plastic straw and sealed with gelatin powder. This sample was cooled at a cooling rate of approximately 1°C/min.
After cooling to 5 to 7°C, it was treated with ice planting, and further cooled to -20°C at a rate of 0.3°C/min to freeze.
−２０℃の最終温度での保持時間は３分間としｔ二 。The holding time at the final temperature of -20°C was 3 minutes.
凍結後、サンプルを２５゜Ｃの温水浴に移して約３０秒
間保持し、加温融解した。融解後、胚をストローより回
収し、前述の培養液を用いて培養し、胚の生存性を調べ
た。After freezing, the sample was transferred to a 25°C hot water bath and held for about 30 seconds to heat and thaw. After thawing, the embryos were collected from the straws, cultured using the above-mentioned culture solution, and the viability of the embryos was examined.
結 果 胚の凍結保存の結果は下表のとおりである。result The results of embryo cryopreservation are shown in the table below.
表１ 胚盤胞 拡大期胚盤胞 ２５／３０（８３．３） ７／１４（５０．０） 盤胞 本凍結条件Table 1 blastocyst Expanding blastocyst 25/30 (83.3) 7/14 (50.0) cyst Main freezing conditions
Claims (1)
特徴とする豚の受精卵の保存方法。 ２）拡大期胚盤胞は豚の受精卵の胚盤胞を培養したもの
であるか、または交配した雌豚の子宮から採取したもの
である請求項１に記載の方法。 ３）拡大期胚盤胞の段階の豚の受精卵を凍結して保存し
、このようにして保存された受精卵を移植することを特
徴とする豚の繁殖方法。[Scope of Claims] 1) A method for preserving fertilized pig eggs, which comprises freezing pig fertilized eggs at the expanding blastocyst stage. 2) The method according to claim 1, wherein the expanded blastocyst is obtained by culturing a blastocyst from a fertilized pig egg, or is obtained from the uterus of a mated sow. 3) A method for breeding pigs, which comprises freezing and preserving fertilized pig eggs at the expanding blastocyst stage and transplanting the thus preserved fertilized eggs.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1059574A JP2636405B2 (en) | 1989-03-14 | 1989-03-14 | How to keep fertilized pigs at low temperatures |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1059574A JP2636405B2 (en) | 1989-03-14 | 1989-03-14 | How to keep fertilized pigs at low temperatures |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02238837A true JPH02238837A (en) | 1990-09-21 |
| JP2636405B2 JP2636405B2 (en) | 1997-07-30 |
Family
ID=13117139
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1059574A Expired - Lifetime JP2636405B2 (en) | 1989-03-14 | 1989-03-14 | How to keep fertilized pigs at low temperatures |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2636405B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06292484A (en) * | 1993-04-06 | 1994-10-21 | Natl Fedelation Of Agricult Coop Assoc | Freeze preservation of fertilized bovine ovum |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02117601A (en) * | 1988-10-27 | 1990-05-02 | Itochu Shiryo Kk | Frozen fertilized ovum of pig and freezing preservation thereof |
-
1989
- 1989-03-14 JP JP1059574A patent/JP2636405B2/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02117601A (en) * | 1988-10-27 | 1990-05-02 | Itochu Shiryo Kk | Frozen fertilized ovum of pig and freezing preservation thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06292484A (en) * | 1993-04-06 | 1994-10-21 | Natl Fedelation Of Agricult Coop Assoc | Freeze preservation of fertilized bovine ovum |
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| Publication number | Publication date |
|---|---|
| JP2636405B2 (en) | 1997-07-30 |
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