JPH03244331A - Culture medium for extracorporeal culture of bovine ovum - Google Patents
Culture medium for extracorporeal culture of bovine ovumInfo
- Publication number
- JPH03244331A JPH03244331A JP2040777A JP4077790A JPH03244331A JP H03244331 A JPH03244331 A JP H03244331A JP 2040777 A JP2040777 A JP 2040777A JP 4077790 A JP4077790 A JP 4077790A JP H03244331 A JPH03244331 A JP H03244331A
- Authority
- JP
- Japan
- Prior art keywords
- serum
- blood serum
- bovine
- medium
- culture
- 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.)
- Pending
Links
- 241000283690 Bos taurus Species 0.000 title claims abstract description 36
- 239000001963 growth medium Substances 0.000 title abstract description 10
- 102000002322 Egg Proteins Human genes 0.000 title description 3
- 108010000912 Egg Proteins Proteins 0.000 title description 3
- 210000004681 ovum Anatomy 0.000 title description 3
- 210000002966 serum Anatomy 0.000 claims abstract description 50
- 230000001605 fetal effect Effects 0.000 claims abstract description 4
- 241001494479 Pecora Species 0.000 claims description 27
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 13
- 239000012091 fetal bovine serum Substances 0.000 claims description 13
- 238000000338 in vitro Methods 0.000 claims description 13
- 230000035800 maturation Effects 0.000 claims description 13
- 210000000287 oocyte Anatomy 0.000 claims description 5
- 230000013020 embryo development Effects 0.000 claims description 4
- 235000019687 Lamb Nutrition 0.000 claims description 3
- 244000309466 calf Species 0.000 claims description 3
- 230000004720 fertilization Effects 0.000 abstract description 12
- 210000002257 embryonic structure Anatomy 0.000 abstract description 10
- 210000002459 blastocyst Anatomy 0.000 abstract description 6
- 230000003325 follicular Effects 0.000 abstract description 3
- 239000004615 ingredient Substances 0.000 abstract 2
- 210000004700 fetal blood Anatomy 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 28
- 235000013601 eggs Nutrition 0.000 description 20
- 230000018109 developmental process Effects 0.000 description 13
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 10
- 102000016938 Catalase Human genes 0.000 description 5
- 108010053835 Catalase Proteins 0.000 description 5
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 5
- 229960002897 heparin Drugs 0.000 description 5
- 229920000669 heparin Polymers 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 210000001672 ovary Anatomy 0.000 description 5
- 229960005322 streptomycin Drugs 0.000 description 5
- 238000012258 culturing Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 235000015278 beef Nutrition 0.000 description 3
- 230000029803 blastocyst development Effects 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- 210000001161 mammalian embryo Anatomy 0.000 description 3
- 210000000582 semen Anatomy 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 206010042573 Superovulation Diseases 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000012173 estrus Effects 0.000 description 1
- 210000004392 genitalia Anatomy 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 230000009027 insemination Effects 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 210000005000 reproductive tract Anatomy 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明は、牛卵子の体外受精に用いる培地、すなわち卵
巣から回収した未成熟卵子を成熟させるための成熟用培
地及び成熟させた卵子を体外で受精して得られる胚を培
養するための発生用培地に関する。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a medium used for in vitro fertilization of bovine eggs, that is, a maturation medium for maturing immature eggs recovered from ovaries, and a method for fertilizing matured eggs in vitro. The present invention relates to a development medium for culturing embryos obtained by
あ班煎責量
近年、牛肉の輸入自由化の情勢にかんがみ、優れた資質
を有する肥育素手を安定して供給するための技術対策と
して受精卵移植の実用化が進められている。しかし、一
方では牛卵子の体外受精及び体外培養技術も注目されて
きている。In recent years, in view of the liberalization of beef imports, the practical use of fertilized egg transplantation has been promoted as a technical measure to ensure a stable supply of high-quality fattened beef. However, on the other hand, in vitro fertilization and in vitro culture techniques for bovine eggs are also attracting attention.
胚移植による技術は、卵子を供給する雌牛（供卵牛）に
対して過剰排卵させるためのホルモン処置を施し、供卵
牛の生殖器内で受精させた胚を子宮潅流により体外に取
り出し、これを性周期を同期化させるためにホルモン処
置を施した、あるいはホルモン処置を施さない他の雌牛
（受卵牛）の生殖器内に移植して子畜を出産させる一連
の技術である。Embryo transfer technology involves hormonal treatment of the female cow that supplies the eggs (donor cow) to cause superovulation, and the embryos fertilized in the donor cow's genitals are removed outside the body through uterine perfusion. This is a series of techniques in which a cow is implanted into the reproductive tract of another cow (receiver cow) that has been treated with hormones to synchronize its oestrous cycle, or that has not been treated with hormones to give birth to a calf.
これに対し、体外受精は、生体あるいは屠体の牛卵巣か
らに未熟成卵子を吸引回収し、成熟用培地にて２０時間
前後培養する０次いで、成熟した時点で凍結精液を融解
し、シャレーの中に入れて卵子と媒精を行う。受精後、
牛卵子を発生培地へ移して培養を行う、この間分割が進
み胚盤胞になったものを受卵牛の発情に合わせて移植す
る技術である。On the other hand, in vitro fertilization involves collecting unripe eggs from the ovaries of live or carcass cows by suction and culturing them in a maturation medium for about 20 hours.Then, when they mature, the frozen semen is thawed and placed in a chalet. Place it inside and inseminate the egg. After fertilization,
This is a technique in which bovine eggs are transferred to a developmental medium and cultured, during which time they undergo division and become blastocysts, which are then transferred to the recipient cow in time for estrus.
上述した受精卵移植による牛の飼育では、供卵牛のホル
モン処置、供卵牛の繁殖管理、胚回収、回収した胚の検
索、受卵牛への移植の５操作に大別されて行われるため
、胚を提供する供卵牛と胚を受は入れる受卵牛が必要と
なり、加うるに採卵と胚移植の操作に熟練を要し、しか
もグループでの作業となるため、綿密な連係プレーが必
要となるなどの問題がある。The above-mentioned breeding of cattle using fertilized egg transplantation is carried out by five operations: hormone treatment of donor cows, reproductive management of donor cows, embryo collection, retrieval of recovered embryos, and transplantation into recipient cows. Therefore, a donor cow to provide the embryos and a recipient cow to receive the embryos are required, and in addition, the operations of egg retrieval and embryo transfer require skill, and since the work must be done in groups, careful coordination is required. There are problems such as the need for
一方、牛の体外受精では、上述したような供卵牛を必要
としない利点があるものの、受精卵の分割率のバラツキ
が大きく、また、受精卵の拡張胚盤胞への発生率が低い
という問題がある。On the other hand, although in vitro fertilization of cattle has the advantage of not requiring donor cows as mentioned above, there is large variation in the division rate of fertilized eggs, and the rate of development of fertilized eggs into expanded blastocysts is low. There's a problem.
が１決しようとする課
牛の体外受精においては、前述した未成熟卵子の成熟の
ための成熟培地及び受精卵の培養のための発生培地には
、−ｉに牛胎児血清が用いられている。ところが、本発
明者は、牛胎児血清に代えて羊血清を含有させた成熟培
地ならびに発生培地を用いる場合、受精卵を培養して得
られる胚の分割率が向上すると共にバラツキが少なくな
り、加うるに拡張胚盤胞への発生率も牛胎児血清を含有
する培地の場合よりも高くなることを見出し、本発明を
なすに至った。したがって、本発明は、牛の体外受精に
おいて一般に用いられている牛胎児血清に比べて牛体外
受精胚の発生に適していて生恥生産に有用である、羊血
清を含有させた前記成熟用培地及び発生培地を提供する
こと課題とする。In in vitro fertilization of cows, which is about to be decided, fetal bovine serum is used in -i for the maturation medium for maturing immature eggs and the development medium for culturing fertilized eggs. . However, the present inventor has found that when a maturation medium and a development medium containing sheep serum instead of fetal bovine serum are used, the division rate of embryos obtained by culturing fertilized eggs is improved and the variation is reduced. The present inventors have discovered that the incidence of expanded blastocysts is also higher than in the case of a medium containing fetal bovine serum, leading to the present invention. Therefore, the present invention provides the maturation medium containing sheep serum, which is more suitable for the development of bovine IVF embryos than the fetal bovine serum commonly used in bovine IVF and is useful for fresh production. The objective is to provide a growth medium and a growth medium.
課 を”°するための
本発明は、牛卵子の体外培養による卵成熟のための培地
及び胚発生のための培地に、羊血清を血清成分として含
有させたものを用いることを特徴とする。The present invention is characterized in that a medium for egg maturation and a medium for embryo development by in vitro culture of bovine eggs contain sheep serum as a serum component.
ここで培地の血清成分として用いる羊血清は、胎児血清
、初生子羊血清、雌成緬羊血清及び雌成緬羊血清を包含
する。The sheep serum used as the serum component of the medium herein includes fetal serum, first-born lamb serum, female adult sheep serum, and adult female sheep serum.
本発明ではこれらの羊血清を従来用いられている牛胎児
血清及び／又は初生子牛血清と併用してもよい。In the present invention, these sheep sera may be used in combination with conventionally used fetal bovine serum and/or primary calf serum.
羊血清は、培地中０．１〜２０％（重量）含有させるが
、胚発生培地では約２％（重量）含有させたものが好ま
しい。Sheep serum is contained in the medium in an amount of 0.1 to 20% (by weight), and in the embryonic development medium, it is preferably contained in an amount of about 2% (by weight).
次に、血清成分として羊血清を含有させた培地の優位性
を試験した結果を示す。Next, the results of testing the superiority of a medium containing sheep serum as a serum component are shown.
成熟培地として、Ｅａｒｌｅ’５１９９に羊血清を１０
重−量％と２０重量％ならびに牛胎児血清を１０重量％
それぞれ添加したものを用い、これに黒毛和種肉用雌牛
の卵巣より回収した未成熟卵子を植えて３９℃の温度で
、Ｃｏ、 ９５％とＡｉｒ ５％から威るＣＯ，雰囲気
培養器内で１９〜２０時間戒熟培養成熟った。As a maturation medium, 10% sheep serum was added to Earle'5199.
20% by weight and 10% by weight of fetal bovine serum.
Using these additives, immature eggs collected from the ovaries of Japanese black beef cows were planted and incubated at a temperature of 39°C with an atmosphere of 95% Co and 5% air in an incubator. It was cultivated for 19-20 hours to reach maturity.
次いで、成熟した卵胞卵子を精液と混合して４時間媒精
を行った後、羊血清（ＳＳ）と牛胎児血清（Ｆｅ２）を
それぞれ２重量％添加した発生培地に移して４６時間培
養を行って胚盤胞の発生率を調べた。Next, the mature follicular ovum was mixed with semen and inseminated for 4 hours, and then transferred to a development medium supplemented with 2% by weight of each of sheep serum (SS) and fetal bovine serum (Fe2) and cultured for 46 hours. The incidence of blastocyst development was investigated.
ここで用いた成熟培地及び発生培地には５０＋ｗＭカタ
ラーゼ、ストレプトマイシン（１００μｇ／ｖａ １
）及び５ ｕ／ｍ ｌヘパリンを添加した。The maturation and development media used here included 50+ wM catalase, streptomycin (100 μg/va 1
) and 5 u/ml heparin were added.
なお、媒精後２日目に分割検査を、また、８〜１０日目
に拡張胚盤胞への発生検査をそれぞれ行った。この試験
は計７回行った。結果は表１にまとめて示すとおりであ
る。A split test was performed on the second day after insemination, and a test for development into expanded blastocysts was performed on days 8 to 10. This test was conducted a total of 7 times. The results are summarized in Table 1.
表
１
以下に実施例を示して本発明とその効果を具体的に説明
する。Table 1 The present invention and its effects will be specifically explained with reference to Examples below.
去−」Ｌ−廻一」−
ホルスタイン種雌乳牛の卵巣より採取した未成熟卵胞卵
子を下記組成の各成熟培地にそれぞれ植えて３９℃の温
度で、ＣＯ□９５％とＡｉｒ５％から戒るＣＯ，雰囲気
培養器内で１９〜２０時間成熟培養を行った。- "L-Kouichi" - Immature follicles and oocytes collected from the ovaries of Holstein female dairy cows were planted in each maturation medium with the following composition and heated at a temperature of 39°C, with a CO concentration of 95% CO□ and 5% Air. , Maturation culture was performed in an atmosphere incubator for 19 to 20 hours.
Ｉｙ、熟培地の組成：
Ｎｏ、 １ ＝４ａｒｌｅ’ｓ培地に１０％ＦＣ５，５
０ｍＭカタラーゼ。Iy, Composition of mature medium: No, 1 = 10% FC5,5 in 4arle's medium
0mM catalase.
ストレプトマイシン（１００μｇ／ ｍ １ ）、５ｕ
／＋ｗ ｊ！ヘパリンを添加。Streptomycin (100μg/m 1 ), 5u
/+w j! Add heparin.
ＮＯ，２・・・Ｅａｒｌｅ’ｓ培地に１０％ＳＳ、 ５
０１５Ｍカタラーゼ。NO, 2...10% SS in Earle's medium, 5
015M catalase.
ストレプトマイシン（１００，ｌＪｇ／ ｍｊ）、５ｕ
／曽ｊ！ヘパリンを添加。Streptomycin (100, lJg/mj), 5u
/ Soj! Add heparin.
Ｆｅ２は牛胎児血清を、ＳＳは羊血清を示す。Fe2 indicates fetal bovine serum, and SS indicates sheep serum.
次に、成熟した卵胞卵子を精液と混合して４時間媒精を
行った後、下記ｍ戒の発生培地に移して４６時間培養を
行って胚盤胞の発生率を調べた。Next, the mature follicular ovum was mixed with semen and inseminated for 4 hours, then transferred to the following development medium and cultured for 46 hours to examine the incidence of blastocyst development.
発生培地の組１′ｌｉ、：
Ｎｏ、 １ ＝４ａｒｌｅ ’ ｓ培地に２％ＦＣＳ、
５０ｍＭカタラーゼ。Set of development media 1'li: No. 1 = 2% FCS in 4arle's medium;
50mM catalase.
ストレプトマイシン＋５ｕ／ｍｌヘパリンを添加。Add streptomycin + 5u/ml heparin.
Ｎ００２−・・Ｅａｒｌｅ ’ ｓ培地に２％ＳＳ、５
０ｍＭカタラーゼ。N002--Earle's medium with 2% SS, 5
0mM catalase.
ストレプトマイシン、 ５ｕ／ｓ＋ １ヘパリンを添加
。Add streptomycin, 5u/s + 1 heparin.
結果は表２に示すとおりである。The results are shown in Table 2.
表 ２
させた発生培地を用いて実施例１に記載したと同様の手
順に従って体外培養を行なって胚盤胞の発生率を調べた
。結果は表３に示すとおりである。Table 2 In vitro culture was carried out in accordance with the same procedure as described in Example 1 using the above-described developmental medium to examine the incidence of blastocyst development. The results are shown in Table 3.
表
表２から、羊血清を血清成分として含有する培地、牛胎
児血清を含有させたものに比べ、牛体外受精胚の発生に
適する傾向が認められる。From Table 2, it can be seen that the culture medium containing sheep serum as a serum component tends to be more suitable for the development of bovine IVF embryos than the culture medium containing fetal bovine serum.
実施例２
黒毛和種（Ｂ）とホルスタイン種（Ｄ）の両雌牛の卵巣
より回収した未成熟卵を、初生子羊血清（Ｂ−２３）、
雌成緬羊血清（Ｄ−３７）、羊胎児血清（Ｄ−４３）、
雄と雌の威緬羊血清（Ｄ４８）及び旗戒緬羊血清（Ｄ−
５０）の各羊血清をそれぞれ１０重量％と、比較として
牛胎児血清（Ｆｅ２）１０重量％を含有させた成熟培地
と、上記各羊血清と牛胎児血清をそれぞれ２重量％を含
有先１１わ匪果
本発明は、生体外受精に用いる培地の血清成分として入
手容易な羊血清を用いることにより、従来、一般に用い
られている牛胎児血清を血清成分とした培地に比べ、牛
卵胞卵子の体外受精による拡張胚盤胞への発生率が優れ
ているので生恥の体外発生は実用化に役立つものである
。Example 2 Immature eggs collected from the ovaries of both Japanese Black (B) and Holstein (D) cows were treated with day-old lamb serum (B-23),
Female adult sheep serum (D-37), fetal sheep serum (D-43),
Male and female Dignified Burmese Sheep Serum (D48) and Flagship Burmese Sheep Serum (D-
Maturation medium containing 10% by weight of each sheep serum (50) and 10% by weight of fetal bovine serum (Fe2) for comparison, and 2% by weight of each of the above sheep serum and fetal bovine serum. The present invention uses readily available sheep serum as a serum component of the culture medium used for in vitro fertilization, thereby improving the in vitro fertilization of bovine follicles and oocytes compared to the conventionally commonly used culture medium containing fetal bovine serum as a serum component. The in vitro development of embryonic embryos is useful for practical use because the rate of development into expanded blastocysts through fertilization is excellent.
Claims (5)
る牛卵子の体外培養による卵子成熟用及び胚発生用培地
。(1) A medium for oocyte maturation and embryo development by in vitro culture of bovine oocytes, characterized by containing sheep serum as a serum component.
項（１）に記載の卵子成熟用培地。(2) The oocyte maturation medium according to claim (1), which contains 0.1 to 20% (weight) of sheep serum.
記載の胚発生用培地。(3) The medium for embryonic development according to claim (1), which contains about 2% (by weight) sheep serum.
及び雌成緬羊血清から成る群から選択される請求項（１
）及至（３）のいずれかに記載の培地。(4) Claim (1) wherein the sheep serum is selected from the group consisting of fetal serum, primary lamb serum, male adult sheep serum and female adult sheep serum.
) to (3).
生子牛血清を含有する請求項（１）に記載の培地。(5) The medium according to claim (1), which contains sheep serum, fetal bovine serum, and/or first-born calf serum as serum components.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2040777A JPH03244331A (en) | 1990-02-21 | 1990-02-21 | Culture medium for extracorporeal culture of bovine ovum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2040777A JPH03244331A (en) | 1990-02-21 | 1990-02-21 | Culture medium for extracorporeal culture of bovine ovum |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH03244331A true JPH03244331A (en) | 1991-10-31 |
Family
ID=12590061
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2040777A Pending JPH03244331A (en) | 1990-02-21 | 1990-02-21 | Culture medium for extracorporeal culture of bovine ovum |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH03244331A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108575886A (en) * | 2018-03-22 | 2018-09-28 | 兰州大学 | A method of feeding Sweet sorghum silage improves functional components in mutton |
-
1990
- 1990-02-21 JP JP2040777A patent/JPH03244331A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108575886A (en) * | 2018-03-22 | 2018-09-28 | 兰州大学 | A method of feeding Sweet sorghum silage improves functional components in mutton |
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