JP5410782B2 - Medium composition for artificial fertilization - Google Patents
Medium composition for artificial fertilization Download PDFInfo
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- JP5410782B2 JP5410782B2 JP2009037625A JP2009037625A JP5410782B2 JP 5410782 B2 JP5410782 B2 JP 5410782B2 JP 2009037625 A JP2009037625 A JP 2009037625A JP 2009037625 A JP2009037625 A JP 2009037625A JP 5410782 B2 JP5410782 B2 JP 5410782B2
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- fertilization
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
本発明は、人工受精用培地組成物、特にウシ胚の培養又は保存の際に使用する体外受精用培地組成物及びこれを用いた体外受精後のウシ胚の培養・保存方法に関する。 The present invention relates to a medium composition for artificial fertilization, in particular, a medium composition for in vitro fertilization used when culturing or storing bovine embryos, and a method for culturing and storing bovine embryos after in vitro fertilization using the composition.
ウシは通常分娩で雌1頭が残せる子孫の数が生涯で最大10頭程度と限定されている。効率よく優良な子牛を生産するために、優良な雌牛に対して排卵誘発剤により排卵を誘発し、優良な雄牛の精子を用いて体内人工授精を行った後、子宮から胚を採取し保存して、他の雌牛の卵管又は子宮に胚を移植するという体内人工授精法が広く用いられており、現在世界で新鮮胚から27万頭、凍結保存胚から28万頭の胚移植が実施されている(非特許文献1)。 Cattle are usually limited to a maximum of about 10 offspring in their lifetimes that can be left by one female for parturition. In order to produce a good calf efficiently, ovulation is induced to a good cow with an ovulation inducer, and after in vivo artificial insemination using a good bull's sperm, an embryo is collected from the uterus. An in-vivo artificial insemination method that preserves and transplants embryos into the fallopian tubes or uterus of other cows is widely used. Currently, 270,000 embryos from fresh embryos and 280,000 embryos from cryopreserved embryos are transplanted worldwide. It has been implemented (Non-patent Document 1).
さらに効率よく優良な子牛を生産するために、食肉市場で肉牛の卵巣から未成熟卵子を採取し、培養液中、in vitroにおいて卵子と精子を受精させ胚を発生させた後、ウシ胚を保存し、乳牛の卵管又は子宮に胚を移植する体外受精法が広く行われている。体外受精法は通常は廃棄される卵巣10個から約100個の未成熟卵子が採取され、約10頭の子ウシが生産できるため、体内人工授精より量産化に適した方法として知られ、日本では年間約2000頭の子ウシが誕生している(非特許文献1)。また、超音波診断装置を用いて生きたウシから採取した卵子を用いた体外受精も実用化段階に入っている(非特許文献1)。 In order to produce better calves more efficiently, immature eggs are collected from the ovary of beef cattle in the meat market, fertilized with eggs and sperm in vitro in a culture medium, and then embryos are developed. In vitro fertilization methods that preserve and transplant embryos into the fallopian tubes or uterus of dairy cows are widely used. In vitro fertilization is generally known as a more suitable method for mass production than in-vivo artificial insemination because about 100 immature eggs are collected from 10 ovaries that are usually discarded and about 10 calves can be produced. Then, about 2000 calves are born each year (Non-patent Document 1). Furthermore, in vitro fertilization using an egg collected from a live cow using an ultrasonic diagnostic apparatus has entered the practical application stage (Non-patent Document 1).
体外受精において胚の培養液での培養及び保存は生産効率に大きく影響を与えることから、体外受精用の培地として23種類のアミノ酸からなる組成物(特許文献1)、メタロプロテナーゼインヒビターを含む培地(特許文献2)、カゼインホスホペプチドを含む培地(特許文献3)、シクロヘキシミドを含有する培地(特許文献4)、グルタミンを実質的に含まない培地(特許文献5)が知られているが、胚の培養、保存時の熱ストレス耐性を付与する培地は知られていない。 In vitro fertilization, the culture and storage of the embryo in the culture medium greatly affects the production efficiency. Therefore, a composition comprising 23 kinds of amino acids as a medium for in vitro fertilization (Patent Document 1), a medium containing a metalloproteinase inhibitor (Patent Document 2), a medium containing casein phosphopeptide (Patent Document 3), a medium containing cycloheximide (Patent Document 4), and a medium substantially free of glutamine (Patent Document 5) are known. There is no known medium that imparts heat stress tolerance during culture and storage.
キサントフィルは、カロテノイドのうち炭素、水素、酸素から構成されるものをいう。アスタキサンチンは、エビ、カニなどに見られる赤色色素であり、アスタキサンチンを含むキサントフィル類は、抗酸化作用をはじめとする優れた生理活性を持つことが明らかになっている。また、家畜の精液生産効果があることから飼料に用いられているが(特許文献6、特許文献7)、受精した胚に関する影響は知られておらず、培地添加物としても知られていない。 Xanthophyll refers to a carotenoid composed of carbon, hydrogen, and oxygen. Astaxanthin is a red pigment found in shrimps, crabs and the like, and it has been clarified that xanthophylls containing astaxanthin have excellent physiological activity including antioxidation. Moreover, although it is used for feed since it has an effect of producing semen from livestock (Patent Document 6, Patent Document 7), the effect on fertilized embryos is not known, and it is not known as a medium additive.
高温による直接的な熱ストレスがウシ初期胚の発生を阻害し、人工受精における胚発生率や受胎率を下げることが問題とされていた。このため、ウシ胚の培養工程、保存工程において熱ストレスの影響を受けにくい新規培地組成物の開発が望まれている。 The direct heat stress caused by high temperature inhibits the development of bovine early embryos, and it has been a problem to reduce the embryo development rate and conception rate in artificial fertilization. For this reason, the development of a novel medium composition that is not easily affected by heat stress in the culture process and preservation process of bovine embryos is desired.
発明者らは鋭意検討することにより、ウシ胚の培養工程において、培養液にキサントフィルを添加することにより、熱ストレスに強く、受胎率の向上に役立つ培地が提供されることを発見した。 The inventors have intensively studied and found that, in the bovine embryo culturing process, by adding xanthophyll to the culture solution, a medium that is resistant to heat stress and useful in improving the conception rate is provided.
本発明は、(1)キサントフィルを含有することを特徴とする人工受精用培地組成物、(2)キサントフィルがアスタキサンチンであることを特徴とする前記(1)記載の培地組成物、(3)人工受精が体外受精であることを特徴とする前記(1)又は(2)記載の培地組成物、並びに(4)人工受精後のウシ胚の培養に用いることを特徴とする前記(1)〜(3)のいずれか1に記載の培地組成物に関する。 The present invention includes (1) a medium composition for artificial fertilization characterized by containing xanthophyll, (2) the medium composition according to (1) above, wherein xanthophyll is astaxanthin, and (3) artificial (1) to (1), wherein the fertilization is in vitro fertilization, and the medium composition according to (1) or (2) above, and (4) the bovine embryo cultured after artificial fertilization The medium composition according to any one of 3).
本発明はまた、(5)人工受精後の胚の培養において、キサントフィルを含有する培地を用いることを特徴とする胚の培養方法、(6)キサントフィルがアスタキサンチンであることを特徴とする前記(5)記載の胚の培養方法、(7)人工受精が体外受精であることを特徴とする前記(5)又は(6)記載の胚の培養方法、並びに(8)胚がウシ初期胚であることを特徴とする前記(5)〜(7)のいずれか1に記載の胚の培養方法に関する。 The present invention also provides (5) a method for culturing an embryo characterized by using a medium containing xanthophyll in the culture of an embryo after artificial fertilization, (6) the xanthophyll is astaxanthin (5) (7) The embryo culture method according to (5) or (6) above, wherein the artificial fertilization is in vitro fertilization, and (8) The embryo is an early bovine embryo The embryo culturing method according to any one of (5) to (7) above,
本発明の培地組成物は、ウシ胚の培養及び保存工程における培養液にキサントフィルを添加することにより、胚が熱ストレスの影響を受けにくくなる効果を有する。したがって、このようなキサントフィルを含有する本発明の培地組成物は、人工受精における受胎率及び胚発生率の向上に有利な効果を奏する。 The medium composition of the present invention has an effect that an embryo becomes less susceptible to thermal stress by adding xanthophyll to a culture solution in the culture and storage process of bovine embryos. Therefore, the medium composition of the present invention containing such xanthophyll has an advantageous effect on improving the conception rate and embryo development rate in artificial fertilization.
本発明において、キサントフィルとしては、ルテイン、ゼアキサンチン、カンタキサンチン、アスタキサンチン等が挙げられ、とりわけアスタキサンチンを用いることが好ましい。アスタキサンチンとしては、化学合成により得られたアスタキサンチン、オキアミ等の水産物から抽出したアスタキサンチン、藻類から抽出したアスタキサンチン、ファフィアロドチーマ等の酵母から抽出したアスタキサンチン等が挙げられる。培地組成物におけるキサントフィルの濃度は、2.5ppm〜1000ppm、好ましくは2.5ppm〜100ppm、とりわけ好ましくは25ppm〜100ppmである。 In the present invention, xanthophylls include lutein, zeaxanthin, canthaxanthin, astaxanthin and the like, and it is particularly preferable to use astaxanthin. Examples of astaxanthin include astaxanthin obtained by chemical synthesis, astaxanthin extracted from marine products such as krill, astaxanthin extracted from algae, astaxanthin extracted from yeasts such as phafiarodozyma, and the like. The concentration of xanthophyll in the medium composition is 2.5 ppm to 1000 ppm, preferably 2.5 ppm to 100 ppm, particularly preferably 25 ppm to 100 ppm.
人工受精用培地とは、卵細胞を培養できる培地、体外における卵細胞の受精に用いることができる培地、又は体内人工授精により採取あるいは体外受精により作出した胚を培養できる培地等をいう。具体的には、199培地、BME培地、CMRL1066培地、MEM培地、マッコイ−5A培地、ウェイマウス培地、トロウェルT−8培地、ハム培地、ライボビッツL−15培地、NCTC培地、ウィリアムス−E培地、ケイン・アンド・フット(Kane and Foote)培地、MCDB104培地、ブリンスター(Brinster)培地、m−タイロード培地、BWW培地、ウィッテン(Whitten)培地、TYH培地、ホップス・アンド・ピット(Hoppes & Pitts)培地、m−KRB培地、BO培地、T6培地、HTF培地、GPM培地、合成卵管液(SOF)、Charles Ronsenkrans培地又はこれらの修正培地等が挙げられる。 The artificial fertilization medium refers to a medium that can culture egg cells, a medium that can be used for fertilization of egg cells outside the body, a medium that can culture embryos collected by in vitro artificial insemination or produced by in vitro fertilization, and the like. Specifically, 199 medium, BME medium, CMRL 1066 medium, MEM medium, McCoy-5A medium, Weymouth medium, Trowell T-8 medium, Ham medium, Leibovitz L-15 medium, NCTC medium, Williams-E medium, Kane -Kane and Foote medium, MCDB104 medium, Brinster medium, m-Tyrode medium, BWW medium, Whitten medium, TYH medium, Hopps & Pitts medium M-KRB medium, BO medium, T6 medium, HTF medium, GPM medium, synthetic fallopian tube fluid (SOF), Charles Ronsenkrans medium, or a modified medium thereof.
前記培地には、必要に応じてアミノ酸、糖質、電解質、ビタミン類、微量金属元素、ホルモン、細胞成長因子、脂質又はその構成成分、担体蛋白質、細胞外基質成分(接着因子)、還元物質等を添加してもよい。 In the medium, amino acids, carbohydrates, electrolytes, vitamins, trace metal elements, hormones, cell growth factors, lipids or constituents thereof, carrier proteins, extracellular matrix components (adhesion factors), reducing substances, etc., as necessary May be added.
アミノ酸としては、L−フェニルアラニン、L−トリプトファン、L−リジン、L−スレオニン、L−バリン、L−メチオニン、L−イソロイシン、L−ロイシン、L−プロリン、グリシン、L−アラニン、L−チロシン、L−ヒスチジン、L−アルギニン、タウリン、L−アスパラギン酸、L−セリン、L−グルタミン酸、L−シスチン、L−グルタミン、L−ヒドロキシプロリン、L−アスパラギン等が挙げられる。 As amino acids, L-phenylalanine, L-tryptophan, L-lysine, L-threonine, L-valine, L-methionine, L-isoleucine, L-leucine, L-proline, glycine, L-alanine, L-tyrosine, Examples include L-histidine, L-arginine, taurine, L-aspartic acid, L-serine, L-glutamic acid, L-cystine, L-glutamine, L-hydroxyproline, L-asparagine and the like.
糖質としては、グルコース、マルトース、フルクトース、キシリトール、ソルビトール、トレハロース等が、電解質としては、塩化ナトリウム、酢酸ナトリウム、クエン酸ナトリウム、塩化カリウム、塩化カルシウム、グルコン酸カルシウム、塩化マグネシウム、硫酸マグネシウム、リン酸二水素ナトリウム、リン酸水素二カリウム、炭酸水素ナトリウム、ピルビン酸ナトリウム、乳酸ナトリウム等が挙げられる。 The carbohydrates include glucose, maltose, fructose, xylitol, sorbitol, trehalose, and the electrolytes include sodium chloride, sodium acetate, sodium citrate, potassium chloride, calcium chloride, calcium gluconate, magnesium chloride, magnesium sulfate, phosphorus Examples include sodium dihydrogen acid, dipotassium hydrogen phosphate, sodium hydrogen carbonate, sodium pyruvate, and sodium lactate.
ビタミン類としては、ビタミンA、ビタミンB類、ビタミンC、ビタミンD類、ビタミンE、ニコチン酸、ビオチン、葉酸等が、微量金属元素としては、亜鉛、鉄、マンガン、銅、ヨウ素、セレン、コバルト等が挙げられる。 Vitamins include vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, nicotinic acid, biotin, folic acid, etc., and trace metal elements include zinc, iron, manganese, copper, iodine, selenium, cobalt Etc.
ホルモンとしては、インスリン、ハイドロコーチゾン、デキサメサゾン、トリヨードサイロニン等が、細胞成長因子としては、上皮成長因子、線維芽細胞成長因子、血小板由来成長因子、インスリン様成長因子、成長ホルモン等が挙げられる。 Hormones include insulin, hydrocortisone, dexamethasone, triiodothyronine, and cell growth factors include epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, growth hormone, etc. .
脂質又はその構成成分としては、オレイン酸、リノール酸又はリノレン酸等の必須不飽和脂肪酸、コレステロール、エタノールアミン、コリン等が、担体蛋白質としては、血清アルブミン、トランスフェリン等が、細胞外基質成分(接着因子)としては、フィブロネクチン、コラーゲン、ゼラチン等が挙げられ、還元物質としては、2−メルカプトエタノール(ME)、ジチオトレイトール、還元型グルタチオン等が挙げられる。 Lipids or constituents thereof include essential unsaturated fatty acids such as oleic acid, linoleic acid or linolenic acid, cholesterol, ethanolamine, choline, etc., and carrier proteins include serum albumin, transferrin, etc. Factors include fibronectin, collagen, gelatin, and the like, and examples of the reducing substance include 2-mercaptoethanol (ME), dithiothreitol, reduced glutathione, and the like.
また前記培地には、ペニシリン、ストレプトマイシン、カナマイシン、ゲンタマイシン、エリスロマイシン等の抗生物質、アンホテリシンB、ナイスタチン等の抗かび剤、メタロプロテアーゼインヒビター、シクロホスファミド、カゼインペプチド等の受精促進剤を適宜添加してもよい。 In addition, fertilizers such as antibiotics such as penicillin, streptomycin, kanamycin, gentamicin and erythromycin, antifungal agents such as amphotericin B and nystatin, metalloprotease inhibitors, cyclophosphamide and casein peptides are appropriately added to the medium. May be.
本発明の培地組成物は、人工受精における胚の培養又は保存に用いることができる。人工受精における胚の培養又は保存とは、体外受精においては動物卵細胞の培養、その受精時又は受精後の胚の培養又は培養後の保存をいい、体内人工授精においては、発生した胚の子宮等から採取した後の培養又は保存をいう。 The medium composition of the present invention can be used for embryo culture or preservation in artificial fertilization. Embryo culture or storage in artificial fertilization refers to the culture of animal egg cells in in vitro fertilization, culture of embryos during or after fertilization, or preservation after culture, and in in vivo artificial insemination, the uterus of the generated embryo, etc. It refers to the culture or preservation after being collected from.
胚は動物胚であればヒト、ウシ等どのような動物でもよいが、哺乳動物が好ましく、ウシ、ブタ、ヒツジ等の家畜動物がより好ましく、とりわけウシが好ましい。 The embryo may be any animal such as a human or a cow as long as it is an animal embryo, but is preferably a mammal, more preferably a livestock animal such as a cow, pig or sheep, and particularly preferably a cow.
本発明における、卵細胞の培養及び受精時の胚の培養においては、人工受精が体外受精の場合は、未成熟卵子に対してキサントフィルを含有する本発明の培地を加え、卵細胞の培養を行う。次いで、一般に37〜39℃、好ましくは38.5℃、5%炭酸ガス/95%空気中あるいは5%炭酸ガス/5%酸素/90%窒素中、加湿環境中で、精子を添加し受精を行い、本発明の培地中で約1日間培養する。 In the culture of the egg cell and the embryo at the time of fertilization in the present invention, when the artificial fertilization is in vitro fertilization, the medium of the present invention containing xanthophyll is added to immature eggs to culture the egg cells. Next, in general, 37-39 ° C, preferably 38.5 ° C, 5% carbon dioxide gas / 95% air, or 5% carbon dioxide gas / 5% oxygen / 90% nitrogen, in a humidified environment, fertilize by adding sperm. And culture for about 1 day in the medium of the present invention.
本発明における、人工受精後の胚の培養において、キサントフィルを含有する培地を用いることを特徴とする胚の培養方法は、以下のように行う。人工受精によって得られた受精卵を、裸化すなわち顆粒膜細胞を除去し、この裸化受精卵を本培地中で培養する。2〜3日毎に培地を置換する。上記の培養は通常37℃〜39℃、好ましくは38.5℃にて、5%炭酸ガス/5%酸素/90%窒素中、加湿環境で行なわれる。通常7〜8日間の培養の後、受精卵は胚盤胞に達する。 In the culture of the embryo after artificial fertilization in the present invention, the embryo culture method characterized by using a medium containing xanthophyll is performed as follows. A fertilized egg obtained by artificial fertilization is naked, that is, granulosa cells are removed, and the naked fertilized egg is cultured in this medium. Replace the medium every 2-3 days. The above culture is usually performed at 37 ° C. to 39 ° C., preferably 38.5 ° C., in a humidified environment in 5% carbon dioxide gas / 5% oxygen / 90% nitrogen. Usually after 7-8 days of culture, the fertilized egg reaches the blastocyst.
発生した胚は、液体窒素中で保存することができる。体内人工授精においては子宮から採取した胚を、本発明の培地に加え、液体窒素中で保存することができる。 The developed embryo can be stored in liquid nitrogen. In in vivo artificial insemination, embryos collected from the uterus can be added to the medium of the present invention and stored in liquid nitrogen.
キサントフィル、胚等の定義は前記と同義である。 The definitions of xanthophyll, embryo, etc. are as defined above.
本明細書における、「受精」とは、雌雄の生殖細胞が接合する生物学的な現象をいい、「授精」とは、受精を人為的に起こさせることをいう。また、本明細書における、「人工授精」とは、人為的に精液を生殖器に注入する、授精のための技術をいい、「体外受精」とは、体外で精子と卵子を融合する、授精のための技術をいい、「人工受精」とは、人工授精及び体外受精を包含する授精のための技術をいう。 In this specification, “fertilization” refers to a biological phenomenon in which male and female germ cells are joined, and “insemination” refers to artificially causing fertilization. Also, in this specification, “artificial insemination” refers to a technique for insemination that artificially injects semen into the genital organs, and “in vitro fertilization” refers to insemination that fuses sperm and eggs outside the body. The term “artificial insemination” refers to a technique for insemination including artificial insemination and in vitro fertilization.
次に、実施例により本発明をさらに具体的に説明する。 Next, the present invention will be described more specifically with reference to examples.
TCM199培地の組成(Kurihara T, Kurome M, Wako N, Ochiai T, Mizuno K, Fujimura T, Takahagi Y, Murakami H, Kano K, Miyagawa S, Shirakura R, Nagashima H. Developmental competence of in vitro matured porcine oocytes after electrical activation. J Reprod Dev. 2002; 48: 271-279)を参考にして、以下の組成の培地(組成物1)を製造する:
CaCl2(無水) 200.00mg/L、Fe(NO3)3・9H2O 0.72mg/L、KCl 400.00mg/L、MgSO4(無水) 97.67mg/L、NaCl 6800.00mg/L、NaH2PO4・H2O 140.00mg/L、アデノシン硫酸 10.00mg/L、ATP(2Na塩) 1.00mg/L、アデニル酸 0.20mg/L、コレステロール 0.20mg/L、デオキシリボース 0.50mg/L、D−グルコース 1000.00mg/L、グルタチオン(GSH) 0.05mg/L、グアニン・HCl 0.30mg/L、ヒポキサンチン(Na塩) 0.354mg/L、フェノールレッド 20.00mg/L、リボース 0.50mg/L、酢酸ナトリウム 50.00mg/L、チミン 0.30mg/L、Tween80(登録商標) 20.00mg/L、ウラシル 0.30mg/L、キサンチン(Na塩) 0.344mg/L、DL−アラニン 50.00mg/L、L−アルギニン・HCl 70.00mg/L、DL−アスパラギン酸 60.00mg/L、L−システイン・HCl・H2O 0.11mg/L、L−シスチン・2HCl 26.00mg/L、DL−グルタミン酸・H2O 150.00mg/L、L−グルタミン 100.00mg/L、グリシン 50.00mg/L、L−ヒスチジン・HCl・H2O 21.88mg/L、L−ヒドロキシプロリン 10.00mg/L、DL−イソロイシン 40.00mg/L、DL−ロイシン 120.00mg/L、L−リジン・HCl 70.00mg/L、DL−メチオニン 30.00mg/L、DL−フェニルアラニン 50.00mg/L、L−プロリン 40.00mg/L、DL−セリン 50.00mg/L、DL−トレオニン 60.00mg/L、DL−トリプトファン 20.00mg/L、L−チロシン(2Na塩) 57.88mg/L、DL−バリン 50.00mg/L、アスコルビン酸 0.05mg/L、α−トコフェロールホスフェート(2Na塩) 0.01mg/L、d−ビオチン 0.01mg/L、カルシフェロール 0.10mg/L、p−パントテン酸カルシウム 0.01mg/L、塩化コリン 0.50mg/L、葉酸 0.01mg/L、i−イノシトール 0.05mg/L、メナジオン 0.01mg/L、ナイアシン 0.025mg/L、ナイアシンアミド 0.025mg/L、p−アミノ安息香酸 0.05mg/L、ピリドキサール・HCl 0.025mg/L、ピリドキシン・HCl 0.025mg/L、リボフラビン 0.01mg/L、チアミン・HCl 0.01mg/L、ビタミンA(アセテート) 0.14mg/L、アスタキサンチン(富士化学工業製) 25mg/L。
Composition of TCM199 (Kurihara T, Kurome M, Wako N, Ochiai T, Mizuno K, Fujimura T, Takahagi Y, Murakami H, Kano K, Miyagawa S, Shirakura R, Nagashima H. Developmental competence of in vitro matured porcine oocytes after With reference to electrical activation. J Reprod Dev. 2002; 48: 271-279), a medium with the following composition (Composition 1) is produced:
CaCl 2 (anhydrous) 200.00mg / L, Fe (NO 3) 3 · 9H 2 O 0.72mg / L, KCl 400.00mg / L, MgSO 4 ( anhydrous) 97.67mg / L, NaCl 6800.00mg / L, NaH 2 PO 4 .H 2 O 140.00 mg / L, adenosine sulfate 10.00 mg / L, ATP (2Na salt) 1.00 mg / L, adenylic acid 0.20 mg / L, cholesterol 0.20 mg / L, Deoxyribose 0.50 mg / L, D-glucose 1000.00 mg / L, Glutathione (GSH) 0.05 mg / L, Guanine / HCl 0.30 mg / L, Hypoxanthine (Na salt) 0.354 mg / L, Phenol red 20.00 mg / L, ribose 0.50 mg / L, sodium acetate 50.00 mg / L, chi 0.30 mg / L, Tween 80 (registered trademark) 20.00 mg / L, uracil 0.30 mg / L, xanthine (Na salt) 0.344 mg / L, DL-alanine 50.00 mg / L, L-arginine · HCl 70.00 mg / L, DL-aspartic acid 60.00 mg / L, L-cysteine · HCl · H 2 O 0.11 mg / L, L-cystine · 2HCl 26.00 mg / L, DL-glutamic acid · H 2 O 150 .00 mg / L, L-glutamine 100.00 mg / L, glycine 50.00 mg / L, L-histidine · HCl · H 2 O 21.88 mg / L, L-hydroxyproline 10.00 mg / L, DL-isoleucine 40 .00 mg / L, DL-leucine 120.00 mg / L, L-lysine · HCl 70.00 mg / DL-methionine 30.00 mg / L, DL-phenylalanine 50.00 mg / L, L-proline 40.00 mg / L, DL-serine 50.00 mg / L, DL-threonine 60.00 mg / L, DL-tryptophan 20 0.000 mg / L, L-tyrosine (2Na salt) 57.88 mg / L, DL-valine 50.00 mg / L, Ascorbic acid 0.05 mg / L, α-tocopherol phosphate (2Na salt) 0.01 mg / L, d -Biotin 0.01 mg / L, Calciferol 0.10 mg / L, Calcium p-pantothenate 0.01 mg / L, Choline chloride 0.50 mg / L, Folic acid 0.01 mg / L, i-Inositol 0.05 mg / L , Menadione 0.01 mg / L, niacin 0.025 mg / L, niacinamide 0.025 mg / L, p-aminobenzoic acid 0.05 mg / L, pyridoxal · HCl 0.025 mg / L, pyridoxine · HCl 0.025 mg / L, riboflavin 0.01 mg / L, thiamine · HCl 0.01 mg / L Vitamin A (acetate) 0.14 mg / L, Astaxanthin (Fuji Chemical Industries) 25 mg / L.
前記組成物1においてアスタキサンチンの濃度を50mg/Lに代える以外は同一の組成をした培地(組成物2)を製造する。 A medium (composition 2) having the same composition is produced except that the concentration of astaxanthin in composition 1 is changed to 50 mg / L.
前記組成物1においてアスタキサンチンの濃度を100mg/Lに代える以外は同一の組成をした培地(組成物3)を製造する。 A medium (composition 3) having the same composition except that the concentration of astaxanthin in the composition 1 is changed to 100 mg / L is produced.
前記組成物1に20%NBS(新生子牛血清)を加えた培地(組成物4)を製造する。 A medium (composition 4) in which 20% NBS (newborn calf serum) is added to the composition 1 is produced.
前記組成物4に0.1mM β−MEを加える以外は同様の方法により培地(組成物5)を製造する。 A medium (composition 5) is produced in the same manner except that 0.1 mM β-ME is added to the composition 4.
基礎培地の作製:修正合成卵管液(Takahashi Y, First NL. In vitro development of bovine one-cell embryos: influence of glucose, lactate, pyruvate, amino acids and vitamins. Theriogenology 1992; 37: 963-978)の組成を参考にして、以下の組成の培地(基礎培地)を作製した:
NaCl 6294.00mg/L、KCl 534.00mg/L、KH2PO4 162.00mg/L、MgCl2(6水和物) 99.60mg/L、CaCl2(2水和物) 251.40mg/L、NaHCO3 2106.00mg/L、L−アルギニン・HCl 21.00mg/L、L−シスチン 12.00mg/L、L−ヒスチジン 8.00mg/L、L−イソロイシン 26.00mg/L、L−ロイシン 26.00mg/L、L−リジン・HCl 36.98mg/L、L−メチオニン 7.50mg/L、L−フェニルアラニン 16.50mg/L、L−トレオニン 24.00mg/L、L−トリプトファン 4.00mg/L、L−チロシン 18.00mg/L、L−バリン 23.50mg/L、L−グルタミン 146.00mg/L、L−アラニン 8.90mg/L、L−アスパラギン(1水和物) 15.00mg/L、L−アスパラギン酸 13.30mg/L、L−グルタミン酸 14.70mg/L、グリシン 7.50mg/L、L−プロリン 11.50mg/L、L−セリン 10.50mg/L、ビルビン酸ナトリウム 55.00mg/L、乳酸ナトリウム 370.00mg/L、ウシ血清アルブミン 3000.00mg/L、ペニシリン100000.00単位/L、ストレプトマイシン 100mg/L、アンホテリシンB 0.25mg/L。
Preparation of basal medium: modified synthetic oviduct fluid (Takahashi Y, First NL. In vitro development of bovine one-cell embryos: influence of glucose, lactate, pyruvate, amino acids and vitamins. Theriogenology 1992; 37: 963-978) With reference to the composition, a medium (basic medium) having the following composition was prepared:
NaCl 6294.00 mg / L, KCl 534.00 mg / L, KH 2 PO 4 162.00 mg / L, MgCl 2 (hexahydrate) 99.60 mg / L, CaCl 2 (dihydrate) 251.40 mg / L, NaHCO 3 2106.00 mg / L, L-arginine · HCl 21.00 mg / L, L-cystine 12.00 mg / L, L-histidine 8.00 mg / L, L-isoleucine 26.00 mg / L, L- 3. Leucine 26.00 mg / L, L-lysine · HCl 36.98 mg / L, L-methionine 7.50 mg / L, L-phenylalanine 16.50 mg / L, L-threonine 24.00 mg / L, L-tryptophan 00 mg / L, L-tyrosine 18.00 mg / L, L-valine 23.50 mg / L, L-glutamine 1 6.00 mg / L, L-alanine 8.90 mg / L, L-asparagine (monohydrate) 15.00 mg / L, L-aspartic acid 13.30 mg / L, L-glutamic acid 14.70 mg / L, glycine 7.50 mg / L, L-proline 11.50 mg / L, L-serine 10.50 mg / L, sodium bibrate 55.00 mg / L, sodium lactate 370.00 mg / L, bovine serum albumin 3000.00 mg / L, Penicillin 10,0000.00 units / L, streptomycin 100 mg / L, amphotericin B 0.25 mg / L.
未成熟卵子の回収と成熟培養:食肉市場において解体されたウシからウシ卵巣を回収し、生理食塩水の入ったデュワー瓶に移し研究室まで輸送した。解体後6時間以内にウシ卵巣表面の直径2〜8mmの卵胞から21G注射針を付けた10mlシリンジを用いて卵胞内容物を吸引し、その内容物から顆粒膜細胞付着卵子を回収した。基礎培地においてウシ血清アルブミンを添加せず、D−グルコース 1000.00mg/L、卵胞刺激ホルモン 200IU/L及びウシ胎仔血清 10%(V/V)を添加する以外は同一の組成をした培地(卵子成熟用培地)を作製し、その培地で、60×15mmペトリディッシュ上に50μlのドロップを作製し、流動パラフィンを被せた。回収した顆粒膜細胞付着卵子を1ドロップあたり10個ずつ移し、38.5℃、飽和湿度、5%炭酸ガス、5%酸素、90%窒素のインキュベーター内で22時間培養した。 Recovery of immature eggs and mature culture: Bovine ovaries were recovered from cattle dismantled in the meat market, transferred to a Dewar bottle containing physiological saline, and transported to the laboratory. Within 6 hours after disassembly, the follicle contents were aspirated from a follicle having a diameter of 2 to 8 mm on the surface of bovine ovary using a 10 ml syringe with a 21G injection needle, and granulosa cell-attached ova were recovered from the contents. Medium with the same composition (egg) except that bovine serum albumin is not added to the basal medium but D-glucose 1000.00 mg / L, follicle stimulating hormone 200 IU / L and fetal bovine serum 10% (V / V) are added. A maturation medium) was prepared, and 50 μl of a drop was prepared on a 60 × 15 mm Petri dish and covered with liquid paraffin. Ten collected granule membrane cell-attached eggs were transferred per drop and cultured in an incubator of 38.5 ° C., saturated humidity, 5% carbon dioxide, 5% oxygen, 90% nitrogen for 22 hours.
成熟卵子の体外受精:基礎培地においてへパリン 20mg/Lを添加する以外は同一の組成をした培地を作製し60×15mmペトリディッシュ上に45μlのドロップを作製し流動パラフィンを被せ、卵子待機用ドロップとした。黒毛和種の凍結精液(0.5ml)を、37℃の温水中で融解させ、15ml Conical Tube内に調製した90%パーコール 2ml、45%パーコール 2mlの重層の上に乗せた。25℃、700g、30分の遠心分離を行い、上清を吸引除去した。残った精子の沈殿を基礎培地10mlに懸濁し、10分の遠心分離後、上清を吸引除去した。これを洗浄精子とし、血球計算板で精子濃度を計測し、精子濃度2×106sperm/mlになるように基礎培地で希釈した。成熟培養後の顆粒膜細胞付着卵子を卵子待機用ドロップに1ドロップあたり10個ずつ移し、精子希釈液を50μlずつ顆粒膜細胞付着卵子の入った卵子待機用ドロップに投入し、38.5℃、飽和湿度、5%炭酸ガス、5%酸素、90%窒素のインキュベーター内で20時間培養した。 In vitro fertilization of mature eggs: Prepare a medium with the same composition except that heparin 20 mg / L is added to the basal medium, make a 45 μl drop on a 60 × 15 mm Petri dish, cover it with liquid paraffin, drop the egg for waiting It was. Japanese black frozen semen (0.5 ml) was thawed in 37 ° C. warm water and placed on a 2 ml of 90% percoll and 2 ml of 45% percoll prepared in a 15 ml conical tube. Centrifugation was performed at 25 ° C. and 700 g for 30 minutes, and the supernatant was removed by suction. The remaining sperm precipitate was suspended in 10 ml of basal medium, centrifuged for 10 minutes, and the supernatant was removed by aspiration. This was used as a washed sperm, the sperm concentration was measured with a hemocytometer, and diluted with a basal medium so that the sperm concentration was 2 × 10 6 sperm / ml. Transfer 10 granule cell-attached eggs after maturation culture to the drop for waiting for ovum, and put 50 μl of each sperm diluted solution into drop for waiting for ovum containing granule membrane cell-attached ovum, 38.5 ° C., The cells were cultured for 20 hours in an incubator of saturated humidity, 5% carbon dioxide, 5% oxygen, and 90% nitrogen.
体外受精胚の培養:基礎培地においてD−グルコース 270.00mg/L、ジメチルスルホキシド 0.025%(v/v)を添加する以外は同一の組成をした培地(組成物6)を作製した。前記組成物6においてアスタキサンチン(マリンケミカル研究所)2.5ppmを添加する以外は同一の組成をした培地(組成物7)を作製した。前記組成物7においてアスタキサンチンの濃度を25ppmに代える以外は同一の組成をした培地(組成物8)を作製した。体外受精開始後20時間に顆粒膜細胞付着卵子から顆粒膜細胞を除去し、1細胞期胚を得た。得られた1細胞期胚を通常処理においては38.5℃の温度条件下で培養し、暑熱処理においては培養開始後0〜10時間及び24〜34時間の2回、40.5℃に温度設定したインキュベーターで培養した。いずれのインキュベーターも飽和湿度、5%炭酸ガス、5%酸素、90%窒素に設定した。それぞれの温度条件下における培地は、組成物6、組成物7あるいは組成物8を4ウェルプレートに1ウェルあたり500μlで入れたものとした。すなわち通常処理かつアスタキサンチン無添加(通常−無添加区)、通常処理かつアスタキサンチン2.5ppmあるいは25ppm添加(通常−Ax2.5区あるいは通常−Ax25区)、暑熱処理かつアスタキサンチン無添加(暑熱−無添加区)、暑熱処理かつアスタキサンチン2.5ppmあるいは25ppm添加(暑熱−Ax2.5区あるいは暑熱−Ax25区)の6区に分けて培養した。体外受精の開始日を0日目とし、3日目に基礎培地においてD−グルコース 270.00mg/Lを添加する以外は同一の組成をした培地に交換した。さらに5日目に基礎培地においてD−グルコース 270.00mg/L、β−ME 0.1mM及びウシ胎仔血清 10%(v/v)を添加する以外は同一の組成をした培地に交換した。3日目以後の培地は、60×15mmペトリディッシュ上に50μlのドロップを作製し、流動パラフィンを被せた状態で用いた。3日目における分割率と5−8細胞期への発生率及び8日目における胚盤胞発生率を計測した。分割率及び発生率は1元配置の分散分析後Sidak法を用いて多重比較を行った。その結果、通常−無添加区と比較して、暑熱−無添加区では分割率、5−8細胞期及び胚盤胞への発生率が有意に低下した。一方、暑熱−Ax2.5区あるいは暑熱−Ax25区では、アスタキサンチンの濃度依存的に胚発生率が回復し、暑熱−Ax25区においては、通常−無添加区との間に有意差が見られなかった。以上の結果より、生理的な暑熱条件によってウシ初期胚発生が直接阻害されることが示され、その発生阻害をアスタキサンチンが緩解しうることが示された(表1)。 Culture of in vitro fertilized embryo: A medium (Composition 6) having the same composition was prepared except that 270.00 mg / L of D-glucose and 0.025% (v / v) of dimethyl sulfoxide were added to the basal medium. A medium (composition 7) having the same composition was prepared except that 2.5 ppm of astaxanthin (Marine Chemical Laboratory) was added to the composition 6. A medium (composition 8) having the same composition was prepared except that the concentration of astaxanthin in composition 7 was changed to 25 ppm. At 20 hours after the start of in vitro fertilization, granulosa cells were removed from the granulosa cell-attached ovum to obtain a 1-cell stage embryo. The obtained 1-cell stage embryo is cultured at a temperature of 38.5 ° C. in the normal treatment, and in the heat treatment, the temperature is increased to 40.5 ° C. twice for 0-10 hours and 24-34 hours after the start of the culture The cells were cultured in a set incubator. All incubators were set to saturation humidity, 5% carbon dioxide, 5% oxygen, and 90% nitrogen. As the medium under each temperature condition, Composition 6, Composition 7, or Composition 8 was put in a 4-well plate at 500 μl per well. That is, normal treatment and no astaxanthin added (usually-no addition), normal treatment and astaxanthin added 2.5 ppm or 25 ppm (usually -Ax2.5 or normal -Ax25), heat treatment and no astaxanthin added (hot-no addition) )), Heat treatment, and astaxanthin 2.5 ppm or 25 ppm added (hot heat-Ax 2.5 ward or hot heat-Ax 25 ward). The start date of in vitro fertilization was defined as day 0, and the medium was replaced with a medium having the same composition except that 270.00 mg / L of D-glucose was added to the basal medium on the third day. Furthermore, on the fifth day, the medium was replaced with a medium having the same composition except that D-glucose 270.00 mg / L, β-ME 0.1 mM and fetal calf serum 10% (v / v) were added in the basal medium. The medium after the third day was used in a state where 50 μl drops were prepared on 60 × 15 mm Petri dishes and covered with liquid paraffin. The division rate on the third day, the incidence to the 5-8 cell stage, and the blastocyst incidence on the eighth day were measured. The division rate and the incidence rate were subjected to multiple comparisons using the Sidak method after a one-way analysis of variance. As a result, compared with the normal-no additive group, the division rate, the 5-8 cell stage, and the incidence of blastocysts were significantly reduced in the hot-additive group. On the other hand, in the heat-Ax2.5 or the heat-Ax25, the embryo development rate recovered depending on the concentration of astaxanthin. In the heat-Ax25, no significant difference was observed between the normal-no-addition group. It was. From the above results, it was shown that the development of bovine early embryos was directly inhibited by physiological heat conditions, and it was shown that astaxanthin can relieve the inhibition of the development (Table 1).
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