JP5904369B2 - Sperm preservation solution, sperm preservation method and artificial insemination method - Google Patents
Sperm preservation solution, sperm preservation method and artificial insemination method Download PDFInfo
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Description
本発明は、ブタの人工授精に用いる精子保存液、精子保存方法及び人工授精方法に関する。 The present invention relates to a sperm preservation solution, a sperm preservation method, and an artificial insemination method used for porcine artificial insemination.
日本において、ブタの年間生産頭数は１，６００万頭程度であり、その出荷額は５，０００億円程度と畜産全体の約２１％を占めている。産子数を増やし、かつ、母豚の空胎期間を縮小させることが養豚業の生産性向上にとって重要となる。しかし、いまだに自然交配による種付けが生産者当たりで約７０％、生産頭数あたりでも凡そ３０％を占めている。このため、種雄ブタの飼育に伴うコスト高、肉質改善などの育種改良の停滞などの問題があり、繁殖技術の向上が望まれている。 In Japan, the annual production of pigs is about 16 million, and the shipment value is about 500 billion yen, accounting for about 21% of the total livestock. Increasing the number of pups and reducing the emptying period of mother pigs are important for improving the productivity of the pig farming industry. However, seeding by natural mating still accounts for about 70% per producer and about 30% per production. For this reason, there are problems such as high costs associated with breeding of breeding boars and stagnation of breeding improvement such as meat quality improvement, and improvement of breeding technology is desired.
このような状況下、近年では、液状精液を用いた人工授精の普及が図られている。各県の試験場や民間の種畜場では、人工授精用の液状精液を販売している。 Under such circumstances, in recent years, artificial insemination using liquid semen has been popularized. In each prefecture's test site and private breeding ground, liquid semen for artificial insemination is sold.
液状精液に用いられる保存液として、例えば、特許文献１に開示されている保存液等があり、採精された精液を保存液に添加して得られた人工精液が養豚業者へ運ばれ、人工授精に利用されている。 As a preservation solution used for liquid semen, for example, there is a preservation solution disclosed in Patent Document 1, and artificial semen obtained by adding the collected semen to the preservation solution is carried to a pig farmer, Used for insemination.
特許文献１の保存液を利用した場合でも、良好な精液では５日以上の保存（利用）が可能な場合もあるが、３日以内に著しく運動性が低下する個体も多く認められている。また、液状精液の輸送中の劣化も問題となっていることから、安定的に精液を液状保存できる保存液の開発が望まれている。 Even when the preservation solution of Patent Document 1 is used, a good semen may be able to be preserved (used) for 5 days or more, but there are many individuals whose motility is remarkably reduced within 3 days. In addition, since deterioration during transport of liquid semen is also a problem, development of a preservation solution that can stably store semen in a liquid state is desired.
本発明は上記事項に鑑みてなされたものであり、その目的は、長期間ブタ精子を液状で保存しても、精子の運動性及び受精能を維持させ得る精子保存液、精子保存方法及び人工授精方法を提供することにある。 The present invention has been made in view of the above-mentioned matters, and the object thereof is to provide a sperm preservation solution, a sperm preservation method, and an artificial product that can maintain sperm motility and fertility even when porcine sperm is preserved in a liquid state for a long period of time. To provide an insemination method.
本発明の第１の態様に係る精子保存液は、
ブタ精子を１０〜２５℃の液状で保存する精子保存液において、
嫌気的解糖系の非基質となる糖を含有し、
前記糖がラクトースであり、
グルコースを更に含有し、
前記ラクトースと前記グルコースとの配合割合がモル比で８：２〜５：５である、
ことを特徴とする。
A sperm preservation solution according to the first aspect of the present invention comprises:
In the sperm preservation solution which preserve | saves a pig sperm in the liquid of 10-25 degreeC ,
Contains a sugar as a non-substrate of anaerobic glycolysis,
The sugar is lactose;
Further containing glucose,
The blending ratio of the lactose and glucose is 8: 2 to 5: 5 in molar ratio.
It is characterized by that.
また、乳酸及び／又はグリシンを含有することが好ましい。 Moreover, it is preferable to contain lactic acid and / or glycine.
また、前記乳酸を４．５ｍＭ〜１０ｍＭ含有することが好ましい。 The lactic acid is preferably contained in an amount of 4.5 mM to 10 mM.
また、前記グリシンを０．５ｍＭ以上含有することが好ましい。 Moreover, it is preferable to contain the said glycine 0.5 mM or more.
本発明の第２の態様に係る精子保存方法は、
本発明の第１の態様に係る精子保存液とブタ精子とを混合し、１０℃〜２５℃で保存する、
ことを特徴とする。
The sperm preservation method according to the second aspect of the present invention comprises:
The sperm preservation solution according to the first aspect of the present invention and porcine sperm are mixed and stored at 10 ° C to 25 ° C.
It is characterized by that.
また、前記ブタ精子を１×１０７〜１×１０９ｓｐｅｒｍ／ｍｌの割合で混合することが好ましい。 Moreover, it is preferable to mix the said porcine sperm in the ratio of 1 * 10 < 7 > -1 * 10 < 9 > sperm / ml.
また、ブタ精液から精漿を除去した前記ブタ精子を用いてもよい。 Moreover, you may use the said pig sperm from which the seminal plasma was removed from the pig semen.
本発明の第３の態様に係る人工授精方法は、
本発明の第１の態様に係る精子保存液とブタ精子とを混合して調製した人工精液をブタの子宮内に注入して人工授精を行う、
ことを特徴とする。
The artificial insemination method according to the third aspect of the present invention comprises:
Artificial insemination is performed by injecting artificial semen prepared by mixing the sperm preservation solution and pig sperm according to the first aspect of the present invention into the uterus of the pig,
It is characterized by that.
また、ブタ精液から精漿を除去した前記ブタ精子を用いてもよい。 Moreover, you may use the said pig sperm from which the seminal plasma was removed from the pig semen.
本発明に係るブタ精子保存液では、嫌気的解糖系の非基質となる糖を含有しており、ブタ精子の酸素呼吸が亢進され、長期間保存後においても精子の運動性及び受精能を維持させることができる。 The porcine sperm preservation solution according to the present invention contains a sugar that is a non-substrate of anaerobic glycolysis, and oxygen respiration of porcine sperm is enhanced, and motility and fertility of sperm are maintained even after long-term storage. Can be maintained.
実施の形態１に係る精子保存液は、ブタ精子を液状で保存する精子保存液であって、嫌気的解糖系の非基質となる糖を含有する。 The sperm preservation solution according to Embodiment 1 is a sperm preservation solution that preserves porcine sperm in a liquid state, and contains a sugar that is a non-substrate of an anaerobic glycolysis system.
嫌気的解糖系の非基質となる糖とは、嫌気条件ではピルビン酸などの有機酸に分解されず、エネルギー産生の栄養源にならない糖をいう。嫌気的解糖系の非基質となる糖として、例えば、ラクトース、ガラクトース、スクロース等が挙げられる。なかでも、後述の実施例及びコストの面から、ラクトースであることが好ましい。 The anaerobic glycolytic non-substrate sugar is a sugar that is not decomposed into an organic acid such as pyruvic acid under anaerobic conditions and does not serve as a nutrient source for energy production. Examples of sugars that are non-substrates for anaerobic glycolysis include lactose, galactose, sucrose, and the like. Of these, lactose is preferable from the viewpoints of Examples and costs described later.
一般的な保存液では、グルコースが添加されている。グルコースは精子の栄養源として機能し、細胞質における嫌気的解糖系で分解されて、一過的にＡＴＰ生産を促進させてしまう。そして、その後、ＡＴＰの生産量が減少に転じてしまい、精子の運動性が低下していく。したがって、長期保存後ではブタ精子の運動性、受精能を担保できない。 In a common preservation solution, glucose is added. Glucose functions as a nutrient source for sperm and is degraded by an anaerobic glycolysis system in the cytoplasm, which temporarily promotes ATP production. After that, the production amount of ATP starts to decrease, and the sperm motility decreases. Therefore, after long-term storage, the motility and fertility of porcine sperm cannot be guaranteed.
一方で、グルコースを除去すると浸透圧が低下してしまうことから、本実施の形態に係る精子保存液では、浸透圧調整のために添加される糖類として、嫌気的解糖系の非基質であるラクトース等の糖を含有している。嫌気的解糖系におけるＡＴＰ生産から、ミトコンドリアにおける酸素呼吸によるＡＴＰ生産へと転換される。これにより、酸素濃度依存的に、長期間に渡り一定量のＡＴＰが継続的に産生される。したがって、ブタ精子の運動性及び受精能が長期間に渡って担保される。 On the other hand, since osmotic pressure decreases when glucose is removed, the sperm preservation solution according to the present embodiment is an anaerobic glycolytic non-substrate as a saccharide added for osmotic pressure adjustment. Contains sugars such as lactose. ATP production in an anaerobic glycolysis system is converted to ATP production by oxygen respiration in mitochondria. Thereby, a certain amount of ATP is continuously produced over a long period of time depending on the oxygen concentration. Therefore, the motility and fertility of porcine sperm are ensured for a long time.
また、精子保存液はグルコースを含有することが好ましく、嫌気的解糖系の非基質となる糖とグルコースとの配合割合がモル比で９：１〜５：５であることが好ましく、９：１〜７：３であることがより好ましい。後述の実施例より、グルコースを全く含有していないよりも、一部含有している方が、長期間保存後の精子の運動性が高まる。 Moreover, it is preferable that a sperm preservation solution contains glucose, and it is preferable that the mixture ratio of the saccharide | sugar which becomes a non-substrate of anaerobic glycolysis type | system | group and glucose is 9: 1-5: 5 by molar ratio, 9: It is more preferable that it is 1-7: 3. From the examples described later, the sperm motility after long-term storage is enhanced when a part of the sperm is contained rather than no glucose.
更に、精子保存液には、精子の代謝物質として乳酸及び／又はグリシンが含有されていることが好ましい。後述の実施例から、良好精漿と不良精漿では、含まれる基礎代謝物質が異なっており、良好なブタ精漿には乳酸、グリシンが多く含有されている。乳酸及び／又はグリシンを含有することで、精子の酸素呼吸によるＡＴＰ生産が促進される。 Furthermore, the sperm preservation solution preferably contains lactic acid and / or glycine as a sperm metabolite. From the examples described later, good seminal plasma and bad seminal plasma contain different basal metabolites, and good porcine seminal plasma contains a large amount of lactic acid and glycine. By containing lactic acid and / or glycine, ATP production by oxygen respiration of sperm is promoted.
精子保存液中の乳酸の含有量は、４．５ｍＭ〜１０ｍＭであることが好ましく、より好ましくは、５ｍＭ〜１０ｍＭである。また、グリシンの含有量は、０．５ｍＭ以上であることが好ましく、より好ましくは、０．５ｍＭ〜１．０ｍＭである。 The content of lactic acid in the sperm preservation solution is preferably 4.5 mM to 10 mM, and more preferably 5 mM to 10 mM. Moreover, it is preferable that content of glycine is 0.5 mM or more, More preferably, it is 0.5 mM-1.0 mM.
精子保存液は、嫌気的解糖系の非基質となる糖と乳酸及び／又はグリシン、更にはグルコースを、他の成分（例えば、クエン酸ナトリウム、炭酸水素ナトリウム、ＥＤＴＡ−２Ｎａ、クエン酸、トリス（ヒドロキシル）アミノメタンなど）と混合することで調製され得る。 The sperm preservation solution contains sugar, lactic acid and / or glycine, which is a non-substrate of anaerobic glycolysis, and glucose, and other components (for example, sodium citrate, sodium bicarbonate, EDTA-2Na, citric acid, tris (Hydroxyl) aminomethane and the like).
また、精子保存液は、公知の保存液を利用して調製することもできる。例えば、公知の保存液に、嫌気的解糖系の非基質となる糖及び乳酸及び／又はグリシンを添加することで調整され得る。用いられる公知の保存液として、この分野で通常用いられる液体であれば特に限定されないが、例えば、モデナ液（０．１５Ｍグルコース、２６．７ｍＭクエン酸ナトリウム、１１．９ｍＭ炭酸水素ナトリウム、１５．１ｍＭクエン酸、６．３ｍＭ ＥＤＴＡ−２Ｎａ、４６．６ｍＭトリス及び１，０００ＩＵ／ｍｌペニシリン）が挙げられる。この場合、モデナ液中のグルコースを除去し、嫌気的解糖系の非基質となる糖、乳酸及び／又はグリシンを所定の割合で添加して混合すればよい。 Moreover, a sperm preservation solution can also be prepared using a well-known preservation solution. For example, it can be adjusted by adding sugar and lactic acid and / or glycine, which are non-substrates of anaerobic glycolysis, to a known preservation solution. The known preservation solution to be used is not particularly limited as long as it is a liquid usually used in this field. For example, modena solution (0.15 M glucose, 26.7 mM sodium citrate, 11.9 mM sodium hydrogen carbonate, 15.1 mM) Citric acid, 6.3 mM EDTA-2Na, 46.6 mM Tris and 1,000 IU / ml penicillin). In this case, glucose in the modena solution may be removed, and sugar, lactic acid and / or glycine, which are non-anaerobic glycolytic non-substrates, may be added and mixed in a predetermined ratio.
実施の形態２に係る精子保存方法では、採取したブタ精液と上述した精子保存液とを混合し、人工精液として保存する。例えば、精子保存液を３０〜３８℃の温度にしておき、これと採取した精液とを混合する。人工精液中の精子の終濃度が、１×１０７ｓｐｅｒｍ／ｍｌ〜１×１０９ｓｐｅｒｍ／ｍｌになるように調製するとよい。そして、調製した人工精液を１０℃〜２５℃、より好ましくは１３℃〜１８℃で保存する。採取した精液を遠心分離等で精漿を除去した後、精子保存液にて上記濃度に希釈してもよい。 In the sperm storage method according to Embodiment 2, the collected pig semen and the sperm storage solution described above are mixed and stored as artificial semen. For example, the sperm preservation solution is kept at a temperature of 30 to 38 ° C., and this is mixed with the collected semen. The final concentration of sperm in the artificial semen may be adjusted to 1 × 10 7 sperm / ml to 1 × 10 9 sperm / ml. And the prepared artificial semen is preserve | saved at 10 to 25 degreeC, More preferably at 13 to 18 degreeC. After removing the seminal plasma from the collected semen by centrifugation or the like, it may be diluted to the above concentration with a sperm preservation solution.
実施の形態３に係るブタの人工授精方法では、上述の精子保存液が用いられる。上述したように調製され、保存された人工精液を用いて雌ブタに人工授精を行う。３０〜３８℃にした人工精液を、発情期を迎えた雌ブタの子宮内に注入することで人工授精を行い得る。その際、子宮頸管注入用のカテーテルを用いて、約５０ｍｌ（約５×１０９ｓｐｅｒｍ／５０ｍｌ）の精液を注入すればよい（子宮頸管内精液注入法）。或いは、子宮角内へ注入できる深部注入カテーテルを用いて子宮角内へ人工精液を注入してもよい（子宮角内精液注入法）。子宮角内精液注入法の場合、逆流漏出や子宮内多核白血球の色作用等による精子の損失が抑えられるので、精子濃度を５×１０９ｓｐｅｒｍ／５０ｍｌ以下に減少させて用いても構わない。なお、雌ブタでは、人工授精後、凡そ１１４日齢で分娩に至る。分娩後、子ブタが２０〜４０日齢のほ乳期間を経て離乳し、離乳して４日後程で雌ブタに発情が再来する。雌ブタの再発情に合わせて、上記と同様に人工授精を行うとよい。このようなサイクルで人工授精を行うことで、一頭の雌ブタの一生涯に、より多くの子ブタを出産させることができる。 In the method for artificial insemination of pigs according to Embodiment 3, the above-described sperm preservation solution is used. Artificial insemination is performed on sows using artificial semen prepared and stored as described above. Artificial insemination can be performed by injecting artificial semen at 30 to 38 ° C. into the uterus of a sow that has reached estrus. At that time, about 50 ml (about 5 × 10 9 sperm / 50 ml) of semen may be injected using a catheter for cervical injection (intracervical semen injection method). Alternatively, artificial semen may be injected into the uterine horn using a deep injection catheter that can be injected into the uterine horn (intrauterine semen injection method). For uterine horns in semen injection method, since the loss of sperm is suppressed due to the color effect such reverse flow leakage or intrauterine polymorphonuclear cells, it may be used to reduce the sperm concentration below 5 × 10 9 sperm / 50ml. In the female pig, after artificial insemination, the baby is delivered at about 114 days of age. After parturition, the piglets wean after 20-40 days of lactation, and estrus reappears in the sows 4 days after weaning. It is advisable to perform artificial insemination according to the recurrence of the sows in the same manner as described above. By performing artificial insemination in such a cycle, more piglets can be born in the lifetime of one sow.
上述したように、精子保存液は、ブタ精子の運動性及び受精能を長期間に渡って維持し得るので、長期保存後でも、高い受胎率をなしえる。 As described above, the sperm preservation solution can maintain the motility and fertility of porcine sperm over a long period of time, so that a high conception rate can be achieved even after long-term storage.
なお、採精したブタ精液をそのまま精子保存液と混合しても、精液を遠心分離等によって精漿と精子とを分離し、精子のみを精子保存液と混合して人工精液を調製してもよい。 Even if the collected porcine semen is directly mixed with the sperm preservation solution, the seminal plasma and sperm are separated by centrifugation or the like, and only the sperm is mixed with the sperm preservation solution to prepare an artificial semen. Good.
以下、実施例に基づき、精子保存液及びこれを用いた人工授精方法について詳細に説明する。以下の実験に用いたブタ精液等について記す。 Hereinafter, based on an Example, a sperm preservation solution and the artificial insemination method using this are demonstrated in detail. The porcine semen used for the following experiment is described.
（ブタ精液）
ブタの精液は、以下のようにして採精して用いた。採精に使用する雄ブタを、それぞれ個々の豚房で別々に飼育し、朝、夕の２回、計２．５ｋｇの種ブタ用飼料を給餌した。ブタには、日本脳炎・ブタパルボウイルス感染症ワクチンの接種を行った。本検討には、ブタ繁殖・呼吸障害症候群（ＰＲＲＳ）、オーエスキー病に対して抗体陰性のブタを選択した。採精は、１週間間隔をあけて行った。採精前は、餌食い、病気の兆候等を確認し、良好なブタであることを確認し、ブタを興奮させないように採精を行った。疑牝台を雄ブタの豚房に入れ、雄ブタを台にのせ、ペニス及び包皮内を生理食塩水で洗浄し、尿を除去した。採精は手圧法で行い、予め滅菌したカップにガーゼをかぶせ、精液と共に出るゼリー状の物質である膠様物を除去しながら、精液をカップに入れた。このように採精した精液を以下の実験に用いた。
また、ブタ精漿は、上記のようにして採精した後、遠心分離によって精漿を精液から分離して用いた。
（精子の運動性及び直進運動速度の測定）
以下における精子運動性及び直進運動速度は、３８℃のプレートの上に、精子を含有するサンプル（精液、精子を精漿で培養したもの、精液を各種精子保存液或いは培地で保存・培養したもの）を５μｌ載せ、運動性解析装置（Ｃｏｍｐｕｔｅｒ Ａｉｄｅｄ Ｓｐｅｒｍ Ａｎａｌｙｓｉｓ（ＣＡＳＡ）ｓｙｓｔｅｍ）を用いて解析した。なお、精子の運動性は、観測される全精子中における動いている精子の割合が１００分率で解析され、直進運動速度は、直進する精子の速度の平均が解析される。
(Pig semen)
Porcine semen was collected and used as follows. Boars used for semen collection were individually raised in individual pig buns and fed with 2.5 kg of seed pig feed twice in the morning and evening. Pigs were vaccinated with Japanese encephalitis / porcine parvovirus infection vaccine. For this study, pigs that were antibody-negative for porcine reproductive and respiratory disorder syndrome (PRRS) and Aujeszky's disease were selected. The semen collection was performed at an interval of one week. Prior to collection, prey was confirmed, signs of illness, etc. were confirmed, the pig was confirmed to be a good pig, and the pig was collected so as not to excite the pig. The suspected female stand was placed in a boar's pig chamber, the male pig was placed on the stand, the penis and the foreskin were washed with physiological saline, and urine was removed. The semen was collected by a manual pressure method, and gauze was put on a previously sterilized cup, and the semen was put into the cup while removing the glue-like substances that are jelly-like substances that come out with the semen. The semen collected in this way was used for the following experiments.
Porcine seminal plasma was collected as described above and then used after separating seminal plasma from semen by centrifugation.
(Measurement of sperm motility and linear motion speed)
The following sperm motility and linear movement speed are the samples containing sperm (semen, sperm cultured in seminal plasma, semen stored and cultured in various sperm preservation solutions or media on a 38 ° C plate. ) Was placed, and analyzed using a motility analyzer (Computer Aided Spam Analysis (CASA) system). The motility of the sperm is analyzed at a rate of 100 minutes of the moving sperm in all the sperm observed, and the average speed of the sperm moving straight is analyzed as the straight movement speed.
（実験１：ブタ精漿が精子の運動性に及ぼす影響）
まず、精子の運動性に及ぼす精漿成分の影響を検討した。精漿に曝されていないマウス精巣上体精子を取り出した。ＨＴＦ培地（株式会社アイエスジャパン）に、６個体のブタ（識別ＩＤ：Ｗ３２７，Ｌ２４１，Ｄ７１７，Ｄ６２１，Ｌ２８４，Ｌ２９１）の精漿をそれぞれ加えて調製した１０％ブタ精漿添加培地で、マウス精子を培養した。そして、経時的にマウス精子の運動性を測定した。
(Experiment 1: Effect of porcine seminal plasma on sperm motility)
First, the effect of seminal plasma components on sperm motility was examined. Mouse epididymal spermatozoa that were not exposed to seminal plasma were removed. Mouse sperm in a medium containing 10% porcine seminal plasma prepared by adding the seminal plasma of 6 pigs (identification ID: W327, L241, D717, D621, L284, L291) to HTF medium (IS Japan Co., Ltd.) Was cultured. Then, the motility of mouse sperm was measured over time.
その結果を図１に示す。ブタ個体の違いにより、精漿添加によりマウス精子の運動性が低下する場合（Ｗ３２７，Ｌ２４１，Ｄ７１７）、高い運動性が維持される場合（Ｄ６２１，Ｌ２８４，Ｌ２９１）の両者が認められた。即ち、ブタ精漿は、個体により精子の運動性を維持することができる良好な精漿と、運動性を抑制或いは向上させる成分を有さない不良な精漿に分けられることがわかった。 The result is shown in FIG. Due to differences in pigs, both mouse sperm motility was reduced by addition of seminal plasma (W327, L241, D717) and high motility was maintained (D621, L284, L291). That is, it has been found that porcine seminal plasma can be divided into good seminal plasma that can maintain sperm motility by individuals and bad seminal plasma that does not have a component that suppresses or improves motility.
（実験２：ブタ精漿の分子量画分が精子の運動性に及ぼす影響）
ブタ精漿中に含まれる精子の運動性を抑制する因子或いは運動性を向上させる因子について明かにすべく、以下の実験を行った。
(Experiment 2: Effect of molecular weight fraction of porcine seminal plasma on sperm motility)
The following experiment was conducted to clarify the factors that suppress or improve the motility of sperm contained in pig seminal plasma.
限界濾過膜を用いた遠心分離にて、精漿を分子量１０万以上、１−１０万、５０００−１万、５０００以下に分子量分画した。いずれの画分についてもＨＴＦ培地に１０％量となるよう添加し、１０％ブタ精漿添加培地を調製した。そして、それぞれの１０％ブタ精漿添加培地でマウス精巣上体精子を培養した。そして、１時間培養後の精子運動性を測定した。 The seminal plasma was fractionated to a molecular weight of 100,000 or more, 1 to 100,000, 5,000 to 10,000, or 5,000 or less by centrifugation using an ultrafiltration membrane. All the fractions were added to the HTF medium so that the amount was 10% to prepare a medium supplemented with 10% porcine seminal plasma. Then, mouse epididymis spermatozoa were cultured in each medium supplemented with 10% porcine seminal plasma. And the sperm motility after 1-hour culture | cultivation was measured.
その結果を図２に示す。分子量５０００−１万、５０００以下の分画において、精子の運動性が低下している。したがって、分子量１万以下の分画に１時間培養後の精子の運動性を低下させる因子が含まれていることがわかった。 The result is shown in FIG. In fractions having a molecular weight of 5,000 to 10,000 or less, the sperm motility is reduced. Therefore, it was found that the fraction having a molecular weight of 10,000 or less contains a factor that decreases the motility of sperm after 1 hour of culture.
（実験３：ブタ精漿が精子の代謝能（ＡＴＰ生産）に及ぼす影響）
ブタ精漿の分子量１万以下の低分子画分が、マウス精子の運動性を低下させることが明らかになったことから、精漿に含まれる代謝基質が精子の運動性に影響を及ぼしていると考えられる。そこで、精子の運動性に直接的に関わるＡＴＰ生産量に及ぼす影響を検討した。
(Experiment 3: Effects of porcine seminal plasma on sperm metabolic capacity (ATP production))
It has been shown that the low molecular weight fraction of porcine seminal plasma with a molecular weight of 10,000 or less reduces the motility of mouse spermatozoa, so the metabolic substrate contained in seminal plasma affects sperm motility. it is conceivable that. Therefore, the effect on ATP production directly related to sperm motility was examined.
マウス精巣上体精子を１０％精漿添加ＴＨＦ培地で１５分、３０分、６０分培養して回収した精子を蛍光発光法にてＡＴＰ含有量の測定を行った。 Sperm of mouse epididymal sperm were cultured for 15 minutes, 30 minutes, and 60 minutes in 10% seminal plasma-added THF medium, and the sperm collected was measured for fluorescence by the ATP content.
その結果を図３に示す。１５分培養後において、不良精漿（Ｗ３２７，Ｌ２４１，Ｄ７１７）において、１５分培養後にＡＴＰ含有量が増加し、６０分後には低下していた。 The result is shown in FIG. After culturing for 15 minutes, ATP content increased after culturing for 15 minutes in poor seminal plasma (W327, L241, D717) and decreased after 60 minutes.
一方、良好精漿（Ｄ６２１，Ｌ２８４，Ｌ２９１）では、１５分、３０分、６０分の培養では、精子のＡＴＰ含有量に大きな変動は見られなかった。 On the other hand, in the fine seminal plasma (D621, L284, L291), the ATP content of sperm did not change greatly in the culture for 15 minutes, 30 minutes and 60 minutes.
すなわち、不良精漿では一過的にＡＴＰ合成が盛んに行われ、精子の運動性が向上するものの、それが持続せず、１時間後には運動性が低下してしまうことが示された。この結果から、長時間に渡って精子の運動性を維持するためには、精子の代謝量を一定に維持させる必要があることがわかった。 That is, it was shown that in poor seminal plasma, ATP synthesis was actively performed transiently, and motility of sperm was improved, but this did not persist, and motility decreased after 1 hour. From these results, it has been found that in order to maintain sperm motility over a long period of time, it is necessary to keep the sperm metabolism constant.
（実験４：ブタ精漿のメタボローム解析（代謝基質の網羅的解析））
精漿を短期間で精子の運動性が低下する不良精漿、中庸な中庸精漿、長期間維持する良好精漿に区分し、それぞれの精漿中に含まれる基質成分を、キャピラリー電気泳動質量分析（ＣＥ−ＭＡＳ）によりメタボローム解析を行った。なお、不良精漿、中庸精漿、良好精漿の区分は、実験１にて、２時間培養後の精子の運動性が３０％以下に低下した精漿を不良精漿（Ｄ７１７）、精子の運動性が３０〜６０％であった精漿を中庸精漿（Ｗ３２７，Ｌ２４１）、精子の運動性が６０％以上であった精漿を良好精漿（Ｄ６２１，Ｌ２８４，Ｌ２９１）と区分して行った。また、測定は、Agilent CE-TOFMS system（Agilent Technologies 社） Capillary : Fused silica capillary i.d. 50 μm × 80 cmを用い、以下の条件１又は２で行った。
＜条件１＞
Run buffer : Cation Buffer Solution (p/n : H3301-1001)
Rinse buffer : Cation Buffer Solution (p/n : H3301-1001)
Sample injection : Pressure injection 50 mbar, 10 sec
CE voltage : Positive, 27 kV
MS ionization : ESI Positive
MS capillary voltage : 4,000 V
MS scan range : m/z 50-1,000
Sheath liquid : HMT Sheath Liquid (p/n : H3301-1020)
＜条件２＞
Run buffer : Anion Buffer Solution (p/n : H3302-1021)
Rinse buffer : Anion Buffer Solution (p/n : H3302-1022)
Sample injection : Pressure injection 50 mbar, 25 sec
CE voltage : Positive, 30 kV
MS ionization : ESI Negative
MS capillary voltage : 3,500 V
MS scan range : m/z 50-1,000
Sheath liquid : HMT Sheath Liquid (p/n : H3301-1020)
(Experiment 4: Metabolomic analysis of porcine seminal plasma (comprehensive analysis of metabolic substrates))
The seminal plasma is divided into poor seminal sperm that decreases sperm motility in a short period of time, moderate intermediate seminal seminal plasma, and good seminal seminal plasma that is maintained for a long time. Metabolomic analysis was performed by analysis (CE-MAS). In addition, in the experiment 1, the seminal plasma in which the sperm motility after 2 hours of culturing was reduced to 30% or less was determined as the poor seminal (D717) The seminal plasma with motility of 30-60% is classified as the intermediate seminal plasma (W327, L241) and the seminal plasma with sperm motility of 60% or more is classified as good seminal plasma (D621, L284, L291). went. The measurement was performed under the following conditions 1 or 2 using an Agilent CE-TOFMS system (Agilent Technologies) Capillary: Fused silica capillary id 50 μm × 80 cm.
<Condition 1>
Run buffer: Cation Buffer Solution (p / n: H3301-1001)
Rinse buffer: Cation Buffer Solution (p / n: H3301-1001)
Sample injection: Pressure injection 50 mbar, 10 sec
CE voltage: Positive, 27 kV
MS ionization: ESI Positive
MS capillary voltage: 4,000 V
MS scan range: m / z 50-1,000
Sheath liquid: HMT Sheath Liquid (p / n: H3301-1020)
<Condition 2>
Run buffer: Anion Buffer Solution (p / n: H3302-1021)
Rinse buffer: Anion Buffer Solution (p / n: H3302-1022)
Sample injection: Pressure injection 50 mbar, 25 sec
CE voltage: Positive, 30 kV
MS ionization: ESI Negative
MS capillary voltage: 3,500 V
MS scan range: m / z 50-1,000
Sheath liquid: HMT Sheath Liquid (p / n: H3301-1020)
その結果を図４に示す。本結果から、良好精漿と不良精漿に含まれる代謝物質が大きく異なっていることがわかる。精漿に含まれる代謝物質の相違から、良好精漿では精子のＴＣＡサイクルが活性化されることが示唆された。そして、本解析からは、良好精漿にはアセチルＣｏＡに変換され得る乳酸、グリシンが高値を示しており、一方、ピルビン酸等の嫌気的解糖系に関わる因子が低値を示していた。なお、良好精漿では、乳酸が５．３０４ｍＭ、グリシンが０．９７ｍＭであり、不良精漿では、乳酸が２．６０１ｍＭ、グリシンが０．５２４ｍＭであった。なお、乳酸については、別途、乳酸測定キット（日東紡績株式会社製）を用いて測定した。 The result is shown in FIG. This result shows that the metabolites contained in good seminal plasma and bad seminal plasma are greatly different. The difference in metabolites contained in seminal plasma suggests that good seminal plasma activates the TCA cycle of sperm. In this analysis, lactic acid and glycine that can be converted to acetyl CoA showed high values in good seminal plasma, while factors related to anaerobic glycolysis such as pyruvate showed low values. In good seminal plasma, lactic acid was 5.304 mM and glycine was 0.97 mM, and in bad seminal plasma, lactic acid was 2.601 mM and glycine was 0.524 mM. In addition, about lactic acid, it measured separately using the lactic acid measuring kit (made by Nittobo Co., Ltd.).
（実験５：ブタ精漿中の乳酸含有量と精子運動性との関係の検証）
ブタ精液中のグルコース及び乳酸のそれぞれの濃度を測定するとともに、その精液を１時間培養した後の精子運動性を測定した。
(Experiment 5: Verification of the relationship between lactic acid content in sperm of pig and sperm motility)
Each concentration of glucose and lactic acid in pig semen was measured, and sperm motility after culturing the semen for 1 hour was measured.
乳酸濃度と精子運動性との関係を図５に示す。乳酸の含有量はサンプル間で大きく異なり、精子の運動性と正の相関関係が認められた（ｒ＝０．５２８６，ｐ＝０．０００８）。図５に示した理論値からは、乳酸濃度が凡そ４．５ｍＭ以上で精子の運動性が７０％程度を越えており、凡そ５ｍＭ以上で精子の運動性が８０％を越えている。このことから、精子保存液中の乳酸濃度は４．５ｍＭ以上であることが好ましく、５ｍＭ以上であることがより好ましいと考えられる。また、図５では、乳酸濃度が高いほど精子運動性は高い傾向にあるが、後述の実験６で乳酸濃度が１５ｍＭの場合に精子運動性が低下していること、及び、図５から乳酸濃度が１０ｍＭを超える精漿の個体が見受けられなかったことから、乳酸濃度は１０ｍＭ以下であることが好ましいと考えられる。なお、グルコースの含有量はいずれの精漿中でも検出限界以下で低濃度であり有意な差はなかった。 The relationship between the lactic acid concentration and sperm motility is shown in FIG. The lactic acid content was greatly different between samples, and a positive correlation with sperm motility was observed (r = 0.5286, p = 0.0008). From the theoretical values shown in FIG. 5, the sperm motility exceeds about 70% when the lactic acid concentration is about 4.5 mM or more, and the sperm motility exceeds 80% at about 5 mM. For this reason, the lactic acid concentration in the sperm preservation solution is preferably 4.5 mM or more, and more preferably 5 mM or more. Further, in FIG. 5, the higher the lactic acid concentration, the higher the sperm motility. However, in Experiment 6 described later, the sperm motility decreases when the lactic acid concentration is 15 mM, and from FIG. However, it was considered preferable that the concentration of lactic acid was 10 mM or less. It should be noted that the glucose content was low below the detection limit in any seminal plasma, and there was no significant difference.
（実験６：乳酸、グリシンを含有する精子保存液が精子運動性に及ぼす影響）
そこで、良好な精漿に多量に含まれていた代謝基質である乳酸、グリシンをモデナ液に添加して精子保存液を調製し、この精子保存液がブタ精子の運動性に及ぼす影響を検証した。
(Experiment 6: Effect of sperm preservation solution containing lactic acid and glycine on sperm motility)
Therefore, we prepared a sperm preservation solution by adding lactic acid and glycine, which are metabolic substrates contained in a large amount in good seminal plasma, to the Modena solution, and verified the effect of this sperm preservation solution on the motility of porcine sperm. .
モデナ液に乳酸を添加し、乳酸含有量が５ｍＭ、１５ｍＭの精子保存液をそれぞれ調製した。また、モデナ液にグリシン含有量が０．５ｍＭ、１．０ｍＭの精子保存液をそれぞれ調製した。 Lactic acid was added to the modena solution to prepare sperm preservation solutions having lactic acid contents of 5 mM and 15 mM, respectively. In addition, sperm preservation solutions having a glycine content of 0.5 mM and 1.0 mM were prepared in the modena solution, respectively.
それぞれの精子保存液でブタ精液を１×１０８ｓｐｅｒｍ／ｍｌに希釈して、１５℃で保存した。また、参照実験として、モデナ液をそのまま用い、モデナ液にブタ精液を加えて同様に培養した。 Porcine semen was diluted to 1 × 10 8 sperm / ml with each sperm storage solution and stored at 15 ° C. As a reference experiment, the modena solution was used as it was, and pig semen was added to the modena solution and cultured in the same manner.
保存開始から三日後に、３７℃に加温後の精子運動性の測定を行った。その結果を図６に示す。 Three days after the start of storage, sperm motility after heating to 37 ° C. was measured. The result is shown in FIG.
保存３日後では、乳酸５ｍＭ添加区、グリシン０．５ｍＭ添加区及びグリシン１．０ｍＭ添加区では、乳酸、グリシンのいずれも添加されていない無添加区（Ｃｏｎｔｒｏｌ）に比べて、精子運動性は有意に高い値を示した。精子保存液がグリシン、乳酸を含有していることによって、ブタ精子は長期間運動性を維持することが証明された。 After 3 days of storage, the sperm motility was significant in the lactic acid 5 mM addition group, glycine 0.5 mM addition group, and glycine 1.0 mM addition group compared to the non-addition group (Control) to which neither lactic acid nor glycine was added. Showed a high value. It has been proved that porcine spermatozoa maintain motility for a long period of time because the sperm preservation solution contains glycine and lactic acid.
（実験７：精子保存液中の溶存酸素量に及ぼす影響）
更に、保存後の精子保存液中の溶存酸素量を測定することで、乳酸、グリシンを含有する精子保存液において、精子の嫌気的解糖系が抑制され酸素呼吸が促進されることを検証した。
(Experiment 7: Effect on dissolved oxygen in sperm preservation solution)
Furthermore, by measuring the amount of dissolved oxygen in the sperm preservation solution after storage, it was verified that sperm anaerobic glycolysis was suppressed and oxygen respiration was promoted in sperm preservation solution containing lactic acid and glycine. .
モデナ液に乳酸及びグリシンを加え、乳酸５ｍＭ、グリシン１ｍＭの精子保存液をそれぞれ調製した。 Lactic acid and glycine were added to the modena solution to prepare sperm preservation solutions of 5 mM lactic acid and 1 mM glycine, respectively.
それぞれの精子保存液にブタ精液を加えて１×１０８ｓｐｅｒｍ／ｍｌに希釈し、３７℃で培養した。また、参照実験として、モデナ液をそのまま用い、モデナ液にブタ精液を加えて同様に培養した。また、良好なブタ精液をそのまま培養した。 Porcine semen was added to each sperm stock solution, diluted to 1 × 10 8 sperm / ml, and cultured at 37 ° C. As a reference experiment, the modena solution was used as it was, and pig semen was added to the modena solution and cultured in the same manner. In addition, good pig semen was cultured as it was.
そして、３０分培養後、それぞれの精子保存液中の酸素分圧を測定し、精子保存液中に溶存している酸素濃度を算出した。 And after culture | cultivating for 30 minutes, the oxygen partial pressure in each sperm preservation solution was measured, and the oxygen concentration dissolved in the sperm preservation solution was computed.
その結果を図７に示す。モデナ液に比べて、乳酸５ｍＭ添加区及びグリシン１ｍＭ添加区では、良好な精液と同様、精子保存液中の酸素濃度が有意に減少していた。即ち、乳酸５ｍＭ添加区及びグリシン１ｍＭ添加区では、精子による酸素消費量（呼吸量）が有意に増加していることがわかった。 The result is shown in FIG. Compared with the modena solution, the oxygen concentration in the sperm preservation solution was significantly decreased in the lactic acid 5 mM addition group and the glycine 1 mM addition group as well as the good semen. That is, it was found that oxygen consumption (respiration volume) by sperm increased significantly in the 5 mM lactic acid addition group and glycine 1 mM addition group.
精子保存液に乳酸、グリシンが添加されていることにより、酸素呼吸が活発化することが示された。即ち、酸素呼吸によるＡＴＰ生産が促され、精子が持続的に運動可能になっていることが関連付けられた。 It was shown that oxygen respiration was activated by adding lactic acid and glycine to the sperm preservation solution. That is, ATP production by oxygen breathing was promoted, and it was associated that sperm can be continuously exercised.
（実施例１：ラクトースを含有する精子保存液が精子運動性に及ぼす影響）
一般的な精子保存液であるモデナ液では、栄養源として、グルコースが１５０ｍＭ程度添加されている。グルコースは嫌気的解糖系の基質であるため、嫌気的解糖系により一過的なＡＴＰ合成が起こり、長期間に渡った運動性の維持に支障をきたしていると考えられる。
(Example 1: Effect of sperm preservation solution containing lactose on sperm motility)
In the Modena solution, which is a general sperm storage solution, about 150 mM glucose is added as a nutrient source. Since glucose is an anaerobic glycolytic substrate, it is considered that transient an ATP synthesis occurs due to the anaerobic glycolytic system, which hinders the maintenance of motility over a long period of time.
そこで、モデナ液に含まれるグルコースを除去し、グルコース除去に伴う浸透圧低下を嫌気的解糖系の非基質となる糖に置換して調整し、嫌気的解糖を抑制して、精子の運動性が長時間維持されるか否か検証した。 Therefore, the glucose contained in the Modena solution is removed, and the decrease in osmotic pressure associated with glucose removal is adjusted by replacing it with a sugar that is a non-substrate of the anaerobic glycolysis, suppressing anaerobic glycolysis, and sperm movement It was verified whether sex was maintained for a long time.
モデナ液から、グルコースを除去した後、これに、ラクトース及びグルコースを添加し、
ラクトースとグルコースとのモル比が１０：０（１０：０区）、９：１（９：１区）、８：２（８：２区）、７：３（７：３区）、５：５（５：５区）である精子保存液を調製した。
After removing glucose from the modena liquor, lactose and glucose are added thereto,
The molar ratio of lactose and glucose is 10: 0 (10: 0 ward), 9: 1 (9: 1 ward), 8: 2 (8: 2 ward), 7: 3 (7: 3 ward), 5: The sperm preservation solution which is 5 (5: 5 section) was prepared.
また、参照実験として、モデナ液に乳酸及びグリシンを加えた精子保存液（０：１０区）を調製した。 In addition, as a reference experiment, a sperm preservation solution (0:10) in which lactic acid and glycine were added to the Modena solution was prepared.
それぞれの精子保存液の組成は表１に示す通りである。なお、精子保存液のｐＨはいずれも７．０〜７．１、浸透圧はいずれも３２８〜３３７ｍＯｓｍｋｇである。 The composition of each sperm preservation solution is as shown in Table 1. The pH of the sperm preservation solution is 7.0 to 7.1, and the osmotic pressure is 328 to 337 mOsmkg.
ブタ精液をそれぞれの精子保存液で１×１０８ｓｐｅｒｍ／ｍｌに希釈し、１５℃で１０日間保存し、１０日後の運動性を測定した。その結果を図８に示す。 Porcine semen was diluted to 1 × 10 8 sperm / ml with each sperm preservation solution, stored at 15 ° C. for 10 days, and motility after 10 days was measured. The result is shown in FIG.
図８を見ると、ラクトースを含有していない０：１０区の精子保存液に比べて、ラクトースを含有する処理区の精子保存液では、いずれも精子運動性は有意に高い値を示している。また、８：２区、７：３区及び５：５区でより高い精子運動性を示している。 Referring to FIG. 8, sperm motility is significantly higher in the sperm preservation solution in the treatment group containing lactose than in the sperm preservation solution in the 0:10 group that does not contain lactose. . Moreover, higher sperm motility is shown in 8: 2, 7: 3 and 5: 5.
さらに、精子の活力を示す直進運動速度を解析した。その結果を図９に示す。 Furthermore, we analyzed the rectilinear movement speed indicating the vitality of sperm. The result is shown in FIG.
図９を見ると、８：２区が最も高い直進運動速度を示している。直進運動速度が高い精子ほど受精能が高いことから、８：２区の精子保存液が最も長期間精子の受精能を高く維持させ得ることがわかった。 When FIG. 9 is seen, 8: 2 section has shown the highest linear movement speed. Since the spermatozoa with higher linear motion speed have higher fertility, it was found that the sperm preservation solution in the 8: 2 section can maintain the fertility of the sperm for the longest period.
（実施例２：その他の嫌気的解糖系の非基質となる糖を含有する精子保存液が精子運動性に及ぼす影響）
ラクトースの他、嫌気的解糖系の非基質となる糖（ガラクトース、スクロース）についても上記と同様に行った。
(Example 2: Effect of sperm preservation solution containing saccharide as non-substrate of other anaerobic glycolysis system on sperm motility)
In addition to lactose, saccharides (galactose and sucrose), which are non-substrates for anaerobic glycolysis, were performed in the same manner as described above.
モデナ液から、グルコースを除去した後、これに、乳酸及びグリシン並びに種々の嫌気的解糖系の非基質となる糖及びグルコースを添加し、乳酸が５ｍＭ、グリシンが１ｍＭ、嫌気的解糖系の非基質となる糖が１２０ｍＭ、グルコースが３０ｍＭである精子保存液を調製した。嫌気的解糖系の非基質となる糖として、ガラクトース、ラクトース、スクロースを用い、それぞれを添加して得られた精子保存液をガラクトース区、ラクトース区、スクロース区と記す。 After removing glucose from the modena solution, lactic acid and glycine, and sugars and glucose that are non-substrates of various anaerobic glycolysis systems are added thereto, and lactic acid is 5 mM, glycine is 1 mM, anaerobic glycolysis system. A sperm preservation solution having a non-substrate sugar of 120 mM and glucose of 30 mM was prepared. A sperm preservation solution obtained by adding galactose, lactose, and sucrose as sugars that are non-substrates of anaerobic glycolysis is referred to as a galactose group, a lactose group, and a sucrose group.
また、参照実験として、モデナ液に乳酸５ｍＭ及びグリシン１ｍＭを加えた精子保存液（グルコース区）を調製した。 As a reference experiment, a sperm preservation solution (glucose group) was prepared by adding 5 mM lactic acid and 1 mM glycine to the Modena solution.
そして、各精子保存液でブタ精液を１×１０８ｓｐｅｒｍ／ｍｌに希釈し、１０日間１５℃で保存して、１０日後の精子の運動性及び直進運動速度を測定した。精子運動性の測定結果を図１０に、直進運動速度の測定結果を図１１にそれぞれ示す。なお、図１０及び図１１におけるＤａｙ０は、用いたブタ精液を精子保存液で希釈する前の精子運動性及び直進運動速度である。 Then, the swine semen was diluted to 1 × 10 8 sperm / ml with each sperm storage solution, stored at 15 ° C. for 10 days, and the sperm motility and linear motion speed after 10 days were measured. The measurement result of sperm motility is shown in FIG. 10, and the measurement result of straight movement speed is shown in FIG. In addition, Day0 in FIG.10 and FIG.11 is the sperm motility and the linear movement speed before diluting the used pig semen with a sperm preservation solution.
図１０を見ると、グルコース区に比べて、嫌気的解糖系の非基質となる糖を含有するガラクトース区、ラクトース区及びスクロース区では、精子の運動性は有意に高い値を示した。 Referring to FIG. 10, the sperm motility was significantly higher in the galactose group, lactose group, and sucrose group containing sugars that are non-substrates for anaerobic glycolysis than in the glucose group.
また、図１１を見ると、精子の直進運動速度についても、グルコース区に比べて、嫌気的解糖系の非基質となる糖を含有するガラクトース区、ラクトース区及びスクロース区では高い値を示した。グルコースから嫌気的解糖系の非基質となる糖に置換することで、ブタ精子を長期間高い活力で保存可能であることが明らかとなった。なお、精子の直進運動速度では、ラクトース区が最も高い値を示しており、嫌気的解糖系の非基質となる糖のなかでラクトースであることが最も好ましいことがわかった。 Moreover, when FIG. 11 is seen, also about the straight movement speed of the sperm, compared with the glucose group, it showed a high value in the galactose group, the lactose group, and the sucrose group containing sugars which are non-substrates of anaerobic glycolysis. . It was revealed that porcine spermatozoa can be preserved with high vitality for a long period of time by substituting glucose as a non-substrate for anaerobic glycolysis. In addition, it was found that the lactose group showed the highest value in the straight movement speed of sperm, and lactose was most preferable among the sugars that are non-substrates of anaerobic glycolysis.
（実施例３：精子のミトコンドリア代謝能）
続いて、ミトコンドリアの代謝機能（酸素呼吸能）が亢進されているかを確認した。
(Example 3: Mitochondrial metabolic capacity of sperm)
Subsequently, it was confirmed whether the mitochondrial metabolic function (oxygen respiration) was enhanced.
実施例１の８：２区（ラクトース：グルコースのモル比が８：２、乳酸５ｍＭ、グリシン１ｍＭ）の精子保存液でブタ精液を精子保存液で１×１０８ｓｐｅｒｍ／ｍｌに希釈した。そして、これにローダミンを添加し染色した。ローダミンはミトコンドリアの膜電位により蛍光を発する。そして、これを７日間培養した。また、参照実験として、モデナ液をそのまま用い、同様に行った。 Swine semen was diluted to 1 × 10 8 sperm / ml with the sperm preservation solution in the sperm preservation solution of Example 1: 8: 2 section (lactose: glucose molar ratio 8: 2, lactate 5 mM, glycine 1 mM). And rhodamine was added and dye | stained to this. Rhodamine fluoresces due to mitochondrial membrane potential. This was cultured for 7 days. As a reference experiment, the modena solution was used as it was, and the same experiment was performed.
その結果を図１２に示す。モデナ液に比べて、８：２区の精子保存液で保存した精子では、ローダミン蛍光が強い精子の割合が増加し、ミトコンドリアの活性が高まっていることがわかる。したがって、ミトコンドリアの代謝機能が亢進され、精子の長期間保存後でも十分な運動性及び受精能が担保されることが明かになった。 The result is shown in FIG. Compared with the modena solution, it can be seen that in the sperm stored in the 8: 2 sperm storage solution, the ratio of sperm with strong rhodamine fluorescence increased and the activity of mitochondria increased. Therefore, it was revealed that the metabolic function of mitochondria is enhanced and sufficient motility and fertility are ensured even after long-term storage of sperm.
（実施例４：人工精液によるブタの人工授精）
実施例１の８：２区（ラクトース：グルコースのモル比が８：２、乳酸５ｍＭ、グリシン１ｍＭ）の精子保存液でブタ精液を１×１０８ｓｐｅｒｍ／ｍｌに希釈し、液状精液を調製した。液状精液は保存５日以内にブタの人工授精に用いた。
(Example 4: Artificial insemination of pigs with artificial semen)
Porcine semen was diluted to 1 × 10 8 sperm / ml with the sperm preservation solution of Example 1: 8: 2 section (lactose: glucose molar ratio 8: 2, lactate 5 mM, glycine 1 mM) to prepare liquid semen. . Liquid semen was used for artificial insemination of pigs within 5 days of storage.
人工授精は、自然発情の雌ブタを用いて行った。上述のように調製した人工精液５０ｍｌ（総精子数５０億）を１９頭の雌ブタの子宮にそれぞれ注入し、人工授精を行った。 Artificial insemination was performed using naturally estrous sows. Artificial insemination was performed by injecting 50 ml of artificial semen prepared as described above (total number of sperm: 5 billion) into the uterus of 19 sows.
人工授精後、２１日目に超音波妊娠鑑定にて胎子をモニターで確認した。２１日目に行うのは、ブタは２１日周期で発情がくるため、受精後２１日目に妊娠鑑定をする際、受精していない、若しくは受精卵が退行した等があれば、陰部は発情の兆候を示すためである。また、同時に２１日目にノンリターン法にて再発情（リターン）がきているかどうかも確認し、妊娠を決定した。 On the 21st day after artificial insemination, the fetus was confirmed on a monitor by ultrasonic pregnancy test. On the 21st day, since pigs estrus in a 21-day cycle, when performing pregnancy test on the 21st day after fertilization, if there is no fertilization or the fertilized egg has regressed, the genital area will be estrus. It is for showing the sign of. At the same time, on the 21st day, it was confirmed whether or not there was a recurrence (return) by the non-return method, and pregnancy was decided.
その結果、ラクトースを含有する精子保存液で調製した人工精液では、１９頭中１７匹が受胎し、受胎率は８９％であった。モデナ液で調製された人工精液で行われた人工授精の統計では受胎率が７６％であるところ、著しく高い値を示した。 As a result, in artificial semen prepared with a sperm preservation solution containing lactose, 17 of 19 animals were conceived, and the conception rate was 89%. According to the statistics of artificial insemination performed with artificial semen prepared with modena solution, the conception rate was 76%, showing a significantly high value.
このように、嫌気的解糖系の非基質となる糖、乳酸、グリシンを含有する精子保存液では、ブタ精子を長期保存した後でも、ブタ精子の受精能が維持され、受胎率の向上に資することを確認した。 In this way, the sperm preservation solution containing sugar, lactic acid, and glycine, which are non-substrates for anaerobic glycolysis, maintains the fertility of porcine spermatozoa even after long-term preservation of porcine sperm, which improves the conception rate. Confirmed that it would contribute.
以上説明したように、本発明に係る精子保存液では、長期間保存後においてもブタ精子の運動性及び受精能を維持させることができるので、養豚業等におけるブタの人工授精に利用可能である。 As described above, the sperm preservation solution according to the present invention can maintain the motility and fertility of pig sperm even after long-term storage, and thus can be used for artificial insemination of pigs in the pig farming industry and the like. .
Claims (9)
嫌気的解糖系の非基質となる糖を含有し、
前記糖がラクトースであり、
グルコースを更に含有し、
前記ラクトースと前記グルコースとの配合割合がモル比で８：２〜５：５である、
ことを特徴とする精子保存液。 In a sperm preservation solution for storing pig sperm in a liquid state at 10 to 25 ° C. ,
Contains a sugar as a non-substrate of anaerobic glycolysis,
The sugar is lactose;
Further containing glucose,
The blending ratio of the lactose and glucose is 8: 2 to 5: 5 in molar ratio.
A sperm preservation solution characterized by that.
ことを特徴とする請求項１に記載の精子保存液。 Containing lactic acid and / or glycine,
Sperm preservation solution according to claim 1, characterized in that.
ことを特徴とする請求項２に記載の精子保存液。 Containing 4.5 mM to 10 mM of the lactic acid,
The sperm preservation solution of Claim 2 characterized by the above-mentioned.
ことを特徴とする請求項２に記載の精子保存液。 Containing 0.5 mM or more of the glycine,
The sperm preservation solution of Claim 2 characterized by the above-mentioned.
ことを特徴とする精子保存方法。 The sperm preservation solution according to any one of claims 1 to 4 and porcine sperm are mixed and stored at 10 ° C to 25 ° C.
A sperm preservation method characterized by the above.
ことを特徴とする請求項５に記載の精子保存方法。 Mixing the porcine sperm at a rate of 1 × 10 7 to 1 × 10 9 superm / ml,
The sperm preservation | save method of Claim 5 characterized by the above-mentioned.
ことを特徴とする請求項５又は６に記載の精子保存方法。 Using the pig sperm from which seminal plasma has been removed from pig semen,
The sperm preservation | save method of Claim 5 or 6 characterized by the above-mentioned.
ことを特徴とする人工授精方法。 Artificial insemination is performed by injecting artificial semen prepared by mixing the sperm preservation solution according to any one of claims 1 to 4 and pig sperm into the uterus of a pig,
An artificial insemination method characterized by that.
ことを特徴とする請求項８に記載の人工授精方法。 Using the pig sperm from which seminal plasma has been removed from pig semen,
The artificial insemination method according to claim 8 , wherein:
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