JP5804379B2 - Artificial insemination medium, artificial insemination method using the same artificial insemination medium, and pregnancy promoting agent - Google Patents

Artificial insemination medium, artificial insemination method using the same artificial insemination medium, and pregnancy promoting agent Download PDF

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JP5804379B2
JP5804379B2 JP2012026496A JP2012026496A JP5804379B2 JP 5804379 B2 JP5804379 B2 JP 5804379B2 JP 2012026496 A JP2012026496 A JP 2012026496A JP 2012026496 A JP2012026496 A JP 2012026496A JP 5804379 B2 JP5804379 B2 JP 5804379B2
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田中　宏光
宏光 田中
正山　征洋
征洋 正山
和田　守正
守正 和田
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Description

本発明は、人工授精用培地及び同人工授精用培地を用いた人工授精方法並びに妊娠促進剤に関するものである。   The present invention relates to an artificial insemination medium, an artificial insemination method using the artificial insemination medium, and a pregnancy promoting agent.

従来より、優良な家畜の安定的な生産や希少動物の保護や不妊治療などのために、動物の雄から採取した精子と雌から採取した卵子とを人工的に授精させる人工授精が行われている。   Conventionally, artificial insemination has been performed to artificially inseminate sperm collected from animal males and ovum collected from females for stable production of excellent livestock, protection of rare animals and infertility treatment, etc. Yes.

この人工授精において、精子や卵子や授精卵は、人工授精用培地を用いて培養されている（たとえば、特許文献１、非特許文献１参照。）。   In this artificial insemination, sperm, ovum and fertilized egg are cultured using a medium for artificial insemination (for example, refer to Patent Document 1 and Non-Patent Document 1).

特開平１１−３４６５９６号公報JP 11-346596 A Toru Takeo,et al,BIOLOGY OF REPRODUCTION 78,546-551(2008)Toru Takeo, et al, BIOLOGY OF REPRODUCTION 78,546-551 (2008)

従来より、人工授精においては、貴重な精子や卵子を用いて人工的に授精させることから、その授精率を向上させて効率良く人工授精を行えるようにすることが望まれている（非特許文献１参照。）。   Conventionally, in artificial insemination, since artificial insemination is performed using precious sperm and eggs, it is desired to improve the insemination rate so that artificial insemination can be performed efficiently (non-patent literature). 1).

そこで、本発明者らは、既存の人工授精用培地を用いた人工授精において、授精率を向上させる培養条件を鋭意研究したところ、甘草エキス水溶液を人工授精用培地として用いることで授精率を向上できることがわかった。そして、更なる研究を行った結果、甘草エキス水溶液に含有される物質のうちで特に授精率の向上に寄与する物質を突き止め、本発明を成すに至った。   Therefore, the present inventors diligently studied the culture conditions for improving the insemination rate in the artificial insemination using the existing insemination medium, and improved the insemination rate by using the licorice extract aqueous solution as the artificial insemination medium. I knew it was possible. As a result of further research, among the substances contained in the licorice extract aqueous solution, substances that particularly contribute to the improvement of the fertilization rate have been identified, and the present invention has been achieved.

請求項１に係る本発明は、イソリクイリチゲニン（Isoliquiritingenin）のみを含有することを特徴とする人工授精用培地を提供するものである。
The present invention according to claim 1 provides an artificial insemination medium characterized by containing only isoliquiritingenin.

また、請求項２に係る本発明は、ホルモノネチン（Formononetin）のみを含有することを特徴とする人工授精用培地を提供するものである。
The present invention according to claim 2 provides a medium for artificial insemination characterized by containing only formononetin.

また、請求項３に係る本発明は、甘草から酢酸エチルでイソリクイリチゲニン（Isoliquiritingenin）又はホルモノネチン（Formononetin）を抽出した抽出液を含有することを特徴とする人工授精用培地を提供するものである。
The present invention according to claim 3 provides an artificial insemination medium characterized by containing an extract obtained by extracting isoliquiritingenin or formononetin from licorice with ethyl acetate. It is.

また、請求項４に係る本発明は、請求項１〜請求項３のいずれかに記載の人工授精用培地を用いて家畜や希少動物の卵子と精子とを授精させることを特徴とする人工授精方法を提供するものである。
Moreover, this invention which concerns on Claim 4 inseminates the egg and sperm of livestock or a rare animal using the culture medium for artificial insemination in any one of Claims 1-3, The artificial insemination characterized by the above-mentioned A method is provided.

また、請求項５に係る本発明は、請求項１〜請求項３のいずれかに記載の人工授精用培地を用いて家畜や希少動物の精子を培養し、培養した精子を人工授精用培地とともに家畜や希少動物の卵子を培養した人工授精用培地に添加して家畜や希少動物の精子と卵子とを授精させることを特徴とする人工授精方法を提供するものである。
Moreover, this invention which concerns on Claim 5 culture | cultivates the sperm of livestock or a rare animal using the culture medium for artificial insemination in any one of Claims 1-3, and culture | cultivated the sperm with the culture medium for artificial insemination. The present invention provides an artificial insemination method characterized by adding fertilized sperm and eggs of domestic animals and rare animals to an artificial insemination medium in which eggs of domestic animals and rare animals are cultured.

また、請求項６に係る本発明は、精子と卵子とが授精する培地に添加して妊娠の確立を向上させるための妊娠促進剤であって、イソリクイリチゲニン（Isoliquiritingenin）を含有することを特徴とする妊娠の確率を向上させるための妊娠促進剤を提供するものである。
Moreover, the present invention according to claim 6 is a pregnancy promoting agent for improving the establishment of pregnancy by adding to a medium in which sperm and ovum fertilize, and contains isoliquiritigenin (Isoliquiritingenin) The present invention provides a pregnancy promoting agent for improving the probability of pregnancy.

また、請求項７に係る本発明は、精子と卵子とが授精する培地に添加して妊娠の確立を向上させるための妊娠促進剤であって、ホルモノネチン（Formononetin）を含有することを特徴とする妊娠の確率を向上させるための妊娠促進剤を提供するものである。
In addition, the present invention according to claim 7 is a pregnancy promoting agent for improving the establishment of pregnancy by adding to a medium for fertilizing sperm and ovum, and contains formononetin. The present invention provides a pregnancy promoting agent for improving the probability of pregnancy.

また、請求項８に係る本発明は、精子と卵子とが授精する培地に添加して妊娠の確立を向上させるための妊娠促進剤であって、甘草から酢酸エチルでイソリクイリチゲニン（Isoliquiritingenin）又はホルモノネチン（Formononetin）を抽出した抽出液を含有することを特徴とする妊娠の確率を向上させるための妊娠促進剤を提供するものである。 The present invention according to claim 8 is a pregnancy promoting agent for improving the establishment of pregnancy by adding to a medium for fertilization of sperm and ovum, comprising from licorice using ethyl acetate with isoliquiritigenin (Isoliquiritingenin) ) Or a formononetin (Formononetin) -extracted solution, and a pregnancy promoting agent for improving the probability of pregnancy.

本発明では、人工授精等における授精率を向上させることができる。   In the present invention, the insemination rate in artificial insemination and the like can be improved.

検体と授精率との関係（表１）を示すグラフ。The graph which shows the relationship (Table 1) with a sample and an insemination rate. 検体と授精率との関係（表２）を示すグラフ。The graph which shows the relationship (Table 2) with a sample and an insemination rate. 検体と授精率との関係（表４）を示すグラフ。The graph which shows the relationship (Table 4) with a sample and an insemination rate.

以下に、本発明に係る人工授精用培地及び同人工授精用培地を用いた人工授精方法並びに妊娠促進剤について説明する。   Hereinafter, the artificial insemination medium, the artificial insemination method using the artificial insemination medium and the pregnancy promoting agent according to the present invention will be described.

本発明では、公知の人工授精方法と同様に、動物から採取した精子と卵子とをそれぞれ人工授精用培地で培養した後に、同一の人工授精用培地で精子と卵子とを培養して、精子と卵子とを授精させるものである。   In the present invention, similarly to the known artificial insemination method, after culturing sperm and ovum collected from an animal on an artificial insemination medium, culturing the sperm and egg on the same artificial insemination medium, Insemination with an egg.

本発明では、人工授精において、使用する人工授精用培地に最も特徴を有している。   In the present invention, the artificial insemination medium is most characterized by the artificial insemination medium used.

本発明者らは、既に、人工授精用培地に甘草エキス水溶液を含有させることで、授精率を向上させることに成功している。   The present inventors have already succeeded in improving the fertilization rate by including an licorice extract aqueous solution in an artificial insemination medium.

その試験結果を表１及び図１に示す。これは、甘草エキス水溶液を含有する人工授精用培地でマウスの精子を培養した後に、培養した精子を甘草エキス水溶液（人工授精用培地）とともにマウスの卵子を培養した人工授精用培地に添加して精子と卵子とを授精させた場合の試験結果である。なお、甘草エキス水溶液を含有する人工授精用培地は、甘草の根から抽出した粉末状の甘草エキスを水に溶かして甘草エキス水溶液を生成し、その甘草エキス水溶液を既存（公知）の人工授精用培地に添加した。   The test results are shown in Table 1 and FIG. After culturing mouse sperm in an artificial insemination medium containing an aqueous solution of licorice extract, the cultured sperm was added to an artificial insemination medium in which mouse eggs were cultured together with an aqueous solution of licorice extract (medium for artificial insemination). It is a test result at the time of fertilizing a sperm and an egg. The artificial insemination medium containing an aqueous solution of licorice extract is prepared by dissolving a powdered licorice extract extracted from licorice root in water to produce an aqueous solution of licorice extract, and the aqueous solution of licorice extract is an existing (known) artificial insemination medium. Added to.

Figure 0005804379
Figure 0005804379

この試験では、比較のために、甘草エキス水溶液を添加していない人工授精用培地と甘草エキス水溶液を添加した人工授精用培地とを用いて同一のマウス（検体ａ〜検体ｅ）から採取した精子と卵子とを人工的に授精させて行った。   In this test, for comparison, sperm collected from the same mouse (sample a to sample e) using an artificial insemination medium to which no licorice extract aqueous solution was added and an artificial insemination medium to which an licorice extract aqueous solution was added. And eggs were artificially inseminated.

その結果、検体ａでは、甘草エキス水溶液を添加していない人工授精用培地を用いた場合には、授精率が0.0%であったのに対して、甘草エキス水溶液を添加した人工授精用培地を用いた場合には、授精率が15.6%に向上した。同様に、検体ｂでは、8.0%から31.3%に、検体ｃでは、43.5%から84.0%に、検体ｄでは、18.5%から38.1%に、検体ｅでは、4.2%から47.1%に向上した。   As a result, in the sample a, when the artificial insemination medium without the licorice extract aqueous solution was used, the insemination rate was 0.0%, whereas the artificial insemination medium added with the licorice extract aqueous solution was When used, the insemination rate improved to 15.6%. Similarly, for sample b, it improved from 8.0% to 31.3%, for sample c from 43.5% to 84.0%, for sample d from 18.5% to 38.1%, and for sample e from 4.2% to 47.1%.

このように、甘草エキス水溶液を含有する人工授精用培地を用いた場合には、いずれの検体でも授精率が著しく増加することがわかった。なお、甘草エキス水溶液を含有する人工授精用培地を用いて精子及び卵子又は卵子のみを培養しても同様に授精率を向上させることができる。   Thus, when the artificial insemination medium containing the licorice extract aqueous solution was used, it was found that the insemination rate was remarkably increased in any specimen. It should be noted that the fertilization rate can be similarly improved by culturing sperm and eggs or eggs only using an artificial insemination medium containing an aqueous solution of licorice extract.

本発明者らは、甘草エキス水溶液に含有される成分の中から授精率の向上に寄与する有効成分を特定するため、更なる試験を行った。   The present inventors conducted further tests in order to identify active ingredients that contribute to the improvement of the fertilization rate among the ingredients contained in the licorice extract aqueous solution.

まず、甘草エキス水溶液をブタノールとエチルアセテートとでそれぞれ抽出した抽出液を生成し、その抽出液を添加した人工授精用培地を用い、上記と同様に、同一のマウス（検体１〜検体６）から採取した精子と卵子とを人工的に授精させた。なお、甘草エキス水溶液をブタノールで抽出した抽出液は、甘草の薬効成分として広く知られたグリチルリチン（Glycyrrhizin）を主に含んでいる。甘草エキス水溶液をエチルアセテートで抽出した抽出液は、リクイリチゲニン（Liquiritingenin）やイソリクイリチゲニン（Isoliquiritingenin）やホルモノネチン（Formononetin）などを含んでいる。   First, an extract obtained by extracting an aqueous solution of licorice extract with butanol and ethyl acetate, respectively, and using an artificial insemination medium to which the extract was added, was used from the same mouse (sample 1 to sample 6) in the same manner as described above. The collected sperm and egg were artificially inseminated. The extract obtained by extracting the licorice extract aqueous solution with butanol mainly contains glycyrrhizin, which is widely known as a medicinal component of licorice. The extract obtained by extracting licorice extract aqueous solution with ethyl acetate contains liquiritigenin, isoliquiritingenin, formononetin, and the like.

その試験結果を表２及び図２に示す。

Figure 0005804379
検体１では、甘草エキス水溶液を添加していない人工授精用培地（基準培地）を用いた場合には、授精率が22%であったのに対して、甘草エキス水溶液をブタノールで抽出した抽出液を添加した人工授精用培地（ブタノール抽出液含有培地）を用いた場合には、授精率が33%に向上し、甘草エキス水溶液をエチルアセテートで抽出した抽出液を添加した人工授精用培地（エチルアセテート抽出液含有培地）を用いた場合には、授精率が92％にも向上した。同様に、検体２では、基準培地で授精率が０％であったのに対して、ブタノール抽出液含有培地で31％、エチルアセテート抽出液含有培地で100％となり、検体３では、基準培地で授精率が０％であったのに対して、ブタノール抽出液含有培地で46％、エチルアセテート抽出液含有培地で62％となり、検体４では、基準培地で授精率が4％であったのに対して、ブタノール抽出液含有培地で10％、エチルアセテート抽出液含有培地で18％となり、検体５では、基準培地で授精率が1％であったのに対して、ブタノール抽出液含有培地で0％、エチルアセテート抽出液含有培地で12％となり、検体６では、基準培地で授精率が25％であったのに対して、ブタノール抽出液含有培地で18％、エチルアセテート抽出液含有培地で44％となった。 The test results are shown in Table 2 and FIG.
Figure 0005804379
 In Sample 1, when an artificial insemination medium (reference medium) to which no licorice extract aqueous solution was added was used, the insemination rate was 22%, whereas the extract obtained by extracting the licorice extract aqueous solution with butanol When using an artificial insemination medium (butanol extract-containing medium) supplemented with fertilizer, the insemination rate is improved to 33%, and an artificial insemination medium (ethyl) with an extract obtained by extracting the licorice extract aqueous solution with ethyl acetate is added. When using an acetate extract-containing medium), the insemination rate was improved to 92%. Similarly, in Sample 2, the fertilization rate was 0% in the reference medium, but 31% in the medium containing butanol extract and 100% in the medium containing ethyl acetate extract, and in Sample 3, the insemination rate was Although the insemination rate was 0%, it was 46% in the medium containing butanol extract and 62% in the medium containing ethyl acetate extract. In specimen 4, the insemination rate was 4% in the reference medium. On the other hand, the medium containing butanol extract contains 10% and the medium containing ethyl acetate extract contains 18%. In specimen 5, the fertilization rate was 1% in the reference medium, whereas 0% in the medium containing butanol extract. %, 12% in the ethyl acetate extract-containing medium, and in the specimen 6, the fertilization rate was 25% in the reference medium, whereas 18% in the butanol extract-containing medium and 44% in the ethyl acetate extract-containing medium. %.

このことから、甘草エキス水溶液をエチルアセテートで抽出した抽出液を人工授精用培地に添加することで、甘草エキス水溶液を添加していない人工授精用培地を用いた場合よりも顕著に授精率が向上することがわかった。なお、甘草エキス水溶液をエチルアセテートで抽出した抽出液を含有する人工授精用培地を用いて精子及び卵子又は卵子のみを培養しても同様に授精率を向上させることができる。   Therefore, by adding an extract obtained by extracting licorice extract aqueous solution with ethyl acetate to the artificial insemination medium, the insemination rate is significantly improved compared to the case of using the artificial insemination medium without the licorice extract aqueous solution. I found out that It should be noted that the fertilization rate can be similarly improved by culturing sperm and eggs or eggs only using an artificial insemination medium containing an extract obtained by extracting an aqueous solution of licorice extract with ethyl acetate.

さらに、授精率を向上させる物質を特定するために、エチルアセテートで抽出した抽出液に含まれる主な成分（表３に示す４種類の成分）をそれぞれ添加した人工授精用培地を用い、上記と同様に、同一のマウス（検体Ａ〜検体Ｅ）から採取した精子と卵子とを人工的に授精させた。   Furthermore, in order to specify the substance that improves the insemination rate, an artificial insemination medium to which main components (four kinds of components shown in Table 3) contained in the extract extracted with ethyl acetate are added, Similarly, sperm and eggs collected from the same mouse (sample A to sample E) were artificially inseminated.

Figure 0005804379
Figure 0005804379

その試験結果を表４及び図３に示す。   The test results are shown in Table 4 and FIG.

Figure 0005804379
Figure 0005804379

検体Ａでは、甘草エキス水溶液を添加していない人工授精用培地（基準培地）を用いた場合には、授精率が4%であったのに対して、培地１を用いた場合には、授精率が2％に低減し、培地２を用いた場合には、授精率が51％に向上し、培地３を用いた場合には、授精率が3％に低減し、培地４を用いた場合には、授精率が22％に向上した。同様に、検体Ｂでは、基準培地で授精率が8％であったのに対して、培地１で7％、培地２で70％、培地３で12％、培地４で63％となり、検体Ｃでは、基準培地で授精率が10％であったのに対して、培地１で0％、培地２で20％、培地３で4％、培地４で15％となり、検体Ｄでは、基準培地で授精率が22％であったのに対して、培地１で20％、培地２で35％、培地３で6％、培地４で45％となり、検体Ｅでは、基準培地で授精率が24％であったのに対して、培地１で26％、培地２で74％、培地３で27％、培地４で90％となった。   In specimen A, the fertilization rate was 4% when using an artificial insemination medium (reference medium) to which no licorice extract aqueous solution was added, while insemination was performed when medium 1 was used. When the rate is reduced to 2% and the medium 2 is used, the insemination rate is improved to 51%. When the medium 3 is used, the insemination rate is reduced to 3% and the medium 4 is used. Insemination rate improved to 22%. Similarly, in the sample B, the insemination rate was 8% in the reference medium, but 7% in the medium 1, 70% in the medium 2, 12% in the medium 3, 63% in the medium 4, and the sample C. In the standard medium, the insemination rate was 10%, while 0% in medium 1, 20% in medium 2, 4% in medium 3, and 15% in medium 4, While the insemination rate was 22%, it was 20% in medium 1, 35% in medium 2, 6% in medium 3, and 45% in medium 4. In sample E, the insemination rate was 24% in the standard medium. In contrast, it was 26% for medium 1, 74% for medium 2, 27% for medium 3, and 90% for medium 4.

このことから、甘草エキス水溶液をエチルアセテートで抽出した抽出液のうちで、イソリクイリチゲニンを添加した人工授精用培地（培地２）やホルモノネチンを添加した人工授精用培地（培地４）を用いた場合には、甘草エキス水溶液を添加していない人工授精用培地を用いた場合よりも顕著に授精率が向上することがわかった。なお、イソリクイリチゲニンを添加した人工授精用培地やホルモノネチンを添加した人工授精用培地を用いて精子及び卵子又は卵子のみを培養しても同様に授精率を向上させることができる。   Therefore, among the extracts obtained by extracting the licorice extract aqueous solution with ethyl acetate, the artificial insemination medium (medium 2) to which isoliquiritigenin is added and the artificial insemination medium (medium 4) to which formononetin is added are used. It was found that the insemination rate was significantly improved when using an artificial insemination medium to which no licorice extract aqueous solution was added. It should be noted that the fertilization rate can be similarly improved by culturing only sperm and eggs or eggs using an artificial insemination medium to which isoliquiritigenin is added or an artificial insemination medium to which formononetin is added.

以上に説明したように、人工授精において、甘草エキス水溶液をエチルアセテートで抽出した抽出液、特に、イソリクイリチゲニンやホルモノネチンを人工授精用培地に含有させることで、授精率を向上させることができる。   As described above, in artificial insemination, an extract obtained by extracting an aqueous solution of licorice extract with ethyl acetate, in particular, isoliqueritigenin and formononetin can be contained in the artificial insemination medium to improve the insemination rate. it can.

そのため、甘草エキス水溶液をエチルアセテートで抽出した抽出液やイソリクイリチゲニンやホルモノネチンを含有する人工授精用培地を用いて動物から採取した貴重な精子と卵子とを人工的に授精させることで、優良な家畜の安定的な生産や希少動物の保護や不妊治療などを行うことができる。   Therefore, by artificially inseminating precious sperm and eggs collected from animals using an extract obtained by extracting licorice extract aqueous solution with ethyl acetate and an artificial insemination medium containing isoliquiritigenin and formononetin, Stable production of excellent livestock, protection of rare animals and infertility treatment can be performed.

なお、本発明では、人工授精において、甘草エキス水溶液をエチルアセテートで抽出した抽出液やイソリクイリチゲニンやホルモノネチンを含有する人工授精用培地を用いて、精子のみ、卵子のみ、精子及び卵子のいずれかを培養すればよく、精子のみを培養する場合に卵子を公知の人工授精用培地で培養し、卵子のみを培養する場合に精子を公知の人工授精用培地で培養してもよく、精子及び卵子を公知の人工授精用培地で培養した後に本発明に係る人工授精用培地で精子と卵子とを培養してもよい。   In the present invention, in artificial insemination, an extract obtained by extracting licorice extract aqueous solution with ethyl acetate or an artificial insemination medium containing isoliqueritigenin or formononetin, and using only sperm, ova only, sperm and ovum Any of the above may be cultured. When only sperm is cultured, the egg is cultured in a known artificial insemination medium. When only the egg is cultured, the sperm may be cultured in a known artificial insemination medium. Alternatively, after culturing the ovum in a known artificial insemination medium, the sperm and ovum may be cultured in the artificial insemination medium according to the present invention.

また、本発明に係るイソリクイリチゲニンやホルモノネチンは、甘草の地下茎や地上茎から抽出したものを用いてもよく、他の生物から抽出したものでもよく、化学合成により生成したものでもよい。   In addition, the isoliquiritigenin and formononetin according to the present invention may be those extracted from licorice rhizome or ground stem, may be extracted from other organisms, or may be generated by chemical synthesis.

さらに、甘草エキス水溶液をエチルアセテートで抽出した抽出液やイソリクイリチゲニンやホルモノネチンは、イソリクイリチゲニンやホルモノネチンの化学構造から体外受精における人工授精用培地に添加して使用する場合に限られず、動物の体内での授精の効率を上げて妊娠の確率を向上させることを目的として座薬や経口薬などの妊娠促進剤として使用することもできる。   In addition, the extract of licorice extract aqueous solution extracted with ethyl acetate, isoliquiritigenin, and formononetin are limited to use when added to the artificial insemination medium for in vitro fertilization due to the chemical structure of isoliquiritigenin and formononetin. However, it can also be used as a pregnancy promoter such as a suppository or oral medicine for the purpose of improving the probability of pregnancy by increasing the efficiency of insemination in the animal body.

Claims (8)

イソリクイリチゲニン（Isoliquiritingenin）のみを含有することを特徴とする人工授精用培地。 An artificial insemination medium characterized by containing only isoliquiritingenin (Isoliquiritingenin). ホルモノネチン（Formononetin）のみを含有することを特徴とする人工授精用培地。 An artificial insemination medium characterized by containing only formononetin. 甘草から酢酸エチルでイソリクイリチゲニン（Isoliquiritingenin）又はホルモノネチン（Formononetin）を抽出した抽出液を含有することを特徴とする人工授精用培地。   An artificial insemination medium comprising an extract obtained by extracting isoliquiritingenin or formononetin from licorice with ethyl acetate. 請求項１〜請求項３のいずれかに記載の人工授精用培地を用いて家畜や希少動物の卵子と精子とを授精させることを特徴とする人工授精方法。   An artificial insemination method, comprising fertilizing eggs and sperm of domestic animals or rare animals using the artificial insemination medium according to any one of claims 1 to 3. 請求項１〜請求項３のいずれかに記載の人工授精用培地を用いて家畜や希少動物の精子を培養し、培養した精子を人工授精用培地とともに家畜や希少動物の卵子を培養した人工授精用培地に添加して家畜や希少動物の精子と卵子とを授精させることを特徴とする人工授精方法。   Artificial insemination in which livestock or rare animal sperm are cultured using the artificial insemination medium according to any one of claims 1 to 3, and the cultured sperm is cultured together with the artificial insemination medium and livestock or rare animal eggs. An artificial insemination method characterized in that it is added to a culture medium and fertilized with sperm and eggs of domestic animals and rare animals. 精子と卵子とが授精する培地に添加して妊娠の確率を向上させるための妊娠促進剤であって、
イソリクイリチゲニン（Isoliquiritingenin）を含有することを特徴とする妊娠の確率を向上させるための妊娠促進剤。 A pregnancy promoter that is added to a medium that fertilizes sperm and ovum to improve the probability of pregnancy,
A pregnancy promoting agent for improving the probability of pregnancy characterized by containing isoliquiritingenin (Isoliquiritingenin). 精子と卵子とが授精する培地に添加して妊娠の確率を向上させるための妊娠促進剤であって、
ホルモノネチン（Formononetin）を含有することを特徴とする妊娠の確率を向上させるための妊娠促進剤。 A pregnancy promoter that is added to a medium that fertilizes sperm and ovum to improve the probability of pregnancy,
A pregnancy promoting agent for improving the probability of pregnancy characterized by containing formononetin. 精子と卵子とが授精する培地に添加して妊娠の確率を向上させるための妊娠促進剤であって、
甘草から酢酸エチルでイソリクイリチゲニン（Isoliquiritingenin）又はホルモノネチン（Formononetin）を抽出した抽出液を含有することを特徴とする妊娠の確率を向上させるための妊娠促進剤。
 A pregnancy promoter that is added to a medium that fertilizes sperm and ovum to improve the probability of pregnancy,
A pregnancy promoting agent for improving the probability of pregnancy, characterized by containing an extract obtained by extracting isoliquiritingenin or formononetin from licorice with ethyl acetate.
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