JP3636609B2 - Pig semen preservation solution and method of use thereof - Google Patents
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Description
【０００１】
【発明の属する技術分野】
本発明は、豚精液を保存する豚精液保存液及びその使用方法に関するものである。
【０００２】
【従来の技術】
近年、優れた豚の産肉を生じるため、遺伝的に産肉能力、繁殖能力の高い雄豚の精液を多数の雌豚に人工的に授精させる豚の人工授精が行われている。
【０００３】
人工授精を行う際には豚精液を豚精液保存液によって希釈し、豚精液量を増加させると共に豚精液の保存時間を延長させている。
【０００４】
豚精液保存液は、ブドウ糖２７．５ｇ、クエン酸三ナトリウム（２水和物）６．９ｇ、炭酸水素ナトリウム１．０ｇ、エチレンジアミン四酢酸二ナトリウム（ＥＤＴＡ．２Ｎａ）２．３５ｇ、クエン酸（１水和物）２．９ｇ、トリス（ヒドロキシル）アミノメタン５．６５ｇ、ゲンタマイシン（アミノ酸糖体抗生物質）６０〜１２０ｍｇを蒸留水で１０００ｍｌにしたものであり、モデナ液として広く知られている。
【０００５】
ここで、豚精液保存液は豚精液と略同じ浸透圧を持つようブドウ糖、緩衝剤により計算調整されており、ブドウ糖は豚精子のエネルギー源となり、炭酸水素ナトリウムは豚精液保存液のｐＨ調整を行っている。なお、豚精液保存液のｐＨは、通常６．８〜７．２である。
【０００６】
豚精液保存液を使用する際には、所定の雄豚から採取した豚精液を直ちに滅菌ガーゼで濾過して膠様物を除去し、豚精液保存液と同じ温度にした後、速やかに豚精液保存液で希釈する。
【０００７】
希釈した豚精液は直ちに人工授精に使用することができ、又、徐々に温度を１５℃に下げ恒温保存することもできる。
【０００８】
このため、豚精液保存液により豚精液量を増加させると共に豚精液の保存時間を延長するので、高能力雄豚精液の安価利用、精液を介した疾病の予防、繁殖成績の高レベル化、交配にかかる時間短縮・コスト低減等の効果を備えている。
【０００９】
【発明が解決しようとする課題】
しかしながら、豚精液保存液内に細菌の発生を防ぐため豚精液保存液を予め加熱滅菌すると、豚精液保存液の組成変化とみられる着色を生じてしまい、結果的に加熱滅菌した豚精液保存液の保存性は非加熱滅菌の豚精液保存液より劣るという問題があり、豚精液の保存時間は２日前後であった。この加熱滅菌は、一般的な凍結保存や用時調製法より劣り、豚精液の保存時間を一層延長するよう豚精液保存液の保存性を高めることが求められていた。
【００１０】
本発明は上述した実情に鑑みてなしたもので、豚精液保存液の保存性を高めることを目的としたものである。
【００１１】
【課題を解決するための手段】
本発明の豚精液保存液は、調製された希釈基液と、豚精液を混合する直前に前記希釈基液に加えるｐＨ調整を行うための添加液とからなり、
前記希釈基液は、蒸留水に対してブドウ糖１〜５％、クエン酸三ナトリウム（２水和物）０．１〜１．５％、エチレンジアミン四酢酸二ナトリウム（ＥＤＴＡ．２Ｎａ）０．０５〜０．５０％、クエン酸（１水和物）０．０５〜０．５０％、トリス（ヒドロキシル）アミノメタン０．１〜１．５％の混合液を用い、
前記添加液は、蒸留水に炭酸水素ナトリウム０ . ０５〜２０．０％を加えて調整されたものを用いたものである。
【００１２】
本発明では、希釈基液に抗生物質を加えてもよい。
【００１３】
又、本発明では滅菌した希釈基液を用いてもよい。
【００１４】
更に本発明では滅菌した添加液を用いてもよい。
【００１７】
更に、本発明の豚精液保存液において、アミカシン、ゲンタマイシン、カナマイシン、ストレプトマイシン、ポリミキシンＢの群から選ばれる１種もしくは２種以上のいずれかを抗生物質として用いることがある。
【００１８】
本発明の豚精液保存液使用方法は、蒸留水に対してブドウ糖１〜５％、クエン酸三ナトリウム（２水和物）０．１〜１．５％、エチレンジアミン四酢酸二ナトリウム（ＥＤＴＡ．２Ｎａ）０．０５〜０．５０％、クエン酸（１水和物）０．０５〜０．５０％、トリス（ヒドロキシル）アミノメタン０．１〜１．５％の混合液を希釈基液にするよう、豚精液を希釈する希釈基液を予め調製し、且つ蒸留水に炭酸水素ナトリウム０ . ０５〜２０．０％を加えて調整された添加液を用いるよう所定濃度のｐＨ調整を行うための添加液を予め調製し、使用直前に前記希釈基液と、添加液とを混合するものである。
【００１９】
以上のように本発明の豚精液保存液及びその使用方法によれば、豚精液の保存時間を延長できることが種々の試験を重ねた結果、明らかになった。
【００２０】
又、希釈基液と添加液を適宜別々に加熱滅菌して混合した際には、豚精液保存液の組成変化とみられる着色を防止すると共に、豚精液保存液の性能に良い結果が報告されているｐＨの低下がみられ、豚精液保存液の保存性は、予め希釈基液と添加液を混合して加熱滅菌した豚精液保存液、及び予め希釈基液と添加液を混合して加熱滅菌しない豚精液保存液より優れているものとなる。
【００２１】
【発明の実施の形態】
以下、本発明の豚精液保存液の実施の形態を説明する。
【００２２】
本発明の豚精液保存液を使用する場合には、豚精液保存液を構成する希釈基液と添加液とを予め調製する。
【００２３】
希釈基液は、蒸留水に対してブドウ糖１〜５％、クエン酸三ナトリウム（２水和物）０．１〜１．５％、エチレンジアミン四酢酸二ナトリウム（ＥＤＴＡ．２Ｎａ）０．０５〜０．５０％、クエン酸（１水和物）０．０５〜０．５０％、トリス（ヒドロキシル）アミノメタン０．１〜１．５％の範囲になるよう調製したものであり、ここでは、ブドウ糖１３．７５ｇ、クエン酸三ナトリウム（２水和物）３．４５ｇ、エチレンジアミン四酢酸二ナトリウム（ＥＤＴＡ．２Ｎａ）１．１７５ｇ、クエン酸（１水和物）１．４５ｇ、トリス（ヒドロキシル）アミノメタン２．８２５ｇを蒸留水で５００ｍｌにしたものである。
【００２４】
又、希釈基液に加えられる抗生物質は、アミカシン、ゲンタマイシン、カナマイシン、ストレプトマイシン、ポリミキシンＢの群から選ばれる１種もしくは２種以上であって、ここでは、ゲンタマイシン３０〜６０ｍｇを選んでいる。
【００２５】
一方、添加液は、蒸留水に炭酸水素ナトリウム０．０５〜２０％を加えて調整されたものであり、ここでは、蒸留水７．１ｍｌ中に炭酸水素ナトリウム０．５ｇを含んだものである。
【００２６】
希釈基液と添加液が調製された後には、希釈基液５００ｍｌと添加液７．１ｍｌを夫々、別々に１００℃で６０分間加熱滅菌し、使用直前に３５℃に加温した希釈基液に添加液を加え、更に抗生物質を混合する。
【００２７】
又、豚精液が採取された場合には、豚精液を直ちに滅菌カーゼで濾過して膠様物を除去し、なるべく速やかに希釈基液と同じ温度とし、希釈基液で希釈する。
【００２８】
希釈された精液はそのまま直ちに人工受精に使用することができ、又、徐々に１５℃に温度を下げ恒温保存することもできる。
【００２９】
以上の豚精液保存液及びその使用方法において豚精液保存液の保存性の試験を行った。
【００３０】
試験は、豚精液保存液の保存性として豚精子の運動力＋＋以上の豚精子が５０％以上存在する日数、すなわち豚精子の活力保持日数を測定比較した。その結果は表１の通りである。
【００３１】
なお、豚精子の運動力は次によって判定した。
＋＋＋：運動激烈で、活発な前進運動を行うもの。壁に接したものは頭部を集め尾部のみ激烈な運動を行う。渦巻きのごとく、また水の流れのごとく見える場合がある。
＋＋：運動やや活発で、緩慢な前進運動、激しい振せん運動及び活発な旋回運動などを行う程度のもの。
＋：運動微弱で、緩慢な振子運動を行う程度のもの。
−：運動停止。
【００３２】
【表１】
本発明の豚精液保存液及びその使用方法によれば、豚精子の活力保持日数を延長するので豚精液保存液の保存性は従来のものより優れていることが明らかである。
【００３４】
又、希釈基液と添加液を適宜別々に加熱滅菌して混合した際には、豚精液保存液の組成変化とみられる着色を防止すると共に、豚精液保存液の性能に良い結果が報告されているｐＨの低下がみられ、豚精液保存液の保存性は、予め希釈基液と添加液を混合して加熱滅菌した豚精液保存液、及び予め希釈基液と添加液を混合して加熱滅菌しない豚精液保存液より優れているものとなる。
【００３５】
なお、本発明の豚精液保存液は、上述の形態例のみに限定されるものではなく、希釈基液に含まれるクエン酸（１水和物）はクエン酸（無水）でもよいこと、又、トリス（ヒドロキシル）アミノメタンはトリス（ヒドロキシルメチル）アミノメタンでもよいこと、添加液はｐＨ調整するものならばどのようなものでもよいこと、その他、本発明の要旨を逸脱しない範囲内において種々変更を加え得ることは勿論である。
【００３６】
【発明の効果】
本発明の豚精液保存液及びその使用方法によれば、従来の豚精液保存液より保存性を延長させることができるという優れた効果を奏し得る。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a pig semen preservation solution for preserving pig semen and a method for using the same.
[0002]
[Prior art]
In recent years, in order to produce excellent pork meat, artificial insemination of pigs has been carried out in which a large number of sows are artificially inseminated with semen of genetically high meat production and breeding ability.
[0003]
When performing artificial insemination, pig semen is diluted with a pig semen preservation solution to increase the amount of pig semen and extend the preservation time of pig semen.
[0004]
Porcine sperm preservation solution was glucose 27.5g, trisodium citrate (dihydrate) 6.9g, sodium hydrogen carbonate 1.0g, ethylenediaminetetraacetic acid disodium (EDTA.2Na) 2.35g, citric acid (1 Hydrate) 2.9 g, tris (hydroxyl) aminomethane 5.65 g, gentamicin (amino acid saccharide antibiotics) 60-120 mg made up to 1000 ml with distilled water, widely known as modena solution.
[0005]
Here, porcine semen preservation solution is calculated and adjusted with glucose and buffer so that it has almost the same osmotic pressure as porcine semen. Glucose is an energy source for pork sperm, and sodium bicarbonate adjusts the pH of pork semen preservation solution. Is going. The pH of the pig semen preservation solution is usually 6.8 to 7.2.
[0006]
When using pork semen stock solution, immediately remove pork semen collected from a given boar with sterilized gauze to remove glue, bring it to the same temperature as pork semen stock solution, and then quickly Dilute with stock solution.
[0007]
The diluted porcine semen can be used immediately for artificial insemination, and the temperature can be gradually lowered to 15 ° C. and stored at a constant temperature.
[0008]
For this reason, the amount of swine semen is increased and the storage time of swine semen is extended by the swine semen preservation solution, so that low-cost use of high-capacity boar semen, prevention of diseases through semen, higher levels of reproductive performance, mating It has effects such as shortening the time required for cost reduction and cost reduction.
[0009]
[Problems to be solved by the invention]
However, pre-heat sterilization of the porcine semen preservation solution to prevent the generation of bacteria in the porcine semen preservation solution will result in a color that appears to be a change in the composition of the porcine semen preservation solution. There is a problem that the storage stability is inferior to the non-heat sterilized porcine semen storage solution, and the storage time of the pig semen was around 2 days. This heat sterilization is inferior to general cryopreservation and preparation methods at the time of use, and it has been required to improve the storage stability of the pig semen preservation solution so as to further extend the preservation time of the pig semen.
[0010]
The present invention has been made in view of the above-described circumstances, and an object thereof is to improve the storage stability of a pig semen preservation solution.
[0011]
[Means for Solving the Problems]
Porcine semen preservation solution of the present invention, a dilute base solution prepared, Ri Do from additive solution for adjusting pH to be added to the diluted base solution just prior to mixing the pig semen,
The diluted base solution is 1 to 5% glucose, 0.1 to 1.5% trisodium citrate (dihydrate), 0.05 to disodium ethylenediaminetetraacetate (EDTA.2Na) based on distilled water. Using a mixed solution of 0.50%, citric acid (monohydrate) 0.05 to 0.50%, tris (hydroxyl) aminomethane 0.1 to 1.5%,
The additive solution is obtained using those adjusted by the addition of sodium bicarbonate from 0.05 to 20.0% in distilled water.
[0012]
In the present invention, an antibiotic may be added to the diluted base solution.
[0013]
In the present invention, a sterilized diluted base solution may be used.
[0014]
Further, in the present invention, a sterilized additive solution may be used.
[0017]
Furthermore, in the pig semen preservation solution of the present invention, one or more selected from the group of amikacin, gentamicin, kanamycin, streptomycin, and polymyxin B may be used as an antibiotic.
[0018]
The method of using the swine semen preservation solution of the present invention is 1-5% glucose, 0.1-1.5% trisodium citrate (dihydrate), disodium ethylenediaminetetraacetate (EDTA.2Na) with respect to distilled water. ) 0.05 to 0.50%, citric acid (monohydrate) 0.05 to 0.50%, tris (hydroxyl) aminomethane 0.1 to 1.5% mixed liquid is used as a diluted base solution. as previously prepared dilution base liquid for diluting the pig semen, and sodium bicarbonate 0 in distilled water. 05 to 20.0% was added for adjusting pH of a predetermined concentration so as to use the additive solution prepared by An additive solution is prepared in advance, and the diluted base solution and the additive solution are mixed immediately before use.
[0019]
As described above, according to the pig semen preservation solution and the method of using the same according to the present invention, it has become clear that the preservation time of pig semen can be extended as a result of various tests.
[0020]
In addition, when the diluted base solution and the additive solution are appropriately heat-sterilized and mixed separately, coloring that appears to be a change in the composition of the swine semen stock solution is prevented, and good performance is reported for the performance of the swine semen stock solution. The storage stability of swine semen stock solution is pre-mixed with diluted base solution and additive solution and sterilized by heating, and pre-mixed diluted base solution and additive solution are heat sterilized. It will be better than the porcine semen preservation solution.
[0021]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the pig semen preservation solution of the present invention will be described.
[0022]
When using the porcine semen preservation solution of the present invention, the diluted base solution and the additive solution constituting the porcine semen preservation solution are prepared in advance.
[0023]
The diluted base solution is 1 to 5% glucose, 0.1 to 1.5% trisodium citrate (dihydrate), 0.05 to 0 disodium ethylenediaminetetraacetate (EDTA.2Na) based on distilled water. .50%, citric acid (monohydrate) 0.05 to 0.50%, tris (hydroxyl) aminomethane 0.1 to 1.5% 13.75 g, trisodium citrate (dihydrate) 3.45 g, disodium ethylenediaminetetraacetate (EDTA.2Na) 1.175 g, 1.45 g citric acid (monohydrate), tris (hydroxyl) aminomethane 2.825 g is made up to 500 ml with distilled water.
[0024]
The antibiotic added to the diluted base solution is one or more selected from the group of amikacin, gentamicin, kanamycin, streptomycin, and polymyxin B. Here, 30 to 60 mg of gentamicin is selected.
[0025]
On the other hand, the additive solution was prepared by adding 0.05 to 20% sodium bicarbonate to distilled water, and here, 0.5 ml of sodium bicarbonate was contained in 7.1 ml of distilled water. .
[0026]
After the diluted base solution and the additive solution are prepared, 500 ml of the diluted base solution and 7.1 ml of the added solution are separately sterilized by heating at 100 ° C. for 60 minutes, and the diluted base solution is heated to 35 ° C. immediately before use. Add additive and mix antibiotics.
[0027]
When porcine semen is collected, the porcine semen is immediately filtered through a sterilized case to remove the glue, and as quickly as possible, the temperature is set to the same as the diluted base solution and diluted with the diluted base solution.
[0028]
The diluted semen can be immediately used for artificial insemination as it is, and the temperature can be gradually lowered to 15 ° C. and stored at a constant temperature.
[0029]
The above-described porcine semen preservation solution and the method of using the same were tested for the preservability of the pig semen preservation solution.
[0030]
The test was conducted by measuring and comparing the number of days in which 50% or more of pig sperm motility + or more of pig sperm existed, that is, the number of days of retention of pig sperm vitality, as the preservation property of the pig semen preservation solution. The results are shown in Table 1.
[0031]
The motility of pig sperm was determined as follows.
++++: Intense movement and active forward movement. Those in contact with the wall collect their heads and perform intense exercise only at the tail. It may look like a swirl or a stream of water.
++: Exercise that is somewhat active, slow forward movement, intense tremor movement, active swing movement, etc.
+: The movement is very weak and the pendulum movement is slow.
−: Stop exercise.
[0032]
[Table 1]
According to the swine semen preservation solution and the method of using the same of the present invention, it is apparent that the preservation property of the swine sperm preservation solution is superior to the conventional one because the vigor retention days of the pig sperm are extended.
[0034]
In addition, when the diluted base solution and the additive solution are appropriately heat-sterilized and mixed separately, coloring that appears to be a change in the composition of the swine semen stock solution is prevented, and good performance is reported for the performance of the swine semen stock solution. The storage stability of swine semen stock solution is pre-mixed with diluted base solution and additive solution and sterilized by heating, and pre-mixed diluted base solution and additive solution are heat sterilized. It will be better than the porcine semen preservation solution.
[0035]
In addition, the pig semen preservation solution of the present invention is not limited only to the above-described embodiments, and citric acid (monohydrate) contained in the diluted base solution may be citric acid (anhydrous), The tris (hydroxyl) aminomethane may be tris (hydroxylmethyl) aminomethane, the additive solution may be any one that adjusts the pH, and various other modifications may be made without departing from the scope of the present invention. Of course, it can be added.
[0036]
【The invention's effect】
According to the pig semen preservation solution and the method of using the same of the present invention, it is possible to achieve an excellent effect that the preservation property can be extended as compared with the conventional pig semen preservation solution.
Claims (6)
前記希釈基液は、蒸留水に対してブドウ糖１〜５％、クエン酸三ナトリウム（２水和物）０．１〜１．５％、エチレンジアミン四酢酸二ナトリウム（ＥＤＴＡ．２Ｎａ）０．０５〜０．５０％、クエン酸（１水和物）０．０５〜０．５０％、トリス（ヒドロキシル）アミノメタン０．１〜１．５％の混合液を用い、
前記添加液は、蒸留水に炭酸水素ナトリウム０ . ０５〜２０．０％を加えて調整されたものを用いることを特徴とする豚精液保存液。A diluent base liquid prepared, Ri Do from additive solution for adjusting pH to be added to the diluted base solution just prior to mixing the pig semen,
The diluted base solution is 1 to 5% glucose, 0.1 to 1.5% trisodium citrate (dihydrate), 0.05 to disodium ethylenediaminetetraacetate (EDTA.2Na) based on distilled water. Using a mixed solution of 0.50%, citric acid (monohydrate) 0.05 to 0.50%, tris (hydroxyl) aminomethane 0.1 to 1.5%,
The additive solution, sodium bicarbonate 0 in distilled water. Porcine semen preservation solution which comprises using those adjusted by the addition of 05 to 20.0%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP04636799A JP3636609B2 (en) | 1999-02-24 | 1999-02-24 | Pig semen preservation solution and method of use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP04636799A JP3636609B2 (en) | 1999-02-24 | 1999-02-24 | Pig semen preservation solution and method of use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2000247801A JP2000247801A (en) | 2000-09-12 |
| JP3636609B2 true JP3636609B2 (en) | 2005-04-06 |
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| Application Number | Title | Priority Date | Filing Date |
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| JP04636799A Expired - Fee Related JP3636609B2 (en) | 1999-02-24 | 1999-02-24 | Pig semen preservation solution and method of use thereof |
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| JP (1) | JP3636609B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008007463A1 (en) * | 2006-07-12 | 2008-01-17 | Nippon Zenyaku Kogyo Co., Ltd. | Composition for diluting and storing sperm |
| CN103843759A (en) * | 2014-03-07 | 2014-06-11 | 四川农业大学 | Chicken seminal fluid diluent applicable to high altitude localities and preparation method and application of diluent |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5980499B2 (en) * | 2011-11-22 | 2016-08-31 | 株式会社ピィアイシィ・バイオ | Preserved semen dilution for pig artificial insemination |
| WO2015193265A1 (en) | 2014-06-16 | 2015-12-23 | Università degli Studi di Parma | Composition for extenders for the long-term conservation of animal seminal material |
| CN107711822A (en) * | 2017-10-09 | 2018-02-23 | 安徽省农业科学院水产研究所 | A kind of Misgurnus auguillicaudatus spermatozoa preservative fluid, activating solution and hatching method |
| CN110115264A (en) * | 2018-02-05 | 2019-08-13 | 天津农学院 | A kind of diluent and preparation method thereof and the application in pig semen cryo-conservation |
| CN112674077A (en) * | 2021-01-13 | 2021-04-20 | 江苏中牧倍康药业有限公司 | External normal temperature preservative for diluting all-round healthy boar semen |
| CN115245161A (en) * | 2022-08-17 | 2022-10-28 | 中国农业大学 | Oregano oil-based long-acting diluent for normal-temperature preservation of boar semen |
-
1999
- 1999-02-24 JP JP04636799A patent/JP3636609B2/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008007463A1 (en) * | 2006-07-12 | 2008-01-17 | Nippon Zenyaku Kogyo Co., Ltd. | Composition for diluting and storing sperm |
| CN103843759A (en) * | 2014-03-07 | 2014-06-11 | 四川农业大学 | Chicken seminal fluid diluent applicable to high altitude localities and preparation method and application of diluent |
| CN103843759B (en) * | 2014-03-07 | 2015-05-20 | 四川农业大学 | Chicken seminal fluid diluent applicable to high altitude localities and preparation method and application of diluent |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2000247801A (en) | 2000-09-12 |
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