凍結・融解過程における活性酸素種および抗酸化剤がブタ精子の活力に及ぼす影響  [in Japanese] Effects of Reactive Oxygen Species and Antioxidants on the Motility of Boar Spermatozoa in the Process of Freezing and Thawing  [in Japanese]

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Abstract

この研究は, ブタ精液の凍結・融解過程で活性酸素種が精子にどのような影響を与えるか, また種々の抗酸化剤の効果を明らかにすることを目的として行った。濃厚部精液に前処理液を添加して3時間放置した後, 6試験区に分割して遠心分離した。沈澱精子はラクトース-卵黄希釈液を基本液とした各試験区の希釈液に浮遊して5℃に冷却後, 2次希釈を行い, 凍結した。次の6試験区を設定した。(1) 無処理区, (2) キサンチン0.6mMおよびキサンチンオキシダーゼ0.05mMを添加 (X-X0区), (3) (2) にカタラーゼ150Uを添加 (X-XO+Cat区), (4) (2) にスーパーオキシドジスムターゼ150Uを添加 (X-XO+SOD区), (5) (2) にグルタチオン (酸化型) 1.5mMを添加 (X-XO+GSSG区), (6) (2) にハイポタウリン10mMを添加 (X-XO+HT区)。45℃で融解後, 融解液で10倍に希釈し, 37℃恒温槽で60分間インキュベートして, 精子の活力を検査した。活力は精子生存指数に換算し, 検定に用いた。<br>X-XO区における凍結・融解後の精子の回復率は無処理区に比べて有意に低く, 活性酸素種の影響が示唆された。これに対しX-XO+Cat区はX-XO区と比較して有意に高い値を示した。また, X-XO+SOD区およびX-XO+HT区では, X-XO区より高い傾向が見られた。精子生存指数で見ると, インキュベート30分後ではX-XO+Cat区およびX-XO+HT区がX-XO区に比べ有意に高い値を示した。<br>以上の結果より, 凍結・融解過程でブタ精子は活性酸素種により活力が損なわれること, その障害がカタラーゼにより抑えられることが明らかにされた。したがって, 精子の活力低下を阻止するために除去すべき活性酸素種は過酸化水素であると考えられた。またその障害の抑制にはハイポタウリンも効果的であることも示された。

The objective of this study was to examine the effects of reactive oxygen species (ROS) produced by a xanthine (X)-xanthine oxidase (XO) generating system on the motility of boar spermatozoa in the process of freezing-thawing, and to evaluate the ability of the antioxidants to protect sperm from this challenge.<br>The sperm rich fraction was diluted with the pre-diluent and stored for 3hrs at room temperature, and then divided into 6 tubes before centrifugation. Precipitated sperm in each tube was suspended in one of the following lactose-egg yolk diluents added antioxidants. 1) Lactose-egg yolk (control), 2) X (0.6mM)-XO (0.05U/m<i>l</i>), 3) X-XO and catalase (Cat) (150U/m<i>l</i>), 4) X-XO and super oxide dismutase (SOD) (150U/ML), 5) X-XO and glutathione (GSSG) (1.5mM) or 6) X-XO and hypotaurine (HT) (10mM). Each group of sperm was cooled to 5°C and then added the 2nd diluent including glycerol and OEP. Finally diluted semen was packaged in the maxi-straw and then frozen in liquid nitrogen vapor. It took about 2hrs from the exposure of ROS to freeze.<br>After the semen was thawed at 45°C, it was diluted with the thawing-diluent and incubated at 37°C for 60 minutes to observe the motility. The results were expressed as the mean±S. E. of 9 ejaculates from 3 boars.<br>Frozen-thawed sperm with X-XO alone was significantly low in revival rate in incubation compared to the control group. The revival rate of the X-XO and catalase group in incubation was significantly high compared to that of the X-XO alone group. Also high revival rates were obtained in both groups of X-XO and SOD, and X-XO and hypotaurine after incubation showed significantly high values in the sperm with X-XO and catalase, and with X-XO and hypotaurine compared to that of the X-XO alone group.<br>These results indicate that ROS adversely affect motility in the process of freezing and thawing of boar sperm. Catalase completely inhibited the decline of motility in the presence of X-XO indicating that hydrogen peroxide is the primary ROS affecting sperm motility. Hypotaurine is also valuable to inhibit adverse effect of ROS.

Journal

  • Nihon Yoton Gakkaishi

    Nihon Yoton Gakkaishi 38(1), 12-19, 2001-03-10

    The Japanese Society of Swine Science

References:  26

Cited by:  1

Codes

  • NII Article ID (NAID)
    10008284189
  • NII NACSIS-CAT ID (NCID)
    AN10202971
  • Text Lang
    JPN
  • Article Type
    Journal Article
  • ISSN
    0913882X
  • NDL Article ID
    5700998
  • NDL Source Classification
    ZR22(科学技術--農林水産--畜産)
  • NDL Call No.
    Z18-1082
  • Data Source
    CJP  CJPref  NDL  J-STAGE  JASI 
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