ブタ新鮮および凍結・融解精子と同種未成熟卵子の培養による精子侵入試験  [in Japanese] In vitro Penetration Assay of Fresh and Frozen/Thawed Porcine Sperm by Co-incubation with Homologous Immature Oocytes  [in Japanese]

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Abstract

ブタ未成熟卵子をもちいた同種精子受精能評価法の開発を目的に新鮮射出 (Fresh) および凍結・融解 (F/T) 精子と未成熟卵子を培養して, 培養後の卵子への精子侵入について調べた。卵胞内の卵丘細胞卵子複合体 (COCs) を採取し, m-M199で洗浄後, 50μ<i>l</i>のm-M199のスポットに30個ずつ導入した。さらに, 0.5%ヒアルロニダーゼ処置とピペッティングによって卵丘細胞を除去した卵子 (DOs) も同様にスポットに導入した。供試精液は手圧法により大ヨークシャー種, デュロック種および雑種の2頭の計4頭の種雄豚より採取した。採取精液をm-M199で洗浄後, 精子数3.0×10<sup>8</sup>/m<i>l</i>に希釈し, 38.5℃の5% CO<sub>2</sub>インキュベーター内で3時間前培養したものを Fresh 精子とした。さらに, Fresh 精子と同じ雄の精液を凍結, 融解後, m-M199で洗浄して, 精子数が3.0×10<sup>8</sup>/m<i>l</i>になるように希釈したものをF/T精子とした。培養時の精子濃度は, Fresh 精子では1.0×10<sup>6</sup>/m<i>l</i>, F/T精子では1.0×10<sup>7</sup>/m<i>l</i>となるようにし, 38.5℃, 5%CO<sub>2</sub>, 95%空気, 湿度飽和の条件下で18-20時間培養した。その後, 卵子のホールマウント標本を作成し, 侵入精子の有無, 侵入精子数を観察した。COCsおよびDOsへの精子侵入率はそれぞれ Fresh 精子で50.9±4.5%および54.6±7.2%, F/T精子で16.7±3.3%および28.0±3.9%であった。F/T精子は卵丘細胞の付着にかかわらず Fresh 精子よりも有意に低い値となった (P<0.001)。COCsとDOsへの卵子あたりの侵入精子数は, Fresh 精子で9.3±0.7および14.5±0.9で, DOsの方が, 有意に多かった (P<0.01)。また, F/T精子ではそれぞれ1.9±0.1, 1.7±0.1で, 有意な差は認められず, 卵丘細胞の付着にかかわらず Fresh 精子よりも有意に少なかった (P<0.001)。以上のことから, ブタ精子の受精能評価法として, 精子と未成熟卵子の培養後の卵子への精子侵入率, 侵入精子数がその精液の受精能を反映することが示唆された。

To develop a method for assessment of porcine sperm fertility using coincubation with immature homologous oocytes, we examined oocyte penetration by fresh and frozen/thawed sperm after co-incubation. Cumulus-oocyte complexes (COCs) were dissected from follicles with a diameter of 2-6mm and washed in m-M 199. COCs were introduced into a droplet of m-M199. Also, COCs denuded by 0.5% hyaluronidase and pipetting (denuded oocytes, DOs) were introduced into a droplet of m-M199. Sperm was collected by gloved-hand method from four boars (one Large white, one Duroc and 2 hybrid). Collected semen was washed and diluted to 3×10<sup>8</sup> cells/m<i>l</i> in m-M199. Semen suspension was pre-incubated for 3 hrs at 38.5°C under 5% CO<sub>2</sub> in humidified air and used as fresh sperm. The collected semen was frozen, thawed, and diluted to 3×10<sup>8</sup> cells/m<i>l</i> in m-M199. This sperm suspension was used as frozen/thawed (F/T) sperm. The sperm concentration in a droplet for co-incubation with immature porcine oocytes was 1×10<sup>6</sup> cells/m<i>l</i> for fresh sperm, 1×10<sup>7</sup> cells/m<i>l</i> for F/T sperm. Co-incubation was performed for 18-20hrs at 38.5°C under 5% CO<sup>2</sup> in humidified air. After co-incubation, oocytes were fixed and stained for assessment of sperm penetration. Rates of COCs or DOs penetration of fresh and F/T sperm were 50.9% or 54.6% and 16.7% or 28.0%, respectively. The penetration rates of F/T sperm were significantly lower (p<0.001) than those of fresh sperm. The mean number of fresh sperm per a penetrated COC and a DO was 9.3 and 14.5, respectively. And the number per a DO was significantly higher (p<0.01) than that per a COC. The mean number of F/T sperm per a penetrated COC and DO was 1.9 and 1.7, respectively. Although there was no significant difference between them, the number of F/T sperm was significantly lower (p<0.001) than that of fresh sperm. The results of the present study indicated that rate of oocyte penetration and number of sperm per a penetrated oocyte after co-incubation with boar sperm and immature oocytes reflected fertility of boar semen.

Journal

  • Nihon Yoton Gakkaishi

    Nihon Yoton Gakkaishi 38(2), 59-66, 2001-06-20

    The Japanese Society of Swine Science

References:  37

Cited by:  1

Codes

  • NII Article ID (NAID)
    10008284281
  • NII NACSIS-CAT ID (NCID)
    AN10202971
  • Text Lang
    JPN
  • Article Type
    Journal Article
  • ISSN
    0913882X
  • NDL Article ID
    5816541
  • 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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