Journal of the Korea Academia-Industrial cooperation Society (한국산학기술학회논문지)

The Korea Academia-Industrial cooperation Society (한국산학기술학회)
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Acrosome staining with Coomassie brilliant blue G or R on the horse spermatozoa

Coomassie brilliant blue G 및 R을 이용한 말 정액 첨체 염색 기법

  • Kim, Sung Woo (Animal Genetic Resources Research Center, National Institute of Animal Science, RDA) ;
  • Shin, Sang Min (Subtropical Livestock Research Institute, National Institute of Animal Science, RDA) ;
  • Yu, Yeonhee (Animal Genetic Resources Research Center, National Institute of Animal Science, RDA) ;
  • Lee, Jae-Yeong (Animal Genetic Resources Research Center, National Institute of Animal Science, RDA) ;
  • Kim, Chan-Lan (Animal Genetic Resources Research Center, National Institute of Animal Science, RDA) ;
  • Ko, Yeoung-Gyu (Animal Genetic Resources Research Center, National Institute of Animal Science, RDA)
  • 김성우 (국립축산과학원 가축유전자원센터) ;
  • 신상민 (국립축산과학원 난지축산연구소) ;
  • 유연희 (국립축산과학원 가축유전자원센터) ;
  • 이재영 (국립축산과학원 가축유전자원센터) ;
  • 김찬란 (국립축산과학원 가축유전자원센터) ;
  • 고응규 (국립축산과학원 가축유전자원센터)
  • Received : 2020.08.10
  • Accepted : 2020.09.04
  • Published : 2020.09.30
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Abstract

To develop simple acrosome staining of horse spermatozoa, this study tested the binding properties of Coomassie brilliant blue G or R on the sperm smears after 3.7% paraformaldehyde (PF) or 35% methanol (MT) fixation. After being fixed with PF and stained with 0.05, 0.1, or 0.2 % of CBB G or R for 2 min, horse spermatozoa were examined for their intact acrosome status. The intact acrosome of fresh horse spermatozoa were 62.6% and 61.5% with 0.05% of the G and R CBB solution, but 80.2 and 79.7% with G type and 78.1 and 76.0% with R type. On the other hand, when MT was used for fixation, the acrosome reacting sperm ratio was 3.5%, but was 9.0% in the case of PF. These results show that the intact acrosome of horse sperm could be judged using a 0.1~0.2% CBB G or R staining technique. PF would be an essential fixative for examining acrosome reacting horse spermatozoa. This method could be used to identify sperm with a damaged acrosome during low-temperature storage or cryopreservation for artificial insemination of horses.

말 정자의 첨체 손상도를 간단하게 분석하는 기술을 개발하기 위하여 Coomassie brilliant blue G 또는 R 염색 시약을 이용하여 정자 도말 슬라이드에 대한 염색도를 조사하였고, 3.7% paraformaldehyde (PF)과 35% methanol (MT) 고정제가 첨체 염색에 미치는 영향을 연구하였다. PF로 고정한 후 말 정자 도말 슬라이드를 0.05, 0.1 및 0.2 % 농도 CBB에 각 2분간 염색을 실시하여 첨체를 관찰하면 0.05%의 경우 완전한 첨체 염색도가 G형에서 62.6%, R형은 61.5%로 관찰되었으나, 0.1 및 0.2%의 경우, 80.2 및 79.7%로 (G 형), 78.1 및 76.0%로 (R 형) 관찰되어 유의적 차이가 나타났다. 그러나 MT 고정제를 이용할 경우, 첨체 외막이 느슨해진 정자의 비율은 3.5%로 관찰되어 PF 고정제를 사용하면 9.0%로 관찰되어 유의적으로 낮게 관찰되었다. 이러한 결과는 말정자첨체손상도를 관찰하기 위하여 CBB G 또는 R형 염색시약을 0.1~0.2% 농도로 이용될 수 있음을 증명한다. 또한, 말 정자 슬라이드 도말을 고정하는데 있어 고정제의 선택이 중요하며 MT보다 PF를 활용하는 것이 첨체 변화 과정 중인 정자를 관찰하는데 중요하다는 보여 준다. 이러한 방법은 말의 인공수정을 위한 정액의 저온저장 또는 동결보존과정 중에서 정자첨체손상율을 정확하게 판별하는데 이용될 수 있음을 증명한다.

Keywords

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