The Effect of Seminal Plasma on Chilling and Freezing of Canine Spermatozoa

개 정액의 정장이 개정자의 냉각과 동결에 미치는 영향

  • You, Myung-Jo (College of Veterinary Medicine & Bio-safety Research Institute, Chonbuk National University) ;
  • Lee, John-Hwa (College of Veterinary Medicine & Bio-safety Research Institute, Chonbuk National University) ;
  • Kim, In-Shik (College of Veterinary Medicine & Bio-safety Research Institute, Chonbuk National University) ;
  • Park, Jin-Ho (College of Veterinary Medicine & Bio-safety Research Institute, Chonbuk National University) ;
  • Kwon, Jung-Kee (College of Veterinary Medicine & Bio-safety Research Institute, Chonbuk National University) ;
  • Kim, Jong-Hoon (College of Veterinary Medicine & Bio-safety Research Institute, Chonbuk National University) ;
  • Kim, Bum-Seok (College of Veterinary Medicine & Bio-safety Research Institute, Chonbuk National University) ;
  • Yu, Il-Jeoung (College of Veterinary Medicine & Bio-safety Research Institute, Chonbuk National University)
  • 유명조 (전북대학교 수의과대학.생체안정성 연구소) ;
  • 이존화 (전북대학교 수의과대학.생체안정성 연구소) ;
  • 김인식 (전북대학교 수의과대학.생체안정성 연구소) ;
  • 박진호 (전북대학교 수의과대학.생체안정성 연구소) ;
  • 권중기 (전북대학교 수의과대학.생체안정성 연구소) ;
  • 김종훈 (전북대학교 수의과대학.생체안정성 연구소) ;
  • 김범석 (전북대학교 수의과대학.생체안정성 연구소) ;
  • 유일정 (전북대학교 수의과대학.생체안정성 연구소)
  • Published : 2007.12.31

Abstract

Seminal plasma(SP) is usually removed from semen that is to be cryopreserved. However, some reports indicate that SP has beneficial effects on spermatozoa during chilling and freezing. The purpose of this study was to determine the effect of SP on sperm survival by adding SP to the extender before cooling and freezing canine spermatozoa. In replicate experiments, ejaculates obtained from four healthy dogs(1-4 years old) of various breeds were pooled, centrifuged at $300{\times}g$ for 10 min at $25^{\circ}C$, and the supernatant of seminal plasma was decanted. Spermatozoa were suspended in egg yolk-Tris(EYT) buffer. The study comprised two experiments: [Exp 1] Sperm were suspended in EYT extender containing either 0, 20, 40, 80 or 100% SP and were slowly cooled to $4^{\circ}C$ for 2h or held at $25^{\circ}C$ as controls. Sperm concentration was adjusted to $2{\times}10^8/ml$. [Exp II] Sperm samples, each of which contained $1{\times}10^8/ml$, were assigned to nine groups to be frozen. In the first four groups, sperm in EYT containing either 20, 40, 80 or 100% SP were cooled to $4^{\circ}C$, then diluted to contain final concentrations of EYT+0.6M glycerol and then were frozen. The final concentrations of SP were 10, 20, 40 or 50%. In the other four groups, sperm in EYT alone were first cooled slowly to $4^{\circ}C$, then diluted to contain final concentrations of EYT+0.6M glycerol plus 10, 20, 40 or 50% SP and then were frozen. Spermatozoa, which chilled in EYT alone and diluted to contain final concentrations of EYT+0.6M glycerol without seminal plasma, and then frozen, was regarded as control. Spermatozoa were frozen at $25^{\circ}C/min$ of cooling rate in plastic straws that were suspended above liquid nitrogen and thawed in water at $38^{\circ}C$ for 1 min. Sperm survival was assayed by determining progressive motility and integrity of plasma and acrosome membranes. Progressive motility was determined by microscopic examination at $200{\times}$ magnification. Membrane integrity was assessed by use of a double fluorescent dye, and acrosome integrity by staining sperm with Pisum sativum agglutinin. The results of the first experiment showed that adding SP did not improve motility of spermatozoa compared to those incubated without SP regardless of temperature. The results of the second experiment showed that spermatozoa suspended in EYT+0.6M glycerol containing SP exhibited the higher progressive motility before being frozen(P<0.05). However, frozen-thawed spermatozoa that had suspended in EYT+0.6M glycerol containing SP showed the similar or lower viability(P<0.05). In summary, although seminal plasma did not affect spermatozoa that were chilled in EYT without cryoprotectant(CPA), addition of seminal plasma to EYT containing CPA did significantly improved progressive motility of canine spermatozoa that were chilled.

개 정액의 정장은 정자를 동결 보존하기전 정액에서 제거되어진다. 그러나 근래 개정장이 정자를 냉각하고 동결하는데 유효한 효과가 있는 것으로 보고되고 있다. 그러므로 본 연구에서는 정자를 냉각과 동결하기 전 정장을 정자 희석액에 첨가하여 정장이 정자의 생존성에 미치는 영향을 알아보고자 하였다. 건강한 4마리의 수캐로부터 정액을 채취 혼합한 후 300g에서 10분간 원심분리하여 얻은 상층액을 다시 원심분리하고 상층액을 회수하여 정장으로 사용하였다. 정자의 희석액으로는 Egg yolk-Tris(EYT)를 사용하였다. 다음과 같이 두 가지 실험을 실시하였다. 실험 1: 정장이 0, 20, 40, 80, 100%비율로 포함된 EYT배지에 정자를 희석하여 두 시간 동안 $4^{\circ}C$$25^{\circ}C$에서 보관하였다. 정자수는 $2{\times}10^8/ml$로 조정하였다. 실험 2: 전체적으로 9개 실험군으로 구분하여 4개 실험군은 정장이 20, 40, 80, 100%포함된 EYT배지에 각각 정자를 희석하여 $4^{\circ}C$까지 냉각한 후 EYT+1.2 M glycerol배지로 2차 희석하여 동결하였다. 나머지 4개의 실험군은 EYT배지에 정자를 희석하여 $4^{\circ}C$까지 냉각한 후 각각 정장이 20, 40, 80, 100%포함된 EYT+1.2 M glycerol배지로 2차 희석하여 동결하였다. 대조군으로서 정장을 포함하지 않은 EYT배지에 정자를 희석하여 $4^{\circ}C$까지 냉각한 후 EYT+1.2 M glycerol배지로 2차 희석하여 동결하였다. glycerol의 최종농도는 0.6M, 정장의 최종농도는 10, 20, 40, 50%이었으며, 정자수는 $1{\times}10^8/ml$로 조정하였다. 정자를 straw에 충진하고 분당 $25^{\circ}C$의 냉각율과 액체질소를 이용하여 동결하였다. 융해는 $38^{\circ}C$에서 1분간 실시하였다. 정자 운동성, 정자 형질막 보존성(생존력), 정자 첨단체의 보존성을 검사하여 정자의 생존성을 검증하였다. 정자의 운동성은 400배율에서 현미경으로 관찰하였으며 생존력과 첨단체 보존성은 각각 이중형광염색과 Pisum sativum agglutinin을 이용하여 검사하였다. 실험 1의 결과는 보존온도와는 관련없이 희석액에 정장을 첨가하는 것은 정장이 포함되지 않은 희석액과 비교하여 정자의 운동성을 향상시키지 못했다. 실험 2 결과, 동결 전 냉각상태에서 정장을 포함한 EYT+0.6 M glycerol배지에 희석되었던 정자가 정장을 포함하지 않은 EYT+0.6 M glycerol배지보다 높은 진행운동성을 보였다(P<0.05). 그러나, 동결 융해 후 정장을 포함한 EYT+0.6 M glycerol배지에 희석되었던 정자의 생존력은 정장을 포함하지 않은 EYT+0.6 M glycerol배지보다 낮거나 비슷하게 나타났다. 본 실험의 결과를 요약하면 비록 정장이 동결보호제가 포함되지 않은 EYT에 혼합되었을 경우는 냉각되어진 정자의 생존성을 향상시키지는 못했으나 동결보호제가 포함된 EYT에 정장을 혼합한 경우 정장은 정자의 냉각 후 정자의 운동성을 향상시켰다.

Keywords

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