Journal of Veterinary Clinics (한국임상수의학회지)

The Korean Society of Veterinary Clinics (한국임상수의학회)
권호 표지

Effect of Thawing Rate on the Function of Cryopreserved Canine Sperm

융해 속도가 동결.융해된 개 정자의 기능에 미치는 영향

  • Kim, Su-Hee (Department of Veterinary Theriogenology, College of Veterinary Medicine, Chonbuk National University) ;
  • Yu, Do-Hyeon (*Department of Veterinary Internal Medicine, College of Veterinary Medicine, Chonbuk National University) ;
  • Kang, Tae-Woon (Department of Veterinary Theriogenology, College of Veterinary Medicine, Chonbuk National University) ;
  • Kim, Yong-Jun (Department of Veterinary Theriogenology, College of Veterinary Medicine, Chonbuk National University)
  • 김수희 (전북대학교 수의과대학) ;
  • 유도현 (전북대학교 수의과대학) ;
  • 강태운 (전북대학교 수의과대학) ;
  • 김용준 (전북대학교 수의과대학)
  • Accepted : 2011.12.15
  • Published : 2011.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

Sperm cryopreservation methods have been improved over the last few decades. However, an optimized thawing rate has not yet been established. Therefore, we investigated the effect of thawing rate on sperm function after cryopreservation. The ejaculates collected from beagle dogs were cryopreserved and then thawed at two different thawing rates ($37^{\circ}C$ for 1 min or $70^{\circ}C$ for 15 sec). The thawed sperm were evaluated for motility, viability, morphology, plasma membrane integrity, phosphatidylserine (PS) translocation, and intracellular $H_2O_2$ level. The sperm thawed rapidly at $70^{\circ}C$ showed improved motility, viability, normal morphology, plasma-membrane integrity and non-PS translocation compared to the sperm thawed slowly at $37^{\circ}C$ (P < 0.05). However, the intracellular $H_2O_2$ levels were not significantly different between the rapid- and slow-thawed sperm (P > 0.05). In conclusion, sperm rapid thawing at $70^{\circ}C$ could improve the function of cryopreserved canine sperm, and the appropriate thawing rate would enhance the quality of the cryopreserved sperm.

정액 동결 방법들은 지속적으로 향상되어 왔지만 융해 속도에 대한 연구는 여전히 정립되어 있지 않다. 따라서, 본 연구의 목적은 동결 후 융해 속도가 정액의 기능에 미치는 영향을 알아보고자 하였다. 비글견으로부터 채취된 정액은 동결 보존 후 다른 융해 속도 ($37^{\circ}C$/1분 or $70^{\circ}C$/15초)에서 융해 되었다. 융해 후, 운동성, 생존성, 정상 형태율, 형질막 온전성, phosphatidylserine (PS) translocation, 세포내 $H_2O_2$ 수준을 평가하였다. $70^{\circ}C$에서 융해된 정자는 $37^{\circ}C$에서 융해된 정자에 비해 향상된 정자 운동성, 생존성, 정상 형태율, 세포막 온전성, non-PS translocation을 보였으나(P < 0.05), $70^{\circ}C$와 $37^{\circ}C$에서 융해된 실험군간 세포내 $H_2O_2$ 수준에서 유의적인 차이는 나타나지 않았다(P > 0.05). 결론으로, $70^{\circ}C$에서의 융해는 개 정자 동결 후 정자 기능을 증진시켰으며, 적절한 융해 온도는 동결 정자의 기능을 향상시킬 수 있을 것으로 판단된다.

Keywords

References

  1. Abe Y, Lee DS, Sano H, Akiyama K, Yanagimoto-Ueta Y, Asano T, Suwa Y, Suzuki H. Artificial insemination with canine spermatozoa frozen in a skim milk/glucose-based extender. J Reprod Dev 2008; 54: 290-294. https://doi.org/10.1262/jrd.19148
  2. Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity. J Androl 1987; 8: 338-348. https://doi.org/10.1002/j.1939-4640.1987.tb00973.x
  3. Alvarez JG, Minaretzis D, Barrett CB, Mortola JF, Thompson IE. The sperm stress test: a novel test that predicts pregnancy in assisted reproductive technologies. Fertil Steril 1996; 65: 400-405. https://doi.org/10.1016/S0015-0282(16)58107-2
  4. Barbas JP, Mascarenhas RD. Cryopreservation of domestic animal sperm cells. Cell Tissue Bank 2009; 10: 49-62. https://doi.org/10.1007/s10561-008-9081-4
  5. Brito LF, Barth AD, Bilodeau-Goeseels S, Panich PL, Kastelic JP. Comparison of methods to evaluate the plasmalemma of bovine sperm and their relationship with in vitro fertilization rate. Theriogenology 2003; 60: 1539-1551. https://doi.org/10.1016/S0093-691X(03)00174-2
  6. Bucak MN, Atessahin A, Varisli O, Yuce A, Tekin N, Akcay A. The influence of trehalose, taurine, cysteamine and hyaluronan on ram semen Microscopic and oxidative stress parameters after freeze-thawing process. Theriogenology 2007; 67: 1060-1067. https://doi.org/10.1016/j.theriogenology.2006.12.004
  7. Buhr MM, Canvin AT, Bailey JL. Effects of semen preservation on boar spermatozoa head membranes. Gamete Res 1989; 23: 441-449. https://doi.org/10.1002/mrd.1120230409
  8. Calamera JC, Doncel GF, Olmedo SB, Kolm P, Acosta AA. Modified sperm stress test: a simple assay that predicts sperm-related abnormal in-vitro fertilization. Hum Reprod 1998; 13: 2484-2488. https://doi.org/10.1093/humrep/13.9.2484
  9. Fiser PS, Ainsworth L, Fairfull RW. Evaluation of a new diluent and different processing procedures for cryopreservation of ram semen. Theriogenology 1987; 28: 599-607. https://doi.org/10.1016/0093-691X(87)90276-7
  10. Glander HJ, Schaller J. Binding of annexin V to plasma membranes of human spermatozoa: a rapid assay for detection of membrane changes after cryostorage. Mol Hum Reprod 1999; 5: 109-115. https://doi.org/10.1093/molehr/5.2.109
  11. Gravance CG, Vishwanath R, Pitt C, Garner DL, Casey PJ. Effects of cryopreservation on bull sperm head morphometry. J Androl 1998; 19: 704-709.
  12. Guthrie HD, Welch GR. Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry. J Anim Sci 2006; 84: 2089-2100. https://doi.org/10.2527/jas.2005-766
  13. Kim SH, Yu DH, Kim YJ. Effects of cryopreservation on phosphatidylserine translocation, intracellular hydrogen peroxide, and DNA integrity in canine sperm. Theriogenology 2010; 73: 282-292. https://doi.org/10.1016/j.theriogenology.2009.09.011
  14. Mahfouz R, Sharma R, Lackner J, Aziz N, Agarwal A. Evaluation of chemiluminescence and flow cytometry as tools in assessing production of hydrogen peroxide and superoxide anion in human spermatozoa. Fertil Steril 2009; 92: 819-827. https://doi.org/10.1016/j.fertnstert.2008.05.087
  15. Mazur P, Koshimoto C. Is intracellular ice formation the cause of death of mouse sperm frozen at high cooling rates? Biol Reprod 2002; 66: 1485-1490. https://doi.org/10.1095/biolreprod66.5.1485
  16. Nothling JO, Shuttleworth R. The effect of straw size, freezing rate and thawing rate upon post-thaw quality of dog semen. Theriogenology 2005; 63: 1469-1480. https://doi.org/10.1016/j.theriogenology.2004.07.012
  17. Olar TT, Bowen RA, Pickett BW. Influence of extender, cryoperservative and seminal processing procedures on postthaw motility of canine spermatozoa frozen in straws. Theriogenology 1989; 31: 451-461. https://doi.org/10.1016/0093-691X(89)90550-5
  18. Pena A, Linde-Forsberg C. Effects of Equex, one- or two step dilution, and two freezing and thawing rates on postthaw survival of dog spermatozoa. Theriogenology 2000; 54: 859-875. https://doi.org/10.1016/S0093-691X(00)00397-6
  19. Ricci G, Perticarari S, Fragonas E, Giolo E, Canova S, Pozzobon C, Guaschino S, Presani G. Apoptosis in human sperm: its correlation with semen quality and the presence of leukocytes. Hum Reprod 2002; 17: 2665-2672. https://doi.org/10.1093/humrep/17.10.2665
  20. Rota A, Strom B, Linde-Forsberg C. Effects of seminal plasma and three extenders on canine semen stored at 4 degrees C. Theriogenology 1995; 44: 885-900. https://doi.org/10.1016/0093-691X(95)00278-G
  21. Rota A, Strom B, Linde-Forsberg C, Rodriguez-Martinez H. Effects of equex STM paste on viability of frozen-thawed dog spermatozoa during in vitro incubation at 38 degrees C. Theriogenology 1997; 47: 1093-1101. https://doi.org/10.1016/S0093-691X(97)00066-6
  22. Schiller J, Arnhold J, Glander HJ, Arnold K. Lipid analysis of human spermatozoa and seminal plasma by MALDI-TOF mass spectrometry and NMR spectroscopy - effects of freezing and thawing. Chem Phys Lipids 2000; 106: 145-156. https://doi.org/10.1016/S0009-3084(00)00148-1
  23. Verheyen G, Pletincx I, Van Steirteghem A. Effect of freezing method, thawing temperature and post-thaw dilution/washing on motility (CASA) and morphology characteristics of highquality human sperm. Hum Reprod 1993; 8: 1678-1684. https://doi.org/10.1093/oxfordjournals.humrep.a137912
  24. Watson PF. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their postthawing function. Reprod Fertil Dev 1995; 7: 871-891. https://doi.org/10.1071/RD9950871