Analysis of Membrane Integrity and Mitochondrial Activity in Fresh and Cryopreserved Boar Sperm Using Flow Cytometry

  • Park C. S. (Division of Animal Science and Resources, Research Center of Transgenic Cloned Pigs, Chungnam National University) ;
  • Li Z. H. (Division of Animal Science and Resources, Research Center of Transgenic Cloned Pigs, Chungnam National University) ;
  • Sung N. D. (Division of Animal Science and Resources, Research Center of Transgenic Cloned Pigs, Chungnam National University) ;
  • Jin D. I. (Division of Animal Science and Resources, Research Center of Transgenic Cloned Pigs, Chungnam National University) ;
  • Cong P. Q. (Division of Animal Science and Resources, Research Center of Transgenic Cloned Pigs, Chungnam National University) ;
  • Kim E. S. (Division of Animal Science and Resources, Research Center of Transgenic Cloned Pigs, Chungnam National University) ;
  • Song E. S. (Division of Animal Science and Resources, Research Center of Transgenic Cloned Pigs, Chungnam National University) ;
  • Yi Y. J. (Division of Animal Science and Resources, Research Center of Transgenic Cloned Pigs, Chungnam National University)
  • Published : 2005.12.01

Abstract

This study was carried out to evaluate the effects of washing medium, breed and washing temperature of fresh and frozen-thawed boar sperm on mitochondrial activity and membrane integrity by flow cytometry. More than $80\%$ of fresh sperm washed with mTLP-PVA medium at $20^{\circ}C$ exhibited an intact membrane and a functional mitochondrion. With frozen-thawed samples, a large number of sperm showed both damaged membrane $(36.4\~46.9\%)$ and nonfunctional mitochondrion $(55.1\~71.1\%)$ in the mTLP-PVA and BTS washing media at $20^{\circ}C$. There were no breed effects of fresh and frozen-thawed sperm on mitochondrial activity and membrane integrity. The percentages of damaged membrane of fresh and frozen sperm, respectively, were higher at $4^{\circ}C$ washing temperature than at $20^{\circ}C$ washing temperature in the mTLP-PVA medium. We found that washing medium and washing temperature of fresh and frozen-thawed boar sperm were important for the analyses of mitochondrial activity and membrane integrity by flow cytometry.

Keywords

References

  1. Almlid T, Johnson LA (1988): Effects of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa frozen in straws. J Anim Sci 66:2899-2905 https://doi.org/10.2527/jas1988.66112899x
  2. Auger J, Ronot X, Dadoune JP (1989): Human sperm mitochondrial function related to motility: a flow and image eytometric assessment. J Androl 10:439-448 https://doi.org/10.1002/j.1939-4640.1989.tb00135.x
  3. Clarke RN, Johnson LA (1987): Effect of liquid storage and cryopreservation of boar spermatozoa on acrosomal integrity and the penetration of zona-free hamster ova in vitro. Gamete Res 16:193-204 https://doi.org/10.1002/mrd.1120160302
  4. Crabo BG, Dial GD (1992): Artificial insemination in swine. Vet Clin North Am Food Anim Pract 8:533-544 https://doi.org/10.1016/S0749-0720(15)30702-7
  5. Evenson DP, Oarzynkiewicz Z, Melamed MR (1982): Simultaneous measurement by flow eytometry of sperm cell viability and mitochondrial membrane potential related to cell motility. J Histochem Cytochem 30: 279-280 https://doi.org/10.1177/30.3.6174566
  6. Gamer DL, Pinkel D, Johnson LA, Pace MM (1986): Assessment of spermatozoal function using dual fluorescent staining and flow cytometric analyses. BioI Reprod 34:127-138 https://doi.org/10.1095/biolreprod34.1.127
  7. Gamer DL, Johnson LA, Allen CH (1988): Fluorometric evaluation of cryopreserved bovine spermatozoa extended in egg yolk and milk. Theriogenology 30:369-378 https://doi.org/10.1016/0093-691X(88)90184-7
  8. Hofmo PO, Almlid T (1991): Recent developments in freezing of boar semen with special emphasis on cryoprotectants. In Boar Semen Preservation Vol. 2, Proceedings of the 2nd International Congress on Boar Semen Preservation. Reprod Domest Anim 1:111-122
  9. Johnson LA (1985): Fertility results using frozen boar spermatozoa: 1970 to 1985 In: Deep Freezing of Boar Semen (Ed. L. A. Johnson and K. Larsson). Swedish University of Agricultural Science, Uppsala pp 199- 222
  10. Kramer RY, Gamer DL, Bruns ES, Ericsson SA, Prins GS (1993): Comparison of motility and flow cytometric assessments of seminal quality in fresh, 24-hour extended and cryopreserved human spermatozoa. J AndroI 14: 374-384
  11. Matyus L, Szabo G Jr, Resli I, Gaspar R Jr, Damjanovich S (1984): Flow cytometric analysis of viability of bull sperm cells. Acta Biochim Biophys Acad Sci Hung 19: 209-214
  12. Morris GJ, Wastson PF, (1984): Cold shock injury - a comprehensive bibliography. Cryo-Letters 5:352-372
  13. Ogier De Baulny B, Le Vern Y, Kerboeuf D, Maisse G (1997): Flow cytometric evaluation of mitochondrial activity and membrane integrity in fresh and cryopreserved rainbow trout (Oncorhynchus mykiss) spermatozoa. Cryobiology 34:141-149 https://doi.org/10.1006/cryo.1996.1992
  14. Pinkel D, Dean P, Lake S, Peters D, Mendelsohn M, Gray J, Van Dilla M, Gledhill B (1979): Flow cytometry of mammalian sperm: progress in DNA and morphology measurement. J Histochem Cytochem 27:353-358
  15. Pursel VG, Park CS (1985): Freezing and thawing procedures for boar spermatozoa, 1st Int. Conf. Deep Freezing of Boar Semen. Swedish University of Agricultural Science, Uppsala pp 147-166
  16. Pursel VG, Johnson LA (1975): Freezing of boar spermatozoa. Fertilizing capacity with concentrated semen and a new thawing procedure. J Anim Sci 40: 99-102 https://doi.org/10.2527/jas1975.40199x
  17. Pursel VG, Johnson LA, Rampacek GB (1972): Acrosome morphology of boar spermatozoa incubated before cold shock. J Anim Sci 34:278-283 https://doi.org/10.2527/jas1972.342278x
  18. SAS Institute Inc (1996): SAS/STAT guide for personal computer 6.12. SAS Inst. Inc., Cary, NC, USA
  19. Watson PF, Plummer JM (1985): The responses of boar sperm membranes to cold shock and cooling. 1st Int. Conf. Deep Freezing of Boar Semen. Swedish University of Agricultural Science, Uppsala pp 113-127
  20. Yi YJ, Ko HJ, Lee SH, Yang CB, Son DS, Kim HK, Park CS (2004): In vitro fertilization and development of pig oocytes inseminated with boar sperm by different sperm washing media after thawing of the frozen straws. Asian-Aust J Anim Sci 17:164-167 https://doi.org/10.5713/ajas.2004.164
  21. Yi YJ, Kwon YA, Ko HJ, Park CS (2002): Effects of diluent component, freezing rate, thawing time and thawing temperature on acrosome morphology and motility of frozen- thawed boar sperm. Asian-Aust J Anim Sci 15:1553-1558 https://doi.org/10.5713/ajas.2002.1553
  22. Yoshida M, Ishigaki K, Pursel VG (1992): Effect of maturation media on male pronucleus formation in pig oocytes matured in vitro. Mol Reprod Dev 31:68-71 https://doi.org/10.1002/mrd.1080310112