Reproductive and Developmental Biology

The Korean Society of Animal Reproduction (한국동물번식학회)
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Effect of Ethylene Glycol Concentration and Freezing Speed on Post-thawed Semen Viability and Acrosome Integrity in Korean Jeju Black Bull

제주흑우 동결정액 제조시 Ethylene Glycol의 농도와 예비 동결 조건이 정자의 생존율 및 첨체양상에 미치는 영향

  • Choi, Sun-Ho (Subtropical Animal Experiment Station, National Institute of Animal Science, RDA) ;
  • Ko, Min-Hee (Subtropical Animal Experiment Station, National Institute of Animal Science, RDA) ;
  • Kang, Tae-Young (Subtropical Animal Experiment Station, National Institute of Animal Science, RDA) ;
  • Cho, Sang-Rae (Subtropical Animal Experiment Station, National Institute of Animal Science, RDA) ;
  • Park, Yong-Sang (Subtropical Animal Experiment Station, National Institute of Animal Science, RDA) ;
  • Oh, Shin-Ae (Subtropical Animal Experiment Station, National Institute of Animal Science, RDA)
  • 최선호 (농촌진흥청 국립축산과학원 난지축산시험장) ;
  • 고민희 (농촌진흥청 국립축산과학원 난지축산시험장) ;
  • 강태영 (농촌진흥청 국립축산과학원 난지축산시험장) ;
  • 조상래 (농촌진흥청 국립축산과학원 난지축산시험장) ;
  • 박용상 (농촌진흥청 국립축산과학원 난지축산시험장) ;
  • 오신애 (농촌진흥청 국립축산과학원 난지축산시험장)
  • Received : 2011.09.09
  • Accepted : 2011.09.22
  • Published : 2011.09.30
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Abstract

The objective of this study was to examine effect of ethylene glycol for semen cryopreservation in Korean Jeju Black Bull. The semen was cryopreserved with extenders containing cryoprotectants (7% glycerol and 3%, 5%, 7% ethylene glycol) and packed to 0.5 ml straws. The semen straws were located above 3 cm of liquid nitrogen for 5 min, 5 cm for 10 min and 8 cm for 10 min. And then frozen straw was plunged into $LN_2$. Post-thawed sperm motility, viability and membrane integrity were significantly higher in 5% ethylene glycol ($72.5{\pm}5.00%$, $54.88{\pm}0.66%$ and $46.00{\pm}2.40%$; p<0.05). Motility and viability were similar between 7% glycerol and 5% ethylene glycol. However, the membrane integrity was significantly higher in 5% ethylene glycol ($34.69{\pm}4.64%$ vs $46.00{\pm}2.40%$; p<0.05). The viability and membrane integrity were significantly higher in 5 cm for 10 min and 8 cm for 10 min than 3 cm for 5 min (viability: $55.81{\pm}2.94$, $55.19{\pm}3.34$ vs $47.94{\pm}3.48%$; p<0.05 and membrane integrity: $44.94{\pm}3.51$, $46.06{\pm}2.25$ vs $40.38{\pm}1.03%$; p<0.05). The percentage of capacitated sperm assessed by CTC staining, percentage of F pattern was higher in 7% glycerol, 5% and 7% ethylene glycerol, and AR pattern was significantly higher in 3% ethylene glycol. F pattern was significantly increased in 5 cm for 10 min and 8 cm for 10 min (p<0.05), but AR pattern was significantly increased in 3 cm for 5 min (p<0.05).

본 연구에서는 제주흑우의 유전자원 보존과 증식을 위한 안정적인 동결정액 제조법을 수립하기 위하여 제주흑우의 정액 동결 제조에 있어 동결 융해 후 ethylene glycol이 정자의 운동성, 생존율 그리고 첨체에 미치는 영향에 대하여 알아보고자 수행하였다. 제주흑우의 동결정액제조시 5%의 ethylene glycol의 첨가가 $72.5{\pm}5.00%$의 활력과 $54.88{\pm}0.66%$의 생존율 그리고 $46.00{\pm}2.40%$의 정자막 온전성을 나타내 3%와 5%의 ethylene glycol을 첨가한 실험구보다 유의적으로 높은 결과를 나타냈다(p<0.05). 또한, 7% glycerol을 사용한 실험구와 비교하였을 때 5% ethylene glycol을 첨가하였을 때 유의적으로 높은 생존율과 정자막 온전성을 나타냈다(p<0.05). 또한, 예비 동결 조건이 정자의 성상에 미치는 영향에 있어서는 3 cm, 5분 예비동결 실험구에서 생존율과 정자막 온전성이 유의적으로 감소하였다(p<0.05). Ethylene glycol이 정자의 첨체 양상에 미치는 영향에 있어서는 5%와 7%의 ethylene glycol을 사용하였을 때 효과적으로 첨체가 보호되어 F pattern의 비율이 유의적으로 높게 나타났으며(p<0.05), 조기 첨체 반응도 유의적으로 낮은 수준을 보였다(p<0.05). 그러나 7%의 glycerol과 비교하였을 때는 유의적인 차이를 나타내지 않았으며, 5%와 7% ethylene glycol 사이의 유의적 차이도 나타나지 않았다. 예비 동결 조건이 정자의 첨체 양상에 미치는 영향에 있어서는 역시 정자의 생존율과 정자막 온전성과 같이 3 cm, 5분 예비동결 시 유의적인 F pattern의 감소와 조기 첨체 반응에 의한 AR paattern의 증가를 볼 수 있었다(p<0.05). 이와 같은 결과는 본 연구에서 사용된 ethylene glycol과 예비동결 조건의 조합으로 나타난 결과를 활용하여, 희소가축의 생식세포 보존 및 유전자원 확립을 위한 중요한 자료가 될 것이며, 생존율과 운동성 증대를 위한 다양한 희석제를 활용한 연구가 필요할 것으로 사료된다.

Keywords

References

  1. Abdel-Khalek EA, Shalaby NA, Eid LN, Gbr AA (2008): Effect of various types and levels of cryoprotectants on sperm motility during freezing processes of Egyptian Buffalo semen. J Agric Sci Mansoura Univ 33:7157-7171.
  2. Alvarenga Ma, Papa FO, Landim-Alvarenga FC, Medeiros AS (2005): Amides as cryoprotectants for freezing stallion semen: a review. Anim Reprod Sci 89:105-113. https://doi.org/10.1016/j.anireprosci.2005.07.001
  3. Amann RP, Pickett BW (1987): Principles of cryopreservation and a review of cryopreservation on stallion spermatozoa. Equine Vet Sci 7:145-176. https://doi.org/10.1016/S0737-0806(87)80025-4
  4. Ashwood-Smith MJ (1987): Mechanisms of cryoprotectant action. Symp Soc Exp Biol 41:395-406.
  5. Ball BA, Vo A (2001): Osmotic tolerance of equine spermatozoa and the effects of soluble cryoprotectants on equine sperm motility, viability and mitochondrial membrane potential. J Androl 22:1061- 1069.
  6. Brisola LBS, Neves JPm, Goncalves PBD, Oliveira JFC, Montagner MM (1999): Integridade das membranas plasmaticas, nuclear e mitocondrial de espermatozoides ovinos criopreservados na vaca. Ciencia Rural 29:527-531. https://doi.org/10.1590/S0103-84781999000300025
  7. Buhr MM, Fiser P, Bailey JL, Curtis EF (2001): Cryopreservation in different concentration of glycerol alters boar sperm and their membranes. J Androl 22:961-969.
  8. Chenier T, Merkies K, Leibo S, Plante C, Johnson W (1998): Evaluation of cryoprotective agents for use in the cryopreservation of equine spermatozoa. Proc. 44th Annual Convention of the American Association of Equine Practitioners, Baltilmore, Maryland, USA, pp 5-6.
  9. Fraser LR, Abeydeera LR, Niwa K (1995): $Ca^{2+}$-regulating mechanisms that modulate bull sperm capacitation and acrosomal exocytosis as determined by chlortetracycline analysis. Mol Reprod Dev 40: 233-241. https://doi.org/10.1002/mrd.1080400213
  10. Gilmore JA, McGann LE, Liu J, Gao DY, Peter AT, Kleinhans FW, Critser JK (1995): Effect of cryoprotectant solutes on water permeability of human spermatozoa. Biol Reprod 53:985-995. https://doi.org/10.1095/biolreprod53.5.985
  11. Gilmore JA, Du J, Tao J, Peter AT, Critser JK (1996): Osmotic properties of boar spermatozoa and their relevance to cryopreservation. J Reprod Fertil 107: 87-95. https://doi.org/10.1530/jrf.0.1070087
  12. Gilmore JA, Liu J, Gao DY, Peter AT, Critser JK (1998): Determination of plasma membrane characteristics of boar spermatozoa and their relevance to cryopreservation. Biol Reprod 58:28-36. https://doi.org/10.1095/biolreprod58.1.28
  13. Gilmore JA, Liu J, Woods EJ, Peter AT, Critser JK (2000): Cryoprotective agent and temperature effects on human sperm membrane permeabilities: convergence of theoretical and empirical approaches for optimal cryopreservation methods. Hum Reprod 15: 335-343. https://doi.org/10.1093/humrep/15.2.335
  14. Guthrie HD, Liu J, Critser JK (2002): Osmotic tolerance limits and effects of cryoprotectants on motility of bovine spermatozoa. Biol Reprod 67:1811- 1816. https://doi.org/10.1095/biolreprod67.6.1811
  15. Hammerstedt RH, Graham JK, Nolan JP (1990): Cryopreservation of mammalian sperm: what we ask them to survive. J Androl 11:73-88.
  16. Hammerstedt RH, ,Graham JK (1992): Cryopreservation of poultry sperm: the enigma of glycerol. Cryobioloby 29:26-38. https://doi.org/10.1016/0011-2240(92)90004-L
  17. Harrison RAP, Ashworth PJC, Miller NGA (1996): Assessment of sperm function under fertilizing conditions. Reprod Dom Anim 31:25-30.
  18. Jeyendran RS, Van der Ven HH, Prez-Pelaez M, Carbo BG, Zaneveld LJ (1984): Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil 70:219-228. https://doi.org/10.1530/jrf.0.0700219
  19. Mantovani R, Rota A, Falomo ME, Bailoni L, Vincenti L (2002): Comparison between glycerol and ethylene glycol for the cryopreservation of equine spermatozoa: sperm quality assessment with standard analyses and with the hypoosmotic swelling test. Reprod Nutr Dev 42:217-226. https://doi.org/10.1051/rnd:2002020
  20. McGonagle LS, Goldstein M, Feldschuh J, Foote RH (2002): The influence of cryoprotective media and processing procedures on motility and migration of frozen- thawed human sperm. Asian J Androl 4:137-141.
  21. Moraes CN, Neves JP, Goncalves PBD, Oliveira JFC, Schweitzer CM (1998): Criopreservac, ao do semen ovino em pellets com etileno glicol (ethylene glycol for freezing ram semen in pellets). Ciencia Rural 28:287-292. https://doi.org/10.1590/S0103-84781998000200018
  22. Pelps MJ, Liu J, Benson JD, Willoughby CE, Gilmore JA, Critser JK (1999): Effects of Percoll separation, cryoprotective agents and temperature on plasma membrane permeability characteristics of murine spermatozoa and their relevance to cryopreservation. Biol Reprod 61:1031-1041. https://doi.org/10.1095/biolreprod61.4.1031
  23. Pena AI, Lopez-Lugilde L, Barrio M, Becerra JJ, Quintela LA, Herradón PG (2003): Studies on the intracellular $Ca^{2+}$ concentration of thawed dog spermatozoa: influence of Equex from different sources, two thawing diluents and post-thaw incubation in capacitating conditions. Reprod Dom Anim 38:27- 35. https://doi.org/10.1046/j.1439-0531.2003.00391.x
  24. Pereira SM, Rigon RM, Mezzalira A, Cecim M (2002): Ethylene glycol on canine semen cryopreservation. Ciencia Rural 32:649-655. https://doi.org/10.1590/S0103-84782002000400017
  25. Polge C, Smith AU, Parkes AS (1945): Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164:666.
  26. Rohilla RK, Tuli Rk, Goyal RL (2005): Comparative study of the effects of cryoprotective agents in freezing Murrah Buffalo bull semen. Indian J Vet Res 14:37-43.
  27. Sieme H, Harrison RA, Petrunkina AM (2008): Cryobiological determinants of frozen semen quality with special reference to stallion. Anim Reprod Sci 107:276-292. https://doi.org/10.1016/j.anireprosci.2008.05.001
  28. Squires EL, Keith SL, Graham JK (2004): Evaluation of alternative cryoprotectants for preserving stallion spermatozoa. Theriogenology 62:1056-1065. https://doi.org/10.1016/j.theriogenology.2003.12.024
  29. Swelum AA, Mansour HA, Elsayed AA, Amer HA (2011): Comparing ethylene glycol with glycerol for cryopreservation of buffalo bull semen in egg-yolk containing extenders. Theriogenology 76:833-842. https://doi.org/10.1016/j.theriogenology.2011.04.015
  30. Szasz F, Sirivaidyapong Sm, Cheng FP, Voorhout WF, Marks A, Colenbrander B, Solti And L, Gadella BM (2000): Detection of calcium ionophore induced membrane changes in dog sperm as a simple method to predict the cryopreservability of dog semen. Mol Reprod Dev 55:289-298. https://doi.org/10.1002/(SICI)1098-2795(200003)55:3<289::AID-MRD7>3.0.CO;2-K
  31. Vidament M, Daire C, Yvon JM, Doligez P, Bruneau B, Magistrini M (2002): Motility and fertility of stallion semen frozen with glycerol and/or dimethyl formamide. Theriogenology 58:249-251. https://doi.org/10.1016/S0093-691X(02)00854-3
  32. Willoughby CE, Mazur P, Peter AT, Critser JK (1996): Osmotic tolerance limits and properties murine spermatozoa. Biol Reprod 55:715-727. https://doi.org/10.1095/biolreprod55.3.715
  33. 이제협, 박 향, 박흠대, 김재명 (2003): 개 정자의 동결 보존에 있어서 Glycerol 농도, 동결 및 융해 속도가 정자의 생존율 및 운동성에 미치는 영향. 한국수정란이식학회지 18:195-201.
  34. 조상래, 최선호, 최창용, 손준규, 김재범, 김성재, 손동수, 김현종 (2009): AndroMed를 이용한 흑우 동결 정액으로 체외수정란 생산 효과. 한국수정란이식학회지24:207-212.
  35. 최선호, 김성재, 조상래, 최창용, 손준규, 유용희, 조영재, 최귀철, 문윤영 (2010): 말 정액 동결시 Glycerol농도와 동결 속도가 생존율에 미치는 영향. 한국동물번식학회지 34:271-274.