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

Effect of Ethylene Glycol Concentration and Freezing Speed on Post-thawed Semen Viability and Acrosome Integrity in Korean Jeju Black Bull

  • 최선호 (농촌진흥청 국립축산과학원 난지축산시험장) ;
  • 고민희 (농촌진흥청 국립축산과학원 난지축산시험장) ;
  • 강태영 (농촌진흥청 국립축산과학원 난지축산시험장) ;
  • 조상래 (농촌진흥청 국립축산과학원 난지축산시험장) ;
  • 박용상 (농촌진흥청 국립축산과학원 난지축산시험장) ;
  • 오신애 (농촌진흥청 국립축산과학원 난지축산시험장)
  • 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)
  • 투고 : 2011.09.09
  • 심사 : 2011.09.22
  • 발행 : 2011.09.30

초록

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.

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).

키워드

참고문헌

  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.