Reproductive and Developmental Biology

The Korean Society of Animal Reproduction (한국동물번식학회)
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Factors Affecting the Survival Rates of Frozen-Thawed Spermatozoa in Equine by Glycerol Concentration and Freezing Speed

말 정액 동결시 Glycerol 농도와 동결 속도가 생존율에 미치는 영향

  • Received : 2010.09.15
  • Accepted : 2010.09.20
  • Published : 2010.09.30
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Abstract

This study was conducted to investigate the survival rate of frozen-thawed spermatozoa in equine by glycerol concentration and freezing speed Two stallions (1 Thoroughbred-13 year old and 1 Arab-7 year old) bred in Korea Racing authority was examined for 1 times in a couple of weeks. Semen was collected by condom method standing heated mare and were centrifuged 650 g for 15 min. and isolated the seminal plasma. Thick fraction of semen was diluted EDTA-Lactose-egg yolk diluents to 1:1 and contained in 0.5 ml straw as $6{\sim}14{\times}10^7\;cells/ml$. Final concentrations of glycerol were 3, 5 and 7% in cryopreseved diluents and added 4 times for 2 hours equilibration. For the freezing, equilibrated straws were located 3 or 5 em above $LN_2$ gas for 5 or 10 min. Survival rates of pre-frozen sperm were $65.0{\pm}13.2%$, $68.3{\pm}10.4%$, $66.7{\pm}11.5%$ and post-frozen were $53.3{\pm}23.1%$, $45.0{\pm}15.0%$, $50.0{\pm}18.0%$ in 3, 5, 7% glycerol concentration, respectively. There was no difference between glycerol concentrations. Survival rates of frozen-thawed sperm on freezing speed were $36.7{\pm}10.4%$, $40.0{\pm}7.1%$, $30.0{\pm}13.2%$ at 3 cm-5 min and $33.3{\pm}11.5%$, $31.7{\pm}2.9%$, $21.7{\pm}10.4%$ at 3 cm-10 min in 3, 5, 7% glycerol concentration, respectively. Survival rates of frozen-thawed sperm on freezing speed were $43.3{\pm}15.3%$, $32.0{\pm}17.9%$, $22.3{\pm}15.7%$ at 5cm-5 min and were $47.5{\pm}15.0%$, $43.3{\pm}12.6%$, $48.3{\pm}15.3%$ at 5cm-10 min in 3, 5, 7% glycerol concentration, respectively. There were significantly different between groups (p<0.05). These results suggest that glycerol concentration did not affect cryopreservation of stallion semen within 3~7% but freezing speed affects. In our experiment, the best cryopreservation condition was at 5 cm above $LN_2$ gas for 10 min for pre-freezing and 7% of glycerol concentration. These results lead to commercial AI with frozen-thawed stallion semen.

Keywords

References

  1. Allen WR (2005): The development and application of the modem reproductive technologies to horse breeding. Reprod Dom Anim 40:310-329. https://doi.org/10.1111/j.1439-0531.2005.00602.x
  2. Backman T, Bruemmer JE, Graham JK, Squire EL (2004): Pregnancy rates of mare inseminated with semen cooled for 18 hours and then frozen. J Anim Sci 82:690-694.
  3. Batallier F, Duchamp G, Vidament M, Arnaud G, Palmer E, Magistrini M (1998): Delayed insemination is successful with a new extender for storing fresh semen at $15^{\circ}C$ under aerobic conditions. Theriogenology 50:229-236. https://doi.org/10.1016/S0093-691X(98)00130-7
  4. Batallier F, Vidament M, Fauquant J, Duchamp G, Arnaud G, Yvon JM (2001): Advances in cooled semen technology. Anim Reprod Sci 68:181-190. https://doi.org/10.1016/S0378-4320(01)00155-5
  5. Bustamante Filho IC, Pederzolli CD, Sgaravatti AM, Gregory RM, Dutra Filho CS, Jobim MIM, Mattos RC (2009): Skim milk-egg yolk based semen extender compensates for non-enzymatic antioxidant activity loss during equine semen cryopreservation. Anim Reprod 6(2):392-399.
  6. Clulow JR, Masfield LJ, Morris LHA, Evans G, Maxwell WMC (2008): A comparison between freezing methods for the cryopreservation of stallion spermatozoa. Anim Reprod Sci 108:298-308. https://doi.org/10.1016/j.anireprosci.2007.08.014
  7. Jasko DJ, Hathaway JA, Schanltenbrand VL, Simper WD, Squire EL (1992): Effect of seminal plasma and egg yolk on motion characteristics of cooled stallion spermatozoa. Theriogenology 37:1241-1252. https://doi.org/10.1016/0093-691X(92)90180-Y
  8. Kenney RM, Bergman RV, Cooper WL, Morse GW (1975): Minimal contamination techniques for breeding mares: thchniques and preliminary findings. Proceedings of 21st Americal Association of Equine Practices pp 327-336.
  9. Loomis PR, Amann RP, Squire EL, Pickett BW (1983): Fertility of unfrozen and frozen stallion spermatozoa extended in EDTA-Lactose-egg yolk and packaged in straw. J Anim Sci 56(3):687-693.
  10. Loomis PR (2001): The equine frozen industry. Anim Reprod Sci 68:191-200. https://doi.org/10.1016/S0378-4320(01)00156-7
  11. Loomis PR, Graham JK (2008): Commercial semen freezing: Individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols. Anim Reprod Sci 105:119-128. https://doi.org/10.1016/j.anireprosci.2007.11.010
  12. Macias Garcia B, Morrell JM, Ortega-Ferrusola C, Gonzalez-Fernandez L, Tapia JA, Rodriguez-Martinez H, Pena FJ (2009): Cetrifugation ona songle layer of colloid selects improved quality spermatozoa from frozen-thawed stallion semen. Anim Reprod Sci 114:193-202. https://doi.org/10.1016/j.anireprosci.2008.08.025
  13. Masuda H, Nanasaki S, Chiba Y (2004): A new extender for preservation of equine spermatozoa at $5^{\circ}C$. J Equine Sci 15:1-5. https://doi.org/10.1294/jes.15.1
  14. Martin JC, Klug E, Gunzel A (1979): Centrifugation of stallion semen and its storage in large volume straw. J Reprod Fertil Suppl 27:47.
  15. Michajilov NN (1950): Sperm dilution in the milk. Czech Vet Mag January 10, abstracted in J Anim Vet Med Assoc 117:337
  16. Pagl R, Aurich JE, Muller-Schlosser F, Kankofer M, Aurich C (2006): Comparison of an extender containing defined milk protein fraction with a skim milk-based extender for storage of equine semen at $5^{\circ}C$. Theriogenology 66:1115-1122. https://doi.org/10.1016/j.theriogenology.2006.03.006
  17. Palmer E (1984): Factors affecting stallion semen survival and fertility. Proceeding of the 10th International Congress on Animal Reproduction, Urbana-Champaign II 3:377.
  18. Pierre A, Fauquant J, Le Graet Y, Piot M, Maubois JL (1992): Preparation de phosphocaseinate natif par microfiltrationsur membrane. Lait 72: 461-474. https://doi.org/10.1051/lait:1992534
  19. Schober D, Aurich C, Nohl H, Gille L (2007): Influence of cryopreservation on mitochondria functions in equine spermatozoa. Theriogenology 68:745-754. https://doi.org/10.1016/j.theriogenology.2007.06.004
  20. Varner DD, Blanchard TL, Love CL, Garcia MC, Kenney RM (1988): Effects of coolingrate and storage temperature on equine spermatozoal motility parameters. Theriogenology 29:1043-1054. https://doi.org/10.1016/S0093-691X(88)80028-1
  21. 박용수, 박흠대, 장용석, 조길재 (2008): 말 동결융해정자의 생존성 및 수정능에 영향을 미치는 인자. 한국수정란이식학회지 23(3):161-166.