L-carnitine Supplemented Extender Improves Cryopreserved-thawed Cat Epididymal Sperm Motility

  • Manee-In, S. (Department of Clinical Sciences and Public Health Medicine, Faculty of Veterinary Science, Mahidol University) ;
  • Parmornsupornvichit, S. (Faculty of Veterinary Science, Mahidol University) ;
  • Kraiprayoon, S. (Faculty of Veterinary Science, Mahidol University) ;
  • Tharasanit, T. (Faculty of Veterinary Science, Chulalongkorn University) ;
  • Chanapiwat, P. (Department of Clinical Sciences and Public Health Medicine, Faculty of Veterinary Science, Mahidol University) ;
  • Kaeoket, K. (Department of Clinical Sciences and Public Health Medicine, Faculty of Veterinary Science, Mahidol University)
  • Received : 2013.09.12
  • Accepted : 2014.02.01
  • Published : 2014.06.01


Cryopreservation of epididymal sperm is an effective technique to preserve genetic materials of domestic cats and wild felids when they unexpectedly die. However, this technique inevitably causes detrimental changes of cryopreserved-thawed spermatozoa, for example, by physical damage and excessive oxidative stress. L-carnitine is an antioxidant that has been used to improve sperm motility in humans and domestic animals. This study aimed to investigate the effects of L-carnitine on cat epididymal sperm quality following cryopreservation and thawing. After routine castration, cauda epididymides were collected from 60 cat testes. The epididymal spermatozoa from 3 cauda epididymides were pooled as 1 replicate. Spermatozoa samples (16 replicates) were examined for spermatozoa quality and then randomly divided into 4 groups: 0 mM L-carnitine (control), 12.5 mM, 25 mM and 50 mM L-carnitine. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, plasma membrane integrity, DNA integrity and acrosome integrity were evaluated. The 25 mM L-carnitine significantly improved sperm motility compared with a control group (p<0.05), although this was not significantly different among other concentrations. In conclusion, supplementation of 25 mM L-carnitine in freezing extender improves cauda epididymal spermatozoa motility. The effects of L-carnitine on the levels of oxidative stress during freezing and thawing remains to be examined.


L-carnitine;Epididymal Spermatozoa;Cryopreservation;Cat


  1. Alvarez, J. G. and B. T. Storey. 1995. Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa. Mol. Reprod. Dev. 42:334-346.
  2. Abd-Allah, A. R., G. K. Helal, A. A. Al-Yahya, A. M. Aleisa, S. S. Al-Rejaie, and S. A. Al-Bakheet. 2009. Pro-inflammatory and oxidative stress pathways which compromise sperm motility and survival may be altered by L-carnitine. Oxid. Med. Cell. Longev. 2:73-81.
  3. Agarwal, A., K. P. Nallella, S. S. Allamaneni, and T. M. Said. 2004. Role of antioxidants in treatment of male infertility: An overview of the literature. Reprod. Biomed. Online 8:616-627.
  4. Aitken, R. J. and A. J. Koppers. 2011. Apoptosis and DNA damage in human spermatozoa. Asian J. Androl. 13:36-42.
  5. Andersen, K. 1975. Insemination with frozen dog semen based on a new insemination technique. Reprod. Domest. Anim. 10:1-4.
  6. Axner, E., U. Hermansson, and C. Linde-Forsberg. 2004. The effect of Equex STM paste and sperm morphology on post-thaw survival of cat epididymal spermatozoa. Anim. Reprod. Sci. 84:179-191.
  7. Axner, E., B. S. Pukazhenthi, D. E. Wildt, C. Linde-Forsberg, and R .E. Spindler. 2002. Creatine phosphokinase in domestic cat epididymal spermatozoa. Mol. Reprod. Dev. 62:265-270.
  8. Cerolini, S., A. Maldjian, F. Pizzi, and T. M. Gliozzi. 2001. Changes in sperm quality and lipid composition during cryopreservation of boar semen. Reproduction 121:395-401.
  9. Bailey, J. L., J. F. Bilodeau, and N. Cormier. 2000. Semen cryopreservation in domestic animals: A damaging and capacitating phenomenon minireview. J. Androl. 21:1-7.
  10. Balercia, G., F. Regoli, T. Armeni, A. Koverech, F. Mantero, and M. Boscaro. 2005. Placebo-controlled double-blind randomized trial on the use of L-carnitine, L-acetylcarnitine, or combined L-carnitine and L-acetylcarnitine in men with idiopathic asthenozoospermia. Fertil. Steril. 84:662-671.
  11. Cavallini, G., A. P. Ferraretti, L. Gianaroli, G. Biagiotti, and G. Vitali. 2004. Cinnoxicam and L-carnitine/acetyl-L-carnitine treatment for idiopathic and varicocele-associated oligoasthenospermia. J. Androl. 25:761-770; discussion 771-762.
  12. Cheng, F. P., A. Fazeli, W. F. Voorhout, A. Marks, M. M. Bevers, and B. Colenbrander. 1996. Use of peanut agglutinin to assess the acrosomal status and the zona pellucida-induced acrosome reaction in stallion spermatozoa. J. Androl. 17:674-682.
  13. Chi, H. J., J. H. Kim, C. S. Ryu, J. Y. Lee, J. S. Park, D. Y. Chung, S. Y. Choi, M. H. Kim, E. K. Chun, and S. I. Roh. 2008. Protective effect of antioxidant supplementation in sperm-preparation medium against oxidative stress in human spermatozoa. Hum. Reprod. 23:1023-1028.
  14. Costa, M., D. Canale, M. Filicori, S. D'Lddio, and A. Lenzi. 1994. L-carnitine in idiopathic asthenozoospermia: A multicenter study. Andrologia 26:155-159.
  15. Donnelly, E. T., N. McClure, and S. E. M. Lewis. 1999. The effect of ascorbate and alpha-tocopherol supplementation in vitro on DNA integrity and hydrogen peroxide-induced DNA damage in human spermatozoa. Mutagenesis 14:505-512.
  16. Donnelly, E. T., N. McClure, and S. E. Lewis. 2000. Glutathione and hypotaurine in vitro: Effects on human sperm motility, DNA integrity and production of reactive oxygen species. Mutagenesis 15:61-68.
  17. Jeulin, C. and L. M. Lewin. 1996. Role of free L-carnitine and acetyl-L-carnitine in post-gonadal maturation of mammalian spermatozoa. Hum. Reprod. Update 2:87-102.
  18. Hammerstedt, R. H., J. K. Graham, and J. P. Nolan. 1990. Cryopreservation of mammalian sperm: What we ask them to survive. J. Androl. 11:73-88.
  19. Hughes, C. M., S. E. Lewis, V. J. McKelvey-Martin, and W. Thompson. 1998. The effects of antioxidant supplementation during Percoll preparation on human sperm DNA integrity. Hum. Reprod. 13:1240-1247.
  20. Jeulin, C., J. L. Dacheux, and J. C. Soufir. 1994. Uptake and release of free L-carnitine by boar epididymal spermatozoa in vitro and subsequent acetylation rate. J. Reprod. Fertil. 100:263-271.
  21. Kim, S. H., D. H. Yu, and Y. J. Kim. 2010a. Apoptosis-like change, ROS, and DNA status in cryopreserved canine sperm recovered by glass wool filtration and Percoll gradient centrifugation techniques. Anim. Reprod. Sci. 119:106-114.
  22. Kim, S. H., D. H. Yu, and Y. J. Kim. 2010b. Effects of cryopreservation on phosphatidylserine translocation, intracellular hydrogen peroxide, and DNA integrity in canine sperm. Theriogenology 73:282-292.
  23. Kitiyanant, Y., B. Chaisalee, and K. Pavasuthipaisit. 2002. Evaluation of the acrosome reaction and viability in buffalo spermatozoa using two staining methods: The effects of heparin and calcium ionophore A23187. Int. J. Androl. 25:215-222.
  24. Kobayashi, D., I. Tamai, Y. Sai, K. Yoshida, T. Wakayama, Y. Kido, J. Nezu, S. Iseki, and A. Tsuji. 2007. Transport of carnitine and acetylcarnitine by carnitine/organic cation transporter (OCTN) 2 and OCTN3 into epididymal spermatozoa. Reproduction 134:651-658.
  25. Kozink, D. M., M. J. Estienne, A. F. Harper, and J. W. Knight. 2004. Effects of dietary L-carnitine supplementation on semen characteristics in boars. Theriogenology 61:1247-1258.
  26. Pena, F. J., F. Saravia, A. Johannisson, M. Walgren, and H. Rodriguez-Martinez. 2005. A new and simple method to evaluate early membrane changes in frozen-thawed boar spermatozoa. Int. J. Androl. 28:107-114.
  27. Lenzi, A., F. Lombardo, P. Sgro, P. Salacone, L. Caponecchia, F. Dondero, and L. Gandini. 2003. Use of carnitine therapy in selected cases of male factor infertility: A double-blind crossover trial. Fertil. Steril. 79:292-300.
  28. Neuman, S. L., T. L. Lin, and P. Y. Heste. 2002. The effect of dietary carnitine on semen traits of white Leghorn roosters. Poult. Sci. 81:495-503.
  29. Pena, F. J., A. Johannisson, M. Wallgren, and H. Rodriguez Martinez. 2003. Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate. Anim. Reprod. Sci. 78:85-98.
  30. Platz, C. C., Jr., and S. W. Seager. 1978. Semen collection by electroejaculation in the domestic cat. J. Am. Vet. Med. Assoc. 173:1353-1355.
  31. Stradaioli, G., L. Sylla, R. Zelli, P. Chiodi, and M. Monaci. 2004. Effect of L-carnitine administration on the seminal characteristics of oligoasthenospermic stallions. Theriogenology 62:761-777.
  32. Thomson, L. K., S. D. Fleming, R. J. Aitken, G. N. De Iuliis, J. A. Zieschang, and A. M. Clark. 2009. Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis. Hum. Reprod. 24:2061-2070.
  33. Thuwanut, P. and K. Chatdarong. 2009. Incubation of post-thaw epididymal cat spermatozoa with seminal plasma. Reprod. Domest. Anim. 44 (Suppl 2):381-384.
  34. Thuwanut, P., K. Chatdarong, A. Johannisson, A. S. Bergqvist, L. Soderquist, and E. Axner. 2010. Cryopreservation of epididymal cat spermatozoa: effects of in vitro antioxidative enzymes supplementation and lipid peroxidation induction. Theriogenology 73:1076-1087.
  35. Zini, A., M. San Gabriel, and A. Baazeem. 2009. Antioxidants and sperm DNA damage: a clinical perspective. J. Assist. Reprod. Genet. 26:427-432.
  36. Tittarelli, C., C. A. Savignone, E. Arnaudin, M. C. Stornelli, M. A. Stornelli, and R. L. de la Sota. 2006. Effect of storage media and storage time on survival of spermatozoa recovered from canine and feline epididymides. Theriogenology 66:1637-1640.
  37. White, I. G. 1993. Lipids and calcium uptake of sperm in relation to cold shock and preservation: A review. Reprod. Fertil. Dev. 5:639-658.
  38. Yeste, M., S. Sancho, M. Briz, E. Pinart, E. Bussalleu, and S. Bonet. 2010. A diet supplemented with L-carnitine improves the sperm quality of Pietrain but not of Duroc and Large White boars when photoperiod and temperature increase. Theriogenology 73:577-586.

Cited by

  1. The Usefulness of Selected Physicochemical Indices, Cell Membrane Integrity and Sperm Chromatin Structure in Assessments of Boar Semen Sensitivity vol.28, pp.12, 2015,
  2. Low-density Lipoprotein Improves Motility and Plasma Membrane Integrity of Cryopreserved Canine Epididymal Spermatozoa vol.29, pp.5, 2015,
  3. ) Epididymal Sperm after Slow Equilibration to 15 or 10°C vol.51, pp.2, 2016,
  4. Cryopreservation of adult primate testes vol.62, pp.5, 2016,
  5. The Effect of L-Carnitine, Hypotaurine, and Taurine Supplementation on the Quality of Cryopreserved Chicken Semen vol.2017, pp.2314-6141, 2017,
  6. Comparison and evaluation of capacitation and acrosomal reaction in freeze-thawed human ejaculated spermatozoa treated with L-carnitine and pentoxifylline pp.03034569, 2017,
  7. ) epididymal sperm subjected to different cooling times (24, 48 and 72 hours) pp.09366768, 2017,