Association Study Analysis of Cluster-of-Differentiation Antigen 9 (CD9) Gene Polymorphism (g.358A>T) for Duroc Boar Post-thawed Semen Motility and Kinematic Characteristics

  • Cho, Eun-Seok (Swine Science Division, National Institute of Animal Science, RDA) ;
  • Sa, Soo-Jin (Swine Science Division, National Institute of Animal Science, RDA) ;
  • Kim, Ki-Hyun (Swine Science Division, National Institute of Animal Science, RDA) ;
  • Lee, Mi-Jin (Swine Science Division, National Institute of Animal Science, RDA) ;
  • Ko, Jun-Ho (Swine Science Division, National Institute of Animal Science, RDA) ;
  • Kim, Young-Ju (Swine Science Division, National Institute of Animal Science, RDA) ;
  • Seol, Kuk-Hwan (Swine Science Division, National Institute of Animal Science, RDA) ;
  • Hong, Joon-ki (Swine Science Division, National Institute of Animal Science, RDA) ;
  • Kim, Kwang-Sik (Swine Science Division, National Institute of Animal Science, RDA) ;
  • Kim, Yong-Min (Swine Science Division, National Institute of Animal Science, RDA) ;
  • Woo, Jae-Seok (Swine Science Division, National Institute of Animal Science, RDA)
  • Received : 2015.06.17
  • Accepted : 2015.06.19
  • Published : 2015.06.30


Cryopreservation of boar semen is continually researched in reproductive technologies and genetic resource banking in breed conservation. For evaluating the boar semen quality, sperm motility (MOT) is an important parameter because the movement of spermatozoa indicates active metabolism, membrane integrity and fertilizing capacity. Various researches have been trying to improve the quality of semen post-thawed in boar. Recently, polymorphism (g.358A>T) of cluster-of-differentiation antigen 9 (CD9) gene reported to be significant association with MOT. Also, CD9 gene was expressed in the male germ line stem cells is crucial for sperm-egg fusion, and was therefore selected as candidate gene for boar semen. This study was conducted to evaluate the pig SNP (g.358A>T) of CD9 gene as a positional controlling for semen parameters of post-thawed boar semen. To results, the g.358A>T SNP of the CD9 gene was significantly associated with the traits such as MOT, curve linear velocity, straight line velocity, average path velocity and amplitude of lateral head displacement. Particularly, the g.358A>T SNP significantly has the highest association with MOT and animals with AA genotype (p<0.001). Therefore, we suggest that the g.358A>T in the intron 6 region of the porcine CD9 may be used as a molecular marker for Duroc boar Post-thawed semen quality, although its functional effect was not defined yet.


Supported by : Rural Development Administration


  1. Vyt PH, Maes D, Dejonckheere E, Castryck F and Van Soom A. 2004. Comparative study on five different commercial extenders for boar semen. Reprod. Domest. Anim. 39(1):8-12.
  2. Estienne M, Harper A and Day J. 2007. Characteristics of sperm motility in boars diluted in different extenders and store for seven days at $18^{\circ}C$. Reprod. Biol. 7(3):221-231.
  3. Boucheix C, Duc GHT, Jasmin C and Rubinstein E. 2001. Tetraspanins and malignancy. Expert Rev. Mol. Med. 3(04):1-17.
  4. Capon F, Allen MH, Ameen M, Burden AD, Tillman D, Barker JN and Trembath RC. 2004. A synonymous SNP of the corneodesmosin gene leads to increased mRNA stability and demonstrates association with psoriasis across diverse ethnic groups. Hum. Mol. Genet. 13(20):2361-2368.
  5. Carvajal G, Cuello C, Ruiz M, Vázquez JM, Martínez EA and Roca J. 2004. Effects of centrifugation before freezing on boar sperm cryosurvival. J. Androl. 25(3):389-396.
  6. Cerolini S, Maldjian A, Pizzi F and Gliozzi T. 2001. Changes in sperm quality and lipid composition during cryopreservation of boar semen. Reproduction 121(3):395-401.
  7. Chen X, Zhu H, Hu C, Hao H, Zhang J, Li K, Zhao X, Qin T, Zhao K and Zhu H. 2014. Identification of differentially expressed proteins in fresh and frozen-thawed boar spermatozoa by iTRAQ-coupled 2D LC-MS/MS. Reproduction 147(3):321-330.
  8. Daghigh-Kia H. 2007. Identification and SNP detection for preimplantation active genes and their association with embryo development and male fertility in cattle. PhD Thesis Institute of Animal Science, Animal Breeding and Husbandry Group, University of Bonn, Bonn, Germany.
  9. Diniz D, Lopes M, Broekhuijse M, Lopes P, Harlizius B, Guimaraes S, Duijvesteijn N, Knol E and Silva F. 2014. A genome-wide association study reveals a novel candidate gene for sperm motility in pigs. Anim. Reprod. Sci. 151(3): 201-207.
  10. Flores E, Taberner E, Rivera M, Pena A, Rigau T, Miro J and Rodriguez-Gil J. 2008. Effects of freezing/thawing on motile sperm subpopulations of boar and donkey ejaculates. Theriogenology 70(6):936-945.
  11. Fraser L, Strzezek J. 2005. Effects of freezing-thawing on DNA integrity of boar spermatozoa assessed by the neutral comet assay. Reprod. Domest. Anim. 40(6):530-536.
  12. Gunawan A, Cinar M, Uddin M, Kaewmala K, Tesfaye D, Phatsara C, Tholen E, Looft C and Schellander K. 2012. Investigation on association and expression of ESR2 as a candidate gene for boar sperm quality and fertility. Reprod. Domest. Anim. 47(5):782-790.
  13. Horejsi V and Vlcek C. 1991. Novel structurally distinct family of leucocyte surface glycoproteins including CD9, CD37, CD53 and CD63. FEBS letters. 288(1):1-4.
  14. Johnson LA. 1985. Fertility results using frozen boar spermatozoa: 1970 to 1985. In: Johnson LA, Larsson K, editors. Deep Freezing of Boar Semen. Uppsala: Swedish University of Agricultural Science, pp. 199-222.
  15. Johnson LA. 1998. Current developments in swine semen: preservation, artificial insemination and sperm sexing. In: Done S, Thomson J, Varley M, editors. Proceedings of the 15th IPVS Congress. Nottingham: University Press, pp. 225-229.
  16. Johnson L, Aalbers J, Willems C and Sybesma W. 1981. Use of boar spermatozoa for artificial insemination. I. Fertilizing capacity of fresh and frozen spermatozoa in sows on 36 farms. J. Anim. Sci. 52(5):1130-1136.
  17. Johnson L, Weitze K, Fiser P and Maxwell W. 2000. Storage of boar semen. Anim. Reprod. Sci. 62(1):143-172.
  18. Kaewmala K, Uddin M, Cinar M, Grobe Brinkhaus C, Jonas E, Tesfaye D, Phatsara C, Tholen E, Looft C and Schellander K. 2012. Investigation into Association and Expression of PLCz and COX 2 as candidate genes for boar sperm quality and fertility. Reprod. Domest. Anim. 47(2):213-223.
  19. Kaewmala K, Uddin MJ, Cinar MU, Grobe-Brinkhaus C, Jonas E, Tesfaye D, Phatsara C, Tholen E, Looft C and Schellander K. 2011. Association study and expression analysis of CD9 as candidate gene for boar sperm quality and fertility traits. Anim. Reprod. Sci. 125(1):170-179.
  20. Kanatsu-Shinohara M, Inoue K, Lee J, Yoshimoto M, Ogonuki N, Miki H, Baba S, Kato T, Kazuki Y and Toyokuni S. 2004. Generation of pluripotent stem cells from neonatal mouse testis. Cell 119(7):1001-1012.
  21. Kurokawa M, Sato K-i, Wu H, He C, Malcuit C, Black SJ, Fukami K and Fissore RA. 2005. Functional, biochemical, and chromatographic characterization of the complete [$Ca^{2+}$i oscillation-inducing activity of porcine sperm. Dev. Biol. 285(2):376-392.
  22. Le Hir H, Nott A and Moore MJ. 2003. How introns influence and enhance eukaryotic gene expression. Trends Biochem. Sci. 28(4):215-220.
  23. Le Naour F, Rubinstein E, Jasmin C, Prenant M and Boucheix C. 2000. Severely reduced female fertility in CD9-deficient mice. Science 287(5451):319-321.
  24. Maes D, Nauwynck H, Rijsselaere T, Mateusen B, Vyt P, de Kruif A and Van Soom A. 2008. Diseases in swine transmitted by artificial insemination: An overview. Theriogenology 70(8):1337-1345.
  25. Mazur P. 1984. Freezing of living cells: mechanisms and implications. Am. J. Physiol. Cell Physiol. 247(3):C125-C142.
  26. Nagase H and Graham E. 1964. Pelleted semen: Comparison of different extenders and processes on fertility of bovine spermatozoa. $5^{th}$. Int. Congr. Anim. Reprod. A. I. Trient. 4: 387-389.
  27. Oka M, Tagoku K, Russell TL, Nakano Y, Hamazaki T, Meyer EM, Yokota T and Terada N. 2002. CD9 is associated with leukemia inhibitory factor-mediated maintenance of embryonic stem cells. Mol. Biol. Cell. 13(4):1274-1281.
  28. Pagani F and Baralle FE. 2004. Genomic variants in exons and introns: Identifying the splicing spoilers. Nat. Rev. Genet. 5(5):389-396.
  29. Polge C, Salamon S and Wilmut I. 1970. Fertilizing capacity of frozen boar semen following surgical insemination. Vet. Rec. 87(15):424-429.
  30. Ren D, Ren J, Xing Y, Guo Y, Wu Y, Yang G, Mao H and Huang L-S. 2009. A genome scan for quantitative trait loci affecting male reproductive traits in a White Duroc Chinese Erhualian resource population J. Anim. Sci. 87(1):17-23.
  31. Yubero N, Jiménez-Marín A, Yerle M, Morera L, Barbancho M, Llanes D and Garrido J. 2003. Molecular cloning, expression pattern and chromosomal mapping of pig CD9 antigen. Cytogenet. Genome Res. 101(2):143-146.
  32. Zeng C, He L, Peng W, Ding L, Tang K, Fang D and Zhang Y. 2014. Selection of optimal reference genes for quantitative RT-PCR studies of boar spermatozoa cryopreservation. Cryobiology 68(1):113-121.