Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
권호 표지

Development of Sperm MTT Assay for Its Application in Boar Semen

  • Jang, Hyun-Yong (College of Animal Life Science, Kangwon National University) ;
  • Lee, Hee-Young (College of Animal Life Science, Kangwon National University) ;
  • Cheong, Hee-Tae (School of Veterinary Medicine, Kangwon National University) ;
  • Kim, Jong-Taek (School of Veterinary Medicine, Kangwon National University) ;
  • Park, In-Chul (School of Veterinary Medicine, Kangwon National University) ;
  • Park, Choon-Keun (College of Animal Life Science, Kangwon National University) ;
  • Yang, Boo-Keun (College of Animal Life Science, Kangwon National University)
  • Received : 2010.11.15
  • Accepted : 2010.11.30
  • Published : 2010.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

The MTT assay is one of superior evaluation methods widely used to analyze the viability of metabolically active cell. It can be used to determine the percentage of viable sperm through measurement of the reduction of MTT granules at mitochondria in sperm tail. The purpose of this study is to determine the optimal condition of a simple and easy MTT assay to validate boar sperm viability and compare the accuracy of this test with microscopic examination. The MTT reduction rate for sperm viability were analyzed in microtiter plates (96 well) from 1 hr to 5 hr incubation periods at $37^{\circ}C$ using spectrophotometer (microplate reader) at 550 nm wavelength. The remainder of semen sample was simultaneously examined to compare the correlation of accuracy between MTT assay and other sperm parameters. Those sperm parameters were included the motility, survival rates, membrane integrity, mitochondria activity and acrosome integrity. The OD values of MTT assay (MTT reduction rates) did not greatly change at 1 hr to 5 hr incubation periods in different proportion of live and freeze-killed sperms (dead sperm). The MTT reduction rates or survival rates were decreased according to the different concentration of live and dead sperm. The linear regression at 1 hr and 4 hr incubation periods in sperm MTT assay was y=291.55x-72.176 and y= 180.64x-44.569, respectively. There are high correlation between 1 hr and 4 hr incubation periods (p<0.001). The results of MTT assay and other sperm parameters has a positive correlation (p<0.01 or 0.05). The correlation coefficients for MTT assay was 0.88115 for motility, 0.89868 for survival rates, 0.91722 for membrane integrity and 0.77372 for acrosome integrity, respectively. In conclusion, the MTT assay can be used as a reliable and efficient evaluation method for boar sperm viability. It can be use practical means to evaluate the quality of boar sperm by a fast, inexpensive and easy method.

Keywords

References

  1. Aziz DM, Ahlswede Land Enbergs H. 2005. Application of MTT reduction assay to evaluate equine sperm viability. Theriogenology 64:1350-1356. https://doi.org/10.1016/j.theriogenology.2005.02.009
  2. Aziz MD. 2006. Assessment of bovine sperm viability by MIT reduction assay. Anim. Reprod. Sci. 92:1-8. https://doi.org/10.1016/j.anireprosci.2005.05.029
  3. Betteridge MV, Herst PM and Tan AS. 2005. Tetrazolium dyes as tools in cell biology: New insights into their cellular deduction. Biotechnol. Annu. Rev. 11:127-152. https://doi.org/10.1016/S1387-2656(05)11004-7
  4. Chandler JE, Harrison CM and Canal AM. 2000. Spermatozoal methylene blue reduction: an indicator of mitochondrial function and its correlation with motility. Theriogenology 54:261-271. https://doi.org/10.1016/S0093-691X(00)00346-0
  5. Gaczarzewicz D, Piasecka M, Udala J, Blaszczyk B, Laszczynska M and Kram A. 2003. Oxidoreductive capability of boar sperm mitochondria in fresh semen and during their preservation in BTS extender. Reprod. Biol. 3:161-172.
  6. Gillan L, Evans G and Maxwell WMC. 2005. Flow cytometric evaluation of sperm parameters in relation to fertility potential. Theriogenology 63:445-457. https://doi.org/10.1016/j.theriogenology.2004.09.024
  7. Iqbal M, Aleem M, Ijaz A, Rehman R and Yousaf MS. 2010. Assessment of buffalo semen with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. J. Anim. Sci. 88:922-925. https://doi.org/10.2527/jas.2009-2344
  8. Jang HY, Kim YH, Kim BW, Park IC, Cheong HT, Kim JT, Park CK, Kong HS, Lee HK and Yang BK. 2010. Ameliorative effects of malatonin against hydrogen peroxid-induced oxidative stress on boar sperm characteristics and subsequent in vitro embryo development. Reprod. Dom. Anim. 45:943-950. https://doi.org/10.1111/j.1439-0531.2009.01466.x
  9. Nasr-Esfahani MH, Aboutorabi R, Esfandiari E and Mardani M. 2002. Sperm MIT viability assay: A new method for evaluation of human sperm viability. J. Assist. Reprod. Genet. 19:477-482. https://doi.org/10.1023/A:1020310503143
  10. Ohtani K, Yamazaki S, Kubota H, Miyagawa M and Saegusa J. 2004. Comparative investigation of several sperm analysis methods for evaluation of spermatotoxicity of industrial chemical:2-bromopropane as an example. Indust. Health 42:219-225. https://doi.org/10.2486/indhealth.42.219
  11. Twentyman PR and Luscombe M. 1987. A study of some viables in a tetrazolium dye(MTT) based assay for cell growth and chemosensitivity. Br. J. Cancer 56:279-285. https://doi.org/10.1038/bjc.1987.190
  12. Zrimsek P, Kunc J, Kosec M and Mrkun J. 2004. Spectrophotometric application of resazurin reduction assay to evaluate boar semen quality. Int. J. Androl. 27:57-62. https://doi.org/10.1046/j.0105-6263.2003.00448.x