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Research Article

volume 50 issue 3 (june 2016) : 305-309,   Doi: 10.18805/ijar.9490

Cysteamine and b-mercaptoethanol supplementation improves the in vitro embryo development in buffaloes (Bubalus bubalis)

V
Vijay Singh1
A
A.K. Misra2
S
Suresh Kumar*3
C
Champak Barman4
1<p>Department of Animal Reproduction, Gynaecology &amp; Obstetrics,&nbsp;College of Veterinary and Animal Science, G.B.P.U.A. &amp; T., Pantnagar-263 145, India.</p>
Cite article:- Singh1 Vijay, Misra2 A.K., Kumar*3 Suresh, Barman4 Champak (2016). Cysteamine and b-mercaptoethanol supplementation improves the in vitroembryo development in buffaloes (Bubalus bubalis) . Indian Journal of Animal Research. 50(3): 305-309. doi: 10.18805/ijar.9490.

ABSTRACT

The objective of the present experiment was to investigate the effect of cysteamine and b-mercaptoethanol supplementation on in -vitro maturation, cleavage of oocytes and development of embryo in buffalo (Bubalus bubalis). Oocytes were aspirated from abattoir ovarian follicles of 3-10 mm diameter followed by maturation in the media in vitro containing cysteamine/b-mercaptoethanol (treatment) and without antioxidant (control). Matured oocytes were co-incubated with sperm (approx.1×106/ml) of Murrah bull in mSOF medium using heparin (10 µg/ml). After 22 h of oocyte-sperm incubation, fertilized oocytes were stripped of cumulus cells and cultured in mSOF medium for 8 days to study embryo development. The oocyte maturation rate improved significantly (P<0.05) following addition of  50 or 100 µM of cysteamine and 10, 50 and 100 µM of  b- mercaptoethanol (ME), respectively as compared to control. The cleavage rate was found to be significantly (P<0.05) higher at 50 and 100 µM of cysteamine and at all concentrations of b-mercaptoethanol as compared to control and development of embryos to morula stage was significantly (P<0.05) improved with 50 µM cysteamine/ b-mercaptoethanol.

REFERENCES


  1. Abeydeera, C. R. , Wang, W. H., Cantley, T. C. , Prather, R. S. and Day, B. N. (1999). Glutathione content and embryo development after in vitro fertilization of pig oocytes matured in the presence of thiol compound and various concentrations of cysteine. Zygote, 7: 203-210. 

  2. Bannai, S. (1984). Transport of cystine and cysteine in mammalian cells. Biochem. Biophysics Acta, 779: 289-306.

  3. Calvin, H. I., Grosshans, K. and Blake, E. J. (1986). Estimation and manipulation of glutathione levels in prepuberal mouse ovaries and ova : relevance to sperm nucleus transformation in the fertilized egg. Gamete Res., 14: 265-75.

  4. Del Corso, A., Cappiello, M. and Mura U. (1994). Thiol dependent oxidation of enzymic : the last chance against oxidative stress. International J. of Biochem., 26: 745-750.

  5. De Matos, D. G. and Furnus, C. C. (2000). The importance of having high glutathione level after bovine in vitro maturation on embryo development: effect of b-mercaptoethanol, cysteine and cystine. Theriogenology, 53: 761-771.

  6. De Matos, D.G., Furnus, C.C. and Moses, D.F. (1997). Glutathione synthesis during in vitro maturation of bovine oocytes: role of cumulus cells. Biol. of Reprod., 57: 1420-1425.

  7. De Matos, D. G., Furnus, C. C., Moses, D. F., Martinez, A .G .and Matkovic, M. (1996). Stimulation of glutathione synthesis of in vitro matured bovine oocytes and its effect on embryo development and freezability. Molecul. Reprod. Develop., 45: 451-457.

  8. Eppig, J. (1996). Coordination of nuclear and cytoplasmic maturation in eutherian mammal. Reprod. Fertility Development, 8: 485-489.

  9. Gardiner, C. S. and Reed, D.J. (1994). Status of glutathione oxidant-induced oxidative stress in the pre-implantation mouse embryo. Biol. of Reprod., 51: 1307-1314.

  10. Gasparrini, B. (2002). In vitro embryo production in buffalo species: state of the art. Theriogenology, 57: 237-256.

  11. Gasparrini, B., Sayond, H., Neglia, G, De Matos, D .G. Donny, I. and Zicarelli, L. (2003). Glutathion synthesis during in vitro maturation of buffalo (Bubalus bubalis) oocytes: effects of cysteamine on embryo development. Theriogenology, 60 : 943-952.

  12. Gasparrini, B., Boccia, L, Marchandise, J., Dipalo, R., George, F., Donnay, I. and Zicarelli, L. (2006). Enrichment of in vitro maturation medium for buffalo (Bubalus bubalis) oocytes with thiol compounds: Effect of cystine on glutathione synthesis and embryo development. Theriogenology, 65: 275-287.

  13. Ishii ,T., Hishimura, I., Bannai, S. and Sugita, Y. (1981). Mechanism of growth promotion of mouse lymphoma L1210 cells in vitro by feeder layer of 2-mercaptoethanol. J. Cell Physiol., 107: 283-293.

  14. Kobayashi, M., Lee, E.S. and Fukui, Y. (2006). Cysteamine or b-mercaptoethanol added to a defined maturation medium improved blastocyst formation of porcine oocytes after intracytoplasmic sperm injection. Theriogenology, 65: 1191-1199.

  15. Lafleur, M.V.M., Hoorweg, J. J., Joenje, H., Westmijze, E. J. and Retel, J. (1994). The ambivalent role of glutathione in the protection of DNA against singlet oxygen. Free Radical. Res., 21 : 9-17.

  16. Meister, A. (1983). Selective modification of glutathione metabolism. Science, 220: 472-477.

  17. Miyamura, M., Yoshida, M., Hamano, S. and Kuwayama, M. (1995). Glutathione concentration during maturation and fertilization in bovine oocytes. Theriogenology, 43: 282.

  18. Nandi, S., Chauhan, M.S. and Palta, P. (1998). Effect of cumulus cells and sperm concentration on cleavage rate and subsequent embryonic development of buffalo (Bubulus bubalis) oocytes matured and fertilized in vitro. Theriogenology, 50: 1251-1262.

  19. Nagai, T. (2001). The improvement of in vitro maturation systems for bovine and porcine oocytes. Theriogenology, 55: 1291-1301.

  20. Park,D.H., Yang, B. K., Kim, J. K., Choung, H. T., Park, C. K., Kim, J. B. and Kim, C. I. (1997). Effect of b-Mercaptoethanol and cysteamine with bovine oviduct epithelial cells on development and intracellular glutathione concentrations of bovine IVM/IVF embryos. Korean J. Embryo Transfer, 12: 269-276.

  21. Perreault, S.D., Barbee, R.R .and Slott, V. I. (1988). Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensation activity in maturing hamster oocytes. Developmental Biol., 125: 181-186.

  22. Sagara, J., Miura, K. and Bannai, S. (1993). Cystine uptake and glutathione level in fetal brain cells in primary culture and in suspense. J. of Neurochem., 61: 1667-1671.

  23. Snedecor, G.W. and Cochran, W.G. (2007) Statistical Methods 8th edition. Iowa State University Press, Ames Iowa.

  24. Takahashi, H., Kuwayama M., Hamano, S., Takahashi, M., Okano, A., Kadokawa , H., Kariya, T. and Nagai,T. (1996). Effect of b-mercaptoethanol on the viability of IVM/IVF/IVC bovine embryos during long-distance transportation in plastic straws. Theriogenology, 46: 1009-1015.

  25. Takahashi, M., Nagai, T., Hamano, S., Kuwayama, M., Okamura, N. and Okano, A. (1993). Effect of thiol compounds on in vitro development and intra-cellular glutathione content of bovine embryos. Biol. of Reprod., 49 : 228-232.

  26. Telford, N. A., Watson, A. J. and Schultz, G. A. (1990). Transition from maternal to embryonic control in early mammalian development : a comparison of several species. Molecul. Reprod. and Develop., 26 : 90-100.

  27. Yoshida, M., Ishigaki, K., Nagai, T., Chikyu, M. and Pursel, V.G. (1993). Glutathione concentration during maturation and after fertilization in pig oocytes: relevance to the ability of oocytes to form male pronucleus. Biol. of Reprod., 49: 89-94.

  28. Zicarelli, L. and Gasparrini, B. (2004). Embryo production in buffalo species. Proceedings of the 7th World Buffalo Congress, Manila, Philippines, 20-23 October, pp. 157-172.
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Indian Journal of Animal Research
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    Research Article

    volume 50 issue 3 (june 2016) : 305-309,   Doi: 10.18805/ijar.9490

    Cysteamine and b-mercaptoethanol supplementation improves the in vitro embryo development in buffaloes (Bubalus bubalis)

    V
    Vijay Singh1
    A
    A.K. Misra2
    S
    Suresh Kumar*3
    C
    Champak Barman4
    1<p>Department of Animal Reproduction, Gynaecology &amp; Obstetrics,&nbsp;College of Veterinary and Animal Science, G.B.P.U.A. &amp; T., Pantnagar-263 145, India.</p>
    Cite article:- Singh1 Vijay, Misra2 A.K., Kumar*3 Suresh, Barman4 Champak (2016). Cysteamine and b-mercaptoethanol supplementation improves the in vitroembryo development in buffaloes (Bubalus bubalis) . Indian Journal of Animal Research. 50(3): 305-309. doi: 10.18805/ijar.9490.

    ABSTRACT

    The objective of the present experiment was to investigate the effect of cysteamine and b-mercaptoethanol supplementation on in -vitro maturation, cleavage of oocytes and development of embryo in buffalo (Bubalus bubalis). Oocytes were aspirated from abattoir ovarian follicles of 3-10 mm diameter followed by maturation in the media in vitro containing cysteamine/b-mercaptoethanol (treatment) and without antioxidant (control). Matured oocytes were co-incubated with sperm (approx.1×106/ml) of Murrah bull in mSOF medium using heparin (10 µg/ml). After 22 h of oocyte-sperm incubation, fertilized oocytes were stripped of cumulus cells and cultured in mSOF medium for 8 days to study embryo development. The oocyte maturation rate improved significantly (P<0.05) following addition of  50 or 100 µM of cysteamine and 10, 50 and 100 µM of  b- mercaptoethanol (ME), respectively as compared to control. The cleavage rate was found to be significantly (P<0.05) higher at 50 and 100 µM of cysteamine and at all concentrations of b-mercaptoethanol as compared to control and development of embryos to morula stage was significantly (P<0.05) improved with 50 µM cysteamine/ b-mercaptoethanol.

    REFERENCES


    1. Abeydeera, C. R. , Wang, W. H., Cantley, T. C. , Prather, R. S. and Day, B. N. (1999). Glutathione content and embryo development after in vitro fertilization of pig oocytes matured in the presence of thiol compound and various concentrations of cysteine. Zygote, 7: 203-210. 

    2. Bannai, S. (1984). Transport of cystine and cysteine in mammalian cells. Biochem. Biophysics Acta, 779: 289-306.

    3. Calvin, H. I., Grosshans, K. and Blake, E. J. (1986). Estimation and manipulation of glutathione levels in prepuberal mouse ovaries and ova : relevance to sperm nucleus transformation in the fertilized egg. Gamete Res., 14: 265-75.

    4. Del Corso, A., Cappiello, M. and Mura U. (1994). Thiol dependent oxidation of enzymic : the last chance against oxidative stress. International J. of Biochem., 26: 745-750.

    5. De Matos, D. G. and Furnus, C. C. (2000). The importance of having high glutathione level after bovine in vitro maturation on embryo development: effect of b-mercaptoethanol, cysteine and cystine. Theriogenology, 53: 761-771.

    6. De Matos, D.G., Furnus, C.C. and Moses, D.F. (1997). Glutathione synthesis during in vitro maturation of bovine oocytes: role of cumulus cells. Biol. of Reprod., 57: 1420-1425.

    7. De Matos, D. G., Furnus, C. C., Moses, D. F., Martinez, A .G .and Matkovic, M. (1996). Stimulation of glutathione synthesis of in vitro matured bovine oocytes and its effect on embryo development and freezability. Molecul. Reprod. Develop., 45: 451-457.

    8. Eppig, J. (1996). Coordination of nuclear and cytoplasmic maturation in eutherian mammal. Reprod. Fertility Development, 8: 485-489.

    9. Gardiner, C. S. and Reed, D.J. (1994). Status of glutathione oxidant-induced oxidative stress in the pre-implantation mouse embryo. Biol. of Reprod., 51: 1307-1314.

    10. Gasparrini, B. (2002). In vitro embryo production in buffalo species: state of the art. Theriogenology, 57: 237-256.

    11. Gasparrini, B., Sayond, H., Neglia, G, De Matos, D .G. Donny, I. and Zicarelli, L. (2003). Glutathion synthesis during in vitro maturation of buffalo (Bubalus bubalis) oocytes: effects of cysteamine on embryo development. Theriogenology, 60 : 943-952.

    12. Gasparrini, B., Boccia, L, Marchandise, J., Dipalo, R., George, F., Donnay, I. and Zicarelli, L. (2006). Enrichment of in vitro maturation medium for buffalo (Bubalus bubalis) oocytes with thiol compounds: Effect of cystine on glutathione synthesis and embryo development. Theriogenology, 65: 275-287.

    13. Ishii ,T., Hishimura, I., Bannai, S. and Sugita, Y. (1981). Mechanism of growth promotion of mouse lymphoma L1210 cells in vitro by feeder layer of 2-mercaptoethanol. J. Cell Physiol., 107: 283-293.

    14. Kobayashi, M., Lee, E.S. and Fukui, Y. (2006). Cysteamine or b-mercaptoethanol added to a defined maturation medium improved blastocyst formation of porcine oocytes after intracytoplasmic sperm injection. Theriogenology, 65: 1191-1199.

    15. Lafleur, M.V.M., Hoorweg, J. J., Joenje, H., Westmijze, E. J. and Retel, J. (1994). The ambivalent role of glutathione in the protection of DNA against singlet oxygen. Free Radical. Res., 21 : 9-17.

    16. Meister, A. (1983). Selective modification of glutathione metabolism. Science, 220: 472-477.

    17. Miyamura, M., Yoshida, M., Hamano, S. and Kuwayama, M. (1995). Glutathione concentration during maturation and fertilization in bovine oocytes. Theriogenology, 43: 282.

    18. Nandi, S., Chauhan, M.S. and Palta, P. (1998). Effect of cumulus cells and sperm concentration on cleavage rate and subsequent embryonic development of buffalo (Bubulus bubalis) oocytes matured and fertilized in vitro. Theriogenology, 50: 1251-1262.

    19. Nagai, T. (2001). The improvement of in vitro maturation systems for bovine and porcine oocytes. Theriogenology, 55: 1291-1301.

    20. Park,D.H., Yang, B. K., Kim, J. K., Choung, H. T., Park, C. K., Kim, J. B. and Kim, C. I. (1997). Effect of b-Mercaptoethanol and cysteamine with bovine oviduct epithelial cells on development and intracellular glutathione concentrations of bovine IVM/IVF embryos. Korean J. Embryo Transfer, 12: 269-276.

    21. Perreault, S.D., Barbee, R.R .and Slott, V. I. (1988). Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensation activity in maturing hamster oocytes. Developmental Biol., 125: 181-186.

    22. Sagara, J., Miura, K. and Bannai, S. (1993). Cystine uptake and glutathione level in fetal brain cells in primary culture and in suspense. J. of Neurochem., 61: 1667-1671.

    23. Snedecor, G.W. and Cochran, W.G. (2007) Statistical Methods 8th edition. Iowa State University Press, Ames Iowa.

    24. Takahashi, H., Kuwayama M., Hamano, S., Takahashi, M., Okano, A., Kadokawa , H., Kariya, T. and Nagai,T. (1996). Effect of b-mercaptoethanol on the viability of IVM/IVF/IVC bovine embryos during long-distance transportation in plastic straws. Theriogenology, 46: 1009-1015.

    25. Takahashi, M., Nagai, T., Hamano, S., Kuwayama, M., Okamura, N. and Okano, A. (1993). Effect of thiol compounds on in vitro development and intra-cellular glutathione content of bovine embryos. Biol. of Reprod., 49 : 228-232.

    26. Telford, N. A., Watson, A. J. and Schultz, G. A. (1990). Transition from maternal to embryonic control in early mammalian development : a comparison of several species. Molecul. Reprod. and Develop., 26 : 90-100.

    27. Yoshida, M., Ishigaki, K., Nagai, T., Chikyu, M. and Pursel, V.G. (1993). Glutathione concentration during maturation and after fertilization in pig oocytes: relevance to the ability of oocytes to form male pronucleus. Biol. of Reprod., 49: 89-94.

    28. Zicarelli, L. and Gasparrini, B. (2004). Embryo production in buffalo species. Proceedings of the 7th World Buffalo Congress, Manila, Philippines, 20-23 October, pp. 157-172.
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