Journal of Reproduction & Infertility

Journal of Reproduction & Infertility

The Effect of Ammonium Chloride Concentration in In Vitro Maturation Culture on Ovine Embryo Development

Authors
Ali Golchin 1
Reza Asadpour 1
Leila Roshangar 2
Raziallah Jafari-Jozani 1
1 Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran
2 Department of Anatomical Sciences, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
Abstract
Background: Ammonium is produced in culture medium due to amino acids degradation and has adverse effect on in vitro culture of embryo. In the current study, the purpose was to evaluate the effects of ammuniom chloride (AC) on in vitro oocyte maturation rate and early embryo development in the sheep and its effect on the expression of Bcl-2. Methods: In vitro maturation (IVM) was performed in the presence of various concentrations (0, 29, 88,132,176 μM/ml) of ammonium chloride (NH4CL) (AC). Meiotic maturation, embryonic development and expression of Bcl2 gene in Blastocyst cells were determined. The data were analyzed by one-way ANOVA and Tukey post HOC test, and values with p<0.05 were considered statistically significant. Results: The highest concentration (176 µM) of AC significantly decreased the rate of fully expanded cumulus cells 24 hr after IVM compared with the control group (p<0.05). Moreover, significantly lower rates of MII oocytes were found in the 176 µM AC group compared with the 29 µM AC group. The percentage of zygotes developing to blastocysts in 176 µM AC was lower than the other group. Also, supplementation of the oocyte maturation media with 176 µM AC decreased Bcl2 expression. Conclusion: Our results suggested that significant increase in IVM rate could be obtained with supplementation maturation medium with AC in a dose dependent manner. Increased AC concentration led to lower blastocyst rate under normal condition. However, regulation of pro–apoptotic (Bcl-2) gene did not change with different concentrations of AC supplementing.
Keywords
Ammonium chloride
Gene expression
Ovine embryo
  1. Alexiou M, Leese HJ. Purine utilisation, de novo synthesis and degradation in mouse preimplantation embryos. Development. 1992;114(1):185-92.
  2. Butler WR, Calaman JJ, Beam SW. Plasma and milk urea nitrogen in relation to pregnancy rate in lactating dairy cattle. J Anim Sci. 1996;74(4):858-65.
  3. Cory S, Adams JM. Matters of life and death: programmed cell death at Cold Spring Harbor. Biochim Biophys Acta. 1998;1377(2):R25-44.
  4. Edwards LJ, Williams DA, Gardner DK. Intracellular pH of the mouse preimplantation embryo: amino acids act as buffers of intracellular pH. Hum Reprod. 1998;13(12):3441-8.
  5. Ellis RE, Yuan JY, Horvitz HR. Mechanisms and functions of cell death. Annu Rev Cell Biol. 1991;7:663-98.
  6. Elrod CC, Butler WR. Reduction of fertility and alteration of uterine pH in heifers fed excess ruminally degradable protein. J Anim Sci. 1993;71 (3):694-701.
  7. Epstein CJ, Smith SA. Amino acid uptake and protein synthesis in preimplanatation mouse embryos. Dev Biol. 1973;33(1):171-84.
  8. Ferguson JD, Blanchard T, Galligan DT, Hoshall DC, Chalupa W. Infertility in dairy cattle fed a high percentage of protein degradable in the rumen. J Am Vet Med Assoc. 1988;192(5):659-62.
  9. Ferguson JD, Galligan DT, Blanchard T, Reeves M. Serum urea nitrogen and conception rate: the usefulness of test information. J Dairy Sci. 1993;76(12):3742-6.
  10. Gardner DK. Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture. Theriogenology. 1998;49(1):83-102.
  11. Gardner DK, Lane M. Amino acids and ammonium regulate mouse embryo development in culture. Biol Reprod. 1993;48(2):377-85.
  12. Gross A, McDonnell JM, Korsmeyer SJ. BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 1999;13(15):1899-911.
  13. Hafez B, Hafez ESE. Reproduction in farm animals. 7th. ed. New York: Wiley; 2000. p. 1-13.
  14. Hammon DS, Wang S, Holyoak GR, Knight-Sherod J, Evans RC. Effects of high ammonia concentrations during IVM on oocyte maturation and in vitro development of bovine embryos. Theriogenology. 1999;51(1):375.
  15. Marei WF, Wathes DC, Fouladi-Nashta AA. The effect of linolenic Acid on bovine oocyte maturation and development. Biol Reprod. 2009;81(6):1064-72.
  16. Cognié Y, Baril G, Poulin N, Mermillod P. Current status of embryo technologies in sheep and goat. Theriogenology. 2003;59(1):171-88.
  17. Lane M. Mechanisms for managing cellular and homeostatic stress in vitro. Theriogenology. 2001;55(1):225-36.
  18. Lindenbaum A. A survey of naturally occurring chelating ligands. Adv Exp Med Biol. 1973;40:67-77.
  19. McEvoy TG, Robinson JJ, Aitken RP, Findlay PA, Robertson IS. Dietary excesses of urea influence the viability and metabolism of preimplantation sheep embryos and may affect fetal growth among survivors. Anim Reprod Sci. 1997;47(1-2):71-90.
  20. Tareq K, Miah AG, Salma U, Yoshida M, Tsujii H. Effect of amino acids and dipeptides on accumulation of ammonia in the medium during in vitro maturation and fertilization of porcine oocytes. Reprod Med Biol. 2007;6(3):165-70.
  21. Papadopoulos S, Lonergan P, Gath V, Quinn KM, Evans AC, O'Callaghan D, et al. Effect of diet quantity and urea supplementation on oocyte and embryo quality in sheep. Theriogenology. 2001;55(5):1059-69.
  22. Schneider M, Marison IW, von Stockar U. The importance of ammonia in mammalian cell culture. J Biotechnol. 1996;46(3):161-85.
  23. Hammon DS, Wang S, Holyoak GR. Effects of ammonia during different stages of culture on development of in vitro produced bovine embryos. Anim Reprod Sci. 2000;59(1-2):23-30.
  24. Yuan Y, Krisher RL. Effect of ammonium during in vitro maturation on oocyte nuclear maturation and subsequent embryonic development in pigs. Anim Reprod Sci. 2010;117(3-4):302-7.
  25. Orsi NM, Leese HJ. Ammonium exposure and pyruvate affect the amino acid metabolism of bovine blastocysts in vitro. Reproduction. 2004;127(1):131-40.
  26. Ruvolo PP, Deng X, May WS. Phosphorylation of Bcl2 and regulation of apoptosis. Leukemia. 2001;15(4):515-22.
  27. Burlacu A. Regulation of apoptosis by Bcl-2 family proteins. J Cell Mol Med. 2003;7(3):249-57.
Journal of Reproduction & Infertility
Volume 17, Issue 3
Summer 2016
Pages 144-150