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

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 (NH 4 CL) (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.