ABSTRACT
Background: Ammonium is produced in culture medium due to amino acids degradationand has adverse effect on in vitro culture of embryo. In the current study, thepurpose was to evaluate the effects of ammuniom chloride [AC] on in vitro oocytematuration rate and early embryo development in the sheep and its effect on the expressionof Bcl-2
Methods: In vitro maturation [IVM] was performed in the presence of various concentrations[0, 29, 88,132,176 M/ml] of ammonium chloride [NH4CL][AC]. Meioticmaturation, embryonic development and expression of Bcl2 gene in Blastocystcells were determined. The data were analyzed by one-way ANOVA and Tukey postHOC test, and values with p<0.05 were considered statistically significant
Results: The highest concentration [176micro M] of AC significantly decreased the rateof 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 176micro M AC group compared with the 29micro M AC group. The percentage of zygotes developingto blastocysts in 176micro M AC was lower than the other group. Also, supplementationof the oocyte maturation media with 176micro M AC decreased Bcl2 expression
Conclusion: Our results suggested that significant increase in IVM rate could be obtainedwith 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 differentconcentrations of AC supplementing
ABSTRACT
Background: Linoleic acid [LA] is a polyunsaturated fatty acid present in high concentrations in follicular fluid, when added to maturation culture media, it affects oocyte competence
Objective: In the present study, we investigated effect of linoleic acid supplementation on in vitro maturation, embryo development and apoptotic related gene expression in ovine
Materials and Methods: The experiments conducted on 450 ovine Cumulus-oocyte complexes [COCs] with homogenous ooplasm and more than two compact layers of cumulus cells. For in vitro maturation COCs were randomly allocated into four treatment groups for 24 hr period. Treatment groups were as follow: control maturation media, 0 micro M LA, 50 micro M LA, 100 micro M LA and 200 micro M LA. The cumulus cell expansion and blastocysts rates were recorded. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to apoptotic gene expression by real-time PCR
Results: Highest concentration [200 micro M/mL] of LA significantly decreased the rate of fully expanded cumulus cells 24 hr after in vitro maturation [IVM] and the percentage of blastocyste rate compared with the control [p<0.05]. These inhibitory effects were associated with an increased in relative mRNA expression of Bax [Bcl-2- associated X] gene compared with controls
Conclusion: Data obtained in present study suggest that low concentration of LA used for maturation had no deleterious effect on subsequent embryonic development compared to high concentration of LA. Relative expression of Bcl-2 [B-cell lymphoma 2] and Bax in embryos seems to be associated with LA concentration