Fig 1.
Hatching rates of bovine embryos cultured in the presence of free or nanoencapsulated melatonin.
Mel = Non-encapsulated melatonin, Mel-NC = melatonin-loaded polymeric nanocapsules, Mel-LNC = melatonin-loaded lipid-core nanocapsules, NC = drug-unloaded nanocapsules, LNC = drug-unloaded lipid-core nanocapsules. Melatonin was used at 10−12 M, 10−9 M or 10−6 M concentrations. Control group was cultured in SOFaa BSA alone. The numerators represent the numbers of hatched blastocysts and the denominators represent total blastocysts in each group. Different letters (a-c) above the bars indicate significant differences between groups (P < 0.05).
Fig 2.
Effects of free and nanoencapsulated melatonin (10−9 M) on total cell number and cell apoptosis in blastocysts.
Effect of treatment on total cell number (A), and apoptotic cell rate in blastocysts (B). Data represent mean ± S.E.M. Different letters above the error bars indicate significant differences between groups (P< 0.05). Representative images of negative control (C), apoptotic cells indicated by arrows (D), total cell number (E), and positive control embryo with both nuclear and TUNEL staining (F). Scale bar = 50 μm. Magnification = 100X.
Fig 3.
ROS levels in 4–8 cell stage embryos cultured in the presence of free and nanoencapsulated melatonin.
Embryos were cultured in the presence of 10−9 M melatonin non-encapsulated (Mel) or encapsulated in nanocapsules (Mel-NC) or lipid-core nanocapsules (Mel-LNC). In the control group embryos were cultured in SOFaa BSA medium alone. Data represent mean ± S.E.M. Different letters above the error bars indicate significant differences between groups (P< 0.05). Representative fluorescent (B, C, D, E) and corresponding bright field (b, c, d, e) images of control, Mel, Mel-NC, and Mel-LNC embryos, respectively. Scale bar = 50 μm. Magnification = 100X.
Fig 4.
Effects of free and nanoencapsulated melatonin (10−9 M) on the relative mRNA abundance of apoptosis-related genes.
Transcript levels of four apoptosis-related genes BAX (A), CASP3 (B), SHC1 (C) and MCL1 (D) were quantified by q-PCR. Data represent mean ± S.E.M. Different letters (a, b and c) indicate significant differences between groups (P< 0.05).
Fig 5.
Effects of free and nanoencapsulated melatonin (10−9 M) on the relative mRNA abundance of oxidative stress-related genes.
Data represent mean ± S.E.M. Different letters (a, b and c) indicate significant differences between groups (P< 0.05).
Fig 6.
Effects of free and nanoencapsulated melatonin (10−9 M) on the relative mRNA abundance of pluripotency-related genes in bovine blastocysts.
Data represent mean ± S.E.M.