92 Boosting in vitro bovine blastocyst production via intermittent administration of nicotinamide

Nicotinamide (NAM), a form of vitamin B3, is a cell culture supplement essential for fetal development, but the effect of intermittent NAM refreshment at both in vitro maturation (IVM) and late embryo culture (IVC3; Day 6 after fertilization treatment) has not yet been scrutinized. In our previous study, we found that the IVM/IVC3 treatment of the antioxidant vanillic acid was associated with the highest yield of blastocyst when compared to either IVM, IVCs (IVC1; IVC2; Day 1 and Day 4 postfertilization treatment respectively), or the combination IVM/IVCs exposure times. In the current study, we first compared the effect of NAM supplementation at IVM, IVC3, IVM+IVC3 on the in vitro development of bovine embryos. In the second experiment, IVM oocytes and Day 6 postfertilization morula cells (at IVC3) were incubated for 24 h and 48 h, respectively, in the presence of NAM and were compared to untreated control. Underlying mechanisms affecting blastocyst metabolism, quality, pluripotency, and mitochondrial bioenergetics were inspected in Day 8 embryos. Differences between NAM-treated and control groups were analysed using Student’s t-test, while the ANOVA test was used for the comparison of more than two experimental groups, and the P-values below 0.05 were considered statistically significant. Results showed that dual NAM exposure (IVM+IVC3) significantly increased blastocyst production rate compared to the sole IVM or IVC3 treatment. Using reverse-transcription quantitative polymerase chain reaction, we displayed a decrease in the mRNA expression of Caspase-3 apoptotic gene, and the up-regulation was observed in antioxidant genes, blastocyst metabolism markers, and mitochondrial bioenergetics key players in the NAM group. In addition, mitochondrial membrane potential and distribution exhibited high polarization and homogeneous pattern in NAM blastocyst. In addition, immunofluorescence of embryos showed a clear increase in the total nuclei number and OCT4 pluripotency-associated factor under NAM treatment unlike the control. Taken together, we recognised the role of intermittent NAM administration during IVM/IVC to enhance blastocyst potential via the direct effect on metabolic flux, quality, mitochondrial dynamics, and pluripotency of developed embryos.