O-070 NAD+ precursor nicotinamide mononucleotide improves the developmental competence of bovine embryos through enhancement of mitochondrial function in morula

Abstract Study question Does the addition of nicotinamide mononucleotide (NMN), an NAD+ precursor, to the culture medium of fertilized ovum increase their ability to develop into blastocysts? Summary answer The addition of NMN to the embryo culture medium increased mitochondrial function at morula stage and increased the ability to develop into blastocysts. What is known already NAD+ and its metabolites function as key regulators to maintain physiological processes, allowing cells to adapt to environmental changes such as nutritional disorders, genotoxic factors, infection, inflammation, and xenobiotics. Culturing isolated cells is predicted to decrease NAD+ levels below the physiological requirement. We have shown that addition of NMN to bovine oocyte culture medium (100 μM) increases oocyte NAD+ levels, increases mitochondrial function and ATP levels, decreases ROS and anaphase lagging, and increases blastocyst formation (Sci Rep 2025). However, it remained unclear whether the addition of NMN to the embryo culture medium would improve the developmental ability of the embryos. Study design, size, duration This prospective study was conducted by randomly allocating 2-cell stage embryos obtained after IVF of bovine oocytes retrieved from slaughterhouses ovaries. Two-cell embryos obtained after IVF were randomly divided into two groups (NMN 0 vs. 100 μM), and the blastulation were examined. Since it was predicted from the morphological changes recorded over time that development would differ after the morula stage, RNA was extracted from the morulae and a comprehensive gene expression analysis was performed. Participants/materials, setting, methods Only normal 2-cell stage embryos that did not undergo abnormal cytoplasmic division were used in this experiment. We used 310 two-cell stage embryos to assess the ability to develop into blastocysts, 125 two-cell embryos to assess morphological changes over time, 486 morulae for next-generation gene expression analysis, and 56 morulae for mitochondrial function analysis by measurement of oxygen consumption rate of each embryo. Main results and the role of chance Addition of NMN significantly increased the blastocyst formation rate (P = 0.0195, NMN: 41% vs. control: 23%) and the morphologically good blastocyst formation rate (P = 0.0018, NMN: 24% vs. control: 11%). No difference was observed in blastocyst formation rate after an additional 24 hours of culture (P = 0.053, NMN: 52.4% vs. control: 32.3%), indicating that NMN improves embryo development rate and embryo quality. We then captured morphological changes of embryos over time at 15-minute intervals using a microscope inside incubator. The morphological changes of embryos over time indicated that developmental retardation of embryos cultured in medium without NMN occurred after morula stage. Therefore, RNA was extracted from morulae cultured with or without NMN, and a comprehensive gene expression analysis was performed, focusing on mitochondria using Mitocarata 3.0. to extract mitochondria-related genes among the differentially-expressed genes. RNA sequence data proposed NMN could significantly affect mitochondrial function in morulae. Therefore, we measured the oxygen consumption rate of individual embryos to evaluate the mitochondrial function of morula and found that NMN significantly increased the oxygen consumption rate of morulae (P = 0.00046, NMN: 3.3 fmol/sec vs. 2.76 fmol/sec). Limitations, reasons for caution Since the data in this study were obtained using bovine fertilized ova, safety should be verified using donated human embryos before use in clinical practice. Wider implications of the findings Our data indicate that the addition of NAD+ precursors to the embryo culture medium improves mitochondrial function and developmental ability to blastocyst. Our data provide a rationale for applying NMN to improve the efficiency of reproductive medicine and IVF zygote production in domestic animals. Trial registration number No