232 The effect of supplementation with nicotinamide mononucleotide on in vitro maturation medium and the impact on bovine embryo development

The efficiency of in vitro embryo production (IVEP) in bovines is critical to the advancement of livestock productivity and genetic preservation. Despite advancements, challenges related to the maturation and development of bovine oocytes endure, affecting the overall success rates. Nicotinamide mononucleotide (NMN), a precursor of NAD⁺, has been associated with enhanced cellular bioenergetics and rejuvenation, potentially mitigating aging-related impairments such as genome instability, reduced mitochondrial bioenergetics, increased reactive oxygen species (ROS), and disturbances during meiotic chromosome segregation. Preliminary studies have indicated that NMN might positively influence oocyte quality and subsequent embryonic development. However, understanding the influence of NMN on non-aged oocytes is a necessary preliminary step. The objective of this study was to explore different concentrations of NMN on the IVM medium and the impact on cleavage and embryo development rates of non-aged bovine oocytes, setting a baseline for further investigations and its anti-aging potential effects. Immature oocytes were harvested from bovine ovaries collected at the local slaughterhouse. Oocytes were in vitro matured for 22–24 h in IVM medium supplemented with 0.33 mM sodium pyruvate, 1 µg mL⁻¹ oestradiol, 10% fetal calf serum (FBS), 83.4 µg mL⁻¹ amikacin, 50 IU mL⁻¹ LH, 1 µg mL⁻¹ FSH, and different concentrations of NMN (5, 1, 0.1, and 0.01 µM). No NMN supplementation was used as a control group. For IVF, in vitro-matured oocytes were co-incubated with 2 × 10⁶ spermatozoa per mL in an atmosphere of 21% O₂ at 38°C in 5% CO₂ humidified air for 18–20 h. Presumptive zygotes were cultured in vitro in 50-μL drops of synthetic oviducal fluid (SOF) medium with 2.5% FBS on 5% CO₂ in air at 38°C. Cleavage was determined 72 h post-fertilization and blastocyst stage was evaluated at Day 6, 7, and 8. Data were analysed by Fisher’s exact test using GraphPad Prism 6.0 (GraphPad Inc.) and differences were considered significant at P < 0.05. Significant differences were found in embryos with more than 4 cells at Day 3 between the 0.01 µM NMN (52/53, 98%) and control groups (58/66, 88%). In terms of blastocyst formation, the 0.01 µM group showed accelerated development to the blastocyst stage at Day 6 (16/29, 55%) compared with the control group (6/27, 22%). Blastocyst development rate showed not significant difference between all groups. However, 0.01 µM exhibited a tendency (P < 0.1466) to higher blastocyst rates compared with the control group (29/80, 36% vs 27/105, 26%). These results suggest a potential impact of NMN on bovine oocyte cleavage and embryonic development. Future research is needed to explore the precise role of NMN in oocyte aging processes.

This work was supported by Universidad CES COD INV.032021.005.