107 Mitigation of bovine oocyte aging through NMN supplementation during in vitro maturation

The quality of the oocyte at the time of fertilization is crucial for the successful development of the mammalian embryo and the production of healthy offspring. Oocytes that are not fertilized in an optimal period undergo postovulatory aging, causing meiotic spindle degradation, zona pellucida hardening, cytoplasmic fragmentation, decreased ATP, increased oxidative stress, altered DNA methylation and gene expression, and dysregulation of intracellular calcium. These factors reduce fertilization rates, increase embryonic abnormalities, and decrease offspring viability, affecting livestock production, profitability and assisted reproduction operations. This study examined the effects of nicotinamide mononucleotide (NMN), a direct NAD⁺ precursor, on bovine oocytes aged through prolonged IVM. Immature oocytes, collected from bovine ovaries at a local slaughterhouse, were divided into control and experimental groups. Control groups were matured for 22–24 h without NMN (non-aged control) and for 30 h without NMN (aged control) in IVM medium supplemented with 0.33 mM sodium pyruvate, 1 µg mL⁻¹ estradiol, 10% fetal calf serum, 83.4 µg mL⁻¹ amikacin, 50 IU mL⁻¹ LH, and 1 µg mL⁻¹ FSH. Experimental groups were matured for 30 h in the same IVM medium with NMN at different concentrations (5 μM, 1 μM, 0.1 μM, and 0.01 μM). For IVF, 1449 in vitro-matured oocytes were co-incubated with 2 × 10⁶ sperm mL⁻¹ 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 oviductal fluid medium with 2.5% FBS at 38.5°C, 5% CO₂ in air. Cleavage was determined at 72 h post-fertilization, and blastocyst stage was evaluated on Days 6, 7, and 8. Data were analyzed by Fisher’s exact test using GraphPad Prism 6.0 (GraphPad Inc.), with differences considered significant at P < 0.05. The results indicated that NMN supplementation significantly mitigated the effects of aging, improving cleavage and blastocyst rates compared with the aged control. Cleavage rates improved to 66% ± 4.51% (P = 0.0005), 62% ± 2.52% (P = 0.0054), 65% ± 3.28% (P = 0.0010), and 67% ± 3.04% (P = 0.0001) for NMN concentrations of 5 µM, 1 µM, 0.1 µM, and 0.01 µM, respectively, compared with 50% ± 2.07% in the aged control. Additionally, the blastocyst rates of 1 μM (58/235, 24% ± 1.79%; P = 0.0035), 0.1 μM (60/260, 23% ± 1.58%; P = 0.0111), and 0.01 μM (66/242, 27% ± 0.75%; P = 0.0004) showed statistically significant improvements compared with the aged control (33/237, 14% ± 0.77%). These findings suggest that NMN supplementation during IVM could offer a promising strategy to improve the efficacy of IVF techniques in the livestock industry, with potential implications in optimizing reproductive efficiency in domestic and nondomestic species conservation, including clinical applications in humans. Additionally, this study contributes to a better understanding of the role of NAD⁺ in ovarian aging and establishes a foundation for future research in this field.

This work was supported by Universidad CES Project No INV.032021.005.