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Supplementing in vitro maturation medium with Nicotinamide mononucleotide (NMN) and Niacin (NA) promotes the maturation of domestic cat oocytes

Chinatsu Nishimoto ^1,2, Mayako Fujihara ², Miho Murayama ².

1. Graduate School of Science, Kyoto University, Kyoto, Japan

2. Wildlife Research Center, Kyoto University, Kyoto, Japan

Abstract Text:

Many wild felid species are threatened with extinction, making assisted reproductive technology (ART) essential for conservation breeding efforts. While ART reproduction has been reported in felids, the practical application of in vitro fertilization (IVF) has not yet been fully investigated. IVF has the advantage of utilizing oocytes from ovaries obtained after ovariohysterectomy or death as genetic resources. However, when a wild or captive felid dies, it takes 1 to 2 days for its ovaries to reach a laboratory and may decrease oocyte quality, reducing the success rate of fertilization. To obtain the fertilizable oocytes for IVF, the optimal culture condition for oocytes in vitro maturation (IVM) is essential. However, the success rate of IVM in felids is still low even with freshly obtained ovaries. Previous studies in mice and pigs have suggested that increasing nicotinamide adenine dinucleotide (NAD⁺) levels enhances oocyte maturation and quality. Using the ovaries of domestic cats (Felis catus) as a model for wild felids, our study aimed to improve the maturation and quality of domestic cat oocytes by increasing NAD⁺ levels.

First, we investigated the changes in oocyte maturation rates after 1 day. Oocytes were collected from domestic cat ovaries on the day of ovariectomy and 1 day after ovariectomy and then cultured for 28 hours to compare maturation rates by timing of ovariectomy. Next, only ovaries 1 day after ovariectomy were used to imitate the ovarian condition of individuals who had died in the wild. We examined the effects of two NAD⁺-boosting compounds, nicotinamide mononucleotide (NMN) and niacin (NA), on the IVM of domestic cat oocytes. Oocytes were cultured in IVM media supplemented with NMN or NA with different concentrations (NMN; 10µM, 100µM, 1000µM, NA; 10µM, 100µM, 400µM) for 28 hours. Also, a combination of 1000µM NMN + 400µM NA was examined on oocyte maturation after 28 hours’ culture. Following culture, the oocyte maturation were examined by immunostaining of the spindle and the chromosomes. The oxidative stress levels were also evaluated by measuring reactive oxygen species (ROS) fluorescence intensity.

Our results showed that oocyte maturation rates were lower after 1 day (13.7 ±3.71 %) compared to on the day of ovariectomy (52.3 ± 14.3 %). In the NMN and NA supplementation experiments using ovaries one day after ovariectomy, the 400µM NA group (29.8 ± 8.62 %) and the NMN + NA combination group (30.0 ± 3.33 %) showed significantly higher maturation rates compared to the control group (P < 0.05). When NMN was added solely, 1000µM had the highest maturation rate (21.7 ± 8.30 %) but there was no significant difference from the control (16.4 ±7.69 %). In addition, ROS levels were significantly reduced in oocytes cultured with 1000µM NMN only (P < 0.01) and with NMN + NA (P < 0.001), compared to the control group. On the contrary, NA supplementation did not reduced ROS levels.

These results indicate that supplementation of NMN and NA on culture medium not only enhances oocyte maturation but also effectively reduces ROS levels in domestic cat oocytes. This study provided the improvement of oocytes IVM medium in domestic cats as a potentially applicable for wild felids.