阅读说明：本技术 烟酸在制备治疗和/或预防肥胖女性生育障碍的药物中的应用 (Application of nicotinic acid in preparing medicine for treating and/or preventing fertility disorders of obese women ) 是由 王强 祝帅 葛娟 于 2021-05-24 设计创作，主要内容包括：本发明公开了烟酸在制备治疗和/或预防肥胖女性生育障碍的药物中的应用,属于医药技术领域。本发明通过体内补充或体外添加烟酸可以有效提高肥胖女性的生育力,降低成熟卵子中减数分裂装置的异常组装比例和氧化应激的水平,促进受精后的早期胚胎发育至囊胚阶段,这对于预防和/或治疗肥胖引起的女性不孕不育问题具有重大意义。烟酸作为机体中一种重要的内源性物质,其结构与性质十分稳定,价格低廉且易于获得,体内补充或体外添加均能发挥作用,因此作为药物或生物制剂的实用性很高。(The invention discloses an application of nicotinic acid in preparing a medicine for treating and/or preventing obesity female fertility disorders, belonging to the technical field of medicines. The invention can effectively improve the fertility of obese women, reduce the abnormal assembly proportion and the level of oxidative stress of a meiosis device in mature ova and promote the development of fertilized early embryos to the blastocyst stage by supplementing nicotinic acid in vivo or adding nicotinic acid in vitro, thereby having great significance for preventing and/or treating the female infertility caused by obesity. Nicotinic acid, an important endogenous substance in the body, is very stable in structure and properties, low in price and easy to obtain, and can play a role in both in vivo supplementation and in vitro addition, so that the practicability of the nicotinic acid as a medicine or a biological agent is very high.)

1. Application of nicotinic acid in preparing a medicament for treating and/or preventing fertility disorders of obese women.

2. Use according to claim 1, characterized in that niacin is used for the preparation of a medicament for the treatment and/or prevention of the reduction in the quality of ova and/or embryos of obese women.

3. The use according to claim 1, characterized by the use of nicotinic acid for the preparation of a medicament for increasing the nicotinamide adenine dinucleotide content in eggs of obese women.

4. The use according to claim 1, characterized in that nicotinic acid is used for the preparation of a medicament for reducing the occurrence of morphological abnormalities of the spindle body, chromosomal alignment disorders and aneuploidy of oocytes from obese women.

5. Use according to claim 1, characterized in that niacin is used for preparing a medicament for reducing the oxidative stress level of oocytes in obese women.

6. Application of nicotinic acid in preparing in-vitro culture solution for improving quality of oocytes and/or early embryos of obese women.

7. An oocyte in-vitro culture solution is characterized by comprising 10-100 mu m of nicotinic acid; preferably, it contains 50 μm of nicotinic acid.

8. The oocyte in vitro culture solution according to claim 7, wherein the oocyte in vitro culture solution is M16 culture solution supplemented with 10-100 μ Μ nicotinic acid.

9. An extra-embryonic body culture solution is characterized by comprising nicotinic acid with the particle size of 20-200 mu m; preferably, it comprises 75 μ Μ of niacin.

10. The in vitro culture solution of embryo according to claim 9, wherein the in vitro culture solution of embryo is KSOM culture solution supplemented with 20-200 μ Μ nicotinic acid.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of nicotinic acid in preventing and/or treating the quality reduction of ova and embryos of obese women.

Background

The world health organization's statistics show that approximately 13% of adults (15% of the female population) worldwide by 2016 have obesity. The obesity population in China reaches more than 2.5 hundred million in 2019, and the chronic metabolic disease caused by multiple factors becomes a great health problem in the current society. Moreover, a large number of studies show that obese women in the population of child bearing age are prone to reproductive problems such as pregnancy failure, premature abortion, birth defects of fetuses and the like. Among them, the degradation of the quality of oocytes and early embryos is an important cause. Obesity can cause a variety of abnormal phenotypes in the ova and in the early embryos after fertilization, including abnormalities in meiotic apparatus, mitochondrial dysfunction, oxidative stress damage, and developmental arrest in early embryos.

Nicotinic Acid (NA), also known as niacin, is an important water-soluble vitamin that plays an important role in maintaining the health and homeostasis of the body. In addition, nicotinic acid can participate in NAD synthesis as a precursor in vivo⁺(nicotinamide adenine dinucleotide), which is used as a coenzyme, is involved in various biological processes such as energy metabolism. In vivo NAD under normal physiological conditions⁺Production and consumption of NAD will be in dynamic equilibrium, and⁺the decrease in the content is also considered to be a cause of various diseases. Nicotinic acid can remarkably improve NAD (nicotinamide adenine dinucleotide) in cells of tissues such as brain⁺And the liver, kidney and intestine can more effectively use nicotinic acid to synthesize NAD (nicotinamide adenine dinucleotide) compared with precursor substances such as nicotinamide⁺。

Disclosure of Invention

Aiming at the defects in the prior art, the technical problems to be solved by the invention are as follows: aiming at the female fertility problems such as the quality reduction of ova and early embryos caused by metabolic diseases such as obesity and the like; provides a new application of nicotinic acid: application of nicotinic acid in preventing and/or treating the quality reduction of ova and embryos of obese women.

The purpose of the invention can be realized by the following technical scheme:

application of nicotinic acid in preparing a medicament for treating and/or preventing fertility disorders of obese women. The nicotinic acid has a structural formula shown in a formula I:

preferably, the nicotinic acid is used for preparing the medicine for treating and/or preventing the quality reduction of ova and/or embryos of obese women.

As a preferable selection of the invention, the application of the nicotinic acid in preparing the medicine for improving the nicotinamide adenine dinucleotide content in the eggs of the obese women.

Preferably, the nicotinic acid is used for preparing the medicine for reducing the abnormal spindle body morphology, the chromosome arrangement disorder and the aneuploidy of the oocytes of the obese female.

Preferably, the present invention relates to the use of niacin for the preparation of a medicament for reducing the oxidative stress level of oocytes in obese women.

Application of nicotinic acid in preparing in-vitro culture solution for improving quality of oocytes and/or early embryos of obese women.

An in vitro oocyte culture solution is characterized by comprising 10-100 mu m of nicotinic acid.

As a preferable mode of the present invention, the oocyte in vitro culture liquid contains 50 μm of nicotinic acid.

More preferably, the oocyte in vitro culture solution is an M16 culture solution added with 10-100 μ M of nicotinic acid.

As a further preferred aspect of the present invention, the oocyte in vitro culture medium is M16 culture medium supplemented with 50 μ M of nicotinic acid.

An extra-embryonic culture solution, which is characterized by comprising 20-200 mu M of nicotinic acid.

Preferably, the embryo culture fluid in vitro contains 75 μ M nicotinic acid.

In a further preferred embodiment of the present invention, the embryo in vitro culture solution is a KSOM culture solution containing 20-200 μ M nicotinic acid.

In a further preferred embodiment of the present invention, the embryo in vitro culture solution is KSOM culture solution containing 75 μ M nicotinic acid.

The invention provides an injection for improving the quality of oocytes and early embryos of obese individuals, which is characterized in that 200-600 mg/kg body weight/day of a nicotinic acid solution is injected into an abdominal cavity, and the injection lasts for 7-14 days. The optimal dosage is 540mg/kg body weight/day, and the injection is continuously performed for 10 days.

The reagents and drugs used in the present invention are commercially available.

Meanwhile, the invention takes an obese mouse fed with HFD (high fat diet) as a research model to find that the nicotinic acid has the following beneficial effects on the fertility of an obese individual:

(1) nicotinic acid is added in the in-vitro maturation culture process, so that in-ovum NAD (nicotinamide adenine dinucleotide) of obese individuals can be remarkably improved⁺(nicotinamide adenine dinucleotide ) content, see FIG. 1.

(2) Nicotinic acid is added in the in vitro maturation culture process, so that the abnormal rate of an ovum meiosis device of an obese individual can be obviously reduced, as shown in figure 2.

(3) Nicotinic acid is added in the in vitro maturation culture process, so that the level of ROS (Reactive oxygen species) in the ovum of an obese individual can be obviously reduced, and the figure 3 shows.

(4) Nicotinic acid is added in the in vitro maturation culture process, so that the developmental capacity of the fertilized ovum of an obese individual can be obviously improved, as shown in figure 4.

(5) The addition of nicotinic acid during the embryo in vitro culture process can significantly increase the proportion of fertilized embryos of obese individuals that develop to the two-cell and blastocyst stage, as shown in fig. 5.

(6) The nicotinic acid is supplemented in vivo in modes of intraperitoneal injection and the like, and the in-ovum NAD (nicotinamide adenine dinucleotide) of obese individuals can be obviously improved⁺See fig. 6.

(7) In vivo supplementation of niacin by intraperitoneal injection or the like significantly reduced the abnormal rate of the egg meiosis apparatus in obese individuals, see figure 7.

(8) In vivo supplementation of niacin by intraperitoneal injection or the like significantly reduced ROS levels in the ova of obese individuals, see fig. 8.

(9) Nicotinic acid, an important endogenous substance in the body, is very stable in structure and properties, low in price and easy to obtain, and can play a role in both in vivo supplementation and in vitro addition, so that the practicability of the nicotinic acid as a medicine or a biological agent is very high.

Drawings

FIG. 1 shows that nicotinic acid is added in vitro to improve intraovular NAD (nicotinamide adenine dinucleotide) of obese mice⁺Results of content, statistical data are expressed as mean ± standard deviation, signifying significance of difference, where P<0.01，***P<0.001；

FIG. 2 is a graph showing the results of in vitro niacin addition for reducing the abnormal rate of the egg meiosis apparatus in obese mice; FIG. 2A is a typical photograph of immunofluorescent staining of spindles and chromosomes of oocytes of each group, with arrows indicating morphological abnormalities of spindles and chromosomal alignment disorders, and a scale of 30 μm; figure 2B is a statistical analysis of the abnormal rates of meiotic devices in each group of oocytes, data expressed as mean ± sd, indicating significance of difference, where P <0.05 and P < 0.001;

FIG. 3 is a graph showing the results of in vitro addition of niacin to reduce ROS levels in the ova of obese mice; FIG. 3A is a typical image of ROS immunofluorescent staining of oocytes of each group, with a scale of 50 μm; figure 3B is a statistic of ROS fluorescence of oocytes in each group, data expressed as mean ± standard deviation, indicating significance of difference, where P <0.05 and P < 0.001;

FIG. 4 is a graph showing the results of in vitro addition of nicotinic acid to enhance the developmental competence of the ova of obese individuals after fertilization; FIG. 4A is a typical image of the developmental to two-cell and blastocyst stage of each group of ova after fertilization, indicating an embryo with dysplasia after fertilization, scale 100 μm; fig. 4B is a statistical result of development to the two-cell stage after fertilization of each group of ova, fig. 4C is a statistical result of development to the blastocyst stage after fertilization of each group of ova, and the data are expressed as mean ± standard deviation, indicating significance of difference, wherein P <0.05, P <0.01, P <0.001, n.s. indicates no significant difference;

FIG. 5 shows the results of in vitro addition of nicotinic acid to promote embryonic development in obese individuals; FIG. 5A is a typical image of the development of each group of fertilized embryos to the two-cell and blastocyst stage, showing dysplastic embryos during culture, scale 100 μm; fig. 5B is a statistical result of the development of each group of fertilized embryos to the two-cell stage, fig. 5C is a statistical result of the development of each group of fertilized embryos to the blastocyst stage, and the data are expressed as mean ± standard deviation, indicating significance of difference, wherein P <0.05, P <0.01, P <0.001, n.s. indicates no significant difference;

FIG. 6 shows that in vivo niacin supplementation increases intraovular NAD in obese mice⁺Results of content, statistical data are expressed as mean ± standard deviation, signifying significance of difference, where P<0.001；

FIG. 7 is a graph showing the results of in vivo niacin supplementation reducing the abnormal rate of the egg meiosis apparatus in obese mice; FIG. 7A is a typical photograph of immunofluorescent staining of spindles and chromosomes of various groups of oocytes, with arrows indicating morphological abnormalities of spindles and chromosomal alignment disorders, and a scale of 30 μm; figure 7B is a statistical analysis of the odds of meiosis apparatus for each group of oocytes, data expressed as mean ± sd, indicating significance of difference, where P <0.001, n.s. indicates no significant difference;

FIG. 8 is a result of in vivo niacin supplementation reducing ROS levels in the ova of obese mice; FIG. 8A is a typical picture of ROS immunofluorescence staining of oocytes of each group, with a scale of 50 μm; figure 8B is a statistic of ROS fluorescence of oocytes in each group, data expressed as mean ± standard deviation, indicating significance of difference, where P <0.001, n.s. indicates no significant difference;

Detailed Description

The present invention will be further described with reference to the following specific examples and drawings, but the scope and the embodiments of the present invention are not limited thereto. The following examples are for illustrative purposes only and changes or modifications may be made without departing from the scope of the invention. Unless otherwise indicated, the pharmaceutical agents referred to in the following examples are commercially available.

Example 1 construction of obese mouse model

Female ICR mice, 3 weeks old, were randomly divided into two groups, and were fed High Fat Diet (HFD) and Normal Diet (ND), respectively, with free access to drinking water. After 16 weeks of feeding, the average body weight of the HFD mice was significantly higher than that of the ND mice (54.3 ± 4.9g vs.38.3 ± 2.7g, P <0.05), and the fasting blood glucose level of the HFD mice was significantly higher than that of the ND mice (8.8 ± 1.4mM vs.4.6 ± 0.9mM, P <0.05), indicating that the obese mice successfully modeled. ND mice served as controls.

EXAMPLE 2 preparation of oocyte in vitro maturation culture solution

The in vitro maturation culture medium of oocytes of obese mice was prepared by adding Nicotinic Acid (NA) at a concentration of 50 μ M to M16 culture medium (Sigma, cat # M7292). M16 broth without added niacin was used as a control.

Example 3 retrieval and in vitro maturation of oocytes from obese mice

Obese mice were euthanized and the ovaries removed 48 hours after intraperitoneal injection of 5IU of pregnant mare serum gonadotropin. GV stage oocytes were obtained by puncturing luminal follicles on the surface of the ovary with a needle tip and cultured in the culture solution described in example 2 for 14 hours.

Example 4 in vitro addition of Niacin to increase intraovular NAD in obese mice⁺In an amount of

Mature ova cultured in vitro in example 3 were collected and in ovo NAD was detected using a commercial kit (Sigma, cat # MAK037)⁺Content, and carrying out data statistics by one-way variance analysis.

The results are shown in FIG. 1: NAD of each ovum in ND group⁺The content was 3.20pmol, whereas the content of each ovum in the HFD group was only 0.52pmol, i.e., NAD in the eggs of obese mice⁺The content is obviously reduced (P)<0.001); and the addition of nicotinic acid (HFD + NA) during maturation culture can make NAD⁺The content reaches 1.65pmol, and is remarkably increased compared with the HFD group (P)<0.01). Therefore, the in-vitro addition of nicotinic acid can effectively improve the in-ovum NAD of the obese mouse⁺The content of (a).

Example 5 in vitro addition of Niacin reduces the abnormal Rate of the Oesophagedemic device in obese mice

Mature ova cultured in vitro in example 3 were collected for immunofluorescent staining: after the oocytes were fixed in 4% paraformaldehyde at room temperature for 30 minutes, they were permeabilized in 0.5% TritonX-100/PBS for 15 minutes, followed by blocking in 1% BSA/PBS at room temperature for 1 hour. FITC-. alpha. -tubulin antibody (Thermo, cat. No. 65-6111) was diluted at 1:200 and incubated overnight at 4 ℃. The chromosomes were labeled and stained with propidium iodide, after plating, spindle morphology and chromosome alignment were observed under an LSM710 zeiss laser confocal microscope, and statistical analysis was performed on the abnormality rates of the groups by one-way anova.

The results are shown in FIG. 2: FIG. 2A is a typical photograph of immunofluorescent staining of each group, wherein most of the oocytes in group ND exhibit a "bipolar barrel" morphology with chromosomes arranged in alignment on the equatorial plate; the spindle bodies in the HFD group can not be assembled normally and the arrangement of chromosomes is disordered; nicotinic acid (HFD + NA) is added during the maturation culture process, so that the spindle morphology and the chromosome arrangement of most ova can be recovered to be normal. FIG. 2B is a statistical graph of the abnormal ratio of spindle in eggs of each group, the abnormal ratio of ND group is 11.8%, and the abnormal ratio of HFD group is 36.1%, i.e., the abnormal ratio of spindle in ovum of obese mice is significantly increased (P < 0.001); whereas the addition of nicotinic acid (HFD + NA) during maturation culture reduced the abnormality rate to 21.0%, significantly lower than in the HFD group (P < 0.001). Therefore, the nicotinic acid added in vitro can effectively reduce the abnormal rate of the fat mouse ovum meiosis device, reduce the occurrence rate of chromosome dislocation and prevent the occurrence of aneuploidy.

Example 6 in vitro addition of Niacin reduces ROS levels in the ova of obese mice

Mature eggs cultured in vitro in example 3 were collected for ROS level detection: the ovum was incubated in M16 culture medium containing 5 μ M of CM-H2DCFDA probe (Invitrogen, cat # C6827) for 30 minutes, then transferred to a live cell observation dish, and the fluorescence signal was observed using an LSM710 Zeiss laser confocal microscope. Fluorescence intensity was calculated using ImageJ software and the fluorescence intensity values for each group were statistically analyzed by one-way anova.

The results are shown in FIG. 3: FIG. 3A is a typical image of ROS fluorescence staining in each group, and FIG. 3B is a statistical result of fluorescence intensity values in each group. ROS fluorescence signals in the ND group are weak, and the oxidative stress level is low; the ROS fluorescence intensity of the HFD group is obviously increased (P <0.001), and the oxidative stress level is higher; whereas the addition of nicotinic acid (HFD + NA) during maturation culture resulted in a decrease of ROS fluorescence signal of about 50%, significantly lower compared to the HFD group (P < 0.001). Therefore, the in vitro addition of nicotinic acid can effectively reduce the ROS level in the ovum of the obese mouse and reduce the damage caused by oxidative stress.

Example 7 in vitro nicotinic acid addition is effective in improving developmental competence of fertilized mouse ovum

The mature ova obtained in each group were incubated with the capacitated sperm in HTF culture for 6 hours to complete in vitro fertilization. The obtained fertilized embryos are continuously cultured in a KSOM culture solution, the proportion of the embryos developed to two cells is observed and recorded 1.5 days after fertilization, the proportion of the embryos developed to blastula is observed and recorded 3.5 days after fertilization, and the two-cell rate and the blastula rate of each group are subjected to statistical analysis through one-factor variance analysis.

The results are shown in FIG. 4: FIG. 4A is a typical picture of the development to the two-cell and blastocyst stage after fertilization of each group of ova. Fig. 4B is a statistical analysis of the post-fertilization development to the two-cell stage of each group of ova, with the proportion of post-fertilization development to the two-cell stage of the ND group of ova being 94.2%, with a significant decrease in HFD (P <0.001) of 71.2%, and with a significant increase in the two-cell rate during in vitro maturation with niacin (HFD + NA) of 89.4% compared to the HFD group (P < 0.001). Fig. 4C is a statistical analysis of the post-fertilization development to blastocyst stage for each group of ova, with a ratio of post-fertilization development to blastocyst stage for the ND group of ova of 87.1%, a significant decrease in HFD group (. about.p <0.01) of 55.5%, and a significant increase in blastocyst rate during in vitro maturation with the addition of niacin (HFD + NA) of 81.2% compared to the HFD group (. about.p < 0.01). Therefore, the addition of the smoke in the in-vitro maturation process can effectively promote the developmental capacity of the fertilized ovum of the obese individual.

EXAMPLE 8 preparation of embryo culture solution in vitro

An in vitro culture solution of obese mouse embryo is prepared by adding Nicotinic Acid (NA) with concentration of 75 μm to KSOM culture solution (Millipore, cat # MR 106D). KSOM medium without nicotinic acid was used as a control.

Example 9 in vitro addition of Niacin to promote Normal development of obese mouse embryos

The obese mice were first intraperitoneally injected with 5IU pregnant mare serum gonadotropin, intraperitoneally injected with 5IU human chorionic gonadotropin 48 hours later, euthanized 14 hours later and the ampulla of the fallopian tube was taken out, and the ova matured in vivo were obtained after laceration with forceps. The obtained in vivo mature ovum and the capacitated sperm were incubated in HTF culture medium for 6 hours to complete in vitro fertilization, thereby obtaining a fertilized embryo. They were cultured in the culture solution described in example 8, and the proportion of embryos developed into two cells was observed and recorded on day 1.5, and the proportion of embryos developed into blastula was observed and recorded on day 3.5, and statistical analysis was performed on the rate of two-cell development and the rate of blastula development of each group of embryos by one-way anova.

The results are shown in FIG. 5: fig. 5A is a typical image of the fertilized embryos of each group developing to a two-cell and blastocyst stage, the two-cell embryos in the ND group have regular cleavage spheres and substantially the same size, the blastocysts have typical morphology, the embryos in the HFD group have abnormal phenotypes such as developmental arrest and cytoplasmic fragmentation, and the embryos can be substantially restored to normal development by adding nicotinic acid (HFD + NA) during the culture process. Fig. 5B is a statistical analysis of the development of the embryos of each group to the two-cell stage, showing that the two-cell development rate of ND embryos was 88.4%, the two-cell development rate of HFD embryos was 62.2%, and a significant decrease (P <0.001) occurred, whereas the two-cell development rate of the group to which niacin was added (HFD + NA) was 82.1%, which was significantly increased compared to the HFD group (P < 0.001). Fig. 5C is a statistical result of the development to blastocyst stage of the embryos of each group, the blastocyst development rate of ND embryos was 75.8%, the blastocyst rate of HFD embryos was 49.5%, and a significant decrease (P <0.01) occurred, whereas the blastocyst rate of the group to which nicotinic acid was added (HFD + NA) was 70.9%, which was significantly increased compared to the HFD group (P < 0.01). Therefore, the normal development of the obese individual embryo can be effectively promoted by adding the tobacco in the culture process.

Example 10 specific regimen for niacin supplementation in obese mice

Obese mice were injected intraperitoneally with niacin solution at a dose of 540mg/kg body weight per day for 10 consecutive days. Obese mice as well as normal mice were injected intraperitoneally with solvent as a control, with the injection dose and cycle consistent with that described above. On day 8 of intraperitoneal injection of nicotinic acid solution or solvent, 5IU of pregnant mare serum gonadotropin is injected into the abdominal cavity of each mouse, 5IU of human chorionic gonadotropin is injected into the abdominal cavity on day 10, and after 14 hours, euthanasia is carried out to obtain mature ova in vivo from the ampulla of the fallopian tube.

Example 11 in vivo Niacin supplementation increases intraovular NAD in obese mice⁺In an amount of

The in vivo matured ova of example 10 were collected and tested for in ovo NAD using a commercial kit (Sigma, cat # MAK037)⁺Content, and carrying out data statistics by one-way variance analysis.

The results are shown in FIG. 6: NAD of each ovum in ND group⁺The content was 2.91pmol, whereas the content of each ovum in the HFD group was only 0.51pmol, i.e., NAD in mature eggs obtained from obese mice⁺Is significantly reduced in content (P)<0.001); while the obese mice receive niacin supplementation (HFD + NA) in vivo, the NAD in the mature ovum⁺The content can reach 1.75pmol, and is remarkably increased compared with the HFD group (P)<0.001). Therefore, the nicotinic acid supplementation in the obese mice can effectively improve the mature in-ovum NAD⁺The content of (a).

Example 12 in vivo Niacin supplementation reduces the abnormal rate of the egg meiosis apparatus in obese mice

The in vivo matured ova from example 10 were collected for immunofluorescent staining, the procedure was the same as described in example 5, spindle morphology and chromosome alignment were observed under LSM710 zeiss laser confocal microscope, and statistical analysis of the abnormality rate for each group was performed by one-way anova.

The results are shown in FIG. 7: FIG. 7A is a typical image of immunofluorescent staining of each group, showing that most of the spindles of mature ova in ND mice are in a "bipolar barrel" shape, and chromosomes are arranged regularly on the equatorial plate; but the spindle morphology of mature ova in HFD mice is abnormal and the chromosome arrangement is disordered; while obese mice, after supplementation with niacin (HFD + NA), their spindle morphology and chromosomal alignment in most mature ova returned to normal. Fig. 7B is a statistical graph of the rate of spindle abnormality in mature ova in vivo, with an abnormality rate of 7.9% in the ND group and 39.7% in the HFD group, i.e., a significant increase in the rate of spindle abnormality in ova expelled from obese mice (P < 0.001); while the abnormality rate of obese mice was reduced to 17.2% after niacin supplementation (HFD + NA), which was significantly reduced compared to the HFD group (P < 0.001). Therefore, the proportion of abnormal spindle bodies in mature ova can be effectively reduced by supplementing nicotinic acid in obese mice.

Example 13 in vivo Niacin supplementation reduces ROS levels in the ova of obese mice

In vivo matured ova were collected for ROS level detection, the procedure was the same as described in example 6, and the ROS fluorescence signal was observed using LSM710 Zeiss laser confocal microscope. Fluorescence intensity was calculated using ImageJ software and the fluorescence intensity values for each group were statistically analyzed by one-way anova.

The results are shown in FIG. 8: FIG. 8A is a typical picture of ROS fluorescence staining of each group, and FIG. 8B is a statistical result of fluorescence intensity values of each group. The ROS fluorescence signal of the mature ovum in the ND mouse is weaker, and the oxidative stress level is lower; the ROS fluorescence intensity of HFD is obviously increased (P <0.001), which indicates that the oxidative stress level is higher; while after the obese mice were supplemented with niacin (HFD + NA), ROS fluorescence signals of the mature ova were reduced by about 50%, significantly reduced compared to the HFD group (P <0.001), and not significantly different compared to the ND group. It can be seen that niacin supplementation in obese mice effectively reduced the level of ROS in mature ova.

In conclusion, the addition of nicotinic acid in the in vitro culture process or the in vivo supplementation of nicotinic acid through intraperitoneal injection can improve the quality reduction of the ovum and embryo of an obese individual, reduce the abnormal assembly proportion and the oxidative stress level of a meiosis device in a mature ovum and promote the development of an early embryo after fertilization to the blastocyst stage.

The above examples are only preferred embodiments of the present invention and are not intended to limit the embodiments of the present invention. Any other substitutions, simplifications, combinations, or alterations made without departing from the spirit and scope of the present invention are also intended to be included therein.