Application of anthocyanin in improving reproductive function of female beef cattle by regulating intestinal flora and reducing apoptosis of follicular granules

Abstract

The invention belongs to the technical field of biological medicine and livestock breeding, and particularly relates to application of anthocyanin in improving the reproductive function of female beef cattle by adjusting intestinal flora and reducing follicular granule cell apoptosis. Specifically, the invention discovers and verifies for the first time through research that the anthocyanin added into the feed can play roles in regulating the reproductive hormone level of the cattle with the ovarian dysfunction, improving the ovarian function and improving the intestinal flora lineage, and meanwhile, the anthocyanin can resist the apoptosis of ovarian granular cells by resisting oxidative stress injury, so that the reproductive function of the cattle is improved. The invention provides a scientific basis for the application of the anthocyanin in the aspect of improving the reproductive capacity of the female beef cattle, and discloses a mechanism for improving the ovarian function by resisting oxidation and regulating intestinal flora, thereby being beneficial to improving the application prospect of the anthocyanin in animal husbandry, and having a good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and animal husbandry, and particularly relates to the application of anthocyanins in improving the reproductive function of female beef cattle by regulating intestinal flora and reducing apoptosis of follicular granulosa cells. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Bovine reproductive disorders refer to a series of problems that affect the reproductive performance of dairy cows or beef cattle, including infertility, miscarriage, dystocia, reproductive system malformations, abnormal sexual behavior, etc. The decline in the fertility of beef cattle has seriously affected the development and economic benefits of the global beef cattle industry. In particular, reproductive disorders caused by abnormal ovarian function, such as persistent corpus luteum, ovulation disorders, follicular development arrest and ovarian cysts, have become a key issue limiting production efficiency. Ovarian function is related to a variety of internal and external factors, among which oxidative stress and intestinal flora imbalance are considered to be important influencing factors. In recent years, studies have found that intestinal flora not only plays a key role in the metabolic regulation of the host, but also affects reproductive health by regulating the endocrine and immune systems. However, the specific relationship between abnormal ovarian function and intestinal flora in beef cattle has not yet been clarified, and there is a lack of effective regulatory strategies.

[0004] Anthocyanins, a powerful antioxidant naturally found in fruits such as blueberries and grape seeds, have attracted widespread attention for their ability to scavenge free radicals and reduce oxidative stress damage. Studies have shown that anthocyanins play an important role in the prevention and treatment of various diseases, such as anti-cancer, cardiovascular protection, and anti-aging. However, there have been no relevant studies on whether anthocyanins can play a regulatory role in ovarian dysfunction in beef cows. Summary of the invention

[0005] In view of the deficiencies in the above-mentioned prior art, the inventors, after long-term technical and practical exploration, provide an application of anthocyanidins in improving the reproductive function of female beef cattle by regulating intestinal flora and reducing apoptosis of follicular granulosa cells. Specifically, the present invention first discovered and confirmed through research that adding anthocyanidins to the feed can regulate the reproductive hormone level of beef cows with ovarian dysfunction, improve ovarian function, and improve the intestinal flora. At the same time, it can resist ovarian granulosa cell apoptosis by resisting oxidative stress damage, thereby improving the reproductive function of female beef cattle. Based on the above research results, the present invention is completed.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides the use of anthocyanins in the preparation of a product for improving the reproductive function of female beef cattle.

[0008] Specifically, the improvement of the reproductive function of female beef cattle is specifically manifested as follows:

[0009] a) Regulate the reproductive hormone level of beef cows with ovarian dysfunction and improve ovarian function;

[0010] b) Improve the intestinal flora of beef cows;

[0011] c) Reduce apoptosis of ovarian granulosa cells by protecting against oxidative stress damage.

[0012] The second aspect of the present invention provides a composition consisting of anthocyanidins and at least one other pharmaceutically active ingredient and / or at least one other non-pharmaceutically active ingredient.

[0013] The composition can be used to improve the reproductive function of beef cows.

[0014] The third aspect of the present invention provides a method for improving the reproductive function of beef cows, which comprises administering anthocyanins or the above composition to the beef cows.

[0015] Compared with the existing technical solutions, the above one or more technical solutions have the following beneficial effects:

[0016] The above technical scheme discovered for the first time that anthocyanins can regulate the reproductive hormone levels of beef cows with ovarian dysfunction, improve ovarian function, and improve intestinal flora. At the same time, they can resist ovarian granulosa cell apoptosis by resisting oxidative stress damage, thereby being used to improve the reproductive function of beef cows. In short, the above technical scheme provides a scientific basis for the application of anthocyanins in improving the reproductive capacity of beef cows, and reveals the mechanism of improving ovarian function through anti-oxidation and regulating intestinal flora, which is conducive to improving its application prospects in animal husbandry and has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0018] Figure 1 This is a diagram of the ovarian morphological observation results of an embodiment of the present invention, wherein A is a control group, B is a low-concentration anthocyanin group, and C is a high-concentration anthocyanin group; the red arrow indicates a persistent corpus luteum, and the green arrow indicates a follicle.

[0019] Figure 2These are the test results of serum reproductive hormone levels in the embodiments of the present invention, wherein A: serum concentration of P4 in the control group; B: serum concentration of P4 after low-concentration anthocyanidin intervention; C: serum concentration of P4 after high-concentration anthocyanidin intervention; D: serum concentration of E2 in the control group; E: serum concentration of E2 after low-concentration anthocyanidin intervention; F: serum concentration of E2 after high-concentration anthocyanidin intervention.

[0020] Figure 3 The OTU Venn diagram (A) and dilution curve diagram (B) in the embodiment of the present invention, a: low anthocyanin concentration group; b: high anthocyanin concentration group; c: control group.

[0021] Figure 4 : This is the PCoA diagram of the intestinal flora of three groups of fecal samples in the embodiment of the present invention, A is the control group, B is the low concentration of anthocyanins group, and C is the high concentration of anthocyanins group.

[0022] Figure 5 The difference species of intestinal flora in three groups of samples at the phylum level (left) and genus level (right) in the embodiment of the present invention, A is the control group, B is the low concentration of anthocyanins group, and C is the high concentration of anthocyanins group.

[0023] Figure 6 This is the example of the present invention where anthocyanin intervention can significantly change granulosa cell oxidative damage, wherein A: heat map; B: volcano map.

[0024] Figure 7 This is the differential gene enrichment analysis in the embodiment of the present invention; wherein, A: GO enrichment analysis; B: KEGG signaling pathway enrichment analysis.

[0025] Figure 8 These are the results of detecting apoptosis and anti-apoptosis gene expression levels in the examples of the present invention; *p<0.05; **p<0.01.

[0026] Fig. 9 The effect of anthocyanin intervention on the level of reactive oxygen species in the examples of the present invention; *p<0.05; **p<0.01; ***p<0.001. DETAILED DESCRIPTION

[0027] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0028] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0029] In a typical embodiment of the present invention, there is provided the use of anthocyanins in the preparation of a product for improving the reproductive function of female beef cattle.

[0030] Specifically, the improvement of the reproductive function of female beef cattle is specifically manifested as follows:

[0031] a) Regulate the reproductive hormone level of beef cows with ovarian dysfunction and improve ovarian function;

[0032] b) Improve the intestinal flora of beef cows;

[0033] c) Reduce apoptosis of ovarian granulosa cells by protecting against oxidative stress damage.

[0034] In the application a), the reproductive hormones include P4 and E2.

[0035] The anthocyanidin addition amount is not less than 200 mg / kg·d anthocyanidin, and further is 400 mg / kg·d anthocyanidin;

[0036] In the application b), the improvement of the intestinal flora of beef cows is specifically manifested as: reducing the abundance of Patellar bacteria, Actinobacteria, Proteobacteria and Chloroflexi in the intestinal flora, and increasing the abundance of Desulfobacteria bacteria; promoting the abundance of unclassified UCG-010 bacteria, Monococcus, unclassified Faecalibacterium, Bacteroides, NK4A214 bacteria and unclassified Ruminococcaceae bacteria.

[0037] In the application c), the oxidative stress damage is induced by H2O2;

[0038] In another specific embodiment of the present invention, the anti-oxidative stress damage reduces the apoptosis of ovarian granulosa cells by down-regulating the expression of apoptotic genes (including P53, Fas, bax) and up-regulating the expression of anti-apoptotic genes (including bcl2, Bcl-xL).

[0039] The product may be a drug or a test reagent, and the test reagent is used for basic research and can be used to construct relevant cell or animal models.

[0040] In another specific embodiment of the present invention, a composition is provided, wherein the composition is composed of anthocyanidins and at least one other pharmaceutically active ingredient and / or at least one other non-pharmaceutically active ingredient.

[0041] The other active ingredients of the medicine include substances that can improve the reproductive function of female beef cattle.

[0042] The non-pharmaceutical active ingredients include pharmaceutically acceptable carriers, excipients and / or diluents.

[0043] In another specific embodiment of the present invention, the non-drug active ingredients include:

[0044] Pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and nonionic surfactants or lipids;

[0045] The administration dosage form of the pharmaceutical preparation includes: liquid dosage form and solid dosage form.

[0046] The composition can be used to improve the reproductive function of beef cows.

[0047] In another specific embodiment of the present invention, a method for improving the reproductive function of beef cows is provided, the method comprising administering anthocyanidins or the above composition to the beef cows.

[0048] It should be noted that although the present invention takes beef cows as an example to provide the application of anthocyanins in improving the reproductive function of beef cows, based on the inventive concept of this application, the application of anthocyanins in other animals, especially livestock animals, also falls within the protection scope of the present invention.

[0049] The present invention is further described below in conjunction with specific examples, which are only for explaining the present invention and do not limit its content. Any simple modification, equivalent changes and modifications made to the implementation methods based on the technical essence of the present invention are within the scope of the technical solution of the present invention.

[0050] Example

[0051] 1. Materials and methods:

[0052] 1.1 Experimental animals and experimental drugs

[0053] In a large beef cattle farm near Yinchuan, B-ultrasound was used to detect the development of ovarian follicles and corpus luteum in beef cows. Ovarian quiescence and persistent corpus luteum were used as screening criteria. Finally, 48 beef cows with abnormal ovarian function (2-4 parities) were screened out and randomly divided into a control group, a low-concentration anthocyanin group, and a high-concentration anthocyanin group, with 16 cows in each group. The control group was fed a basic diet, and the low- and high-concentration anthocyanin groups were added with 200 and 400 mg / kg·d anthocyanin in the basic diet for 60 consecutive days. Anthocyanin was provided by Nanjing Xinhou Biotechnology Co., Ltd. with a purity of ≥95%. After the start of the experiment, blood was collected from the root vein of the tail of the three groups of cattle every 6 days. After centrifugation at 4000rpm / min for 10 minutes, the serum was collected and stored in an ultra-low temperature refrigerator at -80℃ for subsequent determination of serum reproductive hormone indicators. At the end of the experiment, the three groups of cattle were subjected to rectal feces collection, and the collected feces were immediately stored in a liquid nitrogen tank and then transferred to an ultra-low temperature refrigerator at -80℃ for storage.

[0054] The composition of the experimental basic diet is shown in Table 1. This experiment was conducted in a large beef cattle farm near Yinchuan, Ningxia. The selected experimental beef cattle were grouped and housed in three cow sheds for centralized feeding. The sheds were equipped with exercise areas, water troughs, licking bricks, etc., and were centrally fed twice a day (6:30 am and 4:30 pm) and had free access to water.

[0055] Table 1 Composition and nutritional level of experimental diets (dry matter basis, %)

[0056]

[0057]

[0058] 1) 5% cow premix per kg provides the following in the diet: VA 11 500 IU, VD 3 250 IU, VE 58 IU, Fe 92 mg, Cu 13.5 mg, Zn 90 mg, Mn 90 mg.

[0059] 2) Comprehensive net energy is a calculated value.

[0060] 1.2 Isolation and culture of ovarian granulosa cells

[0061] In this study, healthy multiparous cows with 2 to 4 parities and no ovarian dysfunction were selected from a large beef cattle slaughterhouse near Yinchuan. The ovaries were obtained in time after the beef cows were slaughtered, and the obtained ovarian tissue was placed in a culture dish containing PBS to remove the tissue around the ovary and the surface capsule. After washing with PBS several times, it was placed in a new culture dish containing PBS. Under a stereo microscope, the follicles were punctured with a 1mL syringe needle to release the granulosa cells into PBS, blow and mix, and then filter with a 200-mesh cell sieve. The cells and liquid were collected in a centrifuge tube, and then centrifuged at 1000rpm / min for 5min, and the supernatant was discarded. DMEM culture medium was added to the centrifuge tube and gently blown with a pipette to make a cell suspension. The cells were then inoculated into a culture dish and cultured in a 37°C, 5% CO2 incubator. The Yuan-generation cells to be separated grew to about 60%, and the collected cells were divided into four groups, each with replicates. One group was a blank control group, the second group was a H2O2 (oxidative damage) treatment group, the third group was an anthocyanin control group, and the fourth group was an H2O2+anthocyanin intervention group. A blank control group was not treated, and groups two and four were first added with 100mmol / L H2O2, and groups three and four were then treated with 150mmol / L anthocyanin. After the treatment, they were cultured in a 37°C, 5% CO2 incubator. After 24 hours, the cells were collected for the next experiment. That is, they were stored in a liquid nitrogen tank and then transferred to a -80°C ultra-low temperature refrigerator for storage.

[0062] 1.3 Observation of ovarian morphology

[0063] The development of ovarian follicles and corpus luteum of beef cows was observed by using B-ultrasound rectal examination.

[0064] 1.4 Serum reproductive hormone level detection

[0065] The levels of estrogen (E2) and progesterone (P4) in serum were detected by Elisa test kit, and the serum was sent to Wuhan Cloud Clone Company to complete the testing.

[0066] 1.5 Intestinal flora detection and bioinformatics analysis

[0067] use Soil DNA Kit DNA Extraction Kit (Omega, USA) was used to extract total DNA from intestinal feces microorganisms, and then the purity and concentration of DNA were tested by 1% agarose gel electrophoresis. Then, PCR was used to amplify the V3-V4 region of 16S rRNA from a single metagenomic DNA sample. After amplification, the PCR product was stained with QuantiFluor TM-ST blue fluorescence quantitative system (Promega) was used for detection and quantification, and then the corresponding proportions were mixed according to the sequencing requirements of each sample. The Novasek library was constructed using the TruSeqTM DNA Sample Prep Kit. After the library was qualified, the amplified product was sequenced on the NovaSeq6000 platform (Illumina, USA). The PEreads obtained by Novasek sequencing were first spliced ​​according to the overlap relationship, and the sequence quality was quality controlled and filtered. After distinguishing the samples, OTU clustering analysis and species taxonomy analysis were performed.

[0068] 1.6 RNA-sequencing

[0069] The cells collected in 1.2 were treated with H2O2 (oxidative damage) and H2O2 + anthocyanin for 24 hours. A portion of the samples from each group were kept for subsequent experiments, and the rest were transported in dry ice and handed over to Shanghai Meiji Biotechnology Co., Ltd. for RNA-sequencing detection and bioinformatics analysis of the results.

[0070] 1.7 Active oxygen level detection

[0071] After the four groups of cells were treated for 24 hours, the reactive oxygen species stain SOX was added, the cell nuclei were stained with DAPI, and the fluorescence intensity was analyzed by Image J to determine the degree of oxidative damage.

[0072] 1.8 Statistical methods

[0073] SPSS11.5 software was used to analyze the data. The data were expressed as mean±standard deviation (x±s). One-way analysis of variance was used for comparison among the groups. P<0.05 was considered statistically significant.

[0074] 2. Results and Discussion

[0075] 2.1 Observation of ovarian morphology

[0076] Depend on Figure 1 As shown, at the end of the experiment, a small number of large follicles could be detected on the ovaries of the beef cows in the control group ( Figure 1 A), but no significant changes were observed in the persistent corpus luteum; in the low-concentration anthocyanin group, a small number of large follicles and a relatively large number of primordial follicles could be detected on the ovaries ( Figure 1 B), the persistent corpus luteum was improved to a certain extent; in the high concentration anthocyanin group, more large follicles could be detected on the ovaries ( Figure 1 C), the persistent corpus luteum shows signs of regression.

[0077] 2.2 Serum reproductive hormone level detection

[0078] like Figure 2 As shown, there was no significant difference in the change of P4 level after treatment in the control group ( Figure 2 A), while after low-concentration intervention, P4 hormone levels began to decrease significantly at 42 days (P<0.05) ( Figure 2 B), while after high-concentration intervention, P4 hormone levels began to decrease significantly on day 36 (P<0.05) ( Figure 2 C). The estrogen levels in the control group did not change ( Figure 2 D), although the estrogen level increased from 48 days after low concentration anthocyanin intervention, it was not statistically significant ( Figure 2 E), after high-concentration intervention, the estrogen level gradually increased from 42 days, and at 60 days, the estrogen level increased significantly (P<0.05) ( Figure 2 F). Therefore, combined with the results of B-ultrasound, it can be confirmed that high concentration of anthocyanins can significantly improve the ovarian function of beef cattle with persistent corpus luteum.

[0079] 2.3 Analysis of intestinal flora diversity

[0080] 2.3.1 Sequencing analysis and species annotation of intestinal flora in each group of beef cows

[0081] 16S r RNA sequencing was performed on fecal samples of each group of beef cows to obtain the number of OTUs in the intestinal flora of each group. Figure 3 It can be seen that a total of 2,010 OTUs were obtained from the three groups of samples, of which the control group had 1,691 OTUs and 40 unique OTUs; the low-concentration anthocyanin group had 1,833 OTUs and 39 unique OTUs; the high-concentration anthocyanin group had 1,827 OTUs and 95 unique OTUs. There were 1,505 OTUs in total in the three groups. The dilution curve was drawn with the amount of data extracted as the horizontal axis and the number of species as the vertical axis. Figure 3 B shows that as the amount of extracted data increases, the curve tends to be flat, indicating that the amount of sequencing sample data is sufficient to reflect the information of sample microbial species and the results are highly accurate.

[0082] 2.3.2 Effects of anthocyanins on the diversity of intestinal flora of beef cows

[0083] As shown in Table 2, the α diversity index of beef cows in groups B and C, shannon, ace and chao, was significantly higher than that in group A (P<0.01). It can be seen from the PCoA diagram of the three groups of samples that ( Figure 4 ), there were significant differences in the microbial communities of the three groups of samples, indicating that anthocyanins can significantly change the composition of the intestinal flora of beef cows.

[0084] Table 2 Effect of anthocyanins on the Alpha diversity index of intestinal flora of beef cows

[0085]

[0086] 2.3.3 Regulation of Anthocyanins on the Intestinal Flora of Beef Cows

[0087] One-way ANOVA was used to examine the differential flora at the phylum and genus levels among the three groups. At the phylum level, significant differences were found in Patescibacteria, Actinobacteriota, Proteobacteria, Desulfobacterota, and Chloroflexi; at the genus level, there were significant differences in norank_f__UCG-010 of the Ruminococcaceae family

[0088] (norank_f__UCG-010), Monoglobus, norank_f__Eubacterium_coprostanoligenes_group, Paeniclostridium

[0089] (Paeniclostridium), Bacteroides, unclassified_f__Lachnospiraceae, NK4A214_group, norank_f__Ruminococcaceae, norank_f__Muribaculaceae, Lachnospiraceae_NK3A20_group.

[0090] 2.4 Anthocyanin Intervention Significantly Improves Oxidative Damage of Granulosa Cells

[0091] Taking 丨log2(Fold change)丨≥1 and Padj≤0.05 as the conditions for screening differential genes, the heatmap results showed that anthocyanin intervention could significantly distinguish the H2O2 treatment group from the H2O2 + anthocyanin treatment group( Figure 6 A), and the volcano plot results showed that there were 746 down-regulated differential genes and 1221 up-regulated differential genes in the H2O2 + anthocyanin treatment group compared with the H2O2 treatment group. Therefore, anthocyanin intervention can significantly affect the function of ovarian granulosa cells damaged by oxidative stress.

[0092] 2.5 Anthocyanin Intervention Inhibits Granulosa Cell Apoptosis

[0093] GO enrichment analysis of the selected differential gene set revealed that the differential genes were mainly involved in regulating the cell cycle( Figure 7 A), and the KEGG pathway enrichment results revealed that multiple signaling pathways were involved in regulating cell proliferation and apoptosis( Figure 7 B), such as MAPK, PI3K-AKT, FoxO, P53, etc. Therefore, our anthocyanin intervention can reduce the apoptosis of ovarian granulosa cells caused by oxidative damage. Our further RT-PCR results revealed that anthocyanin intervention can significantly downregulate multiple apoptosis genes such as P53, Fas, bax, and upregulate anti-apoptotic genes bcl2, Bcl-xL ( Figure 8 ).

[0094] 2.6 Anthocyanin intervention can reduce oxidative damage

[0095] In order to further explore the effect of anthocyanin intervention on oxidative stress and reactive oxygen levels in granulosa cells, we detected the level of reactive oxygen (SOX, reactive oxygen probe) and used Image J to analyze the fluorescence intensity. The results showed that the reactive oxygen levels in the H2O2 treatment group were significantly increased compared with the control group, while the anthocyanin and H2O2 treatment groups could significantly reduce the reactive oxygen levels compared with the H2O2 treatment group ( Fig. 9 ), reduced ROS levels were accompanied by reduced apoptotic granulosa cells.

[0096] In summary, the present invention explores the role of anthocyanins in improving the fertility of cows with abnormal ovarian function and its potential mechanism. The results show that anthocyanins can regulate intestinal flora and hormone levels, and can also reduce ovarian granulosa cell apoptosis through antioxidant effects, promote follicle development and regulate hormone levels.

[0097] First, the present invention shows that anthocyanins significantly improve the reproductive function of cows with abnormal ovarian function, which is reflected in the increase in the number of large follicles and the reduction of persistent corpora lutea. This means that fertility is improved and ovarian health is improved. In addition, after anthocyanin treatment, the level of progesterone (P4) in the serum of cows decreased significantly, while the level of estrogen (E2) began to increase significantly on the 42nd day in the high-concentration group, indicating that anthocyanins may promote follicle development and ovulation by regulating hormone balance.

[0098] In this experiment, the number of OTUs in the intestinal flora of beef cows in the experimental group given anthocyanins was significantly increased compared with the control group, and the Shannon index, ACE index and Chao index were all significantly higher than those in the control group, indicating that the addition of anthocyanins can significantly increase the abundance and diversity of intestinal flora in beef cows with ovarian dysfunction.

[0099] The analysis of fecal flora showed that the complexity of fecal flora of beef cows fed with anthocyanin-added basic feed was higher than that of the control group, and there were significant differences in Alpha and Bate diversity. The Venn diagram also showed that there were differences in fecal flora, indicating that anthocyanin is beneficial for restoring the number and types of intestinal flora. The experimental results show that anthocyanin can significantly improve the structure of intestinal flora, increase the level of beneficial bacteria, and reduce the content of harmful bacteria, thereby playing a role in restoring ovarian function.

[0100] In addition, RNA-sequencing analysis revealed a significant effect of anthocyanins on transcriptional regulation of ovarian granulosa cells. Oxidative damage usually exacerbates cell damage by inducing the expression of related genes, while anthocyanins enhance the antioxidant capacity of granulosa cells by significantly regulating the expression of antioxidant-related genes. Transcriptome analysis also showed that anthocyanin intervention could significantly downregulate the expression of pro-apoptotic genes (such as P53, Fas, and bax) and upregulate the expression of anti-apoptotic genes (such as bcl2 and Bcl-xL). This further supports the mechanism of action of anthocyanins in reducing granulosa cell apoptosis. In addition, KEGG signaling pathway analysis showed that multiple key signaling pathways (such as MAPK, PI3K-AKT, P53, and FoxO pathways) were involved in the regulation of anthocyanins on granulosa cells, revealing that anthocyanins may protect ovarian cells from damage caused by oxidative stress through multiple pathways.

[0101] In conclusion, the present invention shows that anthocyanins improve the reproductive function of beef cattle by regulating intestinal flora and reducing apoptosis of follicular granulosa cells, and the biological activity of anthocyanins may be dose-dependent.

[0102] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Application of anthocyanins in the preparation of products for improving the reproductive function of female beef cattle; The improvement of the reproductive function of female beef cattle is specifically manifested as follows: a) Regulate the reproductive hormone level of beef cows with ovarian dysfunction and improve ovarian function; b) Improve the intestinal flora of beef cows; c) Reduce apoptosis of ovarian granulosa cells by protecting against oxidative stress damage.

2. The use according to claim 1, characterized in that: In the application a), the reproductive hormones include P4 and E2.

3. The use according to claim 1, characterized in that: In the application a), the amount of anthocyanidin added is not less than 200 mg / kg·d anthocyanidin, and further is 400 mg / kg·d anthocyanidin.

4. The use according to claim 1, characterized in that: In the application b), the improvement of the intestinal flora of beef cows is specifically manifested as: reducing the abundance of Patellar bacteria, Actinobacteria, Proteobacteria and Chloroflexi in the intestinal flora, and increasing the abundance of Desulfobacteria bacteria; promoting the abundance of unclassified UCG-010 bacteria, Monococcus, unclassified Faecalibacterium, Bacteroides, NK4A214 bacteria and unclassified Ruminococcaceae bacteria.

5. The use according to claim 1, characterized in that: In the application c), the oxidative stress damage is induced by H2O2.

6. The use according to claim 1, characterized in that: The anti-oxidative stress damage reduces the apoptosis of ovarian granulosa cells, which is specifically manifested by down-regulating the expression of apoptotic genes (including P53, Fas, bax) and up-regulating the expression of anti-apoptotic genes (including bcl2, Bcl-xL).

7. The use according to claim 6, characterized in that The apoptosis genes include P53, Fas, and bax, and the anti-apoptosis genes include bcl2 and Bcl-xL.

8. The use according to claim 1, characterized in that The product is a drug or a test reagent, and the test reagent is used for basic research.

9. A composition, characterized in that The composition is composed of anthocyanidins and at least one other active pharmaceutical ingredient and / or at least one other non-active pharmaceutical ingredient; The composition is used for improving the reproductive function of beef cows.

10. A method for improving the reproductive function of beef cows, characterized in that: The method comprises administering anthocyanins or the composition of claim 9 to beef cows.

Images