Application of C-phycocyanin and D-glucuronic acid in directly improving sperm motility in vitro and improving sperm motility after freezing recovery

Abstract

The invention discloses application of C-phycocyanin and D-glucuronic acid in directly improving sperm motility in vitro and improving sperm motility after freezing recovery, and belongs to the technical field of biological medicine. According to the present invention, the mixed solution of D-glucuronic acid, C-phycocyanin, and D-phycocyanin has significant effects of in-vitro sperm motility promotion and sperm function protection, can be used for preparing the sperm motility improving drug or the sperm treatment reagent in the assisted reproduction technology, and has good application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the direct in vitro enhancement of sperm motility by C-phycocyanin and D-glucuronic acid and their application in enhancing the motility of frozen and thawed sperm. Background Technology

[0002] C-phycocyanin (C-PC), widely found in cyanobacteria such as Spirulina, is a water-soluble pigment-protein complex. Its molecular structure consists of two subunits, α and β, linked by covalent bonds, with a molecular weight ranging from 70,000 to 110,000 Da. In cyanobacteria, this protein acts as a key light-harvesting pigment-protein complex in the photosynthetic system, participating in core biological processes such as light energy capture, energy transfer, and ATP synthesis. Besides its photosynthetic function, C-phycocyanin exhibits various biological activities. Experimental data have confirmed that C-phycocyanin can efficiently scavenge various reactive oxygen species, including DPPH free radicals, ABTS cation free radicals, and superoxide anion free radicals, demonstrating antioxidant effects and significant functions in anti-tumor, anti-inflammatory, anti-aging, and promoting body repair.

[0003] D-glucuronic acid is a natural sugar acid derived from glucose and plays several key roles in the body. In the liver, many endogenous waste products (such as bilirubin and steroid hormones) and exogenous toxins (such as drugs and environmental toxins) are difficult to excrete in urine due to their poor water solubility. Hepatocytes utilize uridine diphosphate glucuronic acid as an active donor, and under the catalysis of U-glucuronyltransferase, transfer glucuronic acid to specific functional groups (such as -OH, -COOH, -NH2) of these toxins, generating highly water-soluble glucuronide compounds that are easily excreted into the intestines through bile or excreted through urine, thus playing a detoxification role. However, there is currently no research on the effect of D-glucuronic acid on improving sperm motility, and no corresponding products exist. Our findings suggest that D-glucuronic acid can neutralize endogenous metabolic waste and expel it from the cell body, thereby reducing sperm ROS stress in vitro and maintaining sperm motility. The combined use of D-glucuronic acid and C-phycocyanin can significantly improve IVF efficiency.

[0004] In vitro fertilization (IVF), as a core technology of modern assisted reproductive medicine, not only provides an important experimental platform for clinical applications and basic research in human medicine, but also offers a crucial experimental tool in the field of animal model construction (such as rapid propagation technology for mouse IVF). IVF refers to the technique of achieving sperm-egg fusion, completing the fertilization process, and cultivating early embryos in an in vitro simulated physiological environment. Existing research shows that sperm motility is the most critical biological parameter affecting the success rate of IVF. Highly active sperm can more effectively complete chemotaxis, penetrate the cumulus cell complex and zona pellucida structure, and ultimately achieve fertilization. Insufficient sperm motility will significantly reduce the IVF rate, negatively impact embryonic developmental potential, and ultimately lead to a decline in clinical pregnancy success rates. Therefore, improving sperm motility and optimizing sperm quality are crucial aspects of improving the success rate of assisted reproductive technology in IVF procedures.

[0005] In recent years, research teams have discovered that C-phycocyanin has a significant protective effect on the male reproductive system at the animal level (mice, pigs, sheep, etc.), and it shows potential application value in maintaining sperm motility and improving sperm quality parameters. This invention shows that the combined use of C-phycocyanin and D-glucuronic acid can more effectively and directly improve sperm motility and can be used in IVF. Summary of the Invention

[0006] The purpose of this invention is to provide C-phycocyanin and D-glucuronic acid for directly improving sperm motility in vitro and their application in improving the motility of frozen and thawed sperm. This invention discovers that adding C-phycocyanin and D-glucuronic acid to sperm culture in vitro significantly improves sperm motility and also substantially extends sperm survival time.

[0007] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention is the application of C-phycocyanin and D-glucuronic acid in improving sperm motility, wherein the mass ratio of C-phycocyanin to D-glucuronic acid is (2~4):1.

[0008] The second technical solution of the present invention is the application of C-phycocyanin and D-glucuronic acid in the preparation of drugs to improve sperm motility, wherein the mass ratio of C-phycocyanin to D-glucuronic acid is (2~4):1.

[0009] The third technical solution of the present invention is a drug for improving sperm motility, comprising C-phycocyanin and D-glucuronic acid, as well as pharmaceutically acceptable excipients.

[0010] The fourth technical solution of this invention is the application of C-phycocyanin and D-glucuronic acid in the preparation of products for assisted reproduction.

[0011] The fifth technical solution of the present invention is a method for improving sperm motility, which involves culturing sperm in a system containing C-phycocyanin and D-glucuronic acid.

[0012] Based on the above technical solution, the present invention has the following technical effects: This invention is the first to discover that C-phycocyanin and D-glucuronic acid can directly improve sperm motility in vitro, and that they can still effectively enhance sperm motility after cryopreservation and thawing, demonstrating significant value in assisted reproductive applications. Specifically: (1) Significantly improves sperm motility: Experimental results show that after adding C-phycocyanin and D-glucuronic acid to the sperm in vitro culture medium, key motility parameters such as total motility, forward motility, average path speed, linear speed, curvilinear speed, head lateral swing amplitude and whipping frequency of sperm are significantly improved, indicating that the mixture of C-phycocyanin and D-glucuronic acid can effectively enhance sperm motility and fertilization potential. (2) Prolonging sperm survival time: During in vitro culture, the sperm survival time of the C-phycocyanin and D-glucuronic acid treatment groups was significantly prolonged. Especially after 3 hours of culture, the sperm mortality rate of the control group increased significantly, while the C-phycocyanin and D-glucuronic acid groups still maintained a high survival rate, indicating that they have a good sperm protection effect. (3) Improve sperm quality after cryopreservation and thawing: In the sperm cryopreservation and thawing experiment, the sperm motility recovery of the experimental group with added C-phycocyanin and D-glucuronic acid was better than that of the control group, indicating that it can alleviate the damage to sperm caused by the freezing process and improve the thawing quality of frozen sperm.

[0013] In summary, C-phycocyanin and D-glucuronic acid, as two natural active ingredients, when used in combination in a specific ratio, have significant effects on promoting sperm motility and protecting sperm function in vitro. They can be used to prepare drugs to improve sperm motility or sperm treatment reagents in assisted reproductive technologies, and have good application prospects. Attached Figure Description

[0014] Figure 1 The effect of the mixture on the overall sperm motility rate.

[0015] Figure 2 The effect of the mixture on sperm forward motility.

[0016] Figure 3 The effect of the mixture on the average path velocity of sperm.

[0017] Figure 4 The effect of the mixture on sperm linear velocity.

[0018] Figure 5The effect of the mixture on sperm velocity curve.

[0019] Figure 6 The effect of the mixture on the lateral movement of the sperm head.

[0020] Figure 7 The effect of the mixture on sperm whipping frequency.

[0021] Figure 8 For sperm motility trajectory analysis.

[0022] Figure 9 Sperm motility trajectory under a microscope after sperm freezing and thawing (A) and sperm motility test after thawing (B).

[0023] Figure 10 This study describes the in vitro fertilization (IVF) of mouse sperm after cryopreservation and thawing. In this study, A represents embryos from the two-cell and blastocyst stages obtained using the thawed sperm, and B represents the statistical analysis of two-cell and blastocyst stage embryos. Detailed Implementation

[0024] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0025] This invention provides the application of C-phycocyanin and D-glucuronic acid in improving sperm motility, wherein the mass ratio of C-phycocyanin to D-glucuronic acid is (2~4):1.

[0026] In some specific implementations, C-phycocyanin and D-glucuronic acid improve sperm motility by increasing total sperm motility, forward motility, average path velocity, linear velocity, curvilinear velocity, head lateral swing amplitude, whiplash frequency, and survival rate.

[0027] This invention also provides the application of C-phycocyanin and D-glucuronic acid in the preparation of drugs to improve sperm motility, wherein the mass ratio of C-phycocyanin to D-glucuronic acid is (2~4):1.

[0028] In some specific implementations, C-phycocyanin and D-glucuronic acid improve sperm motility by increasing total sperm motility, forward motility, average path velocity, linear velocity, curvilinear velocity, head lateral swing amplitude, whiplash frequency, and survival rate.

[0029] This invention also provides a drug for improving sperm motility, comprising C-phycocyanin and D-glucuronic acid, as well as pharmaceutically acceptable excipients.

[0030] In some specific implementations, the dosage form of the drug is a liquid dosage form, a solid dosage form, or a paste dosage form.

[0031] This invention also provides the application of C-phycocyanin and D-glucuronic acid in the preparation of products for assisted reproduction.

[0032] This invention also provides a method for improving sperm motility by culturing sperm in a system containing C-phycocyanin and D-glucuronic acid.

[0033] In some specific implementations, the concentration of C-phycocyanin in the system is 40 mg / L, and the concentration of D-glucuronic acid in the system is 50 μM.

[0034] Example 1 Materials and reagents: C-Phycocyanin, catalog number: A426071-0005, Sangon Biotech (Shanghai) Co., Ltd.; D-glucuronic acid, catalog number: G105701-1g, Aladdin Reagent. Mouse sperm cryopreservation solution (Nanjing Aibei Biotechnology Co., Ltd., catalog number: R18S3); HTF medium (Nanjing Aibei Biotechnology Co., Ltd., model: M135); male C57 mice, Vitaliva.

[0035] Equipment and instruments: Hamilton Thorne IVOS II sperm analyzer (USA); straw sealing machine; 37℃ CO2 incubator; 37℃ metal hot plate (Nanjing Aibei Biotechnology Co., Ltd., model: ABHOTN01).

[0036] In vitro methods for promoting sperm motility: Sperm suspension preparation: Three SPF-grade C57 male adult mice (provided by Vital River) were selected. The epididymis of both mice was removed and placed in PBS pre-warmed at 37°C to wash away hair and blood. The mixture was then transferred to three EP tubes containing 1 mL of PBS. The epididymal heads were cut open, and after the sperm were fully freed, the bottom tissue was removed. The sperm suspensions from the three EP tubes were mixed (to reduce the influence of individual sperm motility errors) and divided into seven equal portions. The final volume of each EP tube was adjusted to 1 mL with pre-warmed PBS (and stored at 37°C).

[0037] Grouping: The mice were divided into seven groups: normal saline (control) group, 20 mg / L C-phycocyanin single component group, 20 mg / L C-phycocyanin and 25 μM D-glucuronic acid group, 20 mg / L C-phycocyanin and 50 μM D-glucuronic acid group, 40 mg / L C-phycocyanin and 50 μM D-glucuronic acid group, 40 mg / L C-phycocyanin and 100 μM D-glucuronic acid group, and 80 mg / L C-phycocyanin and 100 μM D-glucuronic acid group. For the balanced mouse suspensions in the seven groups, an equal volume of physiological saline was added to the normal group; and an equal volume of C-phycocyanin solution with a matching concentration was added to the experimental groups. After gentle mixing, the suspensions were incubated at 37℃ in a 5% CO2 incubator for 1.0 h, 2.0 h, 3.0 h, and 4.0 h, after which sperm motility was measured.

[0038] Experimental methods for promoting the thawing of frozen sperm: Divide the samples into three groups. Take 6 EP tubes and add 200ul of HTF sperm resuscitation solution to each EP tube. Add an appropriate amount of working solution of C-phycocyanin and D-glucuronic acid to 3 of the tubes to make the C-phycocyanin concentration 40mg / L and the D-glucuronic acid concentration 50μM. Add an equal amount of physiological saline to the other 3 tubes as the control group.

[0039] Adult male C57 rats were euthanized by cervical dislocation. After disinfecting the abdomen with 75% alcohol, the skin and muscles were cut open, and one epididymis was removed and placed in a 120 μL droplet of mouse sperm cryoprotectant (120 μL of mouse sperm cryopreservation solution was used as a droplet on a 6 cm dish). The epididymis was cut open with scissors, and 3 minutes were allowed to ensure sufficient sperm expulsion. During this process, it was recommended to gently mix the semen with a pipette every 1 minute. After 3 minutes, 4-5 drops of 10 μL each were aspirated from the 120 μL sperm cryopreservation solution droplet and placed around it for subsequent aspiration and cryopreservation via straw. The other epididymis was treated in the same manner. The entire experiment was conducted on a hot metal table. Connect a 0.25ml wheat tube to a 1ml syringe via a wheat tube connector (wheat tube connector: remove the needle from the 1ml syringe, insert the tip of the 200ul yellow pipette into the syringe, and insert the wheat tube into the yellow pipette tip). Aspirate approximately 100ul of HTF culture medium, aspirate 1cm of air, then aspirate approximately 10ul of sperm suspension, and finally aspirate air again. Heat-seal the wheat tube using a wheat tube sealing machine. Pour liquid nitrogen into a foam box, ensuring the liquid nitrogen level is >3cm. Place the wheat tube in a wheat tube rack, with the bottom of the rack approximately 6-10cm from the top edge of the liquid nitrogen surface (replenish the liquid nitrogen if it drops before pre-cooling the wheat tube). After pre-cooling with liquid nitrogen vapor for 10 minutes, quickly immerse the wheat tube in the liquid nitrogen using metal tweezers for approximately 1-3 minutes.

[0040] Preheat the water bath to 37°C. Use tweezers to remove the wheat tubes from the liquid nitrogen container and quickly transfer them to the water bath to thaw for 15 minutes. Remove the wheat tubes from the water bath, wipe the surface dry with gauze, cut open the sealed ends, and add the sperm suspension into a drop of 200 μL HTF culture medium that has been preheated and equilibrated (>0.5 h). Place the tubes in a CO2 incubator for capacitation for 1 h, 2 h, and 3 h. Use a Hamilton Thorne IVOS II sperm analyzer to detect sperm motility in the two groups at the three time points.

[0041] Sperm motility testing: Sperm motility was assessed in each group at different time points using a Hamilton Thorne IVOS II sperm analyzer. After incubation for 1.0h, 2.0h, 3.0h, and 4.0h, 25µl of sperm suspension from each group was placed in the chamber of a glass slide, and changes in sperm motility, count, and related movement parameters were analyzed using CASA (Cyclic Acid Separation).

[0042] Experimental results: Total motility (MT, %) refers to the percentage of motile sperm out of the total sperm count. MT is the primary indicator for evaluating sperm motility in IVF technology and a prerequisite for assessing whether sperm can reach the fertilization site (amula of the fallopian tube). Figure 1 The results showed that C-phycocyanin can directly improve sperm motility and prolong the duration of sperm motility. When the C-phycocyanin concentration was 20 mg / L, the group with added D-glucuronic acid showed a significantly higher sperm motility than the group without D-glucuronic acid, and this improvement continued to increase with increasing D-glucuronic acid concentration. The optimal effect was observed when the C-phycocyanin concentration was 40 mg / L and the D-glucuronic acid concentration was 50 μM.

[0043] Progressive motility (PR, %) is the proportion of sperm that move actively in a straight line or large circular motion. Only sperm with good progressive motility can effectively penetrate cervical mucus and swim upstream to meet the egg. PR is most strongly correlated with the success rate of natural pregnancy and intrauterine insemination, and is a key predictive indicator of male sperm fertilization capacity. Figure 2 The results showed that within one hour, at a C-phycocyanin concentration of 20 mg / L, the group with added D-glucuronic acid exhibited a significantly higher sperm forward motility rate compared to the group without D-glucuronic acid, and this increase continued to rise with increasing D-glucuronic acid concentration. The optimal effect was observed when the C-phycocyanin concentration was 40 mg / L and the D-glucuronic acid concentration was 50 μM, resulting in a three-fold increase in sperm PR levels, and the sperm remained active even three hours after treatment.

[0044] Average path velocity (VAP, μm / s) is the velocity calculated from the smoothed average path of the sperm head along its trajectory. It reflects the overall sperm motility; a high VAP value indicates strong sperm motility, which is beneficial for fertilization. Figure 3 The results showed that sperm VAP levels did not differ significantly within the first two hours after treatment with C-phycocyanin and D-glucuronic acid. However, from the third to the fourth hour, the mixture significantly increased the average sperm path velocity. At a C-phycocyanin concentration of 20 mg / L, the group with added D-glucuronic acid showed a significantly higher average sperm path velocity compared to the group without D-glucuronic acid, and this increase continued with increasing D-glucuronic acid concentration.

[0045] Straight-line velocity (VSL, μm / s) is the average velocity of the sperm head as it moves in a straight line from the start to the end of its trajectory. It is also the net velocity at which the sperm overcomes resistance and propels itself toward its target. It reflects the efficiency of sperm forward motility; a higher VSL value indicates stronger sperm penetration ability. Figure 4 The results showed that sperm VSL levels did not differ significantly in the first two hours after treatment with C-phycocyanin and D-glucuronic acid. However, from the third to the fourth hour, the mixture significantly increased sperm linear velocity. At a C-phycocyanin concentration of 20 mg / L, the group with added D-glucuronic acid showed a significantly higher sperm linear velocity compared to the group without D-glucuronic acid, and this increase continued to rise with increasing D-glucuronic acid concentration.

[0046] Curvilinear velocity (VCL, μm / s) is the instantaneous average velocity of the sperm head along its actual trajectory (which is necessarily a curve). A high VCL value indicates vigorous sperm flagellar motility and strong propulsion. Figure 5 The results showed that the sperm VCL levels treated with C-phycocyanin and D-glucuronic acid did not differ significantly in the first hour, with significant differences only detected at 40 mg / L C-phycocyanin and 50 μM D-glucuronic acid. However, from 2 hours to 4 hours, the mixture significantly improved sperm velocity curves, especially at 3-4 hours. The experimental data also showed that the sperm velocity curve was significantly higher in the D-glucuronic acid group than in the group without D-glucuronic acid, and this increase continued to rise with the concentration of D-glucuronic acid.

[0047] The amplitude of lateral head displacement (ALH, μm) is the maximum average width of the sperm head swinging to both sides relative to its average path during motility. A low ALH value may indicate insufficient motility, and ALH is also an important clue for assessing the normality of sperm ultrastructure. Figure 6 The results showed that sperm ALH levels treated with C-phycocyanin and D-glucuronic acid were significantly improved within the first hour. From 2 hours to 4 hours, the mixture significantly increased the lateral movement of sperm heads, especially effectively increasing sperm ALH levels at 3-4 hours. Furthermore, experimental data showed that the sperm velocity curve was significantly higher in the D-glucuronic acid-added group compared to the group without D-glucuronic acid, and this increase continued with increasing D-glucuronic acid concentration.

[0048] Beat cross frequency (BCF, Hz) is the number of times the head trajectory crosses the average path per second. BCF is directly related to sperm propulsion and is an indicator of flagellar motility efficiency. Figure 7 The results showed that treatment with C-phycocyanin and D-glucuronic acid significantly improved sperm BCF levels within 1, 2, 3, and 4 hours. Particularly from 2 hours to 4 hours, the mixture effectively increased sperm BCF. Furthermore, experimental data showed that the sperm curve velocity was significantly higher in the D-glucuronic acid-added group compared to the group without D-glucuronic acid, and this increase continued with increasing D-glucuronic acid concentration.

[0049] Sperm motility trajectory analysis showed no significant difference in trajectory color distribution and number between the experimental and control groups, indicating that the addition of C-phycocyanin and D-glucuronic acid in the early stage of in vitro culture (1-2 hours) had no significant effect on sperm motility parameters. However, with the extension of in vitro culture time, the sperm survival rate and motility of the mixed solution treatment group were significantly better than those of the control group (no addition group). After 3 hours of culture, the number of red markers in the control group samples increased significantly, indicating a sharp increase in sperm mortality at this time. In contrast, the experimental group treated with C-phycocyanin and D-glucuronic acid still showed predominantly purple motility trajectories, indicating that although the sperm in this group showed a decrease in motility, most sperm remained viable. (Note: The experiment used a computer-aided semen analysis system to quantitatively evaluate sperm motility trajectories: blue and cyan trajectories represented forward or highly motile sperm, purple trajectories represented slow-moving sperm, red fluorescent markers indicated stationary or dead sperm, and white and yellow trajectories were not included in the statistical analysis.) Example 2 In vitro fertilization and early embryo culture: Preparation of reagents, culture medium, and oocyte transfer needles: The culture medium and reagents (HTF, mineral oil, KSOM) used in the experiment need to be placed in a 37℃ 5% CO2 incubator overnight (≥6 h) to equilibrate.

[0050] Preparation of in vitro fertilization dishes: Prepare two 35 mm culture dishes, each containing four 100 μL HTF microdrops, cover with mineral oil to prevent evaporation of water from the culture medium, and transfer to a 37°C 5% CO2 incubator for later use. The four drops in one dish are labeled as capacitated sperm after C-phycocyanin treatment in the experimental group, and capacitated sperm after physiological saline treatment in the control group.

[0051] Preparation of in vitro culture dishes: Prepare two 35 mm culture dishes, each containing four 100 μL KSOM microdrops, cover with mineral oil, and then transfer to a 37°C 5% CO2 incubator for later use. One dish serves as the experimental group, and the other as the control group.

[0052] Egg collection droplet preparation: Prepare 4 M2 droplets of 100 μL each in a 35 mm culture dish, cover with mineral oil and preheat on a metal hot plate (≥0.5 h).

[0053] Preparation of the egg transfer needle: Gently heat the Pasteur pipette 2-3 cm from the shoulder over a flame to soften it. Then, remove it from the flame and quickly stretch both ends of the Pasteur pipette to create a tube with an inner diameter of 100-150 μm. Note that you should not stretch the tube while it is still over the flame. Use a small grinding wheel to cut the stretched thin pipette and then quickly break it off. Gently heat it over the flame to ensure that the opening of the tube is flat and smooth. Place it in a suitable place for later use.

[0054] Superovulation in female mice: 5-8 female mice (for each experiment) were injected intraperitoneally with 0.1 mL (5 IU) PMSG per mouse in advance, followed by an intraperitoneal injection of 0.05 mL (5 IU) hCG per mouse 48 h later. Oocyte retrieval was best performed 14-15 h after the hCG injection.

[0055] Oocyte collection and in vitro fertilization: 14 hours after hCG injection, superovulated female mice were euthanized by cervical dislocation. After disinfecting the abdomen with 75% alcohol, the abdominal cavity was opened, and the uterine horns, ovaries, and fallopian tubes were located. The ampullae of both fallopian tubes were cut off with ophthalmic scissors and placed in an M2 container. Under a stereomicroscope, the dilatation of the fallopian tube was located and gently torn with ophthalmic forceps, revealing the outflow of white cumulus-oocyte complexes. The cumulus-oocyte complexes were then transferred into microdroplets in the fertilization dish, with approximately 2-3 cumulus-oocyte complexes per microdroplet. Equal amounts of sperm samples from the experimental and control groups, recovered previously, were added to the microdroplets, adjusting the density to an appropriate level before addition. Finally, the in vitro fertilization dish was placed in a 37°C, 5% CO2 incubator for culture. Five to six hours after fertilization, fertilized eggs were transferred from the fertilization drop to a KSOM culture dish drop using a 100 μm diameter pipette drawn from a 1.5 mm capillary glass tube. The eggs were repeatedly pipetted until virtually no sperm remained on the zona pellucida. They were then transferred to a new drop and cultured for another 4 hours to observe pronucleus formation. The formation of the pronucleus and the expulsion of PB2 were used as markers of fertilization, and the fertilization rate was calculated. Subsequently, fertilized eggs from both groups were continuously observed at 1.5, 2, 2.5, and 4 days post-fertilization, and relevant data on their development to the two-cell and blastocyst stages were recorded. Results showed that the average yield of two-cell embryos was 18.4% for sperm frozen and thawed without the addition of C-phycocyanin and D-glucuronide; the average yield was 22.8% for those with the addition of C-phycocyanin and D-glucuronide, with a statistically significant difference. Two-cell embryos were collected and cultured to the blastocyst stage. In the group without the addition of C-phycocyanin and D-glucuronic acid, 64.7% of the embryos reached the blastocyst stage, while the group with the addition of C-phycocyanin and D-glucuronic acid had a blastocyst rate of 84.7%, showing a significant difference.

[0056] In summary, C-phycocyanin and D-glucuronic acid, as two natural active ingredients, have significant effects in promoting sperm motility and protecting sperm function in vitro. The effective combination of these two natural ingredients exhibits superior sperm motility-promoting and protective effects compared to C-phycocyanin alone. This effective combination of the two natural ingredients has promising applications in the preparation of drugs to improve sperm motility or sperm treatment reagents in assisted reproductive technologies.

[0057] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Use of C-phycocyanin and D-glucuronide for increasing sperm motility, characterized in that, The mass ratio of the C-phycocyanin and D-glucuronidic acid is (2-4):

1.

2. Use according to claim 1, characterized in that, The C-phycocyanin and D-glucuronidic acid improve sperm motility by increasing total sperm motility rate, forward motility rate, average path velocity, straightness velocity, curvilinear velocity, head swing amplitude, beat frequency and survival rate.

3. Use of C-phycocyanin and D-glucuronide in the preparation of a medicament for improving sperm motility, characterized in that, The mass ratio of the C-phycocyanin and D-glucuronidic acid is (2-4):

1.

4. Use according to claim 3, characterized in that, The C-phycocyanin and D-glucuronidic acid improve sperm motility by increasing total sperm motility rate, forward motility rate, average path velocity, straightness velocity, curvilinear velocity, head swing amplitude, beat frequency and survival rate.

5. A medicament for improving the motility of sperm, characterized by comprising the compound of claim 1. The C-phycocyanin and D-glucuronidic acid and pharmaceutically acceptable adjuvants.

6. The medicament according to claim 5, characterized in that, The dosage form of the drug is a liquid dosage form, a solid dosage form or a paste dosage form.

7. Application of C-phycocyanin and D-glucuronidic acid in the preparation of products for assisted reproduction.

8. A method of improving sperm motility, characterized by, Sperm is cultured in a system containing C-phycocyanin and D-glucuronidic acid.

9. The method of claim 8, wherein, The concentration of the C-phycocyanin in the system is 40 mg / L, and the concentration of the D-glucuronidic acid in the system is 50 μM.

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