A sperm protective agent for improving sperm preservation quality and embryo development potential and application thereof

Abstract

The present application relates to the field of animal reproduction technology, and in particular to a sperm protection agent for improving sperm preservation quality and embryo development potential and application thereof, wherein the effective components of the sperm protection agent are selected from any one or several of acetyl carnitine, glycerophosphocholine, tauroursodeoxycholic acid, glutamic acid, sunflower acylcarnitine, pyridoxal phosphate and glyco ursodeoxycholic acid. Based on the regulation of the OR4C13-G13 / Golf signal axis related to sperm capacitation of the effective components, sperm viability can be maintained during preservation, premature capacitation can be inhibited, and the fertilization rate and embryo development potential can be improved. The present application is suitable for the normal temperature preservation of pig semen and can be extended to other mammalian or human assisted reproduction fields.

Description

Technical Field

[0001] This invention relates to the field of animal reproductive technology, specifically to a sperm protectant for improving sperm preservation quality and embryonic development potential, and its application. Background Technology

[0002] Reproductive efficiency in pigs is one of the core limiting factors in modern pig production, and the widespread application of artificial insemination technology has played a crucial role in its advancement. In this technology, the quality of semen preserved in vitro directly determines the fertilization success rate. However, sperm are highly susceptible to premature capacitation during in vitro preservation. This premature, non-physiological activation causes sperm to deplete their energy and lose their fertilization capacity before contacting the egg, representing a long-standing bottleneck restricting the improvement of pig semen quality, especially that preserved at room temperature. How to effectively inhibit or regulate premature capacitation of sperm to extend its functional lifespan and ultimately improve its fertilization rate and subsequent embryonic development potential is a pressing issue in the field of semen preservation. Therefore, exploring a strategy that can effectively maintain and improve the functional state of sperm after long-term preservation is of great significance. Summary of the Invention

[0003] To develop a strategy that can effectively maintain and enhance the functional state of sperm after long-term preservation, this invention provides a sperm preservative and its application for improving sperm preservation quality and embryonic developmental potential. Based on the regulatory effects of acetylcarnitine, glycerophosphate choline, tauroursodeoxycholic acid, glutamate, decanoylcarnitine, pyridoxal phosphate, or glycouroursodeoxycholic acid on the sperm OR4C13–G13 / Golf signaling axis, this invention can simultaneously maintain sperm motility, inhibit premature capacitation, and improve fertilization rate and embryonic developmental potential during preservation. This invention is applicable to the room temperature preservation of porcine semen and can also be extended to other mammalian or human assisted reproductive technologies.

[0004] This invention provides the use of bioactive metabolites in the preparation of formulations for improving sperm preservation quality and embryonic developmental potential, wherein the bioactive metabolites are selected from any one or more of acetylcarnitine, glycerophosphocholine, tauroursodeoxycholic acid, glutamic acid, decanoylcarnitine, pyridoxal phosphate, and glycine-uroursodeoxycholic acid.

[0005] This invention is based on the regulatory effects of acetylcarnitine, glycerophosphocholine, tauroursodeoxycholic acid, glutamic acid, decanocarnitine, pyridoxal phosphate, or glycineuroursodeoxycholic acid on the sperm OR4C13–G13 / Golf signaling axis. It can simultaneously maintain sperm motility, inhibit premature capacitation, and improve fertilization rate and embryonic development potential during preservation.

[0006] Furthermore, the bioactive metabolites are used to inhibit premature sperm capacitation and improve sperm motility, survival rate, and fertilization rate.

[0007] The present invention also provides a sperm protectant for improving sperm preservation quality and embryonic development potential, wherein the effective components of the sperm protectant are selected from any one or more of acetylcarnitine, glycerophosphate choline, tauroursodeoxycholic acid, glutamic acid, decanoylcarnitine, pyridoxal phosphate, and glycine-uroursodeoxycholic acid.

[0008] Furthermore, when the effective component of the sperm protectant is one, the final concentration of acetylcarnitine in the sperm protectant is 100µM to 1000µM, the final concentration of glycerophosphocholine is 20µM to 180µM, the final concentration of tauroursodeoxycholic acid is 20µM to 180µM, the final concentration of glutamate is 1mM to 5mM, the final concentration of decanoylcarnitine is 10µM to 100µM, the final concentration of pyridoxal phosphate is 20µM to 180µM, and the final concentration of glycouroursodeoxycholic acid is 2µM to 18µM.

[0009] Furthermore, the effective components of the sperm protectant are glycoursodeoxycholic acid, acetylcarnitine, and glutamic acid; wherein the final concentration of glycoursodeoxycholic acid is 2µM to 10µM, the final concentration of acetylcarnitine is 100µM to 500µM, and the final concentration of glutamic acid is 1mM to 3mM.

[0010] Furthermore, the effective components of the sperm protectant are decyl carnitine, pyridoxal phosphate, and glycoursodeoxycholic acid; wherein the final concentration of decyl carnitine is 10µM to 50µM, the final concentration of pyridoxal phosphate is 20µM to 100µM, and the final concentration of glycoursodeoxycholic acid is 2µM to 10µM.

[0011] Furthermore, the effective components of the sperm protectant are acetylcarnitine and glycerophosphate choline; wherein the final concentration of acetylcarnitine is 200µM to 250µM, and the final concentration of glycerophosphate choline is 25µM to 50µM.

[0012] The present invention also provides a method for preserving boar semen, wherein the boar semen is resuspended using the aforementioned semen preservative and preserved at 15°C to 25°C.

[0013] Furthermore, the storage time is 1 to 5 days.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a sperm protectant for improving sperm preservation quality and embryonic developmental potential. Its effective components are selected from any one or more of acetylcarnitine, glycerophosphate choline, tauroursodeoxycholic acid, glutamic acid, decanoylcarnitine, pyridoxal phosphate, and glycouroursodeoxycholic acid. Acetylcarnitine, glycerophosphate choline, tauroursodeoxycholic acid, glutamic acid, decanoylcarnitine, pyridoxal phosphate, and glycouroursodeoxycholic acid can effectively inhibit premature capacitation and motility decline in sperm during long-term storage at room temperature. Specifically, sperm motility and viability are significantly maintained, solving the industry problem of short sperm storage periods and rapid quality decline in existing technologies.

[0015] The sperm protectant provided by this invention directly enhances fertilization and embryonic development potential: in vitro fertilization experiments have confirmed that sperm treated with the sperm protectant of this invention show significantly improved fertilization and cleavage rates. This indicates that this invention not only maintains the basic vitality of sperm, but more importantly, protects and enhances its core biological functions, providing a direct guarantee for improving reproductive efficiency.

[0016] The sperm protectant provided by this invention has a clearly defined composition, a clear mechanism, and high safety: The core components of the sperm protectant of this invention are naturally occurring metabolites in the body (acetylcarnitine, glycerophosphate choline, tauroursodeoxycholic acid, glutamic acid, decanoylcarnitine, pyridoxal phosphate, and / or glycine-uroursodeoxycholic acid). Its mechanism of action is based on the regulation of sperm's own energy metabolism and cell membrane stability, avoiding the side effects and safety hazards that may be caused by the introduction of exogenous hormones or chemical stimuli. The composition is clearly defined and easy to control in terms of quality.

[0017] This invention is simple to operate and easy to promote: the sperm protectant of this invention is in the form of a liquid additive, which can be directly integrated into the existing commercial sperm protectant production process without changing the existing production process and equipment. It has low application cost and is easy to promote on a large scale in animal husbandry. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The effect of diluted acetylcarnitine solution on sperm motility; where Total motility represents total sperm motility, Progressive motility represents progressive sperm motility, VCL represents sperm curvilinear motility, and VSL represents sperm linear motility.

[0020] Figure 2 The effect of glycerophosphate choline dilution on sperm motility; where Total motility represents total sperm motility, Progressive motility represents progressive sperm motility, VCL represents sperm curvilinear motility, and VSL represents sperm linear motility.

[0021] Figure 3 The effect of tauroursodeoxycholic acid dilution on sperm motility; where Total motility represents total sperm motility, Progressive motility represents progressive sperm motility, VCL represents sperm curvilinear motility, and VSL represents sperm linear motility.

[0022] Figure 4 The effect of glutamate dilution on sperm motility; where Total motility represents total sperm motility, Progressive motility represents progressive sperm motility, VCL represents sperm curvilinear motility, and VSL represents sperm linear motility.

[0023] Figure 5 The effect of dilute decanoylcarnitine on sperm motility; where Total motility represents total sperm motility, Progressive motility represents progressive sperm motility, VCL represents sperm curvilinear motility, and VSL represents sperm linear motility.

[0024] Figure 6 The effect of pyridoxal phosphate dilution on sperm motility; where Total motility represents total sperm motility, Progressive motility represents progressive sperm motility, VCL represents sperm curvilinear motility, and VSL represents sperm linear motility.

[0025] Figure 7 The effect of diluted glycoursodeoxycholic acid on sperm motility; where Total motility represents total sperm motility, Progressive motility represents progressive sperm motility, VCL represents sperm curvilinear motility, and VSL represents sperm linear motility.

[0026] Figure 8 The effect of sperm protectant mixture A on sperm motility performance; where Total motility represents total sperm motility, Progressive motility represents progressive sperm motility, VCL represents sperm curvilinear motility, and VSL represents sperm linear motility.

[0027] Figure 9 The effect of sperm protectant mixture B on sperm motility performance; where Total motility represents total sperm motility, Progressive motility represents progressive sperm motility, VCL represents sperm curvilinear motility, and VSL represents sperm linear motility.

[0028] Figure 10 The effect of sperm protectant mixture C on sperm motility performance; where Total motility represents total sperm motility, Progressive motility represents progressive sperm motility, VCL represents sperm curvilinear motility, and VSL represents sperm linear motility.

[0029] Figure 11 The effect of the sperm protectant and compound preparation prepared in this invention on cleavage rate. Detailed Implementation

[0030] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0031] The reagent information used in this invention is as follows: Acetylcarnitine (Alcar): In this invention, it refers to acetyl-L-carnitine hydrochloride, brand name McLean, product code A834960-5G, product specification 5g / bottle.

[0032] Glycerylphosphocholine (GPC): Product code C824554-5G, brand name McLean, product specification 5g / bottle.

[0033] Tauroursodeoxycholic acid (TUDCA): Product code S872666-100mg, brand name Maclean, product specification 100mg / bottle.

[0034] Glutamic acid (Glu): In this invention, it refers to D-glutamic acid, brand name Maclean, product code D810325-5g, product specification 5g / bottle.

[0035] Dlcar: In this invention, it refers to decanoyl-L-carnitine, brand name Aladdin, product code D463321-10mg, product specification 10mg / bottle.

[0036] Pyridoxal phosphate (PLP): Product code P815501-5g, brand name McLean, product specification 5g / bottle.

[0037] Glycineursodeoxycholic acid (GUDCA): Product code G135873-250mg, brand name Aladdin, product specification 250mg / bottle.

[0038] Bovine semen basal diluent (BTS) per liter: Mix 37g glucose, 3g sodium citrate, 1.25g Na2-EDTA, 1.25g sodium bicarbonate, and 0.75g potassium chloride, then add deionized water to a final volume of 1L. Adjust the pH to 7.3 to obtain bovine semen basal diluent (BTS), abbreviated as BTS base solution, for later use. The CAS numbers for glucose, sodium citrate, Na2-EDTA, sodium bicarbonate, and potassium chloride are 50-99-7, 6132-04-3, 6381-92-6, 144-55-8, and 7447-40-7, respectively.

[0039] Example 1: A sperm protectant for improving sperm preservation quality and embryonic development potential and its application.

[0040] I. Preparation of Sperm Protectant 1. Mother liquor of key metabolites and its preparation method Accurately weigh high-purity acetylcarnitine (Alcar), glycerophosphocholine (GPC), tauroursodeoxycholic acid (TUDCA), glutamic acid (Glu), decylcarnitine (Dlcar), pyridoxal phosphate (PLP), and glycoursodeoxycholic acid (GUDCA) powders. Dissolve each powder separately in ultrapure water and bring the volume to a final volume to prepare a 100 mM single-component stock solution. Adjust the pH of each stock solution to approximately 7.3 using pH adjusters (HCl and NaOH). After passing sterility testing, label the solutions as acetylcarnitine stock solution, glycerophosphocholine stock solution, tauroursodeoxycholic acid stock solution, glutamic acid stock solution, decylcarnitine stock solution, pyridoxal phosphate stock solution, and glycoursodeoxycholic acid stock solution, respectively. Aliquot and store at 4°C protected from light for later use.

[0041] 2. Sperm protectants and their preparation methods Sperm protectants were prepared by diluting the stock solutions with BTS basal solution. Details are as follows:

[0042] (1) Preparation of acetylcarnitine dilution: The acetylcarnitine stock solution was diluted with PBS solution (Solepro, P1020) to a final concentration of acetylcarnitine of 500 µM, and then filtered through a sterile filter membrane with a pore size of 0.22 µm to obtain the acetylcarnitine dilution.

[0043] (2) Preparation of glycerophosphate choline dilution: Dilute the glycerophosphate choline stock solution with PBS solution (Solepro, P1020) to a final concentration of glycerophosphate choline of 100 µM, and filter it through a sterile filter membrane with a pore size of 0.22 µm to obtain the glycerophosphate choline dilution.

[0044] (3) Preparation of Tauroursodeoxycholic Acid Diluent: The tauroursodeoxycholic acid stock solution was diluted with PBS solution (Solepro, P1020) to a final concentration of 100 µM. The solution was then filtered through a sterile filter membrane with a pore size of 0.22 µm to obtain the tauroursodeoxycholic acid diluent.

[0045] (4) Preparation of glutamic acid diluent: Dilute the glutamic acid mother liquor with PBS solution (Solepro, P1020) to a final glutamic acid concentration of 3 mM, and filter it through a sterile filter membrane with a pore size of 0.22 µm to obtain the glutamic acid diluent.

[0046] (5) Preparation of decyl carnitine dilution: Dilute the decyl carnitine stock solution with PBS solution (Solepro, P1020) to a final concentration of decyl carnitine of 50 µM, and filter it through a sterile filter membrane with a pore size of 0.22 µm to obtain decyl carnitine dilution.

[0047] (6) Preparation of pyridoxal phosphate dilution: The pyridoxal phosphate mother liquor was diluted with PBS solution (Solepro, P1020) to a final concentration of pyridoxal phosphate of 100 µM. The solution was then filtered through a sterile filter membrane with a pore size of 0.22 µm to obtain the pyridoxal phosphate dilution.

[0048] (7) Preparation of glycoursodeoxycholic acid diluent: Dilute the glycoursodeoxycholic acid stock solution with PBS solution (Solepro, P1020) to a final concentration of glycoursodeoxycholic acid of 10 µM, and filter it through a sterile filter membrane with a pore size of 0.22 µm to obtain the glycoursodeoxycholic acid diluent.

[0049] 3. Compound preparations of sperm protectants (1) Sperm protectant mixture A: It is obtained by mixing glycoursodeoxycholic acid dilution, acetylcarnitine dilution and glutamic acid dilution in a volume ratio of 1:1:1, and is denoted as GUDCA+Alcar+Glu.

[0050] (2) Sperm protectant mixture B: It is obtained by mixing decyl carnitine dilution, pyridoxal phosphate dilution and glycine ursodeoxycholic acid dilution in a volume ratio of 1:1:1, and is denoted as Dlcar+PLP+GUDCA.

[0051] (3) Sperm protectant mixture C: It is obtained by mixing acetylcarnitine dilution and glycerophosphate choline in a volume ratio of 1:1, and is denoted as Alcar+ GPC.

[0052] II. Semen Preservation and Quality Assessment Semen was collected from healthy Duroc boars, ensuring that the semen quality met the standards for artificial insemination (sperm density greater than 1×10⁻⁶). 8 Freshly collected boar semen was initially diluted with BTS basal solution at a volume ratio of 1:1 to obtain a semen diluent. The semen diluent was randomly divided into 11 groups: one control group and the remaining 10 experimental groups. 1 mL of semen diluent from each group was centrifuged at 1200 rpm for 5 minutes at 17°C, the supernatant was discarded, and the sperm precipitate was obtained. The sperm precipitate was resuspended in 1 mL of the previously prepared sperm protectant or a combination of sperm protectants. The precipitate was then stored at a constant temperature of 17°C. Details of the specific grouping and treatment for the control and experimental groups are as follows:

[0053] Acetylcarnitine group (Alcar): Sperm precipitate was resuspended in 1 mL of acetylcarnitine dilution and stored at room temperature in a constant temperature refrigerator at 17°C.

[0054] Glycerol-phosphocholine group (GPC): Sperm precipitates were resuspended in 1 mL of glycerol-phosphocholine dilution and stored at room temperature in a constant temperature refrigerator at 17°C.

[0055] Tauroursodeoxycholic acid group (TUDCA): Sperm precipitate was resuspended in 1 mL of tauroursodeoxycholic acid dilution and stored at room temperature in a constant temperature refrigerator at 17°C.

[0056] Glutamate group (Glu): After resuspending the sperm precipitate in 1 mL of glutamate dilution, it was stored at room temperature in a constant temperature refrigerator at 17℃.

[0057] Dlcarnitine group: Sperm precipitate was resuspended in 1 mL of dlcarnitine dilution and stored at room temperature in a constant temperature refrigerator at 17°C.

[0058] Pyridoxal phosphate group (PLP): Sperm precipitates were resuspended in 1 mL of pyridoxal phosphate dilution and stored at room temperature in a constant temperature refrigerator at 17°C.

[0059] Glycineursodeoxycholic acid (GUDCA) group: Sperm precipitate was resuspended in 1 mL of glycoursodeoxycholic acid dilution and stored at room temperature in a constant temperature refrigerator at 17°C.

[0060] Sperm protectant mixture A (GUDCA+Alcar + Glu): After resuspending the sperm precipitate in 1 mL of sperm protectant mixture A, it was stored at room temperature in a constant temperature refrigerator at 17℃.

[0061] Sperm protectant mixture B (Dlcar + PLP + TUDCA): After resuspending the sperm precipitate in 1 mL of sperm protectant mixture B, it was stored at room temperature in a constant temperature refrigerator at 17°C.

[0062] Sperm protectant mixture C group (Alcar + GPC): After resuspending the sperm precipitate in 1 mL of sperm protectant mixture C, it was stored at room temperature in a constant temperature refrigerator at 17℃.

[0063] Control group: Sperm precipitate was resuspended in 1 mL of BTS basal solution and stored at room temperature in a constant temperature refrigerator at 17°C.

[0064] On the fourth day after storage at room temperature in a 17℃ constant temperature refrigerator, semen samples from each treatment group were collected for sperm motility testing and in vitro fertilization experiments. The specific testing methods are as follows:

[0065] 1. Sperm motility detection based on a computer-aided sperm analysis system (CASA) Samples were collected on day 4 of the storage period. Semen samples were preheated on a 37°C stage for 2 minutes before analysis using a computer-aided sperm analysis system (CASA). System parameters were set as follows: sampling frequency of 60 frames / second, analysis of at least 5 non-overlapping fields of view, with at least 200 sperm per field. The system automatically analyzed and recorded sperm motility parameters, including total motility (TM, %), percentage of progressively motile sperm (PR, %), curve velocity (VCL, μm / s), and linear velocity (VSL, μm / s). Each sample was tested three times, and results are expressed as mean ± standard deviation.

[0066] 2. In vitro fertilization (IVF) and embryo fertilization rate detection Mature porcine oocytes were collected, washed three times in in vitro fertilization (IVF) solution, and then placed in four-well culture plates. 50 μL of IVF solution was added to each well, and the plate was covered with mineral oil. Sperm treated with this dilution and stored for a specific time (day 4) were collected, washed twice with IVF solution containing 0.1% BSA, and the sperm concentration was adjusted to 1 × 10⁻⁶. 6 Sperm suspension of 50 μL was added to a fertilization droplet containing oocytes, and the sperm and eggs were incubated together for 6 hours. After incubation, the oocytes were transferred to embryo culture medium and cultured in an incubator at 38.5℃ and 5% CO2. Forty-eight hours after in vitro fertilization, the cleavage rate (number of cleaved embryos / total number of oocytes × 100%) was observed and calculated under an inverted microscope.

[0067] III. Experimental Results 1. The effect of the sperm protectant prepared in this invention on sperm motility performance (1) Effects of acetylcarnitine dilution on sperm motility The results are as follows Figure 1As shown, the total sperm motility in the control group was 71.3%, the forward sperm motility was 61.97%, the curvilinear sperm motility was 91.71 μm / s, and the linear sperm motility was 33.93 μm / s. In the experimental group supplemented with the sperm protectant (acetylcarnitine dilution) prepared in this invention, the total sperm motility was 78.87%, the forward sperm motility was 71.27%, the curvilinear sperm motility was 113.67 μm / s, and the linear sperm motility was 37.95 μm / s.

[0068] The sperm protectant (acetylcarnitine dilution) prepared by this invention can significantly improve the motility of sperm during the preservation process, specifically by significantly improving total sperm motility, forward motility, and motility dynamics parameters; the motility dynamics parameters include curvilinear velocity (VCL) and linear velocity (VSL).

[0069] (2) Effects of glycerophosphate choline dilution on sperm motility The results are as follows Figure 2 As shown, the total sperm motility in the control group was 71.3%, the forward sperm motility was 61.97%, the curvilinear sperm velocity was 91.71 μm / s, and the linear sperm velocity was 33.93 μm / s. In the experimental group supplemented with the sperm protectant (glycerophosphate choline dilution) prepared in this invention, the total sperm motility was 82.49%, the forward sperm motility was 77.79%, the curvilinear sperm velocity was 140.01 μm / s, and the linear sperm velocity was 47.41 μm / s.

[0070] The sperm protectant (glycerophosphate choline dilution) prepared by this invention can significantly improve the motility of sperm during the preservation process, specifically by significantly improving total sperm motility, forward motility, and motility dynamics parameters; the motility dynamics parameters include curvilinear velocity (VCL) and linear velocity (VSL).

[0071] (3) Effects of tauroursodeoxycholic acid dilution on sperm motility The results are as follows Figure 3 As shown, the total sperm motility in the control group was 71.3%, the forward sperm motility was 61.97%, the curvilinear sperm velocity was 91.71 μm / s, and the linear sperm velocity was 33.93 μm / s. In the experimental group supplemented with the sperm protectant (tauroursodeoxycholic acid dilution) prepared in this invention, the total sperm motility was 76.38%, the forward sperm motility was 69.95%, the curvilinear sperm velocity was 120.14 μm / s, and the linear sperm velocity was 46.6 μm / s.

[0072] The sperm protectant (tauroursodeoxycholic acid dilution) prepared by this invention can significantly improve sperm motility during the preservation process, specifically by significantly improving total sperm motility, forward motility, and motility dynamics parameters, including curvilinear velocity (VCL) and linear velocity (VSL).

[0073] (4) Effects of glutamate dilution on sperm motility The results are as follows Figure 4 As shown, the total sperm motility in the control group was 71.3%, the forward sperm motility was 61.97%, the curvilinear sperm velocity was 91.71 μm / s, and the linear sperm velocity was 33.93 μm / s. In the experimental group supplemented with the sperm protectant (glutamate dilution) prepared in this invention, the total sperm motility was 81.04%, the forward sperm motility was 71.06%, the curvilinear sperm velocity was 127.27 μm / s, and the linear sperm velocity was 41.39 μm / s.

[0074] The sperm protectant (glutamate diluent) prepared by this invention can significantly improve the motility of sperm during the preservation process, specifically by significantly improving total sperm motility, forward motility, and motility dynamics parameters; the motility dynamics parameters include curvilinear velocity (VCL) and linear velocity (VSL).

[0075] (5) Effects of dilute carnitine solution on sperm motility The results are as follows Figure 5 As shown, the total sperm motility in the control group was 71.3%, the forward sperm motility was 61.97%, the curvilinear sperm velocity was 91.71 μm / s, and the linear sperm velocity was 33.93 μm / s. In the experimental group supplemented with the sperm protectant (decylanine dilution) prepared in this invention, the total sperm motility was 76.26%, the forward sperm motility was 68.5%, the curvilinear sperm velocity was 110.53 μm / s, and the linear sperm velocity was 36.93 μm / s.

[0076] The sperm protectant (diluted carnitine) prepared by this invention can significantly improve the motility of sperm during the preservation process, specifically by significantly improving total sperm motility, forward motility, and motility dynamics parameters; the motility dynamics parameters include curvilinear velocity (VCL) and linear velocity (VSL).

[0077] (6) Effect of pyridoxal phosphate dilution on sperm motility The results are as follows Figure 6As shown, the total sperm motility in the control group was 71.3%, the forward sperm motility was 61.97%, the curvilinear sperm velocity was 91.71 μm / s, and the linear sperm velocity was 33.93 μm / s. In the experimental group supplemented with the sperm protectant (pyridoxal phosphate dilution) prepared in this invention, the total sperm motility was 82.58%, the forward sperm motility was 80.5%, the curvilinear sperm velocity was 140.11 μm / s, and the linear sperm velocity was 49.61 μm / s.

[0078] The sperm protectant (pyridoxal phosphate diluent) prepared by this invention can significantly improve the motility of sperm during the preservation process, specifically by significantly improving total sperm motility, forward motility, and motility dynamics parameters; the motility dynamics parameters include curvilinear velocity (VCL) and linear velocity (VSL).

[0079] (7) Effects of diluted glycoursodeoxycholic acid solution on sperm motility The results are as follows Figure 7 As shown, the total sperm motility in the control group was 71.3%, the forward sperm motility was 61.97%, the curvilinear sperm velocity was 91.71 μm / s, and the linear sperm velocity was 33.93 μm / s. In the experimental group supplemented with the sperm protectant (diluted glycoursodeoxycholic acid) prepared in this invention, the total sperm motility was 76.94%, the forward sperm motility was 69.36%, the curvilinear sperm velocity was 115.02 μm / s, and the linear sperm velocity was 40.00 μm / s.

[0080] The sperm protectant (diluted glycoursodeoxycholic acid) prepared by this invention can significantly improve the motility of sperm during the preservation process, specifically by significantly improving total sperm motility, forward motility, and motility dynamics parameters, including curvilinear velocity (VCL) and linear velocity (VSL).

[0081] (8) Effects of sperm protectant mixture A on sperm motility The results are as follows Figure 8 As shown, compared with the control group and other sperm protectants, such as glycoursodeoxycholic acid dilution, acetylcarnitine dilution, and glutamate dilution, the total sperm motility, forward motility, and motility parameters (including curvilinear velocity (VCL) and linear velocity (VSL)) were significantly improved. This indicates that the formulation obtained by combining glycoursodeoxycholic acid, acetylcarnitine, and glutamate is more effective than sperm protectants containing a single component.

[0082] (9) Effects of sperm protectant mixture B on sperm motility The results are as follows Figure 9 As shown, compared with the control group and other sperm protectants, such as decyl carnitine dilution, pyridoxal phosphate dilution, and tauroursodeoxycholic acid dilution, the total sperm motility, forward motility, and motility parameters (including curvilinear velocity (VCL) and linear velocity (VSL)) were significantly improved. This indicates that the formulation composed of decyl carnitine, pyridoxal phosphate, and tauroursodeoxycholic acid is more effective than sperm protectants containing a single component.

[0083] (10) Effects of sperm protectant mixture C on sperm motility The results are as follows Figure 10 As shown, compared with the control group and other sperm protectants, such as acetylcarnitine diluent and glycerophosphate choline diluent, total sperm motility, forward motility, and motility parameters (including curvilinear velocity (VCL) and linear velocity (VSL)) were significantly improved. This indicates that the formulation obtained by combining acetylcarnitine and glycerophosphate choline is more effective than sperm protectants containing a single component.

[0084] 2. Effects of the sperm protectant and compound preparation prepared in this invention on cleavage rate The results are as follows Figure 11 As shown, the cleavage rates after treatment in each group are as follows: Contorl: 58.33%, Acetylcarnitine diluent (Alcar): 72.67%, Glycerylphosphocholine diluent (GPC): 78.67%, Tauroursodeoxycholic acid diluent (TUDCA): 75.67%, Glutamate diluent (Glu): 74.33%, Descarnitine diluent (Dlcar): 79.00%, Pyridoxal phosphate diluent (PLP): 77.67%, Glycerylursodeoxycholic acid diluent (GUDCA): 76.67%, Sperm protectant mixture A (GUDCA + Alcar + Glu): 79.767%, Sperm protectant mixture B (Dlcar + PLP + TUDCA): 81.33%, Sperm protectant mixture C (Alcar + GPC): 80.33%.

[0085] As can be seen, compared with the control group, the sperm protectant and compound preparation prepared in this invention significantly improved the cleavage rate. This fully demonstrates that adding exogenous acetylcarnitine (Alcar), glycerolphosphocholine (GPC), tauroursodeoxycholic acid (TUDCA), glutamate (Glu), decanoylcarnitine (Dlcar), pyridoxal phosphate (PLP), glycoursodeoxycholic acid (GUDCA), the combination of glycoursodeoxycholic acid + acetylcarnitine + glutamate (GUDCA + Alcar + Glu), the combination of decanoylcarnitine + pyridoxal phosphate + tauroursodeoxycholic acid (Dlcar + PLP + TUDCA), and the combination of acetylcarnitine + glycerolphosphocholine (Alcar + GPC) to semen effectively improved sperm function and fertilization capacity, ultimately enhancing embryonic development potential.

[0086] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0087] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of bioactive metabolites in the preparation of formulations for improving sperm preservation quality and embryonic developmental potential, characterized in that, The bioactive metabolites are selected from any one or more of acetylcarnitine, glycerophosphocholine, tauroursodeoxycholic acid, glutamic acid, decanocarnitine, pyridoxal phosphate, and glycine-uroursodeoxycholic acid.

2. The application according to claim 1, characterized in that, The bioactive metabolites are used to inhibit premature sperm capacitation and improve sperm motility, survival rate, and fertilization rate.

3. A sperm protectant for improving sperm preservation quality and embryonic developmental potential, characterized in that, The active ingredient of the sperm protectant is selected from any one or more of acetylcarnitine, glycerophosphocholine, tauroursodeoxycholic acid, glutamic acid, decanoylcarnitine, pyridoxal phosphate, and glycouroursodeoxycholic acid.

4. The sperm protectant for improving sperm preservation quality and embryonic developmental potential according to claim 3, characterized in that, When the effective component of the sperm protectant is one, the final concentration of acetylcarnitine in the sperm protectant is 100µM to 1000µM, the final concentration of glycerophosphocholine is 20µM to 180µM, the final concentration of tauroursodeoxycholic acid is 20µM to 180µM, the final concentration of glutamate is 1mM to 5mM, the final concentration of decanoylcarnitine is 10µM to 100µM, the final concentration of pyridoxal phosphate is 20µM to 180µM, and the final concentration of glycouroursodeoxycholic acid is 2µM to 18µM.

5. The sperm protectant for improving sperm preservation quality and embryonic developmental potential according to claim 3, characterized in that, The effective components of the sperm protectant are glycoursodeoxycholic acid, acetylcarnitine and glutamic acid; wherein the final concentration of glycoursodeoxycholic acid is 2µM to 10µM, the final concentration of acetylcarnitine is 100µM to 500µM, and the final concentration of glutamic acid is 1mM to 3mM.

6. The sperm protectant for improving sperm preservation quality and embryonic developmental potential according to claim 3, characterized in that, The effective components of the sperm protectant are decyl carnitine, pyridoxal phosphate, and glycoursodeoxycholic acid; wherein the final concentration of decyl carnitine is 10µM to 50µM, the final concentration of pyridoxal phosphate is 20µM to 100µM, and the final concentration of glycoursodeoxycholic acid is 2µM to 10µM.

7. The sperm protectant for improving sperm preservation quality and embryonic developmental potential according to claim 3, characterized in that, The effective components of the sperm protectant are acetylcarnitine and glycerophosphate choline; wherein the final concentration of acetylcarnitine is 200µM to 250µM and the final concentration of glycerophosphate choline is 25µM to 50µM.

8. A method for preserving boar semen, characterized in that, Bovine sperm were resuspended using the sperm protectant described in any one of claims 3 to 7 and stored at 15°C to 25°C.

9. The method for preserving boar semen according to claim 8, characterized in that, The storage time is 1 to 5 days.

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