Sperm microcapsule preparation and preparation method thereof

Abstract

The invention relates to the technical field of livestock breeding, and particularly discloses a sperm microcapsule preparation and a preparation method thereof. The sperm microcapsule preparation comprises a basic solution, carboxylated fullerene and sodium alginate, wherein the basic solution comprises a sperm diluent, a mycillin mixed solution and egg yolk; the concentration of the carboxylated fullerene is 0-18 [mu] g / mL; the concentration of the sodium alginate is 1%-2%. According to the method, damage to sperms in the microencapsulation process can be reduced, and the sperm quality in the sperm incubation process can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of livestock breeding, and in particular to a sperm microcapsule preparation and a preparation method thereof. Background Art

[0002] The sperm sustained-release technology based on pig sperm microcapsules is a new pig breeding technology that can achieve single insemination of sows. There are two main ways to achieve single insemination of sows, namely, injecting exogenous reproductive hormones to accurately regulate the physiological state of the sow group, achieve synchronous estrus and ovulation of the pig group, and finally timed insemination and use pig sperm microencapsulation technology to extend the time that pig sperm maintains fertilization ability in the reproductive tract of the female animal.

[0003] However, the current porcine sperm microencapsulation technology has problems such as the microencapsulation process greatly damaging the quality of porcine sperm, the rapid quality decline during the incubation of microencapsulated sperm, and the low production efficiency and poor uniformity of sperm microcapsules, which restricts subsequent basic research and actual production applications. Therefore, it is urgent to develop a simple and efficient method for preparing porcine sperm microcapsules. Summary of the invention

[0004] The object of the present invention is to provide a sperm microcapsule preparation and a preparation method thereof, so as to reduce the damage to sperm during the microencapsulation process and improve the sperm quality during the sperm incubation process.

[0005] The present invention is achieved through the following technical solutions:

[0006] A sperm microcapsule preparation comprises a base liquid, carboxylated fullerene and sodium alginate; wherein the base liquid comprises a sperm diluent, a penicillin-streptomycin mixed solution and egg yolk; the concentration of the carboxylated fullerene is 0-18 μg / mL; and the concentration of the sodium alginate is 1%-2%.

[0007] The sperm microcapsule preparation of the present invention is a sperm protectant, which contains sodium alginate. Sodium alginate has good biodegradability and biocompatibility, is stable and non-toxic, is a healthy biological material commonly used in the field of biomedical engineering, and is one of the most commonly used immobilization carriers. Adding egg yolk to a base liquid containing sodium alginate can greatly reduce the damage to sperm during the microencapsulation process; by adding carboxylated fullerene to the base liquid; carboxylated fullerene has excellent antioxidant function, which solves the problem of too fast quality decline during the incubation process of microencapsulated sperm.

[0008] In summary, the present invention can effectively reduce the damage to sperm during the microencapsulation process and improve the sperm quality during sperm incubation by rationally designing the formula of the sperm microcapsule preparation.

[0009] In a preferred embodiment, the concentration of carboxylated fullerene is 6 to 12 μg / mL.

[0010] A method for preparing a sperm microcapsule preparation comprises the following steps:

[0011] S1, adding penicillin-streptomycin mixed solution to sperm diluent, mixing and filtering to obtain filtrate;

[0012] S2, adding egg yolk to the filtrate, centrifuging after magnetic stirring, and obtaining a supernatant, which is the base solution, wherein the amount of egg yolk added is 5-30% based on the volume of the filtrate;

[0013] S3. Add carboxylated fullerene and sodium alginate to the base liquid, mix well, and obtain a sperm microcapsule preparation.

[0014] A sperm microcapsule preparation is used in preparing sperm microcapsules. The sperm microcapsule preparation is mixed with an equal volume of collected semen, and the semen includes pig semen, bovine semen, sheep semen, horse semen, panda semen or primate semen.

[0015] A method for preparing sperm microcapsules comprises the following steps:

[0016] Step 1), mixing the collected semen with an equal volume of the sperm microcapsule preparation to obtain a semen-sperm microcapsule preparation mixed solution; the semen includes pig semen, bovine semen, sheep semen, horse semen, panda semen or primate semen;

[0017] Step 2), adding CaCl2 solution into the centrifuge tube, assembling the syringe and the centrifuge tube to form a centrifugal microfluidic device, and adding the semen-sperm microcapsule preparation mixed solution into the syringe;

[0018] Step 3), placing the centrifugal microfluidic device in a centrifuge and centrifuging at room temperature, during which the sperm-sperm microcapsule preparation mixed solution in the syringe enters a centrifuge tube and mixes with the CaCl2 solution, and after centrifugation, the mixed solution is allowed to stand for a period of time to fully gelate the semen-sperm microcapsule preparation mixed solution;

[0019] Step 4), filtering, washing and collecting the fully gelled semen-sperm microcapsule preparation mixed solution in sequence to obtain sperm microcapsules.

[0020] In a preferred embodiment, in step 2), the concentration of the CaCl2 solution is 50-150 mM; in step S3, the centrifugal acceleration is 25-100 g.

[0021] A centrifugal microfluidic device for preparing sperm microcapsules, the centrifugal microfluidic device comprising a syringe and a centrifuge tube;

[0022] The syringe comprises a tube portion, one end of which is provided with a needle;

[0023] A window for inserting the tube portion is arranged on the sealing cover at the top of the centrifuge tube. The tube portion passes through the sealing cover and the needle is placed in the centrifuge tube.

[0024] A sperm microcapsule prepared by the preparation method.

[0025] A method for incubating sperm microcapsules comprises the following steps:

[0026] Step 1: Cultivation of sperm microcapsules:

[0027] The sperm microcapsules were added into the microcapsule culture medium and cultured at a constant temperature of 37°C;

[0028] Step 2: Incubation of sperm microcapsules:

[0029] After constant temperature incubation, the sperm microcapsules were collected by filtration, and the sperm microcapsules were transferred to a centrifuge tube using a sol solution. The sample was incubated at 37° C. for at least 5 minutes to fully liquefy the solid hydrogel.

[0030] Wherein, the sol liquid includes sperm diluent.

[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0032] The sperm microcapsule preparation of the present invention contains egg yolk, sodium alginate and carboxylated fullerene at the same time; adding egg yolk to the base liquid containing sodium alginate can greatly reduce the damage to sperm during the microencapsulation process; and carboxylated fullerene has excellent antioxidant function, which solves the problem of too fast quality decline during the incubation of microencapsulated sperm and improves the sperm quality during the sperm incubation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0034] Figure 1 This is a flow chart of the preparation of pig sperm microcapsules in Example 1 of the present invention;

[0035] Figure 2 The morphology of the porcine sperm microcapsules prepared under different final concentrations of the sperm microcapsule preparations and different centrifugal acceleration conditions in Example 2 of the present invention is shown in white light photographs taken with an optical microscope, with a scale of 500 μm;

[0036] Figure 3 This is a diagram showing the size of pig sperm microcapsules prepared under different final concentrations of sperm microcapsule preparations and different centrifugal acceleration conditions in Example 2 of the present invention;

[0037] Figure 4The morphology of the porcine sperm microcapsules prepared under different calcium chloride solution concentrations in Example 3 of the present invention is shown; the white light photographs were taken using an optical microscope, and the scale bar is 500 μm;

[0038] Figure 5 This is a size diagram of pig sperm microcapsules prepared under different calcium chloride solution concentrations in Example 3 of the present invention;

[0039] Figure 6 This is a graph showing the effect of different calcium chloride solution concentrations on the viability of microencapsulated sperm in Example 3 of the present invention;

[0040] Figure 7 This is a graph showing the effect of different calcium chloride solution concentrations on the motility of microencapsulated sperm in Example 3 of the present invention;

[0041] Figure 8 This is a graph showing the effect of different incubation times of microcapsules incubated with sol solution on the viability of microencapsulated sperm in Example 4 of the present invention;

[0042] Fig. 9 This is a graph showing the effect of different incubation times of microcapsules incubated with sol solution on the activity of microencapsulated sperm in Example 4 of the present invention;

[0043] Fig.10 This is a graph showing the effect of adding different concentrations of sodium citrate to the sol solution of Example 4 of the present invention on the viability of microencapsulated sperm;

[0044] Fig.11 This is a graph showing the effect of adding different concentrations of sodium citrate to the sol solution of Example 4 of the present invention on the activity of microencapsulated sperm;

[0045] Fig.12 This is a graph showing the effect of adding different concentrations of CF-C60 on the viability of microencapsulated sperm in the pig semen-pig sperm microcapsule preparation of Example 5 of the present invention;

[0046] Fig.13 This is a graph showing the effect of adding different concentrations of CF-C60 on the motility of microencapsulated sperm in the pig semen-pig sperm microcapsule preparation of Example 5 of the present invention;

[0047] Fig.14 This is a graph showing the effect of adding different concentrations of CF-C60 on the linear velocity of microencapsulated sperm in the pig semen-pig sperm microcapsule preparation of Example 5 of the present invention;

[0048] Fig.15 This is a graph showing the effect of adding different concentrations of CF-C60 to the pig semen-pig sperm microcapsule preparation in Example 5 of the present invention on the curve velocity of microencapsulated sperm;

[0049] Fig.16This is a graph showing the effect of adding different concentrations of CF-C60 to the pig semen-pig sperm microcapsule preparation in Example 5 of the present invention on the path velocity of microencapsulated sperm. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments and drawings. The schematic implementation mode of the present invention and its description are only used to explain the present invention and are not intended to limit the present invention. The embodiments described below are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] In the following description, a large number of specific details are set forth in order to provide a thorough understanding of the present invention. However, it is apparent to those of ordinary skill in the art that these specific details do not have to be employed to practice the present invention. In other embodiments, in order to avoid confusing the present invention, known structures, materials or methods are not specifically described. The materials, instruments and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. The technical means used in the examples, unless otherwise specified, are conventional means well known to those skilled in the art.

[0052] Embodiment 1:

[0053] In order to solve the problem that the microencapsulation process in the prior art causes great damage to sperm and the sperm quality decreases rapidly during the sperm incubation process, animal sperm protection has similarities. In order to better illustrate the technology, this embodiment takes the preparation of pig sperm microcapsules as an example for explanation:

[0054] The present embodiment provides a pig sperm microcapsule preparation, which can protect pig sperm during the pig sperm microencapsulation process and the incubation process, including a base liquid, carboxylated fullerene and sodium alginate; wherein the base liquid includes a pig sperm diluent, a penicillin-streptomycin mixed solution and egg yolk; the concentration of the carboxylated fullerene is 0 to 18 μg / mL; and the concentration of the sodium alginate is 1% to 2%.

[0055] Among them, the pig sperm diluent is prepared from pig sperm diluent powder. The pig diluent powder (boar semen diluent powder) used is purchased from Shenzhen Xinzuan Agriculture and Animal Husbandry Technology Co., Ltd., and its main ingredients include glucose, citric acid, sodium bicarbonate, antibiotics, EDTA, BSA, etc. The pig sperm diluent can be prepared according to the instructions.

[0056] Among them, sodium alginate has good biodegradability and biocompatibility, is stable and non-toxic, is a commonly used healthy biomaterial in the field of biomedical engineering, and is one of the most commonly used immobilization carriers.

[0057] Among them, carboxylated fullerene (CF-C 60 ) is one of the important derivatives of carbon nanoparticles, with excellent antioxidant properties. It protects pig sperm mainly by enhancing the antioxidant capacity of sperm and inhibiting cell apoptosis and harmful bacteria. The preferred concentration of carboxylated fullerene in sperm microcapsule preparation is 6-12 μg / mL.

[0058] The applicant has proved through a large number of experiments that adding a certain amount of egg yolk to the base liquid containing sodium alginate can significantly reduce the damage to pig sperm during the microencapsulation process; and studies have found that excessive accumulation of reactive oxygen is an important factor in the quality decline during sperm incubation. Therefore, from the perspective of anti-oxidation, the present invention optimizes the formula of sperm microcapsule preparation by adding carboxylated fullerene, an excellent antioxidant, to solve the problem of rapid quality decline during the incubation of microencapsulated sperm.

[0059] In a preferred embodiment, the porcine sperm microcapsule preparation is prepared by adding 6 μg / mL CF-C to the base solution. 60 and 1% (w / v) sodium alginate powder.

[0060] The experiment proves that the porcine sperm microcapsule preparation prepared in this embodiment can protect porcine sperm from damage during the microencapsulation process to a great extent, and improve the sperm quality at 0h after microencapsulation to the level of the control group. When the porcine sperm is incubated at 37°C for 60h, the sperm motility of the microcapsule group can reach 34.09%, and the motility of the control group is 4.17%. The sperm motility of the microcapsule group is nearly 7 times higher than that of the control group. The porcine sperm microencapsulation technology of this embodiment shows great application potential. At the same time, this embodiment can also provide support for the sperm microencapsulation technology of other animals: the present invention has effectively promoted the quality of sperm after microencapsulation, such as the development of reproductive technology of cattle, sheep, horses, pandas, and primates.

[0061] The preparation method of the sperm microcapsule preparation comprises the following steps:

[0062] S1. Add 1-2% (v / v) penicillin-streptomycin mixture (100X) to the pig sperm diluent, mix well, and filter with a 0.22 μm filter to obtain a filtrate;

[0063] S2. Add fresh egg yolk to the prepared filtrate at a volume ratio of 4:1, use a magnetic stirrer to fully mix the solution, centrifuge at room temperature at a centrifugal acceleration of 3500g for 30 minutes, and use a syringe to extract the supernatant, which is the base solution;

[0064] S3. Add carboxylated fullerene and sodium alginate powder to the base liquid, place in a 4°C refrigerator, shake overnight to mix, and obtain a pig sperm microcapsule preparation.

[0065] In the process of preparing the sperm microcapsules of the porcine sperm microcapsule preparation prepared in this example, the porcine sperm microcapsule preparation is mixed with an equal volume of collected porcine semen.

[0066] A centrifugal microfluidic device is required in the sperm microencapsulation process, which includes a syringe and a centrifuge tube; the syringe includes a tube, one end of which is provided with a needle; a window for inserting the tube is provided on the sealing cover at the top of the centrifuge tube, the tube passes through the sealing cover and the needle is placed in the centrifuge tube.

[0067] A specific manufacturing process of a centrifugal microfluidic device is as follows:

[0068] (1) Material preparation

[0069] A 50mL centrifuge tube, a 5mL syringe tube and a 26G syringe needle. A 1.5cm×1.5cm window was opened in the middle of the lid of the 50mL centrifuge tube using a red-hot iron wire. The 5mL syringe tube was taken from a 5mL syringe produced by Shengguang Medical Products Co., Ltd.; the 26G syringe needle was taken from a 1mL syringe produced by Shengguang Medical Products Co., Ltd., model 26G.

[0070] (2) Assembly of centrifugal microfluidic device

[0071] The tube of the 5mL syringe and the 26G syringe needle are tightly connected to form the "upper" sample layer. The "upper" assembly is passed through the lid of the 50mL centrifuge tube, and then the lid is closed and tightened. In addition, if the "upper" is off-center after assembly, its position can be adjusted by rotating it so that it is in the exact center of the device.

[0072] Preparation of sperm microcapsules:

[0073] (1) Preparation of relevant solutions and materials required for sperm microencapsulation:

[0074] Preheat the porcine sperm microcapsule preparation, 50-100 mM CaCl2 solution, physiological saline, sol solution, microcapsule culture medium and BTS diluent in a 37°C water bath before the experiment. Prepare a centrifugal microfluidic device, tin foil, a cell strainer with a pore size of 70 μm, scissors, centrifuge tubes, etc.

[0075] Among them, the pig sperm microcapsule preparation is prepared by the above method.

[0076] 50-100mM CaCl2 solution: Add calcium chloride powder to pure water (the amount added is determined according to the prepared concentration), mix well, and filter with a 0.22μm filter to prepare CaCl2 solutions of different concentrations.

[0077] Physiological saline: Pure water is added with 0.9% (w / v) sodium chloride powder, mixed, and filtered through a 0.22 μm filter.

[0078] Sol solution: Use commercial diluent of pig sperm stored at room temperature (pig sperm diluent) as the sol solution and prepare it according to the instructions.

[0079] Microcapsule culture medium: The main components are 112.0mM NaCl, 2.7mMKCl, 25.07mM NaHCO3, 0.4mM NaH2PO4·2H2O, 10.0mM Na lactate, 0.5mM MgCl2·6H2O, 5mM HEPES, 3mM CaCl2, 13.9mM glucose and 6mg / mL bovine serum albumin (BSA). Add the above reagents to a beaker and stir until completely dissolved. Use a volumetric flask to make up the volume. After the solution is filtered through a 0.22μm filter, mark, divide, seal, and store at -20℃ for use. Before use, melt at 37℃, add 1% (v / v) penicillin-streptomycin mixture (100X), mix well, filter through a 0.22μm filter, and store for use.

[0080] BTS diluent: weigh 37.0 g glucose, 6.0 g sodium citrate, 1.3 g sodium bicarbonate, 1.3 g sodium ethylenediaminetetraacetic acid, and 0.4 g potassium chloride. After adjusting the pH value to 7.2, use a 1 L volumetric flask to make up the volume. Store the solution in a -20 °C refrigerator and prepare it as needed.

[0081] (2) Collection of pig semen samples

[0082] The traditional handheld method is used to discard the semen in the front and back of ejaculation, and only collect the semen in the middle of ejaculation. Fresh semen is required to be placed in a thermos cup at about 37°C and sent to the laboratory within 20 to 30 minutes.

[0083] like Figure 1 As shown, a method for preparing pig sperm microcapsules comprises the following steps:

[0084] Step 1), preparation of semen-sperm microcapsule preparation mixed solution:

[0085] Take 2mL of fresh pig semen into a 5mL centrifuge tube preheated at 37℃, and then use a pipette to slowly add 2mL of preheated pig sperm microcapsule preparation along the tube wall. Since the pig sperm microcapsule preparation has a certain viscosity, in order to ensure accurate addition, the scale line on the wall of the 5mL centrifuge tube shall be used as the standard. After adding the sample, mix the solution by turning the centrifuge tube upside down; to ensure complete mixing, use a 5mL pipette or sterilized scissors to cut off the 1mL pipette tip, expand the tip diameter, and slowly blow and mix to obtain a semen-sperm microcapsule preparation mixed solution.

[0086] Step 2), loading the sample into the centrifugal microfluidic device:

[0087] Add 2 mL of 100 mM CaCl2 solution to the centrifuge tube of the centrifugal microfluidic device, assemble the syringe and the centrifuge tube to form a centrifugal microfluidic device, and add 1 mL of semen-sperm microcapsule preparation mixed solution to the syringe.

[0088] Step 3), preparing pig sperm microcapsules by centrifugation:

[0089] The centrifugal microfluidic device was placed in a centrifuge and centrifuged at 25g for 10 minutes at room temperature. After centrifugation, the device was placed at room temperature for 5 minutes, and was gently shaken 1 to 2 times during the placement to fully gel the mixture of the pig semen-sperm microcapsule preparation.

[0090] Step 4), Collection and acquisition of pig sperm microcapsules:

[0091] Reference Figure 1 As shown, a "tin foil shovel" was made by folding tin foil disinfected with 75% alcohol. Then, the solution in the centrifugal microfluidic device was poured into a cell strainer with a pore size of 70 μm to collect the microcapsules. Next, the microcapsules were rinsed with saline. Finally, the microcapsules were obtained using a "tin foil shovel" and transferred to a 10 mL centrifuge tube using microcapsule culture medium, with a volume ratio of microcapsules to microcapsule culture medium of approximately 1:8.

[0092] A method for incubating pig sperm microcapsules comprises the following steps:

[0093] Step 1: Cultivation of pig sperm microcapsules:

[0094] Use scissors to cut off the 1mL pipette tip to expand the tip diameter. Use a pipette to blow or turn upside down to mix the microcapsules in the 10mL centrifuge tube, then use a 1mL pipette to transfer the microcapsules to 2mL centrifuge tubes. The volume of each microcapsule-microcapsule culture medium mixture is 1mL. After the transfer, mark the centrifuge tubes and seal them with sealing film. Finally, place the samples in a 37℃ constant temperature incubator for incubation.

[0095] Step 2: Incubation of sperm microcapsules:

[0096] Reference Figure 1The method for collecting and obtaining pig sperm microcapsules shown in the figure is to transfer the microcapsule-microcapsule culture medium mixture in a 2mL centrifuge tube to a cell strainer with a pore size of 70μm, collect the microcapsules using a "tin foil shovel", and transfer the microcapsules to a 1.5mL centrifuge tube using a sol solution. The sample is incubated at 37°C for at least 5 minutes to fully liquefy the solid hydrogel. Among them, the volume ratio of the sol solution to the microcapsule is about 5:1. The fully liquefied sperm was tested using the CASA system, and three groups of repetitions were set up in each group. Three fields of view were randomly selected for each repetition for testing, and the test was repeated three times. Finally, the average value was obtained to obtain the sperm quality index. The sperm quality indicators mainly include: sperm motility, sperm motility, linear speed, curve speed, path speed, etc. The data visualization was displayed using GraphPad Prism software. The final result difference statistics were analyzed using SPSS statistical software.

[0097] Embodiment 2:

[0098] Screening of different sperm microcapsule preparation concentrations and centrifugal acceleration conditions:

[0099] Under the same conditions as in Example 1, different concentrations of sodium alginate powder were added to the preparation of the porcine sperm microcapsule preparation: 1%, 1.5%, and 2%; different centrifugal accelerations were adopted when preparing the porcine sperm microcapsules by centrifugation: 25g, 50g, 75g, and 100g, and the porcine sperm microcapsules were prepared using a centrifugal microfluidic device.

[0100] Under the conditions of sodium alginate concentration (0.5%, 0.75% and 1%) in the mixed solution of semen-sperm microcapsule preparation and different centrifugal accelerations (25g, 50g, 75g, 100g), the morphology of pig sperm microcapsules prepared by using the centrifugal microfluidic device of the present invention is as follows: Figure 2 As shown; the size of sperm microcapsules is Figure 3 As shown, the diameter of the spherical gel or the short diameter of the droplet-shaped gel was measured using OIyVIA software. Three replicates were set up for each group, and the sizes of 30 to 40 microcapsules were counted in each replicate.

[0101] Depend on Figure 2 and Figure 3 It can be seen that:

[0102] When the sodium alginate concentration in the semen-sperm microcapsule preparation mixed solution is the same, the gel particle size decreases with the increase of centrifugal acceleration. Experiments have shown that the larger the gel particle size, the better; therefore, in order to ensure that the gel has a larger particle size, the centrifugal acceleration should not be too large, and the centrifugal acceleration should be controlled at 25-100g, preferably 25-50g.

[0103] Since the sperm microcapsule preparation has a certain viscosity, the preparation efficiency of the device is very low when the sodium alginate concentration in the sperm microcapsule preparation is high and the centrifugal acceleration is low. After comprehensively considering factors such as particle size, sperm microcapsule preparation efficiency and device stability, the two conditions of 25g and 0.5% sperm microcapsule preparation were selected for the next test.

[0104] Embodiment 3:

[0105] Screening of calcium chloride solution concentration conditions:

[0106] (1) Size of pig sperm microcapsules prepared with different calcium chloride concentrations

[0107] Under the same conditions as in Example 1, 50mM, 100mM, and 150mM anhydrous calcium chloride powder were added to pure water during the preparation of calcium chloride solution. After mixing, the solution was filtered with a 0.22μm filter to prepare CaCl2 solutions of different concentrations of 50mM, 100mM, and 150mM. Pig sperm microcapsules were prepared using a centrifugal microfluidic device. The diameter of the spherical gel or the short diameter of the droplet-shaped gel was measured using OIyVIA software. Three replicates were set for each group, and the size of 30 to 40 microcapsules was counted for each replicate. The results are shown in the figure. Figure 4 and Figure 5 shown.

[0108] (2) Sperm quality detection of pig sperm microcapsules prepared with different calcium chloride concentrations

[0109] Under the same conditions as in Example 1, 50 mM sodium citrate was added to the sol solution, dissolved, and filtered with a 0.22 μm filter to prepare a sol solution containing 50 mM sodium citrate. The pig sperm microcapsules prepared at different concentrations were incubated with a sol solution containing 50 mM sodium citrate for 5 min to fully liquefy the solid hydrogel. Finally, the fully liquefied sperm was tested using the CASA system. The results are shown in FIG. Figure 6-Figure 7 shown.

[0110] Depend on Figure 4-Figure 7 It can be seen that:

[0111] There was no significant difference in sperm quality between the concentrations of calcium chloride and the Figure 4 It can be seen that at 50mM, the appearance of sperm microcapsules is mostly "water drop-shaped", indicating that under this condition, the reaction rate of sodium alginate and calcium ions is slow, the stability of the gel is poor, and the microcapsules are easily squeezed to form large pieces of hydrogel during centrifugation, which is not conducive to the stable and efficient production of microcapsules by the device. Therefore, 100mM calcium chloride was selected for the next test.

[0112] The prior art shows that the microencapsulation process of pig sperm will greatly damage the sperm quality. The purpose of the present invention is to improve the quality of pig sperm after microencapsulation to the level of the control group, thereby solving this problem.

[0113] Embodiment 4:

[0114] Screening of the amount of sodium citrate added to the sol and the time of incubation of microcapsules:

[0115] (1) Effect of the incubation time of microcapsules in sol solution on sperm quality

[0116] Under the same conditions as in Example 1, 50 mM sodium citrate was added to the sol solution, dissolved, and filtered with a 0.22 μm filter to prepare a sol solution containing 50 mM sodium citrate. Preparation of pig sperm microcapsules After incubation in the sol solution containing 50 mM sodium citrate for 5 min, 20 min, and 40 min, the fully liquefied sperm was tested using the CASA system. The results are shown in Figure 2. Figure 8-Figure 9 shown.

[0117] Depend on Figure 8-Figure 9 It can be seen that:

[0118] The time for incubating microcapsules with sol solution has no significant effect on sperm quality, so the lowest standard, 5 minutes, is selected to save operation time.

[0119] (2) Effect of sodium citrate addition in sol solution on sperm quality

[0120] Under the same conditions as in Example 1, 0 mM, 25 mM, and 50 mM sodium citrate were added to the sol solution, dissolved, and filtered with a 0.22 μm filter to prepare sol solutions containing different concentrations of sodium citrate. Preparation of pig sperm microcapsules After incubation in sol solutions containing different concentrations of sodium citrate for 5 minutes, the fully liquefied sperm was tested using the CASA system. The results are shown in Figure 2. Figure 10-11 shown.

[0121] Depend on Figure 10-11 It can be seen that:

[0122] The higher the concentration of sodium citrate in the sol, the lower the viability and activity of the sperm. That is, the amount of sodium citrate added to the sol can significantly affect the quality of microencapsulated sperm. When 0 mM sodium citrate is additionally added to the sol, higher quality sperm can be obtained.

[0123] Note: The sol used in this example is a commercial diluent for storing pig sperm at room temperature, and the sodium citrate content in the commercial formula is not disclosed. Therefore, the addition of 0 mM sodium citrate does not mean that the sol does not contain sodium citrate, but that no additional sodium citrate is added.

[0124] Embodiment 5:

[0125] CF-C in the mixture of pig semen and pig sperm microcapsule preparation 60 Concentration screening

[0126] Under the same conditions as in Example 1, when preparing the pig sperm microcapsule preparation, 0 μg / mL, 6 μg / mL, 12 μg / mL, and 18 μg / mL CF-C 60 After mixing, 1% (w / v) sodium alginate powder was added respectively, placed in a 4°C refrigerator, and shaken overnight to mix well to prepare CF-C 60 1% (w / v) pig sperm microcapsule preparation. After the prepared pig sperm microcapsules were incubated in a 37°C constant temperature incubator for different periods of time, samples were taken, the sol solution dissolved the microcapsules, and the fully liquefied sperm was tested using the CASA system. The results are shown in Figure 12-16 shown.

[0127] The experiment was divided into 6 groups: BTS, VM, E0, E3, E6 and E9. BTS was the control group, which was diluted with fresh semen in a ratio of 4:1 and incubated in a 37°C constant temperature incubator after using the classic formula BTS in the experiment of room temperature preservation of pig semen; VM group was diluted with microcapsule culture medium and fresh semen in a ratio of 4:1 and incubated in a 37°C constant temperature incubator; E0 group was a pig sperm microcapsule preparation with 0 μg / mL CF-C 60 After the porcine sperm microcapsule preparation was diluted with fresh semen in a ratio of 1:1, the porcine sperm-porcine sperm microcapsule preparation mixture was added to the centrifugal microfluidic device to prepare porcine sperm microcapsules. The prepared microcapsules were transferred to the microcapsule culture medium and incubated in a constant temperature incubator at 37°C. Group E3 was the porcine sperm microcapsule preparation with 6 μg / mL CF-C 60 , that is, the mixture of pig semen and pig sperm microcapsule preparation contains 3 μg / mL CF-C 60 ; Group E6: pig sperm microcapsule preparation with 12 μg / mL CF-C 60 , that is, the mixture of pig semen and pig sperm microcapsule preparation contains 6 μg / mL CF-C 60 ; Group E9 ​​was a pig sperm microcapsule preparation with 18 μg / mL CF-C 60 , that is, the mixture of pig semen and pig sperm microcapsule preparation contains 9 μg / mL CF-C 60 .

[0128] Depend on Figure 12-16 It can be seen that:

[0129] Alginate hydrogel-CF-C 60 The combined effect can significantly improve the quality of sperm after microencapsulation. Among them, the mixture of pig semen and pig sperm microcapsule preparation contains 3ug / mL CF-C 60The effect was best when the microcapsule was added (E3 group): the sperm quality within 48 hours of incubation at 37°C was significantly higher than that of other experimental groups and the control group; when incubated at 37°C for 60 hours, the sperm motility of the E3 group could reach 34.09%, while that of the control group (BTS) was 4.17%. The sperm motility of the microcapsule group increased nearly 7 times that of the control group.

[0130] Therefore, CF-C in pig sperm microcapsule preparation 60 The optimal addition amount is 6 μg / mL, that is, CF-C 60 The final concentration was 3 μg / mL.

[0131] In summary, through the above experiments we can know:

[0132] The amount of sodium citrate added to the sol has the greatest impact on the microencapsulated sperm. The amount of sodium citrate added can significantly affect the quality of microencapsulated sperm. Therefore, when using it, there is no need to add additional sodium citrate to the sol; CF-C in the pig sperm microcapsule preparation 60 The amount of addition has a great influence on sperm quality.

[0133] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A sperm microcapsule preparation, characterized in that: It comprises a basic liquid, carboxylated fullerene and sodium alginate; wherein the basic liquid comprises a sperm diluent, a penicillin-streptomycin mixed solution and egg yolk; the concentration of the carboxylated fullerene is 0-18 μg / mL; and the concentration of the sodium alginate is 1%-2%.

2. The sperm microcapsule preparation according to claim 1, characterized in that: The concentration of the carboxylated fullerene is 6 to 12 μg / mL.

3. The method for preparing the sperm microcapsule preparation according to claim 1 or 2, characterized in that: The following steps are involved: S1, adding penicillin-streptomycin mixed solution to sperm diluent, mixing and filtering to obtain filtrate; S2, adding the egg yolk to the filtrate, centrifuging after magnetic stirring, and obtaining a supernatant, which is the base solution, wherein the amount of the egg yolk added is 5-30% based on the volume of the filtrate; S3, adding the carboxylated fullerene and the sodium alginate to the base liquid, mixing well, and obtaining the sperm microcapsule preparation.

4. Use of the sperm microcapsule preparation according to claim 1 or 2 in the preparation of sperm microcapsules, characterized in that: The sperm microcapsule preparation is mixed with an equal volume of collected semen, wherein the semen includes pig semen, bovine semen, sheep semen, horse semen, panda semen or primate semen.

5. A method for preparing sperm microcapsules, characterized in that: The following steps are involved: Step 1), mixing the collected semen with the sperm microcapsule preparation according to claim 1 or 2 in equal volumes to obtain a semen-sperm microcapsule preparation mixed solution; the semen includes pig semen, bovine semen, sheep semen, horse semen, panda semen or primate semen; Step 2), adding CaCl2 solution into a centrifuge tube, assembling a syringe and the centrifuge tube to form a centrifugal microfluidic device, and adding the semen-sperm microcapsule preparation mixed solution into the syringe; Step 3), placing the centrifugal microfluidic device in a centrifuge, centrifuging at room temperature, and leaving it for a period of time after centrifugation to allow the semen-sperm microcapsule preparation mixed solution to fully gel; Step 4), filtering, washing and collecting the fully gelled semen-sperm microcapsule preparation mixed solution in sequence to obtain the sperm microcapsules.

6. The preparation method according to claim 5, characterized in that: In step 2), the concentration of the CaCl2 solution is 50-150 mM; in step S3, the centrifugal acceleration is 25-100 g.

7. A centrifugal microfluidic device for use in the preparation method according to claim 5 or 6, characterized in that: The centrifugal microfluidic device comprises a syringe and a centrifuge tube; The syringe comprises a tube portion, one end of which is provided with a needle; A window for inserting the tube portion is arranged on the sealing cover at the top of the centrifuge tube, the tube portion passes through the sealing cover and the needle is placed in the centrifuge tube.

8. Sperm microcapsules prepared by the method according to claim 5 or 6.

9. A method for incubating sperm microcapsules as claimed in claim 8, characterized in that: The following steps are involved: Step 1: Cultivation of sperm microcapsules: Adding the sperm microcapsules into a microcapsule culture medium and culturing at a constant temperature of 37° C.; Step 2: Incubation of sperm microcapsules: After constant temperature incubation, the sperm microcapsules are collected by filtration, and the sperm microcapsules are transferred to a centrifuge tube using a sol solution. The sample is incubated at 37° C. for at least 5 minutes to fully liquefy the solid hydrogel.

10. The incubation method according to claim 9, characterized in that: The sol liquid includes a sperm diluent.

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