Sperm microcapsule preparation and preparation method

Abstract

The application relates to the technical field of livestock reproduction, and particularly discloses a sperm microcapsule preparation and a preparation method; the sperm microcapsule preparation comprises a base liquid, carboxylated fullerene and sodium alginate; the base liquid comprises a sperm diluent, an amikacin mixed solution and egg yolk; the concentration of the carboxylated fullerene is 0-18 mu g / mL; and the concentration of the sodium alginate is 1%-2%. The application can reduce the damage of the microencapsulation process to sperm and improve the sperm quality in the sperm incubation process.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of livestock reproduction, in particular to a sperm microcapsule preparation and a preparation method. BACKGROUND

[0002] The sperm microcapsule technology based on pig sperm is a new type of pig breeding technology for achieving single insemination of sows. There are mainly two ways to achieve single insemination of sows, i.e. injection of exogenous reproductive hormones to precisely control the physiological state of sow groups, to achieve synchronization of estrus and ovulation of pig groups, and finally timed insemination, and adoption of pig sperm microencapsulation technology to prolong the time for maintaining the fertilizing ability of pig sperm in the reproductive tract of sows.

[0003] However, the current pig sperm microencapsulation technology has problems such as significant damage to the quality of pig sperm in the microencapsulation process, too fast decline in the quality of microencapsulated sperm in the incubation process, and low production efficiency and poor uniformity of sperm microcapsules, which restricts the follow-up basic research and practical production application. Therefore, it is urgent to develop a simple and efficient method for preparing pig sperm microcapsules. SUMMARY

[0004] The present application aims to provide a sperm microcapsule preparation and a preparation method to reduce the damage to sperm in the microencapsulation process and improve the quality of sperm in the sperm incubation process.

[0005] The present application is achieved by the following technical solutions:

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

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

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

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

[0010] A preparation method of a sperm microcapsule preparation, comprising the following steps:

[0011] S1, adding a mixture of penicillin and streptomycin to a sperm diluent, mixing and filtering to obtain a filtrate;

[0012] S2, adding egg yolk to the filtrate, stirring magnetically and centrifuging to obtain 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, adding carboxylated fullerene and sodium alginate to the base solution, mixing to obtain a sperm microcapsule preparation.

[0014] The sperm microcapsule preparation is used for preparing a sperm microcapsule, and the sperm microcapsule preparation is mixed with an equal volume of collected semen, which includes pig semen, cow semen, sheep semen, horse semen, panda semen or primate semen.

[0015] A preparation method of a sperm microcapsule, comprising the following steps:

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

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

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

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

[0020] In a preferred mode, in step 2), the concentration of the CaCl2 solution is 50-150 mM; and in step S3, the acceleration of centrifugation 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 the tube portion being provided with a needle;

[0023] A sealing cover is arranged on the top of the centrifugal tube, and a window for inserting a tube part is arranged on the sealing cover, the tube part penetrates through the sealing cover and the needle is arranged in the centrifugal tube.

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

[0025] A hatching method of the sperm microcapsule, comprising the following steps:

[0026] Step one, culture of the sperm microcapsule:

[0027] The sperm microcapsule is added into the microcapsule culture medium and incubated at 37 DEG C.

[0028] Step two, hatching of the sperm microcapsule:

[0029] After the incubation, the sperm microcapsule is collected by filtration, and is transferred to a centrifugal tube by using a sol solution, and the sample is incubated at 37 DEG C for at least 5 min, so that the solid hydrogel is fully liquefied.

[0030] The sol solution comprises a sperm diluent.

[0031] Compared with the prior art, the sperm microcapsule preparation has the following advantages and beneficial effects:

[0032] The sperm microcapsule preparation comprises egg yolk, sodium alginate and carboxylated fullerene; the addition of egg yolk in the base liquid containing sodium alginate can greatly reduce the damage of the microencapsulation process to the sperm; the carboxylated fullerene has excellent antioxidant function, solves the problem of too fast quality decline of the microencapsulated sperm in the hatching process, and improves the sperm quality in the hatching process. BRIEF DESCRIPTION OF DRAWINGS

[0033] The accompanying drawings, which are included to provide a further understanding of the embodiments of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the principles of the application. In the drawings:

[0034] Figure 1 It is a preparation flow chart of the pig sperm microcapsule in the embodiment 1 of the application;

[0035] Figure 2 It is a pig sperm microcapsule morphology chart prepared under different final concentrations of different sperm microcapsule preparations and different centrifugal acceleration conditions in the embodiment 2 of the application, and the white light photo is taken by using an optical microscope, and the scale is 500 μm;

[0036] Figure 3 It is a pig sperm microcapsule size chart prepared under different final concentrations of different sperm microcapsule preparations and different centrifugal acceleration conditions in the embodiment 2 of the application;

[0037] Figure 4Figure 3 is a diagram of the morphology of the pig sperm microcapsules prepared under different calcium chloride solution concentration conditions of Example 3 of the present application; white light photos were taken using an optical microscope, and the scale is 500 μm;

[0038] Figure 5 Figure 4 is a diagram of the size of the pig sperm microcapsules prepared under different calcium chloride solution concentration conditions of Example 3 of the present application;

[0039] Figure 6 Figure 5 is a diagram of the effect of different calcium chloride solution concentration conditions of Example 3 of the present application on the viability of the microencapsulated sperm;

[0040] Figure 7 Figure 6 is a diagram of the effect of different calcium chloride solution concentration conditions of Example 3 of the present application on the motility of the microencapsulated sperm;

[0041] Figure 8 Figure 7 is a diagram of the effect of different incubation times of the microcapsules in the sol solution of Example 4 of the present application on the viability of the microencapsulated sperm;

[0042] Figure 9 Figure 8 is a diagram of the effect of different incubation times of the microcapsules in the sol solution of Example 4 of the present application on the motility of the microencapsulated sperm;

[0043] Figure 10 Figure 9 is a diagram of the effect of the addition of different concentrations of sodium citrate in the sol solution of Example 4 of the present application on the viability of the microencapsulated sperm;

[0044] Figure 11 Figure 10 is a diagram of the effect of the addition of different concentrations of sodium citrate in the sol solution of Example 4 of the present application on the motility of the microencapsulated sperm;

[0045] Figure 12 Figure 11 is a diagram of the effect of the addition of different concentrations of CF-C60 in the pig semen-pig sperm microcapsule preparation of Example 5 of the present application on the viability of the microencapsulated sperm;

[0046] Figure 13 Figure 12 is a diagram of the effect of the addition of different concentrations of CF-C60 in the pig semen-pig sperm microcapsule preparation of Example 5 of the present application on the motility of the microencapsulated sperm;

[0047] Figure 14 Figure 13 is a diagram of the effect of the addition of different concentrations of CF-C60 in the pig semen-pig sperm microcapsule preparation of Example 5 of the present application on the straight-line velocity of the microencapsulated sperm;

[0048] Figure 15 Figure 14 is a diagram of the effect of the addition of different concentrations of CF-C60 in the pig semen-pig sperm microcapsule preparation of Example 5 of the present application on the curvilinear velocity of the microencapsulated sperm;

[0049] Figure 16Figure for the effect of adding different concentrations of CF-C60 in the pig semen-pig sperm microcapsule preparation of embodiment 5 of the present application on the path velocity of the microencapsulated sperm. DETAILED DESCRIPTION

[0050] In order to make the objects, technical solutions and advantages of the present application clearer, further detailed description will be made to the present application in combination with embodiments and drawings, the illustrative embodiments of the present application and the description thereof are only used to explain the present application, and do not limit the present application, the following described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the protection scope of the present application.

[0051] In the following description, a large number of specific details are set forth in order to provide a thorough understanding of the present application. However, it is apparent to those skilled in the art that the present application can be practiced without these specific details. In other embodiments, well-known structures, materials or methods are not specifically described in order to avoid obscuring the present application. The materials, instruments and reagents used in the following embodiments, etc. can be obtained from commercial channels if not specifically stated. The technical means used in the embodiments is well known to those skilled in the art if not specifically stated.

[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 quality of sperm declines rapidly during sperm incubation, animal sperm protection has similar aspects, in order to better illustrate the technology, the present embodiment takes the preparation of pig sperm microcapsules as an example for illustration:

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

[0055] The pig sperm diluent is prepared from pig sperm diluent powder, and the pig diluent powder (boar semen diluent powder) is purchased from Shenzhen Xinzhan Animal Husbandry Technology Co., Ltd., and the main components include glucose, citric acid, sodium bicarbonate, antibiotics, EDTA, BSA, etc. The pig sperm diluent can be prepared according to the instruction manual.

[0056] The 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 immobilized carriers.

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

[0058] The applicant has proved through a large number of tests that adding a certain amount of egg yolk in the sodium alginate-containing base liquid can greatly reduce the damage to pig sperm during the microencapsulation process; and it is found that the excessive accumulation of reactive oxygen species is an important factor for the quality decline of sperm during incubation, so the present application optimizes the sperm microcapsule preparation formula from the perspective of antioxidant by adding carboxyfullerene, an excellent antioxidant, to solve the problem of too rapid quality decline of microencapsulated sperm during incubation.

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

[0060] Tests have proved that the pig sperm microcapsule preparation prepared by the present embodiment can greatly protect pig sperm from damage during the microencapsulation process, and the quality of pig sperm at 0h after microencapsulation is improved to the level of the control group. When the pig sperm is incubated at 37℃ to 60h, the sperm motility of the microencapsulated group can reach 34.09%, and the motility of the control group is 4.17%, the sperm motility of the microencapsulated group is nearly 7 times higher than that of the control group. The pig sperm microencapsulation technology of the present embodiment shows great application potential, and at the same time, it also provides support for the sperm microencapsulation technology of other animals: the present application greatly promotes the quality of microencapsulated sperm, such as the development of bovine, ovine, equine, panda, primate fertility and reproduction technology.

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

[0062] S1, based on the pig sperm diluent, 1-2% (v / v) of penicillin-streptomycin mixture (100X) is added, mixed uniformly, filtered through 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, mix the solution uniformly using a magnetic stirrer, centrifuge at a centrifugal acceleration of 3500g at room temperature for 30 min, and use a syringe to extract the supernatant, which is the base liquid;

[0064] S3, add carboxyfullerene and sodium alginate powder to the base liquid, place it in a 4℃ refrigerator, mix it uniformly on a shaker overnight to obtain a pig sperm microcapsule preparation.

[0065] The pig sperm microcapsule preparation prepared in this embodiment is mixed with the collected pig semen in equal volume in the process of preparing the sperm microcapsule.

[0066] In the process of sperm microcapsule, a centrifugal microfluidic device is needed, which includes a syringe and a centrifugal tube; the syringe includes a tube part, one end of the tube part is provided with a needle; a window for inserting the tube part is arranged on the sealing cover at the top of the centrifugal tube, and the tube part penetrates through the sealing cover and the needle is placed in the centrifugal tube.

[0067] One specific manufacturing process of the centrifugal microfluidic device is as follows:

[0068] (1) Material preparation

[0069] A 50 mL centrifugal tube, a tube part of a 5 mL syringe, and a 26G type syringe needle. Among them, a 1.5 cm x 1.5 cm window is opened in the middle of the cover of the 50 mL centrifugal tube using a red-hot iron wire. The tube part of the 5 mL syringe is taken from the 5 mL syringe produced by Shengguang Medical Products Co., Ltd.; the 26G syringe needle is taken from the 1 mL syringe produced by Shengguang Medical Products Co., Ltd., and the model is 26G.

[0070] (2) Assembly of centrifugal microfluidic device

[0071] The tube part of the 5 mL syringe and the 26G type syringe needle are tightly connected to form the "upper" sample layer, the "upper" component is inserted through the cover of the 50 mL centrifugal tube, and the cover is screwed tightly. In addition, if the "upper" component deviates from the center position after assembly, the position can be adjusted by rotating to make it in the center position of the device.

[0072] Preparation before sperm microcapsule preparation:

[0073] (1) Preparation of related solutions and materials needed in the process of sperm microcapsule:

[0074] Pig sperm microcapsule preparation, 50-100 mM CaCl2 solution, physiological saline, sol solution, microcapsule culture medium, and BTS diluent are preheated in a 37°C water bath before the experiment. Prepare the centrifugal microfluidic device, tin foil paper, cell filter screen with a pore size of 70 μm, scissors, centrifugal tube, etc.

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

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

[0077] Physiological saline: Prepared by adding 0.9% (w / v) of sodium chloride powder to pure water, mixing well, and filtering with a 0.22 μm filter.

[0078] Solvent solution: A commercial diluent for boar sperm cryopreservation (boar sperm diluent) was used as the solvent solution, and was prepared according to the instructions.

[0079] Microcapsule culture medium: The main components were 112.0 mM NaCl, 2.7 mM KCl, 25.07 mM NaHCO3, 0.4 mM NaH2PO4·2H2O, 10.0 mM Na lactate, 0.5 mM MgCl2·6H2O, 5 mM HEPES, 3 mM CaCl2, 13.9 mM glucose, and 6 mg / mL bovine serum albumin (BSA). The above reagents were added to a beaker, stirred well until completely dissolved, and then made up to volume using a volumetric flask. The solution was filtered through a 0.22 μm filter, labeled, aliquoted, sealed, and stored at -20°C for later use. Before use, it was thawed at 37°C, 1% (v / v) of penicillin-streptomycin mixture (100X) was added, mixed well, filtered through a 0.22 μm filter, and stored for later use.

[0080] BTS diluent: 37.0 g of glucose, 6.0 g of sodium citrate, 1.3 g of sodium bicarbonate, 1.3 g of sodium ethylenediaminetetraacetate, and 0.4 g of potassium chloride were weighed, the pH was adjusted to 7.2, and then made up to volume using a 1 L volumetric flask. The solution was stored in a -20°C refrigerator and prepared as needed.

[0081] (2) Collection of boar semen samples

[0082] The traditional handheld method was used, and only the middle part of the ejaculate was collected, with the pre- and post-ejaculate discarded. Fresh semen was required to be placed in a 37°C heat preservation cup and delivered to the laboratory within 20-30 min.

[0083] As shown in Figure 1 , a method for preparing boar sperm microcapsules includes the following steps:

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

[0085] 2 mL of fresh boar semen was taken into a 5 mL centrifuge tube preheated to 37°C, and then 2 mL of preheated boar sperm microcapsule preparation was slowly added along the tube wall using a pipette. Since the boar sperm microcapsule preparation has a certain viscosity, to ensure accurate addition, the scale line of the 5 mL centrifuge tube wall was used as a reference. After the addition was completed, the solution was mixed by inverting the centrifuge tube; to ensure complete mixing, a 1 mL pipette tip was cut off using a 5 mL pipette or a sterilized scissors to enlarge the tip caliber, and the solution was slowly blown and mixed to obtain a semen-sperm microcapsule preparation mixed solution.

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

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

[0088] Step 3), centrifugal preparation of pig sperm microcapsules:

[0089] Put the centrifugal microfluidic device into the centrifuge, and centrifuge the centrifugal microfluidic device at 25 g of centrifugal acceleration for 10 min at room temperature. After centrifugation, the device is placed at room temperature for 5 min, and gently shaken 1-2 times during the placement period to fully gel the pig semen-sperm microcapsule preparation mixture.

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

[0091] Referring to Figure 1 As shown in FIG. 1, a "tin foil shovel" is made by folding the tin foil paper after disinfection with 75% alcohol. Then, the solution in the centrifugal microfluidic device is poured into a cell filter with a pore size of 70 μm to collect the microcapsules. Next, the microcapsules are washed with physiological saline. Finally, the "tin foil shovel" is used to obtain the microcapsules, and the microcapsules are transferred to a 10 mL centrifuge tube using a microcapsule culture medium, and the volume ratio of the microcapsules to the microcapsule culture medium is about 1:8.

[0092] A method for hatching pig sperm microcapsules, comprising the following steps:

[0093] Step 1, culture of pig sperm microcapsules:

[0094] The tip of a 1 mL pipette is cut off using scissors to enlarge the caliber. The microcapsules in the 10 mL centrifuge tube are mixed by blowing or inverting the tube, and then the microcapsules are transferred to a 2 mL centrifuge tube using a 1 mL pipette, with a volume of 1 mL of microcapsule-microcapsule culture medium mixture per tube. After dispensing, the centrifuge tube is labeled and sealed with a sealing film, and the sample is incubated in a 37°C constant temperature incubator.

[0095] Step 2, incubation of sperm microcapsules:

[0096] Referring to Figure 1The method for collecting and obtaining the illustrated pig sperm microcapsules is as follows: the microcapsule-microcapsule medium mixture in a 2 mL centrifuge tube is transferred to a cell filter screen with a pore size of 70 pm, the microcapsules are collected using a "tin foil shovel", and the microcapsules are transferred to a 1.5 mL centrifuge tube using a sol solution, and the sample is incubated at 37°C for at least 5 min to fully liquefy the solid hydrogel. Among them, the volume ratio of sol solution to microcapsule is about 5:1. The CASA system is used to detect the fully liquefied sperm, three sets of repeats are set for each group, three fields of view are randomly selected for detection for each repeat, and the mean value is obtained by repeating three times to obtain the quality index of sperm. The quality index of sperm mainly includes: sperm motility, sperm motility, linear velocity, curved velocity, path velocity, etc. The visualization of data is performed using GraphPad Prism software. Finally, the difference of the results is analyzed using SPSS statistical software.

[0097] Example 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 are added when preparing the pig sperm microcapsule preparation: 1%, 1.5%, and 2%; different centrifugal accelerations are adopted when preparing the pig sperm microcapsule: 25g, 50g, 75g, and 100g, and the centrifugal microfluidic device is used to prepare the pig sperm microcapsule.

[0100] Under the conditions of different concentrations of sodium alginate (0.5%, 0.75%, and 1%) in the semen-sperm microcapsule preparation mixed solution and different centrifugal accelerations (25g, 50g, 75g, and 100g), the pig sperm microcapsule prepared using the centrifugal microfluidic device of the application has the shape as shown in Figure 2 The size of the sperm microcapsule is as shown in Figure 3 The diameter of the spherical gel or the short diameter of the water droplet-shaped gel is measured using OIyVIA software, and three sets of repeats are set for each group, and the size of 30-40 microcapsules is counted for each repeat.

[0101] As can be seen from Figure 2 and Figure 3

[0102] When the concentration of sodium alginate in the semen-sperm microcapsule preparation mixed solution is the same, the gel particle size decreases with the increase of centrifugal acceleration. It has been proved by experiments that the larger the gel particle size is, 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 is controlled at 25-100g, preferably at 25-50g.

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

[0104] Example 3:

[0105] Screening of calcium chloride solution concentration conditions:

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

[0107] Under the same conditions as in Example 1, 50 mM, 100 mM and 150 mM of anhydrous calcium chloride powder were added to pure water when preparing the calcium chloride solution, and after mixing, the solution was filtered with a 0.22 μm filter to prepare 50 mM, 100 mM and 150 mM CaCl2 solutions. The centrifugal microfluidic device was used to prepare pig sperm microcapsules. The diameter of the spherical gel or the short diameter of the water droplet-shaped gel was measured using OIyVIA software, and three sets of repeats were set for each group, with 30-40 microcapsules in each group of repeats. The results are shown in Figure 4 and Figure 5 .

[0108] (2) Quality detection of pig sperm microcapsules prepared by different concentrations of calcium chloride

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

[0110] As can be seen from Figures 4-7 ,

[0111] The concentration of calcium chloride has no significant difference on the quality of sperm, but as can be seen from Figure 4 , when the concentration of calcium chloride is 50 mM, the appearance of the sperm microcapsule is mostly "water droplet-shaped", indicating that the reaction speed of sodium alginate and calcium ions under this condition is slow, the stability of the gel is poor, and the microcapsules are easily squeezed to form large blocks of hydrogel during centrifugation, which is not conducive to the stable and efficient production of microcapsules by the device. Therefore, 100 mM of calcium chloride is selected for the next test.

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

[0113] Example 4:

[0114] Screening of sodium citrate addition in sol solution and incubation time for microcapsules:

[0115] (1) The effect of sol-sol incubation time of microcapsules on sperm quality

[0116] Under the same conditions as in Example 1, 50 mM sodium citrate was added to the sol solution, dissolved, and then filtered through a 0.22 μm filter to prepare a sol solution containing 50 mM sodium citrate. After incubating the porcine sperm microcapsules in the sol solution containing 50 mM sodium citrate for 5 min, 20 min, and 40 min, the fully liquefied sperm were detected using a CASA system. The results are as follows... Figures 8-9 As shown.

[0117] Depend on Figures 8-9 It can be known that:

[0118] Since the incubation time of the microcapsules in the sol solution has no significant impact on sperm quality, the lowest standard of 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. After dissolution, the solutions were filtered through a 0.22 μm filter to prepare sol solutions containing different concentrations of sodium citrate. After incubating the prepared porcine sperm microcapsules in sol solutions containing different concentrations of sodium citrate for 5 min, the fully liquefied sperm were detected using a CASA system. The results are as follows... Figures 10-11 As shown.

[0121] Depend on Figures 10-11 It can be known that:

[0122] The higher the concentration of sodium citrate in the sol, the lower the sperm viability and motility. In other words, the amount of sodium citrate added to the sol can significantly affect the quality of microencapsulated sperm. Adding an additional 0 mM sodium citrate to the sol can yield higher quality sperm.

[0123] Note: The sol used in this example is a commercially available diluted solution of porcine semen stored at room temperature. The sodium citrate content in the commercial formulation is not disclosed. Therefore, the addition of 0 mM sodium citrate does not mean that the sol does not contain sodium citrate, but simply that no additional sodium citrate is added.

[0124] Example 5:

[0125] CF-C in a mixture of boar semen and boar sperm microcapsule formulations 60 Concentration screening

[0126] In the same conditions as in Example 1, the pig sperm microcapsule preparation was prepared by adding 0 pg / mL, 6 pg / mL, 12 pg / mL, 18 pg / mL CF-C 60 to the base solution, mixing, then adding 1% (w / v) sodium alginate powder, placing in a 4°C refrigerator, and mixing overnight on a shaker to prepare 1% (w / v) pig sperm microcapsule preparations containing different concentrations of CF-C 60 . After incubating the prepared pig sperm microcapsules in a 37°C constant temperature incubator for different times, samples were taken, the microcapsules were dissolved in the sol solution, and the fully liquefied sperm were detected using the CASA system. The results are shown in Table 1. Figures 12-16

[0127] The experiment was divided into BTS, VM, E0, E3, E6, and E9 groups. BTS was the control group, in which the classic formula BTS used in the pig semen room temperature preservation experiment was diluted 4:1 with fresh semen and incubated in a 37°C constant temperature incubator; the VM group was incubated in a 37°C constant temperature incubator after the microcapsule culture medium was diluted 4:1 with fresh semen; the E0 group was prepared by adding 0 pg / mL CF-C 60 to the pig sperm microcapsule preparation, diluting the pig sperm microcapsule preparation 1:1 with fresh semen, adding the pig semen-pig sperm microcapsule preparation mixture to the centrifugal microfluidic device to prepare pig sperm microcapsules, and transferring the prepared microcapsules to the microcapsule culture medium and incubating in a 37°C constant temperature incubator; the E3 group was prepared by adding 6 pg / mL CF-C 60 , i.e., the pig semen-pig sperm microcapsule preparation mixture contained 3 pg / mL CF-C 60 ; the E6 group was prepared by adding 12 pg / mL CF-C 60 , i.e., the pig semen-pig sperm microcapsule preparation mixture contained 6 pg / mL CF-C 60 ; and the E9 group was prepared by adding 18 pg / mL CF-C 60 , i.e., the pig semen-pig sperm microcapsule preparation mixture contained 9 pg / mL CF-C 60 .

[0128] As can be seen from Table 2: Figures 12-16

[0129] The alginate hydrogel-CF-C 60 combination can significantly improve the quality of the microencapsulated sperm. Among them, the pig semen-pig sperm microcapsule preparation mixture contains 3 pg / mL CF-C 60 ​​The effect is the best at 37℃ for 48h (E3 group): the sperm quality is significantly higher than that of other experimental groups and the control group; the sperm activity of E3 group can reach 34.09% at 37℃ for 60h, and the activity of the control group (BTS) is 4.17%, and the sperm activity of the microcapsule group is nearly 7 times higher than that of the control group.

[0130] Therefore, the CF-C 60 The optimal addition amount is 6μg / mL, that is, the CF-C 60 The final concentration is 3μg / mL.

[0131] In summary, through the above experiments, it can be known that:

[0132] The addition amount of sodium citrate in the sol solution has the greatest influence on the microencapsulated sperm, and the addition amount of sodium citrate can significantly affect the quality of the microencapsulated sperm, so that in use, it is not necessary to add sodium citrate in the sol solution; the addition amount of CF-C 60 has a great influence on the sperm quality.

[0133] The above specific embodiments further specifically describe the purposes, technical solutions and beneficial effects of the present application, and it should be understood that the above description is only a specific embodiment of the present application, and is not used to limit the protection scope of the present application, and any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A sperm microcapsule preparation, characterized by comprising: The mixture includes a base solution, carboxylated fullerene, and sodium alginate; wherein the base solution includes sperm diluent, penicillin-streptomycin mixture, and egg yolk; the concentration of the carboxylated fullerene is 6-18 µg / mL; and the concentration of the sodium alginate is 1%-2%.

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

3. The method for preparing the sperm microcapsule formulation as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Based on sperm dilution solution, add penicillin-streptomycin mixture, mix well and filter to obtain filtrate; S2. Add the egg yolk to the filtrate, stir magnetically, and centrifuge to obtain the supernatant, which is the base solution. The amount of egg yolk added is 5-30% based on the volume of the filtrate. S3. Add the carboxylated fullerene and the sodium alginate to the base solution, mix well, and obtain the sperm microcapsule formulation.

4. The application of the sperm microcapsule formulation as described in 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, including boar semen, bovine semen, sheep semen, horse semen, panda semen, or primate semen.

5. A method for preparing sperm microcapsules, characterized in that, Includes the following steps: Step 1) Mix the collected semen with an equal volume of the sperm microcapsule formulation as described in claim 1 or 2 to obtain a semen-sperm microcapsule formulation mixed solution; the semen includes pig semen, bovine semen, sheep semen, horse semen, panda semen, or primate semen; Step 2) Add CaCl2 solution to the centrifuge tube and assemble the syringe with the centrifuge tube to form a centrifugal microfluidic device. Add the semen-sperm microcapsule preparation mixture solution to the syringe. Step 3) Place the centrifugal microfluidic device into a centrifuge and centrifuge at room temperature. After centrifugation, let it stand for a period of time to allow the semen-sperm microcapsule preparation mixture to fully gel. Step 4) The fully gelled semen-sperm microcapsule preparation mixture is filtered, washed, and collected sequentially to obtain the sperm microcapsules.

6. The preparation method according to claim 5, characterized in that, In step S2), the concentration of the CaCl2 solution is 50-150 mM; in step S3, the centrifugation acceleration is 25-100 g.

7. A sperm microcapsule prepared by the preparation method as described in claim 5 or 6.

8. A method for incubating sperm microcapsules as described in claim 7, characterized in that, Includes the following steps: Step 1: Cultivation of sperm microcapsules: The sperm microcapsules were added to a microcapsule culture medium and cultured at a constant temperature of 37°C. Step 2: Incubation of sperm microcapsules: After constant temperature incubation, the samples were filtered and collected. The sperm microcapsules were then transferred to centrifuge tubes using a sol solution. The samples were incubated at 37°C for at least 5 minutes to allow the solid hydrogel to fully liquefy.

9. The incubation method according to claim 8, characterized in that, The sol solution includes a sperm diluent.

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