Yak X / Y sperm separation and enrichment method based on TLR7 / 8 agonist R848 and kit

Abstract

The invention relates to the technical field of animal breeding, and discloses a yak X / Y sperm separation and enrichment method based on a TLR7 / 8 agonist R848 and a kit. The method comprises the following steps: treating the yak sperm by using a sperm sorting solution containing a TLR7 / 8 dual agonist R848, incubating under specific conditions, and separating and collecting by using a stratification phenomenon caused by activity difference of X and Y sperms, the concentration of the R848 in the separation liquid is 0.3 [mu] M to 0.7 [mu] M; according to the specific reproductive physiological characteristics of the yak sperms, the problem that the separation efficiency is low or the sperms are seriously damaged due to inapplicable parameters in the prior art is solved, and the technical scheme special for yak sex control is easy and convenient to operate, low in cost, high in separation efficiency and small in influence on the sperm quality.

Description

Technical Field

[0001] This application relates to the field of animal reproduction and breeding, and more specifically, to a method and kit for separating and enriching yak X / Y sperm based on the TLR7 / 8 agonist R848. Background Technology

[0002] yak ( Bos grunniens The yak is the most important economic livestock species in the Qinghai-Tibet Plateau region, providing local herders with necessities such as meat, milk, wool, and hides, playing an irreplaceable role in the plateau's livestock economy and ecosystem. Currently, yak populations generally suffer from problems such as irrational herd structure and severe inbreeding, leading to decreased reproductive efficiency and a significant trend of breed degradation. Furthermore, the sex requirements for yak offspring vary under different production needs: for example, the dairy industry requires a predominance of female offspring, while the meat industry prefers male offspring. Therefore, developing effective sex control technologies is of great significance for optimizing yak population structure, meeting specific production needs, and improving overall reproductive efficiency.

[0003] Pre-fertilization sperm separation is one of the key technologies for sex control. Currently, sperm separation methods reported in species such as cattle and mice are relatively mature. However, yaks, as species adapted to extreme environments, exhibit fundamental differences in their reproductive physiology, sperm metabolism, and stress responses to external stimuli compared to livestock from plains areas. For example, yak sperm exhibit unique characteristics in energy metabolism, membrane stability, and sensitivity to pH and drug concentrations. Directly applying separation methods developed for species like cattle often leads to a sharp decline in yak sperm motility and low separation efficiency.

[0004] While flow cytometry is considered the most precise sperm separation method, its expensive equipment, complex operation, and potential sperm damage during the sorting process make it difficult to implement in the high-altitude, remote pastoral areas where yaks are raised. In contrast, immunoassay is considered a promising alternative due to its lower cost and ease of operation. As a unique livestock species of the Qinghai-Tibet Plateau, yaks exhibit significant differences in reproductive physiology, sperm structure, energy metabolism, and stress response to external stimuli compared to cattle from the plains. Therefore, current technologies lack a sperm enrichment scheme that fully considers the unique biological characteristics of yak sperm while balancing ease of operation, cost control, and high sorting efficiency. Summary of the Invention

[0005] To solve the above problems, the application provides a yak X / Y sperm separation and enrichment method and kit based on TLR7 / 8 agonist R848. The application provides an immune separation scheme specially adapted to the reproductive physiological characteristics of yaks to solve the long-standing technical problems in yak sperm separation. The TLR7 / 8 dual agonist R848 is selected as the separation agent. The influence of the R848 on the sperm motility and separation effect of yaks is determined through systematic experiments, so that the optimal drug concentration, incubation time and matching treatment conditions suitable for yaks are selected, and a complete separation system including a special sorting solution and a kit is constructed based on the above. The scheme effectively overcomes the technical bias that the direct use of general livestock parameters causes damage to yak sperm. Under the premise of keeping the operation simple and the cost controllable, the scheme realizes efficient and specific enrichment of X / Y sperm of yaks, and provides a reliable technical basis for directional sex control of yaks.

[0006] In a first aspect, the application provides a yak X / Y sperm separation and enrichment method, characterized in that the method comprises the following steps: S1: diluting fresh yak semen with a sperm sorting protective solution to resuspend; the sperm sorting protective solution comprises a sperm protective base solution and an agonist R848; S2: incubating the resuspended semen by gradient standing to obtain layered semen; S3: separating the layered semen, collecting the supernatant as a Y sperm-enriched sample, and collecting the precipitate as an X sperm-enriched sample; S4: eluting the Y sperm and X sperm samples to obtain Y sex control semen and X sex control semen.

[0007] The agonist R848 is a TLR7 / 8 dual agonist. The mechanism of action is that it can selectively inhibit the mitochondrial activity and glycolysis metabolic pathway of X chromosome-carrying sperm, thereby significantly reducing the motility of the X chromosome-carrying sperm. However, the agonist R848 has no significant effect on the motility of Y chromosome-carrying sperm. Based on the selective inhibition effect, during the incubation process, the X sperm with inhibited motility gradually settles at the bottom, while the Y sperm with normal motility tends to float and enrich in the upper layer, thereby realizing physical separation.

[0008] As a preferred embodiment, the addition amount of the agonist R848 in the sperm sorting protective solution is 0.1 μM to 1.0 μM, preferably 0.6 μM. The R848 is a pyrido[3,2 d]pyrimidine TLR7 / 8 dual agonist. The molecular formula of the R848 drug is C 17 H 22 N4O2; and the molecular structure formula is: .

[0009] As a preferred embodiment, the sperm protective base solution is Optidyl® Diluted solution of the diluent solution diluted by deionized water at a volume ratio of 1:1~2.

[0010] Preferably, the S2 lower bevel angle is 40~50°, the standing incubation temperature is 35℃~40℃, and the time is 45 min~90 min.

[0011] In the second aspect, the application provides application of yak sex control semen prepared by the yak X / Y sperm separation and enrichment method in yak artificial insemination, in vitro fertilization or intracytoplasmic sperm injection.

[0012] In the third aspect, the application provides a yak X / Y sperm separation and enrichment kit, characterized in that it comprises the sperm sorting protective solution; the kit comprises an enrichment reagent of TLR7 / 8 agonist R848, and 1000 mL of the sperm sorting protective solution contains 105.24 μg~245.56 μg of R848 drug.

[0013] In the fourth aspect, the application provides application of a yak X / Y sperm separation and enrichment kit in yak gender control.

[0014] In summary, the application has at least one of the following beneficial technical effects: 1. The application first determines the optimal concentration of TLR7 / 8 agonist R848 and the matching incubation conditions through systematic optimization experiments according to the unique reproductive physiological characteristics of yak sperm, and realizes effective separation and enrichment of yak X / Y sperm.

[0015] 2. Compared with the flow cytometer sorting method, the application does not require expensive and precise equipment, only requires a conventional constant temperature incubator and a centrifuge, the operation steps are simple, and the technical personnel can easily master it, and it is especially suitable for on-site or nearby processing in high-cold and remote pastoral areas of yak breeding.

[0016] 3. The sorting process of the application does not cause significant negative effects on the plasma membrane integrity, acrosome integrity and reactive oxygen species level of yak sperm, which indicates that the method better maintains the basic structure and functional integrity of sperm while realizing separation. DETAILED DESCRIPTION

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the application, the following will briefly introduce the drawings needed to be used in the description of the embodiments of the application. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0018] Figure 1The following graph shows the effect of the effective concentration of R848 drug provided in the embodiments of this application on the sperm motility of yak. Figure 2 The actual enrichment effect of X sperm and Y sperm provided in the embodiments of this application is shown in the figure; Figure 2 A is a scatter plot of cells used to verify the enrichment effect of yak sex-controlled semen by flow cytometry. Figure 2 B is a cell peak diagram used to verify the enrichment effect of yak sex-controlled semen by flow cytometry. Figure 2 C is a bar chart showing the proportion of X and Y sperm in the upper and lower semen layers of the control and experimental groups, used to verify the enrichment effect of yak sex-controlled semen by flow cytometry. Figure 3 The image shows the results of sperm structure integrity testing after yak semen sorting according to an embodiment of this application. Figure 3 A is a bar chart showing the statistical percentage of plasma membrane integrity in the upper and lower semen layers of the control and experimental groups after yak semen enrichment. Figure 3 B is a bar chart showing the acrosome integrity rate of the upper and lower semen layers in the control and experimental groups after yak semen enrichment. Figure 4 This document shows a graph illustrating the detection results of sperm energy metabolism levels after yak semen sorting, as provided in an embodiment of this application. Figure 4 A is a bar chart showing the mitochondrial membrane potential of the upper and lower semen layers in the control and experimental groups after yak semen enrichment. Figure 4 B is a bar chart showing the ATP content of the upper and lower semen layers in the control and experimental groups after yak semen enrichment. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention. Furthermore, all other embodiments obtained by those skilled in the art without inventive effort are within the protection scope of the present invention.

[0020] Specific experimental steps or conditions are not specified in the embodiments; they can be performed according to the conventional experimental steps or conditions described in the prior art. Reagents and other instruments used, unless otherwise specified, are all commercially available conventional reagent products. Furthermore, the accompanying drawings are merely illustrative diagrams of the embodiments of the present invention and are not necessarily drawn to scale. The same reference numerals in the drawings denote the same or similar parts, and therefore, repeated descriptions of them will be omitted. Some block diagrams shown in the drawings are functional entities and do not necessarily correspond to physically or logically independent entities.

[0021] Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of this specification.

[0022] In the description of this invention, it should be understood that the use of terms such as "first" and "second" to define components is merely for the purpose of distinguishing the corresponding components. Unless otherwise stated, the above terms have no special meaning and therefore should not be construed as limiting the scope of protection of this invention.

[0023] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0024] To enable those skilled in the art to more clearly understand this application, the following examples will provide a detailed description of the method and kit for separating and enriching yak X / Y sperm based on the TLR7 / 8 agonist R848 provided in this application.

[0025] Example Example 1 The purpose of this embodiment is to screen the effect of the TLR7 / 8 agonist R848 on yak sperm motility and determine its optimal concentration range for sperm sorting.

[0026] Semen source: Fresh semen from healthy adult male yaks at Longri Livestock Farm, Hongyuan County, Sichuan Province.

[0027] Main reagents: TLR7 / 8 dual agonist R848, sperm protection base solution (Optidyl) ® Dilute the solution with deionized water at a ratio of 1:1.5.

[0028] Freshly collected yak semen was diluted 2-3 times with a basal diluent, and its initial motility and density were tested. Semen with motility >70% and a density of 5 × 10⁻⁶ was selected. 7 Semen sample of 1 / mL.

[0029] The final concentration gradients of R848 were set to 0 μM (control group NC), 0.3 μM, 0.5 μM, 0.6 μM, 0.7 μM, and 1.0 μM.

[0030] Different concentrations of R848 were added to the sperm protection base solution, mixed with pretreated semen, and incubated at 37°C for 60 minutes to prepare a sperm sorting and protection solution.

[0031] After incubation, the total motility of the middle and lower layers of sperm in each group was detected using a computer-aided sperm analysis system.

[0032] The results are as follows Figure 1 As shown, the total motility of the lower sperm layer gradually decreased with increasing R848 concentration. When R848 was in the range of 0.3–0.7 μM, the motility of the lower sperm layer decreased to about 70% and tended to stabilize; however, when the concentration was further increased to 1.0 μM, the sperm motility continued to decrease significantly, indicating that excessively high concentrations can cause excessive inhibition of sperm activity.

[0033] Based on the above results, and considering the biological basis that the ratio of X to Y sperm in adult male yak semen is close to 1:1, the optimal concentration range of R848 for yak sperm sorting was determined to be 0.3–0.7 μM, with 0.6 μM being the optimal concentration. Within this concentration range, R848 can effectively inhibit the motility of X sperm, promoting their sedimentation, while the activity of Y sperm remains relatively stable, thus providing a reliable concentration basis for achieving the separation and enrichment of X / Y sperm.

[0034] Example 2 The purpose of this embodiment is to verify the actual enrichment effect of X sperm and Y sperm after sorting yak semen using the optimal R848 concentration range (0.3~0.7 μM) determined in Example 1.

[0035] Fresh male yak semen was collected and analyzed using a computer-aided sperm analysis system. Semen with a motility >80% was selected for the experiment. The initial motility of the semen sample used in this example was 84.8%, and the density was 4.2 × 10⁻⁶. 7 per mL.

[0036] S1: Take 3 mL of qualified semen, centrifuge at 4℃ and 5000×g for 5 min, discard the supernatant and retain the sperm precipitate.

[0037] S2: Add 3 mL of pre-cooled yak sperm sorting working solution (in Optidyl) to the sperm precipitate. ® Based on the diluent, add 0.3–0.7 μM of R848 as concluded in Example 1 and gently resuspend. Incubate the resuspended solution at a constant temperature of 37°C for 60 minutes at a 45° angle.

[0038] S3: After incubation, the semen is clearly divided into three layers. Use a pipette to collect the liquid in the top 30% of the volume as the test sample rich in Y sperm; at the same time, collect the sediment and adjacent liquid in the bottom 30% of the volume as the test sample rich in X sperm; discard the middle layer.

[0039] S4: The collected Y-rich clusters and X-rich clusters were washed with an appropriate amount of sperm elution buffer, R848 was removed by centrifugation, and finally resuspended to obtain yak Y-sex control semen and yak X-sex control semen.

[0040] Flow cytometry (Hoechst 33342 staining method) was used to analyze the X / Y sperm ratio in semen samples before and after sorting. The enrichment effect was verified by comparing the proportions of X and Y sperm in the X and Y enriched clusters in semen before sorting (control group) and after sorting. Figure 2 As shown in B.

[0041] like Figure 2 As shown in C, flow cytometry analysis confirmed that the ratio of X to Y sperm in yak semen was close to 1:1 before sorting. After sorting, the proportion of Y sperm in Y-controlled semen (from the upper layer) should be significantly higher than 50%, while the proportion of X sperm in X-controlled semen (from the lower layer) should be significantly higher than 50%.

[0042] This embodiment confirms that by using the 0.3~0.7 μM R848 sorting system, combined with the incubation and collection method, the separation and enrichment of yak X sperm and Y sperm can be effectively achieved, resulting in sex-biased semen.

[0043] Example 3 In this embodiment, flow cytometry was used to analyze the X / Y sperm ratio of yak X-sex controlled semen and Y-sex controlled semen prepared in Example 2, quantitatively verifying the actual enrichment effect of the method of the present invention on yak X / Y sperm.

[0044] Using the method described in Example 2, yak semen X and Y were sorted and used as the experimental group, while unsorted yak semen was used as the control group. Optidyl will be used ® R848 and Optidy prepared as semen diluent l® The semen diluent was preheated at 37°C for 2 minutes. R848 was added to each experimental group to make the final concentration of the sorting solution 0.6 μmol / L. Optidyl® semen diluent was added to each control group (note that when adding the sorting solution and diluent, the pipette tip should be kept close to the surface of the top layer of semen and added slowly).

[0045] Six groups were wrapped in aluminum foil and incubated at a 45° angle in a 37°C incubator in the dark for 45 min, 60 min, and 90 min (the incubator temperature should be adjusted to 37°C before use). After incubation, the upper and lower layers of semen were aspirated into preheated centrifuge tubes wrapped in aluminum foil, and the middle layer was discarded (when aspirating, be careful to keep the pipette tip close to the surface of the semen and let it slowly descend with the surface of the semen). The upper and lower semen layers were labeled, aliquoted, and then used for subsequent experiments.

[0046] Add 10 μL (5 mg / mL diluted 1:1000) of pre-prepared and preheated Hoechst 33342 working solution to each group, mix by inverting the container, and incubate in a 37°C incubator in the dark for 40 min, mixing once every 10 min.

[0047] After incubation, there is no need to wash off the Hoechst 33342 fluorescent dye (unbound free dye exhibits very weak fluorescence when stained at a working concentration of 5-10 μg / mL, minimizing interference with flow cytometry detection and preventing centrifugation from affecting sperm motility and morphology). After staining, equilibrate at room temperature in the dark for 5-10 min, then store at 4°C in the dark before sending the sample to a high-speed sorting flow cytometer to detect the X and Y sperm ratio.

[0048] from Figure 2 As shown in Figure C, the percentages of X sperm and Y sperm in the control group were 50.7% and 49.3%, respectively. The X sperm to Y sperm ratio in the control group's semen was approximately 1:1, consistent with the pre-sorting ratio. In the upper semen layer, X sperm and Y sperm accounted for 36.29% and 63.71%, respectively, while in the sediment, they were 67.85% and 32.15%, respectively.

[0049] The results confirm that the separation method established in this invention (based on 0.6 μM R848, incubation at 37℃ at a 45° angle for 60 minutes) can effectively achieve the separation and enrichment of yak X / Y sperm, providing a reliable experimental basis for sex control.

[0050] Example 4 This embodiment evaluates the impact of the sorting method of the present invention on the structural integrity of yak sperm. Specifically, it tests the integrity of the sperm plasma membrane and acrosome after sorting to confirm whether the method causes significant damage to key sperm structures while achieving effective separation.

[0051] Experimental group: yak X-sex control semen and Y-sex control semen prepared in Example 2 and control group (fresh yak semen that has not been sorted).

[0052] Accurately weigh 0.09 g of fructose and 0.049 g of sodium citrate, dissolve them in 10 mL of distilled water, and mix thoroughly to prepare a hypotonic swelling solution. Take 100 µL of semen samples from the upper (Y-sex control) and lower (X-sex control) layers of the control and experimental groups, respectively, and add 1 mL of the hypotonic swelling solution preheated to 37°C to each sample, then gently mix. Incubate the mixture in a 37°C incubator for 30 minutes. After incubation, drop 10 µL of the mixture onto a glass slide, cover with a coverslip, and immediately observe and photograph under a phase-contrast microscope. Sperm with intact plasma membranes absorb water under hypotonic conditions, resulting in characteristic tail bending or swelling; sperm with damaged plasma membranes do not show this change in the tail. Randomly observe no fewer than 200 sperm from each sample and calculate the percentage of sperm with intact plasma membranes. Results are as follows: Figure 3 As shown in Figure A, the plasma membrane integrity rates of the control group and the experimental group were 74.92%, 73.8%, and 73.1%, respectively.

[0053] The integrity of sperm acrosomes was assessed using FITC-PNA fluorescence staining. The specific steps were as follows: 50 µL of semen samples from each experimental group and control group were taken, and 2.5 µL of a 1:1 mixture of FITC-PNA and Hoechst 33342 staining solution was added and gently mixed by pipetting. The stained samples were incubated at 37°C in the dark for 15 minutes, and then 10 µL was taken to prepare a smear, which was then placed on a 37°C constant temperature plate to air dry in the dark. The samples were observed under a fluorescence microscope at the corresponding excitation wavelength. Hoechst 33342 labeled all sperm cell nuclei (blue fluorescence), and FITC-PNA specifically bound intact acrosomes (green fluorescence). The anterior part of the acrosome of sperm with intact acrosomes showed a bright green fluorescent cap-like structure, while sperm with acrosome reaction or damage did not have this structure or the fluorescence was incomplete. At least 200 sperm were randomly observed and counted from each sample, and the percentage of sperm with intact acrosomes was calculated.

[0054] The results are as follows Figure 3 As shown in B, the acrosome integrity rates of the upper and lower sperm layers in the control group, experimental group, and control group were 72.10%, 69.96%, and 69.41%, respectively, with no significant difference between the groups.

[0055] This embodiment confirms through hypotonic swelling test and fluorescent staining that: after sorting using the method of the present invention, the plasma membrane integrity of sperm in yak X-sex controlled semen and Y-sex controlled semen is not significantly different from that in the unsorted original semen; the acrosome integrity of the sperm is also maintained after sorting, with no significant difference from the control group; the sorting method of the present invention effectively enriches sperm of the target sex without causing significant negative impact on the key structural integrity of yak sperm, indicating that the technology has good biocompatibility and safety, providing quality assurance for subsequent reproductive applications.

[0056] Example 5 This embodiment evaluates the impact of the sorting method of the present invention on the energy metabolism of yak sperm. By detecting mitochondrial membrane potential and ATP content, it reflects the energy preservation and motility of sperm during the sorting process.

[0057] Experimental group: yak X-sex control semen and Y-sex control semen prepared in Example 2; Control group: fresh yak semen that has not been sorted.

[0058] Semen from the control group and experimental group (upper and lower layers) was collected and adjusted to the same sperm concentration. Following the instructions of the Beyotime Mitochondrial Membrane Potential Detection Kit (JC-1), JC-1 working solution was added to each sample, thoroughly mixed, and incubated at 37°C in the dark for 20 minutes. The stained samples were prepared into smears, observed and photographed under a fluorescence microscope. When the mitochondrial membrane potential is high, JC-1 forms a polymer within the mitochondria, emitting red fluorescence; when the membrane potential decreases, JC-1 exists as a monomer, emitting green fluorescence. The membrane potential level was assessed by the red / green fluorescence intensity ratio. The stained samples were transferred to black ELISA plates, and the fluorescence intensity was measured using a fluorescence microplate reader at the excitation / emission wavelengths (Ex / Em=525 / 590 nm, red fluorescence; Ex / Em=490 / 530 nm, green fluorescence), and the red / green fluorescence intensity ratio was calculated. Following the instructions for the Nanjing Jiancheng ATP content assay kit, semen samples were treated with lysis buffer, centrifuged, and the supernatant was collected. Working solution was added and mixed thoroughly, and the mixture was allowed to react at room temperature for a specified time. The absorbance of each well was measured using a microplate reader at 636 nm. The absolute ATP content in each sample was calculated based on the standard curve. The results are shown below. Figure 4 As shown, the red / green fluorescence intensity ratio of the lower layer (X-sex controlled semen) of the experimental group was significantly lower than that of the control group and the upper layer (Y-sex controlled semen) of the experimental group, indicating that the mitochondrial membrane potential of the X-sperm-rich clusters was significantly reduced. There were no significant differences in mitochondrial membrane potential and ATP content between the control group and the upper layer (Y-sex controlled semen) of the experimental group.

[0059] The results show that the sorting method of this invention can significantly reduce the mitochondrial membrane potential and ATP content of X sperm enriched in the lower layer, which is consistent with the mechanism of action of R848 selectively inhibiting the energy metabolism of X sperm. The mitochondrial function and energy levels of Y sperm and unsorted sperm in the upper layer were not significantly affected. These results further verify the specificity of the sorting system of this invention from the perspective of energy metabolism, and indicate that while achieving effective separation, this method has no significant negative impact on the energy status of Y sperm and the overall sperm population, providing a metabolic basis for the practical application of sex-controlled semen.

[0060] In summary, this application systematically verified the feasibility and effectiveness of the yak X / Y sperm separation and enrichment method through a series of examples. Example 1 clarified the optimal concentration range of R848 (0.3~0.7 μM); Example 2 established a complete semen sorting and sex-controlled semen preparation process; Example 3 confirmed by flow cytometry that the system can effectively enrich X and Y sperm (the proportion of X sperm in the lower layer reaches 67.85%, and the proportion of Y sperm in the upper layer reaches 63.71%); Example 4 showed that the sorting process has no significant impact on the integrity of the sperm plasma membrane and acrosome; Example 5 further revealed the mechanism by which R848 selectively inhibits mitochondrial function and ATP synthesis in X sperm from the perspective of energy metabolism. All the above results indicate that this invention provides a simple, cost-effective, efficient, and sperm-structure- and function-friendly yak sex control sorting scheme with good prospects for practical application.

[0061] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0062] Although preferred embodiments of the present application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present application.

[0063] Finally, it should be noted that in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes said element.

[0064] The above provides a detailed description of the method and kit for separating and enriching yak X / Y sperm based on the TLR7 / 8 agonist R848. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the method and its core ideas. At the same time, those skilled in the art will recognize that there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A method for separating and enriching yak X / Y sperm, characterized in that, Includes the following steps: S1: Fresh yak semen was diluted and resuspended using a sperm sorting and preservation solution; the sperm sorting and preservation solution contains a sperm preservation base solution and the agonist R848; S2: The resuspended semen was incubated at an angle to obtain the semen after separation; S3: Separate the layered semen, collect the supernatant as a sample rich in Y sperm, and collect the precipitate as a sample rich in X sperm; S4: The samples of Y sperm and X sperm are eluted to obtain Y-sex control semen and X-sex control semen.

2. The method for separating and enriching yak X / Y sperm according to claim 1, characterized in that, The amount of the dual agonist R848 added to the sperm sorting and protection solution is 0.1 μM to 1.0 μM, preferably 0.6 μM.

3. The method for separating and enriching yak X / Y sperm according to claim 1, characterized in that, The sperm protection base solution is Optidyl ® The diluent is a solution prepared by diluting the solution with deionized water at a volume ratio of 1:1 to 2.

4. The method for separating and enriching yak X / Y sperm according to claim 1, characterized in that, The downward slope angle of S2 is 40~50°, the static incubation temperature is 35℃~40℃, and the time is 45 min~90 min.

5. The application of yak sex-controlled semen prepared using the yak X / Y sperm separation and enrichment method as described in any one of claims 1 to 4 in yak artificial insemination, in vitro fertilization, or intracytoplasmic sperm injection.

6. A kit for separating and enriching yak X / Y sperm, characterized in that, Includes the sperm sorting and protection solution as described in claim 1 or 2.

7. The application of the sperm sorting and protection solution as described in claim 1 or 2 and / or the yak X / Y sperm separation and enrichment kit as described in claim 6 in yak sex control.

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