Yak X / Y sperm separation and enrichment method based on pH value regulation and kit thereof

Abstract

The invention relates to a yak X / Y sperm separation and enrichment method based on pH value regulation and a kit thereof, and belongs to the technical field of animal breeding and sex control. The method comprises the following steps: using an Optidyl diluent as a sperm sorting protection solution, adjusting the pH value of the Optidyl diluent through NaOH or HCl, enriching X sperms in a pH range of 6.2-6.4, and enriching Y sperms in a pH range of 6.8-7.0; the method comprises the following specific steps: diluting yak sperm, centrifugally desizing, re-suspending by using a sorting solution with corresponding pH, standing and incubating for 45-90 minutes at 35-40 DEG C and 45 DEG C, realizing natural layering according to the motility difference of X and Y sperms under different pH conditions, respectively collecting upper or lower sperms, and washing and re-suspending to obtain a sex-controllable sperm sample; the method is easy and convenient to operate, low in cost, small in sperm damage, complete in sperm structure and stable in function after separation, suitable for breeding technologies such as artificial insemination and in-vitro fertilization, capable of meeting the requirement for sexual control sperm in yak production and beneficial for optimizing the population structure and improving the breeding efficiency.

Description

Technical Field

[0001] This application relates to the field of animal reproductive technology, and more specifically, to a method and kit for separating and enriching yak X / Y sperm based on pH adjustment. Background Technology

[0002] yak ( Bos grunniens As a unique livestock species in the Qinghai-Tibet Plateau region, the yak is an indispensable and important component of the region's livestock economy and ecosystem, providing local herders with various necessities such as meat, milk, wool, and hides, and possessing significant economic and social value. However, the current yak population generally faces problems such as imbalanced population structure and prominent inbreeding, leading to decreased reproductive performance, reduced genetic diversity, and an exacerbated trend of breed degradation. Simultaneously, different production needs have placed differentiated requirements on the sex ratio of yak offspring: for example, the dairy industry requires a higher proportion of female offspring, while beef cattle farming prefers male offspring. Therefore, developing efficient and reliable sex control technologies is of great significance for optimizing the yak population structure, adapting to industry needs, and improving overall reproductive efficiency.

[0003] In sex control technology, sperm separation before fertilization is a crucial step in achieving targeted breeding. Currently, various sperm separation methods have been reported in species such as cattle and mice, mainly including flow cytometry sorting, density gradient centrifugation, immunoassay, and pH separation. While flow cytometry sorting offers high precision, it suffers from high equipment costs, complex operation, and potential mechanical and fluorescence damage to sperm, making large-scale application difficult in the actual production environment of yak farming. In contrast, pH separation, due to its simplicity and lower cost, is considered a promising alternative.

[0004] However, directly applying the separation methods established for other species to yak sperm faces significant technical obstacles and uncertainties in applicability. As a high-altitude adapted livestock species, yaks exhibit fundamental differences in reproductive physiology, sperm morphology, metabolic mechanisms, and stress responses to external environmental factors compared to ordinary cattle breeds. In particular, the parameter ranges used in existing pH separation methods are largely derived from species such as ordinary cattle and have not been validated and optimized for yak sperm systems. Therefore, in the field of yak sex control, there is still a lack of sperm donor enrichment technology that is both adaptable to the biological characteristics of yak sperm and possesses ease of operation, cost-effectiveness, and high sorting efficiency. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for separating and enriching yak X / Y sperm based on pH control. By placing yak semen in a specific pH environment, the difference in motility between X and Y sperm under different acidic and alkaline conditions is utilized to achieve natural stratification and separation. Within the pH range of 6.2–6.4, Y sperm activity is selectively inhibited and sediments, while X sperm remain floating; conversely, within the pH range of 6.8–7.0, X sperm are inhibited and sink, while Y sperm are enriched in the upper layer. This method utilizes Optidyl... ® The diluent, used as a sorting and protection solution, adjusts the pH using NaOH or HCl. Combined with specific temperature and incubation angle, it efficiently and cost-effectively obtains sex-controllable semen samples while maintaining sperm motility and structural integrity. This method is suitable for reproductive technologies such as artificial insemination and in vitro fertilization.

[0006] In a first aspect, the present invention provides a method for separating and enriching yak X / Y sperm based on pH adjustment, characterized by comprising the following steps: S1: After thoroughly mixing fresh yak semen with sperm preservation base solution, centrifuge to remove the supernatant and obtain sperm precipitate; S2: Adjust the pH of the sperm protection base solution to a first predetermined range of 6.2~6.4 or a second predetermined range of 6.8~7.0 using a pH adjuster to obtain a sperm sorting solution, and resuspend the sperm precipitate in the sorting solution; S3: The resuspended sperm precipitate was incubated at an angle to obtain layered semen; S4: Collect the upper layer of the layered semen, wherein when the pH value of the sperm sorting solution is within the first predetermined range, the upper layer is collected as an enriched population of X sperm; when the pH value of the sperm sorting solution is within the second predetermined range, the upper layer is collected as an enriched population of Y sperm.

[0007] Preferably, the sperm protection base solution in S1 is Optidyl ® The diluent is a solution prepared by diluting the solution with deionized water at a volume ratio of 1:1 to 2.

[0008] Preferably, the pH adjuster in S2 is a NaOH and / or HCl solution.

[0009] Preferably, the downward slope angle of S3 is 40~50°, the static incubation temperature is 35℃~40℃, and the time is 45 min~90 min.

[0010] Secondly, this invention provides an application of yak sex-controlled semen prepared by a pH-adjusted yak X / Y sperm separation and enrichment method in yak artificial insemination, in vitro fertilization, or intracytoplasmic sperm injection.

[0011] Thirdly, the present invention provides a yak X / Y sperm separation and enrichment kit, characterized in that it includes the sperm sorting solution as described in claim 1.

[0012] Fourthly, this invention provides an application of a yak X / Y sperm separation and enrichment kit in yak sex control.

[0013] In summary, the present invention has at least one of the following beneficial technical effects: 1. This invention is the first to clearly define a specific pH window suitable for yak sperm separation. This parameter system is specially optimized based on the physiological characteristics of yak sperm, overcoming the problems of decreased motility and separation failure caused by the high sensitivity of yak sperm when directly applying ordinary bovine separation methods, and achieving efficient and stable separation in the special species of yak.

[0014] 2. The method provided by this invention only requires conventional laboratory instruments and reagents, and separation can be completed through simple steps such as adjusting pH and constant temperature incubation. It does not rely on expensive complex equipment such as flow cytometers, which greatly reduces the technical threshold and usage cost. It is especially suitable for promotion and application in pastoral areas or under conditions of limited resources, and helps to realize the large-scale production of sex-controlled semen.

[0015] 3. During the separation process under optimized pH conditions, key indicators such as sperm motility, plasma membrane integrity, acrosome status, and mitochondrial function were not significantly affected, indicating that the method causes minimal damage to sperm and can better maintain the natural fertilization ability of sperm, which is beneficial to the success rate of subsequent artificial insemination or in vitro fertilization. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This application provides an example of an analysis diagram showing the effect of different pH values ​​on sperm motility. Figure 2 This paper illustrates the effect of flow cytometry on the enrichment of yak sex-controlled semen according to an embodiment of this application; wherein... 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 2C is a bar chart showing the proportion of X and Y sperm in the upper semen at pH 6.3 and pH 6.9 of the control and experimental groups, respectively, to verify the enrichment effect of yak sex-controlled semen by flow cytometry. Figure 3 This document shows an image illustrating the detection of sperm plasma membrane and acrosome integrity after enrichment of yak sex-controlled semen according to an embodiment of this application; wherein... Figure 3 A is a bar chart showing the plasma membrane integrity rate of the upper semen layer at pH 6.3 and pH 6.9 after yak semen enrichment, comparing the control group and the experimental group. Figure 3 B is a bar chart showing the acrosome integrity rate of the upper semen layer at pH 6.3 and pH 6.9 after yak semen enrichment, comparing the sperm control group and the experimental group. Figure 4 This application provides an embodiment of the graph showing the detection of mitochondrial membrane potential and ATP content in sperm after enrichment of yak sex-controlled semen; wherein... Figure 4 A is a bar chart showing the mitochondrial membrane potential of the upper semen at pH 6.3 and pH 6.9 in the control group and experimental group after yak semen enrichment. Figure 4 B is a bar chart showing the ATP content of the upper semen at pH 6.3 and pH 6.9 in the control and experimental groups after yak semen enrichment. Detailed Implementation

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] To enable those skilled in the art to better understand this application, the following embodiments are provided to illustrate in detail a method for separating and enriching yak X / Y sperm based on pH adjustment and its reagent kit.

[0024] Example Example 1 To investigate the effects of different pH environments on yak sperm motility and to screen the optimal pH range for sperm separation, this embodiment employed the following method: Fresh yak semen samples were collected from Longri Livestock Farm in Hongyuan County, Sichuan Province. After 2-3 times dilution, their initial motility and density were tested. Sperm with motility higher than 70% and density between 4-6 × 10⁻⁶ were selected. 7 Semen samples of 1 / mL were used for subsequent experiments.

[0025] The screened semen samples were placed in sperm enrichment and preservation solutions (Optidyl) adjusted to different pH values ​​(6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0). ® In the preparation of the diluent, the solution was incubated at 37°C for 60 minutes to prepare the component selection solution. The pH 6.6 group served as the control group.

[0026] After incubation, the total sperm viability in the upper semen of each group was detected using a computer-aided sperm analysis system. The results are as follows: Figure 1 As shown.

[0027] The results show that ( Figure 1 A) Under acidic conditions with a pH lower than the control, the sperm motility in the upper layer decreased with decreasing pH; within 60 minutes of incubation, when pH ≥ 6.3, there was no significant difference in sperm motility compared to the control group. Under conditions with a pH higher than the control ( Figure 1 B), sperm motility decreased with increasing pH; within 60 minutes of incubation, when pH ≤ 6.9, sperm motility did not decrease significantly.

[0028] Further observation revealed that within the pH range of 6.2–6.4, the motility of Y sperm was selectively inhibited, causing them to settle to the lower layer, while X sperm maintained good activity and tended to float and accumulate in the upper layer. Within the pH range of 6.8–7.0, the activity of X sperm was inhibited and they settled, while Y sperm maintained good activity and floated and accumulated. Given that the ratio of X to Y sperm in adult male yak semen is approximately 1:1, and considering the above-mentioned activity stability and selective inhibition effect, this embodiment determined that adjusting the pH of the sperm enrichment and protection solution to the range of 6.2–6.4 can achieve effective enrichment of X sperm; adjusting it to the range of 6.8–7.0 can achieve effective enrichment of Y sperm. This pH range is the optimal operating condition for subsequent sorting experiments.

[0029] Example 2 In this embodiment, fresh yak semen was collected, and a computer-aided sperm analysis system was used to detect sperm motility and density. Semen samples with a sperm motility higher than 80% were selected as samples to be sorted, where sperm motility refers to the percentage of rapidly motile and moderately motile sperm in the total sperm count.

[0030] The activity level was 84.8%, and the density was 4.2 × 10⁻⁶. 7 3 mL of fresh semen (sperm count / mL) was placed in a 15 mL centrifuge tube and centrifuged at 4°C and 5000 r / min for 5 minutes. The supernatant containing diluent and seminal plasma was discarded, and the sperm precipitate was retained. 3 mL of yak sex-controlled sperm enrichment solution pre-cooled to 4°C was added to the precipitate, and the mixture was then centrifuged with Optidyl... ® Based on the diluent, the pH value was adjusted to 6.2-6.4 and 6.8-7.0 respectively according to the optimal pH range determined in Example 1, and the sperm was gently resuspended.

[0031] The resuspended semen was incubated at 37°C and tilted at 45° for 60 minutes to allow the sperm to naturally separate into layers. After incubation, the upper layer of semen was collected under different pH conditions. Under pH conditions of 6.2-6.4, the upper semen was collected to obtain a sperm population rich in X chromosomes. This population can fertilize the egg to form an XX-type zygote, thereby obtaining female offspring. Under pH conditions of 6.8–7.0, the upper semen is collected to obtain a sperm population rich in Y chromosomes. This population can fertilize the egg to form an XY zygote, thereby obtaining male offspring.

[0032] The collected upper semen volume accounted for approximately 30% of the total volume. Optidyl was then used. ® The X and Y semen were centrifuged and washed with diluent to remove the enrichment and resuspend the sperm, finally obtaining yak X sex-controlled semen and Y sex-controlled semen, which can be used for artificial insemination, in vitro fertilization and other reproductive technologies.

[0033] Example 3 This embodiment verifies the enrichment effect of yak sex-controlled semen using flow cytometry. Yak X-type sex-controlled semen (pH 6.2-6.4, upper layer) and Y-type sex-controlled semen (pH 6.8-7.0, upper layer) prepared by the aforementioned method were used as the experimental groups. Unsorted yak sex-controlled semen, prepared at a baseline pH of 6.6, was used as the experimental group. ® Yak semen treated in the diluent served as a control group.

[0034] Adjust Optidyl respectively ® The pH of the diluent was adjusted to the appropriate range (experimental group) or maintained at pH 6.6 (control group). After preheating at 37°C, it was used to resuspend or process sperm samples from the corresponding group. Samples from each group were placed in a 37°C incubator at a 45° angle and incubated in the dark for 45, 60, and 90 minutes. After incubation, the supernatant semen was carefully collected and transferred to preheated centrifuge tubes.

[0035] Add 10 μL of preheated Hoechst 33342 staining working solution (stock solution 5 mg / mL, diluted 1:1000) to each tube, mix gently, and incubate at 37°C in the dark for 40 minutes, mixing every 10 minutes during incubation. After staining, do not wash the samples; allow them to equilibrate at room temperature in the dark for 5–10 minutes, then store at 4°C in the dark for later analysis.

[0036] The samples were analyzed using a high-speed sorting flow cytometer to determine the ratio of X sperm to Y sperm. Results are as follows: Figure 2 As shown, the proportions of X sperm and Y sperm in the control group were 49.3% and 50.7%, respectively, which is consistent with the approximately 1:1 sex ratio of yak semen before sorting.

[0037] In the experimental group, the proportion of X sperm in the upper semen collected at pH 6.3 increased to 70.15%, while the proportion of Y sperm decreased to 29.85%, indicating effective enrichment of X sperm. In the upper semen collected at pH 6.9, the proportion of Y sperm increased to 65.4%, while the proportion of X sperm decreased to 34.6%, indicating effective enrichment of Y sperm. These results confirm that the pH-controlled separation method provided by this invention can significantly enrich sperm of the target sex, with a clear separation effect.

[0038] Example 4 This embodiment aims to detect the integrity of sperm plasma membrane and acrosome after enrichment of yak sex-controlled semen. The integrity of the sperm plasma membrane was assessed using a hypotonic swelling experiment. The hypotonic swelling solution was prepared as follows: 0.09 g of fructose and 0.049 g of sodium citrate were added to 10 mL of distilled water and mixed thoroughly. 100 µL each of the yak X-type sex-controlled semen, Y-type sex-controlled semen, and unsorted control group semen (pH 6.6) prepared in Example 2 were added to each sample, and 1 mL of the aforementioned hypotonic swelling solution was added to each sample. The samples were incubated at 37°C for 30 minutes. After incubation, 10 µL of each sample was placed on a glass slide, covered with a coverslip, and the sperm tail morphology was observed and photographed under a microscope. Sperm with intact plasma membranes absorbed water and swelled in the hypotonic environment, exhibiting typical tail curvature; sperm with damaged plasma membranes showed no tail curvature.

[0039] The results are as follows Figure 3 As shown in Figure A, the sperm plasma membrane integrity rates in the control group, the pH 6.3 upper semen group, and the pH 6.9 upper semen group were 74.498%, 69.904%, and 70.326%, respectively. There were no significant differences among the groups, indicating that the separation method described in this invention did not significantly affect the sperm plasma membrane integrity.

[0040] This embodiment further utilizes fluorescence staining to detect sperm acrosome integrity. The staining solution was prepared by mixing FITC-PNA and Hoechst 33342 at a 1:1 volume ratio. 50 µL of each of the three semen samples was taken, and 2.5 µL of the staining solution was added to each sample, which was then gently mixed. 10 µL of the stained sample was prepared into a smear, placed on a 37°C constant-temperature plate, and observed and photographed under a fluorescence microscope to assess acrosome integrity.

[0041] The results are as follows Figure 3 As shown in Figure B, the acrosome integrity rates of sperm in the control group, the upper semen group at pH 6.3, and the upper semen group at pH 6.9 were 67.462%, 66.896%, and 66.261%, respectively. The data from each group were similar, indicating that this separation method did not significantly damage the sperm acrosome structure.

[0042] The above results show that the yak sex-controlled semen obtained by the pH-controlled separation method provided by this invention maintains the integrity of the sperm plasma membrane and acrosome, further confirming that this method can ensure the structural and functional integrity of sperm while achieving sperm sex enrichment.

[0043] Example 5 This embodiment demonstrates, through the detection of mitochondrial membrane potential and ATP content in sperm after enrichment of yak sex-controlled semen, that the method provided by this invention can effectively maintain the physiological function and fertilization potential of sperm while achieving sex enrichment.

[0044] (1) Detection of sperm mitochondrial membrane potential: Yak X sex-controlled semen (pH 6.3 upper layer) and Y sex-controlled semen (pH 6.9 upper layer) prepared in Example 2 were used as experimental groups, and unsorted yak raw semen (pH 6.6) was used as the control group; according to the Beyotime mitochondrial... TM Follow the instructions for the membrane potential assay kit (JC-1 method). The JC-1 dye exists as a polymer (J-aggregates) emitting red fluorescence when the mitochondrial membrane potential is high; it exists as a monomer emitting green fluorescence when the membrane potential decreases. The ratio of red to green fluorescence intensity reflects the relative level of mitochondrial membrane potential. After staining, the samples were observed and photographed using a fluorescence microscope, and the fluorescence intensity of each group was quantitatively detected using a fluorescence microplate reader.

[0045] (2) Detection of sperm ATP content: Yak X sex-controlled semen (pH 6.3 upper layer) and Y sex-controlled semen (pH 6.9 upper layer) prepared in Example 2 were used as experimental groups, and unsorted yak raw semen (pH 6.6) was used as the control group; the operation was carried out according to the instructions of the ATP content test kit of Nanjing Jiancheng Bioengineering Institute. The absorbance value of each sample was measured at a wavelength of 636 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the specific ATP content in sperm was calculated according to the standard curve.

[0046] The statistical results of the detection of mitochondrial membrane potential and ATP content are as follows: Figure 4 As shown, there were no significant differences in mitochondrial membrane potential and ATP content between the experimental groups (upper semen at pH 6.3 and upper semen at pH 6.9) and the control group.

[0047] The above results show that the mitochondrial function and energy metabolism of sperm in the obtained yak sex-controlled semen were not significantly affected after treatment by the pH-controlled separation method provided by the present invention, further confirming that the method can effectively maintain the physiological function and fertilization potential of sperm while achieving sex enrichment.

[0048] In summary, this invention systematically verified the feasibility and effectiveness of pH control for X / Y sperm separation in yaks through a series of examples. Example 1 screened the optimal pH range for sperm motility separation without significantly affecting sperm activity: 6.2–6.4 for enriching X sperm and 6.8–7.0 for enriching Y sperm. Example 2 established a complete sex-controlled semen preparation process based on these parameters. Through centrifugation, pH adjustment, oblique incubation, and stratified collection, semen samples enriched with the target sperm were successfully obtained. Example 3 used flow cytometry to confirm that the proportion of X or Y sperm could be increased to approximately 70% and 65% respectively after sorting, demonstrating a significant enrichment effect. Examples 4 and 5 further demonstrated that this method has no significant negative impact on sperm plasma membrane integrity, acrosome structure, mitochondrial membrane potential, and ATP content, indicating that it can maintain the structural and functional integrity of sperm while achieving efficient sex separation.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] The above provides a detailed description of the method and kit for separating and enriching yak X / Y sperm based on pH adjustment. 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 core ideas of this application. At the same time, for those skilled in the art, 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 based on pH adjustment, characterized in that, Includes the following steps: S1: After thoroughly mixing fresh yak semen with sperm preservation base solution, centrifuge to remove the supernatant and obtain sperm precipitate; S2: Adjust the pH of the sperm protection base solution to a first predetermined range of 6.2~6.4 or a second predetermined range of 6.8~7.0 using a pH adjuster to obtain a sperm sorting solution, and resuspend the sperm precipitate in the sorting solution; S3: The resuspended sperm precipitate was incubated at an angle to obtain layered semen; S4: Collect the upper layer of the layered semen, wherein when the pH value of the sperm sorting solution is within the first predetermined range, the upper layer is collected as an enriched population of X sperm; when the pH value of the sperm sorting solution is within the second predetermined range, the upper layer is collected as an enriched population of Y sperm.

2. The method for separating and enriching yak X / Y sperm based on pH adjustment according to claim 1, characterized in that, The sperm protection base solution in S1 is Optidyl ® The diluent is a solution prepared by diluting the solution with deionized water at a volume ratio of 1:1 to 2.

3. The method for separating and enriching yak X / Y sperm based on pH adjustment according to claim 1, characterized in that, The pH adjuster in S2 is a NaOH and / or HCl solution.

4. The method for separating and enriching yak X / Y sperm based on pH adjustment according to claim 1, characterized in that, The downward slope angle of S3 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 pH-adjusted 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 solution as described in claim 1.

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

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