Method for enriching X sperms of Nubia goats and enrichment kit for X sperms of Nubia goats

Abstract

The invention discloses a method for enriching X sperms of Nubia goats and an enrichment kit for X sperms of Nubia goats, and belongs to the technical field of livestock breeding and biology. The method for enriching the Nubia goat X sperms comprises the following steps: (1) carrying out activity detection on Nubia goat sperms, and washing the Nubia goat sperms which are qualified in activity detection to obtain washed sperm samples; (2) adding a sperm diluent containing R848 into the washed sperm sample, mixing, and incubating at 37 DEG C for 45 minutes; and (3) after incubation is completed, collecting precipitated sperms at the bottom of the container, and washing with a sperm diluent to remove R848 so as to obtain enriched Nubia goat X sperms. According to the method, efficient enrichment of the X sperms of the Nubia goats can be achieved through the steps of incubation, sediment collection and washing, the activity of the X sperms of the Nubia goats obtained through enrichment is not affected, and the method has important value for artificial propagation of the Nubia goats.

Description

Technical Field

[0001] This invention belongs to the field of livestock reproduction and biotechnology, specifically relating to a method for enriching Nubian goat X sperm and a Nubian goat X sperm enrichment kit. Background Technology

[0002] Goat farming is an important part of my country's animal husbandry. Currently, many superior goat breeds suffer from slow growth and low meat yield. Nubian goats, as a dual-purpose breed (dairy and meat), are widely used for crossbreeding and improvement. The pure black Nubian goat, bred by crossing Nubian goats with local black goats, possesses both excellent traits and market acceptance, demonstrating significant potential for industry development.

[0003] In large-scale farming, the sex ratio of offspring directly affects production efficiency and economic benefits. Therefore, developing efficient and low-cost sex control technologies is of great significance for the breeding and propagation of Nubian goats. Currently, flow cytometry is a relatively accurate sperm sex separation technology, but the equipment is expensive, the operation is complex, and it may cause some damage to sperm, limiting its large-scale application in production.

[0004] Existing technologies, such as Chinese patent CN101411722A's "Frozen Mixed Semen for X / Y Sex Control in Dairy Cows and its Preparation Method," mostly focus on other livestock breeds or employ other separation principles. For Nubian goats, there is currently a lack of a simple, low-cost, and stable method for controlling sperm sex.

[0005] Developing a method for enriching Nubian goat X sperm and a Nubian goat X sperm enrichment kit is of great significance for improving the reproduction and economic benefits of Nubian goats. Summary of the Invention

[0006] This invention aims to overcome the shortcomings of existing technologies and provide a method for enriching Nubian goat X sperm and a Nubian goat X sperm enrichment kit. This invention enriches Nubian goat X sperm through incubation with TLR7 / 8 ligand R848, providing a simple, low-cost method with minimal impact on sperm motility and good enrichment effect, thus achieving a method for sex control of Nubian goat sperm.

[0007] The technical solution of this invention is implemented as follows:

[0008] This invention provides a method for enriching Nubian goat X sperm, comprising the following steps:

[0009] (1) Motility testing was performed on Nubian goat semen. Nubian goat semen that passed the motility test was washed to obtain washed semen samples.

[0010] (2) Add sperm diluent containing R848 (requimod) to the washed sperm sample and mix. Incubate at 37°C for 45 minutes.

[0011] (3) After incubation, collect the precipitated sperm at the bottom of the container, wash with sperm dilution solution to remove R848, and obtain enriched Nubian goat X sperm.

[0012] Further, in step (1), the method for detecting sperm motility is as follows: Nubian goat semen is diluted 10 times with a semen diluent, and then sperm motility is detected using a sperm quality computer-aided analysis system (CASA). More preferably, the semen diluent is the French Casu BioXcell type bovine semen diluent.

[0013] Furthermore, in step (1), the qualification of the motility test is a motility test result of sperm motility greater than 80%.

[0014] Further, in step (1), the washing conditions are: after washing at 380×g for 5min, the supernatant is removed to obtain the washed sperm sample.

[0015] Furthermore, the concentration of R848 in the sperm diluent in step (2) is 2 μM / L.

[0016] Furthermore, the sperm diluent mentioned in step (2) is the French Casu BioXcell bovine semen diluent.

[0017] The incubation in step (2) allows R848 to bind to TLR7 and / or TLR8 receptors on the surface of X sperm, thereby inhibiting X sperm motility.

[0018] Further, in step (3), the collection of precipitated sperm at the bottom of the container specifically refers to collecting the precipitated sperm in the bottom 1 / 3 of the container after incubation. Here, the height from the liquid interface in the container to the bottom of the container is defined as 1, and the bottom 1 / 3 refers to the liquid precipitated sperm at the top 1 / 3 position from the bottom of the container.

[0019] Further, in step (3), the washing conditions are: centrifugation at 380×g for 5 minutes, and repeated washing 3 times. The washing in step (3) is to remove residual R848 and avoid the influence of residual R848 on the activity of enriched Nubian goat sperm.

[0020] The Nubian goat X sperm prepared by the above method has a significantly higher proportion of X sperm than the untreated raw Nubian goat semen.

[0021] This invention also provides a Nubian goat X sperm enrichment kit, comprising R848 (Requimod) and sperm diluent. The Nubian goat X sperm enrichment kit can be used in the above-described method for enriching Nubian goat X sperm, thereby achieving the enrichment of Nubian goat X sperm.

[0022] This invention utilizes R848 (Requimod) and sperm diluent treatment. R848 binds to TLR7 and / or TLR8 receptors on the surface of X sperm, selectively inhibiting X sperm motility and thus separating them from Y sperm. Specifically, the R848 compound binds to the specific receptor proteins TLR7 / TLR8 on Nubian goat X sperm, causing impaired X sperm motility and causing them to settle at the bottom, thereby achieving the separation of X and Y sperm.

[0023] This invention utilizes the TLR7 and TLR8 receptors specifically expressed on the surface of X sperm, and treats them with their common synthetic ligand, remiquimod (R848). After binding to the receptors, R848 inhibits glycolysis via the GSK3α / β-hexokinase pathway and affects mitochondrial function, thereby specifically reducing the motility of X sperm, causing them to precipitate at the bottom of the container after static incubation. Y sperm, because they do not express or express low levels of the aforementioned receptors, have no significant impact on their motility, thus achieving the physical separation and enrichment of X and Y sperm.

[0024] Compared with the prior art, the beneficial effects of the present invention include:

[0025] The method for enriching Nubian goat X sperm in this invention is significantly less expensive than flow cytometry sorting, requires no expensive equipment, and is easy to implement in ordinary laboratories or farms. The method is simple to operate, with the main steps being incubation and centrifugation washing, and exhibits good reproducibility. The method has minimal overall impact on sperm motility, and the X sperm retains good motility after separation, which is beneficial for subsequent applications such as artificial insemination. The method is highly specific, utilizing the unique receptor-ligand interaction of X sperm to achieve selective inhibition, resulting in a high proportion of X sperm after enrichment. This invention fills a gap in practical sex control technology for Nubian goats, especially Nubian black goat hybrids. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a comparison of the sperm motility of Nubian goats before and after separation, as shown in the example.

[0028] Figure 2 This is a flow cytometry result diagram showing the X / Y ratio of sperm before separation, analyzed using a focused acoustic flow cytometer in this embodiment.

[0029] Figure 3 The image shows the flow cytometry results of the separated sperm X proportion analyzed using a focused acoustic flow cytometer in this embodiment.

[0030] Figure 4 This is a schematic diagram showing the statistical results of the change in the proportion of X sperm after separation in the example.

[0031] Among them, group E (E-1 and E-2 are two replicate groups) consisted of sorted sperm, and group nc (nc-1 and nc-2 are two replicate groups) served as the control group. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] It should be noted that if the embodiments of the present invention involve directional indicators (such as up, down, left, right, front, back, top, bottom, etc.), the directional indicators are only used to explain the relative positional relationship and movement of the components in a certain specific posture (as shown in the figure). If the specific posture changes, the directional indicators will also change accordingly.

[0034] In this application, unless otherwise expressly specified and limited, the terms "installation," "connection," "joining," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components, unless otherwise expressly limited. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

[0035] It should be noted that when a component is described as "fixed to" or "set on" another component, it can be directly on the other component or there may be an intervening component. When a component is described as "connected to" another component, it can be directly connected to the other component or there may be an intervening component.

[0036] Furthermore, if the embodiments of this invention involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. If the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0037] Currently, there is a lack of a simple, low-cost, and stable method for controlling the sex of Nubian goat sperm. To solve the above technical problems, this invention proposes a method for enriching Nubian goat X sperm and a Nubian goat X sperm enrichment kit.

[0038] Example 1: Collection, motility testing, and washing of Nubian goat semen

[0039] Select healthy, sexually active adult Nubian rams and collect semen using electrostimulation. The specific steps are as follows: restrain the ram, lubricate the electrostimulation rod and slowly insert it into the anus about 20cm. Initially set the frequency to 50Hz and the voltage to 1V, manually stimulate (1 time / second) for 5 seconds, then gradually increase the voltage to about 4V. When the ram shows obvious ejaculation response, collect the semen in a preheated collection cup.

[0040] Freshly collected semen was immediately diluted 10-fold with sperm diluent preheated to 37°C (the semen diluent used was Bovine Semen Diluent, Casu BioXcell, France, batch number: 410501). The diluted semen samples were then subjected to sperm motility testing using a computer-assisted sperm analysis system (CASA). Samples with motility greater than 80% were selected for subsequent experiments. Qualified samples were centrifuged at 380×g for 5 minutes, the supernatant was discarded, and the sperm precipitate was resuspended in fresh diluent for later use.

[0041] Example 2: R848 incubation for enrichment of X sperm

[0042] Prepare a goat sperm dilution containing 2 μM / L requimide (R848); take 3 mL of the R848 dilution and thoroughly resuspend and mix it with the sperm precipitate washed and treated in Example 1; place the mixture in a 37°C constant temperature water bath and incubate in the dark for 45 minutes.

[0043] After incubation, the sample will show stratification. Carefully aspirate about 1 mL of liquid (rich in precipitated sperm) from the bottom of the container and transfer it to a new centrifuge tube. Add 3 mL of fresh sperm diluent free of R848 to the tube, centrifuge at 380×g for 5 minutes, and discard the supernatant. Repeat this washing step 3 times to completely remove any residual R848. Resuspend the final precipitate in an appropriate amount of diluent to obtain the enriched X sperm sample.

[0044] Example 3: Verification of Separation Effect

[0045] 3.1 Sperm motility test: Enriched sperm samples obtained in Example 2 and untreated raw semen samples were used for motility testing using the CASA system. The results are as follows: Figure 1 As shown, the proportion of highly motile sperm in the enriched sperm population changed, but the overall motility remained at a high level, meeting the requirements for artificial insemination.

[0046] 3.2 X / Y Sperm Ratio Analysis: Enriched sperm samples and raw semen samples were stained with DAPI staining solution for 10 minutes in the dark. DNA content analysis of the stained sperm was performed using ultrasound focusing flow cytometry, with at least 10,000 sperm analyzed per sample. FlowJo software was used to analyze the flow cytometry data, distinguishing X and Y sperm based on minute differences in DNA content and calculating their ratio.

[0047] The results are as follows Figure 2 , Figure 3 and Figure 4 As shown in the figure. Flow cytometry revealed that after R848 treatment, the signal peak representing X sperm was significantly enhanced in the collected bottom sperm population. Statistical analysis indicated that the proportion of X sperm in the original semen was approximately 50%, while after enrichment using the method of this invention, the proportion of X sperm significantly increased to over 85%, verifying the effectiveness of this method in efficiently enriching X sperm.

[0048] Example 4: Comparative Experiment of Different Concentrations of R848

[0049] Except for comparing the R848 concentrations of 1 μM / L, 2 μM / L (preferred in this invention), and 4 μM / L, the remaining steps were the same as in Examples 2 and 3. The results showed that 2 μM / L R848 maintained a high X sperm enrichment efficiency (>85%) while having the least negative impact on overall sperm motility. At a concentration of 1 μM / L, the enrichment efficiency decreased somewhat, while at 4 μM / L, although the enrichment efficiency was comparable, the decrease in sperm motility was more significant. The comparison revealed that a R848 concentration of 2 μM / L achieved the optimal enrichment effect and the best obtained sperm motility.

[0050] In summary, this invention provides an efficient, economical, and practical method for enriching Nubian goat X sperm, offering a new technical means for sex-controlled reproduction in Nubian goats.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for enriching Nubian goat X sperm, characterized in that: Includes the following steps: (1) Motility testing was performed on Nubian goat semen. Nubian goat semen that passed the motility test was washed to obtain washed semen samples. (2) Add sperm diluent containing R848 to the washed sperm sample and mix. Incubate at 37°C for 45 minutes. (3) After incubation, collect the precipitated sperm at the bottom of the container, wash with sperm dilution solution to remove R848, and obtain enriched Nubian goat X sperm.

2. The method for enriching Nubian goat X sperm according to claim 1, characterized in that: In step (1), the method for detecting sperm motility is as follows: Nubian goat semen is diluted 10 times with semen diluent, and then sperm motility is detected using a sperm quality computer-aided analysis system. The semen diluent is the French Casu BioXcell bovine semen diluent.

3. The method for enriching Nubian goat X sperm according to claim 1, characterized in that: In step (1), the motility test is qualified when the sperm motility is greater than 80%.

4. The method for enriching Nubian goat X sperm according to claim 1, characterized in that: In step (1), the washing conditions are: after washing at 380×g for 5min, the supernatant is removed to obtain the washed sperm sample.

5. The method for enriching Nubian goat X sperm according to claim 1, characterized in that: The concentration of R848 in the sperm diluent in step (2) is 2 μM / L.

6. The method for enriching Nubian goat X sperm according to claim 1, characterized in that: The sperm diluent mentioned in step (2) is the French Casu BioXcell bovine semen diluent.

7. The method for enriching Nubian goat X sperm according to claim 1, characterized in that: In step (3), the collection of precipitated sperm at the bottom of the container specifically refers to collecting the precipitated sperm in the bottom 1 / 3 of the container after incubation.

8. The method for enriching Nubian goat X sperm according to claim 1, characterized in that: In step (3), the washing conditions are: centrifugation at 380×g for 5 minutes, and repeated washing 3 times.

9. A Nubian goat X sperm enrichment kit, characterized in that: The method used in any one of claims 1-8 includes R848 and sperm diluent.

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