Method for screening high-quality fish sperms and application

Abstract

The invention discloses a method for screening high-quality fish sperms and application. The method comprises the following steps: screening a first sperm of a male fish parent according to the relative content of miR-200a; the suspension is used for screening the first sperms to obtain second sperms, and the suspension is a silica gel solution containing 35% by mass of polyvinylpyrrolidone. According to the method, the problem of low utilization degree caused by large sperm quality difference of male fish groups can be solved, the culture cost is reduced, and the screening and culture efficiency is improved.

Description

Technical Field

[0001] The present application relates to the technical field of fish sperm screening, and specifically to a method and application for screening high-quality fish sperm. Background Art

[0002] With the rapid development of intensive aquaculture, new intensive farming models such as runways, captive farming, land-based farming and factory-scale recirculating aquaculture have emerged. The reproductive activities of bony fish are suggested and regulated by environmental factors. The artificial breeding environment cannot meet the natural reproduction process of farmed fish and needs to rely on exogenous oxytocin to complete the ejaculation or ovulation process. Due to the large number of sperm in male fish, the amount of male fish required for artificial breeding is much lower than that of female fish. Sperm quality is closely related to seedling quality. Therefore, it is urgent to develop a method for screening high-quality sperm in fish. Summary of the invention

[0003] The present application provides a method for screening high-quality sperm of fish. The method extracts total RNA from male sperm with different fertility, screens for differential miRNA, and uses a suspension treated with polyvinyl pyrrolidone to screen for sperm with high chromatin condensation, and combines miRNA markers reflecting male reproductive performance and sperm with high chromatin condensation to obtain high-quality sperm. The method can overcome the problem of low utilization of sperm due to large differences in sperm quality in male fish populations, reduce breeding costs, and improve screening and breeding efficiency.

[0004] Based on this, the embodiments of the present application disclose at least the following technical solutions:

[0005] The embodiment discloses a method for screening high-quality sperm of fish, which includes: screening first sperm of male fish parents according to the relative content of miR-200a; and screening the first sperm using a suspension to obtain second sperm, wherein the suspension is a silica gel solution containing 35% by mass of polyvinyl pyrrolidone.

[0006] Embodiments The relative content of miR-200a can be used to initially obtain high-quality sperm, and further through the suspension, sperm with high chromatin concentration can be obtained, further improving the quality of the screened sperm. The obtained sperm can be used for fish breeding and reproduction to overcome the problem of low utilization caused by large differences in sperm quality in male fish groups, reduce breeding costs, and improve screening and breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 The expression levels of miR-200a and miR-429b and the corresponding fertilization rate and hatching rate provided in the examples.

[0008] Figure 2 The sperm chromatin condensation degree evaluation results provided in the examples. Figure 2A is a real picture of sperm centrifuge tube. Figure 2 B is the flow cytometric image of sperm in the upper layer after CMA3 staining. Figure 2 C is the white light image of the upper sperm. Figure 2 D Fluorescence image of sperm in the upper layer after CMA3 staining. Figure 2 E is the flow cytometric image of the bottom sperm after CMA3 staining. Figure 2 F is the white light image of the bottom layer of sperm. Figure 2 G Fluorescence image of sperm in the bottom layer after CMA3 staining. Figure 2 H Statistics of the CMA3 staining ratio of sperm in the bottom and upper layers. Figure 2 J Statistics of mean fluorescence intensity of CMA3 staining of sperm in the bottom and upper layers.

[0009] Figure 3 The fertilization rate (A), hatching rate (B), seedling weight 30 days after hatching (C), seedling weight 60 days after hatching (D), and seedling survival rate (E) of the second sperm and unscreened sperm provided in the examples. DETAILED DESCRIPTION

[0010] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0011] In order to overcome the disadvantages that sperm quality varies greatly in male fish populations obtained by the prior art, resulting in the inability to fully use high-quality sperm, leading to uneven quality of fish seedlings and increased breeding costs, the embodiment discloses a method for screening high-quality sperm of fish. The method comprises: screening first sperm of male fish parents according to the relative content of miR-200a; using a suspension to screen the first sperm to obtain a second sperm, wherein the suspension is a silica gel solution containing 35% by mass of polyvinyl pyrrolidone.

[0012] Embodiments The relative content of miR-200a can be used to initially obtain high-quality sperm, and further through the suspension, sperm with high chromatin concentration can be obtained, further improving the quality of the screened sperm. The obtained sperm can be used for fish breeding and reproduction to overcome the problem of low utilization caused by large differences in sperm quality in male fish groups, reduce breeding costs, and improve screening and breeding efficiency.

[0013] In some embodiments, the relative expression level of miR-200a is the relative expression level detected by qRT-PCR method.

[0014] In some embodiments, the step of selecting the first sperm comprises:

[0015] The first high fertility sperm and the first low fertility sperm of the male parent are selected according to the motile sperm ratio, average path velocity, linear motion velocity, curvilinear motion velocity, fertilization rate and hatching rate;

[0016] The total RNA of the first high-fertility sperm and the first low-fertility sperm were extracted respectively, an RNA library was established, the RNA library was sequenced, and the miRNA therein was identified;

[0017] If the same miRNA detected in the first high fertility sperm and the first low fertility sperm simultaneously satisfies |log2(FoldChange)|>1 and padj<0.05, it is simply a differential miRNA, and the differential miRNA is miR-200a;

[0018] Sperms with a breeding level lower than 40% and a relative expression level of miR-200a lower than 1.35 were selected from the first high fertility sperm as the first sperm.

[0019] In some embodiments, the preparation step of the suspension comprises:

[0020] Providing 5-50 μm silica gel particles dispersed in water to obtain a primary mixed solution;

[0021] The polyvinyl pyrrolidone K-30 (average molecular weight of about 40,000) and the silica gel particles are added in a mass ratio of 1:10-1:2, and the suspension is obtained by ultrasonic treatment.

[0022] In a specific embodiment, the preparation steps of the suspension include:

[0023] (1) Add 5-50 μm silica particles to the dispersion medium to form an initial suspension system. For example, slowly add a certain mass (e.g., 10 g) of silica particles to 100 mL of deionized water while stirring. The stirring speed can be controlled at about 200-500 rpm to prevent particle agglomeration.

[0024] (2) Add PVP (K-30, average molecular weight of about 40,000) in a mass ratio of 1:10 to 1:2 and dissolve it in the dispersion medium. Slowly add the dissolved PVP solution to the suspension and continue stirring for several hours (such as 3 to 6 hours) to allow the PVP to be fully adsorbed on the surface of the silica gel particles. In order to make the adsorption more uniform, ultrasonic treatment can be used. Place the suspension in an ultrasonic cleaner, set the ultrasonic power between 100 and 300 W, and the ultrasonic time for about 10 to 30 minutes. This can help PVP to be better dispersed and adsorbed on the surface of the silica gel particles.

[0025] The stability of the suspension treated with PVP is significantly improved. This is because PVP forms a protective film on the surface of the silica particles, preventing the particles from aggregating with each other. For example, during long-term storage, untreated suspensions may precipitate within a few hours, while suspensions treated with PVP can remain stable for days or even weeks.

[0026] In some embodiments, the step of selecting the second sperm comprises:

[0027] Adding a 70% PVP-treated suspension to the bottom layer of the sperm centrifuge tube, adding a 35% PVP-treated suspension to the middle layer, and adding a first sperm to the upper layer, wherein the first sperm contains a sperm preservation solution; and

[0028] The sperm centrifuge tubes to which each solution had been added were centrifuged to collect the bottom liquid.

[0029] The method for screening high-quality fish sperm provided by the present application will be described in more detail below, but it does not constitute a limitation to the implementation methods of the present application.

[0030] Example 1

[0031] In order to screen miRNA markers that can reflect the reproductive performance of yellow catfish and establish a breeding method for male yellow catfish with high reproductive performance, the following studies were carried out:

[0032] 1. Screening the first high fertility sperm and the first low fertility sperm

[0033] Before the male yellow catfish reproduces, some sperm are collected for miRNA sequencing and sperm motility analysis, and the remaining sperm is used for large-scale seed production of yellow catfish. Combining the motile sperm ratio and sperm motility parameters of male yellow catfish before artificial reproduction, as well as the fertilization rate and hatching rate after artificial reproduction, the first high fertility sperm and the first low fertility sperm are screened from the sperm of male yellow catfish.

[0034] Table 1 shows the motile sperm ratio, average path velocity, linear motion velocity, curvilinear motion velocity, fertilization rate and hatching rate of the first high fertility sperm and the first low fertility sperm. Different superscript letters in the same row indicate significant differences (P<0.05).

[0035] Table 1 The first high fertility sperm and the first low fertility sperm

[0036] Group The first high fertility sperm First low fertility sperm Motile sperm ratio (%) 81.72±8.16a 70.47±11.36b Average path velocity (μm / s) 158.79±12.06a 124.23±15.8b Linear motion speed (μm / s) 160.9±19.15a 130.49±17.23b Curve movement speed (μm / s) 168.99±15.61a 145.67±17.03b Fertilization rate (%) 83.56±4.41a 71.73±5.2b Hatching rate (%) 91.46±4.33a 81.28±3.25b

[0037] 2. Establishment of miRNA markers related to reproductive performance of male yellow catfish

[0038] Total RNA was extracted from the sperm of the first high-fertility sperm and the first low-fertility sperm male fish, respectively, and each group contained 3 biological replicates after sample detection, library construction, sequencing, miRNA identification and bioinformatics analysis. In relative quantification, if the same miRNA between the two groups simultaneously satisfied |log2(FoldChange)|>1 and padj<0.05, it was identified as a differential miRNA.

[0039] After the above steps, the embodiment screened out that miR-200a and miR-429b, members of the miR-200 family related to sperm motility, were significantly low expressed in the sperm of male fish in the first high fertility sperm group. Further, 20 male yellow catfish were randomly selected to extract sperm RNA for qRT-PCR, and the remaining sperm were artificially inseminated to calculate the fertilization rate and hatching rate. The lowest expression value of miR-200a was taken as 1, and the expression levels of other male fish were used to calculate the expression multiples. The individual order was sorted from high to low according to the fertilization rate. The scatter plot of miR-200a and miR-429b with fertilization rate and hatching rate is shown in Figure 2. Figure 1 As shown, the Pearson correlation coefficients of miR-200a with fertilization rate and hatching rate were -0.76 and -0.75, respectively, while the Pearson correlation coefficients of miR-429b with fertilization rate and hatching rate were -0.67 and -0.59, respectively, showing strong correlation.

[0040] Table 2 Correlation between miRNA and fertilization rate and hatching rate

[0041] Pearson correlation coefficient P-value miR-200a expression level-fertilization rate -0.76 <0.001 miR-200a expression level-hatching rate -0.75 <0.001 miR-429b expression level-fertilization rate -0.67 0.0014 miR-429b expression level-hatching rate -0.59 0.0062

[0042] In order to establish a specific operation method for breeding male fish with high reproductive performance by sperm miRNA markers, we sorted 20 male yellow catfish according to the relative expression content of sperm miR-200a, and compared and analyzed the reproductive performance of yellow catfish according to different breeding levels to provide a reference standard for breeding male yellow catfish with high reproductive performance. The breeding level is the percentage of the number of selected male fish to the total number of male fish. The results are shown in Table 3. As the breeding level decreases, the fertilization rate and hatching rate of male yellow catfish with low expression of miR-200a gradually increase. When the breeding level is 20% and 40%, the expression content of miR-200a in male yellow catfish with low expression is 1.14-1.35, and the fertilization rate and hatching rate are both high. Due to the small amount of male fish used in the production process, the embodiment uses the first high fertility sperm with a breeding level lower than 40% and a relative expression of miR-200a lower than 1.35 as the first sperm, which has high fertility and high production effect.

[0043] Table 3 Effect of breeding male yellow catfish based on relative expression of miR-200a at different selection levels

[0044]

[0045] Example 2

[0046] Since the male fish sperm of the first sperm still has heterogeneity differences, the embodiment also carries out a step of selecting the second sperm from the first sperm, and also obtains high-quality sperm with a high degree of chromatin condensation.

[0047] Second sperm selection steps:

[0048] Prepare solution A: 8g NaCl, 0.4g KCl, 0.14g CaCl2, 0.228g MgSO47H2O, and add 50mL sterile water.

[0049] Prepare solution B: 0.35g NaHCO3, 0.173g Na2HCO4H2O, 0.06g KH2PO4, 1g C6H 12 O6, add 50mL of sterile water.

[0050] Liquid A and liquid B were mixed in a volume ratio of 1:1 as 10× sperm preservation solution, which was diluted 10 times with sterile water to obtain 1× sperm preservation solution. The first high fertility sperm screened above (the sperm of male yellow catfish screened according to the relative expression content of miR-200a and obtained 3 first high fertility sperm according to the above method) was diluted with 1× sperm preservation solution to a concentration of 2×10 7 sperm / mL, and the first sperm was obtained.

[0051] Preparation of sperm screening centrifuge tubes: Prepare a 70% polyvinyl pyrrolidone suspension by mixing polyvinyl pyrrolidone-treated suspension, 10× sperm preservation solution, and sterile water in a volume ratio of 7:1:2. Mix the 70% polyvinyl pyrrolidone suspension (containing 70% PVP by mass) with 1× sperm preservation solution in a volume ratio of 1:1 to obtain a 35% polyvinyl pyrrolidone suspension (containing 35% PVP by mass). Take a 2mL centrifuge tube, add 600μL of the 70% polyvinyl pyrrolidone-treated suspension to the bottom layer, add 600μL of the 35% polyvinyl pyrrolidone-treated suspension to the middle layer, and add 300μL of the first sperm to the upper layer. Centrifuge at room temperature for 15 minutes at 1000g, and collect sperm from the upper and bottom layers ( Figure 2 A). Add 1 mL of 1× sperm preservation solution to wash the sperm, centrifuge at 1000 g for 5 min at room temperature, repeat 3 times, and then add 1× sperm preservation solution to dilute to a concentration of 2×10 7 sperm / mL and proceed to the next step of staining.

[0052] In addition, the embodiment also evaluates the degree of sperm chromatin condensation by CMA3 staining. CMA3 dye evaluates the degree of chromatin condensation by competing with the minor groove of sperm nuclear DNA. When the degree of chromatin condensation is high, CMA3 cannot bind to sperm nuclear DNA, but easily binds to nuclear DNA.

[0053] Take 400 μL of the bottom layer sperm and upper layer sperm (2×10 7 Sperm / mL), then add an equal volume of CMA3 dye (0.25mg / mL), incubate at room temperature for 50min, collect and resuspend in 400μL sperm preservation solution. Use flow cytometry to check the CMA3 staining ratio: at a flow rate of 10μL / min, use a 488nm wavelength laser to excite the sample, and collect the emitted fluorescence through the FITC (525 / 40) channel. Use a confocal microscope to observe the sperm staining, use the FITC channel and bright field illumination for observation, and after collecting the photo results, use Image J software to count the average fluorescence intensity of the sperm. The sorted bottom sperm and upper sperm were artificially inseminated with the same female yellow catfish to calculate the fertilization rate and hatching rate.

[0054] The results of flow cytometry sorting and confocal microscopy are shown in Figure 2 BG. The CMA3 staining ratio of sperm in the bottom layer was 18.73±5.82%, which was significantly lower than that of sperm in the upper layer (63.14±5.51%) (P<0.01). Figure 2 H). The results showed that the average CMA3 fluorescence intensity of sperm in the bottom layer was 6.70±4.18, which was significantly lower than the fluorescence intensity of sperm in the upper layer (29.01±6.38) (P<0.01) Figure 2 I). The fertilization rate and hatching rate of bottom layer sperm are significantly higher than those of top layer sperm ( Figure 2 J and K).

[0055] Example 3

[0056] 10 male yellow catfish were randomly selected and screened by the methods of Examples 1 and 2 to obtain the second sperm, and sperm was obtained by randomly selecting 4 unscreened male yellow catfish. 4 female fish were randomly selected for artificial induced spawning, eggs were collected and mixed evenly, and artificial insemination was performed with the second sperm and the unscreened sperm, respectively, and the fertilization rate and hatching rate were counted. After hatching, 300 fish were randomly selected for a breeding comparison experiment to record the number of deaths per day. At 30d and 60d, the weight of yellow catfish fry in the second sperm and unscreened sperm groups was counted, and a survival curve was drawn.

[0057] The test results are as follows Figure 3As shown, the fertilization rate and hatching rate of the second sperm group were 89.74±2.19% and 93.23±3.32%, respectively, which were significantly higher than those of the unselected sperm group (fertilization rate: 73.64±7.23%, hatching rate: 80.19±5.48%) ( Figure 3 A and B). At 30 and 60 days of culture, the weight of the fry in the second sperm group was higher than that in the unselected sperm group ( Figure 3 C and D). At 60 days, the survival rate of the second sperm group was 79.11%, while the survival rate of the fry in the unselected sperm group was 68.89% ( Figure 3 E).

[0058] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. Methods for screening high-quality sperm of fish, including: The first sperm of male fish parents were screened according to the relative content of miR-200a; The second sperm is obtained by screening the first sperm using a suspension, wherein the suspension is a silica gel solution containing 35% by mass of polyvinyl pyrrolidone.

2. According to the method of claim 1, the relative expression level of miR-200a is detected by qRT-PCR method.

3. The method according to claim 1, wherein the step of selecting the first sperm comprises: The first high fertility sperm and the first low fertility sperm of the male parent are selected according to the motile sperm ratio, average path velocity, linear motion velocity, curved motion velocity, fertilization rate and hatching rate; The total RNA of the first high-fertility sperm and the first low-fertility sperm were extracted respectively, an RNA library was established, the RNA library was sequenced, and the miRNA therein was identified; If the same miRNA detected in the first high fertility sperm and the first low fertility sperm simultaneously satisfies |log2(FoldChange)|>1 and padj<0.05, it is simply a differential miRNA, and the differential miRNA is miR-200a; Sperms with a breeding level lower than 40% and a relative expression level of miR-200a lower than 1.35 were selected from the first high fertility sperm as the first sperm.

4. The method according to claim 1, wherein the first sperm preparation step comprises: Prepare solution A containing 160g / L NaCl, 8g / L KCl, 2.8g / L CaCl2 and 4.56g / L MgSO47H2O; Prepared to contain 7g / LNaHCO3, 3.46g / LNa2HCO4 H2O, 1.2g / L KH2PO4 and 2g / L C6H 12 O6 fluid B; The first high-fertility sperm is prepared with an equal volume of a mixture of the A solution and the B solution or a dilution thereof to obtain a first sperm.

5. The method according to claim 1, wherein the step of selecting the second sperm comprises: Adding a 70% PVP-treated suspension to the bottom layer of the sperm centrifuge tube, adding a 35% PVP-treated suspension to the middle layer, and adding a first sperm to the upper layer, wherein the first sperm contains a sperm preservation solution; and The sperm centrifuge tubes to which each solution has been added are centrifuged to collect sperm at the bottom layer.

6. The method according to claim 1, wherein the step of preparing the suspension comprises: Providing 5-50 μm silica gel particles dispersed in water to obtain a primary mixed solution; Polyvinyl pyrrolidone K-30 (average molecular weight of about 40,000) was mixed with silica gel particles and subjected to ultrasonic treatment to obtain the suspension.

7. The method according to claim 5, wherein the step of selecting the second sperm further comprises: The bottom layer of sperm was diluted with sperm preservation solution to contain 2×10 7 sperm; An equal volume of CMA3 dye solution (0.25 mg / mL) was added, incubated at room temperature for 50 min, and the cells were collected and resuspended in 400 μL sperm preservation solution; CMA3 staining results were examined using flow cytometry; The second sperm was screened based on the CMA3 staining results.

8. The method according to claim 7, wherein the second sperm is obtained by screening according to the CMA3 staining ratio, comprising: The sperm with the lowest CMA3 staining ratio and fluorescence intensity is selected as the second sperm.

9. Use of the method according to claim 1 in screening high-quality sperm of fish.

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