CN111500530A

CN111500530A - Universal animal sperm sorting method and X sperm quality control method

Info

Publication number: CN111500530A
Application number: CN202010363330.2A
Authority: CN
Inventors: 岳昌武; 吕玉红; 崔相宜; 周进科; 孙心悦; 王雪梅; 郭瑞瑞
Original assignee: Yanan University
Current assignee: Yanan University
Priority date: 2020-04-30
Filing date: 2020-04-30
Publication date: 2020-08-07

Abstract

The invention discloses a general animal sperm sorting method which is characterized by comprising the following steps of collecting semen of an animal to be sorted, mixing the semen with sperm diluent, uniformly mixing the semen with the sperm diluent, putting the mixture into a storage bottle, putting the storage bottle into a heat preservation box with the heat preservation temperature of 15-20 ℃, adding sorting liquid into the storage bottle, standing for 1h, layering, pumping out an upper solution by using a water pumping device, wherein the upper solution contains Y sperm, and the rest lower solution contains X sperm, and adding an extraction liquid into the lower solution to extract the X sperm from the sorting liquid, and subpackaging the extracted X sperm in 100m L subpackaging bottles to obtain a solution containing the X sperm.

Description

Universal animal sperm sorting method and X sperm quality control method

Technical Field

The invention belongs to the technical field of biology, and relates to a general animal sperm sorting method and an X sperm quality control method.

Background

Sex control (sex control) refers to a technique in which female reproductive animals are caused to reproduce offspring of a desired sex as desired by human intervention. Sex control techniques are of great significance in animal husbandry. Firstly, the livestock sex control technology is a bioengineering technology which can obviously improve the breeding rate of livestock, control the sex ratio of offspring can increase the seed selection intensity, accelerate the breeding process, and has very important significance for breeding, production and prevention and treatment of genetic diseases of livestock. Secondly, the sex control technology of livestock is also a significant breeding technology in the production of animal husbandry and breeding industry. In animal husbandry, many important economic conditions are associated with sex, for example: meat, eggs, milk, hair, antler and the like can exert the maximum economic benefit of production traits (such as lactation) limited by sex and production traits (such as production speed, meat quality and the like) influenced by sex by controlling the sex ratio of offspring, and especially play an important role in promoting the dairy cow industry. Meanwhile, the strength in fine variety selection can be enhanced, the breeding efficiency can be improved, and the maximum genetic progress can be obtained. In addition, the technology can also avoid twin sterility caused by heterosexual embryo transplantation by controlling the sex of offspring; at the same time, the medicine plays a very important role in preventing the accompanying genetic diseases. As an important aspect of the biology of reproduction, individual sex control is of great importance both theoretically and in animal husbandry.

Over the last 10 years, with the rapid development of molecular biology, cytogenetics, immunohistology and other disciplines, and the advent of modern high-precision instruments, research on sex control has grown significantly. Currently, sex control technology is mainly performed in 2 stages of sex control before fertilization and sex control after fertilization. The most desirable approach for pre-fertilization sex control is to take X and Y sperm separation and then purposefully perform fertilization. The sex control after fertilization is realized by the sex identification of the embryo before implantation. The sperm separation method before fertilization mainly separates X sperm and Y sperm according to their physical and chemical properties, such as density, shape, size, vitality, surface charge and immunogenicity. Hitherto, various sperm separation techniques such as an H-Y antigen immunization method, a flow cytometric method, a sedimentation method, a centrifugal sedimentation method, a density gradient centrifugation method, a gel filtration method, an electrophoresis method, an albumin precipitation method, a staining method, and a method of adjusting pH value of a fertilization environment and a semen deposition time have been used to separate sperm of livestock.

The H-Y antigen immunization method is based on X, Y sperm specific surface antigen, sperm sex identification can be carried out by using an immunological means, recent research shows that the level of the H-Y antigen on the surface of a Y sperm cell membrane is only slightly higher than that of an X sperm, and the method has the defects of relatively few surviving sperms, low vitality, unsuitability for large-scale sperm separation and the like, so the method is difficult to apply and popularize in production. Relevant researches show that the separation effect of the density gradient centrifugation technology is not very ideal and is not commonly used in production practice; the accuracy and repeatability of the separation result of the electrophoresis method are not ideal, and the sperms are not easy to survive in the electrophoresis solution, so the method is not used in production; the technical basis of the albumin column separation method is that the migration speed of Y sperms in an albumin solution is higher than that of X sperms, but the method is not very common because many researchers separate X, Y sperms of human beings or animals by the method and have large disputes on the effect.

At present, the flow cytometry separation method is a method which is relatively efficient, reliable and wide in application range in animal production, and other methods almost have no results. However, there are problems that cannot be avoided by the flow cytometry separation method, such as teratogenicity of nucleic acid dye to gene, damage to sperm by laser when sperm is sorted by the flow cytometry separation method, and expensive equipment.

Disclosure of Invention

The invention aims to provide a universal animal sperm sorting method which can be used for quickly sorting X sperm and Y sperm.

Also relates to an X sperm quality control method.

The invention adopts the technical scheme that a general animal sperm sorting method is specifically carried out according to the following steps:

step 1, collecting semen of an animal to be sorted, mixing the semen with a sperm diluent, uniformly mixing, putting the mixture into a storage bottle, and putting the storage bottle into a heat preservation box with the heat preservation temperature of 15-20 ℃;

step 2, adding the sorting solution into a storage bottle, standing for 1h, layering, pumping out the upper-layer solution by using a water pumping device, wherein the upper-layer solution contains Y sperms, and the rest lower-layer solution contains X sperms;

and 3, adding the extract into the lower layer solution, extracting the X sperms from the sorting solution, subpackaging in subpackaging bottles and obtaining the solution containing the X sperms.

In the step 1, a sperm diluent is prepared from 12% sucrose solution, glycine, sodium citrate, Tris, yolk, glycerol and streptomycin, 0.5g of glycine, 0.5g of sodium citrate and 0.2g of Tris are added into 75ml of 12% sucrose solution and then filtered, the filtrate is boiled for 15 minutes, 20ml of yolk is added after standing to room temperature, 1% glycerol with the final concentration of 1m L and 50-100 mg of streptomycin are added, the mixture is fully stirred uniformly and then the volume is determined, and the final concentration is not lower than 4000 ten thousand/m L.

In the step 2, the sorting solution comprises DMSO and a Toll-like receptor inhibitor, the concentration of the Toll-like receptor inhibitor is 0.3 mu mol, the volume ratio of the DMSO to the Toll-like receptor inhibitor is 1: 1000, and the concentration of sperms after mixing with semen is not lower than 2000 ten thousand/m L.

In the step 3, the volume ratio of the extraction liquid to the lower layer solution is 1:1, the extraction liquid comprises peanut oil, corn oil and olive oil, and the volume ratio of the peanut oil, the corn oil and the olive oil is 3:4: 4.

The invention adopts another technical scheme that the quality control method of the X sperm is specifically carried out according to the following steps:

step 1, extracting a solution containing X sperms obtained by sorting 1m L, grinding the solution in a mortar, sucking a tissue sample by a centrifugal tube until the tissue sample is in a homogenate state, and storing the tissue sample to obtain a crude extraction solution;

step 2, putting the crude extraction liquid obtained in the step 1 of 1 mu L into an enzyme-free PCR tube, adding the extraction-free nucleic acid amplification reaction liquid to obtain a mixed liquid, and putting the mixed liquid into a water bath for 2 min;

step 3, putting the mixed solution obtained in the step 2 into a water bath kettle at 98 ℃ for 30s, then putting the mixed solution into a water bath kettle at 60 ℃ for 30s, putting the mixed solution into a water bath kettle at 68 ℃ for 60s again, and repeating the water bath process for 3-5 times;

and 4, adding L AMP method identification reaction liquid into the mixed liquid processed in the step 3 to estimate the number of X sperms, or adding a color developing agent into the mixed liquid to estimate the DNA content of the X sperms.

In step 2, L AMP primer group is designed, the primer group comprises an outer primer F3 and an outer primer B3, 2 × MightAmp Buffer Ver.3 of 6.25 mu L, an outer primer F3 of 0.376 mu L, an outer primer B3 of 0.374 mu L and sterile water of MightAmp DNA Polymerase Ver.3 and 3.75 mu L of 0.25 mu L and at the concentration of 1.25U/mu L are uniformly mixed to obtain an extraction-free nucleic acid amplification reaction solution, the gene sequences of the outer primer F3 are shown as SEQ ID NO.2 and SEQ ID NO.6, and the gene sequences of the outer primer B3 are shown as SEQ ID NO.3 and SEQ ID NO. 7;

mg in 2 × MightAmp Buffer Ver.3²⁺The concentration was 4mM (2 ×) and the dNTP concentration was 600. mu.M (2 ×).

And 4, putting the mixed solution processed in the step 3 into an L AMP amplification instrument with the temperature of 65 ℃ for water bath for 12min, adding L AMP to identify the reaction solution, and observing the color depth and the color depth of the reaction solution, wherein the larger the number of X sperms is.

In the step 4, the L AMP method identification reaction liquid is any one of L AMP method common identification reaction liquid, L AMP method calcein fluorescence identification reaction liquid, L AMP method hydroxynaphthol blue identification reaction liquid, L AMP method PicoGreen fluorescence identification reaction liquid and L AMP method SYBRGreen I fluorescence identification reaction liquid;

l AMP primer group also includes inner primer FIP and inner primer BIP, the gene sequence of the inner primer FIP is shown in SEQ ID NO.4 and SEQ ID NO.8, the gene sequence of the inner primer BIP is SEQ ID NO.5 and SEQ ID NO. 9;

the L AMP method common identification reaction liquid comprises sterile water with the volume of 4 mu L L02 solution with the volume of 2 mu L1 and the final concentration of 5mM, 10 × Bsm Buffer with the volume of 2.5 mu L mix with the volume of 1 mu L and the concentration of 10mM each, inner primer FIP with the volume of 1 mu L, inner primer BIP with the volume of 1 mu L, Bsm DNA Po L ymease with the volume of 1 mu L and the concentration of 8U/mu L;

the reaction solution for identifying calcein by L AMP method comprises calcein with volume of 2.5 μ L and final concentration of 0.25mM, MnC L₂The volume of the solution was 2 μ L, concentration 5mM, 10 × Bsm Buffer 2.5 μ L mix, 1 μ L, concentration 10mM each, volume of the inner primer FIP 1 μ L, volume of the inner primer BIP 1 μ L, volume of Bsm DNAPO L enzyme 1 μ L, concentration 8U/μ L, volume of sterile water 1.5 μ L;

the identification reaction liquid of the hydroxynaphthol blue by the L AMP method comprises the hydroxynaphthol blue with the volume of 2.5 mu L and MnC L₂The volume of the solution was 2 μ L, final concentration 5mM, 10 × Bsm Buffer 2.5 μ L mix 1 μ L, 10mM each, inner primer FIP 1 μ L, inner primer BIP 1 μ L, Bsm DNA Po L enzyme 1 μ L, concentration 8U/μ L, sterile water 1.5 μ L;

the L AMP method PicoGreen fluorescent identification reaction solution comprises the volume of PicoGreen of 2.5 mu L L₂The volume of the solution was 2 μ L, final concentration 5mM, 10 × Bsm Buffer 2.5 μ L mix 1 μ L, 10mM each, inner primer FIP 1 μ L, inner primer BIP 1 μ L, Bsm DNA Po L enzyme 1 μ L, concentration 8U/μ L, sterile water 1.5 μ L;

the SYBR Green I fluorescent identification reaction solution prepared by the L AMP method comprises SYBR Green I with the volume of 2.5 mu L L₂The volume of the solution was 2. mu. L, final concentration 5mM, 10 × Bsm Buffer 2.5. mu. L mix 1. mu. L, concentration 10mM each, inner primer FIP 1. mu. L, inner primer BIP 1. mu. L, Bsm DNAPO L ymeThe volume of ras was 1 μ L, the concentration was 8U/μ L, and the volume of sterile water was 1.54 μ L.

And 4, putting the mixed solution treated in the step 3 into a 65 ℃ water bath for 12min, adding a color developing agent, uniformly mixing, adding a diphenylamine reagent and distilled water, putting the mixture into a 60 ℃ water bath for 45min, taking out, cooling to room temperature, putting the mixture into a colorimeter, putting the colorimeter into a sample, measuring absorbance at a wavelength of 595nm, comparing the absorbance measured by the sample with the absorbance of a standard curve prepared according to a gradient test, and estimating the DNA content of the X sperms.

In the step 4, the volume ratio of the mixed liquid to the color developing agent is 1:2, the volume ratio of the mixed liquid to the distilled water to the diphenylamine reagent is 1:1:4, the color developing agent comprises a color developing agent A liquid and a color developing agent B liquid, and the volume ratio of the color developing agent A liquid to the color developing agent B liquid is 200: 1;

1.50g of pure diphenylamine is dissolved in 100m L of glacial acetic acid, then 1.5m L of concentrated sulfuric acid is added, and the mixture is stored in a brown bottle to obtain a developer A solution, and 1.6m L of acetaldehyde is dissolved in 100m L of distilled water to obtain a developer B solution.

The invention has the advantages that a series of operations such as sperm collection, sperm dilution, sperm sorting, sperm extraction, sperm quality control and the like can be effectively completed, 199bp and 239bp are respectively obtained by the conservative fragment amplification of JQ664686.1 gene parts by two groups of designed L AMP primer sets, and the animal sperm sorting method is used for controlling the quality of X sperm.

Drawings

FIG. 1 is a standard curve diagram of the X sperm quality control method of the present invention.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

A general animal sperm sorting method is specifically carried out according to the following steps:

and 3, adding the extract into the lower layer solution to extract X sperms from the sorting solution, and subpackaging in 100m L subpackaging bottles to obtain the solution containing the X sperms.

A quality control method of X sperms is specifically carried out according to the following steps:

step 2, putting the crude extraction liquid obtained in the step 1 of 1 mu L into an enzyme-free PCR tube, adding the extraction-free nucleic acid amplification reaction liquid to obtain a mixed liquid, and putting the mixed liquid into a water bath kettle at 98 ℃ for water bath for 2 min;

the SYBR Green I fluorescent identification reaction solution prepared by the L AMP method comprises SYBR Green I with the volume of 2.5 mu L L₂The volume of the solution was 2. mu. L, final concentration 5mM, 10 × Bsm Buffer 2.5. mu. L mix 1. mu. L, concentration 10mM each, inner primer FIP 1. mu. L, inner primer BIP 1. mu. L, Bsm DNAPO L enzymese volume 1U L, concentration 8U/U L, sterile water volume 1.54U L.

In the step 4, the volume ratio of the mixed liquid to the color developing agent is 1:2, the volume ratio of the mixed liquid to the distilled water to the diphenylamine reagent is 1:1:4, the color developing agent comprises a color developing agent A liquid and a color developing agent B liquid, and the volume ratio of the color developing agent A liquid to the color developing agent B liquid is 200:1

Standard curves were prepared according to the gradient test:

taking 6 clean test tubes with the numbers of 1-6, respectively adding DNA standard solution, distilled water and diphenylamine reagents with different contents according to the table 1, uniformly mixing, keeping the temperature in a water bath at 60 ℃ for 45min, cooling to room temperature, placing the standard solution in a colorimeter, measuring the absorbance at the wavelength of 595nm for 3 times, taking the average value, and taking the concentration (ug/ml) of the DNA standard solution as the abscissa and the absorbance as the ordinate to obtain a standard curve chart shown in the figure 1.

TABLE 1 dilution gradiometer for standard solutions

	0	1	2	3	4	5
							DNA Standard solution/m L	0	0.2	0.4	0.6	0.8	1.0
Distilled water/m L	1.0	0.8	0.6	0.4	0.2	0
							Diphenylamine reagent/m L	2.0	2.0	2.0	2.0	2.0	2.0

The sample volume was kept consistent with the standard solution volume, the concentration of the DNA standard solution was 200ug/m L, and the X sperm DNA content was estimated by comparing the absorbance measured for the sample with the absorbance of the standard curve.

Designing a primer:

the gene of the X sperm specific protein common to mammals such as pigs, cows and sheep is searched in an NCBI database, and the JQ664686.1 gene which has high conservation and specificity and can synthesize L AMP primers is obtained by conservative comparison screening, wherein the nucleotide sequence of the JQ664686.1 gene is shown in a sequence table SEQ ID NO. 1.

Utilizing primer design software PrimeExploer V4 to design L AMP primers of JQ664686.1 gene, and screening out two groups of primer groups with high stability, wherein each L AMP primer group comprises an outer primer F3, an outer primer B3, an inner primer FIP and an inner primer BIP, the gene sequence of the outer primer F3 of the first group L AMP primer group is shown as SEQ ID No.2, the gene sequence of the outer primer B3 is shown as SEQ ID No.3, the gene sequence of the inner primer FIP is shown as SEQ ID No.4, and the gene sequence of the inner primer BIP is shown as SEQ ID No. 5;

the gene sequence of an outer primer F3 of a second group L AMP primer group is shown as SEQ ID NO.6, the gene sequence of an outer primer B3 is shown as SEQ ID NO.7, the gene sequence of an inner primer FIP is shown as SEQ ID NO.8, and the gene sequence of an inner primer BIP is shown as SEQ ID NO. 9;

and carrying out hands-free amplification on the crude extract of the sample by using an outer primer F3 and an outer primer B3 to obtain template DNA, then carrying out constant temperature amplification by using an inner primer FIP and an inner primer BIP, and amplifying JQ664686.1 genes by using two groups of primer groups to respectively obtain conserved gene fragments of 199bp and 239bp for controlling the quality of X sperms.

A sample of crude extract 1m L was taken and put into a sperm mass analyzer together with a standard sample, and the number of X sperm was estimated from the turbidity.

The standard substance comprises a positive control substance and a negative control substance, the positive control substance is a nucleotide sequence obtained by carrying out ordinary PCR on plasmid carrying a detection sequence, the negative control substance is sterile water, the positive control substance obtains X sperms through template ordinary PCR, the negative control substance obtains non-X sperms, the negative control substance is firstly put into a sperm quality analyzer for calibration, then the crude extract 1m L of the sample and the positive control substance in the standard substance are together put into the sperm quality analyzer, the number of the X sperms is estimated according to the value obtained in the sperm quality analyzer, and the result is compared with the negative control substance.

Example 1

step 1, collecting semen of an animal to be sorted, mixing the semen with a sperm diluent, uniformly mixing the semen and the sperm diluent, putting the mixture into a storage bottle, and putting the storage bottle into an incubator with the heat preservation temperature of 20 ℃;

In the step 1, the sperm diluent is prepared from 12% sucrose solution, glycine, sodium citrate, Tris, yolk, glycerol and streptomycin, 0.5g of glycine, 0.5g of sodium citrate and 0.2g of Tris are added into 75ml of 12% sucrose solution and then filtered, the filtrate is boiled for 15 minutes, 20ml of yolk is added after the filtrate is stood to room temperature, 1% glycerol with the final concentration of 1m L and 50mg of streptomycin are added, the volume is determined after the mixture is fully and uniformly stirred, and the final concentration is not lower than 4000 ten thousand/m L.

step 3, putting the mixed solution obtained in the step 2 into a water bath kettle at the temperature of 98 ℃ for 30s, then putting the mixed solution into a water bath kettle at the temperature of 60 ℃ for 30s, putting the mixed solution into a water bath kettle at the temperature of 68 ℃ for 60s again, and repeating the water bath process for 5 times;

In step 4, adopting L AMP method to generally identify reaction liquid, wherein the volume of sterile water is 4 mu L L02 solution, the volume is 2 mu L1, the final concentration is 5mM, the volume of 10 × Bsm Buffer is 2.5 mu L mix, the volume is 1 mu L, the concentration is 10mM each, the volume of inner primer FIP is 1 mu L, the volume of inner primer BIP is 1 mu L, the volume of Bsm DNA Po L ymease is 1 mu L, the concentration is 8U/mu L, if the reaction liquid is colorless and transparent, no X sperm exists, if the reaction liquid is white and turbid, X sperm exists, and the larger turbidity indicates that the quantity of the X sperm is larger according to the turbidity of the reaction liquid;

Example 2

step 1, collecting semen of an animal to be sorted, mixing the semen with a sperm diluent, uniformly mixing the semen and the sperm diluent, putting the mixture into a storage bottle, and putting the storage bottle into a heat preservation box with the heat preservation temperature of 15 ℃;

step 3, adding the extract into the lower layer solution, extracting X sperms from the sorting solution, subpackaging in 100m L subpackaging bottles to obtain a solution containing the X sperms;

in the step 1, the sperm diluent is prepared from 12% sucrose solution, glycine, sodium citrate, Tris, yolk, glycerol and streptomycin, 0.5g of glycine, 0.5g of sodium citrate and 0.2g of Tris are added into 75ml of 12% sucrose solution and then filtered, the filtrate is boiled for 15 minutes, 20ml of yolk is added after the filtrate is stood to room temperature, 1% glycerol with the final concentration of 1m L and 100mg of streptomycin are added, the volume is determined after the mixture is fully and uniformly stirred, and the final concentration is not lower than 4000 ten thousand/m L.

step 3, putting the mixed solution obtained in the step 2 into a water bath kettle at the temperature of 98 ℃ for 30s, then putting the mixed solution into a water bath kettle at the temperature of 60 ℃ for 30s, putting the mixed solution into a water bath kettle at the temperature of 68 ℃ for 60s again, and repeating the water bath process for 3 times;

In step 4, the reaction solution for fluorescence identification of calcein by L AMP method comprises calcein with volume of 2.5 μ L and final concentration of 0.25mM, MnC L₂The volume of the solution is 2 mu L, the concentration is 5mM, the volume of 10 × Bsm Buffer is 2.5 mu L mix, the volume is 1 mu L, the concentration is 10mM each, the volume of inner primer FIP is 1 mu L, the volume of inner primer BIP is 1 mu L, the volume of Bsm DNA Po L enzyme is 1 mu L, the concentration is 8U/mu L, the volume of sterile water is 1.5 mu L, if the reaction solution is orange, no X sperm exists, if the reaction solution emits yellow green fluorescence, X sperm exists, and the brighter fluorescence indicates that the number of the X sperm is larger according to the brightness degree of the color of the reaction solution;

Example 3

step 1, collecting semen of an animal to be sorted, mixing the semen with a sperm diluent, uniformly mixing the semen and the sperm diluent, putting the mixture into a storage bottle, and putting the storage bottle into a heat preservation box with the heat preservation temperature of 18 ℃;

In the step 1, the sperm diluent is prepared from 12% sucrose solution, glycine, sodium citrate, Tris, yolk, glycerol and streptomycin, 0.5g of glycine, 0.5g of sodium citrate and 0.2g of Tris are added into 75ml of 12% sucrose solution and then filtered, the filtrate is boiled for 15 minutes, 20ml of yolk is added after the filtrate is stood to room temperature, 1% glycerol with the final concentration of 1m L and 60mg of streptomycin are added, the volume is determined after the mixture is fully and uniformly stirred, and the final concentration is not lower than 4000 ten thousand/m L.

step 3, putting the mixed solution obtained in the step 2 into a water bath kettle at the temperature of 98 ℃ for 30s, then putting the mixed solution into a water bath kettle at the temperature of 60 ℃ for 30s, putting the mixed solution into a water bath kettle at the temperature of 68 ℃ for 60s again, and repeating the water bath process for 4 times;

In step 4, adopting L AMP method to identify the reaction liquid of hydroxynaphthol blue, wherein the volume of hydroxynaphthol blue is 2.5 mu L and MnC L₂The volume of the solution is 2 mu L, the final concentration is 5mM, the volume of the 10 × Bsm Buffer is 2.5 mu L mix, the volume is 1 mu L, the concentration is 10mM each, the volume of the inner primer FIP is 1 mu L, the volume of the inner primer BIP is 1 mu L, the volume of the Bsm DNAPO L ymerase is 1 mu L, the concentration is 8U/mu L, the volume of the sterile water is 1.5 mu L, if the reaction solution is violet, no X sperm exists, if the reaction solution is sky blue, X sperm exists, and the darker the color of the reaction solution indicates that the number of the X sperm is more;

Example 4

In step 4, L AMP method is adopted to identify the reaction solution of PicoGreen by fluorescence, which comprises the volume of PicoGreen is 2.5 mu L L₂The volume of the solution was 2. mu. L, the final concentration was 5mM, the volume of 10 × Bsm Buffer was 2.5. mu. L mix, the volume of 1. mu. L, the concentration was 10mM each, the volume of inner primer FIP was 1. mu. L, the volume of inner primer BIP was 1. mu. L, the volume of Bsm DNAPO L ymerase was 1. mu. L, the concentration was 8U/. mu. L, the volume of sterile water was 1.5. mu. L, if the reaction solution was orange, X sperm was present, if the reaction solution emitted yellow-green fluorescence, X sperm was present, and the brighter fluorescence was indicated by the degree of lightness of the color of the reaction solution, indicating that the number of X sperm was increased.

Example 5

In step 4, SYBR Green I fluorescence identification reaction solution adopting L AMP method comprises SYBR Green I with volume of 2.5 mu L L₂The volume of the solution is 2 mu L, the final concentration is 5mM, the volume of the 10 × Bsm Buffer is 2.5 mu L, the volume of the solution is 1 mu L, the concentration is 10mM each, the volume of the inner primer FIP is 1 mu L, the volume of the inner primer BIP is 1 mu L Po L ymerase, the volume of the inner primer BIP is 1 mu L, the concentration is 8U/mu L, the volume of the sterile water is 1.54 mu L, if the reaction solution is orange, X sperms do not exist, if the reaction solution generates yellow-green fluorescence, X sperms exist, and the brighter fluorescence is determined according to the brightness degree of the color of the reaction solution, the number of the X sperms is increased;

Sequence listing

<110> Yanan university

<120> a general animal sperm sorting method and X sperm quality control method

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aagtattttc tacaagatgc tttccagttg cgacatctgg acctcagctc aaataaaatc 2280

caggttatcc agaagactag ttttccagaa aacgttctca acaatttgca gattttgttt 2340

ttgcatcata atcggtttct gtgcaactgc gacgctgtgt ggcttgtctg gtgggttaac 2400

catacagagg tgaccatccc tttcttggcc acagatgtga cttgtatggg accaggagca 2460

cacaagggcc agagtgtggt ctctctagat ctgtatactt gtgagttaga tctgactaat 2520

tttgttttgt tttcactttc cctatcggca gttctctttc tgattgtaat cacgatagca 2580

aaccatctct atttctggga tgtgtggtac agttaccatt tctgtaaggc caagataaag 2640

gggtatcaac gtctgatatc acccaattcc tgctacgatg cttttattgt ctatgacact 2700

aaagacccag cagtgacaga gtgggttttg gatgagctgg tggccaagtt ggaagatcca 2760

agagagaaac attttaattt atgtctcgaa gaaagggact ggttaccagg gcagccagtt 2820

ctggaaaacc tttcccagag catacagctt agcaaaaaga cggtgtttgt gatgacagac 2880

aagtatgcaa agactgagaa atttaagata gcattttact tgtcccatca gaggctcatg 2940

gatgaaaaag tggatgtcat tatcttgata ttccttgaga agccccttca gaagtccaag 3000

tttttccagc ttcggaagag gctctgtggg agttctgttc ttgagtggcc aacaaatcca 3060

caggctcacc cgtacttctg gcagtgtctg aaaaatgccc tggccacaga caaccacgtt 3120

acctatagtc aggtgttcaa agagacagcc tag 3153

<210>2

<211>22

<212>DNA

<213>Artificial Sequence

<400>2

gattgtaatc acgatagcaa ac 22

<210>3

<211>18

<212>DNA

<213>Artificial Sequence

<400>3

tggatcttcc aacttggc 18

<210>4

<211>46

<212>DNA

<213>Artificial Sequence

<400>4

gatacccctt tatcttggcc ttaccatctc tatttctggg atgtgt 46

<210>5

<211>43

<212>DNA

<213>Artificial Sequence

<400>5

aacgtctgat atcacccaat tcctccaaaa cccactctgt cac 43

<210>6

<211>20

<212>DNA

<213>Artificial Sequence

<400>6

ggatgtgtgg tacagttacc 20

<210>7

<211>20

<212>DNA

<213>Artificial Sequence

<400>7

gggaaaggtt ttccagaact 20

<210>8

<211>46

<212>DNA

<213>Artificial Sequence

<400>8

gacaataaaa gcatcgtagc aggaataagg ccaagataaa ggggta 46

<210>9

<211>40

<212>DNA

<213>Artificial Sequence

<400>9

agtgggtttt ggatgagctg ggtccctttc ttcgagacat 40

Claims

1. A general animal sperm sorting method is characterized by comprising the following steps:

2. The method for sorting universal animal sperm according to claim 1, wherein in step 1, the sperm diluent is prepared from 12% sucrose solution, glycine, sodium citrate, Tris, yolk, glycerol and streptomycin, 0.5g glycine, 0.5g sodium citrate and 0.2g Tris are added into 75ml of 12% sucrose solution, then the mixture is filtered, the filtrate is boiled for 15 minutes, the mixture is left to stand to room temperature, then 20ml of yolk is added, 1m L of glycerol with the final concentration of 1% and 50-100 mg streptomycin are added, the mixture is fully stirred uniformly and the volume is determined, and the final concentration is not lower than 4000 ten thousand/m L.

3. The method as claimed in claim 1, wherein in step 2, the sorting solution comprises DMSO and a Toll-like receptor inhibitor, the concentration of the Toll-like receptor inhibitor is 0.3 μmol, the volume ratio of the DMSO to the Toll-like receptor inhibitor is 1: 1000, and the concentration of the sperm is not lower than 2000 ten thousand/m L after mixing with the semen.

4. The method for sorting universal animal sperm cells according to claim 1, wherein in step 3, the volume ratio of the extraction liquid to the lower layer solution is 1:1, the extraction liquid comprises peanut oil, corn oil and olive oil, and the volume ratio of the peanut oil, the corn oil and the olive oil is 3:4: 4.

5. A quality control method of X sperms is characterized by comprising the following steps:

6. The X sperm quality control method of claim 5, wherein in the step 2, L AMP primer group is designed, the primer group comprises an outer primer F3 and an outer primer B3, 2 × MightAmp Buffer Ver.3 of 6.25 mu L, an outer primer F3 of 0.376 mu L, an outer primer B3 of 0.374 mu L, and MightAmp polymerase Ver.3 and 3.75 mu L of 0.25 mu L and at a concentration of 1.25U/mu L are uniformly mixed with sterile water to obtain the extraction-free nucleic acid amplification reaction solution, the gene sequence of the outer primer F3 is shown as SEQ ID No.2 and SEQ ID No.6, and the gene sequence of the outer primer B3 is shown as SEQ ID No.3 and SEQ ID No. 7;

7. The method for quality control of X sperm according to claim 5, wherein in step 4, the mixed solution processed in step 3 is placed in L AMP expandator at 65 ℃ for 12min in a water bath, L AMP is added to identify the reaction solution, and the dark and light color of the reaction solution is observed, which indicates that the number of X sperm is larger.

8. The X sperm quality control method according to claim 9, wherein in step 4, the L AMP assay reaction solution is any one of a L AMP assay common assay reaction solution, a L AMP assay calcein fluorescence assay reaction solution, a L AMP assay hydroxynaphthol blue assay reaction solution, a L AMP assay PicoGreen fluorescence assay reaction solution, a L AMP assay SYBR Green I fluorescence assay reaction solution;

l the AMP primer group also includes inner primer FIP and inner primer BIP, the gene sequence of the inner primer FIP is shown in SEQ ID NO.4 and SEQ ID NO.8, the gene sequence of the inner primer BIP is shown in SEQ ID NO.5 and SEQ ID NO. 9;

the identification reaction liquid of the hydroxynaphthol blue by the L AMP method comprises the hydroxynaphthol blue with the volume of 2.5 mu L and MnC L₂The volume of the solution was 2 μ L, final concentration 5mM, 10 × Bsm Buffer 2.5 μ L mix 1 μ L, 10mM Meach, inner primer FIP 1 μ L, inner primer BIP 1 μ L, Bsm DNA Po L ymerase 1 μ L, concentration 8U/μ L, sterile water 1.5 μ L;

the SYBR Green I fluorescent identification reaction solution prepared by the L AMP method comprises SYBR Green I with the volume of 2.5 mu L L₂The volume of the solution was 2 μ L, final concentration 5mM, 10 × Bsm Buffer 2.5 μ L mix 1 μ L, 10mM each, inner primer FIP 1 μ L, inner primer BIP 1 μ L, Bsm DNA Po L enzyme 1 μ L, concentration 8U/. mu. L, sterile water 1.54 μ L.

9. The X sperm quality control method of claim 5 wherein in step 4, the mixed liquor treated in step 3 is placed in a 65 ℃ water bath for 12min, the developer is added and mixed uniformly, the diphenylamine reagent and the distilled water are added, the mixed liquor is placed in a 60 ℃ water bath for 45min and then taken out, the mixed liquor is cooled to room temperature and placed in a colorimeter, the sample is placed at a wavelength of 595nm to measure absorbance, the absorbance measured by the sample is compared with the absorbance of a standard curve prepared according to a gradient test, and the X sperm DNA content is estimated.

10. The X sperm quality control method of claim 5 wherein in step 4, the volume ratio of the mixed liquor to the color developing agent is 1:2, the volume ratio of the mixed liquor, the distilled water and the diphenylamine reagent is 1:1:4, the color developing agent comprises a color developing agent A liquid and a color developing agent B liquid, and the volume ratio of the color developing agent A liquid to the color developing agent B liquid is 200: 1;