Formula for preservation of fresh sperms of Brag black sheep

Abstract

The invention provides a formula for preservation of fresh sperms of Brag black sheep, and relates to the technical field of preservation of fresh sperms of sheep. The formula for preserving the fresh sperms of the Brag black sheep comprises a sperm diluent, the sperm diluent comprises a basic diluent, a lycopene concentrated stock solution and a lycopene diluent, the basic diluent comprises 0.68 g of glucose, 0.68 g of fructose, 0.1 g of EDTA, 2.72 g of Tris, 1.53 g of citric acid, 100 thousand units of penicillin, 100 thousand units of streptomycin and 10 ml of yolk, and the lycopene concentrated stock solution is prepared by adding 0.1074 mg of lycopene powder into 1 ml of DMSO (dimethylsulfoxide) and stirring uniformly. The lycopene diluent is prepared by adding 1 microliter of concentrated stock solution into 100 milliliters of basic diluent, and the working concentration of lycopene is 2 micromol / L. The lycopene diluent is prepared by adding 1 microliter of concentrated stock solution into 100 milliliters of basic diluent. Through comparison and detection of a plurality of experiments, the survival rate and activity of the preserved sperms are greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of preservation of fresh semen of sheep, and specifically to a formula for the preservation of fresh semen of Butuo black sheep. Background Art

[0002] Butuo black sheep is a high-quality meat sheep breed, which is famous for its black hair and excellent meat quality. Butuo black sheep has the characteristics of strong adaptability, being able to tolerate roughage, and having delicious meat, and is suitable for being raised in various environments such as mountainous areas and grasslands. Due to its delicate meat and delicious taste, it is loved by consumers.

[0003] The preservation of fresh semen of Butuo black sheep is of great significance for improving breeding efficiency and promoting genetic improvement. By preserving high-quality fresh semen, artificial insemination can be carried out, thereby improving the overall quality of the flock, increasing the breeding efficiency, reducing the risk of disease transmission, and retaining the genes of excellent individuals. In addition, with the increasing demand for high-quality mutton, preserving fresh semen helps farmers better meet market demand, reduce breeding costs, and ensure the effective utilization of resources. Therefore, a method for the preservation of fresh semen of Butuo black sheep is needed. However, the dilution liquid formulas in the existing technologies may not be optimized for specific breeds, resulting in insufficient survival rate and vitality of sperm during dilution and preservation. Summary of the Invention

[0004] (I) Technical Problems to be Solved

[0005] In view of the deficiencies of the existing technology, the present invention provides a formula for the preservation of fresh semen of Butuo black sheep, which solves the problem that the existing dilution liquid formulas are not optimized for specific breeds, resulting in insufficient survival rate and vitality of sperm during dilution and preservation.

[0006] (II) Technical Solutions

[0007] To achieve the above objectives, the present invention is realized through the following technical solutions: A formula for the preservation of fresh semen of Butuo black sheep, including a semen diluent, and the semen diluent includes a basic diluent, a lycopene concentrated stock solution, and a lycopene diluent;

[0008] The basic diluent includes 0.68 g of glucose, 0.68 g of fructose, 0.1 g of EDTA, 2.72 g of Tris, 1.53 g of citric acid, penicillin and streptomycin each 100,000 units, and 10 ml of egg yolk;

[0009] The lycopene concentrated stock solution is prepared by adding 0.1074 mg of lycopene powder to 1 ml of DMSO to form a lycopene concentrated stock solution of 2*105 μmol / L;

[0010] The lycopene diluent is 100 ml of basic diluent plus 1 μL of concentrated stock solution, and the working concentration of lycopene is 2 μmol / L.

[0011] A preservation method realized by a formula for fresh semen preservation of Butuo black sheep includes the following steps:

[0012] S1. Semen collection

[0013] Before semen collection, use a wet towel soaked in warm water to clean the prepuce and its surrounding area of the breeding ram. When collecting semen, fix the teaser ewe on a special semen collection stand, lead the ram close to the teaser ewe, and stimulate the ram's sexual desire with the information emitted by the estrous ewe. When the ram approaches the teaser ewe and is about to mount, gently flick its head with your hand to prevent it from mounting immediately. After blocking 2 - 3 times, the ram's sexual desire will be enhanced, the ejaculation will be sufficient, the semen volume will be large, and the semen quality will also be better. However, mating must be strictly controlled. The semen collector squats behind the right rear of the teaser ewe, holds the artificial vagina in the right hand with the piston downwards, presses the collecting cup with the middle finger and index finger, holds the artificial vagina tightly with other fingers and the palm, with one end of the collecting cup upwards, making it at an angle of about 35 - 40 degrees with the ground. When the ram's front legs step onto the teaser ewe and it extends its penis, quickly guide its penis into the artificial vagina from outside the prepuce of the ram with the left hand. When the ram shows an ejaculation movement, quickly tilt the end with the collecting cup downwards to collect the semen. After ejaculation, erect the collecting bottle, release the air and water, then remove the collecting cup, cover the lid, record the ram number, and place it on the operating table for semen dilution and quality inspection;

[0014] S2. Semen quality inspection

[0015] Conduct routine semen quality inspection of the collected semen in a thermostatic operating box at 35 - 37 °C, which is mainly divided into appearance inspection and microscopic inspection. The semen volume of the ram varies from individual to individual. Generally, the semen volume of a ram is 0.5 - 2 mL; the color of normal semen is milky white; it has a slightly fishy smell or no special smell. Microscopically, mainly observe the cloudy appearance of the semen and check the sperm motility. Only semen with an obvious cloudy appearance and sperm motility above 85% is used for the experiment;

[0016] S3. Preparation of diluent, semen dilution and dilution ratio

[0017] Basic diluent: Weigh 0.68 g of glucose, 0.68 g of fructose, 0.1 g of EDTA, 2.72 g of Tris, 1.53 g of citric acid, 100,000 units each of penicillin and streptomycin, and 10 ml of egg yolk respectively with an electronic analytical balance;

[0018] Lycopene concentrated stock solution: Add 0.1074 mg of lycopene powder to 1 ml of DMSO to prepare a lycopene concentrated stock solution of 2 * 105 μmol / L, wrap it with tin foil and store it at -20 °C for later use;

[0019] Lycopene diluent: Add 1 μL of concentrated stock solution to 100 mL of basic diluent, and the working concentration of lycopene is 2 μmol / L;

[0020] According to the above semen diluent formula, accurately weigh 100 mL of basic solution reagent, add ultrapure water to 100 mL. After the reagent is fully dissolved, filter and sterilize it with a syringe-type bacterial filter, aseptically dispense it, and store it at 5 °C for later use. The storage period shall not exceed one week. When in use, add egg yolk according to the volume ratio of 9:1 of the diluent basic solution to egg yolk, and gently shake and mix well to obtain the semen diluent. The semen diluent should be prepared and used immediately, and heated to about 30 °C to keep it at the same temperature as the collected semen;

[0021] Fresh semen with qualified semen quality inspection should be diluted in time. In the semen diluent formula screening test, the dilution multiple is 10 times. When diluting, first dilute it at a dilution multiple of 1:1 (semen: diluent), and then sequentially dilute it to 1:4 and 1:8, and finally make up the diluent to 1:10. After each step of diluent dilution, the semen motility should be checked;

[0022] When using a simple and portable plastic straw to store semen, it is necessary to store as many sperm as possible to ensure the conception rate of ewes. At the same time, under the condition of a small amount of diluent, it is necessary to avoid the imbalance of the microenvironment caused by too many sperm. Therefore, after screening out the diluent with the optimal formula, four dilution multiples of 4 times, 8 times, 16 times, and 20 times are designed with the optimal diluent for the research on low-temperature storage of diluted semen. The collected semen is divided into four parts, and diluted with the optimal formula diluent at 1:4, 1:8, 1:16, and 1:20 respectively. The diluted semen is respectively dispensed into plastic straws, and 0.4 mL of diluted semen is dispensed into each straw. After the dispensing is completed and the storage starts, record it as 0 h. After observing the sperm motility and plasma membrane integrity rate at 0 h, store it at 5 °C. After that, detect the sperm motility and plasma membrane integrity rate every 24 h. The semen of 3 rams is collected, which is equivalent to repeating three times. Finally, select the dilution multiple that can ensure a long sperm survival time and a high conception rate of ewes when using a plastic straw to store semen for popularization and application;

[0023] S4. Sperm motility inspection, dispensing and low-temperature storage of semen

[0024] Starting from the saved 0d, the sperm motility was detected every 24 hours. The sperm motility refers to the percentage of sperm moving in a straight line at 35 - 37°C among the total number of sperm. During the examination, the diluted semen was dropped on a glass slide, covered with a coverslip, and then observed under a 400 - fold microscope. If 100% of the sperm move in a straight line, it is counted as 1.0; if 90% of the sperm move in a straight line, it is counted as 0.9, and so on; if no sperm move in a straight line, it is counted as 0. When examining the motility of semen stored at low temperature, the glass slide can be waved back and forth above the flame of an alcohol lamp to warm it up and then quickly examined under the microscope. During the examination, several fields of view should be observed. In this experiment, the semen motility in three different fields of view was observed, and finally, the average value was taken as the sperm motility;

[0025] S5. Examination of the integrity rate of sperm plasma membrane in semen preservation

[0026] When sperm are placed in a hypotonic solution, water will enter the sperm to form a balance, finally resulting in an increase in sperm volume, acrosome swelling, and tail bending. The characteristic of tail bending is relatively obvious, and the bent - tail rate can be used as a marker to evaluate the integrity of the sperm plasma membrane. 25 μl was taken from the preserved semen and diluted to 200 μl with a hypotonic solution (0.49 g trisodium citrate, 0.9 g fructose, dissolved in 100 mL of ultrapure water, osmotic pressure 100 mOsm), incubated at 38.5°C for 45 minutes, then 300 μl of 2% glutaraldehyde was added. 10 μl of the mixed solution was smeared, and the number of bent - tail sperm and the total number of sperm were observed in 5 fields of view under a 400 - fold phase - contrast microscope to calculate the percentage. Each time during the detection, at least 200 sperm should be counted with a cell counter. The integrity rate of the plasma membrane = (number of bent - tail sperm / total number of sperm) × 100%;

[0027] (III) Beneficial effects

[0028] The present invention provides a preservation formula for fresh semen of Butuo black sheep. This formula has the following advantages: accurately weighing by an electronic analytical balance, respectively taking 0.68 g of glucose, 0.68 g of fructose, 0.1 g of EDTA, 2.72 g of Tris, 1.53 g of citric acid, 100,000 units each of penicillin and streptomycin, 10 ml of egg yolk, and adding water to a total volume of 100 ml to prepare a basic diluent. In addition, 0.1074 mg of lycopene powder is dissolved in 1 ml of DMSO to prepare a concentrated stock solution of lycopene at 2×10^5 μmol / L. 100 ml of the basic diluent is mixed with 1 μl of the concentrated stock solution to obtain lycopene with a working concentration of 2 μmol / L. After using the finally prepared semen diluent to preserve the fresh semen of Butuo black sheep, comparative tests on sperm motility, sperm survival rate, sperm motility ability, sperm tail plasma membrane integrity, sperm head plasma membrane integrity, acrosome integrity, sperm JC-1 activity, sperm ATP content, and sperm ROS level are carried out, and the results show significant improvements, with a relatively large increase in the survival rate and motility. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a graph showing the change in the motility of the diluted semen of the present invention;

[0030] Figure 2 It is a control chart of sperm motility of the present invention;

[0031] Figure 3 It is a control chart of sperm viability of the present invention;

[0032] Figure 4 It is a detection chart of the tail plasma membrane of the present invention;

[0033] Figure 5 It is a flow cytometry detection chart of sperm plasma membrane integrity of the present invention;

[0034] Figure 6 It is a flow cytometry detection chart of sperm acrosome integrity of the present invention;

[0035] Figure 7 It is a control chart of sperm JC-1 activity of the present invention;

[0036] Figure 8 It is a control chart of sperm ATP of the present invention;

[0037] Figure 9 It is a flow cytometry detection chart of ROS content of the present invention;

[0038] Figure 10 It is a control chart of sperm ROS of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0039] Next, in combination with the accompanying drawings in the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0040] The embodiments of the present invention provide a formula and a preservation method for fresh semen preservation of Butuo black sheep, which specifically include the following steps:

[0041] Semen collection

[0042] Before semen collection, use a wet towel soaked in warm water to wipe clean the prepuce and its surrounding area of the breeding ram. During semen collection, fix the teaser ewe on a special semen collection rack, lead the ram close to the teaser ewe, and stimulate the ram's sexual desire with the information emitted by the estrus ewe. When the ram approaches the teaser ewe and is about to mount, gently flick its head with your hand to prevent it from mounting immediately. After blocking 2 - 3 times, the ram's sexual desire will be enhanced, the ejaculation will be sufficient, the semen collection volume will be large, and the quality of the semen will also be better, but mating must be strictly controlled and prevented. The semen collector squats at the right rear of the teaser ewe, holds the artificial vagina with the right hand, the piston facing downwards, the middle finger and index finger pressing on the semen collection cup, and tightly holds the artificial vagina with other fingers and the palm. One end of the semen collection cup is upward, making an angle of about 35 - 40 degrees with the ground. When the ram's front legs straddle the teaser ewe and the penis extends, quickly introduce the penis into the artificial vagina from outside the ram's prepuce with the left hand. When the ram shows an ejaculation action, quickly tilt the end with the semen collection cup downward to collect the semen. After ejaculation, erect the semen collection bottle, release the air and water, then remove the semen collection cup, cover the lid, record the ram number, and place it on the operating table for semen dilution and quality inspection.

[0043] Semen quality inspection

[0044] Conduct routine semen quality inspection of the collected semen in a thermostatic operating box at 35 - 37 °C, which mainly includes appearance inspection and microscopic inspection. The semen ejaculation volume of rams varies due to breed and individual differences. Generally, the semen ejaculation volume of rams is 0.5 - 2 mL; the color of normal semen is milky white; it has a slightly fishy smell or no special smell. Under the microscope, mainly observe the cloudiness of the semen and check the sperm motility. Only semen with obvious cloudiness and sperm motility above 85% is used for the experiment.

[0045] Semen diluent formula

[0046] Basic diluent: Weigh 0.68 g of glucose, 0.68 g of fructose, 0.1 g of EDTA, 2.72 g of Tris, 1.53 g of citric acid, 100,000 units each of penicillin and streptomycin, 10 ml of egg yolk with an electronic analytical balance, and add water to make up the volume to 100 ml.

[0047] Lycopene concentrated stock solution: 0.1074 mg of lycopene powder was added to 1 ml of DMSO to prepare a lycopene concentrated stock solution with a concentration of 2×10⁵ μmol / L. It was wrapped with tin foil and stored at -20 °C for later use.

[0048] Lycopene dilution solution: 1 μL of the concentrated stock solution was added to 100 ml of the basic dilution solution, and the working concentration of lycopene was 2 μmol / L.

[0049] Preparation of dilution solution, dilution of semen and dilution ratio

[0050] According to the above semen dilution solution formula, the basic solution reagent was accurately weighed at a dose of 100 mL, and ultrapure water was added to 100 mL. After the reagent was fully dissolved, it was filtered and sterilized with a needle-type bacterial filter, aseptically dispensed, and stored at 5 °C for later use, with a shelf life of no more than one week. When in use, egg yolk was added according to the volume ratio of dilution solution basic solution: egg yolk of 9:1, and gently shaken and fully mixed to obtain the semen dilution solution. The semen dilution solution was prepared and used immediately, and heated to about 30 °C to keep it isothermal with the collected semen.

[0051] Fresh semen with qualified semen quality inspection was diluted in time. In the semen dilution solution formula screening test, the dilution ratio was 10 times. During dilution, first, the dilution ratio was 1:1 (semen: dilution solution), and then it was successively diluted to 1:4 and 1:8. Finally, the dilution solution was added to make it 1:10. After each step of dilution with the diluent, the semen motility was checked.

[0052] When using a simple and portable plastic straw to store semen, it is necessary to store as many sperm as possible to ensure the conception rate of ewes, and at the same time, avoid the imbalance of the microenvironment caused by too many sperm under the condition of a small amount of dilution solution. Therefore, after screening out the dilution solution with the optimal formula, four dilution multiples of 4 times, 8 times, 16 times, and 20 times were designed with the optimal dilution solution for the research on low-temperature preservation of diluted semen. The collected semen was divided into four parts, and diluted with the optimal formula dilution solution at 1:4, 1:8, 1:16, and 1:20 respectively. The diluted semen was respectively dispensed into plastic straws, and each straw was dispensed with 0.4 mL of diluted semen. The time when the dispensing ended and the preservation started was recorded as 0 h. After observing the sperm motility and plasma membrane integrity rate at 0 h, it was stored at 5 °C. Then, the sperm motility and plasma membrane integrity rate were detected every 24 h. The semen of 3 rams was collected for the experiment, which was equivalent to repeating three times. Finally, the dilution ratio that can ensure a long sperm survival time and a high conception rate of ewes when using a plastic straw to store semen was selected for popularization and application.

[0053] Sperm motility inspection, dispensing and low-temperature preservation of stored semen

[0054] Starting from the saved 0d, the sperm motility was detected every 24 hours. The motility of sperm refers to the percentage of sperm moving straight forward at 35 - 37°C among the total number of sperm. During the examination, the diluted semen was dropped on a glass slide, covered with a coverslip, and then observed under a 400 - fold microscope. If 100% of the sperm move in a straight line, it is counted as 1.0; if 90% of the sperm move in a straight line, it is counted as 0.9, and so on; if no sperm move in a straight line, it is counted as 0. When examining the motility of semen stored at low temperature, the glass slide can be waved back and forth above the flame of an alcohol lamp to warm it up and then quickly examined under the microscope. During the examination, several fields of view should be observed. In this experiment, the semen motility in three different fields of view was observed, and finally the average value was taken as the sperm motility.

[0055] Examination of the integrity rate of the sperm plasma membrane in stored semen

[0056] The hypoosmotic swelling test (HOST) is a commonly used method for detecting the integrity of the sperm plasma membrane. In 1966, Drevius et al. reported the phenomenon of bent tails in mammalian sperm. When sperm are placed in a hypoosmotic solution, water will enter the sperm to form a balance, eventually leading to an increase in sperm volume, acrosome swelling, and tail bending. The characteristic of tail bending is relatively obvious, and the bent - tail rate can be used as a marker to evaluate the integrity of the sperm plasma membrane. Take 25 μl from the stored semen and dilute it to 200 μl using a hypoosmotic solution (0.49 g trisodium citrate, 0.9 g fructose, dissolved in 100 mL ultrapure water, osmotic pressure 100 mOsm). Incubate at 38.5°C for 45 minutes, and then add 300 μl of 2% glutaraldehyde. Take 10 μl of the mixed solution and smear it. Observe 5 fields of view under a 400 - fold phase - contrast microscope to calculate the number of sperm with bent tails and the total number of sperm, and calculate the percentage. Each time during the detection, at least 200 sperm should be counted using a cell counter. The integrity rate of the plasma membrane = (number of sperm with bent tails / total number of sperm) × 100%.

[0057] The final results of the change in the motility of the diluted semen are as Figure 1 shown.

[0058] The following experiments were carried out on the stored semen in the present invention:

[0059] Example 1: Sperm motility rate

[0060] In this experiment, the motility of the special dilution solution for Butuo (without adding lycopene) was detected, and the semen diluted with skim milk was used as the control group. As Figure 2 can be seen, compared with the sperm motility rate of 36.29 ± 4.2% in the control group after 96 hours of storage, the sperm motility rate of the experimental group was significantly increased to 50.92 ± 8.7% (P < 0.01).

[0061] Example 2: Sperm motility

[0062] The vitality of the special diluent for Butuo was detected in this experiment. The semen diluent without lycopene was used as the control group. It can be seen from Figure 3 that compared with the sperm vitality of 26.00±5% in the control group after 144 hours of storage, the sperm vitality of the experimental group with 2 μmol / L lycopene added increased extremely significantly to 34.82±2.07% (P<0.01).

[0063] Example 3: Sperm motility performance

[0064] After 144 hours of storage, the straight-line velocity (VSL), path velocity (VAP), and curvilinear velocity (VCL) of sperm were detected by CASA. It can be seen from the following table that there was no significant difference in sperm motility parameters between the experimental group and the control group (P<0.05);

[0065] Linear speed (VSL) Path speed (VAP) Curve speed (VCL) Control group 24.18±3.03 31.04±2.66 43.90±3.76 Experimental group 21.76±3.65 29.85±4.06 42.22±5.75

[0066] Example 4: Integrity rate of sperm tail plasma membrane

[0067] After 144 hours of storage, the tail plasma membrane of sperm in the experimental group (added with lycopene) and the control group (not added with lycopene) was detected by the HOST method. It can be seen from Figure 4 that compared with the integrity rate of the tail plasma membrane of 26±0.87% in the control group, the integrity rate of the tail plasma membrane of the experimental group was 28±1.74%, showing an extremely significant increase (P<0.01).

[0068] Example 5: Integrity of sperm head plasma membrane

[0069] Sperm were stained using a sperm plasma membrane integrity fluorescence (SYBR14) detection kit, and the plasma membrane integrity was detected using a flow cytometer. It can be seen from Figure 5 that compared with the integrity rate of the sperm head plasma membrane of 4.84±0.86 in the control group, the integrity rate of the sperm head plasma membrane of the experimental group was 15.27±3.86, showing a significant increase (P<0.05).

[0070] Example 6: Acrosome integrity

[0071] The acrosome integrity of Butuo black sheep sperm was detected using a sperm acrosome morphology peanut agglutinin fluorescence labeling method (PAN-FITC) detection kit in combination with a flow cytometer. It can be seen from Figure 6 that compared with the acrosome integrity rate of 51.98±1.43% in the control group, the acrosome integrity rate of the experimental group was 62.04±0.79%, showing an extremely significant increase (P<0.01).

[0072] Example 7: JC-1 activity of sperm

[0073] The JC-1 activity of sperm from two groups of Butuo black sheep was detected in this experiment. FromFigure 7 It can be seen from [the relevant content] that compared with the control group, the mitochondrial membrane potential of the experimental group was significantly increased (P < 0.05).

[0074] Example 8: Sperm ATP

[0075] In this experiment, the ATP content of the semen of two groups of Butuo black sheep was detected. It can be seen from [the relevant content] that compared with the control group, the addition of lycopene significantly increased the ATP content of the semen of Butuo black sheep (P < 0.01). Figure 8 It can be seen from [the relevant content] that compared with the control group, the addition of lycopene significantly increased the ATP content of the semen of Butuo black sheep (P < 0.01).

[0076] Example 9: Sperm ROS

[0077] In this experiment, the ROS concentration of the semen of two groups of Butuo black sheep was measured. It can be seen from [the relevant content] that compared with the control group, the ROS concentration of the frozen semen of Butuo black sheep in the group added with lycopene was significantly decreased (P < 0.01). Figure 9 It can be seen from [the relevant content] that compared with the control group, the ROS concentration of the frozen semen of Butuo black sheep in the group added with lycopene was significantly decreased (P < 0.01).

[0078] Finally, it can be seen from Examples 1 - 9 that the semen viability after preservation was greatly improved by the semen diluent formula in the present invention.

[0079] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made in these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A formula for preserving fresh semen of Butuo black sheep, comprising a semen diluent, characterized in that: The semen diluent comprises a basic diluent, a lycopene concentrated stock solution and a lycopene diluent; The basic diluent includes 0.68 g glucose, 0.68 g fructose, 0.1 g EDTA, 2.72 g Tris, 1.53 g citric acid, 100,000 units each of penicillin and streptomycin, and 10 ml egg yolk; The lycopene concentrated stock solution is prepared by adding 0.1074 mg lycopene powder into 1 ml DMSO to prepare a 2*105 μmol / L lycopene concentrated stock solution; The lycopene diluent is 100 ml of the basic diluent plus 1 μL of concentrated stock solution, and the working concentration of lycopene is 2 μmol / L.

2. A preservation method based on the recipe for preserving Butuo Black Sheep semen as claimed in claim 1, characterized in that: The steps include: S1. Semen collection Before collecting semen, clean the foreskin and surrounding area of ​​the ram with a wet towel soaked in warm water. When collecting semen, fix the ram on a special semen collection rack, pull the ram close to the ram, and stimulate the ram's sexual desire with the information sent by the estrus ewe. When the ram approaches the ram and is ready to mount, gently flick its head with your hand to prevent it from mounting immediately. After blocking it 2 to 3 times, the ram's sexual desire will be enhanced, ejaculation will be sufficient, the amount of semen collected will be large, and the quality of semen will be better. However, strict control must be exercised to prevent mating. The semen collector squats behind the right side of the ram, holds a fake vagina in his right hand, and pistons into the ram. Down, press the semen collection cup with the middle finger and index finger, hold the false vagina with other fingers and palms, with one end of the semen collection cup facing up, making it at an angle of about 35 to 40 degrees to the ground. When the ram's front legs step onto the platform and stretch out the penis, quickly use the left hand to guide the ram's penis into the false vagina from the outside of the foreskin. When the ram ejaculates, quickly tilt the end with the semen collection cup downward to collect semen. After ejaculation, stand up the semen collection bottle, deflate and drain the water, remove the semen collection cup, cover the lid, record the ram number, and put it on the operating table for semen dilution and quality inspection. S2. Semen quality inspection The semen quality inspection of the collected semen is carried out in a 35-37℃ incubator, which is mainly divided into appearance inspection and microscopic inspection. The ejaculation volume of rams varies with breeds and individuals. Generally, the ejaculation volume of rams is 0.5-2mL. The color of normal semen is milky white, with a slight fishy smell or no special smell. The microscope is mainly used to observe the cloudiness of semen and check sperm motility. Only semen with obvious cloudiness and sperm motility above 85% is used for the test. S3. Preparation of diluent, semen dilution and dilution multiple Basic diluent: weigh 0.68g glucose, 0.68g fructose, 0.1g EDTA, 2.72g Tris, 1.53g citric acid, 100,000 units each of penicillin and streptomycin on an electronic analytical balance, and add water to make up to 100ml; Lycopene concentrated stock solution: add 0.1074 mg lycopene powder into 1 ml DMSO to prepare 2*105 μmol / L lycopene concentrated stock solution, wrap it in tin foil and store it at -20℃ for later use; Lycopene diluent: 100 ml of basic diluent plus 1 μL of concentrated stock solution, the working concentration of lycopene is 2 μmol / L; According to the above-mentioned semen diluent formula, accurately weigh the base liquid reagent according to the dosage of 100mL, add ultrapure water to 100mL, after the reagent is fully dissolved, filter and sterilize with a needle-type bacterial filter, aseptically package, and store at 5°C for standby use. The shelf life shall not exceed one week. When used, add egg yolk according to the volume ratio of diluent base liquid: egg yolk of 9:1, and gently shake and mix thoroughly to obtain semen diluent. The semen diluent is prepared and used immediately, and heated to about 30°C to keep it isothermal with the collected semen; Fresh semen that has passed the semen quality inspection should be diluted in time. The dilution multiple in the semen diluent formula screening test is 10 times. When diluting, first dilute it at a multiple of 1:1 (semen: diluent), then dilute it to 1:4, 1:8, and finally make up the diluent to 1:

10. After each step of dilution, a semen motility test is required. When using a simple and portable plastic straw to preserve semen, it is necessary to preserve as many sperm as possible to ensure the conception rate of ewes, and at the same time, it is necessary to avoid excessive sperm causing microenvironment imbalance under the condition of a small amount of diluent. For this reason, after screening out the diluent with the optimal formula, the optimal diluent was used to design 4 times, 8 times, 16 times, and 20 times of four times to dilute the semen for cryopreservation research. The collected semen was divided into four parts, and diluted with the optimal formula diluent at 1:4, 1:8, 1:16 and 1:20, respectively. The diluted semen was dispensed into plastic tubes, and each tube was dispensed with 0.4mL of diluted semen. The end of dispensing and the start of preservation were recorded as 0h. After observing the sperm motility and plasma membrane integrity rate at 0h, it was cryopreserved at 5℃, and then the sperm motility and plasma membrane integrity rate were detected every 24h. The semen of 3 rams was collected in the experiment, which was equivalent to repeating three times. Finally, the dilution multiple that can ensure the long survival time of sperm and make the ewes have a higher conception rate when the plastic straw is used to preserve semen was selected for promotion and application. S4. Semen storage, sperm motility testing, packaging and cryopreservation Starting from the 0th day of storage, the sperm motility is tested every 24 hours. The motility of sperm refers to the percentage of sperm moving in a straight line to the total number of sperm at 35-37℃. When checking, drop the diluted semen on a glass slide, cover it with a coverslip, and observe it under a 400x microscope. If 100% of the sperm move in a straight line, it is counted as 1.0; if 90% of the sperm move in a straight line, it is counted as 0.9, and so on; if there is no sperm moving in a straight line, it is counted as 0. When checking the motility of low-temperature stored semen, the slide can be swung back and forth above the flame of an alcohol lamp to warm it up and then quickly checked under a microscope. Several fields of view should be observed during the inspection. In this test, the semen motility of three different fields of view was observed, and the average was finally calculated as the sperm motility. S5. Examination of sperm plasma membrane integrity in stored semen When sperm is placed in a hypotonic solution, water will enter the sperm to form a balance, which will eventually lead to an increase in sperm volume, swelling of the acrosome, and bending of the tail. The characteristics of the tail bending are relatively obvious, and the tail bending rate can be used as a sign to evaluate the integrity of the sperm plasma membrane. Take out 25μl from the stored semen, use a hypotonic solution (0.49g trisodium citrate, 0.9g fructose, dissolved in 100mL ultrapure water, osmotic pressure 100mOsm) to dilute to 200μl, incubate at 38.5℃ for 45 minutes, then add 300μl of 2% glutaraldehyde, take 10μl of the mixed solution for smear, observe 5 fields under a 400x phase contrast microscope to calculate the number of sperm with bent tails and the total number of sperm The percentage, at least 200 sperm should be counted with a cell counter for each test, plasma membrane integrity rate = (number of sperm with bent tails / total number of sperm) × 100%.

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