阅读说明：本技术 一种绵羊细管精液的冷冻保存方法 (Cryopreservation method of sheep tubule semen ) 是由 肖广庆 许丹娜 刘欣伟 杨利峰 李成山 杨瑞瑞 谢希玲 于 2021-01-21 设计创作，主要内容包括：本发明涉及一种绵羊细管精液的冷冻保存方法,包括以下步骤：步骤A,将采好的镜检活力在0.7以上的绵羊精液用羊冻精稀释液等温稀释,用细管灌装机灌装后,放入4℃环境中平衡待用；步骤B：将平衡好的绵羊精液细管放入液氮冷冻装置中,所述细管位于液氮面上方、距离液氮面3.5～5cm或8～9cm范围内；步骤C：当绵羊精液细管到达﹣10℃时,盖上液氮冷冻装置的箱盖；步骤D：当绵羊精液细管达﹣100℃后,快速降温至-140℃,结束冷冻,将细管取出迅速放入液氮中保存。本发明能够获得绵羊精液较高的复苏效果,为小规模绵羊冻精生产提供技术支持,为大规模生产提供依据。(The invention relates to a cryopreservation method of sheep tubule semen, which comprises the following steps: step A, carrying out isothermal dilution on the collected sheep semen with microscopic examination activity of more than 0.7 by using sheep frozen semen diluent, filling by using a thin tube filling machine, and then balancing in an environment at 4 ℃ for later use; and B: putting the balanced sheep semen tubule into a liquid nitrogen freezing device, wherein the tubule is positioned above the liquid nitrogen surface and is 3.5-5 cm or 8-9 cm away from the liquid nitrogen surface; and C: when the sheep semen tubule reaches-10 ℃, a box cover of a liquid nitrogen refrigerating device is covered; step D: when the sheep semen tubule reaches-100 ℃, rapidly cooling to-140 ℃, ending freezing, taking out the tubule, and rapidly placing into liquid nitrogen for preservation. The method can obtain higher recovery effect of the sheep semen, provide technical support for small-scale sheep frozen semen production, and provide basis for large-scale production.)

1. The cryopreservation method of the sheep tubule semen is characterized by comprising the following steps:

step A: the collected sheep semen with microscopic examination activity of more than 0.7 is subjected to isothermal dilution by sheep frozen semen diluent, filled by a thin tube filling machine and then put into an environment with the temperature of 4 ℃ for balance for standby;

and B: putting the balanced sheep semen tubule into a liquid nitrogen freezing device, wherein the tubule is positioned above the liquid nitrogen surface and is 3.5-5 cm or 8-9 cm away from the liquid nitrogen surface;

and C: when the sheep semen tubule reaches-10 ℃, a box cover of a liquid nitrogen refrigerating device is covered;

step D: when the sheep semen tubule reaches-100 ℃, rapidly cooling to-140 ℃, ending freezing, taking out the tubule, and rapidly placing into liquid nitrogen for preservation.

2. The cryopreservation method of sheep tubule semen according to claim 1, wherein in the step A, the sheep frozen semen diluent is prepared by uniformly mixing trimethylol-aminomethane, citric acid, sodium citrate, glucose, fructose, EDTA, vitamin B, vitamin C, sodium chloride and double distilled water, and filtering with a 0.2 μm filter membrane, wherein the concentration range of each component is as follows: 35-38 g/L of trihydroxymethyl-aminomethane; 18-19 g/L of citric acid; 5-6 g/L of sodium citrate; 35-45 g/L of glucose; 5-8 g/L of fructose; 1-2 g/L of EDTA; 4-6 g/L of vitamin C; 2-5 g/L of vitamin B; 3-4 g/L of sodium chloride; adding 3000-5000 units of streptomycin to the filtered solution to prepare a sheep frozen semen diluent base solution, taking 80 ml of the base solution, adding 5 ml of glycerol and 15 ml of yolk, and uniformly stirring to obtain the sheep frozen semen diluent.

3. The cryopreservation method of semen from sheep tubules as claimed in claim 1, wherein the equilibration time in step A is 4 h.

4. The cryopreservation method of semen from sheep tubules as claimed in claim 3, wherein the height of the tubule position from the liquid nitrogen surface in step B is 4 cm.

5. The cryopreservation method of the sheep tubule semen according to claim 1, wherein in the step C, the time for cooling the sheep tubule semen from 4 ℃ to-10 ℃ is within the range of 1.08-1.48 min; in the step D, the temperature of the sheep tubule semen is reduced from-10 ℃ to-100 ℃ for 6.36-12.84 min.

6. The cryopreservation method of the sheep tubule semen as claimed in claim 5, wherein in the step C, the time for the temperature of the sheep tubule semen to be reduced from 4 ℃ to-10 ℃ is 1.28 min; in the step D, the temperature of the sheep tubule semen is reduced from-10 ℃ to-100 ℃ for 9.6 min.

7. The cryopreservation method of semen from sheep tubules in step B is characterized in that the liquid nitrogen freezing device in step B comprises a liquid nitrogen tank, a thermometer and a freezing box (1), the liquid nitrogen tank is used for making a low-temperature environment for the freezing box (1), the thermometer is used for detecting the temperature in the freezing box (1), a plurality of tray frames (2) and support frames (4) are placed in the freezing box (1), the tray frames (2) are of a hollow frame structure, the support frames (4) are of a rectangular frame structure, two sides of each support frame (4) are correspondingly provided with fixing teeth (401), the fixing teeth (401) are used for fixing tubules, and the insides of the tray frames (2) are detachably connected with the support frames (4).

8. The cryopreservation method of semen from sheep tubule, according to claim 7, characterized in that the upper end of the outer side wall of the tray frame (2) is provided with an upper rolled edge (203), the lower end of the outer side wall of the tray frame (2) is provided with an arc-shaped clamping piece (204), and the clamping piece (204) is matched with the upper rolled edge (203).

9. A cryopreservation method of semen from sheep tubules, as claimed in claim 7, characterized in that a plurality of feet (201) are provided under the tray rack (2), and the feet (201) are inclined towards the center of the tray rack (2).

10. The cryopreservation method of semen from sheep tubules, characterized in that handles (202) are arranged at both ends of the tray frame (2), and the upper ends of the handles (202) are inclined in the direction away from the center of the tray frame (2).

Technical Field

The invention relates to the field of sheep semen freezing, in particular to a freezing preservation method of sheep tubule semen.

Background

The sheep frozen semen preparation process is similar to the cow frozen semen preparation process, and the sheep frozen semen preparation process can be stored in a liquid nitrogen environment after the process of semen collection, diluent dilution, low-temperature balance at 4 ℃ and freezing. The quality of the diluent can be seen when the semen is diluted and balanced. The freezing needs to control the freezing rate of the diluted semen in a plurality of dangerous areas and the semen passing through the dangerous areas in the descending process, the freezing rate is related to the environment temperature of the semen, the larger the temperature difference is, the faster the cooling speed is, the dangerous temperature for freezing the semen is 4 ℃ to-60 ℃, and the freezing method has two nodes: first, the freezing rate is 4 ℃ to-10 ℃; secondly, the freezing rate is-10 ℃ to-60 ℃. Because the semen is packed in bags and stored in the production, the tubule semen is frozen to-120 ℃ in order to avoid temperature return, and is subpackaged and stored in liquid nitrogen.

In the past freezing process of sheep semen, the freezing process and the cooling rate of cattle frozen semen are mainly referred to, and a freezer is used for freezing the sheep semen, but the freezing effect of the sheep semen is unstable, the microscopic examination activity is low after thawing, the number of effective sperms is small, and batch production cannot be carried out.

Disclosure of Invention

The invention provides a freezing preservation method of sheep tubule semen, which aims to solve the problems of poor freezing effect of the sheep tubule semen and low sperm activity after unfreezing, can obtain higher recovery effect of the sheep semen, provides technical support for small-scale sheep frozen semen production, and provides basis for large-scale production.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the cryopreservation method of the sheep tubule semen is characterized by comprising the following steps:

step A: the collected sheep semen with microscopic examination activity of more than 0.7 is subjected to isothermal dilution by sheep frozen semen diluent, filled by a thin tube filling machine and then put into an environment with the temperature of 4 ℃ for balance for standby;

and B: putting the balanced sheep semen tubule into a liquid nitrogen freezing device, wherein the tubule is positioned above the liquid nitrogen surface and is 3.5-5 cm or 8-9 cm away from the liquid nitrogen surface;

and C: when the sheep semen tubule reaches-10 ℃, a box cover of a liquid nitrogen refrigerating device is covered;

step D: when the sheep semen tubule reaches-100 ℃, quickly cooling to-140 ℃, taking out the tubule and quickly putting into liquid nitrogen for preservation.

Further, the sheep jelly diluent in the step A is prepared by uniformly mixing trihydroxymethyl-aminomethane, citric acid, sodium citrate, glucose, fructose, EDTA, vitamin B, vitamin C, sodium chloride and double distilled water, and filtering with a 0.2 μm filter membrane, wherein the concentration ranges of the components are as follows: 35-38 g/L of trihydroxymethyl-aminomethane; 18-19 g/L of citric acid; 5-6 g/L of sodium citrate; 35-45 g/L of glucose; 5-8 g/L of fructose; 1-2 g/L of EDTA; 4-6 g/L of vitamin C; 2-5 g/L of vitamin B; 3-4 g/L of sodium chloride; adding 3000-5000 units of streptomycin to the filtered solution to prepare a sheep frozen semen diluent base solution, taking 80 ml of the base solution, adding 5 ml of glycerol and 15 ml of yolk, and uniformly stirring to obtain the sheep frozen semen diluent.

Further, the equilibration time in the step A is 4 h.

Further, the height from the position of the thin tube to the liquid nitrogen surface in the step B is 4 cm.

Further, in the step C, the temperature of the sheep tubule semen is reduced from 4 ℃ to-10 ℃ for 1.08-1.48 min; in the step D, the temperature of the sheep tubule semen is reduced from-10 ℃ to-100 ℃ for 6.36-12.84 min.

Further, in the step C, the temperature of the sheep tubule semen is reduced from 4 ℃ to-10 ℃ for 1.28 min; in the step D, the temperature of the sheep tubule semen is reduced from-10 ℃ to-100 ℃ for 9.6 min.

Further, liquid nitrogen refrigerating plant includes liquid nitrogen container, thermometer and freezer chest in the step B, the liquid nitrogen container is used for making low temperature environment for the freezer chest, the thermometer is used for detecting the temperature in the freezer chest, the tray frame and the support frame of having placed a plurality of in the freezer chest, the tray frame is hollow frame construction, the support frame is the support body structure of rectangle, and the both sides correspondence of support frame is provided with fixed tooth, fixed tooth is used for fixed tubule, and tray frame inside can dismantle with the support frame and be connected.

Further, the upper end of the outer side wall of the tray frame is provided with an upper curled edge, the lower end of the outer side wall of the tray frame is provided with an arc-shaped card, and the card is matched with the upper curled edge.

Further, a plurality of support feet are arranged below the tray frame, and the support feet incline towards the center of the tray frame.

Further, both ends of the tray frame are provided with handles, and the upper ends of the handles incline towards the direction far away from the center of the tray frame.

Further, the fixed teeth are right-angled triangles.

Through the technical scheme, the invention has the beneficial effects that:

the invention produces frozen semen by a liquid nitrogen fumigation method, and calculates a more ideal freezing speed by the height of the liquid nitrogen surface and the freezing time of a dangerous area. Placing the semen of the sheep penis in a range of 3.5-4.5 cm or 8-9 cm away from the liquid nitrogen surface, and enabling the semen to safely pass through a dangerous area when the temperature is reduced at a cooling rate within the range. In a further preferred scheme, when the distance is 4cm from the liquid nitrogen surface, the vitality of the semen subjected to microscopic examination after unfreezing is higher, more semen is available, a scientific method is provided for manufacturing the sheep frozen semen, technical support is provided for small-scale sheep frozen semen production, and a basis is provided for large-scale production.

The refrigeration device has the advantages that a plurality of tray frames can be stacked in the refrigeration box of the refrigeration device, the tray frames are provided with rectangular support frames, the tubules are fixedly placed through the fixed teeth on the two sides of each support frame, and one support frame can be used for placing a plurality of tubules, so that the placing operation is simple and convenient, and the time is saved; also can be adapted to the experiment that the fumigation method looked for suitable cooling rate, the process is placed simply to the tubule, can adapt to the contrast experiment of the required not co-altitude data of experiment, has improved experiment efficiency and reliability. The refrigerating device has the advantages of simple structure, low manufacturing cost, simple process of placing the thin tube and improved operation efficiency.

Drawings

FIG. 1 is a schematic diagram of an experimental procedure for a liquid nitrogen freezing apparatus of the present invention;

FIG. 2 is a schematic view of the construction of the tray rack of the present invention;

FIG. 3 is a schematic cross-sectional view of the tray rack of the present invention;

FIG. 4 is one of the schematic structural views of the liquid nitrogen freezing apparatus of the present invention;

FIG. 5 is a schematic view of a stacking arrangement of a plurality of pallet racks of the present invention;

FIG. 6 is a second structural view of the liquid nitrogen freezing apparatus of the present invention;

FIG. 7 is a graph of the frozen semen freezing curve of sheep tubule semen with the liquid nitrogen level being 5-10 cm and 14-40 cm;

figure 8 sheep tubule semen frozen semen freezing curve.

The reference numbers in the drawings are as follows: the refrigerator comprises a freezer 1, a tray frame 2, a supporting foot 201, a handle 202, an upper curled edge 203, a card 204, a supporting frame 4, a fixed gear 401 and a thin tube 5.

Detailed Description

The invention is further described with reference to the following figures and detailed description:

in the description of the present invention, it is to be understood that the terms "front", "rear", "left", "right", "upper", "lower", "lateral", "vertical", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in fig. 1, are only used for convenience in describing the present invention and simplifying the description, and do not indicate or imply that the device or element being referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus, should not be construed as limiting the present invention.

Example one

The embodiment provides a liquid nitrogen freezing device for a cryopreservation method of sheep tubule semen, as shown in fig. 1-6, the liquid nitrogen freezing device comprises a liquid nitrogen tank, a thermometer and a freezing box 1, wherein the liquid nitrogen tank is used for manufacturing a low-temperature environment for the freezing box 1, when the freezing box is used, the liquid nitrogen tank is directly used for dumping to enable the liquid nitrogen to be poured into the freezing box 1, the temperature of the internal environment of the freezing box 1 is reduced, and the more the liquid nitrogen is, the more the temperature of the liquid nitrogen surface is stable; the thermometer is used for detecting the temperature in the freezing box 1, specifically, the thermometer is an ultralow temperature thermometer and comprises an ultralow temperature probe and a temperature data display part; freezer 1 includes the box and covers the case lid on the box, has placed a plurality of's tray frame 2 and support frame 4 in freezer 1, tray frame 2 is hollow frame construction, support frame 4 is the support body structure of rectangle, in this embodiment, two short avris of support frame 4 pass through the bolt and can dismantle with tray frame 2 and be connected, two long avris of support frame 4 correspond and are provided with fixed tooth 401, fixed tooth 401 is used for fixed tubule, and is concrete, and fixed tooth 401's shape is right angled triangle, can place a plurality of tubules through support frame 4, and can be directly place support frame 4 with the both ends level of tubule on to improve the convenience that the tubule was placed, reduce the operating time of placing the tubule step, improved the operating efficiency.

The upper end of 2 lateral walls of tray frame is provided with turn-ups 203, and the lower extreme of 2 lateral walls of tray frame is provided with curved card 204, card 204 and last turn-ups 203 phase-match, as shown in fig. 5, when piling up from top to bottom and placing a plurality of tray frame 2, the card 203 of top tray frame 2 contacts with the last turn-ups of below tray frame 2, and the four sides of tray frame 2 all set up turn-ups 203 and card 204, so the four sides all form the restraint to tray frame 2, prevent that the tray frame 2 from piling up and down and taking place crooked when placing, make a plurality of tray frame 2 pile up from top to bottom more firm.

Further, the below of tray frame 2 is provided with a plurality of stabilizer blades 201, and in this embodiment, tray frame 2 is the rectangle, and the four corners department of tray frame 2 sets up stabilizer blade 201, supports tray frame 2 through the stabilizer blade 201 that sets up, stabilizer blade 201 inclines towards the direction at tray frame 2 center, and when piling up from top to bottom at a plurality of tray frames 2, the stabilizer blade 201 of top tray frame 201 inserts the inner space of below tray frame 2, does not influence the use of tray frame 2 and piles up, makes a plurality of tray frames 2 pile up from top to bottom more firm.

Further, in order to facilitate taking the tray frame 2, handles 202 are arranged at two ends of the tray frame 2, and the upper ends of the handles 202 incline towards the direction far away from the center of the tray frame 2.

In order to realize the experiment of finding the optimal cooling rate by adopting a liquid nitrogen fumigation method in the embodiment, the size of the adopted freezing box 1 is 625 multiplied by 280 multiplied by 300cm, two rows of tray frames 2 can be placed side by side, the height of each tray frame 2 is 4cm, the vertical height of each support leg 201 is 0.5cm, the capacity of a liquid nitrogen tank is 50 liters of liquid nitrogen, the thermometer adopts an ultra-low temperature thermometer with the detection range of-200 to 200 ℃, and the ultra-low temperature thermometer comprises an ultra-low temperature probe and a temperature data display part.

By using the liquid nitrogen freezing device and adopting a liquid nitrogen fumigation method to carry out experiments on the method for freezing and preserving the sheep tubule semen, the experimental operation steps are as follows:

an experiment preparation stage: as shown in fig. 1, two tray frames 2 are stacked and placed in a freezer 1, the height of the two tray frames 2 is 8.5cm, a support frame a is fixed on the second layer of tray frames 2 from bottom to top, another support frame B is separately taken, one end of the support frame B is positioned on a fixed tooth 401 of the support frame a, the contact position is marked as a position C, the other end of the support frame B is obliquely placed by depending on the side wall of the freezer 1, the height of the contact position is marked as a height D, meanwhile, the fixed tooth 401 of the support frame B faces upwards, the right-angle side of the fixed tooth 401 faces upwards, the support frame B is in a stable inclined state, a measuring ruler is used for measuring along the support frame B, the height of the third layer of tray frames 2 is taken as an initial height, the position is marked by a height distance of 0.5cm from bottom to top and vertically, the marking range is 10cm, namely, different height positions E1 required by the experiment, E2, E3, … … and E20, and then taking out the support bracket B; and pouring liquid nitrogen into the freezing box 1 until the height of the liquid nitrogen surface reaches the position of the second layer tray rack, namely the height of the liquid nitrogen surface is 8.5 cm.

And (3) an experimental stage:

the first step is as follows: carrying out isothermal dilution on the sheep semen with microscopic examination activity of more than 0.7 by using sheep frozen semen diluent, filling by using a thin tube filling machine, and then putting into a refrigerator for balancing for 4 hours for later use at 4 ℃;

the second step is that: placing the seminal fluid of the tubule of the sheep at the positions E1, E2, E3, … … and E20 of the support frame B respectively at the temperature of 4 ℃, wherein the probe of the ultra-low temperature thermometer is also at the temperature of 4 ℃;

the third step: obliquely placing the support frame B obtained in the second step into a freezing box to enable the vertical height difference of the thin tubes to be 0.5cm, and simultaneously placing an ultralow-temperature probe of an ultralow-temperature thermometer to enable the probe to be tightly attached to the fine frozen position of the thin tube on the uppermost layer;

the fourth step: when the ultra-low temperature thermometer shows that the temperature reaches-10 ℃, a box cover of the freezer is covered;

the fifth step: when the temperature of the ultra-low temperature thermometer reaches-100 ℃, the temperature is reduced to-140 ℃ within 2min, the freezing is finished, and the thin tube is taken out and is rapidly put into liquid nitrogen for preservation.

And a sixth step: and (4) unfreezing and microscopic examination of the activity of the semen in the tubules.

The sheep frozen semen diluent in the first step is prepared by uniformly mixing trihydroxymethyl-aminomethane, citric acid, sodium citrate, glucose, fructose, EDTA, vitamin B, vitamin C, sodium chloride and double distilled water, and filtering with a 0.2-micron filter membrane, wherein the concentration ranges of the components are as follows: 35-38 g/L of trihydroxymethyl-aminomethane; 18-19 g/L of citric acid; 5-6 g/L of sodium citrate; 35-45 g/L of glucose; 5-8 g/L of fructose; 1-2 g/L of EDTA; 4-6 g/L of vitamin C; 2-5 g/L of vitamin B; 3-4 g/L of sodium chloride; adding 3000-5000 units of streptomycin to the filtered solution to prepare a sheep frozen semen diluent base solution, taking 80 ml of the base solution, adding 5 ml of glycerol and 15 ml of yolk, and uniformly stirring to obtain the sheep frozen semen diluent.

The experimental results are as follows: the experimental data are shown in table 1.

TABLE 1 influence of height from liquid nitrogen surface on sperm motility TABLE I

As can be seen from the data on the table above, the better activity of the frozen semen appears in two ranges above and away from the liquid nitrogen surface, namely the ranges of 3.5-4.5 cm and 8-9 cm away from the liquid nitrogen surface. In the two ranges, experiments for repeating the process of preparing the sheep frozen semen by the liquid nitrogen fumigation method are carried out, and the obtained experimental data are shown in table 2.

TABLE 2 influence of height of sheep frozen semen from liquid nitrogen surface on semen vitality prepared by liquid nitrogen fumigation method II

The experimental data can obtain that the semen recovery effect after freezing at the height of 4cm above the liquid nitrogen surface is the best.

When mass production is considered, the temperature of the thin tube package and the code disc is reduced to-100 ℃, and then the temperature is rapidly reduced to-140 ℃ for two minutes, so that excessive temperature rise of the package and the code disc is avoided. Meanwhile, the influence of the amount of liquid nitrogen in the freezing box and the amount of tubule semen on the freezing speed is considered. Therefore, the following experiment was conducted by placing the thin tube at a distance of 4cm from the liquid nitrogen surface or above the liquid nitrogen surface during freezing.

Experiment one: the amount of the fine tube semen during freezing influences the cooling speed.

The height of liquid nitrogen in the freezing box is 8.5cm, sheep tubule semen with the original semen vitality of 0.9, 0.85 and 0.75 is frozen, the freezing time (T2) of 3-8 tubule semen fumigated and 150 tubule semen full of dishes is recorded, and the vitality of the thawed semen is compared. The specific experimental results are shown in tables 3 and 4.

TABLE 3 statistical table of time for fumigating different numbers of sheep tubules with liquid nitrogen

Table 4 statistics table for activity of frozen tubules with different numbers fumigated by liquid nitrogen

Vigor of original essence	T1 frozen	T2 frozen
			0.90	0.45	0.44
0.85	0.38	0.37
			0.75	0.36	0.37

And (4) conclusion: the number of tubules of the fumigating sheep at the same height influences the fumigating speed, but the activity difference after freezing is not obvious.

Experiment two: the amount of liquid nitrogen is related to the freezing time and activity

Pouring different amounts of liquid nitrogen into a freezing box with the same volume to ensure that the heights of the amounts of the liquid nitrogen are respectively 5-10 cm, 10-14 cm and 14-40 cm, carrying out a freezing experiment on 150 full disks of tubules (3-8 tubules do not have production value) in the number of the fumigating tubules to obtain freezing time, and then carrying out activity comparison after thawing, which is shown in table 5 and table 6.

TABLE 5 amount of liquid nitrogen in the same space and fumigating time

Table 6 statistics of activity after second freezing for experiment

And (4) conclusion: liquid nitrogen somewhat affected the freezing rate, but the post-freezing viability differences were not significant.

And drawing a freezing curve for data with the liquid nitrogen amount of 5-10 cm and the height of 14-40 cm, as shown in figure 7. The vitality difference of the tubule semen after the liquid nitrogen fumigation and freezing is not obvious in the range of the two, and the average value of the two is taken to determine a freezing curve: the average cooling time is shown in table 7, the average cooling rate is calculated, and the average value is taken to draw a sheep tubule semen frozen semen freezing curve as shown in fig. 8.

TABLE 7 mean cool down time and speedometer

Temperature of	Temperature reduction time (min)	Cooling Rate (DEG C/min)
			4 ℃ to-10 DEG C	(1.08-1.48) take 1.28	10.938
From-10 ℃ to-100 DEG C	(6.36-12.84) taking 9.6	9.375
			-100 ℃ to-140 DEG C	2	20

Experiment three

And (3) putting the tubule semen into a freezer according to the tubule semen freezing curve of the sheep shown in the table 7 and the figure 8, and verifying the freezing effect of the frozen semen of the sheep according to the tubule semen freezing curve of the sheep.

As a control group, the sperm of the sheep tubule is frozen according to the freezing speed of the sperm of the cattle, the freezing and cooling time and speed of the sperm of the cattle are shown in Table 8, and the freezing and cooling time and speed of the sperm of the cattle are obtained from the frozen sperm production technology and management training textbook of cattle published by the general livestock breeding station in China, 153 + 158 pages of frozen sperm freezing technology and quality control of cattle. The activities after freezing and thawing by a freezer are shown in Table 9.

TABLE 8 bovine semen freezing speed and timetable

Temperature of	Temperature reduction time (min)	Cooling Rate (DEG C/min)
			4 ℃ to-10 DEG C	3	5
From-10 ℃ to-100 DEG C	2.5	40
			-100 ℃ to-140 DEG C	2	20

TABLE 9 post-freezing activity table after input into the freezing apparatus (compare with using the cattle freezing curve)

The frozen semen frozen by using the sheep freezing curve has good vitality, high acrosome integrity rate and good motion state; the activity of the seminal fluid of the sheep tubules frozen by using the freezing curve of the cattle is reduced to a certain extent, the acrosome integrity rate is also obviously reduced, the difference of the motion states is obvious, and the phenomenon that the sperms are frozen and injured in the freezing process is shown, so that the phenomena of coagulation, rotation, short survival time and the like occur.

In the temperature range of-10 to-100 ℃, the bovine semen is adaptive to rapid cooling due to strong anti-freezing force, and the sheep semen is mild and adaptive to slow cooling. The freezing speed and time of the two are greatly different. The experiment finds out the optimal freezing time and speed of the sheep tubule frozen semen by a simple liquid nitrogen fumigation method, provides reliable freezing data for the future industrial production of the sheep tubule frozen semen, and can realize the industrial production of the sheep tubule frozen semen after the freezing data is input into a freezing instrument.

The above-described embodiments are merely preferred embodiments of the present invention, and not intended to limit the scope of the invention, so that equivalent changes or modifications in the structure, features and principles described in the present invention should be included in the claims of the present invention.