CN111602650A - Semen freezing and diluting method capable of automatically adding liquid and adjusting pre-freezing height - Google Patents
Semen freezing and diluting method capable of automatically adding liquid and adjusting pre-freezing height Download PDFInfo
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Abstract
The invention relates to a semen freezing and diluting method capable of automatically adding liquid and adjusting prefreezing height, which comprises the following steps: putting fresh semen into a 50ml beaker, preheating on a constant-temperature water bath shaking table of an automatic liquid adding dilution device, and setting the rotating speed of the shaking table to be 10-220 rpm; the dropping speed of the diluent is controlled to be 10-200 microliter/second; when the liquid nitrogen is prefrozen in gas phase, the height of the prefrozen device is determined according to animal species, and finally the prefrozen device is put into the liquid nitrogen for preservation. The invention can automatically add diluent, standardize operation, eliminate personal manual operation interference and adjust different liquid nitrogen gas phase prefreezing heights according to different animals. The device used by the method has a simple structure, can be used by simply modifying the existing instruments in a laboratory, does not influence the selling price of the frozen semen, but obviously improves the quality of the frozen semen, can improve the lambing rate by 8-9 percent in a production test, and obviously improves the production benefit of an animal farm.
Description
Technical Field
The invention relates to the field of animal breeding, in particular to a semen freezing and diluting method capable of automatically adding liquid and adjusting the pre-freezing height.
Background
In modern animal husbandry, most of raising cattle and pigs use frozen semen to perform artificial insemination to enable female animals to be pregnant, a few bulls are raised in a farm, the frozen semen is diluted and then made into thin tubes, and one female animal is provided with one thin tube, so that one male animal can be pregnant by a plurality of female animals, and the semen freezing preservation technology has important significance for genetic resource protection and artificial insemination technology popularization. The quality of the frozen semen is related to the efficiency of the pregnant production of the female animals and the robustness of the young animals, and is a key factor influencing the production benefit of the modern animal husbandry. Generally, the semen freezing process comprises the steps of semen collection, quality evaluation, semen dilution, low-temperature balance, liquid nitrogen gas phase prefreezing, liquid nitrogen storage and the like, and for how to improve the quality of the frozen semen, related researches at home and abroad are mainly focused on a diluent formula at present.
However, in actual operation, each link can affect the quality of frozen semen, and particularly 2 links of semen dilution and liquid nitrogen gas phase prefreezing are key links for bringing negative effects to semen freezing. The adding speed of the diluent and the mixing speed of the semen and the diluent have key influence on the quality of the frozen semen, but for a long time, researchers and daily production personnel have insufficient attention to the link, the semen dilution is basically manual and operated by experience, the adding speed of the diluent and the mixing speed of the semen and the diluent cannot be standardized, different operators have large dilution effect difference, and the dilution effect difference is not uniform and standard, so that the method is one of important reasons for causing the quality difference of the frozen semen of different batches.
In addition, when liquid nitrogen gaseous phase prefreezes the operation, present prefreezer is a metal pedestal, put the tubule on the metal pedestal and put into liquid nitrogen gaseous phase prefreezing again, but for a long time, researcher and daily production personnel do not realize that the temperature of liquid nitrogen gaseous phase is different along with the height difference, the animal of different cultivars, the semen temperature that the semen required when prefreezing is actually not the same, if the semen of whatever animal is only with a fixed height metal pedestal when prefreezing, and no matter freeze the height of tubule from the liquid nitrogen liquid level, do so not scientifically, also do not excavate the potential that promotes frozen semen quality completely.
Disclosure of Invention
In order to solve the problems, the invention provides a semen freezing and diluting method capable of automatically adding liquid and adjusting the pre-freezing height, and provides a standardized solution for a diluent adding mode and a pre-freezing mode.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a semen freezing and diluting method capable of automatically adding liquid and adjusting pre-freezing height comprises the following steps:
1) collecting fresh semen for quality inspection, wherein sperm activity is more than 75%, and sperm density is more than 2.5 × 109The product has a distortion rate of less than 10% and can be used for cryopreservation;
2) putting the fresh semen which is obtained in the step 1) and accords with the freezing preservation condition into a 50ml beaker, and putting the beaker on a constant-temperature water bath shaking table of an automatic liquid adding dilution device in advance for preheating, wherein the temperature is set to be 36-40 ℃;
3) opening the shaking table of the automatic liquid adding and diluting device, and setting the rotating speed to be 10-220 rpm;
4) adding the diluent into a burette of the automatic liquid adding diluting device, opening a flow control valve at the lower part of the burette, and controlling the dropping speed of the diluent to be 10-200 microliter/second;
5) after the diluent is completely dripped, closing the shaking table, taking down the beaker, subpackaging the diluted semen in the beaker into 0.25ml plastic thin tubes, and sealing;
6) placing the plastic tubule obtained in the step 5) and the pre-freezing device together in an environment of 5 ℃ for balancing for 4 hours, wherein the plastic tubule is not in contact with the pre-freezing device;
7) quickly placing the plastic tubules completing the step 6) on a pre-freezing device with the height adjusted in advance in an environment of 5 ℃, quickly transferring the pre-freezing device and the plastic tubules to liquid nitrogen steam for fumigating and pre-freezing for 10 minutes, and finally putting the plastic tubules into liquid nitrogen for preservation; the height of the pre-freezing device is the height of a metal net of the pre-freezing device from the liquid level of liquid nitrogen when the pre-freezing device is fumigated in liquid nitrogen steam;
8) when in use, the plastic thin tube is taken out and directly put into a water bath at 37 ℃ for 30-60 seconds for unfreezing.
Further, in the step 2), the beaker which is preheated on the constant temperature water bath shaking table is an empty beaker, or a small amount of diluent is added in advance, and the adding amount does not exceed the amount of fresh semen to be diluted at most.
Further, automatic liquid feeding diluting device includes base (1), be provided with thermostatic water bath shaking table (2) on base (1), install telescopic bracket (4) on base (1), the upper end welding of telescopic bracket (4) has clamp piece (5), the joint has buret (6) in clamp piece (5), the bottom of buret (6) sets up flow control valve (7).
Furthermore, the pre-freezing device is composed of a metal net (13), sawtooth-shaped flanges (14), a frame body (20), a sliding block (17), supporting legs (21) and a handle (16), wherein baffle plates (22) are arranged at two ends of the metal net (13), the baffle plates (22) are fixedly connected to the sliding block (17) through bolts, the sawtooth-shaped flanges (14) are arranged on two side edges of the metal net (13), the sliding block (17) is fixedly connected to two ends of the frame body (20), the sliding block (17) is sleeved on the supporting legs (21), a plurality of height adjusting holes (18) are arranged on the supporting legs (21) at equal intervals, holes with the same size as the height adjusting holes (18) are formed in the sliding block (17), and the supporting legs (21) are fixedly connected through positioning bolts (19); the handle (16) is fixed on the sliding block (17)
Further, the dropping speed of the diluent of the automatic liquid adding and diluting device is controlled to be 50-100 microliter/second.
Furthermore, the rotating speed of the shaking table of the automatic liquid adding and diluting device is set to be 40-120 rpm.
Further, when the semen comes from sheep or goat, the height of the metal mesh of the prefreezing device from the liquid nitrogen level is 4 cm.
Further, when the semen comes from Muscovy ducks, the height of the metal net of the pre-freezing device from the liquid level of the liquid nitrogen is 6 cm.
The invention has the beneficial effects that:
1. the diluent can be automatically added, the standard operation is realized, and the interference of personal manual operation is eliminated: the automatic liquid adding and diluting device provided by the invention utilizes the characteristics of automatic dropping of liquid drops during titration, controllable flow and standardized operation, changes the currently used mode of manually adding diluent into the mode of automatically adding diluent, solves the problem of inconsistent quality of frozen semen of different batches caused by different human operations, and simultaneously verifies that the diluting liquid is automatically added by adopting the titration mode of the invention, so that the diluting effect generated in the semen diluting process can be avoided, and the inactivation probability of the semen in the diluting process is reduced.
2. Different liquid nitrogen gas phase prefreezing heights can be adjusted according to different animals: the gas phase pre-freezing is a small link in the frozen semen preparation process, and is easy to ignore because the time is short, but actually, the air temperatures at different heights on the liquid level of the liquid nitrogen are different, and the resistances of animal sperms of different species to freezing damage are different.
3. The operation is simple, and the cost is low: the automatic liquid adding and diluting device and the height-adjustable pre-freezing device used in the invention have simple structures, can be used by simply modifying the existing instruments in a laboratory, and simultaneously does not change the diluting treatment method before freezing semen in daily work, thereby not influencing the selling price of the frozen semen, but obviously improving the quality of the frozen semen, improving the lambing rate by 8-9% in a production test and obviously improving the production benefit of an animal farm.
Drawings
FIG. 1 is a schematic diagram of an automatic diluting apparatus according to the present invention;
FIG. 2 is a schematic top view of the automatic diluting apparatus;
FIG. 3 is a schematic view of the pre-freezing apparatus according to the present invention;
the various reference numbers in the figures are listed below:
1. a base; 2. a constant-temperature water bath shaking table; 3. a beaker; 4. a telescopic bracket; 5. a clamp member; 6. a burette; 7. a flow control valve; 8. trepanning; 9. a foot pad; 10. a positioning collar; 11. a sleeve; 12. a first adjusting bolt; 13. a metal mesh; 14. a serrated rib; 15. a flange of the supporting leg; 16. a handle; 17. a slider; 18. a height adjustment hole; 19. positioning the bolt; 20. a frame body; 21. a support leg; 22. and a baffle plate.
Detailed Description
The method for diluting semen before freezing will be described in detail with reference to the accompanying drawings.
Example 1 comparative test of the Effect of manually and automatically added Diluent on sheep frozen semen quality
Collecting semen of 10 adult Yunnan semi-fine wool sheep by electric stimulation, and rapidly sending to laboratory for quality inspection of fresh fine product, wherein the semen activity is above 75%, and the semen density is 2.5 × 109Mixing semen meeting the standard uniformly, eliminating the difference between individuals, then averagely dividing the semen into 12 parts, selecting 3 operators, manually adding diluent into 3 parts of semen by each operator, automatically adding diluent into the other 3 parts of semen by 3 automatic liquid adding and diluting devices prepared in a laboratory, and specifically grouping the following components into an operator 1 (conventional manual dilution), an operator 2 (conventional manual operation), an operator 3 (conventional manual dilution) and an automatic adding group (dilution speed 50 microliter/second + shaking table speed 50rpm), setting the final dilution density of 12 parts of semen to be 2.0 × 108/ml。
The dilution treatment steps are as follows: putting the fresh semen into a 50ml beaker 3, placing the beaker in advance into a positioning lantern ring 10 of a constant-temperature water bath shaking table 2 of an automatic liquid adding dilution device for preheating, wherein the beaker is an empty beaker, and the temperature is set to be 36-40 ℃; opening the constant-temperature water bath shaking table 2, and setting the rotating speed to be 50 rpm; adding the diluent into a burette 6 of the automatic liquid adding and diluting device, opening a flow control valve 7 at the lower part of the burette, and controlling the dropping speed of the diluent to be 50 microliters/second; after the diluent is completely dripped, closing the constant temperature water bath shaking table 2, taking down the beaker 3, subpackaging the diluted semen in the beaker into 0.25ml plastic thin tubes, and sealing; placing the plastic thin tube and the pre-freezing device together in an environment with the temperature of 5 ℃ for balancing for 4 hours, wherein the plastic thin tube is not in contact with the pre-freezing device; the height of the pre-freezing device is adjusted in advance, and the distance between the metal net 13 and the liquid level of liquid nitrogen is 4 cm; after the balance is finished, the prefreezing device and the plastic thin tube are quickly transferred into liquid nitrogen steam together by holding the handles 16 at the two sides to fumigate and prefreeze for 10 minutes, and finally the prefreezing device is put into liquid nitrogen to be stored. When in unfreezing, the plastic tubule is directly put into a water bath with the temperature of 37 ℃ for 30-60 seconds for unfreezing, and the activity rate and activity of the unfrozen semen are detected.
TABLE 1 influence of Manual and automatic dilution on frozen semen quality
| Group of | Activity (%) | Activity (%) |
| |
59.38±4.35 | 32.75±2.47 |
| |
58.58±4.52 | 33.38±2.41 |
| |
58.04±3.85 | 33.91±4.19 |
| Automatic dilution set | 61.90±0.56 | 41.79±1.11 |
As can be seen from Table 1, for the same animal species and the same diluent, the semen is diluted by the automatic liquid adding dilution device and then frozen, the frozen semen quality is obviously better than that of the frozen semen by manual dilution, the motility rate of the thawed semen is higher than that of the sperm diluted by manual dilution by 2-3 percentage points, and the motility rate of the sperm is higher than that of the sperm diluted by manual dilution by 8-9 percentage points. The technical scheme adopts a titration mode, liquid is added at a constant speed, only semen exists in a beaker before titration, or a small amount of diluent exists, so that the situation that the liquid adding amount of the diluent is too large in unit time is ensured not to occur, and the sperm inactivation caused by the dilution effect is avoided.
Example 2 Effect of prefreezing device height on sheep jelly vigor
Collecting semen of 5 adult Yunnan semi-fine wool sheep by electrical stimulation, and rapidly sending to laboratory for quality inspection of fresh fine product, wherein the semen activity is above 75%, and the semen density is 2.5 × 109The semen with distortion rate below 10% is frozen and stored, and the automatic liquid adding and diluting device is assembled in advance for standby use, the diluting conditions are that the diluting speed is 50 microliter/second and the shaking table speed is 50rpm, the shaking table temperature is 38 ℃, and the density of the finally diluted semen is 4.0 × 108And/ml. Packaging the diluted semen into 0.25ml plastic fine tube, sealing, slowly cooling to 5 deg.C, and balancing for 4 hr at the temperatureThen (c) is performed. The test components were divided into 4 groups, and the distances between the metal mesh and the liquid nitrogen surface of the prefreezing apparatus were set to 2, 4, 6, and 8cm, respectively. Placing the plastic tubules on the metal mesh of each group of pre-freezing devices, then fumigating and pre-freezing in liquid nitrogen steam for 10 minutes, and finally putting into liquid nitrogen for preservation. The automatic priming dilution unit and prefreezer were operated as in example 1. When unfreezing, the plastic thin tube is directly put into a 38 ℃ water bath for 30 seconds to unfreeze, and the activity of the unfrozen semen is detected. The specific results are shown in Table 2.
TABLE 2 Effect of prefreezing height on sheep Freeze sperm viability (Unit:%)
| Grouping | No. 1 sheep | No. 2 sheep | No. 3 sheep | No. 4 sheep | No. 5 sheep | Mean±SD |
| 2cm | 31.57 | 32.16 | 31.28 | 34.09 | 33.92 | 32.60±1.32 |
| 4cm | 40.57 | 39.58 | 38.12 | 41.68 | 40.23 | 40.04±1.31 |
| 6cm | 38.47 | 39.52 | 37.19 | 39.51 | 38.37 | 38.61±0.97 |
| 8cm | 38.26 | 35.13 | 34.18 | 36.27 | 35.25 | 35.82±1.55 |
It can be seen from table 2 that the quality of liquid nitrogen gaseous phase prefreezing is highly relevant with prefreezing, and for sheep seminal fluid, when the metal mesh was 4cm away from the liquid nitrogen face during prefreezing, the sperm vigor was the highest after unfreezing, and the vigor was the worst when highly being 2cm, because this height was too close to the liquid nitrogen liquid level, gaseous phase temperature was lower, and the freezing damage that produces the sperm is also strongest big, but the vigor also can descend when the prefreezing height was higher, because the prefreezing temperature is not enough, also can influence the guard action that the diluent produced after the sperm dropped into the liquid nitrogen.
Example 3 influence of prefreezing device height on goat frozen sperm viability
The artificial vagina method is adopted to collect the semen of 5 adult black goat rams on the clouds and the semen is rapidly sent to a laboratory for fresh semen quality inspection. Sperm motility is 7Over 5 percent, the sperm density is 2.5 × 109The semen with distortion rate below 10% is frozen and stored, and the automatic liquid adding and diluting device is assembled in advance for standby use, the diluting condition is that the diluting speed is 80 microliter/second and the shaking table speed is 60rpm, the shaking table temperature is set to be 38 ℃, the density of the diluted semen is 4.0 × 108And/ml. And (3) subpackaging the diluted semen into a 0.25ml plastic thin tube, sealing, slowly cooling to 5 ℃, and balancing at the temperature for 4 hours. The test components were divided into 4 groups, and the distances between the metal mesh and the liquid nitrogen surface of the prefreezing apparatus were set to 2, 4, 6, and 8cm, respectively. Placing the plastic tubules on the metal mesh of each group of pre-freezing devices, then fumigating and pre-freezing in liquid nitrogen steam for 7 minutes, and finally putting into liquid nitrogen for preservation. The automatic priming dilution unit and prefreezer were operated as in example 1. When unfreezing, the plastic thin tube is directly put into a 38 ℃ water bath for 30 seconds to unfreeze, and the activity of the unfrozen semen is detected. The specific results are shown in Table 3.
TABLE 3 Effect of prefreezing height on goat Freeze sperm motility (Unit:%)
| Grouping | No. 1 sheep | No. 2 sheep | No. 3 sheep | No. 4 sheep | No. 5 sheep | Mean±SD |
| 2cm | 34.37 | 32.61 | 34.07 | 36.21 | 32.17 | 33.89±1.60 |
| 4cm | 38.93 | 40.22 | 41.38 | 42.57 | 38.91 | 40.40±1.59 |
| 6cm | 38.57 | 38.21 | 41.04 | 40.23 | 35.17 | 38.64±2.26 |
| 8cm | 37.92 | 38.14 | 40.57 | 38.12 | 35.04 | 37.96±1.96 |
As can be seen from Table 3, for goat semen, as with sheep semen, when the distance between the metal mesh and the liquid nitrogen surface is 4cm during prefreezing, the motility of thawed sperm is the highest, the motility is the worst when the height is 2cm, and the motility is also reduced when the prefreezing height is higher.
Example 4
Collecting semen of 5 adult Muscovy ducks by abdominal massage, and rapidly sending to laboratory for quality inspection of fresh refined product, wherein the semen activity is above 75%, and the semen density is 3.0 × 109The semen with distortion rate below 15% is frozen and stored, the automatic liquid adding and diluting device is assembled in advance for standby, the diluting condition is that the diluting speed is 40 microliter/second and the shaking table speed is 40rpm, the shaking table temperature is set to 37 ℃, the density of the finally diluted semen is 1.0 × 109And/ml. And (3) subpackaging the diluted semen into a 0.25ml plastic thin tube, sealing, slowly cooling to 5 ℃, and balancing at the temperature for 4 hours. The distances between the freezing bracket and the liquid nitrogen surface are set to be 2, 4, 6, 8 and 10 cm. Placing the plastic tubules on the metal mesh of each group of pre-freezing devices, then fumigating and pre-freezing in liquid nitrogen steam for 10 minutes, and finally putting into liquid nitrogen for preservation. The automatic priming dilution unit and prefreezer were operated as in example 1. When unfreezing, the plastic thin tube is directly put into a water bath with the temperature of 5 ℃ for 5min to unfreeze. And (5) performing activity detection on the thawed semen. The specific results are shown in Table 4.
TABLE 4 Effect of prefreezing altitude on Muscovy Duck Freeze sperm motility (Unit:%)
| Grouping | No. 1 duck | No. 2 duck | No. 3 duck | No. 4 duck | No. 5 duck | Mean±SD |
| 2cm | 30.13 | 32.51 | 28.29 | 27.17 | 30.86 | 29.79±2.11 |
| 4cm | 33.92 | 34.27 | 31.26 | 35.17 | 26.59 | 32.24±3.48 |
| 6cm | 39.32 | 40.57 | 37.81 | 37.38 | 40.01 | 39.02±1.38 |
| 8cm | 38.53 | 40.16 | 37.27 | 36.21 | 38.83 | 38.20±1.51 |
| 10cm | 36.82 | 37.01 | 35.00 | 32.67 | 34.13 | 35.13±1.83 |
As can be seen from Table 4, when the pre-freezing height is 6cm, the vitality of the frozen Muscovy duck semen after being thawed is the best, which also indicates that the pre-freezing height has a significant influence on the quality of the frozen semen in the liquid nitrogen gas phase pre-freezing step when the semen of different species is subjected to cryopreservation. Therefore, different prefreezing heights need to be set according to species, and the prefreezing device also needs to be prepared so that the height can be adjusted, rather than simply using the current metal pedestal with the fixed height.
EXAMPLE 5 Effect of dilution titration Rate and shaker rotation Rate on goat frozen sperm quality
Collecting semen of 5 black goat rams on adult clouds by using artificial vagina method, rapidly sending to laboratory for quality inspection of fresh fine product, wherein semen activity is above 75%, and semen density is 2.5 × 109The semen with distortion rate below 10% is frozen and stored, and the automatic liquid adding and diluting device is assembled in advance for standby use, and the automatic liquid adding and diluting device is specifically divided into a control group (conventional manual dilution and shaking), a group 1 (dilution speed 10 microliter/second + shaking table speed 10rpm), a group 2 (dilution speed 50 microliter/second + shaking table speed 40rpm), a group 3 (dilution speed 80 microliter/second + shaking table speed 60rpm), a group 4 (dilution speed 100 microliter/second + shaking table speed 120rpm), a group 5 (dilution speed 200 microliter/second + shaking table speed 220rpm), and the density of the finally diluted semen is 2.0 × 108And/ml. And (3) subpackaging the diluted semen into 0.25ml plastic thin tubes, sealing, slowly cooling to 5 ℃, and balancing with the pre-freezing device for 4 hours at the temperature. The height of the pre-freezing device is adjusted to be 4cm away from the liquid level of liquid nitrogen in advance, the thin tube is placed on the metal net after the balance is finished, then the thin tube is fumigated and pre-frozen in liquid nitrogen steam for 7 minutes, and finally liquid nitrogen is added for preservation. The automatic priming dilution unit and prefreezer were operated as in example 1. When unfreezing, the plastic thin tube is directly put into a water bath at 37 ℃ for 30-60 seconds to unfreeze. The thawed semen is tested for viability, acrosome and plasma membrane integrity and mitochondrial activity。
TABLE 5 influence of dilution titration speed and shaker rotation speed on the quality of goat frozen sperm (unit:%)
| Group of | Rate of activity | Vitality of the body | Integrity of roof | Plasma membrane integrity | Mitochondrial Activity |
| Control group | 58.79±4.83 | 35.19±6.23 | 51.67±8.31 | 32.93±8.15 | 47.43±4.96 |
| |
62.39±8.14 | 39.57±5.14 | 55.94±3.87 | 38.67±4.33 | 51.17±6.58 |
| |
67.77±7.51 | 41.69±6.59 | 57.13±7.62 | 39.51±6.17 | 53.91±5.42 |
| |
65.92±6.24 | 44.73±3.96 | 56.41±9.34 | 41.53±4.91 | 53.29±7.14 |
| |
65.67±3.27 | 43.57±4.93 | 57.49±8.37 | 41.87±5.26 | 52.93±5.02 |
| Group 5 | 64.86±7.58 | 42.49±3.27 | 55.92±7.57 | 40.57±6.31 | 51.36±3.95 |
As can be seen from Table 5, the method of the present invention, when used to dilute goat semen followed by freezing and thawing, can increase the motility rate and activity of frozen semen, and can also significantly improve the plasma membrane and acrosome state and increase the mitochondrial activity. When the titration speed is 10-200 microliter/second and the rotating speed of the shaking table is 10-220rpm, the quality of the obtained frozen semen is obviously better than that of the manual dilution group of the contrast, wherein the quality of the frozen semen obtained under the dilution conditions of the group 2, the group 3 and the group 4 is better than that of the group 1 and the group 5.
EXAMPLE 6 Effect of dilution titration Rate and shaker rotation Rate on sheep frozen semen quality
Collecting semen of 6 adult Yunnan semi-fine wool sheep by electric stimulation, and rapidly sending to laboratory for quality inspection of fresh fine product, wherein the semen activity is above 75%, and the semen density is 2.5 × 109The semen with distortion rate below 10% is frozen and stored, and the automatic liquid adding and diluting device is assembled in advance for standby use, and is specifically divided into a control group (conventional manual dilution and shaking), a group 1 (dilution speed 10 microliter/second + shaking table speed 20rpm), a group 2 (dilution speed 50 microliter/second + shaking table speed 50rpm), a group 3 (dilution speed 120 microliter/second + shaking table speed 100rpm), a group 4 (dilution speed 180 microliter/second + shaking table speed 200rpm), and the semen density after final dilution is 2.0 × 108And/ml. And (3) subpackaging the diluted semen into 0.25ml plastic thin tubes, sealing, slowly cooling to 5 ℃, and balancing with the pre-freezing device for 4 hours at the temperature. The height of the pre-freezing device is adjusted to be 4cm away from the liquid level of liquid nitrogen in advance, the thin tube is placed on the metal net after the balance is finished, then the thin tube is fumigated and pre-frozen in liquid nitrogen steam for 10 minutes, and finally liquid nitrogen is added for preservation. The automatic priming dilution unit and prefreezer were operated as in example 1. When unfreezing, the plastic thin tube is directly put into a water bath at 37 ℃ for 30-60 seconds to unfreeze. The thawed semen is tested for viability, acrosome and plasma membrane integrity.
TABLE 6 influence of dilution titration speed and shaker rotation speed on sheep frozen semen quality (Unit:%)
| Group of | Rate of activity | Vitality of the body | Integrity of roof | Plasma membrane integrity |
| Control group | 61.37±8.32 | 34.67±9.02 | 52.03±5.67 | 34.09±5.07 |
| |
64.76±5.79 | 37.85±3.51 | 57.81±7.91 | 39.53±5.73 |
| |
64.53±7.02 | 42.11±4.89 | 56.54±9.00 | 38.96±7.29 |
| |
65.12±7.43 | 42.52±6.57 | 58.04±6.74 | 40.38±3.69 |
| |
64.91±4.75 | 41.83±9.28 | 57.29±3.73 | 38.92±5.13 |
As can be seen from Table 6, when the sheep semen is diluted by the method of the present invention and then frozen and thawed, the motility rate and activity of the frozen semen can be improved, and the plasma membrane and acrosome states can be significantly improved. When the titration speed is 10-200 microliter/second and the rotating speed of the shaking table is 10-220rpm, the quality of the obtained frozen semen is obviously better than that of a control manual dilution group, wherein the sperm motility of the frozen semen obtained under the dilution conditions of the group 2, the group 3 and the group 4 after thawing is better than that of the group 1.
Example 7 production test
Collecting semen of 5 black goat rams on the cloud by using a pseudo-vaginal method, rapidly sending to a laboratory for quality inspection of fresh fine products, wherein the activity of the semen is more than 75%, and the density of the semen is 2.5 × 109The semen with distortion rate below 10% is frozen and stored, and the automatic liquid adding and diluting device is assembled in advance for standby use, and is specifically divided into a control group (conventional manual dilution and shaking), a group 1 (dilution speed of 20 microliter/second + shaking table speed of 40rpm), and a group 2 (dilution speed of 50 microliter/second + shaking table speed of 100rpm), wherein the density of the semen after final dilution is 2.0 × 108And/ml. And (3) subpackaging the diluted semen into 0.25ml plastic thin tubes, sealing, slowly cooling to 5 ℃, and balancing with the pre-freezing device for 4 hours at the temperature. The automatic priming dilution unit and prefreezer were operated as in example 1. The height of the pre-freezing device is adjusted to be 4cm away from the liquid level of liquid nitrogen in advance, the thin tube is placed on the metal net after the balance is finished, then the thin tube is fumigated and pre-frozen in liquid nitrogen steam for 7 minutes, and finally liquid nitrogen is added for preservation.
Carrying out artificial insemination experiments in a Zhuqing sheep farm in Huizhou county, when frozen semen is unfrozen, directly putting a plastic thin tube into a water bath at 37 ℃ for 30-60 seconds for unfreezing, firstly carrying out survival rate and activity detection on the unfrozen semen, then carrying out artificial insemination, wherein 364 local black goats are naturally estrated in all experimental ewes (4-5 months in 2019). Artificial insemination is carried out 18 hours after the ewes have oestrus, cervical deep insemination is carried out 2 times 12 hours later, and lambing rate is counted (calculation: number of ewes for final lambing/total number of sheep multiplied by 100%). The specific results are shown in Table 7.
TABLE 7 Effect of semen dilution method of the present invention on the lambing rate in actual production
| Group of | Activity (%) | Activity (%) | Percentage of lambing (%) |
| Control group | 58.94±6.53 | 35.27±9.15 | 48.33(58/120) |
| |
64.70±5.39 | 40.46±6.59 | 52.46(64/122) |
| |
63.89±5.04 | 41.37±8.41 | 51.64(63/122) |
As can be seen from Table 7, after semen is diluted by the method of the present invention and then frozen and thawed, the activity rate and vitality of frozen semen can be improved, and meanwhile, the lambing rate is improved to more than 50%, which is 8-9% higher than that of the existing semen dilution method.
The semen freezing and diluting method capable of automatically adding liquid and adjusting the prefreezing height provided by the invention is described in detail above. The principles and embodiments of the present invention are explained herein in terms of specific embodiments, the foregoing description being merely included to assist in understanding the method and its core concepts of the present invention. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (8)
1. A semen freezing and diluting method capable of automatically adding liquid and adjusting pre-freezing height is characterized by comprising the following steps:
1) collecting fresh semen for quality inspection, wherein sperm activity is more than 75%, and sperm density is more than 2.5 × 109The product has a distortion rate of less than 10% and can be used for cryopreservation;
2) putting the fresh semen which is obtained in the step 1) and accords with the freezing preservation condition into a 50ml beaker (3), wherein the beaker is placed on a constant-temperature water bath shaking table of an automatic liquid adding dilution device in advance for preheating, and the temperature is set to be 36-40 ℃;
3) opening the shaking table of the automatic liquid adding and diluting device, and setting the rotating speed to be 10-220 rpm;
4) adding the diluent into a burette of the automatic liquid adding diluting device, opening a flow control valve at the lower part of the burette, and controlling the dropping speed of the diluent to be 10-200 microliter/second;
5) after the diluent is completely dripped, closing the shaking table, taking down the beaker, subpackaging the diluted semen in the beaker into 0.25ml plastic thin tubes, and sealing;
6) placing the plastic tubule obtained in the step 5) and the pre-freezing device together in an environment of 5 ℃ for balancing for 4 hours, wherein the plastic tubule is not in contact with the pre-freezing device;
7) quickly placing the plastic tubules completing the step 6) on a pre-freezing device with the height adjusted in advance in an environment of 5 ℃, quickly transferring the pre-freezing device and the plastic tubules to liquid nitrogen steam for fumigating and pre-freezing for 10 minutes, and finally putting the plastic tubules into liquid nitrogen for preservation; the height of the pre-freezing device is the height of a metal net of the pre-freezing device from the liquid level of liquid nitrogen when the pre-freezing device is fumigated in liquid nitrogen steam;
8) when in use, the plastic thin tube is taken out and directly put into a water bath at 37 ℃ for 30-60 seconds for unfreezing.
2. The semen freezing and diluting method capable of automatically adding liquid and adjusting the pre-freezing height as claimed in claim 1, wherein in step 2), the preheated beaker placed on the shaking table with constant temperature water bath is an empty beaker, or a small amount of diluent is added in advance, and the adding amount does not exceed the amount of fresh semen to be diluted at most.
3. The semen freezing and diluting method capable of automatically adding liquid and adjusting the pre-freezing height according to claim 1, wherein the automatic liquid adding and diluting device comprises a base (1), a constant temperature water bath shaking table (2) is arranged on the base (1), a telescopic bracket (4) is installed on the base (1), a clamp member (5) is welded at the upper end of the telescopic bracket (4), a burette (6) is clamped in the clamp member (5), and a flow control valve (7) is arranged at the bottom end of the burette (6).
4. The semen freezing and diluting method capable of automatically adding liquid and adjusting the pre-freezing height according to claim 1, it is characterized in that the pre-freezing device consists of a metal net (13), a saw-toothed flange (14), a frame body (20), a slide block (17), a supporting leg (21) and a handle (16), two ends of the metal net (13) are provided with baffle plates (22), the baffle plates (22) are fixedly connected on the sliding block (17) through bolts, two sides of the metal net (13) are provided with sawtooth flanges (14), two ends of the frame body (20) are fixedly connected with sliding blocks (17), the sliding block (17) is sleeved on the supporting leg (21), a plurality of height adjusting holes (18) are formed in the supporting leg (21) at equal intervals, holes with the same size as the height adjusting holes (18) are formed in the sliding block (17), and the sliding block is fixedly connected with the supporting leg (21) through a positioning bolt (19); the handle (16) is fixed on the sliding block (17).
5. The semen freezing and diluting method capable of automatically adding liquid and adjusting prefreezing height according to claim 1, wherein the dropping speed of the diluent of the automatic liquid adding and diluting device is controlled to be 50-100 microliter/second.
6. The semen freezing and diluting method capable of automatically adding liquid and adjusting prefreezing height according to claim 1, wherein the rotating speed of the shaking table of the automatic liquid adding and diluting device is set to 40-120 rpm.
7. The semen freezing and diluting method capable of automatically adding liquid and adjusting prefreezing height according to claim 1, characterized in that when the semen comes from sheep or goat, the height of the metal mesh (13) of the prefreezing device from the liquid nitrogen level is 4 cm.
8. The semen freezing and diluting method capable of automatically adding liquid and adjusting the prefreezing height according to claim 1, wherein when the semen comes from Muscovy ducks, the height of the metal net (13) of the prefreezing device from the liquid level of the liquid nitrogen is 6 cm.
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| CN202010551672.7A CN111602650B (en) | 2020-06-16 | 2020-06-16 | Semen freezing and diluting method capable of automatically adding liquid and adjusting pre-freezing height |
| AU2021103370A AU2021103370A4 (en) | 2020-06-16 | 2021-06-15 | Semen freezing and diluting method capable of automatic liquid adding and pre-freezing height adjusting |
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| CN202010551672.7A CN111602650B (en) | 2020-06-16 | 2020-06-16 | Semen freezing and diluting method capable of automatically adding liquid and adjusting pre-freezing height |
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| CN112842612A (en) * | 2020-12-31 | 2021-05-28 | 江苏中牧倍康药业有限公司 | Quantitative dripping device of full-function boar-strengthening semen dilution storage machine |
| CN112889808A (en) * | 2021-01-21 | 2021-06-04 | 肖广庆 | Cryopreservation method of sheep tubule semen |
| CN114088519A (en) * | 2021-10-22 | 2022-02-25 | 风帆有限责任公司 | Device and method for testing wet resilience of AGM (absorptive glass mat) separator under different compression amounts |
| CN114348321A (en) * | 2022-01-26 | 2022-04-15 | 江苏集萃药康生物科技股份有限公司 | Automatic tubulation equipment of animal genetic material |
| CN115245402A (en) * | 2022-09-21 | 2022-10-28 | 山东四方新域农牧科技股份有限公司 | Automatic fertilization equipment for rabbit intensive breeding |
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| CN209420750U (en) * | 2019-01-08 | 2019-09-24 | 云南省畜牧兽医科学院 | A kind of height-adjustable frozen semen pre-freeze metal frame |
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| CN203616255U (en) * | 2013-12-10 | 2014-05-28 | 许苗苗 | Chemical titration end point judgment instrument for fertilizer check |
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| CN208809929U (en) * | 2018-09-20 | 2019-05-03 | 深圳市泉晟生物科技有限公司 | A kind of full-automatic semen diluent preparing machine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112842612A (en) * | 2020-12-31 | 2021-05-28 | 江苏中牧倍康药业有限公司 | Quantitative dripping device of full-function boar-strengthening semen dilution storage machine |
| CN112842612B (en) * | 2020-12-31 | 2022-10-11 | 江苏中牧倍康药业有限公司 | Quantitative dripping device of full-function boar-strengthening semen dilution storage machine |
| CN112889808A (en) * | 2021-01-21 | 2021-06-04 | 肖广庆 | Cryopreservation method of sheep tubule semen |
| CN114088519A (en) * | 2021-10-22 | 2022-02-25 | 风帆有限责任公司 | Device and method for testing wet resilience of AGM (absorptive glass mat) separator under different compression amounts |
| CN114348321A (en) * | 2022-01-26 | 2022-04-15 | 江苏集萃药康生物科技股份有限公司 | Automatic tubulation equipment of animal genetic material |
| CN114348321B (en) * | 2022-01-26 | 2023-08-04 | 江苏集萃药康生物科技股份有限公司 | Automatic tubing equipment for animal genetic material |
| CN115245402A (en) * | 2022-09-21 | 2022-10-28 | 山东四方新域农牧科技股份有限公司 | Automatic fertilization equipment for rabbit intensive breeding |
| CN115245402B (en) * | 2022-09-21 | 2022-12-09 | 山东四方新域农牧科技股份有限公司 | Automatic fertilization equipment for rabbit intensive breeding |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111602650B (en) | 2021-12-24 |
| AU2021103370A4 (en) | 2021-08-12 |
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