CN114586771A - Cryopreservation method of pig semen - Google Patents
Cryopreservation method of pig semen Download PDFInfo
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- CN114586771A CN114586771A CN202210259827.9A CN202210259827A CN114586771A CN 114586771 A CN114586771 A CN 114586771A CN 202210259827 A CN202210259827 A CN 202210259827A CN 114586771 A CN114586771 A CN 114586771A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
Abstract
The invention relates to a method for freezing and preserving boar semen, which comprises the steps of firstly preparing a freezing and diluting solution I, a freezing and diluting solution II and a diluent BTS, then carrying out hydrotreating on the three solutions, after semen collection, diluting the boar semen by the hydrotreated diluent BTS with the constant temperature of 1: 1, then balancing for 2-4h at the temperature of 17 ℃, centrifugally removing supernatant, diluting a sperm precipitate by the isothermal hydrotreated freezing and diluting solution I, then cooling and balancing to 4 ℃, finally diluting by the isothermal hydrotreated freezing and diluting solution II with the ratio of 1: 1, subpackaging and sealing the diluted semen by freezing tubules, then placing the sperm precipitate in a box of a program freezing instrument for freezing, and after freezing, transferring the freezing tubules into liquid nitrogen for preservation. The method is simple to operate, can effectively improve the thawing activity of the frozen semen of the pig and the integrity rate of the acrosome of the semen, can reduce the degree of oxidative damage of the semen, and overcomes the problem that the semen is easy to suffer oxidative damage in a longer cooling balance process.
Description
Technical Field
The invention belongs to the technical field of livestock breeding and mammal semen cryopreservation, and relates to a method for cryopreservation of pig semen.
Background
In recent years, the pig semen cryopreservation technology has been developed. The frozen pig semen (abbreviated as frozen pig semen) can achieve the effect of preserving the semen at the temperature close to the normal temperature by adopting a deep-position semen deposition method, and also starts to step into the process of commercial production and popularization and application after cattle.
Compared with other animals, the pig sperms are more sensitive to low temperature, so the frozen sperm production needs longer cooling balance time. However, in a long temperature-reducing balance process, a large amount of Reactive Oxygen Species (ROS) are generated by the respiratory metabolism of the sperms, and the oxidation damage caused by the ROS is a main reason for the sharp reduction of the quality of the frozen sperms of the pigs. Therefore, how to inhibit the oxidation damage problem suffered by the sperms is the key for improving the frozen sperm quality of the pigs.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provide a method for preserving frozen pig semen, which can improve the thawing activity of the frozen pig semen, improve the integrity of the acrosome of the pig semen and reduce the degree of oxidative damage of the pig semen.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
a cryopreservation method of pig semen comprises the following steps:
(1) preparing a freezing base solution TCG, adding 20-25% of egg yolk into the freezing base solution, uniformly stirring to obtain a freezing dilution solution I, adding 6% of glycerol and 0.4-0.6% of OEP (Orvus Es past) into the freezing dilution solution I, and uniformly stirring to obtain a freezing dilution solution II;
(2) respectively placing the diluent BTS, the frozen diluent I and the frozen diluent II in a closed container, and introducing hydrogen for hydrotreating to obtain hydrotreated diluent BTS, frozen diluent I and frozen diluent II;
(3) semen collection, namely diluting the boar semen by using a diluent BTS after hydrotreating at the constant temperature of 1: 1 to obtain pre-diluted semen;
(4) balancing the pre-diluted semen for 2-4h at 17 ℃, centrifuging, removing supernatant, diluting the sperm precipitate with isothermal and hydrotreated frozen diluent I, and diluting until the sperm density is 20 × 108Cooling to 4 ℃, balancing, and finally diluting with isothermal and hydrotreated frozen diluent II in a ratio of 1: 1 to obtain diluted semen;
(5) and subpackaging and sealing the diluted semen by using a freezing tubule, then placing the packaged semen into a box body of a program freezing instrument for freezing, and after the frozen semen is frozen in a cold mode, transferring the freezing tubule into liquid nitrogen for storage.
In the step (1), the preparation method of the freezing base liquid TCG comprises the following steps: 1.1g of glucose, 1.48g of citric acid, 2.42g of Tris, 0.06g of penicillin sodium and 0.1g of streptomycin sulfate are weighed and dissolved in double distilled water, and after stirring and dissolving, the volume is fixed to 100 mL.
In the step (2), the preparation method of the diluent BTS comprises the following steps: 3.7g of glucose, 0.6g of trisodium citrate dihydrate, 0.125g of EDTA0, 0.125g of sodium bicarbonate, 0.075g of potassium chloride, 0.06g of sodium penicillin and 0.1g of streptomycin sulfate are weighed and dissolved in double distilled water, then the volume is fixed to 100mL, the pH value is adjusted to 7.2, and the osmotic pressure is 310 mosm/L.
Further, in the step (2), during the hydrotreatment, the pressure in the container is 0.6-0.8 MPa, and the treatment time is 2-5 h.
Further, in the step (4), the centrifugation conditions are as follows: at 17 ℃, 800-2300 g for 3-15 min.
Further, in the step (5), the freezing procedure is as follows: the temperature is reduced to-5 ℃ at the speed of 6 ℃/min after the temperature is maintained at 4 ℃ for 30min, then reduced to-80 ℃ at the speed of 40 ℃/min after the temperature is maintained at-5 ℃, and then reduced to-145 ℃ at the speed of 60 ℃/min after the temperature is maintained at-80 ℃ for 30s, thus completing the freezing.
The invention has the beneficial effects that:
the invention provides a method for freezing and preserving pig semen, which is simple to operate, can effectively improve the thawing activity of the pig frozen semen and the integrity rate of sperm acrosome, can reduce the degree of sperm oxidative damage, and solves the problems that the preparation of the pig frozen semen needs longer cooling balance time and the sperm is easy to suffer oxidative damage in the prior art.
Detailed Description
The following examples are given solely for the purpose of illustrating the invention more clearly, and it is to be understood that modifications and variations may be made by persons skilled in the art in light of the above teachings and that all such modifications and variations are within the scope of the invention as defined in the appended claims.
Example 1
A cryopreservation method of pig semen comprises the following steps:
(1) preparing a freezing base solution TCG, adding 20-25% of egg yolk into the freezing base solution, uniformly stirring to obtain a freezing dilution solution I, adding 6% of glycerol and 0.4-0.6% of OEP (Orvus Es past) into the freezing dilution solution I, and uniformly stirring to obtain a freezing dilution solution II;
the preparation method of the freezing base liquid TCG comprises the following steps: 1.1g of glucose, 1.48g of citric acid, 2.42g of Tris, 0.06g of penicillin sodium and 0.1g of streptomycin sulfate are weighed and dissolved in double distilled water, and after stirring and dissolving, the volume is fixed to 100 mL.
(2) Preparation of a diluent BTS: 3.7g of glucose, 0.6g of trisodium citrate dihydrate, 0.125g of EDTA0, 0.125g of sodium bicarbonate, 0.075g of potassium chloride, 0.06g of sodium penicillin and 0.1g of streptomycin sulfate are weighed and dissolved in double distilled water, then the volume is determined to be 100mL, the pH value is adjusted to be 7.2, and the osmotic pressure is 310 mosm/L;
respectively placing the diluent BTS, the frozen diluent I and the frozen diluent II in a closed container, introducing hydrogen, and treating for 4 hours under 0.7MPa to obtain the hydrotreated diluent BTS, the frozen diluent I and the frozen diluent II;
(3) collecting semen, wherein the semen is from 3 Duroc breeding boars of 1.5-2 years old and good in reproductive performance, collecting the semen by using a semi-automatic semen collection system, and diluting the semen by using a hydrotreated diluent BTS (BTS) at a constant temperature of 1: 1 (volume ratio) to obtain pre-diluted semen;
(4) equilibrating the pre-diluted semen at 17 deg.C for 3h, centrifuging (17 deg.C, 1100g, 9min), removing supernatant, diluting the sperm precipitate with isothermal, hydrotreated, frozen diluent I, and diluting to sperm density of 20 × 108Cooling to 4 deg.C, and diluting with isothermal and hydrotreated frozen diluent II solution at a volume ratio of 1: 1 to obtain diluted semen;
(5) subpackaging and sealing the diluted semen by using freezing tubules, then placing the packaged semen into a box body of a program freezing instrument for freezing, starting the program freezing instrument for the semen in advance, reducing the temperature in the box body to 4 ℃, and carrying out a freezing program as follows: the temperature is reduced to-5 ℃ at the speed of 6 ℃/min after the temperature is maintained at 4 ℃ for 30min, then reduced to-80 ℃ at the speed of 40 ℃/min after the temperature is maintained at-5 ℃, the temperature is reduced to-145 ℃ at the speed of 60 ℃/min after the temperature is maintained at-80 ℃ for 30s, and after the temperature is frozen, the freezing tubule is moved to liquid nitrogen for storage.
Comparative example 1
And (3) respectively placing the diluent BTS, the frozen diluent I and the frozen diluent II in the step (2) in a closed container, introducing hydrogen, and treating for 4 hours under 0.7MPa to obtain the hydrotreated diluent BTS, the frozen diluent I and the frozen diluent II, deleting the diluent BTS, the frozen diluent I and the frozen diluent II, and performing no hydrotreatment, wherein the rest is the same as that in the example 1.
Unfreezing and evaluating the quality of the frozen semen of the thin tube:
the frozen tubules were quickly removed from the liquid nitrogen tank with forceps, thawed in a 50 ℃ water bath for 16s, the surface beads of the tubules were wiped off with a paper towel, the ends of the tubules were cut, semen (0.5mL) was mixed with the thawing solution (4.5mL, 35 ℃), and then the following indices were tested:
(1) sperm motility
The thawed semen is put in water bath at 37 ℃ for 15min, then 10 mu L of semen is taken and put on a special sperm counting plate, and the sperm motility is detected by utilizing a computer aided analysis system (CASA).
(2) Percentage of sperm acrosome integrity
After dyeing by using FITC-PNA dye solution, observing the shape of a sperm acrosome by using a fluorescence microscope, wherein the specific method comprises the following steps:
adding the unfrozen semen onto the liquid surface of 2mL of 3% polyvinylpyrrolidone, centrifuging at 1500rpm for 6min, and removing the supernatant; washing with phosphate buffer solution for 2 times, centrifuging at 1500rpm for 6min, and removing supernatant; resuspending the sperm with phosphate buffered saline (37 ℃) and adjusting the density to 1-2X 106Per mL; sucking 30 μ L of sperm suspension, smearing, air drying, and fixing with methanol for 10 min; adding 30 mu LFITC-PNA dye solution, and incubating for 30min at 37 ℃ in a dark and humid environment; rinsing with phosphate buffered saline, air drying, dropping a small amount of the coverslipping agent, covering with a cover slip and sealing with colorless nail polish, and observing as soon as possible under a 400 × fluorescence microscope. 200-300 sperms are checked each time, and the process is repeated for 5 times.
(3) Total antioxidant Capacity (T-AOC) assay
And (3) detecting the antioxidant capacity of the semen by using a T-AOC colorimetric detection kit (Nanjing Kangkui, A015-1) (the detection method refers to the instruction).
(4) Sperm mitochondrial Activity assay
The Rh123-PI double staining method is used for detecting the mitochondrial activity of the sperm and comprises the following steps:
taking 50 mu L of unfrozen fresh semen or frozen semen into a 500 mu L centrifuge tube; adding 5 μ L of diluted mixed solution of Rh123 and PI (adding 2 μ L of Rh123 and PI into 50 μ L of PBS respectively and mixing) into semen prepared in advance, and incubating at 37 deg.C for 15 min; and observing after the incubated sperm smear. When more than 200 sperms are counted, the mitochondrial sheath has no fluorescence or weak fluorescence intensity, the mitochondrial activity is poor, the fluorescence intensity of the mitochondrial sheath part is strong, and the mitochondrial activity is high. And counting the proportion of good mitochondrial activity, namely the mitochondrial integrity rate.
The test results are shown in table 1:
TABLE 1
| Treatment of | Sperm motility% | Percentage of intact acrosome% | T-AOC | Sperm mitochondrial Activity |
| Comparative example 1 | 45.72±1.26a | 62.09±7.98a | 114.44±12.95a | 68.67±8.9a |
| Example 1 | 50.00±3.67b | 81.89±4.57b | 208.33±11.67b | 87.83±7.00b |
As can be seen from the test results of table 1: by applying the cryopreservation method disclosed by the invention, the activity of the thawed sperms, the acrosome integrity and the like are obviously higher than those of the method in the application comparative example 1, and the activity of the sperms, T-AOC, mitochondria and the like are also obviously higher than those of the method in the application comparative example 1.
Claims (4)
1. A cryopreservation method of boar semen is characterized by comprising the following steps:
(1) preparing a freezing base solution TCG, adding 20-25% of egg yolk into the freezing base solution, uniformly stirring to obtain a freezing dilution solution I, adding 6% of glycerol and 0.4-0.6% of OEP into the freezing dilution solution I, and uniformly stirring to obtain a freezing dilution solution II;
(2) respectively placing the diluent BTS, the frozen diluent I and the frozen diluent II in a closed container, and introducing hydrogen for hydrotreating to obtain hydrotreated diluent BTS, frozen diluent I and frozen diluent II;
(3) semen collection, namely diluting the boar semen by using a diluent BTS after hydrotreating at the constant temperature of 1: 1 to obtain pre-diluted semen;
(4) balancing the pre-diluted semen for 2-4h at 17 ℃, centrifuging, removing supernatant, diluting the sperm precipitate with isothermal and hydrotreated frozen diluent I, and diluting until the sperm density is 20 × 108Cooling to 4 ℃, balancing, and finally diluting with isothermal and hydrotreated frozen diluent II in a ratio of 1: 1 to obtain diluted semen;
(5) subpackaging and sealing the diluted semen by using a freezing tubule, then placing the packaged semen into a box body of a program freezing instrument for freezing, and after the frozen semen is frozen in a cold state, transferring the freezing tubule into liquid nitrogen for preservation;
in the step (1), the preparation method of the freezing base liquid TCG comprises the following steps: weighing 1.1g of glucose, 1.48g of citric acid, 2.42g of Tris, 0.06g of sodium penicillin and 0.1g of streptomycin sulfate, dissolving in double distilled water, stirring for dissolving, and fixing the volume to 100 mL;
in the step (2), the preparation method of the diluent BTS comprises the following steps: 3.7g of glucose, 0.6g of trisodium citrate dihydrate, 0.125g of EDTA, 0.125g of sodium bicarbonate, 0.075g of potassium chloride, 0.06g of sodium penicillin and 0.1g of streptomycin sulfate are weighed and dissolved in double distilled water, then the volume is determined to be 100mL, the pH value is adjusted to be 7.2, and the osmotic pressure is 310 mosm/L.
2. The cryopreservation method of boar semen according to claim 1, wherein in the step (2), the pressure in the container is 0.6 to 0.8MPa and the processing time is 2 to 5 hours during the hydrogenation.
3. The method for cryopreservation of porcine semen as claimed in claim 1, wherein in step (4), the centrifugation conditions are: at 17 ℃, 800-2300 g for 3-15 min.
4. The method for cryopreservation of porcine semen as defined in claim 1, 2 or 3, wherein in step (5), the procedure of freezing is: the temperature is reduced to-5 ℃ at the speed of 6 ℃/min after the temperature is maintained at 4 ℃ for 30min, then reduced to-80 ℃ at the speed of 40 ℃/min after the temperature is maintained at-5 ℃, and then reduced to-145 ℃ at the speed of 60 ℃/min after the temperature is maintained at-80 ℃ for 30s, thus completing the freezing.
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| CN115024308A (en) * | 2022-06-23 | 2022-09-09 | 上海市农业科学院 | Preparation method of thawing solution containing seminal plasma special for artificial insemination of pig frozen semen |
| CN115024307A (en) * | 2022-06-15 | 2022-09-09 | 上海市农业科学院 | Hydrogen-rich semen diluent, preparation method thereof and semen preservation method |
| CN115141797A (en) * | 2022-07-26 | 2022-10-04 | 华中农业大学 | Frozen pig semen unfreezing agent and preparation method thereof |
| CN115517242A (en) * | 2022-09-07 | 2022-12-27 | 四川农业大学 | Pig sperm cryopreservation kit and application thereof |
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| CN115141797A (en) * | 2022-07-26 | 2022-10-04 | 华中农业大学 | Frozen pig semen unfreezing agent and preparation method thereof |
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| CN115517242A (en) * | 2022-09-07 | 2022-12-27 | 四川农业大学 | Pig sperm cryopreservation kit and application thereof |
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