CN113331179A - Chinese local pig semen cryopreservation agent and application thereof - Google Patents

Abstract

The invention belongs to the technical field of livestock breeding, and particularly relates to a cryopreservation agent for boar semen in China and application thereof. The cryopreservation agent for the boar semen comprises base liquid, egg yolk, glycerol, coenzyme Q10 or vitamin E, and is one of three formulas. The invention is suitable for artificial insemination of frozen semen of local pigs in China, long-term storage after semen freezing and insemination requirements after long-distance transportation, can obviously improve the activity, the mitochondrial activity and the acrosome integrity of the frozen-thawed pig sperm, obviously reduces the sperm aberration rate, and is beneficial to the breeding of pigs.

Description

Chinese local pig semen cryopreservation agent and application thereof

Technical Field

The invention belongs to the technical field of pig breeding, and particularly relates to a cryopreservation agent for boar semen in China and application thereof.

Background

The development of the semen cryopreservation technology solves the problem that fresh semen can not be preserved for a long time, so that semen collection and fertilization can be separated in time and space, the in-vitro storage time of the semen is prolonged to a great extent, the utilization rate of excellent breeding stock is improved, and the preservation of precious genetic materials is facilitated. The sperm is sensitive to temperature change, and is easily subjected to freezing damage in the freezing and cooling process, wherein the freezing damage mainly comprises physical damage, chemical damage and oxidative damage. Compared with fresh semen, the activity, the mitochondrial activity and the acrosome integrity rate of the frozen-thawed sperms are obviously reduced, and the deformity rate is obviously improved. The quality of the sperm is closely related to pregnancy rate, delivery rate, litter size and the like. The application of the frozen semen on cattle is mature, the pig sperm is more sensitive to freezing stimulation, the recovery rate of the thawed sperm is low, abnormal sperm is abundant, the conception rate is lower than the normal conception rate, and therefore the pig sperm freezing is still in the test stage.

Coenzyme Q10 is a fat-soluble retinoid, and has wide biochemical and pharmacological effects, including participation in cellular respiration and metabolism, antioxidant function of cells, stabilization of cell membrane structure, inhibition of apoptosis, etc. It has strong antioxidant effect, and can effectively scavenge ROS generated in cells, prevent lipoprotein and DNA from being damaged by oxidation, and scavenge free radicals in cell bodies. Vitamin E plays an antioxidant role in cells, and can block the transmission of lipid peroxidation on plasma membranes and maintain the integrity of the membranes.

At present, the application of coenzyme Q10 or vitamin E in the cryopreservation agent for the boar semen in China is not seen.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a cryopreservation agent suitable for local pig semen in China and application thereof, so as to solve the problem that the activity of the local pig semen in China is reduced after the local pig semen is frozen and thawed in the prior art.

The technical scheme of the invention is as follows:

a semen cryopreservation agent suitable for local pigs in China, which contains coenzyme Q10 and/or vitamin E, and is one of the following formulas:

(1) the following ingredients were included in each 100ml of semen cryopreservative: coenzyme Q100.5mg-10 mg, tris (hydroxymethyl) aminomethane 2.42g, citric acid 1.48g, glucose 1.1g, penicillin sodium 0.06g, streptomycin sulfate 0.1g, 20% by mass of egg yolk and 5% by mass of glycerin, or

(2) The frozen semen preservative comprises the following components in each 100 ml: vitamin E20 mg-80mg, trihydroxymethyl aminomethane 2.42g, citric acid 1.48g, glucose 1.1g, penicillin sodium 0.06g, streptomycin sulfate 0.1g, 20% of egg yolk by mass and 5% of glycerin by mass, or

(3) The semen cryopreservation agent comprises the following components in each 100ml of semen cryopreservation agent: coenzyme Q100.5mg-2.5 mg, vitamin E40 mg-60mg, tris (hydroxymethyl) aminomethane 2.42g, citric acid 1.48g, glucose 1.1g, penicillin sodium 0.06g, streptomycin sulfate 0.1g, egg yolk with the mass of 20% and glycerol with the mass of 5%.

The frozen pig semen preservative can be applied to the breeding of local pigs in China.

Specific technical scheme

The main component of the boar semen freezing agent comprises coenzyme Q10 and/or vitamin E.

In the freezing process, conventional freezing base liquid, egg yolk, glycerin and the like are required. Particularly, the volume ratio (or quality) of the added glycerol of the pig semen cryopreservation in China is 5%.

In the freezing process of the boar semen freezing agent, the boar semen freezing agent needs to be completely dissolved in the formula of the conventional freezing base liquid, and the formula comprises the following physical quantities: 2.42g of tris (hydroxymethyl) aminomethane, 1.48g of citric acid, 1.1g of glucose, 0.06g of penicillin sodium, 0.1g of streptomycin sulfate, and 100mL of double distilled water.

Specifically, the addition amount of the coenzyme Q10 in each 100mL of the Chinese local boar semen cryopreservation agent is 0.5mg to 10mg, and/or the addition amount of the vitamin E is 20mg to 80 mg.

Further preferably, the addition amount of the coenzyme Q10 in each 100mL of the Chinese local boar semen cryopreservation agent is 0.5mg to 2.5mg, and/or the addition amount of the vitamin E is 40mg to 60 mg.

The invention has the main advantages that:

the coenzyme Q10 and the vitamin E are respectively added independently or jointly to be used as the Chinese local pig semen cryopreservation agent, the effect is reliable, the popularization is easy, the method is suitable for the artificial insemination of the Chinese local pig semen, the long-term preservation after the semen cryopreservation and the insemination after the long-distance transportation, the sperm activity, the mitochondrial activity and the acrosome integrity rate after the local pig semen is frozen and thawed can be obviously improved, the strain sperm aberration rate can be obviously reduced, and the sperm fertilization capability can be maintained. The pig semen cryopreservation agent has low economic cost and is convenient to popularize and apply.

Detailed Description

Example 1:

in this embodiment, a normal temperature diluent (BTS for short) is disclosed, which has a formula: calculated according to the physical quantity: 3.7g of glucose, 0.6g of trisodium citrate dihydrate, 0.125g of ethylenediamine tetraacetic acid, 0.125g of sodium bicarbonate, 0.075g of potassium chloride, 0.06g of penicillin sodium and 0.1g of streptomycin sulfate are uniformly mixed by double distilled water and the volume is fixed to 100 mL. The pH value of the normal-temperature diluent is adjusted to 7.2, and the osmotic pressure is 310 mosm/L.

The main component of the cryopreservation agent for the local boar semen in China is coenzyme Q10, and in the freezing process, as other conventional freezing approaches, the components such as conventional freezing base liquid, egg yolk, glycerol and the like also need to be added. The volume ratio (or called mass) of the glycerol added into the Chinese local boar semen cryopreservation agent is 5%.

The formula of the freezing base liquid is as follows: calculated according to the physical quantity: coenzyme Q100.5mg-10 mg, tris 2.42g, citric acid 1.48g, glucose 1.1g, penicillin sodium 0.06g, streptomycin sulfate 0.1g, and double distilled water 100 mL.

A method for preparing a Chinese local boar semen cryoprotectant comprises the following steps:

step one, preparing a freezing base liquid: weighing trihydroxymethyl aminomethane, citric acid and glucose according to the formula ratio, mixing the trihydroxymethyl aminomethane, the citric acid and the glucose in a beaker filled with 80mL double distilled water, carrying out water bath at 37 ℃ for 20min, taking the diluent after the substances are completely dissolved and cooled, adding penicillin and streptomycin sulfate, adding the double distilled water and keeping the volume to 100 mL; storing in a refrigerator at 4 deg.C for use as freezing base liquid.

And step two, accurately measuring 80mL of the frozen base liquid, adding 20mL of fresh egg yolk, and uniformly mixing to obtain the local pig semen cryopreservation agent I liquid in China.

And step three, adding 5% of glycerol by volume (or called as quality) on the basis of the Chinese local boar semen cryopreservation agent I liquid, filtering, sterilizing, and cooling to room temperature to obtain the boar semen cryopreservation agent (called as Chinese local boar semen cryopreservation agent II liquid for short) of the invention, and placing the boar semen cryopreservation agent II liquid in a refrigerator at 4 ℃ for standby and performing the following test.

The test objects of the invention are Chinese local pig breeds (such as Hubei white pig, Taihu pig, Tongcheng pig and Erhualian pig, which are domestic common local pig breeds, the same applies below), and the following tests are carried out by using the semen cryopreservation diluent prepared by the invention:

(1) collection of porcine semen

Adopting a hand-held method for semen collection: after the boar climbs, the foreskin and the surrounding parts of the boar are cleaned and disinfected, and then the boar is washed by normal saline and wiped dry. The semen collector takes latex gloves for sterilization, when the penis of the boar extends out of the foreskin, the spiral glans of the penis is immediately grasped, the pressure is exerted on the penis in a fist shape rhythmically, after the penis is fully erected, the boar is pulled forwards in a clockwise manner to cause semen ejaculatory, and the semen is manually collected and filtered by the other hand through a semen collecting bottle with 4 layers of gauze.

(2) Boar semen processing and freezing

Adding the qualified boar semen sample into isothermal and equal-volume normal-temperature diluent (namely BTS liquid dilution) according to the formula shown in the embodiment 1, balancing for 2-3h in a thermostat at 17 ℃, and meanwhile, slightly shaking the semen for several times every 20min to prevent the semen from entering a false death state due to long-time standing. And (3) subpackaging the balanced semen into 10m L centrifuge tubes, centrifuging at 17 ℃, centrifuging at the speed of 2400rcf/min for 3min, taking out, and discarding the supernatant.

Diluting the boar semen by adopting a two-step dilution method: slowly adding pre-cooled I solution (80% TCG + 20% yolk) into the centrifuged sediment, diluting according to the volume ratio (mass) of 1:2, wrapping with gauze, and balancing in a 4 ℃ refrigerator for 1.5-2 h to slowly reduce the temperature to 4 ℃. And slowly adding the precooled II solution in an equal volume, wrapping the II solution with gauze, and continuously balancing the II solution for 1.5 to 2 hours at the temperature of 4 ℃. Sucking into 0.25mL tube with self-made injector, fumigating for 10min at 3cm position above liquid nitrogen, and storing in liquid nitrogen.

(3) Thawing of tubule frozen semen and quality evaluation of semen

1) Pig sperm motility assay

Taking out the frozen semen of the thin tube, quickly putting the frozen semen into a water bath at 37 ℃ for thawing for 30s, collecting the frozen semen in a 1.5mL centrifugal tube, continuously incubating for 10min at 37 ℃, taking 10 mu L semen on a glass slide, covering a cover glass, putting the glass slide under a 200X inverted microscope, and analyzing the sperm motility in a sperm density analyzer.

2) Pig sperm mitochondrial Activity assay

Mitochondrial activity was detected using rhodamine 123 and Propidium Iodide (PI) combined staining. Firstly, 100 mu L of HEPES/BSA is sucked, placed at 37 ℃ for preheating for 10min, then 1 mu L of rhodamine 123 and 1 mu L of PI are respectively added, and the two are fully mixed and incubated for 10min under dark condition to be used as sperm dye. 50 mul of unfrozen semen is taken and fully mixed with 100 mul of prepared dye. Placing in a water bath kettle at 37 deg.C, incubating in dark for 30min, and observing sperm staining under fluorescent microscope. PI can enter the cell nucleus of dead sperm and combine with DNA to generate red fluorescence, while rhodamine 123 can specifically combine with mitochondria to emit green fluorescence, so that the sperm with the green fluorescence is considered to have complete mitochondria.

3) Determination of pig sperm acrosome integrity

Adding 500uL of semen of local pig (same breed) in 1mL of acetone (precooled at-20 ℃ in advance), fixing at 4 ℃ for 5min, centrifuging (500r/min, 6min), discarding the supernatant, washing the precipitate with phosphate buffer (PBS, conventional buffer) for 2 times, sealing with HEPES/Bovine Serum Albumin (BSA) for 30min, adding 50uL of isosulfocyanic fluorescein labeled peanut agglutinin (conventional), incubating at 37 ℃ for 30min, centrifuging (500r/min, 6min), washing the precipitate with PBS for 2 times, dripping 10uL of suspension into the center of a glass slide, dripping a small amount of brightener (conventional, commercially available), covering with a cover plate, sealing with colorless oil, observing under a fluorescence microscope, and counting.

4) Determination of pig sperm teratogenesis rate

The pig sperm teratogenesis rate is detected by adopting a conventional Giemsa staining method. And (3) sucking 10uL of the thawed semen, dripping the semen on one end of a glass slide, uniformly smearing the sample, and air-drying. Dripping methanol on the smeared sheet after air drying, fixing for 2-3min, and air drying. And placing the smears after air drying in a Giemsa staining jar, staining for 30min, slowly washing the glass slide with water, and placing on a test bed until the glass slide is naturally dried. The prepared glass slide is observed under a 400X optical microscope, and more than 200 sperms in the left area and the right area are counted.

(5) Evaluation results of pig sperm quality

The evaluation results of the Chinese local boar semen cryoprotectant and the semen freezing-unfreezing method are shown in the table 1.

TABLE 1 evaluation of sperm cryoprotectant and sperm freezing-thawing method for local pig in China

Treatment of	Sperm motility	Mitochondrial Activity	Percentage of acrosomal integrity	Rate of deformity
					Control group	31.09％	30.02％	29.84％	12.56％
Treatment group 1	36.90％	35.42％	36.16％	9.78％
					Treatment group 2	34.64％	37.38％	37.21	9.24％
Treatment group 3	31.46％	33.15％	34.75％	8378％
					Example 1	42.43％	44.29％	39.96％	8.57％

Example 2:

in this embodiment, a normal temperature diluent (BTS) is disclosed, and the formulation thereof is: 3.7g of glucose, 0.6g of trisodium citrate dihydrate, 0.125g of ethylenediamine tetraacetic acid, 0.125g of sodium bicarbonate, 0.075g of potassium chloride, 0.06g of penicillin sodium and 0.1g of streptomycin sulfate are mixed by double distilled water, the volume is determined to be 100mL, the pH value is adjusted to be 7.2, and the osmotic pressure is 310 mosm/L.

The cryopreservation agent for the local boar semen in the China embodiment mainly comprises vitamin E, and then in the freezing process, like other conventional freezing approaches, conventional freezing base liquid, egg yolk, glycerol and the like also need to be added. The volume ratio (mass) of the glycerol added in the cryopreservation agent for the Chinese local boar semen is 5%.

The formula of the frozen base solution comprises 20mg to 80mg of vitamin E, 2.42g of tris (hydroxymethyl) aminomethane, 1.48g of citric acid, 1.1g of glucose, 0.06g of penicillin sodium, 0.1g of streptomycin sulfate and 100mL of double distilled water.

The invention provides a preparation method of a cryoprotectant suitable for local pig semen in China, which comprises the following steps:

step one, preparing a freezing base liquid. The preparation method of the freezing base liquid comprises the following steps: firstly weighing tris (hydroxymethyl) aminomethane, citric acid and glucose, mixing the tris (hydroxymethyl) aminomethane, citric acid and glucose in a beaker filled with 80mL of double distilled water, carrying out water bath at 37 ℃ for 20min, after the substances are completely dissolved and cooled, adding penicillin and streptomycin sulfate into the diluent, adding the double distilled water to a constant volume of 100mL, and storing the mixture in a refrigerator at 4 ℃ for later use.

And secondly, accurately measuring 80mL of frozen base liquid, adding 20mL of fresh egg yolk, and uniformly mixing to obtain the Chinese local boar semen cryopreservation agent (namely cryopreservation agent I liquid).

And step three, adding 5% by volume (mass) of glycerol on the basis of cryopreservation (cryopreservation agent I liquid), filtering, sterilizing, cooling to room temperature to obtain the cryopreservation agent (cryopreservation agent II liquid for short) for the boar semen, and placing the obtained cryopreservation agent in a refrigerator at 4 ℃ for later use to be subjected to the following test.

The using effect of the local boar semen cryopreservation diluent is tested as follows:

(1) collection of porcine semen

Semen collection by hand-held method: after the boar climbs, the foreskin and the surrounding parts of the boar are cleaned and disinfected, and then the boar is washed by normal saline and wiped dry. The semen collector takes the sterilizing latex gloves, holds the spiral glans penis immediately when the penis extends out of the foreskin, applies pressure to the penis in a fist shape with rhythm, draws the penis forwards in a proper posture after the penis is fully erected to cause the ejaculation of the boar, and collects and filters semen by the other hand through a semen collecting bottle hand with 4 layers of gauze.

(2) Processing and freezing of boar semen

Adding isothermal BTS liquid with the same volume into a qualified boar semen sample for dilution, balancing for 2-3h in a thermostat with the temperature of 17 ℃, and meanwhile, slightly shaking the semen for a plurality of times every 20min to prevent the semen from entering a false death state due to long-time standing. And (3) subpackaging the balanced semen into 10m L centrifuge tubes, centrifuging at 17 ℃, centrifuging at the speed of 2400rcf/min for 3min, taking out, and discarding the supernatant.

The semen is diluted by adopting a two-step dilution method: slowly adding pre-cooled cryopreservation agent I solution (80% TCG + 20% yolk) into the centrifuged sediment, diluting according to the mass (volume ratio) of 1:2, wrapping with gauze, and balancing in a refrigerator at 4 ℃ for 1.5-2 h to slowly reduce the temperature to 4 ℃. And then slowly adding the precooled cryopreservation agent II solution into the mixture in an equal volume, wrapping the mixture by using gauze, and continuously balancing the mixture for 1.5 to 2 hours at the temperature of 4 ℃. Sucking into 0.25mL tube with self-made injector, fumigating for 10min at 3cm position above liquid nitrogen, and storing in liquid nitrogen.

(3) Unfreezing of frozen fine pig sperm and quality evaluation of pig sperm

1) Determination of sperm motility

Taking out frozen semen of the pig tubule, rapidly placing the frozen semen into a water bath at 37 ℃ for thawing for 30s, collecting the frozen semen in a 1.5mL centrifugal tube, continuously incubating for 10min at 37 ℃, taking 10 mu L semen on a glass slide, covering a cover glass, placing the glass slide under a 200X inverted microscope, and analyzing the sperm activity in a sperm density analyzer.

2) Measurement of sperm mitochondrial Activity

Mitochondrial activity was measured using rhodamine 123 and Propidium Iodide (PI) combined staining. Firstly, 100 mu L of HEPES/BSA is sucked, placed at 37 ℃ for preheating for 10min, then 1 mu L of rhodamine 123 and 1 mu L of PI are respectively added, and the two are fully mixed and incubated for 10min under the dark condition to be used as sperm dye. 50 mul of unfrozen semen is taken and fully mixed with 100 mul of prepared dye. Placing in a water bath kettle at 37 deg.C, incubating in dark for 30min, and observing sperm staining under fluorescent microscope. PI can enter the cell nucleus of dead sperm and combine with DNA to generate red fluorescence, while rhodamine 123 can specifically combine with mitochondria to emit green fluorescence, so that the sperm with the green fluorescence is considered to have complete mitochondria.

3) Sperm acrosome integrity determination

Adding 500uL of Chinese local pig semen into 1mL of acetone (precooled at-20 ℃) in advance, fixing for 5min at 4 ℃, centrifuging (500r/min, 6min), then discarding the supernatant, washing the precipitate for 2 times by PBS, then sealing for 30min by HEPES/BSA, then adding 50uL of isosulfocyanic fluorescein labeled peanut agglutinin (commercially available), incubating for 30min at 37 ℃, centrifuging (500r/min, 6min), washing the precipitate for 2 times by PBS, taking 10uL of suspension to drop in the center of a glass slide, then dropping a small amount of brightener (conventional and commercially available), covering a sheet, sealing with colorless nail polish, observing and counting under a fluorescence microscope. The acrosome of the intact pig sperm showed green fluorescence with smooth edges.

4) Determination of pig sperm teratogenesis rate

Sperm teratogenesis rate is detected by Giemsa staining method. And (3) sucking 10uL of the thawed semen, dripping the semen on one end of a glass slide, uniformly smearing the sample, and air-drying. Dripping methanol on the smeared sheet after air drying, fixing for 2-3min, and air drying. And placing the smears after air drying in a Giemsa staining jar, staining for 30min, slowly washing the glass slide with water, and placing on a test bed until the glass slide is naturally dried. The prepared glass slide is observed under a 400X optical microscope, and more than 200 sperms in the left area and the right area are counted.

5) Pig sperm quality assessment results

The evaluation results of the semen cryoprotectant and the semen freezing-unfreezing method applied to the local pigs in China are shown in the table 2.

TABLE 2 semen cryoprotectant for local pig in China and semen freezing-thawing method using the same

Treatment of	Sperm motility	Mitochondrial Activity	Percentage of acrosomal integrity	Rate of deformity
					Control group	30.50％	33.39％	30.66％	13.25％
Treatment group 1	31.70％	32.79％	32.89％	13.03％
					Treatment group 2	34.31％	34.56％	33.10％	9.15％
Treatment group 3	30.47％	31.63％	32.74％	10.01％
					Example 2	38.35％	39.1％	38.75％	9.16％

Example 3:

in the technical Solution of this embodiment, a normal temperature diluent (BTS) is involved, and the formulation thereof is: 3.7g of glucose, 0.6g of trisodium citrate dihydrate, 0.125g of ethylenediamine tetraacetic acid, 0.125g of sodium bicarbonate, 0.075g of potassium chloride, 0.06g of penicillin sodium, 0.1g of streptomycin sulfate, and double distilled water, wherein the mixture is uniformly mixed and the volume is adjusted to 100mL, the pH value is adjusted to 7.2, and the osmotic pressure is 310 mosm/L.

The cryopreservation agent for the local boar semen in China of the embodiment is mainly coenzyme Q10 and vitamin E, and secondly, in the freezing process, like other conventional freezing approaches, conventional freezing base liquid, egg yolk, glycerol and the like also need to be added. The volume ratio of the glycerol added in the cryopreservation agent for the Chinese local boar semen is 5%.

The formula of the freezing base liquid is coenzyme Q100.5mg-2.5 mg, vitamin E40 mg-60mg, trihydroxymethyl aminomethane 2.42g, citric acid 1.48g, glucose 1.1g, penicillin sodium 0.06g, streptomycin sulfate 0.1g, and double distilled water 100 mL.

The preparation method of the local boar semen cryoprotectant comprises the following steps:

step one, preparing a freezing base liquid, wherein the preparation method of the freezing base liquid comprises the following steps: firstly weighing trihydroxymethyl aminomethane, citric acid and glucose, mixing the trihydroxymethyl aminomethane, citric acid and glucose in a beaker filled with 80mL double distilled water, carrying out water bath at 37 ℃ for 20min, after the substances are completely dissolved and cooled, adding penicillin (0.06g) and streptomycin sulfate (0.1g) into the diluent, adding the double distilled water to the constant volume of 100mL, and preserving the mixture in a refrigerator at 4 ℃ for later use.

And step two, accurately measuring 80mL of frozen base liquid, adding 20mL of fresh egg yolk, and uniformly mixing to obtain the local boar semen cryopreservation agent I liquid.

And step three, adding 5% by volume (mass) of glycerol on the basis of cryopreservation (cryopreservation agent I liquid), filtering, sterilizing, cooling to room temperature to obtain the cryopreservation agent (cryopreservation agent II liquid) for the boar semen, and placing the obtained cryopreservation agent in a refrigerator at 4 ℃ for later use to perform the following test.

The following tests were carried out on the use of the diluent for cryopreservation of the sperm of the local pig in China of the present invention:

(1) collection of porcine semen

Semen collection by hand-held method: after the boar climbs, the foreskin and the surrounding parts of the boar are cleaned and disinfected, and then the boar is washed by normal saline and wiped dry. The semen collector takes the sterilizing latex gloves, holds the spiral glans penis immediately when the penis extends out of the foreskin, applies pressure to the penis in a fist shape with rhythm, draws the penis forwards in a proper posture after the penis is fully erected to cause the ejaculation of the boar, and collects and filters semen by the other hand through a semen collecting bottle hand with 4 layers of gauze.

(2) Boar semen processing and freezing

Adding isothermal BTS liquid with the same volume into a qualified boar semen sample for dilution, balancing for 2-3h in a thermostat with the temperature of 17 ℃, and meanwhile, slightly shaking the semen for a plurality of times every 20min to prevent the semen from entering a false death state due to long-time standing. And (3) subpackaging the balanced semen into 10m L centrifuge tubes, centrifuging at 17 ℃, centrifuging at the speed of 2400rcf/min for 3min, taking out, and discarding the supernatant.

The semen is diluted by adopting a two-step dilution method: slowly adding pre-cooled I solution (80% TCG + 20% yolk) into the centrifuged sediment, diluting according to the volume (mass) ratio of 1:2, wrapping with gauze, and balancing in a 4 ℃ refrigerator for 1.5-2 h to slowly reduce the temperature to 4 ℃. And slowly adding the precooled II solution in an equal volume, wrapping the II solution with gauze, and continuously balancing the II solution for 1.5 to 2 hours at the temperature of 4 ℃. Sucking into 0.25mL tube with self-made injector, fumigating for 10min at 3cm position above liquid nitrogen, and storing in liquid nitrogen.

(3) Pig sperm quality assessment results

Semen cryoprotectant for local pig in China and semen freezing-unfreezing method, and the evaluation results are shown in Table 3.

TABLE 3 semen cryoprotectant for local pig in China and detection result of semen freezing-thawing method

Treatment of	Sperm motility	Mitochondrial Activity	Percentage of acrosomal integrity	Rate of deformity
					Control group	31.30％	31.56％	30.83％	14.08％
Treatment group 1	42.89％	33.89％	37.20％	11.17％
					Treatment group 2	45.99％	39.75％	36.97％	11.44％
Treatment group 3	44.45％	36.69％	38.24％	10.46％
					Example 3	51.86％	45.16％	43.67％	9.68％

(4) Thawing of tubule frozen semen and quality evaluation of pig semen

1) Determination of pig sperm motility

Taking out frozen semen of the thin-tube pig, quickly putting the frozen semen into a water bath at 37 ℃ for thawing for 30s, collecting the frozen semen into a 1.5mL centrifugal tube, continuously incubating for 10min at 37 ℃, taking 10 mu L semen on a glass slide, covering a cover glass, putting the glass slide under a 200X inverted microscope, and analyzing the activity of the pig semen in a sperm density analyzer.

2) Pig sperm mitochondrial Activity assay

Pig mitochondrial activity was detected using rhodamine 123 and Propidium Iodide (PI) combined staining. Firstly, 100 mu L of HEPES/BSA is sucked, placed at 37 ℃ for preheating for 10min, then 1 mu L of rhodamine 123 and 1 mu L of PI are respectively added, and the two are fully mixed and incubated for 10min under dark condition to be used as sperm dye. 50 mul of unfrozen semen is taken and fully mixed with 100 mul of prepared dye. Placing into a water bath kettle at 37 ℃, incubating for 30min in a dark place, and observing the staining condition of the pig sperms under a fluorescence microscope. PI can enter the cell nucleus of dead pig sperms and is combined with DNA to generate red fluorescence, while rhodamine 123 can be specifically combined with mitochondria to emit green fluorescence, so the pig sperms with the green fluorescence are considered to have complete mitochondria.

3) Determination of pig sperm teratogenesis rate

The pig sperm teratogenesis rate is detected by adopting a Giemsa staining method. Sucking 10uL of thawed boar semen, dripping the boar semen on one end of a glass slide, smearing the sample evenly and drying in the air. Dripping methanol on the smeared sheet after air drying, fixing for 2-3min, and air drying. And placing the smears after air drying in a Giemsa staining jar, staining for 30min, slowly washing the glass slide with water, and placing on a test bed until the glass slide is naturally dried. Observing the prepared glass slide under a 400X optical microscope, and counting more than 200 pig sperms in the left area and the right area.

4) Determination of sperm acrosome integrity

Adding 500uL of pig semen into 1mL of acetone (precooled at-20 ℃) in advance, fixing for 5min at 4 ℃, centrifuging (500r/min, 6min), then discarding the supernatant, washing the precipitate for 2 times by PBS, then sealing for 30min by HEPES/BSA, then adding 50uL of peanut agglutinin marked by isosulfocyanic fluorescein, incubating for 30min at 37 ℃, centrifuging (500r/min, 6min), washing the precipitate for 2 times by PBS, taking 10uL of suspension to drop in the center of a glass slide, then dropping a small amount of brightener, a cover plate, a colorless nail polish sealing plate, and observing and counting under a fluorescence microscope. Intact acrosomal porcine sperm was found, and the acrosomes were found to be green fluorescent and smooth-edged.

Claims (2)

1. The semen cryopreservation agent suitable for the local pigs in China is characterized by being one of the following formulas:

(1) among the cryopreservatives per 100ml of semen are: coenzyme Q100.5mg-10 mg, tris (hydroxymethyl) aminomethane 2.42g, citric acid 1.48g, glucose 1.1g, penicillin sodium 0.06g, streptomycin sulfate 0.1g, 20% by mass of egg yolk and 5% by mass of glycerin, or

(2) The preservative comprises the following components in each 100ml of frozen semen preservative: vitamin E20 mg-80mg, trihydroxymethyl aminomethane 2.42g, citric acid 1.48g, glucose 1.1g, penicillin sodium 0.06g, streptomycin sulfate 0.1g, 20% of egg yolk by mass and 5% of glycerin by mass, or

(3) The semen cryopreservation agent comprises the following components in each 100ml of semen cryopreservation agent: coenzyme Q100.5mg-2.5 mg, vitamin E40 mg-60mg, tris (hydroxymethyl) aminomethane 2.42g, citric acid 1.48g, glucose 1.1g, penicillin sodium 0.06g, streptomycin sulfate 0.1g, egg yolk with the mass of 20% and glycerol with the mass of 5%.

2. The use of the cryopreservation agent for boar semen in China as claimed in claim 1 in boar breeding in China.