CN111771873A - Method for improving quality of thawed pig sperms - Google Patents
Method for improving quality of thawed pig sperms Download PDFInfo
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- CN111771873A CN111771873A CN202010745843.XA CN202010745843A CN111771873A CN 111771873 A CN111771873 A CN 111771873A CN 202010745843 A CN202010745843 A CN 202010745843A CN 111771873 A CN111771873 A CN 111771873A
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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
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
Abstract
The invention discloses a method for improving the quality of thawed pig sperms, which comprises the following operation steps: (1) diluting the collected qualified semen by a semen protection diluent, and then carrying out constant temperature balance treatment; (2) centrifuging the semen, adding the freezing base solution I into the obtained centrifugal precipitate, and carrying out constant-temperature balance treatment in a constant-temperature box after resuspension; (3) adding the freezing base liquid II into the mixture, uniformly mixing, filling the mixture into a thin tube, sealing, fumigating the mixture on liquid nitrogen or freezing the mixture by using a program freezer, and then putting the mixture into the liquid nitrogen for preservation; (4) when the semen is unfrozen, the thin tube is taken out of the liquid nitrogen, incubated in a water bath, added with the unfreezing diluent, and unfrozen. According to the invention, the caffeic acid phenethyl ester and the hydroxyethyl starch with corresponding concentrations are added into the frozen base solution I in a combined manner, and the synergistic effect of the caffeic acid phenethyl ester and the hydroxyethyl starch can effectively improve the activity, the plasma membrane integrity rate, the acrosome integrity rate and the DNA integrity rate of the thawed pig sperms and improve the quality of the frozen pig sperms.
Description
Technical Field
The invention relates to the technical field of pig sperm cryopreservation, in particular to a method for improving the quality of thawed pig sperm.
Background
With the rapid rise of the artificial insemination technology of the pigs, most of breeders in the pig industry adopt the method to breed the piglets so as to reduce the feeding cost of the boars. The semen is frozen and stored, so that the semen can be used without the limitation of time, place and objects, the use time and the use ratio of the semen of a superior boar are obviously increased, and the advantages enable the artificial insemination technology to be more popularized. Therefore, the frozen semen technique plays an important role.
The freezing preservation of the semen can effectively reserve the genetic breeding and germplasm resources of organisms. With the rapid development of the frozen pig semen production process and the artificial insemination technology, the breeding pigs bred by adopting the frozen semen are effectively improved in the aspects of conception rate and piglet delivery number, and are close to the mating level of fresh semen. However, the research on the pig semen freezing technology is still not completely mature due to the reasons of great difficulty in pig frozen semen production, low survival rate after frozen semen-thawing, low conception rate and the like.
The causes of the reduction of the quality of the pig sperms in the freezing process mainly comprise oxidative damage, cell infiltration damage, formation of ice crystals in cells and the like.
Oxidative damage in semen after freezing and thawing is mainly caused by excess Reactive Oxygen Species (ROS). Excess ROS mainly causes sperm lipid Peroxidation (lipid Peroxidation) and DNA fragmentation. The teratospermia rate is increased, the survival rate, the sperm movement rate and the like are reduced, and finally the conception rate and the birth rate are reduced, and the abortion rate and the fetal teratospermia rate are increased.
Cell osmotic damage and intracellular ice crystal formation are primarily due to the freezing rate. Research shows that at the temperature of minus 10 ℃, before the intracellular fluid of the sperm does not form ice crystals, the extracellular fluid of the sperm already forms the ice crystals, if the freezing rate is low, the water in the intracellular fluid of the sperm seeps out of the sperm, the osmotic pressure in the sperm rises, and the organelles and plasma membranes of the sperm are damaged due to high osmotic shrinkage; at this time, if the freezing rate is high, water in the sperm cell fluid cannot seep out, and the water in the sperm cell fluid forms ice crystals to damage sperm organelles and membranes.
In recent years, the prior art has studied in detail on external conditions affecting the quality of frozen semen, and the external conditions can be controlled to be within an appropriate range in the freezing process so as to minimize the influence caused by the external conditions; however, the internal damage mechanism is not clear, and how to avoid the quality loss of the pig sperms in the freezing process to the maximum degree becomes a technical problem to be solved.
Disclosure of Invention
The invention aims to provide a method for improving the quality of thawed pig sperms, which can obviously reduce the damages caused by oxidation damage, cell permeation damage and ice crystal formation in cells of the pig sperms in the freezing process. .
The invention is realized by the following technical scheme:
a method for improving the quality of thawed pig sperms comprises the following operation steps:
(1) diluting the collected qualified semen by a semen protection diluent, and then carrying out constant temperature balance treatment;
(2) centrifuging the semen, adding a freezing base solution I into the obtained centrifugal precipitate, and carrying out constant-temperature balance treatment in a constant-temperature box after resuspension, wherein the freezing base solution I is prepared from the following components in parts by weight: 300 parts of glucose, 300 parts of lactose, 400 parts of sodium bicarbonate, 10-15 parts of sodium bicarbonate, 5-7 parts of penicillin sodium, 8-12 parts of streptomycin sulfate, 2200 parts of yolk 1800, and 9000 parts of triple distilled water 7000, wherein the frozen base liquid I further comprises phenethyl caffeate and hydroxyethyl starch, the molar concentration of the phenethyl caffeate in the frozen base liquid I is 30-50 mu mol/L, and the mass fraction of the hydroxyethyl starch in the frozen base liquid I is 0.8-1.2%;
(3) adding frozen base liquid II to give sperm concentration of 4 × 108-8×108Mixing, filling into a thin tube, sealing, fumigating on liquid nitrogen or freezing by using a program freezing instrument, and then putting into liquid nitrogen for preservation;
(4) when the semen is unfrozen, the thin tube is taken out of the liquid nitrogen, incubated in a water bath kettle at 48-52 ℃ for 15-17s, transferred to a 36.9-37.1 ℃ preheated semen infusion bottle, and unfrozen.
Specifically, after semen is collected in the boar body, the semen is packaged in an incubator, and the semen is diluted by adopting a semen protection diluent within 2 hours.
Specifically, in the step (1), the qualified semen is selected by the following method: selecting slightly fishy and milky semen to perform microscopic examination, wherein the vitality of the semen is 90 percent, and the density of the semen is 2.0-4.0 hundred million/mL, so that the qualified semen is obtained.
Specifically, the semen protection diluent is prepared from the following components in parts by weight: 1.20-1.30 parts of ethylenediamine tetraacetic acid, 1.20-1.30 parts of sodium citrate dihydrate, 0.70-0.80 part of potassium chloride, 1.20-1.30 parts of sodium bicarbonate, 36.00-37.00 parts of glucose, 0.50-0.70 part of penicillin sodium, 0.80-1.20 parts of streptomycin sulfate and 1100 parts of triple distilled water, wherein the pH value of the semen protection diluent is 7.2-7.4, and the osmotic pressure is 300-320 mOsm/L.
Specifically, in the step (1), the specific operation of the constant temperature balance processing is as follows: standing and balancing in a constant temperature box at 25 ℃ for 1h, mixing semen uniformly at an indefinite period, then placing in a constant temperature box at 17 ℃ for standing and balancing for 2h, and mixing uniformly at an indefinite period;
the specific operation of the constant temperature balance treatment in the step (2) is as follows: standing and balancing in a thermostat at 4 ℃ for 3 hours.
Specifically, in the step (3), the frozen base liquid II is prepared from the following components in parts by weight: 300 parts of glucose, 300 parts of lactose, 400 parts of sodium bicarbonate, 10-15 parts of sodium bicarbonate, 5-7 parts of penicillin sodium, 8-12 parts of streptomycin sulfate, 2200 parts of yolk 1800 and 9000 parts of triple distilled water 7000, wherein the freezing base liquid II further comprises amino-sodium-dodecyl sulfate and glycerol, and the final concentration of the amino-sodium-dodecyl sulfate is 0.50 percent and the final concentration of the glycerol is 3.00 percent according to the volume fraction.
Specifically, in the step (3), before the tubule is placed in liquid nitrogen for preservation, the tubule is fumigated for 10min at a position 3cm above the liquid nitrogen surface.
Specifically, in the step (4), the tubule was taken out of the liquid nitrogen and then placed in a water bath for incubation within 8 seconds.
According to the technical scheme, the beneficial effects of the invention are as follows:
the caffeic acid phenethyl ester is a main active carrier of propolis extract, is a phenolic substance, has wide pharmacological activity, such as antimicrobial, anti-tumor, antioxidant, anticoagulant and the like, and has wide significance in medicine and animal production. In the prior art, caffeic acid phenethyl ester is commonly applied to the following aspects: the caffeic acid phenethyl ester can reduce cardiac hypertrophy and cardiac fibrosis, increase the heart pumping function and reduce the change of tissue morphological hypertrophy; the caffeic acid phenethyl ester can remarkably inhibit the oxidative stress of periodontitis of diabetic rats and the generation of osteoclast and alveolar bone loss induced by RANKL; caffeic acid phenethyl ester may be one of the seed compounds that maintain redox homeostasis; caffeic acid phenethyl ester can inhibit HR-induced H9c2 cell apoptosis and ROS generation in vitro by activating SIRT1/eNOS pathway and inhibiting expression of NF-kB. However, no researchers have heretofore applied it to frozen boar semen.
Hydroxyethyl starch is a modified starch obtained by using amylopectin as a raw material and by chemically reacting it with a hydroxyethyl agent such as ethylene oxide or chlorohydrin to introduce a hydrophilic group hydroxyethyl thereto, and has a high-stability ether bond. During the reaction processes of hydrolysis, oxidation, crosslinking, carboxymethylation and the like, the ether bond is stable and cannot be easily broken, and the influence of pH and electrolyte is small. Hydroxyethyl starch is added into the refrigerating fluid, so that the possibility of destructive osmotic stress is low; in addition, it is non-toxic, so cells can be transplanted after thawing without the need for time-consuming washing steps. Hydroxyethyl starch has been extensively demonstrated to provide good protection in freeze-drying of cells and erythrocytes, but to date there has been no data showing its effect on liquid nitrogen freezing of boar semen.
According to the invention, the caffeic acid phenethyl ester and the hydroxyethyl starch with corresponding concentrations are added into the frozen base solution I in a combined manner, and the synergistic effect of the caffeic acid phenethyl ester and the hydroxyethyl starch can effectively improve the activity, the plasma membrane integrity rate, the acrosome integrity rate and the DNA integrity rate of the thawed pig sperms, thereby improving the quality of the frozen pig sperms.
According to the invention, the caffeic acid phenethyl ester and the hydroxyethyl starch are added into the freezing base liquid I, the frozen base liquid I is firstly adopted to carry out constant temperature balance treatment on the boar semen, so that the caffeic acid phenethyl ester, the hydroxyethyl starch and the boar semen are fully mixed, and then the freezing base liquid II is added, so that the adverse effect of amino-sodium-dodecyl sulfate and glycerol in the freezing base liquid II on the combination effect of the caffeic acid phenethyl ester, the hydroxyethyl starch and the boar semen can be effectively avoided, and the effect of the caffeic acid phenethyl ester and the hydroxyethyl starch is further improved.
The semen protection diluent provided by the invention reduces the damage of the centrifugal action on the activity of the pig semen, and the addition of the semen protection diluent also improves the centrifugal effect of the pig semen and avoids the loss of active ingredients in the pig semen.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
A method for improving the quality of thawed pig sperms comprises the following operation steps:
(1) diluting the collected qualified semen by adopting a semen protection diluent within 2 hours, standing and balancing in a constant temperature box at 25 ℃ for 1 hour, uniformly mixing the semen at variable time, then placing in a constant temperature box at 17 ℃ for standing and balancing for 2 hours, uniformly mixing at variable time, and finishing constant temperature balancing; wherein the qualified semen is selected by adopting the following method: selecting semen with slight fishy smell and milky white for microscopic examination, wherein the sperm has the vitality of 90 percent and the density of 2.0-4.0 hundred million/mL and is taken as qualified semen, and the semen protection diluent is prepared from the following components in parts by weight: 1.25 parts of ethylenediamine tetraacetic acid, 1.25 parts of sodium citrate dihydrate, 0.75 part of potassium chloride, 1.25 parts of sodium bicarbonate, 36.50 parts of glucose, 0.60 part of penicillin sodium, 1.00 part of streptomycin sulfate and 1000 parts of triple distilled water, wherein the pH value of the semen protection diluent is 7.3, and the osmotic pressure is 310 mOsm/L;
(2) centrifuging the semen, adding a freezing base solution I into the obtained centrifugal precipitate, and carrying out constant-temperature balance treatment in a constant-temperature box after resuspension, wherein the constant-temperature balance treatment is standing and balancing for 3 hours in the constant-temperature box at 4 ℃, and the freezing base solution I is prepared from the following components in parts by weight: 250 parts of glucose, 350 parts of lactose, 13 parts of sodium bicarbonate, 6 parts of penicillin sodium, 10 parts of streptomycin sulfate, 2000 parts of egg yolk and 8000 parts of triple-distilled water, wherein the frozen base solution I also comprises caffeic acid phenethyl ester and hydroxyethyl starch, the molar concentration of the caffeic acid phenethyl ester in the frozen base solution I is 40 mu mol/L, and the mass fraction of the hydroxyethyl starch in the frozen base solution I is 1.0%;
(3) adding frozen base liquid II to give sperm concentration of 5 × 108Uniformly mixing the components per mL, filling the mixture into a thin tube, sealing the tube, fumigating the thin tube at a position 3cm above a liquid nitrogen surface for 10min, and putting the thin tube into liquid nitrogen for storage, wherein the freezing base liquid II is prepared from the following components in parts by weight: 250 parts of glucose, 350 parts of lactose, 13 parts of sodium bicarbonate, 6 parts of penicillin sodium, 10 parts of streptomycin sulfate, 2000 parts of egg yolk and 8000 parts of triple distilled water, wherein the freezing base liquid II further comprises amino-sodium-dodecyl sulfate and glycerol, and the final concentration of the amino-sodium-dodecyl sulfate is 0.50 percent and the final concentration of the glycerol is 3.00 percent according to the volume fraction;
(4) when the semen is unfrozen, the thin tube is taken out of the liquid nitrogen, the thin tube is placed into a 50 ℃ water bath kettle within 8 seconds to be incubated for 16 seconds, the thin tube is transferred into a 37.0 ℃ preheated semen deposition bottle, and the unfreezing is completed.
Example 2
A method for improving the quality of thawed pig sperms comprises the following operation steps:
(1) diluting the collected qualified semen by adopting a semen protection diluent within 2 hours, standing and balancing in a constant temperature box at 25 ℃ for 1 hour, uniformly mixing the semen at variable time, then placing in a constant temperature box at 17 ℃ for standing and balancing for 2 hours, uniformly mixing at variable time, and finishing constant temperature balancing; wherein the qualified semen is selected by adopting the following method: selecting semen with slight fishy smell and milky white for microscopic examination, wherein the sperm has the vitality of 90 percent and the density of 2.0-4.0 hundred million/mL and is taken as qualified semen, and the semen protection diluent is prepared from the following components in parts by weight: 1.20 parts of ethylenediamine tetraacetic acid, 1.20 parts of sodium citrate dihydrate, 0.70 part of potassium chloride, 1.20 parts of sodium bicarbonate, 36.00 parts of glucose, 0.50 part of penicillin sodium, 0.80 part of streptomycin sulfate and 900 parts of triple distilled water, wherein the pH value of the semen protection diluent is 7.2, and the osmotic pressure is 300 mOsm/L;
(2) centrifuging the semen, adding a freezing base solution I into the obtained centrifugal precipitate, and carrying out constant-temperature balance treatment in a constant-temperature box after resuspension, wherein the constant-temperature balance treatment is standing and balancing for 3 hours in the constant-temperature box at 4 ℃, and the freezing base solution I is prepared from the following components in parts by weight: 200 parts of glucose, 300 parts of lactose, 10 parts of sodium bicarbonate, 5 parts of penicillin sodium, 8 parts of streptomycin sulfate, 1800 parts of egg yolk and 7000 parts of triple-distilled water, wherein the frozen base solution I also comprises caffeic acid phenethyl ester and hydroxyethyl starch, the molar concentration of the caffeic acid phenethyl ester in the frozen base solution I is 30 mu mol/L, and the mass fraction of the hydroxyethyl starch in the frozen base solution I is 0.8%;
(3) adding frozen base liquid II to give sperm concentration of 4 × 108Uniformly mixing the components per mL, filling the mixture into a thin tube, sealing the tube, fumigating the thin tube at a position 3cm above a liquid nitrogen surface for 10min, and putting the thin tube into liquid nitrogen for storage, wherein the freezing base liquid II is prepared from the following components in parts by weight: 200 parts of glucose, 300 parts of lactose, 10 parts of sodium bicarbonate, 5 parts of penicillin sodium, 8 parts of streptomycin sulfate, 1800 parts of egg yolk and 7000 parts of triple distilled water, wherein the freezing base liquid II further comprises amino-sodium-dodecyl sulfate and glycerol, and the final concentration of the amino-sodium-dodecyl sulfate is 0.50 percent and the final concentration of the glycerol is 3.00 percent according to the volume fraction;
(4) when the semen is unfrozen, the thin tube is taken out of the liquid nitrogen, the thin tube is placed into a water bath kettle at 48 ℃ within 8 seconds for incubation for 15 seconds, the thin tube is transferred into a 36.9 ℃ preheated semen deposition bottle, and the unfreezing is completed.
Example 3
A method for improving the quality of thawed pig sperms comprises the following operation steps:
(1) diluting the collected qualified semen by adopting a semen protection diluent within 2 hours, standing and balancing in a constant temperature box at 25 ℃ for 1 hour, uniformly mixing the semen at variable time, then placing in a constant temperature box at 17 ℃ for standing and balancing for 2 hours, uniformly mixing at variable time, and finishing constant temperature balancing; wherein the qualified semen is selected by adopting the following method: selecting semen with slight fishy smell and milky white for microscopic examination, wherein the sperm has the vitality of 90 percent and the density of 2.0-4.0 hundred million/mL and is taken as qualified semen, and the semen protection diluent is prepared from the following components in parts by weight: 1.30 parts of ethylenediamine tetraacetic acid, 1.30 parts of sodium citrate dihydrate, 0.80 part of potassium chloride, 1.30 parts of sodium bicarbonate, 37.00 parts of glucose, 0.70 part of penicillin sodium, 1.20 parts of streptomycin sulfate and 1100 parts of triple distilled water, wherein the pH value of the semen protection diluent is 7.4, and the osmotic pressure is 320 mOsm/L;
(2) centrifuging the semen, adding a freezing base solution I into the obtained centrifugal precipitate, and carrying out constant-temperature balance treatment in a constant-temperature box after resuspension, wherein the constant-temperature balance treatment is standing and balancing for 3 hours in the constant-temperature box at 4 ℃, and the freezing base solution I is prepared from the following components in parts by weight: 300 parts of glucose, 400 parts of lactose, 15 parts of sodium bicarbonate, 7 parts of penicillin sodium, 12 parts of streptomycin sulfate, 2200 parts of egg yolk and 9000 parts of triple distilled water, wherein the frozen base solution I also comprises caffeic acid phenethyl ester and hydroxyethyl starch, the molar concentration of the caffeic acid phenethyl ester in the frozen base solution I is 50 mu mol/L, and the mass fraction of the hydroxyethyl starch in the frozen base solution I is 1.2%;
(3) adding frozen base liquid II to give sperm concentration of 8 × 108Uniformly mixing the components per mL, filling the mixture into a thin tube, sealing the thin tube, freezing the thin tube by using a program freezer, and then putting the thin tube into liquid nitrogen for preservation, wherein the freezing base liquid II is prepared from the following components in parts by weight: 300 parts of glucose, 400 parts of lactose, 15 parts of sodium bicarbonate, 7 parts of penicillin sodium, 12 parts of streptomycin sulfate, 2200 parts of yolk and 9000 parts of triple distilled water, wherein the freezing base liquid II further comprises amino-sodium-dodecyl sulfate and glycerol, and the final concentration of the amino-sodium-dodecyl sulfate is 0.50 percent and the final concentration of the glycerol is 3.00 percent according to the volume fraction;
(4) when the semen is unfrozen, the thin tube is taken out of the liquid nitrogen, the thin tube is placed into a 52 ℃ water bath kettle for incubation for 17s within 8 seconds, the thin tube is transferred into a 37.1 ℃ preheated semen deposition bottle, and the unfreezing is completed.
Comparative example 1
The same procedures as in example 1 were repeated except that the caffeic acid phenethyl ester in the frozen base solution I was replaced with the same amount of chlorogenic acid.
Comparative example 2
The same procedure as in example 1 was repeated except that the hydroxyethyl starch in the frozen base liquid I was replaced with the same amount of polyvinylpyrrolidone.
Comparative example 3
Amino-sodium lauryl sulfate and glycerol were added to the frozen base liquid i in a final concentration of 0.50% by volume and 3.00% by volume, and the remaining operation was exactly the same as in example 1.
The method of each embodiment and the method of the comparative example are respectively used for carrying out thawing treatment after freezing the semen of the same Songliao black pig, and then the sperm motility, the plasma membrane integrity rate, the acrosome integrity rate and the DNA integrity rate of the thawed pig semen are tested, wherein:
pig sperm motility was detected by computer-aided sperm analysis system (CASA).
Many studies have shown that sperm metabolism, acrosome reaction, capacitation and sperm-egg fusion are closely related to the functional integrity of the sperm membrane, and thus one of the important indicators for evaluating sperm fertilization ability is the functional integrity of the sperm membrane, which is an important barrier for isolating the interior and exterior of sperm and also contains an important pathway for sperm to communicate between the interior and exterior, and which affects metabolism and vital functions by receiving signals transmitted from the interior and exterior of sperm cells, and thus, the integrity of the sperm membrane is essential for normal vital functions of sperm6The number of sperms per mL is increased by incubation for 15min at 37 ℃, smear microscopy is carried out, sperms with complete plasma membranes are curled tails, sperms without curled tails are sperms with damaged plasma membranes, at least 200 sperms are counted in the test, the test is repeated for more than 5 times, and the plasma membranes of the sperms are completeThe (%) ratio was × 100% for curved-tailed sperm/total sperm count.
The invention uses fluorescein isothiocyanate labeled peanut agglutinin (FITC-PNA) fluorescence staining method to detect, adding 1mL PBS (phosphate buffer saline solution) preheated at 37 ℃ into thawed semen, centrifuging for 5min at 2400r/min by using a temperature-controlled centrifuge, leaving precipitate, adding PBS solution for resuspension, repeating for three times, finally adjusting semen concentration by using PBS to keep the semen density at 5 × 106About one per mL, uniformly mixing, uniformly coating 30 mu L of the mixture on a glass slide, naturally airing, uniformly smearing with methanol, fixing for 10min at room temperature, uniformly coating 30 mu L of FITC-PNA dye solution on the glass slide in a dark environment, putting the glass slide into a 37 ℃ wet cassette, incubating for 30min, slowly washing with PBS, naturally airing in the dark environment, counting no less than 200 sperms under a 200 × fluorescence microscope, emitting light at 480nm, emitting light at 530nm, emitting green fluorescence from sperms with complete acrosomes, enabling the fluorescence part to be in a cap shape, repeating each group of experiments for more than 5 times, and enabling the sperm acrosome integrity (%) -the sperms with complete acrosomes/the total sperm count to be × 100%.
The invention uses AO fluorescent staining method to detect sperm DNA integrity, sperm pretreatment and acrosome integrity, defrozen semen is washed three times with PBS to keep sperm concentration at 5 × 106About one per mL, uniformly coating 30 mu L of the total sperm on a glass slide, naturally drying the glass slide, fixing the glass slide for 3 hours by absolute ethyl alcohol-glacial acetic acid (3:1), placing the glass slide into newly prepared AO staining solution in a dark environment for 10 minutes, slowly washing by PBS, naturally drying the glass slide in the dark, adding paraffin oil, sealing the glass slide, counting no less than 200 sperms under a 200 × fluorescence microscope, counting no less than 200 sperms under a 480nm fluorescence microscope, emitting light at 530nm, emitting green fluorescence by the sperms with complete DNA, emitting yellow or even red fluorescence by the sperms with damaged DNA, repeating the experiment of each group for more than 5 times, wherein the percent of the DNA integrity of the sperms is × 100 percent of the sperms with complete DNA/total counted sperm.
The test results are shown in table 1:
as can be seen from Table 1, the method for improving the quality of the thawed pig sperms, provided by the invention, can effectively improve the quality of the thawed pig sperms after the caffeic acid phenethyl ester and the hydroxyethyl starch are used in combination; the method comprises the steps of firstly adopting the freezing base solution I to carry out constant-temperature balance treatment on the boar semen so that the caffeic acid phenethyl ester, the hydroxyethyl starch and the boar semen are fully mixed, and then adding the freezing base solution II, so that the adverse effect of amino-sodium-dodecyl sulfate and glycerol in the freezing base solution II on the combination effect of the caffeic acid phenethyl ester, the hydroxyethyl starch and the boar semen can be effectively avoided.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (8)
1. The method for improving the quality of the thawed pig sperms is characterized by comprising the following operation steps:
(1) diluting the collected qualified semen by a semen protection diluent, and then carrying out constant temperature balance treatment;
(2) centrifuging the semen, adding a freezing base solution I into the obtained centrifugal precipitate, and carrying out constant-temperature balance treatment in a constant-temperature box after resuspension, wherein the freezing base solution I is prepared from the following components in parts by weight: 300 parts of glucose, 300 parts of lactose, 400 parts of sodium bicarbonate, 10-15 parts of sodium bicarbonate, 5-7 parts of penicillin sodium, 8-12 parts of streptomycin sulfate, 2200 parts of yolk 1800, and 9000 parts of triple distilled water 7000, wherein the frozen base liquid I further comprises phenethyl caffeate and hydroxyethyl starch, the molar concentration of the phenethyl caffeate in the frozen base liquid I is 30-50 mu mol/L, and the mass fraction of the hydroxyethyl starch in the frozen base liquid I is 0.8-1.2%;
(3) adding frozen base liquid II to give sperm concentration of 4 × 108-8×108Uniformly mixing, filling into a thin tube, sealing, and then putting into liquid nitrogen for preservation;
(4) when the semen is unfrozen, the thin tube is taken out of the liquid nitrogen, incubated in a water bath kettle at 48-52 ℃ for 15-17s, transferred to a 36.9-37.1 ℃ preheated semen infusion bottle, added with unfreezing diluent, and unfrozen to finish the process.
2. The method of claim 1, wherein the semen is collected from the boar and filled in an incubator, and diluted with a semen protection diluent within 2 hours.
3. The method according to claim 1, wherein in the step (1), the qualified semen is selected by the following method: selecting slightly fishy and milky semen to perform microscopic examination, wherein the vitality of the semen is 90 percent, and the density of the semen is 2.0-4.0 hundred million/mL, so that the qualified semen is obtained.
4. The method of claim 1, wherein the semen protection diluent is prepared from the following components in parts by weight: 1.20-1.30 parts of ethylenediamine tetraacetic acid, 1.20-1.30 parts of sodium citrate dihydrate, 0.70-0.80 part of potassium chloride, 1.20-1.30 parts of sodium bicarbonate, 36.00-37.00 parts of glucose, 0.50-0.70 part of penicillin sodium, 0.80-1.20 parts of streptomycin sulfate and 1100 parts of triple distilled water, wherein the pH value of the semen protection diluent is 7.2-7.4, and the osmotic pressure is 300-320 mOsm/L.
5. The method for improving the quality of thawed pig sperm according to claim 1, wherein in the step (1), the specific operation of the constant temperature equilibration treatment is as follows: standing and balancing in a constant temperature box at 25 ℃ for 1h, mixing semen uniformly at an indefinite period, then placing in a constant temperature box at 17 ℃ for standing and balancing for 2h, and mixing uniformly at an indefinite period;
the specific operation of the constant temperature balance treatment in the step (2) is as follows: standing and balancing in a thermostat at 4 ℃ for 3 hours.
6. The method according to claim 1, wherein in the step (3), the frozen base liquid II comprises the following components in parts by weight: 300 parts of glucose, 300 parts of lactose, 400 parts of sodium bicarbonate, 10-15 parts of sodium bicarbonate, 5-7 parts of penicillin sodium, 8-12 parts of streptomycin sulfate, 2200 parts of yolk 1800 and 9000 parts of triple distilled water 7000, wherein the freezing base liquid II further comprises amino-sodium-dodecyl sulfate and glycerol, and the final concentration of the amino-sodium-dodecyl sulfate is 0.50 percent and the final concentration of the glycerol is 3.00 percent according to the volume fraction.
7. The method according to claim 1, wherein in step (3), the tubule is fumigated at a position 3cm above the liquid nitrogen surface for 10min before being stored in liquid nitrogen.
8. The method according to claim 1, wherein in step (4), the thin tube is taken out of the liquid nitrogen and then placed in a water bath for incubation within 8 seconds.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113317312A (en) * | 2021-06-01 | 2021-08-31 | 福建农林大学 | Diluent preparation capable of prolonging normal-temperature survival period of boar semen |
| CN115141797A (en) * | 2022-07-26 | 2022-10-04 | 华中农业大学 | Frozen pig semen unfreezing agent and preparation method thereof |
| CN117158415A (en) * | 2023-11-02 | 2023-12-05 | 黑龙江八一农垦大学 | Pig sperm cryopreservation diluent containing iramate and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113317312A (en) * | 2021-06-01 | 2021-08-31 | 福建农林大学 | Diluent preparation capable of prolonging normal-temperature survival period of boar semen |
| CN113317312B (en) * | 2021-06-01 | 2022-07-12 | 福建农林大学 | Diluent preparation capable of prolonging normal-temperature survival period of boar semen |
| CN115141797A (en) * | 2022-07-26 | 2022-10-04 | 华中农业大学 | Frozen pig semen unfreezing agent and preparation method thereof |
| CN115141797B (en) * | 2022-07-26 | 2024-02-20 | 华中农业大学 | Frozen semen thawing agent for pigs and preparation method thereof |
| CN117158415A (en) * | 2023-11-02 | 2023-12-05 | 黑龙江八一农垦大学 | Pig sperm cryopreservation diluent containing iramate and application thereof |
| CN117158415B (en) * | 2023-11-02 | 2024-02-02 | 黑龙江八一农垦大学 | Pig sperm cryopreservation diluent containing iramate and application thereof |
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| CN111771873B (en) | 2021-12-21 |
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