CN117158414B - Pig sperm cryopreservation diluent containing safflower polysaccharide and application thereof - Google Patents
Pig sperm cryopreservation diluent containing safflower polysaccharide and application thereof Download PDFInfo
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- CN117158414B CN117158414B CN202311442904.5A CN202311442904A CN117158414B CN 117158414 B CN117158414 B CN 117158414B CN 202311442904 A CN202311442904 A CN 202311442904A CN 117158414 B CN117158414 B CN 117158414B
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- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 42
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- 238000005138 cryopreservation Methods 0.000 title claims abstract description 33
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a pig sperm cryopreservation diluent containing safflower polysaccharide and application thereof, belonging to the technical field of sperm preservation and artificial insemination in animal science. The invention aims to solve the technical problem of how to relieve the oxidation injury and the reduction of the oxidation resistance of pig sperm during the cryopreservation. The product consists of individually packaged diluents A, B and C, wherein the diluent A comprises safflower polysaccharide, glucose, lactose, penicillin, streptomycin, yolk and dimethyl sulfoxide, and the diluent B comprises safflower polysaccharide, glucose, lactose, penicillin, streptomycin, yolk, dimethyl sulfoxide and glycerol; the diluent C contains glucose, tris (hydroxymethyl) aminomethane, etc. Experiments prove that the cryopreservation diluent provided by the invention can effectively improve the sperm viability, vitality, kinetic parameters, plasma membrane integrity, high mitochondrial activity, acrosome integrity, DNA integrity, antioxidant capacity, antioxidant enzyme activity, aquaporin expression quantity and egg penetration capacity of pigs.
Description
Technical Field
The invention belongs to the technical field of sperm preservation and artificial insemination in animal science, and particularly relates to a safflower polysaccharide-containing pig sperm cryopreservation diluent and a safflower polysaccharide-containing pig sperm cryopreservation diluent freezing preservation method.
Background
The largest pork production country in China is in the importation country, the pork consumption accounts for the first proportion of national meat consumption, and the pork yield has important importance in meeting the national meat demands and national grain safety. The artificial insemination technology is one of the indispensable basic means in the field of live pig breeding, and has important influence on the aspects of improving the utilization rate of excellent boars, the quality of live pig products, the economic benefits of enterprises and the like.
Pig sperm are highly sensitive to changes in the in vitro preservation environment compared to other livestock, and therefore short-term liquid preservation is often employed for commercial reproduction, although this may be compromised by bacterial and microbial reproduction. However, during the cryopreservation process, severe environmental changes can cause oxidative stress to boar sperm, resulting in reduced sperm motility, plasma membrane and acrosome integrity, further affecting the results of artificial insemination. The main reason for this is that the plasma membrane of porcine sperm is rich in polyunsaturated fatty acids and low in cholesterol and therefore less resistant to changes in extracellular environment.
Reactive oxygen species are byproducts of cellular aerobic metabolism, and suitable concentrations are important for sperm capacitation and fertilization. However, the pig sperm is extremely easy to cause unbalance of the utilization/metabolism system of active oxygen in cells, namely reduction of the oxidation resistance, due to severe change of external environment in the freezing preservation process, so that the active oxygen is excessively accumulated, and finally the sperm is apoptotic.
The freezing preservation of the pig feed has almost infinite preservation time and good transportation tolerance, so that the pig feed becomes a necessary technology for long-distance communication of germplasm resources, and can lead the excellent germplasm resources not to be limited by time and distance any more, thereby effectively promoting the reproduction of excellent pigs and the production of high-quality pig products. Therefore, how to alleviate oxidative damage and reduction of antioxidant capacity of pig sperm during cryopreservation is one of important technical directions for improving cryopreservation quality.
Disclosure of Invention
Aiming at the defects and the defects of the prior art, the invention provides a pig sperm cryopreservation diluent containing safflower polysaccharide and a method for cryopreserving pig sperm.
In order to achieve the above object, according to one aspect of the present invention, there is provided a swine sperm cryopreservation diluent comprising safflower polysaccharide, consisting of diluent a, diluent B and diluent C, individually packaged; wherein the diluent A consists of safflower polysaccharide, glucose, lactose, penicillin, streptomycin, yolk, dimethyl sulfoxide and ultrapure water; the diluent B consists of safflower polysaccharide, glucose, lactose, penicillin, streptomycin, yolk, dimethyl sulfoxide, glycerol and ultrapure water; the diluent C comprises glucose, tris (hydroxymethyl) aminomethane, citric acid, caffeine, sodium bicarbonate, EDTA-2Na, sodium pyruvate and ultrapure water.
The mass ratio of the raw materials of the diluent is as follows:
diluent A: safflower polysaccharide 1.50g, glucose 35g, lactose 35g, penicillin 0.60g, streptomycin 1.00g, 200mL egg yolk, 3.0vol% dimethyl sulfoxide and ultrapure water; the preparation method of the diluent A comprises the following steps: dissolving safflower polysaccharide in dimethyl sulfoxide, adding glucose, lactose, penicillin, streptomycin and yolk, dissolving with ultrapure water, fixing volume to 1000mL, and storing at 3-5deg.C;
diluent B: safflower polysaccharide 1.50g, glucose 35g, lactose 35g, penicillin 0.60g, streptomycin 1.00g, 200mL egg yolk, 3.0vol% dimethyl sulfoxide, 7vol% glycerol and ultrapure water; the preparation method of the diluent B comprises the following steps: dissolving safflower polysaccharide in dimethyl sulfoxide, adding glucose, lactose, penicillin, streptomycin and yolk, dissolving with ultrapure water, fixing volume to 1000mL, and storing at 3-5deg.C;
diluent C: glucose 25g, tris 9g, citric acid 3g, caffeine 4.5g, sodium bicarbonate 3g, disodium ethylenediamine tetraacetate (EDTA-2 Na) 4.3g, sodium pyruvate 0.42g, and ultrapure water. The preparation method of the diluent C comprises the following steps: dissolving with ultrapure water, fixing the volume to 1000mL, and storing at 3-5 ℃.
Further defined, the yolk is egg yolk and is subjected to centrifugation at 10000r/min and precipitation removed.
The dimethyl sulfoxide in the diluent A and the diluent B is not more than 3vol% of the total amount of the diluent.
The invention also provides a method for freezing and preserving pig sperm by using the pig sperm freezing preservation diluent containing safflower polysaccharide, which comprises the following steps:
s1, respectively adjusting the pH values of the diluent A and the diluent B to 7.2-7.4, and then preheating at 35-37 ℃;
s2, removing supernatant after centrifuging the original semen, immediately diluting the semen to 4 hundred million/mL by using a diluent A, and placing the semen in an environment of 16-18 ℃ for balancing for 30 minutes;
s3, adding a diluent B which is equal to the diluent A to dilute the sperms to 2 hundred million/mL, and standing for 60 minutes in an environment of 3-5 ℃;
s4, filling the diluted sperms into the tubules by using a frozen fine tube filling and sealing machine, cooling to-115 ℃ to-125 ℃ in a gradient way, maintaining for 55-65 seconds, and then rapidly putting into liquid nitrogen for preservation;
s5, pre-heating the diluent C at 35-37 ℃, quickly placing the frozen fine tube at 48-52 ℃ for 15 seconds after the frozen fine tube is taken out of liquid nitrogen, taking out the frozen fine tube, uniformly mixing the frozen fine tube with the diluent C with the same amount, and temporarily storing the frozen fine tube at 35-37 ℃ for waiting use.
Further defined, in step S1, the pH is adjusted with dilute hydrochloric acid or dilute NaOH.
Further defined, the sperm concentration is determined in step S2 using a blood count plate.
Further defined, step S4 uses a programmable freezer to gradient cool.
Further defined, the gradient cooling process in step S4 is as follows: reducing the temperature to-4.5 ℃ to-5.5 ℃ at the speed of-5.5 ℃/min to-6.5 ℃/min and maintaining the temperature for 55 seconds to 65 seconds, and reducing the temperature to-115 ℃ to-125 ℃ at the same speed and maintaining the temperature for 55 seconds to 65 seconds.
Active oxygen is one of the key factors which obviously influence the in-vitro preservation quality of pig sperms, the cytoplasmic structure of the sperms is highly concentrated and specialized, and meanwhile, the resistance of the pig sperms to the severe change of the external environment in the freezing preservation process is lower than that of other domestic animals, so that the intracellular antioxidant system of the pig sperms cannot timely remove excessive active oxygen, thereby causing oxidative stress and finally reducing the preservation quality of the sperms.
In general, the above technical solutions conceived by the present invention have the following beneficial effects compared with the prior art:
the pig sperm freezing diluent has synergistic effect, wherein, safflower polysaccharide generates hydrogen ions, and combines with free radicals to terminate oxidation chain reaction, or directly combines with free radical ions to terminate oxidation chain reaction, thereby improving sperm quality and antioxidation capability; glucose and lactose can provide sufficient energy for the sperm to maintain the sperm energy requirement; penicillin and streptomycin can inhibit bacterial growth in the dilution treatment process, and avoid the reduction of sperm motility caused by the bacterial growth; yolk and glycerol can protect sperm cell membranes from ice crystals; the tris (hydroxymethyl) aminomethane, the citric acid, the sodium bicarbonate and the sodium pyruvate together form an acid-base buffer system, so that the pH value of the external environment of the sperm is maintained to be stable; EDTA-2Na reduces the formation size and number of ice crystals and protects the sperm plasma membrane structure; the caffeine is beneficial to the rapid recovery of sperm motility after thawing; dimethyl sulfoxide facilitates the full dissolution of safflower polysaccharide in the diluent.
Experiments show that the pig sperm cryopreservation diluent containing safflower polysaccharide can effectively relieve the reduction level of indexes such as the survival rate, the activity, the exercise performance, the plasma membrane integrity, the acrosome integrity, the DNA integrity, the mitochondrial activity, the total antioxidant capacity, the glutathione peroxidase activity, the catalase, the superoxide dismutase activity, the aquaporin and the like of the cryopreserved pig sperm, and can obviously reduce the deformity rate, the malondialdehyde level, the H and the like of the cryopreserved pig sperm 2 O 2 Horizontal.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and technical advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
Example 1: the swine sperm cryopreservation diluent containing safflower polysaccharide of the embodiment consists of diluent A, diluent B and diluent C which are packaged independently; wherein, the mass ratio of the raw materials of the diluent A, the diluent B and the diluent C is as follows:
diluent A: safflower polysaccharide 1.50g, glucose 35g, lactose 35g, penicillin 0.60g, streptomycin 1.00g, 200mL egg yolk, 3vol% dimethyl sulfoxide and ultrapure water. The preparation method of the diluent A comprises the following steps: dissolving safflower polysaccharide in dimethyl sulfoxide, adding glucose, lactose, penicillin, streptomycin and yolk, dissolving with ultrapure water, fixing volume to 1000mL, and standing at 4deg.C;
diluent B: safflower polysaccharide 1.50g, glucose 35g, lactose 35g, penicillin 0.60g, streptomycin 1.00g, 200mL egg yolk, 3vol% dimethyl sulfoxide, 7vol% glycerol and ultrapure water. The preparation method of the diluent B comprises the following steps: dissolving safflower polysaccharide in dimethyl sulfoxide, adding glucose, lactose, penicillin, streptomycin and yolk, dissolving with ultrapure water, fixing volume to 1000mL, and standing at 4deg.C;
diluent C: 25g of glucose, 9g of tris (hydroxymethyl) aminomethane, 3g of citric acid, 4.5g of caffeine, 3g of sodium bicarbonate, 4.3g of disodium ethylenediamine tetraacetate (EDTA-2 Na), 0.42g of sodium pyruvate and ultrapure water. The preparation method of the diluent C comprises the following steps: dissolving with ultrapure water, fixing volume to 1000mL, and standing at 4deg.C for preservation;
wherein the yolk is egg yolk, and is subjected to 10000r/min centrifugation to remove precipitate.
In the formula, the safflower polysaccharide can improve sperm quality and oxidation resistance; glucose and lactose can provide sufficient energy for sperm; penicillin and streptomycin can inhibit bacterial growth in the dilution process; yolk and glycerol can protect sperm cell membranes from ice crystals; the tris (hydroxymethyl) aminomethane, the citric acid, the sodium bicarbonate and the sodium pyruvate together form an acid-base buffer system, so that the pH value of the external environment of the sperm is maintained to be stable; EDTA-2Na reduces the formation size and number of ice crystals and protects the sperm lipid membrane structure; the caffeine is beneficial to the rapid recovery of sperm motility after thawing; dimethyl sulfoxide facilitates the full dissolution of safflower polysaccharide in the diluent.
The application method of the swine sperm cryopreservation diluent containing safflower polysaccharide, namely the method for cryopreserving swine sperm, comprises the following steps:
s1, respectively adjusting the pH values of a diluent A and a diluent B to 7.2, and then preheating at 35 ℃;
s2, determining the concentration of sperms in original semen by using a blood cell counting plate, centrifuging the original semen, removing supernatant, immediately diluting the sperms to 4 hundred million/mL by using a diluent A, and standing in a 16 ℃ environment for 30 minutes for balancing;
s3, after finishing the balancing in the step S2, adding a diluent B which is equal to the diluent A to dilute the sperms to 2 hundred million/mL, and standing in an environment of 3 ℃ for balancing for 60 minutes;
s4, filling diluted sperms into the tubules by using a frozen fine tube filling and sealing machine after balancing in the step S3, cooling to-120 ℃ according to a preset program gradient by using a program-controlled freezer for 60 seconds, and then rapidly putting into liquid nitrogen for preservation;
s5, pre-heating the diluent C at 35 ℃, quickly placing the frozen fine tube at 50 ℃ for 15 seconds after taking out the frozen fine tube from liquid nitrogen, taking out the frozen fine tube, uniformly mixing the frozen fine tube with the diluent C subjected to equivalent pre-heating, and temporarily storing the frozen fine tube at 35 ℃ for waiting use.
The program-controlled freezer preset program in step S4 is as follows: reducing from 5 ℃ to-5 ℃ at a rate of-6 ℃/min and maintaining for 60 seconds; reduced from-5 ℃ to-120 ℃ at a rate of-6 ℃/min and maintained for 60 seconds.
The freezing time is up to 30 days, and the physiological activity of the sperm is almost completely stopped in an ultralow temperature state; thus, the length of freezing has little effect on sperm quality.
The following experiments are adopted to verify the effect of the invention:
preparing pig sperm cryopreservation diluent according to the above description, wherein the concentration of safflower polysaccharide is as follows: test group 1 (0 g/L), test group 2 (0.50 g/L), test group 3 (1.00 g/L), test group 4 (1.50 g/L) and test group 5 (2.00 g/L), the specific compositions are shown in tables 1-3, respectively, followed by freezing and thawing of porcine sperm.
Thawing mode: the diluent C is preheated at 35 ℃, the frozen fine tube is quickly placed at 50 ℃ for 15 seconds after being taken out of liquid nitrogen, then taken out and evenly mixed with the diluent C subjected to equivalent preheating, and placed at 35 ℃ for temporary storage and waiting for use.
Table 1 shows the A component of the cryopreservation diluent for pig sperm of each test group
Table 2 shows the B component of the cryopreservation diluent for pig sperm of each test group
Table 3 shows the C component of the cryopreservation dilutions of porcine sperm for each test group
Analyzing the activity rate, the vitality, the deformity rate and the kinetic parameters of the thawed sperm by using a computer-aided sperm analysis system; JC-1/SYBR-14 combined staining was used to assess sperm plasma membrane integrity and high mitochondrial activity; the sperm acrosome integrity was assessed using FITC-PNA staining; using acridine orange staining to evaluate sperm DNA integrity; evaluating sperm antioxidant capacity and antioxidant enzyme activity using an antioxidant capacity assay kit; estimating the expression quantity of sperm aquaporin by using a Western Blot technology; using syrian hamster @ sMesocricetus auratus) The adhesion index test evaluates the ability of sperm to pass through eggs, the results are expressed as mean ± standard deviation,Pa difference of < 0.05 was significant.
Table 4 shows the effect of cryopreservation diluents on pig sperm viability, vigor and deformity.
In the same column numberThe difference is obvious according to different shoulder mark lettersP＜0.05）。
As can be seen from Table 4, the sperm viability of test groups 2, 3, 4 and 5 was significantly higher than that of test group 1 #P< 0.05), test group 4 was significantly higher than the other test groupsPLess than 0.05), test group 5 is significantly higher than test group 2 and 3 #P< 0.05); the sperm motility of the test group 4 is obviously higher than that of other test groupsP< 0.05), test groups 3 and 5 were significantly higher than test groups 1 and 2; the sperm malformation rate of the test groups 4 and 5 is obviously lower than that of other test groupsP< 0.05), test group 1 was significantly higher than each of the other test groupsP＜0.05）。
Table 5 shows the effect of cryopreservation diluents on kinetic parameters of porcine sperm
The difference between the different expression of the same column data shoulder mark letters is obviousP＜0.05）。
As can be seen from Table 5, the sperm path velocity of test group 4 is significantly higher than those of test groups 1, 2 and 3 #P< 0.05); the linear velocity of sperm in test group 4 and 5 is obviously higher than that in test group 1, 2 and 3%PLess than 0.05), the linear velocity of sperm of the test group 2 and 3 is obviously higher than that of the test group 1%P< 0.05); the curve speeds of the sperms in the test groups 4 and 5 are obviously higher than those in the test groups 1, 2 and 3P< 0.05); the whipping frequency of the sperm in the test group 4 is obviously higher than that of other test groupsPLess than 0.05), the whipping frequency of the sperm in the test groups 3 and 5 is obviously higher than that in the test groups 1 and 2%P＜0.05）。
Table 6 shows the effect of cryopreservation diluents on porcine sperm acrosome integrity, plasma membrane integrity, high DNA integrity and high mitochondrial activity.
The difference between the different expression of the same column data shoulder mark letters is obviousP＜0.05）。
As can be seen from Table 6, the test group 4 sperm acrosome has significantly higher integrityIn test groups 1, 2 and 3%P< 0.05); the sperm plasma membrane integrity rate of the test group 4 is obviously higher than that of the test groups 1, 2 and 5%P< 0.05); the DNA integrity rate of the sperm of the test group 4 is obviously higher than that of the test groups 1, 2 and 3%P< 0.05); the high mitochondrial activity rate of the sperm of the test group 4 is obviously higher than that of other test groupsP＜0.05）。
Table 7 shows the effect of cryopreservation diluents on antioxidant capacity of porcine sperm.
The difference between the different expression of the same column data shoulder mark letters is obviousP＜0.05）。
As can be seen from Table 7, the total antioxidant capacity of the sperm of the pig in the test group 4 is significantly higher than that of the sperm of the pig in other test groupsP< 0.05); the hydrogen peroxide level of the sperm of the test group 4 pigs is obviously lower than that of the test group 1, the test group 2 and the test group 3P< 0.05); the level of malondialdehyde in test group 4 is significantly lower than that in test groups 1, 2 and 5%P＜0.05）。
Table 8 shows the effect of cryopreservation diluents on swine sperm antioxidant enzyme activity.
The difference between the different expression of the same column data shoulder mark letters is obviousP＜0.05）。
As can be seen from Table 8, the activity of the sperm catalase of the 4 pigs in the test group is significantly higher than that of the sperm catalase of the 4 pigs in other test groupsPLess than 0.05), the activity of the catalase of the sperm of the test groups 3 and 5 is obviously higher than that of the test groups 1 and 2 #P< 0.05); the glutathione peroxidase activity and the superoxide dismutase activity of the sperm of the test group 4 are obviously higher than those of the test groups 1, 2 and 5%P< 0.05); test group 4 porcine sperm superoxide dismutase activity was significantly higher than test groups 1, 2, and 5.
Table 9 shows the effect of the freeze diluent on the expression level of porcine sperm aquaporin
As shown in Table 9, the expression level of aquaporin 3 and aquaporin 7 in the test group 4 is significantly higher than that in the test group 1 #P＜0.05）。
Table 10 shows the effect of the freeze diluent on the ability of porcine sperm to pass through eggs
The difference between the different expression of the same column data shoulder mark letters is obviousP＜0.05）。
As can be seen from Table 10, the adhesion index of the sperm of the test group 4 is significantly higher than that of the test group 1 #P＜0.05）。
Thus, the addition of safflower polysaccharide to the cryopreservation diluent has beneficial effects on the sperm viability, vitality, kinetic parameters, plasma membrane integrity, high mitochondrial activity, acrosome integrity, DNA integrity, antioxidant capacity, antioxidant enzyme activity, aquaporin expression level and egg penetration capacity of pigs.
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, or improvements made within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (8)
1. A swine sperm cryopreservation diluent comprising safflower polysaccharide, wherein the preservation diluent consists of individually packaged diluents A, B and C;
the diluent A consists of safflower polysaccharide, glucose, lactose, penicillin, streptomycin, yolk, dimethyl sulfoxide and ultrapure water;
the diluent B consists of safflower polysaccharide, glucose, lactose, penicillin, streptomycin, yolk, dimethyl sulfoxide, glycerol and ultrapure water;
the diluent C consists of glucose, tris (hydroxymethyl) aminomethane, citric acid, caffeine, sodium bicarbonate, EDTA-2Na, sodium pyruvate and ultrapure water;
wherein, in the diluent A, the concentration of safflower polysaccharide is 1.50g/L, the concentration of glucose is 35g/L, the concentration of lactose is 35g/L, the concentration of penicillin is 0.60g/L, the concentration of streptomycin is 1.0g/L, the concentration of yolk is 200mL/L, and the concentration of dimethyl sulfoxide is 3vol%;
in the diluent B, the concentration of safflower polysaccharide is 1.50g/L, the concentration of glucose is 35g/L, the concentration of lactose is 35g/L, the concentration of penicillin is 0.60g/L, the concentration of streptomycin is 1.0g/L, the concentration of yolk is 200mL/L, the concentration of dimethyl sulfoxide is 3vol%, and the concentration of glycerol is 7vol%;
in the diluent C, the concentration of glucose is 25g/L, the concentration of tris (hydroxymethyl) aminomethane is 9g/L, the concentration of citric acid is 3g/L, the concentration of caffeine is 4.5g/L, the concentration of sodium bicarbonate is 3g/L, the concentration of EDTA-2Na is 4.3g/L, and the concentration of sodium pyruvate is 0.42g/L.
2. The safflower polysaccharide-containing swine sperm cryopreservation diluent as set forth in claim 1, wherein diluent a, diluent B and diluent C are stored at 3-5 ℃.
3. The safflower polysaccharide containing swine sperm cryopreservation diluent of claim 1, wherein the yolk is chicken yolk, which is centrifuged and sediment removed.
4. The safflower polysaccharide containing swine sperm cryopreservation diluent of claim 1, wherein the method for preparing diluent a and diluent B comprises: dissolving safflower polysaccharide in dimethyl sulfoxide, adding other components, dissolving with ultrapure water, and fixing volume.
5. A method for cryopreserving porcine sperm cells using a safflower polysaccharide containing porcine sperm cryopreservation diluent as described in any one of claims 1-4, wherein said method is performed by the steps of:
s1, respectively adjusting the pH values of the diluent A and the diluent B to 7.2-7.4, and then preheating at 35-37 ℃;
s2, removing supernatant after centrifuging the original semen, immediately diluting the semen to 4 hundred million/mL by using a diluent A, and placing the semen in an environment of 16-18 ℃ for balancing for 30 minutes;
s3, adding a diluent B which is equal to the diluent A to dilute the sperms to 2 hundred million/mL, and standing for 60 minutes in an environment of 3-5 ℃;
s4, filling the diluted sperms into the tubules by using a frozen fine tube filling and sealing machine, cooling to-115 ℃ to-125 ℃ in a gradient way, maintaining for 55-65 seconds, and then rapidly putting into liquid nitrogen for preservation;
s5, pre-heating the diluent C at 35-37 ℃, quickly placing the frozen fine tube at 48-52 ℃ for 15 seconds after the frozen fine tube is taken out of liquid nitrogen, taking out the frozen fine tube, uniformly mixing the frozen fine tube with the diluent C with the same amount, and temporarily storing the frozen fine tube at 35-37 ℃ for waiting use.
6. The method for cryopreserving porcine sperm cells using a safflower polysaccharide containing porcine sperm cryopreservation diluent as described in claim 5, wherein the sperm cell concentration is determined using a blood cell counter plate.
7. The method for cryopreserving porcine sperm cells using a carthamin polysaccharide-containing cryopreservation diluent according to claim 5, wherein the temperature is reduced using a programmable freezer.
8. The method for cryopreserving porcine sperm cells using a safflower polysaccharide containing porcine sperm cryopreservation diluent according to claim 5, wherein the gradient cooling process is as follows: reducing the temperature to-4.5 ℃ to-5.5 ℃ at the speed of-5.5 ℃/min to-6.5 ℃/min and maintaining the temperature for 55 seconds to 65 seconds, and reducing the temperature to-115 ℃ to-125 ℃ at the same speed and maintaining the temperature for 55 seconds to 65 seconds.
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