CN111149795A - Cryoprotectant and application thereof, sperm freezing liquid and preparation method thereof - Google Patents
Cryoprotectant and application thereof, sperm freezing liquid and preparation method thereof Download PDFInfo
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- CN111149795A CN111149795A CN202010037287.0A CN202010037287A CN111149795A CN 111149795 A CN111149795 A CN 111149795A CN 202010037287 A CN202010037287 A CN 202010037287A CN 111149795 A CN111149795 A CN 111149795A
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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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
Abstract
The invention relates to the technical field of assisted reproduction, and discloses a cryoprotectant and application thereof, and a sperm refrigerating fluid and a preparation method thereof, wherein the sperm refrigerating fluid comprises the cryoprotectant, a composition and a basic culture solution; wherein the cryoprotectant comprises 0.3-1.1 mg/L of paclitaxel, 16-22 g/L of trehalose, 7-13 g/L of sucrose and 80-250 mL/L of glycerol; the composition comprises 12-26 mg/L of resveratrol, 0.2-1.9 mg/L of reduced glutathione, 436-775 mg/L of lecithin, 0.5-1.6 mg/L, ATP mg/L of cholesterol, 9-17 mg/L of sodium salt, 11-28 mg/L of heparin, 93-299 mg/L of phosphatidylserine and 105-25 mg/L of coenzyme Q; the sperm freezing liquid can reduce the damage of the sperm in the processes of low-temperature freezing and freezing recovery, improve the activity of the sperm after freezing recovery, prolong the survival time of the sperm after freezing recovery, promote sperm capacitation and enhance the penetrating fertilization capability of the sperm to egg cells, further improve the fertilization success rate of the ovum and the blastocyst formation rate, and finally achieve the effect of improving the pregnancy success rate of the in vitro fertilization assisted reproduction technology.
Description
Technical Field
The invention relates to the technical field of assisted reproduction, in particular to a cryoprotectant and application thereof, a sperm refrigerating fluid and a preparation method thereof.
Background
In Vitro Fertilization (IVF), also known as tube infant, is a method of assisted reproductive therapy, and specifically refers to taking out an ovum and a sperm from the body, combining and fertilizing the sperm and the ovum under in vitro specific environmental conditions to form a fertilized ovum, and transplanting the embryo back to a maternal uterus for pregnancy to develop into a fetus after the fertilized ovum is continuously cultured and developed in vitro to a blastocyst stage embryo.
In the in vitro fertilization assisted reproduction technology, based on the consideration of future bearing and rearing plan, a male can freeze self sperm in a medical institution with corresponding qualification so as to prevent future fertility risks; the frozen preservation of qualified sperm is required by various large sperm banks and assisted reproduction medical institutions to serve as the assisted reproduction requirement for in vitro fertilization. Meanwhile, in order to prevent the patients with severe oligospermia and asthenospermia from developing azoospermia in the future to completely lose fertility, the sperms can be preserved in a frozen way for a long time in the process of treating the oligospermia in advance and used for treating infertility by an assisted reproduction technology when needed in the later period, so that the aim of smoothly breeding the next generation is fulfilled.
Currently, the sperm freezing liquids available on the market are mainly divided into two main categories, namely those containing yolk and those without yolk, but all have some disadvantages: on one hand, as the yolk contains a plurality of complex components besides lecithin, granular substances can appear after the sperm freezing recovery is carried out by utilizing the sperm freezing solution containing the yolk, the further processing process is influenced, and meanwhile, as the yolk belongs to heterogeneous source substances, certain biological risk also exists; on the other hand, the sperm freezing solution without yolk only contains basic salt ions, a small amount of amino acid, a single sperm freezing protective agent and the like, so that the sperm freezing protection effect is limited, and a great promotion space exists.
Therefore, there is a need for a sperm freezing solution without impurities and with biosafety and functional diversity.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a sperm freezing liquid and a preparation method thereof so as to achieve the effects of reducing damage of sperm in the processes of low-temperature freezing and freezing recovery, improving the activity of the sperm after freezing recovery, prolonging the survival time of the sperm after freezing recovery, promoting sperm capacitation, enhancing the penetrating fertilization capability of the sperm on egg cells, and further improving the fertilization success rate of the egg and the blastocyst formation rate.
It is another object of the present invention to overcome the deficiencies of the prior art and to provide a cryoprotectant and its use.
The purpose of the invention is realized by the following technical scheme: a cryoprotectant comprises paclitaxel, trehalose, sucrose and glycerol.
The use of a cryoprotectant for the cryoprotective of germ cells, preferably sperm.
A sperm freezing solution comprises a basal culture solution, a cryoprotectant and a composition, wherein the cryoprotectant comprises paclitaxel, trehalose, sucrose and glycerol, and the composition comprises resveratrol, reduced glutathione, lecithin, cholesterol, ATP sodium salt, heparin, phosphatidylserine and coenzyme Q10.
The paclitaxel can be used as a cytoskeleton stabilizer, enhances the close relation of α and β -tubulin dimers, enhances the crosslinking of microtubules, promotes the polymerization and the stabilization of the microtubules, and has dependence and reversibility on the combination of microtubules with an ultramicro-skeleton structure.
The trehalose is a safe and reliable natural saccharide, and is a non-reducing saccharide formed by two glucose molecules through 1, 1-glycosidic bonds. Under severe environmental conditions of high temperature, high cold, high osmotic pressure, dry dehydration and the like, the trehalose can form a unique protective film on the cell surface, and protein molecules are effectively protected from invariance and inactivation, so that the trehalose has an important protection effect on organisms and various bioactive substances.
The resveratrol (3-4' -5-trihydroxystilbene) is a non-flavonoid polyphenol compound, and the chemical name of the resveratrol is 3,4', 5-trihydroxy-1, 2-diphenylethene (3,4', 5-stilbenetriol). In vitro experiments and animal experiments show that resveratrol has antioxidant, antiinflammatory, anticancer, antibacterial, antiasthmatic, immunoregulatory and cardiovascular protecting effects. By adopting the technical scheme, the resveratrol is used in the sperm refrigerating fluid, and can remove active oxygen free radicals in the solution by utilizing the antioxidation effect of the resveratrol, so that the effect of reducing the oxidative damage of the active oxygen free radicals to sperm cells is achieved.
The reduced glutathione can participate in the oxidation-reduction process in cells, active oxygen free radicals generated in the movement process of sperms are removed, and the effect of reducing the oxidative damage of the active oxygen free radicals to the sperms is achieved.
The lecithin has an anti-oxidation effect, and unsaturated bonds in molecules of the lecithin are combined with active oxygen free radicals to remove the active oxygen free radicals generated in the movement process of sperms, so that sperms are protected and repaired from being oxidized and damaged, and the effect of improving the sperm activity is achieved; in addition, the lecithin can also stabilize the cell membrane skeleton structure of sperms, and the effects of preventing acrosome membrane from cracking and resisting low-temperature shock are achieved.
The heparin can mildly and effectively improve the energy production function of mitochondria in sperm cells and promote the calcium ion inflow by combining a receptor on the surface of the sperm and freezing a signal conduction path coupled with the receptor, thereby inducing acrosome reaction and achieving the effect of improving the fertilization activity of the sperm.
The ATP sodium salt not only can directly provide energy substances required by movement for sperms, but also can contribute to the energy production of mitochondria in the middle part of the spermatid.
The phosphatidylserine is a ubiquitous phospholipid, is located in the inner layer of a cell membrane, is one of cell membrane structural components, and contributes to cell membrane freezing protection and rapid repair of freezing damage.
The coenzyme Q10 has the functions of resisting oxidation and scavenging free radicals, and the coenzyme Q10 is required to participate in the energy production of human cells so as to drive the human cells to generate energy; the coenzyme Q10 can accelerate cell renewal, stimulate cell activity and maintain mitochondrial morphological structure, thereby achieving the effect of remarkably promoting the ability of cell to take in nutrient substances.
Further, the basic culture solution comprises a solvent and a solute, wherein the solvent comprises water, preferably ultrapure water; the solute comprises 5-12 g/L of sodium chloride, 311-433 mg/L of potassium chloride, 110-230 mg/L of calcium chloride, 22-36 mg/L of magnesium sulfate, 46-55 mg/L of monopotassium phosphate, 322-396 mg/L of sodium bicarbonate, 0.51-2.34 g/L of glucose, 46-88 mg/L of amino acid or salt thereof, 204-247 mg/L of alanylglutamine, 2.31-2.46 mg/L of sodium lactate, 34.1-38.9 mg/L of sodium pyruvate, 9.82-12.36 mg/L, HEPES 1.8.8-2.9 g/L of taurine and 1.6-2.7 g/L of MOPS.
Through the technical scheme, the ultrapure water is free of endotoxin and heavy metal and is suitable for being used as a solvent.
The taurine exists in a free state in vivo as a sulfur-containing non-protein amino acid, belongs to a free amino acid, does not participate in the biosynthesis of in vivo protein, can adjust the osmotic pressure between cells and a solution, and is closely related to the metabolism of cystine and cysteine; meanwhile, the taurine can also interact with insulin receptor protein, promote the uptake and the utilization of glucose by cells and accelerate glycolysis; in addition, it has the function of maintaining the normal fertilization function of the sperm.
The HEPES is added, so that the effects of effectively buffering the pH of the solution and supporting fertilization and embryo development are achieved; the MOPS is added, so that the effects of maintaining the stable pH of the basic culture solution, prolonging the standing time of the solution and reducing the production cost of the solution are achieved.
Further, the amino acids include essential amino acids and non-essential amino acids;
the essential amino acid or salt thereof comprises at least one of L-valine, L-leucine, L-isoleucine, L-phenylalanine, L-tryptophan, L-methionine, L-lysine and L-threonine;
the non-essential amino acid or a salt thereof includes at least one of glycine, L-alanine, L-proline, L-serine, L-tyrosine, L-cysteine, L-asparagine, L-glutamine, L-aspartic acid, L-glutamic acid, L-arginine, and L-histidine.
Through the technical scheme, the essential amino acid and the nonessential amino acid provide basic substances required by synthesizing protein and nucleic acid for cells, further provide various energy and nitrogen sources required by the cells, participate in cell signal transduction pathways, maintain the intracellular environment stability, regulate the osmotic pressure balance and acid-base balance of the solution, chelate heavy metals, and achieve the effect of providing comprehensive protection effect for the cells.
Further, the content of each component in the cryoprotectant is as follows: 0.3-1.1 mg/L of paclitaxel, 16-22 g/L of trehalose, 7-13 g/L of sucrose and 80-250 mL/L of glycerol;
the content of each component in the composition is as follows: 12-26 mg/L of resveratrol, 0.2-1.9 mg/L of reduced glutathione, 436-775 mg/L of lecithin, 0.5-1.6 mg/L, ATP of sodium salt 9-17 mg/L of cholesterol, 11-28 mg/L of heparin, 93-299 mg/L of phosphatidylserine and 105-24 mg/L of coenzyme Q.
Further, the sperm refrigerating fluid also comprises 5-15 g/L of human serum albumin and 7-14 mg/L of gentamicin sulfate.
By adopting the technical scheme, the human serum albumin can chelate heavy metals, so that the effect of avoiding the toxic influence of the heavy metals in the sperm activating solution on the sperms is achieved; the gentamicin sulfate is used as a spectrum antibiotic, and the effect of effectively inhibiting the growth of bacteria is achieved, so that the bacterial pollution of the sperm refrigerating fluid is avoided.
A preparation method of sperm refrigerating fluid comprises the following steps:
s1, weighing the reduced glutathione, the ATP sodium salt, the heparin, the trehalose, the sucrose, the sodium chloride, the potassium chloride, the calcium chloride, the magnesium sulfate, the monopotassium phosphate, the glucose, the amino acid, the alanyl glutamine, the sodium lactate, the sodium pyruvate, the taurine, the HEPES and the MOPS according to the proportion under hundred-grade conditions, and adding water to uniformly mix to obtain a solution I;
s2, weighing the resveratrol, the coenzyme Q10, the paclitaxel and the cholesterol according to the proportion, and adding the resveratrol, the coenzyme Q10, the paclitaxel and the cholesterol into ethanol for uniform mixing to obtain a solution II;
s3, weighing the lecithin, the phosphatidylserine and the glycerol according to the proportion, and uniformly mixing to obtain a solution III;
s4, stirring and dropwise adding the solution II into the solution III to obtain a solution IV;
s5, weighing the sodium bicarbonate, the human serum albumin and the gentamicin sulfate according to the proportion;
s6, uniformly mixing the solution IV, sodium bicarbonate, human serum albumin, gentamicin sulfate and the solution I to obtain a solution V;
s7, mixing the solution V with a pH regulator to obtain a solution VI with the pH value of 7.10-7.20;
and S8, carrying out sterile filtration treatment on the solution VI to obtain the sperm refrigerating fluid.
Furthermore, the environmental temperature and the environmental humidity of S1-S7 are 18-26 ℃ and 40-60%.
Through the technical scheme, the environmental temperature and the environmental humidity are limited, and the effects of effectively ensuring the smooth running of the solution preparation process and reducing the risk caused by the environmental change difference in the solution production process are achieved.
Further, in S2, the ethanol is absolute ethanol, the concentration of resveratrol in the solution II is 12-26 g/L, the concentration of coenzyme Q10 is 5-24 g/L, the concentration of paclitaxel is 0.3-1.1 g/L, and the concentration of cholesterol is 0.5-1.6 g/L.
Through the technical scheme, the absolute ethyl alcohol has toxic effect on cells, the concentrations of the resveratrol, the coenzyme Q10, the paclitaxel and the cholesterol in the solution II are limited, the amount of the absolute ethyl alcohol in the solution II is further limited, and the effects of fully dissolving components and ensuring the biological safety are achieved.
Further, in S7, the pH adjusting agent is a sodium hydroxide solution, and the concentration of the sodium hydroxide solution is 1 mol/L.
Further, in S8, the sterile filtration process specifically includes: the solution VI is filtered using a filter with a pore size of 0.22 μm or 0.45. mu.m.
Furthermore, the pore size of the filter membrane is preferably 0.22 μm to achieve better sterilization effect.
The paclitaxel, the lecithin, the coenzyme Q10, the cholesterol and the like are easy to denature under the conditions of high temperature, high pressure, ultraviolet or ordinary light long-term irradiation to generate peroxide, so that the oxidative damage is caused to sperm cells; calcium ions and magnesium ions contained in the basic culture solution are easy to separate out to form insoluble precipitates after high-temperature and high-pressure sterilization; meanwhile, in the high-temperature and high-pressure sterilization process, the cell wall of the bacteria is cracked to release endotoxin, and the high content of the endotoxin can cause certain toxic effect on spermatids, thereby further influencing the subsequent fertilization process.
Through above-mentioned technical scheme, select for use aseptic filtration to handle, can avoid the emergence of above-mentioned condition when reaching the degerming effect.
The invention has the beneficial effects that:
1. according to the sperm refrigerating fluid, the components are added into the sperm refrigerating fluid at the same time, so that a sperm energy metabolism path can be actively excited to promote sperm capacitation and protect sperms from oxidative damage, the vitality of recovered sperms is improved, the survival time is prolonged, the fertilization effect is promoted, the fertilization success rate of ova and the blastocyst formation rate of fertilized ova developing into usable embryos are improved, the pregnancy success rate of an in vitro fertilization assisted reproduction technology is finally improved, and the effect of greatly improving the functional diversity of the sperm refrigerating fluid is achieved.
2. The sperm freezing liquid has no biological risk, can play a role in freezing protection on a sperm cell structure, prevents ice crystals formed by freezing from damaging the sperm cell structure, and further protects the integrity of the ultrastructure of a sperm, such as a cell membrane, mitochondria, acrosome, DNA and the like.
3. The sperm refrigerating fluid is effectively suitable for low-temperature cryopreservation of sperm in the in vitro fertilization assisted reproduction technology.
Detailed Description
The technical solutions of the present invention are described in further detail below, but the scope of the present invention is not limited to the following.
The following 3 groups of examples were carried out using the sperm freezing solution of the present invention.
1. The components and the contents of the sperm freezing solution (the solvent is ultrapure water and ethanol) in the examples 1-3 are shown in the following table:
2. the preparation method of the sperm refrigerating fluid in the embodiments 1 to 3 comprises the following steps:
s1, weighing reduced glutathione, ATP sodium salt, heparin, trehalose, sucrose, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, monopotassium phosphate, glucose, amino acid, alanyl glutamine, sodium lactate, sodium pyruvate, taurine, HEPES and MOPS according to a ratio under hundred-grade conditions, and adding water to mix uniformly to obtain a solution I;
s2, weighing resveratrol, coenzyme Q10, paclitaxel and cholesterol according to a ratio, and adding the weighed materials into ethanol to be uniformly mixed to obtain a solution II; wherein the concentration of resveratrol, coenzyme Q10, paclitaxel and cholesterol in the solution II is 1000 times of the final concentration;
s3, weighing lecithin, phosphatidylserine and glycerol according to the proportion, and uniformly mixing to obtain a solution III;
s4, stirring and dropwise adding the solution II into the solution III to obtain a solution IV;
s5, weighing sodium bicarbonate, human serum albumin and gentamicin sulfate according to the proportion;
s6, uniformly mixing the solution IV, sodium bicarbonate, human serum albumin, gentamicin sulfate and the solution I to obtain a solution V;
s7, mixing the solution V with a sodium hydroxide solution with the concentration of 1mol/L to obtain a solution VI with the pH value of 7.10-7.20;
s8, filtering the solution VI by using a filter membrane with the aperture of 0.22 mu m to obtain a sperm refrigerating fluid;
wherein the environmental temperature is 18-26 ℃ and the environmental humidity is 40-60% in S1-S7.
Comparative example 1
The sperm freezing solution in the example 1 of the invention is compared with the sperm freezing solution in the comparative example 1, and the composition in the comparative example 1 is as follows: lecithin, cholesterol, ATP sodium salt, heparin, phosphatidylserine and coenzyme Q10, and other components such as the dosage, steps and conditions are the same as those in example 1 of the present invention (compared with example 1, resveratrol and reduced glutathione are not added to the composition in this comparative example, which is used to prove that the sperm freezing solution of the present invention has a better effect).
Comparative example 2
The sperm freezing solution in the embodiment 1 of the invention is compared with the sperm freezing solution in the comparison example 2, and the composition in the comparison example 2 is as follows: resveratrol, reduced glutathione, lecithin, cholesterol, phosphatidylserine, and other components such as the amount, steps, and conditions are the same as those in example 1 of the present invention (in this comparative example, compared to example 1, no ATP sodium salt, heparin, and coenzyme Q10 are added to the composition, which is used to prove that the sperm freezing solution of the present invention has a better effect).
Comparative example 3
The sperm freezing solution of the invention in the example 1 is compared with the sperm freezing solution of the comparison example 3, and the composition of the comparison example 3 is as follows: resveratrol, reduced glutathione, cholesterol, ATP sodium salt, heparin and coenzyme Q10, and other components such as the dosage, steps and conditions are the same as those in example 1 of the present invention (compared with example 1, lecithin, phosphatidylserine and cholesterol are not added in the composition, which is used for proving that the sperm freezing solution of the present invention has better effect).
Comparative example 4
The sperm refrigerating fluid in the embodiment 1 of the invention is compared with the sperm refrigerating fluid in the comparison example 4, and the cryoprotectant in the comparison example 4 is as follows: glycerol and sucrose, and other components such as dosage, steps and conditions are the same as those in example 1 of the present invention (in this comparative example, trehalose and paclitaxel are not added to the cryoprotectant, compared to example 1, and are used to prove that the sperm freezing solution of the present invention has better effect).
Test effects
1. In order to verify the effect of the sperm refrigerating fluid of the present invention, the sperm refrigerating fluids obtained in examples 1 to 3 and comparative examples 1 to 4 were tested, and the test items include pH, osmotic pressure, and bacterial endotoxin, and the test method was as follows:
-pH detection: detecting a certain amount of finished products by using a blood gas analyzer, recording the three detection values, taking the average value as the final pH value of the sperm freezing liquid, and determining that the pH value is qualified within the range of 7.20-7.40;
-osmotic pressure test: taking a proper amount of sperm refrigerating fluid, diluting the sperm refrigerating fluid by 10 times with water for injection, taking 25 mu L of diluted liquid, dripping the diluted liquid into a 0.5mL EP tube, putting the EP tube into a probe of an osmometer (freezing point osmometer) to wait for the stability of a detection value, and reading out a numerical value; the average value of the sperm freezing solution is obtained by the same method for detecting three times, and the osmotic pressure of the sperm freezing solution is qualified within the range of 299-348 mOsM;
-bacterial endotoxin detection: the method is carried out by adopting a limulus reagent gel method in the detection method of the endotoxin in Chinese pharmacopoeia, and the test result is qualified, wherein the limulus reagent gel method is less than 0.2 EU/mL;
the results are shown in the following table:
| group of | pH | Osmolarity mOsM | Bacterial endotoxins |
| Example 1 | 7.31 | 331 | <0.005 |
| Example 2 | 7.26 | 314 | <0.005 |
| Example 3 | 7.37 | 346 | <0.005 |
| Comparative example 1 | 7.25 | 328 | <0.005 |
| Comparative example 2 | 7.27 | 326 | <0.005 |
| Comparative example 3 | 7.34 | 329 | <0.005 |
| Comparative example 4 | 7.30 | 319 | <0.005 |
As can be seen from the above table, the pH, osmotic pressure and bacterial endotoxin values of the sperm freezing solutions obtained in examples 1 to 3 and comparative examples 1 to 4 were close to each other and all of them were acceptable.
2. In order to verify the influence of the sperm refrigerating fluid on fertilization and blastocyst formation, in-vitro mouse embryo tests were performed on the sperm refrigerating fluids in examples 1 to 3 and comparative examples 1 to 4, and the method included the following steps:
A1. superovulation/aspiration: selecting 6-8 weeks old female mice, and injecting 10 IU/female mouse by abdominal cavity; after 48 hours, 10 IU/mouse of hCG is injected into the abdominal cavity; after 14h, washing the oviduct of the ampulla of the mouse, taking out mature eggs, placing the mature eggs in a culture solution balanced overnight in advance, and removing abnormal eggs to obtain normal eggs;
A2. the method comprises the following steps of (1) taking sperm/freezing sperm/reviving sperm from a mouse, wherein the steps comprise:
A21. directly taking sperms from epididymis and vas deferens of a male rat through an operation, mixing the sperms to obtain high-activity sperm concentrated solution through an upstream method and sperm refrigerating solution balanced at room temperature in advance (the ratio of the sperm concentrated solution to the sperm refrigerating solution is 3:1) to obtain mixed solution; transferring the mixed solution into a freezing tube, and directly putting the tube into liquid nitrogen for freezing for a certain time;
A22. taking out the frozen tube, thawing rapidly in 37 deg.C water bath, adding balanced sperm-washing receptor solution (37 deg.C, 6% CO)2) Cleaning and centrifuging to obtain revived sperms;
A3. in vitro fertilization/blastocyst culture: the density is 5 to 10 x 106Co-culturing 80 mu L of sperms and 100 mature ova in 400 mu L of sperm-washing and fertilization solution droplets for 6-8 h to finish fertilization; after washing the fertilized eggs twice with the embryo culture solution, the fertilized eggs are washed and transferred to new embryo culture solution microdroplets balanced in advance, and then the fertilized eggs are washed and transferred to 6 percent CO at 37 DEG C2、5％O2And culturing in an incubator with saturated humidity to 96h of a blastocyst stage, and performing morphological observation and analysis on the development condition of the embryo to obtain the blastocyst formation rate.
The results are shown in the following table (three replicates):
as can be seen from the above table, the rate of blastocyst formation in example 1 was as high as 88%, whereas the rate of blastocyst formation in comparative examples 1 to 4 was significantly lower than that in example 1.
Therefore, in the process of freezing sperms, the components of resveratrol, reduced glutathione, lecithin, cholesterol, ATP sodium salt, heparin, phosphatidylserine, coenzyme Q10, paclitaxel and trehalose supplement each other, and the deletion of any one or more components can directly influence the blastocyst formation rate, so that the blastocyst formation rate is obviously reduced. Therefore, resveratrol, reduced glutathione, lecithin, cholesterol, ATP sodium salt, heparin, phosphatidylserine, coenzyme Q10, paclitaxel and trehalose have obvious effect of improving the blastocyst formation rate of fertilization after the frozen sperm is recovered.
In conclusion, the sperm freezing liquid provided by the invention can reduce damage of sperm in the processes of low-temperature freezing and freezing recovery, improve the activity of the sperm after freezing recovery, prolong the survival time of the sperm after freezing recovery, promote sperm capacitation and enhance the penetrating fertilization capability of the sperm on egg cells, thereby improving the fertilization success rate of eggs and the blastocyst formation rate.
The foregoing is illustrative of the preferred embodiments of this invention, and it is to be understood that the invention is not limited to the precise form disclosed herein and that various other combinations, modifications, and environments may be resorted to, falling within the scope of the concept as disclosed herein, either as described above or as apparent to those skilled in the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A cryoprotectant comprising paclitaxel, trehalose, sucrose and glycerol.
2. Use of a cryoprotectant according to claim 1, wherein the cryoprotectant is used for cryoprotection of sperm cells.
3. A sperm freezing solution is characterized by comprising a basic culture solution, a cryoprotectant and a composition, wherein the cryoprotectant comprises paclitaxel, trehalose, sucrose and glycerol, and the composition comprises resveratrol, reduced glutathione, lecithin, cholesterol, ATP sodium salt, heparin, phosphatidylserine and coenzyme Q10.
4. A sperm freezing solution according to claim 3, wherein the basal medium comprises a solvent and a solute, the solvent comprises water, and the solute comprises 5-12 g/L of sodium chloride, 311-433 mg/L of potassium chloride, 110-230 mg/L of calcium chloride, 22-36 mg/L of magnesium sulfate, 46-55 mg/L of monopotassium phosphate, 322-396 mg/L of sodium bicarbonate, 0.51-2.34 g/L of glucose, 46-88 mg/L of amino acid or salt thereof, 204-247 mg/L of alanylglutamine, 2.31-2.46 mg/L of sodium lactate, 34.1-38.9 mg/L of sodium pyruvate, 9.82-12.36 mg/L, HEPES 1.8.8-2.9 g/L of taurine and 1.6-2.7 g/L of MOPS.
5. A sperm freezing fluid as described in claim 4, wherein the cryoprotectant comprises the following components: 0.3-1.1 mg/L of paclitaxel, 16-22 g/L of trehalose, 7-13 g/L of sucrose and 80-250 mL/L of glycerol;
the content of each component in the composition is as follows: 12-26 mg/L of resveratrol, 0.2-1.9 mg/L of reduced glutathione, 436-775 mg/L of lecithin, 0.5-1.6 mg/L, ATP of sodium salt 9-17 mg/L of cholesterol, 11-28 mg/L of heparin, 93-299 mg/L of phosphatidylserine and 105-24 mg/L of coenzyme Q.
6. A sperm freezing solution according to claim 5, further comprising 5-15 g/L human serum albumin and 7-14 mg/L gentamicin sulfate.
7. A method of preparing the sperm freezing fluid of claim 6 comprising the steps of:
s1, weighing the reduced glutathione, ATP sodium salt, heparin, trehalose, sucrose, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, monopotassium phosphate, glucose, amino acid or salt thereof, alanyl glutamine, sodium lactate, sodium pyruvate, taurine, HEPES and MOPS according to a ratio under hundred-grade conditions, adding water, and uniformly mixing to obtain a solution I;
s2, weighing the resveratrol, the coenzyme Q10, the paclitaxel and the cholesterol according to the proportion, and adding the resveratrol, the coenzyme Q10, the paclitaxel and the cholesterol into ethanol for uniform mixing to obtain a solution II;
s3, weighing the lecithin, the phosphatidylserine and the glycerol according to the proportion, and uniformly mixing to obtain a solution III;
s4, stirring and dropwise adding the solution II into the solution III to obtain a solution IV;
s5, weighing the sodium bicarbonate, the human serum albumin and the gentamicin sulfate according to the proportion;
s6, uniformly mixing the solution IV, sodium bicarbonate, human serum albumin, gentamicin sulfate and the solution I to obtain a solution V;
s7, mixing the solution V with a pH regulator to obtain a solution VI with the pH value of 7.10-7.20;
and S8, carrying out sterile filtration treatment on the solution VI to obtain the sperm refrigerating fluid.
8. The method according to claim 7, wherein the ambient temperature and the ambient humidity in S1-S7 are 18-26 ℃ and 40-60%.
9. The method according to claim 7, wherein in S7, the pH regulator is sodium hydroxide solution.
10. The method according to claim 7, wherein in S8, the sterile filtration process is specifically: the solution VI is filtered using a filter with a pore size of 0.22 μm or 0.45. mu.m.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010037287.0A CN111149795A (en) | 2020-01-14 | 2020-01-14 | Cryoprotectant and application thereof, sperm freezing liquid and preparation method thereof |
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| CN114208817A (en) * | 2022-01-18 | 2022-03-22 | 上海市农业科学院 | Application of combined action of cholesterol and soybean lecithin in chicken semen after being frozen |
| CN114514924A (en) * | 2021-03-23 | 2022-05-20 | 上海慧存医疗科技有限公司 | Ovarian tissue preservation method |
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| CN113016779A (en) * | 2021-02-07 | 2021-06-25 | 内蒙古大学 | Method for freezing and preserving semen of milk sheep and diluent thereof |
| CN114514924A (en) * | 2021-03-23 | 2022-05-20 | 上海慧存医疗科技有限公司 | Ovarian tissue preservation method |
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