CN106818712B - Sperm drying liquid and sperm preservation method - Google Patents
Sperm drying liquid and sperm preservation method Download PDFInfo
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- CN106818712B CN106818712B CN201710168610.6A CN201710168610A CN106818712B CN 106818712 B CN106818712 B CN 106818712B CN 201710168610 A CN201710168610 A CN 201710168610A CN 106818712 B CN106818712 B CN 106818712B
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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
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Abstract
A sperm drying liquid and a sperm preservation method relate to the field of biological cell preservation, and can simulate the environmental state of a sperm growth state, provide the basic needs of the sperm survival in vitro and maintaining a certain metabolic activity, better protect the sperm and improve the integrity of the sperm. The invention provides a sperm preservation method, which adopts a centrifugal method to remove the spermatic clear in the semen, then mixes the spermatic clear liquid with the sperm drying liquid, and finally adopts an evaporation drying method to dry. The whole process is simple to operate, complex freezing and drying equipment is not needed, the processed sperms can be stored for a long time, high integrity is still kept, high cleavage rate and blastocyst development rate can be still obtained after the sperms are combined with egg cells after long-term storage, and the sperm collecting device is particularly suitable for field collection and collection of wild animal semen.
Description
Technical Field
The invention relates to the field of biological cell preservation, in particular to a sperm drying solution and a sperm preservation method.
Background
Semen is preserved to prolong the survival time of sperm in vitro and maintain the fertilization ability, so that sperm can be transported for a long time and in a long distance without being limited by time and regions. Is convenient for developing inter-provincial and international communication cooperation, is beneficial to improving the utilization rate of excellent sires and the long-term preservation of genetic resources, and has important significance in the aspects of conservation, variety improvement, introduction, human reproductive medicine and the like.
In a natural state, the life of the sperm after being separated from the body is only a few hours, and long-distance transportation can be realized only after certain treatment. The traditional sperm preservation method is to store the frozen semen in liquid nitrogen or dry ice, the low-temperature freezing preservation technology needs the liquid nitrogen or the dry ice, the liquid nitrogen needs a special steel tank and has great danger, which brings great inconvenience to transportation, and the dry ice is easy to melt at room temperature and is not beneficial to the long-time transportation of the sperm at room temperature.
Since the use of intracytoplasmic sperm injection (ICSI), many simpler methods of preserving semen have been investigated, including freeze-drying and evaporative drying. After the semen is dried, the semen can be stored for a long time at 4 ℃, and can be transported and stored for a short time at room temperature, and meanwhile, the dry storage method does not need liquid nitrogen, has small storage space and low transportation cost, so that the semen is more economical and practical than the conventional low-temperature freezing storage. Although the dried sperm have lost motility and natural fertilization, they have a high rate of chromosomal integrity and can still associate with an ovum and develop into an embryo by intracytoplasmic sperm injection techniques.
Compared with a freeze-drying method, the evaporation drying method has the advantages that a freeze-drying machine required by freeze-drying is not required in the drying process, the semen collection and treatment process is convenient, and the like. However, the integrity of sperm genetic material is a prerequisite for fertilization and embryo development, and in the prior art, the integrity of sperm after being dried by an evaporation drying method and the fertilization rate and embryo development rate after intracytoplasmic injection are all lower than those of a freeze drying method.
Disclosure of Invention
The invention aims to provide a sperm drying liquid which has the advantages of high efficiency, convenience, no toxicity to cells and the like, can effectively protect sperms, reduce physical damage and chemical damage to the sperms in the drying process, obviously improve the sperm integrity rate, effectively activate ova after the sperms are injected into cytoplasm, and improve the embryo development rate.
Another object of the present invention is to provide a method for preserving sperm, which is simple and easy to operate, is inexpensive, does not require refrigeration equipment, and is particularly suitable for collecting and collecting sperm of wild animals in the field. The processed sperms are convenient to store and transport for a long time and a long distance. The sperm preserved by the method for a long time can still keep higher integrity rate and embryo development rate.
The embodiment of the invention is realized by the following steps:
a sperm drying fluid comprising, per liter of sperm drying fluid: 6-7 g of sodium chloride, 0.2-0.3 g of potassium chloride, 0.3-0.6 g of calcium chloride, 0.05-0.1 g of magnesium chloride, 2-3 g of sodium bicarbonate, 0.03-0.05 g of sodium phosphate, 1-1.2 g of glucose, 2.5-3 g of 4-hydroxyethyl piperazine ethanesulfonic acid, 10-50 mmol of raffinose and the balance of water.
A method of sperm preservation comprising: centrifuging the diluted semen to remove the semen, and mixing with a sperm drying solution to obtain a sperm suspension; the sperm suspension was evaporated to dryness and stored.
The embodiment of the invention has the beneficial effects that: the sperm drying liquid provided by the invention can simulate the environmental state of sperm in a growth state, has the functions of maintaining osmotic pressure, controlling acid-base balance, supplying energy and inorganic salt components necessary for cell survival and metabolism and the like, and can provide basic requirements for sperm survival in vitro and certain metabolic activity maintenance. In the invention, a certain amount of raffinose is added into the sperm drying liquid, and the raffinose is not only a main non-electrolyte and can moderate the ionization degree of seminal plasma, but also a main energy substance after sperm separation, and is one of important substances for regulating osmotic pressure in a diluent. Meanwhile, the molecular weight of raffinose is larger, and compared with micromolecular sugar, the raffinose can better protect sperms and improve the integrity of the sperms.
The invention provides a sperm preservation method, which adopts a centrifugal method to remove the spermatic clear in the semen, then mixes the spermatic clear liquid with the sperm drying liquid, and finally adopts an evaporation drying method to dry. The whole process is simple to operate, complex freezing and drying equipment is not needed, the processed sperms can be stored for a long time, and high integrity is still kept, so that the method is particularly suitable for field collection and collection of wild animal semen.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.
The following will specifically describe a sperm drying solution and a sperm preservation method according to an embodiment of the present invention.
A sperm drying fluid comprising, per liter of sperm drying fluid: 6-7 g of sodium chloride, 0.2-0.3 g of potassium chloride, 0.3-0.6 g of calcium chloride, 0.05-0.1 g of magnesium chloride, 2-3 g of sodium bicarbonate, 0.03-0.05 g of sodium phosphate, 1-1.2 g of glucose, 2.5-3 g of 4-hydroxyethyl piperazine ethanesulfonic acid, 10-50 mmol of raffinose and the balance of water.
The semen drying solution is rich in various inorganic salts, and has functions of maintaining osmotic pressure and controlling acid-base balance. Meanwhile, the sperm drying solution also contains saccharide components such as glucose, raffinose and the like, and can supply energy necessary for cell survival and metabolism. Under the coexistence of inorganic salt components and carbohydrate components, the sperm drying liquid can simulate the environmental state of the sperm growth state in vivo, and provides the basic requirement of the sperm survival in vitro and the maintenance of certain metabolic activity.
In the present invention, a certain amount of raffinose is added to the sperm drying solution. Raffinose (Raffinose) is the best known trisaccharide in nature and is formed by combining galactose, fructose and glucose. It has good solubility, is soluble in water, slightly soluble in polar solvent such as ethanol, and insoluble in nonpolar solvent such as petroleum ether. Meanwhile, the composite material has better thermal stability and acid stability, and can not be decomposed at a certain high temperature and under a certain strong acidity. Unlike other oligosaccharides, raffinose crystal powder contains 5 molecules of crystal water, does not absorb moisture and cake even in an environment with a relative humidity of 90%, and is more hygroscopic than other oligosaccharide powders.
In the process of preserving sperm, raffinose is not only a main non-electrolyte which can ease the ionization degree of seminal plasma, but also a main energy substance after sperm separation, and is also one of important substances for regulating osmotic pressure in diluent. Meanwhile, the molecular weight of raffinose is larger, and compared with micromolecular sugar, the raffinose can more effectively stabilize protein-lipid complex of sperm plasma membrane, protect the sperm plasma membrane from oxidation, and thus improve the integrity of sperm.
Preferably, the sperm drying solution comprises, per liter: 6.5-7 g of sodium chloride, 0.25-0.3 g of potassium chloride, 0.35-0.5 g of calcium chloride, 0.07-0.1 g of magnesium chloride, 2.4-2.8 g of sodium bicarbonate, 0.04-0.05 g of sodium phosphate, 1-1.1 g of glucose, 2.6-2.9 g of 4-hydroxyethyl piperazine ethanesulfonic acid, 10-50 mmol of raffinose and the balance of water. The sperm drying liquid under the optimal value has better sperm preservation effect and can effectively maintain the integrity of the sperm.
The pH value of the sperm drying liquid is 7.2-7.6, and preferably 7.4. The pH range is the pH value of the sperm in the organism in the growth state, can effectively prolong the survival time of the sperm in vitro, and is beneficial to ensuring the preservation of the sperm.
A method of sperm preservation comprising: and centrifuging the diluted semen to remove the semen, and mixing with the semen drying solution to obtain the semen suspension.
The semen is preferably newly collected fresh semen, the sperm motility is about 75-98%, the sperm motility is gradually reduced and gradually dies as time goes on, the newly collected semen needs to be diluted by a diluent, wherein the diluent comprises, by weight, 5-10 parts of glucose, 0.5-1 part of sodium citrate and 80-100 parts of water, in the invention, the semen and the diluent are mixed according to the mass ratio of 1: 0.8-1.2, the ratio is an optimal value obtained by combining self experience and creative work of an inventor, the semen is diluted within the ratio range, and the concentration of the sperm in the diluted semen can be 1 × 106～1.2×106In the range of one/mL, to obtain the best dilution effect.
The semen is diluted to reduce the metabolism rate of the sperm, and meanwhile, the sugar in the diluent can also provide nutrition for the sperm. In addition to providing nutrients, sugars can also replace water molecules in hydrophilic tissues, and this property can act to protect cell membranes during rapid temperature changes experienced by cells. Sugars can also alter the effectiveness of the diluent by increasing its viscosity, which can prevent ice crystal formation and facilitate vitrification. Furthermore, the sugar can also prevent membrane damage caused by volume and osmotic pressure changes accompanying extreme dehydration.
Further, the semen after being separated is centrifuged to remove the semen. The semen cleaning component is very suitable for the existence of sperms and plays a role in nutrition and support of the sperms, but researches suggest that a plurality of protein components which are not known by human beings exist in the semen cleaning, and a plurality of anti-fertility factors such as a defunctional factor, a human anti-fertility factor, a rabbit acrosome stabilizing factor, bovine seminal plasma and goat yolk coagulase and the like are found in the semen cleaning of various animals, and the factors influence the sperm motility through mechanisms of inhibiting the capacitation of the sperms, the occurrence of acrosome reactions, inducing the early release of the acrosome enzymes and the like. Most researches consider that semen preservation needs to be performed with semen removal treatment, and mainly that substances such as phospholipase or phospholipase-like substances in the semen are very unfavorable for the quality of the semen.
There are many ways to remove the essence, among them, centrifugation is the simplest and most direct method to remove the essence, and the equipment needed is simple, the operation is convenient, and the removing effect is good. Preferably, the diluted semen is centrifuged for 2-5 min under the condition that the separation factor is 600-800 g. Centrifugation under these conditions can achieve the best separation without undue damage to the sperm. Too long centrifugation time or too high rotation speed can cause damage to the sperms due to mutual overstocking, thereby affecting the sperm motility.
The seminal fluid after the removal of the seminal fluid needs to be mixed with a sperm drying solution and a sperm suspension is formed. The salinity and the sugar contained in the sperm drying liquid can simulate the living environment of the sperm in the body, maintain the osmotic pressure and the pH value of the living environment of the sperm and effectively improve the integrity of the sperm. Meanwhile, the sugar content in the sperm drying solution is similar to that in the diluted solutionAnd also functions to provide energy and protect the cell membrane of the sperm cell, wherein the concentration of sperm in the sperm suspension is 1 × 106～2×106one/mL. The range value is an optimal value obtained by combining self experience with creative work, the sperm suspension in the range has good drying effect in the subsequent drying process, and the content of salt and sugar is proper, so that the integrity of the sperm can be ensured to the maximum extent.
The invention provides a sperm preservation method, which further comprises the following steps: the sperm suspension was evaporated to dryness and stored.
Preferably, the evaporation drying condition of the sperm suspension is selected to be natural evaporation drying at room temperature for 20-40 min. The natural evaporation process is simple to operate, does not need any complex equipment, has low cost and high efficiency, has instantaneity, is not limited by regional conditions, and is particularly suitable for collecting and collecting the semen of wild animals in the field.
Furthermore, the sperm suspension can be dispersed into 10-30 mu L suspension drops, and then natural evaporation drying is carried out. The contact area between the dispersed sperm suspension and the air is increased, and the drying effect can be increased. The volume size of the suspension liquid drop is an optimal value obtained by combining self experience and creative work of the inventor, the drying speed of the suspension liquid drop in the volume range is high, the drying effect is good, and the density of the dried sperms is moderate, so that the suspension liquid drop is suitable for long-term storage of the sperms.
In practical operation, a liquid-transfering gun can be adopted to extract 10-30 mu L of sperm suspension liquid, the sperm suspension liquid is dripped on a glass slide to form suspension liquid drops, then the suspension liquid drops are naturally evaporated and dried, and then the glass slide is placed in a glass slide box for unified storage.
The method for preserving sperm can be used for short-term preservation for one month at room temperature, thereby ensuring higher integrity of sperm. Under the condition of 4 ℃, the preservation time of half a year to one year can be reached, and the temperature is reduced to-80 ℃ or liquid nitrogen, so that the long-term preservation of the sperms can be realized. Further, the preservation is preferably performed in the absence of light. If the conditions allow, the vacuum preservation is carried out, and the obtained preservation effect is better.
The features and properties of the present invention are described in further detail below with reference to examples. It should be noted that, for the comparability of experimental data, the semen used in the following examples is from the college of agricultural sciences of Jilin province, Duroc boars aged 18-22 months, but the method for preserving sperm according to the present invention is not limited thereto. In other preferred embodiments of the present invention, the sperm preservation method can also achieve good preservation effect on sperm of other organisms.
Example 1
This embodiment provides a sperm drying solution, each liter of the sperm drying solution includes: 6.82g of sodium chloride, 0.28g of potassium chloride, 0.35g of anhydrous calcium chloride, 0.09g of magnesium chloride hexahydrate, 2.51g of sodium bicarbonate, 0.048g of sodium phosphate, 1.08g of glucose, 2.76g of 4-hydroxyethyl piperazine ethanesulfonic acid, 10mmol of raffinose and the balance of water.
The present embodiments also provide a method for preserving sperm, comprising:
s1, diluting the collected fresh semen with a diluent until the density of the semen in the semen is 1 × 106one/mL.
S2, centrifuging the diluted semen by using a centrifuge to remove the semen, wherein the centrifugation factor of the centrifuge is 700g, and the centrifugation time is 3 min.
S3, mixing the centrifuged semen with the sperm drying liquid to obtain a sperm suspension, wherein the density of the sperm in the sperm suspension is 1-2 × 106one/mL.
S4, dripping the sperm suspension liquid on a glass slide by a pipette with a small drop of every 20 mu L to form a suspension liquid drop, and naturally evaporating and drying for 30min at room temperature.
S5, placing the glass slide into a glass slide box for preservation.
Example 2
This example provides a sperm drying solution having substantially the same composition as the sperm drying solution provided in example 1, except that raffinose is included in an amount of 20mmol per liter of the sperm drying solution.
This example also provides a method for preserving sperm, which is the same as the method for preserving sperm provided in example 1, and for details, reference is made to example 1.
Example 3
This example provides a sperm drying solution having substantially the same composition as the sperm drying solution provided in example 1, except that the sperm drying solution contained raffinose in an amount of 50mmol per liter.
This example also provides a method for preserving sperm, which is the same as the method for preserving sperm provided in example 1, and for details, reference is made to example 1.
Example 4
This embodiment provides a sperm drying solution, each liter of the sperm drying solution includes: 6g of sodium chloride, 0.25g of potassium chloride, 0.5g of anhydrous calcium chloride, 0.1g of magnesium chloride hexahydrate, 2g of sodium bicarbonate, 0.05g of sodium phosphate, 1g of glucose, 2.5g of 4-hydroxyethyl piperazine ethanesulfonic acid, 20mmol of raffinose and the balance of water.
The present embodiments also provide a method for preserving sperm, comprising:
s1, diluting the collected fresh semen with a diluent until the density of the semen in the semen is 1.2 × 106one/mL.
S2, centrifuging the diluted semen by using a centrifuge to remove the semen, wherein the centrifugation factor of the centrifuge is 600g, and the centrifugation time is 5 min.
S3, mixing the centrifuged semen with the sperm drying liquid to obtain a sperm suspension, wherein the density of the sperm in the sperm suspension is 1-2 × 106one/mL.
S4, dripping the sperm suspension liquid on a glass slide by a pipette with a small drop of 30 mu L to form a suspension liquid drop, and naturally evaporating and drying for 40min at room temperature.
S5, placing the glass slide into a glass slide box for preservation.
Example 5
This embodiment provides a sperm drying solution, each liter of the sperm drying solution includes: 6.5g of sodium chloride, 0.3g of potassium chloride, 0.6g of anhydrous calcium chloride, 0.05g of magnesium chloride hexahydrate, 2.4g of sodium bicarbonate, 0.03g of sodium phosphate, 1.2g of glucose, 3g of 4-hydroxyethyl piperazine ethanesulfonic acid, 20mmol of raffinose and the balance of water.
The present embodiments also provide a method for preserving sperm, comprising:
s1, diluting the collected fresh semen with a diluent until the density of the semen in the semen is 1.2 × 106one/mL.
S2, centrifuging the diluted semen by using a centrifuge to remove the semen, wherein the centrifugation factor of the centrifuge is 800g, and the centrifugation time is 2 min.
S3, mixing the centrifuged semen with the sperm drying liquid to obtain a sperm suspension, wherein the density of the sperm in the sperm suspension is 1-2 × 106one/mL.
S4, dripping the sperm suspension liquid on a glass slide by a pipette with a small drop of every 10 mu L to form a suspension liquid drop, and naturally evaporating and drying for 20min at room temperature.
S5, placing the glass slide into a glass slide box for preservation.
Example 6
This embodiment provides a sperm drying solution, each liter of the sperm drying solution includes: 7g of sodium chloride, 0.2g of potassium chloride, 0.3g of anhydrous calcium chloride, 0.1g of magnesium chloride hexahydrate, 3g of sodium bicarbonate, 0.05g of sodium phosphate, 1g of glucose, 2.9g of 4-hydroxyethyl piperazine ethanesulfonic acid, 20mmol of raffinose and the balance of water.
The present embodiments also provide a method for preserving sperm, comprising:
s1, diluting the collected fresh semen with a diluent until the density of the semen in the semen is 1 × 106one/mL.
S2, centrifuging the diluted semen by using a centrifuge to remove the semen, wherein the centrifugation factor of the centrifuge is 700g, and the centrifugation time is 3 min.
S3, mixing the centrifuged semen with the sperm drying liquid to obtain a sperm suspension, wherein the density of the sperm in the sperm suspension is 1-2 × 106one/mL.
S4, dripping the sperm suspension liquid on a glass slide by a pipette with a small drop of every 20 mu L to form a suspension liquid drop, and naturally evaporating and drying for 30min at room temperature.
S5, placing the glass slide into a glass slide box for preservation.
Example 7
This embodiment provides a sperm drying solution, each liter of the sperm drying solution includes: 6.5g of sodium chloride, 0.25g of potassium chloride, 0.35g of anhydrous calcium chloride, 0.07g of magnesium chloride hexahydrate, 2.8g of sodium bicarbonate, 0.04g of sodium phosphate, 1.1g of glucose, 2.6g of 4-hydroxyethyl piperazine ethanesulfonic acid, 20mmol of raffinose and the balance of water.
This example also provides a method of preserving sperm, which is the same as the method of preserving sperm provided in example 6, and for further details, reference may be made to example 6.
Comparative example
This comparative example provides a sperm drying solution, comprising per liter of the sperm drying solution: 6.82g of sodium chloride, 0.28g of potassium chloride, 0.35g of anhydrous calcium chloride, 0.09g of magnesium chloride hexahydrate, 2.51g of sodium bicarbonate, 0.048g of sodium phosphate, 1.08g of glucose, 2.76g of 4-hydroxyethyl piperazine ethanesulfonic acid and the balance of water.
This comparative example also provides a sperm cell preservation method that is the same as the sperm cell preservation method provided in example 1, and reference may be made to example 1 for specific details.
Test example 1
And (3) taking the sperm drying solution provided by the embodiments 1-3 and the comparative example, processing the semen according to the sperm preservation method provided by the embodiment 1 to obtain a dried semen sample, preserving the dried semen sample at room temperature for 1 month, and performing the following tests on the dried semen sample.
1. Each dried semen sample was tested by TUNEL (in situ end labeling of DNA fragmentation) and the amount of apoptotic sperm was detected by flow cytometry, 10000 sperm tested. The percentage of cells that were positive in the test results, i.e., DNA-disrupted cells, was calculated and the results are shown in table 1.
2. And (2) combining each dry semen sample with an egg cell by using an ICSI (intracytoplasmic sperm injection technology), culturing the obtained embryo in vitro, and observing the cleavage rate when the embryo is cultured for 48 hours, wherein the cleavage rate is the number of cleavage embryos/the number of tested eggs multiplied by 100%, and the blastocyst rate is the number of blastocysts/the number of tested eggs multiplied by 100% when the embryo is cultured for 168 hours. The test was repeated 8 times for each dried semen sample and the calculation results are shown in table 2.
TABLE 1 TUNEL test Positive cell ratios
| Positive cell ratio/% | |
| Example 1 | 74 |
| Example 2 | 40 |
| Example 3 | 69 |
| Comparative example | 78 |
| Fresh semen | 8 |
TABLE 2 cleavage Rate and blastocyst development Rate
As can be seen from Table 1, after 1 month of storage at room temperature, the proportion of DNA-disrupted cells in the dried sperm sample was reduced and the integrity of the cells was higher in the case where raffinose was added to the sperm drying solution (examples 1-3) than in the case where raffinose was not added to the sperm drying solution (comparative example); wherein, when the content of the raffinose is 20mmol/L, the effect is most remarkable, and the proportion of the DNA broken cells is only 40%. Meanwhile, compared with the dry sperm sample of the comparative example, the dry sperm samples of the examples 1-3 have the advantages that the cleavage rate and the blastocyst development rate are improved after the dry sperm sample is combined with the egg cells, and the improvement effect is also optimal when the content of the raffinose is 20 mmol/L. The sperm drying liquid provided by the invention has a relatively obvious protective effect on sperm cells, and can obtain an optimal protective effect when the content of raffinose is 20 mmol/L.
Test example 2
The sperm drying solution provided in example 2 was taken, semen was processed according to the sperm preservation method provided in example 1 to obtain dried semen samples, which were stored at room temperature, 4 ℃, -20 ℃ and-80 ℃, respectively, and the dried semen samples were sampled at 1 month, 3 months, 1 year and 2 years, respectively, and tested as follows.
1. Dried semen samples were tested by TUNEL method (in situ end labeling of DNA fragmentation) and the amount of apoptotic sperm was detected by flow cytometry, 10000 sperm tested. The percentage of cells that were positive in the test results, i.e., DNA-disrupted cells, was calculated and the results are shown in table 3.
2. Combining a dry semen sample with an egg cell by an ICSI (intracytoplasmic sperm injection technology), culturing the obtained embryo in vitro, and observing the cleavage rate when the embryo is cultured for 48 hours, wherein the cleavage rate is equal to the number of cleavage embryos/the number of tested eggs multiplied by 100 percent, and the blastocyst rate is equal to the number of blastocysts/the number of tested eggs multiplied by 100 percent when the embryo is cultured for 168 hours. The test was repeated 8 times for each dried semen sample and the calculations are shown in table 4.
TABLE 3 TUNEL test Positive cell ratios
TABLE 4 cleavage Rate and blastocyst development Rate
As can be seen from Table 3, the dried sperm cell samples provided in example 2 were stored for a short period of time at room temperature and maintained high integrity for periods of 1 month or even 3 months. While when the temperature is lowered to 4 ℃, the dried sperm sample can be stored for 1 year while still maintaining high integrity. And when the temperature is continuously reduced to-20 ℃ or even-80 ℃, the long-term storage can be carried out. Meanwhile, as can be seen from table 4, the dried sperm samples provided in example 2 can be stored for a short period at room temperature, still bind to the egg cells, and maintain a high cleavage rate and blastocyst development rate. When the temperature is reduced to 4 ℃, the dried sperm sample still can reach more than 75 percent of cleavage rate and more than 30 percent of blastocyst development rate after being stored for 1 year. When the temperature is continuously reduced to-20 ℃ or even-80 ℃, the good activity can be still kept after long-term storage.
In summary, the sperm drying fluid provided by the invention can simulate the environmental state of sperm in the growth state, has the functions of maintaining osmotic pressure, controlling acid-base balance, supplying energy and inorganic salt components necessary for cell survival and metabolism, and the like, and can provide the basic requirements for sperm survival in vitro and certain metabolic activity maintenance. In the invention, a certain amount of raffinose is added into the sperm drying liquid, and the raffinose is not only a main non-electrolyte and can moderate the ionization degree of seminal plasma, but also a main energy substance after sperm separation, and is one of important substances for regulating osmotic pressure in a diluent. Meanwhile, the molecular weight of raffinose is larger, and compared with micromolecular sugar, the raffinose can better protect sperms and improve the integrity of the sperms. The invention provides a sperm preservation method, which adopts a centrifugal method to remove the spermatic clear in the semen, then mixes the spermatic clear liquid with the sperm drying liquid, and finally adopts an evaporation drying method to dry. The whole process is simple to operate, complex freezing and drying equipment is not needed, the processed sperms can be stored for a long time, and high integrity is still kept, so that the method is particularly suitable for field collection and collection of wild animal semen.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A sperm drying fluid comprising, per liter: 6-7 g of sodium chloride, 0.2-0.3 g of potassium chloride, 0.3-0.6 g of calcium chloride, 0.05-0.1 g of magnesium chloride, 2-3 g of sodium bicarbonate, 0.03-0.05 g of sodium phosphate, 1-1.2 g of glucose, 2.5-3 g of 4-hydroxyethyl piperazine ethanesulfonic acid, 20mmol of raffinose and the balance of water.
2. The sperm drying fluid of claim 1, comprising, per liter of the sperm drying fluid: 6.5 to 7g of sodium chloride, 0.25 to 0.3g of potassium chloride, 0.35 to 0.5g of calcium chloride, 0.07 to 0.1g of magnesium chloride, 2.4 to 2.8g of sodium bicarbonate, 0.04 to 0.05g of sodium phosphate, 1 to 1.1g of glucose, 2.6 to 2.9g of 4-hydroxyethylpiperazine ethanesulfonic acid, 20mmol of raffinose, and the balance of water.
3. The sperm drying solution of claim 1, wherein the pH of the sperm drying solution is between 7.2 and 7.6.
4. A method of sperm preservation comprising: centrifuging the diluted semen to remove the semen, and mixing the semen with the sperm drying solution as described in any one of claims 1 to 3 to obtain a sperm suspension; the sperm suspension was evaporated to dryness and stored.
5. A method of sperm preservation according to claim 4, characterised in that said semen is diluted by: mixing the semen and the diluent according to the mass ratio of 1: 0.8-1.2, and the diluent comprises the following components in parts by weight: 5-10 parts of glucose, 0.5-1 part of sodium citrate and 80-100 parts of water.
6. The method of preserving sperm cells as described in claim 4, wherein said concentration of sperm cells in said diluted semen is 1 × 106～1.2×106one/mL.
7. A sperm cell preservation method according to claim 4, wherein said diluted semen is centrifuged for 2-5 min at a separation factor of 600-800 g to remove the semen.
8. The method of preserving sperm cells of claim 4, wherein said sperm cell suspension has a sperm cell concentration of 1 × 106～2×106one/mL.
9. A sperm cell preservation method according to claim 4, wherein the conditions for the evaporation drying of the sperm suspension are natural evaporation drying at room temperature for 20-40 min.
10. The method for preserving sperm according to claim 9, wherein the sperm suspension is dispersed into 10 to 30 μ L of suspension droplets, and the suspension droplets are evaporated and dried for preservation.
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| "Desiccation tolerance in bovine sperm: A study of the effect of intracellular sugars and the supplemental roles of an antioxidant and a chelator";Ranjan Sitaula et al.;《Cryobiology》;20090324;第58卷(第3期);第322页右栏第2段，第323页右栏第1-3段，表1，第324页左栏第1-2段，第325页左栏第2段，图3，图12，第328页左栏第2段 * |
| "The effects of different sugars on motility, morphology and DNA damage during the liquid storage of rat epididymal sperm at 4 ℃";Serpil Sarıozkan et al.;《Cryobiology》;20120523;第65卷(第2期);第93页右栏第1段，第94页左栏第3段，右栏第2-5段，表1-2 * |
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